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An improved synthesis of α‐ 13 C glycine and heteronuclear NMR studies of its incorporation into thioredoxin
Author(s) -
Wishart David S.,
Sykes Brian D.,
Richards Frederic M.
Publication year - 1992
Publication title -
journal of labelled compounds and radiopharmaceuticals
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.432
H-Index - 47
eISSN - 1099-1344
pISSN - 0362-4803
DOI - 10.1002/jlcr.2580311209
Subject(s) - heteronuclear molecule , chemistry , glycine , heteronuclear single quantum coherence spectroscopy , yield (engineering) , hydrolysis , carbon 13 nmr , cyanide , amidine , combinatorial chemistry , nuclear magnetic resonance spectroscopy , stereochemistry , biochemistry , organic chemistry , amino acid , materials science , metallurgy
We present an improved method to easily prepare gram quantities of α‐ 13 C glycine beginning from K 13 CN. The four step synthesis involves the production of an N, N‐diphenyl‐cyanoformamidine intermediate through the coupling of cyanide to N, N‐diphenylcarbodiimide. Subsequent reduction by LiAlH 4 and hydrolysis of the resulting amidine produces fully enriched α‐ 13 C labelled glycine with a 45‐50% yield. This relatively fast and simple synthesis uses only commonly available compounds and requires no special equipment, making the process easy to perform in any well equipped biochemistry laboratory. We further demonstrate that the product may be used, without extensive purification, to specifically label bacterially expressed proteins ( E. coli thioredoxin) through standard biosynthetic procedures. We also show that the 13 C glycine‐labelled protein may be readily analyzed using commonly available heteronuclear NMR techniques. Complete assignments for all 9 glycines of native E. coli thioredoxin are presented.