Synthesis and biological evaluation of analogues of bacterial lipid I

Article abstract of DOI:10.1016/S0960-894X(00)00583-7

Bacterial Lipid I analogues containing different anomeric groups at the muramic acid moiety were synthesized and screened in MurG enzyme assays run in the presence and absence of cell wall membranes. The results obtained in this study help elucidate the role of the lipid diphosphate in the recognition of Lipid I by MurG. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00583-7

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2811±2813
Synthesis and Biological Evaluation of Analogues of
Bacterial Lipid I
Domingos J. Silva,a,* Caryn L. Bowe,a Arthur A. Branstrom,b Eugene R. Baizmanb
and Michael J. So®aa
aDepartment of Chemistry, Incara Research Laboratories, 8 Cedar Brook Drive, Cranbury, NJ 08536, USA
bDepartment of Biological Research, Incara Research Laboratories, 8 Cedar Brook Drive, Cranbury, NJ 08536, USA
Received 8 August 2000; accepted 6 October 2000
AbstractÐBacterial Lipid I analogues containing di€erent anomeric groups at the muramic acid moiety were synthesized and
screened in MurG enzyme assays run in the presence and absence of cell wall membranes. The results obtained in this study help
elucidate the role of the lipid diphosphate in the recognition of Lipid I by MurG. # 2000 Elsevier Science Ltd. All rights reserved.
The available arsenal of antibiotics was once thought to
be powerful enough to ®ght most bacterial infections.
However, identi®cation of deadly multiple drug resis-
tant bacterial strains in the last decades has created the
need for novel, more potent antibiotics. One of the
prime targets for drug discovery is cell wall biosynthesis,
an essential process in the bacterial life cycle.1,2 Unfor-
tunately, enzymatic steps that take place late in the
pathway have not been fully characterized to date,
mainly because the enzymes are either membrane-asso-
ciated or membrane-bound, complicating isolation and
biochemical studies.3 Furthermore, the substrates for
these enzymes are not readily isolated because of low
abundance in cellular systems and unfavorable physical
properties.4 Such problems must be overcome in order
to allow a clearer understanding of the enzymatic pro-
cesses and the discovery of novel enzyme inhibitors for
this pathway.
Lipid I, the sugar acceptor of MurG, could not be iso-
lated in large amounts from natural sources. In 1998
Walker et al. showed that 3, a simpler version of Lipid I
with a short citronellol lipid (Fig. 2), was eciently
processed by MurG in solution.6 This readily accessible
substrate allowed the authors to study the kinetic prop-
erties of the enzyme in the absence of membranes.
Walker et al. evaluated the ability of phosphorylated
Lipid I analogues incorporating changes in the lipid and
peptide portions to act as alternative substrates or dead-
end inhibitors for MurG.7 In these studies the anomeric
phosphate (4) inhibited MurG with an IC50 of
(50Æ25 mg mL 1), but had no acceptor ability, suggest-
ing that the anomeric group in Lipid I played a key role
in acceptor recognition.
We have recently described a coupled MraY/MurG
assay, designed to identify and characterize novel inhi-
bitors of these enzymes.8 This assay uses biotinylated
UDP-MurNAc-pentapeptide and UDP-[14C]-GlcNAc
as substrates. The assay utilizes E. coli cell wall mem-
brane fragments as enzyme source, and is based on the
speci®c capture of biotinylated Lipid II by avidin-
coated beads. This coupled MraY/MurG assay di€ers
considerably from the assay developed by Walker et al.
since the latter uses 3 as the substrate for soluble MurG
in the absence of membranes, circumventing issues rela-
ted to solution-membrane partition of the substrate. In
an e€ort to obtain more detailed information about the
substrate requirements of MurG, we decided to study
the behavior of analogues of Lipid I in the MraY/MurG
assay. In these compounds the anomeric diphosphoryl
lipid unit was replaced by a simple phosphate group (4)
MurG (UDP-N-acetylglucosamine: undecaprenyl-pyro-
phosphoryl-N-acetylmuramoyl-pentapeptide N-acetyl
glucosaminyl transferase) [GenBank P17443] is a cyto-
plasmatic membrane-associated GlcNAc transferase,
responsible for the conversion of Lipid I (1) to Lipid II
(2), the substrate for transglycosylases and transpepti-
dases.5 Although the MurG enzyme was ®rst described
in 1965, studies of its mechanism have lagged because
*Corresponding author at present address: SmithKline Beecham
Pharmaceuticals, 709 Swedeland Road, PO Box 1539, UW2421, King
of Prussia, PA 19406, USA. Tel.: +1-610-270-6537; fax: +1-610-270-
4490; e-mail: domingos_j_silva@sbphrd.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00583-7

2812
D. J. Silva et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2811±2813
Figure 1.
or noncharged groups, such as the methoxy group (5a)
and the polarizable hydrophobic thiophenoxy group
(5b).9 Screening of these compounds in MurG assays
should potentially shed further light on the role of the
lipid diphosphate moiety in acceptor recognition and
glycosylation, and help establish the mechanism of
MurG catalysis.
Lipid analogues 5a and 5b were tested in an assay
modeled after Walker et al. using 3 as substrate and
either puri®ed soluble MurG or E. coli membranes as
enzyme source. In either case, no inhibition of MurG
was detected even at 200 mg mL 1 of the compounds.
Analogues 4, 5a, and 5b were tested in the coupled
MraY/MurG assay for their ability to inhibit the activ-
ity of either enzyme. The analogues were tested at con-
Lipid I derivatives 5a±b were prepared by synthesizing
the carbohydrate and peptide moieties separately, cou-
pling them through an amide linkage and performing
®nal deprotection. The protected muramic acids were
synthesized from the common intermediate 6 (Fig. 3),
obtained in three steps from glucosamine hydrochlo-
ride.
1
centrations up to 200 mg mL (238, 256, and 234 mM,
respectively), but no inhibition of Lipid II synthesis was
observed. Under the same assay conditions, tunicamy-
cin13 and ramoplanin14 (known inhibitors of MraY and
MurG, respectively) had IC50 values lower than
1
1 mg mL
.
Compounds 6 and 7 (prepared from 6 via the reactive b-
bromide) were deacetylated, protected as 4,6-benzyli-
dene acetals and alkylated with methyl-(1S)-O-tri-
¯uoromethanesulfonyl lactate.10 The corresponding
esters 9a±b were saponi®ed and neutralized, yielding the
muramic acids 10a±b. Pentapeptide 11 (Fig. 4) was syn-
thesized from commercially available N-Boc protected
amino acids, using a sequence of HATU-promoted
amide couplings and TFA acidic deprotections.
Lipid I analogues 4, 5a, and 5b were further tested in a
peptidoglycan formation assay, which measures the
formation of peptidoglycan by assessing incorporation
of 14C-GlcNAc (contained in Lipid II) into TCA-inso-
luble, ®lter-capturable material.15 Once again, no inhi-
bition was observed for compound 4, 5a, or 5b at
1 16
concentrations of 200 mg mL
,
suggesting that these
analogues do not a€ect downstream processing of Lipid
II to peptidoglycan polymer.
Muramic acids 10a±b were coupled to the pentapeptide
11 using HATU (Fig. 4). Glycopeptides 12a±b were
reduced to the free amines with PMe3 and then acety-
lated. The benzylidene groups were selectively removed
using TFA in CH3CN±CHCl3 to produce diols 13a±b.
Finally, treatment with 0.1 M LiOH in THF±MeOH±
H2O removed all base sensitive protective groups to
yield ®nal products 5a11 and 5b.12
These results provide interesting insights into the role of
the lipid diphosphate in MurG activity. Whereas 4
behaves as an inhibitor for MurG in the soluble enzyme
assay,7 this compound was found to be inactive in the
coupled MraY/MurG assay, suggesting that 4 cannot
compete eciently for the active site of membrane-bound
MurG. Compounds 5a and 5b were inactive in both
Figure 2. Lipid I analogues.
Figure 3. Synthesis of protected muramic acids.

D. J. Silva et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2811±2813
2813
Figure 4. Coupling and deprotection of analogues 5a and 5b.
8. Branstrom, A. A.; Midha, S.; Longley, C. B.; Han, K.;
Baizman, E. R.; Axelrod, H. R. Anal. Biochem. 2000, 280, 315.
9. Compound 4 was obtained as a kind gift from Prof.
Suzanne Walker (Princeton University).
10. (a) Synthesis of the alkylating reagent was adapted from:
Vedejs, E.; Engler, D. A.; Mullins, M. J. J. Org. Chem. 1977,
42, 3109. (b) Alkylation procedure was adapted from: Kinzy,
W.; Schmidt, R. R. Liebigs. Ann. Chem. 1987, 407.
assays. Taken together, our studies indicate that the
anomeric substituent of the muramic acid in Lipid I
plays a key role in substrate recognition and processing.
The lipid phosphate may not only help determine the
substrate speci®city of the MurG enzyme (1 and 3 are
substrates, 4 is an inhibitor, and 5a and 5b are inactive)
but also may localize the Lipid I substrate into the cell
membrane, avoiding the processing of other possible
solution-based substrates or inhibitors (4 is an inhibitor
in the solution assay but not in the membrane-based
coupled assay). The chemistry described here allowed us
to synthesize the desired Lipid I analogues in an ecient
and convergent manner. The synthetic route to these
glycopeptides is ¯exible and can be extended to the
synthesis of Lipid I derivatives containing di€erent
substituents at the anomeric center. Present work con-
centrates on the synthesis of lipid analogues containing
pyrophosphate mimics at the anomeric center of the
muramic acid and testing of these compounds in the
soluble MurG and coupled MraY/MurG assays.
11. Analytical data for 5a: 1H NMR (300 MHz, CD3OD) d
7.43±7.40 (m, 2H), 7.28±7.20 (m, 3H), 5.81 (d, 1H, J=5.4 Hz),
4.60 (qapp, 1H, J=6.6 Hz), 4.43 (qapp, 1H, J=7.5 Hz), 4.35±
4.30 (m, 2H), 4.17 (br, 2H), 4.02 (m, 2H), 3.77 (m, 2H), 3.59
(m, 2H), 2.89 (br, 2H), 2.33 (br, 3H), 1.92 (s, 3H), 1.90±1.80
(m, 3H), 1.63 (br, 2H), 1.50±1.30 (m, 14H); 13C NMR
(75.4 MHz, CD3OD) d 176.65, 174.22 (br), 173.60, 135.75,
133.13, 130.06, 128.46, 88.96, 79.88, 77.63, 75.23, 72.11, 62.21,
55.78, 54.99, 50.80, 44.94, 40.48, 33.07, 32.21, 29.60, 28.16,
23.49, 22.91, 20.18, 18.66, 18.20, 17.93. MS (ES) m/z obsd 856
[M+H+].
12. Analytical data for 5b: 1H NMR (300 MHz, CD3OD) d
4.68 (d, 1H, J=3.6 Hz), 4.37±4.31 (m, 6H), 3.97 (dd, 1H,
J=3.0 Hz, 10.2 Hz), 3.80 (t, 1H, J=11.4 Hz), 3.71 (dd, 1H,
J=4.8 Hz, 12.3 Hz), 3.63±3.45 (m, 3H), 3.35 (s, 3H), 2.94 (br,
2H), 2.32 (br, 3H), 1.94 (s, 3H), 2.00±1.82 (m, br, 3H), 1.68
(br, 2H), 1.51±1.30 (m, 14H); 13C NMR (75.4 MHz, CD3OD)
d 176.14, 175.47, 174.27, 174.15, 173.36, 99.73, 80.80, 78.31,
73.82, 71.21, 62.53, 55.52, 55.07, 54.86, 50.80, 50.10, 40.48,
32.95, 32.24, 29.63, 28.15, 23.46, 22.91, 19.75, 18.62, 18.22,
17.90. MS (ES) m/z obsd 777 [M+H+].
13. Brandish, P. E.; Kimura, K. I.; Inukai, M.; Southgate, R.;
Lonsdale, J. T.; Bugg, T. D. Antimicrob. Agents Chemother.
1996, 40, 1640.
14. (a) Somner, E. A.; Reynolds, P. E. Antimicrob. Agents
Chemother. 1990, 34, 413. (b) Lo, M.-C.; Men, H.; Branstrom,
A.; Helm, J.; Yao, N.; Goldman, R.; Walker, S. J. Am. Chem.
Soc. 2000, 122, 3540. Ramoplanin was received as a gift from
Biosearch Italia (Gerenzano, Italy).
15. (a) Allen, N. E.; Hobbs, J. N.; Richardson, J. M.; Riggin,
R. M. FEMS Microbiol. Lett. 1992, 92, 109. (b) Goldman, R.
C.; Baizman, E. R.; Branstrom, A. A.; Longley, C. B. FEMS
Microbiol. Lett. 2000, 183, 209.
References and Notes
1. Rogers, H. J.; Perkins, H. R.; Ward, J. B. Biosynthesis of
Peptidoglycan; Chapman and Hall: London, 1980.
2. Bugg, T. D. H.; Walsh, C. T. Nat. Prod. Rep. 1992, 9, 199.
3. Goldman, R. C.; Branstrom, A. A. Curr. Pharm. Des. 1999,
5, 229.
4. van Heijenoort, Y.; Gomez, M.; Derrien, M.; Ayala, J.; van
Heijenoort, J. J. Bacteriol 1992, 174, 3549.
5. (a) Bupp, K.; van Heijenoort, J. J. Bacteriol. 1993, 175,
1841. (b) The 1.9 A crystal structure of E. coli MurG was
recently reported: Ha, S.; Walker, D.; Shi, Y.; Walker, S.
Protein Sci. 2000, 9, 1045.
6. Men, H.; Park, P.; Ge, M.; Walker, S. J. Am. Chem. Soc.
1998, 120, 2484.
16. Under these conditions the drug moenomycin, a known
1
7. Ha, S.; Chang, E.; Lo, M.-C.; Men, H.; Park, P.; Ge, M.;
Walker, S. J. Am. Chem. Soc. 1999, 121, 8415.
transglycosylase inhibitor, had an IC50 of 0.004 mg mL
.