Photoactivable bioluminescent probes for imaging luciferase activityw

The firefly luciferase (fLuc) has been widely utilized for optical reporter gene imaging in biochemical assays, cell culture and living animals. fLuc can catalyze the oxidation of its substrate D-luciferin in the presence of O2, ATP, and Mg 2+ to generate the bioluminescence that can be imaged by a CCD camera. The luciferase based bioluminescence imaging technique displays low background and high sensitivity compared to fluorescence imaging modality, since fLuc nonexpression cells and tissues do not produce significant bioluminescence during normal cellular processes. Therefore, it has been extensively applied for noninvasive imaging of gene expression, studying in vivo cell trafficking and detecting enzyme activities such as for b-galactosidase, caspases, monoamine oxidase and b-lactamase. Normally, cellular structures exhibit complex spatiotemporal organization. The precise tracking of the dynamic properties of cellular functions and repetitive monitoring of reporter genes at a desired time and/or location in intact cells, tissues or living animals will be crucial for many biomedical applications including cell trafficking, gene therapy studies and transgenic model engineering. The most notable and promising strategy for this purpose is the photolysis of photoactivable or ‘‘caged’’ molecules, by which the activation process can be readily modulated by a beam of light with high spatial and temporal precision. In line with this direction, several photocaged fluorophores have been reported for fluorescent labeling of proteins, monitoring the gene silencing and real-time imaging dynamics of cell–cell coupling in vivo. However, simple and specific photoactivable luciferase probes for efficient imaging of bioluminescence reporter gene in living animals are unavailable, since most of the current ‘‘caged’’ luminescence substrates are less stable, unable to be applied in the living animals or only limited to in vitro fluorescent detection. Here we present one set of stable and novel photo-releasable luciferin derivatives and report the first-time study of imaging luciferase activity using photocage technology in living mice. These compounds were designed by masking the 6-hydroxy group with different cage groups (3a–c, Scheme 1), and all of them showed rapid uncaging and significant bioluminescence enhancement upon UV irradiation. Their inherent fluorescent properties were studied and their bioluminescent properties were further investigated by using fLuc as reporter enzyme in buffer, cell and living animals. As shown in Scheme 2, the chemical synthesis of caged bioluminescent derivatives was started from alkylation of 2-cyano-6-hydroxybenzothiazole under basic conditions with different cages: 2-nitrobenzyl (NB, 3a), 4,5-dimethoxy2-nitrobenzyl (DMNB, 3b) and 1-(2-nitrophenyl)ethyl (NPE, 3c). Then the alkylated transmediates were directly condensed with D-cysteine, followed by ring cyclization to provide compounds 3a–c with reaction yields of 83.4, 87.6 and 75.8%, respectively (see ESIw). Care needs to be taken to minimize the free luciferin as trace amounts of luciferin contamination would result in a significant bioluminescent background. After obtaining the precursors, the photochemical properties of the ‘‘caged’’ luciferin derivatives were evaluated. These compounds did not show fluorescence and bioluminescence signals before photolysis, indicating the substrates were Scheme 1 The structure of the ‘‘caged’’ D-luciferin derivatives and proposed photolysis reaction for detection of fLuc.