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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°C in benzonitrile affords the crystalline multimetal porphyrin system TPPCu/TPPCr in good yield. The X-ray single crystal structural analysis reveals a random distribution of TPPCu and TPPCr, with a Cu:Cr ratio of 71(2):29(2)%. Exploratory DFT calculations of TPPCu/TPPCr indicate little if any electron transfer. In contrast, calculations of a hypothetical cationic TPPCu/TPPRu system indicates the possibility of self-doping.

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 can resist unfolding with concomitant detrimental aggregation. Fourier transform (FT) allows us to take into account the order of the protein sequence and the nonlinear interactions between positions, and thus to grasp epistatic phenomena. The innov'SAR approach is interpolative, extrapolative and makes outside‐the‐box, predictions not found in other state‐of‐the‐art ML or deep learning approaches. Equally significant is the finding that our approach to ML in the present context, flanked by advanced molecular dynamics simulations, uncovers the connection between epistatic mutational interactions and protein robustness., A quick learner: Machine learning based on the innov'SAR algorithm leads to efficient selection of highly robust limonene epoxide hydrolase mutants with enhanced unfolding stability and resistance to aggregation by recognizing epistatic mutational interactions.

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 C7 is spatially far away from any functional groups that could direct C−H activation, these transformations are not readily accessible using modern synthetic organic techniques. Reported here are P450‐BM3 mutants that catalyze the oxidative hydroxylation of six different steroids with pronounced C7 regioselectivities and β stereoselectivities, as well as high activities. These challenging transformations were achieved by a focused mutagenesis strategy and application of a novel technology for protein library construction based on DNA assembly and USER (Uracil‐Specific Excision Reagent) cloning. Upscaling reactions enabled the purification of the respective steroidal alcohols in moderate to excellent yields. The high‐resolution X‐ray structure and molecular dynamics simulations of the best mutant unveil the origin of regio‐ and stereoselectivity., One mutant, six targets: The regio‐ and stereoselective C−H activation for hydroxylation of six different steroids with formation of C7β alcohols was accomplished using a single P450 mutant evolved by protein library construction based on a special DNA assembly and cloning procedure. The C7β steroidal alcohols, not readily accessible by synthetic reagents or catalysts, are of intense interest as therapeutic drugs.

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 intermediates. In the present study, we consider the transformation of isosteric substrates via sulfoxidation and oxidative hydroxylation, respectively, catalyzed by regio- and enantioselective mutants of P450-BM3 which were constructed by directed evolution. 1-Thiochromanone and 1-tetralone were used as the isosteric substrates because, unlike previous studies involving fully flexible compounds such as thia-fatty acids and fatty acids, respectively, these compounds are rigid and cannot occur in a multitude of different conformations and binding modes in the large P450-BM3 binding pocket. The experimental results comprising activity and regio- and enantioselectivity, flanked by molecular dynamics computations within a time scale of 300 ns and QM/MM calculations of transition-state energies, unequivocally show that heme-Fe=O (Cpd I) is the common catalytically active intermediate in both sulfoxidation and oxidative hydroxylation.

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 prochiral precursors in enzyme catalyzed reactions has been systematically neglected. We designate these elusive species as fleeting chiral intermediates and analyze such crucial questions as "Do such intermediates occur in homochiral form?" If so, what is the absolute configuration, and why did Nature choose that particular stereoisomeric form, even when the isolable final product may be achiral? Does the absolute configuration of a chiral product depend in any way on the absolute configuration of the fleeting chiral precursor? How does this affect the catalytic proficiency of the enzyme? If these issues continue to be unexplored, then an understanding of the mechanisms of many enzyme types remains incomplete. We have systematized the occurrence of these chiral intermediates according to their structures and enzyme types. This is followed by critical analyses of selected case studies and by final conclusions and perspectives. We hope that the fascinating concept of fleeting chiral intermediates will attract the attention of scientists, thereby opening an exciting new research field.

Directed evolution of stereo-, regio-, and chemoselective enzymes has enriched the toolbox of synthetic organic chemistry. Among the different gene mutagenesis techniques, saturation mutagenesis (S...

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 Protein Data Bank, can be used to identify the flexibility of atoms, side chains, or even whole regions. This requires a technique for obtaining normalized B-factors. Since then the exploitation of B-factors has been extensively elaborated and applied in a variety of studies with quite different goals, all having in common the identification and interpretation of rigidity, flexibility, and/or internal motion which are crucial in enzymes and in proteins in general. Importantly, this review includes a discussion of limitations and possible pitfalls when using B-factors. A second research area, which likewise exploits B-factors, is also reviewed, namely, the development of the so-called B-FIT-directed evolution method for increasing the thermostability of enzymes as catalysts in organic chemistry and biotechnology. In both research areas, a maximum of structural and mechanistic insights is gained when B-factor analyses are combined with other experimental and computational techniques.

Transition 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 different approach, namely, embedding achiral ligand/metal moieties covalently or noncovalently in protein hosts with formation of artificial metalloenzymes. While structurally fascinating, this approach led in each case only to a single (bio)catalyst, with its selectivity and activity being a matter of chance. In order to solve this fundamental problem, my group proposed in 2000-2002 the idea of directed evolution of artificial metalloenzymes. In earlier studies, we had already demonstrated that directed evolution of enzymes constitutes a viable method for enhancing and inverting the stereoselectivity of enzymes as catalysts in organic chemistry. We speculated that it should also be possible to manipulate selectivity and activity of artificial metalloenzymes, which would provide organic chemists with a tool for optimizing essentially any transition metal catalyzed reaction type. In order to put this vision into practice, we first turned to the Whitesides system for artificial metalloenzyme formation, comprising a biotinylated diphosphine/Rh moiety, which is anchored noncovalently to avidin or streptavidin. Following intensive optimization, proof of principle was finally demonstrated in 2006, which opened the door to a new research area. This personal Account critically assesses these early studies as well as subsequent efforts from my group focusing on different protein scaffolds, and includes briefly some of the most important current contributions of other groups. Two primary messages emerge: First, since organic chemists continue to be extremely good at designing and implementing man-made transition metal catalysts, often on a large scale, those scientists that are active in the equally intriguing field of directed evolution of artificial metalloenzymes should be moderate when generalizing claims. All factors required for a truly viable catalytic system need to be considered, especially activity and ease of upscaling. Second, the most exciting and thus far very rare cases of directed evolution of artificial metalloenzymes are those that focus on selective transformations that are not readily possible using state of the art transition metal catalysts.

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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 landscape for a cytochrome P450 monooxygenase that was engineered for the regio- and stereoselective hydroxylation of a steroid. We develop a computational program to automatically quantify non-additive effects among all possible mutational pathways, finding pervasive cooperative signs and magnitude epistasis on multiple catalytic traits. By using quantum mechanics and molecular dynamics simulations, we show that these effects are modulated by long-range interactions in loops, helices and β-strands that gate the substrate access channel allowing for optimal catalysis. Our work highlights the importance of conformational dynamics on epistasis in an enzyme involved in secondary metabolism and offers insights for engineering P450s., Connecting conformational dynamics and epistasis has so far been limited to a few proteins and a single fitness trait. Here, the authors provide evidence of positive epistasis on multiple catalytic traits in the evolution and dynamics of engineered cytochrome P450 monooxygenase, offering insights for in silico protein design.

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 hypothetical cationic TPPCu/TPPRu system, in contrast to TPPCu/TPPCr.

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 libraries have been developed, including the combinatorial active-site saturation test (CAST) which involves saturation mutagenesis at appropriate residues surrounding the binding pocket, and iterative saturation mutagenesis (ISM). Nevertheless, even CAST/ISM mutant libraries require a formidable screening effort. Thus far, rational design as the alternative protein engineering technique has had only limited success when aiming for stereoselectivity. Here, we highlight a recent methodology dubbed focused rational iterative site-specific mutagenesis (FRISM), in which mutant libraries are not involved. It makes use of the tools that were previously employed in traditional rational enzyme design, but, inspired by CAST/ISM, the process is performed in an iterative manner. Only a few predicted mutants need to be screened, a fast process which leads to the identification of highly enantioselective and sufficiently active mutants.

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 prevented ecologically and economically viable processes from being developed. In the present study, directed evolution was applied to the thermostable alcohol dehydrogenase TbSADH in the successful quest to obtain mutants that are effective in the dynamic reductive kinetic resolution (DYRKR) of racemic arylpropanals. Using rac-2-phenyl-1-propanal in a model reaction, (S)- and (R)-selective mutants were evolved which catalyzed DYRKR of this racemic substrate with formation of the respective (S)- and (R)-alcohols in essentially enantiomerically pure form. This was achieved on the basis of an unconventional form of iterative saturation mutagenesis (ISM) at randomization sites lining the binding pocket using a reduced amino acid alphabet. The best mutants were also effective in the DYRKR of several other structurally related racemic aldehydes.

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 now report that this long-standing dogma no longer pertains, provided additional mutations are introduced by directed evolution. By employing Candida antarctica lipase B (CALB) as the model enzyme with the Ser-His-Asp catalytic triad, a highly active cysteine-lipase having a Cys-His-Asp catalytic triad and additional mutations W104V/A281Y/A282Y/V149G can be evolved, showing a 40-fold higher catalytic efficiency than wild-type CALB in the hydrolysis of 4-nitrophenyl benzoate, and tolerating bulky substrates. Crystal structures, kinetics, MD simulations and QM/MM calculations reveal dynamic features and explain all results, including the preference of a two-step mechanism involving the zwitterionic pair Cys105−/His224+ rather than a concerted process., Candida antarctica lipase B (CALB) is a serine lipase. Here, the authors use directed evolution to exchange serine with cysteine in the catalytic triad of the enzyme, thereby obtaining a highly active CALB variant that — unlike the wild type — accommodates bulky substrates.

The quality and efficiency of any PCR-based mutagenesis technique may not be optimal due to, among other things, 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-free manner. First, it involves plasmid digestion by utilizing a complex of Cas9 with specific single guide RNA (sgRNA) followed by degradation with T5 exonuclease to generate a 15 nt homologous region. Second, primers containing the desired mutations are annealed to form the double-stranded DNA fragments, which are then ligated into the linearized plasmid. In theory, neither the size of the target plasmid nor the unavailable restriction enzyme site poses any problems that may arise in traditional techniques. In this study, single and multiple site-directed mutagenesis were successfully performed even for a large size plasmid (up to 9.0 kb). Moreover, a PCR-free site-saturation mutagenesis library on single site and two adjacent sites of a green fluorescent protein was also generated with promising results. This demonstrates the great potential of the ICM system for creating high-quality mutant libraries in directed evolution as an alternative to PCR-based saturation mutagenesis, thus facilitating research on synthetic biology.

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 simulations demonstrate that the high affinity of polar molecules to the wet 1-butanol phase is associated with its nanostructure. Small inverse micelles of water are able to accommodate polar solutes and locally mimic an aqueous environment. We verify the simulations based on partition coefficients between water and 1-octanol, and include a blind prediction of the water/1-butanol partition coefficient of cyclohexane-1,2-diol. The calculations are in excellent agreement with experiment, reaching root-mean-square deviations below 0.7 kcal/mol. Actual extractions of cyclohexane-1,2-diol from buffer solutions that mimic cell lysates and suspensions in biocatalytic reactions further exemplify our findings. The yields highlight that extractions with 1-butanol can be significantly more efficient than the conventional protocol based on ethyl acetate.

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. From the computed activation energies of various pathways, we discover the underlying stereochemistry for favorable reactions. Surprisingly, some of the most enantioselective mutants that rapidly convert cyclohexene oxide do not catalyze the analogous transformation of the structurally similar cyclopentene oxide, as shown by additional X-ray structures of the variants harboring this slightly smaller substrate. We explain this puzzling observation on the basis of computational calculations which reveal a disrupted alignment between nucleophilic water and cyclopentene oxide due to the pronounced flexibility of the binding pocket. In contrast, in the stereoselective reactions of cyclohexene oxide, reactive conformations are easily reached. The unique combination of structural and computational data allows insight into mechanistic details of this epoxide hydrolase and provides guidance for future protein engineering in reactions of structurally different substrates.

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 wild-type P450-BM3 shows in the case of the parent compound poor enantioselectivity in slight favor of the (S)-sulfoxide (er=75:25), (S)-selectivity was enhanced to er=93:7, while reversal of enantioselectivity favoring the (R)-sulfoxide was also achieved (er=7:93). Two derivatives of the parent substrate underwent similar stereoselective sulfoxidation reactions. Sulfoxides of this type are of potential pharmaceutical interest. This biocatalytic approach nicely complements synthetic methods.

A series of SN 1-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 compressed gases. High-pressure UV/Vis spectroscopic measurements were carried out to quantify the degree of carbocation formation of a highly SN 1-active alkyl halide as a function of the applied solvent. No measureable concentration of carbocations could be detected in scCO2 , just like in other low polarity solvents. Taken together, these results do not support the previously claimed activating effect via enhanced SN 1 ionization due to the quadrupolar moment of the supercritical fluid.

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 catalyst preparation. Moreover, even at low catalyst loadings (0.5 %), excellent yields are usually achieved.

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 discovered thermally stable BVMO, TmCHMO from Thermocrispum municipale, was studied. This revealed that the wild-type (WT) enzyme catalyzes the oxidation of a variety of structurally different ketones with notable activity and enantioselectivity, including the desymmetrization of 4-methylcyclohexanone (99% ee, S). In order to induce the reversal of enantioselectivity of this reaction as well as the transformations of other substrates, directed evolution based on iterative saturation mutagenesis (ISM) was applied, leading to (R)-selectivity (94% ee) without affecting the thermostability of the biocatalyst.

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 niger (MAO-N) in the reaction of sterically demanding substrates which are of interest in the synthesis of chiral pharmaceuticals based on the benzo-piperidine scaffold. Reversal of enantioselectivity of Turner-type deracemization was achieved in the synthesis of (S)-1,2,3,4-tetrahydro-1-methyl-isoquinoline, (S)-1,2,3,4-tetrahydro-1-ethylisoquinoline and (S)-1,2,3,4-tetrahydro-1-isopropylisoquinoline. Extensive molecular dynamics simulations indicate that the altered catalytic profile is due to increased hydrophobicity of the entrance tunnel acting in concert with the altered shape of the binding pocket.

Enzymatic stereodivergent synthesis to access all possible product stereoisomers bearing multiple stereocenters is relatively undeveloped, 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 individually accessing all four stereoisomers would be ideal. Although significant success has been achieved in directed evolution of enzymes in general, stereodivergent engineering of one enzyme into four highly stereocomplementary variants for obtaining the full complement of stereoisomers bearing multiple stereocenters remains a challenge. Using Candida antarctica lipase B (CALB) as a model, we report the protein engineering of this enzyme into four highly stereocomplementary variants needed for obtaining all four stereoisomers in transesterification reactions between racemic acids and racemic alcohols in organic solvents. By generating and screening less than 25 variants of each isomer, we achieved >90% selectivity for all of the four possible stereoisomers in the model reaction. This difficult feat was accomplished by developing a strategy dubbed "focused rational iterative site-specific mutagenesis" (FRISM) at sites lining the enzyme's binding pocket. The accumulation of single mutations by iterative site-specific mutagenesis using a restricted set of rationally chosen amino acids allows the formation of ultrasmall mutant libraries requiring minimal screening for stereoselectivity. The crystal structure of all stereodivergent CALB variants, flanked by MD simulations, uncovered the source of selectivity.

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 reactions, they also ensure an internal in situ H2O2 recycle mechanism that avoids its accumulation and thus prevents possible toxic effects. By directed evolution of P450-BM3 as the catalyst in the enantioselective epoxidation of the styrene-intermediate, formed from 3-phenyl propionic acid, and the epoxide hydrolase ANEH for final hydrolytic ring opening, ( R)-phenyl glycol and 9 derivatives thereof were synthesized from the respective carboxylic acids in one-pot processes with high enantioselectivity.

Directed evolution of stereo-, regio-, and chemoselective enzymes constitutes a unique way to generate biocatalysts for synthetically interesting transformations in organic chemistry and biotechnology. In order for this protein engineering technique to be efficient, fast, and reliable, and also of relevance to synthetic organic chemistry, methodology development was and still is necessary. Following a description of early key contributions, this review focuses on recent developments. It includes optimization of molecular biological methods for gene mutagenesis and the design of efficient strategies for their application, resulting in notable reduction of the screening effort (bottleneck of directed evolution). When aiming for laboratory evolution of selectivity and activity, second-generation versions of Combinatorial Active-Site Saturation Test (CAST) and Iterative Saturation Mutagenesis (ISM), both involving saturation mutagenesis (SM) at sites lining the binding pocket, have emerged as preferred approaches, aided by in silico methods such as machine learning. The recently proposed Focused Rational Iterative Site-specific Mutagenesis (FRISM) constitutes a fusion of rational design and directed evolution. On-chip solid-phase chemical gene synthesis for rapid library construction enhances library quality notably by eliminating undesired amino acid bias, the future of directed evolution?

Biocatalytic cascade reactions using isolated stereoselective enzymes or whole cells in one-pot processes lead to value-added chiral products in a single workup. The concept has been restricted mainly to starting materials and intermediate products that are accepted by the respective wild-type enzymes. In the present study, we exploited directed evolution as a means to create E. coli whole cells for regio- and stereoselective cascade sequences that are not possible using man-made catalysts. The approach is illustrated using P450-BM3 in combination with appropriate alcohol dehydrogenases as catalysts in either two-, three-, or four-step cascade reactions starting from cyclohexane, cyclohexanol, or cyclohexanone, respectively, leading to either (R,R)-, (S,S)-, or meso-cyclohexane-1,2-diol. The one-pot conversion of cyclohexane into (R)- or (S)-2-hydroxycyclohexanone in the absence of ADH is also described.