## 2 Results and Discussion
### Design and Synthesis of Cathelicidin-PY Analogues  
With the successfully optimised protocol in hand, these conditions were adapted to prepare a linearised analogue with alanine substituted for Cys\({}^{3}\) and Cys\({}^{7}\) (**2**) and a further constrained head-to-tail bicyclised peptide (**3**). Accordingly, linear crude peptide **2** was afforded a respectable crude purity (51.6%), (Supplementary Materials Figure S15). With the NMR structure calculations of [PERSON] and co-workers in hand [33], we envisioned that head-to-tail cyclisation of cathelicidin-PY could offer a favourable rigidifying effect to its folded structure. While the respective termini of the peptide are in relatively close proximity to one another to facilitate this, the terminal regions also appeared to possess the greatest RMSD, or degree of flexibility compared to the rest of the more well-defined peptide structure. To prepare bicyclic peptide **3**, we adapted our synthesis to incorporate a highly acid sensitive 4-(4-hydroxymethyl-3-methoxyphenoxy) butyric acid (HMPB) linker (Scheme 5). Under otherwise analogous conditions to peptide **1**, the linear peptide was prepared on-resin, and disulfide cyclisation between Cys\({}^{3}\) and Cys\({}^{7}\) was effected with iodine (3 equiv.) in DMF prior to liberating the fully protected peptide from the resin with 20% HFIP in CH\({}_{2}\)Cl\({}_{2}\) (\(v/v\)). The fully side-chain-protected linear precursor was then cyclised under high dilution conditions (0.5 mM) in DMF, effected by PyAOP (5 equiv.) and DIPEA (1% _v/v_ ) [56].  
**Scheme 5.** Synthesis of bicyclic cathelcidin-PY analogue **3** on continuous flow synthesis at 65 \({}^{\circ}\)C with a flow rate of 15 mL/min and a resonance time of 40 s. 6-Cl-HOBt-1-hydroxy-6-chloro-benz-otriazole, DIC-_N_,_N_-diisopropylcarbodiimide, DIPEA-_N_,_N_-disopropylthymamine, DMP-4-dimethylaminopyridine, FA-formic acid, HATU-hexafluorophosphate azabenzotriazole tetramethyl uranium, HFIP-1,1,3,3,3, hexafluoro-2-propanol, NMM-_N_-methylmorpholine, PyAOP-7-azabenzotriazole-1-yloxy)tripyrrolidinophosphonium, hexafluorophosphate, TFA-trifluoroacetic acid, TIS-trisisopropylsilane.  
Curious to probe the disulfide further, we envisaged that a more rigid lactam bridge may offer improved antimicrobial activity and plasma stability. We believed this would additionally offer interesting insights into the structure-activity relationship with respect to tolerances of the _N_-terminal cyclic loop of cathelcidin-PY and likely related amphibian AMPs [57; 58; 59]. Accordingly, a series of lactam rings containing mimetics (**4-9**) were prepared (Scheme 6). Implementing Fmoc-Glu(O-2-PhiPr)-OH or Fmoc-Asp(O-2-PhiPr)-OH and Fmoc-Lys(Mtt)-OH in position of Cys\({}^{3}\) and Cys\({}^{7}\), as required, enabled an on-resin macrolatamisation of various ring size analogues (peptides **4-7**) upon orthogonal deprotection by 1% _v/v_ TFA in CH\({}_{2}\)Cl\({}_{2}\), followed by immediate on-resin macrocyclisation mediated by PyAOP and DIPEA in DMF for 4 h, removal of the terminal \(N^{\alpha}\)-Fmoc protection and resin cleavage. Guided by preliminary assay results (vide infra), further ring contractions were enabled by implementing Fmoc-2,4-diaminobutyric acid (Dab) in place of lysine to prepare macrocyclic peptides **8** and **9**. With Fmoc-Dab(Dde)-OH commercially available, the \(N^{\alpha}\)-Fmoc group of the terminal Arg residue was exchanged on-resin for a Boc group, thus accommodating on-resin removal of the Dde protecting group by 2% _v/v_ hydrazine in DMF (2 \(\times\) 4 min), without unmasking the terminal \(N^{\alpha}\)-amino group [60]. With cyclisation requiring up to 24 h for completion for the more constrained lactams (**8** and **9**), DIPEA was substituted for _sym_-collidine during on-resin macrocyclisation. Upon global deprotection and liberation from the respective resins with TFA, lactam-bridged peptides **4-9** were afforded in satisfactory crude purity (ranging 44-54%) and purified to homogeneity by RP-HPLC.