Aldolase-catalyzed synthesis of β-D-Galp-(1→9)-D-KDN: A novel acceptor for sialyltransferases

Article abstract of DOI:10.1021/ol060736m

β-D-Galp-(1→9)-D-KDN, a disaccharide component of the cell wall of Streptomyces sp. MB-8, was synthesized from β-D-Galp-(1→6)-D-Manp and pyruvate using a sialic acid aldolase. The obtained KDN-containing compound was a novel acceptor for bacterial sialylt

Full text of DOI:10.1021/ol060736m

ORGANIC
LETTERS
2
006
Vol. 8, No. 11
393-2396
Aldolase-Catalyzed Synthesis of
-Galp-(1 9)- -KDN: A Novel
Acceptor for Sialyltransferases
2
â
-D
f
D
Hai Yu and Xi Chen*
Department of Chemistry, UniVersity of California, One Shields AVenue,
DaVis, California 95616
chen@chem.ucdaVis.edu
Received March 26, 2006
ABSTRACT
â
-
D
-Galp-(1
and pyruvate using a sialic acid aldolase. The obtained KDN-containing compound was a novel acceptor for bacterial sialyltransferases.
Unusual 2,3- and 2,6-linked sialyltrisaccharides and a tetrasaccharide were synthesized using a one-pot two-enzyme system containing a
f9)-D-KDN, a disaccharide component of the cell wall of Streptomyces sp. MB-8, was synthesized from â-D-Galp-(1f6)-D-Manp
r
r
Neisseria meningitidis CMP-sialic acid synthetase and
a Pasteurella multocida sialyltransferase or a Photobacterium damsela r2,6-
sialyltransferase.
Sialic acids are negatively charged nine-carbon monosac-
charides that play pivotal roles in many physiologically and
pathologically important processes, including cellular rec-
(e.g., cell wall components, capsular polysaccharides or
lipopolysaccharides) of some pathogenic bacteria. These
3
ognition and communication, bacterial and viral infection,
(
3) (a) McSweegan, E. F.; Pistole, T. G. Biochem. Biophys. Res. Commun.
and tumor metastasis, etc.1,2 Although having been predomi-
1982, 106, 1390-1397. (b) Dutton, G. G.; Parolis, H.; Parolis, L. A.
Carbohydr. Res. 1987, 170, 193-206. (c) Krauss, J. H.; Himmelspach, K.;
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(d) Kogan, G.; Shashkov, A. S.; Jann, B.; Jann, K. Carbohydr. Res. 1993,
nantly found as terminal carbohydrate units on glycoproteins
and glycolipids of vertebrates or as components of capsular
polysaccharides and lipooligosaccharides of pathogenic
bacteria in the forms of polysialic acids or side-chain end
2
38, 261-270. (e) Vinogradov, E. V.; Knirel, Y. A.; Shashkov, A. S.;
Paramonov, N. A.; Kochetkov, N. K.; Stanislavsky, E. S.; Kholodkova, E.
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(h) Shashkov, A. S.; Kosmachevskaya, L. N.; Streshinskaya, G. M.;
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brandt, E. Eur. J. Biochem. 2002, 269, 6020-6025. (i) Shashkov, A. S.;
Streshinskaya, G. M.; Kosmachevskaya, L. N.; Evtushenko, L. I.; Naumova,
I. B. MendeleeV Commun. 2000, 5, 167-168. (j) Yanagidaira, S.; Yabe,
K.; Matsuyama, H.; Kato, T. Jpn. Kokai Tokkyo Koho 1998, JP 10029944.
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T.; Isobe, R.; Higuchi, R. Eur. J. Org. Chem. 1998, 11, 2519-2525.
1
units, sialic acids have also been found as internal residues
that link to other carbohydrate units in polysaccharide or
glycoconjugate forms.
Carbohydrate structures containing nonterminal sialic acid
residues have been found mainly in the surface molecules
(
1) (a) Schauer, R. Glycoconjugate J. 2000, 17, 485-499. (b) Angata,
T.; Varki, A. Chem. ReV. 2002, 102, 439-469.
(
2) (a) Paulson, J. C. Trends Biochem. Sci. 1989, 14, 272-276. (b) Lasky,
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Biol. 2002, 12, 609-615.
1
0.1021/ol060736m CCC: $33.50
© 2006 American Chemical Society
Published on Web 05/04/2006

8
ionic molecules are believed to be essential for the efficient
6
His -tagged fusion protein. When expressed at 37 °C for 3
3h
attachment of the bacteria to host cells and play important
h with the induction of 0.1 mM IPTG (isopropyl-1-thio-â-
D-galactopyranoside), the majority of the recombinant aldo-
lase presented in cell lysate as a soluble form, and it can be
easily purified by an affinity column packed with Ni -NTA-
agarose (nickel-nitrilotriacetic acid-agarose) resin. In agree-
4
roles in the virulence of the pathogenic organisms. For
example, KDN (deoxyneuraminic acid or 2-keto-3-deoxy-
D-glycero-D-galacto-nonulosonic acid), one of three basic
2+
5
sialic acid forms, was first found in rainbow trout eggs. It
6
a
has recently been detected in the cell wall of Streptomyces
ment with previous reports, the recombinant aldolase can
tolerate a wide range of modifications (even bulky groups)
at various positions on the ManNAc substrate or mannose
and it has been used successfully in one-pot multiple-enzyme
systems for the efficient synthesis of CMP-sialic acid
3
i
sp. VKM Ac-2090 and strain Streptomyces sp. VKM
3
h
Ac-2124 as â2,4-linked KDN polymers branched with
1
Glcâ1,8-linked side chains. Using the combination of H and
1
3
C NMR as well as MALDI-TOF mass spectrometry
8
9
analyses, KDN-containing oligomers have also been found
as cell wall components of Streptomyces sp. MB-8. They
are tetrasaccharide components with two â-D-Galp-(1f9)-
D-KDN disaccharides linked through an R-D-KDN-(2f4)-
â-D-KDN linkage, with some of the galactose residues being
derivatives and sialosides.
On the basis of the extremely flexible substrate specificity
of the sialic acid aldolase, we hypothesize that the disac-
charide structure â-D-Galp-(1f9)-D-KDN observed in the
cell wall of Streptomyces sp. MB-8 (Figure 1) can be
synthesized by an aldolase-catalyzed reaction from pyruvate
and a simpler disaccharide â-D-Galp-(1f6)-D-Manp, in
which the Gal residue can be considered as a substituent
replacing the H atom in the 6-O-hydroxyl group of the
mannose.
3
g
3
-O-methylated (Figure 1).3g
To test our hypothesis, disaccharide â-D-Galp-(1f6)-D-
Manp 1 was synthesized using a conventional synthetic
approach (Scheme 2). Direct tritylation followed by acety-
Scheme 2. Synthesis of Disaccharide â-D-Galp-(1f6)-D-Manp
1
Figure 1. Tetrasaccharide components found in the cell wall of
Streptomyces sp. MB-8.3g
Sialic acid aldolase (EC 4.1.3.3.) catalyzes a reversible
condensation of pyruvate with D-N-acetylmannosamine
(
ManNAc) to form D-N-acetylneuraminic acid (Neu5Ac) with
6
the equilibrium favoring the aldol cleavage (Scheme 1).
Scheme 1. Reaction Catalyzed by Sialic Acid Aldolases
lation of mannose 210 afforded acetyl 2,3,4-tri-O-acetyl-6-
O-trityl-R-D-mannopyranoside 3 in 90% yield. Although it
(7) (a) Schauer, R.; Kamerling, J. P. In Glycoproteins II; Montreuil, J.,
Vliegenthart, J. F. G., Schachter, H., Eds.; Elsevier: Amsterdam, The
Netherlands, 1997; pp 243-402. (b) Gijsen, H. J. M.; Wong, C.-H. J. Am.
Chem. Soc. 1995, 117, 7585-7591. (c) Mahmoudian, M.; Noble, D.; Drake,
C. S.; Middleton, R. F.; Montgomery, D. S.; Piercey, J. E.; Ramlakhan,
D.; Todd, M.; Dawson, M. J. Enzyme Microb. Technol. 1997, 20, 393-
Sialic acid aldolase has flexible substrate specificity and has
been widely used in the enzymatic synthesis of naturally
occurring and structurally modified sialic acids.1a,7 We have
cloned a full-length sialic acid aldolase (NanA) from
Escherichia coli K-12 and overexpressed it as a C-terminal
400. (d) Kim, M. J.; Hennen, W. J.; Sweers, H. M.; Wong, C.-H. J. Am.
Chem. Soc. 1988, 110, 6481-6486. (e) Simon, E. S.; Bednarski, M. D.;
Whitesides, G. M. J. Am. Chem. Soc. 1988, 110, 7159-7163. (f) Kragl,
U.; Gygax, D.; Ghisalba, O.; Wandrey, C. Angew. Chem., Int. Ed. 1991,
30, 827-828. (g) Halcomb, R. L.; Fitz, W.; Wong, C. H. Tetrahedron:
Asymmetry 1994, 5, 2437-2442. (h) Kong, D. C. M.; von Itzstein, M.
Carbohydr. Res. 1997, 305, 323-329. (i) Lin, C. C.; Lin, C.-H.; Wong,
C.-H. Tetrahedron Lett. 1997, 38, 2649-2652. (j) Pan, Y.; Ayani, T.; Nadas,
J.; Wen, S.; Guo, Z. Carbohydr. Res. 2004, 339, 2091-2100. (k) Fitz, W.;
Schwark, J.-R.; Wong, C.-H. J. Org. Chem. 1995, 60, 3663-3670.
(8) Yu, H.; Yu, H.; Karpel, R.; Chen, X. Bioorg. Med. Chem. 2004, 12,
6427-6435.
(
4) Bardotti, A.; Averani, G.; Berti, F.; Berti, S.; Galli, C.; Giannini, S.;
Fabbri, B.; Proietti, D.; Ravenscroft, N.; Ricci, S. Vaccine 2005, 23, 1887-
899.
5) Nadano, D.; Iwasaki, M.; Endo, S.; Kitajima, K.; Inoue, S.; Inoue,
Y. J. Biol. Chem. 1986, 261, 11550-11557.
6) (a) Wong, C.-H. In Enzymes in Synthetic Organic Chemistry; Wong,
1
(
(
(9) Yu, H.; Chokhawala, H.; Karpel, R.; Yu, H.; Wu, B.; Zhang, J.;
Zhang, Y.; Jia, Q.; Chen, X. J. Am. Chem. Soc. 2005, 127, 17618-17619.
(10) Reynolds, D. D.; Evans, W. L. J. Am. Chem. Soc. 1940, 62, 66-
69.
C.-H., Whitesides, G. M., Eds.; Elsevier Science Ltd: Oxford, U.K., 1994;
pp 215-219. (b) Auge, C.; David, S.; Gautheron, C. Tetrahedron Lett. 1984,
2
5, 4663-4664.
2394
Org. Lett., Vol. 8, No. 11, 2006

was reported that the trityl ether can be easily removed by
acid-catalyzed hydrolysis, removal of the trityl at C6 of 3
a potential acceptor for sialyltransferases, disaccharide 7 was
incubated with Neu5Ac and CTP, as well as two enzymes
including the N. meningitidis CMP-sialic acid synthetase
(NmCSS) and a sialyltransferase (an R2,3- or R2,6-sialyl-
transferase) in a Tris-HCl buffer (pH ) 8.5) containing 20
mM Mg . In this system, Neu5Ac was activated by NmCSS
to form CMP-Neu5Ac, an activated sugar nucleotide donor
for sialyltransferases, from which the Neu5Ac moiety can
be transferred to the galactose residue in 7 (a Gal-terminated
acceptor structure) to form an R2,3- or R2,6-linked sialoside
depending on the sialyltransferase used. We found that di-
saccharide 7 indeed was a good acceptor for both the Photo-
bacterium damsela R2,6-sialyltransferase (Pd2,6ST) and the
Pasteurella multocida multifunctional sialyltransferase
under standard conditions (HCl/MeOH, HOAc/H
2
O, or HF/
CH Cl ) was problematic and produced a mixture containing
2
2
a byproduct formed by 4f6 migration of the O-acetyl at
2+
C-4 to newly deprotected C-6. Instead, using hydrogen
bromide1
0,11
in acetic acid was proven to be a satisfactory
alternative approach for selective deprotection of the C-6
hydroxyl group. The desired compound 4 was obtained in
85% yield under this condition. Promoted by trimethylsilyl
trifluoromethane sulfonate (TMSOTf) in dichlorometh-
ane, Schmidt glycosylation of 4 with donor 2,3,4,6-tetra-
1
2
O-acetyl-R-D-galactopyranosyl trichloroacetimidate 5 af-
forded the peracetylated disaccharide 6 in 75% yield.
Deacetylation of 6 by Z e´ mplen reaction in sodium methoxide
and methanol produced the desired disaccharide 1 in
quantitative yield.
9
,15
16
(PmST1). Using the one-pot two-enzyme approach, we
obtained novel trisaccharides R-D-Neu5Ac-(2f6)-â-D-Galp-
(1f6)-D-KDN 8 and R-D-Neu5Ac-(2f3)-â-D-Galp-(1f6)-
D-KDN 9 in 90% and 89% yields, respectively (Scheme 4).
To our delight, the obtained disaccharide â-D-Galp-(1f6)-
D-Manp 1 was a good substrate for the recombinant sialic
acid aldolase. In fact, disaccharide â-D-Galp-(1f9)-D-KDN
7
was obtained in an excellent (85%) yield from 1 and 5
Scheme 4. One-Pot Two-Enzyme Synthesis of Novel R2,3-
and R2,6-Linked Sialyltrisaccharides and an Unusual
equiv of pyruvate in Tris-HCl buffer (100 mM, pH 7.5) by
incubating with the aldolase at 37 °C for 24 h followed by
a Bio-gel P-2 gel filtration column purification step (Scheme
1
6
Tetrasaccharide Containing Three Sialic Acid Residues
3). The structure of the product was confirmed by NMR and
Scheme 3. Aldolase-Catalyzed Synthesis of Disaccharide
â-D-Galp-(1f9)-D-KDN
high-resolution mass spectrometry. To our knowledge, the
synthesis of this type of oligosaccharides which contain a
sialic acid residue at the reducing end has only been achieved
enzymatically by using the trans-glycosylation activity of a
Bacillus circulans â-galactosidase with lactose as the ga-
1
3
lactosyl donor. An earlier attempt to convert â-D-Glcp-
1f6)-D-ManpNAc to â-D-Glcp-(1f6)-D-Neu5Ac using
(
1
4
aldolase was not successful. The enzymatic synthesis we
report here, thus, is the very first example to show that the
reducing-terminal mannose in a disaccharide can be con-
verted to KDN by a sialic acid aldolse.
Examining the structure of 7 identified a terminal galactose
at the nonreducing end of the disaccharide. The galactose
residue in 7, thus, would be a potential acceptor candidate
for sialyltransferases. A one-pot multiple-enzyme system
These results further confirmed the flexible acceptor speci-
ficity of both Pd2,6ST and PmST1 as reported earlier. Using
9
the similar one-pot two-enzyme approach, we can obtain
unusual tetrasaccharide 10 containing three sialic acid
residues from 9 in 87% yield using the NmCSS and the
9
established in our lab was used to test this hypothesis. As
(
11) Lee, G. S.; Lee, Y.-J.; Choi, S. Y.; Park, Y. S.; Yoon, K. B. J. Am.
Chem. Soc. 2000, 122, 12151-12157.
12) Ren, T.; Zhang, G.; Liu, D. Bioorg. Med. Chem. 2001, 9, 2969-
978.
13) Yanahira, S.; Yabe, Y.; Nakakoshi, M.; Miura, S.; Matsubara, N.;
Ishikawa, H. Biosci. Biotechnol. Biochem. 1998, 62, 1791-1794.
14) Auge, C.; David, S.; Gautheron, C.; Malleron, A.; Cavaye, B. New
1
7
Pd2,6ST. An attempt to synthesize 10 from 8 using the
(
one-pot two-enzyme system containing PmST1, however,
2
(
(15) (a) Yamamoto, T.; Nakashizuka, M.; Terada, I. J. Biochem. 1998,
123, 94-100. (b) Ni, L.; Sun, M.; Yu, H.; Chokhawala, H.; Chen, X.; Fisher,
A. J. Biochemistry 2006, 45, 2139-2148.
(
J. Chem. 1988, 12, 733-744.
Org. Lett., Vol. 8, No. 11, 2006
2395

was not successful. This indicates that a Neu5Ac residue
linked to the C-3 of the galactose residue in 7 does not block
the C-6 site for sialylation by Pd2,6ST. The Neu5Ac residue
on C-6 of the galactose residue in 7, however, blocks the
C-3 site for PmST1-catalyzed sialylation.
In conclusion, we report herein the very first example of
an aldolase-catalyzed synthesis of an unusual disaccharide
â-D-Galp-(1f9)-D-KDN which is a component of the cell
wall of Streptomyces sp. MB-8. Using the disaccharide
obtained from the aldolase reaction as a novel sialyltrans-
ferase acceptor, we have also synthesized two novel sialyl-
trisaccharides and an unusual sialyltetrasaccharide. We have
demonstrated here that the recombinant E. coli sialic acid
aldolase has extremely flexible substrate flexibility. Together
with the one-pot multiple-enzyme system established in our
lab, it is a powerful catalyst in the efficient synthesis of sialic
acid-containing structures.
(16) General experimental procedures for the preparation of sialosides
using a one-pot two-enzyme system: Reactions were typically carried out
in a 50 mL centrifuge tube in 10 mL of Tris-HCl buffer (100 mM, pH
8
.5) containing an acceptor substrate (disaccharide 7 or trisaccharide 9)
(
50-100 mg), Neu5Ac (1.5 equiv), CTP (1.5 equiv), MgCl2 (20 mM),
NmCSS (0.8 mg), PmST1 (0.04 mg for preparing R2,3-linked sialosides),
or Pd2,6ST (0.2 mg for preparing R2,6-linked sialosides). The reaction
mixture was incubated at 37 °C for 12 h with shaking (120 rpm). After the
addition of 10 mL of ice-cold methanol to stop the reaction, precipitates
were removed by centrifugation. The supernatant was concentrated by rotor
evaporation and purified by Bio-gel P-2 gel filtration chromatography.
Lyophilized sialoside products were characterized by NMR and high-
resolution mass spectrometry (HRMS). Notice that under the reaction
conditions used (pH 8.5), the R2,6-sialyltransferase activity of the PmST1
was not observed. The PmST1, thus, was used as an R2,3-sialyltransferase
here.
Acknowledgment. This work was supported by NSF
CAREER Award CHE-0548235 and startup funds from the
Regents of the University of California.
(17) The sialosides 8-10 could also be prepared from disaccharide 7 or
trisaccharide 9 in a one-pot three-enzyme system containing the E. coli
sialic acid aldolase, NmCSS, and a sialyltransferase (PmST1 or Pd2,6ST).
In this system, Neu5Ac was generated in situ from ManNAc and pyruvate
by the aldolase, activated to form CMP-Neu5Ac by NmCSS, and
transferred to acceptors by a sialyltransferase. As the aldolase-catalyzed
reactions are reversible, a large excess of pyruvate (10 equiv) has to be
used in the one-pot three-enzyme system to prevent the KDN residue at
the reducing end in 7 and 9 from converting back to mannose.
Supporting Information Available: Experimental pro-
cedures and NMR spectra for compounds 1 and 4-10. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL060736M
2396
Org. Lett., Vol. 8, No. 11, 2006