Enzymatic functionalization of carbon-hydrogen bonds

Abstract

The development of new catalytic methods to functionalize carbon-hydrogen (C-H) bonds continues to progress at a rapid pace due to the significant economic and environmental benefits of these transformations over traditional synthetic methods. In nature, enzymes catalyze regio- and stereoselective C-H bond functionalization using transformations ranging from hydroxylation to hydroalkylation under ambient reaction conditions. The efficiency of these enzymes relative to analogous chemical processes has led to their increased use as biocatalysts in preparative and industrial applications. Furthermore, unlike small molecule catalysts, enzymes can be systematically optimized via directed evolution for a particular application and can be expressed in vivo to augment the biosynthetic capability of living organisms. While a variety of technical challenges must still be overcome for practical application of many enzymes for C-H bond functionalization, continued research on natural enzymes and on novel artificial metalloenzymes will lead to improved synthetic processes for efficient synthesis of complex molecules. In this critical review, we discuss the most prevalent mechanistic strategies used by enzymes to functionalize non-acidic C-H bonds, the application and evolution of these enzymes for chemical synthesis, and a number of potential biosynthetic capabilities uniquely enabled by these powerful catalysts (110 references).

Figure 1

Typical biological routes to C-H bond cleavage include A) deprotonation and B) dehydrogenation. C) Mechanism of alcohol dehydrogenation using NAD(P)H as a hydride acceptor.

Figure 2

Representative mechanisms for enzymatic functionalization of non-acidic C-H bonds.,, A) Electrophilic aromatic substitution. B) Electron abstraction followed by radical coupling. C) H abstraction followed by radical recombination. D) H abstraction followed by radical reaction.

Figure 3

A) Structures of riboflavin, FAD, and FMN. B) Mechanism of nucleophilic and electrophilic oxygenation.

Figure 4

A) Primary reactions of p-hydroxybenzoate 3-hydroxylase (PHBH) and B) a newly-characterized long-chain alkane hydroxylase, LadA.

Figure 5

Proposed mechanism of FAD-dependent halogenases (PrnA/RebH) catalyzing halogenation of tryptophan. A hydrogen-bonded HOX oxidant is depicted.

Figure 6

Structures of AdoMet (A) and AdoCbl (B) and mechanism for generation of Ado• in each case.

Figure 7

Examples of Ado• reactions. A) 1,2-rearrangement catalyzed by lysine-2,3-aminomutase. B) Double C-H abstraction and sulfur insertion catalyzed by biotin synthase (BioB). C) Radical methylation catalyzed by Fom3 in the biosynthesis of fosfomycin. D) Hydroalkylation of fumarate catalyzed by benzylsuccinate synthase.

Figure 8

A) General mechanism for hydroxylation of C-H bonds on substrates (R-H) by αKG-dependent dioxygenases. B) Hydroxylation reaction catalyzed by clavaminate synthase (CAS). C) Chlorination reaction catalyzed by halogenase CytC3. D) Oxidative cyclization reaction catalyzed by an isopenicillin-N synthase (IPNS).

Figure 9

A) Representative reactions catalyzed by each of the four classes of bacterial multicomponent monooxygenases (BMMs). B) The catalytic cycle for the soluble diiron methane monooxygenase.

Figure 10

Toluene monooxygenase (aromatic oxidation) vs xylene monooxygenase (benzylic oxidation) and subsequent dehydrogenation for the synthesis of phenols, alcohols, aldehydes, and acids.

Figure 11

A) The catalytic cycle for tyrosinase. Reactions catalyzed by B) dopamine β-monooxygenase (DβM) and C) peptidylglycine α-hydroxylating monooxygenase (PHM) (AscH₂ = ascorbic acid, Pep = D-Tyr-Val).,

Figure 12

A) Structure of a heme cofactor and general schemes for its conversion to compound I in peroxidases (HRP and CPO) and cytochromes P450 (CYPs). B) Oxidative coupling catalyzed by HRP. C) Halogenation catalyzed by CPO. D) Hydroxylation catalyzed by CYP 102A1. Synthesis of substituted phenols using A) 2-hydroxybiphenyl 3-monooxygenase (HbpA) and B) p-hydroxybenzoate 3-hydroxylase (PHBH).

Figure 12

A) Structure of a heme cofactor and general schemes for its conversion to compound I in peroxidases (HRP and CPO) and cytochromes P450 (CYPs). B) Oxidative coupling catalyzed by HRP. C) Halogenation catalyzed by CPO. D) Hydroxylation catalyzed by CYP 102A1. Synthesis of substituted phenols using A) 2-hydroxybiphenyl 3-monooxygenase (HbpA) and B) p-hydroxybenzoate 3-hydroxylase (PHBH).

Figure 13

Substrate scope and regioselectivity of indole halogenation using the halogenase PrnA.

Figure 14

Production of 1,3-propane diol by E. coli expressing an AdoCbl-dependent dehydratase and an oxidoreductase. A) Synthesis of phenol derivatives using recombinant toluene-4-monooxygenase (T4MO). B) Synthesis of substituted benzyl alcohols using cells expressing a xylene monooxygenase (XMO) with reaction rates shown. C). Lonza synthesis of (hetero)arylcarboxylic acids using XMO and additional dehydrogenases.

Figure 14

Production of 1,3-propane diol by E. coli expressing an AdoCbl-dependent dehydratase and an oxidoreductase. A) Synthesis of phenol derivatives using recombinant toluene-4-monooxygenase (T4MO). B) Synthesis of substituted benzyl alcohols using cells expressing a xylene monooxygenase (XMO) with reaction rates shown. C). Lonza synthesis of (hetero)arylcarboxylic acids using XMO and additional dehydrogenases.

Figure 15

A) Hydroxylation of 11-deoxycortisol using cytochrome P450_lun in Curvularia lunata. B) Hydroxylation of compactin using cytochrome 105A3 in Streptomyces sp. Y-110. C) Six sequential oxidations on n-dodecane catalyzed by cytochrome 52A1 in Candida tropicalis.

Figure 16

A) Various hydroxylation and halogenation reactions catalyzed by chloroperoxidase (CPO)., B) Oxidative coupling between arbutin and gentisate catalyzed by HRP followed by spontaneous lactonization.

Figure 17

A) Total turnovers for variants along the P450_PMO lineage on propane and ethane. B) Activity of variants along the P450_PMO lineage on C_n (n = 1–10) alkanes.

Figure 18

A) Hydroxylation of phenylacetic esters. B) Regioselectivity of hydroxylation reactions catalyzed by BM3 chimeras. C) Tandem P450_BM3-catalyzed hydroxylation/DAST deoxyfluorination. D) Heteroatom demethylation for regioselective deprotection of monosaccharides.

Figure 19

Regioselective hydroxylation of toluene using mutants of toluene para-monooxygenase (TpMO).

Figure 20

A) General scheme for in vitro or in vivo biocatalytic cascades. Engineered metabolic pathways for production of B) hydrocortisone, C) artemisinic acid, and D) 1,3-propanediol.