Vl. Boyd et al., ACTIVATION OF THE CARBOXY-TERMINUS OF A PEPTIDE FOR CARBOXY-TERMINAL SEQUENCING, Journal of organic chemistry, 60(8), 1995, pp. 2581-2587
Previously, we reported [Anal. Biochem. 1992, 206, 344-352] a new meth
od of sequencing proteins from the carboxy terminus (C-terminus). The
carboxyl group at the C-terminus is activated and derivatized into a t
hiohydantoin (TH). We reported that, by alkylating the TH formed at th
e C-terminus, the TH is converted into a readily displaced leaving gro
up. Reaction with {NCS}(-) under acidic conditions cleaves the alkylat
ed thiohydantoin (ATH) and derivatizes the freshly exposed C-terminus
into a new proteinyl-TH. The efficiency of the initial activation of t
he carboxy group at the C-terminus is critical to the initial yield of
the first ATH residue. In order to directly observe the intermediates
that form during activation of the C-terminus, a model tripeptide, ac
etylalanine-alanine-alanine-OH (Ac-Ala-Ala-Ala-OH) was subjected to th
e reagents used to form the peptidyl-TH, Ac-Ala-Ala-Ala-TH. The reacti
on was monitored by nuclear magnetic resonance spectroscopy. An oxazol
one was observed to form immediately at the C-terminus during the reac
tion with diphenyl chlorophosphate (DPCP), tetraphenyl pyrophosphate (
TPPP), or tetramethylchlorouronium chloride (TMU-Cl). The oxazolone wa
s observed to react with an excess of the carboxy group-activating rea
gents while under basic conditions. Diketopiperazine formation at the
C-terminus was also observed. These side reactions prevent or retard t
he reaction of (NCS)- to form a peptidyl-TH and correlate with a reduc
ed initial yield observed during automated C-terminal protein sequenci
ng. The carboxylic acid-reactive reagents react with the side-chain ca
rboxylic acid groups of aspartic and glutamic acid residues as well as
the C-terminus. We found that the sidechain carboxylic acid groups in
a protein could be selectively amidated in the presence of the protei
nyl-oxazolone at the C-terminus.