Manfred T. Reetz's research while affiliated with Chinese Academy of Sciences and other places

Publications (708)

Article
This invited essay outlines how the idea of directed evolution of stereoselective enzymes was born and implemented experimentally at the Max‐Planck‐Institut für Kohlenforschung in Mülheim/Germany during the period 1994‐1998, a time when Andreas Pfaltz was present in the Institute. As the new and sole Director of the MPI, I initiated new research pr...
Article
This review outlines recent developments in protein engineering of stereo‐ and regioselective enzymes, which are of prime interest in organic and pharmaceutical chemistry as well as biotechnology. The widespread application of enzymes was hampered for decades due to limited enantio‐, diastereo‐ and regioselectivity, which was the reason why most or...
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Water is the solvent of choice in terms of greenness and sustainability. However, it can be difficult to isolate very polar products from water. Here, we discuss the use of n ‐butanol as a solvent to extract polar compounds from aqueous solution. n ‐Butanol produces high yields and can also be sourced sustainably from biomass, which makes it attrac...
Chapter
In the quest to produce by directed evolution chemo-, stereo-, and regioselective enzymes as catalysts for the sustainable production of chiral compounds needed in modern society, focused saturation mutagenesis at sites surrounding the binding pocket has emerged as a particularly effective strategy. This chapter focuses on the genesis and recent pr...
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Decades of extensive research efforts by biochemists, organic chemists, and protein engineers have led to an understanding of the basic mechanisms of essentially all known types of enzymes, but in a formidable number of cases an essential aspect has been overlooked. The occurrence of short-lived chiral intermediates formed by symmetry-breaking of p...
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In this short essay, I re‐emphasize the inter‐relationship between mentoring doctoral students or postdocs and the never ending problem of assessing the quality of scientific outputs. It includes the plea to mentees and mentors not to employ an overblown language in papers, posters and lectures, which constitute a gross exaggeration of the true sta...
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Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. To date, such efforts usually focus on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. Here, we report a multiparametric fitness land...
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An intriguing advance in the rational protein engineering of a newly identified glycosyltransferase from Siraitia grosvenorii (UGT74AC2) has been accomplished by Zhoutong Sun, Yuanxia Sun and coworkers for the targeted pharmaceutically significant glucosylation of the polyhydroxy substrate silybin. Using an advanced version of Focused Rational Iter...
Article
An approach for the possible production of novel bimetallic self-doped porphyrin-based compounds of potential interest in material science is reported. Heating Cu(II)tetraphenylporphyrin (TPPCu) with chromocene at 120[Formula: see text]C in benzonitrile affords the crystalline multimetal porphyrin system TPPCu/TPPCr in good yield. The X-ray single...
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Machine learning (ML) has pervaded most areas of protein engineering, including stability and stereoselectivity. Using limonene epoxide hydrolase as the model enzyme and innov’SAR as the ML platform, comprising a digital signal process, we achieved high protein robustness that may resist unfolding with concomitant detrimental aggregation. Fourier t...
Chapter
Directed evolution has emerged as the most productive enzyme engineering method, with stereoselectivity playing a crucial role when evolving mutants for application in synthetic organic chemistry and biotechnology. In order to reduce the screening effort (bottleneck of directed evolution), improved methods for the creation of small and smart mutant...
Preprint
p>Heating Cu(II)tetraphenylporphyrin (TPPCu) with chromocene at 120 °C affords the crystalline self-doped multimetal porphyrin system TPPCu/TPPCr in good yield. The X-ray structural analysis reveals a random configuration of TPPCu and TPPCr with a Cu:Cr ratio of about 71:29 %. Exploratory DFT calculations indicate significant electron transfer in a...
Preprint
Full-text available
Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. Yet such efforts are rare and focus only on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. Here, we report the first multiparametric...
Article
Full-text available
Steroidal C7β alcohols and their respective esters have shown significant promise as neuroprotective and anti‐inflammatory agents to treat chronic neuronal damage like stroke, brain trauma and cerebral ischemia. Since position C7 is spatially far away from any functional groups that could direct C–H activation, these transformations are not readily...
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Steroidal C7β alcohols and their respective esters have shown significant promise as neuroprotective and anti‐inflammatory agents to treat chronic neuronal damage like stroke, brain trauma and cerebral ischemia. Since position C7 is spatially far away from any functional groups that could direct C–H activation, these transformations are not readily...
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While the mechanism of the P450-catalyzed oxidative hydroxylation of organic compounds has been studied in detail for many years, less is known about sulfoxidation. Depending upon the structure of the respective substrate, heme-Fe═O (Cpd I), heme–Fe(III)–OOH (Cpd 0), and heme–Fe(III)–H2O2 (protonated Cpd 0) have been proposed as reactive intermedia...
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Arbutin (also called β-arbutin) is a natural product occurring in the leaves of a variety of different plants, the bearberries of the Ericaceae and Saxifragaceae families being prominent examples. It is a β-glucoside derived from hydroquinone (HQ; 1,4-dihydroxybenzene). Arbutin has been identified in traditional Chinese folk medicines as having, in...
Article
Directed evolution of stereo-, regio- and chemoselective enzymes has enriched the toolbox of synthetic organic chemistry. Among the different gene mutagenesis techniques, saturation mutagenesis (SM) at sites lining the enzyme’s binding pocket has emerged as a particularly viable approach to control selectivity and activity. Traditionally, NNK codon...
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Engineering artificial enzymes with high activity and catalytic mechanism different from naturally occurring enzymes is a challenge in protein design. For example, many attempts have been made to obtain active hydrolases by introducing a Ser → Cys exchange at the respective catalytic triads, but this generally induced a breakdown of activity. We no...
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Die gerichtete Evolution von stereo‐, regio‐ und chemoselektiven Enzymen ist ein einzigartiges Verfahren zur Herstellung von Biokatalysatoren für synthetisch interessante Transformationen. Methodenentwicklung war und bleibt unerlässlich, um diese Technik des Protein‐Engineerings effizient, schnell, zuverlässig und als relevant für die organische Ch...
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Directed evolution of stereo‐, regio‐ and chemoselective enzymes provides a means to generate biocatalysts for synthetically interesting transformations. In order for this protein engineering technique to be efficient, fast and reliable, and of relevance to organic chemistry, methodology development was and still is necessary. Following a descripti...
Article
Enzymatic stereodivergent synthesis to access all possible product stereoisomers bearing multiple stereocenters is relatively undevel-oped, although enzymes are being increasingly used in both academic and industrial areas. When two stereocenters and thus four stereoisomeric products are involved, obtaining stereodivergent enzyme mutants for indivi...
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A unique P450 monooxygenase-peroxygenase mutual benefit system was designed as the core element in the construction of a biocatalytic cascade reaction sequence leading from 3-phenyl propionic acid to (R)-phenyl glycol. In this system, P450 monooxygenase (P450-BM3) and P450 peroxygenase (OleTJE) not only function as catalysts for the crucial initial...
Article
Machine learning as a form of artificial intelligence consists of algorithms and statistical models for improving computer performance for different tasks. Training data is utilized for making decisions and predictions. Since directed evolution of enzymes produces huge amounts of potential training data, machine learning seems to be ideally suited...
Article
The term B-factor, sometimes called the Debye-Waller factor, temperature factor, or atomic displacement parameter, is used in protein crystallography to describe the attenuation of X-ray or neutron scattering caused by thermal motion. This review begins with analyses of early protein studies which suggested that B-factors, available from the Protei...
Article
ConspectusTransition metal catalysts mediate a wide variety of chemo-, stereo-, and regioselective transformations, and therefore play a pivotal role in modern synthetic organic chemistry. Steric and electronic effects of ligands provide organic chemists with an exceedingly useful tool. More than four decades ago, chemists began to think about a di...
Chapter
Cytochrome P450 monooxygenases (CYPs) have been used in synthetic organic chemistry and biotechnology for decades, especially in CH-activating oxidative hydroxylation. However, the control of both the regio- and stereoselectivity on a broad basis, a prerequisite for ecologically and economically viable transformations, has remained elusive until re...
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Die enzymatische Benzol‐Dihydroxylierung zu Hydrochinon im Eintopfverfahren wird von A. Li, M. T. Reetz et al. in ihrer Zuschrift (DOI: 10.1002/ange.201812093) vorgestellt. Eine Mutante einer Cytochrom‐P450‐Monooxygenase, die durch halbgezieltes Protein‐Engineering erhalten wurde, katalysiert die anspruchsvolle Umwandlung ohne Überoxidation, sodass...
Article
Hydroquinone (HQ) is produced commercially from benzene by multi‐step Hock‐type processes with equivalent amounts of acetone as side‐product. We describe an efficient biocatalytic alternative using the cytochrome P450‐BM3 monooxygenase. Since the wildtype enzyme does not accept benzene, a semi‐rational protein engineering strategy was developed. Hi...
Article
Hydroquinone (HQ) is produced commercially from benzene by multi‐step Hock‐type processes with equivalent amounts of acetone as side‐product. We describe an efficient biocatalytic alternative using the cytochrome P450‐BM3 monooxygenase. Since the wildtype enzyme does not accept benzene, a semi‐rational protein engineering strategy was developed. Hi...
Article
Unique enzymatic one‐pot dihydroxylation of benzene to hydroquinone is achieved as described by A. Li, M. T. Reetz et al. in their Communication (DOI: 10.1002/anie.201812093). A mutant of a cytochrome P450 monooxygenase obtained by a semi‐rational protein engineering strategy catalyzes this challenging transformation with no overoxidation, enabling...
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Directed evolution is an important research activity in synthetic biology and biotechnology. Numerous reports describe the application of tedious mutation/screening cycles for the improvement of proteins. Recently, knowledge-based approaches have facilitated the prediction of protein properties and the identification of improved mutants. However, e...
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Oil and water, apples and pears: Mixing things that should not be mixed can result in scientifically unsound conclusions. A recent directed‐evolution study compared iterative saturation mutagenesis (ISM) with the OmniChange version of saturation mutagenesis (SM). ISM is a semirational strategy in directed evolution that is independent of the partic...
Article
Chiral arylpropanols are valuable components in important pharmaceuticals and fragrances, which is the motivation for previous attempts to prepare these building blocks enantioselectively in asymmetric processes using either enzymes or transition metal catalysts. Thus far, enzymes used in kinetic resolution proved to be best, but several problems p...
Article
The quality and efficiency of any PCR-based mutagenesis technique may not be optimal due to, inter alia, amino acid bias, which means that the development of efficient PCR-free methods is desirable. Here, we present a highly efficient in vitro CRISPR/Cas9-mediated mutagenic (ICM) system that allows rapid construction of designed mutants in a PCR-fr...
Article
Manipulating the regioselectivity of Baeyer-Villiger (BV) reactions remains an ongoing issue in organic chemistry, be it by synthetic catalysts or enzymes of the type Baeyer-Villiger monooxygenases. Herein, we address the challenging problem of switching normal to abnormal BVMO regioselectivity by directed evolution using three linear ketones as su...
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Site-saturation mutagenesis (SSM) has been used in directed evolution of proteins for a long time. As a special form of saturation mutagenesis, it involves individual randomization at a given residue with formation of all 19 amino acids. To date, the most efficient embodiment of SSM is a one-step PCR-based approach using NNK codon degeneracy. Howev...
Article
The extraction of polar molecules from aqueous solution is a challenging task in organic synthesis. 1-Butanol has been used sporadically as an eluent for polar molecules, but it is unclear which molecular features drive its efficiency. Here, we employ free energy simulations to study the partitioning of 15 solutes between water and 1-butanol. The s...
Article
Dimeric disulfide-linked peptides are formed by the regioselective oxidative folding of thiol precursors containing the CX3CX2CX3C tetra-cysteine motif. Here, we investigate the general applicability of this peptide as a dimerization motif for different proteins. By recombinant DNA technology, the peptide CHWECRGCRLVC was loaded with proteins and f...
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Cytochrome P450 monooxygenases play a crucial role in the biosynthesis of many natural products and in the human metabolism of numerous pharmaceuticals. This has inspired synthetic organic and medicinal chemists to exploit them as catalysts in regio- and stereoselective CH-activating oxidation of structurally simple and complex organic compounds su...
Article
The front cover picture shows symbolically a high-throughput silicon-chip-based DNA solid-phase synthetic platform (background) for preparing a library of DNA mutants that encode epoxide hydrolase (center) variants. Massive sequencing revealed that 97 % of the theoretically possible DNA mutants were formed, compared to only 56 % when applying conve...
Article
Chiral alcohols are important building blocks for the production of pharmaceuticals, catalytic access being possible by the use of enzymes, transition metal catalysts or organocatalysts. Herein, we report the use of cytochrome P450-BM3 mutants for the oxidative hydroxylation of 6-iodo-tetralone regio- and enantioselectively at the C4-position with...
Article
Directed evolution of stereo- or regioselective enzymes as catalysts in asymmetric transformations is of particular interest in organic synthesis. Upon evolving these biocatalysts, screening is the bottleneck. To beat the numbers problem most effectively, methods and strategies for building “small but smart” mutant libraries have been developed. He...
Chapter
Directed evolution has emerged as one of the most effective protein engineering methods in basic research as well as in applications in synthetic organic chemistry and biotechnology. The successful engineering of protein activity, allostery, binding affinity, expression, folding, fluorescence, solubility, substrate scope, selectivity (enantio-, ste...
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Directed evolution of limonene epoxide hydrolase (LEH), which catalyzes the hydrolytic desymmetrization reactions of cyclopentene oxide and cyclohexene oxide, results in (R,R)- and (S,S)-selective mutants. Their crystal structures combined with extensive theoretical computations shed light on the mechanistic intricacies of this widely used enzyme....
Article
This essay constitutes a personal assessment of two quite different projects that the author has initiated during the last two decades, both involving combinatorial libraries of catalysts. The first encounter with combinatorial catalysis involved the use of a mixture of two different monodentate ligands in asymmetric transition metal catalysis, esp...
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Baeyer–Villiger monooxygenases (BVMOs) and evolved mutants have been shown to be excellent biocatalysts in many stereoselective Baeyer–Villiger transformations, but industrial applications are rare which is partly due to the insufficient thermostability of BVMOs under operating conditions. In the present study, the substrate scope of the recently d...
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The long-standing problem of achieving high activity of a thermophilic enzyme at low temperatures and short reaction times with little tradeoff in thermostability has been solved by directed evolution, an...
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The possibility of a double Walden inversion mechanism of the fluoracetate dehalogenase FAcD (RPA1163) has been studied by subjecting rac-2-fluoro-2-phenyl acetic acid to the defluorination process. This stereochemical probe led to inversion of configura-tion in a kinetic resolution with an extremely high selectivity factor (E >500), showing that t...
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Correction for ‘Simultaneous engineering of an enzyme’s entrance tunnel and active site: the case of monoamine oxidase MAO-N’ by Guangyue Li et al., Chem. Sci., 2017, 8, 4093–4099.
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A new directed evolution approach is presented to enhance the activity of an enzyme and to manipulate stereoselectivity by focusing iterative saturation mutagenesis (ISM) simultaneously on residues lining the entrance tunnel and the binding pocket. This combined mutagenesis strategy was applied successfully to the monoamine oxidase from Aspergillus...
Article
Enzymes have been used for a long time as catalysts in the asymmetric synthesis of chiral intermediates needed in the production of therapeutic drugs. However, this alternative to man-made catalysts has suffered traditionally from distinct limitations, namely the often observed wrong or insufficient enantio- and/or regioselectivity, low activity, n...
Article
Directed evolution utilizing an unconventional approach to saturation mutagenesis has been applied to cytochrome P450-BM3 as a catalyst in the asymmetric sulfoxidation of 1-thiochroman-4-one and two derivatives thereof with complete chemoselectivity as well as (S)- and (R)-selectivity on an optional basis. Whereas wildtype P450-BM3 shows in the cas...
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The acid/base-catalysed Kemp elimination of 5-nitro-benzisoxazole forming 2-cyano-4-nitrophenol has long served as a design platform of enzymes with non-natural reactions, providing new mechanistic insights in protein science. Here we describe an alternative concept based on redox catalysis by P450-BM3, leading to the same Kemp product via a fundam...
Data
Supplementary Figures, Supplementary Tables, Supplementary Methods and Supplementary References.
Article
Biocatalytic site-selective (regioselective) organic transformations have been practiced for decades, but the traditional limitations of enzymes regarding narrow substrate acceptance and the often observed insufficient degree of selectivity have persisted until recently. With the advent of directed evolution, it is possible to engineer site-selecti...
Chapter
Directed evolution of enzymes provides a prolific source of biocatalysts for asymmetric reactions in organic chemistry and biotechnology. Nowadays, the real challenge in this research area is the development of mutagenesis methods and strategies which ensure the formation of small and highest-quality mutant libraries requiring minimal screening eff...
Article
Enzymes active at low temperature are of great interest for industrial bioprocesses due to their high efficiency at a low energy cost. One of the particularities of naturally evolved cold-active enzymes is their increased enzymatic activity at low temperature, however the low thermostability presented in this type of enzymes is still a major drawba...
Article
We describe herein an air stable phosphorus ligand for both Suzuki-Miyaura and Buchwald-Hartwig palladium-catalyzed cross coupling reactions. The versatility of the Pd(dba)2-di-tert-butyl N,N-diethylphosphoramidite catalytic system is demonstrated for a variety of aryl and heteroaryl halides. This ligand is ‘’bench stable’’ which allows easy cataly...
Article
A series of SN1-type reactions has been studied under various conditions to clarify the role of supercritical carbon dioxide (scCO2) as reaction medium for this kind of transformations. The application of scCO2 did not result in higher yields in any of the experiments in comparison to those under neat conditions or in the presence of other inert co...
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Background Recent methodology development in directed evolution of stereoselective enzymes has shown that various mutagenesis strategies based on saturation mutagenesis at sites lining the binding pocket enable the generation of small and smart mutant libraries requiring minimal screening. Methods In this endeavor, limonene epoxide hydrolase (LEH)...
Article
With the advent of directed evolution of stereoselective enzymes almost 20 years ago and the rapid development of this exciting area of research, the traditional limitations of biocatalysts in organic chemistry have been eliminated. It is now possible to enhance or invert enantioselectivity, broaden the substrate scope and increase the activity of...
Chapter
Enzymes have been used as catalysts in organic chemistry for more than a century. The use of biocatalysis in academia and, particularly, in industry has some of the limitations such as limited substrate scope, insufficient activity, insufficient or wrong stereoselectivity, insufficient or wrong regioselectivity, and insufficient robustness under op...