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:
bThe School of Chemical Sciences, University of Auckland, 23 Symonds St, Auckland 1010, New Zealand. E-mail:
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
1. P.W.R.Harris,S.H.YangandM.A.Brimble,TetrahedronLett.,2011,52,6024-6026.2. K.J.Jensen,M.MeldalandK.Bock,J.Chem.Soc.,PerkinTrans.1,1993,2119-2129.3. T.Inazu,Synlett,1993,11,869-870.4. R.J.BaileyandD.L.Hay,Peptides,2006,27,1367-1375.5. J.J.Gingell,T.Qi,R.J.BaileyandD.L.Hay,Peptides,2010,31,1400-1404.6. J.J.Gingell,E.R.BurnsandD.L.Hay,Endocrinology,2014,155,21-26.