Disaccharides as sialic acid aldolase substrates: Synthesis of disaccharides containing a sialic acid at the reducing end

Article abstract of DOI:10.1002/anie.200604799

(Chemical Equation Presented) Synthesis with an unfussy enzyme: Escherichia coli sialic acid aldolase has been found to be unusually flexible in accepting disaccharides containing a mannose (Man) or N-glycolylmannosamine (ManNGc) at the reducing end as su

Full text of DOI:10.1002/anie.200604799

Angewandte
Chemie
DOI: 10.1002/anie.200604799
Chemoenzymatic Synthesis
Disaccharides as Sialic Acid Aldolase Substrates: Synthesis of
Disaccharides Containing a Sialic Acid at the Reducing End**
Shengshu Huang, Hai Yu, and Xi Chen*
Sialic acid aldolases (EC 4.1.3.3), or N-acetylneuraminate
lyases (Neu5Ac lyases or NALs), catalyze the reversible aldol
cleavage of N-acetylneuraminic acid (Neu5Ac) to form
A wild-type sialic acid aldolase has been cloned from
E. coli K-12 (substrain MG1655) and used in the one-pot
multiple-enzyme synthesis of cytidine 5’-monophosphate–
[
6f–h]
pyruvate
Scheme 1). They have been extensively studied and the
reverse reaction has been widely applied in the enzymatic
and
N-acetyl-d-mannosamine
(ManNAc)
sialic acid and sialoside derivatives in our laboratory.
Our
[
1]
(
previous work has shown that a disaccharide, Galb1,6Man,
can also be used by the recombinant aldolase for the high-
yield synthesis of the corresponding disaccharide,
Galb1,9KDN, containing a deaminoneuraminic acid (KDN)
[
1a]
synthesis of sialic acids and their derivatives.
[
8]
at the reducing end. The resultant KDN-containing dis-
accharide is a part of the tetrasaccharide component found in
[
8]
the cell wall of Streptomyces sp. MB-8. In this case, the
disaccharide Galb1,6Man can be considered as a mannose
derivative in which the hydrogen atom on the C6 hydroxy
group of the mannose is substituted with a galactose residue.
In order to investigate whether disaccharides containing a
mannose residue at the reducing end are generally tolerated
by the sialic acid aldolase, we chemically synthesized a list of
disaccharides with a galactose (Gal), a glucose (Glc), or a
mannose (Man) residue a or b linked to the hydroxy group at
the C2, C4, C5, or C6 positions of a mannose residue. Two
Scheme 1. Reaction catalyzed by sialic acid aldolases (NALs).
NALs are Schiff base forming type I aldolases. The NAL-
catalyzed reaction proceeds through ring-opening of the a
[
2]
anomer of Neu5Ac (Scheme 1) and a Schiff base inter-
mediate is formed between a strictly conserved catalytic
lysine residue in the enzyme and the ketone carbonyl group at
disaccharides
containing
an
N-glycolylmannosamine
(ManNGc) at the reducing end, Gala1,2ManNGc (23) and
Galb1,2ManNGc (24, Table 1), were also synthesized. We
demonstrate here that the two ManNGc-containing disac-
charides and most of the disaccharides containing a mannose
at the reducing end can be used as unusual substrates for the
sialic acid aldolase (Scheme 2). This type of sialic acid
[
3,4]
the C2 position in the open ring form of Neu5Ac. Although
quite restricted in its donor substrate (for example, pyruvate),
Escherichia coli aldolase is flexible about using a variety of
hexoses, pentoses, and their derivatives as acceptor substrates
for the synthesis of sialic acids and their analogues. C2-, C4-,
C5-, and/or C6-substituted ManNAc or mannose are well
[
5,6]
tolerated.
can also be different from those of ManNAc or mannose.
The configurations at the C4 and C5 positions
[
1a,4,5]
Protein crystal-structure-based site-directed mutagenesis,
structure-guided saturation mutagenesis, and directed evolu-
tion have been carried out to generate aldolase mutants with
altered substrate specificity or broader substrate tolerance for
[
7]
the synthesis of novel products.
[
+]
[+]
[
*] S. Huang, Dr. H. Yu, Prof. Dr. X. Chen
Scheme 2. Sialic acid aldolase catalyzed synthesis of disaccharides
Department of Chemistry
containing a sialic acid residue at the reducing end.
University of California, Davis
One Shields Avenue, Davis, CA 95616 (USA)
Fax: (+1)530-752-8995
E-mail: chen@chem.ucdavis.edu
Homepage: http://chemgroups.ucdavis.edu/~chen/
] These authors contributed equally to this work.
aldolase catalyzed chemoenzymatic reaction can thus be
widely applied as a general and efficient method for
producing disaccharides containing sialic acid at the reducing
end. The resultant disaccharides can be used as precursors for
the synthesis of naturally occurring carbohydrates containing
nonterminal sialic acid residues, which have been found
mainly as surface polysaccharides (for example, capsular
polysaccharides or lipopolysaccharides) in some pathogenic
+
[
[
**] This work was supported by a National Sciences Foundation
CAREER Award (grant no.: CHE-0548235)and start-up funds from
the Regents of the University of California.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Communications
[
9]
[10]
bacteria. The synthesis of these molecules is essential to the
investigation of their biological roles.
donors.
The desired b-linked disaccharides Galb1,2Man
(13, 72%; Table 1, entry b), Galb1,4Man (15, 59%; entry d),
Glcb1,4Man (16, 51%; entry e), Galb1,5Man (18, 41%;
entry g), Glcb1,6Man (22, 68%; entry k), and Galb1,2-
ManNGc (24, 82%; entry m), as well as the a-linked
disaccharide Mana1,6Man (20, 71%; entry i) were obtained
by incubating the corresponding donor and acceptor in the
presence of trimethylsilyl triflate (TMSOTf) promoter, fol-
lowed by deprotection steps. The reactions proceeded with
excellent stereoselectivity due to the assistance of the acetyl
group at the C2 position of the glycosyl donors (neighboring-
group participation).
The chemical synthesis of disaccharides with a mono-
saccharide (selected from a galactose, a glucose, or a
mannose) a or b linked to a mannose or a ManNGc residue
at the reducing end was achieved by two general approaches.
Both approaches used partially protected mannose deriva-
tives 6–10 or partially protected ManNGc 11 as glycosyl
acceptors (Table 1).
For the production of disaccharides with the glycosidic
bond trans to the C2 group on the glycosyl donor, peracety-
lated d-galacto- (2), d-gluco- (3) and d-mannopyranosyl (4)
trichloroacetimidates were synthesized and used as glycosyl
Table 1: Chemical synthesis of disaccharides containing mannose or ManNGc at the reducing end and sialic acid aldolase catalyzed enzymatic
synthesis of disaccharides containing KDN or Neu5Gc at the reducing end.
Entry
Chemical synthesis
product
Aldolase-catalyzed reaction
[
a]
[b]
donor
acceptor
yield [%]
50
product
yield [%]
0
a
b
c
72
44
0
[
c]
1
2
ca. 5
d
e
f
8
8
59
51
60
38
35
62
1
g
h
2
1
9
41
62
85
81
2
250
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Chemie
Table 1: (Continued)
Entry
Chemical synthesis
product
Aldolase-catalyzed reaction
[
a]
[b]
donor
acceptor
yield [%]
product
yield [%]
78
i
j
10
71
10
10
59
68
65
83
k
l
3
1
65
82
36
34
m
2
11
[a] The products of the chemical glycosylation are the potential substrates for the sialic acid aldolase catalyzed enzymatic reaction. [b] Yields for the
reaction steps including glycosylation and deprotection. [c] Yield was estimated by thin-layer chromatography.
For the synthesis of disaccharides with the glycosidic bond
cis to the C2 group on the glycosyl donor, thioethyl glycoside
donors bearing a nonparticipating substituent at the C2
position were used. Coupling of perbenzylated thiogalacto-
side donor 1 with acceptors 6 and 8–11 by using N-
iodosuccinimide/trifluoromethanesulfonic acid (NIS/TfOH)
as the catalyst, followed by appropriate deprotection steps,
gave the expected a-linked disaccharides Gala1,2Man (12,
(Gala1,5Man (17) and Galb1,5Man (18); Table 1, entries f
and g) or C6 (Gala1,6Man (19), Mana1,6Man (20), Glca1,6-
Man (21), and Glcb1,6Man (22); Table 1, entries h–k) hy-
droxy group of the mannose were good substrates for the
aldolase. The corresponding KDN-containing disaccharides
could be obtained with synthetic yields of 62–85%. Disac-
charides with a monosaccharide linked to the C4 hydroxy
group of the mannose (Gala1,4Man (14), Galb1,4Man (15),
and Glcb1,4Man (16); Table 1, entries c–e) were acceptable
substrates for the aldolase, with yields varying from 5–38%.
Disaccharides containing a galactose residue directly linked
to the C2 hydroxy group of the mannose (Gala1,2Man (12)
and Galb1,2Man (13); Table 1, entries a and b) were not
acceptable substrates for the aldolase.
[
11]
5
0%; Table 1, entry a), Gala1,4Man (14, 44%; entry c),
Gala1,5Man (17, 60%; entry f), Gala1,6Man (19, 62%;
entry h), and Gala1,2ManNGc (23, 65%; entry l), respec-
tively. Similarly, glycosylation of thioglucoside donor 5 and
acceptor 10 followed by deprotection afforded the disacchar-
ide Glca1,6Man (21) in 59% yield (Table 1, entry j).
All of the disaccharides obtained by chemical glycosyla-
tion were tested as potential substrates for the recombinant
E. coli sialic acid aldolase. As shown in Table 1, except for
The length of the linkage between the two monosacchar-
ides in the disaccharides could also be important to the
aldolase activity. For example, although disaccharides with a
galactose directly linked to the C2 position on the mannose
were not aldolase substrates, disaccharides containing a
galactose residue linked through an elongated N-glycolyl
linkage to the C2 position of ManNGc (Gala1,2ManNGc (23)
and Galb1,2ManNGc (24); Table 1, entries l and m) were
acceptable to the aldolase for the synthesis of the corre-
sponding disaccharides Gala1,5Neu5Gc (36) and Galb1,5-
Neu5Gc (37) in 36 and 34% yields, respectively.
(
1!2)-linked disaccharides Gala1,2Man (12) and Galb1,2-
Man (13; Table 1, entries a and b) which were not acceptable
to the aldolase, all of the disaccharides tested (Table 1,
entries c–m) could be used as substrates for the sialic acid
aldolase for the formation of disaccharides containing a sialic
acid residue at the reducing end.
The yield of the aldolase-catalyzed disaccharide synthesis
was closely related to the position of the glycosidic linkage in
the disaccharide substrates (or the position of the mono-
saccharide substituent on the reducing mannose). In general,
disaccharides with a monosaccharide linked to the C5
The aldolase activity was affected moderately by the a- or
b-glycosidic linkage in the disaccharides. Overall, b-linked
disaccharides were preferred by the aldolase. For example,
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Communications
only about 5% of Gala1,4Man (14) could be processed by the
centrifugation and discarded. The supernatant was concentrated by
rotary evaporation and the product was purified on a Bio-Gel P-2 gel
filtration column.
aldolase for the formation of Gala1,7KDN (27; Table 1,
entry c), while about 38% of the corresponding disaccharide
with a b-linkage, Galb1,4Man (15), was converted into the
product Galb1,7KDN (28) by the aldolase (Table 1, entry d).
Similarly, a lower yield (62%) was observed for the aldolase-
catalyzed formation of Gala1,8KDN (30) from Gala1,5Man
Received: November 27, 2006
Published online: February 19, 2007
Keywords: aldolases · carbohydrates ·
.
(17; Table 1, entry f) compared to that (85%) for the
chemoenzymatic synthesis · enzyme catalysis · sialic acids
formation of Galb1,8KDN (31) from Galb1,5Man (18;
Table 1, entry 9). Another example is that the yield for the
formation of Glca1,9KDN (34) from Glca1,6Man (21) was
[
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5% (Table 1, entry j), while the yield for the formation of the
corresponding b-linked disaccharide, Glcb1,9KDN (35), from
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No significant effect on the aldolase activity was observed
for disaccharides containing different monosaccharides at the
nonreducing terminus. For example, the yields for the
aldolase-catalyzed synthesis of Galb1,7KDN (28, 38%;
Table 1, entry d) and Glcb1,7KDN (29, 35%; Table 1,
entry e) were almost identical. Similarly, the yields for the
aldolase-catalyzed synthesis of Gala1,9KDN (32, 81%;
Table 1, entry h), Mana1,9KDN (33, 78%; Table 1, entry i),
and Glca1,9KDN (34, 65%; Table 1, entry j) were close.
Except for the formation of Gala1,7KDN (27) from
Gala1,4Man (14) (Table 1, entry c) for which the yield was
too low to obtain purified product, all of the other disacchar-
ides, 15–24 (Table 1, entries d–m), were used with an excess
amount of sodium pyruvate for the aldolase-catalyzed
preparative-scale synthesis of disaccharides 28–37 containing
KDN or Neu5Gc at the reducing terminus. These disacchar-
ide products have been purified and characterized by NMR
spectroscopy and high-resolution mass spectrometry
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the nonreducing end is insignificant. This chemoenzymatic
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[
Experimental Section
General procedures for aldolase-catalyzed reaction: A disaccharide
(50–100 mg, 1 equiv) and sodium pyruvate (5 equiv) were dissolved in
H O (5 mL). Tris(hydroxymethyl)aminomethane (Tris)/HCl buffer
2
(1 mL, 1m, pH 7.5) was added, followed by addition of the E. coli
aldolase (5 mg). The reaction solution was brought to 10 mL in
volume by adding H O. The reaction mixture was incubated at 378C
2
with agitation at 140 rpm. The reaction was monitored by thin-layer
chromatographic analysis with developing solvent (EtOAc:MeOH:
H O:HOAc 5:3:2:0.1, by volume) and stained with p-anisaldehyde
2
sugar stain. When no additional products were observed (the general
reaction time was 24 h), an equal volume (10 mL) of 95% EtOH was
added to the reaction mixture. The precipitates were separated by
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