Flow-Based Fmoc-SPPS Preparation and SAR Study of Cathelicidin-PY Reveals Selective Antimicrobial Activity

Abstract

Antimicrobial peptides (AMPs) hold promise as novel therapeutics in the fight against multi-drug-resistant pathogens. Cathelicidin-PY (NH2-RKCNFLCKLKEKLRTVITSHIDKVLRPQG-COOH) is a 29-residue disulfide-cyclised antimicrobial peptide secreted as an innate host defence mechanism by the frog Paa yunnanensis (PY) and reported to possess broad-spectrum antibacterial and antifungal properties, exhibiting low cytotoxic and low hemolytic activity. Herein, we detail the total synthesis of cathelicidin-PY using an entirely on-resin synthesis, including assembly of the linear sequence by rapid flow Fmoc-SPPS and iodine-mediated disulfide bridge formation. By optimising a synthetic strategy to prepare cathelicidin-PY, this strategy was subsequently adapted to prepare a bicyclic head-to-tail cyclised derivative of cathelicidin-PY. The structure-activity relationship (SAR) of cathelicidin-PY with respect to the N-terminally positioned disulfide was further probed by preparing an alanine-substituted linear analogue and a series of lactam-bridged peptidomimetics implementing side chain to side chain cyclisation. The analogues were investigated for antimicrobial activity, secondary structure by circular dichroism (CD), and stability in human serum. Surprisingly, the disulfide bridge emerged as non-essential to antimicrobial activity and secondary structure but was amenable to synthetic modification. Furthermore, the synthetic AMP and multiple analogues demonstrated selective activity towards Gram-negative pathogen E. coli in physiologically relevant concentrations of divalent cations.

Biocompatible strategies for peptide macrocyclisation

“…71 Alternatively, using iodine as an oxidising agent, cysteine-rich peptides can rapidly cyclise within only a few minutes. 72–74 The liability of disulfide bonds to reducing conditions has motivated chemists to explore various bioisosteric crosslinks. 75–78…”

“…71 Alternatively, using iodine as an oxidising agent, cysteine-rich peptides can rapidly cyclise within only a few minutes. [72][73][74] The liability of disulde bonds to reducing conditions has motivated chemists to explore various bioisosteric crosslinks. [75][76][77][78] An area of signicant importance where disulde bonds have been used routinely to form macrocyclic peptides under biocompatible conditions is phage display (Scheme 2C).…”

Non‐canonical amino acid bioincorporation into antimicrobial peptides and its challenges

“…SPPS also enabled post-synthesis modifications either resembling that in nature or new to nature to further introduce complexities in the peptides produced [189][190][191] . This time-efficient synthesis has enhanced structure-activity relationship studies in AMPs 85,[192][193][194] .…”

A Portable and Disposable Electrochemical Sensor Utilizing Laser-Scribed Graphene for Rapid SARS-CoV-2 Detection

“…The lysine residue of this sequence was incorporated with orthogonal side chain protection as Fmoc-Lys(Dde)-OH. Upon completion of the linear peptide sequence, the N-terminal Fmoc group was exchanged for Boc protection on-resin by treatment with Boc 2 O in DMF [24], followed by removal of the Dde protecting group from the N ε amino group of the most C-terminal lysine with a 2% hydrazine solution in DMF [25]. The free amino group was coupled 4-(pyren-1-yl)butanoic acid (5 equiv., AK Scientific, Union City, CA, USA) with HATU (4.8 equiv.)…”