US 20170218346A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/021834.6 A1 Kan et al. (43) Pub. Date: Aug. 3, 2017

(54) PROTEIN CATALYSTS FOR Publication Classification CARBON-SILCON BOND FORMATION IN (51) Int. Cl. VITRO AND IN VIVO CI2N 9/02 (2006.01) (71) Applicant: California Institute of Technology, C07F 7/08 (2006.01) Pasadena, CA (US) (52) U.S. Cl. CPC. CI2N 9/0042 (2013.01); C12Y 114/14001 (72) Inventors: Sek Bik Jennifer Kan, Pasadena, CA (2013.01); C12Y 106/02004 (2013.01); C12N (US); Russell D. Lewis, Pasadena, CA 9/0071 (2013.01); C07F 7/0896 (2013.01); (US); Kai Chen, Pasadena, CA (US); C07F 7/0818 (2013.01); C07F 7/0829 Frances H. Arnold, Pasadena, CA (US) (2013.01) (57) ABSTRACT (73) Assignee: California Institute of Technology, The present invention provides compositions and methods Pasadena, CA (US) for catalyzing the formation of carbon-silicon bonds using heme proteins. In certain aspects, the present invention (21) Appl. No.: 15/422,360 provides heme proteins, including variants and fragments thereof, that are capable of carrying out in vitro and in vivo (22) Filed: Feb. 1, 2017 carbene insertion reactions for the formation of carbon silicon bonds. In other aspects, the present invention pro vides methods for producing an organosilicon product, the Related U.S. Application Data method comprising providing a silicon-containing reagent, a (60) Provisional application No. 62/290,211, filed on Feb. carbene precursor, and a heme protein; and combining the 2, 2016, provisional application No. 62/365,797, filed components under conditions sufficient to produce an on Jul. 22, 2016, provisional application No. 62/409, organosilicon product. Host cells expressing the heme pro 137, filed on Oct. 17, 2016. teins are also provided by the present invention.

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HEME PROTEIN CATALYSTS FOR one or more mutations comprise a M100 mutation and an CARBON-SILICON BOND FORMATION IN M103 mutation relative to the sequence set forth VITRO AND IN VIVO in SEQ ID NO:1. In yet other instances, the one or more mutations comprise a V75 mutation, a M100 mutation, and CROSS-REFERENCES TO RELATED an M103 mutation relative to the amino acid sequence set APPLICATIONS forth in SEQ ID NO:1. In particular instances, the V75 0001. The present application claims priority to U.S. mutation is a V75T mutation. In other instances, the M100 Provisional Application No. 62/290,211 filed on Feb. 2, mutation is an M100D or M100E mutation. In still other 2016; U.S. Provisional Application No. 62/365,797 filed on instances, the M103 mutation is an M103E mutation. Jul. 22, 2016; and U.S. Provisional Application No. 62/409, 0005. In other embodiments, the heme of the 137 filed on Oct. 17, 2016, the disclosures of which are c protein variant is a non-native cofactor. incorporated herein by reference in their entirety for all 0006. In some embodiments, the cytochrome c protein purposes. variant has a higher total turnover number (TTN) compared to the wild-type protein. In some instances, the TTN is STATEMENT AS TO RIGHTS TO INVENTIONS greater than about 70. In particular instances, the TTN is MADE UNDER FEDERALLY SPONSORED greater than about 1,800. RESEARCH AND DEVELOPMENT 0007. In other embodiments, the cytochrome c protein variant has a higher turnover frequency (TOF) compared to 0002 This invention was made with government support the wild-type protein. In some instances, the TOF is at least under Grant No. CBET1403077 awarded by the National about 2-fold greater than the wild-type protein. In particular Science Foundation and Grant No. GMO7616 awarded by instances, the TOF is at least about 7-fold greater than the the National Institutes of Health. The government has cer wild-type protein. In some instances, the TOF is at least tain rights in the invention. about 10 min'. In particular instances, the TOF is at least about 45 min. BACKGROUND OF THE INVENTION 0008. In some embodiments, the cytochrome c protein 0003 Organic compounds containing carbon-silicon variant produces an organosilicon product with a % ee of at bonds are of great interest in the fields of synthetic chem least about 75%. In some instances, the cytochrome c istry, drug discovery, nuclear medicine, biotechnology, and protein variant produces an organosilicon product with a % materials science. As a result, chemical methods available ee of at least about 95%. In particular instances, the for introducing silicon to the carbon framework of organic cytochrome c protein variant produces an organosilicon molecules have improved in recent years. Among these product with a % ee of at least about 99%. methods, however, only a small fraction are suitable for the 0009. In a second aspect, the invention provides a cell preparation of chiral organosilicon compounds. One comprising a cytochrome c protein variant, or a fragment approach for asymmetric carbon-silicon bond formation is thereof, of the present invention. In some embodiments, the via carbenoid insertion into silicon-hydrogen bonds, which cell is a bacterial, archaeal, yeast, or fungal cell. In some can be achieved using chiral rhodium, iridium, or copper instances, the bacterial cell is an Escherichia coli cell. catalysts. While these transition metal catalysts have dem 0010. In a third aspect, the invention provides a method onstrated utility in preparing highly selective products, their for producing an organosilicon product. In some embodi turnovers are poor (i.e., none exceeds a total turnov