Synthesis of an RNA-Peptide Conjugate by Orthogonal Ligation

Abstract

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...

Conjugates of Oligonucleotides and Analogues with Cell Penetrating Peptides as Gene Silencing Agents

“…Another method of chemoselective conjugation is called "native ligation" [77] which has been applied to the synthesis of OPCs [78,79] and PPCs [80,81]. The principle of conjugation is based on a reversible trans-thioesterification followed by SàN acyl migration that leads to an amide linkage between the biomolecules (Fig.…”

Conjugation Approaches for Peptide-Mediated Delivery of Oligonucleotides Therapeutics

“…[41] An alternative approach involves the conjugation of unprotected peptides to oligonucleotides using native chemical ligation (NCL). [42][43][44][45][46][47][48][49] NCL utilises a chemoselective S-N acyl transfer to form an amide linkage. [50] Initially, an unprotected peptide with an activated C-terminal thioester forms a trans-thioester with the N-terminal thiol of the ligating oligonucleotide.…”

“…4b). [46,48] With this approach, the 5 0 -ends of DNA oligonucleotides were modified with a cysteine residue, which allows efficient S-N acyl transfer owing to the close proximity of both a thiol and an amino residue. Additionally, an S-benzyl succinyl moiety at the site of ligation was used to generate N-terminal peptide thioesters.…”

Modern methods for the synthesis of peptide–oligonucleotide conjugates

“…A more recent report 43 describes selective non-matrix chemical ligation of modified RNA containing the 5 H -S-thioester group and polypeptides with N-terminal cysteine residues which affords peptide ± 5 H -RNA conjugates (Scheme 13).…”

“…However, even now it is quite clear that the method in question 45 can be used for the preparation of oligonucleotide conjugates with a very broad range of peptides with small restrictions for amino acid sequences. This approach has obvious advantages over the method considered above for the synthesis of peptide ± RNA conjugates, 43 since it allows the use of peptides with thioesters at their C-and N-ends and a 5 H -cysteinyl-substituted oligonucleotide prepared by a standard phosphoramidite procedure. The method 43 allows the use of exclusively N-terminal cysteine-containing peptides and RNA modified with 5 H -phosphothioate.…”