Amino acids thiohydantoin derivatives

HPLC Separation of the Amino Acid Thiohydantoins. Reverse phase HPLC separation of the thiohydantoin amino acid derivatives (400 pmol) was performed on a C-18 (3 x, lOOA)... 

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... 

Benzylthio or 2-benzyloxy derivatives of A-2-thiazoline-5-one (224) are readily opened by amines to give the amide derivatives (225) (Scheme 115) (459. 471). Compound 225 can be cyclized thermally to the corresponding thiohydantoins (459). Similarly, treatment of 4-substituted-2-phenylthiazol-5(4H)-ones (226) with amino acids, peptides, or hydrazine affords the corresponding Nfcti-thiobenzamidoacetylated derivatives (227) (Scheme 116) (455). 

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-Thiazolyl a-amino acids 56 have been prepared. The preferred route to these compounds would utilise the Hantzsch synthesis, however in this case the in situ formation of the required thiourea derivatives of a-aminoacids 52 failed. A variety of isothiocyanate reagents were tried, with the result being either no reaction, decomposition or the corresponding thiohydantoin 53. A modified version of the Hantzsch synthesis was developed. If the bromoketone 54 is initially treated with sodium thiocyanate an a-thiocyanatoketone 55 is formed, subsequent addition of the amino acid ester 51 yields A-thiazolyl a-amino acids 56 <00T3161>. 

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. 

A comparative study was carried out of the effectiveness of three commercially available chiral columns and nonchiral derivatives of amino acids such as A-(3,5-dinitrobenzoyl) esters (119), phenylurea esters (120), hydantoins (121) and thiohydantoins (98). Although good separations were obtained, no column was universally effective294. 

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. 

Fairwell, T., S. Ellis, and R.E. Lovins, Quantitative protein sequencing using mass spectrometry thermally induced formation of thiohydantoin amino acid derivatives from N-methyl- and N-phenylthiourea amino acids and peptides in the mass spectrometer. Anal Biochem, 1973. 53(1) 115-23. 

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. 

Eyem and Sjoquist have suggested the use of short (4.5 m) glass capillary columns and reported the separation of 19 of 20 silylated methyl thiohydantoin (MTH) derivatives of the amino acids (62). The histidine derivative can be separated on the same column by starting at a higher temperature. Separation of some of the silylated PTH derivatives was also demonstrated, though not in as much detail as the MTH 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. 

Thiohydantoins (see Scheme 4.25, p. 78) play an important role as derivatives of amino acids, particularly in the sequential analysis of peptides. When the sequence is being determined from the carboxyl end, ammonium thiocyanate is dissolved in acetic acid and acetic anhydride and this mixture is allowed to react with the carboxyl end of the peptide with the formation of l-acyl-2-thiohydantoin. 2-Thiohydantoin is released from the peptide by the action of a base, and a new carboxyl end of the amino acid is exposed. Prior to GC analysis, thiohydantoins must be further modified, e.g., by silylation [271], as follows. A 25-/A volume of ethyl acetate and 25 /d of BSA are added to 500 nmol of 2-thiohydantoin and the mixture is shaken until it is homogeneous. The mixture is then heated in a stoppered test-tube at 80°C for 5 min, cooled and centrifuged before injection. 

Protein samples were obtained from Sigma Chemical Co. (St.Louis,MO). Mouse immunoglobulin G was a generous gift of Dr. J. Bailey, City of Hope. Thiohydantoin-amino acid derivatives were prepared according to published procedures and quality-controlled by HP (3). The HP C-terminal chemical sequencing method was developed for automation using a chemistry licensed by HP from the City of Hope, Duarte, CA. 

The dry membrane is inserted into a C-terminal sequencer column (inert Kel-F columns fitted with inert endfiits) and installed in any one of the four sample positions on the HP G1009A sequencer. The sequencer column reactions that occur on the Zitex membrane include the chemical coupling and cyclization of the C-terminal residue and the cleavage and extraction that releases the thiohydantoin-amino acid derivative. The thiohydantoin-amino acids are extracted off the Zitex... 

D. HPLC Analysis of Thiohydantoin-Amino Acid Derivatives... 

The thiohydantoin-amino acid standard HPLC chromatogram (Figure 1) shows the elution times for each of the 20 common amino acid derivatives (approximately 50 pmols) including thiohydantoin-Pro (P) and the common peak designated S/C, identifying Ser and Cys residues. The relative retention time for the S-carboxymethyl derivative of cysteine is indicated by the arrow. 

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. 

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. 

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). 

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). 

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