1 / 20

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

A SEMI-SYNTHETIC STRATEGY TO FUCOSYLATED CHONDROITIN SULFATE POLYSACCHARIDES FROM MICROBIAL-SOURCED CHONDROITIN. Laezza Antonio 1 , Iadonisi Alfonso 1 , De Castro Cristina 2 , De Rosa Mario 3 , Schiraldi Chiara 3 , Parrilli Michelangelo 4 , Bedini Emiliano 1

samueljackson
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related