Phenylthiohydantoin amino acids

Hayakawa, K., Hirano, M., Yoshikawa, K., Katsumata, N, and Tanaka, T., Separation of phenylthiohydantoin-amino acids by temperature-controlled reversed-phase high-performance liquid chromatography, /. Chromatogr. A, 846, 73, 1999. 

Figure 7.5 The Edman degradation method, by which the sequence of a peptide/polypeptide may be elucidated. The peptide is incubated with phenylisothiocyanate, which reacts specifically with the N-terminal amino acid of the peptide. Addition of 6 mol l-1 HCl results in liberation of a phenylthiohydantoin-amino acid derivative and a shorter peptide, as shown. The phenylthiohydantoin derivative can then be isolated and its constituent amino acid identified by comparison to phenylthiohydantoin derivatives of standard amino acid solutions. The shorter peptide is then subjected to a second round of treatment, such that its new amino terminus may be identified. This procedure is repeated until the entire amino acid sequence of the peptide has been established...

Sun, T., and R.E. Lovins, Quantitative protein sequencing using mass spectrometry use of low ionizing voltages in mass spectral analysis of methyl- and phenylthiohydantoin amino acid derivatives. Anal Biochem, 1972. 45(1) 176-91. 

The N-terminal amino acid of a protein can be determined by reacting the protein with dansyl chloride or fluorodinitrobenzene prior to acid hydrolysis. The amino acid sequence of a protein can be determined by Edman degradation which sequentially removes one residue at a time from the N terminus. This uses phenyl isothiocyanate to label the N-terminal amino acid prior to its release from the protein as a cyclic phenylthiohydantoin amino acid. 

An ultraviolet-laser based thermo-optical absorbance detector for micrometer capillaries was used by Qi et al. [76] to monitor the separation of a mixture of 13 phenylthiohydantoin-amino acids. A modulated pump laser beam periodically illuminated the capillary at a point near its end. Complex deflection and diffraction effects occur at the capillary-solution interface. Perturbation of the refractive index at this interface changes the intensity of the probe beam that is measured using a small photodiode. 

Certain fundamental characteristics of MECC that influence retention have been investigated (5). The technique has been used in the analysis of a variety of samples including phenolic compounds (1), phenylthiohydantoin—amino acids (6), and metabolites of vitamin Bg (7). In related electrokinetic separation techniques, substituted benzene compounds have been separated based on the formation of inclusion complexes with an ionic cyclodextrin derivative in the mobile phase (8) and polyaromatic hydrocarbons have been separated based on solvophobic interactions with a tetraakyl— ammonium ion in the mobile phase (9). The effects of injection procedures on efficiency have also been studied (10). 

The changes in retention and selectivity can also be exploited in the thermally tuned tandem column concept by Mao and Carr [25], in which the temperatures of two sequentially linked columns containing different stationary phases can be altered to provide the optimum separation. The technique was applied to the separation of barbiturates, phenylthiohydantoin amino acids [26], and selected basic pharmaceuticals, such as antihistamines (Figure 18-1) [27]. 

Y. Mao and P. W. Carr, Separation of barbiturates and phenylthiohydantoin amino acids using the thermally tuned tandem column concept. Anal. Chem. 73 (2001), 1821-1830. 

The DNA strands were first reported by Brenner and co-workers to define the sequence of a peptide constructed on a solid support. Upon completion of the synthesis, an on-bead assay was performed. Whereas each bioactive peptide was defined by a unique DNA sequence, the decoding process simply involved amplification of the code by the polymerase chain reaction followed by sequencing. This technique marked the beginning of the tagging method for encoded split synthesis. Sequenceable peptide strands are an alternative to DNA encoding. The code is read by HPLC of the Edmon degradation phenylthiohydantoin amino acid derivatives, a well-developed microsequencing method. 

H- and 13C-NMR data have been reported for diagnostic purposes in direct analysis of phenylthiohydantoin amino acid derivatives (PTH) produced in the Edman degradation of peptides and proteins.189-193 The insensitivity of 3H-NMR spectroscopy constitutes a major hurdle for its application in the sequence study of peptides.194,195 Alternatively, identification of the cleaved amino acids in the automated Edman degradation has been solved in some cases by using IR,196-198 mass,199 and gas chromatographic techniques.200... 

Figure 6.5. Illustration of the zone refocusing mechanism in multiple development (left) and its application to the separation of a mixture of phenylthiohydantoin-amino acid derivatives (right). The broken line represents the change in zone size due to the expansion and contraction stages in multiple development and the solid line depicts the expected zone width for a zone migrating the same distance in a single development. (From ref. [71] Research Institute for Medicinal Plants).

Otsuka, K., Terabe, S., and Ando, T. Electrokinetic chromatography with miceUar solutions separation of phenylthiohydantoin amino acids, J. Chromatogr., 332, 219, 1985. 

Mueller, T., Posch, U.C., and Lindner, H.H., Separation of phenylthiohydantoin amino acids by capillary electrochromatography. Electrophoresis, 25, 578, 2004. 

Waldron, K. C. and Dovichi, N. J., Sub-femtomole determination of phenylthiohydantoin-amino acids Capillary electrophoresis and thermooptic detection. Ana/. Chem., 64,1396,1992. 

The phenylthiohydantoin amino acids are extracted and identified spectroscopically or chromatographically. 

Zhou J, Hefta S, and Lee TD (1997) High sensitivity analysis of phenylthiohydantoin amino acid derivatives by electrospray mass spectrometry. Journal of the American Society for Mass Spectrometry 8 1165-1168. 

Steiner, W. E. Clowers, B. HiU, H. H. Rapid separation of phenylthiohydantoin amino acids Ambient pressure ion-mobiUty mass spectrometry (IM-MS). Anal. Bioanal. Chem. 2003, 375,99-102. 

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