Phenyl thiohydantoins

The combined ether extract (which may be washed with 0.01 N HCl to remove peptide material) is evaporated to dryness with a stream of nitrogen and 50 /il of 1 N HCl is added. After mixing, the thiazolinone derivative of the NH2 terminal residue is converted to the phenyl-thiohydantoin by incubation at 80°C (temperature block) for 10 min. If Asx or Glx residues are expected, 80°C for 3 min may give better results and for threonine, proline and serine, 50°C for 10 min may be better (Li and Yanofsky 1972). After cooling, the phenylthiohydantoin is extracted into ethyl acetate (100-200 /rl for each of 3 extractions), which is then evaporated with a stream of nitrogen. The phenylthio-hydantoins of arginine, histidine and cysteic acid will usually remain in the aqueous phase and may be recovered by lyophilization and dissolving the residue in methanol. 

Sjoquist, J., Paper strip identification of phenyl thiohydantoins. Acta Chem. Scand. 7, 447-448 (1953). 

S30a. Sjoquist, J., Determination of amino acids as phenyl thiohydantoin derivatives. III. Quantitative determination of 3-phenyl-2-thiohydantoins from paper chromatograms. Biochim. Biophys. Acta 41, 20-30 (1960). 

Smith, G. F., and Murray, M., Direct spectrophotometric quantitation of phenyl-thiohydantoin derivatives of amino acids from thin layers of silica gel. Anal. Biochem. 23, 183-195 (1968). 

Sun and Lovins [212] studied the elimination of neutral fragments, obtained from amino acids liberated during the Edman degradation, which are transformed into derivatives of methyl- (or phenyl-) thiohydantoin (Fig. 59). 

Figure 59. Production of methyl (or phenyl) thiohydantoin derivatives from polypeptides.

A typical example of single-stage derivatization of amino acids is based on their reaction with methyl or phenyl isothiocyanates with the formation of 3-methyl (phenyl) thiohydantoins. The optimal analytical method for the analysis of semivolatile organic compounds of this class is RP HPLC, but derivatives of the simplest amino acids can be objects of GC analysis as well (Fig. 3). 

When peptide 179 reacts with 186, the isothiocyanate reacts with the amide unit (at pH 8-9) of the alanine residue to give 187. This is not isolated, however, because the sulfur atom attracts the amide carbonyl to release the phenyl-thiohydantoin derivative of alanine (188) and the original peptide minus the alanine residue (see 189), which is a new N-terminus. The phenylthiohydantoin is soluble in organic solvents, so it is easily removed from the peptide and can be identified using various techniques. 

Amino acids, and phenyl thiohydantoin (PTH) derivatized amino acids D- and... 

Bhushan, R. and Ali, I., Resolution of enantiomeric mixtures of phenyl-thiohydantoin amino acids on (-l-)-tartaric acid-impregnated silica gel, J. Chromatogr., 392, 460-463, 1987. 

Mass spectral fragmentation patterns of alkyl and phenyl hydantoins have been investigated by means of labeling techniques (28—30), and similar studies have also been carried out for thiohydantoins (31,32). In all cases, breakdown of the hydantoin ring occurs by a-ftssion at C-4 with concomitant loss of carbon monoxide and an isocyanate molecule. In the case of aryl derivatives, the ease of formation of Ar—NCO is related to the electronic properties of the aryl ring substituents (33). Mass spectrometry has been used for identification of the phenylthiohydantoin derivatives formed from amino acids during peptide sequence determination by the Edman method (34). 

Free amino acids can be derivatized with isothiocyanates to phenyl- or methyl-thiohydantoin derivatives. The thiohydantoins can be separated on a CSP with poly-[Af-acryloyl-L-phenylalanine ethylester] (Chiraspher ) as a chiral selector [25]. This CSP offers a known selectivity for many five-membered heterocyclic rings. 

A study was made of RP-HPLC with constant-potential (1.2 V vs SCE) and pulsed-potential amperometric detection using platinum or gold electrodes, of the derivatives of the common amino acids, obtained from phenyl and methyl isothiocyanates. All the thiohydantoins (98) were oxidized at both electrodes LOD was less than 0.2 pM for lysine and glycine, for 50 pL injection268. 

The thiohydantoin derivatives of amino acids obtained from 4-(4-dimethyaminophenyl-azo)phenyl isothiocyanate (141) and fluorescein isothiocyanate (133) can be separated by CZE. Lowering the absolute detection limits of thiohydantoin derivatives of the amino acids is a basic requirement for the development of highly sensitive protein sequencer based on Edman-like processes. Thus, the absolute LOD of thiohydantoin derivatives are at present of the order of 1CT16 mol for 141 and 10-21 mol for 133331. 

PTH = 3-phenyl-2-thiohydantoin DMAA = dimethylallylamine CNBr = cyanogen bromide... 

A powerful method of sequencing a peptide from the TV-terminal end is the Edman degradation in which phenyl isothiocyanate, C6HsN=C=S, reacts selectively with the terminal amino acid under mildly basic conditions. If the reaction mixture is then acidified, the terminal amino acid is cleaved from the peptide as a cyclic thiohydantoin, 8 ... 

In spite of its toxicity, derivatives of phenyl mercuric acetate having substituents such as thiohydantoins in the aromatic ring are still being studied as fungicides and bactericides.144"147... 

A wider range of possibilities of preparing cyclic derivatives is offered by the presence of carboxyl and a-amino groups in the molecule. Substituted 5-oxazolinone (Scheme 4.23) is prepared by refluxing with TFA anhydride, and substituted 5-oxazolidinone (Scheme 4.24)by reaction with (halogenated) acetone [117,118]. Thiohydantoins are formed by reaction with isothiocyanate (Scheme 4.25). If R is methyl or phenyl, then the corresponding methyl- or phenylthiohydantoin is produced. Thiohydantoins are usually not volatile enough and for the purposes of GC analyses must be further modified, e.g., by trimethylsilylation [119,120]. 

Many N-amino add derivatives have been proposed to this purpose and the most commonly studied by TLC are 2,4-dinitrophenyl (DNP)- and 5-dimethylamino-l-naphthalene-sulfonyl (Dansyl,Dns)-amino adds, and 3-phenyl-2-thiohydantoins (PTH-amino adds). 

The 3-phenyl-2-thiohydantoins can be separated and identified by TLC on silica gel. If the latter contains a fluorophore, the thiohydantoins quench the fluorescence... 

Obviously, incomplete reaction and losses during manipulation prevent the yield of 3-phenyl-2-thiohydantoin from reaching 100%. With each cycle of the Edman method, the yield of product derived from the newly exposed TV-terminus will decrease. In addition, small amounts of the 3-phenyl-2-thiohydantoins corresponding to earlier positions in the sequence will be formed as a consequence of incomplete reaction at each cycle. If the fraction of peptide that reacts with phenyl isothiocyanate and gives the relevant 3-phenyl-2-thiohydantoin is x, then the yield at any stage is... 

Unfortunately, the 3-phenyl-2-thiohydantoins formed in the Edman stepwise degradation suffer racemisation (Davies and Mohammed, 1984) so that the method cannot be used to determine the configuration of amino acids in a peptide. This is not usually a serious limitation, but enantiomerisation is a perpetual hazard in peptide synthesis (Chapter 7). It is therefore desirable to determine if enantiomerisation has occurred at any residue. Such information could be important, for example, in dating bone proteins obtained in archaeological excavations. Examination of the chiral purity of the amino acids in a total acid hydrolysate is not satisfactory. 

B23. Brenner, M., Niederwieser, A., and Pataki, G., Thin layer chromatography of amino acid derivatives using Kieseigel G. N-(2,4-dinitrophenyl)-amino acid and 3-phenyl-2-thiohydantoin). Experientia 17, 145-153 (1961). 

Use, D., and Edman, P., The formation of 3-phenyl-2-thiohydantoins from phenylthiooarbamoyl amino acids. Aust. J. Chem. 16, 411-416 (1963). 

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