Cysteinyl peptide inhibitors of Bacillus cereus zinc β-lactamase

Article abstract of DOI:10.1016/S0968-0896(00)00257-1

Several cysteinyl peptides have been synthesised and shown to be reversible competitive inhibitors of the Bacillus cereus metallo-β-lactamase. The pH dependence of pK_i indicates that the thiol anion displaces hydroxide ion from the active site zinc(II). D,D-Peptides bind to the enzyme better than other diastereoisomers, which is compatible with the predicted stereochemistry of the active site.

Full text of DOI:10.1016/S0968-0896(00)00257-1

Bioorganic & Medicinal Chemistry 9 (2001) 503±510
Cysteinyl Peptide Inhibitors of Bacillus cereus Zinc ꢀ-Lactamase
Sakina Bounaga,a Moreno Galleni,b Andrew P. Lawsa and Michael I. Pagea,*
aDepartment of Chemical and Biological Sciences, University of Hudders®eld, Queensgate, Hudders®eld, HD1 3DH, UK
b
Â
Universite de Liege, Centre d'Ingenierie des Proteines, Institut de Chimie B6, Sart Tilman B4000, Liege 1, Belgium
Â
Á
Received 17 July 2000; accepted 4 October 2000
AbstractÐSeveral cysteinyl peptides have been synthesised and shown to be reversible competitive inhibitors of the Bacillus cereus
metallo-b-lactamase. The pH dependence of pKi indicates that the thiol anion displaces hydroxide ion from the active site zinc(II).
d,d-Peptides bind to the enzyme better than other diastereoisomers, which is compatible with the predicted stereochemistry of the
active site. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
greater than that shown by ``classical'' class A b-lacta-
mases such as TEM-1. A review of the catalytic pro-
perties of the well-characterised class B b-lactamases
showed that the A. hydrophila enzyme clearly exhibits
the most speci®c substrate pro®le, while the other
enzymes are rather broad-spectrum.4
The major mechanism of bacterial resistance to the nor-
mally lethal action of b-lactam antibiotics is the micro-
organism's production of b-lactamase enzymes which
catalyse the hydrolysis of the b-lactam. There are two
major types of b-lactamase Ð serine enzymes and those
requiring zinc(II) ions for their activity.1 In 1985 there
were just two microorganisms which had been identi®ed
as producing metallo-b-lactamases but to date there are
over 20 bacterial sources of the metallo-enzyme, including
those found in Bacillus cereus, Pseudomonas aeruginosa,
Stenotrophomonas maltophilia, Aeromonas hydrophila and
Bacteroides fragilis,2 all of which are classi®ed as class B
metallo-b-lactamases.
The sequences of the metallo-b-lactamases have been
established and they all contain a single peptide chain,
composed of 220±230 residues.5 A comparison of these
amino acid sequences indicates that they appear to
constitute a fairly homogeneous group and exhibit up to
37% sequence identity. The enzymes from B. cereus,
Aeromonas hydrophila, B. fragilis and L1 from S. mal-
tophilia have the capacity to bind up to 2 mole equiva-
lents of metal ion per mole of protein.6À10 However,
despite these structural similarities the functional role of
the second metal ion is controversial11À17 and the dif-
ferent metallo-enzymes may have di€erent metal ion
requirements. Although the B. cereus enzyme can bind
two metal ions,7À9 it appears that only one is required
for maximum catalytic activity.18 For the other enzymes
the e€ect of binding the second metal ion is not clear
and it may either increase or even decrease activity.11À14
The role of the ®rst zinc ion appears to be to provide a
metal bound, but nucleophilic, hydroxide ion to attack
the b-lactam carbonyl carbon, which generates a tetra-
hedral intermediate with the oxyanion stabilised by the
metal ion.13,18
Although these metallo-enzymes were initially thought to
be clinically unimportant, some pernicious strains have
been shown to owe their antibiotic resistance to their abil-
ity to produce zinc-b-lactamases.3 Of particular concern is
the ability of these metallo-b-lactamases to catalyse the
hydrolysis of some of the newer b-lactam antibiotics
such as the carbapenems. The mechanism-based inacti-
vators which have been used against the serine enzymes
are generally ine€ective against the Zn(II)-dependent
enzymes, and, at present, there are no clinically useful
inhibitors known of the metallo-b-lactamases.
The metallo-b-lactamases are extremely ecient at cat-
alysing the hydrolysis of imipenem with values of kcat
s
/
Km of ca. 106 MÀ1 À1, which is at least 1000-fold
There have been several recent reports of inhibitors
which inhibit the metallo-b-lactamases.18À22 A series of
thioester substrates of IMP-1 metallo-b-lactamases are
reversible competitive inhibitors of the enzyme, probably
by producing thiols as hydrolytic products.22 In contrast
*Corresponding author. Tel.: +44-1484-472169; fax: +44-1484-472182;
e-mail: m.i.page@hud.ac.uk
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00257-1

504
S. Bounaga et al. / Bioorg. Med. Chem. 9 (2001) 503±510
thioesters were thought to irreversibly inhibit B. cereus
metallo-b-lactamases by covalent modi®cation of the
active site by mercaptoacetic acid,20 but in a separate
study,21 thioesters of mercaptophenylacetic acid were
shown to be weak competitive inhibitors of the B. cereus
enzyme as the unreacted thioesters.
Table 1. Inhibitor dissociation constants for the b-lactamase II cata-
lysed hydrolysis of b-lactams, at 30 ꢀC, pH 7.0 and ionic strength
1.0 M (NaCl). Enzyme concentration 0.1 mM
Inhibitor
Ki mM
(ZnSO4
2Â10À6 M)
N(2-Mercaptoethyl)phenylacetamide (1)
N-Phenylacetylglycine (2)
56.2Æ11
1000Æ150
108Æ3
Whatever the detailed mechanism of action of the
metallo-b-lactamases,13,18 it is likely that zinc(II) stabi-
lises the tetrahedral intermediate presumed to be formed
during the catalytic process. Thiols are well known
inhibitors of metallo-proteases because of their ability
to coordinate to the active site zinc. We have reported
earlier1,18 the use of thiol derivatives, with structures
containing suitable sites for molecular recognition, to
inhibit the class B metallo-b-lactamases from Bacillus
cereus 5/B/6 and herein we develop that theme, includ-
ing the importance of stereochemistry.
N-Phenylacetyl-l-cysteine (3)
N-Phenylacetyl-dl-penicillamine (4)
515Æ38
N-Carbobenzoxy-l-cysteine (5)
97Æ5
N-Carbobenzoxy-d-cysteine (5)
20.1Æ1.6
42.2Æ10
46.3Æ0.1
62.0Æ9.3
48.3Æ2.3
135.5Æ17
72.5Æ0.5
84Æ4
N-Carbobenzoxy-l-cysteinyl-glycine (6)
N-Carbobenzoxy-dl-cysteinyl-glycine (6)
N-Carbobenzoxy-l-cysteinyl-dl-alanine (7)
N-Carbobenzoxy-l-cysteinyl-dl-valine (8)
N-Carbobenzoxy-l-cysteinyl-l-leucine (9)
N-Carbobenzoxy-l-cysteinyl-dl-serine (10)
N-Carbobenzoxy-dl-cysteinyl-l-serine (10)
N-Carbobenzoxy-d-cysteinyl-d-penicillamine (11)
N-Carbobenzoxy-l-cysteinyl-dl-phenylalanine (12)
N-Carbobenzoxy-l-cysteinyl-l-phenylalanine (12)
N-Carbobenzoxy-l-cysteinyl-d-phenylalanine (12)
N-Carbobenzoxy-d-cysteinyl-l-phenylalanine (12)
N-Carbobenzoxy-d-cysteinyl-d-phenylalanine (12)
N-Carbobenzoxy-l-cysteinyl-dl-proline (13)
Captopril
Chelates Zn++
17.3Æ0.2
26.9Æ0.5
9.3Æ0.4
11.0Æ1
Results and Discussion
3.0Æ0.1
A series of substituted thiols and cysteinyl dipeptides
were synthesised as outlined in Scheme 1 and then tested
as inhibitors of the B. cereus 569/H/9 class B metallo-b-
lactamase. Even the simple thiol, N(2-mercaptoethyl)-
phenylacetamide (1), was found to be a competitive
inhibitor of the enzyme and the e€ects of substituents
and their stereochemistry in various mercapto dipeptides
on Ki were determined (Table 1).
95.0Æ1.5
41.6Æ9
zinc-bound water is thought to have a pKa of 5.718 and
so it will be fully deprotonated above pH 6. If the thiol
binds to the metal ion as the thiolate anion then the
measured binding constant of the neutral undissociated
thiol would be pH independent between pH 6 and 9, as
seen in Figure 1.
A plot of the logarithm of the inhibition constant, pKi, for
N-carboxy-l-cysteinyl-glycine (6) against pH is pH inde-
pendent between pH 6 and 9 but decreases in both acidic
and basic solution (Fig. 1). The in¯ections are very similar
to those observed for enzyme catalysed hydrolysis of
penicillins and cephalosporins, with calculated pKas of
5.66Æ0.4 and 9.93Æ0.1, which indicates that the ionisa-
tion states of the enzyme required for catalysed hydro-
lysis are also those required for binding the inhibitor.
The thiol group is necessary for inhibition and, for exam-
ple, N-phenylacetylglycine (2) is a very poor inhibitor.
Conversely, the addition of a carboxylate residue to the
simple thiol, 1, as in N-phenylacetyl-l-cysteine 3, slightly
decreases potency, probably simply as a result of increas-
ing the pKa of the thiol group and indicating that a
negatively charged carboxylate anion in this position
does not facilitate binding. There is an order of magni-
tude decrease in binding when two gem-dimethyl groups
are a-substituted to the thiol, as in N-phenylacetyl-dl-
The pKa of the thiol 6 is 9.3 so the predominant species
at pHs below this is the undissociated neutral form. The
Scheme 1.

S. Bounaga et al. / Bioorg. Med. Chem. 9 (2001) 503±510
505
which does not contain a thiol group, is practically
ine€ective.
There are two asymmetric centres in the cysteinyl
dipeptides and the e€ect of the stereochemistry at each
centre on inhibition was determined. For example, the
d-enantiomer of the N-carbobenzoxy-cysteine (5) is ®ve
times more potent than the l-enantiomer. The four ste-
reoisomers of N-carbobenzoxy-cysteinyl-phenylalanine
(12) display stereoselectivity in their inhibition of b-lac-
tamase II. As with other cysteinyl derivatives, the d-
centre is preferred, and this con®guration is also
favoured at the carboxy terminal residue. Thus the d,d-
enantiomer is an order of magnitude better than the
l,l-one.
It is concluded that the best interactions with the
enzyme are obtained with compounds with a thiol resi-
due and a hydrophobic side chain a to carboxyl ter-
minus. The d,d isomers exhibit the highest potency, N-
carbobenzoxy-d-cysteinyl-d-phenylalanine (12) with a
Ki of 3.0 mM being the most ecient in vitro inhibitor of
the B. cereus b-lactamase.
Figure 1. The pH dependence of pKi for B. cereus b-lactamase class B
inhibited by N-carboxy-l-cysteinyl-glycine at 30ꢀC and 1.0 M (NaCl).
The values of pKi were determined using cephaloridine as the substrate.
penicillamine (4). This is indicative of some selectivity in
binding and suggests that thiol co-ordination is impor-
tant and can be sterically inhibited.
The most e€ective thiol peptide inhibitors have the d
con®guration at the cysteinyl chiral centre and the d con-
®guration at the carboxyl terminus. By contrast, the ste-
reochemistry of penicillin is l at C6 and d at C3.
However, this is compatible with similar binding interac-
tions at the active site with the carboxylate anion of peni-
cillin and of the thiol inhibitor binding to His 210 or Lys
171 and the b-lactam carbonyl oxygen or the thiolate
anion of the inhibitor binding to the active-site zinc
(Scheme 2).
The e€ect of substituents on the inhibition was investi-
gated in various N-carbobenzoxy-cysteinyl amino acids,
although the dipeptide cbzCysGly (6) shows no signi®cant
increased binding compared with the simple thiol 1,
despite potential increased recognition. However, hydro-
phobic substituents at the a-carbon of the C-terminal
amino acid do increase binding, and Ki decreases as the
size of the R group increases from CH3 in N-carbo-
benzoxy-l-cysteinyl-dl-alanine (7) to CH(CH3)2 in N-
carbobenzoxy-l-cysteinyl-dl-valine (8), but increases
again if the alkyl substituent becomes too large (9). When
the a-substituent is CH2-Ph as in N-carbobenzoxy-l-
cysteinyl-dl-phenylanine (12), the Ki of 17 mM indicates
a more potent inhibitor, presumably due to the hydro-
phobic e€ect of the aromatic group. An a-hydroxymethyl
substituent as in N-carbobenzoxy-l-cysteinyl-serine (10)
has little e€ect upon binding and a proline residue (13)
does not improve the inhibition.
The thiol inhibitors do not act as chelating agents. The
measured inhibition constants were independent of zinc
ion concentration and the enzyme activity was not restored
by the addition of zinc ions. However, the enzyme was
completely inactivated by the dithiol, N-carbobenzoxy-
d-cysteinyl-d-penicillamine (11), but the activity was, in
this case, mostly recovered upon addition of excess zinc,
indicating that the dithiol was acting as a chelating agent
and removed the zinc ion from the protein. As expected,
other dithiol compounds, such as 2,3-dimercaptosuccinic
acid and 2,3-dimercapto-1-propane sulfonic acid, exhibit
a similar phenomenon.
Captopril and enalapril are potent inhibitors of the zinc-
dependent angiotensin converting enzyme (ACE), and
with the B. cereus zinc b-lactamase captopril was found to
inhibit the enzyme with a Ki of 41 mM, whereas enalapril,
Scheme 2.

506
S. Bounaga et al. / Bioorg. Med. Chem. 9 (2001) 503±510
Summary
13C NMR dC (DMSO) 171.6, 170.4, 136.3, 129.1, 128.2,
126.4, 54.5, 41.9, 25.7. IR (nujol) n 3342, 2550, 1731,
Several cysteinyl peptides have been synthesised and
shown to be reversible competitive inhibitors of the B.
cereus metallo-b-lactamase. The pH dependence of pKi
indicates that the thiol anion probably displaces
hydroxide ion from the active site zinc(II) and that the
terminal carboxylate binds to His 210 or Lys 171. d,d-
Peptides bind to the enzyme better than other diaster-
eoisomers, which is compatible with the predicted
stereochemistry from the binding of penicillin substrates
to the active site.
1623 cmÀ1. ES MS [MÀH]À m/z 238.
N-Phenylacetyl-DL-penicillamine (4). Obtained from
phenylacetyl chloride (0.98 mmoles) and penicillamine
(6.7 mmoles). Yield 42%. Mp 118±120 ꢀC. H NMR dH
1
(DMSO) 8.34 (1H, d, J=9.2), 7.32 (5H, m), 4.44 (1H,
d), 3.58 (2H, dd, JAB=13.8), 2.86 (1H, s) and 1.38 and
1.36 (6H, 2 s). 13C NMR dC (DMSO) 171.2, 170.3, 136.3,
129.0, 128.1, 126.3, 61.3, 45.5, 41.8, 29.7, 29.6. IR (nujol) n
3365, 2571, 1728, 1635cmÀ1. ES MS [MÀH]À m/z 266.
N-Carbobenzoxy-L- and -D-cysteine (5). Obtained from
cystine (0.0125 moles) and carbobenzoxy chloride
1
(0.0375 moles). Yield 50%. H NMR dH (DMSO) 7.77
Experimental
Synthesis
(1H, d, J=8.4), 7.35 (5H, m), 5.07 (2H, s), 4.36 (1H, m),
3.20 and 2.97 (2H, m, J=4.1, J=10.1, J=13.7). 13C
NMR dC (DMSO) 172.3, 156.2, 136.9, 128.4, 128.1.1,
127.9, 65.7, 53.1, 39.6. IR (nujol) n 3329, 1730,
1655 cmÀ1. ES MS [MÀH]À m/z 509. The disul®de
(2.18 mmoles) was then reduced with sodium borohy-
1H and 13C NMR spectra were recorded at 270 MHz on a
Bruker AC-270 spectrometer. The chemical shifts (dH) are
quoted in p.p.m. and referenced to TMS. Infrared spectra
were obtained on a Perkin Elmer 1600 Series FTIR, as
nujol mulls or chloroform solutions. The mass spectra
were recorded on a VG (Fisons Instruments) Quattro
(II) S Q mass spectrometer.
1
dride (8.74 mmoles). Yield 90%. H NMR dH (DMSO)
7.64 (1H, d, J 7.9), 7.36 (5H, m), 5.08 (2H, s), 4.14 (1H,
m), 2.91 and 2.75 (2H, m), 2.51 (1H, m). 13C NMR dC
(DMSO) 172.3, 156.2, 136.9, 128.5, 127.9.1, 127.8, 65.7,
56.6, 25.6. IR (nujol) n 3327, 2571, 1719, 1640 cmÀ1. ES
MS [MÀH]À m/z 254.
N(2-Mercaptoethyl)phenylacetamide (1). Phenylacetyl
chloride (5.5 mmoles) was added dropwise to a solution
of cystamine (2.5 mmoles) in water (40 mL) containing
sodium hydroxide (11.25 mmoles) and was cooled to
0 ꢀC. At the end of the reaction, the white precipitate
formed was ®ltered and washed with water. The solid
was recrystallised from hot methanol. The disul®de thus
obtained was reduced by adding sodium borohydride
(88 mmoles) to a solution of the disul®de (4.4 mmoles)
in ethanol:water (5:3, v/v), which was heated to 90 ꢀC
for 1 h, cooled, then acidi®ed to pH 2 with dilute
hydrochloric acid. The solvent was evaporated, ®ltered
and the ®ltrate extracted with chloroform, washed with
water and dried over anhydrous sodium sulfate. The
solvent was evaporated and the solid recrystallised.
N-Carbobenzoxy-L-cysteinyl amino acids. Synthesised
using the following general procedure. A solution of
N,N0-dicarbobenzoxycystine (6 mmoles) in dichloro-
methane (50 mL), cooled to 0 ꢀC, was treated with dicy-
clohexylcarbodiimide (12 mmoles), a solution of tri-
ethylamine (12 mmoles) and amino acid methylester
hydrochloride (12.6 mmoles) in dichloromethane
(50 mL). The mixture was stirred at 0 ꢀC for 1 h and then
for 24 h at room temperature. The precipitated dicyclo-
hexylurea was ®ltered, the ®ltrate washed twice with
1 M sulfuric acid (30 mL) and twice with 5% sodium
bicarbonate (30 mL). The organic layer was dried over
anhydrous sodium sulfate and evaporated. The residue
was dissolved in acetone, the insoluble part was
removed by ®ltration and the solvent evaporated to give
a white product. Yield 90%. To a solution of (Z-Cys-
AaOMe)2 (5 mmoles) in 50 mL of methanol, cooled to
5 ꢀC, was added dropwise lithium hydroxide (10.2
mmoles) in water (17 mL). After 18h, 50mL of water
were added, the methanol evaporated and the solution
washed with diethyl ether (2Â25mL). The aqueous phase
was acidi®ed to pH 2 with hydrochloric acid and the pro-
duct ®ltered, washed with water, and dried. Yield 70%.
Yield 87%. Mp 48±50 ꢀC. H NMR dH (CDCl3) 7.30
1
(5H, m), 5.75 (1H, m), 3.55 (2H, s), 3.35 (2H, m), 2.52
(2H, dt, J=6.6 and 8.5, CH2-S), 1.18 (1H, t, J=8.5,
SH). IR (nujol) n 3253, 2544, 1637 cmÀ1. A pKa of
9.5Æ0.10 of the thiol was determined from the depen-
dence of the absorbance of an aqueous 2.52Â10À4
M
solution at 238 nm as a function of pH.
N-Phenylacetyl-L-cysteine (3). The disul®de was obtained
from phenylacetyl chloride (13 mmoles) and cystine (5.9
mmoles). Yield 80%. 1H NMR dH (DMSO) 8.54 (1H, d,
J=7.9), 7.26 (5H, m), 4.50 (1H, m), 3.50 (2H, s), 3.16
and 2.95 (2H, m, J=4.6, J=9.5, J=13.6). 13C NMR dC
(DMSO) 171.87, 170.25, 136.0, 129.0, 128.1, 126.3, 51.3,
41.9, 40.3. IR (nujol) n 3319, 1720, 1660 cmÀ1. ES MS
[MÀH]À m/z 475.
To a solution of (Z-Cys-AaOH)2 (2 mmoles) in ethanol
(20 mL) at 68±70 ꢀC was added dropwise a solution
containing 4 equivalents of sodium borohydride (8.74
mmoles) in ethanol (10 mL) and the mixture heated at
75±80 ꢀC for 1 h. After cooling to 25 ꢀC, the solution
was added to 50 mL iced water, to which was added
6 M hydrochloric acid to give pH 3. After evaporating
the ethanol, the solution was extracted three times with
ethyl acetate, washed three times with distilled water
and dried over anhydrous sodium sulfate. Removal of
The disul®de (5 mmoles) was then reduced with sodium
borohydride (100 mmoles). Yield 90%. Mp 85±88ꢀC. 1H
NMR dH (DMSO) 8.47 (1H, d, J=7.8), 7.25 (5H, m),
4.40 (1H, m), 3.53 (2H, s), 2.82 (2H, m), 2.45 (1H, m).

S. Bounaga et al. / Bioorg. Med. Chem. 9 (2001) 503±510
507
the solvent gave an oil which was puri®ed by chroma-
tography. Yield 97%.
136.9, 128.4, 127.7, 127.1, 65.6, 57.2, 53.9, 40.6, 30.1,
19.2, 17.9. IR (nujol) n 3309, 1715, 1661 cmÀ1
.
1
(Z-Cys-GlyOMe)2. H NMR dH (DMSO) 8.50 (1H, t,
Z-L-Cys-DL-ValOH (8). 1H NMR dH (DMSO) 8.15
(1H, m), 7.60 (1H, m), 7.35 (5H, m), 5.05 (2H, s), 4.19
(2H, m), 2.80 (2H, m), 2.05 (1H, m), 0.86 (6H, d). 13C
NMR dC (DMSO) 172.9, 170.5, 156.1, 137.1, 128.5,
128.2, 127.9, 65.7, 57.2, 30.2, 26.9, 19.2 18.0. IR (nujol) n
3317, 2625, 1718, 1668 cmÀ1. ES MS [MÀH]À m/z 353.
Mp 37±40 ꢀC.
J=5.6), 7.66 (1H, d, J=8.5), 7.36 (5H, m), 5.05 (2H, s),
4.36 (1H, m), 3.85 (2H, d), 3.58 (3H, s), 3.14 and 2.85
(2H, m, J=4.0, J=10.1, J=13.3). 13C NMR dC
(DMSO) 170.8, 170.1, 156.1, 136.9, 128.4, 127.8, 127.7,
65.6, 53.6, 51.7, 40.8, 38.6. IR (nujol) n 3303, 1740, 1687
and 1659 cmÀ1. ES MS [M+Cl]À m/z 685.
1
(Z-Cys-GlyOH)2. H NMR dH (DMSO) 8.31 (1H, t,
(Z-L-Cys-L-LeuOMe)2. 1H NMR dH (DMSO) 8.39 (1H,
m), 7.62 (1H, m), 7.35 (5H, m), 5.05 (2H, s), 4.33 (2H,
m), 3.61 (3H, s), 3.13 and 2.89 (2H, m), 1.55 (3H, m),
0.85 (6H, m). 13C NMR dC (DMSO) 172.7, 170.4, 156.0,
136.9, 128.3, 127.7, 127.2, 65.6, 53.7, 51.9, 50.4, 40.4,
39.5, 24.2, 22.8, 21.3. IR (nujol) n 3266, 1746, 1703,
J=5.9), 7.63 (1H, d, J=8.5), 7.36 (5H, m), 5.05 (2H, s),
4.36 (1H, m), 3.76 (2H, d), 3.16 and 2.85 (2H, m). 13C
NMR dC (DMSO) 170.8, 170.6, 156.1, 136.9, 128.4,
127.7, 127.2, 65.6, 53.6, 40.9, 40.0. IR (nujol) n 3309,
1729, 1689, 1653 cmÀ1. ES MS [MÀH]À m/z 621,
[MÀ312]À m/z 310.
1655 cmÀ1
.
1
1
Z-Cys-GlyOH (6). H NMR dH (DMSO) 12.63 (1H, s),
(Z-L-Cys-L-LeuOH)2. H NMR dH (DMSO) 8.20 (1H,
d, J=7.9), 7.60 (1H, d, J=8.1), 7.35 (5H, m), 5.05 (2H,
s), 4.34 (1H, m), 4.23 (1H, m), 3.13 and 2.87 (2H, m),
1.59 (3H, m), 0.86 (6H, m); 13C NMR dC (DMSO)
173.7, 170.2, 156.0, 136.9, 128.4, 128.0, 127.8, 65.6, 53.8,
50.5, 40.1, 39.8, 24.3, 22.9 and 21.4; IR (nujol) n 3312,
8.34 (1H, t), 7.51 (1H, d), 7.36 (5H, m), 5.05 (2H, s),
4.18 (1H, m), 3.77 (2H, m), 2.83 and 2.67 (2H, m), 2.37
(1H, t, J=8.5). 13C NMR dC (DMSO) 171.3, 170.7,
156.2, 137.1, 128.5, 127.9, 127.2, 65.8, 57.4, 41.0, 26.6.
IR (nujol) n 3285, 3215, 2500, 1728, 1686, 1635 cmÀ1
.
ES MS [MÀH]À m/z 311.
1693, 1655 cmÀ1
.
1
(Z-L-Cys-DL-AlaOMe)2. 1H NMR dH (DMSO) 8.49
(1H, m), 7.60 (1H, m), 7.36 (5H, m), 5.05 (2H, s), 4.29
(2H, m), 3.62 (3H, s), 3.11 and 2.88 (2H, m), 1.27 (3H, d,
J=7.3). 13C NMR dC (DMSO) 172.7, 170.2, 156.1,
136.9, 128.5, 128.4, 128.1, 65.9, 53.6, 51.9, 47.8, 40.1,
Z-L-Cys-L-LeuOH (9). H NMR dH (DMSO) 8.22 (1H,
m), 7.53 (1H, m), 7.35 (5H, m), 5.05 (2H, s), 4.25 (2H, m),
2.85 (2H, m), 1.58 (3H, m), 0.88 (6H, m); 13C NMR dC
(DMSO) 174.9, 170.2, 156.0, 136.9, 128.4, 127.8, 127.7,
65.6, 57.18, 50.5, 40.1, 26.45, 24.5, 22.9 and 21.4; IR
(nujol) n 3308, 2503, 1714, 1661 cmÀ1; ES MS [MÀH]À
m/z 361.
17.0, 16.8. IR (nujol) n 3420, 3333, 1743, 1690, 1651cmÀ1
ES MS [MÀH]À m/z 677.
.
1
(Z-L-Cys-DL-AlaOH)2. H NMR dH (DMSO) 8.32 (1H,
m), 7.59 (1H, m), 7.35 (5H, m), 5.05 (2H, s), 4.38 (1H,
m), 4.23 (1H, m), 3.15 and 2.87 (2H, m), 1.29 (3H, m).
13C NMR dC (DMSO) 174.1, 170.2, 156.2, 137.0, 128.5,
128.0, 128., 65.8, 53.8, 47.9, 40.7, 17.5. IR (nujol) n
3326, 1718, 1669, 1659 cmÀ1. ES MS [MÀH]À m/z 649.
(Z-L-Cys-DL-SerOMe)2, (Z-DL-Cys-L-SerOMe)2. 1H
NMR dH (DMSO) 8.30 (1H, m), 7.65 (1H, m), 7.36 (5H,
m), 5.06 (2H, s), 4.42 (2H, m), 3.72 (2H, m), 3.63 (3H, s),
3.15 and 2.88 (2H,m); 13C NMR dC (DMSO) 170.8,
170.5, 156.1, 136.9, 128.4, 128.1, 127.8, 65.6, 61.2, 54.9,
53.7, 52.0, 40.5; IR (nujol) n 3505, 1738, 1700, 1651 cmÀ1
;
ES MS [MÀH]À m/z 709.
Z-L-Cys-DL-AlaOH (7). mp 40±44ꢀC 1H NMR dH
(DMSO) 8.33 (1H, m), 7.44 (1H, m), 7.34 (5H, m), 5.06
(2H, s), 4.26 (2H, m), 2.73 (2H, m), 2.25 (1H, m), 1.29 (3H,
m). 13C NMR dC (DMSO) 174.4, 170.3, 156.2, 137.2,
128.7, 128.4, 128.0, 66.0, 57.3, 48.1, 27.0, 17.7. IR (nujol) n
3413, 3322, 2571, 1719, 1671, 1659 cmÀ1. ES MS [MÀH]À
m/z 325.
1
(Z-L-Cys-DL-SerOH)2, (Z-DL-Cys-L-SerOH)2. H NMR
dH (DMSO) 8.10 (1H, m), 7.65 (1H, m), 7.35 (5H, m),
5.05 (2H, s), 4.42 (1H, m), 4.28 (1H, m), 3.67 (2H, m),
3.16 and 2.86 (2H, m); 13C NMR dC (DMSO) 171.8,
170.3, 156.2, 136.9, 128.5, 127.9, 127), 65.7, 61.4, 54.9,
53.8, 40.5; IR (nujol) n 3298, 1718, 1703 and 1650 cmÀ1
;
ES MS [MÀH]À m/z 681.
(Z-L-Cys-DL-ValOMe)2. 1H NMR dH (DMSO) 8.30 (1H,
m), 7.60 (1H, m), 7.35 (5H, m), 5.05 (2H, s), 4.40 (1H, m),
4.21 (1H, m), 3.64 (3H, s), 3.09 and 2.90 (2H, m), 2.05
(1H, m), 0.86, 0.85 (6H, d, J=4.6). 13C NMR dC
(DMSO) 171.8, 170.5, 155.9, 136.9, 128.4, 127.8, 127.6,
65.6, 57.5, 53.7, 51.8, 40.5, 30.1, 29.9, 19.0, 18.1. IR
(nujol) n 3303, 1739, 1693, 1656 cmÀ1. ES MS [MÀH]À
m/z 733.
1
Z-L-Cys-DL-SerOH, Z-DL-Cys-L-SerOH (10). H NMR
dH (DMSO) 8.10 (1H, m), 7.53 (1H, m), 7.37 (5H, m),
5.06 (2H), 4.30 (2H, m), 3.68 (2H, m), 3.15 and 2.87
(2H, m), 2.29 (1H, m); 13C NMR dC (DMSO) 171.8,
171.7, 170.2, 170.1, 156.0, 136.9, 128.4, 127.8, 127.7,
65.6, 61.4, 61.2, 57.5, 55.2, 55.1, 26.6; IR (nujol) n 3301,
2609, 1718, 1663; ES MS [MÀH]À m/z 341.
1
1
(Z-L-Cys-DL-ValOH)2. H NMR dH (DMSO) 8.17 (1H,
(Z-D-Cys-D-PenicillamineOMe)2. H NMR dH (DMSO)
m), 7.65 (1H, m), 7.35 (5H, m), 5.05 (2H, s), 4.43 (1H,
m), 4.17 (1H, m), 3.13, 2.90 (2H, m), 2.06 (1H, m), 0.86
(6H, d). 13C NMR dC (DMSO) 172.7, 170.3, 155.9,
8.25 (1H, m), 7.60 (1H, m), 7.35 (5H, m), 5.04 (2H, s),
4.50 (2H, m), 3.63 (3H, s), 3.04 and 2.73 (2H, m), 1.33
(6H, m); 13C NMR dC (DMSO) 170.5, 169.9, 156.0,

508
S. Bounaga et al. / Bioorg. Med. Chem. 9 (2001) 503±510
136.9, 128.4, 127.9, 127.7, 65.6, 61.5, 54.9, 51.9, 45.21,
40.4, 29.5 and 24.19; IR (nujol) n 3324, 1734, 1675 cmÀ1
ES MS [M+Cl]À m/z 835.
Z-L-Cys-DL-PheOH (12). 1H NMR dH (DMSO) 8.28
(1H, m), 7.53 (1H, m), 7.35 (5H, m), 7.21 (5H, m,), 5.04
(2H, s), 4.50 (1H, m), 4.19 (1H, m), 2.90 (4H, m); 13C
NMR dC (DMSO) 173.0, 170.4 and 170.1, 156.3, 137.7,
137.3, 129.6, 128.7, 128.2, 128.0, 126.8, 126, 66.0, 57.5,
57.4, 53.9, 53.0, 37.3, 37.0, 26.8 and 26.7; IR (nujol) n
3314, 2570, 1719, 1669 cmÀ1; ES MS [MÀH]À m/z 401
;
1
(Z-D-Cys-D-PenicillamineOH)2. H NMR dH (DMSO)
8.20 (1H, m), 7.57 (1H, m), 7.35 (5H, m), 5.04 (2H, s),
4.67 (1H, m), 4.34 (1H, m), 3.63 (3H, s), 2.89 (2H, m),
1.35 (6H, m); 13C NMR dC (DMSO) 171.4, 170.2, 155.,
136.9, 128.4, 128.1, 127.9, 65.6, 59.4, 53.0, 45.21, 40.2,
Z-L-Cys-L-PheOH, Z-D-Cys-D-PheOH. 1H NMR dH
(DMSO) 8.21 (1H, d, J=7.7), 7.36 (6H, m), 7.23 (5H,
m), 5.04 (2H, s), 4.44 (1H, m), 4.14 (1H, m), 3.00, 2.68
(4H, m), 2.25 (1H, m); 13C NMR dC (DMSO) 172.6,
170.0, 155.9, 137.3, 136.9, 129.1, 128.3, 128.1, 127.8,
127.7, 126.4, 65.5, 57.1, 53.5, 36.5, 26.3; IR (Z-l-Cys-l-
26.8 and 19.7; IR (nujol) n 3416, 2500, 1719, 1671 cmÀ1
.
1
Z-D-Cys-D-PenicillamineOH (11). H NMR dH (CDCl3)
8.31 (1H, d), 7.35 (6H, m), 5.11 (2H, s), 4.82 (1H, m),
4.68 (1H, m), 2.96 (2H, m), 1.41 (6H, m); 13C NMR dC
(CDCl3) 173.1, 171.8, 155.2, 135.9, 128.5, 128.3, 128.1,
67.2, 59.5, 53.6, 57.0, 41.6, 26.7 and 19.8; IR (nujol) n
3303, 2570, 1719, 1669 cmÀ1; ES MS [MÀ3H]À m/z 383.
Phe-OH) (nujol) n 3312, 2569, 1717, 1692 and 1658 cmÀ1
;
IR (Z-d-Cys-d-Phe-OH) (nujol) n 3314, 2567, 1715, 1693
and 1658 cmÀ1; ES MS [MÀH]À m/z 401; mp 143±146 ꢀC.
(Z-L-Cys-DL-PheOMe)2. 1H NMR dH (DMSO) 8.50
(1H, m), 7.60 (1H, m), 7.35 (5H, m), 7.24 (5H, m), 5.05
(2H, s), 4.52 (1H, m), 4.34 (1H, m), 3.61 (3H, m), 2.87
(4H, m); 13C NMR dC (DMSO) 171.6, 170.2, 155.9,
137.0, 136.9, 129.2, 129.1, 127.8, 127.0, 126.6, 65.6, 53.7
and 53.5, 52.0 and 51.9, 40.4, 40.0, 36.8 and 36.5; IR
Z-L-Cys-D-PheOH, Z-D-Cys-L-PheOH. 1H NMR dH
(DMSO) 8.28 (1H, d, J=8.2), 7.34 (6H, m), 7.23 (5H,
m), 5.04 (2H, s), 4.49 (1H, m), 4.15 (1H, m), 2.99 and
2.52 (4H, m), 2.03 (1H, m); 13C NMR dC (DMSO)
172.3, 169.7, 155.9, 137.3, 136.9, 129.2, 128.4, 128.2,
127.9, 127.8, 127.7, 65.76, 57.0, 53.3, 36.9, 26.5; IR
(nujol) n 3308, 2568, 1718, 1669 cmÀ1; ES MS [MÀH]À
m/z 401; mp 120±122 ꢀC.
(nujol) n 3321, 1743, 1717 and 1669 cmÀ1
.
(Z-L-Cys-L-PheOMe)2, (Z-D-Cys-D-PheOMe)2.
1H
NMR dH (DMSO) 8.45 (1H, d, J=7.9), 7.62 (1H, d,
J=8.5 ), 7.34 (5H, m), 7.21 (5H, m), 5.06 (2H, s), 4.53 (1H,
m), 4.39 (1H, m), 3.59 (3H, s), 3.05 and 2.85 (4H, m); 13C
NMR dC (DMSO) 171.7, 170.4, 156.0, 137.0, 136.9, 129.2,
128.4, 128.3, 127.9, 127.8, 127.7, 65.8, 53.8, 52.0, 40.4,
39.5.
Z-L-Cys-DL-ProOH (13). 1H NMR dH (DMSO) 7.64
(1H, m), 7.34 (5H, m), 7.21 (5H, m), 5.05 (2H, m), 4.22
and 3.66 (2H, m), 2.88, 2.70, 2.12 and 1.88 (8H, m); 13C
NMR dC (DMSO) 173.5, 173.4, 169.1, 156.2, 137.3,
127.7, 128.4, 128.1, 127.9, 65.9, 60.7, 57.2, 56.9, 46.9,
29.1, 25.7, 24.4, 24.7; IR (nujol) n 3295, 2606, 1716,
1630 cmÀ1; ES MS [MÀH]À m/z 351; mp 60±65 ꢀC.
(Z-L-Cys-D-PheOMe)2,
(Z-D-Cys-L-PheOMe)2.
1H
NMR dH (DMSO) 8.51 (1H, d, J=7.9), 7.56 (1H, d,
J=8.5 ), 7.34 (5H, m), 7.21 (5H, m), 5.04 (2H, s), 4.52 (1H,
m), 4.33 (1H, m), 3.62 (3H, s), 3.00 and 2.75 (4H, m); C
NMR dC (DMSO) 171.7, 170.3, 156.0, 137.0, 136.9, 129.3,
128.4, 128.3, 127.9, 127.8, 127.7, 65.7, 53.6, 52.0, 39.9,
36.8.
Kinetic studies
Benzylpenicillin, cefuroxime and cephaloridine were
gifts from GlaxoWellcome. Deionised water was used for
preparation of bu€ers and other aqueous solutions. The
bu€ers were acetate (pKa 4.75), MES (pKa 6.15), MOPS
(pKa 7.20), TAPS (pKa 8.40), CAPSO (pKa 9.60), hexa-
¯uoroisopropanol (pKa 9.30) and CAPS (pKa 10.4). The
ionic strength was maintained at a constant level with
sodium chloride or potassium chloride (AnalaR grade).
Kinetic studies were carried out using the class B b-lacta-
mase enzyme from Bacillus cereus 569/H which was iso-
lated using the previously described procedure.18 UV±Vis
wavelength scans were performed using a Cary 1/3 (Var-
ian) spectrophotometer. The temperature was kept con-
stant at 30 ꢀC with a water circulator and a peltier system.
The pHs of solutions were measured using either a JEN-
WAY 3020 or a Beckman È40 pH meter ®tted with Ag/
AgCl, orHg/Hg2Cl2 Russell semi-micro electrodes, which
were calibrated prior to use.
1
(Z-L-Cys-DL-PheOH)2. H NMR dH (DMSO) 8.28 (1H,
m), 7.53 (1H, m), 7.35 (5H, m), 7.21 (5H, m), 5.03 (2H, s),
4.50 (1H, m), 4.32 (1H, m), 2.90 (4H, m); 13C NMR dC
(DMSO) 172.6, 170.1, 170.0, 156.0, 137.3, 136.9, 129.3,
129.2, 128.4, 128.2, 127.9, 127.7, 127.1, 126.5, 65.7, 53.8,
53.6, 53.4, 40.0, 36.9 and 36.6; IR (nujol) n 3308, 1712,
1651 cmÀ1; ES MS [MÀH]À m/z 801.
(Z-L-Cys-L-PheOH)2, (Z-D-Cys-D-PheOH)2. 1H NMR
dH (DMSO) 8.13 (1H, d, J=7.8), 7.54 (1H, d, J=8.7),
7.32 (5H, m,), 7.22 (5H, m), 5.04 (2H, s), 4.44 (1H, m),
4.31 (1H, m), 3.06 and 2.88 (4H, m); 13C NMR dC
(DMSO) 172.4, 169.9, 155.9, 137.2, 136.8, 129.1, 128.3,
128.1, 127.7, 127.6, 126.4, 65.6, 53.8, 53.4, 40.4, 36.5.
(Z-L-Cys-D-PheOH)2, (Z-D-Cys-L-PheOH)2. 1H NMR
dH (DMSO) 8.21 (1H, d, J=8.2), 7.48 (1H, d, J=8.7),
7.32 (5H, m), 7.20 (5H, m), 5.04 (2H, s), 4.48 (1H, m),
4.32 (1H, m), 3.00 and 2.74 (4H, m); 13C NMR dC
(DMSO) 172.5, 169.9, 155.9, 137.2, 136.8, 129.2, 128.3,
128.1, 127.8, 127.6, 126.4, 65.6, 53.7, 53.3, 39.9, 36.8
Generally, 25 ml of a stock solution of substrate was
injected into 2.5 mL of bu€er solution (0.05 M, I=1.0 M,
30 ꢀC) and stirred. 25 ml of a stock solution of enzyme
was then added to the system. Usually, the substrate and
enzyme concentrations in the cell were 0.12 to 1.1 mM
and 0.04±2.0 mM respectively. The experiment was repe-

S. Bounaga et al. / Bioorg. Med. Chem. 9 (2001) 503±510
509
ated by adding more substrate at the end of the reaction.
Hydrolysis of the substrate was followed by measuring the
decrease in absorbance, at 260 nm for cephaloridine and
cefuroxime, and at 230 nm for benzylpenicillin, as a func-
tion of time. Enzyme catalysed reactions followed
Michaelis±Menten kinetics, but the kinetic constants were
generally measured by following the entire course of the
reaction. Complete time curves were studied because of
increased accuracy and there was no evidence of either
product inhibition or enzyme denaturation. Repeat
kinetics runs, performed by injecting more substrate into
the cell after the ®rst reaction was complete, gave identical
rate constants. Furthermore, rate constants obtained
from initial rate studies also gave identical rate constants.
The rate constants have an estimated error of Æ3%.
Curve ®ttings were achieved using ENZFITTER
(Elsevier Biosoft, Cambridge, UK) or SCIENTIST
(MicroMath Scienti®c Software, Utah, USA) software.
strate at the end of the hydrolysis reaction. Zinc sulfate
was also added to the mixture in order to investigate
metal chelating reactions, and restoration of the enzyme
activity.
The kinetic parameters were determined from the sec-
ond-order rate constant kcat/Km. Inhibition constants
Ki were calculated using the equation for competitive
inhibition:
kcat Á‰EŠÁ‰SŠ
ꢀ
ꢁ
Rate ˆ
Ki ‡ ‰IŠ
‰SŠ ‡ Km
Ki
References
The zinc ion content of the b-lactamase II stock solution
was determined by atomic absorption spectrometry on a
Perkin Elmer AAnalyst 100 Atomic Absorption Spec-
trometer. The hollow cathode lamp wavelength was set
at 213.9 nm, with a current of 7 mA, and a slit width of
0.7 nm. Zinc nitrate solutions of di€erent concentrations
were used as standards.
1. Page, M. I., Laws, A. P. Chem. Commun. 1998, 1609.
2. Saino, Y.; Kobayashi, F.; Inoue, M.; Mitsuhashi, S., Anti-
microb. Agents Chemother. 1982, 34, 1590. Bicknell, R.;
Emmanuel, E. L.; Gagnon J.; Waley, S. G. Biochem. J. 1985,
229, 791. Iaconis, J. P.; Sanders, C. C.; Antimicrob. Agents
Chemother. 1990, 34, 44. Cuchural, G. J. Jr.; Malamy, M. H.;
Tally, F. P. Antimicrob. Agents Chemother. 1986, 30, 645.
3. Payne, D. J., J. Med. Microbiol., 1993, 39, 93. Cornaglia,
G., Riccio, M. L.; Mazzariol, A.; Lauretti, L.; Fontana, R.;
Rossolini, G. M. Lancet 1999, 353, 899.
Inhibition studies
4. Felici, A.; Amicosante, G.; Oratore, A.; Strom, R.; Ledent,
P.; Joris, B.; Fanuel, L.; Frere, J.-M. Biochem. J. 1993, 291,
151.
Values of Ki were determined by the e€ect of varying
concentrations of the thiol on the value of the second-
order rate constant kcat/Km for the hydrolysis of two
substrates Ð benzylpenicillin and cephaloridine Ð using
Eq. (1).
5. Lim, H. M.; Pene, J. J.; Shaw, R. J. Bacteriol. 1988, 170,
2873; Frere, J.-M. Mol. Microbiol. 1995, 16, 385. Massidda,
O.; Rossolini, G. M.; Salta, G. J. Bacteriol. 1991, 173, 4611.
Rasmussen, B. A.; Gluzman, Y.; Tally, F. P. Antimicrob.
Agents Chemother. 1990, 34, 1590. Thompson, J. S.; Malamy,
M. H. J. Bacteriol. 1990, 172, 2584. Hussain, M.; Carlino, A.;
Madonnam, M. J.; Lampen, J. O. J. Bacteriol. 1985, 164, 223.
Ambler, R. P.; Daniel, M.; Fleming, J.; Hermoso, J.-M.; Pang,
C.; Waley, S. G. FEBS Lett. 1986, 189, 207
ꢀ
ꢁ
ꢀ
ꢁ ꢀ
ꢁ
kcat
Km
kcat
Km
Ki
ˆ
:
ꢀ1†
‰IŠ ‡ Ki
I
O
6. Sutton, B. J.; Artymiuk, P. J.; Cordero-Borboa, A. E.; Little,
C; Phillips, D. C.; Waley, S. G. Biochem. J. 1987, 248, 181.
7. Car®, A.; Pares, S.; Duee, E.; Galleni, M.; Duez, C.; Frere,
J.-M.; Dideberg, O. EMBO J. 1995, 14, 4914.
8. Car®, A.; Duee, E.; Galleni, M.; Frere, J.-M.; Dideberg, O.
Acta Crystallogr. 1998, D54, 313.
9. Fabiane, S. M.; Sohi, M. K.; Wan, T; Payne, D. J.; Bateson,
J. H.; Mitchell, T.; Sutton, B. J. Biochemistry 1998, 37, 2404.
10. Concha, N. O.; Rasmussen, B. A.; Bush, K.; Herzberg, O,
Structure, 1996, 4, 823. Concha, N. O.; Rasmussen, B. A.;
Bush, K.; Herzberg, O. Protein Science 1997, 6, 2671.
11. Crowder, M. W.; Wang, Z; Franklin, S. C.; Zovinka, E.
P.; Benkovic, S. J. Biochemistry 1996, 35, 12126.
where (kcat/Km)I and (kcat/Km)O are the observed second-
order rate constants for hydrolysis of cephaloridine or
benzylpenicillin in the presence or absence, respectively,
of a given concentration of the inhibitor, I, and Ki is the
dissociation constant of the enzyme±inhibitor complex.
The steady-state kinetics were obtained using con-
centrations of the thiol inhibitors ranging from 1 to
103 mM and similar values of Ki were obtained whether
cephaloridine or benzylpenicillin was used as a substrate
to monitor enzyme activity.
The stock solutions of the inhibitors were prepared in
ethanol. Unless otherwise stated, 25 ml of solution of
inhibitor at a given concentration were added to 2.5 mL
of bu€er containing the substrate. The substrate was
shown to be stable in the presence of inhibitor. The
hydrolysis of the substrate was initiated by the addition
of 25 ml of enzyme solution and enzyme activity was
measured as described in the previous section by fol-
lowing the complete time course of the reaction. The
time dependence of the inhibition was studied either by
incubating the enzyme with inhibitor for di€erent times
before adding the substrate and by adding more sub-
12. Li, Z.; Rasmussen, B. A.; Herzberg, O. Protein Science
1999, 8, 249.
13. Wang, Z.; Fast W.; Benkovic, S. J. Biochemistry 1999, 38,
10013. Wang Z.; Fast W.; Valentine A. M.; Benkovic, S. J.;
Curr. Opin. Chem. Biol. 1999, 3, 614.
14. Car®, A.; Paul-Soto, R.; Martin, L.; Petillot, Y.; Frere,
J.-M.; Dideberg, O. Acta Crystallogr. Sect. D: Biol. Crystal-
logr. 1997, D53, 485. Car®, A.; Duee, E.; Paul-Soto, R.;
Galleni, M.; Frere J.-M.; Dideberg, O. Acta Crystallogr. 1998,
D54, 47.
15. Paul-Soto, R.; Hernandez-Valladores, M.; Galleni, M.;
Bauer, R.; Zeppezaur, M.; Frere, J.-M.; Adolph, H.-W. FEBS
Lett. 1998, 438, 137.

510
S. Bounaga et al. / Bioorg. Med. Chem. 9 (2001) 503±510
16. Wendelstorf, C.; Warzeska, S.; Kovari, E.; Krame, R. J.
Chem. Soc. Dalton Trans. 1996, 3087
17. Ullah, J. H.; Walsh, T. R.; Taylor, I. A.; Emery, D. C.;
Verma, C. S.; Gamblin, S. J.; Spencer, J. J. Mol. Biol. 1998,
284, 125.
Toney, J. H.; Cleary, K. A.; Hammond, G. G.; Yuan, X.;
May, W.J.; Hutchins, S. M.; Ashton, W. T.; Vanderwall, D. E.
Biorg. Med. Chem. Lett. 1999, 9, 2741. Walter, M. W.; Valla-
deres, M. H.; Adington, R. M.; Amicosante, G.; Baldwin, J.
E.; Frere, J.-M.; Galleni, M.; Rossolini, G. M.; Scho®eld, C.
J. Bioorg. Chem. 1999, 27 35. Nagano, R.; Adachi, Y.; Ima-
mara, H.; Yamada, K.; Hashizume, T.; Morishima, H.; Anti-
microb. Agents Chemother. 1999, 43, 2497. Yang, K. W.;
Crowder, M. W. Arch. Biochem. Biophys. 1999, 368, 1.
20. Payne, D. J.; Bateson, J. H.; Gasson, B. C.; Proctor, D.;
Khushi, T.; Farmer, T. H.; Tolson, D. A.; Bell, D.; Skett, P. W.;
Marshall, A. C.; Reid, R.; Ghosez, L.; Combret, Y.; Marchand-
Brynaert, J. Antimicrob. Agents Chemother. 1997, 41, 135.
21. Payne, D.; Bateson, J. H.; Gasson, B. C.; Khushi, T.;
Proctor, D.; Pearson, S. C.; Reid, R. FEMS Microbiol. Lett.
1997, 157, 171.
18. Bounaga, S.; Laws, A. P.; Galleni, M.; Page, M. I.; Bio-
chem. J. 1998, 331
19. Gilpin, M. L.; Fulston, M.; Payne, D.; Cramp, R.; Hood,
I. J. Antibiot. 1995, 48, 1081. Walter, M. W.; Felici, A, Gal-
leni, M., Soto, R. P.; Adlington, R. M.; Baldwin, J. E.; Frere,
J.-M.; Gololobov, M and Scho®eld, C. J. Bioorg. Med. Chem
Lett. 1996, 6, 2455; Goto, M.; Takahashi, T.; Yamashita, F.;
Koreeda, A.; Mori, H.; Ohta, M.; Arakawa, Y. Biol. Pharm.
Bull. 1997, 20, 1136. Toney, J. T.; Fitzgerald, P. M. D.; Gro-
ver-Sharma, N.; Olsen, S. H.; May, W. J.; Sundelot, J. G.;
Vanderwall, D. E.; Cleary, K. A.; Grant, S. K.; Wu, J. K.;
Kozarich, J. W.; Pompliano, D. L.; Hammond, G. H. Chem.
Biol. 1998, 5, 185. Greenlee, M. L.; Lamb, J. B.; Balkovic, J.
M.; Hammond, M. L.; Hammond, G. G.; Pompliano, D. L.;
Epstein-Toney, J. H. Biorg. Med. Chem. Lett. 1999, 2549;
22. Hammond, G. G.; Huber, J. L.; Greenlee, M. C.; Laub, J.
B.; Young, K.; Silver, L. L.; Balkovec, J. M.; Pryor, K. D.;
Wu, J. K.; Leiting, B.; Pompliano, D. C.; Toney, J. H. FEMS
Microbiol. Lett. 1999, 459, 289.