Over the last couple of years there have been several reports by various research groups describing fluorous photoaffinity labeling agents (PALs). These molecules have been aziridines with a fluorous tag attached such that upon exposure to UV light a carbene is formed which can then react with a variety of functionality which might be nearby to form a new covalent bond. The affinity tag can then be used to enrich a sample for the newly formed molecule. Photoaffinity labeling is used extensively in the study of intermolecular interactions particularly protein-protein and protein-ligand interactions. The highly reactive nature of the carbene formed means that just about anything that is nearby is going to react and a bond will be formed.
Another application of fluorous methods that has been steadily gaining popularity over the last 5 years or so is microarray formation by fluorous immobilization. In this strategy the partitioning of fluorous tagged molecules onto a fluorous modified surface is used to form microarrays of peptides, small molecules, and oligosaccharides. Fluorous immobilization has quite a number of advantages over other immobilization strategies including lower background signal, low non-specific binding, display-specific orientation, and simpler workflows through the elimination of multiple washing nad blocking steps. Fluorous microarrays have been used in binding and activity assays with purified proteins and with cell lysates.
A just published paper in Tetrahedron Letters from Mark Bradley and co-workers at the University of Southampton combines a fluorous PAL with fluorous immobilization. The goal of the work is to develop a method to form small molecule micrarrays which have all the advantages of fluorous immobilization without having to construct who libraries of fluorous tagged molecules. The authors strategy is relatively straightforward; spot a solution containing a fluorous PAL onto a fluorous glass slide and overlay that spot with a solution of a small molecule , let the solvent evaporate, and irradiate the slide to form the carbene that reacts with the small molecule resulting in fluorous immobilization.
The main advantage to this is that existing libraries and compound collections could be easily immobilized into a microarray format without having to individually tag each one prior to immobilization. The major downside is that the since the tagging via the carbene is non-selective, the substrates are not displayed in a specific orientation, but rather randomly. This may not be that big of a deal, however, since there will probably be enough molecules displayed properly to provide the information needed.
Tactically, the authors made three fluorous PALs differing in the length of linker between the aziridine and the fluorous chain. They also prepared two fluorous tagged molecules as positive controls; a fluorous biotin and a fluorous fluorescein. Using these fluorous tagged substrates they then ran a series of array experiments to demonstrate their strategy.
They first spotted the fluorous fluorescein and found that washing with water resulted in no compound diffusion, while any organic solvent resulted in desorption of the compound from the surface. They then the spotted the three different PALs and followed that by spotting a solution of carboxyfluorescein at different concentrations on the PAL spots. Drying and irradiation for 40 min at 365 nm was followed by washing and scanning. The results, shown below, are interesting that they observed that the shorter linker provided the best results. Note the lack of fluorescence in the controls (red boxes where only carboxyfluorescein was spotted and green boxes where only the fluorous PAL was spotted). This shows that the observed signals are due only to carboxyfluorescein that has been captured and immobilized through the fluorous PAL.
The authors then went one step further by immobilizing biotin in the same fashion through photoaffinity labeling using an ink jet printer. The immobilized biotin was then dectected through Cy-3 labeled streptavidin. Once again the negative control, no fluorous PAL, showed no signal. The positive control was the directly tagged fluorous biotin which was spotted at 2 mM vs. the fluorous PAL which was spotted at 10 mM and one can see the increased intensity of fluorescence due to the higher amount of immobilized biotin.
Overall, this communication describes a general method by which existing content can be easily arrayed onto a fluorous surface. This will allow researchers to make full use of all the advantages of fluorous immobilization, i.e. simpler workflows, high signal to noise ratios, low non-specific binding, etc. with a minimal amount of effort.