*3.7. Three-Dimensional Solution Structure Calculations*

The distance restraints used in calculating the structure for talarolide A (**1**) in DMSO-*d*<sup>6</sup> were derived from ROESY spectra (recorded at 298 K) using mixing time (spin-lock) of 300 ms with 41 NOEs (see Supplementary Materials). NOE cross-peak volumes were classified manually as strong (upper distance constraint ≤ 2.7Å), medium (≤3.5Å), weak (≤5.0Å), or very weak (≤6.0Å). Standard pseudoatom distance corrections were applied for nonstereospecifically assigned protons. To address the possibility of conformational averaging, intensities were classified conservatively and only upper distance limits were included in the calculations to allow the largest possible number of conformers to fit the experimental data. Backbone dihedral angle restraints were inferred from <sup>3</sup>*J*NHCH<sup>α</sup> coupling constants in 1D spectra, using the Karplus equation [15] with angle ± 30◦. There was one *cis*-amides bond between *N*-Me-L-Ala6-*N*-Me-L-Tyr<sup>7</sup> (i.e., strong CHα-CHα (*i*, *i+1*) NOEs); the rest were in *trans* configuration (ψ-angles were set to *trans* (ψ = 180◦)). Starting structures with randomized φ and ψ angles and extended side chains were generated using an ab initio simulated annealing protocol. The calculations were performed using the standard forcefield parameter set (PARALLHDG5.2.PRO) and topology file (TOPALLHDG5.2.PRO) in XPLOR-NIH with in-house modifications to generated N-methylated and *N*-hydroxylated residues. Refinement of structures was achieved using the conjugate gradient Powell algorithm with 4000 cycles of energy minimization and a refined forcefield based on the program CHARMM [12]. Structures were visualized with Pymol and analyzed for distance (>0.2Å) and dihedral angle (>2◦) violations using noe.inp files. 1H NMR (DMSO-*d*6) variable temperatures for **1** was obtained from 298 K to 318 K in five degrees stepwise on a Bruker Avance III 600 MHz NMR spectrometer. The chemical shift differences of amide NH and *N*-OH were plotted against temperature to generate the temperature coefficient (Δδ/T) using Prism version 10.0.2 (Figure 8).
