Electrophilic RNA for Peptidyl‐RNA Synthesis and Site‐Specific Cross‐Linking with tRNA‐Binding Enzymes

Abstract

RNA functionalization is challenging due to the instability of RNA and the limited range of available enzymatic reactions. We developed a strategy based on solid phase synthesis and post-functionalization to introduce an electrophilic site at the 3' end of tRNA analogues. The squarate diester used as an electrophile enabled sequential amidation and provided asymmetric squaramides with high selectivity. The squaramate-RNAs specifically reacted with the lysine of UDP-MurNAc-pentapeptide, a peptidoglycan precursor used by the aminoacyl-transferase FemX for synthesis of the bacterial cell wall. The peptidyl-RNA obtained with squaramate-RNA and unprotected UDP-MurNAc-pentapeptide efficiently inhibited FemX . The squaramate unit also promoted specific cross-linking of RNA to the catalytic Lys of FemX but not to related transferases recognizing different aminoacyl-tRNAs. Thus, squaramate-RNAs provide specificity for cross-linking with defined groups in complex biomolecules due to its unique reactivity.

Squaramate‐Modified Nucleotides and DNA for Specific Cross‐Linking with Lysine‐Containing Peptides and Proteins

“…[13] Diamides (squaramides) have been used as ap hosphate surrogate in nucleotide [14] or oligonucleotide (ON) analogues. [15] Ac hemically synthesized 2'-sugar-linked squaramate-RNAc onjugate,p repared through reaction of 2'amino-modified RNAwith diethyl squarate,w as reported to cross-link to aminoacyl-transferase FemX Wv , [16] as the only example of its use in nucleic-acid conjugation. Within the framework of our program aimed at base-functionalized nucleic acids for applications in chemical biology, [17] we designed novel squaramate-linked cytosine 2'-deoxyribonucleoside triphosphate (dNTP) for the enzymatic synthesis of modified DNAa nd cross-linking with proteins.…”

Squaramate‐Modified Nucleotides and DNA for Specific Cross‐Linking with Lysine‐Containing Peptides and Proteins

“…Diamides (squaramides) have been used as a phosphate surrogate in nucleotide or oligonucleotide (ON) analogues . A chemically synthesized 2′‐sugar‐linked squaramate–RNA conjugate, prepared through reaction of 2′‐amino‐modified RNA with diethyl squarate, was reported to cross‐link to aminoacyl‐transferase FemX Wv , as the only example of its use in nucleic‐acid conjugation. Within the framework of our program aimed at base‐functionalized nucleic acids for applications in chemical biology, we designed novel squaramate‐linked cytosine 2′‐deoxyribonucleoside triphosphate (dNTP) for the enzymatic synthesis of modified DNA and cross‐linking with proteins.…”

Small-Molecule Acetylation by GCN5-Related N -Acetyltransferases in Bacteria

“…Mutational analysis of S. pneumoniae MurM chimeras with substitutions in a 30-amino-acid region in the coil-coil domain demonstrates that this region determines residue specificity (Ala versus Ser) (223). Analogues of aminoacyl-tRNA substrates that inhibit Fem proteins have been reported (224)(225)(226). More work is needed to understand the mechanisms and specificity of Fem proteins in order for these proteins to be a possible future drug target.…”

“…Acetylation of numerous small molecules modulates many cellular processes specific to bacteria. Thus, GNATs may serve as good therapeutic targets, and recent work has focused on inhibitors of GNATs (80,226). For example, mycobacteria exclusively use mycothiol as a redox-balancing molecule, and mycothiol synthesis is dependent on the GNAT MshD ( 175).…”