TLC of Phenylthiohydantoins of Amino Acids: A Review

Abstract

Proteins are among the most important components of all living systems. Their function range from catalysts 9enzymes) to regulators to structural components. The building blocks and language of proteins are about 20 amino acids (H₂N CHR COOH), linked together By peptide bonds (sbnd]COsbnd]NHsbnd]) in chains that may consist of a few dozen to more than 1000 amino acides. The determination of primary structure of proteins, namely the sequence (arrangement) of the various amino acids along the chain is still a challenging tast. Edman reaction lies virtually at the core of all modern sequencing strategies 1,2]. The N-terminal polypeptide is first coupled to phenyl isothiocyanate to from the phenylthio carbamyl peptide; this derivative is then cleaved with anhydrous acid to expose a new N-terminus and to release the original N-terminal amino acid as a 5′thiazolinone (Scheme-1). The excess reagents and by products are extracted by an organic solvent wash. The extract of thiazolinone amino acid (obtained either from liquid-phase or solid-phase degration) is evaporated and converted to the phenylhtiohydantion derivative by 5 n HCL/CH₃COOH (1:2 v/v) at 52°C for 50 min. The sample is extracted with ethyl acetate, dried and redissolved in a suitable volume of ethanol for TLC identification. Repetition of this process with identification of the released PTH-amino acidsfro the N-terminal end. For smaller peptides PITC may be used to remove the amino terminal amino acid while a chromphore or fluorophore such as dansyl chloride or DABITC, which react with the newly exposed amino terminus, is used to identify the new amino terminus. Both manual and automated methodologies are currently used for small and large polypeptides which rely upon identification of amino terminal amino acid as PTH derivative. A large number of papers have been and continue to be publihsed on the analysis of the PTH derivatives of amino acides.