Synthesis of peptides with cysteine sulfinic acid via the cysteine methoxybenzyl sulfone

Abstract

Cysteine sulfinic acid is a protein posttranslational modification that is formed under oxidative conditions and is regulated both enzymatically and nonenzymatically. Cysteine oxidation to the sulfinic acid has been observed broadly throughout the proteome and can induce activation or inhibition of function in proteins. Recently, wide‐scale, reversible regulation of the sulfinic acid state of cysteine within proteins was identified, posing new questions in cysteine sulfinic acid biology. Existing methods to synthesize peptides with cysteine sulfinic acid can suffer from low yield, due to the formation of side products in the disulfide, sulfenic acid, and/or sulfonic acid oxidation states. Herein, a method for the synthesis of peptides with cysteine sulfinic acids was developed, via protection of cysteine sulfinic acid as the methoxybenzyl (Mob) sulfone. Cysteine Mob sulfone was synthesized as an Fmoc amino acid in one step from the commercially available Mob‐protected Fmoc‐cysteine (Fmoc‐Cys(Mob)‐OH). This amino acid was directly incorporated into peptides via solid‐phase peptide synthesis. Alternatively, peptides were synthesized using Fmoc‐Cys(Mob)‐OH, followed by subsequent oxidation within peptides of the thioether to the Mob sulfone via H2O2 and catalytic niobium carbide. Deprotection of peptides under strongly acidic conditions (50% triflic acid, 45% trifluoroacetic acid, 5% water) generated peptides with cysteine sulfinic acid. This approach was applied to the synthesis of peptides containing cysteine sulfinic acid within diverse peptide sequence contexts.

Synthesis and conformational preferences of peptides and proteins with cysteine sulfonic acid

“…We previously developed a method for the high-yield synthesis of peptides containing cysteine sulfinic acid using the commercially available methoxybenzyl (Mob)-protected Fmoc-cysteine. 44 In that work, the protected amino acid was oxidized in solution to the sulfone, and then incorporated into peptides via standard solid-phase peptide synthesis. After resin cleavage and side chain deprotection, the peptide containing the cysteine Mob sulfone was then purified and subjected to strong-acid-mediated Mob deprotection.…”

“…HPLC analysis was conducted using an analytical C18 column with a linear gradient of 0-25% buffer B (20% H 2 O, 80% CH 3 CN, and 0.05% TFA) in buffer A (98% H 2 O, 2% CH 3 CN, and 0.06% TFA) over 60 minutes. Peak broadening has previously been observed for peptides in the Ac-GPPXPPGY-NH 2 context, including in some peptides the presence of multiple peaks of the same molecular weight that are in equilibrium, apparently due to proline cis-trans isomerization at the four X-Pro amide bonds 44,56,58,59.…”

“…For example, in our work to understand the structural effects of cysteine oxidation to the sulfinic acid and to develop fluorescent sensors of the oxidation to the sulfinate, 36,44,45 we observed that subjection of a peptide containing cysteine to oxidation with any of H 2 O 2 , NaOCl, or peroxynitrite yielded a mixture of products in various cysteine oxidation states, with the major product (usually the sulfinic acid, but the results were dependent on the local peptide sequence) typically obtained in less than 50% yield. In peptides that lack oxidation-sensitive functional groups, iron-oxo chemistry (generated in situ from Fe(II) and H 2 O 2 ) has been employed in superstoichiometric quantities to generate the cysteine sulfonate (Fig.…”

“…Peak broadening has previously been observed for peptides in the Ac-GPPXPPGY-NH 2 context, including in some peptides the presence of multiple peaks of the same molecular weight that are in equilibrium, apparently due to proline cis-trans isomerization at the four X-Pro amide bonds. 44,56,58,59 Fig. 4 Solution-phase oxidation of an α-helical model peptide with MeReO 3 /H 2 O 2 .…”

Precise size-control and functionalization of gold nanoparticles synthesized by plasma–liquid interactions: using carboxylic, amino, and thiol ligands

“…In addition, the available H 2 O 2 also oxidized L-cysteine into cysteine sulfinic acid or sulfonic acid, with the peak of around 168 eV (see Fig. 3)a 43 . These acids can adsorb on the surface of GNPs via the bond NH 2 -Au, see Glu molecules in aqueous solutions are present in both pyran-ring and straight-chain forms (see in Fig.…”