Improved chemical synthesis of UDP-galactofuranose

Article abstract of DOI:10.1021/ol016170d

(Equation presented) A reliable and efficient synthetic route to UDP-α-D-galactofuranose (UDP-Galf) has been developed. Reaction of UMP-N-methylimidazolide with Galf 1-phosphate proceeds rapidly to provide UDP-Galf with excellent reproducibility and in a yield approximately twice as high as those reported previously.

Full text of DOI:10.1021/ol016170d

ORGANIC
LETTERS
2001
Vol. 3, No. 16
2517-2519
Improved Chemical Synthesis of
UDP-Galactofuranose
,†,‡
Allison L. Marlow† and Laura L. Kiessling*
Departments of Chemistry and Biochemistry, UniVersity of WisconsinsMadison,
Madison, Wisconsin 53706
kiessling@chem.wisc.edu
Received May 24, 2001
ABSTRACT
A reliable and efficient synthetic route to UDP-r-D-galactofuranose (UDP-Galf) has been developed. Reaction of UMP-N-methylimidazolide with
Galf 1-phosphate proceeds rapidly to provide UDP-Galf with excellent reproducibility and in a yield approximately twice as high as those
reported previously.
D-Galactose is a critical constituent of polysaccharides,
glycoproteins, and glycolipids. This hexose can exist in either
the six-membered ring pyranose or the five-membered ring
furanose form. The more stable pyranose form is the only
one found in mammalian glycoproteins and glycolipids.
Intriguingly, galactofuranose (Galf) residues occur in various
polysaccharides and glycoconjugates produced by infectious
bacteria, protozoa, and fungi.1 Polysaccharides and glyco-
conjugates that contain Galf residues are essential for the
survival and pathogenicity of many microorganisms. The
enzymes that mediate the incorporation of this moiety are
therefore attractive targets for the development of antimi-
crobial agents.
from the action of the enzyme UDP-galactopyranose mutase
(UDP-Galp mutase) on UDP-R-D-galactopyranose (UDP-
Galp) (1) (Figure 1).3
Figure 1. UDP-Galp mutase catalyzes the interconversion of UDP-
Galp (1) and UDP-Galf (2). The pyranose isomer 1 is favored.
The biosynthetic glycosyl donor used in the incorporation
of Galf residues is uridine 5′-diphospho-R-D-galactofuranose
(UDP-Galf) (2) (Figure 1). Presumably, galactofuranosyl
transferases use this donor to incorporate Galf residues into
cell-surface carbohydrates.2 Glycosyl donor 2 is generated
To investigate the processes involved in Galf incorporation,
access to UDP-Galf 2 is essential. This biosynthetic inter-
mediate is required to identify putative glycosyltransferases
and to study the action of UDP-Galp mutase. The mutase
† Department of Chemistry.
‡ Department of Biochemistry.
(3) (a) Ko¨plin, R.; Brisson, J.-R.; Whitfield, C. J. Biol. Chem. 1997,
272, 4121-4128. (b) Lee, R.; Monsey, D.; Weston, A.; Duncan, K.; Rithner,
C.; McNeil, M. Anal. Biochem. 1996, 242, 1-7. (c) Nassau, P. M.; Martin,
S. L.; Brown, R. E.; Weston, A.; Monsey, D.; McNeil, M. R.; Duncan, K.
J. Bacteriol. 1996, 178, 1047-1052.
(1) De Lederkremer, R. M.; Colli, W. Glycobiology 1995, 5, 547-552.
(2) (a) Dearruda, M. V.; Colli, W.; Zingales, B. Eur. J. Biochem 1989,
182, 413-421. (b) Mikusova, K.; Yagi, T.; Stern, R.; McNeil, M. R.; Besra,
G. S.; Crick, D. C.; Brennan, P. J. J. Biol. Chem. 2000, 275, 33890-33897.
10.1021/ol016170d CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/14/2001

catalyzes the conversion of UDP-Galp 1 to UDP-Galf 2 with
the low efficiencies dictated by thermodynamics (5-8%),
but it efficiently isomerizes UDP-Galf 2 to UDP-Galp 1 (92-
95% yield). Thus, UDP-Galf 2 is needed to analyze the
reaction in the favored direction.2b Given the low yields of
the enzymatic reaction, chemical synthesis is the only route
to obtain UDP-Galf 2 in sufficient quantities for biological
and biochemical studies.
Although the structure of UDP-Galf 2 has been known
for 30 years,4 its synthesis, via two similar routes, has been
reported only recently.5 Both of the published routes suffer
from low yields arising from the final key step: the coupling
of R-D-Galf 1-phosphate (3) to an activated 5′-UMP deriva-
tive. The difficulties in synthesizing substrate 2 are likely
due to its lability. The conformational preferences and
increased flexibility of the five-membered ring of UDP-Galf
2 compared to those of the six-membered ring of UDP-Galp
1 facilitate decomposition reactions of the galactofuranose
donor. Specifically, cyclization to yield R-D-Galf 1,2-cyclic
phosphodiester and 5′-UMP2a and glycosidic bond hydrolysis
to afford D-galactose and 5′-UDP are reactions of 2 that occur
readily.6 These degradative processes complicate the syn-
thesis of this important biosynthetic intermediate. Here, we
report an effective coupling procedure that results in the
preparation of UDP-Galf 2 reproducibly, in a short reaction
time (2 h at 0 °C), and in an improved yield (35%) (Scheme
1).
morpholidate (in the presence of 1H-tetrazole)5a,b and the
UMP-imidazolide5a were tested as sources of activated 5′-
UMP. Unfortunately, the reactions of these substrates were
slow (1-2 days), proceeded only to 50% completion, and
afforded low yields (19-23%). The major side reaction
observed was base-catalyzed degradation of product 2 to the
R-D-Galf 1,2-cyclic phosphate and 5′-UMP.5a
To develop an efficient route to the target, UDP-Galf 2,
we explored alternative conditions for the coupling of R-D-
Galf 1-phosphate (3) and 5′-UMP. Trifluoroacetic anhydride
has been shown to be effective for the activation of
nucleoside monophosphates en route to nucleoside triphos-
phates.8 All four nucleoside triphosphates can be obtained
in 89-92% yields in very short reaction times (2-5 min).
Although this method has been used only for triphosphate
synthesis, we reasoned that it might be adapted for the
synthesis of the target compound 2. We hypothesized that
the mild conditions and the short reaction times employed
would minimize the decomposition of the labile UDP-Galf
2 during the course of reaction.
The synthesis of the substrate R-D-Galf 1-phosphate (3)
was accomplished by a strategy analogous to that described
recently (Scheme 2).5a D-Galactose was converted to a
Scheme 2
Scheme 1
mixture of anomeric methyl furanosides 5 via ferric chloride-
catalyzed Fischer glycosylation.9 The remaining hydroxyl
groups were protected as benzoyl esters, and acetolysis of
the resulting methyl glycoside provided 6. Treatment of 6
with hydrogen bromide afforded the appropriate glycosyl
donor for phosphorylation. Reaction of this glycosyl bromide
with dibenzyl phosphate produced the diastereomeric ga-
lactofuranosyl derivatives 7R and 7â (2.5:1 ratio), which
were separated by flash chromatography. Contrary to reports
that 7â is unstable and decomposes rapidly,10 this compound
was isolated as a white crystalline solid that was stable for
months at room temperature. Our attempts to isomerize 7â
to 7R, using either catalytic BF3‚OEt2 or the phosphorylation
reaction conditions, have been unsuccessful to date. The 7R
phosphoryl protecting groups were removed to yield tetra-
The formation of nucleoside diphospho sugars from a
sugar 1-phosphate and an activated 5′-UMP derivative is a
challenging synthetic operation; the yields are typically
moderate. Strategies for improving the efficiency of the
coupling step have been reported. For example, increased
product yields have been obtained using a nucleophilic
catalyst.7 In the synthesis of UDP-Galf 2, both the UMP-
(4) Garcia Trejo, A.; Haddock, J. W.; Chittenden, G. J. F.; Baddiley, J.
Biochem. J. 1971, 122, 49-57.
(5) (a) Tsvetkov, Y. E.; Nikolaev, A. V. J. Chem. Soc, Perkin Trans. 1
2000, 889-891. (b) Zhang, Q.; Liu, H.-w. J. Am. Chem. Soc. 2000, 122,
9065-9070.
(8) Bogachev, V. S. Russ. J. Bioorg. Chem. 1996, 22, 599-604.
(9) Lubineau, A.; Fischer, J.-C. Synth. Commun. 1991, 21, 815-818.
(10) De Lederkremer, R. M.; Nahmad, V. B.; Varela, O. J. Org. Chem.
1994, 59, 690-692.
(6) Collins, P.; Ferrier, R. Monosaccharides: Their Chemistry and Their
Roles in Natural Products; Wiley & Sons: New York, 1995.
(7) Wittmann, V.; Wong, C. H. J. Org. Chem. 1997, 62, 2144-2147.
2518
Org. Lett., Vol. 3, No. 16, 2001

O-benzoyl-R-D-Galf 1-phosphate triethylammonium salt us-
ing a reported protocol.5a The benzoyl groups were subse-
quently removed using a triethylamine-methanol-water
solution to afford phosphorylated monosaccharide 3. Puri-
fication was accomplished by ion-exchange chromatography
to provide 3 as the bisammonium salt.
Both the model coupling and the key reaction were
followed using 31P NMR spectroscopy. Each component of
the reaction mixture, including intermediates and products,
could be distinguished.11 We found that when the coupling
reactions were conducted at 0 °C, only a slight excess of
electrophile 4 (1.2 equiv) was needed for complete consump-
tion of the sugar 1-phosphate. Under these conditions, little
or no degradation of the product to the cyclic phosphodiester
and 5′-UMP was observed.
Our UDP-Galf 2 coupling procedure consists of several
steps. The electrophilic UMP-N-methylimidazolide 4 is
formed by the reaction of the triethylammonium salt of 5′-
UMP with an excess of trifluoroacetic anhydride in the
presence of triethylamine and N,N-dimethylaniline in aceto-
nitrile.8 Excess trifluoroacetic anhydride is added to increase
the solubility of the electrophile in the reaction medium; its
addition results in acylation of the 2′- and 3′-hydroxyl groups
as well as the phosphoryl group. The nucleophilic catalyst,
N-methylimidazole, is added to provide the electrophilic
derivative 4. This compound can be detected by 31P NMR
spectroscopy, HPLC, and TLC. An excess of activated 4 is
treated with the tributylammonium salt of R-D-Galf 1-phos-
phate (3). To minimize side reactions of the product, the
reaction is quenched with aqueous ammonium acetate buffer
(pH 7), which also promotes the removal of the O-
trifluoroacetyl groups. After workup, the product 2 is isolated
by HPLC purification.
A number of parameters were found to affect the yield of
the coupling reaction. Various reaction conditions were tested
in the model coupling of commercially available R-D-Galp
1-phosphate to form UDP-Galp 1. We altered the counterion
for R-D-Galp 1-phosphate, the number of equivalents of
activated 5′-UMP 4, the reaction duration, and the reaction
temperature.
With regard to counterion, bisammonium salts of both 3
and R-D-Galp 1-phosphate were found to be insoluble in
acetonitrile. The tributylammonium salt of R-D-Galp 1-phos-
phate was completely soluble in acetonitrile, but the corre-
sponding triethyl- and tetraethylammonium derivatives were
not. Thus, we employed the tributylammonium salt of 3 in
the key coupling reaction.
Under optimized conditions, the tributylammonium salt
of R-D-Galf 1-phosphate (3) was treated with UMP-N-
methylimidazolide 4 (1.2 equiv) at 0 °C. The sugar 1-phos-
phate 3 was completely consumed after 2 h. According to
the 31P NMR spectrum, the proportion of UDP-Galf 2 at this
stage was 83% and the amount of cyclic phosphodiester
produced was low (17%). This ratio compares well with that
observed using other methods; the highest yield of UDP-
Galf 2 reported previously was 50% after 18-19 h. With
the less reactive UMP-imidazolide, degradation of the
product to the cyclic phosphodiester and 5′-UMP is a more
significant side reaction.5a We found that ammonium acetate
buffer (pH 7, 250 mM) is the most effective solution for
quenching the reaction. The product 2 was purified by ion-
exchange HPLC. Slight degradation (loss of ca. 4%) of the
product to R-D-Galf 1,2-cyclic phosphate and 5′-UMP (1:1
ratio) occurs during purification. This protocol affords UDP-
Galf 2 reproducibly in 35% yield.
In conclusion, we devised an effective and reproducible
coupling method to prepare UDP-Galf 2. Our protocol
involves reaction of a sugar 1-phosphate and the activated
5′-N-methyl phosphorylimidazolide nucleoside. The in-
creased reactivity of the electrophilic phosphoryl group used
here eliminates the long reactions times typically required
for nucleoside diphospho sugar synthesis. We found that this
method is effective for preparing a highly labile biosynthetic
intermediate. We anticipate that it may also facilitate the
preparation of diphosphoryl bonds embedded in other reac-
tive or unstable products.
The extent of completion of the coupling reaction de-
pended on the number of equivalents of activated UMP-N-
methylimidazolide 4 added. For nucleoside triphosphate
synthesis, the pyrophosphate nucleophile is used in excess
(2-fold) relative to the electrophile.8 This ratio results in a
suppression of the reaction of the product nucleoside
triphosphate with the activated NMP-N-methylimidazolide,
a process that produces the symmetrical dinucleoside tetra-
phosphate. In the formation of nucleoside diphospho sugars,
however, it is desirable to use the electrophile in excess,
rather than the valuable sugar 1-phosphate. The disadvantage
of this approach is that any hydrolysis of UMP-N-meth-
ylimidazolide 4 produces 5′-UMP, which can react with the
electrophile 4 to form the dimer, UPPU. The presence of
this species complicates purification of the desired product.
Thus, conditions were sought that would maximize the
formation of the product while minimizing formation of
UPPU.
Acknowledgment. This research was supported by the
NSF and the Dreyfus Foundation. A.L.M. thanks the NIH
for a postdoctoral fellowship (GM20582). We thank D.
Perreault and C. Sherrill for conducting preliminary experi-
ments.
Supporting Information Available: Experimental pro-
cedure for the coupling to produce UDP-Galf 2. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL016170D
(11) Relative to external aqueous 85% H3PO4: R-D-galactose 1,2-cyclic
phosphate (10.0 ppm for Galp and 16.9 ppm for Galf2a), 5′-UMP (0.5-1.1
ppm), galactose 1-phosphate (-0.2 ppm for Galp 11 and 0.3 ppm for Galf
3), UMP-N-Me-imidazolide (-10.9 ppm), product UDP-galactose (-11.1
(d), -12.6 (d) ppm for Galp 1 and -11.3 (d), -12.6 (d) ppm for Galf 2),
and side product UPPU (-11.2 ppm).
Org. Lett., Vol. 3, No. 16, 2001
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