Facile Preparation of PNA-Peptide Conjugates with a Polar Maleimido-Thioether Linkage

Conjugation of a delivery peptide containing a thiol functionality (e.g., a cysteine residue) with a PNA oligomer with a single unprotected aliphatic primary amine (e.g., the N-terminus or a C-terminal lysine residue) can be achieved via a one-pot modification with a bisfunctional maleimide linker having a reactive N-hydroxysuccinimidyl ester group (e.g., Mal-PEG2-OSu; PEG2 = eg1). Here, an optimized protocol with respect to ratios between the reactants, as well as recommended reaction time, is presented. Progress in formation and subsequent conversion of the maleimide-PNA intermediate was monitored by analytical HPLC, as exemplified by a conjugation involving (KFF)₃K-Cys. In addition, reaction time required for direct conversion of a preformed Mal-(CH₂)₂-(C=O)-PNA oligomer in the presence of a slight excess of thiol-modified peptides (with a varying degree of sterical hindrance: HS-(CH₂)₂-CONH-(KFF)₃K-NH₂, (KFF)₃K-hCys and (KFF)₃K-Cys) is provided.