Extending the biocatalytic scope of regiocomplementary flavin-dependent halogenase enzymes

Flavin-dependent halogenases are potentially valuable biocatalysts for the regioselective halogenation of aromatic compounds. These enzymes, utilising benign inorganic halides, offer potential advantages over traditional non-enzymatic halogenation chemistry that often lacks regiocontrol and requires deleterious reagents. Here we extend the biocatalytic repertoire of the tryptophan halogenases, demonstrating how these enzymes can halogenate a range of alternative aryl substrates. Using structure guided mutagenesis we also show that it is possible to alter the regioselectivity as well as increase the activity of the halogenases with non-native substrates including anthranilic acid; an important intermediate in the synthesis and biosynthesis of pharmaceuticals and other valuable products.     

A High‐Throughput Fluorescence Assay to Determine the Activity of Tryptophan Halogenases

Biocatalytic halogenation with tryptophan halogenases is hampered by severe limitations such as low activity and stability. These drawbacks can be overcome by directed evolution, but for screening large mutant libraries, a facile high‐throughput method is required. Therefore, we developed a quantitative halogenase assay based on a Suzuki–Miyaura cross‐coupling towards the formation of a fluorescent aryltryptophan. The technique was optimized for application in crude E. coli lysate without intermediary purification steps, and was used for quantitatively monitoring the formation of halogenated tryptophans with high specificity by facile fluorescence screening in microtiter plates. This novel screening approach was exploited to engineer a thermostable tryptophan 6‐halogenase. Libraries were constructed by error‐prone PCR and selected for improved thermal resistance simply by fluorogenic cross‐coupling. Our method led to an enzyme variant with substantially increased thermal stability and 2.5‐fold improved activity.     

Halogenase‐Inspired Oxidative Chlorination Using Flavin Photocatalysis

Chlorine gas or electropositive chlorine reagents are used to prepare chlorinated aromatic compounds, which are found in pharmaceuticals, agrochemicals, and polymers, and serve as synthetic precursors for metal‐catalyzed cross‐couplings. Nature chlorinates with chloride anions, FAD‐dependent halogenases, and O₂ as the oxidant. A photocatalytic oxidative chlorination is described based on the organic dye riboflavin tetraacetate mimicking the enzymatic process. The chemical process allows within the suitable arene redox potential window a broader substrate scope compared to the specific activation in the enzymatic binding pocket.     

黄素为辅基的色氨酸卤化酶及其在生物合成色氨酸衍生物中的应用

黄素为辅基的色氨酸卤化酶在催化合成有机氯化物和溴化物过程中的起着非常重要的作用。在这篇论文我们将阐述一种通过在假单胞菌株中表达外源色氨酸卤化酶基因来催化合成3-（2-氨基-4-氯苯基）吡咯的新组合生物合成方法——组合培育法。3-（2-氨基-4-氯苯基）吡咯是分析和诱导性基因突变硝吡咯菌素生物合成途径的重要中间产物。研究表明，这种新的组合培育法可以用于改变和分析一些重要的二级代谢途径。     

Fluorogenic Diversification of Unprotected Bromotryptophan by Aqueous Mizoroki–Heck Cross-Coupling

Fluorescent styryl-tryptophans have been synthesized by a Mizoroki–Heck cross-coupling from unprotected bromotryptophan in aqueous medium showing promising spectrophotometric properties for possible application in fluorescence labelling of biomolecules. Moreover, this strategy permits a modular combination of biocatalytic halogenation by using immobilized FAD-dependent tryptophan halogenases and Pd-mediated chemocatalysis in a multistep one-pot process.     

Evolved Aliphatic Halogenases Enable Regiocomplementary C−H Functionalization of a Pharmaceutically Relevant Compound

Non‐heme iron halogenases are synthetically valuable biocatalysts that are capable of halogenating unactivated sp³‐hybridized carbon centers with high stereo‐ and regioselectivity. The reported substrate scope of these enzymes, however, is limited primarily to the natural substrates and their analogues. We engineered the halogenase WelO5* for chlorination of a martinelline‐derived fragment. Using structure‐guided evolution, a halogenase variant with a more than 290‐fold higher total turnover number and a 400‐fold higher apparent k_cat compared to the wildtype enzyme was generated. Moreover, we identified key positions in the active site that allow direction of the halogen to different positions in the target substrate. This is the first example of enzyme engineering to expand the substrate scope of a non‐heme iron halogenase beyond the native indole‐alkaloid‐type substrates. The highly evolvable nature of WelO5* underscores the usefulness of this enzyme family for late‐stage halogenation.     

A Collaborative Journey towards the Late-Stage Functionalization of Added-Value Chemicals Using Engineered Halogenases

In 2017, two companies, Novartis AG and Syngenta AG, joined forces with the group of Prof. Buller, head of the Competence Center for Biocatalysis (CCBIO), to tackle the challenge of enzymatic late-stage halogenation. This biocatalytic method was considered to provide a more sustainable approach to late-stage halogenation of complex molecules than traditional synthetic approaches. Using machine-learning guided protein engineering, α-ketoglutarate dependent halogenases were evolved into versatile catalysts capable of selectively chlorinating inactivated C−H bonds. Structurally diverse molecules, namely an analogue of martinelline as well as two members of the soraphen natural product family, were enzymatically chlorinated at two distinct positions in a regio- and stereoselective manner, thus demonstrating the synthetic usefulness of such a strategy. As part of our three-year collaboration, flavin-dependent halogenases were also studied.     

Identification and Spectroscopic Characterization of Nonheme Iron(III) Hypochlorite Intermediates

Fe^III–hypohalite complexes have been implicated in a wide range of important enzyme‐catalyzed halogenation reactions including the biosynthesis of natural products and antibiotics and post‐translational modification of proteins. The absence of spectroscopic data on such species precludes their identification. Herein, we report the generation and spectroscopic characterization of nonheme Fe^III–hypohalite intermediates of possible relevance to iron halogenases. We show that Fe^III‐OCl polypyridylamine complexes can be sufficiently stable at room temperature to be characterized by UV/Vis absorption, resonance Raman and EPR spectroscopies, and cryo‐ESIMS. DFT methods rationalize the pathways to the formation of the Fe^III‐OCl, and ultimately Fe^IV?O, species and provide indirect evidence for a short‐lived Fe^II‐OCl intermediate. The species observed and the pathways involved offer insight into and, importantly, a spectroscopic database for the investigation of iron halogenases.     

卤过氧化物酶在绿色卤化反应中的研究进展

卤化反应是一类极其重要的有机合成反应,在实验室研究和化工生产领域占据重要地位.传统卤化反应因存在使用有毒有害试剂、反应缺乏选择性等问题而亟待改进,生物酶催化策略则为突破上述瓶颈提供了可能.自然界已经进化出多种可对有机物中催化引入卤素的卤化酶.酶催化卤化反应的突出优势在于常温常压下,可使用来源温和的卤素进行高效的催化反应.催化范围包括卤化、羟卤化、卤环合和氧化脱羧等多种具有挑战性的反应.鉴于酶催化卤化反应展示出巨大的潜力,从催化活性、酶稳定性、底物浓度、催化范围等几个方面着重介绍了卤过氧化物酶在绿色卤化反应中的最新研究进展,为进一步开发绿色的卤化酶催化卤化反应提供参考.     

Exploring the Biocatalytic Potential of Fe/α-Ketoglutarate-Dependent Halogenases

Freestanding Fe/α-ketoglutarate-dependent halogenases are oxidoreductases that catalyze the installation of halogen atoms into unactivated sp³-hybridized carbon centers with high stereo- and regioselectivity. Since their discovery in 2014, a small number of indole alkaloid and amino acid halogenases have been identified and characterized. First enzyme engineering examples suggest that the accessible substrate range of these enzymes may be expanded through the use of rational enzyme design and directed evolution. Structural investigations of non-heme iron halogenases acting on freestanding as well as tethered substrates are beginning to inform about the principles of the underlying halogenation mechanism.     

RadH: A Versatile Halogenase for Integration into Synthetic Pathways

Flavin‐dependent halogenases are useful enzymes for providing halogenated molecules with improved biological activity, or intermediates for synthetic derivatization. We demonstrate how the fungal halogenase RadH can be used to regioselectively halogenate a range of bioactive aromatic scaffolds. Site‐directed mutagenesis of RadH was used to identify catalytic residues and provide insight into the mechanism of fungal halogenases. A high‐throughput fluorescence screen was also developed, which enabled a RadH mutant to be evolved with improved properties. Finally we demonstrate how biosynthetic genes from fungi, bacteria, and plants can be combined to encode a new pathway to generate a novel chlorinated coumarin “non‐natural” product in E. coli.     

Nicotinamide Adenine Dinucleotides Arrest Photoreduction of Class II DNA Photolyases in FADH˙ State

All light‐sensitive members of the photolyase/cryptochrome family rely on FAD as catalytic cofactor. Its activity is regulated by photoreduction, a light‐triggered electron transfer process from a conserved tryptophan triad to the flavin. The stability of the reduced flavin depends on available external electron donors and oxygen. In this study, we show for the class II photolyase of Methanosarcina mazei , Mm CPDII , that it utilizes physiologically relevant redox cofactors NADH and NADPH for the formation of the semiquinoid FAD in a light‐dependent reaction. Using redox‐inert variants Mm CPDII /W388F and Mm CPDII /W360F, we demonstrate that photoreduction by NADH and NADPH requires the class II ‐specific tryptophan cascade of Mm CPDII . Finally, we confirmed that mutations in the tryptophan cascade can be introduced without any substantial structural disturbances by analyzing crystal structures of Mm CPDII /W388F, Mm CPDII /W360F and Mm CPDII /Y345F.     

Factor analysis of spectroelectrochemical reduction of FAD reveals the pKa of the reduced state and the reduction pathway

The free flavin adenine dinucleotide (FAD) cofactor is known to exhibit a pH‐dependent midpoint potential involving a simultaneous two‐electron transfer step (n = 2). Uv‐vis spectroelectrochemical reductions of FAD at constant pH, ranging from 5 to 9, were recorded and analyzed by factor analysis. Principal factor analysis was used to determine the number of species present at each pH. The results indicate that only two composite forms of FAD are present: the oxidized and the reduced forms. Window factor analysis was used to extract the concentration profiles of the controlling species. The oxidized form was found to be a single pH‐independent species, whereas the reduced form consists of two species. The pH‐dependent spectroscopic changes of reduced FAD were best modeled by a single proton transfer step involving two different ionization states with an apparent pK_a = 6.3. This value compares favorably with those obtained from NMR and from midpoint potential measurements. At pH 6, the reduction of FAD was found to be first order, whereas at pH 9 the reduction is zero order; these observations are explained in terms of the reaction pathway involving xanthine oxidase, its substrate, and the pH. Copyright © 2007 John Wiley & Sons, Ltd.     

Ionic Liquids as Performance Additives for Electroenzymatic Syntheses

Electroenzymatic syntheses combine oxidoreductase‐catalysed reactions with electrochemical reactant supply. The use of ionic liquids as performance additives can contribute to overcoming existing limitations of these syntheses. Here, we report on the influence of different water‐miscible ionic liquids on critical parameters such as conductivity, biocatalyst activity and stability or substrate solubility for three typical electroenzymatic syntheses. In these investigations promising ionic liquids were identified and have been used as additives for batch electrolyses on preparative scale for the three electroenzymatic systems. It was possible to improve the space‐time‐yield for the electrochemical regeneration of NADPH by a factor of three. For an amino acid oxidase catalysed resolution of a methionine racemate with ferrocene‐mediated electrochemical regeneration of the enzyme‐bound cofactor FAD a 50 % increase in space time yield and 140 % increase in catalyst utilisation (TTN) were achieved. Furthermore, for the chloroperoxidase‐catalysed synthesis of (R)‐phenylmethylsulfoxide with electrochemical generation of the required cosubstrate H₂O₂ the space time yield and the catalyst utilisation were improved by a factor of up to 4.2 depending on the ionic liquids used.     

Regioselective Dichlorination of a Non‐Activated Aliphatic Carbon Atom and Phenolic Bismethylation by a Multifunctional Fungal Flavoenzyme

The regioselective functionalization of non‐activated carbon atoms such as aliphatic halogenation is a major synthetic challenge. A novel multifunctional enzyme catalyzing the geminal dichlorination of a methyl group was discovered in Aspergillus oryzae (Koji mold), an important fungus that is widely used for Asian food fermentation. A biosynthetic pathway encoded on two different chromosomes yields mono‐ and dichlorinated polyketides (diaporthin derivatives), including the cytotoxic dichlorodiaporthin as the main product. Bioinformatic analyses and functional genetics revealed an unprecedented hybrid enzyme (AoiQ) with two functional domains, one for halogenation and one for O‐methylation. AoiQ was successfully reconstituted in vivo and in vitro, unequivocally showing that this FADH₂‐dependent enzyme is uniquely capable of the stepwise gem‐dichlorination of a non‐activated carbon atom on a freestanding substrate. Genome mining indicated that related hybrid enzymes are encoded in cryptic gene clusters in numerous ecologically relevant fungi.     

Reengineering a tryptophan halogenase to preferentially chlorinate a direct alkaloid precursor

Installing halogens onto natural products can generate compounds with novel or improved properties. Notably, enzymatic halogenation is now possible as a result of the discovery of several classes of halogenases; however, applications are limited because of the narrow substrate specificity of these enzymes. Here we demonstrate that the flavin-dependent halogenase RebH can be engineered to install chlorine preferentially onto tryptamine rather than the native substrate tryptophan. Tryptamine is a direct precursor to many alkaloid natural products, including approximately 3000 monoterpene indole alkaloids. To validate the function of this engineered enzyme in vivo, we transformed the tryptamine-specific RebH mutant (Y455W) into the alkaloid-producing plant Madagascar periwinkle ( Catharanthus roseus ) and observed the de novo production of the halogenated alkaloid 12-chloro-19,20-dihydroakuammicine. While wild-type (WT) RebH has been integrated into periwinkle metabolism previously, the resulting tissue cultures accumulated substantial levels of 7-chlorotryptophan. Tryptophan decarboxylase, the enzyme that converts tryptophan to tryptamine, accepts 7-chlorotryptophan at only 3% of the efficiency of the native substrate tryptophan, thereby creating a bottleneck. The RebH Y455W mutant circumvents this bottleneck by installing chlorine onto tryptamine, a downstream substrate. In comparison with cultures harboring RebH and WT RebF, tissue cultures containing mutant RebH Y455W and RebF also accumulate microgram per gram fresh-weight quantities of 12-chloro-19,20-dihydroakuammicine but, in contrast, do not accumulate 7-chlorotryptophan, demonstrating the selectivity and potential utility of this mutant in metabolic engineering applications.     

Room‐Temperature ortho‐Alkoxylation and ‐Halogenation of N‐Tosylbenzamides by Using Palladium(II)‐Catalyzed CH Activation

The N‐tosylcarboxamide group can direct the room‐temperature palladium‐catalyzed C?H alkoxylation and halogenation of substituted arenes in a simple and mild procedure. The room‐temperature stoichiometric cyclopalladation of N‐tosylbenzamide was first studied, and the ability of the palladacycle to react with oxidants to form C?X and C?O bonds under mild conditions was demonstrated. The reaction conditions were then adapted to promote room‐temperature ortho‐alkoxylations and ortho‐halogenations of N‐tosylbenzamides using palladium as catalyst. The scope and limitation of both alkoxylations and halogenations was studied and the subsequent functional transformation of the N‐tosylcarboxamide group through nucleophilic additions was evaluated. This methodology offers a simple and mild route to diversely functionalized arenes.     

Planar Chiral Organoboranes with Thermoresponsive Emission and Circularly Polarized Luminescence: Integration of Pillar[5]arenes with Boron Chemistry

Enantiopure molecules based on macrocyclic architecture are unique for applications in enantioselective host‐guest recognition, chiral sensing and asymmetric catalysis. Taking advantage of the chiral transfer from the intrinsically planar chirality of pillar[5]arenes, we herein present an efficient and straightforward approach to achieve early examples of highly luminescent chiral systems ( P5NN and P5BN ). The optical resolution of their enantiomers has been carried out via preparative chiral HPLC, which was ascribed to the molecular functionalization of pillar[5]arenes with π‐conjugated, sterically bulky triarylamine (Ar₃N) as an electron donor and triarylborane (Ar₃B) as an acceptor. This crucial design enabled investigations of the chiroptical properties, including circular dichroism (CD) and circularly polarized luminescence (CPL) in the solid state. The intramolecular charge transfer (ICT) nature in P5BN afforded an interesting thermochromic shift of the emission over a wide temperature range.     

An Ethenoadenine FAD Analog Accelerates UV Dimer Repair by DNA Photolyase

Reduced anionic flavin adenine dinucleotide (FADH^?) is the critical cofactor in DNA photolyase (PL) for the repair of cyclobutane pyrimidine dimers (CPD) in UV‐damaged DNA. The initial step involves photoinduced electron transfer from *FADH^? to the CPD. The adenine (Ade) moiety is nearly stacked with the flavin ring, an unusual conformation compared to other FAD‐dependent proteins. The role of this proximity has not been unequivocally elucidated. Some studies suggest that Ade is a radical intermediate, but others conclude that Ade modulates the electron transfer rate constant (k_ET) through superexchange. No study has succeeded in removing or modifying this Ade to test these hypotheses. Here, FAD analogs containing either an ethano‐ or etheno‐bridged Ade between the AN1 and AN6 atoms (e‐FAD and ε‐FAD, respectively) were used to reconstitute apo‐PL, giving e‐PL and ε‐PL respectively. The reconstitution yield of e‐PL was very poor, suggesting that the hydrophobicity of the ethano group prevented its uptake, while ε‐PL showed 50% reconstitution yield. The substrate binding constants for ε‐PL and rPL were identical. ε‐PL showed a 15% higher steady‐state repair yield compared to FAD‐reconstituted photolyase (rPL). The acceleration of repair in ε‐PL is discussed in terms of an ε‐Ade radical intermediate vs superexchange mechanism.     

Stereochemical control of calixarenes useful as rigid and conformationally diversiform platforms for molecular design

Stereochemical problems and related functions of calix[4]arenes, calix[6]arenes and their chiral derivatives have been reviewed. In p-tert-butylcalix[4]arene (1H₄) and its mono-, di-, tri-, and tetra-O-alkyl derivatives (1H₃R, 1H₂R₂,1HR₃, and 1R₄, respectively), 23 different homologues can exist (including 1H₄). We found that the OH group in the unmodified phenol unit is permeable through the calix[4]arene ring. Thus, several conformational isomers become equivalent after the ‘oxygen-through-the-annulus’ rotation of the OH group and the number of possible homologues is reduced to 13 (including 1H₃). We report in this paper the syntheses of all of these possible conformational isomers using a protection-deprotection method with a benzyl group and metal template effects. On the other hand, all possible chiral isomers that can be derived from calix[4]arene by modification of the OH groups have been systematically classified. Molecular asymmetry can be generated not only by different substituents but also by conformational isomerism. The numbers of chiral isomers are 17 for tetra-O-substituted calix[4]arenes, 9 for tri-O-substituted calix[4]arenes, 3 for di-O-substituted calix[4]arenes, and 0 for mono-O-substituted calix[4]arenes. Chiral calix[4]arenes can also be designed by the introduction of a substituent into the m-position of a phenol unit or by the use of a dissymmetric ‘stapling reaction’ in proximal phenol units. In p-tert-butylcalix[6]arene, the conformational behaviour is totally different from that in p-tert-butylcalix[4]arene. A large degree of conformational freedom remains in the framework, and both ‘oxygen-through-the-annulus rotation’ and ‘para-substituent-through-the-annulus rotation’ can take place. However, when metal cations are bound to calix[6]aryl esters, the conformation is changed to a cone type. Bridging and capping are powerful methods to immobilize the conformation of calix[6]arenes. In addition, definitive evidence for ring immobilization was obtained from the absence of racemization in the chiral calix[6]arene. A successful example of chiral recognition for α-amino acid derivatives was achieved by using chiral homooxacalix[3]arene which has ‘pseudo C₂ symmetry’. These examples indicate that calixarenes serve as rigid and conformationally diversiform platforms for the design of novel functional supramolecules.