Efficient terpene hydroxylation catalysts based upon P450 enzymes derived from actinomycetes

The hydroxylation of alpha-ionone 1 and beta-ionone 2 to their corresponding mono-hydroxylated derivatives has been examined using a recombinant E. coli whole cell system, in which cytochromes P450 SU1 and SU2, and P450 SOY were over-expressed with their cognate ferrodoxins. Both substrates are hydroxylated with a high degree of regioselectivity and for alpha-ionone 1 the reaction is highly diastereoselective yielding the anti-isomer.     

A parallel colorimetric method for the rapid discovery and optimization of heterogeneous hydrodesulfurization catalysts

A spectrophotometric based, parallel approach to screen heterogeneous catalysts for hydrodesulfurization has been developed. The method utilizes optical changes resulting from the hydrodesulfurization of 1,1'-binaphthothiophene to 1,1'-binaphthyl to determine catalyst activity. Hydrodesulfurization of 1,1'-binaphthothiophene to 1,1'-binaphthyl results in a loss of conjugation in the binaphthyl ring system. This results in a blue shift of more than 60 nm in the UV-vis spectrum. Since only small amounts of the HDS catalyst and 1,1'-binaphthothiophene are required, a large number of catalysts may be screened simultaneously. The synthesis of the 1,1'-binaphthothiophene and conditions for HDS catalyst screening are described.     

New combined model for the prediction of regioselectivity in cytochrome P450/3A4 mediated metabolism

Cytochrome P450 3A4 metabolizes nearly 50% of the drugs currently in clinical use with a broad range of substrate specificity. Early prediction of metabolites of xenobiotic compounds is crucial for cost efficient drug discovery and development. We developed a new combined model, MLite, for the prediction of regioselectivity in the cytochrome P450 3A4 mediated metabolism. In the model, the ensemble catalyticphore- based docking method was implemented for the accessibility prediction, and the activation energy estimation method of Korzekwa et al. was used for the reactivity prediction. Four major metabolic reactions, aliphatic hydroxylation, N-dealkylation, O-dealkylation, and aromatic hydroxylation reaction, were included and the reaction data, metabolite information, were collected for 72 well-known substrates. The 47 drug molecules were used as the training set, and the 25 well-known substrates were used as the test set for the ensemble catalyticphore-based docking method. MLite predicted correctly about 76% of the first two sites in the ranking list of the test set. This predictability is comparable with that of another combined model, MetaSite, and the recently published QSAR model proposed by Sheridan et al. MLite also offers information about binding configurations of the substrate-enzyme complex. This may be useful in drug modification by the structure-based drug design.     

A rapid method for catalyst testing. Zeolite catalysts for methanol conversion to hydrocarbon

A rapid method for testing catalysts is suggested, which permits a 30-fold reduction in the time of their examination. The method is recommended for studying catalytic processes taking place with either evolution or absorption of heat.

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Convenient method for the rapid generation of highly active and enantioselective yttrium catalysts for asymmetric hydroamination

A facile method for the preparation of highly active and enantioselective yttrium precatalysts for asymmetric hydroamination of gem-disubstituted aminoalkenes, from the combination of YCl(3) or YCl(3)(THF)(3.5) with ligand (R)- and n-BuLi is described.     

Optimization of a direct analysis in real time/time-of-flight mass spectrometry method for rapid serum metabolomic fingerprinting

Metabolomic fingerprinting of bodily fluids can reveal the underlying causes of metabolic disorders associated with many diseases, and has thus been recognized as a potential tool for disease diagnosis and prognosis following therapy. Here we report a rapid approach in which direct analysis in real time (DART) coupled with time-of-flight (TOF) mass spectrometry (MS) and hybrid quadrupole TOF (Q-TOF) MS is used as a means for metabolomic fingerprinting of human serum. In this approach, serum samples are first treated to precipitate proteins, and the volatility of the remaining metabolites increased by derivatization, followed by DART MS analysis. Maximum DART MS performance was obtained by optimizing instrumental parameters such as ionizing gas temperature and flow rate for the analysis of identical aliquots of a healthy human serum samples. These variables were observed to have a significant effect on the overall mass range of the metabolites detected as well as the signal-to-noise ratios in DART mass spectra. Each DART run requires only 1.2 min, during which more than 1500 different spectral features are observed in a time-dependent fashion. A repeatability of 4.1% to 4.5% was obtained for the total ion signal using a manual sampling arm. With the appealing features of high-throughput, lack of memory effects, and simplicity, DART MS has shown potential to become an invaluable tool for metabolomic fingerprinting.     

P450 monooxygenase in biotechnology. I. Single-step, large-scale purification method for cytochrome P450 BM-3 by anion-exchange chromatography.

An efficient single-step purification protocol for recombinant cytochrome P450 BM-3 from Bacillus megaterium, expressed in E. coli, was developed. Functional crude protein was obtained by disintegrating induced E. coli DH5 alpha and removing cell debris by centrifugation. After investigating different anion-exchange matrices, elution salts and the elution procedures involving an AKTAexplorer system, adsorption of the crude extract from lysed E. coli to Toyopearl DEAE 650M anion exchanger, followed by a two-step elution using NaCl, proved sufficient to isolate almost pure protein without inactivation (up to 93% P450 BM-3 content) in yields that ranged between 79-86%. The purification method could be scaled up 1500-fold and higher without further optimization to a 6-1 production-scale column containing Toyopearl DEAE 650M anion exchanger.     

Cycloisomerization of dienes with carbophilic lewis acids: mimicking terpene biosynthesis with Pt(II) catalysts

[reaction: see text]. Dicationic Pt(II) complexes containing triphosphine pincer ligands are excellent catalysts for the cycloisomerization of 1,6- and 1,7-dienes into bicyclopropane carbocycles. In analogy to the biosynthetic route to these monoterpene-like compounds, carbocation intermediates are proposed and supported by trapping experiments. Reactivation of the trapped intermediates indicates that cation generation by C-C bond formation is both rapid and reversible.     

Automated affinity selection for rapid discovery of peptide binders

In-solution affinity selection (AS) of large synthetic peptide libraries affords identification of binders to protein targets through access to an expanded chemical space. Standard affinity selection methods, however, can be time-consuming, low-throughput, or provide hits that display low selectivity to the target. Here we report an automated bio-layer interferometry (BLI)-assisted affinity selection platform. When coupled with tandem mass spectrometry (MS), this method enables both rapid de novo discovery and affinity maturation of known peptide binders with high selectivity. The BLI-assisted AS-MS technology also features real-time monitoring of the peptide binding during the library selection process, a feature unattainable by current selection approaches. We show the utility of the BLI AS-MS platform toward rapid identification of novel nanomolar (dissociation constant, K_D < 50 nM) non-canonical binders to the leukemia-associated oncogenic protein menin. To our knowledge, this is the first application of BLI to the affinity selection of synthetic peptide libraries. We believe our approach can significantly accelerate the use of synthetic peptidomimetic libraries in drug discovery.

This work reports an automated affinity selection-mass spectrometry (AS-MS) approach amenable to both de novo peptide binder discovery and affinity maturation of known binders in a high-throughput and selective manner.     

Combinatorial chemistry: a tool for the discovery of new catalysts

The discovery of new reactions and catalysts has always presented an intriguing challenge to scientists. With the rise of combinatorial chemistry, a new method has emerged that holds considerable promise to facilitate the task since it allows for the simultaneous generation and testing of a large number of compounds. The crucial difficulty lies in establishing general technologies for rapid and reliable screening of libraries to determine the catalytic activity of their members. Several recent publications have addressed this question by using infrared thermography, colorimetric assays and fluorescence spectroscopy. These techniques have not only been applied successfully to the high-throughput screening of parallel compound arrays but also to the screening of one-bead-one-compound libraries. This demonstrates that combinatorial chemistry possesses indeed the potential to establish itself as a powerful tool for the discovery of new catalysts. This review describes the methodologies used so far for the detection of catalytic events and will place particular emphasis on the on-bead screening of one-bead-one-compound libraries.     

Application of carbon nanotubes for in-capillary incubations with cytochrome P450 enzymes

The utility of multi-walled carbon nanotubes (MWNTs) to decrease the interaction between cytochrome P450 enzymes and the capillary wall during in-capillary enzymatic incubation was investigated. First, 18 surfactants were screened to determine their MWNT-dispersing capacity. A probe sonication procedure was developed in order to attain homogeneous MWNT dispersions within a reasonable time. Next, the influence of surfactants and MWNTs on P450 activity was studied, employing verapamil and CYP3A4 as model substrate and P450 isoform, respectively. MWNTs dispersed in Brij 35 did not affect CYP3A4 activity significantly and were selected for subsequent in-capillary tests. An in-line CE assay, involving electrophoretic mixing of reagents and zero potential amplification in the thermostatted part of the capillary, was developed. In-capillary incubations without MWNTs caused adsorption of enzyme to the capillary wall and a concomitant decline of capillary lifetime, even when extensive between-run rinsing was applied. Addition of MWNTs to the enzyme solution entailed substantial improvement of migration time and peak shape repeatability. The performance of three types of MWNTs was compared.     

High-throughput approaches for the discovery and optimization of new olefin polymerization catalysts

The discovery of new olefin polymerization catalysts is currently a time-intensive trial-and-error process with no guarantee of success. A fully integrated high-throughput screening workflow for the discovery of new catalysts for polyolefin production has been implemented at Symyx Technologies. The workflow includes the design of the metal-ligand libraries using custom-made computer software, automated delivery of metal precursors and ligands into the reactors using a liquid-handling robot, and a rapid primary screen that serves to assess the potential of each metalligand-activator combination as an olefin polymerization catalyst. "Hits" from the primary screen are subjected to secondary screens using a 48-cell parallel polymerization reactor. Individual polymerization reactions are monitored in real time under conditions that provide meaningful information about the performance capabilities of each catalyst. Rapid polymer characterization techniques support the primary and secondary screens. We have discovered many new and interesting catalyst classes using this technology.     

ansa-Rare-earth-metal catalysts for rapid and stereoselective polymerization of renewable methylene methylbutyrolactones

Two ansa-half-sandwich rare-earth-metal (REM) dialkyl complexes supported by an ethylene-bridged fluorenyl (Flu)-N-heterocyclic carbene (NHC) ligand, [M{C(2)H(4)(η(5)-Flu-κ(1)-NHC)}(CH(2)SiMe(3))(2)] (M=Y, 1; Lu, 2), and a chiral ansa-sandwich samarocene incorporating a C(2) ligand, [Sm(η(5)-C(12)H(8))(2)(thf)(2)] (3), have been investigated for the coordination-addition polymerization of renewable methylene butyrolactones, α-methylene-γ-butyrolactone (MBL) and γ-methyl-α-methylene-γ-butyrolactone ((γ)MMBL). Both ansa-half-sandwich complexes 1 and 2 exhibit exceptional activity for the polymerization of (γ)MMBL at room temperature in dimethylformamide (DMF); with a 0.25 mol% catalyst loading, quantitative monomer conversion can be achieved under 1 min, giving a high turn-over frequency (TOF) of 24,000 h(-1). This TOF value represents a rate enhancement, by a factor of 8, 22, or 2400, over the polymerizations by unbridged samarocene [Sm(Cp*)(2)(thf)(2)] (Cp*=η(5) -pentamethylcyclopentadienyl), by bridged ansa-samarocene 3 with C(2) ligation, or by the corresponding REM trialkyls without the ansa-Flu-NHC ligation, respectively. Complexes 1 and 2 are also highly active for the polymerization of β-methyl-α-methylene-γ-butyrolactone ((β)MMBL), realizing the first example of the metal-mediated coordination polymerization of this monomer and its copolymerization with (γ)MMBL. More remarkably, the resulting P(β)MMBL homopolymer is highly stereoregular (91% mm) and exhibits a high T(g) of 290 °C. In sharp contrast, catalysts 1 and 2 have poor activity and efficiency in the polymerization of the parent MBL or the acyclic analog methyl methacrylate. Polymerization and kinetic studies using the most active catalyst (1) of the series have uncovered characteristics of its (γ)MMBL polymerization and yielded a unimolecular propagation mechanism. A surprising chain-initiation pathway for the polymerization in DMF by 1 has been revealed, and catalytic polymerization in the presence of an organoacid has also been examined.     

制备方法对添加钇(Y)的非负载型Ni2P催化剂的影响

采用程序升温还原(T)法和低温次磷酸盐法(L)制备了Y-Ni₂P-T和Y-Ni₂P-L催化剂,并采用XRD、BET、CO吸附、XPS等手段对催化剂进行了表征。以二苯并噻吩(DBT)为模型化合物,研究了稀土Y对不同方法制备得到的催化剂加氢脱硫(HDS)性能的影响。结果表明,对T法制备的催化剂,添加稀土Y可以抑制Ni₅P₄杂晶的生成,从而促进活性相Ni₂P的生成;添加稀土Y能显著提高催化剂的比表面积,促进小粒径、高度分散的Ni₂P晶粒的生成。Y-Ni₂P-T催化剂的DBT转化率达到91.0%,比Ni₂P-T催化剂提高了29%。对L法制备的催化剂,添加稀土Y能抑制其他杂晶的生成,提高了Y-Ni₂P-L催化剂对联苯(BP)的选择性,但催化剂的总HDS活性比Ni₂P-L催化剂略有降低。     

A sensitive method for determination of allergenic fragrance terpene hydroperoxides using liquid chromatography coupled with tandem mass spectrometry

Different compositions of monoterpenes are utilized for their pleasant scent in cosmetics and perfumes. However, the most commonly used fragrance terpenes easily oxidize upon contact with air, forming strongly skin‐sensitizing hydroperoxides. Due to their thermolability and low UV absorbance, detection methods for hydroperoxides are scarce. For the first time, a simple and sensitive method using LC/ESI‐MS/MS was developed to quantitatively determine hydroperoxides from the common fragrance compounds linalool, linalyl acetate, and limonene. The method was applied to autoxidized petitgrain oil and sweet orange oil. A separation was accomplished using a C₃ column. The method LOD for the investigated hydroperoxides in the essential oils was below 0.3 μg/mL, corresponding to 0.3 ppm. For prevention purposes and according to EU regulations, concentrations in cosmetics exceeding 100 ppm in “rinse‐off” and 10 ppm in “stay‐on” products of linalool and limonene must be labeled. However, the products may still contain allergens, such as hydroperoxides, formed by oxidative degradation of their parent terpenes. The sensitivity and selectivity of the presented LC/MS/MS method enables detection of hydroperoxides from the fragrance terpenes linalool, linalyl acetate, and limonene. However, for routine measurements, the method requires further validation.     

A fully integrated high-throughput screening methodology for the discovery of new polyolefin catalysts: discovery of a new class of high temperature single-site group (IV) copolymerization catalysts

For the first time, new catalysts for olefin polymerization have been discovered through the application of fully integrated high-throughput primary and secondary screening techniques supported by rapid polymer characterization methods. Microscale 1-octene primary screening polymerization experiments combining arrays of ligands with reactive metal complexes M(CH(2)Ph)(4) (M = Zr, Hf) and multiple activation conditions represent a new high-throughput technique for discovering novel group (IV) polymerization catalysts. The primary screening methods described here have been validated using a commercially relevant polyolefin catalyst, and implemented rapidly to discover the new amide-ether based hafnium catalyst [eta(2)-(N,O)[bond](2-MeO[bond]C(6)H(4))(2,4,6-Me(3)C(6)H(2))N]Hf(CH(2)Ph)(3) (1), which is capable of polymerizing 1-octene to high conversion. The molecular structure of 1 has been determined by X-ray diffraction. Larger scale secondary screening experiments performed on a focused 96-member amine-ether library demonstrated the versatile high temperature ethylene-1-octene copolymerization capabilities of this catalyst class, and led to significant performance improvements over the initial primary screening discovery. Conventional one gallon batch reactor copolymerizations performed using selected amide-ether hafnium compounds confirmed the performance features of this new catalyst class, serving to fully validate the experimental results from the high-throughput approaches described herein.     

Oxidation of acyclic monoterpenes by P450 BM-3 monooxygenase: influence of the substrate E/Z-isomerism on enzyme chemo- and regioselectivity

Oxidized terpenes and terpenoids are highly valuable compounds for organic chemistry. Cytochrome P450 monooxygenase P450 BM-3 from Bacillus megaterium is able to catalyze oxidation of terpenes with high efficiency. Mutations at the amino acid positions 47, 51, and 87 resulted in significantly enhanced activity and regioselectivity of the enzyme during oxidation of geranylacetone and related compounds. The activity of the mutant R47L/Y51F/F87V was in the order of ketone>alcohol>aldehyde>acid. An effect of the substrate cis/trans-isomerism on the enzyme chemo- and regioselectivity was studied. P450 monooxygenase demonstrated similar NADPH turnovers with cis/trans isomers, nerylacetone/geranylacetone (1.9×10³/2.1×10³ min⁻¹) and nerol/geraniol (5.7×10²/5.9×10² min⁻¹), however, resulted in different number of products and product distribution. The Z-isomers, nerylacetone and nerol, were oxidized resulting in several products (five and three, respectively), including allylic alcohols. In contrast, E-isomers were epoxidized exclusively. Geranylacetone was converted with high activity (2080 min⁻¹) and enantioselectivity (97% ee) to 9,10-epoxygeranylacetone, while geraniol was enantioselectively epoxidized to the 6,7-epoxide (250 min⁻¹, 90% ee) with 90% regioselectivity.