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Biocompatible click cycloladdition reactions in chemical biology
[edit]Recent advances in technology have allowed scientists to view substructures of cells at levels of unprecedented detail. Unfortunately these “aerial” pictures offer little information about the mechanics of the biological system in question. To be fully effective, precise imaging systems require a complementary technique that better elucidates the machinery of a cell. By attaching tracking devices (optical probes) to biomolecules in vivo, one can learn far more about cell metabolism, molecular transport, cell-cell interactions and many other processes[1]
Bioorthogonal Reactions
[edit]Successful labeling of a molecule of interest requires specific functionalization of that molecule to react chemospecifically with an optical probe. For a labeling experiment to be considered robust, that functionalization must minimally perturb the system[2]. Unfortunately, these requirements can often be extremely hard to meet. Many of the reactions normally available to organic chemists in the laboratory are unavailable in living systems. Water- and redox- sensitive reactions would not proceed, reagents prone to nucleophilic attack would offer no chemospecificity, and any reactions with large kinetic barriers would not find enough energy in the relatively low-heat environment of a living cell. Thus, chemists have recently developed a panel of “bioorthogonal reactions” that proceed chemospecifically, despite the milieu of distracting reactive materials in vivo.
Design of Bioorthogonal Reagents
[edit]The coupling of an optical probe to a molecule of interest must occur within a reasonably short time frame; therefore, the kinetics of the coupling reaction should be highly favorable. Click chemistry is well suited to fill this niche, since click reactions are, by definition, rapid, spontaneous, selective, and high-yielding[3]. Unfortunately, the most famous “click reaction,” a [3+2] cycloaddition between an azide and an acyclic alkyne, is copper-catalyzed, posing a serious problem for use in vivo due to copper’s toxicity[4]. To bypass the necessity for a catalyst, the lab of Dr. Carolyn Bertozzi introduced inherent strain into the alkyne species by using a cyclic alkyne. In particular, cyclooctyne reacts with azido-molecules with distinctive vigor[5]. Further optimization of the reaction led to the use of difluorinated cyclooctynes (DIFOs), which increased yield and reaction rate[6]. Other coupling partners discovered by separate labs to be analogous to cyclooctynes include trans cyclooctene[7], norbornene[8], and a cyclobutene-functionalized molecule[9].
Use in Biological Systems
[edit]As mentioned above, the use of bioorthogonal reactions to tag biomolecules requires that one half of the reactive “click” pair is installed in the target molecule, while the other is attached to an optical probe. When the probe is added to a biological system, it will selectively conjugate with the target molecule. The most common method of installing bioorthogonal reactivity into a target biomolecule is through metabolic labeling. Cells are immersed in a medium where access to nutrients is limited to synthetically-modified analogues of standard fuels such as sugars. Consequently, these altered biomolecules are incorporated into the cells in the same manner as their wild-type bretheren. The optical probe is then incorporated into the system to image the fate of the altered biomolecules. Other methods of functionalization include enzymatically inserting azides into proteins[10], crosslinking modified peptide domains[11], and synthesizing phospholipids conjugated to cyclooctynes[12]
Future Directions As these bioorthogonal reactions are further optimized, they will likely be used for increasingly complex interactions involving multiple different classes of biomolecules. More complex interactions have a smaller margin for error, so increased reaction efficiency is paramount to continued success in optically probing cellular machinery. Also, by minimizing side reactions, the experimental design of a minimally perturbed living system is closer to being realized.
- ^ Jewett, J. C., and Bertozzi, C. R. (2010) Cu-free click cycloaddition reactions in chemical biology, Chemical Society Reviews 39, 1272-1279. [DOI:[1]
- ^ Sletten, E. M., and Bertozzi, C. R. (2009) Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality, Angewandte Chemie-International Edition 48, 6974-6998. [DOI:[2]
- ^ Kolb, H. C., Finn, M. G., and Sharpless, K. B. (2001) Click Chemistry: Diverse Chemical Function from a Few Good Reactions, Angewandte Chemie International Edition 40, 2004-2021. [DOI:<2004::AID-ANIE2004>3.0.CO;2-5
- ^ Rostovtsev, V. V., Green, L. G., Fokin, V. V., and Sharpless, K. B. (2002) A stepwise Huisgen cycloaddition process: Copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes, Angewandte Chemie-International Edition 41, 2596-+. [DOI:<2596::AID-ANIE2596>3.0.CO;2-4
- ^ Agard, N. J., Prescher, J. A., and Bertozzi, C. R. (2004) A strain-promoted 3+2 azide-alkyne cycloaddition for covalent modification of blomolecules in living systems, Journal of the American Chemical Society 126, 15046-15047. [DOI:[3]
- ^ Baskin, J. M., Prescher, J. A., Laughlin, S. T., Agard, N. J., Chang, P. V., Miller, I. A., Lo, A., Codelli, J. A., and Bertozzit, C. R. (2007) Copper-free click chemistry for dynamic in vivo imaging, Proceedings of the National Academy of Sciences of the United States of America 104, 16793-16797. [DOI:[4]
- ^ Blackman, M. L., Royzen, M., and Fox, J. M. (2008) Tetrazine ligation: Fast bioconjugation based on inverse-electron-demand Diels-Alder reactivity, Journal of the American Chemical Society 130, 13518-+. [DOI:[5]
- ^ Devaraj, N. K., Weissleder, R., and Hilderbrand, S. A. (2008) Tetrazine-Based Cycloadditions: Application to Pretargeted Live Cell Imaging, Bioconjugate Chemistry 19, 2297-2299. [DOI:[6]
- ^ Pipkorn, R., Waldeck, W., Didinger, B., Koch, M., Mueller, G., Wiessler, M., and Braun, K. (2009) Inverse-electron-demand Diels-Alder reaction as a highly efficient chemoselective ligation procedure: Synthesis and function of a BioShuttle for temozolomide transport into prostate cancer cells, Journal of Peptide Science 15, 235-241. [DOI:[7]
- ^ Fernandez-Suarez, M., Baruah, H., Martinez-Hernandez, L., Xie, K. T., Baskin, J. M., Bertozzi, C. R., and Ting, A. Y. (2007) Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes, Nat Biotech 25, 1483-1487. [DOI:[8]
- ^ Hur, G. H., Meier, J. L., Baskin, J., Codelli, J. A., Bertozzi, C. R., Marahiel, M. A., and Burkart, M. D. (2009) Crosslinking Studies of Protein-Protein Interactions in Nonribosomal Peptide Biosynthesis, Chemistry & Biology 16, 372-381. [DOI:[9]
- ^ Neef, A. B., and Schultz, C. (2009) Selective Fluorescence Labeling of Lipids in Living Cells, Angewandte Chemie International Edition 48, 1498-1500. [DOI:[10]