# Zidovudine-β-Lactam Pronucleoside Strategy for Selective Delivery into Gram-Negative Bacteria Triggered by β-Lactamases

Miyanou Rosales-Hurtado, Filomena Sannio, Lindita Lari, Federica Verdirosa, Georges Feller, Elodie Carretero, Yen Vo-Hoang, Patricia Licznar-Fajardo, Jean-Denis Docquier, Laurent Gavara

► To cite this version:

Miyanou Rosales-Hurtado, Filomena Sannio, Lindita Lari, Federica Verdirosa, Georges Feller, et al.. Zidovudine-β-Lactam Pronucleoside Strategy for Selective Delivery into Gram-Negative Bacteria Triggered by β-Lactamases. ACS Infectious Diseases, 2023, 9 (8), pp.1546-1557. 10.1021/acsin-fecdis.3c00110. hal-04321173

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# Zidovudine-β-lactam pronucleoside strategy for selective delivery into Gram-negative bacteria triggered by β-lactamases

Miyanou Rosales-Hurtado,ª Filomena Sannio,ᵇ Lindita Lari,ª Federica Verdirosa,ᵇ Georges Feller,ᶜ Elodie Carretero,ª Yen Vo-Hoang,ᵈ Patricia Licznar-Fajardo,ᵈ Jean-Denis Docquier,ᵇ,e
Laurent Gavaraª,\*

Corresponding author's email address: laurent.gavara@umontpellier.fr

**ABSTRACT:** Addressing antibacterial resistance is a major concern of the modern world. The development of new approaches to meet this deadly threat is a critical priority. In this article, we investigate a new approach to negate bacterial resistance: exploit the β-lactam bond cleavage by β-lactamases to selectively trigger antibacterial prodrugs into the bacterial periplasm. Indeed, multidrug-resistant Gram-negative pathogens commonly produce several β-lactamases able to inactivate β-lactam antibiotics, our most reliable and widely used therapeutic option. The chemical structure of those prodrugs is based on a monobactam promoiety, covalently attached to the active antibacterial substance, the zidovudine (AZT). We describe the synthesis of 10 prodrug analogs (5a-h) in 4-9 steps and their biological activity. Selective enzymatic activation by a panel of β-lactamases is demonstrated and subsequent structure-activity relationships (SARs) are discussed. The best compounds are further evaluated for their activity on both laboratory strains and clinical isolates, preliminary stability, and toxicity.

**KEYWORDS:** prodrug, β-lactamase, bacterial resistance, Gram-negative pathogen, zidovudine

<figure><img src="image_2.png" /></figure>

<footnote>ª Institut des Biomolécules Max Mousseron, Univ Montpellier, CNRS, ENSCM, 34293 Montpellier, France</footnote>

<footnote>ᵇ Dipartimento di Biotecnologie Mediche, Università di Siena, I-53100 Siena, Italy</footnote>

<footnote>ᶜ Laboratoire de Biochimie, Centre d'Ingénierie des Protéines-InBioS, Université de Liège, B-4000 Liège, Belgium</footnote>

<footnote>ᵈ HSM, Univ Montpellier, CNRS, IRD, CHU Montpellier, 34090 Montpellier, France</footnote>

<footnote>ᵉ Laboratoire de Bactériologie Moléculaire, Centre d'Ingénierie des Protéines-InBioS, Université de Liège, B-4000 Liège, Belgium</footnote>
In early 1900's, bacterial infections represented one of the most significant causes of death worldwide. This plague has been circumvented by the development of several classes of antibiotics, including the β-lactams, which show optimal efficacy and selectivity.¹ Almost immediately after the introduction of penicillin, penicillin-resistant strains of *Staphylococcus aureus* emerged in the clinical setting. More generally, due to the natural selective pressure when exposed to exogenous agents, bacteria developed an arsenal of mechanisms to escape the therapeutic effect of antibiotics.² Although the fast discovery and development of new antibacterial drugs allowed to adequately address the problem of resistance (a period known as the golden age of antibiotic discovery), the situation drastically changed in the late 90's with the emergence of multi-drug resistance bacteria and the progressive disinterest of pharmaceutical companies in antibiotic R&D, leading to a substantial erosion of available therapeutic options.³ Nowadays, besides multidrug-resistant bacteria, the prevalence of isolates showing extensively-drug or pan-drug resistance phenotypes is increasing in the clinical setting.⁴,⁵ Currently, global deaths attributable to antimicrobial resistance is estimated to exceed 1.2 million, raising worldwide awareness.⁶ Among other responses (antibiotic stewardship, better and faster diagnosis), the development of new antibiotics remains urgent, especially against the so-called "critical priority" pathogens, as established by WHO, which include carbapenem-resistant Gram-negative bacteria (*Enterobacterales*, *Acinetobacter*, and *Pseudomonas*).⁷ β-lactam antibiotics still represent the mainstay of antibacterial therapy, accounting for more than 50% of antibiotics prescribed worldwide.⁸ Numerous families were developed such as penicillins, cephalosporins, monobactams, and carbapenems, and are all characterized by a four membered β-lactam ring, crucial for their antibacterial activity. A major mechanism of β-lactam resistance in Gram-negative pathogens is the production of enzymes able to hydrolyze the amide bond of the β-lactam ring, called β-lactamases.⁹ They are classified into 4 molecular classes (A, B, C and D) but can be divided into two main families based on their mechanism of action. The Serine-β-Lactamases (SBLs, classes A, C and D) are related to serine hydrolase enzymes (SBLs)¹⁰ and the Metallo-β-Lactamases (MBLs, class B) are characterized by the presence of one or two zinc atoms into the active site.¹¹ Their hydrolytic action results from nucleophilic attack of serine residue or water molecule, according to β-lactamase type, on the β-lactam carbonyl. β-lactamases show an extraordinary diversity (with > 7,800 unique enzymes) and evolutionary potential, leading to the emergence of clinical variants (by either mutation a or the acquisition of novel from the environmental resistome) with peculiar substrate profile, such as extended-spectrum β-lactamases (ESBLs) or carbapenem-hydrolyzing enzymes (e.g., KPC or OXA-type carbapenemases).¹² The latter represent a major threat as they confer resistance to the last-resort and life-saving carbapenems, a major therapeutic resource for the treatment of infections caused by MDR isolates. Furthermore, resistant bacterial strains commonly produce several β-lactamases, sometimes both serine- and metallo- carbapenemases. In this context, the development of new effective β-lactam-based therapies is still necessary but extremely challenging.¹³ One strategy is represented by the development of combinations with β-lactamase inhibitors, such as clavulanate or, more recently, avibactam or vaborbactam. Nevertheless, resistance to such new combinations is already observed in the clinical setting, highlighting the need for further innovation in the field.¹⁴ The development of new drugs based on original strategies is fundamental to tackle this growing major threat.¹⁵

In this context, the design of compounds able to reverse bacterial resistance to β-lactam agents, by exploiting the specific β-lactam fission mechanism leads to a new type of hybrid antibacterial drugs.¹⁶⁻¹⁸ A first generation of dual agents was developed based on a fissile covalent combination of two antibiotics (Scheme 1). Several studies have explored the association of cephalosporin with a fluoroquinolone agent (such as Ro 23-9424, 1).¹⁹ With such compounds, the penicillin-binding proteins (PBPs) are initially irreversibly inactivated through the formation of a covalent adduct with the β-lactam moiety, which, after subsequent spontaneous rearrangement release fluoroquinolone moiety,
inhibiting bacterial DNA gyrase (Scheme 1). The chemical codrug activation has been widely studied and different leaving groups have been introduced as well as self-immolative linkers to improve stability and other biological properties.²⁰⁻²² Due to the ever-expanding heterogeneity and simultaneous and increased production of β-lactamases in MDR clinical isolates, inactivation of PBPs mediated by such codrugs is seriously compromised. Prodrug 2, based on a similar approach by exploiting the β-lactamase-mediated release of a fluoroquinolone from a β-lactam scaffold, was also developed.²³ In this case, inactivation of PBPs was not desirable, and only β-lactamases producers were targeted. Recently, specific prodrugs to fight MBLs were designed with a cephalosporin-MBL inhibitor conjugate 3.²⁴⁻²⁶ The hydrolysis of the β-lactam ring is mediated by the MBL enzymes that will then be inhibited by the released entity. This adjuvant strategy needs a combination with a β-lactam agent (e.g., meropenem) to provide bacterial inhibition.

<figure><img src="image_3.png" /><figcaption>**Scheme 1.** Some dual β-lactam agents described in the literature enzymatically activated (Enz: PBPs or β-lactamases)</figcaption></figure>

All these approaches are mainly based on cephalosporin core as the promoiety. The use of clinically valuable β-lactam antibiotic scaffolds, such as especially third-generation cephalosporin (e.g., ceftazidime, ceftriaxone, cefotaxime), which still represents the most widely used β-lactam subclass, is expected to maintain and even increase global bacterial resistance and compromise the antibacterial activity of potentially life-saving new β-lactam agents (e.g., cefiderocol).²⁷ We here propose an alternative approach to disrupt this domino effect leading to worsening resistance by protecting crucial β-lactam therapeutic subclasses through the investigation of a novel design of β-lactamase-activated prodrugs.

## ■ RESULTS AND DISCUSSION

**Prodrugs design.** Antibiotic therapies are never specific to the bacterial pathogens and gut microbiota disturbance,²⁸ or superinfection phenomenon²⁹ are very commonly observed side effects. To provide selective treatments, it is necessary to specifically target (opportunistic) pathogens, especially multidrug-resistant strains, which represent a common cause of difficult-to-treat hospital-acquired
infections. Our approach relies on the consideration that, in Gram-negative bacteria, the production
of one- or more β-lactamases, more importantly carbapenem-hydrolyzing enzymes, represents a
common resistance mechanism found in resistant strains, where the level of β-lactamase production
often correlates with the level of resistance.⁹ Therefore, the use of the β-lactam motif as a prodrug
carrier will provide a new way to hijack one of the most common bacterial resistance mechanisms and
offer the opportunity to remodulate the spectrum of activity of antibacterial compounds, limited to β-
lactamase producers, with potentially beneficial effects such as (a) reducing the selective pressure
contributing to the dissemination of resistant strains and (b) limiting the undesirable effect of broad-
spectrum antibiotics on the gut microbiota and the damaging consequences of antibiotic-associated
dysbiosis. We decided to focus on the structurally simplest β-lactam core, thus leading to a novel
monobactam-based prodrug approach. Interestingly, this β-lactam subclass is underexploited and
aztreonam is the only monobactam agent, thus sharing this structural minimalism (although with some
structural specificities), to be approved for the treatment of bacterial infections in humans.³⁰ To
prevent any anti-PBP activity and avoid β-lactamase stability (as the efficient cleavage of the β-lactam
is actually desired here), all usual β-lactam designs, such as aminothiazole ring, oxime moiety, or β-
lactam substitutions, will be avoided.³¹⁻³³ At position 3 (R part, Scheme 2) of the azetidinone ring, we
focused on first generation side chains of β-lactam agents to combine optimal β-lactamase recognition,
while protecting the most effective substitutions present in marketed drugs. Thus, a list of side chains
was drawn up from known derivatives (Scheme 2): penicillin V (a), clavulanic acid (c), penicillin G (g),
cephalothin (h), reported SBL inhibitors (d and e),³⁴ inspired from serine-protease inhibitors (f)³⁵ or
chemical intermediate (b). The next step consisted in the selection of the antibacterial agent to be
released. Drug repurposing, i.e., the use of existing drugs for a different therapeutic application is a
common strategy presenting potential advantages, such as decreased costs and minimized risks during
drug development.³⁶ In the complicated field of antibiotics, numerous active substances have been
considered and one of the most promising class is represented by biomolecules belonging to the
nucleoside analog class.³⁷ Nucleoside Analogs (NAs) are indeed a major therapeutic class, mainly
known as antiviral and anticancer agents. They display pleiotropic effects in all fundamental genetic
information processes in both eukaryotic and prokaryotic cells. For now, the most relevant NA studied
for its antibacterial activity is zidovudine (AZT, 4, Scheme 2). Initially developed as an anticancer agent,
it is currently intensively used in AIDS treatment.³⁸ Several studies show it is a potent antibacterial
agent against relevant opportunistic pathogens, such as *E. coli*, one of the first causes of hospital-
acquired infections, including on β-lactamase-producing isolates.³⁹,⁴⁰ To obtain the desired
pronucleoside 5, AZT has to be covalently attached to the monobactam promoiety. This linker will be
achieved by an ether function between the 5' hydroxyl group of AZT and the β-lactam moiety. By
masking the phosphorylation site at position 5' of AZT, all potential intrinsic biological activities of the
corresponding pronucleoside 5a-h are prevented (Scheme 2). Lactamase-mediated hydrolysis of the
β-lactam ring will lead to the unstable intermediate 6, which will spontaneously rearrange to release
AZT through a rearrangement and elimination process similar to that described previously.
<figure><img src="image_4.png" /><figcaption>**Scheme 2.** Main envisioned substitutions and targeted pronucleosides 5a-h and their activation mechanisms by β-lactamase enzymes (β-L).</figcaption></figure>

Thus, pronucleosides 5a-h will be evaluated as substrates on a range of different β-lactamases and their antibacterial activity investigated β-lactamase-producing strains. Some ADME aspects will be also explored such as stability, solubility, and toxicology. This study represents a new attractive approach to fight bacterial resistance by exploiting the major mechanism of β-lactam resistance in relevant pathogens, leading to an antibacterial drug delivery system with a selective activity on β-lactamase-producing isolates.

**Chemical synthesis.** Due to the structural relevance of the 2-azetidinone core, retrosynthetic analyses have been extensively studied in the literature. Numerous synthetic pathways are available according to the desired substitutions on the β-lactam ring.⁴¹ We explored several of them such as Reformatsky reaction, β-amino-acids intramolecular cyclization or Mitsunobu reaction. Unfortunately, most of them failed at the β-lactam cyclization step. We deeply explored retrosynthesis based on the hydroxamate approach described by Miller (Scheme 3).⁴²,⁴³ The carboxylic function of a properly acylated serine residue 7 was activated to react with hydroxylamine and form the corresponding hydroxamate compound 8. Several experimental conditions were described to carry on the subsequent β-lactam cyclization through a Mitsunobu reaction such as CCl₄/PPh₃ or DEAD/PPh₃.⁴⁴,⁴⁵ All these conditions led to a weak conversion rate and a complex mixture to separate. Finally, the best conditions have been identified and consist of activation of the hydroxyl group by a mesyl group, followed by cyclization in basic media to get the key cyclized intermediate 9.⁴⁶ After removal of the protecting group (PG), the NH of the β-lactam function was substituted by a glyoxalate reagent, which was acetylated to give the corresponding promoiety 10. The introduction of the AZT drug will be performed thanks to the N-acylium chemistry providing final pronucleoside compounds 5a-h.

<figure><img src="image_5.png" /><figcaption>**Scheme 3.** Retrosynthetic analysis</figcaption></figure>
Two sequential approaches were implemented according to the nature of R moiety. First, in order to explore, optimize and develop the synthetic pathway, the side chain of penicillin V was introduced by acylation reaction of L-serine compound **11** to get the starting material **7a** (Scheme 4). Second, the same synthesis was performed with a Boc residue as the protecting group (from **7b**) allowing access to a broad range of acyl substituents on this position at the end of the synthetic pathway. Carboxylic acid functions of **7a-b** were activated by EDCI/HOBt coupling condition, then benzylhydroxylamine was introduced to give corresponding hydroxamate **8a-b**. The hydroxyl functions were replaced by a mesyl leaving group, and then the cyclization step was performed in presence of K₂CO₃ to provide β-lactam compounds **9a-b**. The deprotection of hydroxamate function needed the N-O bond cleavage. The use of Raney-Ni amalgam⁴⁷ or a two-step process based on hydrogenation on Pd/C and then hydroxylamine deprotection by TiCl₃⁴⁸ were shown to be ineffective conditions in our case. Finally, deprotected β-lactam rings **12a-b** were obtained by a solution of Sml₂ in THF in good yields.⁴⁹ However, this step was highly sensitive to dissolved oxygen level and quality of commercial solution of samarium.

<figure><img src="image_6.png" /><figcaption>**Scheme 4.** Synthetic pathway of pronucleosides **5a-g**. <sup>a</sup> Not isolated, <sup>b</sup> yield over two steps, <sup>c</sup> yield over three steps, <sup>d</sup> Not isolated except for **17c** with 20% yield. Reagents and conditions: (a) Phenoxyacetyl chloride (1.3 eq.), Na₂CO₃, CH₃CN-H₂O, RT; (b), Benzylhydroxylamine (1.3 eq.), EDCI.HCl (1.1 eq.), HOBt (1.1 eq.), DIPEA (1.5-3 eq.), THF, 0°C; (c), MsCl (3 eq.), pyridine, 0°C, Ar; (d) K₂CO₃ (1.5-3 eq.), acetone, 60-80°C; (e) Sml₂ [0.1M] (5 eq.), THF-H₂O, 0°C, Ar; (f) TBDMSCl (1.15 eq.), DIEA, DCM anhy., RT, Ar; (g) TMSCI (1.2 eq.), LDA [1 M], THF anhy., -78°C, Ar; (h) Electrophile (1.2 eq.), LDA [1 M], THF, -78°C, Ar; (i) TBAF [1 M], THF anhy., RT, Ar; (j) Allyl glyoxalate (3 eq.), DMF anhy., 90°C; (k) Ac₂O (10 eq.), pyridine, RT; (l) BF₃-Et₂O (anhy.), DCM anhy., 0°C, Ar; (m) AlCl₃ (2 eq.), THF anhy., RT, Ar; (n) TFA 50%, DCM, RT; (o) carboxylic acid compound (1.3 eq.), EDCI.HCl (1.1 eq.), HOBt (1.1 eq.), DIPEA (3.5 eq.), THF, 0°C; (p) NaOH [0.1 M], THF, H₂O, RT.</figcaption></figure>

Non-acyl substituents were also introduced at position 3 (Substituent R). The commercially available azetidinone **12c** was protected by a TBDMS group to provide the corresponding β-lactam **13**. The enolate **14** was obtained after the action of LDA and anion-trapping by the addition of
chlorotrimethylsilane reagent. This intermediate was not isolated and directly engaged in the next step in presence of LDA to trap an electrophile reagent.⁵⁰ First, the 2-pyridinecarboxaldehyde was used to condense on the enolate form to give a separable mixture of isomers **15d** (Z) and **15e** (E). In the same way, allyl bromide electrophile was also selected to decorate the β-lactam ring by a substitution reaction to provide the allyl derivative **15f** in good yield. Finally, the silyl ether protecting group was removed by a solution of TBAF in THF to give corresponding β-lactam compounds **12d-f**.⁵¹ The correct identification between the two Z and E isomers was based on the ¹H NMR chemical shift of the vinylic proton and confirmed by NOE experiments (see SI for detailed spectrum of **12d**).⁵² The next stage of the synthesis was done on the different synthesized or commercially available β-lactam precursors **12a-f**. To prepare intermediates **16a-f**, the nitrogen atom has to be condensed on glyoxalate moiety. Allyl group on the ester function as protecting group has been chosen and the corresponding glyoxalate reagent was prepared in two steps according to the literature method.⁵³ Hydroxyl intermediates **16a-f** can be purified or directly engaged in the next step. The acylation reaction was performed by acetic anhydride to give corresponding protected compounds **10a-f** in good yields. The last reaction consisted in Lewis acid activation of acetyl function to generate *N*-acyl-iminium intermediate able to react with AZT.⁵⁴ Several experimental conditions were investigated: Lewis acid (AlCl₃, BF₃-Et₂O, ZnCl₂, LiCl, and Mg(OCl₄)₂), solvents (THF, DCM, and DMF) or temperatures (-20°C to reflux). Initially, acetylated compound **10a** was used as a reference. Two procedures quickly emerged as being the more efficient based on BF₃-Et₂O in CH₂Cl₂ at 0°C or AlCl₃ in THF at RT. In the first case, the use of BF₃-Et₂O leads to the *N*-Acyl iminium intermediate that was trapped by the hydroxyl group of the AZT to provide corresponding conjugate **17a** in the form of an inseparable diastereomeric mixture. Nevertheless, in presence of AlCl₃, only one diastereomer was obtained with a good yield and next synthesis steps will be done on it. This specificity was valid only for **10a** and with other acetylated compounds **10b-f**, only BF₃-Et₂O Lewis acid proved to be efficient, leading each time to a mixture of diastereomers when possible. The last stage of the synthetic pathway consisted in a sequence of successive deprotection reactions with a diversity step on the amino function at position 3 of the β-lactam heterocycle. The Boc group of **17b** was removed by a solution of TFA in CH₂Cl₂ at RT to give the deprotected compound **18** with a low yield over two steps. To introduce the *N*-acyl side chain, the carboxylic acid function of the corresponding substituent was activated by EDCI/HOBt mixture and then condensed on amino compound **18** to provide conjugates **17g-h**. It was initially considered the use of a palladium catalyst to remove the allyl protecting group. Finally, the more economical and convenient based-catalyzed ester hydrolysis was successfully applied to give final pronucleosides **5a-h** without any significant side product formation.³¹ To prevent any solubility issue, all final compounds **5a-h** were purified in reverse phase in presence of ammonium acetate to provide the corresponding sodium salt. During this purification step, some diastereoisomeric mixtures have proven to be separable. In this context, compounds **5f** and **5g** led to pure diastereoisomers **5f', 5f''**, **5g'**, and **5g''** for further biological evaluations. Inspired by described methods coupled with the *N*-acyl iminium chemistry strategy, a straightforward and original synthetic pathway was developed. That allowed the obtention of 10 final pronucleosides **5a-h** in 4-9 steps with an overall yield in 5-37% range. All compounds were fully characterized by NMR and mass spectroscopy with a UV purity ≥95% (see supporting information for all spectra). All the pronucleosides compounds obtained were subjected to biological evaluation.

**Enzyme assays and reactivity towards various β-lactamases.** Initially, kinetic experiments to monitor compound hydrolysis by UV spectroscopy were carried out. β-lactams are well-known to absorb light in the UV range (220-300), allowing the development of direct spectrophotometric assays.⁵⁵ Sadly, AZT also displayed an even stronger absorbance at these wavelengths, thus preventing any discernible measurement (see SI figures S1-S4). Furthermore, such method would not unambiguously identify the
nature of the released product as AZT. To overcome this issue, an indirect approach was implemented by preincubating the compounds with various β-lactamases, the release of AZT being measured as a result of bacterial growth inhibition in a disk diffusion assay of *E. coli* XL-1 susceptible laboratory strain (MIC value of 0.015 mg.mL⁻¹ for AZT), with suitable negative control (compound incubated without β-lactamase). As a positive control of growth inhibition of *E. coli* XL-1 strain, AZT was spotted on blank disks (quantities, 0.005-10 µg), showing a dose-dependent effect. The final control, performed in parallel in all subsequent experiments, consisted in 0.625µg AZT, resulting in a growth inhibition zone diameter of 40 ± 2 mm (See supporting information Table S1). A representative panel of 12 β-lactamases was assayed with all possible molecular classes including various substrate specificities and different modes of action (Table 1). As a model, the pronucleoside **5a** was chosen and preincubated for 30 min at 2 µM concentration with each β-lactamase (tested at concentrations 0.5 and 0.01 mg.mL⁻¹). Results are reported in table 1 (all pictures of diffusion disks are provided in SI, figure S5).

<table><caption>**Table 1.** β-lactamase-mediated release of AZT measured by disk-diffusion after incubation in the presence of β-lactamases representative of the four structural classes.</caption><thead><tr><th rowspan="3">[Enzyme]<br>mg.mL<sup>-1</sup></th><th colspan="10">E. coli growth inhibition zone diameter (mm) after preincubation of 5a with a β-lactamase<sup>a</sup></th></tr><tr><th colspan="3">Class A</th><th colspan="3">Class B</th><th colspan="3">Class C</th><th colspan="3">Class D</th></tr><tr><th>KPC-2</th><th>SHV-12</th><th>TEM-1</th><th>IMP-1</th><th>NDM-4</th><th>VIM-2</th><th>AmpC</th><th>CMY-4</th><th>FOX-7</th><th>FUS-1</th><th>OXA-46</th><th>OXA-48</th></tr></thead><tbody><tr><td>0.5</td><td>38</td><td>36</td><td>35</td><td>28</td><td>36</td><td>32</td><td>24</td><td>25</td><td>-</td><td>19</td><td>31</td><td>33</td></tr><tr><td>0.01</td><td>38</td><td>28</td><td>19</td><td>-<sup>b</sup></td><td>-</td><td>13</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

The highest antibacterial activity, i.e., reflecting the optimal release of AZT from the prodrug, was obtained with class A β-lactamases (19-38 mm). Interestingly, significant compound conversion was also observed at low enzyme concentrations (0.01 mg.mL⁻¹), particularly with the KPC-2 carbapenemase (Table 1). Almost similar inhibition levels were obtained at 0.5 mg.mL⁻¹ for class B metallo-β-lactamases although AZT conversion was drastically reduced at lower concentrations, except, to some extent, in the presence of the VIM-2 enzyme. This result is consistent with the substrate profile of metallo-β-lactamases for which monobactams are poor substrates.⁵⁶ Preferred substrates of class C enzymes (AmpC-type) are generally cephalosporins over monobactams or carbapenems (cephalosporinase activity). Nonetheless, weak **5a** conversion was detected with *Enterobacter cloacae* AmpC and the plasmid-encoded CMY-4 β-lactamase. Among class D enzymes, the narrow-spectrum FUS-1 (OXA-85) yielded a limited release of AZT, but the activity of OXA-46 and, more interestingly, the carbapenem-hydrolyzing OXA-48 β-lactamase, allowed for a conversion rate similar to that observed with other enzymes, although no conversion could be observed in the presence of lower enzyme concentrations. Since class A β-lactamases, and notably the epidemiologically relevant KPC-2 carbapenemase, appeared as the most active enzymes on compound **5a**, further biochemical experiments were carried out to further characterize the interaction and KPC-2-mediated hydrolysis of this compound.

**Characterization of beta-lactamase-mediated hydrolysis by ITC.** Based on previous panel results, the KPC-2 enzyme was chosen as a model for the determination of the kinetic parameters for the hydrolysis of pronucleosides **5a-h** using Isothermal Titration Calorimetry (ITC).⁵⁷ Based on the enthalpy value of β-lactam amide bond hydrolysis, *k*<sub>cat</sub> and *K*<sub>M</sub> values were determined.⁵⁸ The method was validated by determining the kinetic parameters of hydrolysis of meropenem, yielding values of *k*<sub>cat</sub>

<footnote><sup>a</sup>performed in duplicate with 30 min of preincubation at RT between **5a** and the corresponding β-lactamase and transfer onto *E. coli* XL1-inoculated agar plate (see Materials and Methods for details). <sup>b</sup>No growth inhibition detected.</footnote>
and K_M comparable to that reported in the literature (Table 2).⁵⁹ All considered prodrugs are structurally equivalent except for the nature of the side chain at the 3-position of the β-lactam ring. These experiments allowed to obtain relevant SAR data. First, the effect of this substitution was investigated on the affinity and hydrolytic activity of the KPC-2 enzyme. No enzymatic activity was highlighting the requirement of a more complex side chain for enzyme recognition and hydrolysis. Substitutions by allyl groups, such as in the diastereoisomers 5f' or 5f'', did not improve compounds reactivity towards KPC-2. The introduction of a pyridine moiety seemed more favorable. Indeed, with the *E*-configuration of the carbon-carbon double bond, compound 5e displayed low but measurable turnover rates, although with a relatively high K_M value, leading to an overall poor catalytic efficiency (kcat/KM). However, the *Z*-configurated pyridine 5d showed a 10-fold higher kcat and 10-fold lower K_M values, compared to *E*-configuration, allowing a significant improvement of the catalytic efficiency. Based on literature data, only *E*-configuration was explored to design β-lactamase inhibitors (due to the necessary intramolecular cyclization step for the inhibition mechanism),³⁴ but *Z*-configurated substrates seemed better suited to accommodate themselves into the active site of KPC-type enzymes. The main type of explored substitution at 3-position was acyl-side chain like most marketed β-lactam agents. The Boc-protected compound 5b displayed unexpected pretty good recognition even, to the best of our knowledge, it was never described in the literature as an appropriate side chain. The two isolated epimers 5g' and 5g" inspired by the benzylic penicillin G side chain were evaluated separately to understand the significance of the stereochemistry of the hemiaminal carbon atom. As expected, only one epimer, the compound 5g", was strongly active whereas the other one 5g' displayed almost no activity. It is a consistent result because, in the β-lactam agent series, it is well known that only one stereochemistry is allowed, the *R* configuration. The pure epimer 5a with the side chain of the penicillin V showed a similar activity profile than that of 5g", probably with the same carbon stereochemistry. Unfortunately, the other epimer of 5a was never obtained in pure form to confirm this hypothesis. Finally, the best result was achieved by the analog of the cephalothin 5h characterized by a thiophene residue. The K_M value was in the same range as the other best compounds (around 1-4 mM) but the kcat was significantly better (350 s⁻¹). The resulting catalytic efficiency for 5h hydrolysis was interestingly similar to that of the substrate meropenem (≈250 mM⁻¹s⁻¹), indicating that a significant rate of conversion of our compounds should be expected with at least some clinically relevant β-lactamases. However, this series of pronucleosides 5a-g is characterized by high values of K_M when compared to that of β-lactam substrates, which is probably due to the presence of the monobactam core, generally known to display lower affinity against KPC-type β-lactamase. Hopefully, this is compensated by significantly high turnover rates, exceeding that of meropenem by up to 60-fold.
<table><caption>**Table 2.** Kinetic parameters of KPC-2 and NDM-1 for the hydrolysis of pronucleosides, determined by ITC.</caption><thead><tr><th rowspan="2">Cpd</th><th colspan="3">KPC-2</th></tr><tr><th>k<sub>cat</sub> (s<sup>-1</sup>)<sup>a</sup></th><th>K<sub>M</sub> (mM)<sup>a</sup></th><th>k<sub>cat</sub>/K<sub>M</sub> (mM<sup>-1</sup>s<sup>-1</sup>)</th></tr></thead><tbody><tr><td>Meropenem<sup>b</sup></td><td>5.8 ± 0.1<br>(4.1)</td><td>0.023 ± 0.002<br>(0.016)</td><td>252.2<br>(256)</td></tr><tr><td>5a<sup>e</sup></td><td>105 ± 3</td><td>4.1 ± 0.2</td><td>25.6</td></tr><tr><td>5b</td><td>96.1 ± 2.2</td><td>5.3 ± 0.2</td><td>18.1</td></tr><tr><td>5c</td><td>&lt;0.003<sup>c</sup></td><td>-</td><td>-</td></tr><tr><td>5d</td><td>3.2 ± 0.1</td><td>0.45 ± 0.04</td><td>7.1</td></tr><tr><td>5e</td><td>0.21 ± 0.01</td><td>3.1 ± 0.3</td><td>0.07</td></tr><tr><td>5f<sup>e</sup></td><td>0.02<sup>d</sup></td><td>-</td><td>-</td></tr><tr><td>5f<sup>II</sup><sup>e</sup></td><td>0.09<sup>d</sup></td><td>-</td><td>-</td></tr><tr><td>5g<sup>e</sup></td><td>≥0.06<sup>d</sup></td><td>≥3.8<sup>d</sup></td><td>-</td></tr><tr><td>5g<sup>II</sup><sup>e</sup></td><td>127.6 ± 2.1</td><td>3.3 ± 0.1</td><td>38.7</td></tr><tr><td>5h</td><td>351.4 ± 8.8</td><td>1.5 ± 0.08</td><td>234.3</td></tr></tbody></table><table><thead><tr><th rowspan="2">Cpd</th><th colspan="3">NDM-1</th></tr><tr><th>k<sub>cat</sub> (s<sup>-1</sup>)<sup>a</sup></th><th>K<sub>M</sub> (mM)<sup>a</sup></th><th>k<sub>cat</sub>/K<sub>M</sub> (mM<sup>-1</sup>s<sup>-1</sup>)</th></tr></thead><tbody><tr><td>Meropenem<sup>b</sup></td><td>109 ± 1<br>(144.9)</td><td>0.06 ± 0.003<br>(0.06)</td><td>1816.7<br>(2415)</td></tr><tr><td>5a<sup>e</sup></td><td>15.5 ± 0.4</td><td>3.6 ± 0.1</td><td>4.3</td></tr></tbody></table>

Considering that metallo-β-lactamases showed a surprising ability to convert our monobactam-based pronucleoside, the hydrolysis of compound **5a** was also further investigated in the presence of NDM-1 (Table 2). As previously, kinetic parameters of meropenem hydrolysis were measured and compared to that reported in the literature, showing a satisfactory agreement.<sup>60</sup> Although high $K_M$ values were obtained, an expected data based on our current knowledge on MBL spectrum of activity, a significantly high turnover rate (15 s<sup>-1</sup>) could be measured, in agreement with what was observed in earlier disk diffusion-based experiments. This is relevant because it might translate into a moderate, though potentially sufficient, conversion rate to observe AZT release, which exerts its antibacterial activity at very low concentrations.

<footnote><sup>a</sup>Values are mean ± SD; <sup>b</sup>KPC-2 data are from Wang *et al.*,<sup>59</sup> NDM-1 data are from Ali *et al.*,<sup>60</sup>. <sup>c</sup>Not detected, below detection limit; <sup>d</sup>approximate values due to the very low activity; <sup>e</sup>pure diastereomer compound.</footnote>
**Chemical and enzymatic stability of pronucleosides.** Before any microbiological evaluations, the chemical stability of conjugates in aqueous solution has to be carefully determined. Indeed, zidovudine (AZT) displays a very strong antibacterial activity with MIC values well under 0.5 µg.ml⁻¹ on susceptible stains.³⁹ Thus, even a minor chemical instability with subsequent AZT release may represent a significant interference biasing the interpretation of *in vitro* antibacterial susceptibility testing. To monitor the chemical stability, a two-step process was implemented. In the first step, a representative panel of prodrugs (**5a**, **5d-e**, and **5h**) were evaluated by tandem mass spectroscopy coupled to HPLC. A 0.9 % NaCl physiological solution, previously used in enzyme assays (see Table 1), was used as the aqueous media and the compounds were further incubated at 37 °C. Calibration curves were obtained for each compound and AZT, and then, stability was monitored over a period of 16h. Almost all considered compounds proved to be completely stable, with no detectable AZT formation, except for compound **5e**. In the case of *E*-pyridine **5e**, a quick degradation was observed with the detection of AZT from the first sample associated with decreasing intensity of starting compound (all chromatograms and result tables are provided in SI, Table S4). The underlying degradation mechanism is unknown, but it seems specific for the *E* configuration (intensity of Z isomer **5d** decreases slightly but with no detection of AZT release). To fully demonstrate the specific enzymatic activation of the prodrugs, the same experiments were conducted in presence of CTX-M-15 class A β-lactamase. The same preincubation conditions were used as those described in Table 1, and a full conversion of **5a** into AZT was observed after 30 min. To confirm these stability data, the two best compounds **5a** and **5h** and the presumably unstable **5e** were selected for a further NMR-based study. Compounds were solubilized in deuterium water at 37 °C and several NMR spectra were generated for 14 h. NMR characteristic peaks of AZT were carefully monitored, and no significant degradation was observed with **5a** and **5h** (all spectra are provided in SI, Figures S15-S16). In the case of **5e**, AZT peaks emerged after 20 min only, confirming its propensity to self-degradation. Based on NMR integration of proton 5' of AZT, 50% of starting compound **5e** was hydrolyzed with subsequent release of corresponding AZT after a period of 6.7 h (see SI Figures S17-S18).

**In vitro antibacterial activity of pronucleosides.** Only best and most stable compounds **5a** and **5h** were selected for further microbiological evaluations. AZT was used as the reference compound in such assays. MIC values were first determined using a set of isogenic *E. coli* BL21 (DE3)-derived laboratory strains producing a single β-lactamase (Table 3, entries 1-5). Based on ITC results and epidemiology relevance, strains producing the KPC-3, NDM-1, IMP-1, and *Enterobacter cloacae* AmpC β-lactamases were used. It was also verified that all corresponding *E. coli* strains were highly susceptible to AZT (MIC ranging from 0.03-0.5 µg.mL⁻¹). No significant antibacterial activity (16-32 µg.mL⁻¹) could be measured for compounds **5a** and **5h** with the parent strain in the absence of the vector. In agreement with our previous results (including ITC), the strain producing the KPC-3 carbapenemase was highly susceptible to both prodrugs **5a** and **5h**, with MIC values very similar to that of AZT, indicating a significant rate of conversion of the prodrug. This further confirms the ability of KPC-type β-lactamases to accommodate and efficiently hydrolyze our β-lactam-based carrier moiety also in the context of the bacterial periplasm. Metallo-β-lactamase-producing laboratory strains (IMP-1 and NDM-1) were less susceptible to the pronucleosides, although a moderate level of compound conversion could be observed, more particularly **5a** with NDM-1 and **5h** with IMP-1. Although difficult to interpret, these data may also reflect the different functional properties of these two enzyme subtypes. Similarly, the *E. cloacae* AmpC-producing strain showed a strong dependence on the nature of the compound, being able to efficiently convert prodrug **5h** only whereas acyl-side-chain equivalent β-lactam agents, amoxicillin and cephalothin, are both known to be good substrates.⁶¹ To rule out any spontaneous activation of the prodrug in the culture medium (matrix effect), MIC values of **5a** were also determined on all *E. coli* BL21 (DE3) strains after 6h of incubation at 37°C in the culture medium. No significant modification of
MIC value was observed compared to provided values in Table 3 without the incubation step, confirming that lower MICs were relying specifically on the β-lactamase-mediated release of AZT. These results, as previously mentioned, showed that the prodrug compounds were also able to enter into the bacterial periplasm and that these β-lactamases could exert their hydrolytic activity on such substrates in a cellular context. To further confirm this aspect, 2 types of strains were selected with no or very weak β-lactamase production (Table 3, entries 6 and 7). The prodrug 5a showed no significant growth inhibition on both strains demonstrating the necessity of β-lactamase enzymes to trigger it. The narrower antibacterial spectrum of conjugates allows for a specific targeting of β-lactamase-producing opportunistic pathogens and not antibiotic-susceptible bacteria found in the healthy gut microbiota. Finally, the activity of such prodrugs was also determined with a small panel of carbapenemase-producing MDR clinical isolates (Table 2, entries 8-11). Although these isolates were susceptible to AZT, the MIC values of the prodrugs were significantly higher. However, we found it extremely encouraging to observe MIC values as low as 4-8 µg.mL⁻¹ on a NDM-1-producing clinical isolate, further supporting the rationale and validity of our approach. In the case of KPC-producing bacteria, only the K. pneumoniae SI-353 strain displayed a moderate inhibition with MIC values of 32 and 16 µg.mL⁻¹ with 5a and 5h, respectively, reflecting the importance of other factors in the susceptibility to our prodrug, such as modifications of the outer membrane permeability. Indeed, these clinical isolates are genetically heterogeneous and thus characterized by a different combination of mechanism of resistance and potentially unknown other factors compromising the activity of our prodrugs. To rule out any potential permeability-related issue, the MICs of the prodrugs were determined in the presence of a sub-inhibitory concentration of colistin, a polymyxin antibiotic known for its membrane permeabilizing activity, but no modification was observed. Thus, outer membrane diffusion does not seem to be the main factor involved. It is likely to result from a combination of factors, which deserves further detailed investigation. Nevertheless, in almost every case, the thiophene substitution of 5h seems slightly favorable compared to the penicillin V side chain of 5a.
<table><caption>**Table 3.** MIC determination by Broth micro-dilution method<sup>a</sup></caption><thead><tr><th rowspan="2">Entry</th><th rowspan="2">Bacterial strain</th><th colspan="3">MIC<sup>b</sup> (µg.mL<sup>-1</sup>)</th></tr><tr><th>AZT<sup>f</sup></th><th>5a</th><th>5h</th></tr></thead><tbody><tr><td>1<sup>c</sup></td><td><i>E. coli</i> BL21 (DE3)</td><td>0.06</td><td>32</td><td>16</td></tr><tr><td>2<sup>d</sup></td><td><i>E. coli</i> BL21 (DE3) pET9a-KPC-3</td><td>0.5</td><td>1</td><td>0.25</td></tr><tr><td>3<sup>d</sup></td><td><i>E. coli</i> BL21 (DE3) pET9a-IMP-1</td><td>0.03</td><td>8</td><td>2</td></tr><tr><td>4<sup>d</sup></td><td><i>E. coli</i> BL21 (DE3) pET9a-NDM-1</td><td>0.06</td><td>2</td><td>4</td></tr><tr><td>5<sup>d</sup></td><td><i>E. coli</i> BL21 (DE3) pET9a-AmpC</td><td>0.125</td><td>32</td><td>2</td></tr><tr><td>6<sup>c</sup></td><td><i>E. coli</i> ATCC25922</td><td>4</td><td>>256</td><td>ND</td></tr><tr><td>7<sup>c</sup></td><td><i>K. pneumoniae</i> ATCC13833</td><td>0.25</td><td>128</td><td>ND</td></tr><tr><td>8<sup>e</sup></td><td><i>E. coli</i> SI44 KPC-3</td><td>2</td><td>64</td><td>64</td></tr><tr><td>9<sup>e</sup></td><td><i>K. pneumoniae</i> SI-353 KPC-3</td><td>4</td><td>32</td><td>16</td></tr><tr><td>10<sup>e</sup></td><td><i>K. pneumoniae</i> SI-109 KPC-3</td><td>2</td><td>>128</td><td>>128</td></tr><tr><td>11<sup>e</sup></td><td><i>E. coli</i> B28 NDM-1</td><td>0.25</td><td>8</td><td>4</td></tr></tbody></table>

**Solubility and preliminary toxicity evaluations.** To consider further potential clinical developments, the solubility and toxicity profiles of prodrugs **5a-h** were investigated. Final compounds were initially isolated in neutral form (e.g., with carboxylic acid function) displaying a moderate aqueous solubility capacity. Sodium salts were later obtained, which showed a significantly high solubility (≥ 100 µM). The potential cytotoxicity of compounds AZT, **5a**, and **5h** was evaluated on human HeLa cells. No membrane-damaging activity or cytotoxic effects could be detected at concentrations up to 128 µg.mL<sup>-1</sup>, indicating that our prodrugs are comparable to AZT. Those preliminary results are favorable for further optimizations and developments.

## ■ CONCLUSION

The development of new approaches leading to antibacterial agents is a crucial step towards addressing and minimizing the potentially dramatic of antimicrobial resistance, already accounting for 1.3 M deaths worldwide. This study addresses this expectation to use bacterial resistance to trigger β-lactam conjugates: the higher will be the level of β-lactamase expression, the more vulnerable will be the bacteria. First, based on the literature, a synthetic pathway was developed to obtain prodrugs in good yields. AZT was chosen as the antibacterial agent while several substitutions on the β-lactam ring were considered. Finally, 10 final compounds **5a-h** were obtained and characterized. It was possible in some cases to separate the diastereomers allowing to measure the impact of the configuration on the biological activity. Nevertheless, it is still necessary to optimize this separation step or to develop a

<footnote><sup>a</sup> Triplicate experiment; <sup>b</sup> Minimal Inhibitory Concentration; <sup>c</sup> reference isolate; <sup>d</sup> isogenic β-lactamase producer, <sup>e</sup> clinical isolate; <sup>f</sup> positive control experiment with AZT alone; ND, Not Determined</footnote>
stereoselective synthesis. The unambiguously determination of absolute configuration of this
asymmetric center also remains to be established.

Second, the reactivity of the various synthesized prodrugs towards a representative panel of β-lactamases was investigated. To this aim, indirect detection of released AZT after pre-incubation with different β-lactamases provided interesting preliminary information. KPC-2 was identified as the β-lactamase able to catalyze compound conversion more efficiently, although some metallo-β-lactamases also surprisingly, considering our use of a monobactam core, showed this ability. A calorimetric titration study was used to determine the enzymatic parameters of KPC-2 for the hydrolysis of our prodrugs and allowed to establish some interesting SAR considerations. Different pharmacomodulations were evaluated showing the necessity of specific substitution motifs in particularly based on acyl side chains of regular β-lactam agents (penicillin V and G, and cephalothin). Into class A β-lactamase-β-lactam binary complex, these substituents are generally oriented toward a Ω-loop of the enzyme involved in the stabilization of the acyl-enzyme intermediate.⁶² This crucial loop is located next to the catalytic pocket, and it is described to well accommodate small aromatic side chains, especially in the case of KPC variants.⁶³ In this context, compound 5c, with no substituents, is not able to favorably interact with this Ω-loop and probably explains the lack of activity. Several hydrogen bond networks define the interaction between the Ω-loop and a β-lactam substrate, although these may vary in different β-lactamases. However, a conserved Asn residue is involved in a crucial hydrogen bond with the carbonyl of the β-lactam acyl amide function.⁶⁴ The necessity of the carbonyl motif is well illustrated with the better affinities of acylated compounds 5a, 5g and 5h compared to that exhibited by carbon-carbon derivatives 5d-f. Finally, thiophene moiety of 5h seems to be slightly favorable than other acyl-derivatives which is consistent with literature data.⁶⁵ Moreover, specific chirality of stereogenic center proved to be crucial for the activity. Even if the affinities were globally lower compared to that of meropenem, two compounds, 5a and 5h, emerged with interesting values, especially because of their high turnover rates. Even in the case of the metallo-enzyme NDM-1, the monobactam prodrug remained an appropriate substrate contrary to aztreonam. That was demonstrated with a good MIC value on clinical isolate expressing NDM-1 enzyme. The chemical stability was also evaluated, showing that most compounds were not prone to self-conversion or degradation in aqueous medium. Microbiological assays, first performed using isogenic laboratory strains producing different β-lactamases, resulted in low MIC values, confirming the specific and β-lactamase-mediated release of AZT in a cellular context. Nevertheless, the results were more contrasted on clinical isolates, although undoubtfully interesting. Preliminary solubility and toxicity studies showed that the compounds 5a and 5h also presented a favorable profile for future clinical development.

This pioneering study demonstrated that it was possible to target pathogenic bacteria by using the expression of β-lactamases to selectively release antibacterial agents, this obtaining compounds specifically targeting antibiotic-resistant bacteria. This has led to the possibility of developing targeted antibiotic therapies leading to a potentially drastic reduction of off-target effects, particularly on intestinal microbiota. A larger study is underway to incorporate other active substances than AZT as well as to modify the nature of the transporter to increase affinity towards β-lactamases.

■ METHODS

**General methods.** Nuclear Magnetic Resonance (NMR) spectra were recorded with a Bruker Avance 400 or 500 or 600 spectrometer (for ¹H and ¹³C NMR) and Bruker Avance 400 spectrometer (376.5 for ¹⁹F). Chemical shifts (δ) and coupling constants (J) are given in ppm and Hz, respectively, using residual solvent signals as reference for the ¹H and ¹³C. The following abbreviations are used: s = singlet, d = doublet, t = triplet, q = quartet, br s = broad signal, dd = double doublet, dt = double of triplet, m =
multiplet. High-resolution mass spectra (HRMS) were registered on a JEOL JMS-SX-102A mass spectrometer. Reactions were monitored with Merck Kieselgel 60F254 precoated aluminum silica gel plates (0.25 mm thickness) which were visualized under 254 nm light or by charring with a ninhydrin solution in butanol or with a vanillin solution in ethanol followed by heating or iodine stains. Purification experiments were carried out on Silica Gel premium Rf grade (40-63 µm) or were performed on a Biotage Isolera One using a 20-50 µm packed silica cartridge or using reverse phase preparative HPLC on Akta pure C18 column (Luna 21.2 x 100 mm, 5 µm, 100 Å). Eluent A: Water (10 mM AcONH₄ or 0.1% TFA or Neutral); B: Acetonitrile (Neutral or 0.1% TFA, respectively) with flow rate: 20 mL/min. LC-MS analyses were obtained on the Waters Alliance 2690 HPLC, coupled to a Waters Micromass Quattro micro API (electrospray ionization mode, ESI+). Conditions: RP C18 Chomolith High-Resolution column (25 x 4.6 mm) using a linear gradient (0-100%) of water and acetonitrile (each containing 0.1% of formic acid) over 3 min and at a flow rate of 3 mL/min, mono λ detection (214 nm). Positive ion electrospray mass spectra were acquired at a solvent flow rate of 0.15 µL.min⁻¹. Nitrogen was used as both the nebulizing and drying gas. The data were obtained in a scan mode in 0.7 s intervals. UPLC analyses were obtained on the Waters Acquity H-Class UPLC, coupled to a Waters Zspray (electrospray ionization mode, ESI+). Conditions: Acquity UPLC® peptide HSS T3 column (2.1 x 50 mm, 1.8 µm, 100 Å) using a linear gradient (0-100%) of water and acetonitrile (each containing 0.1% of formic acid) over 5 min and at a flow rate of 0.8 mL.min⁻¹ flow rate, photodiode array detection (TUV – 214 nm). Positive ion electrospray mass spectra were acquired at a solvent flow rate of 0.8 µL.min⁻¹. Nitrogen was used as both the nebulizing and drying gas. The data were obtained in a scan mode in 0.2 s intervals. The purity of all synthetic compounds was determined by UPLC analysis and was greater than 95%.

**Enzyme assays and release of AZT from prodrugs.** The β-lactam derivative 5a (2.5 µL) at 5 mg·mL⁻¹ was pre-incubated with 8µL of different enzymes at a concentration of 0.5 mg/mL (Deposit A), 0.01 mg·mL⁻¹ (Deposit B), and no enzyme (Deposit C) in 9.5 µL of 0.9% of NaCl solution. After 30 minutes at room temperature, 2 µl were plated on Müller-Hinton (MH) agar containing *E. coli* XL1 strain and incubated at 37 °C for 24 hours. The same method is used for MICs determination described below.

**Determination of kinetic parameters of hydrolysis by ITC.** Enzyme activity was recorded by ITC on a MicroCal ITC200 (GE-Malvern) equipped with a 200 µl Hastelloy sample cell and an automated 40 µl glass syringe rotating at 1000 rpm. Compounds were solubilized in 10 mM Hepes-NaOH, 0.15 M NaCl, pH 7.5 and both enzymes were diluted to the desired concentrations with the same buffer. For compounds 5a-h, the buffer also contained 1% DMSO and KPC-2 was brought to the same final DMSO concentration. The activity was recorded and calculated as described, using a molar enthalpy value for β-lactam amide bond hydrolysis of 12 kcal/mol at 25°C. The reaction rate constant $k_{cat}$ (s⁻¹) is defined here as the number of amide bonds hydrolyzed per second and per active site. For $K_m$ determination, activities were recorded at decreasing substrate concentrations, and the data points were fitted to the Michaelis-Menten equation.

**MICs determination.** The minimum inhibitory concentrations (MICs) of considered inhibitors were determined in triplicate using cation-supplemented Mueller-Hinton broth and a bacterial inoculum of 5 × 10⁴ CFU/well, as recommended by the CLSI and the European Committee on Antimicrobial Susceptibility Testing (EUCAST document "Terminology relating to methods for the determination of susceptibility of bacteria to antimicrobial agents", 2000). The MIC values of compounds were determined on *E. coli* BL21(DE3)-derivative laboratory strains hyperproducing a single β-lactamase and carbapenemase-producing clinical isolates part of our collection.

**Stability assays by ultra-performance liquid chromatography separation and tandem mass spectrometry.** Identification and stability experiments were carried out by reverse phase liquid
chromatography on a Waters Acquity Iclass UPLC system coupled to a high-resolution mass spectrometer (Xevo TQD Waters), equipped with an electrospray ionization source and controlled by MassLynx v.4.2 software for data acquisition. All the analyses were carried out using a Acquity UPLC BEH C18 2.1 x 50 mm column at 40°C. A flow rate of 0.8 mL.min⁻¹ and a gradient of (0-100%) B over 3.5 min were used: eluent A, water/0.1% HCO₂H; eluent B, acetonitrile/0.1% HCO₂H. The mass spectrometer operated in positive or negative ionization mode and parameters were set as follows: source block temperature, 150 °C, nebulizer nitrogen flow rate, 50 L/hour, capillary voltage, 2.0 or 2.5 kV; desolvation temperature, 550 or 600 C; desolvation nitrogen gas flow, 900 or 1000 L/hour. The different investigated parameters were cone voltage (CV), collision energy (CE), ionization mode, and MRM channels. Specific settings for each compound are provided in Supporting Information (Table S4). All samples were prepared by dilution in a commercial physiological solution (0.9% NaCl). In case of enzymatic assay, a filtration on Amicon Ultra-0.5 Centrifugal Filter Unit, MWCO 3 kDa is performed before injection. A calibration curve was determined for each compound as follows: samples used for the calibration were injected (1µl) in triplicate from the lowest concentration (0.0001mg.ml⁻¹) to the highest one (0.01 mg.ml⁻¹) and the calibration curves were plotted with the Targetlynx tool from Masslynx. Then, samples used for stability experiments were injected (1µl) at 0.005mg/mL at 37°C and monitored every hour over 24 hours. The stability curves were plotted with the Targetlynx tool from Masslynx. The MRM channel monitoring of AZT apparition and molecule degradation were plotted at the same time to check the release.

**Stability assays by NMR.** Pronucleosides were dissolved in 600 µL of D₂O at a concentration of 10 mM. The ¹H NMR spectra were measured on a Bruker 600 MHz spectrometer at various time points at 310 K.

**Toxicity assays.** The potential cytotoxic activity of compounds was evaluated using the commercially available integrity assay (CytoTox 96 nonradioactive cytotoxicity assay, Promega). The compounds were tested for their ability to induce the lysis of HeLa cells by measuring the release of lactate dehydrogenase (LDH) after incubating the HeLa cell cultures (20,000 cells/well) for 24 h (37 °C, 5% CO₂) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 4.5 mg.mL⁻¹ glucose, and 2 mM L-glutamine in the absence and presence of varying concentrations of the compounds (up to 250 µM). Further controls included samples containing the medium only or in which cell lysis was induced by the addition of 9% Triton X-100 (maximum LDH release control). The percentage of cytotoxicity was computed as 100 × (sample LDH release) / (maximum LDH release). The variation in the percentage of cytotoxicity allowed us to compute an IC₅₀ value (compound concentration inducing 50% cytotoxicity).

## ASSOCIATE CONTENT

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/xxxx

Detailed synthetic procedures with all NMR spectrum associated. Supporting figures and schemes cited in the text. Miscellaneous data from biological and stability evaluations.

## AUTHOR INFORMATION

### Corresponding author

Laurent Gavara - Institut des Biomolécules Max Mousseron, Univ Montpellier, CNRS, ENSCM, 34293 Montpellier, France; orcid.org/0000-0003-0146-1848; Email: laurent.gavara@umontpellier.fr
Author

Miyanou Rosales-Hurtado - Institut des Biomolécules Max Mousseron, Univ Montpellier, CNRS, ENSCM, 34293 Montpellier, France; orcid.org/0000-0003-2988-4244

Filomena Sannio - Dipartimento di Biotecnologie Mediche, Università di Siena, I-53100 Siena, Italy

Lindita Lari - Institut des Biomolécules Max Mousseron, Univ Montpellier, CNRS, ENSCM, 34293 Montpellier, France

Federica Verdirosa - Dipartimento di Biotecnologie Mediche, Università di Siena, I-53100 Siena, Italy

Georges Feller - Laboratoire de Biochimie, Centre d'Ingénierie des Protéines-InBioS, Université de Liège, B-4000 Liège, Belgium

Elodie Carretero - Institut des Biomolécules Max Mousseron, Univ Montpellier, CNRS, ENSCM, 34293 Montpellier, France

Yen Vo-Hoang - HSM, Univ Montpellier, CNRS, IRD, CHU Montpellier, 34090 Montpellier, France

Patricia Licznar-Fajardo - HSM, Univ Montpellier, CNRS, IRD, CHU Montpellier, 34090 Montpellier, France

Jean-Denis Docquier - Dipartimento di Biotecnologie Mediche, Università di Siena, I-53100 Siena, Italy;
orcid.org/0000-0001-9483-4476

Author contributions

M.R-H. synthesized, purified, and characterized compounds, conducted stability assays, and wrote the supporting information, F.S. and F.V. were in charge of enzymatic, microbiological, and toxicity assays, L.L. synthesized, purified, and characterized compounds, G.F. performed and interpreted ITC measurements, E.C. monitored LCMSMS stability assays, Y.V-H. and P. L-F. performed and interpreted microbiological assays, J-D. D. designed and interpreted biological evaluations and contributed to project design, L.G. designed, funded, and supervised the project, and wrote the draft. All authors contributed to edit this manuscript.

Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

This work, the ABR-Breaker project, was supported by Agence Nationale de la Recherche (ANR-19-
CE18-0003-01). We also want to thank analytical and SynBio3 platforms and in particular Mr. Luc
Brunel, Dr. Pascal Verdié and Mr. Pierre Sanchez for mass spectrometry analyses. Background image
of the graphical abstract is designed by macrovector_official / Freepik.

■ ABBREVIATIONS

AIDS, Acquired ImmunoDeficiency Syndrome; AmpC, Ampicillin C; AZT, Zidovudine; β-L, Beta-Lactamase; DEAD, DiEthyl AzoDicarboxylate; EDCI, 1-Ethyl-3-(3-Dimethylaminopropyl)Carbodlimide; ESBL, Extended-Spectrum Beta-Lactamase; HOBt, 1-Hydroxybenzotriazole; IMP, Imipenemase; ITC, Isothermal Titration Calorimetry; KPC, *Klebsiella pneumoniae* Carbapenemases; LDA, Lithium DiisopropylAmide; MBL, Metallo-Beta-Lactamase; NA, Nucleoside analog; NDM, New Delhi Metallo-beta-lactamase; OXA, Oxacillinase; PBPs, penicillin-binding proteins; SARs, Structure-Activity relationships; SBL, Serine-Beta-Lactamase; TBDMS, *Tert*-ButylDiMethylSilyl; TFA, TriFluoroacetic Acid.
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Zidovudine-β-lactam pronucleoside strategy for selective delivery
into Gram-negative bacteria triggered by β-lactamases

Miyanou Rosales-Hurtado,ª Filomena Sannio,ᵇ Lindita Lari,ª Federica Verdirosa,ᵇ Georges Feller,ᶜ Elodie Carretero,ª Yen Vo-Hoang,ᵈ Patricia Licznar-Fajardo,ᵈ Jean-Denis Docquier,ᵇ,e
Laurent Gavaraª,\*

**Synopsis:** The TOC graphic displays the specific prodrug activation of synthesized conjugates into β-lactamase producers to deliver the antibacterial AZT, and also provides an overview of the different biological evaluations performed.

<figure><img src="image_7.png" /></figure>