Structure-based optimization of cephalothin-analogue boronic acids as β-lactamase inhibitors

Article abstract of DOI:10.1016/j.bmc.2007.10.075

Boronic acids have proved to be promising selective inhibitors of β-lactamases, acting as transition state analogues. Starting from a previously described nanomolar inhibitor of AmpC β-lactamase, three new inhibitors were designed to gain interactions with highly conserved residues, such as Asn343, and to bind more tightly to the enzyme. Among these, one was obtained by stereoselective synthesis and succeeded in placing its anionic group into the carboxylate binding site of the enzyme, as revealed by X-ray crystallography of the complex inhibitor/AmpC. Nevertheless, it failed at improving affinity, when compared to the lead from which it was derived. The origins of this structural and energetic discrepancy are discussed.

Full text of DOI:10.1016/j.bmc.2007.10.075

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1195–1205
Structure-based optimization of cephalothin-analogue boronic
acids as b-lactamase inhibitors
Stefania Morandi,a Federica Morandi,a,b Emilia Caselli,a
Brian K. Shoichetb and Fabio Pratia,*
a
`
Dipartimento di Chimica, Universita degli studi di Modena e Reggio Emilia, via Campi 183, 41100 Modena, Italy
bDepartment of Pharmaceutical Chemistry, University of California San Francisco, 1700 4th Street, Byers Hall Room 508D,
San Francisco, CA 94158, USA
Received 14 June 2007; revised 11 October 2007; accepted 23 October 2007
Available online 7 November 2007
Abstract—Boronic acids have proved to be promising selective inhibitors of b-lactamases, acting as transition state analogues. Start-
ing from a previously described nanomolar inhibitor of AmpC b-lactamase, three new inhibitors were designed to gain interactions
with highly conserved residues, such as Asn343, and to bind more tightly to the enzyme. Among these, one was obtained by ster-
eoselective synthesis and succeeded in placing its anionic group into the carboxylate binding site of the enzyme, as revealed by X-ray
crystallography of the complex inhibitor/AmpC. Nevertheless, it failed at improving affinity, when compared to the lead from which
it was derived. The origins of this structural and energetic discrepancy are discussed.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
(phosphonates, aldehydes, trifluoromethylketones and
boronic acids)3 that can covalently modify the nucleo-
philic catalytic serine. Substrate-analogues bearing such
electrophilic ‘warheads’ have found extensive use as
mechanistic probes4 and as leads for drug design.1
Structure-based design is widely used to discover new
enzyme inhibitors, often by mimicking interactions
observed between the target enzyme and its natural sub-
strates.1 Once a lead compound is identified, structure-
based optimization can improve affinity on the basis of
the structures of inhibitor–enzyme complexes. When
leads are substrate-analogues, these structures can also
provide insight into enzyme mechanism and molecular
recognition.
Among these classes, the boronic acids5 and the phos-
phonates6 have proven to be the most effective inhibitors
of b-lactamases, with the former arguably having great-
er in-cell efficacies. b-Lactamases are the most wide-
spread resistance mechanism to the b-lactam class of
antibiotics, such as the penicillins and cephalosporins.
These enzymes catalyze the hydrolysis of the b-lactam
bond in these antibiotics, rendering them inactive. In
boronic acid inhibitors, the boron atom acts as an elec-
trophile that mimics the carbonyl carbon of the b-lac-
tam ring, and forms with the catalytic serine a
tetrahedral adduct that closely resembles one of the
transition states of the hydrolytic mechanism (Scheme
1a and b).7
Great interest is focused on inhibition of serine-ami-
dases, a class of enzymes that mediate several patholog-
ical conditions, such as thrombosis (thrombin, factor
Xa, factor VIIa), inflammation and emphysema (elas-
tase), hepatitis C (proteases involved in replication)
and bacterial resistance against b-lactam antibiotics (b-
lactamases).2 The structure of many of these enzymes
has been exhaustively mapped and their mechanism of
action carefully investigated. These studies have been
advanced by inhibitors bearing an electrophilic centre
Our previous works in inhibition of the class C b-lacta-
mase AmpC by boronic acids, based on a biomimetic
approach, highlighted that the closer the boronic acid
resembles the natural substrate in its interaction with
the enzyme, the better its inhibition (Scheme 1c). Thus,
as one moves from 1 to 5, a growing mimesis of the b-
lactam cephalothin is displayed and higher inhibition to-
Keywords: b-Lactamase; Inhibition; Boronic acids; X-ray
crystallography.
*
Corresponding author. Tel.: +39 059 205 5056; fax: +39 059 373
543; e-mail: prati.fabio@unimore.it
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.075

1196
S. Morandi et al. / Bioorg. Med. Chem. 16 (2008) 1195–1205
c
a
b
S
H
N
H
N
R
B
H
N
S
7
CH3
B
S
H3C
O
HO
OH
N
HO
OH
O
B
O
B
CH2OCOCH3
O
HO
OH
4
βL-Ser-OH
HO
OH
COOH
3
1
2
βL-Ser-OH
103 μM
0.320 μM
18.5 μM
H2O
H
N
H
N
CH2OCOCH3
COOH
S
S
S
COOH
H
N
R
S
O
B
O
B
N
H
B
HO
HO
OH
HO
OH
O
O
HO
4
5
O
HO
HO
Ser-βL
0.035 μM
0.001 μM
Ser-βL
Scheme 1. Mechanism of action of a b-lactamase (a) with a b-lactam (the antibiotic cephalothin) and (b) with a boronic acid. (c) Growing mimesis of
cephalothin by boronic acids and their activity against the b-lactamase AmpC.
wards the b-lactamase measured. In fact, if methanebo-
ronic acid 1 (Ki 1000 lM) offers to the b-lactamase the
sole interaction of the boron with the serine residue,
compound 2 (Ki 30 lM), characterized by the presence
of the acetamide moiety, gains the additional hydrogen
bond with Asn152, Gln120 and Ala318, as also dis-
played by the amide at C7 of the natural substrate. A
further improvement in inhibition is observed by inser-
tion of more complex amide side chains on the boronic
acid, and among these we selected the cephalothin one
as a model, being a good compromise between complex-
ity and inhibition (compound 3, Ki 0.32 lM).8 In addi-
tion, the stereo-controlled introduction of a phenyl
group, mimicking the dihydrothiazine ring as well as
the configuration at the C7 of cephalosporins, led us
to identify a hydrophobic binding pocket in the active
site of AmpC b-lactamase, formed by Leu119 and
Leu293, which accounts for 10-fold improvement in
affinity (inhibitor 4, Ki 0.035 lM). Finally, the insertion
of a m-carboxyphenyl moiety meant to gain the interac-
tion of the carboxy group at C4 and further improved
affinity led to the discovery of the most potent boronic
inhibitor of AmpC b-lactamase ever tested (inhibitor
5, Ki 0.001 lM).9
5. For many design projects, such accidental successes—
interaction with a non-conserved and unintentional res-
idue—would have occasioned little worry. Since b-lacta-
mases are resistance enzymes, subject to rapid molecular
evolution in the face of inhibitor pressure,12 we were
concerned that this interaction could easily be overcome
by mutation at Asn289, which is not necessary for
AmpC function. Indeed, experiments with other mem-
bers of the class C b-lactamase family that lack
Asn289, such as the P99 b-lactamase from Enterobacter
cloacae, suggested that such a substitution would reduce
inhibitor affinity.
We wanted to find an analogue of 5 to exploit the carbox-
ylate binding site implicated in substrate recognition,
improving affinity and diminishing the ease of resistance
substitutions. We reasoned that increasing the length of
the anionic side chain would allow us to reach this sub-
site. Here we describe the design, synthesis and biological
and crystallographic evaluation of such inhibitors,
resulting in the development of a new inhibitor of AmpC.
This inhibitor succeeds in placing its anionic group into
the carboxylate binding site of the enzyme, largely avoid-
ing interaction with the mutable Asn289. Nevertheless, it
fails at improved affinity compared to the lead from
which it was derived. The origins of this structural and
energetic discrepancy will be considered.
A surprising feature of the crystal structure of 5 in com-
plex with AmpC b-lactamase was the observation of a
hydrogen bond between the carboxylate group of the
inhibitor and the amide of Asn289. This was unex-
pected, because this asparagine is not conserved among
other members of the class C enzymes. In designing 5,
we had hoped that the carboxylate would interact with
the conserved site that in b-lactam complexes interacts
with the C4 carboxylates of these substrates (Asn343,
Asn346 and Thr316).10 Instead,11 in the AmpC/5 com-
plex, these residues make no direct hydrogen bond with
2. Results
2.1. Design
In the design of new boronic acids as b-lactamase inhib-
itors we chose to modify the lead structure of compound
5 following two main strategies. The first was to explore
the effect on affinity of a different anionic group on the
phenyl ring, replacing the carboxy moiety with a bio-
isosteric group such as the tetrazole (compound 6,
Scheme 2), which should enhance the lipophilicity of
the molecule and increase the distance of the anionic
charge from the phenyl ring, allowing it to interact with
more canonical anion recognition residues.
˚
the inhibitor—the closest of them, Asn343, is 3.43 A
away from the inhibitor in the complexed structure.
Thermodynamic cycle experiments confirmed that the
Asn289 was critical to the recognition of compound
5—substitution of this residue with an Ala reduced affin-
ity by 17-fold, and overall this interaction was found to
contribute 2.1 kcal/mol to the overall binding affinity of

S. Morandi et al. / Bioorg. Med. Chem. 16 (2008) 1195–1205
1197
H
N
H
H
N
N
COOH
S
N
S
S
COOH
N
O
B
N
O
B
O
B
NH
HO
OH
HO
OH
HO
OH
6
7
8
O
N
COOMe
O
O
B
B
B
COOMe
N
O
N
N
O
O
9
CPh3
10
11
Scheme 2. Newly designed potential inhibitors of b-lactamases and (+)-pinanediol arylboronates, respectively, needed as their synthetic precursors.
The second was to insert different spacers between the
carboxy group and the phenyl ring; this too would place
the anionic moiety closer to the carboxylate sub-binding
site, targeting its more conserved residues. To do so, we
explored a methylene (compound 7) and a vinyl spacer
(compound 8), distancing the anionic group from the
transesterification with (+)-pinanediol afforded 9 in
48% overall yield.
(+)-Pinanediol (3-methoxycarbonylmethyl)phenylboro-
nate (10) was obtained from pinacol (3-cyanomethylphe-
nyl)boronate by treatment with gaseous hydrochloric
acid at 0 ꢁC in anhydrous diethyl ether followed by alco-
holysis of the intermediate iminoester with methanol; fi-
nal transesterification with (+)-pinanediol afforded 10 in
57% overall yield.17
˚
phenyl ring by 2 and 4 A, respectively.
2.2. Molecular modelling
Compounds 6–8 were designed and built in the active
site of AmpC using Sybyl13 and Moloc.14 Each molecule
was minimized by using the MAB force field allowing
(+)-Pinanediol (E)-3-(2-methoxycarbonylvinyl)phenyl-
boronate (11) was synthesized by cross-coupling reac-
˚
for flexibility only to protein residues within 7 A dis-
tion
of
methyl
trans-3-bromocinnamate
with
tance to the active site catalytic serine.15 During minimi-
zation the boron and Oc of serine 64 were covalently
linked. The process was driven by shape and polar com-
plementarity between ligand and protein surface and the
absence of unfavourable van der Waals interactions. As
shown in Figure 1, these efforts suggested that com-
pound 6, bearing the tetrazole moiety, would interact
with Asn343, whereas the spacer-bearing compounds 7
and 8 would reach both the non-conserved residue
Asn289 and the well-conserved Asn343.
bispinacolate diboron, using (1,10-Bis-(diphenylphos-
phino)-ferrocene)-palladium dichloride [Pd(dppf)Cl2] as
the catalyst in dry dimethylsulfoxide at 80 ꢁC (59%
yield, 65% conversion).18 The resulting pinacol ester
was quantitatively converted to pinanediol ester by sim-
ple transesterification with (+)-pinanediol at rt.
Compounds 9–11 were then subjected to Matteson’s
homologation protocol (Scheme 3).16 Pinanediol esters
I were treated with in situ generated dichloromethyllithi-
um from dichloromethane and n-butyllithium at
ꢀ100 ꢁC and the reaction mixture was warmed gradu-
ally to 0 ꢁC. The presence of the a-chloroboronate II
was investigated by means of 1H NMR analysis. Homol-
ogation resulted feasible for compounds 9 and 11: in
fact, successful insertion of a chlorine-bearing carbon
atom into the C–B bond was confirmed in the 1H
NMR spectrum by the presence of a broad singlet at
about 4.6 ppm. Compound 10, on the other hand, ended
up in almost complete decomposition, probably due to
the incompatibility of the benzylic enolizable protons
of the methylcarboxylate moiety with strong nucleo-
philes such as dichloromethyllithium.
2.3. Synthesis
As with inhibitor 5, all these molecules display a stereo-
genic carbon atom, which duplicates the stereochemistry
on the correspondent C7 of cephalothin. In view of the
general strategy developed by Matteson for the intro-
duction of stereocentres a to the boron atom through
highly stereoselective homologation of boronic esters,
suitable arylboronates 9–11 were prepared from com-
mercially available starting material as key intermedi-
ates for the synthesis of 6–8. (+)-Pinanediol was
chosen as the chiral auxiliary as it is well known to in-
duce the desired S configuration at the newly inserted
chlorine-bearing carbon atom with very high diastere-
oselectivity (>98%).16
It is worth to mention that although the homologation
reaction generally proceeds with very high diastereose-
lection (>98%), the benzylic stereocentre is prone to epi-
merization even for short exposure of the reaction
mixture at rt.19 This annoying problem can be overcome
by never allowing the a-chloroboronate to reach tem-
peratures over 0 ꢁC,16c but treating in situ with lithi-
umhexamethyldisilylamide [LiN(TMS)2] at ꢀ78 ꢁC.
(+)-Pinanediol 3-(2-trityltetrazol-5-yl)phenylboronate
(9) was synthesized in two steps from 5-(3-bromophe-
nyl)-2H-tetrazole. Protection of the tetrazole ring as a
tritylate followed by boronation with n-butyllithium
and trimethylborate at ꢀ78 ꢁC and successive in situ

1198
S. Morandi et al. / Bioorg. Med. Chem. 16 (2008) 1195–1205
Figure 1. Stereo view of the interactions observed between AmpC and compounds 6–8 in the modelled conformation. (A) Compound 6. (B)
Compound 7. (C) Compound 8. Carbon atoms are coloured grey for the protein and orange for the ligands; oxygen atoms, red; nitrogen atoms, blue;
sulfur atoms, yellow; boron atoms, magenta. Atomic interactions within hydrogen bonding distance are shown as dashed yellow lines. This figure was
generated with Pymol (htpp://pymol.sourceforge.net/).
Substituted intermediate III was not isolated, but di-
rectly desilylated by treatment with methanol at 0 ꢁC
and immediately acylated at ꢀ78 ꢁC with 2-thiophenace-
tyl chloride.
low (1 nM) Kd value.21 Like compound 5, compound 8
had a time-dependent component to its inhibition—we
presume the origins of such behaviour were similar,
reflecting the very high affinity of this inhibitor. Neverthe-
less, compound 8 remained reversible, and the inhibitor
could be competed off by increasing substrate concentra-
tion. We have accounted for this time-dependent effect in
the Ki value reported for this inhibitor (see Section 4).
Compound 11 afforded the desired amide IV, whereas
several attempts to pursue the same one-pot procedure
with 9 only produced variable quantities of oxidation
by-products, as the nucleophilic displacement at the hal-
ogenated carbon atom probably was most likely ham-
pered by the steric hindrance of the trityl protective
group, as also suggested by studies of molecular model-
ling. Final hydrolysis of both the chiral auxiliary and the
methoxy protective group was accomplished by reflux-
ing in 3 N degassed hydrochloric acid to afford the free
boronic inhibitor 8 in 25% yield.
To investigate the effect of adding a spacer between the
carboxylate group and the phenyl ring we determined
the potency of compound 8. Compound 8 inhibited
AmpC with a Ki of 1 nM. While this inhibition is potent,
it is no greater than that of the analogue compound 5
from which it was derived (Table 1). Comparison of 8
with compound 4 suggests that the m-vinylcarboxylate
improves the binding energy by 2.1 kcal/mol, as did
the m-carboxylic acid moiety of compound 5.23
2.4. Enzymology and binding affinities
Almost all boronic acid inhibitors of AmpC b-lactamase
previously studied are reversible, fast-on, fast-off, com-
petitive inhibitors.20 The one important exception to this
rule is compound 5, which has a time-dependent compo-
nent to it; this can, at least partially, be attributed to its
A key question was whether the affinity of compound 8
derived was from the hydrogen bond to Asn289, as was
the case for 5 (based on thermodynamic double-pertur-
bation analyses),22 or whether the affinity was derived
through interactions with the conserved residues

S. Morandi et al. / Bioorg. Med. Chem. 16 (2008) 1195–1205
1199
R2
H
Cl
(TMS)2N
R1
MeOH
R1COCl,
N
R2
R2
R2
H
N
R1
LiN(TMS)2
B
LiCHCl2
HCl
B
I
B
O
B
R2
O
O
O
O
O
O
O
O
O
B
HO
OH
II
III
IV
V
Scheme 3. Stereoselective synthesis of chiral boronic acids.
Table 1. Ki values of the cephalothin-analogue boronic acids versus
AmpC
Table 3. Crystallographic summary for the complex AmpC
AmpC/8 complex
Compound
Ki (lM)
DDGb versus compound 3 (kcal/mol)
˚
Cell constants (A; ꢁ)
a = 118.38, b = 76.94,
c = 97.25, b = 115.87
C2
Cephalothin
0.320a
0.035
0.001
0.001
0
4
5
8
1.31
3.41
3.41
Space group
Resolution (A)
˚
2.00
53061
Unique reflections
Total observation
Rmerge (%)
740048
6.9 (49.2)a
99.9 (99.4)a
18.5 (2.0)a
20.0–2.00 (2.07–2.00)
716
a These Ki were determined in 50 mM KPi, pH 7.0.7 The values in
TRIS are typically 2-fold lower.
b Differential free energy of binding relative to compound 1, calculated
at 298 K. Positive values indicate improved affinity.
Completeness (%)
hIi/hrIi
˚
Resolution range for refinement (A)
Number of protein molecules
Number of water molecules
482
0.013
˚
RMSD bond lengths (A)
Table 2. Ki values for compounds 5 and 8 against various b-lactamases
RMSD bond lengths (ꢁ)
Rcryst (%)
Rfree (%)
1.482
17.6
Compound
Ki (nM)
TEM-1
AmpC
N289A
TEM-30
21.9
27.2b
38.3b
34.5
2
˚
Average B-factor, protein atoms (A )
5
8
1
1
17
64
106
7800
3800
2
Average B-factor, inhibitor atoms (A )
˚
2.8
2
Average B-factor, water atoms (A )
˚
a Values in parentheses are for the highest resolution shell.
b Values cited were calculated for both molecules in the asymmetric
unit.
Asn343 or Asn346, as had been designed. To investigate
this question, we measured the affinity of 8 versus the
AmpC N289A mutant (Table 2). Whereas this substitu-
tion reduces affinity of compound 5 by 17-fold (1.7 kcal/
mol), it led to only a 2.8-fold (0.6 kcal/mol) decrease in
affinity for compound 8, consistent with the idea that
this compound, even though it retains the affinity of
the parent structure, no longer does so via an important
hydrogen-bond with the non-conserved Asn289.
b-lactams. The major differences emerge in the point of
substitution of this molecule, that is, the vinylcarboxy-
late: as designed, the carboxylic acid group is observed
˚
to interact only with Nd2 of Asn343, and is 3.3 A from
the side chain of Asn289.
3. Discussion
2.5. X-ray crystallographic structure determination
Our goal was to obtain an inhibitor of AmpC b-lacta-
mase that could interact with the carboxylate subsite
formed by Asn343, Ans346 and Thr316, replacing the
interaction with Asn289 experienced by compound 5.
Our hope was that such a molecule would bind more
tightly than the lead inhibitor 5, and would be less sub-
ject to resistance substitutions of the non-conserved
Asn289. Compound 8 was the synthesized product of
this design strategy. A key result of this study is that
the design strategy was structurally successful—the X-
ray crystal structure of the AmpC/8 complex reveals
the new inhibitor to hydrogen-bond with Asn343, as
called for by design. It is clear from studies of the mu-
tant N289A enzyme that, in contrast with the lead inhib-
itor 5, the carboxylate of 8 no longer interacts
significantly with Asn289, but instead is likely to owe
its interaction energy to the new hydrogen bond with
Asn343, also as designed. On the other hand, the affinity
The crystal structure of AmpC in complex with 8 was
˚
determined at 2.0 A resolution (Table 3 and Fig. 1A,
B). The inhibitor was unambiguously identified in the
initial Fo ꢀ Fc difference map contoured at 3r. Electron
density connects the Oc of the catalytic Ser64 to the bor-
on atom of the inhibitor. The inhibitor was modelled at
full occupancy in molecule B of the asymmetric unit,
and at 0.6 occupancy in molecule A of the asymmetric
unit. Excluding proline and glycine residues, 91.4% of
the amino acids are in the most favoured regions of
the Ramachandran plot (8.6% in the additionally al-
lowed regions).23
Overall, the AmpC/8 complex resembles that of AmpC/5
(Table 4). The boron geometry is tetrahedral, as ex-
pected, key hydrogen bond interactions are maintained
and closely resemble those typically observed in b-lacta-
mase structures with transition state analogues and with

1200
S. Morandi et al. / Bioorg. Med. Chem. 16 (2008) 1195–1205
Table 4. Interactions in inhibitor bound and native AmpC b-
lactamase
the predicted and observed structures for the AmpC/8
complex correspond fairly well, the differences between
them may be sufficient to erase any improvement in
affinity.
17
17
16
10
18
20
16
10
18
19
H
H
4
S
4
S
24
6
6
O21
O21
5
N
9
7
7
20
5
1
N
9
14
14
19
3
15
3
15
23
O-
22
8O
B
O-
22
8 O
B
1
The possibility that the hydrogen-bond with Asn289 of-
fers as much to the carboxylate as the canonical carbox-
ylate site of the enzyme is also possible, though it seems
harder to credit. Not only is Asn289 not conserved, but
its interaction with the carboxylate of 5 occurs in a sur-
face-exposed region. Asn343 is, by comparison, much
more buried, forming a clear pocket along with
Thr316 and Asn346 that typically accommodates sub-
strate carboxylates. Of course, one might argue that as
the ion–dipole interaction is buried, it will lose in solva-
tion what it gains in interaction energy, and in some
sense this must be true. Nevertheless, it seems surprising
that one might gain so much from this interaction on the
surface (the carboxylate with Asn289), and no more
from an analogous interaction occurring in a pocket
pre-formed over evolutionary time to accommodate it.
Finally, it must be admitted that the vinyl carboxylate
of 8 is not exactly equivalent to the direct carboxylate
substituent of 5, and the former may pay entropic or
strain energy costs in binding.24
2
2
HO 11 OH
HO 11 OH
12
12
13
13
5
8
˚
Interaction
S64N–O12
Distance (A)
AmpC/8b
AmpC/5a
Apob,c
3.1
2.7
3.3
2.7
2.8
3.0
2.9
3.0
3.3
2.8
2.6
3.4
2.6
2.7
3.0
3.1
2.8
6.5
2.6
7.1
3.0
3.1
N.P.
2.9
3.1
2.7
3.2
2.5
2.7
2.8
2.9
3.1
3.2
2.8
3.2
3.7
2.9
2.6
3.0
3.1
2.9
4.1
2.6
3.2
N.P.
N.P.
3.0
3.3
N.P.d
N.P.
N.P.
N.P.
N.P.
N.P.
2.5
A318N–O12
A318O–O12
Y150OH–O13
Wat402–O12
Wat402–O13
Y150OH–K315Nn
Y150OH–S64Oc
Y150OH–K67Nn
K67Nn–A220O
K67Nn–S64Oc
Wat402–T316Oc1
Wat402–Wat403
Wat403–N346Od1
Wat403–R349Ng1
A318O–N9
3.2
3.1
3.5
3.5
3.8
2.9
2.7
2.9
N.P.
N.P.
N.P.
2.7
N152Nd2–O8
Q120Ne2-O8
N152Od1–K67Nn
N152Nd2–Q120Oe1
Wat181–O21
Definitive answers to these questions are possible, but
they must await further thermodynamic cycle analy-
ses,22,25b involving site-directed mutagenesis and ligand
derivatization. We remain convinced that the affinity
in this series of inhibitors can be substantially improved
by judicious choice of substitution, and the results of
this study provide useful guidance in that direction.
They also provide a cautionary lesson: even when com-
putational design is structurally successful, with the
appropriate ligand groups binding as predicted in subse-
quent experimental structures, it does not guarantee that
affinity will improve. Predicting binding affinity changes
remains challenging, with increases in interaction ener-
gies balanced by losses in solvation, entropic and strain
energies. In this case, the balance was exact.
3.0
N.P.
N.P.
N.P.
N.P.
Wat469–O22
N343Nd2–O22
N289Nd2–O21
a Distances are for monomer 1 of the asymmetric unit.
b Distances are for monomer 2 of the asymmetric unit.
c For the apo structures, see ref. 20b (PDB code: 1KE4).
d Not present.
of 8 was no better than the lead compound 5. How can
this apparent contradiction between a structurally suc-
cessful design and lack of energetic improvement be rec-
onciled? What lessons can we draw for future inhibitors
of this antibiotic resistant enzyme?
4. Experimental
The discrepancy between apparent structural ‘success’
and energetic failure has two possible origins. First,
the structure-based design may not have succeeded in
every detail, and small discrepancies can foil energetic
improvement. Second, the design may have been mis-
placed, with the canonical carboxylate site offering no
real advantage over the fortuitous Asn289 interaction.
We cannot discount either of these possibilities at this
time. Comparing the overlap between the predicted
and experimental structures (Fig. 2C), it seems clear that
the inhibitor is placed largely as designed. In the pre-
dicted structure, the carboxylate group interacts with
Asn343, as well as Asn289. The distance between the
4.1. Molecular modelling
The modelling was performed with SYBYL 6.8 and
MAB/Moloc13–15 on a Linux workstation. Ligands were
minimized in the pocket by using the MAB force field.
During minimization the default parameters were ap-
plied and flexibility was allowed only for residues in
˚
7 A distance from the active serine.
4.2. Synthesis and analysis
4.2.1. General. All reactions were performed under ar-
gon using oven-dried glassware and dry solvents. Anhy-
drous tetrahydrofuran (THF) and diethyl ether were
prepared by standard methods and freshly distilled un-
der argon from sodium benzophenone ketyl prior to
use. Methanol was dried by storage upon 4 molecular
sieves. The ꢀ100 ꢁC bath was prepared by addition of
˚
carboxylate and Asn343 is 2.8 A, and the distance be-
˚
tween the carboxylate and Asn289 is 3.0 A. In the exper-
imental structure, conversely, the interaction between
˚
the carboxylate of 8 and Asn343 is 3.0 A, and no inter-
action with Asn289 is observed. Thus, whereas overall

S. Morandi et al. / Bioorg. Med. Chem. 16 (2008) 1195–1205
1201
˚
Figure 2. Stereo view of the active site region of the AmpC/8 complex determined to 2.0 A resolution. (A) The 2Fo ꢀ Fc electron density map is
shown in blue, contoured at 1.0 r. (B) Interactions observed between AmpC and compound 8 in the crystallographic complex. Carbon atoms are
coloured grey for the protein and cyan for the ligand; oxygen atoms, red; nitrogen atoms, blue; sulfur atoms, yellow; boron atoms, magenta. Dashed
yellow lines represent key hydrogen bonds. Red spheres represent water molecules. (C) Overlay of the modelled and crystallographic conformations
of compound 8 in the AmpC site. Carbon atoms of compound 8 in the modelled conformation, orange; carbon atoms of compound 8 in the crystal
structure, cyan. This figure was generated with Pymol (htpp://pymol.sourceforge.net/).
liquid nitrogen to a pre-cooled (ꢀ78 ꢁC) mixture of eth-
anol/methanol (1:1). Degassed 3 N hydrochloric acid
was prepared by diluting 37% hydrochloric acid with de-
gassed water. All reactions were monitored by TLC on
pre-loaded (0.25 mm) glass supported silica gel plates
(Merk, Kieselgel 60, F254) and compounds were visual-
ized by exposure to UV light and by the Hanessian’s cer-
ium molybdate stain. Chromatographic purification of
compounds was performed on silica gel (particle size
recorded on a Bruker Advance-400. Mass spectra were
determined on a Finnigan MAT SSQ A mass spectrom-
eter (EI, 70 eV). Elemental analyses were performed on
a Carlo Erba Elemental Analyzer 1110.
The catalyst (1,10-bis-(diphenylphosphino)-ferrocene)-
palladium dichloride [Pd(dppf)Cl2] was purchased from
Sigma–Aldrich.
0.05–0.20 mm). Melting points were measured on a Bu-
¨
chi 510 apparatus. Optical rotations were recorded at
4.3. Synthesis of (+)-pinanediol 3-(2-trityltetrazol-5-
yl)phenylboronate (9)
20 ꢁC on a Perkin-Elmer 241 polarimeter and are ex-
1
pressed in 10ꢀ1 deg cm2 gꢀ1. H and 13C NMR spectra
A solution of commercially available 5-(3-bromophe-
nyl)-2H-tetrazole (1.07 g, 4.75 mmol) and Et3N
(0.70 mL, 4.99 mmol) in THF (15 mL) was heated at
40 ꢁC and trityl chloride (1.391 g, 4.99 mmol) in THF
(6 mL) was added dropwise. A white abundant precipi-
tate of ammonium salt formed immediately. After
30 min the solid was filtered off, the solvent was removed
under reduced pressure and the resulting oil was crystal-
lized from diethyl ether (20 mL) to afford 5-(3-bromo-
phenyl)-2-trityltetrazole as a white solid (2.214 g, 100%
were recorded on a Bruker DPX-200 spectrometer at
200 and 50 MHz, respectively; chemical shifts (d) are re-
ported in ppm downfield from TMS as internal stan-
dard. When peak multiplicities are given the following
abbreviations are used: s, singlet; d, doublet; t, triplet;
q, quartet; m, multiplet; br, broadened signal. Two-
dimensional NMR techniques (heteronuclear single
quantum coherence and heteronuclear multiple bond
correlation) were used to aid signal assignment and were

1202
S. Morandi et al. / Bioorg. Med. Chem. 16 (2008) 1195–1205
1
yield). Mp 128–130 ꢁC. H NMR (CDCl3): d 7.10–7.50
(16H, m, H5, CPh3), 7.58 (1H, ddd, J = 8.0, 1.9,
1.1 Hz, H6), 8.10 (1H, ddd, J = 7.7, 1.7, 1.1 Hz, H4),
8.29 (1H, dt, J = 1.7, 0.3 Hz, H2),. 13C NMR (CDCl3):
d 122.8, 125.6, 127.8, 128.4, 129.9, 126.7, 130.3,
130.32, 133.2, 141.3. EI-MS: m/z 468 (M++2, <1%),
466 (M+, <1), 412 (<1), 410 (<1), 334 (<1), 332 (<1),
253 (1), 243 (100), 228 (5), 135 (38). Anal. Calcd for
C26H19BrN4: C, 66.82; H, 4.10; N, 11.99. Found: C,
66.80; H, 4.07; N, 12.02.
diluted with diethyl ether, thus causing the precipitation
of ammonium chloride. After filtration, the clear solu-
tion was concentrated in vacuo affording pinacol 3-
(methoxycarbonylethyl)phenylboronate as a pale yellow
oil which was used as such for the following step
1
(1.242 g, 75% overall yield). H NMR (CDCl3): d 1.35
(12H, s, CH3), 3.64 (2H, s, PhCH2), 3.69 (3H, s,
OCH3), 7.25–7.47 (2H, m, H5,6), 7.66–7.78 (2H, m,
H2,4). 13C NMR (CDCl3): d 26.2, 42.4, 53.3, 85.2,
129.3, 133.5, 134.7, 134.9, 137.0, 173.4. C–B not seen.
EI-MS: m/z 275 (M+ꢀ1, 14%), 260 (25), 215 (100), 200
(11), 190 (5), 176 (23), 131 (7), 117 (35), 91 (5), 59 (7).
Anal. Calcd for C15H21BO4: C, 65.24; H, 7.67. Found:
C, 65.23; H, 7.69.
In a 50 mL three-necked flask 5-(3-bromophenyl)-2-tri-
tyltetrazole (0.972 g, 2.08 mmol) was dissolved in THF
(20 mL) and cooled at ꢀ78 ꢁC. n-Butyllithium
(0.92 mL of a 2.5 M solution in hexanes, 2.29 mmol)
was added dropwise over 10 min whereupon the solu-
tion turned red. The mixture was gradually allowed to
reach ꢀ20 ꢁC and stirred for 1 h, and a yellow solution
with an abundant precipitate was obtained. The reaction
mixture was then re-cooled at ꢀ78 ꢁC and a solution of
freshly distilled trimethyl borate (0.234 mL, 2.08 mmol)
in THF (1 mL) was added. After 1 h at ꢀ78 ꢁC, the reac-
tion mixture was treated with chlorotrimethylsilane
(0.264 mL, 2.08 mmol) and the resulting clear solution
was warmed to rt and stirred overnight. Finally,
(+)-pinanediol (0.354 g, 2.08 mmol) was added at rt
and the solution stirred for 1 h. The crude mixture was
partitioned between water (20 mL) and ethyl acetate
(100 mL) and the aqueous phase was extracted with
ethyl acetate (2· 50 mL). Combined organic layers were
dried over MgSO4, filtered and concentrated in vacuo to
give a white residue. Column chromatography using
light petroleum/ethyl acetate (9:1) as the eluant afforded
9 as a white crystalline solid (0.560 g, 48% yield). Mp
185 ꢁC. [a]D +1.75 (aD +0.031, c 1.8%, CHCl3). 1H
NMR (CDCl3): d 0.91 (3H, s, pinanyl CH3), 1.23 (1H,
d, J = 10.4 Hz, pinanyl Hendo), 1.33 (3H, s, pinanyl
CH3), 1.51 (3H, s, pinanyl CH3), 1.80–2.60 (5H, m,
pinanyl protons), 4.48 (1H, dd, J = 8.5, 2.0 Hz, CHOB),
7.10–7.45 (15H, m, CPh3), 7.48 (1H, t, J = 7.6 Hz, H5),
7.91 (1H, dt, J = 7.6, 1.1 Hz, H6), 8.24 (1H, dt,
J = 7.6, 1.1 Hz, H4), 8.59 (1H, s, H2). 13C NMR
(CDCl3): d 25.4, 27.9, 28.5, 30.1, 36.9, 39.6, 40.9, 52.8,
79.8, 87.8, 128.6, 129.1, 129.6, 131.2, 131.7, 134.7,
138.0, 142.8, 148.3, 165.5. C–B not seen. EI-MS: m/z
510 (M+ꢀ2N2, 100%), 132 (8), 357 (11), 330 (26), 280
(19), 253 (47), 243 (72), 178 (25), 165 (41), 135 (28),
107 (14), 93 (39). Anal. Calcd for C19H25BO4: C,
69.53; H, 7.68. Found: C, 69.55; H, 7.71.
The pinacol ester (1.242 g, 4.5 mmol) was dissolved in
anhydrous THF (5mL) and (+)-pinanediol (0.766 g,
4.5 mmol) was added. After stirring for 3 h at rt, the
crude mixture was concentrated in vacuo and purified
by column chromatography using light petroleum/ethyl
acetate (7:3) as the eluant, affording 10 as a viscous oil
(1.123 g, 76% yield). [a]D +2.27 (aD +0.031, c 1.4%,
CHCl3). 1H NMR (CDCl3): d 0.89 (3H, s, pinanyl
CH3), 1.22 (1H, d, J = 1.06 Hz, pinanyl Hendo), 1.30
(3H, s, pinanyl CH3), 1.48 (3H, s, pinanyl CH3), 1.80–
2.61 (5H, m, pinanyl protons), 3.64 (2H, s, PhCH2),
3.68 (3H, s, OCH3), 4.45 (1H, dd, J = 8.8, 1.9 Hz,
CHOB), 7.25–7.45 (2H, m, H5, H6), 7.65–7.80 (2H, m,
H2, H4). 13C NMR (CDCl3): d 25.4, 27.9, 28.5, 30.1,
36.9, 39.6, 40.9, 42.4, 52.8, 53.3, 79.7, 87.7, 129.4,
133.4, 134.7, 134.9, 137.0, 173.3. C–B not seen. EI-
MS: m/z 328 (M+, 31%), 313 (28), 287 (20), 269 (33),
259 (100), 245 (30), 232 (82), 173 (20), 134 (19), 117
(57), 91 (41), 83 (52), 67 (48). Anal. Calcd for
C19H25BO4: C, 69.53; H, 7.68. Found: C, 69.55; H, 7.71.
4.5. Synthesis of (+)-pinanediol 3-(2-methoxycarbonylvi-
nyl)phenylboronate (11)
In a three-necked flask, oven-dried potassium acetate
(0.488 g, 4.98 mmol), bispinacolate diboron (0.463 g,
1.82 mmol) and commercially available trans-3-bromo-
cinnamic acid (0.400 g, 1.66 mmol) were dissolved in
anhydrous DMSO (5 mL). Pd(dppf)Cl2 catalyst
(0.048 g, 0.066 mmol, 4%) was added to the solution,
which turned red, and the mixture was heated at 80 ꢁC
for 1 h, thus leading to a black solution. As TLC analy-
sis (light petroleum/diethyl ether 9:1) showed only
incomplete conversion, additional catalyst (4%) was
added and the mixture was heated for 1 h. The crude
mixture was partitioned between water (50 mL) and
ethyl acetate (200 mL) and the aqueous phase was ex-
tracted with ethyl acetate (2· 100 mL). The organic lay-
ers were washed with brine, dried (MgSO4), filtered and
concentrated in vacuo. The brown solid residue was
purified by gradient column chromatography using light
petroleum/diethyl ether (100% to 96:4) as the eluant and
affording the desired coupling product pinacol 3-(2-
methoxycarbonylvinyl)phenylboronate (0.280 g, 59%
yield, 65% conversion). Attempts to scale-up the reac-
tion always resulted in poorer conversions. Mp 86 ꢁC.
1H NMR (CDCl3): d 1.36 (12H, s, CCH3), 3.80 (3H, s,
COOCH3), 6.50 (1H, d, J = 16.0 Hz, Ph–CH@CH),
4.4. Synthesis of (+)-pinanediol 3-(methoxycarbonyleth-
yl)phenylboronate (10)
Commercially available pinacol 3-(cyanomethyl)phenyl-
boronate (1.460 g, 6.0 mmol) was dissolved in anhy-
drous diethyl ether (4 mL) and dry methanol (0.29 mL,
7.18 mmol) was added. The solution was cooled at
0 ꢁC and gaseous hydrochloric acid was bubbled. Upon
storage in the refrigerator for 48 h the intermediate imi-
noester precipitated as a white solid and was recovered
by quick filtration, washing with cooled diethyl ether
(1.680 g, mp 92–96 ꢁC). The crude solid was then stirred
with methanol (2.5 mL) at rt for 24 h and subsequently

S. Morandi et al. / Bioorg. Med. Chem. 16 (2008) 1195–1205
1203
7.38 (1H, t, J = 7.4 Hz, H5 arom), 7.60 (1H, dt, J = 7.4,
1.2 Hz, H6 arom), 7.72 (1H, d, J = 16.0 Hz, Ph–
CH@CH), 7.83 (1H, dt, J = 7.4, 1.2 Hz, H4 arom),
8.00 (1H, br, H2 arom). 13C NMR (CDCl3): d 25.0,
51.5, 83.4, 117.9, 128.2, 130.7, 133.7, 134.4, 136.5,
144.8, 167.3. C–B not seen. EI-MS: m/z 288 (M+, 4%),
273 (2), 254 (2), 239 (17), 213 (2), 171 (4), 155 (6), 129
(5), 113 (6), 84 (100), 69 (20). Anal. Calcd for
C16H21BO4: C, 66.69; H, 7.35. Found: C, 66.71; H, 7.38.
tracted with ethyl acetate (2· 50 mL). Combined organic
phases were washed with satd. NaHCO3 (50 mL), dried
over MgSO4, filtered and concentrated in vacuo. The
resulting brown oil was purified by column chromatog-
raphy using diethyl ether as the eluant to afford
(+)-pinanediol (1R)-1-(2-thiophen-2-yl-acetylamino)-1-
(3-(2-methoxycarbonylvinyl)phenyl)methylboronate as
a pale yellow solid which was used for the final step
(0.113 g, 11% yield). Mp 56 ꢁC, [a]D ꢀ1.27 (aD ꢀ0.030,
c 1.18%, CHCl3). 1H NMR (CDCl3): d 0.81 (3H, s, pina-
nyl CH3), 1.15 (1H, d, J = 10.1 Hz, pinanyl Hendo), 1.24
(3H, s, pinanyl CH3), 1.35 (3H, s, pinanyl CH3), 1.50–
2.30 (5H, m; pinanyl protons), 3.81 (3H, s, COOCH3),
3.99 (2H, br, CH2CONH), 4.08 (1H, br, CHB), 4.21
(1H, dd, J = 8.5, 2.0 Hz, CHOB), 6.38 (1H, d,
J = 16.0 Hz, Ph–CH@CH), 6.75 (1H, br, CONH),
6.96-7.08 (2H, m, C@CH–CH), 7.17 (1H, dt, J = 6.5,
1.9 Hz, H6 arom), 7.23–7.39 (4H, m, H2,4,5 arom, S–
CH@CH), 7.63 (1H, d, J = 16.0 Hz, Ph–CH@CH). 13C
NMR (CDCl3): d 25.4, 27.7, 28.6, 30.0, 35.8, 37.4,
39.5, 41.1, 48.7 (br, C–B), 53.0, 53.4, 78.7, 86.3, 119.2,
127.0, 127.5, 127.7, 129.1, 129.6, 129.7, 130.3, 135.2,
135.8, 142.5, 146.3, 168.8, 175.4. EI-MS: m/z 493 (M+,
59%), 462 (2), 424 (2), 395 (2), 358 (4), 341 (13), 314
(7), 297 (8), 257 (40), 234 (12), 216 (8), 190 (19), 175
(13), 158 (7), 135 (15), 124 (24), 97 (100), 93 (36). Anal.
Calcd for C27H32BNO5S: C, 65.72; H, 6.54; N, 2.84; S
6.50. Found: C, 65.70; H, 6.56; N, 2.87; S, 6.55.
The pinacol ester (0.940 g, 3.26 mmol) was dissolved in
anhydrous THF (3 mL), (+)-pinanediol (0.555 g,
3.26 mmol) was added and the mixture was stirred at
rt. After 24 h the solvent was removed under reduced
pressure and the crude yellow residue was purified on sil-
ica using light petroleum/ethyl acetate (8:2) as the elu-
ant, thus affording 11 as a pale yellow oil which
crystallizes on standing (1.111 g, 100% yield). Mp
1
113 ꢁC, [a]D ꢀ2.51 (aD ꢀ0.024, c 0.95%, CHCl3). H
NMR (CDCl3): d 0.91 (3H, s, pinanyl CH3), 1.22 (1H,
d, J = 10.6 Hz, pinanyl Hendo), 1.33 (3H, s, pinanyl
CH3), 1.50 (3H, s, pinanyl CH3), 1.90–2.55 (5H, m,
pinanyl protons), 3.81 (3H, s, COOCH3), 4.48 (1H,
dd, J = 8.6, 1.9 Hz, CHOB), 6.50 (1H, d, J = 16.0 Hz,
Ph–CH@CH), 7.41 (1H, t, J = 7.4 Hz, H5 arom), 7.62
(1H, dt, J = 7.4, 1.2 Hz, H6 arom), 7.73 (1H, t,
J = 16.1 Hz, Ph–CH@CH–COOCH3), 7.84 (1H, dt,
J = 7.4, 1.2 Hz, H4 arom), 8.00 (1H, br, H2 arom). 13C
NMR (CDCl3): d 24.0, 26.5, 27.1, 28.7, 35.5, 38.2,
39.5, 51.4, 51.6, 78.4, 86.5, 117.9, 128.3, 130.7, 133.8,
134.4, 136.6, 144.8, 167.4. C–B not seen. EI-MS: m/z
340 (M+, 82%), 325 (30), 309 (14), 299 (22), 284 (26),
271 (100), 257 (27), 244 (67), 213 (16), 189 (15), 157
(32), 134 (45), 109 (21), 93 (22), 83 (66). Anal. Calcd
for C20H25BO4: C, 70.61; H, 7.41. Found: C, 70.60; H,
7.38.
The (+)-pinanediol boronic ester (0.113 g, 0.23 mmol)
was refluxed for 1 h in 3 N degassed hydrochloric acid
(5 mL). The solution was extracted with diethyl ether
(20 mL) and the aqueous layer was concentrated under
reduced pressure to afford 0.030 g of a whitish solid res-
idue. Insoluble impurities were precipitated with metha-
nol and filtered off. The resulting clear solution was
evaporated to dryness to give a pale yellow film which
solidified into a white solid (25 mg) upon treatment with
4.6. Synthesis of (1R)-1-(2-thiophen-2-yl-acetylamino)-1-
(3-(2-carboxyvinyl)phenyl)methyl boronic acid (8)
1
a single drop of water. H NMR analysis confirmed the
presence of the desired product (25% yield) along with a
deboronated by-product as impurity (22%), which dis-
played a doublet at 3.8 ppm corresponding to benzylic
protons. The two products were chromatographically
inseparable under all conditions tested. Under milder
conditions and shorter reaction times removal of both
protective groups failed. Mp 240 ꢁC (dec.), [a]D ꢀ62.7
(aD ꢀ0.320, c 0.51%, MeOH, corrected). 1H NMR
A solution of dichloromethane (0.209 mL, 3.26 mmol)
in THF (4 mL) was cooled at ꢀ100 ꢁC and was treated
with n-butyllithium (0.98 mL of a 2.5 M solution in hex-
anes, 2.45 mmol) under argon flow and mechanical stir-
ring. After 30 min, a solution of 11 (0.694 g, 2.04 mmol)
in THF (3 mL) was slowly added and the temperature
was allowed to gradually reach 0 ꢁC during 5 h. The
clear solution became yellow and darkened upon warm-
ing. After stirring for 1 h at 0 ꢁC, TLC analysis (light
petroleum/ethyl acetate 8:2) showed complete conver-
sion of the starting material to the a-chloroboronate.
Hence the mixture was cooled at ꢀ78 ꢁC and treated
with lithium (hexamethyldisilyl)amide (2.24 mL of an
1 M solution in THF, 2.24 mmol) and allowed to warm
to rt overnight. Dry methanol (0.980 mL of a 2.5 M
solution in dry THF, 2.45 mmol) was then added at
0 ꢁC and the resulting mixture was stirred for 1 h at
0 ꢁC and at rt for one additional hour. Final acylation
was accomplished at ꢀ78 ꢁC by adding a solution of
2-thiophenacetylchloride (0.302 mL, 2.45 mmol) in
THF (2 mL) and stirring at rt overnight. The crude mix-
ture was partitioned between ethyl acetate (100 mL) and
water (25 mL) and the acidic aqueous layer was ex-
(CD3OD):
d 3.84 (1H, s, CHB), 4.16 (2H, s,
CH2CONH), 6.39 (1H, d, J = 16.0 Hz, Ph–CH@CH),
6.85–7.80 (8H, m, Ph–CH@CH, H2,4ꢀ6 arom, S–
CH@CH–CH–C). 13C NMR (CD3OD): d 32.1, 52.2
(br, C–B), 119.3, 126.1, 126.7, 127.1, 128.4, 129.1,
129.2, 129.8, 134.6, 135.6, 143.2, 146.4, 176.7, 179.2.
4.7. Enzymology
Inhibitors were initially dissolved in DMSO at a concen-
tration of 50 mM; more dilute stocks (10 mM to 1 lM)
were subsequently prepared as necessary. Kinetic mea-
surements were performed in 50 mM Tris buffer, pH
7.0, and monitored in an HP8453 UV/vis spectropho-
tometer. The concentration of each enzyme was deter-
mined spectrophotometrically; the enzyme was

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S. Morandi et al. / Bioorg. Med. Chem. 16 (2008) 1195–1205
expressed and purified as described.26 For AmpC,
0.5 nM enzyme was used with 200 lM nitrocefin (Km
127 lM) or 50 lM centa (Km 19 lM) as initial substrate
concentrations. For N289A, 1 nM enzyme was used
with 400 lM nitrocefin (Km 27 lM) as initial substrate
concentration. The hydrolysis of nitrocefin was moni-
tored at 480 nm, and that of centa at 405 nm. To deter-
mine Ki values, inhibitor and enzyme were incubated
together at their final concentration for 5 min before
the reaction was initiated by the addition of substrate.
The Ki values for compound 8 were obtained by com-
parison of progress curves in the presence and absence
of inhibitor, using the method described by Waley27
which has been widely used for boronic acid inhibitors
of b-lactamases. In these analyses, sufficient inhibitor
was used to give at least 50% inhibition; the Ki values re-
ported are the average calculated from reactions at five
different inhibitor concentrations, each of which was re-
peated three times. In all reactions, rates were measured
after reactions had overcome their initial lag phase and
had reached a steady state.
Acknowledgments
This work was supported by GM63815 (to B.K.S. and
F.P.). We thank Dr. Paolo Davoli for reading this
manuscript.
References and notes
1. Babine, R. E.; Bender, S. L. Chem. Rev. 1997, 97, 1359–
1472.
2. Yang, W.; Gao, X.; Wang, B. In Boronic Acids. Prepara-
tion, Applications in Organic Synthesis and Medicine; Hall,
D. G., Eds.; Wiley-VCH Verlag GmbH & Co., 2005;
Chapter 13, p 481.
3. Pechenov, A.; Stefanova, M. E.; Nicholas, R. A.; Peddi,
S.; Gutheil, W. G. Biochemistry 2003, 42, 579–588.
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437; (b) Fromm, H. J. Methods Enzymol.: Enzyme Kinet.
Mech., Part D 1995, 249, 123–143.
5. Ness, S.; Martin, R.; Kindler, A. M.; Paetzel, M.; Gold,
M.; Jensen, S. E.; Jones, J. B.; Strynadka, N. J. C.
Biochemistry 2000, 39, 5312–5321.
6. Silvaggi, N. R.; Anderson, J. W.; Brinsmade, S. R.; Pratt,
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7. Powers, R. A.; Caselli, E.; Focia, P. J.; Prati, F.; Shoichet,
B. K. Biochemistry 2001, 40, 9014–9207.
8. Caselli, E.; Powers, R. A.; Blasczcak, L. C.; Wu, C. Y.;
Prati, F.; Shoichet, B. K. Chem. Biol. 2001, 8, 17–31.
9. Morandi, F.; Caselli, E.; Morandi, S.; Focia, P. J.;
Blasquez, J.; Stoichet, B. K.; Prati, F. J. Am. Chem. Soc.
2003, 125, 685–695.
10. (a) Beadle, B. M.; Trehan, I.; Focia, P. J.; Shoichet, B. K.
Structure 2002, 10, 413–424; (b) Patera, A.; Blaszczak, L.
C.; Shoichet, B. K. J. Am. Chem. Soc. 2000, 122, 10504–
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11. Lobkovsky, E.; Billings, E. M.; Moews, P. C.; Rahil, J.;
Pratt, R. F.; Knox, J. R. Biochemistry 1994, 33, 6762–
6777.
4.8. Crystal growth and structure determination
Co-crystals of AmpC in complex with compound 8 were
grown by vapour diffusion in hanging drops equilibrated
over 1.7 M potassium phosphate buffer (pH 8.7) using
micro-seeding techniques. The initial concentration of
the protein in the drop was 2.8 mg/mL, and the concen-
tration of compound 8 was 588 lM. The compound was
added to the crystallization drops in a 1.2% DMSO, 1 M
potassium phosphate buffer (pH 8.7) solution. Crystals
appeared in 7–10 days after equilibration at 21 ꢁC. Be-
fore data collection, crystals were immersed in a cryo-
protectant solution of 20% sucrose, 1.7 M potassium
phosphate, pH 8.7, for about 30 s, and flash-cooled in
liquid nitrogen. Diffraction data were collected on fro-
zen crystals at the Advance Light Source (ALS, Law-
rence Berkley Lab, CA). The data set was measured
from single crystal. Reflections were indexed, integrated
and scaled using the HKL software package.28 The
space group was C2, with two molecules in the asym-
metric unit, each containing 358 residues. The initial
phasing model was an AmpC apo structure (PDB entry
1KE4), with water molecules and ions removed. The
model was positioned by rigid body refinement and its
position further refined using the maximum likelihood
target in CNS29 including simulated annealing, posi-
tional minimization and individual B-factor refinement,
with a bulk solvent correction. Sigma A-weighted elec-
tron density maps were calculated using CNS, with the
program Coot used for manual model rebuilding and
placement of water molecules.30 The inhibitors were
built into the 2|Fo| ꢀ |Fc| and |Fo| ꢀ |Fc| difference density
maps in each active site of the asymmetric unit. Subse-
quent refinement consisted of positional minimization
and B-factor refinement in CNS.
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