New β-phospholactam as a carbapenem transition state analog: Synthesis of a broad-spectrum inhibitor of metallo-β-lactamases

Article abstract of DOI:10.1016/j.bmcl.2013.08.098

In an effort to test whether a transition state analog is an inhibitor of the metallo-β-lactamases, a phospholactam analog of carbapenem has been synthesized and characterized. The phospholactam 1 proved to be a weak, time-dependent inhibitor of IMP-1 (70%), CcrA (70%), L1 (70%), NDM-1 (53%), and Bla2 (94%) at an inhibitor concentration of 100 μM. The phospholactam 1 activated ImiS and BcII at the same concentration. Docking studies were used to explain binding and to offer suggestions for modifications to the phospholactam scaffold to improve binding affinities.

Full text of DOI:10.1016/j.bmcl.2013.08.098

Accepted Manuscript
New β-phospholactam as a carbapenem transition state analog: synthesis of a
broad-spectrum inhibitor of metallo-β-lactamases
Ke-Wu Yang, Lei Feng, Shao-Kang Yang, Mahesh Aitha, Alecander E.
LaCuran, Peter Oelschlaeger, Michael W. Crowder
PII:
S0960-894X(13)01047-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.08.098
BMCL 20836
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
19 July 2013
21 August 2013
26 August 2013
Please cite this article as: Yang, K-W., Feng, L., Yang, S-K., Aitha, M., LaCuran, A.E., Oelschlaeger, P., Crowder,
M.W., New β-phospholactam as a carbapenem transition state analog: synthesis of a broad-spectrum inhibitor of
metallo-β-lactamases, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.
2013.08.098
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New β-phospholactam as a carbapenem transition state analog: synthesis of
a broad-spectrum inhibitor of metallo-β-lactamases
Ke-Wu Yanga,*, Lei Fenga, Shao-Kang Yanga, Mahesh Aithab, Alecander E. LaCuranc, Peter
Oelschlaegerc, and Michael W. Crowderb,*
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,
b
P. R. China; Department of Chemistry and Biochemistry, Miami University, 160 Hughes
c
Hall, Oxford, OH 45056, USA; Department of Pharmaceutical Sciences, College of
Pharmacy, Western University of Health Sciences, 309 E. Second St., Pomona, CA 91766,
USA.
Corresponding authors: Tel/Fax: +86-29-8830-2429 (K.-W.Y.); +1-513-529-7274 (M.W.C.).
E-mail address: kwyang@nwu.edu.cn (K.-W. Yang); crowdemw@MiamiOH.edu (M.W.
Crowder)

Abstract
In an effort to test whether a transition state analog is an inhibitor of the
metallo-β-lactamases, a phospholactam analog of carbapenem has been synthesized and
characterized. The phospholactam 1 proved to be a weak, time-dependent inhibitor of IMP-1
(70%), CcrA (70%), L1 (70%), NDM-1 (53%), and Bla2 (94%) at an inhibitor concentration
of 100 μM. The phospholactam 1 activated ImiS and BcII at the same concentration. Docking
studies were used to explain binding and to offer suggestions for modifications to the
phospholactam scaffold to improve binding affinities.

Since the antibiotic properties of penicillin were first discovered in the beginning of
the last century, antibiotics have been well developed as miracle drugs in the treatment of
2
bacterial infections in clinics.1, However, overuse of antibiotics has resulted in a large
number of bacteria that produce β-lactamases, and the resulting bacteria are resistant to many
commonly-used β-lactam antibiotics.3-5 β-Lactamases catalyze the hydrolysis of the β-lactam
ring of these antibiotics. They are divided into classes A, B, C, and D.6 The B class enzymes,
called metallo-β-lactamases (MβLs), are Zn(II)-dependent and hydrolyze nearly all known
β-lactam-containing antibiotics, including penicillins, cephalosporins and carbapenems
(Figure 1). There are no known clinical inhibitors of the MβLs.4 The MβLs are further
divided into B1, B2, and B3 subclasses.6 Both B1 and B3 subclasses have a broad-spectrum
substrate profile including penicillins, cephalosphorins and carbapenems. In contrast, the B2
subclass enzymes primarily hydrolyze carbapenems.
Given the enormous biomedical importance of MβLs, there has been a large amount of
effort in identifying novel inhibitors of these enzymes.4, 7 Succinic acid derivatives were
shown to be inhibitors of IMP-1 with IC50 values in the nanomolar range.8 Lienard et al.
reported that D-captopril inhibits L1 and CcrA,9 and thiomandelic acid was tested as an
inhibitor for nine different MβLs.10 Picolinic acid derivatives were shown to inhibit CphA
with Ki values in the micromolar range,11 while hydroxamic acid derivatives inhibit Fez-1 but
not L1, IMP-1, BcII, or CphA.12 A mechanism-based inhibitor of IMP-1 was reported that
exhibited irreversible inhibition.13 Penicillin-based inhibitors have been shown to be
micromolar inhibitors of L1, BcII, and Bla2,14 and some N-arylsulfonyl hydrazones are

inhibitors of IMP-1.15 A series of pyrrole-based inhibitors of IMP-1 have recently been
reported.16 However, most of the inhibition reports have involved studies on one or two of
the MβLs, and to the best of our knowledge, only three classes of inhibitors, thiomandelic
17-19
acid,10 thiols,9,
and mercaptophosphonate compounds,20 have been reported to be
broad-spectrum inhibitors of the MβLs. Our goal is to develop broad-spectrum, transition
state analog inhibitors of MβLs and to use these inhibitors as drug/inhibitor combinations to
combat bacterial infections in which the bacteria produce a MβL.
Previous mechanistic studies have suggested a ring-opened intermediate, whose
breakdown is rate-limiting, for L1, CcrA, and NDM-1 when using nitrocefin or chromacef as
substrate.21-23 When using other MβLs or substrates, nucleophilic attack or β-lactam ring
cleavage appears to be rate-limiting.24-26 Previous studies on peptidases, many of which
exhibit rate-limiting bond cleavage, suggested that a tetrahedral transition state forms during
the reaction.27-29 The fact that several phosphinate, phosphonate, and phosphoramidate peptide
analogs proved to be very tight binding inhibitors (one with a reported Ki of 10-15 M) strongly
supported the existence of the tetrahedral transition state in these peptidases.30-37 Since
β-lactam-containing antibiotics are peptide mimics and since β-lactamases catalyze peptide
bond cleavage, we hypothesize that a tetrahedral transition state may form during the reaction
(Figure 2) and that a chemically-stable β-phospholactam may be a very tight binding
inhibitor of the MβLs.
Toward this goal, a β-phospholactam analog of a carbapenem transition state (Figure
2) was synthesized by using a 12-step protocol, and the resulting compound 1 was
characterized by NMR and MS. The inhibitory activities of the β-phospholactam 1 were

evaluated using MβLs from the three subclasses B1 (IMP-1, CcrA, Bla2, NDM-1), B2 (ImiS),
and B3 (L1). In the absence of experimental structures of MβL-ligand complexes, docking
studies can provide insights into possible binding modes of inhibitors38 and β-lactam
substrates.39, 40 These studies have shown that substituents with high electron density, such as,
thiols, carboxylates, and carbonyl groups interact electrostatically with the zinc ions and the
positively-charged Lys224 conserved in many B1 and B2 ΜβLs.41, 42 Here we assessed the
binding mode of β-phospholactam 1 to the different MβLs using docking. Previously,
non-cyclic phosphinates43 and monocyclic β-phospholactams44 were tested as inhibitors of
MβLs; however, none of these compounds inhibited the tested enzymes, most likely due to
the fact that these compounds did not have the correct structure to be recognized by the MβLs.
Page and coworkers reported a number of studies using β-sultams and β-phospholactams as
inhibitors of a serine β-lactamase, and these studies, along with a theoretical study,45 explored
the stability of these compounds at different pH’s.44, 46-48 None of these compounds tested
were bicyclic. Rees and coworkers reported the synthesis of a 1,2-azaphosphetidine, which is
a bicyclic β-phospholactam;49 however, the compound was not tested as an inhibitor of any of
the MβLs. Our efforts to synthesize this compound were unsuccessful using the published
procedure; therefore, we developed a novel synthetic approach to obtain a bicyclic
β-phospholactam 1 (Scheme 1). Dimethyl 2,5-dibromoadipate 3 as white solid was prepared
by acylation, α-bromination, and esterification of adipic acid, using previously reported
methods.50 Adipate 3 was reacted with benzylamine in alkaline medium, and the resulting
product was treated by acidification and alkalization to afford pure pyrrole dicarboxylate 4.
Compound 4 has a highly symmetric structure, which required harsh reduction conditions to

obtain the pyrrole monocarboxylate 5. After attempting various reaction conditions, sodium
borohydride was identified as the optimum reductant, and ethanol was identified as the
optimum solvent. Alcohol 5 was converted to aldehyde 6 by Swern oxidation, and this
product was used in the next step without further purification. Intermediate 6 was reacted
with dimethyl phosphate in the presence of catalyst 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) at 0 °C to afford dimethyl phosphonate 7. Dimethyl phosphonate was converted to
monomethyl phosphonate 8 by treatment of 7 with NaI in acetone. The α-hydroxy was
protected by treatment of 8 in pyridine with acetic anhydride in the presence of
4-dimethylaminopyridine (DMAP) to afford 9. The removal of the benzyl protecting group
was very challenging. A series of bases, including triethylamine, diisopropylethylamine,
potassium acetate, sodium hydroxide, and NaH, and solvents were tested in an effort to
remove the benzyl group. Fortunately, intermediate 9 was hydrogenated with 10% Pd/C in
the presence of HCO2NH4 to afford 10. The ring-closure step to yield 11 was carried out
using NaH. Finally, the target compound β-phospholactam 1 was obtained by hydrolysis of
11 with LiOH to remove the methyl protecting groups. The overall yield was 0.68%. The
product and intermediates were characterized and confirmed by NMR and MS.
The inhibitory activity of β-phospholactam 1 on MβLs was evaluated, and percent
inhibition values are listed in Table 1. Without pre-incubation with enzymes, the
β-phospholactam 1 activated CcrA and NDM-1 and showed 48, 22, and 37% inhibition
against IMP-1, Bla2, and L1, respectively. After incubation with enzymes for 30 min,
β-phospholactam 1 exhibited a 70% inhibition against IMP-1, CcrA, and L1, a 53%
inhibition for NDM-1, and a 94% inhibition of Bla2, suggesting that β-phospholactam 1 is a

time-dependent inhibitor. At this point, it is unclear whether the time-dependent inhibition is
caused by intact β-phospholactam 1, a covalently-modified enzyme-inhibitor complex, or by
a hydrolyzed product. Previous studies with a β-phospholactam and class C β-lactamase P99
revealed time-dependent inhibition was caused by a phosphonylated enzyme.46 Nonetheless,
stability studies revealed a relatively fast hydrolysis of monocyclic β-phospholactam in
water.44, 46 We believe that the different trend exhibited by the enzymes with and without
pre-incubation is due to the enzymes behaving differently in DMSO, which is used as a
solvent for the inhibitor. Unexpectedly, the inhibitor activates ImiS and BcII. The mechanism
of this activation is unknown, but it could be due to an allosteric effect as a result of
phosphonylation of solvent-exposed hydroxyl groups by β-phospholactam 1.
In docking calculations (computational details can be found in the Supporting
Information) the majority of conformations (between 42 and 50 out of 50 per complex) were
found in clusters that correspond to the expected binding mode, which is the way that the
hypothetical carbapenem transition state would bind. Since no crystal structure of any
MβL-carbapenem transition state complex is available, the lowest-energy conformations
docked to NDM-1 and L1 are compared to enzyme-hydrolyzed β-lactam complexes in Figure
3 and Table 2. In all complexes, one phosphinate oxygen, which corresponds to the oxygen
derived from the β-lactam carbonyl, coordinates Zn1, while the β-lactam nitrogen coordinates
Zn2. In some complexes (IMP-1 and CcrA), the phosphinate oxygen additionally coordinates
Zn2 (Figure S1), which may be due to the short Zn1-Zn2 distance of 3.6 Å and 3.5 Å,
respectively, in these crystal structures. The carboxylate attached to C7 of the five-membered
ring forms a salt bridge with the Lys224 side chain, as is seen in the enzyme-hydrolyzed

β-lactam complexes, except in L1, which does not contain Lys224. Interestingly, in the
L1-β-phospholactam 1 complex, which is compared to an L1-hydrolyzed moxalactam
complex51 in Table 2 and Figure S2, one phosphinate oxygen occupies the site occupied by
the carboxylate oxygen coordinating Zn1, while the other oxygen occupies the site of a water
molecule that was localized between the two zinc ions, which are also very close (3.7 Å) in
the crystal structure. As seen in Figure 3, the conformations of the β-phospholactam 1 docked
to NDM-1 structures correspond very well to the co-crystallized hydrolyzed ampicillin and
meropenem,52 consistent with the similarity of the inhibitor with both the penicillin and
carbapenem core. The more substrate-like binding mode in NDM-1 (Figure 3), where the
Zn1-Zn2 distance is increased to 4.6 (PDB entry 4HL2) and 4.1 Å (PDB entry 4EYL),
consistent with previous findings that the Zn1-Zn2 distance varies during the catalytic cycle53,
54
as opposed to the binding mode with phosphinate oxygens coordinating both zinc ions
when they are closer (Figures S1 and S2) could account for the slightly lower inhibition of
NDM-1 versus that of IMP-1, CcrA, and L1 (Table 1). Based on the docking calculations, we
cannot exclude the possibility that the inhibiting species is hydrolyzed β-phospholactam.
When docking this product to NDM-1 (PDB entry 4EYL), the resulting phosphonate
coordinated both zinc ions, but the carboxylate at C7 did not form a salt bridge with Lys244
(data not shown). The most favorable binding energy was -12.4 kcal/mol, not significantly
lower than -11.2 kcal/mol observed for the intact β-phospholactam 1.
These structures also suggest how the binding affinity of this inhibitor scaffold could
be improved. Currently, it only occupies the area close to the zinc ions. However, the active
site of MβLs is rather a cleft and the inhibitor could be designed to better fill out that cleft by

extending the substituent at C3 to something similar to R in penicillins (Figure 3A) and
adding a substituent to C7 that resembles R’ of carbapenems (Figure 3C).
We have developed a prototype β-phospholactam analog of a carbapenem transition
state, which exhibits broad-spectrum and time-dependent inhibitory activity against MβLs,
with an inhibition percentage of 70% for IMP-1, CcrA, and L1, 53% for NDM-1, and 94%
for Bla2. This study opened a way to develop β-phospholactam compounds as
broad-spectrum inhibitors of MβLs.
Acknowledgements
This work was supported by grants (21272186 to K.W.Y) from the National Natural
Science Fund of China and from the National Institutes of Health (GM093987 to M.W.C.)
and National Science Foundation (CHE1151658 to M.W.C.). The Center for Macromolecular
Modeling and Materials Design (CM3D) at California State Polytechnic University, Pomona,
where docking calculations were conducted, is supported by the W. M. Keck Foundation.
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Table 1: Percent inhibition of MβLs by β-phospholactam 1
Enzyme
L1
% inhibition
70 + 7
IMP-1
CcrA
70 + 6
69 + 8
NDM-1
Bla2
53 + 6
94 + 4
The concentration of inhibitor was 100 µM.

Table 2: Summary of geometries of lowest-energy docked MβL-β-phospholactam 1
complexes (bold) in comparison to available MβL-hydrolyzed β-lactam complexes (italic).
Binding O(P2)-Zn1 O(P2)-Zn2
Carboxylate(C7)
Energy
(kcal/mol)
-10.8
O(C2)-Zn1 O(C2)-Zn2 N1-Zn2
-Lys244 Nζ
Subclass
Complex
(Å)e
(Å)e
1.8
1.9
-
(Å)
3.1
3.1
2.4
2.2
2.3
2.2
2.8
-
(Å)f
2.9
2.7
2.9
2.9
2.9
3.2
-
IMP-1(1DD6)-βPLa
CcrA(1A7T)-βPL
NDM-1(4HL2)-βPL
NDM-(4HL2)-hAMPb
NDM-1(4EYL)-βPL
NDM-1(4EYL)-hMERc
L1(2AIO)_βPL
1
1
1
1
1
1
3
3
3
1.9
-11.9
1.8
-12.6
1.7/2.6
2.4
-
-11.2
-11.8
1.8/2.6
2.3/2.7
2.0/2.1
2.0
2.7
2.5
1.9
2.2
-
L1(2AIO)-HOH600
L1(2AIO)-hMOXd
-
2.4
2.4
-
a βPL = β-phospholactam 1
b hAMP = hydrolyzed ampicillin
c hMER = hydrolyzed meropenem
d hMOX = hydrolyzed moxalactam
e O(P2) and O(C2) designate the oxygen atoms bound to P2 in the β-phospholactam 1 and C2
in the β-lactams. Only atoms that are within a distance of 3.2 Å are reported. If both are
within that distance, both are reported starting with the closer one.
f Average distance between the two carboxylate oxygens and Lys244 Nζ.

R
O
R
O
R
S
S
R'
N
N
N
R'
O
-
-
CO2
-
CO2
CO2
penicillin
cephalosporin
carbapenem
Figure 1. Structures of β-lactam-containing antibiotics.

Figure 2. Structures of a hypothetical carbapenem transition state and β-phospholactam 1
synthesized in this study.

Figure 3. Complexes of NDM-1 co-crystalized with hydrolyzed β-lactams (A and C) and
with the lowest-energy docked β-phospholactam 1 conformation (B and D). The coordinates
of NDM-1 in panels A and B as well as hydrolyzed ampicillin in panel A are taken from PDB
entry 4HL2. The coordinates of NDM-1 in panels C and D as well as hydrolyzed meropenem
in panel C are taken from PDB entry 4EYL. The images were generated with VMD.55 The
protein backbone is shown as a green cartoon and zinc ions as black spheres (Zn1 on the left
and Zn2 on the right). The β hairpin loop in the back is loop 3; loop 10 was removed for
clarity. The hydrolyzed substrates as well as the β-phospholactam 1 inhibitor and the Lys224
side chain are shown as sticks colored by atom (C, gray; O, red, N, blue; S, yellow; P,
orange). Key distances summarized in Table 2 are indicated by dashed lines.

O
O
O
Br
O
O
O
OH
b
c
a
COOMe
COOMe
COOH
COOH
N
N
Br
2
3
4
5
O
O
O
OH
O
OH
d
e
OMe
OMe
OH
f
O
H
O
P
O
P
N
N
OMe
N
O
O
8
7
6
O
O
O
O
O
O
O
O
i
g
h
OH
OH
O
P
O
P
P
N
OMe
OMe
N
H
N
O
O
O
MeO
COOMe
10
11
9
O
O
j
P
N
O
HO
CO H
2
1
Scheme 1. Synthetic route of β-phospholactam 1: a: (1) acetyl chloride, (2) Br2, (3) MeOH; b:
benzylamine, K2CO3, CH3CN; c: NaBH4, EtOH; d: oxalyl chloride, DMSO, dichloromethane,
-78 °C; e: dimethyl phosphonate, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), anhydrous THF;
f: NaI, acetone; g: acetic anhydride, pyridine; h: Pd-C, H2; i: NaH, 15-crown-5,
dichloromethane; j: LiOH, H2O, MeOH.

Supporting Information
New β-phospholactam as a carbapenem transition state analog: synthesis of
a broad-spectrum inhibitor of metallo-β-lactamases
Ke-Wu Yanga,*, Lei Fenga, Shao-Kang Yanga, Mahesh Aithab, Alecander E. LaCuranc, Peter
Oelschlaegerc, and Michael W. Crowderb,*
aKey Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of
Education, College of Chemistry and Materials Science, Northwest University, Xi’an 710069,
b
P. R. China; Department of Chemistry and Biochemistry, Miami University, 160 Hughes
c
Hall, Oxford, OH 45056, USA; Department of Pharmaceutical Sciences, College of
Pharmacy, Western University of Health Sciences, 309 E. Second St., Pomona, CA 91766,
USA.
Corresponding authors: Tel/Fax: +8629-8830-2429 (K.-W.Y.); +1-513-529-7274 (M.W.C.).
E-mail address: kwyang@nwu.edu.cn (K.-W. Yang); crowdemw@MiamiOH.edu (M.W.
Crowder)

Experiments
General methods
All starting materials were purchased from commercial sources and purified using
standard methods. Analytical Thin Layer Chromatography (TLC) was carried out on silica
gel F254 plates with visualization by ultraviolet radiation. 1H, 31P, and 13C NMR spectra were
recorded on a Varian INOVA400 MHz NMR spectrometer. Chemical shifts were given in
part per million (ppm) on the delta scale. The peaks patterns are indicated as follows: s,
singlet; d, doublet; t, triplet; q, quartet; dd, doublet doublet; m, multiplet. The spectra were
recorded with TMS as internal standard or with 42.5% phosphoric acid as external reference.
Coupling constants (J) were reported in hertz (Hz). Mass spectra were obtained on a micro
TOF-Q (BRUKER) mass spectrometer. UV-visible spectra were recorded on an Agilent
UV8453 spectrometer. Activity evaluation of inhibitors was performed on an Agilent 8453
UV-Vis spectrometer.
Dimethyl 2,5-dibromoadipate (3). Thionyl chloride (323 g, 2.71 mol) was added in 70 ml
portions over 2 h to adipic acid (197 g, 1.35 mol) heated at 80 °C in a three-neck round
bottom flask equipped with a reflux condenser and a constant pressure dropping funnel. The
mixture was stirred until gas evolution ceased and partial solid adipic acid still remained. An
additional 100 ml of thionyl chloride was added in 7 h, and heating was continued until gas
evolution ceased. Bromine (473 g, 2.96 mol) was added dropwise to the pale yellow reaction
mixture over an ice bath, and a white precipitate formed during the addition. The white
precipitate was collected by filtration and recrystallized from MeOH to offer 253 g of 3 as

1
white power with a yield of 53%. H NMR (400 MHz, CDCl3, δ ppm): 2.00~2.05 (m, 2H),
2.28~2.35 (m, 2H), 3.80 (s, 6H), 4.24~4.26 (t, 2H).
Dimethyl N-benzylpyrrolidine-2,5-dicarboxylate (4). A mixture of compound 3 (66.6 g, 0.2
mol), benzylamine (22.3 ml, 0.2 mol), potassium carbonate (33.4 g, 0.24 mol), toluene (140
ml), and H2O (66 ml) was refluxed for 24 h. After cooling, the organic layer was separated,
and the aqueous layer was extracted with hexane (2×100 mL). The organic layers were
combined, washed with brine (2×100 ml), and dried over Na2SO4. The solvent was removed
1
to give 4 as a colorless oil with a yield of 87%. H NMR (400 MHz, CDCl3, δ ppm): δ
2.02~2.07 (m, 4H), 3.41~3.45 (t, 2H), 3.56 (s, 6H), 3.91 (s, 2H).
Methyl N-benzyl-5-hydroxymethylproline ester (5). NaBH4 (7.0 ml, 69.3 mmol) was added
to a stirred solution of the dimethyl ester 4 (10.0 g, 66 mmol) in MeOH (150 ml). The
reaction mixture was refluxed for 30 min, cooled to room temperature, and quenched by
addition of cold water (50 ml). The mixture was extracted with dichloromethane (50 ml) and
washed with a saturated solution of NaHCO3 (3×20 ml) and brine (3×20ml), respectively.
The organic phases were dried over Na2SO4, filtered, and evaporated in vacuo to give 5 as a
pale-yellow oil in 45% yield. 1H NMR (400 MHz, CDCl3, δ ppm): 1.73~2.08 (m, 1H), 3.01
(m, 1H), 3.32 (t, J = 10.8 Hz, 1H), 3.43 (s, 3H), 3.47 (d, 2H), 3.66~3.84 (dd, J = 36 Hz, 2H).
Methyl N-benzyl-5-(dimethoxyphosphoryl)(hydroxy)methylproline ester (7). To oxalyl
chloride (2.7 ml, 27.6 mmol) in dry CH2Cl2 (20 ml) at -78 °C under argon was added

dropwise DMSO (4 ml, 55.2 mmol) in CH2Cl2 (5 ml). The mixture was stirred for 15 min,
and compound 5 (5.0 g, 23 mmol) in CH2Cl2 (5 ml) was added over 30 min. After the
reaction mixture was stirred at -78 °C for 30 min, triethylamine (9.0 ml, 59.8 mmol) was
added, and the reaction was stirred for 30 min. The resulting mixture was warmed to
room-temperature, poured into water, and extracted with CH2Cl2 (100 ml×3). The organic
layer was washed sequentially with 1% HCl, water, 5% Na2CO3, water, and brine and dried
over anhydrous Na2SO4. The solvents were removed in vacuo to afford aldehyde 6 as pale
yellow oil, which was used directly in the next step reaction without further purification.
To dimethyl phosphite (1.5 ml, 16.5 mmol) in dry THF (20 ml) under argon was added
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (3 ml, 24 mmol). The reaction mixture was stirred
for 1 h and cooled to 0 °C. Aldehyde 6 (4.7 g, 16 mmol) in THF (5 ml) was added, and the
reaction was stirred at 0 °C for 48 h. The resulting mixture was poured into saturated NH4Cl
and extracted with ethyl acetate. The organic layer was washed twice with water and brine
and dried over anhydrous MgSO4. The solvents were removed in vacuo, and the residue was
purified by column chromatography, eluting with ethyl acetate, to afford 5.2 g phosphonates
1
7 as a pale yellow oil, yield 91%. 31P NMR (400 MHz, CDCl3, δ ppm): 25.24, 25.78. H
NMR (400 MHz, CDCl3, δ ppm): 1.94~2.12 (m, 4H), 3.48 (s, 3H), 3.50~3.55 (m, 1H),
3.73~3.75 (dd, 2H), 3.78 (s, 3H), 3.83(s, 3H), 3.85~3.86 (t, 1H), 4.24~4.27 (d, J=12Hz, 1H),
7.22~7.36 (m, 5H). 13C NMR (400 MHz, CDCl3, δ ppm): 25.1, 30.2~30.9, 52.1, 53.3, 57.6,
60.1, 65.0~64.3, 66.4, 68.9, 70.5, 127.5, 128.8, 129.3, 128.4, 138.3, 176.1. HRMS(ESI+) m/z:
380.1088 (Calcd for [M+Na+]+: 380.1341 m/z).

Methyl N-benzyl-5-(hydroxyl(hydroxy)(methoxy)phosphoryl)methylproline ester (8). A
stirred mixture of compound 7 (0.43 g, 1.25 mmol), sodium iodide (0.19 g, 1.25 mmol), and
acetone (3.0 g) was refluxed, and a clear solution was observed after 5 min. A white
precipitate appeared after 45 min. The reaction mixture was cooled to 4 °C, and a white solid
was filtered, washed with cooled acetone, and dried in the presence of P2O5 to offer
phosphonic acid 8 as white powder, yield 60%. 31P NMR (400 MHz, CDCl3, δ ppm)：23.78.
1H NMR (400 MHz, CDCl3, δ ppm): 1.82~1.92 (m, 4H), 3.35 (m, 1H), 3.40 (s, 3H),
3.48~3.49 (t, 1H), 3.61~3.67 (t, 3H), 3.77~3.78 (d, J=4Hz), 3.97~4.16 (dd, J=76Hz, 2H),
7.16~7.33 (m, 5H). 13C NMR (400 MHz, CDCl3, δ ppm): 24.9, 30.0, 30.8~30.0, 50.2,
51.8~52.0, 57.2, 58.5, 64.0, 64.8, 66.1, 69.7, 71.2, 127.3, 128.3, 129.5, 130.5, 137.4, 177.0.
HRMS(ESI+) m/z: 366.0955 (Calcd for [M+Na+]+: 366.1185)
Methyl N-benzyl-5-(acetoxy(hydroxy)(methoxy)phosphoryl)methylproline ester (9).
Phosphonic acid 8 (300 mg, 0.44 mmol) in dry pyridine (5 ml) was treated with acetic
anhydride (0.5 ml, 5.3 mmol) in the presence of 4-dimethylaminopyridine (DMAP) (50 mg),
and the mixture was stirred for 24 h. The solvents were removed in vacuo, and the resulting
residue was purified by column chromatography, eluting with ethyl acetate, to afford
compound 9 as colorless waxy solid, 57% yield. 31P NMR (400 MHz, CDCl3, δ ppm)：16.91.
1H NMR (400 MHz, CDCl3, δ ppm): 1.67~1.99 (m, 4H), 2.30 (s, 3H), 2.77 (t, 1H), 3.99 (s,
3H), 3.51~3.54 (d, J=12Hz, 6H), 3.77~3.80 (t, 1H), 3.85~3.86 (d, J=4Hz, 1H), 4.52~5.32 (dd,
J=40Hz, 2H), 7.25~7.42 (m, 5H).

Methyl 5-(acetoxy(hydroxyl)(methoxy)phosphoryl)methylproline ester (10). A solution of
compound 9 (0.44 g, 0.65 mmol) in MeOH (9 ml) was hydrogenated for 24 h in presence of
H2, 10% Pd/C (0.891 mg) and HCO2NH4 (2 × 0.286 g). The catalyst was removed by
filtration through Celite, and the pad was washed with MeOH (5 × 10 mL). The solvent was
removed in vacuo to afford crude product as a sodium salt. The crude product was purified by
chromatographic separation on basic aluminum oxide and cation exchange resin and
lyophilized to give the intermediate amine 10 as white powder, in 42% yield, which was
1
hygroscopic. 31P NMR (400 MHz, CDCl3, δ ppm): 23.91. H NMR (400 MHz, CDCl3, δ
ppm): 1.94~2.43 (m, 4H), 2.23 (s, 3H), 3.62~3.64 (m, 1H), 3.67~3.70 (d, J=6Hz, 3H), 3.77 (s,
3H), 4.33~4.40 (m, 1H), 4.48~4.52 (t, 1H).
Methyl 3-acetoxy-2-methoxy-1-aza-2-phosphabicyclo [3.2.0] heptane-7-carboxylate
2-oxide (11). Phosphonic acid monoester 10 (5.0 mmol) was suspended in dry chloroform
o
(50 ml), thionyl chloride was added (0.6 ml, 7.5 mmol) at 0 C, and the mixture was stirred
for 12 h at room temperature. The volatile components of the reaction mixture were
evaporated under reduced pressure, and the resulting oily residue was redissolved in
chloroform and evaporated again in order to remove hydrogen chloride and unreacted thionyl
chloride. This step was repeated three times using dry chloroform. The resulting
phosphonochloridate was dissolved in dry cooled CH3CN (30 ml), and NaH was added. The
mixture was stirred for two days at room temperature. The mixture was filtered to remove the
undissolved salt, and the filtrate was evaporated in vacuo and purified by HPLC, eluting with
1:9 water/acetonitrile (flow rate 1 ml/min), to give β-phospholactam ester 11 as an oil, in

1
38% yield. 31P NMR (400 MHz, D2O, δ ppm): 15.48. H NMR (400 MHz, D2O, δ ppm):
1.95~2.45 (m, 4H), 2.21 (s, 3H), 3.59~3.62 (d, 3H), 3.69 (s, 3H), 3.91~3.96 (t, 1H),
4.35~4.40 (d, 1H), 4.45~4.50 (m, 1H). HRMS(ESI-) m/z: 276.0625 (Calcd for [M-H+]-:
276.0715)
3-Acetoxy-2-hydroxy-1-aza-2-phosphabicyclo [3.2.0] heptane-7-carboxylic acid 2-oxide
(1). Removal of the methyl groups of compound 11 was achieved by alkaline hydrolysis of
the methyl ester in a 1.5 M LiOH/MeOH solution (2 equiv of LiOH was used). The solvent
was removed by freeze-drying, and the mixture was treated with cation exchange resin to
afford the β-phospholactam 1 as white solid, in 66% yield. 31P NMR (400 MHz, D2O, δ ppm):
17.10, 17.93. 1H NMR (400 MHz, D2O, δ ppm):1.94~2.45 (m, 4H), 2.13 (s, 3H), 3.85~3.95 (t,
1H), 4.08~4.11 (m, 1H), 4.27~4.29 (d, 1H). HRMS(ESI-) m/z: 248.0351 (Calcd for [M-H+]-:
248.0402 m/z)
Determination of inhibition percentage
Hydrolysis of nitrocefin was monitored at 482 nm using an Uvikon 8453
spectrophotometer. IMP-1, CcrA, L1, Bla2, and NDM-1 enzymes were used at fixed
concentrations between 0.03 and 0.7 nM. β-Phospholactam 1 was dissolved in 50 mM
cacodylate, pH 7.0, containing 50 μM ZnCl2 and 15% DMSO; this was also the buffer used
in the assays. In reactions that we did not pre-incubate the enzyme with inhibitor, we added
the inhibitor (to a concentration of 100 μM) to a buffered solution of enzyme, then quickly
added the substrate (at a concentration of 100 μM), and followed the hydrolysis of nitrocefin

at 482 nm. In the samples that we pre-incubated the enzyme with inhibitor, we added
inhibitor to a stock of enzyme and allowed the enzyme/inhibitor mixture to incubate for 30
minutes at room temperature. We then took an aliquot of this mixture and added it to a
buffered solution of nitrocefin and followed the hydrolysis of nitrocefin at 482 nm. All assays
were conducted in triplicate, and data are reported as mean percentages of the rate without
inhibitor present.
Docking calculations
The β-phospholactam 1 was docked into the active sites of the MβLs that it inhibited and
for which high resolution structures are available: IMP-1 (PDB code 1DD6),1 CcrA (PDB
code 1A7T),2 NDM-1 (PDB code 4HL2 (unpublished) and 4EYL,3 and L1 (PDB code
2AIO).4 The program AutoDock 4.25 and previously used charges (+1.4)6 and van der Walls
parameters (σ = 1.95 Å, ε = 0.25 kcal/mol)7 for the zinc ions8 were used and 50
conformations were generated for each complex. The flexible ligand was docked into each
rigid monomeric receptor using a grid box with dimensions of 40 x 40 x 40 grid points
equally spaced at 0.375 Å per grid and centered between the two active-site zinc ions. The
maximum number of energy evaluations and generations were set to 2,500,000 and 27,000,
respectively, and the mutation and crossover rates were set to 0.02 and 0.8, respectively. The
rest of the parameters were set at their default values and all docking calculations were
performed without constraints. Binding energies were calculated via the Lamarckian genetic
algorithm and the conformations that constitute each cluster were defined by a root mean
square deviation tolerance of 2.0 Å. The conformations with the lowest binding energy were

used for Figures 3, S1 and S2.

Figure S1. Complexes of IMP-1 (A) CcrA (B) with the lowest-energy docked
β-phospholactam 1 conformation. The coordinates of IMP-1 and CcrA are taken from PDB
entries 1DD6 and 1A7T, respectively. The images were generated with VMD.9 The protein
backbone is shown as a green cartoon and zinc ions as black spheres (Zn1 on the left and Zn2
on the right). The β hairpin loop in the back is loop 3; loop 10 was removed for clarity. The
β-phospholactam 1 inhibitor and the Lys224 side chain are shown as sticks colored by atom
(C, gray; O, red, N, blue; S, yellow; P, orange). Key distances below 3.0 Å summarized in
Table 2 are indicated by dashed lines.