A convergent strategy for the synthesis of an RNApeptide conjugate is presented. Regioselective ligation between a 5'-modified RNA harboring a 5'-thioester group and a peptide carrying an N-terminal cysteine afforded an RNA-peptide conjugate under physiological conditions without the need for protecting groups.We have been interested in the synthesis of covalent nucleic acid-peptide conjugates for the construction of encoded peptide libraries. 1 In particular, we wanted to develop a convergent ligation strategy for the synthesis of RNA-protein conjugates. Ideally, this ligation reaction should take place between the native protein and the RNA with minimal chemical manipulation and under physiological conditions. A review of the literature revealed several step-wise synthetic schemes using protecting group strategies to prepare oligonucleotide-peptide conjugates. 2 Alternatively, convergent schemes have been devised for attaching unprotected peptides to oligonucleotides using a covalent linkage, such as disulfide, 3 thioether, 4 amide 5,6 or by Schiff base formation between an e-amino group of lysine and an oligonucleotide 2'-(3')-dialdehyde. 7 Analogous schemes have been developed for attaching RNA to proteins. 8 A major drawback in using protein thiols or amines for conjugation is the lack of regiospecific ligation upon reaction with proteins containing multiple cysteine or lysine residues.We now report a highly regiospecific method for the preparation of RNA-peptide conjugates. Our strategy is based on orthogonal ligation chemistry, which has proven highly successful in the ligation of large, unprotected peptide fragments in solution. 9,10 Chemoselective ligation between a 5'-modified RNA harboring a 5'-thioester group and a polypeptide bearing a cysteine residue at the amino terminus yields a well defined 5'-RNA-peptide conjugate (Scheme). Ligation is initiated via a transthioesterification reaction followed by a spontaneous intramolecular rearrangement to form an amide bond between the RNA and the peptide.We initially developed a simplified model to study the ligation chemistry (Figure 1). Guanosine-5'-phosphorothioate 1 (GMPS) was used as a mimic for RNA carrying a 5'-phosphorothioate group. A phenyl thioester group was introduced via S-alkylation of the phosphorothioate moiety of 1 with phenyl a-bromothioacetate 2. 11 Alkylation of 1 with an excess of 2 was quantitative and complete in 5 min as monitored by reversed phase HPLC. 12 A single product 3 was observed and alkylation of the phosphorothioate group was confirmed by 31 P NMR. 13 No N-or O-alkylated 1 was detected ( 1 H NMR, MS) after prolonged reaction times (1h). The product 3 was then treated with a stoichiometric amount of Cys-Gly and analysis of the reaction mixture by RP-HPLC showed the disappearance of 3 (R t = 14.8 min) and the formation of a new peak at R t = 11.2 min (see HPLC chromatogram, Figure 1). 14 The structure of the nucleotide-dipeptide conjugate 4 was confirmed by NMR and mass spectroscopy. 15 Following efficient ligation in our...