5.6- Dihydroxyindoles, fluorescence

Freshly prepared solutions of pure samples of the aminochromes should not exhibit any fluorescence. However, the ease with which they are converted into highly fluorescent 5,6-dihydroxyindoxyls (see Section IV, B) and 5,6-dihydroxyindoles (see Sections IV, B and IV, C) might lead to some confusion, since solutions of aminochromes contaminated with such compounds would undoubtedly fluoresce (cf. ref. 112). 

Subsequent investigations have shown that Raper s suggestion that dopachrome (4) and related aminochromes decompose by an internal oxidation-reduction process forming 5,6-dihydroxyindoles was essentially correct.73,118,120,184-137 The 5,6-dihydroxyindoles obtained from aminochromes such as dopachrome (4) and epino-chrome (27) (i.e. with no substitution in the 3-position) show only a relatively weak blue to blue-mauve fluorescence.118,120 The intense yellow-green fluorescence shown by the rearrangement products of aminochromes with a 3-hydroxyl group is due to the formation of... 

Reduction of the 7-iodo- and 7-bromo-aminochromes with this reagent gives more complex mixtures of products. The reduction process is accompanied by a considerable amount of dehalogenation in each case, and both the expected halogeno-5,6-dihydroxyindole and the corresponding 5,6-dihydroxyindoles are produced.155 Traces of products, similar to the unidentified fluorescent product obtained from adrenochrome, were usually also detected chromatographically, together with several minor unidentified products.155... 

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... 

A characteristic subgroup of 5,6-dihydroxyindoles is represented by the 3,5,6-trihydroxyindoles (e.g. 4b), also known as 5,6-dihydroxyindoxyls or lutins , which exist almost exclusively in the 3-keto form (i.e. 4a). These are crystalline solids which form deep-yellow solutions in water and exhibit a typical intense green fluorescence that has provided the basis of several tests for catecholamines in biological fluids (59CR181). When pure, both anhydrous 3,5,6-trihydroxy-1-methylindole 4... 

The 3-alkoxyaminochromes rearrange to form non-fluorescent compounds, probably 3-alkoxy-5,6-dihydroxyindoles [160] (e.g. adrenochrome methyl ether (51) would be expected to rearrange to 5,6-dihydroxy-3-methoxy-Af-methylindole (70)). However, these indole derivatives appear to be readily oxidis in air and have not yet been isolated in pure form. 

Aminochromes without a substituent in the 3-position rearrange with great facility to 5,6-dihydroxyindoles [e.g. epinochrome (71) to 5,6-dihy-droxy-W-methylindole (44)], which exhibit a characteristic blue fluorescence. As a result of a study of the kinetics of the rearrangement, Heacock and Mattok proposed the mechanism of the reaction to be as shown in Scheme 6.2 [161]. 

The specificity of this method is such that it requires a catechol nucleus, a -hydroxy substituent and an alkylamine on the a-carbon. Since compounds without a /5-hydroxyl, however, undergo similar reactions to produce dihydroxyindoles with similar fluorescence properties, the presence of DA or DOPA may interfere with the assay. 

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