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

Conjugation of a delivery peptide containing a thiol functionality (e.g., a cysteine residue) with a PNA oligomer displaying 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 also displaying a reactive N-hydroxysuccinimidyl ester group (e.g., Mal-PEG2-OSu). Here, an optimized protocol with respect to ratios between the reactants as well as recommended reaction times is presented. Formation and conversion of the maleimide-PNA intermediate was followed by analytical HPLC as exemplified by its conjugation to (KFF)₃K-Cys-NH₂. In addition, the 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 peptide (with a varying degree of sterical hindrance: HS-(CH₂)₂-CONH-(KFF)₃K-NH₂, (KFF)₃K-hCys-NH₂ and (KFF)₃K-Cys-NH₂) is provided.