US010208322B2 (12 ) United States Patent ( 10 ) Patent No. : US 10 ,208 , 322 B2 Coelho et al. (45 ) Date of Patent: * Feb . 19, 2019 ( 54 ) IN VIVO AND IN VITRO OLEFIN ( 56 ) References Cited CYCLOPROPANATION CATALYZED BY HEME ENZYMES U . S . PATENT DOCUMENTS 3 , 965 ,204 A 6 / 1976 Lukas et al. (71 ) Applicant: California Institute of Technology , 4 , 243 ,819 A 1 / 1981 Henrick Pasadena , CA (US ) 5 ,296 , 595 A 3 / 1994 Doyle 5 , 703 , 246 A 12 / 1997 Aggarwal et al. 7 , 226 , 768 B2 6 / 2007 Farinas et al. ( 72 ) Inventors : Pedro S . Coelho , Los Angeles, CA 7 , 267 , 949 B2 9 / 2007 Richards et al . (US ) ; Eric M . Brustad , Durham , NC 7 ,625 ,642 B2 12 / 2009 Matsutani et al. (US ) ; Frances H . Arnold , La Canada , 7 ,662 , 969 B2 2 / 2010 Doyle et al. CA (US ) ; Zhan Wang , San Jose , CA 7 ,863 ,030 B2 1 / 2011 Arnold (US ) ; Jared C . Lewis , Chicago , IL 8 ,247 ,430 B2 8 / 2012 Yuan 8 , 993 , 262 B2 * 3 / 2015 Coelho . .. .. .. • * • C12P 7 /62 (US ) 435 / 119 9 ,399 , 762 B26 / 2016 Farwell et al . (73 ) Assignee : California Institute of Technology , 9 , 493 ,799 B2 * 11 /2016 Coelho .. C12P 7162 Pasadena , CA (US ) 2006 / 0030718 AL 2 / 2006 Zhang et al. 2006 / 0111347 A1 5 / 2006 Askew , Jr . et al. 2007 /0276013 AL 11 /2007 Ebbinghaus et al . ( * ) Notice : Subject to any disclaimer , the term of this 2009 /0238790 A2 9 /2009 Sommadosi et al. patent is extended or adjusted under 35 2010 / 0056806 A1 3 / 2010 Warren U . S . C . 154 (b ) by 0 days . 2010 /0168463 A1 7 /2010 Hirata et al. This patent is subject to a terminal dis 2010 /0240106 AL 9 /2010 Wong et al . claimer . 2011/ 0196086 A18 / 2011 Matsushita et al. ( Continued ) (21 ) Appl. No. : 15 / 278 , 561 FOREIGN PATENT DOCUMENTS (22 ) Filed : Sep . 28, 2016 EP 0 200 638 B1 4 / 1986 (65 ) Prior Publication Data WO 2007 / 144599 A2 12 / 2007 US 2017 / 0247725 A1 Aug. 31 , 2017 (Continued ) OTHER PUBLICATIONS Related U . S . Application Data Adams, P . et al ., “ Phenix : a comprehensive Python -based system for (63 ) Continuation of application No . 14 /625 , 449, filed on macromolecular structure solution ,” Acta Crystallogr ., Sect . D , Feb . 18 , 2015 , now Pat . No . 9 ,493 ,799 , which is a Biol. Crystallogr ., 2010 , D66 ( 2 ) : 213 -221 . continuation of application No . 14 / 185 , 861 , filed on ( Continued ) Feb . 20 , 2014 , now Pat . No . 8 , 993 , 262 , which is a continuation of application No . PCT /US2013 / 063577 , filed on Oct . 4 , 2013 . Primary Examiner — Pancham Bakshi (74 ) Attorney, Agent, or Firm — Kilpatrick Townsend & (60 ) Provisional application No . 61/ 711 ,640 , filed on Oct. Stockton LLP 9 , 2012 , provisional application No . 61 /740 , 247 , filed on Dec . 20 , 2012 , provisional application No . ABSTRACT 61 /784 ,917 , filed on Mar . 14 , 2013 , provisional (57 ) application No . 61/ 838 , 167 , filed on Jun . 21, 2013 , The present invention provides methods for catalyzing the provisional application No. 61/ 815 ,997 , filed on Apr . conversion of an olefin to any compound containing one or 25 , 2013 , provisional application No . 61/ 818 , 329 , more cyclopropane functional groups using heme enzymes . filed on May 1 , 2013 , provisional application No . In certain aspects , the present invention provides a method 61/ 856 ,493 , filed on Jul. 19 , 2013 . for producing a cyclopropanation product comprising pro (51 ) Int. CI. viding an olefinic substrate , a diazo reagent, and a heme C12P 13 / 02 ( 2006 .01 ) enzyme; and admixing the components in a reaction for a C12P 7762 ( 2006 . 01 ) time sufficient to produce a cyclopropanation product. In C12N 9 /02 ( 2006 . 01 ) other aspects , the present invention provides heme enzymes (52 ) U . S . CI. including variants and fragments thereof that are capable of CPC . .. .. C12P 13 /02 (2013 . 01 ) ; C12N 9 / 0004 carrying out in vivo and in vitro olefin cyclopropanation (2013 .01 ) ; C12N 9 /0042 (2013 . 01 ) ; C12P 7762 reactions . Expression vectors and host cells expressing the ( 2013 . 01 ) ; C12Y 106 / 02004 ( 2013 .01 ) ; YO2P heme enzymes are also provided by the present invention . 20 /52 (2015 . 11 ) (58 ) Field of Classification Search CPC .. .. C12P 13 /02 , C12P 7 /62 ; C12N 9 /0042 7 Claims, 46 Drawing Sheetsts See application file for complete search history . Specification includes a Sequence Listing . US 10 , 208 , 322 B2 Page 2 References Cited Davies , H . and Venkataramani, C . , “ Dirhodium tetraprolinate ( 56 ) catalyzed asymmetric cyclopropanations with high turnover num U . S . PATENT DOCUMENTS bers , ” Org . Lett ., 2003 , 5 ( 9 ) : 1403 - 1406 . 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