Amino acids, isoindole derivatives

The method finds increasing application for the determination of amino acids. Factors affecting the retention of the isoindole derivatives by reversed phase columns have been investigated [335, 337]. Usually columns with Ci8 brushes are used [350]. Numerous gradient elution systems suitable for the separation of amino acid-derived isoindoles have been published their number is still increasing with the availability of new column types. The various references [184, 337, 339-365] are a representative selection of those available, most of which involve minor variations of the basic method. 

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 interactions of the amino acid derivatives with a modified C18 column and micellar mobile phases of the anionic SDS are mainly of hydrophobic and electrostatic nature. The basic structure of the amino acid isoindoles is the same. Consequently, in the absence of electrostatic interactions, the hydrophobic character of the Rj substituent should be responsible of the retention. The nitrogen atom in the isoindole heterocycle is nonprotonated at pH 3, and thus, electrostatic interactions can only exist with ionizable groups giving a positive charge to R in the molecule. This occurs with arginine and histidine, which have amino groups that do not react with OPA. 

Scheme 3.113 Bergman cyclization of acyclic amino acid derived enediynes leading to 2,3-dihydrobenzo[/]isoindoles.

The reaction of ort/io-phthalaldehyde and a thiol compound with an amino acid to form an isoindole derivative can be used to enhance the detection sensitivity for the normally only weakly UV-detectable amino acid compounds, and to introduce an... 

One method that combines the good chromatographic properties with improved limit of detection is the separation of isoindole derivatives of amino acids that may be detected fluorimetrically. This method may be applied to protein hydrolysates, and used in automated format in routine analyses [22]. 

The mixture of free amino acids is reacted with OPA (Fig. 7-8) and a thiol compound. When an achiral thiol compound is used, a racemic isoindole derivative results. These derivatives from different amino acids can be used to enhance the sensitivity of fluorescence detection. Figure 7-9 shows the separation of 15 amino acids after derivatization with OPA and mercaptothiol the racemic amino acids may be separated on a reversed-phase column. If the thiol compound is unichiral, the amino acid enantiomers may be separated as the resultant diastereomeric isoindole compound in the same system. Figure 7-10 shows the separation of the same set of amino acids after derivatization with the unichiral thiol compound Wisobutyryl-L-cysteine (IBLC). 

A number of drawbacks in the application of the 0PA/2-ME reagent system include the instability of the fluorescent isoindole derivative (5-7) the use of the noisome reagent 2-mercaptoethanol the low and solvent-dependent fluorescence efficiencies (8,9) of the isoindole and—perhaps the most limiting—the effective restriction of the OPA assay to primary aliphatic amines and to amino acids. 

In the presence of mercaptoethanol [3, 12] o-phthalaldehyde reacts with primary amines and amino acids to yield fluorescent isoindole derivatives. 

NDA derivatization has also been automated for analysis of amino acids in brain tissue and microdialysates (Shah et al, 1999). NDA reacts with primary amines in the presence of cyanide to form a highly stable N-substituted l-cyanobenz[/] isoindole (GBI) derivative. Addition of a nucleophile, such as cyanide, hydrogen sulphite, isothiocyanate, or 2-mercaptoethanol, is essential for the formation of the derivative. 

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. 

If only very small samples of amino acids are available for analysis, fluorescence is used for detection. One of the most sensitive methods of microanalysis is based on the reaction of amino acids with o-phthalaldehyde and /3-mercaptoethanol (Equation E2.1). The isoindole derivative is fluorescent and amounts as small as 10-12 mole may be measured. 

Figure 27.17 Separation and determination of amino acids by reversed phase gradient LCEC of isoindole derivatives. The sample was obtained from an awake monkey using a microdialysis sampling probe to collect amino acids from the extracellular fluid of the brain.

Fonnation of Isoindoks. In 1971 Roth described a sensitive analytical method for amino adds based on their reaction with o-phthaldia dehyde (OPA) and a thiol, 2-mercaptoethanol (117). The reaction produces an intensely fluorescent isoindole derivative of the amino acid and is specific for the primary amino group (118,119). Subsequently, the OPA method was extended to the enantiospedfic LC analysis of amino adds by substituting an optically active (single enantiomer) thiol for 2-mercaptoethanol in the derivatization reaction (120-122). This results in the formation of two... 

Fig. 5. Structure of fluorescent isoindole derivatives of amino acids. R = amino-acid carbon chain.

Use of cyclic amino acids instead of sarcosine to generate the corresponding dipole as well as the subsequent [3+2]-cycloaddition allowed to obtain condensed isoindoles [135]. Thus, tricyclic derivatives 173 were obtained in the case of proline (Scheme 84). 

The poor fluorescence of the cysteine, lysine, and ornithine derivatives may be a drawback of the technique. Cysteine yields weakly fluorescent properties due to its sulfydryl group (Cooper and Turnell, 1982). However, these sulfydryl groups can be blocked with iodoacetic acid, lodoacetamine, or acrylonitrile, with the result that fluorescent isoindoles can then be formed with OPA (Cooper and Turnell, 1982). Cysteine may also be oxidized to cysteic acid, which forms a highly fluorescent product with OPA. Following the oxidation of cysteine, however, it may be difficult to obtain high reproducibility with the OPA derivatization of amino acids, because the conditions for these two reac-... 

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]. 

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]. 

M.C. Garcia Alvarez-Coque, M.J. Medina Hernandez, R.M. Villanueva Camanas and C. Mongay Fernandez, Studies on the Formation and Stability of Isoindoles derived from Amino Acids, o-Phthalaldehyde andN-Acetyl-L-cysteine, Anal Biochem., 180 172 (1989). 

OPA can also be employed for the detection of mixtures of amino acids. It reacts with primary amines in the presence of mercaptoethanol or other thiols to produce highly fluorescent isoindole derivatives (excitation wavelength 340 nm, emission 455 nm). In contrast to that with ninhydrin, this... 

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