ABSTRACT
The methodology enabling enzymatic and nonenzymatic information transfer with FNAs is described. This methodology includes the chemical synthesis of fNTPs and fN phosphoramidites, in addition to protocols for the enzymatic and nonenzymatic transfer of information.
Subject(s)
DNA-Directed DNA Polymerase/metabolism , Nucleic Acids/chemistry , RNA/chemistry , DNA Replication , Nucleic Acid Conformation , Oligonucleotides/chemistry , Polymerization , Templates, GeneticABSTRACT
We report here the synthesis of stable Phe-tRNA(Phe) and Leu-tRNA(Leu) analogues containing a 1,2,3-triazole ring instead of the ribose-amino acid ester bond. The 1,2,3-triazole ring is generated by dipolar cycloaddition of alkyne Phe and Leu analogues to 3'-azido-3'-deoxyadenosine via the Cu(I)-catalysed Huisgen, Meldal, Sharpless 1,3-cycloaddition. The corresponding triazoyl pdCpA dinucleotides, obtained by classical phosphoramidite chemistry, were enzymatically ligated to 22-nt or 74-nt RNA generating stable Phe-tRNA(Phe) analogues containing the acceptor stem or full tRNA moieties, respectively. These molecules represent useful tools to study the contribution of the RNA and amino acid moieties in stabilization of aminoacyl-tRNA/protein complexes.
Subject(s)
Nucleotides/chemical synthesis , RNA, Transfer, Leu/chemistry , RNA, Transfer, Phe/chemistry , Triazoles/chemistry , Models, Molecular , Molecular Conformation , Nucleotides/chemistry , RNA, Transfer, Leu/chemical synthesis , RNA, Transfer, Phe/chemical synthesis , Triazoles/chemical synthesisABSTRACT
Aminoacyl-tRNAs serve as amino acid donors in many reactions in addition to protein synthesis by the ribosome, including synthesis of the peptidoglycan network in the cell wall of bacterial pathogens. Synthesis of analogs of aminoacylated tRNAs is critical to further improve the mechanism of these reactions. Here we have described the synthesis of two non-isomerizable analogues of Ala-tRNA(Ala) containing an amide bond instead of the isomerizable ester that connects the amino acid with the terminal adenosine in the natural substrate. The non-isomerizable 2' and 3' regioisomers were not used as substrates by FemX(Wv), an alanyl-transferase essential for peptidoglycan synthesis, but inhibited this enzyme with IC50 of 5.8 and 5.5 µM, respectively.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Nitrogenous Group Transferases/antagonists & inhibitors , RNA, Transfer, Ala/chemical synthesis , RNA, Transfer, Ala/pharmacology , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Models, Molecular , Molecular Conformation , Nitrogenous Group Transferases/metabolism , RNA, Transfer, Ala/chemistry , Structure-Activity RelationshipABSTRACT
Aminoacyl-tRNAs have important roles in a variety of biological processes. Here, we describe the synthesis of stable aminoacyl-tRNA analogs containing 1,4-substituted 1,2,3-triazole rings. The procedure involves (i) copper-catalyzed cycloadditions of 3'-or 2'-azido-adenosine and alkynes, (ii) coupling between the resulting triazole-deoxyadenosine derivatives and a deoxycytidine phosphoramidite, and (iii) the enzymatic ligation of the 2'- or 3'-triazole-dinucleotides with a 22-nt RNA microhelix that mimics the acceptor arm of tRNA. Each nucleoside and nucleotide intermediate was characterized by MS spectrometry and (1)H, (31)P, and (13)C NMR spectroscopy, and the tRNA-analogs were assayed for inhibition of FemXWv, an alanyl-transferase essential for the formation of the peptidoglycan network of Gram-positive bacterial pathogens. The low IC(50) values obtained (2 to 4 µM) indicate that the five-membered triazole rings acted as an isosteres of esters and can be used for the design of stable aminoacyl-tRNA analogs.
Subject(s)
RNA, Transfer, Amino Acyl/chemical synthesis , Anti-Bacterial Agents/chemical synthesis , RNA, Transfer, Amino Acyl/chemistry , Spectrum Analysis , Structure-Activity Relationship , Transferases/antagonists & inhibitors , Triazoles/chemistryABSTRACT
Transferases of the Fem family catalyse peptide-bond formation by using aminoacyl-tRNAs and peptidoglycan precursors as donor and acceptor substrates, respectively. The specificity of Fem transferases is essential since mis-incorporated amino acids could act as chain terminators thereby preventing formation of a functional stress-bearing peptidoglycan network. Here we have developed chemical acylation of RNA helices with natural and non-proteinogenic amino acids to gain insight into the specificity of the model transferase FemX(Wv). Combining modifications in the RNA and aminoacyl moieties of the donor substrate revealed that unfavourable interactions of FemX(Wv) with the acceptor arm of tRNA(Gly) and with L-Ser or larger residues quantitatively accounts for the preferential transfer of L-Ala observed with complete aminoacyl-tRNAs. The main FemX(Wv) identity determinant was identified as the penultimate base pair (G(2)-C(71)) of the acceptor arm instead of G(3)*U(70) for the alanyl-tRNA synthetase. FemX(Wv) tolerated a configuration inversion of the Calpha of L-Ala but not the introduction of a second methyl on this atom. These results indicate that aminoacyl-tRNA recognition by FemX(Wv) is distinct from other components of the translation machinery and relies on the exclusion of bulky amino acids and of the sequence of tRNA(Gly) from the active site.
Subject(s)
Bacteria/enzymology , Cell Wall/chemistry , Nitrogenous Group Transferases/metabolism , RNA, Transfer, Amino Acyl/chemistry , RNA, Transfer, Amino Acyl/metabolism , Alanine/metabolism , Cell Wall/metabolism , Glycine/metabolism , RNA, Transfer, Ala/chemistry , RNA, Transfer, Ala/metabolism , RNA, Transfer, Amino Acyl/chemical synthesis , Serine/metabolism , Stereoisomerism , Substrate Specificity , Uridine Diphosphate N-Acetylmuramic Acid/analogs & derivatives , Uridine Diphosphate N-Acetylmuramic Acid/metabolismABSTRACT
Aminoacyl-tRNAs have important roles in a variety of biological processes, including protein synthesis by ribosomes, targeting of proteins for degradation by the proteasome, and bacterial cell wall synthesis. Here we describe the synthesis of stable aminoacyl-tRNA analogues containing 1,4- and 1,5-substituted 1,2,3-triazole rings. The procedure involves i) Cu- and Ru-catalysed cycloadditions of 3'-azidoadenosine and alkynes, which produced the 1,4 and 1,5 regioisomers of the triazoles, respectively, ii) coupling between the resulting triazole-deoxyadenosine derivatives and a deoxycytidine phosphoramidite, and iii) the enzymatic ligation of the substituted dinucleotides with a 22 nt RNA microhelix that mimics the acceptor arm of tRNA. Nucleoside and nucleotide compounds were characterized by MS spectrometry and (1)H, (31)P and (13)C NMR spectroscopy and were assayed for inhibition of FemX(Wv), an alanyltransferase essential for the formation of the peptidoglycan network of gram-positive bacterial pathogens. The low IC(50) values obtained (2 to 4 microM) indicate that the five-membered triazole rings acted as bioisosters of esters and can be used for the design of stable aminoacyl-tRNA analogues.
Subject(s)
Aminoacyltransferases/antagonists & inhibitors , RNA, Transfer, Amino Acyl/chemical synthesis , Triazoles/chemistry , Base Sequence , Esters , Inhibitory Concentration 50 , Models, Biological , Models, Chemical , Molecular Structure , RNA, Transfer, Amino Acyl/chemistry , RNA, Transfer, Amino Acyl/pharmacology , Structure-Activity RelationshipABSTRACT
The FemX(Wv) aminoacyl transferase of Weissella viridescens initiates the synthesis of the side chain of peptidoglycan precursors by transferring l-Ala from Ala-tRNA(Ala) to UDP-MurNAc-pentadepsipeptide. FemX(Wv) is an attractive target for the development of novel antibiotics, since the side chain is essential for the last cross-linking step of peptidoglycan synthesis. Here, we show that FemX(Wv) is highly specific for incorporation of l-Ala in vivo based on extensive analysis of the structure of peptidoglycan. Comparison of various natural and in vitro-transcribed tRNAs indicated that the specificity of FemX(Wv) depends mainly upon the sequence of the tRNA although additional specificity determinants may include post-transcriptional modifications and recognition of the esterified amino acid. Site-directed mutagenesis identified cytosines in the G1-C72 and G2-C71 base pairs of the acceptor stem as critical for FemX(Wv) activity in agreement with modeling of tRNA(Ala) in the catalytic cavity of the enzyme. In contrast, semi-synthesis of Ala-tRNA(Ala) harboring nucleotide substitutions in the G3-U70 wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemX(Wv). The different modes of recognition of the acceptor stem indicate that specific inhibition of FemX(Wv) could be achieved by targeting the distal portion of tRNA(Ala) for the design of substrate analogues.
