Amino acids, acetyl derivatives detection

The chromatographic analysis of amino acids with spectrophotometric detection usually requires the formation of derivatives, because of httle absorption of UV light above 210 run. Precolumn deiivatization is usually preferable. o-Phthalaldehyde (OPA) is the deiivatization reagent that probably has the best characteristics. It reacts with primary amino groups in the presence of a thiol at pH 9.5 and room temperature to form l-alkylthio-2-alkyl substituted isoindoles (Fig. 10.5). The derivatives show maximum absorption at 335 nm and are highly fluorescent, with excitation wavelength at 340 nm and emission at 445 nm. Mercaptoethanol has been more extensively used than other thiols for the derivatization, but the OPA-mercaptoethanol isoindoles are unstable. The stability of isoindoles is improved when A-acetyl-L-cysteine (NAC) is used instead of mercaptoethanol [12]. 

OPA in combination with chiral thiols is one method used to determine amino acid enantiomers. A highly fluorescent diastereomeric isoindole is formed and can be separated on a reverse-phase column. Some of these chiral thiols include N-acetyl-L-cysteine (NAC), N-tert-butyloxy-carbonyl- L-cysteine (Boc-L-Cys), N-isobutyryl- L-cysteine (IBLC), and N-isobutyryl- D -cysteine (IBDC). Replacing OPA-IBLC with OPA-IBDC causes a reversal in the elution order of the derivatives of D- and L-amino acids on an ODS column (Hamase et al., 2002). Nimura and colleagues (2003) developed a novel, optically active thiol compound, N-(tert-butylthiocarbamoyl)- L-cysteine ethyl ester (BTCC). This reagent was applied to the measurement of D-Asp with a detection limit of approximately 1 pmol, even in the presence of large quantities of L-ASP. 

Colistin (COL) is a multicomponent antibiotic (polymyxins E) that is produced by strains of inverse Bacillus polymyxa. It consists of a mixture of several closely related decapeptides with a general structure composed of a cyclic heptapeptide moiety and a side chain acetylated at the N-terminus by a fatty acid. Up to 13 different components have been identified. The two main components of colistin are polymyxins El and E2 they include the same amino acids but a different fatty acid (216). A selective and sensitive HPLC method was developed for the determination of COL residues in milk and four bovine tissues (muscle, liver, kidney, and fat). The sample pretreatment consists of protein precipitation with trichloracetic acid (TCA), solid-phase purification on Cl 8 SPE cartridges, and precolumn derivatization of colistin with o-phthalaldehyde and 2-mercaptoethanol in borate buffer (pH 10.5). The last step was performed automatically, and the resulting reaction mixture was injected into a switching HPLC system including a precolumn and the reversed-phase analytical column. Fluorescence detection was used. The structural study of El and E2 derivatives was carried out by HPLC coupled with an electrospray MS. Recoveries from the preseparation procedure were higher than 60%. 

The reduction of 2, either catalytically or with zinc in acetic acid, leads to 3-amino-4-hydroxy-2-quinolone 20 [72TH000], These amino compounds are rather unstable they dimerize with loss of ammonia to "bis-amines", which in turn are readily oxidized to dyes similar to those obtained from ninhydrin and primary amines [68M1205] [68M1543], The amino derivatives 20 are therefore conveniently converted into 0,N-diacetyl derivatives, the N-acetyl derivative 21, or its dehydrated form, the oxazolo derivative 22 [95MI000], The variety of biological activity of oxazolo-quinolines of type 22 has been detected only in recent years [94JHC1647],... 

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). 

Similarly, conversion of A -acetyl amino acid esters into the related A/ -Boc-protected derivatives is performed with B0C2O/DMAP which in the first step leads to acylation of the acetyl amide group. This is then in turn hydrolyzed with hydrazine or lithium hydroxide to produce the A -Boc-protected amino add ester or free carboxy derivative, respectively (Scheme 32). No racemization was detected under these reaction conditions.t ... 

Thus, treating readily available (Z)-A-acetyl a-enamides, obtained via Erlenmeyer eondensation, with di-ZezV-butyldicarbonate followed directly by hydrazine or LiOH, affords the corresponding A -Boc a-enamide ester or acid, respectively. Hydrogenation of substrates 3 was found to proceed with enantiose-lectivities of > 98% ee by the use of the Et-DuPHOS-Rh catalyst. Alternatively, A-Boc amino acids may be obtained by initial hydrogenation of the A -acctyl a-enamides, followed by transmutation to the A -Boc derivative as previously outlined [18]. The mild nature of this method is evinced by the absence of detectable racemization in the A-Ac to A -Boc conversion performed after hydrogenation. 

Isoindoles of o-phthalaldehyde (OPA)/N acetyl-L-cysteine (NAC) are commonly used for detection of amino acids (RiCH(COOH)NH2) RPLC. A particular hydrophobicity scale was established with the retention data of these derivatives in mobile phases of SDS at pH 3, using the glycine derivative as a reference [20], Linear relationships were obtained between the ratios of log k of each amino acid derivative to log k of the glycine derivative (which was called quantitation of hydrophobicity index, QH), and log Pq for the Ri substituents (tTri). 

The substitution of sodium acetate or N-methyl-imldazole for pyridine in the preparation of alditol acetate derivatives for the capillary g.c. analysis of neutral and amino-sugars using selected ion monitoring m.s. detection has been examined. While N-methyl-imidazole effected full acetylation of alditols in the presence of borates and water, it led to a higher detector background than the more tedious use of sodium acetate. Muramic acid produced two alditol acetate derivatives, the lactams (1) and (2), in a reproduceable fashion only in the sodium acetate-catalyzed procedure... 

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