Synthesis and inhibitory activity of acetamidophosphonic acids against metallo-β-lactamases

Article abstract of DOI:10.1080/10426507.2016.1225741

Metallo-β-lactamases (MβLs) are the target enzymes of antibiotic resistance and the phosphonic drugs make great influence to the development of contemporary medicine. Eleven acetamidophosphonic compounds were prepared and evaluated as inhibitors of the MβLs. Compounds 4, 5, 7, 9, and 10 exhibited specific inhibitory activity against the MβL NDM-1 and CcrA with an IC50 value range of 17 to 354 μM. Analysis of the structure–activity relationship showed that both the acetamido linker and the position of the substituent on the phenyl ring played an important role in the inhibitory abilities of the inhibitors against MβLs.

Full text of DOI:10.1080/10426507.2016.1225741

Phosphorus, Sulfur, and Silicon and the Related Elements
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Synthesis and inhibitory activity of
acetamidophosphonic acids against metallo-β-
lactamases
Yi-Lin Zhang, Yue-Juan Zhang, Wen-Ming Wang & Ke-Wu Yang
To cite this article: Yi-Lin Zhang, Yue-Juan Zhang, Wen-Ming Wang & Ke-Wu Yang
(2016): Synthesis and inhibitory activity of acetamidophosphonic acids against
metallo-β-lactamases, Phosphorus, Sulfur, and Silicon and the Related Elements, DOI:
10.1080/10426507.2016.1225741
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Date: 22 November 2016, At: 23:13

PHOSPHORUS, SULFUR, AND SILICON
http://dx.doi.org/./..
SHORT COMMUNICATION
Synthesis and inhibitory activity of acetamidophosphonic acids against
metallo-β-lactamases
Yi-Lin Zhanga,b, Yue-Juan Zhanga, Wen-Ming Wanga, and Ke-Wu Yanga
aKey Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Chemical Biology Lab, College of Chemistry and
Materials Science, Northwest University, Xi’an, P. R. China; bCollege of Biology Pharmacy and Food Engineering, Shangluo University, Shangluo, P. R.
China
ABSTRACT
ARTICLE HISTORY
Received  June 
Accepted  August 
Metallo-β-lactamases (MβLs) are the target enzymes of antibiotic resistance and the phosphonic drugs
make great influence to the development of contemporary medicine. Eleven acetamidophosphonic com-
pounds were prepared and evaluated as inhibitors of the MβLs. Compounds 4, 5, 7, 9, and 10 exhibited
specific inhibitory activity against the MβL NDM-1 and CcrA with an IC50 value range of 17 to 354 μM. Anal-
ysis of the structure–activity relationship showed that both the acetamido linker and the position of the
substituent on the phenyl ring played an important role in the inhibitory abilities of the inhibitors against
MβLs.
KEYWORDS
Metallo-β-lactamase;
acetamidophosphonic acid;
inhibitor; synthesis
GRAPHICAL ABSTRACT
in their active site. However, the class B enzymes, also called
metallo-β-lactamases (MβLs), require one or two Zn ions in
the active sites to exhibit their activity. MβLs are further divided
into three subclasses: B1, B2, and B3.9–10 New Delhi metallo-β-
lactamase-1 (NDM-1), a B1 subclass enzyme and discovered in
2008, has become a formidable threat to human health, prompt-
ing the World Health Organization to issue a global warning.11
MβLs inactivate almost all β-lactam antibiotics, but there is no
inhibitor of MβLs available for clinical purpose to date.9
We are interested in MβLs inhibitor templates that can be
developed into broad-spectrum inhibitors of the MβLs. The
phosphorus compounds are important substrates in the study
of biochemical processes, and tetracoordinate pentavalent phos-
phorus compounds are widely used as biologically active com-
pounds.12,13 By nature, the phosphonic group [R-PO(OH)2] is
Introduction
β-lactam antibiotics, as one of the three largest antibiotic classes
(the others being the macrolides and fluoroquinolones), have
been widely used in clinical applications for a long time. There
are no words to describe the important role that these antibiotics
play in human health.1,2 However, during the past 70 years, β-
lactam antibiotic resistance of gram-negative bacteria caused by
the overuse of these drugs has caused worldwide attention. The
resistance threatens the lives of hundreds of thousands of people
every year.2–4
β-lactamases are the primary cause of β-lactam antibiotic
resistance because they catalyze the hydrolysis of β-lactam ring
of the drugs.5 β-lactamases are subdivided into four classes:
A, B, C, and D.6–8 The classes A, C, and D enzymes are also
called serine β-lactamases (SβLs) for an essential serine residue
CONTACT Ke-Wu Yang
kwyang@nwu.edu.cn
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Chemical Biology
Lab, College of Chemistry and Materials Science, Northwest University, Xi’an , P. R. China.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/gpss.
©  Taylor & Francis Group, LLC

2
Y. -L. ZHANG ET AL.
Scheme . Synthetic route to acetamidophosphonic acids.
introduced into a vast number of structural diverse molecules,
which are involved in numerous biological functions. Several
clinical drugs also comprise a phosphonic acid with promi-
nent examples being anticancer, antibacterial, and anti-HIV
agents.14–16 In this study, the acetamidophosphonic acids were
synthesized and evaluated as potential inhibitors of MβLs.
Results and discussion
Eleven acetamidophosphonic acids were synthesized by a syn-
thetic route shown in Scheme 1 and characterized by NMR and
MS. The compounds 9, 10, and 11 have not been reported. The
acetamidophosphonic acids were prepared by the Arbuzov reac-
tion, and the phosphonic acids were obtained by conversion of
the phosphate esters in the presence of bromotrimethylsilane.
The structures of the synthesized phosphonic acids are shown
in Figure 1.
In vitro, the synthesized acetamidophosphonic acids were
tested as inhibitors of MβL NDM-1 (subclass B1b), CcrA (sub-
class B1a), ImiS (subclass B2), and L1 (subclass B3) by employ-
ing the spectrometric method as described by Bush et al. using
cefazolin (imipenem for ImiS) as the substrate.17 The deter-
mined IC50 values of the phosphonic acids against MβLs are
listed in Table 1. The results reveal most of these compounds
to be potential inhibitors of the B1 subclass MβL NDM-1 and
CcrA.
Figure . Structures of acetamidophosphonic acids.
Table . IC values of acetamidophosphonic acids against MβLs (μM).
Compounds
NDM-∗
CcrA∗
1
—

—
—
—

2
3
4
5
6
7
8
9
10
11



—

—




—

—


—
Compounds 2, 4, 5, 7, 9, 10, and 11 showed a certain
inhibitory activity on NDM-1 with an IC50 value range from 17
to 354 μM. Compounds 3, 4, 5, 7, 9, and 10 can inhibit CcrA
with an IC50 value range from 30 to 140 μM. We can see com-
pounds 4, 5, 7, 9, and 10 showed inhibitory activity to defense
NDM-1 and CcrA. Among these phosphonic acids, 2 and 9
∗The antibiotic used was cefazolin;“-”: no inhibitory activity.

PHOSPHORUS, SULFUR, AND SILICON
3
exhibited high inhibitory activities against NDM-1 with the IC50 General procedure for the preparation of phosphate esters
value of 30 and 17 μM, and 4 showed a slightly higher inhibitory
Under the protection of N2, triethyl phosphate (0.012 mol) was
activity for CcrA with IC50 value of 30 μM.
added to the corresponding chloride (0.01 mol) at room tem-
The acetamidophosphonic acids 2, 4, 7, and 9 showed an out-
perature; the resulting mixture was refluxed at 120°C for 12 h,
standing inhibitory activity on NDM-1, with 2 and 9 being even
during which time the reaction was monitored by TLC (Rf =
more active than 4 and 7. This is probably due to the structural
0.5, AcOEt/petroleumether 2:1). The cooled mixture was puri-
difference. Both 2 and 9 contain a benzimidazole group, whereas
fied by flash chromatography (CHCl3/AcOEt 4:1) to yield the
4 and 7 exhibit an acetamido linker and halogenophenyl or
phosphonates Q1, Q2, and Q3.
nitrophenyl ring. Moreover, 2 and 11 do not inhibit CcrA
like 1, 6, and 8 although 2 has a benzimidazole group but no
acetamido linker, and 11 has an acetamido linker and, in addi-
General procedure for the preparation of phosphate acids
tion, a sulfydrylphenyl ring. Furthermore, compounds 3, 4,
and 7 showed better inhibitory activity against CcrA, with 4
exhibiting the best inhibitory activity. All three compounds con-
tain an acetamido linker without a heterocyclic group.
The acetamidophosphonic acids 1, 6, and 8 do not exhibit
inhibitory activity against all tested MβLs, in which 1 contains
benzyl without an acetamido linker or heterocyclic group, while
6 and 8 contain an acetamido linker and halogeno or nitro sub-
stituent. Compound 3 has a similar structure as 6 and 8 but with-
out a substituent at the phenyl ring and showed inhibitory activ-
ity on CcrA.
In conclusion, the aromatic phosphonic acids with acetamido
and heterocyclic substituents exhibit inhibitory activity against
NDM-1 with the benzimidazole derivatives showing the best
inhibitory activity. Regarding CcrA, the acetamido linker is
essential for the inhibitory activities of the compounds, and a
substituent in the ortho- or meta-position of the phenyl ring
improves the inhibitory activity of the compounds compared
with the para-substituted ones.
To a stirred solution of the phosphonates Q1, Q2, and Q3
(14.0 mmol) in DCM (30 mL) NaI (0.03 mol) was added within
10 min. Then, Me3SiBr (0.03 mol) was added dropwise to the
reaction mixture and the mixture was heated to 60°C for 5 h.
The solid target products 1–11 are obtained after cooling.
Spectroscopic data of products
Dimethyl benzylphosphonate (Q1): Colorless oil, 93%. 1H NMR
(CDCl3): δ 9.39 (s, 1H), 7.54–7.47 (m, 2H), 7.21 (t, J = 7.6 Hz,
2H), 7.02 (s, 1H), 3.83 (d, J = 11.2 Hz, 6H), 3.13 (d, J = 8.4 Hz,
2H). 31P NMR (CDCl3): δ 25.64.
Ethyl 2-[(dimethoxyphosphoryl)methyl]-1H-benzo[d]imi-
1
dazole-1-carboxylate (Q2): Yellow oil, 79%. H NMR (MHz,
CDCl3): δ 7.79–7.54 (m, 2H), 7.19 (s, 2H), 4.46–4.36 (m, 2H),
3.98–3.85 (m, 2H), 3.76–3.62 (m, 6H), 1.46–1.27 (m, 3H). 13
C
NMR (CDCl3): δ 149.92, 146.97, 141.78, 132.54, 124.49, 124.07,
119.38, 114.65, 64.13, 52.81, 29.18, 27.80, 13.70. 31P NMR
(CDCl3): δ 24.42.
Conclusion
Dimethyl [2-oxo-2-(phenylamino)ethyl]phosphonate (Q3–
3): Yellow oil, 85%.1H NMR (CDCl3): δ 9.39 (s, 1H), 7.54–7.47
(m, 2H), 7.21 (t, J = 7.7 Hz, 2H), 7.02 (t, J = 7.4 Hz, 1H), 3.83 (d,
J = 11.2 Hz, 6H), 3.13 (d, J = 8.4 Hz, 2H). 13C NMR (CDCl3):
Eleven acetamidophosphonic acids have been synthesized, char-
acterized, and tested as inhibitors of MβLs. The results indicated
that partial phosphonic acids displayed specific inhibitory abil-
ity on the B1 subclass of MβLs NDM-1 and CcrA. The analysis
of the structure–activity relationship reveals that the acetamido
linker plays an important role in the inhibitory activity of the
enzymes, and substitution in ortho-position of the phenyl ring
improves the inhibitory activity of the compounds.
δ 162.18, 137.98, 128.78, 124.27, 119.80, 53.40, 36.07, 34.77. 31
NMR (CDCl3): δ 25.61.
P
Dimethyl 2-[(2-chlorophenyl)amino]-2-oxoethylphosphon-
ate (Q3–4): Yellow oil, 88%. 1H NMR (CDCl3): δ 8.93 (s, 1H),
8.23 (dd, J = 8.2, 1.4 Hz, 1H), 7.36 (dd, J = 8.0, 1.4 Hz, 1H),
7.24 (td, J = 8.2, 1.4 Hz, 1H), 7.09–7.03 (m, 1H), 3.83 (d, J =
11.2 Hz, 6H), 3.15 (d, J = 9.0 Hz, 2H). 13C NMR (CDCl3):
δ 162.32, 134.44, 129.19, 127.46, 125.18, 123.67, 122.38, 53.33,
36.11, 34.81. 31P NMR (CDCl3): δ 25.64.
Experimental
Dimethyl {2-[(3-chlorophenyl)amino]-2-oxoethyl}phosph-
Materials and methods
onate (Q3–5): Yellow oil, 83%. 1H NMR (CDCl3): δ 9.80 (s, 1H),
Chemicals were purchased from Aladin and were used without 7.60 (t, J = 2.0 Hz, 1H), 7.32–7.25 (m, 1H), 7.06 (t, J = 8.1 Hz,
further purification. The reaction progress was monitored by 1H), 6.98–6.92 (m, 1H), 3.86 (d, J = 11.2 Hz, 6H), 3.14 (d, J =
thin-layer chromatography (TLC) using Merck silica gel 60 alu- 21.8 Hz, 2H).31P NMR (CDCl3): δ 25.57.
minum sheets (F254). NMR spectra were recorded with a Bruker
Dimethyl {2-[(4-chlorophenyl)amino]-2-oxoethyl}phosph-
1
DRX 400 MHz spectrometer. H (400 MHz), 13C (101 MHz), onate (Q3–6): Yellow oil, 90%. 1H NMR (CDCl3): δ 9.67 (s, 1H),
and 31P NMR (162 MHz) spectra were recorded with TMS as 7.38 (d, J = 8.8 Hz, 2H), 7.10 (d, J = 8.8 Hz, 2H), 3.85 (d, J =
internal standard. The peak patterns are indicated as follows: s, 11.2 Hz, 6H), 3.13 (d, J = 8.6 Hz, 2H). 13C NMR (CDCl3): δ
singlet; d, doublet; t, triplet; sept, septuplet; m, multiplet. HRMS 162.29, 136.79, 129.14, 128.74, 120.79, 53.58, 36.21, 34.91. 31P
spectra were recorded on a BrukerMicrOTOF-Q II mass spec- NMR (CDCl3): δ 25.54.
trometer. Inhibition studies were performed on an Agilent-8453
UV-visible spectrometer. All antibiotics used were purchased nate (Q3–7): Yellow oil, 86%. 1H NMR (CDCl3): δ 10.14 (s, 1H),
from Sigma-Aldrich.
8.25 (d, J = 2.0 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.76–7.70 (m,
Dimethyl {2-[(3-nitrophenyl)amino]-2-oxoethyl}phospho-

4
Y. -L. ZHANG ET AL.
1H), 7.24 (s, 1H), 3.93 (d, J = 11.2 Hz, 6H), 3.22–3.13 (m, 2H). 1H), 7.64 (d, J = 8.7 Hz, 2H), 7.34–7.29 (m, 2H), 2.82 (dd, J =
31P NMR (CDCl3): δ 25.30.
8.7, 12.2 Hz, 2H). 13C NMR (DMSO-d6): δ 166.97, 138.22,
Dimethyl {2-[(4-nitrophenyl)amino]-2-oxoethyl}phospho- 128.50, 126.69, 120.78, 51.60. 31P NMR (DMSO-d6): δ 18.15.
nate (Q3–8): Yellow oil, 86%. 1H NMR (CDCl3): δ 9.92 (s, 1H), HRMS [M-H]−m/z for C7H9ClNO4P: calcd. 247.9858, obsd.
8.04 (d, J = 9.1 Hz, 2H), 7.61 (d, J = 9.1 Hz, 2H), 3.89 (d, J = 247.9912.
11.3 Hz, 6H), 3.15 (d, J = 8.4 Hz, 2H). 13C NMR (CDCl3):
[N-(3-Nitrophenyl)carbamoyl]methylphosphonic acid (7):
δ 162.52, 148.16, 139.23, 129.41, 124.61, 118.34, 113.94, 53.68, White solid, yield 81%.1H NMR (CD3OD-d4): δ 8.74 (s, 1H),
36.34, 35.05. 31P NMR (CDCl3): δ 25.09.
7.95 (dd, J = 13.4, 7.9 Hz, 2H), 7.59 (t, J = 8.2 Hz, 1H), 3.02–
Dimethyl {2-[(1H-benzo[d]imidazol-2-yl)amino]-2-oxoet- 2.91 (m, 2H). 13C NMR (CD3OD-d4): δ 175.36, 157.53, 150.51,
hyl}phosphonate (Q3–9): Yellow oil, 77%. 1H NMR (CDCl3): δ 139.81, 134.72, 127.38, 122.74, 47.97. 31P NMR (CD3OD-d4):
7.33 (s, 2H), 7.31–7.28 (m, 2H), 7.26 (dd, J = 4.4, 2.4 Hz, 1H), δ 15.80. HRMS [M-H]−m/z for C8H9N2O6P: calcd. 259.0098,
3.64 (s, 6H), 3.21–3.14 (m, 2H). 31P NMR (CDCl3): δ 23.26.
obsd. 259.0165.
Dimethyl [2-(benzo[d]thiazol-2-ylamino)-2-oxoethyl]pho-
[N-(4-Nitrophenyl)carbamoyl]methylphosphonic acid (8):
sphonate (Q3–10): yellow oil, 83%. H NMR (CDCl3): δ 7.68 White solid, yield 80%.1H NMR (CD3OD-d4): δ 8.35–8.25 (m,
(d, J = 7.8 Hz, 1H), 7.49 (t, J = 7.8 Hz, 1H), 7.39–7.31 (m, 2H), 2H), 7.93 (dd, J = 9.1, 2.6 Hz, 2H), 4.93 (s, 2H), 3.20–3.04 (m,
3.84 (d, J = 11.2 Hz, 6H), 3.72–3.69 (m, 3H), 3.31 (d, J = 21.6 Hz, 2H).31P NMR (CD3OD-d4): δ 18.22. HRMS [M-H]−m/z for
1
2H). 31P NMR (CDCl3): δ 26.04.
C8H9N2O6P: calcd. 259.0098, obsd. 259.0169.
Dimethyl {2-[(4-mercaptophenyl)amino]-2-oxoethyl}pho-
[N-Benzimidazolylcarbamoyl]methylphosphonic acid (9):
sphonate (Q3–11): yellow oil, 81%. 1H NMR (CDCl3): δ 9.60 (s, White solid, yield 65%.1H NMR (DMSO-d6): δ 7.96 (dd, J = 6.2,
1H), 7.46 (d, J = 8.7 Hz, 2H), 7.15 (d, J = 8.7 Hz, 2H), 3.91 (d, J = 3.1 Hz, 2H), 7.67 (dd, J = 6.2, 3.1 Hz, 2H), 3.89 (s, 1H), 3.11 (d,
11.2 Hz, 6H), 3.20 (d, J = 8.6 Hz, 2H). 13C NMR (CDCl3): δ J = 6.3 Hz, 1H). 31P NMR (DMSO-d6): δ 15.30. HRMS [M-H]−
162.03, 135.67, 133.30, 127.57, 120.18, 53.42, 36.05, 34.76, 16.55. (m/z) for C9H10N3O4P: calcd. 255.0414, obsd. 255.2366.
31P NMR (CDCl3): δ 25.73.
[N-Benzothiazolylphenylcarbamoyl]methylphosphonic
Benzylmethylphosphonic acid (1): White solid, yield 92%. acid (10): White solid, yield 69%. 1H NMR (DMSO-d6): δ 7.98
1H NMR (DMSO-d6): δ 8.13 (s, 2H), 7.30–7.23 (m, 4H), 7.20 (d, J = 7.9 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.1 Hz,
(dd, J = 5.6, 2.8 Hz, 1H), 2.97 (d, J = 6.0 Hz, 2H). 13C 1H), 7.32 (d, J = 7.3 Hz, 1H), 3.72 (s, 2H), 3.15 (d, J = 8.5 Hz,
NMR (DMSO-d6): δ 134.19, 134.10, 129.94, 129.88, 128.20, 2H). 31P NMR (DMSO-d6): δ 19.04.
128.17, 126.15, 126.12, 35.99, 34.67. 31P NMR (DMSO-d6): δ
[N-(4-Sulfydrylphenyl)carbamoyl]methylphosphonic acid
22.10. HRMS [M-H]−m/z for C7H9O3P: calcd. 171.0189, obsd. (11): White solid, yield 55%.1H NMR (DMSO-d6): δ 10.21 (s,
171.0027.
1H), 7.63–7.56 (m, 2H), 7.42 (d, J = 8.5 Hz, 1H), 7.28 (d, J =
Benzimidazolyl-2-methylphosphonic acid (2): White solid, 8.7 Hz, 1H), 3.81 (s, 3H), 2.86 (d, J = 7.8 Hz, 1H), 2.57 (s, 3H).
1
yield 87%. H NMR (CD3OD-d4): δ 8.23–8.20 (m, 1H), 7.85– 31P NMR (DMSO-d6): δ 16.80.
7.82 (m, 1H), 7.68 (t, J = 6.3 Hz, 2H), 4.74 (q, J = 7.1 Hz, 2H),
1.59 (t, J = 7.1 Hz, 3H). 13C NMR (CD3OD-d4): δ148.24, 127.57,
127.28, 126.17, 116.23, 114.51, 113.37, 66.88, 12.90. 31P NMR
Enzyme preparations
(CD3OD-d4): δ13.49.
All MβLs used were purified as described in the references for
[N-(Phenylcarbamoyl)methyl]phosphonicacid (3): White
L1,17 ImiS,18 CcrA,19 and NDM-1.20
1
solid, yield 87%. H NMR (DMSO-d6): δ 9.97 (d, J = 9.1 Hz,
1H), 7.61–7.53 (m, 2H), 7.28 (t, J = 7.9 Hz, 2H), 7.02 (s,
1H), 2.83 (dd, J = 6.3, 3.5 Hz, 2H).13C NMR (DMSO-d6):
δ 160.80, 130.57, 120.15, 115.39, 111.61, 31.52, 30.34. 31P
Over-expression and purification of L
NMR(DMSO-d6):δ17.34. HRMS [M-H]−m/z for C8H10NO4P:
L1 was over-expressed and purified as previously described.
The gene that encodes L1 was ligated into pET26b, and E.
coli DH5R cells were transformed with the resulting plasmid
pET26b(+)L1. Presence of the insert was confirmed with DNA
sequencing. BL21(DE3) E. coli cells were then transformed with
the pET26b(+)L1 plasmid, and the resulting cells were used for
over-expression of protein. L1 was determined quantitatively by
monitoring the absorbance at 280 nm and using an extinction
calcd. 214.0247, obsd. 214.0049.
[N-(2-Chlorophenyl)carbamoyl]methylphosphonic
acid
1
(4): White solid, yield 82%. H NMR (DMSO-d6): δ 9.57 (s,
1H), 8.01 (d, J = 7.1 Hz, 1H), 7.49 (dd, J = 8.0, 1.4 Hz, 1H),
7.35–7.29 (m, 1H), 7.14 (td, J = 7.8, 1.5 Hz, 1H), 2.95–2.93
(m, 2H). 13C NMR (DMSO-d6): δ 164.85, 135.13, 129.46,
127.56, 125.46, 124.20, 123.70, 37.62. 31P NMR (DMSO-d6): δ
17.35.HRMS [M-H]−m/z for C7H9ClNO4P: calcd. 247.9858,
obsd. 247.9904.
coefficient of 54,614 M−1 cm−1
.
[N-(3-Chlorophenyl)carbamoyl]methylphosphonic
acid
(5): White solid, yield 71%. 1H NMR (DMSO-d6): δ 9.81
(s, 1H), 7.76 (d, J = 8.7 Hz, 2H), 7.40 (s, 2H), 2.89 (d, J =
6.8 Hz, 2H). 13C NMR (DMSO-d6): δ 166.97, 138.22, 128.50,
126.69, 120.78, 51.60. 31P NMR (DMSO-d6): δ 18.15. HRMS
[M-H]−m/z for C7H9ClNO4P: calcd. 247.9858, obsd. 247.9911.
Over-expression and purification of ImiS
ImiS was over-expressed and purified as previously described.
BL21(DE3) E. coli cells were transformed with a plasmid con-
taining the gene for ImiS, pET-26b-ImiS. A culture of these cells
in LB medium was used for over-expression of protein. The
concentration of ImiS was determined using Beer’s law and an
extinction coefficient of 37,250 M−1 cm−1 at 280 nm.
[N-(4-Chlorophenyl)carbamoyl]methylphosphonic
acid
(6): White solid, yield 82%.1HNMR (DMSO-d6): δ 10.47 (s,

PHOSPHORUS, SULFUR, AND SILICON
5
Over-expression and purification of CcrA
References
CcrA was over-expressed and purified as previously described.
BL21(DE3) E. coli cells were transformed with plasmid
pMSZ02-CcrA and a culture of these cells in LB medium was
used for protein over-expression. Protein concentration was
determined using Beer’s law and an extinction coefficient of
39,000 M−1 cm−1 at 280 nm.
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Over-expression and purification of NDM-
The over-expression plasmid, pET26b-NDM-1, was used for
expression of NDM-1 as previously described. The over-
expression plasmid, pET26b-NDM-1, was used to transform
BL21(DE3) E. coli cells. The crude protein NDM-1 was fur-
ther purified by running through a G75 column. Protein purity
was ascertained by sodium dodecyl sulfate (SDS) polyacry-
lamide gel electrophoresis. The protein concentration was deter-
mined using Beer’s law and an extinction coefficient of 27,960
M−1 cm−1 at 280 nm.
Determination of IC50 values
The inhibition studies were conducted at 25°C using imipenem
as substrate of ImiS and cefazolin V as substrate of CcrA,
NDM-1, and L1.21 Compounds were dissolved in a small
volume of DMSO and diluted with 50 mM Tris, pH 7.0.
The final DMSO concentration in the reaction mixture was
less than 0.5%, which did not alter the enzyme activ-
ity. The substrate concentrations were varied between 20
and 140 μM, inhibitor concentrations were varied between
0 and 10 μM, and the enzyme concentrations were var-
ied between 3 and 60 nM. The enzyme and inhibitor
were preincubated for 30 min before starting the kinetic
experiments.
Funding
20. Thomas, P. W.; Zheng, M.; Wu, S. S.; Guo, H.; Liu, D.; Xu, D. G.; Fast,
W. Biochemistry. 2011, 50, 10102-10113.
21. Bush, K.; Macalintal, C.; Rasmussen, B. A.; Lee, V. J.; Yang, Y. Antimi-
crob. Agents Chemother. 1993, 37, 851-858.
This work was financially supported by grants 81361138018, 21272186, and
21572179 (to K.W.Y.) from the National Natural Science Foundation of
China.