Synthesis and reactivity with β-lactamases of a monobactam bearing a retro-amide side chain

Article abstract of DOI:10.1016/j.bmcl.2005.11.006

The monobactam sodium 3-benzylcarbamoyl-2-oxo-1-azetidinesulfonate, bearing a retro (vs classical β-lactam)-amide side chain, has been synthesized and the kinetics of its reaction with typical β-lactamases studied. The new compound is generally a poorer substrate than the analogous compound with a normal side chain but its formation of a transiently stable complex with a class C β-lactamase sustains the retro-amide side-chain concept.

Full text of DOI:10.1016/j.bmcl.2005.11.006

Bioorganic & Medicinal Chemistry Letters 16 (2006) 869–871
Synthesis and reactivity with b-lactamases of a
monobactam bearing a retro-amide side chain
S. A. Adediran,a J.-F. Lohier,b D. Cabaret,b M. Wakselmanb,* and R. F. Pratta,*
aDepartment of Chemistry, Wesleyan University, Middletown, CT 06459, USA
ˆ
SIRCOB, UMR CNRS 8086, Universite´ de Versailles, Bat. Lavoisier, 45, avenue des Etats Unis F-78075, Versailles, France
b
Received 13 September 2005; revised 2 November 2005; accepted 3 November 2005
Available online 21 November 2005
Abstract—The monobactam sodium 3-benzylcarbamoyl-2-oxo-1-azetidinesulfonate, bearing a retro (vs classical b-lactam)-amide
side chain, has been synthesized and the kinetics of its reaction with typical b-lactamases studied. The new compound is generally
a poorer substrate than the analogous compound with a normal side chain but its formation of a transiently stable complex with a
class C b-lactamase sustains the retro-amide side-chain concept.
ꢀ 2005 Elsevier Ltd. All rights reserved.
b-Lactamases catalyze the hydrolysis of b-lactam antibi-
otics and are thereby responsible for much of the bacte-
rial resistance toward these drugs. At present, the most
important b-lactamases in medicine are the serine-based
classes A, C, and D.1 Much research has been and is still
directed toward the discovery of new molecular entities
that react with the active site of these enzymes and thus
may serve as lead compounds in antibiotic development.
reactions with any relevant enzymes have not. In this
communication, we report the synthesis of the retro-am-
ide monobactam 3 and describe its reactivity with typi-
cal class A, C, and D b-lactamases. The classical
monobactam 4 was also studied to allow a direct com-
parison of the side-chain effects.
PhCH2NHCO
PhCH2CONH
N
Recently, we showed that acyclic depsipeptides such as
1, containing an atypical retro-amide side chain, reacted
covalently with typical b-lactamases.2 In some cases,
these reactions were more rapid than those of analogous
compounds containing the normal amide side chain
such as 2 (and as found in the classical b-lactams).
N
4
3
-
-
O
O
SO3
SO3
The synthesis of 3 is shown in outline in Scheme 1. Thus,
the method of Southwick4 was applied to p-methoxyb-
enzylamine to yield the dioxopyrrolidine 5. Acid hydro-
lysis and decarboxylation afforded 6. Then, the
Rapoport periodate-induced rearrangement4c,5 led to
the ring-contracted b-lactam 7, which has a carboxylic
acid group at C-3. The coupling of 7 with benzylamine
readily afforded the amide 8. Oxidative removal of the
N-p-methoxybenzyl-protecting group6 yielded the 1-
unsubstituted azetidinone 9. Finally, sulfation7 of the
ring NH of the b-lactam 9 yielded the racemic monobac-
tam 3, which was isolated as the sodium salt.8 The mon-
obactam 4 was obtained from Bristol-MyersSquibb.
X
1: X = PhCH2NHCO-
O
O
2: X = PhCH2CONH-
-
CO2
It was clearly of interest, therefore, to examine the effect
of this side-chain reorientation on the reactivity of b-lac-
tams themselves. Although a small number of such
compounds have been described in the literature,3 their
The reactions of 3 and 4 in aqueous buffer were moni-
tored spectrophotometrically at 240 nm (De = 75 cmÀ1
M
À1). The spontaneous hydrolysis of 3 and 4 in
Keywords: b-Lactam; Antibiotic; b-Lactamase; Enzyme; Kinetics;
Retro-amide.
100 mM MOPS, pH 7.5, was quite slow, with rate con-
stants of 6.7 · 10À6 and 1.1 · 10À5 sÀ1, respectively, and
*
Corresponding authors. E-mail: rpratt@wesleyan.edu
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.11.006

870
S.A. Adediran et al. / Bioorg. Med. Chem. Lett. 16 (2006) 869–871
CO Me
O
2
O
HO C
2
+
ArCH NH
2
2
H O
3
NaIO
4
CO Me
2
N
O
O
N
N
(CO Me)
2
2
O
5
6
7
Ar
Ar
Ar
Ar = p-OMe-Ph-
PhCH NH
DCC
2
2
PhCH NHCO
PhCH NHCO
PhCH NHCO
2
2
2
SO
K S O
2 2 4
3
N
NH
N
-
O
SO
O
O
3
DMF
3
9
8
Ar
Scheme 1. Synthesis of 3.
thus could be ignored in initial rate measurements with
the various enzymes. Steady state parameters for the
hydrolysis of 3 and 4, catalyzed by several b-lactamases
and derived from the initial rate measurements, are
given in Table 1.
other enzymes in that it involved a slowly equilibrating
side branch (Schemes 2a or b, which could not be distin-
guished by the data at hand).
The smallest rate constant, k3, was obtained from a re-
turn of activity experiment, where aliquots of a reaction
mixture of 3 and the enzyme were diluted into a solution
of a good substrate at saturating concentration (cepha-
lothin, 200 lM) and the return of activity was monitored
spectrophotometrically. A value of 0.013 sÀ1 was thus
obtained for k3. The other rate constants were obtained
from total progress curve analysis, where k3 was fixed at
the value obtained as described above. In both experi-
ments, the data were analyzed by the Dynafit program.11
Values of the other constants thus obtained (Scheme 2a)
were K1 = 0.21 0.04 mM, k2 = 0.023 0.002 sÀ1, and
k4 = 2.2 0.15 sÀ1; steady state parameters were then
calculated to be kcat = k4/(1 + k2/k3) = 0.82 sÀ1 and
Km = K1/(1 + k2/k3) = 0.075 mM. Analysis in terms of
Scheme 2b yielded the same values for the steady state
parameters kcat and Km.
Compound 4 had been prepared9 in the reactive 3b-ami-
do configuration shown in the structural diagram above.
Compound 3, however, was a racemic mixture of enan-
tiomers, as derived from the synthesis of Scheme 1. A 1H
NMR experiment with 3 showed that proton exchange
with solvent and hence epimerization at C3 was faster
than the enzyme-catalyzed reactions. It was assumed,
in accordance with precedent,10 that the 3b-enantiomer
reacted with b-lactamases, while the 3a-enantiomer was
inert. Under these conditions, the real Km for the reactive
3b-enantiomer is half the apparent value.10c The Km and
kcat/Km values of Table 1 include this adjustment.
The reaction of 3 with the Enterobacter cloacae P99
b-lactamase differed qualitatively from that with the
Table 1. Steady state kinetics parameters for the hydrolysis of 3 and 4 by b-lactamases
Enzymea
kcat (sÀ1 b
)
Km (mM)c
kcat/Km (sÀ1MÀ1 c
)
3
4
3
4
3
4
P99
0.82d
43
410 90
0.075d
1.0 0.4
6.8 0.4
1.1 · 104
1.7 · 104
1.5 · 103
7.0 · 102
4.1 · 105
2.4 · 105
5.8 · 103
2.1 · 105
TEM
PC1
OXA-1
1
1600 570
0.38 0.01
6.0 0.1
2.5 0.2
0.30 0.03
2.3 0.4
0.46 0.03
1.6 0.2
0.07 0.02e
0.029 0.001f
a P99, the class C b-lactamase of Enterobacter cloacae P99; TEM, the class A b-lactamase from the TEM-2 plasmid; PC1, the class A b-lactamase of
Staphylococcus aureus PC1; OXA-1, the class D b-lactamase from Escherichia coli.
b Reaction conditions.15
c The values for 3 were adjusted as described in the text.
d See text.
e Determined from competitive inhibition of nitrocefin hydrolysis.
f Determined from competitive inhibition of cephalothin hydrolysis.
a
b
k4
k4
ES
E + P
E + S
ES
E + P
E + P
E + S
K1
K1
k3
k2
k2
k3
ES'
ES'
Scheme 2.

S.A. Adediran et al. / Bioorg. Med. Chem. Lett. 16 (2006) 869–871
871
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The results of Table 1 show that, in general, 3 is a poorer
substrate of the enzymes tested than is 4. It is also
noticeable that, even in cases where enzyme deacylation
is usually rate-determining in the kcat step (P99, PC1,
and OXA-1 b-lactamases), there is no evidence of for-
mation of a tightly bound acyl-enzyme intermediate
(low Km), although this phenomenon is evident in the
parameters for the PC1 and OXA-1 enzymes with 4. It
seems, therefore, that the normal amide group in the
side chain interacts considerably more favorably with
these enzymes. Crystal structures indicate that the nor-
mal amide side chain of b-lactam substrates generally
forms hydrogen bonds with an asparagine carboxamide
NH and a protein backbone CO,12 but modeling sug-
gested that the retro-amide of 3 might also be able to
interact in a favorable way.2 Although the latter may
be true in some cases with acyclic substrates, it does
not appear to be true with b-lactams.
´
3049; (e) Mastalerz, H.; Menard, M. Heterocycles 1991,
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H. J. Org. Chem. 1975, 40, 1264.
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Fritz, A. W.; Floyd, D. M.; Kissick, T.; Koster, W. H.;
Kronenthal, D.; Massa, F.; Mueller, R. H.; Pluscek, J.;
Slusarchyk, W. A.; Sykes, R. B.; Taylor, M.; Weaver, E.
R. Tetrahedron 1983, 39, 2577.
8. White powder (47% yield); mp > 250 ꢁC; Rf (acid form)
0.24 (ethyl acetate/methanol, 85:15); 1H NMR (D2O) d
3.84 (d, J = 4.0 Hz, 2H), 4.29 (t, J = 4.0 Hz, 1H), 4.45 (s,
2H), 7.36–7.41 (m, 5H); 13C NMR (D2O) d 46.03, 46.36,
56.16, 129.99, 130.28, 131.57, 140.27, 165.74, 170.18; Anal.
Calcd for C, 41.91; H, 3.84; N, 8.89. Found: C, 41.69; H,
3.78; N, 8.98.
9. Breuer, H.; Cimarusti, C. M.; Denzel, T.; Koster, W. H.;
Slusarchyk, W. A.; Treuner, U. D. J. Antimicrob. Chemo-
ther. 1981, 8(Suppl E), 21.
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J.; Pratt, R. F.; Wakselman, M. J. Org. Chem. 1999,
64, 713; (b) Adediran, S. A.; Cabaret, D.; Pratt, R. F.;
Wakselman, M. Bioorg. Med. Chem. Lett. 1999, 9, 341;
(c) Adediran, S. A.; Cabaret, D.; Drouillat, B.; Pratt,
R. F.; Wakselman, M. Bioorg. Med. Chem. 2001, 9,
1175.
The most interesting feature of the results appears to be
the observation that the retro-amide promotes or allows
partition of a complex of 3 with the P99 b-lactamase,
most likely the acyl-enzyme intermediate, into a more
inert complex (t1/2 = 54 s). This phenomenon is not
unusual with b-lactamases,13 although it is not generally
seen with the simple benzyl side chain. It is possible that
incorporation of a third generation side chain into 3
might generate much more inert complexes and thus
effective inhibitors. Neither 3 nor 4 inhibited the Strep-
tomyces R61 DD-peptidase or Escherichia coli pbp5 to
any significant extent although, again, this situation
may also be changed by the presence of appropriate side
chains, as in aztreonam, for example.14
Acknowledgments
11. Kuzmic, P. Anal. Biochem. 1996, 237, 260.
12. (a) Strynadka, N. C. J.; Adachi, H.; Jensen, S. E.; Johns,
K.; Sielecki, A.; Betzel, C.; Sutoh, K.; James, M. N. G.
Nature 1992, 359, 700; (b) Beadle, B. M.; Trehan, I.;
Focia, P. J.; Shoichet, B. K. Structure 2002, 10, 413.
13. Pratt, R. F. In The Chemistry of b-Lactams; Page, M. I.,
Ed.; Chapman and Hall: London, 1992, Chapter 7.
14. Sykes, R. B.; Bonner, D. P. Am. J. Med. 1985, 78(2A), 2.
15. Stock solutions of 3 and 4 were prepared in DMSO. The
buffer employed for enzyme kinetics was 100 mM MOPS,
pH 7.5, except in the case of the OXA-1 enzyme where
50 mM NaHCO3 was also included. The reaction temper-
ature was 25 ꢁC and reaction solutions contained 5%
DMSO. The enzymes were obtained as previously
described.2
This research was supported by the US National Insti-
tute of Health (R.F.P.). We are grateful to Bristol-
MyersSquibb for the generous gift of 4 (SQ-026324)
and to Dr. Michiyoshi Nukaga of Jyosai International
University, Japan, for the OXA-1 b-lactamase.
References and notes
1. Rice, L. B.; Bonomo, R. A. Drug Resist. Updat. 2000, 3,
178.
2. Cabaret, D.; Adediran, S. A.; Pratt, R. F.; Wakselman, M.
Biochemistry 2003, 42, 6719.