CatalyticpromiscuityofglycopeptideN‐methyltransferasesenablesbio‐orthogonal labeling

ofbiosyntheticintermediates

ClaraBrieke,GraceYim,MadeleinePeschke,GerardD.Wright,MaxJ.Cryle

ElectronicalSupplementaryInformation

Content:

GeneralMethods

CloningandExpressionofMtfadbvandMtfapek

AlignmentofMtfadbvandMtfapekwithMtfacle

Synthesisofcompounds1,2a‐c

Mtaseinvitroactivityassayswithteicoplaninaglycone1

Loadingof3‐CoAontoapo‐PCP‐Xtei

CoupledP450/Mtaseinvitroactivityassay

IEDDAofmodifiedteicoplaninaglycone1cwithtetrazines

HPLC,HPLC‐MS‐andMS/MS‐Characterisationofpeptidesisolatedfrominvitroactivitystudies

Chemicalstructuresoftype‐IVandtype‐IGPAs

Antibioticactivityassaysof1,1b,1c

NMRcharacterizationof1,1b,1c

References

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

GeneralMethods

All chemicals and solvents were obtained from commercial suppliers (Sigma‐Aldrich; VWR) and

usedwithoutfurtherpurification.

HPLC analysis and purifications were carried out using a High Performance Liquid

Chromatograph/MassSpectrometerLCMS‐2020(ESI,operatingbothinpositiveandnegativemode)

equippedwith a SPD‐M20A Prominence Photo Diode Array Detector in preparativemode and a

SPD‐20A Prominence Dual Wavelength UV Detector in analytical mode, all from Shimadzu. For

analytical analyses the solvent deliverymodule LC‐20ADwas used; for preparative purifications

twoLC‐20APunitswereused.AnalyticalseparationswereperformedonWatersXBridgeBEH300

C18 columns (5 or 10 µm, 4.6 x 250mm). Preparative separations were performed on aWaters

XBridgeBEH300PrepC18column(5µm,19x150mm)ataflowrateof20mL/min.Thesolvents

usedwereHPLC‐grade acetonitrile + 0.1% formic acid (solventA) andwater + 0.1% formic acid

(solvent B). UHPLC‐MS measurements were performed on a Shimadzu Nexera X2 LCMS 8050

system(triplequadrupoleESI,operatingbothinpositiveandnegativemode)usinganAcquityUPLC

peptideBEHC18column(130Å,1.7µm,2.1x100mm)andMS‐gradeacetonitrile+0.1%formic

acid(solventA)andwater+0.1%formicacid(solventB)assolvents.ElectrosprayIonizationMass

Spectrometry(ESI‐MS)andESI‐MS/MSmeasurementswereperformedwithHPLC‐purifiedsamples

on a Bruker maXis ultra‐high resolution time‐of‐flight (TOF) mass spectrometer. Samples were

analysed in positive mode and fragmented by collision induced dissociation (CID). NMR studies

were performed at the Institute for Pharmacy and Molecular Biotechnology of the University of

HeidelbergusingaVarian500MHzNMRspectrometer.

The synthesis of the teicoplanin‐like heptapeptide‐Coenzyme A conjugate 3‐CoA has been

previouslyreported.1,2TheteicoplaninproducingP450enzymesOxyBtei(ProteinID:Q70AY8),3and

OxyAtei(ProteinID:Q6ZZI8)4werealsogeneratedaspreviouslydescribed.Cloning,expressionand

purification for thePCP‐Xdi‐domainproteinasaGB1(IgGbindingB1domainofStreptococcus)5‐

fusion proteinwith anN‐terminal hexahistidine tag and a C‐terminal Strep‐II‐tagwas performed

according to references.6 Redox partner proteinswere obtained fromDr. StephenBell (Adelaide,

Australia).7

CloningofmethyltransferasesMtfadbvandMtfapek

Genes were PCR amplified from genomic DNA using Phusion High‐Fidelity DNA Polymerase

(Thermo Fisher Scientific, USA). The primers MtfA WAC4229 NdeI F (5' ‐

TTTCATATGAGTGATCAGCTGGAGCACG ‐3') and MtfA WAC4229 HindIII R (5' ‐

TTTAAGCTTCATGCGGGACCGGTCTTC ‐3') were used to amplify mtfapek from the pekiskomycin

producer S. malachitospinus WAC4229 11. The oligonucleotides MtfA dbv HindIII R (5' ‐

TTTAAGCTTCTAATGCGCGTCTTCCAC ‐3') and MtfA dbv NdeI F (5' ‐

AAACATATGATAAGCAAAGCAATGCATG ‐3’) were employed to amplify mtfadbv from the A4096

producerActinomadurasp.ATCC39727.PCRproductsandthevectorpET28a(Novagen,USA)were

digestedwithNde IandHind IIIand ligatedtogetherwithT4DNA ligase.Ligationreactionswere

transformedintoE.coliTOP10(ThermoFisherScientific,USA).Error‐freeconstructswerevalidated

bySangersequencingusingstandardT7promoterandterminatorprimers.

ExpressionandPurificationofmethyltransferasesMtfadbvandMtfapek

The methyltransferase proteins were expressed in the E.coli BL21Gold (DE3) strain (Agilent,

Waldbronn,Germany)withanN‐terminalhexahistidinetag.2LLBmedium(+50mg/Lkanamycin)

wereinoculatedwithanovernightstartercultureoftransformedE.coliBL21(DE3)andgrownat37

°CuntilOD600reached0.6.Aftercoolingdownto20°Cproteinexpressionwasinducedbyadding

100 mM IPTG and the cell culture was incubated at 20 °C overnight. Cells were harvested by

centrifugationat4°C(5000xg),resuspendedinlysisbuffer(50mMTrispH8.0,50mMNaCl,10

mMimidazole)includingproteaseinhibitorcocktail(Sigma‐Aldrich,München,Germany)andlysed

bypassingtreetimesthroughafluidizer(Microfluidics,Newton,USA).Aftercentrifugation(50000x

g, 4 °C,25min) the soluble fractionwas incubated for1h at 4°Cwith4mLofNi‐NTA resinpre‐

equilibratedwith2x10columnbedvolumes(CV)ofwashbuffer(50mMTrispH8.0,300mMNaCl,

10mMimidazole).Afterseparatingtheresinfromthesupernatantbycentrifugationat1000gitwas

transferredintoacolumnandwashedwith10CVofwashbuffer.Proteinswereelutedwith3CVof

elution buffer (50mM Tris pH 8.0, 300mM NaCl, 300mM imidazole). The elution fraction was

further purified by size exclusion chromatography on a Superose‐12 column (GE Healthcare,

München,Germany)connectedtoanÄktaFPLCusingexchangebuffer(20mMTris.HClpH7.4,20

mMNaCl,5mMDTE).ElutionfractionswereanalysedbySDS‐PAGE,appropriatefractionspooled

and concentrated using centrifugal concentrators with a 10,000 MW cut‐off (Sartorius Stedim

Biotech, Göttingen, Germany). Aliquotswere flash frozen in liquid nitrogen and stored at ‐80 °C.

Proteinswereobtainedinayieldof15.8mg(0.5µmol)/LcultureforMtfadbvand32mg(1µmol)/L

cultureforMtfapek.

FigS1.Gelfiltrationchromatogramsandelectrophoreticanalysis(10%SDS‐PAGEgel)ofpurified

Mtfadbv (left side, marked with a star) and Mtfapek (right side, marked with a star). As marker

“PrecisionPulsProteinAllBlueStandards(BioRad)wasused;stainingwasperformedusingInstant

Blue(Expedeon).

*  * 

Fig.S2:AlignmentofMtfadbvandMtfapekwiththealreadycharacterisedMtfacle fromthechloroeremomycin

producingA.orientalis.SecondarystructureelementsaretakenfromMtfacleandshownabovethealignment.

Thesequencesdisplay46%identity.AlignmentwasperformedwithClustalOmega.8

Synthesisofcompounds

Teicoplanin aglycone 1 was obtained as describe in literature.9 Briefly, teicoplanin (45 mg,

SantaCruz,Heidelberg,Germany)wassonicatedinamixtureofconc.HCl(1.12mL)andaceticacid

(10.2mL) until a clear solutionwas obtained. Thismixturewas heated to 80 °C under vigorous

shaking.After2 h ice‐colddiethyl ether (35mL)was added andawhite solidprecipitated.After

keeping themixture at ‐24 °C overnight, the solidwas separated and purified using preparative

HPLC.AfterlyophilisationoftherespectiveHPLCfractionsteicoplaninaglycone1wasobtainedas

whitefoam.Stocksolutions(1mM)werepreparedin20%DMSOandstoredat‐24°C.

AdoPropen 2b and AdoViBenz 2c were synthesized according to literature:10,11 S‐adenosyl‐L‐

homocysteine(20mg,52μmol)wasdissolvedin3mLofa1:1mixtureofformicandaceticacidand

cooledto0°C.3‐Bromo‐1‐propene(264μL,3.1mmol)or4‐vinylbenzylchloride(533μL,3.7mmol)

was slowly added to the stirred solution.The reactionmixturewas allowed towarmup to room

temperature and stirred for 4 days.By adding the reactionmixture into10mL cooledwater the

reactionwasquenchedandthewaterphasewasextractedwith5mLdiethyletherforfourtimes.

After lyophilisation the crude product was purified by preparative reversed‐phase HPLC using

following gradient: 0 ‐15 min up to 10 % solvent A. The obtained diastereomeric mixture was

concentrated by lyophilisation. Stock solutions (10 mM) in water were aliquoted and stored at

−80°Cbeforeuse.

Fig.S3:ReversedphaseHPLCchromatogramsandMSspectraofpurified2b(leftside)and2c(right

side)monitoredatλ=260nm.DuetodegradationofthecofactortracesofS‐adenosylhomocysteine

(*)andtherespectiveallyl‐orbenzvinyl‐thioadenosinecanbedetected(°).Gradient:0–22.5min

0%‐upto15%solventA;flowrate1.0mL/min;WatersCortecsC18+column,2.7µm,4.6x150mm.

Methyltransferaseinvitroactivityassaywithteicoplaninaglycone

 

SchemeS1:ReactionschemeofMtase‐catalysedtransalkylationreactions.

Methyltransferase catalysed transfer reactions were performed in 50mM Hepes pH 7.4, 50mM

NaClbuffer:forthatteicoplaninaglycone(50µM)wasusedtogetherwithMtfadbvorMtfapek(5µM)

and the reaction was started by the addition of S‐adenosylmethionine 2a (500 µM) or cofactor

analoguesAdoPropen2bandAdoViBenz2c(500µM).Themixturewasincubatedfor1hat30°C

undergentleshaking(350rpm).Thereactionwasstoppeduponadditionofanequalvolumeofice‐

cold methanol and stored at ‐24 °C for 30 min. After removal of precipitated protein by

centrifugation at 15000 rpm (10 min), the supernatant was concentrated under vacuum and

analysedusingUHPLC‐MS(gradient:0–0.5min5%solventA,0.5–0.7minupto30%solventA,

0.7–6minupto80%solventA;flowrate0.5mL/min).

LargescaletransalkylationassaysforantibioticactivityandNMRstudieswereperformedinatotal

volumeof1mL50mMHepespH7.4,50mMNaClbuffercontaining500µMteicoplaninaglycone1,

MtfadbvorMtfapek(10µM)andcofactoranaloguesAdoPropen2bandAdoViBenz2c(750µM).The

reaction was incubated overnight at 30°C under gentle shaking (350 rpm). The reaction was

stopped upon addition of an equal volume of ice‐coldmethanol and stored at ‐24 °C for 30min.

After removal of precipitated protein by centrifugation, the supernatantwas concentrated under

vacuumandpurifiedusinganalyticalHPLC(gradient:0–4min5%solventA,4–5min,upto20%

solventA,5–25minupto75%solventA;flowrate1mL/min).

Loadingofteicoplanin‐like‐CoApeptide(3‐CoA)ontoapo‐PCP‐Xtei

Thepeptide‐CoAconjugatewas loadedontothePCP‐domainoftheteicoplaninPCP‐Xteidi‐domain

protein catalyzed by the Sfp variant R4‐412 by incubating amixture of 60 µMPCP‐Xtei, 6 µM Sfp,

120µM3‐CoA,50mMHepespH7.0,50mMNaCl,10mMMgCl2for1hat30°C.Afterwards,excess

of free peptidewas removed by a dilution‐concentration procedurewith low salt buffer (50mM

HepespH7.0 forP450activityassayor50mMHepespH7.4 formethyltransferaseactivityassay,

50mM NaCl, 4 x 1:5 dilution) using centrifugal filter units with a 10,000MW cut‐off (Merck

Millipore, Darmstadt, Germany). The generatedpeptidyl‐PCP‐Xtei constructwas immediately used

forfurtherenzymaticinvitroactivityassays.

CoupledP450/methyltransferaseinvitroactivityassay

Invitrooxidationexperimentswereperformedin50mMHepes,pH7.0,50mMNaClbufferat30°C.

Understandardturnoverconditions2µMofOxyBteiorOxyBteiandOxyAteiwereusedtogetherwith

50µM peptidyl‐PCP‐Xtei immediately after the PCP loading reaction. Palustrisredoxin B variant

A105V (PuxB) together with palustrisredoxin reductase (PuR) were used as a redox system7 in

5:1:50(PuxB:PuR:peptidyl‐PCP‐X)ratio.Theturnoverreactionwasstarteduponadditionof2mM

NADH (Gerbu, Biotechnik, Heidelberg, Germany). For NADH regeneration 0.33% glucose and

9U/mL glucose dehydrogenase (Sorachim, Lausanne, Switzerland)were included in the reaction

mixture.Theturnoverreactionwasincubatedfor1hwithgentleshaking(350rpm).

Forthesubsequentmethyltransferaseassayeitherprotocola)orb)wasused.

Protocola)MethyltransferaseassaywithPCP‐Xtei‐boundpeptidesassubstrates

Tostoptheoxidationreaction in thecytochromeP450turnovermixtureNADHandglucosewere

removedbyadilution‐concentrationprocedurewithbuffer(50mMHepespH7.4,50mMNaCl,3x

1:5 dilution) using centrifugal filter unitswith a 10,000MW cut‐off (MerckMillipore, Darmstadt,

Germany).Followingthat,themixturewasseparatedintotriplicatesandpeptidyl‐PCP‐Xtei(50µM)

wasincubatedwithmethyltransferaseMtfadbvorMtfapek(5µM)andS‐adenosylmethionine2a(500

µM)for1hat30°C(350rpm).Controlreactionswereperformedwithoutmethyltransferaseorco‐

substrate.Forworkup,peptideswerereleasedfromthePCPaddingmethylamine(40%solutionin

H2O, 15min incubation at RT), resulting in peptidemethylamides. For the following purification

procedure ice‐cold diluted formic acidwas added to neutralise the turnovermixture,whichwas

thenpurifiedviasolidphaseextraction(Strata‐Xpolymericreversedphase3mg/mL,Phenomenex,

Aschaffenburg, Germany): 1. loading sample using gravity flow, 2. washing column with 5 %

methanol in water, 3. elution of peptidewith 100%methanol + 0.1% formic acid. The elution

fraction was concentrated under vacuum and analysed by analytical UHPLC‐MS using single ion

monitoring in negative mode. After integration of the signals obtained for the different peptide

speciestheMtfaactivitieswerecalculatedbasedonthepercentageofalkylatedpeptiderelativeto

therespectivesubstrateobtainedfromtheP450turnover.

Protocolb)MethyltransferaseassaywithPCP‐X‐cleavedpeptidesassubstrates

ThecytochromeP450turnovermixturewastreatedwithmethylaminetocleavethepeptidefrom

thePCP‐Xteidi‐domainproteinasdescribedinprotocola).Afterneutralisationwithdilutedformic

acidandpurificationviasolidphaseextractiontheelutionfractionwasconcentratedundervacuum

anddissolvedin50mMHepespH7.4,50mMNaCl(equalvolumeasforcytochromeP450turnover

assay). The mixture was split into triplicates and Mtfadbv or Mtfapek (5 µM) and co‐substrate

derivatives 2a‐c (500 µM) were added. Control reactions were performed without

methyltransferase.Afterincubationfor1hat30°C(350rpm)thereactionwasquenchedwithan

equal volume of ice‐cold methanol and stored at ‐24 °C for 30 min. Precipitated protein was

removed by centrifugation and the supernatant was concentrated under vacuum followed by

UHPLC‐MSanalysis.Dataanalysiswasperformedasdescribedinprotocola).

Forenzymaticmodificationswithco‐substrate2aand2bthefollowinggradientwasused:0–0.5

min5%solventA,0.5–0.7minupto20%solventA,0.7–6minupto70%solventA;flowrate0.5

mL/min. For reactionswith co‐substrate2c: 0 – 0.5min 5% solventA, 0.5 – 0.7min up to 20%

solventA,0.7–7minupto90%solventA;flowrate0.5mL/min.

Enzymaticmodificationofteicoplaninaglyconefollowedby inverseelectron‐demandDiels‐

Alderreaction

Teicoplaninaglycone1 (500µM)was incubated togetherwithMtfapek (50µM)andAdoViBenz2c

(600µM)in50mMHepes,pH7.4,50mMNaClbufferfor2hat30°C(350rpm).Aftermethanol

precipitation and removal of precipitated protein by centrifugation the supernatantwas purified

usinganalyticalHPLCtoseparate4‐vinylbenzylN‐terminallymodifiedteicoplaninaglycone1cfrom

unreactedcofactorspecies.AfterremovaloftheHPLCsolvent1cwasdissolvedin50mMHepes,pH

7.4/ acetonitrile (1:1) and incubatedwith6‐methyl‐tetrazine‐5‐TAMRA6 (1mMof10mMstock

solutioninDMSO,JenaBioscience,Jena,Germany)ortetrazine‐PEG4‐biotin7(1mMof50mMstock

solutioninDMSO,JenaBioscience,Jena,Germany)at37°Cfor24hexcludinglightexposure(strong

shaking at 2000 rpm). The reactionwas analysed using analytical HPLC and labelled teicoplanin

aglycones8and9werecharacterisedusingMSfragmentation(seeFig.S10‐S12).

Fig.S4:RepresentativeUHPLC‐MSchromatogramsofmethyltransferaseturnoverreactionswithteicoplanin

aglycone 1 and different co‐substrates 2a‐c. Chromatograms were recorded using single monitoring for

unmodifiedteicoplaninaglycone(blackline),mono‐(redline)andbisalkylatedaglycone(blueline)inpositive

mode.

Fig.S5:HPLCchromatogramsofinverseelectrondemandingDielsAlderreactionsmonitoredatλ=260and

280nm.A)HPLCtraceofpurifiedteicoplaninaglyconederivative1c;B)HPLCtraceofreactionfrom1cwith

tetrazine‐TAMRAderivative6yielding80%ofproduct8after18hreactiontime;C)HPLCtraceofreaction

from1cwithtetrazine‐PEGderivative7toproduct9.

Fig.S6:Mtfawacturnoverreactionswithteicoplaninaglycone1anddifferentco‐substrates2a‐canalysedwith

analyticalUV/VISHPLC(leftside,monitoredat260and280nm)andUHPLC‐MS(rightside,monitoredusing

single ion detection in positive mode). UV/VIS‐HPLC monitoring confirms full conversion of teicoplanin

aglycone1asshownbyUHPLC‐MSanalysis.Peaksrepresentingco‐substrates2a‐c,co‐substrateproductof

Mtase reactionS‐adenosylhomocysteine (#) and co‐substratedegradationproducts allyl‐thioadenosine (for

2b,*)orvinylbenzyl‐thioadenosine(2c,*)aremarked.

Fig.S7:Mtfadbvturnoverreactionswithteicoplaninanddifferentco‐substrates2a‐canalysedwithanalyticalUV/VISHPLC(leftside,monitoredat260and280nm)andUHPLC‐MS(rightside,monitoredusingsingleiondetectionofdoublychargedteicoplaninspeciesinpositivemode).TurnoveryieldsdeterminedintriplicatesforMtfadbvandMtfapekarelisted.Peaksrepresentingco‐substrates2a‐c,co‐substrateproductofMtasereactionS‐adenosylhomocysteine(#)andco‐substratedegradationproductsallyl‐thioadenosine(for2b,*)orvinylbenzyl‐thioadenosine(2c,*)aremarked.Turnoverreactionswereperformedwithcommercialteicoplanin(Sigma‐Aldrich,Germany)usingthesameconditionsasforteicoplainaglycone1(50mMHepes,pH7.4,50mMNaCl,50µMteicoplanin,5µMMtfadbvorMtfapek,500µMco‐substrate2a‐c,1hat30°C).

   

   

   

   

1198.23

1199.23

1200.231+

1201.231+

1202.231+

1203.231+

+MS, 0.1‐0.6min #3‐35

0.0

0.5

1.0

1.5

2.0

2.5

5x10Intens.

1196 1198 1200 1202 1204 m/z

1198.231+

1199.231+

1200.231+

1201.231+

1202.231+

1203.231+

C₅₈H₄₆Cl₂N₇O₁₈, , 1198.23

0.0

0.5

1.0

1.5

2.0

2.5

5x10Intens.

1196 1198 1200 1202 1204 m/z

1212.24

1214.241+

+MS, 0.0‐0.8min #2‐50

0

1000

2000

3000

4000

5000

6000

Intens.

1210.0 1212.5 1215.0 1217.5 m/z

1212.241+

1214.241+

C₅₉H₄₈Cl₂N₇O₁₈, , 1212.24

0

1000

2000

3000

4000

5000

Intens.

1210.0 1212.5 1215.0 1217.5 m/z

1226.261+

1228.261+

+MS, 0.0‐0.7min #2‐44

0

250

500

750

1000

1250

1500

Intens.

1225.0 1227.5 1230.0 1232.5 m/z

1226.261+

1228.261+

C₆₀H₅₀Cl₂N₇O₁₈, M, 1226.26

0

250

500

750

1000

1250

1500

Intens.

1225.0 1227.5 1230.0 1232.5 m/z

1238.261240.26

1+ +MS, 0.1‐0.8min #3‐50

0.0

0.5

1.0

1.5

2.0

4x10Intens.

1236 1238 1240 1242 1244 m/z

1238.261+

1240.261+

C₆₁H₅₀Cl₂N₇O₁₈, , 1238.26

0.0

0.5

1.0

1.5

2.0

4x10Intens.

1236 1238 1240 1242 1244 m/z

A

B

C

D

   

   

Fig.S8:ESI‐TOFanalysisofteicoplaninaglyconederivatives1(A),1a1(B),1a2(C),1b1(D),1b2(E),1c(F).MSspectra inthemiddlerepresentthemeasuredMSprofileshowingthecharacteristic isotopedistributionforteicoplaninderivatives.OntherightsidecalculatedMSprofilesareshown.MeasuredandcalculatedspectrawereobtainedusingBrukerCompassMSSoftwareDataAnalysis4.1.

1278.29

1279.29

1280.29

1281.29

1282.28

1283.28

+MS, 0.1‐2.9min #2‐44, 

0

1000

2000

3000

4000

Intens.

1276 1278 1280 1282 1284 m/z

1278.291+

1279.291+

1280.291+

1281.291+

1282.291+

1283.291+

1284.291+

C₆₄H₅₄Cl₂N₇O₁₈, , 1278.29

0

1000

2000

3000

4000

Intens.

1276 1278 1280 1282 1284 m/z

1314.291316.29

1+ +MS, 0.0‐1.0min #2‐58

0.0

0.5

1.0

1.5

4x10Intens.

1312.5 1315.0 1317.5 1320.0 m/z

1314.291+

1316.291+ C₆₇H₅₄Cl₂N₇O₁₈, , 1314.29

0.0

0.5

1.0

1.5

4x10Intens.

1312.5 1315.0 1317.5 1320.0 m/z

E

F

NH

HN

NH

HN

NH

O

O

O

O

O O Cl

ClHO

H

H

O

HNH

O

HOOH

HO2C

H

HO

H

OH

HO

HO OH

NH

HN

NH

HN

NH

O

O

O

O

O O Cl

ClHO

H

H

O

HNH

O

HOOH

HO2C

H

HO

H

O

HO O

H

NH

HN

NH

HN

NH

O

O

O

O

O O Cl

ClHO

H

H

O

HNH

O

HOOH

H

HO

H

OH

HO

HO OH

NH3

NH

HN

NH

HN

NH

O

O

O

O

O O Cl

ClHO

H

H

O

HNH

O

HOOH

H

HO

H

OH

HO

HO OH

NH2

H2O

NH

HN

NH

HN

NH

O

O

O

O

O O Cl

ClHO

H

H

O

HNH

O

HOOH

H

HO

H

HO

HO OH

NH2

NH

HN

NH

HN

NH

O

O

O

O

O O Cl

ClHO

H

H

O

HNH

O

HOOH

H

HO

H

OH

HO

HO OH

NH

+ H2O

NH

HN

NH

HN

NH

O

O

O

O

O O Cl

ClHO

H

H

O

HNH

O

HOOH

H

HO

H

OH

HO

HO OH

HN

HO2C

NH

HN

NH

HN

NH

O

O

O

O

O O Cl

ClHO

H

H

O

HNH

O

HOOH

HO2C

H

HO

H

OH

HO

HO OH

NH

NH

HN

NH

HN

NH

O

O

O

O

O O Cl

ClHO

H

H

O

HNH

O

HOOH

H

HO

H

OH

HO

HO OH

NH2

NH

HN

NH

HN

NH

O

O

O

O

O O Cl

ClHO

H

H

O

HNH

O

HOOH

H

HO

H

OH

HO

HO OH

NH

NH

HN

NH

HN

NH

O

O

O

O

O O Cl

ClHO

H

H

O

HNH

O

HOOH

HO2C

H

HO

H

OH

HO

HO OH

HN

C58H41Cl2N6O17+

Exact Mass: 1163,19

C58H43Cl2N6O18+

Exact Mass: 1181,20

C57H46Cl2N7O172+

Exact Mass: 1170,23

C57H45Cl2N7O16•+

Exact Mass: 1153,23

C57H43Cl2N7O15•+

Exact Mass: 1135,22

C60H50Cl2N7O17+

Exact Mass: 1210,26

C62H52Cl2N7O18+

Exact Mass: 1252,27

C60H47Cl2N7O18•+

Exact Mass: 1223,23

C65H50Cl2N7O18+

Exact Mass: 1286,26

C66H53Cl2N7O16•+

Exact Mass: 1269,29

C64H49Cl2N7O16•+

Exact Mass: 1241,26

m/z: 1286.28

m/z: 1269.25

m/z: 1252.26

m/z: 1241.25

m/z: 1223.20

m/z: 1210.25

m/z: 1181.20

m/z: 1170.24

m/z: 1163.20

m/z: 1153.23

m/z: 1135.23

 

Fig.S9:ESI‐MS/MSanalysisofteicoplaninaglyconederivatives1(A),1a1(B),1a2(C),1b1(D),1b2(E),1c(F).Datawereobtained inpositivemode.G)Proposed structures for characteristic fragments observedduringtandemmassspectrometry.

G

 

 

 

 

FigS10:ESI‐TOFanalysisoflabeledteicoplaninaglyconederivatives8(A)and9(B).Left:measuredspectrashowing the characteristic isotope distribution for teicoplanin derivatives; on the right side calculatedMSprofiles are shown. Measured and calculated spectra were obtained using Bruker Compass MS SoftwareDataAnalysis4.1.

949.262+

949.762+

950.262+

950.762+

951.262+

951.762+

952.26

+MS, 0.0‐1.0min #1‐58

0.0

0.5

1.0

1.5

2.05x10

Intens.

949 950 951 952 m/z

949.262+ 949.76

2+950.262+

950.762+

951.262+

951.762+

952.262+

C₁₀₂H₈₄Cl₂N₁₂O₂₂, , 949.2595

0.0

0.5

1.0

1.5

5x10Intens.

949 950 951 952 m/z

972.792+973.29

2+

973.792+

974.292+

974.792+

975.292+

975.792+

+MS, 0.1‐0.6min #3‐33

0.0

0.2

0.4

0.6

0.8

4x10Intens.

973 974 975 976 m/z

972.792+973.29

2+973.792+

974.292+

974.792+

975.292+

975.792+

C₉₇H₉₇Cl₂N₁₃O₂₅S, , 972.7903

0.0

0.2

0.4

0.6

0.8

4x10Intens.

973 974 975 976 m/z

A

B

NH

HN

NH

HN

NH

O

O

O

O

O O Cl

ClHO

H

H

O

HNH

O

OHOH

H

HO

H

OH

HOHO O

H

HN

NN

NHO

O NHN

C101H82Cl2N12O202+

Exact Mass: 1852,51

O

O

H3N

NN

NHO

O NN

O

O

C44H41N6O4+

Exact Mass: 717,32

NH

HN

NH

HN

NH

O

O

O

O

O O Cl

ClHO

H

H

O

HNH

O

OHOH

H

HO

H

HOHO O

H

H2N

O

HO

NN

NHO

O NH3N

O

O

C100H80Cl2N12O222+

Exact Mass: 1870,49

NH

HN

NH

HN

NH

O

O

O

O

O O Cl

ClHO

H

H

O

HNH

O

OHOH

H

HO

H

HOHO O

H

H2N

O

NN

NHO

O NN

O

O

C102H82Cl2N12O212+

Exact Mass: 1880,51

OH

OH

m/z = 926,26

m/z = 935,25

m/z = 940,26

m/z = 717,32

 

Fig S11: ESI‐MS/MS analysis of labeled teicoplanin aglycone derivative 8 (positive mode) with proposedstructuresforcharacteristicfragmentsobservedduringtandemmassspectrometry.

 

Fig S12: ESI‐MS/MS analysis of labeled teicoplanin aglycone derivative 9 (positive mode) with proposedstructuresforcharacteristicfragmentsobservedduringtandemmassspectrometry.   

 

 

 

FigS13:Overviewaboutlinear(3),monocyclic(4)andbicyclic(5)methylamidepeptidederivativesobtainedfromcoupledP450/methyltransferaseinvitroactivitystudieswiththeircorrespondingexactmasses.

 

Fig.S14:RepresentativeUHPLC‐MSchromatogramsforcoupledP450/Mtaseactivityassayswithdifferentco‐substratesusingsingleionmonitoringinnegativemode.A)ControlreactionsoftheP450activityassaysjustwith OxyBtei (left side) or OxyBtei / OxyAtei (right side). B)‐E) Representative chromatograms for coupledP450/MtaseactivityassayswithMtfadbv(leftside)andMtfapek(rightside)usingsolelyOxyBtei(B)orOxyBtei/OxyAtei (C‐E). As co‐substrates AdoMet 2a (B & C), AdoPropen 2b (D) and AdoViBenz 2c (E) were used.

Turnover reactions represent a mixture of linear (black lines), monocyclic (red lines) and bicyclic (greenlines)peptidemethylamidespecies.Traces fornon‐alkylatedpeptidesarepresentedasdotted lines,mono‐alkylatedpeptidesassolidlinesandbis‐alkylatedpeptidesasdashedlines.Multiplepeaksforonespeciescanbeascribedtothesubstratepeptide3,whichisusedasadiastereomericmixture (racemization of Hpg on position 7 can´t be prevented during synthesis).4 For linear peptidemethylamide3thediastereomersarenotresolvedyetbutformonocyclicpeptidemethylamide4clearlytwopeakscanbedistinguished(seeA).Forthebicyclicpeptide5multiplepeaksareobservedwhichweascribetoother peptide conformations such as cis‐trans amide bond rotamers. Through the second P450 catalysedcrosslinksuchrotamersarepossiblylocked.Foronebicyclicspecieselutingatearlierretentiontimes(4‐4.5min)weobservedgenerallynoconversionthroughMtases.WehavepreviouslyobservedthesamebehaviorforP450enzymes followingOxyAtei in thecrosslinkingcascade(unpublisheddata), thereforethispeakwasexcludedfromcalculationstodeterminetheyieldofalkylatedbicyclicpeptidemethylamides.Forquantitativeanalysiswe assume that thedifferentpeptide species showsimilar ionizationbehavior (basedonpreviousexperimentsperformedwithaninternalstandard).2,4,6

NH

HN

NH

HN

NH

O

O

O

O

O

O

O

OH

O

HN

O

OH

NH

OH OH

H2N

C68H62N8O13•+

Exact Mass: 1198,44

NH

HN

NH

HN

NH

O

O

O

O

O

O

OOH

OH

O

HN

O

OH OH OH

NH C64H59N7O14•+

Exact Mass: 1149,41

NH

HN

NH

HN

NH

O

O

O

O

O

O

OOH

OH

O

HN

O

OH

H2N

OH OH

NH C64H61N8O14+

Exact Mass: 1165,43

NH

HN

NH

HN

NH

O

O

O

O

O

O

OOH

OH

O

HN

O

OH

NH

OH OH

C59H52N7O14+

Exact Mass: 1082,36

NH

HN

NH

HN

NH

O

O

O

O

O

O

O

OH

O

HN

O

OH

NH

OH OH

C59H51N7O13•+

Exact Mass: 1065,35

NH

HN

NH

HN

NH

O

O

O

O

OO

OOH

OH

O

N

O

HO

OH OH

C58H47N6O14+

Exact Mass: 1051,31

NH

HN

NH

HN

NH

O

O

O

O

O

O

O

OH

O

N

OH OH OH

H2N C58H51N7O12

•+

Exact Mass: 1037,36

NH

HN

NH

HN

NH

O

O

O

O

O

O

OOH

OH

O

N

OH OH OH

C57H47N6O13+

Exact Mass: 1023,32

NH

HN

NH

HN

NH

O

O

O

O

O

OOH

OH

O

HN

O

OH

NH2

OH

C51H47N7O12•+

Exact Mass: 949,33

HN

NH

HN

NH

O

O

O

O

O

OOH

OH

O

HN

O

OH

NH

OH

C51H45N6O12+

Exact Mass: 933,31

NH

HN

NH

HN

NH

O

O

O

O

O

O

OOH

OH

O

H2N

OH OH

C50H43N6O12+

Exact Mass: 919,29

NH

HN

NH

HNN

O

O

O

OOO

OOH

OH

O

OH OH

C50H40N5O12+

Exact Mass: 902,27

NH

HN

NH

HNN

O

O

O

OOO

O

OH

O

OH OH

C50H39N5O11•+

Exact Mass: 885,26

HN

NH

HN

NH

O

O

O

O

O

OOH

OH

O

N

OH OH

C49H40N5O11+

Exact Mass: 874,27

NH

HN

NH

HN

N

O

O

O

O

O

OOH

OH

O

OH OH

C49H40N5O11+

Exact Mass: 874,27

NH

HN

NH

HN

N

O

O

O

O

O

O

OH

O

OH OH

C49H39N5O10•+

Exact Mass: 857,27

m/z: 1198.42

m/z: 1165.34

m/z: 1149.36

m/z: 1082.36

m/z: 1065.33

m/z: 1051.31

m/z: 1037.33

m/z: 1023.32

m/z: 949.31

m/z: 933.31

m/z: 919.29

m/z: 902.27

m/z: 885.25

m/z: 874.27

m/z: 874.27

m/z: 857.25

 

Fig.S15: ESI‐MS/MSanalysis of teicoplanin aglyconederivatives5a1 (A),5a2 (B),5b1 (C),5b2 (D),5c (E).Datawereobtained inpositivemode.G)Proposed structures for characteristic fragments observedduringtandemmass spectrometry. Due to low amounts ofmonocyclic peptide species during turnover reactions,MS/MSanalysisforthesepeptideswasabandoned.

F

 

Fig.S16:Structuresofthetype‐IVGPAteicoplaninandA40926–theprecursorofsemisyntheticdalbavancin‐‐andthetype‐IGPAschloroeremomycinandpekiskomycin.

TableS1.Minimalinhibitoryconcentration(MIC)ofcompounds1,1b,1c

Compound No.     vancomycin 1  1b  1c 

MIC in BHI (ug/ml)    

Enterococcus faecalis (ATCC 29212) 

4  4  32  2 

Enterococcus faecalis VRE B (ATCC 51299) 

64  32  >64  4 

     

MIC in MHB (ug/ml)    

Bacillus subtilis 168 (ATCC 23857)    

0,25  0,5  8  1 

MICs were determined in three independent experiments in duplicates for each strain and compound.EnterococcusMICsweredeterminedusingthebrothmicrodilutionmethodinBBLBrainHeartInfusionbrothaccordingtoNCCLSprotocols.BacillusMICsweredeterminedusingthebrothmicrodilutionmethodinBBLCation‐adjustedMuellerHintonbrothaccordingtoNCCLSprotocols.

Fig.S17:1H‐NMRofteicoplaninaglycone1

1HNMR(500MHz,1H‐1H‐COESY,1H‐1H‐ROESY,DMSO‐d6)δ=9.70(s,1H,OH),9.56(s,1H,OH),9.51(s,1H,OH),9.39(s,1H,OH),9.17(s,1H,OH),8.85(s,1H,OH),8.46(d,J=5.2Hz,1H,Hpg5‐NH),8.41(d,J=6.0Hz,1H,Dpg7‐NH),8.16(s,1H),7.78(d,J=1.9Hz,1H,Tyr6‐Har),7.71–7.67(m,2H,Hpg4‐NH,Har),7.60(d,J=8.4Hz,1H,Dpg3‐NH),7.55(m,1H,Tyr2‐NH),7.44(dd,J=8.3,2.0Hz,1H,Har),7.25(d,J=3.2Hz,1H,Har),7.24(d,J=3.3Hz,1H,Har),7.22(d,J=1.9Hz,1H,Har),7.14–7.07(m,2H,Har),6.94(d,J=8.3Hz,1H,Har),6.73(d,J=11.8Hz,1H,Tyr6‐NH),6.71–6.57(m,4H,Har),6.38(m,2H,Har),6.34–6.31(m,2H,Har),6.24(d,J=2.3Hz,1H,Har),5.89(d,J=6.6Hz,1H,β‐OH),5.66(d,J=8.2Hz,1H,Dpg3‐Hα),5.50(d,J=2.1Hz,1H,Hpg4‐Har),5.33(d,J=10.4Hz,1H,Hpg4‐Hα),5.13–5.05(m,2H,Tyr6‐Hβ,Hpg4‐Har),4.97(m,1H,Tyr2‐Hα),4.77(bs,1H,Hpg1‐Hα),4.40(d,J=5.8Hz,1H,Dpg7‐Hα),4.33(d,J=5.5Hz,1H,Hpg5‐Hα),4.12(d,J=11.8Hz,1H,Tyr6‐Hα),3.35(m,underwatersignal,1H,Tyr2‐Hβ1),2.84(m,1H,Tyr2‐Hβ2)ppm.

Fig.S18:1H‐NMRofteicoplaninaglyconederivative1b

1HNMR(500MHz,1H‐1H‐COESY,DMSO‐d6)δ9.69(d,J=5.1Hz,1H,OH),9.56(s,1H,OH),9.42–9.37(m,2H,OH),9.17(s,1H,OH),8.87(s,1H,OH),8.44(m,2H,Hpg5‐NH,Dpg7‐NH),8.13(s,1H),7.78(d,J=1.9Hz,1H,Tyr6‐Har),7.70–7.61(m,3H,Hpg4‐NH,Dpg3‐NH,Har),7.55(m,1H,Tyr2‐NH),7.45(dd,J=8.4,2.0Hz,1H,Har),7.25–7.15(m,3H,Har),7.12–7.06(m,2H,Har),6.95(m,1H,Har),6.91–6.85(m,1H),6.77–6.61(m,3H,Har,Tyr6‐NH),6.50–6.43(m,1H),6.41–6.34(m,3H,Har),6.27–6.21(1H),5.93–5.84(m,2H,allyl‐H),5.67–5.62(m,1H,Dpg3‐Hα),5.47(m,1H,Hpg4‐Har),5.39–5.20(m,4H,Hpg4‐Hα,allyl‐H),5.13‐5.04(m,2H,Tyr6‐Hβ,Hpg4‐Har),4.91(m,1H,Tyr2‐Hα),4.41(m,1H,Dpg7‐Hα),4.32(d,J=5.5Hz,1H,Hpg5‐Hα),4.10(d,J=11.4Hz,1H,Tyr6‐Hα),3.35(m,underwatersignal,1H,Tyr2‐Hβ1),2.87(m,1H,Tyr2‐Hβ2)ppm.

Fig.S19:1H‐NMRofteicoplaninaglyconederivative1c

1HNMR(500MHz,1H‐1H‐COESY,DMSO‐d6)δ9.70(s,1H,OH),9.56(s,1H,OH),9.42(s,1H,OH),9.38(s,1H,OH),9.15(s,1H,OH),8.82(s,1H,OH),8.44(d,J=5.2Hz,1H,Hpg5‐NH)8.37(d,J=5.8Hz,1H,Dpg7‐NH),8.26(s,1H),7.78(d,J=1.9Hz,1H,Tyr6‐Har),7.61(d,J=10.3Hz,1H,Hpg4‐NH),7.56(d,J=8.5Hz,1H,Dpg3‐NH),7.49–7.40(m,6H,Tyr2‐NH,Har),7.24(d,J=8.3Hz,1H,Har),7.21(d,J=1.9Hz,1H,Har),7.16–7.08(m,3H,Har),6.93(d,J=8.3Hz,1H,Har),6.74(dd,J=10.9,17.6Hz,Ar‐CH=CH2),6.71–6.64(m,3H,Tyr6‐NH,Har),6.38(d,J=2.3Hz,1H,Har),6.35–6.31(m,3H,Har),6.26(s,1H,Har),5.88(d,J=6.7Hz,1H,β‐OH),5.81(dd,J=17.6,1.1Hz,1H,Ar‐CH=CH2‐trans),5.65(d,J=8.3Hz,1H,Dpg3‐Hα),5.47(d,J=2.1Hz,1H,Hpg4‐Har),5.35–5.32(m,1H,Hpg4‐Hα),5.23(dd,J=10.9,1.1Hz,1H,Ar‐CH=CH2‐cis),5.11–5.06(m,2H,Tyr6‐Hβ,Hpg4‐Har),5.00–4.94(m,1H,Tyr2‐Hα),4.42–4.38(m,1H,Dpg7‐Hα),4.32(d,J=5.4Hz,1H,,Hpg5‐Hα),4.11(d,J=11.8Hz,1H,Tyr6‐Hα),3.86–3.70(m,2H,Hpg1‐Hα,Hpg1‐NH),3.35(m,underwatersignal,1H,Tyr2‐Hβ1),2.84(d,J=13.9Hz,1H,Tyr2‐Hβ2),2.28–2.16(m,NH‐CH2‐Ar)ppm.

References

1 C.BriekeandM.J.Cryle,Org.Lett.,2014,16,2454–2457.2 C.Brieke,V.Kratzig,K.Haslinger,A.WinklerandM.J.Cryle,Org.Biomol.Chem.,2015,13,2012–2021.3 K.Haslinger,E.Maximowitsch,C.Brieke,A.KochandM.J.Cryle,ChemBioChem,2014,15,2719–2728.4 C.Brieke,M.Peschke,K.HaslingerandM.J.Cryle,Angew.Chem.Int.Ed.,2015,54,15715–15719.5 J.Bogomolovas,B.Simon,M.SattlerandG.Stier,ProteinExpr.Purif.,2009,64,16–23.6 K.Haslinger,M.Peschke,C.Brieke,E.MaximowitschandM.J.Cryle,Nature,2015,521,105–109.7 S.G.Bell,A.B.H.Tan,E.O.D.JohnsonandL.‐L.Wong,Mol.Biosyst.,2010,6,206–214.8 F.Sievers,A.Wilm,D.Dineen,T.J.Gibson,K.Karplus,W.Li,R.Lopez,H.McWilliam,M.Remmert,J.Soding,

J.D.ThompsonandD.G.Higgins,Mol.Syst.Biol.,2014,7,539–539.9 H.Shimamura,S.P.Breazzano,J.Garfunkle,F.S.Kimball,J.D.TrzupekandD.L.Boger,J.Am.Chem.Soc.,

2010,132,7776–7783.10C.Dalhoff,G.Lukinavičius,S.KlimašauskasandE.Weinhold,Nat.Protoc.,2006,1,1879–1886.11B.J.C.Law,A.‐W.Struck,M.R.Bennett,B.WilkinsonandJ.Micklefield,Chem.Sci.,2015,6,2885–2892.12M.Sunbul,N.J.Marshall,Y.Zou,K.ZhangandJ.Yin,J.Mol.Biol.,2009,387,883–898.