###### Abstract  
Antimicrobial peptides (AMPs) hold promise as novel therapeutics in the fight against multi-drug-resistant pathogens. Cathelicidin-PY (NH\({}_{2}\)-RKCNFLCLKKEKLRTVITISHIDKVLRPQGCOOH) is a 29-residue disulfide-cyclised antimicrobial peptide secreted as an innate host defence mechanism by the frog _Paa yunnensis_ (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. c 20232819931 2023281931 2023281931 2023281931 2023281931 2023281931 202328193 2  
a foreign bacterial invasion [3; 6]. Phagocytes initiate proteolytic cleavage of the inactive pro-sequence via serine proteases and elastase to readily form and release the mature cathelicidin peptide as an innate protective mechanism [7]. Cathelicidin peptides typically demonstrate a broad spectrum of antimicrobial activity towards Gram-positive bacteria (e.g., _Staphylococcus aureus_[8], _Enterococcus faecalis_[9], and _Bacillus cereus_[10]), Gram-negative bacteria (e.g., _Escherichia coli_[11], _Pseudomonas aeruginosa_[12], and _Salmonella enterica_[13]), and fungi (e.g., _Candida albicans_[14]). Furthermore, these have shown promising anti-inflammatory and specific immune modulatory capabilities in immunocytes [15], as well as antiviral activity against human immunodeficiency virus (HIV) [16], influenza A virus (IAV) [17], respiratory syncytial virus (RSV) [18], human rhinovirus (HRV) [19], herpes simplex virus (HSV) [20], Zika virus (ZIKV), and hepatitis C virus (HCV) [21; 22; 23]. They are highly cationic (between +4 and +13 charges in physiological conditions) and exhibit membrane-lytic mechanisms of action [11; 24]. Cathelicidin-TK, isolated from the salamander _Tylotortion [PERSON]_, is the only known anionic cathelicidin, exhibiting wound healing properties rather than typical antimicrobial activity [25]. Since the first reported cathelicidin peptide in 1989 by [PERSON] et al. [26], more than 150 cathelicidin peptides have been isolated from over 30 different vertebrate species and are found in almost all species of mammals [27; 28]. The occurrence of cathelicidins in amphibians, however, was not reported until 2012 by [PERSON] et al. [29] from the frog skin of _Anolops loolensis_ in the Yunnan Province of China. Since then, _ca._ twenty new cathelicidins have been identified from only _ca._ ten amphibian species [25; 27; 30; 31; 32; 33; 34; 35; 36; 37; 38; 39], despite 8516 amphibian species reported on AmphibiaWeb Database Search, thus highlighting our relatively poor understanding of both the ecological role and medicinal potential of these amphibian-derived AMPs. Existing structure-activity relationship (SAR) studies of mammalian-derived cathelicidins indicate that the highly cationic \(N\)-terminal region is essential for antimicrobial activity and a minimum of 16-18 residues are required [40; 41; 42].