Insights into E.†coli Cyclopropane Fatty Acid Synthase (CFAS) Towards Enantioselective Carbene Free Biocatalytic Cyclopropanation.

Cyclopropane fatty acid synthases (CFAS) are a class of S-adenosylmethionine (SAM) dependent methyltransferase enzymes able to catalyse the cyclopropanation of unsaturated phospholipids. Since CFAS enzymes employ SAM as a methylene source to cyclopropanate alkene substrates, they have the potential to be mild and more sustainable biocatalysts for cyclopropanation transformations than current carbene-based approaches. This work describes the characterisation of E. coli CFAS (ecCFAS) and its exploitation in the stereoselective biocatalytic synthesis of cyclopropyl lipids. ecCFAS was found to convert phosphatidylglycerol (PG) to methyl dihydrosterculate 1 with up to 58 % conversion and 73 % ee and the absolute configuration (9S,10R) was established. Substrate tolerance of ecCFAS was found to be correlated with the electronic properties of phospholipid headgroups and for the first time ecCFAS was found to catalyse cyclopropanation of both phospholipid chains to form dicyclopropanated products. In addition, mutagenesis and in silico experiments were carried out to identify the enzyme residues with key roles in catalysis and to provide structural insights into the lipid substrate preference of ecCFAS. Finally, the biocatalytic synthesis of methyl dihydrosterculate 1 and its deuterated analogue was also accomplished combining recombinant ecCFAS with the SAM regenerating AtHMT enzyme in the presence of CH3I and CD3I respectively.

Chemoenzymatic Cascades for the Enantioselective Synthesis of ß-Hydroxysulfides Bearing a Stereocentre at the C-O or C-S Bond by Ketoreductases.

Chiral ß-hydroxysulfides are an important class of organic compounds which find broad application in organic and pharmaceutical chemistry. Herein we describe the development of novel biocatalytic and chemoenzymatic methods for the enantioselective synthesis of ß-hydroxysulfides by exploiting ketoreductase (KRED) enzymes. Four KREDs were discovered from a pool of 384 enzymes identified and isolated through a metagenomic approach. KRED311 and KRED349 catalysed the synthesis of ß-hydroxysulfides bearing a stereocentre at the C-O bond with opposite absolute configurations and excellent ee values by novel chemoenzymatic and biocatalytic-chemical-biocatalytic (bio-chem-bio) cascades starting from commercially available thiophenols/thiols and α-haloketones/alcohols. KRED253 and KRED384 catalysed the enantioselective synthesis of ß-hydroxysulfides bearing a stereocentre at the C-S bond with opposite enantioselectivities by dynamic kinetic resolution (DKR) of racemic α-thioaldehydes.

Unconventional Biocatalytic Approaches to the Synthesis of Chiral Sulfoxides.

Sulfoxides are a class of organic compounds that find wide application in medicinal and organic chemistry. Several biocatalytic approaches have been developed to synthesise enantioenriched sulfoxides, mainly by exploiting oxidative enzymes. Recently, the use of reductive enzymes such as Msr and Dms has emerged as a new, alternative method to obtain enantiopure sulfoxides from racemic mixtures. In parallel, novel oxidative approaches, employing nonclassical solvents such as ionic liquids (ILs) and deep eutectic solvents (DESs), have been developed as greener and more sustainable biocatalytic synthetic pathways. This minireview aims highlights the recent advances made in the biocatalytic synthesis of enantioenriched sulfoxides by employing such unconventional approaches.

A mild and chemoselective CALB biocatalysed synthesis of sulfoxides exploiting the dual role of AcOEt as solvent and reagent.

A mild, chemoselective and sustainable biocatalysed synthesis of sulfoxides has been developed exploiting CALB and using AcOEt with a dual role of more environmentally friendly reaction solvent and enzyme substrate. A series of sulfoxides, including the drug omeprazole, have been synthesised in high yields and with excellent E-factors.

Synthesis and Biological Evaluation of Amidinourea Derivatives against Herpes Simplex Viruses.

Current therapy against herpes simplex viruses (HSV) relies on the use of a few nucleoside antivirals such as acyclovir, famciclovir and valacyclovir. However, the current drugs are ineffective against latent and drug-resistant HSV infections. A series of amidinourea compounds, designed as analogues of the antiviral drug moroxydine, has been synthesized and evaluated as potential non-nucleoside anti-HSV agents. Three compounds showed micromolar activity against HSV-1 and low cytotoxicity, turning to be promising candidates for future optimization. Preliminary mode of action studies revealed that the new compounds act in an early stage of the HSV replication cycle, just after the viral attachment and the entry phase of the infection.

Enantioselective Synthesis of α-Thiocarboxylic Acids by Nitrilase Biocatalysed Dynamic Kinetic Resolution of α-Thionitriles.

The enantioselective synthesis of α-thiocarboxylic acids by biocatalytic dynamic kinetic resolution (DKR) of nitrile precursors exploiting nitrilase enzymes is described. A panel of 35 nitrilase biocatalysts were screened and enzymes Nit27 and Nit34 were found to catalyse the DKR of racemic α-thionitriles under mild conditions, affording the corresponding carboxylic acids with high conversions and good-to-excellent ee. The ammonia produced in situ during the biocatalytic transformation favours the racemization of the nitrile enantiomers and, in turn, the DKR without the need of any external additive base.

Synthesis, biological evaluation and mode of action studies of novel amidinourea inhibitors of hepatitis C virus (HCV).

Novel amidinourea derivatives have been synthesised and evaluated for their antiviral activity against Hepatitis C Virus (HCV). A compound with an amidinourea-spermine chemical structure, different from that of standard anti-HCV drugs, showed micromolar activity against HCV and excellent viability. Studies on the mode of action revealed that the new compound may act against HCV through the inhibition of IRES-mediated translation.

A facile and regioselective multicomponent synthesis of chiral aryl-1,2-mercaptoamines in water followed by monoamine oxidase (MAO-N) enzymatic resolution.

A facile microwave assisted three-component protocol allows the synthesis of chiral aryl-1,2-mercaptoamines in water in a few minutes with high yields, bypassing the use of toxic aziridine intermediates. The chiral 1,2-mercaptoamines were then deracemized through enzymatic resolution of the racemates using monoamine oxidase (MAO-N) biocatalysts.

Synthesis and Reactivity of Propargylamines in Organic Chemistry.

Propargylamines are a versatile class of compounds which find broad application in many fields of chemistry. This review aims to describe the different strategies developed so far for the synthesis of propargylamines and their derivatives as well as to highlight their reactivity and use as building blocks in the synthesis of chemically relevant organic compounds. In the first part of the review, the different synthetic approaches to synthesize propargylamines, such as A3 couplings and C-H functionalization of alkynes, have been described and organized on the basis of the catalysts employed in the syntheses. Both racemic and enantioselective approaches have been reported. In the second part, an overview of the transformations of propargylamines into heterocyclic compounds such as pyrroles, pyridines, thiazoles, and oxazoles, as well as other relevant organic derivatives, is presented.

Photo-biocatalytic One-Pot Cascades for the Enantioselective Synthesis of 1,3-Mercaptoalkanol Volatile Sulfur Compounds.

The synthesis of enantiomerically pure 1,3-mercaptoalkanol volatile sulfur compounds through a one-pot photo-biocatalytic cascade reaction is described. Two new KRED biocatalysts with opposite enantioselectivity were discovered and proved to be efficient on a wide range of substrates. The one-pot cascade reaction combining photocatalytic thio-Michael addition with biocatalytic ketoreduction in an aqueous medium provides a green and sustainable approach to enantiomerically pure 1,3-mercaptoalkanols in high yields with excellent enantioselectivity.

Synthesis of 1,2,3-Substituted Pyrroles from Propargylamines via a One-Pot Tandem Enyne Cross Metathesis-Cyclization Reaction.

Enyne cross metathesis of propargylamines with ethyl vinyl ether enables the one-pot synthesis of substituted pyrroles. A series of substituted pyrroles, bearing alkyl, aryl, and heteroaryl substituents, has been synthesized in good yields under microwave irradiation. The reactions are rapid and procedurally simple and also represent a facile entry to the synthetically challenging 1,2,3-substituted pyrroles. The value of the methodology is further corroborated by the conversion of pyrroles into 3-methyl-pyrrolines and the derivatization of the 3-methyl-substituent arising from the metathesis reaction.

Rethinking the old antiviral drug moroxydine: Discovery of novel analogues as anti-hepatitis C virus (HCV) agents.

The discovery of a novel class of HCV inhibitors is described. The new amidinourea compounds were designed as isosteric analogues of the antiviral drug moroxydine. The two derivatives 11g and 11h showed excellent HCV inhibition activity and viability and proved to inhibit a step(s) of the RNA replication. The new compounds have been synthesized in only three synthetic steps from cheap building blocks and in high yields, thus turning to be promising drug candidates in the development of cheaper HCV treatments.

Enantioselective carbolithiation of S-alkenyl-N-aryl thiocarbamates: kinetic and thermodynamic control.

The addition of n-butyllithium to alkenylthiocarbamates in the presence of (-)-sparteine or the (+)-sparteine surrogate leads to asymmetric carbolithiation, and returns enantiomerically enriched thiocarbamate derivatives of secondary thiols. In THF, with the (+)-sparteine surrogate, in situ aryl migration leads to an enantiomerically enriched tertiary thiol derivative. Remarkably, the two pseudoenantiomeric chiral ligands do not always give enantiomeric products, probably as a result of a complex interplay of kinetic and thermodynamic control. In situ IR and NMR studies of a stable, hindered lithiated thiocarbamate demonstrated its chemical and configurational stability over a period of hours at 0 °C.

BacPROTACs targeting Clp protease: a promising strategy for anti-mycobacterial drug discovery.

Tuberculosis (TB) has claimed more lives over the course of two millennia than any other infectious disease worldwide. In 2021, the World Health Organization (WHO) estimated that 10.6 million people were diagnosed with TB, resulting in the deaths of 1.4 million HIV-negative individuals. The emergence of multidrug-resistant TB (MDR-TB), defined as resistance to at least rifampicin (RIF) and isoniazid (INH), and extensively drug-resistant TB (XDR-TB), poses the primary challenge to overcome in the coming years. We have recently conducted an extensive analysis of investments and research endeavours in the field, with the overarching objective of achieving the established milestone of TB eradication by the year 2030. Over the past several years, there has been notable progress in advancing a multitude of promising compounds, each possessing distinct mechanisms of action, into clinical phases of development. However, it is worth noting that strains of mycobacteria resistant to current antitubercular drugs have already emerged for some of these compounds The exploration of the innovative Proteolytic Target Chimeras (PROTACs) protein degradation approach has emerged as a viable avenue for the discovery of novel antimicrobials. While the ubiquitin system is exclusive to eukaryotic cells, certain bacteria use a similar degradation system that relies on the recognition of phosphorylated arginine residues (pArg) by the ClpC:ClpP (ClpCP) protease, thereby leading to protein degradation. In this opinion article, we have described and analized the advances in the use of PROTACs that leverage bacterial proteolytic machinery (BacPROTACs) to design new antitubercular agents. Scope Statement. The development of novel pharmaceuticals for tuberculosis treatment is deemed urgently necessary due to the emergence of resistant strains. In this context, the introduction of new technologies capable of alleviating the disease and attaining the objectives outlined by the World Health Organization is imperative. Among the innovative strategies, the degradation of proteins that are crucial for the survival of the bacillus holds promise for generating new medications, particularly those that are effective at treating latent (non-replicating) Mycobacterium tuberculosis. Within this perspective, we present the advancements and obstacles encountered in the exploration of new BacPROTAC compounds, with the intention of encouraging research and illuminating challenges associated with the implementation of BacPROTACs to address to the global tuberculosis crisis.

Development of Novel Membrane Disrupting Lipoguanidine Compounds Sensitizing Gram-Negative Bacteria to Antibiotics.

A new class of amphiphilic molecules, the lipoguanidines, designed as hybrids of guanidine and fatty acid compounds, has been synthesized and developed. The new molecules present both a guanidine polar head and a lipophilic tail that allow them to disrupt bacterial membranes and to sensitize Gram-negative bacteria to the action of the narrow-spectrum antibiotics rifampicin and novobiocin. The lipoguanidine 5g sensitizes Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli to rifampicin, thereby reducing the antibiotic minimum inhibitory concentrations (MIC) up to 256-fold. Similarly, 5g is able to potentiate novobiocin up to 64-fold, thereby showing a broad spectrum of antibiotic potentiating activity. Toxicity and mechanism studies revealed the potential of 5g to work synergistically with rifampicin through the disruption of bacterial membranes without affecting eukaryotic cells.

Liposome-siderophore conjugates loaded with moxifloxacin serve as a model for drug delivery against Mycobacterium tuberculosis.

Tuberculosis (TB) is an infectious disease that annually affects millions of people, and resistance to available antibiotics has exacerbated this situation. Another notable characteristic of Mycobacterium tuberculosis, the primary causative agent of TB, is its ability to survive inside macrophages, a key component of the immune system. In our quest for an effective and safe treatment that facilitates the targeted delivery of antibiotics to the site of infection, we have proposed a nanotechnology approach based on an iron chelator. Iron chelators are the primary mechanism by which bacteria acquire iron, a metal essential for their metabolism. Four liposomes were synthesized and characterized using the dynamic light scattering technique (DLS), nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). All of these methods revealed the presence of spherical particles, approximately 200 nm in size. NTA indicated a concentration of around 1011 particles/mL. We also developed and validated a high-performance liquid chromatography method for quantifying Moxifloxacin to determine encapsulation efficiency (EE) and release profiles (RF). The EE was 51.31 % for LipMox and 45.76 % for LipIchMox. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) confirmed the phagocytosis of liposomal vesicles by macrophages. Functionalizing liposomes with iron chelators can offer significant benefits for TB treatment, such as targeted drug delivery to intracellular bacilli through the phagocytosis of liposomal particles by cells like macrophages.

Discovery and synthesis of novel benzofurazan derivatives as inhibitors of influenza A virus.

The identification of a novel hit compound inhibitor of the protein-protein interaction between the influenza RNA-polymerase PA and PB1 subunits has been accomplished by means of high-throughput screening. A small family of structurally related molecules has been synthesized and biologically evaluated with most of the compounds showing micromolar potency of inhibition against viral replication.

Pyrimidine derivatives with antitubercular activity.

Small molecules with antitubercular activity containing the pyrimidine motif in their structure have gained more attention after three drugs, namely GSK 2556286 (GSK-286), TBA-7371 and SPR720, have entered clinical trials. This review provides an overview of recent advances in the hit-to-lead drug discovery studies of antitubercular pyrimidine-containing compounds with the aim to highlight their structural diversity. In the first part, the review discusses the pyrimidine compounds according to their targets, pinpointing the structure-activity relationships of each pyrimidine family. The second part of this review is concentrated on antitubercular pyrimidine derivatives with a yet unexplored or speculative target, dividing the compounds according to their structural types.

Oxazolidinones as versatile scaffolds in medicinal chemistry.

Oxazolidinone is a five-member heterocyclic ring with several biological applications in medicinal chemistry. Among the three possible isomers, 2-oxazolidinone is the most investigated in drug discovery. Linezolid was pioneered as the first approved drug containing an oxazolidinone ring as the pharmacophore group. Numerous analogues have been developed since its arrival on the market in 2000. Some have succeeded in reaching the advanced stages of clinical studies. However, most oxazolidinone derivatives reported in recent decades have not reached the initial stages of drug development, despite their promising pharmacological applications in a variety of therapeutic areas, including antibacterial, antituberculosis, anticancer, anti-inflammatory, neurologic, and metabolic diseases, among other areas. Therefore, this review article aims to compile the efforts of medicinal chemists who have explored this scaffold over the past decades and highlight the potential of the class for medicinal chemistry.

Biocatalytic and Chemo-Enzymatic Synthesis of Quinolines and 2-Quinolones by Monoamine Oxidase (MAO-N) and Horseradish Peroxidase (HRP) Biocatalysts.

The oxidative aromatization of aliphatic N-heterocycles is a fundamental organic transformation for the preparation of a diverse array of heteroaromatic compounds. Despite many attempts to improve the efficiency and practicality of this transformation, most synthetic methodologies still require toxic and expensive reagents as well as harsh conditions. Herein, we describe two enzymatic strategies for the oxidation of 1,2,3,4-tetrahydroquinolines (THQs) and N-cyclopropyl-N-alkylanilines into quinolines and 2-quinolones, respectively. Whole cells and purified monoamine oxidase (MAO-N) enzymes were used to effectively catalyze the biotransformation of THQs into the corresponding aromatic quinoline derivatives, while N-cyclopropyl-N-alkylanilines were converted into 2-quinolone compounds through a horseradish peroxidase (HRP)-catalyzed annulation/aromatization reaction followed by Fe-mediated oxidation.