Scheme 1. SPPS and macrocyclization for the synthesis of ramoplanin aglycone analogues. (A) Native chemical ligation and desulfurization (site highlighted in purple – R = –H); (B) macrolactamization (site highlighted in blue – R = –H or –Cl). Protocol: (i) (a) DBU/DMF (v/v′; 1 : 99), 3 × 30 s, RT; (b) Fmoc-AA-OH (3 eq.), DIC (3 eq.), Oxyma (6 eq.), DMF, 10 min, 50 °C or for Fmoc-arylglycine 3 (compounds 11–14) (b)′ Fmoc-AA-OH (3 eq.), DIC (3 eq.), Oxyma (6 eq.), DMF, 1 h, RT; (ii) Pd(PPh₃)₄ (0.1 eq.), phenylsilane (5 eq.), TEA (0.2 eq.), DCM, 2 × 45 min, RT; (iii) cocktail K: TFA/TIPS/water (v/v′/v′′; 95 : 2.5 : 2.5), ^a TFA/DODT/TIPS/H₂O (v/v′/v′′/v′′′; 95 : 2: 2 : 1), 1 h, RT; (iv) HFIP/DCM (v/v′; 30 : 70), 3 × 30 min, RT; (v) (a) NaNO₂ (5 eq.), GnHCl pH 3, 15 min, −15 °C; (b) (4-amino)thiophenol (40 eq.), GnHCl pH 7, 15 min, −15 °C; then NaOH (1 M) until pH 7, 1 h, −15 °C to RT; (d) TCEP pH 7, 15 h, RT; (vi) VA-044, tBuSH, TCEP, GnHCl pH 7, 4 h, 37 °C; (vii) PyBOP, TEA, DCM/DMF (50 : 50), 2 h, RT. The site of macrocyclization used in total synthesis is highlighted in green. *Represents Alloc removal position and timing. Hpg residues are highlighted in grey and orange in the structure and sequence respectively.