*3.4. Marfey's Analysis of Talarolides A–D*

3.4.1. Standard Marfey's Hydrolysis and Derivatization Method #1

A sample analyte (50 μg) in 6 M HCl (100 μL) was heated to 100 ◦C in a sealed vial for 12 h, after which the hydrolysate was concentrated to dryness at 40 ◦C under a stream of dry N2. The hydrolysate was then treated with 1 M NaHCO3 (20 μL) and L-FDAA (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide) or D-FDAA (1-fluoro-2,4-dinitrophenyl-5-D-alanine amide) as a 1% (*w*/*v*) solution in acetone (40 μL) at 40 ◦C for 1 h, after which the reaction was neutralized with 1 M HCl (20 μL), diluted with MeCN (200 μL) and filtered (0.45 μm PTFE) prior to analysis.

#### 3.4.2. Standard Marfey's HPLC Method #2

An aliquot of Marfey's derivatized analyte (3 μL) (see method #1) was subjected to HPLC-DAD-MS analysis using a binary solvent system (Phase A: 95% H2O: 5% MeCN: 0.1% formic acid; Phase B: 95% MeOH: 5% MeCN: 0.1% formic acid) on an Agilent Poroshell 120 SB-C8 2.7 μm, 3.0 × 150 mm column, at 50 ◦C with a 0.8 mL/min linear gradient over 29 min from 16% to 63% Phase B in A, and with DAD (340 nm) and ESI(±)MS monitoring, supported by single ion extraction (SIE) methodology, and with comparison to authentic standards of Marfey's derivatized amino acids.

### 3.4.3. Marfey's HPLC Method #3 Optimized for Resolving N-Me-Ala Derivatives

An aliquot of Marfey's derivatized analyte (3 μL) (see method #1) was subjected to UPLC-DAD-MS analysis using the same binary solvent system and detection as described above (method #2), but with an isocratic 0.8 mL/min elution at 23% Phase B in A using an Agilent Poroshell 120 EC-C18 2.7 μm, 3.0 × 150 mm column at 50 ◦, with comparison to authentic standards of Marfey's derivatized amino acids (Figures 3 and S38).

#### 3.4.4. Marfey's HPLC Method #4 Optimized for Resolving Leu, Ile and *allo*-Ile Derivatives

An aliquot of Marfey's derivatized analyte (3 μL) (see method #1) was subjected to UPLC-DAD-MS analysis using the same binary solvent system and detection as described above (method #2), but with an isocratic 0.8 mL/min elution at 37% Phase B in A, with comparison to authentic standards of Marfey's derivatized amino acids (Figures 4 and S39).
