Characterization of a three-component coupling reaction on proteins by isotopic labeling and nuclear magnetic resonance spectroscopy.
A three-component Mannich-type electrophilic aromatic substitution reaction was previously developed to target the phenolic side chain of tyrosine residues on proteins. This reaction proceeds under mild conditions and provides a convenient alternative to lysine-targeting strategies. However, the use of reactive aldehydes, such as formaldehyde, warrants careful inspection of the reaction products to ensure that other modifications have not occurred. Through the use of isotopically enriched reagents, nuclear magnetic resonance (NMR)-based studies were used to obtain structural confirmation of the tyrosine-modification products. These experiments also revealed the formation of a reaction byproduct arising from the indole ring of tryptophan residues. Cysteine residues were shown to not participate in the reaction, except in the case of a reduced disulfide, which formed a dithioacetal. We anticipate that this analysis method will prove useful for the detailed study of a number of bioconjugation reactions.
McFarland
Jesse M
JM
Department of Chemistry, University of California, Berkeley, California 94720-1460, USA.
Joshi
Neel S
NS
Francis
Matthew B
MB
D002250:Carbon Radioisotopes / Q000737:chemistry; D002919:Chymotrypsinogen / Q000737:chemistry; D003545:Cysteine / Q000032:analysis; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D007553:Isotope Labeling; D009113:Muramidase / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010206:Papain / Q000737:chemistry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D013879:Thioredoxins / Q000737:chemistry; D014364:Tryptophan / Q000032:analysis*; D014443:Tyrosine / Q000032:analysis*
D002250:Carbon Radioisotopes; D011506:Proteins; D005557:Formaldehyde; D014443:Tyrosine; D013879:Thioredoxins; D014364:Tryptophan; D002919:Chymotrypsinogen; D009113:Muramidase; D010206:Papain; D003545:Cysteine