Aminofluoresceins Versus Fluorescein: Ascertained New Unusual Features of Tautomerism and Dissociation of Hydroxyxanthene Dyes in Solution

Abstract

Within the course of this spectroscopic research, we revealed novel features of the protolytic behavior, which extend the knowledge of the chemistry of xanthene dyes and rationalize the utilization of these compounds. In addition to the well-known tautomerism of the molecular form, H 2 R, of fluorescein dyes, new aspects of tautomeric transformation of anions are disclosed. First, for the dyes bearing the substituents in the phthalic acid residue, 4′-and 5′-aminofluoresceins and 4′-fluorescein isothiocyanate, the monoanion HR − exists in non-hydrogen-bond donor solvents not only as a tautomer with the ionized carboxylic and nonionized OH group but also as a "phenolate" ion with a nonionized COOH group. Such state of HR − ions is typical for dyes bearing halogen atoms or NO 2 groups in the xanthene moiety but was not observed until now in the case of substitution in the phthalic residue. Second, the possibility of the existence of the HR − species in DMSO in the form of colorless lactone is deduced for the 5′-aminofluorescein using the visible and infrared spectra. This results in a dramatic difference in medium effects. For instance, whereas for fluorescein in DMSO, the inversion of the stepwise ionization constants takes place and the K a1 /K a2 value equals 0.08, the same ratio for 5′-aminofluorescein is as high as ∼800. In addition, the pK a values of sulfonefluorescein, erythrosin, methyl ether of fluorescein, and phenol red were obtained to verify the acidity scale in DMSO and to support the detailed scheme of protolytic equilibria of fluorescein dyes.

Protolytic Equilibria in Organized Solutions: Ionization and Tautomerism of Fluorescein Dyes and Related Indicators in Cetyltrimethylammonium Chloride Micellar Solutions at High Ionic Strength of the Bulk Phase

“…Much more complicated is the equilibrium in the case of the main group of fluorescein dyes with the free COOH group. Basing on a series of our previous studies [7,15,31,[37][38][39][40][41][42]47,48,57], we propose the following generalized scheme of a detailed protolytic equilibrium (Scheme 4). The symbols H3R + , H2R, HR − , and R 2− designate the stoichiometric composition irrespective of the molecular (ionic) structure.…”

“…The H2Z ± tautomer was observed mainly for fluorescein in water [7,57,61]. The fraction of the H2Q tautomer decreases while that of H2L increases on going from water to organic solvents [13,30,37,[39][40][41]57,60] or different kinds of organized solutions, such as micellar solutions of colloidal surfactants [7,13,16,17,39], direct or reversed microemulsions [15,39,62], solutions of dendrimers [63], calixarenes [64], and cyclodextrins [65]. Monoanions HR − of HQ − type are usually observed for fluorescein and its derivatives bearing halogen atoms in the phthalic acid residue [15,62] In the case of alkyl fluorescein, alkyl eosin, and some other model compounds the ionization occurs in two steps.…”

“…Accordingly, new works appear on the protolytic and spectral properties of both previously known [28][29][30][31][32] and new [25,[33][34][35] or previously little studied [36][37][38][39][40][41][42] fluorescein compounds, including data analysis in the gas phase [43][44][45].…”

“…Normally, 15 working solutions with various pH values and 20 analytical wave lengths were used for determining the a a pK values of "overlapping" equilibria, Equations (4) and (5). The same procedure was used by us earlier for data processing in different systems [15,37,[39][40][41]. The stepwise ionization constants were calculated by using the dependences A vs. pH at fixed selected wave length and constant total dye concentration and optical path length (Equation ( 6)):…”

“…Normally, 15 working solutions with various pH values and 20 analytical wave lengths were used for determining the pK a a values of "overlapping" equilibria, Equations ( 4) and ( 5). The same procedure was used by us earlier for data processing in different systems [15,37,[39][40][41]. Note, that in the case of 6-hydroxy-9-phenylfluorone (Figure 1a) and ethyl fluorescein and other esters of fluorescein and eosin, the pK a a0 and pK a a1 refer to the dissociation of H 2 R + and HR, respectively.…”