Cytochrome P450-catalyzed insertion of carbenoids into N–H bonds

Expanding nature’s catalytic repertoire to include reactions important in synthetic chemistry will open new opportunities for ‘green’ chemistry and biosynthesis. We demonstrate the first enzyme-catalyzed insertion of carbenoids into N–H bonds. This type of bond disconnection, which has no counterpart in nature, can be mediated by variants of the cytochrome P450 from Bacillus megaterium. The N–H insertion reaction takes place in water, provides the desired products in 26–83% yield, forms the single addition product exclusively, and does not require slow addition of the diazo component

P450-catalyzed asymmetric cyclopropanation of electron-deficient olefins under aerobic conditions

A variant of P450 from Bacillus megaterium five mutations away from wild type is a highly active catalyst for cyclopropanation of a variety of acrylamide and acrylate olefins with ethyl diazoacetate (EDA). The very high rate of reaction enabled by histidine ligation allowed the reaction to be conducted under aerobic conditions. The promiscuity of this catalyst for a variety of substrates containing amides has enabled synthesis of a small library of precursors to levomilnacipran derivatives

Analysis of different methods for wave generation and absorption in a CFD-based numerical wave tank

In this paper, the performance of different wave generation and absorption methods in computational fluid dynamics (CFD)-based numerical wave tanks (NWTs) is analyzed. The open-source CFD code REEF3D is used, which solves the Reynolds-averaged Navier-Stokes (RANS) equations to simulate two-phase flow problems. The water surface is computed with the level set method (LSM), and turbulence is modeled with the k-\u3c9 model. The NWT includes different methods to generate and absorb waves: the relaxation method, the Dirichlet-type method and active wave absorption. A sensitivity analysis has been conducted in order to quantify and compare the differences in terms of absorption quality between these methods. A reflection analysis based on an arbitrary number of wave gauges has been adopted to conduct the study. Tests include reflection analysis of linear, second- and fifth-order Stokes waves, solitary waves, cnoidal waves and irregular waves generated in an NWT. Wave breaking over a sloping bed and wave forces on a vertical cylinder are calculated, and the influence of the reflections on the wave breaking location and the wave forces on the cylinder is investigated. In addition, a comparison with another open-source CFD code, OpenFOAM, has been carried out based on published results. Some differences in the calculated quantities depending on the wave generation and absorption method have been observed. The active wave absorption method is seen to be more efficient for long waves, whereas the relaxation method performs better for shorter waves. The relaxation method-based numerical beach generally results in lower reflected waves in the wave tank for most of the cases simulated in this study. The comparably better performance of the relaxation method comes at the cost of larger computational requirements due to the relaxation zones that have to be included in the domain. The reflections in the NWT in REEF3D are generally lower than the published results for reflections using the active wave absorption method in the NWT based on OpenFOAM

Periprosthetic joint infection of a total hip arthroplasty with Candida parapsilosis

INTRODUCTION: Fungal periprosthetic joint infection (PJI) is a disruptive and complex complication of joint arthroplasty. We present a case of a fungal PJI with Candida parapsilosis after a total hip arthroplasty (THA). PRESENTATION OF CASE: A 73-year-old woman with a history of ovarian cancer with peritoneal metastases, was treated with a THA, due to symptomatic arthritis of the right hip. One month after surgery, she had difficulties walking. Inflammatory parameters were mildly increased. Aspiration of a subcutaneous abscess diagnosed Candida parapsilosis. A two-stage revision arthroplasty without spacer was performed. During a six-week prosthesis-free interval, intravenous fluconazole 400 mg was given. After reimplantation, fluconazole was continued for two weeks intravenously and life-long perorally. Follow-up of the patient after six months showed no recurrence of infection. DISCUSSION: This case revealed that when PJI is suspected, a low treshold for joint aspiration is important. Two-stage revision with systematic antifungal therapy is the preferred treatment of fungal PJI. Our case demonstrated a good result with a prosthesis-free interval. Fluconazole is the preferred antifungal treatment and it should be applied for at least six months or longer. CONCLUSION: To our knowledge, this is the first case of a fungal PJI with Candida parapsilosis after a THA treated with a two-stage revision arthroplasty without spacer and a life-long fluconazole treatment. (C) 2020 The Authors. Published by Elsevier Ltd on behalf of IJS Publishing Group Ltd

Improved Cyclopropanation Activity of Histidine-Ligated Cytochrome P450 Enables the Enantioselective Formal Synthesis of Levomilnacipran

Engineering enzymes capable of modes of activation unprecedented in nature will increase the range of industrially important molecules that can be synthesized through biocatalysis. However, low activity for a new function is often a limitation in adopting enzymes for preparative-scale synthesis, reaction with demanding substrates, or when a natural substrate is also present. By mutating the proximal ligand and other key active-site residues of the cytochrome P450 enzyme from Bacillus megaterium (P450-BM3), a highly active His-ligated variant of P450-BM3 that can be employed for the enantioselective synthesis of the levomilnacipran core was engineered. This enzyme, BM3-Hstar, catalyzes the cyclopropanation of N,N-diethyl-2-phenylacrylamide with an estimated initial rate of over 1000 turnovers per minute and can be used under aerobic conditions. Cyclopropanation activity is highly dependent on the electronic properties of the P450 proximal ligand, which can be used to tune this non-natural enzyme activity

Nitrogen in water-Portugal and Denmark: two contrasting realities

ReviewAgricultural activities are responsible for most of the nitrogen (N) inputs that degrade water quality. To elucidate the drivers leading to N pressures on water, we examined the resulting state of surface waters in terms of N concentrations, the impact of this on water quality status and policy responses to these constraints across different climatic and management conditions. Portugal and Denmark were chosen as contrasting case studies for the Driver-Pressure-State-Impact-Response (DPSIR) analysis. Our results showed reductions of 39% and 25% in the use of mineral fertilizer in Portugal and Denmark, respectively, between 2000 and 2010. The N surplus in Portugal varied between 15 and 30 kg N ha -1 between 1995 and 2015. In Denmark, in 2015, this amount was 70 kg N ha -1, representing a 53% decrease from the 1990 value. The average amount of total N discharged to surface waters was 7 kg ha -1 for mainland Portugal in 2015 and 14.6 kg ha -1 for Denmark in 2014. These reductions in the N surplus were attributed to historical policies aimed at N pressure abatement. In Denmark, N losses are expected to decline further through the continuation or improvement of existing national action plans. In Portugal, they are expected to decline further due to the expansion of Nitrate Vulnerable Zones and the introduction of targeted policies aimed at improving N use effciency and reducing losses to waterinfo:eu-repo/semantics/publishedVersio

Highly Stereoselective Biocatalytic Synthesis of Key Cyclopropane Intermediate to Ticagrelor

Extending the scope of biocatalysis to important non-natural reactions such as olefin cyclopropanation will open new opportunities for replacing multistep chemical syntheses of pharmaceutical intermediates with efficient, clean, and highly selective enzyme-catalyzed processes. In this work, we engineered the truncated globin of Bacillus subtilis for the synthesis of a cyclopropane precursor to the antithrombotic agent ticagrelor. The engineered enzyme catalyzes the cyclopropanation of 3,4-difluorostyrene with ethyl diazoacetate on a preparative scale to give ethyl-(1R, 2R)-2-(3,4-difluorophenyl)-cyclopropanecarboxylate in 79% yield, with very high diastereoselectivity (>99% dr) and enantioselectivity (98% ee), enabling a single-step biocatalytic route to this pharmaceutical intermediate