Fluorescence derivatization thiols

A new cyanide dye for derivatizing thiols has been reported (65). This thiol label can be used with a visible diode laser and provide a detection limit of 8 X 10 M of the tested thiol. A highly sensitive laser-induced fluorescence detector for analysis of biogenic amines has been developed that employs a He—Cd laser (66). The amines are derivatized by naphthalenedicarboxaldehyde in the presence of cyanide ion to produce a cyanobenz[ isoindole which absorbs radiation at the output of He—Cd laser (441.6 nm). Optimization of the detection system yielded a detection limit of 2 x 10 M. 

For those without native fluorescence, two common approaches have been employed, namely, derivatization and indirect fluorescence. Derivatizing agents should be pure, low fluorescent, and stable, as well as react quickly and uniquely with the analytes and, thus, formed compounds should be strongly fluorescent and stable. These include dansyl chloride, fluorescamine, 4-clair-7-nitro-benz-2-oxa-l,3-diazole (NBD), o-phthaldialdehyde (OPA), fluorescein isothiocyanate (FITC), and naphthalene-2,3-dicarboxaldehyde (NDA), which have been used for the analyses of amino acids, peptides, proteins, thiols, and sugars with LOD in 1-100 nM range. Compared to LINF, approaches based on derivatization provide the advantages of relatively low cost and versatility in instrumentation, but they may suffer from contamination and loss of temporal information. 

Izquierdo-Alvarez A, Martinez-Ruiz A (2011) Thiol redox proteomics seen with fluorescent eyes the detection of cysteine oxidative modifications by fluorescence derivatization and 2-DE. J Proteomics 75(2) 329-338. doi 10.1016/ j.jprot.2011.09.013... 

A wide range of fluorescent derivatization reagents and techniques have been developed to utilize the analytical advantages that fluorescence offers. These derivatization reagents are usually specific to functional groups (e.g., amine, hydroxy, thiol), and their specificity offers yet another opportunity for discriminating against interferents. For excellent reviews, the reader is referred to references [38] and [39]. 

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). 

At this point, the anaiyte may not be amenabie to UV, FL, or EC detection. In this case, the best course of action may be to choose LC/MS (see Section 4.2). However, one other option is to use a pre- " or post-coiumn derivatization step to increase the detectabiiity of the anaiyte with respect to FL or UV. Fluorescent or UV labels are available for carboxylic acids," amines, phenols, and thiols. The decision to use pre- or post-column derivatization is predicated upon the functionality of the analyte available for derivatization and the rate and extent of the reaction between each derivatizing agent and the analyte. 

Fluorescent probes containing sulfhydryl-reactive groups can be coupled to DNA molecules containing thiol modification sites. The chemical derivatization methods outlined in Section 2.2 (this chapter) may be used to thiolate the oligo for subsequent modification with a fluorophore. Appropriate fluorescent compounds and their reaction conditions may be found in Chapter 9. The protocol discussed in the previous section can be used as a general guide for labeling DNA molecules. 

The microspheres—synthesised via a two-step process (acid-catalysed hydrolysis and condensation of 3-mercaptopropyltrimethoxysilane (MPS) in aqueous solution, followed by condensation catalysed by triethanolamine)—have a narrow size distribution (Figure 5.16) and are considerably more stable than polystyrene divinylbenzene microspheres as shown in phosphoramidite oligonucleotide synthesis by the excellent retention of fluorescence intensity in each of the reagent steps involved in phosphoramidite DNA synthesis (Figure 5.17, in which the organo-silica microsphere free thiol groups are derivatized with ATTO 550 maleimide coupled to the entrapped dye). 

Since BAs occurring in food do not exhibit satisfactory absorbance or fluorescence in the visible or ultraviolet range, chemical derivatization, either pre- (35-37) or postcolumn (38), is usually used for their detection in HPLC. The most frequently employed reagents for precolumn derivatization are fluorescamine, aminoquinolyl-lV-hydroxysuccinimidyl carbamate (AQC) (39, 40), 9-fluorenylmethyl chloroformate (FMOC) (41-43), 4-dimethylaminoazobenzene-4 -sul-fonyl chloride (dabsylchloride, DBS) (44), N-acetylcysteine (NAC) (45,46), and 5-dimethyl-amino-1-naphthalene-1-sulfonyl chloride (dansylchloride, DNS) (47,48), phthalaldehyde (PA), and orf/to-phthaldialdehyde (OPA) (49-51), together with thiols such as 3-mercaptopropionic acid (MPA) (37) and 2-mercaptoethanol (ME) (35,49). 

Further derivatization of DTPA bis-anhydride has led to the introduction of ferric-selective moieties (20)116-1. Other derivatives of DTPA produced are DTPA - bis(-3-ami-do-4-thiol-benzotrifluoride), a chelating agent which can be monitored by 19F-NMR116, and a fluorescent DTPA derivative formed by reacting the bisanhydride with 7-amino-4-methyl coumarin116. Reaction of ethylene sulphide with HPDTA leads to the 0-2-ethanethiol derivative (21)116. ... 

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... 

Derivatization is a technique that is most commonly performed prior to UV absorption or fluorescence detection. It is not restricted to these detection modes, however, and postcolumn electrochemical31 and postcolumn chemiluminescence32 detection have also been reported. Table 3.6 provides a list of some of the more common derivatizing agents and the compounds with which they react.33 Derivatization has also been used to aid in the detection of compounds such as amino acids,34 amines,35 saccharides,36 thiols,37 carboxylic acids,38 steroids,39 alcohols,40 fatty acids 41 and several inorganic species.42... 

Park, S.K., Boulton, R.B., Noble A.C. (2000). Automated HPLC analysis of glutathione and thiol-containing compounds in grape juice and wine using pre-column derivatization with fluorescence detection. Food Chem., 68, 475-480. 

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... 

Reversed-phase HPLC with fluorescence detection, after derivatization of plasma thiols with ammonium 7-fluorobenzo-2-oxa-l,3-diazole-4-sulphonate (SBD-F), is the most widely used method to determine total plasma amino thiols (cysteine, cysteinylglycine, and homocysteine). The time required for sample preparation (thiolic reduction, deproteinization, and precolumn derivatization g with SBD-F) and for thiol derivatives separation is nearly 2 h per sample. =... 

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