Fluorophore-forming reactions

The fluorescence of a good acid-base indicator should be in the visible region, and either the indicator H I or its ionized form I (charges are omitted) may be the fluorophore (see reaction [III]). 

Cascade Blue cadaverine and Cascade Blue ethylenediamine both contain a carboxamide-linked diamine spacer off the 8-methoxy group of the pyrene trisulfonic acid backbone. The cadaverine version contains a 5-carbon spacer, while the ethylenediamine compound has only a 2-carbon arm. Both can be coupled to carboxylic acid-containing molecules using a carbodiimide reaction (Chapter 3, Section 1). Since Cascade Blue derivatives are water-soluble, the carbodiimide EDC can be used to couple these fluorophores to proteins and other carboxylate-containing molecules in aqueous solutions at a pH range of 4.5-7.5. The reaction forms amide bond linkages (Figure 9.39). 

A potentially more sensitive and selective approach involves reaction of formic acid with a reagent to form a chromophore or fluorophore, followed by chromatographic analysis. A wide variety of alkylating and silylating reagents have been used for this purpose. Two serious drawbacks to this approach are that inorganic salts and/or water interfere with the derivatisation reaction, and these reactions are generally not specific for formic acid or other carboxylic acids. These techniques are prone to errors from adsorption losses, contamination, and decomposition of the components of interest. Enzymic techniques, in contrast, are ideal for the analysis of non-saline water samples, since they are compatible with aqueous media and involve little or no chemical or physical alterations of the sample (e.g., pH, temperature). 

The brilliant emissions resulting from the oxidation of certain oxalic acid derivatives, especially in the presence of a variety of fluorophores, are the bases of the most active area of current interest in CL. This group of chemiluminescent reactions has been classified as peroxyoxalate chemistry because it derives from the excited states formed by the decomposition of cyclic peroxides of oxalic acid derivatives called dioxetanes, dioxetanones, and dioxetanediones. 

For a reaction to produce detectable CL emission, it must fulfill the following conditions (1) it should be exothermic so that sufficient energy for an electronically excited state to be formed (at least 180 kJ/mol for emission in the visible region) can be provided (2) there should be a suitable reaction pathway for the excited state to be formed and (3) a radiactive pathway (either direct or via energy transfer to a fluorophore) for the excited state to lose its excess energy should exist. 

Peroxyoxalate-based CL reactions are related to the hydrogen peroxide oxidation of an aryl oxalate ester, producing a high-energy intermediate. This intermediate (l,2-dioxetane-3,4-dione) forms, in the presence of a fluorophore, a charge transfer complex that dissociates to yield an excited-state fluorophore, which then emits. This type of CL reaction can be used to determine hydrogen peroxide or fluorophores including polycyclic aromatic hydrocarbons, dansyl- or fluores-camine-labeled analytes, or, indirectly, nonfluorescers that are easily oxidized (e.g., sulfite, nitrite) and quench the emission. The most widely used oxalate... 

Extrinsic fluors are produced via a chemical reaction where the added reagent either enhances emission of a weak emitter through association or the analyte is derivatized with a fluor tag. 8-Hydroxyquinoline (HQS) is an example of an extrinsic complexing reagent (Reaction 11.1) where the native ligand is a marginal fluorophore but forms intense emitting metal chelates. This approach affords sensitive detection of... 

One of the first fluorescence-based ee assays uses umbelliferone (14) as the built-in fluorophore and works for several different types of enzymatic reactions 70,86). In an initial investigation, the system was used to monitor the hydrolytic kinetic resolution of chiral acetates (e.g., rac-11) (Fig. 8). It is based on a sequence of two coupled enzymatic steps that converts a pair of enantiomeric alcohols formed by the asymmetric hydrolysis under study (e.g., R - and (5)-12) into a fluorescent product (e.g., 14). In the first step, (R)- and (5)-ll are subjected separately to hydrolysis in reactions catalyzed by a mutant enzyme (lipase or esterase). The goal of the assay is to measure the enantioselectivity of this kinetic resolution. The relative amount of R)- and ( S)-12 produced after a given reaction time is a measure of the enantioselectivity and can be ascertained rapidly, but not directly. 

Assay procedures for dopamine which are superficially similar to the lutin procedure described above have been reported recently.266-268 The chemistry of the production of the fluorophore from dopamine is, however, somewhat different since the fluorophore is not a 5,6-dihydroxyindoxyl, it is incorrect to refer to the trihy-droxyindole fluorophore of dopamine (cf. ref. 252). Oxidation of the extracted catecholamine is usually carried out with iodine,266-268 presumably with the formation of 7-iodonorepinochrome. The aminochrome is subsequently rearranged to 5,6-dihydroxyindole (it is probable that deiodination accompanies the rearrangement in this case) by a solution of sodium sulfite in aqueous alkali the solution is acidified before measuring the fluorescence of the product (which is said to form relatively slowly and to be very stable).266-268 Irradiation of the reaction mixture with ultraviolet light accelerates the maximal development of fluorescence.266 Since acidification will produce sodium bisulfite in the reaction mixture, it is probable that the fluorophore is a 5,6-dihydroxyindole-sodium bisulfite addition complex. Complexes of this type are known to be both fluorescent and relatively stable in dilute acid solution.118 123,156 265 They also form relatively slowly.255... 

In this expression, aB is the probability that a collision leads to reaction and the term u(E — E ) is added to require that the energy which a pair of reactants A and B have prior to collision [E = 2m(t>f + vl)] is greater than a minimum energy, E, before reaction can occur that is the chemical reaction is activated. After reaction, the deactivated fluorophor, C, can be regarded as a solvent molecule, S, for all intents and purposes, because the reaction is irreversible. However, this means that no A is formed from C. The first of the two delta functions in the expression for TrB may be dropped, i.e. the term involving b12 J AC can be dropped. When this is done, the forward reactive T operator is... 

Fluorescamine reacts with primary amines to form fluorophores (see Fig. B2.2.4) that are excited at 390 nm and fluoresce at 475 nm. Peptides react with fluorescamine at pH 7.0, giving higher fluorescence than amino acids, which have maximum fluorescence at pH 9. The reaction proceeds rapidly with primary amines at 25°C. The resulting fluorescence is proportional to the amine concentration. The fluorophores are stable for several hours. A negligible interference is produced with ammonia. 

AMCA-HPDP is JV-[6-(7-amino-4-methylcoumarin-3-acetamido)hexyl]-3 -(2 -pyridyldithio)propionamide. It is formed from AMCA plus a 1,6-diaminohexyl spacer off the carboxylate that has been additionally modified at its other end with SPDP (Chapter 5, Section 1.1). The result is a long spacer arm terminating in a pyridyl disulfide group reactive toward free sulfhydryl residues. The reaction of this group with a thiol creates a disulfide bond between the AMCA fluorophore and the molecule being modified. Thus, the fluorescent tag can be specifically cleaved by reduction with DTT or other disulfide reducing agents (Fig. 222). 

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