Chemoenzymatic synthesis of C8-modified sialic acids and related α2-3- and α2-6-linked sialosides

Article abstract of DOI:10.1016/j.bmcl.2011.04.083

Naturally occurring 8-O-methylated sialic acids, including 8-O-methyl-N-acetylneuraminic acid and 8-O-methyl-N-glycolylneuraminic acid, along with 8-O-methyl-2-keto-3-deoxy-d-glycero-d-galacto-nonulosonic acid (Kdn8Me) and 8-deoxy-Kdn were synthesized fro

Full text of DOI:10.1016/j.bmcl.2011.04.083

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5037–5040
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Bioorganic & Medicinal Chemistry Letters
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Chemoenzymatic synthesis of C8-modified sialic acids and related
a2–3-and a2–6-linked sialosides
Hai Yu a, Hongzhi Cao b, Vinod Kumar Tiwari c, Yanhong Li a, Xi Chen a,
⇑
a Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, United States
b National Glycoengineering Research Center, Shandong University, Jinan, Shandong 250012, China
c Banaras Hindu University, Chemistry, Faculty of Science, Varanasi, UP 221 005, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 24 April 2011
Naturally occurring 8-O-methylated sialic acids, including 8-O-methyl-N-acetylneuraminic acid and
8-O-methyl-N-glycolylneuraminic acid, along with 8-O-methyl-2-keto-3-deoxy- -glycero- -galacto-
nonulosonic acid (Kdn8Me) and 8-deoxy-Kdn were synthesized from corresponding 5-O-modified
six-carbon monosaccharides and pyruvate using sialic acid aldolase cloned from Pasteurella
multocida strain P-1059 (PmNanA). In addition, 2–3- and 2–6-linked sialyltrisaccharides containing
Neu5Ac8Me and Kdn8Deoxy were also synthesized using a one-pot multienzyme approach. The strat-
egy reported here provides an efficient approach to produce glycans containing various C8-modified
sialic acids for biological evaluations.
D
D
Keywords:
a
Chemoenzymatic synthesis
C8-modification
Sialic acid
Sialoside
Sialyltransferase
a
a
Ó 2011 Elsevier Ltd. All rights reserved.
Sialic acids have been widely found in higher animals and some
microorganisms. They are commonly found at the termini of the gly-
can chains on glycoproteins and glycolipids.1 Sialic acids constitute a
structurally diverse family of nine-carbon acidic monosaccharides
with more than 50 members having been identified. N-Acetylneu-
raminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), and
enzyme-catalyzed reactions often offer great advantages and are
considered attractive and practical approaches for the synthesis
of sialosides including those containing uncommon sialic acid
forms. Recently, Withers et al. reported the chemical synthesis of
C8-modified sialic acids and their application in the CMP-sialic acid
synthetase-catalyzed synthesis of CMP-sialic acid derivatives.
2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn) are the
Campylobacter jejuni
a2–3-sialyltransferase Cst-I-catalyzed forma-
three basic forms of sialic acids which are distinguished from
one another by different substituents at carbon-5.2–4 Additional
modifications at different hydroxyl groups of sialic acids include
O-acetylation as the most frequently occurring modification. 8-O-
Methylation of sialic acids is also commonly observed. For example,
8-O-methylated sialic acids have been reported in starfish Asterias
rubens as the components of gangliosides5–8 and glycoproteins.9
Several 8-O-methylated sialic acid forms observed include 8-O-
methyl-N-acetylneuraminic acid (Neu5Ac8Me 1, Fig. 1), 8-O-
methyl-N-glycolylneuraminic acid (Neu5Gc8Me 2, Fig. 1), and their
O-acetylated derivatives.10,11 Neu5Ac8Me has also been found in
the sperm and eggs of teleost fish,12 in human red blood cell mem-
brane,13 and in mouse tissues.14 As 8-O-methylated sialic acids are
resistant to sialidases,15 8-O-methylation of sialic acid may play
important biological roles. Nevertheless, the significance of naturally
occurring C8-modified sialic acid derivatives is currently unclear.
Only a few methods have been reported for chemical synthesis
of sialosides containing Neu5Ac8Me.16–18 These methods,
however, are inefficient, lengthy, and tedious. In comparison,
tion of a2–3-linked sialyllactose containing C8-modified sialic
acids was also achieved from CMP-sialic acid derivatives.19 Never-
theless, both chemical and enzymatic syntheses of sialosides
containing C8-modified sialic acids so far have been limited to
Neu5Ac-based structures and with a2–3-sialyl linkage.
Here, we report a facile chemoenzymatic approach for prepara-
tive synthesis of Neu5Ac8Me (1), Neu5Gc8Me (2), Kdn8Me (3), and
Kdn8Deoxy (4) from chemically synthesized C5-modified N-acetyl-
mannosamine (ManNAc), N-glycolylmannosamine (ManNGc), and
mannose derivatives using a sialic acid aldolase-catalyzed reaction.
The use of 5-O-methyl-ManNAc and 5-deoxy-mannose as donor
substrates in a one-pot three-enzyme system for preparing both
a2–3- and a2–6-linked sialosides containing Neu5Ac8Me and
Kdn8Deoxy are also described. These compounds are important
probes for studying the importance of C8-hydroxy group at the sia-
lic acid residue in the interaction of sialylated carbohydrates and
sialic acid binding proteins.
Sialic acid aldolases are enzymes involved in the metabolism of
sialic acids. They catalyze the aldol cleavage reaction in nature but
the reaction is reversible and the enzymes can be used syntheti-
cally in the aldol addition direction for the formation of Neu5Ac
from pyruvate and ManNAc. The sialic acid aldolase from
⇑
Corresponding author. Tel.: +1 530 754 6037; fax: +1 530 752 8995.
E-mail address: chen@chem.ucdavis.edu (X. Chen).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.083

5038
H. Yu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5037–5040
MeO
MeO
MeO
OH
OH
OH
OH
OH
HO
OH
HO
OH
HO
OH
HO
HO
HO
HO
HO
-
-
-
-
CO2
CO2
CO2
CO2
O
O
O
O
AcHN
GcHN
HO
HO
Neu5Ac8Me (1)
Neu5Gc8Me (2)
Kdn8Me (3)
Kdn8Deoxy (4)
Figure 1. Structures of C8-modified sialic acid forms.
Pasteurella multocida P-1059 (PmNanA) recently cloned in our lab
has shown flexible substrate specificity.20 It is a more efficient en-
zyme than the E. coli sialic acid aldolase reported previously21 for
using 5-O-methyl ManNAc as a substrate.20 Based on the extremely
flexible substrate specificity of PmNanA, we hypothesize that the
5-O-methyl ManNGc, 5-O-methyl mannose, and 5-deoxy-mannose
are also potential substrates for this enzyme to produce the
corresponding C8-modified sialic acids 2–4.
ester (glycolyl-NHS ester),21 leading to the formation of compound
15. Debenzylation by hydrogenolysis with H2 and Pd/C in methanol
produced the desired 5-O-methyl-ManNGc 16 in 76% yield.
The preparation of 5-O-methyl mannose 22 is outlined in
Scheme 3. Commercially available 2,3:5,6-di-O-isopropylidene-
a-D
-mannofuranose 17 was treated with sodium hydride and
benzyl bromide to produce the corresponding benzyl - and b-gly-
a
cosides 18 in 57% and 42% yields, respectively. Partial regioselec-
tive hydrolysis of 18 was achieved at 30 °C for 20 h using 70%
aqueous acetic acid to produce the desired diol 19. Selective 6-O-
benzylation was achieved by formation of a dibutylstannylene
derivative followed by alkylation with benzyl bromide. Product
20 was treated with iodomethane and sodium hydride to produce
the methylation product 21, which was then treated with 75% TFA
followed by hydrogenolytic removal of the benzyl groups to
produce desired 5-O-methyl mannose 22 in 90% yield.
5-O-Methyl ManNAc 13 was synthesized from readily accessi-
ble and inexpensive 1,2:5,6-di-O-isopropylidene-a-D-glucopyra-
nose (5).22 As shown in Scheme 1, after benzylation of C3-OH,
the 5,6-isopropylidene protecting group was selectively removed
by mild acid hydrolysis and the resulting intermediate diol 6 was
treated with methyl chloroformate to produce carbonate 7.23
Treatment of 7 with benzyl alcohol in the presence of acidic ion ex-
change resin24 produced benzyl
a
- and b-anomers of furanoside 8
in 88% yield with a ratio of approximately 1.2:125, which can be
separated by flash chromatography. The C2-OH of the -anomer
For the preparation of 5-deoxy-mannose 27 (Scheme 4), diol 23
a
was obtained from D
-mannose in three steps.29 Regioselective ben-
8 was converted to triflate ester by treating with Tf2O. It was then
reacted with NaN3 in DMF to produce 2-azido-2-deoxy-manopyr-
anoside 9. The carbonate protecting group was removed by
treating 9 with sodium methoxide in methanol. Selective 6-O-ben-
zylation of the azido diol was then achieved by formation of a
dibutylstannylene derivative followed by alkylation with benzyl
bromide.26 The product 10 was treated with iodomethane and so-
dium hydride to produce methylation product 11 as the required
key intermediate. The 2-azido group of 11 was converted to acet-
amido group by treating with AcSH in pyridine to produce
12.27,28 After hydrogenation in the presence of H2 and Pd/C,
5-O-methyl-ManNAc 13 was produced in 90% yield.
As shown in Scheme 2, to synthesize 5-O-methyl ManNGc 16,
the azido group in compound 11 was reduced to an amino group
in the presence of 1,3-dithiopropanol and Et3N in pyridine/H2O.28
The resulting amino group in 14 was readily converted to N-glyco-
lyl by coupling with N-hydroxysuccinamide-activated glycolyl
zoylation of 23 produced partially benzoated compound 24 in 90%
yield. Treatment of benzoate 24 with phenyl chlorothionoformate
and pyridine produced the corresponding thiocarbonyl derivative
25 in good yield (95%). Reaction of 25 with tri-n-butylstannane
and AIBN provided the deoxy intermediate 26 in 71% yield. Subse-
quent removal of the isopropylidene group using TFA/H2O fol-
lowed by debenzoylation produced the target compound 5-deoxy
mannose 27 in 91% yield.
With these C-5 modified monosaccharides (13, 16, 22, 27) on
hands, the substrate specificity of PmNanA was examined. Despite
of several reported unsuccessful aldolase-catalyzed enzymatic
reaction of 5-O-methyl ManNAc with pyruvate25 or the observa-
tion of trace amount of product,30 our results showed that all of
the C5-modified monosaccharides tested can be tolerated by the
recombinant PmNanA as the substrates. The PmNanA-catalyzed al-
dol addition of 13, 16, 22, and 27 with five equivalents of sodium
pyruvate in Tris-HCl buffer (100 mM, pH 7.5) at 37 °C for 24 h
O
O
HO
HO
O
OH
OBn
O
O
OBn
O
O
Methyl chloroformate,
i) NaH, BnBr, DMF
O
ii) 50% HOAc, 50 o
C
Et3N, CH2Cl2
81%
O
O
5
6
7
O
98% ( two steps)
O
O
O
O
BnOH,
88%
α β
Dowex H+ resin,
80 oC, 4h
:
= 1.2:1
O
i) NaOMe, MeOH
ii) Bu2SnO,BnBr,
Bu4NI, toluene
O
O
BnO
O
OBn
O
N3
BnO
N3
BnO
O
O
i) Tf2O, Py, CH2Cl2
HO
10
O
ii) NaN3, DMF
OBn 91% (two steps)
86% (two steps)
OBn
9
8
OBn
OH
quantitive NaH, MeI, DMF
BnO
BnO
MeO
12
HO
MeO
13
NHAc
OBn
BnO
H2, 10% Pd/C, EtOH
BnO N3
AcSH, Py
87%
NHAc
OH
O
O
MeO
11
O
90%
OH
OBn
Scheme 1. Synthesis of 5-O-methyl ManNAc 13.

H. Yu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5037–5040
5039
O
BnO
BnO
1,3-propanodithiol,
BnO
OH
N3
NH2
BnO
BnO
Py, H2O, Et3N
100%
HN
BnO
Glycolyl-NHS ester,
DMF 86%
O
O
MeO
11
MeO
14
O
MeO
15
OBn
OBn
OBn
O
HO
OH
HN
HO
H2, 10% Pd/C, EtOH
76%
MeO
16
O
OH
Scheme 2. Synthesis of 5-O-methyl ManNGc 16.
O
O
HO
70% HOAc, 30oC
95%
NaH, BnBr, DMF
99%
O
O
O
O
O
O
O
O
O
O
O
HO
19
OH
OBn
OBn
17
18
Bu2SnO,BnBr,
Bu4NI, toluene
86%
HO
i) TFA;
BnO
BnO
ii) H2, 10% Pd/C, EtOH
NaH, MeI, DMF
OH
OH
O
O
O
O
O
O
MeO
HO
20
O
quantitive
MeO
21
90%
OBn
OBn
OBz
OH
22
Scheme 3. Synthesis of 5-O-methyl-D-mannose 22.
HO
BzO
PhO(C=S)Cl,
CH2Cl2, Py
BzO
BzCl, Py/CH2Cl2
90%
O
O
O
O
O
O
O
O
O
HO
23
HO
24
95%
(PhO)SO
25
OBz
OBz
i) 60% TFA
HO
BzO
OH OH
O
O
O
ii) NaOCH3, CH3OH
O
n-Bu3SnH, AIBN, toluene
OBz
OH
91%
26
27
71%
Scheme 4. Synthesis of 5-deoxy-D-mannose 27.
to be used by NmCSS as a good substrate. These results indicate
that the activity of NmCSS is affected by certain modifications on
C-8 and/or C-5 of sialic acids.
R2
R1
HO
R2
O
OH
OH
HO
HO
1
2
3
4
R1
O-
PmNanA
pH 7.5
-
OH
+
OH
CO2
O
O
It turned out that both 5-O-methyl ManNAc 13 and 5-deoxy-D-
mannose 27 can be used as sialic acid precursors for efficient
O
R1 = NHAc, R2 = OMe
R1 = NHAc, R2 = OMe 89%
13
one-pot three-enzyme synthesis31–33 of
a2–3- and a2–6-linked
16 R1 = NHGc, R2 = OMe
22 R1 = OH, R2 = OMe
R1 = NHGc, R2 = OMe 86%
R1 = OH, R2 = OMe
R1 = OH, R2 = H
85%
90%
sialosides containing C-8 modified sialic acids including 8-O-
methyl Neu5Ac 1 and 8-deoxy-Kdn 4. As shown in Scheme 6,
R1 = OH, R2 = H
27
a
2–3-linked sialosides Neu5Ac8Me
Kdn8Deoxy 2–3LacbProN3 30 were synthesized in excellent
yields (93% and 91%, respectively) from 3-azidopropyl b- -galacto-
pyranosyl-(1 ? 4)-b- -glucopyranoside (LacbProN3) 28 catalyzed
by Pasteurella multocida multifunctional 2–3-sialyltransferase
PmST133 in the presence of PmNanA and NmCSS. Similarly,
2–
6-linked sialosides Neu5Ac8Me 2–6LacbProN3 31 and Kdn8Deox-
2–6LacbProN3 32 were obtained highly efficiently (95% and 92%
yields, respectively) catalyzed by a Photobacterium damselae 2–6-
a2–3LacbProN3 29 and
Scheme 5. PmNanA-catalyzed enzymatic synthesis of C8-modified sialic acids 1–4
(54–84 mg).
a
D
D
a
followed by the combination of anion exchange and gel filtration
column purifications produced corresponding sialic acids and
derivatives (1, 2, 3, and 4, respectively) in excellent yields
(Scheme 5). The structures of the products were confirmed by
NMR and high resolution mass spectrometry (HRMS). The PmNanA,
thus, is a highly efficient enzyme for synthesizing C8-modified sialic
acids.
The obtained C8-modified sialic acids 1–4 were used in sub-
strate specificity study of a recombinant N. meningitidis CMP-sialic
acid synthetase (NmCSS).21 Neu5Ac8Me 1 was an excellent sub-
strate for NmCSS, which was in consistent with the previous report
for CMP-sialic acid synthetase from Neisseria.19 However, to our
surprise, Neu5Gc8Me 2 and Kdn8Me 3 were not substrates for
NmCSS. Interestingly, unlike Kdn8Me 3, Kdn8Dexoy 4 was able
a
a
ya
a
sialyltransferase (Pd2,6ST)31,34 in the presence of PmNanA and
NmCSS. All sialoside products were purified by Bio-Gel P-2 gel fil-
tration chromatography and the structures were characterized by
1H and 13C NMR as wells as high resolution mass spectrometry
(HRMS). These compounds are valuable probes for investigating,
at a molecular level, the involvement of the C-8 group of sialic
acids in the interaction of sialosides and sialic acid-binding
proteins. The azido group at the reducing end of the synthesized
sialosides can be easily reduced to form a primary amino group,
which can be used to link to proteins or other molecules.35–38

5040
H. Yu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5037–5040
R2
R1
HO
O
OH
O
-O2C
O
OH
O
OH
HO
HO
HO
R2
13 or 27
pyruvate, CTP, pH8.5
PmNanA, NmCSS
O
O
N3
HO
with PmST1
with Pd2,6ST
OH R1
O
HO
HO
R1 = NHAc, R2 = OMe
HO
HO
OH
O
29
30
93%
91%
OH
OH
O
R
1 = OH, R2 = H
O
O
N3
HO
28
R2
HO
-O2C
OH
HO
OH
HO
O
O
OH
O
R1
O
HO
O
HO
O
N3
HO
HO
HO
1 = NHAc, R2 = OMe
31
32
95%
92%
R
R1 = OH, R2 = H
Scheme 6. One-pot three-enzyme synthesis of
a2–3- and
a
2–6-linked sialosides containing Neu5Ac8Me and Kdn8Deoxy (54–82 mg). PmNanA, Pasteurella multocida sialic
acid aldolase;20 NmCSS, N. meningitidis CMP-sialic acid synthetase;21 PmST1, Pasteurella multocida multifunctional
a
2–3-sialyltransferase;33 Pd2,6ST, Photobacterium
damselae a
2–6-sialyltransferase.31,34
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jugation with molecules containing a terminal or strained alkyne
group via Huisgen’s [3+2] cyclization reaction with or without
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In conclusion, we report here
a convenient and efficient
sialic acid aldolase-catalyzed enzymatic approach for producing
C8-modified sialic acids. Using chemically synthesized 5-O-meth-
ylated monosaccharides and pyruvate, natural occurring 8-O-
methylated sialic acids Neu5Ac8Me and Neu5Gc8Me as well as
non-natural sialic acids Kdn8Me and Kdn8Deoxy were synthesized
in excellent yields using a promiscuous recombinant Pasteurella
multocida P-1059 sialic acid aldolase (PmNanA). In addition,
a2–3- and a2–6-linked sialosides containing Neu5Ac8Me and
Kdn8Deoxy were also synthesized using an efficient one-pot
three-enzyme system. The obtained 8-O-methylated sialic acids
and sialosides are important probes for understanding the signifi-
cance of O-methyl modification of sialic acids in nature.
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Acknowledgments
This work was supported by the Beckman Young Investigator
Award from the Arnold and Mabel Beckman Foundation and the
National Institutes of Health Grant R01GM076360.
Supplementary data
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Supplementary data (experimental procedures and character-
ization of compounds) associated with this article can be found,
in the online version, at doi:10.1016/j.bmcl.2011.04.083.
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