Preloaded fmoc protected wang resin

All natural and uniformly labeled ¹³C and ¹⁵N amino acids were purchased from Sigma Aldrich (St. Louis, MO). Preloaded Fmoc-protected Wang resin was purchased from EMD Millipore (Billerica, MA). All other reagents were purchased from Sigma Aldrich (St. Louis, MO) and used without purification. Peptides were synthesized on a CEM Liberty Blue peptide synthesizer using a standard 9-fluorenylmethoxycarbonyl (FMOC) and tert-butyl protection scheme. Preloaded Fmoc-protected Wang resin was used for solid phase synthesis. Peptides were cleaved from the resin in a 10 mL solution of 95:2.5:2.5 trifluoroacetic acid: triisopropylsilane (TRIS): water mixture per 1.0 gram of peptide/resin. The resulting filtrate was added dropwise into cold tert-butyl methyl ether, followed by centrifugation and three rinses of the resulting solids with 40 mL of cold tert-butyl methyl ether. Peptides were purified using RP-HPLC (Varian ProStar HPLC, Alltima WP C4 column, 5 mL/min, eluent A: water with 0.2% TFA, eluent B: acetonitrile with 0.2% TFA), using a gradient of 15–50% B over 40 minutes. Chromatograms were generated by observing the UV absorbance at 254 nm, and the analyte was verified by mass spectrometry. The fractions were then lyophilized, resulting in the pure peptide.

All natural and uniformly labeled ¹³C and ¹⁵N amino acids were purchased from Sigma Aldrich (St. Louis, MO). Preloaded Fmoc-protected Wang resin was purchased from EMD Millipore (Billerica, MA). All other reagents were purchased from Sigma Aldrich (St. Louis, MO) and used without purification.