Constructing Cross-linked Nanofibrous Scaffold via Dual-Enzyme-Instructed Hierarchical Assembly - Zhang, Cortes, Zhang - 1919 - Unknown

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ConstructingCross-linkedNanofibrousScaffoldviaDual-Enzyme-InstructedHierarchicalAssemblyShijinZhang,WilliamCortes,YeZhang*BioinspiredSoftMatterUnit,OkinawaInstituteofScienceandTechnologyGraduateUniversity,1919-1Tancha,Onna-son,Okinawa,904-0495Japan.Email:ye.zhang@oist.jp1

1ContentsSynthesis........................................................................................................................................................3Materialsandinstruments.......................................................................................................................3Compound1a............................................................................................................................................4Compound1..............................................................................................................................................5Compound2a............................................................................................................................................7Compound2b............................................................................................................................................8Compound2..............................................................................................................................................9ALPcatalyzedhydrolysis...........................................................................................................................11FigureS1......................................................................................................................................................11CEScatalyzedhydrolysis...........................................................................................................................12FigureS2......................................................................................................................................................12Stage-by-stagetreatmentwithenzyme.....................................................................................................13UV-Visabsorptionandemissionspectroscopy.........................................................................................13FigureS3......................................................................................................................................................13FigureS4......................................................................................................................................................132

2SynthesisMaterialsandinstruments:Fmoc-Aminoacid,4-dimethylaminopyridine(DMAP)and2-chlorotritylchlorideresinwerepurchasedfromGLBiochem(Shanghai,China);Dimethylformamide(DMF,99.5%),Dichloromethane(DCM,98.0%),N,N-diisopropylethylamine(DIEA),trifluoroaceticacid(TFA,98.0%),N-(3-dimethylaminopropyl)-N'-ethylcarbodiimidehydrochloride(EDC),piperidine(99.0%),Meldrum'sacid(98%),methanol(99.0%),ethanol(99.5%),hexane(95.0%)werepurchasedfromNacalaiTesqueInc.,Japan;2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate(HBTU)(97.0%)waspurchasedfromMatrixScientific;N,N'-diisopropylcarbodiimide(DIC,99.0%)waspurchasedfromFUJIFILMWakoPureChemicals;ethyleneglycol(99.8%)waspurchasedfromSigmaAldrich.Piperidiniumacetate(98.0%),N-hydroxysuccinimide(NHS,98.0%)andbiotin(98.0%)wereobtainedfromTokyoChemistryIndustry(TCI).4-(diethylamino)salicylaldehyde(98.0%)waspurchasedfromBLDPharmatechLtd;2-Naphthylaceticacid(NapOH,98.0%)waspurchasedfromCombi-Blocks;Organicsolventsweredehydratedbeforeexperiment.HPLCpurificationwasperformedonaAgilent1260InfinityPreparativePumpwithAgilent1260InfinityDiodeArrayDetector(Eluent:Acetonitrileandwater(0.1%TFA)).MassspectrawererecordedbyThermoLTQ-ETDmassspectrometer(ESI-MS).1HNMRand13CNMRspectrawererecordedonaJEOL400(400MHzand101MHz,respectively)spectrometer.3

3SchemeS1.Synthesisofcompound1a.Compound1a:Compound1awasobtainedviasolidphasepeptidesynthesis(SPPS).2-Chlorotritylchlorideresin(2.0g,2mmol)wasswelledinanhydrousDCMfor20min,L-Fmoc-Lys(Boc)-OH(2.34g,5mmol)dissolvedinanhydrousDMFwasconjugatedtoresinwithDIEAin30min.ThenwashedwithanhydrousDMFfor3times,unreactedsitesintheresinwereblockedwithDCM/MeOH/DIEA(80:15:5)for20minandwashedbyanhydrousDMFfor5times.Thenpiperidinesolution(20%inDMF)wasaddedintoreactionfor30mintoremovetheFmocprotectinggroupontheaminoacid.WecarriedoutthepeptideelongationaccordingtostandardSPPSprotocoltogetthedesiredcompound.1awascleavedofftheresinusingTFAfor2h.Afterremovingthesolvent,anhydrousetherwasaddedintotheconcentratedproductandwhiteprecipitationof1awasobtained,theyieldis82.1%.1HNMR(400MHz,DMSO-d6)δ=8.89(d,J=7.5Hz,1H),8.64(s,1H),8.50(d,J=8.3Hz,1H),8.42(d,J=7.8Hz,1H),7.68(d,J=9.1Hz,1H),7.33–7.21(m,5H),7.18–7.12(m,1H),7.03–6.91(m,4H),6.84–6.78(m,1H),6.60(s,1H),4.82–4.49(m,2H),4.30–4.26(m,1H),3.07–2.94(m,4H),2.85–2.67(m,6H),1.82–1.72(m,2H),1.67–1.51(m,4H),1.44–1.36(m,2H),1.14(t,J=7.0Hz,6H)ppm.MS:calculatedforC38H46N5O11Pis779.2931,found780.2991,[M+H]+.4

4CouYpFK_191010154029#65-111RT:0.96-1.63AV:47NL:2.05E6T:FTMS+pESIFullms[150.00-2000.00]780.29911009080706050782.30584030RelativeAbundance2010537.2093663.4523055060065070075080085090095010001050110011501200m/zSchemeS2.Synthesisofcompound1.Compound1:Biotin(0.41mmol,100mg),NHS(0.45mmol,51.8mg)andEDC(0.45mmol,86.3mg)weretransferredintoa25mLroundbottomflask,thendissolvedin5mLanhydrousDMF.Stirredatroomtemperatureforovernight.TLCwasappliedtoconfirmthegenerationofactivatedester,then1a(0.38mmol,300mg)wasaddingintothereactionmixture,DIEA5

5(155.8mg,1.2mmol)wasslowlydroppingintothesolution,stirringatroomtemperaturefor24h.PreparationHPLCwasappliedtopurifythetargetcompound.Theyieldis62.3%.1HNMR(400MHz,DMSO-d6)δ8.82(d,J=7.7Hz,1H),8.55(s,1H),8.38(d,J=8.3Hz,1H),8.26(d,J=7.6Hz,1H),7.75(t,J=5.4Hz,1H),7.62(d,J=8.7Hz,1H),7.27–6.92(m,9H),6.75(dd,J=9.1,1.6Hz,1H),6.56(d,J=1.4Hz,1H),6.38(d,J=28.5Hz,2H),4.70–4.50(m,2H),4.30–4.04(m,5H),3.43(dd,J=13.8,6.8Hz,5H),3.08–2.88(m,5H),2.88–2.66(m,3H),2.00(t,J=7.3Hz,2H),1.75–1.19(m,12H),1.09(t,J=7.0Hz,6H)ppm.13CNMR(101MHz,DMSO-d6)δ=174.01,172.50,171.70,170.74,163.30,162.23,157.81,153.08,150.58,150.52,148.43,138.26,133.07,132.21,130.86,129.76,129.69,128.54,126.77,120.09,110.71,109.21,108.11,96.37,61.56,59.72,55.94,54.09,52.51,44.87,35.73,28.74,28.52,25.84,23.38,18.59,17.24,12.84ppm.MS:calculatedforC+48H60N7O13PSis1005.3707,found1006.3771,[M+H].6

6L-CouYpFK-Bio_200227152317#147RT:2.30AV:1NL:5.52E6T:FTMS+cESIsid=35.00Fullms[150.00-2000.00]1006.3771z=110090807060504030851.3362RelativeAbundancez=11022.371820712.2429z=1926.410510z=1z=106007008009001000110012001300140015001600m/zSchemeS3.Synthesisofcompound2a.Compound2a:Synthesiswascompletedaccordingtopublishedmethod.7

71HNMR(400MHz,DMSO-d6)δ=8.28(dd,J=25.7,8.2Hz,2H),7.87–7.76(m,1H),7.75–7.64(m,2H),7.53(s,1H),7.46–7.38(m,2H),7.23–7.08(m,11H),4.55–4.48(m,1H),4.45–4.35(m,1H),3.04–2.63(m,6H)ppm.MS:calculatedforC30H28N2O4is480.2049,found503.1931,[M+Na]+.NapFF#115-134RT:2.16-2.46AV:20NL:1.08E6T:FTMS+pESIsid=35.00Fullms[150.00-2000.00]503.193110090807060504.1947618.18335040481.2096684.336430534.1318RelativeAbundance20288.1372316.1320535.1293619.1887685.3399603.208010790.30710200250300350400450500550600650700750800850900m/zSchemeS4.Synthesisofcompound2b.Compound2bwassynthesizedbyfollowingthepublishedmethod.[2]8

81HNMR(400MHz,CDCl3)δ=8.63(d,J=0.6Hz,1H),7.44(d,J=9.1Hz,1H),6.71(dd,J=9.1,2.5Hz,1H),6.53(dd,J=2.4,0.4Hz,1H),3.48(q,J=7.2Hz,4H),1.25(t,J=7.2Hz,6H)ppm.MS:calculatedforC+14H15NO4is261.1001,found284.0886,[M+Na].Coumarin#211-247RT:4.08-4.66AV:37NL:2.60E6T:FTMS+pESIsid=35.00Fullms[150.00-2000.00]284.08861009080706050300.062340RelativeAbundance30576.1274486.201020438.0903598.1095875.1745100200400600800100012001400160018002000m/zSchemeS5.Synthesisofcompound2.Compound2:Synthesiswascompletedaccordingtothepublishedmethod.1HNMR(400MHz,DMSO-d6)δ=8.57(dd,J=27.6,7.7Hz,1H),8.47(d,J=15.0Hz,1H),8.30–8.18(m,1H),7.82–7.77(m,1H),7.74–7.65(m,2H),7.51(dd,J=14.9,9.2Hz,2H),9

97.46–7.36(m,2H),7.20–7.05(m,10H),6.75–6.60(m,1H),6.51–6.41(m,1H),4.60–4.43(m,2H),4.40–4.22(m,4H),3.57–3.49(m,1H),3.41(q,J=7.2Hz,4H),3.30(s,2H),3.09–2.99(m,1H),2.96–2.83(m,1H),2.70–2.59(m,1H),1.08(t,J=7.0Hz,6H)ppm.13CNMR(101MHz,DMSO-d6)δ=171.89,170.24,163.54,163.53,158.63,157.47,153.45,153.44,150.05,138.19,138.12,137.64,137.51,134.44,133.40,132.20,129.76,129.66,129.58,128.75,128.34,128.08,127.93,127.83,127.73,126.47,125.94,110.31,107.50,107.16,96.35,63.20,62.70,54.06,44.89,42.67,38.36,37.00,12.84ppm.MS:calculatedforC+46H45N3O8is767.3207,found790.3104,[M+Na].10

10NapFFOCH2CH2OCou#159RT:2.97AV:1NL:8.40E5T:FTMS+pESIsid=35.00Fullms[150.00-2000.00]790.3104100908070605040806.281830808.2914RelativeAbundance20768.3255684.3369900.223410244.0960453.20020200300400500600700800900100011001200130014001500m/zALPcatalyzedhydrolysisAlkalinephosphatase(ALP)fromcalfintestinalwaspurchasedfromInvitrogen(Catno.18009-019).Followingtheinstruction,ALPstocksolution(1u/L)waspreparedwiththedilutionbuffer.Stocksolutionsofcompound1ormixtureofcompound1and2(40mMinDMSO)weredilutedinboratebuffertothedesiredconcentration.PropervolumeofALPstocksolutionwasaddedinto200Lofcompoundsolutionreachingfinalconcentrationof1U.HPLCandLCMSwereappliedtoidentifythereactionproductsandmonitortheprogressofhydrolysisatroomtemperature.11

11FigureS1.(a)KineticprofilesofALPcatalyzedhydrolysisofcompound1(200M)inboratebufferatroomtemperature;(b)HPLCspectrumofhydrolysisof1at3min;(c)MSspectrumobtainedfromthefractionofpeak1ain(b).CEScatalyzedhydrolysisCarboxylesterase(CES)fromrabbitliverlyophilizedpowderwaspurchasedfromSigma-Aldrich(E0887-500UN).Followingtheinstruction,CESstocksolution(1u/5L)waspreparedinboratebuffer(pH8.0).Stocksolutionsofcompound2ormixtureofcompound1and2(40mMinDMSO)weredilutedinboratebuffertothedesiredconcentration.PropervolumeofCESstocksolutionwasaddedinto200Lofcompoundsolutionreachingfinalconcentrationof1U.HPLCandLCMSwereappliedtoidentifythereactionproductsandmonitortheprogressofhydrolysisatroomtemperature.FigureS2.(a)KineticprofilesofCEScatalyzedhydrolysisofcompound2(200M)inboratebufferatroomtemperature;(b)HPLCspectrumofhydrolysisof2at2h;(c)MSspectrumobtainedfromthefractionofpeak2bin(b).12

12Stage-by-stagetreatmentwithenzymeInthemixtureofcompound1and2inboratebuffer,regulatedhydrolysisprocesswasachievedbyaddingALPandCESindifferentorder,thusresultingindifferentnanostructures.Firstly,bothALPandCEScanbeaddedinatimetotriggerhydrolysisofthemixtureatthesametime;secondly,ALPwasaddedtohydrolyzecompound1,andaddedCESintothemixtureaftercompletelyhydrolysisof1;thirdly,CESwasaddedtohydrolyzecompound2,andnotaddedALPintothemixtureuntilcompletelyhydrolysisof2.UV-VisabsorptionandemissionspectroscopyUV-VisabsorptionmeasurementsofspecimensolutionsarecarriedoutonThermoNanodrop2000Cspectrophotometer.Thedetectionpathlengthofthecuvetteis1cm.Thedetectionrangewassetto250–700nmwithaspectralresolutionat1.0nm.Emissionspectrawerecollectedonahome-madeHamamatsufluorescencespectrometer.Theexcitationwavelengthwassetto405nmandemissioncollectionlengthwasbetween415and750nm.(424)(472)Coumarin1.01.0).0.80.8nu.oisasi(emcn0.60.6Eadebrzoilsab0.40.4AmroN0.20.20.00.0300400500600700Wavelength(nm)FigureS3.UV-visabsorptionandemissionspectra(excitedat405nm)ofmolecule2binboratebufferat25C.13

13FigureS4.HeLacellviabilityuponthetreatmentofmolecule1(A)andmolecule2(B)atvariousconcentrations.14