A novel fluorogenic substrate for dinuclear Zn(II)-containing metallo-β-lactamases

Article abstract of DOI:10.1016/j.bmcl.2013.01.071

In an effort to prepare a fluorogenic substrate to be used in activity assays with metallo-β-lactamases, (6R,7R)-8-oxo-7-(2-oxo-2H-chromene-3- carboxamido)-3-((4-(2-oxo-2H-chromene-3-carboxamido)-phenylthio)methyl) -5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (CA) was synthesized and characterized. CA exhibited a fluorescence quantum yield (φ) of 0.0059, two fluorescence lifetimes of 3.63 × 10^-10 and 5.38 × 10 ^-9 s, and fluorescence intensity that is concentration-dependent. Steady-state kinetic assays revealed that CA is a substrate for metallo-β-lactamases (MβLs) L1 and CcrA, exhibiting K_m and k_cat values of 18 μM and 5 s^-1 and 11 μM and 17 s^-1, respectively.

Full text of DOI:10.1016/j.bmcl.2013.01.071

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1676–1679
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel fluorogenic substrate for dinuclear Zn(II)-containing
metallo-b-lactamases
a
a
Yin-Lin Zhang a, , Jian-Min Xiao a, , Ji-Li Feng a, , Ke-Wu Yang a, , Lei Feng , Li-Sheng Zhou ,
⇑
Michael W. Crowder b,
⇑
a Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an 710069,PR
China
b Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In an effort to prepare a fluorogenic substrate to be used in activity assays with metallo-b-lactamases,
(6R,7R)-8-oxo-7-(2-oxo-2H-chromene-3-carboxamido)-3-((4-(2-oxo-2H-chromene-3-carboxamido)-phe
nylthio)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (CA) was synthesized and charac-
Received 12 December 2012
Revised 8 January 2013
Accepted 16 January 2013
Available online 26 January 2013
terized. CA exhibited a fluorescence quantum yield (
u) of 0.0059, two fluorescence lifetimes of 3.63 ꢀ 10
ꢁ10
and 5.38 ꢀ 10ꢁ9 s, and fluorescence intensity that is concentration-dependent. Steady-state kinetic
assays revealed that CA is a substrate for metallo-b-lactamases (MbLs) L1 and CcrA, exhibiting Km and kcat
Keywords:
b-Lactamase
Substrate
Enzyme
values of 18 l l
M and 5 sꢁ1 and 11 M and 17 sꢁ1, respectively.
Ó 2013 Elsevier Ltd. All rights reserved.
Enzymes that hydrolyze b-lactam-containing antibiotics, such
as penicillins, cephalosporins, and carbapenems, are called b-lacta-
mases, and the production of b-lactamases is the most common
way that bacteria become antibiotic-resistant. The b-lactam-con-
taining compounds account for over 50% of the available, effective
antibiotics used in the clinic,1–4 and bacterial resistance to these
compounds has become a serious public health issue.5,6 Therefore,
the characterization of b-lactamases is very important so that no-
vel inhibitors can be identified. There are more than 1000 distinct
b-lactamases known, and these enzymes have been categorized
into four classes: A, B, C, and D.2,7 The class B enzymes (metallo-
b-lactamases, MbLs) are Zn(II)-dependent and hydrolyze nearly
all known b-lactam containing antibiotics.4,8–11 Clinical inhibitors
for these enzymes are not known. The MbLs have been further cat-
egorized into the B1, B2, and B3 subclasses based on their amino
acid sequences.12 The B1 and B3 enzymes are most active when
bound to two Zn(II) ions, while the B2 enzymes are most active
when bound to one Zn(II).10,11 Determining catalytic activities
and screening of inhibitors for b-lactamases have been most often
accomplished using chromogenic substrates, such as nitrocefin,
chromacef, and PADAC.13–15 In addition, some fluorogenic and
hydrogel-based substrates have been developed as reporters for
imaging gene expression of b-lactamases in vitro and in vivo.16–
19 Fluorogenic substrates could also be used in steady state kinetic
studies and for high-throughput screening of inhibitors. These
alternative substrates may be especially needed for screening
inhibitors that have maximum absorbances in the same vis region
as nitrocefin, chromacef, or PADAC; vis-based activity assays
would require observing small changes in a large absorbance sig-
nal. In the current study, we describe the synthesis of a fluorogenic
substrate, based on 7-amino-3-chloromethyl-3-cephem-4-carbox-
ylic acid p-methoxybenzyl ester (ACLE) coupled to coumarin-3-
carboxylic acid (coumarin-COOH).
The fluorescent substrate, CA, was obtained in three steps as
shown in Scheme 1. The initial reagent, ACLE, was modified with
amino thiophenol to afford compound 3. Coumarin-3-carboxylic
acid was linked to the two amino groups of modified ACLE to afford
compound 4. The final product was obtained by removing the
4-methoxybenzyl protecting group using phenol, and the three
step synthesis had an overall yield of 14%. CA was characterized
by mass spectrometry and 1H NMR and UV–vis spectroscopies. In
order to confirm the target compound, the samples were subjected
to electrospray ionization mass spectral (ESI-MS) analysis, and a
peak at m/z 702.0615 [M+Na+]+ corresponding to CA was clearly
Abbreviations: ACLE, 7-amino-3-chloromethyl-3-cephem-4-carboxylic acid
p-methoxybenzyl ester; CA, (6R,7R)-8-oxo-7-(2-oxo-2H-chromene-3-carboxamido)
-3-((4-(2-oxo-2H-chromene-3-carboxamido)-phenylthio)methyl)-5-thia-1-azabicy
clo[4.2.0]oct-2-ene-2-carboxylic acid; PADAC, pyridinium-2-azo-p-dimethylaniline
chromophore.
⇑
Corresponding authors. Tel./fax: +86 29 8830 2429 (K.-W.Y.); tel.: +1 513 529
7274 (M.W.C.).
E-mail addresses: kwyang@nwu.edu.cn (K.-W. Yang), crowdemw@muohio.edu
(M.W. Crowder).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.071

Y.-L. Zhang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1676–1679
1677
O
O
O
O
O
Ac2O,H2SO4
Acetone
O
O
Salicylaldehyde
OH
O
HO
OH
O
2
1
SH
H2N
S
ClH2N
O
S
N
O
S
O
N
O
Cl
NH2
O
NH2
O
OCH3
OCH3
3
HCl
ACLE
O
O
O
OH
HATU, DIPEA
O
O
O
S
H
N
S
N
N
H
O
O
O
O
O
O
OCH3
4
Phenol
O
O
O
S
H
S
N
N
N
H
O
O
O
O
OH
CA
O
Scheme 1. Synthesis of fluorescence cephalosporin CA. ACLE = 7-amino-3-chloromethyl-3-cephem-4-carboxylic acid p-methoxybenzyl ester; DIPEA = N,N0-diisopropyleth-
ylamine; HATU = O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate.
observed. Peaks in 1H NMR spectra of intermediates and the final
compound confirmed the structure of CA (see Supplementary data
for details). UV–vis spectra of CA and coumarin-COOH are similar,
as shown in Figure 1a, and both compounds exhibited a major peak
centered at 291 and a shoulder at 325 nm.
To evaluate whether CA is a substrate for MbLs, we over-ex-
pressed and purified CcrA and L1, using previously published pro-
cedures.23,24 CcrA and L1 are representative B1 and B3 MbLs,
respectively. Initially, we used UV–vis spectrophotometry to probe
the hydrolysis of the b-lactam bond of CA by the MbLs. CcrA and L1
The fluorescence properties including spectra, lifetime, and
quantum yields of CA were determined. CA displayed a fluores-
cence emission spectrum centered at 413 nm (excitation at
305 nm), and the emission intensity was nearly seven-fold less
than that of coumarin-COOH (Fig. 1b). The fluorescence quantum
exhibited Km values of 18 8 and 13
9 lM, respectively, and kcat
values of 12 1 and 5 1 sꢁ1, respectively, when using CA as the
substrate and UV–vis spectrophotometry for detection (Table 1).
These steady state kinetic values are very similar to those deter-
mined using fluorescence emission. CcrA and L1 exhibited Km val-
yield (
u
) of CA was 50% lower than that of coumarin (0.0027–
ues of 11 1 and 18
2
l
M, respectively, and kcat values of 17
4
0.0059), which supports the hypothesis that the two coumarin
groups are in close proximity and quenching each other.20 The
fluorescence lifetime of CA was determined using a concentration
and 5 1 sꢁ1, respectively, when using CA as the substrate and
fluorescence emission for detection. In the fluorescence emission
assays, the fluorescence emission increased as the enzymes hydro-
lyzed CA, suggesting that the coumarin groups do not quench each
other in the product. By using ample precedent in the litera-
ture,25,26,27 we hypothesize that the hydrolysis reaction proceeds
with a loss of the thiophenol substituent (Scheme 2).
of 100 lM CA in 50 mM Tris, pH 7.0, containing 1% DMSO and
using 50 mM Tris, pH 7.0, containing 1% DMSO as a blank. The data
were best fitted using a double exponential equation with decay
times of 3.63 ꢀ 10ꢁ10 and 5.38 ꢀ 10ꢁ9 s, which suggests that one
coumarin group is quenched slightly faster than the other.21,22
The fluorescence emission intensity is a function of the concentra-
tion of CA, and fluorescence emission intensity was linear using CA
In this Letter, we successfully designed and synthesized a novel
fluorescent cephalosporin, which was characterized by 1H NMR,
UV–vis and fluorescence spectra and confirmed by MS. Before
hydrolysis, CA exhibits low fluorescence emission most likely due
concentrations from 20 to 100 lM (Fig. 2).

1678
Y.-L. Zhang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1676–1679
Table 1
Steady state kinetic constants of CA hydrolysis by metallo-b-lactamases using UV–vis
and fluorescence spectrum methodsa
MbLs
UV–vis
Fluorescence
M) )
kcat (sꢁ1
Km
(lM)
kcat (sꢁ1
)
Km
(l
L1
CcrA
13
18
9
8
5
12
1
1
18
11
2
1
5
17
1
4
a
Steady-state kinetic studies were conducted in 50 mM Tris, pH 7.0, and at 25 °C.
The concentrations of L1 and CcrA were 0.33 and 0.25 M, respectively. The kinetic
l
constants were determined on at least three different preparations of the enzymes.
Scheme 2. Proposed mechanism of CA cleavage by b-lactamases.
to mutual quenching of the coumarin groups. However upon
hydrolysis, there is an increase in fluorescence due most likely to
the loss of a substituent containing one of the coumarin groups
(Scheme 2). CA can be used in UV–vis and in fluorescence assays
for steady-state kinetic and inhibition studies; therefore, it would
be amenable for high-throughput experiments, even in assays with
an inhibitor that absorbs strongly in the UV–vis region.
Figure 1. UV–vis spectra (a) and fluorescence spectra (b) of 50 lM CA and
coumarin-COOH in methanol.
Acknowledgments
This work was supported (to K.W.Y.) by National Science Fund
of China (20972127, 21272186), Doctoral Fund of China
(200806970005), National Science Fund of Shaanxi Province
(2009JM2002), Key Fund for International Cooperation of Shaanxi
Province (2010KW-16) and Shaanxi Provincial Bureau of Foreign
Expert Funds (SLZ2008013, 20106100080) and from the National
Science Foundation (CHE-1151658 to M.W.C.).
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
01.071.
References and notes
Figure 2. Fluorescence emission spectra of 20–100 mM CA. Inset: fluorescence
emission intensity at 413 nm as a function of CA concentration. The buffer used in
these spectra was 50 mM Tris, pH 7.0, containing 1% DMSO and at 25 °C.
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