The Synthesis of Peptide-Based Tools for Drug Discovery and Chemical-Biology Applications

Abstract

Peptidyl mono-fluoromethyl ketones (mFMK) are a class of compounds that have shown potential as protease inhibitors for the treatment of a range of diseases. They have also found application as chemical-biology probes that can be used in the interrogation of cellular processes. Despite the aforementioned applications, the number of synthetically viable routes reported for accessing these compounds is currently rather limited.

To address this issue, the work herein reports the efforts that have been made towards developing a novel synthetic pathway for preparing mFMKs. Initial attempts (Chapter 2) involved the formation of a tri-carbonyl system derived from a Boc-protected amino acid and Meldrum’s acid, with the intention of subsequently fluorinating, converting to the corresponding β-ketoester and coupling to a peptide of choice in solution. The idea was that the resulting substrate could then be saponified and decarboxylated, allowing access to the fluoromethyl ketone (FMK). However, the inherent reactivity of the moisture-sensitive fluorinated tri-carbonyl systems made them difficult to handle and isolate.

Attention was therefore turned towards the direct electrophilic fluorination of β-ketoesters, derived from either an N-Fmoc (Chapter 3) or N-Boc-protected (Chapter 4) amino acid of choice and formed as the tert-butyl ester. Selective mono-fluorination of the resulting 1,3-dicarbonyl was then achieved using CpTiCl3 in conjunction with Selectfluor, through utilisation of adapted literature protocols. The resulting N-Fmoc-protected substrate was carefully transformed to the β-ketoacid (Chapter 3) before attempted resin loading was performed, with the intention of allowing on-resin peptide growth followed by concomitant resin cleavage, global deprotection and decarboxylation to the FMK. However, unsuccessful resin loading attempts, coupled with evidence for premature FMK formation having occurred during these endeavours, suggested the presence of the fluorine atom had significantly reduced the nucleophilicity of the carboxylate, thus favouring decarboxylation over resin loading.

Given the challenges encountered with resin loading, the application of N-Boc-protected mono-fluorinated β-ketoesters in solution-phase peptide chemistry was explored (Chapter 4). Selective removal of the N-Boc group in the presence of the tert-butyl ester, through the modification of conditions reported in the literature, enabled coupling to a peptide of choice. Subsequent deprotection and decarboxylation led to the desired FMK. Whilst some epimerisation did appear to occur during peptide coupling, this route represents a straightforward approach for accessing these desirable and highly expensive molecular warheads in only 5 steps.

A slightly adapted approach, in which the N-Boc protected β-ketoester was first decarboxylated to the corresponding FMK prior to peptide coupling in solution, was also trialled (Chapter 4). However, this appeared to lead to challenges in purification of the resultant mFMKs, along with an accompanying low yield. Nonetheless, this methodology was successfully adapted and utilised for the synthesis of a peptidyl mono-chloromethyl ketone (mCMK) in only four steps (Chapter 5), although epimerisation was apparent. Furthermore, it is believed that extension of this methodology could allow access to other peptide-based C-terminal modified species, such as peptidyl bromomethyl ketones and di-fluoromethyl ketones.

In summary, the methodology developed for accessing both peptidyl mono-fluoromethyl ketones and peptidyl mono-chloromethyl ketones offers ready access to chemical-probes that can be used to interrogate cellular functions in a range of disease relevant systems.