4,5‐Dinitrosulfonefluorescein and related dyes: Kinetics of reversible rupture of the pyran ring and their interaction with lysozyme

Abstract

The rupture of the pyran ring of fluorescein and rhodamine dyes is well documented. [1][2][3][4][5] For the mother compound fluorescein (1), the process occurs in extremely alkaline media. According to Orndorff et al, 3 in cold 33% aqueous potassium hydroxide (KOH) first the absorption band at 496 nm is only observed, whereas a new band with λ max = 583 nm (2) appears only after 5-10 weeks (Scheme 1). (It should be taken into account that, according to Lewis, these λ max values are 10-20 nm overestimated. 6 ) However, in hot aqueous KOH of the same concentration the last-named band appears immediately.Accordingly, the intensity of the first band decreases, but over time the solution is discoloured owing to formation of carbinol 3. 3 The latter is a result of nucleophilic attack of the hydroxide (HO -) on the nodal carbon atom. Even earlier, Meyer and Fischer observed the spectrum of the "violet fluorescein" after boiling fluorescein with sodium hydroxide (NaOH). 2 Analogous reactions were described for rhodamine dyes; further research is summarised in the corresponding review. 1 For eosin, the opening of the heterocycle occurs at markedly lower alkali concentration. 4 Contrary to halogen derivatives of triphenylmethine and hydroxyxanthene dyes, the nitro derivatives are largely ignored. However, as early as 1900-1902 it was already known that in alkaline solutions, 2,7-dibromo-4,5-dinitrofluorescein and 4,5-dinitrofluorescein gradually turned from yellow to deep blue, and the last dye becomes colourless after action of hot alkali over time. 7,8 El'tsov et al 9 proved the results for the last-named dye spectroscopically. In a 0.1 mol/L sodium hydrogen carbonate (NaHCO 3 ) aqueous solution, the initial dye exists in the form of a double-charged anion (λ max = 482 nm), while at pH > 11 the pyran cycle opens; λ max = 580 nm. The