The labeling of biomolecules with exogenous reagents is generally done for one of two purposes either enrichment or detection. Enrichment labeling is generally in the form of some sort of affinity tag such as biotin, a His tag, and yes, fluorous tag and is designed to allow one to separate the components of interest from molecules. The complexity of biological mixtures can make the analysis of all components impractical, so enrichment of the sample in only targeted analytes is often necessary. Detection labeling on the other hand allows one to easily determine which components in a mixture are labeled. Detection methods include fluorescence, radiometric, or luminescence. Sometimes indirect methods are used also. For example, a biomolecule may be biotinylated as the primary label which is then detected using fluorescently labeled avidin.
So what do you do if you want to enrich and detect? You make a trifunctional reagent, of course, containing a protein reactive group, a detection tag, and an enrichment tag. Trifunctional reagents are pretty common, but most contain an enrichment or detection tag in conjunction with a cross-linker, such a photoaffinity tag in order to investigate protein-protein interactions, not an enrichment and detection tag.
A new paper from the Lo group at City University of Hong Kong, the site of the upcoming ISoFT ’11 conference, describes just such a reagent though. In this instance they prepared a trifunctional reagent with a isothiocyanate as the protein reactive group, a fluorous chain as the enrichment tag, and a rhenium complex as the detection tag. The authors write that they chose rhenium(I) complexes due to the ease by which they can be color tuned and their long lived excited states which could lead to multi-color probes. Previously in 2008, the same authors had made a similar trifunctional probe using a biotin tag, which they have now replaced with a fluorous tag. The advantages of the fluorous tag over biotin include less non-specific binding, easier separation by fluorous solid phase extraction(FSPE), and excellent mass spec characteristics.
The researchers first prepared the trifunctional reagent 2 from aminopyridine 1 and found that it reacted well with ethylamine to form 3. The measured adsorption and emission spectra of each of these were also found to be as expected. They then labeled a protein, BSA, and a tripeptide, gluthione (GSH), with the reagent 2. Both resulted in labeled compounds with long-lived emissions; green colored in the case of Re-BSA and yellow for Re-GSH. They then demonstrated that FSPE could be used for enrichment by separating the the tagged GSH from a mixture with 20 amino acids.
Finally, they measured the cellular uptake and cytotoxicity of 3 upon incubation with HeLa cells and found that 3 was predominantly found associated with the mitochondria of the cells as seen by confocal microscopy. The authors conclude by stating that having demonstrated the use of such reagents in labeling, enrichment, and detection that they foresee the design and use of such reagents in the isolation and identification of intracellular receptors.