Use of Nalpha-Fmoc-cysteine(S-thiobutyl) derivatized oligodeoxynucleotides for the preparation of oligodeoxynucleotide-peptide hybrid molecules.

Abstract

The chemical modification of antisense oligodeoxynucleotides (ODNs) by conjugating synthetic peptides of known membranotropic activities to the 3' and/or 5' terminus of ODNs may serve two functions that are important for increasing their bioavailability by protecting the ODNs from exonuclease digestion and facilitated delivery into cells. We have previously reported the preparation of ODN-peptide conjugates by the total synthesis approach. However, by such technology the preparation of ODN-peptide conjugates in amounts sufficient for in vitro functional analysis is at present limited to the syntheses of peptides containing residues without acidolytic deprotection. Requisite to the alternative method of site-specific conjugation, the segment coupling approach is the derivatization of an ODN with a nucleophilic moiety. In this paper, we describe a novel method of functionalizing synthetic ODNs by incorporating S-thiobutyl-protected Nalpha-Fmoc-cysteine to aminopropyl-functionalized CPG by standard Nalpha-Fmoc SPPS methodology. The derivatized solid support can be used to synthesize an ODN of any sequence by the phosphoramidite chemistry, and the removal of the S-thiobutyl side chain function can be conveniently affected by the standard amminolytic deprotection of ODNs containing 1 M DTT. The purified cysteine-derivatized ODN was shown to react specifically and efficiently with two types of synthetic peptides corresponding to regions within the glycoprotein (gp) of HIV that have been shown to have membranotropic activities: a 18 residue maleimide-derivatized Tat peptide of the transactivator (tat) of HIV and a 22 residue peptide corresponding to the carboxyl terminus of gp41(Ca-gp41).