A perfluoroaryl-cysteine S(N)Ar chemistry approach to unprotected peptide stapling

Abstract

We report the discovery of a facile transformation between perfluoroaromatic molecules and a cysteine thiolate, which is arylated at room temperature. This new approach enabled us to selectively modify cysteine residues in unprotected peptides, providing access to variants containing rigid perfluoroaromatic staples. This stapling modification performed on a peptide sequence designed to bind the C-terminal domain of an HIV-1 capsid assembly polyprotein (C-CA) showed enhancement in binding, cell permeability, and proteolytic stability properties, as compared to the unstapled analog. Importantly, chemical stability of the formed staples allowed us to use this motif in the native chemical ligation-mediated synthesis of a small protein affibody that is capable of binding the human epidermal growth factor 2 receptor.

Figure 1

(A) S_NAr reaction between thiolate and a perfluroaromatic species. (B) Model reactions carried out between protected cysteine derivative 1 and two commercially available perfluoroaromatic reagents 2a and 2b. In both cases the reaction yields are > 95% as determined by in situ ¹⁹F NMR spectroscopy – (see SI).

Figure 2

(A) Perfluoroarylation of cysteine residues in an unprotected peptide 4. (B) LC-MS traces of the crude reaction mixtures (214 nm, UV) and mass spectra (inset) of the stapled peptides 4a (bottom) and 4b (top).

Figure 3

(A) CD spectra of 6 and 6a, b and (B) of 3a, b; (C) Biacore binding sensograms of 6 and 6a, b with immobilized C-CA (adsorption/desorption time – 300 sec); (D) Flow cytometry data for FITC-based peptides incubated with HEK293T cells (25 μM; fluorescence response is averaged for each sample and normalized to 7b); (E) Z-stack accumulated fluorescent confocal microscopy images (DNA – blue; cell membrane – red; peptides – green (FITC)) of the HEK293T cells treated with peptides 7, 7a–b (5 μM), unstapled controls 8a and 8b and NYAD-2 – 9.

Figure 4

(A) Representation of the HER-2 affibodies (PDB code: 2B89) with chemically modified Cys positions [Cys⁴², Cys⁴⁶] via stapling (10a) and alkylation (10); (B) CD spectra of 10 and 10a, and the corresponding temperature-dependent melting curves measured from the CD spectra at 222 nm (inset); (C) Biacore binding sensograms of 10, 10a with immobilized HER-2 extra-celluar binding region (adsorption/desorption time - 300 sec).

Scheme 1

Peptides studied in the context of the C-CA model.