biocatalysis enzymatic construction of highly strained ......biocatalysis enzymatic construction of...

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BIOCATALYSIS Enzymatic construction of highly strained carbocycles Kai Chen, Xiongyi Huang, S. B. Jennifer Kan, Ruijie K. Zhang, Frances H. Arnold* Small carbocycles are structurally rigid and possess high intrinsic energy due to their ring strain. These features lead to broad applications but also create challenges for their construction. We report the engineering of hemeproteins that catalyze the formation of chiral bicyclobutanes, one of the most strained four-membered systems, via successive carbene addition to unsaturated carbon-carbon bonds. Enzymes that produce cyclopropenes, putative intermediates to the bicyclobutanes, were also identified. These genetically encoded proteins are readily optimized by directed evolution, function in Escherichia coli, and act on structurally diverse substrates with high efficiency and selectivity, providing an effective route to many chiral strained structures. This biotransformation is easily performed at preparative scale, and the resulting strained carbocycles can be derivatized, opening myriad potential applications. I n cyclic organic molecules, ring strain arises from distortions of bond angle and bond length, steric clashes of nonbonded substituents, and other effects (1). The simplest carbocycles, cy- clopropanes and cyclobutanes, possess ring strains of 26 to 28 kcal/mol ( 2). Introducing carbon- carbon multiple bonds or bridges to these small ring systems induces additional strain as well as structural rigidity. For example, cyclopropenes with an endo-cyclic double bond bear a strain of 54 kcal/mol, whereas bicyclo[1.1.0]butanes, folded into puckered structures, distinguish them- selves as one of the most strained four-membered systems, with strain of ~66 kcal/mol (fig. S1) (2). These carbocycles are particularly attractive inter- mediates in chemical and materials synthesis, because they can undergo strain-release trans- formations to furnish a myriad of useful scaffolds (36). The structural rigidity imparted by strained rings in supramolecular materials can lead to in- teresting physical properties, such as mechanical stability (7) and high glass transition temperature (8). The intrinsic energy of these strained struc- tures can also be relieved in response to exog- enous force, which leads to radical changes in physical properties (e.g., conductivity), a feature highly desirable for stimulus-responsive materials (9, 10). High ring strain, however, greatly increases the difficulty of synthesis. A commonly used method for preparing bicyclobutanes starts from dibromo- 2-(bromomethyl)cyclopropane substructures and uses organolithium reagents for lithium-halogen exchange, followed by nucleophilic substitution under rigorously anhydrous and cryogenic con- ditions (3). An alternative route relies on the double transfer of a carbene to alkynes, but the few examples in the literature are mostly limited to methylene carbene (1113). Asymmetric bicy- clobutane construction is particularly challeng- ing, with multiple chiral centers generated at the same time (14, 15) (fig. S2). Cyclopropene syn- thesis through enantioselective single-carbene addition to alkynes also requires chiral transition metal catalysts based on rhodium (16, 17), irid- ium (18), and cobalt (19). Development of a sus- tainable catalytic system that performs with high efficiency and selectivity under ambient condi- tions would be a major advance for construction of these useful, highly strained carbocycles. Enzymes, the catalytic workhorses of biology, are capable of accelerating chemical transforma- tions by orders of magnitude while exhibiting exquisite control over selectivity (20). Although na- ture synthesizes various cyclopropane-containing products (21), cyclopropene or bicyclobutane frag- ments are extremely rare (fig. S3) (22, 23). This may be attributed to the lack of biological machin- ery for synthesizing these motifs and/or the in- stability of these structures under biological or natural product isolation/purification conditions. Nonetheless, we envisioned that existing enzymes could be repurposed to forge strained carbocycles by taking advantage of their catalytic promiscuity (24, 25) in the presence of non-natural substrates and by using directed evolution to optimize the ac- tivity and selectivity of these starting enzymes ( 26). In the past several years, we and others have engineered natural hemeproteins to catalyze re- actions not known in nature (2732). We hypoth- esized that carbene transfer to triple bonds with a heme-dependent enzyme might afford highly strained cyclopropene and bicyclobutane struc- tures and might do so enantioselectively. We anticipated several challenges at the outset, es- pecially in chiral bicyclobutane formation, as it involves two sequential carbene additions to the alkyne substrate: (i) The enzyme would need to bind the alkyne in a specific conformation in order to transfer the carbene enantioselective- ly; (ii) the high-energy cyclopropene interme- diate generated by the first carbene addition would need to be accepted and stabilized by the protein; (iii) relative to methylene carbene used previously, a substituted carbene (e.g., with an ester group) might hinder access of the cyclo- propene to the iron-carbenoid; and (iv) the protein would also need to exert precise stereocontrol over the second carbene transfer step, regard- less of structural differences between the ini- tial alkyne and the cyclopropene intermediate. Despite these challenges, we decided to inves- tigate whether a starting enzyme with this unusual and non-natural activity could be identified, and whether its active site could be engineered to create a suitable environment for substrate bind- ing, intermediate stabilization, and selective pro- duct formation. We first tested whether free heme [with or without bovine serum albumin (BSA)], which is known to catalyze styrene cyclopropanation (27), could transfer carbene to an alkyne. Reac- tions using ethyl diazoacetate (EDA) and phenyl- acetylene (1a) as substrates in neutral buffer (M9-N minimal medium, pH 7.4) at room temperature, however, gave no cyclopropene or bicyclobutane product. Next, a panel of hemeproteinsincluding variants of cytochrome P450, cytochrome P411 (P450 with the axial cysteine ligand replaced by serine), cytochrome c, and globins in the form of E. coli whole-cell catalystswere tested for the desired transformation under anaerobic conditions ( 32), but none were fruitful (Fig. 1C and table S1). Interestingly, a P411 variant obtained in a previous cyclopropanation study, P411- S1 I263W (see sup- plementary materials for sources, sequences, and mutations), afforded a furan product (3b) with a total turnover number (TTN) of 210. Because other furan analogs have been identified as ad- ducts of carbenes and alkynes (33), we were curious as to how furan 3b was generated. Pre- liminary kinetic study of the enzymatic reaction suggested that the enzyme first synthesized an unstable cyclopropene (3a), which subsequently rearranged to the furan either spontaneously or with assistance from the enzyme (Fig. 1B and fig. S5). This result provided strong evidence that the P411 hemeprotein is capable of transferring a car- bene to an alkyne, which is, to our knowledge, an activity not previously reported for any protein or even any iron complex. To divert the enzymatic reaction to bicyclobu- tane formation, the enzyme would have to trans- fer a second carbene to cyclopropene intermediate 3a before the cyclopropene rearranges to the undesired furan product (Fig. 1B). We thus tested P411 variants closely related to P411-S1 I263W. We reasoned that amino acid residue 263, which resides in the distal pocket above the heme co- factor, might modulate the rate of this step, and that the bulky tryptophan (Trp) side chain at this site may be blocking the second carbene transfer. A P411-S1 variant with phenylalanine (Phe) in- stead of Trp at this position (263F) in fact cata- lyzed bicyclobutane formation at a very low level (<5 TTN) (table S1). Variant P4 with three ad- ditional mutations relative to P411-S1 I263F (V87A, A268G, and A328V) (28) synthesized the desired bicyclobutane 2a with 80 TTN and with the for- mation of furan adduct substantially suppressed (2a:3b > 50:1; Fig. 1C). Another related P411 var- iant, E10 (= P4 A78V A82L F263L), which was RESEARCH Chen et al., Science 360, 7175 (2018) 6 April 2018 1 of 5 Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA. *Corresponding author. Email: [email protected]

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Page 1: BIOCATALYSIS Enzymatic construction of highly strained ......BIOCATALYSIS Enzymatic construction of highly strained carbocycles Kai Chen, Xiongyi Huang, S. B. Jennifer Kan, Ruijie

BIOCATALYSIS

Enzymatic construction of highlystrained carbocyclesKai Chen, Xiongyi Huang, S. B. Jennifer Kan, Ruijie K. Zhang, Frances H. Arnold*

Small carbocycles are structurally rigid and possess high intrinsic energy due to their ringstrain. These features lead to broad applications but also create challenges for theirconstruction. We report the engineering of hemeproteins that catalyze the formation ofchiral bicyclobutanes, one of the most strained four-membered systems, via successivecarbene addition to unsaturated carbon-carbon bonds. Enzymes that producecyclopropenes, putative intermediates to the bicyclobutanes, were also identified. Thesegenetically encoded proteins are readily optimized by directed evolution, functionin Escherichia coli, and act on structurally diverse substrates with high efficiency andselectivity, providing an effective route to many chiral strained structures. Thisbiotransformation is easily performed at preparative scale, and the resulting strainedcarbocycles can be derivatized, opening myriad potential applications.

In cyclic organic molecules, ring strain arisesfromdistortionsof bondangle andbond length,steric clashes of nonbonded substituents, andother effects (1). The simplest carbocycles, cy-clopropanes and cyclobutanes, possess ring

strains of 26 to 28 kcal/mol (2). Introducing carbon-carbon multiple bonds or bridges to these smallring systems induces additional strain as well asstructural rigidity. For example, cyclopropeneswith an endo-cyclic double bond bear a strainof 54 kcal/mol, whereas bicyclo[1.1.0]butanes,folded into puckered structures, distinguish them-selves as one of the most strained four-memberedsystems, with strain of ~66 kcal/mol (fig. S1) (2).These carbocycles are particularly attractive inter-mediates in chemical and materials synthesis,because they can undergo strain-release trans-formations to furnish amyriad of useful scaffolds(3–6). The structural rigidity imparted by strainedrings in supramolecular materials can lead to in-teresting physical properties, such asmechanicalstability (7) and high glass transition temperature(8). The intrinsic energy of these strained struc-tures can also be relieved in response to exog-enous force, which leads to radical changes inphysical properties (e.g., conductivity), a featurehighly desirable for stimulus-responsivematerials(9, 10).High ring strain, however, greatly increases the

difficulty of synthesis. A commonly used methodfor preparing bicyclobutanes starts from dibromo-2-(bromomethyl)cyclopropane substructures anduses organolithium reagents for lithium-halogenexchange, followed by nucleophilic substitutionunder rigorously anhydrous and cryogenic con-ditions (3). An alternative route relies on thedouble transfer of a carbene to alkynes, but thefew examples in the literature are mostly limitedto methylene carbene (11–13). Asymmetric bicy-clobutane construction is particularly challeng-

ing, with multiple chiral centers generated at thesame time (14, 15) (fig. S2). Cyclopropene syn-thesis through enantioselective single-carbeneaddition to alkynes also requires chiral transitionmetal catalysts based on rhodium (16, 17), irid-ium (18), and cobalt (19). Development of a sus-tainable catalytic system that performswith highefficiency and selectivity under ambient condi-tions would be a major advance for constructionof these useful, highly strained carbocycles.Enzymes, the catalytic workhorses of biology,

are capable of accelerating chemical transforma-tions by orders of magnitude while exhibitingexquisite control over selectivity (20). Although na-ture synthesizes various cyclopropane-containingproducts (21), cyclopropene or bicyclobutane frag-ments are extremely rare (fig. S3) (22, 23). Thismay be attributed to the lack of biologicalmachin-ery for synthesizing these motifs and/or the in-stability of these structures under biological ornatural product isolation/purification conditions.Nonetheless, we envisioned that existing enzymescould be repurposed to forge strained carbocyclesby taking advantage of their catalytic promiscuity(24, 25) in the presence of non-natural substratesand by using directed evolution to optimize the ac-tivity and selectivity of these starting enzymes (26).In the past several years, we and others have

engineered natural hemeproteins to catalyze re-actions not known in nature (27–32). We hypoth-esized that carbene transfer to triple bonds witha heme-dependent enzyme might afford highlystrained cyclopropene and bicyclobutane struc-tures and might do so enantioselectively. Weanticipated several challenges at the outset, es-pecially in chiral bicyclobutane formation, as itinvolves two sequential carbene additions tothe alkyne substrate: (i) The enzyme would needto bind the alkyne in a specific conformation inorder to transfer the carbene enantioselective-ly; (ii) the high-energy cyclopropene interme-diate generated by the first carbene additionwould need to be accepted and stabilized bythe protein; (iii) relative to methylene carbeneused previously, a substituted carbene (e.g., with

an ester group) might hinder access of the cyclo-propene to the iron-carbenoid; and (iv) the proteinwould also need to exert precise stereocontrolover the second carbene transfer step, regard-less of structural differences between the ini-tial alkyne and the cyclopropene intermediate.Despite these challenges, we decided to inves-tigatewhether a starting enzymewith this unusualand non-natural activity could be identified, andwhether its active site could be engineered tocreate a suitable environment for substrate bind-ing, intermediate stabilization, and selective pro-duct formation.We first tested whether free heme [with or

without bovine serum albumin (BSA)], whichis known to catalyze styrene cyclopropanation(27), could transfer carbene to an alkyne. Reac-tions using ethyl diazoacetate (EDA) and phenyl-acetylene (1a) as substrates inneutral buffer (M9-Nminimal medium, pH 7.4) at room temperature,however, gave no cyclopropene or bicyclobutaneproduct. Next, a panel of hemeproteins—includingvariants of cytochrome P450, cytochrome P411(P450 with the axial cysteine ligand replaced byserine), cytochrome c, and globins in the formof E. coliwhole-cell catalysts—were tested for thedesired transformationunder anaerobic conditions(32), but none were fruitful (Fig. 1C and table S1).Interestingly, a P411 variant obtained in a previouscyclopropanation study, P411-S1 I263W (see sup-plementary materials for sources, sequences, andmutations), afforded a furan product (3b) witha total turnover number (TTN) of 210. Becauseother furan analogs have been identified as ad-ducts of carbenes and alkynes (33), we werecurious as to how furan 3b was generated. Pre-liminary kinetic study of the enzymatic reactionsuggested that the enzyme first synthesized anunstable cyclopropene (3a), which subsequentlyrearranged to the furan either spontaneously orwith assistance from the enzyme (Fig. 1B and fig.S5). This result provided strong evidence that theP411 hemeprotein is capable of transferring a car-bene to an alkyne, which is, to our knowledge, anactivity not previously reported for any proteinor even any iron complex.To divert the enzymatic reaction to bicyclobu-

tane formation, the enzyme would have to trans-fer a second carbene to cyclopropene intermediate3a before the cyclopropene rearranges to theundesired furan product (Fig. 1B).We thus testedP411 variants closely related to P411-S1 I263W.We reasoned that amino acid residue 263, whichresides in the distal pocket above the heme co-factor, might modulate the rate of this step, andthat the bulky tryptophan (Trp) side chain at thissitemay be blocking the second carbene transfer.A P411-S1 variant with phenylalanine (Phe) in-stead of Trp at this position (263F) in fact cata-lyzed bicyclobutane formation at a very low level(<5 TTN) (table S1). Variant P4 with three ad-ditionalmutations relative to P411-S1 I263F (V87A,A268G, and A328V) (28) synthesized the desiredbicyclobutane 2a with 80 TTN and with the for-mation of furan adduct substantially suppressed(2a:3b > 50:1; Fig. 1C). Another related P411 var-iant, E10 (= P4 A78V A82L F263L), which was

RESEARCH

Chen et al., Science 360, 71–75 (2018) 6 April 2018 1 of 5

Division of Chemistry and Chemical Engineering 210-41,California Institute of Technology, Pasadena, CA 91125, USA.*Corresponding author. Email: [email protected]

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engineered from P4 for nitrene transfer reac-tions (29), catalyzed the desired transformationwith a factor of >6 higher activity (530 TTN, Fig.1E). Nuclear magnetic resonance (NMR) analysisrevealed an exo, endo-configuration of the enzy-matically produced bicyclobutane 2a, which isdistinct from the only reported achiral endo, endo-isomer, made using an osmium-porphyrin com-plex (34, 35). We chose this P411-E10 variant asthe starting template for directed evolution of aneven more efficient bicyclobutane-constructingenzyme.Because the side chain of residue 263 influ-

enced formation of the bicyclobutane product,we performed site-saturationmutagenesis (SSM)of variantE10 at position 263 and screenedwholeE. coli cells expressing the mutated proteins forimproved production of bicyclobutane 2a. Theenzyme having leucine at this position (263L) wasthe most active; other amino acid residues eitherlowered the reactivity toward bicyclobutane for-mation and/or delivered more furan product. Inparallel, two additional residues inE10, Val78 andSer438, were also targeted by SSM. Aromatic re-sidues were found to be activating at position 78,with a phenylalanine or tyrosine mutation givinga factor of 1.5 to 2 improvement over E10. Thisbeneficial effect may stem from a p-p stacking in-teraction between the side chain and the alkynesubstrate or the cyclopropene intermediate. Asingle S438A mutation on a loop residing abovethe heme also increased the activity, giving a fac-tor of >2.5 increase in turnover. Finally, recombi-nation of V78F/Y and S438Amutations led to thediscovery of even more powerful biocatalysts forbicyclobutane formation (e.g., 1880 TTNwithE10V78F S438A) (Fig. 1E and fig. S9).With the evolved E10 V78F S438A variant in

hand,we next assayed the bacterial catalyst againsta panel of aromatic alkyne coupling partners. Bio-transformations with 10 different substrates wereperformed on a scale of 0.1 to 0.2 mmol. Thesepreparative-scale reactions proceeded smoothlyto furnish the corresponding bicyclobutanes withup to 1760 TTN and 80% yield (Fig. 2A). Addi-tionally, three alkynes, 1k, 1l, and 1m, were trans-formed at mmol scale, and bicyclobutanes wereisolated in hundred-milligram quantities, dem-onstrating that the biocatalytic transformation isreadily scalable. Among the 13 different sub-strates, the engineered P411 hemeprotein did notexhibit strong preference toward specific elec-tronic or steric features. Electron-deficient halides(2b–2d),which can be used as prefunctionalitiesfor further transformations, were accepted bythe enzyme, as were electron-rich alkyl or alkoxylgroups (2e–2h and 2k) at the meta or para po-sition of the phenyl group. Even heterocyclic sub-strates such as thiophene (2j) served as suitablealkyne partners, albeit with lower reactivity.Free functionalities, including alcohols (2i and

2m) and a second alkyne (2l), are well preserved,providing an additional opportunity for deriva-tization of these products. A terminal alkyne al-lows copper-catalyzed click chemistry, throughwhich bicyclobutane 2l can be modified with asimple sulfonyl azide (4a) or even decorated with

Chen et al., Science 360, 71–75 (2018) 6 April 2018 2 of 5

Fig. 1. Hemeprotein-catalyzed bicyclobutane formation. (A) Overall reaction of carbene transfer to analkyne catalyzed by an engineered hemeprotein (Et, ethyl; Ph, phenyl). (B) Proposed catalytic cycle of carbenetransfer to phenylacetylene to form cyclopropene and bicyclobutane structures. (C) Screening of heminand hemeprotein catalysts for bicyclobutane formation (BSA, bovine serum albumin;WT,wild type;TDE,V75TM100D M103E; H*, C400H). See supplementary materials for sources, sequences, and mutations in Bacillusmegaterium P411-S1 and other proteins. (D) X-ray crystal structure of P411-E10 (PDB ID: 5UCW) (29)and view of its distal heme region.The P411 heme axial ligand is S400; amino acid residues Val78, Leu263, andSer438 are shown as sticks. (E) Directed evolution of P411-E10 for bicyclobutane formation [usingphenylacetylene and EDA as substrates; numbers refer to total turnovers to product (TTN) measured].Experiments were performed at analytical scale using suspensions of E. coli expressing P411-E10 variants[optical density at 600 nm (OD600) = 10 to 30], 10 mM phenylacetylene, 10 mM EDA, 5 vol % EtOH, andM9-N buffer (pH 7.4) at room temperature under anaerobic conditions for 6 hours. Reactions were performedin quadruplicate.TTN refers to the total desired product, as quantified by gas chromatography (GC), dividedby total hemeprotein. (Note: Because bicyclobutane formation requires two carbene transfers, the numberof carbene transfers the hemeprotein catalyzes is 2 × TTN in these reactions.) See supplementarymaterials forfurther details of reaction conditions and data analysis. Single-letter amino acid abbreviations (here or inFig.3):A,Ala;C,Cys;D,Asp;E,Glu;F,Phe;G,Gly;H,His; I, Ile; L, Leu;M,Met;P,Pro;S,Ser;T,Thr;V,Val;W,Trp;Y,Tyr.

RESEARCH | REPORT

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biologically relevant fragments, such as a phenyl-alanine derivative (4b). An unprotected hydroxylgroup could also offer the possibility of linkage touseful structures. Additionally, to probe the en-antiopurity of bicyclobutane products, we deriv-atized 2l and 2m with L-azido-phenylalanineand (R)-Mosher’s acid, respectively. The diaster-eomeric excess of these derivatized products wouldinform us of the enantiomeric ratio (e.r.) of thebicyclobutanes. In fact, we observed only one di-astereomer of derivatized bicyclobutanes 4b and4c by NMR. Furthermore, the dicarboxylic esterson the bicyclobutane structure can be reducedeasily with a mild reducing reagent, LiBH4, togive diol product 4dwith the strained ring struc-ture preserved. The diol product 4d allowed for

the unequivocal confirmation of the bicyclobu-tane structure and determination of the absoluteconfiguration through x-ray crystallography.We next asked whether the enzyme could stop

at the cyclopropene product if less reactive ali-phatic alkynes were used. To this end, we exam-ined enzyme variants from the P411-S1 lineagefor cyclopropene formation, using phenylbutyne(5a) and EDA as starting reagents. We were en-couraged to see that P4 catalyzed the desiredtransformation with 260 TTN and 95.5:4.5 e.r.Further evolution was performed on P4 to im-prove its catalytic efficiency. We first targetedposition 87, known for its importance to sub-strate recognition in P450-catalyzed oxidations(36). A87F (290 TTN, 3.0:97.0 e.r.) and A87W

(240 TTN, 97.1:2.9 e.r.) were found to exert theopposite enantiopreference, suggesting that re-sidue 87 also controls substrate orientation fornon-native carbene chemistry. Single- and double-site-saturation mutagenesis conducted sequen-tially on P4 A87F and P4 A87W improved bothreactivity and selectivity (Fig. 3A and figs. S11 andS13). The final K10 and C6 variants performedwith higher activity by a factor of >10 relative tothe initial P4 variant and with excellent stereo-control (99.55:0.45 and 99.95:0.05 e.r., respectively).To evaluate the substrate range of the evolved

P411 variants for cyclopropene construction, wefocused on P411-C6 and examined structurally di-verse aliphatic alkynes. Enzymatic reactions with12 alkynes at preparative scale (up to 5.0 mmol)

Chen et al., Science 360, 71–75 (2018) 6 April 2018 3 of 5

Fig. 2. Scope of bicyclobutaneformation and derivatization.(A) Scope of P411-E10 V78FS438A–catalyzed bicyclobutaneformation. Standard conditions ofpreparative-scale reactions (0.1- to0.2-mmol scale unless otherwiseindicated): suspension of E. coli(OD600 = 15 to 20) expressingP411-E10 V78F S438A, 1.0 equivaromatic alkyne, 2.0 to 4.0 equiv EDA,10 to 15 mM D-glucose, 1 to 5 vol %EtOH, and M9-N buffer (pH 7.4) atroom temperature under anaerobicconditions for 12 hours. TTNs weredetermined on the basis of theisolated yields shown. (B) Derivatizationof bicyclobutane products: (a) and(b), copper-catalyzed click cyclizationof 2l with azide substrates (Ac, acetyl; CuTc,copper(I) thiophene-2-carboxylate; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride); (c), esterification of 2mwith Mosher’s acid; (d), reduction of 2k todiol with LiBH4. See supplementary materialsfor further details of reaction conditionsand data analysis.

RESEARCH | REPORT

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afforded the desired cyclopropenes, with TTNsranging from hundreds to thousands and goodto excellent stereoselectivities (Fig. 3, B and C).Alkynes with a linear carbon chain (5b) or cyclicfragments (5g, 5h, and 5j) all served as goodsubstrates. Different functional groups, includ-ing ether (5f, 5i, and 5l), ester (5d), acetal (5e),chloride (5k), and free hydroxyl (5m), were welltolerated. Further optimization of reaction con-ditions with slow addition of EDA, for example,would likely improve the isolated yields, as wedemonstrated for cyclopropene 6h (66% yield,Fig. 3B; 94% yield, Fig. 3C).Cyclopropenes are used as synthetic building

blocks (4, 37), bio-orthogonal imaging precursors(38), and monomers in polymer synthesis (39).Our ability to construct these motifs using bac-teria at scale allows us to further explore theirpotential utility in diverse fields. We carried out

two simple transformations of cyclopropenesto build amultisubstituted cyclopropane 7a anda fused ring system, [4.1.0]heptene 7b (Fig.3C), both of which are substructures commonin pharmaceutical candidates and bioactivenatural products (21).Our results constitute a biocatalytic platform

for the construction of highly strained bicyclo-butanes and cyclopropenes through directed evo-lution of a serine-ligated cytochrome P450 (P411)enzyme. That the protein could be quickly adaptedto produce these highly strained structures (threeto six rounds ofmutagenesis and screening) high-lights the evolvability of the P411 scaffold and itspotential to direct the construction of complexmotifs. The protein enabled the desired trans-formations through activation of iron-carbenoidfor carbene addition to alkynes, stabilization ofthe reactive cyclopropene intermediate (in bicy-

clobutane formation), and precise stereocontrolof the carbene transfer processes. Biotransforma-tions with the evolved enzymes have a surpris-ingly broad substrate scope with high reactivityand selectivity, providing a route to more than25 products in preparative scale. This biocatalyticsystem grants facile access to versatile moleculararchitectures rarely seen in nature, expandingthe set of chemical structures available to bio-logical systems.

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Chen et al., Science 360, 71–75 (2018) 6 April 2018 4 of 5

Fig. 3. Engineering P411 enzymes forstereodivergent cyclopropeneformation: Scope and derivatization.(A) Evolutionary trajectory ofP411-P4 variants for stereodivergentcyclopropenation of aliphatic alkynes.(B) Scope of P411-C6–catalyzedcyclopropene formation. Standardconditions of preparative-scale reactions(0.08- to 0.4-mmol scale): suspensionof E. coli (OD600 = 10 to 32) expressingP411-C6 or K10, 1.0 equiv alkyne,1.0 to 4.0 equiv EDA (6.0 equiv for 5m),10 to 15 mM D-glucose, 1 to 5 vol %EtOH, and M9-N buffer (pH 7.4) atroom temperature under anaerobicconditions for 12 hours (iPr, isopropyl).TTNs were determined on the basisof the isolated yields shown; e.r. values weredetermined by chiral high-performanceliquid chromatography (HPLC). (C) Enzymaticcyclopropenation at mmol scale andderivatization of corresponding products:(a), copper-catalyzed addition to cyclopropene6a for synthesizing a multisubstitutedcyclopropane; (b), Diels-Alder reactionof cyclopropene 6h with 2,3-diMe-buta-1,3-diene to form a fused-ring system. Seesupplementary materials for further details ofreaction conditions and data analysis.

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ACKNOWLEDGMENTS

We thank D. K. Romney, S. C. Hammer, and S.-Q. Zhang forhelpful discussions and comments on the manuscript; C. K. Pier,O. F. Brandenberg, and A. M. Knight for sharing hemeproteinvariants; K. Ding (D. J. Anderson Lab, Caltech) and J. Li (R. H. GrubbsLab, Caltech) for generous donation of materials and reagents;S. C. Virgil and the Caltech Center for Catalysis and ChemicalSynthesis, N. Torian and the Caltech Mass Spectrometry Laboratory,

and M. K. Takase, L. M. Henling, and the Caltech X-rayCrystallography Facility for analytical support; and B. M. Stoltzfor use of polarimeter and chiral gas chromatography equipment.Funding: Supported by NSF Division of Molecular and CellularBiosciences grant MCB-1513007; Ruth L. Kirschstein NIHPostdoctoral Fellowship F32GM125231 (X.H.); and NSF GraduateResearch Fellowship grant DGE-1144469 and the Donna andBenjamin M. Rosen Bioengineering Center (R.K.Z.). R.K.Z. is atrainee in the Caltech Biotechnology Leadership Program. Anyopinions, findings, and conclusions or recommendations expressed inthis material are those of the author(s) and do not necessarilyreflect the views of the funding organizations. Authorcontributions: conceptualization, K.C.; methodology, K.C.; validation,K.C. and X.H.; formal analysis, K.C., X.H., S.B.J.K., and R.K.Z.; writing(original draft), K.C. and F.H.A.; writing (review and editing), X.H., S.B.J.K., and R.K.Z.; funding acquisition, F.H.A.; supervision, F.H.A.Competing interests: K.C., X.H., and S.B.J.K. are inventors on patentapplication (CIT-7744-P) submitted by California Institute ofTechnology that covers biocatalytic synthesis of strained carbocycles.Data and materials availability: All data are available in the maintext or the supplementary materials. Plasmids encoding the enzymesreported in this study are available for research purposes from F.H.A.under a material transfer agreement with the California Instituteof Technology. Crystallographic coordinates and structure factorshave been deposited with the Cambridge Crystallographic Data Centre(www.ccdc.cam.ac.uk) under reference number 1815089 forcompound 4d.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/360/6384/71/suppl/DC1Materials and MethodsFigs. S1 to S15Tables S1 to S9References (40–71)

8 November 2017; accepted 6 February 201810.1126/science.aar4239

Chen et al., Science 360, 71–75 (2018) 6 April 2018 5 of 5

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