The structure of Mycobacterium tuberculosis peptidoglycan is atypical since it contains a majority 3→3 cross-links synthesized by l,d-transpeptidases that replace 4→3 formed the d,d-transpeptidase activity classical penicillin-binding proteins. Carbapenems inactivate these l,d-transpeptidases, and meropenem combined with clavulanic acid bactericidal against extensively drug-resistant M. tuberculosis. Here, we used mass spectrometry stopped-flow fluorimetry to investigate kinetics mechanisms...

Active-site serine D,D-transpeptidases belonging to the penicillin-binding protein family (PBPs) have been considered for a long time as essential peptidoglycan cross-linking in all bacteria. However, bypass of PBPs by an L,D-transpeptidase (Ldtfm) conveys high-level resistance β-lactams penam class Enterococcus faecium with minimal inhibitory concentration (MIC) ampicillin >2,000 µg/ml. Unexpectedly, Ldtfm does not confer carbapenem (imipenem MIC = 0.5 µg/ml) whereas cephems display...

The bacterial cell wall peptidoglycan contains unusual l- and d-amino acids assembled as branched peptides. Insight into the biosynthesis of polymer has been hampered by limited access to substrates suitable polymerization assays. Here we report full synthesis peptide stem precursors from two pathogenic bacteria, Enterococcus faecium Mycobacterium tuberculosis, development a sensitive post-derivatization assay for their cross-linking l,d-transpeptidases. Access series peptides showed that...

1,2,3-triazole is an important building block in organic chemistry. It now well known as a bioisostere for various functions, such the amide or ester bond, positioning it key pharmacophore medicinal chemistry and has found applications fields including life sciences. Attention was first focused on synthesis of 1,4-disubstituted molecules however 1,4,5-trisubstituted 1,2,3-triazoles have emerged valuable due to possibility expand structural modularity. In last decade, methods mainly derived...

Ribonucleic acid (RNA) molecules play a crucial role in nearly every cellular process, with their function closely tied to three-dimensional (3D) structure. As result, determining the precise 3D structure of RNAs is essential understand biological functions. However, obtaining high-resolution structures remains significant challenge using traditional biophysical techniques. To address this, chemical probing methods such as SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension)...

Molecular mimicry is an essential part of the development drugs and molecular probes. In chemical glycobiology field, although many glycomimetics have been developed in past years, it has considered that failures their use are related to lack anomeric effects these analogues. Additionally, origin still subject virulent scientific debates. Herein, by combining synthesis, NMR methods, theoretical calculations, we show possible restore effect for acetal when replacing one oxygen atoms a CF2...

A new generation of surface-enhanced Raman scattering encoded-nanoparticles has been designed by combining aryl diazonium salt chemistry and gold nanoparticles.

Human malignant glioblastoma (GBM) is a highly invasive and lethal brain tumor. Targeting of integrin downstream signaling mediators in GBM such as focal adhesion kinase (FAK) seems reasonable recently demonstrated promising results early clinical studies. Herein, we report the structure-guided development series covalent inhibitors FAK. These new compounds displayed potent inhibitory potency against FAK enzymatic activity with IC50 values nanomolar range. Several retarded tumor cell growth...

Aminoacyl-tRNAs have important roles in a variety of biological processes, including protein synthesis by ribosomes, targeting proteins for degradation the proteasome, and bacterial cell wall synthesis. Here we describe stable aminoacyl-tRNA analogues containing 1,4- 1,5-substituted 1,2,3-triazole rings. The procedure involves i) Cu- Ru-catalysed cycloadditions 3'-azidoadenosine alkynes, which produced 1,4 1,5 regioisomers triazoles, respectively, ii) coupling between resulting...

To gain insight into the catalytic mechanism of non-ribosomal amino acid transferases, peptidyl-RNA conjugates were synthesized for co-crystallization with FemXWv Weissella viridescens, which transfers L-Ala from Ala-tRNAAla to peptidoglycan precursor UDP-MurNAc-pentapeptide. The structure resulting complex and mutational studies revealed by binds its substrates substrate-assisted catalysis stabilization tetrahedral intermediate. As a service our authors readers, this journal provides...

There is a renewed interest for β-lactams treating infections due to Mycobacterium tuberculosis and M. abscessus because their β-lactamases are inhibited by classical (clavulanate) or new generation (avibactam) inhibitors, respectively. Here, access an azido derivative of the diazabicyclooctane (DBO) scaffold avibactam functionalization Huisgen-Sharpless cycloaddition reaction reported. The amoxicillin-DBO combinations were active, indicating that triazole ring compatible with drug...

RNA methyltransferases (MTases) catalyse the transfer of a methyl group to their substrates using most-often S-adenosyl-L-methionine (SAM) as cofactor. Only few RNA-bound MTases structures are currently available due difficulties in crystallising RNA:protein complexes. The lack complex results poorly understood recognition patterns and methylation reaction mechanisms. On contrary, many cofactor-bound MTase available, resulting well-understood protein:cofactor recognition, that can guide...

Chemical synthesis of RNA conjugates has opened new strategies to study enzymatic mechanisms in biology. To gain insights into poorly understood nucleotide methylation processes, we developed a method synthesize RNA-conjugates for the recognition and methyl-transfer SAM-dependent m6A methyltransferases. These contain SAM cofactor analogue connected at N6-atom an adenosine within dinucleotides, trinucleotide or 13mer RNA. Our chemical route is chemo- regio-selective allows flexible...

The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the major enzyme that deposits N6-methyladenosine (m6A) modifications on messenger RNA (mRNA) in humans. METTL3-14 plays key roles various biological processes through its methyltransferase (MTase) activity. However, little known about substrate recognition methyl transfer mechanism from cofactor donor S-adenosylmethionine (SAM). Here, we study MTase by a combined experimental multiscale simulation approach using...

RNA methyltransferases (RNA MTases) are a family of enzymes that catalyze the methylation using cofactor S-adenosyl-L-methionine. While MTases promising drug targets, new molecules needed to fully understand their roles in disease and develop effective drugs can modulate activity. Since suitable for bisubstrate binding, we report an original strategy synthesis m6A analogues. Six compounds containing S-adenosyl-L-methionine (SAM) analogue unit covalently tethered by triazole ring N-6 position...

Focal Adhesion Kinase signaling pathway and its functions have been involved in the development aggressiveness of tumor malignancy, it then presents a promising cancer therapeutic target. Several reversible FAK inhibitors developed are being conducted clinical trials. On other hand, irreversible covalent would bring many desirable pharmacological features including high potency increased duration action. Herein we report structure-guided first highly potent inhibitor kinase. This showed very...

The FemX(Wv) aminoacyl transferase of Weissella viridescens initiates the synthesis side chain peptidoglycan precursors by transferring l-Ala from Ala-tRNA(Ala) to UDP-MurNAc-pentadepsipeptide. is an attractive target for development novel antibiotics, since essential last cross-linking step synthesis. Here, we show that highly specific incorporation in vivo based on extensive analysis structure peptidoglycan. Comparison various natural and vitro-transcribed tRNAs indicated specificity...

Transferases of the Fem family catalyse peptide-bond formation by using aminoacyl-tRNAs and peptidoglycan precursors as donor acceptor substrates, respectively. The specificity transferases is essential since mis-incorporated amino acids could act chain terminators thereby preventing a functional stress-bearing network. Here we have developed chemical acylation RNA helices with natural non-proteinogenic to gain insight into model transferase FemX(Wv). Combining modifications in aminoacyl...

Combinations of β-lactams the carbapenem class, such as meropenem, with clavulanate, a β-lactamase inhibitor, are being evaluated for treatment drug-resistant tuberculosis. However, carbapenems approved human use have never been optimized inactivation unusual β-lactam targets Mycobacterium tuberculosis or escaping to hydrolysis by broad-spectrum BlaC. Here, we report three routes synthesis modification two side chains carried and five-membered rings core. In particular, show that...

Natural selection: Replacement of the 3′-OH group Ala-tRNAAla with 3′-H affected FemXWv-catalyzed aminoacyl transfer from 2′-position, but not substrate binding. The ability FemXWv to bind and transacylate 3′-O-Ala isomer initially formed by alanyl-tRNA synthetase (AlaRS) may be crucial for efficient competition ribosome (see scheme). Detailed facts importance specialist readers are published as "Supporting Information". Such documents peer-reviewed, copy-edited or typeset. They made...

Abstract Peptidyl–RNA conjugates have various applications in studying the ribosome and enzymes participating tRNA‐dependent pathways such as Fem transferases peptidoglycan synthesis. Herein a convergent synthesis of peptidyl–RNAs based on Huisgen–Sharpless cycloaddition for final ligation step is developed. Azides alkynes are introduced into tRNA UDP‐MurNAc‐pentapeptide, respectively. Synthesis 2′‐azido RNA helix starts from 2′‐azido‐2′‐deoxyadenosine that coupled to deoxycytidine by...