Surprising bacterial nucleotidyltransferase selectivity in the conversion of carbaglucose-1-phosphate

Article abstract of DOI:10.1021/ja045522j

The drive to understand the molecular determinants of carbohydrate binding as well as the search for more chemically and biochemically stable sugar derivatives and carbohydrate-based therapeutics has led to the synthesis of a variety of analogues that rep

Full text of DOI:10.1021/ja045522j

Published on Web 09/23/2004
Surprising Bacterial Nucleotidyltransferase Selectivity in the Conversion of
Carbaglucose-1-phosphate
Kwang-Seuk Ko, Corbin J. Zea, and Nicola L. Pohl*
Department of Chemistry and the Plant Sciences Institute, Gilman Hall, Iowa State UniVersity,
Ames, Iowa 50011-3111
Received July 26, 2004; E-mail: npohl@iastate.edu
Herein we report the first synthesis of the carbocyclic version
of the most common naturally occurring sugar-1-phosphate,
glucose-1-phosphate, and its evaluation with a bacterial and a
eukaryotic sugar nucleotidyltransferase. In contrast to results with
the eukaryotic enzyme, the carbocyclic glucose-1-phosphate serves
as a substrate for the bacterial enzyme to provide the carbocyclic
Figure 1. Sugar nucleotidyltransferases catalyze the formation of activated
nucleotide diphosphate sugars that are substrates for, when X ) O, but
inhibitors of, when X ) CH2, Leloir pathway glycosyltransferases.
uridinediphosphoglucose. This result demonstrates not only the first
chemoenzymatic strategy to this class of glycosyltransferase inhibi-
tors but also the possibility of using sugar nucleotidyltransferases
in vivo to convert prodrug forms of glycosyltransferase inhibitors.
In addition, we report several general microwave-assisted reactions
that serve to accelerate the synthesis of carbasugars for further
studies.
Scheme 1
Sugars mediate a large variety of protein-protein and cell-cell
interactions implicated in disease states, thereby making carbohydrate-
based therapeutics attractive.1 The drive to understand the molecular
determinants of these carbohydrate binding interactions as well as
the search for more chemically and biochemically stable sugar
derivatives has led to the synthesis of a variety of analogues that
replace the glycosidic oxygen with sulfur or carbon.2 In contrast,
the effect of substitution of the ring oxygen on the conformations
and biological activity of pyranose sugars has been largely
neglected, in part because of the difficulty in obtaining these
analogues. The small amount of existing biological data outside
glycosidase inhibitors has shown sulfur versions of activated
nucleotidediphosphosugars to be poor substrates for glycosyltrans-
ferases,3 whereas the carbocyclic versions serve as inhibitors of
these enzymes.4 These substrates are difficult to synthesize chemi-
cally but led us to consider strategies to form such analogues
biologically. However, no data are available for the tolerance of
sugar nucleotidyltransferases to ring oxygen substitutions.
The ubiquitous Leloir pathway glycosyltransferases require
activated sugars that are produced by sugar nucleotidyltransferases
(Figure 1). The latter enzymes have been proposed as possible
antibiotic targets,5a,5g,6 but facile screening assays and knowledge
of differences in carbohydrate substrate recognition between bacteria
and humans are needed to design compounds with the necessary
selectivity. Presently, only a few structures of sugar nucleotidyl-
transferases are known.5 No sequence homology between enzymes
of similar function from eukaryotes and prokaryotes is apparent;
therefore, differences in substrate recognition and turnover could
be expected for exploitation in antibiotic design.
Scheme 2
mercury is often used and the rearrangements require hours. Many
reaction times, especially for transition metal-mediated reactions,
can be significantly shortened with microwave assistance.8 Indeed,
we found that not only could the Ferrier rearrangement be carried
out in less time and with higher yields in the presence of palladium
dichloride but that the synthesis of the necessary precursor 5 could
also be hastened by the application of microwave-assisted reactions
rather than conventional heating (Scheme 1 and Supporting
Information). Iodination9 of the selectively protected glucose
precursor 3 took place in a minute; the subsequent elimination
reaction took place in a half hour under microwave irradiation
without any competing side reactions. The development of this
series of microwave-assisted reactions significantly shortened the
time to form the core carbocyclic structure from a protected
D-glucose, thereby providing a route that should be applicable to a
variety of other sugars for studies with sugar nucleotidyltransferases.
To complete the synthesis of the desired carbaglucose-1-
phosphate (2), the free hydroxyl group of the Ferrier product 6
was silylated and methylation with Tebbe’s reagent yielded the
corresponding exo-methylene derivative 7 (Scheme 2). Hydrobo-
ration/oxidation of alkene 7 resulted in alcohol 8 with the desired
equatorial configuration in 87% overall yield. Alcohol protection
by benzylation followed by silyl protecting group removal produced
free hydroxyl 9. Treatment of 9 with dibenzyl diisopropyl phos-
phoramidite in the presence of a catalytic amount of N-(phenyl)-
imidazolium triflate10 produced a phosphite intermediate that was
Discovery of the tolerance of sugar nucleotidyltransferases to
carbocyclic sugar substrates first required the synthesis of carba-
sugar-1-phosphates. Several strategies have been applied to the
synthesis of carbasugars, including radical cyclizations, conversion
of quinic acid or bacterial metabolites, zirconium-mediated ring
contractions, ring-closing metathesis, Cope rearrangements, and
anionic or transition metal-mediated cyclizations.7,4b The Ferrier
rearrangement is the most common approach to carbasugars, but
9
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J. AM. CHEM. SOC. 2004, 126, 13188-13189
10.1021/ja045522j CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
Supporting Information Available: Experimental details for the
synthesis of 2 with 1H and 13C NMR spectra, details of the mass
spectrometry analysis, including calibration curves, and Michaelis-
Menten plots (PDF). This material is available free of charge via the
Internet at http://pubs.acs.org.
oxidized in situ to the phosphate. Global debenzylation afforded
the desired carbasugar 2.
With the desired carbocyclic analogue in hand, we next compared
its interactions with representative bacterial and eukaryotic sugar
nucleotidyltransferases. A major obstacle in testing nonnatural
substrates and inhibitors with this class of enzymes has been the
lack of a rapid assay to determine kinetic parameters for a variety
of compounds; however, the recent development of an electrospray
ionization mass spectrometry (ESI-MS)-based assay11 circumvents
these difficulties. The carbasugar 2 was first incubated with a
glucose-1-phosphate uridylyltransferase from Escherichia coli,
which is known to also accept thymidine triphosphate and is
homologous to a range of bacterial sugar nucleotidyltransferases.12
Surprisingly, the analogue was turned over to produce the carbo-
cyclic version of UDP-glucose. In fact, carbasugar 2 exhibited Km
values (17 ( 2 µM) similar to those of the natural substrate 1
(12 ( 2 µM). However, a lower turnover rate meant that kcat/Km
values (s-1 µM-1) were 0.0020 for the analogue compared to 1.45
for the natural substrate. In contrast, the corresponding sugar
nucleotidyltransferase from yeast, which is also homologous to the
human enzyme, showed no evidence for carbocyclic UDP-glucose
formation even with 5-fold higher enzyme concentrations.
These data provide the first evidence that carbocyclic sugar
analogues can serve to inhibit the class of enzymes that provide
sugar nucleotide donors to glycosyltransferases, which make
compounds such as the cyclic glucans that render some bacteria
resistant to standard antibiotics.13 The relatively weak inherent
affinity of glycosyltransferase substrates has been a large hurdle
in the design of potent and, most importantly, selective inhibitors
for this class of enzymes. This difficulty stems in part from the
fact that a large portion of the protein binding energy of these
charged sugar substrates comes from the phosphates and not from
the carbohydrate itself. However, the incorporation of a catalytic
step in addition to a binding step can create a more prominent
distinction between prokaryotes and eukaryotes. Differences in
substrate turnover that have been exploited in the design of cancer
drugs (cancer cells often upregulate enzymes that convert pro-
drugs)14 also can serve as a potential strategy to increase the
selectivity of drugs targeted for carbohydrate biosynthetic pathways.
Compounds can be designed to make use of not only the inherent
differences in bacterial versus eukaryotic substrate binding pockets
but also the differences in substrate turnover. Finally, we have
shown that sugar nucleotidyltransferases provide means for the
facile chemoenzymatic synthesis of carbocyclic versions of activated
sugars for further studies of the effects of this substitution on the
conformations and properties of carbasugars and for cocrystalliza-
tion studies with glycosyltransferases and their respective glycosyl
acceptors.
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Acknowledgment. We are thankful for an NSF CAREER award
and a Shimadzu University Research Grant and thank the Herman
Frasch Foundation (American Chemical Society) for its support of
this research. N.L.P. is a Cottrell Scholar of Research Corporation.
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