Amine Detection Reagents

A succinylated casein derivative that has nearly all its amines blocked can be used as a substrate in protease assays (Hatakeyama etal., 1992). As the casein is degraded by a protease, free amines are created from a-chain cleavage and release of a-amino groups. The creation of amines can be monitored by an amine detection reagent such as trinitrobenzene sulfonic acid (TNBS Section 4.3). The procedure forms the basis for a highly sensitive assay for protease activity. 

Succinylated derivatives of nucleic acids may be prepared by reaction of the anhydride with available —OH groups. The reaction forms relatively stable ester derivatives that create carboxylates on the nucleotide for further conjugation or modification (Fig. 75). This method has been used in nucleic acid synthesis (Matteucci and Car-uthers, 1980) and to derivatize nucleotide analogs such as AZT (Tadayoni et al., 1993). 

Succinic anhydride also is a convenient extender for creating spacer arms on chromatography supports. Supports derivatized with amine-terminal spacers may be succinylated to block totally the amine functional groups and form terminal carboxylic acid linkers for coupling amine-containing affinity ligands (Cuatrecasas, 1970). 

Molecules modified with succinic anhydride to create terminal carboxylate functional groups may be further conjugated to amine-containing molecules by use of amide bond-forming reagents such as carbodiimides (Chapter 3, Section 1). 

Add a quantity of succinic anhydride to the reaction medium to provide at least a 5-10 molar excess of reagent over the amount of amines to be modified. Even greater molar excesses may be required for total blocking of all the amines of some proteins. When adding solid succinic anhydride, multiple additions may 

There are several methods available for the detection or measurement of amine groups in proteins and other molecules. Accurate determination of target amine groups in molecules before or after modification may be important for assessing reaction yield or suitability for subsequent crosslinking procedures. The following methods use commercially available reagents and are easily employed to detect primary amines with simple spectrophotometric measurement. 

TNBS has been used to measure the free amino groups in proteins (Habeeb, 1966 Kakade and Liener, 1969), as a qualitative check for the presence of amines, sulfhydryls, or hydrazides (Inman and Dintzis, 1969), and to specifically determine the number of s-amino groups of L-lysine in carrier proteins (Sashidhar et al., 1994). 

The following protocol may be used for the measurement of amines in soluble molecules, such as proteins or other macromolecules. 

Dissolve or dialyze the molecule to be assayed into 0.1M sodium bicarbonate, pH 8.5, at a concentration of 20-200 pg/ml (for large molecules like proteins) or 2-20 pg/ml (for small molecules like amino acids). 

Molecules containing primary amines or hydrazide groups can react with 2,4,6-trinitrobenzenesulfianate (TNBS) to form a highly chromogenic derivative (Fig. 90). This reaction may be used to assay the amine content of compounds by measuring the absorbance of the orange-colored product at 335 nm. 

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O-Phthaldialdehyde (OPA) is an amine detection reagent that reacts in the presence of 2-mercaptoethanol to generate a fluorescent product (for preparation, see Section 4.1, 2-mercaptoethanol) (Fig. 91). The resultant fluorophore has an excitation wavelength of 360 nm and an emission point at 455 nm. OPA can be used as a sensitive detection reagent for the HPLC separation of amino acids, peptides, and proteins (Fried et al., 1985). It is also possible to measure the amine content in proteins and other molecules using a test tube or microplate format assay with OPA. Detection limits are typically in the microgram per milliliter range for proteins. 

Blocking of amine groups on proteins also has been used to create a sensitive reagent for measuring protease activity (Hatakeyama etal., 1992). With nearly all the primary amines of casein blocked, an amine detection reagent such as trinitrobenzene sulfonic acid (TNBS) will only minimally react with the protein and form its typical orange derivative. As proteases cleave the protein, however, primary a-amines are created from cleavage of the a-chain peptide bonds, and TNBS can react with them. The more protease activity present, the more color is formed. 

Note It is reported that the use of chlorobenzene as solvent is essential when the reagent is to be used to detect aromatic amines [1]. In the case of steroids, penicillins, diuretics and alkaloids the reaction should be accelerated and intensified by spraying afterwards with dimethylsulfoxide (DMSO) or dimethylformamide (DMF), indeed this step makes it possible to detect some substances when this would not otherwise be possible [5,9-11] this latter treatment can, like heating, cause color changes [5,9]. Penicillins and diuretics only exhibit weak reactions if not treated afterwards with DMF [10, 11]. Steroids alone also yield colored derivatives with DMSO [9]. Tlreatment afterwards with diluted sulfuric acid (c = 2 mol/L) also leads to an improvement in detection sensitivity in the case of a range of alkaloids. In the case of pyrrolizidine alkaloids it is possible to use o-chloranil as an alternative detection reagent however, in this case it is recommended that the plate be treated afterwards with a solution of 2 g 4-(dimethyl-amino)-benzaldehyde and 2 ml boron trifluoride etherate in 100 ml anhydrous ethanol because otherwise the colors initially produced with o-chloranil rapidly fade [12]. 

Fluorobenzene-type compounds have been used as functional groups in homobifunctional crosslinking agents (Chapter 4, Section 4). Their reaction with amines involves nucleophilic displacement of the fluorine atom with the amine derivative, creating a substituted aryl amine bond (Reaction 9). Detection reagents incorporating reactive aryl chemistry include 2,4-dinitrofluorobenzene and trinitrobenzenesulfonate (Eisen et al., 1953). These compounds form... 

Indications. An orange, red-orange, or brown-orange precipitate suggests the presence of an alkaloidal base (precipitated as the alkaloidal bismuth iodide). Positive for primary, secondary, tertiary, and quaternary amines. This reagent is commonly used as a spray for detecting alkaloids on thin-layer chromatographic plates. 

Goto et al. (67) synthesized the sucdnimidyl ester [14] of (—)- -methoxy-a-methyl-l-naphthaleneacetic acid for the normal-phase LC resolution of chiral amines. The reagent permitted the determination of the enantiomers of an amphetamine derivative in blood plasma after administration of racemic drug to rabbits. With detection at 280 nm, the lower limit of sensitivity was 5 ng/mL for each enantiomer (67). Several chiral acids from the "profen" group of nonsteroidal antiinflammatory drugs have been adapted as CDAs. One of these, naproxen, [15], is the S enantiomer and is commercially available as the resolved acid several of these acids have the advantage of providing fluorescent derivatives (68,69). 

In addition to application by exposure to vapor (see Section III.A.l), dipping, or spraying, detection reagents can be incorporated in the mobile phase if they spread uniformly over the layer and move with the solvent front during development. Reagents successfully added to the mobile phase include acids for detection of quinine alkaloids and fluorescamine for biogenic amines (Kovar and Morlock,... 

TLC studies on amino acids can be performed on a variety of sorbents. Blackburn (1989) has provided 56 tables showing TLC data and separations of amino acids and amines using various combinations of layers, mobile phases, and detection reagents. Information on both free amino acids and amino acid derivatives was included. 

Many alkaloids are derivatives of tertiary amines and, as the name implies, are alkaline in nature. Numerous drugs of abuse as well as abused drugs are alkaloids. An extensive list of alkaloids, their sample preparation, solvent systems, and detection reagents have been given by Wagner et al. (1984). 

Other solvent systems and detection reagents are recommended for the identification and confirmation of drugs and their metabolites. These include ethyl acetate-methanol-concentrated ammonium hydroxide (90 7 3) with detection by fluorescamine, diphenylcarbazone, and iodoplatinate reagents for D-amphet-amine, barbiturates, and acidic drugs (e.g., phenobarbital, Dilantin, Mellaril, Valium, chlorpromazine, Lomotil, and Talwin) and ethyl acetate-methanol-concentrated ammonium hydroxide-water (85 13.5 0.5 1.0) with detection by... 

The silver nitrate detection reagent functions by reduction of Ag to metallic silver by the UV light at the location of the chlorinated herbicides on the layer. Chlorpropham is detected by hydrolysis with HCl to a primary amine, followed by diazotization with NaN02 and coupling with the Bratton-Marshall reagent, A-( 1 -naphthyl)ethylenediamine. 

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