Synthesis of nitrogen-containing furanose sugar nucleotides for use as enzymatic probes

Article abstract of DOI:10.1021/ol4032073

The sugar nucleotides UDP-2-acetamido-2-deoxy-α-d-galactofuranose (UDP-GalfNAc) and UDP-2-azido-2-deoxy-α-d-galactofuranose (UDP-GalfN ₃) have been synthesized in preparative scale for the first time. These compounds are useful probes for study

Full text of DOI:10.1021/ol4032073

Letter
pubs.acs.org/OrgLett
Synthesis of Nitrogen-Containing Furanose Sugar Nucleotides for
Use as Enzymatic Probes
Ryan B. Snitynsky and Todd L. Lowary*
Alberta Glycomics Centre and Department of Chemistry, Gunning-Lemieux Chemistry Centre, University of Alberta, Edmonton,
Alberta, Canada T6G 2G2
S
* Supporting Information
ABSTRACT: The sugar nucleotides UDP-2-acetamido-2-deoxy-α-D-galactofuranose (UDP-Galf NAc) and UDP-2-azido-2-
deoxy-α-D-galactofuranose (UDP-Galf N3) have been synthesized in preparative scale for the first time. These compounds are
useful probes for studying the biosynthesis of glycans containing galactofuranose and/or 2-acetamido-2-deoxy-α-D-
galactofuranose residues.
ugar nucleotides are widespread in nature, being the
substrates for many glycosyltransferase enzymes.1 The six-
analogue of 1, UDP-2-azido-2-deoxy-α-D-galactofuranose
(UDP-GalfN3, 2). We expect 2 to be an efficient probe of
both Galf NAc and Galf biosynthesis, similar to how other
azido-substituted carbohydrate derivatives have been useful in
probing biosynthetic pathways by which complex glycans are
assembled.6
The synthetic precursor to 1, 2-acetamido-2-deoxy-α-D-
galactofuranose-1-phosphate (GalfNAc-1-P, 3), was synthe-
sized as shown in Scheme 1. 2-Azido-2-deoxy-D-galactopyr-
anose (4), obtained through either the azidonitration of 3,4,6-
tri-O-acetyl-D-galactal7 or diazo transfer with galactosamine and
TfN3,8 was converted to its furanose form in the presence of
2,2-dimethoxypropane and p-TsOH,9 followed by deprotection
and acetylation to produce 5 in 78% overall yield. Acetolysis
yielded a fully acetylated compound 6 in 67% yield, suitable for
phosphorylation.
Phosphorylation was accomplished by first converting the
fully acetylated compound 6 into the corresponding bromide,
followed by reaction with dibenzyl phosphate under basic
conditions to yield 7 in 61% yield as a 5:1 α/β mixture. For the
first step in this transformation, due to the incompatibility of
the azido group with HBr, a previously described method10
employed successfully on the corresponding galactofuranose
derivative could not be used. Instead, TiBr4 was used to form
the glycosyl bromide.11 With the target compound 7 in hand,
debenzylation, azide reduction, and amine acetylation were
carried out in one step through hydrogenation over Pd/C with
acetic anhydride as the solvent. Subsequent removal of the ester
protecting groups in a methanol−water−triethylamine solution
produced Galf NAc-1-P (3) as a triethylammonium salt in 74%
yield.
S
membered ring pyranose forms of sugar nucleotides predom-
inate in most biological systems. In contrast, the five-membered
ring furanose sugar nucleotides are less common and are absent
in mammals.2 As a result, glycosyltransferases from pathogenic
organisms that use furanose sugar nucleotides for cell wall
biosynthesis have been proposed as potential drug targets.2b
Campylobacter jejuni, one of the leading causes of bacterial
gastroenteritis worldwide,3 features a 2-acetamido-2-deoxy-α-D-
galactofuranose (Galf NAc) moiety within its capsular poly-
saccharide (CPS), occasionally modified with a methyl
phosphoramidate group at C-3.4 To understand the pathways
that C. jejuni uses to construct its CPS, and potentially study
the inhibition of CPS formation, milligram quantities of the
sugar nucleotide precursors are required. We describe here the
first preparative-scale synthesis of UDP-2-acetamido-2-deoxy-α-
D-galactofuranose (UDP-Galf NAc, 1) (Figure 1), the donor
species for Galf NAc transferases. The only previously reported
preparation of 1 is an enzyme-mediated reaction that
interconverts the pyranose and furanose forms of this sugar
nucleotide.5 At equilibrium, the enzymatic reaction produces a
93:7 pyranose/furanose mixture, necessitating a more viable
way of producing 1. We also report the synthesis of an azido
Figure 1. Structure of UDP-Galf NAc (1, R = NHAc) and UDP-
Galf N3 (2, R = N3).
Received: November 6, 2013
Published: December 16, 2013
© 2013 American Chemical Society
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Letter
Scheme 1. Synthesis of 2-Acetamido-2-deoxy-α-D-
galactofuranose-1-phosphate (GalfNAc-1-P, 3)
Scheme 2. Synthesis of 2-Azido-2-deoxy-α-D-
galactofuranose-1-phosphate (Galf N3-1-P, 9)
Scheme 3. Chemical Coupling of Sugar-1-phosphates 3 and
9 to UMP
It was thought that preserving the azide moiety could
produce an analogue of 1 that would be a useful probe of Galf/
Galf NAc biosynthesis. To protect the phosphate moiety with
groups that could be removed without concomitant azide
reduction, diallyl phosphate was prepared from methyl
dichlorophosphate12 and then reacted with 6 to produce 8
(Scheme 2) in a yield of 45%. Deallylation of 8 with Pd(OAc)2
in acetic acid, followed by removal of the ester protecting
groups, gave the desired azide analogue 9 in 69% yield.
as a source of UDP-Galf NAc (1) for enzyme-catalyzed
reactions.13
It is well established that the enzymatic synthesis of sugar
nucleotides can, in many cases, give better yields, selectivity,
and shorter reaction times as compared to chemical
syntheses.2c,14,15 For example, the highest yielding chemical
synthesis of UDP-galactofuranose (UDP-Galf, 10, Scheme 4)
gives the desired product in 35% yield,16 whereas the enzymatic
synthesis of this compound carried out by Errey et al. produces
UDP-Galf in 79% yield.17
We explored the possibility of enzymatically synthesizing 1
and 2 using an optimized procedure similar to that reported by
Errey et al.18,19 and outlined in Scheme 4. Unfortunately, this
three-enzyme system failed to produce any of the desired sugar
nucleotides when incubated with either Galf NAc-1-P (3) or
GalfN3-1-P (9). GalPUT is known to possess relaxed substrate
specificity,17 and it is somewhat surprising that 11 is a substrate
whereas 3 and 9 are not. In previous studies, a 2-methoxy
derivative of 11 could be converted to the corresponding UDP-
Galf derivative, albeit in low yield.20
With furanose-1-phosphates 3 and 9 in hand, their coupling
with uridine-5′-monophosphate (UMP) was investigated.
Several methods exist for achieving similar transformations,
summarized by Wagner and co-workers in 2009;1b however, we
chose to use a recent method developed by Tanaka et al.13
(Scheme 3). This method relies on the initial formation of an
imidazole-activated nucleotide monophosphate (NMP)
through the action of imidazole and 2-chloro-1,3-dimethylimi-
dazolinium chloride (DMC) on a NMP in D2O. Subsequent
addition of a sugar-1-phosphate and overnight reaction
produces the desired sugar nucleotide. Using UMP and 3 or
9, both UDP-Galf NAc 1 (16% yield) and UDP-GalfN3 2 (23%
yield) were formed and isolated using this method. Although
modest, the isolated yields of these compounds are comparable
to those reported with similar substrates.1b Analysis of the H
To further probe the effect that the C-2 substituent has on
the ability of these furanose-1-phosphates to serve as GalPUT
substrates, GalfN3-1-P (9) was reduced using Pd/C under an
H2 atmosphere to produce 2-amino-2-deoxy-α-D-galactofur-
anose-1-phosphate (Galf NH2-1-P, see the Supporting Informa-
tion). This compound was tested as a GalPUT substrate as
1
and 31P NMR spectra indicated that, upon complete
consumption of UMP-imidazole, 50% of the starting material
remained in solution (see the Supporting Information).
Nevertheless, as noted by Tanaka et al., the reaction mixture
for the UDP coupling may be used directly without purification
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Letter
a
transferase from Escherichia coli K-12 that contributes to O-
antigen biosynthesis.24 Although no product formation was
observed when 2 was incubated with WbbI and a model
acceptor, GlfT2 did attach 2 to the trisaccharide acceptor 12 to
form the tetrasaccharide 13, shown in Scheme 5. No product
formation was observed in similar experiments using 1 as the
donor substrate. Despite the fact that GlfT2 is a processive
polymerase,25 the resulting product contains only a single
GalfN3 residue, suggesting that it could be useful as a chain
terminating substrate in biochemical studies of this enzyme.20,26
In summary, we report here the first preparative-scale
synthesis of UDP-GalfNAc (1) and its azido analogue 2
using a chemical approach. A previously described enzymatic
method, which could be used to produce UDP-Galf (10) from
Galf-1-P (11), failed to convert either Galf NAc-1-P (3) or
GalfN3-1-P (9) into the corresponding sugar nucleotides. The
glycosyltransferase GlfT2 accepts 2 as a substrate, raising the
possibility of using this compound for in vitro labeling studies
of Galf-containing glycans. Access to milligram quantities of 1
will greatly facilitate investigations of C. jejuni CPS biosyn-
thesis.5,27
Scheme 4. Enzymatic Synthesis of UDP-Galf, 10
a
Reactions performed at rt on an 11 μmol scale, using 1 equiv of UTP,
5 U GalU, 1.25 U inorganic pyrophosphatase, and 300 μL of
immobilized GalPUT containing 5 U of enzyme buffered with 50 mM
HEPES (pH = 8.0) containing 10 mM MgCl2 and 5 mM KCl. Total
reaction volume was 500 μL. Reaction initiated by the addition of 0.05
μmol UDP-Glc and analyzed after 24 h incubation. PPi = inorganic
pyrophosphate; Pi = inorganic phosphate.
ASSOCIATED CONTENT
■
described above but was similarly found not to be a substrate.
This suggests that the enzyme, although generally promiscuous,
is sensitive to the nature of the C-2 group in these furanose
phosphate derivatives. Further studies on the origin of this
specificity are underway.
S
* Supporting Information
Spectroscopic data and experimental details of all new
compounds are provided. This material is available free of
charge via the Internet at http://pubs.acs.org.
Both 1 and 2 could prove useful for a number of biological
investigations. For example, 1 is a substrate for UDP-N-
acetylgalactopyranose mutase (UNGM), an enzyme from C.
jejuni that catalyzes the interconversion of UDP-GalpNAc and
UDP-Galf NAc (1).5 Additionally, incorporation of the sugar
moiety of UDP-Galf N3 (2) into C. jejuni CPS, or into other
molecules containing Galf or Galf NAc residues, would present
attractive opportunities for labeling these glycans with a
fluorescent tag via “click” chemistry21 or Staudinger ligation.22
However, to date the transferase that carries out this
transformation has not been identified.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: tlowary@ualberta.ca.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NSERC and the Alberta
Glycomics Centre. R.B.S. thanks NSERC for a CGS-M
scholarship and Alberta Innovates Technology Futures for a
Graduate Student Scholarship.
To demonstrate the potential utility of 2, we incubated it
with two different glycosyltransferases: GlfT2, a galactofur-
anosyltransferase that synthesizes the cell wall galactan in
Mycobacterium tuberculosis,20,23 and WbbI, a β-(1→6)-Galf
a
Scheme 5. Coupling of Acceptor 12 with UDP-Galf N3 Donor 2 by the Glycosyltransferase Enzyme GlfT2
a
Reaction performed at rt over 3 days using final concentrations of 0.5 mM 12, 4 mM 2, 150 μg of GlfT2, and 2 U alkaline phosphatase in MOPS
(pH = 7.6) containing 20 mM MgCl2 and 5 mM β-mercaptoethanol.
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Wren, B. W.; Muggleton, S. H. J. Mol. Biol. 2013, 425, 186−197.
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