Deuterium-Labeled Peptide Synthesis

[D₄]L-alanine from Cambridge Isotope Laboratories (Tewksbury, MA) was modified with an N-terminal Fmoc protecting group and was recrystallized from Ethyl acetate/hexane (80:20). Other protected amino acids and Rink amide resin, including arginine whose side chain was additionally protected with 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) protecting group, were purchased from Anaspec (Freemont, CA), Bachem (Torrance, CA), Sigma Aldrich and NovaBiochem (MillaporeSigma, Burlington, MA). Peptides were synthesized on a model 433A solid-phase peptide synthesizer (Applied Biosystems/Thermo Fisher Sci.; Foster City, CA) using a 0.1 mmol scale FastMoc chemistry. Two deuterium-labeled alanines were incorporated per peptide, with 50% and 100% isotope abundance levels, to distinguish the ²H NMR signals based on relative intensities. Following cleavage from the resin using a trifluoroacetic acid (TFA) based cocktail, the resulting C-termini amidated peptides were precipitated with methyl-tert-butyl-ether/hexane (50:50). The peptides were purified by reversed-phase high-performance liquid chromatography on a 5 μm octylsilica Zorbax SB-C8 or SB300-C3 column (9.4 mm × 250 mm) (Agilent Technologies, Santa Clara, CA), as described in ^{[32 (link), 49 (link)]}. The molecular mass of peptide and distributions of deuterium were confirmed by MALDI-TOF mass spectrometry (Figure S1).