Use of N^α-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).

Specific 3′‐Terminal Modification of DNA with a Novel Nucleoside Analogue that Allows a Covalent Linkage of a Nuclear Localization Signal and Enhancement of DNA Stability

“…fied with a thiol and then conjugated with peptides functionalized with maleimide. [30][31][32] Our method has many advantages over these: i) the yield is almost quantitative and 200 pmol of DNAs were modified with only 4 U of TdT, so even milligram quantities of modified DNA can easily be prepared in the laboratory, ii) our method does not involve any extra modification of the DNA, such as the preparation of alkylamino-containing DNA, and iii) our method can be applied to any unmodified double-stranded DNA (dsDNA), prepared by PCR or by other methods, and there is no limit to the length of the DNA that can be modified.…”

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

“…thioether) or unstable (disulfide) linkage. Two variants of thioether linkage formation are available: nucleophilic substitution of haloacetamides ( [54,55], oligonucleotides [56][57][58][59] or siRNA [60] in solution by use of maleimide activated esters [50,[57][58][59] or haloacetic anhydrides [56]. Also solid-phase synthesis of N-bromoacetamide peptides has been described [61].…”

Peptide-Oligonucleotide Hybrids in Antisense Therapy

“…Thiols afford specific and rapid conjugation and react with a wide variety of substrates [11]. In addition, conjugation to maleimido peptides gives rise to better results as there are fewer side products [12].…”

“…The latter conjugation produced six antisense molecules per protein. Conjugation via the 3' end of the oligonucleotide is also possible, although more difficult; 3' thiols have been conjugated to a maleimido peptide [11]. An advantage to the latter conjugation is that ligation of a peptide to the 3' terminus of the oligonucleotide will also protect it from 3' exonucleases [11].…”