Open-chain Ketones Research Articles

The steroid monooxygenase from Rhodococcus rhodochrous; a versatile biocatalyst

The substrate scope of a steroid monooxygenase (STMO) from Rhodococcus rhodochrous DSM 43269 was investigated for a large range of different ketone substrates. These studies revealed that this enzyme not only oxygenates steroids, but also ketone moieties of a series of other open-chain ketones, such as cyclohexyl methyl ketone, cyclopentyl methyl ketone, and 3-acetylindole. Furthermore, the STMO catalyzed the oxygenation of cyclobutanone derivatives. Comparative biotransformations with recombinant Escherichia coli resting cells harboring the STMO, the cycloalkanone monooxygenase (CAMO) from Cylindrocarpon radicicola or the cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus revealed that the STMO is enantiodivergent compared to the CHMO-type. Moreover, the STMO resulted in a higher enantiomeric excess of the product lactones compared to the known BVMOs of the same enantiopreference, such as cyclopentanone monooxygenases.

Structural Basis for Substrate Specificity and Mechanism of N-Acetyl-d-neuraminic Acid Lyase from Pasteurella multocida

N-Acetylneuraminate lyases (NALs) or sialic acid aldolases catalyze the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac, the most common form of sialic acid) to form pyruvate and N-acetyl-d-mannosamine. Although equilibrium favors sialic acid cleavage, these enzymes can be used for high-yield chemoenzymatic synthesis of structurally diverse sialic acids in the presence of excess pyruvate. Engineering these enzymes to synthesize structurally modified natural sialic acids and their non-natural derivatives holds promise in creating novel therapeutic agents. Atomic-resolution structures of these enzymes will greatly assist in guiding mutagenic and modeling studies to engineer enzymes with altered substrate specificity. We report here the crystal structures of wild-type Pasteurella multocida N-acetylneuraminate lyase and its K164A mutant. Like other bacterial lyases, it assembles into a homotetramer with each monomer folding into a classic (β/α)₈ TIM barrel. Two wild-type structures were determined, in the absence of substrates, and trapped in a Schiff base intermediate between Lys164 and pyruvate, respectively. Three structures of the K164A variant were determined: one in the absence of substrates and two binary complexes with N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Both sialic acids bind to the active site in the open-chain ketone form of the monosaccharide. The structures reveal that every hydroxyl group of the linear sugars makes hydrogen bond interactions with the enzyme, and the residues that determine specificity were identified. Additionally, the structures provide some clues for explaining the natural discrimination of sialic acid substrates between the P. multocida and Escherichia coli NALs.

Cloning, expression and characterization of a eukaryotic cycloalkanone monooxygenase from Cylindrocarpon radicicola ATCC 11011

In this study, we have cloned and characterized a cycloalkanone monooxygenase (CAMO) from the ascomycete Cylindrocarpon radicicola ATCC 11011 (identical to Cylindrocarpon destructans DSM 837). The primary structure of this Baeyer-Villiger monooxygenase (BMVO) revealed 531 residues with around 45% sequence identity to known cyclohexanone monooxygenases. The enzyme was functionally overexpressed in Escherichia coli and investigated with respect to substrate spectrum and kinetic parameters. Substrate specificity studies revealed that a large variety of cycloaliphatic and bicycloaliphatic ketones are converted by this CAMO. A high catalytic efficiency against cyclobutanone was observed and seems to be a particular property of this BVMO. The thus produced butyrolactone derivatives are valuable building blocks for the synthesis of a variety of natural products and bioactive compounds. Furthermore, the enzyme revealed activity against open-chain ketones such as cyclobutyl, cyclopentyl and cyclohexyl methyl ketone which have not been reported to be accepted by typical cyclohexanone monooxygenases. These results suggest that the BVMO from C. radicicola indeed might be rather unique and since no BVMOs originating from eukaryotic organisms have been produced recombinantly so far, this study provides the first example for such an enzyme.

Enantioselective Intermolecular Aldehyde–Ketone Cross‐Coupling through an Enzymatic Carboligation Reaction

Happy new YerE: The first example of the title reaction is presented using a ThDP-dependent enzyme catalyst. The substrate tolerance of the enzyme is very broad and includes cyclic and open-chain ketones, as well as diketones and α- and β-ketoesters as acceptor substrates. The absolute configurations of two enzymatic products were determined by single-crystal structure analysis.

Cloning, expression, characterization, and biocatalytic investigation of the 4-hydroxyacetophenone monooxygenase from Pseudomonas putida JD1.

While the number of available recombinant Baeyer-Villiger monooxygenases (BVMOs) has grown significantly over the last few years, there is still the demand for other BVMOs to expand the biocatalytic diversity. Most BVMOs that have been described are dedicated to convert efficiently cyclohexanone and related cyclic aliphatic ketones. To cover a broader range of substrate types and enantio- and/or regioselectivities, new BVMOs have to be discovered. The gene encoding a BVMO identified in Pseudomonas putida JD1 converting aromatic ketones (HAPMO; 4-hydroxyacetophenone monooxygenase) was amplified from genomic DNA using SiteFinding-PCR, cloned, and functionally expressed in Escherichia coli. Furthermore, four other open reading frames could be identified clustered around this HAPMO. It has been suggested that these proteins, including the HAPMO, might be involved in the degradation of 4-hydroxyacetophenone. Substrate specificity studies revealed that a large variety of other arylaliphatic ketones are also converted via Baeyer-Villiger oxidation into the corresponding esters, with preferences for para-substitutions at the aromatic ring. In addition, oxidation of aldehydes and some heteroaromatic compounds was observed. Cycloketones and open-chain ketones were not or poorly accepted, respectively. It was also found that this enzyme oxidizes aromatic ketones such as 3-phenyl-2-butanone with excellent enantioselectivity (E >>100).

Cloning, expression and characterization of a Baeyer-Villiger monooxygenase from Pseudomonas putida KT2440

The gene encoding a Baeyer-Villiger monooxygenase and identified in Pseudomonas putida KT2440 was cloned and functionally expressed in Escherichia coli. The highest yield of soluble protein could be achieved by co-expression of molecular chaperones. In order to determine the substrate specificity, biocatalyses were performed using crude cell extract, growing and resting cells. Examination of aromatic, cyclic and aliphatic ketones revealed a high specificity towards short-chain aliphatic ketones. Interestingly, some open-chain ketones were converted to the alkylacetates, while for others formation of the ester products with oxygen on the other side of the keto group could also be detected yielding the corresponding methyl or ethyl esters.

Synthesis of and NMR studies on the four diastereomeric 1-deoxy- d-ketohexoses

The four 1-deoxy- d-ketohexoses—1-deoxy- d-psicose, 1-deoxy- d-fructose, 1-deoxy- d-sorbose and 1-deoxy- d-tagatose—were synthesised by methyl lithium addition to suitably protected and readily available pentonolactones. The 1-deoxy- l-ketohexoses are available from the enantiomeric lactones. The NMR studies on aqueous solutions of each diastereomer show that the relative amounts of open chain ketones, α- and β-pyranoses, and α- and β-furanoses vary considerably; at least four different species are identifiable from each equilibrium.

Cloning, expression, and characterization of a Baeyer–Villiger monooxygenase from Pseudomonas fluorescens DSM 50106 in E. coli

A gene encoding a Baeyer-Villiger monooxygenase (BVMO) identified in Pseudomonas fluorescens DSM 50106 was cloned and functionally expressed in Escherichia coli JM109. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis showed an estimated 56 kDa-size protein band corresponding to the recombinant enzyme. Expression in BL21 (DE3) resulted mainly in the formation of inclusion bodies. This could be overcome by coexpression of molecular chaperones, especially the DnaK/DnaJ/GrpE complex, leading to increased production of soluble BVMO enzyme in recombinant E. coli. Examination of the substrate spectra using whole-cell biocatalysis revealed a high specificity of the BVMO for aliphatic open-chain ketones. Thus, octyl acetate, heptyl propionate, and hexyl butyrate were quantitatively formed from the corresponding ketone substrates. Several other esters were obtained in conversion >68%. Selected esters were also produced on preparative scale.

Characterization of a Lipid Hydroperoxide-Derived RNA Adduct in Rat Intestinal Epithelial Cells

Five major products (adducts A(1a), A(1b), A(2), A(3,) and B) from the reaction of guanosine (Guo) with 4-oxo-2(E)-nonenal (ONE) were detected by liquid chromatography-mass spectrometry (LC-MS). Tandem MS (MS/MS) analysis of these compounds suggested that modifications to the nucleoside had been introduced. Adducts A(1a), A(1b), A(2), and A(3) were heptanone-ethano-2'-Guo adducts that all decomposed to adduct B. Adducts A(1a) and A(1b) were isomeric hemi-ketal forms. Adducts A(2) and A(3) were diastereomers of the open chain ketone form. The structure of adduct B was shown by LC-MS/MS and NMR spectroscopy to be the heptanone-etheno-Guo (HepsilonGuo) adduct, 3-(D-erythropentafuranosyl)imidazo-7-(heptane-2' '-one)-9-hydroxyl[1,2-alpha]purine. The overall reaction of Guo with ONE was very similar to its reaction with 2'-deoxyguanosine. Reaction of ONE with yeast transfer RNA also resulted in the formation of HepsilonGuo. Finally, HepsilonGuo was detected and quantified in the RNA from rat intestinal epithelial cells that stably express cyclooxygenase-2. These data show that RNA is modified by the same bifunctional reactive electrophiles derived from lipid peroxidation that covalently modify DNA.

Synthesis of monoprotected 1,4-diketones by photoinduced alkylation of enones with 2-substituted-1,3-dioxolanes

Photosensitized hydrogen abstraction from 2-alkyl-1,3-dioxolanes by triplet benzophenone gives the corresponding 1,3-dioxolan-2-yl radicals and these are trapped by α,β-unsatured ketones yielding monoprotected 1,4-diketones. With open chain ketones (3-buten-2-one and 4-penten-3-one) the yields are low and competitive pathways in part consume the radicals. With cyclic enones however, yields are good as tested with cyclopentenone, cyclohexenone and 4-hydroxy-cyclopentenone. More generally, this is a viable alternative for the synthesis of 1,4-diketones via radicals while the thermal initiation gives only low yield. The reaction cannot be extended to strongly stabilized radicals, such as the 2-phenyl-1,3-dioxolanyl radical.

Covalent modifications to 2'-deoxyguanosine by 4-oxo-2-nonenal, a novel product of lipid peroxidation.

Two major products (adducts A and B) from the reaction of 2-deoxyguanosine (dGuo) with 13-hydroperoxylinoleic acid were detected by liquid chromatography/mass spectrometry (LC/MS). Adducts A and B were also the major products formed enzymatically when dGuo was incubated in the presence of linoleic acid and lipoxygenase. The mass spectral fragmentation patterns of adducts A and B suggested that unique modifications to the nucleoside had been introduced. This resulted in the characterization of a novel bifunctional electrophile, 4-oxo-2-nonenal, as the principal breakdown product of linoleic acid hydroperoxide. In subsequent studies, adduct A was found to be a substituted ethano dGuo adduct that was a mixture of three isomers (A(1)-A(3)) that all decomposed to form adduct B. Adduct A(1) was the hemiacetal form of 3-(2-deoxy-beta-D-erythropentafuranosyl)-3,5,6, 7-tetrahydro-6-hydroxy-7-(heptane-2-one)-9H-imidazo[1, 2-alpha]purine-9-one. Adducts A(2) and A(3) were the diastereomers of the open chain ketone form. Adduct B was the substituted etheno dGuo adduct, 3-(2-deoxy-beta-D-erythropentafuranosyl)imidazo-7-(heptane-2 -one)-9-hydroxy[1,2-alpha]purine, the dehydration product of adducts A(1)-A(3). Identical covalent modifications to dGuo were observed when calf-thymus DNA was treated with 4-oxo-2-nonenal. These data illustrate the diversity of reactive electrophiles produced from the peroxidative decomposition of lipids and have implications in fully assessing the role of lipid peroxidation in mutagenesis and carcinogenesis.

1-Deoxy-D-xylulose

1-Deoxy-D-xylulose (= 1-deoxy-D-theropentulose) is a precursor of thiamin (Vitamin B1) and of pyridoxine (Vitamin B6) in bacteria. The synthesis of a [2,3-13C2] bond-labeled sample of the compound, to be used for investigations of the biosynthesis of the two vitamins, is described. In aqueous solution 1-deoxy-D-xylulose exists mainly as the open chain ketone. In methanol solution the compound exists as a mixture of the open chain ketone and the two corresponding epimeric furanoses. In acid solution the compound yields a dimeric anhydride, di-β-1-deoxy-D-xylulofuranose 2,3′:3,2′-dianhydride, whose structure was established by X-ray crystallography. Keywords: [2,3-13C2]-1-deoxy-D-xylulose, di-β-1-deoxy-D-xylulofuranose 2,3′:3,2′-dianhydride, 5-O-benzyl-3,4-O-isopropylidene-1-deoxy-D-xylulose, 4-O-benzyl-2,3-O-isoproylidene-D-threose.

Donor-acceptor effects on the stability of radicals derived from .alpha.-dialkylamino ketones

The bond dissociation energies (BDEs) for the acidic C−H bonds in CH 3 COCH 2 NMeNMe 2 and CH 3 COCH 2 NEt 2 ketones, estimated in DMSO from pK HA and oxidation potential data, are 3-4 kcal/mol higher than that for the acidic C−H bond in PhCOCH 2 NMe 2 . It is suggested that the electronic and steric effects of the Ph group dictate a Z structure for the precursor enolate ion and the generation of a radical that is strongly stabilized by electrostatic effects, which are responsible for the apparent synergistic effects of the PhCO and NMe 2 groups in stabilizing the PhCOCHNMe 2 radical. This suggestion was supported by the observation that the BDEs of the acidic C−H bonds in ketocyclic analogues of the α-dialkylamino open-chain ketones, wherein the enolate ion is locked in an E structure, have higher BDEs by 9-11.5 kcal/mol

The oxidation of ketones with a heteropolyacid, H5[PMo10V2O40] and dioxygen

Substituted cycloalkanones, 1-phenylalkanones, and open-chain ketones are oxidatively cleaved by the title compound under very mild conditions.

The Mutarotation of Fructose and the Invertase Hydrolysis of Sucrose

Abstract Studies by GLC and GLC/MS of the mutarotation of fructose in water have been made to determine equilibrium composition as a function of temperature. The major components are β-fructo-pyranose, β-fructofuranose and α-fructofuranose in agreement with studies in the literature. There are small amounts of the α-fructopyranose and the open-chain ketone form. The major change during mutarotation is the ring size change for β-fructopyranose → β-fructofuranose, but other changes contribute. For this reason, polarimetric rate studies of this system as a simple first-order equilibrium process are not valid. Hydrolysis of sucrose catalyzed by invertase has been accomplished to the extent of 99% in 1 min. The mutarotation of glucose and fructose were then studied without the complication of further production of the products from sucrose. The enzymatic cleavage is stereo-specific to provide retained configuration in α-glucopyranose and β-fructofuranose which each mutarotate to equilibrium. The mutarotation l...

Ring size effect on the proton affinity of cycloalkanones — a MINDO/3 theoretical study

The proton affinities of cycloalkanones and some open chain ketones have been calculated by means of the MINDO/3 method. The marked ring size effect on the proton affinities of cyclic ketones as opposed to their open chain counterparts is rationalized in terms of a model for protonation of carbonyl compounds proposed by Del Bene, as well as in terms of the energy partitioning scheme within MINDO/3.

Preliminary examination of an electrochemical process for converting olefins to ketones

Olefins higher than 3-C were converted to ketones using aqueous chloropalladate ion as oxidant. The chloride ion content of the solution was kept at very low levels relative to normal usage to increase significantly the reaction rates of these higher olefins. The reduced palladium was reoxidized electrochemically using the ferric/ferrous couple as the transfer agent. Using cyclopentene as a model compound the ionic strength, the acidity, the concentration of palladium, the means of extracting product and the means of adding olefin were all adjusted as to achieve about 70% current selectivity with minimal palladium inventory. There were indications of even better behaviour with several open chain ketones.

Lanthanide Shift Reagents. Paper 19. Equilibrium Binding Constants for Alicyclic Ketones, Ethers and Epoxides

Abstract Previous measurements of the binding constants of the lanthanide reagent, Eu(thd)3, with (mainly) open chain ketones showed that moderately strong complexes were formed1,2. We have now extended these studies using nine alicyclic ketones to examine the effects of ring size and carbonyl conjugation. The influence of Europium and Ytterbium fluroinated shift reagents on five ethers and epoxides is also discussed.

Influnce des interactions de torsion dans la reduction des cetones par les hydrures interpretation generale du deroulement sterique de la reduction des cetones acycliques et de l'addition des organomagnesiens sur les aldehydes acycliques

Reduction of ketones L-CHMe-CO-R [L=Ph and Cy(Cy = cyclohexyl) and R=Me, Et, iPr and tBu] affords pairs of diastereoisomeric alcohols L-CHMe-CHOH-R. The predominant diastereoisomer is always that predicted by Cram's rule, and the stereoselectivity of the reaction generally increases as R is made more bulky. Thus, with LiAlH 4 in ether at 35°, the diastereoisomer ratios are respectively 2.8:1, 3.2:1, 5.0:1, and 49:1 when L=Ph and 1.6:1, 2.0:1, 4.1:1, and 1.6:1 when L=Cy. With NaBH 4 in isopropanol at 50°, these ratios are respectively 1.6:1, 2.0:1, 2.7:1, and 7.3:1 when L=Ph, and 1.2:1, 1.6:1, 3.2:1, and 3.5:1 when L=Cy. It is suggested that an important factor determining the steric course of the reduction of both open-chain ketones and cyclohexanones is torsional strain in the transition state, and that torsional strain involving partial bonds can represent a substantial fraction of the strain between fully-formed bonds, even when the degree of bonding is quite low. This postulate has been made the basis of an internally consistent interpretation of the steric course of the reaction between carbonyl compounds and nucleophilic reagents such as hydrides and Grignard reagents.

Reissert compound chemistry. Part 5. Formation of pyrrolo[1,2-a]-quinolines and pyrrolo[2,1-a]isoquinolines

Treatment of a quinoline Reissert compound carrying a blocking group at the 4-position with base in the presence of acrylonitrile can lead in modest yield to the pyrrolo[1,2-a]quinoline system (8) and to an open chain ketone (11) derived via an intermediate tetrahydropyrrolo[1,2-a]quinoline. This behaviour contrasts with the more ready formation of the pyrrolo[2,1-a]isoquinoline system (3) by a similar process.