Novel derivatives of UDP-glucose: Concise synthesis and fluorescent properties

Article abstract of DOI:10.1039/b805216f

A series of novel 5-substituted UDP-glucose derivatives with interesting fluorescent properties and potential applications as sensors for carbohydrate-active enzymes is reported. An efficient synthesis of the target molecules was developed, centred around the Suzuki-Miyaura reaction of (hetero)arylboronic acids with 5-iodo UDP-glucose. Interestingly, the optimised cross-coupling conditions could also be applied successfully to 5-bromo UMP, but not to 5-bromo UDP-glucose. The Royal Society of Chemistry.

Full text of DOI:10.1039/b805216f

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Volume 6 | Number 16 | 21 August 2008 | Pages 2829–3016
ISSN 1477-0520
FULL PAPER
John Robinson et al.
FULL PAPER
Thomas Pesnot and Gerd Wagner
Novel derivatives of UDP-glucose:
concise synthesis and fluorescent
properties
Exploring the substrate specificity
of OxyB, a phenol coupling P450
enzyme involved in vancomycin
biosynthesis

PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Novel derivatives of UDP-glucose: concise synthesis and fluorescent
properties†
Thomas Pesnot and Gerd K. Wagner*
Received 27th March 2008, Accepted 6th May 2008
First published as an Advance Article on the web 18th June 2008
DOI: 10.1039/b805216f
A series of novel 5-substituted UDP-glucose derivatives with interesting fluorescent properties and
potential applications as sensors for carbohydrate-active enzymes is reported. An efficient synthesis
of the target molecules was developed, centred around the Suzuki–Miyaura reaction of
(hetero)arylboronic acids with 5-iodo UDP-glucose. Interestingly, the optimised cross-coupling
conditions could also be applied successfully to 5-bromo UMP, but not to 5-bromo UDP-glucose.
preparation of these analogues is usually carried out by multi-
Introduction
step synthesis from a fluorescently labelled precursor. Frequently,
this approach involves lengthy protection–deprotection sequences,
suffers from low overall yields, and is lacking the synthetic
flexibility required for efficient analogue synthesis. In addition, the
steric bulk of many conventional fluorophores may compromise
the biological activity of the labelled UDP-sugars.6
UDP-Sugars‡ like UDP-glucose (Fig. 1) and UDP-galactose are
the natural donor substrates for a large number of carbohydrate-
active enzymes, including glycosyltransferases and epimerases.1
Many of these UDP-sugar-dependent enzymes are involved in
important biological processes, such as the biosynthesis of blood
group determinants,2 the assembly of cell-surface glycoconjugates
and cell wall components in (myco)bacteria3a,b and fungi,3c and
galactose metabolism in trypanosomes.4 Structural analogues of
naturally occurring UDP-sugars are therefore sought after as
inhibitor candidates and molecular tools for the investigation of
these enzymes and processes.
As part of an ongoing effort to develop fluorescence-based
glycosyltransferase assays, we have designed a novel type of
fluorescent UDP-sugar with a compact, fluorogenic substituent
at the uracil base (Fig. 1). Our target design was inspired by the
recent discovery that uracil nucleosides and nucleotides with a
small, heterocyclic substituent in position 5 possess interesting
fluorescent properties.7 We reasoned that this concept can also be
applied to UDP-sugars, providing sugar-nucleotides sufficiently
“auto-fluorescent” as to obviate the need for a bulky fluorophore.
Our synthetic strategy for the target 5-substituted UDP-sugars
was devised with maximum structural flexibility in mind (Fig. 1).
The fluorogenic aryl and heteroaryl substituents can be installed at
the uracil base of a suitable halogenated precursor using Suzuki–
Miyaura cross-coupling chemistry.8 Taking advantage of recent
advances in Pd-catalysed reactions in aqueous media,9 we sought
to carry out the cross-coupling directly on a preassembled 5-
halo UDP-sugar substrate, in the final step of the synthesis. This
approach facilitates analogue synthesis, thus allowing the rapid
optimisation of biological and fluorescent properties.
Fig. 1 UDP-glucose (X = H), and general synthetic strategy towards
novel, fluorescent analogues.
Previously, we and others have described the cross-coupling
of various unprotected purine10 and pyrimidine11 nucleotides.
Recently, we have also reported the first example for the Suzuki–
Miyaura reaction of an unprotected GDP-sugar.12 However, to
the best of our knowledge no cross-coupling reactions with
unprotected UDP-sugars are known in the literature. This gap
illustrates the substantial challenge associated with the use of
UDP-sugars as cross-coupling substrates, due to the intrinsic
susceptibility of e.g. the glycosyl phosphate linkage to hydrolytic
cleavage.
Herein, we report the first successful Suzuki–Miyaura reaction
of an unprotected 5-halo UDP-sugar. We describe the develop-
ment of cross-coupling conditions compatible with the use of
sugar-nucleotide substrates and investigate the parameters impor-
tant for cross-coupling reactivity. We show, for the first time, that
these conditions can also be applied to the efficient cross-coupling
Of particular interest in this context are fluorescently labelled
UDP-sugars, which can be used in operationally simple glyco-
syltransferase bioassays. Previously, several UDP-sugars with a
fluorophore connected via a linker to either the sugar5,6 or, in
one case, the nucleobase6 have been described. The synthetic
Centre for Carbohydrate Chemistry, School of Chemical Sciences &
Pharmacy, University of East Anglia, Norwich, NR4 7TJ, England.
E-mail: g.wagner@uea.ac.uk; Fax: +44 (0)1603 592003; Tel: +44 (0)1603
593889
1
† Electronic supplementary information (ESI) available: H, 13C and 31P
NMR spectra for sugar-nucleotides 6a, 6b, 7a–d and selected nucleosides
and nucleotides; UV absorbance spectra for 6b and 7a–d. See DOI:
10.1039/b805216f
‡ Abbreviations: GDP guanosine diphosphate; UDP uridine diphosphate;
UMP uridine monophosphate; TPPTS tris(3-sulfonatophenyl)phosphine
trisodium salt.
2884 | Org. Biomol. Chem., 2008, 6, 2884–2891
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©

Table 1 Suzuki–Miyaura coupling of 5-bromouridine 2 and 5-bromo
of 5-bromouracil nucleotides. Using this methodology, we have
prepared a series of four 5-(hetero)aryl UDP-glucose derivatives
from a single synthetic precursor. These novel UDP-glucose
analogues show interesting and differential fluorescent properties
and may be useful for the development of glycosyltransferase
bioassays. The synthetic method is robust and flexible, and may be
applicable to the efficient preparation of other fluorescent UDP-
sugars also.
a
UMP 4a with various arylboronic acids R-B(OH)2
Entry
Substrate
Product
R
Yieldb (%)
1
2
3
4
5
6
2
3a
3b
3c
5a
5b
5c
Phenyl
45
46
43
57
27
26
2
4-Methylphenyl
4-Chlorophenyl
Phenyl
4-Methylphenyl
4-Chlorophenyl
2
4a
4a
4a
a See Scheme 1 for structures. b Isolated yields.
Results and discussion
An important objective of the present study was the identification
of suitably mild reaction conditions for the Suzuki–Miyaura cross-
coupling of unprotected 5-halo UDP-sugars in aqueous solution.
We decided to establish a suitable cross-coupling protocol using
uracil nucleosides and nucleotides first, before attempting the
more challenging cross-coupling of the corresponding sugar-
nucleotides. We were particularly interested in using 5-bromouracil
nucleosides and (sugar-)nucleotides as cross-coupling substrates,
despite their potentially lower cross-coupling reactivity compared
to the 5-iodo congeners, as the uracil base can be brominated
selectively in position 5 under mild, aqueous conditions.13 In
principle, this would allow us to combine bromination and cross-
coupling reaction in a one-pot, two-step procedure12 in aqueous
media, thus opening a very efficient synthetic route towards the
target 5-(hetero)aryl UDP-glucose derivatives from commercially
available UDP-glucose. However, while there is precedent for
the cross-coupling of unprotected 5-iodouracil nucleotides11 and
nucleosides,14 no examples have been reported for the successful
use of the corresponding 5-bromo derivatives as Suzuki–Miyaura
substrates.
The substrates required for the cross-coupling reaction, 5-
bromouridine 2 and 5-bromo UMP 4a, were prepared from uridine
via Kumar’s bromination method13 and an adaptation of the
original Yoshikawa protocol for the 5ꢀ-selective phosphorylation
of unprotected nucleosides (Scheme 1).15 The use of proton
sponge16 during this reaction helped accelerate the phosphoryla-
tion reaction, which is known to require prolonged reaction times
for uracil nucleosides,15 as well as suppress a potential bromine–
chlorine exchange reaction in position 5.16b
Na2Cl4Pd, the water-soluble phosphine ligand TPPTS‡14b and
K2CO3 as the base. Reactions were carried out at 60 C for 3 h,
◦
as we were concerned that elevated temperature and prolonged
reaction times may promote decomposition under the basic
conditions (e.g. by cleavage of the glycosidic bond). Under these
conditions, the cross-coupling of both nucleoside 2 and nucleotide
4a did afford the desired 5-aryl derivatives (Table 1). However,
none of the cross-coupling reactions went to completion, and the
cross-coupled products 3a–c and 5a–c were isolated in generally
low to moderate yields. The problem of unsatisfactory yields due
to incomplete conversion was further exacerbated by the loss
of material during purification of the polar reaction products
by chromatography. In order to improve the isolated yields, we
therefore set out to identify a more reactive catalytic system for
the cross-coupling of 5-bromo UMP 4a.
The cross-coupling of 4a with phenylboronic acid was chosen
as a model reaction for the optimisation of ligand and catalytic
base (Table 2). Neither an increase in the amount of K2CO3
(entries 1–3) nor use of a stronger base (entries 4–6) improved
cross-coupling efficiency. However, replacement of K2CO3 with
Cs2CO3 resultedinasubstantiallyhigher yieldof5-phenyl UMP 5a
(entry 7). No decomposition or side reactions were observed under
these conditions, and any loss of material occurred only during
purification by ion-pair chromatography. The use of additional
equivalents of Cs2CO3 gave a reduced isolated yield (entry 8), as
previously observed for K2CO3 and NaOH. This finding may be
due to the deprotonation of the 5-bromouracil ring in position 3 in
the presence of a large excess of base, deactivating the heterocycle
for the cross-coupling reaction and/or facilitating coordination of
the palladium catalyst.14b The superiority of Cs2CO3 over K2CO3
in the cross-coupling of 5-bromo UMP appears to be independent
Table 2 Optimisation of conditions for the Suzuki–Miyaura coupling of
5-bromo UMP 4a with phenylboronic acida
Entry
Ligandb
Base
Yieldc (%)
1
2
3
4
5
6
7
8
9
TPPTS
TPPTS
TPPTS
TPPTS
TPPTS
TPPTS
TPPTS
TPPTS
K2CO3 (2 eq.)
K2CO3 (5 eq.)
K2CO3 (10 eq.)
NaOH (2 eq.)
NaOH (5 eq.)
NaOH (10 eq.)
Cs2CO3 (2 eq.)
Cs2CO3 (10 eq.)
Cs2CO3 (2 eq.)
Cs2CO3 (2 eq.)
57
23
0
0
17
0
71
18
53
34
Scheme 1 Reagents and conditions: i) NBS, NaN3, 1,2-DME–H2O, rt,
5 h; ii) Na2Cl4Pd (1 mol%), TPPTS (2.5 mol%), R-B(OH)2, K2CO3, H2O,
60 ◦C, 3 h; iii) POCl3, proton sponge, MeCN, −5 ◦C, 4 h. For substituents
R and individual yields see Table 1.
Buchwald ligand
Kirschning ligand
10
For the Suzuki–Miyaura cross-coupling of 5-bromouracil nu-
cleosides and nucleotides, we initially investigated the suitability
of a catalytic system composed of the water-soluble Pd source
a See Scheme 1 for structures; other conditions: Na2Cl4Pd (1 mol%), H2O,
60 ◦C, 2 h. b 2.5 mol%. c Isolated yields.
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©

Table 3 Suzuki–Miyaura coupling of 4b, 6a, and 6b with various
from the nature of the leaving group, as similar observations have
previously been reported for the cross-coupling of 5-iodouridine
nucleotides.11b
a
arylboronic acids R-B(OH)2
Entry
Substrate
Product
R
Yieldb (%)
Next, we investigated the influence of alternative ligands, in
combination with Na2Cl4Pd as the Pd source, on cross-coupling ef-
ficiency. Neither a water-soluble, phosphine-based ligand recently
introduced by the Buchwald group for the cross-coupling of aryl
chlorides and sterically hindered aryl bromides in water,17a nor a
water-insoluble, 4-pyridine-aldoxime-based ligand developed by
Kirschning and coworkers17b offered any advantage over TPPTS
(Table 2, entries 9 and 10). From these reactions, 5a was isolated
in slightly or clearly lower yield.
1
2
3
4
5
6
7
6a
6b
6b
6b
6b
4b
4b
7a
7a
7b
7c
7d
5a
5b
Phenyl
Phenyl
4-Methoxyphenyl
4-Chlorophenyl
2-Furyl
Phenyl
4-Methylphenyl
0
64
58
57
40
71
66
a See Scheme 2 for structures. b Isolated yields.
Following optimisation of the catalytic system, we required
5-bromo UDP-glucose 6a as the sugar-nucleotide substrate for
the Suzuki–Miyaura cross-coupling (Scheme 2). We envisaged to
prepare 6a from the corresponding nucleoside monophosphate
4a and glucose-1-phosphate. This approach involves activation
of the phosphate group in 4a in the form of e.g. the phos-
phoromorpholidate, followed by acid-catalysed formation of the
pyrophosphate bond. Previously, we have successfully used MnCl2
as a catalyst for this type of transformation in the synthesis of
GDP-sugars.12 However, while the conversion of nucleotide 4a
into its morpholidate proceeded smoothly, all attempts to react this
morpholidate under MnCl2-catalysis with glucose-1-phosphate to
UDP-sugar 6a failed. We finally resorted to activating nucleotide
4a with the classical carbonyldiimidazole method.18 Thus, 4a was
converted into the corresponding imidazolide and reacted in situ
with glucose-1-phosphate, to provide sugar-nucleotide 6a in 38%
yield.
With both the optimised catalytic system and the required
substrate for the cross-coupling in hand, we attempted the direct
Suzuki–Miyaura reaction of UDP-sugar 6a with phenylboronic
acid (Scheme 2). Surprisingly, this reaction was unsuccessful under
our optimised conditions (Table 3, entry 1). No cross-◦coupling
product could be detected by TLC after 4 hours at 50 C when
decomposition of the sugar-nucleotide set in and the reaction was
stopped. The lack of cross-coupling reactivity of sugar-nucleotide
6a was particularly intriguing as the corresponding 5-bromouracil
nucleoside 2 and nucleotide 4a had both proved to be sufficiently
reactive under these conditions.
bromide analogues.19 Faced with the non-reactivity of 6a, we
therefore selected 5-iodo UDP-glucose 6b as an alternative cross-
coupling substrate, in order to gain access to the target 5-
(hetero)aryl UDP-glucose derivatives (Scheme 2). In analogy to
the synthesis of 6a, 6b was prepared from the corresponding
nucleoside monophosphate 4b and glucose-1-phosphate using the
carbonyldiimidazole method. Gratifyingly, and in contrast to the
unsuccessful cross-coupling of 6a, the reaction of 5-iodo UDP-
glucose 6b with phenylboronic acid proceeded smoothly to afford
5-phenyl UDP-glucose 6a in 64% yield after 1 h (Table 3, entry 2).
To determine the scope of the reaction conditions, we then syn-
thesised a small family of UDP-glucose derivatives with different
aromatic and heteroaromatic substituents at position 5 (Table 3,
entries 3–5). The cross-coupling reactions were successful with
both electron-rich and electron-deficient (hetero)aromatic boronic
acids, and the desired 5-substituted UDP-glucose derivatives 7b–d
were obtained in 40–58% isolated yield.
At this point, we decided to carry out further experiments
in order to better understand the differential cross-coupling
reactivity of sugar-nucleotides 6a and 6b. It seemed to us that
in the light of the successful cross-couplings with 5-bromouracil
derivatives 2 and 4a, the lack of reactivity observed for 6a could
not be explained by the potentially limited leaving group quality
of the bromo substituent alone. Therefore, 5-iodo UMP 4b was
prepared from 5-iodouridine and reacted with phenylboronic acid,
for direct comparison both with its 5-bromo congener 4a and with
the corresponding sugar-nucleotide 6b (Table 3, entry 6). From
this reaction, 5-phenyl UMP 5a was obtained in 71% yield after
3 h, similar to the cross-coupling results obtained with both 4a
It is well known that (hetero)aryl iodides are generally more
reactive in cross-coupling reactions than the corresponding
Scheme 2 Reagents and conditions: i) carbonyldiimidazole, DMF, rt, 3 h; ii) MeOH; iii) glucose-1-phosphate, DMF, rt, 24 h; iv) Na2Cl4Pd (2.5 mol%),
TPPTS (6.25 mol%), Cs2CO3, R-B(OH)2, H2O, 50 ◦C, 1 h. For substituents R and individual yields see Table 3.
2886 | Org. Biomol. Chem., 2008, 6, 2884–2891
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Table 4 Fluorescence spectroscopic properties of UDP-Glc derivatives
and 6b. The similar cross-coupling behaviour of 5-bromo UMP
4a, 5-iodo UMP 4b, and 5-iodo UDP-glucose 6b suggests that
neither a bromo leaving group, nor the sugar-nucleotide nature of
the substrate are per se an obstacle to cross-coupling success.
It is known that the outcome of Suzuki–Miyaura reactions is
strongly influenced by steric parameters, e.g. the accessibility of the
substrate for the palladium–ligand catalytic complex.19a In order to
investigate the possibility that 6a, but not 6b, may in solution adopt
a conformation which restricts access of the palladium–ligand
catalytic complex, we carried out a comparative conformational
analysis of both sugar-nucleotides. In NOESY experiments with
6a, NOE contacts were observed between protons H-6 and
protons H-3ꢀ (major NOE), H-1ꢀ and H-5ꢀ (Fig. 2). The close
spatial proximity between H-6 and H-5ꢀ strongly suggests a global
conformation for this sugar-nucleotide in which the uracil base
is oriented anti to the ribose ring, similar to the conformation of
the 5-unsubstituted parent UDP-glucose.20 In contrast, NOESY
results for 6b indicate that in solution, the 5-iodo congener
exists predominantly in the syn conformation. For 6b, a strong
NOE signal was observed between protons H-6 and H-2ꢀ and a
weaker one between H-6 and H-1ꢀ, while no NOE contact was
seen between H-6 and H-3ꢀ or H-5ꢀ. In the syn conformation,
the 5-iodo substituent in 6b is pointing away from the glucose
diphosphate group, and the 5 position is easily accessible for
the Pd catalyst. Such easy access may not be granted in the anti
conformation preferred by the 5-bromo analogue 6a, where the 5-
bromo substituent is facing towards the bulky glucose diphosphate
moiety. Thus, the superior cross-coupling reactivity of 6b may
be attributed, at least in part, to the capacity of the bulky iodo
substituent in 6b to induce a conformation which is favourable
for the insertion of the reactive Pd species during the oxidative
addition step.
Compound
kex/nm
kem/nm
Stokes shifta/cm−1
6b
7a
7b
7c
7d
287
278
278
281
314
377
403
444
398
437
8318
11157
13449
10462
8963
a Stokes shift = (1/kex − 1/kem).
almost non-fluorescent, the 5-(hetero)aryl-substituted derivatives
are weakly (7a and 7c) or strongly (7b and 7d) fluorescent.
Among the three phenyl-substituted derivatives, the electron-
rich 4-methoxy analogue 7b stands out with its relatively large
Stokes shift and strong fluorescence emission. Only 7d, whose
furan substituent is known as a strong fluorescence emitter7 but
more difficult to install than a phenyl substituent (cf. Table 3 for
yields), can compare with 7b in terms of fluorescence intensity.
Of the new UDP-glucose derivatives presented herein, 7b and
7d may therefore be particularly useful as biological sensors
for carbohydrate-active enzymes, and investigations into their
biological activity are currently under way.
Fig. 3 Fluorescence emission spectra for UDP-glucose derivatives 6b and
7a–d in H2O (c = 100 lM in H2O).
Fig. 2 Diagnostic NOE interactions, suggesting an anti conformation for
6a, and a syn conformation for 6b (R = glucosyl-1-diphosphate).
Conclusions
In this study, we have shown that installation of an aromatic
or heteroaromatic substituent at position 5 of UDP-glucose
yields a novel type of fluorescent analogue, whose fluorescent
properties can be modulated by the nature of the fluorogenic
substituent. We have developed an efficient synthetic route towards
the target molecules via Suzuki–Miyaura cross-coupling of 5-iodo
UDP-glucose 6b, which may be applicable to the preparation of
other fluorescent UDP-sugars also. Interestingly, under otherwise
identical conditions the cross-coupling reaction was unsuccessful
with 5-bromo UDP-glucose 6a as the substrate. Results from
NOESY experiments suggest that the differential cross-coupling
reactivity of 5-bromo UDP-glucose 6a and its 5-iodo congener 6b
is due at leastinparttoconformationaldifferences betweenthetwo
sugar-nucleotides in solution. Investigations into the biological
properties of the novel UDP-sugars described herein are currently
While 5-substituted uracil nucleosides and nucleotides have
been used previously as fluorescent probes,7 no fluorescent
sugar-nucleotides of this type have so far been reported. In
order to assess the suitability of the new 5-substituted UDP-
glucose derivatives as fluorescent sensors, we have recorded UV
absorbance and fluorescence spectra for 6b and 7a–d. The UV
absorbance spectra of all 5-substituted UDP-glucose analogues
show a low energy absorbance band, which lies at ∼280 nm
for the 5-phenyl derivatives (7a–c), at 287 nm for 5-iodo UDP-
glucose 6b, and at 314 nm for 5-(2-furyl) UDP-glucose 7d.† In
aqueous solution, all analogues were fluorescence emissive upon
excitation at the absorbance maximum (Fig. 3). As expected, their
fluorescent properties are strongly influenced by the nature of the
substituent in position 5 (Table 4). While 5-iodo UDP-glucose 6b is
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©

under way, and results from these studies will be reported in due
course.
gave 5-bromouridine 2 as white crystals in 80% yield (2.11 g). dH
(400 MHz, DMSO-d6) 3.49–3.71 (2H, m, H-5ꢀ), 3.82–3.86 (1H, m,
H-4ꢀ), 3.92 (1H, t, J = 5.6 Hz, H-3ꢀ), 4.03 (1H, dd, J = 3.6 and
5.2 Hz, H-2ꢀ), 5.05 (1H, d, J = 5.1 Hz, OH-3ꢀ), 5.26 (1H, t, J =
4.6 Hz, OH-5ꢀ), 5.41 (1H, d, J = 4.2 Hz, OH-2ꢀ), 5.70 (1H, d, J =
3.9 Hz, H-1ꢀ), 8.46 (1H, s, H-6), 11.80 (1H, s, NH); dC (75.5 MHz,
DMSO-d6) 60.2 (C-5ꢀ), 69.3 (C-3ꢀ), 74.0 (C-2ꢀ), 84.8 (C-4ꢀ), 88.6
(C-1ꢀ), 95.9 (C-5), 140.4 (C-6), 150.3 (C-2), 159.5 (C-4). m/z (ESI)
322.9872 [M + H]+, C9H12O6N2Br79 requires 322.9873.
Experimental
General
All chemicals were obtained commercially and used as received
unless stated otherwise. TLC was performed on precoated alu-
minium plates (Silica Gel 60 F254, Merck). Compounds were
visualized by exposure to UV light. NMR spectra were recorded
at 298 K on a Varian VXR 400 S spectrometer or on a Bruker
Avance DPX-300 spectrometer. Chemical shifts (d) are reported
in ppm and, for solutions in D2O, are referenced to methanol
(dH 3.34, dC 49.50). Coupling constants (J) are reported in Hz.
Resonance allocations were made with the aid of COSY and
HSQC experiments. NOESY measurements were carried out with
a relaxation delay of 1.000 s and a mixing time of 0.200 s. High
resolution electrospray ionisation mass spectra (HR ESI-MS) were
obtained on a Finnigan MAT 900 XLT mass spectrometer at the
EPSRC National Mass Spectrometry Service Centre, Swansea.
UV absorbance measurements were carried out on a Perkin
Elmer Lambda 25 UV/VIS spectrometer. Fluorescence spectra
were recorded on a PerkinElmer LS-45 spectrometer at ambient
temperature. Samples were measured in aqueous solution at a
concentration of 100 lM in a quartz micro fluorescence cell (path
length 1.0 cm). Preparative chromatography was performed on a
Biologic LP chromatography system equipped with a peristaltic
pump and a 254 nm UV Optics Module under the following
conditions.
General method A: Suzuki–Miyaura cross-coupling of
5-bromouridine
A 2-necked round bottom flask with 5-bromouridine 2 (1 eq.),
arylboronic acid (1.5 eq.) and K2CO3 (2 eq.) was purged with N2.
Upon addition of TPPTS (0.025 eq.), Na2Cl4Pd (0.01 eq.) and
degassed H2O (5 mL) the mixture turned orange in colour. The
reaction was stirred under N2 for 3 h at 60 ◦C, turning brown–
black. Once the starting material was fully consumed, the reaction
mixture was cooled to room temperature. The pH was adjusted
to 7 with HCl 1% and the reaction mixture was filtered through
Celite. The solvents were removed to give a white powder, which
was purified by column chromatography (silica; gradient of 0–15%
methanol in chloroform).
5-Phenyluridine (3a). The title compound was prepared from
2 (50 mg, 155 lmol) and phenylboronic acid according to general
method A. After recrystallisation from absolute ethanol, 3a
was obtained in colourless crystals in 45% yield (22.4 mg). dH
(400 MHz, DMSO-d6) 3.52–3.68 (2H, m, H-5ꢀ), 3.84–3.88 (1H,
m, H-4ꢀ), 3.97–4.14 (2H, m, H-2ꢀ, H-3ꢀ), 5.07 (1H, d, J = 4.8 Hz,
OH-3ꢀ), 5.20 (1H, t, J = 4.5 Hz, OH-5ꢀ), 5.41 (1H, d, J = 5.4 Hz,
OH-2ꢀ), 5.82 (1H, d, J = 3.0 Hz, H-1ꢀ), 7.24–7.56 (5H, m, Ph), 8.25
(1H, s, H-6), 11.5 (1H, br s, NH); dC (75.5 MHz, DMSO-d6) 60.5
(C-5ꢀ), 69.3 (C-3ꢀ), 74.7 (C-2ꢀ), 84.6 (C-4ꢀ), 88.2 (C-1ꢀ), 108.3 (C-5),
127.7, 127.9, 128.0 (Ph), 137.6 (C-6), 147.2 (C-2), 162.0 (C-4). m/z
(ESI) 321.1081 [M + H]+, C15H17O6N2 requires 321.1087.
Purification method 1. Ion-pair chromatography was per-
formed using Lichroprep RP-18 resin, gradient 0–10% MeCN
against 0.05 M TEAB (triethylammonium bicarbonate) over
480 mL, flow rate 5 mL min−1. Product-containing fractions were
combined and reduced to dryness. The residue was co-evaporated
repeatedly with methanol to remove residual TEAB.
Purification method 2. Anion exchange chromatography was
performed using a Macro prep 25Q resin, gradient 0–100% 1 M
TEAB (pH 7.3) against H2O over 480 mL, flow rate 5 mL min−1.
Product-containing fractions were combined and reduced to
dryness. The residue was co-evaporated repeatedly with methanol
to remove residual TEAB.
5-(4-Methylphenyl)uridine (3b). The title compound was ob-
tained from 2 (50 mg, 155 lmol) and 4-methylphenylboronic
acid according to general method A in 46% yield (24.0 mg). dH
(400 MHz, DMSO-d6) 2.30 (3H, s, Me), 3.50–3.72 (2H, m, H-5ꢀ),
3.82–3.90 (1H, m, H-4ꢀ), 3.96–4.05 (1H, m, H-3ꢀ), 4.07–4.15 (1H,
m, H-2ꢀ), 5.10 (1H, s, OH-3ꢀ), 5.22 (1H, s, OH-5ꢀ), 5.44 (1H, s,
OH-2ꢀ), 5.86 (1H, d, J = 4.5 Hz, H-1ꢀ), 7.17 (2H, d, J = 6.6 Hz,
mPh), 7.43 (2H, d, J = 8.4 Hz, oPh), 8.24 (1H, s, H-6ꢀ), 11.42
(1H, br s, NH). dC (75.5 MHz, DMSO-d6) 20.6 (Me), 60.3 (C-5ꢀ),
69.6 (C-3ꢀ), 73.9 (C-2ꢀ), 84.7 (C-4ꢀ), 88.2 (C-1ꢀ), 113.4 (C-5), 127.8,
128.7, 130.3, 136.5 (Ph), 137.7 (C-6), 150.3 (C-2), 162.3 (C-4). m/z
(ESI) 335.1236 [M + H]+, C16H19O6N2 requires 335.1238).
5-Bromouridine (2)
N-Bromosuccinimide (1.6 g, 9.0 mmol) was added to a stirred
suspension of uridine 1 (2.0 g, 8.2 mmol) in 1,2-dimethoxyethane
(100 mL) at 25 ◦C. The mixture was stirred for 5 minutes. To
the clear solution was added a solution of sodium azide (2.15 g,
32 mmol) in water (6.0 mL). The reaction turned bright yellow,
accompanied by the separation of a colourless oil. The yellow
colour disappeared slowly while the oily material remained. After
stirring for 2 h, TLC (30% methanol in chloroform, Rf 0.62;
RfSM 0.45) showed complete conversion of starting material. The
solvents were removed under vacuum to give a pale yellow,
amorphous solid. The crude material was purified by column
chromatography (silica; 15% methanol in CHCl3). Initially, a
colourless oil was obtained which was dried under vacuum
overnight to give a white solid. Recrystallisation from dry ethanol
5-(4-Chlorophenyl)uridine (3c). The title compound was ob-
tained from 2 (20 mg, 62 lmol) and 4-chlorophenylboronic
acid according to general method A in 43% yield (9.4 mg). dH
(400 MHz, DMSO-d6) 3.50–3.75 (2H, m, H-5ꢀ), 3.84–3.91 (1H,
m, H-4ꢀ), 4.00–4.07 (1H, m, H-3ꢀ), 4.08–4.18 (1H, m, H-2ꢀ), 5.10
(1H, d, J = 3.9 Hz, OH-3ꢀ), 5.26 (1H, s, OH-5ꢀ), 5.46 (1H, d, J =
4.8 Hz, OH-2ꢀ), 5.82 (1H, d, J = 4.5 Hz, H-1ꢀ), 7.36–7.62 (4H, 2d,
J = 8.4 and 8.7 Hz, Ph), 8.38 (1H, s, H-6ꢀ), 11.6 (1H, br s, NH);
dC (75.5 MHz, DMSO-d6) 60.1 (C-5ꢀ), 69.3 (C-3ꢀ), 74.0 (C-2ꢀ), 84.6
(C-4ꢀ), 88.5 (C-1ꢀ), 112.1 (C-5), 128.2, 129.6, 131.8, 132.1 (Ph),
2888 | Org. Biomol. Chem., 2008, 6, 2884–2891
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The Royal Society of Chemistry 2008
©

138.6 (C-6), 150.2 (C-2), 162.1 (C-4). m/z (ESI) 355.0693 [M +
117.2 (C-5), 128.2, 129.4, 131.7 (Ph), 139.6 (C-6), 152.7 (C-2),
165.8 (C-4); dP (121.5 MHz, D2O) 7.6. m/z (ESI) 399.0593 [M −
H]−, C15H16O9N2P requires 399.0599.
H]+, C15H16O6N2Cl35 requires 355.0691.
5-Bromouridine-5ꢀ-monophosphate (4a)
5-(4-Methylphenyl)uridine-5ꢀ-monophosphate (5b). The tri-
ethylammonium salt of the title compound was obtained as a
glassy solid in 27% yield (5.5 mg) from 3a (20 mg, 49.6 lmol)
and 4-methylphenylboronic acid according to general method B.
dH (400 MHz, D2O) 2.35 (3H, s, Me), 4.04–4.09 (2H, m, H-5ꢀ),
4.24–4.28 (1H, m, H-4ꢀ), 4.32 (1H, t, J = 4.6 Hz, H-3ꢀ), 4.44 (1H,
t, J = 4.5 Hz, H-2ꢀ), 6.00 (1H, d, J = 4.5 Hz, H-1ꢀ), 7.29 (2H, d,
J = 6.0, Ph), 7.39 (2H, d, J = 6.0 Hz, Ph), 7.80 (1H, s, H-6); dC
(75.5 MHz, D2O) 20.9 (Me), 65.0 (C-5ꢀ), 70.8 (C-3ꢀ), 74.1 (C-2ꢀ),
84.4 (C-4ꢀ), 89.4 (C-1ꢀ), 116.8 (C-5), 129.4, 130.1, 138.8 (Ph), 139.5
(C-6), 154.1 (C-2), 165.5 (C-4); dP (121.5 MHz, D2O) 7.6. m/z
(ESI) 413.0753 [M − H]−, C16H18O9N2P requires 413.0755.
5-(4-Chlorophenyl)uridine-5ꢀ-monophosphate (5c). The triethy-
lammonium salt of the title compound was obtained as a glassy
solid in 26% yield (5.5 mg) from 3a (20 mg, 49.6 lmol) and
4-chlorophenylboronic acid according to general method B. dH
(400 MHz, D2O) 3.97–4.03 (2H, m, H-5ꢀ), 4.20–4.25 (1H, m, H-
4ꢀ), 4.29 (1H, t, J = 4.7 Hz, H-3ꢀ), 4.43 (1H, t, J = 5.4 Hz, H-2ꢀ),
5.99 (1H, d, J = 5.7 Hz, H-1ꢀ), 7.40–7.55 (4H, m, Ph), 7.86 (1H, s,
H-6); dC (75.5 MHz, D2O), 65.0 (C-5ꢀ), 70.9 (C-3ꢀ), 74.3 (C-2ꢀ), 82.5
(s, C-4ꢀ), 89.6 (C-1ꢀ), 101.6 (C-5), 129.6, 129.9, 131.2 (Ph), 139.6
(C-6), 152.4 (C-2), 165.8 (C-4); dP (121.5 MHz, D2O) 7.6. m/z
(ESI) 433.0210 [M − H]−, C15H15O9N2Cl35P requires 433.0209.
A suspension of 2 (50 mg, 0.15 mmol) and proton sponge (199 mg,
0.9 mmol) in dry acetonitrile (3 mL) was cooled to −5 ◦C, and
POCl3 (58 ll, 0.6 mmol) was added dropwise. The orange-coloured
reaction was stirred at −5 ◦C for 4 h, at which time TLC indicated
near complete conversion (IPA–H2O–NH3 6 : 3 : 1; Rf 0.31; RfSM
0.71). The reaction was quenched with 50 mL of ice cold 0.2 M
TEAB buffer. The pale orange solution was stirred for 1 h at
0 ◦C. After warming to 25 ◦C, the aqueous layer was washed with
ethyl acetate (3×) and concentrated under reduced pressure. The
crude residue was purified sequentially by purification methods
1 and 2 to provide 4a in its triethylammonium salt form as a
colourless, glassy solid in 50% yield (31.2 mg). dH (400 MHz, D2O)
4.03–4.16 (2H, m, H-5ꢀ), 4.23–4.27 (1H, m, H-4ꢀ), 4.30 (1H, t, J =
4.8, H-3ꢀ), 4.34 (1H, t, J = 5, H-2ꢀ), 5.93 (1H, d, J = 4.8 Hz, H-1ꢀ),
8.24 (1H, s, H-6); dC (75.5 MHz, D2O) 64.9 (C-5ꢀ), 70.3 (C-3ꢀ), 74.7
(C-2ꢀ), 84.2 (C-4ꢀ), 89.7 (C-1ꢀ), 97.7 (C-5), 141.3 (C-6), 152.0 (C-2),
162.7 (C-4); dP (121.5 MHz, D2O) 7.5. m/z (ESI) 400.9391 [M −
H]−, C9H11O9N2Br79P requires 400.9393.
5-Iodouridine-5ꢀ-monophosphate (4b)
The title compound was prepared from 5-iodouridine21 (480 mg,
1.3 mmol) by the procedure used in the synthesis of 4a. The
triethylammonium salt of 4b was obtained as a colourless, glassy
solid in 53% yield (307 mg). dH (400 MHz, D2O) 3.96–4.10 (2H,
m, H-5ꢀ), 4.20–4.27 (1H, m, H-4ꢀ), 4.31 (1H, t, J = 3.9 Hz, H-3ꢀ),
4.38 (1H, t, J = 5.1 Hz, H-2ꢀ), 5.92 (1H, d, J = 5.1 Hz, H-1ꢀ),
8.27 (1H, s, H-6); dC (75.5 MHz, D2O) 64.8 (C-5ꢀ), 69.6 (C-5), 70.8
(C-3ꢀ), 74.6 (s, C-2ꢀ), 84.8 (d, J = 9.1 Hz, C-4ꢀ), 89.6 (C-1ꢀ), 147.1
(C-6), 152.8 (C-2), 164.3 (C-4); dP (121.5 MHz, D2O) 7.6. m/z
(ESI) 448.9255 [M − H]−, C9H11O9N2IP requires 448.9252.
5-Bromouridine-5ꢀ-diphosphate-a-D-glucose (6a)
4a (43 mg, 0.11 mmol) was coevaporated with dry DMF (3×).
Carbonyldiimidazole (35 mg, 0.21 mmol) was added and the flask
was purged with N2. Dry DMF (2 mL) was added, and the mixture
was stirred at room temperature for 1 h. Once the formation of the
phosphoroimidazolide was complete dry methanol (350 lL) was
added to destroy excess carbonyldiimidazole. The reaction was
stirred for 20 min, and the solvents were removed under reduced
pressure. a-D-Glucose monophosphate (triethylammonium salt;
51 mg, 0.13 mmol) was coevaporated with DMF (3×) and
dissolved in dry DMF (2 mL). Under N2, this solution was
added to the 4a-imidazolide, and the reaction was stirred at room
temperature for 24 h. Once TLC indicated complete consumption
of the 4a-imidazolide (IPA–H2O–NH3 6 : 3 : 1; Rf 0.40; RfSM
0.37; Rfimidazolide 0.71) all solvents were removed under reduced
pressure, and the crude residue was purified sequentially by
purification methods 1 and 2. The title compound was obtained
in its triethylammonium salt form as a colourless, glassy solid in
38% yield (26 mg). dH (400 MHz, D2O) 3.40–3.46 (1H, m, H-4ꢀꢀ),
3.46–3.56 (1H, m, H-2ꢀꢀ), 3.57–3.78 (4H, m, H-3ꢀꢀ, H-5ꢀꢀ and H-
6ꢀꢀ), 4.20–4.26 (1H, m, H-5ꢀ), 4.26–4.30 (1H, m, H-4ꢀ), 4.34–4.40
(2H, m, H-2ꢀ, H-3ꢀ), 5.43 (1H, dd, J = 3.6 and 9.6 Hz, H-1ꢀꢀ), 5.78
(1H, d, J = 4.4 Hz, H-1ꢀ), 8.06 (1H, s, H-6); dC (75.5 MHz, D2O)
61.2 (C-6ꢀꢀ), 65.9 (C-5ꢀ), 70.1 (C-4ꢀꢀ), 70.4 (C-3ꢀ), 73.4 (C-2ꢀꢀ), 73.7,
73.8 (C-3ꢀꢀ/C-5ꢀꢀ), 74.7 (C-2ꢀ), 84.2 (C-4ꢀ), 89.8 (C-1ꢀ), 96.6 (C-1ꢀꢀ),
118.6 (C-5), 141.8 (C-6), 152.3 (C-2), 163.1 (C-4); dP (121.5 MHz,
D2O) −9.07 (d, J = 20.0 Hz), −7.57 (d, J = 22.2 Hz). m/z (ESI)
642.9586 [M − H]−, C15H22O17N2Br79P2 requires 642.9583.
General method B: Suzuki–Miyaura cross-coupling of 5-bromo
UMP
A 2-necked round bottom flask with 5-bromouridine-5ꢀ-mono-
phosphate 4a (1 eq.), K2CO3 (2 eq.) and arylboronic acid (1.5 eq.)
was purged with N2. TPPTS (0.025 eq.), Na2Cl4Pd (0.01 eq.) and
degassed H2O (4 mL) were added, and the reaction was stirred
under N2 for 3 h at 60 ◦C. The reaction was cooled to room
temperature, and the pH was adjusted to 7 with 1% HCl. The black
suspension was concentrated under reduced pressure, and the
residue was taken up in MeOH. The methanolic suspension was
filtered through Celite, and the residue was washed with methanol.
The combined filtrates were evaporated under reduced pressure,
and the residue was purified using purification method 1.
5-Phenyluridine-5ꢀ-monophosphate (5a). The triethylammo-
nium salt of the title compound was obtained as a glassy solid in
57% yield (11.5 mg) from 3a (20 mg, 49.6 lmol) and phenylboronic
acid according to general method B. dH (400 MHz, D2O) 4.07–4.11
(2H, m, H-5ꢀ), 4.26–4.30 (1H, m, H-4ꢀ), 4.23 (1H, t, J = 4.8 Hz,
H-3ꢀ), 4.45 (1H, t, J = 6.0 Hz, H-2ꢀ), 6.04 (1H, d, J = 5.6 Hz,
H-1ꢀ), 7.42–7.54 (5H, m, Ph), 7.89 (1H, s, H-6); dC (75.5 MHz,
D2O) 65.1 (C-5ꢀ), 71.2 (C-3ꢀ), 74.6 (C-2ꢀ), 85.8 (C-4ꢀ), 89.8 (C-1ꢀ),
5-Iodouridine-5ꢀ-diphosphate-a-D-glucose (6b). The title com-
pound was prepared from 4b (137 mg, 0.30 mmol) by the procedure
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 2884–2891 | 2889
©

used in the synthesis of 6a. The triethylammonium salt of 6b was
obtained as a colourless, glassy solid in 37% yield (78 mg). dH
(400 MHz, D2O) 3.40–3.58 (2H, m, H-2ꢀꢀ and H-4ꢀꢀ), 3.75–3.92
(4H, m, H-3ꢀꢀ, H-5ꢀꢀ and H-6ꢀꢀ), 4.18–4.25 (2H, m, H-5ꢀ), 4.25–4.29
(1H, m, H-4ꢀ), 4.32–4.42 (2H, m, H-2ꢀ, H-3ꢀ), 5.61 (1H, dd, J = 3.6
and 9.6 Hz, H-1ꢀꢀ), 5.93 (1H, d, J = 4.4 Hz, H-1ꢀ), 8.25 (1H, s, H-6);
dC (75.5 MHz, D2O) 59.9 (C-5), 61.3 (C-6ꢀꢀ), 66.0 (d, J = 3.8 Hz,
C-5ꢀ), 70.2 (C-4ꢀꢀ), 70.6 (C-3ꢀ), 72.6 (C-2ꢀꢀ), 73.7, 73.8 (C-3ꢀꢀ/C-5ꢀꢀ),
74.7 (C-2ꢀ), 84.3 (d, J = 9.1 Hz, C-4ꢀ), 89.7 (C-1ꢀ), 96.6 (d, J =
6.8 Hz, C-1ꢀꢀ), 147.0 (C-6), 152.8 (C-2), 164.4 (C-4); dP (121.5 MHz,
D2O) −9.21 (d, J = 20.9 Hz), −7.70 (d, J = 21.1 Hz). m/z (ESI)
690.9431 [M − H]−, C15H22O17N2IP2 requires 690.9444.
(d, J = 21.9 Hz). m/z (ESI) 671.0909 [M − H]−, C22H29O18N2P2
requires 671.0896.
5-(4-Chlorophenyl)uridine-5ꢀ-diphosphate-a-D-glucose
(7c).
The triethylammonium salt of the title compound (TLC: IPA–
H2O–NH3 6 : 3 : 1; Rf 0.58, RfSM 0.37) was obtained as a glassy
solid in 57% yield (14.9 mg) from 6b (20 mg, 29 lmol) and
4-chlorophenylboronic acid according to general method C. dH
(400 MHz, D2O) 3.39–3.44 (1H, m, H-4ꢀꢀ), 3.42–3.47 (1H, m,
H-2ꢀꢀ), 3.71–3.92 (4H, m, H-3ꢀꢀ, H-5ꢀꢀ and H-6ꢀꢀ), 4.17–4.22 (2H,
m, H-5ꢀ), 4.28–4.32 (1H, m, H-4ꢀ), 4.39 (1H, t, J = 4.4 Hz, H-3ꢀ),
4.47 (1H, t, J = 5.6 Hz, H-2ꢀ), 5.54 (1H, dd, J = 2.8 and 5.2 Hz,
H-1ꢀꢀ), 6.03 (1H, d, J = 6.0 Hz, H-1ꢀ), 7.45–7.58 (4H, m, Ph), 7.90
(1H, s, H-6); dC (75.5 MHz, D2O) 61.1 (C-6ꢀꢀ), 65.9 (C-5ꢀ), 69.9
(C-4ꢀꢀ), 70.6 (C-3ꢀ), 72.3 (C-2ꢀꢀ), 73.4, 73.6 (C-3ꢀꢀ/C-5ꢀꢀ), 74.1 (C-2ꢀ),
84.3 (C-4ꢀ), 89.3 (C-1ꢀ), 96.3 (C-1ꢀꢀ), 115.9 (C-5), 129.5, 130.9,
134.4 (Ph), 139.3 (C-6), 152.3 (C-2), 165.4 (C-4); dP (121.5 MHz,
D2O) −9.27 (d, J = 20.2 Hz), −7.68 (d, J = 21.8 Hz). m/z (ESI)
675.0402 [M − H]−, C21H26O17N2Cl35P2 requires 675.0401.
5-(2-Furyl)uridine-5ꢀ-diphosphate-a-D-glucose (7d). The tri-
ethylammonium salt of the title compound (TLC: IPA–H2O–NH3
6 : 3 : 1; Rf 0.58, RfSM 0.37) was obtained as a glassy solid in 40%
yield (10.4 mg) from 6b (20 mg, 29 lmol) and 2-furylboronic acid
according to general method C. dH (400 MHz, D2O) 3.39–3.44
(1H, m, H-4ꢀꢀ), 3.42–3.47 (1H, m, H-2ꢀꢀ), 3.70–3.93 (4H, m, H-3ꢀꢀ,
H-5ꢀꢀ and H-6ꢀꢀ), 4.23–4.28 (2H, m, H-5ꢀ), 4.30–4.34 (1H, m, H-4ꢀ),
4.42 (1H, t, J = 4.6 Hz, H-3ꢀ), 4.49 (1H, t, J = 5.6 Hz, H-2ꢀ), 5.60
(1H, dd, J = 3.6 and 7.8 Hz, H-1ꢀꢀ), 6.05 (1H, d, J = 6.0 Hz, H-1ꢀ),
6.55 (1H, d, J = 3.2 Hz, H-3 fur), 6.90 (1H, d, J = 3.6 Hz, H-2
fur), 7.60 (1H, s, H-4 fur), 8.21 (1H, s, H-6); dC (75.5 MHz, D2O)
61.0 (C-6ꢀꢀ), 66.3 (C-5ꢀ), 69.9 (C-4ꢀꢀ), 70.6 (C-3ꢀ), 72.3 (C-2ꢀꢀ), 73.5,
73.6 (C-3ꢀꢀ/C-5ꢀꢀ), 74.3 (C-2ꢀ), 84.3 (C-4ꢀ), 89.4 (C-1ꢀ), 96.3 (C-1ꢀꢀ),
109.7 (C-2 fur), 112.2 (C-3 fur), 121.5 (C-5), 133.4 (C-6), 143.5
(C-4 fur), 146.1 (C-1 fur), 151.7 (C-2), 163.3 (C-4); dP (121.5 MHz,
D2O) −9.20 (d, J = 20.2 Hz), −7.62 (d, J = 20.1 Hz). m/z (ESI)
631.0586 [M-H]−, C19H25O18N2P2 requires 631.0583.
General method C: Suzuki–Miyaura cross-coupling of
5-iodouridine-5ꢀ-diphosphate-a-D-glucose
A 2-necked round bottom flask with 5-iodouridine-5ꢀ-diphos-
phate-a-D-glucose 6b (20 mg, 0.029 mmol), Cs2CO3 (19 mg,
0.058 mmol) and arylboronic acid (0.043 mmol) was purged with
N2. TPPTS (0.9 mg, 1.8 lmol), Na2Cl4Pd (0.2 mg, 0.7 lmol) and
degassed H2O (4 mL) were added, and the reaction was stirred
under N2 for 1 h at 50 ◦C. Upon completion, the reaction was
cooled to room temperature, and the pH was adjusted to 7 with
1% HCl. The black suspension was filtered through a membrane
filter (0.45 lm). The filter was washed with H2O, and the combined
filtrates were evaporated under reduced pressure. The residue was
purified sequentially by purification methods 2 and 1.
5-Phenyluridine-5ꢀ-diphosphate-a-D-glucose (7a). The triethy-
lammonium salt of the title compound (TLC: IPA–H2O–NH3 6 :
3 : 1; Rf 0.58, RfSM 0.37) was obtained as a glassy solid in 64%
yield (15.9 mg) from 6b (20 mg, 29 lmol) and phenylboronic acid
according to general method C. dH (400 MHz, D2O) 3.38–3.46
(2H, m, H-2ꢀꢀ and H-4ꢀꢀ), 3.71–3.92 (4H, m, H-3ꢀꢀ, H-5ꢀꢀ and H-6ꢀꢀ),
4.17–4.20 (2H, m, H-5ꢀ), 4.26–4.30 (1H, m, H-4ꢀ), 4.39 (1H, t, J =
4.6 Hz, H-3ꢀ), 4.47 (1H, t, J = 5.4 Hz, H-2ꢀ), 5.55 (1H, dd, J =
3.2 and 7.2 Hz, H-1ꢀꢀ), 6.03 (1H, d, J = 6.0 Hz, H-1ꢀ), 7.4–7.6 (5H,
m, Ph), 7.87 (1H, s, H-6); dC (75.5 MHz, D2O) 61.0 (C-6ꢀꢀ), 66.1
(C-5ꢀ), 69.9 (C-4ꢀꢀ), 70.6 (C-3ꢀ), 72.2 (C-2ꢀꢀ), 73.4, 73.6 (C-3ꢀꢀ/C-
5ꢀꢀ), 74.0 (C-2ꢀ), 84.2 (C-4ꢀ), 89.3 (C-1ꢀ), 96.3 (C-1ꢀꢀ), 117.0 (C-5),
129.2, 129.6, 132.4 (Ph), 139.3 (C-6), 152.4 (C-2), 165.6 (C-4); dP
(121.5 MHz, D2O) −9.26 (d, J = 22.0 Hz), −7.68 (d, J = 20.4 Hz).
m/z (ESI) 641.0793 [M − H]−, C21H27O17N2P2 requires 641.0790.
5-(4-Methoxyphenyl)uridine-5ꢀ-diphosphate-a-D-glucose (7b).
The triethylammonium salt of the title compound (TLC: IPA–
H2O–NH3 6 : 3 : 1; Rf 0.58, RfSM 0.37) was obtained as a glassy
solid in 58% yield (15.5 mg) from 6b (20 mg, 29 lmol) and
4-methoxyphenylboronic acid according to general method C.
dH (400 MHz, D2O) 3.38–3.43 (1H, m, H-4ꢀꢀ), 3.40–3.45 (1H, m,
H-2ꢀꢀ), 3.70–3.87 (4H, m, H-3ꢀꢀ, H-5ꢀꢀ and H-6ꢀꢀ), 3.86 (3H, s, Me),
4.16–4.21 (2H, m, H-5ꢀ), 4.27–4.31 (1H, m, H-4ꢀ), 4.39 (1H, t, J =
4.6 Hz, H-3ꢀ), 4.47 (1H, t, J = 5.6 Hz, H-2ꢀ), 5.54 (1H, dd, J =
3.2 and 7.2 Hz, H-1ꢀꢀ), 6.03 (1H, d, J = 6.0 Hz, H-1ꢀ), 7.07 (2H,
d, J = 8.8 Hz, mPh), 7.49 (2H, d, J = 8.8 Hz, oPh), 7.81 (1H, s,
H-6); dC (75.5 MHz, D2O) 56.0 (Me), 61.0 (C-6ꢀꢀ), 66.1 (C-5ꢀ), 69.9
(C-4ꢀꢀ), 70.6 (C-3ꢀ), 72.4 (C-2ꢀꢀ), 73.4, 73.6 (C-3ꢀꢀ/C-5ꢀꢀ), 74.0 (C-2ꢀ),
84.2 (C-4ꢀ), 89.31 (C-1ꢀ), 96.3 (C-1ꢀꢀ), 115.0 (mPh), 116.6 (C-5),
125.2 (iPh), 130.9 (oPh), 138.5 (C-6), 152.4 (C-2), 159.7 (pPh),
165.6 (C-4); dP (121.5 MHz, D2O) −9.25 (d, J = 20.2 Hz), −7.62
Acknowledgements
Financial support by the EPSRC (First Grant EP/D059186/1)
and by the Royal Society (Research Grant Scheme) is gratefully
acknowledged. We thank the EPSRC National Mass Spectrometry
Service Centre, Swansea, for the recording of mass spectra, and
Colin MacDonald and Dr Joseph Wright for assistance with NMR
experiments.
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