Enantiomeric Substrates Research Articles

Enzymatic Resolution of cis‐Dimethyl‐1‐acetylpiperidine‐2,3‐dicarboxylate for the Preparation of a Moxifloxacin Building Block

AbstractThis work presents the enantioselective resolution of a water‐soluble racemic mixture of cis‐dimethyl 1‐acetylpiperidine‐2,3‐dicarboxylate catalyzed by Candida antarctica lipase B. The separation of the carboxylic acid product in its (2R,3S) configuration from non‐reacted substrate in the (2S,3R) configuration was easily obtained by organic phase extraction. The latter molecule can be used as an enantiomerically pure precursor in the synthesis of (4aS,7aS)‐octahydro‐1H‐pyrrolo[3,4‐b]pyridine, an expensive building block in the production of the antibiotic Moxifloxacin. On the other hand, the hydrolyzed product in the (2R,3S) configuration can be used for the preparation of a neuromediator analogue. The lipase demonstrated enantioselectivity towards the (2R,3S) substrate enantiomer and regioselectivity towards the ester in position 3 of the molecule. Studies of hydrolysis kinetics and the use of the immobilized enzyme were performed to evaluate its industrial application.

Photochemical Deracemization at sp3-Hybridized Carbon Centers via a Reversible Hydrogen Atom Transfer.

A photochemical deracemization of 5-substituted 3-phenylimidazolidine-2,4-diones (hydantoins) is reported (27 examples, 69%-quant., 80-99% ee). The reaction is catalyzed by a chiral diarylketone which displays a two-point hydrogen bonding site. Mechanistic evidence (DFT calculations, radical clock experiments, H/D labeling) suggests the reaction to occur by selective hydrogen atom transfer (HAT). Upon hydrogen binding, one substrate enantiomer displays the hydrogen atom at the stereogenic center to the photoexcited catalyst allowing for a HAT from the substrate and eventually for its conversion into the product enantiomer. The product enantiomer is not processed by the catalyst and is thus enriched in the photostationary state.

An ATP-responsive metabolic cassette comprised of inositol tris/tetrakisphosphate kinase 1 (ITPK1) and inositol pentakisphosphate 2-kinase (IPK1) buffers diphosphosphoinositol phosphate levels

Inositol polyphosphates are ubiquitous molecular signals in metazoans, as are their pyrophosphorylated derivatives that bear a so-called ‘high-energy’ phosphoanhydride bond. A structural rationale is provided for the ability of Arabidopsis inositol tris/tetrakisphosphate kinase 1 to discriminate between symmetric and enantiomeric substrates in the production of diverse symmetric and asymmetric myo-inositol phosphate and diphospho-myo-inositol phosphate (inositol pyrophosphate) products. Simple tools are applied to chromatographic resolution and detection of known and novel diphosphoinositol phosphates without resort to radiolabeling approaches. It is shown that inositol tris/tetrakisphosphate kinase 1 and inositol pentakisphosphate 2-kinase comprise a reversible metabolic cassette converting Ins(3,4,5,6)P4 into 5-InsP7 and back in a nucleotide-dependent manner. Thus, inositol tris/tetrakisphosphate kinase 1 is a nexus of bioenergetics status and inositol polyphosphate/diphosphoinositol phosphate metabolism. As such, it commands a role in plants that evolution has assigned to a different class of enzyme in mammalian cells. The findings and the methods described will enable a full appraisal of the role of diphosphoinositol phosphates in plants and particularly the relative contribution of reversible inositol phosphate hydroxykinase and inositol phosphate phosphokinase activities to plant physiology.

Thiourea Derivative of 2-[(1R)-1-Aminoethyl]phenol: A Flexible Pocket-like Chiral Solvating Agent (CSA) for the Enantiodifferentiation of Amino Acid Derivatives by NMR Spectroscopy.

Thiourea derivatives of 2-[(1R)-1-aminoethyl]phenol, (1S,2R)-1-amino-2,3-dihydro-1H-inden-2-ol, (1R,2R)-(1S,2R)-1-amino-2,3-dihydro-1H-inden-2-ol, and (R)-1-phenylethanamine have been compared as chiral solvating agents (CSAs) for the enantiodiscrimination of derivatized amino acids using nuclear magnetic resonance (NMR) spectroscopy. Thiourea derivative, prepared by reacting 2-[(1R)-1-aminoethyl]phenol with benzoyl isothiocyanate, constitutes an effective CSA for the enantiodiscrimination of N-3,5-dinitrobenzoyl (DNB) derivatives of amino acids with free or derivatized carboxyl functions. A base additive 1,4-diazabicyclo[2.2.2]octane(DABCO)/N,N-dimethylpyridin-4-amine (DMAP)/NBu4OH) is required both to solubilize amino acid derivatives with free carboxyl groups in CDCl3 and to mediate their interaction with the chiral auxiliary to attain efficient differentiation of the NMR signals of enantiomeric substrates. For ternary systems CSA/substrate/DABCO, the chiral discrimination mechanism has been ascertained through the NMR determination of complexation stoichiometry, association constants, and stereochemical features of the diastereomeric solvates.

Efficient synthesis of L-phosphinothricin using a novel aminoacylase mined from Stenotrophomonas maltophilia

L-phosphinothricin (L-PPT) is a competitive and environmentally friendly herbicide. To develop an efficient approach for synthesis of l-PPT, a kinetic resolution route with a novel aminoacylase (SmAcy) mined from Stenotrophomonas maltophilia using N-acetyl-PPT as a substrate was first constructed. This SmAcy exhibited high hydrolytic activity and excellent enantioselectivity (E > 200) toward N-acetyl-PPT. Optically pure l-PPT (> 99.9 % eep) was acquired with high conversion (> 49 %) within 4 h by the whole cells. On the basis of the docking analysis, a main reason for high enantioselectivity (E > 200) of SmAcy towards l-enantiomer would be that the D-N-acetyl-PPT cannot interact with the key general acid-base residue and the metal ions. A low-cost and simple preparation process of the substrate from commercially available racemic PPT for production of L-PPT was provided. A chemical racemization method of the unreacted D-enantiomer of substrate was also provided to recycle the unwanted substrate enantiomer. This study provides a potential route for the industrial production of L-PPT.

Dynamic covalent kinetic resolution

ABSTRACTImplemented with the highly efficient concept of Dynamic Kinetic Resolution (DKR), dynamic covalent chemistry can be a useful strategy for the synthesis of enantioenriched compounds. This gives rise to dynamic covalent kinetic resolution (DCKR), a subset of DKR that over the last decades has emerged as increasingly fruitful, with many applications in asymmetric synthesis and catalysis. All DKR protocols are composed of two important parts: substrate racemization and asymmetric transformation, which can lead to yields of >50% with good enantiomeric excesses (ee) of the products. In DCKR systems, by utilizing reversible covalent reactions as the racemization strategy, the substrate enantiomers can be easily interconverted without the presence of any racemase or transition metal catalyst. Enzymes or other chiral catalysts can then be adopted for the resolution step, leading to products with high enantiopurities. This tutorial review focuses on the development of DCKR systems, based on different reversible reactions, and their applications in asymmetric synthesis.

Molecular basis for enantioselective herbicide degradation imparted by aryloxyalkanoate dioxygenases in transgenic plants.

The synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) is an active ingredient of thousands of commercial herbicides. Multiple species of bacteria degrade 2,4-D via a pathway initiated by the Fe(II) and α-ketoglutarate (Fe/αKG)-dependent aryloxyalkanoate dioxygenases (AADs). Recently, genes encoding 2 AADs have been deployed commercially in herbicide-tolerant crops. Some AADs can also inactivate chiral phenoxypropionate and aryloxyphenoxypropionate (AOPP) herbicides, albeit with varying substrate enantioselectivities. Certain AAD enzymes, such as AAD-1, have expanded utility in weed control systems by enabling the use of diverse modes of action with a single trait. Here, we report 1) the use of a genomic context-based approach to identify 59 additional members of the AAD class, 2) the biochemical characterization of AAD-2 from Bradyrhizobium diazoefficiens USDA 110 as a catalyst to degrade (S)-stereoisomers of chiral synthetic auxins and AOPP herbicides, 3) spectroscopic data that demonstrate the canonical ferryl complex in the AAD-1 reaction, and 4) crystal structures of representatives of the AAD class. Structures of AAD-1, an (R)-enantiomer substrate-specific enzyme, in complexes with a phenoxypropionate synthetic auxin or with AOPP herbicides and of AAD-2, which has the opposite (S)-enantiomeric substrate specificity, reveal the structural basis for stereoselectivity and provide insights into a common catalytic mechanism.

First biological conversion of chiral heterophosphonate derivative – Scaling and paths of conversion discussion

Presented work describes the first approach for the biocatalytic resolution of racemic mixtures of heterophosphonate derivative. Penicillium funiculosum and Rhodotorula mucilaginosa were successfully applied for the biological conversion of racemic mixture of 1-amino-1-(3′-pyridyl)methylphosphonic acid 3. Both microorganisms carried out the kinetically driven process leading to conversion of one from the substrate enantiomers, leaving the second one unreacted. Application of R. mucilaginosa allowed obtaining pure enantiomer of the substrate (yield 100%, e.e 100% - unreacted isomer) after 24 h of biotransformation of 3 in the laboratory scale process (Method E), applying biocatalyst pre-treatment step − 24 h of starvation. In case of other biocatalyst, application of whole cells of P. funiculosum in laboratory scale process, also resulted in conversion of the racemic mixture of substrate 3via oxidative deamination into ketone derivative, which was then bioreduced (second step of the process) into 1-hydroxy-1-(3′-pyridyl)methylphosphonic acid 4. This time two products were isolated: unreacted substrate and hydroxy compound 4. Conversion degree ranged from 30% (standard procedure, method A) to even 70% (with extra addition of sodium pyruvate - method B2). However, in this case, bioconversion was not enantioselective – products: amino- and hydroxyderivative were obtained as racemic mixtures. Both biocatalysts were also tested towards the scaling so other biocatalytic procedures were introduced - with immobilized fungal mycelium. In case of Rhodotorula mucilaginosa this approach failed (data not shown) but Penicillium funiculosum turned out to be active and also selective. Thus, application of this biocatalyst in the half-preparative scale, continuous-flow bioprocess (Method C2) resulted in the obtaining of pure S-3 (100% e.e.) isomer with the 100% of conversion degree, without any side products. Recorded NMR spectra allowed confirming the reaction progress and its selectivity and also postulating possible mechanism of conversion.

α-Amine Desaturation of d-Arginine by the Iron(II)- and 2-(Oxo)glutarate-Dependent l-Arginine 3-Hydroxylase, VioC.

When challenged with substrate analogues, iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases can promote transformations different from those they enact upon their native substrates. We show here that the Fe/2OG enzyme, VioC, which is natively an l-arginine 3-hydroxylase, catalyzes an efficient oxidative deamination of its substrate enantiomer, d-Arg. The reactant complex with d-Arg retains all interactions between enzyme and substrate functional groups, but the required structural adjustments and opposite configuration of C2 position this carbon more optimally than C3 to donate hydrogen (H•) to the ferryl intermediate. The simplest possible mechanism, C2 hydroxylation followed by elimination of ammonia, is inconsistent with the demonstrated solvent origin of the ketone oxygen in the product. Rather, the reaction proceeds via a hydrolytically labile C2-iminium intermediate, demonstrated by its reductive trapping in solution with NaB2H4 to produce racemic [2H]Arg. Of two alternative pathways to the iminium species, C2 hydroxylation followed by dehydration versus direct desaturation, the latter possibility appears to be more likely, because the former mechanism would be expected to result in detectable incorporation of 18O from 18O2. The direct desaturation of a C-N bond implied by this analysis is analogous to that recently posited for the reaction of the l-Arg 4,5-desaturase, NapI, thus lending credence to the prior mechanistic proposal. Such a pathway could also potentially be operant in a subset of reactions catalyzed by Fe/2OG N-demethylases, which have instead been purported to enact C-N bond cleavage by methyl hydroxylation and elimination of formaldehyde.

Dynamic Kinetic Resolution of Phosphinic Acid Derivatives via Nucleophilic­ Substitution at Phosphorus Center

Reaction of racemic phosphinic acid derivatives with chiral alcohols proceeds with predominant formation of one diastereomer. The highest level of enrichment has been obtained for transesterfication of racemic methyl benzylphenylphosphinate (64% de). The outcome of the reaction depends on both the structure of chiral alcohol and the starting organophosphorus compound. The results strongly suggest that the nature of the observed phenomena is not a classical equilibration of intermediates found in dynamic kinetic resolution process but is a result of a different reactivity of both enantiomers of racemic substrate towards the same chiral nucleophile.

Prediction of enantioselectivity of lipase catalyzed kinetic resolution using umbrella sampling

Enantiopure intermediates are preferred for drug synthesis in pharmaceutical industry. Lipases are widely used for chiral resolution of optically active compounds based on kinetic resolution. In the kinetic resolution, two enantiomers react with different rates in presence of a chiral catalyst or reagent, resulting in obtaining one enantiomer of great excess compared to other. The enantiomeric ratio (E) is closely related to the free energy difference (ΔΔG) of the activated state of substrate enantiomers. Molecular dynamic (MD) simulations with umbrella sampling technique can be used for estimation of activation free energy change of enantiomers. In this work, lipase-catalyzed transesterification of racemic alcohols with single and double hydroxyl groups have been studied. The umbrella sampling studies have been carried out for Candida rugosa lipase and Burkholderia cepacia lipase in n-hexane. The distance between serine residue in the catalytic triad and the ligand has been considered as a reaction coordinate and various conformations have been selected for MD simulation. The E value has been estimated based on free energy change from umbrella sampling. The estimated E values are in good agreement with experimental data. The work highlights changes in lipase conformation in n-hexane, ligand-protein interaction as well as free energy curve as a function of the reaction coordinate.

Remote Cooperative Group Strategy Enables Ligands for Accelerative Asymmetric Gold Catalysis.

An accelerative asymmetric gold catalysis is achieved for the first time via chiral ligand metal cooperation. An asymmetrically positioned remote amide group in the designed chiral binaphthyl-based ligand plays the essential role of a general base catalyst and selectively accelerates the cyclizations of 4-allen-1-ols into one prochiral allene face. The reactions are mostly highly enantioselective with achiral substrates, and due to the accelerated nature of the catalysis catalyst loadings as low as 100 ppm are allowed. With a pre-existing chiral center at any of the backbone sp3-carbons, the reaction remained highly efficient and most importantly maintained excellent allene facial selectivities regardless of the substrate stereochemistry. By using different combinations of ligand and substrate enantiomers, it is now possible to access all four stereoisomers of versatile 2-vinyltetrahydrofurans with exceedingly high selectivity. The underpinning design of this chemistry reveals a novel and conceptually distinctive strategy to tackle challenging asymmetric gold catalysis, which to date has relied on decelerative asymmetric steric hindrance approaches.

Controlling the Regioselectivity of Baeyer-Villiger Monooxygenases by Mutation of Active-Site Residues.

Baeyer-Villiger monooxygenase (BVMO)-mediated regiodivergent conversions of asymmetric ketones can lead to the formation of "normal" or "abnormal" lactones. In a previous study, we were able to change the regioselectivity of a BVMO by mutation of the active-site residues to smaller amino acids, which thus created more space. In this study, we demonstrate that this method can also be used for other BVMO/substrate combinations. We investigated the regioselectivity of 2-oxo-Δ3 -4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase from Pseudomonas putida (OTEMO) for cis-bicyclo[3.2.0]hept-2-en-6-one (1) and trans-dihydrocarvone (2), and we were able to switch the regioselectivity of this enzyme for one of the substrate enantiomers. The OTEMO wild-type enzyme converted (-)-1 into an equal (50:50) mixture of the normal and abnormal products. The F255A/F443V variant produced 90 % of the normal product, whereas the W501V variant formed up to 98 % of the abnormal product. OTEMO F255A exclusively produced the normal lactone from (+)-2, whereas the wild-type enzyme was selective for the production of the abnormal product. The positions of these amino acids were equivalent to those mutated in the cyclohexanone monooxygenases from Arthrobacter sp. and Acinetobacter sp. (CHMOArthro and CHMOAcineto ) to switch their regioselectivity towards (+)-2, which suggests that there are hot spots in the active site of BVMOs that can be targeted with the aim to change the regioselectivity.

Roles of the Active Site Zn(II) and Residues in Substrate Discrimination by Threonyl-tRNA Synthetase: An MD and QM/MM Investigation.

Threonyl-tRNA synthetase (ThrRS) is a Zn(II) containing enzyme that catalyzes the activation of threonine and its subsequent transfer to the cognate tRNA. This process is accomplished with remarkable fidelity, with ThrRS being able to discriminate its cognate substrate from similar analogues such as serine and valine. Molecular dynamics (MD) simulations and hybrid quantum mechanics/molecular mechanics (QM/MM) methods have been used to elucidate the role of Zn(II) in the aminoacylation mechanism of ThrRS. More specifically, the role of Zn(II) and active site residues in ThrRS's ability to discriminate between its cognate substrate l-threonine and the noncognate l-serine, l-valine, and d-threonine has been examined. The present results suggest that a role of the Zn(II) ion, with its Lewis acidity, is to facilitate deprotonation of the side chain hydroxyl groups of the aminoacyl moieties of cognate Thr-AMP and noncognate Ser-AMP substrates. In their deprotonated forms, these substrates are able to adopt a conformation preferable for aminoacyl transfer from aa-AMP onto the Ado-3'OH of the tRNAThr cosubstrate. Relative to the neutral substrates, when the substrates are first deprotonated with the assistance of the Zn(II) ion, the barrier for the rate-limiting step is decreased significantly by 42.0 and 39.2 kJ mol-1 for l-Thr-AMP and l-Ser-AMP, respectively. An active site arginyl also plays a key role in stabilizing the buildup of negative charge on the substrate's bridging phosphate oxygen during the mechanism. For the enantiomeric substrate analogue d-Thr-AMP, product formation is highly disfavored, and as a result, the reverse reaction has a very low barrier of 16.0 kJ mol-1.

Chiral Nanozymes-Gold Nanoparticle-Based Transphosphorylation Catalysts Capable of Enantiomeric Discrimination.

Enantioselectivity in RNA cleavage by a synthetic metalloenzyme has been demonstrated for the first time. Thiols containing chiral Zn(II) -binding head groups have been self-assembled on the surface of gold nanoparticles. This results in the spontaneous formation of chiral bimetallic catalytic sites that display different activities (kcat ) towards the enantiomers of an RNA model substrate. Substrate selectivity is observed when the nanozyme is applied to the cleavage of the dinucleotides UpU, GpG, ApA, and CpC, and remarkable differences in reactivity are observed for the cleavage of the enantiomerically pure dinucleotide UpU.

Exploring the substrate specificity and catalytic mechanism of imidazolonepropionase (HutI) from Bacillus subtilis

Imidazolonepropionase (HutI, EC 3.5.2.7) catalyzes the hydrolytic cleavage of carbon-nitrogen bond in 4-imidazolone-5-propionic acid (IPA) to yield l-formiminoglutamic acid, which is the third step in the universal histidine degradation pathway. In this article, using a combined quantum mechanics and molecular mechanics (QM/MM) approach, the specificity and catalytic mechanism of HutI from Bacillus subtilis have been explored by considering the four isomers of (S)- and (R)-enantiomers of IPA (SIPA-1, SIPA-2, RIPA-1 and RIPA-2). Our calculations reveal that the activation of hydrolytic water (a zinc-bound water) is performed by residue E252 via a "bridging" water molecule, which occurs before binding of the substrate. After the substrate binding, this activation channel is blocked by the substrate, and the other two residues (D324 and H272) cannot act as the general base to activate the hydrolytic water. For the two (S)-enantiomers of IPA, HutI can specifically convert one isomer of (S)-enantiomer (SIPA-1) to l-formiminoglutamic acid with an energy barrier of 16.6 kcal mol-1. The conversion of another (S)-enantiomer (SIPA-2) corresponds to the energy barrier of 21.9 kcal mol-1. However, for the two isomers of (R)-enantiomer, RIPA-1 corresponds to a higher energy barrier (21.8 kcal mol-1), and RIPA-2 is associated with a weak binding in the active site compared to SIPA-2. Thus, on the basis of our calculations, SIPA-1 is suggested to be the most favorable substrate for HutI, whereas the hydrolytic cleavage of SIPA-2 may require a preliminary isomerization to SIPA-1.

Zwitterionic phosphorylated quinines as chiral solvating agents for NMR spectroscopy.

Because of their unique 3D arrangement, naturally occurring Cinchona alkaloids and their synthetic derivatives have found wide-ranging applications in chiral recognition. Recently, we determined the enantioselective properties of C-9-phosphate mixed triesters of quinine as versatile chiral solvating agents in nuclear magnetic resonance (NMR) spectroscopy. In the current study, we introduce new zwitterionic members of this class of molecules containing a negatively charged phosphate moiety (i.e., ethyl, n-butyl and phenyl hydrogen quininyl phosphate). An efficient approach for synthesizing these compounds is elaborated, and full characterization, including conformational and autoaggregation phenomena studies, was performed. Therefore, their ability to induce NMR anisochrony of selected enantiomeric substrates (i.e., primarily N-DNB-protected amino acids and their methyl esters) was analyzed compared to uncharged diphenyl quininyl phosphate and its positively charged quaternary ammonium hydrochloride salt. In addition, (1) H and (13) C NMR experiments revealed their enantiodiscrimination potential toward novel analytes, such as secondary amines and nonprotected amino acids.

Enantioselective Photochemical Rearrangements of Spirooxindole Epoxides Catalyzed by a Chiral Bifunctional Xanthone

The title compounds were shown to undergo an enantioselective photochemical rearrangement to 3-acylindolin-2-ones (16–33 % ee). A xanthone, which is tethered via an anellated oxazole to a chiral 1,5,7-trimethyl-3-azabicyclo[3.3.1]nonan-2-one scaffold, efficiently catalyzed this reaction at λ 366 nm, presumably by triplet sensitization. The observed enantioselectivity can be explained by hydrogen bonding of the oxindole substrate and the putative 1,3-diradical intermediate to the lactam part of the catalyst. Although one substrate enantiomer is processed with minor preference over the other, it was shown that the reaction is not stereospecific. Rather, the main reason for the observed selectivity is the enantioselective migration step.

E. coli cells expressing the Baeyer-Villiger monooxygenase 'MO14' (ro03437) from Rhodococcus jostii RHA1 catalyse the gram-scale resolution of a bicyclic ketone in a fermentor.

The Baeyer-Villiger monooxygenase (BVMO) 'MO14' from Rhodococcus jostii RHA1, is an enantioselective BVMO that catalyses the resolution of the model ketone substrate bicyclo[3.2.0]hept-2-en-6-one to the (1S,5R)-2-oxa lactone and the residual (1S,5R)-substrate enantiomer. This regio-plus enantioselective behaviour is highly unusual for BVMOs, which often perform enantiodivergent biotransformations of this substrate. The scaleability of the transformation was investigated using fermentor-based experiments, in which variables including gene codon optimisation, temperature and substrate concentration were investigated. E. coli cells expressing MO14 catalysed the resolution of bicyclo[3.2.0]hept-2-en-6-one to yield (1S,5R)-2-oxa lactone of >99% ee and (1S,5R)-ketone of 96% ee after 14 h at a temperature of 16 °C and a substrate concentration of 0.5 g L(-1) (4.5 mM). MO14 is thus a promising biocatalyst for the production of enantio-enriched ketones and lactones derived from the [3.2.0] platform.

ChemInform Abstract: Our Recent Progress on the Intramolecular Diels—Alder Reaction Approach in Natural Products Synthesis: Synthetic Studies of the Octahydronaphthalene Substructure of Versipelostatins and the A/B/C‐Tricyclic Substructure of GKK1032s

AbstractReview: [66 refs.