Amines, fluorescent derivatives

An excellent discussion on derivatization techniques has been given by Lawrence (17) including a detailed discussion on pre-column derivatization (18) and post-column derivatization (19). Probably, the more popular procedures are those that produce fluorescing derivatives to improve detector sensitivity. One of the more commonly used reagents is dansyl chloride (20), 5-dimethylamino-naphthalene-1-sulphonyl chloride (sometimes called DNS-chloride or DNS-C1). The reagent reacts with phenols and primary and secondary amines under slightly basic conditions forming sulphonate esters or sulphonamides. 

Another reagent that readily forms fluorescent derivatives with primary amines is o-phthalaldehyde (trade name "Fluoropa"). The reaction proceeds in aqueous solution in the presence of a mercaptan at a pH of 9-11 producing an isoindole. 

Reports on the use of fluorescent derivatives abound (5). Some reagents have become widely used. The dansyl group is probably the most thoroughly studied. Dansyl chloride has been widely used as a fluorescent derivatizing reagent for HPLC (6,7). It reacts readily with primary and secondary amino groups (7) and with phenols (8), but forms derivatives of alcohols very slowly (9). The lower detection limit for dansyl derivatives of aliphatic amines is in the range of 300 femtomoles per injection. 

This fluorescent acid chloride can be used to form derivatives of alcohols, amines, and phenols. Using these fluorescent derivatives, an analysis of a series of n-alcohols from Ci to C4 was developed. A chromatogram produced by this technique is shown in Figure 3. Derivatives were also formed from ammonia, dimethylamine, and phenol. A derivative was formed from pentachlorophenol but was not fully characterized. The quantum yields of fluorescence of the alcohol derivatives of V were lower than those of the alkyl halide derivatives of III. 

Primary aliphatic and aromatic amines [3, 14], yield fluorescent derivatives. Primary [15] aromatic amines yield stable yellow-colored derivatives that can be eluted from the TLC layer [16]. 

In the first step tin(Il) chloride in acetic acid solution reduces the aromatic nitro groups to amino groups. The aromatic amines produced then react with fluorescamine in weakly basic medium to yield fluorescent derivatives (cf. reagent monograph Fluorescamine Reagent , Volume la). 

Fluorescamine reacts directly with primary amines to yield fluorescent derivatives of the general formula 1. On the other hand, secondary amines react in weakly basic medium to yield nonfluorescent derivatives of type II after the hydrolysis of excess fluorescarrtine, these are converted to fluorescent products of type I by reaction with a primary amine, e.g. taurine. 

Note Primary amines yield fluorescent chromatogram zones even before the application of reagent 3. Secondary amines do not yield fluorescent derivatives until they have been treated with reagent 3. Hence, the reagent sequence allows the stepwise detection of primary and secondary amines. Taurine is preferred as the essential component of reagent 3 over the multiplicity of other possibilities because it produces intense fluorescence it is also not very volatile and is readily available. Amides and substances with peptide linkages, eg. hippuric acid, are not detected, neither are secondary amines that are volatile at high temperatures. 

Two general forms of amine-reactive fluorescein probes are available. Both of them react under alkaline conditions with primary amines in proteins and other molecules to form stable, highly fluorescent derivatives. 

Three main forms of amine-reactive AMCA probes are commonly available. One of them is simply the free acid form of AMCA, which can be used to couple to amine-containing molecules using the carbodiimide reaction (Chapter 3, Section 1.1). The other two are active-ester derivatives of AMCA—the water-insoluble NHS ester and the water-soluble sulfo-NHS ester forms—both of which spontaneously react with amines to create stable amide linkages. All of them react under mild conditions with primary amines in proteins and other molecules to form highly fluorescent derivatives. 

A number of BODIPY derivatives that contain reactive groups able to couple with amine-containing molecules are commonly available. The derivatives either contain a carboxy-late group, which can be reacted with an amine in the presence of a carbodiimide to create an amide bond, or an NHS ester derivative of the carboxylate, which can react directly with amines to form amide linkages. The three discussed in this section are representative of this amine-reactive BODIPY family. The two NHS ester derivatives react under alkaline conditions with primary amines in molecular targets to form stable, highly fluorescent derivatives. The carboxylate derivative can be coupled to an amine using the EDC/sulfo-NHS reaction discussed in Chapter 3, Section 1.2. 

BODIPY 530/550 C3 is insoluble in aqueous solution, but it may be dissolved in DMF or DMSO as a concentrated stock solution prior to addition of a small aliquot to a reaction. Coupling to amine-containing molecules may be done using the EDC/sulfo-NHS reaction as discussed in Chapter 3, Section 1.2 (Figure 9.29). However, modification of proteins with this fluorophore probably won t yield satisfactory results, since BODIPY fluorophores are easily quenched if substitutions on a molecule exceed a 1 1 stoichiometry. For labeling molecules which contain only one amine group, such as DNA probes modified at the 5 end to contain an amine (Chapter 27, Section 2.1), BODIPY 530/550 C3 will give intensely fluorescent derivatives. 

Amines are another important group of analytes. Mellbin and Smith [72] compared three different fluorescent reagents, dansyl chloride, 4-chloro-7-nitrobenzo-1,2,5-oxadiazole, and o-phthaldialdehyde, for derivatization of alkylamines. The dansyl tag was found to be the most effective. Hamachi et al. [73] described the application of an HPLC-POCL method for determination of a fluorescent derivative of the synthetic peptide ebiratide. Another comparative study was done by Kwakman et al. [74], where naphthalene-2,3-dialdehyde and anthracene-2,3-dial-dehyde were evaluated as precolumn labeling agents for primary amines. The anthracene-2,3-dialdehyde derivatives were not stable, especially in the presence of hydrogen peroxide, and the POCL detection of these derivatives was therefore... 

N-methylcarbamate and N,N -dimethylcarbamates have been determined in soil samples by hydrolyses with sodium bicarbonate and the resulting amines reacted with 4-chloro-7-nitrobenzo-2,l,3-Oxadiazole in isobutyl methyl ketone solution to produce fluorescent derivatives [81]. These derivatives were separated by thin layer chromatography on silica gel G or alumina with tetrahydrofuran-chloroform (1 49) as solvent. The fluorescence is then measured in situ (excitation at 436 nm, emission at 528 and 537nm for the derivatives of methylamine and dimethylamine respectively). The... 

Dansyl Chloride (afluorotag) is invariably used to obtain fluorescent derivatives of proteins, amines and phenolic compounds, the excitation and emission wavelengths being 335 to 365 nm and 520 nm respectively. 

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

The separation mechanism in the LC analysis of aminoglycosides is usually highly dependent on the applied derivatization technique, either precolumn or postcolumn. This is due to the fact that a prerequisite of aminoglycosides analysis is most often suitable derivatization to produce fluorescent derivatives the presence of primary amine groups in most of the aminoglycoside antibiotics enables a number of derivatives to be readily formed. 

A procedure for the fluorimetric determination of 3-methoxytyra-mine was described by Carlsson and Waldeck in 1964.304 This procedure, which resembled the dopamine assay procedures previously described (cf. Section V, E, 3), involved the oxidation of the amine with iodine, presumably to an aminochrome (cf. the oxidation of meta-nephrine (Section II, D, 1)] which was subsequently rearranged to a fluorescent derivative.304... 

Figure 3.16—Flow cell of a fiuorimetric detector (reproduced by permission of Hewlett-Packard Inc.). One example of a reagent that is used to form fluorescent derivatives with compounds containing primary amines. Reaction of OPA in the presence of monothioglycol.

Figure 15. Open tubular liquid chromatography of amine-NBD derivatives using on-column fluorescence detection. Peaks correspond from left to right to ethylamine, n-propylamine, n-butylamine, cyclohexylamine, and n-hexylamine. Conditions 20-p.m X 8.3-m column with C-18 bonded phase 20% acetonitrile and 80% water (v/v) mobile phase at a linear velocity of 0.50 cm/s on-column injection of 5 nL. (Reproduced from reference 59. Copyright 1984 American Chemical Society.)...

Since BODIPY fluorophores are easily quenched if substitutions on a molecule exceed a 1 1 stoichiometry, modification of proteins with this fluorophore probably will not yield satisfactory results. However, for labeling molecules that contain only one amine group, BODIPY FL C3-SE will give intensely fluorescent derivatives. 

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