Amino acids, acetyl derivatives chromatography

Corr, L. T., Berstan, R. and Evershed, R. P. (2007a) Development of N acetyl methyl ester derivatives for the determination of 813C values of amino acids using gas chromatography combustion isotope ratio mass spectrometry. Analytical Chemistry 79, 9082 9090. 

Mengerink Y, Kutlan D, Toth F, Csampai A, and Molnar-Perl I (2002) Advances in the evaluation of the stability and characteristics of the amino acid/amine derivatives obtained with the o-phthaldialdehyde (OPA)/3-merca-ptopropionic acid and OPA/N-acetyl-L-cysteine reagents HPLC/MS study. Journal of Chromatography A 949 99-124. 

Nonvolatile analytes must be chemically converted to a volatile derivative before analysis. For example, amino acids are not sufficiently volatile to analyze directly by gas chromatography. Reacting an amino acid with 1-butanol and acetyl chloride produces an esterfied amino acid. Subsequent treatment with trifluoroacetic acid gives the amino acid s volatile N-trifluoroacetyl- -butyl ester derivative. 

The original Garner preparation3 of 5 involves the conversion of serine into the protected methyl ester 3 and controlled reduction of the latter by DIBAL. The reaction sequence employed for the preparation of 3 involves the protection of the amino acid as N-Boc derivative using di-tert-butyl dicarbonate, esterification with methyl iodide or diazomethane, and acetonization with 2,2-dimethoxypropane under acid catalysis. The N-Boc methyl serinate and the ester 3 require purification by vacuum distillation or chromatography. In a modification to this procedure reported by McKillop,2 the esterification reaction of serine is carried out first by methanol/acetyl chloride. The resulting ester is then converted into the N-Boc derivative 2 with di-tert-butyl dicarbonate and the latter transformed into 3 by acetonization. This procedure avoids... 

Monotrifluoroacetylated diaminopyrazole was first reacted with the free Kemp s triacid to produce the imide, followed by N-Boc protection and amide-coupling with a m-substituted aniline derivative. Final Boc-deprotection occurred on the chromatography column leading directly to the new receptor modules. The recognition site X was chosen to be ethyl as a neutral reference, acetyl for polar side-chains, nitro for electron-rich aromatic residues and carboxylate for basic amino acids (Figure 2.4.4). 

While the a-amino group of ornithine could also react with acetyl -P, this possibility was eliminated in a number of ways. Chromatography in the automatic recording amino acid analyzer by the procedure of Spackman, Stein, and Moore (41) yields excellent separation of 6-acetylornithine and a-acetylornithine. Only traces of a -acetylornithine have been detected with ornithine transcarbamylase and acetyl-P the product is always better than 95% 6-acetylornithine. The separation of a- and 6-acetylornithine by paper chromatography is not practically feasible, although both acetyl derivatives are very readily separated from ornithine (15). 6-Acetylornithine is a natural product first isolated from a Siberian plant 26 years ago (28) and now known to be present in many plants (39). 

The scheme summarises all modern analytical and preparative chromatography protocols, such as high-performance liquid chromatography (HPLC) and gas-liquid chromatography (GLC), with all their conceivable variations. Reverse-phase HPLC or GLC , in which a non-polar liquid is adsorbed onto the solid - the stationary phase - is more appropriate for the analysis of mixtures of derivatives of amino acids and peptides. Cellulose in the above scheme would be replaced by a less-polar medium, such as acetylated cellulose, silanised silica gel, etc. in standard reversed-phase HPLC. 

Three histone-specific acetyltransferases have been partially purified and characterized from rat thymus nuclei (225). The enzymes were extracted from rat thymus nuclei by sonication in the presence of 1M ammonium sulfate and separated into two active fractions (A and B) by DEAE-cellulose chromatography. Fraction B was further separated into two active fractions (Bi and B2) by gel filtration on Sephadex G-200. Each fraction was then purified further by chromatography on hydroxyapatite. The molecular weights, determined by Sephadex G-200 and by sucrose density gradient centrifugation, were 99,000, 110,000, and 92,000 for enzymes A, Bi, and B2, respectively. All three enzymes required acetyl CoA as acetate donor, and the activity of the enzymes was inhibited by p-chloromercuribenzoate. Acetyltransferase A preferentially acetylated histone I (FI) and also poly-L-lysine. Acetyltransferase Bi and B2 preferred histone H4 (other names IV, F2al) and did not acet-ylate poly-L-lysine and histone H3 (III, F3). In addition to c-N-acetyl-lysine, two other unidentified amino acid derivatives were obtained from a digest of histone H4 acetylated by the two B enzymes. 

Using chiral thiols for derivative formation, isoindoles are formed from amino acids and amino alcohols. BOC-L-cysteine, N-acetyl-L-cysteine, Af-acetyl-D-penicillamine [340] and l-thio-jS-D-glucose [341] have been shown to be suitable reagents which allow the separation of most amino acid enantiomers using reversed phase column chromatography. Thus, OPA/N-acetyl-L-cysteine has been used among others for the separation of enantiomers of aspartate [342], baclofen [343], norepinephrine, dopa [344] and lombricine [345]. 

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