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. Crystallogr., 2010, Biol. Chem., 2009, 284(3): 1337-1342. D66(2):133-144. Caballero, A. et al., “Metal-catalyzed olefin cyclopropanation with Kataoka, M. et al., “Novel bioreduction system for the production of ethyl diazoacetate: control of the diastereoselectivity.” Eur, J. lnorg. chiral alcohols.” Appl. Microbiol. Biotechnol., 2003, 62(5-6):437 Chem., 2009(9): 1137-1144. 45. Chen, M.S. and M.C. White, "A predictably selective aliphatic C-H Lebel, H. et al., “Stereoselective cyclopropanation reactions.” Chem. oxidation reaction for complex molecule synthesis.” Science, 2007. Rev., 2003, 103(4):977-1050. 318(5851):783-787. Lewis, J.C. and F.H. Arnold, “Catalysts on demand: selective oxida Chen, Y. and X.P. Zhang, "Asymmetric cyclopropanation of styrenes tions by laboratory-evolved cytochrome P450 BM3.” Chimia, 2009, catalyzed by metal complexes of D-symmetrical chiral porphyrin: 63(6):309-312. Superiority of cobalt over iron.” J. Org. Chem., 2007, 72(15):5931 Lewis, J.C. et al., "Chemoenzymatic elaboration of monosaccharides 5934. using engineered cytochrome P450 demethylases.” Proc. Natl. Cirino, P.C. and F.H. Arnold, “A self-sufficient peroxide-driven Acad. Sci. U. S. A., 2009, 106(39): 16550-16555. hydroxylation biocatalyst.” Angew. Chem. Int. Ed., 2003, Lewis, J.C. et al., “Combinatorial alanine substitution enables rapid 42(28):3299-3301. optimization of cytochrome P450 for selective hydroxylation of Clark, J.P. et al., “The role of Thr268 and Phe393 in cytochrome P large substrates.” Chembiochem, 2010, 11(18):2502-05. 450 BM3.” J. Inorg. Biochem., 2006, 100(5-6):1075-1090. Maas, G., “Ruthenium-catalyzed carbenoid cyclopropanation reac Coelho, P.S. et al., “A serine-substituted P450 catalyzes highly effi tions with diazo compounds.” Chem. Soc. Rev., 2004, 33(3):183 cient carbene transfer to olefins in vivo.” Nat. Chem. Biol., 2013, 190. 9(8):485-487. Mansuy, D. et al., “Reaction of carbon tetrachloride with 5,10,15,20 Coelho, P.S. et al., “Olefin cyclopropanation via carbene transfer tetraphenyl-porphinato iron(II) (TPP)Fe'l: evidence for the forma catalyzed by engineered cytochrome P450 enzymes.” Science, 2013, tion of the carbene complex (TPP)Fe"(CCI)),” J. Chem. Soc., 339(6117):307-310. Chem. Commun., 1977(18):648-9. Davies, H.M.L. and C. Venkataramani, “Dirhodium tetraprolinate Meinhold, P. et al., “Engineering cytochrome P450 BM3 for terminal catalyzed asymmetric cyclopropanations with high turnover num alkane hydroxylation.” Adv. Synth. Catal., 2006, 348(6):763-772. bers.” Org. Lett., 2003, 5(9): 1403-1406. Morandi, B. and E.M. Carreira, “Iron-catalyzed cyclopropanation in Davies, H.M.L. and J.R. Manning, “Catalytic C-H functionalization 6 M KOH with in situ generation of diazomethane.” Science, 2012, by metal carbenoid and nitrenoid insertion.” Nature, 2008, 335(6075): 1471-1474. 451(7177):417-424. Mouzin, G. et al., “A convenient synthesis of bifunctional vicinal Davies, H.M.L. and R.E.J. Beckwith, “Catalytic enantioselective cyclopropanes.” Synthesis, 1978, 4:304-305. C-H activation by means of metal-carbenoid-induced C-H insertion.” Murshudov, G.N. et al., “Refinement of macromolecular structures Chem. Rev., 2003, 103(8):2861-2903. by the maximum-likelihood method.” Acta Cryst., Sect. D, Biol. Dawson, J.H., “Probing structure-function relations in heme-con Crystallogr., 1997, D53(3):240-255. taining oxygenases and peroxidases.” Science, 1988,240(4851):433 Nakagawa, S. et al., "Construction of catalase deficient Escherichia 9. coli strains for the production of uricase.” Biosci. Biotechnol. Donaldson, W.A., “Synthesis of cyclopropane containing natural Biochem., 1996, 60(3):415-20. products.” Tetrahedron, 2001, 57(41):8589-8627. Narhi, L.O. and A.J. Fulco, “Characterization of a catalytically self Doyle, M.P. et al., “Dirhodium(II) tetrakismethyl 2-oxaazetidine-4- sufficient 119,000-dalton cytochrome P-450 monooxygenase carboxylate: a chiral dirhodium(II) carboxamidate of exceptional induced by barbiturates in Bacillus megaterium.” J. Biol. Chem. reactivity and selectivity.” Org. Lett. 2000, 208): 1145-7. 1986, 261 (16): 7160-9. Dunford, A.J. et al., “Probing the molecular determinants of coen Nelson, D.R., “The cytochrome P450 homepage.” Hum. Genomics, zyme selectivity in the P450 BM3 FAD/NADPH domain.” 2009, 4(1):59-65. Biochimica Biophysica Acta, 2009, 1794(8): 1181-1189. Nicolas, I. et al., “Asymmetric catalytic cyclopropanation reactions Emsley, P. and K. Cowtan, "Coot: model-building tools for molecular in water.” Coord. Chem. Rev., 2008, 252(5-7):727-735.