As evidenced by the large body of work on click chemistry, bioorthogonal reactions that can be performed in live cells are quite useful.1 Besides the application of in vivo chemistry to help study biological processes, there is also the application in engineered biosynthesis – the modification of enzymatic processes to make new molecules.2
While nature has an impressive arsenal of synthetic methodology that organic chemists continue to study and reproduce,3 the fact that biorthogonal chemistry exists tells us that there are a few reactions evolution didn’t uncover on its own. Olefin metathesis is one of these reactions, along with several other platinum group metal (PGM)-catalyzed processes (we haven’t discovered a natural Suzuki-ase, either). The relative scarcity of these metals in the environment may explain their absence from the corpus of natural metalloenzymes. There are plenty of enzymes that make use of iron, cobalt, even molybdenum and tungsten, but not so many incorporate ruthenium.
Ward’s group has been working for some time to bring some of these PGM-catalyzed reactions into the world of enzymes using the well-studied biotin affinity of avidin and streptavidin (SAV) to pull transition metal complexes into the binding pocket.4 Then, through point mutations and directed evolution, the newly created “active site” is tailored to the particular chemistry for which that transition metal complex is useful. This has yielded some pretty interesting artificial metalloenzymes, catalyzing asymmetric reactions ranging from transfer hydrogenation5 to C-H activation.6
Artificial metalloenzymes that promote metathesis reactions in water have been reported by Ward’s group7 and others,8 but there is a world of difference between activity in water and activity in cells. The activity of Ward’s metathesis enzyme is completely shut down by the presence of glutathione, which is present at millimolar concentration in a cell’s cytosol. That glutathione acts as a catalyst poison may not come as a surprise to savvy readers, as it contains a cysteine residue with a free thiol group. Cysteine is an excellent chelator, and as a lone amino acid has been used specifically to coordinate to the ruthenium center of a metathesis catalyst and pull it away from a precious organic product.9
To get around this issue, the authors tailored the cells to express the streptavidin host in the periplasm, the space between the inner and outer membranes of the gram-negative E. coli. As it turns out, the concentration of glutathione in its thiol form is very low in the periplasm (the oxidized, disulfide form is there, but that doesn’t interfere with the enzyme). So, the artificial metalloenzyme is free to go about its business.
Once they had established baseline activity of the biot-Ru complex within wild-type SAV, they set up an assay using umbelliferone as a fluorescent signal to tell them which mutations on the enzyme had positive effects on metathesis activity,10 and it was off to the directed evolution races. In the end, they identified a streptavidin variant with 5 amino acid residues mutated that displayed superior performance in the umbelliferone transformation. So, in addition to demonstrating activity in a cell, the authors also showed they can start from biot-Ru-SAVwt and optimize the metalloenzyme around a given metathesis transformation.
Understandably, this achievement has garnered a bit of attention in the chemistry community.11 Demonstrating metathesis in vivo is a significant step toward harnessing the power of olefin metathesis for both engineered biosynthesis and bioconjugation. So, our hats are off to Prof. Ward, Prof. Panke and the teams at ETH and the University of Basel for illuminating the potential for olefin metathesis to be added to the in vivo chemistry toolbox.
 Prescher, J. A.; Bertozzi, C. R. Nature Chemical Biology 2005, 1, 13.
 McDaniel, R.; Ebert-Khosla, S.; Hopwood, D. A.; Khosla, C. Science, 1993, 262, 1546.
 (a) Breslow, R. J. Biol. Chem. 2009, 284, 1337. (b) Engelmann, X.; Monte-Pérez, I.; Ray, K. Angew. Chem. Int. Ed., 2016, 55, 7632.
 Ward, T. R. “Artificial Metalloenzymes Based on the Biotin-Avidin Technology: Enantioselective Catalysis and Beyond”, Acc. Chem. Res., 2011, 44, 47.
 Collot, J.; Gradinaru, J.; Humbert, N.; Skander, M.; Zocchi, A; Ward, T. R. J. Am. Chem. Soc. 2003, 195, 9030.
 Hyster, T.; Knörr, L.; Ward, T. R; Rovis, T. Science, 2012, 338, 500.
 Kajetanowicz, A.; Chatterjee, A.; Reuter, R.; Ward, T. R. Catal. Lett. 2014, 144, 373.
 Sauer, D. F.; Himiyama, T.; Tachikawa, K.; Fukumoto, K.; Onoda, A.; Mizohata, E.; Inoue, T.; Bocola, M.; Schwaneberg, U.; Hayashi, T.; Okuda, J. ACS Catal. 2015, 5, 7519.
 Wang, H.; Matsuhashi, H.; Doan, B. D.; Goodman, S. N.; Ouyang, X.; Clark Jr. W. M. Tetrahedron 2009, 65, 6291.
 Reuter, R.; Ward, T. R. Beilstein, J. Org. Chem, 2015, 11, 1886.
 (a) Borman, S. “Organometallic reaction catalyzed in living cells” C&E News, September 5, 2016, 94(35), 11. (b) Lowe, D. “New Metalloenzymes Made to Order” In The Pipeline, September 7, 2016.