Chemoenzymatic total synthesis of rotigotine via IRED-catalyzed reductive amination.

A short and chemoenzymatic synthesis of rotigotine using an IR-36-M5 mutant is reported. Focusing on the residues that directly contact the 2-tetralone moiety, we applied structure-guided semi-rational design to obtain a double-mutant F260W/M147Y, which showed a good isolated yield and S-stereoselectivity >99% toward 2-aminotetralin synthesis. Furthermore, the utility of this biocatalytic protocol was successfully demonstrated in the enantioselective synthesis of rotigotine via enzymatic reductive amination as the key step.

An unexpected role of EasDaf: catalyzing the conversion of chanoclavine aldehyde to chanoclavine acid.

Ergot alkaloids (EAs) are a diverse group of indole alkaloids known for their complex structures, significant pharmacological effects, and toxicity to plants. The biosynthesis of these compounds begins with chanoclavine-I aldehyde (CC aldehyde, 2), an important intermediate produced by the enzyme EasDaf or its counterpart FgaDH from chanoclavine-I (CC, 1). However, how CC aldehyde 2 is converted to chanoclavine-I acid (CC acid, 3), first isolated from Ipomoea violacea several decades ago, is still unclear. In this study, we provide in vitro biochemical evidence showing that EasDaf not only converts CC 1 to CC aldehyde 2 but also directly transforms CC 1 into CC acid 3 through two sequential oxidations. Molecular docking and site-directed mutagenesis experiments confirmed the crucial role of two amino acids, Y166 and S153, within the active site, which suggests that Y166 acts as a general base for hydride transfer, while S153 facilitates proton transfer, thereby increasing the acidity of the reaction. KEY POINTS: • EAs possess complicated skeletons and are widely used in several clinical diseases • EasDaf belongs to the short-chain dehydrogenases/reductases (SDRs) and converted CC or CC aldehyde to CC acid • The catalytic mechanism of EasDaf for dehydrogenation was analyzed by molecular docking and site mutations.

Dual-purpose isocyanides produced by Aspergillus fumigatus contribute to cellular copper sufficiency and exhibit antimicrobial activity.

The maintenance of sufficient but nontoxic pools of metal micronutrients is accomplished through diverse homeostasis mechanisms in fungi. Siderophores play a well established role for iron homeostasis; however, no copper-binding analogs have been found in fungi. Here we demonstrate that, in Aspergillus fumigatus, xanthocillin and other isocyanides derived from the xan biosynthetic gene cluster (BGC) bind copper, impact cellular copper content, and have significant metal-dependent antimicrobial properties. xan BGC-derived isocyanides are secreted and bind copper as visualized by a chrome azurol S (CAS) assay, and inductively coupled plasma mass spectrometry analysis of A. fumigatus intracellular copper pools demonstrated a role for xan cluster metabolites in the accumulation of copper. A. fumigatus coculture with a variety of human pathogenic fungi and bacteria established copper-dependent antimicrobial properties of xan BGC metabolites, including inhibition of laccase activity. Remediation of xanthocillin-treated Pseudomonas aeruginosa growth by copper supported the copper-chelating properties of xan BGC isocyanide products. The existence of the xan BGC in several filamentous fungi suggests a heretofore unknown role of eukaryotic natural products in copper homeostasis and mediation of interactions with competing microbes.

Chemoenzymatic Synthesis of Selegiline: An Imine Reductase-Catalyzed Approach.

(R)-Homobenzylic amines are key structural motifs present in (R)-selegiline, a drug indicated for the treatment of early-stage Parkinson's disease. Herein, we report a new short chemoenzymatic approach (in 2 steps) towards the synthesis of (R)-selegiline via stereoselective biocatalytic reductive amination as the key step. The imine reductase IR36-M5 mutant showed high conversion (97%) and stereoselectivity (97%) toward the phenylacetone and propargyl amine substrates, offering valuable biocatalysts for synthesizing alkylated homobenzylic amines.

Peroxygenase-Enabled Reductive Kinetic Resolution for the Enantioenrichment of Organoperoxides.

Enantiomerically pure organoperoxides serve as valuable precursors in organic transformations. Herein, we present the first examples of unspecific peroxygenase catalyzed kinetic resolution of racemic organoperoxides through asymmetric reduction. Through meticulous investigation of the reaction conditions, it is shown that the unspecific peroxygenase from Agrocybe aegerita (AaeUPO) exhibits robust catalytic activity in the kinetic resolution reactions of the model substrate with turnover numbers up to 60000 and turnover frequency of 5.6 s-1. Various aralkyl organoperoxides were successfully resolved by AaeUPO, achieving excellent enantioselectivities (e.g., up to 99 % ee for the (S)-organoperoxide products). Additionally, we screened commercial peroxygenase variants to obtain the organoperoxides with complementary chirality, with one mutant yielding the (R)-products. While unspecific peroxygenases have been extensively demonstrated as a powerful oxidative catalysts, this study highlights their usefulness in catalyzing the reduction of organoperoxides and providing versatile chiral synthons.

Semi-rational design of an imine reductase for asymmetric synthesis of alkylated S-4-azepanamines.

Although imine reductase (IRED)-catalyzed reductive amination is promising for the synthesis of alkylated chiral amines, precisely regulating the stereoselectivity of IRED remains a great challenge. Herein, focusing on the residues directly in contact with the ketone moiety, we applied structure-guided semi-rational design to obtain the triple-mutant I149Y/L200H/W234K. This mutant showed high stereoselectivity, of up to >99% (S), toward reductive amination of N-Boc-4-oxo-azepane and different amines, and to the best of our knowledge is the first biocatalyst developed for asymmetric synthesis of chiral azepane-4-amines.

Tuning an Imine Reductase for the Asymmetric Synthesis of Azacycloalkylamines by Concise Structure-Guided Engineering.

Although imine reductases (IREDs) are emerging as attractive reductive aminases (RedAms), their substrate scope is still narrow, and rational engineering is rare. Focusing on hydrogen bond reorganization and cavity expansion, a concise strategy combining rational cavity design, combinatorial active-site saturation test (CAST), and thermostability engineering was designed, that transformed the weakly active IR-G36 into a variant M5 with superior performance for the synthesis of (R)-3-benzylamino-1-Boc-piperidine, with a 4193-fold improvement in catalytic efficiency, a 16.2 °C improvement in Tm , and a significant increase in the e.e. value from 78 % (R) to >99 % (R). M5 exhibits broad substrate scope for the synthesis of diverse azacycloalkylamines, and the reaction was demonstrated on a hectogram-scale under industrially relevant conditions. Our study provides a compelling example of the preparation of versatile and efficient IREDs, with exciting opportunities in medicinal and process chemistry as well as synthetic biology.

Total Synthesis and Target Identification of the Curcusone Diterpenes.

The curcusone natural products are complex diterpenes featuring a characteristic [6-7-5] tricyclic carbon skeleton similar to the daphnane and tigliane diterpenes. Among them, curcusones A-D demonstrated potent anticancer activity against a broad spectrum of human cancer cell lines. Prior to this study, no total synthesis of the curcusones was achieved and their anticancer mode of action remained unknown. Herein, we report our synthetic and chemoproteomics studies of the curcusone diterpenes which culminate in the first total synthesis of several curcusone natural products and identification of BRCA1-associated ATM activator 1 (BRAT1) as a cellular target. Our efficient synthesis is highly convergent, builds upon cheap and abundant starting materials, features a thermal [3,3]-sigmatropic rearrangement and a novel FeCl3-promoted cascade reaction to rapidly construct the critical cycloheptadienone core of the curcusones, and led us to complete the first total synthesis of curcusones A and B in only 9 steps, C and D in 10 steps, and dimericursone A in 12 steps. The chemical synthesis of dimericursone A from curcusones C and D provided direct evidence to support the proposed Diels-Alder dimerization and cheletropic elimination biosynthetic pathway. Using an alkyne-tagged probe molecule, BRAT1, an important but previously "undruggable" oncoprotein, was identified as a key cellular target via chemoproteomics. We further demonstrate for the first time that BRAT1 can be inhibited by curcusone D, resulting in impaired DNA damage response, reduced cancer cell migration, potentiated activity of the DNA damaging drug etoposide, and other phenotypes similar to BRAT1 knockdown.

Flow Chemistry-Enabled Divergent and Enantioselective Total Syntheses of Massarinolin†A, Purpurolides†B, D, E, 2,3-Deoxypurpurolide†C, and Structural Revision of Massarinolin†A.

Massarinolin A and purpurolides are bioactive bergamotane sesquiterpenes condensed with a variety of synthetically challenging ring systems: a bicyclo[3.1.1]heptane, an oxaspiro[3.4]octane, and a dioxaspiro[4.4]nonane (oxaspirolactone). Herein, we report the first enantioselective total syntheses of massarinolin A, purpurolides B, D, E, and 2,3-deoxypurpurolide C. Our synthesis and computational analysis also led to a structural revision of massarinolin A. The divergent approach features an enantioselective organocatalyzed Diels-Alder reaction to install the first stereogenic center in high ee, a scalable flow photochemical Wolff rearrangement to build the key bicyclo[3.1.1]heptane, a furan oxidative cyclization to form the oxaspirolactone, a late-stage allylic C-H oxidation, and a Myers' NBSH-promoted sigmatropic elimination to install the exo methylene group of massarinolin A.

A comparative study of patients' satisfaction with different levels of hospitals in Beijing: why do patients prefer high-level hospitals?

BACKGROUND: To promote the integration of medical resources, Beijing has built medical alliances since 2012, but this reform has not changed the state of disordered medical treatment. Patients are still willing to go to high-level hospitals for medical treatment. What causes patients to prefer high-level hospitals? To explore the reasons behind this preference for high-level medical treatment among patients and to guide patients to make visits in an orderly manner, we conducted this study and compared patients' satisfaction with hospitals of different levels in the context of medical resource integration. METHODS: This study conducted a questionnaire survey among 1250 patients who were selected in 18 medical alliances in Beijing from October to December 2016. The study type is a comparative study based on cross-sectional data. Patient satisfaction was the main outcome, and descriptive analysis, chi-square tests, nonparametric tests and binary logistic regression analysis were used. The level of statistical significance was set at p < 0.05. RESULTS: The overall satisfaction score of the medical alliances was 3.375, and the satisfaction scores of core hospitals and cooperative hospitals were 2.77 and 3.07, respectively. The overall patient satisfaction rate was 44.62%, and the satisfaction rates of core hospitals and cooperative hospitals were 34.34 and 50.43%, respectively. The type of hospital and understanding of medical alliance policy were the factors associated with patient satisfaction with the medical alliance. CONCLUSIONS: The patients' satisfaction with cooperative hospitals was higher than their satisfaction with core hospitals. Although the patients were more satisfied with the service attitude of the cooperative hospitals, they still preferred core hospitals due to their higher expectations for their medical treatment and greater trust in their medical technology. It is necessary to explore the establishment of closed medical alliances under the unified management of human and financial resources to enable medical alliances to become a community of common interests and provide integrated medical services for patients.

The willingness of patients to make the first visit to primary care institutions and its influencing factors in Beijing medical alliances: a comparative study of Beijing's medical resource-rich and scarce regions.

BACKGROUND: To improve the efficiency of the use of medical resources, China has implemented medical alliances (MAs) to implement a hierarchical diagnosis and treatment system. The willingness to undertake a first visit to primary care institutions (PCIs) is an important indicator of the effect of this system. Beijing has also built MAs since 2013, but to date, there have been few studies on the first visit to PCIs in Beijing. The purpose of this study is to analyze patients' willingness to make their first visit to PCIs and its influencing factors to provide references for the realization of a hierarchical diagnosis and treatment system. METHODS: Two relatively different districts with large differences in resources in Beijing, D and F, were selected, and a self-reported questionnaire and convenience sampling method were applied. A cross-sectional survey was administered to 1221 patients of MAs. The chi-square test and binary logistic regression were used to analyze the influencing factors of patients' willingness to undertake a first visit to a PCI. RESULTS: Fewer patients in District D received medical alliance services (44.42%) than those in District F (59.25%), but patients in District D had a higher degree of satisfaction with the services they received (72.04%) than those in District F (28.96%). Patients in District D had a higher willingness to undertake a first visit (64.00%) than those in District F (58.18%). Patients of an older age, low medical expenses, participation in urban employees' basic medical insurance, a high understanding of MAs and high satisfaction with medical services were indicators of being more willing to choose primary care institutions for their first visit. CONCLUSIONS: The different medical resources and MA constructions in the two districts have resulted in a difference between the two districts in terms of the willingness of individuals to make their first visit to PCIs. Strengthening the service capabilities of PCIs remains a priority. The government should propose solutions to solve the problems encountered in practice and actively promote the realization of MAs and hierarchical diagnosis and treatment.

Microwave-Assisted Intramolecular Ullmann Diaryl Etherification as the Post-Ugi Annulation for Generation of Dibenz[b,f][1,4]oxazepine Scaffold.

A sequence of the Ugi four-component reaction (U-4CR) and microwave-assisted intramolecular Ullmann etherification has been established for efficient generation of a dibenz[b,f][1,4]oxazepine scaffold. The U-4CR, using 2-aminophenols and 2-bromobenzoic acids or 2-bromobenzaldehydes as the inputs, was carried out in MeOH at 50-60 °C for 2-3 days to form a collection of 22 linear products in 46-90% yields. A microwave-assisted intramolecular Ullmann etherification was then used to transform these acyclic U-4CR products into the cleft-shaped 6/7/6-fused tricyclic heterocycles. The intramolecular Ullmann diaryl ether formation was catalyzed by 10 mol % of CuI and 30 mol % of N,N-dimethylglycine hydrochloride (DMG·HCl) in the presence of Cs2CO3 with microwave irradiation (150 °C, 30 min) to furnish dibenz[b,f][1,4]oxazepin-11(10H)-ones and dibenz[b,f][1,4]oxazepin-11(10H)-carboxamides in 64-100% yields.

Impact of the Essential Public Health Service program on financial protection and health outcomes among hypertensive patients: A quasi-experimental study in China.

BACKGROUND: The Chinese government launched the Essential Public Health Service (EPHS) program nationwide in 2009. However, prior studies have not provided clear and integrated evidence on whether the EPHS program improves health outcomes and prevents financial risks among individuals. Because hypertension is the chronic disease with the highest prevalence, this study evaluated the impact of the EPHS program among hypertensive patients to provide evidence for the progress of the program. METHODS: A cohort of hypertensive patients was identified from the 2011-2018 China Health and Retirement Longitudinal Study (CHARLS). The outcomes assessed included hospitalization expenditure, outpatient expenditure and cardiovascular disease (heart attack and stroke). The key independent variable was whether an individual received EPHS-covered blood pressure measurements in 2013-2015. Based on the International Health Partnership+ (IHP+) common monitoring and evaluation (M&E) framework, a difference-in-differences (DID) method with propensity score matching (PSM) was used to examine the impact of the EPHS program on hypertensive patients. RESULTS: The results showed that among hypertensive patients covered by the EPHS program, outpatient total costs/OOP costs were reduced by 29.8% and 30.8%, respectively, and hospitalization total costs/OOP costs were reduced by 34.9% and 35.6%, respectively. The EPHS program reduced the probability of heart attack and stroke among hypertensive patients by 3.5% and 2.7%, respectively. Mechanistic tests showed that the EPHS program improved health outcomes by reducing alcohol consumption and increasing physical activity, thereby further reducing health expenditure among hypertensive patients. The impacts of the EPHS program on hypertensive patients varied by age, educational attainment, residential region, and alcohol consumption status. CONCLUSION: The EPHS program in China significantly improved health outcomes and prevented financial risks for hypertensive patients. This evidence provides a valuable reference for low- and middle-income countries with their essential public health service programs.

Associations Among Organizational Capabilities, Organizational Performance and the Medical Alliance Implementation Effect in Community Health Centers in China: A Moderated Mediation Model.

Purpose: Community health centers (CHCs) are an important part of the healthcare system worldwide. Based on the dual process model of organizational capabilities, this study explores the relationship between organizational capabilities and the organizational performance of CHCs, as well as the role played by the medical alliance implementation effect. Methods: In this study, whole-group sampling was used to extract CHCs. All 135 CHCs in 8 of 16 districts of Beijing were selected as subjects. The organizational capabilities of the CHCs and the medical alliance implementation effect were evaluated using a questionnaire survey of 1957 managers and 3622 medical staff, respectively. A pathway analysis of the mediating role of the organizational capabilities of CHCs and the moderating role of the medical alliance implementation effect was conducted using Mplus 8.0. Results: The development capabilities had a positive impact on basic capabilities (ß = 0.878, P < 0.001), and core capabilities (ß = 0.952, P < 0.001), but had no direct impact on organizational performance. Basic capabilities positively affected organizational performance (ß = 1.163, P < 0.001), and core capabilities negatively affected organizational performance (ß =- 0.886, P = 0.004). Both basic capabilities (ß =1.022, P < 0.001) and core capabilities (ß =- 0.843, P = 0.005) played a mediating role in the relationship between development capabilities and organizational performance. The moderating role of the medical alliance implementation effect was not significant. Conclusion: This study found that strengthening the organizational capabilities of CHCs can effectively improve their performance, with the development of basic capabilities being a primary concern. The medical alliance implementation effect has not had a significant impact on organizational performance, and the cooperation between CHCs and high-level hospitals should be further promoted to give full play to the medical alliance's role and improve the organizational performance of CHCs.

What factors affect Beijing residents' contracts with family doctors? A comparative study of Beijing's urban and suburban areas.

Objective: To improve the health of residents and promote hierarchical diagnosis and treatment to achieve an orderly pattern of medical treatment, Beijing implemented family doctor contract services (FDCSs) in 2011. The aims of this study were to analyze the current status of Beijing residents' contracts with family doctors (FDs), compare the differences in contracting between urban and suburban residents, and explore the factors that affect residents' contract behavior. Methods: From August 2020 to October 2020, a stratified sampling method was adopted to select residents from community health centers (CHCs) in districts D (urban area) and S (suburb) of Beijing to conduct a questionnaire survey. Chi-square tests, rank sum tests and logistic regression analyzes were used to analyze the current status and influencing factors of residents' contracting with FDs. Results: A total of 4,113 valid questionnaires were included in the final analysis. District D was rich in medical resources, and the FD contract rate of residents there (93.09%) was significantly higher than that of residents in district S (78.06%; p < 0.05). Residents' district (OR = 1.55, 95% CI = 1.18-2.05), understanding of FDCS policies (OR = 4.13, 95% CI = 3.63-4.69), preferred medical institutions (OR = 0.58, 95% CI = 0.42-0.79 for tertiary hospitals in the district; OR = 0.36, 95% CI = 0.22-0.59 for urban medical institutions in Beijing), age, education level, average annual medical expenses and medical insurance type were factors that influenced residents' contracts with FDs (p < 0.05). Conclusion: This study shows that residents who are located in districts with rich medical resources, prefer CHCs as their first choice, have a better understanding of FDCS policies, and are more inclined to contract with FDs than other residents. It is recommended that the number and quality of FDs in suburban areas be increased and that medical staff strengthen publicity about FDCSs and actively encourage residents to contract with FDs.

Structure-guided semi-rational design of an imine reductase for enantio-complementary synthesis of pyrrolidinamine.

In this study, engineered imine reductases (IREDs) of IRED M5, originally from Actinoalloteichus hymeniacidonis, were obtained through structure-guided semi-rational design. By focusing on mutagenesis of the residues that directly interact with the ketone donor moiety, we identified two residues W234 and F260, playing essential roles in enhancing and reversing the stereoselectivity, respectively. Moreover, two completely enantio-complementary variants S241L/F260N (R-selectivity up to 99%) and I149D/W234I (S-selectivity up to 99%) were achieved. Both variants showed excellent stereoselectivity toward the tested substrates, offering valuable biocatalysts for synthesizing pyrrolidinamines. Its application was demonstrated in a short synthesis of the key intermediates of potential drug molecules leniolisib and JAK1 inhibitor 4, from cheap and commercially available pro-chiral N-Boc-piperidone 1 (2 and 3 steps, respectively).

Removable, fully covered, self-expandable metal stents for the treatment of refractory benign esophagogastric anastomotic strictures.

The use of metal stents for malignant esophageal strictures for palliation is well accepted. However, utilization of metal stents for benign esophageal diseases has been controversial. Given the availability of removable, fully covered, self-expandable metal stents (RFCSEMSs), this study was undertaken to evaluate the effectiveness and safety of RFCSEMSs in patients with refractory benign esophagogastric anastomotic strictures. Twenty-four patients with RFCSEMSs were enrolled in this study. All patients had undergone endoscopic Savary-Gilliard bougie dilatation five times or more but there was no significant improvement in symptoms. For all 24 patients, the symptom of dysphagia was alleviated significantly while the stent was in place and for a short time after stent removal, and dysphagia scores decreased from 3-4 to 0-1. After 12 months of follow-up, 18 patients were free from dysphagia but the other 6 patients still suffered obvious dysphagia. RFCSEMSs are still not perfect and can induce some complications. The treatment failure rate of restenting was remarkably high after the first failure. Given that effective methods for treating refractory stricture have not been found, RFCSEMSs could be considered for treating refractory benign esophagogastric anastomotic stricture.

A hybrid system for the overproduction of complex ergot alkaloid chanoclavine.

Synthetic biology-based methods (Sbio) and chemical synthesis (Csyn) are two independent approaches that are both widely used for synthesizing biomolecules. In the current study, two systems were combined for the overproduction of chanoclavine (CC), a structurally complex ergot alkaloid. The whole synthetic pathway for CC was split into three sections: enzymatic synthesis of 4-Br-Trp (4-Bromo-trptophan) using cell-lysate catalysis (CLC), chemical synthesis of prechanoclavine (PCC) from 4-Br-Trp, and overproduction CC from PCC using a whole-cell catalysis (WCC) platform. The final titer of the CC is over 3 g/L in this Sbio-Csyn hybrid system, the highest yield reported so far, to the best of our knowledge. The development of such a combined route could potentially avoid the limitations of both Sbio and Csyn systems and boost the overproduction of complex natural products.

Chemoenzymatic Hunsdiecker-Type Decarboxylative Bromination of Cinnamic Acids.

In this contribution, we report chemoenzymatic bromodecarboxylation (Hunsdiecker-type) of α,ß-unsaturated carboxylic acids. The extraordinarily robust chloroperoxidase from Curvularia inaequalis (CiVCPO) generated hypobromite from H2O2 and bromide, which then spontaneously reacted with a broad range of unsaturated carboxylic acids and yielded the corresponding vinyl bromide products. Selectivity issues arising from the (here undesired) addition of water to the intermediate bromonium ion could be solved by reaction medium engineering. The vinyl bromides so obtained could be used as starting materials for a range of cross-coupling and pericyclic reactions.

A combined strategy for the overproduction of complex ergot alkaloid agroclavine.

Microbial cell factories (MCFs) and cell-free systems (CFSs) are generally considered as two unrelated approaches for the biosynthesis of biomolecules. In the current study, two systems were combined together for the overproduction of agroclavine (AC), a structurally complex ergot alkaloid. The whole biosynthetic pathway for AC was split into the early pathway and the late pathway at the point of the FAD-linked oxidoreductase EasE, which was reconstituted in an MCF (Aspergillus nidulans) and a four-enzyme CFS, respectively. The final titer of AC of this combined system is 1209 mg/L, which is the highest one that has been reported so far, to the best of our knowledge. The development of such a combined route could potentially avoid the limitations of both MCF and CFS systems, and boost the production of complex ergot alkaloids with polycyclic ring systems.