A Synthetic Dual Drug Sideromycin Induces Gram-Negative Bacteria to Commit Suicide with a Gram-Positive Antibiotic

Article abstract of DOI:10.1021/acs.jmedchem.8b00218

Many antibiotics lack activity against Gram-negative bacteria because they cannot permeate the outer membrane or suffer from efflux and, in the case of β-lactams, are degraded by β-lactamases. Herein, we describe the synthesis and studies of a dual drug conjugate (1) of a siderophore linked to a cephalosporin with an attached oxazolidinone. The cephalosporin component of 1 is rapidly hydrolyzed by purified ADC-1 β-lactamase to release the oxazolidinone. Conjugate 1 is active against clinical isolates of Acinetobacter baumannii as well as strains producing large amounts of ADC-1 β-lactamase. Overall, the results are consistent with siderophore-mediated active uptake, inherent activity of the delivered dual drug, and in the presence of β-lactamases, intracellular release of the oxazolidinone upon cleavage of the cephalosporin to allow the freed oxazolidinone to inactivate its target. The ultimate result demonstrates that Gram-positive oxazolidinone antibiotics can be made to be effective against Gram-negative bacteria by β-lactamase triggered release.

Full text of DOI:10.1021/acs.jmedchem.8b00218

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A Synthetic Dual Drug Sideromycin Induces Gram-Negative
Bacteria to Commit Suicide with a Gram-Positive Antibiotic
Rui Liu, Patricia A. Miller, Sergei B. Vakulenko, Nichole K. Stewart, William C. Boggess, and Marvin J. Miller
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00218 • Publication Date (Web): 19 Mar 2018
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A Synthetic Dual Drug Sideromycin Induces Gramꢀ
Negative Bacteria to Commit Suicide with a Gramꢀ
Positive Antibiotic
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Rui Liu, Patricia A. Miller, Sergei B. Vakulenko, Nichole K. Stewart, William C. Boggess and
Marvin J. Miller*
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556.
ABSTRACT. Many antibiotics lack activity against Gramꢀnegative bacteria because they cannot
permeate the outer membrane or suffer from efflux and, in the case of βꢀlactams, are degraded by
βꢀlactamases. Herein, we describe the synthesis and studies of a dual drug conjugate (1) of a
siderophore linked to a cephalosporin with an attached oxazolidinone. The cephalosporin
component of 1 is rapidly hydrolyzed by purified ADCꢀ1 βꢀlactamase to release the
oxazolidinone. Conjugate 1 is active against clinical isolates of Acinetobacter baumannii as well
as strains producing large amounts of ADCꢀ1 βꢀlactamase. Overall, the results are consistent
with siderophoreꢀmediated active uptake, inherent activity of the delivered dual drug and, in the
presence of βꢀlactamases, intracellular release of the oxazolidinone upon cleavage of the
cephalosporin to allow the freed oxazolidinone to inactivate its target. The ultimate result
demonstrates that Gramꢀpositive oxazolidinone antibiotics can be made to be effective against
Gramꢀnegative bacteria by βꢀlactamase triggered release.
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INTRODUCTION
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The need for new antibiotics and strategies for treating multiꢀdrug resistant bacterial infections
is urgent!1 While bacterial resistance has developed to essentially all of our current antibiotics,
few new antibiotics have been developed over the last several decades. A primary cause of drug
resistance is the overuse of antibiotics that can result in alteration of microbial permeability,
alteration of drug target binding sites, induction of enzymes that destroy antibiotics (ie., βꢀ
lactamases) and even induction of efflux mechanisms.2,3,4 Herein, we report that a dual drug
sideromycin (siderophoreꢀcephalosporinꢀoxazolidinone conjugate 1, Figure 1) circumvents these
resistance mechanisms by siderophoreꢀmediated active uptake promoting inherent activity of the
delivered antibiotic combination. Furthermore, βꢀlactamase induced destructive hydrolysis of the
cephalosporin component initiates intracellular release of the oxazolidinone warhead (3) thus
allowing this normally Gram positive only class of antibiotics to be effective against targeted
Gramꢀnegative bacteria, including cephalosporinaseꢀproducing Acinetobacter baumannii. The
World Health Organization (WHO) recently labeled infections due to βꢀlactamase producing
strains of A. baumannii and Pseudomonas aeruginosa as being of critical concern worldꢀwide.5
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Figure 1. Siderophoreꢀcephalosporinꢀoxazolidinone conjugate 1 and proposed cephalosporinase
triggered release of oxazolidinone 3.
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In order to develop and sustain an infection, bacteria must sequester iron that is needed for
essential physiological processes.6 In fact, bacteria require micromolar concentrations of iron yet
the concentration of free iron (Fe+3) in an aerobic, neutral pH environment is ~10ꢀ18 molar.6
Thus, bacteria synthesize and excrete iron chelating agents called siderophores that bind ferric
iron with high affinity.7 The resulting complex is then recognized by specific outer membrane
proteins that initiate internalization by an active transport of the ferric siderophore. Subsequent
reductive or hydrolytic release of the iron then makes it available to the bacteria. Since mammals
do not utilize siderophores, exploitation of this essential bacterial reliance on iron uptake for
virulence has potential for development of selective new antibiotic therapies.8
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Studies of natural and synthetic siderophoreꢀantibiotic conjugates (sideromycins) are of
interest for development of potential Trojan Horse antibiotics to especially target multidrug
resistant bacterial infections.9 Most reported synthetic sideromycins consist of a siderophore or
siderophore mimic directly attached to a βꢀlactam antibiotic. Many such constructs, including
representatives (4-11) shown in Figure 2,10,11,12,13,14,15 show enhanced and strainꢀselective
antibacterial activity because of siderophoreꢀreceptor mediated active transport and reduced
susceptibility to antibiotic efflux mechanisms.10,16 While some of these conjugates also are less
prone to deactivation by βꢀlactamases, the activity of other more susceptible conjugates is
augmented by coꢀadministration of βꢀlactamase inhibitors.
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Figure 2. Selected representative examples of synthetic sideromycins containing βꢀlactam
warheads.
To further circumvent problems associated with βꢀlactamases, siderophore conjugates with
nonꢀβꢀlactam antibiotics have been designed and synthesized. Some siderophore conjugates with
nonꢀreleasable linkers to other antibiotics, especially those with cytoplasmic targets, might not be
recognized by the targets while the warhead remains attached to the siderophore and thus do not
have enhanced activity.17 However, representative compounds shown in Figure 3 have
remarkably potent activity against bacteria targeted by the bacteriaꢀselective siderophore
component. In fact, this strategy allows delivery of antibiotics, regardless of size or charge, into
microbial cells and induces efficacy of these drugs against pathogens that are normally not
susceptible to the antibiotic alone. For example, while artemisinin, a small uncharged
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antimalarial drug, has no antiꢀtuberculosis activity itself, its mycobactin conjugate 12, is a potent
antiꢀTB agent that is completely selective for Mycobacterium tuberculosis (Mtb).18 It is not
active against any other strain of mycobacteria nor any other Gramꢀnegative or Gramꢀpositive
bacteria because the siderophore component is specific for Mtb. In another case, the mixed
ligand, bisꢀcatechol, monohydroxamate, conjugate (13) of daptomycin, a large, polyanionic
antibiotic, confers potent and selective in vitro and in vivo activity of daptomycin against multiꢀ
drug resistant strains of A. baumannii, even though the parent daptomycin itself is only active
against Gramꢀpositive bacteria.19
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Figure 3. Non βꢀlactam synthetic and highly targeted sideromycins.
In all cases shown in Figures 2 and 3, the synthetic sideromycins were made by direct coupling
of the siderophore component to the antibiotic warhead and no intracellular drug release process
was necessary since the intact sideromycin is recognized by the antibiotic target. However, as
shown in Figure 4, most natural sideromycins require an intracellular process that allows release
of the antibiotic component once the conjugate is sequestered and actively transported into the
bacterial cell.20 Albomycins (14)21 and salmycins (15)22 are the most well studied natural
sideromycins.23 The tRNA synthetase inhibitor portion of albomycin and the aminoglycoside
component of salmycin have no antibiotic activity alone, presumably since they cannot reach
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their cytoplasmic targets. However, the natural siderophore conjugates are potently active. In
the case of the albomycins, bacterial peptidases cleave the seryl tRNA synthetase inhibitor from
the conjugate after active transport allowing the inhibitor to exert its toxic antibiotic effect both
in vitro and in vivo. Studies of albomycin have demonstrated clinical efficacy.21,24,25 Intracellular
reductive removal of the iron from salmycins to desferri salmycins (16) may trigger
intramolecular cyclization with concomitant release of the aminoglycoside antibiotic. Thus,
further expansion of the sideromycinꢀbased Trojan Horse approach must consider the nature of
the linker between the iron binding component and the warhead to be delivered.
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O
O
O
NH
N
O
N
N
O
O
NH
N
O
O
Fe
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OH
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HN
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- Fe
OH
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(CH2)n-OH
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Peptidase required
for drug release
OH
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OH
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OH
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16, desferri salmycins
Figure 4. The natural sideromycins, albomycins and salmycins, require drug release.
Early attempts to incorporate releasable linkers included introduction of randomly
hydrolysable linkers, but these are prone to premature release before bacterial assimilation.26
More recent efforts include examples of esteraseꢀtriggered27 and reductiveꢀcyclization28 ꢀinduced
release following bacterial internalization of the synthetic sideromycin. As indicated earlier, βꢀ
lactams conjugated to siderophores can still be susceptible to hydrolysis by destructive βꢀ
lactamase enzymes. Many studies have demonstrated that nonꢀsiderophore derivatives of
appropriate covalent combinations of cephalosporins or penicillins, themselves, with other drugs
create dual action agents with activity due to the βꢀlactam component but, if that is susceptible to
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a βꢀlactamase, the destructive ring opening results in release of the additional attached
drug.29,30,31,32,33 βꢀLactamaseꢀtriggered hydrolysis of cephalosporinꢀantitumor conjugates has
also been widely studied,34 indicating that cephalosporinases can recognize and cleave
cephalosporins even when conjugated to large molecules. Thus, we reasoned that a siderophore
conjugate of a cephalosporin with an additional antibiotic attached to the 3’ꢀmethyl position
would create a new class of multiple action sideromycin (1) that could target both, bacteria that
are susceptible to its βꢀlactam component and the βꢀlactamaseꢀproducing bacteria which would
hydrolyze the βꢀlactam thus releasing the additional antibiotic warhead (3) capable of killing
bacteria. Thus, when susceptible to βꢀlactamases, the cephalosporin component should serve as
a releasable linker. Essentially, the normally deleterious βꢀlactamase enzyme would be utilized
to induce the targeted bacteria to commit suicide.
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In order to test this, we decided to incorporate an oxazolidinone as the antibiotic that would
ultimately be released upon hydrolytic opening of the cephalosporin linker. Oxazolidinones, like
eperezolid,35,36 have a ribosomal target and either cannot permeate Gramꢀnegative outer
membranes or, if they do get into Gramꢀnegative bacteria, they are rapidly effluxed.36 Thus,
while oxazolidinone antibiotics are effective against clinically important Gramꢀpositive bacteria,
they are not effective against most Gramꢀnegative bacteria. We anticipated that siderophoreꢀ
mediated transport of oxazolidinones would allow for the efficient passage of these drugs
through the bacterial outer membrane. We also hypothesized that incorporation of a
cephalosporin linker that is prone to hydrolysis by bacterial βꢀlactamases would allow efficient
release of oxazolidinone so it can reach its intracellular target. Enhanced concentration of the
delivered oxazolidinone might also help to overwhelm efflux pumps to allow the drug to exert its
lethal effect.
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RESULTS AND DISCUSSION
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The specific siderophoreꢀcephalosporinꢀoxazolidinone 1 was designed to test our hypothesis
related to siderophoreꢀmediated dual drug delivery and βꢀlactamase triggered release of an
oxazolidinone to Gramꢀnegative bacteria. The synthesis of 1 required access to all three
components, the siderophore, an appropriately functionalized cephalosporin and an
oxazolidinone with suitable pendant functionality for coupling to the cephalosporin moiety. As
shown in detail below (Scheme 1), the primary alcohol of commercially available eperezolid (17)
was converted to the mesylate which was then displaced with potassium phthalimide to provide
protected amine (18). Standard deprotection with MeNH2 provided oxazolidinone 3 with a free
amine for subsequent reaction with an appropriately activated cephalosporin (21).35
Cephalosporin 21 was prepared in four steps from readily available 3ꢀaminocephalosporanic acid
(19). Thus, 19 was first converted to the tꢀbutyl ester by BF3ꢀcatalyzed reaction with tꢀbutyl
acetate. DꢀPhenylglycine, a common side chain in cephalosporins, including cefaclor,37 was
protected with a Boc group and then coupled to the free amine of the tꢀbutyl cephalosporinate.
Finally, the 3’ꢀacetoxy group was removed enzymatically using Candida lipase B38 to give
protected cephalosporin 20 with a free 3’ꢀhydroxyl group. Reaction of the hydroxyl group with
1,2,2,2ꢀtetrachloroethyl carbonochloridate gave the isolable activated cephalosporin 21.
Treatment of cephalosporin carbonate 21 with aminooxazolidinone 3 followed by reaction with
TFA to remove the Boc group and tꢀbutyl ester gave cephalosporin oxazolidinone 22.
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Protected bisꢀcatechol 2410 was first saponified to give the free acid which was
hydrogenolytically debenzylated and then treated with EDC/NHS to give active ester 25. Direct
reaction of the active ester with cephalosporin oxazolidinone 22 gave the final conjugate 1.
Separate coupling of the activated siderophore with Dꢀphenylglycyl cephalosporin (23) and
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aminooxazolidinone (3) gave the corresponding siderophoreꢀcephalosporin (26) and siderophoreꢀ
oxazolidinone (27) conjugates for use as controls in subsequent bioassays.
Scheme 1. Syntheses of siderophore conjugates.
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With the final siderophoreꢀcephalosporinꢀoxazolidinone conjugate (1) along with the separate
cephalosporin and oxazolidinone conjugates (26 and 27) as well as the corresponding
cephalosporin (23), oxazolidinone (3) and eperezolid (17) available, we performed enzymatic
and antiꢀbacterial assays to test our hypothesis. Our plan and design critically depended on the
susceptibility of conjugate 1 to the hydrolytic activity of βꢀlactamases from Gramꢀnegative
bacteria. Thus, we evaluated whether the oxazolidinone could be efficiently released from the
final siderophoreꢀcephalosporinꢀoxazolidinone conjugate (1) as a result of cephalosporin
hydrolysis by a βꢀlactamase. For this purpose, we chose one of the ADCꢀtype βꢀlactamases
(ADCꢀ1), as ADC enzymes are intrinsic to A. baumannii, an important clinical pathogen, and are
capable of hydrolyzing cephalosporin antibiotics. As anticipated, incubation of the purified
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ADCꢀ1 with conjugate 1 resulted in its rapid and complete hydrolysis and release of the
oxazolidinone, as determined by LC/MS (Figure 5).
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Figure 5. Cephalosporinase (ADCꢀ1)ꢀinduced hydrolytic release of oxazolidinone 3 from
conjugate 1. LC/MS Base Peak Chromatograms showing pure conjugate (A), product formed
following reaction with ADCꢀ1 (B) and ADCꢀ1 only (C).
Next, we tested whether the siderophoreꢀcephalosporinꢀoxazolidinone conjugate (1) exhibits
antimicrobial activity against A. baumannii producing the ADCꢀ1 βꢀlactamase. For this purpose,
we used A. baumannii ATCC 17978 which produces a small amount of the chromosomallyꢀ
encoded enzyme and the same strain that produces a large amount of ADCꢀ1 from the pNT255
vector (Table 1). As expected, the nonꢀconjugated oxazolidinones (3 and 17) themselves as well
as the cephalosporinꢀoxazolidinone (22) and simple cephalosporin (23) were not active against
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this strain of A. baumannii, due to inadequate delivery or efflux. The siderophoreꢀoxazolidinone
(27, with a nonꢀreleasable linkage between the transport agent and antibiotic component) also
was not active, suggesting that without release from the siderophore, the oxazolidione either
could not reach its target or was not recognized by its target. The siderophoreꢀcephalosporin (26)
was remarkably active against A. baumannii producing a low amount of ADCꢀ1. Dual conjugate,
1, retained similarly potent activity which indicates that activity of both compounds (26 and 1) is
due to the cephalosporin. The dramatic improvement in MICs (more than 125ꢀfold) of the
cephalosporin or cephalosporinꢀoxazolidinone when conjugated to the siderophore indicates that
the antibiotics are very efficiently delivered inside the bacterial cell.
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When the production of the enzyme was significantly increased by the introduction of the
plasmidꢀencoded βꢀlactamase (pNT255:adc1), this A. baumannii was rendered highly resistant to
the siderophoreꢀcephalosporin (26), an indication that large amounts of ADCꢀ1 protected
bacteria by hydrolyzing the incoming cephalosporin antibiotic, as anticipated from our studies
that showed that the isolated ADC enzyme rapidly hydrolyzed the cephalosporin component.
Addition of the oxazolidinone warhead to the siderophoreꢀcephalosporin to result in final
conjugate 1 significantly decreased the MIC (at least 8ꢀfold) when compared to the siderophoreꢀ
cephalosporin. These results are consistent with the scenario in which cleavage of the
cephalosporin by the ADCꢀ1 βꢀlactamase results in release of the oxazolidinone that can then
reach its target, perhaps minimize the influence of efflux pumps and allow the oxazolidinone to
exert a lethal effect on targeted Gramꢀnegative bacteria.
Table 1. Activity (in µM) of tested compounds against A. baumannii ATCC 17978 without and
with plasmidꢀencoded βꢀlactamase.
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17
27
22
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1
siderophore
siderophore
+
oxazolidinone oxazolidinone
cephalosporin
+
siderophore
+
cephalosporin
+
oxazolidinone eperezolid
cephalosporin
cephalosporin
+
oxazolidinone
A. baumannii
ATCC 17978
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>500
>500
>50
>50
>50
>50
>50
>50
>50
>50
0.4
0.4
0.4
A. baumannii
ATCC 17978
pNT255
0.4
A. baumannii
ATCC 17978
pNT225:
>500
>50
>50
>50
>50
>50
6
ADCꢀ1
Subsequently, we evaluated activity of our compounds against four clinical isolates of A.
baumannii (ATCC 17961, ATCC BAA1793, ATCC BAA1797 and ATTC BAA1800). All four
A. baumannii isolates were resistant to compounds 3, 22, 23, and 27. Strain ATCC 17961 was
susceptible to both 26 and 1, indicating lack of or low βꢀlactamase content and/or activity.
Conjugate 26, with the cephalosporin alone, had detectable activity against two clinical isolates
(ATCC BAA1793 and 1800). Notably, the final siderophoreꢀcephalosporinꢀoxazolidinone
conjugate 1 was very active against all of the clinical isolates, especially the siderophoreꢀ
cephalopsorin (26) resistant clinical strain ATCC BAA 1797 (Table 2).
Table 2. Activity (in µM) of compounds against A. baumannii and other bacteria tested.
strain
3
22
23
27
26
1
siderophore
cephalosporin
+
oxazolidinone
siderophore
+
oxazolidinone cephalosporin
siderophore
+
+
oxazolidinone
cephalosporin
cephalosporin
+
oxazolidinone
A. baumannii
ATCC 17961
>50
>50
>50
>50
0.8
0.8
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A. baumannii
ATCC BAA
1793
A. baumannii
ATCC BAA
1797
>50
>50
>50
>50
>50
>50
>50
>50
12.5
>50
0.8ꢀ1.6
6.25
9
A. baumannii
ATCC BAA
1800
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12
13
>50
>50
>50
>50
>50
>50
>50
50
>50
>50
>50
25
<0.025
0.4
0.8
E. coli DC0
<0.025
0.2ꢀ0.4
P. aeruginosa
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>50
KW799/wt
Further studies with two other Gramꢀnegative bacteria (Escherichia coli DC0 and
Pseudomonas aeruginosa KW799) revealed that nonꢀconjugated oxazolidinone 3,
cephalosporinꢀoxazolidinone 22, cephalosporin 23, or the siderophoreꢀoxazolidinone 27 were not
active against the E. coli and Pseudomonas aeruginosa tested. However, the siderophoreꢀ
cephalosporin (26, MIC: < 0.025 µM vs E. coli DC0 and 0.4 µM vs P. aeruginosa KW799/wt)
and siderophoreꢀcephalosporinꢀoxazolidinone (1, MIC: < 0.025 µM vs E. coli DC0 and 0.2ꢀ0.4
µM vs P. aeruginosa KW799/wt) were remarkably active.
Conclusion
Overall, these results are consistent with siderophoreꢀmediated active transport that facilitates
uptake of the cephalosporin (conjugate 26) to improve its activity relative to the cephalosporin
(23) itself. The lack of activity of the siderophoreꢀoxazolidinone conjugate (27) reflects previous
studies discussed earlier that indicate cytoplasmic targets do not tolerate intact conjugates to
other drugs without releasable linkers. Notably, the activity and cephalosporinase susceptibility
of the final siderophoreꢀcephalosporinꢀoxazolidinone conjugate (1) is consistent with
siderophoreꢀmediated active uptake, inherent activity of the delivered dual drug and, in the
presence of βꢀlactamase, intracellular release of the oxazolidinone upon cleavage of the
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cephalosporin to allow the freed oxazolidinone to inactivate its target. The final result
demonstrates that oxazolidinone antibiotics are active against Gramꢀnegative bacteria when they
are efficiently delivered to their target. Importantly, we chose to demonstrate this approach by
targeting βꢀlactamase producing A. baumannii, which is a pathogen of major concern worldꢀ
wide.5,39
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These studies demonstrate that βꢀlactams, and cephalosporins in particular, can serve as βꢀ
lactamaseꢀtriggered releasable linkers to allow intracellular delivery of Gramꢀpositive antibiotics
to Gramꢀnegative bacteria and thereby provide new methods to inhibit proliferation of multiꢀdrug
resistant bacteria.
Experimental Section
General Methods. All solvents and reagents were obtained from commercial sources and used
without further purification unless otherwise stated. Silica gel (230−400 mesh) was purchased
from Silicycle, Quebec City, Canada. All compounds are >98% pure by HPLC analysis, and
MIC values reported are the average of three individual measurements. All compounds were
1
13
characterized by H and C NMR using Bruker 400 MHz, 500 MHz NMR, and/or Varian 600
MHz NMR spectrometers. Chemical shifts are reported in ppm (δ) relative to the residual solvent
peak in the corresponding spectra, and coupling constants (J) are reported in hertz (Hz). The
mass spectra values are reported as m/z, and ultrahigh pressure liquid chromatography mass
spectrometry (UPLCMS) analyses of the reaction of 1 with ADC-1 were carried out with a
Bruker MicrOTOFꢀQII, quadrupole timeꢀofꢀflight mass spectrometer coupled via electrospray
ionization with a Dionex Ultimate 3000 RSLC system. A 20 minute binary gradient separation
on a Zorbax® RXꢀC8 narrow bore 2.1x150 mm 5 micron column kept at 37 C was run under the
following conditions: solvent A = water with 0.1% formic acid, solvent B = acetonitrile with
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0.1% formic acid, A:B = 90:10 for 2 minutes followed by a 16 minute linear ramp to A:B 0:100
before returning to initial conditions for 2 minutes. Additional liquid chromatography mass
spectrum (LC/MS) analyses for all compounds, except 1, 26, and 27, were carried out on a
Waters ZQ instrument consisting of chromatography module Alliance HT, photodiode array
detector 2996, and mass spectrometer Micromass ZQ, using a 3 × 50 mm Pro C18 YMC reverse
phase column. Mobile phases: 10 mM ammonium acetate in HPLC grade water (A) and HPLC
grade acetonitrile (B). A gradient was formed from 5% to 80% of B in 10 min at 0.7 mL/min.
The MS electrospray source operated at capillary voltage 3.5 kV and a desolvation temperature
of 300 °C. For compounds 1 and 26, LCMS analysis was performed on a Waters 2695
Separations Module integrated 3100 Mass Detector system employing positive ion ESI mode,
equipped with a Waters 996 Photodiode Array Detector, using a Waters XBridge BEH C18 2.5
µm column (3.0 × 30 mm), DAD detected at 254 nm, at 50 °C. The operation software is
MassLynx Software License V4.1 (Waters, Milford, MA, USA). Mobile phases: 0.1% TFA in
HPLC grade water (A) and 0.06% TFA in HPLC grade acetonitrile (B). A gradient was formed
from 3% to 97% of B in 4 min at 0.5 mL/min. For compound 27, LCMS analysis was performed
on a Waters 2695 Separations Module integrated 3100 Mass Detector system employing positive
ion ESI mode, equipped with a Waters 996 Photodiode Array Detector, using a Waters XBridge
BEH C18 2.5 µm column (3.0 × 30 mm), DAD detected at 254 nm, at 50 °C. The operation
software is MassLynx Software License V4.1 (Waters, Milford, MA, USA). Mobile phases:
0.1% TFA in HPLC grade water (A) and 0.06% TFA in HPLC grade acetonitrile (B). A gradient
was formed from 3% to 97% of B in 4 min at 0.5 mL/min. Reverse phase chromatographic
purification was performed on a Waters 1525 Binary pump equipped with a Waters 2998
Photodiode Array Detector, Waters 2707 Autosampler, and Water Fraction Collector III utilizing
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Empower 3 Chromatography Manager software (Waters, Milford, MA, USA). A Waters
SymmetryPrep C18 7 ꢁm OBD column (300 × 7.8 mm) was used for separation. DAD was
detected at 254 nm at 20 °C, eluting with a linear gradient of 0.5−20% acetonitrile in 0.1%
aqueous TFA (flow rate 5 mL/min).
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(6R,7R)-3-((((2-(4-(4-((S)-5-(Acetamidomethyl)-2-oxooxazolidin-3-yl)-2-
fluorophenyl)piperazin-1-yl)-2-oxoethyl)carbamoyl)oxy)methyl)-7-(2-(2-(N-(4-(2,3-
dihydroxybenzamido)butyl)-2,3-dihydroxybenzamido)acetamido)-2-phenylacetamido)-8-
oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (1).
To a suspension of 22 (0.1 mmol, 88 mg) in anhydrous acetonitrile (5 mL), was added
diisoprpylethylamine (0.2 mmol, 35 µL) at room temperature. To this mixture was added a DMF
solution of 25 (0.1 mmol) and the mixture was stirred for 4h. The reaction was then concentrated
under reduced pressure. The residue was purified using a C18 reverse phase silica gel column,
eluting with a gradient of water and acetonitrile to give 1 (0.01 mmol, 11 mg) as a white solid in
1
10% yield. H NMR (500 MHz, DMSOꢀd6): δ 1.23ꢀ1.29 (m, 3H), 1.46ꢀ1.56 (m, 3H), 1.83 (s,
3H), 2.92ꢀ2.96 (m, 4H), 3.08ꢀ3.16 (m, 2H), 3.40 (t, 2H, J = 5 Hz), 3.5ꢀ3.59 (m, 4H), 3.68ꢀ3.71
(m, 1H), 3.87ꢀ3.93 (m, 2H), 4.08 (t, 1H, J = 10 Hz), 4.14ꢀ4.19 (m, 1H), 4.57ꢀ4.71 (m, 2H), 4.84
(s, 1H), 4.94ꢀ5.12 (m, 2H), 5.46 (s, 1H), 5.59 (s, 1H), 5.70 (brs, 1H), 6.46ꢀ6.77 (m, 5H), 6.87ꢀ
6.90 (m, 1H), 7.07 (t, 1H, J = 10 Hz), 7.17ꢀ7.29 (m, 5H), 7.46ꢀ7.52 (m, 2H), 8.01 (s, 1H), 8.24 (t,
1H, J = 4 Hz), 8.64 (brs, 2H), 8.80ꢀ8.83 (m, 1H), 9.07ꢀ9.11 (m, 1H), 9.40ꢀ9.48 (m, 2H), 12.79ꢀ
13
12.90 (m, 1H), 13.64 (brs, 1H). C NMR (500 MHz, DMSOꢀd6): δ 22.30, 24.68, 25.26, 41.34,
41.92, 43.93, 47.26, 50.12, 50.42, 55.43, 57.39, 58.66, 62.62, 71.40, 106.50, 110.04, 113.65,
114.06, 117.02, 117.40, 117.64, 118.00, 119.73, 124.71, 125.70, 126.72, 126.99, 127.35, 128.05,
133.60, 135.13, 138.02, 149.83, 150.90, 151.06, 151.16, 153.74, 153.92, 155.19, 155.36, 155.95,
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156.14, 157.79, 158.00, 162.60, 164.14, 166.95, 168.86, 169.06, 169.85, 170.47. HRMS (m/z):
[M+Na]+ calcd for C55H59FN10NaO17S, 1205.3665; found, 1205.3656. LC/MS retention time
6.32 min.
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(S)-N-((3-(3-Fluoro-4-(4-glycylpiperazin-1-yl)phenyl)-2-oxooxazolidin-5-
yl)methyl)acetamide (3).
A mixture of 18 (0.59 mmol, 308 mg) and 0.52 mL of 40% methylamine in water (5.9 mmol, 10
eq) and 30 mL of EtOH was heated at reflux for 3h. When the starting material 18 was
consumed, the mixture was concentrated under reduced pressure and the residue was purified
using a silica gel column, eluting with DCM/MeOH (5/1, v/v) to give 3 as a yellow foamy solid
in 43% yield (99 mg, 0.25 mmol). 1H NMR (400 MHz, DMSOꢀd6): δ 1.81 (s, 3H), 2.94ꢀ2.98 (m,
4H), 3.38 (t, 2H, J = 8.0 Hz), 3.65ꢀ3.70 (m, 4H), 3.91ꢀ3.92 (m, 2H), 4.06 (t, 2H, J = 8.0 Hz),
4.67ꢀ4.71 (m, 1H), 7.06 (t, 1H, J = 8.0 Hz), 7.16ꢀ7.19 (m, 1H), 7.47 (d, 1H, J = 12 Hz), 8.05 (brs,
13
2H), 8.24 (t, 1H, J = 8.0 Hz). C NMR (400 MHz, DMSOꢀd6): δ 23.13, 42.12, 42.27, 44.88,
48.03, 50.83, 51.07, 72.27, 107.25ꢀ107.51, 114.86, 120.59, 134.52ꢀ134.62, 135.74ꢀ135.83,
154.16, 154.76, 156.58, 165.33, 170.70. LC/MS (m/z): [m+H]+ calcd for C18H25FN5O4, 394.19;
found, 394.25; retention time: 1.23 min.
(S)-N-((3-(4-(4-(2-(1,3-Dioxoisoindolin-2-yl)acetyl)piperazin-1-yl)-3-fluorophenyl)-2-
oxooxazolidin-5-yl)methyl)acetamide (18)
To a solution of Eperezolid (17) (394 mg, 1 mmol) and Et3N (418 µL, 3 mmol) in 20 mL of
anhydrous DCM at 0oC was added methanesulfonyl chloride (155 µL, 2 mmol) dropwise. The
mixture was then stirred at 0oC for 1h and then at room temperature for 1h. When the starting
material was disappeared, the mixture was washed with 100 mL water, and the aqueous layer
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was extracted with DCM (30 mL). The combined organic layers were dried with Na2SO4,
filtered, then concentrated under reduced pressure to give the corresponding mesylate as a yellow
7
8
1
foamy solid (472 mg, 1 mmol) which was used directly without further purification. H NMR
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(400 MHz, DMSOꢀd6): δ 1.83 (s, 3H), 2.96ꢀ3.00 (m, 4H), 3.07ꢀ3.14 (m, 1H), 3.27 (s, 3H), 3.40
(t, 2H, J = 8.0 Hz), 3.51ꢀ3.62 (m, 4H), 3.69ꢀ3.72 (m, 1H), 4.08 (t, 1H, J = 8.0 Hz), 4.69ꢀ4.73 (m,
1H), 5.07 (s, 1H), 7.08 (t, 1H, J = 8.0 Hz), 7.18ꢀ7.20 (m, 1H), 7.48ꢀ7.53 (m, 1H), 8.24 (t, 1H, J =
4.0 Hz). 13C NMR (400 MHz, DMSOꢀd6): δ 23.13, 38.48, 42.12, 43.78, 44.77, 46.52, 48.02,
50.80ꢀ51.01, 67.43, 72.25, 107.21ꢀ107.47, 114.83, 120.59, 134.44, 135.83, 154.14, 154.75,
164.51, 170.68. LC/MS (m/z): [m+H]+ calcd for C19H26FN4O7S, 473.15; found, 473.29; retention
time: 4.30 min.
To a solution of the mesylate in CH3CN (50 mL) and H2O (0.25 mL) was added potassium
phthalimide (556 mg, 3 mmol) and then the reaction was refluxed for 48h. After the reaction was
complete as monitored by TLC, it was filtered to remove the solid, and the solvent was
concentrated under reduced pressure. The residue was purified by silica gel column, eluting with
1
DCM / MeOH (100/1, v/v) to give 18 as a white solid in 59% yield (308 mg, 0.59 mmol). H
NMR (400 MHz, DMSOꢀd6): δ 1.83 (s, 3H), 2.96 (brs, 2H), 3.06 (brs, 2H), 3.40 (t, 2H, J = 8.0
Hz), 3.60 (brs, 2H), 3.69ꢀ3.72 (m, 3H), 4.09 (t, 1H, J = 8.0 Hz), 4.59 (s, 2H), 4.68ꢀ4.74 (m, 1H),
7.11 (t, 1H, J = 8.0 Hz), 7.18ꢀ7.20 (m, 1H), 7.49 (dd, 1H, J1 = 4 Hz, J2 =16 Hz), 7.87ꢀ7.94 (m,
4H), 8.24 (t, 1H, J = 8.0 Hz). 13C NMR (400 MHz, DMSOꢀd6): δ 23.14, 42.13, 42.41, 44.92,
48.02, 50.84, 51.30, 72.26, 107.20ꢀ107.46, 114.81, 120.61ꢀ120.65, 123.96, 132.38, 134.47ꢀ
134.57, 135.38, 135.87ꢀ135.96, 154.18, 154.75, 156.60, 164.87, 168.31, 170.68. LC/MS (m/z):
[m+H]+ calcd for C26H27FN5O6, 524.19; found, 524.35; retention time: 5.53 min.
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tert-Butyl
(6R,7R)-7-(2-((tert-butoxycarbonyl)amino)-2-phenylacetamido)-3-
5
6
(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate (20)
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To a flame dried pressure tube under argon was added tꢀbutyl acetate (18.5 mL, 138 mmol).
Argon was bubbled through the tꢀbutyl acetate for 5 min and compound 19 (1.00 g, 3.67 mmol)
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.
was added. To this suspension was added BF3 Et2O (2.8 mL, 22.0 mmol), the tube was capped,
and the reaction was stirred until homogeneous (~1.25 h). The reaction was poured into 50 mL of
stirring ice water and the organic layer was removed. The aqueous layer was washed with
EtOAc/hexanes (1:1, v/v) and this organic layer was also discarded. The aqueous layer was
transferred to a flask, placed in an ice bath, and EtOAc (75 mL) was added. To this stirring
suspension was slowly added Na2CO3 until the pH was 8.5. The organic layer was removed and
the aqueous layer was extracted with EtOAc (3 times). The pooled organic layers were dried
with Na2SO4, filtered, and concentrated under reduced pressure to give a yellow oil that was
stirred in EtOAc/hexanes (1:10, v/v) for 10 min. Most of the solvent was decanted and the
product was dried under reduced pressure to give the tꢀbutyl ester as a lightꢀyellow solid in 68%
yield (822 mg, 2.5 mmol). 1H NMR (400 MHz, DMSOꢀd6): δ 1.49 (s, 9H), 2.02 (s, 3H), 2.28 (s,
2H), 3.40 (d, 1H, J = 20 Hz), 3.56 (d, 1H, J = 20 Hz), 4.59 (d, 1H, J = 12 Hz), 4.79 (s, 1H), 4.87
13
(d, 1H, J = 12 Hz), 4.98 (d, 1H, J = 4 Hz). C NMR (400 MHz, DMSOꢀd6): δ 21.25, 26.00,
28.11, 59.90, 63.41, 64.46, 83.15, 122.66, 127.74, 161.42, 170.68, 170.89.
A solution of BocꢀDꢀphenylglycine (126 mg, 0.5 mmol) and Et3N (70 µL, 0.5 mmol) in THF (10
o
mL) was cooled to ꢀ10 C. While stirring, 65 µL (0.5 mmol) isobutyl chloroformate was added
and the temperature was maintained at ꢀ10 oC for 10 min. A cold solution of the tꢀbutyl ester in 3
mL of THF was added with stirring to the mixed anhydride solution. The mixture was stirred at 5
oC for 1h and then at room temperature for 1h. When the reaction was finished, as monitored by
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TLC, the THF was evaporated and the residue was purified using a silica gel column eluting with
hexanes / EtOAc (4:1, v/v) to give tert-Butyl (6R,7R)-3-(acetoxymethyl)-7-(2-((tert-
butoxycarbonyl)amino)-2-phenylacetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
1
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carboxylate as a white solid in 77% yield (216 mg, 0.38 mmol). H NMR (400 MHz, DMSOꢀ
d6): δ 1.39 (s, 9H), 1.47 (s, 9 H), 2.01 (s, 3H), 3.42 (d, 1H, J = 20 Hz), 3.55 (d, 1H, J = 20 Hz),
4.61 (d, 1H, J = 12 Hz), 4.89 (d, 1H, J = 12 Hz), 5.04 (d, 1H, J = 4 Hz), 5.35 (d, 1H, J = 8 Hz),
5.73 (brs, 1H), 7.29ꢀ7.44 (m, 5H), 9.17ꢀ9.18 (d, 1H, J = 4 Hz). 13C NMR (400 MHz, DMSOꢀd6):
δ 21.21, 26.32, 28.10, 28.86, 58.07, 58.31, 59.43, 63.22, 79.16, 83.46, 122.89, 127.52, 127.91,
128.33, 128.40, 128.87, 129.02, 138.75, 155.58, 161.01, 164.91, 170.81, 171.73. LC/MS (m/z):
[MꢀH]+ calcd for C27H34N3O8S, 560.20; found, 560.36; retention time: 8.88 min.
To an Erlenmeryer flask was added tert-butyl (6R,7R)-3-(acetoxymethyl)-7-(2-((tert-
butoxycarbonyl)amino)-2-phenylacetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
carboxylate (0.2 mmol, 112 mg), 1 mL of THF, and 10 mL of hexanes. The flask was swirled to
give a suspension. To this suspension was added CAB Lipase (44 mg), 4Å molecular sieves (90
mg) and sꢀbutanol (0.37 mL). The flask was stopped with a septum (with a small air vent) and
o
the reaction was shaken in an incubated shaker at 50 C for 3ꢀ4 days. The reaction was diluted
with DCM and filtered using vacuum filtration. The filtrate was concentrated under reduced
1
pressure to give 20 as a white solid in 85% yield (88 mg, 0.17 mmol). H NMR (400 MHz,
DMSOꢀd6): δ 1.39 (s, 9H), 1.47 (s, 9 H), 3.39ꢀ3.50 (m, 2H), 4.11ꢀ4.21 (m, 2H), 4.98 (d, 1H, J = 4
Hz), 5.04 (brs, 1H), 5.34 (d, 1H, J = 8 Hz), 5.66ꢀ5.69 (m, 1H), 7.27ꢀ7.44 (m, 5H), 9.14 (d, 1H, J
= 8 Hz). 13C NMR (400 MHz, DMSOꢀd6): δ 26.05, 28.18, 28.86, 58.02, 58.30, 59.20, 60.53,
79.14, 82.85, 124.42, 127.89, 128.30, 128.86, 138.80, 155.63, 161.49, 164.79, 171.74. LC/MS
(m/z): [MꢀH]+ calcd for C25H32N3O7S, 518.19; found, 518.20; retention time: 7.72 min.
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tert-Butyl
(6R,7R)-7-(2-((tert-butoxycarbonyl)amino)-2-phenylacetamido)-8-oxo-3-
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((((1,2,2,2-tetrachloroethoxy)carbonyl)oxy)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
carboxylate (21).
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To a solution of compound 20 (0.1 mmol, 52 mg) in anhydrous DCM at room temperature was
added 1,2,2,2ꢀtetrachloroethyl carbonochloridate (0.11 mmol, 17 µL), pyridine (0.13 mmol, 11
µL) and 2 mg of DMAP. The mixture was stirred for 2h. When the starting material was
consumed based on TLC analysis, the reaction was concentrated under reduced pressure. The
residue was dissolved in 1 mL of EtOAc, and the crude compound, 21, was precipitated as a light
pink solid in 77% yield by adding 5 mL of hexanes. Compound 21 was used directly for the next
step without further purification.
(6R,7R)-3-((((2-(4-(4-((S)-5-(Acetamidomethyl)-2-oxooxazolidin-3-yl)-2-
fluorophenyl)piperazin-1-yl)-2-oxoethyl)carbamoyl)oxy)methyl)-7-(2-amino-2-
phenylacetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (22).
To a solution of 3 (0.1 mmol, 39 mg) in anhydrous THF (3 mL) was added DIPEA (0.2 mmol,
34 µL) at room temperature. The mixture was stirred for 2 min, then a solution of crude 21 (0.1
mmol, 73 mg) in THF (2 mL) was added. The reaction was stirred at room temperature for 6h.
After evaporation, the residue was purified using a silica gel column eluting with DCM/MeOH
(40:1, v/v) to give the corresponding protected cephalosporin oxazolidinone as a white solid in
1
50% yield (0.05 mmol, 47 mg). H NMR (400 MHz, DMSOꢀd6): δ 1.39 (s, 9H), 1.47 (s, 9H),
1.81 (s, 3H), 2.93 (d, 4H, J = 12 Hz), 3.40ꢀ3.70 (m, 5H), 3.89 (brs, 2H), 4.08 (t, 1H, J = 8.0 Hz),
4.56ꢀ4.61 (m, 1H), 4.70 (s, 1H), 4.84 (d, 1H, J = 8.0 Hz), 5.03 (s, 1H), 5.33 (s, 1H), 5.71 (brs,
1H), 7.07 (t, 1H, J = 8.0 Hz), 7.17 (d, 1H, J = 4.0 Hz), 7.27ꢀ7.31 (m, 2H), 7.43ꢀ7.51 (m, 5H),
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8.17 (s, 1H), 8.24 (s, 1H), 9.16 (s, 1H). 13C NMR (400 MHz, DMSOꢀd6): δ 23.13, 26.78, 28.14,
28.86, 42.12, 42.70, 44.72, 48.01, 50.93, 51.28, 58.30, 59.40, 63.30, 72.25, 79.18, 83.43, 107.20ꢀ
107.46, 114.81, 120.50, 124.02, 126.98, 127.91, 128.22, 128.87, 129.88, 134.40ꢀ134.51, 135.90ꢀ
135.99, 137.05, 138.75, 154.14, 154.75, 156.56ꢀ156.92, 161.05, 164.90, 167.77, 169.27, 170.68,
171.74. LC/MS (m/z): [M+H]+ calcd for C44H56FN8O12S, 939.37; found, 939.61; retention time:
7.72 min.
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To 5 mL of anhydrous trifluoroacetic acid at room temperature was added 40 mg of the white
solid from the last step (0.04 mmol) with stirring. After 30 min, pouring the reaction solution
into anhydrous ether afforded the trifluoroacetic salt of 22 (30 mg, 0.03 mmol) as a white solid in
1
75% yield. H NMR (500 MHz, DMSOꢀd6): δ 1.83 (s, 3H), 2.92ꢀ2.96 (m, 4H), 3.40 (brs, 2H),
3.48ꢀ3.59 (m, 5H), 3.70 (t, 2H, J = 8.0 Hz), 3.87 (d, 2H, J = 4.0 Hz), 4.08 (t, 1H, J = 8.0 Hz),
4.62 (d, 1H, J = 16.0 Hz), 4.71 (t, 1H, J =8.0 Hz), 4.93 (d, 1H, J = 12.0 Hz), 5.04 (d, 2H, J = 4.0
Hz), 5.78 (t, 1H, J = 8.0 Hz), 7.07 (t, 1H, J = 12.0 Hz), 7.17 (d, 1H, J = 4.0 Hz), 7.28 (t, 1H, J =
4.0 Hz), 7.43ꢀ7.52 (m, 5H), 8.12 (brs, 1H), 8.27 (brs, 1H), 8.78 (brs, 2H), 9.56 (d, 1H, J = 8.0
Hz). 13C NMR (400 MHz, DMSOꢀd6): δ 23.12, 25.98, 42.11, 42.69, 44.68, 50.93, 51.24, 56.03,
57.88, 59.32, 63.39, 72.23, 107.21ꢀ107.47, 114.82, 120.53, 125.37, 126.37, 128.45, 129.43,
130.00, 134.14, 134.40ꢀ134.50, 135.90ꢀ135.99, 154.14, 154.76, 156.57ꢀ156.99, 158.63ꢀ158.94,
163.38, 164.41, 165.33, 167.78, 168.93, 170.72. LC/MS (m/z): [M+H]+ calcd for
C35H40FN8O10S, 782.25; found, 783.41; retention time: 4.17 min.
(6R,7R)-3-(Acetoxymethyl)-7-(2-(2-(N-(4-(2,3-dihydroxybenzamido)butyl)-2,3-
dihydroxybenzamido)acetamido)-2-phenylacetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-
2-ene-2-carboxylic acid (26).
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Compound 23 was synthesized according to the literature reported method.40 To a suspension of
23 (0.1 mmol, 50 mg) in anhydrous acetonitrile (5 mL), was added diisopropylethylamine (0.2
mmol, 35 µL) at room temperature. To this mixture was added a DMF solution of 25 (0.1 mmol)
and the mixture was stirred for 4h. The reaction was then concentrated under reduced pressure.
The residue was purified using a C18 reverse phase silica gel column, eluting with a gradient of
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water and acetonitrile to give 26 as a white solid in 12% yield (0.012 mmol, 10 mg). H NMR
(500 MHz, DMSOꢀd6): δ 1.23ꢀ1.32 (m, 2H), 1.45ꢀ1.56 (m, 2H), 2.01 (s, 3H), 3.09ꢀ3.17 (m, 2H),
3.30ꢀ3.54 (m, 4H), 3.92ꢀ3.94 (m, 1H), 4.17ꢀ4.20 (m, 1H), 4.64 (d, 1H, J = 10 Hz), 4.95ꢀ5.02 (m,
2H), 5.59ꢀ5.71 (m, 2H), 6.46ꢀ6.77 (m, 5H), 6.87ꢀ6.90 (m, 1H), 7.22ꢀ7.31 (m, 5H), 7.46 (brs, 1H),
8.62ꢀ8.80 (m, 2H), 9.12 (s, 1H), 9.36ꢀ9.49 (m, 2H), 12.79 (d, 1H, J = 50.0 Hz), 13.73 (s, 1H). 13C
NMR (500 MHz, DMSOꢀd6): δ 20.37, 25.42, 25.83, 28.44, 28.83, 47.47, 48.74, 55.69, 57.37,
58.67, 62.52, 114.94, 115.68, 115.89, 117.03, 117.45, 117.69, 117.83, 118.62, 119.17, 123.34,
126.20, 126.72, 126.91, 127.54, 128.10, 137.79, 141.25, 145.21, 146.05, 149.53, 162.59, 164.11,
168.13, 169.20, 169.56, 170.00, 170.30. HRMS (m/z): [M+Na]+ calcd for C38H39N5NaO13S,
828.2167; found, 828.2157. LC/MS retention time 6.18 min.
(S)-N-(2-((2-(4-(4-(5-(Acetamidomethyl)-2-oxooxazolidin-3-yl)-2-fluorophenyl)piperazin-1-
yl)-2-oxoethyl)amino)-2-oxoethyl)-N-(4-(2,3-dihydroxybenzamido)butyl)-2,3-
dihydroxybenzamide (27).
To a suspension of 3 (0.1 mmol, 39 mg) in anhydrous acetonitrile (5 mL), was added
diisoprpylethylamine (0.2 mmol, 35 µL) at room temperature. To this mixture was added a DMF
solution of 25 (0.1 mmol) and the mixture was stirred for 4h. The reaction was then concentrated
under reduced pressure. The residue was purified using a C18 reverse phase silica gel column,
eluting with a gradient of water and acetonitrile to give 27 (0.01 mmol, 8 mg) as a white solid in
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10% yield. H NMR (500 MHz, DMSOꢀd6): δ 1.23ꢀ1.32 (m, 2H), 1.48ꢀ1.58 (m, 2H), 1.83 (s,
3H), 2.93ꢀ2.98 (m, 4H), 3.11ꢀ3.20 (m, 2H), 3.38ꢀ3.52 (m, 4H), 3.58 (s, 4H), 3.69 (dd, 1H, J1 =
10.0 Hz, J2 = 5.0 Hz), 3.86ꢀ3.96 (m, 2H), 4.06ꢀ4.14 (m, 2H), 4.67ꢀ4.73 (m, 1H), 6.58ꢀ6.67 (m,
3H), 6.77 (dd, 1H, J1 = 20.0 Hz, J2 = 10.0 Hz), 6.89 (d, 1H, J = 5.0 Hz), 7.06 (brs, 1H), 7.16 (d,
1H, J = 10.0 Hz), 7.21ꢀ7.30 (m, 1H), 7.49 (dd, 1H, J1 = 15.0 Hz, J2 = 5.0 Hz), 8.06 (s, 1H), 8.24
(t, 1H, J = 5.0 Hz), 8.67 (t, 1H, J = 15.0 Hz), 8.80ꢀ8.97 (m, 1H), 9.13 (s, 1H), 9.53 (s, 1H), 12.77
(d, 1H, J = 55.0 Hz). 13C NMR (500 MHz, DMSOꢀd6): δ 23.12, 24.82, 25.97, 26.56, 26.89,
29.70, 39.10, 39.46, 41.24, 42.08, 44.78, 45.33, 47.95, 48.16, 49.44, 50.89, 51.19, 51.55, 72.22,
107.15ꢀ107.36, 114.74, 115.58, 116.28, 116.61, 117.75, 118.25, 118.51, 118.67, 119.40, 119.96,
120.15, 120.48, 125.22, 125.66, 134.37ꢀ134.45, 135.88ꢀ135.95, 141.92, 145.95, 146.88, 150.38,
154.34, 154.73, 156.28, 158.44, 158.81, 167.33, 169.30, 169.93, 170.37, 170.69. HRMS (m/z):
[M+Na]+ calcd for C38H44FN7NaO11, 816.2982; found, 816.2975. LC/MS retention time 2.08
min.
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Enzymatic hydrolysis experiments
The ADCꢀ1 βꢀlactamase was expressed and purified as previously described.41 Conjugate 1 (10
mM in DMSO) was diluted 5ꢀfold in DMSO and then another 10ꢀfold in 50 mM sodium
phosphate, pH 7.4 to give a 200 µM solution with 10% DMSO. Subsequently, a 60 µL reaction
was prepared consisting of 20 µM of 1 (6 µL of a 200 µM solution) and 20 µM ADCꢀ1 βꢀ
lactamse (8.1 µL of 148 µM enzyme) in 50 mM sodium phosphate, pH 7.4 (the final
concentration of DMSO was 1%). The reaction was incubated at room temperature for 40 min
prior to analysis by LC/MS. Two control reactions, one containing only 1 (20 µM) and the other
only ADCꢀ1 (1 µM enzyme), were also prepared using the same conditions.
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Antimicrobial susceptibility assays
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The gene encoding the ADCꢀ1 enzyme from A. baumannii with its leader (GenBank accession
number NG_048633.1) was custom synthesized (GenScript) and cloned into the NdeIꢀHindIII
restriction sites of the pNT255 shuttle vector, which is a derivative of the pNT221 shuttle vector
in which the ISAba3 promoter sequence has been replaced with that of ISAba1.42 To remove two
HindIII sites within the ADCꢀ1 nucleotide sequence, silent mutations were made using the codon
usage of A. baumannii. This plasmid was then introduced by electroporation into A. baumannii
ATCC 17978 for MIC measurements. MIC analyses were performed using iron depleted media
as described previously.19
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information (1H NMR, 13C NMR, LC/MS, MS, EIMS of ADC, Charge
Deconvoluted Electrospray Mass Spectrum of ADC and Molecular Formula Strings) is available
free of charge on the ACS Publications website at DOI: …….
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: mmiller1@nd.edu (M.J.M.). Phone 574 631 7571.
ORCID
Marvin J. Miller: 0000ꢀ0002ꢀ3704ꢀ8214
Author Contributions
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The manuscript was written through contributions of all authors. RL performed the syntheses and
PAM and NS performed the bioassays. All authors have given approval to the final version of
the manuscript.
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Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
This work was supported in part by the George and Winifred Clark Chair of Chemistry and
Biochemistry, University of Notre Dame, the NIH (1R01AI114668 to S. B. Vakulenko) and the
NSF (CHEꢀ0741793). We thank V. Krchnak and N. Sevova for LC/MS assistance and J. Zajicek
of the NMR facility at Notre Dame.
ABBREVIATIONS
ADC, Acinetobacterꢀderived cephalosporinase; Mtb, Mycobacterium tuberculosis; Boc, tertꢀ
butyloxycarbonyl;
TFA,
trifluoroacetic
acid;
EDC,
1ꢀethylꢀ3ꢀ(3ꢀ
dimethylaminopropyl)carbodiimide; NHS, Nꢀhydroxysuccinimide; MIC, minimum inhibitory
concentration; Et3N, triethylamine; DCM, dichloromethane; THF, tetrahydrofuran; DIPEA, N,Nꢀ
diisopropylethylamine; CAB Lipase, Candida lipase B; DMF, N,Nꢀdimethylformamide; TLC,
thin layer chromatography; DMSO, dimethyl sulfoxide.
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