3.4.5. Marfey's Analysis of Talarolides A–D (**1**–**4**)

Samples of talarolides A–D (**1**–**4**) (50 μg) were subjected to standard Marfey's hydrolysis and derivatization (method #1), after which individual aliquots of Marfey's derivatized analytes (3 μL) were subjected to each of methods #2, #3, and #4 to unambiguously identify the following amino acid constituents:

Talarolide A (**1**): L-Ala, *N*-Me-D-Leu, D-*allo*-Ile, D-Ala, *N*-Me-L-Ala, *N*-Me-L-Tyr (see Figures 3, 4 and S34)

Talarolide B (**2**): Gly, L-Ala, *N*-Me-D-Leu, D-*allo*-Ile, D-Ala, *N*-Me-L-Ala, *N*-Me-L-Tyr (see Figure S35)

Talarolide C (**3**): L-Ala, *N*-Me-D-Leu, D-Val, D-Ala, *N*-Me-L-Ala, *N*-Me-L-Tyr (see Figure S36)

Talarolide D (**4**): L-Ala, *N*-Me-D-Leu, D-*allo*-Ile, D-Ala, L-Ala, *N*-Me-L-Tyr (see Figure S37)

## *3.5. Two-Dimensional Marfey's Analysis of Talarolide B*

3.5.1. Two-Dimensional Marfey's Method #5 Partial Hydrolysis of Talarolide B

A sample of talarolide B (**2**) (50 μg) was subjected to the standard Marfey's hydrolysis conditions (method #1) but with a reduced reaction time from 12 to 3 h, and after derivatization yielded a dipeptide attributed to L-FDAA-D-*allo*-Ile-Ala (unspecified Ala configuration) based on an HPLC-DAD-MS (Marfey's method #2) [*t*<sup>R</sup> 22.3 min, *m*/*z* 455 (M + H)] (Figure S40).

#### 3.5.2. Synthesis of Dipeptides L-FDAA-D-*allo*-Ile-L-Ala and L-FDAA-D-*allo*-Ile-D-Ala

Syntheses were performed using standard peptide synthesis on a 2-chlorotrityl chloride (2-CTC) resin (substitution ratio: 1.55 mmol/g, 0.1 mmol scale, 64.5 mg) using hexafluorophosphate azabenzotriazole tetramethyl uranium (HATU) and *N,N*-diisopropylethylamine (DIPEA) coupling, and fluorenylmethoxycarbonyl (Fmoc) protection chemistry.
