3.6.4. Cyclization of Linear Protected Peptide

A solution of the linear protected peptide (0.5 mg/mL in DMF (128 mL, 64 mg, 0.082 mmol) was stirred vigorously and treated by dropwise addition over 30 min with a mixture of 0.4 M HATU (618 μL, 0.247 mmol, 3 eq.), hydroxybenzotriazole (HOBT) (34 mg, 0.247 mmol, 3 eq.) and collidine (33 μL, 30 mg, 0.247mmol, 3 eq.) in DMF (2 mL). After 14 h, HPLC-DAD-MS analysis of the mixture showed the cyclization was completed. The DMF was evaporated, and the residue dissolved in MeCN (10 mL), filtered (0.45 μm filter), and purified by preparative HPLC (Agilent Zorbax Rx-C8 7 μm, 21.2 × 250 mm column, with a 20 min gradient elution at 20 mL/min from 90% H2O/MeCN to 100% MeCN with an isocratic 0.01% trifluoroacetic acid/MeCN modifier). After lyophilization, the protected cyclic peptide was obtained as an amorphous powder. The product was confirmed by HPLC-DAD-MS (method as described in general experimental section): *t*<sup>R</sup> = 5.8 min, ESI(+)MS *m*/*z* 758.5 [M + H]+

#### 3.6.5. Cyclic Peptide Deprotection

The protected cyclic peptide was mixed with an aqueous solution of 90% formic acid (3 mL) for 40 min, after which it was concentrated under a stream of nitrogen gas and the residue dissolved in MeCN (2 mL) and purified by preparative HPLC (Agilent Zorbax Rx-C8 7 μm, 21.2 × 250 mm column, with a 20 min gradient elution at 20 mL/min from 90% H2O/MeCN to 100% MeCN with an isocratic 0.01% trifluoroacetic acid/MeCN modifier) to yield synthetic talarolide B (16 mg, 23% overall yield); NMR (DMSO-*d*6), see Figures S50–S52; HRESI(+)MS *m*/*z* 724.4008 [M + Na]+ (calcd for C35H55N7O8Na 724.4004); identical with natural talarolide B (**2**), including by co-injection HPLC-DAD-MS (Figure S48).
