An improved synthesis of α-13C glycine and heteronuclear NMR studies of its incorporation into thioredoxin

✍ Scribed by David S. Wishart; Brian D. Sykes; Frederic M. Richards

• Publisher: John Wiley and Sons
• Year: 1992
• Tongue: French
• Weight: 502 KB
• Volume: 31
• Category: Article
• ISSN: 0022-2135
• DOI: 10.1002/jlcr.2580311209

✦ Synopsis

Abstract

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.