Serendipitous discovery of α-hydroxyalkyl esters as β-lactamase substrates

Article abstract of DOI:10.1021/bi101071r

O-(1-Carboxy-1-alkyloxycarbonyl) hydroxamates were found to spontaneously decarboxylate in aqueous neutral buffer to form O-(2-hydroxyalkylcarbonyl) hydroxamates. While the former molecules do not react rapidly with serine β-lactamases, the latter are quite good substrates of representative class A and C, but not D, enzymes, and particularly of a class C enzyme. The enzymes catalyze hydrolysis of these compounds to a mixture of the α-hydroxy acid and hydroxamate. Analogous compounds containing aryloxy leaving groups rather that hydroxamates are also substrates. Structure-activity experiments showed that the α-hydroxyl group was required for any substantial substrate activity. Although both d- and l-α-hydroxy acid derivatives were substrates, the former were preferred. The response of the class C activity to pH and to alternative nucleophiles (methanol and d-phenylalanine) suggested that the same active site functional groups participated in catalysis as for classical substrates. Molecular modeling was employed to explore how the α-hydroxy group might interact with the class C β-lactamase active site. Incorporation of the α-hydroxyalkyl moiety into novel inhibitors will be of considerable interest.

Full text of DOI:10.1021/bi101071r

10496 Biochemistry 2010, 49, 10496–10506
DOI: 10.1021/bi101071r
Serendipitous Discovery of R-Hydroxyalkyl Esters as β-Lactamase Substrates†
Ryan B. Pelto and R. F. Pratt*
Department of Chemistry, Wesleyan University, Middletown, Connecticut 06459, United States
Received July 5, 2010; Revised Manuscript Received November 8, 2010
ABSTRACT: O-(1-Carboxy-1-alkyloxycarbonyl) hydroxamates were found to spontaneously decarboxylate in
aqueous neutral buffer to form O-(2-hydroxyalkylcarbonyl) hydroxamates. While the former molecules do not react
rapidly with serine β-lactamases, the latter are quite good substrates of representative class A and C, but not D,
enzymes, and particularly of a class C enzyme. The enzymes catalyze hydrolysis of these compounds to a mixture of
the R-hydroxy acid and hydroxamate. Analogous compounds containing aryloxy leaving groups rather that
hydroxamates are also substrates. Structure-activity experiments showed that the R-hydroxyl group was required
for any substantial substrate activity. Although both
were preferred. The response of the class C activity to pH and to alternative nucleophiles (methanol and
-phenylalanine) suggested that the same active site functional groups participated in catalysis as for classical
D
- and -R-hydroxy acid derivatives were substrates, the former
L
D
substrates. Molecular modeling was employed to explore how the R-hydroxy group might interact with the class C
β-lactamase active site. Incorporation of the R-hydroxyalkyl moiety into novel inhibitors will be of considerable interest.
The unexpected discovery of a new class of substrates for an
enzyme opens up a period of recollection and reflection. How does
the newly discovered structural motif facilitate catalysis; i.e., how
does it interact with the enzyme active site? Does the enzyme
catalyze the reaction of the new substrate in the same way as that of
classical substrates, and how might it be incorporated into new
inhibitors? These questions arise with particular immediacy for
enzymes with medical implications such as the β-lactamases, which
continue to represent a serious barrier to future clinical application
of the β-lactam antibiotics (1). The discovery of acyclic depsipep-
tide substrates of the β-lactamases (2), for example, led directly to
the development of phosphonate inhibitors (3).
doing so form R-hydroxyalkyl esters 3 that are substrates of
β-lactamases. Extension of 3 to 4 also yielded β-lactamase sub-
strates. The ability of R-hydroxyalkyl esters to react with serine
β-lactamases has not been reported previously, to the best of our
knowledge. In this paper, we describe an initial survey of the
reactivity of 3 and 4 with serine β-lactamases.
MATERIALS AND METHODS
Synthetic reagents were, in general, purchased from Sigma-
Aldrich. tert-Butyl
-lactic acid from Bachem, Boc-
ester from Chem-Impex, (R)-(-)-2-hydroxy-4-phenylbutyric acid
from AnaSpec Inc., -4-biphenylalanine from PepTech Corp.,
N-methylhydroxylamine hydrochloride from Acros, and
15N]hydroxylamine hydrochloride from Isotec. Phosgene was
D-lactate was purchased from Fluka, anhydrous
D
D-phenylalanine 4-nitrophenyl
D
[
purchased as a 20% solution in toluene from Sigma-Aldrich.
The class C P99 β-lactamase from Enterobacter cloacae and the
class A TEM-2 β-lactamase from Escherichia coli W3310 were
purchased from the Centre for Applied Microbiology and
Research (Porton Down, Wiltshire, U.K.). The class D OXA-1
β-lactamase was generously provided by M. Nukaga (Jyosai
International University, Chiba, Japan) and the class C ampC
enzyme by B. Shoichet (University of California, San Francisco,
CA). The Streptomyces R61 and Actinomadura R39 DD-pepti-
Recently, we described a new class of β-lactamase inhibitors,
the O-aryloxycarbonyl hydroxamates, 1. These molecules were
found to be effective against all serine β-lactamases, although
particularly so against representative class C enzymes (4, 5).
ꢀ
dases were generous gifts from J.-M. Frere and P. Charlier
ꢀ
ꢀ
(University of Liege, Liege, Belgium).
A Varian Gemini-300 MHz NMR1 spectrometer was used to
1
collect H NMR spectra, and a Perkin-Elmer 1600 FTIR instru-
As an extension of this structural class, we prepared the
analogues 2, which also incorporate the carboxylate moiety that
is found in good β-lactamase substrates and which interacts with
specific active site residues (6-8). As we found and describe in
this paper, compounds of structure 2 rearrange spontaneously in
solution more rapidly than they inhibit β-lactamases but on
ment was used to obtain IR spectra. Elemental analyses were
1Abbreviations: AMPSO, 3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-
hydroxypropanesulfonic acid; BSA, bovine serum albumin; CAPS, 3-(cyclo-
hexylamino)-1-propanesulfonic acid; Centa, 7β-[(thien-2-yl)acetamido]-
3-[(4-nitro-3-carboxyphenylthio)methyl]-3-cephem-4-carboxylic acid; DCC,
N,N0-dicyclohexylcarbodiimide; DMSO, dimethyl sulfoxide; ESMS, electro-
spray mass spectrometry; IR, infrared; MES, 2-(N-morpholino)ethanesulfo-
nic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; NMR, nuclear
magnetic resonance; PBP, penicillin-binding protein; TAPS, N-tris-
(hydroxymethyl)methyl-3-aminopropanesulfonic acid; TBDMS, tert-butyl-
dimethylsilyl; TFA, trifluoroacetic acid.
†This research was supported by National Institutes of Health Grant
R01 AI-17986.
*To whom correspondence should be addressed. Telephone: (860)
685-2629. E-mail: rpratt@wesleyan.edu. Fax: (860) 685-2211.
r
pubs.acs.org/Biochemistry
Published on Web 11/18/2010
2010 American Chemical Society

Article
Biochemistry, Vol. 49, No. 49, 2010 10497
conducted by the Desert Analytics Laboratory. Routine ESI
mass spectra were recorded using a Thermo LCQ Advantage
instrument.
Syntheses. (i) N-(Benzyloxycarbonyl)-O-(1-D-carboxy-
ethoxycarbonyl)hydroxylamine (2, R = CH2Ph, R0 =
Me). (a) 1-D-(tert-Butoxycarbonyl)ethyl Chlorofor-
mate. Phosgene as a 20% solution in toluene (7 mL, 14 mmol)
yellow oil. The oil was dissolved in a mixture of methanol (3.5 mL)
and water (5 mL), followed by addition of potassium carbonate
(1.24 g, 9 mmol), and the mixture stirred vigorously for 12 h at
room temperature. The methanol was removed by rotary evapora-
tion and the remaining aqueous layer extracted with methylene
chloride (2 ꢀ 10 mL). The combined washings were dried over
MgSO4, and the solvent was evaporated to yield 398 mg (58%
overall) of a pale yellow oil.
was stirred under nitrogen at 0 °C, and tert-butyl
D-lactate (0.98 g,
6.6 mmol) in methylene chloride (5 mL) was added dropwise,
followed by DMAP (0.81 g, 6.6 mmol), dissolved in methylene
chloride (5 mL). The mixture was stirred for 20 min and the
precipitate of DMAP hydrochloride removed by filtration. The
resulting chloroformate solution was used directly in the follow-
ing acylation step.
1-
then prepared by the method described above for 1-
butoxycarbonyl) ethyl chloroformate, yielding a colorless oil
(65% yield): IR (neat) 1781, 1744 cm-1
D-(tert-Butoxycarbonyl)-2-phenylethyl chloroformate was
D-(tert-
.
(b) N-(Benzyloxycarbonyl)-O-[1-D-(tert-butoxycarbonyl)-
2-phenylethoxycarbonyl]hydroxylamine. The procedure for 2
(R=CH2Ph, R0 = Me), described above, was also followed for
this synthesis. The crude oil obtained from the reaction of benzyl
(b) N-(Benzyloxycarbonyl)-O-(1-D-carboxyethoxycar-
bonyl)hydroxylamine. Benzyl N-hydroxycarbamate (0.43 g,
2.5 mmol) in methylene chloride (4 mL) was stirred at 0 °C under
an atmosphere of dry nitrogen. Anhydrous pyridine (0.21 mL,
2.5 mmol) was added and the mixture stirred for 10 min. This was
followed by dropwise addition of the chloroformate (0.43 mg, 2.8
mmol). The ensuing mixture was stirred for a further 25 min and
filtered and the solvent evaporated from the filtrate. The crude
product was collected as a colorless oil (0.48 g) that was purified
on silica gel with a hexane/ethyl acetate mixture (2:1) as the
eluent. The colorless solid product was recrystallized from a
benzene/cyclohexane mixture to yield 0.28 g (33%) of colorless
needlelike crystals (mp 79 °C): 1H NMR (CDCl3) δ 8.38 (s, 1H),
7.34 (s, 5H), 5.19 (s, 2H), 4.90 (q, 1H), 1.50 (d, 3H), 1.43 (s, 9H);
IR (KBr) 1792, 1759, 1702 cm-1. Anal. Calcd for C16H21NO7: C,
56.63; H, 6.24; N, 4.13. Found: C, 56.42; H, 6.23; N, 4.04.
(c) Deprotection. The tert-butyl-protected derivative de-
scribed above (265 mg, 0.75 mmol) was dissolved in trifluoroa-
cetic acid (10 mL) and the mixture stirred for 30 min. The solvent
was evaporated and the free acid obtained quantitatively as a
colorless oil, which proved to be recalcitrant to crystallization
attempts. The oil was used directly in kinetic and analytical
N-hydroxycarbamate with 1-D-(tert-butoxycarbonyl)-2-pheny-
lethyl chloroformate was purified by chromatography on silica
gel preparative plates with a hexane/ethyl acetate mixture (2:1) as
1
the eluent, yielding 152 mg (33%) of a colorless oil: H NMR
(CDCl3) δ 7.86 (s, 1H), 7.35 (m, 5H), 7.26 (m, 5H), 5.21 (s, 2H),
5.07 (dd, 1H, J = 5.1 Hz), 3.18 (t, 2H, J = 5.1 Hz), 1.40 (s, 9H); IR
(neat) 1794, 1732 cm-1 (broad, two peaks); ES(þ)MS m/z 438.0
(M þ Naþ).
(c) Deprotection. The tert-butyl-protected compound (20 mg,
0.05 mmol) was dissolved in trifluoroacetic acid (4 mL) and
the mixture stirred for 30 min under a flow of nitrogen. The
trifluoroacetic acid was then evaporated, quantitatively yielding
the free acid as an oil: 1H NMR (CD3CN) δ 9.10 (s, 1H), 7.35 (m,
5H), 7.26 (m, 5H), 5.20 (m, 1H), 5.17 (s, 2H), 3.22 (m, 2H); IR
(neat) 1792, 1732, 1665 cm-1; ES(-)MS m/z 358.07 (M - Hþ).
(iii) R-Hydroxy Esters. Enantiomerically pure R-hydroxy
esters (6-20) were synthesized from their corresponding lactate
derivatives by a common method, as reported below for 7,
except where otherwise noted. The hydroxyl and carboxyl
groups of lactic acid derivatives were first protected as tert-
butyldimethylsilyl (TBDMS) ethers and esters, respectively.
The protected acids were converted to the corresponding acid
chlorides as reported by Weinger (10). Coupling of the acid
chlorides to either a N-hydroxycarbamate or alcohol and
phenol was accomplished with pyridine as the base. The silyl
ethers were then deprotected with KHSO4 in aqueous metha-
nol (11). The products were generally initially purified on
silica gel with a hexane/ethyl acetate mixture (3:1) as the
eluent and finally recrystallized from a benzene/cyclohexane
mixture (1:1).
1
assays. H NMR (CD3CN) δ 9.15 (s, 1H), 7.41 (s, 5H), 5.21
(s, 2H), 5.06 (q, 1H, J = 5.7 Hz), 1.54 (d, 3H, J = 6.3 Hz); IR (neat)
1790, 1736, 1721 cm-1; ES(-)MS m/z 282.3 (M - Hþ).
The 15N isotopomer of this compound was prepared as
described above but using benzyl [15N]-N-hydroxycarbamate (4)
as the starting material.
(ii) N-(Benzyloxycarbonyl)-O-(1-D-carboxy-2-pheny-
lethoxycarbonyl)hydroxylamine (2, R = R0 = CH2Ph).
(a) tert-Butyl 2-D-Hydroxy-3-phenylpropanoate. tert-Butyl
2-
lactic acid following the procedure of Yang et al. (9). Thus, acetyl
chloride (2.47 mL) was added dropwise to solid -3-phenyllactic
D-hydroxy-3-phenylpropanoate was prepared from D-3-phenyl-
(iv) N-(Benzyloxycarbonyl)-O-(2-hydroxy-3-phenylpro-
panoyl)hydroxylamine (7) (and 8). (a) 2-D-TBDMSO-3-
D
acid (500 mg, 3 mmol) at 0 °C. The mixture was brought to reflux
at 60 °C and stirred for 4 h. The reaction mixture was then dried by
rotary evaporation, leaving a pale yellow oil that was taken into
diethyl ether (15 mL) and washed with water (2 ꢀ 15 mL). The
ether layer was dried over Na2SO4 and the solvent evaporated. The
resulting oil was dissolved in methylene chloride (7.5 mL) and
stirred at 0 °C. tert-Butyl alcohol (0.45 g) and DMAP (0.122 g,
1 mmol) were added, followed by DCC (0.83 g, 4 mmol) dissolved
in methylene chloride (2.5 mL). The reaction mixture was allowed
to warm to room temperature as it was stirred for 12 h. The
mixture was filtered and the filter cake washed with methylene
chloride. The combined solutions were washed with water (2 ꢀ
10 mL), and the organic layer was dried over MgSO4. The solvent
was evaporated under reduced pressure, yielding 636 mg of a pale
phenylpropionyl Chloride. A solution of D-3-phenyllactic acid
(500 mg, 3 mmol) in DMF (7.5 mL) was stirred at room
temperature. TBDMS chloride (0.9 g, 6 mmol) and imidazole
(0.61 g, 8.9 mmol) were added, and the reaction mixture was
stirred overnight. The reaction mixture was then diluted with
hexanes and washed sequentially with water (25 mL), saturated
NaHCO3 (25 mL), and brine (25 mL). The organic layer was then
dried over MgSO4 and the solvent removed under reduced
pressure. The protected product was taken into dry methylene
chloride (4 mL) containing one drop of catalytic DMF and
stirred at 0 °C. Oxalyl chloride (1.2 mL, 14 mmol) was added
dropwise and the mixture stirred for 1 h at 0 °C, followed by 1 h at
room temperature. The solvent was removed by rotary evapora-
tion and the acid chloride used directly for acylation.

10498 Biochemistry, Vol. 49, No. 49, 2010
Pelto and Pratt
(b) AcylationReaction. The protected acid chloride (181 mg,
0.61 mmol) in methylene chloride (1.5 mL) was added
dropwise to a stirred solution of benzyl N-hydroxycarbamate
(95 mg, 0.61 mmol) and pyridine (49 μL, 0.61 mmol), dissolved in
a mixture of ethyl acetate (6 mL) and methylene chloride (3 mL)
at 0 °C, under dry nitrogen. The reaction mixture was then stirred
for a further 1 h at 0 °C, the mixture filtered, and the solvent
removed under vacuum. The resulting oil was fractionated on
silica gel with a hexane/ethyl acetate mixture (3:1) as the eluent,
yielding 160 mg (62%) of a clear oil. This silyl ether (94 mg) was
then dissolved in a mixture of methanol (10 mL) and water
(3 mL) containing 15 mg of potassium bisulfate. The mixture was
stirred for 5 days and filtered, and the methanol was removed by
rotary evaporation. The aqueous mixture was extracted with
ethyl acetate and the organic portion dried over MgSO4. The
resulting product was then purified further on silica gel with a
hexane/ethyl acetate mixture (3:1) as the eluent, yielding a
colorless solid that was recrystallized from a benzene/cyclohexane
mixture (1:1). Thus, 33 mg (48%) of 7 as colorless crystals
trifluoroacetic acid (4 mL) was stirred for 30 min. The trifluoro-
acetic acid was removed by rotary evaporation, yielding a clear
colorless oil that was further dried by means of an oil pump. The
resulting free acid was taken into CD3CN (1 mL) and then
diluted into D2O (10 mL) containing 20 mM MOPS buffer
adjusted to an apparent pH of 7.1 (pD 7.5). The subsequent
spontaneous reaction was monitored by proton NMR, to deter-
mine the time of maximum accumulation of the intermediate.
The methylene resonance was conveniently monitored for this
purpose. At this time, the sample was extracted with methylene
chloride (2 ꢀ 4 mL), the combined extracts were washed with
cold distilled water (3 ꢀ 3 mL), and the organic fraction was dried
over Na2SO4. The solvent was then removed by rotary evapora-
1
tion, yielding 5 mg of a colorless semisolid: H NMR (CDCl3)
δ 8.08 (s, 1H, NH), 7.35 (s, 5H), 5.22 (s, 2H), 4.49 (q, 1H, J =
7.1 Hz), 1.50 (d, 3H, J = 7.1 Hz); IR 1735, 1798 cm-1; ES(-)MS
m/z 238.1 (M - Hþ).
(v) P99 β-Lactamase Steady State Kinetics. The enzyme
(final concentration of 0.1-2.0 μM) was added to a buffered
solution of the substrate (0.01-2.5 mM) and the subsequent
hydrolysis monitored spectrophotometrically at an appropriate
wavelength (Table S1 of the Supporting Information). Initial
rates were measured and corrected for spontaneous hydrolysis by
subtraction, where necessary, and the data were fit by means of a
nonlinear least-squares program to the Michaelis-Menten equa-
tion to determine kcat and Km. Alternatively, where saturation
was not achieved, the data were fit linearly to determine kcat/Km.
(vi) Km Values by Competition. The P99 β-lactamase (with
the stock solution containing 1 mg/mL BSA) was diluted into a
buffered solution (final concentration of 1 nM) containing the
substrate cephalothin (50 μM; Km=15 μM) and an R-hydroxy
ester (0-1 mM). The hydrolysis of cephalothin was monitored at
278 nm (Δε = 4000 M-1 cm-1), and the initial rates were
determined. The initial rates were plotted as a function of the
R-hydroxy ester concentration and the data fit to the steady state
equation for competitive inhibition (eq 1) by means of a non-
linear least-squares program. The Ki value obtained should
correspond to the Km value of the R-hydroxy ester as a substrate.
1
(mp 110-112 °C) was obtained: H NMR (CD3CN) δ 8.97 (s,
1H), 7.39 (m, 5H), 7.30 (m, 5H), 5.19 (s, 1H), 4.52 (dd, 1H), 3.01
(m, 2H); 13C NMR δ 173.97, 157.80, 138.26, 137.26, 129 (m),
71.88, 71.45, 69.02; IR (KBr) 1804, 1721 cm-1; ES(-)MS m/z
314.33 (M - Hþ). Anal. Calcd for C17H17NO5: C, 64.75; H, 5.44;
N, 4.44. Found: C, 64.33; H, 5.44; N, 4.43.
Analytical and Kinetic Methods. Absorption spectra and
spectrophotometric reaction rates were measured with a Hewlett-
Packard 8453 UV-vis spectrophotometer. Steady state kinetics
were assessed at 25 °C, buffered in 20 mM 3-morpholinopropa-
nesulfonic acid (MOPS) at pH 7.5, unless otherwise noted. The
substrates were prepared in concentrated acetonitrile stock
solutions and diluted to e5% acetonitrile in assays. Acetonitrile
alone at these concentrations had no effect on initial rate
measurements.
(i) 15N gHSQC Spectroscopy. Two-dimensional (2D) 15
N
gHSQC spectra of [15N]2 (R = PhCH2, R0 = Me) (10 mM) and
its reaction products in d6-DMSO were recorded at 25 °C on a
Varian Unityplus 500 spectrometer. The data acquisition param-
eters were as follows: 10 kHz spectral width, 2 kHz 2D spectral
width, 1 s acquisition time, 1 s delay time, 2 ꢀ 64 increments, and
8 repetitions. Chemical shifts were referenced to formamide
(108.5 ppm) as an internal standard (12).
v0 ¼ VmaxS0=½S0 þ Kmð1 þ I=KiÞꢁ
ð1Þ
(vii) Methanolysis of the P99 β-Lactamase Acyl Enzyme.
The effect of methanol on the initial rates of solvolysis of sub-
strate 7 (700 μM; Km=71 μM) by the P99 β-lactamase (0.1 μM)
was determined spectrophotometrically (233 nm), under close to
saturating substrate conditions. Methanol concentrations of up
to 2.5 M in aqueous MOPS buffer (20 mM) were employed; this
concentration of methanol has been shown to have a negligible
effect on enzyme activity (13). The effect of methanol (0-2.5 M)
on the initial rates of solvolysis of 14 (800 μM; Km ∼ 700 μM) in
the presence of the P99 β-lactamase (0.5 μM) was similarly
determined spectrophotometrically at 230 nm. The initial rates of
the enzyme-catalyzed solvolysis of 7 were plotted as a function of
methanol concentration and fit to eq 2 [derived from Scheme 4,
Results and Discussion (14)] by means of a nonlinear least-
squares program.
(ii) Spontaneous Hydrolysis. The spontaneous hydrolysis
rates of the compounds were measured spectrophotometrically in
triplicate at concentrations of 50-500 μM at appropriate wave-
lengths as reported in Table S1 of the Supporting Information.
The full progress curves were fit to a pseudo-first-order rate
equation by means of a nonlinear least-squares program and the
triplicate rate constants averaged.
(iii) pKa Determination. The pKa of ester 7 (100 μM) was
determined spectrophotometrically by monitoring the change in
absorbance of the molecule at 233 nm as a function of pH. The
pH was varied from 4.0 to 10.0 in increments of 0.5 at 25 °C. A
mixed buffer system containing sodium acetate, MES, MOPS,
AMPSO, TAPS, and CAPS (20 mM each) was employed. A
constant ionic strength of 1.0 was maintained with sodium
chloride.
Rβð1 þ S0=KmÞ
v=v0
¼
ð2Þ
Rβ þ ½H2Oꢁ0ðk2=k3 þ βÞðS0=KmÞ
(iv) Isolation of the Reaction Intermediate from Sponta-
neous Hydrolysis of N-(Benzyloxycarbonyl)-O-(1-D-car-
boxyethoxycarbonyl)hydroxylamine. N-(Benzyloxycarbonyl)-
where R = k2/k3 þ [H2O]0 and β = [H2O] þ (k4/k3)[MeOH].
(viii) Aminolysis of the P99 β-Lactamase Acyl Enzyme.
To investigate the aminolysis of the acyl enzyme derived from
O-(1-D-tert-butylcarboxyethoxycarbonyl)hydroxylamine (30 mg,
88 μmol) was freshly deprotected when a solution of it in

Article
Biochemistry, Vol. 49, No. 49, 2010 10499
Scheme 1
compound 20, two complementary methods were employed.
First, the turnover of 20 (1 mM, ∼5Km) by the P99 β-lactamase
monitoring the turnover of m-carboxyphenyl N-phenylacetyl-
-alaninate (3 mM) by the R39 enzyme (0.1 μM) at 305 nm.
(xi) Molecular Modeling. Simulations were performed on a
D,
L
(0.1 μM), in the presence of -phenylalanine (0-40 mM), was
D
monitored by following the release of m-hydroxybenzoate at
SGI workstation running InsightII (Accelrys). The crystal struc-
ture of a phosphonate-bound P99 β-lactamase (Protein Data
Bank entry 1BLS) (16), after removal of the phosphonate ligand,
was the starting point for building the tetrahedral intermediate of
acylation (21/22). In this construct, Tyr 150 was neutral and both
Lys 67 and Lys 315 were cationic. Partial charges on the atoms of
the substrate, as the anionic intermediate, were calculated using a
model of the adduct, which included the nucleophilic Ser 64 but
not the rest of the protein. The model was subjected to a 1000-
step steepest gradient energy minimization prior to the use of
MNDO level calculations from the MOPAC module of InsightII,
to calculate the partial charges.
290 nm. Second, at a constant concentration of -phenylalanine
D
(20 mM), the aminolysis of the substrate (0-3 mM) by the
β-lactamase (0.1 μM) was monitored (290 nm). Initial rates were
measured in each case, and the two data sets were fit simulta-
neously to Scheme 4 by means of Dynafit (15).
(ix) pH-Rate Profiles. pH-rate profiles were obtained in a
mixed buffer system of sodium acetate, MES, MOPS, AMPSO,
TAPS, and CAPS (20 mM each) with a constant ionic strength of
1.0 maintained with NaCl. Experiments were conducted at 25 °C.
Compound 7 was hydrolyzed over a pH range of 6.0-9.5, and
kcat/Km was determined by initial rates as described above, with
the spontaneous hydrolysis taken into account. For pH 6.0-8.0
and 8.5-9.5, hydrolysis was monitored at 233 and 250 nm,
respectively, because of pH-dependent changes in the extinction
coefficients. Because the solubility of 7 diminished at pH values
below its pKa (7.6), and the change in the extinction coefficient
also decreased, rate measurements were not possible below pH 6.
Values of kcat/Km in the obtained range, however, were plotted
against the pH to obtain a curve, which was fit to eq 3 by means of
a nonlinear least-squares program. The more soluble compound
20 was employed to obtain a full pH profile (pH range of
4.0-9.5) in the same manner.
Both R and S isomers of the chiral reaction center were
constructed. Hydrogens were added to the Protein Data Bank
structure, and the pH of the enzyme complex was set to 7.5.
The partial charges of the protein residues were assigned with
InsightII. The total charge on each complex was -1.0. The active
˚
site was hydrated with a 15 A sphere of water centered on Oγ of
the nucleophilic Ser 64. The models were energy-minimized with
1000 steepest gradient steps followed by molecular dynamics of
10000 equilibration steps and 90000 production steps. Confor-
mations that were found to contain specific hydrogen bonding
interactions with the R-hydroxyl group of the substrate were then
energy minimized by 1000 steepest descent steps, followed by
2000 conjugate gradient steps. Relative quantitative evaluation of
the models was achieved by means of calculations of ligand
interaction energies, Eint (17).
max
kobs ¼ k1 Ka1h=ðKa1h þ Ka1Ka2 þ h2Þ
ð3Þ
(x) Kinetics with Other Enzymes. Experiments for deter-
mining steady state parameters for turnover of the R-hydroxy
esters (50-1000 μM) by the TEM-2 (0.01- 2.0 μM) and OXA-1
β-lactamases were performed as described above for the P99
enzyme. In the case of the OXA-1 enzyme, the buffer consisted of
20 mM MOPS, 50 mM NaHCO3 and 0.1% gelatin, maintained
at pH 7.5. Hydrolyses of compounds 7 (e1 mM) and 20 (e1 mM)
in the presence of this enzyme (0.5 and 1.0 μM, respectively) were
monitored as described above. Competitive inhibition experi-
ments were performed as described above with compound 20
(3 mM) and the OXA-1 enzyme (0.1 μM) using the substrates
benzylpenicillin (100 μM, monitored at 230 nm) and Centa (10 μM,
monitored at 410 nm) by the techniques described above.
Compound 15 (5 μM) was also tested for competitive inhibition
against the substrate cephalothin (50 μM) with the OXA-1
enzyme (0.2 μM).
RESULTS AND DISCUSSION
The synthesis of compounds of general structure 2, as their
tert-butyl esters, was achieved largely in the manner successfully
employed for 1, from the reaction of a hydroxamic acid with a
chloroformate in the presence of a tertiary amine base (Scheme 1)
(5). The tert-butyl ester carboxyl protecting group was removed
by trifluoroacetic acid treatment immediately before use because
it was found that the free acids were unstable, to decarboxylation
as subsequently demonstrated (see below). That compound 5
(R0 = Me) was indeed an O-acyl hydroxamate, rather than N-acyl,
was demonstrated by 15N gHSQC spectroscopy, in which direct
coupling between a 15N nucleus resonating at 158.8 ppm and a
proton at 11.8 ppm was observed.
Incubation of 2 (R = CH2Ph, R0 = Me) with the P99
β-lactamase led to the data presented in Figure 1 which shows an
initial loss of enzyme activity, measured against the good sub-
strate, cephalothin, followed by a slower restoration of activity.
This result is very different from that obtained with the aryl
derivatives, 1, in which the enzyme became irreversibly inactiv-
ated (4, 5); the mechanism of the reaction of 2 with the enzyme,
Hydrolysis of compounds 7 (500 μM), 8 (500 μM), and 20
(3.0 mM), in the presence of the Streptomyces R61 DD-peptidase
(0.5 μM), was monitored spectrophotometrically as described
above. Hydrolysis of 7 (500 μM), 8 (500 μM), and 20 (1.0 mM)
was also studied in the presence of the Actinomadura R39 DD-
peptidase (0.4, 0.4, and 1.0 μM, respectively). Competitive
inhibition experiments were performed with 20 (1.0 mM),

10500 Biochemistry, Vol. 49, No. 49, 2010
Pelto and Pratt
F
IGURE 3: 1H NMR spectral changes in the methylene region on
spontaneous reaction of 2 (R = PhCH2, R0 = Me) (S) at pH 7.5.
Resonance peaks from intermediate I and final product P (benzyl
N-hydroxycarbamate) appeared as the reaction proceeded. The signal
corresponding to the former subsequently disappeared as that of the
latter continued to rise.
F
IGURE 1: Activity of the P99 β-lactamase (0.25 μM) as a function of
time after being mixed with 2 (R = PhCH2, R0 = Me) (100 μM).
1
A H NMR experiment (Figure 3) showed that during the
reaction of 2 (R = CH2Ph, R0 = Me) in neutral aqueous buffer
in the absence of an enzyme, there was an intermediate, I,
between the starting material, S, and the final hydrolysis product(s),
P. The final product spectrum showed the expected mixture of
benzyl N-hydroxycarbamate and lactate. The intermediate was
isolated by extraction from a reaction mixture, as described in
Materials and Methods, and identified on the basis of its spectra
(see Materials and Methods) as 6, an R-hydroxycarboxylic acid
anhydride with benzyl N-hydroxycarbamate. This identification
was confirmed by independent synthesis (Materials and Methods).
The rates of spontaneous and enzyme-catalyzed reactions of
the isolated intermediate and the independently synthesized
material (6) were identical. These kinetics were also in accord
with the observations of Figure 1, which indicated that reaction
of 2 (R=PhCH2, R0=Me) in the presence of the P99 β-lactamase
involves a transiently stable acyl enzyme species, probably
derived from reaction of the enzyme with 6. Reaction of 2
(R=R0 =PhCH2) was similarly shown to yield 7.
F
IGURE 2: Absorbance changes at 250 nm upon reaction of 2 (R =
PhCH2, R0 = Me) (0.4 mM) in the absence (---) or presence of the
P99 β-lactamase [(---) 0.25, (;;;) 0.5, and (;) 1.25 μM].
which exhibits a transiently stable intermediate, must therefore be
different from that of 1, despite the common O-acyl hydroxamate
structure. Thus, we obtained an immediate indication of new
active site chemistry and therefore a new class of substrates
and/or inhibitors.
Thus, in aqueous solution, 2 rearrange to R-hydroxyalkyl
esters 3. A likely rearrangement path from 2 (R=PhCH2, R0=Me)
to 6 is shown in Scheme 2. We are unaware of a strong precedent
for the first step, although the probably more thermodynamically
favorable reverse reaction has been described previously (18). It is
likely that 23 would be a very transient intermediate, and we did
not observe it in the experiments described in this paper.
Precedent for the second step is found in the rearrangement of
mixed anhydrides of carboxylic and carbonic acids to esters with
loss of carbon dioxide (19, 20).
If interpreted correctly, the results described above suggested
that R-hydroxycarboxylic acid esters are β-lactamase substrates.
Because the R-hydroxyalkyl moiety has not previously been
noticed to have β-lactamase affinity, we proceeded to investigate
further. Synthesis of 3 and 4 (Scheme 3) was achieved by reaction
of R-hydroxy acid chlorides, where the R-hydroxy group was
protected by tert-butyldimethylsilylation, with either hydroxamic
acids or alcohols and phenols in the presence of pyridine.
Desilylation of the product was affected by its treatment with
KHSO4 in aqueous methanol. Thus, compounds 6-20 were
prepared. They were purified by recrystallization if they were
solids or by flash chromatography on a silica gel if not. As noted
Direct spectrophotometric observation of the reaction be-
tween the P99 enzyme and 2 (R = CH2Ph, R0 = Me) gave data
such as those depicted in Figure 2. In the absence of enzyme, a
burst phase is observed followed by a slower reaction. In the
presence of enzyme, a second, enzyme-catalyzed, phase of the
reaction is inserted between the two phases described in the
previous sentence. The first phase, observed in both the absence
and presence of enzyme, is not enzyme-catalyzed. The nature of
the slower spontaneous reaction is unknown at present. It
appeared to require the hydroxamate leaving group and repre-
sents a process with a large extinction coefficient change at 250 nm,
but one that does not produce an amount of product that can be
detected by 1H NMR. One interpretation of these data, and one
that we pursued, was that 2 in aqueous buffer was not an effective
enzyme substrate or inhibitor, at least over several minutes, but
spontaneously rearranged to a molecule that is a β-lactamase
substrate.

Article
Biochemistry, Vol. 49, No. 49, 2010 10501
Scheme 2
above, compounds 6 and 7 were identical to the intermediates
isolated from the reaction of 2 (R = CH2Ph, R0 = Me) and 2 (R =
R0 = CH2Ph), respectively. Compounds 6-20 were then examined
for their reactivity with serine β-lactamases.
Most of the compounds in the group of 6-20 were found to be
quite effective substrates of the P99 β-lactamase, not as effective as
the best β-lactam substrates (kcat/Km g 106 s-1 M-1), but compar-
able to previously described depsipeptides (2, 13, 14). Steady state
rate parameters for the new compounds are listed in Table 1. It can
be seen that the best of these substrates is 7 with a kcat/Km value
of 1.2 ꢀ 105 s-1 M-1. This compound is a considerably better
substrate than 6, presumably because of addition of the hydro-
phobic phenyl group. Further hydrophobic extension of the acyl
moiety, as seen in 13, did not lead to a rate constant larger than that
of 7. Although 15 was prepared, the details of its interaction with the
P99 β-lactamase could not be obtained directly because of a
combination of its low water solubility (e50 μM) and the small
absorption change accompanying hydrolysis. An experiment in
which its inhibition (at 20 μM) of cephalothin turnover by the
enzyme was assessed showed that its Km value must be higher than
that of 7. Aggregation in solution might, of course, also be a
problem with 15. Comparison of the parameters for 16 and 7
suggests that some degree of hydrophobic bulk in the leaving group
is also favorable for reaction with the enzyme.
All of the compounds 6-20 are susceptible to spontaneous
hydrolysis in a neutral aqueous solution. 1H NMR studies showed
that the hydrolysis products were the R-hydroxy acid and the
leaving group hydroxyl compound. Rate constants for this process
in 20 mM MOPS (pH 7.5) are listed in Table 1 and can be seen to
range between 10-5 and 10-4 s-1 (excluding the very reactive
amino acid ester 11). For perspective on these rate constants, it
might be noted that the spontaneous hydrolysis rate constants of
benzylpenicillin and clavulanic acid under similar conditions are
1.5 ꢀ 10-5 (21) and 8.0 ꢀ 10-5 s-1 [the hydrolysis of clavulanic acid
(85 μM) in 25 mM MOPS buffer (pH 7.5, 25 °C) was monitored
spectrophotometrically at 260 nm; the resulting trace was fitted to a
first-order reaction scheme], respectively.
The importance of the R-hydroxy group to the recognition and
turnover of these compounds by the β-lactamase is illustrated by
the results with compounds 9 and 10. Compound 9, a direct
analogue of 7, but lacking the hydroxyl group, was not detectably
a substrate at enzyme concentrations of e2 μM. No time-
dependent inactivation of the enzyme (0.25 μM) by 9 (200 μM)
was detected, eliminating the possibility that a refractory acyl
enzyme formed. An experiment for detecting inhibition of
cephalothin turnover by 9 showed that its Ki value, and thus
the Km value of 9 as a substrate, must be g0.5 mM. Along the
same lines, it was found that 10, where the hydroxyl group of 6 is
replaced with a methoxy group, is a much poorer substrate than 6.
Finally, compound 11, in which the hydroxyl group of 7 is
replaced with an amine (11 would probably be mainly in the neutral
form at pH 7.5) and a better leaving group is present [pKa values of
benzyl N-hydroxycarbamate and p-nitrophenol of 14.3 and 9.4,
respectively, in a dioxane/water mixture (22)], is clearly a much
poorer substrate than 7. Only an upper limit to kcat/Km for 11 could
be obtained because no enzyme-catalyzed reaction was observed
over the rapid spontaneous hydrolysis. Taken together, these results
attest to the benefits of the R-hydroxy group in determining the
ability of these esters to be P99 β-lactamase substrates. The results
described above also suggest that the hydroxyl group may act as a
hydrogen bond donor in its productive complex with the enzyme.
Comparison of the data for 7 and 8 shows that the
D
-R-
hydroxy enantiomer is preferred to the , although the latter
L
retains significant activity, suffering mainly in the Km parameter.
Previous investigations of class C β-lactamases with acyclic
substrates have generally shown preferences for the L enantiomer
in the acyl fragment adjacent to the scissile bond (23, 24), for
structural reasons that have been discussed (24). The preference
As O-acyl hydroxamates, 6-10, 12, 13, 15, and 16 would be
expected to have an acidic NH proton (5). Spectrophotometric
titration of 7 indeed yielded a pKa value of 7.58 ( 0.03, slightly
higher than those of 1 [6.8-7.2 (5)]. Thus, at pH 7.5, roughly equal
amounts of the neutral and anionic forms of 7, and presumably of
its analogues, 8-10, 12, 13, 15, and 16, would be present in solution.
for the
D enantiomer observed in this work suggests that the acyl
substituents of 3 may interact with the active site rather differ-
ently than those of, say, 24 (see below).
The electronic quality of the leaving group is also important.
The aliphatic ester 17, even when bearing a carboxylate and a

10502 Biochemistry, Vol. 49, No. 49, 2010
Pelto and Pratt
Scheme 3
Scheme 4
Table 1: Rate Parameters for Spontaneous Hydrolysis of R-Hydroxy
Esters and Their Steady State Turnover by the P99 β-Lactamase
k
sp (s-1
)
Km (μM)
kcat (s-1
)
kcat/Km (s-1 M-1
)
6
7
8
9
(8.6 ( 1.9) ꢀ 10-5
(8.2 ( 0.8) ꢀ 10-5
NDa
520 ( 140
71 ( 10
340 ( 60
NRb
1.5 ( 0.2
8.7 ( 0.3
5.9 ( 0.3
2.8 ꢀ 103
1.2 ꢀ 105
1.74 ꢀ 104
(6.5 ( 0.2) ꢀ 10-5
10 (1.64 ( 0.02) ꢀ 10-5 g103
g0.05
46 ( 1
11 (6.8 ( 1.7) ꢀ 10-3
e3 ꢀ 103
7.5 ꢀ 103
5.5 ꢀ 104
3.2 ꢀ 104
12 (6.0 ( 0.7) ꢀ 10-5
13 (6.4 ( 2.1) ꢀ 10-5
14 (9.0 ( 0.6) ꢀ 10-5
15 NDa
460 ( 240
20 ( 2
660 ( 340
insoluble/NRb
480 ( 100
NRb
3.5 ( 0.7
1.5 ( 0.1
21.0 ( 5.7
Support for rate-determining deacylation in these cases was
obtained from measurement of the kinetic effect of alternative
nucleophiles. At substrate concentrations where a significant
amount of the acyl enzyme intermediate would be expected to
accumulate, i.e., gKm, alternative nucleophiles, Nu, would
directly accelerate turnover if enzyme deacylation were rate-
determining. This effect has been observed with many substrates
of the P99 β-lactamase, including β-lactams (13), in the presence
of methanol, and acylic substrates in the presence of methanol
16 (5.6 ( 2.2) ꢀ 10-5
17 (2.6 ( 0.1) ꢀ 10-5
18 (2.0 ( 0.2) ꢀ 10-5
19 (9.0 ( 0.5) ꢀ 10-5
20 (2.0 ( 0.5) ꢀ 10-5
7.4 ( 0.7
1.5 ꢀ 104
1010 ( 120
320 ( 60
172 ( 16
10.4 ( 0.7
10.9 ( 0.8
6.9 ( 0.2
1.0 ꢀ 104
3.4 ꢀ 104
3.9 ꢀ 104
aNot determined. bNo reaction observed.
and D-amino acids (26). In this case, turnover of 7 was linearly
phenyl ring in the leaving group, which might have been expected
to enhance specificity, was not observably a substrate. On the
other hand, the aryl esters 18-20 were comparable in reactivity to
the hydroxamates. The limited effect of nitro group substitution
(19 vs 18) may reflect either a steric problem with the nitro group
or the presence of significant electrophilic catalysis. The latter has
been noted previously in acylation of the P99 β-lactamase active
site (14, 25) and, indeed, may be present with 1 (5). The result
with compound 20 is distinctive. The addition of the m-carbox-
ylate to 18 has an effect on kcat/Km much weaker than that found
in acyclic substrates such as 24 (see below); in the latter, the
m-carboxylate is placed to interact with the same active site
functional groups as the carboxylate of β-lactams (6-8). It seems
likely that the leaving group in acylation of the enzyme by 4 is
placed differently compared to that in acylation by 24.
If, as is likely, the hydrolysis of R-hydroxy esters is catalyzed by
the P99 β-lactamase by a double-displacement mechanism invol-
ving an acyl enzyme intermediate (Scheme 4), the observation of
quite similar kcat values for 7, 16, and 18-20, which have the
same acyl group, suggests that deacylation may be rate-determin-
ing in turnover of these molecules under substrate saturation
conditions. This would not be surprising because rate-determin-
ing deacylation is a common feature of catalysis by the P99
β-lactamase (13, 14).
accelerated by addition of methanol (Figure S1 of the Supporting
Information), from which data the partition ratio k4/k3 was
calculated to be 22.6 ( 0.8. This value is interestingly similar to
those for more classical substrates, 25 ( 10 (8, 14, 24, 27).
Similarly, D-phenylalanine (but not L-phenylalanine) accelerated
turnover of 20 quite dramatically (Figure 4). From these data and
with the assumption of Scheme 4, the following kinetic constants
were obtained by curve fitting: Ks = 2.85 mM, k2 = 113 s-1
,
k3 = 7.3 s-1, and k4 = 1600 s-1 M-1. These data support the
proposition that Scheme 4 describes the turnover of R-hydroxy
esters by the P99 β-lactamase and suggests that the deacylation
step may often be the slow one.
A pH-rate profile for hydrolysis of 20 by the P99 β-lactamase
yielded the bell-shaped curve in Figure 5 and the associated
pKa values of 5.75 ( 0.20 and 9.37 ( 0.19. These values are
comparable to those from classical substrates (27-30) and
indicate that the same active site functional group assembly is
probably responsible for catalysis. The profile for hydrolysis of 7,
which could be studied spectrophotometrically only at pH values
above the pKa because of the absence of a measurable spectral
change at lower pH, yielded a pKa for decreasing activity of
7.6 ( 0.2 (Figure 5). This correlates well with the pKa obtained
with 1 (5) and strongly suggests that the reactive form of 7 is the

Article
Biochemistry, Vol. 49, No. 49, 2010 10503
F
IGURE 5: pH-rate profiles for hydrolysis of 7 (b) and 20 (0) in the
presence of the P99 β-lactamase. The fitted lines were calculated as
described in the text.
the normal acyl (side chain) site (16, 31, 32) and the hydroxamate
in the leaving group site (31, 33). A variety of starting conforma-
tions of the R-hydroxyacyl group were chosen and stable con-
formers sought by a combination of molecular dynamics and
energy minimization computations. In all of the thereby derived
structures, the usual positioning of active site residues was
observed; viz., the Lys 67 and Tyr 150 side chain functional
groups closely associated with either Ser 64 Oγ or the leaving
group (hydroxamate or phenolic) oxygen, and placement of the
oxyanion in its usual hole composed of the backbone NH groups
of Ser 64 and Ser 318 (31, 34). The reactivity of 3 and 4 would still,
therefore, be controlled by the pKa values of these residues.
F
IGURE 4: Initial rates of reaction of 20 in the presence of the P99
β-lactamase (0.1 μM) and D-phenylalanine. (A) Variation of the rate
at a fixed concentration of 20 (1.0 mM) and varying D-phenylalanine
concentrations. (B) Effect of varying the concentration of 20 at a
fixed D-phenylalanine concentration (20 mM). The fitted lines were
calculated as described in the text.
The most favorable (by the criterion of ligand interaction
energy values, Eint) conformations obtained showed the
R-hydroxyl group hydrogen bonded as an acceptor to the Lys
67 terminal ammonium group (Figure 6A) or, more favorably, as
a donor to the Ser 318 carbonyl oxygen (Figure 6B). In the former
case, the phenyl group of the substrate was situated in such a way
that it formed an amide NH-π complex (35, 36) with the side
chain of Asn 152 and, in the latter, hydrophobic contact with Tyr
221. Another apparently stable structure, similar to that depicted
in Figure 6B but in which the R-hydroxyl group is hydrogen-
bonded to Ser 318 Oγ, was also noted. This structure, however,
seems unlikely to be generally important because 7 was also
found to be a good substrate of the ampC β-lactamase, another
class C enzyme, but in which Ser 318 is replaced with Ala. In none
of these structures does there appear to be any particular very
specific interaction with the hydroxamate leaving group except
that of Tyr 150 with the oxygen more proximal to the reaction
center (see Figure 6B, for example).
neutral ester rather than the nitrogen-based anion. In further
support of this conclusion, the data in Table 1 indicate that 14,
the N-methylated derivative of 7, is also an effective substrate.
The apparently rather higher kcat value of 14 versus what would
be expected on the basis of the discussion given above (14 has the
same acyl group as 7 and 20) may be an artifact; because of the
low solubility of 14 (e0.8 mM), rate measurements at concen-
trations greater than Km were not possible, and hence, the kcat
and Km values should be seen as rather uncertain. Deacylation of
the acyl enzyme derived from 14, like that from 7, is likely to be
rate-determining at saturation because methanol acceleration of
the reaction of the former was observed at a concentration close
to Km (data not shown).
Structural Considerations. Molecular modeling was used to
explore possible interactions of the R-hydroxyl group with active
site residues and thus, perhaps, rationalize the activity of these
compounds as substrates. Tetrahedral intermediate 21 was con-
structed at the P99 β-lactamase active site. This represents the R
configuration at the tetrahedral carbon generated by nucleophilic
attack by the active site serine hydroxyl group at the substrate
carbonyl. The R configuration has the R-hydroxyacyl moiety in
The available structure-activity data can then be assessed in
terms of these structures. First, if the R-hydroxyl group is acting
as a hydrogen bond donor, as suggested by the experimental data
given above, the model in Figure 6B would be more likely. The
limited effect of the m-carboxylate in 20, when compared with 18
and 19, for example, is, however, rather striking. The m-carbox-
ylate in phenaceturate substrates such as 24 has been shown to

10504 Biochemistry, Vol. 49, No. 49, 2010
Pelto and Pratt
FIGURE 6: Stereoviews of energy-minimized tetrahedral intermediate structures formed upon reaction of 7 with the P99 β-lactamase. Only heavy
atoms are shown. Panels A and B show alternative conformations of the R adduct (21).
enhance catalysis rather more markedly (13), probably by its
interaction with hydrogen bond donors adjacent to the active
site (8). Also, although the phenyllactate 25 is a substrate of the
P99 β-lactamase (13), the R-hydroxyacyl analogue 17 is not.
These observations rather suggest that the orientation of the
leaving groups of the two series of substrates when bound to the
enzyme may be different. One further possibility with respect to
this issue is, of course, that the S tetrahedral intermediate (22)
rather than the R intermediate (21) is formed, where the relative
positions of the acyl group and leaving group are reversed.
Models of the S configuration were constructed (e.g., Figure
S1 of the Supporting Information), but none led to structures in
which the R-hydroxyl group interacted directly with the enzyme.
versus the
L
enantiomer 8, which is rather unusual if the reaction
is thought to take place through the positioning of 3 as for
classical substrates (Figure 6A,B). Another abnormal result,
relating to this last point, is the fact that 7, 8, and 20 (the other
compounds were not tested) were not substrates of the Strepto-
myces R61 and Actinomadura R39 DD-peptidases. The R61
peptidase, in particular, has a protein fold and active site
structure very similar to those of the P99 β-lactamase (37, 38).
Phenaceturates such as 24 (R = H) are substrates of this enzyme,
as are R-substituted analogues such as 24 (R = Me or OMe),
where there is an absolute stereochemical preference for one
enantiomer at the R-position, thought to be
structure of natural substrates of this enzyme (24, 33). This
enzyme, unlike the β-lactamase, has a specific binding pocket for
D on the basis of the
small
however, reflecting a preference for the methyl group of its
natural substrate, an acyl- -alanyl- -alanine peptide. The place-
D-R-substituents (33, 39, 40). The pocket is hydrophobic,
D
D
ment of a hydroxyl group in this site may well be unfavorable.
The β-lactamase, which does not have such a pocket (33, 39, 41),
is forced to react with
bound in a different conformation (24). In the case presented
here, the polar -substituent (OH) is most likely hydrogen
D
-R-substituted substrates, which it does,
A final point relating to structure was also noted above, the
clear preference of the P99 β-lactamase for the
D
enantiomer 7
D

Article
Biochemistry, Vol. 49, No. 49, 2010 10505
bonded to either Lys 67 (Figure 6A) or Ser 318 (Figure 6B) of the
β-lactamase. The structures of Figure 6 can each accommodate
5. Pelto, R. B., and Pratt, R. F. (2008) Kinetics and mechanism of
inhibition of a serine β-lactamase by O-aryloxycarbonyl hydroxa-
mates. Biochemistry 47, 12037–12046.
an
L
-R-hydroxy substrate by redirection of the alkyl group.
6. Strynadka, N. C. J., Adachi, H., Jensen, S. E., Johns, K., Sielecki, A.,
Betzel, C., Sutoh, K., and James, M. N. G. (1992) Molecular structure
Although the discussion given above seems to favor occupa-
˚
of the acyl-enzyme intermediate in β-lactam hydrolysis at 1.7 A
resolution. Nature 359, 700–705.
tion of the active site by R-hydroxy esters as shown in Figure 6B,
direct evidence of the orientation of these substrates during
reaction with the enzyme will most likely come from the crystal
structure of an acyl enzyme derived from 7 or from a poorer
substrate or inhibitor containing the R-hydroxylalkyl moiety.
Other β-Lactamases. The class A TEM-2 β-lactamase did
catalyze hydrolysis of the R-hydroxy esters 3 and 4, although less
efficiently than the class C enzymes. For example, kcat/Km values
for 7, also the best substrate of the group of 6-20, and 20 were
2.4 ꢀ 103 and 460 s-1 M-1, respectively; in both cases, Km values
were >1 mM. This result may just reflect the generally lower
reactivity of this class A enzyme with acyclic substrates (13, 24).
7. Beadle, B. M., Trehan, I., Focia, P. J., and Shoichet, B. K. (2002)
Structural milestones in the reaction pathway of an amide hydrolase:
Substrate, acyl and product complexes of cephalothin with amp C
β-lactamase. Structure 10, 413–424.
8. Ahn, Y.-M., and Pratt, R. F. (2004) Kinetic and structural conse-
quences of the leaving group in substrates of a class C β-lactamase.
Bioorg. Med. Chem. 12, 1537–1542.
9. Yang, D., Li, B., Ng, F., Yan, Y., Qu, J., and Wu, Y. (2001) Synthesis
and characterization of chiral N-O turns induced by R-aminoxy acids.
J. Org. Chem. 66, 7303–7312.
10. Weinger, J. S., Kitchen, D., Scaringe, S. A., Strobel, S. A., and Muth,
G. W. (2004) Solid phase synthesis and binding affinity of peptidyl
transferase transition state mimics containing 20-OH at P-site position
A76. Nucleic Acids Res. 32, 1502–1511.
11. Arumugam, P., Karthikeyan, G., and Perumal, P. T. (2004) A mild,
efficient, and inexpensive protocol for the selective deprotection of
TBDMS ethers using KHSO4. Chem. Lett. 33, 1146–1147.
12. Someswara Rao, N., Babu Rao, G., Murthy, B. N., Maria Das, M.,
Prabhakar, T., and Lalitha, M. (2002) Natural abundance nitrogen-15
nuclear magnetic resonance spectral studies on selected donors.
Spectrochim. Acta, Part A 58, 2737–2757.
13. Govardhan, C. P., and Pratt, R. F. (1987) Kinetics and mechanism of
the serine β-lactamase catalyzed hydrolysis of depsipeptides. Bio-
chemistry 26, 3385–3395.
14. Xu, Y., Soto, G., Hirsch, K. H., and Pratt, R. F. (1996) Kinetics and
mechanism of the hydrolysis of depsipeptides catalyzed by the
β-lactamase of Enterobacter cloacae P99. Biochemistry 35, 3595–3603.
15. Kuzmic, P. (1996) Program DYNAFIT for the analysis of enzyme
kinetic data: Application to HIV proteinase. Anal. Biochem. 237, 260–
273.
16. Lobkovsky, E., Billings, E. M., Moews, P. C., Rahil, J., Pratt, R. F.,
and Knox, J. R. (1994) Crystallographic structure of a phosphonate
derivative of the Enterobacter cloacae P99 cephalosporinase: Mechan-
istic interpretation of a β-lactamase transition state analog. Biochem-
istry 33, 6762–6772.
17. Curley, K., and Pratt, R. F. (1997) Effectiveness of tetrahedral
adducts as transition-state analogs and inhibitors of the class C
β-lactamase of Enterobacter cloacae P99. J. Am. Chem. Soc. 119, 1529–
1538.
18. Kashiwagi, T., Kozuka, S., and Oae, S. (1970) The decomposition of
diacyl peroxides. I. The thermal decomposition of primary and
secondary diacyl peroxide. Tetrahedron 26, 3619–3629.
19. Hyun, M. H., Kang, M. H., and Han, S. C. (1999) Use of 2-ethoxy-
1-(ethoxycarbonyl)-1,2-dihydroquinoline as a convenient reagent for
the selective protection or derivatization of 2-hydroxycarboxylic
acids. Tetrahedron Lett. 40, 3435–3438.
20. Guzzo, P. R., Dinn, S. R., Lu, J., and Oettinger-Loomis, S. (2002)
Preparation of optically active (acyloxy)alkyl esters from optically
active O-acyl-R-hydroxy acids. Tetrahedron Lett. 43, 5685–5689.
21. Cabaret, D., Adediran, S. A., Garcia Gonzalez, M. J., Pratt, R. F.,
and Wakselman, M. (1999) Synthesis and reactivity with β-lactamases
of “penicillin-like” cyclic depsipeptides. J. Org. Chem. 64, 713–720.
22. Pless, J. (1963) Use of some new active esters in peptide synthesis. In
Peptides: Proceedings of the Fifth European Symposium, pp 69-72,
MacMillan, New York.
The TEM-2 β-lactamase, as did the P99 enzyme, preferred the
enantiomer 7 to the enantiomer 8; the kcat/Km value for 8 was
540 s-1 M-1
D
L
.
A class D β-lactamase, OXA-1, did not catalyze hydrolysis of 7
and 20 at all. This enzyme is, however, an even poorer catalyst of
the hydrolysis of acyclic substrates in general than class A (TEM)
and C (P99) enzymes (27). It is possible that interaction of the
R-hydroxy group with the carboxylated active site lysine, which is
believed to be an essential general acid/base in substrate turn-
over (42, 43), precludes reaction; no significant fast reversible or
slow inhibition of the OXA-1 enzyme by 7 or by the more
hydrophobic 15 was, however, detected.
Conclusions. In distinction from their aryl analogues 1, the
O-acyloxycarbonyl hydroxamates 2 were not good irreversible
inhibitors of β-lactamases. An investigation of the reactions of
2 (R = CH2Ph, R0 = Me or CH2Ph) in aqueous solution,
however, led to the serendipitous discovery that R-hydroxy esters
3 and 4 are new substrates of class C β-lactamases. The class A
TEM-2 β-lactamase also catalyzes their turnover, although less
efficiently. The hydroxamate leaving group appears to be supe-
rior to simple aryloxy leaving groups in these substrates. It seems
likely that the R-hydroxyl group enforces a specific orientation of
these compounds at the β-lactamase active site that may differ
from that of classical substrates. Incorporation of the R-hydro-
xyalkyl moiety into other platforms, for example, β-lactams and
phosphonates, will be interesting.
SUPPORTING INFORMATION AVAILABLE
Synthetic details for the preparation of 6 and 9-20, analytical
absorption data for hydrolysis of 7-20, kinetic data for the P99
β-lactamase-catalyzed methanolysis of 7, and a stereoview of an
energy-minimized S tetrahedral intermediate structure formed
upon reaction of 7 with the P99 β-lactamase (22). This material is
available free of charge via the Internet at http://pubs.acs.org.
23. Damblon, C., Zhao, G.-H., Jamin, M., Ledent, P., Dubus, A.,
ꢀ
Vanhove, M., Raquet, X., Christiaens, L., and Frere, J.-M. (1995)
Breakdown of stereospecificity of DD-peptidases and β-lactamases
with thioester substrates. Biochem. J. 309, 431–436.
24. Adediran, S. A., Cabaret, D., Flavell, R. R., Sammons, J. A., Wakselman,
M., and Pratt, R. F. (2006) Synthesis and β-lactamase reactivity of
R-substituted phenaceturates. Bioorg. Med. Chem. 14, 7023–7033.
25. Majumdar, S., and Pratt, R. F. (2009) Inhibition of class A and class C
β-lactamases by diaroyl phosphates. Biochemistry 48, 8285–8292.
26. Pazhanisamy, S., Govardhan, C. P., and Pratt, R. F. (1989)
β-Lactamase-catalyzed aminolysis of depsipeptides: Amine specificity
and steady state kinetics. Biochemistry 28, 6863–6870.
27. Cabaret, D., Adediran, S. A., Pratt, R. F., and Wakselman, M. (2003)
New substrates for β-lactam-recognizing enzymes: Aryl malona-
mates. Biochemistry 42, 6719–6725.
28. Page, M. I., Vilanova, B., and Layland, N. J. (1995) pH dependence of
and kinetic solvent isotope effects on the methanolysis and hydrolysis
REFERENCES
1. Livermore, D. M. (2009) β-Lactamases: The threat renews. Curr.
Protein Pept. Sci. 10, 397–400.
2. Pratt, R. F., and Govardhan, C. P. (1984) β-Lactamase-catalyzed
hydrolysis of acyclic depsipeptides and acyl transfer to specific amino
acid acceptors. Proc. Natl. Acad. Sci. U.S.A. 81, 1302–1306.
3. Pratt, R. F. (1989) Inhibition of a class C β-lactamase by a specific
phosphonate monoester. Science 246, 917–919.
4. Wyrembak, P. N., Babaoglu, K., Pelto, R. B., Shoichet, B. K., and
Pratt, R. F. (2007) O-Aryloxycarbonyl hydroxamates: New β-lacta-
mase inhibitors that cross-link the active site. J. Am. Chem. Soc. 129,
9548–9549.

10506 Biochemistry, Vol. 49, No. 49, 2010
Pelto and Pratt
of β-lactams catalyzed by class C β-lactamase. J. Am. Chem. Soc. 117,
12092–12095.
29. Rahil, J., and Pratt, R. F. (1993) Kinetics and mechanism of
β-lactamase inhibition by phosphonamidates: The quest for a proton.
Biochemistry 32, 10763–10772.
36. Ottinger, P., Pfaffen, C., Leist, R., Leutweiler, S., Bachorz, R. A., and
Klopper, W. (2009) Strong N-H π hydrogen bonding in amide-
benzene interactions. J. Phys. Chem. B. 113, 2937–2943.
37. Kelly, J. A., and Kuzin, A. P. (1995) The refined crystallographic
3 3 3
˚
structure of a DD-peptidase penicillin-target enzyme at 1.6 A resolu-
30. Adediran, S. A., and Pratt, R. F. (1999) β-Secondary and solvent
deuterium isotope effects on catalysis by the Streptomyces R61 DD-
peptidase: Comparisons with a structurally similar class C β-lacta-
mase. Biochemistry 38, 1469–1477.
tion. J. Mol. Biol. 254, 223–236.
38. Knox, J. R., Moews, P. C., and Frere, J.-M. (1996) Molecular
evolution of bacterial β-lactam resistance. Chem. Biol. 3, 937–947.
39. Adediran, S. A., Zhang, Z., Nukaga, M., Palzkill, T., and Pratt, R. F.
ꢀ
31. Oefner, C., Darcy, A., Daly, J. J., Gubernator, K., Charnas, R. L.,
Heinze, I., Hubschwerlen, C., and Winkler, F. K. (1990) Refined
crystal structure of β-lactamase from Citrobacter freundii indicates a
mechanism for β-lactam hydrolysis. Nature 343, 284–288.
32. Beadle, B. M., Trehan, I., Focia, P. J., and Shoichet, B. K. (2002)
Structural milestones in the reaction pathway of an amide hydrolase:
Substrate, acyl, and product complexes of a cephalothin with ampC
β-lactamase. Structure 10, 413–424.
(2005) The D-methyl group in β-lactamase evolution: Evidence from
the Y221G and GCl mutants of the class C β-lactamase of Enter-
obacter cloacae P99. Biochemistry 44, 7543–7552.
40. McDonough, M. A., Anderson, J. W., Silvaggi, N. R., Pratt, R. F.,
Knox, J. R., and Kelly, J. A. (2002) Structures of two kinetic
intermediates reveals species specificity of penicillin-binding proteins.
J. Mol. Biol. 332, 111–122.
41. Pratt, R. F. (2002) Functional evolution of the β-lactamase active site.
J. Chem. Soc., Perkin Trans. 2, 851–861.
42. Golemi, D., Maveyraud, L., Vakuleuko, S., Samama, J.-P., and
Mobashery, S. (2001) Critical involvement of a carbanylated lysine
in catalytic function of class D β-lactamases. Proc. Natl. Acad. Sci. U.
S.A. 98, 14280–14285.
43. Schneider, K. D., Bethel, C. R., Distler, A. M., Hujer, A. M.,
Bonomo, R. A., and Leonard, D. A. (2009) Mutation of the active
site carboxy-lysine (K70) of OXA-1 β-lactamase results in a deacyla-
tion-deficient enzyme. Biochemistry 48, 6136–6145.
33. Bernstein, N. J., and Pratt, R. F. (1999) On the importance of a methyl
group in β-lactamase evolution: Free energy profiles and molecular
modeling. Biochemistry 38, 10499–10510.
ꢁ
34. Morandi, F., Caselli, E., Morandi, S., Focia, P. J., Blazquez, J.,
Shoichet, B. K., and Prati, F. (2003) Nanomolecular inhibitors of
AmpC β-lactamase. J. Am. Chem. Soc. 125, 685–695.
35. Steiner, T., and Koellner, G. (2001) Hydrogen bonds with π-acceptors
in proteins: Frequencies and role in stabilizing local 3D structures.
J. Mol. Biol. 305, 535–557.