Thiohydantoin derivatives

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. 

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. 

GLC is an important adjunct to protein sequence determination. Automatic "sequenators" based upon the approach developed by Edman are available and have been described in detail by Niall (60). The Edman degradation, summarized in Equation 9.5, makes use of methyl or phenylisothiocyanate which reacts with the N-terminus of a peptide. Exposure of the isothiocyanate derivative of the protein to acid results in cleavage of the terminal amino acid as a thiaxolinones and exposure of the next amine group on the peptide. Thus, the process can be repetitively carried out, each amino acid removed from the peptide, in a sequential manner. Thiazolinones rearrange in acid medium to form thiohydantoin derivatives of amino acids, some of which may be directly gas chromatographed others must be derivatized typically as trimethylsilyl derivatives. 

PITC). The mixture is warmed at 40 °C for 1 h, then diluted with 1 ml of water and the excess of MITC (or PITC) is removed by extracting four times with 2-ml volumes of benzene. The aqueous layer is evaporated and dried in a vacuum desiccator over sodium hydroxide. The general scheme for the formation of PTH-amino acids is illustrated in Fig.4.1. For peptide hydrolysis, 1.5 ml of a mixture consisting of equal volumes of 3 N hydrochloric acid and 60% acetic acid are added and the reaction is incubated in a nitrogen atmosphere for 30 min at 40 °C. The mixture is diluted with 2 ml of water and the thiohydantoin derivative is extracted with 2 ml of ethyl acetate followed by 2 ml of benzene. The combined extracts are used for chromatography. 

Phenylimidazole was synthesized by treatment with Raney Ni of the corresponding thiohydantoin derivative. This desulfurization was considered to take place via the tautomeric dimercaptoimidazole, as shown in eq. 13.67.143... 

The inability of C-terminal proline to be derivatized to a thiohydantoin has been a major impediment to the development of a routine method for the C-terminal sequence analysis of proteins and peptides. Since the method was first described in 1926 (2), the derivatization of C-terminal proline has been problematic. While over the years a few investigators have reported the derivatization of proline, either with the free amino acid or on a peptide, to a thiohydantoin (6-8), others have been unable to obtain any experimental evidence for the formation of a thiohydantoin derivative of proline (9-12). Recently, utilizing a procedure similar to that described by Kubo et al. (6), Inglis et al. (13) have described the successful synthesis of thiohydantoin proline from N-acetylproline. This was done by the one-step reaction of acetic anhydride, acetic acid, trifluoroacetic acid, and ammonium thiocyanate with N-acetyl proline. We have reproduced this synthesis and further developed it to a large scale synthesis of TH-Proline. 

We have described a simple procedure for the large scale (200 mg) synthesis of thiohydantoin proline from N-acet proline and extensively characterized this analogue. The thiohydantoin derivative of proline is conveniently obtained as a white powder which is stable to long term storage. The availability of a thiohydantoin proline standard is critical for the evaluation of automated sequencing results. 

Figure 3. Automated C-Terminal Sequencing of the Tripeptide, LAP (15 nmol), Covalently Attached to Carboxylic Acid Modified Polyethylene. Each thiohydantoin derivative is identified by comparison to the retention time of an authentic standard. Unlabeled peaks are background produced by reaction side products.

Grassmann and Hermann (1953) determined the COOH-terminal amino acids of collagen and gelatin using a modification of the thiohydantoin method of Schlack and Kumpf (1926). They first benzoylated the amino groups and then reacted the proteins with ammonium thiocyanate in acetic anhydride and acetic acid to form thiohydantoin derivatives. These could... 

The N-methyl- and IV-phenylthiourea derivatives of the N-terminal amino acid of a peptide thermally rearrange in the MS ion source to give the thiohydantoin derivative of the terminal amino acid and the shortened peptide [189]. This has been suggested as a sequencing method for the first four acids after which point interference from side products becomes excessive. 

Herbicidal activity is retained to a high degree in 1-amino-2-thiohydantoin derivatives. 

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). 

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

The ot-halogen acids are often used in reactions with primary amines and urea,27,28 as well as in cyclocondensations of chloroacetates and ureas. Some 3-(benzothiazol-2-yl)-2-thiohydantoins derivatives have been obtained by this fashion.29... 

Racemic and optically active hydantoins have been prepared through isocyanate derivatives, starting from chiral disubstituted cyanoacetic acids 71.123 Similarly, N2,N2-disubstituted thiocyanatocarboxylic hydrazides give 3-amino-2-thiohydantoin derivatives 72,124 and l-phenyl-3-aminohydantoin is obtained in excellent yield in the reaction of ethyl N-phenyl-N-carbethoxyglycinate and hydrazine hydrate.123... 

The tautomerism of some 5-arylazo-2-thiohydantoin derivatives as a criterion of their acidity has been studied, and the apparent pKa values have been discussed in terms of Hammett correlations.288... 

Synthesis and antinociceptive activity of thiohydantoin derivatives Xu, Guoyou Yu, Zhengwei Peng, Sixun... 

A series of poly(amide-imide)s have been obtained by polycondensation of hydantoin and thiohydantoin derivatives of pyromellitic acid chlorides with [N,N -(4,4 -carbonyldiphthaloyl)] bisalanine diacid chloride [57], N,N -(pyromellitoyl)-bis-i-phenylalanine diacid chloride [58], and N,N -(4,4 -diphenyl ether) bistrimellitide... 

Schlack-Kumpf method, also called the isothiocyanate method, a method for C-terminal sequence analysis of peptides and proteins. The C-terminus is converted with acetic anhydride into a 5(4H) oxa-zolone. Upon reaction with ammonium thiocyanate or with the new derivatizing reagent triphenylgermanyl isothiocyanate (TPG-ITC), Tacyl-2-thiohydantoins are formed, with the peptidyl residue being the acyl group. The peptidyl residue is then cleaved to give the 2-thiohydantoin derived from the C-terminal amino acid, which is subsequently analyzed [P. Schlack,... 

S-containing amino acids such as cystine, methionine [3], cysteine [8] thiohydantoin derivatives [17], PTH amino acids [9-12J Penicillin derivatives and cephalosporins e.g. penicillic acid, oxacillin, clonadllin,... 

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. 

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