Synthesis and Amylin Receptor Activity of Glycomimetics of Pramlintide using Click

Chemistry

LaurenR.Yule,a,b,cRebekahL.Bower,aHarveenKaur,b,cRenataKowalczyk,a,b,cDebbieL.Hay,a,cMargaretA.Brimble.*a,b,c

aThe School of Biological Sciences, University of Auckland, 3 Symonds St, Auckland 1010, New Zealand. E-mail:

dl.hay@auckland.ac.nz

bThe School of Chemical Sciences, University of Auckland, 23 Symonds St, Auckland 1010, New Zealand. E-mail:

m.brimble@auckland.ac.nz

cMaurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Private Bag 92019, Auckland 1010, New

Zealand.

1.Materials

All reagents were purchased as reagent grade and used without further purification. O-(6-Chlorobenzotriazol-1-yl)-

N,N,N',N'-tetramethyluronium hexafluorophosphate (HCTU), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium

hexafluorophosphate(HATU),N-(9-fluorenylmethoxycarbonyloxy)succinimide(FmocOSu),4-[(R,S)-α-[1-(9H-fluoren-9-yl)]-

methoxycarbonylamino]-2,4-dimethoxy]phenoxyacetic acid (Fmoc-Rink amide linker) 19 and Fmoc-amino acids were

purchasedfromGLBiochem(Shanghai,China).Fmoc-aminoacidsweresuppliedwiththefollowingside-chainprotection:

Fmoc-Tyr(tBu)-OH,Fmoc-Thr(tBu)-OH,Fmoc-Ser(tBu)-OH,Fmoc-Asn(Trt)-OH,Fmoc-His(Trt)-OH,Fmoc-Arg(Pbf)-OH,Fmoc-

Gln(Trt)-OH,Fmoc-Cys(Trt)-OH,Fmoc-Lys(Boc)-OH.

Fmoc-Ser(tBu)-Ser(ΨMe,Mepro)-OH 21 was purchased from Aapptec (Louisville, Kentucky). N,N-Diisopropylethylamine

(iPr2NEt), 2,4,6-collidine, piperidine, N,N’-diisopropylcarbodiimide (DIC), 3,6-dioxa-1,8-octane-dithiol (DODT),

triisopropylsilane (iPr3SiH), 1-methyl-2-pyrrolidinone (NMP), 6-chloro-1-hydroxybenzotriazole (6-Cl-HOBt), ninhydrin,

phenol,potassiumcyanide(KCN),methanol(MeOH),ethanol(EtOH),diethylether(Et2O),hydrazinehydrate(NH2NH2·1.5

H2O), and copper(II) sulphate pentahydrate (CuSO4·5 H2O) were purchased from Sigma-Aldrich (St. Louis, Missouri).

Dichloromethane (CH2Cl2), disodiumhydrogenphosphate (Na2HPO4),magnesium sulphate (MgSO4), sodiumbicarbonate

(NaHCO3),sodiumcarbonate(Na2CO3),ethylacetate(EtOAc)andhexanewerepurchasedfromECPlimited(Auckland,New

Zealand). Hydrochloric acid (HCl), sodium hydroxide (NaOH), N,N-dimethylformamide (DMF) (synthesis grade), and

acetonitrile (MeCN),were purchased from Scharlau (Barcelona, Spain). Dimethyl sulfoxide (DMSO)was purchased from

Romil Limited (Cambridge, United Kingdom). Tris(2-carboxethyl)-phosphine hydrochloride (TCEP·HCl) and L-

propargylglycine(L-Pra)werepurchasedfromAKScientific (UnionCity,California).Tetrahydrofuran(THF)waspurchased

fromAvantorPerformanceMaterials(CentreValley,Pennsylvania).Guanidiniumchloride(Gu·HCl)waspurchasedfromMP

Biomedicals(SantaAna,California).Trifluoroaceticacid(TFA)waspurchasedfromHalocarbon(RiverEdge,NewJersey).

Aminomethyl polystyrene resin 18 (AMPS)1 and Fmoc-propargylglycine20 (Fmoc-L-Pra-OH)2were synthesised following

literature procedures. 2-acetamido-2-deoxy-β-D-glucopyranosyl azide (β-GlcNAcN3) 22 and N4-(2-Acetamido-3,4,6-tri-O-

acetyl-2-deoxy-β-D-glucopyranosyl)-N2-(9-fluorenylmethylcarbonyl)asparagine (Fmoc-Asn(GlcNAc(OAc)3)-OH) 233 were

suppliedfromProfessorAntonyFairbanksfromtheUniversityofCanterbury,NewZealand.

Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2016

2.Generalprocedureforpeptidesynthesis

Peptides were synthesised by automated 9-fluorenylmethoxycarbonyl solid phase peptide synthesis (Fmoc-SPPS) using

eitheramicrowaveenhancedBiotage®initiator+alstraoraroomtemperature(rt)TributeTMpeptidesynthesiserona0.1

mmolscale.

Using the TributeTM peptide synthesiser, all amino acid couplings were performed as single coupling cycles. Protected

aminoacidswere incorporatedusingFmoc-AA-OH (5.0eq.,0.5M),HCTU (4.5eq.,0.45M)and iPr2NEt (10eq.,2M) in

DMF,for45min.Fmoc-[Asn[GlcNAc(OAc)3]-OH23(2eq.),Fmoc-L-Pra-OH20(2eq.)andFmoc-Ser(tBu)-Ser(ΨMe,Mepro)-OH

(2eq.)21werecoupledfor1.5hatroomtemperatureinthepresenceofHATU(1.9eq.)andcollidine(6eq.)inDMF.The

Fmocgroupwasremovedusing20%piperidineinDMF(2x5min).

UsingtheBiotage®initiator+alstrapeptidesynthesiser,allaminoacidcouplingswereperformedassinglecouplingcycles,

with theexceptionof Fmoc-Arg(Pbf)-OH, Fmoc-His(Trt)-OHand Fmoc-Cys(Trt)-OHwhere adoubling coupling cycle was

performed. Protected amino acids were incorporated using Fmoc-AA-OH (5.0 eq., 0.2M), HCTU (4.5 eq., 0.23M) and

iPr2NEt(10eq.,2M)inDMF,for5minatamaximumtemperatureof75°Candat25W,exceptFmoc-Arg(Pbf)-OHwhich

wascoupledfor25minatrtfollowedbyasecondcouplingfor3minatamaximumtemperatureof72°Candat25W,and

Fmoc-His(Trt)-OHandFmoc-Cys(Trt)-OHwhichwerecoupledfor10minatrtfollowedbyasecondcouplingfor5minata

maximumtemperatureof47°Candat25W.Fmoc-Pra-OH20(2eq.),andFmoc-Ser(tBu)-Ser(ΨMe,Mepro)-OH(2eq.)21and

Fmoc-Asn[GlcNAc(OAc)3]-OH233(2eq.)werecoupledfor15minatamaximumtemperatureof75°Candat25Winthe

presenceofHATU(1.9eq.)andcollidine(6eq.).TheFmocgroupwasremovedusing20%piperidineinDMF(2x3minata

maximumtemperatureof70°Candat62W).

Peptides were cleaved from the resin by treatment with trifluoroacetic acid/triisopropylsilane/water/3,6-dioxa-1,8-

octanedithiol (TFA/TIS/H2O/DODT) (v/v/v/v; 94/1/2.5/2.5) for 2.5 h at rt The crude peptides were precipitated and

trituratedwithcolddiethylether(40mL),isolated(centrifugation),anddissolvedinMeCN/H2O(1:1)containing0.1%TFA

andlyophilised.

3.Generalprocedureforpurificationandanalysis

Analytical reversephasehigh-performance liquid chromatography (RP-HPLC)wasperformedon aDionexultimate3000

usingthefollowingcolumns:VydacDiphenyl300Å,3µm,4.6mmx250mm,AgilentZorbax300SB-C3,3.0mmx150mm;

3.5µm,AgilentZorbax300SB-C3,4.6mmx150mm;5µm.Analytical liquid-chromatography-massspectrometry(LCMS)

wasperformedonanAgilentTechnologies1120CompactLCconnectedtoaHPSeries1100MSDspectrometerusingan

Agilent Zorbax 300SB-C3, 3.0mm x 150mm; 3.5 µm column. Semi-preparative reverse phase high-performance liquid

chromatography (RP-HPLC) was performed using either a Waters 600E System with a Waters 2487 dual wavelength

absorbancedetectororaDionexUltimate3000usingthefollowingcolumns:PhenomenexGeminiC18110Å,10.0mmx

250mm; 5 µm (5mL/min) or Vydac Diphenyl 300 Å, 10.0mm x 250mm; 5 µm (5mL/min). A linear gradient of 0.1%

trifluoroaceticacid/water (A)and0.1%trifluoroaceticacid/acetonitrile (B)wasusedwithdetectionat210nm.Gradient

systemsused for semi-preparativeRP-HPLCwereadjustedaccording to theelutionandpeakprofilesobtained fromthe

analyticalRP-HPLCchromatograms,andarespecifiedintheexperimentalproceduressection.

4.Generalprocedurefordisulfidebondformation(Cys-2/Cys-7)forpramlintide1andanalogues5-7

Thefinalpeptideconcentrationusedforeachdisulfidebondformationreactionwas3mMinamixtureofGu·HCl (6M)

andNa2HPO4buffer(adjustedtothefinalconcentrationof0.2M).Themixturewasthenagitatedatrtfor2h.Thecrude

productwaslyophilisedandpurifiedbyRP-HPLCusingconditionsasspecifiedingeneralproceduressection3.

5.GeneralprocedureforCu(I)mediatedazide-alkynecycloaddition“click”reactionwithsimultaneousdisulfidebondformation(Cys-2/Cys-7)foranalogues2-4and10-11

Thefinalpeptideconcentrationusedforeach“click”reactionwas3mMinasolutionofGu·HCl(6M)andNa2HPO4buffer

(adjustedtothefinalconcentrationof0.2M).ThefinalconcentrationsofTCEP·HClandCuSO4·5H2Owereadjustedto20

mM.

0.5MStock solutionsofTCEP·HClandCuSO4·5H2Owereprepared.100µlof the0.5Mstock solutionofTCEP·HClwas

basifiedtopH7usingsolidNaOH.Therequiredvolumeof0.5MCuSO4·5H2O(6.7eq.)wasaddedtotherequiredvolume

of0.5M,pH7TCEP·HCl(6.7eq.),mixed,andshakenfor1minuntilablueprecipitateformed.Partialdisappearanceofthe

blue precipitate was observed over the next 2-3min. The peptide containing a propargylglycine residue (1.0 eq.) was

dissolved in thedeoxygenated (Ar,30min)buffer solutionofGu·HClandNa2HPO4. ThecloudymixtureofCuSO4·5H2O/

TCEP·HClwasthenaddedtothepeptidesolutionportion-wisetofurthersolubilisetheprecipitate.Themixturewasthen

incubated for30minat 60 °Cafterwhich timea clear, faintblue solutionwasobtained.GlcNAcN322 (6 eq.)was then

addedtothemixtureandthesolutionwaspurgedwithAr,whichwasthensubjectedtomicrowaveirradiationfor2hat60

°Cand20WandthereactionprogresswasmonitoredbyRP-HPLC.Thecrudeproductwasdiluted(H2O,1mL),acidifiedto

pH1(TFA),lyophilised,andpurifiedbyRP-HPLCusingconditionsasspecifiedingeneralproceduressection3.

6.Generalprocedureforacetateremovalwithsimultaneousdisulfidebondformation(Cys-2/Cys-7)foranalogues8-13

Thecrudeacetateprotectedglycopeptidewasdissolvedin5%NH2NH2·1.5H2OinDMSOtoreachafinalconcentrationof3

mg/ml.Themixturewasagitatedatrtfor3handDMSOwasremovedbyRP-HPLCusinganisocraticmethodof80%Bfor

15 minutes. The crude peptide was then lyophilised, and purified by RP-HPLC using conditions specified in general

procedures3.

7.Generalprocedureformeasuringtheagonisteffectofpramlintide1andpramlintideanalogues2-13attheAMY1(a)receptor

Pramlintide1 andglycopeptides2-13werescreenedat theAMY1(a) receptor.Pramlintide1was includedasacontrol in

each experiment. Cos 7 cells were transiently transfected with the necessary receptor components, and cyclic AMP

productionwasmeasuredaccordingtoourpublishedmethods.4,5,6ThehCT(a)constructusedwastheinsertnegativehCT(a)

receptorwithleucineatthepolymorphicaminoacidposition447andanN-terminalhemagglutinintaginpcDNA3.1vector

(fromProfessorPatrickSexton,MonashInstituteofPharmaceuticalSciences,Monash,Australia).HumanRAMP1withan

N-terminalmyctaginapcDNA3vectorwasused(fromStevenFoord,GlaxoSmithKline).Peptides1-13wereweighedout

andstocksolutionsmadeat1mMor10mM(dilutedinsterilewater),onthebasisofpeptideweight.80%peptidecontent

wasassumedandtakenintoaccountinthesecalculations.AllpeptidestocksolutionswerestoredinsiliconisedorLobind

(Eppendorf,Hamburg,Germany)microcentrifugetubesat-30°Cin2-6μLaliquotstominimisefreeze-thawcycles.

SynthesisofPramlintide1

AutomatedFmoc-SPPSusingTributeTMrtpeptidesynthesiserwasusedforthesynthesisofthelinearpramlintide14,which

wasfollowedbyresincleavageusingtheconditionsoutlinedingeneralprocedure2toaffordcrudereduced14asawhite

solid (172mg,19%yieldbasedon43%puritybyLCMS), (FigureS1). Thecrude linearpeptide14 (9.62mg,2.43×10-3

mmol)was dissolved in amixture of 6MGu·HCl (0.81mL) andNa2HPO4 (20mg, 0.16mmol) to form a disulfide bond

betweenCys-2andCys-7accordingtogeneralprocedure4(FigureS2)toaffordcrudepramlintide1.

The crude pramlintide 1 was purified by semi-preparative RP-HPLC using Dionex Ultimate 3000 on a Vydac Diphenyl

column,usingagradientof0%Bto13%Bover13min(ca.1%B/min)then13%Bto60%Bover313min(ca.0.15%B/min).

Fractionswerecollectedat0.5minintervalsandanalysedbyESI-MSandRP-HPLC.Fractionsidentifiedwiththecorrectm/z

werecombinedandlyophilisedtoaffordthetitlecompound1asawhiteamorphoussolid(2.75mg,67%yield,96%purity);

Rt30.06min;m/z(ESI-MS)987.7([M+4H]4+requires988.4),FigureS3.

FigureS1LCMSprofileof crude linearpramlintide14 (ca 43%asanalysedbypeakareaofRP-HPLCat214nm); lineargradientof5%Bto65%Bover20min(ca.3%B/min)at40°C,0.3mL/min.

FigureS2LCMSprofileofcrudepramlintide1; lineargradientof5%Bto65%Bover20min, (ca.3%B/min)at40°C,0.3

mL/min.

FigureS3LCMSprofileofpurepramlintide1(97%);lineargradientof5%Bto65%Bover60min,(ca.1%B/min)at40°C,

0.3mL/min.

Synthesisofpramlintideanalogue2

AutomatedFmoc-SPPSusingTributeTMrtpeptidesynthesiserwasusedforthesynthesisofreducedpramlintideprecursor

15,whichwas followedby resin cleavageusing the conditionsoutlined in generalprocedure2 toafford crude reduced

pramlintideanalogue15asawhitesolid(170mg,29%yieldbasedon68%puritybyLCMS)(FigureS4).Thecrudelinear

peptide15(20mg,4.7×10-3mmol)underwentaCu(I)mediatedcycloadditionreactionandsimultaneousdisulfidebond

formation(betweenCys-2andCys-7)withGlcNAcN322(7.45mg,3.1×10-2mmol)accordingtogeneralprocedure5(Figure

S5).Thisreactionwascarriedoutusing0.5MTCEP·HCl(68µL,3.4×10-2mmol,pH7),0.5MCuSO4·5H2O(68µL,3.4×10-2

mmol)andNa2HPO4(48.3mg,0.34mmol)in6MGu·HCl(1.56mL)toaffordcrudepramlintideanalogue2.

The crudepramlintide analogue2waspurifiedby semi-preparativeRP-HPLCusingDionexUltimate3000onaDiphenyl

Vydac column, using a gradient of 0%B to 13%B over 13 min (ca. 1%B/min) then 13%B to 60%B over 313 min (ca.

0.15%B/min).Thisaffordedthetitlecompound2asawhiteamorphoussolid(3.3mg,23%yield,95%purity);Rt15.23min;

m/z(ESI-MS)1045.0([M+4H]4+requires1045.1),FigureS6.

FigureS4LCMSprofileofcrudelinearpramlintideanalogue15(ca68%asanalysedbypeakareaofRP-HPLCat214nm);lineargradientof5%Bto65%Bover20min,(ca.3%B/min)at40°C,0.3mL/min.

FigureS5:LCMSprofileofcrudepramlintideanalogue2;lineargradientof5%Bto65%Bover20min,(ca.3%B/min)at40°C,0.3mL/min.

FigureS6:LCMSprofileofpurepramlintideanalogue2(95%);lineargradientof5%Bto65%Bover20min,(ca.3%B/min)at40°C,0.3mL/min.

Synthesisofpramlintideanalogue3

AutomatedFmoc-SPPSusingTributeTMrtpeptidesynthesiserwasusedforthesynthesisofreducedpramlintideprecursor

16whichwas followedby resin cleavage using the conditions outlined in general procedure 2 to afford crude reduced

pramlintideanalogue16asawhitesolid(222mg,30%yieldbasedon53%puritybyLCMS)(FigureS7).Thecrudelinear

peptide16(20mg,4.7×10-3mmol)underwentaCu(I)mediatedcycloadditionreactionandsimultaneousdisulfidebond

formation(betweenCys-2andCys-7)withGlcNAcN322(7.45mg,3.1×10-2mmol)accordingtogeneralprocedure5(Figure

S8).Thisreactionwascarriedoutusing0.5MTCEP·HCl(68µL,3.4×10-2mmol,pH7),0.5MCuSO4·5H2O(68µL,3.4×10-2

mmol)andNa2HPO4(48.3mg,0.34mmol)in6MGu·HCl(1.56mL)toaffordcrudepramlintideanalogue3.

The crudepramlintide analogue3waspurifiedby semi-preparativeRP-HPLCusingDionexUltimate3000onaDiphenyl

Vydac column, using a gradient of 0%B to 13%B over 13 min (ca. 1%B/min) then 13%B to 60%B over 313 min (ca.

0.15%B/min).Thisaffordedthetitlecompound3asawhiteamorphoussolid(1.1mg,10%yield,97%purity);Rt39.88min;

m/z(ESI-MS)1045.1([M+4H]4+requires1045.1),FigureS9.

FigureS7LCMSprofileofcrudelinearpramlintideanalogue16(ca53%asanalysedbypeakareaofRP-HPLCat214nm);

lineargradientof5%Bto65%Bover20min,(ca.3%B/min)at40°C,0.3mL/min.

FigureS8LCMSprofileofcrudepramlintideanalogue3;lineargradientof5%Bto65%Bover20min,(ca.3%B/min)at40°C,0.3mL/min.

FigureS9:AnalyticalRP-HPLCandESI-MSprofileofpurepramlintideanalogue3 (97%); lineargradientof5%Bto65%B

over60min(ca.1%B/min)at40°C,1mL/min.

Synthesisofpramlintideanalogue4

AutomatedFmoc-SPPSusingTributeTMrtpeptidesynthesiserwasusedforthesynthesisoflinearpramlintideanalogue17

which was followed by resin cleavage using the conditions outlined in general procedure 2 to afford crude linear

pramlintide17asawhitesolid(156mg,19%yieldbasedon48%puritybyLCMS)(FigureS10).Thecrudelinearpeptide17

(20mg,5.1×10-3mmol)underwentaCu(I)mediatedcycloadditionreactionandsimultaneousdisulfidebondformation

(betweenCys-2andCys-7)withGlcNAcN322(7.55mg,3.1×10-2mmol)accordingtogeneralprocedure5(FigureS11).This

reactionwascarriedoutusing0.5MTCEP·HCl(136µL,6.8×10-2mmol,pH7),0.5MCuSO4·5H2O(136µL,6.8×10-2mmol)

andNa2HPO4(48.3mg,0.34mmol)in6MGu·HCl(1.43mL)toaffordthecrudepramlintideanalogue4.

The crudepramlintide analogue4waspurifiedby semi-preparativeRP-HPLCusingDionexUltimate3000onaDiphenyl

Vydac column, using a gradient of 0%B to 13%B over 13 min (ca. 1%B/min) then 13%B to 60%B over 313 min (ca.

0.15%B/min).Thisaffordedthetitlecompound4asawhiteamorphoussolid(2.6mg,24%yield,96%purity);Rt15.32min;

m/z(ESI-MS)1101.8([M+4H]4+requires1101.8),FigureS12.

FigureS10LCMSprofileofcrudelinearpramlintideanalogue17(ca48%asanalysedbypeakareaofRP-HPLCat214nm);lineargradientof5%Bto65%Bover20min,(ca.3%B/min)at40°C,0.3mL/min.

FigureS11:LCMSprofileofcrudepramlintideanalogue4;lineargradientof5%Bto65%Bover20min,(ca.3%B/min)at40°C,0.3mL/min.

FigureS12LCMSprofileofpurepramlintideanalogue4(98%);lineargradientof5%Bto65%Bover20min,(ca.3%B/min)at40°C,0.3mL/min.

Synthesisofpramlintideanalogue5

Thecrudelinearpeptide15(seesynthesisofpramlintideanalogue2,FigS4)(20mg,4.7×10-3mmol)wasdissolvedina

solutionof 6MGu·HCl (1.56mL) andNa2HPO4 (44mg, 0.31mmol), to forma disulfidebondbetweenCys-2 andCys-7

accordingtogeneralprocedure4toaffordcrudepramlintideanalogue5(FigureS13).

The crudepramlintide analogue5waspurifiedby semi-preparativeRP-HPLCusingDionexUltimate3000onaDiphenyl

Vydac column using a gradient of 0%B to 14%B over 14 min (ca. 1%B/min) then 14%B to 60%B over 307 min (ca.

0.15%B/min).Fractionswerecollectedat0.5minintervalsandanalysedbyESI-MSandRP-HPLC.Fractionswiththecorrect

m/zwerecombinedand lyophilised toafford thetitlecompound5asawhiteamorphoussolid (3.6mg,26%yield,99%

purity);Rt16.28min;m/z(ESI-MS)983.5([M+4H]4+requires983.6),FigureS14.

FigureS13LCMSprofileofcrudepramlintideanalogue5;lineargradientof5%Bto65%Bover20min,(ca.3%B/min)at40°C,0.3mL/min.

FigureS14:LCMSprofileofpurepramlintideanalogue5(98%);lineargradientof5%Bto65%Bover20min,(ca.3%B/min)at40°C,0.3mL/min.

Synthesisofpramlintideanalogue6

Thecrudelinearpeptide16(seesynthesisofpramlintideanalogue3,FigS7)(20mg,4.7×10-3mmol)wasdissolvedina

solutionof 6MGu·HCl (1.56mL) andNa2HPO4 (44mg, 0.31mmol), to forma disulfidebondbetweenCys-2 andCys-7

accordingtogeneralprocedure4toaffordcrudepramlintideanalogue6(FigureS15).

Thecrudepramlintideanalogue6waspurifiedbysemi-preparativeRP-HPLCusingWaters600ESystemonaGeminiC18

column,usingagradientof0%Bto20%Bover20min(ca.1%B/min)then20%Bto60%Bover267min(ca.0.15%B/min).

Thisaffordedthetitlecompound6asawhiteamorphoussolid(3.5mg,33%yield,96%purity);Rt16.10min;m/z(ESI-MS)

983.5([M+4H]4+requires983.6),FigureS16.

FigureS15LCMSprofileofcrudepramlintideanalogue6;lineargradientof5%Bto65%Bover20min,(ca.3%B/min)at40

°C,0.3mL/min.

FigureS16LCMSprofileofpurepramlintideanalogue6(96%);lineargradientof5%Bto65%Bover20min,(ca.3%B/min)

at40°C,0.3mL/min.

Synthesisofpramlintideanalogue7

Thecrudelinearpeptide17(seesynthesisofpramlintideanalogue4,FigS10)(20mg,5.1×10-3mmol)wasdissolvedina

solutionof 6MGu·HCl (1.70mL) andNa2HPO4 (48mg, 0.34mmol), to forma disulfidebondbetweenCys-2 andCys-7

accordingtogeneralprocedure4toaffordcrudepramlintideanalogue7(FigureS17).

Thecrudepramlintideanalogue7waspurifiedbysemi-preparativeRP-HPLCusingDionexUltimate3000onaGeminiC18

column,usingagradientof0%Bto23%Bover23min(ca.1%B/min)then23%Bto60%Bover247min(ca.0.15%B/min).

Thisaffordedthetitlecompound7asawhiteamorphoussolid(6.90mg,8%yield,99%purity);Rt34.97min;m/z(ESI-MS)

978.5([M+4H]4+requires978.9),FigureS18.

FigureS17LCMSprofileofcrudepramlintideanalogue7;lineargradientof5%Bto65%Bover20min,(ca.3%B/min)at40°C,0.3mL/min.

FigureS18:AnalyticalRP-HPLCandESI-MSprofileofpurepramlintideanalogue7(99%);5%Bto65%Bover60min(ca.1%B/min)at40°C,1mL/min.

Synthesisofpramlintideanalogue8

AutomatedFmoc-SPPSusingTributeTMrtpeptidesynthesiserwasusedforthesynthesisofthecrudereducedandacetate

protectedpramlintideanalogue24whichwas followedbyresincleavageusingtheconditionsasoutlined in thegeneral

procedure2toaffordcrudereducedandacetateprotectedpramlintide24asawhitesolid(186mg,45%yieldbasedon

45%puritybyLCMS)(FigureS19).Acetateprotectinggroupswereremovedfrompeptide24 (30mg,7.22×10-3mmol)

along with simultaneous disulfide bond formation between Cys-2 and Cys-7 using conditions described in general

procedure6usingNH2NH2·1.5H2O(0.5mL)inDMSO(9.5mL),toaffordcrudepramlintideanalogue8(FigureS20).

Thecrudepramlintideanalogue8waspurifiedbysemi-preparativeRP-HPLCusingDionexUltimate3000onaGeminiC18

column,usingagradientof0%Bto25%Bover25min(ca.1%B/min)then25%Bto60%Bover233min(ca.0.15%B/min).

Thisaffordedthetitlecompound8asawhiteamorphoussolid(5.5mg,42%yield,99%purity);Rt30.17min;m/z(ESI-MS)

1038.9([M+4H]4+requires1039.1),FigureS21.

FigureS19:LCMSprofileofcrudelinearandacetateprotectedpramlintideanalogue24(ca45%asanalysedbypeakarea

ofRP-HPLCat214nm);lineargradientof5%Bto65%Bover20min,(ca.3%B/min)at40°C,0.3mL/min.

FigureS20:LCMSprofileofcrudepramlintideanalogue8;lineargradientof5%Bto65%Bover20min,(ca.3%B/min)at40

°C,0.3mL/min.

FigureS21:LCMSprofileofpurepramlintideanalogue8(98%);lineargradientof5%Bto65%Bover60min,(ca.1%B/min)

at40°C,0.3mL/min.

Synthesisofpramlintideanalogue9

AutomatedFmoc-SPPSusingBiotage®microwaveenhancedpeptide synthesiserwasused for the synthesisof thecrude

reducedandacetateprotectedpramlintide25whichwas followedby resincleavageusing theconditionsasoutlined in

generalprocedure2 toafford crude reducedandacetateprotectedpramlintide25asawhite solid (256mg,23%yield

basedon41%purityoffullyacetateprotectedproductbyLCMS)(FigureS22).Acetateprotectinggroupswereremoved

frompeptide25 (32mg,7.34×10-3mmol)alongwith simultaneousdisulfidebond formationbetweenCys-2andCys-7

using conditions described in general procedure 6 using NH2NH2·1.5 H2O (0.5 mL) in DMSO (9.5 mL) to afford crude

pramlintideanalogue9(FigureS23).

Thecrudepramlintideanalogue9waspurifiedbysemi-preparativeRP-HPLCusingDionexUltimate3000onaGeminiC18

column,usingagradientof0%Bto25%Bover25min(ca.1%B/min)then25%Bto60%Bover233min(ca.0.15%B/min).

Thisaffordedthetitlecompound9asawhiteamorphoussolid(4.5mg,36%yield,98%purity);Rt29.80min;m/z(ESI-MS)

1089.8([M+4H]4+requires1089.8),FigureS24.

FigureS22:LCMSprofileofcrudelinearandacetateprotectedpramlintideanalogue25; lineargradientof5%Bto65%B

over20min,(ca.3%B/min)at40°C,0.3mL/min.

FigureS23:LCMSprofileofcrudepramlintideanalogue9;lineargradientof5%Bto65%Bover20min,(ca.3%B/min)at40

°C,0.3mL/min.

FigureS24:LCMSprofileofpurepramlintideanalogue9(98%);lineargradientof5%Bto65%Bover60min,(ca.1%B/min)

at40°C,0.3mL/min.

Synthesisofpramlintideanalogue10

Crudepramlintide12(20mg,4.8×10-3mmol)underwentaCu(I)mediatedcycloadditionreaction(betweenCys-2andCys-

7)withGlcNAcN322 (7.15mg,2.9×10-2mmol)accordingtogeneralprocedure5(FigureS25).Thisreactionwascarried

outusing0.5MTCEP·HCl(64µL,3.2×10-2mmol),0.5MCuSO4·5H2O(64µL,3.2×10-2mmol)andNa2HPO4(45.7mg,0.32

mmol)in6MGu·HCl(1.48mL)toaffordcrudepramlintideanalogue10.

Thecrudepeptide10wasthenpurifiedbysemi-preparativeRP-HPLCusingDionexUltimate3000onaGeminiC18column,

using a gradient of 0%B to 15%B over 15min (ca.1%B/min) then 15%B to 60%B over 300min (ca.0.15%B/min). This

afforded the title compound10 asawhiteamorphoussolid (2.8mg,25%yield,99%purity).Rt38.43min;m/z (ESI-MS)

1095.8([M+4H]4+requires1095.8),FigureS26.

FigureS25:LCMSprofileofcrudepramlintideanalogue10;lineargradientof5%Bto65%Bover20min,(ca.3%B/min)at

40°C,0.3mL/min.

FigureS26:AnalyticalRP-HPLCandESI-MSprofileofpurepramlintideanalogue10(96%);5%Bto65%Bover63min(ca.

1%B/min)at40°C,1mL/min.

Synthesisofpramlintideanalogue11

Crudepramlintideanalogue13(20mg,4.8×10-3mmol)underwentaCu(I)mediatedcycloadditionreaction(betweenCys-

2andCys-7)withGlcNAcN322(7.15mg,2.9×10-2mmol)accordingtogeneralprocedure5(FigureS27).Thisreactionwas

carriedoutusing0.5MTCEP·HCl(64µL,3.2×10-2mmol),0.5MCuSO4·5H2O(64µL,3.2×10-2mmol)andNa2HPO4(45.7

mg,0.32mmol)in6MGu·HCl(1.48mL)toaffordcrudepramlintideanalogue11.

The crude pramlintide analogue 11 was then purified by semi-preparative RP-HPLC using Dionex Ultimate 3000 on a

GeminiC18 column,usingagradientof0%B to15%Bover15min (ca.1%B/min) then15%B to60%Bover300min (ca.

0.15%B/min).Thisafforded the title compound11 asawhiteamorphoussolid (3.1mg,28%yield,99%purity).Rt26.52

min;m/z(ESI-MS)1095.8[M+4H]4+([M+4H]4+requires1096.0),FigureS28.

FigureS27:LCMSprofileofcrudepramlintideanalogue11;lineargradientof5%Bto65%Bover20min,(ca.3%B/min)at

40°C,0.3mL/min.

FigureS28:AnalyticalRP-HPLCandESI-MSprofileofpurepramlintideanalogue11(99%);5%Bto65%Bover63min(ca.

1%B/min)at40°C,1mL/min,usingAgilentZorbax300SB-C3,4.6mmx150mm,5µmcolumn.

Synthesisofpramlintideanalogue12

AutomatedFmoc-SPPSusingTributeTMrtpeptidesynthesiserwasusedforthesynthesisofthecrudereducedandacetate

protectedpramlintide26whichwasfollowedbyresincleavageusingtheconditionsasoutlinedinthegeneralprocedure2

toaffordcrudereducedandacetateprotectedpramlintide26asawhitesolid(220mg,27%yieldbasedon52%purityby

LCMS) (Figure S 29). Acetate protecting groups were removed from peptide 26 (21 mg, 4.7 × 10-3 mmol) along with

simultaneousdisulfidebondformationbetweenCys-2andCys-7usingconditionsdescribedingeneralprocedure6using

NH2NH2·1.5H2O(0.88mL)inDMSO(16.7mL)toaffordcrudepramlintideanalogue12.

Thecrudepramlintideanalogue12wasthenpurifiedbysemi-preparativeRP-HPLCusingDionexultimate3000onaGemini

C18 column, using a gradient of 0%B to 15%B over 15 min (ca. 1%B/min) then 15%B to 60%B over 300 min (ca.

0.15%B/min).Thisafforded the title compound12 asawhiteamorphoussolid (3.8mg,36%yield,99%purity).Rt32.82

min;m/z(ESI-MS)1034.2([M+4H]4+requires1034.4),FigureS30.

FigureS29:LCMSprofileofcrudepramlintideanalogue26(ca52%asanalysedbypeakareaofRP-HPLCat214nm);linear

gradientof5%Bto65%Bover20min,(ca.3%B/min)at40°C,0.3mL/min.

FigureS30:AnalyticalRP-HPLCandESI-MSprofileofpurepramlintideanalogue12(98%);5%Bto65%Bover60min(ca.

1%B/min)at40°C,1mL/min.

Synthesisofpramlintideanalogue13

AutomatedFmoc-SPPSusingBiotage®microwaveenhancedpeptide synthesiserwasused for the synthesisof thecrude

reducedandacetateprotectedpramlintide27whichwasfollowedbyresincleavageusingtheconditionsasoutlinedinthe

generalprocedure2toaffordcrudereducedandacetateprotectedpramlintide27(240mg,30%yieldbasedon41%purity

offullyacetateprotectedproductbyLCMS)(FigureS31).Acetateprotectinggroupswereremovedfrompeptide27(22

mg, 4.9 × 10-3 mmol) along with simultaneous disulfide bond formation between Cys-2 and Cys-7 using conditions

describedingeneralprocedure6usingNH2NH2·1.5H2O(0.88mL)inDMSO(16.7mL)toaffordcrudepramlintideanalogue

13(FigureS32).

The crude pramlintide analogue 13 was then purified by semi-preparative RP-HPLC using Dionex Ultimate 3000 on a

GeminiC18 column,usingagradientof0%B to15%Bover15min (ca.1%B/min) then15%B to60%Bover300min (ca.

0.15%B/min).Thisafforded the title compound13 asawhiteamorphoussolid (4.1mg,36%yield,99%purity).Rt31.68

min;m/z(ESI-MS)1034.0([M+4H]4+requires1034.4),FigureS33.

FigureS29:LCMSprofileofcrudepramlintideanalogue31;lineargradientof5%Bto65%Bover20min,(ca.3%B/min)at

40°C,0.3mL/min.

FigureS30:LCMSprofileofcrudepramlintideanalogue32;lineargradientof5%Bto65%Bover20min,(ca.3%B/min)at

40°C,0.3mL/min.

FigureS31:AnalyticalRP-HPLCandESI-MSprofileofpurepramlintideanalogue33(99%);5%Bto65%Bover63min(ca.

1%B/min)at40°C,1mL/min.

References

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