Overriding Traditional Electronic Effects in Biocatalytic Baeyer-Villiger Reactions by Directed Evolution.

Controlling 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 (BVMOs). Herein, we address the challenging problem of switching normal to abnormal BVMO regioselectivity by directed evolution using three linear ketones as substrates, which are not structurally biased toward abnormal reactivity. Upon applying iterative saturation mutagenesis at sites lining the binding pocket of the thermostable BVMO from Thermocrispum municipale DSM 44069 (TmCHMO) and using 4-phenyl-2-butanone as substrate, the regioselectivity was reversed from 99:1 (wild-type enzyme in favor of the normal product undergoing 2-phenylethyl migration) to 2:98 in favor of methyl migration when applying the best mutant. This also stands in stark contrast to the respective reaction using the synthetic reagent m-CPBA, which provides solely the normal product. Reversal of regioselectivity was also achieved in the BV reaction of two other linear ketones. Kinetic parameters and melting temperatures revealed that most of the evolved mutants retained catalytic activity, as well as thermostability. In order to shed light on the origin of switched regioselectivity in reactions of 4-phenyl-2-butanone and phenylacetone, extensive QM/MM and MD simulations were performed. It was found that the mutations introduced by directed evolution induce crucial changes in the conformation of the respective Criegee intermediates and transition states in the binding pocket of the enzyme. In mutants that destabilize the normally preferred migration transition state, a reversal of regioselectivity is observed. This conformational control of regioselectivity overrides electronic control, which normally causes preferential migration of the group that is best able to stabilize positive charge. The results can be expected to aid future protein engineering of BVMOs.

P450-Catalyzed Regio- and Stereoselective Oxidative Hydroxylation of 6-Iodotetralone: Preparative-Scale Synthesis of a Key Intermediate for Pd-Catalyzed Transformations.

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-iodotetralone regio- and enantioselectively at C4 with formation of the ( R)-alcohol. This CH activation is not possible using modern synthetic catalysts. The synthetic utility of this valuable synthon was explored in palladium-catalyzed coupling reactions that occur in the absence of undesired racemization.

Investigating Substrate Scope and Enantioselectivity of a Defluorinase by a Stereochemical Probe.

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 configuration in a kinetic resolution with an extremely high selectivity factor (E > 500), showing that the classical mechanism involving SN2 reaction by Asp110 pertains. The high preference for the (S)-substrate is of synthetic value. Wide substrate scope of RPA1163 in such hydrolytic kinetic resolutions can be expected because the reaction of the even more sterically demanding rac-2-fluoro-2-benzyl acetic acid proceeded similarly. Substrate acceptance and stereoselectivity were explained by extensive molecular modeling (MM) and molecular dynamics (MD) computations. These computations were also applied to fluoroacetic acid itself, leading to further insights.

Manipulating the stereoselectivity of the thermostable Baeyer-Villiger monooxygenase TmCHMO by directed evolution.

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.

Whole-Cell-Catalyzed Multiple Regio- and Stereoselective Functionalizations in Cascade Reactions Enabled by 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.

Biocatalytic route to chiral acyloins: P450-catalyzed regio- and enantioselective α-hydroxylation of ketones.

P450-BM3 and mutants of this monooxygenase generated by directed evolution are excellent catalysts for the oxidative α-hydroxylation of ketones with formation of chiral acyloins with high regioselectivity (up to 99%) and enantioselectivity (up to 99% ee). This constitutes a new route to a class of chiral compounds that are useful intermediates in the synthesis of many kinds of biologically active compounds.

Metallophilic bonding and agostic interactions in gold(I) and silver(I) complexes bearing a thiotetrazole unit.

Gold(I) and silver(I) complexes of 1-methyl-5-thio-tetrazole (1) have been prepared and the coordination chemistry of this ligand toward metal-phosphine frameworks has been explored. As indicated by IR and Raman data, ligand 1 is deprotonated and the resulted anion acts as a bidentate (S,N)-tetrazole-5-thiolato unit in the new gold(I) complexes, [Au(SCN(4)Me)(PPh(3))] (2), [{Au(SCN(4)Me)}(2)(µ-dppm)] (3), and [{Au(SCN(4)Me)}(2)(µ-dppe)] (4), while it is coordinated only through the sulfur atom as its neutral tetrazole-5-thione form in the silver(I) derivative, [Ag(HSCN(4)Me)(PPh(3))](2)(OTf)(2) (5). Further characterization of the new compounds was performed using multinuclear ((1)H, (13)C, (31)P, (19)F) NMR spectroscopy, mass spectrometry, and DSC measurements. Single-crystal X-ray diffraction studies revealed basically linear P-M-S arrangements in complexes 3-5. The bidentate (S,N) coordination pattern results in a T-shaped (S,N)PAu core in 3 and 4, whereas, in 5, a similar coordination geometry is achieved in the dimer association based on S-bridging ligand 1. Herein, weak (C)H···Au and (C)H···Ag agostic interactions were observed. An intramolecular Au···Au contact occurs in 3, while in 4 intermolecular aurophilic bonds lead to formation of a chain polymer. An intermolecular Ag···Ag contact is also present in the dimer unit of 5. Low-temperature (31)P NMR data for 5 evidenced the presence of monomer and dimer units in solution. Theoretical calculations on model of the complexes 2 and 4 are consistent with the geometries found by X-ray diffraction studies.

Bis(8-methyl-2,8-dicarba-closo-dodeca-boran-2-yl) tris-elenide.

In the title compound, C(6)H(26)B(20)Se(3), the geometry around the central Se atom is V-shaped, with the Se-Se-Se angle being 105.60 (4)°. The Se-Se bond lengths are consistent with single covalent bonds.

A redox-mediated Kemp eliminase.

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 fundamentally different mechanism. QM/MM computations show that it involves coordination of the substrate's N-atom to haem-Fe(II) with electron transfer and concomitant N-O heterolysis liberating an intermediate having a nitrogen radical moiety Fe(III)-N· and a phenoxyl anion. Product formation occurs by bond rotation and H-transfer. Two rationally chosen point mutations cause a notable increase in activity. The results shed light on the prevailing mechanistic uncertainties in human P450-catalysed metabolism of the immunomodulatory drug leflunomide, which likewise undergoes redox-mediated Kemp elimination by P450-BM3. Other isoxazole-based pharmaceuticals are probably also metabolized by a redox mechanism. Our work provides a basis for designing future artificial enzymes.

Increasing the activity and enantioselectivity of lipases by sol-gel immobilization: further advancements of practical interest.

The entrapment of lipases in hydrophobic silicate matrices formed by sol-gel mediated hydrolysis of RSi(OCH3)3/Si(OCH3)4 as originally reported in 1996 has been improved over the years by a number of modifications. In the production of second-generation sol-gel lipase immobilizates, a variety of additives during the sol-gel process leads to increased activity and enhanced stereoselectivity in esterifying kinetic resolution. Recent advances in this type of lipase immobilization are reviewed here, in addition to new results regarding the sol-gel entrapment of the lipase from Burkholderia cepacia. It constitutes an excellent heterogeneous biocatalyst in the acylating kinetic resolution of two synthetically and industrially important chiral alcohols, rac-sulcatol and rac-trans-2-methoxycyclohexanol. The observation that the catalyst can be used 10 times in recycling experiments without losing its significant activity or enantioselectivity demonstrates the practical viability of the sol-gel approach.

CH-activating oxidative hydroxylation of 1-tetralones and related compounds with high regio- and stereoselectivity.

Mutants of P450-BM3 evolved by directed evolution are excellent catalysts in the CH-activating oxidative hydroxylation of 1-tetralone derivatives and of indanone, with unusually high regio- and enantioselectivity being observed. Similar results were achieved in the oxidative hydroxylation of tetralin and indane. The products are useful building blocks in the synthesis of a number of biologically active compounds.

Diselenolate- and ditellurolate-carborane gold complexes.

Gold complexes with selenolate and tellurolate carborane ligands [E2C2B10H10](2-) (E = Se, Te) have been synthesized by reaction of a freshly prepared solution of [1,2-(LiE)2C2B10H10] (E = Se, Te) with [AuClL] (L = PPh3, PPh2Me, PPh2py) in a 1 : 2 molar ratio or [Au2Cl2(P∼P)] [P∼P = dppf, 1,1'-bis(diphenylphosphano)ferrocene; dppe, 1,2-bis(diphenylphosphano)ethane; dppc, 1,2-bis(diphenylphosphano)-o-carborane] in a 1 : 1 molar ratio affording complexes [Au2(µ-1,2-E2C2B10H10)L2] or [Au2(µ-1,2-E2C2B10H10)(P∼P)], respectively. The gold(III) species PPN[Au(E2C2B10H10)2] (PPN = bis(triphenylphosphano)iminium) have been afforded by reaction of PPN[AuCl4] with [1,2-(LiE)2C2B10H10]. Complex [Au4(µ-1,2-Se2C2B10H10)2(µ-dppc)2] displays a tetranuclear structure, different from the dinuclear cyclic arrangement proposed for other complexes with diphosphanes of [Au2(µ-1,2-Se2C2B10H10)(P∼P)] stoichiometry.