Synthesis of 3'-triazoyl-dinucleotides as precursors of stable Phe-tRNA(Phe) and Leu-tRNA(Leu) analogues.

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.

Efficient access to peptidyl-RNA conjugates for picomolar inhibition of non-ribosomal FemX(Wv) aminoacyl transferase.

Peptidyl-RNA conjugates have various applications in studying the ribosome and enzymes participating in tRNA-dependent pathways such as Fem transferases in peptidoglycan synthesis. Herein a convergent synthesis of peptidyl-RNAs based on Huisgen-Sharpless cycloaddition for the final ligation step is developed. Azides and alkynes are introduced into tRNA and UDP-MurNAc-pentapeptide, respectively. Synthesis of 2'-azido RNA helix starts from 2'-azido-2'-deoxyadenosine that is coupled to deoxycytidine by phosphoramidite chemistry. The resulting dinucleotide is deprotected and ligated to a 22-nt RNA helix mimicking the acceptor arm of Ala-tRNA(Ala) by T4 RNA ligase. For alkyne UDP-MurNAc-pentapeptide, meso-cystine is enzymatically incorporated into the peptidoglycan precursor and reduced, and L-Cys is converted to dehydroalanine with O-(mesitylenesulfonyl)hydroxylamine. Reaction of but-3-yne-1-thiol with dehydroalanine affords the alkyne-containing UDP-MurNAc-pentapeptide. The Cu(I)-catalyzed azide alkyne cycloaddition reaction in the presence of tris[(1-hydroxypropyl-1H-1,2,3-triazol-4-yl)methyl]amine provided the peptidyl-RNA conjugate, which was tested as an inhibitor of non-ribosomal FemX(Wv) aminoacyl transferase. The bi-substrate analogue was found to inhibit FemX(Wv) with an IC(50) of (89±9) pM, as both moieties of the peptidyl-RNA conjugate contribute to high-affinity binding.

Synthesis and biological evaluation of non-isomerizable analogues of Ala-tRNA(Ala).

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.

Total synthesis of quinolizidine (-)-217A.

We report here the construction of quinolizidine ring systems by intramolecular cyclization of suitable functionalized piperidines via a reductive amination sequence. This reaction proceeds with a total stereocontrol at C4. The preparation of the piperidine precursors is based on a chain elongation of a piperidine aldehyde either by aldolization or by Wittig reaction. We applied this second route to the total synthesis of quinolizidine (-)-217A from (S)-methyl 2-((S)-1-((R)-1-phenylethyl)piperidin-2-yl)propanoate 5.

Formal total synthesis of (+)-gephyrotoxin.

An efficient formal total synthesis of (+)-gephyrotoxin is described. The key step of our strategy relies on the diastereoselective reduction of a chiral pyrrolidine beta-enamino ester obtained by condensation of ( S)-phenylglycinol on a protected 8-hydroxy-3,6-dioxooctanoate.