Scheme 2. Deuterium Labeling of isoAsp and isoGlu.
In the presence of pyridine and acetic anhydride, acetylpyridinium is formed, which then reacts with the α-carboxyl group of isoX (X = Asp when n = 1; X = Glu when n = 2) to form mixed anhydride IV. The α-hydrogen (α-H) of VI in the keto form is abstracted by pyridine in step a1 to form an enolate anion represented by two resonance hybrids VII and VIII. The subsequent reaction of this enolate with deuterium (red) in D2O in step a2 gives a racemic oxazolone VIV incorporating a deuteron at the α-position. The yield of deuterium labeling is significantly enhanced by the repetitive process of generating an oxazolone VI from IsoX through step b, as long as a large excess of acetic anhydride remains in the reaction mixture. The labile amide, hydroxy, and carboxy protons (blue) being exchanged with deuterons in D2O are exchanged back to protons in H2O, but the deuterons incorporated into the α-position of isoX remain unexchanged.