Amino acids thiohydantoins

Diisopropylethylamine (sequenal grade), trifluoroacetic add (sequenal grade), and 1,3-dicydohexylcarbodiimide (DCC) were obtained from Pierce. The carboxylic add modified polyethylene membranes were from the Pall Corporation (Long Island, NY). Zitex G-110 was from Norton Performance Plastics (Wayne, NJ). The amino acid thiohydantoins used in this study were synthesized as described (9). The Reliasil HPLC columns used in this study were obtained from Column Engineering (Ontario, CA). 

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)... 

Figure 1. HPLC Separation of the Amino Acid Thiohydantoins.

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. 

The mechanism involves the initial formation of a substituted urea followed by ring closure to form the thiohydantoin. The amino acid is dissolved in 60% aqueous pyridine containing the phenylisothiocyanate... 

In a separate study, Ohberg and Westman applied the same PS-DMAP in a one-pot microwave-induced base-catalyzed reaction of N-aryl and N-alkyl amino acids (or esters) and thioisocyanates for the library synthesis of thiohydantoins (Scheme 7.115) [136]. Thiohydantoins are of interest due to their ease of preparation and the range of biological properties associated with this heterocyclic ring system. The use of PS-DMAP as the base in this reaction gave slightly lower yields compared to when triethylamine (TEA) was used, but it resulted in a cleaner reaction mixture and an easier purification procedure. Cyclizations of a number of N-substituted... 

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. 

Isothioureas can be prepared on insoluble supports by S-alkylation or S-arylation of thioureas (Entry 7, Table 14.6). Further methods for the preparation of isothioureas on insoluble supports include the N-alkylation of polystyrene-bound, A/,/V -di(alkoxy-carbonyl)isothioureas with aliphatic alcohols by Mitsunobu reaction (Entry 7, Table 14.6) and the addition of thiols to resin-bound carbodiimides [7]. Resin-bound dithio-carbamates, which can easily be prepared from Merrifield resin, carbon disulfide, and amines [76], react with phosgene to yield chlorothioformamidines, which can be converted into isothioureas by treatment with amines (Entry 8, Table 14.6). The conversion of support-bound a-amino acids into thioureas can be accompanied by the release of thiohydantoins into solution (see Section 15.9). The rate of this cyclization depends, however, on the type of linker used and on the nucleophilicity of the intermediate thiourea. 

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 ... 

Microwave-induced imine formation, subsequent reduction with NaBH(OAc)3 and cyclisation of the resulting amino acid with isothiocyanates was used in an efficient one-pot multi-step synthesis of thiohydantoins (Scheme 4.26). The reductive animation was conducted as a two-step procedure to avoid direct reduction of the aldehyde at high temperatures48. 

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. 

To increase the number of diversities, the hydantoin (or thiohydantoin) formation reaction was performed starting from N-alkylated dipeptides (Fig. 3). In the last synthesis step, the hydantoin (or thiohydantoin) ring was alkylated followed by the cleavage from the resin. Using 54 different amino acids for the first position of diversity (R1), 60 different amino acids for the second position of diversity (R2), and four different alkylating... 

The selected example by Sim and Ganesan [19] reported the design of a 3078-member discrete thiohydantoin library, prepared without purification of intermediates, with a three-step synthesis, which is shown in Figure 7.2. Nine amino acid esters were reacted with eighteen aromatic aldehydes to produce imines, which were reduced by sodium triacetoxyborohydride. The resulting amines were treated with nineteen isothiocyanates in the presence of triethylamine (TEA), producing intermediate isoureas, which eventually cyclized to the final thiohydantoins. 

Thiohydantoins of amino acids Silica gel, activated at 100°C for 1 h (1) n-Heptane-l-butanol-anhydrous formic acid (10 7 3) (2) Chloroform-95% ethanol-acetic acid (100 50 15) 17... 

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. 

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