Synthesis and biochemical evaluation of cephalosporin analogues equipped with chemical tethers

Article abstract of DOI:10.1039/d0ra04893c

Molecular probes typically require structural modifications to allow for the immobilisation or bioconjugation with a desired substrate but the effects of these changes are often not evaluated. Here, we set out to determine the effects of attaching functio

Full text of DOI:10.1039/d0ra04893c

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Synthesis and biochemical evaluation of
cephalosporin analogues equipped with chemical
Cite this: RSC Adv., 2020, 10, 36485
tethers†
a
Reyme Herman,b Ivan Gyulev,b Thomas F. Krauss,c
*
Lisa M. Miller,
Gavin H. Thomasb and Anne-Kathrin Duhme-Klaira
Molecular probes typically require structural modifications to allow for the immobilisation or
bioconjugation with a desired substrate but the effects of these changes are often not evaluated. Here,
we set out to determine the effects of attaching functional handles to a first-generation cephalosporin. A
series of cephalexin derivatives was prepared, equipped with chemical tethers suitable for the site-
selective conjugation of antibiotics to functionalised surfaces. The tethers were positioned remotely
from the b-lactam ring to ensure minimal effect to the antibiotic's pharmacophore. Herein, the activity of
the modified antibiotics was evaluated for binding to the therapeutic target, the penicillin binding
proteins, and shown to maintain binding interactions. In addition, the deactivation of the modified drugs
by four b-lactamases (TEM-1, CTX-M-15, AmpC, NDM-1) was investigated and the effect of the tethers
on the catalytic efficiencies determined. CTX-M-15 was found to favour hydrolysis of the parent
antibiotic without a tether, whereas AmpC and NDM-1 were found to favour the modified analogues.
Furthermore, the antimicrobial activity of the derivatives was evaluated to investigate the effect of the
structural modifications on the antimicrobial activity of the parent drug, cephalexin.
Received 3rd June 2020
Accepted 25th September 2020
DOI: 10.1039/d0ra04893c
rsc.li/rsc-advances
analogues could reveal valuable information. The b-lactam
antibiotics are widely used and are typically considered to be
one of the safest classes of antibiotics.4 Since their discovery,
there has been extensive research carried out into the derivati-
sation of the b-lactam scaffold, resulting in the successful
development of numerous antibiotics.5,6
Recently, we demonstrated the ability of surface bound b-
lactam drugs to be recognised by the therapeutic target proteins
as well as enzymes produced by resistant bacteria.7 In order to
attach the antibiotic molecule to the surface, an analogue of
cephalexin (1) was prepared featuring a maleimide group
attached via a PEG linker (2), Fig. 1. Studies of the surface-
bound antibiotic demonstrated that b-lactamases and a peni-
cillin binding protein (PBP) were able to recognise and bind the
immobilised drugs. Here, we set out to investigate the effect of
the addition of a chemical tether on the properties of the parent
compound, thus contributing new SAR information.
Introduction
The controlled functionalisation of surfaces is imperative for
the preparation of functional materials. This a key step in the
preparation of many (bio)sensors, for example, which are
designed for selective and sensitive detection of analytes. The
immobilisation or bioconjugation of a molecular probe oen
requires structural modication to introduce a functional
handle, able to react with a desired substrate. A common
approach is to attach a linker to the probe, such as a bifunc-
tional polyethylene glycol (PEG), with orthogonal functional
groups that allows for controlled reaction with the probe and
with the desired substrate.1 However, the effect of such modi-
cations on the function of a probe are oen not evaluated,
even though binding interactions are likely to be affected.
Consequently, opportunities to gain insights into the structure–
activity relationships (SAR) are missed.
With the difficulties faced in the development of novel
antibiotics and the increasing challenges of ghting against
antibiotic resistance,2,3 investigations into the SAR of antibiotic
Results and discussion
Chemistry
Compound design. The modications to 1 described herein
were performed via the amine of the molecule. This position
was chosen to minimise the effect of the tether on the phar-
macophore, the b-lactam ring. The amine motif provided
a functional handle for the addition of the desired chemical
tether through amidation reactions, allowing access to
aDepartment of Chemistry, University of York, Heslington, York, YO10 5DD, UK.
E-mail: lisa.miller@york.ac.uk
bDepartment of Biology, University of York, Heslington, York, YO10 5DD, UK
cDepartment of Physics, University of York, Heslington, York, YO10 5DD, UK
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra04893c
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Therefore, compound 10 was included to determine the effects
of introducing an amide and a short exible tether, while
maintaining an amine group.
Compound synthesis. The two maleimide analogues (2 and
3) were prepared as previously reported.7 All analogues were
prepared by addition of the tethers to the parent antibiotic, as
described in Scheme 1. Compounds 4 and 5 were prepared from
reaction of 1 with the NHS-esters of the corresponding tether,
intermediates 16 and 18. In the preparation of compound 6, the
previously reported acid (20)12 was used to allow for the desired
amidation via the acid chloride, while maintaining the NHS-
ester. Compounds 7, 8, and 9 were prepared by reaction with
the required anhydride reagent: glutaric anhydride, acetic
anhydride and Boc anhydride, respectively. Finally, compound
10 was prepared in three steps from the N-Boc protected 5-
aminovaleric acid 21, Scheme 1. All compounds were charac-
1
terised by H, 13C NMR, IR spectroscopy, and HRMS. Purity of
compounds for in vitro and in vivo testing was determined using
HPLC or QNMR.
Fig. 1 Chemical structures of the parent antibiotic cephalexin (1) and
the nine analogues (2–10) evaluated for the effects of addition of
a chemical tether.
a selection of compounds with reactive pendant groups. In
addition to the previously reported compounds 2 and 3,7
a further seven analogues were synthesised, Fig. 1. Compounds
2–6 were designed to include commonly employed functional
groups that are used in biocompatible reactions with surfaces
and other substrates of interest.
Compounds 2 and 3 both feature a maleimide; this motif is
a prevalent functional handle used for selective reaction
through conjugation with thiol groups.8 Another commonly
used bio-compatible reaction is the copper-catalysed click
reaction in which an alkyne, such as in compound 4, reacts with
an azide to form a triazole.9 For the direct attachment to gold
surfaces disuldes, such as the lipoic acid tether of compound
5, are ubiquitous.10 Whereas, for the attachment to a protein, or
another source of amine functional groups, N-hydrox-
ysuccinimide (NHS) esters are common.11 Compounds with this
activated ester, as in compound 6, react readily with lysine
residues and other amines, providing attachment through the
formation of an amide bond.
NHS-esters, such as compound 6, are known to have a short
half-life in aqueous media;11 thus, the hydrolysis product with
the carboxylic acid was prepared for comparison, compound 7.
Three further control compounds were synthesised: one to test
the effect of a small modication with the acetyl group of
compound 8, whereas the tert-butyloxycarbonyl (Boc) group of
compound 9 was designed to test the effect of a large sterically
demanding group in this position. Lastly, compound 10 was
included featuring a small aliphatic tether with a terminal
amine group. By attaching the tethers via an amide linker in
compounds 2–9, the amine of the parent antibiotic is lost.
Scheme 1 Synthesis of tethered analogues. (a) NHS, DCC, DCM; (b) 1,
DIPEA, MeCN; (c) NHS, DMAP, THF; (d) (i) (ClCO)2, DMF, DCM, (ii) 1,
anhydrous pyridine, MeCN; (e) 1, Et3N, MeCN; (f) TFA, TES, DCM.
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Biological evaluation
PBP binding assay. A thermal shi assay was used to inves-
tigate the binding of the cephalexin analogues to the thera-
peutic target, PBP, in vitro. This assay provides a fast and
inexpensive method of detecting a ligand binding to a protein of
interest. Briey, the protein is gradually heated in the presence
of a uorescent dye that binds non-specically to the hydro-
phobic surfaces. As the protein unfolds, hydrophobic surfaces
within the protein are revealed, causing the dye to uoresce. A
change in the melting temperature (Tm) when the protein is
heated in the presence of a ligand, indicates a protein–ligand
interaction. Fig. 2 shows the results of the thermal shi assay
carried out using recombinant PBP3 and PBP4 with compounds
1–10. The PBPs can be divided into two main categories: high
molecular mass (HMM) and low molecular mass (LMM).13 PBP3
was chosen as a representative HMM PBP, and PBP4 as
a representative LMM PBP. Cephalexin (1) has been previously
reported to show good binding affinities for both PBP3 and
PBP4.14 Penicillin (11) and cefpodoxime (12) were included as
positive controls.
The results from this assay showed that all of the analogues
tested caused an increase in the Tm with PBP3, thus increasing
the thermostability of this particular PBP upon binding.
Whereas, with PBP4 there was either no shi or a small decrease
in the Tm observed, indicating the ligands are facilitating the
unfolding of the protein. Previously reported thermal shi
assays with PBPs have noted both increases and decreases in Tm
of the protein aer the binding of different b-lactam
analogues.15–17
Fig. 3 Relative affinities for compounds 1–12 with PBP3. Ligand
equivalents required to cause >Tm1/2 shift. Lower equivalents conveys
higher affinity. The assay protocol is provided in the Experimental
section.
that 75 equivalents of compound 1 was required to achieve
>Tm1/2 but with analogues 2–10, fewer equivalents were
required. This study also showed that penicillin (11) has a great
affinity for PBP3 than cephalexin (1), which is consistent with
previously reported data.14 The results, as shown in Fig. 3,
suggest that addition of the tethers has improved the binding
affinity of analogues 2–10 with PBP3, compared to that of the
parent antibiotic 1.
b-Lactamase kinetics studies. Previously reported SAR
around the b-lactam class of antibiotics has shown that
increased steric bulk at the 7-position of cephalosporins can
reduce the rates of drug hydrolysis by the b-lactamases.18 Many
of the b-lactam antibiotics from the later generations, for
example cefpodoxime (12, Fig. 4), which are stable in the pres-
ence of many b-lactamases, feature sterically demanding groups
in this position. The SAR of cephalosporins suggested that
increasing the steric bulk at the 7-position of cephalexin (1) will
provide resistance to b-lactamase-mediated hydrolysis. There-
fore, it was hypothesised that analogues 2–10, which all feature
tethers at this position, would have improved stability,
compared to that of the parent drug 1.
Further thermal shi studies were carried out to approxi-
mate the affinities of compounds 1–12 with PBP3. PBP3 was
selected for this study as this particular PBP is essential for cell
division in E. coli, making PBP3 an important target for b-lac-
tam antibiotics.13 By measuring the Tm values of PBP3 with
compounds 1–12 at a range of ligand concentrations, aer
a constant incubation time, the equivalents of each ligand
required to cause a shi greater than half the Tm value (Tm1/2
)
were determined, Fig. 3. Using this assay, it was determined
To investigate the effect that the addition of the chemical
tethers reported herein had on the rate of b-lactamase-mediated
hydrolysis,
a previously reported absorbance assay was
employed.19 The rates of hydrolysis were measured by the
decrease in absorbance at 260 nm caused by hydrolysis of the b-
lactam ring. The rates of hydrolysis were measured and the initial
Fig. 2 Thermal shift assay of compounds 1–12 with PBP3 and PBP4.
Melting temperatures (Tm) were calculated from the average of three
measurements, with ꢀs.d. error. DTm was calculated by subtracting the Fig. 4 The cephalosporin structure. Cefpodoxime (12), has increased
Tm of the PBP. The assay protocol is provided in the Experimental stability in the presence of b-lactamases due to the increased steric
section.
bulk at the 7-position.
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velocity of each determined. The high Km values prevented resistance to the cephalosporins such as cephalexin (1), as well
determinations of the Vmax values, therefore the catalytic effi- as cephamycins and carbapenems.22 Compound 2 was less
ciencies (kcat/Km) were determined using v ¼ (kcat/Km)[E][S].20,21 favoured than 1, but all other analogues had a signicantly
The relative catalytic efficiencies, with respect to the (kcat/Km) higher catalytic efficiency than the parent antibiotic, particu-
value for the parent antibiotic cephalexin (1), were used to larly compound 7, which featured a glutaric acid tether. This
compare the reaction specicities of four selected b-lactamases observation suggests that modications to this point of the
with the tethered analogues, Table 1.
antibiotic results in reduced stability to AmpC mediated
The b-lactamase enzymes are well studied, with over 2500 hydrolysis. This is in contrast to the trend observed with CTX-M-
unique proteins identied.22 This vast family of enzymes can be 15, showing that the effect of these structural modications, to
categorised into subsets (class A, B, C, or D), based on their the 7-position of cephalexin (1), is specic to the b-lactamase
protein sequence. Initial tests were carried out using TEM-1, under investigation.
one of the most common b-lactamases found in Gram-
Further tests were carried out to investigate the effects of the
negative bacteria.23 This class A b-lactamase is able to hydro- tethers on the hydrolysis by a metallo-b-lactamase, NDM-1. The
lyse penicillins and the early generations of cephalosporins. Of metallo-b-lactamases are known to hydrolyse penicillins, ceph-
the ten analogues tested, compound 9 was the preferred alosporins and even last resort carbapenems. These class B b-
substrate with TEM-1, suggesting that the large Boc group lactamases hydrolyse b-lactams by an alternative mechanism
attached to this analogue does not hinder the hydrolysis. The that relies on one or two zinc ions present in the active site.27
least preferred substrate with TEM-1 was compound 2, which is These enzymes are oen produced by clinical strains with
the largest of the compounds tested with a PEG tether. However, multiple forms of resistance that are only susceptible to last line
there was no clear trend in the relative catalytic efficiencies of antibiotics.28 The relative catalytic efficiencies of compounds 1–
compounds 1–10 with TEM-1.
10 with NDM-1 demonstrated that introduction of the tethers to
As with TEM-1, compound 2 was also observed to be the least the antibiotic resulted in increased substrate activity, the same
preferred substrate with CTX-M-15, an extended spectrum b- trend that was observed with AmpC. These results suggest that
lactamase (ESBL) from class A. ESBLs are plasmid-encoded modications to this point of the antibiotic results in reduced
enzymes that confer increased antibiotic resistance to stability to NDM-1 mediated hydrolysis and should be avoided
commonly used antibiotics.24 CTX-M-14 and CTX-M-15 are the in developing cephalexin (1) analogues stable to NDM-1.
most prevalent ESBLs and known to contribute towards many
In vivo assays. The in vitro assays demonstrated that the
cases of multidrug resistant infections.25,26 The relative catalytic modied analogues were able to interact with the therapeutic
efficiencies showed that the two more preferred substrates of target proteins (PBP3 and PBP4) and four selected b-lactamases
CTX-M-15 had no tether (compound 1) and the smallest tether (TEM-1, CTX-M-15, AmpC, and NDM-1), as shown in Fig. 2, 3
tested, the acetyl group (compound 8). This trend suggests that and Table 1. Further testing was carried out in vivo to determine
modications to this point of the antibiotic has a direct effect whether the tethered compounds had any antibacterial activity.
on the stability against hydrolysis by CTX-M-15. Thus structural Table 2 summarises the MIC values determined for 1–12, MBC50
modications to this point of the antibiotic could be an effective data is included in the ESI.†
strategy in the production of antibiotics with increased stability
to this ESBL.
The activity of 1–12 was investigated with growth assays
against Staphylococcus aureus (NCTC 6571). S. aureus is a viru-
The relative catalytic efficiencies of compounds 1–10 with lent Gram-positive pathogen that can cause a wide range of
AmpC demonstrated that introduction of the tethers resulted, infections in humans.29,30 Each compound was tested at 400–6
surprisingly, in increased substrate activity against the modi- mM concentrations, with lower concentrations tested as
ed b-lactam. AmpC is a class C b-lactamase known to confer required. All tethered analogues were still able to inhibit the
growth of this strain of S. aureus; however, the tethers were
detrimental to the antimicrobial activity against this Gram-
Table 1 Relative catalytic efficiencies (kcat/Km)a of compounds 1–10
with TEM-1, CTX-M-15, AmpC, and NDM-1
positive bacteria, with all but one MIC value (compound 9)
determined to be greater than that of the parent antimicrobial
1.
Compound
TEM-1
CTX-M-15
AmpC
NDM-1
Further growth assays tested 1–12 against Escherichia coli
BW25113, a K-12 strain. E. coli K-12 is the workhorse of many
microbiology laboratories; however, E. coli is a versatile Gram-
negative bacterium and the evolution of pathogenic E. coli can
cause a number of harmful infections in humans.31 Only the
parent antibiotic 1 and the two control compounds 11 and 12
were observed to have antimicrobial activity against E. coli. Thus
showing that the addition of the chemical tethers has a signi-
cant detrimental effect on the activity of the antibiotic against E.
coli.
1
2
3
4
5
6
7
8
9
10
1.0
0.2
1.5
1.2
0.6
0.4
2.2
1.8
2.6
0.3
1.0
0.2
0.3
0.3
0.8
0.5
0.5
1.0
0.3
0.6
1.0
0.7
5.7
17
11
4.3
19
11
4.7
13
1.0
1.1
1.7
4.9
3.7
1.2
5.2
4.7
1.3
3.5
a
Physicochemical properties. Since the abilities of 2–10 to
bind the therapeutic target had been conrmed in vitro (Fig. 2),
Catalytic efficiencies (kcat/Km) were determined using v ¼ (kcat/Km)[E]
[S].20,21 Relative values calculated as a ratio with respect to cephalexin, 1.
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Table 2 MIC results for compounds 1–12
Table 3 Score for the likelihood of accumulation inside Gram-nega-
tive bacteria
MICa
Compound
NoAa
Globb
NoRBc
Scored
S. aureus
E. coli
Compound
(NCTC 6571)
(BW25113)
1
2
3
4
5
6
7
8
1
0
0
0
0
0
0
0
0
1
0
0
0.057
0.022
0.064
0.098
0.083
0.035
0.085
0.134
0.133
0.088
0.095
0.068
4
3
1
1
1
1
1
1
2
1
2
2
1
30
11
7
10
11
9
5
7
9
4
1
2
3
4
5
6
7
8
18.9 mM
92.6 mM
55.8 mM
35.0 mM
24.9 mM
82.9 mM
81.4 mM
59.0 mM
11.0 mM
51.4 mM
97.7 nM
20.9 mM
94.9 mM
>400 mMb
>400 mMb
>400 mMb
>400 mMb
>400 mMb
>400 mMb
>400 mMb
>400 mMb
>400 mMb
371 mM
9
10
11e
12e
9
10
11c
12c
7
a
b
c
Number of primary amines (NoA). Globularity (Glob). Number of
7.7 mM
d
rotatable bonds (NoRB). Score out of three based on the following
criteria: NoA $ 1; Glob # 0.25; NoRB # 5. Penicillin (11) and
e
a
MIC values were determined aer 16 h incubation. Dose–response
cefpodoxime (12) included for comparison. NoRB determined using
Marvin 17.21.0, ChemAxon. Glob determined using https://entry-
way.org.
curves are provided in the ESI, MIC data was analysed using
b
GraphPad Prism (version 8.3.0).
MIC not determined in
c
concentration range tested (up to 400 mM). Penicillin (11) and
cefpodoxime (12).
However, the criteria compared in Table 3 fail to predict the
measured antimicrobial activity of 11 and 12 against E. coli. A
reliable method of predicting antimicrobial activity is very
much coveted, but it is yet to be achieved due to the complex
factors involved.32–35
the in vivo results suggest that the tethered analogues may be
unable to access the PBPs within the periplasm of E. coli. To
gain a further insight into the effects of the chemical tethers,
the physicochemical properties of each compound was evalu-
ated. The compounds were scored on the properties favourable
for accumulation in Gram-negative bacteria, based on the
observations of Richter et al. who reported that there are three
key properties in predicting the accumulation: a primary amine,
low globularity, and high rigidity.32,33 In order to score the
likelihood of the compounds accumulating, each was scored
based on the “eNTRy rules” reported by Richter et al.: number of
primary amines (NoA) $ 1; globularity (Glob) # 0.25; number of
rotatable bonds (NoRB) # 5, Table 3. The predicted accumula-
tion scores were determined to be below ideal for all of the
tethered analogues (2–10), primarily due to the loss of the
amine group used in the introduction of the tethers via an
amide bond. Compound 10, which maintained an amine group,
failed based on the increased NoRB. Chemical tethers are oen
designed with exibility to favour the subsequent reactions with
other substrates, particularly in examples that form a mono-
layer on a surface. However, this exibility increases the NoRB,
reducing the rigidity of these analogues, which reduces the
likelihood of accumulation. Based on the predicted accumula-
tion scores of Table 3, all of the tethered analogues are expected
to have lower activity than that of 1 against Gram-negative
bacteria. As shown in Table 2, this was found to be true for all
tethered analogues, 2–10. Compounds 8 and 10 both scored two
but for different reasons: compound 8 lacked the required
amine whereas compound 10 exceeded the limit for NoRB. The
growth assay results showed that both compounds were inactive
against E. coli demonstrating the importance of these two
criteria of the “eNTRy rules”. By using more rigid tethers and
including an amine group, it may be possible to produce teth-
ered analogues that are active against Gram-negative bacteria.
Summary and conclusions
A series of cephalexin (1) analogues equipped with chemical
tethers was evaluated for binding to the therapeutic target, the
penicillin binding proteins, and shown to maintain binding
interactions in vitro. Further investigations with four b-lacta-
mases (TEM-1, CTX-M-15, AmpC, and NDM-1) were carried out
and revealed that the modications affected each enzyme's
catalytic rates differently. With TEM-1, there was no clear trend
in the catalytic efficiencies of 1–10. CTX-M-15 was found to
favour hydrolysis of the parent antibiotic without a tether, thus
demonstrating that modications to this position of 1 could
produce antibiotics with increased stability to this ESBL.
Conversely, both AmpC and NDM-1 were found to favour the
modied analogues suggesting that these types of structural
modications should be avoided in the design of analogues
stable to AmpC and/or NDM-1. The tethers were found to lower
the antimicrobial activities when testing against S. aureus and
cause complete loss of activity against E. coli. The loss of activity
against E. coli was consistent with previously reported obser-
vations linking the globularity, rigidity, and amine functionality
of antibiotics with accumulation in Gram-negatives.
These results show that the addition of the tethers directly
affected the properties of the antibiotic, thus highlighting the
importance of evaluating the changes that occur from modi-
fying molecular probes. Most notably, the effect of the tethers
on the rate of b-lactamase-mediated hydrolysis was specic to
the b-lactamase under investigation. This suggests that
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modifying the 7-position of 1 could be key in the development room temperature. Aer 16 h, the urea precipitate formed during
of surface-bound antibiotics for the selective detection of b- the reaction was ltered off, and the lter cake washed with DCM.
lactamases associated with multidrug resistant infections, such The ltrate was concentrated under reduced pressure to afford
as NDM-1.
2,5-dioxopyrrolidin-1-yl pent-4-ynoate (16), which was used
without further purication. The residue was dissolved in anhy-
drous MeCN (10 mL) and cephalexin monohydrate (338 mg,
0.927 mmol, 0.9 equiv.) was added. The mixture was cooled in an
ice bath followed by the slow addition of DIPEA (443 mL,
Experimental
Chemistry
General experimental. Analytical thin layer chromatography 2.550 mmol, 2.5 equiv.). Once the addition was complete, the ice
(TLC) was performed with EM Science silica gel 60 F254 bath was removed, and the reaction was allowed to stir at room
aluminium plates. Visualisation was carried out using a UV temperature for 2 h at which time the reaction appeared complete
lamp (254 nm) and by immersion in potassium permanganate by TLC. The reaction mixture was concentrated under reduced
(KMnO4), followed by heating using a heat gun. Organic solu- pressure, dissolved in EtOAc (20 mL), and washed with 0.1 M HCl
tions were concentrated by rotary evaporation at 40–45 ꢁC. (2 ꢂ 20 mL). The organic layer was collected and concentrated to
Purication of reaction products by ash column chromatog- a cream solid, which was the puried by trituration from diethyl
ꢀ
raphy was carried out using Fluka Silica, pore size 60 A, 220–440 ether to afford (6R,7R)-3-methyl-8-oxo-7-[(2R)-2-(pent-4-ynamido)-
mesh, 35–75 mm.
2-phenylacetamido]-5-thia-1-azabicyclo[4.2.0]
Materials. Unless otherwise noted, all purchased materials oct-2-ene-2-carboxylic acid (4) (277 mg, 70% yield) as a white solid.
were used without purication. All standard solvents were 1H NMR (400 MHz, DMSO-d6) d 9.32 (d, J ¼ 8.4 Hz, 1H), 8.65 (d, J ¼
purchased from Sigma Aldrich. All standard acids, bases, and 8.2 Hz, 1H), 7.44 (d, J ¼ 7.0 Hz, 2H), 7.37–7.25 (m, 3H), 5.71 (d, J ¼
drying agents were purchased from Fisher Scientic. NHS and 8.2 Hz, 1H), 5.62 (dd, J ¼ 8.3, 4.7 Hz, 1H), 4.96 (d, J ¼ 4.7 Hz, 1H),
N,N-diisopropylethylamine (DIPEA) were purchased from Acros 3.46 (d, J ¼ 18.5 Hz, 1H), 3.28 (d, J ¼ 18.4 Hz, 1H), 2.76 (t, J ¼
Organics. Cephalexin monohydrate and Boc2O were purchased 2.6 Hz, 1H), 2.47–2.40 (m, 2H), 2.39–2.31 (m, 2H), 1.98 (s, 3H). 13
C
from
Fluorochem.
Pentynoic
acid,
N,N0-dicyclohex- NMR (101 MHz, DMSO-d6) d 170.8, 170.2, 164.1, 163.6, 138.3,
ylcarbodiimide (DCC), lipoic acid, glutaric anhydride, oxalyl 129.7, 128.2, 127.6, 127.1, 122.8, 83.8, 71.4, 58.4, 57.2, 55.6, 33.7,
chloride, and Et3N were purchased from Sigma Aldrich. DMAP 28.9, 19.4, 14.1. HRMS: exact mass calculated for [M ꢃ H]ꢃ
and 5-(Boc-amino)pentanoic acid were purchased from TCI.
(C21H20N3O5S) requires m/z 426.1129, measured m/z 426.1148. IR
Instrumentation. 1H and 13C NMR spectra were recorded on (neat): 3280, 3010, 1762, 1721, 1650, 1643, 1539, 1377, 1218, 1108,
1
a Jeol ECS 400 (400 MHz for H, 101 MHz for 13C) at ambient 1070 cmꢃ1. HPLC purity (254 nm): 96%.
temperature. Chemical shis are reported relative to residual
Preparation of 5. (6R,7R)-7-[(2R)-2-[5-(1,2-Dithiolan-3-yl)
solvent peaks and coupling constants (J) are given in hertz. pentanamido]-2-phenylacetamido]-
High-resolution ESI mass spectra were recorded on a Bruker 3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic
microTOF electrospray mass spectrometer. Infrared (IR) spectra acid. To a stirred solution of lipoic acid (17) (1 g, 4.854 mmol, 1
were recorded on a PerkinElmer Spectrum Two (ATIR). Analyt- equiv.) in anhydrous DCM (30 mL, 0.2 M) at 0 ꢁC was added NHS
ical HPLC measurements were performed on a Shimadzu HPLC (614 mg, 5.340 mmol, 1.1 equiv.). A solution of DCC (1.3 g,
system (Prominence) equipped with a LC-20AD pump, SIL-20A 6.311 mmol, 1.3 equiv.) in DCM (10 mL) was added slowly. The
autosampler, DGU-20AS degasser, CTO-20AC column oven, reaction mixture was then allowed to warm to room tempera-
CBM-20A communication bus module and SPD-M20A diode ture. Aer 16 h, the urea precipitate formed during the reaction
ꢀ
array detector using an Athena C18-WP column (100 A, 4.6 ꢂ was ltered off, and the lter cake washed with DCM. The
250 mm, 5 mm). Eluent gradient: 5–95% MeCN/H2O with a 0.1% ltrate was concentrated under reduced pressure to afford
formic acid modier, over 15 minutes.
a yellow residue. Recrystallisation from EtOAc : hexane (1 : 1)
Compound synthesis
afforded 2,5-dioxopyrrolidin-1-yl 5-(1,2-dithiolan-3-yl)penta-
Preparations of 2 and 3. Compound 2 (6R,7R)-7-((R)-30-(2,5- noate (18) (978 mg, 66%) as an off-white solid. H NMR (400
1
Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,8,25-trioxo-2-phenyl-
6,12,15,18,21-pentaoxa-3,9,24-triazatriacontanamido)-3-methyl-
MHz, chloroform-d) d 3.58 (dq, J ¼ 8.2, 6.4 Hz, 1H), 3.25–3.06
(m, 2H), 2.84 (d, J ¼ 4.1 Hz, 4H), 2.63 (t, J ¼ 7.3 Hz, 2H), 2.47
8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid and 3, (dtd, J ¼ 13.1, 6.6, 5.4 Hz, 1H), 1.93 (dq, J ¼ 12.7, 6.9 Hz, 1H),
(6R,7R)-7-((R)-2-(6-(2,5-dioxo-2,5-dihyrdo-1H-pyrrol-1-yl)
hexanamido)-2-phenylacetamido)-3-methyl-8-oxo-5-thia-1-
1.79 (p, J ¼ 7.4 Hz, 2H), 1.77–1.65 (m, 2H), 1.63–1.52 (m, 2H).
13C NMR (101 MHz, chloroform-d) d 169.3, 168.6, 56.2, 40.3,
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid were previously 38.7, 34.6, 30.9, 28.5, 25.7, 24.5. 1H and 13C are consistent with
reported.7
previously reported data.36 HRMS: exact mass calculated for [M
Preparation of 4. (6R,7R)-3-Methyl-8-oxo-7-[(2R)-2-(pent-4-yn + Na]+ (C12H17NNaO4S2) requires m/z 326.0491, measured m/z
amido)-2-phenylacetamido]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
326.0490. IR (neat): 2934, 2915, 2861, 1809, 1780, 1729, 1630,
carboxylic acid. To a stirred solution of pentynoic acid (15) 1574 cmꢃ1. Mp 93–94 C, consistent with reported data.37
ꢁ
(100 mg, 1.020 mmol, 1 equiv.) in anhydrous DCM (1 mL, 1 M) at
2,5-Dioxopyrrolidin-1-yl
5-(1,2-dithiolan-3-yl)pentanoate
0 ꢁC was added NHS (123 mg, 1.071 mmol, 1.05 equiv.). A solution (18) (294 mg, 0.971 mmol, 1 equiv.) was dissolved in anhydrous
of DCC (221 mg, 1.071 mmol, 1.05 equiv.) in DCM (1 mL) was MeCN (20 mL, 0.05 M) and cephalexin monohydrate (235 mg,
added slowly. The reaction mixture was then allowed to warm to 0.647 mmol, 0.66 equiv.) was added. The mixture was cooled in
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an ice bath followed by the slow addition of DIPEA (169 mL, d 9.27 (dd, J ¼ 8.3, 3.1 Hz, 1H), 8.57 (dd, J ¼ 21.1, 8.1 Hz, 1H),
0.971 mmol, 1 equiv.). Once the addition was complete, the ice 7.43 (d, J ¼ 7.1 Hz, 1H), 7.38–7.25 (m, 3H), 5.67 (d, J ¼ 8.0 Hz,
bath was removed and the reaction was allowed to stir at room 1H), 5.61 (dd, J ¼ 8.3, 4.6 Hz, 1H), 4.95 (d, J ¼ 4.6 Hz, 1H), 3.46
temperature for 6 h. The reaction mixture was then concen- (d, J ¼ 18.5 Hz, 1H), 3.27 (d, J ¼ 18.3 Hz, 1H), 2.81 (s, 4H), 2.73–
trated under reduced pressure and triturated using 5% DCM/ 2.63 (m, 2H), 2.33 (t, J ¼ 7.3 Hz, 2H), 2.27–2.16 (m, 2H), 1.98 (s,
diethyl ether to afford (6R,7R)-7-[(2R)-2-[5-(1,2-dithiolan-3-yl) 3H). 13C NMR (101 MHz, DMSO-d6) d 171.2, 170.9, 170.3, 168.8,
pentanamido]-2-phenylacetamido]-
168.5, 164.1, 163.5, 138.2, 129.8, 128.2, 127.6, 127.2, 122.7, 58.4,
57.2, 55.7, 33.4, 29.7, 28.9, 25.5, 20.5, 19.4. HRMS: exact mass
3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic
acid (5) (190 mg, 55%) as a white solid. 1H NMR (400 MHz, calculated for [M
+
Na]+ (C25H26N4NaO9S) requires m/z
DMSO-d6) d 9.29 (d, J ¼ 8.3 Hz, 1H), 8.55 (d, J ¼ 8.3 Hz, 1H), 581.1313, measured m/z 581.1322. IR (neat): 3286, 2945, 1776,
7.48–7.39 (m, 2H), 7.35–7.23 (m, 3H), 5.70 (d, J ¼ 8.3 Hz, 1H), 1728, 1643, 1524, 1360, 1204, 1158, 1066 cmꢃ1. QNMR purity
5.61 (dd, J ¼ 8.4, 4.7 Hz, 1H), 4.95 (d, J ¼ 4.7 Hz, 1H), 3.65–3.54 (1H NMR, maleic acid reference, DMSO-d6): 90%.
(m, 1H), 3.46 (d, J ¼ 18.2 Hz, 1H), 3.27 (d, J ¼ 18.2 Hz, 1H), 3.22–
Preparation of 7. (6R,7R)-7-[(2R)-2-(4-Carboxybutanamido)-
3.06 (m, 2H), 2.40 (dt, J ¼ 12.5, 6.2 Hz, 1H), 2.22 (t, J ¼ 7.4 Hz, 2-phenylacetamido]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]
2H), 1.98 (s, 3H), 1.90–1.79 (m, 1H), 1.76–1.57 (m, 2H), 1.57–1.46 oct-2-ene-2-carboxylic acid. A solution of cephalexin mono-
(m, 2H), 1.34 (q, J ¼ 7.4, 6.9 Hz, 2H). 13C NMR (101 MHz, DMSO- hydrate (100 mg, 0.274 mmol, 1 equiv.) and Et3N (46 mL,
d6) d 171.9, 170.9, 164.0, 163.6, 138.4, 128.2, 127.6, 127.1, 58.4, 0.329 mmol, 1.2 equiv.) was stirred at room temperature for
57.2, 56.2, 55.5, 38.1, 34.7, 34.1, 33.4, 28.9, 28.3, 25.3, 25.0, 24.5, 10 min. Glutaric anhydride (19) (38 mg, 0.329 mmol, 1.2 equiv.)
19.4. HRMS: exact mass calculated for [M + Na]+ (C24H29N3- was then added and the resultant reaction mixture stirred for
NaO5S3) requires m/z 558.1162, measured m/z 558.1152. IR 3 h at room temperature. The reaction mixture was then
(neat): 3287, 2927, 1766, 1642, 1513, 1360, 1219 cmꢃ1. HPLC concentrated under reduced pressure to afford a yellow solid,
purity (254 nm): 95%.
which was then triturated using 10% DCM/diethyl ether to
Preparation of 6. (6R,7R)-7-[(2R)-2-{5-[(2,5-Dioxopyrro afford the Et3N salt of (6R,7R)-7-[(2R)-2-(4-carboxybutanamido)-
lidin-1-yl)oxy]-5-oxopentanamido}-2-phenylacetamido]-
3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic
2-phenylacetamido]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]
oct-2-ene-2-carboxylic acid (7) (144 mg, 94%), as a cream solid.
acid. NHS (1 g, 8.772 mmol, 1 equiv.) and DMAP (1 g, 1H NMR (400 MHz, acetonitrile-d3) d 8.18 (t, J ¼ 8.4 Hz, 1H), 7.58
8.772 mmol, 1 equiv.) were dissolved in anhydrous THF (50 mL) (t, J ¼ 8.5 Hz, 1H), 7.44–7.36 (m, 2H), 7.35–7.27 (m, 3H), 5.77
and cooled in an ice bath. Glutaric anhydride (19) (1.6 g, (dd, J ¼ 7.7, 4.3 Hz, 1H), 5.61 (dd, J ¼ 9.2, 4.7 Hz, 1H), 4.83 (d, J ¼
13.158 mmol, 1.5 equiv.) was then added portion-wise and the 4.7 Hz, 1H), 3.39 (d, J ¼ 17.4 Hz, 1H), 3.11–2.98 (m, 7H), 2.37–
resulting reaction mixture was allowed to warm to room 2.24 (m, 4H), 1.87 (s, 3H), 1.84–1.73 (m, 2H), 1.22 (t, J ¼ 7.3 Hz,
temperature. Aer a further 4 h at room temperature, the 9H). 13C NMR (101 MHz, DMSO-d6) d 174.4, 171.6, 170.9, 165.4,
reaction was concentrated under reduced pressure. The residue 162.8, 138.4, 128.2, 127.9, 127.5, 127.2, 58.1, 56.9, 55.6, 45.0,
was dissolved in EtOAc (60 mL) and washed with 0.1 M HCl (2 ꢂ 34.1, 33.3, 28.5, 20.8, 19.3, 9.0. HRMS: exact mass calculated for
30 mL) followed by brine (1 ꢂ 30 mL). The organic layer was [M + H]+ (C21H24N3O7S) requires m/z 462.1329, measured m/z
concentrated to afford 5-[(2,5-dioxopyrrolidin-1-yl)oxy]- 462.1331. IR (neat): 3260, 2987, 1770, 1682, 1635, 1532, 1383,
5-oxopentanoic acid (20) (592 mg) as a colourless oil, which was 1351, 1276, 1208, 1154 cmꢃ1. HPLC purity (254 nm): 95%.
used without further purication. The prepared NHS ester (20)
Preparation
of
8.
(6R,7R)-7-[(2R)-2-Acetamido-2-phenyl
(500 mg, 2.183 mmol, 1 equiv.) was then dissolved in anhydrous acetamido]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
DCM (10 mL) under an atmosphere of N2 and cooled in an ice carboxylic acid. A solution of cephalexin monohydrate (200 mg,
bath. Oxalyl chloride (225 mL, 2.621 mmol, 1.2 equiv.) was then 0.548 mmol, 1 equiv.) and DIPEA (143 mL, 0.822 mmol, 1.5 equiv.)
added followed by one drop of DMF. The resultant reaction was stirred at room temperature for 10 min. Acetic anhydride (62 mL,
mixture was allowed to return to room temperature and stirred 0.658 mmol, 1.2 equiv.) was then added and the resultant reaction
for 16 h, aer which it was concentrated under reduced pres- mixture was stirred at room temperature for 4 h. The reaction
sure to afford the acid chloride. In a separate ask, cephalexin mixture was then concentrated under reduced pressure to afford
monohydrate (598 mg, 1.637 mmol, 0.75 equiv.) was suspended a cream solid, which was then triturated using diethyl ether to
in anhydrous MeCN (20 mL) then anhydrous pyridine (329 mL, afford
(6R,7R)-7-[(2R)-2-acetamido-2-phenylacetamido]-3-met
4.093 mmol, 1.9 equiv.) was added. The previously prepared hyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (8)
acid chloride was then dissolved in anhydrous MeCN (10 mL) (220 mg, quantitative) as a white solid. 1H NMR (400 MHz, DMSO-
and added to the cephalexin/pyridine mixture. The reaction was d6) d 9.26 (d, J ¼ 8.3 Hz, 1H), 8.59 (d, J ¼ 8.4 Hz, 1H), 7.43 (d, J ¼
then stirred at room temperature for 24 h aer which it was 6.9 Hz, 2H), 7.36–7.22 (m, 3H), 5.69 (d, J ¼ 8.3 Hz, 1H), 5.55 (dd, J ¼
concentrated under reduced pressure and the residue was trit- 8.3, 4.6 Hz, 1H), 4.91 (d, J ¼ 4.7 Hz, 1H), 3.41 (d, J ¼ 17.5 Hz, 1H),
urated in EtOAc to afford a cream solid. The solid was then 3.19 (d, J ¼ 17.9 Hz, 1H), 1.94 (s, 3H), 1.91 (s, 3H). 13C NMR (101
recrystallised from MeCN to afford (6R,7R)-7-[(2R)-2-{5- MHz, DMSO-d6) d 170.9, 169.0, 164.0, 163.5, 138.4, 128.2, 127.6,
[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxo
pentanamido}-2-phenylacetamido]-3-methyl-8-oxo-5-thia-1-
127.2, 58.3, 57.1, 55.6, 28.7, 22.4, 19.4. Two carbon resonances not
observed/coincident. HRMS: exact mass calculated for [M + H]+
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (6) (501 mg, 52% (C18H20N3O5S) requires m/z 390.1116, measured m/z 390.1115. IR
1
over 3 steps) as a cream solid. H NMR (400 MHz, DMSO-d6)
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(neat): 3283, 1761, 1640, 1538, 1497, 1374, 1298, 1188, 1125 cmꢃ1
HPLC purity (254 nm): 98%.
.
carbonyl]amino}pentanoate (22) (258 mg, 0.822 mmol, 1.5
equiv.) was then added and the reaction was allowed to stir at
Preparation of 9. (6R,7R)-7-[(2R)-2-{[(Tert-butoxy)carbonyl] room temperature for 16 h. The reaction mixture was concen-
amino}-2-phenylacetamido]-
trated under reduced pressure and the residue dissolved in
3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic
EtOAc. The organic solution was washed with 0.01 M HCl (3 ꢂ
acid.
A solution of cephalexin monohydrate (500 mg, 20 mL), then dried (MgSO4), and concentrated under reduced
1.370 mmol, 1 equiv.) and Et3N (190 mL, 1.370 mmol, 1 equiv.) pressure to afford a pale yellow solid. Trituration in diethyl
was stirred at room temperature for 10 min. Boc2O (388 mg, ether afforded (6R,7R)-7-[(2R)-2-(5-{[(tert-butoxy)carbonyl]
1.781 mmol, 1.3 equiv.) was then added and the resultant amino}pentanamido)-2-phenylacetamido]-
reaction mixture was stirred at room temperature for 3 h. The 3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic
1
reaction mixture was then concentrated under reduced pressure acid (23) (169 mg, 56%) as a cream solid. H NMR (400 MHz,
and the residue dissolved in DCM (20 mL). This organic solu- DMSO-d6) d 9.27 (d, J ¼ 8.3 Hz, 1H), 8.49 (d, J ¼ 8.3 Hz, 1H), 7.41
tion was then washed with 0.1 M HCl (2 ꢂ 20 mL) and brine (1 (d, J ¼ 7.2 Hz, 2H), 7.34–7.22 (m, 3H), 6.77 (t, J ¼ 5.7 Hz, 1H),
ꢂ 20 mL). The organic layer was collected, dried (MgSO4) and 5.67 (d, J ¼ 8.2 Hz, 1H), 5.60 (dd, J ¼ 8.3, 4.6 Hz, 1H), 4.94 (d, J ¼
concentrated under reduced pressure. The residue was then 4.6 Hz, 1H), 3.45 (d, J ¼ 18.3 Hz, 1H), 3.27 (d, J ¼ 18.3 Hz, 1H),
triturated using diethyl ether to afford (6R,7R)-7-[(2R)-2- 2.86 (q, J ¼ 6.6 Hz, 2H), 2.18 (t, J ¼ 7.3 Hz, 2H), 1.97 (s, 3H), 1.44
{[(tert-butoxy)carbonyl]amino}-2-phenylacetamido]-
3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic
(p, J ¼ 7.0 Hz, 2H), 1.35 (s, 9H), 1.33–1.27 (m, 2H). 13C NMR (101
MHz, DMSO-d6) d 172.0, 170.9, 164.1, 163.5, 155.6, 138.4, 129.8,
acid (9) (608 mg, 93%) as a cream solid. 1H NMR (400 MHz, 128.2, 127.6, 127.1, 122.7, 77.4, 58.4, 57.2, 55.5, 47.5, 34.6, 29.2,
chloroform-d) d 9.57 (s, 1H), 7.45–7.23 (m, 5H), 7.18 (s, 1H), 6.07 28.9, 28.3, 22.7, 19.4. HRMS: exact mass calculated for [M + Na]+
(d, J ¼ 7.2 Hz, 1H), 5.65 (s, 1H), 5.34 (d, J ¼ 7.3 Hz, 1H), 4.90 (d, J (C26H34N4NaO7S) requires m/z 569.2040, measured m/z
¼ 4.5 Hz, 1H), 3.38 (d, J ¼ 18.3 Hz, 1H), 3.03 (d, J ¼ 18.4 Hz, 1H), 569.2041. IR (neat): 3284, 2931, 1764, 1639, 1520, 1452, 1364,
2.09 (s, 3H), 1.40 (s, 9H). 1H consistent with previously reported 1221, 1164, 1067, 1040 cmꢃ1
.
data.38 13C NMR (101 MHz, chloroform-d) d 171.1, 164.2, 163.7,
To a solution of (6R,7R)-7-[(2R)-2-(5-{[(tert-butoxy)carbonyl]
155.8, 138.0, 131.9, 129.1, 128.6, 127.4, 122.6, 81.1, 59.2, 57.9, amino}pentanamido)-2-phenylacetamido]-3-methyl-8-oxo-5-thia-
57.6, 30.3, 28.4, 20.1. HRMS: exact mass calculated for [M + H]+ 1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (23) (100 mg,
(C21H26N3O6S) requires m/z 448.1537, measured m/z 448.1546. 0.183 mmol, 1 equiv.) in DCM (4 mL, 0.04 M) was added and
IR (neat): 3324, 2977, 1771, 1682, 1496, 1454, 1366, 1237, 1160, 0.1 mL triethyl silane followed by 0.5 mL TFA. The reaction mixture
1049 cmꢃ1. HPLC purity (254 nm): 98%.
was stirred at room temperature for 3 h, aer which it was
Preparation of 10. (6R,7R)-7-[(2R)-2-(5-Aminopentanamido)- concentrated to an orange oil. The residue was then azeotroped
2-phenylacetamido]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]
with DCM (3 ꢂ 10 mL) to afford a cream solid. This solid was
oct-2-ene-2-carboxylic acid. To a stirred solution of 5-(Boc- triturated with diethyl ether to afford (6R,7R)-7-[(2R)-
amino)pentanoic acid (21) (1 g, 4ꢁ.608 mmol, 1 equiv.) in anhy- 2-(5-aminopentanamido)-2-phenylacetamido]-3-methyl-8-oxo-
drous DCM (30 mL, 0.2 M) at 0 C, was added NHS (583 mg, 5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (10) (77 mg,
5.069 mmol, 1.1 equiv.). A solution of DCC (1.2 g, 5.991 mmol, 94%) as a cream solid. 1H NMR (400 MHz, DMSO-d6) d 9.31 (d, J ¼
1.3 equiv.) in DCM (10 mL) was added slowly. The reaction 8.4 Hz, 1H), 8.57 (d, J ¼ 8.2 Hz, 1H), 7.67 (br. s, 2H), 7.47–7.41 (m,
mixture was then allowed to warm to room temperature. Aer 2H), 7.35–7.26 (m, 3H), 5.69 (d, J ¼ 8.2 Hz, 1H), 5.62 (dd, J ¼ 8.4,
16 h, the reaction was ltered and the lter cake washed with 4.6 Hz, 1H), 4.96 (d, J ¼ 4.7 Hz, 1H), 3.47 (d, J ¼ 18.8 Hz, 1H), 3.28
DCM. The ltrate was concentrated under reduced pressure and (d, J ¼ 18.2 Hz, 1H), 2.77 (br. s, 2H), 2.31–2.22 (m, 2H), 1.98 (s, 3H),
the residue was puried by column chromatography, eluting 1.59–1.48 (m, 4H). 13C NMR (101 MHz, DMSO-d6) d 171.6, 170.9,
with 10–60% EtOAc/petroleum ether, to afford a white oil. 164.1, 163.5, 138.3, 129.6, 128.2, 127.6, 127.1, 122.7, 58.4, 57.2,
Trituration in diethyl ether afforded 2,5-dioxopyrrolidin-1-yl 5- 55.5, 38.7, 34.1, 28.9, 26.7, 22.1, 19.4. HRMS: exact mass calculated
{[(tert-butoxy)carbonyl]amino}pentanoate (22) (1.37 g, 95%) as for [M + H]+ (C21H27N4O5S) requires m/z 447.1697, measured m/z
1
a white solid. H NMR (400 MHz, chloroform-d) d 4.59 (br. s, 447.1698. IR (neat): 3250, 3050, 2939, 1763, 1639, 1525, 1363, 1184,
1H), 3.16 (q, J ¼ 6.7 Hz, 2H), 2.84 (d, J ¼ 3.7 Hz, 4H), 2.64 (t, J ¼ 1130, 1070 cmꢃ1. HPLC purity (254 nm): 97%.
7.3 Hz, 2H), 1.78 (p, J ¼ 7.4 Hz, 2H), 1.60 (p, J ¼ 7.0 Hz, 2H), 1.44
(s, 9H). H is consistent with previously reported data.39 13C
NMR (101 MHz, chloroform-d) d 169.3, 168.6, 156.1, 79.4, 40.0,
1
Biology
34.1, 29.2, 28.5, 25.7, 25.1, 21.9. HRMS: exact mass calculated
Materials. AmpC (Uniprot ID: P00811) was purchased from
for [M + H]+ (C14H22N2NaO6) requires m/z 337.1370, measured Abcam plc (Cambridge, UK). PBP4 (Uniprot ID: P32959) was
m/z 337.1371. IR (neat): 3318, 2975, 2932, 2851, 1812, 1777, purchased from Generon (Slough, UK).
1726, 1682, 1626, 1572, 1514, 1364, 1274, 1201, 1170, 1069,
1056, 1008 cmꢃ1
Expression and purication of PBP3. A truncated version of
the sI gene formed of residues W44-S588, encoding only the
.
Et3N (114 mL, 0.822 mmol, 1.5 equiv.) was added to soluble domain, was amplied from E. coli BW25113. The
a suspension of cephalexin monohydrate (200 mg, 0.548 mmol, resulting construct was inserted into the vector pBADnLIC2005,40
1 equiv.) in MeCN (50 mL). Aer 5 min dissolution occurred to introducing an N-terminal deca-histidine tag when expressed.
give a yellow solution. 2,5-Dioxopyrrolidin-1-yl 5-{[(tert-butoxy) The resulting vector was transformed into E. coli MC1061 for
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expression. Cultures for protein expression were grown in 1 L of an OD600 of between 0.4–0.6 before induction with 0.01% (w/v)
Luria–Bertani (LB) broth at 37 ꢁC on an orbital shaker. Expression L-arabinose. The induced culture was allowed to grow for 18 h at
ꢁ
was induced by addition of 0.01% L-arabinose during mid-log 30 C. The resulting culture was harvested by centrifugation at
phase of growth. Cultures were further incubated for 16 h at 5000g. The pellet was then resuspended in 30 mL of sterile SET
20 ꢁC, then the cells were harvested by centrifugation. Cell pellets buffer (0.5 M sucrose, 5 mM EDTA, 50 mM Tris–HCl pH 7.8).
were resuspended in 50 mM KPi pH 7.8, 200 mM NaCl, 10 mM 13 mg of lysozyme was added to the mixture before incubation
imidazole, 20% glycerol with 1 mM phenylmethylsulfonyl uo- for 1 h at 30 ꢁC. To isolate the periplasmic fraction, the treated
ride followed by sonication. The lysate was claried by centrifu- sample was then claried by centrifugation at 27 000g. The
gation before loading onto
a HisTrap HF column (GE supernatant was dialysed into 50 mM KPi pH 7.8, 200 mM NaCl
Healthcare). To remove any pre-bound ligands, refolding puri- over 18 hours at 4 ꢁC. Aer dialysis, the periplasmic fraction was
cation was performed by initially washing with the protein loaded onto an equilibrated 5 mL HisTrap column. The bound
unfolding buffer [2 M guanidine HCl, 50 mM KPi (pH 7.8), His-tagged TEM-1 were washed with 10 column volumes of
200 mM NaCl, 20% glycerol, and 20 mM imidazole] and then wash buffer (50 mM KPi pH 7.8, 200 mM NaCl, 20% glycerol,
performing a gradient wash to the protein refolding buffer 40 mM imidazole). To elute the bound protein, 5 column
[50 mM KPi (pH 7.8), 200 mM NaCl, 20% glycerol, and 20 mM volumes of elution buffer (50 mM KPi pH 7.8, 200 mM NaCl,
imidazole] before the elution of the protein using the elution 20% glycerol, 500 mM imidazole) was owed through the
buffer [50 mM KPi (pH 7.8), 200 mM NaCl, 20% glycerol, and column while collecting the ow through. For downstream
500 mM imidazole]. The eluted protein was buffer-exchanged to analysis, the protein was buffer exchanged into 50 mM KPi pH
the buffer 50 mM KPi (pH 7.8), 200 mM NaCl using a HisTrap 7.8, 200 mM NaCl using the HisTrap desalting column.
Desalting (GE Healthcare) column.
Expression and purication of CTX-M-15. CTX-M-15 (Uni-
Thermal shi assay. The thermal shi assay was carried out prot ID: Q9EXV5) was synthesised using the protocol described
using the Protein Thermal Shi™ assay kit (Applied Bio- above in the expression and purication of TEM-1, with the
systems). 3 mM of puried E. coli PBP3 was incubated with the b- following deviations: the gene coding for NDM-1 was syn-
lactam analogues at 300 mM concentration in a mixture con- thesised as a gBlock (IDT). Following induction with L-arabi-
ꢁ
taining the Protein Thermal Shi™ Dye. The samples were then nose, 1 L cultures were incubated for 20 h at 20 C.
heated in a StepOnePlus™ Real-Time PCR System from 25 to
Expression and purication of NDM-1. NDM-1 (Uniprot ID:
95 ꢁC at a rate of 1 ꢁC minꢃ1. Tests were carried out in triplicate C7C422, residues G29-R270) was synthesised using the protocol
and the averages plotted as the negative rst derivative vs. described above in the expression and purication of TEM-1,
temperature. Reference wells, i.e. solutions consisting only of with the following deviations: the gene coding for NDM-1 was
only PBP3 with dye, PBP3 only, and dye only, were used as synthesised as a gBlock (IDT). Following induction with L-
ꢁ
controls. Melting temperature (Tm) values were determined with arabinose, 1 L cultures were incubated for 20 h at 20 C.
and without each compound, and the change in melting
Enzyme kinetics. The rate of b-lactamase-mediated hydro-
temperature (DTm) was obtained, Fig. 2. The thermal shi assay lysis was montiored using a previously reported absorbance
with PBP4 was carried out using the protocol described for assay.19 In a 96-well plate, 200 mL of the test compounds at the
PBP3, with the follow deriviatisation: 500 nM of puried Bacillus desired concentrations with the relevant b-lactamases were
subtilis PBP4 was incubated with 50 mM of the b-lactam incubated and the absorbance at 260 nm was monitored.
analogues.
Experiments using NDM-1 included 1 equivalent of ZnCl2. All
To determine the approximate affinity of PBP3 for studies were carried out using one batch of puried enzyme.
compounds 1 to 12, 2 mM of puried E. coli PBP3 was incubated Experiments were carried out at 37 ꢁC and measurements were
with a range of concentrations of each compound. The mixtures taken every 60 s (Epoch 2 Microplate Spectrophotometer, Bio-
were incubated at room temperature for 25 minutes prior to the Tek) for 1 h. Each sample was carried out in triplicate and the
start of the protein thermal shi assay program on the qPCR average was used for further calculations. kcat/Km was deter-
machine. The signals from the hydrophobic uorescent dye mined from v ¼ (kcat/Km)[E][S]. The initial velocity for each
were monitored as the mixtures were heated from 35 to 70 ꢁC at compound was determined and used as “v”.
a ramp rate of 0.3%. The Tm values of PBP3 incubated with each
MIC growth assay. Stock solutions of each compound were
compound at the various concentrations were recorded. The prepared in 50% DMSO/water at ꢂ100 the nal concentration.
relative changes in the Tm values, relative to the highest Tm In a 96-well plate, 2 mL of the test compounds were added to
change seen for each compound, are reported as a ratio in the each well. Each plate included the positive control (750 mg mLꢃ1
ESI.† The concentration at which the Tm rose to >Tm1/2 was used chloramphenicol), the negative control (50% DMSO/water) and
to approximate the relative affinities.
media only wells. To each test well was then added 198 mL of the
Expression and purication of TEM-1. TEM-1 (Uniprot ID: required bacteria stock (OD 0.05). E. coli assays were carried out
A5PHA6) was cloned into pBKR, a pBADcLIC2005 derivative using LB broth, S. aureus were carried out using tryptic soy
ꢁ
with a kanamycin resistance cassette in place of the ampicillin broth (TSB). Plates were incubated at 37 C and the OD600 was
resistance cassette. The resulting plasmid, pBKR-TEM1, was measured (Epoch 2 Microplate Spectrophotometer, BioTek)
transformed into the expression strain E. coli MC1061. Starting every 30 min over a 17 h period. Tests were carried out in trip-
cultures were grown in 10 mL LB broth at 37 ꢁC overnight with licate, the background absorbance was removed using reference
shaking. A litre of expression culture was prepared and grown to wells, and the averages were determined. Dose curves were
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plotting using the OD600 aer 16 h growth vs. compound 16 S. Sainsbury, L. Bird, V. Rao, S. M. Shepherd, D. I. Stuart,
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Funding
This work was funded by EPSRC (EP/P02324X/1).
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Conflicts of interest
There are no conicts to declare.
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We gratefully acknowledge nancial support from the EPSRC
(EP/P02324X/1). We thank Heather Fish for her assistance in
obtaining NMR spectra. We thank Karl Heaton for his assis-
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36494 | RSC Adv., 2020, 10, 36485–36494
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