*3.5. Determination of the Absolute Configuration of Wenchangamide A (1)*

Wenchangamide A (**1**, 1.0 mg) was treated with 6 M HCl (100 μL) for 24 h at 110 ◦C. The hydrolyzed product was evaporated to dryness for purification of the individual structural components. Using HPLC separation and a Cosmosil 5C18-PAQ column [(φ4.6 × 250 mm); flow rate, 1.0 mL/min; UV detection at 215 nm; solvent H2O], the components; Ser (*t*<sup>R</sup> = 2.6), Leu and *N*-Me-Ile (*t*<sup>R</sup> = 5.2), and *N*-Me-Phe (*t*<sup>R</sup> = 12.3) were collected. In another HPLC separation using the Cosmosil 5C18-PAQ column [(φ4.6 × 250 mm); flow rate, 1.0 mL/min; UV detection at 215 nm; solvent 0.1% aqueous TFA], *N*-Me-Glu (*t*<sup>R</sup> = 3.4) was obtained. In a final HPLC separation using the Cosmosil 5C18-PAQ column [(φ4.6 × 250 mm); flow rate, 1.0 mL/min; UV detection at 215 nm; solvent 40% aqueous MeOH], Amp (*t*<sup>R</sup> = 3.7) was collected.

Each hydrolyzed fraction, except for Amp, was dissolved in H2O and analyzed by chiral HPLC, and the retention times were compared to those of authentic standards. For this analysis, a DAICEL CHIRALPAK MA(+) column [(φ4.6 × 50 mm); flow rate 1 mL/min; detection, UV 254 nm; solvent 2.0 mM CuSO4, 2.0 mM CuSO4/MeCN = 95/5] was used. With 2.0 mM CuSO4 as a solvent, the retention times of *N*-Me-Glu and Leu hydrolyzed from **1** matched those of the authentic standards of *N*-Me-L-Glu (20.2 min; *N*-Me-D-Glu, 18.6 min), D-Leu (9.6 min) and L-Leu (18.7 min). With 2.0 mM CuSO4/MeCN = 95/5 as a solvent, the retention time of *N*-Me-Phe hydrolyzed from **1** matched that of the authentic standard of *N*-Me-L-Phe (19.8 min; *N*-Me-D-Phe, 16.5 min). Increased resolution was required for the Ser residue, and a series of two DAICEL CHIRALPAK MA(+) columns [(φ4.6 × 50 mm); flow rate 1 mL/min; detection, UV 254 nm; solvent 2.0 mM CuSO4] was used. The retention time of Ser hydrolyzed from **1** matched those of the authentic standards of D-Ser (2.5 min; L-Ser, 3.2 min).

For analysis of the Amp and *N*-Me-Ile residues, Marfey's method was used to clarify the absolute configurations. To each isolated residue was added 1.0% L-FDLA acetone sol. (100 μL) and 1 M NaHCO3 (25 μL). The mixtures were heated at 80 ◦C for 3 min,

cooled to room temperature, and neutralized with 1 M HCl. The products were analyzed by HPLC and the retention time was compared with those of authentic standards. A Cosmosil Cholester column [(φ4.6 × 250 mm); flow rate 1 mL/min; detection, UV 340 nm; solvent MeCN/H2O/TFA = 70/30/0.1] was used to evaluate the Amp derivatives. The retention time of Amp-L-FDLA from hydrolysate of **1** matched that of the authentic standard (*S*)-Amp-L-FDLA (t*<sup>R</sup>* = 4.9 min; (*S*)-Amp-D-FDLA, 5.6 min). A Cosmosil PBr column [(4.6 × 250 mm); flow rate 1 mL/min; detection, UV 340 nm; solvent MeCN/H2O/TFA = 55/45/0.1] was used to evaluate the *N*-Me-Ile derivatives. The retention time of *N*-Me-Ile-L-FDLA from hydrolysate of **1** matched that of the authentic standard *N*-Me-D-*allo*-Ile-L-FDLA (t*<sup>R</sup>* = 17.8 min; *N*-Me-L-Ile-L-FDLA 14.2 min; *N*-Me-D-Ile-L-FDLA 17.3min; *N*-Me-D-*allo*-Ile-D-FDLA 14.7 min).
