Vinylphosphonites for Staudinger-induced chemoselective peptide cyclization and functionalization

Abstract

In this paper, we introduce vinylphosphonites for chemoselective Staudinger-phosphonite reactions (SPhR) with azides to form vinylphosphonamidates for the subsequent modification of cysteine residues in peptides and proteins. An electron-rich alkene is turned into an electron-deficient vinylphosphonamidate, thereby inducing electrophilic reactivity for a following thiol addition. We show that by varying the phosphonamidate ester substituent we can fine-tune the reactivity of the thiol addition and even control the functional properties of the final conjugate. Furthermore, we observed a drastic increase in thiol addition efficiency when the SPhR is carried out in the presence of a thiol substrate in a one-pot reaction. Hence, we utilize vinylphosphonites for the chemoselective intramolecular cyclization of peptides carrying an azide-containing amino acid and a cysteine in high yields. Our concept was demonstrated for the stapling of a cell-permeable peptidic inhibitor for protein-protein interaction (PPI) between BCL9 and beta-catenin, which is known to create a transcription factor complex playing a role in embryonic development and cancer origin, and for macrocyclization of cell-penetrating peptides (CPPs) to enhance the cellular uptake of proteins.

Scheme 1. Vinylphosphonites for intramolecular peptide cyclization and cysteine selective protein modification.

Fig. 1. (a) Synthesis of substituted vinylphosphonites from phosphorus trichloride (route I) or bis(diisopropylamino)chlorophosphine (route II). Stated are isolated yields. (b) One-pot SPhR with vinylphosphonites and azides. Synthesis from borane protected vinylphosphonites (route III) or diethyl chlorophosphite (route IV). Stated are isolated yields.

Fig. 2. (a) Reaction of glutathione (GSH) with differently substituted vinylphosphonamidates. 10 mM vinylphosphonamidates were reacted with 20 mM GSH at pH 8.5 (NH₄HCO₃ buffer). Concentration of the starting materials over time, mean and error from three independent measurements, monitored by UPLC-UV. (b) N-Phenyl vs. N-alkyl comparison. (c) Comparison of different O-substituents. Dotted line depicts 50% conversion.

Fig. 3. Cetuximab modification with 6. (a) Reduction and alkylation principles and structure of 6. (b) Gel analysis: reactions were carried out with varying equivalents of 6: Lane 1: untreated antibody. Lane 2–4: control reactions without prior DTT treatment. Lane 5–7: prior DTT treatment. HC: antibodies' heavy chain, LC: light chain.

Scheme 2. Principle of the intramolecular peptide cyclization of an azido containing cysteine-BCL-9 derived peptide. Investigations with a capped cysteine peptide (9) revealed an accelerated thiol addition when SPhR is performed in the presence of thiols. SPhR on peptides is always carried out with the crude diethyl phosphonite, synthesized from diethyl chlorophosphite (route III).

Fig. 4. (a) Stability of 9a at different pH values. Shown are mean and error from three independent measurements, monitored by UPLC-UV. (b) UPLC-UV spectra of 9a upon incubation at pH 3.1 over several days. *P–N-bond cleavage is the only degradation product observed. (c) Structures of differently cyclized BCL-9 peptides. Stated are isolated yields from the cyclization reaction. (d) CD-measurement of peptides. (e) HTRF assay shows disruption of PPI by peptidic inhibitors. (f) Cellular uptake studies of 10 μM mixture of 10% carboxy-fluorescein (CF)-labelled and 90% non-labelled peptides in HeLa cells. Confocal images with scale bar = 20 μm. Depicted is the overlay of fluorescein-fluorescence and Hoechst nucleus stain at different time points of laser (405/488 nm) irradiation.

Fig. 5. (a) Principle of the Staudinger induced peptide cyclization with R₁₀ and subsequent attachment of a thiol reactive handle. (b) Conjugation of cR₁₀ to eGFP C70M S147C. Shown are MALDI-TOF-MS spectra before and after the reaction with 23. (c) Cellular uptake studies of 30 μM eGFP-14 and eGFP-18 in HeLa cells. Confocal images with scale bar = 10 μm.