Efficient Conjugation of Peptides to Oligonucleotides by “Native Ligation”

Abstract

A new strategy has been developed for conjugation of peptides to oligonucleotides. The method is based on the "native ligation" of an N-terminal thioester-functionalized peptide to a 5'-cysteinyl oligonucleotide. Two new reagents were synthesized for use in solid-phase peptide and oligonucleotide synthesis, respectively. Pentafluorophenyl S-benzylthiosuccinate was used in the final coupling step in standard Fmoc-based solid-phase peptide assembly. Deprotection with trifluoracetic acid generated in solution peptides substituted with an N-terminal S-benzylthiosuccinyl moiety. O-trans-4-(N-alpha-Fmoc-S-tert-butylsulfenyl-L-cysteinyl)aminoc yclohe xyl O-2-cyanoethyl-N,N-diisopropylphosphoramidite was used in the final coupling step in standard phosphoramidite solid-phase oligonucleotide assembly. Deprotection with aqueous ammonia solution generated in solution 5'-S-tert-butylsulfenyl-L-cysteinyl functionalized oligonucleotides. Functionalized peptides and oligonucleotides were used without purification in native ligation conjugation reactions in aqueous/organic solution using tris-(2-carboxyethyl)phosphine to remove the tert-butylsulfenyl group in situ and thiophenol as a conjugation enhancer. A range of peptide-oligonucleotide conjugates were prepared by this route and purified by reversed-phase HPLC.

Synthesis of peptide-oligonucleotide conjugates: Application to basic peptides

“…After deprotection and release from the support, the two molecules are conjugated (in either crude or purified form) in buffered aqueous/DMF solution in the presence of a reducing agent (TCEP) and thiophenol as a thiol transfer promoting agent (5).…”

PNA zipper as a dimerization tool: Development of a bZip mimic

“…and coupled for 60 min at room temperature. 24 The resin was then washed with DMF, MeOH, and diethyl ether and dried. Cleavage from the support and deprotection of the side chain protecting groups were carried out in a TFA/benzylmercaptan/phenol/water (90/5/2.5/2.5 v/v/v/v) mixture for 4 h at room temperature.…”

“…The Mtt protecting group on the lysine side chain amino group was selectively removed at the end of the peptide synthesis; the amine was reacted with the mono benzyl succinimidyl thioester. 24 The PNA strands were obtained by standard solid phase techniques; a lysine residue was appended respectively to the C and N-terminus of complementary strands. At the end of the synthesis, the lysine side chain was selectively deprotected; the free amine was reacted with a cysteine.…”

Organic Synthesis and Antisense Effects of Oligonucleotide-Peptide Conjugates

“…J. Gait and his colleagues successfully reported the synthesis of a variety of DNA-peptide conjugates by "native ligation" [4]. By their method, oligonucleotide modified with S-tert-butylsulfenyl L-cystein moiety at 5'-end can be reacted site-specifically with thioester of a peptide fragment to give a covalent conjugate (Scheme 2).…”