Amino acids methylthiohydantoins

Amino acid Methylthiohydantoin derivatives Phenylthiohydantoin derivatives ... 

Fig. 5.20. GC separation of TMS-methylthiohydantoins of amino acids. Conditions borosilicate-glass column, 165 cm X 4 mm I.D., 2% (w/w) OV-17 on Gas-Chrom Q (80-100 mesh) nitrogen flow-rate, 50 ml/min temperature programme as indicated. (Reproduced from Anal. Biochem., 58 (1974) 549, by courtesy of Academic Press.)...

Lamkin et al. [276] studied in detail the GC analysis of silylated methylthiohydantoins of all protein amino acids. They effected the silylation with BSA-acetonitrile (1 3) at 100°C for 10 min. They separated the products in a simple column packed with 2% of OV-17 on Gas-Chrom Q at 145—230°C, and Fig. 5.20 illustrates the results. The authors used a flame photometric detector, sensitive to sulphur-containing compounds, in order to ensure sensitive and selective detection. Minor incidental peaks that were often noticed during the analysis of the samples obtained by the Edman degradation of proteins with the use of an FID did not appear and the peak of the solvent was not detected. The baseline stability was good and the response was linear over a range of two orders of magnitude of concentration. Asn and Phe were the only unresolved pair Arg, as in previous instances, did not form a volatile derivative. 

The sequencing methods and determination of C-terminal and N-terminal amino acids are now widely used in biochemical research. The identification and quantitation of the characteristic degradation products can be accomplished by the gas-phase analytical methods. Thus, GC of both dinitrophenyl and various hydantoin amino acid derivatives has now been widely documented. Separation of thiohydantoins [244,245], phenylthiohydantoins [490,491] and methylthiohydantoins [492] generally requires additional silylation for the sake of volatility. Furthermore, acyl derivatives of similar substances have also been reported [493,494]. The most obvious advantage of GC for determination of the Edman degradation products is sensitivity which is particularly important in the sequence analysis of only minute amounts of proteins and peptide hormones. 

Racemic dinitropyridyl, dinitrophenyl, and dinitrobenzoyl amino acids were resolved on C-18W/UV TLC and HPTLC plates (Macherey-Nagel) developed with 2% aqueous isopropanol containing 2-5% BSA. Development times were 1-2 h, and visualization was under 254 nm UV hght. Ri differences ranged from 0.06 to 0.49 [34]. The same plates with mobile phases composed of isopropanol -E BSA-E sodium tetraborate, acetic acid, or sodium carbonate served to separate enantiomeric D,L-methylthiohydantoin andphenylthiohydantoin derivatives of amino acids, kynureyne, 3-(l-naphthyl)alanine, lactic acid derivatives, alanine and leucine p-nitroanilides, and 2,2,2-trifluoro-l-(9-anthryl)ethanol [35], and with mobile phases composed of water with 5-7% BSA -E 2% isopropanol to separate dansyl amino acid derivatives [36]. 

Bearing in mind the considerations outlined above, the lack of resolution of methylthiohydantoin-DL-leucine (MTH-DL-Leu) could be attributed to the absence of an aromatic ring in the side chain of the amino acid. This behavior is common to all the methylthiohydantoin derivatives of aliphatic amino acids. 

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