Inhibition of Escherichia coli glycosyltransferase MurG and Mycobacterium tuberculosis Gal transferase by uridine-linked transition state mimics

Article abstract of DOI:10.1016/j.bmc.2010.02.026

Glycosyltransferase MurG catalyses the transfer of N-acetyl-d-glucosamine to lipid intermediate I on the bacterial peptidoglycan biosynthesis pathway, and is a target for development of new antibacterial agents. A transition state mimic was designed for MurG, containing a functionalised proline, linked through the α-carboxylic acid, via a spacer, to a uridine nucleoside. A set of 15 functionalised prolines were synthesised, using a convergent dipolar cycloaddition reaction, which were coupled via either a glycine, proline, sarcosine, or diester linkage to the 5′-position of uridine. The library of 18 final compounds were tested as inhibitors of Escherichia coli glycosyltransferase MurG. Ten compounds showed inhibition of MurG at 1 mM concentration, the most active with IC₅₀ 400 μM. The library was also tested against Mycobacterium tuberculosis galactosyltransferase GlfT2, and one compound showed effective inhibition at 1 mM concentration.

Full text of DOI:10.1016/j.bmc.2010.02.026

Bioorganic & Medicinal Chemistry 18 (2010) 2651–2663
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of Escherichia coli glycosyltransferase MurG and Mycobacterium
tuberculosis Gal transferase by uridine-linked transition state mimics
Amy E. Trunkfield a, Sudagar S. Gurcha b, Gurdyal S. Besra b, Timothy D. H. Bugg a,
*
a Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom
b Department of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Glycosyltransferase MurG catalyses the transfer of N-acetyl-D-glucosamine to lipid intermediate I on the
Received 21 November 2009
Revised 7 January 2010
Accepted 16 February 2010
Available online 19 February 2010
bacterial peptidoglycan biosynthesis pathway, and is a target for development of new antibacterial
agents. A transition state mimic was designed for MurG, containing a functionalised proline, linked
through the
a-carboxylic acid, via a spacer, to a uridine nucleoside. A set of 15 functionalised prolines
were synthesised, using a convergent dipolar cycloaddition reaction, which were coupled via either a gly-
cine, proline, sarcosine, or diester linkage to the 50-position of uridine. The library of 18 final compounds
were tested as inhibitors of Escherichia coli glycosyltransferase MurG. Ten compounds showed inhibition
of MurG at 1 mM concentration, the most active with IC50 400 lM. The library was also tested against
Mycobacterium tuberculosis galactosyltransferase GlfT2, and one compound showed effective inhibition
Keywords:
Glycosyltransferase
Inhibition
Peptidoglycan biosynthesis
MurG
at 1 mM concentration.
Ó 2010 Elsevier Ltd. All rights reserved.
Mycobacterium tuberculosis
1. Introduction
cycle of reactions is responsible for transformation of cytoplasmic
precursor UDPMurNAc-L-Ala-c-D-Glu-m-DAP-D-Ala-D-Ala into pep-
Glycosyltransferase enzymes are involved in a wide range of
biosynthetic pathways responsible for the formation of polysac-
charides, glycoproteins, glycolipids and glycosylated natural prod-
tidoglycan, via a series of lipid-linked reactions,7 shown in Figure 2.
The first step, catalysed by translocase MraY, is the reaction of
UDPMurNAc-pentapeptide with undecaprenyl phosphate to form
lipid intermediate I: this reaction is inhibited by several nucleo-
side-containing natural products, upon which some structure–
activity studies have been undertaken.8 The second step, catalysed
by glycosyltransferase MurG, is the reaction of lipid intermediate I
with UDPGlcNAc to form lipid intermediate II. Escherichia coli
MurG is an extrinsic membrane protein,9 for which a crystal struc-
ture has been solved, in complex with UDPMurNAc.10 The E. coli
enzyme has been overexpressed,11 and is able to accept synthetic
lipid I analogues containing shortened prenyl chains.12,13 Using a
fluorescent binding assay, several small molecule inhibitors of
MurG have been identified by screening of combinatorial li-
braries.14 The active compounds are structurally unrelated to the
enzyme substrates, and it is not known exactly how they bind to
the enzyme.
Examination of the MurG structure reveals that there is are a
series of specific interactions with the uracil base, and hydrogen-
bonding interactions with the GlcNAc C-3 and C-4 hydroxyl
groups, but no electrostatic interactions with the diphosphate
bridge (see Fig. 3A).10 In order to prepare a substrate-based inhib-
itor for MurG, we have therefore designed a cyclic mimic for the
oxonium ion transition state, linked via an uncharged spacer to a
uridine nucleoside, shown in Figure 1. Adjacent to the GlcNAc
ucts.1 The glycosyl transfer reaction involves transfer of
a
monosaccharide from a nucleotide mono/diphosphate donor to
an acceptor alcohol, usually with inversion of stereochemistry at
the anomeric centre, and the transition state for the reaction is
thought to possess oxonium ion character,1 as shown in Figure 1.
Although a number of glycosyltransferases are potentially inter-
esting targets for chemotherapeutic intervention, there are rela-
tively few documented inhibitors for glycosyltransferases,2–5
compared with many examples of glycosidase enzyme inhibitors.6
Known inhibitors of glycosyltransferases in general contain the
nucleoside found in the donor substrate: synthetic inhibitors in-
clude analogues of UDP-galactose in which the diphosphate link-
age has been replaced by methylenediphosphate,2 or the ring
oxygen replaced by a methylene group,3 or the glycosidic linkage
replaced by a hydroxymethylene linkage.4 C-Glycosides have also
been prepared, containing a linker to a uridine nucleoside, as inhib-
itors of chitin synthase.5
Enzymes of the bacterial peptidoglycan biosynthetic pathway
are well-established targets for antibacterial action. A lipid-linked
* Corresponding author. Tel.: +44 2476 573018; fax: +44 2476 524112.
E-mail address: T.D.Bugg@warwick.ac.uk (T.D.H. Bugg).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.026

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A. E. Trunkfield et al. / Bioorg. Med. Chem. 18 (2010) 2651–2663
mimic δ+ charge in transition state by amine substituent
OH
OH
OH
O
OH
HO
HO
O
δ+
HO
HO
O
HO
HO
O
HO
HO
NHAc
OR
NHAc
O-
O-
P
O-
P
OR
NHAc
O-
P
O
NHAc
O
OUr
O
O
P
OUr
O
O
O
O
presence of nucleoside
required for inhibition of
glycosyltransferases
UDP
OH
OH
O
O
HO
HO
O
HO
NHAc
NHAc
OR
Transition state mimic:
R2
H
H
H2
N
O
H
R2
R2
N
H
R1
N
R1
O
N
Ur
R1
O
CO2H
O
O
CO2P
O
O
O
O
N
O
N
Ar
HO OH
N
Ar
Ar
Figure 1. Glycosyl transfer reaction, showing presumed transition state, transition state mimic, and retrosynthetic disconnection.
OH
OH
OH
OH
O
O
O
HO
HO
O
HO
HO
O
O
O
O
AcNH
OUDP
NHAc
AcNH
AcNH
O
O
O
MurG
O
MraY
O
O-
O-
O
O
P
O
P
O
O-
O-
HN
HN
O
O
P
O
P
O
HN
UDPGlcNAc
undecaprenyl
phosphate
L-Ala
γ-D-Glu
m-DAP
D-Ala
L-Ala
γ-D-Glu
m-DAP
D-Ala
L-Ala
γ-D-Glu
m-DAP
D-Ala
D-Ala
D-Ala
D-Ala
lipid intermediate I
lipid intermediate II
Figure 2. Reactions of the lipid-linked cycle of bacterial peptidoglycan biosynthesis catalysed by MraY and MurG.
binding site is a large cavity, lined with hydrophobic residues,
therefore some members of the inhibitor set included an aromatic
substituent able to bind to this site.
2. Results
2.1. Docking of transition state analogues into E. coli MurG
In this paper, we report the synthesis and screening of a set of
transition state analogues using this design. The functionalised
proline transition state mimic is readily assembled using a 1,3-dipo-
lar cycloaddition sequence developed by Grigg and co-workers,15
which allows the convergent assembly of analogues containing
a range of R1 groups (see Fig. 1). We have included a range of
hydrogen-bond acceptor groups in the analogue set (methoxy-aryl
substituents, ethane-1,2-diol substituent), in order to interact with
the MurG active site. This approach could be used to inhibit other
UDP-sugar glycosyltransferases, therefore we also report the
screening of the inhibitor set against Mycobacterium tuberculosis
galactosyl transferase.16,17
active site
Several of the transition state analogue structures were energy
minimised, and docked into the E. coli MurG active site (PDB file
1NLM) using eHiTS software.18 Analogues containing a glycine lin-
ker were found to be a suitable length to fit into the MurG active
site, as shown in Figure 3B. Binding of the substrate UDPGlcNAc re-
quires a twisted conformation at the GlcNAc-phosphate glycosidic
linkage, in order to access the GlcNAc binding site, as shown in
Figure 3A, and in several cases the docked proline substituent
was found not to lie in the GlcNAc binding site. Therefore, con-
formationally flexible linkers were also included in the inhibitor

A. E. Trunkfield et al. / Bioorg. Med. Chem. 18 (2010) 2651–2663
2653
H
N
CO2P
R
O
OBn
a
R
N
O
O
O
N
2a-e P = Bn
3a-e P = H
b
1a-e
O
Ph
N
Ph
H
N
MeO2C
CO2P
R
a
1a-e
4a-e P = Bn
5a-e P = H
b
CO2Me
MeO2C
CO2Me
CO2P
H
N
R
CO2Me
a
MeO2C
1a-e
6a-e P = Bn
7a-e P = H
b
MeO2C
CO2Me
Sidechains (R):
a
b
c
d
e
O
O
OMe
OMe MeO
OMe
OMe
Scheme 1. Preparation of substituted prolines via 1,3-dipolar cycloaddition.
Reagents and conditions: (a) AgOAc, KOH, toluene, rt, 24 h; (b) H2, Pd/C.
10% palladium on carbon to give free acid 3a, as shown in Scheme 1.
Dipolar cycloadditions under the same reaction conditions with di-
methyl fumarate and dimethyl maleate gave the benzyl esters 4a
and 6a in 69% and 73%, respectively, which were debenzylated in
94% and 96% yield, respectively, to give free acids 5a, and 7a, as
shown in Scheme 1.
In order to prepare enzyme inhibitors containing hydrogen
bond acceptors in the sidechain R, imines of
were prepared from -glyceraldehyde acetonide, o-anisaldehyde,
2,3-dimethoxybenzaldehyde, and 3,4-dimethoxybenzaldehyde.
Dipolar cycloadditions with -alanine benzyl ester, using the above
conditions, proceeded well in each case to give a single major dia-
stereoisomer, with yields of cycloadducts 2a–e, 4a–e, and 6a–e in
most cases in the range 50–75%, the highest yield being 96% for
cycloadduct 2b. Debenzylation proceeded smoothly, in most cases
in 90–100% yield, to give the free acids 3a–e, 5a–e, and 7a–e, as
shown in Scheme 1.
L-alanine benzyl ester
D
Figure 3. (A) Binding of UDPGlcNAc to E. coli MurG, showing twisted substrate
conformation. (B) Example of docked inhibitor structure, showing positioning of
proline substituent
L
collection, containing N-methyl glycine, proline amide linkers, or
ester linkages, as described below.
In the case of diethyl fumarate cycloadducts 4a–e, the major
diastereomer obtained was the endo-isomer, as found by Casas
et al.,20 with smaller amounts of the exo-diastereoisomer (epimer
at C-300/C-400). Casas et al. reported a 94:6 ratio of endo:exo prod-
ucts, using the benzaldehyde imine of alanine iso-propyl ester;20
using the corresponding benzyl ester, we observed a 97:3 ratio of
endo:exo products. For compounds with aryl R groups, only small
amounts of the exo-diastereoisomer were formed (4a 3%; 4b not
detected; 4c 16%; 4d 13%); in the case of 4e, the exo-isomer was
formed as 40% of the isolated product. With the exception of 4e,
the minor exo-diastereoisomer was not observed in subsequent
coupled products, following purification.
2.2. Preparation of substituted prolines via 1,3-dipolar
cycloaddition
Grigg and co-workers have previously demonstrated that imi-
nes formed between amino acid methyl esters and a range of aryl
and alkyl aldehydes can undergo a 1,3-dipolar cycloaddition with
N-phenyl maleimide,15 or with dimethyl maleate or dimethyl
fumarate,19 to give a single major diastereoisomer product in each
case. Using the imine formed between benzaldehyde and L-alanine
methyl ester, cycloaddition reactions with N-phenyl maleimide
were found to proceed with variable yield and product purity using
the thermal cyclisation method,19 or using a one-pot acid-cata-
lysed procedure also reported.19
2.3. Coupling of glycine linker
A procedure involving 0.1 equiv of silver(I) acetate, carried out
using the benzaldehyde imine of
L
-alanine isopropyl ester,20 was
Since docking studies had indicated that a glycine linker was a
suitable length for binding to MurG, each of the acids 3a–e, 5a–e,
and 7a–e was coupled with glycine benzyl ester, using either car-
bodi-imide EDCI in the presence of hydroxybenzotriazole (HOBt),
or uronium coupling agent HATU, in the presence of HOBt, which
in most cases proceeded in 60–90% yield. Debenzylation was
achieved by hydrogenation using 10% palladium on carbon, in high
yield, as illustrated in Scheme 2.
found to proceed in 40–88% yield and with high product purity
after chromatography, however, subsequent hydrolysis of the iso-
propyl ester was found in our hands to be problematic. This latter
procedure was found to work well using the benzaldehyde imine
of L-alanine benzyl ester, to give cycloadduct 2a with N-phenyl
maleimide in 59% yield after flash chromatography. Benzyl ester
2a was debenzylated in quantitative yield by hydrogenation using

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A. E. Trunkfield et al. / Bioorg. Med. Chem. 18 (2010) 2651–2663
In the case of the dimethyl maleate cycloadducts 7a–e, the cou-
O
N
O
N
pled products were found to contain variable amounts of a bicyclic
product, in which the nitrogen atom of the glycine linker had cyc-
lised onto the cis-substituted methyl ester to form a cyclic imide.
No cyclisation was observed in compound 10e containing an ali-
phatic sidechain R, whereas compounds containing aromatic side-
chains had cyclised to the extent of 50% for 10a/11a, 67% for 10b/
11b, and 100% for 10c/11c and 10d/11d. The bicyclic compounds
were not separable by silica chromatography from the uncyclised
material, so compounds 10a/11a and 10b/11b were taken forward
as mixtures.
NH
NH
HO
N3
O
O
a,b
O
O
O
O
O
O
c,d
O
N
O
2.4. Synthesis of uridine-containing analogues
NH
NH
Attempts to couple acid 8a to the 50-hydroxyl group of 20,30-iso-
propylidene-uridine, using a range of coupling methods, gave none
of the desired ester-linked product after chromatography. In sev-
eral reactions a new product was observed in the reaction mixture,
using thin-layer chromatography, which was not observed after
work-up and chromatography, suggesting that an ester linkage in
this series of compounds may be unstable. Therefore, a 50-amino,
50-deoxyuridine derivative 12 containing TBDMS 20-O and 30-O pro-
tecting groups was prepared, as shown in Scheme 3. As observed
previously,21 we found that in the presence of the 20,30-isopropyli-
dene protecting group, a 50-amino substituent was prone to intra-
molecular cyclisation onto the uracil base, but when re-protected
as the 20,30-OTDBMS derivative, was quite stable.
Coupling of acid 8a with 12 was found to proceed in 56% yield
using HATU, in the presence of HOAt, to give the amide product,
which was deprotected to give 10a. The same procedure was used
to couple the other analogues (see Scheme 4). After deprotection,
the final products were purified by semi-preparative reverse phase
HPLC. In the case of 13b, two diastereoisomers were separated by
HPLC.
H2N
N3
e
O
N
O
O
O
TBSO
OTBS
12
TBSO
OTBS
Scheme 3. Preparation of 50-amino,20,30-OTBDMS uridine. Reagents and yields: (a)
TsCl, pyr, 49%; (b) NaN3, DMF, 50 °C, 100%; (c) CF3COOH, 95%; (d) TBSCl, imidazole,
DMF, 81%; (e) H2, Pd/C, 100%.
The cyclisation of the nitrogen atom of the glycine linker to
form a bicyclic imide was once again observed in the reaction
products after coupling: for the dimethyl maleate adducts 10, all
of the reaction products 16a–d contained only a bicyclic imide.
Cyclisation was also observed to varying extents for the dimethyl
fumarate adducts 9b (partial cyclisation, products 14b and 15b
separated by HPLC) and 9e (product 15e wholly cyclised), but
not in the case of 9a (product 14a). The slower cyclisation of the
O
NH
O
O
H
N
O
H
N
H
N
H
R
O
N
H
CO2H
N
O
H
R
O
N
H
O
CO2H
N
R
O
N
a,b
R
O
N
H
CO2H
O
a,b
8
O
HO
OH
N
O
N
13a-c,e
8a-e
Ph
Ph
O
O
N
O
O
N
O
NH
O
3a-e
Ph
Ph
H
N
H
N
H
N
R
R
N
O
N
H
N
H
CO2H
O
a,b
H
N
H
N
O
CO2H
R
R
9
a,b
a,b
N
H
CO2H
9a-c,e
MeO2C
CO2Me
MeO2C
CO2Me
HO
OH
O
14a,b
O
H
N
MeO2C
CO2Me
MeO2C
CO2Me
O
O
R
5a-e
NH
N
N
H
N
O
H
N
H
N
O
MeO2C
CO2H
R
O
R
N
CO2H
10a,b,e
H
O
H
HO
OH
15b,e
N
R
N
H
CO2H
MeO2C
CO2Me
7a-e
MeO2C
R
CO2Me
a,b
a,b
10
O
H
O
N
R
MeO2C
CO2Me
O
NH
H
N
O
N
N
H
N
O
H
N
O
MeO2C
O
R
O
N
CO2H
11a-d
N
CO2H
11
MeO2C
HO
OH
MeO2C
16a-d
O
O
Scheme 2. Coupling of glycine linker. Reagents and conditions: (a) H2NCH2CO2Bn,
HATU, DIPEA, THF, 0 °C; (b) H2, Pd/C. R = Ph (a), 2-MeOPh (b), 2,3-(MeO)2Ph (c), 3,4-
(MeO)2Ph (d), (isopropylidene)ethane-1,2-diol (e).
Scheme 4. Coupling to 50-amino, 50-deoxyuridine. Reagents and conditions: (a) 9,
HATU, HOAt, DIPEA, THF, 0 °C; (b) CF3COOH, H2O/CH2Cl2. R = Ph (a), 2-MeOPh (b),
2,3-(MeO)2Ph (c), 3,4-(MeO)2Ph (d), ethane,1-2-diol (e).

A. E. Trunkfield et al. / Bioorg. Med. Chem. 18 (2010) 2651–2663
2655
OMe
OMe
N
OMe
OMe
O
H
N
Me
H
N
H
N
CO2Bn
O
i
N
CO2H
CO2 Pr
OH
a-d
Me
a
O
O
N
O
N
O
O
Ph
2f
O
Ph
O
N
N
18
Ph
Ph
2g
21
OMe
O
N
e,f
O
O
N
MeO
Me
O
N
NH
O
n
OMe
Me
NH
N
O
H
N
HO2C
O
N
O
N
O
O
O
Me
O
MeO2C
CO2Me
22 n = 1
23 n = 2
b,c
17
O
HO
OH
O
N
O
O
Ph
19
Scheme 5. Synthesis of analogues containing N-substituted linkers. Reagents and
conditions: (a) Me2SO4, NaH, THF/H2O, 35%; (b) H2, Pd/C, 100%; (c) H2NCH2CO2Bn,
HATU, HOBt, DIPEA, THF, 0 °C, 49%; (d) H2, Pd/C, 100%; (e) 9, HATU, HOAt, DIPEA,
THF, 0 °C; (f) CF3COOH, H2O/CH2Cl2, 31% over two steps.
O
NH
OMe
O
O
H
N
N
O
O
O
O
n
dimethyl fumarate adducts can be explained by formation of the
less favourable trans-fused bicyclic imide in these cases. Com-
pounds 9e and 15e contained 40% of the C-300/C-400 epimer arising
from the earlier cycloaddition reaction, whereas compounds 9a
and 9b (and hence 14a, 14b, and 15b) contained only a single
diastereoisomer.
HO
OH
O
O
N
24 n = 1
25 n = 2
Ph
Scheme 7. Synthesis of analogues containing ester linkages. Reagents and condi-
tions: (a) DIBAL, THF, ꢀ78 °C, 26%; (b) EDCI/DMAP or HATU, THF, 0 °C; (c) CF3COOH/
H2O, 16–24% over two steps.
2.5. Synthesis of compounds containing conformationally
flexible linkers
N-methyl amide group. Similar intramolecular reactions were ob-
served using acids 3c, 7c, and 5b,
Since the earlier docking studies had indicated that the confor-
mation of the bound UDPGlcNAc substrate contained a twisted
conformation at the GlcNAc-phosphate glycosidic linkage, several
analogues were synthesised containing conformationally flexible
linkers. Coupling of acid 5c with sarcosine (N-methyl glycine) ben-
zyl ester, using the HATU/HOBt coupling used above, gave the
unexpected reaction product 17, in which the benzyl ester had
undergone an intramolecular reaction with the endocyclic nitrogen
atom. This reaction was unexpected, since earlier studies had
found that the endocyclic nitrogen of derivatives 2a–e was extre-
mely unreactive towards acylation or alkylation reactions, how-
ever, such a cyclisation would be assisted by the presence of an
Therefore, methylation of the endocyclic nitrogen of derivative
2b was undertaken, in order to prevent intramolecular cyclisation.
Derivative 2b was methylated in 38% yield using dimethyl sulphate
using the method of Prashad et al.,22 however, after debenzylation,
coupling of the free acid with sarcosine benzyl ester was unsuc-
cessful. Therefore, compound 2f lacking the
a-methyl substituent
was prepared by dipolar cycloaddition of the o-anisaldehyde imine
of glycine benzyl ester with N-phenyl maleimide in 86% yield, fol-
lowed by debenzylation. N-Methylation of 2f using the method of
Prashad et al.22 proceeded in 35% yield, and subsequent debenzy-
lation proceeded in quantitative yield. Coupling with sarcosine
benzyl ester, using HATU/HOBt, gave the desired amide product
in 49% yield, which was debenzylated to give 18 (see Scheme 5).
Coupling with 50-amino, 50-deoxy, 20,30-OTBDMS uridine 12 was
carried out on a small scale, using the above method, and after
deprotection and HPLC purification gave the sarcosine-linked ana-
logue 19.
O
NH
O
H
N
O
H
N
Several compounds containing an
prepared, which would possess a low energy barrier for cis/trans-
amide bond interconversion. Acid 3b was coupled to -proline ben-
L-proline linker were also
N
O
R1
O
N
L
zyl ester using HATU/HOBt in 91% yield, and debenzylated in 92%
yield. Coupling with uridine derivative 12, followed by deprotec-
tion, gave analogue 20a. Using the same procedure, proline-con-
taining analogues 20b–d were prepared. Mixtures of cis- and
trans-amide rotamers were observed for 20a–d by NMR spectros-
copy (Scheme 6).
Two compounds containing a diester linker were also prepared.
Alcohol 21 was prepared by reduction of the isopropyl ester 2g
with DIBAL at ꢀ78 °C, in 26% yield. The 50-O-succinyl uridine deriv-
ative 22 was prepared by treatment of 20,30-isopropylidene-uridine
with succinic anhydride, while the 50-O-malonyl derivative 23
was prepared by HATU/HOBt coupling of benzyl malonate with
R2
R3
R1
HO
R2, R3
OH
20a
2-MeOPh
2-MeOPh
2-MeOPh
N-phenyl-succinimide
β-CO2Me, β-CO2Me
β-CO2Me, α-CO2Me
20b
20c
20d
ethane-1,2-diol N-ethyl-succinimide
Scheme 6. Analogues containing proline linker. Compound 20a, R1 2-MeOPh, R2/R3
N-phenylsuccinimide; 20b, R1 2-MeOPh, R2 b-CO2Me, R3 b-CO2Me; 20c, R1 2-
MeOPh, R2 b-CO2Me, R3
a-CO2Me; 20d, R1 ethane-1,2-diol, R2/R3 N-
ethylsuccinimide.

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A. E. Trunkfield et al. / Bioorg. Med. Chem. 18 (2010) 2651–2663
20,30-isopropylidene-uridine, followed by debenzylation. Coupling
of alcohol 21 with acid 22 was achieved using carbodi-imide EDCI,
in the presence of DMAP, in 42% yield, and deprotected using aque-
ous trifluoroacetic acid, to give the succinyl diester 24. Coupling of
alcohol 21 with acid 23 using the same methods gave the malonyl
diester 25, as shown in Scheme 7.
>5 mM, while compound 16d showed IC50 4 mM, and compound
13b gave an IC50 value of 400 M.
These compounds were tested for antibacterial activity against
Gram-negative strains E. coli and Pseudomonas putida, and Gram-
positive strains Micrococcus luteus and B. subtilis. Compound 13b
l
showed 50% growth inhibition of M. luteus at 100 lg/ml, but no ef-
fects upon the other strains.
2.6. Assay of compounds as inhibitors of E. coli MurG
Recombinant E. coli MurG-C-His6 was purified from E. coli strain
C43 transformed with a pET3a vector containing the murG gene.12
From 2 l of cell culture, enzyme was purified by Ni2+ affinity Hi-
sTrap FPLC, yielding 19 mg of homogeneous protein. UDPMur-
NAc-pentapeptide was prepared from Bacillus subtilis W23, as
previously described.23 MurG was assayed using a coupled radio-
chemical assay, as described by Zawadzke et al.,24 in which lipid
intermediate I is generated in situ by membranes containing trans-
locase MraY, and is then converted in the presence of [3H]-UDPGlc-
NAc and MurG to give radiolabelled lipid intermediate II, which is
extracted into n-butanol and quantitated by scintillation counting.
A time-course in the absence of inhibitor revealed that the product
release was linear for 15–20 min, therefore, assays were recorded
over 15 min. Treatment with 20 lM ramoplanin resulted in >90%
inhibition, consistent with literature data.25
Eighteen compounds were tested as inhibitors of MurG, each at
1 mM final concentration. The results are shown in Table 1. Ten of
the 18 compounds showed inhibition of MurG, the highest levels of
inhibition by compounds 13b (85% inhibition) and 14b (32% inhi-
bition). Selected compounds were then assayed at variable concen-
trations, and in each case, concentration-dependent inhibition was
observed. Compounds 14b, 15e, and 20 each gave IC50 values of
Figure 4. (A) TLC/autoradiogram of reactions products produced through the
inclusion of acceptor Galf(b1-6)Galf-O-C8, mycobacterial membranes and UDP[14C]-
Galf. Lane 1, no acceptor; lane 2, 0.4 mM acceptor; and lane 3, 0.4 mM acceptor and
1 mM 16a. Assay carried out as described in Section 4.
Table 1
Percentage inhibition of Escherichia coli MurG and Mycobacterium tuberculosis galactosyl transferase by compounds 13–25, at 1 mM concentration
O
O
N
H
N
O
NH
R
O
NH
X
H
N
N
O
linker
N
R1
O
N
H
O
O
MeO2C
O
bicyclic
R2
R3
HO
OH
HO
OH
R1
R2
R3
linker
E. coli MurG
Mt GTase
13a
13b
13c
13e
14a
14b
15b
15e
16a
16b
16c
16d
19
Ph
2-MeOPh
2,3-(MeO)2Ph
Ethane-1,2-diol
Ph
2-MeOPh
2-MeOPh
Ethane-1,2-diol
Ph
2-MeOPh
2,3-(MeO)2Ph
3,4-(MeO)2Ph
2-MeOPh
2-MeOPh
2-MeOPh
2-MeOPh
Ethane-1,2-diol
2-MeOPh
N-Phenyl succinimide
N-Phenyl succinimide
N-Phenyl succinimide
N-Phenyl succinimide
-CO2Me
-CO2Me
-CO2Me
-CO2Me
-CO2Me
-CO2Me
-CO2Me
-CO2Me
N-Phenyl succinimide
N-Phenyl succinimide
-CO2Me
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Sar
Pro
Pro
Pro
Pro
C3
NT
85
0
0
0
32
22
27
0
0
0
26
31
0
0
22
0
NT
0
0
0
NT
0
a
a
–CO2Me
–CO2Me
Bicyclic
Bicyclic
Bicyclic
Bicyclic
Bicyclic
Bicyclic
0
NT
80
0
NT
0
NT
0
0
0
0
0
0
20a
20b
20c
20d
24
b–CO2Me
–CO2Me
-CO2Me
a
N-Ethyl succinimide
N-Phenyl succinimide
N-Phenyl succinimide
17
15
25
2-MeOPh
C4
Linkers: glycine (Gly), sarcosine (Sar), proline (Pro), C-3 diester (C3), C-4 diester (C4). NT, not tested. Assays carried out as described in Section 4.

A. E. Trunkfield et al. / Bioorg. Med. Chem. 18 (2010) 2651–2663
2657
2.7. Assay of compounds against M. tuberculosis
galactosyltransferase
strates. This strategy could in principle be used to design inhibitors
for other glycosyltransferase enzymes that use a UDP-sugar sub-
strate. Screening of this set of compounds against M. tuberculosis
galactosyltransferase GlfT2 revealed that compound 16a (which
did not inhibit MurG) acted as an inhibitor for this enzyme. This
is the first inhibitor that has been identified for this enzyme, which
is a potential target for the development of new anti-TB drugs.
Therefore, this strategy could be used to design inhibitors for other
glycosyltransferase enzymes of therapeutic importance.
The arabinogalactan cell wall polymer of M. tuberculosis is bio-
synthesised through the action of a series of glycosyltransferases,16
several of which use UDP-sugar substrates, and therefore might be
inhibited by this class of compound. We have characterised a UDP-
galactose-dependent galactosyltransferase activity GlfT2, which
can be assayed using specific neoglycolipid acceptors in the pres-
ence of membranes and Galf(b1?6)Galf-O-C8 resulting in excellent
[
14C]Galf incorporation from UDP-[14C]Galp, following endogenous
4. Experimental
4.1. Materials
conversion to UDP-[14C]Galf and transferase activity (Fig. 4).17 TLC/
autoradiography (Fig. 4) demonstrated the enzymatic conversion
of the acceptor to both a trisaccharide product [14C]Gal to the 50-
OH of Galf(b1?6)Galf-O-C8 and a second, slower migrating band
(Fig. 4) which based on relative migration profiles would be antic-
ipated to be a tetrasaccharide product resulting from further elon-
gation of the above trisaccharide precursor at the 60OH consistent
with the alternating linkage pattern of arabinogalactan. The com-
plete chemical characterisation of the enzymatically synthesised
products along with the identity of the galactosyltransferase gene
product were previously reported.17
D-Glyceraldehyde acetonide was prepared from D-mannitol
using literature procedures,26,27 via bis-acetonide protection with
SnCl2 and 2,2-dimethoxypropane, followed by oxidative cleavage
by NaIO4. Di-tert-butyldimethylsilyl-50-amino-50-deoxyuridine
(12) was prepared (see Scheme 3) by sodium azide displacement
of 50-tosyl-20,30-isopropylidene uridine, as described by Wang
et al.,28 followed by deprotection of the acetonide as described
by Winans and Bertozzi,29 followed by TBDMS protection and
hydrogenation, as described by Dempcy et al.30 UDPMurNAc-
L-
Assays were carried out in the presence of 14 of the compounds,
at 0.5–1 mM concentration, as described in Table 1. Compound 16a
showed effective inhibition galactosyltransferase activity in the as-
say (Fig. 4).
Ala-c-D-Glu-m-DAP-D-Ala-D-Ala was either prepared by the proce-
dure previously described,23 or was purchased from the BACWAN
synthesis facility (University of Warwick).
3. Discussion
4.2. General procedure for 1,3-dipolar cycloaddition reaction to
form 2a–g, 4a–e, 6a–e, hydrogenation to form 3a–g, 5a–e, 7a–e
In this paper, we describe the synthesis and assay of a new fam-
ily of glycosyltransferase transition state analogues, in which a uri-
dine nucleoside is attached, via a variable length spacer, to a
substituted proline analogue, which acts as an oxonium ion mimic.
Using the 1,3-dipolar cycloaddition synthetic methodology devel-
oped by Grigg and co-workers,19,20 followed by a range of coupling
methods, we have prepared a library of 19 analogues. One unex-
pected side-reaction was the intramolecular cyclisation of 10 to
11, resulting in a number of bicyclic analogues.
Imines 1a–e were formed using the method of Casas et al.20
-Alanine benzyl ester p-tosylate (0.50 g, 1.48 mmol), aldehyde
L
(1.48 mmol), sodium carbonate (0.16 g, 1.48 mmol) were sus-
pended in water (30 ml) and stirred vigorously at room tempera-
ture for 20 h. The mixture was extracted with ethyl acetate
(3 ꢁ 30 ml), dried (Na2SO4) and then concentrated in vacuo to yield
the crude imino acyl ester, which was used immediately. The 1,3-
dipolar cycloaddition was carried out using the method of Casas
et al.20: The imino ester 1a (est 0.26 g, 0.97 mmol), N-phenyl malei-
mide (0.19 g, 1.07 mmol), and silver(I) acetate (16 mg, 0.09 mmol)
were suspended in toluene (15 ml). Potassium hydroxide (5 mg,
0.09 mmol) was then added and the mixture was stirred vigorously
at room temperature for 24 h. The solvent was removed in vacuo
and the residue was dissolved in ethyl acetate and percolated
through a plug of silica gel, eluting with ethyl acetate. The filtrate
was concentrated and the resulting material was purified by flash
chromatography (eluent 3:2, hexane/ethyl acetate) to yield a white
solid, 2a (0.25 g, 0.57 mmol, 59%).
Assay of the compound library as inhibitors of recombinant
E. coli glycosyltransferase MurG revealed that 10 compounds
showed some level of inhibition at 1 mM concentration, the high-
est activity being shown by compound 13b (IC50 400 lM). Most
compounds showing inhibition of MurG contained a 2-methoxy-
phenyl R1 substituent, whilst no compound containing a phenyl
R1 substituent inhibited MurG, consistent with a favourable hydro-
gen-bonding interaction between the 2-methoxy substituent and
the MurG active site. Although the compounds were able to bind
to MurG, the potency of inhibition was not as high as expected
for binding of a transition state mimic, therefore it is suspected
that the compounds do not adopt the desired conformation in
the active site.
The crystal structure of E. coli MurG in complex with UDPGlc-
NAc shows that there is a significant twist in the conformation of
the bound substrate between the diphosphate bridge and the ano-
meric centre. In order to encourage potential inhibitors to bind in
such a conformation, a number of compounds containing conform-
ationally flexible linkers were synthesised. Although compound 31
containing a sarcosine linker did bind to MurG, it bound no more
effectively than the glycine analogue 13b, whilst compounds
20a–d containing a proline linker did not bind at all, implying that
the MurG active site is unable to accommodate a more bulky sub-
stituent at this position. Compounds 24 and 25 containing a diester
linker showed weak activity, but no improvement upon 13b.
This set of compounds represents a new set of ligands for MurG,
and the first inhibitors related in structure to the natural sub-
Data for 2a: Melting point 183–184 °C. 1H NMR (CD3CN,
400 MHz) d 7.49–7.09 (15H, m, Ar-H), 5.27 (1H, d, J = 12.0 Hz,
CHHPh), 5.18 (1H, d, J = 12.0 Hz, CHHPh), 5.02–4.98 (1H, m, H500),
3.86 (1H, dd, J = 9.5, 9.5 Hz, H400), 3.65 (1H, d, J = 9.5 Hz, H300),
2.84–2.83 (1H, m, NH100), 1.69 (3H, s, 200-CH3); 13C NMR (CDCl3,
100 MHz) d (not all quaternary C’s seen) 138.7, 136.2, 128.8,
128.3, 128.1, 127.9, 127.9, 127.6, 127.3, 126.6, 66.9, 66.5, 61.1,
55.2, 49.4, 22.7; m/z (ESI, +ve ion) 479.0 (MK)+, 463.1 (MNa)+,
441.2 (MH)+; mmax 3335, 1726, 1702 cmꢀ1
; HRMS calcd for
C27H24N2O4 (M+H+) 441.1814; found 441.1818. Data for 4a: colour-
less oil (0.34 g, 0.82 mmol, 69%). 1H NMR (CDCl3, 400 MHz) d 7.43–
7.23 (10H, m, Ar-H), 5.33 (1H, d, J = 12.5 Hz, CHHPh), 5.25 (1H, d,
J = 12.5 Hz, CHHPh), 4.83 (1H, d, J = 9.5 Hz, H500), 4.02 (1H, d,
J = 9.5 Hz, H300), 3.88 (1H, dd, J = 9.5, 9.5 Hz, H400), 3.61 (3H, s,
CO2CH3), 3.17 (3H, s, CO2CH3), 1.42 (3H, s, 200-CH3); 13C NMR
(CDCl3, 100 MHz) d 172.4, 171.9, 171.4, 139.2, 135.5, 128.6,
128.4, 128.3, 127.9, 127.7, 127.3, 67.6, 67.2, 62.9, 53.7, 52.5, 52.1,

2658
A. E. Trunkfield et al. / Bioorg. Med. Chem. 18 (2010) 2651–2663
51.5, 21.3; m/z (ESI, +ve ion) 434.1 (MNa)+, 412.2 (MH)+;
m
max 2950,
on carbon was added. The solution was stirred under a hydrogen
atmosphere at room temperature for 3 h. The solution was then fil-
tered through Celite and concentrated in vacuo to yield 8a as a
white solid (80 mg, 0.2 mmol, 100%). Melting point 149–153 °C.
1H NMR (CD3OD, 400 MHz) d 7.29 (2H, d, J = 7.5 Hz, Ar-H), 7.18–
7.15 (2H, m, Ar-H), 7.09–7.07 (1H, m, Ar-H), 7.05–7.00 (2H, m,
Ar-H), 6.91–6.87 (3H, m, Ar-H), 4.87 (1H, d, J = 6.0 Hz, H500), 4.14
(1H, d, J = 17.5 Hz, NHCHH of glycine linker), 4.08 (1H, d,
J = 17.5 Hz, NHCHH of glycine linker), 3.64 (1H, dd, J = 6.0, 8.5 Hz,
H400), 3.52 (1H, d, J = 8.5 Hz, H300), 1.59 (CH3); 13C NMR (CD3OD,
100 MHz) d 179.1, 176.6, 170.7, 170.7, 138.5, 137.5, 129.5, 129.4,
129.1, 127.9, 125.7, 122.2, 68.8, 68.7, 56.8, 55.2, 40.7, 23.1; m/z
(ESI, +ve ion) 430.1 (MNa)+, 408.1 (MH)+, 392.2 (MH–Me)+; vmax
1728 cmꢀ1; HRMS calcd for C23H25NO6 (M+H)+ 412.1760; found
412.1777. Data for 6a: colourless oil (0.26 g, 0.63 mmol, 73%). 1H
NMR (CDCl3, 400 MHz) d 7.38–7.25 (10H, m, Ar-H), 5.26 (1H, d,
J = 12.0 Hz, CHHPh), 5.15 (1H, d, J = 12.0 Hz, CHHPh), 4.63 (1H, d,
J = 6.5 Hz, H500), 3.46–3.42 (4H, m, H400 and CO2CH3), 3.28 (1H, d,
J = 7.0 Hz, H300), 3.25 (3H, s, CO2CH3), 1.71 (3H, s, 200-CH3); 13C
NMR (CDCl3, 100 MHz) d 174.1, 171.1, 170.7, 137.4, 135.4, 128.8,
128.5, 128.4, 128.3, 127.8, 126.8, 68.4, 67.6, 63.7, 57.7, 53.1, 51.8,
51.3, 28.6; m/z (ESI, +ve ion) 450.1 (MK)+, 434.1 (MNa)+, 412.2
(MH)+; mmax 3032, 2949, 1729 cmꢀ1; HRMS calcd for C23H25NO6
(M+H)+ 412.1760; found 412.1768. Data for 2b–g, 4b–e, 6b–e in
Supplementary data.
3301, 1701, 1697 cmꢀ1
; HRMS (LSIMS, +ve ion) calcd for
4.3. Method for hydrogenation
C22H21N3O5 (M+H)+ 408.1559; found 408.1564.
The benzyl ester (0.20 g, 0.45 mmol) was dissolved in THF
(20 ml), and 10 mol % of 10% palladium on carbon was added.
The solution was stirred under a hydrogen atmosphere at room
temperature for 3 h. The solution was then filtered through Celite
and concentrated in vacuo to yield the deprotected acid.
4.4.2. Method for HATU coupling
The cycloadduct 3b (0.11 g, 0.29 mmol) was dissolved in dry
THF (10 ml) and cooled to 0 °C. HATU (0.14 g, 0.38 mmol) was then
added followed by HOAt (52 mg, 0.38 mmol) or HOBt. The mixture
was stirred for 10 min at 0 °C and then DIPEA (75 mg, 0.58 mmol)
was added, followed by glycine benzyl ester tosylate (0.11 g,
0.32 mmol) and dry THF (10 ml). The mixture was stirred at 0 °C
for 2 h and then allowed to warm to room temperature and stirred
for a further 24 h. The solvent was removed in vacuo and the res-
idue was dissolved in ethyl acetate (10 ml) and water (10 ml). The
organic phase was washed with brine (3 ꢁ 10 ml), dried (MgSO4)
and concentrated in vacuo to yield the crude product. The product
was purified by flash chromatography (4:1, ethyl acetate/hexane)
Data for 3a16: White solid (0.16 g, 0.45 mmol, 100%). 1H NMR
(CD3OD, 400 MHz) d 7.46–7.14 (10H, m, Ar-H), 4.97 (1H, d,
J = 9.5 Hz, H500), 3.84 (1H, dd, J = 7.5, 9.5 Hz, H400), 3.56 (1H, d,
J = 7.5 Hz, H300), 1.68 (3H, s, Me); 13C NMR (CD3OD, 100 MHz),
175.2, 172.1, 172.0, 138.1, 133.5, 129.9, 129.6, 129.5, 129.2,
128.5, 127.9, 66.9, 63.6, 57.2, 51.7, 23.8; m/z (ESI, +ve ion) 395.2
(MNa2)+, 389.0 (MK)+, 373.1 (MNa)+, 351.1 (MH)+, 305.2
(MHꢀCO2H)+; mmax 2964, 1707 cmꢀ1. Data for 5a: white solid
(0.21 g, 0.65 mmol, 96%). Melting point 198–200 °C (dec). 1H
NMR (CD3CN, 400 MHz) d 7.32–7.16 (5H, m, Ar-H), 4.87 (1H, d,
J = 9.0 Hz, H500), 3.72 (1H, d, J = 7.5 Hz, H300), 3.65 (1H, dd, J = 7.5,
9.0 Hz, H400), 3.61 (3H, s, CO2CH3), 3.08 (3H, s, CO2CH3), 1.30 (3H,
s, 200-CH3); 13C NMR (CD3CN, 100 MHz) d (quaternary C’s not seen)
127.9, 127.5, 127.1, 62.3, 52.8, 51.9, 51.6, 50.9, 20.1; m/z (ESI, +ve
ion) 344.1 (MNa)+, 322.1 (MH)+. Data for 7a: white solid (0.18 g,
0.57 mmol, 94%). Melting point 200–202 °C (dec). 1H NMR (CD3OD,
400 MHz) d 7.44–7.39 (5H, m, Ar-H), 5.10 (1H, d, J = 7.5 Hz, H500),
3.83 (1H, dd, J = 7.5, 7.5 Hz, H400), 3.74 (3H, s, CO2CH3), 3.55 (1H,
d, J = 7.5 Hz, H300), 3.32 (3H, s, CO2CH3), 1.81 (3H, s, 200-CH3); 13C
NMR (CD3OD, 100 MHz) d (not all quaternary C’s seen) 171.6,
134.1, 130.2, 130.0, 128.0, 72.4, 62.7, 56.0, 52.9, 52.0, 51.9, 25.1;
m/z (ESI, +ve ion) 344.1 (MNa)+, 322.1 (MH)+, 276.2 (MHꢀCO2H)+;
to yield the coupled benzyl ester as
a white solid (0.15 g,
0.29 mmol, 99%). The benzyl ester (0.13 g, 0.25 mmol) was dis-
solved in methanol (10 ml) and 10 mol % of 10% palladium on car-
bon was added. The solution was stirred under a hydrogen
atmosphere at room temperature for 3 h. The solution was then fil-
tered through Celite and concentrated in vacuo, to give 8b as a
white amorphous solid (0.10 g, 0.23 mmol, 93%). 1H NMR (CD3OD,
400 MHz) d 7.68–7.66 (0.6H, d, J = 7.5 Hz, Ar-H), 7.36–6.81 (8.4H,
m, Ar-H), 5.15–5.13 (1H, m, H500), 4.20 (0.4H, d, J = 17.0 Hz, NHCH2
of glycine linker), 4.14 (0.4H, d, J = 17.0 Hz, NHCH2 of glycine lin-
ker), 4.03–3.95 (1H, m, NHCH2 of glycine linker, H400), 3.91–3.83
(4.2H, m, OCH3, NHCH2 of glycine linker, H400), 3.66 (0.4H, d,
J = 9.0 Hz, H300), 3.43 (0.6H, d, J = 8.0 Hz, H300), 1.69 (1.2H, s, 200-
CH3), 1.65 (1.8H, s, 200-CH3); 13C NMR (CD3OD, 100 MHz) d 176.9,
176.0, 159.0, 158.0, 138.3, 133.5, 130.4, 129.9, 129.6, 129.6,
128.0, 127.6, 125.9, 122.5, 121.5, 121.4, 111.3, 68.4, 63.9, 57.9,
56.6, 56.3, 56.2, 56.1, 52.7, 48.5, 42.5, 23.0, 22.8; m/z (ESI, +ve
ion) 482.1 (MNa2)+, 460.1 (MNa)+, 438.2 (MH)+, 395.2
mmax 3006, 2951, 1743, 1724, 1655 cmꢀ1
; HRMS calcd for
C16H19NO6 (M+H)+ 322.1291; found 322.1280. Data for 3b–g, 5b–
e, 7b–e given in Supplementary data.
4.4. General procedure for coupling of glycine linker to form
8a–c,e, 9a–e, 10a,b,e, 11a–d
(MHꢀCO2H)+; vmax 3374, 3303, 2944, 1701, 1685, 1654 cmꢀ1
;
HRMS (LSIMS, +ve ion) calcd for C23H23N3O6 (M+H)+ 438.1665;
found 438.1680.
4.4.1. Method for EDCI coupling
Data for 9a: white solid, 56 mg (EDCI coupling 57%, deprotec-
tion 71%). Melting point 144–146 °C. 1H NMR (CD3OD, 400 MHz)
d 7.30 (2H, d, J = 8.5 Hz, Ar-H), 7.20–7.13 (3H, m, Ar-H), 4.87 (peak
hidden behind D2O, H500), 3.88 (2H, s, NHCH2 of glycine linker), 3.73
(1H, dd, J = 8.5, 8.5 Hz, H400), 3.62–3.61 (4H, m, H300 and CO2CH3),
3.04 (3H, s, CO2CH3), 1.30 (3H, s, 200-CH3); 13C NMR (CD3OD,
100 MHz) d 177.2, 173.2, 173.2, 173.0, 141.9, 129.1, 128.8, 128.7,
67.8, 63.4, 54.3, 53.8, 52.7, 52.0, 42.2, 21.3; m/z (ESI, +ve ion)
417.1 (MK)+, 401.1 (MNa)+, 379.1 (MH)+; vmax 3345, 3302, 1719,
1658 cmꢀ1; HRMS calcd for C18H22N2O7 (M+H)+ 379.1505; found
379.1516.
Data for 10a/11a: white solid, 0.16 g (EDCI coupling 32%, depro-
tection 97%), containing 10a and bicyclic 11a in a 1:1 molar ratio.
1H NMR (CD3OD, 400 MHz) d 7.48 (2H, d, J = 7.5 Hz, Ar-H) 7.22
(3H + 5H, m, Ar-H), 5.01 (1H, d, J = 6.5 Hz, H500 11a), 4.95 (1H, d,
J = 7.0 Hz, H500 10a), 4.26 (1H, d, J = 17.5 Hz, NHCHH of glycine
The cycloadduct 3a (0.10 g, 0.29 mmol) was dissolved in dry
DCM (5 ml) and cooled to 0 °C. EDCIꢂHCl (60 mg, 0.32 mmol) was
then added followed by HOBt (49 mg, 0.32 mmol). The mixture
was stirred for 10 min at 0 °C and then a solution of glycine benzyl
ester tosylate (0.11 g, 0.32 mmol) and triethylamine (44 ll,
0.32 mmol) in dry DCM was added. The mixture was stirred at
0 °C for 2 h and was then allowed to warm to room temperature
and stirred for a further 24 h. The solvent was removed in vacuo
and the residue was dissolved in ethyl acetate (10 ml) and water
(10 ml). The organic phase was washed with brine (3 ꢁ 10 ml),
dried (MgSO4) and concentrated in vacuo to yield the crude prod-
uct. The product was purified by flash chromatography (1:1, hex-
ane/ethyl acetate) to yield the coupled benzyl ester as a white
solid (0.11 g, 0.23 mmol, 79%). The benzyl ester (0.10 g, 0.20 mmol)
was dissolved in methanol (10 ml) and 10 mol % of 10% palladium

A. E. Trunkfield et al. / Bioorg. Med. Chem. 18 (2010) 2651–2663
2659
linker 11a), 4.18 (1H, d, J = 17.5 Hz, NHCHH of glycine linker 11a),
4.02 (1H, d, J = 18.0 Hz, NHCHH of glycine linker 10a), 3.97 (1H, d,
J = 18.0 Hz, NHCHH of glycine linker 10a), 3.75 (1H, dd, J = 6.5,
8.0 Hz, H400 11a), 3.70 (3H, s, CO2CH3 11a), 3.56 (2H, m, H300 11a
and H400 10a), 3.46 (1H, d, J = 7.0 Hz, H300 10a), 3.18 (3H, s, CO2CH3
10a), 3.13 (3H, s, CO2CH3 11a), 1.73 (3H, s, 200-CH3 11a), 1.65 (3H, s,
200-CH3 11a); 13C NMR (CD3OD, 100 MHz) d 178.7, 176.4, 172.9,
172.6, 172.5, 170.5, 138.5, 137.4, 129.4 129.4, 128.5, 127.8, 68.0,
67.9, 63.3, 56.8, 55.7, 53.7, 53.2, 52.8, 52.2, 51.9, 50.0, 42.4, 40.7,
27.2, 22.42; m/z (ESI, +ve ion) 401.1 (M(10a)Na)+, 379.1
(M(10a)H)+, 347.1 (M(11a)H)+; vmax 3328, 2989, 1746,
1711 cmꢀ1; HRMS (LSIMS, +ve ion) calcd for C18H22N2O7 (Ma+H)+
379.1497; found 379.1505. Data for 8c, 8e, 9b–e, 10b/11b, 10e,
11c–d given in Supplementary data.
Compound 13b.1: Melting point 197–200 °C.1H NMR (CD3OD,
500 MHz) d 7.61 (1H, d, J = 8.0 Hz, H6), 7.55 (1H, d, J = 7.5 Hz, Ar-
H), 7.53–7.36 (4H, m, Ar-H), 7.23–7.17 (2H, m, Ar-H), 7.12–7.06
(2H, m, Ar-H), 5.75 (1H, d, J = 4.5 Hz, H10), 5.71 (1H, d, J = 8.0 Hz,
H5), 5.46 (1H, d, J = 10.0 Hz, H500), 4.38–3.97 (7H, m, H20, H30,
H40, H400, H300, NHCH2 of glycine linker), 3.83 (3H, s, OCH3), 3.57
(2H, d, J = 5.0 Hz, CH2 50), 1.97 (3H, s, 200-CH3); 13C NMR (CD3OD,
125 MHz) d 174.3, 173.1, 172.4, 170.0, 169.0, 167.3, 164.7, 157.5,
156.4, 150.8, 141.6, 141.3, 136.5, 131.7, 130.9, 130.7, 128.6,
128.4, 126.2, 126.1, 124.9, 121.2, 120.7, 120.3, 118.3, 110.9,
110.6, 101.7, 101.5, 90.5, 90.1, 82.4, 82.3, 73.4, 70.8, 70.5, 69.4,
67.4, 62.8, 55.1, 55.0, 54.3, 48.2, 48.1, 43.2, 41.4, 40.7, 40.4, 24.9,
19.7; m/z (ESI, +ve ion) 701.2 (MK)+, 685.2 (MNa)+, 663.2 (MH)+;
vmax 3345, 1668, 1179, 1130 cmꢀ1; HRMS (micrOTOF, +ve ion)
calcd for C32H35N6O10 (M+H)+ 663.2409; found 663.2424.
Compound 13b.2: Melting point 160–164 °C. 1H NMR (CD3OD,
500 MHz) d 7.60 (0.5H, d, J = 8.0 Hz, H6), 7.56–7.51 (1H, m, Ar-H),
7.46 (0.5H, d, J = 8.0 Hz, H6), 7.41–7.36 (1H, m, Ar-H), 7.23–7.12
(2H, m, Ar-H), 7.12–7.05 (4H, m, Ar-H), 6.99–6.93 (1H, m, Ar-H),
5.78–5.77 (1H, m, H5, H10), 5.75 (0.5H, d, J = 4.5 Hz, H10), 5.68–
5.67 (1.5H, m, H5, H500), 4.36 (0.5H, d, J = 16.5 Hz, NHCHH of gly-
cine linker), 4.25 (1.5H, m, NHCHH of glycine linker), 4.20–4.17
(1H, m, H400), 4.14 (0.5H, dd, J = 4.5, 5.5 Hz, H20), 4.11 (0.5H, dd,
J = 4.5, 5.5 Hz, H20), 4.07–4.05 (1H, m, H300), 4.02–3.95 (5H, m,
H30, H40, OCH3), 3.53 (2H, m, CH2 50), 1.97 (3H, s, 200-CH3); 13C
NMR (CD3OD, 125 MHz) d 173.1, 172.8, 172.4, 169.1, 168.9,
167.3, 167.1, 164.6, 164.4, 156.4, 150.9, 150.8, 141.7, 141.3,
136.5, 136.5, 130.7, 128.4, 128.3, 126.3, 126.2, 125.0, 124.9,
120.9, 120.7, 120.3, 118.3, 110.6, 101.7, 101.7, 90.7, 90.2, 82.4,
82.1, 73.4, 73.3, 70.9, 70.6, 67.4, 67.4, 62.9, 62.8, 55.0, 52.9, 49.4,
49.2, 41.4, 41.1, 40.5, 40.4, 19.7, 19.6; m/z (ESI, +ve ion) 701.2
(MK)+, 685.3 (MNa)+, 663.2 (MH)+; vmax 3309, 1663, 1176,
1139 cmꢀ1; HRMS (mircrOTOF, +ve ion) calcd for C32H35N6O10
(M+H)+ 663.2421; found 663.2409.
4.5. General procedure for uridine coupling and deprotection
The cycloadduct-linker (8b, 70 mg, 0.16 mmol) was dissolved in
dry THF (10 ml) and cooled to 0 °C. HATU (80 mg, 0.21 mmol) was
then added followed by HOAt (28 mg, 0.21 mmol). The mixture
was stirred for 10 min at 0 °C and then DIPEA (40 mg, 0.32 mmol)
was added followed by 50-amino, 50-deoxy-20, 30-O-bis(tert-butyl-
dimethylsilyl) uridine (12, 80 g, 0.18 mmol). The mixture was stir-
red at 0 °C for 2 h and then allowed to warm to room temperature
and stirred for a further 48 h. The solvent was removed in vacuo
and the residue was dissolved in ethyl acetate (10 ml) and water
(10 ml). The organic phase was washed with brine (3 ꢁ 10 ml),
dried (MgSO4) and concentrated in vacuo to yield the crude prod-
uct. The product was purified by flash chromatography (1:1 hex-
ane/ethyl acetate, 100% ethyl acetate) to yield the coupled
product as a pure white solid, (50 mg, 0.06 mmol, 36%) (1:1 ratio
of diastereomers a and b). Melting point 174–179 °C. 1H NMR
(CDCl3, 400 MHz) d 8.10 (1H, br t, J = 11.4 Hz, NHCH2 a of glycine
linker), 7.99–7.95 (1H, m, NHCH2 b of glycine linker), 7.46 (1H,
br d, J = 8.5 Hz, H6 b), 7.31 (1H, d, J = 9.0 Hz, H6 a), 7.29–6.71
(9H + 9H, m, Ar-H a and b), 5.74–5.49 (2H + 2H, m, H5 a and b,
H10 a and b), 5.11–5.08 (1H, m, H500 a), 5.00–4.99 (1H, m, H500 b),
4.23–3.04 (10H + 10H, m, H20 a and b, H30 a and b, H40 a and b,
CH2 50 a and b, OCH3 a and b, H400 a and b, H300 a and b), 1.64
(3H, s, 200-CH3 a), 1.59 (3H, s, 200-CH3 b), 0.83, 0.81 (2 ꢁ 9H, s,
C(CH3)3 a), 0.77, 0.73 (2 ꢁ 9H, s, C(CH3)3 b), 0.04, 0.03, 0.02,
ꢀ0.02, ꢀ0.04, ꢀ0.06, ꢀ0.07, ꢀ0.11 (8 ꢁ 3H, s, Si-CH3 a and b); 13C
NMR (CDCl3, 100 MHz) d 177.6, 177.5, 175.3, 173.4, 172.7, 168.5,
167.9, 167.5, 157.9, 156.0, 142.4, 141.4, 136.1, 131.3, 129.2,
128.9, 128.6, 128.5, 126.6, 126.4, 126.2, 125.9, 125.1, 124.6,
124.3, 121.0, 120.9, 120.5, 120.1, 110.2, 109.9, 102.7, 102.3, 90.5,
89.8, 86.8, 85.5, 84.7, 73.9, 73.7, 73.6, 72.9, 72.6, 66.4, 62.5, 62.4,
55.6, 55.4, 54.5, 42.2, 41.9, 41.6, 41.5, 25.8, 25.8, 0.97, ꢀ4.5, ꢀ4.7,
ꢀ4.8; m/z (ESI, +ve ion) 913.4 (MNa)+, 891.4 (MH)+, 779.4 (MꢀUr)+,
647.3 (Mꢀ2 ꢁ TBS–Me)+, 474.2 (MNaꢀuracil–ribose–2 ꢁ TBS),
452.2 (MH+ꢀuracil–ribose–2 ꢁ TBDMS); vmax 3313, 2931, 2856,
Data for 14a: White solid (16 mg, coupling 31%, deprotection
74%) isolated as a 1:1 ratio of diastereoisomers a and b). HPLC
retention time 19.77 min. 1H NMR (D2O, 400 MHz)
d 7.70
(1H + 1H, d, J = 8.0 Hz, H6 a and b), 7.55–7.53 (3H + 3H, m, Ar-H
a and b), 7.47–7.45 (2H + 2H, m, Ar-H a and b), 5.91 (1H + 1H, d,
J = 8.0 Hz, H5 a and b), 5.82 (1H + 1H, d, J = 4.0 Hz, H10 a and b),
5.48 (1H + 1H, d, J = 10.0 Hz, H500 a and b), 4.46 (1H + 1H, dd,
J = 10.0, 10.0 Hz, H400 a and b), 4.39 (1H + 1H, dd, J = 4.0, 4.0 Hz,
H20 a and b), 4.27 (1H + 1H, d, J = 10.0 Hz, H300 a and b), 4.21–
4.16 (3H + 3H, m, NHCHH of glycine linker a and b, H30 a and b,
H40 a and b), 4.09 (1H + 1H, d, J = 17.0 Hz, NHCHH of glycine linker
a and b), 3.92 (3.5H + 3.5H, s, CO2CH3 a and b, CHH 50 a and b),
3.88–3.87 (0.5H + 0.5H, m, CHH 50 a and b), 3.56 (1H + 1H, dd,
J = 7.5, 14.5 Hz, CHH 50 a and b), 3.35 (3H + 3H, s, CO2CH3 a and
b), 1.84 (3H + 3H, s, 200-CH3 a and b); 13C NMR (D2O, 100 MHz) d
170.7, 170.7, 169.7, 166.2, 166.2, 151.3, 141.9, 132.1, 129.9,
129.2, 127.2, 101.8, 90.6, 81.9, 73.4, 70.4, 68.8, 61.3, 53.7, 52.6,
52.5, 50.0, 43.4, 40.9, 17.9; m/z 642.1 (MK)+, 626.2 (MNa)+, 604.3
(MH)+; vmax 3293, 2980, 1662 cmꢀ1; HRMS calcd for C27H33N5O11
(M+H)+ 604.2255; found 604.2242.
Data for 16a: White solid (62 mg, coupling 33%, deprotection
75%) isolated as 1:1 ratio of diastereoisomers a and b. HPLC reten-
tion time 20.21 min. 1H NMR (CD3OD, 500 MHz) d 7.65–7.63
(1H + 1H, 2 ꢁ overlapping d, J = 7.5 and 7.5 Hz, H6 c and d), 7.42–
7.36 (5H + 5H, m, Ar-H c and d), 5.79 (1H + 1H, d, J = 4.5 Hz, H10 c
and d), 5.77 (1H, d, J = 7.5 Hz, H5 c or d), 5.73 (1H, d, J = 8.0 Hz,
H5 c or d), 5.32–5.29 (1H + 1H, 2 ꢁ overlapping d, J = 7.5 and
7.5 Hz, H500 c and d), 4.33–4.17 (3H + 3H, m, NCH2 c and d, H20 c
and d), 4.05–3.99 (2H + 2H, m, H30 c and d, H40 c and d), 3.94–
3.90 (1H + 1H, m, H400 c and d), 3.81 (1H, d, J = 9.5 Hz, H300 c or
d), 3.79 (1H, d, J = 9.5 Hz, H300 c or d), 3.64–3.56 (2H + 2H, m, CH2
50 c and d), 3.25 (3H, s, CO2CH3 c or d), 3.24 (3H, s, CO2CH3 c or
1707 cmꢀ1
; HRMS (LSIMS, +ve ion) calcd for C44H62N6O10Si2
(M+H)+ 891.4144; found 891.4137.
The 50-cycloadduct-aminoacyl-20,30-O-bis(tert-butyldimethylsi-
lyl) uridine compound (80 mg) was dissolved in a mixture of
DCM, water and TFA in a 4:2:3 ratio, respectively. The mixture
was stirred at room temperature for 24 h and was then diluted
with water (10 ml) and DCM (10 ml). The aqueous portion was ex-
tracted with DCM (3 ꢁ 10 ml) and then concentrated in vacuo to
yield the crude product. The product was purified by reverse phase
HPLC on
(250 ꢁ 10.0 mm, 4
a
Phenomenex Synergi 4u fusion-RP 80A column
) using a water/ethanol gradient (0–100% eth-
l
anol over 35 min, 3.5 ml/min). Isomer 13b.1 (32 mg, 0.04 mmol,
46%) was eluted at 19.45 min and 13b.2 (29 mg, 0.04 mmol, 42%)
was eluted at 21.17 min.

2660
A. E. Trunkfield et al. / Bioorg. Med. Chem. 18 (2010) 2651–2663
d), 1.80 (3H + 3H, s, 200-CH3 c and d); 13C NMR (CD3OD, 125 MHz) d
176.2, 176.0, 175.4, 175.2, 172.7, 172.6, 168.8, 168.8, 166.2, 152.4,
143.6, 143.3, 134.9, 134.6, 130.2, 130.1, 129.9, 129.8, 128.6, 127.8,
103.2, 103.1, 92.2, 92.1, 83.9, 83.9, 74.7, 74.7, 72.2, 72.1, 68.3, 68.3,
68.0, 54.8, 52.8, 52.7, 49.5, 49.3, 42.5, 42.4, 42.1, 41.9, 21.4, 21.3;
m/z (ESI, +ve ion) 610.1 (MK)+, 594.2 (MNa)+, 572.2 (MH)+; vmax
3294, 2956, 1665, 1560 cmꢀ1; HRMS (LSIMS, +ve ion) calcd for
C26H29N5O10 (M+H)+ 572.1993; found 572.1997. Data for 13C,
13e, 14b, 15e, 16b–d given in Supplementary data.
65.1, 55.6, 55.4, 54.6, 51.6, 51.5, 51.3, 45.7, 44.6, 41.2, 40.7, 40.4,
40.3, 38.9, 38.6, 35.6, 35.3, 34.9, 34.6; m/z (ESI, +ve ion) 715.1
(MK)+, 699.2 (MNa)+, 677.3 (MH)+; vmax 3350, 1713, 1661, 1167,
1130 cmꢀ1. HRMS (MicrOTOF, +ve ion) calcd for C33H37N6O10
(M+H)+ 677.2566; found 677.2580.
4.7. Preparation of proline-linked compounds
Cycloadducts 3b, 5b, and 5b were coupled with
L-proline benzyl
ester, and deprotected by hydrogenation, using the methods de-
4.6. Preparation of N-methylated sarcosine-linked compound
19
scribed above for compound 8, to give the L-prolyl adducts. These
compound were coupled with 50-amino-uridine derivative 12,
and deprotected as described above for compounds 13–16.
Data for 20a: The product was purified by HPLC using a water/
methanol gradient and 20a was eluted at 17.89 min and concen-
A
solution of 2f (0.43 g, 0.91 mmol) and water (3.40 ll,
0.21 mmol) in dry THF (4 ml) was added dropwise over a period
of 20 min to a suspension of sodium hydride (82 mg, 3.40 mmol)
in dry THF (6 ml), whilst a temperature of 0 °C was maintained.
The mixture was stirred for 10 min and then dimethyl sulfate
(0.29 ml, 3.06 mmol) was added dropwise at 0 °C. The mixture
was stirred for 3 h and then allowed to warm to room temperature
and stirred for a further 21 h. The reaction mixture was quenched
by the addition of 30% ammonium hydroxide (5 ml) over a period
of 10 min, maintaining a temperature of below 20 °C, and stirring
was continued for a period of 1 h. The mixture was diluted with
toluene (20 ml) and water (10 ml). The organic phase was sepa-
rated and washed with water (10 ml) and concentrated in vacuo.
The product was purified by flash chromatography (4:1, hexane/
ethyl acetate) to yield the N-methylated product as a pure white
solid (0.31 g, 0.31 mmol, 38%). Melting point 108–111 °C. 1H
NMR (CDCl3, 400 MHz) d 7.46 (1H, dd, J = 1.5, 7.5 Hz, Ar-H), 7.27–
7.15 (9H, m, Ar-H), 6.88–6.81 (4H, m, Ar-H), 5.21 (1H, d,
J = 13.0 Hz, CHHPh), 5.17 (1H, d, J = 13.0 Hz, CHHPh), 4.46 (1H, d,
J = 10.0 Hz, H500), 3.82 (3H, s, OCH3), 3.66 (1H, dd, J = 8.0, 10.0 Hz,
H400), 3.36 (1H, d, J = 8.0 Hz, H300), 2.21 (3H, s, N100-CH3), 1.45 (3H,
s, 200-CH3); 13C NMR (CDCl3, 100 MHz) d 174.9, 173.9, 171.7,
158.1, 136.1, 131.7, 129.0, 128.9, 128.5, 128.4, 128.3, 128.1,
127.1, 126.2, 125.4, 120.6, 110.4, 70.1, 67.1, 62.3, 55.6, 54.9, 46.5,
34.4, 15.6; m/z (ESI, +ve ion) 523.1 (MK)+, 507.2 (MNa)+, 485.2
(MH)+; vmax 2958, 2834, 1738, 1712 cmꢀ1; HRMS (LSIMS, +ve
ion) calcd for C29H28N2O5 (M+H)+ 485.2076; found 485.2057. This
benzyl ester was deprotected by hydrogenation (100% yield), cou-
pled with glycine benzyl ester (49% yield), and further deprotected
by hydrogenation (100% yield), as described above for compounds
8a–e, to give compound 18 (32 mg). 1H NMR (CD3OD, 400 MHz) d
7.61–6.91 (9H, m, Ar-H), 4.69–4.61 (1H, m, H500), 4.24–4.06 (1H, m,
H200), 3.88–3.67 (7H, m, OCH3, H400, H300, CH2 of sarcosine linker),
3.34 (3H, s, NCH3 of sarcosine linker), 2.18 (3H, s, N100-CH3); 13C
NMR (CD3OD, 100 MHz) d not all quaternary C’s were seen)
177.1, 176.4, 159.3, 133.6, 130.3, 130.1, 129.9, 129.6, 129.6,
129.5, 127.5, 111.4, 69.4, 66.6, 56.0, 51.1, 46.9, 40.3, 36.6; m/z
(ESI, +ve ion) 496.1 (MNa2–H)+, 490.1 (MK)+, 474.2 (MNa)+, 452.2
(MH)+. Coupling to uridine derivative 12 and deprotection was car-
ried out as described above, to give compound 19 as a white solid
(9 mg, 9%). HPLC retention time 20.43 min. A mixture of cis and
trans N-methyl amide rotamers was observed by NMR spectros-
copy. 1H NMR (CD3OD, 500 MHz) d7.72–7.30 (7.5H, m, Ar-H, H6),
7.21–7.13 (2H, m, Ar-H), 7.00 (0.5H, m, Ar-H), 5.83–5.73 (1.5H,
m, H5, H10), 5.41–5.38 (0.5H, m, H10, H500), 5.27–5.19 (1H, m,
H500), 4.66–3.49 (13H, m, H20, H30, H40, CH2 50, H200, H500, H300,
OCH3, N(CH3)CH2), 3.40–3.38 (2H, m, NCH3 of sarcosine linker),
3.15 (1H, s, NCH3 of sarcosine linker), 2.94–2.93 (2H, m, N100-
CH3), 2.27–2.26 (1H, m, N100-CH3); 13C NMR (CD3OD, 125 MHz) d
171.3, 165.4, 164.7, 157.9, 157.1, 150.8, 132.4, 131.6, 128.8,
128.7, 128.3, 128.0, 127.5, 126.6, 126.1, 125.9, 122.1, 111.5,
110.0, 101.9, 101.6, 101.5, 91.6, 90.8, 90.4, 89.5, 82.9, 82.6, 82.5,
82.1, 81.9, 73.6, 73.3, 72.9, 72.1, 71.6, 71.1, 70.9, 70.8, 70.5, 68.1,
trated to yield a white solid (25 mg; L-Pro coupling 91%, deprotec-
tion 100%, uridine coupling/deprotection 15%) isolated as a 1:1
ratio of diastereoisomers a and b. 1H NMR (CD3OD, 500 MHz) d
7.66 (1H + 1H, d, J = 8.0 Hz, H6 a and b), 7.50–7.39 (5H + 5H, m,
Ar-H a and b), 7.25–7.23 (2H + 2H, m, Ar-H a and b), 7.13–7.07
(2H + 2H, m, Ar-H a and b), 5.76–5.70 (2H + 2H, m, H10 a and b,
H5 a and b), 5.53 (1H + 1H, d, J = 10.0 Hz, H500 a and b), 4.44
(1H + 1H, dd, J = 7.5, 9.5 Hz, NCH of proline linker a and b), 4.29–
4.16 (3H + 3H, m, H20 a and b, H30 a and b, H400 a and b), 4.15–
3.95 (3H + 3H, m, NCH2 of proline linker a and b, H40 a and b),
3.77 (1H + 1H, m, H300 a and b), 3.74 (3H + 3H, s, OCH3 a and b),
3.60–3.50 (2H + 2H, m, CH2 50 a and b), 2.43–2.38 (1H + 1H, m,
CH2 of proline linker a and b), 2.17–2.12 (2H + 2H, m, CH2 of pro-
line linker a and b), 2.10 (3H + 3H, s, 200-CH3 a and b), 1.94–1.86
(1H + 1H, m, CH2 of proline linker a and b); 13C NMR (CD3OD,
125 MHz) d174.4, 174.3, 173.3, 173.1, 168.7, 166.1, 158.8, 152.4,
152.3, 143.6, 143.4, 133.2, 133.0, 132.7, 130.1, 129.9, 127.3,
123.1, 117.9, 112.8, 103.1, 103.0, 92.8, 83.9, 74.6, 72.4, 72.3, 64.7,
62.0, 56.9, 53.6, 51.6, 42.4, 30.7, 26.9, 21.3; m/z (ESI, +ve ion)
725.3 (MNa)+, 703.3 (MH)+; HRMS (ESI, +ve ion) calcd for
C35H38N6O10 (M+H)+ 703.2722; found 703.2741. Data for 20b–d gi-
ven in Supplementary data.
4.8. Preparation of ester-linked compounds 25 and 26
Cycloadduct isopropyl ester 2g (0.20 g, 0.47 mmol) was dis-
solved in dry THF (5 ml) and cooled to ꢀ78 °C under a nitrogen
atmosphere. A 1 M solution of DIBAL-H in hexane (1.00 ml,
0.99 mmol) was added dropwise. The mixture was stirred at
ꢀ78 °C for 1 h and was then quenched by the cautious addition
of methanol (1 ml). The THF was removed in vacuo and the residue
was dissolved in diethyl ether (10 ml). A saturated solution of
potassium sodium tartrate (10 ml) was then added and the mix-
ture was left to stir overnight until two layers had separated. The
organic portion was washed with brine (2 ꢁ 10 ml), dried (MgSO4)
and concentrated in vacuo. The product was purified by flash chro-
matography (3:1, hexane/ethyl acetate) to yield alcohol 22 as a
white crystalline solid (44 mg, 0.12 mmol, 26%). Melting point
104–107 °C. 1H NMR (CDCl3, 300 MHz) d 7.78 (1H, d, J = 7.5 Hz,
Ar-H), 7.53–7.50 (2H, m, Ar-H), 7.33–7.23 (3H, m, Ar-H), 7.16–
7.11 (1H, m, Ar-H), 7.06–7.01 (1H, m, Ar-H), 6.86 (1H, d,
J = 8.5 Hz, Ar-H), 5.04 (1H, dd, J = 2.0, 3.5 Hz), 4.90 (1H, d,
J = 12.0 Hz), 4.67 (1H, br d, J = 9.5 Hz), 4.49 (1H, br d, J = 12.0 Hz),
3.81 (3H, s, OCH3), 3.72 (1H, dd, J = 3.5, 5.5 Hz), 3.01 (1H, dd,
J = 2.0, 5.5 Hz), 1.92 (2H, br s, NH2), 1.39 (3H, s, 200-CH3); 13C NMR
(CDCl3, 75 MHz) d 171.9, 156.2, 137.2, 128.9, 128.7, 127.9, 127.0,
125.4, 120.9, 120.5, 109.2, 95.1, 88.1, 65.1, 57.1, 56.5, 55.2, 50.4,
19.5; m/z (ESI, +ve ion) 405.1 (MK)+, 389.1(MNa)+, 367.1 (MH)+,
349.1 (M–OH)+; vmax 3343, 2924, 1708, 1045, 753 cmꢀ1. HRMS
(LSIMS, +ve ion) calcd for C21H22N2O4 (M+H)+ 367.1645; found
367.1658.

A. E. Trunkfield et al. / Bioorg. Med. Chem. 18 (2010) 2651–2663
2661
4.9. Malonyl diester 25
(8 mg, 0.01 mmol, 11%) as a 1:1 ratio of diastereoisomers a and b.
1H NMR (CD3OD, 500 MHz) d 7.72–7.66 (3H + 3H, m, Ar-H a and b,
H6 a and b), 7.53–7.43 (4H + 4H, m, Ar-H a and b), 7.31 (1H + 1H,
dt, J = 1.5, 7.5 Hz, Ar-H a and b), 7.16 (1H + 1H, d, J = 7.5 Hz, Ar-H a
and b), 7.12 (1H + 1H, d, J = 8.5 Hz, Ar-H a and b), 6.42 (1H + 1H, s),
6.00 (1H + 1H, dd, J = 6.5, 6.5 Hz), 5.86–5.85 (1H + 1H, m, H10 a and
b), 5.79–5.77 (1H + 1H, m, H5 a and b), 5.53 (1H + 1H, d, J = 6.5 Hz,
H500 a and b), 4.50–4.43 (1H + 1H, m, CHH 50 a and b), 4.36–4.31
(1H + 1H, m, CHH 50 a and b), 4.24 (1H + 1H, dd, J = 4.5, 4.5 Hz,
H20 a and b), 4.18–4.14 (2H + 2H, m, H30 a and b, H40 a and b),
3.87–3.80 (2H + 2H, m, H400 a and b, H300 a and b), 3.77 (3H + 3H,
50-Malonyl 20,30-isopropylidene uridine 23 was prepared by
EDCI coupling of monobenzyl malonate with 20,30-isopropylidene
uridine (23% yield), followed by hydrogenation (100% yield). Com-
pound 23 (87 mg, 0.23 mmol) was dissolved in dry THF (5 ml) and
cooled to 0 °C. HATU (128 mg, 0.34 mmol) was added and the mix-
ture was stirred at 0 °C for 10 min. DIPEA (60 mg, 0.46 mmol) was
added followed by alcohol 22 (100 mg, 0.26 mmol). The mixture
was stirred at 0 °C for 4 h and was then allowed to warm to room
temperature and stirred for 24 h. The solvent was removed in va-
cuo and the residue was partitioned between ethyl acetate and
water. The organic portion was washed with saturated sodium
bicarbonate (10 ml), brine (2 ꢁ 10 ml) and then dried (MgSO4)
and concentrated in vacuo. The crude yellow oil (90 mg) was dis-
solved in DCM (2 ml). Sixty-six percentage of TFA (3 ml) was then
added and the mixture was stirred at room temperature for 24 h.
DCM (5 ml) and water (5 ml) was then added and the mixture
was allowed to separate. The aqueous phase was extracted with
DCM (2 ꢁ 5 ml) and then concentrated in vacuo. The residue was
then purified by HPLC using a water/methanol gradient and 25
was eluted at 24.80 min and concentrated to yield a white solid
(29 mg, 0.11 mmol, 48%), isolated as a 1:1 ratio of diastereoisomers
a and b. 1H NMR (CD3OD, 400 MHz) d 9.97 (1H + 1H, s, NH3 a and
b), 7.70 (1H, d, J = 8.0 Hz, H6 a or b), 7.60 (1H, d, J = 8.0 Hz, H6 a or
b), 7.40–7.34 (2H + 2H, m, Ar-H a and b), 7.22–7.12 (5H + 5H, m,
Ar-H a and b), 6.99–6.94 (1H + 1H, m, Ar-H a and b), 6.72–6.69
(1H + 1H, m, Ar-H a and b), 5.98 (1H, d, J = 10.5 Hz, H500 a or b),
5.96 (1H, d, J = 10.5 Hz, H500 a or b), 5.89–5.87 (1H + 1H, m, H10 a
and b), 5.76 (1H, d, J = 8.0 Hz, H5 a or b), 5.66 (1H, d, J = 8.0 Hz,
H5 a or b), 4.78 (1H + 1H, d, J = 4.0 Hz), 4.36–4.06 (5H + 5H, m,
H20 a and b, H30 a and b, H40 a and b, CH2 50 a and b), 4.02 (3H,
s, OCH3 a or b), 4.00 (3H, s, OCH3 a or b), 3.70 (1H + 1H, d,
J = 7.5 Hz, H300 a and b), 3.65 (1H + 1H, ddd, J = 3.0, 7.5, 10.5 Hz,
H400 a and b), 3.25 (1H + 1H, dd, J = 3.0, 16.0 Hz, CHHOCO a and
b), 3.02 (1H + 1H, dd, J = 3.0, 16.0 Hz, CHHOCO a and b), 1.71 (3H,
s, 200-CH3 a or b), 1.69 (3H, s, 200-CH3 a or b); 13C NMR (CD3OD,
125 MHz) d 199.3, 199.2, 171.5, 169.0, 167.7, 166.0, 157.8, 152.3,
142.6, 142.3, 137.4, 131.1, 129.6, 128.5, 128.1, 126.1, 125.8,
124.7, 122.7, 112.3, 111.7, 103.2, 91.2, 91.1, 86.7, 86.7, 83.0, 82.9,
75.1, 75.0, 74.9, 72.1, 71.1, 65.6, 65.5, 59.2, 59.0, 57.3, 57.3, 56.5,
56.3, 48.9, 20.0; m/z (micrOTOF, +ve ion) 701.2 (MNa)+; vmax
3392, 1679, 1399 cmꢀ1; HRMS (micrOTOF, +ve ion) calcd for
C33H34N4O12Na, 701.2065; found 701.2069.
s, OCH3 a and b), 2.87–2.80 (4H + 4H, m, CH 1000 a and b, CH2 2000
2
a and b), 1.86 (3H + 3H, s, 200-CH3 a and b); 13C NMR (CD3OD,
125 MHz) d 172.2, 170.4, 170.4, 170.1, 164.7, 157.9, 150.8, 141.1,
137.2, 131.5, 130.0, 128.7, 126.4, 122.2, 122.1, 121.5, 116.7,
111.5, 101.5, 98.3, 90.8, 90.4, 90.3, 81.5, 73.6, 73.6, 69.8, 64.7,
63.6, 55.3, 50.5, 49.2, 28.4, 28.1, 17.2; m/z (ESI, +ve ion) 731.1
(MK)+, 715.2 (MNa)+, 693.2 (MH)+; vmax 2972, 1674, 1130 cmꢀ1
;
HRMS (ESI, +ve ion) calcd for C34H36N4O12 (M+H)+ 693.2402; found
693.2412.
4.11. Purification of recombinant E. coli MurG
Freshly transformed E. coli C43 expressing E. coli murG from a
pET3a vector were grown in 6L of LB medium supplemented with
100 lg/ml ampicillin, and induced with IPTG (1 mM) at OD600 0.5.
The induced cell culture was grown for another 3 h and then the
cells were centrifuged at 10,000 g for 10 min. The cell pellets were
resuspended in 50 mM Tris–HCl pH 7.9 (50 ml) containing 2.5 mg/ml
lysozyme, PMSF (0.2 mM), leupepsin (0.2 mM) and pepstatin
(0.02 mM) were added and the suspension was sonicated. The sus-
pension was centrifuged at 10,000g for 20 min and then the super-
natant was transferred to fresh tubes and centrifuged at 50,000g
for 1 h. The pellet was resuspended in 50 mM HEPES pH 7.6
(15 ml) containing 2 mM MgCl2, 0.5% CHAPS, 10% glycerol, 0.5 M
NaCl and 5 mM imidazole and stirred at 4 °C for 1 h. The suspen-
sion was then centrifuged again at 50,000g for 1 h, and the super-
natant was retained. The pellet was suspended again in 15 ml of
50 mM HEPES pH 7.6, 2 mM MgCl2, 0.5% CHAPS, 10% glycerol,
0.5 M NaCl and 5 mM imidazole and stirred at 4 °C for 1 h. The sus-
pension was then centrifuged at 50,000g for 1 h and the superna-
tant was pooled with the supernatant from the previous
centrifugation. The supernatant was loaded onto a Ni affinity Hi-
sTrap fast flow column (5 ml) equilibrated with 50 mM HEPES
pH 7.6, 2 mM MgCl2, 0.5% CHAPS, 10% glycerol, 0.5 M NaCl and
5 mM imidazole (Buffer A). The column was washed with 30 ml
of Buffer A. The column was eluted with buffer solutions based
upon Buffer A containing 50 mM imidazole (30 ml) and 250 mM
imidazole (50 ml). MurG eluted with the 250 mM wash. The mate-
rial was then loaded onto a Superdex 200 HR 16/60 column (Phar-
macia Biotech) at a flow rate of 2 ml/min of 20 mM Tris–HCl pH
7.9, 150 mM NaCl, 50 mM EDTA, 4 mM DTT and 0.5% CHAPS. The
protein eluted as a symmetrical peak at 180–250 ml. Fractions con-
taining MurG were pooled and concentrated using Centricon tubes
to a final volume of 4.5 ml. The protein concentration was esti-
mated to be 4.15 mg/ml and the protein was seen to be homoge-
neous by a Coomassie blue-stained SDS–polyacrylamide gel. The
purified enzyme was stored at ꢀ20 °C and was stable for at least
three months.
4.10. Succinyl diester 26
50-Succinyl 20,30-isopropylidene uridine 24 was prepared by
reaction of succinic anhydride with 20,30-isopropylidene uridine
(40% yield). Compound 24 (35 mg, 0.09 mmol) was dissolved in
dry THF (5 ml) and cooled to 0 °C. HATU (45 mg, 0.12 mmol) was
then added. The mixture was stirred for 10 min at 0 °C and then DI-
PEA (23 mg, 0.18 mmol) was added followed by alcohol 19 (40 mg,
0.10 mmol). The mixture was stirred at 0 °C for 2 h and then al-
lowed to warm to room temperature and stirred for a further
48 h. The solvent was removed in vacuo and the residue was dis-
solved in ethyl acetate (10 ml) and water (10 ml). The organic
phase was washed with brine (3 ꢁ 10 ml), dried (MgSO4) and con-
centrated in vacuo to yield the crude product. The crude product
was dissolved in a mixture of DCM, water and TFA in a 4:2:3 ratio,
respectively. The mixture was stirred at room temperature for 24 h
and was then diluted with water (10 ml) and DCM (10 ml). The
aqueous portion was extracted with DCM (3 ꢁ 10 ml) and then
concentrated in vacuo to yield the crude product. The product
was purified by HPLC using a water/methanol gradient. Compound
26 was eluted at 22.49 min and concentrated to yield a white solid
4.12. Kinetic assays of E. coli MurG
A preparation of solubilised translocase MraY was prepared
from Micrococcus flavus membranes (100
stock) added to 150 l of solubilisation buffer (50 mM Tris–HCl
pH 7.5, 1 mM MgCl2, 2 mM 2-mercaptoethanol, 0.5% CHAPS). The
ll of 19 mg protein/ml
l

2662
A. E. Trunkfield et al. / Bioorg. Med. Chem. 18 (2010) 2651–2663
mixture was shaken at 4 °C for 30 min and was then centrifuged at
13,000 rpm for 30 min. The protein concentration of the superna-
tant was estimated by Biorad assay to be 1.5 mg/ml and was used
directly in the radiochemical assay. Freshly solubilised MraY
37 °C for 1 h. A CHCl3/CH3OH (1:1, 533 ll) solution was then added
to the incubation tubes and the entire contents centrifuged at
18,000g. The supernatant was recovered and dried under a stream
of argon and re-suspended in C2H5OH/H2O (1:1, 1 ml) and loaded
(12.5
(0.25
l
l
l) was added directly to undecaprenyl phosphate
g). 12.5 l of buffer (400 mM Tris–HCl pH 7.5, 100 mM
l), UDP-MurNAc-penta-
l, 110 g protein/ml)
l of aqueous inhibitor solution. The mixture was incubated
at 35 °C for 15 min, and then 5 M,
l of UDP-[3H]GlcNAc (10
500 mCi/mmol) was added, and the mixture was incubated for a
further 15 min. The reaction was stopped by the addition of 50
onto a pre-equilibrated (C2H5OH/H2O [1:1]) 1 ml Whatmann
l
strong anion exchange (SAX) cartridge which was washed with
3 ml of ethanol. The eluate was dried and the resulting products
partitioned between the two phases arising from a mixture of n-
butanol (3 ml) and H20 (3 ml). The resulting organic phase was
recovered following centrifugation at 3,500g and the aqueous
phase was again extracted twice with 3 ml of n-butanol saturated
water, the pooled extracts were back-washed twice with water
saturated with n-butanol (3 ml). The n-butanol–saturated water
MgCl2) was added, followed by water (9
peptide solution (5 l, 1 mM), MurG (1
and 5
l
l
l
l
l
l
l
l
l
of 6 M pyridinium acetate pH 4.6. Hundred microlitres of n-butanol
were added, and the layers were mixed and separated by centrifu-
gation. Hundred microlitres of the top n-butanol phase were re-
moved and counted for radioactivity.
fraction was dried and re-suspended in 200 ll of n-butanol. The to-
tal cpm of radiolabeled material extractable into the n-butanol
phase was measured by scintillation counting using 10% of the la-
belled material and 10 ml of EcoScintA (National Diagnostics,
Atlanta). The incorporation of [14C]Gal was determined by sub-
tracting counts present in control assays (incubation of the reac-
tion components in the absence of the compounds). Another 10%
of the labelled material was subjected to thin-layer chromatogra-
phy (TLC) in CHCl3/CH3OH/NH4OH/H2O (65:25:0.5:3.6) on alumin-
ium backed Silica Gel 60 F254 plates (E. Merck, Darmstadt,
Germany). Autoradiograms were obtained by exposing TLCs to X-
ray film (Kodak X-Omat) for 4–5 days. Competition based experi-
ments were performed by mixing compounds together at various
concentrations (acceptor, 0.4 mM with inhibitors at 0.5–1.0 mM)
followed by thin-layer chromatography/autoradiography as de-
scribed earlier to determine the extent of product formation.
4.13. Antibacterial testing
The following procedure was performed using P. putida (ATCC
33015), M. luteus (ATCC 13513), E. coli BL21 and B. subtilis W23.
A single colony of the bacterial strain was picked from an LB agar
plate and grown in 10 ml of sterile LB medium overnight at
37 °C. 1 ml of this culture was then used to inoculate 50 ml of ster-
ile LB medium at 2%. Hundred microlitres of this 2% culture was
pipetted into each well of a 96-well plate. Hundred microlitres of
sterile water or sterile aqueous inhibitor solution (200 lg/ml or
1 mg/ml) were then pipetted into each well and the 96-well plates
were incubated at 37 °C. Bacterial growth was monitored by mea-
suring the OD at 595 nm at 3, 6 and 24 h.
Acknowledgements
4.14. Preparation of M. smegmatis membrane and cell wall
enzyme fractions
We thank Professor S. Walker (Harvard Medical School) for the
recombinant clone bearing the E. coli murG gene, and the BaCWAN
Network for supply of UDPMurNAc-pentapeptide. We thank EPSRC
and the University of Warwick for a studentship (AET). G.S.B.
acknowledges support in the form of a Personal Research Chair
from Mr. James Bardrick, Royal Society Wolfson Research Merit
Award, as a former Lister Institute-Jenner Research Fellow, the
Medical Research Council and The Wellcome Trust (081569/Z/06/
Z).
Liquid cultures of Mycobacterium smegmatis mc2155 were
grown at 37 °C in Luria Bertoni (LB) broth medium (Difco) supple-
mented with 0.05% Tween 80, biomass harvested, washed with
phosphate buffered saline (PBS) and stored at ꢀ20 °C until further
use. M. smegmatis cells (10 g wet weight) were washed and re-sus-
pended in 30 ml of buffer A, containing 50 mM MOPS (adjusted to
pH 8.0 with KOH), 5 mM b-mercaptoethanol and 10 mM MgCl2 at
4 °C and subjected to probe sonication (Soniprep 150, MSE Sanyo
Gallenkamp, Crawley, Sussex, UK; 1 cm probe) for a total time of
10 min in 60 s pulses and 90 s cooling intervals between pulses.
The sonicate centrifuged at 27,000g for 60 min at 4 °C. The result-
ing mycobacterial cell wall pellets were re-suspended in buffer A.
Percoll (Pharmacia, Sweden) was added to yield a 60% suspension
and centrifuged at 27,000g for 1 h at 4 °C. The upper, particulate
diffuse cell wall enzymatically active (P60) band was collected
and washed three times with buffer A and re-suspended in buffer
A at a final protein concentration of 10 mg/ml. Membrane fractions
were obtained by centrifugation of the 27,000g supernatant at
100,000g for 1 h at 4 °C. The supernatant was carefully removed
and the membranes gently re-suspended in buffer A at a protein
concentration of 20 mg/ml. Protein concentrations were deter-
mined using the BCA Protein Assay Reagent kit (Pierce Europe,
Oud-Beijerland, Netherlands).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.02.026.
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