## 2 Results and Discussion  
### Total Synthesis of Cathelicidin-PY  
The total synthesis of cathelicidin-PY is yet to be detailed; therefore, a feasible and efficient synthetic route was developed by implementing Fmoc-SPPS. Microwave irradiation is known to be highly efficient in preparing larger or so-called \"difficult\" peptides [44; 45; 46; 47]. Thus, expecting a problem-free synthesis, the preparation of cathelicidin-PY (**1**) was first attempted by Fmoc-SPPS under microwave irradiation (75 \({}^{\circ}\)C temp) with low-cost aminomethyl polystyrene (AM-PS) resin, with a substitution rate of 1.26 mmolg\({}^{-1}\) and implementing O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyl hyaluron hexafluorophosphate (HCTU) as the coupling reagent (Scheme 1a). Figure 1: Sequence alignment of recently discovered amphibian cathelicidins, mature peptides shown. Blue denotes cationic residues and pink denotes the cysteine residues forming a disulfide bond. Figure 2: Structure of the cathelicidin-PY peptide, **1**, and a series of analogues, **2–9** of cathelicidin-PY. Dab = 2,4-diaminobutyric acid. Progression of the synthesis at cathelicidin-PY20-29 was monitored by cleavage of a small amount of peptidyl resin and determination of its purity by RP-HPLC (214 nm), which indicated the presence of the desired compound in satisfactory purity (82%; Figure 3a). However, upon continuing the synthesis to cathelicidin-PY10-29 and to completion, the RP-HPLC chromatogram became increasingly complex, failing to afford the final 29-mer (Figure 3b,c). The high substitution resin (1.26 mmolg\({}^{-1}\)) may have contributed to steric hinderance and intra- and intermolecular aggregation, ultimately resulting in the failed synthesis [48]. Somewhat surprised by this failure, we attempted automated synthesis at room temperature with increased coupling times from 5 min to 1 h and otherwise identical conditions (Scheme 1b). Pleasingly, analysis by cleavage of a small amount of peptidyl resin and determination of its purity by RP-HPLC (214 nm) revealed the successful assembly of the full linear sequence, albeit with an exceedingly poor overall purity of 4% (Figure 3d and Supplementary Materials Figure S2). The modest improvement, despite the reduced temperature, was possibly afforded by the increased coupling times throughout the synthesis. With the need to perform an additional synthetic step (disulfide cyclisation) and the desire to prepare multiple analogues, we were unsatisfied with this result. We speculated that the poor performance of both microwave and room temperature synthesis could be attributed to the high resin substitution rate of the relatively poor swelling 1% DVB cross-linked PS resin [48]. Thus, AM-PS (1.26 mmolg\({}^{-1}\)) resin was substituted for polyethylene glycol (PEG) grafted resin, TentaGel HL NH\({}_{2}\) resin, which has greater swelling capacity and is loaded at a lower substitution rate (0.48 mmolg\({}^{-1}\)). Repeating the synthesis at room temperature with this resin and double coupling of the final seven residues (Scheme 2a) afforded a remarkably improved crude purity (44%) for **12** (Supplementary Materials Figure S3). Pleased with this improvement, we sought to further optimise the preparation by implementing TentaGel S NH\({}_{2}\) resin (Scheme 2b) with a further reduced substitution rate (0.26 mmolg\({}^{-1}\)). Furthermore, both Cys\({}^{3}\) and Cys\({}^{7}\) were manually coupled with PyAOP and the sterically hindered weak organic base, _sym_-collidine, to minimise epimerisation [49; 50]. To further streamline the synthesis, and assuming the lower resin substitution would minimise aggregation, double couplings were abandoned for the final seven residues. To determine the effectiveness of this newly optimised synthetic protocol, a small sample of the peptidyl resin was cleaved upon completion of the synthesis and analysed by RP-HPLC, which indicated further improvement in the synthesis, affording the full linear peptide **12** in crude purity of 49% (Supplementary Materials Figure S4).