Zorbax sb c8

[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).

Aliquots of 1–4 (0.1 mg) were dissolved in either 0.1% TFA in MeCN (0.5 mL) or 0.1% TFA in MeOH (0.5 mL) and heated at 40 °C for 24 h, after which the solutions were analysed by HPLC-DAD-MS (Agilent Zorbax SB-C₈, 5 μm, 4.6 × 150 mm column, 15 min gradient elution at 1 mL min⁻¹ from 90% H₂O–MeCN to 100% MeCN with isocratic 0.05% formic acid modifier). Samples of 1–4 (0.1 mg) dissolved in MeOH (1.0 mL) were used as authentic standards, and analysed by the same HPLC method. Analytical results, as illustrated in the ESI Fig. S12–S14,^† demonstrate that whereas 1 and 4 were stable under these conditions, 2 and 3 equilibrate, and transform through 5 and 6, to 7 and 8, and finally to 4.