Biocatalytic Friedel–Crafts Alkylation Using a Promiscuous Biosynthetic Enzyme

Schultz, Erica E.; Braffman, Nathaniel R.; Luescher, Michael U.; Hager, Harry H.; Balskus, Emily P.

doi:10.1002/ange.201814016

Cited by 30 publications

(13 citation statements)

References 33 publications

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“… 103 The enzyme CylK has also been shown to be highly promiscuous, carrying out alkylation of a variety of resorcinol derivatives with secondary alkyl halides. 104 Biocatalytic Friedel–Crafts alkylation has also been carried out to synthesize podophyllotoxin lignans. 105 , 106 For example, the NHI enzyme 2-ODD-PH has been utilized to carry out the biocatalytic synthesis of deoxypodophyllotoxin ( 20 ) and related analogs.…”

Section: State-of-the-art Biocatalysismentioning

confidence: 99%

State-of-the-Art Biocatalysis

et al. 2021

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The use of enzyme-mediated reactions has transcended ancient food production to the laboratory synthesis of complex molecules. This evolution has been accelerated by developments in sequencing and DNA synthesis technology, bioinformatic and protein engineering tools, and the increasingly interdisciplinary nature of scientific research. Biocatalysis has become an indispensable tool applied in academic and industrial spheres, enabling synthetic strategies that leverage the exquisite selectivity of enzymes to access target molecules. In this Outlook, we outline the technological advances that have led to the field’s current state. Integration of biocatalysis into mainstream synthetic chemistry hinges on increased access to well-characterized enzymes and the permeation of biocatalysis into retrosynthetic logic. Ultimately, we anticipate that biocatalysis is poised to enable the synthesis of increasingly complex molecules at new levels of efficiency and throughput.

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Section: State-of-the-art Biocatalysismentioning

confidence: 99%

State-of-the-Art Biocatalysis

et al. 2021

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“… 1 − 4 The use of enzymes as catalysts for this transformation is of great appeal because of their mild, efficient, benign, and highly selective nature; however, relatively few enzymes are known to perform this chemistry. 4 − 7 Notable examples are tryptophan synthase (TrpB), 8 a promiscuous enzyme involved cylindrocyclophane biosynthesis (CylK), 9 , 10 as well as enzymes which catalyze Friedel–Crafts acylation ( Pp ATase) ( Figure 1 a). 11 , 12 Catalytic machinery in these enzymes includes nucleophilic cysteine residues as well as pyridoxal phosphate, each powerful chemical motifs which have inspired biomimetic catalysis strategies.…”

Section: Introductionmentioning

confidence: 99%

Unlocking Iminium Catalysis in Artificial Enzymes to Create a Friedel–Crafts Alkylase

et al. 2021

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The construction and engineering of artificial enzymes consisting of abiological catalytic moieties incorporated into protein scaffolds is a promising strategy to realize non-natural mechanisms in biocatalysis. Here, we show that incorporation of the noncanonical amino acid para-aminophenylalanine (pAF) into the nonenzymatic protein scaffold LmrR creates a proficient and stereoselective artificial enzyme (LmrR_pAF) for the vinylogous Friedel–Crafts alkylation between α,β-unsaturated aldehydes and indoles. pAF acts as a catalytic residue, activating enal substrates toward conjugate addition via the formation of intermediate iminium ion species, while the protein scaffold provides rate acceleration and stereoinduction. Improved LmrR_pAF variants were identified by low-throughput directed evolution advised by alanine-scanning to obtain a triple mutant that provided higher yields and enantioselectivities for a range of aliphatic enals and substituted indoles. Analysis of Michaelis–Menten kinetics of LmrR_pAF and evolved mutants reveals that different activities emerge via evolutionary pathways that diverge from one another and specialize catalytic reactivity. Translating this iminium-based catalytic mechanism into an enzymatic context will enable many more biocatalytic transformations inspired by organocatalysis.

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“…Previously, Balskus and co-workers reported that CylK could also accept other secondary alkyl halide as electrophiles, and the reactivity of alkyl chloride is higher than alkyl bromide and alkyl iodide. 24 We reasoned that this abnormal trend was due to the hydrogen bonding network is more suitable to bind to the Cl atom than the Br and I atoms, thus making the C-Cl bond more susceptible to activation.…”

Section: Resultsmentioning

confidence: 99%

“…In addition, Balskus and co-workers demonstrated that CylK could catalyze stereospecific Friedel-Crafts alkylation reactions of other resorcinol rings with various secondary alkyl halides. 24 These findings indicate the potential of using CylK as a mild biocatalyst for stereospecific and regioselective aryl-alkyl C-C linkage. Balskus and co-workers proposed a putative structural model for CylK based on structural prediction.…”

Section: Introductionmentioning

confidence: 83%

Structural Basis for the Friedel-Crafts Alkylation in Cylindrocyclophane Biosynthesis

Wang

Mou

Xiao

et al. 2021

Preprint

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The Lewis acid-catalyzed Friedel-Crafts alkylation of an aromatic ring with an alkyl halide is extensively used in organic synthesis. However, its biological counterpart was not reported until the elucidation of the cylindrocyclophane biosynthetic pathway in Cylindrospermum licheniforme ATCC 29412 by Balskus and co-workers. CylK is the key enzyme to catalyze the formation of the cylindrocyclophane scaffold through the Friedel-Crafts alkylation reactions with regioselectivity and stereospecificity. Further research demonstrates that CylK can accept other resorcinol rings and secondary alkyl halides as substrates. To date, the crystal structure of CylK has not been disclosed and the catalytic mechanism remains obscure. Herein we report the crystal structures of CylK in its apo form and its complexes with the analogues of its substrate and reaction intermediate. Combining the crystal structures, free energy simulations and the mutagenesis experiments, we proposed a concerted double-activation mechanism, which could explain the regioselectivity and stereospecificity. This work provides a foundation for engineering CylK as a biocatalyst to expand its substrate scope and applications in organic synthesis.

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Biocatalytic Friedel–Crafts Alkylation Using a Promiscuous Biosynthetic Enzyme

Abstract: This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record.

Cited by 30 publications

References 33 publications

State-of-the-Art Biocatalysis

State-of-the-Art Biocatalysis

Unlocking Iminium Catalysis in Artificial Enzymes to Create a Friedel–Crafts Alkylase

Structural Basis for the Friedel-Crafts Alkylation in Cylindrocyclophane Biosynthesis

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