Copper catalyzed cycloaddition for the synthesis of non isomerisable 2' and 3'-regioisomers of arg-tRNAarg.

In this report, non-isomerisable analogs of arginine tRNA (Arg-triazole-tRNA) have been synthesized as tools to study tRNA-dependent aminoacyl-transferases. The synthesis involves the incorporation of 1,4 substituted-1,2,3 triazole ring to mimic the ester bond that connects the amino acid to the terminal adenosine in the natural substrate. The synthetic procedure includes (i) a coupling between 2'- or 3'-azido-adenosine derivatives and a cytidine phosphoramidite to access dinucleotide molecules, (ii) Cu-catalyzed cycloaddition reactions between 2'- or 3'-azido dinucleotide in the presence of an alkyne molecule mimicking the arginine, providing the corresponding Arg-triazole-dinucleotides, (iii) enzymatic phosphorylation of the 5'-end extremity of the Arg-triazole-dinucleotides with a polynucleotide kinase, and (iv) enzymatic ligation of the 5'-phosphorylated dinucleotides with a 23-nt RNA micro helix that mimics the acceptor arm of arg-tRNA or with a full tRNAarg. Characterization of nucleoside and nucleotide compounds involved MS spectrometry, 1H, 13C and 31P NMR analysis. This strategy allows to obtain the pair of the two stable regioisomers of arg-tRNA analogs (2' and 3') which are instrumental to explore the regiospecificity of arginyl transferases enzyme. In our study, a first binding assay of the arg-tRNA micro helix with the Arginyl-tRNA-protein transferase 1 (ATE1) was performed by gel shift assays.

The catalytic mechanism of the RNA methyltransferase METTL3.

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 in various biological processes through its methyltransferase (MTase) activity. However, little is known about its substrate recognition and methyl transfer mechanism from its cofactor and methyl donor S-adenosylmethionine (SAM). Here, we study the MTase mechanism of METTL3-14 by a combined experimental and multiscale simulation approach using bisubstrate analogues (BAs), conjugates of a SAM-like moiety connected to the N6-atom of adenosine. Molecular dynamics simulations based on crystal structures of METTL3-14 with BAs suggest that the Y406 side chain of METTL3 is involved in the recruitment of adenosine and release of m6A. A crystal structure with a BA representing the transition state of methyl transfer shows a direct involvement of the METTL3 side chains E481 and K513 in adenosine binding which is supported by mutational analysis. Quantum mechanics/molecular mechanics (QM/MM) free energy calculations indicate that methyl transfer occurs without prior deprotonation of adenosine-N6. Furthermore, the QM/MM calculations provide further support for the role of electrostatic contributions of E481 and K513 to catalysis. The multidisciplinary approach used here sheds light on the (co)substrate binding mechanism, catalytic step, and (co)product release, and suggests that the latter step is rate-limiting for METTL3. The atomistic information on the substrate binding and methyl transfer reaction of METTL3 can be useful for understanding the mechanisms of other RNA MTases and for the design of transition state analogues as their inhibitors.

Halo-1,2,3-triazoles: Valuable Compounds to Access Biologically Relevant Molecules.

1,2,3-triazole is an important building block in organic chemistry. It is now well known as a bioisostere for various functions, such as the amide or the ester bond, positioning it as a key pharmacophore in medicinal chemistry and it has found applications in various fields including life sciences. Attention was first focused on the synthesis of 1,4-disubstituted 1,2,3-triazole molecules however 1,4,5-trisubstituted 1,2,3-triazoles have now emerged as valuable molecules due to the possibility to expand the structural modularity. In the last decade, methods mainly derived from the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction have been developed to access halo-triazole compounds and have been applied to nucleosides, carbohydrates, peptides and proteins. In addition, late-stage modification of halo-triazole derivatives by metal-mediated cross-coupling or halo-exchange reactions offer the possibility to access highly functionalized molecules that can be used as tools for chemical biology. This review summarizes the synthesis, the functionalization, and the applications of 1,4,5-trisubstituted halo-1,2,3-triazoles in biologically relevant molecules.

The catalytic mechanism of the RNA methyltransferase METTL3.

The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the major enzyme that deposits N6-methyladenosine (m6A) modifications on mRNA in humans. METTL3-14 plays key roles in various biological processes through its methyltransferase (MTase) activity. However, little is known about its substrate recognition and methyl transfer mechanism from its cofactor and methyl donor S-adenosylmethionine (SAM). Here, we study the MTase mechanism of METTL3-14 by a combined experimental and multiscale simulation approach using bisubstrate analogues (BAs), conjugates of a SAM-like moiety connected to the N6-atom of adenosine. Molecular dynamics simulations based on crystal structures of METTL3-14 with BAs suggest that the Y406 side chain of METTL3 is involved in the recruitment of adenosine and release of m6A. A crystal structure with a bisubstrate analogue representing the transition state of methyl transfer shows a direct involvement of the METTL3 side chains E481 and K513 in adenosine binding which is supported by mutational analysis. Quantum mechanics/molecular mechanics (QM/MM) free energy calculations indicate that methyl transfer occurs without prior deprotonation of adenosine-N6. Furthermore, the QM/MM calculations provide further support for the role of electrostatic contributions of E481 and K513 to catalysis. The multidisciplinary approach used here sheds light on the (co)substrate binding mechanism, catalytic step, and (co)product release catalysed by METTL3, and suggests that the latter step is rate-limiting. The atomistic information on the substrate binding and methyl transfer reaction of METTL3 can be useful for understanding the mechanisms of other RNA MTases and for the design of transition state analogues as their inhibitors.

Synthesis of Bisubstrate Analogues for RNA Methylation Studies using two Transition-Metal-Catalyzed Reactions.

RNA methyltransferases (RNA MTases) are a family of enzymes that catalyze the methylation of RNA using the cofactor S-adenosyl-L-methionine. While RNA MTases are promising drug targets, new molecules are needed to fully understand their roles in disease and to develop effective drugs that can modulate their activity. Since RNA MTases are suitable for bisubstrate binding, we report an original strategy for the synthesis of a new family of m6A MTases bisubstrate analogues. Six compounds containing a S-adenosyl-L-methionine (SAM) analogue unit covalently tethered by a triazole ring to the N-6 position of an adenosine were synthesized. A procedure using two transition-metal-catalyzed reactions was used to introduce the α-amino acid motif mimicking the methionine chain of the cofactor SAM. First, a copper(I)-catalyzed alkyne-azide iodo-cycloaddition (iCuAAC) reaction afforded the 5-iodo-1,4-disubstituted-1,2,3-triazole which was functionalized by palladium-catalyzed cross-coupling to connect the α-amino acid substituent. Docking studies of our molecules in the active site of the m6A ribosomal MTase RlmJ show that the use of triazole as a linker provides additional interactions and the presence of the α-amino acid chain stabilizes the bisubstrate. The synthetic method developed here enhances the structural diversity of bisubstrate analogues to explore the active site of RNA modification enzymes and to develop new inhibitors.

Traceless Staudinger Ligation to Access Stable Aminoacyl- or Peptidyl-Dinucleotide.

Aminoacyl- and peptidyl-tRNA are specific biomolecules involved in many biological processes, from ribosomal protein synthesis to the synthesis of peptidoglycan precursors. Here, we report a post-synthetic approach based on traceless Staudinger ligation for the synthesis of a stable amide bond to access aminoacyl- or peptidyl-di-nucleotide. A series of amino-acid and peptide ester phenyl phosphines were synthetized, and their reactivity was studied on a 2'-N3 di-nucleotide. The corresponding 2'-amide di-nucleotides were obtained and characterized by LC-HRMS, and mechanistic interpretations of the influence of the amino acid phenyl ester phosphine were proposed. We also demonstrated that enzymatic 5'-OH phosphorylation is compatible with the acylated di-nucleotide, allowing the possibility to access stable aminoacylated-tRNA.

Ruthenium-Catalyzed Cycloaddition for Introducing Chemical Diversity in Second-Generation ß-Lactamase Inhibitors.

Ruthenium(II) alkyne azide cycloaddition (RuAAC) is an attractive reaction to access 1,5-triazole derivatives and is applicable to internal alkynes. Here, we explore RuAAC to introduce molecular diversity on the diazabicyclooctane (DBO) scaffold of ß-lactamase inhibitors. The methodology presented is fully regioselective and enabled synthesis of a series of 1,5-triazole DBOs and trisubstituted analogues. Molecular modelling and biological evaluation revealed that the DBO substituents provided putative stabilizing interactions in the active site of broad-spectrum ß-lactamase KPC-2 and promising activity against a hyperpermeable strain of Escherichia coli producing KPC-2.

Amino-acyl tXNA as inhibitors or amino acid donors in peptide synthesis.

Xenobiotic nucleic acids (XNAs) offer tremendous potential for synthetic biology, biotechnology, and molecular medicine but their ability to mimic nucleic acids still needs to be explored. Here, to study the ability of XNA oligonucleotides to mimic tRNA, we synthesized three L-Ala-tXNAs analogs. These molecules were used in a non-ribosomal peptide synthesis involving a bacterial Fem transferase. We compared the ability of this enzyme to use amino-acyl tXNAs containing 1',5'-anhydrohexitol (HNA), 2'-fluoro ribose (2'F-RNA) and 2'-fluoro arabinose. L-Ala-tXNA containing HNA or 2'F-RNA were substrates of the Fem enzyme. The synthesis of peptidyl-XNA and the resolution of their structures in complex with the enzyme show the impact of the XNA on protein binding. For the first time we describe functional tXNA in an in vitro assay. These results invite to test tXNA also as substitute for tRNA in translation.

Modulation of the Specificity of Carbapenems and Diazabicyclooctanes for Selective Activity against Mycobacterium tuberculosis.

Treatment of multidrug-resistant tuberculosis with combinations of carbapenems and ß-lactamase inhibitors carries risks for dysbiosis and for the development of resistances in the intestinal microbiota. Using Escherichia coli producing carbapenemase KPC-2 as a model, we show that carbapenems can be modified to obtain drugs that are inactive against E. coli but retain antitubercular activity. Furthermore, functionalization of the diazabicyclooctanes scaffold provided drugs that did not effectively inactivate KPC-2 but retained activity against Mycobacterium tuberculosis targets.

Synthesis of RNA-cofactor conjugates and structural exploration of RNA recognition by an m6A RNA methyltransferase.

Chemical synthesis of RNA conjugates has opened new strategies to study enzymatic mechanisms in RNA biology. To gain insights into poorly understood RNA nucleotide methylation processes, we developed a new method to synthesize RNA-conjugates for the study of RNA recognition and methyl-transfer mechanisms of SAM-dependent m6A RNA methyltransferases. These RNA conjugates contain a SAM cofactor analogue connected at the N6-atom of an adenosine within dinucleotides, a trinucleotide or a 13mer RNA. Our chemical route is chemo- and regio-selective and allows flexible modification of the RNA length and sequence. These compounds were used in crystallization assays with RlmJ, a bacterial m6A rRNA methyltransferase. Two crystal structures of RlmJ in complex with RNA-SAM conjugates were solved and revealed the RNA-specific recognition elements used by RlmJ to clamp the RNA substrate in its active site. From these structures, a model of a trinucleotide bound in the RlmJ active site could be built and validated by methyltransferase assays on RlmJ mutants. The methyl transfer by RlmJ could also be deduced. This study therefore shows that RNA-cofactor conjugates are potent molecular tools to explore the active site of RNA modification enzymes.

Traceless Staudinger Ligation To Introduce Chemical Diversity on ß-Lactamase Inhibitors of Second Generation.

We explored the traceless Staudinger ligation for the functionalization of the C2 position of second generation ß-lactamase inhibitors based on a diazabicyclooctane (DBO) scaffold. Our strategy is based on the synthesis of phosphine phenol esters and their ligation to an azido-containing precursor. Biological evaluation showed that this route provided access to a DBO that proved to be superior to commercial relebactam for inhibition of two of the five ß-lactamases that were tested.

Click and Release Chemistry for Activity-Based Purification of ß-Lactam Targets.

ß-Lactams, the cornerstone of antibiotherapy, inhibit multiple and partially redundant targets referred to as transpeptidases or penicillin-binding proteins. These enzymes catalyze the essential cross-linking step of the polymerization of cell wall peptidoglycan. The understanding of the mechanisms of action of ß-lactams and of resistance to these drugs requires the development of reliable methods to characterize their targets. Here, we describe an activity-based purification method of ß-lactam targets based on click and release chemistry. We synthesized alkyne-carbapenems with suitable properties with respect to the kinetics of acylation of a model target, the Ldtfm L,D-transpeptidase, the stability of the resulting acylenzyme, and the reactivity of the alkyne for the cycloaddition of an azido probe containing a biotin moiety for affinity purification and a bioorthogonal cleavable linker. The probe provided access to the fluorescent target in a single click and release step.

Traceless Staudinger Ligation for Bioconjugation of RNA.

Staudinger ligation is an attractive bioorthogonal reaction for use in studying biomolecules due to its capacity to form a native amide bond between a tag and a biomolecule. Here, we explore the traceless variant of the Staudinger ligation for 3'-end modification of oligoribonucleotides. The procedure involves (i) synthesis of phosphine-containing reactive groups, affinity purification tags, or photoactivatable benzophenone probe, (ii) synthesis of 2'-azido dinucleotides and 24-nt RNA, and (iii) traceless Staudinger ligation experiments. Each phosphine was characterized by 1 H, 13 C, and 31 P NMR and high-resolution spectrometry and the functionalized nucleotides were characterized by LC/MS. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of phosphines Basic Protocol 2: Synthesis of dinucleotides 4 and 5 Basic Protocol 3: Synthesis of modified RNA 6 Basic Protocol 4: Traceless Staudinger reactions on a dinucleotide Basic Protocol 5: Traceless Staudinger reaction on RNA.

Synthesis of Carbapenems Containing Peptidoglycan Mimetics and Inhibition of the Cross-Linking Activity of a Transpeptidase of l,d Specificity.

The carbapenem class of ß-lactams has been optimized against Gram-negative bacteria producing extended-spectrum ß-lactamases by introducing substituents at position C2. Carbapenems are currently investigated for the treatment of tuberculosis as these drugs are potent covalent inhibitors of l,d-transpeptidases involved in mycobacterial cell wall assembly. The optimization of carbapenems for inactivation of these unusual targets is sought herein by exploiting the nucleophilicity of the C8 hydroxyl group to introduce chemical diversity. As ß-lactams are structure analogs of peptidoglycan precursors, the substituents were chosen to increase similarity between the drug and the substrate. Fourteen peptido-carbapenems were efficiently synthesized. They were more effective than the reference drug, meropenem, owing to the positive impact of a phenethylthio substituent introduced at position C2 but the peptidomimetics added at position C8 did not further improve the activity. Thus, position C8 can be modified to modulate the pharmacokinetic properties of highly efficient carbapenems.

Partition of tRNAGly isoacceptors between protein and cell-wall peptidoglycan synthesis in Staphylococcus aureus.

The sequence of tRNAs is submitted to evolutionary constraints imposed by their multiple interactions with aminoacyl-tRNA synthetases, translation elongation factor Tu in complex with GTP (EF-Tu•GTP), and the ribosome, each being essential for accurate and effective decoding of messenger RNAs. In Staphylococcus aureus, an additional constraint is imposed by the participation of tRNAGly isoacceptors in the addition of a pentaglycine side chain to cell-wall peptidoglycan precursors by transferases FmhB, FemA and FemB. Three tRNAGly isoacceptors poorly interacting with EF-Tu•GTP and the ribosome were previously identified. Here, we show that these 'non-proteogenic' tRNAs are preferentially recognized by FmhB based on kinetic analyses and on synthesis of stable aminoacyl-tRNA analogues acting as inhibitors. Synthesis of chimeric tRNAs and of helices mimicking the tRNA acceptor arms revealed that this discrimination involves identity determinants exclusively present in the D and T stems and loops of non-proteogenic tRNAs, which belong to an evolutionary lineage only present in the staphylococci. EF-Tu•GTP competitively inhibited FmhB by sequestration of 'proteogenic' aminoacyl-tRNAs in vitro. Together, these results indicate that competition for the Gly-tRNAGly pool is restricted by both limited recognition of non-proteogenic tRNAs by EF-Tu•GTP and limited recognition of proteogenic tRNAs by FmhB.

Phosphine-Mediated Bioconjugation of the 3'-End of RNA.

Staudinger ligation is an attractive bio-orthogonal reaction that has been widely used to tag proteins, carbohydrates, and nucleic acids. Here, we explore the traceless variant of the Staudinger ligation for 3'-end modification of oligoribonucleotides. An azido-containing dinucleotide was used to study the ligation. Nine phosphines containing reactive groups, affinity purification tags, or photoswitch probes have been successfully obtained. The corresponding modified dinucleotides were synthesized and characterized by LC/MS. Mechanistic interpretations of the reaction are proposed, in particular, the unprecedented formation of an oxazaphospholane nucleotide derivative, which was favored by the vicinal position of 2'-N3 and 3'-OH functional groups on the terminal ribose has been observed. The post-functionalization of a 24-nt RNA with a photoactivable tag is also reported.

Synthesis of Triazole-Linked SAM-Adenosine Conjugates: Functionalization of Adenosine at N-1 or N-6 Position without Protecting Groups.

More than 150 RNA chemical modifications have been identified to date. Among them, methylation of adenosine at the N-6 position (m6A) is crucial for RNA metabolism, stability and other important biological events. In particular, this is the most abundant mark found in mRNA in mammalian cells. The presence of a methyl group at the N-1 position of adenosine (m1A) is mostly found in ncRNA and mRNA and is mainly responsible for stability and translation fidelity. These modifications are installed by m6A and m1A RNA methyltransferases (RNA MTases), respectively. In human, deregulation of m6A RNA MTases activity is associated with many diseases including cancer. To date, the molecular mechanism involved in the methyl transfer, in particular substrate recognition, remains unclear. We report the synthesis of new SAM-adenosine conjugates containing a triazole linker branched at the N-1 or N-6 position of adenosine. Our methodology does not require protecting groups for the functionalization of adenosine at these two positions. The molecules described here were designed as potential bisubstrate analogues for m6A and m1A RNA MTases that could be further employed for structural studies. This is the first report of compounds mimicking the transition state of the methylation reaction catalyzed by m1A RNA MTases.

Raman reporters derived from aryl diazonium salts for SERS encoded-nanoparticles.

Surface-enhanced Raman scattering (SERS) tags are usually prepared by immobilizing Raman reporters on plasmonic nanoparticles (NPs) via thiol-based self-assembled monolayers. We describe here the first example of SERS tags obtained by combining gold NPs and aryl diazonium salts. This strategy results in robust Au-C covalent bonds between the Raman reporter and the NPs, thus ensuring a high stability of the nanohybrid interface.

Diazabicyclooctane Functionalization for Inhibition of ß-Lactamases from Enterobacteria.

Second-generation ß-lactamase inhibitors containing a diazabicyclooctane (DBO) scaffold restore the activity of ß-lactams against pathogenic bacteria, including those producing class A, C, and D enzymes that are not susceptible to first-generation inhibitors containing a ß-lactam ring. Here, we report optimization of a synthetic route to access triazole-containing DBOs and biological evaluation of a series of 17 compounds for inhibition of five ß-lactamases representative of enzymes found in pathogenic Gram-negative bacteria. A strong correlation (Spearman coefficient of 0.87; p = 4.7 × 10-21) was observed between the inhibition efficacy of purified ß-lactamases and the potentiation of ß-lactam antibacterial activity, indicating that DBO functionalization did not impair penetration. In comparison to reference DBOs, avibactam and relebactam, our compounds displayed reduced efficacy, likely due to the absence of hydrogen bonding with a conserved asparagine residue at position 132. This was partially compensated for by additional interactions involving certain triazole substituents.

Bisubstrate analogues as structural tools to investigate m6A methyltransferase active sites.

RNA methyltransferases (MTases) catalyse the transfer of a methyl group to their RNA substrates using most-often S-adenosyl-L-methionine (SAM) as cofactor. Only few RNA-bound MTases structures are currently available due to the difficulties in crystallising RNA:protein complexes. The lack of complex structures results in poorly understood RNA recognition patterns and methylation reaction mechanisms. On the contrary, many cofactor-bound MTase structures are available, resulting in well-understood protein:cofactor recognition, that can guide the design of bisubstrate analogues that mimic the state at which both the substrate and the cofactor is bound. Such bisubstrate analogues were recently synthesized for proteins monomethylating the N6-atom of adenine (m6A). These proteins include, amongst others, RlmJ in E. coli and METLL3:METT14 and METTL16 in human. As a proof-of-concept, we here test the ability of the bisubstrate analogues to mimic the substrate:cofactor bound state during catalysis by studying their binding to RlmJ using differential scanning fluorimetry, isothermal titration calorimetry and X-ray crystallography. We find that the methylated adenine base binds in the correct pocket, and thus these analogues could potentially be used broadly to study the RNA recognition and catalytic mechanism of m6A MTases. Two bisubstrate analogues bind RlmJ with micro-molar affinity, and could serve as starting scaffolds for inhibitor design against m6A RNA MTases. The same analogues cause changes in the melting temperature of the m1A RNA MTase, TrmK, indicating non-selective protein:compound complex formation. Thus, optimization of these molecular scaffolds for m6A RNA MTase inhibition should aim to increase selectivity, as well as affinity.