6 publications

6 publications

Abiological Catalysis by Artificial Haem Proteins Containing Noble Metals in Place of Iron

Hartwig, J.F.

Nature 2016, 534, 534-537, 10.1038/nature17968

Enzymes that contain metal ions—that is, metalloenzymes—possess the reactivity of a transition metal centre and the potential of molecular evolution to modulate the reactivity and substrate-selectivity of the system1. By exploiting substrate promiscuity and protein engineering, the scope of reactions catalysed by native metalloenzymes has been expanded recently to include abiological transformations2,3. 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 sites1,4,5. 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 protein6,7,8. 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 metalloenzymes9,10. 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.


Metal: Ir
Ligand type: Methyl; Porphyrin
Host protein: Myoglobin (Mb)
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: C-H activation
Max TON: 7260
ee: 68
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Methyl; Porphyrin
Host protein: Myoglobin (Mb)
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: C-H activation
Max TON: 92
ee: 84
PDB: ---
Notes: ---

An Artificial Metalloenzyme with the Kinetics of Native Enzymes

Hartwig, J.F.

Science 2016, 354, 102-106, 10.1126/science.aah4427

Natural enzymes contain highly evolved active sites that lead to fast rates and high selectivities. Although artificial metalloenzymes have been developed that catalyze abiological transformations with high stereoselectivity, the activities of these artificial enzymes are much lower than those of natural enzymes. Here, we report a reconstituted artificial metalloenzyme containing an iridium porphyrin that exhibits kinetic parameters similar to those of natural enzymes. In particular, variants of the P450 enzyme CYP119 containing iridium in place of iron catalyze insertions of carbenes into C–H bonds with up to 98% enantiomeric excess, 35,000 turnovers, and 2550 hours−1 turnover frequency. This activity leads to intramolecular carbene insertions into unactivated C–H bonds and intermolecular carbene insertions into C–H bonds. These results lift the restrictions on merging chemical catalysis and biocatalysis to create highly active, productive, and selective metalloenzymes for abiological reactions.


Metal: Ir
Ligand type: Methyl; Porphyrin
Host protein: Cytochrome P450 (CYP119)
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: C-H activation
Max TON: 582
ee: 98
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Methyl; Porphyrin
Host protein: Cytochrome P450 (CYP119)
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: C-H activation
Max TON: 35129
ee: 91
PDB: ---
Notes: ---

Beyond Iron: Iridium-Containing P450 Enzymes for Selective Cyclopropanations of Structurally Diverse Alkenes

Hartwig, J.F.

ACS Cent. Sci. 2017, 3, 302-308, 10.1021/acscentsci.6b00391

Enzymes catalyze organic transformations with exquisite levels of selectivity, including chemoselectivity, stereoselectivity, and substrate selectivity, but the types of reactions catalyzed by enzymes are more limited than those of chemical catalysts. Thus, the convergence of chemical catalysis and biocatalysis can enable enzymatic systems to catalyze abiological reactions with high selectivity. Recently, we disclosed artificial enzymes constructed from the apo form of heme proteins and iridium porphyrins that catalyze the insertion of carbenes into a C–H bond. We postulated that the same type of Ir(Me)-PIX enzymes could catalyze the cyclopropanation of a broad range of alkenes with control of multiple modes of selectivity. Here, we report the evolution of artificial enzymes that are highly active and highly stereoselective for the addition of carbenes to a wide range of alkenes. These enzymes catalyze the cyclopropanation of terminal and internal, activated and unactivated, electron-rich and electron-deficient, conjugated and nonconjugated alkenes. In particular, Ir(Me)-PIX enzymes derived from CYP119 catalyze highly enantio- and diastereoselective cyclopropanations of styrene with ±98% ee, >70:1 dr, >75% yield, and ∼10,000 turnovers (TON), as well as 1,2-disubstituted styrenes with up to 99% ee, 35:1 dr, and 54% yield. Moreover, Ir(Me)-PIX enzymes catalyze cyclopropanation of internal, unactivated alkenes with up to 99% stereoselectivity, 76% yield, and 1300 TON. They also catalyze cyclopropanation of natural products with diastereoselectivities that are complementary to those attained with standard transition metal catalysts. Finally, Ir(Me)-PIX P450 variants react with substrate selectivity that is reminiscent of natural enzymes; they react preferentially with less reactive internal alkenes in the presence of more reactive terminal alkenes. Together, the studies reveal the suitability of Ir-containing P450s to combine the broad reactivity and substrate scope of transition metal catalysts with the exquisite selectivity of enzymes, generating catalysts that enable reactions to occur with levels and modes of activity and selectivity previously unattainable with natural enzymes or transition metal complexes alone.


Metal: Ir
Ligand type: Methyl; Porphyrin
Host protein: Cytochrome P450 (CYP119)
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: Cyclopropanation
Max TON: 10181
ee: 98
PDB: ---
Notes: Selectivity for cis product (cis/trans = 90:1)

Chemoselective, Enzymatic C−H Bond Amination Catalyzed by a Cytochrome P450 Containing an Ir(Me)-PIX Cofactor

Hartwig, J.F.

J. Am. Chem. Soc. 2017, 139, 1750-1753, 10.1021/jacs.6b11410

Cytochrome P450 enzymes have been engineered to catalyze abiological C–H bond amination reactions, but the yields of these reactions have been limited by low chemoselectivity for the amination of C–H bonds over competing reduction of the azide substrate to a sulfonamide. Here we report that P450s derived from a thermophilic organism and containing an iridium porphyrin cofactor (Ir(Me)-PIX) in place of the heme catalyze enantioselective intramolecular C−H bond amination reactions of sulfonyl azides. These reactions occur with chemoselectivity for insertion of the nitrene units into C−H bonds over reduction of the azides to the sulfonamides that is higher and with substrate scope that is broader than those of enzymes containing iron porphyrins. The products from C−H amination are formed in up to 98% yield and ∼300 TON. In one case, the enantiomeric excess reaches 95:5 er, and the reactions can occur with divergent site selectivity. The chemoselectivity for C–H bond amination is greater than 20:1 in all cases. Variants of the Ir(Me)-PIX CYP119 displaying these properties were identified rapidly by evaluating CYP119 mutants containing Ir(Me)-PIX in cell lysates, rather than as purified enzymes. This study sets the stage to discover suitable enzymes to catalyze challenging C–H amination reactions.


Metal: Ir
Ligand type: Methyl; Porphyrin
Host protein: Cytochrome P450 (CYP119)
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: C-H activation
Max TON: 294
ee: 26
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Methyl; Porphyrin
Host protein: Cytochrome P450 (CYP119)
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: C-H activation
Max TON: 192
ee: 95
PDB: ---
Notes: ---

Noble−Metal Substitution in Hemoproteins: An Emerging Strategy for Abiological Catalysis

Review

Hartwig, J.F.

Acc. Chem. Res. 2019, 52, 326-335, 10.1021/acs.accounts.8b00586

Enzymes have evolved to catalyze a range of biochemical transformations with high efficiencies and unparalleled selectivities, including stereoselectivities, regioselectivities, chemoselectivities, and substrate selectivities, while typically operating under mild aqueous conditions. These properties have motivated extensive research to identify or create enzymes with reactivity that complements or even surpasses the reactivity of small-molecule catalysts for chemical reactions. One of the limitations preventing the wider use of enzymes in chemical synthesis, however, is the narrow range of bond constructions catalyzed by native enzymes. One strategy to overcome this limitation is to create artificial metalloenzymes (ArMs) that combine the molecular recognition of nature with the reactivity discovered by chemists. This Account describes a new approach for generating ArMs by the formal replacement of the natural iron found in the porphyrin IX (PIX) of hemoproteins with noble metals. Analytical techniques coupled with studies of chemical reactivity have demonstrated that expression of apomyoglobins and apocytochrome P450s (for which “apo-” denotes the cofactor-free protein) followed by reconstitution with metal−PIX cofactors in vitro creates proteins with little perturbation of the native structure, suggesting that the cofactors likely reside within the native active site. By means of this metal substitution strategy, a large number of ArMs have been constructed that contain varying metalloporphyrins and mutations of the protein. The studies discussed in this Account encompass the use of ArMs containing noble metals to catalyze a range of abiological transformations with high chemoselectivity, enantioselectivity, diastereoselectivity, and regioselectivity. These transformations include intramolecular and intermolecular insertion of carbenes into C−H, N−H, and S−H bonds, cyclopropanation of vinylarenes and of internal and nonconjugated alkenes, and intramolecular insertions of nitrenes into C−H bonds. The rates of intramolecular insertions into C−H bonds catalyzed by thermophilic P450 enzymes reconstituted with an Ir(Me)−PIX cofactor are now comparable to the rates of reactions catalyzed by native enzymes and, to date, 1000 times greater than those of any previously reported ArM. This reactivity also encompasses the selective intermolecular insertion of the carbene from ethyl diazoacetate into C−H bonds over dimerization of the carbene to form alkenes, a class of carbene insertion or selectivity not reported to occur with small-molecule catalysts. These combined results highlight the potential of well-designed ArMs to catalyze abiological transformations that have been challenging to achieve with any type of catalyst. The metal substitution strategy described herein should complement the reactivity of native enzymes and expand the scope of enzyme-catalyzed reactions.


Notes: ---

Site‐Selective Functionalization of (sp3)C-H Bonds Catalyzed by Artificial Metalloenzymes Containing an Iridium‐Porphyrin Cofactor

Hartwig, J.F.

Angew. Chem. Int. Ed. 2019, 58, 13954-13960, 10.1002/anie.201907460

The selective functionalization of one C-H bond over others in nearly identical steric and electronic environments can facilitate the construction of complex molecules. We report site-selective functionalizations of C-H bonds, differentiated solely by remote substituents, catalyzed by artificial metalloenzymes (ArMs) that are generated from the combination of an evolvable P450 scaffold and an iridium-porphyrin cofactor. The generated systems catalyze the insertion of carbenes into the C-H bonds of arange of phthalan derivatives containing substituents that render the two methylene positions in each phthalan inequivalent. These reactions occur with site-selectivity ratios of up to 17.8:1 and, in most cases, with pairs of enzyme mutants that preferentially form each of the two constitutional isomers. This study demonstrates the potential of abiotic reactions catalyzed by metalloenzymes to functionalize C-H bonds with site selectivity that is difficult to achieve with small-molecule catalysts.


Metal: Ir
Ligand type: Porphyrin
Host protein: Cytochrome P450 (CYP119)
Anchoring strategy: Reconstitution
Optimization: Genetic
Max TON: 2286
ee: 94
PDB: ---
Notes: ---