A semi-synthetic strategy - PowerPoint Presentation

1. A semi-synthetic strategy to fucosylated chondroitin sulfate polysaccharides from microbial sourced chondroitinLaezza Antonio1, Iadonisi Alfonso1, De Castro Cristina2,De Rosa Mario3, Schiraldi Chiara3, Parrilli Michelangelo4, Bedini Emiliano11 Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario Monte S.Angelo, via Cintia 4, I-80126 Napoli, Italy2Dipartimento di Agraria, Università di Napoli Federico II,Via Università 100, I-80055, Portici, Italy3Dipartimento di Medicina Sperimentale, Seconda Università di Napoli, via De Crecchio 7, I-80138 Napoli, Italy4 Dipartimento di Biologia, Università di Napoli Federico II, Complesso Universitario Monte S.Angelo, via Cintia 4, I-80126 Napoli, Italy7th Baltic Meeting on Microbial Carbohydrates, Gϋstrow, 25-29 September

2. SUMMARYR = H or SO3-+NaR’ = H or variably sulfated L-FucoseFucosylated chondroitin sulfate structure similar to polysaccharide isolated from sea cucumberMicrobial sourced chondroitinR= H , SO3-+Na or variably sulfated L-fucoseAnticoagulant and antithrombotic activity assays

3. FUCOSYLATED CHONDROITIN SULFATEFucosylated chondroitin sulfate (fCS) is a glycosaminoglycan (GAG) from sea cucumber[1]R, R’’ = H or SO3-+NaR’ = H, SO3-+Na or FucSTRUCTURE:Trisaccharide repeating unitß-D-glucuronic acid(GlcA)N-Acetyl-ß-D-galactosamine(GalNAc)α-L-fucose(Fuc)Responsible for biological activities of fCS[1] Pomin, V.H. Mar. Drugs 2014, 12, 232-254Sulfated α-fucose branches depends on the species of sea cucumber[2][3][4][5][2] Chen et al. Carbohydr. Polym. 2010, 83, 688-696[3] Chen et al. Biochim. Biophys. Acta 2013, 1830, 3054-3066[4] Luo et al. Mar. Drugs 2013, 11, 399-417[5] Wu et al. Eur. J. Biol. Chem. 2015, 87, 862-868

4. BIOLOGICAL ACTIVITIESfCS may exhibit activities related to: Coagulation and thrombosis[1] Atherosclerosis[6] Cancer metastasis and inflammation[7][1] Pomin, V.H. Mar. Drugs 2014, 12, 232-254[6] Tovar et al. Altherosclerosis 1996, 26, 185-195[7] Borsig et al. J. Biol. Chem. 2007, 282, 14984-14991 Viral infection[8][9][8] Lian et al. Biochim. Biophys. Acta 2013, 1830, 4681-4691[9] Huang et al. Carbohydr. Res. 2013, 380, 64-69

5. COAGULATION AND THROMBOSISSerpin-dependent mechanism driven by Heparin Cofactor II (HCII)[10]Serpin-independent mechanism due to inhibition of Factor Xa and thrombin generation[11]Blood coagulation relies on: 1) Vasoconstriction2) Platelet plug3) Clot formation[10] Zancan et al. Blood Coagul Fibrinolysis, 2004, 15, 45-54 [11] Glauser et al. Thromb. Haemost., 2008, 100, 420-428

6. CHONDROITIN DERIVATIZATIONEscherichia coli O5:K4:H4Biotechnological productionPolysaccharide purificationDefructosylationIN COLLABORATION WITH THE RESEARCH GROUP OFPROF. M. DE ROSA ATTHE SECOND UNIVERSITY OF NAPLES (SUN)[12][12] Cimini et al. Appl. Microbiol. Biotechnol 2010, 85, 1779-1787chondroitinpurity grade: 89-94%weight-averaged Mw: 45.0 kDa polydispersity: 1.40

7. CHONDROITIN DERIVATIZATION1a: X = COOH2345CH3COCl/CH3OH 0.58 MCH3OH, RT, 2 days1b: X = COO-(n-Bu)4N+n-CH3(CH2)10CH2ITBAF, DMF, T = 80°C, overnight [13]PrecipitationPhCH(OCH3)2CSA, DMFT = 80°C, overnightPhCH(OCH3)2CSA, DMFT = 80°C, overnightPrecipitation[13] Pawar et al. Carbohydr. Polym. 2013, 98, 1288-1296

8. SEMI-SYNTHETIC STRATEGY4: R = CH35: R = CH2(CH2)10CH3Et3N, Ac2ONaBrO3, Na2S2O4 DMAP, CH3CNRT, overnightH2O-AcOEtRT, overnight1) SO3.py, DMFT = 50°C, overnight2) NaOHpH~ 13,RT, 6hfCS(I-V): R = per-O-sulfated-Fuc or H, (R’ = H and R’’ = SO3-Na+) or (R’ = SO3-Na+ and R’’ = H)Capping unreacted –OH as -OAcCleavage Bn and benzylidene groupsSulfation free -OHGlobal deprotection[14]fCSAcceptorDonorYield Degree of fucosylationα/βO-3-linked-: O-2- linked-: (O-4,6)-linked-α-Fuc-ratioI4698%0.772.940% : 22% : 38%II4676%0.322.338% : 62% : 0%III4776%1.152.633% : 24% : 43%IV5656%0.60n.d.n.d.V5758%0.87n.dn.d[14] Laezza et al. Biomacromolecules, 2015, 16(7), 2237-2245 Glycosylation

9. O-GLYCOSYLATIONEntryAcceptorDonorSolventProduct1a465:3 v/vCH2Cl2-DMF[15]8I2b465:3 v/vCH2Cl2-DMF8II3c475:3 v/vCH2Cl2-DMF8III4a563:1 v/vTHF/CH2Cl29IV6d575:3 v/vCH2Cl2-DMF9Va Reaction conditions: acceptor, donor (5 eq with respect to the acceptor), NIS (5.5 eq), TMSOTf (5.5 eq), AW-300 4Å MS, rt, 4hb Reaction conditions: acceptor, donor (5 eq with respect to the acceptor), NIS (5.5 eq), TMSOTf (1.7 eq), AW-300 4Å MS, T = -20°C 4hc Reaction conditions: acceptor, donor (5 eq with respect to the acceptor) ,TMSOTFf(0.1 eq), AW-300 4Å MS, rt, 4h, (inverse procedure) d Reaction conditions: acceptor, donor (5 eq with respect to the acceptor), TMSOTf (0.5 eq) AW-300 4Å MS, rt, 4h, (inverse procedure) 4 5 6 7Table 1: Glycosylation reactions of acceptors 4-5 with donors 6-7[15] Lu et al., Angew. Chem. Int. Ed. 2011, 50, 7315-7320

10. O-GLYCOSYLATION1H-NMR of polysaccharide 88 (I-III): R = CH3, R’ = per-O-Bn-Fuc or H9 (IV-VI): R = CH2(CH2)10CH3, R’ = per-O-Bn-Fuc or H1H-NMR of polysaccharide 9

11. 4: R = CH35: R = CH2(CH2)10CH3Et3N, Ac2ONaBrO3, Na2S2O4 DMAP, CH3CNRT, overnightH2O-AcOEtRT, overnight1) SO3.py, DMFT = 50°C, overnight2) NaOHpH~ 13,RT, 6hfCS(I-V): R = per-O-sulfated-Fuc or H, (R’ = H and R’’ = SO3-Na+) or (R’ = SO3-Na+ and R’’ = H)Capping unreacted –OH as -OAcCleavage Bn and benzylidene groupsSulfation free -OHGlobal deprotection[14]fCSAcceptorDonorYield Degree of fucosylationα/βO-3-linked-: O-2- linked-: (O-4,6)-linked-α-Fuc-ratioI4698%0.772.940% : 22% : 38%II4676%0.322.338% : 62% : 0%III4776%1.152.633% : 24% : 43%IV5656%0.60n.d.n.d.V5758%0.87n.dn.dGlycosylation conducted at rtGlycosylation conducted at -20°C[14] Laezza et al. Biomacromolecules, 2015, 16(7), 2237-2245 SEMI-SYNTHETIC STRATEGYGlycosylation

12. A library of semi-synthetic fCS with different Fuc branching site and/or sulfation pattern:Fuc donorschondroitin acceptorsFuc sulfation / Branching positionO-2 and/or O-3 of GlcA unitsO-4 or O-6 of GalNAc unitsFuc 2,3,4 SFuc 2,4 SFuc 2,3 SFuc O Sbranching sitesulfation site (protected with orthogonal cleavable groups)Anti-coagulant tests for SAR investigation of natural and non-natural (semi-synthetic) fCS polysaccharidesMw (natural) ~ 70-80 kDaMw (semi-synthetic) ~ 8-12 kDa1011 6412

13. SEMI-SYNTHETIC STRATEGYEt3N, Ac2ONaBrO3, Na2S2O4 DMAP, CH3CNRT, overnightH2O-AcOEtRT, overnight1) SO3.py, DMFT = 50°C, overnight2) NaOHpH~ 13,RT, 6hfCS I: R = per-O-sulfated-Fuc or H, (R’ = H and R’’ = SO3-Na+) or (R’ = SO3-Na+ and R’’ = H)[16]6 or10 or 11Capping unreacted –OH as -OAcCleavage Bn and benzylidene groupsSulfation free -OHGlobal deprotection5:3 v/vCH2Cl2-DMFAW-300 MST = -20°C, 4hfCSAcceptorDonor YieldDegree offucosylationα/βO-3-linked-: O-2-linked-α-Fuc-ratioI4641%0.513.60.9VI41041%0.435.50.6VII41117%0.442.81.1fCS VI: R = 2,3-di-O-sulfated-Fuc or H, (R’ = H and R’’ = SO3-Na+) or (R’ = SO3-Na+ and R’’ = H)fCS VII: R = 2,4-di-O-sulfated-Fuc or H, (R’ = H and R’’ = SO3-Na+) or (R’ = SO3-Na+ and R’’ = H)[16] Laezza et al. Chem. Eur. J. 2016 accepted4

14. Fucosylated chondroitin sulfateHSQC-DEPT NMR of fCS VII.600 MHz, D2O, T = 298 KfCS VII

15. SEMI-SYNTHETIC STRATEGYEt3N, Ac2OH2O-AcOEtRT, overnight12: (R = Bz and R’ = H) or (R = H and R’ = Bz)DMAP, CH3CNRT, overnightNaBrO3, Na2S2O412: (R = Bz and R’ = H) or (R = H and R’ = Bz)5:3 (v/v)CH2Cl2/DMF AW-300 MS RT, 4h6 or 10 or 11NaBrO3, Na2S2O41) SO3.py, DMFT = 50°C, overnightH2O-AcOEtRT, overnight2) NaOHpH~ 13,RT, 6hCleavage Bn and benzylidene groupsGlobal deprotectionSulfation free -OHfCS VIII: (R = H and R’ = persulfated-Fuc) or (R = persulfated-Fuc and R’ = H)fCS IX: (R = H and R’ = 2,4-disulfated-Fuc) or (R = 2,4-disulfated-Fuc and R’ = H)fCS X (R = H and R’ = 2,3-disulfated-Fuc) or (R = 2,3-disulfated-Fuc and R’ = H)fCSAcceptorDonor YieldDegree of fucosylationα/βO-4-linked-: O-6-linked-α-Fuc-ratioVIII4647%0.454.31.3IX41038%0.434.02.1X41140%0.501.42.4[16][16] Laezza et al. Chem. Eur. J. 2016 accepted4

16. Fucosylated Chondroitin sulfateHSQC-DEPT NMR of fCS VIII.600 MHz, D2O, T = 298 KfCS VIII

17. SEMI-SYNTHETIC STRATEGY5:3 (v/v)CH2Cl2/DMF AW-300 MS RT, 4h65:3 (v/v)CH2Cl2/DMF AW-300 MS RT, 4h612: (R = Bz and R’ = H) or (R = H and R’ = Bz)Et3N, Ac2ODMAP, CH3CNRT, overnightNaBrO3, Na2S2O4H2O-AcOEtRT, overnightNaBrO3, Na2S2O4H2O-AcOEtRT, overnightNaOHpH~ 13,RT, 6hNaOHpH~ 13,RT, 6hfC-I: R = Fuc or H, (R’ = R’’ = H)fC-II: (R = H and R’ = Fuc) or (R = Fuc and R’ = H)[16][16] Laezza et al. Chem. Eur. J. 2016 accepted[16]4

18. fCAcceptorDonor YieldDegree of fucosylation α/βO-2-linked-: O-3-linked-: O-4,6-linked-α-Fuc-ratioI4633%0.345.20.9 (O-2-, O-3-linked)II4639%0.383.52.1 (O-4-, O-6-linked)SEMI-SYNTHETIC STRATEGYHSQC-DEPT NMR of fC I.600 MHz, D2O, T = 298 KfC-I: R = Fuc or H, (R’ = R’’ = H)HSQC-DEPT NMR of fC II.600 MHz, D2O, T = 298 KfC-II: (R = H and R’ = Fuc) or (R = Fuc and R’ = H)

19. ANTICOAGULANT ACTIVITYfC-IfC-IIfCS-IfCS-VIfCS-VIIfCS-VIIIfCS-IXfCS-XHeparinActivity[IU/mg]0.200.250.180.27n.d.0.260.330.23198Table 2:AT-dependent activity against factor Xa020406080100120140160fC-IfC-IIfCS-IfCS-VIfCS-VIIfCS-VIIIfCS-IXfCS-XheparinHC-II concentration [ng/mL]HC-II-mediated anti-factor IIa activity[17] a) Wu et al. Eur. J. Med. Chem. 2015, 92, 257-269 b) Panagos et al. J. Biol. Chem. 2014, 289, 28284-28298 c) Mourão et al. J. Biol. Chem. 1996, 271, 23973-23984AT-dependent activity is very similar to that reported for low molecular mass fCS[17]

20. FUTURE PERSPECTIVES Glycomar LTD1) Anticoagulant assays2) dfCS-selectins interactions University of Edinburgh (Prof. Dusan Uhrin) Different protection patterns on chondroitin intermediates Different glycosyl activators

21. CONCLUSIONSFirst O-glycosylation of secondary hydroxyls of polysaccharidesSemi-synthetic strategies from microbial sourced chondroitin to fucosylated chondroitin sulfateLibrary of different O-glycosylated chondroitin polysaccharides

22. L.297 project “Produzione biotecnologica di condroitina”Thanks toUniversity of Naples Federico II- Prof. M.Parrilli- Dr. E.BediniProf. A.IadonisiProf. C.De CastroSecond University of Naples (SUN)- Prof. M.De Rosa- Prof. C.SchiraldiUniversity of Edinburgh- Dr. Dusan Uhrin

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24. DERIVATIZATION ON FUCOSYL DONORSAc2OPy, RTI2, Et3SiHCH2Cl2, T=50°C reflux (NH2)2CS1) CH3CN T = 60°C Et3N 2) EtI RTNa(s)MeOH, RTTBDMSiCl, ImHDMF, RTDMF, RTBnBrTHF, RTTBAFPy, RTBzClEthyl 2,4-di-O-benzyl-3-O-benzoyl-β-L-fucopyranosil thioglycoside151617181920212223

25. DERIVATIZATION ON FUCOSYL DONORS(CH3)2C(OCH3)2CSA, RTBnBrDMF, RTTHF/H2O4:1(v/v) RT Bu2SnO1) C6H6/MeOH T = 60°C BnBr/Bu4NBr2) Toluene T = 65°C Py, RTBzClEthyl 2,3-di-O-benzyl-4-O-benzoyl-β-L-fucopyranosil thioglycoside192425262728