We have developed a strategy for the synthesis of novel oligodeoxynucleotide (ODN)-peptide conjugates on a scale suitable for the investigation of their potential as antisense inhibitors of gene expression. These conjugates have the 3'-terminus of the antisense oligodeoxynucleotide linked covalently to the N-terminus of a peptide. This strategy allows the preparation of conjugates containing a peptide segment designed to facilitate intracellular delivery of the antisense oligodeoxynucleotide as well as providing protection against 3'-exonuclease digestion. To illustrate the synthetic approach we describe the preparation of a series of conjugates comprising antisense oligonucleotides to human immunodeficiency virus type 1 (HIV) linked to fusion peptides derived from the HIV transmembrane glycoprotein gp41. The conjugates were prepared by the total synthesis method, in which the peptide is assembled first by the N-(fluorenylmethoxycarbonyl) (Fmoc) solid-phase methodology. This is followed by derivatization of the amino terminus by reaction with an alpha,omega-hydroxycarboxylic acid derivative which converts the terminus to a protected aliphatic hydroxy group on which standard solid phase DNA synthesis by the phosphoramidite method is performed. The purified conjugates were characterized extensively by several analytical techniques including ion spray mass spectrometry. Thermal denaturation studies showed that the interaction of the ODN-peptide conjugate with its complementary strand was similar to that of unmodified oligonucleotides. Preparation by the total synthesis method gave the purified conjugate with overall yields in the range of 6-14%.

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).