Stereoselective chemical synthesis of sugar nucleotides via direct displacement of acylated glycosyl bromides

Article abstract of DOI:10.1021/ol063068d

Figure presented The use of Leloir glycosyltransferases to prepare biologically relevant oligosaccharides and glycoconjugates requires access to sugar nucleoside diphosphates, which are notoriously difficult to efficiently synthesize and purify. We report a novel stereoselective route to UDP- and GDPα-D-mannose as well as UDP- and GDP-β-L-fucose via direct displacement of acylated glycosyl bromides with nucleoside 5′- diphosphates.

Full text of DOI:10.1021/ol063068d

ORGANIC
LETTERS
2007
Vol. 9, No. 7
1227-1230
Stereoselective Chemical Synthesis of
Sugar Nucleotides via Direct
Displacement of Acylated Glycosyl
Bromides
Shannon C. Timmons† and David L. Jakeman*,†,‡
Department of Chemistry, Dalhousie UniVersity, Halifax, N.S., Canada B3H 4J3, and
College of Pharmacy, Dalhousie UniVersity, Halifax, N.S., Canada B3H 3J5
daVid.jakeman@dal.ca
Received December 19, 2006
ABSTRACT
The use of Leloir glycosyltransferases to prepare biologically relevant oligosaccharides and glycoconjugates requires access to sugar nucleoside
diphosphates, which are notoriously difficult to efficiently synthesize and purify. We report a novel stereoselective route to UDP- and GDP-
r-D-mannose as well as UDP- and GDP-â-L-fucose via direct displacement of acylated glycosyl bromides with nucleoside 5′-diphosphates.
Oligosaccharides and glycoconjugates play a critical role in
many biochemical recognition processes.1,2 Through estab-
lishing structure-function relationships, glycobiology aims
to further our understanding of the roles of complex
carbohydrates in both eukaryotes and prokaryotes. In Nature,
Leloir glycosyltransferases3 catalyze the transfer of a monosac-
charide unit from a nucleoside diphosphate donor to an
acceptor substrate bearing a free hydroxyl group such as an
amino acid, a carbohydrate, or a natural product aglycon.
The use of glycosyltransferases to synthesize biologically
relevant oligosaccharides has become an attractive alternative
to total synthesis in recent years. Glycosyltransferase-
catalyzed syntheses generally offer the advantages of both
regio- and stereospecificity in addition to eliminating the need
for laborious protection/deprotection procedures.4,5
In mammalian systems, Leloir glycosyltransferases pri-
marily utilize the following seven sugar nucleotide donors:
UDP-R-D-Glc,6 UDP-R-D-Gal, UDP-R-D-GlcNAc, UDP-R-
D-GalNAc, GDP-R-D-Man, GDP-â-L-Fuc, and CMP-â-D-
NeuAc.7 In order to effectively use glycosyltransferases to
prepare complex oligosaccharides for biological study,
efficient methodologies are required to access sugar nucle-
otide substrates. Although chemoenzymatic strategies to
prepare sugar nucleotides are emerging,8,9 and several new
(4) Uchiyama, T.; Hindsgaul, O. J. Carbohydr. Chem. 1998, 17, 1181.
(5) Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T. Angew.
Chem., Int. Ed. Engl. 1995, 34, 521.
(6) Abbreviations: UDP, uridine 5′-diphosphate; GDP, guanosine 5′-
diphosphate; CMP, cytidine 5′-monophosphate; Glc, glucose; Gal, galactose;
GlcNAc, N-acetyl-glucosamine; GalNAc, N-acetylgalactosamine; Man,
mannose; Fuc, fucose; NeuAc, N-acetylneuraminic acid.
† Department of Chemistry.
‡ College of Pharmacy.
(1) Dove, A. Nat. Biotechnol. 2001, 19, 913.
(2) Varki, A. Glycobiology 1993, 3, 97.
(3) Leloir, L. F. Science 1971, 172, 1299.
(7) Palcic, M. M. Methods Enzymol. 1994, 230, 300.
10.1021/ol063068d CCC: $37.00
© 2007 American Chemical Society
Published on Web 03/06/2007

Scheme 1. Stereoselective Synthesis of Sugar Nucleotides via Direct Displacement of Acylated Glycosyl Bromides
in vitro approaches have recently been described to help
circumvent this requirement,10,11 chemical synthesis remains
a robust and versatile method to prepare these important
substrates.
benzylated epoxides derived from glycals.21,22 These meth-
odologies have generally resulted in similarly moderate yields
in the key coupling step but eliminate the need for sugar
1-phosphates, using instead more easily accessible glycosyl
donors. Purification of these reaction mixtures is often more
straightforward as residual nucleoside 5′-diphosphates can
easily be degraded to their respective nucleoside bases and
inorganic phosphate using alkaline phosphatase before pass-
ing reaction mixtures through a reversed-phase or ion-
exchange column.4 The major drawback to these direct
coupling approaches is the lack of stereoselectivity obtained
in the coupling of glycosyl donors with nucleoside 5′-
diphosphates. In the majority of cases R/â selectivities are
approximately 1/1 and even in cases where couplings are
more selective it is difficult to predict which diastereomer
will predominate. It has been suggested that R/â diastereo-
meric mixtures of sugar nucleotides are not problematic since
glycosyltransferases are believed to select for the required
diastereomer and not be significantly inhibited by sugar
nucleotides of opposite anomeric configuration.4,19 This being
said, it is obviously advantageous to develop synthetic routes
with high levels of stereocontrol to improve the yields of
desired sugar nucleotide diastereomers, and to allow kinetic
studies without any possibility of interference from unwanted
diastereomers.
The majority of chemical syntheses of sugar nucleotides
involve the coupling of sugar 1-phosphates with activated
nucleoside 5′-monophosphates. Although some common
sugar 1-phosphates are commercially available, others must
be prepared via multistep syntheses. The nucleoside 5′-
monophosphate is frequently activated as a morpholidate,12,13
and the coupling reaction with the sugar 1-phosphate
typically takes several days and results in only moderate
yields.14,15,16 More recently, the use of an N-methylimida-
zolide nucleoside 5′-monophosphate donor has improved
reaction times (2 h),17 but overall yields remain moderate
and tedious purifications often result. Purifications are often
difficult because of the dinucleoside diphosphate (NppN)
byproducts that result from the self-condensation of activated
nucleoside 5′-monophosphates and, as a result, purification
protocols vary greatly from laboratory to laboratory.17-19
In attempts to improve the synthesis and purification of
sugar nucleoside diphosphates, the direct coupling of various
glycosyl donors with nucleoside 5′-diphosphates has been
explored. Examples of glycosyl donors used in this approach
include benzylated glycosyl bromides,19 trimethylsilylated
glycosyl iodides,4 2-(1,2-trans-glycopyranosyloxy)-3-meth-
oxypyridines (MOP glycosides),20 and triethylsilylated and
Herein, we present the first stereocontroled synthesis of
four sugar nucleotides using a direct coupling approach.
Using this methodology, UDP-R-D-Man, GDP-R-D-Man,
UDP-â-L-Fuc, and GDP-â-L-Fuc were efficiently prepared
in only four synthetic steps from their respective reducing
sugars (Scheme 1). Through the use of acyl protecting
groups, neighboring group participation was employed
resulting in the exclusive preparation of sugar nucleotides
with the desired anomeric configurations (Figure 1). Yields,
determined by both mass and UV absorbance, are presented
along with key NMR chemical shifts and coupling constants
in Table 1. Although the assignment of L-fuco-linked
(8) Timmons, S. C.; Mosher, R. H.; Knowles, S. A.; Jakeman, D. L.
Org. Lett. 2007, 9, 857, and references therein (ol0630853).
(9) Errey, J. C.; Mukhopadhyay, B.; Kartha, K. P. R.; Field, R. A. Chem.
Commun. 2004, 2706 and references therein.
(10) Lairson, L. L.; Wakarchuk, W. W.; Withers, S. G. Chem. Commun.
2007, 365.
(11) Zhang, C.; Griffith, B. R.; Fu, Q.; Albermann, C.; Fu, X.; Lee, I.-
K.; Li, L.; Thorson, J. S. Science 2006, 313, 1291.
(12) Moffatt, J. G. Methods Enzymol. 1966, 8, 136.
(13) Moffatt, J. G.; Khorana, J. J. Am. Chem. Soc. 1961, 83, 659.
(14) Chang, C.-W. T.; Liu, H.-w. Bioorg. Med. Chem. Lett. 2002, 12,
1493.
(15) Zhang, Q.; Liu, H.-w. Bioorg. Med. Chem. Lett. 2001, 11, 145.
(16) Wittmann, V.; Wong, C.-H. J. Org. Chem. 1997, 62, 2144.
(17) Marlow, A. L.; Kiessling, L. L. Org. Lett. 2001, 3, 2517.
(18) Dinev, Z.; Wardak, A. Z.; Brownlee, R. T. C.; Williams, S. J.
Carbohydr. Res. 2006, 341, 1743.
(20) Hanessian, S.; Lu, P.-P.; Ishida, H. J. Am. Chem. Soc. 1998, 120,
13296.
(21) Ernst, C.; Klaffke, W. J. Org. Chem. 2003, 68, 5780.
(22) Ernst, C.; Klaffke, W. Tetrahedron Lett. 2001, 42, 2973.
(19) Arlt, M.; Hindsgaul, O. J. Org. Chem. 1995, 60, 14.
1228
Org. Lett., Vol. 9, No. 7, 2007

molecular sieves and triethylamine. Triethylamine (1 equiv)
was added to reaction mixtures to help neutralize any acid
produced from the reaction and limit degradation of the
glycosyl bromide. Higher numbers of equivalents of triethyl-
amine produced no further benefit. Reactions at rt were very
sluggish, and, after several days, low conversions to product
of approximately 5-10% were obtained by integration of
31P{1H} signals. The addition of extra equivalents of glycosyl
bromide improved yields only slightly. In an attempt to
expediate the reaction rate and improve the yield of the
coupling reaction, experiments were conducted wth 1 equiv
of glycosyl bromide and 1 equiv of nucleoside 5′-diphosphate
at 80 °C in acetonitrile and monitored closely by TLC and
HPLC. After 30 min, all glycosyl bromide had been
consumed or degraded by TLC and the presence of a sugar
nucleotide product was detected by HPLC. It should be noted
that the addition of extra equivalents of glycosyl bromide
resulted in substantially more degradation of nucleoside 5′-
diphosphates and sugar nucleotide products, thus producing
lower overall conversions to product as observed by HPLC.
A 1/1/1 ratio of glycosyl bromide/nucleoside 5′-diphosphate/
triethylamine was therefore found to be optimal in the
coupling step.
Figure 1. Influence of neighboring group participation on the
trajectory of NDP attack for D-manno- (11) and L-fuco-configured
(12) acylated sugars.
products as â configured sugar nucleotides was straightfor-
ward from large vicinal H1′′-H2′′ J values, the anomeric
configuration of D-manno-linked sugar nucleotides was more
difficult to assign. As with L-rhamnose, both R and â
anomers of glycosides derived from D-mannose have similar
small H1′′-H2′′ J values differing by ∼1 Hz. To unambigu-
ously determine the anomeric configuration of D-manno-
containing products, the 13C1-H1 J values23-25 of all sugar
nucleotides was determined. The measured 13C1-H1 J values
(Table 1) were consistent with literature expectations for R
and â-linked glycosides.
This synthetic methodology begins with D-mannose (1)
and L-fucose (6), which were first protected with acetyl and
benzoyl groups, respectively, to produce acylated compounds
2 and 7. Both acetyl and benzoyl groups were employed to
investigate the generality of this approach with respect to
acyl protecting groups. Second, a bromo substituent was
easily installed at the anomeric center using phosphorus
tribromide to give R-linked glycosyl bromides 3 and 8, which
were obtained in stereopure form due to the anomeric effect.
To facilitate the efficient purification of sugar nucleotide
products, reaction mixtures were concentrated and redis-
solved in H2O. The pH was then adjusted to 8 using
triethylamine and 50 EU26 of alkaline phosphatase was added
to degrade unreacted nucleoside 5′-diphosphates. The deg-
radation process was monitored by HPLC and was typically
complete after stirring at rt for approximately 24 h. After
concentration, reaction mixtures were redissolved in a 2/2/1
mixture of MeOH/H2O/Et3N and stirred overnight (∼16 h)
at rt as previously described27 to deprotect acyl groups present
on the monosaccharides. The reaction mixtures were sub-
sequently subjected to purification via C18 ion-pair reversed-
phase chromatography using 10 mM tributylammonium
bicarbonate as the ion-pair reagent of choice.28
Prior to the coupling reaction, nucleoside 5′-diphosphates
were converted to free acids by passing through a column
of Amberlite IR-120 (H+) ion-exchange resin. The free acids
were subsequently titrated with an aqueous solution of
tetrabutylammonium hydroxide solution (40% w/v) to pH 6
and lyophilized. Care should be taken during the titration
because titrations to pH 5 and 7 have resulted in product
degradation and reduced nucleophilicity/lower yields, re-
spectively, in coupling reactions.21,22
We have determined that acetylated and benzoylated
glycosyl bromides work equally well to give exclusively the
desired anomeric stereochemistry (a 1,2-trans relationship)
upon coupling with nucleoside 5′-diphosphates. This coupling
procedure also worked well with both pyrimidine and purine
bases, illustrating the general applicability of this approach
The key synthetic step, coupling of the acylated glycosyl
bromides with nucleoside 5′-diphosphates, was initially
attempted at room temperature in acetonitrile with 3 Å
Table 1. Yields and NMR Characterization of Sugar Nucleotide Products 4, 5, 9, and 10
nucleoside
5′-diphosphate (NDP)
yield by
UVa,b (%)
yield by
massa (%)
H1′′ chemical
shift (ppm)
3JH1′′-H2′′
(Hz)
1J C1′′-H1′′
(Hz)
product
sugar
4
5
9
10
D-mannose
D-mannose
L-fucose
uridine 5′-diphosphate
guanosine 5′-diphosphate
uridine 5′-diphosphate
guanosine 5′-diphosphate
29
35
26
31
33
38
31
35
5.49
5.49
4.84
4.90
n/oc
n/oc
8.0
170
173
163
160
L-fucose
8.0
a Yields are reported for purified products over two steps (coupling and deprotection). b UV yields were determined at λmax 261 nm ) 1.01 × 104
3
M-1cm-1 for products containing uridine and λmax 253 nm ) 1.37 × 104 M-1cm-1 for products containing guanosine. c Not observed (br d, JH1′′-P ) 8.0
Hz).
Org. Lett., Vol. 9, No. 7, 2007
1229

with D-mannose and L-fucose, two prominent sugar residues
in many biologically relevant oligosaccharides and glyco-
conjugates.
In summary, this novel methodology, employing the well-
known concept of neighboring group participation, represents
a simple, efficient procedure for preparing diastereomerically
pure R-D-manno- and â-L-fuco-linked sugar nucleoside
diphosphates. Further investigations are currently underway
to extend this approach to prepare various R-D-manno- and
â-L-fuco-linked sugar nucleotide derivatives synthetically
modified at any position except C2 for use as glycosyltrans-
ferase probes. Similarly, this procedure is also amenable for
use in the synthesis of R-D-manno- and â-L-fuco-linked sugar
nucleotide derivatives modified at the nucleobase.29
Acknowledgment. This work was supported by a grant
from the Canadian Institutes of Health Research. We thank
Dr. Charles Borissow (University of Leicester, UK) for
helpful discussions.
(23) Duus, J. Ø.; Gotfredsen, C. H.; Bock, K. Chem. ReV. 2000, 100,
4589.
Supporting Information Available: Experimental pro-
cedures and spectral data for compounds 2-5 and 7-10;
1H, 13C, and 31P{1H} spectra as well as HPLC traces for sugar
nucleotides 4, 5, 9, and 10. This material is available free of
charge via the Internet at http://pubs.acs.org.
(24) Bock, K.; Pedersen, C. J. Chem. Soc., Perkin Trans. 2 1974, 293.
(25) Bock, K.; Lundt, I.; Pedersen, C. Tetrahedron Lett. 1973, 13, 1037.
(26) 1 EU (enzyme unit) ) the amount of enzyme needed to catalyze
the transformation of 1 mmol of substrate per minute.
(27) Oberthu¨r, M.; Leimkuhler, C.; Kahne, D. Org. Lett. 2004, 6, 2873.
(28) Detailed procedures describing ion-pair buffer preparation and C18
reversed-phase purification conditions can be found in the Supporting
Information.
(29) Collier, A.; Wagner, G. Org. Biomol. Chem. 2006, 4, 4526.
OL063068D
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Org. Lett., Vol. 9, No. 7, 2007