Peptide Stapling via Thiophosphoramidate Intermediates

Abstract

Through the adaptation of 'click'-like aqueous N-thiophosphorylation chemistry established previously by the Hodgson group, we were able to staple a bis-lysine model peptide substrate in a novel one-pot, two-step stapling methodology via a di-thiophosphoramidate intermediate.
In order to reach the goal of obtaining a peptide stapled via a di-thiophosphoramidate intermediate, work first involved the optimisation of aqueous N-thiophosphorylation of a chromophore-labelled lysine residue model and a lysine-containing chromophore-labelled tetra peptide model using thiophosphoryl chloride as the thiophosphorylation agent and sodium hydroxide solution as the base. Chromophore labelling allowed for the development of LC-MS DAD-based analytical assays to determine conversion from amine substrate to thiophosphoramidate product.
A study on the selectivity go the N-thiophosphorylation reaction was conducted on a series of unprotected tetra peptides, designed with different N-terminal amino acids, following optimisation of aqueous N-thiophosphorylation. Through the implementation of a phosphorus-31 NMR spectroscopy-based assay, it was found that aqueous N-thiophosphorylation is selective for thiophosphorylation at an ε-amino group rather than an α-amino group. It was also noted that the size of the N-terminal amino acid side chain had a greater impact on thiophosphorylation selectivity that the pKaH of the tetrapeptide N-terminus.
Brief studies were also conducted on the use of an alternative thiophosphorylating agent (potassium thiophosphorodichloridate) and/ or an alternative base (triethylammonium bicarbonate solution).
Finally, a bis-lysine-containing peptide was designed and synthesised for stapling experiments. Through the use of corroborative LC-MS DAD and proton-phosphorus coupled HMBC NMR spectroscopy analyses, peptide stapling was achieved using a one-pot, two-step procedure via a di-thiophosphoramidate intermediate using cheap and readily available reagents.