Synthesis and properties of lysosome-specific photoactivatable probes for live-cell imaging

We describe the synthesis and application of a new class of large Stokes shift lysosome-specific photoactivatable probes for live-cell imaging.


Introduction
Fluorescence microscopy is a powerful tool that is universally employed to study biological processes at the cellular level. [1][2][3] Many uorescent dyes targeting a multitude of organelles and subcellular targets have been developed. 4,5 Photoactivatable dyes are an important but rare class of probes allowing for spatial and temporal control during imaging studies. [6][7][8][9][10][11] Photoactivatable dyes can be grouped into two broad categories, the rst switching from a dark state to a uorescent state and the second converting from one uorescent state to another uorescent state. [12][13][14] The later are oen referred to as photoconvertible dyes. Each category has its own merit depending on the experimental conditions. Photoconvertible dyes have the added advantage of being able to track the pre-activated state, although few examples of useful dyes in this category currently exist. 15,16 A combined Cy5-Cy3 probe was introduced by Johnsson et al. in 2010 as a photoconvertible protein label. 14 In 2013, cell tracking experiments were performed using a commercial membrane stain DiR. 17 Herein, we report a new photoconvertible lysosomal dye based on a diazaxanthilidene scaffold. The uorescent probe is water-soluble, cell permeable, and noncytotoxic with a large Stokes shis for both the pre-and post-activated forms.
In previous studies, we determined that the molecular structure of the natural product xylopypridine A was inconsistent with that of diazaxanthilidene (E)-1. 18 During these studies we made several important observations about the photophysical and photochemical properties of (E)-1/(Z)-1. 18 We also discovered that methylation of the pyridine ring led to water-soluble derivatives, facilitating biological experiments. In this report, we show that a simple switch in solvent produces two different derivatives, both of which can be used as lysosomal uorescent probes for live cell imaging experiments. Importantly, we show that the monomethylated derivative can be photoactivated in cells, allowing for spatial and temporal control during the imaging process. Additionally, these new uorescent probes are cell permeable and non-cytotoxic with good photostability and large Stokes shis, facilitating applications in biological imaging experiments.

Results and discussion
We developed an efficient 5 step synthesis of (E)-1/(Z)-1 resulting in a 41% overall yield. 18 Treatment of (E)-1/(Z)-1 with an excess of dimethyl sulfate in chloroform provided dimethylated derivatives (E)-2/(Z)-2 in 64% yield, while one equivalent of dimethyl sulfate in toluene gave rise to the monomethylated derivatives (E)-3/(Z)-3 in 55% yield (Scheme 1). Both methylated forms are isolated as a mixture of dynamic equilibrating E and Z isomers.
Live cell imaging studies were performed using (E)-2/(Z)-2 and (E)-3/(Z)-3. Both compounds are water-soluble and can be dosed in water or buffer without the addition of organic solvents, which can be problematic for live cell imaging. Aer a 3 hour incubation in a humidied atmosphere with 5% CO 2 at 37 C, both dyes were found to be cell permeable and exhibited punctate staining patterns in HeLa cells, consistent with lysosomes (Fig. 2). Unlike (E)-2/(Z)-2, (E)-3/(Z)-3 was found to be photoconvertible, a property that allowed for sequential labelling of individual cells (Fig. 5 and 6).
To study the photoreaction, a solution of (E)-3/(Z)-3 in water was irradiated with visible light (26 W uorescent light bulb) for 24 hours (Fig. 4A). Photoproduct 4 was isolated in 64% yield. The photoproduct 4 showed a bathochromic shi of 89 nm and  a large Stokes shi (98 nm) (Fig. 4B). Photoactivation experiments were performed in live HeLa cells incubated with (E)-3/ (Z)-3. The pre-activated (E)-3/(Z)-3 can be observed using a 405 nm excitation wavelength and a 525 (AE25) nm emission wavelength. The post-activated species can be excited at 488 nm and observed at 675 (AE25) nm emission. Alternating 2.5 second pulses with 488 nm and 405 nm laser were used to investigate the photoconversion of (E)-3/(Z)-3. The photoproduct signal quickly increased, approaching its maximum at 180 seconds followed by a slow decrease to 80% aer 400 seconds. The emission signal of the pre-activated state is reduced slowly followed by a plateau around 200 seconds at 60% of the original brightness (Fig. 5). The sharp increase in uorescence signal of the post-activated form but slow decrease in uorescence signal of the pre-activated form is a result of the relative brightness of the photoproduct compared to (E)-3/(Z)-3. Only 40% of (E)-3/(Z)-3 was photoconverted but the increase in brightness of the photoproduct still allows for good signal detection over background. The remaining 60% of (E)-3/(Z)-3 serves as a reference. This allows the ability to image both pre-and post-activated regions during imaging studies using two different uorescent states as compared to traditional photoactivatable dyes with a pre-activated dark state and a post-activated uorescent state.   Similar experiment using 444 nm laser instead of 405 nm laser for (E)-3/(Z)-3 excitation also showed photoactivation (Fig. S5 †).
To investigate spatial selectivity, sequential activation was carried out with a dense population of HeLa cells. A 405 nm laser was used for photoconversion and the 488/675 channel was used to observe the post-activated state. Five individual cells (cell 1, 2, 3, 4, and 5) can be sequentially activated by 40 seconds of irradiation using a 405 nm laser (Fig. 6).

Conclusions
In conclusion, a new class of water-soluble dixanthilidene uorescent probe has been synthesized and used for lysosomal imaging in live cells. The monomethylated derivative can be photoconverted to a new uorescent state, allowing precise spatiotemporal control during imaging experiments. These new uorescent probes are cell permeable and photostable displaying large Stokes shis and low cytotoxicity. More studies are under way in order to expand this new class of uorophores, developing more organelle specic probes for live cell imaging and exploring further biological applications.