Highly efficient chemoenzymatic synthesis of naturally occurring and non-natural α-2,6-linked sialosides: A P. damsela α-2,6- sialyltransferase with extremely flexible donor-substrate specificity

Article abstract of DOI:10.1002/anie.200600572

(Chemical Equation Presented) Just relax: A one-pot, three-enzyme chemoenzymatic synthetic approach is highly efficient for obtaining complex sialosides that contain diverse naturally occurring sialic acid modifications. α-2,6-Linked sialosides containing

Full text of DOI:10.1002/anie.200600572

Angewandte
Chemie
DOI: 10.1002/anie.200600572
Communications
a-2,6-Linked sialosides that contain both natural and non-natural sialic
acid forms have been prepared through a one-pot, three-enzyme
chemoenzymatic synthesis. In the Communication on the following
pages, X. Chen and co-workers show that relaxed substrate specificity
of biosynthetic enzymes is the key.
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Enzyme Catalysis
DOI: 10.1002/anie.200600572
Highly Efficient Chemoenzymatic Synthesis of
Naturally Occurring and Non-Natural a-2,6-
Linked Sialosides: A P. damsela a-2,6-
Sialyltransferase with Extremely Flexible
Donor–Substrate Specificity**
Figure 2. Naturally occurring sialic acid modifications.
are developmentally regulated and are believed to be closely
related to their biological functions.[1] Nevertheless, a clear
understanding of the mechanism and the significance of
natureꢀs sialic acid structural diversity is currently missing.
This is mainly owing to the difficulties in obtaining homoge-
nous sialosides or sialylglycoconjugates, especially those that
contain diverse naturally occurring sialic acid modifications.
These structures are extremely difficult to isolate in homo-
genous forms from natural sources[3] and chemical sialylation
remains challenging.[4] Although sialyltransferase-catalyzed
synthesis offers great advantages,[5] it suffers from the low
expression level and the narrow substrate specificity of many
sialyltransferases, especially those from mammalian sources.[6]
Current chemical[4,7] and enzymatic[5,8] sialylation activities
have been focusing on structures containing non-natural sialic
acid derivatives and a limited number of natural sialic acid
forms (e.g. Neu5Ac, Neu5Gc, KDN, Neu5Ac8Me, and
Neu5,9Ac2).[3–9] Further development of the synthetic meth-
odology, thus, is required to obtain structurally defined
naturally occurring sialosides for better understanding of
their biological roles.
Hai Yu, Shengshu Huang, Harshal Chokhawala,
Mingchi Sun, Haojie Zheng, and Xi Chen*
Sialic acids are a family of a-keto acids with a nine-carbon
backbone. They have been predominantly found as terminal
carbohydrate units on glycoproteins and glycolipids of
vertebrates or as components of capsular polysaccharides
and lipooligosaccharides of pathogenic bacteria.[1] Sialic acid
containing structures play pivotal roles in many physiologi-
cally and pathologically important processes, including cell-
ular recognition and communication, bacterial and viral
infection, and tumor metastasis, etc.[1] Currently, more than
50 structurally distinct forms of sialic acids have been found in
nature,[1] more than 15of which have been found on human
red-blood-cell (RBC) surfaces, saliva proteins, and gastro-
intestinal mucins.[2] Three basic forms of sialic acids (Figure 1)
Sialic acid modifications, such as O-acetylation, O-meth-
ylation, O-lactylation, and O-sulfation, are believed to occur
after the formation of sialylglycoconjugates or polysialic acids
in mammals,[10] group C meningococci,[11] and E. coli.[12] There
is indication, however, that O-acetylation may occur on free
Neu5Ac in the biosynthesis of Group B Streptococcus (GBS)
capsular polysaccharide.[10b] Only a few enzymes involved in
the sialic acid modifications have been discovered, including
9-O-acetyltransferases from rat liver,[10a] bovine submandib-
ular gland,[13] E. coli K1,[1b,14] and C. jejuni;[15] 4-O-acetyl-
transferases from guinea-pig liver[16] and equine submandib-
ular gland;[17] a sialic acid 8-O-methyltransferase from starfish
A. rubens.[18] Cytidine monophosphate (CMP)-Neu5Ac
hydroxylase[19] and C. jejuni 9-O-acetyltransferase[15] are the
only proteins in this category that have ever been cloned.
Owing to the unavailability of these sialic acid modifying
enzymes, it is impractical to synthesize naturally occurring
sialosides by totally following their biosynthetic pathways.
Instead of natureꢀs way of introducing sialic acid modifi-
cations after oligosaccharide formation, we have established a
highly efficient and convenient one-pot, three-enzyme che-
moenzymatic approach for the synthesis of sialosides con-
taining naturally occurring as well as non-natural sialic acid
modifications at C5, C7, C8, and/or C9. In this method, sialic
acid modifications can be chemically introduced at the very
beginning, onto the six-carbon sugar precursors (ManNAc or
mannose) of sialic acids. These ManNAc or mannose
analogues can then be directly converted to the correspond-
ing sialosides in one pot by using three enzymes, including a
sialic acid aldolase, a CMP-sialic acid synthetase, and a
sialyltransferase without the isolation of intermediates
Figure 1. Three basic forms of naturally occurring sialic acids.
Ac=acetyl.
are N-acetylneuraminic acid (Neu5Ac), N-glycolylneura-
minic acid (Neu5Gc), and deaminoneuraminc acid (KDN).
Based on these three forms, single or multiple substitutions
can occur at the hydroxyl group on C4, C5, C7, C8, and/or C9
positions, including O-acetylation and the less frequent O-
methylation, O-lactylation, O-sulfation, and O-phosphoryla-
tion (Figure 2).[1]
Modifications of sialic acids and cell-surface presentation
of modified sialic acids are species and tissue specific. They
[*] Dr. H. Yu, S. Huang, H. Chokhawala, M. Sun, H. Zheng,
Prof. Dr. X. Chen
Department of Chemistry
University of California, Davis
One Shields Avenue, Davis, CA 95616 (USA)
Fax: (+1)530-752-8995
E-mail: chen@chem.ucdavis.edu
[**] The authors thank Prof. Ajit Varki for providing plasmids containing
the P. damsela a-2,6-sialyltransferase gene in a TOPO vector. This
work was supported by Mizutani Foundation for Glycoscience and
NIH R01M076360.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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(Scheme 1). Depending on the type of the sialyltransferase
(Tris)-Cl buffer solution at pH 8.5for 2–10 h at 25 8C. When
this condition was used for the synthesis of sialosides
containing O-acetyl groups, de-O-acetylation was observed.
Therefore, a Tris-Cl buffer solution (pH 7.5) with a lower pH
value was used for synthesizing sialosides containing O-
acetylated or O-lactylated sialic acid residues to avoid the
hydrolysis of the esters. After observing the product forma-
tion on TLC, preparative scale syntheses were carried out.
Sialoside products were purified by gel filtration chromatog-
raphy and characterized by NMR spectroscopy and HRMS.
As shown in Table 1, by using 3-azidopropyl lactoside 20
as an acceptor for Pd2,6ST, naturally occurring a-2,6-linked
sialosides containing Neu5Ac or its C5-substituted analogues
(26–32) were firstly synthesized in 75–99% yields (Table 1,
entries a–g) from ManNAc, mannose, or their C2-modified
analogues (1–7) as sialic acid precursors. 5-O-Acetyl-KDN
(KDN5Ac) (the sialic acid form in 29) has been found on the
eggs of Rana temporaria, Rana arvalis, and Pleurodeles
waltii;[2b] 5-O-methyl-KDN (KDN5Me) (the sialic acid form
in 30) has been observed in Sinorhizobium fredii;[25] N-
acetylglycolyl-neuraminic acid (Neu5GcAc) (the sialic acid
form in 31) has been detected in rat peritoneal macro-
phages.[26]
Naturally occurring a-2,6-linked sialosides containing a
C9-substituted Neu5Ac or KDN (33–35) were synthesized in
72–84% yields (Table 1, entries h–j) from the corresponding
C6-modified ManNAc or mannose (8–10). 9-O-Acetylation
has been found in nearly all higher animals and certain
bacteria and is believed to play a pivotal role in modulating
various biological processes.[27] For example, 9-O-acetyl-N-
acetylneuraminic acid (Neu5,9Ac2) (the sialic acid form in 33)
is an essential determinant of the cell surface receptors for the
influenza C virus, but prevents the recognition of influenza A
and B viruses.[28] It is also presented in an antigenic ganglio-
side found in developing rat embryonic cells.[29] 9-O-Lactyl-
Neu5Ac (Neu5Ac9Lt) (the sialic acid form in 34) has been
found in bovine submandibular gland glycoprotein[30] and
human RBC membranes.[2a] 9-O-Acetyl-KDN (KDN9Ac)
(the sialic acid form in 35) has been found in O. nerka adonis
polysialoglycoproteins.[31]
used, a-2,3- or a-2,6-linked sialosides can be synthesized
conveniently. Combining the diversity of organic synthesis
and the highly efficient, regio- and stereoselective enzymatic
approaches, this chemoenzymatic system is an attractive
synthetic method for complex sialic acid containing struc-
tures.
Two major challenges for highly effective chemoenzy-
matic synthesis are 1) the availability and efficiency of
biosynthetic enzymes; and 2) the extent of substrate modifi-
cation that can be tolerated by the enzymes. We have
successfully found solutions for synthesizing complex sialo-
sides by matching these two challenges.
Previously, we reported the high-level expression of a
recombinant sialic acid aldolase from E. coli K-12 and a CMP-
sialic acid synthetase from N. meningitidis (NmCSS).[20] Both
enzymes have flexible substrate specificity. They have been
used successfully in one-pot, two-enzyme preparative syn-
theses of CMP-sialic acid derivatives[20] and one-pot, three-
enzyme syntheses of a-2,3-linked sialoside libraries.[21] To
prepare a-2,6-linked sialosides with naturally occurring or
non-natural sialic acid modifications, we investigated the
donor–substrate specificity of a recombinant Photobacterium
damsela a-2,6-sialyltransferase (Pd2,6ST) and explored its
application in the one-pot, three-enzyme synthesis.
Pd2,6ST was the first bacterial a-2,6-sialyltransferase that
has ever been cloned.[22] The isolation of this sialyltransferase
was firstly reported in 1996.[23] Subsequently, its extremely
flexible acceptor–substrate specificity was described.[8c,24] No
study, however, has been reported on the donor–substrate
specificity of this enzyme.
To facilitate the protein purification, a truncated Pd2,6ST
encoding for amino acid residues 16–497 of the full-length
protein was cloned into a pET15b vector and expressed as a
N-His6-tagged protein. The recombinant Pd2,6ST was puri-
fied by a nickel-nitrilotriacetic acid (Ni-NTA) agarose affinity
column. The tolerance of donor–substrate modification of the
purified Pd2,6ST was firstly tested by TLC analysis (EtOAc/
MeOH/H2O/HOAc = 4:2:1:0.1) in the one-pot, three-enzyme
system shown in Scheme 1, in which CMP-sialic acid deriv-
atives were generated in situ from sialic acid precursors
catalyzed by the aldolase and NmCSS. For sialosides that do
not contain O-acetyl or O-lactyl groups, reactions were
typically performed in a tris(hydroxymethyl)aminomethane
Naturally occurring a-2,6-linked sialosides containing
disubstituted sialic acid residues 36 and 37 have also been
synthesized successfully in 78% and 65% yields (Table 1,
entries k and l), respectively. 9-O-Acetyl-Neu5Gc (Neu9-
Scheme 1. An efficient chemoenzymatic approach for the synthesis of sialosides containing sialic acid modifications. PPi=inorganic pyrophos-
phate, SiaT=sialyltransferase.
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Table 1: Synthesis of a-2,6-linked sialosides using the one-pot, three-enzyme system shown in Scheme 1.[a]
Entry
Donor Precursors
Acceptor
Product
Yield [%]
a
98
b
c
d
e
f
20
20
20
20
20
20
20
97
95
75
76
87
99
84
g
h
i
20
72
j
20
20
20
20
20
75
78
65
92
99
93
k
l
m
n
o
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Table 1: (Continued)
Entry
Donor Precursors
Acceptor
Product
Yield [%]
p
21
91
q
r
21
21
21
90
86
87
87
s
t
u
v
61
92
w
91
[a] All reactions were carried out in preparative (50–150 mg) scales.
Ac5Gc) (the sialic acid form in 36) has been found in bovine
submandibular gland glycoprotein.[30] 5,9-Di-O-acetyl-KDN
(KDN5,9Ac2) (the sialic acid form in 37) has also been found
in nature.[1a]
sialosides tagged with an azide or alkyne group can be
easily linked to other molecules through a Staudinger
ligation[32] or click chemistry.[33]
Acceptor specificity of the recombinant Pd2,6ST was
explored by using an N-acetyl-b-galactosaminide 22, an N-
acetyl-a-galactosaminide 23, an a-galactoside (methyl-a-d-
galactopyranoside), b-galactosides 20, 21, and 24, and an a-
2,3-linked sialoside 25. Similar to that reported previous-
ly,[8c,23] Pd2,6ST showed a much broader acceptor specificity
compared to mammalian sialyltransferases.[24] The recombi-
nant Pd2,6ST could use both a- or b-linked GalNAc
derivatives. Furthermore, b-galactosides and a-2,3-linked
sialosides were all excellent acceptors for the sialyltransfer-
ase. In contrast to that reported,[23] however, a-galactosides
were not acceptors for the enzyme.
In conclusion, we have demonstrated that the recombi-
nant P. damsela a-2,6-sialyltransferase does not only have
relaxed acceptor–substrate specificity, but also extremely
flexible donor–substrate specificity. We have also proved the
practical application of the one-pot, three-enzyme chemo-
enzymatic approach in effective preparative-scale synthesis of
diverse sialosides with naturally or non-naturally occurring
sialic acid modifications. This system can be easily extended
By using the same 3-azidopropyl lactoside 20 as the
acceptor for Pd2,6ST, non-natural sialosides containing a 4,6-
bis-epi-KDO (38) and an N-(benzyloxycarboxyamido) glyci-
nylamido-neuraminic acid (NeuGlyCbz) (39) were success-
fully synthesized in high yields (Table 1, entries m and n). The
successful synthesis of the KDO analogue containing trisac-
charide 38 demonstrates that Pd2,6ST can transfer sialic acids
with carbon backbones shorter than nine carbons to galacto-
side with high efficiency. Furthermore, a bulky benzyloxycar-
bonyl (Cbz) group at C9 of the sialic acid residue of the donor
does not affect the activity of Pd2,6ST. This further demon-
strates the extremely flexible donor–substrate specificity of
this enzyme.
By using GalbOMe (21) as an acceptor for Pd2,6ST, a-2,6-
linked sialosides containing sialic acid modified with non-
natural substitutents (40–44), such as an azido or an acetylene
group, were also synthesized in excellent yields (86–93%)
from C2- or C6-modified ManNAc or mannose that bears
azide or alkyne functional groups 15–19. The resulting
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Experimental Section
Preparative scale (50–150 mg) synthesis: Reactions were typically
carried out in a 50 mL centrifuge tube of Tris-Cl buffer solution
(10 mL, 100 mm; pH 8.5or pH 7.5) containing an acceptor (lactose,
GalNAc, or galactose derivatives, 50–100 mg), ManNAc or mannose
derivatives (1.5equiv), sodium pyruvate (7.5equiv), cytidine-5 ’-
triphosphate (CTP; 1.5equiv), MgCl 2 (20 mm), aldolase, NmCSS,
and Pd2,6ST. The reaction mixture was incubated at 258C for 12 h
with shaking (120 rpm). After adding the same volume of ice-cold
methanol to stop the reaction, insoluble material was removed by
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Received: February 13, 2006
Keywords: carbohydrates · chemoenzymatic synthesis ·
enzyme catalysis · sialic acids · transferases
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