WO2016086015A1 - Catalyseurs à base de myoglobine pour réactions de transfert de carbène - Google Patents

Catalyseurs à base de myoglobine pour réactions de transfert de carbène Download PDF

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WO2016086015A1
WO2016086015A1 PCT/US2015/062478 US2015062478W WO2016086015A1 WO 2016086015 A1 WO2016086015 A1 WO 2016086015A1 US 2015062478 W US2015062478 W US 2015062478W WO 2016086015 A1 WO2016086015 A1 WO 2016086015A1
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myoglobin
substituted
catalyst
aryl
aliphatic
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Rudi Fasan
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University Of Rochester
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P11/00—Preparation of sulfur-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/002—Preparation of hydrocarbons or halogenated hydrocarbons cyclic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P9/00—Preparation of organic compounds containing a metal or atom other than H, N, C, O, S or halogen

Definitions

  • the present invention relates to engineered variants of myoglobin and their use as biocatalysts for catalyzing carbene transfer reactions.
  • the invention relates to myoglobin-based catalysts having capability to catalyze olefin cyclopropanation reactions, carbene insertion reaction into N— H, S— H, and Si— H bonds, sigmatropic rearrangement reactions, and/or aldehyde olefination reactions.
  • the invention also relates to methods for carrying out these transformations in vitro and in whole cells comprising providing a carbene acceptor substrate, a carbene donor reagent, and a myoglobin-based catalyst in an isolated form or contained within a host cell.
  • Enzymes and other protein-based biocatalysts constitute an attractive alternative to traditional chemical catalysts due to their ability to operate in aqueous media and under very mild reactions conditions such as ambient temperature and pressure (Bornscheuer, Huisman et al. 2012). These properties combined with the proteinaceous nature of these catalysts make them particularly relevant toward the design and implementation of sustainable and environmentally friendly procedures for chemical synthesis and manufacturing (Bornscheuer, Huisman et al. 2012). Notably, no naturally occurring enzymes are known to catalyze the aforementioned carbene transfer reactions in biological systems. Recent studies have shown that cytochrome P450 enzymes (e.g., P450BM3, a.k.a.
  • CYP102A1 can react with diazocompounds and promote reactions such as the cyclopropanation of styrene derivatives (Coelho, Housead et al. 2013; Coelho, Wang et al. 2013; Wang, Renata et al. 2014) and carbene N— H insertion in aniline derivatives in vitro and in vivo (Wang, Peck et al. 2014). See also Coehlo et al. US Pat. 8,993,262 B2 and Coehlo et al. Pat. Appl. WO2014058729.
  • P450-based catalysts include their large size (5-115 kDa) and limited stability, in particular at elevated temperatures and in the presence of organic cosolvents.
  • these P450-based catalysts are often characterized by modest catalytic efficiency, limited substrate scope, and/or moderate diastereo- and enantio/stereoselectivity (Coelho, Housead et al. 2013; Coelho, Wang et al. 2013; Wang, Peck et al. 2014; Wang, Renata et al. 2014). 3.
  • An engineered myoglobin catalyst having an improved capability, as compared to the myoglobin of SEQ ID NO: 1, to catalyze a carbene transfer reaction, wherein the engineered myoglobin catalyst comprises an amino acid sequence that is at least 60% identical to SEQ ID NO: 1, 112, 113, 114, 115, and 116.
  • the improved capability of the myoglobin catalyst is an improvement in its catalytic activity, regioselectivity,
  • the myoglobin catalyst comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and comprises an amino acid substitution at a position selected from the group consisting of position X29, X32, X33, X39, X44, X45, X46, X64, X67, X68, X93, X107, and XI 11 of SEQ ID NO: 1.
  • the amino acid sequence of the myoglobin catalyst comprises at least one of the features selected from the group consisting of: X29 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, U, V, W, or Y; X32 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, U, V, W, or Y; X33 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, U, V, W, or Y; X39 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, U, V, W, or Y; X43 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S,
  • the myoglobin catalyst is selected from the group consisting of SEQ ID NOS: 2 through 110.
  • the myoglobin catalyst contains a metal-binding cofactor selected from the group consisting of a heme analog, a metalloporphyrin, and a porphyrin analog.
  • the metal-binding cofactor is selected from the group consisting of mesoporphyrin, protoporphyrin, bisglycolporphyrin, corrole, phthalocyanine, phlorin, chlorin, 5-isocorrole, 10-isocorrole, and porphycene.
  • the metal bound by the metal-binding cofactor is selected from the group consisting of iron, manganese, cobalt, ruthenium, rhodium, and osmium.
  • the amino acid residue that coordinates the metal atom at the axial position of the metal-containing cofactor in the myoglobin catalyst is selected from the group consisting of serine, threonine, cysteine, tyrosine, histidine, aspartic acid, glutamic acid, selenocysteine, para-amino-phenylalanine, meta-amino- phenylalanine, para-mercaptomethyl-phenylalanine, meto-mercaptomethyl-phenylalanine, parci- (isocyanomethyl)-phenylalanine, meto-(isocyanomethyl)-phenylalanine, 3-pyridyl-alanine, and 3-methyl-histidine.
  • a method for catalyzing a carbene insertion reaction comprising:
  • R] a and R 2a are independently selected from H, halo, cyano (— CN), nitro (— N0 2 ), trifluoromethyl (— CF 3 ), optionally substituted C 1-18 alkyl, optionally substituted C 6 -io aryl, optionally substituted 5- to 10-membered heteroaryl, — C(0)OR lb , — C(0)N(Ri b )(Ric), — C(0)R lb , — Si(R lb )(R lc )(R ld ), and — S0 2 (R lb ), where each R] b , R] Ci and R ⁇ are independently selected from H, optionally substituted CMS alkyl, optionally substituted C 6 -io aryl, and optionally substituted 6- to 10-membered heteroaryl. (b) providing a myoglobin-based catalyst;
  • R 2 is independently selected from optionally substituted C 6 -i5 aryl, optionally substituted 5- to 15-membered heteroaryl, and optionally substituted C 1-18 aliphatic
  • R3 is independently selected from H, halo, cyano, optionally substituted C 1-18 aliphatic, optionally substituted C 6 -io aryl, optionally substituted 5- to 10-membered heteroaryl,— C(0)ORi b ,— C(0)N(Ri b )(Ri c ), and— C(0)Ri b , where each R lb and R lc are independently selected from H, optionally substituted C 1-18 aliphatic, optionally substituted C 6 -io aryl, and optionally substituted 5- to 10-membered heteroaryl;
  • R 4 and R5 are independently selected from H, halo, cyano, optionally substituted C 1-18 aliphatic, optionally substituted C 6 -io aryl, and optionally substituted 5- to
  • R 6 is independently selected from optionally substituted C 1-18 aliphatic, optionally substituted C 6 -i6 aryl, optionally substituted 5- to 10-membered heteroaryl, optionally substituted C 4 -Ci 6 cyclic aliphatic, and optionally substituted C 4 -Ci 6 heterocyclic group;
  • R7 is independently selected from H, optionally substituted C 1-18 aliphatic, optionally substituted C 6 -i6 aryl, optionally substituted 5- to 10-membered heteroaryl; or where R 6 and R7 are connected to form an optionally substituted C 4 -Ci 6 cyclic aliphatic or heterocyclic group;
  • Rs is selected from optionally substituted C 1-18 aliphatic, optionally substituted C 6 -i6 aryl, optionally substituted 5- to 10-membered heteroaryl, optionally substituted C 4 - Ci6 cyclic aliphatic, and optionally substituted C 4 -Ci 6 heterocyclic group;
  • R9 is independently selected from optionally substituted C 1-18 aliphatic, optionally substituted C 6 -i6 aryl, optionally substituted 5- to 10-membered heteroaryl, optionally substituted C 4 -Ci 6 cyclic aliphatic, and optionally substituted C 4 -Ci 6 heterocyclic group; Rio and R n are optionally substituted Ci_ 6 aliphatic groups. (d) contacting the diazo-containing carbene precursor and the carbene acceptor substrate with the myoglobin-based catalyst, optionally in the presence of a reducing agent; and
  • R] a , R 2a , R 2 , R3, R4, R5, R 6 , R7, Rs, R9, Rio, and Rn are as defined above.
  • the myoglobin comprises an amino acid sequence that is at least 60% identical to SEQ ID NO: 1, 112, 113, 114, 115, and 116.
  • the myoglobin catalyst comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and comprises an amino acid substitution at a position selected from the group consisting of position X29, X32, X33, X39, X44, X45, X46, X64, X67, X68, X93, X107, and XI 11 of SEQ ID NO: 1.
  • the amino acid sequence of the myoglobin catalyst comprises at least one of the features selected from the group consisting of: X29 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, U, V, W, or Y; X32 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, U, V, W, or Y; X33 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, U, V, W, or Y; X39 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, U, V, W, or Y; X43 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S,
  • the myoglobin catalyst is selected from the group consisting of SEQ ID NOS: 2 through 110.
  • the myoglobin catalyst contains a metal-binding cofactor selected from the group consisting of a heme analog, a metalloporphyrin, and a porphyrin analog.
  • the metal-binding cofactor is selected from the group consisting of mesoporphyrin, protoporphyrin, bisglycolporphyrin, corrole,
  • the metal bound by the metal-binding cofactor is selected from the group consisting of iron, manganese, cobalt, ruthenium, rhodium, and osmium.
  • the amino acid residue that coordinates the metal atom at the axial position of the metal-containing cofactor in the myoglobin catalyst is selected from the group consisting of serine, threonine, cysteine, tyrosine, histidine, aspartic acid, glutamic acid, selenocysteine, para-amino-phenylalanine, meto-amino-phenylalanine, para- mercaptomethyl-phenylalanine, meto-mercaptomethyl-phenylalanine, /?ara-(isocyanomethyl)- phenylalanine, meto-(isocyanomethyl)-phenylalanine, 3-pyridyl-alanine, and 3-methyl-histidine.
  • the myoglobin catalyst is tethered to a solid support.
  • the myoglobin catalyst is contained in a host cell.
  • the host cell is selected from the group consisting of Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris.
  • the carbene insertion product of formula (III) is selected from the group of consisting of:
  • Ar is independently selected from optionally substituted C 6 -i5 aryl and optionally substituted 6 to 15 membered heteroaryl;
  • Alk is an optionally substituted C 1-18 aliphatic.
  • the diazo-containing carbene precursor and the carbene acceptor substrate are part of the same molecule.
  • a method for catalyzing a sigmatropic rearrangement reaction comprising:
  • R] a and R 2a are independently selected from H, halo, cyano (— CN), nitro (— N0 2 ), trifluoromethyl (— CF 3 ), optionally substituted C 1-18 alkyl, optionally substituted C 6 -io aryl, optionally substituted 5- to 10-membered heteroaryl, — C(0)OR lb , — C(0)N(Ri b )(Ric), — C(0)R lb , — Si(R lb )(R lc )(R ld ), and — S0 2 (R lb ), where each R] b , R] C and R w are independently selected from H, optionally substituted C 1-18 alkyl, optionally substituted C 6 -io aryl, and optionally substituted 6- to 10-membered heteroaryl.
  • R12 is selected from optionally substituted C 1-18 aliphatic, optionally substituted C 6 -i6 aryl, optionally substituted 5- to 10-membered heteroaryl, and optionally substituted C4-C16 heterocyclic group;
  • Ri 3 , Ri4, and R15 are independently selected from H, optionally substituted Ci_ 6 aliphatic groups, optionally substituted C 6 -i6 aryl, or where R1 3 and R14 are connected to form an optionally substituted C4-C16 cyclic aliphatic or heterocyclic group;
  • Ri6 is independently selected from optionally substituted C 1-18 aliphatic, optionally substituted C 6 -i6 aryl, optionally substituted 5- to 10-membered heteroaryl, and optionally substituted C4-C16 heterocyclic group;
  • Ri7 is independently selected from optionally substituted Ci_6 aliphatic, optionally substituted C 6 aryl, optionally substituted 5- to 6-membered heteroaryl; or where R] 6 and Ri7 are connected together to form an optionally substituted C4-C16 cyclic aliphatic or heterocyclic group;
  • Ri 8 , R19, and R 2 o are independently selected from H, optionally substituted Ci_6 aliphatic groups, optionally substituted C 6 -i6 aryl, or where Ris and R19 are connected together to form an optionally substituted C4-C16 cyclic aliphatic or heterocyclic group;
  • R] a , R 2a , R9, R12, R13, R14, R15, R1 ⁇ 2, R17, Ri8, R19, and R 2 o are as defined above.
  • the myoglobin comprises an amino acid sequence that is at least 60% identical to SEQ ID NO: 1, 112, 113, 114, 115, and 116.
  • the myoglobin catalyst comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and comprises an amino acid substitution at a position selected from the group consisting of position X29, X32, X33, X39, X44, X45, X46, X64, X67, X68, X93, X107, and XI 11 of SEQ ID NO: 1.
  • the amino acid sequence of the myoglobin catalyst comprises at least one of the features selected from the group consisting of: X29 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, U, V, W, or Y; X32 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, U, V, W, or Y; X33 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, U, V, W, or Y; X39 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, U, V, W, or Y; X43 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S,
  • the myoglobin catalyst is selected from the group consisting of SEQ ID NOS: 2 through 110.
  • the myoglobin catalyst contains a metal-binding cofactor selected from the group consisting of a heme analog, a metalloporphyrin, and a porphyrin analog.
  • the metal-binding cofactor is selected from the group consisting of mesoporphyrin, protoporphyrin, bisglycolporphyrin, corrole,
  • the metal bound by the metal-binding cofactor is selected from the group consisting of iron, manganese, cobalt, ruthenium, rhodium, and osmium.
  • the amino acid residue that coordinates the metal atom at the axial position of the metal-containing cofactor in the myoglobin catalyst is selected from the group consisting of serine, threonine, cysteine, tyrosine, histidine, aspartic acid, glutamic acid, selenocysteine, para-amino-phenylalanine, meto-amino-phenylalanine, para- mercaptomethyl-phenylalanine, meto-mercaptomethyl-phenylalanine, /?ara-(isocyanomethyl)- phenylalanine, meto-(isocyanomethyl)-phenylalanine, 3-pyridyl-alanine, and 3-methyl-histidine.
  • the myoglobin catalyst is tethered to a solid support.
  • the myoglobin catalyst is contained in a host cell.
  • the host cell is selected from the group consisting of Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris.
  • the diazo-containing carbene precursor and the carbene acceptor substrate are part of the same molecule.
  • a method for catalyzing an aldehyde olefination reaction comprising:
  • R] a and R 2a are independently selected from H, halo, cyano (— CN), nitro (— N0 2 ), trifluoromethyl (— CF 3 ), optionally substituted C 1-18 alkyl, optionally substituted C 6 -io aryl, optionally substituted 5- to 10-membered heteroaryl, — C(0)OR lb , — C(0)N(Ri b )(Ric), — C(0)R lb , — Si(R lb )(R lc )(R ld ), and — S0 2 (R lb ), where each R] b , R] Ci and R ⁇ are independently selected from H, optionally substituted C 1-18 alkyl, optionally substituted C 6 -io aryl, and optionally substituted 6- to 10-membered heteroaryl.
  • R 2 i is selected from optionally substituted C 1-18 aliphatic, optionally substituted C 6 -i6 aryl, optionally substituted 5- to 10-membered heteroaryl, and optionally substituted C4-C16 heterocyclic group;
  • nucleophilic reagent selected from the group consisting of triphenylphosphine, triphenylarsine, and triphenylstilbine;
  • the myoglobin comprises an amino acid sequence that is at least 60% identical to SEQ ID NO: 1, 112, 113, 114, 115, and 116.
  • the myoglobin catalyst comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and comprises an amino acid substitution at a position selected from the group consisting of position X29, X32, X33, X39,
  • the amino acid sequence of the myoglobin catalyst comprises at least one of the features selected from the group consisting of: X29 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, U, V, W, or Y; X32 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, U, V, W, or Y; X33 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, U, V, W, or Y; X39 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, U, V, W, or Y; X43 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S,
  • the myoglobin catalyst is selected from the group consisting of SEQ ID NOS: 2 through 110.
  • the myoglobin catalyst contains a metal-binding cofactor selected from the group consisting of a heme analog, a metalloporphyrin, and a porphyrin analog.
  • the metal-binding cofactor is selected from the group consisting of mesoporphyrin, protoporphyrin, bisglycolporphyrin, corrole,
  • the metal bound by the metal-binding cofactor is selected from the group consisting of iron, manganese, cobalt, ruthenium, rhodium, and osmium.
  • the amino acid residue that coordinates the metal atom at the axial position of the metal-containing cofactor in the myoglobin catalyst is selected from the group consisting of serine, threonine, cysteine, tyrosine, histidine, aspartic acid, glutamic acid, selenocysteine, para-amino-phenylalanine, meto-amino-phenylalanine, para- mercaptomethyl-phenylalanine, meto-mercaptomethyl-phenylalanine, /?ara-(isocyanomethyl)- phenylalanine, meto-(isocyanomethyl)-phenylalanine, 3-pyridyl-alanine, and 3-methyl-histidine.
  • the myoglobin catalyst is tethered to a solid support.
  • the myoglobin catalyst is contained in a host cell.
  • the host cell is selected from the group consisting of Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris.
  • the diazo-containing carbene precursor and the aldehyde substrate are part of the same molecule.
  • FIG. 1 Crystal structure of sperm whale myoglobin (pdb 1A6K).
  • the heme cof actor, the heme-coordinating proximal histidine, and various amino acid residues lining the active site ('distal pocket') of the hemoprotein are displayed as stick models.
  • FIG. 2 Carbene transfer reactions catalyzed by the myoglobin catalysts provided herein: (a) olefin cyclopropanation; (b) carbene N— H insertion; (c) carbene S— H insertion; (d) carbene Si— H insertion.
  • FIG. 3 Additional reactions catalyzed by the myoglobin catalysts provided herein.
  • FIG. 4 Activity and selectivity of wild-type sperm whale myoglobin and engineered variants thereof toward cyclopropanation of styrene in the presence of ethyl diazoacetate.
  • FIG. 5 Mechanistic model for myoglobin-catalyzed cyclopropanation of styrene with diazo esters.
  • FIGS. 6A-B Plots of initial rates (v 0 ) for Mb(H64V,V68A)-catalyzed
  • FIG. 7 Optimization of styrene: EDA ratio for Mb(H64V,V68A)-catalyzed reactions. Turnover numbers (TON) for the cyclopropane product and carbene dimerization byproduct (diethyl fumarate + diethyl maleate) are plotted against the different styrene : EDA ratios used in the reaction.
  • FIG. 8 Yields and turnovers numbers for Mb(H64V,V68A)-catalyzed styrene cyclopropanation in the presence of EDA at varying reagents and catalyst loadings using a constant styrene : EDA ratio of 1 : 2 and after one hour reaction time.
  • FIG. 9 Substrate scope for Mb(H64V,V68A)-catalyzed cyclopropanation.
  • FIGS. 10A-B Representative chiral GC chromatograms corresponding to the products 3a, 3b, 3c, and 3d (a) as authentic racemic standards obtained from the reaction with styrene and EDA in the presence of Rh 2 (OAc) 4 as the catalyst, and (b) as produced from the reaction with Mb(H64V,V68A) as the catalyst.
  • FIG. 11 Catalytic activity of hemin, wild-type sperm whale myoglobin (Mb), and the Mb(H64V,V68A) variant toward promoting carbene N— H insertion reaction in the presence of aniline and EDA.
  • FIG. 12 Yields and total turnover numbers (TTN) for Mb(H64V,V68A)-catalyzed carbene N— H insertion with various aryl amines. Reaction conditions: 10 mM amine, 10 mM EDA, 10 mM Na 2 S 2 0 4 with (a) 20 ⁇ (0.2 mol ) and (b) 1 ⁇ (0.01 mol ) hemoprotein.
  • FIG. 13 Total turnovers supported by the different Mb variants for formation of N— H insertion products 32b (N-methyl aniline + EDA) and 35 (aniline + iBDA).
  • FIG. 14 Catalytic turnovers (TON) and enantioselectivity exhibited by representative Mb catalysts for formation of carbene N— H insertion products starting from aniline and various carbene donor reagents.
  • FIG. 15 Catalytic turnovers (TON) and enantioselectivity exhibited by representative Mb catalysts for formation of carbene N— H insertion products starting from various alkyl amines and carbene donor reagents.
  • FIG. 16 Catalytic activity of sperm whale myoglobin (Mb) for the carbene S— H insertion reaction with thiophenol and EDA. Reaction conditions: 400 ⁇ -scale reactions, 12 hours, room temperature, anaerobic conditions.
  • FIGS. 17A-B Mb-catalyzed S-H reaction.
  • A GC chromatogram corresponding to the Mb-catalyzed S-H insertion reaction with thiophenol and EDA. The peaks corresponding to the S-H insertion product, oc-(phenylthio)acetate (53), and the internal standard (ISTD) are labelled. Thiophenol elutes at 2.42 min and is completely consumed in the reaction. Trace amounts of diphenyldisulfide (labeled with *) are observed in the reaction mixture.
  • FIG. 18 Total turnover numbers (TTN) supported by various engineered Mb variants for conversion of thiophenol and EDA to 53. Reaction conditions: 2.5 ⁇ Mb variant, 10 mM PhSH, 20 mM EDA, 10 mM Na 2 S 2 0 4 in KPi buffer (pH 8.0), 16 h. WT: wild-type.
  • FIG. 19 Yields and total turnover numbers (TTN) for Mb(L29A,H64V)-catalyzed carbene S— H insertion with various aryl mercaptans and oc-diazo esters. Reaction conditions: 10 mM thiol, 20 mM EDA, 10 mM Na 2 S 2 0 4 with (a) 20 ⁇ (0.2 mol ) and (b) 2.5 ⁇ (0.025 mol ) hemoprotein, 16 hours. * Buffer added with 20% (v/v) methanol.
  • FIG. 20 Substrate scope and catalytic activity of Mb(L29A,H64V) toward carbene S— H insertion in the presence of different alkyl mercaptans and oc-diazo esters. Reaction conditions: 10 mM thiol, 20 mM diazo ester, 20 ⁇ Mb(L29A,H64V) (0.2 mol%), 10 mM Na 2 S 2 0 4 in oxygen- free phosphate buffer (pH 8.0), 12 hours. * Total turnover numbers (TTN) were measured using 0.025 mol% protein (2.5 ⁇ ).
  • FIG. 21 Enantioselectivity of myoglobin (Mb) and variants thereof for the carbene S— H insertion reaction in the presence of ethyl oc-diazopropanoate (52e).
  • Reaction conditions 400 ⁇ -scale reactions, 20 ⁇ protein, 10 mM Na 2 S 2 0 4 , 12 hours, room temperature, anaerobic conditions.
  • Enantiomeric excess (% e.e.) was determined based on chiral gas chromatography using racemic standards for calibration.
  • FIG. 22 Representative chiral GC chromatograms corresponding to product 71 (a) as authentic racemic standard synthesized using Rh 2 (OAc) 4 as the catalyst, (b) as produced from the reaction with Mb(F43V) (Entry 3, FIG. 21), (c) as produced from the reaction with
  • FIG. 23 Myoglobin-catalyzed conversion of allyl(phenyl)sulfane to the [2,3]- sigmatropic rearrangement product 92 in the presence of EDA.
  • the table describes the catalytic activity (TON) and enantioselectivity of different engineered Mb variants. Reaction conditions: 10 mM thiol, 20 mM diazo reagent, 10 ⁇ Mb catalyst, KPi pH 8.0, room temperature, 12 hours.
  • FIGS. 24-25 Representative [2,3] sigmatropic rearrangement reactions involving different sulfane substrates and carbene donor reagents as catalyzed by the myoglobin catalysts provided herein.
  • S.r.c. standard reactions conditions (10 mM thiol, 20 mM diazo reagent, 10 ⁇ Mb catalyst, KPi pH 8.0, room temperature, 12 hours).
  • FIGS. 26A-D Metallo-substituted Mb variants. Overlay plot of the electronic absorption spectrum of (A) wild-type Mb, (B) the Mn-containing Mb variant, and (C) Co- containing Mb variant, in oxidized (solid line) and reduced form (dotted line). The Q band regions are enlarged in the inserts. (D) Overlay of the electronic absorption spectra of H93S and H93pAmF variants of sperm whale myoglobin.
  • FIG. 27 Relative yield of Mb(H64V,V68A)-catalyzed styrene cyclopropanation with ethyl 2-diazoacetate in whole-cell systems under anaerobic or aerobic conditions and in the presence or absence of glucose. Reaction conditions: 30 mM styrene, 60 mM EDA,
  • FIGS. 28A-B Whole-cell reactions involving E. coli cells expressing
  • % ee ⁇ Positive and negative values refer to the formation of the trans- (IS,2S) (3a) and trans-(lR,2R) (3b) stereoisomer, respectively.
  • % eez Positive and negative values refer to the formation of the cis-(lR,2S) (3d) and cis-(lS,2R) (3c) stereoisomer, respectively.
  • FIG. 30 Catalytic activity of hemin and wild-type sperm whale myoglobin (Mb) in the olefination of benzaldehyde with ethyl oc-diazoacetate (EDA). Reaction conditions: 10 mM 111a, 10 mM 112a, 20 ⁇ catalyst, 10 mM Na 2 S 2 0 4 , and 10 mM Y in oxygen-free phosphate buffer (pH 8.0) for 12 hours at room temperature.
  • EDA ethyl oc-diazoacetate
  • FIG. 31 Catalytic activity and selectivity of myoglobin variants in benzaldehyde olefination with EDA. Reaction conditions: same as described in legend of FIG. 30.
  • FIG. 32 Catalytic activity and selectivity of Mb(F43V,V68F) variants in
  • FIG. 33 Substrate scope for Mb(F43V,V68F)-catalyzed aldehyde olefination.
  • Reaction conditions 10 mM aryl aldehyde, 1 ⁇ Mb(F43V,V68F), 10 mM cyclohexyl cc-diazo- acetate (112d), 10 mM AsPh 3 , 10 mM Na 2 S 2 0 4 .
  • Myoglobin is a small (about 150 amino acid residues), oxygen-binding hemoprotein found in the muscle tissue of vertebrates. The physiological role of myoglobin is to bind molecular oxygen (0 2 ) with high affinity, providing a reservoir and source of oxygen to support the aerobic metabolism of muscle tissue.
  • Myoglobin contains a heme group (iron- protoporphyrin IX) which is coordinated at the proximal site via the imidazolyl group of a conserved histidine residue (e.g., His93 in sperm whale myoglobin).
  • a distal histidine residue (e.g., His64 in sperm whale myoglobin) is present on the distal face of the heme ring, playing a role in favoring binding of 0 2 to the heme iron center.
  • Myoglobin belongs to the globin superfamily of proteins and consists of multiple (typically eight) alpha helical segments connected by loops. In biological systems, myoglobin does not exert any catalytic function.
  • engineered variants of sperm whale myoglobin can provide robust, efficient, and selective biocatalysts for promoting a variety of carbene-mediated reactions of high synthetic utility.
  • engineered variants of sperm whale myoglobin can react with diazo-containing reagents and catalyze a variety of synthetically valuable reactions which include alkene cyclopropanation, carbene insertion into a N— H, S— H, or Si— H bond, the [2,3]-sigmatropic rearrangements of thioether and tertiary amine substrates, and aldehyde olefination.
  • the inventor has discovered methods, involving sperm whale myoglobin and engineered variants thereof, to catalyze a variety of other reactions, including carbene S— H insertion, carbene Si— H insertion, sigmatropic rearrangements of thioether/tertiary amine substrates, and aldehyde olefination, for which no natural or engineered biocatalysts have been reported.
  • the myoglobin-based catalysts provided herein constitute valuable and efficient catalysts for the synthesis of a variety of organic molecules, including cyclopropanes, amines, ethers, thioethers, silanes, and olefins.
  • FIG. 2 shows carbene transfer reactions catalyzed by the myoglobin catalysts provided herein: (a) olefin cyclopropanation; (b) carbene N— H insertion; (c) carbene S— H insertion; (d) carbene Si— H insertion.
  • FIG. 3 shows additional reactions catalyzed by the myoglobin catalysts provided herein: (a) [2,3] sigmatropic rearrangement of allylic thioethers; (b) [2,3] sigmatropic rearrangement of propargylic thioethers; (c) [2,3] sigmatropic rearrangement of allylic amines; (d) [2,3] sigmatropic rearrangement of propargylic amines; (e) aldehyde olefination.
  • a method for catalyzing an alkene cyclopropanation reaction to produce a product having two new C— C bonds comprising:
  • a method for catalyzing a carbene N— H insertion reaction to produce a product having a new C— N bond comprising: (a) providing an N— H containing substrate, a diazo-containing reagent as carbene precursor, and an engineered myoglobin variant as the catalyst;
  • a method for catalyzing a carbene S— H insertion reaction to produce a product having a new C— S bond comprising:
  • a method for catalyzing a carbene Si— H insertion reaction to produce a product having a new C— Si bond comprising:
  • a method for catalyzing a [2,3] sigmatropic rearrangement reaction to produce a product having a new C— S bond comprising:
  • a method for catalyzing a [2,3] sigmatropic rearrangement reaction to produce a product having a new C— N bond comprising:
  • Cytochrome P450s and engineered variants thereof have been reported to catalyze a carbene N— H insertion reaction with aniline derivatives and EDA (Wang, Peck et al. 2014).
  • these P450-based biocatalysts exhibit only modest catalytic efficiencies ( ⁇ 500 TON) and no enantioselectivity (e.g., in the reaction of aniline with EDP) in these reactions.
  • they have limited substrate scope, exhibiting no reactivity in the presence of alkyl amine substrates such as benzyl amine or morpholine (Wang, Peck et al. 2014).
  • sperm whale myoglobin can catalyze these carbene N— H insertion reactions with much greater efficiency (up to 7,000 TON with aniline and EDA).
  • these myoglobin-derived biocatalysts can react with alkyl amines (e.g., benzyl amine, cyclohexyl amine, morpholine) and are capable of catalyzing carbene N-H insertion reactions in a stereoselective manner (e.g., 50% e.e. with benzyl amine and EDP), thus exhibiting a broader scope and reactivity.
  • alkyl amines e.g., benzyl amine, cyclohexyl amine, morpholine
  • sperm whale myoglobin and engineered variants thereof can catalyze a number of other chemical transformations for which no natural or engineered biocatalysts have been reported to date. These transformations include carbene S— H insertion, carbene Si— H insertion reactions, [2,3] sigmatropic rearrangement reactions of thioether and tertiary amine substrates, and aldehyde olefination reactions.
  • these myoglobin-catalyzed reactions can be performed, if desired, in aqueous solvents in the presence of large amounts (e.g., up to 40%) of an organic cosolvent (e.g., acetonitrile, tetrahydrofuran, ethanol, dimethylformamide) and/or elevated temperatures (e.g., up to 60-70°C).
  • organic cosolvent e.g., acetonitrile, tetrahydrofuran, ethanol, dimethylformamide
  • elevated temperatures e.g., up to 60-70°C.
  • these myoglobin-catalyzed reactions can be performed, if desired, in the presence of high substrate loadings (e.g., 0.1-0.3 M substrate and diazo- containing reagents).
  • substrate loadings e.g., 0.1-0.3 M substrate and diazo- containing reagents.
  • the possibility to conduct reactions in the presence of high substrate loadings is convenient toward minimizing the volume of reaction and solvent waste associated with it.
  • these myoglobin-catalyzed reactions can be carried out in whole-cell systems that is, employing cells expressing the myoglobin catalyst instead of purified protein. This capability is important toward eliminating the costs and time associated with the purification of the protein and thus toward optimizing the cost- and time-effectiveness of the biocatalytic process.
  • aliphatic or "aliphatic group” as used herein means a straight or branched Ci-15 hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic C 3 _s hydrocarbon, or bicyclic Cs-i2 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as "cycloalkyl”).
  • suitable aliphatic groups include, but are not limited to, linear or branched alkyl, alkenyl, alkynyl groups or hybrids thereof such as
  • alkyl, alkenyl, or alkynyl group may be linear, branched, or cyclic and may contain up to 15, up to 8, or up to 5 carbon atoms.
  • alkyl groups include methyl, ethyl, propyl, cyclopropyl, butyl, cyclobutyl, pentyl, or cyclopentyl groups.
  • alkenyl groups include propenyl, butenyl, or pentenyl groups.
  • alkynyl groups include propynyl, butynyl, or pentynyl groups.
  • aryl and aryl group refers to an aromatic substituent containing a single aromatic or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such as linked through a methylene or an ethylene moiety).
  • An aryl group may contain from 5 to 24 carbon atoms, 5 to 18 carbon atoms, or 5 to 14 carbon atoms.
  • heteroatom means nitrogen, oxygen, or sulphur, and includes any oxidized forms of nitrogen and sulfur, and the quaternized form of any basic nitrogen.
  • Heteroatom further includes Se, Si, and P.
  • heteroaryl refers to an aryl group in which at least one carbon atom is replaced with a heteroatom.
  • a heteroaryl group is a 5- to 18-membered, a 5- to 14-membered, or a 5- to 10-membered aromatic ring system containing at least one heteroatom selected from the group consisting of oxygen, sulphur, and nitrogen atoms.
  • heteroaryl groups include pyridyl, pyrrolyl, furyl, thienyl, indolyl, isoindolyl, indolizinyl, imidazolyl, pyridonyl, pyrimidyl, pyrazinyl, oxazolyl, thiazolyl, purinyl, quinolinyl, isoquinolinyl, benzofuranyl, and benzoxazolyl groups.
  • a heterocyclic group may be any monocyclic or polycyclic ring system which contains at least one heteroatom and may be unsaturated or partially or fully saturated.
  • heterocyclic thus includes heteroaryl groups as defined above as well as non-aromatic heterocyclic groups.
  • a heterocyclic group is a 3- to 18- membered, a 3- to 14-membered, or a 3- to 10-membered, ring system containing at least one heteroatom selected from the group consisting of oxygen, sulphur, and nitrogen atoms.
  • heterocyclic groups include the specific heteroaryl groups listed above as well as pyranyl, piperidinyl, pyrrolidinyl, dioaxanyl, piperazinyl, morpholinyl, thiomorpholinyl, morpholinosulfonyl, tetrahydroisoquinolinyl, and tetrahydrofuranyl groups.
  • a halogen atom may be a fluorine, chlorine, bromine, or iodine atom.
  • substituted sulfhydryl refers to a contiguous group of atoms.
  • substituted sulfhydryl include, without limitation: alkoxy, aryloxy, alkyl, heteroatom-containing alkyl, alkenyl, heteroatom-containing alkenyl, alkynyl, heteroatom-containing alkynyl, aryl, heteroatom-containing aryl, alkoxy, heteroatom-containing alkoxy, aryloxy, heteroatom- containing aryloxy, halo, hydroxyl (— OH), sulfhydryl (— SH), substituted sulfhydryl, carbonyl (— CO— ), thiocarbonyl, (— CS— ), carboxy (— COOH), amino (— NH 2 ), substituted amino, nitro (— N0 2 ), nitroso (—NO), sulfo (— S0 2 — OH), cyano (— C ⁇ N), cyanato (—
  • substituents include, without limitation, halogen atoms, hydroxyl (— OH), sulfhydryl (— SH), substituted sulfhydryl, carbonyl (—CO—), carboxy (—COOH), amino (— NH 2 ), nitro (— N0 2 ), sulfo (— S0 2 — OH), cyano (— C ⁇ N), thiocyanato (— S— C ⁇ N), phosphono (— P(0)OH 2 ), alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, heterocyclic, alkylthiol, alkyloxy, alkylamino, arylthiol, aryloxy, or arylamino groups.
  • optionally substituted modifies a series of groups separated by commas (e.g., “optionally substituted A, B, or C”; or “A, B, or C optionally substituted with”), it is intended that each of the groups (e.g., A, B, or C) is optionally substituted.
  • heteroatom-containing aliphatic refers to an aliphatic moiety where at least one carbon atom is replaced with a heteroatom, e.g., oxygen, nitrogen, sulphur, selenium, phosphorus, or silicon, and typically oxygen, nitrogen, or sulphur.
  • alkyl and alkyl group refer to a linear, branched, or cyclic saturated hydrocarbon typically containing 1 to 24 carbon atoms, 1 to 18 carbon atoms or 1 to 12 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl and the like.
  • heteroatom-containing alkyl refers to an alkyl moiety where at least one carbon atom is replaced with a heteroatom, e.g., oxygen, nitrogen, sulphur, phosphorus, or silicon, and typically oxygen, nitrogen, or sulphur.
  • alkenyl and alkenyl group refer to a linear, branched, or cyclic hydrocarbon group of 2 to 24 carbon atoms, 2 to 18 carbon atoms, or 2 to 12 carbon atoms, containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, and the like.
  • heteroatom-containing alkenyl refers to an alkenyl moiety where at least one carbon atom is replaced with a heteroatom.
  • alkynyl and alkynyl group refer to a linear, branched, or cyclic hydrocarbon group of 2 to 24 carbon atoms, 2 to 18 carbon atoms, or 2 to 12 carbon atoms, containing at least one triple bond, such as ethynyl, n-propynyl, and the like.
  • heteroatom-containing alkynyl refers to an alkynyl moiety where at least one carbon atom is replaced with a heteroatom.
  • heteroatom-containing aryl refers to an aryl moiety where at least one carbon atom is replaced with a heteroatom.
  • alkoxy and alkoxy group refer to an aliphatic group or a heteroatom-containing aliphatic group bound through a single, terminal ether linkage.
  • aryloxy and aryloxy group refer to an aryl group or a heteroatom-containing aryl group bound through a single, terminal ether linkage.
  • the term "contact” as used herein with reference to interactions of chemical units indicates that the chemical units are at a distance that allows short range non-covalent interactions (such as Van der Waals forces, hydrogen bonding, hydrophobic interactions, electrostatic interactions, dipole-dipole interactions) to dominate the interaction of the chemical units. For example, when a protein is 'contacted' with a chemical species, the protein is allowed to interact with the chemical species so that a reaction between the protein and the chemical species can occur.
  • polypeptide and “protein” as used herein refers to any chain of two or more amino acids bonded in sequence, regardless of length or post-translational modification. According to their common use in the art, the term “protein” refers to any polypeptide consisting of more than 50 amino acid residues. These definitions are however not intended to be limiting.
  • metal protein refers to a protein that contains one or more metal ions.
  • the metal ion(s) confers the protein with catalytic activity (e.g., iron atom in cytochrome P450 enzymes) or other properties such as that of binding other molecules (e.g., iron atom in myoglobin and hemoglobin).
  • hemoprotein or "heme-containing protein” refers to a protein containing a heme group (iron-protoporphyrin IX).
  • enzyme refers to a protein capable of catalyzing a reaction as part of its native biological function.
  • a cytochrome P450 monoxygenase whose native function is typically that of catalyzing an oxygenation reaction (e.g., hydroxylation)
  • Myoglobin whose native function is that of binding and releasing oxygen, is a hemoprotein but not an enzyme (or heme enzyme).
  • carrier equivalent or “carbene precursor” refers to a molecule that can be decomposed in the presence of a transition metal catalyst or a metalloprotein catalyst to a structure that contain at least one divalent carbon with only 6 valence shell electrons and that can be transferred to carbon-carbon double bonds to form cyclopropanes or to carbon— hydrogen or heteroatom— hydrogen bonds to form products with new C— C or C— heteroatom bonds.
  • Diazo-containing reagents can serve as carbene precursor molecules for the carbene transfer reactions encompassed in this disclosure.
  • Non limiting examples of diazo-containing reagents are oc-diazo-esters, oc-diazo-amides, oc- diazo-ketones, oc-cyano- oc-diazo-esters, oc-nitro-oc-diazo-esters, and oc-keto-oc-diazo-esters.
  • carbene transfer refers to a chemical transformation where a carbene equivalent is added to a carbon-carbon double bond, a carbon-heteroatom double bond or inserted into carbon-hydrogen or heteroatom— hydrogen bond.
  • carbene acceptor substrate refers to any compound that can be made react with a carbene precursor reagent in a myoglobin-catalyzed reaction according to the methods provided herein, thereby forming a product carrying one or more new C— C, C— N, C— S, and/or C— Si bond(s).
  • Representative examples of carbene acceptor substrate are compounds of formula (II), (IV), (VI), (VIII), (X), (XI), (XIV) or (XV) as defined below.
  • heme refers to iron-protoporphyrin IX.
  • heme analog and "metalloporphyrin” as used herein refer to any metal- containing porphyrin molecule other than iron-protoporphyrin IX.
  • heme analogs include but are not limited to iron-deuteroporphyrin, iron-mesoporphyrin, iron-protoporphyrin, iron-bisglycolporphyrin, etc.
  • These porphyrin molecules may contain metals other than Fe, including but not limited to Mn, Co, Ni, Cu, Rh, Ru, and Os.
  • metal ion e.g., Fe, Mn, Co, Rh, Ru, or Os
  • Examples of porphyrin analogs include but are not limited to corroles,
  • anaerobic when used in reference to a reaction, culture or growth condition, refers to a condition in which the concentration of oxygen is less than about 25 ⁇ , less than about 5 ⁇ , or less than 1 ⁇ .
  • the term is also intended to include sealed chambers of liquid or solid medium maintained with an atmosphere of less than about 1% oxygen.
  • anaerobic conditions are achieved by sparging a reaction mixture with an inert gas such as nitrogen or argon.
  • heterologous indicates molecules that are expressed in an organism other than the organism from which they originated or are found in nature, independently of the level of expression that can be lower, equal or higher than the level of expression of the molecule in the native microorganism.
  • homolog refers to distinct enzymes or genes of a second family or species which are determined by functional, structural or genomic analyses to be an enzyme or gene of the second family or species which corresponds to the original enzyme or gene of the first family or species. Homologs most often have functional, structural, or genomic similarities. Techniques are known by which homologs of an enzyme or gene can readily be cloned using genetic probes and PCR. Identity of cloned sequences as homolog can be confirmed using functional assays and/or by genomic mapping of the genes. [00145] A protein has "homology" or is "homologous" to a second protein if the amino acid sequence encoded by a gene has a similar amino acid sequence to that of the second gene.
  • a protein has homology to a second protein if the two proteins have "similar" amino acid sequences.
  • the term “homologous proteins” is intended to mean that the two proteins have similar amino acid sequences.
  • the homology between two proteins is indicative of its shared ancestry, related by evolution.
  • analogs and “analogous” include nucleic acid or protein sequences or protein structures that are related to one another in function only and are not from common descent or do not share a common ancestral sequence. Analogs may differ in sequence but may share a similar structure, due to convergent evolution.
  • mutant or “variant” as used herein with reference to a molecule such as polynucleotide or polypeptide, indicates that such molecule has been mutated from the molecule as it exists in nature.
  • mutate and “mutation” as used herein indicates any modification of a nucleic acid and/or polypeptide which results in an altered nucleic acid or polypeptide.
  • Mutations include any process or mechanism resulting in a mutant protein, enzyme, polynucleotide, or gene.
  • a mutation can occur in a polynucleotide or gene sequence, by point mutations, deletions, or insertions of single or multiple nucleotide residues.
  • a mutation in a polynucleotide includes mutations arising within a protein-encoding region of a gene as well as mutations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences.
  • a mutation in a coding polynucleotide such as a gene can be "silent", i.e., not reflected in an amino acid alteration upon expression, leading to a "sequence-conservative" variant of the gene.
  • a mutation in a polypeptide includes but is not limited to mutation in the polypeptide sequence and mutation resulting in a modified amino acid.
  • Non-limiting examples of a modified amino acid include a glycosylated amino acid, a sulfated amino acid, a prenylated (e.g., farnesylated, geranylgeranylated) amino acid, an acetylated amino acid, an acylated amino acid, a PEGylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, and the like.
  • engine refers to any manipulation of a molecule that result in a detectable change in the molecule, wherein the manipulation includes but is not limited to inserting a polynucleotide and/or polypeptide heterologous to the cell and mutating a polynucleotide and/or polypeptide native to the cell.
  • myoglobin-based catalyst or simply “myoglobin catalyst” as used herein refer to any polypeptide which shares at least 60% sequence identity to SEQ. ID NO: l and exhibits carbene transfer reactivity within the scope of the disclosed compositions and methods.
  • Myoglobin catalysts also comprise engineered variants of sperm whale myoglobin (SEQ. ID NO: l), in which the naturally occurring heme cofactor is substituted for a heme analog, a metalloporphyrin (e.g., Co- or Mn-protoporphyrin IX), or a metalloporphyrin analog.
  • Myoglobin catalysts also comprise engineered variants of sperm whale myoglobin (SEQ.
  • Myoglobin catalysts further comprise polypeptides that share at least 60% sequence identity to SEQ. ID NOS: 112, 113, 114, 115, or 116, or engineered variants thereof.
  • nucleic acid molecule refers to any chain of two or more nucleotides bonded in sequence.
  • a nucleic acid molecule can be a DNA or a RNA.
  • a common type of vector is a "plasmid”, which generally is a self-contained molecule of double- stranded DNA that can be readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell.
  • plasmid which generally is a self-contained molecule of double- stranded DNA that can be readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell.
  • vectors including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts.
  • Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego, Calif.), or pMAL plasmids (New England Biolabs, Beverly, Mass.), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art.
  • the terms “express” and “expression” refer to allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence.
  • a DNA sequence is expressed in or by a cell to form an "expression product" such as a protein.
  • the expression product itself e.g., the resulting protein, may also be the to be “expressed” by the cell.
  • a polynucleotide or polypeptide is expressed recombinantly, for example, when it is expressed or produced in a foreign host cell under the control of a foreign or native promoter, or in a native host cell under the control of a foreign promoter.
  • fused means being connected through one or more covalent bonds.
  • bound means being connected through non-covalent interactions. Examples of non-covalent interactions are van der Waals, hydrogen bond, electrostatic, and hydrophobic interactions.
  • tethered as used herein means being connected through covalent or non-covalent interactions.
  • a polypeptide tethered to a solid support refers to a polypeptide that is connected to a solid support (e.g., surface, resin bead) either via non-covalent interactions or through covalent bonds.
  • Myoglobin catalysts are provided that are capable of promoting carbene transfer reactions with high efficiency and/or selectivity and across a broader range of substrates.
  • Myoglobin catalysts are provided having the capability to catalyze a carbene transfer reaction, wherein the myoglobin catalyst comprises an amino acid sequence having at least 60%, 80% or 90% sequence identity to SEQ. ID NOS:l, 112, 113, 114, 115, or 116.
  • the capability to catalyze a carbene transfer reaction corresponds to the capability of the myoglobin catalyst to react with a diazo-containing reagent and catalyze a carbene addition to an alkene group of an alkene-containing molecule.
  • such capability corresponds to the capability of the myoglobin catalyst to react with a diazo-containing reagent and catalyze a carbene insertion into the N— H bond of an N— H bond containing molecule.
  • such capability corresponds to the capability of the myoglobin catalyst to react with a diazo-containing reagent and catalyze a carbene insertion into the S— H bond of an S— H bond containing molecule.
  • such capability corresponds to the capability of the myoglobin catalyst to react with a diazo-containing reagent and catalyze a carbene insertion into the Si— H bond of a Si— H bond containing molecule. In other embodiments, such capability corresponds to the capability of the myoglobin catalyst to react with a diazo-containing reagent and catalyze [2,3] sigmatropic rearrangement in the presence of a thioether substrate to give a molecule with a new C— S bond.
  • such capability corresponds to the capability of the myoglobin catalyst to react with a diazo-containing reagent and catalyze [2,3] sigmatropic rearrangement in the presence of a tertiary amine substrate to give a molecule with a new C— N bond.
  • Myoglobin catalysts are provided that are capable of catalyzing the aforementioned reactions, and which have an improved property compared with a reference myoglobin such as wild-type sperm whale myoglobin (SEQ ID NO: 1), or when compared to another hemoprotein such as CYP102A1 (P450 BM 3) from Bacillus megaterium (SEQ ID NO: 111).
  • a reference myoglobin such as wild-type sperm whale myoglobin (SEQ ID NO: 1)
  • another hemoprotein such as CYP102A1 (P450 BM 3) from Bacillus megaterium (SEQ ID NO: 111).
  • the polypeptides can be described in reference to the amino acid sequence of a naturally occurring myoglobin or another engineered myoglobin variant.
  • the amino acid residue is determined in the myoglobin polypeptide beginning from the first amino acid after the initial methionine (M) residue (i.e., the first amino acid after the initial methionine M represents residue position 1).
  • M methionine
  • the initiating methionine residue may be removed by biological processing machinery such as in a host cell or in vitro translation system, to generate a mature protein lacking the initiating methionine residue.
  • the amino acid residue position at which a particular amino acid or amino acid change is present is sometimes described herein as "Xn", or "position n", where n refers to the residue position.
  • the myoglobin catalysts provided herein are characterized by an improved property as compared to the wild-type sperm whale myoglobin (SEQ ID NO: 1) or another reference hemoprotein (e.g., SEQ ID NO: 111). Changes to such properties can include, among others, improvements in catalytic efficiency, number of catalytic turnovers supported by the biocatalyst, regioselectivity, diastereoselectivity, enantioselectivity and/or reduced substrate or product inhibition.
  • the altered properties are based on engineered myoglobin polypeptides having residue differences at specific residue positions as compared to wild-type sperm whale myoglobin (SEQ ID NO: 1)
  • the myoglobin catalyst is an engineered variant of sperm whale myoglobin (SEQ ID NO: 1), the variant comprising an amino acid change at one or more of the following positions of SEQ ID NO: 1: X29, X32, X33, X39, X44, X45, X46, X64, X67, X68, X93, X107, and XI 11.
  • the myoglobin catalysts can have additionally one or more residue differences at residue positions not specified by an X above as compared to the sequence SEQ ID NO: 1.
  • the differences can be 1-2, 1-5, 1-10, 1-20, 1-30, 1-40, 1- 50, 1-75, or 1-90, residue differences at other amino acid residue positions not defined by X above.
  • the myoglobin catalysts having one or more of the improved enzyme properties described herein can comprise an amino acid sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 99% or more identical to the sequence SEQ ID NO: 1.
  • the improved myoglobin catalyst can comprise an amino acid sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 99% or more identical to a sequence corresponding to SEQ ID NO: 112, 113, 114, 115, or 116.
  • the improved myoglobin catalyst comprises an amino acid sequence corresponding to a sequence selected from the group consisting of SEQ ID NOS: 2 - 110.
  • the improved property of the myoglobin catalyst is with respect to its catalytic activity, regioselectivity, diastereoselectivity, and/or enantioselectivity.
  • the improvement in catalytic activity can be manifested by an increase in the number of catalytic turnovers (TON) supported by the myoglobin catalyst for the carbene transfer reaction, as compared to wild- type sperm whale myoglobin (SEQ ID NO: 1), or other reference sequences (e.g., SEQ ID NO: 111).
  • the myoglobin catalysts are capable of supporting a number of catalytic turnovers (TON) that is at least 1.1-fold, 2-fold, 5-fold, 10- fold, 100-fold, 200-fold, 500-fold, or more higher than the number of catalytic turnovers supported by the polypeptide having sequence SEQ ID NO: 1.
  • the improvement in catalytic activity can be also manifested by an increase in the catalytic efficiency for the carbene transfer reaction, this catalytic efficiency being
  • the myoglobin catalysts exhibit a catalytic efficiency that is at least 1.1-fold, 2-fold, 5-fold, 10-fold, 100-fold, 200-fold, 500-fold, or more higher than the catalytic efficiency of the polypeptide with sequence SEQ ID NO: 1.
  • the myoglobin catalysts having improved catalytic activity toward alkene cyclopropanation, toward carbene Y— H insertion, where Y is S, N, or Si, toward [2,3] sigmatropic rearrangement of a thioether or tertiary amine substrate, and/or toward aldehyde olefination comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 2 - 110.
  • the degree of diastereoselectivity can be conventionally described in terms of diasteromeric excess (d. e.).
  • the improvement in diastereoselectivity exhibited by the myoglobin catalyst is with respect to producing the (E) diastereomer of the cyclopropanation product (i.e., diastereomer in which the configuration of the cyclopropane ring is trans or (£)). In some embodiments, such improvement in
  • the myoglobin catalysts are capable of cyclopropanating an alkene-containing substrate with a (Z)- or (£)-diastereoselectivity (i.e., diastereomeric excess) that is at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 98%, 99% or more higher than that exhibited by the wild-type parental sequence SEQ ID NO: 1, or the reference sequence SEQ ID NO: 111.
  • the degree of stereoselectivity can be conventionally described in terms of stereomeric excess, that is in terms of enantiomeric excess (e. e. ) or diasteromeric excess (d. e. ) depending on the nature of the substrate.
  • the improvement in enantioselectivity exhibited by the myoglobin catalyst is with respect to producing the (IS,2S) stereoisomer of the cyclopropanation product. In some embodiments, such improvement in stereoselectivity is with respect to producing the (IR,2R), (IS,2R), or (IR,2S) stereoisomer of the cyclopropanation product.
  • the myoglobin catalysts are capable of cyclopropanating an alkene-containing substrate with a stereoselectivity (i.e., stereomeric excess) that is at least 1 %, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 98%, 99% or more higher than that exhibited by the wild-type parental sequence SEQ ID NO: 1 , or the reference sequence SEQ ID NO: 111.
  • a stereoselectivity i.e., stereomeric excess
  • the improvement in enantioselectivity can be manifested by an increase in the enantioselectivity by which a Y— H bond, where Y is S, N, or Si, is functionalized via a carbene insertion reaction by action of the myoglobin catalyst, as compared to the wild-type parental sequence SEQ ID NO: 1.
  • the degree of stereoselectivity can be conventionally described in terms of stereomeric excess, that is in terms of enantiomeric excess (e. e. ) or diasteromeric excess (d. e.) depending on the nature of the substrate.
  • the improvement in enantioselectivity exhibited by myoglobin catalyst is with respect to producing the (S) stereoisomer of the carbene insertion product.
  • such improvement in stereoselectivity is with respect to producing (R) stereoisomer of the carbene insertion product.
  • the engineered myoglobin catalysts are capable of catalyzing a carbene Y— H insertion reaction, where Y is S, N, or Si, with a stereoselectivity (i.e., stereomeric excess) that is at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 98%, 99% or more higher than that exhibited by the wild-type parental sequence SEQ ID NO: 1, or the reference sequence SEQ ID NO: 111.
  • the improvement in enantioselectivity can be manifested by an increase in the enantioselectivity by which a [2,3] sigmatropic rearrangement of thioether or amine substrate is catalyzed by action of the myoglobin catalyst, as compared to the wild-type parental sequence SEQ ID NO: 1.
  • the degree of stereoselectivity can be conventionally described in terms of stereomeric excess, that is in terms of enantiomeric excess (e. e. ) or diasteromeric excess (d. e. ) depending on the nature of the substrate.
  • the improvement in enantioselectivity exhibited by the myoglobin catalyst is with respect to producing the (S) stereoisomer of the rearrangement product. In some embodiments, such improvement in stereoselectivity is with respect to producing (R) stereoisomer of the rearrangement product.
  • the myoglobin catalysts are capable of catalyzing the [2,3] sigmatropic rearrangement of thioether or amine substrate, with a stereoselectivity (i.e., stereomeric excess) that is at least 1 %, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 98%, 99% or more higher than that exhibited by the wild-type parental sequence SEQ ID NO: 1 , or the reference sequence SEQ ID NO: 111.
  • a stereoselectivity i.e., stereomeric excess
  • the myoglobin catalysts having improved catalytic activity toward alkene cyclopropanation, and/or toward carbene Y— H insertion— where Y is S, N, or Si— , and/or toward [2,3] sigmatropic rearrangement of a thioether or tertiary amine substrate comprise an amino acid sequence corresponding to SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13.
  • the degree of diastereoselectivity can be conventionally described in terms of diasteromeric excess (d.e. ).
  • the improvement in diastereoselectivity exhibited by the myoglobin catalyst is with respect to producing the (E) diastereomer of the aldehyde olefination product (i.e., diastereomer in which the configuration of the alkene is trans or (£)). In some embodiments, such improvement in diastereoselectivity is with respect to producing the (Z) diastereomer of the aldehyde olefination product.
  • the myoglobin catalysts are capable of olefinating an aldehyde substrate with a (Z)- or ( ⁇ -diastereoselectivity (i.e., diastereomeric excess) that is at least 1 %, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 98%, 99% or more higher than that exhibited by the wild-type parental sequence SEQ ID NO: 1 , or the reference sequence SEQ ID NO: 111.
  • a (Z)- or ( ⁇ -diastereoselectivity i.e., diastereomeric excess
  • the capability of the myoglobin catalysts to catalyze any of the aforementioned carbene transfer reactions can be established according to methods well known in the art. Most typically, such capability can be established by contacting the substrate with the myoglobin catalyst under suitable reaction conditions in which the myoglobin catalyst is functional (e.g., under reducing and anaerobic conditions), and then determining the formation of the desired product (e.g., cyclopropanation, carbene Y— H insertion, rearrangement, or aldehyde olfination product) by standard analytical methods such as, for example, thin-layer chromatography, HPLC, GC, LC-MS, and/or GC-MS.
  • suitable reaction conditions in which the myoglobin catalyst is functional
  • the desired product e.g., cyclopropanation, carbene Y— H insertion, rearrangement, or aldehyde olfination product
  • standard analytical methods such as, for example, thin-layer chromatography, HPLC,
  • Such catalytic activity of the myoglobin catalysts can be measured and expressed in terms of number of catalytic turnovers, product formation rate, catalytic efficiency (k cat /KM ratio), and the like.
  • substrate activity can be measured and expressed in terms of turnover numbers (TON) or total turnover numbers (TTN), the latter corresponding to the total number of catalytic turnovers supported by the myoglobin catalyst in the presence of a given carbene acceptor substrate (e.g., styrene or aniline) and carbene donor (e.g., ethyl diazoacetate, ethyl oc-diazopropanoate).
  • the diastereo- and stereoselectivity of the myoglobin catalysts for any of the aforementioned carbene transfer reactions can be measured by determining the relative distribution of stereoisomeric products generated by the reaction using conventional analytical methods such as, for example, (chiral) normal phase liquid chromatography, (chiral) reverse- phase liquid chromatography, or (chiral) gas chromatography.
  • the improved myoglobin catalysts comprise deletions of the myoglobin catalyst provided herein. Accordingly, for each of the embodiment of the myoglobin catalysts provided herein, the deletions can comprise 1, 2, 5, 10, 30, or more amino acids, as long as the functional activity and/or improved properties of the myoglobin catalyst is maintained.
  • the myoglobin catalysts are fused to a polypeptide that can serve as an affinity tag in order to facilitate the isolation and purification of the myoglobin polypeptide.
  • affinity tags include but are not limited to a polyhistidine affinity tag, a FLAG tag, and a glutathione-S-transferase tag.
  • the myoglobin catalysts can comprise one or more non- natural amino acids in their primary sequence.
  • the non-natural amino acid can be present at one or more of the positions defined by "Xn" above for the purpose of modulating the catalytic or selectivity properties of the myoglobin catalyst.
  • the non-natural amino acid can be introduced in another position of the myoglobin catalyst sequence for the purpose, for example, of linking the myoglobin catalyst to another protein, another biomolecule, or a solid support.
  • Several methods are known in the art for introducing an unnatural amino acid into a polypeptide. These include the use of the amber stop codon suppression methods using engineered tRNA/aminoacyl-tRNA synthetase (AARS) pairs such as those derived from AARS.
  • AARS engineered tRNA/aminoacyl-tRNA synthetase
  • Methanocaldococcus sp. and Metanosarcina sp. (Liu and Schultz 2010).
  • natural or engineered frameshift suppressor tRNAs and their cognate aminoacyl-tRNA synthetases can also be used for the same purpose (Rodriguez, Lester et al. 2006; Neumann, Wang et al. 2010).
  • an unnatural amino acid can be incorporated in a polypeptide using chemically (Dedkova, Fahmi et al. 2003) or enzymatically (Bessho, Hodgson et al.
  • non-natural amino acids include but are not limited to, para-ammo- phenylalanine, para-acetyl-phenylalanine, meta-acetyl-phenylalanine, para-mercaptomethyl- phenylalanine, 3-pyridyl-alanine, 3-methyl-histidine, /?ara-butyl-l,3-dione-phenylalanine, O- allyl-tyrosine, O-propargyl-tyrosine, para-azido-phenylalanine, para-borono-phenylalanine, /?ara-bromo-phenylalanine, para-iodo-phenylalanine, 3-iodo-tyrosine, para-benzoyl- phenylalanine, para-benzoyl-phenylalanine, ⁇ -N-allyloxycarbonyl-lysine, ⁇ -N- propargyloxycarbonyl-lysine
  • the myoglobin catalyst comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and comprises at least one of the features selected from the group consisting of:
  • X29 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, U, V, W, or Y;
  • X32 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, U, V, W, or Y;
  • X33 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, U, V, W, or Y;
  • X39 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, U, V, W, or Y;
  • X43 is A, R, N, D,
  • the amino acid residue that coordinates the iron atom at the axial position of the heme cofactor in the myoglobin catalyst is a naturally occurring amino acid selected from the group consisting of serine, threonine, cysteine, tyrosine, histidine, aspartic acid, glutamic acid, and selenocysteine.
  • this non- naturally occurring oc-amino acid amino is para-amino-phenylalanine, meta-ammo- phenylalanine, para-mercaptomethyl-phenylalanine, meto-mercaptomethyl-phenylalanine, para- (isocyanomethyl)-phenylalanine, meta-(isocyanomethyl)-phenylalanine, 3-pyridyl-alanine, or 3- methyl-histidine.
  • the heme cofactor in the myoglobin catalyst is substituted for a heme analog, a metalloporphyrin, or a metalloporphyrin analog.
  • the heme cofactor in the myoglobin catalyst is substituted for a heme analog selected from the group consisting of iron-mesoporphyrin, iron-protoporphyrin, or iron-bisglycolporphyrin.
  • the heme cofactor in the myoglobin catalyst is substituted for a Mn-, Co-, Ru-, Rh-, or Os-porphyrin.
  • the heme cofactor in the myoglobin catalyst is substituted for a metalloporphyrin analog selected from the group consisting of corrole, phthalocyanine, phlorin, chlorin, 5-isocorrole, 10-isocorrole, and porphycene derivatives.
  • metalloporphyrin analog selected from the group consisting of corrole, phthalocyanine, phlorin, chlorin, 5-isocorrole, 10-isocorrole, and porphycene derivatives.
  • cofactor-substituted myoglobin catalysts can be prepared according to methods known in the arts, which include, for example, removal of the heme cofactor from the myoglobin polypeptide followed by refolding of the apoprotein in the presence of the heme analog, metalloporphyrin, or porphyrin analog (Yonetani and Asakura 1969; Yonetani, Yamamoto et al.
  • these cofactor-substituted myoglobin catalysts can be obtained via recombinant expression of the myoglobin polypeptide in bacterial strains that are capable of uptaking the heme analog or another metalloporphyrin from the culture medium (Woodward, Martin et al. 2007; Bordeaux, Singh et al. 2014).
  • the amino acid residue that coordinates the metal atom at the axial position of the protein-bound cofactor (e.g., heme, heme analog, metalloporphyrin, or metalloporphyrin analog) in the myoglobin catalyst is a naturally occurring amino acid selected from the group consisting of serine, threonine, cysteine, tyrosine, histidine, aspartic acid, glutamic acid, and selenocysteine.
  • this non-naturally occurring oc-amino acid amino is para-amino-phenylalanine, meto-amino-phenylalanine, para- mercaptomethyl-phenylalanine, meto-mercaptomethyl-phenylalanine, /?ara-(isocyanomethyl)- phenylalanine, meto-(isocyanomethyl)-phenylalanine, 3-pyridyl-alanine, or 3-methyl-histidine.
  • kits may contain an individual myoglobin catalyst or a plurality of myoglobin catalysts.
  • the myoglobin catalysts contained in the kit may be in lyophilized form, in solution, or tethered to a solid support.
  • the kits can further include reagents for carrying out the myoglobin-catalyzed reactions, substrates for assessing the activity of the myoglobin catalysts, and reagents for detecting the products.
  • the kits can also include instructions for the use of the kits.
  • the myoglobin catalysts described herein can be covalently or non-covalently linked to a solid support for the purpose, for example, of screening the myoglobin catalysts for activity on a range of different substrates or for facilitating the separation of reactants and products from the myoglobin catalyst after the reactions.
  • solid supports include but are not limited to, organic polymers such as polystyrene, polyacrylamide, polyethylene, polypropylene, poly ethylenegly cole, and the like, and inorganic materials such as glass, silica, controlled pore glass, metals.
  • the configuration of the solid support can be in the form of beads, spheres, particles, gel, a membrane, or a surface.
  • polynucleotide molecules are provided that encode for the myoglobin polypeptides disclosed herein.
  • the polynucleotides may be linked to one or more regulatory sequences controlling the expression of the myoglobin polypeptide-encoding gene to form a recombinant polynucleotide capable of expressing the polypeptide.
  • codons are selected to fit the host cell in which the polypeptide is being expressed.
  • codons used in bacteria are used to express the polypeptide in a bacterial host.
  • the polynucleotide molecule comprises a nucleotide sequence encoding for a myoglobin polypeptide with an amino acid sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 99% or more identical to SEQ ID NO:l.
  • the polynucleotide molecule comprises a nucleotide sequence encoding for a myoglobin polypeptide with an amino acid sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 99% or more identical to SEQ ID NO: 112, 113, 114, 115, or 116.
  • the polynucleotide molecule encoding for the myoglobin polypeptide is comprised in a recombinant expression vector.
  • Suitable recombinant expression vectors include but are not limited to, chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, pseudorabies, adenovirus, adeno-associated viruses, retroviruses and many others. Any vector that transduces genetic material into a cell, and, if replication is desired, which is replicable and viable in the relevant host can be used.
  • expression vectors and expression hosts are known in the art, and many of these are commercially available.
  • a person skilled in the art will be able to select suitable expression vectors for a particular application, e.g., the type of expression host (e.g., in vitro systems, prokaryotic cells such as bacterial cells, and eukaryotic cells such as yeast, insect, or mammalian cells) and the expression conditions selected.
  • the type of expression host e.g., in vitro systems, prokaryotic cells such as bacterial cells, and eukaryotic cells such as yeast, insect, or mammalian cells
  • an expression host system comprising a polynucleotide molecule encoding for the myoglobin polypeptides disclosed herein.
  • Expression host systems that may be used include any systems that support the transcription, translation, and/or replication of a polynucleotide molecule provided herein.
  • the expression host system is a cell.
  • Host cells for use in expressing the polypeptides encoded by the expression vector disclosed herein are well known in the art and include but are not limited to, bacterial cells (e.g., Escherichia coli, Streptomyces); fungal cells such as yeast cells (e.g., Saccharomyces cerevisiae, Pichia pastoris); insect cells; plant cells; and animal cells.
  • the expression host systems also include lysates of prokaryotic cells (e.g., bacterial cells) and lysates of eukaryotic cells (e.g., yeast, insect, or mammalian cells). These systems also include in vitro
  • transcription/translation systems many of which are commercially available.
  • the choice of the expression vector and host system depends on the type of application intended for the methods provided herein and a person skilled in the art will be able to select a suitable expression host based on known features and application of the different expression hosts.
  • the engineered myoglobin polypeptides can be prepared via mutagenesis of the polynucleotide encoding for the naturally occurring sperm whale myoglobin (SEQ ID NO: 1) or for an engineered variant thereof.
  • the engineered myoglobin polypeptides can be prepared via mutagenesis of the polynucleotide encoding for the naturally occurring myoglobins corresponding to SEQ ID NO: 112, 113, 114, 115, or 116, or an engineered variant thereof.
  • mutagenesis methods include, but are not limited to, site-directed mutagenesis, site-saturation mutagenesis, random mutagenesis, cassette- mutagenesis, DNA shuffling, homologous recombination, non-homologous recombination, site- directed recombination, and the like.
  • Detailed description of art-known mutagenesis methods can be found, among other sources, in U.S. Pat. No. 5,605,793; U.S. Pat. No. 5,830,721 ; U.S. Pat. No. 5,834,252; WO 95/22625; WO 96/33207; WO 97/20078; WO 97/35966; WO
  • oligonucleotide primers having a predetermined or randomized sequence can be prepared chemically by solid phase synthesis using commercially available equipment and reagents. Polynucleotide molecules can then be synthesized and amplified using a polymerase chain reaction, digested via endonucleases, ligated together, and cloned into a vector according to standard molecular biology protocols known in the art (e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (Third Edition), Cold Spring Harbor Press, 2001).
  • Engineered myoglobin polypeptides expressed in a host expression system can be isolated and purified using any one or more of the well-known techniques for protein purification, including, among others, cell lysis via sonication or chemical treatment, filtration, salting-out, and chromatography (e.g., ion-exchange chromatography, gel- filtration chromatography, etc.).
  • the recombinant myoglobin polypeptides obtained from mutagenesis of a parental myoglobin sequence can be screened for identifying engineered myoglobin polypeptides having improved catalytic and/or selectivity properties, such as improvements with respect to their catalytic activity, regioselectivity, diastereoselectivity and/or enantioselectivity for any of the
  • a method for catalyzing an alkene cyclopropanation reaction to produce a product having two new C— C bonds comprising:
  • R] a and R 2a are independently selected from H, halo, cyano (— CN), nitro (— N0 2 ), trifluoromethyl (— CF 3 ), optionally substituted C 1-18 alkyl, optionally substituted C 6 -io aryl, optionally substituted 5- to 10-membered heteroaryl, —
  • R] b , R] Ci and R ⁇ are independently selected from H, optionally substituted C MS alkyl, optionally substituted C 6 -io aryl, and optionally substituted 6- to 10-membered heteroaryl.
  • R 2 is independently selected from optionally substituted C 6 -i5 aryl, optionally substituted 5- to 15-membered heteroaryl, and optionally substituted C MS aliphatic
  • R 3 is independently selected from H, optionally substituted C MS aliphatic, optionally substituted C 6 -io aryl, optionally substituted 5- to 10-membered heteroaryl, — C(0)ORi b , — C(0)N(Rib)(Ric), and — C(0)Ri b , where each R lb and R lc are independently selected from H, optionally substituted C MS aliphatic, optionally substituted C 6 -io aryl, and optionally substituted 5- to 10-membered heteroaryl;
  • R 4 and R5 are independently selected from H, halo, cyano, optionally substituted C 1-18 aliphatic, optionally substituted C 6 -io aryl, and optionally substituted 5- to 10- membered heteroaryl.
  • R] a , R 2a , R 2 , R3, R 4 and R5 are as defined above.
  • a method for catalyzing a carbene N— H insertion reaction to produce a product having a new C— N bond comprising:
  • R ]a and R 2a are as defined above.
  • R 6 is independently selected from optionally substituted C 1-18 aliphatic, optionally substituted C 6 -i6 aryl, optionally substituted 5- to 10-membered heteroaryl, optionally substituted C4-C16 cyclic aliphatic, and optionally substituted C4-C16 heterocyclic group;
  • R 7 is independently selected from H, optionally substituted C 1-18 aliphatic, optionally substituted C 6 -i6 aryl, optionally substituted 5- to 10-membered heteroaryl; or where R 6 and R7 are connected to form an optionally substituted C4-C16 cyclic aliphatic or heterocyclic group.
  • R] a , R 2a , R 6 , and R7 are as defined above.
  • a method for catalyzing a carbene S— H insertion reaction to produce a product having a new C— S bond comprising:
  • R] a and R 2a are as defined above,
  • Rs is selected from optionally substituted C 1-18 aliphatic, optionally substituted C 6 -i6 aryl, optionally substituted 5- to 10-membered heteroaryl, optionally substituted C4-C16 cyclic aliphatic, and optionally substituted C4-C16 heterocyclic group.
  • R] a , R 2a , and Rs are as defined above.
  • a method for catalyzing a carbene Si— H insertion reaction to produce a product having a new C— Si bond comprising:
  • R ]a and R 2a are as defined above.
  • R9 is independently selected from optionally substituted CMS aliphatic, optionally substituted C 6 -i6 aryl, optionally substituted 5- to 10-membered heteroaryl, optionally substituted C4-C16 cyclic aliphatic, and optionally substituted C4-C16 heterocyclic group; Rio and Rn are optionally substituted Ci_6 aliphatic groups, (c) providing an engineered myoglobin variant as the catalyst; (d) contacting the S— H containing substrate and the diazo-containing reagent with the engineered myoglobin variant under appropriate reaction conditions; and
  • R] a , R 2a , R9, Rio, and Rn are as defined above.
  • a method for catalyzing a sulfur ylide [2,3] sigmatropic rearrangement to produce a product having a new C— S bond comprising:
  • R] a and R 2a are as defined above,
  • R] a , R 2a , R12, R13, Ri 4 , and R15 are as defined above.
  • a method for catalyzing a nitrogen ylide [2,3] sigmatropic rearrangement to produce a product having a new C— N bond comprising:
  • R] a and R 2a are as defined above,
  • R] 6 is independently selected from optionally substituted C 1-18 aliphatic, optionally substituted C 6 -i6 aryl, optionally substituted 5- to 10-membered heteroaryl, and optionally substituted C4-C16 heterocyclic group
  • Rn is independently selected from optionally substituted Ci_ 6 aliphatic, optionally substituted C 6 aryl, optionally substituted 5- to 6-membered heteroaryl; or where R] 6 and Rn are connected together to form an optionally substituted C4-C16 cyclic aliphatic or heterocyclic group
  • Ris, R19, and R 2 o are independently selected from H, optionally substituted Ci_ 6 aliphatic groups, optionally substituted C 6 -i6 aryl, or where Ris and R19 are connected together to form an optionally substituted C4-C16 cyclic aliphatic or heterocyclic group.
  • R] a , R 2a , R1 ⁇ 2, R17, Ri8, R19, and R 2 o are as defined above.
  • R] a and R 2a are as defined above.
  • nucleophilic reagent selected from the group consisting of triarylphosphine, triarylarsine, and triarylstilbine;
  • the diazo-containing carbene precursor in the methods described above is selected from the group consisting of ethyl 2-diazo-acetate, ieri-butyl 2- diazo-acetate, ethyl 2-diazo-2-phenylacetate, ethyl 2-diazo-propanoate, ieri-butyl 2-diazo- propanoate, ethyl 2-diazo-3,3,3-trifluoropropanoate, ethyl 2-cyano-2-diazoacetate, ethyl 2-diazo- 2-nitroacetate, diazomethane, diazo(nitro)methane, 2-diazoacetonitrile,
  • the myoglobin catalyst used in the methods described above comprises a polypeptide with an amino acid sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NO: 1 - 110.
  • the methods provided herein include forming reaction mixtures that contain the myoglobin catalyst, the carbene donor reagent (e.g., an alkyl a-diazoester) , the carbene acceptor substrate (e.g., alkene-containing substrate for the cyclopropanation reaction), and other additives (e.g., a reducing agent).
  • the carbene donor reagent e.g., an alkyl a-diazoester
  • the carbene acceptor substrate e.g., alkene-containing substrate for the cyclopropanation reaction
  • other additives e.g., a reducing agent
  • the myoglobin polypeptides may be added to the reaction mixture in the form of purified proteins, whole cells containing the myoglobin polypeptide, and/or cell extracts and/or lysates of such cells.
  • reactions are conducted under conditions sufficient to catalyze the formation of the desired products.
  • the reaction time and concentration of the myoglobin polypeptide in the reaction mixture can vary widely, in large part depending on the catalytic rate and efficiency of the myoglobin catalyst. Typically, reaction times range from 10 min to 24 hours. For example, the reaction time can be 30 min or 12 hours.
  • the amount of the myoglobin catalyst in the reaction mixture is also variable. Typically, the reaction mixtures contain between 0.001 mol% and 20 mol% myoglobin catalyst with respect to the diazo-containing reagent and/or the carbene acceptor substrate.
  • the reaction mixtures contain between 0.01 mol% and 2 mol% myoglobin catalyst with respect to the diazo-containing reagent and/or the carbene acceptor substrate.
  • concentration of the diazo-containing reagent and carbene acceptor substrate in the reaction mixtures can also vary. In an embodiment, the concentration of these compounds in the reaction mixture is between 100 ⁇ and 2 M. In another embodiment, the concentration of these compounds in the reaction mixture is between 1 mM and 500 mM.
  • the myoglobin-catalyzed reactions are carried out in a buffered aqueous solution.
  • buffering agents that can be used include sodium phosphate, sodium acetate, 2-amino-2-hydroxymethyl-propane-l,3-diol (TRIS), 3-morpholinopropane-l- sulfonic acid (MOPS), 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES), and 2- (N-morpholino)ethanesulfonic acid (MES).
  • additives can be present in these solutions, which include salts (e.g., NaCl, KC1, CaCl 2 ), detergents (e.g., sodium dodecylsulfate and Triton-X 100), chelators (e.g., 2-( ⁇ 2-[Bis(carboxymethyl)amino]ethyl ⁇
  • salts e.g., NaCl, KC1, CaCl 2
  • detergents e.g., sodium dodecylsulfate and Triton-X 100
  • chelators e.g., 2-( ⁇ 2-[Bis(carboxymethyl)amino]ethyl ⁇
  • EDTA (carboxymethyl)amino)acetic acid
  • EGTA ethylene glycol-bis(2-aminoethylether)-N,N,N',N'- tetraacetic acid
  • organic cosolvents such as, for example, methanol, ethanol, dimethylsulfoxide (DMSO), acetonitrile, dimethylformamide (DMF), and tetrahydrofuran (THF).
  • Buffers, cosolvents, salts, detergents, and chelators can be used at any suitable concentration, which can be readily determined by a person skilled in the art.
  • Cosolvents in particular, can be included in the reaction mixtures in amounts ranging from about 1 % v/v to about 70% v/v, or higher.
  • the myoglobin catalysts provided herein maintain carbene transfer reactivity in the context of the reactions described herein in the presence of a concentration of DMF, THF, acetonitrile, methanol, or ethanol in buffer as high as 50% v/v, or higher.
  • most proteins and heme-containing enzymes e.g., P450 undergo denaturation under these conditions.
  • the reactions can be conducted at any suitable temperature which is compatible with the catalytic function of the myoglobin polypeptides within the scope of the disclosed compositions and methods. Typically, the reactions are conducted at a temperature ranging from about 2°C to about 70°C. The reactions can be conducted, for example, at about 25 °C or about 50°C. The reactions can be conducted at any suitable pH which is compatible with the catalytic function of the myoglobin polypeptides within the scope of the disclosed compositions and methods. In general, the reactions are conducted at a pH ranging from about 6 to about 10. The reactions can be conducted, for example, at a pH of 6, 7, 8, or 9.
  • the reduced form of the myoglobin catalyst e.g., ferrous form vs. ferric form for heme-containing myoglobin catalysts
  • the reactions are conducted in the presence of a reducing agent, in particular in vitro reactions.
  • the reducing agent is sodium dithionite (Na 2 S 2 C>4).
  • reducing agents include, but are not limited to, ascorbic acid, enzymatic redox systems comprising of a myoglobin reductase enzyme and the cognate reduced nicotinamide adenine dinucleotide cofactor (NADPH or NADH), and non-enzymatic redox systems comprising of a reduced nicotinamide adenine dinucleotide cofactor (NADPH or NADH) or an NADH mimic (Paul, Arends et al.
  • the concentration of the ultimate reducing agent in the reaction mixtures can vary, ranging from substoichiometric amounts (e.g., 0.2, 0.5, 0.8 equiv.) to stoichiometric (1 equiv.) and overstoichiometric amounts (e.g., 2, 5, 10, 100 equiv.) with respect to the myoglobin catalyst.
  • the myoglobin catalyst can be reduced or maintained in the reduced ferrous form electrochemically by means of an electrode.
  • the reactions are conducted under anaerobic conditions.
  • Anaerobic conditions can be achieved by conducting the reactions under an inert atmosphere, such as a nitrogen atmosphere or argon atmosphere, and using solvents from which molecular oxygen has been removed via degassing.
  • the myoglobin-catalyzed reactions are allowed to proceed until a substantial amount of the substrate is transformed into the product.
  • Product formation or substrate consumption
  • can be monitored using standard analytical methods such as, for example, thin-layer chromatography, GC, HPLC, or LC-MS.
  • the methods provided herein can be assessed in terms of diastereoselectivity and/or enantioselectivity, that is the extent to which the reaction produces a particular isomer, be it a diastereomer or enantiomer.
  • a perfectly selective reaction produces a single isomer, such that the isomer constitutes 100% of the product.
  • a reaction producing a particular enantiomer constituting 95% of the total product can be said to be 95% enantioselective.
  • a reaction producing a particular diastereomer constituting 40% of the total product meanwhile, can be said to be 40% diastereoselective.
  • the methods provided herein include reactions that are from about 1 % to about 99.9% diastereoselective.
  • the reaction can be about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or about 95% diastereoselective.
  • the methods provided herein also include reactions that are from about 1% to about 99.9% enantioselective.
  • the reaction can be about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or about 95% enantioselective.
  • some embodiments disclosed herein provide methods wherein the reaction is at least 20% to at least 99% diastereoselective. In some embodiments, the reaction is at least 20% to at least 99% enantioselective.
  • two stereoisomeric products containing a new chiral carbon atom with an (R) or (5) absolute configuration are formed from the reactions described herein.
  • four or three stereoisomeric products containing two chiral carbon atoms each having an (R) or (5) absolute configuration are formed.
  • two "trans" or "£"' isomers and "cis” or “Z” isomers can be formed.
  • the two cis isomers are enantiomers with respect to one another, in that the structures are non- superimposable mirror images of each other.
  • the two trans isomers are enantiomers.
  • stereochemical configuration of certain of the products herein will depend on factors including the structures of the particular carbene acceptor substrate and diazo-containing reagent used in the reaction, as well as the nature and identity of the myoglobin catalyst. Accordingly, the distribution of the stereoisomeric products formed in the reactions described herein will also depend on such factors.
  • the distribution of a product mixture can be described in terms of the enantiomeric excess, or "% e.e. ".
  • the diasteromeric excess (% d.e.) can be calculated in the same manner.
  • the distribution of the (E) and (Z) isomers can be described in terms of the E : Z (or trans : cis) ratio.
  • the methods provided herein include reactions that lead to product mixtures exhibiting % e.e. values which range from about 1% to about 99.9%, or from about - 1% to about -99.9%.
  • the methods provided herein also include reactions that lead to product mixtures exhibiting % d.e. values which range from about 1% to about 99.9%, or from about - 1% to about -99.9%.
  • the methods provided herein include reactions that lead to product mixtures exhibiting a Z : E ratios ranging from about 1 : 99.9 to about 99.9: 1.
  • some embodiments provide methods that lead to a product mixture exhibiting at least 20% to at least 99% e.e.. In some embodiments, the product mixture exhibits at least 20% to at least 99% d.e.. Some embodiments also provide methods that lead to a mixture of cyclopropanation products with a Z : E ratio of at least 90 : 10 to at least 99 : 1. In other embodiments, the mixture of cyclopropanation products exhibit a Z : E ratio of at least 10 : 90 to at least 1 : 99.
  • the reactions can be conducted with intact cells expressing a myoglobin polypeptide provided herein (also referred to as "whole-cell reactions"). These whole-cell reactions can be carried out with any of the host cells used for expression of the myoglobin polypeptide, as described above.
  • the host cells are bacterial cells such as, for example, Escherichia coli cells.
  • the host cells are yeast cells such as, for example, Saccharomyces cerevisiae or Pichia pastoris cells.
  • suspension of cells expressing a myoglobin polypeptide provided herein can be formed in a suitable medium (e.g., phosphate buffer, M9 medium, Luria- Bertani medium, Terrific Broth medium) supplemented with nutrients such as, for example, mineral micronutrients (e.g., CoCl 2 , Q1SO 4 , MnCl 2 ), vitamins (e.g., thiamine, riboflavin, pantothenate) cofactor precursors (e.g., delta-aminolevulinic acid, p-aminobenzoic acid), sugars (e.g., glucose), and other energy sources (e.g., glycerol).
  • a suitable medium e.g., phosphate buffer, M9 medium, Luria- Bertani medium, Terrific Broth medium
  • nutrients such as, for example, mineral micronutrients (e.g., CoCl 2 , Q1SO 4 , MnCl 2 ), vitamins (e.g
  • the medium is also supplemented with a heme analog or a metalloporphyrin other than heme (e.g., Co- or Mn-protoporphyrin IX) with the purpose of incorporating such heme analog or metalloporphyrin in the myoglobin catalyst.
  • a heme analog or a metalloporphyrin other than heme e.g., Co- or Mn-protoporphyrin IX
  • Whole-cell reactions using cells expressing a myoglobin polypeptide provided herein can be carried under aerobic conditions or anaerobic conditions.
  • whole-cell reactions using cells expressing a myoglobin polypeptide provided herein can be carried without the addition of an exogenous reductant to the cell suspension.
  • a typical host cell e.g., E. coli
  • the intracellular concentration of oxygen in a typical host cell is sufficiently low to enable the myoglobin polypeptide provided herein to operate as carbene transfer catalyst.
  • the yield and rate of the whole-cell reactions can be controlled, at least in part, by varying the cell density of the cell suspension used in these reactions.
  • the cell density can be determined by measuring the absorbance at 600 nm and can be expressed as optical density at 600 nm (OD 6 oo)- Alternatively, the cell density can be expressed in gram cell dry weight per liter (g cdw L "1 ).
  • the whole-cell reactions can be conducted using cell suspensions that have an optical density (OD 6 oo) ranging from about 0.1 to about 100 or that have a cell density ranging from about 0.02 g cdw L "1 to about 20 g cdw L "1 .
  • Other cell densities can be useful, depending on the nature of the host cell, myoglobin catalyst, carbene acceptor substrate and diazo-containing reagent.
  • the concentration of the myoglobin catalyst in the cell suspensions used for the whole-cell reactions can be adjusted by varying the protein expression conditions (e.g., type of growth medium, temperature, concentration of the inducer of expression (e.g., ITPG, arabinose), and expression time) according to procedures well known in the art.
  • the number of catalytic turnovers supported by the myoglobin catalyst in whole-cell systems can be expressed in the form of amount of product (e.g., in mmol) per gram cell dry weight.
  • whole-cell reactions involving the myoglobin catalysts provided herein exhibit turnovers ranging from about 0.1 mmol (g cdw) -1 to about 20 mmol (g cdw) -1 .
  • the compounds provided herein may contain one or more chiral centers.
  • the compounds are intended to include racemic mixtures, diastereomers, enantiomers, and mixture enriched in one or more stereoisomer.
  • a group of substituents is disclosed herein, all the individual members of that group and all subgroups, including any isomers, enantiomers, and diastereomers are intended to be included in this disclosure.
  • cyclopropanation reaction involves formation of a heme-bound carbene intermediate upon reaction of EDA with the protein in its reduced, ferrous state.
  • 'End- on' (Wolf, Hamaker et al. 1995; Che, Huang et al. 2001 ; Li, Huang et al. 2002; Nowlan, Gregg et al. 2003) attack of the styrene molecule to this heme-carbenoid species would then lead to the cyclopropanation product (FIG. 5).
  • Positions 43 or 68 were substituted with amino acids carrying a larger (i.e., Mb(F43W) and Mb(V68F)) or smaller apolar side chain (i.e., Mb(F43V) and Mb(V68A)), in order to affect the catalyst selectivity in the cyclopropanation reaction by varying the steric bulk on either side of the heme (FIGS. 1 and 5).
  • Mb(F43W) and Mb(V68F) a larger i.e., Mb(F43W) and Mb(V68F)
  • Mb(F43V) and Mb(V68A) apolar side chain
  • the H64V mutation resulted in a two-fold increase in the turnover numbers, the highest among this set of single mutants, while having marginal effect on diastereo- and enantioselectivity.
  • all the mutations at the level of Phe43 and Val68 dramatically improved the enantioselectivity of the Mb variant as compared to wild-type Mb, resulting in formation of the (IS,2S) stereoisomer (3a) with e.e. (E values ranging from 44% to 99.9%.
  • the V68 substitutions also resulted in an appreciable increase in both catalytic activity (TON) and E- diastereoselectivity of the catalyst (FIG. 4).
  • the H64V mutation was found to be particularly effective in enhancing Mb-dependent cyclopropanation activity, whereas the mutations at the level of V68 and F43 were beneficial toward tuning its diastereo- and enantioselectivity.
  • a series of Mb double mutants were prepared and tested (FIG. 4).
  • Variant Mb(H64V,V68A) was found to exhibit high activity as well as excellent E-diastereoselectivity (>99.9% de) and (lS ⁇ -enantioselectivity (>99.9% ee) (FIG. 10B vs. FIG. 10A), and it was thus selected for further investigations.
  • Mb(H64V,V68A) was found to support TTNs ranging from 7,700 to 14,500 on these substrates.
  • Substrates such as oc-methylstyrene (lh) and N-methyl-3-vinyl-indole (li) could be also converted with to the corresponding cyclopropanation products 10a and 11a with high selectivity, albeit the efficiency of the reaction with the latter (li) was compromised by the instability of this substrate in water.
  • Mb(H64V,V68A)-catalyzed styrene cyclopropanation with ieri-butyl diazoacetate (12) and ethyl diazopropanoate (13) yielded the corresponding (IS,2S) cyclopropane products, i.e., iert-butyl 2-phenylcyclopropane-l-carboxylate (14) and ethyl l-methyl-2- phenylcyclopropane-l-carboxylate (15), respectively, with good diastereoselectivity (82% d.e. and 74% d.e.
  • Ethyl 2-diazo-2-phenylacetate was also accepted by the Mb variant although cyclopropanation of styrene with this diazo reagent proceeded with low efficiency (TON ⁇ 10).
  • Mb(H64V,V68A) and other engineered Mb variants were found to retain between 50 and 90% of their carbene transfer activity in the presence of up to 30-40% of an organic cosolvent (MeOH, THF, DMF, or CH 3 CN). Similarly, they were found to retain between 50 and 90% of their carbene transfer activity at elevated temperatures up to 60°C. These results further highlight the operational robustness of these biocatalysts for carbene transfer reactions.
  • engineered variants of sperm whale myoglobin can provide highly reactive and selective olefin cyclopropanation catalysts.
  • the engineered Mb variant Mb(H64V,V68A) is capable of catalyzing the
  • Tetramethylsilane (TMS) served as the internal standard (0 ppm) for ] H NMR and CDC1 3 was used as the internal standard (77.0 ppm) for 13 C NMR.
  • Silica gel chromatography purifications were carried out using AMD Silica Gel 60 230-400 mesh. Preparative thin layer chromatography was performed on TLC plates (Merck).
  • Gas chromatography (GC) analyses were carried out using a Shimadzu GC-2010 gas chromatograph equipped with a FID detector and a Shimadzu SHRXI-5MS column (15 m x 0.25 mm x 0.25 ⁇ film).
  • Enantiomeric excess was determined by chiral gas chromatography (GC) using a Shimadzu GC-2010 gas chromatograph equipped with a FID detector, and a Cyclosil-B column (30 m x 0.25 mm x 0.25 ⁇ film).
  • GC chiral gas chromatography
  • Wild-type Mb and the engineered Mb variants were expressed in E. coli BL21(DE3) cells as described previously (Bordeaux, Singh et al. 2014). Briefly, cells were grown in TB medium (ampicillin, 100 mg L _1 ) at 37 °C (150 rpm) until OD 6 oo reached 0.6. Cells were then induced with 0.25 mM ⁇ -d-l-thiogalactopyranoside (IPTG) and 0.3 mM ⁇ -aminolevulinic acid (ALA). After induction, cultures were shaken at 150 rpm and 27 °C and harvested after 20 h by centrifugation at 4000 rpm at 4 °C.
  • IPTG 0.25 mM ⁇ -d-l-thiogalactopyranoside
  • ALA ⁇ -aminolevulinic acid
  • the proteins were purified by Ni-affinity chromatography using the following buffers: loading buffer (50 mM Kpi, 800 mM NaCl, pH 7.0), wash buffer 1 (50 mM Kpi, 800 mM NaCl, pH 6.2), wash buffer 2 (50 mM Kpi, 800 mM NaCl, 250 mM glycine, pH 7.0) and elution buffer (50 mM Kpi, 800 mM NaCl, 300 mM L-histidine, pH 7.0).
  • buffer exchange 50 mM Kpi, pH 7.0
  • reactions were initiated by addition of 10 ⁇ of styrene (from a 1.2 M stock solution in methanol), followed by the addition of 10 ⁇ of EDA (from a 0.4 M stock solution in methanol) with a syringe, and the reaction mixture was stirred for 18 h at room temperature, under positive argon pressure. Reaction with hemin were carried out using an identical procedure with the exception that the purified Mb was replaced by 80 ⁇ of a hemin solution (100 ⁇ in
  • Rh 2 (OA)4-catalyzed cyclopropanation reactions were carried out according to the following general procedure. To a flame dried round bottom flask was added olefin (5 equiv.) and Rh 2 (OAC) 4 (2 mol ) in CH 2 C1 2 (2 mL) under argon. To this solution was added a solution of diazo compound (1 equiv.) in CH 2 C1 2 (3-5 mL) via slow addition over 30-40 minutes. The resulting mixture was stirred at room temperature for another 30 min to 1 hour.
  • Ethyl 2-(l-methyl-lH-indol-3-yl)cyclopropane-l-carboxylate (11) This product was obtained following the standard Rh-catalyzed cyclopropanation protocol starting from 1 -methyl - 3-vinyl-lH-indole, which was synthesized according to a published procedure (Waser, Caspar et al. 2006).
  • Mb(H64V,V68A) was found to exhibit also significantly higher N— H insertion reactivity than wild-type Mb (>500 vs. 210 TON, FIG. 11).
  • Mb(H64V,V68A) was found to remain active in the presence of the amine substrate and EDA at a concentration as high as 0.16 M, which corresponds to -15 g aniline per L (FIG. 11).
  • Mb(H64V,V68A) catalyzes nearly -3,000 turnovers. With 10 mM aniline and a catalyst loading of 0.001 mol , over 6,000 total turnovers (TTN) were supported by this Mb variant. This value is an order of magnitude higher than that recently reported engineered P450BM3 variants (Wang, Peck et al. 2014) and ranks among the highest TTNs reported for catalytic N— H insertion reactions with acceptor- only diazo compounds (Aviv and Gross 2006).
  • the Mb(H64V,V68A)-catalyzed reaction is also remarkably fast, proceeding at an initial rate of 740 and 174 turnovers min 1 over the first minute and first 10 min, respectively. .
  • a range of substituted anilines (24a-32a) and other arylamines (33a, 34a) were subjected to Mb(H64V,V68A)- catalyzed N— H functionalization in the presence of EDA. As summarized in FIG.
  • the Mb variants were capable to catalyze the reactions with oc-substituted diazo compounds (i.e., ethyl 2-diazopropanoate, ieri-butyl 2-diazopropanoate) in an enantioselective manner (15-30% e.e.)
  • oc-substituted diazo compounds i.e., ethyl 2-diazopropanoate, ieri-butyl 2-diazopropanoate
  • Chiral GC analyses were carried out using a Shimadzu GC-2010 gas chromatograph equipped with a FID detector, and a Cyclosil-B column (30 m x 0.25 mm x 0.25 ⁇ film). Separation method: 1 ⁇ L ⁇ injection, injector temp.: 200 °C, detector temp: 300 °C. Gradient: column temperature set at 140 °C for 3 min, then to 160 °C at 1.8 °C/min, then to 165 °C at 1 °C/min, then to 245 °C at 25 °C/min. Total run time was 28 min.
  • N-H insertion reactions were typically carried out at a 400 ⁇ scale using 20 ⁇ myoglobin, 10 mM aniline, 10 or 5 mM EDA, and 10 mM sodium dithionite.
  • a solution containing sodium dithionate (100 mM stock solution) in potassium phosphate buffer (50 mM, pH 8.0) was degassed by bubbling argon into the mixture for 4 min in a sealed vial.
  • a buffered solution containing myoglobin was carefully degassed in a similar manner in a separate vial. The two solutions were then mixed together via cannula.
  • Reactions were initiated by addition of 10 ⁇ of aniline (from a 0.4 M stock solution in methanol), followed by the addition of 10 ⁇ or 5 ⁇ of EDA (from a 0.4 M stock solution in methanol) with a syringe, and the reaction mixture was stirred for 12 h at room temperature, under positive argon pressure.
  • FIG. 16 Upon optimization of the reaction conditions, nearly quantitative conversion of thiophenol to 3 (68 ⁇ 98%), and correspondingly higher catalytic turnovers (TON: 170 ⁇ 492), could be achieved using a two-fold excess of EDA over the thiol substrate at a catalyst loading of 0.2 mol% (Entry 3, FIGS. 16 and 17A). Notably, comparable yields in this transformation have been obtained using transition metal complexes at 5- to 25-fold higher catalyst loadings
  • this mutation is likely to enhance the catalytic efficiency of Mb by increasing the accessibility of the heme pocket to the diazo ester and thiol reactants.
  • the initial rate for Mb(L29A,H64V)- catalyzed formation of the S-H insertion product 53 was determined to be 35 turnovers per minute.
  • Mb(L29A,H64V) was found to readily functionalize benzyl mercaptan (72), substituted benzyl mercaptan derivatives (73-75), and alkyl mercaptans such as cyclohexanethiol (76) and octane- 1 -thiol (77), providing conversions in the range of 30-50% and supporting between 930 and 2,550 total turnover numbers (Entries 1-6, FIG. 20).
  • 52c or 52d
  • Mb(F43V,V68A) showed appreciable enantioselectivity in this transformation (21-22% ee, Entries 3 and 5 in FIG. 21; FIG. 22). Since Mb(V68A) exhibited only 6% ee, the beneficial effect in terms of enantioselectivity can be mainly attributed to the substitution at the level of Phe43, which is located in close proximity to the heme cofactor (FIG. 1).
  • results demonstrate the amenability of the Mb catalysts to promote asymmetric carbene S-H insertions, the possibility to tune this property via active site engineering, and the scalability of Mb-catalyzed S-H insertion reactions, further highlighting the utility of these biocatalysts for synthetic applications.
  • Enantiomeric excess for product 71 was determined using the following method: 1 ⁇ injection, injector temp.: 200 °C, detector temp: 300 °C. Gradient: column temperature set at 80 °C for 3 min, then to 180 °C at 1.00 °C/min, then to 200 °C at 2 °C/min, then to 245 °C at 25 °C/min.
  • Reactions were initiated by addition of 10 ⁇ of thiophenol (from a 0.4 M stock solution in methanol), followed by the addition of 10 ⁇ L ⁇ of EDA (from a 0.2 M stock solution in methanol) with a syringe, and the reaction mixture was stirred for 12 h at room temperature, under positive argon pressure.
  • the preparative-scale reaction was carried out using solution containing sodium dithionate (100 mM stock solution, 1 mL, 10 mM) in potassium phosphate buffer (50 mM, pH 8.0, 5.87 mL) and 466 ⁇ of MeOH (>5 of reaction volume) was degassed by bubbling argon into the mixture for 20 min in a sealed vial.
  • the sulfonium ylide likely arises from nucleophilic attack of the sulfane substrate to the heme-bound carbene intermediate generated upon reaction of the diazo compound with the hemoprotein (FIG. 5).
  • the Mb(L29A,H64V)-catalyzed formation of 92 was also found to occur with a certain degree of enantioselectivity (15% e.e. , FIG. 23), as determined by chiral GC analysis.
  • Upon screening additional Mb variants it was possible to identify Mb catalysts with improved catalytic efficiency and enantioselectivity for the conversion of 90 to 92 (FIG. 23).
  • the Mb variant Mb(F43V,V68F) was found to have complementary enantioselectivity as compared to
  • EXAMPLE 3 (sulfanes) and EXAMPLE 2 (amines). Authentic standards for the rearrangement products were prepared according to general procedure described below.
  • the carbene transfer reactivity of the myoglobin catalysts is dependent upon the presence of a heme cofactor (iron-protoporphyrin IX) bound to the protein. Accordingly, it was envisioned that varying the nature of this cofactor, e.g., via using an alternative
  • metalloporphyrin cofactor could provide a means to modulate the carbene transfer reactivity of these catalysts.
  • catalytic properties of metallo- substituted Mb variants, in which the heme cofactor is substituted for a Mn- or Co-protoporphyrin IX was investigated.
  • Mn- and Co-substituted Mb variants have been previously obtained by reconstitution of apomyoglobin with the corresponding metallo-protoporphyrins IX (Yonetani and Asakura 1969; Yonetani, Yamamoto et al. 1974; Heinecke, Yi et al. 2012). While remaining viable, this approach however involves laborious and time-consuming refolding procedures. To overcome this issue, a convenient and practical strategy was implemented for the recombinant expression of metallo-substituted Mb variants by using E. coli cells, which express a heterologous, outer- membrane heme transporter (ChuA) (Varnado and Goodwin 2004).
  • ChuA heterologous, outer- membrane heme transporter
  • wild-type sperm whale Mb and the heme transporter ChuA from 0157:H7 E. coli were initially expressed in BL21(DE3) cells using a dual plasmid system in which the Mb and ChuA genes are under an IPTG-inducible promoter.
  • Cells were grown in M9 minimal medium supplemented with Mn m (ppIX). Under these conditions, Mn-substituted Mb
  • Mb(Mn m ) could be successfully isolated with a yield of approximately 5 mg / L of culture.
  • a second plasmid encoding for both ChuA and the chaperone complex, GroEL/ES was prepared. The latter was expected to increase the fraction of the desired protein in correctly folded and soluble form. Indeed, this system led to a significant increase (2.5-fold) in the yield of Mb(Mn m ) (13 vs. 5 mg/L culture).
  • Mb(Mn) and Mb(Co) The purified Mn- and Co-containing myoglobin variants, referred to as Mb(Mn) and Mb(Co), were characterized by electron absorption spectroscopy in both oxidized and reduced form (FIGS. 26A-C). As shown in FIG. 26B, Mb(Mn m ) shows a split Soret band with X max at 375 and 469 nm in phosphate buffer at pH 7.0. Upon addition of dithionite, a single Soret band with max at 438 nm becomes apparent, indicating complete reduction of the protein to
  • Mb(Mn n ) Mb(Mn n ).
  • the visible spectrum of Mb(Co m ) shows a prominent absorption band at 422, which shifts to 401 nm under reducing conditions, thus evidencing the formation of the reduced form, Mb(Co n ) (FIG. 26C).
  • E. coli BL21(DE3) (or C41(DE3) (Lucigen)) cells were co-transformed with the Mb-encoding plasmid (pET22_MYO) and the ChuA-encoding vectors pHPEX2 or pGroES/EL-ChuA. Cells were grown in M9 minimal media supplemented with micronutrients and the appropriate antibiotics at 37 °C until the OD 6 oo reached 0.6.
  • Example 6 Preparation and carbene transfer reactivity of myoglobin- based catalysts with alternative proximal ligands
  • proximal ligand Mb variants SEQ ID NO: 14 through 27
  • Mb(H64V,V68A) SEQ ID NO: 11
  • residues i.e., Cys, Asp, Glu, Tyr, Ser
  • residues have a side-chain group capable of coordinating the metal ion of the heme group (or other metalloporphyrin/metalloporphyrin analog) in the Mb catalyst, while others (Ala, Gly) do not, leaving the proximal site available for coordination by other species (e.g., solvent).
  • proximal ligand Mb variants were prepared by replacing His93 residue in wild-type sperm whale myoglobin (SEQ ID NO: l) and in one of the most promising cyclopropanation catalyst, Mb(H64V,V68A) (SEQ ID NO: 11), with the unnatural amino acid p- amino-phenylalanine (pAmF) (SEQ ID NOS: 28 and 31), 3-pyridyl-alanine (3PyA) (SEQ ID NOS: 29 and 32), and 3-methyl-histidine (3MeH) (SEQ ID NOS: 30 and 33) via amber stop codon suppression (Liu and Schultz 2010).
  • pAmF unnatural amino acid p- amino-phenylalanine
  • 3PyA 3-pyridyl-alanine
  • 3MeH 3-methyl-histidine
  • this gene was then expressed in BL21(DE3) cells containing a second plasmid encoding for an engineered, orthogonal Methanocaldococcus jannaschii tRNA/aminoacyl-tRNA synthetase (AARS) pair capable of suppressing an amber stop codon with p-amino-phenylalanine, 3-pyridyl-alanine, or 3-methyl-histidine.
  • AARS orthogonal Methanocaldococcus jannaschii tRNA/aminoacyl-tRNA synthetase
  • the unnatural amino acid-containing Mb variants were purified by Ni-affinity chromatography as described in EXAMPLE 1. All the variants were able to bind and retain the heme cofactor as indicated by the presence of a Soret band in their UV-VIS electronic absorption spectra (FIG. 26D).
  • His93Asp, His93Glu, His93Tyr, His93Cys, His93Ser, His93Gly, His93(pAmF), His93(3PyA), or His93(3MeH) mutation were tested for their carbene transfer reactivity using representative reactions for olefin cyclopropanation (styrene + EDA), carbene N— H insertion (aniline + EDA; aniline + EDP), and carbene S— H insertion (thiophenol + EDA; thiophenol + EDP).
  • the cells were resuspended in phosphate buffer (50 mM KPi, pH 8.0) to a final OD 6 oo of 40 and the substrate (styrene) and carbene donor (EDA) were added to the cell suspension.
  • phosphate buffer 50 mM KPi, pH 8.0
  • EDA carbene donor
  • Example 8 Gram-scale synthesis of drugs and advanced pharmaceutical intermediates using engineered myoglobin catalysts.
  • Example 9 Myoglobin catalysts with altered selectivity via active site mutagenesis.
  • EXAMPLES 1-4 illustrate how the catalytic activity and/or selectivity of myoglobin-based polypeptides as carbene transfer catalysts can be modulated via mutagenesis of amino acid residues defining the active site of the hemoprotein according to the methods provided herein.
  • a library of Mb variants was prepared starting from Mb(H64V) by mutating one or more additional active site residues in sperm whale myoglobin (FIG. 1) by site-directed mutagenesis.
  • Mb variants carrying double mutations at positions H64/V68, L29/H64, H64/I107, and F43/H64, and carrying triple mutations at positions L29/H64/V68 were prepared and then tested to identify Mb-based cyclopropanation catalysts with altered diastereo- and stereoselectivity as compared to the (IS- 25)-selective Mb(H64V,V68A) catalyst, using a model reaction with styrene and EDA (FIG. 29).
  • Mb(H64V,V68L) and Mb(H64V,V68F) were found to exhibit excellent trans -diastereoselectivity (>99.9% de) and high (IR,2R)- stereoselectivity, thus complementing the scope of Mb(H64V,V68A).
  • Mb catalysts were identified that can produce the cis product 3d with high stereoselectivity (e.g., 95- 99% ee) such as, for example, Mb(H64V,V68G).
  • Mb(H64V,V68G) Mb(H64V,V68G)
  • further mutagenesis can be applied to these and/or other Mb variants in order to further optimize the catalytic and selectivity properties of the myoglobin catalysts in the context of olefin cyclopropanation and/or the other carbene transfer reactions described herein.
  • Example 10 Aldehyde olefination reactions catalyzed by myoglobin-based catalysts.
  • hemin reaction is much less chemoselective, yielding larger amounts of the carbene dimerization byproducts, diethyl fumarate and diethyl maleate (TON ( 3 a )/TON(4 a) : 0.4 vs. 2.8 with Mb, FIG. 30).
  • Mb(F43V,V68F) used in combination with AsPh 3 , emerged as the most promising catalyst for this reaction, exhibiting 3 -fold higher TON compared to wild-type Mb, excellent diasteroselectivity (>99.9% de), and high chemoselectivity toward aldehyde olefination over carbene dimerization.
  • Mb(F43V,V68F) was determined to support over 1,100 catalytic turnovers for the conversion of 111 to E-113a, featuring an initial rate of 320 and 40 turnovers min "1 over the first minute and first 15 minutes, respectively.
  • Paquet 2004 Cao, Li et al. 2007; Lebel and Davi 2008; Lebel and Ladjel 2008).
  • Aldehyde olefination reaction Typically, reactions were carried out at a 400 ⁇ scale using 20 ⁇ myoglobin, 10 mM benzaldehyde, 10 mM EDA, 10 mM triphenylphosphine (or trialkyl phosphines, AsPh 3 , SbPh 3 , BiPh 3 ) and 10 mM sodium dithionite.
  • a solution containing sodium dithionate (100 mM stock solution) in potassium phosphate buffer (50 mM, pH 8.0) was degassed by bubbling argon into the mixture for 4 min in a sealed vial.
  • a buffered solution containing myoglobin was carefully degassed in a similar manner in a separate vial. The two solutions were then mixed together via cannula. Reactions were initiated by addition of 10 ⁇ L ⁇ of benzaldehyde (from a 0.4 M stock solution in DMSO), 10 ⁇ L ⁇

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Abstract

Cette invention concerne des procédés permettant de mettre en œuvre des transformations par transfert de carbène telles que des réactions de cyclopropanation d'oléfines, des réactions d'insertion d'hétéroatomes de carbène-H (hétéroatome = N, S, Si), des réactions de réarrangement sigmatropique, et des réactions d'oléfination d'aldéhydes à une efficacité et à une sélectivité élevées à l'aide d'une nouvelle classe de biocatalyseurs à base de myoglobine. Ces procédés sont utiles pour la synthèse de divers composés organiques qui contiennent une ou plusieurs nouvelles liaisons carbone-carbone ou carbone-hétéroatome (N, S, ou Si). Les procédés peuvent être appliqués pour conduire ces transformations in vitro (à savoir, à l'aide du biocatalyseur sous forme isolée) et in vivo (à savoir, à l'aide du biocatalyseur dans un système cellulaire entier).
PCT/US2015/062478 2014-11-25 2015-11-24 Catalyseurs à base de myoglobine pour réactions de transfert de carbène WO2016086015A1 (fr)

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WO2016191612A3 (fr) * 2015-05-26 2017-01-19 California Institute Of Technology Catalyseurs à base d'hémoprotéines permettant d'améliorer la synthèse énantiosélective enzymatique du ticagrelor
US9732080B2 (en) 2006-11-03 2017-08-15 Vertex Pharmaceuticals Incorporated Azaindole derivatives as CFTR modulators
US10071979B2 (en) 2010-04-22 2018-09-11 Vertex Pharmaceuticals Incorporated Process of producing cycloalkylcarboxamido-indole compounds
US10081621B2 (en) 2010-03-25 2018-09-25 Vertex Pharmaceuticals Incorporated Solid forms of (R)-1(2,2-difluorobenzo[D][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide
US10206877B2 (en) 2014-04-15 2019-02-19 Vertex Pharmaceuticals Incorporated Pharmaceutical compositions for the treatment of cystic fibrosis transmembrane conductance regulator mediated diseases
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Cited By (10)

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US9732080B2 (en) 2006-11-03 2017-08-15 Vertex Pharmaceuticals Incorporated Azaindole derivatives as CFTR modulators
US10081621B2 (en) 2010-03-25 2018-09-25 Vertex Pharmaceuticals Incorporated Solid forms of (R)-1(2,2-difluorobenzo[D][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide
US10071979B2 (en) 2010-04-22 2018-09-11 Vertex Pharmaceuticals Incorporated Process of producing cycloalkylcarboxamido-indole compounds
US10208322B2 (en) 2012-10-09 2019-02-19 California Institute Of Technology In vivo and in vitro olefin cyclopropanation catalyzed by heme enzymes
US11008596B2 (en) 2012-10-09 2021-05-18 California Institute Of Technology Cytochrome P450 BM3 enzyme variants for preparation of cyclopropanes
US10206877B2 (en) 2014-04-15 2019-02-19 Vertex Pharmaceuticals Incorporated Pharmaceutical compositions for the treatment of cystic fibrosis transmembrane conductance regulator mediated diseases
WO2016191612A3 (fr) * 2015-05-26 2017-01-19 California Institute Of Technology Catalyseurs à base d'hémoprotéines permettant d'améliorer la synthèse énantiosélective enzymatique du ticagrelor
US11518768B2 (en) 2015-10-14 2022-12-06 The Regents Of The University Of California Artificial metalloenzymes containing noble metal-porphyrins
CN114479110A (zh) * 2022-02-11 2022-05-13 云南大学 以三苯基锑为骨架的共价有机框架及其制备方法和应用
CN114479110B (zh) * 2022-02-11 2023-02-28 云南大学 以三苯基锑为骨架的共价有机框架及其制备方法和应用

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