Facile and General Synthesis of Photoactivatable Xanthene Dyes**

Photoactivatable “caged” fluorophores enable numerous advanced biological imaging experiments, including photoactivated localization microscopy (PALM) and related super-resolution imaging techniques. Of the extant fluorophore scaffolds, caged rhodamines and fluoresceins display properties that are exceptionally well suited for superresolution microscopy, exhibiting high contrast and photon yields. The utility of these probes in PALM imaging has been hampered, however, by inefficient syntheses. For example, the existing route to caged Q-rhodamine, a promising PALM probe, requires harsh, strongly basic conditions and is reported to “generate many products” with yields given as “poor” and “variable”. Such difficult, inefficient syntheses have rendered these important caged molecules unavailable to the scientific community. We set out to develop a general synthesis of photoactivatable xanthene fluorophores and evaluate these dyes as PALM labels. We recognized the synthetic difficulties of caged xanthenes are inextricably linked with the fluorogenic mechanism of the dyes. Rhodamines and fluoresceins exist in equilibrium between a brightly fluorescent, “open” quinoid structure and a colorless, “closed” lactone. This equilibrium can be controlled in a light-dependent manner using several strategies with the most versatile involving attachment of electron withdrawing photolabile groups to the aniline nitrogens of rhodamine or phenolic oxygens of fluorescein. While the open–closed equilibrium is essential for the fluorogenic properties of the caged dyes, this attribute also complicates their syntheses. The quinoid form of the dye, which predominates under the basic conditions required for functionalization, exhibits poor solubility and low reactivity thereby frustrating the installation of caging groups. Since the open–closed equilibrium is both the basis for fluorogenicity and the cause of synthetic difficulties, we envisioned eliminating this process in a reversible manner. Rhodamines and fluoresceins can be reduced to “leuco” derivatives, which are widely used sensors for reactive oxygen species but essentially unexplored as synthetic intermediates for fluorogenic derivatives. We surmised reduction of the xanthene core would increase the reactivity of the aniline nitrogens in rhodamines and the phenolic oxygens in fluoresceins allowing installation of caging groups using mild conditions. Here, we establish the use of leuco-dyes as an effective method to prepare caged fluorophores. This efficient and general route enables the preparation of the elusive caged Q-rhodamine (RhQ) with exceptional ease, and can be extended to rhodamine 110 (Rh110) and 2’,7’-difluorofluorescein (Oregon Green) derivatives bearing free carboxyl groups for bioconjugation. This synthetic method facilitated the evaluation of these probes for super-resolution microscopy, culminating in the first PALM imaging of DNA in a cellular context. Our synthesis of a caged Q-rhodamine is shown in Scheme 1. Condensation of trimellitic anhydride (1) and 7hydroxy-1,2,3,4-tetrahydroquinoline (2) gave a crude isomeric mixture of 5(6)-carboxy-RhQ. [1a] To install base-labile protecting groups on the nitrogen substitutents we treated this material with trifluoroacetic anhydride (TFAA), affording 5(6)-carboxy-RhQ-bis(trifluoroacetamide). The trifluoroacetamides also facilitated isolation of 5-carboxy-RhQ-bis(trifluoroacetamide) (3) by straightforward crystallization. Compound 3 was reduced to the leuco-rhodamine by catalytic