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(12) United States Patent (10) Patent No.: US 8,993,262 B2 Coelho Et Al

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(12) United States Patent (10) Patent No.: US 8,993,262 B2 Coelho Et Al USOO8993262B2 (12) United States Patent (10) Patent No.: US 8,993,262 B2 Coelho et al. (45) Date of Patent: Mar. 31, 2015 (54) IN VIVO AND IN VITRO OLEFIN 2006/0030718 A1* 2/2006 Zhang et al. .................. 548,954 CYCLOPROPANATION CATALYZED BY 2006/01 11347 A1 5/2006 Askew, Jr. et al. 2007/0276013 A1 1 1/2007 Ebbinghaus et al. HEMIE ENZYMES 2009/0238790 A2 9/2009 Sommadossi et al. 2010.00568.06 A1 3/2010 Warren (71) Applicant: California Institute of Technology, 2010/0168463 A1 7, 2010 Hirata et al. Pasadena, CA (US) 2010/024010.6 A1 9/2010 Wong et al. 2011 0196086 A1 8, 2011 Matsushita et al. (72) Inventors: Pedro S. Coelho, Los Angeles, CA (US); 2012/0237591 A1 9, 2012 Cullis et al. Eric M. Brustad, Durham, NC (US); Frances H. Arnold, La Canada, CA FOREIGN PATENT DOCUMENTS (US); Zhan Wang. San Jose, CA (US); EP O 200 638 B1 4, 1986 Jared C. Lewis, Chicago, IL (US) WO 2007 144599 A2 12/2007 WO 2011, 1595.50 A3 12/2011 (73) Assignee: California Institute of Technology, Pasadena, CA (US) OTHER PUBLICATIONS (*) Notice: Subject to any disclaimer, the term of this Adams, P.D. et al., “PHENIX: a comprehensive Python-based system patent is extended or adjusted under 35 for macromolecular structure solution.” Acta Crystallogr., Sect. D, U.S.C. 154(b) by 0 days. R.JKumar, F.K.R. et R.O.al., 1soprenoid painwayNavonimation optimization Ioror taxoto (21) Appl. No.: 14/185.861 precursor overproduction in Escherichia coli,” Science, 2010, y x- - - 9 330:70-74. (22) Filed: Feb. 20, 2014 Alliot, J. et al., “Enantioselective synthesis of levomilnacipran.” e a? V.9 Chem. Commun., 2012, 48(65):811 1-8113. (65) Prior Publication Data Altschul, S.F. et al., “Basic local alignment search tool.” J. Mol. Biol. 1990, 215(3):403-10. US 2014/0242647 A1 Aug. 28, 2014 Bailey, S., “The CCP4 Suite: programs for protein crystallography.” Acta Cryst., Sect. D Biol. Crystallogr., 1994, D50(5):760-763. Related U.S. Application Data titly chemity chasiation" (63) Continuation of application No. PCT/US2013/ Bloom, J.D. et al., “Protein stability promotes evolvability.” Proc. 063577, filed on Oct. 4, 2013. Natl. Acad. Sci. U. S. A., 2006, 103(15):5869-5874. Bonnaud, B. et al., “1-Aryl-2-(aminomethyl)cyclo (60) Provisional application No. 61/711,640, filed on Oct. propanecarboxylic acid derivatives. A new series of potential antide 9, 2012, provisional application No. 61/740.247, filed pressants.” J. Med. Chem., 1987, 30(2):318-25. on Dec. 20, 2012, provisional application No. Bornscheuer, U.T. and R.J. Kazlauskas, “Reaction specificity of 61/784,917, filed on Mar. 14, 2013, provisional enzymes: Catalytic promiscuity in biocatalysis: Using old enzymes application No. 61/838,167, filed on Jun. 21, 2013, to form new bonds and follow new pathways.” Angew. Chem. Int. provisional application No. 61/815,997, filed on Apr. Ed., 2004, 43(45):6032-6040. 25, 2013, provisional application No. 61/818,329, Boyce, M. and C.R. Bertozzi. "Bringing chemistry to life.” Nat. filed on May 1, 2013, provisional application No. Methods, 2011, 8(8):638-642. 61/856,493, filed on Jul. 19, 2013. (Continued) 51) Int. Cl. ( ) CI2P I/O (2006.01) Primary Examiner — Fereydoun G Saadi CI2P 13/02 (2006.01) Assistant Examiner — Pancham Bakshi CI2P 7/62 (2006.01) (74) Attorney, Agent, or Firm — Kilpatrick Townsend & (52) U.S. Cl. Stockton LLP CPC. CI2P 13/02 (2013.01): CI2P 7/62 (2013.01) USPC .............................. 435/41; 435/119; 435/166 (57) ABSTRACT (58) Field of Classification Search The present invention provides methods for catalyzing the CPC ..................................... C12P 7/04; C12P1/00 conversion of an olefin to any compound containing one or USPC . 435/41, 119, 166 more cyclopropane functional groups using heme enzymes. See application file for complete search history. In certain aspects, the present invention provides a method for roducing a cVclopropanation product comprising providin (56) References Cited p g a cycloprop p p gp 9. an olefinic Substrate, a diazo reagent, and a heme enzyme; and admixing the components in a reaction for a time Sufficient to U.S. PATENT DOCUMENTS produce a cyclopropanation product. In other aspects, the 3,965,204 A 6, 1976 Lukas et al. present invention provides heme enzymes including variants 5,296,595 A 3/1994 Doyle and fragments thereofthat are capable of carrying out in vivo 5,703,246 A 12/1997 Aggarwal et al. and in vitro olefin cyclopropanation reactions. Expression 7,267,949 B2 9, 2007 Richards et al. 7.625,642 B2 12/2009 Matsutani et al. vectors and host cells expressing the heme enzymes are also 7,662.969 B2 2/2010 Doyle et al. provided by the present invention. 7,863,030 B2 1/2011 Arnold et al. 8,247.430 B2 8, 2012 Yuan 30 Claims, 46 Drawing Sheets US 8,993,262 B2 Page 2 (56) References Cited Isin, E.M. and F.P. Guengerich, "Complex reactions catalyzed by cytochrome P450 enzymes.” Biochim. Biophys. Acta, Gen. Subj. OTHER PUBLICATIONS 2007, 1770(3):314-329. Kabsch, W., “Integration, Scaling, space-group assignment and post Breslow, R., "Biomimetic chemistry: Biology as an inspiration.” J. refinement.” Acta Crystallogr., Sect. D, Biol. 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