Chemo-enzymatic synthesis of antagonists of the Myelin-associated glycoprotein (MAG)

Article abstract of DOI:10.2533/000942904777678019

Ganglioside GQ1bα is the most potent antagonist of the Myelin-associated glycoprotein (MAG) identified so far. For the efficient synthesis of the partial structure of GQ1bα and derivatives thereof, a chemo-enzymatic strategy using the α(2→3)-sialyltransfe

Full text of DOI:10.2533/000942904777678019

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CHIMIA 2004, 58, No. 4
Chimia 58 (2004) 215–218
© Schweizerische Chemische Gesellschaft
ISSN 0009–4293
Chemo-Enzymatic Synthesis of
Antagonists of the Myelin-associated
Glycoprotein (MAG)
Gan-Pan Gao*, Oliver Schwardt, Tamara Visekruna, Said Rabbani, and Beat Ernst
Abstract: Ganglioside GQ1bα is the most potent antagonist of the Myelin-associated glycoprotein (MAG) identified
so far. For the efficient synthesis of the partial structure of GQ1bα and derivatives thereof, a chemo-enzymatic strat-
egy using the α(2→3)-sialyltransferase ratST3Gal ΙΙΙ (EC 2.4.99.6) was applied. Besides the natural substrates
Galβ(1→3)GlcNAc (19) and Galβ(1→4)GlcNAc (20), the disaccharides Galβ(1→3)GalNTCAβ-OSE (9),
Galβ(1→3)GalNAcβ-OSE (11), and Galβ(1→3)Galβ-OSE (14) were also tolerated by the enzyme and were trans-
formed to the target structures in preparative scale.
Keywords: Chemo-enzymatic synthesis · Myelin-associated glycoprotein (MAG) · ratST3Gal ΙΙΙ ·
α(2→3)-Sialyltransferase
Introduction
Axons of the adult mammalian central
nervous system (CNS) do not regenerate af-
ter injury, largely due to inhibitors in the
myelin sheath, an insulator that wraps
around axons [1]. To date, the myelin-asso-
ciated glycoprotein (MAG) [2] has been
identified as one of the neurite outgrowth-
inhibitory proteins, together with Nogo-A
and the oligodendrocyte myelin glycopro-
tein (OMgp) [3]. All of them bind to the
same receptor NgR, which interacts with
p75NTR to transduce the inhibitory signal
across the membrane (Fig. 1) [4]. In addi-
tion, MAG – the only member of the siglec
(sialic acid binding immunoglobulin lectin)
family in the mammalian CNS – was found
to bind with high specificity and affinity to
brain gangliosides like GT1b, GD1a and
GQ1bα [5].
*Correspondence: G.-P. Gao
University of Basel
Institute of Molecular Pharmacy
Klingelbergstrasse 50
CH–4056 Basel
Tel.: +41 61 267 15 57
Fax: +41 61 267 15 52
E-Mail: Ganpan.Gao@unibas.ch
Fig. 1. Myelin-associated inhibitors of axonal regeneration in the adult mammalian CNS

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The ganglioside GQ1bα (Fig. 2), which
is the most potent MAG antagonist identi-
fied so far [5], consists of an octasaccharide
and a ceramide at its reducing end. Based
on a structure–activity relationship (SAR)
study with numerous glycosphingolipids,
the branched tetrasaccharide Neu5Acα−
(2→3)Galβ(1→3)[Neu5Acα(2→6)]GalN
Ac (Fig. 2, in grey) was found to make the
major contribution to MAG-binding,
whereas the α(2→3)-linked sialic acid
residue (Fig. 2, in box) was a prerequisite
for affinity [5].
C13H27
HO
C17H35
O
OH
OH
N
H
O
O
HO
OH
AcHN
CO2H
O
HO
HO
O
OH
OH
O
HO
O
HO
O
HO
OH
HO
O
OH
For a refined SAR study, we decided to
synthesize modified sialylated oligosaccha-
rides. Despite considerable recent progress
[6], the stereoselective synthesis of α-sialo-
sides by chemical sialylation remains a sig-
nificant challenge due to the sterically hin-
dered tertiary anomeric centre, the presence
of an electron-withdrawing carboxylate and
the lack of a participating neighbouring
group in the 3-position of the sialic acid
moiety [7]. Enzymatic syntheses employ-
ing sialyltransferases as biocatalysts offer
an alternative approach to sialylated
oligosaccharides with excellent stereose-
lectivity and generally good to excellent
yields [8].
O
O
AcHN
O
O
OH
AcHN
O
HO
HO2C
OH
CO2H
O
HO
AcHN
OH
CO2H
O
O
OH
OH
OH
HO
AcHN
OH
Previous studies showed that the
α(2→3)-sialyltransferases ST3Gal I [9], II
[10], and IV [11] are the physiological en-
zymes which transfer sialic acid residues to
the 3-OH group of the terminal galactose
moiety of the type III disaccharide
Galβ(1→3)GalNAc. The natural substrates
of ST3Gal III [12], however, are type Ι
Fig. 2. The ganglioside GQ1bα
[Galβ(1→3)GlcNAc]
and
type
ΙΙ
[Galβ(1→4)GlcNAc] disaccharides. In ad-
dition, it was shown that ST3Gal III accepts
a wide variety of substituents on the glu-
cosamine nitrogen [8b], as well as lactal,
lactose and 2-O-pivaloyllactose [13]. Here,
we report that ratST3Gal III (EC 2.4.99.6)
can also be used for the enzymatic synthe-
sis of partial structure of GQ1bα and deriv-
atives thereof.
Results and Discussion
The type ΙΙΙ substrates Galβ(1→3)GalN
and Galβ(1→3)Gal were chemically syn-
thesized as shown in Schemes 1 and 2 [14].
By using the trichloroacetyl (TCA) protect-
ing group, Galβ(1→3)GalNTCAβ-OSE (9)
and Galβ(1→3)GalNAcβ-OSE (11) could
easily be obtained (Scheme 1).
For this purpose, per-O-acetylated N-
trichloroacetylglucosamine (1) [15] was
transferred into the (trimethylsilyl)ethyl
glycoside 2 via the corresponding bromide.
Subsequent O-deacetylation and selective
3,6-O-bis-pivaloylation gave 3 in 89%
yield. After transformation of the 4-hy-
droxy group into the corresponding triflate,
the galactosamides 4 and 5 were obtained in
Scheme 1. a) i. HBr/AcOH, rt, 17 h, ii. HOSE, Hg(CN)2, MS 3Å, toluene, rt, 3 d (89%); b) cat.
NaOMe/MeOH, rt, 16 h (quant); c) 2.5 equiv. PivCl, cat. DMAP, pyridine, –30 °C, 20 h (89%); d) Tf2O,
DCE/pyridine 2:1, 0 °C, 4 h, then H2O, 80 °C, 2 h (83%); e) 0.1 M NaOMe/MeOH, rt, 7 h, (96%); f)
PhCH(OMe)2, cat. p-TsOH, MeCN, rt, 4 h (86%); g) ethyl 2,3,4,6-tetra-O-benzoyl-1-thio-β-D-galac-
topyranoside (7), NIS, TfOH, CH2Cl2, –25 °C, 1.5 d (64% β); h) i. cat. NaOMe/MeOH, rt, 18 h, ii. 80%
aq. AcOH, 80 °C, 2 h (46%); i) Bu3SnH, AIBN, benzene, 80 °C, 3 h (93%); j) i. 80% aq. AcOH, 50 °C,
4.5 h, ii. cat. NaOMe/MeOH, rt, 21 h (64%).

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CHIMIA 2004, 58, No. 4
ural substrates 19 [22][23] and 20 [23][24]
OH
OH
O
were then incubated with cytidine-5’-
HO
HO
HO
O
O
monophospho-N-acetylneuraminic
acid
OSE
(CMP-Neu5Ac) and recombinant rST3Gal
III (9 U/L) (Scheme 3) in preparative scale
[14]. After 17–24 h of incubation, one addi-
tional aliquot of transferase was added (ex-
cept for the natural substrates 19 and 20).
The addition of another aliquot of rST3Gal
III and CMP-Neu5Ac did not further in-
crease the yields.
OH
OH
14
b)
OBz
O
OBn
O
OBz
OBn
O
OH
O
HO
HO
BzO
BzO
BzO
BzO
HO
O
HO
O
a)
c)
O
OSE
OSE
OSE
OBn
12
OBz
OBz
OBn
OH
13
15
The natural substrates 19 (type Ι) and 20
(type ΙΙ) were converted quantitatively into
the corresponding trisaccharides 21 and 22
[12] (entries 1 and 2 in the Table). In addi-
tion, the disaccharides 9, 11 and 14 were al-
so sialylated by rST3Gal III affording the
corresponding trisaccharides 23–25 in ac-
ceptable yields (entries 3–5, Table). In all
three cases, the unreacted substrates could
be recovered almost quantitatively. The ki-
netic data for the sialylation reactions indi-
cate that the activity of rST3Gal III towards
the substrates 9, 11 and 14 is reduced about
10-fold compared to the one for its natural
substrates 19 and 20. As expected, also the
transfer efficiency Vmax/Km is much lower
for the unnatural substrates. This explains
the incomplete, but still preparatively use-
ful conversion of the type III disaccharides.
Probably, due to the bulky substituent in the
6-position of galactose, the 6-O-sialylated
trisaccharide 18 was not tolerated as sub-
strate by rST3Gal III.
OAc
OAc
CO2Me
SMe
16
AcO
d)
O
AcHN
AcO
OAc
OH
OAc
OAc
OH
OH
AcHN
AcO
AcHN
CO2Me
CO2Na
O
O
HO
OH
OBz
O
O
O
BzO
BzO
HO
HO
HO
HO
e)
O
O
O
O
OSE
O
OSE
OBz
OH
OH
OH
18
17
Scheme 2. a) 7, DMTST, CH2Cl2, 7 °C, 16 h (87% β); b) i. NaOMe, MeOH, rt, 2 h, ii. 10% Pd/C, H2,
MeOH, rt, 3 h (75%); c) 10% Pd/C, H2 (4 bar), MeOH/dioxane, rt, 9 d (67%); d) 16, NIS, TfOH, CH2Cl2,
–30 °C, 16 h (45% α); e) NaOMe, MeOH, rt, 7 h, then aq. NaOH, rt, 16 h (90%).
NH2
O
O
N
O
O
P
The introduction of a sialic acid unit
was indicated by signals in 13C NMR at ap-
proximately 100 ppm and 40 ppm, which
are characteristic for the C(2) and C(3) of
an α-linked sialic acid [12][13][24]. In ad-
dition, down-field shifts (~4 ppm) of the
galactose C(3) in 13C NMR and approxi-
mately 0.6 ppm of the galactose H(3) in 1H
NMR confirmed the regioselective intro-
duction of sialic acid in the 3-position of the
galactose moiety [12].
O
OH
OH
N
O
HO
O
O
AcHN
OH
O
OH
O
OH
OH
HO
HO
HO
O
HO2C
O
HO
HO
OH HO
OH
O
O
O
OR
O
OR
AcHN
OH
OH
rST3Gal III, pH 6.5, BSA, CIAP,
sodium cacodylate buffer, MnCl2
HO
Scheme 3. Enzymatic sialidation using rST3Gal ΙΙΙ and CMP-Neu5Ac
a 6:1 ratio by epimerization at 80 °C. By
transesterification, the pivaloates were re-
moved and the 4- and 6-OHs were protect-
ed as benzylidene (→6). The subsequent cou-
pling reaction with donor 7 [16] was carried
out using NIS/TfOH [17] as promotor, re-
sulting in pure β-disaccharide 8 in 64% yield.
Removal of the benzoyl groups and subse-
quent cleavage of the benzylidene group gave
Galβ(1→3)GalNTCAβ-OSE (9) in 46%
yield. From 8, Galβ(1→3)GalNAcβ-OSE
(11) was obtained by reduction of the N-
trichloroacetyl group with Bu3SnH/AIBN in
refluxing benzene (→10), followed by O-
debenzoylation (→11).
rivative 12 [18] (Scheme 2). For this
purpose, 12 was coupled with donor 7 using
Conclusion
dimethyl(methylthio)sulfonium
triflate
The MAG antagonists 23, 24, and 25
were prepared by the chemical synthesis of
the type ΙΙΙ disaccharides 9, 11, and 14, and
subsequent enzymatic sialylation using
rST3Gal III for the transfer of sialic acid
from CMP-Neu5Ac. The determination of
the MAG affinity in comparison with
GQ1bα is currently ongoing.
(DMTST) [19] as promoter to afford 13 in
excellent yield and stereoselectivity. After
removal of the benzoyl and benzyl protect-
ing groups by transesterification and hy-
drogenolysis respectively, Galβ(1→3)
Galβ-OSE (14) was isolated in 75% yield.
18 was obtained starting from 13, which
was partially deprotected by catalytic hy-
drogenation (→15), followed by coupling
with the sialic acid donor 16 [20] in the
presence of NIS/TfOH affording the corre-
sponding α-sialoside 17 in 45% yield. Sub-
sequent saponification gave trisaccharide
18 in 90%.
Acknowledgements
We thank Markus Streiff (Novartis Pharma
Inc., Basel) for supplying us with the rST3Gal
ΙΙΙ construct. Financial support by the Volks-
wagen Stiftung is kindly acknowledged.
The other two starting materials for
the enzymatic sialylation experiments,
Galβ(1→3)Galβ-OSE (14) and Galβ(1→3)
[Neu5Acα(2→6)]Galβ-OSE (18), were
synthesized starting from the galactose de-
Received: February 5, 2004
Following our standard sialylation pro-
tocol [12][21], the oligosaccharides 9, 11,
14 and 18, and, as positive controls, the nat-
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