Other derivatives of amino acids

Pre-column derivatization—RPC analysis of phenylisothiocyanate (PITC), o-phthaldehyde (OPA), 9-fluorenylmethyl chloroformate (FMOC), or other derivatives of amino acids with UV or fluorescence detection is most common. This is the preferred methodology for life science research because of its higher sensitivity. More examples of precolumn derivatization of amino acids are shown in the life science section of this chapter. 

A broad study concerning other derivatives of amino acids and peptides, such as triazine and benzimidazole derivatives, has been started (Maekawa et al., 1975). Of the triazine derivatives, few have shown any decisive fungicidal action. However,... 

Naudet [23], Tsubone [24], Heitmann [25], Kester [26], Fieser [27], Komatsu [28], Takehara [29], Imanaka [30], Seguer [31], Hatsutori [32], Abramzon [33], and others. Other derivatives of amino acids have also been prepared by reacting epoxidized fatty acid with ammonium or amines [34],... 

Studies to date indicate that transaminases are specific for L-amino acids and a-keto acids. Peptides and other derivatives of amino acids, such as amides and A -substituted amino acids, are not active. Recently it has been reported that E. coli extracts catalyze a transamination reaction between adenine and a-ketoglutaric acid. ... 

Diamide Chiral Separations. The first chiral stationary phase for gas chromatography was reported by GH-Av and co-workers in 1966 (113) and was based on A/-trifluoroacetyl (A/-TFA) L-isoleucine lauryl ester coated on an inert packing material. It was used to resolve the tritiuoroacetylated derivatives of amino acids. Related chiral selectors used by other workers included -dodecanoyl-L-valine-/-butylamide and... 

In the 1950s, a group of coryneform bacteria which accumulate a large amount of L-glutamic acid in the culture medium were isolated (21). The use of mutant derivatives of these bacteria offered a new fermentation process for the production of many other kinds of amino acids (22). The amino acids which are produced by this method are mostiy of the T.-form, and the desired amino acid is singly accumulated. Therefore, it is very easy to isolate it from the culture broth. Rapid development of fermentative production and en2ymatic production have contributed to the lower costs of many protein amino acids and to their availabiUty in many fields as economical raw materials. 

The enolic form of 2 was confirmed by a ferric chloride color reaction and by its acidity and ultraviolet spectrum, A-Aroyl derivatives of amino acids other than glycine fail to form such azlactones, probably because the stabilization afforded by enolization cannot occur. 

Obviously, the main purpose for the introduction of CL detection coupled to CE separations is inherent to the development and improvement of sensitive and uncomplicated devices to achieve a decrease of the band broadening caused by turbulence at the column end, together with the attractive separation efficiency of CE setups. With this purpose in mind, Zhao et al. [83] designed a postcolumn reactor for CL detection in the capillary electrophoretic separation of isoluminol thiocarbamyl derivatives of amino acids, because, like other isothiocyanates, isoluminol isothiocyanate has potential applications in the protein-sequencing area. 

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. 

Amino acids cannot be analysed by GC unless suitable derivatives are prepared, which obviously is a disadvantage over other methods, such as paper, thin-layer and ion-exchange chromatography. On the other hand, the GC analysis of derivatives of amino acids is rapid, instrumentation can be used for a wide range of applications, the sensitivity of the analysis if high and there is the possibility of working with small amounts of samples. 

The silylation of amino acids with BSTFA was studied in detail by Gehrke and coworkers [254—256]. BSTFA—acetonitrile (1 1) was applied first and fourteen amino acids were silylated at 135°C for 15 min. Glu, Arg, Lys, Trp, His and Cys, however, require up to 4 h, in order for measurable peaks to be obtained in the chromatogram. Despite such a long reaction, Gly and Glu gave two peaks and also it was difficult to separate the tris-TMS derivative of Gly from the derivatives of lie and Pro. The influence of polar and non-polar solvents was demonstrated later and was decisive mainly with respect to uniformity of the products. Only the bis-TMS derivative was produced in hexane, methylene chloride, chloroform and 1,2-dichloroethane bis- and tris-derivatives were produced in six more polar solvents. On the other hand, Arg did not provide any peak in the less polar solvents that were used and only one peak in the six more polar solvents. The best and most reproducible results were obtained when silylating seventeen amino acids with BSTFA—acetonitrile (1 1) at 150°C for 15 min 2.5 h at 150°C were necessary for the reproducible derivatization of Gly, Arg, and Glu. These reaction conditions were recommended for the analysis of all twenty amino acids. The TMS derivatives of amino acids were found to be stable on storing them in a sealed vial at room temperature for 8 days, with no decomposition. 

TMS derivatives of amino acids were also combined with other procedures and some difficulties were thus avoided. N-TMS-methyl and -ethyl esters of most protein amino acids were prepared by the action of TMSDEA on alkyl esters of amino acids and were chromatographed on methylsilicone stationary phases [246], Their retention times were found to be 15—20% lower than those of the corresponding TMS derivatives. Despite having an additional step in comparison with direct silylation, the procedure was applied by Hardy and Kerrin [259] to the GC analysis of twenty protein amino acids, including Hypro and CysH. Amino acids were esterified with a 3 N HC1 solution in n-butanol at 150°C for 15 min with subsequent silylation with BSTFA for 90 min at the same temperature. Acetonitrile and methylene chloride were used as solvents for the silylation. In the former solvent double derivatives of Gly and Lys (bis- and tris-) were produced, whereas in the latter the less silylated form only was produced. As Arg, in contrast to direct silylation, also leads to one peak in this instance, methylene chloride is recommended as the silylation solvent. The separation of all twenty amino acids was achieved on a simple column with 2% of OV-7 on GLC-110 textured glass beads (100—120 mesh). 

Other reactions conducted in pyridine solution include the esterification of simple sugars by N-benzyloxycarbonyl derivatives of amino acids in the presence of N,N - dicyclohexylcarbodiimide339 to produce essentially the 6-O-aminoacyl derivatives, and formation of carbo-... 

Enzymatic Kinetic Resolution of N-Acyl Amino Acids Coupled with Racemization by N-Acyl Amino Acid Racemase Acylases are enzymes hydrolysing the N-acetyl derivatives of amino acids. They require the free carboxylate for activity and have long been used for the kinetic resolution of amino acids. The unreacted enantiomer is usually racemized in a separate step by treatment with acetic anhydride. While acylases from hog kidney have an L-specificity, bacterial acylases with L- and D-specificity of various origins have been isolated and used for the kinetic resolution of N-acetyl amino acids. An industrial process for the production of L-Met and other proteinogenic and non-proteinogenic L-amino acids such as L-Val, L-Phe, L-Norval, or L-aminobutyric acid has been established. Currently, several hundred tons per year of L-methionine are produced by this enzymatic conversion using an enzyme membrane reactor [46]. 

Some hormones are proteins or polypeptides (oxytocin, TSH, insulin). Others, while neither proteins nor polypeptides, arc derivatives of amino acids (thyroxine, epinephrine). Others (steroids) are produced in Lipidland as... 

Cooper G. and Cronin J. R. (1995) Einear and cyclic aliphatic carboxamides of the Murchison meteorite hydrolyzable derivatives of amino acids and other carboxylic acids. Geochim. Cosmochim. Acta 59, 1003-1015. 

A simpler way to restrict the conformation of an enolate is to coniine it in aheterocycle and an important group of chiral enolates come from various derivatives of amino acids. The hrst successful such compounds were Schollkopf s bislactim ethers 41 derived from the diketopiperazines 40 formed when an amino acid such as alanine 39 condenses with itself.4 Treatment of 41 with butyl lithium creates a lithium enolate on one position in the ring the methyl group in the other position keeps the chirality intact. Alkylation occurs selectively on the opposite side to the remaining methyl group 42 and hydrolysis releases a new tertiary amino acid 43 and one of the original alanines. 

Transamination reactions require the coenzyme pyridoxal-5 -phosphate (PLP), which is derived from pyridoxine (vitamin B6). PLP is also required in numerous other reactions of amino acids. Examples include racemizations, decarboxylations, and several side chain modifications. (Racemizations are reactions in which mixtures of l- and D-amino acids are formed.) The structures of the vitamin and its coenzyme form are illustrated in Figure 14.2. 

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