Highly Diastereoselective and Enantioselective Olefin Cyclopropanation Using Engineered Myoglobin‐Based Catalysts

Bordeaux, Mélanie; Tyagi, Vikas; Fasan, Rudi

doi:10.1002/ange.201409928

Cited by 107 publications

(85 citation statements)

References 38 publications

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“…2b for the cyclopropanation of β -methylstyrene 3 with ethyl diazoacetate 4 (EDA). In agreement with literature reports 10 , Fe-PIX enzymes did not catalyse this reaction (Fig. 2b).…”

Section: Letter Researchsupporting

confidence: 93%

“…In particular, mutants of Physeter macrocephalus myoglobin (Myo) and Bacillus megaterium cytochrome P450 BM3h (P450) with and without an mOCR stability tag were overexpressed in high yields and purified (up to 70 mg l −1 of protein; Fig. 1a, Supplementary Tables 1 and 2) 9, 10,27 . Circular dichroism spectroscopy revealed that these apo-proteins retain the fold of their native Fe-PIX analogues (Fig.…”

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confidence: 99%

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Abiological catalysis by artificial haem proteins containing noble metals in place of iron

Key

Dydio

Clark

et al. 2016

Nature

378

298

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Enzymes that contain metal ions-that is, metalloenzymespossess the reactivity of a transition metal centre and the potential of molecular evolution to modulate the reactivity and substrateselectivity of the system 1 . By exploiting substrate promiscuity and protein engineering, the scope of reactions catalysed by native metalloenzymes has been expanded recently to include abiological transformations 2,3 . However, this strategy is limited by the inherent reactivity of metal centres in native metalloenzymes. To overcome this limitation, artificial metalloproteins have been created by incorporating complete, noble-metal complexes within proteins lacking native metal sites 1,4,5 . The interactions of the substrate with the protein in these systems are, however, distinct from those with the native protein because the metal complex occupies the substrate binding site. At the intersection of these approaches lies a third strategy, in which the native metal of a metalloenzyme is replaced with an abiological metal with reactivity different from that of the metal in a native protein 6-8 . This strategy could create artificial enzymes for abiological catalysis within the natural substrate binding site of an enzyme that can be subjected to directed evolution. Here we report the formal replacement of iron in Fe-porphyrin IX (Fe-PIX) proteins with abiological, noble metals to create enzymes that catalyse reactions not catalysed by native Fe-enzymes or other metalloenzymes 9,10 . In particular, we prepared modified myoglobins containing an Ir(Me) site that catalyse the functionalization of C-H bonds to form C-C bonds by carbene insertion and add carbenes to both β-substituted vinylarenes and unactivated aliphatic α-olefins. We conducted directed evolution of the Ir(Me)-myoglobin and generated mutants that form either enantiomer of the products of C-H insertion and catalyse the enantio-and diastereoselective cyclopropanation of unactivated olefins. The presented method of preparing artificial haem proteins containing abiological metal porphyrins sets the stage for the generation of artificial enzymes from innumerable combinations of PIX-protein scaffolds and unnatural metal cofactors to catalyse a wide range of abiological transformations.To create artificial metalloenzymes formed by combining abiological metals and natural metalloprotein scaffolds, we focused on haem proteins, which contain Fe-porphyrin IX (Fe-PIX) as a metal cofactor. Native haem enzymes catalyse reactions that include C-H oxidation and halogenation 11 , and they have been successfully evolved to oxidize abiological substrates 12,13 . Fe-PIX proteins have also been shown to catalyse abiological reactions involving the addition and insertion of carbenes and nitrenes to olefins and X-H bonds 2,3,9,14 . However, the reactivity of the Fe-centre in haem proteins limits the scope of these transformations. For example, Fe-PIX proteins catalyse the cyclopropanation of activated terminal vinylarenes 9,10 , but they do not catalyse reactions with internal vinylarenes ...

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“…2b for the cyclopropanation of β -methylstyrene 3 with ethyl diazoacetate 4 (EDA). In agreement with literature reports 10 , Fe-PIX enzymes did not catalyse this reaction (Fig. 2b).…”

Section: Letter Researchsupporting

confidence: 93%

mentioning

confidence: 99%

Abiological catalysis by artificial haem proteins containing noble metals in place of iron

Key

Dydio

Clark

et al. 2016

Nature

378

298

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show abstract

“…Fasan's group recently reported that myoglobin, which naturally uses histidine as the axial ligand to the iron-heme, can also be engineered for cyclopropanation (Bordeaux et al 2015), at least in vitro. Both labs have now demonstrated that optimized cyclopropanating enzymes catalyze tens of thousands of turnovers for various olefins, with substrate range and product selectivity that are tunable by protein engineering (Bordeaux et al 2015;Coelho et al 2013b).…”

Section: New Enzymes From Old: Adding To the Cytochrome P450's (Alreamentioning

confidence: 99%

“…Both labs have now demonstrated that optimized cyclopropanating enzymes catalyze tens of thousands of turnovers for various olefins, with substrate range and product selectivity that are tunable by protein engineering (Bordeaux et al 2015;Coelho et al 2013b). The P450-derived enzyme functions very well in whole bacterial cells, where a His-ligated variant has been used for preparative-scale synthesis of a pharmaceutical intermediate (Wang et al 2014a).…”

Section: New Enzymes From Old: Adding To the Cytochrome P450's (Alreamentioning

confidence: 99%

The nature of chemical innovation: new enzymes by evolution

Arnold

2015

Quart. Rev. Biophys.

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Abstract. I describe how we direct the evolution of non-natural enzyme activities, using chemical intuition and information on structure and mechanism to guide us to the most promising reaction/enzyme systems. With synthetic reagents to generate new reactive intermediates and just a few amino acid substitutions to tune the active site, a cytochrome P450 can catalyze a variety of carbene and nitrene transfer reactions. The cyclopropanation, N-H insertion, C-H amination, sulfimidation, and aziridination reactions now demonstrated are all well known in chemical catalysis but have no counterparts in nature. The new enzymes are fully genetically encoded, assemble and function inside of cells, and can be optimized for different substrates, activities, and selectivities. We are learning how to use nature's innovation mechanisms to marry some of the synthetic chemists' favorite transformations with the exquisite selectivity and tunability of enzymes.

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“…Up to 40 000 TTN were obtained at higher substrate or lower catalyst loadings (see Figure S4 in the Supporting Information), which places P411-VAC cis among the most active carbene transfer hemoproteins described to date. 31,33,44 For the trans-selective variant P411 BM3 -CIS I263G L437F, further site-saturation mutagenesis and screening yielded variant P411 BM3 -CIS I263G L437F V87L L181R, which we term P411-VAC trans . In whole E. coli cells, this variant catalyzes cyclopropanation of N-vinylphthalimide with EDA in 86% yield with 980 TTN, 3:97 dr, and 92% ee ( Figure 1B).…”

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confidence: 99%

Stereoselective Enzymatic Synthesis of Heteroatom-Substituted Cyclopropanes

2018

Synfacts

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The repurposing of hemoproteins for non-natural carbene transfer activities has generated enzymes for functions previously accessible only to chemical catalysts. With activities constrained to specific substrate classes, however, the synthetic utility of these new biocatalysts has been limited. To expand the capabilities of non-natural carbene transfer biocatalysis, we engineered variants of Cytochrome P450 BM3 that catalyze the cyclopropanation of heteroatom-bearing alkenes, providing valuable nitrogen-, oxygen-, and sulfur-substituted cyclopropanes. Four or five active-site mutations converted a single parent enzyme into selective catalysts for the synthesis of both cis and trans heteroatomsubstituted cyclopropanes, with high diastereoselectivities and enantioselectivities and up to 40 000 total turnovers. This work highlights the ease of tuning hemoproteins by directed evolution for efficient cyclopropanation of new substrate classes and expands the catalytic functions of iron heme proteins.

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Highly Diastereoselective and Enantioselective Olefin Cyclopropanation Using Engineered Myoglobin‐Based Catalysts

Cited by 107 publications

References 38 publications

Abiological catalysis by artificial haem proteins containing noble metals in place of iron

Abiological catalysis by artificial haem proteins containing noble metals in place of iron

The nature of chemical innovation: new enzymes by evolution

Stereoselective Enzymatic Synthesis of Heteroatom-Substituted Cyclopropanes

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