O-aryloxycarbonyl hydroxamates: New β-lactamase inhibitors that cross-link the active site

Article abstract of DOI:10.1021/ja072370u

O-Aryloxycarbonyl hydroxamates represent a new class of β-lactamase inhibitors. N-Benzyloxycarbonyl-O-(phenoxycarbonyl) hydroxylamine, for example, inactivates the class C Enterobacter cloacae P99 β-lactamase with a rate constant of 6.1 × 10³ s

Full text of DOI:10.1021/ja072370u

Published on Web 07/12/2007
O-Aryloxycarbonyl Hydroxamates: New â-Lactamase Inhibitors That
Cross-Link the Active Site
Pauline N. Wyrembak,† Kerim Babaoglu,‡ Ryan B. Pelto,† Brian K. Shoichet,‡ and R. F. Pratt*,†
Department of Chemistry, Wesleyan UniVersity, Middletown, Connecticut 06459, and Department of Pharmaceutical
Chemistry, UniVersity of California, San Francisco, 1700 4th Street, San Francisco, California 94158-2330
Received April 4, 2007; E-mail: rpratt@wesleyan.edu
The effective lifetime of â-lactams as antibiotics can be extended
by concurrent treatment of patients with â-lactamase inhibitors.1,2
The â-lactamase inhibitors in commercial production at present,
however, are of limited spectrum and are largely specific to the
class A enzymes.1 New classes of inhibitor with broader specificity
covering classes B, C, and D â-lactamases would be welcome since
the prevalence of these enzymes, and thus â-lactam resistance
among bacteria, continues to grow. We describe in this communica-
tion a new type of â-lactamase inhibitor with an unusual mechanism
of action involving the covalent cross-linking of active side residues.
The depsipeptides of general structure 1 are â-lactamase
substrates.3,4 Although the aza analogues 2 display little or no
substrate activity,5 we were encouraged to try the oxa analogues 3
because of the inhibitory properties of vanadate/ hydroxamic acid
complexes.6 Compounds 4 and 5 were therefore obtained from
careful reaction of the appropriate hydroxamic acid and chlorofor-
mate (the latter carboxyl-protected in the case of 5) in the presence
Figure 1. Activity of the P99 â-lactamase (0.25 µM) as a function of time
in the presence of 4 (0 µM, [; 0.5 µM, 9; 2.5 µM, b).
of imidazole (Supporting Information). An alkoxy side chain was
chosen since the alkyl or aryl analogues were unstable to the Lo¨ssen
rearrangement.7 An NMR spectrum of 15N-4 in DMSO-d6 showed
a
15N resonance at 161.1 ppm coupled (J ) 93 Hz) to a proton at
11.75 ppm. This clearly identifies the product as the O-acyl rather
than N-acyl hydroxamic acid. Compounds 4 and 5 hydrolyzed in
aqueous buffer (20 mM MOPS, pH 7.5), yielding benzyl N-
hydroxycarbamate, the phenol, and, presumably, bicarbonate;
pseudo-first-order rate constants (k0) of 2.5 × 10-4 and 2.78 ×
10-4 s-1, respectively, were obtained.
Figure 2. Activity of the P99 â-lactamase (0.25 µM) after complete reaction
with 4 (b) and 5 (O) at various concentrations (0-1.0 µM).
Compound 4 inhibited, essentially irreversibly, the class C
â-lactamase of Enterobacter cloacae P99 in a time-dependent
fashion, as evident from Figure 1. At low inhibitor/enzyme
concentration ratios, the final activity of the enzyme was not zero,
which suggested that some turnover accompanied the inhibition
reaction (background hydrolysis of 4 was not sufficient to explain
the final activity). A greater excess of inhibitor did completely
inactivate the enzyme (Figure 1). A plot of residual activity versus
concentration of 4 (Figure 2) suggested that about two turnovers
accompanied inhibition. These data were fitted to Scheme 1, where
EI is likely to be a hydrolyzable acyl enzyme4 which can also
partition to a dead end complex EI′. These fits, shown as solid
Scheme 1
lines in the figures, yielded k1 and k2/k3 values of 6.1 ( 0.2 × 103
s-1 M-1 and 2.0 ( 0.1, respectively.
Compound 5 was also an inhibitor of the P99 enzyme (Figure
2). Experiments analogous to those described above yielded values
of k1 and k2/k3 of 5.4 ( 0.3 × 103 s-1 M-1 and 1.00 ( 0.05,
respectively. It is interesting that 5, bearing the m-carboxy sub-
† Wesleyan University.
‡ University of California, San Francisco.
9
9548
J. AM. CHEM. SOC. 2007, 129, 9548-9549
10.1021/ja072370u CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2
S1, Supporting Information). Lys73 remains hydrogen bonded to
Tyr150 Oú. Coordinates of the structure have been deposited in
the RCSB protein data bank as entry 2P9V.
The mechanism of inhibition of the P99 â-lactamase by 4 and 5
can thus, from the data available at present, be represented by the
sequence shown in Scheme 2. This represents a novel cross-linking
of the active site and a previously unobserved specific modification
of one of the two conserved lysine residues of the â-lactamase active
site. Inhibition of class A â-lactamases by clavulanic acid and
penicillin sulfones has been shown to involve cross-linking of the
active site serine to the conserved Ser130.11,12
We have also observed that 4 and 5, and other derivatives of
these compounds, inhibit the class A TEM â-lactamase. We plan
further experiments to determine the scope of these compounds
against â-lactam-recognizing enzymes.
Figure 3. Top: crystal structure of the active site of the AmpC â-lactamase
after inhibition by 4, showing the carbamate cross-link between Ser64 and
Lys315. The electron density is contoured at the 3σ level. Bottom: the
same view of the wild-type enzyme,10 showing a clear gap between Ser64
and Lys315.
Acknowledgment. This research was supported by the National
Institutes of Health through Grant AI 17986 to R.F.P. and GM
63815 to B.K.S. K.B. is supported by a Ruth L. Kirschstein National
Research Service Award fellowship (GM 076883).
stituent, is not a better inhibitor than 4. This result is contrary to
what would be expected from comparable substitution in the
depsipeptides 1. A m-carboxy group in 1 is thought to interact
specifically with the P99 active site.8 The results suggest that 1
and 5 may not bind to the active site in the same way. It should be
noted, however, that inactivation of the enzyme by 5 was
competitively inhibited by p-nitrobenzene boronic acid, which is
itself a competitive inhibitor of the P99 enzyme.9
Supporting Information Available: Synthetic procedures for
compounds 4 and 5 and the kinetics methods. Details of the crystal-
lographic procedures and statistics are also provided. This material is
available free of charge via the Internet at http://pubs.acs.org.
An electrospray mass spectrum of the inhibited enzyme was
obtained. Enzyme (10 µM) and 4 (5 mM) were incubated together
in MOPS buffer (above) for 5 min, after which time the enzyme
was inactive. The protein was then precipitated with trichloroacetic
acid, washed, and dried, and an ES+ mass spectrum obtained. The
spectrum showed an increase in protein mass of 29, in good
agreement with the mechanism of inactivation described below.
A 1.8 Å resolution crystal structure of the inhibited AmpC class
C â-lactamase was also obtained, as described in the Supporting
Information. The only observable difference from the structure of
the native enzyme10 was at the active site. In monomer A of the
structure, the Oγ oxygen of Ser64 is flipped some 180°
(Ser64CRCâOγC) and forms part of an unprecedented carbamate
bridge to Nê of Lys315 (Figure 3). Tyr150 has moved aside slightly
to accommodate insertion of a carbonyl, but Oú remains within
hydrogen-bonding distance of the inserted carbonyl oxygen (Figure
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