Using ¹⁹F NMR and two‐level factorial design to explore thiol‐fluoride substitution in hexafluorobenzene and its application in peptide stapling and cyclisation

Abstract

Hexafluorobenzene undergoes 1,4‐selective thiol‐fluoride disubstitution and is an attractive disulfide crosslinking reagent for peptide cyclisation and stapling. Little attention has been directed toward understanding the scope of this reaction. Traditional reaction optimisation relies on a one‐variable‐at‐a‐time approach, which can exclude important combined effects of reaction variables. This study initially explored base and solvent effects to inform a subsequent two‐level factorial design approach to understand how to control the reactivity and product selectivity in a model reaction of hexafluorobenzene. We describe new conditions that selectively afford higher order substitution products for example, 1,2,4,5‐tetrasubstitution, making hexafluorobenzene a possible suitable scaffold for future branched or multicyclic peptide systems. Moreover, our new conditions provide an improved rapid (<1 minute) and selective peptide disulfide stapling and cyclisation approach under peptide‐compatible conditions.

Perfluoroaryl and Perfluoroheteroaryl Reagents as Emerging New Tools for Peptide Synthesis, Modification and Bioconjugation

“… [54] Advantageously, perfluoroaromatics afford the distinct ability to monitor reactions and to identify substitution patterns around the aromatic ring using 19 F NMR. [35] …”

“…Perfluoroaromatics react with natural amino acids to form stable and non‐reversible conjugates under physiological conditions, unlike other reagents, such as maleimides [54] . Advantageously, perfluoroaromatics afford the distinct ability to monitor reactions and to identify substitution patterns around the aromatic ring using 19 F NMR [35] …”

“…[54] Advantageously, perfluoroaromatics afford the distinct ability to monitor reactions and to identify substitution patterns around the aromatic ring using 19 F NMR. [35] In general, perfluoroaromatic bioconjugation can enhance the pharmacokinetic properties for example, serum stability and conformational stability through stapling of bioactive synthetic peptides (many examples above). However, perfluoroarylation can add significant lipophilicity to a biomolecule and may affect its solubility or cellular penetration in the case of for example, stapled or macrocyclic peptides, albeit, this probably is more dependent on the physicochemical properties of the starting peptide.…”

“…This transformation has since been employed in several different peptide systems under related conditions, typically employing DMF as solvent and either TRIS‐base, Et 3 N or DIPEA as base, with reaction times ranging from 30 min up to 18 h. [34] In one example, the reaction of hexafluorobenzene 40 with N‐acetyl cysteine was explored and optimised using a 2‐level factorial design experiment (varying: reaction time, temperature, concentration and equivalents of base/thiol) and monitoring the reaction outcomes by the distinct spectra obtained using 19 F NMR analysis of crude mixtures. [35] Application of the optimal mild conditions (DMSO/Cs 2 CO 3 , 21 °C or MeCN/DBU, 21 °C) to a model unprotected peptide system rapidly (<1 minute and < 1 h, respectively,) afforded the quantitative disubstitution (stapled) product. The reaction was also possible on‐resin (DMF, DIPEA, 21 °C, 18 hours), making this applicable to solid phase modification.…”

Covalent Scrambling in Porous Polyarylthioethers through a Stepwise SNAr for Tunable Bandgap and Porosity

“…Hence, the multi‐substitution of aryl halide with metal sulfide as a nucleophile is rare, especially with suitable regioselectivity. If the extent of substitution on aryl halides is controllable, [13] polycondensation using metal sulfides may be a viable way to synthesize POPs with multi‐dimensions.…”