Enzymatic asymmetric sulfoxidation using molecular oxygen as the oxidant is a promising green chemistry approach to chiral sulfoxide production. Despite the broad substrate spectrum of cyclohexanone monooxygenases (CHMOs), some unnatural substrates with bulky functional groups, such as the pharmaceutically relevant omeprazole sulfide, cannot be effectively accepted by CHMOs. Herein, we describe a set of variants derived from an Acinetobacter calcoaceticus CHMO (AcCHMO), whose active sites adjacent to the substrate tunnel were altered to shift the substrate specificity from cyclohexanone monooxygenation toward omeprazole sulfide sulfoxidation. We performed homologous modeling and molecular docking to identify key residues that might affect the substrate specificity. Two libraries of residues lining the active center of AcCHMO were then constructed and screened by an effective halo-based selection method using the solubility difference between the substrate (omeprazole sulfide) and product (esomeprazole). Functional evaluation of the resultant variants showed that the substrate specificity of AcCHMO was markedly altered from the small natural substrate (cyclohexanone) toward the desired bulky substrate (omeprazole sulfide) despite the extremely poor activity detected even for the best variant, M2 (0.61 U/ g prot ). The crystal structure of M2 complexed with a flavin adenine dinucleotide (FAD) prosthetic group was determined, which provided insight into the altered substrate specificity. To improve the activity of enzyme M2 toward pharmaceutical precursor omeprazole sulfide, we performed both local and global protein engineering among the two CASTing libraries surrounding FAD + and NADP + prosthetic groups and an error-prone PCR library of the full-length AcCHMO. As a result, variant M6 was obtained, giving a 50-fold higher activity compared to M2. This structure-guided protein engineering of AcCHMO provided a promising candidate for converting omeprazole sulfide into (S)-omeprazole using a green biocatalytic method.

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Aims Propofol may result in hypotension, bradycardia and loss of protective reflexes, especially in elderly patients, while esketamine, a N‐methyl‐D‐aspartate receptor antagonist, has analgesic, anaesthetic and sympathomimetic properties and is known to cause less cardiorespiratory depression. We hypothesized that esketamine may reduce the median effective concentration (EC50) of propofol and coadministration is less likely to produce hypotension during gastrointestinal endoscopy in elderly patients. Methods Ninety elderly patients, aged 65–89 years, undergoing gastrointestinal endoscopy were randomly assigned into 3 groups: SK0 (control) group (0 mg/kg esketamine); SK0.25 group (0.25 mg/kg esketamine); and SK0.5 group (0.5 mg/kg esketamine). Anaesthesia was achieved by plasma target‐controlled infusion of propofol with different bolus doses of esketamine. The EC50 of propofol for gastrointestinal endoscopy was determined by using the up‐and‐down method of Dixon. The initial plasma target concentration is 2.5 μg/mL and the adjacent concentration gradient is 0.5 μg/mL. Cardiovascular variables were also measured. Results Propofol EC50s and its 95% confidence interval for gastrointestinal endoscopy in elderly patients were 3.69 (2.59–4.78), 2.45 (1.85–3.05) and 1.71 (1.15–2.27) μg/mL in the SK0, SK0.25 and SK0.5 groups, respectively (P < .05). The average percent change from baseline mean arterial pressure was −19.7 (7.55), −15.2 (7.14) and −10.1 (6.73), in the SK0, SK0.25 and SK0.5 groups, respectively (P < .001). Conclusion Combination medication of propofol with esketamine reduced the propofol EC50 during gastrointestinal endoscopy in elderly patients compared with administration of propofol without esketamine. Increasing doses of SK with propofol are less likely to produce hypotension with shorter recovery time.

Esomeprazole is the most popular proton pump inhibitor (PPI) for treating gastroesophageal reflux disease. Enzymatic asymmetric sulfoxidation is a green approach to produce chiral sulfoxides. In this report, we focused on optimizing asymmetric sulfoxidation catalyzed by prazole sulfide monooxygenase (AcPSMO). The costly redox cofactor NADPH utilized by AcPSMO was regenerated by formate dehydrogenase with CO 2 as the coproduct, which can be removed easily. During the scale-up process, oxygen supply was found to be the main limiting factor during the early phase of the reaction, while the instability of AcPSMO and the lack of the cofactor NADPH hindered progress during the middle and late phases of the 0.6 L reaction. Finally, by adjusting oxygen mass transfer and increasing the dissolved oxygen, the enzymatic reaction was stepwise amplified to a 120 L scale using a 300 L thermostatic stirred reactor, affording 95.9% conversion and 99.9% enantiomeric excess after 12 h. Extraction and refinement of the product resulted in 0.39 kg of the isolated esomeprazole (sodium salt), with 57.8% overall yield (73.4% before the salt-forming reaction) and 99.1% purity. Thus, a green-by-design system was constructed for the efficient and precise oxidation of omeprazole sulfide into esomeprazole with molecular O 2 as the green cosubstrate and CO 2 and H 2 O as byproducts.

The serine threonine kinase Akt1 has been implicated in the control of cellular metabolism, survival and growth. Herein, disruption of the ubiquitously expressed member of the Akt family of genes, Akt1, in the mouse, demonstrates a requirement for Akt1 in miRNA-mediated cellular apoptosis. The miR-17/20 cluster is known to inhibit breast cancer cellular proliferation through G1/S cell cycle arrest via binding to the cyclin D1 3'UTR. Here we show that miR-17/20 overexpression sensitizes cells to apoptosis induced by either Doxorubicin or UV irradiation in MCF-7 cells via Akt1. miR-17/20 mediates apoptosis via increased p53 expression which promotes Akt degradation. Akt1 −/− mammary epithelial cells which express Akt2 and Akt3 demonstrated increased apoptosis to DNA damaging agents. Akt1 deficiency abolished the miR-17/20-mediated apoptosis. These results demonstrated a novel pathway through which miR17/20 regulate p53 and Akt controlling breast cancer cell apoptosis.

Two Baeyer-Villiger monooxygenases (BVMOs), designated BVMO andBVMO, were discovered from and, respectively. Both monooxygenases displayed novel features for catalyzing the asymmetric sulfoxidation of bulky and pharmaceutically relevant thioethers. Evolutionary relationship and sequence analysis revealed that the two BVMOs belong to the family of typical type I BVMOs and the subtype ethionamide monooxygenase. Both BVMOs are active toward medium- and long-chain aliphatic ketones as well as various thioether substrates but are ineffective toward cyclohexanone, aromatic ketones, and other typical BVMO substrates. BVMO andBVMO showed the highest activities (0.117 and 0.025 U/mg protein, respectively) toward thioanisole among the tested substrates. Furthermore, these BVMOs exhibited distinct activity and excellent stereoselectivity toward bulky and prochiral prazole thioethers, which is a unique feature of this family of BVMOs. No native enzyme has been reported for the asymmetric sulfoxidation of bulky prazole thioethers into chiral sulfoxides. The identification of BVMO andBVMO provides an important scaffold for discovering enzymes capable of asymmetrically oxidizing bulky thioether substrates by genome mining. Baeyer-Villiger monooxygenases (BVMOs) are valuable enzyme catalysts that are an alternative to the chemical Baeyer-Villiger oxidation reaction. Although BVMOs display broad substrate ranges, no native enzymes were reported to have activity toward the asymmetric oxidation of bulky prazole-like thioether substrates. Herein, we report the discovery of two type I BVMOs from (BVMO) and (BVMO) which are able to catalyze the asymmetric sulfoxidation of bulky prazole thioethers (proton pump inhibitors [PPIs], a group of drugs whose main action is a pronounced and long-lasting reduction of gastric acid production). Efficient catalysis of omeprazole oxidation by BVMO was developed, indicating that this enzyme is a promising biocatalyst for the synthesis of bulky and pharmaceutically relevant chiral sulfoxide drugs. These results demonstrate that the newly identified enzymes are suitable templates for the discovery of more and better thioether-converting BVMOs.

An efficient aryl to vinyl 1,4-palladium migration/Heck sequence was developed for the stereoselective synthesis of 1,3-dienes. High stereoselectivity was observed not only for 1,3-dienes bearing two similar aryl groups at terminal positions, but also for those with configurations shown to be unfavorable with previous methods.

[structure: see text] Three strands of natural collagen are linked by covalent bonds prior to their folding into a triple helix. We report on a synthetic collagen in which the strands are pendent on a rigid macrocyclic scaffold of C(3) symmetry. The scaffold confers substantial conformational stability upon the collagen triple helix and makes its folding independent of concentration, both desirable attributes for exploring and exploiting synthetic collagens.