Laezza Antonio 1 Iadonisi Alfonso 1 De Castro Cristina 2 De Rosa Mario 3 Schiraldi Chiara 3 Parrilli Michelangelo 4 Bedini Emiliano 1 1 Dipartimento di Scienze Chimiche Università di Napoli Federico II Complesso Universitario Monte ID: 914093
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Slide1
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 Emiliano11 Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario Monte S.Angelo, via Cintia 4, I-80126 Napoli, Italy2 Dipartimento di Agraria, Università di Napoli Federico II, via Università 100, I-80055 Portici, Italy3 Dipartimento 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, Italy
4th EPNOE International
Polysaccharide Conference, Warsaw, 19-22 Oct 2015
Slide2FUCOSYLATED CHONDROITIN SULFATE
Fucosylated chondroitin sulfate (fCS) is a glycosaminoglycan (GAG) from sea cucumber[1]R, R’’ = H or SO3-+NaR’ = H, SO3- or FucSTRUCTURE:Trisaccharide repeating unit
ß-
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, 232-254
Sulfated α-fucosed 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
Slide3BIOLOGICAL ACTIVITIES
fCS 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
Slide4COAGULATION AND THROMBOSIS
Serpin-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
Slide5CHONDROITIN DERIVATIZATION
Escherichia 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
, 1779-1787
chondroitinpurity
grade
: 89-94%
weight-averaged
M
w
: 45.0
kDa
polydispersity
: 1.40
See
OP133 (
E.Bedini
)
“A MICROBIOLOGICAL-CHEMICAL ACCESS TO CHONDROITIN SULFATE POLYSACCHARIDES”
Bedini et al. Angew. Chem. Int. Ed. 2011, 50, 6160-6163
Slide6CHONDROITIN DERIVATIZATION
1a: X = COO-H+2345CH3COCl/CH3OH 0.58 M
CH3
OH, RT, 2 days1b: X = COO-(n-Bu)
4N+
n
-CH
3
(CH
2
)
10
CH
2
I
TBAF, DMF,
T = 80°C, overnight
[13]
Precipitation
PhCH
(OCH
3)
2CSA, DMFT = 80°C, overnightPhCH(OCH3)2CSA, DMFT = 80°C, overnight
Precipitation
[13] Pawar et al. Carbohydr. Polym. 2013, 98, 1288-1296
Slide7CHONDROITIN DERIVATIZATION
HSQC-DEPT NMR of polysaccharide 2(400 MHz, D2O, 298 K)DS = 0.92 2 4HSQC-DEPT NMR of polysaccharide 4(400 MHz, D2O, 298 K)DS = 0.99c
Slide8CHONDROITIN DERIVATIZATION
3HSQC-DEPT NMR of polysaccharide 3(400 MHz, DMSO-d6, 298 K)DS = 1.0 5HSQC-DEPT NMR of polysaccharide 5(400 MHz, DMSO-d6, 298 K)DS = 0.96
Slide9SEMI-SYNTHETIC STRATEGY
Glycosylation Et3N, Ac2O, DMAP, CH3CNRT, overnightPrecipitationNaBrO3, Na2S2O47:3 v/v H2O-AcOEtRT, overnight
Precipitation
2)
NaOH
pH~
13,RT, 6h
Dialysis
1) SO
3
.
py, DMF
T = 50°C, overnight
Precipitation
4: R = CH
3
5: R = CH
2
(
CH
2
)
10
CH
38(I-III): R = CH3, R’ =
per-O-Bn-Fuc or H9(IV-VI): R = CH2(CH2)10CH3, R’ = per-O-Bn-Fuc or H10(I-III): R = CH3
, R’ = per-O-Bn-Fuc or OAc11(IV-VI): R = CH2(CH2)10CH3, R’ = per-O-Bn-Fuc or
OAc12(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
)
14(I-VI): R =
per-O-sulfated-Fuc
or H, (
R’ = H and R’’ = SO
3
-
Na
+
)
or (
R’ = SO
3
-
Na
+
and R’’ = H
)
[14]
Laezza
et
al.
Biomacromolecules
,
2015,
16(7),
2237-2245
[14]
Slide10SEMI-SYNTHETIC STRATEGY
14(I) and 14(III)14(II)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)
Slide11Product
YieldaDFbα/βcO-3-linked- : O-2-linked- :(O-4,6-linked-α-Fuc ratio)d8I
98%
0.77
2.9 (2.6)
40% : 22% : 38%
8II
76%
0.32
f
2.3 (4.0)
38% : 62% : 0%
8III
76%
1.15
2.6 (2.7)
33% : 24% : 43%
9IV
56%
0.60
n.d.
e
n.d.
e
9V
---
---
---
---
9VI
58%
0.87
n.d.
e
n.d.
e
SEMI-SYNTHETIC STRATEGY
a
Mass
yield
determined
with
respect
to
starting
glycosyl
acceptor
(4-5)
over
five
steps
.
b
Determined
by
1H-NMR
integration
of
Fuc
methyl
and
GalNAc
acetyl
signals
.
c
Determined
as
difference
between
1H-NMR
integration
of
Fuc
methyl
signal
and
α
-
anomeric
signals
d
Determined
by
HSQC-DEPT
integration
of
related
anomeric
signals
e
Not
determined
f
Determined
by
HSQC-DEPT
integration
of
Fuc
methyl
and
GalNAc
acetyl
signals
Table
2
:
Yield
and
structural
data
of
fCS
8I, 8II, 8III
Slide12DERIVATIZATION ON
CHONDROITIN ACCEPTORS4Et3N, Ac2O, DMAP, CH3CNRT, overnightPrecipitation1516a: R = Bz and R’ = H16b: R = H and R’ = BzNaBrO
3, Na
2S2O47:3 v/v H2O-AcOEtRT, overnight
17
Precipitation
?
Slide13DERIVATIZATION ON FUCOSYL DONORS
L-fucoseEthyl 2,4-di-O-benzyl-3-O-benzoyl-β-L-fucopyranosil thioglycosideEthyl 2,3-di-O-benzyl-4-O-benzoyl-β-L-fucopyranosil thioglycoside
fCS
: R
= 2,4-di-
O
-sulfated-Fuc
or H,
(R’ = H and R’’ = SO
3
-
Na
+
) or (R’ = SO
3
-
Na
+
and R’’ = H)
fCS
: R = 2,3-di-
O-sulfated-Fuc or H, (R’ = H and R’’ = SO
3-Na+) or (R’ = SO3-Na+ and R’’ = H)
Slide14CONCLUSIONS
First O-glycosylation of secondary hydroxyls of polysaccharidesFirst seven-step semi-synthetic strategy from microbial source chondroitin to fucosylated chondroitin sulfate HIGH GLOBAL YIELD RESEMBLANCE WITH NATURAL fCS CHEAPNESS OF THE USED REAGENTS
Slide15FUTURE PERSPECTIVES
Different protection patterns on Fuc donorsDifferent protection patterns on chondroitin intermediatesSynthesis of a library of fCS polysaccharidesAnticoagulant tests on fCS polysaccharides
Slide16ACKNOWLEDGMENT
University 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.Schiraldi
MIUR
L.297 project “Produzione biotecnologica di condroitina”
BioTekNet
Slide17Slide18O-GLYCOSYLATION
EntryAcceptorDonorSolventProduct1a465:3 v/vCH2Cl2-DMF8I2b465:3 v/vCH2
Cl2
-DMF8II3c
4
7
5:3 v/
v
CH
2
Cl
2
-DMF
8III
4
a
5
6
3:1 v/
v
THF/CH
2
Cl2
9IV
5c573:1 v/v
THF/CH2Cl29V6d5
75:3 v/vCH2Cl2-DMF9VI
a Reaction conditions: acceptor, donor (5 eq with
respect to the acceptor), NIS (5.5 eq), TMSOTf (5.5 eq), AW-300 4Å 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 4Å MS, T = -20°C 4h
c
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
7
Table
1
:
Glycosylation
reactions
of
acceptors
4-5
with
donors
6-7
Slide19O-GLYCOSYLATION
HSQC-DEPT NMR of polysaccharide 99 (I-III): R = CH3, R’ = per-O-Bn-Fuc or H10 (IV-VI): R = CH2(CH2)10CH3, R’ = per-O-Bn-Fuc or HHSQC-DEPT NMR of polysaccharide 10
Slide20