A Diazido Mannose Analogue as a Chemoenzymatic Synthon for Synthesizing Di-N-acetyllegionaminic Acid-Containing Glycosides

Article abstract of DOI:10.1002/anie.201712022

A chemoenzymatic synthon was designed to expand the scope of the chemoenzymatic synthesis of carbohydrates. The synthon was enzymatically converted into carbohydrate analogues, which were readily derivatized chemically to produce the desired targets. The

Full text of DOI:10.1002/anie.201712022

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Angewandte
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Accepted Article
Title: A diazido mannose analog as a chemoenzymatic synthon for
synthesizing di-N-acetyllegionaminic acid-containing glycosides
Authors: Abhishek Santra, An Xiao, Hai Yu, Wanqing Li, Yanhong Li,
Linh Ngo, John B. McArthur, and Xi Chen
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201712022
Angew. Chem. 10.1002/ange.201712022
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10.1002/anie.201712022
Angewandte Chemie International Edition
COMMUNICATION
A diazido mannose analog as a chemoenzymatic synthon for
synthesizing di-N-acetyllegionaminic acid-containing glycosides
Abhishek Santra,[a] An Xiao,[a] Hai Yu,[a] Wanqing Li,[a] Yanhong Li,[a] Linh Ngo,[a] John B. McArthur,[a]
and Xi Chen*[a]
Abstract: A novel strategy is developed to expand the scope of
chemoenzymatic synthetic products by designing a chemoenzymatic
synthon. The synthon is enzymatically converted to carbohydrate
analogs which are readily derivatized chemically to produce desired
targets. The strategy is demonstrated for synthesizing glycosides
containing 7,9-di-N-acetyllegionaminic acid (Leg5,7Ac2), a bacterial
nonulosonic acid (NulO) analog of sialic acid. A versatile library of 2–
3/6-linked Leg5,7Ac2- glycosides is built using chemically synthesized
2,4-diazido-2,4,6-trideoxy mannose as a chemoenzymatic synthon for
highly efficient one-pot multienzyme (OPME) sialylation followed by a
downstream chemical conversion of the azido groups to acetamido
groups. The overall yields of the syntheses are 34–52% in 10 steps
from commercially available D-fucose, representing significant
bacteria.[2, 4] Pathogenic bacterial LPSs containing Leg5,7Ac2 and
derivatives are attractive targets for developing bacterial
polysaccharide vaccines and diagnostic tools (e.g. anti-
carbohydrate antibodies).
Figure 1. Structures of 5,7-di-N-acetyllegionaminic acid (Leg5,7Ac2, 1),
legionaminic acid (Leg, 2), N-acetylneuraminic acid (Neu5Ac, 3), and 2,4-
diacetamido-2,4,6-trideoxy-D-mannose (6deoxyManNAc4NAc, 4) which is the
six-carbon biosynthetic precursor of Leg5,7Ac2 (1).
improvements
over
previous
methods.
Free
Leg5,7Ac2
monosaccharide is also synthesized using a sialic acid aldolase-
catalyzed reaction.
Several examples of chemical synthesis of Leg5,7Ac2 and its
simple glycosides have been reported. Condensation of
oxalacetic acid with chemically synthesized 2,4-diacetamido-
2,4,6-trideoxy-D-mannose (6deoxyManNAc4NAc, 4, Figure 1)
produced both Leg5,7Ac2 and its C4-epimer with 7% and 10%
yields, respectively.[5] De novo synthesis of a -glycoside (not the
desired -glycoside) of legionaminic acid from D-threonine was
achieved with a yield of 7% in 17 steps.[6] From Neu5Ac (3), a
protected thio-adamentyl diazido-legionaminic acid donor was
obtained with a yield of 17% in 15 steps which was used for
chemical glycosylation followed by additional chemical
transformation to produce an 2–3-Leg5,7Ac2-terminated methyl
-galactoside with an overall yield of 7% in 19 steps.[7] All these
efforts resulted in low yields and demonstrated challenges for
synthesizing Leg5,7Ac2-glycosides including low stereo and
regio-selectivities.[6-7]
The high efficiency of enzymatic and chemoenzymatic synthetic
approaches in carbohydrate synthesis has been increasingly
recognized. The methods, however, rely heavily on the access to
related enzymes and can be limited by the substrate specificity of
the enzymes that are available.
Here we develop a novel strategy to expand the scope of
chemoenzymatic synthetic products using chemoenzymatic
synthons, which are designed to be used by enzymatic reactions
to produce carbohydrate analogs that can be readily converted to
desired targets by chemical derivatization. The strategy is
demonstrated for the synthesis of a versatile library of glycosides
containing
a
terminal
2–3
or
2–6-linked
di-N-
acetyllegionaminic acid (Leg5,7Ac2, 1) (Figure 1). Leg5,7Ac2, or
5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-
ulosonic acid, is a bacterial nine-carbon -keto acid (nonulosonic
acid, NulO)[1] that is part of serological specificity defining
structures of pathogenic bacteria.[2] It is the di-N-acetyl derivative
of legionaminic acid (Leg, 2) (5,7-diamino-3,5,7,9-tetradeoxy-D-
glycero-D-galacto-non-2-ulosonic acid) and has the exact the
same stereochemistry of N-acetylneuraminic acid (Neu5Ac, 3),
the most common sialic acid form in nature.[1b] It differs from
Neu5Ac at two sites where the C-7 and C-9 hydroxyl groups of
Neu5Ac are substituted by C-7 acetamido group and C-9
hydrogen, respectively.[1b, 3]
Biosynthetically,
Leg5,7Ac2
is
produced
from
6deoxyManNAc4NAc (4) and phosphoenolpyruvate (PEP)
catalyzed by a Leg5,7Ac2 synthase.[8] 6deoxyManNAc4NAc itself
is formed from uridine 5’-diphosphate-N-acetylglucosamine
(UDP-GlcNAc, e.g. in Legionella penumophila) or guanosine 5’-
diphosphate-N-acetylglucosamine
(GDP-GlcNAc,
e.g.
in
Campylobacter jejuni) by a process involving four reactions
catalyzed by different enzymes.[8] Leg5,7Ac2 is activated by a
cytidine
synthetase[3]
5’-monophosphate-Leg5,7Ac2
(CMP-Leg5,7Ac2)
as the
to provide CMP-Leg5,7Ac2
glycosyltransferase donor for the synthesis of desired structures.
Although a native Leg5,7Ac2-glycosylltransferase has yet to
be identified, several mammalian and bacterial sialyltransferases
have been tested for catalyzing the transfer of Leg5,7Ac2 from
CMP-Leg5,7Ac2 to galactosides. Porcine ST3Gal I, human
ST6Gal I,[9] Pasteurella multocida sialyltransferase 1 (PmST1),[10]
and Neisseria meningitides MC58 2–3-sialyltransferase[11]
showed reasonable activity in forming Leg5,7Ac2-glycosides.
Nevertheless, these enzymatic syntheses relied on a complex
process of producing CMP-Leg5,7Ac2 either from UDP-GlcNAc
by multiple enzymes[12] in vitro with chemical acetylation of the 4-
Leg5,7Ac2 (1) and its N-acyl derivatives and related 4- or 8-
epimers have been found in lipopolysaccharides (LPSs) or
extracellular polysaccharides of many pathogenic Gram-negative
[a]
Dr. A. Santra, A. Xiao, Dr. H. Yu, W. Li, Dr. Y. Li, L. Ngo, Dr. J. B.
McArthur, *Prof. Dr. X. Chen
Department of Chemistry, University of California, Davis
One Shields Avenue, Davis, CA 95616 (USA)
E-mail: xiichen@ucdavis.edu
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201712022
Angewandte Chemie International Edition
COMMUNICATION
amino group[10] or from Leg5,7Ac2 produced de novo using
Escherichia coli engineered with combined biosynthetic pathways
from Saccharomyces cerevisiae, Campylobacter jejuni,[13] and
Legionella pneumophila.[14]
preparative-scale synthesis of Leg5,7Ac2 (1) with a 71% yield
(Scheme 2).
Here we show that a versatile library of 2–3 and 2–6-linked
Leg5,7Ac2-glycosides can be produced readily from chemically
synthesized
2,4-diazido-2,4,6-trideoxy
mannose
(6deoxyMan2,4diN3) as a chemoenzymatic synthon in highly
efficient one-pot multienzyme (OPME) sialylation systems[15]
using commercially available enzymes with downstream chemical
derivatization.
Scheme 2. PmAldolase-catalyzed synthesis of Leg5,7Ac2 (1) from
6deoxyManNAc4NAc (4) and sodium pyruvate.
We started by developing an efficient method for the
production of 6deoxyManNAc4NAc (4), the six-carbon precursor
of Leg5,7Ac2 (1). Inspired by a method reported recently by the
Kulkarni group,[16] commercially available D-fucose (5) was
chosen as the starting material to allow simultaneous inversion of
its stereochemistry at C-2 and C-4 to form the desired mannose
derivative. As shown in Scheme 1, per-O-acetylation of D-fucose
(5) followed by BF3·Et2O-catalyzed nucleophilic displacement of
the anomeric acetate with p-methoxyphenol in CH2Cl2 produced
intermediate 6 in 84% yield. De-O-acetylation with sodium
methoxide in methanol produced triol. Dimethyltin chloride
(Me2SnCl2)-catalyzed regio-selective benzoyl protection[16] of 3-
OH formed D-fucosyl-2,4-diol (7) in two steps in 94% yield.
Compound 7 was then treated with trifluoromethanesulfonic
anhydride (Tf2O) and pyridine to form the corresponding 2,4-
bistriflate[16] which upon treating with 2.5 equivalent of
tetrabutylammonium azide (TBAN3) in anhydrous toluene at 70 °C
to reflux,[17] 6-deoxy-D-mannose derivative 8 was formed in 2
hours in 93% yield. Debenzoylation and ceric ammonium nitrate-
catalyzed removal of the p-methoxyphenyl group[18] produced 2,4-
diazido-2,4,6-trideoxy mannose (6deoxyMan2,4diN3, 9) in 81%
yield. Overall, the production of 6deoxyMan2,4diN3 (9) from D-
fucose (5) was achieved in eight steps with an overall yield of 60%.
ManNAc derivative 6deoxyManNAc4NAc (4) was obtained readily
from 9 in 73% yield by treating with thioacetic acid in pyridine[19]
at room temperature for 20 hours.
Nevertheless, the resulting Leg5,7Ac2 (1) was not a suitable
substrate for Neisseria meningitidis CMP-sialic acid synthetase
(NmCSS)[20] for the synthesis of the corresponding CMP-
Leg5,7Ac2.
As azido derivatives of N-acetylmannosamine (ManNAc) and
mannose have been shown to be suitable starting materials for
OPME sialylation systems[15] for the synthesis of various 2–3/6-
linked sialosides[22] including those containing 7-azido-[23] or 9-
deoxy-derivative[24] of Neu5Ac, the diazido compound 9 was
tested for the synthesis of glycosides. To our delight, 9 was well
tolerated by the OPME 2–3/6-sialylation systems as
demonstrated using three different acceptors including para-
nitrophenyl -galactoside (GalpNP,10), thiotolyl -galactoside
(GalSTol, 11), and lactosyl -propylchloride (LacProCl, 12). In
these systems, 9 was coupled with pyruvate to form the diazido-
derivative of Leg (Leg5,7diN3) by a PmAldolase-catalyzed
reaction. Leg5,7diN3 was reacted with cytidine 5'-triphosphate
(CTP) using an NmCSS-catalyzed reaction to form CMP-
Leg5,7diN3 which was used by
a sialyltransferase (e.g.
PmST1_M144D[25] or Psp2,6ST[26]) to produce 2–3/6-linked
Leg5,7diN3-containing glycosides (13–18) (Table 1).
Using PmST1_M144D[25] as the sialyltransferase in the OPME
sialylation system containing PmAldolase and NmCSS (Table 1),
2–3-linked Leg5,7diN3-containing glycosides (13, 15, and 17)
were obtained in good (71%) to excellent (98% and 91%) yields
with GalpNP (10), GalSTol (11), and LacProCl (12) as the
sialyltransferase acceptors, respectively. Similarly, using
Psp2,6ST[26] as the sialyltransferase, 2–6-linked Leg5,7diN3-
containing glycosides (14, 16, and 18) were obtained in good
(73%) to excellent (93% and 97%) yields. Compared to GalSTol
(11) and LacProCl (12), GalpNP (10) was a less effective
sialyltransferase acceptor, resulting in lower sialylation yields in
OPME sialylation systems.
Different strategies were tested to convert the azido groups in
the glycosides synthesized (13–18) to N-acetyl groups to form
desired Leg5,7Ac2-containing glycosides (19–24). A conventional
Perlman catalyst-mediated reduction of azide to amine[27] followed
by selective acetylation of the amine worked for 13, 15, 16, and
18 with ~50% yields in two steps but hydrogenation also
converted the aromatic nitro group of 13 and 14 to the
corresponding amine which was undesirable. PMe3-mediated
Staudinger reaction[28] worked well for all compounds and
quantitatively produced the corresponding diamine derivatives.
However, selective acetylation of amine by a combination of
acetylchloride and triethyl amine in tetrahydrofuran and water (4:1
v/v) produced the di-N-acetyl derivative in poor yields (~40%). An
alternative N-acetylation strategy using thioacetic acid and
Scheme 1. Chemical synthesis of 6deoxyManNAc4NAc (4) from commercially
available D-fucose (5) via a diazido intermediate 2,4-diazido-2,4,6-trideoxy
mannose (6deoxyMan2,4diN3, 9).
To our delight, 6deoxyManNAc4NAc (4) was a suitable
substrate for both recombinant Escherichia coli (EcAldolase)[20]
and Pasteurella multocida (PmAldolase)[21] sialic acid aldolases.
PmAldolase was found to be more efficient and was used for
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10.1002/anie.201712022
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COMMUNICATION
catalytic copper sulfate in methanol[29] produced Leg5,7Ac2-
glycosides with very poor yields (<20%). Finally, thioacetic acid-
mediated one-pot conversion of azido to acetamido group[19]
using saturated sodium bicarbonate in water was found to be the
optimal condition and the Leg5,7Ac2-containing glycosides (19–
24) were produced in 69–88% yields (Table 1). In comparison,
poorer yields (45–70%) were obtained when pyridine was used
as the solvent in the same method (data not shown).
Table 1. Production of Leg5,7Ac2-containing glycosides (19–24) by one-pot multienzyme (OPME) synthesis of Leg5,7diN3-containing glycosides (13–18) followed
by chemical conversion of the azido group to N-acetyl group.
Acceptor
Product
Yield (%)
Product
Yield (%)
71
81
GalpNP (10)
Leg5,7Ac22–3GalpNP (19)
Leg5,7Ac22–6GalpNP (20)
Leg5,7diN32–3GalpNP (13)
73
78
Leg5,7diN32–6GalpNP (14)
Leg5,7diN32–3GalSTol (15)
98
93
88
84
Leg5,7Ac22–3GalSTol (21)
GalSTol (11)
Leg5,7Ac22–6GalSTol (22)
Leg5,7Ac22–3LacProCl (23)
Leg5,7diN32–6GalSTol (16)
Leg5,7diN32–3LacProCl (17)
91
97
72
69
LacProCl (12)
Leg5,7diN32–6LacProCl (18)
Leg5,7Ac22–6LacProCl (24)
It is worth to note that Leg5,7Ac22–3Gal- and Leg5,7Ac22–
6Gal-containing structures have been found in O-antigens[4b, 4c, 30]
and an extracellular polysaccharide fraction,[4e] respectively, of
various opportunistic pathogens. Therefore, the obtained thiotolyl
-glycosides (compounds 15–16 and 21–22) can be used to form
building blocks for efficient chemical synthesis of more complex
glycosides similar to those described before for Neu5Ac-
containing sialosides.[31]
Figure 2. Structures of Leg5,7Ac22–3LacProN3 (25) and Leg5,7Ac22–
6LacProN3 (26).
The propyl chloride aglycon in compounds 23 and 24 was
readily converted to propyl azide by treating them with sodium
azide (NaN3) and a catalytic amount of sodium iodide (NaI) in
dimethylformamide (DMF) at 60 °C for 12 hours[32] to produce
Leg5,7Ac22–3LacProN3 (25) and Leg5,7Ac22–6LacProN3
(26) in 85% and 92% yields, respectively (Figure 2). The azido
group in the final products can be easily reduced to an amine
group in future for microarray study and for synthesizing
glycoconjugates.
Leg5,7diN32–3/6GalpNP (13–14) and Leg5,7Ac22–
3/6GalβpNP (19–20) were tested as potential substrates for
recombinant human cytosolic sialidase hNEU2[33] and several
bacterial sialidases including three commercially available
sialidases from Arthrobacter ureafaciens, Vibrio cholerae, and
Clostridium perfringens (CpNanH), as well as five recombinant
bacterial sialidases such as the 2–3-sialidase activity of
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10.1002/anie.201712022
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COMMUNICATION
multifunctional PmST1,[22a] Bifidobacterium infantis sialidase
BiNanH2,[34] Streptococcus pneumoniae sialidases SpNanA,[35]
SpNanB,[35] and SpNanC).[36] It was interesting to note that
Leg5,7Ac22–3GalpNP (19), but not Leg5,7diN32–3GalpNP
(13), was a substrate for the 2–3-sialidase activity of PmST1.[22a]
Compared to Neu5Ac2–3GalpNP (kcat/KM = 117 min-1 mM-1),
the 2–3-sialidase activity of PmST1 (in the presence of 0.4 mM
CMP) for Leg5,7Ac22–3GalpNP (19) (kcat/KM = 4 min-1 mM-1)
was about 30-fold less efficient (Table S1, ESI). Other sialidases
tested did not show any activity for the compounds used (13, 14,
19, and 20) indicating the sialidase activity-blocking effect of 7-
NAc substitution in Leg5,7Ac2-glycosides as 9-deoxy substitution
of Neu5Ac-glycosides was tolerated by several sialidases.[24] The
lack of PmST1 2–3-sialidase activity for Leg5,7diN32–
3GalpNP (13) is advantageous for synthesizing 2–3-linked
Leg5,7diN3-glycosides. Indeed, commercially available PmST1
was found to be as effective as its M144D mutant (with decreased
sialidase activity) in synthesizing 2–3-linked Leg5,7Ac2-
glycosides 13, 15, and 17. Gram-scale (1.85 g) synthesis of
Leg5,7diN32–3GalSTol (15) was readily achieved in an
excellent 93% yield using the OPME sialylation system containing
PmST1. Psp2,6ST A336G mutant[37] with a higher expression
level and commercially available Photobacterium damselae 2–
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sialyltransferases for OPME synthesis of 2–6-linked Leg5,7diN3-
glycosides 14, 16, and 18 (data not shown).
In
conclusion,
2,4-diazido-2,4,6-trideoxy
mannose
(6deoxyMan2,4diN3) has been designed as an easy-to-obtained
and highly effective chemoenzymatic synthon. It was readily
synthesized from commercially available D-fucose by chemical
methods in eight steps with an overall yield of 60% and was
successfully used for highly efficient chemoenzymatic synthesis
of a library of 2–3- and 2–6-linked di-N-acetyllegionaminic acid
(Leg5,7Ac2)-containing glycosides in 57–86% yields. The
chemoenzymatic method described here allows high-yield
synthesis of a diverse array of biologically important Leg5,7Ac2-
containing glycosides using commercially available enzymes. The
method of designing chemoenzymatic synthons for enzymatic
formation of glycosides followed by chemical derivatization can be
a general strategy for producing complex N-acetyl-containing
glycosides.
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Detailed synthetic procedures, nuclear magnetic resonance (NMR)
spectroscopy and high-resolution mass spectrometry (HRMS) data, and
NMR spectra for products are available in the supporting information.
Acknowledgements
This work is supported by National Institutes of Health (NIH)
grants R01AI130684 and U01GM125288. Bruker Avance-800
NMR spectrometer was funded by NSF grant DBIO-722538.
Keywords: carbohydrates • chemoenzymatic synthesis •
glycosylation • legionaminic acid • sialic acid
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This article is protected by copyright. All rights reserved.

10.1002/anie.201712022
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
Layout 2:
COMMUNICATION
Abhishek Santra, An Xiao, Hai Yu,
Wanqing Li, Yanhong Li, Linh Ngo, John
B. McArthur, and Xi Chen*
Page No. – Page No.
A diazido mannose analog as a
chemoenzymatic synthon for
synthesizing di-N-acetyllegionaminic
acid-containing glycosides
Chemoenzymatic synthesis: A versatile library of 2–3/6-linked Leg5,7NAc2-
containing glycosides has been built by highly efficient one-pot multienzyme
(OPME) sialylation systems using chemically synthesized 6deoxyMan2,4diN3 as a
chemoenzymatic synthon followed by facile chemical converting the azido groups to
acetamido groups.
This article is protected by copyright. All rights reserved.