A one-pot Pd- and P450-catalyzed chemoenzymatic synthesis of a library of oxyfunctionalized biaryl alkanoic acids leveraging a substrate anchoring approach.

A one-pot chemoenzymatic approach was developed by combining Palladium-catalysis with selective cytochrome P450 enzyme oxyfunctionalization. Various iodophenyl alkanoic acids could be coupled with alkylphenyl boronic acids to generate a series of alkyl substituted biarylalkanoic acids in overall high yield. The identity of the products could be confirmed by various analytical and chromatographic techniques. Addition of an engineered cytochrome P450 heme domain mutant with peroxygenase activity upon completion of the chemical reaction resulted in the selective oxyfunctionalization of those compounds, primarily at the benzylic position. Moreover, in order to increase the biocatalytic product conversion, a reversible substrate engineering approach was developed. This involves the coupling of a bulky amino acid such as L- phenylalanine or tryptophan, to the carboxylic acid moiety. The approach resulted in a 14 to 49% overall biocatalytic product conversion increase associated with a change in regioselectivity of hydroxylation towards less favored positions.

Insights into an efficient light-driven hybrid P450 BM3 enzyme from crystallographic, spectroscopic and biochemical studies.

BACKGROUND: In order to perform selective CH functionalization upon visible light irradiation, Ru(II)-diimine functionalized P450 heme enzymes have been developed. The sL407C-1 enzyme containing the Ru(bpy)2PhenA (bpy=2,2'-bipyridine and PhenA=5-acetamido-1,10-phenanthroline) photosensitizer (1) covalently attached to the non-native single cysteine L407C of the P450BM3 heme domain mutant, displays high photocatalytic activity in the selective CH bond hydroxylation of several substrates. METHODS: A combination of X-ray crystallography, site-directed mutagenesis, transient absorption measurements and enzymatic assays was used to gain insights into its photocatalytic activity and electron transfer pathway. RESULTS: The crystal structure of the sL407C-1 enzyme was solved in the open and closed conformations revealing a through-space electron transfer pathway involving highly conserved, F393 and Q403, residues. Several mutations of these residues (F393A, F393W or Q403W) were introduced to probe their roles in the overall reaction. Transient absorption measurements confirm rapid electron transfer as heme reduction is observed in all four hybrid enzymes. Compared to the parent sL407C-1, photocatalytic activity was negligible in the dF393A-1 enzyme while 60% increase in activity with total turnover numbers of 420 and 90% product conversion was observed with the dQ403W-1 mutant. CONCLUSIONS: In the sL407C-1 enzyme, the photosensitizer is ideally located to rapidly deliver electrons, using the naturally occurring electron transfer pathway, to the heme center in order to activate molecular dioxygen and sustain photocatalytic activity. GENERAL SIGNIFICANCE: The results shed light on the design of efficient light-driven biocatalysts and the approach can be generalized to other members of the P450 superfamily.

Regio- and stereoselective hydroxylation of 10-undecenoic acid with a light-driven P450 BM3 biocatalyst yielding a valuable synthon for natural product synthesis.

We report herein the selective hydroxylation of 10-undecenoic acid with a light-activated hybrid P450 BM3 enzyme. Under previously developed photocatalytic reaction conditions, only a monohydroxylated product is detected by gas chromatography. Hydroxylation occurs exclusively at the allylic position as confirmed from a synthesized authentic standard. Investigation into the stereochemistry of the reaction indicates that the R enantiomer is obtained in 85% ee. The (R)-9-hydroxy-10-undecenoic acid obtained enzymatically is a valuable synthon en route to various natural products further expanding the light-activated P450 BM3 biocatalysis and highlighting the advantages over traditional methods.

An efficient light-driven P450 BM3 biocatalyst.

P450s are heme thiolate enzymes that catalyze the regio- and stereoselective functionalization of unactivated C-H bonds using molecular dioxygen and two electrons delivered by the reductase. We have developed hybrid P450 BM3 heme domains containing a covalently attached Ru(II) photosensitizer in order to circumvent the dependency on the reductase and perform P450 reactions upon visible light irradiation. A highly active hybrid enzyme with improved stability and a modified Ru(II) photosensitizer is able to catalyze the light-driven hydroxylation of lauric acid with total turnover numbers of 935 and initial reaction rate of 125 mol product/(mol enzyme/min).

Light-triggered modulation of cellular electrical activity by ruthenium diimine nanoswitches.

Ruthenium diimine complexes have previously been used to facilitate light-activated electron transfer in the study of redox metalloproteins. Excitation at 488 nm leads to a photoexcited state, in which the complex can either accept or donate an electron, respectively, in the presence of a soluble sacrificial reductant or oxidant. Here, we describe a novel application of these complexes in mediating light-induced changes in cellular electrical activity. We demonstrate that RubpyC17 ([Ru(bpy)(2)(bpy-C17)](2+), where bpy is 2,2'-bipyridine and bpy-C17 is 2,2'-4-heptadecyl-4'-methyl-bipyridine), readily incorporates into the plasma membrane of cells, as evidenced by membrane-confined luminescence. Excitable cells incubated in RubpyC17 and then illuminated at 488 nm in the presence of the reductant ascorbate undergo membrane depolarization leading to firing of action potentials. In contrast, the same experiment performed with the oxidant ferricyanide, instead of ascorbate, leads to hyperpolarization. These experiments suggest that illumination of membrane-associated RubpyC17 in the presence of ascorbate alters the cell membrane potential by increasing the negative charge on the outer face of the cell membrane capacitor, effectively depolarizing the cell membrane. We rule out two alternative explanations for light-induced membrane potential changes, using patch clamp experiments: (1) light-induced direct interaction of RubpyC17 with ion channels and (2) light-induced membrane perforation. We show that incorporation of RubpyC17 into the plasma membrane of neuroendocrine cells enables light-induced secretion as monitored by amperometry. While the present work is focused on ruthenium diimine complexes, the findings point more generally to broader application of other transition metal complexes to mediate light-induced biological changes.

Light-initiated hydroxylation of lauric acid using hybrid P450 BM3 enzymes.

We have developed hybrid P450 BM3 enzymes consisting of a Ru(II)-diimine photosensitizer covalently attached to non-native single cysteine residues of P450 BM3 heme domain mutants. These enzymes are capable, upon light activation, of selectively hydroxylating lauric acid with 40 times higher total turnover numbers compared to the peroxide shunt.

Enantiomer-specific binding of ruthenium(II) molecular wires by the amine oxidase of Arthrobacter globiformis.

The copper amine oxidase from Arthrobacter globiformis (AGAO) is reversibly inhibited by molecular wires comprising a Ru(II) complex head group and an aromatic tail group joined by an alkane linker. The crystal structures of a series of Ru(II)-wire-AGAO complexes differing with respect to the length of the alkane linker have been determined. All wires lie in the AGAO active-site channel, with their aromatic tail group in contact with the trihydroxyphenylalanine quinone (TPQ) cofactor of the enzyme. The TPQ cofactor is consistently in its active ("off-Cu") conformation, and the side chain of the so-called "gate" residue Tyr296 is consistently in the "gate-open" conformation. Among the wires tested, the most stable complex is produced when the wire has a -(CH2)4- linker. In this complex, the Ru(II)(phen)(bpy)2 head group is level with the protein molecular surface. Crystal structures of AGAO in complex with optically pure forms of the C4 wire show that the linker and head group in the two enantiomers occupy slightly different positions in the active-site channel. Both the Lambda and Delta isomers are effective competitive inhibitors of amine oxidation. Remarkably, inhibition by the C4 wire shows a high degree of selectivity for AGAO in comparison with other copper-containing amine oxidases.

Structural and spectroscopic characterization of copper(II) complexes of a new bisamide functionalized imidazole tripod and evidence for the formation of a mononuclear end-on Cu-OOH species.

A new polyimidazole tripod N,N-bis((1-methyl-4-pivalamidoimidazol-2-yl)methyl)-N'-((1-methylimidazol-2-yl)methyl)amine (L2) has been synthesized and shown to form intramolecular hydrogen bonds with different axial ligands bonded to Cu(II) in the solid state. The same hydrogen-bonding property of L2 appears responsible for the stabilization of a Cu(II)-OOH species in solution. The crystal structures of L2 and three of its Cu(II) complexes are reported. The [Cu(L2)X]ClO4 complexes, 4-6 (X- = Cl-, OH-, or N3-) have distorted trigonal bipyramidal geometries in the solid state and have been characterized further by UV-vis absorption, electron paramagnetic resonance (EPR) spectroscopy, and cyclic voltammetry. The reaction of [Cu(L2)OH](ClO4) (5) with H2O2 and tert-butyl hydroperoxide in methanol generates [Cu(L2)OOH](ClO4) (7) and [Cu(L2)OO(t)Bu](ClO4) (8) which have been characterized by different spectroscopic methods. The compound [Cu(L2)OO(t)Bu]+ displays a band at 395 nm (epsilon = 950 M(-1) cm(-1)) assigned to an alkylperoxo pi*(sigma) --> Cu ligand-to-metal charge transfer (LMCT) transition, while [Cu(L2)OOH]+ displays a peroxo pi*(sigma) --> Cu charge-transfer transition at 365 nm with epsilon = 1300 M(-1) cm(-1), a mass ion at m/z 593.4, and nu(O-O) stretch (resonance Raman) at 854 cm(-1) that shifts to lower energy by 46 cm(-1) upon 18O substitution.

5,10-dihydroxy-5H,10H-diimidazo[1,2-a:1',2'-d]pyrazine.

Crystallization of the title compound, C8H8N4O2, results in the formation of one-dimensional chains of imidazole (im) molecules linked together by strong hydrogen bonds. The O...N(im) separation and O-H(...N) distance are 2.6906 (17) and 1.74 (2) A, respectively, and the O-H...N angle is 173 (2) degrees. The one-dimensional chains are weakly pi stacked along the b axis, with centroid-to-centroid separations of 3.678 (2) A between five- and six-membered rings and 3.963 (2) A between six-membered rings. Each molecule is arranged around an inversion center.

A supramolecular assembly of side-by-side polyimidazole tripod coils stabilized by pi-pi stacking and unique boric acid templated hydrogen bonding interactions.

The self assembly of (bis(1-methyl-imidazol-2-yl)methyl)(1-methyl-4-nitroimidazol-2-yl)methyl)amine and boric acid results in a supramolecular structure containing bundled antiparallel imidazole-boric acid helices and boric acid filled one-dimensional channels.

Structure and properties of an Fe(III) complex containing a novel amide functionalized polyimidazole ligand.

A novel amide functionalized polyimidazole tripod ligand has been synthesized and used to prepare a mononuclear Fe(III) complex that has been characterized by X-ray crystallography and other physical methods.