US 20140242647A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0242647 A1 Coelho et al. (43) Pub. Date: Aug. 28, 2014 (54) IN VIVO AND IN VITRO OLEFIN application No. 61/815,997, filed on Apr. 25, 2013, CYCLOPROPANATION CATALYZED BY provisional application No. 61/818.329, filed on May HEMIE ENZYMES 1, 2013, provisional application No. 61/856,493, filed on Jul. 19, 2013. (71) Applicant: California Institute of Technology, Pasadena, CA (US) Publication Classification (72) Inventors: Pedro S. Coelho, Los Angeles, CA (US); (51) Int. Cl. Eric M. Brustad, Durham, NC (US); CI2P 13/02 (2006.01) Frances H. Arnold, La Canada, CA CI2P 7/62 (2006.01) (US); Zhan Wang, San Jose, CA (US); (52) U.S. Cl. Jared C. Lewis, Chicago, IL (US) CPC. CI2P 13/02 (2013.01): CI2P 7/62 (2013.01) USPC ............................ 435/129; 435/189: 435/135 (73) Assignee: California Institute of Technology, Pasadena, CA (US) (57) ABSTRACT (21) Appl. No.: 14/185.861 The present invention provides methods for catalyzing the conversion of an olefin to any compound containing one or (22) Filed: Feb. 20, 2014 more cyclopropane functional groups using heme enzymes. In certain aspects, the present invention provides a method for Related U.S. Application Data producing a cyclopropanation product comprising providing an olefinic Substrate, a diazo reagent, and a heme enzyme; and (63) Continuation of application No. PCT/US2013/ admixing the components in a reaction for a time Sufficient to 063577, filed on Oct. 4, 2013. produce a cyclopropanation product. In other aspects, the (60) Provisional application No. 61/711,640, filed on Oct. present invention provides heme enzymes including variants 9, 2012, provisional application No. 61/740,247, filed and fragments thereofthat are capable of carrying out in vivo on Dec. 20, 2012, provisional application No. 61/784, and in vitro olefin cyclopropanation reactions. Expression 917, filed on Mar. 14, 2013, provisional application vectors and host cells expressing the heme enzymes are also No. 61/838,167, filed on Jun. 21, 2013, provisional provided by the present invention. Patent Application Publication Aug. 28, 2014 Sheet 1 of 46 US 2014/0242647 A1 Monooxygenation (Oxene transfer) Compound N N FIG. 1 Patent Application Publication Aug. 28, 2014 Sheet 2 of 46 US 2014/0242647 A1 O N s OEt ©N GN catalvst ( 0.2 cis y mol%) trans RMS RAR S/VS Phvv COOEt S/N Ph' COOEt Ph 5A,COOEt Ph COOEt Hemin Y P450BM3-T268A C3C FIG. 2 Patent Application Publication Aug. 28, 2014 Sheet 3 of 46 US 2014/0242647 A1 0.1 M KPipH 8.0 O 5% MeOH COOEt s + l oE P450 10 mMNa2S2O4 H2A1 Oholo -er oN Anaerobic Ph eN 20 uM Variable 10 mM 0.2% moleq (wrt EDA) 4.00 3.50 0 3.OO 2.50 s 2.00 H. () 150 1.OO OOO O 1 O 20 30 40 50 60 70 8O 90 100 styrene] 1 mM FIG. 3 Patent Application Publication Aug. 28, 2014 Sheet 4 of 46 US 2014/0242647 A1 0.1 M KPipH 8.0 O 5% MeOH 10 mM Na2S2O4 COOEt is GNroE +5H2A10holo Anaerobic A Ph eN 30 mM 10 mM Variable 6 5 4 0 3 OR { 5, 2 C 2., -n. 0 - O 20 40 60 80 100 120 P450 / uM FIG. 4 Patent Application Publication Aug. 28, 2014 Sheet 5 of 46 US 2014/0242647 A1 FIG. 5A 50 40 30 O 5 10 15 20 25 (EDA (mM) O 2 4 6 8 10 12 14 16 styrene / mM Patent Application Publication Aug. 28, 2014 Sheet 6 of 46 US 2014/0242647 A1 c g c O S. Ol O 2 () Ca Z H How H. W. 1 3-CS BM3-CS BM3-CIS-C4OOS BM3-CS-C40OS anaerobic aerobic anaerobic aerobic FIG. 6 Patent Application Publication Aug. 28, 2014 Sheet 7 of 46 US 2014/0242647 A1 Cyclopropanes | styrene Oxide r . BM3-CS BM3-CIS BM3-CIS-C400S BM3-CS-C4OOS anaerobic aerobic anaerobic aerobic FIG. 7 Patent Application Publication Aug. 28, 2014 Sheet 8 of 46 US 2014/0242647 A1 FIG. 8A O 5 10 15 2O 25 (EDA) (mM) O 5 10 15 2O 25 styrene (mM) Patent Application Publication Aug. 28, 2014 Sheet 9 of 46 US 2014/0242647 A1 3 : i FIG. 9 Patent Application Publication Aug. 28, 2014 Sheet 10 of 46 US 2014/0242647 A1 7 0. 6 O O 0.200 ABC anaerobic ABC aerobic BM3-CIS BM3-CIS aerobic anaerobic Catalyst Conditions Total turnovers (mmol/g cdw) BM3-cs. Aerobic 0.13900s s 4s 2.79-49-12 "Diastereomeric ratios and enantiomeric excess were determined by GC analysis. (RS)- (S,R). C (R,R)v. - (SS) FIG. 10 Patent Application Publication Aug. 28, 2014 Sheet 11 of 46 US 2014/0242647 A1 |N | No. | N No. No.?3 + -3 }}|-C) <> (1) |- FIG. 11 Patent Application Publication Aug. 28, 2014 Sheet 13 of 46 US 2014/0242647 A1 2 mM glucose M9 1S + EDA + ABC -O- Pn Ph H OD variable 5% MeOH 500 L COOEt 20 mM 10 mM 2 hrs 20 30 40 Cell density (OD 600mm) Cell density (OD 60mm) FIG. 13 Patent Application Publication Aug. 28, 2014 Sheet 14 of 46 US 2014/0242647 A1 cooE P411 BM-CS 2 mM SS'CuCOSe A, Ph1 S + N2 whole-cells 5%. MeOH. Ph COOEt variable 10 mM OD600 = 22 2 hrs 2O 30 40 styrene (mM) FIG. 14 Patent Application Publication Aug. 28, 2014 Sheet 15 of 46 US 2014/0242647 A1 M9 Ph11SN N + EDA + wholeOD = cells25 5%--> MedH Ph 500 ul COOEt 20 mM 10 mM 0.4 0.O0. 132 ABC ABC no induction P450-less pCWOri ABC-glucose + Na2S2O4 Catalyst ABC + glucose 60 ABC no induction 17 P450-less pcwori 11 ABC + Na2S2O4 46 54 -87 FIG. 15 Patent Application Publication Aug. 28, 2014 Sheet 16 of 46 US 2014/0242647 A1 M9 1S EDA + ABC -o- Ph Ph1 N + OD = 25 5% MeOH 500 UL COOEt 20 mM 10 mM ------- 80.0 () 700 60,0 0. 50.0 Š {) 40,0 () > 30.0 () 200 10.0 O 50 100 150 200 250 300 Time (min) FIG 16 Patent Application Publication Aug. 28, 2014 Sheet 17 of 46 US 2014/0242647 A1 Patent Application Publication Aug. 28, 2014 Sheet 18 of 46 US 2014/0242647 A1 0.3 O.25 O2 BM3cis BM3 CIS. 0.15 C400S O. O.5 O ROA'st 350 400 450 500 550 600 650 700 Wavelength (nm) FIG. 18 Patent Application Publication Aug. 28, 2014 Sheet 19 of 46 US 2014/0242647 A1 F.G. 19A l Edithionite Ph1S Y - ten 12Fell S. S Of 30(e) StVreney Scys S3? E"Fe-Cyss ENAD(P)H Fe(I EDA FIG. 19B FIG. 19C 100 a 98 3 0.5 O.4" 8O 93 3 0.2 sc O - 60 As 8.U, SS 0.5 o 40 2 0.4 < 0.3 O.2 0.1 20 Orrara w Wavelength (nm) y -500 -400 -300 -200 - 1 OO O Potential (mV vs SHE) Patent Application Publication Aug. 28, 2014 Sheet 20 of 46 US 2014/0242647 A1 FIG. 20A FIG. 20B Patent Application Publication Aug. 28, 2014 Sheet 21 of 46 US 2014/0242647 A1 0.8 O.7 O6 0.5 0.4 1 - Fe(III) 0.3 2 a Fe(II) O2 3 - Fe(II)CO 0.1 350 400 450 500 550 600 650 700 Wavelength (nm) FIG. 21 Patent Application Publication Aug. 28, 2014 Sheet 22 of 46 US 2014/0242647 A1 O. 2 S. d U 0.1 O O CC 350 400 450 500 550 600 650 7OO Wavelength (nm) FIG. 22 Patent Application Publication Aug. 28, 2014 Sheet 23 of 46 US 2014/0242647 A1 FIG. 23A -0.3 429 Wavelength (nm) 0.15 O Wavelength (nm) FIG. 23B Patent Application Publication Aug. 28, 2014 Sheet 24 of 46 US 2014/0242647 A1 100 80 S O 8 CC 60 aS s 400 500 6OO O 40 Wavelength (nm) S. So so -400 so-20 -o Open Circuit Potential (mV vs SHE) FIG. 24 Patent Application Publication Aug. 28, 2014 Sheet 25 of 46 US 2014/0242647 A1 CD c) s O se O f) O CC 400 5OO 600 700 Wavelength (nm) -360 -400 -300 -200 -100 O Open Circuit Potential (mV vs SHE) FIG. 25 Patent Application Publication Aug. 28, 2014 Sheet 26 of 46 US 2014/0242647 A1 100 0.4 0.35 3 0.3 80 30.25' i 0.2 < 0.15 0.1 a 60 5 O t 400 500 600 700 SS Wavelength (nm) o 40 20 -soo -500 -400 -300 -200 -100 Open Circuit Potential (mV vs SHE) FIG. 26 Patent Application Publication Aug. 28, 2014 Sheet 27 of 46 US 2014/0242647 A1 Cld O (s O m O O O <C 60 St. O 350 400 450 500 550 600 650 P Wavelength (nm) g 40 -360 -400 -300 -200 -100 0 Open Circuit Potential (mV vs SHE) FIG. 27 Patent Application Publication Aug. 28, 2014 Sheet 28 of 46 US 2014/0242647 A1 COOEt 10 mM Na2S2O4. Phs + N2 + P450 0.1 M KPipH 8.0 Ph COOEt 5% MeOH CIS 30 mM 10 mM (0.15%15 moleq)M O Wrt EDA -- A. phriS Ph CHOOEt styrene oxide tranS not formed When Na2S2O4 is the reductant anaerobic aerobic anaerobic aerobic O cis:transt %ee %ee inhibition cist transs ABC- Anaerobic 5 342 - 2 CISheme ABC- Aerobic 2 -797 CISheme * Based on EDA.