Metallo-β-lactamase (MBL) production in Gram-negative bacteria is an important contributor to β-lactam antibiotic resistance. Combining β-lactams with β-lactamase inhibitors (BLIs) is a validated route to overcoming resistance, but MBL inhibitors are not available in the clinic. On the basis of zinc utilization and sequence, MBLs are divided into three subclasses, B1, B2, and B3, whose differing active-site architectures hinder development of BLIs capable of "crossclass" MBL inhibition. We previously described 2-mercaptomethyl thiazolidines (MMTZs) as B1 MBL inhibitors (e.g., NDM-1) and here show that inhibition extends to the clinically relevant B2 (Sfh-I) and B3 (L1) enzymes. MMTZs inhibit purified MBLs in vitro (e.g., Sfh-I, K i 0.16 μM) and potentiate β-lactam activity against producer strains. X-ray crystallography reveals that inhibition involves direct interaction of the MMTZ thiol with the mono-or dizinc centers of Sfh-I/L1, respectively. This is further enhanced by sulfur-π interactions with a conserved active site tryptophan. Computational studies reveal that the stereochemistry at chiral centers is critical, showing less potent MMTZ stereoisomers (up to 800-fold) as unable to replicate sulfur-π interactions in Sfh-I, largely through steric constraints in a compact active site. Furthermore, in silico replacement of the thiazolidine sulfur with oxygen (forming an oxazolidine) resulted in less favorable aromatic interactions with B2 MBLs, though the effect is less than that previously observed for the subclass B1 enzyme NDM-1. In the B3 enzyme L1, these effects are offset by additional MMTZ interactions with the protein main chain. MMTZs can therefore inhibit all MBL classes by maintaining conserved binding modes through different routes.

Bisthiazolidines (BTZ) are bicyclic compounds considered as penicillin analogs that inhibit the full range of Metallo‐β‐Lactamases (MBLs) and potentiate β‐lactam activity against resistant bacteria. Herein, we present a new methodology to prepare 2‐substituted bisthiazolidines by aldehyde exchange. Thirteen new bisthiazolidines were prepared using this methodology, with yields ranging from 31 to 75 %. The reaction is based on in situ imines formation, which are able to exchange side chains. The reaction intermediates were studied based on NMR experiments, and a key imine 1b‐II could be detected in the reaction mixture. Furthermore, a DFT computational analysis was performed to gain insights into the reaction mechanism, allowing us to unveil the different pathways and their activation barriers within the synthetic route. The results suggest that the most favorable route involve the formation of the thiazolidine 1b‐III by i) a N‐assisted N–C bond cleavage, and ii) a thiol‐mediated 5 endo‐trig cyclization followed by a C–N bond cleavage. In contrast with previously reported evidence, the imine metathesis was discarded as a plausible pathway. Finally, the reaction of 1b‐III with aldehyde 2a leads to bicycle 4a via the iminium ion 1b‐V.

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The synthesis of new oxazolidinylthiazolidines bicycles, oxygen analogues of bisthiazolidines, also known as metallo-β-lactamase inhibitors is described. The reaction of β-aminoalcohols and 2,5-dihydroxy-1,4-dithiane led to oxazolidinylthiazolidines and/or dithia-azabicycles as the main products. The distribution pattern depends mainly on the aminoalcohol substituents. In a one-pot reaction, four new bonds are formed in good yields and with high atom efficiency. When the oxazolidinylthiazolidines are formed, two stereogenic centres are generated with high enantiospecificity. The reaction mechanism is discussed based on crystallographic data and interconversion studies. Two oxazolidinylthiazolidines were evaluated as inhibitors of the potent lactamase NDM-1 and compound 4f displayed competitive inhibition with Ki = 1.6 ± 0.6 µM.

The reversibility of the thiol-thioester linkage has been broadly employed in many fields of biochemistry (lipid synthesis) and chemistry (dynamic combinatorial chemistry and material science). When the transthioesterification is followed by a S-to-N acyl transfer to give an amide bond, it is called Native Chemical Ligation (NCL), a high-yield chemoselective process used for peptide synthesis. Recently, we described thioglycolic acid (TGA) as a useful reagent for thioester deprotection both in solution and anchored to a solid-support under mild conditions. Inspired by NCL, in this work, we extended this approach and explored the use of 2-aminothiols for the deprotection of thiols bearing an acyl group. The best results were obtained using cysteamine or L-cysteine in an aqueous buffer pH 8 at room temperature for 30 min. The described approach was useful for S-acetyl, S-butyryl, and S-benzoyl heterocycles deprotection with yields up to 84%. Employing this methodology, we prepared six new analogs 2 of mercaptomethyl bisthiazolidine 1, a useful inhibitor of a wide-range of Metallo-β-Lactamases (MBLs). Compared with the previous methodologies (TGA polymer supported and TGA in solution), the biomimetic deprotection herein described presents better performance with higher yields, shorter reaction times, less time-consuming operations, easier setup, and lower costs.