Adenosine phosphonate inhibitors of lipid II: Alanyl tRNA ligase MurM from Streptococcus pneumoniae

Article abstract of DOI:10.1016/j.bmcl.2007.05.071

Adenosine and 2′-deoxyadenosine phosphonate transition state analogues act as the first inhibitors for the MurMN/FemABX family of tRNA-dependent ligases implicated in high-level penicillin resistance in Gram-positive bacteria.

Full text of DOI:10.1016/j.bmcl.2007.05.071

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4654–4656
Adenosine phosphonate inhibitors of lipid II: Alanyl tRNA
ligase MurM from Streptococcus pneumoniae
Elena Cressina,a Adrian J. Lloyd,b Gianfranco De Pascale,a,b David I. Roper,b
Christopher G. Dowsonb and Timothy D. H. Bugga,*
aDepartment of Chemistry, University of Warwick, Coventry CV4 7AL, UK
bDepartment of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
Received 7 May 2007; revised 20 May 2007; accepted 22 May 2007
Available online 25 may 2007
Abstract—Adenosine and 20-deoxyadenosine phosphonate transition state analogues act as the first inhibitors for the MurMN/Fem-
ABX family of tRNA-dependent ligases implicated in high-level penicillin resistance in Gram-positive bacteria.
Ó 2007 Elsevier Ltd. All rights reserved.
High-level penicillin resistance in Streptococcus pneumo-
niae is associated with the presence of additional -Ala-
Ala- and -Ser-Ala- cross-links in the peptidoglycan
structure,1 as shown in Figure 1. The presence of these
cross-links is associated with the presence of particular
sequences of the murM and murN genes,2 related to
the femABX genes responsible for formation of the
(Gly)5 cross-link in Staphylococcus aureus.3 The encoded
MurMN proteins catalyse the addition of Ala(Ser) and
Ala, respectively, to lipid intermediate II in peptidogly-
can biosynthesis,4 using Ala-tRNA and Ser-tRNA,
respectively, analogous to the addition of Gly-tRNA
by FemABX in S. aureus.5 Deletion of the murM gene
in S. pneumoniae leads to loss of the high-level resistance
phenotype,2 therefore MurM is a potential target for
anti-resistance agents that could be applied synergisti-
cally with penicillin, against antibiotic-resistant bacteria.
and 20-deoxy-adenosine phosphonates as MurM
inhibitors.
In the 20-deoxy series, a protected 20-deoxyadenosine
derivative (1)6 was coupled with aminoethylphosphonic
monomethyl ester 27 using DCC, in 54% yield. Depro-
tection was achieved in 52% yield, to give phosphonate
3, as shown in Figure 3.
In the ribo-series, attempts to couple 2 to a 20,50-
TBDMS-protected adenosine to obtain selectively the
30 phosphonate (6a) were unsuccessful, using a range
of coupling agents. Therefore, an improved version
of the procedure reported by Zemlicka8 to obtain a
mixture of 20 and 30 regioisomers was followed. A
50-TBDMS-protected adenosine (4)6 was coupled with
aminoethylphosphonic acid 57 to give a mixture of
20- and 30-phosphonates 6a and 6b, in a 3:1 ratio,
The catalytic mechanism of the MurM-catalysed reac-
tion is likely to proceed via a tetrahedral transition state,
shown in Figure 2. Since adenosine is found at the 30-ter-
minus of tRNA nucleic acids, a possible transition state
mimic for this family of enzymes is an adenosine 30-
phosphonate, shown in Figure 2. This paper describes
the synthesis and in vitro assay of a series of adenosine
MurNAc
L-Ala
MurNAc
L-Ala
MurNAc
L-Ala
MurNAc
L-Ala
D-Gln
L-Lys
D-Gln
D-Gln
L-Lys
D-Gln
L-Lys
D-Ala
L-Lys L-Ala(Ser) L-Ala D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
Keywords: MurM; Inhibitor; Peptidoglycan biosynthesis; Antibacte-
rial; Streptococcus pneumoniae.
Figure 1. (a) Direct peptidoglycan cross-link found in normal
S. pneumoniae; (b) indirect peptidoglycan cross-link found in
penicillin-resistant S. pneumoniae.
*
Corresponding author. Tel.: +44 02476 573018; fax: +44 02476
524112; e-mail: T.D.Bugg@warwick.ac.uk
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.071

E. Cressina et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4654–4656
4655
NH2
NH2
N
N
N
N
N
tRNA
O
tRNA
O
C55-OPP
MurNAc GlcNAc
C55-OPP
MurNAc GlcNAc
N
N
N
O
O
L-Ala
L-Ala
O
MurM
O
γ-D-Glu
O
OH
O
OH
γ-D-Glu
L-Lys
N
L-Lys
NH2
H
D-Ala
D-Ala
NH3
NH3
D-Ala
D-Ala
tRNA
C55-OPP
NH2
Phosphonate
analogues:
MurNAc GlcNAc
L-Ala
N
N
N
HO
O
N
γ-D-Glu
O
NH3
L-Lys
N
H
-O
D-Ala
D-Ala
O
R
O
P
NH3
Figure 2. The MurM-catalysed ligation of lipid intermediate II with alanyl-tRNA, showing the likely tetrahedral oxyanion intermediate, and the
structures of the phosphonate analogues. R = OH or H; C55, undecaprenyl.
which were not separable. Deprotection gave a 3:1
mixture of 20- and 30-phosphonates 7a and 7b (see
Fig. 4). Anion exchange FPLC of the Cbz-protected
intermediate yielded a small amount of pure 7b for
enzyme assays.
S. pneumoniae alanyl tRNA synthetase, and tRNA iso-
lated from Micrococcus flavus.9 The MurM reaction
NHBz
N
N
Phosphonates 3, 7b, and the 3:1 mixture of 7a/7b were
assayed as inhibitors of MurM from S. pneumoniae
strain 159, whose expression and purification is de-
scribed elsewhere.9 Lipid intermediate II was prepared
enzymatically, using the method of Breukink et al.10
[3H]-Ala-tRNA was prepared enzymatically, using
N
TBSO
N
O
O
CbzNH
P
OH
HO OH
4
OH
5
a
NHBz
NHBz
N
NHBz
N
N
N
N
N
N
N
N
O
TBSO
TBSO
N
O
O
N
TBSO
N
O
CbzNH
P
OMe
O
O
O
OH
OH O
OH
HO
P
6a 6b
3 : 1
P
OH
OH
1
2
NHCbz
NHCbz
a
b,c,d
NHBz
N
NH2
NH2
NH2
N
N
N
N
N
N
N
N
N
HO
N
TBSO
N
N
HO
N
HO
N
N
O
O
O
O
O
b,c,d
O
O
O
O
OH
O
O
OH O
-O
P
-O
P
HO
P
P
OH
7a 7b
3 : 1
3
NH3
NHCbz
NH3
NH3
Figure 3. Synthesis of 20-deoxyadenosine phosphonate 3. Reagents
and yields: (a) DCC, pyridine, Dowex (pyr+), 54%; (b) NH3, MeOH,
85%; (c) Et3NÆHF, THF, 99%; (d) H2/Pd/C, MeOH, 62%.
Figure 4. Synthesis of ribo-phosphonates 7a and 7b. Reaction condi-
tions: (a) EDC, pyridine, Dowex (pyr+), 70%; (b) NH3, MeOH, 97%;
(c) Et3NÆHF, THF, 99%; (d) H2/Pd/C, MeOH, 86%.

4656
E. Cressina et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4654–4656
100
logue 7a is equatorial, implying that an axial
7b
3
phosphonate group is required for efficient binding to
MurM. This is supported by the observation that 20-
phosphonate 7b, which contains an axial phosphonate
at C-20, still shows modest enzyme inhibition.
90
80
70
60
50
40
30
20
10
0
Phosphonate 3, the first in vitro inhibitor of the Mur-
MN/FemABX ligase family, was tested for antibacterial
activity against penicillin-resistant S. pneumoniae strain
159, but showed no growth inhibition at 1 mM concen-
tration (380 lg/ml), and no effect upon penicillin MIC,
perhaps due to lack of transport across the cell mem-
brane. Further derivatives of 3 are being synthesised
for in vitro and antibacterial testing.
0.0
0.2
0.4
0.6
0.8
1.0
Inhibitor conc (mM)
Figure 5. Inhibition of S. pneumoniae 159 MurM by 3 and 7b.
Acknowledgment
was then monitored radiochemically, by extraction of
the lipid II-[3H]-Ala product into n-butanol, followed
by scintillation counting.ꢀ
This work was supported by the European Commission
COBRA Network (Contract LSHM-CT-2003-503335),
and a BBSRC studentship (to G.P.).
The 20-ribo-phosphonate 7b showed moderate levels of
inhibition, yielding an IC50 value of 780 lM, whereas
the 3:1 mixture of 7a/7b showed no inhibition of MurM
at 1 mM concentration. The 20-deoxyadenosine phos-
phonate 3 showed much higher levels of inhibition, as
shown in Figure 5, giving an IC50 value of 100 lM.
References and notes
1. Garcia-Bustos, J.; Tomasz, A. Proc. Natl. Acad. Sci. USA
1990, 87, 5414.
2. Filipe, S. R.; Tomasz, A. Proc. Natl. Acad. Sci. USA 2000,
97, 4891.
3. Rohrer, S.; Berger-Bachi, B. Antimicrob. Agents Chemo-
ther. 2003, 47, 837.
4. Filipe, S. R.; Pinho, M. G.; Tomasz, A. J. Biol. Chem.
2000, 275, 27768.
5. Schneider, T.; Senn, M. M.; Berger-Bachi, B.; Tossi, A.;
Sahl, H.-G.; Wiedemann, I. Mol. Microbiol. 2004, 53, 675.
6. Ti, G. S.; Gaffney, B. L.; Jones, R. A. J. Am. Chem. Soc.
1982, 104, 1316; Ogilvie, K. K.; Beaucage, S. L.; Schifman,
A. L.; Theriault, N. Y.; Sadana, K. L. Can. J. Chem. 1978,
56, 2768.
7. Yuan, C.; Wang, G. Synthesis 1990, 256.
8. Zemlicka, J.; Chladek, S. Collection Czechoslov. Chem.
Commun. 1969, 34, 1007.
9. Lloyd, A. J.; De Pascale, G.; Roper, D. I.; Bugg, T. D. H.;
Dowson, C. G., submitted for publication, 2007.
10. Breukink, E.; van Heusden, H. E.; Vollmerhaus, P. J.;
Swiezewska, E.; Brunner, L.; Walker, S.; Heck, A. J.; de
Kruijff, B. J. Biol. Chem. 2003, 278, 19898.
11. Saenger, W. Principles of Nucleic Acid Structure; Springer:
New York, 1983, pp 55–61.
Assay of synthetic precursors containing either an N-
Cbz protecting group or 50-O-TBDMS-protecting group
showed no enzyme inhibition at 1 mM concentration.
Puromycin, an adenosine analogue containing a 30-ami-
no-tyrosyl substituent,11 also showed no inhibition of
MurM at 1 mM concentration, indicating the impor-
tance of the phosphonate group for enzyme inhibition.
One possible rationalisation of the observed inhibition
data is that the potency of inhibition depends upon
the conformation of the furanose ring. Ribo-nucleoside
derivatives are known to prefer the 30-endo conforma-
tion, in which the 20-substituent is axial and 30-substitu-
ent equatorial, whereas 20-deoxy derivatives prefer the
20-endo conformation, in which the 30-substituent is
axial.12 1H–1H NMR coupling constants from the
NMR spectrum of 20-deoxy analogue 3 are consistent
with a 20-endo conformation, bearing an axial 30-phos-
phonate group, whereas the 30-phosphonate of ribo-ana-
12. Nathans, D. Nature 1963, 197, 1076.
ꢀ
Assays contained 50 lM lipid intermediate II, 0.2 lM [3H]-Ala-
tRNA, 0.01–1 mM inhibitor and 25 nM S. pneumoniae 159 MurM in
50 mM MOPS buffer, pH 6.8, containing 30 mM KCl, 10 mM
MgCl2, 1.5% CHAPS, 1 mM L-Ala and 2 mM DTT.