A minor structure modification serendipitously leads to a highly carbapenemase-specific fluorogenic probe

Article abstract of DOI:10.1039/d0ob00114g

Reported herein is a fluorogenic probe for the detection of carbapenemase activity. This reagent features carbapenem as an enzyme recognition motif and a carbon-carbon double bond between carbapenem and the fluorophore, exhibiting high specificity to all carbapenemases, including metallo carbapenemases and serine carbapenemases, over other β-lactamases.

Full text of DOI:10.1039/d0ob00114g

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PAPER
I . J. Dmochowski et al.
Oligonucleotide modifi cations enhance probe stability for
single cell transcriptome in vivo analysis (TIVA)
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Minor structure modification serendipitously leads to a highly
carbapenemase-specific fluorogenic probe
Received 00th January 20xx,
‡
‡
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Jie Wang, Weipan Xu, Shuyuan Xue, Tao Yu and Hexin Xie*
Reported herein is a fluorogenic probe for the detection of lactamases, a large number of -lactamases fluorescent
carbapenemase activity. This reagent features with carbapenem probes have been developed for the detection of resistant
7
as enzyme recognition motif and a carbon-carbon double bond bacteria. In spite of these, fluorescent reagents with high
between carbapenem and fluorophore, exhibiting high specificity specificity to carbapenemase were less explored. Rao and
to all carbapenemases, including metallo carbapenemases and colleagues employed trans-substituted cephalosporin as
serine carbapenemases, over other -lactamases.
carbapenemase recognition moiety and developed a number
of carbapenemase-selective fluorogenic probes. With the core
structure of carbapenem as enzyme recognition motif and
alkenyl-linked boron dipyrromethene (BODIPY) dye as
8
Bacterial resistance to -lactam antibiotics, the most
widely used antibiotics in clinical practice over the past several
decades, has posed a severe threat to public health globally.
1
activatable fluorophore, we have reported
a
highly
The major mechanism for bacteria to survive in the presence
of -lactam antibiotics involves the production of a class of
highly efficient -lactam-degrading enzymes, named as -
carbapenemase-specific reagent for the rapid detection of
9
CPOs. Moreover, we recently integrated carbapenem and
10
umbelliferone to develop a fluorogenic probe CPC-1.
2
lactamases (blas), in bacteria. These enzymes, on the basis of
However, to our surprise, this carbapenem-based reagent is
selective to metallo carbapenemases, yet barely responses to
serine carbapenemases. Following our continuing interest on
Ambler classification, are divided into four classes, class A,
3
class B, class C, and class D -lactamases. Class A, C, and D -
lactamases are known as serine--lactamases (SBLs) while
class B -lactamases are metallo--lactamases (MBLs).
Carbapenems (e.g., Imipenem, Meropenem, Doripenem,
and Ertapenem), with the unusual trans-substituents on the -
lactam rings, were relatively resistant to the hydrolysis by most
7h, 7j, 9-11

-lactamase fluorescent probe,
we herein present a
structurally similar carbapenem-based probe, but with
unexpectedly high specificity to both metallo carbapenemases
and serine carbapenemases.
Inspired by the cephalosporin-based fluorescent probes
CC1,12 which equips with an extra carbon-carbon double bond

-lactamases and thus remained effective to these -
lactamase-expressing bacterial pathogens; carbapenems are
between enzyme recognition motif and fluorophore compared
4
considered as “antibiotics of last resort”. Nevertheless,
carbapenem-hydrolyzing -lactamases (carbapenemases),
which include all MBLs and a number of SBLs, for instance,
Klebsiella pneumoniae carbapenemase (KPC), have been
a) Rao's works
Bn
H
N
S
O
O
O
most
-lactamases
O
5
N
identified in pathogenic bacteria. The increasing occurrence
O
n
n = 0, CDC-1
of these carbapenemase-producing organisms (CPOs) has
significantly undermined the efficacy of these life-saving
molecules.4
COOH
(
dark) n = 1, CC1
b) Our previous work
OH
H H
metallo carbapenemases
(class B) only
O
O
O
O
Early identification is crucial to prevent rapid spreading of
N
O
6
O
CPOs. Taking advantage of the high catalytic efficiency of -
CPC-1
a
COOH
O
(dark)
strong fluorescent
c) This study
OH
metallo and serine
carbapenemases
O
H H
a. State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of New
Drug Design, School of Pharmacy, East China University of Science and
Technology, Shanghai 200237, China. E-mail: xiehexin@ecust.edu.cn
O
O
N
CPC-2
(dark)
O
COOH
‡
These authors contributed equally.
Electronic Supplementary Information (ESI) available: [details of any Figure 1 Design of fluorogenic probe for the detection of
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
carbapenemase activities.
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the double bonding-bearing CC1 exhibits very similar enzyme
OTBS
H H
a
OTBS
H H
View Article Online
O
DO
I:
10.
10-239
/D
0
O
B00li1
1
4G
kinetics as CDC-1, we expected CPC-1 a
nd
CP
C
w
ou
ld
kely
b
CPC-2
O
have very close enzymatic hydrolysis profile.
N
N
O
O
O
As outlined in Scheme 1, starting from readily available
carbapenem 1, CPC-2 was easily prepared after a cross-
metathesis reaction in presence of second generation of
Hoveyda-Grubbs catalyst, followed by removal of TBS- and
PNB-protecting groups, subsequently. This fluorogenic
molecule was purified by reverse-phase high-performance
liquid chromatography (RP-HPLC) and the structure was
unambiguously confirmed by nuclear magnetic resonance
COOPNB
COOPNB
1
2
Scheme 1 Preparation of CPC-2. a) 7-allyloxycoumarin, Hoveyda-
Grubbs 2nd catalyst, CHCl
, reflux; b) (i) (NH ) HF , DMF/NMP, rt; (ii)
Zn, THF/PB (0.35M, pH 6.0), rt. TBS: tert-butyldimethylsilyl; PNB: p-
3
4
2
nitrobenzyl.
to CDC-1,7c we designed a carbon-carbon double bond-
(NMR) and high-resolution mass spectrometry (HR-MS).
containing carbapenem-based probe CPC-2 in order to simplify
the synthesis of carbapenem-based probe (Fig. 1). As the
double bond is far away from the enzyme recognition site and
With CPC-2 available, we first examined its absorbance
spectra before and after incubation with metallo
a
b
6
3
c
6
3
CPC-2
CPC-2 + VIM-27
CPC-2
CPC-2 + VIM-27
CPC-2 + VIM-27
CPC-2 + TEM-1
0
.6
0
.3
0
.0
2
0
6
0
50
300
350
400
450
400
450
500
550
600
0
10
20
30
Wavelength (nm)
Wavelength (nm)
Time (min)
d 8
e
f
i
10
CPC-2 + VIM-27
CPC-2 + KPC-3
CPC-1 + VIM-27
CPC-1 + KPC-3
w/o inhibitor
PBA
EDTA
4
3
5
0
0
0
0
10
20
30
0
10
Time (min)
20
30
VIM-27
KPC-3
Time (min)
g1
6
h 16
10
1
2
3
4
5
6
7
8
9
: NDM-1
: VIM-27
: CphA
: L1
: KPC-2
: KPC-3
: TEM-1
: AmpC
1: NDM-1
Y = 177507X + 5.77 E7
CPC-2
CPC-1
2: VIM-27
3: CphA
4: L1
5: KPC-2
6: KPC-3
7: TEM-1
8: AmpC
9: CTX-M-9
2
R = 0.999
8
6
8
0
8
0
: CTX-M-9
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
0
50
100
150
200
250
KPC-3 (p)
Figure 2 Detection of β-lactamase activities with CPC-2. UV/Vis spectra (a) and fluorescent spectra (b) of CPC-2 (10 M in PBS,
pH 7.4) before and after incubation with VIM-27 β-lactamase (500 nM) for 3 min. (c-e) Time course fluorescence intensity of
CPC-2 (10 M in PBS, pH 7.4) with indicated β-lactamases (0.5 nM). (f) Fluorescent enhancement of CPC-2 (10 M in PBS, pH 7.4)
after incubation of indicated β-lactamases (0.5 nM) for 30 min in the presence of PBA (2 mM) or EDTA (400 M). (g, h)
Fluorescent enhancement of CPC-2 and CPC-1 (10 M in PBS, pH 7.4) upon incubation with series of β-lactamases (1 nM) for 30
min. (i) Linear correlation between fluorescent intensity of CPC-2 (10 M in PBS, pH 7.4) and concentration of KPC-3 β-
lactamase. ex/em =365/460 nm; error bars represent standard deviation of three experiments.
2
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carbapenemase VIM-27.13 As expected, these spectra are
similar to that of CPC-1. In addition, the fluorescence spectra
of CPC-2 were also recorded with or without incubation with
same enzyme and, again, these show high similarity with those
of CPC-1: both probes alone are barely fluorescent and turn
strong fluorescent after treatment with VIM-27.
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DOI: 10.1039/D0OB00114G
4
2
0
Having demonstrated the high fluorescent response of
CPC-2 to metallo carbapenemase, we thus turned our
attention to the selectivity of this reagent to different -
lactamases. TEM-1 (a narrow-spectrum class A -lactamase
1
4
commonly found in Gram-negative bacteria) and VIM-27
were first selected in this study. As depicted in Fig. 2c, VIM-27
1
2
3
4
5
6
elicited massive enhancement in fluorescence intensity while Figure 3 Enhanced fluorescence intensity of CPC-2 (15 M in PBS,
TEM-1 hardly turned on the fluorescence of CPC-2, indicating pH 7.4) upon incubation at room temperature for 60 min with β-
the carbapenem-based CPC-2 is highly selective to VIM-27 lactamase-negative and clinically important antibiotic-resistant
over TEM-1.
bacteria. 1: bla-negative E. coli (ATCC 25922, 4×10
5
CFU); 2: TEM-1
Moreover, KPC-3,15 a class A serine carbapenemase, was
5
E. coli (ATCC 35218, 4×10 CFU); 3: KPC-2 E. coli (ATCC BAA 2340,
×10 CFU); 4: VIM-1 K. pneumoniae (NCTC 13440, 4×10 CFU); 5:
VIM-1 K. pneumoniae (NCTC 13440, 4×10 CFU) + TEM-1 E. coli
ATCC 35218, 4×10 CFU); 6: VIM-1 K. pneumoniae (NCTC 13440,
×105 CFU) + TEM-1 E. coli (ATCC 35218, 4×105 CFU); ex/em
65/460 nm; error bars represent standard deviation of three
also investigated and, to our surprise, this enzyme is highly
efficient in the hydrolysis of CPC-2, significantly increasing
fluorescence intensity. By contrast, CPC-1 was only activated
by VIM-27; this molecule is obviously a poor substrate of KPC-3
and led to almost no fluorescence enhancement, which is in
agreement with previous observations. We further applied
HPLC and LC-MS to analyze the KPC-3-incubated CPC-2 and it
turned out umbelliferone (a) and hydrolyzed -lactam (b) were
formed as the major products, which are identical with those
from VIM-27-treated CPC-2 (Fig. S1).
Additionally, we performed an inhibition assay to validate
whether the fluorescence enhancement of CPC-2 is attributed
solely to the activity of enzyme. We employed metal ion
chelator ethylenediaminetetraacetic acid (EDTA) as inhibitor
for metallo -lactamase and phenylboronic acid (PBA) as
inhibitor for serine -lactamase (PBA). As illustrated in Fig. 2f,
EDTA selectively suppressed the fluorescence enhancement by
VIM-27, leaving the KPC-3-containing sample unaffected. On
the other hand, PBA inhibited only the fluorescence
enhancement mediated by KPC-3, having little impact on the
activity of VIM-27. These results further demonstrate the
fluorescence enhancement of CPC-2 was mediated only by -
lactamase, which thus allows to monitor the activity of
carbapenemase by simply measuring the increasement in
fluorescence intensity.
5
5
4
5
4
(
4
3
=
1
0a
experiments.
circumstance, CPC-1 was activated only by metallo -
lactamases whereas all of the others serine -lactamases,
including serine carbapenemases (KPC-2 and KPC-3) are much
less efficient to trigger the enhancement in fluorescence
intensity; CPC-1 is a metallo -lactamase-selective fluorogenic
reagent. Given the high structural similarity of CPC-1 and CPC-
2
(
, their totally different responses to serine carbapenemases
KPC-2 and KPC-3) suggest divergent mechanisms may involve
in this serine carbapenemase-mediated hydrolysis process
though exact reason remains unknown at current stage.
In general, carbapenems are less stable compared to
cephalosporin-based antibiotics. To assess the stability of
carbapenem-based CPC-2, we measured its spontaneous
hydrolysis rate in PBS (pH 7.4) at room temperature and it was
-5 -1
estimated to be 4.7 × 10 s (Fig. S2). This result indicates the
introduction of extra carbon-carbon double bond decreases its
aqueous stability. Nevertheless, the effect of the spontaneous
hydrolysis on the detection of carbapenemases might
somehow be compensated by the incubation of CPC-2 alone at
the same time.
To obtain information on the detection sensitivity of CPC-2,
we performed further investigation on the determination of
limit of detection (LOD). We incubated CPC-2 with a range
concentration of carbapenemases (VIM-27, KPC-2, and KPC-3)
in PBS for 30 minutes before measuring their fluorescence
intensity with microplate reader. After plotting fluorescence
intensity versus concentrations of enzymes, the LOD (3N/k; N:
standard deviation of blank sample; k: slope) to VIM-27, KPC-2,
and KPC-3 were calculated to be 18.6, 40.6, and 34.4 pM,
respectively. These data support CPC-2 as a sensitive reagent
for the detection of carbapenemases in spite of its relatively
low aqueous stability.
To further reveal the specificity of CPC-2, we tested more

-lactamases with this imaging reagent, including class A -
1
6
17
lactamases (KPC-2, KPC-3, TEM-1, and CTX-M-9, etc), class
B -lactamases (NDM-1, VIM-27, L1, and CphA, etc), and
1
8
19
20
2
1
class C -lactamases (AmpC ). Among these -lactamases, all
class B -lactamases (or metallo -lactamases) and KPC-2 and
KPC-3 are known as carbapenemases, whereas AmpC (class C)
and CTX-M-9 (class A) belong to extended-spectrum -
lactamase (ESBL). As shown in Fig. 2g, all carbapenemases,
including metallo carbapenemases and serine carbapenemases
(KPC-2 and KPC-3) are capable of activating CPC-2 and lead to
strong fluorescence signal; all of other -lactamases, even
ESBLs (AmpC and CTX-M-9), are ineffective to turn on
fluorescence of CPC-2. In other word, CPC-2 is a highly
carbapenemase-specific fluorogenic sensor. Under identical
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With the detection sensitivity and specificity of CPC-2 to 54, 969.
Journal Name
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DOI: 10.1039/D0OB00114G
4
5
4
. M. Mckenna, Nature, 2013, 499, 394.
carbapenemases validated, we moved to apply this molecule
in the detection of resistant pathogenic bacteria. As proof of
concept, we selected four strains of clinically occurring
pathogenic bacteria for this test, including TEM-1-expressing
Escherichia coli (E. coli, ATCC 35218), KPC-2-expressing E. coli
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(ATCC BAA 2340), and VIM-1-expressing Klebsiella pneumonia
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6
. (a) Y. Matsumura and J. D. Pitout, Expert review of molecular
diagnostics, 2016, 16, 783; (b) W. Mao and H. Xie, Future Microbiol.,
017, 12, 1027.
(
E. coli (ATCC 25922). We incubated CPC-2 with these bacteria
2
respectively for 60 minutes and then measured fluorescence 7. (a) G. Zlokarnik, P. A. Negulescu, T. E. Knapp, L. Mere, N. Burres, L.
intensity with a microplate reader. As depicted in Fig. 3, all of Feng, M. Whitney, K. Roemer and R. Y. Tsien, Science, 1998, 279, 84;
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Thornton, J. C. Sacchettini, J. D. Cirillo and J. Rao, Nat. Chem., 2012,
, 802; (d) J. Zhang, Y. Shen, S. L. May, D. C. Nelson and S. Li, Angew.
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Tang, X. Yan and B. Xing, Chem. Eur. J., 2013, 19, 10903; (f) Y. Cheng,
H. Xie, P. Sule, H. Hassounah, E. A. Graviss, Y. Kong, J. D. Cirillo and J.
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pneumonia) led to significant enhancement in fluorescence
intensity whereas other bacteria, susceptible E. coli or TEM-1-
expressing E. coli, remained barely fluorescent. These results
demonstrate CPC-2 as a potential sensor in the identification
of carbapenemase-producing bacterial pathogens, even
though the detection sensitivity of this reagent is somewhat
limited by its relatively low stability. Moreover, to investigate
the interference of carbapenemase-negative bacteria in the
(
4
test, we incubated CPC-2 with VIM-1-expressing K. pneumonia Xia, Y. Wang and H. Xie, Chem. Asian J., 2016, 11, 3493; (i) J. Aw, F.
5
4
(
4x10 CFU) in the presence of TEM-1-expressing E. coli (4x10
Widjaja, Y. Ding, J. Mu, Y. Liang and B. Xing, Chem. Commun., 2017,
53, 3330; (j) W. Mao, X. Qian, J. Zhang, L. Xia and H. Xie,
Chembiochem, 2017, 18, 1990; (k) Y. Cheng, J. Xie, K.-H. Lee, R. L.
Gaur, A. Song, T. Dai, H. Ren, J. Wu, Z. Sun, N. Banaei, D. Akin and J.
Rao, Sci. Transl. Med., 2018, 10, eaar4470.
5
CFU or 4x10 CFU). It turned out the enhancement of these
samples in fluorescence intensity is comparable with the
sample without E. coli, suggesting the presence of E. coli has
insignificant impact on the test of carbapenemase-producing
bacteria with CPC-2.
8
. (a) H. Shi, Y. Cheng, K. H. Lee, R. F. Luo, N. Banaei and J. Rao,
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N. Banaei and J. Rao, Chem. Sci., 2017, 8, 7669.
In summary, with
a carbon-carbon double bond-
conjugated carbapenem as enzymatic recognition motif, we
have developed a fluorogenic reagent to visualize the activity
of carbapenemases. This probe can be selectively activated by
9
1
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. W. Mao, L. Xia and H. Xie, Angew. Chem. Int. Ed., 2017, 56, 4468.
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and H. Xie, Chem. Commun., 2019, 55, 9919.
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even extended-spectrum -lactamases. Further test of this
reagent with carbapenemase-producing bacteria has
demonstrated this compound as a promising probe in the
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Acknowledgements
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Organic & Biomolecular Chemistry
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DOI: 10.1039/D0OB00114G
A carbapenem-based fluorogenic reagent has been
developed to detect the activity of carbapenemases,
exhibiting high specificity to all carbapenemases, including
both metallo carbapenemases and serine carbapenemases,
over other -lactamases.
OH
H H
both metallo and serine
carbapenemases
O
O
O
O
O
N
O
O
CPC-2
COOH (dark)
(strong fluorescent)