Methionine is one of the amino acids most prone to oxidation. In biological systems, it can be attacked by reactive oxygen species (ROS) and free radicals, such as the superoxide anion or the hydroxyl radical. During Fmoc/^tBu solid phase peptide synthesis and purification, the thioether function may also be oxidized. Exposure to atmospheric oxygen or acid-catalyzed oxidation can lead to a considerable accumulation of methionine sulfoxide Met(O) and/or methionine sulfone Met(O₂). Both processes can be analyzed via mass spectrometry showing a shift of +16 Da for Met(O), and +32 Da for Met(O₂), respectively, in the mass spectrum. Typically, the percentage of oxidized peptide also depends on the position of the methionine within the sequence. The closer it is to the C-terminus, the longer its exposure to atmospheric oxygen and thus the higher the degree of oxidation.

The first oxidation of the Methionine sulfur leads to a mixture of two diastereomers – Methionine-S-sulfoxide and Methionine-R-sulfoxide. In biological systems, this process can be reverted by enzymes of the methionine sulfoxide reductase family. This indicates that the oxidation/reduction process is a tool to regulate protein activities in vivo. The second oxidation step leads to the formation of methionine sulfone Met(O₂). Both oxidation processes might alter the protein’s structure and stability and cause aggregation and/or loss of biological function. However, there are also reports highlighting the functional activation of proteins by Met oxidation.

The use of cleavage cocktails during SPPS may help to prevent oxidation of the Methionine sulfur during TFA cleavage. Most known is Reagent H, which is based on the combination of ammonium iodide (NH₄I) and dimethyl sulfide (Me₂S, DMS). Another example is the use of tetra-n-butylammonium bromide (Bu₄NBr, TBB) in Reagent R.

Exemplary reduction mechanism of methionine sulfoxide.

Another option to prevent oxidation of Methionine is the incorporation of Norvaline (Nva) or Azidohomoalanine (Aha) as surrogate. The azide moiety of Aha can even be further selectively modified using Staudinger ligation or Click-chemistry, or be reduced to the amine. Iris Biotech offers variously protected derivatives of both Met(O) and Met(O₂), as well as of the methionine analogs Nva and Aha, which can be used for the investigation of structure activity relationships. Furthermore, L-Methionine sulfoxide acts as structural analog of glutamate and may be used as substrate to study glutamate synthase and γ-glutamyl transpeptidase.

Overview of related Iris Biotech products. Find many more Met derivatives in our webshop.

References:

• Antiinflammatory activity of methionine, methionine sulfoxide and methionine sulfone; M. K. Unnikrishnan and M. N. A. Rao; Agents and Actions 1990; 31: 110-112. https://doi.org/10.1007/BF02003229.
• Selective Oxidation of Methionine and Tryptophan Residues in a Therapeutic lgG1 Molecule; E. Folzer, K. Diepold, K. Bomans, C. Finkler, R. Schmidt, P. Bulau, J. Huwyler, H.-C. Mahler, A. V. Koulov; J. Pharm. Sci. 2015; 104: 2824-2831. https://doi.org/10.1002/jps.24509.
• Characterization of methionine oxidation and methionine sulfoxide reduction using methionine-rich cysteine-free proteins; BMC Biochem. 2012; 13. https://doi.org/10.1186/1471-2091-13-21.
• Methionine oxidation by reactive oxygen species: reaction mechanisms and relevance to Alzheimer’s disease; C. Schöneich; Biochim Biophys Acta. 2005; 17: 111-119. https://doi.org/10.1016/j.bbapap.2004.09.009.
• Convenient reduction of S-oxides in synthetic peptides, lipopeptides and peptide libraries; W. Beck and G. Jung; Lett. Pept. Sci. 1994; 1: 31-37. https://doi.org/10.1007/BF00132760.
• Stability of Methionyl Residues towards Oxidation during Solid Phase Peptide Synthesis; K. Norris, J. Halstrøm, K. Brunfeldt; Acta Chem. Scand. 1971; 25: 945-954. https://doi.org/10.3891/acta.chem.scand.25-0945.
• Oxidation of methionyl residues in proteins: Tools, targets, and reversal; W. Vogt; Free Radic. Biol. Med. 1995; 18: 93-105. https://doi.org/10.1016/0891-5849(94)00158-G.