USE OF N-ALPHA-FMOC-CYSTEINE(S-THIOBUTYL) DERIVATIZED OLIGODEOXYNUCLEOTIDES FOR THE PREPARATION OF OLIGODEOXYNUCLEOTIDE-PEPTIDE HYBRID MOLECULES

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 imp ortant for increasing their bioavailability by protecting the ODNs fro m exonuclease digestion and facilitated delivery into cells. We have p reviously reported the preparation of ODN-peptide conjugates by the to tal 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 alternativ e method of site-specific conjugation, the segment coupling approach i s the derivatization of an ODN with a nucleophilic moiety. In this pap er, we describe a novel method of functionalizing synthetic ODNs by in corporating S-thiobutyl-protected N-alpha-Fmoc-cysteine to aminopropyl -functionalized CPG by standard N-alpha-Fmoc SPPS methodology. The der ivatized solid support can be used to synthesize an ODN of any sequenc e by the phosphoramidite chemistry, and the removal of the S-thiobutyl side chain function can be conveniently affected by the standard ammi nolytic deprotection of ODNs containing 1M DTT. The purified cysteine- derivatized ODN was shown to react specifically and efficiently with t wo types of synthetic peptides corresponding to regions within the gly coprotein (gp) of HIV that have been shown to have membranotropic acti vities: a 18 residue maleimide-derivatized Tat peptide of the transact ivator (tat) of HIV and a 22 residue peptide corresponding to the carb oxyl terminus of gp41(Ca-gp41).