5 publications

5 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: ---

Biotinylated Rh(III) Complexes in Engineered Streptavidin for Accelerated Asymmetric C–H Activation

Rovis, T.; Ward, T.R.

Science 2012, 338, 500-503, 10.1126/science.1226132

Enzymes provide an exquisitely tailored chiral environment to foster high catalytic activities and selectivities, but their native structures are optimized for very specific biochemical transformations. Designing a protein to accommodate a non-native transition metal complex can broaden the scope of enzymatic transformations while raising the activity and selectivity of small-molecule catalysis. Here, we report the creation of a bifunctional artificial metalloenzyme in which a glutamic acid or aspartic acid residue engineered into streptavidin acts in concert with a docked biotinylated rhodium(III) complex to enable catalytic asymmetric carbon-hydrogen (C–H) activation. The coupling of benzamides and alkenes to access dihydroisoquinolones proceeds with up to nearly a 100-fold rate acceleration compared with the activity of the isolated rhodium complex and enantiomeric ratios as high as 93:7.


Metal: Rh
Ligand type: Amino acid; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Reaction: C-H activation
Max TON: 95
ee: 82
PDB: ---
Notes: ---

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: ---

Intramolecular C(sp3)-H Amination of Arylsulfonyl Azides with Engineered and Artificial Myoglobin-Based Catalysts

Fasan, R.

Bioorg. Med. Chem. 2014, 22, 5697-5704, 10.1016/j.bmc.2014.05.015

The direct conversion of aliphatic CH bonds into CN bonds provides an attractive approach to the introduction of nitrogen-containing functionalities in organic molecules. Following the recent discovery that cytochrome P450 enzymes can catalyze the cyclization of arylsulfonyl azide compounds via an intramolecular C(sp3)H amination reaction, we have explored here the CH amination reactivity of other hemoproteins. Various heme-containing proteins, and in particular myoglobin and horseradish peroxidase, were found to be capable of catalyzing this transformation. Based on this finding, a series of engineered and artificial myoglobin variants containing active site mutations and non-native Mn- and Co-protoporphyrin IX cofactors, respectively, were prepared to investigate the effect of these structural changes on the catalytic activity and selectivity of these catalysts. Our studies showed that metallo-substituted myoglobins constitute viable CH amination catalysts, revealing a distinctive reactivity trend as compared to synthetic metalloporphyrin counterparts. On the other hand, amino acid substitutions at the level of the heme pocket were found to be beneficial toward improving the stereo- and enantioselectivity of these Mb-catalyzed reactions. Mechanistic studies involving kinetic isotope effect experiments indicate that CH bond cleavage is implicated in the rate-limiting step of myoglobin-catalyzed amination of arylsulfonyl azides. Altogether, these studies indicate that myoglobin constitutes a promising scaffold for the design and development of CH amination catalysts.


Metal: Mn
Ligand type: Amino acid; Porphyrin
Host protein: Myoglobin (Mb)
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: C-H activation
Max TON: 142
ee: ---
PDB: ---
Notes: ---