Changing the regioselectivity of a P450 from C15 to C11 hydroxylation of progesterone

CYP106A2 is known as a 15β‐hydroxylase, but also shows minor 11α‐hydroxylase activity for progesterone. 11α‐Hydroxyprogesterone is an important pharmaceutical compound with anti‐androgenic and blood‐pressure‐regulating activity. This work therefore focused on directing the regioselectivity of the enzyme towards hydroxylation at position 11 in the C ring of the steroid through a combination of saturation mutagenesis and rational site‐directed mutagenesis. With the aid of data from a homology model of CYP106A2 containing docked progesterone, together with site‐directed mutagenesis of active‐site residues (Lisurek et al. ChemBioChem 2008 , 9, 1439–1449), a saturation mutagenesis library at positions A395 and G397 was created. Screening of the library identified the mutants A395I and A395W/G397K as having 11α‐hydroxylase activities 8.9 and 11.5 times higher than that of the wild type (WT). In the next step, additional mutations were integrated by a rational site‐directed mutagenesis approach to increase the catalytic efficiency. Of the 40 candidates analyzed, the mutants A106T/A395I, A106T/A395I/R409L, and T89N/A395I turned out to display increased 11α‐hydroxylase selectivities and activities relative to the WT (14.3‐, 12.6‐, and 11.8‐fold increases in selectivity and 39.3‐, 108‐, and 24.4‐ in k_cat/K_m). In the last step of the study, the best mutants were applied in a whole‐cell biotransformation. In these experiments the production (percentage) of 15β‐hydroxyprogesterone decreased from 50.4 % (wild type) to 4.8 % (mutant T89N/A395I), whereas that of 11α‐hydroxyprogesterone increased from 27.7 to 80.9 %, thus demonstrating an impressive regioselectivity.     

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. (CHMO_Arthro and CHMO_Acineto) 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.     

Relationship between the Bond Dissociation Energies and Impact Sensitivities of Some Nitro‐Explosives

The bond dissociation energy (BDE) for removal of the NO₂ group for eleven CHNO nitro‐containing explosive molecules is studied to find its correlation with impact sensitivity. The BDE for removal of the NO₂ group in nitroaromatic molecules with nitro alkyl, and esters with nitro alkyl, is calculated using the B3LYP method of Density Functional Theory with the 6‐31G* basis set. The relationship between the impact sensitivities and the weakest C‐NO₂ bond dissociation energy values is examined. The results indicate a nearly linear correlation between the impact sensitivity and the ratio of the BDE value to the total molecular energy.     

On the Chemical Reactions of Diphenylamine and its Derivatives with Nitrogen Dioxide at Normal Storage Temperature Conditions

The chemical reactions between nitrogen dioxide vapour and diphenylamine (DPA), some of its nitro/nitroso derivatives and between their consecutive products have been studied at temperatures of about 23 °C, whereby cellulose was used as substrate material. The following stabilizer compounds were used as starting components: DPA, N‐NO‐DPA, 2‐NO₂‐DPA and 4‐NO₂‐DPA. Concentration profiles as function of added nitrogen dioxide have been measured by HPLC. These profiles are similar to the ones obtained by accelerated aging, and the typical consecutive stabilizer products have been formed. The profiles are interpreted in terms of possible reactions. Furthermore, the influences of time and light on the decomposition of the mono‐nitro‐N‐nitroso compounds have been investigated. The consequences of these reactions are discussed to explain differences between the storage aging at ambient temperatures and the higher temperature induced accelerated aging. The reactivity towards nitrogen dioxide for each stabilizer compound was obtained by a kinetic model for the decrease of the starting component.     

On the nature of the burning rate-controlling reaction of energetic materials for the gas-phase model

For model systems with known kinetics of elementary reactions (CH₃NO₂ and HN₃), temperature ranges are established in which the rate-controlling reactions are the initial endothermic decomposition of the starting material or the subsequent secondary reactions. Heat release in reactions of NO₂, NO, and N₂O with various fuels, such as CH₂O, CO, H₂, and HCN, is modeled to establish the kinetic parameters and nature of the rate-controlling reactions in gas flames of nitro compounds. It is shown that the activation energy of the heat-release reaction due to the interaction of NO₂ with a hydrocarbon fuel (which is characteristic of the first flame of nitro compounds) is in the range of 29–33 kcal/mole, depending on the type of fuel. According to the calculations performed, the activation energy of the rate-controlling heat-releasing process due to the deoxidation of NO and N₂O (which is typical of the second flame of nitro compounds) is 43–58 kcal/mole. In the range of high pressures, where the flames merge, the kinetic parameters of heat release are determined by the reactions of the most reactive nitrogen oxide NO₂. __________ Translated from Fizika Goreniya i Vzryva, Vol. 43, No. 3, pp. 59–71, May–June, 2007.     

Preparation of organo‐soluble poly[(2,2′‐m‐phenylene)‐5,5′‐bibenzimidazole] with high yield by homogeneous nitration reaction

To prepare organo‐soluble poly[(2,2,′‐m‐phenylene)‐5,5′‐bibenzimidazole] (PBI) with high yield, a homogeneous nitration of PBI was attempted. Nitro‐substituted PBI (NO₂‐PBI) was synthesized through the homogeneous reaction of the PBI powder with nitric acid in sulfuric acid. The degree of substitution (DS) of this NO₂‐PBI is higher than that of the NO₂‐PBI prepared through the heterogeneous reaction of the PBI fiber. The viscosity of the NO₂‐PBI prepared through the homogeneous reaction decreased with increasing amount of nitric acid added. The DS of the NO₂‐PBI reached the maximum value of 2. The substitution efficiency of nitro groups decreased as the amount of nitric acid added increased. When a small quantity of nitric acid was added, the substitution of the sulfonic acid group was confirmed as well as that of the nitro group. The solubility of the NO₂‐PBI depended strongly on the DS. The NO₂‐PBI having the DS of about 2 was completely soluble in dimethylacetamide and almost soluble in N‐methylpyrrolidone. At an elevated temperature, it was also soluble in other polar aprotic solvents such as dimethylformamide and dimethylsulfoxide. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 78: 438–445, 2000     

Protein Arginine N‐Methyltransferase Substrate Preferences for Different Nη‐Substituted Arginyl Peptides

Protein arginine N‐methyltransferases (PRMTs) catalyze methyl‐group transfer from S‐adenosyl‐L ‐methionine onto arginine residues in proteins. In this study, modifications were introduced at the guanidine moiety of a peptidyl arginine residue to investigate how changes to the PRMT substrate can modulate enzyme activity. We found that peptides bearing Nη‐hydroxy or Nη‐amino substituted arginine showed higher apparent k_cat values than for the monomethylated substrate when using PRMT1, whereas this catalytic preference was not observed for PRMT4 and PRMT6. Methylation by compromised PRMT1 variants E153Q and D51N further supports the finding that the N‐hydroxy substitution facilitates methyl transfer by tuning the reactivity of the guanidine moiety. In contrast, Nη‐nitro and Nη‐canavanine substituted substrates inhibit PRMT activity. These findings demonstrate that methylation of these PRMT substrates is dependent on the nature of the modification at the guanidine moiety.     

Biological Nutrient Removal Using a Novel Laboratory‐Scale Twin Fluidized‐Bed Bioreactor

The capability of biological nutrient removal from wastewater of a novel laboratory‐scale twin fluidized‐bed bioreactor (TFBBR) was studied. The work showed approximately 96 % organic matter, 84 % nitrogen, and 12 % phosphorus removal efficiencies in the first three phases of the study at influent synthetic municipal wastewater (SMW) flow rates of 150, 190, and 240 L/d, with corresponding organic loading rates of 1.3, 1.7, and 2.3 kg COD m^–3 d^–1 and nitrogen loading rates of 0.14, 0.18 and 0.25 kg N m^–3 d^–1. The TFBBR effluent was characterized by <1.0 mg NH₄‐N/L, <4.3 mg NO₃‐N/L, <6 mg TN/L, <6 mg SBOD/L, and 6–10 mg VSS/L. For the three phases, biomass yields of 0.06, 0.066, and 0.071 g VSS/g COD were observed, respectively, which was a significant further reduction in yield compared to the liquid‐solid circulating fluidized‐bed bioreactor technology developed and patented by this research group, of 0.12–0.16 g VSS/g COD. The very low yield was due to a longer solid retention time of 72–108 d.     

Effect of H2 admixture on the adsorption of NO,NO2 and propane at Ag/Al2O3 catalyst as examined by in situ FTIR

The interaction of DeNO_x components comprising NO, NO₂, propane, O₂ and H₂ with a selected Ag/Al₂O₃ catalyst was studied by in situ FTIR spectroscopy at a fixed temperature where a promoting effect of H₂ admixture on the catalytic NO_x reduction has been reported to occur. The significant enhancement of nitrato and acetate ad-species from NO and propane, respectively, could be confirmed. New findings are the relative inertness of these species for further reaction progress. Instead, nitrite, nitrito, and nitro species are reactive, and the H₂ co-fed favours the formation of those species. Nevertheless, the way H₂ interferes with the kind of species formed includes the promotion of oxidative reaction steps evidenced by different effect of H₂ on the interaction of the catalyst with NO/O₂ and NO₂, respectively. During NO₂/H₂ adsorption adsorbed NO_x species and Ag⁺ ions at the surface are reduced. This prevents abundant formation of stable nitrato species and favour the formation of largely unstable but reactive nitro and nitrito species. Otherwise, NO₂ is able to oxidize pre-reduced Ag/Al₂O₃. Furthermore, indications were found for minor propene formation from propane in the presence of hydrogen.     

Study of the Daughter Products of Akardite‐II

In this work, five akardite‐II (AK‐II) daughter products were produced and isolated. Their respective MS, NMR, IR and UV spectra were recorded. These five products were positively identified as: N‐NO‐AK‐II, 2‐NO₂‐AK‐II, 4‐NO₂‐AK‐II, N‐NO‐2‐NO₂‐AK‐II and N‐NO‐4‐NO₂‐AK‐II. An HPLC method giving baseline separation of 6 AK‐II family products and 10 diphenylamine (DPA) family products is also described. Finally, preliminary results concerning the stability of AK‐II daughter products are discussed.     

Non‐natural Peptide Triazole Antagonists of HIV‐1 Envelope gp120

We investigated the derivation of non‐natural peptide triazole dual receptor site antagonists of HIV‐1 Env gp120 to establish a pathway for developing peptidomimetic antiviral agents. Previously we found that the peptide triazole HNG‐156 [R‐I‐N‐N‐I‐X‐W‐S‐E‐A‐M‐M‐CONH₂, in which X=ferrocenyltriazole‐Pro (FtP)] has nanomolar binding affinity to gp120, inhibits gp120 binding to CD4 and the co‐receptor surrogate mAb 17b, and has potent antiviral activity in cell infection assays. Furthermore, truncated variants of HNG‐156, typified by UM‐24 (Cit‐N‐N‐I‐X‐W‐S‐CONH₂) and containing the critical central stereospecific ^LX‐^LW cluster, retain the functional characteristics of the parent peptide triazole. In the current work, we examined the possibility of replacing natural with unnatural residue components in UM‐24 to the greatest extent possible. The analogue with the critical “hot spot” residue Trp 6 replaced with L ‐3‐benzothienylalanine (Bta) (KR‐41), as well as a completely non‐natural analogue containing D ‐amino acid substitutions outside the central cluster (KR‐42, ^DCit‐^DN‐^DN‐^DI‐X‐Bta‐^DS‐CONH₂), retained the dual receptor site antagonism/antiviral activity signature. The results define differential functional roles of subdomains within the peptide triazole and provide a structural basis for the design of metabolically stable peptidomimetic inhibitors of HIV‐1 Env gp120.     

Nitrogen Production Efficiency for Acetaldehyde and Propionaldehyde in Lean NO x Reduction over Anatase Titania

The nitrogen formation efficiencies, defined as the number of nitrogen molecules formed for every reductant molecule consumed were determined for acetaldehyde and propionaldehyde in the reduction of NO₂ under lean conditions over anatase TiO₂. The efficiency increased with increasing NO₂/reductant in the feed, reaching 0.61 ± 0.05 for NO₂/acetaldehyde between 1.5 and 3.0 and 0.85 ± 0.08 for NO₂/propionaldehyde between 3.8 and 5.7. Simultaneously, the CO/CO₂ ratio as well as the small concentration of N₂O in the product stream increased. The results suggested that at higher NO₂/reductant ratios, the reaction between the reductant and adsorbed NO₂ accounted for a large majority of the reaction. The results were consistent with the IR results, which showed that surface nitro groups reacted readily with acetaldehyde. The implications of these results on the NO₂ reduction mechanism were discussed.     

Computational Studies on Nitro,Nitramino, and Dinitramino Derivatives of 1‐Aminoazadiboridine as High Energetic Material

Computational studies were performed to determine the thermodynamic and explosive characteristics of high energy materials formed by placing explosophores such as nitro (−NO₂), nitramino (−NHNO₂), and dinitramino (−N(NO₂)₂) groups on 1‐aminoazadiboridine. G3 level calculations were made to determine the gas phase heat of formation of the designed species. In addition to the above, condensed phase heat of formation was also determined by evaluating the sublimation enthalpy. Crystal densities of title compounds were predicted with the help of a wave function analysis (WFA) program and were found to be in the range of 1.55–1.83 g cm⁻³. Bond dissociation energies of various possible bond rupture routes of the designed molecules were calculated at DFT‐B3LYP/6‐311G(d,p) level and attempt was made to identify the trigger linkage. Impact sensitivity was evaluated theoretically by employing a method based on statistical parameters determined from electrostatic potential data. Results show that the designed molecules are highly energetic and their corresponding detonation properties place them in the category of safe and high performance explosive materials.     

The existence of dual Cu site involved in the selective catalytic reduction of NO with propene on Cu/ZSM-5

In order to establish the role of surface species in the selective catalytic reduction (SCR), in situ IR studies were carried out using a DRIFT (diffuse reflectance infrared Fourier transform) cell in gas mixtures of various C₃H₆/NO ratios containing excess oxygen. The location and mobility of Cu ions were investigated by recording the relevant bands of CO adsorbed on Cu/ZSM-5. The nitro species coordinated on Cu²⁺ and the -NCO surface complex as possible intermediates were observed in the reduction of NO with propene on Cu/ZSM-5 between 350 and 400°C. The reactivities of these species toward NO, O₂ and propene were examined. The nitro species can react with propene very rapidly to form N₂ without the formation of NCO species. NCO also reacts with NO₂ and/ or NO at 350°C. IR spectra of CO adsorbed on cuprous ions show that two kinds of Cu ions, which are responsible for the activation of NO and propene respectively, exist on Cu/ZSM-5. From these results, a dual site mechanism involving nitro species and -NCO species as intermediates is suggested.     

Fischer–Tropsch synthesis in slurry-phase reactors over Mn- and Zr-modified Co/SiO2 catalysts

Fischer–Tropsch (F–T) synthesis was carried out in a gas-flowed slurry-phase reaction system over Mn- and Zr-modified Co/SiO₂ catalysts. A 0.5 L stirred tank slurry reactor (STSR) was used for catalyst screening and a 12.5 L slurry bubble column reactor (SBCR) was used for trial pilot operation. While using the 0.5 L reactor for catalyst screening, Co supported on the SiO₂ with an average pore size of 10 nm showed a high catalytic performance for the F–T synthesis due to the suitable Co particle size in the catalyst. Zr promoter improved the activity and Mn promoter improved the stability of Co/SiO₂ catalyst for the F–T synthesis. H₂-TPR profiles indicated that Zr and Mn promoters improved the reduction degree of Co₃O₄ particles (on SiO₂ surface) to Co⁰ active species in H₂ flow at low temperature. While using the 12.5 L reactor for trial pilot operation over Mn–Zr–Co/SiO₂ catalyst, the space-time yield (STY) of C₅₊ hydrocarbons (liquid fuel) showed almost the same values when various solvents (n-C₁₆H₃₄, n-C₁₄H₃₀, diesel from petrol station, F–T crude oil) were used. Diesel and F–T crude oil are suitable for using in a large-scaled F–T synthesis plant owing to the low prices. Mn–Zr–Co/SiO₂ catalyst achieved a STY of C₅₊ hydrocarbons larger than 1000 g-C₅₊ kg-cat^? 1 h^? 1 in the 12.5 L reactor. The production capacity of liquid fuel from the 12.5 L reactor reached to 15.6 L per day (assumed for 24 h continuous operation). The stirring was very important for the F–T synthesis both reaction in the 0.5 L reactor and reaction in the 12.5 L reactor. The shape of slurry reactor also influenced the CO conversion for the F–T synthesis: reaction in the 12.5 L SBCR gave a higher CO conversion than that of reaction in the 0.5 L STSR (at the same W/F value under the same stirring speed) because the slender column reactor (SBCR) extended the residue time of reaction gas in the slurry-phase containing catalyst.     

Effect of Silver Loading on the Lean NOx Reduction with Methanol Over Ag–Al2O3

The influence of silver loading on the lean NO_x reduction activity using methanol as reductant has been studied for alumina supported silver catalysts. In general, increasing the silver loading (0–3 wt%), in Ag–Al₂O₃, shifts or extends the activity window, for lean NO_x reduction towards lower temperatures. In particular Ag–Al₂O₃ with 3 wt% silver is active for NO_x reduction under methanol-SCR conditions in a broad temperature interval (200–500 °C), with high activity in the low temperature range (maximum around 300 °C) typical for exhaust gases from diesel and other lean burn engines. Furthermore, increasing the C/N molar ratio enhances the reduction of NO_x. However, too high C/N ratios results in poor selectivity to N₂.     

Single Step Synthesis of Nitro‐Functionalized Hydroxyl‐Terminated Polybutadiene

The paper reports the energization of Hydroxyl‐Terminated Polybutadiene (HTPB) by functionalizing explosophore  NO₂ over the HTPB backbone, resulting in the formation of conjugated nitro‐alkene derivative of HTPB. A convenient, inexpensive and efficient “one pot” procedure of synthesizing Nitro‐Functionalized Hydroxyl‐Terminated Polybutadiene (Nitro‐HTPB) is reported. The reaction was carried out with sodium nitrite and iodine. To retain the unique physico‐chemical properties of HTPB, functionalization by  NO₂ group was restricted to 10 to 15 % of double bonds. The Nitro‐HTPB was characterized by FTIR, ¹H NMR, VPO, DSC, TGA etc. The polymer has shown good thermal stability for practical applications. The kinetic parameters for the decomposition of Nitro‐HTPB at 150–300 °C were obtained from non‐isothermal DSC data.     

An Overview on the Synthetic Routes and Properties of Ammonium Dinitramide (ADN) and other Dinitramide Salts

In recent years, there has been a considerable interest in the development of novel type of high performance propellants for use in solid rocket motors. Ammonium salt of dinitramidic acid NH₄N(NO₂)₂ (ADN) has attracted wide interest as a potentially useful energetic oxidizer for rocket propellants because of its clean and environment‐friendly exhaust products during burning. ADN contains one N (NO₂)₂ group and its synthesis requires new type of N‐nitration. The present paper reviews the general synthetic methods used for the synthesis of inorganic, organic and metal dinitramide salts and their properties, with a special emphasis on ammonium dinitramide. The salient features with reference to the extent of conversion and ease of separation of the products of the various synthetic methodologies are also addressed.     

Potential reaction intermediates of NOx reduction with propane over Cu/ZSM-5

Adsorption complexes that are formed by exposing Cu/ZSM-5 to nitric oxides have been identified by FTIR. Some of them are thermally decomposed below 200°C, but others, though much more stable thermally, are reduced at low temperature by chemical interaction with gaseous propane. The FTIR spectra suggest that the latter complexes are nitro and/or nitrate groups associated with Cu ions. Their formation and reduction when Cu/ZSM-5 is exposed to NO + O₂ + C₃H₈ seems consistent with the assumption that they are intermediates in catalytic NO_x reduction. Mass spectrometric analysis of the gases that are released when these complexes react with propane reveals formation of N₂, whereas thermal decomposition at higher temperatures only yields NO₂, NO, H₂O, traces of O₂, but no N₂. The results suggest that adsorbed NO_y, groups (y ? 2) are able to abstract H atoms from hydrocarbon molecules.     

Nitro-Oleic Acid Reduces J774A.1 Macrophage Oxidative Status and Triglyceride Mass: Involvement of Paraoxonase2 and Triglyceride Metabolizing Enzymes

Nitro‐fatty acids possess anti‐atherogenic properties, but their effects on macrophage oxidative status and lipid metabolism that play important roles in atherosclerosis development are unclear. This study compared the effects of nitro‐oleic acid (OLA‐NO₂) with those of native oleic acid (OLA) on intracellular reactive oxygen species (ROS) generation, anti‐oxidants and metabolism of triglycerides and cholesterol in J774A.1 macrophages. Upon incubating the cells with physiological concentrations of OLA‐NO₂ (0–1 µM) or with equivalent levels of OLA, ROS levels measured by 2, 7‐dichlorofluorescein diacetate, decreased dose‐dependently, but the anti‐oxidative effects of OLA‐NO₂ were significantly augmented. Copper ion addition increased ROS generation in OLA treated macrophages without affecting OLA‐NO₂ treated cells. These effects could be attributed to elevated glutathione levels and to increased activity and expression of paraoxonase2 that were observed in OLA‐NO₂vs OLA treated cells. Beneficial effects on triglyceride metabolism were noted in OLA‐NO₂vs OLA treated macrophages in which cellular triglycerides were reduced due to attenuated biosynthesis and accelerated hydrolysis of triglycerides. Accordingly, OLA‐NO₂ treated cells demonstrated down‐regulation of diacylglycerol acyltransferase1, the key enzyme in triglyceride biosynthesis, and increased expression of hormone‐sensitive lipase and adipose triglyceride lipase that regulate triglyceride hydrolysis. Finally, OLA‐NO₂vs OLA treatment resulted in modest but significant beneficial effects on macrophage cholesterol metabolism, reducing cholesterol biosynthesis rate and low density lipoprotein influx into the cells, while increasing high density lipoprotein‐mediated cholesterol efflux from the macrophages. Collectively, compared with OLA, OLA‐NO₂ modestly but significantly reduces macrophage oxidative status and cellular triglyceride content via modulation of cellular anti‐oxidants and triglyceride metabolizing enzymes.