Non-Native Site-Selective Enzyme Catalysis

Abstract

The ability to site-selectively modify equivalent functional groups in a molecule has the potential to streamline syntheses and increase product yields by lowering step counts. Enzymes catalyze site-selective transformations throughout primary and secondary metabolism, but leveraging this capability for non-native substrates and reactions requires a detailed understanding of the potential and limitations of enzyme catalysis and how these bounds can be extended by protein engineering. In this review, we discuss representative examples of site-selective enzyme catalysis involving functional group manipulation and C-H bond functionalization. We include illustrative examples of native catalysis, but our focus is on cases involving non-native substrates and reactions often using engineered enzymes. We then discuss the use of these enzymes for chemoenzymatic transformations and target-oriented synthesis and conclude with a survey of tools and techniques that could expand the scope of non-native site-selective enzyme catalysis.

Scheme 1.. (A) Definitions of Regioselectivity and Site Selectivity with Representative Examples of Each;^a (B) Examples of Site-Selective Reactions in the Biosynthesis of Paclitaxel^b

^aAdapted with permission from ref . Copyright 1996 Georg Thieme Verlag. ^bThroughout this review, crimson is used to highlight functional groups (A/B) or sites (B, filled circles) undergoing site-selective reactions and mint is used to highlight the functionalized sites.

Scheme 2.. Simplified Mechanism of Serine Hydrolases

Scheme 3.. (A) Hydrolysis of 1 in 90/10 v/v t-BuOH/H₂O Using Novozym 435 To Give 2; (B) Monohydrolysis of Triethylcitrate Using Novozym 435 To Give 4

Scheme 4.. (A) PLE-Catalyzed Hydrolysis of (Z)-2-Methylbutenedioic Acid Esters (B) PLE- and Subtilisin-Catalyzed Hydrolysis of 8; (C) Hydrolysis of (R)-Aspartate Dimethyl Ester 11 Using PLE

Scheme 5.. (A and B) Selective Hydrolysis of Peracylated Sugars from the Pyranose and Furanose Series by A. niger Lipase (ANL) and C. cylindracea Lipase (CCL); (C) R. toruloides Esterase-Catalyzed Hydrolysis of Peracetylated Compounds 17 and 19^,

Scheme 6.. (A) Activated Carboxylic Acid Intermediates; (B) Amidation of Diamine Substrate by CfaL and Related Enzymes; (C) PbCfaL-Catalyzed Coupling of Acids 25 and 26

Scheme 7.. Site-Selective Macrocyclization of Peptidic Substrate 28 to Surugamide 29

Scheme 8.. (A) Lipase-Catalyzed Site-Selective Acylation of Vicinal Diols of Steroid Molecules; (B) Site-Selective Acylation of Deoxynucleosides by CALB; (C) Site-Selective Esterification of the Sugar Moiety of Naringin Using Immobilized Lipase from C. antarctica

Scheme 9.. (A) ArmB-Catalyzed Site-Selective Acylation To Generate Melleolide; (B) Representative Orsellinic Acid Esters Found in Fungi; (C) Evolved Acyltransferase LovD Catalyzes the Acylation of Monacolin J Acid (MJA) To Generate Lovastatin

Scheme 10.. (A) Mechanism for Methylation by the SAM Cofactor; (B) Methylation of Naringenin to Sakuranetin; (C) Methylation by OMT in Reticuline Biosynthesis

Scheme 11.. (A and B) Methylation of Phenols Catalyzed by OMTs to Give Meta (51) and Para (53) Products

Scheme 12.. (A) Methylation of Novel Benzenediol Lactones Catalyzed by HsOMT and LtOMT Variants; (B) Selective Methylation of 60–62 by RnCOMT and MxSafC Using a SAM Regeneration System

Scheme 13.. (A) Site-Selective Carboxymethylation of 63 Using Carboxy-S-adenosyl-l-methionine (cxSAM) Catalyzed by COMT; (B) Regeneration of Alkyl-S-adenoxyl-l-methionines Using HMT, and Representative Ethyl and Allyl Products Generated by IOMT and COMT Variants, Respectively

Scheme 14.. (A) Site-Selective Alkylation Catalyzed by NMT Variant v36 and Haloalkanes; (B) Site-Selective Methylation by Variants Obtained through In Silico Mutational Studies; (C) Representative Examples of Site-Selective Alkylation Catalyzed by Variants of the NMTs

Scheme 15.. Evolved Variants of (A) E. coli Phosphopentomutase (PPM), (B) E. coli Pantothenate Kinase (PanK), and (C) Klebsiella sp. 5-S-Methylthioribose (MTR) Kinase Catalyze Site-Selective Phosphorylation of Different Non-Native Substrates

Scheme 16.. A) Glycosylation of 84 by Glycosyl Transferase UGT71A15; Representative Scope of (B) Glycosyl Acceptors and (C) Glycosyl Donors for Glycosylation of Oleandomycin with OleD, OleI, and MGT^a

^aUDP = uridine diphosphate, GDP = guanosine diphosphate, and TDP = thymidine diphosphate glucose.

Scheme 17.. Site-Selective Glycosylation by (A) β-1,3-Galactosyltransferase CgtB and (B) CgtB Variant S42; (C) Substrate Scope for Glycosylation of Non-Natural Sugar Acceptors and Uridine 5′-Diphosphogalactose (UDP Gal) Donors Catalyzed by LgtC

Scheme 18.. (A) Glycosylation of the Open Chain Form of 2-Hydroxyflavanone (108) by C-Glucosyltransferase; (B) Site-Selective Catalytic Promiscuity of TcCGT1

Scheme 19.. Site-Selective Glycosidase Catalysis: (A) Synthesis of Glycoflavonoids Catalyzed by Cel7B–E197S with Disaccharide Donor Lactosyl Fluoride (LacF); (B) Structures of Saponins QS-21, QS-18, and QS-17

Scheme 20.. Epimerization of Abundant Carbohydrates To Furnish Rare Sugars: (A) Evolved d-Fructose Epimerases for d-Psicose and l-Tagatose Production; (B) Wild-Type C-3 d-Frucuronate Epimerase and Evolved C-4 d-Fructose Epimerase

Scheme 21.. One-Pot Synthesis of 12-Ketoursodeoxycholic Acid from Cholic Acid Using HSDHs

Scheme 22.. Site-Selective Reduction of (A) Sterically and Electronically Similar Ketones in 127 and (B) Either Ketone in 129 Using Engineered KREDs

Scheme 23.. Site-Selective Carbonyl Reductions: (A) Reduction of 3-Methyl-2,4-hexadione; (B) Formation of Dehydroepiandrosterone (DHEA) by a KRED from S. wittichii; (C) Reduction of a Diketone as Part of the Chemoenzymatic Synthesis of Navoximod; (D) Kinetic Resolution of tert-Butyl 4-Methyl-3,5-dioxohexanoate with Alcohol Dehydrogenases^,

Scheme 24.. ω-Transaminase-Catalyzed Cyclization of Diketones: (A) General Scheme for Cyclization with TAs; (B) Cyclization of Nonane-2,6-dione with a Panel of ω-TAs

Scheme 25.. (A) Coupling of ω-TA with Nonselective Ammonia Borane Reduction and trans-Pyrrolidine-Selective MAO-N Oxidation To Accumulate Product 154; (B) ω-TA-Catalyzed Amination Coupled to IRED-Catalyzed Reduction of Simple Diketone 155

Scheme 26.. Reversible (De)hydration of the Tertiary Alcohol (S)-Linalool to β-Myrcene and Its Isomerization to the Primary Alcohol Geraniol Catalyzed by LinD

Scheme 27.. Alkene Epoxidation by P450 Enzymes: (A) Epoxidation of Parthenolide with Evolved BM3 Variant III-D4; (B) Epoxidation of a Terminal Alkene by a TamI Variant; (C) Selective Epoxidation of Alkenes Appended to Theobromine by CYP 3A4

Scheme 28.. Chemoenzymatic Cascade to Compound 174 Enabled by Site-Selective Hydration of Polynitrile Precursor

Scheme 29.. Site-Selective Demethylation of Papaverine and rac-Yatein by Cobalamin-Dependent Methyltransferase MT-vdmB

Scheme 30.. Simplified Scheme of the Catalytic Cycle for the Hydroxylation of a Substrate R─H by a Cytochrome P450

Scheme 31.. (A) Site-Selective Hydroxylation of n-Alkanes by P450_BM3 Variants; (B) Demethylation of Protected Monosaccharides by P450_BM3 Variants Shown as the Major Product for Each Reaction

Scheme 32.. Site Selectivity Expressed as Percent in the Product Distribution in the Hydroxylation of Steroids by Engineered P450 Enzymes^a

^aRemaining products consist of hydroxylation at different sites and other oxidation products. Reactions shown include (A) 11α-hydroxyprogesterone (185) with P450_BM3 F1, (B) testosterone (186) with P450_BM3 variants KSA-1 and KSA-14, and (C) progesterone (187) with CYP106A2 T89N/A395I.

Scheme 33.. Site-Selective Hydroxylation Reactions Carried out with Engineered P450_BM3 Enzymes and (A) Propylbenzene (188), (B) Cyclohexene-1-carboxylic Acid Methyl Ester (189), and Cyclopentene-1-carboxylic Acid Methyl Ester (191)^a

^aProduct distribution is presented as the percentage of the major product in each reaction.

Scheme 34.. (A) Selectivity in the Product Distribution for the Hydroxylation of Artemisinin (193) by Engineered P450_BM3 Variants; (B) Site Selectivity of Hydroxylation Shown as the Ratio of C-10:C-12 Hydroxylated Products for YC-17 Analogues with Varying Anchoring Groups with PikC

Scheme 35.. (A) Product Distribution in the Hydroxylation of Monosubstituted Benzenes by P450_BM3 M2; (B) Selective Hydroxylation of 4-Phenylbenzoic Acid (202) by Variant CYP199A4 F182L with Percentages Depicting the Product Distribution

Scheme 36.. Simplified Scheme of the Catalytic Cycle for the Hydroxylation of Substrate R–H by a FeDO

Scheme 37.. Examples of Site-Selective Hydroxylation of Amino Acids with FeDOs: (A) Proline and Pipecolic Acid Hydroxylases; (B) Lysine Hydroxylases; (C) Engineered SadA Variants^a

^aPercentages correspond to relative amounts in the product distribution.

Scheme 38.. (A) Flavonoid Site-Selective Aromatic Hydroxylation by Heme Peroxidase AaeUPO; (B) Hydroxylation of Saturated Fatty Acids by AaeUPO Variants^a

^aProducts are shown as percentages of the total product pool, with remaining products being ω-hydroxylated fatty acids and overoxidation products.

Scheme 39.. (A) Site Selectivity in Hydroxylation Expressed as Percentage of Major Product in the Product Distribution for Rieske Dioxygenases NDO and CDO; (B) Site-Selective Monooxygenation of Saxitoxin-Derived Natural Products by Rieske Monooxygenases SxtT and GxtA

Scheme 40.. Major Products in Hydroxylation as Percentage in the Product Pool for (A) Bacterial Multicomponent Monooxygenase T4MO and (B) Methane Monooxygenases sMMO^, and pMMO

Scheme 41.. Catalytic Cycle for Hydroxylation of a Phenolic Compound by a Single-Component FMO Triggered by Substrate Binding

Scheme 42.. Hydroxylase Activity of Native and Engineered FMOs with Strict Site Selectivity: (A) Mutation M321A in HbpA Increases Substrate Scope; (B) Site-Selective Hydroxylation by Engineered HpaB Variants in Non-Native Substrates;^a (C) SorbC Catalyzed Oxidative Dearomatization

^aHydroxylation sites are denoted with arrows.

Scheme 43.. (A) Simplified FDH Mechanism; (B) PrnA and RebH Natively Chlorinate L-Tryptophan Site Selectively at C7 of the Indole Ring

Scheme 44.. (A) Biocatalysis with Wild-Type RebH and Rdc2 for Halogenation of Non-Native Substrates;^a(B) Directed Evolution of a Thermostable RebH Variant for Halogenation of Biologically Active Molecules; (C) Site-Selective and Atroposelective Catalysis with FDHs

^aNumber refers to HPLC yield when provided.

Scheme 45.. (A) Use of a Deuterated Tryptamine Probe–Substrate Allows for MALDI Screening Based on m/z Values; (B) Directed Evolution of RebH into 5- and 6-Selective Chlorinases; (C) Using the Crystal Structures of Thal and RebH Substitution of Key Active Site Residues from RebH into Thal Results in Modified Site Selectivity

Scheme 46.. Genome Mining for FDHs Enables Site-Selective Catalysis on Biologically Active Compounds^a

^aNumbers represent isolated yields.

Scheme 47.. Substrate Scope of AetF for Site-Selective (A) Bromination and (B) Iodination

Scheme 48.. Site-Selective Halogenation of Fisherindole (285), Hapalindole (287 and 288), and Ambiguine (289) Substrates by WelO5 and AmbO5; (B) WelO5* and Evolved Variants Catalyze Halogenation of a Martinelline-Derived Substrate; (C) Site-Selective Halogenation of Soraphen A by Evolved WelO5* Variants

Scheme 49.. (A) Natural Product Pathway of BesD Results in Alkyne Formation; (B) Homologues of BesD Site Selectively Halogenate Amino Acid Substrates; (C) Fluorogenic Click Assay Developed for High-Throughput Screening of Chimera Hydox/Hal Libraries

Scheme 50.. Site-Selective Azidation by Engineered SadX Variants

Scheme 51.. (A) P411 Enzymes Evolved for Regiodivergent Intramolecular Sulfamidation; (B) Sulfamidation Catalyzed by Iridium–Heme Bearing Artificial Metalloenzymes

Scheme 52.. (A) P411_BPA and (B) uAMD9 Expressed in Whole Cells Are Capable of Site-Selective Functionalization Amination and Amidation of Benzyl C─H Bonds

Scheme 53.. Evolution of P450 Variants for Site-Selective Amidation and Amination of Unactivated C─H Bonds

Scheme 54.. Biocatalytic Deuteration of Amino Acids: (A) General Site-Selective Deuteration by DsaD and DsaE; (B) Synthetic Outline for Production of α-, β-, and αβ-Deuterated Amino Acid Reaction Outcomes Are Formatted as % Recovery,% Deuteration (Site), and $ ee

Scheme 55.. (A) Percent of Product Pool in the Synthesis of the Arylomycin Core by P450 MG-AR Variant; (B) Intermolecular Cross-Coupling of Nonidentical Monomers by KtnC; (C) Reactions of 7-Demethylsiderin (347) with Enzymes KtnC and DesC

Scheme 56.. (A) General Scheme of the Native Prenyltransferase Reaction by Dimethylallyltryptophan Synthases (DMATSs) and Representative Non-Native Products for DMATSs with Different Site Selectivity;^, (B) Geranylation Reactions on Genistein (359) by AtaPT Single Mutants

Scheme 57.. (A) Sequential Methylation of Carbapenem Substrate 362 by SAM-Dependent Methylase; (B) Alkylation of the Glutamine Residue in a 24-Mer Polypeptide Substrate Employing a One-Pot SAM Regeneration System^a

^aSAE: S-adenosylethionine. DOA: 5′-deoxyadenosine. SAH: S-adenosylhomocysteine. Met: l-methionine. Eth: l-ethionine.

Scheme 58.. (A) Radical Heterocoupling of Coniferyl Alcohol Analogues by Laccase TvLac and the Effect of Dirigent Protein PhDIR in the Product Ratio;^a (B) Non-Native Heteroarene Alkylation by Ene-Reductases^b

^aPercentages represent the amount of 8–8′ heterodimer product compared to the 5–8′ heterodimer product. ^bSite selectivity expressed as the percentage of the shown compound in the total product pool.

Scheme 59.. (A) Alkylation of the 2 Position of the Tetrahydroquinoline Moiety of N-Methyltetrahydroquinoline (376) with the Remaining Product Pool Being the Alkylation of the Methyl Group; (B) P411 Variants Selective for Methyl and Ethyl Alkylation of N-Ethyl-N-methylaniline (379)

Scheme 60.. (A) Distribution of Para and Meta Products in the Alkylation of Several Benzofurans by Artificial Iridium-Containing P450s; (B) Lactam Formation via Intramolecular Cyclization by Engineered Artificial Copper–Streptavidin Metalloenzymes with Major Products Displayed with the Corresponding Percent of the Product Pool

Scheme 61.. Representative Examples of (A) Site-Selective Chemoenzymatic Hydroxylation/Deoxy Fluorination and (B) Halogenation/Cross-Coupling

Scheme 62.. (A) BM3 MERO1 and KSA 15 Are Both Highly Selective for the C3 Hydroxylation of Sclareolide and Episclareolide, Respectively; (B) Sequential Gram-Scale Oxidation of Sclareol with BM3 Variants LG-23 and MERO1 Provide a Precursor for the Core of (+)-Pallavincin

Scheme 63.. Site-Selective Oxidation of a Complex Diterpenoid Using P450 and FeDO Enzymes

Scheme 64.. (A) Hydroxylation with UscF and GetF Is Used To Construct a Core Unnatural Amino Acid Fragment in Polyoxpeptin A; (B) GriE Hydroxylation of an Azidated Substrate; (C) KDO1 Catalyzed the Hydroxylation of Lysine in the Total Synthesis of Tambromycin

Scheme 65.. Chemoenzymatic Synthesis of Cepafungin I^a

^aA lysine-derived fragment can be modified with various lysine hydroxylases to study the impact of modification. For KDO1- and KDO3-derived peptides, yield refers to the Boc-protected monomer. Two-step enzymatic synthesis of 422 combining SadA hydroxylation with LasA decuccinylation; yield for 422 refers to the Fmoc-protected monomer.

Scheme 66.. Hydroxylation of Cotylenol and Brassicicene I Precursors with Evolved Variants of MoBsc9, a Homologue of the Native Hydroxylase

Scheme 67.. Oxidative Coupling of Etoposide Precursors with FeDOs: (A) Native Reaction; (B) Closely Related Non-Native Substrate Shows Altered Selectivity for the Ring Closure

Scheme 68.. (A) FasV Was Used as the Final Step in the Total Synthesis of Fasamycin A To Affect Site-Selective Halogenation of Napthacemycin B1; (B) SorbC Was Used in the First Total Synthesis of Sorbicillactone A

Scheme 69.. Toolbox for Insulin Modification with Penicillin Acylases