Practical synthesis of sugar monophosphonucleotides

Article abstract of DOI:10.1080/00397910701471543

A reliable procedure for the preparation of sugar nucleoside monophosphates is presented, which involves condensation of an activated glycosyl-1-H- phosphonate with an appropriately protected nucleoside and simple end-product isolation via lithium perchlo

Full text of DOI:10.1080/00397910701471543

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Practical Synthesis of Sugar
Monophosphonucleotides
Wjatschesslaw A. Wlassoff a , Richard M. J. Finlay a & Chris J. Hamilton a
a School of Chemistry and Chemical Engineering, Queens University Belfast ,
Belfast, U.K.
Published online: 30 Aug 2007.
To cite this article: Wjatschesslaw A. Wlassoff , Richard M. J. Finlay & Chris J. Hamilton (2007) Practical
Synthesis of Sugar Monophosphonucleotides, Synthetic Communications: An International Journal for Rapid
Communication of Synthetic Organic Chemistry, 37:17, 2927-2934, DOI: 10.1080/00397910701471543
To link to this article: http://dx.doi.org/10.1080/00397910701471543
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Synthetic Communicationsw, 37: 2927–2934, 2007
Copyright # Taylor & Francis Group, LLC
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910701471543
Practical Synthesis of Sugar
Monophosphonucleotides
Wjatschesslaw A. Wlassoff, Richard M. J. Finlay, and
Chris J. Hamilton
School of Chemistry and Chemical Engineering, Queens University
Belfast, Belfast, U.K.
Abstract: A reliable procedure for the preparation of sugar nucleoside monophos-
phates is presented, which involves condensation of an activated glycosyl-1-H-phos-
phonate with an appropriately protected nucleoside and simple end-product isolation
via lithium perchlorate–induced precipitation. The utility of these methods is demon-
strated by the preparation of a number of purine- and pyrimidine-based sugar nucleo-
side monophosphate derivatives.
Keywords: acyl deprotection, glycosyl-1-H-phosphonates, sugar nucleotides
In vivo, Leloir-type glycotransferases play a central role in mediating the bio-
synthesis of oligosaccharides and glycosylated natural products. These
enzymes catalyze the transfer of sugar moieties from activated donor sugar sub-
strates [most commonly a sugar nucleoside diphosphate (sugar-NDP)] (Fig. 1)
to specific acceptor molecules (e.g., mono/oligosaccharides), forming glycosi-
dic bonds.[1,2] Instead of a sugar-NDP, sialytransferases utilize a sugar
nucleoside monophosphate (sugar-NMP), namely cytidine monophosphate-N-
acetylneuraminic acid (CMP-Neu5Ac), as the donor substrate for enzymatic
sialyltransfer (Fig. 1). The chemical and enzymatic syntheses of many
natural, and modified, sugar-NDPs have received considerable attention
Received in the U.K. September 12, 2006
Address correspondence to Chris J. Hamilton, School of Chemistry and Chemical
Engineering David Keir Building, Queens University Belfast, Stranmillis Rd.,
Belfast BT9 5AG, U.K. E-mail: c.hamilton@qub.ac.uk
2927

2928
W. A. Wlassoff, R. M. J. Finlay, and C. J. Hamilton
Figure 1. Some sugar nucleotide structures.
because of the expanding use of glycosyltransferases as biocatalysts for prepara-
tive-scale biotransformations.[3,4] Conversely, the synthesis of hexose-derived
sugar-NMPs has been less extensively addressed, and to our knowledge, the
potential of native or engineered sialyltransferases as an alternative subset of
biotransformation tools, beyond their use with CMP-sialic acid analogues,[5]
remains unexplored. To be able to pursue such studies requires routine access
to numerous sugar-NMP conjugates.
Other motives for exploring the preparation of sugar-NMPs include the
following. (i) There is some precedence for hexose conjugated NMPs (e.g.,
Man-6-O-AZTMP, Fig. 1) being used to facilitate the intracellular delivery
of antiviral nucleoside monophosphates.[6] (ii) The competitive nature of
UMP-Glc (with respect to UDP-Glc) as an inhibitor of UDP-glucose pyropho-
sphorylase[7] suggests that sugar NMPs might also provide access to inhibitors
of other sugar nucleotide processing enzymes.
Such studies would be greatly facilitated by a general and reliable
synthetic route to easily purified purine-/pyrimidine-based sugar-NMPs so
that the libraries of different sugar-NMP conjugates can be readily prepared
for evaluation with appropriate enzyme targets. Toward this goal, a method
is described herein that involves the coupling of a readily activated sugar-1-
H-phosphonate with an appropriately protected nucleoside component
followed by oxidation of the phosphite to the phosphate-diester and global
deprotection to give the sugar-NMP conjugate (Scheme 1). The utility of
this method is demonstrated by the preparation of a number of dTMP-,
AMP-, and CMP-sugar derivatives. Before discussing these optimized pro-
cedures, the problems encountered with other previously reported protocols
for sugar-NMP synthesis are briefly discussed.
A simple procedure for the preparation of nucleoside 50-(b-D-glucopyrano-
syl monophosphates) via NMP coupling with sugar ortho esters has previously
been used to access beta-configured conjugates of glucose with various purine
and pyrimidne NMPs.[8] Unfortunately, the nature of this reaction excludes the
use of certain monosaccharides such as 2-deoxy-sugars. Sugar-NMPs have also

Sugar Monophosphonucleotides
2929
Scheme 1.
been prepared by the reaction of glycosyl trichloroacetimidates with appropri-
ately protected nucleoside 50-monophosphates,[9] and a series of sialyl-NMPs
has been prepared via the reaction of sialyl phosphites with protected ribonu-
cleoside 50-monophosphates.[10] These methods involve too many preparative
and/or purification steps to be suitable for our needs.
We initially repeated a reported preparation of UMP-glucose 2 via the
silver carbonate–promoted reaction of 20,30,40,60-tetra-O-acetyl-a-D-glucopyra-
nosyl bromide 1 with uridine monophosphate (Scheme 2a)[7] but were unable to
improve on the low yields (,25%) that were originally reported for this
reaction. Reactions with the analogous glycosyl iodides were equally proble-
matic. A quick route to UDP-sugars has previously been reported that
involves the reaction of per-O-silylated hexoses 3 with trimethylsilyl iodide
and reaction of the in situ generated glycosyl iodide with UDP.[11] However,
in our hands it proved difficult to obtain acceptable and reproducible yields of
sugar-NMP 4 when dTMP was used instead of UDP (Scheme 2b). Others
have experienced similar difficulties with this approach to sugar-nucleotide
synthesis.[12] Coupling reactions of cyanoethyl ether protected nucleoside phos-
phoramidite 5 with an appropriately protected sugar 6[13] provided access to high
yields of protected coupling product 7 (80%) under very mild conditions
(Scheme 2c). However, the basic conditions required for efficient removal of
the cyanoethyl group [conc. NH3(aq), 608C, 12 h] also cleaved the glycosyl
phosphate linkage, whereas milder reaction conditions [i.e., 1,8-diazabicyclo
(5.4.0)undec-7-Ene (DBU), tetrabutyl ammonium fluoride, or conc. NH3(aq),
room temperature] yielded complex mixtures due to incomplete deprotection
and partial product degradation. An alternative phosphoramidite strategy has
previously been employed in a synthesis of CMP-Neu5Ac using an allyl protect-
ing group instead of cyanoethyl for the phosphate moiety to achieve deprotection
under very mild conditions.[14] Although very effective, the preparation of the
activated nucleotide component is convoluted, which presents a problem when
the derivatives of several different nucleotides should be made.

2930
W. A. Wlassoff, R. M. J. Finlay, and C. J. Hamilton
Scheme 2. Early attempts at preparing sugar-NMPs.
Previously, a limited number of 30-azido-20,30-dideoxythymidine (AZT)-
derived sugar-NMPs have been prepared via the reaction of appropriately
protected glycosyl-1-H-phosphonates with the 50-hydroxyl group of AZT in
the presence of pivaloyl chloride, as a condensing agent followed by
oxidation of the resulting sugar-nucleotide-H-phosphonate.[15] We have
further modified and developed this route as a practical and general
procedure for the preparation of readily purified purine- and pyrimidine-
based sugar-NMPs in consistent and reproducible yields (Scheme 1). Of par-
ticular note, the relative chemical instability of the final sugar-NMP conju-
gates puts certain restrictions on the nature of protecting groups that can be
used in their synthesis. They are unstable in acidic and strongly basic con-
ditions, which therefore require the use of sugar/nucleoside protecting
groups that can be removed under neutral or mildly basic conditions in the
final stages of the synthesis.
A general strategy for preparation of glycosyl-1-H-phosphonates 10a–d
is outlined in Scheme 3.[16] The readily obtained per-benzoylated sugars
8a–d can be selectively debenzoylated at the anomeric position under
mildly basic conditions (i.e., with THF/methanolic ammonia solutions). The
mono-debenzoylated products 9a–d are stable when stored as dry solids but
are prone to benzoyl group migration when stored in solution. The apparent
isomerization was particularly fast for 20,30,40-tribenzoyl xylopyranoside 9c.

Sugar Monophosphonucleotides
2931
Scheme 3. Syntheses of glycosyl-1-H-phosphonates.
Glycosyl-1-H-phosphonates 10a–d are synthesized in good yields by
reaction of 9a–d with phosphorus tri-imidazolide (generated in situ) followed
by hydrolysis in aqueous tetraethylammonium bicarbonate solution (TEAB).
Following an aqueous workup, the products 10a–d were sufficiently pure (by
proton NMR) to be carried through the subsequent reaction step without
further purification. Once prepared, compounds 10a–d were kept at 2208C
but were not stable enough for long-term storage. For the subsequent prep-
arations of sugar-NMPs 14–16 (Scheme 1), the best results are obtained
when using glycosyl-1-H-phosphonates 10a–d that are less than 3 weeks old.
Older samples produced significantly lower yields.
The coupling of sugar and nucleoside components (Scheme 1) involves
activation of the glycosyl-H-phosphonates 10a–d as the pivaloyl mixed
anhydride followed by treatment with the selectively protected nucleosides
11–13. Oxidation of the subsequently formed phosphite diester gives the
per-benzoyl protected sugar-NMP. Others have previously used methanolic
sodium methoxide to remove the acyl protecting groups from such conjugates,
which then required purification by ion-exchange chromatography.[15] Under
these conditions we often observed partial product decomposition (hence the
need for chromatographic purification) and a significant decrease in isolated
yield. Cleaner deprotection conditions were therefore established to improve
yields and simplify the end-product purification. A variety of basic deprotec-
tion conditions were investigated, and product degradation was not observed
when sodium methoxide was replaced by concentrated aqueous ammonia for
removal of the acyl protecting groups. Note that it is important to use
N-benzoyl protecting groups with cytidine and adenosine nucleosides 12
and 13 as the N-acetyl protecting group could only be removed under

2932
W. A. Wlassoff, R. M. J. Finlay, and C. J. Hamilton
conditions that also degrade the sugar-NMP product. The fully deprotected
products 14–16 (Table 1) can then be readily isolated as the lithium salts
(after the removal of ammonia on a rotary evaporator) by precipitation from
2% (w/v) lithium perchlorate in acetone. Using this method, a number of
sugar-NMP conjugates containing glucose, galactose, mannose, and xylose
as sugar components and 20-dTMP 14a–d, CMP 15a–c, and AMP 16a and
b as nucleotide components have been prepared (Table 1). As their dry
lithium salts, 14–16 are stable when stored at 2208C for at least 12 months
but undergo slow hydrolysis when stored in the freezer as aqueous solutions.
To summarize, a simple, efficient method for the preparation of purine and
pyrimidine-based sugar-NMPs, which can be isolated by simple precipitation,
has been established. A number of representative sugar-NMP conjugates have
been prepared using several common sugars and natural purine and pyrimidine
nucleosides. The method is now being utilized for the preparation of parallel
libraries of sugar-NMP conjugates to be screened in appropriate enzymatic
systems. Results of such studies are ongoing and will be reported in due course.
EXPERIMENTAL
General Procedure for Preparation of Benzoylated Glycosyl 1-H-
Phosphonates 10a–d
Phosphorus trichloride (0.38 mL, 4.3 mmol) and triethylamine (2.1 mL,
15.1 mmol) were added to an ice-cold solution of imidazole (970 mg,
14.3 mmol) in acetonitrile (25 mL). The mixture was stirred for 15 min, and
then a solution of 1-monounprotected benzoylated sugar 9a–d (1 mmol) in
acetonitrile (25 mL) was added dropwise over 30 min. The mixture was
removed from ice, and after 5 min 1M TEAB (7 mL) was added. The
solution was stirred at room temperature for 15 min and then co-evaporated
Table 1. Sugar-NMPs 14–16 produced via Scheme 3
Compound
NMP
Sugar
a:b ratioa
Yield (%)
14a
14b
14c
14d
15a
15b
15c
16a
16b
dTMP
dTMP
dTMP
dTMP
CMP
Glc
Gal
Xyl
Man
Glc
Gal
Xyl
Glc
Gal
87:13
81:19
85:15
76:24
88:12
81:19
84:16
77:23
69:31
56
45
65
70
70
75
65
70
72
CMP
CMP
AMP
AMP
aDetermined by 31P NMR and anomeric proton integrals (1H NMR).

Sugar Monophosphonucleotides
2933
with a mixture of pyridine and triethylamine (4:1, 100 mL). The residue was
dissolved in chloroform (200 mL) and extracted with ice-cold water
(2 ꢀ 200 mL) and 0.5 M TEAB (2 ꢀ 200 mL). The organic layer was dried
(Na2SO4), and the solvent removed in vacuo to give the glycosyl-1-H-phospo-
nate (70–80% yield), which was used in the next step without further
purification.
General Procedure for Preparation of Sugar–NMP Conjugates
14–16
Benzoylated glycosyl 1-H-phosphonate 10a–d (1 mmol) and 50-monounpro-
tected nucleoside 11–13 (0.66 mmol) were co-evaporated with anhydrous
pyridine (3 ꢀ 10 mL) and dissolved in 8 mL of anhydrous pyridine.
Pyvaloyl chloride (0.216 mL, 1.65 mmol) was added to the mixture, and
after 10 min a solution of iodine (0.5 g, 2 mmol) in pyridine/water (95:5.
5.8 mL) was added. After 15 min, the solution was diluted with chloroform
(200 mL), washed with 0.5 M Na2S2O3 (2 ꢀ 100 mL) and 0.5 M TEAB
(2 ꢀ 100 mL), dried (Na2SO4), and evaporated. The residue was purified by
chromatography (0–15% methanol in dichloromethane). The product was
dissolved in methanol (2–4 mL), added to an excess of concentrated
aqueous ammonia (50 mL), left at room temperature overnight, and then evap-
orated. The residue was suspended in 2 mL of water and centrifuged to remove
the nonsoluble components, and the product was precipitated as a lithium salt
by the addition of 2% (w/v) LiClO4 in acetone (20 mL), then washed with
acetone and dried in vacuo to give the desired product as a white solid,
which was stored at 2208C.
Cytidine 50-(a-D-Glucopyranosyl Phosphate), Lithium Salt 15a
This was prepared (in 70% isolated yield) according to the general procedure
from 20,30,N4-tribenzoylcytidine 12 and 2,3,4,6-tetra-O-benzoyl-D-glucopyra-
nosyl 1-H-phosphonate 10a. a-Anomer: dH (500 MHz, D2O): 3.39 (1H, t, J
9.7, H-400), 3.50 (1H, dt, J 9.7, 2.7, H-200), 3.66 (1H, t, J 9.7, H-300), 3.69
(2H, s, H-600), 3.72–3.74 (1H, m, H-500), 4.07–4.09 (1H, m, H-40), 4.14–
4.19 (2H, m, H-50), 4.21–4.24 (2H, m, H-20, H-30), 5.43 (1H, dd, J 7.1, 3.4,
H-100), 5.89 (1H, d, J 4.5, H-10), 6.01 (1H, d, J 7.6, H-5), 7.83 (1H, d, J 7.6,
H-6): dC NMR (125 MHz, D2O): 60.58 (C-600), 64.99 (d, J 5.3, C-50), 69.44
(C-400), 69.64 (C-30), 71.66 (d, J 8.8, C-200), 72.91, 73.24 (C-20, C-300), 74.56
(C-500), 82.98 (d, J 9.1, C-40), 89.78 (C-10), 95.84 (d, J 6.5, C-100), 96.92
(C-5), 141.69 (C-6), 158.07 (C-2), 166.50 (C-4): dP (121 MHz, D2O):
20.36. b-Anomer: dH (500 MHz, D2O): 5.38 (1H, dd, J 7.4, 3.5, H-100); dC
(125 MHz, D2O): 89.69 (C-100, obscured by C-10); dP (121 MHz, D2O):
20.59. MS (ES2) m/z ¼ 484.0969 (calc. for C15H25N3O13P, 484.0974).

2934
W. A. Wlassoff, R. M. J. Finlay, and C. J. Hamilton
ACKNOWLEDGMENTS
This work was supported by a Wellcome Trust Showcase Award (grant ref.
067525/Z/02/Z). We also thank the Engineering and Physical Sciences
Research Council (EPSRC) Mass Spectrometry Service Centre, Swansea,
for invaluable support.
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