Thiols, fluorescent deriv

Chloro-7-nitrobenz-2,l,3-oxadiazole (NBD-C1) reacts with primary and secondary aliphatic amines to produce intensely fluorescent derivatives. Although anilines, phenols and thiols react with NBD-C1, they produce derivatives which do not fluoresce or which are only weakly fluorescent. This limits the types of compounds which can be analyzed with this reagent and thus makes the technique more selective. This selectivity is desirable in trace analysis because of a lower degree of interference from co-extractives. The general reaction scheme for formation of the fluorescent amine derivatives is shown in Fig.4.50. 

The OPA method is based on the formation of a highly fluorescent isoindole derivative by reaction with o-phthalaldehyde and 2-aminoethanol in mildly basic aqueous solution (331. The advantage of this technique is that a large variety of thiols and other reduced sulfur compounds can be detected at subnanomolar to nanomolar concentrations. Its disadvantage is that the fluorescent derivative, which preserves the thiol in its reduced stage, is unstable and must be formed just prior to injection. These characteristics preclude the delayed analysis (e.g., in the laboratoiy) of large numbers of samples collected in the field. 

Used for the precolumn preparation of fluorescent derivatives of thiols Reference 1... 

Used for the precolumn preparation of fluorescent derivatives of primary and secondary amines, phenols, and thiols (4-chloro-7-nitrobenzofuran)... 

Used for the precolumn preparation of fluorescent derivatives of thiols optimum derivatization conditions were found to be pH 8.2 with a minimum of a 2.7-fold molar reagent excess using a reaction time of 1 h at 60°C under these conditions only free sulfhydryl groups are derivatized Reference 16... 

Maleimides react, as shown in Figure 17, by addition of thiols to the 3,4-double bond. By introduction of a fluorigenic substituent on the imide nitrogen, numerous fluorescent maleiirtides have been prepared in the past, and they usually form derivatives with high quantum yields [443—445]. However, the derivatives cire mostly unstable they react by rearrangement, and thus form more than one fluorescent derivative. 

Phenols, thiols 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-chloride) Formation of fluorescent derivatives... 

The OPA reagent was first reported in 1971 by Roth as a postcolimm fluorogenic reagent for amines [5] and has been widely used for the sensitive determination of primary amino compounds. However, the fluorescent derivatives are not sufficiently stable, and it is sometimes difficult to obtain reproducible results using the postcolumn derivatization system. A precolumn derivatization technique has also been developed using OPA in the presence of alkylthiol compounds such as 2-mercaptoethanol. OPA rapidly reacts with primary amino compounds within 2 min at room temperature, and the derivatives can be separated by reversed-phase liquid chromatography [20]. Fluorescence detection of the derivatives is performed at 440 nm (emission wavelength) with excitation at 330 nm. Because OPA does not react with secondary amino compounds, proline and hydroxyproline can not be determined by this method. Replacement of 2-mercaptoethanol with other thiols, such as 2-ethanethiol [21] and 3-mercaptopropionic acid [22], produced more stable fluorescent derivatives. 

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). 

Fluorescent 4-nitrobenzofuran derivatives are produced. Primary and secondary aromatic amines and thiols also react. 

SAMSA-fluorescein, 5- [2(and 3)-5-(acetylmercapto)-succinoyl]amino fluorescein, is a fluorescent probe containing a protected sulfhydryl group. In its protected state, the compound is unre-active. The acetyl-protecting group can be removed by treatment with dilute NaOH at pH 10.0 (Figure 9.9). The resulting free sulfhydryl derivative can be used to label thiol-reactive crosslinkers or to couple with sulfhydryl residues on proteins and other molecules. After activating... 

Conjugates of (strept)avidin with these fluorescent probes may be prepared by activation of the phycobiliprotein with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) to create a sulf-hydryl-reactive derivative, followed by modification of (strept)avidin with 2-iminothiolane or SATA (Chapter 1, Section 4.1) to create the free sulfhydryl groups necessary for conjugation. The protocol for SATA modification of (strept)avidin can be found in Section 3.1, this chapter. The procedure for SPDP activation of phycobiliproteins can be found in Chapter 9, Section 7. Reacting the SPDP-activated phycobiliprotein with thiol-labeled (strept)avidin at a molar ratio of 2 1 will result in highly fluorescent biotin binding probes. 

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. 

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