Novel methodologies for the conjugation and cyclisation of polyamides

[eng] An alternative allowing diene-derivatised polyamides to be used in Diels-Alder conjugation reactions has been explored and developed. In this methodology, conjugation takes place between a fully protected, resin-linked diene-derivatised polyamide and a soluble maleimide- containing compound. The acid-labile diene is thus not exposed to acids, and the acidic deprotection treatment that follows does not affect the cycloadduct. In contrast with previously described alternatives, this methodology has no limitations in terms of sequence and does not require special protecting groups. (E)-4,6-Heptadienoyl-polyamide-resins are used due to the good yields observed in the Diels- Alder reaction and the stability of the generated compounds. Polyamides are assembled using the standard, commercially available building blocks on water-swelling solid matrixes, and no special building blocks need to be synthesised. Both water and organic solvent/water mixtures can be used for the on-resin cycloaddition, and simultaneous maleimide deprotection and on- resin Diels-Alder cycloaddition is feasible, allowing conjugates with different linking sites to be prepared. The on-resin Diels-Alder reaction is also compatible with the wide-spread maleimide- thiol reaction, and combination of both transformations is feasible, making it possible to synthesise double conjugates. However, diene-derivatised oligonucleotides do not withstand the reaction conditions required for the solid-phase Diels-Alder conjugation, meaning that peptide- oligonucleotide conjugates cannot be prepared using this methodology. In a different project, 2,2-disubstituted cyclopent-4-ene-1,3-diones (CPDs), which were chosen as non-hydrolysable maleimide analogs, have been found to possess an unexpected reactivity. While maleimides react in an irreversible manner with all types of thiols, the Michael-type reaction between CPDs and cysteines placed at internal or C-terminal positions, which do not possess a free amine, is reversible. In contrast, cysteines with a free amine (at the N-terminus of peptides) react with CPDs to end up furnishing a stable product with a mass 20 Da lower (M- 20 Da) than the Michael-type adduct (weighing M Da). Formation of the M-20 Da adduct plausibly takes place through the following steps: First, conjugate addition of the N-terminal cysteine thiol to the CPD double bond yields a Michael-type adduct. This product immediately undergoes intramolecular imine formation, by reaction betwe