Fucosylated Chondroitin Sulfate Polysaccharides Strategy

A semi-synthetic strategy to fucosylated chondroitin sulfate polysaccharides from microbial sourced chondroitin Laezza Antonio1, Iadonisi Alfonso1, De Castro Cristina2, De Rosa Mario3, Schiraldi Chiara3, Parrilli Michelangelo4, Bedini Emiliano1 1 Dipartimento di Scienze Chimiche, Universit di Napoli Federico II, Complesso Universitario Monte S.Angelo, via Cintia 4, I-80126 Napoli, Italy 2Dipartimento di Agraria, Universit di Napoli Federico II, Via Universit 100, I-80055, Portici, Italy 3Dipartimento di Medicina Sperimentale, Seconda Universit di Napoli, via De Crecchio 7, I-80138 Napoli, Italy 4 Dipartimento di Biologia, Universit di Napoli Federico II, Complesso Universitario Monte S.Angelo, via Cintia 4, I- 80126 Napoli, Italy 7thBaltic Meeting on Microbial Carbohydrates, G strow, 25-29 September

SUMMARY RO OR H OR O O RO Na+-OOC O O Microbial sourced chondroitin O O O HO NHAc OH R= H , SO3-+Na or variably sulfated L-fucose OR RO Na+-OOC O O O O O O NHAc OR' O R = H or SO3-+Na OR R = H or variably sulfated L-Fucose RO Fucosylated chondroitin sulfate structure similar to polysaccharide isolated from sea cucumber OR

FUCOSYLATED CHONDROITIN SULFATE Fucosylated chondroitin sulfate (fCS) is a glycosaminoglycan (GAG) from sea cucumber[1] [1] Pomin, V.H. Mar. Drugs 2014, 12, 232-254 STRUCTURE: Sulfated -fucose branches depends on the species of sea cucumber[2][3][4][5] Trisaccharide repeating unit [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 N-Acetyl- -D-galactosamine (GalNAc) -L-fucose (Fuc) -D-glucuronic acid (GlcA) R, R = H or SO3-+Na R = H, SO3-+Naor Fuc Responsible for biological activities of fCS

BIOLOGICAL ACTIVITIES fCS may exhibit activities related to: Coagulation and thrombosis[1] [1] Pomin, V.H. Mar. Drugs 2014, 12, 232-254 Atherosclerosis[6] [6] Tovar et al. Altherosclerosis 1996, 26, 185-195 Cancer metastasis and inflammation[7] [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

COAGULATION AND THROMBOSIS Blood coagulation relies on: 1) Vasoconstriction 2) Platelet plug 3) Clot formation Serpin-dependent mechanism driven by Heparin Cofactor II (HCII)[10] Serpin-independent mechanism due to inhibition of Factor Xa and thrombin generation[11] [10] Zancan et al. Blood Coagul Fibrinolysis, 2004, 15, 45-54 [11] Glauser et al. Thromb. Haemost., 2008, 100, 420-428

CHONDROITIN DERIVATIZATION Biotechnological production Polysaccharide purification Defructosylation Escherichia coli O5:K4:H4 chondroitin IN COLLABORATION WITH THE RESEARCH GROUP OF PROF. M. DE ROSA AT THE SECOND UNIVERSITY OF NAPLES (SUN)[12] purity grade: 89-94% weight-averaged Mw: 45.0 kDa polydispersity: 1.40 [12] Cimini et al. Appl. Microbiol. Biotechnol2010, 85, 1779-1787

CHONDROITIN DERIVATIZATION n-CH3(CH2)10CH2I TBAF, DMF, T = 80 C, overnight [13] CH3COCl/CH3OH 0.58 M CH3OH, RT, 2 days 1a: X = COOH 1b: X = COO-(n-Bu)4N+ 3 2 PhCH(OCH3)2 CSA, DMF T = 80 C, overnight PhCH(OCH3)2 CSA, DMF T = 80 C, overnight Precipitation Precipitation 4 5 [13] Pawar et al. Carbohydr. Polym.2013, 98, 1288-1296

SEMI-SYNTHETIC STRATEGY Sulfation free -OH Capping unreacted OH as -OAc 1) SO3.py, DMF T = 50 C, overnight [14] Glycosylation Et3N, Ac2O DMAP, CH3CN RT, overnight NaBrO3, Na2S2O4 H2O-AcOEt RT, overnight 2) NaOH pH~ 13,RT, 6h 4: R = CH3 5: R = CH2(CH2)10CH3 fCS(I-V): R = per-O-sulfated-Fuc or H, (R = H and R = SO3-Na+) or (R = SO3-Na+ and R = H) Cleavage Bn and benzylidene groups [14] Laezza et al. Biomacromolecules, 2015, 16(7), 2237-2245 Global deprotection fCS Acceptor Donor Yield Degree of fucosylation / O-3-linked-: O-2- linked-: (O-4,6)- linked- -Fuc-ratio I 4 6 98% 0.77 2.9 40% : 22% : 38% II 4 6 76% 0.32 2.3 38% : 62% : 0% III 4 7 76% 1.15 2.6 33% : 24% : 43% IV 5 6 56% 0.60 n.d. n.d. V 5 7 58% 0.87 n.d n.d

O-GLYCOSYLATION Table 1: Glycosylation reactions of acceptors 4-5 with donors 6-7 Entry Acceptor Donor Solvent Product 4 1a 4 6 5:3 v/v CH2Cl2- DMF[15] 5:3 v/v CH2Cl2-DMF 8I 2b 4 6 8II 5 3c 4 7 5:3 v/v CH2Cl2-DMF 8III SEt O 4a 5 6 OBn 3:1 v/v THF/CH2Cl2 9IV 6 OBn OBn 6d 5 7 5:3 v/v CH2Cl2-DMF 9V O CF3 O a Reaction conditions: acceptor, donor (5 eq with respect to the acceptor), NIS (5.5 eq), TMSOTf (5.5 eq), AW-300 4 MS, rt, 4h b Reaction conditions: acceptor, donor (5 eq with respect to the acceptor), NIS (5.5 eq), TMSOTf (1.7 eq), AW-300 4 MS, T = -20 C 4h c Reaction conditions: acceptor, donor (5 eq with respect to the acceptor) ,TMSOTFf(0.1 eq), AW-300 4 MS, rt, 4h, (inverse procedure) d Reaction conditions: acceptor, donor (5 eq with respect to the acceptor), TMSOTf (0.5 eq) AW-300 4 MS, rt, 4h, (inverse procedure) OBn NPh 7 OBn OBn [15] Lu et al., Angew. Chem. Int. Ed.2011, 50, 7315-7320

O-GLYCOSYLATION 8 (I-III): R = CH3, R = per-O-Bn-Fuc or H 9 (IV-VI): R = CH2(CH2)10CH3, R = per-O-Bn-Fuc or H 1H-NMR of polysaccharide 8 1H-NMR of polysaccharide 9

SEMI-SYNTHETIC STRATEGY Sulfation free -OH Capping unreacted OH as -OAc 1) SO3.py, DMF T = 50 C, overnight [14] Glycosylation Et3N, Ac2O DMAP, CH3CN RT, overnight NaBrO3, Na2S2O4 H2O-AcOEt RT, overnight 2) NaOH pH~ 13,RT, 6h 4: R = CH3 5: R = CH2(CH2)10CH3 fCS(I-V): R = per-O-sulfated-Fuc or H, (R = H and R = SO3-Na+) or (R = SO3-Na+ and R = H) Cleavage Bn and benzylidene groups [14] Laezza et al. Biomacromolecules, 2015, 16(7), 2237-2245 Global deprotection fCS Acceptor Donor Yield Degree of fucosylation / O-3-linked-: O-2- linked-: (O-4,6)- linked- -Fuc-ratio Glycosylation conducted at rt I 4 6 98% 0.77 2.9 40% : 22% : 38% II 4 6 76% 0.32 2.3 38% : 62% : 0% Glycosylation conducted at -20 C III 4 7 76% 1.15 2.6 33% : 24% : 43% IV 5 6 56% 0.60 n.d. n.d. V 5 7 58% 0.87 n.d n.d

A library of semi-synthetic fCS with different Fuc branching site and/or sulfation pattern: branching site sulfation site (protected with orthogonal cleavable groups) ( (H HO O) )( (O OB Bz z) ) OH BzO COOCH3 O O O 6 O O AcO NHAc OAc 12 O SEt SEt Ph O O O OBn OBn COOCH3 O O O OBn OBz 10 11 O O HO OBz OBn NHAc OH 4 Fuc donors chondroitin acceptors Fuc sulfation / Branching position O-2 and/or O- 3 of GlcA units O-4 or O-6 of GalNAc units Anti-coagulant tests for SAR investigation of natural and non-natural (semi-synthetic) fCS polysaccharides Fuc 2,3,4 S Fuc 2,4 S Mw (natural) ~ 70-80 kDa Mw (semi-synthetic) ~ 8-12 kDa Fuc 2,3 S Fuc O S

SEMI-SYNTHETIC STRATEGY Sulfation free -OH Capping unreacted OH as -OAc 1) SO3.py, DMF T = 50 C, overnight [16] O Ph O COOCH3 6 or 10 or 11 5:3 v/v CH2Cl2-DMF AW-300 MS T = -20 C, 4h O O Et3N, Ac2O DMAP, CH3CN RT, overnight NaBrO3, Na2S2O4 H2O-AcOEt RT, overnight O O O HO 2) NaOH pH~ 13,RT, 6h OH NHAc 4 fCS I: R = per-O-sulfated-Fuc or H, (R = H and R = SO3-Na+) or (R = SO3-Na+ and R = H) fCS VI: R = 2,3-di-O-sulfated-Fuc or H, (R = H and R = SO3-Na+) or (R = SO3-Na+ and R = H) Cleavage Bn and benzylidene groups fCS VII: R = 2,4-di-O-sulfated-Fuc or H, (R = H and R = SO3-Na+) or (R = SO3-Na+ and R = H) Global deprotection fCS Acceptor Donor Yield Degree of fucosylation / O-3-linked-: O-2-linked- -Fuc-ratio 4 6 41% 0.51 3.6 0.9 I 4 10 41% 0.43 5.5 0.6 VI 4 11 17% 0.44 2.8 1.1 VII [16] Laezza et al. Chem. Eur. J. 2016 accepted

Fucosylated chondroitin sulfate S or or S OH COO-Na+ HO O O O O O HO Fuc 2,4 S or or OH NHAc Fuc 2,4 S fCS VII HSQC-DEPT NMR of fCS VII. 600 MHz, D2O, T = 298 K

SEMI-SYNTHETIC STRATEGY Sulfation free -OH NaBrO3, Na2S2O4 Et3N, Ac2O DMAP, CH3CN RT, overnight H2O-AcOEt RT, overnight 4 12: (R = Bz and R = H) or (R = H and R = Bz) OR 1) SO3.py, DMF T = 50 C, overnight COO-+Na R'O [16] O O 6 or 10 or 11 NaBrO3, Na2S2O4 O O O HO 5:3 (v/v) CH2Cl2/DMF AW-300 MS RT, 4h OH NHAc 2) NaOH pH~ 13,RT, 6h H2O-AcOEt RT, overnight fCS 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) 12: (R = Bz and R = H) or (R = H and R = Bz) Cleavage Bn and benzylidene groups Global deprotection [16] Laezza et al. Chem. Eur. J. 2016 accepted fCS Acceptor Donor Yield Degree of fucosylation / O-4-linked-: O-6-linked- -Fuc-ratio 4 6 47% 0.45 4.3 1.3 VIII 4 10 38% 0.43 4.0 2.1 IX 4 11 40% 0.50 1.4 2.4 X

Fucosylated Chondroitin sulfate F Fu uc c 2 2, ,3 3, ,4 4 S S o or r o or r F Fu uc c 2 2, ,3 3, ,4 4 S S OH COO-Na+ HO O O O O O HO OH NHAc fCS VIII HSQC-DEPT NMR of fCS VIII. 600 MHz, D2O, T = 298 K

SEMI-SYNTHETIC STRATEGY O Ph O COOCH3 O O O O O HO 4 OH NHAc 12: (R = Bz and R = H) or (R = H and R = Bz) 5:3 (v/v) CH2Cl2/DMF AW-300 MS RT, 4h 5:3 (v/v) CH2Cl2/DMF AW-300 MS RT, 4h 6 6 DMAP, CH3CN RT, overnight H2O-AcOEt RT, overnight Et3N, Ac2O NaBrO3, Na2S2O4 H2O-AcOEt RT, overnight NaOH pH~ 13,RT, 6h COO-+Na NaBrO3, Na2S2O4 OR [16] R'O NaOH pH~ 13,RT, 6h O O O O O HO OH NHAc [16] fC-II: (R = H and R = Fuc) or (R = Fuc and R = H) fC-I: R = Fuc or H, (R =R = H) [16] Laezza et al. Chem. Eur. J. 2016 accepted

SEMI-SYNTHETIC STRATEGY fC Acceptor Donor Yield Degree of fucosylation / O-2-linked-: O-3-linked-: O-4,6-linked- -Fuc-ratio 4 6 33% 0.34 5.2 0.9 (O-2-, O-3-linked) I 4 6 39% 0.38 3.5 2.1 (O-4-, O-6-linked) II OR COO-+Na R'O O O O O O HO OH NHAc fC-I: R = Fuc or H, (R =R = H) fC-II: (R = H and R = Fuc) or (R = Fuc and R = H) HSQC-DEPT NMR of fC I. 600 MHz, D2O, T = 298 K HSQC-DEPT NMR of fC II. 600 MHz, D2O, T = 298 K

ANTICOAGULANT ACTIVITY fC- I fC- II fCS- I fCS- VI fCS- VII fCS- VIII fCS- IX fCS-X Heparin Activity [IU/mg] 0.20 0.25 0.18 0.27 n.d. 0.26 0.33 0.23 198 Table 2:AT-dependent activity against factor Xa AT-dependent activity is very similar to that reported for low molecular mass fCS[17] [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) Mour o et al. J. Biol. Chem.1996, 271, 23973-23984 160 140 HC-II concentration [ng/mL] 120 100 80 60 40 20 0 fC-I fC-II fCS-I fCS-VI fCS-VII fCS-VIII fCS-IX fCS-X heparin HC-II-mediated anti-factor IIa activity

FUTURE PERSPECTIVES Different protection patterns on chondroitin intermediates Different glycosyl activators Glycomar LTD 1) Anticoagulant assays University of Edinburgh (Prof. Dusan Uhrin) 2) dfCS-selectins interactions

CONCLUSIONS Semi-synthetic strategies from microbial sourced chondroitin to fucosylated chondroitin sulfate First O-glycosylation of secondary hydroxyls of polysaccharides Library of different O-glycosylated chondroitin polysaccharides

Thanks to University of Naples Federico II Second University of Naples (SUN) University of Edinburgh - Dr. Dusan Uhrin - Prof. M.Parrilli - Dr. E.Bedini -Prof. A.Iadonisi -Prof. C.De Castro - Prof. M.De Rosa - Prof. C.Schiraldi L.297 project Produzione biotecnologica di condroitina

DERIVATIZATION ON FUCOSYL DONORS I OAc OH I2, Et3SiH O O Ac2O O OAc CH2Cl2, T=50 C reflux OAc OH Py, RT AcO AcO HO OAc 15 16 17 OAc OH Et3N 2) EtI RT (NH2)2CS 1) CH3CN T = 60 C SEt SEt SEt O O TBDMSiCl, ImH O Na(s) OH OH OAc DMF, RT MeOH, RT HO AcO OTBDMS OH OH 18 OAc 20 19 BnBr DMF, RT SEt O SEt SEt O O TBAF BzCl OBn OBn OBn THF, RT Py, RT OBz HO OTBDMS OBn OBn OBn 23 21 22 Ethyl 2,4-di-O-benzyl-3-O-benzoyl- -L-fucopyranosil thioglycoside

DERIVATIZATION ON FUCOSYL DONORS SEt (CH3)2C(OCH3)2 BnBr SEt O O OBn OH CSA, RT DMF, RT O HO 19 24 25 O OH THF/H2O 4:1(v/v) RT Bu2SnO SEt 1) C6H6/MeOH T = 60 C BnBr/Bu4NBr 2) Toluene T = 65 C O BzCl SEt O SEt O OBn Py, RT OBn OBn HO BnO OH OBn 27 26 OH 28 OBz Ethyl 2,3-di-O-benzyl-4-O-benzoyl- -L-fucopyranosil thioglycoside