IMPROVED SYNTHETIC METHODS FOR THE SELECTIVE DEUTERATION OF AROMATIC-AMINO-ACIDS - APPLICATIONS OF SELECTIVE PROTONATION TOWARDS THE IDENTIFICATION OF PROTEIN-FOLDING INTERMEDIATES THROUGH NUCLEAR-MAGNETIC-RESONANCE

In this report we describe several novel methods for the preparation o f selectively deuterated aromatic amino acids. New syntheses for [2,3, 5,6-H-2(4)]phenylalanine and [2,4,6,7-H-2(4)]tryptophan, as well as im proved catalytic exchange methods for [2,3,5,6-H-2(4)]tyrosine and [2, 3,4,5,6-H-2(5)]phenylalanine are presented. Isotopic substitution leve ls for all compounds are generally found to be greater than 95%. Biosy nthetic incorporation of these amino acids is also shown to be possibl e with little or no evidence of isotopic scrambling. The products from these new syntheses, in combination with other selectively deuterated aromatic amino acids, are found to permit group-specific 'single-prot on' labelling of proteins. This highly-efficient and very cost-effecti ve method of selective protonation is shown to produce greatly simplif ied H-1-NMR spectra of the aromatic region of proteins. The utility of this approach to isotopic editing is demonstrated with the identifica tion of a transient folding intermediate of Escherichia coli thioredox in which is undetectable by standard 2-D NMR techniques.