## 2 Results and Discussion
### Total Synthesis of Cathelicidin-PY
With the recent addition of fast-flow SPPS to our laboratory [49], we wished to compare our optimised room-temperature synthesis protocol to flow conditions at 65 \({}^{\circ}\)C. The  
Figure 3: RP-HPLC chromatograms (214 nm) monitoring key intermediates during on-resin preparation of linear cathelicidin-PY (**12**) under microwave irradiation according to Scheme 1a. (**a**) confirms the desired product Fmoc-protected linear cathelicidin-PY2\({}^{\text{--}}\)29 (\(\text{\text{\text{t}}}_{\text{R}}\) = 21.5 min) (**b**) confirms the desired product Fmoc-protected linear cathelicidin-PY10\({}^{\text{--}}\)29 (\(\text{\text{\text{t}}}_{\text{R}}\) = 18.6 min) (**c**) the desired product, linear cathelicidin-PY (**12**), was not present (**d**) confirms the presence of the desired linear cathelicidin-PY (**12**) (\(\text{\text{\text{t}}}_{\text{R}}\) = 18.5 min) under automated synthetic conditions according to Scheme 1b. continuous flow approach to Fmoc-SPPS has offered significant advancement in the field, expediting peptide synthesis, whereby the coupling time is reduced to mere seconds [51; 52; 53]. However, reports of flow-chemistry preparation of disulfide-bridged peptides remain minimal. We recently demonstrated the applicability of flow chemistry towards the synthesis of capitellacin, a two disulfide containing AMP, finding that using altered cysteine coupling conditions and on-resin iodine-mediated orthogonal disulfide formation could rapidly yield the desired bicyclic peptide in respectable overall yield and purity [49]. Accordingly, we were eager to further explore the generality of this preparation by applying it to cathelicidin-PY [54]. In our first attempt, linear cathelicidin-PY was prepared on ChemMatrix resin, and a small-scale resin cleavage was carried out to analyse the product by RP-HPLC and ESI-MS. As envisioned, linear peptide **12** was successfully afforded, albeit at a crude purity of only 38% (Supplementary Materials Figure S6). Somewhat disappointingly, in comparison to room temperature, batch-wise coupling cycles (Scheme 2b) and flow chemistry synthesis (as per Scheme 3a) failed to provide a superior result. At this point, it was decided to review conditions such as the Fmoc removal solution and the coupling conditions. In accordance with our previously optimised protocol for epimersion-prone cysteine residues, modified coupling conditions were also applied to His20 [55; 50]. Lower-substituted TentaGel S NH\({}_{2}\) resin (0.25 mmolg\({}^{-1}\)) was also implemented in place of ChemMatrix (0.6 mmolg\({}^{-1}\)). In addition, an acetic anhydride capping process was introduced after each fourth residue coupling to mask any unreacted amino groups. These slight adjustments (Scheme 3b), in line with our recent work [49], afforded a marked improvement, with **12** affording a crude purity of 79% (Supplementary Materials Figure S8), after having removed the terminal Fmoc-protection and cleaving a small portion from the resulting resin **17** (Scheme 4). **Scheme 3. ** Synthesis of linear cathelicidin-PY on continuous flow synthesis at 65 \({}^{\circ}\)C with a flow rate of 15 mL/min and a resonance time of 40 s using different resin substitutions (**a**) ChemMatrix (0.6 mmolg\({}^{-1}\)) and (**b**) TentaGel S NH\({}_{2}\) (0.25 mmolg\({}^{-1}\)); 6-Cl-HObt--1-hydroxy-6-chloro-benzotriazole, HATU-hexafluorophosphate azabenzotriazole tetramethyl uronium, DIC-_N_/_N_-diisopropylcarbodiimide, DMAP-4-methyldiaminopyridine, DODT-2,2-(ethylenedioxy)diethanethiol, NMM-_N_-methylmorpholine, PyAOP-7-azabenzotriazole1-lyoxy)tripyrylrolidinophosphonium hexafluorophosphate, FA-formic acid, TFA-trifluoroacetic acid, TIS-trisiopropylsilane. With resin-bound peptide **17** in hand (Scheme 4), the maximally protected linear peptide was subjected to iodine (3 equiv.) mediated simultaneous deprotection and disulfide formation between trityl-protected Cys\({}^{3}\) and Cys\({}^{7}\) residues in DMF for 2 \(\times\) 5 min.