Products for DNA Research · dna damage/repair 61 click dna and rna ligation 64 5’-labeling of...

Products for DNA Research2020 Catalog

glenresearch.com

Oligo synthesis success. The first time and every time.

TABLE OF CONTENTS

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INTRODUCTION 5ABOUT US 5CATALOG 6

STERLING 7QUALITY AND PERFORMANCE ASSURED 7

APPLIED BIOSYSTEMS INSTRUMENTS 8STERLING CE PHOSPHORAMIDITES 8STERLING SOLVENTS/REAGENTS 8STERLING SUPPORTS 9ABI 3900 POLYSTYRENE MODIFIER COLUMNS 11

EXPEDITE™ INSTRUMENTS 12STERLING CE PHOSPHORAMIDITES 12STERLING SOLVENTS/REAGENTS 12STERLING SUPPORTS 13

DNA PHOSPHORAMIDITES - SPECIAL PACKAGING 15

MERMADE INSTRUMENTS 16STERLING CE PHOSPHORAMIDITES 16STERLING SOLVENTS/REAGENTS 16STERLING SUPPORTS 17

GE HEALTHCARE LIFE SCIENCES INSTRUMENTS 18STERLING CE PHOSPHORAMIDITES 18STERLING SOLVENTS/REAGENTS 19

DR. OLIGO INSTRUMENTS 20STERLING CE PHOSPHORAMIDITES 20STERLING SOLVENTS/REAGENTS 20STERLING SUPPORTS 21OLIGONUCLEOTIDE PURIFICATION 21

ALTERNATIVE PROTECTING GROUPS 22DEPURINATION RESISTANT CE PHOSPHORAMIDITES 22ULTRAMILD CE PHOSPHORAMIDITES 23ULTRAMILD SUPPORTS 23ULTRAMILD SOLVENTS/REAGENTS 23

ULTRAMILD DNA SYNTHESIS 23

SUPPORTS 24GLEN UNYSUPPORT 24GLEN UNYSUPPORT FC 25UNIVERSAL SUPPORT III 26Q-SUPPORTS 27HIGH LOAD CPG 29

REAGENTS 30ALTERNATIVE SOLVENTS/REAGENTS 30CSOFORNON-AQUEOUSOXIDATION 32UNICAP PHOSPHORAMIDITE 32

BACKBONE MODIFICATION 33SULFURIZING REAGENTS 335’-CEPHOSPHORAMIDITES 345’-SUPPORTS 35METHYL PHOSPHONAMIDITES 36PACE PHOSPHORAMIDITES 37METHYL PHOSPHORAMIDITES 38

TABLE OF CONTENTS

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ULTRAMILD SOLVENTS/REAGENTS 38H-PHOSPHONATEMONOMERS 39H-PHOSPHONATEREAGENTS 39BETA-L-DNAMONOMERS 40LOCKED ANALOG PHOSPHORAMIDITES 41

OLIGONUCLEOTIDE-DIRECTED MUTAGENESIS 42TRIMER PHOSPHORAMIDITES 42

DUPLEX STABILITY MODIFICATION 46BASESAFFECTINGDUPLEXSTABILITY 46ZIPNUCLEICACIDS(ZNA®) 48CDPI

3 MGB™ LABELING 48SELECTIVELYBINDINGCOMPLEMENTARY(SBC)OLIGOS 48UNNATURAL BASE PAIRS 48CAPSFORINCREASEDDUPLEXSTABILITYANDBASE-PAIRINGFIDELITY 49

EPIGENETICS 50DNA METHYLATION 50

PCR/SEQUENCING APPLICATIONS 51DUPLEXEFFECTS 51Tm MODULATION 53CLEANAMP® MONOMERS 54CHAIN TERMINATORS 55

STRUCTURAL STUDIES 57STRUCTURE/ACTIVITY RELATIONSHIP 57HALOGENATED NUCLEOSIDES 60DNA DAMAGE/REPAIR 61CLICK DNA AND RNA LIGATION 645’-LABELINGOFMicroRNAs 642’-5’LINKEDOLIGONUCLEOTIDES 65MUTAGENESIS 66IN SITU SYNTHESIS OF DNA ANALOGS 67PROBING DNA STRUCTURE WITH FLUORESCENT NUCLEOSIDES 68PHOTO-REGULATIONOFDNAFUNCTION 70INHIBITION OF DNA METHYLTRANSFERASES 71LARGE SCALE SYNTHESIS 71NON-CANONICALSTRUCTURES 72G-QUADRUPLEX 72TRIPLEX-FORMINGOLIGONUCLEOTIDES 72i-MOTIFDNASTRUCTURES 72APTAMER DEVELOPMENT 73

MODIFIERS 74TERMINUS MODIFIERS 74SEQUENCE MODIFIERS 773’-MODIFIERS 79CHEMICAL PHOSPHORYLATION 82ALDEHYDE MODIFICATION 83SPACER MODIFIERS 84DENDRIMERS 85BRANCHING PHOSPHORAMIDITE 85PHOTOCLEAVABLE MONOMERS 86CONJUGATION USING CLICK CHEMISTRY 87OLIGO-CLICKKITS 90COPPER-FREECLICKCHEMISTRY 91SERINOL REAGENTS FOR MODIFICATION AND LABELING 94COT SERINOL PHOSPHORAMIDITE 97DABCYL LABELING 98BIOTIN LABELING 99FLUORESCEIN LABELING 102FLUORESCEINLABELING(SIMA) 105

TABLE OF CONTENTS

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CYANINE LABELING 106ELITECHGROUP DYES AND QUENCHER 108BLACK HOLE QUENCHER DYES 110BLACKBERRY®QUENCHER(BBQ-650®) 112RHODAMINE(TAMRA)LABELING 113ACRIDINE LABELING 114DNP LABELING 114CHOLESTEROL LABELING 115TOCOPHEROL LABELING 115STEARYL LABELING 115N-ACETYLGALACTOSAMINE(GalNAc)LABELING 116CDPI3 MGB™ LABELING 117PSORALEN LABELING 118EDTA LABELING 118FERROCENE LABELING 119METHYLENE BLUE LABELING 119LABELING WITH THIAZOLE ORANGE 120LABELING WITH METAL CHELATES 121LABELING WITH POLYAROMATIC HYDROCARBONS 121PUROMYCIN CPG 122QUENCHEDAUTOLIGATION(QUAL)PROBES 122LABELINGFORPHOTO-REGULATIONOFOLIGONUCLEOTIDES 123LABELINGWITHULTRAFASTPHOTOCROSS-LINKER 124

RNA SUPPORTS 125RNA SUPPORTS FOR 3’ MODIFICATION 125

RNA SYNTHESIS 126TOM-PROTECTEDRNAPHOSPHORAMIDITES 126RNA SUPPORTS FOR TOM RNA SYNTHESIS 126TBDMS-PROTECTEDRNAPHOSPHORAMIDITES 128RNAPHOSPHORAMIDITES-SPECIALPACKAGING 128ULTRAMILD TBDMS RNA PHOSPHORAMIDITES 129TBDMS RNA SUPPORTS 129ULTRAMILD SOLVENTS/REAGENTS 130

MINOR RNA BASES 131MINORRNAPHOSPHORAMIDITES(TOMPROTECTED) 131RNASEQUENCEMODIFIER(TOMPROTECTED) 132MINORRNAPHOSPHORAMIDITES(TBDMSPROTECTED) 133MINOR RNA TRIPHOSPHATES 136

2’-OME-RNA SYNTHESIS 1372’-OME-RNAPHOSPHORAMIDITES 137ULTRAMILD2’-OME-RNA 138ULTRAMILD SOLVENTS/REAGENTS 1382’-OME-RNASUPPORTS 139MINOR2’-OME-RNAPHOSPHORAMIDITES 1402’-OME-THIOPHOSPHORAMIDITES 141

2’-MOE-RNA PHOSPHORMIDITES 1422’-MOERNAPHOSPHORAMIDITES 142

2’-F RNA SYNTHESIS 1432’-F-RNAPHOSPHORAMIDITES 143

2’-F ANA SYNTHESIS 1442’-F-ARABINONUCLEICACID(2’-F-ANA) 144

2’-OME-RNA-PACE SYNTHESIS 1452’-OME-RNA-PACEPHOSPHORAMIDITES 145

PURIFICATION 147GLEN-PAK™PURIFICATION 147

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POLY-PAK™PURIFICATION 149GLENGEL-PAK™DESALTING 150OLIGO-AFFINITYSUPPORT 151

PHYSICAL DATA 152PHYSICAL DATA 152

INDEX 162

GENERAL INFORMATION 172ORDERING 172DISCOUNTS 172TERMS AND CONDITIONS OF SALE 172PATENTS 172

INTRODUCTION

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GABOUT US

GlenResearchdevelops,manufacturesandmarkets reagents foroligonucleotide synthesis,modification, labelingandpurification.Thecompanyservescustomersworldwideinvolvedinbasicresearch,diagnosticsandtherapeutics.AlthoughGlenResearch’soriginalmissionwastoprovidestate-of-the-artreagentstoresearchers,thecompanyalsobeganofferingstandardreagentsforoligonucleotidesynthesisbutwiththeinnovationthateverybatchwasaccompaniedbyaCertificateofAnalysis.Theanalyticaltechniquesandqualitycriteriausedfortheevaluationandacceptanceofthesereagentsweretobecomeanindustrystandardyearslater.ThecompanyisheadquarteredinSterling,Virginia.Aprivatelyheldcompany,GlenResearchwasacquiredbyMaravaiLifeSciencesinDecember2017.

OVER 30 YEARS OF ASSURED QUALITY FOR OLIGO SYNTHESIS

1987 GlenResearchwasincorporatedintheCommonwealthof Virginia

1991Company awarded SBIR grant for the investigationof large scale oligonucleotide synthesis usingH-phosphonatechemistry1993

Glen Research introduced the Sterling line of products, anewstandardofqualityforoligonucleotidesynthesis 1995

GlenResearchnegotiatedanexclusiveagreementtosupply5’-biotinphosphoramiditeworldwide

1996 CompanynegotiatedanexclusivelicensewithGileadSciencestosupplyC5-propynylpyrimidinenucleosidesandG-Clampphosphoramidites 1997

GlenResearchmoves intoacustombuiltbuilding inSterling, Virginia

1999 Company awarded patents for a chemica lphosphorylation reagent compatiblewithDMT-ONpurification 2002

CompanymadeanagreementwithEpochBiosciences,Inc.tosupplytheirproprietarydyesandnucleosidesto the research market2003

Glen Research negotiated an agreementwithGEHealthcareBiosciencesCorp.tosupplyCyanineDyesto the research market 2004

Companyawardedpatentsforatrulyuniversalsupportforoligonucleotidesynthesis-USIII.

2006In collaborationwithBerry&Associates, Inc.,GlenResearch awardedpatents for pyrrolo-C analogues(fluorescentCanalogues). 2008

GlenResearchobtainedalicenseforthesaleofGlenUnySupportfromIonisPharmaceuticals

2013In collaborationwithNelsonBiotechnologies, Inc.,companyawardedpatentforserinolphosphoramiditesand supports

2019GlenResearchreceivesitsISO9001:2015certificationforQualityManagementSystems

2017GlenResearchisacquiredbyMaravaiLifeSciences

CATALOG

WelcometotheGlenResearchCatalogcontainingthemostcompleteselectionofproductsforDNAandRNAresearch. TheTableofContentsatthebeginningandtheIndexattheendoftheCatalogarethemostcomprehensivewehaveproduced.Therearealwayslimitationstoprintedcatalogsinafast-movingtechnologysectorandacompleteandup-to-datecatalogisalsomaintainedonourwebsite.

Allminorbases,modifiersandRNAproductsarepackagedforAppliedBiosystemsinstruments.Wecanprovidevialsandcolumnsforawidevarietyofotherinstruments.Asshowninthetabletotheleft,wecanaccommodatecatalognumbersforunusualproductstofitallpopularinstruments.Thetabletotheleftisreproducedonallrelevantspreadsofthiscatalog.

WeareuniqueinconductingaQCtestforsupportstoshowthelengthofoligothatcanbepreparedbeforeadrop-offincouplingduetostericeffectsbeginstooccur.Thedrop-offpointisrecordedintheCertificateofAnalysisorAnalyticalReport.Unlessotherwisespecified,ourminorbaseandmodificationsupportsare1000ÅCPG,whichresultsinimprovedperformanceandtheabilitytomakemuchlongeroligos.Polystyrenesupportsarealsoavailableforsomeofourmostpopularitems.

Forreasonsofqualityassurance,wedonottransferpowdersoroilsfromstockAppliedBiosystemsvialstovialsforotherinstruments.Powdersmaybehygroscopicandelectrostatic,makingtransferdifficult,andoilshavetobedissolvedandthesolventevaporated.Forbestperformance,itispreferableforthecustomertodissolvetheproductandimmediatelytransferthesolutiontothecorrectinstrumentvial.Consequently,theproductwillbedeliveredinanindustry-standardseptum-cappedvialalongwithacleandryvialfortheappropriateinstrument.

GlenResearchwillonlyguaranteeproductspurchasedthroughourofficialdistributors.Acompletelistingofauthorizeddistributorscanbefoundonourwebsiteat:https://www.glenresearch.com/international-distributors.

OTHER INSTRUMENT TYPES

Allminorbases,RNAproductsandmodifiersarepackagedinseptum-cappedvialssuitableforABIandotherinstruments.Ifyouwouldlikeanothertypeofvial/columnaddthefollowingtotheendof thecatalognumber.

MonomersFor Instrument type Add

Expedite EMerMade M

ColumnsFor Instrument type Add

Expedite E AppliedBiosystems3900 A MerMade M

(Please inquire for availability of vials and columns for other instrument types.)

INTRODUCTION

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https://www.glenresearch.com/international-distributors

Excellence Since 1987

QUALITY AND PERFORMANCE ASSURED

GlenResearchhasdevelopedandimplementedaQualityManagementSystem(QMS)designedtoenhance

customersatisfactionbyfocusingonprocessesforcontinualimprovementandonassuranceofconformityto

customer needs, withfullconsiderationofapplicableregulatoryrequirements.

STERLING PERFORMANCE

The standard of accomplishment for DNA and

RNAsynthesis.EverybatchofSterlingreagentsis

analyzedbytitrationtoconfirmexactformulation.

EverybatchofSterlingmonomers,supportsand

activators is synthesis-tested toensureoptimal

performance.CertificatesofAnalysisprovideyour

guaranteeofSterlingPerformance.

STERLINGisatrademarkofGlenResearchCorporation.

Glen Research offersthehighestlevelofQualityAssuranceforreagentsforDNAandRNAsynthesis-Sterling

QualityandPerformance.WenowapplytheSterlingcriteriaofqualityandperformancetoallofGlenResearch’s

establishedproducts.

Thecommonmonomersandsupports,whosestructuresareillustratedbelow,areavailableforthevarietyof

synthesizerslistedonthefollowingpages.

dA-CE Phosphoramidite dC-CE Phosphoramidite Ac-dC-CE Phosphoramidite dG-CE Phosphoramidite dT-CE Phosphoramidite

STERLING QUALITY

The benchmark for excellence in DNA and

RNA synthesis. All Sterlingmaterialsmust

passstringentpurityand identitytestspriorto

acceptance. Sterlingproductsare formulated,

filtered,andpackagedinoptimalenvironments

using specially cleaned and dried glassware

and columns. Color-coded labeling andpost-

packaging analysis guarantee accuracy and

SterlingQuality.

dT-lcaa-CPGdG-lcaa-CPGAc-dC-lcaa-CPGdC-lcaa-CPGdA-lcaa-CPG

STERLING

O

O P N(iPr)2O CNEt

DMTO

NHBz

N

N

N

N

O

O P N(iPr)2O CNEt

DMTO

NHBz

O N

N

O

O P N(iPr)2O CNEt

DMTO

NHAc

O N

N

O

O P N(iPr)2O CNEt

DMTOiBuHN

O

N

N

N

HN

O

O P N(iPr)2O CNEt

DMTOO

O

N

HNCH 3

ODMTO

OOlcaaCPGCCH 2CH 2CO

NHBz

N

N

N

N

ODMTO

NHBz

O N

N


ODMTO

NHAc

O N

N


ODMTO


iBuHN

O

N

N

N

HN

ODMTO


O

O

N

HNCH 3

STERLING

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QUALITY ASSURANCE

EverybatchoftheseCEPhosphoramiditesistestedasfollows:

1. HPLCa) Identityisconfirmedbycomparison

withareferencesample.b)PurityisdeterminedbyHPLCtobe

≥98.0%.2. TLC

PurityisverifiedbyTLC.3. 31P NMR

Purityisdeterminedby31P NMR to be≥98%.

4. Coupling Test Couplingefficiencyisdeterminedtobe≥99%.

5. Solution Test A0.1Msolutionisdeterminedtobeclearandfreeofparticulatecontamination.

6. Loss on Drying Volatilecontaminantsaredeterminedtobe≤2%.

dmf-dG-CE Phosphoramidite

O

O P N(iPr)2O CNEt

DMTO

(Me)2NN

O

N

N

N

HN

ABI INSTRUMENTS

1. 60mLseptum-cappedvialsusedon oldest ABI 380, 381 and 391 instruments.200mLoxidizerand450mLdeblockscrew-cappedbottlesalso used on ABI 380, 381 and 391 instruments.

2. Smallscrew-cappedvialsusedonABI392and394instruments.

3. Largerscrew-cappedvialsusedonABI392.394and3400instruments.

4. LargebottlesusedonABI3900instruments.

STERLING CE PHOSPHORAMIDITES

GlenResearchCE(β-cyanoethyl)PhosphoramiditesareproducedandpackagedtoensurethehighestperformanceonDNAsynthesizers.EveryGlenResearchproductisaccompaniedbyaCertificateofAnalysisandHPLCtrace,showingtheresultsofourQCtesting.EveryGlenResearchmonomervialisspeciallycleanedtoeliminateparticulatecontaminationandtestedtoensureatightfitonsynthesizers.

Item Catalog No. Pack

dA-CEPhosphoramidite 10-1000-02 0.25g 10-1000-05 0.5g 10-1000-10 1.0g 10-1000-20 2.0g 10-1000-40 4.0gdC-CEPhosphoramidite 10-1010-02 0.25g 10-1010-05 0.5g 10-1010-10 1.0g 10-1010-20 2.0g 10-1010-40 4.0gAc-dC-CEPhosphoramidite 10-1015-02 0.25g 10-1015-05 0.5g 10-1015-10 1.0g 10-1015-20 2.0g 10-1015-40 4.0gdG-CEPhosphoramidite 10-1020-02 0.25g 10-1020-05 0.5g 10-1020-10 1.0g 10-1020-20 2.0g 10-1020-40 4.0gdmf-dG-CEPhosphoramidite 10-1029-02 0.25g 10-1029-05 0.5g 10-1029-10 1.0g 10-1029-20 2.0g 10-1029-40 4.0gdT-CEPhosphoramidite 10-1030-02 0.25g 10-1030-05 0.5g 10-1030-10 1.0g 10-1030-20 2.0g 10-1030-40 4.0g

STERLING SOLVENTS/REAGENTS

Allsolventsandreagentsarepreparedtoourexactingspecificationstoensurethehighestsynthesisefficiencyandarepassedthrougha0.2micronfilterduringpackagingtoeliminateparticulatecontamination.GlenResearchusesfreshlysublimed1H-tetrazoleforpremiumperformanceonAppliedBiosystemssynthesizers.

Item Catalog No. PackActivatorTetrazoleinAcetonitrile 30-3100-451 45mL 30-3100-522 200mL 30-3100-573 450mL 30-3100-624 2000mL

DiluentAcetonitrile,anhydrous 40-4050-45 60mL 40-4050-50 100mL

APPLIED BIOSYSTEMS INSTRUMENTS

RELATED

DepurinationResistantdA........22

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ABBREVIATIONS

Ac2O=AceticAnhydride CE=Cyanoethyl CPG = Controlled Pore Glass DCM = Dichloromethane dmf=dimethylformamidine I2 = Iodine lcaa=longchainalkylamino MeIm=1-Methylimidazole µm=micromole(s) nm=nanomole(s) TCA=TrichloroaceticAcid THF=Tetrahydrofuran

dmf-dG-CPG

O

O

DMTO

(Me)2NN

O

N

N

N

HN

C CH 2CH 2C lcaaO O

CPG

STERLING CE PHOSPHORAMIDITES (CONT.)


Cap Mix ATHF/Pyridine/Ac2O 40-4110-451 45mL 40-4110-522 200mL 40-4110-573 450mL 40-4110-624 2000mL

Cap Mix B16%1-MeIminTHF 40-4220-451 45mL (This Cap B solution is identical to the 40-4220-522 200mL formulation produced by Applied Biosystems.) 40-4220-624 2000mL

Oxidizing Solution0.02MI2inTHF/Pyridine/H2O 40-4330-521,2 200mL 40-4330-573 450mL 40-4330-624 2000mL

Deblocking Mix3%TCA/DCM 40-4140-571,2 450mL 40-4140-623,4 2000mL

STERLING SUPPORTS

All Glen Research CPG supportsusethestandardlongchainalkylamino(lcaa)linkerbutdifferintheglassporesize,500Å,1000Åor2000Å.The500Åsupport isappropriateforshortersequences,whilethe1000Åsupportsperformbetter inthesynthesisoflonger(>30-mer)DNAsequences.The2000Åsupportisbestforverylong(>150-mer)oligonucleotides. WehaveinstitutedanadditionalQCtestforsupportstoshowthelengthofoligothatcanbepreparedbeforeadrop-offincouplingduetostericeffectsbeginstooccur.Thedrop-offpointisrecordedintheCertificateofAnalysis.AllGlenResearchsupportsarefullyend-cappedtoensurethattheCPGsurfaceistotallyinert,therebyavoidingtheintroductionofimpuritysequencescontainingdeletionsatthe3’-terminus.

Catalog No. Catalog No. Catalog No. Catalog No. Catalog No. Catalog No. Catalog No. Pack dA dC dG dT dA,dC,dG,dT Ac-dC d m f - d G (1columnof) eachbase)

500Å Columns

20-2100-42 20-2110-42 20-2120-42 20-2130-42 20-2140-42 20-2113-42 4x0.2µm20-2100-41 20-2110-41 20-2120-41 20-2130-41 20-2140-41 20-2113-41 4x1.0µm20-2100-13 20-2110-13 20-2120-13 20-2130-13 20-2113-13 1x10µm

1000Å Columns

20-2101-45 20-2111-45 20-2121-45 20-2131-45 20-2141-45 20-2115-45 20-2129-45 4x40nm20-2101-42 20-2111-42 20-2121-42 20-2131-42 20-2141-42 20-2115-42 20-2129-42 4x0.2µm20-2101-41 20-2111-41 20-2121-41 20-2131-41 20-2141-41 20-2115-41 20-2129-41 4x1.0µm20-2101-13 20-2111-13 20-2121-13 20-2131-13 20-2115-13 20-2129-13 1x10µm


RELATED

AlternativeSolvents..................30

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ABI 3900 1000Å CPG COLUMNS

GlenResearch’sABI39001000ÅCPGcolumnsbringthelowercostofCPGtothisplatformwhilemaintainingthehighsynthesisefficiencyof1000ÅCPG.Ourcolumnsofferthefollowingkeyattributes:• Noneedtochangeinstrumentsettings• Noneedtochangesoftware

parameters• Easierhandlingpost-synthesis

compared to PS• Highquality1000ÅCPGforoptimalsynthesisresults

BULK CPG LOADING

500Åsupports 35-50µmoles/g 1000Åsupports 25-40µmoles/g

STERLING SUPPORTS (CONT.)

Catalog No. Catalog No. Catalog No. Catalog No. Catalog No. Catalog No. Catalog No. Pack dA dC dG dT dA,dC,dG,dT Ac-dC d m f - d G (1columnof) eachbase)

2000Å Columns

20-2102-42 20-2112-42 20-2122-42 20-2132-42 20-2142-42 4x0.2µm

Low Volume (LV) Polystyrene Columns

26-2100-45 26-2110-45 26-2120-45 26-2130-45 26-2140-45 4x40nm26-2100-42 26-2110-42 26-2120-42 26-2130-42 26-2140-42 4x0.2µm

ABI 3900 Polystyrene Columns

26-2600-65 26-2610-65 26-2630-65 26-2629-65 200x40nm26-2600-62 26-2610-62 26-2630-62 26-2629-62 200x200nm

ABI 3900 1000Å CPG Columns

20-2101-65 20-2131-65 20-2115-65 20-2129-65 200x40nm20-2101-62 20-2131-62 20-2115-62 20-2129-62 200x200nm20-2101-61 20-2131-61 20-2115-61 20-2129-61 200x1.0µm

500Å Bulk CPG

20-2000-01 20-2010-01 20-2020-01 20-2030-01 20-2013-01 0.1g20-2000-02 20-2010-02 20-2020-02 20-2030-02 20-2013-02 0.25g20-2000-10 20-2010-10 20-2020-10 20-2030-10 20-2013-10 1.0g

1000Å Bulk CPG

20-2001-01 20-2011-01 20-2021-01 20-2031-01 20-2015-01 20-2029-01 0.1g20-2001-02 20-2011-02 20-2021-02 20-2031-02 20-2015-02 20-2029-02 0.25g20-2001-10 20-2011-10 20-2021-10 20-2031-10 20-2015-10 20-2029-10 1.0g

2000Å Bulk CPG

20-2002-01 20-2012-01 20-2022-01 20-2032-01 0.1g20-2002-02 20-2012-02 20-2022-02 20-2032-02 0.25g20-2002-10 20-2012-10 20-2022-10 20-2032-10 1.0g


EmptySynthesisColumns-TWIST40nm,0.2umor1um 20-0030-00 Packof10EmptySynthesisColumns-TWIST10um/15um 20-0040-00 Packof10ReplacementFrits-TWIST10um/15um 20-0040-0F Packof20

TWISTisatrademarkofGlenResearchCorporation.


RELATED

Universal Supports....................24Q-Supports................................27High Load Supports...................29

1010

ABI 3900 1000Å CPG COLUMNS

GlenResearch’sABI39001000ÅCPGcolumnsbringthelowercostofCPGtothisplatformwhilemaintainingthehighsynthesisefficiencyof1000ÅCPG.Ourcolumnsofferthefollowingkeyattributes:• Noneedtochangeinstrumentsettings• Noneedtochangesoftwareparameters• Easierhandlingpost-synthesis

compared to PS• Highquality1000ÅCPGforoptimalsynthesisresults

ABI 3900 POLYSTYRENE MODIFIER COLUMNS

SomeofourmorepopularminorbaseandmodifiersupportsareavailableonpolystyreneincolumnsfullycompatiblewiththeAppliedBiosystems3900synthesizer.TheseincludeourpopularUniversalSupportIII,whichwillallowDNA,RNAorLNAoligostobeproducedonthe3900withANYbaseatthe3’terminus.Atthesametime,weareoffering1μmolecolumnsofUniversalSupport III forthe3900instrument.Structuresandmorecompletedescriptionsarefoundintherelevantcatalogsectionsforeachitem.ABI3900columnscanbepreparedwithvirtuallyanyoftheCPGsupportsinthiscatalog. ItisnolongernecessarytoadjusttheflowusingourABI3900CPGcolumns,asnotedintheboxtotheright.ModifiedCPGcolumnsareonlyavailablein200nmolesize-simpleadd‘A’totheregularcatalognumbertoorder.


Universal Support III PS 200nmolecolumns 26-5110-52 Packof10 40nmolecolumns(ABI3900Format) 26-5110-55 Packof10

GlenUnySupport™PS 200nmolecolumns 26-5140-52 Packof10 40nmolecolumns 26-5140-55 Packof10

3’-PhosphatePS 200nmolecolumns 26-2900-52 Packof10 40nmolecolumns 26-2900-55 Packof10

3’-PT-Amino-ModifierC6PS 200nmolecolumns 26-2956-52 Packof10 40nmolecolumns 26-2956-55 Packof10

3’-(6-FAM)PS 200nmolecolumns 26-2961-52 Packof10 40nmolecolumns 26-2961-55 Packof10

3’-DabcylPS 200nmolecolumns 26-5912-52 Packof10 40nmolecolumns 26-5912-55 Packof10

3’-TAMRAPS 200nmolecolumns 26-5910-52 Packof10 40nmolecolumns 26-5910-55 Packof10

3’-BiotinTEGPS 200nmolecolumns 26-2955-52 Packof10 40nmolecolumns 26-2955-55 Packof10


RELATED

Universal Supports....................24

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QUALITY ASSURANCE




≥98.0%.2. TLC






EXPEDITE INSTRUMENTS

1. ForuseonExpedite8905instruments.

2. ForuseonExpedite8909instruments.


GlenResearchCE(β-cyanoethyl)PhosphoramiditesareproducedandpackagedtoensurethehighestperformanceonDNAsynthesizers.EveryGlenResearchproductisaccompaniedbyaCertificateofAnalysisandHPLCtrace,showingtheresultsofourQCtesting.EveryGlenResearchmonomervialisspeciallycleanedtoeliminateparticulatecontamination.


dA-CEPhosphoramidite 10-1000-C2 0.25g 10-1000-C5 0.5g 10-1000-1C 1.0g 10-1000-2C 2.0g

dC-CEPhosphoramidite 10-1010-C2 0.25g 10-1010-C5 0.5g 10-1010-1C 1.0g 10-1010-2C 2.0g

Ac-dC-CEPhosphoramidite 10-1015-C2 0.25g 10-1015-C5 0.5g 10-1015-1C 1.0g 10-1015-2C 2.0g

dG-CEPhosphoramidite 10-1020-C2 0.25g 10-1020-C5 0.5g 10-1020-1C 1.0g 10-1020-2C 2.0g

dmf-dG-CEPhosphoramidite 10-1029-C2 0.25g 10-1029-C5 0.5g 10-1029-1C 1.0g 10-1029-2C 2.0g

dT-CEPhosphoramidite 10-1030-C2 0.25g 10-1030-C5 0.5g 10-1030-1C 1.0g 10-1030-2C 2.0g


All solventsandreagentsarepreparedtoourexactingspecificationstoensurethehighestsynthesisefficiencyandarepassedthrougha0.2micronfilterduringpackagingtoeliminateparticulatecontamination.GlenResearchusesfreshlysublimed1H-tetrazoleforpremiumperformanceonExpeditesynthesizers.


ActivatorTetrazoleinAcetonitrile 30-3102-661 60mL 30-3102-522 200mL 30-3100-572 450mL


EXPEDITE™ INSTRUMENTS

RELATED


1212

BULK CPG LOADING

500Åsupports 35-50µmoles/g 1000Åsupports 25-40µmoles/g

ABBREVIATIONS

Ac2O=AceticAnhydride CE=Cyanoethyl CPG = Controlled Pore Glass DCM = Dichloromethane dmf=dimethylformamidine I2 = Iodine lcaa=longchainalkylamino MeIm=1-Methylimidazole µm=micromole(s) nm=nanomole(s) TCA=TrichloroaceticAcid THF=Tetrahydrofuran

STERLING SOLVENTS/REAGENTS (CONT.)


Anhydrous WashAcetonitrile,anhydrous 40-4050-531 300mL 40-4050-572 450mL

Cap Mix ATHF/Ac2O 40-4012-661 60mL 40-4012-522 200mL 40-4012-572 450mL

Cap Mix B10%1-MeIminTHF/Pyridine 40-4122-661 60mL 40-4122-522 200mL 40-4122-572 450mL

Oxidizing Solution0.02MI2inTHF/H2O/Pyridine 40-4132-661 60mL 40-4132-522 200mL 40-4132-572 450mL

Deblocking Mix3%TCA/DCM 40-4140-681 180mL 40-4140-712 1L

STERLING SUPPORTS

All Glen Research supportsusethestandard longchainalkylamino(lcaa) linkerbutdiffer intheglassporesize,500Å,1000Åor2000Å.The500Åsupport isappropriateforshortersequences,whilethe1000Åsupportsperformbetter inthesynthesisoflonger(>30-mer)DNAsequences.The2000Åsupportisbestforverylong(>150-mer)oligonucleotides. WehaveinstitutedanadditionalQCtestforsupportstoshowthelengthofoligothatcanbepreparedbeforeadrop-offincouplingduetostericeffectsbeginstooccur.Thedrop-offpointisrecordedintheCertificateofAnalysis.AllGlenResearchsupportsarefullyend-cappedtoensurethattheCPGsurfaceistotallyinert,therebyavoidingtheintroductionofimpuritysequencescontainingdeletionsatthe3’-terminus.

Catalog No. Catalog No. Catalog No. Catalog No. Catalog No. Catalog No. Catalog No. Pack dA dC dG dT dA,dC,dG,dT Ac-dC dmf-dG (1 column of eachbase)

500Å Columns

20-2200-42 20-2210-42 20-2220-42 20-2230-42 20-2240-42 20-2213-42 4x0.2µm20-2200-41 20-2210-41 20-2220-41 20-2230-41 20-2240-41 20-2213-41 4x1.0µm20-2200-14 20-2210-14 20-2220-14 20-2230-14 20-2213-14 1x15µm

1000Å Columns

20-2201-45 20-2211-45 20-2221-45 20-2231-45 20-2241-45 20-2215-45 20-2229-45 4x40nm20-2201-42 20-2211-42 20-2221-42 20-2231-42 20-2241-42 20-2215-42 20-2229-42 4x0.2µm20-2201-41 20-2211-41 20-2221-41 20-2231-41 20-2241-41 20-2215-41 20-2229-41 4x1.0µm20-2201-14 20-2211-14 20-2221-14 20-2231-14 20-2215-14 20-2229-14 1x15µm


RELATED


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STERLING SUPPORTS (CONT.)

Catalog No. Catalog No. Catalog No. Catalog No. Catalog No. Catalog No. Catalog No. Pack dA dC dG dT dA,dC,dG,dT Ac-dC dmf-dG (1 column of eachbase)

2000Å Columns

20-2202-42 20-2212-42 20-2222-42 20-2232-42 20-2242-42 4x0.2µm

500Å Bulk CPG

20-2000-01 20-2010-01 20-2020-01 20-2030-01 20-2013-01 0.1g20-2000-02 20-2010-02 20-2020-02 20-2030-02 20-2013-02 0.25g20-2000-10 20-2010-10 20-2020-10 20-2030-10 20-2013-10 1.0g

1000Å Bulk CPG

20-2001-01 20-2011-01 20-2021-01 20-2031-01 20-2015-01 20-2029-01 0.1g20-2001-02 20-2011-02 20-2021-02 20-2031-02 20-2015-02 20-2029-02 0.25g20-2001-10 20-2011-10 20-2021-10 20-2031-10 20-2015-10 20-2029-10 1.0g

2000Å Bulk CPG

20-2002-01 20-2012-01 20-2022-01 20-2032-01 0.1g20-2002-02 20-2012-02 20-2022-02 20-2032-02 0.25g20-2002-10 20-2012-10 20-2022-10 20-2032-10 1.0g


EmptySynthesisColumns,40nm,0.2umExpediteStyle 20-0021-02 Packof10EmptySynthesisColumns,1umExpediteStyle 20-0021-01 Packof10ReplacementFilters-Expedite 20-0021-0F Packof20

EmptySynthesisColumns-TWIST10um/15um 20-0040-00 Packof10ReplacementFrits-TWIST10um/15um 20-0040-0F Packof20

TWISTisatrademarkofGlenResearchCorporation.ExpediteisatrademarkofAppliedBiosystems.


RELATED

Universal Supports....................24Q-Supports................................27High Load Supports...................29

1414

INSTRUMENT TYPES

Glen Research packages these monomersinavarietyofindustry-standardvialsandbottles.Pleaseprovidetheexactspecificationofthebottlerequiredpriortoreceivingaquotation.

DNA PHOSPHORAMIDITES - SPECIAL PACKAGING

WeofferourhighqualityDNAphosphoramidites specificallypackaged forhigh throughputand large-scale synthesiscustomers.Thesecustomersnormallyrequirehighqualitymaterialsproducedundertheguidelinesofavalidatedqualitymanagementsystemwhilestillbeingpricedaggressively.TheseproductsincludetheusualGlenResearchcertificationandguaranteesandtheyareavailableinlargerpacksorinbulk.ThecorecatalognumbersforregularDNAphosphoramiditesareshownbelow.Fortheseproducts,pleaserequestaquote.

Item Catalog No.

dA-CEPhosphoramidite 10-1000-SPdC-CEPhosphoramidite 10-1010-SPAc-dC-CEPhosphoramidite 10-1015-SPdG-CEPhosphoramidite 10-1020-SPdmf-dG-CEPhosphoramidite 10-1029-SPdT-CEPhosphoramidite 10-1030-SP

DNA PHOSPHORAMIDITES - SPECIAL PACKAGING

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QUALITY ASSURANCE




≥98.0%.2. TLC







MerMadesynthesizersbelongtoafamilyofsynthesizers,includingthecolumn-basedMerMade4,MerMade6and12instrumentsand theparallel array synthesizers,MerMade192andMerMade192E,manufacturedbyBioAutomationCorporation.Theirwebsitecanbefoundat:http://www.BioAutomation.com.Phosphoramiditemonomersarepackagedin30mLand240mLamberbottlesfordissolvingataconcentrationof1g/20mLandareconnecteddirectlytotheinstrument.SomeinstrumentsmayalsobeconfiguredtoacceptAppliedBiosystemsserumvials,asshownonpage6.


dA-CEPhosphoramidite 10-1000-02M 0.25g 10-1000-05M 0.5g 10-1000-10M 1.0g 10-1000-5S 5.0g 10-1000-1S 10.0gdC-CEPhosphoramidite 10-1010-02M 0.25g 10-1010-05M 0.5g 10-1010-10M 1.0g 10-1010-5S 5.0g 10-1010-1S 10.0gAc-dC-CEPhosphoramidite 10-1015-02M 0.25g 10-1015-05M 0.5g 10-1015-10M 1.0g 10-1015-5S 5.0g 10-1015-1S 10.0gdG-CEPhosphoramidite 10-1020-02M 0.25g 10-1020-05M 0.5g 10-1020-10M 1.0g 10-1020-5S 5.0g 10-1020-1S 10.0gdmf-dG-CEPhosphoramidite 10-1029-02M 0.25g 10-1029-05M 0.5g 10-1029-10M 1.0g 10-1029-5S 5.0g 10-1029-1S 10.0gdT-CEPhosphoramidite 10-1030-02M 0.25g 10-1030-05M 0.5g 10-1030-10M 1.0g 10-1030-5S 5.0g 10-1030-1S 10.0g


All solventsandreagentsarepreparedtoourexactingspecificationstoensurethehighestsynthesisefficiencyandarepassedthrougha0.2micronfilterduringpackagingtoeliminateparticulatecontamination.Parallelsynthesizerstypicallyuse5-ethylthio-1H-tetrazole(ETT)asactivatortominimizethechanceofcrystallization.ETTisusedataconcentrationof0.25Minacetonitrile,whichisfarbelowthelevelatwhichcrystallizationmayoccur.


Activator0.25M5-Ethylthio-1H-TetrazoleinAcetonitrile 30-3140-57 450mL 30-3140-61 960mL 30-3140-62 2000mL

MERMADE INSTRUMENTS

RELATED

DepurinationResistantdA........22AlternativeActivators...............30

1616

http://www.BioAutomation.com

ABBREVIATIONS

Ac2O=AceticAnhydride CE=Cyanoethyl CPG = Controlled Pore Glass DCM = Dichloromethane dmf=dimethylformamidine I2 = Iodine MeIm=1-Methylimidazole TCA=TrichloroaceticAcid THF=Tetrahydrofuran



DiluentAcetonitrile,anhydrous 40-4050-50 100mL

Cap Mix ATHF/2,6-Lutidine/Ac2O 40-4010-57 450mL 40-4010-61 960mL 40-4010-62 2000mL

Cap Mix B16%1-MeIminTHF 40-4220-57 450mL 40-4220-61 960mL 40-4220-62 2000mL

Ozidizing Solution0.02MI2inTHF/Pyridine/H2O 40-4330-57 450mL 40-4330-61 960mL 40-4330-62 2000mL

Deblocking Mix3%DichloroaceticacidinDCM 40-4040-57 450mL 40-4040-61 960mL 40-4040-62 2000mL3%TCA/DCM 40-4140-57 450mL 40-4140-61 960mL 40-4140-62 2000mL

STERLING SUPPORTS

Columnscontaining1000ÅCPGareavailableinpacksof200tofitMerMadeplates.Regular500Åor1000Åsupportsmayalsobeusedtofillthewellsofregular96wellplates.However,thisrequireseachplatetobepreparedwitheachnucleosideaccuratelyinallwells.Auniversalsupportclearlyremovestheneedforfourspecificsupportsandmakespreparingplatesstraightforward.GlenUnySupport™40nmolefritscanalsobeused.

Catalog No. Catalog No. Catalog No. Catalog No. Catalog No. Catalog No. Pack dA dC dG dT Ac-dC dmf-dG

Mermade 1000Å Columns20-2001-65 20-2021-65 20-2031-65 20-2015-65 20-2029-65 200x50nm20-2001-62 20-2021-62 20-2031-62 20-2015-62 20-2029-62 200x200nm20-2001-61 20-2021-61 20-2031-61 20-2015-61 20-2029-61 48x1.0µm


Glen UnySupport™ 1000 1µmolecolumns 20-5141-91 Packof96 200nmolecolumns 20-5141-92 Packof96 40nmolecolumns 20-5141-95 Packof96

Empty MerMade Columns EmptyMerMadeColumns(50nm) 20-0050-05 Packof48 EmptyMerMadeColumns(200nmand1µm) 20-0050-02 Packof48

MERMADE INSTRUMENTS

RELATED

AlternativeSolvents..................30Universal Supports....................24Q-Supports................................27High Load Supports...................29

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QUALITY ASSURANCE




≥98.0%.2. TLC







GlenResearchCE(β-cyanoethyl)PhosphoramiditesareproducedandpackagedtoensurethehighestperformanceonDNAsynthesizers.EveryGlenResearchproductisaccompaniedbyaCertificateofAnalysisandHPLCtrace,showingtheresultsofourQCtesting.EveryGlenResearchmonomervialisspeciallycleanedtoeliminateparticulatecontamination.


ÄKTA oligopilotdA-CEPhosphoramidite 10-1000-20 2.0g 10-1000-50 5.0g

dC-CEPhosphoramidite 10-1010-20 2.0g 10-1010-50 5.0g

Ac-dC-CEPhosphoramidite 10-1015-20 2.0g 10-1015-50 5.0g

dG-CEPhosphoramidite 10-1020-20 2.0g 10-1020-50 5.0g

dmf-dG-CEPhosphoramidite 10-1029-20 2.0g 10-1029-50 5.0g

dT-CEPhosphoramidite 10-1030-20 2.0g 10-1030-50 5.0g

GE HEALTHCARE LIFE SCIENCES INSTRUMENTS

RELATED


1818

ABBREVIATIONS

Ac2O=AceticAnhydride CE=Cyanoethyl

CPG = Controlled Pore Glass DCA=DichloroaceticAcid

DCM = Dichloromethane I2 = Iodine MeIm=1-Methylimidazole

µm=micromole(s)

*CapMixBisatwopartformulationthatiscombinedimmediatelybeforeshipment.


All solventsandreagentsarepreparedtoourexactingspecificationstoensurethehighestsynthesisefficiencyandarepassedthrougha0.2micronfilterduringpackagingtoeliminateparticulatecontamination.



ÄKTA oligopilot

Activator0.40MTetrazoleinAcetonitrile 30-3105-71 1L

Cap Mix AAcetonitrile/MeIm 40-4015-71 1L

Cap Mix B*Acetonitrile/Ac2O/Lutidine 40-4028-71 1L

Oxidizing Solution0.05MI2inPyridine/H2O 40-4035-71 1L

Deblocking Mix3%DichloroaceticacidinDCM 40-4040-71 1L3%TCA/DCM 40-4140-71 1L3%DCAinToluene 40-4240-71 1L

GE HEALTHCARE LIFE SCIENCES INSTRUMENTS

RELATED


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QUALITY ASSURANCE




≥98.0%.2. TLC







Dr.Oligosynthesizersbelongtoafamilyofsynthesizers,includingtheparallelarraysynthesizers,Dr.Oligo96,Dr.Oligo192,Dr.Oligo384andDr.Oligo768,manufacturedbyBiolytic®LabPerformance,Inc.inFremont,CA.Theirwebsitecanbefoundat:http://www.biolytic.com.Phosphoramiditemonomersarepackagedin30mLand240mLamberbottlesfordissolvingataconcentrationof1g/20mLandareconnecteddirectlytotheinstrument.SomeinstrumentsmayalsobeconfiguredtoacceptAppliedBiosystemsserumvials.


dA-CEPhosphoramidite 10-1000-02M 0.25g 10-1000-05M 0.5g 10-1000-10M 1.0g 10-1000-5S 5.0g 10-1000-1S 10.0g

dC-CEPhosphoramidite 10-1010-02M 0.25g 10-1010-05M 0.5g 10-1010-10M 1.0g 10-1010-5S 5.0g 10-1010-1S 10.0g

Ac-dC-CEPhosphoramidite 10-1015-02M 0.25g 10-1015-05M 0.5g 10-1015-10M 1.0g 10-1015-5S 5.0g 10-1015-1S 10.0g

dG-CEPhosphoramidite 10-1020-02M 0.25g 10-1020-05M 0.5g 10-1020-10M 1.0g 10-1020-5S 5.0g 10-1020-1S 10.0g

dmf-dG-CEPhosphoramidite 10-1029-02M 0.25g 10-1029-05M 0.5g 10-1029-10M 1.0g 10-1029-5S 5.0g 10-1029-1S 10.0g

dT-CEPhosphoramidite 10-1030-02M 0.25g 10-1030-05M 0.5g 10-1030-10M 1.0g 10-1030-5S 5.0g 10-1030-1S 10.0g


All solventsandreagentsarepreparedtoourexactingspecificationstoensurethehighestsynthesisefficiencyandarepassedthrougha0.2micronfilterduringpackagingtoeliminateparticulatecontamination.Parallelsynthesizerstypicallyuse5-ethylthio-1H-tetrazole(ETT)asactivatortominimizethechanceofcrystallization.ETTisusedataconcentrationof0.25Minacetonitrile,whichisfarbelowthelevelatwhichcrystallizationmayoccur.


Activator0.25M5-Ethylthio-1H-TetrazoleinAcetonitrile 30-3140-57 450mL 30-3140-62 2000mL

DR. OLIGO INSTRUMENTS

RELATED

DepurinationResistantdA........22AlternativeActivators...............30

2020

http://www.biolytic.com

ABBREVIATIONS

Ac2O=AceticAnhydride CE=Cyanoethyl CPG = Controlled Pore Glass DCM = Dichloromethane dmf=dimethylformamidine I2 = Iodine MeIm=1-Methylimidazole TCA=TrichloroaceticAcid THF=Tetrahydrofuran



DiluentAcetonitrile,anhydrous 40-4050-50 100mL

Cap Mix ATHF/2,6-Lutidine/Ac2O 40-4010-57 450mL 40-4010-62 2000mL

Cap Mix B16%1-MeIminTHF 40-4220-57 450mL 40-4220-62 2000mL

Oxidizing Solution0.02MI2inTHF/Pyridine/H2O 40-4330-57 450mL 40-4330-62 2000mL

Deblocking Mix3%DichloroaceticacidinDCM 40-4040-57 450mL 40-4040-62 2000mL3%TCA/DCM 40-4140-57 450mL 40-4140-62 2000mL

STERLING SUPPORTS

Dr.Oligo instrumentsaredesigned forflexibility in theuseof supportsandcolumns.Theycanuse frittedplateswith looseCPGandABI3900stylepolystyreneandCPGcolumns.GlenUnySupport™40nmolefritscanalsobeused.

Catalog No. Catalog No. Catalog No. Catalog No. Catalog No. Catalog No. Pack dA dC dG dT Ac-dC dmf-dG

ABI 3900 Polystyrene Columns26-2600-65 26-2610-65 26-2630-65 26-2629-65 200x40nm26-2600-62 26-2610-62 26-2630-62 26-2629-62 200x200nm

ABI 3900 1000Å CPG Columns20-2101-65 20-2131-65 20-2115-65 20-2129-65 200x40nm20-2101-62 20-2131-62 20-2115-62 20-2129-62 200x200nm20-2101-61 20-2131-61 20-2115-61 20-2129-61 200x1.0µm

OLIGONUCLEOTIDE PURIFICATION

BiolyticLabsalsoofferstheinnovativeDr.OligoProcessorforhighthroughputpurificationofoligonucleotidesusingGlen-Pak™DNAPurificationCartridges:https://www.biolytic.com/p-6814-dr-oligo-processor-fully-automated.aspx.

DR. OLIGO INSTRUMENTS

RELATED

AlternativeSolvents..................30Universal Supports....................24Q-Supports................................27High Load Supports...................29Glen-Pak™DNA.......................147

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https://www.biolytic.com/p-6814-dr-oligo-processor-fully-automated.aspx

DEPURINATION RESISTANT CE PHOSPHORAMIDITES

Depurination is defined as the cleavage of the glycosidic bond attaching a purine base to the sugarmoiety. Electronwithdrawingacylprotectinggroups likebenzoyl and isobutyrylon thepurineaminogroup(s)destabilize theglycosidicbond,whereaselectrondonatingformamidineprotectinggroupsstabilizetheglycosidicbond.Theconsequenceofdepurinationduringoligonucleotidesynthesisisthelossofthepurinebasetoformaninternucleotidelinkagecontainingtheabasicsugaratthatposition.Thissiteisstableduringfurthersynthesiscyclesbut,upondeprotectionwithbasicreagents,theoligonucleotideiscleavedatthatpositionleadingtotwoshorterfragments.Thefragmenttowardsthe5’terminusstillcontainstheDMTgroup.IfDMT-ONpurificationisbeingused,thedepurinatedfragmentsareco-purifiedalongwiththefulllengthproductastruncatedoligonucleotides.

ThemostcommonlyuseddA-CEPhosphoramiditecontainingbenzoylprotectinggroupssufferssubstantialdegradationbydepurinationafterexcessiveexposuretoTCA.Atthesametime,twodepurinationresistantdAmonomers,protectedwithdiethylformamidine (def)anddimethylacetamidine (dma),areessentiallystable todepurinationduring thesameexposuretoTCA.

BothnewdepurinationresistantdAmonomers(defanddmaprotected),wererapidlydeprotectedinammoniumhydroxideandarefullycompatiblewithregulardeprotectionstrategies.Def-protected-dAwasrapidlydeprotectedwithAMAat65°in20minutes,whichmakesitfullycompatiblewithregularAMAdeprotection.Incontrast,thedma-protected-dArequired80minuteswithAMAat65°forcompletedeprotection.

Dmf-dGisalsoadepurinationresistantCEPhosphoramiditewiththeisobutyrylgroupoftheoriginalmonomerreplacedwithdimethylformamidine(dmf).

Althoughdepurinationdoesoccur in regularoligonucleotide synthesis, thedegradation is at anextremely low level. Howeverincertainothercircumstances,depurinationmaybecomemoresignificant,suchassynthesisoflongoligos,chip-basedsynthesis,andlarge-scalesynthesis.


def-dA-CEPhosphoramidite 10-1504-02 0.25g 10-1504-05 0.5g 10-1504-10 1.0g

dmf-dG-CEPhosphoramidite 10-1029-02 0.25g 10-1029-05 0.5g 10-1029-10 1.0g 10-1029-20 2.0g 10-1029-40 4.0g

def-dA

dma-dA

N

N N

N

N

ODMTO

O P N(iPr)2

O CNEt

N(Me)2

Me

N

N N

N

N

ODMTO

O P N(iPr)2

O CNEt

N(Et)2

dmf-dG

O

O P N(iPr)2O CNEt

DMTO

(Me)2NN

O

N

N

N

HN


All minor bases, RNA products andmodifiers are packaged in septum-capped vials suitable forABI andotherinstruments. If youwould like anothertypeofvial/columnaddthefollowingtotheendofthecatalognumber.


Expedite EMerMade M




ALTERNATIVE PROTECTING GROUPS

2222

ULTRAMILD CE PHOSPHORAMIDITES

AnalternativeprotectingschemeforthenormalCEphosphoramiditesshouldallowUltraMILDdeprotectionandshouldnotreactwithawidervarietyoftagsandlabels.Asetofmonomersusingphenoxyacetyl(Pac)protecteddAand4-isopropyl-phenoxyacetyl(iPr-Pac)protecteddG,alongwithacetylprotecteddC,metthedesiredcriteriaforUltraMILDdeprotection.

Werecommendtheuseofphenoxyaceticanhydride(Pac2O)inCapA.ThismodificationremovesthepossibilityofexchangeoftheiPr-PacprotectinggrouponthedGwithacetatefromtheaceticanhydridecappingmix.Cleavageanddeprotectioncanbecarriedoutin2hoursatroomtemperaturewithammoniumhydroxideor4hourswith0.05Mpotassiumcarbonateinmethanol.


Pac-dA-CEPhosphoramidite 10-1601-02 0.25g 10-1601-05 0.5g 10-1601-10 1.0g

Ac-dC-CEPhosphoramidite 10-1015-02 0.25g 10-1015-05 0.5g 10-1015-10 1.0g

iPr-Pac-dG-CEPhosphoramidite 10-1621-02 0.25g 10-1621-05 0.5g 10-1621-10 1.0g

ULTRAMILD SUPPORTS

Item Catalog No. Catalog No. Catalog No. Pack Pac-dA Ac-dC iPr-Pac-dG

UltraMildCPG(Bulk) 20-2601-01 Listed 20-2621-01 0.1g 20-2601-02 on 20-2621-02 0.25g 20-2601-10 Page8 20-2621-10 1.0gABIColumns 20-2701-45 20-2115-45 20-2721-45 4X40nm 20-2701-42 20-2115-42 20-2721-42 4X0.2µm 20-2701-41 20-2115-41 20-2721-41 4X1µm 20-2701-13 20-2115-13 20-2721-13 10µmExpediteColumns 20-2801-45 20-2215-45 20-2821-45 4X40nm 20-2801-42 20-2215-42 20-2821-42 4X0.2µm 20-2801-41 20-2215-41 20-2821-41 4X1µm 20-2801-14 20-2215-14 20-2821-14 15µm

ULTRAMILD SOLVENTS/REAGENTS


Cap Mix ATHF/Pyridine/Pac2O 40-4210-52 200mL (Applied Biosystems) 40-4210-57 450mL THF/Pac2O 40-4212-52 200mL (Expedite) 40-4212-57 450mL

Deprotection Solution0.05MPotassiumCarbonateinMethanol 60-4600-30 30mL

ULTRAMILD DNA SYNTHESIS




Expedite EMerMade M




Pac-dA Ac-dC

iPr-Pac-dG

O

O P N(iPr)2O CNEt

DMTO

NHAc

O N

N

O

O P N(iPr)2O CNEt

DMTON

N

N

N

NHAcOPh

O

O P N(iPr)2O CNEt

DMTOiPrPhOAcHN

O

N

N

N

HN

RELATED

Universal Support III..................26

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Me-

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MIS

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REFERENCES

(1)A.P.Guzaev,andM.Manoharan,J Am Chem Soc, 2003, 125,2380-2381.

(2)R.K.Kumar,A.P.Guzaev,C.Rentel,andV.T.Ravikumar,Tetrahedron, 2006, 62, 4528.

ELIMINATION CONDITIONS

Reagent Conditions

Ammoniumhydroxide 80°C/2h 55°C/8h

Ammoniumhydroxide/ 80°C/0.5h40%Methylamine(AMA) 65°C/1h 55°C/8h MethylamineGas 65°C/0.5h/30psi PotassiumCarbonate RT/17h in Methanol

t-Butylamine/Water(1:3v/v) 60°C/4h

Glen UnySupport

GLEN UNYSUPPORT

Arecentdevelopmenthasbeentheuseofasupportbasedonamoleculewhichis“conformationallypreorganized”toacceleratethedephosphorylationreaction.1,2Byusingarigidbicyclicmoleculeonthesupport,therateofeliminationismarkedlyfasterthantheoriginalUniversalSupport.ThestructureofGlenUnySupport™isshownbelow.TheN-phenylversion,developedatIsisPharmaceuticalsasUnyLinker™,isavailablefromseveralcompaniesforlargescaleoligosynthesis.GlenUnySupportistheN-methylversion,whichispreferredforhighthroughputoligonucleotidesynthesissincemethylamineratherthananilineisformedondeprotection.WearehappytoofferGlenUnySupportinavarietyofpopularformatsunderlicensefromIonisPharmaceuticals.


Bulk SupportsGlenUnySupport 20-5040-01 0.1g (500ÅCPG) 20-5040-02 0.25g 20-5040-10 1.0g

GlenUnySupport 20-5041-01 0.1g (1000ÅCPG) 20-5041-02 0.25g

20-5041-10 1.0g

HighLoadGlenUnySupport 25-5040-01 0.1g 25-5040-02 0.25g 25-5040-10 1.0g

GlenUnySupportPS 26-5040-01 0.1g 26-5040-02 0.25g 26-5040-10 1.0g

ColumnsThe1000Åcolumnsandfritsbelowareroutinelystocked.

ABI Format (not LV) 1µmolecolumns 20-5141-41 Packof4 0.2µmolecolumns 20-5141-42 Packof4 40nmolecolumns 20-5141-45 Packof4 10µmolecolumn(TWISTFormat) 20-5141-13 Packof1 40nmolefrits 20-5441-95 Packof96

Female-FemaleLuerAdapterfor40nmolefrits 20-0060-00 Packof10

ABI 3900 FormatGlenUnySupportPS 200nmolecolumns 26-5140-52 Packof10 40nmolecolumns 26-5140-55 Packof10

Expedite Format 1µmolecolumns 20-5241-41 Packof4 0.2µmolecolumns 20-5241-42 Packof4 40nmolecolumns 20-5241-45 Packof4 15µmolecolumn(TWISTFormat) 20-5241-14 Packof1

96 Well Format (MerMade, etc.) 1µmolecolumns 20-5141-91 Packof96 200 nmolecolumns 20-5141-92 Packof96 40nmolecolumns 20-5141-95 Packof96

INTELLECTUAL PROPERTY

ThisproductiscoveredbyUSPatent7,202,264ownedbyIonisPharmaceuticals,Inc..

NH

O

O

O

O ODMT

N

O

O

Me




Expedite EMerMade M




SUPPORTS

2424

GLEN UNYSUPPORT FC

TheextendedtimerequiredtocleavethesuccinatelinkageoftheoriginalGlenUnySupportcanbeproblematical,especiallyinhigh-throughputproductionofoligos,duetotheoutgassingofammoniaand/ormethylamine.ThisreductioninconcentrationofgascannecessitatetheevaporationofthecleavagesolutionandadditionoffreshAmmoniumHydroxide:MethylAmine1:1(AMA)orammoniumhydroxide(NH4OH)toensurecompletedeprotectionanddephosphorylationoftheproductoligos.UsingadiglycolatelinkageinGlenUnySupportFCinsteadofthesuccinateinGlenUnySupport,asignificantincreaseintherateofcleavagehasbeenachieved.Theminimumcleavagetimesforbothversionsareasfollows: AMA NH4OHGlenUnySupport 10min. 40min.GlenUnySupportFC 2min. 5min.

WiththecleavagetimeofGlenUnySupportFCreducedtolessthan5minutes,thereisminimallossofvolatilegasand,therefore,noneedtoevaporatethecleavagesolutionandreplenishwithfreshAMAorammoniumhydroxidesolutions.

WeofferGlenUnySupportFCattachedto1000ÅCPGinavarietyofformatssuitedtohighthroughputsynthesis,aswellasinbulkformoreroutineuse.


Bulk SupportGlenUnySupportFC 22-5041 Discontinued (1000ÅCPG)

SUPPORTS

ELIMINATION CONDITIONS

Reagent Conditions

Ammoniumhydroxide 80°C/2h 55°C/8h

Ammoniumhydroxide/ 80°C/0.5h40%Methylamine(AMA) 65°C/1h 55°C/8h MethylamineGas 65°C/0.5h/30psi PotassiumCarbonate RT/17h in Methanol

t-Butylamine/Water(1:3v/v) 60°C/4h


ThisproductiscoveredbyUSPatent7,202,264ownedbyIonisPharmaceuticals,Inc..

Glen UnySupport FC

O

O

O ODMT

N

O

O

MeO

HN

O

25

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SUPPORTS

UNIVERSAL SUPPORT III

Thekeystepintheuseofanyuniversalsupportinoligonucleotidesynthesisisthedephosphorylationofthe3’-phosphategrouptoformthedesired3’-hydroxylgroup.Azhayev1,2hasexcelledintheinvestigationofneighboringgroupassistanceinthedephosphorylationreaction.AmidegroupsmaybeconsideredtobeweakN-Hacidsandcandisplaybasicpropertiesinammoniumhydroxideoraqueousmethylamine.Intheoriginalwork1,2,(±)-3-amino-1,2-propanediolwasusedtoformanoveluniversalsupport(1).Asuccinatelinkerattachesthe3-aminogrouptothesupportandthe2-OHisprotectedwithabase-labilegrouptosetupanamideassistedeliminationinmildlybasicconditions.Inthisway,thedephosphorylationreactionwouldeliminatethedesired3’-OHoligonucleotideintosolutionandtheproductofanyß-eliminationcompetingsidereactionwouldremainboundtothesupport.Afurtherimprovementhasbeenachievedbyusingacarbamategrouptoconnecttheuniversal linkertothesupport,as inourproductUniversalSupport III (2). UsingUniversalSupport III, anoligoyieldof>80%canbeachievedonpolymericsupports,withpurityequivalenttothesameoligopreparednormally.

ConditionsforCleavageandDeprotectionareoutlinedinthetableopposite. UniversalSupport IIIhasbeenshowntogenerateoligonucleotideswiththesameefficacyinpolymeraseextensionreactionsasregularoligos.Despitethemildeliminationreaction,oligonucleotidesupto75merinlengthcanbepreparedroutinelywithoutlossofoligoduringthesynthesiscycles.ThissupportisalsousedfortheproductionofsiRNAoligos.


Bulk SupportUniversalSupportIIIPS 26-5010-01 0.1g 26-5010-02 0.25g 26-5010-10 1.0g

ABI Format (not LV) Universal Support III PS 1µmolecolumns 26-5110-41 Packof4 0.2µmolecolumns 26-5110-42 Packof4 40nmolecolumns 26-5110-45 Packof4

10µmolecolumn(TWISTFormat) 26-5110-13 Packof1

Expedite Format 1µmolecolumns 26-5210-41 Packof4 0.2µmolecolumns 26-5210-42 Packof4 40nmolecolumns 26-5210-45 Packof4

15µmolecolumn(TWISTFormat) 26-5210-14 Packof1

96 Well Format (MerMade, etc.)Universal Support III PS 1µmolecolumns 26-5110-91 Packof96 200nmolecolumns 26-5110-92 Packof96 40nmolecolumns 26-5110-95 Packof96

ABI 3900 FormatUniversal Support III PS 200nmolecolumns 26-5110-52 Packof10 40nmolecolumns 26-5110-55 Packof10

Universal Support (1)

CLEAVAGE AND DEPROTECTION

1. Cleavage F o r s t a n d a r d a n d U l t ra Fa s t

deprotectionprotocols, cleave theoligo from the support using 2M ammonia in methanol at room temperature for30minutes. (Onlyfor oligonucleotides greater than 50nucleotides in length, rinse thesupportwith a further volume ofwater.Combinethetwowashesandevaporatetodryness.)

2. Deprotection Standard Add 1 volume of 30% ammonium

hydroxide,sealanddeprotectusingthe conditions appropriate for removaloftheprotectinggroupsonthenucleobases.

UltraFast Add 1 volume of AMA (ammonium

hydroxide/40%aqueousmethylamine1:1)sealanddeprotectat65°Cfor10minutes.

UltraMild Using Ammonium Hydroxide Add 1 vo lume of ammonium

hydroxide, seal and leave at roomtemperaturefor8hours.

UltraMild Cleavage and Deprotection Using Potassium Carbonate in Methanol Cleave the oligo from the support

using50mMpotassiumcarbonateinmethanol at room temperature for 30 minutes.Sealandleaveovernightatroomtemperature.

O

ODMTO

HNO

NHO

CHCl 2

O


ThisproductiscoveredbyUSPatentNo.:6,770,754andEuropeanPatentNo.:1404695.

DTMO

HNHN

O

CHCl2

O

OUniversal Support III (2)

REFERENCES

(1)A.V.Azhayev,Tetrahedron, 1999, 55, 787-800.

(2)A.V.AzhayevandM.Antopolsky,Tetrahedron, 2001, 57, 4977-4986.

2626

Q/SUCCINATE COMPARISON

Q-Support Succinate (2 minutes (60 minutes cleavage) cleavage)

132 ODU* 125 ODU*

*Average crude yield from eight 1µmole columns deprotected normally.

REFERENCE

(1)R.T.PonandS.Y.Yu,Tetrahedron Lett, 1997, 38, 3327-3330.

SUPPORTS

Q-SUPPORTS

Oligonucleotidesare routinelypreparedon supports towhich thefirstnucleoside is attachedviaa succinate linkage. Overtheyears,thesuccinatelinkagehasdemonstratedstabilityduringthesynthesisprocessbuthassufficientlabilitytobecleavedquicklyinthedeprotectionstep.However,ifthecleavagestepiscarriedoutwithammoniumhydroxidemanuallyoron the synthesizer, it consumesonehourofprecioustimewhile releasingonlyabout80%of theoligonucleotide. Thisstepis,therefore,abottleneckintheproductivityofmanysynthesisgroups.

Isitpossibletofindareplacementtothesuccinategroupwhichoffersgoodstabilitytothesynthesisreagentswhileofferingamuchfastercleavagestep?Theoxalategrouphasbeenshowntobeverylabileduringcleavagebutitsstabilitytothenormalsynthesisreagentsisnotgood,requiringchangesforsuccessfuluse.Inapracticalbutelegantstudy1 of various bifunctional carboxylic acids,RichardPon’s group identifiedhydroquinone-O,O’-diaceticacidas themost satisfactoryalternativetothesuccinategroup.Nucleosideswiththis linkerarm(Q-linker)areattachedtosupportswiththesameeaseasthesuccinyllinkerarm.

Thecleavagetimeinammoniumhydroxideatroomtemperaturewasfoundtobe2minutes,butwhataboutthestabilityduringsynthesis? Howsignificantwasprematurecleavageofoligonucleotideonthesynthesizerbecauseof thebasicreagentsinthecappingmixesandoxidizer?PonshowedthattheQ-linkerisstabletothecappingreagentsbutveryslightlylabiletotheoxidizer(8%cleavageinovernightexposurewhichwouldcorrespondtoabout2,000normalsynthesiscycles).

Wetestedthesignificanceofprematurecleavagebypreparingsixteen20meroligonucleotidesona0.2µmolescale,eightwithsuccinateandeightwithQ-linkers.Thesuccinatesupportedoligoswerecleavedfor1houratroomtemperature,whilethoseontheQ-supportwerecleavedfor2minutes.Bothsetswerethendeprotectednormallywithammoniumhydroxide. TheQ-supportsactuallygave5%betteryieldsofproductthanthesuccinatesupports. Oligopuritieswereequivalentinbothsets.

TheQ-linkerisabsolutelycompatiblewithallhydrolyticcleavageprocedures,butespeciallymildprocedureslikepotassiumcarbonateinmethanol.PonalsoshowedthatitispreferableforRNAsupports,improvingthecleavagetimefor2’-silylprotectednucleosidesupportsfrom2hours(60-65%cleavage)to5minutes(95%cleavage).

WeareofferingQ-linkersofthefourregularnucleosideson500ÅCPGin0.2and1µmolescales.




Expedite EMerMade M




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Q-SUPPORTS (CONT.)

Catalog No. Catalog No. Catalog No. Catalog No. Catalog No. Pack dA dC Ac-dC dmf-dG dT

500Å Bulk Support21-2000-01 21-2010-01 21-2013-01 21-2029-01 21-2030-01 0.1g21-2000-02 21-2010-02 21-2013-02 21-2029-02 21-2030-02 0.25g21-2000-10 21-2010-10 21-2013-10 21-2029-10 21-2030-10 1.0g

ABI Format (not LV)21-2100-41 21-2110-41 21-2113-41 21-2129-41 21-2130-41 4X1µm21-2100-42 21-2110-42 21-2113-42 21-2129-42 21-2130-42 4X0.2µm

Expedite Format21-2200-41 21-2210-41 21-2213-41 21-2229-41 21-2230-41 4X1µm21-2200-42 21-2210-42 21-2213-42 21-2229-42 21-2230-42 4X0.2µm

dA-Q dC-Q

dmf-dG-Q dT-Q

SUPPORTS

Ac-dC-Q

OO

DMTOO

OO O

O

NH

NHBz

N

N

N

N

OO

DMTOO

OO O

O

NH

NHBz

O N

N

O

ODMTO

NHAc

O N

N

O

O

O OO

NH

NH

OOO

O

O

Me2NN

O

N

N

N

HN

ODMTO

O O

ODMTO

O

OO O

O

NH

O

O

N

HNCH 3

2828

SUPPORTS

HIGH LOAD CPG

Ourhighloadingsupportisbasedoncontrolledporesilicaanditretainstheusual500Åpores.Thespacerisalsoconventional.Theonlysignificantdifferenceistheloadingwhichisintherange80-130µmoles/gorabout2.5timestheloadingofnormal500ÅCPG.TypicalloadingsforourhighloadCPGareinthe100-120µmoles/grange.Asaconsequenceofthehighloading,thissupportshouldnotbeusedforsequenceslongerthan40mers.Thishighloadingsupportisavailableincolumnsformostsynthesizers.The2.5µmolecolumnisidenticaltoourstandard1µmolecolumn(withtheexceptionoftheloading).Itshouldbeusedonoccasionswhengreaterthan1µmole isdesiredbutwhena10or15µmolesynthesis istoohigh. Itshouldberunusingthe1µmolecycle.The25µmolecolumnisidenticaltothe10µmolecolumnusedonAppliedBiosystemssynthesizers.Itisrunusingthe10µmolecycle.The35µmolecolumnisusedasanalternativetothe15µmoleExpeditecolumn.Againnochangestothestandardcyclearerecommended.Thesupportisofcourseavailableinbulkforuseonlarge-scalesynthesizers.Awordofcautionisinorder.Whenusingacolumnwithahigherloadthanrecommendedbytheinstrumentmanufacturer,thereisamuchsmallermarginforerror.Allreagentsmustbefreshandanhydrousdiluentandactivatormustbeused.Shouldyoudecidetopreparehigher-loadingcolumns,ensurethatthemolarexcessofmonomertosupportnucleosideisatleast5Xandpreferably10X.

Item Catalog No. Catalog No. Catalog No. Catalog No. Pack dA dC dG dT

Columns (ABI) 25-2100-46 25-2110-46 25-2120-46 25-2130-46 4X2.5µm 25-2100-17 25-2110-17 25-2120-17 25-2130-17 1X25µm

(Expedite) 25-2200-46 25-2210-46 25-2220-46 25-2230-46 4X2.5µm 25-2200-18 25-2210-18 25-2220-18 25-2230-18 1X35µm

Bulk

25-2000-02 25-2010-02 25-2020-02 25-2030-02 0.25g 25-2000-10 25-2010-10 25-2020-10 25-2030-10 1.0g

dT-CPGdG-CPGdC-CPGdA-CPG

ODMTO


NHBz

N

N

N

N

ODMTO

NHBz

O N

N


ODMTO


iBuHN

O

N

N

N

HN

ODMTO


O

O

N

HNCH 3




Expedite EMerMade M




RELATED

GlenUnySupport.......................24

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REAGENTS

5-Ethylthio-1H-tetrazole

ALTERNATIVE SOLVENTS/REAGENTS

GlenResearchoffersalternativesolventsandreagentsinsuitablebottlesandformulationsforuseonvariousDNAsynthesizers.Allsolventsandreagentsarepreparedtoourexactingspecificationstoensurethehighestcouplingefficienciesandarepassed througha0.2micronfilterduringpackaging toeliminateparticulatecontamination. GlenResearchoffers theactivatorsbelowinpowderformforlaterdissolutioninanhydrousacetonitrileorasapreparedsolution.


Activator5-Ethylthio-1H-tetrazole(ETT) 30-3040-10 1g (Dissolve 1g in 31mL anhydrous 30-3040-20 2g acetonitrile for a 0.25M solution) 30-3040-25 25g

0.25M5-Ethylthio-1H-tetrazoleinAcetonitrile 30-3140-45 45mL (Applied Biosystems) 30-3140-52 200mL 30-3140-57 450mL 30-3140-62 2L (Expedite) 30-3142-52 200mL 30-3140-57 450mL

4,5-Dicyanoimidazole(DCI),crystalline 30-3050-10 1g (Dissolve 1g in 34mL anhydrous 30-3050-25 25g acetonitrile for a 0.25M solution) 4,5-Dicyanoimidazole(DCI) 30-3060-50 5g (Dissolve 1g in 34mL anhydrous 30-3060-30 30g acetonitrile for a 0.25M solution) 30-3060-K5 500g 30-3060-1K 1000g

0.25MDCIinAcetonitrile 30-3150-45 45mL (Applied Biosystems) 30-3150-52 200mL 30-3150-57 450mL 30-3150-62 2L (Expedite) 30-3152-52 200mL 30-3150-57 450mL

5-Benzylthio-1H-tetrazole(BTT) 30-3070-10 1g (Dissolve 1g in 21.3mL anhydrous 30-3070-20 2g acetonitrile for a 0.25M solution) 30-3070-25 25g

0.25M5-Benzylthio-1H-tetrazoleinAcetonitrile 30-3170-45 45mL (Applied Biosystems) 30-3170-52 200mL 30-3170-57 450mL 30-3170-62 2L (Expedite) 30-3172-52 200mL 30-3170-57 450mL

Saccharin1-Methylimidazole(SMI) 30-3080 Discontinued 30-3180 Discontinued 30-3182 Discontinued

ABBREVIATIONS

Ac2O=AceticAnhydride DCA=DichloroaceticAcid DCM = Dichloromethane DMAP=Dimethylaminopyridine I2 = Iodine MeIm=1-Methylimidazole TCA=TrichloroaceticAcid THF=Tetrahydrofuran

DCI

NN

NN

HCH 3CH 2S

N

NH

CN

CN

N

NN

N

H

PhCH2S

5-Benzylthio-1H-tetrazole

NS

O O

O-

H+N

N

Saccharin 1-Methylimidazole


SMI is sold under license from Avecia BiotechnologyInc.

3030

ALTERNATIVE SOLVENTS/REAGENTS (CONT.)


Cap Mix ATHF/Lutidine/Ac2O 40-4010-52 200mL 40-4010-57 450mL 40-4010-62 2L

THF/Ac2O(9:1) 40-4012-62 2L

Cap Mix B6.5%DMAPinTHF 40-4020-52 200mL (Cap B solutions containing DMAP are preferred by some researchers for preparing long oligos.)

10%MeIminTHF 40-4120-52 200mL 40-4120-57 450mL 40-4120-62 2L

10%MeIminTHF/Pyridine(8:1) 40-4122-62 2L

Oxidizing Solution0.02MI2inTHF/Pyridine/H2O 40-4132-62 2L

Deblocking Mix3%DCA/DCM 40-4040-57 450mL (DCA solutions are more mildly acidic than 40-4040-62 2L the TCA equivalents, possibly causing less depurination of dA sites.)

2.5%DCA/DCM 40-4042-57 450mL 40-4042-62 2L

REAGENTS

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REAGENTS


This capping reagent is supplied under license.

OO

OCNEtON(iPr)2P

UniCap Phosphoramidite

ON

CH3H3C

SO O

CSO

CSO FOR NON-AQUEOUS OXIDATION

Iodine-basedoxidizershavebeenthestandardforDNAandRNAsynthesissincetheadventofautomatedsynthesizers.Theyarefastandefficientoxidizers,typicallyrequiringlessthan30secondsforcompleteoxidationofphosphitetriesterstophosphatetriesters.However,whileiodine-basedoxidizersworkwellformostapplications,therearesomecircumstanceswherenon-aqueousoxidizersmaybeadvantageous,especiallywherethebasesorlinkagesbeingproducedaresensitivetothepresenceofwaterand/oriodineduringsynthesis.

Theuseof(1S)-(+)-(10-camphorsulfonyl)-oxaziridine(CSO)hasbeeninvestigatedasanon-aqueousoxidizerinDNAsynthesis.Forexample,wefoundthata0.5MsolutionofCSOinacetonitrileworkedwellasanoxidizerforthesynthesisofoligoscontainingmultipleincorporationsof7-deaza-dG,comparedwithiodineoxidationwhichcausedsubstantialdegradation.CSOhasalsoworkedwellinthesynthesisofalongpoly-dIoligo,whichcouldnotbepreparedusingiodineoxidationduetothesensitivityofthebase.

CSOhasbeenusedforsynthesizingoligosthatincorporatethephosphonoacetatemodification.Asolutionof0.1MCSOisrecommendedfortheoxidationofPACEmodificationsasthephosphoniteinternucleotidelinkageismoreeasilyoxidizedthanthephosphiteinternucleotidelinkage.WhensynthesizingDNA-phosphonoacetatechimericoligos,a0.5MCSOsolutionisrecommended.


0.5MCSOinAnhydrousAcetonitrile(ABI) 40-4632-52 200mL0.5MCSOinAnhydrousAcetonitrile(Expedite) 40-4632-52E 200mL0.5MCSOinAnhydrousAcetonitrile 40-4632-57 450mL 40-4632-62 2L(A minimum oxidation time of 3 minutes is required on small scales.)

UNICAP PHOSPHORAMIDITE

Thephosphoramiditeof diethyleneglycolmonoethyl ether,UniCap, is thebasis for an alternative capping reagent. TouseUniCapasacappingamiditeontheExpedite8909orABsynthesizers,diluteittothestandardamiditeconcentrationandplacethevialinposition5ontheinstrument.Cyclescanbemodifiedbyaddingcouplingstepsforamiditereservoir5afterthelastcolumncouplingstep.Thestandardcappingstepscanbeleftoutofthecycle.UniCapPhosphoramiditewasoriginallydevelopedforoligosynthesisonthesurfaceofchipsandisthecappingreagentofchoiceforthisapplication.

Item Catalog No. Pack UniCapPhosphoramidite 10-4410-02 0.25g 10-4410-05 0.5g 10-4410-10 1.0g 10-4410-20 2.0g

RELATED

0.1MCSOinPACEChemistry......37

3232

BACKBONE MODIFICATION

SULFURIZING REAGENTS

GlenResearch’sSulfurizingReagentsareusedtopreparephosphorothioatelinkagesusingCEphosphoramiditechemistry.Eachreagentexhibitsthefollowingattributes:1)Reliablysoluble,makingthemsafetouseonautomatedsynthesizers.2)Reactionisfast(30seconds),makingtheprocessconvenientonsmallscalesandreadilyamenabletoscale-up.3)Processisefficient,withbetterthan96%ofthelinkagesbeingphosphorothioateandtheremainderbeingphosphodiester.

SulfurizingReagentII(3-((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione,DDTT)exhibitsallthepropertiesofBeaucageReagentwhileaddingstabilityinsolutiononthesynthesizerANDofferingstrongabilitytosulfurizeRNAlinkages.SulfurizingReagentIIisavailableinpowderformandasastablesolution.


SulfurizingReagentII(DDTT) 40-4037-10 1g (Dissolveataconcentrationof1g/100mL 40-4037-20 2g toformanapproximate0.05Msolution)

0.05MSulfurizingReagentIIinpyridine/acetonitrile 40-4137-51 100mL 40-4137-52 200mL 40-4137-57 450mL

SS

N SN N

Sulfurizing Reagent II

33

BACK

BON

E M

OD

IFIE

RS

5’-CE PHOSPHORAMIDITES

GlenResearch5’-CE(ß-cyanoethyl)Phosphoramiditesaredesignedfortheproductionof5’-5’or3’-3’linkages,usefulinantisensestudies,ortosynthesizeoligonucleotidesegmentsintheoppositesensefromnormalsynthesis(ReverseSynthesis),forstructuralstudies.ThesemonomersarepackagedinABI-stylevials(seenotebox).


dA-5’-CEPhosphoramidite 10-0001-02 0.25g 10-0001-05 0.5g 10-0001-10 1.0g

dC-5’-CEPhosphoramidite 10-0101-02 0.25g 10-0101-05 0.5g 10-0101-10 1.0g

dmf-dG-5’-CEPhosphoramidite 10-9201-02 0.25g 10-9201-05 0.5g 10-9201-10 1.0g

dT-5’-CEPhosphoramidite 10-0301-02 0.25g 10-0301-05 0.5g 10-0301-10 1.0g

dA-5’-CE Phosphoramidite dC-5’-CE Phosphoramidite dT-5’-CE Phosphoramidite

P(iPr)2NOCNEt

O

ODMT

O

NHBz

N

N

N

N

P(iPr)2NOCNEt

O

ODMT

O

NHBz

N

NOP(iPr)2NOCNEt

O

CH 3HN

N

O

O

ODMT

O

Dmf-dG-5’-CE Phosphoramidite

HN

N

N

O

N

O

ODMT

N(Me)2N

OPN(iPr)2

OCNEt




Expedite EMerMade M





34

dG-5’-CPG dmf-dG-5’-CPG dT-5’-CPG

OO

ODMT

NHBz

N

N

N

N

CPG -lcaa-succinyl

5’-SUPPORTS

Thefollowingsupportsareusedtoproduceoligonucleotideswithnucleaseresistant3’-3’linkagesatthe3’terminus(byattachingregular3’-CEphosphoramidites)ortoproduceoligonucleotidesectionsintheoppositesense(byattaching5’-CEphosphoramidites).ABI-stylecolumnsaresuppliedunlessotherwiserequested(seenotebox).


dA-5’-CPG 20-0002-01 0.1g 20-0002-10 1.0g1µmolecolumns 20-0012-41 Packof40.2µmolecolumns 20-0012-42 Packof410µmolecolumn(ABI) 20-0012-13 Packof115µmolecolumn(Expedite) 20-0012-14 Packof1

dC-5’-CPG 20-0102-01 0.1g 20-0102-10 1.0g1µmolecolumns 20-0112-41 Packof40.2µmolecolumns 20-0112-42 Packof410µmolecolumn(ABI) 20-0112-13 Packof115µmolecolumn(Expedite) 20-0112-14 Packof1

dG-5’-CPG 20-0202-01 0.1g 20-0202-10 1.0g1µmolecolumns 20-0212-41 Packof40.2µmolecolumns 20-0212-42 Packof410µmolecolumn(ABI) 20-0212-13 Packof115µmolecolumn(Expedite) 20-0212-14 Packof1

dmf-dG-5’-CPG 20-9202-01 0.1g 20-9202-10 1.0g1µmolecolumns 20-9212-41 Packof40.2µmolecolumns 20-9212-42 Packof410µmolecolumn(ABI) 20-9212-13 Packof115µmolecolumn(Expedite) 20-9212-14 Packof1

dT-5’-CPG 20-0302-01 0.1g 20-0302-10 1.0g1µmolecolumns 20-0312-41 Packof40.2µmolecolumns 20-0312-42 Packof410µmolecolumn(ABI) 20-0312-13 Packof115µmolecolumn(Expedite) 20-0312-14 Packof1

OO

ODMT

O N

N

NHBz

CPG -lcaa-succinylO

O

ODMT

iBuHN

O

N

N

N

HN

CPG -lcaa-succinyl

HN

N

N

O

N

O

ODMT

N(Me)2N

OCPG-lcaasuccinyl OO

ODMT

O

O

N

HNCH 3

CPG -lcaa-succinyl

dA-5’-CPG dC-5’-CPG


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dA-Me Phosphonamidite dT-Me PhosphonamiditedG-Me PhosphonamiditeAc-dC-Me Phosphonamidite

REFERENCE

(1)M.P.Reddy,F.Farooqui,andN.B.Hanna,TetrahedronLett.,1996,37,8691-8694.

METHYL PHOSPHONAMIDITES

MethylPhosphonamiditesmaybeusedinDNAsynthesizersfollowingconventionalCEPhosphoramiditeprotocolstoproduceoligonucleotidescontainingoneormoremethylphosphonatelinkages.However,deprotectionandpurificationtechniquesdifferandadescriptionoftheproceduresisincludedintheTechnicalBulletin.WealsoofferthedCmonomerwithacetylbaseprotection.1 Thisprotectinggroup is removedwithammoniumhydroxideduring the cleavage step,eliminatingmodificationatthedCsitesduringthedeprotectionstepusingethylenediamineinethanol.


dA-MePhosphonamidite 10-1100-02 0.25g 10-1100-05 0.5g

Ac-dC-MePhosphonamidite 10-1115-02 0.25g 10-1115-05 0.5g

dG-MePhosphonamidite 10-1120-02 0.25g 10-1120-05 0.5g

dT-MePhosphonamidite 10-1130-02 0.25g 10-1130-05 0.5g

O

O P N(iPr)2CH 3

DMTO

N

N

N

N

NHBz

O

O P N(iPr)2CH 3

DMTO

NHAc

O N

N

O

O P N(iPr)2CH 3

DMTO

HN

N

N

N

O

iBuHN

O

O P N(iPr)2CH 3

DMTO

HN

N

O

O

CH 3




Expedite EMerMade M





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PACE PHOSPHORAMIDITES

Phosphonoacetate(PACE)modifiedoligonucleotidesshowgreatpotentialasbiologicalmodifiersinawidevarietyofresearchapplications. PACEmonomersarepartofafamilyofPhosphonocarboxylatemonomers. Themonomerscanbeeasilyincorporatedintocomplexoligonucleotidesandarecompatiblewithawidevarietyofothersugarorheterobasemodifications.PACEDNAcanbeconjugatedthroughthecarboxylicacidfunctionalgroup.TheyhavebeenshowntobeactiveinsiRNAduplexesandacceleratetheinitialrateofcleavagebyRNaseH-1whenincorporatedwithphosphorothioates.However,themostinterestingobservationtodateisthattheyexhibitanunprecedentedenhancementinpenetrationofculturedcells.

PACEmonomersarefullysolubleinacetonitrileatarecommendedconcentrationof0.1MandarecompatiblewithstandardDNAsynthesizers.Asanoptimalcycle,werecommendusingDCIasanactivator(30-3150-XX)anda15minutecouplingtime.Followingcoupling,capusingUnicap(10-4410-XX)witharegularcouplingtimeandthenoxidizeusing0.5MCSOfor3minutes.Alternatively,a33minutecouplingtimeusing0.45Mtetrazole,oxidationusinglow-wateriodine(40-4032-XX)followedbycappingwith6.5%DMAPasCapBwillgiveacceptableresults.Fordeprotection,pre-treatthesynthesiscolumnwith1.5%DBUinanhydrousacetonitrilefor60minutesatroomtemperaturetoremove1,1-dimethyl-2-cyanoethylprotectinggroups.Rinsethecolumnwithacetonitrile,dryunderargonandcompletethedeprotectionwith40%aqueousmethylaminefor2hoursatroomtemperature.


dA-PACEPhosphoramidite 10-1140-02 0.25g 10-1140-05 0.5g 10-1140-10 1.0g

Ac-dC-PACEPhosphoramidite 10-1150-02 0.25g 10-1150-05 0.5g 10-1150-10 1.0g

dG-PACEPhosphoramidite 10-1160-02 0.25g 10-1160-05 0.5g 10-1160-10 1.0g

dT-PACEPhosphoramidite 10-1170-02 0.25g 10-1170-05 0.5g 10-1170-10 1.0g

dA-PACE Phosphoramidite dT-PACE PhosphoramiditedG-PACE PhosphoramiditeAc-dC-PACE Phosphoramidite

N

N N

N

NHBz

ODMTO

O P N(iPr)2

OCN

O

ODMTON

N

NHAc

O

O P N(iPr)2

OCN

O

HN

N

N

O

N

ODMTO

O P N(iPr)2

iBuHN

OCN

O

OCN

O

ODTMO

N

HN

O

O

CH3

O P N(iPr)2


Theseproductsarecoveredbypatents, US 6,693,187 and 7,067,641, andpatentspendingownedbyMetasenseTechnologies.Purchaseofalloranyoftheseproductsincludes a limited license to use the productssolelyforthemanufactureofoligonucleotidesforresearchuseonly.Thislicensespecificallyexcludestheuseoftheproductoroligonucleotidescontainingtheproductfor:(a)therapeuticordiagnosticapplications(includingkits,pools,librariesandotherproducts or services that incorporate oligonucleotidescontainingtheproduct),(b)anyinvivotoxicity/safetystudyinsupportofaninvestigationalnewdrugapplication(orforeigncounterpart),or(c)resale (including sale of kits, pools, librariesandotherproductsorservices that incorporate the product oroligonucleotidescontainingtheproduct).Ifsuchactivitieshavecommercialapplication,aseparatelicenseisrequiredfromMetasenseTechnologies.Neithertheproductnoranyproductcreatedthroughitsusemaybeusedinhumanclinicaltrials.

Asimpleagreementmustbesignedbeforeend-usersandcustomoligoservicesmaypurchasetheseproductsforuseasdefinedabove.

https://www.glenresearch.com


RELATED

DCI..............................................30UniCap.......................................320.5MCSO...................................322’-OMe-PACE...........................145

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https://www.glenresearch.com/media/productattach/import/technical_note/PACE.pdf

METHYL PHOSPHORAMIDITES

Formanyyears,GlenResearchhassuppliedmethylphosphoramiditesinadditiontoß-cyanoethyl(CE)phosphoramiditesforthefewsituationswherethemore labilecyanoethylgroupisnotanadvantage. Someofourcustomers,probablyrememberingthatthemethylgroupwasremovedspecificallywiththiophenol,havetriedtousethesemonomerstopreparetheinteresting,uncharged,andnuclease-resistantmethylphosphotriesterlinkage.Unfortunately,thislinkageislabiletoammoniumhydroxideandtheregularphosphodiester linkageisformed(alongwithasmallamountofchainscission). WeofferUltraMildmethylphosphoramiditesforthisapplication.Oligosproducedfromthesemonomerscanbedeprotectedwithpotassiumcarbonateinmethanoltoproducemethylphosphotriesterlinkages.Sincetheselinkagesarediastereomericanduncharged,theoligosmaybehardtohandle.Consequently,itislikelythatchimeraswillbeproducedusingthesemonomersalongwiththeregularUltraMildCEphosphoramidites.IfmanydGresiduesareincludedintheoligonucleotide,werecommendtheuseofphenoxyaceticanhydride(Pac2O)inCapA.Thismodificationremovesthepossibilityofexchangeoftheisopropyl-phenoxyacetate(iPr-Pac)protectinggrouponthedGwithacetatefromtheaceticanhydridecappingmix.


Pac-dA-MePhosphoramidite 10-1301-02 0.25g 10-1301-05 0.5g 10-1301-10 1.0g

Ac-dC-MePhosphoramidite 10-1315-02 0.25g 10-1315-05 0.5g 10-1315-10 1.0g

iPr-Pac-dG-MePhosphoramidite 10-1321-02 0.25g 10-1321-05 0.5g 10-1321-10 1.0g

dT-MePhosphoramidite 10-1330-02 0.25g 10-1330-05 0.5g 10-1330-10 1.0g






Pac-dA-Me Phosphoramidite dT-Me PhosphoramiditeiPr-Pac-dG-Me PhosphoramiditeAc-dC-Me Phosphoramidite

O

O P N(iPr)2O

DMTO

CH 3

N

N

N

N

NHAcOPh

O

O P N(iPr)2O

DMTO

CH 3

NHAc

O N

N

O

O P N(iPr)2O CH 3

DMTO

HN

N

N

N

O

iPr-PhOAcHN

O

O P N(iPr)2O CH 3

DMTO

CH 3

O

O

N

HN

38

dA-H-Phosphonate dC-H-Phosphonate dG-H-Phosphonate dT-H-Phosphonate

H-PHOSPHONATE MONOMERS

OurH-Phosphonatelinehasbeendiscontinued.PleasecontactGlenSupport.


dA-H-Phosphonate,TEASalt 10-1200 Discontinued

dC-H-Phosphonate,DBUSalt 10-1210 Discontinued

dG-H-Phosphonate,TEASalt 10-1220 Discontinued

dT-H-Phosphonate,TEASalt 10-1230 Discontinued

H-PHOSPHONATE REAGENTS

OurH-Phosphonatesolventsandreagentshavebeendiscontinued.H-Phosphonatereagentsareeasilypreparedusinghighpurityproductsandtheformulationsshownbelow.

Item

1-AdamantanecarbonylchlorideisavailablefromAldrich,CatalogNo.117722.Diluteto0.1M. (Activator for monomers and capping reagent)

Acetonitrile/Pyridine(50:50),anhydrous (Monomer Diluent)

Acetonitrile/Pyridine(95:5),anhydrous (Activator Diluent)

1%IsopropylPhosphiteinAcetonitrile/Pyridine(50:50) (Capping Reagent)

Acetonitrile/Pyridine(50:50) (Neutralizer and Wash Solvent)

4%I2inPyridine/H2O/THF(10:10:80)

THF/H2O/TEA(80:10:10) (Both reagents are required for oxidation of H-phosphonate linkages)


O

O

P

DMTO

HOO- TEA+

NHBz

N

N

N

N

O

O

P

DMTO

HO

NHBz

O N

N

O- DBU+

O

O

P

DMTO

HN

N

O

O

CH 3

HOO- TEA+

O

O

P

DMTO

HOO- TEA+

iBuHN

O

N

N

N

HN




Expedite EMerMade M




ABBREVIATIONS

I2 = Iodine TEA=Triethylamine THF=Tetrahydrofuran

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REFERENCES

(1)J.Nielsen,W.K.D.Brill,andM.H.Caruthers, Tetrahedron Letters, 1988, 29,2911-2914.

(2)L.Cummins,D.Graff,G.Beaton,W.S.Marshall,andM.H.Caruthers,Biochemistry, 1996, 35,8734-41.

(3)X.Yang,andD.G.Gorenstein,Curr Drug Targets, 2004, 5,705-15.

(4)W.S.Marshall,andM.H.Caruthers,Science, 1993, 259,1564-70.

(5)J.L.Tonkinson,etal.,Antisense Research and Development, 1994, 4, 269-278.

(6)X.Yang,etal.,Bioorg Med Chem Lett, 1999, 9,3357-62.

(7)X.Yang,etal.,Ann N Y Acad Sci, 2006, 1082,116-9.

(8)X.Yang,etal.,Nucleic Acids Res, 2002, 30,e132.


BETA-L-DNA MONOMERS

betaL-DNAisthemirrorimageversionofnaturallyoccurringD-DNA.L-DNAandD-DNAshareidenticalstructuresthatdifferonlyintermsofstereochemistryandgenerallyhaveidenticalphysicalandchemicalproperties.Thedifferenceintheirstereochemistryresultsindifferencesintheirinteractionswithchiralmolecules,D-DNAwillonlybindtoitsD-DNAcomplementtoformright-handedhelices,andlikewise,L-DNAwillonlybindtoitsL-DNAcomplementtoformleft-handedhelices.Forthisreason,enzymesthatinteractwithD-DNA,includingnucleases,typicallywon’tinteractwithL-DNA.TheuniquepropertiesofL-DNAshavemadethemattractiveformanybiologicalapplicationssuchasAptamers,MolecularBeacons,MolecularTagging,andDrugNanocarriers.NotethattheprocedureforsynthesizingL-DNAoligonucleotidesisverysimilartothatofD-DNAoligonucleotides.PleaseseeourGlenReportversion31.2formoredetails.


beta-L-Pac-dA-CEPhosphoramidite 10-2101-02 0.25g 10-2101-05 0.5g 10-2101-10 1.0gbeta-L-Ac-dC-CEPhosphoramidite 10-2115-02 0.25g 10-2115-05 0.5g 10-2115-10 1.0gbeta-L-iPr-Pac-dG-CEPhosphoramidite 10-2121-02 0.25g 10-2121-05 0.5g 10-2121-10 1.0gbeta-L-dT-CEPhosphoramidite 10-2130-02 0.25g 10-2130-05 0.5g 10-2130-10 1.0g

beta-L-Pac-dA beta-L-Ac-dC beta-L-iPr-dG beta-L-dT




Expedite EMerMade M




RELATED

2’-OMe-RNAThiophosphoramidites..............35

N

N N

N

NHAcOPh

OODMT

O P N(iPr)2O CNEt

OODMT

N

O P N(iPr)2O CNEt

N

NHAc

O

OODMT

N

O P N(iPr)2O CNEt

HN

O

O

HN

N

N

O

NiPrPhOAcHN

OODMT

O P N(iPr)2O CNEt

40

LOCKED ANALOG PHOSPHORAMIDITES

LockedNucleicAcid(LNA)wasfirstdescribedbyWengelandco-workers in19981asanovelclassofconformationallyrestrictedoligonucleotideanalogues.LNAisabicyclicnucleicacidwherearibonucleosideislinkedbetweenthe2’-oxygenandthe4’-carbonatomswithamethyleneunit.OligonucleotidescontainingLNAexhibitunprecedentedthermalstabilitiestowardscomplementaryDNAandRNA2,whichallowsexcellentmismatchdiscrimination.Infact,thehighbindingaffinityofLNAoligosallowsfortheuseofshortprobesin,forexample,SNPgenotyping3,allelespecificPCRandmRNAsamplepreparation.LNAisrecommendedforuseinanyhybridizationassaythatrequireshighspecificityand/orreproducibility,e.g.,duallabelledprobes,insituhybridizationprobes,molecularbeaconsandPCRprimers.Furthermore,LNAoffersthepossibilitytoadjustTmvaluesofprimersandprobesinmultiplexassays.LNAcanbemixedwithDNAandRNA,aswellasothernucleicacidanalogues,modifiersandlabels.LNAoligonucleotidesarewatersoluble,andcanbeseparatedbygelelectrophoresisandprecipitatedbyethanol.

GlenResearchispleasedtoofferthesehighlyusefulreagents-LockedAnalog(LA)Phosphoramidites-astoolsforthistechnology.


Bz-A-LA-CEPhosphoramidite 10-2000-05 0.5g 10-2000-10 1.0g

5-Me-Bz-C-LA-CEPhosphoramidite 10-2011-05 0.5g 10-2011-10 1.0g

dmf-G-LA-CEPhosphoramidite 10-2029-05 0.5g 10-2029-10 1.0g

T-LA-CEPhosphoramidite 10-2030-05 0.5g 10-2030-10 1.0g


Bz-A-LNA 5-Me-Bz-C-LNA dmf-G-LNA T-LNA

N

N N

N

NHBz

ODMTO

O

P

O

N(iPr)2

O CNEt

ODMTO

O

P

O

N(iPr)2

O CNEt

N

N

O

NHBz

CH3

ODMTO

O

P

O

N(iPr)2

O CNEt

HN

N

N

O

NN(Me)2N

ODMTO

O

P

O

N(iPr)2

O CNEt

N

HN

O

O

CH3

REFERENCES

(1a)A.A.Koshkin,S.K.Singh,P.Nielsen,V.K.Rajwanshi,R.Kumar,M.Meldgaard,C.E.Olsen,andJ.Wengel, Tetrahedron,1998, 54, 3607-3630.

(1b)S.K.Singh,P.Nielsen,A.A.Koshkin,andJ.Wengel,Chem. Comm.,1998,(4),455-456.

(2) L.KværnøandJ.Wengel,Chem. Comm.,1999,(7),657-658.

(3)P.Mouritzen,A.T.Nielsen,H.M.Pfundheller,Y.Choleva,L.Kongsbak,andS.Møller,Expert Review of Molecular Diagnostics, 2003, 3(1),27-38.

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TRIMER PHOSPHORAMIDITES

Trimer phosphoramidites1-4haveproventobeextremelyvaluablebecausetheyallowcodon-basedmutagenesis,whichcircumventsthecommonproblemsofcodon-bias,frame-shiftmutations,andtheintroductionofnonsenseorstopcodons.5

Thisisaccomplishedbyintroducingamixtureofall20aminoacidcodons(orsubsetthereof)atanylocationwithinthesequencedtobemutated.Thisleadstotheproductionofclonallibrariesofexceptionaldiversitywithorder-of-magnitudeincreasesinaminoacidsequencevariancewhileeithermaintainingauniformaminoaciddistribution6oronethatisbiasedtowardadesiredsetofaminoacids.7

However, difficulties arisewhen trying to introducemutations inmultiple distal regionsof a gene simultaneously. Thesynthesisoflongoligonucleotidesisrequired,whichinevitablyleadstolowersequencefidelityduetodeletionmutants,depurinationeventsand,toalesserextent,mutationsarisingfromdeaminationofcytidine,forexample.

Anelegant solution to thisproblem is theuseofAntisenseTrimerPhosphoramidites. These trimers are the reversecomplementof the cannonical ‘sense’ codons.When theseantisensecodonsareput into thenoncoding strandof atemplateDNAandamplifiedbyPCR,theywillcodeforthesensecodonintheoppositestrandofDNA.ThisallowsthepowerfultechniqueofPCRAssembly8togeneratenotonlykilobase-sizedgenesfromshort50meroligonucleotides,buttosimultaneouslymutatemultipledistalregionsofthatgene,asshowninFigure1.

ThesenseandtheircorrespondingantisensecodonsarelistedinTable1.Conveniently,manyofourexistingsensetrimerscanactasantisensecodons.Forexample,AAC,whichcodesforasparagine,hastheanticodonGTT,whichisthesensecodonforvaline.However,someoftheexistingtrimers,whiletheycanactasanantisensecodon,arenotgoodchoicesforuse. Forexample,TGG,whichcodesfortryptophan,couldbeusedasanantisensecodonforprolinebecauseCCAisoneofproline’ssynonymouscodons.However,CCAhasarelativelylowCodonAdaptationIndex(CAI)value9inE.coli,whichcouldlimitproteinexpression inthatcommonlyusedorganism.Forthisreason,theanticodonCGGwaschosenforoptimalexpressioninE.coli,asweretheothernewantisensecodonsshowninboldinTable1.

IncludedinTable1arethereactionfactors(RFs)foreachofthesenseandantisensetrimers.Thereactionfactoriscriticalsincethetrimerswill likelybemixedandtheyexhibitdifferentratesofreactionwhencouplingduringoligonucleotidesynthesis.AnexamplewheretheRFisusedtocompensatefordifferingratesofcouplingfollows.TheRFforAACis1.0andforTACis1.6.Therefore,1.6equivalentsofTACareneededforevery1.0equivalentofAACforequalcouplingrates. Sotoobtain25umolesoftrimermixthatyields,onaverage,a1:1ratioofAAC/TACatthemutationsite,9.6umolesofAACwouldbeaddedto15.4umolesofTAC.

Allofthetrimersareavailableindividuallysotheresearcherscanpreparecustomtrimermixes.Twopre-madecatalogtrimermixesareavailable:13-1991-xx,forincorporatingall20aminoacidcodonsequallyintoasequenceand13-1992-xx,forincorporating19aminoacidcodons(-Cys).Foracustomtrimermixofaparticularsubsetofcodonsoratrimermixthatrepresentsasetoftrimersthatisbiasedtowardaparticularcodonorcodons,[email protected] foraquotationandprojecteddeliverydate.

Thereisaconcernthatthesequenceofthetrimershastobeverified.Forexample,CATcodingforhistidine,hastobedifferentiatedfromTAC,codingfortyrosine.ThesetwotrimershavevirtuallyidenticallipophilicityandtheiridentitycannotbeclearlyconfirmedbyHPLC.Thisproblemhasbeensolved4usingHPLCelectrospraymassspectrometricanalysisofthetrimers,whichprovidesdataconfirmingmolecularweightandsequence.

REFERENCES

(1)A.L.Kayushin,M.D.Korosteleva,A.I.Miroshnikov,W.Kosch,D.Zubov,andN.Piel,Nucleic Acids Research, 1996, 24, 3748-3755.

(2)A.Kayushin,etal.,Nucleos Nucleot, 1999, 18,1531-1533.

(3)A.Kayushin,M.Korosteleva,andA.Miroshnikov,Nucleos Nucleot Nucleic Acids, 2000, 19,1967-1976.

(4)T.Mauriala,S.Auriola,A.Azhayev,A.Kayushin,M.Korosteleva,andA.Miroshnikov, J Pharm Biomed Anal, 2004, 34,199-206.

(5)C.Neylon,Nucleic Acids Res, 2004, 32,1448-59.

(6)L.R.Krumpe,K.M.Schumacher,J.B.McMahon,L.Makowski,andT.Mori,BMC Biotechnol, 2007, 7,65-72.

(7)F.A.Fellouse,etal.,J Mol Biol, 2007, 373,924-40.

(8)W.P.Stemmer,A.Crameri,K.D.Ha,T.M.Brennan,andH.L.Heyneker,Gene, 1995, 164, 49-53.

(9)P.M.Sharp,andW.H.Li,Nucleic Acids Res, 1987, 15,1281-95.

DMTO O P O O P OO O

OClPh OClPh

OCEPB1 B2 B3

General Structure of Trimer Phosphoramidites,

where B=Abz, Cbz, Gibu, T

OLIGONUCLEOTIDE-DIRECTED MUTAGENESIS

42

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Figure 1: Simultaneous Mutation of Multiple Distal Regions of Gene

TABLE 1: RF of Trimer Phosphoramidites

Sense codons Reaction Antisense codons Reaction(5'->3') Factor (RF) (5’->3’) Factor (RF)

AAA(Lys) 1.10 TTT 1.70AAC(Asn) 1.00 GTT 1.90ACT(Thr) 1.60 GGT 1.10ATC(Ile) 1.50 GAT 1.40ATG(Met) 1.30 CAT 1.30CAG(Gln) 2.00 CTG 1.20CAT(His) 1.30 ATG 1.30CCG(Pro) 1.80 CGG 0.80CGT(Arg) 1.40 GCG 0.60CTG(Leu) 1.20 CAG 2.00GAA(Glu) 1.40 TTC 1.30GAC(Asp) 1.60 ATC 1.50GCT(Ala) 1.50 TGC 1.50GGT(Gly) 1.10 ACC 0.90GTT(Val) 1.90 AAC 1.00TAC(Tyr) 1.60 GTA 1.50TCT(Ser) 1.30 AGA 1.40TGC(Cys) 1.50 GCA 1.00TGG(Trp) 1.10 CCA 1.10TTC(Phe) 1.30 GAA 1.40

OO

Cl

ClO

O

O P N(iPr)2O CNEt

O

OP

O

OOP

O

DMTO O

HN

N

O

O

CH 3

N

NO

NHBz

NHBz

N

N

N

N

ATC Trimer

Pool of Oligos

PCR

43

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BASE

S



Sense TrimersAAATrimerPhosphoramidite 13-1000-95 50µm (Lys) 13-1000-90 100µm

AACTrimerPhosphoramidite 13-1001-95 50µm (Asn) 13-1001-90 100µm

ACTTrimerPhosphoramidite 13-1013-95 50µm (Thr) 13-1013-90 100µm

ATCTrimerPhosphoramidite 13-1031-95 50µm (Ile) 13-1031-90 100µm

ATGTrimerPhosphoramidite 13-1032-95 50µm (Met) 13-1032-90 100µm

CAGTrimerPhosphoramidite 13-1102-95 50µm (Gln) 13-1102-90 100µm

CATTrimerPhosphoramidite 13-1103-95 50µm (His) 13-1103-90 100µm

CCGTrimerPhosphoramidite 13-1112-95 50µm (Pro) 13-1112-90 100µm

CGTTrimerPhosphoramidite 13-1123-95 50µm (Arg) 13-1123-90 100µm

CTGTrimerPhosphoramidite 13-1132-95 50µm (Leu) 13-1132-90 100µm

GAATrimerPhosphoramidite 13-1200-95 50µm (Glu) 13-1200-90 100µm

GACTrimerPhosphoramidite 13-1201-95 50µm (Asp) 13-1201-90 100µm

GCTTrimerPhosphoramidite 13-1213-95 50µm (Ala) 13-1213-90 100µm

GGTTrimerPhosphoramidite 13-1223-95 50µm (Gly) 13-1223-90 100µm

GTTTrimerPhosphoramidite 13-1233-95 50µm (Val) 13-1233-90 100µm

TACTrimerPhosphoramidite 13-1301-95 50µm (Tyr) 13-1301-90 100µm

TCTTrimerPhosphoramidite 13-1313-95 50µm (Ser) 13-1313-90 100µm

TGCTrimerPhosphoramidite 13-1321-95 50µm (Cys) 13-1321-90 100µm

TGGTrimerPhosphoramidite 13-1322-95 50µm (Trp) 13-1322-90 100µm

TTCTrimerPhosphoramidite 13-1331-95 50µm (Phe) 13-1331-90 100µm

TrimerPhosphoramiditeMix1 13-1991-95 50µm (Mixofabove20trimers) 13-1991-90 100µm

TrimerPhosphoramiditeMix2 13-1992-95 50µm (Mixofabove20trimerslessTGC-Cys) 13-1992-90 100µm




Expedite EMerMade M




44



Antisense TrimersAACTrimerPhosphoramidite 13-1001-95 50µm (Anti Val) 13-1001-90 100µm

ACCTrimerPhosphoramidite 13-1011-95 50µm (Anti Gly) 13-1011-90 100µm

AGATrimerPhosphoramidite 13-1020-95 50µm (Anti Ser) 13-1020-90 100µm

ATCTrimerPhosphoramidite 13-1031-95 50µm (Anti Asp) 13-1031-90 100µm

ATGTrimerPhosphoramidite 13-1032-95 50µm (Anti His) 13-1032-90 100µm

CAGTrimerPhosphoramidite 13-1102-95 50µm (Anti Leu) 13-1102-90 100µm

CATTrimerPhosphoramidite 13-1103-95 50µm (Anti Met) 13-1103-90 100µm

CCATrimerPhosphoramidite 13-1110-95 50µm (Anti Trp) 13-1110-90 100µm

CGGTrimerPhosphoramidite 13-1122-95 50µm (Anti Pro) 13-1122-90 100µm

GAATrimerPhosphoramidite 13-1200-95 50µm (Anti Phe) 13-1200-90 100µm

GATTrimerPhosphoramidite 13-1203-95 50µm (Anti Ile) 13-1203-90 100µm

GCATrimerPhosphoramidite 13-1210-95 50µm (Anti Cys) 13-1210-90 100µm

GCGTrimerPhosphoramidite 13-1212-95 50µm (Anti Arg) 13-1212-90 100µm

GGTTrimerPhosphoramidite 13-1223-95 50µm (Anti Thr) 13-1223-90 100µm

GTATrimerPhosphoramidite 13-1230-95 50µm (Anti Tyr) 13-1230-90 100µm

TGCTrimerPhosphoramidite 13-1321-95 50µm (Anti Ala) 13-1321-90 100µm

TTCTrimerPhosphoramidite 13-1331-95 50µm (Anti Glu) 13-1331-90 100µm

TTTTrimerPhosphoramidite 13-1333-95 50µm (Anti Lys) 13-1333-90 100µm

45

MIN

OR

BASE

S

BASES AFFECTING DUPLEX STABILITY

SubstitutionofC-5propynyl-dC(pdC)fordCandC-5propynyl-dU(pdU)fordTareeffectivestrategiestoenhancebasepairing.Usingthesebasesubstitutions,duplexstabilityandmeltingtemperaturesareraisedbythefollowingamounts:C-5propynyl-C2.8°persubstitution;C-5propynyl-U1.7°persubstitution.AP-dC(G-clamp)substitutesfordCandisanotherveryimportantmodifiednucleosidethatenhanceshybridizationby7-21°persubstitutiondependinguponthesequenceandlocationoftheAP-dC.Theabilityofthesemodifiedbasestoenhancebindingwhilemaintainingspecificityhasprovenusefulinantisenseresearchandinthesynthesisofhighaffinityprobes.AP-dCisalsoafluorescentnucleosideandshouldfindusesinDNAstructuralresearch.

dWisaC-nucleosidethatactsasastrongadeninebaseparinganalog.InadditiontothetypicaltwohydrogenbondsfoundbetweenTandA,dWcanalsointeractwithAviavanderWaalsforces.TheresultisadW–AinteractionthatapproachesthestrengthofaC–Gbasepairwhilealsoexhibitingenhancedbase-pairingfidelity.dWcanbeusedinplaceofTasasinglesubstitutionoracompletereplacementforoligonucleotidehybridizationapplications.


pdC-CEPhosphoramidite 10-1014-90 100µmole 10-1014-02 0.25g 10-1014-05 0.5g

pdU-CEPhosphoramidite 10-1054-90 100µmole 10-1054-02 0.25g 10-1054-05 0.5g

AP-dC-CEPhosphoramidite 10-1097-95 50µmole (G-Clamp) 10-1097-90 100µmole 10-1097-02 0.25g

dW-CEPhosphoramidite 10-1527-95 50µmole 10-1527-90 100µmole 10-1527-02 0.25g

C-5methylpyrimidinenucleosidesare known to stabilizeduplexes relative to thenon-methylatedbases. Therefore,enhancedbindingcanbeachievedusing5-methyl-dCinplaceofdC,duplexmeltingtemperaturebeingincreasedby1.3°.Ac-5-Me-dC-CEPhosphoramiditeisfullycompatiblewithAMAdeprotectionandnoneoftheN4-Metransaminationmutantisobservedondeprotection.


5-Me-dC-CEPhosphoramidite 10-1060-90 100µmole 10-1060-02 0.25g

Ac-5-Me-dC-CEPhosphoramidite 10-1560-90 100µmole 10-1560-02 0.25g

DUPLEX STABILITY MODIFICATION

5-Me-dC

O

O P N(iPr)2O CNEt

DMTO

CH 3

NHBz

O N

N

O

O P N(iPr)2O CNEt

DMTON

HN

O

O

CH 3

ODMTO

O P N(iPr)2

O CNEt

N

TIPS

OPiv

O

O P N(iPr)2O CNEt

DMTON

N

O

CH 3

NN(iBu)2

pdC pdU dW

ONHF3C

OHN

DMTO

O P N(iPr)2O CNEt

N

NO

O

O

AP-dC

ODMTON

N

NHAc

O

O P N(iPr)2

O CNEt

CH3

Ac-5-Me-dC

46


BASES AFFECTING DUPLEX STABILITY (CONT.)

ThesimplestapproachtothedesignofhighaffinityprimersandprobesistosubstituteAsiteswith2-amino-A,sincethe2-amino-A-TbasepairisequivalentinstrengthtotheG-Tbasepair.2-Amino-AalsodestabilizesA-Gwobblemismatches,thusincreasingspecificity.In1998,weintroduceda2-amino-dAmonomerwhichexhibitsfastandeffectivedeprotectioninammoniumhydroxideanditisstabilizedtodepurinationduringsynthesis.Wenowrecommendtheuseof0.5MCSOinanhydrousacetonitrile (40-4632-xx) forbest resultswithmultipleadditionsof2-amino-dA. This isbecause thebisformamidineprotected2-amino-dA leads to significant strand scissionwhen standard iodineoxidation isusedduringsynthesis.Forthisreason,wehavealsoaddedPac-2-Amino-dA,amonomerwithoptimizedprotectiontomeetthefollowingcriteria:stableduringoligonucleotidesynthesis,oxidation,anddetritylation;labiletowardscommondeprotectionconditions(NH3, AMA, MeNH2);andthenucleobaseprotectinggroupsarecleavedunderfairlymildconditions.


2-Amino-dA-CEPhosphoramidite 10-1085-95 50µmole (2,6-diaminopurine) 10-1085-90 100µmole 10-1085-02 0.25g

Pac-2-Amino-dA-CEPhosphoramidite 10-1585-95 50µmole (2,6-diaminopurine) 10-1585-90 100µmole 10-1585-02 0.25g

SequenceswithhighGCcontentmaycontainmismatchesandstillhybridizebecauseofthehighstabilityoftheG-Cbasepair.TheN4-ethylanalogueofdC(N4-Et-dC)hybridizesspecificallytonaturaldGbutthestabilityofthebasepairisreducedtoaboutthelevelofanATbasepair.

CouplingN6-Me-dA(10-1003)andN4-Et-dC(10-1068)with1H-tetrazoleleadstoatraceofbranchingatthesecondaryaminepositions,whileDCIleadstoaround15%branching.IncollaborationwithBerryandAssociates,theacetylprotectedmonomerswereprepared. Acetylprotectionwaschosen since itwouldblockbranching reactions. OligonucleotidessynthesizedusingthesemonomersprovedtobecompatiblewithallpopulardeprotectionstrategiesfromUltraMildtoUltraFast.WhentheacetylprotectedmonomerswerecomparedwiththeunprotectedmonomersusingDCIasactivator,branchingwasreducedfrom15%tozero.


N4-Et-dC-CEPhosphoramidite 10-1068-95 50µmole 10-1068-90 100µmole 10-1068-02 0.25g

N4-Ac-N4-Et-dC-CEPhosphoramidite 10-1513-95 50µmole 10-1513-90 100µmole 10-1513-02 0.25g

N6-Me-dA-CEPhosphoramidite 10-1003-90 100µmole 10-1003-02 0.25g

N6-Ac-N6-Me-dA-CEPhosphoramidite 10-1503-90 100µmole 10-1503-02 0.25g

N4-Et-dC

Et

O

O P N(iPr)2O CNEt

DMTO

N

NO

NH

ODMTON

N

NAc

O

O P N(iPr)2

O CNEt

Et

N4-Ac-N4-Et-dC N6-Me-dA

O

O P N(iPr)2O CNEt

DMTO

NHMe

N

N

N

NN

N N

N

NAc

ODMTO

O P N(iPr)2

O CNEt

Me

N6-Ac-N6-Me-dA2-Amino-dA

O

O P N(iPr)2O CNEt

DMTO

N

N

N

N

N

N

N(iBu)2

(iBu)2N

ODMTO

O P N(iPr)2

O CNEt

N

N N

N

N

PhOAcHN

N(nBu)2

Pac-2-Amino-dA




Expedite EMerMade M




RELATED

0.5MCSO...................................32N6-Me-dA..................................35

47

MIN

OR

BASE

S


”Sperminephosphoramidite”synthonisthesubjectmatterof U.S.DivisionalPatentApplicationNo.14/745,871,EuropeanPatentNo.1973927andforeignequivalentsforwhichPolyplus-transfectionistheco-owner.Productissoldforresearchpurposesonly.Productshallnotbeusedtomanufactureoligospermine-oligonucleotideconjugatesforuseindiagnostics,clinicalorcommercialapplicationsincludinguseinhumans.Thereisno implied license to manufacture oligospermine-oligonucleotideconjugatesfordiagnostic,clinical, orcommercialapplications,includingbutnotlimitedtocontractresearch.PleasecontactPolyplus-transfectionatlicensing@polyplus-transfection.comtoobtainalicenseforsuchuse.

ZNA®isaregisteredtrademarkofPolyplus-transfectionSA.


ZIP NUCLEIC ACIDS (ZNA®)

Sperminephosphoramiditeisusedtoproduceoligospermine-oligonucleotideconjugates-ZipNucleicAcids(ZNA®)Oligos.Thenamereflectsthepresumedmodeofaction.Theconjugatesarebelievedtousetheoligosperminetoseekoutandmovealong(scan)oligonucleotidestrandsuntiltheprobecomplementarysequenceislocated.Theoligosperminethenperformsthefunctionofstabilizingtheformedduplexbyreducingelectrostaticrepulsion,therebyleadingtosignificantlyincreasedbindingaffinities. ZNA®Oligoshave founduse in the followingapplications:MultiplexPCR;PCRofAT-richRegions;RTqPCR;DetectionofMicroRNA;ImprovedSNPDiscrimination;andAntisenseandAntigeneEffects.Sperminephosphoramiditeissimpletouseinoligonucleotidesynthesisandcanbeaddedmultipletimesatthe3’or5’terminus.Deprotectionandisolationarealsostraightforward. HPLCanalysisoftheconjugatesrequireshighpHtosuppresstheionizationofthespermineresidues.


SperminePhosphoramidite 10-1939-95 50µmole 10-1939-90 100µmole 10-1939-02 0.25g

CDPI3 MGB™ LABELING

Syntheticoligonucleotideswithcovalently-attachedCDPI3haveenhancedDNAaffinityand improved thehybridizationpropertiesofsequence-specificDNAprobes.ShortCDPI3-oligonucleotideshybridizewithsingle-strandedDNAtogivemorestableDNAduplexesthanunmodifiedODNsofsimilarlength.ThesimplestapproachtoMGBprobedesignistouseanMGBsupport,addaquenchermoleculeasthefirstadditionandcompletethesynthesiswitha5’-fluorophore.Alternatively,afluorophoresupportcouldbeusedwiththe5’terminuscontainingaquenchermoleculefollowedbyafinalMGBadditionatthe5’terminus.GlenResearchoffers5’-CDPI3 MGB™ Phosphoramidite and 3’-CDPI3MGB™CPG.

SELECTIVELY BINDING COMPLEMENTARY (SBC) OLIGOS

SBColigosexhibithighaffinity fornaturaloligonucleotidesbut they show littleaffinity forotherSBColigosevenofacomplementarysequence.OligosinwhichAhasbeenreplacedwith2-amino-AandTwith2-thio-TrepresentanexcellentexampleofSBColigos.While2-amino-AformsaverystablebasepairwithTcontainingthreehydrogenbonds,thestabilityofthebasepairwith2-thio-Tisgreatlydiminished.However,2-thio-TbasepairsperfectlywellwithA.Asanexample,SBC20mersannealedagainstaDNA20mertargetexhibitedTmvalues10°ChigherthanthecorrespondingDNA-DNAhybrid,whereastheSBC-SBChybridyieldedTmvalues30°Clower.

UNNATURAL BASE PAIRS

UnnaturalbasepairsdisplayuniqueabilitiesinduplexDNAandinnucleicacidandproteinbiosyntheses.AstandardWatsonandCrickbasepairisformedbetweeniso-Candiso-G,butthehydrogenbondingpatternisquitedifferentfromthenaturalbasepairsA-TandC-G.Iso-basescan,therefore,increasespecificityofnucleicacidhydridizationwhenintroducedasathirdbasepair.Ithasalsobeendemonstratedthatiso-bases5-Me-iso-dCandiso-dGcanfunctionasdegeneratepyrimidineandpurinebases,respectively.Iso-dGfurtherfunctionedasadegeneratebaseoppositeB(C,T,andG)ambiguoussites.

DMTONTFA

NTFA

TFAN

TFAN

O P N(iPr)2

O CNEtSpermine Phosphoramidite

RELATED

CDPI3MGB™Labeling............1172-Amino-dA...............................47Pac-2-Amino-dA........................472-Thio-dT...................................58dmf-5-Me-isodC........................53dmf-isodG..................................53

48

mailto:licensing%40polyplus-transfection.com?subject=

mailto:licensing%40polyplus-transfection.com?subject=


CAPS FOR INCREASED DUPLEX STABILITY AND BASE-PAIRING FIDELITY

Newcapstructuresallowforthepreparationofhybridizationprobeswithincreasedaffinityforcomplementarysequences.ThemonomersusedtopreparecappedoligonucleotidesarephosphoramiditesthatcanbereadilyintroducedviaautomatedDNAsynthesisattheendofsolidphasesyntheses.ThecapsfavortheformationofstableWatson-Crickduplexesbystackingontheterminalbasepair(Figures1and2).

Meltingpointincreasesofover10°Cpermodificationcanberealizedforshortduplexes.1,2ThecapsfitcanonicalWatson-Crickbasepairsanddonotstackwellonmismatchedbasepairs.Thisleadstoincreasedbasepairingselectivityattheterminalandthepenultimatepositionofoligonucleotidesfeaturingthecaps.Basepairingfidelityisusuallylowatthetermini,wherefrayingoccursfrequentlyintheabsenceofcaps.Thebeneficialeffectsofthecapsarealsorealizedwhenlongertargetstrandsarebound,sothereisnoneedforbluntendsfortheduplexesformed.1,2Thecaps,whenattachedtothe5’terminusofanoligonucleotide,alsofacilitatepurificationastheirlipophilicityleadstoprolongedretentiononreversedphasecolumnsorcartridges.Finally,cappingofterminimaydiscouragethedegradationofoligonucleotidesbyexonucleases.

3’-UaqCapCPG,aUridinesupportmodifiedwitha2’-anthraquinoneresidue,isthemosteffectiveoligonucleotidecapknowntodate.3,4ForshorthybridduplexesbetweenDNAprobesandRNAtargetstrands,theincreaseinTmisupto18°CandthemodificationiseffectiveinincreasingtheTmofDNA:DNA,RNA:RNA,andDNA:RNAhybridduplexes.3’-UaqCapalsoincreasesprobespecificitybydepressingthemeltingpointofterminalmismatches.


5’-TrimethoxystilbeneCapPhosphoramidite 10-1986-90 100µmole 10-1986-02 0.25g

5’-PyreneCapPhosphoramidite 10-1987-90 100µmole 10-1987-02 0.25g

3’-UaqCapCPG 20-2980-01 0.1g 20-2980-10 1.0g 1µmolecolumns 20-2980-41 Packof4 0.2µmolecolumns 20-2980-42 Packof4 10µmolecolumn(ABI) 20-2980-13 Packof1 15µmolecolumn(Expedite) 20-2980-14 Packof1

REFERENCES

(1)Dogan,Z.;Paulini,R.;RojasStütz,J.A.;Narayanan,S.;Richert,C.J. Amer. Chem. Soc. 2004, 126,4762-4763.

(2)Narayanan,S.;Gall,J.;Richert,C.Nucleic Acids Res. 2004, 32,2901-2911.

(3)A.Patra,C.Richert,J. Amer. Chem. Soc., 2009, 131,12671-12681.

(4)C.Ahlborn,K.Siegmund,C.Richert,J. Amer. Chem. Soc., 2007, 129, 15218-15232.

NH

O

CNEtON(iPr)2PO

MeOOMe

MeO

N

CNEtON(iPr)2PO

5’-Trimethoxystilbene Cap 5’-Pyrene Cap

cap

Unmodified DNA:fraying and wobbling

at the terminus

Duplex with cap-bearing probe

FIGURE 1: STACKING OF CAP ON 5’ TERMINAL BASE PAIR

O

PO2

N

NH

O

O

HO

O

O

O

HN

OO B

OH or OH

O

chain

terminal base

Uaq cap

Cap

capped duplex

ODMT-O N

NH

O

O

O

O

O

O

HNO

HNO

Uaq controlled pore glass oligonucleotide

FIGURE 2: STACKING OF Uaq CAP ON 3’ TERMINAL BASE PAIR

ODMTO

N

HN

O

O

OHN

O

OO

succinyl-CPG3’-Uaq Cap CPG




Expedite EMerMade M




49

MIN

OR

BASE

S

EPIGENETICS

DNA METHYLATION

Oneofthefastestgrowingfieldsinbiologyandcancerresearchisepigenetics.Whiletheunderlyinggeneticcodedefineswhichproteinsandgeneproductsaresynthesized,itisepigeneticcontrolthatdefineswhenandwheretheyareexpressed.ThisdynamiccontrolofgeneexpressionisessentialforXchromosomeinactivation,embryogenesis,cellulardifferentiationandappearsintegraltomemoryformationandsynapticplasticity.

In2009,tworeports1,2describedthediscoveryof5-hydroxymethyl-2’-deoxyCytidine(hmdC),anoveldCmodificationinPurkinjeneuronsandembryonicstemcells.Later,athirdreportfoundthismodificationtobestronglyenrichedinbraintissuesassociatedwithhighercognitive functions.3ThisdCmodification isgeneratedby theactionofa-ketoglutaratedependentteneleventranslocation(TET)enzymes,whichoxidizes5-Me-dCtohmdC.Thisfindingstimulateddiscussionaboutactivedemethylationpathwaysthatcouldoccur,e.g.,viabaseexcisionrepair(BER),withthehelpofspecializedDNAglycosylases.Alternatively,onecouldenvisionaprocessinwhichthehydroxymethylgroupofhmdCisfurtheroxidizedto5-formyl-dC(fdC)or5-carboxy-dC(cadC)followedbyeliminationofeitherformicacidorcarbondioxide4,5.

GlenResearchhas supported this research since its inceptionbyproviding thebuildingblocks for the synthesis ofoligonucleotidescontainingallthenewdCderivatives-hmdC,fdCandcadC.ThefirstgenerationhmdCphosphoramiditewasfairlyverywellacceptedbutrequiresfairlyharshdeprotectionconditions.Therefore,asecondgenerationbuildingblock (5-Hydroxymethyl-dC II)developedbyCarellandco-workers that iscompatiblewithUltraMilddeprotectionwasintroduced.65-Formyl-dCIIIhasbeendesignedtomeetalloftherequirementstoprepareanoligocontainingallofthemethylatedvariants.7


5-Hydroxymethyl-dC-CEPhosphoramidite 10-1062-95 50µmole 10-1062-90 100µmole 10-1062-02 0.25g

5-Carboxy-dC-CEPhosphoramidite 10-1066-95 50µmole 10-1066-90 100µmole 10-1066-02 0.25g

5-Formyl-dC-CEPhosphoramidite 10-1514-95 50µmole 10-1514-90 100µmole 10-1514-02 0.25g

5-Hydroxymethyl-dCII-CEPhosphoramidite 10-1510-95 50µmole 10-1510-90 100µmole 10-1510-02 0.25g

5-Formyl-dCIII-CEPhosphoramidite 10-1564-95 50µmole 10-1564-90 100µmole 10-1564-02 0.25g

REFERENCES

(1)S.Kriaucionis,andN.Heintz,Science, 2009, 324,929-30.

(2)M.Tahiliani,etal.,Science, 2009, 324,930-935.

(3)M.Münzel,etal.,Angewandte Chemie-International Edition, 2010, 49,5375-5377.

(4)D.Globisch,etal.,PLoS One, 2010, 5,e15367.

(5)S.C.Wu,andY.Zhang,Nat Rev Mol Cell Biol, 2010, 11,607-20.

(6)M.Münzel,D.Globisch,C.Trindler,andT.Carell,Org Lett, 2010, 12, 5671-3.

(7)A.S.Schroder,etal.,Angewandte Chemie-International Edition, 2014, 53,315-318.

ODMTON

N

NHBz

O

O P N(iPr)2

O CNEt

CH2-O-CNEt

ODMTON

N

NHBz

O

O P N(iPr)2

O CNEt

CO2Et

ODMTON

N

NHAc

O

O P N(iPr)2

O CNEt

OAc OAc

5-Hydroxymethyl-dC 5-Formyl-dC5-Carboxy-dC

ODMTON

N

O

O P N(iPr)2

O CNEt

OHN

O

5-Hydroxymethyl-dC II

ODMTON

N

NH

O

O P N(iPr)2

O CNEt

O

O

O

MeO

5-Formyl-dC III

RELATED

5-Me-dC.....................................465-hmdU......................................61

50

PCR/SEQUENCING APPLICATIONS

DUPLEX EFFECTS

Thedesignofprimersisfrequentlycomplicatedbythedegeneracyofthegeneticcode.Threestrategiesarenowavailabletoconfrontthisproblem.Inthefirst,amixedbaseaddition(N)isusedtoformthedegeneratesite.Thisapproachisbestifthenumberofdegeneratesitesissmall.Asecondoptionistheuseof2’-deoxyInosineor2’-deoxyNebularinewhichexhibitlow,butunequal,hydrogenbondingtotheotherfourbases.Thethirdoptionistheuseofauniversalnucleoside.Inthisstrategy,thebaseanalogdoesnothybridizesignificantlytotheotherfourbasesandmakesupsomeoftheduplexdestabilizationbyactingasan intercalatingagent. 3-Nitropyrrole2’-deoxynucleoside (M) is thefirstexampleofa setofuniversalbases.Subsequently,5-nitroindolewasdeterminedtobeaneffectiveuniversalbaseandtobesuperiorto3-nitropyrrole,basedonduplexmeltingexperiments.

ThemodifiedbasesdesignatedPandKshowconsiderablepromiseasdegeneratebases.ThepyrimidinederivativeP,whenintroducedintooligonucleotides,basepairswitheitherAorG,whilethepurinederivativeKbasepairswitheitherCorT.AdP+dKmixalsocanserveasamixedbasewithmuchlessdegeneracythandA+dC+dG+dT(N).


dA+dG-CEPhosphoramidites 10-1002-02 0.25gdC+dT-CEPhosphoramidites 10-1013-02 0.25gdA+dC+dG+dT-CEPhosphoramidites 10-1023-02 0.25g

Other packsizes,mixedbasecombinationsandcustomdopingofindividualmonomersareavailableonrequest.Also,mixedbasecolumnsareavailablein0.2and1.0µmolesizesonrequest.

dI-CEPhosphoramidite 10-1040-90 100µmole 10-1040-02 0.25g

dI-CPG500 20-2040-01 0.1g 1µmolecolumns 20-2190-41 Packof4 0.2µmolecolumns 20-2190-42 Packof4

dI-CPG1000 20-2041-01 0.1g 1µmolecolumns 20-2191-41 Packof4 0.2µmolecolumns 20-2191-42 Packof4

dU-CEPhosphoramidite 10-1050-90 100µmole 10-1050-02 0.25g

dU-CPG500 20-2050-01 0.1g 1µmolecolumns 20-2150-41 Packof4 0.2µmolecolumns 20-2150-42 Packof4

dU-CPG1000 20-2051-01 0.1g 1µmolecolumns 20-2151-41 Packof4 0.2µmolecolumns 20-2151-42 Packof4

2’-deoxyInosine

O

O P N(iPr)2O CNEt

DMTO

O

N

N

N

HN




Expedite EMerMade M




2’-deoxyUridine

O

O P N(iPr)2O CNEt

DMTOO

O

N

HN

51

MIN

OR

BASE

S

DUPLEX EFFECTS (CONT.)


2’-DeoxyNebularine-CEPhosphoramidite 10-1041-90 100µmole (Purine) 10-1041-02 0.25g

5-Nitroindole-CEPhosphoramidite 10-1044-90 100µmole 10-1044-02 0.25g

dP-CEPhosphoramidite 10-1047-90 100µmole 10-1047-02 0.25g

dK-CEPhosphoramidite 10-1048-90 100µmole 10-1048-02 0.25g

dP+dK-CEPhosphoramidite 10-1049-90 100µmole 10-1049-02 0.25g

5-Nitroindole3-Nitropyrrole2’-deoxyNebularine


O

O P N(iPr)2O CNEt

DMTON

N

N

N

O

O P N(iPr)2O CNEt

DMTON

NO2

O

O P N(iPr)2O CNEt

DMTON

NO2

dP dK

O

O P N(iPr)2O CNEt

DMTO

O

O

N

N

HN

O

O P N(iPr)2O CNEt

DMTO

MeO

Me2NN

N

N

N

N

HN




Expedite EMerMade M




52


DUPLEX EFFECTS (CONT.)

Unnaturalbasepairsdisplayuniqueabilities induplexDNAand innucleicacidandproteinbiosyntheses. A standardWatsonandCrickbasepairisformedbetweeniso-Candiso-G,butthehydrogenbondingpatternisquitedifferentfromthenaturalbasepairsA-TandC-G.(The5-methylanaloguewaschosenasthesynthetictargetduetothereportedinstabilityof2’-deoxyisocytidinecausedbydeaminationduringoligonucleotidesynthesisordeprotection.)


dmf-5-Me-isodC-CEPhosphoramidite 10-1065-90 100μmole 10-1065-02 0.25g

dmf-isodG-CEPhosphoramidite 10-1078-90 100μmole 10-1078-02 0.25g

Tm MODULATION

Anytechniquethatinvolveshybridizationofmultiplesequencessimultaneously,asinDNAchipandreversehybridizationtechnologies,issubjecttoinaccuraciesduetodifferencesinGCcontent.SequenceswithhighGCcontentmaycontainmismatchesandstillhybridize,whereasalowGCcontentprobemaymatchperfectlyandyetdisassociatefromthetarget,leadingtofalsepositivesandnegatives,respectively.

AnelegantwayofcircumventingthisproblemwouldbetouseamodifiedbasethatnormalizedthestabilityoftheGCandATbasepairs.TheN4-ethylanalogue(N4-Et-dC)hybridizesspecificallytonaturaldGbutthestabilityofthebasepairisreducedtoaboutthelevelofanATbasepair.InaseriesofprobeswhoseGCcontentrangedfrom0to100%,therangeinTmvalueswhenN4-Et-dCwasusedwasonly7°C;whendCwasused,thatrangewas39°C.

dmf-isodGdmf-5-Me-isodC

N

N

OCH 3

N(Me)2N

DMTO

CNEtON(iPr)2PO

O

N

N

N

N

N

DMTO

CNEtON(iPr)2PO

O

DPCO

N(Me) 2

RELATED

N4-Et-dC.....................................47N4-Ac-N4-Et-dC.........................47

53

MIN

OR

BASE

S

CLEANAMP® MONOMERS

CleanAmp®PrimersofferanalternativetootherHotStarttechnologiesandallowgreatercontrolofprimerhybridizationandextensionduringPCR.IthasbeendemonstratedthatCleanAmp®Primersoutperformothertechnologiesinmultipleapplications.Indeed,overabroadrangeofapplications,CleanAmp®Primersreduceoreliminateoff-targetamplification.Greaterampliconyieldisalsoachieved,duetoimprovementinspecificityandsensitivity.Byusingeithertheslow-releasingPrecisionprimerswithtwoCleanAmp®phosphotriesterlinkagesorthefaster-releasingTurboPrimerswithasingleCleanAmp®phosphotriesterlinkage,therateofformationofunmodifiedprimercanbecontrolledtosuitreactionneeds. TurboPrimers PrecisionPrimers

Fastcycling StandardcyclingMultiplexPCR One-stepreverse-transcriptionPCRImprovesampliconyield ImprovedspecificityandlimitofdetectionReducesmis-priming/primerdimerformation Greatestreductioninmis-priming/primerdimerformation

SynthesisofCleanAmp®PrimersrequirestheuseofUltraMildChemistry.

CleanAmp®PrimersandmonomersareavailablefromTriLinkBioTechnologies.



CleanAmp®isatrademarkofTriLinkBioTechnologies,Inc.CleanAmp®productsorportionsthereofarecoveredbyTriLinkpendingPatentApplications,US2007281308andWO2007139723, US Provisional PatentApplicationSerial#61/056, 324 and US Patent 6762298 licensed from the Department of Health andHumanServices.CleanAmp®productsaresoldexclusivelyforR&Dusebythepurchaser.Theymaynotberesold,distributedorre-packaged.Nolicenseisgrantedorimpliedwiththepurchaseofthisproduct.AmplificationmethodsusedinconnectionwithPolymeraseChainReaction(“PCR”)Processarecoveredbymanypatents.Itmaybenecessarytoobtainaseparatelicenseforcertainpatentedapplicationsin whichtheproductisused.CleanAmp®LicensesareavailabledirectlyfromTriLinkBioTechnologies.www.trilinkbiotech.com

N

N N

N

ODMTO

OP

O(CH2)9CH3

N(iPr)2

O

HNO

O

ODMTON

N

NHAc

O

OP

O(CH2)9CH3

N(iPr)2

O

Chemical Formula: C52H73N4O9PExact Mass: 928.51

Molecular Weight: 929.13m/z: 928.51 (100.0%), 929.51 (57.7%), 930.52 (18.0%), 931.52

(4.3%), 929.52 (1.2%)Elemental Analysis: C, 67.22; H, 7.92; N, 6.03; O, 15.50; P, 3.33

HN

N N

N

O

ODMTO

OP

O(CH2)9CH3

N(iPr)2

O

NH

O

O

Chemical Formula: C59H77N6O10PExact Mass: 1060.54

Molecular Weight: 1061.25m/z: 1060.54 (100.0%), 1061.55 (65.1%), 1062.55 (22.9%), 1063.55 (6.0%), 1061.54 (2.2%), 1062.54

(1.4%)Elemental Analysis: C, 66.77; H, 7.31; N, 7.92; O, 15.08; P, 2.92

ODMTON

HN

O

O

OP

O(CH2)9CH3

N(iPr)2

O

CleanAmp™-Pac-dA CleanAmp™-dTCleanAmp™-Pac-dGCleanAmp™-Ac-dC

RELATED

UltraMildDNASynthesis..........23

54

http://www.trilinkbiotech.com

CHAIN TERMINATORS

Insituationswhereligationmustbeblockedatthe5’terminus,5’-OMe-dTmaybeused.5’-OMemodificationofastrandofsiRNAusing5’-OMe-Tcancontrolguidestrandselectionandtargetingspecificity.15’-Amino-dTterminatesanoligonucleotidewitha5’-aminogroupwhichmaybeusedforattachingapeptideoraPNAsequence.Toavoidpolymeraseextensionatthe3’terminus,2’,3’-dideoxynucleosideand3’-deoxynucleosideCPGshaveprovedtobeeffective.2’,3’-Phosphoramiditesaredesignedtobeusedwiththe5’-phosphoramiditesandsupports.SincethesephosphoramiditeshavenoDMTgroup,theyarenotcompatiblewithpurificationbytheDMT-ontechnique.IonexchangeHPLCorPAGEshouldbeusedtopurifythesedideoxyterminatedoligostoensurethatshortersequences(containing3’-OH)groupsareremoved.(3’-Terminationcanalsobeeffectedusinga3’-3’linkageformedusing5’-supports,or3’-spacerC3CPG.)


5’-OMe-dT-CEPhosphoramidite 10-1031-90 100µmole 10-1031-02 0.25g

5’-Amino-dT-CEPhosphoramidite 10-1932-90 100µmole 10-1932-02 0.25g 3’-dA-CPG 20-2004-01 0.1g 1µmolecolumns 20-2104-41 Packof4 0.2µmolecolumns 20-2104-42 Packof4

3’-dC-CPG 20-2064-01 0.1g 1µmolecolumns 20-2164-41 Packof4 0.2µmolecolumns 20-2164-42 Packof4 3’-dG-CPG 20-2074-01 0.1g 1µmolecolumns 20-2174-41 Packof4 0.2µmolecolumns 20-2174-42 Packof4 3’-dT-CPG 20-2084-01 0.1g 1µmolecolumns 20-2184-41 Packof4 0.2µmolecolumns 20-2184-42 Packof4


3’-dA-CPG 3’-dC-CPG 3’-dG-CPG 3’-dT-CPG 5’-OMe-Thymidine 5’-Amino-dT

O

O P N(iPr)2O CNEt

MeO

CH 3

O

O

N

HN

O

O P N(iPr)2O CNEt

O

O

N

HNCH 3

MMTNH

ODMTO

O succinylCPG

N

N

N

N

NHBz

ODMTO

O succinylCPG

N

NO

NHBz

ODMTO

O succinylCPG

N

O

N

N

N

HNMe2N

ODMTO

O succinylCPG

CH 3

O

O

N

HN

REFERENCE

(1)P.Y.Chen,etal.,RNA, 2008, 14,263-274..




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RELATED

5’-Phosphoramidites.................345’-Supports................................353’-SpacerC3CPG......................84

55

MIN

OR

BASE

S

CHAIN TERMINATORS (CONT.)


2’,3’-ddC-CPG 20-2017-01 0.1g 1µmolecolumns 20-2117-41 Packof4 0.2µmolecolumns 20-2117-42 Packof4

2’,3’-ddA-CEPhosphoramidite 10-7001-90 100µmole 10-7001-02 0.25g

2’,3’-ddC-CEPhosphoramidite 10-7101-90 100µmole 10-7101-02 0.25g

2’,3’-ddG-CEPhosphoramidite 10-7201-90 100µmole 10-7201-02 0.25g

2’,3’-ddT-CEPhosphoramidite 10-7301-90 100µmole 10-7301-02 0.25g


2’,3’-ddC-CPG 2’,3’-ddA 2’,3’-ddC 2’,3’-ddG 2’,3’-ddT

succinylCPG

ODMTO

NH

O N

N

OP(iPr)2NOCNEt

N

N

N

N

N

O

N(iBu)2

OP(iPr)2NOCNEt

O

N

O N

N

N(iBu)2

OP(iPr)2NOCNEt

O

Me2NN

O

N

N

N

HN

O

CH 3

O

O

N

HN

CNEt O(iPr)2N P O




Expedite EMerMade M




56

STRUCTURAL STUDIES

7-Deaza-2’-deoxyAdenosine 7-Deaza-2’-deoxyGuanosine

STRUCTURE/ACTIVITY RELATIONSHIP

Thefollowingproductsareusedtoinvestigatetheeffectontheactivityofanoligonucleotidewhenkeystructuralelementsarechanged.The7-deazapurinemonomerslackgroupscriticalforhydrogenbonding.7-Deaza-8-aza-Aand7-deaza-8-aza-G(PPG)monomersareisomersofAandGandhavesimilarelectrondensity.TheirpresenceinoligosisslightlystabilizingrelativetoAandG.UnlikeG,PPGdoesnotleadtoaggregationandG-richoligoscanbeeasilypreparedandisolated.5’-FluoresceinoligoswithPPGatthe5’-terminusaremuchlessquenchedthantheequivalentGoligos.AsapurineanalogueofThymidine,7-deaza-2’-deoxyXanthosine(7-deaza-dX)promisestohaveinterestingeffectsonDNAstructureoftriplexes.7-Deaza-dXalsoformsanon-standardbasepairwitha2,4-diaminopyrimidinenucleosideanalogue.StandardnucleobaseshaveanunsharedpairofelectronsthatprojectintotheminorgrooveofduplexDNA.EnzymesthatinteractwithDNA,polymerases,reversetranscriptases,restrictionenzymes,etc.,mayuseahydrogenbonddonatinggrouptocontactthehydrogenbondacceptorintheminorgroove.3-Deaza-2’-deoxyadenosineisveryinterestinginthatitmaintainstheabilityforregularWatson-CrickhydrogenbondingtoTbutislackingtheelectronpairatthe3-positionnormallyprovidedbyN3.


7-Deaza-dA-CEPhosphoramidite 10-1001-95 50µmole 10-1001-90 100µmole 10-1001-02 0.25g

7-Deaza-8-aza-dA-CEPhosphoramidite 10-1083-95 50µmole 10-1083-90 100µmole 10-1083-02 0.25g

7-Deaza-dG-CEPhosphoramidite 10-1021-95 50µmole 10-1021-90 100µmole 10-1021-02 0.25g

7-Deaza-8-aza-dG-CEPhosphoramidite 10-1073-95 50µmole (PPG) 10-1073-90 100µmole 10-1073-02 0.25g

7-Deaza-dX-CEPhosphoramidite 10-1076-95 50µmole 10-1076-90 100µmole 10-1076-02 0.25g

3-Deaza-dA-CEPhosphoramidite 10-1088-95 50µmole 10-1088-90 100µmole 10-1088-02 0.25g

O

O P N(iPr)2O CNEt

DMTO

NHBz

NN

N

O

O P N(iPr)2O CNEt

DMTO

Me2NN

O

NN

HN HN

NN

N

O

N

DMTO

CNEtON(iPr)2PO

O

Me2N

7-Deaza-8-Aza-2’-deoxyAdenosine 7-Deaza-8-Aza-2’-deoxyGuanosine


TheuseofPPGissubjecttoproprietaryrightsofELITechGroupand it is sold under license from ELITechGroup.

(1)I.V.Kutyavin,etal.,Nucleic Acids Res., 2002, 30, 4952-4959.

STABILITY NOTES

7-Deaza-dGisunstabletoiodineoxidation.Addamaximumof2timeswhenusingiodineoxidationoruse0.5M(10-camphorsulfonyl)-oxaziridine(CSO)inanhydrousacetonitrileand3min.oxidationtime.(SeeGlenReport-Vol.9,No.1,1996,page8.)

O

O P N(iPr)2O CNEt

DMTO

Me2NN

N

NN

N

7-deaza-dX

O

O P N(iPr)2O CNEt

DMTO

O

O

NN

HN

H

N N

N

NHBz

O

O P N(iPr)2O CNEt

DMTO

3-Deaza-dA

57

MIN

OR

BASE

S

STRUCTURE/ACTIVITY RELATIONSHIP (CONT.)

TheC-nucleoside2’-deoxypseudouridine,incontrasttodU,formsstableC:pseudoU-Atriplets.2-Aminopurinelacksgroupscriticalforhydrogenbondingandisamildlyfluorescentbase.

Demandforsulfurmodifiedbasescontinuestoexpandforinvestigationsofoligonucleotidestructure,butprimarilyforcross-linkingpurposes. 6-Thio-dG,4-Thio-dTand4-thio-dUare veryusefulmodifications forphoto cross-linkingandphotoaffinitylabelingexperiments.Oligoscontaining2-thio-dTareusefulinexaminingprotein-DNAinteractionbyactingasphotosensitizingprobes.Thethiocarbonylgroupin2-thio-dTisespeciallyinterestinginthatitisavailabletoreactwithcompoundsassociatingwiththeminorgrooveofDNA.2-Amino-AformsaverystablebasepairwithTcontainingthreehydrogenbondsbutthestabilityofthebasepairwith2-thio-Tisgreatlydiminished.Duetostericinteractionsbetweenthe2-thiogroupofthymidineandthe2-aminogroupof2-amino-A,thebasepaircontainsonlyasinglehydrogenbond.Oligos containing 2-amino-dAand2-thio-dTexhibithighaffinityfornaturaloligonucleotidesbutshowlittleaffinityforothersimilaroligosevenofacomplementarysequence.


2’-deoxypseudoU-CEPhosphoramidite 10-1055-95 50µmole 10-1055-90 100µmole 10-1055-02 0.25g

2-Aminopurine-CEPhosphoramidite 10-1046-90 100µmole 10-1046-02 0.25g

6-Thio-dG-CEPhosphoramidite 10-1072-95 50µmole 10-1072-90 100µmole 10-1072-02 0.25g

4-Thio-dT-CEPhosphoramidite 10-1034-95 50µmole 10-1034-90 100µmole 10-1034-02 0.25g

4-Thio-dU-CEPhosphoramidite 10-1052-95 50µmole 10-1052-90 100µmole 10-1052-02 0.25g

2-Thio-dT-CEPhosphoramidite 10-1036-95 50µmole 10-1036-90 100µmole 10-1036-02 0.25g

STRUCTURAL STUDIES

4-Thio-dT 2-Thio-dT2-Aminopurine 4-Thio-dU6-Thio-dG2’-dpseudoU

O

O P N(iPr)2O CNEt

DMTOO

O

NHHN

O

O P N(iPr)2O CNEt

DMTO

Me2NN

N NN

N

TFAHN

N

N

N

N

S

DMTO

CNEtON(iPr)2PO

O

CN

O

O P N(iPr)2O CNEt

DMTON

N

O

S

CN

CH 3

O

O P N(iPr)2O CNEt

DMTON

N

O

S

CN

CH 3

O

O P N(iPr)2O CNEt

DMTON

N

O

S

H3CO

STABILITY NOTES

6-Thio-dG,4-Thio-dTand4-thio-dUareprotectedastheS-cyanoethyletherwhichisstableduringsynthesisandreadilyremovedbyammoniumhydroxide.Itiscriticaltoadd50mMsodiumhydrosulfide(NaSH)totheammoniumhydroxideusedfordeprotection.Especiallyifroomtemperaturedeprotectioniscarriedout,thistechniqueradicallyreducesthelevelofammonolysiswhichwouldleadtoundesiredaminatedbases.Moreover,itisalsodesirabletoremovethecyanoethylprotectinggroup(1MDBUinacetonitrile,2-5h/RT)priortotheammoniumhydroxidecleavageanddeprotection.




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58

STRUCTURE/ACTIVITY RELATIONSHIP (CONT.)

8-Amino-dAand8-amino-dGareusefulintriplexformationduetothepresenceoftheadditionalaminogroups.

2’-DeoxyXanthosine (dX) is a naturally occurring nucleoside thatmay be derived fromoxidative deamination of2’-deoxyGuanosine(dG).dXhasasimilarbondingpatterntothymidineanditmaybasepairwithdA,withsuchpurine-purineinteractionscausingduplexdistortion.dXalsofeaturedinattemptstoextendthegeneticalphabetwithanewbasepairofdXandpyrimidine-2,4-diaminenucleoside.dXhasalsointerestedresearchersinthefieldofDNAdamageandrepairsinceitisaproductofnitricoxide-inducedmutagenesis.


8-Amino-dA-CEPhosphoramidite 10-1086-95 50µmole 10-1086-90 100µmole 10-1086-02 0.25g

8-Amino-dG-CEPhosphoramidite 10-1079-95 50μmole 10-1079-90 100μmole 10-1079-02 0.25g

2’-dX-CEPhosphoramidite 10-1537-95 50µmole 10-1537-90 100µmole 10-1537-02 0.25g

O

N

N

N

N

NN

NMe 2

NMe 2

DMTO

O P N(iPr)2O CNEt

O P N(iPr)2O CNEt

N(Me 2)(Me2)N N

HN

N

N

N

O

N

DMTO O

8-Amino-dA 8-Amino-dG

STABILITY NOTE

Syntheticoligonucleotidescontaining8-amino-dGmustbecleavedanddeprotectedwithammoniumhydroxidecontaining0.25M2-mercaptoethanoltoavoidoxidativedegradationof8-amino-dGsites.

STRUCTURAL STUDIES

N

N N

N

O

ODMTO

O P N(iPr)2

O CNEt

O

NO2

O2N

dX

59

MIN

OR

BASE

S

HALOGENATED NUCLEOSIDES

Brominated and iodinated nucleosides are used in X-raycrystallographystudiesofoligonucleotidestructure.Theyarealsophotolabileandareusedforcross-linkingstudiestoprobethestructureofprotein-DNAcomplexes.AntibodiesexisttoBr-dUandoligonucleotidescontainingBr-dUcanbeusedasprobes.5-Fluoro-dUcanbeusedasanon-photoreactivealternativeto5-Br-dUwithsimilarelectrondensity.5-F-dUbasepairsmorestronglythanTtobothdAandthedGmismatch.ItisalsousefulforprobingDNAstructureusing19FNMRspectroscopy.


8-Br-dA-CEPhosphoramidite 10-1007-90 100µmole 10-1007-02 0.25g

8-Br-dG-CEPhosphoramidite 10-1027-90 100µmole 10-1027-02 0.25g

5-Br-dC-CEPhosphoramidite 10-1080-90 100µmole 10-1080-02 0.25g

5-I-dC-CEPhosphoramidite 10-1081-90 100µmole 10-1081-02 0.25g

5-Br-dU-CEPhosphoramidite 10-1090-90 100µmole 10-1090-02 0.25g

5-I-dU-CEPhosphoramidite 10-1091-90 100µmole 10-1091-02 0.25g

5-F-dU-CEPhosphoramidite 10-1092-90 100µmole 10-1092-02 0.25g

5-Br-dU-CPG 20-2090-01 0.1g1µmolecolumns 20-2090-41 Packof40.2µmolecolumns 20-2090-42 Packof4

STRUCTURAL STUDIES

5-Iodo-2’-deoxyCytidine 5-Bromo-2’-deoxyUridine 5-Iodo-2’-deoxyUridine 5-Fluoro-2’-deoxyUridine

O

O P N(iPr)2O CNEt

DMTO

NHBz

O N

NI

O

O P N(iPr)2O CNEt

DMTOO

O

N

HNBr

O

O P N(iPr)2O CNEt

DMTOO

O

N

HNI

O

O P N(iPr)2O CNEt

DMTOO

O

N

HNF




Expedite EMerMade M




8-Bromo-2’-deoxyGuanosine8-Bromo-2’-deoxyAdenosine

O P N(iPr)2O CNEt

DMTO

N

N

N

N

N

Br

NCH 3

CH 3

O O

O P N(iPr)2O CNEt

DMTO

HN

N

N

N

O

NBrMe2N

O

O P N(iPr)2O CNEt

DMTO

NHBz

O N

NBr

5-Bromo-2’-deoxyCytidine

STABILITY NOTE

Oligonucleotidescontainingabromooriodogrouparepreparedconventionallywiththeexceptionthatdeprotectioniscarriedoutinammoniumhydroxideatroomtemperaturefor24hours.Undertheseconditions,degradationof thehalogengroupwaslessthan2%.

60

DNA DAMAGE/REPAIR

CellularDNA isconstantlybeingdamagedbyoxidationandalkylation,by free radicals,andbyultravioletand ionizingradiation. Thebodyhas thereforeevolved anumberof repair enzyme systems to excise and repair these lesions. The8-oxopurinemonomersallowinvestigationofthestructureandactivityofoligonucleotidescontainingan8-oxomutationwhichisformednaturallywhenDNAissubjectedtooxidativeconditionsorionizingradiation.5,6-DihydropyrimidinesarenaturallyoccurringcompoundsthatarestructuralcomponentsofalaninetransferRNA.DihydrouracilandthehydroxypyrimidinesaremajorbasedamageproductsformedbyexposureofDNAtoionizingradiation.


8-Oxo-dA-CEPhosphoramidite 10-1008-90 100µmole 10-1008-02 0.25g

8-Oxo-dG-CEPhosphoramidite 10-1028-95 50µmole 10-1028-90 100µmole 10-1028-02 0.25g

5,6-Dihydro-dT-CEPhosphoramidite 10-1530-90 100µmole 10-1530-02 0.25g

5,6-Dihydro-dU-CEPhosphoramidite 10-1550-90 100µmole 10-1550-02 0.25g

5-OH-dC-CEPhosphoramidite 10-1063-90 100µmole 10-1063-02 0.25g

5-OH-dU-CEPhosphoramidite 10-1053-90 100µmole 10-1053-02 0.25g

5-Hydroxymethyl-dU-CEPhosphoramidite 10-1093-90 100µmole 10-1093-02 0.25g

STRUCTURAL STUDIES

8-oxo-2’-deoxyAdenosine 8-oxo-2’-deoxyGuanosine

5,6-Dihydro-dU

5,6-Dihydro-dT

5-OH-dU 5-Hydroxymethyl-dU

STABILITY NOTES

Syntheticoligonucleotidescontaining8-oxo-dGmustbecleavedanddeprotectedwithammoniumhydroxidecontaining0.25M2-mercaptoethanoltoavoidoxidativedegradationof8-oxo-dGsites.

Oligonucleotidessynthesizedusing5,6-dihydro-dUor5,6-dihydro-dTandUltraMILDmonomerscanbecleaved using either concentrated ammoniumhydroxideor50mMpotassiumcarbonateinanhydrousmethanol.Completecleavageanddeprotectioncanbeaccomplishedatroomtemperaturein2-4hourswithoutdamagingeitherthedihydro-dUordihydro-dTbases.

5-OH-dC

N

N N

NHO

NHBz

O P N(iPr)2O CNEt

DMTOO

HN

N

NH

N

O

iBuHNO

O P N(iPr)2O CNEt

DMTOO O

O P N(iPr)2O CNEt

DMTOH

CH 3

O

O

N

HN

O

O P N(iPr)2O CNEt

DMTOO

O

N

HN

Bz

N

NO

NH

OBz

O

O P N(iPr)2O CNEt

DMTOO

O P N(iPr)2O CNEt

DMTO

OAc

O

O

N

HN

O

O P N(iPr)2O CNEt

DMTO O

O

N

HN OAc

RELATED

5-Hydroxymethyl-dC.................50dX...............................................59

61

MIN

OR

BASE

S

DNA DAMAGE/REPAIR (CONT.)

8-Amino-Gisformedalongwith8-oxo-GasthemajormutageniclesionsformedinDNAdamagecausedby2-nitropropane.2-Nitropropaneisanindustrialsolventandacomponentofpaints,dyesandvarnishes,andisalsopresentincigarettesmoke. Thymineglycol (5,6-dihydroxy-5,6-dihydrothymine) is formedwhen thymine is subjected tooxidative stress,includingionizingradiation.Oxidationofthe5,6doublebondofThymidinegeneratestwochiralcentersatC5andC6.The cis-5R,6S form is generatedas thepredominantproduct alongwith theotherdiastereomer, the cis-5S,6R form. Thepresenceofthymidineglycol inDNAhassignificantbiologicalconsequencesandmanyorganismspossessspecificrepairenzymesfortheexcisionofthislesion.

HydrolysisofnucleosideresiduesinDNAoccurstogenerateabasicsites.Mostcommonly,dAsitesarehydrolyzedcausingdepurinationandleadingtoabasicresidues.ForresearcherstryingtodetermineiftheirsourceofdepurinationinchemicalsynthesisofDNAisreagent,fluidicsorprotocol-based,weofferadepurination-resistantdAmonomer.Anewchemicalmethodallows thegenerationof abasic sites indoubleand single strandedoligonucleotidesusing verymild specificconditionsandwithverylowprobabilityofsidereactions.AbasicIIPhosphoramidite1hastheadvantageofsimplicityinthatthesilylgroupisremovedpost-synthesisusingaqueousaceticacid.dSpacerhasalsobeenusedsuccessfullyasamimicofthehighlybase-labileabasicsite.


8-Amino-dG-CEPhosphoramidite 10-1079-95 50µmole 10-1079-90 100µmole 10-1079-02 0.25g

ThymidineGlycolCEPhosphoramidite 10-1096-95 50µmole 10-1096-90 100µmole 10-1096-02 0.25g

AbasicIIPhosphoramidite 10-1927-95 50µmole (dRPrecursor) 10-1927-90 100µmole 10-1927-02 0.25g

Abasic II Phosphoramidite

O P N(iPr)2O CNEt

N(Me 2)(Me2)N N

HN

N

N

N

O

N

DMTO ODMTO

O

OTBDMSOTBDMS

H

CH 3

O

O

N

HN

CNEtON(iPr)2PO

8-Amino-dG Thymidine Glycol

STRUCTURAL STUDIES

ODMTO

O P N(iPr)2

O CNEt

OTBDMS

REFERENCE

(1)K.Groebke,andC.J.Leumann,Helv

Chim Acta, 1990, 73,608-617.

STABILITY NOTES

Syntheticoligonucleotidescontaining8-amino-dGmustbecleavedanddeprotectedwithammoniumhydroxidecontaining0.25M2-mercaptoethanoltoavoidoxidativedegradationof8-amino-dGsites.

OligonucleotidessynthesizedusingThymidineGlycolandUltraMILDmonomerscanbecleaved using either concentrated ammoniumhydroxideor50mMpotassiumcarbonateinanhydrousmethanol.Completecleavageanddeprotectioncanbeaccomplishedatroomtemperaturein2-4hourswithoutdamagingThymidineGlycolbase.ThebestmethodtoremovetheTBDMSgroupswasachievedusingTEA.3HFat40°Covernight.




Expedite EMerMade M




RELATED

dSpacer......................................84Pyrrolidine.................................63

62

DNA DAMAGE/REPAIR (CONT.)

OneofthemajorsourcesofDNAdamageinallorganismsistheUVcomponentofsunlight.Thepredominantreactioninduced byUV light onDNA is dimerization of adjacent pyrimidine bases leading to cyclobutane dimers (CPDs). Thedimersformedinthemostsignificantquantityarethecis-syncyclobutanedimeroftwothyminebases.Althoughformedroutinely,thesedimerproductsareefficientlyexcisedandrepairedenzymaticallybynucleotideexcisionrepair(NER)orthedimerizationisreversedbyphotolaseenzymes.Afurthermodeofoxidativedamageisradiation-induceddamageofDNA,whichhasbeenshowntoleadtobridgedcyclonucleosides.Thepurines,cyclo-dAandcyclo-dG,arepredominantlyformed,althoughthecyclopyrimidineshavealsobeendetected.Cyclo-dAisdoublyintriguingsinceitcontainsbothdamagedbaseanddamagedsugarresiduesand,assuch,shouldhaveaconsiderablebiologicalimpact.Inamanneranalogoustothyminedimer,cyclopurinescausesignificantdistortionoftheregularDNAhelixandtheselesionsarerepairednotbybaseexcisionrepair(BER)butbyNER.


Cis-synThymineDimerPhosphoramidite 11-1330-95 50µmole 11-1330-90 100µmole 11-1330-02 0.25g 5’,8-Cyclo-dACEPhosphoramidite 10-1098 Discontinued

5’,8-Cyclo-dGCEPhosphoramidite 10-1598 Discontinued

Baseexcisionrepair (BER) isoneof themoststudiedrepairmechanisms. In thispathway,DNAglycosylases recognizethedamagedbasesandcatalyzetheirexcisionthroughhydrolysisoftheN-glycosidicbond.AttemptstounderstandthestructuralbasisforDNAdamagerecognitionbyDNAglycosylaseshavebeenhamperedbytheshort-livedassociationoftheseenzymeswiththeirDNAsubstrates.Toovercomethisproblem,theVerdinegroupatHarvardsynthesizedapyrrolidineanalogthatmimicsthechargedtransitionstateoftheenzyme-substratecomplex.Whenincorporatedintodouble-strandedDNA, they found thepyrrolidineanalog (PYR), introducedas thePyrrolidine-CEPhosphoramidite, formsanextremelystablecomplexwiththeDNAglycosylaseAlkA,exhibitingadissociationconstantinthepMrangeandpotentlyinhibitedthereactioncatalyzedbytheenzyme.


Pyrrolidine-CEPhosphoramidite 10-1915-95 50µmole (PYR) 10-1915-90 100µmole 10-1915-02 0.25g

STRUCTURAL STUDIES

ODMTO

O P O

NH

N

O

O

OCH 3

N(iPr)2PO

O

O

O

N

HNCH 3

CH 3H

HO

OCH 3

Cis-syn Thymine Dimer

INSTRUMENT TYPES

Fortheseveryexpensivephosphoramidites, an ABI septum vialisthestandardvial.AddEtothecatalogno.foranExpeditevialorVtothecatalogno.foranExpediteVvial.

5’,8-Cyclo-dA

N

NN

N

NHBz

ODMTO

O P N(iPr)2

O CNEt

5’,8-Cyclo-dG

O

THPO

O P N(iPr)2

O CNEt

NH

N

N

O

N NH

O

NDMTO

O P N(iPr)2

O CNEt

OO

Pyrrolidine 63

MIN

OR

BASE

S

CLICK DNA AND RNA LIGATION

Ligationofanoligocontaininga5’-azidewithanoligocontaininga3’-propargylgroupusingClickChemistryleadstoatriazolelinkagethathasbeenshowntohavein vivobiocompatibility.ThistechniquehasbeenusedtosynthesizeDNAconstructsupto300basesinlength.WhentheresultanttriazolelinkagewasplacedinaPCRtemplate,variouspolymeraseswereabletocopythesequencecorrectly.Thelinkagehasalsobeenshowntobecompatiblewithtranscriptionandrollingcircleamplification,aswellasgeneexpressioninE. coli.IntheRNAworld,ahammerheadribozymecontainingthetriazolelinkageatthesubstratecleavagesitehasbeenshowntoretainitsactivity.Alargevarietyofapplicationsisenvisagedforthis biocompatiblechemicalligation.SupportforthistechnologyisofferedwiththehelpofTomBrown’sgroupattheUniversityofSouthampton.


3’-Propargyl-5-Me-dCCPG 20-2982-01 0.1g 20-2982-10 1.0g 1µmolecolumns 20-2982-41 Packof4 0.2µmolecolumns 20-2982-42 Packof4 10µmolecolumn(ABI) 20-2982-13 Packof1 15µmolecolumn(Expedite) 20-2982-14 Packof1

5’-LABELING OF MicroRNAs

SeveralmethodshavebeendevelopedforthedetectionofmiRNAs,however,fewallowthesimultaneousdetectionofmultiplemiRNAs. Toovercome thisanalyticaldeficiency, theRichertgroupat theUniversityofStuttgarthas recentlydevelopedaningeniousmethodtoselectivelydetectmiRNAsonmicroarrayswithoutinterferencefromendogenouspre-mRNAs,mRNAsandotherRNAspecies.Inthismethod,ashortoligonucleotidecontaining3’-amino-dTanda5’reportermolecule ischemically ligatedtothemicroRNA inaone-stepprocedureby in situactivationofthemicroRNA. This isspecificallyachievedbytakingadvantageofthefactthatmiRNAs,unlikeotherRNAs,are5’-phosphorylated.Thereactionistemplate-directed(andthussequencespecific)andcanbeperformedtogetherwithenzymatic3’-extension/labeling,eitherinsolutionoronasupport.TheshortDNAlabelingstrandmayfeatureoneofavarietyofdifferentlabels,suchasabiotingrouporafluorophore.


3’-Amino-dTCPG 20-2981-01 0.1g 20-2981-10 1.0g 1µmolecolumns 20-2981-41 Packof4 0.2µmolecolumns 20-2981-42 Packof4 10µmolecolumn(ABI) 20-2981-13 Packof1 15µmolecolumn(Expedite) 20-2981-14 Packof1

STRUCTURAL STUDIES

ODMTON

HN

O

O

HN

CH3

CF2

F2C CF2 HN

O

O

ODMTON

N

NH

O

O

CH3

HN

O

O

3’-Propargyl-5-Me-dC CPG 3’-Amino-dT CPG

OO

O

base

DNA

ON3

O

base

DNA

+

OO

O

base

DNA

O

O

base

DNA

NN

N

OO

O

base

DNA

O

O

base

DNA

P O

O

-O

BIOCOMPATIBLE TRIAZOLE LINKAGE

REFERENCES - CLICK LIGATION

(1)A.H.El-Sagheer,A.P.Sanzone,R.Gao,A.Tavassoli,andT.Brown,Proc Natl Acad Sci U S A, 2011, 108,11338-43.

(2)A.H.el-Sagheer,andT.Brown,Chem Commun (Camb), 2011, 47,12057-8.

(3)A.P.Sanzone,A.H.El-Sagheer,T.Brown,andA.Tavassoli,Nucleic Acids Res,2012.

(4)A.Dallmann,etal.,Chemistry, 2011, 17,14714-7.

(5)A.H.El-Sagheer,andT.Brown,Proc Natl Acad Sci U S A, 2010, 107, 15329-34.

REFERENCES - MicroRNA Labeling

(1)H.Vogel,andC.Richert,ChemBioChem, 2012, 13,1474-82.

(2)R.Eisenhuth,andC.Richert,Journal of Organic Chemistry, 2008, 74,26-37.

(3)E.Kervio,A.Hochgesand,U.E.Steiner,andC.Richert,Proc Natl Acad Sci U S A, 2010, 107,12074-9.

RELATED

5’-I-dTinClickChemistry..........90ClickChemistry..........................88

64

2’-5’ LINKED OLIGONUCLEOTIDES

CellularDNAandRNAaremadeupofribo-and2’-deoxyribonucleicacidslinkedtogethervia3’-5’phosphodiesterlinkagesandbyfarcomprisethebulkofpolynucleicacidsfoundincells.Muchlesscommonareoligonucleotideswhichhave2’-5’linkages.However,auniquefeatureof2’-5’linkedoligonucleotidesistheirabilitytobindselectivelytocomplementaryRNA. These features suggest anumberof interestinguses for2’-5’ linkedoligos suchas theiruseasRNA specificprobesor in antisenseoligos. Recently, oligoshavebeen synthesizedusing3’-deoxy-2’-phosphoramidites and2’-deoxy-3’-phosphoramiditestoproducechimeraswith2’-5’ linkedendsand3’-5’ linkedcentralregions. Itwasfoundthat2’-5’phosphorothioateoligos:1)bindselectivelytocomplementaryRNAwiththesameaffinityasphosphodiesteroligos;2)exhibitmuchlessnonspecificbindingtocellularproteins;3)donotactivateRNaseH.A3’-deoxynucleosideatthe3’-terminusofanotherwisenormaloligonucleotideeffectivelyblockspolymeraseextension.


3’-dA-CEPhosphoramidite 10-1004-95 50µmole 10-1004-90 100µmole 10-1004-02 0.25g

3’-dC-CEPhosphoramidite 10-1064-95 50µmole 10-1064-90 100µmole 10-1064-02 0.25g

3’-dG-CEPhosphoramidite 10-1074-95 50µmole 10-1074-90 100µmole 10-1074-02 0.25g

3’-dT-CEPhosphoramidite 10-1084-95 50µmole 10-1084-90 100µmole 10-1084-02 0.25g

3’-dA-CPG 20-2004-01 0.1g 1µmolecolumns 20-2104-41 Packof4 0.2µmolecolumns 20-2104-42 Packof4

3’-dC-CPG 20-2064-01 0.1g 1µmolecolumns 20-2164-41 Packof4 0.2µmolecolumns 20-2164-42 Packof4

3’-dC 3’-dG 3’-dT

STRUCTURAL STUDIES

3’-dA

ODMTO

CNEtON(iPr)2PO

NHBz

N

N

N

NBz

ODMTO

CNEtON(iPr)2PO

NH

O N

NiBu

ODMTO

CNEtON(iPr)2PO

HN

O

N

N

N

HN

ODMTO O

O

N

HN

CNEtON(iPr)2PO

CH 3

3’-dA-CPG 3’-dC-CPG

ODMTO

O succinylCPG

N

N

N

N

NHBz

ODMTO

O succinylCPG

N

NO

NHBz




Expedite EMerMade M




RELATED

3’-deoxynucleosideCPG...........35

65

MIN

OR

BASE

S

2’-5’ LINKED OLIGONUCLEOTIDES (CONT.)


3’-dG-CPG 20-2074-01 0.1g 1µmolecolumns 20-2174-41 Packof4 0.2µmolecolumns 20-2174-42 Packof4 3’-dT-CPG 20-2084-01 0.1g 1µmolecolumns 20-2184-41 Packof4 0.2µmolecolumns 20-2184-42 Packof4

MUTAGENESIS

Cellularpolynucleotidesarealkylatedbyendogenouscomponents,suchasS-adenosylmethionine,orafterreactingwithtwogeneralclassesofenvironmentalandlaboratorychemicals.SN1chemicalagentsincludealkylnitrosoureaandN-alkyl-N-nitro-N-nitrosoguanidinethatreactwiththeN7positionofguanine,N3ofadenine,O6ofguanine,O2orO4ofpyrimidines,andthenon-phosphodiesteroxygenatomsofthephosphatebackbone.Incontrast,SN2chemicalagentssuchasmethylmethanesulfonateanddimethylsulfatereactprimarilywiththeN1positionofadenine(1-Methyl-2’-deoxyadenosine)andN3ofcytosine.ToavoidchainbranchingduringsynthesiswhenusingDCIasactivator,N6-Me-dAisofferedwithacetylprotection.


O6-Me-dG-CEPhosphoramidite 10-1070-90 100µmole 10-1070-02 0.25g

N6-Me-dA-CEPhosphoramidite 10-1003-90 100µmole 10-1003-02 0.25g

N6-Ac-N6-Me-dA-CEPhosphoramidite 10-1503-90 100µmole 10-1503-02 0.25g

O4-Me-dT-CEPhosphoramidite 10-1032 Discontinued

1-Me-dA-CEPhosphoramidite 10-1501-95 50µmole 10-1501-90 100µmole 10-1501-02 0.25g

O6-Me-2’-deoxyGuanosine N6-Methyl-2’-deoxyAdenosine O4-Me-Thymidine

STRUCTURAL STUDIES

3’-dG-CPG 3’-dT-CPG

ODMTO

O succinylCPG

N

O

N

N

N

HNMe2N

ODMTO

O succinylCPG

CH 3

O

O

N

HN

O

O P N(iPr)2O CNEt

DMTO

OMe

iBuHN N

N

N

N

O

O P N(iPr)2O CNEt

DMTO

NHMe

N

N

N

N

O

O P N(iPr)2O CNEt

DMTOO N

N

OMeCH 3

1-Methyl-2’-deoxyAdenosine

N

N N

N

NAc

ODMTO

O P N(iPr)2

O CNEt

Me

N6-Ac-N6-Me-dA

RELATED

N6-Me-dA..................................47

66

IN SITU SYNTHESIS OF DNA ANALOGS

The convertiblenucleosidestrategyisoneofthemostversatilemethodsforproducingmodificationsinbasestoexaminetheireffectsonDNAstructureandactivity.Insomecases,withversatilitycomesdifficultyinthattheconvertiblebaseismodifiedafteroligonucleotidesynthesis.Thechemistryissometimescomplexandbasecompositionanalysisofthefinaloligonucleotideisrequiredtoverifystructure.TheconvertibledUmonomercanbeusedtointroduceavarietyofmodificationsattheconvertibleposition,includingN,OandSmodifications.ConvertibleF-dCisbyfarthesimplestapproachtothepreparationofoligonucleotidescontainingF-dC-normalammoniumhydroxidetreatmenteffectstheconversiontoF-dC. ConvertibledAhasbeenusedtoprepareoligonucleotidescontainingmultiplepointsforattachmenttosolidsupports.Inthisway,highcapacityaffinitysupportsforthepurificationofDNAbindingproteinshavebeenprepared.2-F-dIisaconvertiblenucleosideforthepreparationof2’-dGderivativesfollowingthedisplacementofthe2-fluorinebyprimaryamines.


TMP-F-dU-CEPhosphoramidite 10-1016-90 100µmole (ConvertibleF-dC) 10-1016-02 0.25g

O6-Phenyl-dI-CEPhosphoramidite 10-1042-90 100µmole (ConvertibledA) 10-1042-02 0.25g

O4-Triazolyl-dU-CEPhosphoramidite 10-1051-90 100µmole (ConvertibledU) 10-1051-02 0.25g

2-F-dI-CEPhosphoramidite 10-1082-95 50µmole (ConvertibledG) 10-1082-90 100µmole 10-1082-02 0.25g

O6-Phenyl-dI O4-Triaz.-dU

STRUCTURAL STUDIES

TMP-F-dU

ABBREVIATION

TMP=2,4,6-trimethylphenyl

O

O P N(iPr)2O CNEt

DMTOO N

N

OF

O

O P N(iPr)2O CNEt

DMTO

OPh

N

N

N

N

O

O P N(iPr)2O CNEt

DMTOO N

N

NN

N

O

O P N(iPr)2O CNEt

DMTO

N

N

N

N

F

O

NO2

2-F-dI




Expedite EMerMade M




67

MIN

OR

BASE

S

STRUCTURAL STUDIES

Etheno-2’-deoxyAdenosine

O

O P N(iPr)2O CNEt

DMTON

N

N

N

N

PROBING DNA STRUCTURE WITH FLUORESCENT NUCLEOSIDES

Etheno-dAisafluorescentnucleosidewhichisespeciallyusefulinobservingthetransitionbetweenDNAstructuraltypes. Itisquitebaselabileandshouldbedeprotectedwithammoniumhydroxideatroomtemperaturefor24hours.Alternatively,UltraMildchemistrycanbeused.2-AminopurineandAP-dC(G-Clamp)arealsousefulfluorescentnucleosides.

Pyrrolo-dCisafluorescentdeoxycytidineanalogthatisanidealprobeofDNAstructureanddynamics.1,2Itbase-pairsasanormaldCnucleotide.Anoligofullysubstitutedwithpyrrolo-dChasthesameTmasthecontroldColigowiththesamespecificityfordG.ItssmallsizedoesnotperturbthestructureoftheDNAhelixanditiswelltoleratedbyanumberofDNAandRNApolymerases.Itishighlyfluorescentanditsexcitationandemissionarewelltotheredofmostfluorescentnucleotideanalogs,whicheliminatesorreducesbackgroundfluorescencefromproteins.Pyrrolo-dCTPhaspotentialusesinbiologicalassaydevelopment.


Etheno-dA-CEPhosphoramidite 10-1006-90 100µmole 10-1006-02 0.25g

Pyrrolo-dC-CEPhosphoramidite 10-1017-95 50µmole 10-1017-90 100µmole 10-1017-02 0.25g

Pyrrolo-dCTP 81-1017 Discontinued (10mM)

O

O P N(iPr)2O CNEt

DMTON

N

O

HN

Pyrrolo-dC

SPECTRAL PROPERTIES

Thespectralpropertiesofpyrrolo-dC,coupledwithitsuniquebase-pairingability,makethisfluorescentanalogextremelyvaluableinprobingDNAstructure.Whenthepyrrolo-dCisbase-paired,itsfluorescenceissignificantlyquenchedthroughwhatismostlikelybasestackingordGinteractions.Thequantumyieldoffluorescenceforpyrrolo-dCisquitesensitivetoitshybridizationstate,makingitideallysuitedforprobing thedynamicstructureofDNA.

QY l e (L/mol.cm)

single-stranded 0.07 260nm 4000 347nm 3700

double-stranded 0.02 (QYdeterminedrelativetoquinine

sulfatein0.5MH2SO4)

O

OH

ON

N

O

HN

POPOPNaOO O O

ONa ONa ONa

Pyrrolo-dCTP

REFERENCES

1. D.A.Berry,etal.,Tetrahedron Lett, 2004, 45,2457-2461.

2. TheGlenReport,2007,19,8-9.3. P.Sandin,etal.,Nucleic Acids Res.,

2008, 36,157-167.4. P.Sandin,etal.,Nucleic Acids Res.,

2005, 33,5019-5025.5. K.C.Engman,etal.,Nucleic Acids Res.,

2004, 32,5087-5095.


Pyrrolo-dCisajointdevelopmentprojectofBerry&Associates,Inc.andGlenResearchCorporation.Pyrrolo-dCiscoveredbyUSPatentNo.:7,144,995.

RELATED

2-Aminopurine..........................58AP-dC(G-Clamp).......................46UltraMildChemistry..................23Pyrrolo-C..................................132Pyrrolo-CTP..............................136

68

mol.cm

PROBING DNA STRUCTURE WITH FLUORESCENT NUCLEOSIDES (CONT.)

Byattachingpyreneorperylenetothe5positionofdeoxyuridinethroughatriplebond,thefluorophoreiselectronicallycoupled to thedeoxyuridinebase. Thiselectronic couplingof thebaseand thefluorophoremakes thefluorescencesensitivetothebasepairingofthedUportionofthemolecule,allowingthediscriminationbetweenperfectandonebasemismatchedtargets.


Pyrene-dU-CEPhosphoramidite 10-1590-95 50µmole 10-1590-90 100µmole 10-1590-02 0.25g

Perylene-dU-CEPhosphoramidite 10-1591-95 50µmole 10-1591-90 100µmole 10-1591-02 0.25g

ODMTON

HN

O

O

O P N(iPr)2

O CNEt

ODMTO

N

HN

O

O

O P N(iPr)2

O CNEt

Perylene-dUPyrene-dU

SPECTRAL PROPERTIES

Absorbance Emission Maximum Maximum

Pyrene-dU 402nm 472nmPerylene-dU 473nm 490nm




Expedite EMerMade M




STRUCTURAL STUDIES

69

MIN

OR

BASE

S

PROBING DNA STRUCTURE WITH FLUORESCENT NUCLEOSIDES (CONT.)

Thetricyclicfluorescentnucleosideanalogues,1,3-diaza-2-oxophenothiazine,tC,and1,3-diaza-2-oxophenoxazine,tCo, are deoxycytidineanalogsthathavebeenshowntobasepairfaithfullywithdGwithvirtuallynodisruptionofthenormalduplexstructure.3-5ThismeansthatthestabilityoftheDNAduplexisnotcompromisedascomparedtothecontrolregardlessofDNAsequence.ThefluorescencequantumyieldoftCisessentiallyunchangedbetweensinglestrandedanddoublestrandedDNA-0.21forsinglestrandedDNAand0.19forduplexDNA.Also,thefluorescencecharacteristicsoftCarenotsensitivetoneighboringbasecombinations.tCohasbeenshowntobethebrightestfluorescentnucleosideanalogueinduplexcontextreportedsofarandevenretainsthemajorityofitsfluorescencewhensurroundedbyguanineresidues.Indeed, tCohasbeenreportedtobe25-50timesbrighterthan2-aminopurine.

ThebaseanaloguetCnitroisaFRET-acceptortogetherwithtCO(ortC)asthedonormolecule.Thisconstitutesthefirstever

descriptionofanucleobaseFRET-pair.ThisnovelFRET-pairprovidesauniquetoolforinvestigationsofnucleicacidcontainingsystems.tCnitroisvirtuallynon-fluorescentundernormalconditions.


tC-CEPhosphoramidite 10-1516-95 50µmole 10-1516-90 100µmole 10-1516-02 0.25g

tC°-CEPhosphoramidite 10-1517-95 50µmole 10-1517-90 100µmole 10-1517-02 0.25g

tCnitro-CEPhosphoramidite 10-1518-95 50µmole 10-1518-90 100µmole 10-1518-02 0.25g

PHOTO-REGULATION OF DNA FUNCTION

GlenResearch’s interest lies inthepreparationofcagedoligonucleotideswhosefunctionisrestoredafteruncagingbyUVlightatawavelengththatcausesnoDNAdamage.TheDeitersgroupatNorthCarolinaStateUniversityhasdescribedNPOM-Caged-dT,wherethenucleobaseiscagedwiththephotolabilegroup,6-nitropiperonyloxymethyl(NPOM),whichcanberemovedusingUVlightat365nm.OligonucleotidescontainingNPOM-Caged-dTeveryfiveorsixbasesdonothybridizetotheircomplementarystrand.Photo-uncagingofthecagedoligonucleotideistheneasilycarriedoutwithUVlightat365nmforsecondstominutestorestoretheactivityoftheoligonucleotide.


NPOM-Caged-dT-CEPhosphoramidite 10-1534-95 50µmole 10-1534-90 100µmole 10-1534-02 0.25g

SPECTRAL PROPERTIES

AbsorptionandemissiondatafortC and tCoarecollectedbelow:

tC QY l e (L/mol.cm)

single-stranded 0.21 385nm 4000

double-stranded 0.19

tCo QY l e (L/mol.cm)

single-stranded 0.30 360nm 9000

double-stranded 0.21 (QYdeterminedrelativetoquinine

sulfatein0.5MH2SO4)

ODMTON

N

O

O P N(iPr)2

O CNEt

S

HN

ODMTON

N

O

O P N(iPr)2

O CNEt

O

HN

tCotC

O

N

N

O

O P N(iPr)2

O CNEt

S

HN

DMTO

NO2

tCnitro


TheseproductsareofferedincollaborationwithModyBaseHB.

ODMTO

N

N

O

O

H3C

O P N(iPr)2

O CNEt

O

NO2

OO

CH3

NPOM-Caged-dT

STRUCTURAL STUDIES

RELATED

Ribo-tCo...................................136

70

mol.cm

mol.cm

STRUCTURAL STUDIES

Ara-C

O

O P N(iPr)2O CNEt

DMTOOAc

NHAc

O N

N

INHIBITION OF DNA METHYLTRANSFERASES

Zebularine(pyrimidin-2-oneribonucleoside)isacytidineanaloguethatactsasaDNAdemethylaseinhibitor,aswellasacytidinedeaminaseinhibitor.ThisstructureisveryactivebiologicallyandZebularineisnowusedasapotentanti-cancerdrug. A2’-deoxynucleosideanalogueof Zebularine,5-methyl-pyrimidin-2-one,2’-deoxynucleoside,hasbeenused toprobetheinitiationofthecellularDNArepairprocessbymakinguseofitsmildlyfluorescentproperties.Thiscombinationofbiological activityandfluorescencepropertieswouldmake5-Me-2'-deoxyZebularinea strongaddition toourarrayofnucleosideanalogues.

Cytosine-5-methyltransferases are found ineverything fromarchaebacteria tomammals andwhen the regulationofcytosine-5-methyltransferasesgoesawry,cancercanresult.ThemechanismofactionforthisfamilyofenzymesinvolvesattackofacysteinethiolgroupontheC6positionofcytosine,leadingtoatransientdihydrocytosineintermediate,whichthenfacilitatesthenucleophilicattackbyC5ontheactivatedmethylgroupoftheS-adenosyl-L-methioninecofactor.Aswithmanyenzymes,theintermediatecanbetrappedusingasuicidesubstrateand5-fluoro-cytosinehasbeenusedextensivelyinthisrole.Analternatestrategyistouseatransition-statemimicthatbindstotheactivesitewithhighaffinity.Anexcellentcandidatewasfoundin5-aza-5,6-dihydrocytosine.Despitenotbeingcovalentlyboundtotheenzyme,itwasfound1,2tobeamorepotentinhibitorofcytosine-5-methyltransferasesthan5-fluoro-cytosine.5-Aza-5,6-dihydro-dCiscompatiblewithstandardoligonucleotidesynthesisanddeprotectionconditionsandisanexcellenttoolforuseinmethyltransferaseresearch.


5-Me-2'-deoxyZebularine-CEPhosphoramidite 10-1061-95 50µmole 10-1061-90 100µmole 10-1061-02 0.25g

5-Aza-5,6-dihydro-dC-CEPhosphoramidite 10-1511-95 50µmole 10-1511-90 100µmole 10-1511-02 0.25g

LARGE SCALE SYNTHESIS

ThemostcommonsidereactionduringdeprotectionofoligonucleotidesonalargescaleisthealkylationofdTresiduesbyacrylonitrile,formedbyß-eliminationofthecyanoethylphosphateprotectinggroups,togenerateN3-cyanoethyl-dT.


N3-Cyanoethyl-dT 10-1531-90 100µmole 10-1531-02 0.25g

5-Me-2'-deoxyZebularine

N

NODMTO

CNEtON(iPr)2PO

O O

O P N(iPr)2O CNEt

DMTO

N

NO

N

NH

N

5-Aza-5,6-Dihydro-dC

REFERENCES

(1)G.Sheikhnejad,etal.,J Mol Biol, 1999, 285,2021-2034.

(2)V.E.Marquez,etal.,Antisense Nucleic Acid Drug D, 1999, 9,415-421.

i

N3-Cyanoethyl-dT




Expedite EMerMade M




RELATED

ConvertibleF-dC........................355-Fluoro-2’-deoxyUridine.........60Pyrrolidine.................................63

71

MIN

OR

BASE

S

STRUCTURAL STUDIES

NON-CANONICAL STRUCTURES

DNAandRNAstructuresaredefinedbyWatson-Crickrulesofhybridization.However,avarietyofDNAandRNAstructureshavebeendefinedwhichdonotrelyonsimpleA-T/UandG-Cbinding.SincethesestructuresdisobeytheWatson-Crickcanon,theyaredescribedasnon-canonical.Non-canonicalDNAandRNAsegmentsareformedasaresultofsecondarystructures.TheseincludeG-quadruplexes,triplexformingoligos,hairpins,cruciforms,andi-Motifstructures.

G-QUADRUPLEX

Oligonucleotide structural analysis hasdemonstrated thatDNAandRNAnucleic acid sequences containingG-tractsseparatedbyotherbasesspontaneouslyfoldintoG-quadruplexstructures.G-quadruplexesareformedwhenfouradjacentguanineresiduesstackinacyclicHoogsteenhydrogen-bondingarrangementleadingtofour-strandedhelicalstructures.ThestudyofG-quadruplexesinbasicgeneticprocessesisanactiveareaofresearchintelomeraseactivity,generegulation,andfunctionalgenomics.Guanineanaloguesthathavedifferenthydrogenbondingcharacteristics-7-deaza-8-aza-dGand7-deaza-dG-haveprovedusefulinanalyzingG-quadruplexstructures.Similarly,commonDNAlesions-8-oxo-dGandabasicsites-havebeenusedtoinvestigatetheireffectonG-quadruplexstructureandactivity.

TRIPLEX-FORMING OLIGONUCLEOTIDES

Triplex-formingoligonucleotides(TFO)bindinthemajorgrooveofduplexDNAinasequence-specificmannerthroughtheformationofnonWatson-Crick(Hoogsteen)hydrogenbonds.Theformationofatriplexalongthemajorgroovecompeteswiththebindingoftranscriptionfactorsandotherproteinsthatarenecessaryfortranscription,thereby inhibitingtheexpressionofparticulargenes.AvarietyofnucleosideanalogueshavebeenusedinTFO-8-amino-dG,8-amino-dA,6-thio-dGanddeoxypseudouridine.

i-MOTIF DNA STRUCTURES

IntercalatedMotif(i-Motif)DNAstructuresmaybeformedinregionsrichin2’-deoxyCytidine.EspeciallyatacidicpH,thesestructurescouldbedescribedasC-Quadruplexeswithtwoparallelstrandedsequencesalsoheldtogetherinanantiparallelorientationbycytosine-cytosinebasepairs.SincethesestructuresarestableatacidicpH,theycanactasnanoswitchesbychangeinpH.AstheywerenotconsideredtobestableatphysiologicalpH,theywerenotinitiallyconsideredtoberelevanttobiologicalsystems.However,thestabilityofthecytosine-cytosinebasepairisenhancedbyintercallatingligandsandsoavarietyofi-Motifstructuresarenowconsideredtobebiologicallysignificant.Sincei-Motifstructureshavenowbeenobservedforminganddissolvinginlivingcells,thesestructuresarenowthesubjectofactiveinvestigationofthemeaningoftheiractivityinhumancells.ResearchisalsobeingdirectedtotheeffectofcommonDNAlesions,likedepurinatedsites,8-oxo-dGand5-hydroxymethyl-dC,onthesetransientstructures.

RELATED

7-Deaza-8-Aza- 2’-deoxyGuanosine...................578-oxo-2’-deoxyGuanosine........617-Deaza-2’-deoxyGuanosine....57AbasicIIPhosphoramidite........62dSpacer......................................848-Amino-dG...............................628-Amino-dA...............................596-Thio-dG...................................582’-deoxypseudoU-CEPhosphoramidite.......................585-Hydroxymethyl-dC.................50

72

STRUCTURAL STUDIES

APTAMER DEVELOPMENT

Aptamers,generatedthroughrepetitiveselectionusingSELEXoranequivalent in vivo procedure, are chosen for their abilitytobinddesiredtargetmolecules,whicharefrequentlysmallmoleculesusefulintherapeutics.Insomeways,theymaybedescribedaschemicallyengineeredversionsofantibodies.Ofcourse,nucleicacidaptamershaveadvantagesoverantibodiesinthattheycanbedevelopedrapidlybyin vitromethods,withthereproducibilityofchemicalsynthesisandinherentstabilityofmodifiedoligonucleotides.Afullbatteryofbase,sugarandinternucleotidemodificationsisavailableforaptamerdevelopment.

2’-F-RNAhasbeenusedextensivelyinaptamerdevelopment,aswellas2’-F-ANAmorerecently.AnarticleinTheGlenReportbyJeffCarter,Director,ProcessChemistry,SomaLogic,Inc.described1theuseofaDNAbackbonewith5-substituteddUanaloguesaslowoff-ratemodifiedaptamer(SOMAmer®)reagentstoenablemultiplexedscreeningofthousandsofserumorplasmaproteins.TheseaptamersalsoincludePCBiotinalongwithafluorophore,inthiscaseCyanine3,forsubsequentdetection.

REFERENCE

(1)J.Carter,The Glen Report, 2015, 27.1, 6-8.

RELATED

2’-F-RNAPhosphoramidites...1432’-F-Arabinonucleic Acid(2’-F-ANA)........................144PCBiotinPhosphoramidite....100Cyanine3Phosphoramidite...106

73

MIN

OR

BASE

S

MMTNH

CNEtON(iPr)2PO

MODIFIERS

TERMINUS MODIFIERS

GlenResearch5’-Modifiers aredesigned for use inDNA synthesizers to functionalize the5’-terminusof the targetoligonucleotide.The5’-Amino-Modifiersareavailablewithavarietyofchainlengthstofitexactlythedesiredapplication.

TheDMS(O)MT-protectedaminogroupiseasiertodeprotectcomparedtotheMMT-protectedone.Thesulfoxyderivativesurvivesconditionsofoligonucleotidesynthesisandcaneitherbecleavedwithstandarddeblocksolution,orleftintactforHPLCpurification.Atthesametime,theDMS(O)MTgroupisfullycompatiblewithcartridgepurification.Whendetritylationonacartridge iscarriedout,theDMS(O)MT+,which ismorestablethanMMT+,doesnotreattachitselftoanamine. Wenowoffer5’-DMS(O)MT-Amino-ModifierC6utilizingthisnewtritylbasedprotectinggroup.

5’-Amino-ModifierTEG,ahydrophilictriethyleneglycolethylaminederivative,is12atomsinlengthandfullysolubleinaqueousmedia.

MethacrylateC6Phosphoramiditeisaterminusmodifierthatattachesamethacrylatefunctionalgrouptoanoligonucleotide.


5’-Amino-ModifierC3-TFA 10-1923-90 100µmole 10-1923-02 0.25g

5’-Amino-ModifierC6 10-1906-90 100µmole 10-1906-02 0.25g

5’-Amino-ModifierC6-TFA 10-1916-90 100µmole 10-1916-02 0.25g

5’-Amino-ModifierC12 10-1912-90 100µmole 10-1912-02 0.25g

5’-Amino-Modifier5 10-1905-90 100µmole 10-1905-02 0.25g

5’-DMS(O)MT-Amino-ModifierC6 10-1907-90 100µmole 10-1907-02 0.25g

5’-Amino-ModifierTEG 10-1917-90 100µmole 10-1917-02 0.25g

MethacrylateC6Phosphoramidite 10-1891-90 100µmole 10-1891-02 0.25g

ABBREVIATIONS

CNEt=Cyanoethyl CPG = Controlled Pore Glass DMT=4,4’-Dimethoxytrityl Fmoc=Fluorenylmethoxycarbonyl iPr=Isopropyl MMT=4-Monomethoxytrityl T=Trityl TFA=Trifluroacetyl

5’-Amino-Modifier C3-TFA 5’-Amino-Modifier C6

5’-Amino-Modifier C12

5’-Amino-Modifier C6-TFA

5’-Amino-Modifier 5

TFANH

CNEtON(iPr)2PO

MMTNH

CNEtON(iPr)2PO

TFANHO P N(iPr)2

O CNEt

CNEtON(iPr)2POMMTNH

O


5’-Carboxy-ModifierC10isofferedfor sale under license from TriLink BioTechnologies,Inc.Itisintendedfor research and development purposesonly,andmaynotbeusedforcommercial,clinical,diagnosticoranyotheruse.ItiscoveredunderUSPatentNo.6,320,041.

i

5’-DMS(O)MT-Amino-Modifier C6

F3C

O

HN

OO

OO P N(iPr)2

O CNEt

O P N(iPr)2

O CNEt

HN

O

5’-Amino-Modifier TEG

Methacrylate C6 Phosphoramidite

RELATED

PCmodifiers..............................86

74

TERMINUS MODIFIERS (CONT.)

Ourmorerecent5’-aminomodifiers,protectedbyanovelphthalicaciddiamide(PDA)protectinggroup,arestablesolids.IncontrasttotheTFAprotectedaminomodifiers,whichareviscousoils,theanalogousPDAprotectedcompoundsaregranularpowders.Thisimportantpropertyofthesecompoundsallowsstraightforwardhandling,storageandaliquotingandleadstoasignificantincreaseinstability.

DeprotectionwithmethylamineingasphaseoraqueoussolutionorAMAleadstofastandcompleteremovalofthePDAprotectinggroup.However,ammoniumhydroxidewillnotdrivetheequilibriumreactiontocompletionandonlypartialdeprotectionoccurs-overnightdeprotectionwithammoniumhydroxidewillyieldaround80%activeamine.

WeareofferingthreePDAAmino-Modifiers:

•5’-Amino-ModifierC6-PDA •Hydrophobic5’-Amino-ModifierC12-PDA •Hydrophilic5’-Amino-Modifier-TEG-PDA


5’-Amino-ModifierC6-PDA 10-1947-90 100µmole 10-1947-02 0.25g

5’-Amino-ModifierC12-PDA 10-1948-90 100µmole 10-1948-02 0.25g

5’-Amino-Modifier-TEG-PDA 10-1949-90 100µmole 10-1949-02 0.25g

MODIFIERS


PDAamino-modifierswereevelopedbyStefanPitschandReseaChemGmbH(S.Berger),Patentpending.




Expedite EMerMade M




O

O

HN

HN

O P N(iPr)2

O CNEt

O

O

HN

HN

O P N(iPr)2

O CNEt

O

O

HN

HN

OO

OO P N(iPr)2

O CNEt

5’-Amino-Modifier C6-PDA

5’-Amino-Modifier-TEG-PDA5’-Amino-Modifier C12-PDA

75

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MODIFIERS

TERMINUS MODIFIERS (CONT.)

Thedisulfidethiolmodifiermaybeusedforintroducing3’-or5’-thiollinkages.Dithiol Serinol, produced from lipoic acid andourpatentedserinolbackbone,allowseasyconnectionofmultiplydithiol-labeledoligostogoldsurfaces.5’-Carboxy-ModifierC10isauniquelinkerdesignedtobeaddedattheterminusofanoligonucleotidesynthesis. ItgeneratesanactivatedcarboxylicacidN-hydroxysuccinimide(NHS)estersuitableforimmediateconjugationonthesynthesiscolumnwithmoleculescontainingaprimaryamine,resultinginastableamidelinkage.Analternativecarboxylateprotectinggroupisthe2-chlorotritylgroup,whichissimplyremovedusingthestandarddeblockcycletogenerateafreecarboxylgrouponanotherwisefullyprotectedoligonucleotide.The2-chlorotritylgroupisalsoremovedduringoligodeprotectionwithammoniumhydroxideorAMAandisincompatiblewithRPpurificationtechniques.PCAmino-ModifierisaphotocleavableC6amino-modifier,partofourlineofphotocleavable(PC)modifiers.5’-AminoOxy-Modifier11isbasedonatetraethyleneglycollinkageforimprovedsolubilityandforreducingthepotentialnegativeimpactonhybridizationoftheoligo.Theoximeformedfromthereactionofalkyloxyamineswithaldehydescreatesastablecovalentbond.Incomparison,theimineformedbytheconjugationofprimaryamineswithaldehydesisnotstabletoacidicorbasicconditionsandrequiressubsequentreductionwithborohydridetoformstableamineconjugates.5’-MaleimideModifierPhosphoramidite,developedattheUniversityofBarcelona,incorporatesamaleimidecycloadductthatisstabletoammoniumhydroxideatroomtemperature.Thisphosphoramidite canbe incorporated intoDNAandRNAwithbothphosphateandphosphorothioate linkages. Aretro–Diels-Alderreactiondeprotectsthemaleimideimmediatelypriortoconjugation.


5’-Thiol-ModifierC6 10-1926-90 100µmole 10-1926-02 0.25g

Thiol-ModifierC6S-S 10-1936-90 100µmole 10-1936-02 0.25g

DithiolSerinolPhosphoramidite 10-1991-95 50µmole 10-1991-90 100µmole 10-1991-02 0.25g

PCAmino-ModifierPhosphoramidite 10-4906-90 100µmole 10-4906-02 0.25g

5’-Carboxy-ModifierC10 10-1935-90 100µmole 10-1935-02 0.25g

5’-Carboxy-ModifierC5 10-1945-90 100µmole 10-1945-02 0.25g

5’-AminoOxy-Modifier11 10-1919-95 50µmole 10-1919-90 100µmole 10-1919-02 0.25g

5’-Maleimide-ModifierPhosphoramidite 10-1938-90 100µmole 10-1938-02 0.25g

5’-Thiol-Modifier C6 Thiol-Modifier C6 S-S

TS

CNEtON(iPr)2PO

5’-Carboxy-Modifier C10

DMTOS

SO P N(iPr)2

OCNEt

N

O

O

O

OO P N(iPr)2

O CNEt

TFAHNNH

ONO2

H3C

CNEtON(iPr)2PO

PC Amino-Modifier




Expedite EMerMade M




OO

O P N(iPr)2

O CNEt

OO

HNDMT

5’-AminoOxy-Modifier 11


5’-MaleimideModifierPhosphoramiditeisprotectedbyapatentapplicationandisofferedbyGlenResearchunderanon-exclusivelicenseagreementfromtheUniversityofBarcelona.

O

HH

N

O

O

CH2CH2O P N(iPr)2

O CNEt

5’-Maleimide-Modifier

O

O

Cl

O P N(iPr)2

O CNEt

5’-Carboxy-Modifier C5

SS

HN

HN

O O

ODMT

O P N(iPr)2

O-CNEtDithiol Serinol

76

SEQUENCE MODIFIERS

SequenceModifiersaredesignedforuseinautomatedsynthesis.Thecarboxy-dTishydrolyzedduringdeprotectionandcanbecoupleddirectlytoamoleculecontainingaprimaryaminogroupbyastandardpeptidecouplingorviatheintermediateN-hydroxysuccinimide (NHS)ester. Amino-ModifierdA,Amino-ModifierdC,N2-Amino-ModifierdGandbothAmino-ModifierdTproductscanbeaddedinplaceofadA,dC,dGanddTresidue,respectively,duringoligonucleotidesynthesis.CorrespondingAmino-Modifiersupportscanreplacetheirrespectivedeoxynucleosidesupports.Afterdeprotection,theprimaryamineontheC6analoguesisseparatedfromtheoligonucleotidebyaspacerarmwithatotalof7-10atomsandcanbelabeledorattachedtoanenzyme.TheC2analogueismoresuitablefortheattachmentofmoleculesdesignedtoreactwiththeoligonucleotide.


Amino-ModifierC6dA 10-1089-90 100µmole 10-1089-02 0.25g

Amino-ModifierC6dC 10-1019-90 100µmole 10-1019-02 0.25g

N2-Amino-ModifierC6dG 10-1529-95 50µmole 10-1529-90 100µmole 10-1529-02 0.25g

Carboxy-dT 10-1035-90 100µmole 10-1035-02 0.25g

Amino-ModifierC2dT 10-1037-90 100µmole 10-1037-02 0.25g 10-1037-05 0.5g

Amino-ModifierC6dT 10-1039-90 100µmole 10-1039-02 0.25g 10-1039-05 0.5g

MODIFIERS

Carboxy-dT Amino-Modifier C2 dT Amino-Modifier C6 dT

Amino-Modifier C6 dC

HN

N

O

O

O

OMe

O

O P N(iPr)2O CNEt

DMTO

Amino-Modifier C6 dA

O

CNEtON(iPr)2PO

DMTON

N

N

NNH

NHCCF 3

ONHBz

NHNHCCF 3

O O

O

CNEtON(iPr)2PO

N(CH 3)2

N

O N

N

DMTO

N2-Amino-Modifier C6 dG

NHCCF 3

O

HN

N

O

O

NH

O

O

O P N(iPr)2O CNEt

DMTO

HN

N

O

O

NHNHCCF 3

O O

O

O P N(iPr)2O CNEt

DMTO

HN

N

N

O

N

ODMTO

O P N(iPr)2

O CNEt

NH

CF3CNH

O

RELATED

Amino-Modifiersupports.........79

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SEQUENCE MODIFIERS (CONT.)

OurrepertoireofNHSesterderivativeshasbeenexpandedtoincludetheNHS-Carboxy-dT-CEPhosphoramidite.BymakingadTanalogoftheCarboxy-ModifierC10,itispossibletolabeloneormultiplesiteswithinanoligonucleotide.Thisopensupthepossibilitytolabelanynumberofdifferentdyesormoleculeswithinanoligonucleotidewhenthephosphoramiditeisunavailable.Doingsoisstraightforwardandmaybedonemanuallyoffthesynthesizeroreveninafully-automatedmannerontheDNAsynthesizer.

WehaveneverfoundconditionswhichallowtheTFAgrouptoberemovedfromanamino-modifierwhiletheoligonucleotideremainsattachedtothesupport.Weareabletosolvethisproblembyusinga9-fluorenylmethoxycarbonyl(Fmoc)protectinggroup.TheFmocgroupisremovedusingatwostepprocedure,thefirsttoremovethecyanoethylprotectiongroupsandflushtheformedacrylonitrilefromthesynthesiscolumnusing1%diisopropylamineinacetonitrile,andthesecondtoremovetheFmocgroupusing10%piperidineinDMF.Theaminogroupsoformedonthecolumncanbereactedwithavarietyofactivatedesters.WeofferFmoc-Amino-ModifierC6dTPhosphoramiditeasanucleosidicoptionandAmino-ModifierSerinolPhosphoramiditeasanon-nucleosidicalternative.WealsoofferS-Bz-Thiol-ModifierC6-dTtojointheranksofthiol-modifiersforoligonucleotidesynthesis.Thiol-ModifierC6-dTcanbeaddedasusualatthedesiredlocationswithinasequence.


NHS-Carboxy-dT 10-1535-90 100µmole 10-1535-02 0.25g

Fmoc-Amino-ModifierC6dT 10-1536-90 100µmole 10-1536-02 0.25g

S-Bz-Thiol-ModifierC6-dT 10-1538-95 50µmole 10-1538-90 100µmole 10-1538-02 0.25g

Amino-ModifierSerinolPhosphoramidite 10-1997-95 50µmole 10-1997-90 100µmole 10-1997-02 0.25g

i

ODMTO

N

HN

O

O

O P N(iPr)2

O CNEt

NH

OHN O

O

NHS-Carboxy-dT Fmoc-Amino-Modifier C6 dT

MODIFIERS




Expedite EMerMade M




ODMT

O P N(iPr)2

O CNEt

HN

HN

Fmoc

OODMTO

N

HN

O

O

O P N(iPr)2

O CNEt

NH

OHN

O

SBz

S-Bz-Thiol-Modifier C6-dT Amino-Modifier Serinol Phosphoramidite

RELATED

Carboxy-Modifiers.....................76

78

3’-MODIFIERS

3’-Amino-ModifierCPGs,containingaminogroupsprotectedwiththebase-labileFmocgroup,aredesignedtofunctionalizethe3’-terminusofthetargetoligonucleotidebytheintroductionofaprimaryamine.Inanalternativeapproach,thenitrogendestinedtobecomethe3’-aminogroupisincludedinaphthalimide(PT)groupwhichisattachedtothesupportthroughanamidegroupattachedtothearomaticring.Thissimplelinkageisverystabletoallconditionsofoligonucleotidesynthesisandcontainsnochiralcenter.Usinganextendedammoniumhydroxidetreatment(55°Cfor17hours),thecleavageoftheaminefromthephthalimideisaccomplishedalongwiththedeprotectionoftheoligonucleotide.ABI-stylecolumnsaresuppliedunlessotherwiserequested.

Item Cat. No. Pack

3’-Amino-ModifierC7CPG1000 20-2958-01 0.1g 20-2958-10 1.0g 1µmolecolumns 20-2958-41 Packof4 0.2µmolecolumns 20-2958-42 Packof4 10µmolecolumn(ABI) 20-2958-13 Packof1 15µmolecolumn(Expedite) 20-2958-14 Packof1

3’-Amino-ModifierSerinolCPG 20-2997-01 0.1g 20-2997-10 1.0g 0.2µmolecolumns 20-2997-42 Packof4 1µmolecolumns 20-2997-41 Packof4 10µmolecolumn(ABI) 20-2997-13 Packof1 15µmolecolumn(Expedite) 20-2997-14 Packof1

3’-PT-Amino-ModifierC3CPG 20-2954-01 0.1g 20-2954-10 1.0g 1µmolecolumns 20-2954-41 Packof4 0.2µmolecolumns 20-2954-42 Packof4 10µmolecolumn(ABI) 20-2954-13 Packof1 15µmolecolumn(Expedite) 20-2954-14 Packof1

3’-PT-Amino-ModifierC6CPG 20-2956-01 0.1g 20-2956-10 1.0g 1µmolecolumns 20-2956-41 Packof4 0.2µmolecolumns 20-2956-42 Packof4 10µmolecolumn(ABI) 20-2956-13 Packof1 15µmolecolumn(Expedite) 20-2956-14 Packof1

3'-PT-Amino-ModifierC6PS 26-2956-01 0.1g 26-2956-10 1.0g 200nmolecolumns(ABI3900) 26-2956-52 Packof10 40nmolecolumns(ABI3900) 26-2956-55 Packof10

MODIFIERS

3’-Amino-Modifier C7 CPG 3’-PT Amino-Modifier C3 CPG 3’-PT Amino-Modifier C6 CPG

-succinyl-lcaa-CPG

FmocNHODMT

O

N ODMTNH

O

O

CPG

O

NNH

O

O

ODMT

CPG

O

ODMT

O-succinyl-CPG

HN

HN

Fmoc

O

3’-Amino-Modifier Serinol CPG

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3’-MODIFIERS (CONT.)

The3’-Thiol-Modifier S-SCPG supports areused to introduce3’-thiol linkageswith threeand six atomspacers intooligonucleotides.3’-DithiolSerinolCPGisusedtointroduceadithiolgroupatthe3’-terminus.InconjunctionwithDithiolSerinolPhosphoramidite,itissimpletoproduceoligonucleotideswithmultiplethiolgroupsatthe3’terminus,whichisidealforconjugationtogoldsurfaces.WithGlycerylCPGthe3’-terminusofanoligonucleotideisreadilyoxidizedbysodiumperiodatetoforma3’-phosphoglycaldehyde.Thealdehydemaybefurtheroxidizedtothecorrespondingcarboxylicacid.Eitherthealdehydeorthecarboxylatemaybeusedforsubsequentconjugationtoamine-containingproducts.

Item Cat. No. Pack

3’-Thiol-ModifierC3S-SCPG 20-2933-01 0.1g 20-2933-10 1.0g 0.2µmolecolumns 20-2933-42 Packof4 1µmolecolumns 20-2933-41 Packof4 10µmolecolumn(ABI) 20-2933-13 Packof1 15µmolecolumn(Expedite) 20-2933-14 Packof1

3’-Thiol-Modifier6S-SCPG 20-2938-01 0.1g 20-2938-10 1.0g 0.2µmolecolumns 20-2938-42 Packof4 1µmolecolumns 20-2938-41 Packof4 10µmolecolumn(ABI) 20-2938-13 Packof1 15µmolecolumn(Expedite) 20-2938-14 Packof1

3’-DithiolSerinolCPG 20-2991-01 0.1g 20-2991-10 1.0g 0.2µmolecolumns 20-2991-42 Packof4 1µmolecolumns 20-2991-41 Packof4 10µmolecolumn(ABI) 20-2991-13 Packof1 15µmolecolumn(Expedite) 20-2991-14 Packof1

3’-GlycerylCPG 20-2902-01 0.1g 20-2902-10 1.0g 0.2µmolecolumns 20-2902-42 Packof4 1µmolecolumns 20-2902-41 Packof4 10µmolecolumn(ABI) 20-2902-13 Packof1 15µmolecolumn(Expedite) 20-2902-14 Packof1

MODIFIERS

3’-Glyceryl CPG

3’-Thiol-Modifier C3 S-S CPG

CPGNH

O ODMTO

O

OAc

-succinyl-lcaa-CPGDMTO S

S O




Expedite EMerMade M




3’-Dithiol Serinol CPG

DMTOO S

S OO-succinyl-CPG

3’-Thiol-Modifier 6 S-S CPG

SS

HN

HN

O O

ODMT

O succinoyl-CPG

RELATED

Dithiol Serinol............................76

80

3’-MODIFIERS (CONT.)

3’-Amino-ModifierC6dCCPGand3’-Amino-ModifierC6dTCPG replaceadCandT, respectively, at the3’-terminus. Theseproductsallowconvenientlabelingatthe3’withoutblockingtheterminusfromdesiredenzymaticactivity.

Item Cat. No. Pack

3’-Amino-ModifierC6dCCPG 20-2019-01 0.1g 20-2019-10 1.0g 1µmolecolumns 20-2019-41 Packof4 0.2µmolecolumns 20-2019-42 Packof4 10µmolecolumn(ABI) 20-2019-13 Packof1 15µmolecolumn(Expedite) 20-2019-14 Packof1

3’-Amino-ModifierC6dTCPG 20-2039-01 0.1g 20-2039-10 1.0g 1µmolecolumns 20-2039-41 Packof4 0.2µmolecolumns 20-2039-42 Packof4 10µmolecolumn(ABI) 20-2039-13 Packof1 15µmolecolumn(Expedite) 20-2039-14 Packof1

Amino-Modifier C6 dC CPG Amino-Modifier C6 dT CPG

NHNHCCF 3

O O

O

O

DMTO

succinyl-lcaa-CPG

N

NO

N

N(CH 3)2

ODMTO

O succinyl-lcaa-CPG

HN

N

O

O

NHNHCCF 3

O O

MODIFIERS

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CHEMICAL PHOSPHORYLATION

Chemical PhosphorylationReagent ismost commonlyused tophosphorylate the5’-terminusof anoligonucleotide. Althoughthisproductisalsosuccessfulin3’-phosphorylation,3’-PhosphateCPGallowsdirectpreparationofoligonucleotideswitha3’-phosphategroup.ChemicalPhosphorylationReagentIIcontainsaDMTgrouponasidechainwhichisstabletobasecleavageandcanbeleftontheoligonucleotideforuseinRPpurification.TheDMTgroupislaterremovedwithaqueousacidandthesidechainiseliminatedafterbrieftreatmentwithaqueousammoniumhydroxidetoyieldthe5’-phosphate.1 Solid CPR II is similar in performance to CPRIIbutitiseasiertopreparealiquotssinceitisapowder.Manyresearcherstreatsynthesissupportswithahinderedbase(e.g.,diethylamine,diisopropylethylamine,orDBU)post-synthesistoeliminateandremovethecyanoethylphosphategroups.Inthisway,theacrylonitrileformedinsituisremovedfromthesupportandisnotavailabletoalkylatedTresiduesattheN3positionintheoligos.Sincethesulfonylethylgroupin3’-PhosphateCPGisalsosusceptibletoß-eliminationleadingtooligocleavage,thistechniqueisnotcompatiblewith3’-phosphateCPG.UsingCPRIICPG,whichisbaselabilebutdoesnotsupportß-elimination,thecyanoethylgroupscanberemovedfromtheoligopriortocleavageandbasedeprotection.ABI-stylevialsandcolumnsaresuppliedunlessotherwiserequested.

Item Cat. No. Pack

ChemicalPhosphorylationReagent 10-1900-90 100µmole 10-1900-02 0.25g

3’-PhosphateCPG 20-2900-01 0.1g 20-2900-10 1.0g 1µmolecolumns 20-2900-41 Packof4 0.2µmolecolumns 20-2900-42 Packof4 10µmolecolumn(ABI) 20-2900-13 Packof1 15µmolecolumn(Expedite) 20-2900-14 Packof1

3'-PhosphatePS 26-2900-01 0.1g 26-2900-10 1.0g 200 nmole columns(ABI3900) 26-2900-52 Packof10 40 nmole columns(ABI3900) 26-2900-55 Packof10

3’-PhosphateCPG 25-2900-01 0.1g (HighLoad) 25-2900-10 1.0g 2.5µmolecolumns 25-2900-46 Packof4

ChemicalPhosphorylationReagentII 10-1901-90 100µmole (CPRII) 10-1901-02 0.25g

SolidChemicalPhosphorylationReagentII 10-1902-90 100µmole (SolidCPRII) 10-1902-02 0.25g

3’-CPRIICPG 20-2903-01 0.1g 20-2903-10 1.0g 0.2µmolecolumns 20-2903-42 Packof4 1µmolecolumns 20-2903-41 Packof4 10µmolecolumn(ABI) 20-2903-13 Packof1 15µmolecolumn(Expedite) 20-2903-14 Packof1

MODIFIERS

Chemical Phosphorylation Reagent II

Chemical Phosphorylation Reagent

3’-Phosphate CPG

DMTOS

O O CNEtON(iPr)2PO

DMTOS

O

O O

-succinyl-lcaa-CPG

DMTOEtO2C CO 2Et

P N(iPr)2O CNEt

O


SolidChemicalPhosphorylationReagent II and related supports arecoveredbyEuropeanPatent:EP0816368.

(1)A.Guzaev,H.Salo,A.Azhayev,andH.Lonnberg,Tetrahedron, 1995, 51, 9375-9384.

Solid Chemical Phosphorylation Reagent II

DMTO O P N(iPr)2

O CNEt

CONHMeMeHNOC

DMTO OCONHMeMeHNOC

succinyl-CPG

3’-CPR II CPG

RELATED

High load supports....................29

82

H.Salo

ALDEHYDE MODIFICATION

AldehydemodifierswouldbeattractiveelectrophilicsubstitutionsinoligonucleotidessincetheyareabletoreactwithaminogroupstoformaSchiff’sbase,withhydrazinogroupstoformhydrazones,andwithsemicarbazidestoformsemi-carbazones.TheSchiff’sbaseisunstableandmustbereducedwithsodiumborohydridetoformastablelinkagebuthydrazonesandsemicarbazidesareverystablelinkages.

Our collaborationwith ELITechGroup, formerly EpochBiosciences, has allowedus tooffer5'-Aldehyde-ModifierC2Phosphoramidite.TheacetalprotectinggroupissufficientlyhydrophobicforuseinRPHPLCandcartridgepurificationandisreadilyremovedafteroligonucleotidesynthesisunderstandardoligonucleotidedetritylationconditionswith80%aceticacid/20%wateror2%aqueoustrifluoroaceticacidduringcartridgepurification.

A formylindolenucleosideanaloguehasbeenused to introducealdehydegroupswithinanoligonucleotideorat the5’ terminus. Thisproducthasnoprotectinggroupon thealdehyde,whichmeans thatdeprotectionof themodifiedoligonucleotidecanbedonewithoutchangingpreferredconditions.

Item Cat. No. Pack

5'-Aldehyde-ModifierC2Phosphoramidite 10-1933-90 100µmole 10-1933-02 0.25g

FormylindoleCEPhosphoramidite 10-1934-90 100µmole 10-1934-02 0.25g

5'-Aldehyde-Modifier C2

CH 2CH 2CN

ONPO

OO

O


These Products are for research purposesonly,andmaynotbeusedforcommercial,clinical,diagnosticoranyotheruse.TheseProductsaresubjecttoproprietaryrightsofELITechGroup and are made and sold underlicensefromELITechGroup.There is no implied license for commercialusewithrespecttotheseProductsandalicensemustbeobtaineddirectlyfromELITechGroupwithrespecttoanyproposedcommercialuseoftheseProducts.“Commercialuse”includesbutisnotlimited to the sale, lease, license or othertransferoftheProductoranymaterial derived or produced from it, the sale, lease, license or other grant ofrightstousetheProductoranymaterial derived or produced from it, or the use of the Product to perform servicesforafeeforthirdparties(includingcontractresearch).

Asimpleagreementmustbesignedbeforeend-usersandcustomoligoservicesmaypurchasetheseproductsforuseasdefinedabove. https://www.glenresearch.com/media/productattach/import/technical_note/ELITechGroupProducts.pdf

MODIFIERS

Formylindole

N

ODMTO

O P N(iPr)2

O CNEt

CHO




Expedite EMerMade M




83

MO

DIF

ICAT

ION

/LAB

ELIN

G

SPACER MODIFIERS

ThespacerphosphoramiditesC3,9,C12and18areusedtoinsertaspacerarminanoligonucleotide.Thecompoundsmaybeaddedinmultipleadditionswhenalongerspacerisrequired.3’-SpacerC3CPGmayalsoactasablockerofexonucleaseandpolymeraseactivityatthe3’-terminus.dSpacerisusedtointroduceastableabasicsitewithinanoligonucleotide. PCSpacerisaphotocleavableC3spacermodifier,partofourlineofphotocleavable(PC)modifiers.

Item Cat. No. Pack

SpacerPhosphoramidite9 10-1909-90 100µmole 10-1909-02 0.25g

SpacerPhosphoramiditeC3 10-1913-90 100µmole 10-1913-02 0.25g

dSpacerCEPhosphoramidite 10-1914-90 100µmole 10-1914-02 0.25g

SpacerPhosphoramidite18 10-1918-90 100µmole 10-1918-02 0.25g

SpacerC12CEPhosphoramidite 10-1928-90 100µmole 10-1928-02 0.25g

3’-SpacerC3CPG 20-2913-01 0.1g 20-2913-10 1.0g 1µmolecolumns 20-2913-41 Packof4 0.2µmolecolumns 20-2913-42 Packof4 10µmolecolumn(ABI) 20-2913-13 Packof1 15µmolecolumn(Expedite) 20-2913-14 Packof1

PCSpacerPhosphoramidite 10-4913-90 100µmole 10-4913-02 0.25g

MODIFIERS

Spacer 18

dSpacerSpacer 9 Spacer C3

Spacer C12 PC Spacer

DMTO

CNEtON(iPr)2PO O P N(iPr)2

O CNEt

DMTOO

DMTOO

O

CNEtON(iPr)2PO

DMTOO

OO

OO

O P N(iPr)2O CNEt

DMTOO P N(iPr)2

O CNEt NH

ONO2

H3C

CNEtON(iPr)2PO

DMTO

RELATED

PCModifiers..............................86Pyrrolidine.................................63

84

Symmetric Doubler

Trebler

DENDRIMERS

Dendrimersarediscrete,highlybranched,monodispersedpolymersthatpossesspatternsreminiscentofthebranchingoftrees.Plainandmixedoligonucleotidedendrimerscanbesynthesizedusingnoveldoublingandtreblingphosphoramiditesynthons.1,2Dendrimersofferthefollowingadvantages.Incorporationoflabelusingγ-32P-ATPandpolynucleotidekinaseincreasesinproportiontothenumberof5’-ends.Fluorescentsignalalsoincreasesinproportiontothenumberof5’-ends,ifspacersareincorporatedbetweenthelabelsandtheendsofthebranches.WhenusingadendrimericoligonucleotideasaPCRprimer,thestrandbearingthedendrimerisresistanttodegradationbyT7Gene6exonucleasemakingiteasytoconvertthedouble-strandedproductofthePCRtoamultiplylabeled,single-strandedprobe.EnhancedstabilityofDNAdendrimersmakesthemusefulasbuildingblocksforthe‘bottomup’approachtonano-assembly.ThesefeaturesalsosuggestapplicationsinDNAchiptechnologywhenhighertemperaturesarerequired,forexample,tomeltsecondarystructureinthetarget.


SymmetricDoublerPhosphoramidite 10-1920-90 100µmole 10-1920-02 0.25g

AsymmetricDoubler(LEV)Phosphoramidite 10-1981-90 100µmole 10-1981-02 0.25g

TreblerPhosphoramidite 10-1922-90 100µmole 10-1922-02 0.25g

LongTreblerPhosphoramidite 10-1925-90 100µmole 10-1925-02 0.25g

BRANCHING PHOSPHORAMIDITE

Abranchingmonomerisrequiredtoconstructcomb-likeoligonucleotideprobes.ThedevelopersofthecombsystemfromChironCorporationevaluated3severalprotectinggroupsforthebranchpointandchoselevulinyl(LEV),whichisspecificallyremovedusingareagentcontaininghydrazinehydrate,aceticacidandpyridine.


5-Me-dCBrancherPhosphoramidite 10-1018-90 100µmole 10-1018-02 0.25g

MODIFIERS

REFERENCES

(1)M.S.Shchepinov,I.A.Udalova,A.J.Bridgman,andE.M.Southern,Nucleic Acids Res, 1997, 25, 4447-4454.

(2)M.S.Shchepinov,K.U.Mir,J.K.Elder,M.D.Frank-Kamenetskii,andE.M.Southern,Nucleic Acids Res, 1999, 27, 3035-41.

(3)T.Horn,C.A.Chang,andM.S.Urdea, Nucleic Acids Res, 1997, 25, 4842-4849.

NH

NH

DMTO

DMTOO

O

CNEtON(iPr)2PO

ODMTOCNEtON(iPr)2PO

DMTOO

DMTO O

O

O P N(iPr)2O CNEt

DMTO

O

O

ONH

O N

N CH 3

5-Me-dC Brancher

i

Long Trebler




Expedite EMerMade M




ONH

DMTONH

O

O

O

OO

P

O

N(iPr)2

CNEt

Asymmetric Doubler (LEV)

85

MO

DIF

ICAT

ION

/LAB

ELIN

G

MODIFIERS

PHOTOCLEAVABLE MONOMERS

PCBiotinPhosphoramiditecanbeusedtoprepare5’-biotinylatedoligonucleotidessuitableforcapturebystreptavidininamodesimilartoourpopular5’BiotinPhosphoramidite.Amino-andthiol-modifiedoligonucleotideshaveproventobeveryusefulfortheattachmentofavarietyofhaptensandfluorophores,aswellasforthetetheringoftheoligonucleotidestoadiversityofbeadsandsurfaces.PCAmino-ModifierPhosphoramiditeisusedtoprepare5’-amino-modifiedoligonucleotidessuitableforsubsequentphotocleavage.PCSpacerPhosphoramiditecanbeusedasanintermediarytoattachanymodificationreagent, availableasaphosphoramidite, to the terminusofoligonucleotides. Afterphotocleavage,a5’-phosphate isgeneratedontheDNA,renderingitsuitableforfurtherbiologicaltransformations,suchasgeneconstructionandcloningafterligation.

AversatilephotocleavableDNAbuildingblockhasbeendescribedbyresearchersinWashingtonUniversity,Missouriandusedinphototriggeredhybridization.1 ThisreagenthasalsobeenusedinthedesignofmultifunctionalDNAandRNAconjugates2 for the in vitroselectionofnewmoleculescatalyzingbiomolecularreactions.ResearchersatBrukerDaltonikinGermanyhavealsodevelopedgenoSNIP,amethodforsingle-nucleotidepolymorphism(SNP)genotypingbyMALDI-TOFmassspectrometry.3Thismethodusessizereductionofprimerextensionproductsbyincorporationofthephotocleavablelinkerforphototriggeringstrandbreaksneartothe3’endoftheextensionprimer.PCLinkercanbeincorporatedintooligonucleotidesatanypositionby standardautomatedDNAsynthesismethodology. PCLinkerPhosphoramiditehastheaddedadvantage inthatphotocleavageresults inmonophosphatefragmentsatboththe3’-and5’-terminioftheoligonucleotidefragments.


PCBiotinPhosphoramidite 10-4950-95 50µmole 10-4950-90 100µmole 10-4950-02 0.25g

PCAmino-ModifierPhosphoramidite 10-4906-90 100µmole 10-4906-02 0.25g

PCSpacerPhosphoramidite 10-4913-90 100µmole 10-4913-02 0.25g

PCLinkerPhosphoramidite 10-4920-90 100µmole 10-4920-02 0.25g

PC Biotin

PC Amino-Modifier PC Spacer

NHDMTN

SNH

O

ONH

ONO2

H3C

CNEtON(iPr)2PO

TFAHNNH

ONO2

H3C

CNEtON(iPr)2PO

NH

ONO2

H3C

CNEtON(iPr)2PO

DMTO


GlenResearchoffersPCBiotin,PCAmino-ModifierandPCSpacerproductsinassociationwithAmberGen,Inc.andLinkTechnologies,Ltd.Foracommercialapplicationlicense,pleasecontactAmberGen,Inc.,+617-923-9990,([email protected]), https://www.ambergen.com

PC Linker phosphoramidite is availablefromGlenResearchinassociationwithLinkTechnologies Ltd(Scotland).

REFERENCES

(1) P.OrdoukhanianandJ-S.Taylor,J. Am. Chem. Soc., 117,9570-9571,1995.

(2a)F.HauschandA.Jäschke,NucleicAcids Research, 2000, 28,e35.

(2b)F.HauschandA.Jäschke,Tetrahedron, 2001, 57,1261-1268.

(3) T.Wenzel,T.Elssner,K.Fahr,J.Bimmler,S.Richter,I.Thomas,andM.Kostrzewa,Nucleosides, Nucleotides & Nucleic Acids, 2003, 22,1579-1581.

PC Linker

NO2

CNEtON(iPr)2PODMTO




Expedite EMerMade M




RELATED

5’-Biotin.....................................99

86

mailto:sales%40ambergen.com?subject=

CONJUGATION USING CLICK CHEMISTRY

Thecopper(I)-catalyzedazide-alkynecycloaddition(CuAAC)reactionbetweenazidesandalkynestoform1,2,3-triazoles,as reported1bySharpless,wasfoundtobesoexquisitelyregioselectiveandefficientateventhemostmildconditionsthatSharplesscoinedtheterm‘ClickChemistry’todescribeit.TheuseofthismethodforDNAmodificationhasbeensomewhatdelayedbythefactthatcopperionsdamageDNA,typicallyyieldingstrandbreaks.2Astheseproblemshavenowbeenovercomebytheuseofcopper(I)-stabilizingligands(e.g.,tris(benzyltriazolylmethyl)amine,TBTA3),Carelletal.andSeelaetal.discoveredthattheCuAACreactioncanbeusedtofunctionalizealkyne-modifiedDNAnucleobaseswithextremelyhighefficiency.4

OligonucleotidesbearingasinglenucleosidicalkynegroupcanbepreparedusingaC8-Alkyne-dCordT-CEPhosphoramidite.Purifiedoligonucleotidesareusuallymodifiedwith2-5equivalentsofthecorrespondingmarker-azide(e.g.,fluorescent-dyeazides).AftertheadditionofprecomplexedCu(I),completeconversiontothelabeledoligoisobservedinatimespanbetween30minand4hours.Afterasimpleprecipitationstep,labeledoligonucleotidescanberecoveredinnearquantitativeyields.UsingacombinationofC8-Alkyne,C8-TIPS-AlkyneandC8-TMS-Alkyne,itispossibletolabeloligonucleotidesinuptothreeseparateclickreactions.Thealkynegroupsonthelasttwomonomersareprotected,respectively,withtriisopropylsilyl(TIPS)andtrimethylsilyl(TMS)protectinggroups.5,6ThefirstclickreactiononsolidphaseonaC8-AlkyneyieldsthesinglymodifiedoligonucleotidewithfullretentionoftheTIPSand/orTMSprotectinggroup.Fordoubleclick,aC8-TIPS-AlkyneisusedasthesecondnucleosideandtheTIPSprotectinggroupiscleavedwithtetrabutylammoniumfluoride(TBAF)withoutcausinganydamagetotheDNA.Thesecondclickreactioninsolutionyieldsthedoublymodifiedoligonucleotideinexcellentyield.Fortheintroductionofthreedifferentlabels,allthreenucleosidesareintroducedintooligonucleotides.Thefirstclickreaction isperformeddirectlyontheresin.Thesinglymodifiedoligonucleotide issubsequentlycleavedfromthesupportwithconcomitantcleavageoftheTMSgroupandretentionoftheTIPSprotectinggroup.Thesecondclickreactionisperformedinsolution.Precipitationofthedoublymodifiedoligonucleotide,cleavageoftheTIPSgroupwithTBAF,andasubsequentthirdclickreactioninsolutionfurnishesthedesiredtriplymodifiedoligonucleotideinexcellentoverallyield.


C8-Alkyne-dT-CEPhosphoramidite 10-1540-95 50µmole 10-1540-90 100µmole 10-1540-02 0.25g

C8-TIPS-Alkyne-dC-CEPhosphoramidite 10-1541 Discontinued

C8-TMS-Alkyne-dC-CEPhosphoramidite 10-1542 Discontinued

C8-Alkyne-dC-CEPhosphoramidite 10-1543-95 50µmole 10-1543-90 100µmole 10-1543-02 0.25g

MODIFIERS

REFERENCES

[1]C.W.Tornoe,C.Christensen,M.Meldal, J. Org. Chem. 2002, 67, 3057-3064;V.V.Rostovtsev,L.G.Green,V.V.Fokin,K.B.Sharpless,Angew. Chem. 2002, 114,2708-2711;Angew. Chem. Int. Ed. 2002, 41,2596-2599.

[2]C.J.Burrows,J.G.Muller,Chem. Rev. 1998, 98,1109–1151.

[3]T.R.Chan,R.Hilgraf,K.B.Sharpless,V.V.Fokin,Org. Lett. 2004, 6,2853–2855.

[4]J.Gierlich,G.A.Burley,P.M.E.Gramlich,D.M.Hammond,T.Carell, Org. Lett. 2006, 8,3639-3642.F.Seela,V.R.Sirivolu,Chem. Biodiversity 2006, 3,509-514.

[5]P.M.E.Gramlich,S.Warncke,J.Gierlich,T.Carell,Angew. Chem. 2008, 120,3491–3493;Angew. Chem. Int. Ed. 2008, 47,3442–3444.

[6]P.M.E.Gramlich,C.T.Wirges,A.Manetto,T.Carell, Angew. Chem. Int. Ed. 2008, 47,8350-8358.


baseclickGmbHhasbeengrantedthefollowingpatents(1-3)besidesitsfurtherpatentapplications(4-5).

1. WO2006/117161(Newlabelingstrategiesforthesensitivedetectionofanalytes)

2. WO2008/952775(Clickchemistryfortheproductionofreportermolecules)

3. WO2010/115957(ClickChemistryonheterogeneouscatalysts)

4. PCT/EP2013/064610(Anandamide-modifiednucleicmolecules)

5. PCT/EP2015/056007(Self-assemblyofDNAOrigami:adiagnostictool)

baseclickGmbHholdsaworldwideexclusivelicenseforgrantedpatentapplicationWO03/101972(Copper-catalysedligationofazidesandacetylenesforthenucleicacidfield)intheareaofdiagnosticsandresearch.

AsGlenResearchandbaseclickarepartners,GlenResearchisnowabletohelpinsublicensingthisoutstandingtechnology.

ODMTO

N

HN

O

O

O P N(iPr)2

O CNEt

ODMTO

N

N

NHBz

O

O P N(iPr)2

O CNEt

TIPS

ODMTO

N

N

NHBz

O

O P N(iPr)2

O CNEt

TMS

C8-Alkyne-dT C8-TMS-Alkyne-dCC8-TIPS-Alkyne-dC

ODMTO

N

N

NHBz

O

O P N(iPr)2

O CNEt

C8-Alkyne-dC

87

MO

DIF

ICAT

ION

/LAB

ELIN

G

CONJUGATION USING CLICK CHEMISTRY (CONT.)

5-Ethynyl-dUoffersconvenientclickconjugationwithanazidetogeneratealabelrigidlyattachedtooneoftheoligonucleotidebases.5-Ethynyl-dUissubjecttobase-catalyzedhydrationduringcleavageanddeprotection,especiallywhenusingastrongbaseorheat.Hydrationofanethynylgroupformsamethylketonewhichsubsequentlyblockspotentialclickreactions.Milddeprotectionconditionsarenecessarywhenusing5-Ethynyl-dU-CEPhosphoramiditetopreventthissidereaction.TIPS-5-Ethynyl-dU-CEPhosphoramidite,containingaprotectedalkyne,offersbroadercompatibilitywitholigonucleotidesynthesisanddeprotection.Protectingthe5-ethynylgroupwithatriisopropylsilyl(TIPS)protectinggrouppreventsacidorbasecatalyzedhydrationduringoligonucleotidesynthesisandworkup.AquicktreatmentwithTBAFremovestheTIPSprotectinggroup.


C8-TIPS-Alkyne-dT-CEPhosphoramidite 10-1544 Discontinued

C8-TMS-Alkyne-dT-CEPhosphoramidite 10-1545-95 50µmole 10-1545-90 100µmole 10-1545-02 0.25g

5-Ethynyl-dU-CEPhosphoramidite 10-1554-95 50µmole 10-1554-90 100µmole 10-1554-02 0.25g

TIPS-5-Ethynyl-dU-CEPhosphoramidite 10-1555-95 50µmole 10-1555-90 100µmole 10-1555-02 0.25g

THPTALigand 50-1004-92 25µmole (Watersoluble) 50-1004-90 100µmole

Click-Solution(DMSO/t-BuOH) 50-1002-11 10x1.0mL

ODMTO

N

HN

O

O

O P N(iPr)2

O CNEt

TIPS

ODMTO

N

HN

O

O

O P N(iPr)2

O CNEt

TMS

C8-TMS-Alkyne-dTC8-TIPS-Alkyne-dT

MODIFIERS




Expedite EMerMade M




ODMTO

N

HN

O

O

O P N(iPr)2

O CNEt

5-Ethynyl-dU

ODMTO

N

HN

O

O

O P N(iPr)2

O CNEt

TIPS

TIPS-5-Ethynyl-dU

RELATED

3’-Propargyl-5-Me-dCCPG.......64

88

REFERENCES

(1)R.Kumar,etal.,Journal of the American Chemical Society, 2007, 129,6859-6864.

(2)J.Lietard,A.Meyer,J.J.Vasseur,andF.Morvan,Tetrahedron Letters, 2007, 48,8795-8798.


Oligonucleotides prepared using 5’-Hexynyl Phosphoramidite are stable to standard deprotection conditions andexhibitaslightlyincreasedretentiontimeonRPHPLC.Azidesarenotcompatiblewitholigonucleotidesynthesisusingphosphoramiditessoapost-synthesisreactionisrequired.AzidobutyrateNHSEsterisused1forazido-modificationofaminesateitherthe3’-endorthe5’-endofanoligoanditcanevenbeusedforinternalmodificationonanAmino-Modifier-C6dXresiduewithinthesequence.Specifictothe5’-terminus,5’-BromohexylPhosphoramiditeisaddedinthelastcycle. Thismodifier can thenbeeasily transformed intoa5’-azidogroupbydisplacementofbromideusing sodiumazide.2 AlkyneNHSester allows the functionalizationof anaminomoiety ina varietyofmolecules, includingDNAandRNAoligonucleotidesaswellaspeptidesorproteins.WealsooffertwoproductsforuseinClickChemistrybaseduponour1,3-diolproductportfoliowiththeserinolbackbone-aphosphoramiditeforaddinganalkynegroupatthe5’terminusorwithinthesequence,andasynthesissupportforlabelingthe3’terminusofoligonucleotideswithanalkynegroup.


5’-HexynylPhosphoramidite 10-1908-90 100µmole 10-1908-02 0.25g

AzidobutyrateNHSEster 50-1904-23 2.3mg (Dissolve2.3mgin60µLofDMSO) 50-1904-24 23mg

5’-BromohexylPhosphoramidite 10-1946-90 100µmole 10-1946-02 0.25g

Alkyne-NHSEster 50-1905-23 2.3mg (Dissolve2.3mgin60µLofDMSO) 50-1905-24 23mg

Alkyne-ModifierSerinolPhosphoramidite 10-1992-95 50µmole 10-1992-90 100µmole 10-1992-02 0.25g

3’-Alkyne-ModifierSerinolCPG 20-2992-01 0.1g 20-2992-10 1.0g 0.2µmolecolumns 20-2992-42 Packof4 1µmolecolumns 20-2992-41 Packof4 10µmolecolumn(ABI) 20-2992-13 Packof1 15µmolecolumn(Expedite) 20-2992-14 Packof1

O P N(iPr)2

O CNEt

5’-Hexynyl Phosphoramidite

BrO P N(iPr)2

O CNEt

N3O N

O

OO

Azidobutyrate NHS Ester 5’-Bromohexyl Phosphoramidite

O

O

NH

NH

O

OODMT

succinoyl CPGO

O

ON

O

O

Alkyne-NHS Ester 3’-Alkyne-Modifier Serinol CPG

MODIFIERS

O

NH

NH

O

OODMT

P N(iPr)2

O CNEt

Alkyne-Modifier Serinol Phosphoramidite

RELATED

Serinol Products........................94

89

MO

DIF

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/LAB

ELIN

G


1-Ethynyl-dSpacerCEPhosphoramiditecanbeusedinanypositionwithinanoligonucleotidewhilestillretainingthehighefficiencyofclickchemistry.Themodifierisefficientlyincorporatedintooligonucleotidesusingstandardphosphoramiditechemistry,isstabletocommondeprotectionconditions,andiscompatiblewithGlen-Pak™purification.1-Ethynyl-dSpacergeneratesasubstituted1,2,3-triazolepseudo-nucleobaseafterclickchemistryconjugationwithanazide.The1-ethynyl-dSpacermodificationexhibitssimilarduplexstabilitytothestandarddSpacer(10-1914)anddestabilizestheduplexwheninternallyincorporated.Uponcycloaddition,theduplexstabilityismoderatedbytheresultingstructureofthemodification.Simple1,2,3-triazolesweredestabilizing,asweremodificationsthatincorporatedTEGlinkers(6-FAM-TEGandAmino-TEG).Modificationsthatincorporatedaromaticfunctionalgroupsrestoredduplexstabilitytovaryingdegreeswithcoumarinandpsoralensignificantlyrestoringstability.A5’-iodo-modifiedoligonucleotide(preparedusing5’-Iodo-dT)canbequantitativelyconvertedtothecorresponding5’-azide.


1-Ethynyl-dSpacerCEPhosphoramidite 10-1910-95 50µmole 10-1910-90 100µmole 10-1910-02 0.25g

5’-I-dT-CEPhosphoramidite 10-1931-90 100µmole 10-1931-02 0.25g

OLIGO-CLICK KITS

Oligo-ClickKitshasbeendiscontinued.Pleasecontacttechnicalsupport.


baseclickOligo-Click-M-Reload 50-2100 Discontinued

baseclickOligo-Click-M-Biotin 50-2101 Discontinued

baseclickOligo-Click-M-Fluorescein 50-2102 Discontinued

baseclickOligo-Click-M-TAMRA 50-2103 Discontinued

MODIFIERS

STABILITY NOTES

Oligonucleotidescontaininga5’-iodogrouparepreparedconventionallywiththeexceptionthatdeprotectionis carried out in ammonium hydroxideatroomtemperaturefor24hours.Undertheseconditions,degradationoftheiodogroupwaslessthan2%.

5’-I-dT

O

O P N(iPr)2O CNEt

IO

O

N

HNCH 3

ODMTO

O P N(iPr)2

O CNEt

1-Ethynyl-dSpacer

RELATED

dSpacer......................................84

90

COPPER-FREE CLICK CHEMISTRY

AtGlenResearch,ourgoalwastoofferacopper-freeclickphosphoramiditereagentwiththefollowingproperties:

• Simple to use •Stableinsolutiononthesynthesizer •StabletoammoniumhydroxideandAMA •Excellentclickperformancein17hoursorlessatroomtemperature

Fromthevarietyofcyclooctyne-basedcopper-freeclickreagentssofardescribed,wehavechosentooffercompoundsbasedonadibenzo-cyclooctyne(DBCO)structure.Weareoffering5’-DBCO-TEGPhosphoramiditeforpreparingoligoswitha5’-DBCOmodificationandDBCO-dT-CEPhosphoramiditeforinsertingaDBCOgroupatanypositionwithintheoligonucleotide.Inaddition,weofferafurtherDBCOphosphoramidite–DBCO-SerinolPhosphoramidite. Usingourproprietaryserinolbackboneasanon-nucleosidicspacerallowstheDBCOgrouptobeplacedatanylocationwithinasequencewithmultipleadditionsclearlypossible.DBCO-sulfo-NHSEsterisalsoofferedforpost-synthesisconjugationreactions.DBCO-modifiedoligosmaybeconjugatedwithazidesinorganicsolvents,suchasDMSO,oraqueousbuffers.Dependingontheazideused,thereactionwillgotocompletionin4-17hoursatroomtemperature.SimpledesaltingonaGlenGel-Pak™leadstoaproductwithvirtuallyquantitativeconjugationefficiency.

Note:Wenow recommend that synthesisofoligos containingDBCO-dTbecompletedusing0.5MCSO inanhydrousacetonitrile(40-4632-xx).AcceptableresultscanbeachievedwithiodineoxidationifDBCO-dTissubjectedtonomorethan8-10cycles.


5’-DBCO-TEGPhosphoramidite 10-1941-95 50µmole 10-1941-90 100µmole 10-1941-02 0.25g

DBCO-dT-CEPhosphoramidite 10-1539-95 50µmole 10-1539-90 100µmole 10-1539-02 0.25g

DBCO-sulfo-NHSEster 50-1941-23 5.2mg (Dissolve5.2mgin60µLwaterorDMSO) 50-1941-24 52mg

DBCO-SerinolPhosphoramidite 10-1998-95 50µmole 10-1998-90 100µmole 10-1998-02 0.25g

N HN

OO

OO

O

O P N(iPr)2

O CNEt

NO

OO

N

O

OSO3Na

DBCO-sulfo-NHS Ester

5’-DBCO-TEGODMTO

N

HN

O

O

O P N(iPr)2

O CNEt

NH

OHN

N

OO

DBCO-dT

MODIFIERS




Expedite EMerMade M




N

OO

HN

OP

NiPr2

ODMT

OCNEt

DBCO-Serinol

RELATED

0.5MCSO...................................32Serinol Products........................94

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MODIFIERS

HN

OS

NHHN

O

OO

ON3

HN

O

NHHN

O

OO

ON3

O

HN

O

OO

O

O

OO

OO

ON3

O

HN

O

OHHO

O

O

OO

ON3

BiotinTEG Azide DesthiobiotinTEG Azide

Dipivaloyl 6-FAM-TEG Azide 6-FAM-TEG Azide

REFERENCE

(1)J.Gierlich,G.A.Burley,P.M.Gramlich,D.M.Hammond,andT.Carell,Org Lett, 2006, 8,3639-42.

O

N3

OHO

Coumarin Azide

O

HN

O

OHHO

O

O

N3

Cl

Cl

Cl

ClCl

Cl

O

HN

O

OHHO

O

O

N3

Cl

Cl

ClCl

6-TET Azide6-HEX Azide


GlenResearchisofferingfirstourmostpopularlabelsforgeneralinterestand,subsequently,wewilladdazideproductsthatarenotcompatiblewithphosphoramiditechemistry.

BiotinisstillourmostcommonlyusedlabelandbiotinTEG,withitshydrophilictriethyleneglycolspacer,isthemostpopularbiotinproduct.Desthiobiotinisabiotinanaloguethatiswellcapturedbystreptavidinbutthecapturedproductcanbeeasilyreleasedbyapplyingabiotinsolutiontothestreptavidinbeads.6-FAMisourmostpopularfluoresceinderivativeandweofferazidesofboth6-FAMandpivaloyl-protected6-FAMforsituationswheresubsequentreactionsrequirethe6-FAMtobeprotected. Inboth6-FAMproducts,thehydrophilicTEGspacerisagainused.Theazidesareofferedin25and100µmolepacksforconvenientoligonucleotidelabeling.

7-Hydroxycoumarin,alsoknownasumbelliferone,isahighlyfluorescent,pH-sensitivefluorophorethatemitsintheblueregionofthespectrum.However,itsfluorescenceisstronglyquenchedifthehydroxylisalkylatedorphosphorylated,makingitusefulinhigh-throughputscreeningforphosphatasesandlipases.Interestingly,itwasfoundthatthe3-azidoderivativeisalsohighlyquenchedbut,uponreactionwithanalkyneinthepresenceofcoppertoformthetriazole,thefluorescenceisrestored.1Theclickedcoumarinemitsatalambdamaxof480nmandabsorbsat358nm.

HEXandTETaretwoofourmostpopularfluorescein-baseddyesforlabelingoligonucleotides.Wearehappytooffer6-HEXand6-TETAzidesforuseinclickconjugations.


BiotinTEGAzide 50-2000-92 25µmole 50-2000-90 100µmole

DesthiobiotinTEGAzide 50-2001-92 25µmole 50-2001-90 100µmole

Dipivaloyl6-FAM-TEGAzide 50-2002-92 25µmole 50-2002-90 100µmole

6-FAM-TEGAzide 50-2003-92 25µmole 50-2003-90 100µmole

CoumarinAzide 50-2004-92 25µmole 50-2004-90 100µmole

6-HEXAzide 50-2005-92 25µmole 50-2005-90 100µmole

6-TETAzide 50-2006-92 25µmole 50-2006-90 100µmole

92


Twonitroxidespinlabels,TEMPOAzideandTEMPO-TEGAzide,forsitedirectedspinlabeling(SDSL)arenowoffered.

ClickChemistrywithpsoralenazideandoneofourmanynucleosidicandnon-nucleosidic alkynederivativeshas thepotentialtogenerateavarietyofpracticalcross-linkers.Thewellknownreversiblecross-linkingbehaviorofpsoralenwithanadjacentthymidineresiduecouldbeveryuseful.

Tobetteraddressapplicationsinnear-infrared(NIR)imaging,GlenResearchisofferingawatersolubleDisulfo-Cyanine7azidethatcanbeeasilyconjugatedtoDNAandRNAthroughstandardclickchemistry.Thislongwavelengthdyeoffersthebenefitsofimprovedsolubility,reducedaggregation,andimprovedstabilityinthenear-infraredspectrumalongwiththeconvenienceofclickchemistry.


TEMPOAzide 50-2007-92 25µmole 50-2007-90 100µmole

TEMPO-TEGAzide 50-2008-92 25µmole 50-2008-90 100µmole

PsoralenAzide 50-2009-92 25µmole 50-2009-90 100µmole

Disulfo-Cyanine7Azide 50-2010 Discontinued

O O

N3

O

Psoralen Azide

N

SO3-K+

N+

-O3S

HNN3

O

Disulfo-Cyanine 7 Azide

N•O N3 N•O O

O

O

O

N3

TEMPO Azide TEMPO-TEG Azide

MODIFIERS

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LABELING

SERINOL REAGENTS FOR MODIFICATION AND LABELING

Mostpopularnon-nucleosidicphosphoramiditesformodificationandlabelingarebasedontwostructuraltypes:1,2-diolsand1,3-diols.Productsbasedona1,2-diolbackbonewerefirstdescribedtoallowamino-modificationandbiotinlabeling.Technically, the1,2-diolbackbonehas somedrawbacks relative to the1,3-diolbackbone. The1,2-diolbackbonecanparticipateinadephosphorylationreactionsincethe1,2-diolcanformafavored5-memberedcyclicphosphateintermediate.Thisreactioniscompetitivewithsimplehydrolysisoftheprotectinggroupsandleadstosomelossoflabel.However,thedegreeoflossatthe3’terminuscanbelimitedbytheremovalofthecyanoethylprotectinggroupusingDBUordiethylaminepriortothecleavageanddeprotectionsteps.Similarly,lossatthe5’terminuscanbeeliminatedbyretainingtheDMTgroupuntiltheoligoisfullydeprotected.Fortunately,theeliminationreactionisvirtuallynon-existentinthe1,3-diolbackbonesincethecyclicintermediatewouldbea6-memberedringwhichisnotfavoredforacyclicphosphateintermediate.

IVDcustomershaverequestedanewbackbonebasedona1,3-diolthatwouldovercomeanytechnicalorIPissuessurroundingourcurrentproducts.Wenowofferalineofproductsbasedontheserinolbackbone,whichhavebeendevelopedinclosecollaborationbetweenGlenResearchandNelsonBiotechnologies.ProtectedBiotinSerinolPhosphoramiditeandCPGareprotectedwithat-butylbenzoylgrouponthebiotinring.Thisgroupisdesignedtostopanyphosphoramiditereactionsatthisactivepositioninbiotin.ThisprotectionavoidsbranchingwhenusingnucleophilicactivatorslikeDCI.Theprotectinggroupiseasilyremovedduringoligonucleotidecleavageanddeprotection.TheBiotinLCversionsaresimilarlyprotectedandshouldbeusefulforthesynthesisofhighlysensitivebiotinylatedprobes.6-FluoresceinSerinolPhosphoramiditeandCPGaredesignedtoprepareoligonucleotidescontainingoneorseveral6-Fluorescein(6-FAM)residues.Amino-ModifierSerinolPhosphoramiditeandCPGareusedtoaddaminogroupsintooneorseveralpositionsinoligonucleotides.TheaminogroupisprotectedwithFmoc,whichmayberemovedonthesynthesiscolumnpriortosolid-phaseconjugationtotheaminogroups,orwhichmayberemovedduringdeprotectionforsubsequentsolutionphaseconjugationtotheaminogroups.

Combininglipoicacidandourpatentedserinolbackbone,wenowofferDithiolSerinolPhosphoramiditeandtherelated3’-DithiolSerinolCPG.Thisuniquearchitecturemovesthebulkydithiolawayfromthephosphatebackbone,makingitsuitablefor conjugationtogoldsurfaces.ThelongspacerarmofDithiolSerinolalsoallowsmultipleconsecutiveincorporationsofthemodifierwithouttheneedforintermediatespacerphosphoramiditeadditionstoachieveoptimalstepwisecouplingefficiency.

WeofferthreeproductsforuseinClickChemistrybaseduponour1,3-diolproductportfoliowiththeserinolbackbone-aphosphoramiditeforaddinganalkynegroupatthe5’terminusorwithinthesequence,asynthesissupportforlabelingthe3’terminusofoligonucleotideswithanalkynegroup,andDBCO-Serinolphosphoramiditeasacopper-freeclickreagent.


ProtectedBiotinSerinolPhosphoramidite 10-1993-95 50µmole 10-1993-90 100µmole 10-1993-02 0.25g

6-FluoresceinSerinolPhosphoramidite 10-1994-95 50µmole 10-1994-90 100µmole 10-1994-02 0.25g

ODMT

O P N(iPr)2

O CNEt

HN

HN

OOS

NHN

O O

O

HN

O

OO

O

O

OO

ODMT

O P N(iPr)2

O CNEt

HN

O

Protected Biotin Serinol Phosphoramidite 6-Fluorescein Serinol Phosphoramidite




Expedite EMerMade M





SerinolReagentsforModificationandLabelingarecoveredbyUSPatentNo.:8,394,948.

94

SERINOL REAGENTS FOR MODIFICATION AND LABELING (CONT.)


ProtectedBiotinLCSerinolPhosphoramidite 10-1995-95 50µmole 10-1995-90 100µmole 10-1995-02 0.25g

Amino-ModifierSerinolPhosphoramidite 10-1997-95 50µmole 10-1997-90 100µmole 10-1997-02 0.25g

DithiolSerinolPhosphoramidite 10-1991-95 50µmole 10-1991-90 100µmole 10-1991-02 0.25g

Alkyne-ModifierSerinolPhosphoramidite 10-1992-95 50µmole 10-1992-90 100µmole 10-1992-02 0.25g

DBCO-SerinolPhosphoramidite 10-1998-95 50µmole 10-1998-90 100µmole 10-1998-02 0.25g

LABELING

O

NH

NH

O

OODMT

P N(iPr)2

O CNEt

Alkyne-Modifier Serinol Phosphoramidite

HN

OS

NHN

O O

OO

O

O P N(iPr)2

O CNEt

HN

O

O

HN

O

ODMT

ODMT

O P N(iPr)2

O CNEt

HN

HN

Fmoc

O

Protected BiotinLC Serinol Phosphoramidite

Amino-Modifier Serinol Phosphoramidite

SS

HN

HN

O O

ODMT

O P N(iPr)2

O-CNEt

Dithiol Serinol

N

OO

HN

OP

NiPr2

ODMT

OCNEt

DBCO-Serinol

RELATED

DBCO..........................................91

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3’-ProtectedBiotinSerinolCPG 20-2993-01 0.1g 20-2993-10 1.0g 0.2µmolecolumns 20-2993-42 Packof4 1µmolecolumns 20-2993-41 Packof4 10µmolecolumn(ABI) 20-2993-13 Packof1 15µmolecolumn(Expedite) 20-2993-14 Packof1

3’-6-FluoresceinSerinolCPG 20-2994-01 0.1g 20-2994-10 1.0g 0.2µmolecolumns 20-2994-42 Packof4 1µmolecolumns 20-2994-41 Packof4 10µmolecolumn(ABI) 20-2994-13 Packof1 15µmolecolumn(Expedite) 20-2994-14 Packof1

3’-ProtectedBiotinLCSerinolCPG 20-2995-01 0.1g 20-2995-10 1.0g 0.2µmolecolumns 20-2995-42 Packof4 1µmolecolumns 20-2995-41 Packof4 10µmolecolumn(ABI) 20-2995-13 Packof1 15µmolecolumn(Expedite) 20-2995-14 Packof1

3’-Amino-ModifierSerinolCPG 20-2997-01 0.1g 20-2997-10 1.0g 0.2µmolecolumns 20-2997-42 Packof4 1µmolecolumns 20-2997-41 Packof4 10µmolecolumn(ABI) 20-2997-13 Packof1 15µmolecolumn(Expedite) 20-2997-14 Packof1

Protected Biotin Serinol CPG Amino-Modifier Serinol CPG 6-Fluorescein Serinol CPG

HN

OS

NHN

O O

ODMT

O-succinyl-CPG

HN

O

O

HN

O

OO

O

O

OO

ODMT

O-succinyl-CPG

HN

O

HN

OS

NHN

O O

OO

O ODMT

O-succinyl-CPG

HN

O

O

HN

O

ODMT

O-succinyl-CPG

HN

HN

Fmoc

O

Protected BiotinLC Serinol CPG




Expedite EMerMade M




96

LABELING



3’-DithiolSerinolCPG 20-2991-01 0.1g 20-2991-10 1.0g 0.2µmolecolumns 20-2991-42 Packof4 1µmolecolumns 20-2991-41 Packof4 10µmolecolumn(ABI) 20-2991-13 Packof1 15µmolecolumn(Expedite) 20-2991-14 Packof1

3’-Alkyne-ModifierSerinolCPG 20-2992-01 0.1g 20-2992-10 1.0g 0.2µmolecolumns 20-2992-42 Packof4 1µmolecolumns 20-2992-41 Packof4 10µmolecolumn(ABI) 20-2992-13 Packof1 15µmolecolumn(Expedite) 20-2992-14 Packof1

COT SERINOL PHOSPHORAMIDITE

COTSerinolPhosphoramiditeshasbeendiscontinued.Pleasecontacttechnicalsupport.


COTSerinolPhosphoramidite 10-1996 Discontinued

O

O

NH

NH

O

OODMT

succinoyl CPG

3’-Alkyne-Modifier Serinol CPG3’-Dithiol Serinol CPG

SS

HN

HN

O O

ODMT

O succinoyl-CPG

HN

HN

O O

ODMT

O P N(iPr)2

O-CNEt

COT Serinol


This product is covered under US Patent8,945,515B2.

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Dabsyl CPG

Dabcyl-dT 5’-Dabcyl Phosphoramidite

Dabcyl CPG

REFERENCE

(1)S.TyagiandF.R.Kramer,Nature Biotechnology, 1996, 4, 303-308.

DABCYL LABELING

Amolecularbeaconprobe1hasitsnaturalfluorescencequenchedinsolutionunlessitishybridizedtothetargetsequence.Consequently,thedesignofamolecularbeaconrequiresafluorophoretobeinonepartofthesequenceandthequenchermolecule tobe in another,withbothmoleculesbeing separated from theoligonucleotidebyahydrocarbon spacer. TheDabcylgrouphasbeenfoundtobeauniversalquencher.3’-DabsylCPGand3’-DabcylCPGareusedtoprepareprobeswith thequencherblocking the3’-terminus. 5’-DabcylPhosphoramidite locates thequencherat the5’-terminusandDabcyl-dTplacesitwithinthesequence,leavingthe3’-terminusavailableforpolymeraseextension.


3’-DabsylCPG 20-5911-01 0.1g 20-5911-10 1.0g 1µmolecolumns 20-5911-41 Packof4 0.2µmolecolumns 20-5911-42 Packof4 10µmolecolumn(ABI) 20-5911-13 Packof1 15µmolecolumn(Expedite) 20-5911-14 Packof1

3’-DabcylCPG 20-5912-01 0.1g 20-5912-10 1.0g 1µmolecolumns 20-5912-41 Packof4 0.2µmolecolumns 20-5912-42 Packof4 10µmolecolumn(ABI) 20-5912-13 Packof1 15µmolecolumn(Expedite) 20-5912-14 Packof1

3'-DabcylPS 26-5912-01 0.1g 26-5912-10 1.0g 200 nmole columns(ABI3900) 26-5912-52 Packof10 40 nmole columns(ABI3900) 26-5912-55 Packof10

Dabcyl-dT 10-1058-95 50µmole 10-1058-90 100µmole 10-1058-02 0.25g

5’-DabcylPhosphoramidite 10-5912-95 50µmole 10-5912-90 100µmole 10-5912-02 0.25g

O

NH

O

NHHN

N

O

ODMTO

CNEtON(iPr)2PO

O

NN N(Me) 2

O

NH CPGO

OHNODMTO

N(Me) 2NNSO 2

O

NH CPGO

OHNODMTO

NN N(Me) 2O

O

HNN

N(Me)2NCNEtON(iPr)2PO

LABELING

300 400 500 600 700 800

wavelength (nm)

____ Dabcyl____ Eclipse____ BHQ-1____ BHQ-2____ BHQ-3

FAM

TET

HEX Cy3

TMR

Cy3

.5

Cy5

Cy5

.5

____ BBQ-650

DYE QUENCHER PLOT

https://www.glenresearch.com/spectral-characteristics-of-fluorescent-dyes

98



BIOTIN LABELING

GlenResearchbiotinphosphoramiditesfordirectlabelingofsyntheticoligonucleotidesexhibitthefollowingfeatures:1. AllaresolubleinacetonitrileatconcentrationsusefulforDNAsynthesis.2. AllincludeaDMTgroupforcartridgepurificationswhichisessentialforthepreparationofbiotinylatedPCRprimers

becauseofthepotentialforcrosscontaminationinHPLCpurifications.3. Forthedevelopmentofdiagnosticprobes,biotinphosphoramiditeiscapableofbranchingtoallowmultiplebiotinsto

beintroducedatthe3’-or5’-terminus.BiotinTEGPhosphoramiditecontainsa15atommixedpolarityspacerarmbasedonatriethyleneglycol.

4. ProtectedBiotinSerinol Phosphoramidite andCPGareprotectedwith a t-butylbenzoyl groupon thebiotin ring. Thisgroupisdesignedtostopanyphosphoramiditereactionsatthisactivepositioninbiotin.ThisprotectionavoidsbranchingwhenusingnucleophilicactivatorslikeDCI.Theprotectinggroupiseasilyremovedduringoligonucleotidecleavageanddeprotection.TheBiotinLCversionsaresimilarlyprotectedandshouldbeusefulforthesynthesisofhighlysensitivebiotinylatedprobes.


BiotinPhosphoramidite 10-1953-95 50µmole 10-1953-90 100µmole 10-1953-02 0.25g

BiotinTEGPhosphoramidite 10-1955-95 50µmole 10-1955-90 100µmole 10-1955-02 0.25g

ProtectedBiotinSerinolPhosphoramidite 10-1993-95 50µmole 10-1993-90 100µmole 10-1993-02 0.25g

ProtectedBiotinLCSerinolPhosphoramidite 10-1995-95 50µmole 10-1995-90 100µmole 10-1995-02 0.25g

Biotin Phosphoramidite BiotinTEG Phosphoramidite

LABELING

O

O

S

NH NH

NHODMT

O P N(iPr)2O CNEt

NH OO

OO ODMT

O

CNEtON(iPr)2PO

O

S

NH NH




Expedite EMerMade M




ODMT

O P N(iPr)2

O CNEt

HN

HN

OOS

NHN

O O

HN

OS

NHN

O O

OO

O

O P N(iPr)2

O CNEt

HN

O

O

HN

O

ODMT

Protected Biotin Serinol Phosphoramidite

Protected BiotinLC Serinol Phosphoramidite

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BIOTIN LABELING (CONT.)

Biotin-dTcanreplacedTresidueswithintheoligonucleotidesequence.5’-BiotinphosphoramiditecanbeaddedONLYONCEtothe5’-terminusofanoligonucleotide.However,theDMTgrouponthebiotincanbeusedinRPcartridgeandHPLCpurificationtechniques.PCBiotinisaphotocleavable5’-biotinphosphoramidite.BiotinTEGCPGandProtectedBiotinLCSerinolCPGaredesignedforthedirectsynthesisofoligonucleotidescontainingbiotinatthe3’terminus.

Desthiobiotinisabiotinanaloguethatexhibitslowerbindingtobiotin-bindingproteinssuchasstreptavidin.Thisbiotinanalogueislackingthesulfurgroupfromthemoleculeandhasadissociationconstant(Kd)severalordersofmagnitudelessthanbiotin/streptavidin.Asaresult,biomoleculescontainingdesthiobiotinaredissociatedfromstreptavidinsimplyinthepresenceofbufferedsolutionsofbiotin.WeofferdesthiobiotinTEGphosphoramiditeandthecorrespondingCPG.

ABI-stylevialsandcolumnsaresuppliedunlessotherwiserequested(seenotebox).

IItem Catalog No. Pack

5’-BiotinPhosphoramidite 10-5950-95 50µmole 10-5950-90 100µmole 10-5950-02 0.25g

Biotin-dT 10-1038-95 50µmole 10-1038-90 100µmole 10-1038-02 0.25g

PCBiotinPhosphoramidite 10-4950-95 50µmole 10-4950-90 100µmole 10-4950-02 0.25g

DesthiobiotinTEGPhosphoramidite 10-1952-95 50µmole 10-1952-90 100µmole 10-1952-02 0.25g

LABELING

PC Biotin Phosphoramidite

NHDMTN

SNH

O

ONH

ONO2

H3C

CNEtON(iPr)2PO

DesthiobiotinTEG Phosphoramidite

NH OO

OO ODMT

O

CNEtON(iPr)2PO

O

NH NH

Biotin-dT

HN

S

O

O

N

O

O

O P N(iPr)2O CNEt

DMTO

HN

N

O

O

NH

ONH

5’-Biotin Phosphoramidite

NHDMTN

SNH

O

O

O P N(iPr)2O CNEt

RELATED

PCBiotin....................................86

100

BIOTIN LABELING (CONT.)


3’-BiotinTEGCPG 20-2955-01 0.1g 20-2955-10 1.0g 0.2µmolecolumns 20-2955-42 Packof4 1µmolecolumns 20-2955-41 Packof4 10µmolecolumn(ABI) 20-2955-13 Packof1 15µmolecolumn(Expedite) 20-2955-14 Packof1

3’-BiotinTEGPS 26-2955-01 0.1g 26-2955-10 1.0g 200 nmole columns(ABI3900) 26-2955-52 Packof10 40 nmole columns(ABI3900) 26-2955-55 Packof10

3’-ProtectedBiotinSerinolCPG 20-2993-01 0.1g 20-2993-10 1.0g 0.2µmolecolumns 20-2993-42 Packof4 1µmolecolumns 20-2993-41 Packof4 10µmolecolumn(ABI) 20-2993-13 Packof1 15µmolecolumn(Expedite) 20-2993-14 Packof1

3’-ProtectedBiotinLCSerinolCPG 20-2995-01 0.1g 20-2995-10 1.0g 0.2µmolecolumns 20-2995-42 Packof4 1µmolecolumns 20-2995-41 Packof4 10µmolecolumn(ABI) 20-2995-13 Packof1 15µmolecolumn(Expedite) 20-2995-14 Packof1

DesthiobiotinTEGCPG 20-2952-01 0.1g 20-2952-10 1.0g 0.2µmolecolumns 20-2952-42 Packof4 1µmolecolumns 20-2952-41 Packof4 10µmolecolumn(ABI) 20-2952-13 Packof1 15µmolecolumn(Expedite) 20-2952-14 Packof1

LABELING




Expedite EMerMade M




-succinyl-lcaa-CPG

NH OO

OO ODMT

O O

O

S

NH NH

NH OO

OO ODMT

O O-succinyl-lcaa-CPG

O

NH NH

HN

OS

NHN

O O

ODMT

O-succinyl-CPG

HN

O

HN

OS

NHN

O O

OO

O ODMT

O-succinyl-CPG

HN

O

O

HN

O

BiotinTEG CPG

Protected Biotin Serinol CPG

Protected BiotinLC Serinol CPG

DesthiobiotinTEG CPG

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FLUORESCEIN LABELING

5’-Fluoresceinphosphoramiditecontainsno4,4’-dimethoxytrityl(DMT)groupandcanbeaddedonlyonceatthe5’-terminus,thereby terminating synthesis. Thisproduct ispreparedusing the6-carboxyfluoresceinderivative. The tetrachloro-,hexachloro-anddichloro-dimethoxy-fluorescein (TET,HEX and JOE, respectively) phosphoramidites aredesigned totakeadvantageof themulticolordetectioncapabilityofmodernDNAsequencersandgeneticanalyzers. Fluoresceinphosphoramidite isdesigned toproduce the samefluorescein-type structureashadbeenpreviouslypreparedusingfluoresceinisothiocyanate(FITC).OurfluoresceinphosphoramiditealsocontainsaDMTgrouptoallowquantificationofcoupling.Theanalogousstructure,6-FluoresceinPhosphoramidite,preparedusing6-FAM,isalsoavailable,alongwith6-FluoresceinSerinolPhosphoramidite.Fluorescein-dTcanbeinsertedintothedesiredsequenceasareplacementforadTresidue.

Weofferfivefluoresceinsupports.FluoresceinCPGhastraditionallybeenusedtoaddthefluoresceinlabelatthe3’-terminus.Theanalogousstructure,3’-(6-Fluorescein)CPG,preparedusing6-FAM,isnowalsoavailable,alongwith6-FluoresceinSerinolCPG. Wealsooffer3’-(6-FAM)CPGandFluorescein-dTCPG,bothderivativesof6-carboxyfluorescein(6-FAM). Botharesingleisomersanduseanamidelinkagewhichisstableduringcleavageanddeprotectionanddoesnotallowisomerformation.3’-(6-FAM)CPGallowseffectiveblockageofthe3’-terminusfrompolymeraseextensionaswellasexonucleasedigestion.Fluorescein-dTCPGallowsbothoftheseenzymaticactivitiestoproceed.Normalcleavageanddeprotectionwithammoniumhydroxidereadilygeneratesthefluoresceinlabeledoligos.

Thespectralcharacteristicsofthesedyesaredetailedonthefollowingpage.

Item Cat. No. Pack

5’-FluoresceinPhosphoramidite 10-5901-95 50µmole (6-FAM) 10-5901-90 100µmole 10-5901-02 0.25g

5’-Hexachloro-Fluorescein 10-5902-95 50µmole Phosphoramidite 10-5902-90 100µmole (HEX) 10-5902-02 0.25g

5’-Tetrachloro-Fluorescein 10-5903-95 50µmole Phosphoramidite 10-5903-90 100µmole (TET) 10-5903-02 0.25g

5’-Dichloro-dimethoxy-FluoresceinPhosphoramiditeII 10-5906-95 50µmole (JOE) 10-5906-90 100µmole 10-5906-02 0.25g

5’-Fluorescein Phosphoramidite 5’-Tetrachloro-FluoresceinPhosphoramidite

5’-Hexachloro-Fluorescein Phosphoramidite

O

O

O

OO

O O

HN

O PN

O

OCN

O

O

O

OO

O O

HN

O PN

O

OCN

Cl

Cl

Cl

ClCl

Cl

O

O

O

OO

O O

HN

O PN

O

OCN

Cl Cl

Cl

Cl

LABELING

O

HN

O

MeO

OO

O

O

OO

OP

N

O

CN

OMe

Cl Cl

5’-Dichloro-dimethoxy-FluoresceinPhosphoramidite II

300 400 500 600 700 800

wavelength (nm)


FAM

TET

HEX Cy3

TMR

Cy3

.5

Cy5

Cy5

.5

____ BBQ-650

DYE QUENCHER PLOT


102



FLUORESCEIN LABELING (CONT.)

Item Cat. No. Pack

FluoresceinPhosphoramidite 10-1963-95 50µmole 10-1963-90 100µmole 10-1963-02 0.25g

6-FluoresceinPhosphoramidite 10-1964-95 50µmole 10-1964-90 100µmole 10-1964-02 0.25g

6-FluoresceinSerinolPhosphoramidite 10-1994-95 50µmole 10-1994-90 100µmole 10-1994-02 0.25g

Fluorescein-dTPhosphoramidite 10-1056-95 50µmole 10-1056-90 100µmole 10-1056-02 0.25g

Fluorescein Phosphoramidite Fluorescein dT

LABELING

6-Fluorescein Phosphoramidite

OO

O O

O

O

O

P N(iPr)2O CNEt

O

ODMT

NH

O

O

O

O

OO

O O

HNNH

ODMTS

OCNEtON(iPr)2P

�

O

O P N(iPr)2O CNEt

DMTO O

O

N

HNNH

O

NH

O

OO

O O

O

O

O

O

HN

O

OO

O

O

OO

ODMT

O P N(iPr)2

O CNEt

HN

O

6-Fluorescein Serinol Phosphoramidite




Expedite EMerMade M




FLUORESCENT DYES

Absorbance Maximum

Emission Maximum Color

Fluorescein 494nm 525nm Green

Tetrachloro- 521nm 536nm Orange

Fluorescein

Hexachloro- 535nm 556nm Pink

Fluorescein

SIMA (HEX) 538nm 551nm Pink

Dichloro- 525nm 548nm Orange/ Pinkdimethoxy-

Fluorescein

TAMRA 565nm 580nm Rose

Cy3 546nm 563nm Red

Cy3.5 588nm 604nm Purple

Cy5 646nm 662nm Violet

Cy5.5 683nm 707nm Dark Blue

Yakima Yellow 530nm 549nm Yellow

Redmond Red 579nm 595nm Red

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Item Cat. No. Pack

3’-FluoresceinCPG 20-2963-01 0.1g 20-2963-10 1.0g 1µmolecolumns 20-2963-41 Packof4 0.2µmolecolumns 20-2963-42 Packof4 10µmolecolumn(ABI) 20-2963-13 Packof1 15µmolecolumn(Expedite) 20-2963-14 Packof1

3’-(6-Fluorescein)CPG 20-2964-01 0.1g 20-2964-10 1.0g 1µmolecolumns 20-2964-41 Packof4 0.2µmolecolumns 20-2964-42 Packof4 10µmolecolumn(ABI) 20-2964-13 Packof1 15µmolecolumn(Expedite) 20-2964-14 Packof1

3’-(6-FAM)CPG 20-2961-01 0.1g 20-2961-10 1.0g 1µmolecolumns 20-2961-41 Packof4 0.2µmolecolumns 20-2961-42 Packof4 10µmolecolumn(ABI) 20-2961-13 Packof1 15µmolecolumn(Expedite) 20-2961-14 Packof1

3’-(6-FAM)PS 26-2961-01 0.1g 26-2961-10 1.0g 200nmolecolumns(ABI3900) 26-2961-52 Packof10 40nmolecolumns(ABI3900) 26-2961-55 Packof10

3’-6-FluoresceinSerinolCPG 20-2994-01 0.1g 20-2994-10 1.0g 0.2µmolecolumns 20-2994-42 Packof4 1µmolecolumns 20-2994-41 Packof4 10µmolecolumn(ABI) 20-2994-13 Packof1 15µmolecolumn(Expedite) 20-2994-14 Packof1

3’-Fluorescein CPG

O-succinyl-CPG

O

O

O

OO

O O

HN

S

NH

ODMT

LABELING

3’-(6-FAM) CPG 3’-(6-Fluorescein) CPG

OO

O O

O

O

O

O

ODMT

NH

O

-succinyl-CPG

NH

O

ODMT

OO

NHCPG

O

O

O

O

O

OO

O O

O

HN

O

OO

O

O

OO

ODMT

O-succinyl-CPG

HN

O

3’-6-Fluorescein Serinol CPG

300 400 500 600 700 800

wavelength (nm)


FAM

TET

HEX Cy3

TMR

Cy3

.5

Cy5

Cy5

.5

____ BBQ-650

DYE QUENCHER PLOT


104




Item Cat. No. Pack

3’-Fluorescein-dTCPG 20-2056-01 0.1g 20-2056-10 1.0g 1µmolecolumns 20-2056-41 Packof4 0.2µmolecolumns 20-2056-42 Packof4 10µmolecolumn(ABI) 20-2056-13 Packof1 15µmolecolumn(Expedite) 20-2056-14 Packof1

FLUORESCEIN LABELING (SIMA)

Dichloro-diphenyl-fluorescein,SIMA(HEX)exhibitsvirtuallyidenticalabsorbanceandemissionspectratoHEX.SIMA(HEX)ismuchmorestabletobasicdeprotectionconditionsthanHEXandoligonucleotidescanbedeprotectedusingammoniumhydroxideatelevatedtemperaturesandevenammoniumhydroxide/methylamine(AMA)atroomtemperatureor65°Cfor10minutes.SIMAabsorptionmaximumwas3nmblue-shiftedcomparedtoHEXatpH7.Theabsorbanceisbroader,sotheextinctioncoefficientissmallerthanthatofHEX,butwhenexcitingat500nmwheretheabsorbancewasnormalized,theemissionwasstill90%ofHEXandtheemissionwasred-shiftedby5nm.AsecondSIMA(HEX)product,SIMA(HEX)-dT,canbeusedtointroduceSIMA(HEX)inthesyntheticoligonucleotidesequence,usuallyasareplacementforthenativedT linkage. Again,thisproduct is fullycompatiblewithdeprotectionschemesusingammoniumhydroxideatelevatedtemperaturesorAMAatroomtemperatureand65°C.

Item Cat. No. Pack

SIMA(HEX)Phosphoramidite 10-5905-95 50µmole 10-5905-90 100µmole 10-5905-02 0.25g

SIMA(HEX)-dTPhosphoramidite 10-5945-95 50µmole 10-5945-90 100µmole 10-5945-02 0.25g

LABELING

3’-Fluorescein-dT CPG

O

O

DMTO O

O

N

HNNH

O

NH

O

OO

O O

O

O

O

-Succinyl-lcaa-CPG




Expedite EMerMade M




O

HN

O

OO

O

O

OO

OP

N

O

CN

Cl

Cl

ODMTO

N

HN

O

O

O P N(iPr)2

O CNEt

NH

HN

O

O OO

O

O

OO

Cl

Cl

O

SIMA (HEX) Phosphoramidite SIMA (HEX)-dT Phosphoramidite

300 400 500 600 700 800

wavelength (nm)


FAM

TET

HEX Cy3

TMR

Cy3

.5

Cy5

Cy5

.5

____ BBQ-650

DYE QUENCHER PLOT


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CYANINE LABELING

Twocyaninederivatives,Cyanine3andCyanine5,whichdiffer in structure simplyby thenumberof carbons in theconjugatedpolyenelinkage,arejoinedbythecloselyrelatedanalogues,Cyanine3.5andCyanine5.5,andareavailableasphosphoramidites.Cyaninedyesarenormallyaddedonceatthe5’-terminusandtheMMTgroupshouldberemovedonthesynthesizer.TheabsorbanceoftheMMTcation(yellow)isnoticeablydifferentfromtheDMTcation(orange),andso,absorbance-basedtritylmonitorswilldetectitincorrectlyasalowcoupling.Ontheotherhand,conductivitydetectorswill interpretthereleasemorecorrectly. Cyaninedyephosphoramiditeshavealsobeenusedsuccessfullyadjacenttothe3’-terminus.Cyanine3andCyanine5supportsarealsoofferedtoallowsimplerproductionof3’cyaninedye-labeledoligonucleotides.

Deprotectionofoligos containingCyaninedyesmaybecarriedoutwithammoniumhydroxideat room temperature,regardlessofthebaseprotectinggroupsonthemonomersused.Ifthereisaneedtouseammoniumhydroxideatelevatedtemperature,Cyanine3andCyanine3.5aremorestablethanCyanine5andCyanine5.5.However,itisalwaysprudenttousemonomerswithbaselabileprotectinggroupstolimittheexposuretimeto2hoursorlessat65°Cduringdeprotection.

Tobetteraddressapplicationsinnear-infrared(NIR)imaging,GlenResearchisofferingawatersolubleDisulfo-Cyanine7azidethatcanbeeasilyconjugatedtoDNAandRNAthroughstandardclickchemistry.Thislongwavelengthdyeoffersthebenefitsofimprovedsolubility,reducedaggregation,andimprovedstabilityinthenear-infraredspectrumalongwiththeconvenienceofclickchemistry.

Item Cat. No. Pack

Cyanine3Phosphoramidite 10-5913-95 50µmole 10-5913-90 100µmole 10-5913-02 0.25g

Cyanine3.5Phosphoramidite 10-5914-95 50µmole 10-5914-90 100µmole 10-5914-02 0.25g

Cyanine5Phosphoramidite 10-5915-95 50µmole 10-5915-90 100µmole 10-5915-02 0.25g

Cyanine5.5Phosphoramidite 10-5916-95 50µmole 10-5916-90 100µmole 10-5916-02 0.25g

LABELING

Cyanine 5 PhosphoramiditeCyanine 3 Phosphoramidite Cyanine 5.5 PhosphoramiditeCyanine 3.5 Phosphoramidite

N N

OMMT

Cl

O P N(iPr)2

O CNEt

N N

OMMT

Cl

O P N(iPr)2

O CNEt

N N

OMMT

Cl

O P N(iPr)2

O CNEt

N N

OMMT O P N(iPr)2

O CNEt

I-

300 400 500 600 700 800

wavelength (nm)


FAM

TET

HEX Cy3

TMR

Cy3

.5

Cy5

Cy5

.5

____ BBQ-650

DYE QUENCHER PLOT


SPECTRAL DATA FOR CYANINE DYES

Absorbance Maximum


Cyanine 3 546nm 563nm Red

Cyanine 3.5 588nm 604nm Purple

Cyanine 5 646nm 662nm Violet

Cyanine 5.5 683nm 707nm Dark Blue

Cyanine 7 750nm 773nm Dark Green

(Measuredinanoligoin0.1MTEAAbuffer,pH7.)

106



LABELING

CYANINE LABELING (CONT.)

Item Cat. No. Pack

Cyanine3CPG 20-5913-01 0.1g 20-5913-10 1.0g 1µmolecolumns(TWISTformatonly) 20-5913-41 Packof4 0.2µmolecolumns 20-5913-42 Packof4

Cyanine5CPG 20-5915-01 0.1g 20-5915-10 1.0g 1µmolecolumns(TWISTformatonly) 20-5915-41 Packof4 0.2µmolecolumns 20-5915-42 Packof4

Disulfo-Cyanine7Azide 50-2010-92 25µmole 50-2010-90 100µmole

N N

OMMT OO

ON

H

Cl

N N

OMMT OO

ON

H

Cl

Cyanine 5 CPGCyanine 3 CPG

N

SO3-K+

N+

-O3S

HNN3

O

Disulfo-Cyanine 7 Azide




Expedite EMerMade M




300 400 500 600 700 800

wavelength (nm)


FAM

TET

HEX Cy3

TMR

Cy3

.5

Cy5

Cy5

.5

____ BBQ-650

DYE QUENCHER PLOT


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LABELING

ELITECHGROUP DYES AND QUENCHER

GlenResearch’sagreementwithELITechGroup,formerlyEpochBiosciences,allowsustoofferseveraloftheirproprietaryproductsdesignedforthesynthesisofnovelDNAprobes.WearepleasedtoofferproductsbasedonELITechGroup’sRedmondRed®,YakimaYellow®andAquaPhluor®593fluorophoresandEclipse®non-fluorescentquencher.UnderouragreementwealsosupplyPPG,amodifiednucleoside,and5’-Aldehyde-ModifierC2Phosphoramidite.Thefluorescentdyes,YakimaYellow,RedmondRedandAquaPhluor593,areavailableasphosphoramiditesandsupports.YakimaYellowhasanabsorbancemaximumat530nmandemissionmaximumat549nm,RedmondRed’sabsorbanceandemissionmaximaareat579nmand595nm,respectively,andAquaPhluor593hasanabsorbancemaximumat593nmandemissionmaximumat613nm.

TheEclipsequencherfromELITechGroupsolvesmostoftheproblemsinherentinthesynthesisofmolecularbeaconandFRETprobes. TheEclipsemolecule ishighlystableandcanbeusedsafely inallcommonoligodeprotectionschemes. TheabsorbancemaximumforEclipseQuencherisat522nm,comparedto479nmfordabcyl.Inaddition,thestructureoftheEclipseQuencherissubstantiallymoreelectrondeficientthanthatofdabcylandthisleadstobetterquenchingoverawiderrangeofdyes,especiallythosewithemissionmaximaatlongerwavelengths(redshifted)suchasRedmondRedandCyanine5.Inaddition,withanabsorptionrangefrom390nmto625nm,theEclipseQuencheriscapableofeffectiveperformanceinawiderangeofcoloredFRETprobes.

Item Cat. No. Pack

RedmondRed®Phosphoramidite 10-5920-95 50µmole 10-5920-90 100µmole 10-5920-02 0.25g

YakimaYellow®Phosphoramidite 10-5921-95 50µmole 10-5921-90 100µmole 10-5921-02 0.25g

5’-AquaPhluor®593Phosphoramidite 10-5923-95 50µmole 10-5923-90 100µmole 10-5923-02 0.25g

Eclipse®QuencherPhosphoramidite 10-5925-95 50µmole 10-5925-90 100µmole 10-5925-02 0.25g


These Products are for research purposesonly,andmaynotbeusedforcommercial,clinical,diagnosticoranyotheruse.TheseProductsaresubjecttoproprietaryrightsofELITechGroup and are made and sold underlicensefromELITechGroup.There is no implied license for commercialusewithrespecttotheseProductsandalicensemustbeobtaineddirectlyfromELITechGroupwithrespecttoanyproposedcommercialuseoftheseProducts.“Commercialuse”includesbutisnotlimited to the sale, lease, license or othertransferoftheProductoranymaterial derived or produced from it, the sale, lease, license or other grant ofrightstousetheProductoranymaterial derived or produced from it, or the use of the Product to perform servicesforafeeforthirdparties(includingcontractresearch).

Asimpleagreementmustbesignedbeforeend-usersandcustomoligoservicesmaypurchasethese productsforuseasdefinedabove. https://www.glenresearch.com/media/productattach/import/technical_note/ELITechGroupProducts.pdf AquaPhluor®, Yakima Yellow®, Redmond Red® and Eclipse®, are registered Trademarks of ELITechGroup.

Redmond Red® Yakima Yellow®

Epoch Eclipse™ Quencher

O

NO

O DMT

O P NO

CH 2CH 2CNN

O

O O

Yakima Yellow Phosphoramidite

C49H60Cl4N3O10P1023.81

1021.277044C 57.5% H 5.9% Cl 13.9% N 4.1% O 15.6% P 3.0%

Cl

Cl

O

O

Cl

ClCH 3

O

OOO

O

CH 2CH 2CN

ONPO

O

NH

OCH 3

N

NN

ClO2N

N CH 2CH 2CN

ONPO

DMTO

ON+ N

N

PO

O

O

O O

PO N

NH

N

F3C O

PF6-

5’-AquaPhluor® 593

FLUORESCENT DYES

Absorbance Maximum


Yakima Yellow 530nm 549nm Yellow

Redmond Red 579nm 595nm Red

AquaPhluor 593 593nm 613nm Red

RELATED

PPG.............................................575’-Aldehyde-ModifierC2..........83

108

https://www.glenresearch.com/media/productattach/import/technical_note/ELITechGroupProducts.pdf




LABELING

Redmond Red® CPG

Yakima Yellow® CPG

Eclipse® Quencher CPG

Redmond Red CPG

NHO

O

CPG

OO

O

N

O

DMTOO

N

O

O

Cl

Cl

Cl

ClCH 3

O

OO

O

O

ONH CPG

O

O

O

O

N

O DMT

OCH 3

N

NN

ClO2N

O

O

O

NH CPG

N

DMTO




Expedite EMerMade M




300 400 500 600 700 800

wavelength (nm)


FAM

TET

HEX Cy3

TMR

Cy3

.5

Cy5

Cy5

.5

____ BBQ-650

DYE QUENCHER PLOT


O N+N

N

PO

N

DMTO

O

NH-CPG

O

O

O

O

O-O

AquaPhluor® 593 CPG

ELITECHGROUP DYES AND QUENCHER (CONT.)

Item Cat. No. Pack

RedmondRed®CPG 20-5920-01 0.1g 20-5920-10 1.0g 1µmolecolumns 20-5920-41 Packof4 0.2µmolecolumns 20-5920-42 Packof4 10µmolecolumn(ABI) 20-5920-13 Packof1 15µmolecolumn(Expedite) 20-5920-14 Packof1

YakimaYellow®CPG 20-5921-01 0.1g 20-5921-10 1.0g 1µmolecolumns 20-5921-41 Packof4 0.2µmolecolumns 20-5921-42 Packof4 10µmolecolumn(ABI) 20-5921-13 Packof1 15µmolecolumn(Expedite) 20-5921-14 Packof1

AquaPhluor®593CPG 20-5923-01 0.1g 20-5923-10 1.0g 1µmolecolumns 20-5923-41 Packof4 0.2µmolecolumns 20-5923-42 Packof4 10µmolecolumn(ABI) 20-5923-13 Packof1 15µmolecolumn(Expedite) 20-5923-14 Packof1

Eclipse®QuencherCPG 20-5925-01 0.1g 20-5925-10 1.0g 1µmolecolumns 20-5925-41 Packof4 0.2µmolecolumns 20-5925-42 Packof4 10µmolecolumn(ABI) 20-5925-13 Packof1 15µmolecolumn(Expedite) 20-5925-14 Packof1

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BLACK HOLE QUENCHER DYES

Withthegrowingpopularityofredandnear-infrareddyes,weareofferingtheBlackHoleQuencherTMdyes(BHQs),whosephysicalpropertiesaredetailedinTable1.BHQdyesarerobustdarkquenchersthatverynicelycomplementourexistingproductline.Theyarecompatiblewithammoniumhydroxidedeprotection,exhibitexcellentcouplingefficiencies,havelargeextinctioncoefficientsandarecompletelynon-fluorescent.Theirabsorbancesarewell-tunedtoquenchavarietyofpopularfluorophores–eventhosefarintothered,suchasCy3andCy5.ThedarkquenchermosttypicallyusedinaMolecularBeaconisDabcyl.BecausethequenchingdoesnotinvolveFRET,thereislittle,ifany,dependenceupondonor-acceptorspectraloverlap.InacomprehensivepaperbyMarras,KramerandTyagi,1 theabilityofBHQ-1andBHQ-2toquench22differentfluorophoreswasevaluated.Forshorterwavelengthfluorophoressuchasfluorescein,thequenchingefficiencywasroughlythesameasDabcyl(91%–93%).However,fordyesemittinginthefarred,suchasCy5,theBHQdyeswerefarsuperior–quenchingtheCy5with96%efficiency,comparedto84%withDabcyl.ThismayreflecttheBHQ’sabilitytoformstable,non-fluorescentcomplexeswhichcanbeapluseveninFRETprobes.Indeed,recentworksuggeststhatthesenon-fluorescentcomplexeswillformevenintheabsenceofahairpinstemstructureusedbyMolecularBeacons.2

Item Cat. No. Pack

5’-BHQ-1Phosphoramidite 10-5931-95 50µmole 10-5931-90 100µmole 10-5931-02 0.25g

5’-BHQ-2Phosphoramidite 10-5932-95 50µmole 10-5932-90 100µmole 10-5932-02 0.25g

BHQ-1-dT 10-5941-95 50µmole 10-5941-90 100µmole 10-5941-02 0.25g

BHQ-2-dT 10-5942-95 50µmole 10-5942-90 100µmole 10-5942-02 0.25g


"BlackHoleQuencher","BHQ-0","BHQ-1","BHQ-2"and"BHQ-3"are trademarks of Biosearch Technologies,Inc.,Novato,CA.TheBHQdyetechnologyisthesubjectofpendingpatentsandislicensed and sold under agreement withBiosearchTechnologies,Inc..ProductsincorporatingtheBHQdyemoietyaresoldexclusivelyforR&Dusebytheend-user.Theymaynotbeusedforclinicalordiagnosticpurposesandtheymaynotbere-sold,distributedorre-packaged.

TABLE 1: BLACK HOLE QUENCHERS

Quencher lmax E260 Emax (nm) (L/mol.cm) (L/mol.cm)

BHQ-1 534 8,000 34,000BHQ-2 579 8,000 38,000BHQ-3 672 13,000 42,700

REFERENCES

(1)S.A.E.Marras,F.R.Kramer,andS.Tyagi,Nucleic Acids Res., 2002, 30, E122.

(2)M.K.Johansson,H.Fidder,D.Dick,andR.M.Cook,J Am Chem Soc, 2002, 124,6950-6956.

BHQ-1-dT BHQ-2-dT

NN N

N N

CNEtON(iPr)2PO

OCH 3

H3COO2NN

N NN N

CNEtON(iPr)2PONO2

H3C

OCH 3

H3C

O

NH

O

NHHN

N

O

ODMTO

CNEtON(iPr)2PO

O

CH 3

CH 3O

CH 3

O

N NN N

N

O2N

O

NH

O

NHHN

N

O

ODMTO

CNEtON(iPr)2PO

O

O

NO2

CH 3O

N NN N

NOCH 3

5’-BHQ-1 5’-BHQ-2

LABELING

RELATED

Dabcyl........................................98Eclipse™...................................109BBQ-650®................................112

110

mol.cm

mol.cm

BLACK HOLE QUENCHER DYES (CONT.)

Item Cat. No. Pack

3'-BHQ-1CPG 20-5931-01 0.1g 20-5931-10 1.0g 1µmolecolumns 20-5931-41 Packof4 0.2µmolecolumns 20-5931-42 Packof4 10µmolecolumn(ABI) 20-5931-13 Packof1 15µmolecolumn(Expedite) 20-5931-14 Packof1



3'-BHQ-2 CPG 3'-BHQ-3 CPG

NN N

N

NO2

H3C

OCH 3

H3CODMT

O-glycolate-CPG

N

NN N

N

OCH 3

H3COO2N N

O-glycolate-CPG

ODMTN

N+N

NN

ODMT

O-glycolate-CPG

N

X-

3'-BHQ-1 CPG

LABELING




Expedite EMerMade M




300 400 500 600 700 800

wavelength (nm)


FAM

TET

HEX Cy3

TMR

Cy3

.5

Cy5

Cy5

.5

____ BBQ-650

DYE QUENCHER PLOT


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BLACKBERRY® QUENCHER (BBQ-650®)

WearehappytoofferseveralproductscontainingtheBlackBerry®Quencher(BBQ-650®),whichexhibitsabroadabsorptionprofilefrom550nmto750nm,centeredat650nm.ThisrangeoffersmoreeffectivequenchingofsomeofourpopularlongwavelengthdyeslikeTAMRA,RedmondRed,CydyesandDyLightdyes.WeofferBBQ-650productsforthe3’and5’termini,aswellasBBQ-650-dTforinclusionwithintheoligonucleotidesequence,withthefollowingproperties:

• Quenchesthefluorescenceoflongwavelengthdyes• Quenches in FRET and contact mode• Absorbancemaximumat~650nm• Quenchingrange–550-750nm• Compatiblewithstandardoligosynthesischemistry• CompatiblewithregulardeprotectionbutrequiresmilddeprotectionwithAMAatroomtemperature• Availablefor3’,5’,andinternalsubstitution• MorestablethanBHQ-3

Item Cat. No. Pack

5’-BBQ-650®Phosphoramidite 10-5934-95 50µmole 10-5934-90 100µmole 10-5934-02 0.25g

BBQ-650®-dT 10-5944-95 50µmole 10-5944-90 100µmole 10-5944-02 0.25g

3’-BBQ-650®CPG 20-5934-01 0.1g 20-5934-10 1.0g 1µmolecolumns 20-5934-41 Packof4 0.2µmolecolumns 20-5934-42 Packof4 10µmolecolumn(ABI) 20-5934-13 Packof1 15µmolecolumn(Expedite) 20-5934-14 Packof1


BlackBerry®Quenchertechnology:USPatent7,879,986.ThepurchaseofBlackBerry®Quencherreagentsincludes a limited license to usethesereagentsexclusivelyfor research and development purposes.Theymaynotbeusedforclinicalordiagnosticpurposesandtheymaynotbere-sold,distributed,orre-packagedwithoutprioragreementandconsentofBerry&Associates,Inc.Subsequentsaleof products that are derived from BlackBerry®Quencherreagentsispermittedsolongasthefollowingwrittendisclaimerisincludedinwrittenandelectroniccatalogs,incommercialadvertisement,andinpackageswithcontainersofsuchderivativeproducts:“BlackBerry is a trademark of Berry & Associates, Inc. Products derived from BlackBerry® Quencher reagents are sold exclusively for research and development use by the purchaser. They may not be used for clinical or diagnostic purposes without prior agreement and consent of Berry & Associates, Inc.”

BBQ-650™-dT

N O

NN

NN

NO2

OCH3

H3CO

O P N(iPr)2

O CNEt

5’-BBQ-650™ 3’-BBQ™-650™ CPG

LABELING

NO

NN

NN

O2N

H3CO

OCH3

O

DMTO

glycolate-lcaa-CPG

NO

NN

NN

O2N

H3CO

OCH3ODMTO

N

HN

O

O

O P N(iPr)2

O CNEt

NH

HN

O

O

112

RHODAMINE (TAMRA) LABELING

Rhodaminederivativesarenotsufficientlystable tosurviveconventionaldeprotectionandthesemustbeattachedtoamino-modifiedoligonucleotidesusingpost-synthesislabelingtechniques.BecauseTetramethylRhodamine(TAMRA)isnotbasestable,theproceduretocleaveanddeprotectthelabeledoligonucleotidemustbecarefullyconsidered.UsingtheUltraMILDmonomersanddeprotectionwithpotassiumcarbonateinmethanol,TAMRAoligonucleotidescanbefairlyconvenientlyisolated.TostreamlinethepreparationofTAMRAoligos,weoffer3’-TAMRACPGfor3’labelingandTAMRA-dTforlabelingwithinthesequence.WealsoofferTAMRANHSesterforlabelingamino-modifiedoligonucleotides.

Item Cat. No. Pack

3’-TAMRACPG 20-5910-01 0.1g 20-5910-10 1.0g 1µmolecolumns 20-5910-41 Packof4 0.2µmolecolumns 20-5910-42 Packof4

3'-TAMRAPS 26-5910-01 0.1g 26-5910-10 1.0g 200 nmole columns(ABI3900) 26-5910-52 Packof10 40 nmole columns(ABI3900) 26-5910-55 Packof10

TAMRA-dT 10-1057-95 50µmole 10-1057-90 100µmole 10-1057-02 0.25g

TAMRANHSEster 50-5910-66 60µL (SolutioninanhydrousDMSO)

LABELING

TAMRA NHS Ester

TAMRA CPG TAMRA-dT

O N+N

NHS

O

-O2C

O

NH CPGO

OHNODMTO

O

N+

O

NCO 2-

N+

O

NCO 2-

O

NH

O

NHHN

N

O

ODMTO

CNEtON(iPr)2PO

O




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UltraMILD monomers...............23

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ACRIDINE LABELING

Acridinephosphoramiditeisdesignedtoproduceanoligonucleotidecontainingacridineatanypositioninthemolecule.AcridineCPGisusedtolabelthe3’-terminus.Acridineisaneffectiveintercalatingagent.

Item Cat. No. Pack

AcridinePhosphoramidite 10-1973-95 50µmole 10-1973-90 100µmole 10-1973-02 0.25g

3’-AcridineCPG 20-2973-01 0.1g 20-2973-10 1.0g 1µmolecolumns 20-2973-41 Packof4 0.2µmolecolumns 20-2973-42 Packof4 10µmolecolumn(ABI) 20-2973-13 Packof1 15µmolecloumn(Expedite) 20-2973-14 Packof1

DNP LABELING

Ananalyticaltestbasedondetectionof2,4-dinitrophenyl(DNP)labeledoligonucleotideswithanti-DNPantibodieshasbeenproposed.Wehavechosenthebranchedtriethyleneglycol(TEG)spacerinourversionofDNPphosphoramiditesinceitcanbeaddedonceorseveraltimestothe3’or5’terminus.


DNP-TEGPhosphoramidite 10-1985-95 50µmole 10-1985-90 100µmole 10-1985-02 0.25g

LABELING

Acridine Acridine CPG DNP-TEG

O-CNEt

O-P-N( iPr)2

ODMT

MeO

Cl

NH

N

-succinyl-lcaa-CPG

N

NH

Cl

MeO

O

ODMTNO2

O2N

O P N(iPr)2O CNEt

ODMTOO

OONH




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CHOLESTEROL LABELING

Potentialtherapeuticoligonucleotidesmustpermeatethecellmembraneforoptimalactivity.Theadditionoflipophilicgroupstoanoligonucleotidewouldbeexpectedtoenhancecellularuptake/membranepermeation.Theuseofcholesteryloligosandtheconsequentimprovementinactivityhasbeendescribed.WehavedesignedourCholesterylproductswithtriethyleneglycol(TEG)spacersformaximumsolubility.


Cholesteryl-TEGPhosphoramidite 10-1975-95 50µmole 10-1975-90 100µmole 10-1975-02 0.25g

5’-Cholesteryl-TEGPhosphoramidite 10-1976-95 50µmole 10-1976-90 100µmole 10-1976-02 0.25g

3’-Cholesteryl-TEGCPG 20-2975-01 0.1g 20-2975-10 1.0g 1µmolecolumns 20-2975-41 Packof4 0.2µmolecolumns 20-2975-42 Packof4 10µmolecolumn(ABI) 20-2975-13 Packof1 15µmolecolumn(Expedite) 20-2975-14 Packof1

TOCOPHEROL LABELING

VitaminEisbothlipophilicandnon-toxicevenathighdosessowouldbeanexcellentcandidateasalipophiliccarrierforoligonucleotides.Therefore,asanadditiontoourcholesterylproductline,weoffersimplea-tocopheryl(vitaminE)labeling.Totallysynthetica-tocopherolisracemicatitsthreechiralcentersandisusedtopreparethisproduct.


a-Tocopherol-TEGPhosphoramidite 10-1977-95 50µmole 10-1977-90 100µmole 10-1977-02 0.25g

STEARYL LABELING

WenowofferasimpleC18lipidasaneconomicalandeffectivecarriermolecule.Weenvisagethatthe5’-stearylgroupwillbecomeafavoredlipophiliccarrierforexperimentationwithsyntheticoligonucleotides.


5’-StearylPhosphoramidite 10-1979-90 100µmole 10-1979-02 0.25g

LABELING

3’-Cholesteryl-TEG CPG

Cholesteryl-TEGON

HO

OO

O

ODMTO

O P N(iPr)2

O CNEt

ONH

OO

O

O

ODMTO

O

HN CPGO

O

ONH

OO

O

O

O

P N(iPr)2

O CNEt

5’-Cholesteryl-TEG

OO

OODMTO

O P N(iPr)2

O CNEtO

a-Tocopherol-TEG

O P N(iPr)2

O CNEt5’- Stearyl

RELATED

Spermine...................................48

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LABELING

N-ACETYLGALACTOSAMINE (GalNAc) LABELING

Adirected approach to thedeliveryof therapeuticoligonucleotides specifically to the liver hasbeen to target theasialoglycoprotein receptor (ASGPR)usingasuitableglycoconjugate. Indeed,ASGPR is the ideal target fordeliveryoftherapeuticoligonucleotidestotheliversinceitcombinestissuespecificity,highexpressionlevelsandrapidinternalizationandturnover.Theuseofoligonucleotideglycoconjugateshasledtosignificantadvancesintherapeuticdeliveryasevidencedby theworkofAlnylamPharmaceuticalsand IonisPharmaceuticalsusingmultivalentN-acetylgalactosamine (GalNAc)oligonucleotideconjugates.

GlenResearch isdelighted to introduceaGalNAcmodification strategyusingamonomericGalNAc support and theequivalentGalNAcphosphoramidite. Ourexperimentalworkhasshownthattheseproductsarefullycompatiblewithregularoligonucleotidesynthesisanddeprotection.OligonucleotidescontainingGalNAccanbedeprotectedusingstandardproceduresduringwhichtheacetylprotectinggroupsonGalNAcareremoved.Wehavedemonstratedthat5’-GalNAcC3phosphoramiditecanbeusedtoprepareoligonucleotideswithmultipleconsecutiveGalNAcadditionsatthe5’terminus.GlenResearchofferstheseGalNAcC3productsunderanagreementwithAMChemicalsLLC.


5’-GalNAcC3Phosphoramidite 10-1974-95 50µmole 10-1974-90 100µmole 10-1974-02 0.25g

GalNAcC3CPG 20-2974-01 0.1g 20-2974-10 1.0g 1µmolecolumns 20-2974-41 Packof4 0.2µmolecolumns 20-2974-42 Packof4 10µmolecolumn(ABI) 20-2974-13 Packof1 15µmolecolumn(Expedite) 20-2974-14 Packof1

OP

N(iPr)2

O CNEt

NNH

O

OO

OAc OAc

AcHN

AcO O OTMT

ONN

HO

OO

OAc OAc

AcHN

AcO O OTMT

NH

O

O CPG

5’-GalNAc C3 Phosphoramidite

GalNAc C3 CPG




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LABELING

CDPI3 MGB™ LABELING

The tripeptideofdihydropyrroloindole-carboxylate (CDPI3) is aminorgroovebinding (MGB)moietyderived from thenaturalproductCC-1065withstrongDNAbindingproperties.Syntheticoligonucleotideswithcovalently-attachedCDPI3 haveenhancedDNAaffinityandhaveimprovedthehybridizationpropertiesofsequence-specificDNAprobes.ShortCDPI3-oligonucleotideshybridizewithsingle-strandedDNAtogivemorestableDNAduplexesthanunmodifiedODNsofsimilarlength.CDPI3MGB-oligonucleotideconjugateshavebeenfoundtobeusefulinthefollowingapplications:

• ArrestofprimerextensionandPCRblockers• ShortandfluorogenicPCRprimers• Real-timePCRprobes• miRNAInhibitors

ThesimplestapproachtoMGBprobedesignistouseanMGBsupport,addaquenchermoleculeasthefirstadditionandcompletethesynthesiswitha5’-fluorophore.Alternatively,afluorophoresupportcouldbeusedwiththe5’terminuscontainingaquenchermoleculefollowedbyafinalMGBadditionatthe5’terminus.GlenResearchoffers5’-CDPI3 MGB™ Phosphoramiditeand3’-CDPI3MGB™CPG.

5’-CDPI3MGBphosphoramiditewasfoundtobehydrophobicenoughthatitrequired10%THFinACNtogocompletelyintosolutionata0.1Mconcentrationandrequireda3minutecouplingtime.DeprotectioncanbecarriedoutinEtOH/NH4OH1:3(v/v)17hrat55°CandCDPI3MGBiscompatiblewithGlenPak™purification.

With the CDPI3MGBCPG,optimalresultsareobtainedifUltraMildmonomersandCapAareusedduringsynthesisalongwith0.5MCSOoxidizer.However,theuseofstandardmonomerswithiodineoxidationfollowedbydeprotectionwithEtOH/NH4OH1:3(v/v)for17hrat55°Cwillgiveacceptableresults.


5’-CDPI3MGB™Phosphoramidite 10-5924-95 50µmole 10-5924-90 100µmole 10-5924-02 0.25g

CDPI3MGB™CPG 20-5924-01 0.1g 20-5924-10 1.0g 1µmolecolumns 20-5924-41 Packof4 0.2µmolecolumns 20-5924-42 Packof4 10µmolecolumn(ABI) 20-5924-13 Packof1 15µmolecolumn(Expedite) 20-5924-14 Packof1

LEGAL NOTICE

NOTICE TO PURCHASER: LIMITED LICENSE

This product is sold under licensing

arrangementsbetweenELITechGroupInc.andGlenResearch.Thepurchaseprice of this product includes limited, nontransferablerightstousetheproductsolelyforactivitiesofthepurchaserwhicharedirectlyrelatedtohumandiagnostics.Otheruses,includingincorporationoftheproduct into another commercial product,areprohibitedwithoutadditionallicenserights.Forinformationonpurchasingalicenseto this product for purposes other thanthosestatedabove,contact:

ELITechGroupMolecularDiagnostics,21720 23rd Drive SE, Suite 150,

Bothell, WA 98021 Phone(425)482-5555 Fax(425)482-5550 Email: [email protected]

This limited license permits the personorlegalentitytowhichthisproducthasbeenprovidedtousetheproduct,andthedatageneratedbyuseoftheproduct,onlyforhumandiagnostics.NeitherELITechGroupInc.noritslicensorsgrantsanyotherlicenses,expressedorimpliedforanyotherpurposes.

Some components of nucleic acid analysis,suchasspecificmethodsandcompositionsformanipulatingorvisualizingnucleicacidsforanalysis,maybecoveredbyoneormorepatentsownedbyotherparties.Similarly,nucleicacidscontainingspecificnucleotidesequencesmaybepatented.Making,using,offeringfor sale, or selling such components ornucleicacidsmayrequireoneormorelicenses.Nothinginthisdocumentshouldbeconstruedasanauthorizationorimplicitlicensetomake,useorsellanysocoveredcomponentornucleicacidunderanysuchpatents.

5’-CDPI3 MGB™ Phosphoramidite CDPI3 MGB™ CPG

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mailto:mdx%40elitechgroup.com?subject=

LABELING

PSORALEN LABELING

PsoralenC2at the5’-terminusof anoligonucleotide serveseffectively as a cross-linking reagent indouble-strandedoligonucleotides. The6 atom spacer armof PsoralenC6 allows cross-linkingwith a triplexoligonucleotide strand. ClickChemistrywithpsoralenazideandoneofourmanynucleosidicandnon-nucleosidic alkynederivativeshas thepotentialtogenerateavarietyofpracticalcross-linkers.Thewellknownreversiblecross-linkingbehaviorofpsoralenwithanadjacentthymidineresiduecouldbeveryuseful.

Item Cat. No. Pack

PsoralenC2Phosphoramidite 10-1982-90 100µmole 10-1982-02 0.25g

PsoralenC6Phosphoramidite 10-1983-90 100µmole 10-1983-02 0.25g

PsoralenAzide 50-2009-92 25µmole 50-2009-90 100µmole

EDTA LABELING

EDTA-C2-dTphosphoramiditecontainsthetriethylesterofEDTAwhichallowssequence-specificcleavageofsingle-anddouble-strandedDNAandRNA.ThecleavagereactionisonlyinitiatedonceFe(II)anddithiothreitolareaddedandsoisreadilycontrolled.CouplingofEDTA-dTisnormalbutcleavageanddeprotectionshouldbecarriedoutwithsodiumhydroxideinaqueousmethanol(0.4MNaOHinmethanol/water4:1)overnightatroomtemperature.

Item Cat. No. Pack

EDTA-C2-dT-CEPhosphoramidite 10-1059-95 50µmole 10-1059-90 100µmole 10-1059-02 0.25g




Expedite EMerMade M




Psoralen C2 Psoralen C6

OO

CH 3

CH 3

CH 3

O

OO P N(iPr)2

O CNEtOO

CH 3

CH 3

CH 3

O

O

CNEtON(iPr)2PO

O O

N3

O

Psoralen Azide EDTA-C2-dT

O

O P N(iPr)2O CNEt

DMTO

NNH

NH

O

O

N

HN O OEt

O

NOEt

O

O

EtOO

118

ODMTO

N

HN

O

O

O P N(iPr)2

O CNEt

NH

HN

O

O Fe

Ferrocene-dT

S+

N

N N

OO N

O

OClO4

-

Methylene Blue NHS Ester

S

N

ClN N

DMTO O P N(iPr)2

O CNEt

Methylene Blue II


MethyleneBlueIIiscoveredunderEuropean patent EP2820003 and US patent US9540405 and is sold under licensefromtheUniversityofLyon.

FERROCENE LABELING

Withanexcellentstabilityprofile,ferrocenehasalwaysattractedconsiderableinterestforDNAlabelingtogenerateprobesforelectrochemicaldetection.BasedonourAmino-ModifierC6-dTstructure,Ferrocene-dTiseasilyaddedtooligonucleotideswithnodisruptionofregularhybridizationbehavior.Multipleincorporationsintoanoligonucleotideprobearealsosimplyachieved.Oligonucleotidesaredeprotectedusingstandardtechniques.FerroceneoligonucleotidesshouldbestoredunderArgonandaqueoussolutionsshouldbedegassedimmediately.

Item Cat. No. Pack

Ferrocene-dT-CEPhosphoramidite 10-1576-95 50µmole 10-1576-90 100µmole 10-1576-02 0.25g

METHYLENE BLUE LABELING

MethyleneBlue,whichbelongstothephenothiazinefamilyofdyes,isauniquedyewithavarietyofusefulproperties.Despiteitshighextinctioncoefficientinthevisibleregion(81,000L/mol•cm),itisweaklyfluorescentduetoitshighrateofintersystemcrossingfromtheS1excitedstatetotheT1tripletstate.Thispropertymakesitanexcellentphotosensitizer,andithasbeenusedextensivelytoproducehighlyreactivesingletoxygen.MethylenebluehastheabilitytobothintercalateinduplexDNA,preferringG:CoverT:Abasepairs,andcanactasanelectrochemicalredoxprobe.Methylenebluehasalsobeenshowntobeunmatchedinperformanceasaredox-activereporterforelectrochemicalbiosensors.

Earlier,we introducedMethyleneBlueC3Phosphoramiditebutthisproductprovedtohavequite limitedstabilityandhasbeendiscontinued.Asanalternativeoption,weintroducedMethyleneBlueNHSEstertoallowresearcherstolabelamino-modifiedoligonucleotideswiththisinterestingdye.WiththeencouragementandtechnicalexpertiseofCaroleChaixandhercolleaguesattheUniversityofLyon,wedecidedtoprepareanalternativestructurethatseemedtohaveamuchsuperiorstabilityprofile-MethyleneBlueIIPhosphoramidite.Fortunately,thisstructuredidindeedprovemorestableandwearenowabletoofferagainaMethyleneBluePhosphoramidite.

Item Cat. No. Pack

MethyleneBlueNHSEster 50-1960-23 5.4mg (Dissolve5.4mgin60µLofDMSO)

MethyleneBlueIIPhosphoramidite 10-5961-95 50µmole 10-5961-90 100µmole 10-5961-02 0.25g

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LABELING

Thiazole Orange NHS Ester

LABELING WITH THIAZOLE ORANGE

Thiazoleorangeisanasymmetriccyaninedyewhosefluorescencecanbequitedependentonitslocalenvironment.Whenanoligonucleotidelabeledwiththiazoleorangeishybridizedtoitscomplementarysequence,thethiazoleorangeactsasanintercalator.Inadditiontoprovidingenhancedthermalstability,thedyeadoptsamostlyplanarconfigurationresultinginsignificantlyenhancedfluorescence.This“lightup”effectcanbeashighas34-folddependingonthesequenceandhowthedyeisattached.ThisNHSesterwillallowsimplefunctionalizationofinternallylocatedaminomodificationssuchasthosegeneratedwithamino-modifierC6dT(10-1039).

Item Cat. No. Pack

ThiazoleOrangeNHSEster 50-1970-23 5.4mg

LABELING

120

FLUORESCENT DYES

Absorbance Maximum

Emission Maximum Excimer

Pyrene-dU 402nm 472nm 486nm

Perylene-dU 473nm 490nm Not Determined

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LABELING WITH METAL CHELATES

2,2’-DipicolylaminePhosphoramiditehasbeendiscontinued.ThisproductwasmanufacturedanddevelopedbySyntrixBiosystemsInc.Forfurtherinformation,pleasecontact:

DeanY.Maeda,Ph.D.,M.B.A.Director,ChemistryandPreclinicalDevelopmentSyntrixBiosystems215ClayStNWSteB5Auburn,WA98001tel:253-833-8009ext.23fax:[email protected]

LABELING WITH POLYAROMATIC HYDROCARBONS

Pyreneandperylenearefluorescentpolycyclicaromatichydrocarbonsthathavetheabilitytoform‘excitedstatedimers’knownasexcimers.Thisunstructured,long-wavelengthemissionarisesfromtheformationofacharge-transfercomplexbetweentheexcitedstateandthegroundstateoftwofluorescentmolecules.InPyrene-dUandperylene-dU,thehydrocarbonisattachedatthe5positionofdeoxyuridinethroughatriplebondandiselectronicallycoupledtothedeoxyuridinebase.ThiselectroniccouplingofthebaseandthehydrocarbonmakesthefluorescencesensitivetothebasepairingofthedUportionofthemolecule,allowingthediscriminationbetweenperfectandonebasemismatchedtargets.

Item Cat. No. Pack

Pyrene-dU-CEPhosphoramidite 10-1590-95 50µmole 10-1590-90 100µmole 10-1590-02 0.25g

Perylene-dU-CEPhosphoramidite 10-1591-95 50µmole 10-1591-90 100µmole 10-1591-02 0.25g

Perylene-dUPyrene-dU

ODMTON

HN

O

O

O P N(iPr)2

O CNEt

ODMTO

N

HN

O

O

O P N(iPr)2

O CNEt

N

NN

DMTO

O P N(iPr)2

O CNEt

2,2’-Dipicolylamine




Expedite EMerMade M




LABELING

mailto:Dmaeda%40syntrixbio.com?subject=

RELATED

3’-PhosphateCPG.....................82SulfurizingReagent...................35Fluorescein-dT.........................103

LABELING

122

PUROMYCIN CPG

Oneofthemostchallengingrequirementsassociatedwithcombinatorialchemistryistherecoveryofsequenceinformationoftheoligonucleotideorpeptideselectedbythescreeningassay.Amethod1hasbeendevelopedtogenerateafusionproductbetweenmRNAandthepolypeptideitencodesusing in vitrotranslationofsyntheticRNAs3’-labeledwithpuromycin,anantibioticthatmimicstransferRNA.Puromycinbindsintheribosome’sAsite,formsapeptidebondwiththegrowingpeptidechain,andblocksfurtherpeptideelongation.BylinkingpuromycintomRNA,apeptide-RNAfusionproductresultsfromthetranslationofthemessagelinkingtheencodingmRNAwithitspeptideproduct.


PuromycinCPG 20-4040-01 0.1g 20-4040-10 1.0g 1µmolecolumns 20-4140-41 Packof4 0.2µmolecolumns 20-4140-42 Packof4 10µmolecolumn(ABI) 20-4140-13 Packof1 15µmolecolumns(Expedite) 20-4140-14 Packof1

QUENCHED AUTOLIGATION (QUAL) PROBES

QUALprobes2consistoftwooligonucleotides,thefirstcontaininganucleophilicgroupatthe3’-terminus,whilethesecondhasanelectrophilicgroupatthe5’-terminus.Whentheprobepairfindsthetarget,theoligoslineupwiththe3’-terminusofthefirstdirectlyadjacenttothe5’-terminusofthesecond.Anautoligationreactionthentakesplacetocombinethetwooligosintoasingleprobe.Asusual,the3’nucleophilicgroupisthe3-thiophosphate,easilypreparedusing3’-phosphateCPGwithasulfurizingstepinthefirstcycle.Inthiscase,theelectrophilicgroupisa5’-dabsylgroup,whichisanexcellentleavinggroupaswellasafinequencheroffluorescence.Thesecondoligo,therefore,containsafluorophorewhichisquenchedbythedabsylgroup.Apopularchoiceforfluorophoreisfluorescein-dTbutitiseasytoimaginethatavarietyoffluorophorescouldbeattachedtoanyofthecommerciallyavailableamino-modifiednucleosidephosphoramidites.


5’-Dabsyl-dT-CEPhosphoramidite 10-1532-90 100µmole 10-1532-02 0.25g

Puromycin CPG

OCH 3

succinyl-lcaa-CPG

ODMTO

O

N

N

N

N

N(CH 3)2

ONHCOCF 3

H

NH

REFERENCES

(1)R.W.RobertsandJ.W.Szostak,Proc. Natl. Acad. Sci. USA, 1997, 94,12297-302.

(2)S.SandoandE.T.Kool,J Amer Chem Soc, 2002, 124,2096-2097.

i

5’-Dabsyl-dT

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LABELING FOR PHOTO-REGULATION OF OLIGONUCLEOTIDES

Photo-control,theuseofultravioletorvisiblelighttocontrolareaction,hasanumberofadvantagesoverotherexternalstimuli:

• Lightdoesnotintroducecontaminantsintothereactionsystem,• Excitationwavelengthcanbecontrolledthroughthedesignofthephoto-responsivemolecule,and• Itisnowstraightforwardtocontrolirradiationtimeand/orlocalexcitation. Whenaphoto-responsivemoleculeisdirectlyattachedtoDNAasareceptor,photo-regulationofthebioprocessregulatedbythatDNAmoleculecould,inprinciple,beachieved.Suchphoto-responsiveDNAcouldalsobeusedasaswitchinaDNA-basednano-machine.ProfessorHiroyukiAsanumaandhisgroupatthedepartmentofMolecularDesignandEngineeringoftheGraduateSchoolofEngineeringoftheNagoyaUniversity(Japan)havedevelopedanefficientmethodtoachievethisgoal.TheyhaveattachedazobenzenetoDNAandmadeitphoto-responsive1,2.Azobenzeneisatypicalphoto-responsivemoleculethatisomerizesfromitsplanartrans-formtothenon-planarcis-formafterUV-lightirradiationwithawavelengthbetween300nmand400nm(lmaxisaround330nm).Interestingly,thesystemrevertsfromthecis-formtothetrans-formafterfurtherirradiationwithvisiblelight(wavelengthover400nm).Thisprocessiscompletelyreversible,andtheazobenzenegroupdoesnotdecomposeorinduceundesirablesidereactionsevenonrepeatedtrans-cis isomerization.ByintroducingazobenzenesintoDNAthroughD-threoninolasalinker,Asanumaandco-workerssucceededinachievingphoto-regulationof:

• FormationanddissociationofaDNAduplex3,4 and • TranscriptionbyT7-RNApolymerasereaction5,6,7


AzobenzenePhosphoramidite 10-5800-95 50µmole 10-5800-90 100µmole 10-5800-02 0.25g

REFERENCES

(1)H.Asanuma,etal.,Angew Chem Int Ed, 2001, 40,2671-2673.

(2)T.Takarada,etal.,Chem Lett., 2001, 30,732.

(3)H.Asanuma,X.G.Liang,T.Yoshida,andM.Komiyama,Chembiochem, 2001, 2,39-44.

(4)H.Asanuma,D.Matsunaga,andM.Komiyama,NUCLEIC ACIDS SYMP SER (OXF), 2005, 49,35.

(5)H.Asanuma,etal.,Chembiochem, 2002, 3,786.

(6)M.Liu,H.Asanuma,andM.Komiyama,J. Amer. Chem. Soc., 2006 , 128,1009.

(7)H.Asanuma,etal.,Nature Protocols, 2007, 2,203-212.

NN

O

NH

DMTO

H3CO

P

O

N(iPr)2

CNEt

Azobenzene Phosphoramidite




Expedite EMerMade M




LABELING

N

ODMTO

O P N(iPr)2

O CNEt

CN

3-Cyanovinylcarbazole

REFERENCES

(1)Y.Yoshimura,andK.Fujimoto,Org Lett, 2008, 10,3227-30.

(2)K.Fujimoto,K.Konishi-Hiratsuka,T.Sakamoto,andY.Yoshimura,ChemBioChem, 2010, 11,1661-4.

(3)Y.Yoshimura,T.Ohtake,H.Okada,andK.Fujimoto,ChemBioChem, 2009, 10, 1473-6.

LABELING WITH ULTRAFAST PHOTO CROSS-LINKER

When3-cyanovinylcarbazolenucleoside(CNVK)isincorporatedintoanoligonucleotide,veryrapidphoto cross-linkingtothecomplementarystrandcanbeinducedatonewavelengthandrapidreversalofthecross-linkispossibleatasecondwavelength.NeitherwavelengthhasthepotentialtocausesignificantDNAdamage.IrradiationofaduplexcontainingasingleincorporationofCNVKat366nmledto100%cross-linkingtothyminebasein1second,althoughcompletecross-linkingtocytosinetakes25seconds.1A30secondirradiationtimeshouldcoverallsituations.Inaddition,itwasdemonstratedthatthepurinebaseswereunreactivetocross-linking,allowingdifferentiationbetweenpyrimidinesandpurinesatthetargetsite.TheauthorsalsodeterminedtheeffectofsequencecontextsaroundtheCNVK site and demonstrated that the identityofbasesoneithersideofthecross-linkingsitehaslittleeffectonthereaction.Oncecross-linked,theUVmeltingtemperatureoftheduplexwasraisedbyaround30°Crelativetotheduplexbeforeirradiation.Completereversalofthecross-linktakesplaceat312nmin3minutes.Thisfacilereversalreactionis,therefore,accomplishedwithnodamagetonormalDNA.

Inalaterpublication,afurtherapplicationofthiscross-linkingtechniquewasinvestigated.2 When CNVKwascross-linkedwithadCresidueinduplexDNA,heatingat90°Cfor3.5hoursledtodeaminationofthecytosinebasetoformuracilinthecomplementarystrand.Reversalofthecross-linkat312nmledtoaDNAstrandinwhichdChadbeenconvertedtodU. TheauthorsshowedthatthistransformationisspecificforthedCresidueoppositetheCNVKandanyfurtheradjacentdCresiduesareunaffected.Similarly,theauthorshaveshownthatCNVKcanbecross-linkedtoanadjacentRNAstrand.3

Item Cat. No. Pack

3-CyanovinylcarbazolePhosphoramidite 10-4960-95 50µmole (CNVK) 10-4960-90 100µmole 10-4960-02 0.25g

124

LABELING

125

RNA

AND

2’-O

Me-

RNA

RNA SUPPORTS

ABBREVIATIONS

Ac=Acetyl Bz=Benzoyl CNEt=Cyanoethyl CPG = Controlled Pore Glass DMT=4,4’-Dimethoxytrityl

RNA SUPPORTS FOR 3’ MODIFICATION

GlenResearchoffersRNAsupportsinwhichprotectedribonucleosidesareattachedtoCPG.With5’-DMTprotection,andallotherprotectinggroupsbase-labile,theuseofthesesupportsisidenticaltoDNAsupports.Thesesupportsaresuitableforuseinproducingoligodeoxynucleotidesmodifiedatthe3’-terminusoroligoribonucleotides.ABI-stylecolumnsaresuppliedunlessotherwiserequested(seenotebox).


Bz-A-RNA-CPG 20-3303-01 0.1g 20-3303-02 0.25g 20-3303-10 1.0g 1µmolecolumns 20-3403-41 Packof4 0.2µmolecolumns 20-3403-42 Packof4 10µmolecolumns(ABI) 20-3403-13 Packof1 15µmolecolumn(Expedite) 20-3403-14 Packof1

Ac-C-RNA-CPG 20-3315-01 0.1g 20-3315-02 0.25g 20-3315-10 1.0g 1µmolecolumns 20-3415-41 Packof4 0.2µmolecolumns 20-3415-42 Packof4 10µmolecolumn(ABI) 20-3415-13 Packof1 15µmolecolumn(Expedite) 20-3415-14 Packof1 Ac-G-RNA-CPG 20-3324-01 0.1g 20-3324-02 0.25g 20-3324-10 1.0g 1µmolecolumns 20-3424-41 Packof4 0.2µmolecolumns 20-3424-42 Packof4 10µmolecolumn(ABI) 20-3424-13 Packof1 15µmolecolumn(Expedite) 20-3424-14 Packof1

U-RNA-CPG 20-3330-01 0.1g 20-3330-02 0.25g 20-3330-10 1.0g 1µmolecolumns 20-3430-41 Packof4 0.2µmolecolumns 20-3430-42 Packof4 10µmolecolumn(ABI) 20-3430-13 Packof1 15µmolecolumn(Expedite) 20-3430-14 Packof1

Bz-A-CPG Ac-C-CPG U-CPG

ODMTO

O OAc

NHBz

N

N

N

N

succinyl-CPG succinyl-CPG

ODMTO

O OAc

NHAc

O N

N

ODMTO

O OAc

O

O

N

HN

succinyl-CPG

ODMTO

O

succinyl-CPG

OAc

HN

N

N

O

NAcHN

Ac-G-CPG

126

RNA SYNTHESIS

TOM-Protecting-Group™

O OSi

2'-

TOM-Protecting-Group is a trademarkofQIAGEN.

TOM-PROTECTED RNA PHOSPHORAMIDITES

RNAsynthesisusingmonomers containing the2’-O-TriisopropylsilylOxyMethyl (TOM)group (TOM-Protecting-Group™) ischaracterizedbyveryhighcouplingefficiencyalongwithfast,simpledeprotection.Highcouplingefficiencyisachievedbecause theTOM-Protecting-Groupexhibits lower sterichindrance than the2’-O-t-butyldimethylsilyl (TBDMS)groupusedinouralternativeRNAmonomers.Fastandreliabledeprotectionisachievedusingmethylamineinethanol/wateratroomtemperature.AfurtherfeatureoftheTOM-Protecting-Groupisthatduringbasicstepsitcannotundergo2’to3’migration.Thismigrationunderbasicconditionsleadstonon-biologicallyactive2’-5’linkageswhenusingtheTBDMSgroup. ThesefeaturesallowtheTOM-Protectedmonomerstoproducelongeroligonucleotides.TOM-ProtectedRNAmonomersarealsofullycompatiblewithminorbaseswith2’-O-TBDMSprotection.


A-TOM-CEPhosphoramidite 10-3004-02 0.25g 10-3004-05 0.5g 10-3004-10 1.0g C-TOM-CEPhosphoramidite 10-3014-02 0.25g 10-3014-05 0.5g 10-3014-10 1.0g

G-TOM-CEPhosphoramidite 10-3024-02 0.25g 10-3024-05 0.5g 10-3024-10 1.0g

U-TOM-CEPhosphoramidite 10-3034-02 0.25g 10-3034-05 0.5g 10-3034-10 1.0g

RNA SUPPORTS FOR TOM RNA SYNTHESIS


Ac-A-RNA-CPG 20-3304-01 0.1g 20-3304-02 0.25g 20-3304-10 1.0g 1µmolecolumns 20-3404-41 Packof4 0.2µmolecolumns 20-3404-42 Packof4 10µmolecolumn(ABI) 20-3404-13 Packof1 15µmolecolumn(Expedite) 20-3404-14 Packof1

A-TOM C-TOM G-TOM U-TOM

O

O P N(iPr)2O CNEt

DMTO

OTOM

NHAc

N

N

N

N

O

O P N(iPr)2O CNEt

DMTO

OTOM

NHAc

O N

N

O

O P N(iPr)2O CNEt

DMTO

OTOM

AcHN

O

N

N

N

HN

O

O P N(iPr)2O CNEt

DMTO

OTOM

O

O

N

HN





Expedite EMerMade M




127

RNA

AND

2’-O

Me-

RNA

RNA SUPPORTS FOR TOM RNA SYNTHESIS (CONT.)


Ac-C-RNA-CPG 20-3315-01 0.1g 20-3315-02 0.25g 20-3315-10 1.0g 1µmolecolumns 20-3415-41 Packof4 0.2µmolecolumns 20-3415-42 Packof4 10µmolecolumn(ABI) 20-3415-13 Packof1 15µmolecolumn(Expedite) 20-3415-14 Packof1

Ac-G-RNA-CPG 20-3324-01 0.1g 20-3324-02 0.25g 20-3324-10 1.0g 1µmolecolumns 20-3424-41 Packof4 0.2µmolecolumns 20-3424-42 Packof4 10µmolecolumn(ABI) 20-3424-13 Packof1 15µmolecolumn(Expedite) 20-3424-14 Packof1


RNA SYNTHESIS

128Ac-C-CE PhosphoramiditeBz-A-CE Phosphoramidite

RNA SYNTHESIS

ABBREVIATIONS

Bz=Benzoyl CNEt=Cyanoethyl CPG = Controlled Pore Glass dmf=Dimethylformamidine DMT=4,4’-Dimethoxytrityl iPr=Isopropyl lcaa=longchainalkylamino Pac=Phenoxyacetyl PhOAc=Phenoxyacetyl TBDMS=t-Butyl-dimethylsilyl

TBDMS-PROTECTED RNA PHOSPHORAMIDITES

Glen Research CE (ß-cyanoethyl) Phosphoramidites for RNA synthesis are produced and packaged to ensure the highest performance on commercial synthesizers. Every batch is accompanied by a Certificate of Analysis and an HPLC trace, showing the results of our QC testing. RNA Phosphoramidites are synthesis-tested with a minimum coupling efficiency of 97%. Glen Research RNA monomers are packaged in industry standard vials which are specially cleaned to eliminate particulate contamination. These monomers are available in a variety of packs, including high throughput (HT) and low cost (LC). An UltraMild set is also available for situations where sensitive bases are in use. Dmf-G (10-3029) has been discontinued and may be substituted with Ac-G (10-3025).


Bz-A-CE Phosphoramidite 10-3003-02 0.25g 10-3003-05 0.5g 10-3003-10 1.0g Ac-C-CE Phosphoramidite 10-3015-02 0.25g 10-3015-05 0.5g 10-3015-10 1.0g

Ac-G-CE Phosphoramidite 10-3025-02 0.25g 10-3025-05 0.5g 10-3025-10 1.0g

U-CE Phosphoramidite 10-3030-02 0.25g 10-3030-05 0.5g 10-3030-10 1.0g

RNA PHOSPHORAMIDITES - SPECIAL PACKAGING

We offer our high quality DNA phosphoramidites specifically packaged for high throughput and large-scale synthesis customers. These customers normally require high quality materials produced under the guidelines of a validated quality management system while still being priced aggressively. These products include the usual Glen Research certification and guarantees and they are available in larger packs or in bulk. The core catalog numbers for regular DNA phosphoramidites are shown below. For these products, please request a quote.

Item Catalog No.

Bz-A-CE Phosphoramidite 10-3003-SPAc-C-CE Phosphoramidite 10-3015-SPAc-G-CE Phosphoramidite 10-3025-SPU-CE Phosphoramidite 10-3030-SP

N

N

N

N

NHBz

ODMTO

O

CNEtON(iPr)2P

OTBDMS

ODMTO

OTBDMSO

NHAc

O N

N

P N(iPr)2O CNEt

INSTRUMENT TYPES

Glen Research packages these monomersinavarietyofindustry-standardvialsandbottles.Pleaseprovidetheexactspecificationofthebottlerequiredpriortoreceivingaquotation.

U-CE Phosphoramidite

DMTO O

O

O

N

HN

O

P N(iPr)2O CNEt

OTBDMS

Ac-G-CE Phosphoramidite

ODMTO

O P N(iPr)2

O-CNEt

OTBDMS

HN

N

N

O

NAcHN

129

RNA

AND

2’-O

Me-

RNA

ULTRAMILD TBDMS RNA PHOSPHORAMIDITES


Pac-A-CEPhosphoramidite 10-3000-02 0.25g 10-3000-05 0.5g 10-3000-10 1.0g Ac-C-CEPhosphoramidite 10-3015-02 0.25g 10-3015-05 0.5g 10-3015-10 1.0g

iPr-Pac-G-CEPhosphoramidite 10-3021-02 0.25g 10-3021-05 0.5g 10-3021-10 1.0g

U-CEPhosphoramidite 10-3030-02 0.25g 10-3030-05 0.5g 10-3030-10 1.0g

TBDMS RNA SUPPORTS

ABI-stylecolumnsaresuppliedfor1µmoleand0.2µmolescalesunlessotherwiserequested(seenotebox).


Pac-A-RNA-CPG 20-3300-01 0.1g 20-3300-02 0.25g 20-3300-10 1.0g 1µmolecolumns 20-3400-41 Packof4 0.2µmolecolumns 20-3400-42 Packof4 10µmolecolumn(ABI) 20-3400-13 Packof1 15µmolecolumn(Expedite) 20-3400-14 Packof1

Bz-A-RNA-CPG 20-3303-01 0.1g 20-3303-02 0.25g 20-3303-10 1.0g 1µmolecolumns 20-3403-41 Packof4 0.2µmolecolumns 20-3403-42 Packof4 10µmolecolumn(ABI) 20-3403-13 Packof1 15µmolecolumn(Expedite) 20-3403-14 Packof1

RNA SYNTHESIS

U-CE Phosphoramidite

DMTO O

O

O

N

HN

O

P N(iPr)2O CNEt

OTBDMS

Pac-A-CE Phosphoramidite

DMTO O

NHAcOPh

N

N

N

N

O

P N(iPr)2O CNEt

OTBDMS

Ac-C-CE Phosphoramidite iPr-Pac-G-CE Phosphoramidite

ODMTO

OTBDMSO

NHAc

O N

N

P N(iPr)2O CNEt

HN

N

N

N

O

HNDMTO O

O

P N(iPr)2O CNEt

iPr-PhOAc

OTBDMS




Expedite EMerMade M




130

TBDMS RNA SUPPORTS (CONT.)


Ac-C-RNA-CPG 20-3315-01 0.1g 20-3315-02 0.25g 20-3315-10 1.0g 1µmolecolumns 20-3415-41 Packof4 0.2µmolecolumns 20-3415-42 Packof4 10µmolecolumn(ABI) 20-3415-13 Packof1 15µmolecolumn(Expedite) 20-3415-14 Packof1

iPr-Pac-G-RNA-CPG 20-3321-01 0.1g 20-3321-02 0.25g 20-3321-10 1.0g 1µmolecolumns 20-3421-41 Packof4 0.2µmolecolumns 20-3421-42 Packof4 10µmolecolumn(ABI) 20-3421-13 Packof1 15µmolecolumn(Expedite) 20-3421-14 Packof1

Ac-G-RNA-CPG 20-3324-01 0.1g 20-3324-02 0.25g 20-3324-10 1.0g 1µmolecolumns 20-3424-41 Packof4 0.2µmolecolumns 20-3424-42 Packof4 10µmolecolumn(ABI) 20-3424-13 Packof1 15µmolecolumn(Expedite) 20-3424-14 Packof1






RNA SYNTHESIS




Expedite EMerMade M




RELATED

Minor TBDMS monomers......133Pyrrolo-CTP..............................136rSpacer TBDMS.......................134

131

RNA

AND

2’-O

Me-

RNA

MINOR RNA PHOSPHORAMIDITES (TOM PROTECTED)

GlenResearchoffersminorRNAphosphoramiditeswitheitherTOMorTBDMSprotectinggroups.4-Thio-U,5-Methyl-Cytidine,and2-Amino-AdenosineareusefulforanalyzingRNAstructureandactivityrelationships,forexample,inribozymestudies.

Pyrrolo-CisafluorescentnucleosidewhosefluorescenceissensitivetoitsenvironmentandisidealforprobingRNAstructure.Itbase-pairsasanormalCnucleotide.Itishighlyfluorescentanditsexcitationandemissionarewellsuitedtotheredofmostfluorescentnucleotideanalogs,whicheliminatesorreducesbackgroundfluorescencefromproteins.Pyrrolo-CTPhaspotentialusesinbiologicalassaydevelopment.

rSpacerisusedtointroduceanabasicsitetoanRNAsequence.TheTOMprotectedversionhasbeendiscontinuedandisreplacedwiththeTBDMSversion.

Theprotectingschemefor2,6-Diaminopurinehasbeenchangedandtheoriginalproduct(10-3084)hasbeenreplacedwiththeoptimizedproduct(10-3085)below.


4-Thio-U-TOM-CEPhosphoramidite 10-3052-95 50µmole 10-3052-90 100µmole 10-3052-02 0.25g

5-Me-C-TOM-CEPhosphoramidite 10-3064-95 50µmole 10-3064-90 100µmole 10-3064-02 0.25g 2,6-Diaminopurine-TOM-CEPhosphoramidite 10-3085-95 50µmole (2-amino-A) 10-3085-90 100µmole 10-3085-02 0.25g

MINOR RNA BASES

2,6-diaminopurine-TOM5-Me-C-TOM4-Thio-U-TOM

ODMTO

N

N

S

O

O P N(iPr)2

O CNEt

OTOM

CN

O

O P N(iPr)2O CNEt

DMTO

OTOM

NHAc

O N

NMe

ODMTO

O P N(iPr)2

O CNEt

OTOM

N

N

N

NMeOAcHN

NHiBu

RELATED

Pyrrolo-dC..................................68Pyrrolo-CTP..............................136

132

MINOR RNA PHOSPHORAMIDITES (TOM PROTECTED) (CONT.)


Pyrrolo-C-TOM-CEPhosphoramidite 10-3017-95 50µmole 10-3017-90 100µmole 10-3017-02 0.25g

RNA SEQUENCE MODIFIER (TOM PROTECTED)

Amino-ModifierC6-UhasbeenaddedtothegrowingfamilyofsequencemodifiersandweenvisageapplicationsinRNAstructuralstudiesaswellasforlabelingsiRNAtoprobeuptakeandcellulardistribution.


Amino-ModifierC6-UPhosphoramidite 10-3039-95 50µmole 10-3039-90 100µmole 10-3039-02 0.25g

O

O P N(iPr)2O CNEt

DMTO O N

N

OTOM

HN

O

O P N(iPr)2O CNEt

DMTO

OTOM

rSpacerPyrrolo-C-TOM

MINOR RNA BASES

HN

N

O

O

NHNHCCF 3

O O

DMTO

CNEtON(iPr)2PO

O

OTOM

Amino-Modifier C6-U




Expedite EMerMade M




RELATED

Minor TOM monomers..........131

133

RNA

AND

2’-O

Me-

RNA

MINOR RNA BASES

Br-U I-U

O

OP N(iPr)2O CNEt

DMTO

Br

O

O

N

HN

OTBDMS

I

O

O

DMTON

HN

O

O

OTBDMS

P N(iPr)2O CNEt

REFERENCES

(1)C.J.Adams,J.B.Murray,M.A.Farrow,J.R.P.Arnold,andP.G.Stockley,Tetrahedron Lett., 1995, 36,5421-5424.

(2)D.A.Berry,etal.,Tetrahedron Lett, 2004, 45,2457-2461.

iBuHN

N

N

N

N

S

DMTO

CNEtON(iPr)2PO

O

CN

OTBDMS

6-Thio-G

MINOR RNA PHOSPHORAMIDITES (TBDMS PROTECTED)

Inosineand5-Methyl-Uridineareuseful for analyzingRNAstructureandactivity relationships. 5-Bromo-Uridineand5-Iodo-Uridinehavebeenusedforcrystallographystudiesandcross-linkingexperiments.6-Thioguanosine(6-thio-G)hasapplicationsinribozymeandsiRNAresearch,aswellasinRNA-proteininteractions.Theremovalofthesilylprotectinggroupwithoutinterferingwiththesulfuriscritical.Thisisremoved1cleanlybytriethylaminetrihydrofluorideinDMSObut t-butylammoniumfluoride (TBAF) leads todegradationof the thio-nucleotideanalogueand shouldnotbeused.2-AminopurineribosideisusefulforanalyzingRNAstructureandactivityrelationships,forexample,inribozymestudies.


I-CEPhosphoramidite 10-3040-95 50µmole 10-3040-90 100µmole 10-3040-02 0.25g 5-Me-U-CEPhosphoramidite 10-3050-95 50µmole(T) 10-3050-90 100µmole 10-3050-02 0.25g

Br-U-CEPhosphoramidite 10-3090-95 50µmole 10-3090-90 100µmole 10-3090-02 0.25g

I-U-CEPhosphoramidite 10-3091-95 50µmole 10-3091-90 100µmole 10-3091-02 0.25g

6-Thio-G-CEPhosphoramidite 10-3072-95 50µmole 10-3072-90 100µmole 10-3072-02 0.25g

2-Aminopurine-CEPhosphoramidite 10-3070-95 50µmole 10-3070-90 100µmole 10-3070-02 0.25g

5-Me-U

HN

N

O

O

CH 3

DMTO

CNEtON(iPr)2PO

O

OTBDMS

Inosine

O

O P N(iPr)2O CNEt

DMTO

O

N

N

N

HN

OTBDMS

N

N N

N

ODMTO

O P N(iPr)2

O CNEt

NH

O

OTBDMS

2-Aminopurine

134

MINOR RNA (TBDMS PROTECTED) (CONT.)

8-Aza-7-deaza-AdenosineisanisomerofAdenosinewithvirtuallyidenticalelectrondensity.TheN7nitrogenisnotavailableforhydrogenbonding.

Ribozymeactivityissubstantiallyaffectedbythesubstitutionofmodifiedpyrimidinebases.Zebularine(pyrimidin-2-oneribonucleoside)mayberegardedasaCytidinederivativelackingtheexocyclicaminogroup.ZebularineandPyridin-2-oneRibonucleoside, the3-deazaanalogueofZebularine,areprimecandidates foruse inevaluating ribozymeactivityandfunction.ItshouldbenotedthatZebularineismildlyfluorescent,absorbingat298nmandemittingat367nm.

PseudoUridineisoneofthemostcommonmodifiednucleosidesfoundinRNA.TheavailabilityofaphosphoramiditewillallowdetailedresearchintotheeffectsofthismodifiedbaseonRNAstructureandactivity.

rSpacerisusedtointroduceanabasicsitetoanRNAsequence.


Zebularine-CEPhosphoramidite 10-3011-95 50µmole 10-3011-90 100µmole 10-3011-02 0.25g

Pyridin-2-one-CEPhosphoramidite 10-3012 Discontinued

PseudoUridine-CEPhosphoramidite 10-3055-95 50µmole 10-3055-90 100µmole 10-3055-02 0.25g 8-Aza-7-deaza-A-CEPhosphoramidite 10-3083 Discontinued

rSpacerTBDMSCEPhosphoramidite 10-3915-95 50µmole 10-3915-90 100µmole 10-3915-02 0.25g

Pyridin-2-one PseudoUridineZebularine 8-Aza-7-deaza-A

ODMTO

O P N(iPr)2

O CNEt

OTBDMS

N

NOODMTO

O P N(iPr)2

O CNEt

OTBDMS

NOODMTO

O P N(iPr)2

O CNEt

OTBDMS

HN NH

O

O

ODMTO

O P N(iPr)2

O CNEt

OTBDMS

N

N NN

N N




Expedite EMerMade M




MINOR RNA BASES

ODMTO

O P N(iPr)2

O CNEt

OTBDMS

rSpacer TBDMS

RELATED

5-Me-C.....................................131Pseudouridine.........................134

135

RNA

AND

2’-O

Me-

RNA


Methylationofadenosineatposition1producesadrasticfunctional changeinthenucleobase.1-Methyladenosine(pKa 8.25)isa muchstrongerbasethanadenosine(pKa3.5). N-1methylation excludesparticipationoftheadeninebaseincanonicalWatson–Crick basepairingandprovidesapositivechargetothenucleobase.Thismodificationalsoaltersthehydrophobicityofthebase,thestackingproperties,theorderingofwatermoleculesandthechelationproperties. Thebasemaybecomeinvolvedinnon-canonicalhydrogenbonding, inelectrostaticinteractionsand,ingeneral,itmaycontribute to theconformationaldynamicsofthetRNA.

Inthecentraldogmaofmolecularbiology,geneticinformationflowsfromDNAtoRNAandthentoprotein.ReversibleepigeneticmodificationsongenomicDNAandhistonehavebeenknowntosubstantiallyregulategeneexpression.Ontheotherhand,thereexistsmorethan100naturallyoccurringchemicalmodificationsinRNA;however,thefunctionsoftheseRNAmodificationsarelargelyunknown.WhethersomeofthesemodificationsinRNAcanbereversedandcouldimpactgeneexpressioninthecentraldogmawasunknownuntiltherecentdiscoveryofN6-methyladenosine(N6-Me-A)asthefirstexampleofreversibleRNAmethylation.1WeoffertheN6-Me-ARNAmonomerwithaphenoxyacetylprotectinggrouptominimizepotentialbranching.WehaveshownN6-Me-A-CEPhosphoramiditetobecompletelycompatiblewithallpopularRNAsynthesisanddeprotectionmethods,fromUltraMildtothemostpopularprocedureusingAMAfordeprotection.


1-Me-A-CEPhosphoramidite 10-3501-95 50µmole 10-3501-90 100µmole 10-3501-02 0.25g

N6-Me-A-CEPhosphoramidite 10-3005-95 50µmole 10-3005-90 100µmole 10-3005-02 0.25g

RNAmethylationoccursinalargeselectionofRNAnucleosidesandthisposttranscriptionalmodificationofRNA,carriedoutbyavarietyofRNAmethyltransferases,appearsinawidevarietyofRNAspecies-includingtRNA,mRNA,miRNAandRNAviruses.Over90methylatednucleosideshavebeenfoundintRNAandtheseplaymanysignificantrolesintRNAstructure.Inaddition,methylationappearstomarkthetRNAasmature,preventingitsdegradationaswellasdirectinglocalizationwithinthecell.mRNA,modifiedwith1-methylpseudouridine(1-Me-Y)aloneor incombinationwith5-methylcytidine (5-Me-C),significantlyincreasesproteinexpressionincellsandmousemodels.1-Me-YisalsoamodifiednucleobasethatcangreatlyenhancethepropertiesofmRNAbyreducingimmunogenicityandincreasingstability.


1-Me-PseudouridinePhosphoramidite 10-3056-95 50µmole 10-3056-90 100µmole 10-3056-02 0.25g

1-Me-A

REFERENCE

(1)Y.Fu,D.Dominissini,G.Rechavi,andC.He, Nat Rev Genet, 2014, 15,293-306.

N

N N

N

N

ODMTO

O

P N(iPr)2

O CNEt

OTBDMS

PhO

O

CH3

N6-Me-A

MINOR RNA BASES

ODMTO

O P N(iPr)2

O CNEt

OTBDMS

HN N

O

O

CH3

1-Me-Pseudouridine

RELATED

tCO...............................................70Pyrrolo-dC..................................68Pyrrolo-C..................................132

136

OH

CH 3

N

NO

NH

OO

PO

PO

PHO

O O O

OH OH OH

OH

Pyrrolo-CTP


ThebrightfluorescenttricycliccytosineanaloguestCandtCOstandoutamongfluorescentbasesduetotheirvirtuallyunquenchedfluorescenceinsidesingle-ordouble-strandedDNA. Untilrecently,thisfamilyoftricycliccytosineshadonlybeenstudiedandusedinDNAcontextsand,importantly,introducedaspossibledonorsofthefirstDNAbaseanalogueFRET-pairwithtCnitro.FluorescentbaseanaloguesforRNAare limited innumbercomparedtotheirDNAcounterparts. Tofacilitatetheapplicationofsuchanalogues,characterizationoftheirstructuralanddynamicsbehaviorinRNAcomparedto the correspondingnatural nucleoside is important. Wenow introduce the tCO ribonucleoside,whichhas beenincorpratedintoarangeofRNAsequences,whereitwasshowntobeaverypotentandusefulfluorophoreinthiscontext.1 GlenResearchoffersthisusefulfluorescentribonucleosideanalogueincooperationwithModyBaseHB.


Ribo-tCO-CEPhosphoramidite 10-3517-95 50µmole 10-3517-90 100µmole 10-3517-02 0.25g

MINOR RNA TRIPHOSPHATES

Pyrrolo-dCisafluorescentnucleosidethatcodesasdCandbasepairsefficientlywithdG.Preliminaryevidenceindicatesthatpyrrolo-dCtriphosphateisanexcellentsubstrateforTaq,PfuandVentpolymerasesandisincorporatedspecificallyoppositedG.Pyrrolo-dCTPhasbeenavailableforsometimeandisinuseinbiologicalassays.Pyrrolo-CTPisafluorescentribonucleotidewithfluorescenceexquisitelysensitivetoitsenvironmentandisofgreatinterestforRNAstructuralresearch.Thepyrrolo-CprojectisajointdevelopmentbyBerryandAssociates,Inc.andGlenResearchCorporation.


Pyrrolo-CTP 81-3017-01 Discontinued 10mM

REFERENCE

(1)Füchtbauer,A.F.,Preus,S.,Börjesson,K.,McPhee,S.A.,LilleyD.M.J.,Wilhelmsson,L.M.,Sci. Rep., 2017, 7, 2393.


TheseproductsareofferedincollaborationwithModyBaseHB.

O

N

N

O

O P N(iPr)2

O CNEt

O

HN

DMTO

OTBDMS

Ribo-tC°

MINOR RNA BASES

137

RNA

AND

2’-O

Me-

RNA

2’-OMe-Ac-C 2’-OMe-U2’-OMe-G2’-OMe-C2’-OMe-A

2’-OME-RNA PHOSPHORAMIDITES

GlenResearch2’-OMe-RNACE (ß-cyanoethyl) Phosphoramidites aredesigned toproduce syntheticoligonucleotidescontainingnucleaseresistant2’-O-methylribonucleotidelinkages. Deprotection, isolationandhandlingof2’-O-methyloligonucleotidesareidenticaltotheproceduresforoligodeoxynucleotides.


2’-OMe-A-CEPhosphoramidite 10-3100-90 100µmole 10-3100-02 0.25g 10-3100-05 0.5g 10-3100-10 1.0g

2’-OMe-Ac-C-CEPhosphoramidite 10-3115-90 100µmole 10-3115-02 0.25g 10-3115-05 0.5g 10-3115-10 1.0g

2’-OMe-iBu-G-CEPhosphoramidite 10-3120-90 100µmole 10-3120-02 0.25g 10-3120-05 0.5g 10-3120-10 1.0g

2’-OMe-G-CEPhosphoramidite 10-3121-90 100µmole 10-3121-02 0.25g 10-3121-05 0.5g 10-3121-10 1.0g

2’-OMe-U-CEPhosphoramidite 10-3130-90 100µmole 10-3130-02 0.25g 10-3130-05 0.5g 10-3130-10 1.0g

ODMTO

OMeO

NHBz

N

N

N

N

P N(iPr)2O CNEt

ODMTO

OMeO

NHBz

O N

N

P N(iPr)2O CNEt

P N(iPr)2O CNEt

NHAc

O N

N

ODMTO

OMeO

ODMTO

OMeO

(Me)2NN

O

N

N

N

HN

P N(iPr)2O CNEt

ODMTO

OMeO

O

O

N

HN

P N(iPr)2O CNEt

ODMTO

O

P N(iPr)2

O CNEt

OMe

HN

N

N

NNH

O

O

2’-OMe-iBu-G




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2’-OME-RNA SYNTHESIS

138

ULTRAMILD 2’-OME-RNA

TheuseofUltraMildmonomersinoligonucleotidesynthesishasallowedverysensitivedyeslikeTAMRA,HEXandCy5tobeusedvirtuallyroutinely.TheDNAandRNAmonomersarecurrentlyavailableandwealsoprovidethissetof2’-OMe-RNAmonomers.Inourversionofthischemistry,weuseasprotectinggroupsphenoxyacetyl(Pac)forA,acetyl(Ac)forC,andisopropyl-phenoxyacetyl(iPr-Pac)forG.

Ithasbecomeclearthataceticanhydrideintheconventionalcappingmixcancausetransamidationinsituationswhereanamineprotectinggroupisquitelabile.Thisleadstoacetylprotectionontheaminogroupthatmaybeslowtoberemoved.Consequently,ifmanydGresiduesareincludedintheoligonucleotide,werecommendtheuseofphenoxyaceticanhydride(Pac2O)inCapA.ThismodificationremovesthepossibilityofexchangeoftheiPr-PacprotectinggrouponthedGwithacetatefromtheaceticanhydridecappingmix.


2’-OMe-Pac-A-CEPhosphoramidite 10-3601-02 0.25g 10-3601-05 0.5g 10-3601-10 1.0g

2’-OMe-Ac-C-CEPhosphoramidite 10-3115-02 0.25g 10-3115-05 0.5g 10-3115-10 1.0g

2’-OMe-iPr-Pac-G-CEPhosphoramidite 10-3621-02 0.25g 10-3621-05 0.5g 10-3621-10 1.0g




P N(iPr)2O CNEt

NHAc

O N

N

ODMTO

OMeO

O

O P N(iPr)2O CNEt

OMe

O

N

N

N

HN

DMTO

iPr-PhOAcHN

OMe

DMTO

CNEtON(iPr)2PO

O

NHAcOPh

N

N

N

N

2’-OMe-Pac-A 2’-OMe-Ac-C 2’-OMe-iPr-Pac-G


139

RNA

AND

2’-O

Me-

RNA

2’-OME-RNA SUPPORTS

ABI-stylecolumnsaresuppliedfor1µmoleand0.2µmolescalesunlessotherwiserequested(seenotebox).


2’-OMe-A-RNA-CPG 20-3600-01 0.1g 20-3600-02 0.25g 20-3600-10 1.0g 1µmolecolumns 20-3700-41 Packof4 0.2µmolecolumns 20-3700-42 Packof4 10µmolecolumn(ABI) 20-3700-13 Packof1 15µmolecolumn(Expedite) 20-3700-14 Packof1

2’-OMe-C-RNA-CPG 20-3610-01 0.1g 20-3610-02 0.25g 20-3610-10 1.0g 1µmolecolumns 20-3710-41 Packof4 0.2µmolecolumns 20-3710-42 Packof4 10µmolecolumn(ABI) 20-3710-13 Packof1 15µmolecolumn(Expedite) 20-3710-14 Packof1

2’-OMe-Ac-C-RNA-CPG 20-3615-01 0.1g 20-3615-02 0.25g 20-3615-10 1.0g 1µmolecolumns 20-3715-41 Packof4 0.2µmolecolumns 20-3715-42 Packof4 10µmolecolumn(ABI) 20-3715-13 Packof1 15µmolecolumn(Expedite) 20-3715-14 Packof1

2’-OMe-G-RNA-CPG 20-3621-01 0.1g 20-3621-02 0.25g 20-3621-10 1.0g 1µmolecolumns 20-3721-41 Packof4 0.2µmolecolumns 20-3721-42 Packof4 10µmolecolumn(ABI) 20-3721-13 Packof1 15µmolecolumn(Expedite) 20-3721-14 Packof1

2’-OMe-U-RNA-CPG 20-3630-01 0.1g 20-3630-02 0.25g 20-3630-10 1.0g 1µmolecolumns 20-3730-41 Packof4 0.2µmolecolumns 20-3730-42 Packof4 10µmolecolumn(ABI) 20-3730-13 Packof1 15µmolecolumn(Expedite) 20-3730-14 Packof1




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140

MINOR 2’-OME-RNA PHOSPHORAMIDITES

Toaid in theevaluationof the structuresof2’-OMe-RNAcomplexes,weoffer theCEphosphoramidites listedbelow.2’-OMe-Tisusefulintriplexstudieswhilethe2-aminopurinederivativemaybetestedinribozymestudies.Bysupportinganadditionalhydrogenbond,2,6-diaminopurine(2-amino-adenosine)bindsmorestronglywithuridinethandoesadenosine.Oligonucleotidescontaining2’-OMe-5-Me-Cand2’-OMe-Iwouldbeofinteresttoresearchersinvolvedintriplexandantisensestudiesusing2’-OMe-RNA.Theusesof2’-OMe-5-bromo-Uphosphoramiditerangefromcrystallographicstudiesduetotheheavyatomtocross-linkingbecauseofitsphotolability.5-Fluoro-pyrimidinenucleosideshavebeenusefulastherapeuticagentsandtheireffectonthestructureandactivityofoligonucleotidesmaybeexaminedusingthe2’-OMe-RNAderivatives.

ABI-stylevialsaresuppliedunlessotherwiserequested(seenotebox).


2’-OMe-2-Aminopurine-CEPhosphoramidite 10-3123 Discontinued

2’-OMe-2,6-Diaminopurine- 10-3124-95 50µmoleCEPhosphoramidite 10-3124-90 100µmole (2-amino-A) 10-3124-02 0.25g

2’-OMe-5-Me-U-CEPhosphoramidite 10-3131-90 100µmole (2’-OMe-T) 10-3131-02 0.25g

2’-OMe-I-CEPhosphoramidite 10-3140-90 100µmole 10-3140-02 0.25g

2’-OMe-5-Me-C-CEPhosphoramidite 10-3160-90 100µmole 10-3160-02 0.25g

2’-OMe-5-Br-U-CEPhosphoramidite 10-3190-90 100µmole 10-3190-02 0.25g

2’-OMe-5-F-U-CEPhosphoramidite 10-3132 Discontinued

2’-OMe-5-Me-U2’-OMe-2-amino-A


O

O

P N(iPr)2O CNEt

DMTO

OMe

iBuHN N

N

N

N

NNMe 2

P N(iPr)2O CNEt

ODMTO

OMeO

O

O

N

HNCH 3

2’-OMe-5-Br-U2’-OMe-5-Me-C2’-OMe-I 2’-OMe-TMP-5-F-U 2’-OMe-3-deaza-5-aza-C

O

O

P N(iPr)2O CNEt

DMTO

OMe

O

N

N

N

HN

ODMTON

N

NHAc

O

O

CH3

P N(iPr)2

O CNEt

OMe

O

OP N(iPr)2O CNEt

DMTO

Br

O

O

N

HN

OMe

O N

NF

O

P N(iPr)2O CNEt

O

O

DMTO

OMe

ODMTO

O

CNEtON(iPr)2P

OMe

NMe 2N

O

N

N




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141

RNA

AND

2’-O

Me-

RNA


2’-OME-THIOPHOSPHORAMIDITES

Thephosphorodithioatelinkage(PS2)isbothachiralandessentiallyresistanttonucleases.PreviousstudieshaveshownveryinterestingresultswhichincludeobservationsthatDNAwithPS2linkagesactivatesRNaseHin vitro,stronglyinhibitshumanimmunodeficiencyvirus(HIV)reversetranscriptase,inducesB-cellproliferationanddifferentiation,andiscompletelyresistanttohydrolysisbyvariousnucleases.2’-OMe-RNAThiophosphoramiditesareRNAmonomersdesignedtoproduceoligoscombiningthePS2linkagewiththe2’-O-methylribosemodification.ThesePS2-modifiedRNAoligoshavepotentialforuseinsiRNAsanddithiophosphateaptamers(thioaptamers).


2’-OMe-A-Thiophosphoramidite 10-3170-90 100µmole 10-3170-02 0.25g

2’-OMe-C-Thiophosphoramidite 10-3171-90 100µmole 10-3171-02 0.25g

2’-OMe-G-Thiophosphoramidite 10-3172-90 100µmole 10-3172-02 0.25g

2’-OMe-U-Thiophosphoramidite 10-3173-90 100µmole 10-3173-02 0.25g




Expedite EMerMade M




N

N N

N

NHBz

ODMTO

O

PN SCH2CH2S C

O

COCH3

OMe

ODMTO

O

PN SCH2CH2S C

O

PN SCH2CH2S COCH3

N

N

NHAc

O

OMe

ODMTO

O

PN SCH2CH2S C

O

HN

N

N

O

NiBuHN

OMe

ODMTO

O

PN SCH2CH2S C

O

N

HN

O

O

OMe

2'-OMe-C-Thiophosphoramidite2'-OMe-A-Thiophosphoramidite 2'-OMe-U-Thiophosphoramidite2'-OMe-G-Thiophosphoramidite

142

2’-MOE-RNA PHOSPHORMIDITES

2’-MOE RNA PHOSPHORAMIDITES

Liketheverysimilar2’-OMebackbone,the2’-O-methoxyethyl-RNA(2’-MOE)backboneprovidesenhancedduplexstability,significantnucleaseresistanceandrelativelylowtoxicity.Asaresult,2’-MOEhasbeenanattractivebackboneformanytherapeuticcandidates, severalofwhichhavebeenapprovedby theFDA.Thesedrugshave included1)2’-MOE/DNAchimerastofacilitateRNaseHcleavageoftargetRNAsequencesaswellas2)stericblockingoligonucleotidestoalterthesplicingofmRNA.Thestandard2’-MOEnucleotidesareA,5-Me-C,Gand5-Me-U.

ABI-stylevialsaresuppliedunlessotherwiserequested(seenotebox).


A-2'-MOE-Phosphoramidite 10-3200-05 0.5g 10-3200-10 1.0g 10-3200-20 2.0g

5-Me-C-2'-MOE-Phosphoramidite 10-3211-05 0.5g 10-3211-10 1.0g 10-3211-20 2.0g

G-2'-MOE-Phosphoramidite 10-3220-05 0.5g 10-3220-10 1.0g 10-3220-20 2.0g

5-Me-U-2'-MOE-Phosphoramidite 10-3231-05 0.5g 10-3231-10 1.0g 10-3231-20 2.0g

5-Me-C-2'-MOEA-2'-MOE 5-Me-U-2'-MOEG-2'-MOE




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143

RNA

AND

2’-O

Me-

RNA

2’-F-RNA PHOSPHORAMIDITES

2’-Deoxy-2’-fluoro-nucleosidesadoptanRNA-typesugarconformation,presumablyduetothehighelectronegativityoffluorine.Becauseofthissugarconformation,RNAduplexes(A-form)aregenerallymorethermodynamicallystablethanDNAduplexes(B-form).Asexpected,theadditionof2’-F-RNAresiduestooligodeoxynucleotidesprogressivelyincreasesthethermalstabilityoftheirduplexeswithRNA.Thestabilizationisadditiveatapproximately2°perresidue.Thiscomparesfavorablywith2’-OMe-RNAataround1.5°andRNAat1.1°perresidue.Inthemeantime,basepairspecificityremainsintact.

2’-F-RNAphosphodiesterlinkagesarenotnucleaseresistant,althoughthecorrespondingphosphorothioatelinkagesarehighlyresistant.ResearchersusuallydesignantisenseoligonucleotidestoformduplexeswithRNA,whicharethensubstratesforRNaseH.Uniformlymodified2’-F-RNA/RNAduplexesarenotsubstratesforRNaseH.However,itisstraightforwardtopreparechimeric2’-F-RNA/DNAphosphorothioateoligonucleotideswhichexhibitenhancedbindingtotheRNAtarget,aresubstratesforRNaseH,andarehighlynucleaseresistant.


2'-F-A-CEPhosphoramidite 10-3400-02 0.25g 10-3400-05 0.5g

2'-F-Ac-C-CEPhosphoramidite 10-3415-02 0.25g 10-3415-05 0.5g

2'-F-G-CEPhosphoramidite 10-3420-02 0.25g 10-3420-05 0.5g

2'-F-U-CEPhosphoramidite 10-3430-02 0.25g 10-3430-05 0.5g

2'-F-I-CEPhosphoramidite 10-3440-90 100µmole 10-3440-02 0.25g

2’-F-U 2’-F-I

2’-F RNA SYNTHESIS

O

O P N(iPr)2O CNEt

DMTO

F

NHAc

O N

N

O

O P N(iPr)2O CNEt

DMTO

F

O

O

N

HNN

N

N

N

NHBz

O

O P N(iPr)2O CNEt

DMTO

F

HN

N

N

N

O

iBuHN

F

DMTO

CNEtON(iPr)2PO

O

2’-F-G2’-F-Ac-C2’-F-A

STABILITY NOTE

Syntheticoligonucleotidescontaining2’-F-RNAlinkagesmaybedeprotectedwithammoniumhydroxideasnormal.DeprotectionusingAMAat65°Cleadstosomedegradationandsowerecommendthe use of AMA at room temperaturefor2hours.




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144

2’-F ANA SYNTHESIS

2’-F-ARABINONUCLEIC ACID (2’-F-ANA)

Arabinonucleosidesareepimersofribonucleosideswiththechiralswitchbeingatthe2’positionofthesugarresidue.2’-F-ANAadoptsamoreDNA-likeB-typehelixconformation,notthroughthetypicalC2’-endoconformationbut,rather,throughanunusualO4’-endo(east)pucker.However,thepresenceoftheelectronegativefluorineleadstoastillsignificantincrease (DTm1.2°C/mod)inmeltingtemperaturepermodification.12’-F-ANA-containingoligonucleotidesexhibitveryhighbindingspecificitytotheirtargets.Indeed,asinglemismatchina2’-F-ANA–RNAduplexleadstoaDTmof-7.2°Candina2’-F-ANA-DNAduplexaDTmof-3.9°C.

2

Thepresenceoffluorineatthe2’positionin2’-F-ANAleadstoincreasedstabilitytohydrolysisunderbasicconditionsrelativetoRNAandeven2’-F-RNA.1,3Thestabilityof2’-F-ANAtonucleasesalsomakesthisausefulmodificationforenhancingthestabilityofoligonucleotidesinbiologicalenvironments.22’-F-ANAhybridizesstronglytotargetRNAand,unlikemost2’modifications,inducescleavageofthetargetbyRNaseH.Phosphorothioate(PS)2’-F-ANAisroutinelyusedintheseapplicationsdueto its increasednucleaseresistance. Alternating2’-F-ANAandDNAunitsprovideamongthehighestpotencyRNaseH-activatingoligomers. Boththe“altimer”and“gapmer”strandarchitecturesconsistentlyoutperformPS-DNAandDNA/RNAgapmers.4

siRNAoligoswere found to tolerate thepresenceof 2’-F-ANA linkages verywell. Highpotency gene silencingwasdemonstrated5withsiRNAchimerascontaining2’-F-RNAand/orLNAand2’-F-ANA.ThehighefficacyofthesechimeraswasattributedtothecombinationoftherigidRNA-likepropertiesof2’-F-RNAandLNAwiththeDNA-likepropertiesof2’-F-ANA.


2’-F-A-ANACEPhosphoramidite 10-3800-90 100µmole 10-3800-02 0.25g

2’-F-Bz-C-ANACEPhosphoramidite 10-3810 Discontinued

2’-F-Ac-C-ANACEPhosphoramidite 10-3815-02 0.25g 10-3815-05 0.5g

2’-F-G-ANACEPhosphoramidite 10-3820-90 100µmole 10-3820-02 0.25g

2’-F-U-ANACEPhosphoramidite 10-3830-02 0.25g 10-3830-05 0.5g

2’-F-Me-U-ANACEPhosphoramidite 10-3850-02 0.25g 10-3850-05 0.5g

ODMTO

N

HN

O

O

O P N(iPr)2

O CNEt

F

N

N N

N

NHBz

O

O P N(iPr)2

O

F

CNEt

DMTO ODMTON

N

NHBz

O

O P N(iPr)2

O CNEt

F

HN

N

N

O

N

ODMTO

O P N(iPr)2

O CNEt

iBuHN

F

REFERENCES

1.E.Viazovkina,M.M.Mangos,M.I.Elzagheid,andM.J.Damha,Curr Protoc Nucleic Acid Chem, 2002, Chapter 4,Unit415.

2.J.K.Watts,andM.J.Damha,Can. J. Chem., 2008, 86,641-656.

3.J.K.Watts,A.Katolik,J.Viladoms,andM.J.Damha,Org Biomol Chem, 2009, 7,1904-10.

4.A.Kalota,et al., Nucleic Acids Res., 2006, 34,451.

5.G.F.Deleavey, et al., Nucleic Acids Res., 2010, 38,4547-4557,J.K.Watts,et al., Nucleic Acids Res., 2007, 35,1441-1451,T.Dowler, et al., Nucleic Acids Res., 2006, 34, 1669-1675.

STABILITY NOTE

Syntheticoligonucleotidescontaining2’-F-RNAlinkagesmaybedeprotectedwithammoniumhydroxideasnormal.DeprotectionusingAMAat65°Cleadstosomedegradationandsowerecommendthe use of AMA at room temperaturefor2hours.


2’-F-ANAiscoveredbyintellectualproperty.KeypatentscoveringsiRNAandantisenseapplicationsareasfollows:

WO/2009/146556(siRNA);WO03064441 and WO 0220773 (antisense).

2'-F-A-ANA 2'-F-U-ANA 2'-F-Me-U-ANA 2'-F-G-ANA 2'-F-Bz-C-ANA 2'-F-Ac-C-ANA

ODMTON

N

NHAc

O

O P N(iPr)2

O CNEt

F

Chemical Formula: C41H49FN5O8PExact Mass: 789.33

Molecular Weight: 789.83m/z: 789.33 (100.0%), 790.33 (46.5%), 791.34 (10.0%), 791.33 (2.5%), 792.34 (2.2%)

Elemental Analysis: C, 62.35; H, 6.25; F, 2.41; N, 8.87; O, 16.21; P, 3.92

RELATED

DNA PACE...................................37DCI..............................................30UniCap.......................................320.5MCSO...................................32

145

RNA

AND

2’-O

Me-

RNA


Theseproductsarecoveredbypatents, US 6,693,187 and 7,067,641, andpatentspendingownedbyMetasenseTechnologies.Purchaseofalloranyoftheseproductsincludes a limited license to use the productssolelyforthemanufactureofoligonucleotidesforresearchuseonly.Thislicensespecificallyexcludestheuseoftheproductoroligonucleotidescontainingtheproductfor:(a)therapeuticordiagnosticapplications(includingkits,pools,librariesandotherproducts or services that incorporate oligonucleotidescontainingtheproduct),(b)anyin vivotoxicity/safetystudyinsupportofaninvestigationalnewdrugapplication(orforeigncounterpart),or(c)resale (including sale of kits, pools, librariesandotherproductsorservices that incorporate the product oroligonucleotidescontainingtheproduct).Ifsuchactivitieshavecommercialapplication,aseparatelicenseisrequiredfromMetasenseTechnologies.Neithertheproductnoranyproductcreatedthroughitsusemaybeusedinhumanclinicaltrials.

Asimpleagreementmustbesignedbeforeend-usersandcustomoligoservicesmaypurchasetheseproductsforuseasdefinedabove.

https://www.glenresearch.com/media/productattach/import/technical_note/PACE.pdf

2’-OME-RNA-PACE SYNTHESIS

REFERENCES

1. A.Hendel, et al., Nat Biotechnol, 2015, 33,985-989.

2. D.E.Ryan, et al., Nucleic Acids Res, 2018, 46,792-803.

N

N N

N

NHBz

ODMTO

O P N(iPr)2

OCN

O

OMe

ODMTO

O P N(iPr)2

OCN

O

OMe

N

N

NHAc

O

ODMTO

O P N(iPr)2

OCN

O

OMe

HN

N

N

O

NiBuHN

ODMTO

O P N(iPr)2

OCN

O

OMe

N

HN

O

O

2’-OMe-A-PACE 2’-OMe-U-PACE2’-OMe-Ac-C-PACE 2’-OMe-G-PACE

2’-OME-RNA-PACE PHOSPHORAMIDITES

PACEmodifications have enjoyed a resurgence in interest as applied to the field of CRISPR gene editing. In aninitial publication, itwas shown that single guideRNAs (sgRNA)provided significantly higher activity in cellswhen2‘-O-methylthiophosphonoacetateswereincorporatedontheendsoftheguideRNAtoprotectagainstcellularnucleases.1 Insubsequentstudies,2’-OMePACEmodifiedsgRNAswerealsoshowntosignificantlyincreaseon-targetspecificityoftheCRISPR-Cas9DNAcleavageineukaryoticcells.Inarecentpaper,theincorporationof2’-OMePACEmodifiednucleotidesinthe20-nucleotideguideregionofthesgRNAwasshowntodecreaseoff-targetcuttingbyoveranorderofmagnitudewhileinmostcasesincreasingtheoverallon-targetefficiencyascomparedtounmodifiedsingleguideRNA.2

Asanoptimalcycle,werecommendusingDCIasanactivator(30-3150-XX)anda15minutecouplingtime.Followingcoupling,capusingUnicap(10-4410-XX)witharegularcouplingtimeandthenoxidizeusing0.5MCSOfor3minutes.Alternatively,a33minutecouplingtimeusing0.45Mtetrazole,oxidationusinglow-wateriodine(40-4032-XX)followedbycappingwith6.5%DMAPasCapBwillgiveacceptableresults.Fordeprotection,pre-treatthesynthesiscolumnwith1.5%DBUinanhydrousacetonitrilefor60minutesatroomtemperaturetoremove1,1-dimethyl-2-cyanoethylprotectinggroups.Rinsethecolumnwithacetonitrile,dryunderargonandcompletethedeprotectionwith40%aqueousmethylaminefor2hoursatroomtemperature.


2’-OMe-A-PACEPhosphoramidite 10-3150-02 0.25g 10-3150-05 0.5g 10-3150-10 1.0g

2’-OMe-Ac-C-PACEPhosphoramidite 10-3151-02 0.25g 10-3151-05 0.5g 10-3151-10 1.0g

2’-OMe-G-PACEPhosphoramidite 10-3152-02 0.25g 10-3152-05 0.5g 10-3152-10 1.0g

2’-OMe-U-PACEPhosphoramidite 10-3153-02 0.25g 10-3153-05 0.5g 10-3153-10 1.0g

146

NOTES

RELATED

Poly-PakReagents...................149

147

MIS

CELL

ANEO

US

GLEN-PAK™ PURIFICATION

Glen-Pak™DNAandRNAcartridgeshaveadvantagesoverPoly-Pakcartridgesinthatasingleloadingofthedilutedcrudedeprotectionsolutionisallthatisnecessary.Also,therangeofpurificationhasbeenextendedto100+usingDMT-ONoligos.Inaddition,Glen-Pakcartridgesallowpurificationofvirtuallythecompleterangeofdyesandmodifiers.

TheGlen-PakDNACartridge3gisalargecartridgecapableofpurifying10-20µmoleoligonucleotidesynthesesusingthestandardDMT-ONprocedureandGlen-PakDNA30mg96-WellPlatesareforparallelpurificationofupto50nmolescalesyntheses.TheGlen-PakDNA3mg384-WellPlateisdesignedforusewith384-wellplatecompatiblevacuummanifoldsystemsandcanpurifyuptoa20nmolescalesynthesis.Eachwellcontains3mgofGlen-PakDNAresin,whichbindsabout15nmolesoffulllength40-merDMT-ONoligo.

ScalesuggestionsfortheGlen-PakDNAproductlineareshownbelow:

Glen-Pak DNA Product Catalog Number Synthesis Scale Compatibility

Glen-PakDNA50mgPurificationCartridge 60-5000-96 10nmole–200nmoleGlen-PakDNAPurificationCartridge 60-5100-XXand60-5200-XX 10nmole–1.0µmoleGlen-PakDNACartridge3G 60-5300-01 5µmole–20µmoleGlen-PakDNA30mg96-WellPlate 60-5400-01 10nmole–50nmoleGlen-PakDNA3mg384-WellPlate 60-5500-XX Upto20nmole

A User Guide to Glen-Pak™ PurificationdescribesindetailtheprocessandseveralapplicationsforDNAandRNApurification.Thisbookletisavailableonlineat:https://www.glenresearch.com/media/productattach/g/l/glen-pak_2.9_1.pdf.


DNA Purification CartridgesGlen-Pak™50mgDNAPurificationCartridge 60-5000-96 Packof96 (For use in vacuum manifolds andhigh-throughputdevices)

Glen-Pak™DNAPurificationCartridge 60-5100-10 Packof10 (Foruseinvacuummanifolds 60-5100-30 Packof30 andhigh-throughputdevices) 60-5100-96 Packof96

Glen-Pak™DNAPurificationCartridge 60-5200-01 Packof1 (Forusewithdisposablesyringes) 60-5200-10 Packof10

Glen-Pak™DNACartridge3g 60-5300-01 Packof1

Glen-Pak™DNA30mg96-WellPlate 60-5400-01 Packof1

Glen-Pak™DNA3mg384-WellPlate 60-5500-01 Packof1 60-5500-10 Packof10

PURIFICATION

Poly-Pak™andGlen-Pak™aretrademarksofGlenResearchCorporation

https://www.glenresearch.com/media/productattach/g/l/glen-pak_2.9_1.pdf

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GLEN-PAK™ PURIFICATION (CONT.)


RNA Purification CartridgesGlen-Pak™RNAPurificationCartridge 60-6100-10 Packof10 (Foruseinvacuummanifolds 60-6100-30 Packof30 andhigh-throughputdevices) 60-6100-96 Packof96

Glen-Pak™RNAPurificationCartridge 60-6200-01 Packof1 (Forusewithdisposablesyringes) 60-6200-10 Packof10

ReagentsRNAQuenchingBuffer 60-4120-82 250mL 60-4120-80 1L

Racks and Seals AdapterRack 60-0010-01 each (Forusewith96wellmanifolds)SealforAdapterRack 60-0020-01 each (Foruseon96welladapterrack)

PURIFICATION

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Poly-Pak Cartridge Used Manually

POLY-PAK™ PURIFICATION

TheuseofPoly-Pak™packingsincartridgesorbarrelsovercomesseveraldisadvantagesusuallyassociatedwithreversephase(RP)cartridges.ThepackingisstableinthepHrange1-13,thustheammoniumhydroxidesolution,dilutedwithwater,isloadeddirectlyontothepacking.Also,afterelutionoffailuresequences,thetritylgroupisremovedandwashedfromthesupport-boundoligonucleotide.Thefullydeprotectedproductcanthenbeelutedandisolatedbylyophilization.Poly-Pak™Cartridgesmayalsobeusedfordesaltingnormalorlabeledoligonucleotides.TheoriginalPoly-Pakcartridgeandbarrelaredesignedfor0.2µmolesynthesesorless.Poly-PakIIcartridgesandbarrelsaredesignedforusewith1µmolesyntheses.Abooklet,UserGuideToPoly-Pak™CartridgePurification,describesindetailtheprocessandseveralapplications. Thisbookletisavailableonlineat:https://www.glenresearch.com/media/productattach/import/tbn/PolyPakBooklet.pdf


Packing, Cartridges and Barrels

Poly-Pak™Packing 60-1000-05 5g 60-1000-25 25g

Poly-Pak™Cartridge 60-1100-01 Packof1 60-1100-10 Packof10

Poly-Pak™IICartridge 60-3100-01 Packof1 60-3100-10 Packof10

Reagents

2.0MTriethylamineAcetate(TEAA) 60-4110-52 200mLHPLCGrade 60-4110-57 450mL 60-4110-60 960mL 60-4110-62 2L 2%AqueousTrifluoroaceticAcid 60-4040-57 450mL

PURIFICATION

Poly-Pak™isatrademarkofGlenResearchCorporation

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PURIFICATION

GLEN GEL-PAK™ DESALTING

TheprincipleoftheGlenResearchgelfiltrationcolumn,GlenGel-Pak™,isbasedonsizeexclusionchromatographythatseparatesmoleculesaccordingtothehydrodynamicvolumeofthemoleculeinaqueoussolutions.Ingelfiltration,themobilephaseforsizeexclusionisanaqueoussolutionandthestationaryphaseisaporousresin.Theporesoftheresinaresizedsuchthattheyallowsmallmoleculestoenterthepores,yetexcludelargermoleculesfromthepores.Thesmallmolecules,suchassaltsandhydrolyzedprotectinggroups,diffuseintotheporesoftheresinandmoveslowlythroughthecolumn.Thelargermolecules,suchasDNAorproteins,areexcludedfromtheporesandmovequicklythroughthecolumn.Theendresultisthatthelargermoleculeselutefirstinthecolumnvoidvolumewhilethesmallmoleculesarestillflowingthroughtheresinofthecolumn.

GlenGel-Pakcolumnsare ideal fordesaltingandreactioncleanup. Theycanbeusedforremovalof theammoniumhydroxidedeprotectionsolutionandhydrolyzedprotectinggroupsafterdeprotection.ThecolumnscanalsobeusedforthecleanupofNHS-labelingreactionstoseparatethelabeledoligoandunlabeledoligofromtheunreactedNHSester,thehydrolyzedlabel,andn-hydroxysuccinimide,therebygreatlysimplifyingthedownstreampurificationsteps.

TherearemanybenefitstoGlenGel-Pakcolumns:

Versatility:

• Ability to directly desalt oligonucleotides deprotected ineither 30% ammonium hydroxideOR 50:50 ammoniumhydroxide/40%aqueousmethylamine(AMA)

• Easilyexchangebuffers• Simpleclean-upoflabelingreactions• Mildmethodforpurificationfromsaltsandsolventssuchas

DMSO and DMF

Capacity:

• Multiplecolumnsizes(0.2mL,1.0mLand2.5mL)areavailabletomatchsynthesisscale• Abilitytoefficientlydesaltshortandlongoligosatdifferentscalesusingthesameprotocol• Suitableforoligos>10merinlength


GlenGel-Pak™0.2DesaltingColumn 61-5002-05 Packof5 (0.2mLCapacity) 61-5002-50 Packof50GlenGel-Pak™1.0DesaltingColumn 61-5010-05 Packof5 (1.0mLCapacity) 61-5010-50 Packof50GlenGel-Pak™2.5DesaltingColumn 61-5025-05 Packof5 (2.5mLCapacity) 61-5025-25 Packof25

Glen Gel-Pak 0.2 Glen Gel-Pak 2.5 Glen Gel-Pak 1.0

GlenGel-Pak™isatrademarkofGlenResearchCorporation

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PURIFICATION

OLIGO-AFFINITY SUPPORT

Oligo-affinitysupports(OAS)shouldideallybecompatiblewithautomatedsynthesis,shouldbenon-friable,shouldnotshrinkorswell,andshouldhavelownon-specificbindingoftheproteinsorDNA.OnthesupportshownbelowisanAdenosineresidueattachedthroughtheexocyclicaminogroup.Inthisway,synthesisprogressesregularlyonremovalofthe5’-DMTgroup.However,ontreatmentwithammoniumhydroxide,theoligoisnotcleavedfromthesupport.ThismatrixcanthenbeusedasanaffinitysupportforacomplementarysegmentofDNAorRNA.Alternatively,thecomplementarystrandcanbeannealedtothesupportandthedoublestrandedDNAcanbeusedasanaffinitysupportforpurifyingDNAbindingproteins.

WeexpectthatOASPSwillbeusedforpurificationofcomponentsfrombiologicalfluids.


Oligo-AffinitySupport(PS) 26-4001-01 0.1g (OASPS) 26-4001-02 0.25g 26-4001-10 1.0g

Oligo-AffinitySupport(PS) 1µmoleTWISTcolumns 26-4101-41 Packof4

OAS PS

O

ODMTO

OAc OAc

NH

N

N

N

N

PS




Expedite EMerMade M




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PHYSICAL DATA

PHYSICAL DATA

Thephysicaldatatablecontainsinformationwhichisuniquetoeachmonomerphosphoramidite.Themolecularweight(MW)istheformulaweightofthefully-protectedmonomerphosphoramidite.TheMWisusedtocalculatethevolumeofsolventrequiredtodilute0.25gofthemonomertogiveafinal0.1Mconcentration.Thisfigureisalsoshowninthetable.Theunitmolecularweight(UnitFW)istheformulaweightofeachmonomeronceinsertedintoanoligonucleotidewithallprotectinggroupsremoved.Toobtainthemolecularweightofaspecificoligonucleotide,thefollowingformulaisused:OligonucleotideMW=SumofUnitFW-61.96

Cat. No. Item Phosphoramidite MW Unit FW Dilution (0.1M)

10-0001 dA-5’-CEPhosphoramidite 857.95 313.21 0.25g/2.91mL10-0101 dC-5’-CEPhosphoramidite 833.93 289.18 0.25g/3.00mL10-0301 dT-5’-CEPhosphoramidite 744.83 304.2 0.25g/3.36mL10-1000 dA-CEPhosphoramidite 857.95 313.21 0.25g/2.91mL10-1001 7-Deaza-dA-CEPhosphoramidite 856.96 312.22 0.25g/2.92mL10-1003 N6-Me-dA-CEPhosphoramidite 767.86 327.24 0.25g/3.26mL10-1004 3’-dA-CEPhosphoramidite 857.95 313.21 0.25g/2.91mL10-1006 Etheno-dA-CEPhosphoramidite 777.86 337.23 0.25g/3.21mL10-1007 8-Br-dA-CEPhosphoramidite 887.81 392.11 0.25g/2.82mL10-1008 8-oxo-dA-CEPhosphoramidite 873.95 329.21 0.25g/2.86mL10-1010 dC-CEPhosphoramidite 833.93 289.18 0.25g/3.00mL10-1014 pdC-CEPhosphoramidite 907.1 327.23 0.25g/2.76mL10-1015 Ac-dC-CEPhosphoramidite 771.85 289.18 0.25g/3.24mL10-1016 TMP-F-dU-CEPhosphoramidite 866.97 307.18 0.25g/2.88mL10-1017 Pyrrolo-dC-CEPhosphoramidite 767.85 327.23 0.25g/3.26mL10-1018 5-Me-dCBrancherPhosphoramidite 942.1 402.36 0.25g/2.65mL10-1019 Amino-ModifierC6dC 1049.14 457.42 0.25g/2.38mL10-1020 dG-CEPhosphoramidite 839.92 329.21 0.25g/2.98mL10-1021 7-deaza-dG-CEPhosphoramidite 823.93 328.22 0.25g/3.03mL10-1027 8-Br-dG-CEPhosphoramidite 903.9 408.1 0.25g/2.77mL10-1028 8-oxo-dG-CEPhosphoramidite 855.93 345.21 0.25g/2.92mL10-1029 dmf-dG-CEPhosphoramidite 824.92 329.21 0.25g/3.03mL10-1030 dT-CEPhosphoramidite 744.83 304.2 0.25g/3.36mL10-1031 5’-OMe-dT-CEPhosphoramidite 456.48 318.22 0.25g/5.48mL10-1032 O4-Me-dT-CEPhosphoramidite 758.85 318.22 0.25g/3.29mL10-1034 4-Thio-dT-CEPhosphoramidite 813.95 320.26 0.25g/3.07mL10-1035 Carboxy-dT 814.88 360.22 0.25g/3.07mL10-1036 2-Thio-dT-CEPhosphoramidite 879.02 320.26 0.25g/2.84mL10-1037 Amino-ModifierC2dT 938.94 402.3 0.25g/2.66mL10-1038 Biotin-dT 1285.55 684.7 0.25g/1.94mL10-1039 Amino-ModifierC6dT 995.05 458.41 0.25g/2.51mL10-1040 dI-CEPhosphoramidite 754.79 314.19 0.25g/3.31mL10-1041 2’-DeoxyNebularine-CEPhosphoramidite(Purine) 738.82 298.19 0.25g/3.38mL10-1042 O6-Phenyl-dI-CEPhosphoramidite 830.92 Varies 0.25g/3.01mL10-1044 5-Nitroindole-CEPhosphoramidite 780.86 340.23 0.25g/3.20mL10-1046 2-Aminopurine-CEPhosphoramidite 809.01 313.21 0.25g/3.09mL10-1047 dP-CEPhosphoramidite 771.85 330.23 0.25g/3.24mL10-1048 dK-CEPhosphoramidite 853.96 358.25 0.25g/2.93mL10-1050 dU-CEPhosphoramidite 730.8 290.17 0.25g/3.42mL10-1051 O4-Triazolyl-dU-CEPhosphoramidite 781.84 varies 0.25g/3.20mL10-1052 4-Thio-dU-CEPhosphoramidite 799.93 306.23 0.25g/3.13mL10-1053 5-OH-dU-CEPhosphoramidite 788.83 306.17 0.25g/3.17mL10-1054 pdU-CEPhosphoramidite 768.85 328.22 0.25g/3.25mL

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10-1055 2’-deoxypseudoU-CEPhosphoramidite 730.8 290.17 0.25g/3.42mL10-1056 Fluorescein-dTPhosphoramidite 1425.57 815.71 0.25g/1.75mL10-1057 TAMRA-dT 1311.48 870.85 0.25g/1.91mL10-1058 Dabcyl-dT 1150.32 709.7 0.25g/2.17mL10-1059 EDTA-C2-dT-CEPhosphoramidite 1201.32 676.53 0.25g/2.08mL10-1060 5-Me-dC-CEPhosphoramidite 847.9 303.21 0.25g/2.95mL10-1061 5-Me-2’-deoxyZebularine-CEPhosphoramidite 728.82 288.19 0.25g/3.43mL10-1062 5-Hydroxymethyl-dC-CEPhosphoramidite 917 319.21 0.25g/2.73mL10-1063 5-OH-dC-CEPhosphoramidite 954.03 305.18 0.25g/2.62mL10-1064 3’-dC-CEPhosphoramidite 833.92 289.18 0.25g/3.00mL10-1065 dmf-5-Me-isodC-CEPhosphoramidite 798.91 303.21 0.25g/3.13mL10-1066 5-Carboxy-dC-CEPhosphoramidite 905.97 333.19 0.25g/2.76mL10-1068 N4-Et-dC-CEPhosphoramidite 757.87 317.42 0.25g/3.30mL10-1070 O6-Me-dG-CEPhosphoramidite 853.97 343.24 0.25g/2.93mL10-1072 6-thio-dG-CEPhosphoramidite 934.97 345.26 0.25g/2.67mL10-1073 7-Deaza-8-aza-dG-CEPhosphoramidite(PPG) 824.91 329.2 0.25g/3.03mL10-1074 3’-dG-CEPhosphoramidite 824.92 329.21 0.25g/3.03mL10-1076 7-deaza-dX-CEPhosphoramidite 769.83 329.21 0.25g/3.25mL10-1078 dmf-isodG-CEPhosphoramidite 1020.13 329.21 0.25g/2.45mL10-1079 8-Amino-dG-CEPhosphoramidite 895.01 344.22 0.25g/2.79mL10-1080 5-Br-dC-CEPhosphoramidite 912.82 368.08 0.25g/2.74mL10-1081 5-I-dC-CEPhosphoramidite 959.83 415.08 0.25g/2.60mL10-1082 2-F-dI-CEPhosphoramidite 921.96 varies,2F=332.18 0.25g/2.71mL10-1083 7-deaza-8-aza-dA-CEPhosphoramidite 808.91 313.2 0.25g/3.09mL10-1084 3’-dT-CEPhosphoramidite 744.83 304.2 0.25g/3.36mL10-1085 2-Amino-dA-CEPhosphoramidite 1047.33 328.22 0.25g/2.39mL10-1086 8-Amino-dA-CEPhosphoramidite 879.01 328.22 0.25g/2.84mL10-1088 3-deaza-dA-CEPhosphoramidite 856.95 312.22 0.25g/2.92mL10-1089 Amino-ModifierC6dA 1068.14 427.4 0.25g/2.34mL10-1090 5-Br-dU-CEPhosphoramidite 809.69 369.07 0.25g/3.09mL10-1091 5-I-dU-CEPhosphoramidite 856.69 416.07 0.25g/2.92mL10-1092 5-F-dU-CEPhosphoramidite 748.79 308.16 0.25g/3.34mL10-1093 5-Hydroxymethyl-dU-CEPhosphoramidite 802.86 320.19 0.25g/3.11mL10-1096 ThymidineGlycolCEPhosphoramidite 1007.36 338.21 0.25g/2.48mL10-1097 AP-dC-CEPhosphoramidite 974.97 438.33 0.25g/2.56mL10-1098 8,5’-Cyclo-dACEPhosphoramidite 855.92 311.19 0.25g/2.92mL10-1100 dA-MePhosphonamidite 802.91 311.24 0.25g/3.11mL10-1115 Ac-dC-MePhosphonamidite 716.81 287.21 0.25g/3.49mL10-1120 dG-MePhosphonamidite 784.89 327.24 0.25g/3.19mL10-1130 dT-MePhosphonamidite 689.79 302.23 0.25g/3.62mL10-1140 dA-PACEPhosphoramidite 928.02 354.24 0.25g/2.69mL10-1150 Ac-dC-PACEPhosphoramidite 841.93 330.21 0.25g/2.97mL10-1160 dG-PACEPhosphoramidite 910.01 370.24 0.25g/2.75mL10-1170 dT-PACEPhosphoramidite 814.9 345.22 0.25g/3.07mL10-1200 dA-H-Phosphonate,TEASalt 822.9 313.21 0.25g/3.04mL10-1210 dC-H-Phosphonate,DBUSalt 849.35 289.18 0.25g/2.94mL10-1220 dG-H-Phosphonate,TEASalt 804.88 329.21 0.25g/3.11mL10-1230 dT-H-Phosphonate,TEASalt 709.78 304.2 0.25g/3.52mL10-1301 Pac-dA-MePhosphoramidite 848.93 327.23(Methyltriester) 0.25g/2.94mL10-1315 Ac-dC-MePhosphoramidite 732.81 303.21(Methyltriester) 0.25g/3.41mL10-1321 iPr-Pac-dG-MePhosphoramidite 907.01 343.23(Methyltriester) 0.25g/2.76mL10-1330 dT-MePhosphoramidite 705.79 318.22(Methyltriester) 0.25g/3.54mL

PHYSICAL DATA

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10-1440 CleanAmp™-Pac-dA-CEPhosphoramidite 1045.25 523.56(triester) 0.25g/2.39mL10-1450 CleanAmp™-Ac-dC-CEPhosphoramidite 929.13 499.54(triester) 0.25g/2.69mL10-1460 CleanAmp™-Pac-dG-CEPhosphoramidite 1061.25 539.56(triester) 0.25g/2.36mL10-1470 CleanAmp™-dT-CEPhosphoramidite 902.11 514.55(triester) 0.25g/2.77mL10-1501 1-Me-dA-CEPhosphoramidite 814.31 328.24 0.25g/3.07mL10-1503 N6-Ac-N6-Me-dA-CEPhosphoramidite 809.89 327.23 0.25g/3.09mL10-1504 def-dA-CEPhosphoramidite 836.97 313.21 0.25g/2.99mL10-1510 5-Hydroxymethyl-dCII-CEPhosphoramidite 785.82 319.21 0.25g/3.18mL10-1511 5-aza-5,6-dihydro-dC-CEPhosphoramidite 787.89 292.18 0.25g/3.17mL10-1513 N4-Ac-N4-Et-dC-CEPhosphoramidite 799.89 317.24 0.25g/3.13mL10-1514 5-Formyl-dC-CEPhosphoramidite 915.96 317.19(formyl) 0.25g/2.73mL 349.23(diol)10-1516 tC-CEPhosphoramidite 835.95 395.33 0.25g/2.99mL10-1517 tCO-CEPhosphoramidite 819.88 379.26 0.25g/3.05mL10-1518 tCnitro-CEPhosphoramidite 880.94 440.32 0.25g/2.84mL10-1527 dW-CEPhosphoramidite 992.30 311.23 0.25g/2.52mL10-1529 N2-Amino-ModifierC6dG 965.01 428.38 0.25g/2.59mL10-1530 5,6-Dihydro-dT-CEPhosphoramidite 746.84 306.21 0.25g/3.35mL10-1531 N3-Cyanoethyl-dT 797.88 357.26 0.25g/3.13mL10-1532 5’-Dabsyl-dT-CEPhosphoramidite 729.78 591.53 0.25g/3.43mL10-1534 N-POMCaged-dT-CEPhosphoramidite 967.99 527.38(N-POM-dT) 0.25g/2.58mL10-1535 NHS-Carboxy-dT 897.91 varies,-CO2H=360.22 0.25g/2.78mL10-1536 FmocAmino-ModifierC6dT 1121.28 458.41(NH2) 0.25g/2.23mL10-1537 dX-CEPhosphoramidite 1069.1 330.19 0.25g/2.34mL10-1538 S-Bz-Thiol-ModifierC6-dT 1091.26 546.53 0.25g/2.29mL10-1539 DBCO-dT-CEPhosphoramidite 1214.57 773.77 0.25g/2.06mL10-1540 C8-Alkyne-dT-CEPhosphoramidite 834.94 394.32 0.25g/2.99mL10-1541 C8-TIPS-Alkyne-dC-CEPhosphoramidite 1094.4 393.33 0.25g/2.28mL10-1542 C8-TMS-Alkyne-dC-CEPhosphoramidite 1010.24 393.33 0.25g/2.47mL10-1543 C8-Alkyne-dC-CEPhosphoramidite 938.06 393.33 0.25g/2.67mL10-1544 C8-TIPS-Alkyne-dT-CEPhosphoramidite 991.28 394.32 0.25g/2.52mL10-1545 C8-TMS-Alkyne-dT-CEPhosphoramidite 907.12 394.32 0.25g/2.76mL10-1550 5,6-Dihydro-dU-CEPhosphoramidite 732.81 292.19 0.25g/3.41mL10-1554 5-Ethynyl-dU-CEPhosphoramidite 754.81 314.19 0.25g/3.31mL10-1555 TIPS-5-Ethynyl-dU-CEPhosphoramidite 911.15 314.19 0.25g/2.74mL10-1560 Ac-5-Me-dC-CEPhosphoramidite 785.86 303.21 0.25g/3.18mL10-1564 5-FormyldCIIICEPhosphoramidite 950.02 317.19 0.25g/2.63mL 375.27(acetal)10-1576 Ferrocene-dT-CEPhosphoramidite 1125.07 684.45 0.25g/2.22mL10-1585 Pac-2-Amino-dA-CEPhosphoramidite 1042.21 328.22 0.25g/2.40mL10-1590 Pyrene-dU-CEPhosphoramidite 955.04 514.42 0.25g/2.62mL10-1591 Perylene-dU-CEPhosphoramidite 1005.1 564.48 0.25g/2.49mL10-1598 8,5’-Cyclo-dG-CEPhosphoramidite 619.65 327.19 0.25g/4.03mL10-1601 Pac-dA-CEPhosphoramidite 887.97 313.21 0.25g/2.82mL10-1621 iPr-Pac-dG-CEPhosphoramidite 946.05 329.21 0.25g/2.64mL10-1700 dA-Thiophosphoramidite 955.09 345.34(dithioate) 0.25g/1.75mL10-1710 dC-Thiophosphoramidite 931.07 321.31(dithioate) 0.25g/1.79mL10-1720 dG-Thiophosphoramidite 937.07 361.34(dithioate) 0.25g/1.78mL10-1730 dT-Thiophosphoramidite 841.97 336.32(dithioate) 0.25g/1.98mL10-1891 MethacrylateC6Phosphoramidite 385.48 247.23 0.25g/6.49mL10-1900 ChemicalPhosphorylationReagent 656.77 79.98 0.25g/3.81mL10-1901 ChemicalPhosphorylationReagentII 722.82 79.98 0.25g/3.46mL10-1902 SolidChemicalPhosphorylationReagentII 692.79 79.98 0.25g/3.61mL10-1905 5’-Amino-Modifier5 577.71 167.1 0.25g/4.33mL

PHYSICAL DATA

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10-1906 5’-Amino-ModifierC6 589.76 179.16 0.25g/4.24mL10-1907 5’-DMS(O)MT-Amino-ModifierC6 681.34 179.16 0.25g/3.67mL10-1908 5’-HexynylPhosphoramidite 298.36 160.11 0.25g/8.38mL10-1909 SpacerPhosphoramidite9 652.77 212.14 0.25g/3.83mL10-1910 1-Ethynyl-dSpacerCEPhosphoramidite 644.74 204.12 0.25g/3.88mL10-1912 5’-Amino-ModifierC12 673.92 263.32 0.25g/3.71mL10-1913 SpacerPhosphoramiditeC3 578.69 138.06 0.25g/4.32mL10-1914 dSpacerCEPhosphoramidite 620.73 180.1 0.25g/4.03mL10-1915 Pyrrolidine-CEPhosphoramidite 841.97 178.1 0.25g/2.97mL10-1916 5’-Amino-ModifierC6-TFA 413.42 179.16 0.25g/6.05mL10-1917 5’-Amino-ModifierTEGCE-Phosphoramidite 489.47 255.21 0.25g/5.11mL10-1918 SpacerPhosphoramidite18 784.93 344.3 0.25g/3.18mL10-1919 5’-Aminooxy-Modifier-11-CEPhosphoramidite 711.82 271.21 0.25g/3.51mL10-1920 SymmetricDoublerPhosphoramidite 1095.32 351.31 0.25g/2.28mL10-1922 TreblerPhosphoramidite 1417.72 370.33 0.25g/1.76mL10-1923 5’-Amino-ModifierC3-TFA 371.34 137.08 0.25g/6.73mL10-1925 LongTreblerPhosphoramidite 1475.78 428.41 0.25g/1.69mL10-1926 5’-Thiol-ModifierC6 576.78 196.2 0.25g/4.33mL10-1927 AbasicIIPhosphoramidite 750.98 196.1 0.25g/3.33mL10-1928 SpacerC12CEPhosphoramidite 704.93 264.3 0.25g/3.55mL10-1931 5’-I-dT-CEPhosphoramidite 552.35 414.09 0.25g/4.53mL10-1932 5’-Amino-dT-CEPhosphoramidite 713.81 303.21 0.25g/3.50mL10-1933 5’-Aldehyde-ModifierC2Phosphoramidite 480.58 228.14 0.25g/5.20mL10-1934 5-Formylindole-CEPhosphoramidite 763.86 323.24 0.25g/3.27mL10-1935 5’-Carboxy-ModifierC10 485.56 varies,-CO2H=250.23 0.25g/5.15mL10-1936 Thiol-ModifierC6S-S 769.05 328.4(disulfide) 0.25g/3.25mL 196.2(thiol)10-1938 5’-Maleimide-ModifierPhosphoramidite 437.47 299.22(pre-retro-DA) 0.25g/5.71mL 203.09(maleimide)10-1939 SperminePhosphoramidite 1233.17 408.52 0.25g/2.03mL10-1941 5’-DBCO-TEGPhosphoramidite 708.82 570.57 0.25g/3.53mL10-1945 5’-Carboxy-ModifierC5 595.11 180.1 0.25g/4.20mL10-1946 5’-BromohexylPhosphoramidite 381.29 243.04(bromide) 0.25g/6.56mL 205.15(azide)10-1947 5’-Amino-ModifierC6-PDA 478.57 179.15 0.25g/5.22mL10-1948 5’-Amino-ModifierC12-PDA 562.7 263.32 0.25g/4.44mL10-1949 5’-Amino-ModifierTEGPDA 554.62 255.21 0.25g/4.51mL10-1952 DesthiobiotinTEGPhosphoramidite 980.19 539.56 0.25g/2.55mL10-1953 BiotinPhosphoramidite 876.1 435.48 0.25g/2.85mL10-1955 BiotinTEGPhosphoramidite 1010.24 569.61 0.25g/2.47mL10-1963 FluoresceinPhosphoramidite 1207.5 598.56 0.25g/2.07mL10-1964 6-FluoresceinPhosphoramidite 1176.35 566.48 0.25g/2.13mL10-1973 AcridinePhosphoramidite 891.53 450.86 0.25g/2.80mL10-1974 5’-GalNAcC3Phosphoramidite 1206.38 609.61 0.25g/2.07mL10-1975 Cholesteryl-TEGPhosphoramidite 1196.6 755.97 0.25g/2.09mL10-1976 5’-Cholesteryl-TEGPhosphoramidite 820.13 682.89 0.25g/3.05mL10-1977 a-Tocopherol-TEGPhosphoramidite 1139.56 698.91 0.25g/2.19mL10-1979 StearylPhosphoramidite 470.71 332.46 0.25g/5.31mL10-1981 AsymmetricDoubler(Lev)Phosphoramidite 891.04 352.32 0.25g/2.81mL10-1982 PsoralenC2Phosphoramidite 502.55 364.29 0.25g/4.97mL10-1983 PsoralenC6Phosphoramidite 558.65 420.4 0.25g/4.48mL10-1985 DNP-TEGPhosphoramidite 950.00 509.41 0.25g/2.63mL

PHYSICAL DATA

156

PHYSICAL DATA


10-1986 5’-TrimethoxystilbeneCapPhosphoramidite 571.65 433.39 0.25g/4.37mL10-1987 5’-PyreneCapPhosphoramidite 501.6 363.35 0.25g/4.98mL10-1991 DithiolSerinolPhosphoramidite 853.08 412.46 0.25g/2.93mL10-1992 Alkyne-ModifierSerinolPhosphoramidite 758.88 318.26 0.25g/3.29mL10-1993 ProtectedBiotinSerinolPhosphoramidite 1051.28 450.45 0.25g/2.38mL10-1994 6-FluoresceinSerinolPhosphoramidite 1191.3 582.45 0.25g/2.10mL10-1995 ProtectedBiotinLCSerinolPhosphoramidite 1298.57 697.74 0.25g/1.93mL10-1996 COTSerinolPhosphoramidite 822.97 382.35 0.25g/3.04mL10-1997 Amino-ModifierSerinolPhosphoramidite 887.01 224.15 0.25g/2.82mL10-1998 DBCO-SerinolPhosphoramidite 909.08 468.45 0.25g/2.75mL10-2000 Bz-A-LA-CEPhosphoramidite 885.96 341.22 0.25g/2.82mL10-2011 5-Me-Bz-C-LA-CEPhosphoramidite 875.96 331.22 0.25g/2.85mL10-2029 dmf-G-LA-CEPhosphoramidite 852.93 357.22 0.25g/2.93mL10-2030 T-LA-CEPhosphoramidite 772.84 332.20 0.25g/3.23mL10-3000 Pac-A-CEPhosphoramidite 1018.23 329.21 0.25g/2.46mL10-2101 beta-L-Pac-dA-CEPhosphoramidite 887.97 313.21 0.25g/2.82mL10-2115 beta-L-Ac-dC-CEPhosphoramidite 771.85 289.18 0.25g/3.24mL10-2121 beta-L-iPr-dG-CEPhosphoramidite 946.05 329.21 0.25g/2.64mL10-2130 beta-L-dT-CEPhosphoramidite 744.83 304.20 0.25g/3.36mL10-3003 Bz-A-CEPhosphoramidite 988.21 329.21 0.25g/2.53mL10-3004 A-TOM-CEPhosphoramidite 998.24 329.21 0.25g/2.50mL10-3005 N6-Methyl-A-CEPhosphoramidite 1032.25 343.23 0.25g/2.42mL10-3011 Zebularine-CEPhosphoramidite 845.05 290.17 0.25g/2.96mL10-3012 Pyridin-2-one-CEPhosphoramidite 844.06 289.18 0.25g/2.96mL10-3014 C-TOM-CEPhosphoramidite 974.22 305.18 0.25g/2.57mL10-3015 Ac-C-CEPhosphoramidite 902.11 305.18 0.25g/2.77mL10-3017 Pyrrolo-C-TOM-CEPhosphoramidite 970.23 343.27 0.25g/2.58mL10-3021 iPr-Pac-G-CEPhosphoramidite 1076.31 345.21 0.25g/2.32mL10-3024 G-TOM-CEPhosphoramidite 1014.24 345.21 0.25g/2.46mL10-3025 Ac-G-CEPhosphoramidite 941.43 345.21 0.25g/2.66mL10-3030 U-CEPhosphoramidite 861.06 306.17 0.25g/2.90mL10-3034 U-TOM-CEPhosphoramidite 933.17 306.17 0.25g/2.68mL10-3039 Amino-ModifierC6-UPhosphoramidite 1197.41 474.4 0.25g/2.09mL10-3040 I-CEPhosphoramidite 885.08 330.19 0.25g/2.82mL10-3050 5-Me-U-CEPhosphoramidite 875.08 320.19 0.25g/2.86mL10-3052 4-Thio-U-TOM-CEPhosphoramidite 1002.29 322.22 0.25g/2.49mL10-3055 PseudoUridine-CEPhosphoramidite 861.05 306.17 0.25g/2.90mL10-3056 1-Methyl-PseudoUridinePhosphoramidite 875.07 320.19 0.25g/2.86mL10-3064 5-Me-C-TOM-CEPhosphoramidite 988.25 319.21 0.25g/2.53mL10-3070 2-Aminopurine-TBDMS-CEPhosphoramidite 954.19 329.21 0.25g/2.62mL10-3072 6-Thio-G-CEPhosphoramidite 1039.31 361.26 0.25g/2.41mL10-3083 8-Aza-7-deaza-A-CEPhosphoramidite 939.16 329.21 0.25g/2.66mL10-3085 2,6-Diaminopurine-TOM-CEPhosphoramidite 1113.36 344.22 0.25g/2.25mL10-3090 Br-U-CEPhosphoramidite 939.96 385.06 0.25g/2.66mL10-3091 5-I-U-CEPhosphoramidite 986.96 432.07 0.25g/2.53mL10-3100 2’-OMe-A-CEPhosphoramidite 887.97 343.24 0.25g/2.82mL10-3110 2’-OMe-C-CEPhosphoramidite 863.95 319.21 0.25g/2.89mL10-3111 2’-OMe-TMP-5-F-U-CEPhosphoramidite 897.08 337.2 0.25g/2.79mL10-3115 2’-OMe-Ac-C-CEPhosphoramidite 801.88 319.21 0.25g/3.12mL10-3116 2’-OMe-3-deaza-5-aza-C-CEPhosphoramidite 816.91 319.21 0.25g/3.06mL

157

MIS

CELL

ANEO

US


10-3120 2’-OMe-ibu-G-CEPhosphoramidite 869.97 359.24 0.25g/2.87mL10-3121 2’-OMe-G-CEPhosphoramidite 854.93 359.24 0.25g/2.92mL10-3123 2’-OMe-2-Aminopurine-CEPhosphoramidite 839.04 343.24 0.25g/2.98mL10-3124 2’-OMe-2,6-Diaminopurine-CEPhosphoramidite 924.05 358.25 0.25g/2.71mL10-3130 2’-OMe-U-CEPhosphoramidite 760.82 320.2 0.25g/3.29mL10-3131 2’-OMe-5-Me-U-CEPhosphoramidite 774.84 334.22 0.25g/3.23mL10-3132 2’-OMe-5-F-U-CEPhosphoramidite 778.78 338.19 0.25g/3.21mL10-3140 2’-OMe-I-CEPhosphoramidite 784.85 344.22 0.25g/3.19mL10-3150 2’-OMe-A-PACEPhosphoramidite 958.07 385.27 0.25g/2.61mL10-3151 2’-OMe-Ac-C-PACEPhosphoramidite 871.97 361.25 0.25g/2.87mL10-3152 2’-OMe-G-PACEPhosphoramidite 940.05 401.27 0.25g/2.66mL10-3153 2’-OMe-U-PACEPhosphoramidite 830.92 362.23 0.25g/3.01mL10-3160 2’-OMe-5-Me-C-CEPhosphoramidite 815.9 333.24 0.25g/3.06mL10-3170 2’-OMe-A-Thiophosphoramidite 985.12 375.36 0.25g/1.69mL10-3171 2’-OMe-C-Thiophosphoramidite 899.02 351.34 0.25g/1.85mL10-3172 2’-OMe-G-Thiophosphoramidite 967.1 391.36 0.25g/1.72mL10-3173 2’-OMe-U-Thiophosphoramidite 857.97 352.32 0.25g/1.94mL10-3190 2’-OMe-5-Br-U-CEPhosphoramidite 839.72 399.09 0.25g/2.98mL10-3200 A-2'-MOE-Phosphoramidite 932.03 387.29 0.25g/2.68mL10-3211 5-Me-C-2'-MOE-Phosphoramidite 922.03 377.29 0.25g/2.71mL10-3220 G-2'-MOE-Phosphoramidite 914.01 403.29 0.25g/2.74mL10-3231 5-Me-U-2'-MOE-Phosphoramidite 818.90 378.27 0.25g/3.05mL10-3400 2’-F-A-CEPhosphoramidite 875.93 331.2 0.25g/2.85mL10-3415 2’-F-Ac-C-CEPhosphoramidite 789.84 307.18 0.25g/3.17mL10-3420 2’-F-G-CEPhosphoramidite 857.91 347.19 0.25g/2.91mL10-3430 2’-F-U-CEPhosphoramidite 748.79 308.16 0.25g/3.34mL10-3440 2’-F-I-CEPhosphoramidite 772.82 332.18 0.25g/3.23mL10-3501 1-Me-A-CEPhosphoramidite 944.57 344.24 0.25g/2.65mL10-3517 Ribo-tC°Phosphoramidite 950.16 395.26 0.25g/2.63mL10-3601 2’-OMe-Pac-A-CEPhosphoramidite 917.99 343.24 0.25g/2.72mL10-3621 2’-OMe-iPr-Pac-G-CEPhosphoramidite 976.07 359.24 0.25g/2.56mL10-3800 2’-FANA-A-CEPhosphoramidite 875.93 331.2 0.25g/2.85mL10-3815 2’-FANA-Ac-C-CEPhosphoramidite 789.83 307.17 0.25g/3.16mL10-3820 2’-FANA-G-CEPhosphoramidite 857.91 347.19 0.25g/2.91mL10-3830 2’-FANA-U-CEPhosphoramidite 748.79 308.16 0.25g/3.34mL10-3850 2’-F-5-Me-U-ANA-CEPhosphoramidite 762.80 322.18 0.25g/3.28mL10-3914 rSpacerCEPhosphoramidite 823.09 196.09 0.25g/3.04mL10-3915 rSpacerTBDMSCEPhosphoramidite 750.99 196.09 0.25g/3.33mL10-4410 UniCapPhosphoramidite 334.39 0.25g/7.48mL10-4906 PCAmino-ModifierPhosphoramidite 605.59 371.32 0.25g/4.13mL10-4913 PCSpacerPhosphoramidite 784.88 344.26 0.25g/3.19mL10-4920 PCLinkerPhosphoramidite 699.78 259.15 0.25g/3.57mL10-4950 PCBiotinPhosphoramidite 1038.25 597.62 0.25g/2.41mL10-4960 3-CyanovinylcarbazolePhosphoramidite(CNVK) 836.95 396.33 0.25g/2.99mL10-5800 AzobenzenePhosphoramidite 815.94 375.32 0.25g/3.06mL10-5901 5’-FluoresceinPhosphoramidite 843.95 537.46 0.25g/2.96mL10-5902 5’-Hexachloro-FluoresceinPhosphoramidite 1050.62 744.13 0.25g/2.38mL10-5903 5’-Tetrachloro-FluoresceinPhosphoramidite 981.73 675.24 0.25g/2.55mL10-5905 SIMA(HEX)Phosphoramidite 1065.02 759.54 0.25g/2.35mL10-5906 5’-Dichloro-dimethoxy-FluoresceinPhosphoramiditeII972.88 666.4 0.25g/2.57mL10-5912 5’-DabcylPhosphoramidite 568.69 430.18 0.25g/4.40mL10-5913 Cyanine3Phosphoramidite 953.64 507.59 0.25g/2.62mL

PHYSICAL DATA

158


10-5914 Cyanine3.5Phosphoramidite 1053.76 607.7 0.25g/2.37mL10-5915 Cyanine5Phosphoramidite 979.68 533.63 0.25g/2.55mL10-5916 Cyanine5.5Phosphoramidite 1171.25 633.74 0.25g/2.13mL10-5920 RedmondRed®Phosphoramidite 971.09 445.34 0.25g/2.57mL10-5921 YakimaYellow®Phosphoramidite 1023.81 718.33 0.25g/2.44mL10-5923 5’-AquaPhluor®593CEPhosphoramidite 1239.17 787.82 0.25g/2.02mL10-5924 5’-CDPI3MGB™Phosphoramidite 1323.42 872.96 0.25g/1.89mL10-5925 Eclipse®QuencherPhosphoramidite 978.5 537.89 0.25g/2.55mL10-5931 5’-BHQ-1Phosphoramidite 676.75 538.49 0.25g/3.69mL10-5932 5’-BHQ-2Phosphoramidite 678.72 540.47 0.25g/3.68mL10-5934 5’-BBQ-650®-CEPhosphoramidite 802.9 665.65 0.25g/3.11mL10-5941 BHQ-1-dT 1401.56 960.93 0.25g/1.78mL10-5942 BHQ-2-dT 1403.53 962.91 0.25g/1.78mL10-5944 BBQ-650®-dT-CEPhosphoramidite 1441.57 1000.95 0.25g/1.73mL10-5945 SIMA(HEX)-dTPhosphoramidite 1646.64 1037.79 0.25g/1.52mL10-5950 5’-BiotinPhosphoramidite 846.08 405.45 0.25g/2.95mL10-5961 MethyleneBlueIIPhosphoramidite 967.67 489.57 0.25g/2.58mL10-7001 2’,3’-ddA-CEPhosphoramidite 574.7 297.21 0.25g/4.35mL10-7101 2’,3’-ddC-CEPhosphoramidite 550.68 273.18 0.25g/4.54mL10-7201 2’,3’-ddG-CEPhosphoramidite 506.54 313.2 0.25g/4.94mL10-7301 2’,3’-ddT-CEPhosphoramidite 426.45 288.19 0.25g/5.86mL10-9201 dmf-dG-5’-CEPhosphoramidite 824.92 329.21 0.25g/3.03mL11-1330 Cis-synThymineDimerPhosphoramidite 1024.01 608.39 0.25g/2.44mL13-1000 AAATrimerPhosphoramidite 1911.5 0.25g/1.31mL13-1001 AACTrimerPhosphoramidite 1887.5 0.25g/1.32mL13-1011 ACCTrimerPhosphoramidite 1863.5 0.25g/1.34mL13-1013 ACTTrimerPhosphoramidite 1774.5 0.25g/1.41mL13-1020 AGATrimerPhosphoramidite 1893.5 0.25g/1.32mL13-1031 ATCTrimerPhosphoramidite 1774.5 0.25g/1.41mL13-1032 ATGTrimerPhosphoramidite 1780.5 0.25g/1.40mL13-1102 CAGTrimerPhosphoramidite 1869.5 0.25g/1.34mL13-1103 CATTrimerPhosphoramidite 1774.5 0.25g/1.41mL13-1110 CCATrimerPhosphoramidite 1863.5 0.25g/1.34mL13-1112 CCGTrimerPhosphoramidite 1845.5 0.25g/1.35mL13-1122 CGGTrimerPhosphoramidite 1851.5 0.25g/1.35mL13-1123 CGTTrimerPhosphoramidite 1756.5 0.25g/1.42mL13-1132 CTGTrimerPhosphoramidite 1756.5 0.25g/1.42mL13-1200 GAATrimerPhosphoramidite 1893.5 0.25g/1.32mL13-1201 GACTrimerPhosphoramidite 1869.5 0.25g/1.34mL13-1203 GATTrimerPhosphoramidite 1780.5 0.25g/1.40mL13-1210 GCATrimerPhosphoramidite 1869.5 0.25g/1.34mL13-1212 GCGTrimerPhosphoramidite 1851.5 0.25g/1.35mL13-1213 GCTTrimerPhosphoramidite 1756.5 0.25g/1.42mL13-1223 GGTTrimerPhosphoramidite 1762.5 0.25g/1.42mL13-1230 GTATrimerPhosphoramidite 1780.5 0.25g/1.40mL13-1233 GTTTrimerPhosphoramidite 1667.5 0.25g/1.50mL13-1301 TACTrimerPhosphoramidite 1774.5 0.25g/1.41mL13-1313 TCTTrimerPhosphoramidite 1661.4 0.25g/1.50mL13-1321 TGCTrimerPhosphoramidite 1756.5 0.25g/1.42mL

PHYSICAL DATA

159

MIS

CELL

ANEO

US


13-1322 TGGTrimerPhosphoramidite 1762.5 0.25g/1.42mL13-1331 TTCTrimerPhosphoramidite 1661.4 0.25g/1.50mL13-1333 TTTTrimerPhosphoramidite 1572.4 0.25g/1.59mL20-0002 dA-5’-CPG 313.2120-0102 dC-5’-CPG 289.1820-0202 dG-5’-CPG 329.2120-0302 dT-5’-CPG 304.220-2000 dA-CPG500 313.2120-2001 dA-CPG1000 313.2120-2002 dA-CPG2000 313.2120-2004 3’-dA-CPG 313.2120-2010 dC-CPG500 289.1820-2011 dC-CPG1000 289.1820-2012 dC-CPG2000 289.1820-2013 Ac-dC-CPG500 289.1820-2015 Ac-dC-CPG1000 289.1820-2017 2’,3’-ddC-CPG 273.1920-2019 3’-Amino-ModifierC6dCCPG 457.4220-2020 dG-CPG500 329.2120-2021 dG-CPG1000 329.2120-2022 dG-CPG2000 329.2120-2029 dmf-dG-CPG 329.2120-2030 dT-CPG500 304.220-2031 dT-CPG1000 304.220-2032 dT-CPG2000 304.220-2040 dI-CPG500 314.1920-2041 dI-CPG1000 314.1920-2050 dU-CPG500 290.1720-2051 dU-CPG1000 290.1720-2056 3’-Fluorescein-dTCPG 815.7120-2064 3’-dC-CPG 289.1820-2074 3’-dG-CPG 329.2120-2084 3’-dT-CPG 304.220-2090 5-Br-dU-CPG 369.0720-2101-61 dA-CPG1000 313.2120-2101-62 dA-CPG1000 313.2120-2101-65 dA-CPG1000 313.2120-2115-61 Ac-dC-CPG1000 289.1820-2115-62 Ac-dC-CPG1000 289.1820-2115-65 Ac-dC-CPG1000 289.1820-2129-61 dmf-dG-CPG 329.2120-2129-62 dmf-dG-CPG 329.2120-2129-65 dmf-dG-CPG 329.2120-2131-61 dT-CPG1000 304.220-2131-62 dT-CPG1000 304.220-2131-65 dT-CPG1000 304.220-2601 Pac-dA-CPG 313.2120-2621 iPr-Pac-dG-CPG 329.2120-2900 3’-PhosphateCPG 79.9820-2902 3’-GlycerylCPG 154.0620-2903 3’-CPRIICPG 79.98

PHYSICAL DATA

160


20-2913 3’-SpacerC3CPG 138.0620-2933 3’-Thiol-ModifierC3S-SCPG 154.12(thiol),244.27(disulfide)20-2938 3’-Thiol-Modifier6S-SCPG 198.18(thiol),332.37(disulfide)20-2952 DesthiobiotinTEG-CPG 539.5620-2954 3’-PT-Amino-ModifierC3CPG 137.0720-2955 3’-BiotinTEGCPG 569.6120-2956 3’-PT-Amino-ModifierC6CPG 179.1520-2958 3’-Amino-ModifierC7CPG1000 209.1820-2961 3’-(6-FAM)CPG 569.4620-2963 3’-FluoresceinCPG 598.5620-2964 3’-(6-Fluorescein)CPG 566.4820-2973 3’-AcridineCPG 450.8620-2974 GalNAcC3CPG 609.6120-2975 3’-Cholesteryl-TEGCPG 755.9720-2980 3’-UaqCapCPG 539.3920-2981 3’-Amino-dTCPG 303.2120-2982 3’-Propargyl-5-Me-dCCPG 341.2620-2991 3’-DithiolSerinolCPG 412.4620-2992 3’-Alkyne-ModifierSerinolCPG 334.2620-2993 3’-ProtectedBiotinSerinolCPG 450.4520-2994 3’-6-FluoresceinSerinolCPG 584.4720-2995 3’-ProtectedBiotinLCSerinolCPG 697.7420-2997 3’-Amino-ModifierSerinolCPG 224.1520-3300 Pac-A-RNA-CPG 329.2120-3303 Bz-A-RNA-CPG 329.2120-3304 Ac-A-RNA-CPG 329.2120-3315 Ac-C-RNA-CPG 305.1820-3321 iPr-Pac-G-RNA-CPG 345.2120-3324 Ac-G-RNA-CPG 345.2120-3330 U-RNA-CPG 306.1720-3600 2’-OMe-A-RNA-CPG 343.2420-3610 2’-OMe-C-RNA-CPG 319.2120-3615 2’-OMe-Ac-C-RNA-CPG 319.2120-3621 2’-OMe-G-RNA-CPG 359.2420-3630 2’-OMe-U-RNA-CPG 320.220-4040 Puromycin-CPG 533.4820-5910 3’-TAMRACPG 623.620-5911 3’-DabsylCPG 498.4920-5912 3’-DabcylCPG 462.4420-5913 Cyanine3CPG 507.5920-5915 Cyanine5CPG 533.6320-5920 RedmondRed®CPG 445.3420-5921 YakimaYellow®CPG 718.3320-5923 AquaPhluor®593CPG 900.9320-5924 CDPI3MGB™CPG 831.8720-5925 Eclipse®QuencherCPG 537.8920-5931 3’-BHQ-1CPG 554.4920-5932 3’-BHQ-2CPG 556.4720-5933 3’-BHQ-3CPG 597.6320-5934 BBQ-650®CPG 667.6320-9202 dmf-dG-5’-CPG 329.21

PHYSICAL DATA

161

MIS

CELL

ANEO

US

PHYSICAL DATA

Cat. No. Item MW Unit FW Dilution (0.1M)

21-2000 dA-Q-CPG500 313.2121-2010 dC-Q-CPG500 289.1821-2013 Ac-dC-Q-CPG500 305.1821-2029 dmf-dG-Q-CPG500 329.2121-2030 dT-Q-CPG500 304.225-2000 dA-HighLoad-CPG 313.2125-2010 dC-HighLoad-CPG 289.1825-2020 dG-HighLoadCPG 329.2125-2030 dT-HighLoad-CPG 304.225-2900 3’-PhosphateCPG(HighLoad) 79.9826-2600 dAPS 313.2126-2610 dCPS 289.1826-2629 dmf-dGPS 329.2126-2630 dT-PS 304.226-2900 3’-PhosphatePS 79.9826-2955 3’-BiotinTEGPS 569.6126-2956 3’-PT-Amino-ModifierC6PS 179.1526-2961 3’-(6-FAM)PS 569.4626-5910 3’-TAMRAPS 623.626-5912 3’-DabcylPS 462.4450-1904 AzidobutyrateNHSEster 226.19 113.1250-1905 Alkyne-NHSEster 225.2 110.1150-1941 DBCO-sulfo-NHSEster 532.5 316.3750-1960 MethyleneBlueNHSEster 538.96 425.8950-1970 ThiazoleOrangeNHSEster 538.06 386.5150-2000 BiotinTEGAzide 444.55 50-2001 DesthiobiotinTEGAzide 414.5 50-2002 Dipivaloyl6-FAM-TEGAzide 744.79 50-2003 6-FAM-TEGAzide 576.55 50-2004 CoumarinAzide 203.15 50-2005 6-HEXAzide 665.09 50-2006 6-TETAzide 596.2 50-2007 TEMPOAzide 197.26 50-2008 TEMPO-TEGAzide 373.47 50-2009 PsoralenAzide 283.28 50-2010 Disulfo-Cyanine7Azide 829.08 50-5910 TAMRANHSEster 527.53 413.45

Index

Symbols2'-5' Linkages3’-dA-CEPhosphoramidite653’-dA-CPG653’-dC-CEPhosphoramidite653’-dC-CPG653’-dG-CEPhosphoramidite653’-dG-CPG663’-dT-CEPhosphoramidite653’-dT-CPG66

2’-5’ Linked Oligonucleotides 64, 665’ -> 3’ SYNTHESIS5’-CEPhosphoramidites34

AA1-Me-A-CEPhosphoramidite1351-Me-dA-CEPhosphoramidite662’,3’-ddA562’-F-A-ANACEPhosphoramidite1442'-F-A-CEPhosphoramidite1432'-OMe-A-CEPhosphoramidite1372’-OMe-A-PACEPhosphoramidite1452'-OMe-A-RNA1392'-OMe-Pac-A-CEPhosphoramidite1383'-dA-CEPhosphoramidite653'-dA-CPG55, 667-Deaza-dA-CEPhosphoramidite578-Amino-dA-CEPhosphoramidite598-Br-dA-CEPhosphoramidite608-Oxo-dA-CEPhosphoramidite61A-2'-MOE-Phosphoramidite142Ac-A-RNA-CPG126Amino-ModifierC6dA77A-TOM-CEPhosphoramidite126Bz-A-CEPhosphoramidite128Bz-A-LNA-CEPhosphoramidite41Bz-A-RNA-CPG125, 129dA-CEPhosphoramidite8, 12, 15, 16, 18, 20dA-H-Phosphonate39dA-MePhosphonamidite36dA-MePhosphoramidite38dA-PACEPhosphoramidite37dA-Thiophosphoramidite141def-dA-CEPhosphoramidite22N6-Ac-N6-Me-dA-CEPhosphoramidite47, 66N6-Me-A-CEPhosphoramidite135N6-Me-dA-CEPhosphoramidite47, 66Pac-A-CEPhosphoramidite129Pac-A-RNA-CPG129Pac-dA-CEPhosphoramidite23

A-2-Amino2-Amino-A-TOM-CEPhosphoramidite1312-Amino-dA-CEPhosphoramidite472'-OMe-2-Amino-A-CEPhosphoramidite140

Pac-2-Amino-dA-CEPhosphoramidite47

Abasic Site 62, 84AbasicIIPhosphoramidite62dSpacer Phosphoramidite 62Pyrrolidine-CEPhosphoramidite63

Acridine Labelling3’-AcridineCPG114Acridine Phosphoramidite 114

Activator4,5-Dicyanoimidazole305-Benzylthio-1H-tetrazole305-Ethylthio-1H-tetrazole26, 30, 70, 110, 112Activator(Powder)30Saccharin1-Methylimidazole30Tetrazole12

Adamantane Carbonyl Chloride 39Affinity Chromatography 151ÄKTA oligopilot 18, 19Aldehyde Modifier5'-Aldehyde-ModifierC2Phosphoramidite835-Formyl-dC-CEPhosphoramidite50FormylindoleCEPhosphoramidite83

Alternative Solvents and Reagents 30Amino-dA8-Amino-dA-CEPhosphoramidite59

Amino-dG8-Amino-dG-CEPhosphoramidite62

Amino-Modifiers3'-Amino-ModifierC6dCCPG813'-Amino-ModifierC6dTCPG813’-Amino-ModifierSerinolCPG793'-PT-Amino-ModifierC3CPG793'-PT-Amino-ModifierC6CPG793'-PT-Amino-ModifierC6PS795'-Amino-dT-CEPhosphoramidite555'-Amino-Modifier5745'-Amino-ModifierC3-TFA74, 755'-Amino-ModifierC674, 755’-Amino-ModifierC6-PDA755'-Amino-ModifierC6-TFA74, 755'-Amino-ModifierC12745’-Amino-ModifierC12-PDA755’-Amino-ModifierTEG745’-Amino-Modifier-TEG-PDA755’-DMS(O)MT-Amino-ModifierC674Amino-ModifierC2dT77Amino-ModifierC6dA77Amino-ModifierC6dC77Amino-ModifierC6dT77Amino-ModifierC6-UPhosphoramidite132Amino-ModifierSerinolPhosphoramidite78Fmoc-Amino-ModifierC6dT78N2-Amino-ModifierC6dG77PCAmino-ModifierPhosphoramidite76, 86, 95

AminoOxy-Modifier

162

INDEX

5’-AminoOxy-Modifier1176

Aminopurine2'-OMe-2-Aminopurine-CEPhosphoramidite140

Anthraquinone3’-UaqCapCPG49

Applied Biosystems InstrumentsAB39001000ÅCPGColumns10AB3900PolystyreneColumns10AB3900PolystyreneModifierColumns11CE Phosphoramidites 8Solvents/Reagents 8Supports and Columns 9

Aptamer Development 73AquaPhluor® 5935’-AquaPhluor®593Phosphoramidite108AquaPhluor®593CPG109

Aza-dC5-Aza-5,6-dihydro-dC-CEPhosphoramidite71

Azides6-FAM-TEGAzide926-HEXAzide926-TETAzide92BiotinTEGAzide92CoumarinAzide92DesthiobiotinTEGAzide92Dipivaloyl6-FAM-TEGAzide92Disulfo-Cyanine7Azide93PsoralenAzide93TEMPOAzide93TEMPO-TEGAzide93

Azidobutyrate NHS Ester 89AzobenzeneAzobenzenePhosphoramidite123

BBenzylthio-1H-tetrazole 30Biocompatible Chemical Ligation 64Biotin Labelling3’-BiotinTEGCPG1013’-BiotinTEGPS1013’-ProtectedBiotinLCSerinolCPG96, 1013’-ProtectedBiotinSerinolCPG95, 1015’-BiotinPhosphoramidite100Biotin-dT100BiotinPhosphoramidite99BiotinTEGAzide92BiotinTEGPhosphoramidite99DesthiobiotinTEGAzide92DesthiobiotinTEG-CPG101DesthiobiotinTEGPhosphoramidite100PCBiotinPhosphoramidite86, 100ProtectedBiotinLCSerinolPhosphoramidite95, 99ProtectedBiotinSerinolPhosphoramidite94, 99

BlackBerry® Quencher

3’-BBQ-650®CPG1125’-BBQ-650®Phosphoramidite112BBQ-650®-dT112

Black Hole Quencher™ Dyes3'-BHQ-1CPG1113'-BHQ-2CPG1113'-BHQ-3CPG111, 1125’-BHQ-1Phosphoramidite1105’-BHQ-2Phosphoramidite70, 110, 112BHQ-1-dT110, 112BHQ-2-dT110

Brancher PhosphoramiditedC Brancher Phosphoramidite 85

Br-dA8-Br-dA-CEPhosphoramidite60

Br-dC5-Br-dC-CEPhosphoramidite60

Br-dG8-Br-dG-CEPhosphoramidite60

Br-dU5-Br-dU-CEPhosphoramidite605-Br-dU-CPG60

Bromohexyl Phosphoramidite 89Br-U2'-OMe-5-Br-U-CEPhosphoramidite1405-Br-U-CEPhosphoramidite133

CC2’,3’-ddC562’,3'-ddC-CPG562’-F-Bz-C-ANACEPhosphoramidite1442'-OMe-5-Me-C-CEPhosphoramidite1402'-OMe-Ac-C-CEPhosphoramidite137, 1382’-OMe-Ac-C-PACEPhosphoramidite1452'-OMe-Ac-C-RNA1392'-OMe-C-RNA1393'-Amino-ModifierC6dCCPG813'-dC-CEPhosphoramidite653'-dC-CPG55, 655-Br-dC-CEPhosphoramidite605-Carboxy-dC-CEPhosphoramidite505-Formyl-dC-CEPhosphoramidite505-Hydroxymethyl-dC-CEPhosphoramidite505-Hydroxymethyl-dCII-CEPhosphoramidite505-I-dC-CEPhosphoramidite605-Me-Bz-C-LNA-CEPhosphoramidite415-Me-C-2'-MOE-Phosphoramidite1425-Me-dC-CEPhosphoramidite465-OH-dC-CEPhosphoramidite61Ac-C-CEPhosphoramidite128Ac-C-RNA-CPG125, 127, 130Ac-dC-CEPhosphoramidite8, 12, 15, 16, 18, 20, 23Ac-dC-MePhosphonamidite36Ac-dC-PACEPhosphoramidite37Amino-ModifierC6dC77

163

MIS

CELL

ANEO

US

INDEX

AP-dC68AP-dC-CEPhosphoramidite46C8-Alkyne-dC-CEPhosphoramidite87, 90C8-TMS-Alkyne-dC-CEPhosphoramidite87C-TOM-CEPhosphoramidite126dC Brancher Phosphoramidite 85dC-CEPhosphoramidite8, 12, 15, 16, 18, 20dC-H-Phosphonate39dC-MePhosphoramidite38dC-Thiophosphoramidite141N4-Et-dC-CEPhosphoramidite47pdC-CEPhosphoramidite46Pyrrolo-dCTP68tC-CEPhosphoramidite70tC°-CEPhosphoramidite70

Camphorsulfonyloxaziridine (CSO) 32Cap CPG3’-UaqCapCPG49, 64

Cap Phosphoramidite5’-PyreneCapPhosphoramidite495’-TrimethoxystilbeneCapPhosphoramidite49

Capping ReagentUniCap Phosphoramidite 32

Carboxy-dC5-Carboxy-dC-CEPhosphoramidite50

Carboxy-Modifiers5’-Carboxy-ModifierC5765'-Carboxy-ModifierC1076Carboxy-dT77

Chain Terminators 56ChelatesEDTA-C2-dT-CEPhosphoramidite118

Chemical Phosphorylation 82Cholesterol Labelling3’-Cholesteryl-TEGCPG1155’-Cholesteryl-TEGPhosphoramidite115Cholesteryl-TEGPhosphoramidite75, 115

CleanAmp™ TechnologyCleanAmp™ Primers 54

Click Chemistry 871,2,3-triazoles871-Ethynyl-dSpacerCEPhosphoramidite903’-Alkyne-ModifierSerinolCPG80, 89, 973’-Propargyl-5-Me-dCCPG645’-BromohexylPhosphoramidite895-Ethynyl-dU-CEPhosphoramidite885’-HexynylPhosphoramidite895’-I-dT-CEPhosphoramidite89Alkyne-ModifierSerinolPhosphoramidite89, 95Alkyne-NHSEster89Azides89AzidobutyrateNHSEster89baseclickOligo-Click-M-Biotin90baseclickOligo-Click-M-Fluorescein90baseclickOligo-Click-M-Reload90baseclickOligo-Click-M-TAMRA90

C8-Alkyne-dC-CEPhosphoramidite87, 90C8-Alkyne-dT-CEPhosphoramidite87C8-TIPS-Alkyne-dC-CEPhosphoramidite88C8-TIPS-Alkyne-dT-CEPhosphoramidite88C8-TMS-Alkyne-dC-CEPhosphoramidite87, 88C8-TMS-Alkyne-dT-CEPhosphoramidite88ClickDNAandRNALigation64Copper-freeClickChemistry89THPTA Ligand 88TIPS-5-Ethynyl-dU-CEPhosphoramidite88

Convertible 2-dG2-F-dI-CEPhosphoramidite67

Convertible dAO6-Phenyl-dI-CEPhosphoramidite67

Convertible dUO4-Triazolyl-dU-CEPhosphoramidite67

Convertible F-dCTMP-F-dU-CEPhosphoramidite67

Convertible Nucleosides 67Copper-free Click Chemistry 905’-DBCO-TEGPhosphoramidite91DBCO-dT-CEPhosphoramidite91DBCO-sulfo-NHSEster91

Cross-linking 58, 60, 93, 118, 124Custom Doping 51Cyanine LabellingCyanine3.5Phosphoramidite106Cyanine3CPG107Cyanine3Phosphoramidite106Cyanine5.5Phosphoramidite106Cyanine5CPG107Cyanine5Phosphoramidite106Disulfo-Cyanine7Azide107

Cyanovinylcarbazole3-CyanovinylcarbazolePhosphoramidite124CNVK 124

Cyclo-dA5’,8-Cyclo-dACEPhosphoramidite63

Cyclo-dG5’,8-Cyclo-dGCEPhosphoramidite63

CyclooctatetraeneCOT Serinol Phosphoramidite 97

DDabcyl Labelling3'-DabcylCPG983'-DabcylPS983'-DabsylCPG70, 98, 110, 1125’-DabcylPhosphoramidite98Dabcyl-dT98

DBCO5’-DBCO-TEGPhosphoramidite91DBCO-dT-CEPhosphoramidite91DBCO-SerinolPhosphoramidite91

164

INDEX

DBCO-sulfo-NHSEster91

DCI (4,5-Dicyanoimidazole) 30Deaza-8-aza-A7-deaza-8-Aza-A-CEPhosphoramidite1347-Deaza-8-aza-dA-CEPhosphoramidite57

Deaza-8-aza-G7-deaza-8-Aza-dG-CEPhosphoramidite57

Deaza-A3-Deaza-dA-CEPhosphoramidite577-Deaza-dA-CEPhosphoramidite57

Deaza-G7-Deaza-dG-CEPhosphoramidite57

Deaza-X7-Deaza-dX-CEPhosphoramidite5057

DendrimersAsymmetricDoubler(LEV)Phosphoramidite85LongTreblerPhosphoramidite85SymmetricDoublerPhosphoramidite85TreblerPhosphoramidite85

Depurination Resistant CE Phosphoramidites 22DesthiobiotinDesthiobiotinTEG-CPG101DesthiobiotinTEGPhosphoramidite100

Deuterated Nucleosides 60Diaminopurine2,6-Diaminopurine-TOM-CEPhosphoramidite1312-Amino-dA-CEPhosphoramidite472’-OMe-2,6-DiaminopurineCEPhosphoramidite140Pac-2-Amino-dA-CEPhosphoramidite47

Dicyanoimidazole 30Dideoxynucleoside, 2',3'- 55Dideoxynucleosides2’,3’-ddA-CEPhosphoramidite562’,3’-ddC-CEPhosphoramidite562’,3'-ddC-CPG562’,3’-ddG-CEPhosphoramidite562’,3’-ddT-CEPhosphoramidite56

Dihydro-dT5,6-Dihydro-dT-CEPhosphoramidite61

Dihydro-dU5,6-Dihydro-dU-CEPhosphoramidite61

Dithiol3’-DithiolSerinolCPG80Dithiol Serinol Phosphoramidite 76

DNA Damage/Repair 62–63, 63DNA Methylation5-Carboxy-dC-CEPhosphoramidite505-Formyl-dC-CEPhosphoramidite505-Me-dC-CEPhosphoramidite46

DNA Methyltransferases 71DNP LabellingDNP-TEGPhosphoramidite114

Doubler PhosphoramiditeSymmetric85

Dr. Oligo SynthesizersCE Phosphoramidites 20Solvents and Reagents 20

Duplex StabilizationBasesAffectingDuplexStability46CapsforIncreasedDuplexStability/Base-PairingFidelity49DuplexStabilityModification46

EEclipse® QuencherEclipse®QuencherCPG109Eclipse®QuencherPhosphoramidite108

EDTA-dTEDTA-C2-dT-CEPhosphoramidite118

EdU5-Ethynyl-dU-CEPhosphoramidite88TIPS-5-Ethynyl-dU-CEPhosphoramidite88

ELITechGroup Dyes and Quencher 108Epigenetics 50, 135EstersAlkyne-NHSEster89AzidobutyrateNHSEster89BCO-sulfo-NHSEster91MethyleneBlueNHSEster119TAMRA NHS Ester 113

Et-dC-CE Phosphoramidite 47Etheno-AEtheno-dA-CEPhosphoramidite68

Ethylthiotetrazole 30Excimers 121Expedite™ Instruments

CE Phosphoramidites 12Solvents and Reagents 12Supports and Columns 13

FFAM3’-(6-FAM)CPG1043'-(6-FAM)PS1046-FAM1026-FAM-TEGAzide92Dipivaloyl6-FAM-TEGAzide92

F-ANA Monomers2’-F-A-ANACEPhosphoramidite1442’-F-Ac-C-ANACEPhosphoramidite1442’-F-Bz-C-ANACEPhosphoramidite1442’-F-G-ANACEPhosphoramidite1442’-F-U-ANACEPhosphoramidite144

165

MIS

CELL

ANEO

US

INDEX

F-C2'-F-Ac-C-CEPhosphoramidite49F-dCPrecursor67

F-dI2-F-dI-CEPhosphoramidite67

F-dU5-F-dU-CEPhosphoramidite605-Fluoro-dU60

Ferrocene LabellingFerrocene-dT-CEPhosphoramidite119

Fluorescein Labelling3’-(6-FAM)CPG1043’-(6-FAM)PS1043’-(6-Fluorescein)CPG104, 1073’-6-FluoresceinSerinolCPG96, 1043’-FluoresceinCPG1043'-Fluorescein-dTCPG1045’-Dichloro-dimethoxy-Fluorescein1025’-FluoresceinPhosphoramidite1025’-Hexachloro-Fluorescein1025’-Tetrachloro-Fluorescein1026-FluoresceinPhosphoramidite1036-FluoresceinSerinolPhosphoramidite94, 103Dichloro-diphenyl-fluorescein105Fluorescein-dTPhosphoramidite103SIMA(HEX)105

Fluorescent Nucleosides 682-Aminopurine-CEPhosphoramidite585-Me-2'-deoxyZebularine-CEPhosphoramidite71AP-dC-CEPhosphoramidite46Etheno-dA-CEPhosphoramidite68Perylene-dU-CEPhosphoramidite69Pyrrolo-C-TOM-CEPhosphoramidite132Pyrrolo-dC-CEPhosphoramidite68Pyrrolo-dCTP68tC-CEPhosphoramidite70tC°-CEPhosphoramidite70

Formyl-dC5-Formyl-dC-CEPhosphoramidite505-Formyl-dCIII-CEPhosphoramidite50

Formylindole CE Phosphoramidite 83Free Radicals 61F-RNA Monomers2'-F-Ac-C-CEPhosphoramidite142, 1432'-F-A-CEPhosphoramidite1432'-F-G-CEPhosphoramidite1432'-F-U-CEPhosphoramidite143

F-U2'-OMe-5-F-U-CEPhosphoramidite140

GG2’,3’-ddG562’-F-G-ANACEPhosphoramidite144

2'-F-G-CEPhosphoramidite1432'-OMe-G-CEPhosphoramidite1372’-OMe-G-PACEPhosphoramidite1452'-OMe-G-RNA1392'-OMe-iPr-Pac-G-CEPhosphoramidite1383'-dG-CEPhosphoramidite653'-dG-CPG55, 666-Thio-dG-CEPhosphoramidite586-Thio-G-CEPhosphoramidite1337-Deaza-8-aza-dG-CEPhosphoramidite577-Deaza-dG-CEPhosphoramidite578-Amino-dG-CEPhosphoramidite628-Br-dG-CEPhosphoramidite608-Oxo-dG-CEPhosphoramidite61Ac-G-CEPhosphoramidite128Ac-G-RNA-CPG127, 130dG-CEPhosphoramidite8, 12, 15, 16, 18, 20dG-H-Phosphonate39dG-MePhosphonamidite36dG-MePhosphoramidite38dG-PACEPhosphoramidite37dG-Thiophosphoramidite141dmf-dG-5’-CEPhosphoramidite34dmf-dG-CEPhosphoramidite8, 12, 15, 16, 18, 20dmf-G-LNA-CEPhosphoramidite41G-2'-MOE-Phosphoramidite142G-TOM-CEPhosphoramidite126iPr-Pac-dG-CEPhosphoramidite23iPr-Pac-G-RNA-CPG130N2-Amino-ModifierC6dG77O6-Me-dG-CEPhosphoramidite66

GalNAc5’-GalNAcC3Phosphoramidite116GalNAc C3 CPG 116

G-Clamp 46, 68GE Healthcare LIfe Sciences Instruments

CE Phosphoramidite 18Solvents and Reagents 19

Glen Gel-Pak™ PurificationGlenGel-Pak™150

Glen-Pak™ PurificationAdapter Rack 148Glen-Pak™DNAPurificationCartridge147Glen-Pak™RNAPurificationCartridge148RNAQuenchingBuffer148Seal for Adapter Rack 148

Glen UnySupport™GlenUnySupportCPG24, 25GlenUnySupportFCCPG25GlenUnySupportPS24

Glyceryl CPG 80GoldConjugationtogoldsurfaces94

G-Quadruplex 72

166

INDEX

HHalogenated Nucleosides2'-OMe-5-F-U-CEPhosphoramidite1405-Br-dC-CEPhosphoramidite605-Br-dU-CEPhosphoramidite605-Br-dU-CPG605-Br-U-CEPhosphoramidite1335-F-dU-CEPhosphoramidite605-I-dC-CEPhosphoramidite605-I-dU-CEPhosphoramidite605-I-U-CEPhosphoramidite1338-Br-dA-CEPhosphoramidite608-Br-dG-CEPhosphoramidite60

HEX 1026-HEXAzide92, 93

Hexynyl Phosphoramidite 89High Load CPG 29H-Phosphonate Chemistry

Monomers 39, 40ReagentsforABIsynthesizers39

Hydrogen Bonding 57Hydroxy-C5-OH-dC-CEPhosphoramidite61

Hydroxymethyl-dC5-Hydroxymethyl-dC-CEPhosphoramidite505-Hydroxymethyl-dCII-CEPhosphoramidite50

Hydroxymethyl-dU5-Hydroxymethyl-dU-CEPhosphoramidite61

Hydroxy-U5-OH-dU-CEPhosphoramidite61

II2-F-dI-CEPhosphoramidite672'-OMe-I-CEPhosphoramidite141dI-CEPhosphoramidite51dI-CPG51I-CEPhosphoramidite133O6-Phenyl-dI-CEPhosphoramidite67

I-dC5-I-dC-CEPhosphoramidite60

I-dT5'-I-dT-CEPhosphoramidite89

I-dU5-I-dU-CEPhosphoramidite60

i-Motif DNA structures 72Introduction 1, 3, 5, 6, 8, 10, 12, 14, 16, 18, 20Ionizing Radiation 61isodCdmf-5-Me-isodC-CEPhosphoramidite53

isodGdmf-isodG-CEPhosphoramidite53

Isopropyl Phosphite 39I-U5-I-U-CEPhosphoramidite133

JJOE5’-Dichloro-dimethoxy-FluoresceinPhosphoramiditeII102

KKdK-CEPhosphoramidite52

LLabelling of MicroRNAs 64Large Scale SynthesisN3-Cyanoethyl-dT71

beta-L-DNA monomers 40Locked Analog Phosphoramidites5-Me-Bz-C-LA-CEPhosphoramidite41Bz-A-LA-CEPhosphoramidite41dmf-G-LA-CEPhosphoramidite41T-LA-CEPhosphoramidite41

Locked Nucleic Acid (LNA) 41

M2’-MOE RNA 142Maleimide-Modifier5’-Maleimide-ModifierPhosphoramidite76

Me-C2'-OMe-5-Me-C-CEPhosphoramidite1403’-Propargyl-5-Me-dCCPG645-Me-C-TOM-CEPhosphoramidite1315-Me-dC-CEPhosphoramidite46Ac-5-Me-dC-CEPhosphoramidite46

MerMade InstrumentsCE Phosphoramidites 16Solvents and Reagents 16Supports and Columns 17

Methacrylate C6 Phosphoramidite 74Methylated Nucleosides1-Me-A-CEPhosphoramidite1351-Me-dA-CEPhosphoramidite661-Me-PseudouridinePhosphoramidite135N6-Ac-N6-Me-dA-CEPhosphoramidite47, 66N6-Me-A-CEPhosphoramidite135N6-Me-dA-CEPhosphoramidite47, 66O4-Me-dT-CEPhosphoramidite47, 66

167

MIS

CELL

ANEO

US

INDEX

O6-Me-dG-CEPhosphoramidite66

Methylene BlueMethyleneBlueIIPhosphoramidite119MethyleneBlueNHSEster119

Methyl Phosphonamidites 36Me-U2'-OMe-5-Me-U-CEPhosphoramidite1405-Me-U-CEPhosphoramidite133

MGB3’-CDPI3MGB™CPG485’-CDPI3MGB™Phosphoramidite48, 117CDPI3 MGB™ CPG 117

MicroRNA 64Minor 2’-OMe-RNA Phosphoramidites 140–142Minor Groove 58Minor Groove Binder (MGB) 117Mixed Base Combinations 51Modifiers 74, 75, 78Mutagenesis 66

NNebularine2'-DeoxyNebularine-CEPhosphoramidite51

Nitroindole5-Nitroindole-CEPhosphoramidite52

Non-canonical Structures 72

OOligo-Affinity Support

OAS PS 151

OMe-RNA Synthesis2’-OMe-RNAPhosphoramidites1372’-OMe-RNASupports139Minor2’-OMe-RNAPhosphoramidites140

OMe-T 140Oxo-dA8-Oxo-dA-CEPhosphoramidite61

Oxo-dG8-Oxo-dG-CEPhosphoramidite61

PPdP-CEPhosphoramidite52, 54

PACE Phosphoramidites2’-OMe-RNA-PACEPhosphoramidites145DNA PACE phosphoramidites 37

PCR/Sequencing Utilities 51PerylenePerylene-dU-CEPhosphoramidite69, 121

PhenothiazinetC-CEPhosphoramidite70

PhenoxazinetC°-CEPhosphoramidite70

Phosphonocarboxylate Monomers 37Phosphorylation3’-CPRIICPG823'-PhosphateCPG823'-PhosphateCPG-HighLoad823'-PhosphatePS82ChemicalPhosphorylationReagent82ChemicalPhosphorylationReagentII82CPR II 82Solid CPR II 82

Photoaffinity Labelling 58Photocleavable MonomersPCAmino-ModifierPhosphoramidite76, 86, 95PCBiotinPhosphoramidite86, 100PC Linker Phosphoramidite 86PC Spacer Phosphoramidite 84, 86

Photo cross-linking 58, 124Photo-Regulation of DNA Function 70NPOM-Caged-dT-CEPhosphoramidite70

Photo-responsive DNAAzobenzenePhosphoramidite123

Phthalimide (PT)3'-PT-Amino-ModifierC3CPG793'-PT-Amino-ModifierC6CPG793'-PT-Amino-ModifierC6PS79

Poly-Pak™ PurificationPoly-Pak™Cartridge149Poly-Pak™IICartridge149Poly-Pak™Packing148, 149Reagents 148, 149

Polystyrene Supports3'-(6-FAM)PS1043'-BiotinTEGPS1013'-DabcylPS983'-PhosphatePS823'-PT-Amino-ModifierC6PS793'-TAMRAPS113GlenUnySupportPS24Universal Support III PS 26

PPG 57Propyne DerivativespdC-CEPhosphoramidite46pdU-CEPhosphoramidite46

Protein-DNA Interaction 58PseudoU1-Me-PseudouridinePhosphoramidite1352’-deoxypseudoU-CEPhosphoramidite58PseudoUridine-CEPhosphoramidite134

Psoralen LabellingPsoralenAzide93, 118

168

INDEX

Psoralen C2 Phosphoramidite 118Psoralen C6 Phosphoramidite 118

PurificationGlen-Pak™Purification147, 148Poly-Pak™Purification149

PurinePurine 52

PuromycinPuromycinCPG122

Pyrene5’-PyreneCapPhosphoramidite49Pyrene-dU-CEPhosphoramidite69, 70, 121

Pyridin-2-one-CE Phosphoramidite 134Pyrrolidine-CE Phosphoramidite 63Pyrrolo-CPyrrolo-C-TOM-CEPhosphoramidite132Pyrrolo-dC-CEPhosphoramidite68Pyrrolo-dCTP68

QQ-Supports 27, 28Quenched Autoligation (QUAL) Probes 1225’-Dabsyl-dT-CEPhosphoramidite122

RRedmond Red®RedmondRed®CPG109RedmondRed®Phosphoramidite108

Repair Enzyme 61Reverse Synthesis 34Rhodamine 1132’-MOE RNA Phosphoramidites2’-MOERNAPhosphoramidites142

RNA Supportsfor3’DNAModification125

RNA SynthesisMinor RNA Phosphoramidites 130, 135RNA Phosphoramidites 128, 129RNA Supports 129, 130RNASupportsforTOM-RNASynthesis126, 127TOM-ProtectedMinorRNAPhosphoramidites127, 131,

132, 133TOM-ProtectedRNAPhosphoramidites126

SSaccharin 1-Methylimidazole 30SBC Oligos 48Sequence Modifiers 78Serinol Backbone3’-6-FluoresceinSerinolCPG96, 1043’-Alkyne-ModifierSerinolCPG80, 89, 97

3’-Amino-ModifierSerinolCPG79, 963’-DithiolSerinolCPG80, 973’-ProtectedBiotinLCSerinolCPG96, 1013’-ProtectedBiotinSerinolCPG95, 1016-FluoresceinSerinolPhosphoramidite94, 103Alkyne-ModifierSerinolPhosphoramidite89, 96Amino-ModifierSerinolPhosphoramidite78, 95COT Serinol Phosphoramidite 97Dithiol Serinol Phosphoramidite 76, 95ProtectedBiotinLCSerinolPhosphoramidite95, 100ProtectedBiotinSerinolPhosphoramidite94, 100

SIMASIMA(HEX)-dTPhosphoramidite105SIMA(HEX)Phosphoramidite105

SMI 30Spacer Modifiers1-Ethynyl-dSpacerCEPhosphoramidite903'-SpacerC3CPG84dSpacer CE Phosphoramidite 84PC Spacer Phosphoramidite 84, 86rSpacer TBDMS CE Phosphoramidite 134Spacer C12 CE Phosphoramidite 84Spacer Phosphoramidite 9 84Spacer Phosphoramidite 18 84Spacer Phosphoramidite C3 84

Spermine Phosphoramidite 48Spin LabelsTEMPOAzide93TEMPO-TEGAzide93

Stearyl Labelling 1155’-StearylPhosphoramidite115

SterlingIntroduction7

Structural Studies 57Structure/Activity Relationship 57Sulfurizing Reagent 33Sulfurizing Reagent II 33

TT2’,3’-ddT562-Thio-dT-CEPhosphoramidite583’-Amino-dTCPG643'-Amino-ModifierC6dTCPG813'-dT-CEPhosphoramidite653'-dT-CPG55, 663'-Fluorescein-dTCPG1044-Thio-dT-CEPhosphoramidite585,6-Dihydro-dT-CEPhosphoramidite615'-Amino-dT-CEPhosphoramidite555’-Dabsyl-dT1225'-I-dT-CEPhosphoramidite895'-OMe-dT-CEPhosphoramidite55Amino-ModifierC2dT77Amino-ModifierC6dT77

169

MIS

CELL

ANEO

US

INDEX

C8-Alkyne-dT-CEPhosphoramidite87C8-TIPS-Alkyne-dT-CEPhosphoramidite88C8-TMS-Alkyne-dT-CEPhosphoramidite88DBCO-dT-CEPhosphoramidite91dT-CEPhosphoramidite8, 12, 15, 16, 18, 20dT-H-Phosphonate39dT-MePhosphonamidite36dT-MePhosphoramidite38dT-PACEPhosphoramidite37EDTA-C2-dT-CEPhosphoramidite118Ferrocene-dT-CEPhosphoramidite119Fluorescein-dT103N3-Cyanoethyl-dT71NPOM-Caged-dT70O4-Me-dT-CEPhosphoramidite47, 66S-Bz-Thiol-ModifierC6-dT78TAMRA-dT113ThymidineGlycolCEPhosphoramidite62T-LNA-CEPhosphoramidite41

TAMRA Labelling3'-TAMRACPG1133'-TAMRAPS113TAMRA-dT113TAMRA NHS Ester 113

tCtC-CEPhosphoramidite70

tCnitrotCnitro-CEPhosphoramidite70

tCoRibo-tC°-CEPhosphoramidite136tC°-CEPhosphoramidite70

TEMPOTEMPOAzide93TEMPO-TEGAzide93

Termination, 3'2’,3’-ddA562’,3’-ddC562’,3'-ddC-CPG562’,3’-ddG562’,3’-ddT563'-3'linkage35, 553'-dA-CPG553'-dC-CPG553'-dG-CPG553'-dT-CPG553'-SpacerC3CPG84

Termination, 5'5'-OMe-dT-CEPhosphoramidite55

Terminus Modifiers 74TET 1026-TETAzide92, 93

Thio-dT2-Thio-dT-CEPhosphoramidite584-Thio-dT-CEPhosphoramidite58

Thio-dU4-Thio-dU-CEPhosphoramidite58

Thio-G6-Thio-dG-CEPhosphoramidite586-Thio-G-CEPhosphoramidite133

Thiol-Modifiers3’-DithiolSerinolCPG803’-Thiol-Modifier6S-SCPG805’-Thiol-ModifierC676Dithiol Serinol Phosphoramidite 76S-Bz-Thiol-ModifierC6-dT78Thiol-ModifierC6S-S76

Thiophosphoramidites2’-OMe-RNAThiophosphoramidites141

Thio-U4-Thio-U-TOM-CEPhosphoramidite131

Thymidine GlycolThymidineGlycolCEPhosphoramidite62

Thymine DimerCis-synThymineDimerPhosphoramidite63

Tm Modulation 53Tocopherola-Tocopherol-TEGPhosphoramidite115

TOM-Protecting-GroupAc-A-RNA-CPG126Ac-C-RNA-CPG127Ac-G-RNA-CPG127A-TOM-CEPhosphoramidite126C-TOM-CEPhosphoramidite126G-TOM-CEPhosphoramidite126U-RNA-CPG127U-TOM-CEPhosphoramidite126

Trebler PhosphoramiditeTrebler85

Trimer phosphoramidites 42Trimethoxystilbene5’-TrimethoxystilbeneCapPhosphoramidite49

Triphosphate NucleotidesPyrrolo-dCTP68

Triplex 57Triplex-forming oligonucleotides 72

UU2’-F-U-ANACEPhosphoramidite1442'-OMe-5-Br-U-CEPhosphoramidite1402'-OMe-5-F-U-CEPhosphoramidite1402'-OMe-5-Me-U-CEPhosphoramidite1402'-OMe-U-CEPhosphoramidite1372’-OMe-U-PACEPhosphoramidite1452'-OMe-U-RNA1393’-UaqCapCPG49, 644-Thio-dU-CEPhosphoramidite585,6-Dihydro-dU-CEPhosphoramidite61

170

INDEX

5-Br-dU-CEPhosphoramidite605-Br-dU-CPG605-Ethynyl-dU-CEPhosphoramidite885-F-dU-CEPhosphoramidite605-Hydroxymethyl-dU-CEPhosphoramidite615-I-dU-CEPhosphoramidite605-I-U-CEPhosphoramidite1335-Me-U-2'-MOE-Phosphoramidite1425-OH-dU-CEPhosphoramidite61Amino-ModifierC6-UPhosphoramidite132Br-U-CEPhosphoramidite133dU-CEPhosphoramidite51dU-CPG50051dU-CPG100051O4-Triazolyl-dU-CEPhosphoramidite67pdU-CEPhosphoramidite46Perylene-dU-CEPhosphoramidite69Pyrene-dU-CEPhosphoramidite69, 70TIPS-5-Ethynyl-dU-CEPhosphoramidite88TMP-F-dU-CEPhosphoramidite67U-CEPhosphoramidite128, 129U-RNA-CPG125, 127, 130U-TOM-CEPhosphoramidite126

UltraMILD Deprotection2'-OMe-Ac-C-CEPhosphoramidite1382'-OMe-iPr-Pac-G-CEPhosphoramidite1382'-OMe-Pac-A-CEPhosphoramidite138Ac-C-CEPhosphoramidite129Ac-dC-CEPhosphoramidite23CapMixA23, 38, 130, 138iPr-Pac-dG-CEPhosphoramidite23iPr-Pac-G-CEPhosphoramidite129Pac-A-CEPhosphoramidite129Pac-dA-CEPhosphoramidite23PotassiumCarbonateinMethanol23, 38, 130, 138

UniCap Phosphoramidite 32Universal Support III

Universal Support III PS 26

Unnatural base pairs 48Unnatural Base Pairs5-Me-isodC53isodG 53

VVitamin E 115

WdW-CE Phosphoramidite 46

XX2’-dX-CEPhosphoramidite597-deaza-dX-CEPhosphoramidite57

X-ray crystallography 60

YYakima Yellow®YakimaYellow®CPG109YakimaYellow®Phosphoramidite108

ZZebularine5-Me-2'-deoxyZebularine-CEPhosphoramidite71Zebularine-CEPhosphoramidite134

Zip Nucleic Acid 48Spermine Phosphoramidite 48

ZNA® 48Spermine Phosphoramidite 48

171

MIS

CELL

ANEO

US

INDEX

172

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