Rhodamines NN: a novel class of caged fluorescent dyes.

Caged (that is, masked) fluorescent dyes are maintained in their nonfluorescent state by the incorporation of a photochemical labile group. The photosensitive masking group or “molecular cage” can be cleaved-off by irradiation with nearUV light, thereby rendering the dye fluorescent. Caged fluorescent dyes are of enormous interest for biological imaging because they may be used, for example, for the analysis of protein dynamics, multicolor fluorescence microscopy, and far-field optical nanoscopy. o-Nitrobenzyl groups are often used as masking groups; however, the use of these dyes is limited because of their rather complex synthesis and the unwanted by-products liberated by photolysis. Herein we report on the synthesis and characterization of a novel class of caged compounds—rhodamine NN dyes, which have a 2-diazoketone (COCNN) caging group incorporated into a spiro-9H-xanthene fragment (compounds 3 and 9-R in Schemes 1 and 3, respectively). This very simple and small caging group is the core element of a new class of masked rhodamines that have remarkable properties. The rhodamine NN dyes can be easily prepared and conjugated with biomolecules, they undergo rapid uncaging under standard irradiation conditions (with wavelengths 420 nm) with formation of highly fluorescent rhodamine derivatives, and they can be used in aqueous buffers, as well as in various embedding media utilized in imaging applications. In microscopy, these novel rhodamines may be used as labels alone or in combination with conventional fluorescent dyes and switchable rhodamine spiroamides. In the latter case, they enable new imaging protocols based on the stepwise activation and detection of several fluorescent markers. The combination of the new rhodamine NN derivative (9-R) with the photochromic spiroamide of rhodamine S and a normal (uncaged) N,N,N’,N’-tetramethylrhodamine resulted in a monochoromatic multilabel imaging scheme with low cross-talk, despite using three fluorophores with very similar absorption and emission spectra. Rhodamines are very photostable and bright fluorescent dyes which can readily be chemically modified and caged. Coumarines and fluorescein have also been used as caged fluorescent dyes. As a photocleavable unit, most of these caged compounds contain a 2-nitrobenzyl group or a derivative with an alkyl or a carboxy group in the a position to the phenyl ring (at the CH2 group) and/or one or two methoxy groups in the aromatic ring. Compounds with a free carboxy group are required for bioconjugation. However, the synthesis of caged rhodamines with a free (“second”) carboxy group is difficult and their yield is low. The 2-nitrobenzyl group and its substitutes are bulky and generate toxic, colored, and highly reactive 2-nitrosobenzaldehyde or 2-nitrosobenzophenone derivatives upon photolysis. These compounds or their oligomers are expected to be poisonous to living cells, and they are also colored and interfere with optical measurements. Other modern caging groups with the required absorption in the near-UV region are also bulky, rather lipophilic, and the procedures for their synthesis and introduction are often complex. For example, 2-(N,N-dimethylamino)-5-nitrophenol was reported to give photocleavable phenyl esters. 7-Diethylamino-4-(hydroxymethyl)-2H-chromen-2-one is known to form esters which can be cleaved easily by irradiation at 412 nm. Derivatives of 8-bromo-7-hydroxyquinoline and 6-bromo-7-hydroxycoumarines have also been proposed as light-sensitive protecting groups. The photolysis of these caged compounds generates light-absorbing by-products. We set out to prepare masked fluorescent dyes without bulky caging groups. A very small 2-diazoketone fragment would be an ideal caging group, provided that it is still possible to integrate this group into the colorless form of a fluorescent dye and then restore the fluorescent state by photolysis. Rhodamines are ideal for this purpose, because they contain a carboxy group, which is known to form colorless and nonfluorescent lactones or lactams with the spiro-9H-xanthene fragment. Furthermore, this carboxy group may be transformed into a 2-diazoketone residue. For the practical realization of this caging strategy, we used rhodamine B as a model compound and performed the reaction of diazomethane with its acid chloride 1. The yellow crystalline diazoketone 3 was obtained in high yield (Scheme 1). In the course of the facile caging reaction, the positively charged C9 atom of the xanthene fragment attacks the negatively charged carbon atom of the diazomethane residue in the intermediate 2. The simultaneous abstraction of a proton furnishes the stable five-membered ring. [*] Dr. V. N. Belov, Dr. C. A. Wurm, Dr. V. P. Boyarskiy, Dr. S. Jakobs, Prof. Dr. S. W. Hell Department of NanoBiophotonics Max Planck Institute for Biophysical Chemistry Am Fassberg 11, 37077 G ttingen (Germany) Fax: (+49)551-201-2506 E-mail: [email protected] [email protected] [email protected] Homepage: http://www.mpibpc.gwdg.de/abteilungen/200/