Synthesis and evaluation of mimetics of UDP and UDP-α-D-galactose, dTDP and dTDP-α-D-glucose with monosaccharides replacing the key pyrophosphate unit

Article abstract of DOI:10.1039/b500418g

A series of 5′-O-glycosyl-uridine and thymidine derivatives have been prepared as potential mimics of sugar nucelotides and nucleotide-diphosphates. These compounds proved not to be inhibitors of bovine β-1,4- galactosyltransferase although some showed mo

Full text of DOI:10.1039/b500418g

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A R T I C L E
Synthesis and evaluation of mimetics of UDP and
UDP-a-D-galactose, dTDP and dTDP-a-D-glucose with
monosaccharides replacing the key pyrophosphate unit
Lluis Ballell,a Robert J. Youngb and Robert A. Field*a,c
a Centre for Carbohydrate Chemistry, School of Chemical Sciences and Pharmacy, University of
East Anglia, Norwich, UK NR4 7TJ. E-mail: r.a.field@uea.ac.uk; Fax: +44-1603-592003
b CVU UK Medicinal Chemistry, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood
Road, Stevenage, UK SG1 2NY
c Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich,
UK NR4 7UH
Received 1st January 2005, Accepted 27th January 2005
First published as an Advance Article on the web 21st February 2005
A series of 5ꢀ-O-glycosyl-uridine and thymidine derivatives have been prepared as potential mimics of sugar
nucelotides and nucleotide-diphosphates. These compounds proved not to be inhibitors of bovine
b-1,4-galactosyltransferase although some showed moderate inhibition of Salmonella dTDP-a-D-glucose
4,6-dehydratase (RmlB).
the pyrophosphate bridge of its substrate through hydrogen
Introduction
bonding, rather than with the aid of a metal ion).3 Reflecting the
stereochemistry evident in the central unit of tunicamycin, 5ꢀ-O-
b-L-rhamnosyl thymidine 4 might serve as a dTDP mimetic and
might also inhibit RmlB (Fig. 2). In order to investigate this no-
tion, and to further investigate the reported inhibition of b-1,4-
galactosyltransferase by lactosyl-uridine,12 glycosyl-nucleosides
1–4 were synthesised and assessed as potential inhibitors of
bovine b-1,4-galactosyltransferase and Salmonella RmlB.
In connection with studies on the dTDP-b-L-rhamnose biosyn-
thetic pathway,1 a potential target for the development of new
anti-tuberculosis agents,2 we had a need to develop inhibitors of
sugar-nucleotide processing enzymes. In particular, we wished
to devise small molecules capable of inhibiting RmlB, a key
dTDP-a-D-glucose 4,6-dehydratase enzyme that converts dTDP-
a-D-glucose to dTDP-6-deoxy-a-D-xylo-4-hexulose en route to
dTDP-b-L-rhamnose. Although much crystallographic3 and
mechanistic information4 is available about RmlB, to date few
molecules have been described that inhibit this or other enzymes
involved in dTDP-rhamnose biosynthesis.5
The development of NDP/NDP-sugar mimetics is not new
to medicinal chemistry6 and many examples based on nat-
urally occurring antibiotics have been noted. These include
nucleocidin,7 an antitumor compound active against a wide
variety of gram positive bacteria; uracil/thymine polyoxins,8
active against phytopathogenic fungi and useful therapeutically
against Candida albicans; ascamycin,9 with broad antibacterial
activity against various gram-negative and gram-positive bac-
teria; and nikkomycin Z,10 an anti-fungal agent based on it’s
ability to serve as a competitive inhibitor of chitin synthase.
An inherent feature of these types of molecules is their lack
of charge with respect to NDP/NDP-sugars, rendering them
cell membrane permeable at physiological pH. Amongst many
recent efforts to prepare NDP/NDP-sugar mimetics,11 we were
intrigued by a report from Wong and coworkers12 noting lactosyl
uridine 1 (Fig. 1) to be an inhibitor (Ki 119.6 lM) of the b-
1,4-galactosyltransferase activity present in L1210 leukaemia
ascites fluid. The corresponding galactosyl-uridine proved to
be a very poor inhibitor (Ki >1 mM) of the same system.
Based on analogy with the mode of action of tunicamycin
(Fig. 1),13 it seemed reasonable that the central glucose unit
in lactosyl-uridine 1 might also serve as a surrogate for a
pyrophosphate–metal ion complex, or as a spacer between sugar
and nucleoside units (Fig. 1).
Results and discussion
Lactosyl-uridine 1 was prepared essentially as described by
Wong and co-workers,12 using an approach described earlier
by Lichtenthaler,14 from the known building blocks 2ꢀ,3ꢀ-O-
isopropylidene uridine15 and acetobromolactose .12 Cellobiosyl-
thymidine 2 was prepared in a similar fashion, using the
known donor acetylated cellobiosyl bromide 516 and acceptor
3ꢀ-O-benzoyl-thymidine 6,17 which were coupled together using
AgOTf as an activating agent in the presence of 2,4,6-collidine.
For ease of purification, after work-up the crude product
was treated with sodium methoxide to effect complete de-
O-acetylation. The deprotected product was purified by gel
filtration on Sephadex LH-20 in methanol to give cellobiosyl
thymidine 2 in a modest 25% yield (Scheme 1). The beta stereo-
chemistry of the newly formed glycosidic linkage was confirmed
by 1H NMR spectroscopy (Glc dH−1 4.39 ppm; J1,2 8 Hz).
The poor glycosylation yield obtained during the preparation
of compound 2 is likely due to two factors: internal hydrogen
bonding between the heterocyclic base and the alcohol nu-
cleophile in the acceptor nucleoside, and donor consumption
through acetate transfer from the donor to the acceptor hydroxyl
group. The former has been addressed in a modification of
the Koenigs–Knorr reaction18 whereby a 5ꢀ-O-trityl-nucleoside
is employed as an acceptor instead of the nucleoside per se.
The problem of acetyl transfer is overcome by substituting
acetates with less prone to migrate benzoate groups.19 Hence,
in the synthesis of glucosyl-thymidine 3 known 3ꢀ-O-acetyl-5ꢀ-
O-trityl thymidine 720 was coupled with benzobromoglucose 816
in the presence of AgOTf to give protected glucosyl-thymidine
9 in 68% yield (Scheme 2). The use of benzoate in place of
acetate protection in the donor improved the coupling yield
Considering the model explaining the activity of lactosyl
uridine (Fig. 1), 5ꢀ-O-b-cellobiosyl-thymidine 2 might serve as a
dTDP-a-D-glucose analogue and, similarly, 5ꢀ-O-b-D-glucosyl-
thymidine 3 might serve as a dTDP analogue (Fig. 2), both
potentially with the ability to inhibit RmlB (which recognises
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 1 0 9 – 1 1 1 5
1 1 0 9
©

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Fig. 1 Monosaccharides as pyrophosphate–metal ion complex mimetics: reaction catalysed by dolichol-pyrophosphoryl-a-D-GlcNAc synthase and
its relationship to tunicamycin structure; UDP-a-D-galactose–manganese cation complex and its relationship to 5ꢀ-O-b-lactosyl-uridine 1.
Fig. 2 Reaction catalysed by RmlB, putative dTDP-glucose and dTDP analogues 2–4.
substantially.21 Deacetylation of 9, silica column chromatogra-
phy and crystallisation then gave a 64% yield of the deprotected
5ꢀ-O-b-D-glucopyranosyl thymidine 3,21 the structure of which
thymidines by Schmid and co-workers22 provides diagnostic
information about regioisomers (glycosylation of O-5ꢀ results
in resolved H-5ꢀa/b 1H NMR signals; O-3ꢀ glycosylation results
in distinctive shifts of 13C NMR signals of C-3ꢀ to lower field
and C-5ꢀ to higher field).
1
was confirmed by H NMR spectroscopy (Glc dH−1ꢀꢀ 4.34 ppm,
J1ꢀꢀ ꢀꢀ 7.7 Hz) and through comparison with literature data.21
,2
In the preparation of glycosyl-nucleosides 2 and 3 there is
the potential issue of regiocontrol arising from the possibility of
O-3ꢀ-ester migration under (acidic) glycosylation conditions to
O-5ꢀ, followed by O-3ꢀ glycosylation. However, extensive NMR
studies on the related deprotected 3ꢀ- and 5ꢀ-b-D-galactosyl
Synthesis of b-L-rhamnosyl-thymidine 4 presents the classical
1,2-cis-b-glycosylation problem evident in b-mannoside and b-
rhamnoside chemistry. Incisive work from Crich and Sun,23
exploiting torsional control of reactivity,24 has led to a practical
solution to the synthesis the former. A recent elegant variation
1 1 1 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 1 0 9 – 1 1 1 5

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Scheme 1 i). AgOTf–collidine, 4 A molecular sieves, DCM, −20 ◦C; ii). NaOMe–MeOH.
˚
Scheme 2 i). AgOTf–collidine, 4 A molecular sieves, DCM, −78→0 ◦C; ii). NaOMe–MeOH.
˚
on this theme by Crich and Yao also provides access to b-
rhamnosides.25 In the context of our work, a report from
Silva and Sofia26 on the 5-O-L-mannosylation of a uridine
derivative with an appropriately 4,6-O-benzylidenated mannosyl
sulfoxide donor gave an a/b selectivity of only a 1 : 1.8.
These authors suggest that the lack of stereoselectivity may
be due to particular stereoelectronic characteristics of the
uridyl acceptor.26 We were therefore discouraged from preparing
elaborate donor molecules and considered other options. In
recent work on the synthesis of b-L-rhamnosyl apiose, we have
exploited 2,3-carbonate protection in a rhamosyl donor27 and,
in other studies, we have demonstrated apparent SN2 reactions
of a benzylated a-mannosyl sulfoxide donor, giving rise to b-
mannosides in excess in (some) glycosylation reactions.28 Based
on the related work of Silva and Sofia,26 the separability of
anomeric rhamnosyl-nucleosides seemed likely, encouraging
us to employ a benzylated a-L-rhamnosyl sulfoxide donor in
glycosylation studies. Suitably protected uridine was initally
investigated as an acceptor, followed by thymidine.
To generate the required donor, known benzyl protected
L-thiorhamnoside 1229 was oxidised with hydrogen peroxide–
acetic anhydride–silica in dichloromethane30 to give 2,3,4-tri-
O-benzyl-1-(phenylsulfinyl)-a-L-rhamnose 13, in 81% yield. As
expected for a reaction that is likely to proceed via an SN1-like
fragmentation, and where the b-face of the resulting oxocarbe-
nium ion is more hindered, reaction of donor 13 with the known
glycosyl acceptor 3-N-benzoyl-2ꢀ,3ꢀ-di-O-benzoyl uridine 1431
in the presence of Tf2O–DTBMP23 gave a-linked disaccharide
15 in 63% yield with no sign of the b-anomer. The anomeric
configuration of 15 was confirmed using the distinct JC1−H1
coupling constants of a and b glycosides.32 In contrast, iodine-
promoted reaction of sulfoxide donor 13 and uridine acceptor
14 gave a/b-disaccharides 15/16 in a 3 : 5 ratio and a combined
59% yield. The a and b anomers were easily separable by
column chromatography to give pure 15 (H-1a JC1ꢀꢀ ꢀꢀ 167 Hz)
−H1
and 16 (H-1b JC1ꢀꢀ ꢀꢀ 157 Hz). Methanolic liquid ammonia
−H1
deesterification and palladium-mediated hydrogenation gave the
deprotected counterparts 10 (H-1aJC1ꢀꢀ ꢀꢀ 169 Hz) and 11 (H-1b
−H1
JC1ꢀꢀ
ꢀꢀ 159 Hz) in 72% and 77% yield, respectively (Scheme 3).
−H1
The same glycosylation conditions employed for the synthesis
of rhamnosyl uridines 15 and 16 were used for synthesis of
the related thymidine derivatives 18 and 19. Reaction of known
acceptor 1719 with the benzylated rhamnosyl sulfoxide 13 in the
presence of iodine gave a mixture of a- and b-rhamnosides (2 :
3) in a 71% combined yield. The separated stereoisomers 18
(H-1a JC1ꢀꢀ ꢀꢀ 167 Hz) and 19 (H-1b JC1ꢀꢀ ꢀꢀ 157 Hz) were fully
−H1
−H1
deprotected as described above, giving a-rhamnoside 20 (H-1a
ꢀꢀ 171 Hz) and b-rhamnoside 4 (H-1b JC1ꢀꢀ ꢀꢀ 161 Hz),
JC1ꢀꢀ
−H1
−H1
respectively (Scheme 4).
Synthetic glycosyl-nucleosides were assessed for their ability
to inhibit bovine b-1,4-galactosyltransferase.33 None of the
compounds showed more than 10% inhibition at 500 lM
concentration, where UDP showed 90% inhibition. The same
compounds were also assessed as inhibitors of recombinant
Salmonella enterica serovar typhimurium LT2.34,35 Some mod-
erate inhibition was found at the concentration assayed (1 mM)
for some compounds, although unexpectedly for an enzyme that
utilises a dTDP-sugar substrate, uridine derivatives 1 and 11
proved more active than the thymidine derivatives investigated
in this study (Table 1).
Conclusion
In contrast to the observations of Wong et al. on the in-
hibition of the b-1,4-galactosyltransferase activity present in
L1210 leukaemia ascites fluid by lactosyl-uridine,12 none of the
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 1 0 9 – 1 1 1 5
1 1 1 1

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Scheme 3 i). 30% H2O2–Ac2O–SiO2 in DCM; ii). Tf2O–DTBMP in DCM at −78 ◦C; iii). I2–K2CO3 in DCM; iv). MeOH–NH3; v). H2–10% Pd/C
in EtOH.
Scheme 4 i). I2–K2CO3 in DCM; ii). MeOH–NH3; iii). H2–10% Pd/C in EtOH.
Table 1 Inhibition of RmlB by glycosyl-nucleosides (data are accurate
to ∼10%)
in progress, a report on attempts to prepare chitin synthase
inhibitors using sugars to replace the pyrophosphate group in
UDP-GlcNAc analogues also demonstrated only very weak
inhibition of chitin synthase.36 We conclude that the notion that
sugars might serve as generic surrogates for pyrophosphate–
metal ion complexes, or as spacers between sugar and nucleoside
moieties in sugar-nucleotide mimics, should be treated with
caution. However, in relation to RmlB inhibition, in particular,
the moderate inhibition observed warrants further investigation.
Compound
Inhibition at 1 mM concentration (%)
b-Lactosyl-uridine 1
43
3
27
7
27
47
0
b-Cellobiosyl-thymidine 2
b-D-Glucosyl-thymidine 3
b-L-Rhamnosyl-thymidine 4
a-L-Rhamnosyl-uridine 10
b-L-Rhamnosyl-uridine 11
a-L-Rhamnosyl-thymidine 20
Experimental
General information
compounds prepared in this study showed significant inhibition
of the bovine b-1,4-galactosyltransferase. This may highlight
structural differences between b-1,4-galactosyltransferases from
different species. Although reasonable inhibition of Salmonella
RmlB was noted for b-lactosyl-uridine 1 and b-L-rhamnosyl-
uridine 11, none of the compounds reported in this study gave
>50% inhibition at 1 mM concentration. Whilst our work was
TLC was performed on Silica Gel 60 F254 (Merck) detected by
immersion in a 5% ethanolic solution of H2SO4, followed by
heating (>100 ◦C). Column chromatography was performed
using Silica Gel 60 (0.063–0.200 mm). Concentration of organic
extracts was typically carried out below 40 ◦C and at water pump
pressure. Unless otherwise stated, NMR spectra were obtained
1 1 1 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 1 0 9 – 1 1 1 5

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in CDCl3 (referenced to d 77.0 or residual CHCl3 at d 7.27 ppm
for 13C and 1H, respectively) or D2O (referenced to added acetone
at d 31.00 or 2.25 ppm for 13C and 1H, respectively).
5ꢀ-O-b-D-Galactopyranosyl-(1→4)-O-b-D-glucopyranosyl-uri-
dine 1. This compound was prepared essentially as described
in the literature.12
was stirred until tlc showed the reaction to be complete. Dowex-
50X8 (H+) resin was then added to neutralise the reaction
mixture. Removal of the resin by filtration, concentration in
vacuo and column chromatography (DCM : MeOH, 10 : 3) gave
glucopyranosyl-thymidine 3 (92 mg, 64%) as a white solid; mp
◦
◦
118–120 C (lit.21 119–120 C); [a]D20 12.5 (c 0.5, MeOH) (lit.21
12.2); dH (CD3OD): 7.83 (1 H, s, H-6), 6.32 (1 H, m, H-1ꢀ), 4.51
5ꢀ-O-b-D-Glucopyranosyl-(1→4)-O-b-D-glucopyranosyl-thymi-
dine 2. a-D-Hepta-O-acetylcellobiosyl bromide 516 (302 mg,
0.43 mmol) and 3ꢀ-O-benzoyl thymidine 817 (100 mg, 0.29 mmol)
(1 H, m, H-3ꢀ), 4.34 (1 H, d, J1ꢀꢀ ꢀꢀ 7.7 Hz, H-1ꢀꢀ), 4.23 (1 H, dd,
,2
J4 ,5a
ꢀ
ꢀ
3 Hz, J5a ,5b
ꢀ
ꢀ
11 Hz, H-5aꢀ), 4.08 (1 H, dd, J3 ,4
ꢀ
ꢀ
5.3, Hz, J4 ,5
ꢀ
ꢀ
3 Hz, H-4ꢀ), 3.87 (1 H, dd, J5ꢀꢀ ꢀꢀ 2 Hz, J6aꢀꢀ ꢀꢀ 12 Hz, H-6aꢀꢀ),
,6a
,6b
were suspended in dry DCM (4 ml) together with 4 A molecular
3.71 (1 H, dd, J4 ,5b
ꢀ
ꢀ
3, Hz, J
5a ,5b , H-5bꢀ), 3.65 (1 H, dd, J5ꢀꢀ ꢀꢀ
ꢀ
ꢀ
˚
,6b
sieves and the reaction mixture was cooled to −20 ◦C. 2,4,6-
Collidine (64 mg, 0.53 mmol) and AgOTf (133 mg, 0.52 mmol)
were added and the mixture was allowed to warm slowly to
room temperature whilst protected from light. The mixture
was then diluted with DCM (50 ml) and washed with equal
volumes of saturated NaHCO3 solution (2 ×) and H2O (2 ×).
The organic extract was dried (MgSO4), concentrated in vacuo
and subjected to column chromatography (DCM : acetone,
6 : 1). Without further characterisation, the resulting crude,
fully protected trisaccharide was dissolved in anhydrous MeOH
(4 ml), NaOMe (35 mg, 0.576 mmol) was added and the mixture
was stirred at room temperature for 1 h. When tlc showed
deprotection to be complete, the mixture was concentrated in
vacuo and the crude mixture was purified on a Sephadex LH-20
column eluted with MeOH. The required cellobiosyl-thymidine
2 was obtained as a white powder (40 mg, 25%); mp 146–148 ◦C
(MeOH–Et2O); [a]2D5 4.7 (c 0.42, MeOH); dH (MeOD): 7.57 (1 H,
s, H-6), 6.21 (1 H, t, J1,2 7 Hz, H-1ꢀ), 4.63 (1 H, d, J 8.5 Hz,
5.9 Hz, J6aꢀꢀ ꢀꢀ , H-6bꢀ), 3.26–3.36 (3 H, m, H-2ꢀ, H-3ꢀꢀ,4ꢀꢀ), 3.20
,6b
(1 H, m, H-5ꢀꢀ), 2.55 (2 H, m, H-2aꢀꢀ, 2bꢀꢀ), 1.89 (3 H, s, CH
3
Thy); dC (CDCl3): 165.5 (C-4), 151.5 (C-2), 137.3 (C-6), 110.5
(C-5), 103.4 (C-1ꢀꢀ), 86.6, 85.6 (C-1ꢀ, C-4ꢀ), 77.2 (C-5ꢀꢀ), 74.2 (C-
2ꢀꢀ), 72.1, 70.7, 69.4, 69.2 (C-3ꢀꢀ, C-4ꢀꢀ,C-3ꢀ, C-5ꢀ), 61.8 (C-6ꢀꢀ),
40.1 (C-2ꢀ), 11.5 (CH Thy). NMR data are in accordance with
3
literature data;21 ES-MS C16H25N2O10 requires 405.1509, found
(M + H)+ 405.1514.
2,3,4-Tri-O-benzyl-1-(phenylsulfinyl)-a-L-rhamnopyranoside
13. To a stirred solution of known thioglycoside 1229 (5 g,
9.5 mmol), Ac2O (1.05 ml, 11 mmol) and silica 220–240 mesh
(2 g) in DCM (40 ml) was added aqueous 30% H2O2 solution
(2 g, 1.80 ml). The resulting mixture was stirred vigorously until
tlc (EtOAc : hex, 1 : 1) showed the reaction to be complete.
The mixture was then diluted with DCM (150 ml), washed with
aqueous NasS2O5 solution (2 × 100 ml) and brine (2 × 100 ml),
dried (MgSO4) and concentrated in vacuo. The resulting crude
mixture was subjected to column chromatography (EtOAc :
hex, 1 : 2) to give rhamnosyl-sulfoxide 13 as a colourless oil
(4.22 g, 82%); [a]2D5 57.6 (c 1.14, CHCl3); dH (CDCl3): 7.48–7.57
(5H, m, Ar SPh), 7.21–7.39 (15H, m, Ar Bn), 5.27 (1 H, d, J1,2
1.4 Hz, H-1), 4.97 (1 H, d, J4,5 11 Hz, H-4), 4.48–4.67 (6 H, m,
H-1ꢀꢀ), 4.44 (1 H, d, J1ꢀꢀꢀ ꢀꢀꢀ 8 Hz, H-1ꢀꢀꢀ), 4.42 (1 H, m, H-3ꢀ),
,2
4.10 (2 H, m), 3.87 (2 H, m), 3.79 − 3.50 (8 H, m), 3.39 (2 H,
m), 3.29 (1 H, m, H-5ꢀꢀ or H-5ꢀꢀꢀ) 3.22 (2 H, m), 2.29 (2H, m,
H-2aꢀ,2bꢀ), 1.81 (3 H, s, CH Thy); dC (MeOD): 164.6 (C-4),
3
150.5 (C-2), 137.7 (C-6), 112.5 (C-5), 102.6, 102.3 (C-1ꢀꢀ, C-1ꢀꢀꢀ),
85.5, 85.3 (C-1ꢀ, C-4ꢀ), 78.9, 76.0, 75.5, 74.8, 74.4, 73.2, 71.5,
71.0 (C-2ꢀꢀ-C-5ꢀꢀ, C-2ꢀꢀꢀ-C-5ꢀꢀꢀ), 69.5, 69.3 (C-3ꢀ, C-5ꢀ), 61.8, 60.6
3 × −CH -), 4.16 (1 H, m, H-3), 3.97 (1 H, m, H-5), 3.71 (1H,
2
m, H-2), 1.30 (3 H, d, J5,6 6.3 Hz, 6-CH ); dC (CDCl3): 142.3,
3
(C-6ꢀꢀ, C-6ꢀꢀꢀ), 38.2 (C-2ꢀ), 11.6 (CH3 Thy); ES-MS C28H35N2O15
138.5, 138.3, 137.8 (4 × quat. Ar), 131.6, 131.0, 129.5, 129.1,
128.9, 128.8, 128.7, 128.4, 128.3, 128.1, 128.0, 127.9, 127.8,
127.1, 124.6 (Ar), 96.9 (C-1), 79.8, 79.4, 75.6, 74.6, 72.9, 72.4,
+
requires 567.2037, found (M + H)+ 567.2041.
3ꢀ-O-Acetyl-5ꢀ-O-(2,3,4,6-tetra-O-benzoyl-b-D-glucopyranosyl)-
thymidine 9. 3ꢀ-O-Acetyl-5ꢀ-O-trityl-thymidine 720 (400 mg,
0.76 mmol) and 2,3,4,6-tetra-O-benzoyl-a-D-glucopyranosyl
bromide 816 (752 mg, 1.14 mmol) were dissolved in 5 ml of
72.1 (C-2-5 and 3 × -CH –Ph), 18.5 (C-6); CI C33H35O5S+
2
requires 543.2205, found (M + H+) 542.2211.
3-N-Benzoyl-2ꢀ,3ꢀ-di-O-benzoyl-5ꢀ-O-(2,3,4-tri-O-benzyl-a-L-
˚
dry DCM and 4 A molecular sieves were added (500 mg). The
rhamnopyranosyl)-uridine
15. Rhamnosyl-sulfoxide
13
reaction mixture was then cooled to −78 ◦C under N2. AgOTf
(475 mg, 1.14 mmol) was then added and the reaction mixture
was allowed to slowly warm −10 ◦C, when tlc showed the
reaction to be complete. The reaction was then quenched with
collidine (200 ll), concentrated in vacuo and the crude product
was subjected to column chromatography (EtOAc : hex, 1 :
1) to give glycoside 9 (446 mg, 68%) as a colourless syrup; dH
(CDCl3): 7.20–8.02 (20 H, m, Ar), 7.64 (1 H, s, H-6), 6.34 (1H,
(347 mg, 0.64 mmol) and 2,6-di-tert-butyl-4-methyl pyridine
(395 mg, 1.9 mmol) were dissolved in dry DCM (5 ml) and the
resulting solution was cooled to −78 ◦C. Tf2O (90 mg, 54 ll,
0.32 ◦mmol) was added and the reaction was allowed to reach
−60 C over a period of 15 min. 3-N-Benzoyl-2ꢀ,3ꢀ-di-O-benzoyl
uridine 1431 (88 mg, 0.16 mmol) in dry DCM (5 ml) was added
drop-wise and the resulting solution was stirred for another
10 min. The reaction mixture was then allowed to warm to
−5 ◦C over a period of 1 h, at which point the reaction was
quenched by the addition of saturated NaHCO3 solution
(1 ml). The organic extract was separated, dried (MgSO4),
concentrated in vacuo and the resulting crude product was
subjected to column chromatography (acetone : hex, 1 : 5→1 :
3) to protected rhamnosyl-uridine 15 (97 mg, 63%); dH (CDCl3):
dd, J1 ,2a
ꢀ
ꢀꢀ 6.0 Hz, J1 ,2b
ꢀ
ꢀ
9 Hz
,4
ꢀ
, H-1ꢀ), 5.97 (1 H, t, J3ꢀꢀ ꢀꢀ 9.7 Hz,
,4
H-3ꢀꢀ), 5.69 (1 H, t, J3ꢀꢀ ꢀꢀ , H-4ꢀꢀ), 5.50 (1 H, dd, J1,2 5.9 Hz J2,3
8.1 Hz, H-2), 4.90 (2 H, m, H-1ꢀꢀb, H-5:ꢀꢀ), 4.67 (1 H, dd, J5ꢀꢀ ꢀꢀ
,6b
3.1 Hz, J6aꢀꢀ ꢀꢀ 12.2 Hz, H-6aꢀꢀ), 4.50 (1 H, dd, J5ꢀꢀ ꢀꢀ 5.2 Hz,
,6b
,6b
J6aꢀꢀ ꢀꢀ , H-6bꢀꢀ), 4.32 (1 H, dd, J4ꢀꢀ ꢀꢀ 2.1 Hz, J5aꢀꢀ ꢀꢀ 10.5 Hz,
,6b
,5a
,5b
H-5aꢀꢀ), 4.20 (1 H, m, H-4ꢀ), 4.08 (1 H, br s, H-3ꢀ), 3.78 (1 H, dd,
J4ꢀꢀ ꢀꢀ 1.8 Hz, J5aꢀꢀ ꢀꢀ , H-5bꢀ), 2.08 (2 H, m, H-2aꢀ, H-2bꢀ), 1.20
,5b
,5b
7.12–7.98 (31 H, 3 × Ph and H-6), 6.6 (1 H, d, J1 ,2
ꢀ
ꢀ
7.3 Hz,
(3 H, s, CH Thy); dC (CDCl3): 171.2 (COCH3), 165.7, 166.0,
H-1ꢀ), 5.73 (1 H, m, H-3ꢀ), 5.63 (1 H, d, J5,6 8.2 Hz, H-5), 5.37
3
166.4, 166.7 (4 × COPh), 164.1 (C-4), 150.9 (C-6), 133.8, 133.9,
134.1, 134.3 (quat. Ar) 128.9–130.4 (Ar), 112.0 (C-5), 101.9
(C-1ꢀꢀ), 85.0 (C-1ꢀ), 84.0 (C-4ꢀ), 75.9, 73.1, 72.8, 72.4, 70.3, 70.1
(1 H, dd, J
ꢀ
1 ,2 , J
2 ,3 , 5.8 Hz, H-2ꢀ), 4.91 (1 H, s, H-1ꢀꢀ), 4.44–4.85
ꢀ
ꢀ
(6 H, m, 3 ×ꢀ CH –Bn), 4.58 (1H, m, H-4ꢀ), 3.98–4.15 (2H, m,
2
H-3ꢀꢀ, H-4ꢀꢀ), 3.61–3.82 (4 H, m, H-5aꢀ, 5bꢀ and H-2ꢀꢀ, H-5ꢀꢀ), 1.36
(C-3ꢀ-5ꢀ and C-2ꢀꢀ-5ꢀꢀ), 63.3 (C-6ꢀꢀ), 36.9 (C-2ꢀ), 21.2 (COCH ),
(3H, d, J5ꢀꢀ ꢀꢀ 4.8 Hz, 6ꢀꢀ-CH ); dC (CDCl3): 168.4, 165.7, 165.6,
3
,6
+
3
12.9 (CH Thy); FAB-MS C46H42N2NaO15 requires 885.2483,
3
164.0, 161.7 (CO), 149.6 (C-6), 126.7–138.7 (aromatic), 104.5
found (M + Na)+ 885.2486. The compound was used in the
(C-5), 99.2 (C-1ꢀꢀ JC1−H1 167 Hz), 85.8 (C-1ꢀ), 82.6 (C-4ꢀ), 80.2
synthesis of 3 without further characterisation.
(C-2ꢀꢀ), 78.7 (C-4ꢀꢀ), 76.0 (–CH –Ph 74.2 (C-3ꢀꢀ), 74.1 (C-2ꢀ),
2
5ꢀ-O-b-D-Glucopyranosyl-thymidine 321. The esterified glu-
copyranosyl thymidine 9 (300 mg, 0.35 mmol) was dissolved in
dry MeOH (5 ml), NaOMe (20 mg) was added and the mixture
73.8 (–CH –Ph 72.3 (C-3ꢀ), 71.8 (–CH –Ph 69.3 (C-5ꢀ), 67.8
2
2 +
(C-5ꢀ), 18.3 (C-6ꢀꢀ); ES-MS C57H56N3O13 requires 990.3813,
found (M + NH4+) 990.3809.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 1 0 9 – 1 1 1 5
1 1 1 3

View Article Online
3-N-Benzoyl-2ꢀ,3ꢀ-di-O-benzoyl-5ꢀ-O-(2,3,4-tri-O-benzyl-a-L-
rhamnopyranosyl)-uridine 15 and 3-N-benzoyl-2ꢀ,3ꢀ-di-O-
benzoyl-5ꢀ -O-(2,3,4-tri-O-benzyl-b-L-rhamnopyranosyl)-uridine
16. 3-N-Benzoyl-2ꢀ,3ꢀ-di-O-benzoyl uridine 1431 (88 mg,
0.16 mmol) and 2,3,4-tri-O-benzyl-1-(phenylsulfinyl)-a-L-
rhamnose 13 (347 mg, 0.64 mmol) were dissolved in dry
DCM (5 ml). K2CO3 (23 mg, 0.16 mmol) and iodine (61 mg,
0.24 mmol) were added to the solution and the reaction mixture
was flushed under N2. The reaction was monitored by tlc
(acetone : hex, 1 : 2) until it was complete, concentrated in vacuo
to ∼1 ml and subjected to column chromatography (acetone :
hex, 1 : 5→1 : 3). The first compound to elute from the column
was the a-anomer 15 (34 mg, 22%) followed by the b-anomer 16
(57 mg, 37%). a-anomer 15: data as reported above. b-anomer
16: dH (CDCl3): 7.12–8.04 (31 H, 6 × Ph, and H-6), 6.41 (1 H,
and subjected to column chromatography (acetone : hex, 1 :
2). The mixture was then concentrated to ∼1 ml in vacuo and
subjected to column chromatography (acetone : hex, 1 : 5→1 :
3) for purification. The first compound to be eluted from the
column was the a-anomer 18 (33 mg, 29%), followed by the
b-anomer 19 (48 mg, 42%). a-anomer 18: dH (CDCl3): 9.02 (1H,
br s, NH), 7.24–7.41 (15 H, 3 × Ph), 7.08 (1 H, s, H-6), 6.22
(1 H, m, H-1ꢀ), 5.07 (1 H, m, H-3ꢀ), 4.57–4.98 (8 H, m, 3 ×
−CH –Bn, H-1ꢀꢀ,2ꢀꢀ), 4.09 (1 H, br s, H-4ꢀ), 3.93 (1H, dd, J4 ,5a
ꢀ
ꢀ
2
2.2 Hz, J5a ,5b
ꢀ
ꢀ
11.0 Hz, H-5aꢀ), 3.72 (1H, br s, H-4ꢀꢀ), 3.65 (2
H, m, H-3ꢀꢀ,5ꢀꢀ), 3.53 (1H, dd, J4 ,5b
ꢀ
ꢀ
3.4 Hz J
5a ,5b , H-5bꢀ), 2.26
ꢀ
ꢀ
(1H, m, H-2aꢀ), 2.10 (3H, s, COCH ), 1.75 (3H, s, CH Thy),
3
3
1.69 (3H, m, H-2bꢀ), 1.33 (3H, d, J5ꢀꢀ ꢀꢀ 4.9 Hz, 6ꢀꢀ-CH ); dC
,6
3
(CDCl3): 170.7, 163.7, 150.5 (CO), 138.4, 138.2, 138.1 (quat.
Ar), 134.2 (C-6), 127.8, 127.9, 128.0, 128.1, 128.2, 128.4, 128.5,
1
1
d, J1 ,2
ꢀ
ꢀ
7.0 Hz, H-1ꢀ), 5.94 (1 H, dd, J2 ,3
ꢀ
ꢀ
5.6 J3 ,4
ꢀ
ꢀ
2.2 Hz, H-3ꢀ),
128.6 (Ar), 111.9 (quat. Thy), 99.2 (C-1ꢀꢀ JC
-
167 Hz), 84.4
H
5.67 (2 H, m, H-5 and H-2ꢀ), 4.63–4.98 (6 H, m, 3 × -CH –Bn),
(C-1ꢀ), 83.2 (C-4ꢀ), 80.2, 78.7 (C-3ꢀꢀ,5ꢀꢀ), 76.6 (CH Bn), 75.5,
2
2
4.55 (1 H, s, H-1ꢀꢀ), 4.50 (1H, d, J
ꢀ
ꢀ
3 ,4 ,H-4ꢀ), 4.28 (1H, dd, J4 ,5a
ꢀ
ꢀ
74.8 (2 × −CH –Bn), 74.5, 72.9, 71.9 (C-3ꢀ and C-2ꢀꢀ,4ꢀꢀ), 37.5
2
2.2 Hz J5a ,5b
dd, J4 ,5b 2.5 Hz J
ꢀ
ꢀ
11.4 Hz, H-5aꢀ), 3.96 (1 H, m, H-2ꢀꢀ), 3.91 (1H,
(C-2ꢀ), 20.9 (COCH ), 17.9 (C-6ꢀꢀ), 12.4 (CH Thy); ES-MS
3
3
5a ,5b , H-5bꢀ), 3.68 (1H, d, J3ꢀꢀ ꢀꢀ 8.8 Hz H-4ꢀꢀ),
C39H48N2O10 requires 718.3340, found (M + NH4+) 718.3344.
b-anomer 19: dH (CDCl3): 8.84 (1H, br s, NH), 7.22–7.39 (16 H,
3 × Ph and H-6), 6.27 (1 H, m, H-1ꢀ), 5.35 (1 H, m, H-3ꢀ), 4.93
(2H, m, −CH –Bn), 4.61–4.77 (4H, m, 2 × −CH –Bn), 4.27
+
ꢀ
ꢀ
ꢀ
ꢀ
,4
3.60 (1H, dd, J2ꢀꢀ ꢀꢀ 2.9 Hz J3ꢀꢀ ꢀꢀ , H-3ꢀꢀ), 3.45 (1H, m, H-5ꢀꢀ),
,3
,4
(3H, d, J5ꢀꢀ ꢀꢀ 6.2 Hz, 6ꢀ-CH ); dC (CDCl3): 168.4, 165.7, 165.6,
,6
3
164.0, 161.7 (CO), 149.6 (C-6), 126.5–138.8 (aromatic), 103.3
2
2
(C-5), 100.5 (C-1ꢀꢀ JC1−H1 157 Hz), 83.0 (C-1ꢀ), 82.3 (C-4ꢀ), 80.2
(1 H, s, H-1ꢀꢀ), 4.19 (1 H, br s, H-4ꢀ), 4.13 (1H, dd, J4 ,5a
3.1 Hz,
ꢀ
ꢀ
(C-2ꢀꢀ), 75.9, 75.7, 74.5, 74.4, 73.5, 73.0, 72.8, 72.3 (–CH –Ph ×
J5a ,5b
ꢀ
ꢀ
10.8 Hz, H-5aꢀ), 3.90 (1H, d, J2ꢀꢀ ꢀꢀ 2.5 Hz, H-2ꢀꢀ), 3.72
,3
2
3, C-2ꢀ, 3ꢀ, 5ꢀ and C-3ꢀꢀ,4ꢀꢀ), 69.0 (C-5ꢀꢀ), 18.4 (C-6ꢀꢀ), ESI-MS
(1H, dd, J4 ,5a
ꢀ
ꢀ
2.4 Hz, J
ꢀ
ꢀ
5a ,5b , H-5bꢀ), 3.64 (1H, t, J3ꢀꢀ ꢀꢀ 9.2 Hz,
,4
C57H56N3O13 requires 990.3813, found (M + NH4+) 990.3810.
H-4ꢀꢀ), 3.52 (1H, dd, J2ꢀꢀ ꢀꢀ J3ꢀꢀꢀ ꢀꢀ , H-3ꢀꢀ), 3.35 (1H, m, H-5ꢀꢀ),
+
,3
,4
2.30 (1H, m, H-2aꢀ), 2.10 (3H, s, COCH ), 1.81 (3H, s, CH
3
3
5ꢀ-O-a-L-Rhamnopyranosyl-uridine 10. Protected a-rha-
mnosyl-uridine 15 (80 mg, 0.08 mmol) was dissolved in methanol
(2 ml) and aqueous ammonia was added (2 ml). After 48 h, tlc
(DCM : MeOH, 5 : 1) showed the reaction to be complete.
The solution was then concentrated in vacuo, the resulting crude
product was dissolved in ethanol (5 ml) and AcOH–H2O (1 ml
of 1 : 1 v/v solution) and 10% Pd/C (50 mg) were added
and the mixture was placed under a hydrogen atmosphere for
16 h. The mixture was then filtered, neutralised with NH4OAc
and concentrated to dryness. Purificaton on a Sephadex LH-20
column eluted with MeOH gave the◦a-glycoside 10 as a white
powder (21 mg, 65%); mp 122–124 C (DCM : MeOH); [a]D25
−31.9 (c 0.68, MeOH); dH (DMSO): 10.24 (1H, br s, NH),
7.84 (1H, d, J5,6 7.1 Hz, H-6), 7.42 (1H, d, J5,6 7.1 Hz, H-5),
5.71 (1H, s, H-1ꢀ), 4.55 (1H, s, H-1ꢀꢀ), 3.02–3.94 (9H, m, H-2ꢀꢀ-
Thy), 1.69 (3H, m, H-2bꢀ), 1.35 (3H, d, J5ꢀꢀ ꢀꢀ 6.0 Hz, 6ꢀꢀ-CH );
,6
3
dC (CDCl3): 170.5, 163.7, 150.6 (CO), 138.4, 138.1 (quat. Ar),
135.4 (C-6), 127.7, 127.8, 127.9, 128.0, 128.3, 128.4, 128.5, 128.7
(Ar), 111.2 (quat. Thy), 101.2 (C-1ꢀꢀ JC1−H1 157 Hz), 85.4 (C-1ꢀ),
83.8 (C-4ꢀ), 82.9 (C-3ꢀꢀ), 80.1 (C-4ꢀꢀ), 76.7, 76.1, 76.0, 75.4, 74.3
(3 × −CH –Bn and C-2ꢀꢀ,3ꢀ), 72.4 (C-5ꢀꢀ), 69.1 (C-5ꢀ), 37.8
2
(C-2ꢀ), 21.4 (COCH ), 18.3 (C-6ꢀꢀ), 13.2 (CH Thy); ES-MS
3
3
C39H48N2O10 requires 718.3340, found (M + NH4+) 718.3343.
+
5ꢀ-O-a-L-Rhamnopyranosyl-thymidine 20. Protected a-rha-
mnosyl-thymidine 18 (80 mg, 0.1 mmol) was deprotected and
purified as described for compound 10 to give a-rhamnosyl-
thymidine 20 as a colourless syrup (31 mg, 80%); [a]2D5 −7.0 (c
0.41, MeOH); dH (MeOD): 7.43 (1H, br s, H-6), 6.19 (1H, s,
H-1ꢀ), 4.62 (1H, s, H-1ꢀꢀ), 3.02–4.13 (9H, m, H-2ꢀꢀ-5ꢀꢀ and H-3ꢀ-
5aꢀ,5bꢀ), 2.11 (1H, m, H-2aꢀ), 1.95 (1 H, m, H-2bꢀ), 1.91 (3H, s,
5ꢀꢀ and H-2ꢀ-4ꢀ, 5aꢀ,5bꢀ), 1.10 (1H, d, J5ꢀꢀ ꢀꢀ 5.9 Hz, 6-CH ); dC
,6
3
(DMSO): 171.9, 154.3 (2 × NHCO), 129.4, 128.7 (C-5,6), 101.4
(C-1ꢀꢀ JC1−H1169 Hz), 88.7 (C-1ꢀ), 82.5 (C-2ꢀ), 73.0, 71.9, 71.7,
71.5, 71.4, 69.5, 68.1 (C-3ꢀ-5ꢀ and C-2ꢀꢀ-5ꢀꢀ), 20.0 (C-6ꢀꢀ); ES-MS
C15H22N2NaO10+ requires 413.1172, found (M + Na+) 413.1181.
CH Thy), 1.19 (1H, br s, J5ꢀꢀ ꢀꢀ 5.9 Hz, 6-CH ); dC (MeOD):
,6
3
3
166.1, 152.1 (2 × NHCO), 137.0 (C-6), 111.5 (quat. Thy), 101.9
(C-1ꢀꢀ JC1−H1171 Hz), 86.5, 85.7, 73.3, 71.9, 71.7, 71.4 (C-1ꢀ, C-,4ꢀ
and C-2ꢀꢀ-5ꢀꢀ), 69.7, 68.1 (C-3ꢀ, C-5ꢀ), 40.2 (C-2ꢀ), 17.4 (C-6ꢀꢀ),
5ꢀ-O-b-L-Rhamnopyranosyl-uridine 11. Protected b-rham-
nsoyl-uridine 16 (80 mg, 0.1 mmol) was deprotected and purified
as described in the preparation of the a-anomer 10 to give the
b-glycoside 11 as a colourless syrup (30 mg, 77%); [a]2D5 <5 (c
0.11, MeOH); dH (DMSO): 10.21 (1H, br s, NH), 7.87 (1H, d,
J5,6 7.7 Hz, H-6), 7.42 (1H, d, J5,6 7.6 Hz, H-5), 5.74 (1H, s, H-
1ꢀ), 4.35 (1H, s, H-1ꢀꢀ), 3.02–3.94 (8H, m, H-2ꢀꢀ-4ꢀꢀ and H-2ꢀ-4ꢀ,
+
12.1 (CH3 Thy): ES-MS C16H28N3O9 requires 406.1826, found
(M + NH4+) 406.1827.
5ꢀ-O-b-L-Rhamnopyranosyl thymidine 4. Protected b-rham-
nosyl-thymidine 19 (70 mg, 0.09 mmol) was deprotected and
purified as described for compound 10 to give b-rhamnosyl-
thymidine 4 as a colourless syrup (25 mg, 74%); [a]2D5 +8.2 (c
5aꢀ,5bꢀ), 3.01 (1H, m, H-5ꢀꢀ), 1.13 (1H, d, J5ꢀꢀ ꢀꢀ 5.9 Hz, 6-CH ); dC
,6
3
(DMSO): 171.7, 154.1 (2 × NHCO), 129.2, 128.5 (C-5,6), 102.4
(C-1ꢀꢀ JC1−H1159 Hz), 88.4 (C-1ꢀ), 81.5 (C-2ꢀ), 73.5, 72.1, 71.7,
71.4, 71.3, 69.1, 67.5 (C-3ꢀ-5ꢀ and C-2ꢀꢀ-5ꢀꢀ), 19.8 (C-6ꢀꢀ); ES-MS
C15H22N2NaO10+ requires 413.1172, found (M + Na+) 413.1181.
0.37, MeOH); dH (MeOD): 7.60 (1H, s, H-6), 6.23 (1H, dd, J1 ,2a
ꢀ
ꢀ
8 Hz J1 ,2b
6.2 Hz, H-1ꢀ), 4.47 (1H, s, H-1ꢀꢀ), 4.41 (1H, m, H-3ꢀ),
ꢀ
ꢀ
3.94 (2H, m, H-4ꢀ,5aꢀ), 3.81 (1H, d, J2ꢀꢀ ꢀꢀ 2.9 Hz, H-2ꢀꢀ), 3.70 (1H,
,3
3ꢀ -O-Acetyl-5ꢀ-O-(2,3,4-tri-O-benzyl-a-L-rhamnopyranosyl)-
thymidine 18 and 3ꢀ-O-acetyl-5ꢀ-O-(2,3,4-tri-O-benzyl-b-L-
rhamnopyranosyl)-thymidine 19. 3ꢀ-O-Acetyl-thymidine 1719
(42 mg, 0.16 mmol) and 2,3,4-tri-O-benzyl-1-(phenylsulfinyl)-
a-L-rhamnose 13 (347 mg, 0.64 mmol) were dissolved in dry
DCM (5 ml). K2CO3 (23 mg, 0.16 mmol) and iodine (61 mg,
0.24 mmol) were added to the solution and the reaction mixture
was flushed with N2. The reaction was monitored by tlc (acetone :
hex, 1 : 2) until it was complete, concentrated in vacuo to ∼1 ml
dd, J4 ,5a
ꢀ
ꢀꢀ 2.3 Hz J5a ,5b
ꢀꢀ 10.1 Hz, H-5bꢀ), 3.36 (1H, dd, J2ꢀꢀ ꢀꢀ J3ꢀꢀ ꢀꢀ
ꢀ
,3
,4
9.1 Hz, H-3ꢀꢀ), 3.24 (2H, m, H-4ꢀꢀ,5ꢀꢀ), 2.24 (1H, ddd, J1 ,2a
ꢀ
ꢀ
J2a ,2b
2.7 Hz,
ꢀ
ꢀ
13 Hz J2a ,3
ꢀ
ꢀ
6 Hz, H-2aꢀ), 2.24 (1H, ddd, J1 ,2b
ꢀ
ꢀ
J2a ,2b
ꢀ
ꢀ
J2b ,3
ꢀ
ꢀ
H-2bꢀ), 1.81 (3H, s, CH Thy), 1.23 (1H, br s, J5ꢀꢀ ꢀꢀ 5.9 Hz, 6-
,6
3
CH ), dC (MeOD) 166.1, 152.1 (2 × NHCO), 137.6 (C-6), 111.3
3
(q Thy), 101.0 (C-1ꢀꢀ JC1−H1161 Hz), 86.9 (C-4ꢀ), 86.0 (C-1ꢀ), 74.5,
73.3, 72.7, 72.3, 72.0 (C-2ꢀ,C-2ꢀꢀ-5ꢀꢀ), 69.5, 67.8 (C-3ꢀ, C-5ꢀ), 40.2
(C-2ꢀ) 17.4 (C-6ꢀꢀ), 11.9 (CH3 Thy); ES-MS C16H28N3O9+ requires
406.1826, found (M + NH4+) 406.1828.
1 1 1 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 1 0 9 – 1 1 1 5

View Article Online
Enzyme assays
11 P. Compain and O. R. Martin, Bioorg. Med. Chem., 2001, 9, 3077–
3092.
12 R. Wang, D. H. Steensma, Y. Takoaka, J. W. Yun, T. Kajimoto and
C.-H. Wong, Bioorg. Med. Chem., 1997, 5, 661–672.
13 Reviewed in:K. Kimura and T. D. H. Bugg, Nat. Prod. Rep., 2003,
20, 252–273.
14 F. W. Lichtenthaler and K. Kitahara, Angew. Chem., Int. Edn. Engl.,
1975, 14, 815–816.
15 K. K. Ogilvie and D. Iwacha, Can. J. Chem., 1974, 52, 1787–1797.
16 S. A. Mitchell, M. R. Pratt, V. J. Hruby and R. Polt, J. Org. Chem,
2001, 66, 2327–2342.
Bovine b-1,4-galactopyranosyltransferase33 (Sigma) was assayed
essentially as described previously, with both donor and acceptor
substrates concentrations at their respective KM values. Under
these conditions IC50 ∼ 2Ki for a competitive inhibitor.
Recombinant Salmonella enterica serovar typhimurium LT2
RmlB34,35 was also assayed essentially as described previously,
with [S] ∼ 3KM. Under these conditions IC50 ∼ 4Ki for a
competitive inhibitor.
17 T. Wada, A. Ohkubo, A. Mochizuki and M. Sekine, Tetrahedron
Lett., 2001, 42, 1069–1072.
18 H. Bredereck, A. Wagner, H. Kuhn and H. Ott, Chem. Ber., 1960,
93, 1201–1206.
19 D. M. Whitfield, S. P. Douglas, T.-H. Tang, I. G. Csizmadia, H. Y. S.
Pang, F. L. Moolten and J. J. Krepinsky, Can. J. Chem., 1994, 72,
2225–2238.
Acknowledgements
These studies were supported by the GlaxoSmithKline Action
TB programme and the Weston Foundation. We thank Dr
James Errey for assistance with enzyme assays, Professor Jim
Naismith (University of St Andrews) for kindly providing the
RmlB expression construct and the EPSRC Mass Spectrometry
Service Centre, Swansea for invaluable support.
20 N. C. Bar, R. Patra, B. Achari and S. B. Mandal, Tetrahedron, 1997,
53, 4727–4738.
21 N. D. Chkanikov, V. N. Tolkachev and M. N. Preobrazhenskaya,
Bioorg. Khim., 1978, 4, 1620–1626.
22 W. H. Binder, H. Ka¨hlig and W. Schmid, Tetrahedron: Asymmetry,
1995, 6, 1703–1710.
References
1 M.-F. Giraud and J. H. Naismith, Curr. Opin. Chem. Biol., 2000, 10,
687–696.
2 Y. F. Ma, F. Pan and M. McNeil, J. Bacteriol., 2002, 184, 3392–3395;
J. A. Mills, K. Motichka, M. Jucker, H. P. Wu, B. C. Uhlik, R. J.
Stern, M. S. Scherman, V. D. Vissa, F. Pan, M. Kundu, Y. Fang Ma
and M. McNeil, J. Biol. Chem., 2004, 279, 43540–43546.
23 D. Crich and S. Sun, J. Org. Chem., 1996, 61, 4506–4507; D. Crich
and S. Sun, J. Org. Chem., 1997, 62, 1198–1199.
24 B. Fraser-Reid, Z. F. Wu, C. W. Andrews, E. Skowronski and J. P.
Bowen, J. Am. Chem. Soc., 1991, 113, 1434–1435.
25 D. Crich and Q. L. Yao, J. Am. Chem. Soc., 2004, 126, 8232–
8236.
3 S. T. M. Allard, M.-F. Giraud, C. Whitfield, M. Graninger, P. Messner
and J. H. Naismith, J. Mol. Biol., 2001, 307, 283–295.
26 D. J. Silva and M. J. Sofia, Tetrahedron Lett., 2000, 41, 855–
858.
4 K. Beis, S. T. M. Allard, A. D. Hegeman, G. Murshudov, D. Philp and
J. H. Naismith, J. Am. Chem. Soc., 2003, 125, 11872–11878; S. T. M.
Allard, K. Beis, M. F. Giraud, A. D. Hegeman, J. W. Gross, R. C.
Wilmouth, C. Whitfield, M. Graninger, P. Messner, A. G. Allen, D. J.
Maskell and J. H. Naismith, Structure, 2002, 10, 81–92.
27 A.-L. Chauvin, S. A. Nepogodiev and R. A. Field, Carbohydr.
Res., 2004, 339, 21–27; for a recent thorough investigation of
carbonate-protected rhamnosides in glycosylation chemistry see:; D.
Crich, A. U. Vinod and J. Picione, J. Org. Chem., 2003, 68, 8453–
8458.
5 For instance: K. Babaoglu, M. A. Page, V. C. Jones, M. R. McNeil,
C. J. Dong, J. H. Naismith and R. E. Lee, Bioorg. Med. Chem. Lett.,
2003, 13, 3227–3230; Y. F. Ma, R. J. Stern, M. S. Scherman, V. D.
Vissa, W. X. Yan, V. C. Jones, F. Q. Zhang, S. G. Franzblau, W. H.
Lewis and M. R. McNeil, Antimicrob. Agents Chemother., 2001, 45,
1407–1416; J. P. Shilvock, J. R. Wheatley, R. J. Nash, A. A. Watson,
R. C. Griffiths, T. D. Butters, M. Mu¨ller, D. J. Watkin, D. A. Winkler
and G. W. J. Fleet, J. Chem. Soc., Perkin Trans. 1, 1999, 45, 2735–2745.
6 S. Rachakonda and L. Cartee, Curr. Med. Chem., 2004, 11, 775–793.
7 A. R. Maguire, W. D. Meng, S. M. Roberts and A. J. Willets, J. Chem.
Soc., Perkin Trans. 1, 1993, 1795–1808 and references cited therein.
8 H. A. Dondas, C. W. G. Fishwick, R. Grigg and C. Kilner,
Tetrahedron, 2004, 60, 3473–3485 and references cited therein.
9 D. Heacock, C. J. Forsyth, K. Shiba and K. Musier-Forsyth, Bioorg.
Chem., 1996, 24, 273–289 and references cited therein.
28 S. J. Marsh, K. P. R. Kartha and R. A. Field, Synlett, 2003, 1376–
1379.
29 J. Furukawa, S. Kobayashi, M. Nomizu, N. Nishi and N. Sakairi,
Tetrahedron Lett., 2000, 41, 3453–3457.
30 R. Kakarla, R. G. Dulina, N. T. Hatzenbuhler, Y. W. Hui and M. J.
Sofia, J. Org. Chem, 1996, 61, 8347–8349.
31 X. Liu and C. B. Reese, J. Chem. Soc., Perkin Trans. 1, 2000, 2227–
2236.
32 K. Bock and C. Pedersen, J. Chem. Soc., Perkin Trans. 1, 1974, 293–
297.
33 R. A. Field, D. C. A. Neville, R. W. Smith and M. A. J. Ferguson,
Bioorg. Med. Chem. Lett., 1994, 4, 391–394.
34 M. Graninger, B. Nidetzky, D. E. Heinrichs, C. Whitfield and P.
Messner, J. Biol. Chem., 1999, 274, 25069–25077.
35 J. A. Vara and C. R. Hutchinson, J. Biol. Chem., 1988, 263, 14992–
14995.
10 J. D. More and N. S. Finney, Synlett, 2003, 1307–1310; A. Suda, A.
Ohta, M. Sudoh, T. Tsukuda and N. Shimma, Heterocycles, 2001,
55, 1023–1028 and references cited therein.
36 J.-B. Behr, T. Gourlain, A. Helimi and G. Guillerm, Bioorg. Med.
Chem. Lett., 2003, 13, 1713–1716.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 1 0 9 – 1 1 1 5
1 1 1 5