The Pennsylvania Green Fluorophore: A Hybrid of Oregon Green and Tokyo Green for the Construction of Hydrophobic and pH-Insensitive Molecular Probes

Abstract

Fluorescent small molecules are powerful tools for exploring cellular biology. As a more hydrophobic, photostable, and less pH sensitive alternative to fluorescein, we synthesized Pennsylvania Green, a bright, monoanionic fluorophore related to Oregon Green and Tokyo Green.Comparison of membrane probes comprising N-alkyl-3 -cholesterylamine linked to 4-carboxyTokyo Green (pKa ~ 6.2) and 4-carboxy-Pennsylvania Green (pKa ~ 4.8)revealed that only Pennsylvania Green was highly fluorescent in acidic early and recycling endosomes within living mammalian cells.Molecular probes derived from fluorescein (1, Figure 1) are widely used as tools for studies of cellular biology. This green fluorophore is particularly suited for cellular analysis by confocal laser scanning microscopy and flow cytometry due to its excitation maximum at 490 nm, closely matching the 488 nm spectral line of the argon-ion laser. In addition, fluorescein has a high molar absorptivity and excellent quantum yield (0.92 at pH 9). Under physiological conditions (pH 7.4), fluorescein is predominantly a highly hydrophilic dianion. However, the monoanionic form of fluorescein exhibits the relatively high pKa of 6.5, rendering this dye much less fluorescent in acidic solutions. 1 Fluorescein is also relatively susceptible to photobleaching, which is thought to involve reactions with molecular oxygen and proximityinduced reactions of the dye. 2 Oregon Green, a more acidic 2', 7'-difluoro derivative of fluorescein (2, Figure 1), was developed as a less pH-sensitive fluorophore. 3 The appended fluorine atoms reduce the pKa of this dye to 4.8, substantially improving fluorescence at low pH. This compound is also significantly more photostable than fluorescein. However, the high cost and high polarity of Oregon Green limits its utility as a building block for hydrophobic molecular probes.As a more hydrophobic alternative to fluorescein, recent pioneering work by Urano, Nagano, and coworkers replaced the carboxylate of fluorescein with a methyl group. 4 This structural modification yielded a highly fluorescent monoanionic fluorophore termed Tokyo Green (3, Figure 1). This analogue of fluorescein provides a new platform for the design of fluorescent probes.We report here the synthesis of a novel fluorophore termed Pennsylvania Green (4). This fluorophore melds the pH-insensitivity and photostability of Oregon Green with the hydrophobicity of Tokyo Green. To demonstrate the utility of the Pennsylvania Green fluorophore, we compared cellular membrane probes derived from 4-carboxy-Tokyo Green brpeters@chem.psu.edu. (5) and 4-carboxy-Pennsylvania green (6). The lower pKa of the Pennysylvania Green-derived probe enables visualization of early / recycling endosomes within living mammalian cells, and this fluorophore provides a usful tool for analysis of these and related acidic intracellular compartments. NIH Public AccessThe synthesis of 4-carboxy-Pennsylvania Green (6) was accomplished in 10-steps from commercially available 1,2,4-trifluoro-5-nitrobenzene (7)...

Synthesis of fluorinated oxygen- and sulfur-containing heteroaromatics

“…Heating 131a-i with aqueous sodium hydroxide in a sealed tube at 200ºC effected displacement of fluoride which was followed by cyclisation to the target xathones 132a-j in good to excellent yields. It is noteworthy that when R 2 was fluorine, it could be displaced by the hydroxide ion through an addition elimination mechanism presumably facilitated by the para-ketone, whereas the two remaining fluorines remained intact [90,91]. Synthesis of the related thioxanthones was also reported in this paper.…”

Aarhus green: a tetrafluoro-substituted derivative of fluorescein

“…This has been explicitly shown, for example, with the difluorinated derivatives Pennsylvania Green 4 and Oregon Green 3 (see structures in Figure 2). Thus, the fluorinated derivatives become a more useful probe simply because they are strongly fluorescent over a larger pH range.…”

“…For the addition of the aryl group to compound 5, we modified and optimized a procedure originally published by Mottram, et al 4 (Scheme 2). The subsequent ring closure to make the xanthene-related moiety was achieved using a slight modification of a procedure published by Yang, et al 9 In this latter case, we prefer the more easily-handled HBr/AcOH instead of BBr 3 for the ring closure (Scheme 2) despite the need for several additions of HBr/AcOH (possibly due to leakage of HBr from the reaction vessel).…”

“…For these fluorescein derivatives, a carboxyl group often works well. 4,10 As such, we set out to also prepare 5-carboxy-Aarhus Green.…”

“…[2][3][4][5][6][7] Specifically, the quantum efficiency of fluorescence, φ f , for the neutral molecule is small (~ 0.3 -0.4). 4,6 However, under alkaline conditions, where the xanthene-related chromophore/fluorophore exists as the anion, φ f is much larger (i.e., 0.92 for fluorescein). 3,6,8 This point is illustrated in Figure 1.…”