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Laezza Antonio 1 , Iadonisi Alfonso 1 , De Castro Cristina 2 , De Rosa Mario 3 ,

Explore a semi-synthetic strategy to produce fucosylated chondroitin sulfate polysaccharides from microbial-sourced chondroitin. Discover their structure and potential biological activities. Collaborating with the University of Naples, this innovative approach offers derivatization methods and characterization techniques.

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Laezza Antonio 1 , Iadonisi Alfonso 1 , De Castro Cristina 2 , De Rosa Mario 3 ,

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  1. 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, Università di Napoli Federico II, Complesso Universitario Monte S.Angelo, via Cintia 4, I-80126 Napoli, Italy 2 Dipartimento di Agraria, Università di Napoli Federico II, via Università 100, I-80055 Portici, Italy 3 Dipartimento di Medicina Sperimentale, Seconda Università di Napoli, via De Crecchio 7, I-80138 Napoli, Italy 4 Dipartimento di Biologia, Università di Napoli Federico II, Complesso Universitario Monte S.Angelo, via Cintia 4, I-80126 Napoli, Italy 4th EPNOE International PolysaccharideConference, Warsaw, 19-22 Oct 2015

  2. FUCOSYLATED CHONDROITIN SULFATE Fucosylatedchondroitinsulfate (fCS) is a glycosaminoglycan (GAG) fromseacucumber[1] [1] Pomin, V.H.Mar. Drugs2014, 12, 232-254 STRUCTURE: Sulfatedα-fucosedbranchesdepends on the speciesofseacucumber[2][3][4][5] Trisacchariderepeatingunit [2]Chenet al. Carbohydr. Polym.2010, 83, 688-696 [3]Chenet al. Biochim. Biophys. Acta2013, 1830, 3054-3066 [4]Luoet al. Mar. Drugs2013, 11, 399-417 [5]Wuet al. Eur. J. Biol. Chem. 2015, 87, 862-868 N-Acetyl-ß-D-galactosamine (GalNAc) α-L-fucose (Fuc) ß-D-glucuronic acid (GlcA) R, R’’ = H or SO3-+Na R’ = H, SO3- or Fuc Responsibleforbiological activitiesoffCS

  3. BIOLOGICAL ACTIVITIES • fCSmayexhibitactivitiesrelatedto: • Coagulation and thrombosis[1] [1] Pomin, V.H.Mar. Drugs2014, 12, 232-254 • Atherosclerosis[6] [6]Tovaret al.Altherosclerosis1996, 26, 185-195 • Cancermetastasis and inflammation[7] [7]Borsiget al. J. Biol. Chem. 2007, 282, 14984-14991 • Viralinfection[8][9] [8]Lianet al.Biochim. Biophys. Acta2013, 1830, 4681-4691 [9] Huang et al.Carbohydr. Res. 2013, 380, 64-69

  4. COAGULATION AND THROMBOSIS Bloodcoagulationrelies on: 1) Vasoconstriction 2) Plateletplug 3) Clotformation Serpin-dependentmechanismdrivenbyHeparinCofactor II (HCII)[10] Serpin-independentmechanism due toinhibitionofFactorXa and thrombin generation[11] [10] Zancanet al. BloodCoagulFibrinolysis, 2004, 15, 45-54 [11] Glauseret al. Thromb. Haemost., 2008, 100, 420-428

  5. CHONDROITIN DERIVATIZATION Biotechnological production Polysaccharide purification Defructosylation Escherichia coli O5:K4:H4 IN COLLABORATION WITH THE RESEARCH GROUP OF PROF. M. DE ROSA AT THE SECOND UNIVERSITY OF NAPLES (SUN)[12] chondroitin puritygrade: 89-94% weight-averagedMw: 45.0 kDa polydispersity: 1.40 SeeOP133 (E.Bedini) “A MICROBIOLOGICAL-CHEMICAL ACCESS TO CHONDROITIN SULFATE POLYSACCHARIDES” Bediniet al. Angew. Chem. Int. Ed.2011, 50, 6160-6163 [12] Ciminiet al. Appl. Microbiol. Biotechnol2010, 85, 1779-1787

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

  7. CHONDROITIN DERIVATIZATION 2 c HSQC-DEPT NMR ofpolysaccharide 2 (400 MHz, D2O, 298 K) DS = 0.92 HSQC-DEPT NMR ofpolysaccharide 4 (400 MHz, D2O, 298 K) DS = 0.99 4

  8. CHONDROITIN DERIVATIZATION 3 HSQC-DEPT NMR ofpolysaccharide 3 (400 MHz, DMSO-d6, 298 K) DS = 1.0 HSQC-DEPT NMR ofpolysaccharide 5 (400 MHz, DMSO-d6, 298 K) DS = 0.96 5

  9. SEMI-SYNTHETIC STRATEGY Glycosylation Et3N, Ac2O, DMAP, CH3CN RT, overnight Precipitation 4: R = CH3 5: R = CH2(CH2)10CH3 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 NaBrO3, Na2S2O4 7:3 v/v H2O-AcOEt RT, overnight 1) SO3.py, DMF T = 50°C, overnight Precipitation 2) NaOH pH~ 13,RT, 6h Dialysis Precipitation 12(I-III): R = CH3, R’ = Fuc or OAc, (R’’ = H and R’’’ = Bz) or (R’’ = Bz and R’’’ = H) 13(IV-VI) R = CH2(CH2)10CH3, R’ = Fuc or OAc, (R’’ = H and R’’’ = Bz) or R’’ = Bz and R’’’ = H) 10(I-III): R = CH3, R’ = per-O-Bn-Fuc or OAc 11(IV-VI): R = CH2(CH2)10CH3, R’ = per-O-Bn-Fuc or OAc [14] 14(I-VI): R = per-O-sulfated-Fuc or H, (R’ = H and R’’ = SO3-Na+)or (R’ = SO3-Na+and R’’ = H) [14] Laezzaet al. Biomacromolecules, 2015, 16(7), 2237-2245

  10. SEMI-SYNTHETIC STRATEGY 14(II) 14(I) and 14(III) HSQC-DEPT NMR of polysaccharide 14(I) (600 MHz, D2O, 298 K) HSQC-DEPT NMR of polysaccharide 14(III) (600 MHz, D2O, 298 K) HSQC-DEPT NMR of polysaccharide 14(I) (600 MHz, D2O, 298 K)

  11. SEMI-SYNTHETIC STRATEGY Table 2: Yield and structural data offCS 8I, 8II, 8III a Mass yielddeterminedwithrespecttostartingglycosylacceptor (4-5) overfivesteps. bDeterminedby 1H-NMR integrationofFucmethyl and GalNAcacetylsignals. cDeterminedasdifferencebetween 1H-NMR integrationofFucmethylsignal and α- anomericsignals dDeterminedby HSQC-DEPT integrationofrelatedanomericsignals eNotdetermined fDeterminedby HSQC-DEPT integrationofFucmethyl and GalNAcacetylsignals

  12. DERIVATIZATION ON CHONDROITIN ACCEPTORS Et3N, Ac2O, DMAP, CH3CN RT, overnight NaBrO3, Na2S2O4 7:3 v/v H2O-AcOEt RT, overnight Precipitation 4 Precipitation 15 ? 16a: R = Bz and R’ = H 16b: R = H and R’ = Bz 17

  13. DERIVATIZATION ON FUCOSYL DONORS L-fucose Ethyl 2,4-di-O-benzyl-3-O-benzoyl-β-L-fucopyranosilthioglycoside Ethyl 2,3-di-O-benzyl-4-O-benzoyl-β-L-fucopyranosilthioglycoside fCS: R = 2,4-di-O-sulfated-Fuc or H, (R’ = H and R’’ = SO3-Na+) or (R’ = SO3-Na+ and R’’ = H) fCS: R = 2,3-di-O-sulfated-Fuc or H, (R’ = H and R’’ = SO3-Na+) or (R’ = SO3-Na+ and R’’ = H)

  14. CONCLUSIONS First seven-stepsemi-syntheticstrategyfrommicrobial source chondroitintofucosylatedchondroitinsulfate First O-glycosylationofsecondaryhydroxylsofpolysaccharides • HIGH GLOBAL YIELD • CHEAPNESS OF THE USED REAGENTS • RESEMBLANCE WITH NATURAL fCS

  15. FUTURE PERSPECTIVES Differentprotectionpatterns on Fucdonors Differentprotectionpatterns on chondroitinintermediates Synthesisof a libraryoffCSpolysaccharides Anticoagulanttests on fCSpolysaccharides

  16. ACKNOWLEDGMENT UniversityofNaples “Federico II” SecondUniversity ofNaples (SUN) MIUR L.297 project “Produzione biotecnologica di condroitina” • - Prof. M.Parrilli • - Dr. E.Bedini • Prof. A.Iadonisi • Prof. C.De Castro - Prof. M.De Rosa - Prof. C.Schiraldi BioTekNet

  17. O-GLYCOSYLATION Table 1: Glycosylationreactionsofacceptors 4-5withdonors6-7 4 5 6 7 aReactionconditions: acceptor, donor (5 eqwithrespectto the acceptor), NIS (5.5 eq), TMSOTf (5.5 eq), AW-300 4Å MS, rt, 4h bReactionconditions: acceptor, donor (5 eqwithrespectto the acceptor), NIS (5.5 eq), TMSOTf (1.7 eq), AW-300 4Å MS, T = -20°C 4h cReactionconditions: acceptor, donor (5 eqwithrespectto the acceptor) ,TMSOTFf(0.1 eq), AW-300 4Å MS, rt, 4h, (inverse procedure) dReactionconditions: acceptor, donor (5 eqwithrespectto the acceptor), TMSOTf (0.5 eq) AW-300 4Å MS, rt, 4h, (inverse procedure)

  18. O-GLYCOSYLATION 9 (I-III): R = CH3, R’ = per-O-Bn-Fuc or H 10 (IV-VI): R = CH2(CH2)10CH3, R’ = per-O-Bn-Fuc or H HSQC-DEPT NMR ofpolysaccharide 9 HSQC-DEPT NMR ofpolysaccharide 10

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