Preparation of (S)-epichlorohydrin using a novel halohydrin dehalogenase by selective conformation adjustment.

Chiral epichlorohydrin (ECH) is an attractive intermediate for chiral pharmaceuticals and chemicals preparation. The asymmetric synthesis of chiral ECH using 1,3-dicholoro-2-propanol (1,3-DCP) catalyzed by a haloalcohol dehalogenase (HHDH) was considered as a feasible approach. However, the reverse ring opening reaction caused low optical purity of chiral ECH, thus severely restricts the industrial application of HHDHs. In the present study, a novel selective conformation adjustment strategy was developed with an engineered HheCPS to regulate the kinetic parameters of the forward and reverse reactions, based on site saturation mutation and molecular simulation analysis. The HheCPS mutant E85P was constructed with a markable change in the conformation of (S)-ECH in the substrate pocket and a slight impact on the interaction between 1,3-DCP and the enzyme, which resulted in the kinetic deceleration of the reverse reactions. Compared with HheCPS, the catalytic efficiency (kcat(S)-ECH/Km(S)-ECH) of the reversed reaction dropped to 0.23-fold (from 0.13 to 0.03 mM-1 s-1), while the catalytic efficiency (kcat(1,3-DCP)/Km(1,3-DCP)) of the forward reaction only reduced from 0.83 to 0.71 mM-1 s-1. With 40 mM 1,3-DCP as substrate, HheCPS E85P catalyzed the synthesis of (S)-ECH with the yield up to 55.35% and the e.e. increased from 92.54 to >99%. Our work provided an effective approach for understanding the stereoselective catalytic mechanism as well as the green manufacturing of chiral epoxides.

Nitric oxide synthase and its function in animal reproduction: an update.

Nitric oxide (NO), a free radical labile gas, is involved in the regulation of various biological functions and physiological processes during animal reproduction. Recently, increasing evidence suggests that the biological role and chemical fate of NO is dependent on dynamic regulation of its biosynthetic enzyme, three distinct nitric oxide synthase (NOS) according to their structure, location and function. The impact of NOS isoforms on reproductive functions need to be timely elucidated. Here, we focus on and the basic background and latest studies on the development, structure, importance inhibitor, location pattern, complex functions. Moreover, we summarize the exactly mechanisms which involved some cell signal pathways in the regulation of NOS with cellular and molecular level in the animal reproduction. Therefore, this growing research area provides the new insight into the important role of NOS male and female reproduction system. It also provides the treatment evidence on targeting NOS of reproductive regulation and diseases.

Designing a novel (R)-ω-transaminase for asymmetric synthesis of sitagliptin intermediate via motif swapping and semi-rational design.

The application of (R)-ω-transaminases as biocatalysts for chiral amine synthesis has been hampered by inadequate stereoselectivity and narrow substrate spectrum. Herein, an effective evolution strategy for (R)-ω-transaminase designing for the asymmetric synthesis of sitagliptin intermediate is presented. Since natural transaminases lack activity toward bulky prositagliptin ketone, transaminase scaffolds with catalytic machinery and activity toward the truncated prositagliptin ketone were firstly screened based on substrate walking principle. A transaminase chimera was established synchronously conferring catalytic activity and (R)-selectivity toward prositagliptin ketone through motif swapping, followed by stepwise evolution. The process resulted in a "best" engineered variant MwTAM8, which exhibited 79.2-fold higher activity than the chimeric scaffold MwTAMc. Structural analysis revealed that the heightened activity is mainly due to the enlarged and adaptive substrate pocket and tunnel. The novel (R)-transaminase exhibited unsatisfied industrial operation stability, which is expected to further modify the protein to enhance its tolerance to temperature, pH, and organic solvents to meet sustainable industrial demands. This study underscores a useful evolution strategy of engineering biocatalysts to confer new properties and functions on enzymes for synthesizing high-value drug intermediates.

Efficient biosynthesis of Vibegron intermediate using a novel carbonyl reductase based on molecular modification of hydrogen bonding network regulation.

Vibegron is a novel, potent, highly selective ß3-adrenergic receptor agonist for the treatment of overactive bladder with higher therapeutic capacity and lower side effects. Methyl(2S,3R)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-phenylpropanoate ((2S,3R)-aminohydroxy ester) is a key chiral intermediate for the synthesis of Vibegron. A novel carbonyl reductase from Exiguobacterium sp. s126 (EaSDR6) was isolated using data mining technology from GenBank database with preferable catalytic activity. Hydrogen bond network regulation was performed using site-directed saturation mutagenesis and combination mutagenesis. The mutant EaSDR6A138L/S193A was obtained with the activity improvement by 4.58 folds compared with the wild type EaSDR6. The Km of EaSDR6A138L/S193A was decreased from 1.57 mM to 0.67 mM, kcat was increased by 2.17 folds, and the overall catalytic efficiency kcat/Km was increased by 5.07 folds. The organic-aqueous biphasic bioreaction system for the asymmetric synthesis of (2S,3R)-aminohydroxy ester was constructed for the first time. Under the substrate concentration of 150 g/L, the yield of (2S,3R)-aminohydroxy ester was > 99.99%, the e.e. was > 99.99%, and the spatiotemporal yield was 1.55 g/(L·h·g DCW) after 12 h reaction. While the substrate concentration was increased to 200 g/L and the reaction lasted for 36 h, the yield of (2S,3R)-aminohydroxy ester was > 99.99%, the e.e. was > 99.99% and the spatiotemporal yield was 1.05 g/(L·h·g DCW). The substrate concentration and spatiotemporal yield were higher than ever reported.

Tuning an efficient Escherichia coli whole-cell catalyst expressing l-pantolactone dehydrogenase for the biosynthesis of d-(-)-pantolactone.

d-(-)-Pantolactone (DPL) is a key intermediate for the production of d-(+)-pantothenate (vitamin B5). Deracemization of d,l-pantolactone (D,L-PL) through oxidizing l-(+)-pantolactone (LPL) to ketopantoyl lactone (KPL) and subsequently reducing KPL to DPL is a promising route for synthesizing DPL. Herein, a newly mined l-pantolactone dehydrogenase from Rhodococcus hoagie (RhoLPLDH) was used for the oxidative dehydrogenation of LPL. To alleviate inclusion bodies formed by membrane-bound RhoLPLDH intracellular expression in E. coli, strategies involving chaperone assistance and decreasing induction temperature were used to achieve RhoLPLDH soluble expression. To enhance its activity, directed evolution and hydrophilicity-based engineering yielded increased catalytic activity and thermostability. 1 M LPL was efficiently converted to KPL by engineering strain CM5 co-expressing RhoLPLDHL254I/V241I/I156L/F224Q/N164K and chaperone. A "two stages in one-pot" method was employed in deracemization of 1 M D,L-PL with 91.2% yield. These results demonstrated that CM5 catalyst exhibits great potential in enzyme cascade deracemization for the production of DPL.

Integrated pharmacokinetics and pharmacometabolomics to reveal the synergistic mechanism of a multicomponent Chinese patent medicine, Mailuo Shutong pills against thromboangiitis obliterans.

BACKGROUND: Mailuo Shutong Pills (MLST) have displayed pharmacological activity against thromboangiitis obliterans (TAO). However, the active ingredients and therapeutic mechanism of MLST against TAO remained to be further clarified. PURPOSE: The aim of this study was to explore the active components of MLST and their synergistic mechanism against TAO by integrating pharmacokinetics (PK) and pharmacometabolomics (PM). METHODS: TAO model rats were established by sodium laurate solution. Firstly, the efficacy of MLST was evaluated by gangrene score, blood flow velocity, and hematoxylin-eosin (H&E) staining. Secondly, PK research was conducted on bioavailable components to characterize their dynamic behaviors under TAO. Thirdly, multiple plasma and urine metabolic biomarkers for sodium laurate-induced TAO rats were found by untargeted metabolomics, and then variations in TAO-altered metabolites following MLST treatment were analyzed utilizing multivariate and bioinformatic analysis. Additionally, metabolic pathway analysis was performed using MetaboAnalyst. Finally, the dynamic link between absorbed MLST-compounds and TAO-associated endogenous metabolites was established by correlation analysis. RESULTS: MLST significantly alleviated gangrene symptoms by improving the infiltration of inflammatory cells and blood supply in TAO rats. Significant differences in metabolic profiles were found in 17 differential metabolites in plasma and 24 in urine between Sham and TAO rats. The 10 bioavailable MLST-compounds, such as chlorogenic acid and paeoniflorin, showed positive or negative correlations with various TAO-altered metabolites related to glutamate metabolism, histidine metabolism, arachidonic acid metabolism and so on. CONCLUSION: This study originally investigated the dynamic interaction between MLST and the biosystem, providing unique insight for disclosing the active components of MLST and their synergistic mechanisms against TAO, which also shed light on new therapeutic targets for TAO and treatment.

Roles of lncRNAs in pancreatic ductal adenocarcinoma: Diagnosis, treatment, and the development of drug resistance.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers, primarily due to its late diagnosis, high propensity to metastasis, and the development of resistance to chemo-/radiotherapy. Accumulating evidence suggests that long non-coding RNAs (lncRNAs) are intimately involved in the treatment resistance of pancreatic cancer cells via interacting with critical signaling pathways and may serve as potential diagnostic/prognostic markers or therapeutic targets in PDAC. DATA SOURCES: We carried out a systematic review on lncRNAs-based research in the context of pancreatic cancer and presented an overview of the updated information regarding the molecular mechanisms underlying lncRNAs-modulated pancreatic cancer progression and drug resistance, together with their potential value in diagnosis, prognosis, and treatment of PDAC. Literature mining was performed in PubMed with the following keywords: long non-coding RNA, pancreatic ductal adenocarcinoma, pancreatic cancer up to January 2022. Publications relevant to the roles of lncRNAs in diagnosis, prognosis, drug resistance, and therapy of PDAC were collected and systematically reviewed. RESULTS: LncRNAs, such as HOTAIR, HOTTIP, and PVT1, play essential roles in regulating pancreatic cancer cell proliferation, invasion, migration, and drug resistance, thus may serve as potential diagnostic/prognostic markers or therapeutic targets in PDAC. They participate in tumorigenesis mainly by targeting miRNAs, interacting with signaling molecules, and involving in the epithelial-mesenchymal transition process. CONCLUSIONS: The functional lncRNAs play essential roles in pancreatic cancer cell proliferation, invasion, migration, and drug resistance and have potential values in diagnosis, prognostic prediction, and treatment of PDAC.

Population total and unbound pharmacokinetics and pharmacodynamics of ciprofol and M4 in subjects with various renal functions.

AIMS: The aim of this study was to develop a population pharmacokinetic (PK) model to simultaneously describe both total and unbound concentrations of ciprofol and its major glucuronide metabolite, M4, and to link it to the population pharmacodynamics (PD) model in subjects with various renal functions. METHODS: A total of 401 and 459 pairs of total and unbound plasma concentrations of ciprofol and M4, respectively, as well as 2190 bispectral index (BIS) data from 24 Chinese subjects with various renal functions were available. Covariates that may potentially contribute to the PK and PD variability of ciprofol were screened using a stepwise procedure. The optimal ciprofol induction dosing regimen was determined by model-based simulations. RESULTS: The PK of unbound ciprofol could best be described by a three-compartment model, while a two-compartment model could adequately describe unbound M4 PK. The concentrations of total and unbound ciprofol and M4 were linked using a linear protein binding model. The relationship between plasma concentrations of ciprofol and BIS data was best described by an inhibitory sigmoidal Emax model with a two-compartment biophase distribution compartment. Hemoglobin was the identified covariate determining the central compartment clearance of ciprofol; uric acid was a covariate affecting the central compartment clearance of M4 and protein binding rate, kB . The included covariates had no effect on the PD of ciprofol. Simulation results indicated that the label-recommended dose regimen was adequate for anaesthesia induction. CONCLUSIONS: The developed model fully characterized the population PK and PD profiles of ciprofol. No dose adjustment is required in patients with mild and moderate renal impairment.

Population pharmacokinetics and clinical outcomes of polymyxin B in paediatric patients with multidrug-resistant Gram-negative bacterial infections.

BACKGROUND: Current polymyxin B dosing in children relies on scant data. OBJECTIVES: To build a population pharmacokinetic (PK) model for polymyxin B in paediatric patients and assess the likely appropriateness of different dosages. METHODS: A total of 19 paediatric patients were enrolled to receive intravenous polymyxin B (1.33-2.53 mg/kg/day), and the median age was 12.5 (range 3.2-17.8) years. Serial plasma samples were collected at steady-state and modelled by population PK analysis. Clinical efficacy and nephrotoxicity of polymyxin B treatment were also assessed. RESULTS: PK data were adequately described by a two-compartment model with first-order elimination, and weight was a significant covariate of polymyxin B clearance. Clinical success occurred in 14 of 19 patients (73.7%) and only one patient developed acute kidney injury. The 28 day mortality was 10.5% (2/19). The steady-state polymyxin B exposure was 36.97 ±â€Š9.84 mg·h/L, lower than the therapeutic exposure of 50-100 mg·h/L. With the AUC24h/MIC target of 50, the dosage of 1.5-3.0 mg/kg/day had a probability of target attainments over 90% when MICs were <0.5 mg/L. CONCLUSIONS: Dose adjustment of polymyxin B needs to consider the MIC of infecting pathogens. Current polymyxin B dosing for paediatric patients may be acceptable when MICs are <0.5 mg/L.

Identifying quality markers of Mailuoshutong pill against thromboangiitis obliterans based on chinmedomics strategy.

BACKGROUND: Mailuoshutong pill (MLSTP) is a traditional Chinese medicine (TCM) for the treatment of Thromboangiitis obliterans (TAO, Buerger's disease) which is a segmental non-atherosclerotic inflammatory occlusive disorder. However, the mechanism and quality standards of MLSTP have not been sufficiently studied. PURPOSE: This work aims to investigate the potential mechanisms and quality markers (Q-markers) of MLSTP treating TAO based on the chinmedomics strategy. METHODS: The therapeutical effect of MLSTP on TAO rats was evaluated by changes in body weight and clinical score, regional blood flow velocity and perfused blood vessel distribution, hematoxylin-eosin (H&E) staining, serum metabolic profile. Moreover, both endogenous metabolites and exogenous components were simultaneously detected in serum based on ultra-high performance liquid chromatography coupled with a Q Exactive hybrid quadrupole-orbitrap high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS), and multivariate analysis was applied to identify the biomarkers, as well as the dynamic changes of metabolites were observed to explore the mechanism of action of MLSTP. In addition, the pharmacodynamic material basis were identified by correlation analysis between biomarkers and absorbed constituents. Finally, the Q-markers of MLSTP were determined according to the screening principles of Q-marker and validated the measurability. RESULTS: MLSTP treatment alleviated disease severity of TAO, reduced inflammatory infiltration, and ameliorated vascular function. 26 potential biomarkers associated with glutamate metabolism, linoleic acid metabolism, arachidonic acid metabolism and so on were identified. Besides, 27 prototypical components were identified in serum, 16 of which were highly correlated with efficacy and could serve as the pharmacodynamic material basis of MLSTP against TAO. In addition, 7 compounds, namely, sweroside, chlorogenic acid, calycosin-7-glucoside, formononetin, paeoniflorin, liquiritigenin and 3-butylidenephthalide, were considered as potential Q-markers of MLSTP. Ultimately, the measurability of the seven Q-markers was validated by rapid identifcation and quantifcation. CONCLUSION: This study successfully clarified the therapeutic effect and Q-markers of MLSTP by chinmedomics strategy, which is of great significance for the establishment of quality standards. Furthermore, it provides a certain reference for the screening of Q-markers in TCM prescriptions.

Efficient enzymatic synthesis of L-ascorbyl palmitate using Candida antarctica lipase B-embedded metal-organic framework.

The Candida antarctica lipase B (CALB) was embedded in the metal-organic framework, zeolitic imidazolate framework-8 (ZIF-8), and applied in the enzymatic synthesis of L-ascorbic acid palmitate (ASP) for the first time. The obtained CALB@ZIF-8 achieved the enzyme loading of 80 mg g-1 with 11.3 U g-1 (dry weight) unit activity, 59.8% activity recovery, and 92.7% immobilization yield. Under the optimal condition, ASP was synthesized with over 75.9% conversion of L-ascorbic acid in a 10-batch reaction. Continuous synthesis of ASP was subsequently performed in a packed bed bioreactor with an outstanding average space-time yield of 58.1 g L-1  h-1 , which was higher than ever reported continuous ASP biosynthesis reactions.

Enabling biocatalysis in high-concentration organic cosolvent by enzyme gate engineering.

Biocatalysis in high-concentration organic solvents (OSs) offers many advantages, but realizing this process remains a huge challenge. An R-selective ω-amine transaminase variant (AcATAM2 ) exhibited high activity toward 50 g/L pro-sitagliptin ketone 1-[1-piperidinyl]-4-[2,4,5-trifluorophenyl]-1,3-butanedione (PTfpB). However, AcATAM2 displayed unsatisfactory organic-cosolvent resistance against high-concentration dimethyl sulfoxide (DMSO), which is required to enhance the solubility of the hydrophobic substrate PTfpB. Located in the substrate-binding tunnel, enzyme gates are structural elements that undergo reversible conformational transitions, thus affecting the accessibility of the binding pocket to solvent molecules. Depending on the conformation of the enzyme gates, one can define an open or closed conformation on which the enzyme activity in OSs may depend. To enhance the DMSO resistance of AcATAM2 , we identified the beneficial residues at the "enzyme gate" region via computational analysis, alanine scanning, and site-saturation mutagenesis. Two beneficial variants, namely, AcATAM2F56D and AcATAM2F56V , not only displayed improved enzyme activity but also exhibited enhanced DMSO resistance (the half-life value increased from 25.71 to 42.49 h under 60% DMSO). Molecular dynamic simulations revealed that the increase in DMSO resistance was mainly caused by the decrease in the number of DMSO molecules in the substrate-binding pocket. Moreover, in the kilogram-scale experiment, the conversion of 80 g/L substrate was increased from 50% (AcATAM2 ) to 85% (M2F56D in 40% DMSO) with a high e.e. of >99% within 24 h.

[Study on metabolic dynamics,metabolic enzyme phenotype and species difference of hepatic and intestinal microsome of psoralidin].

This study aims to investigate metabolic activities of psoralidin in human liver microsomes( HLM) and intestinal microsomes( HIM),and to identify cytochrome P450 enzymes( CYPs) and UDP-glucuronosyl transferases( UGTs) involved in psoralidin metabolism as well as species differences in the in vitro metabolism of psoralen. First,after incubation serial of psoralidin solutions with nicotinamide adenine dinucleotide phosphate( NADPH) or uridine 5'-diphosphate-glucuronic acid( UDPGA)-supplemented HLM or HIM,two oxidic products( M1 and M2) and two conjugated glucuronides( G1 and G2) were produced in HLM-mediated incubation system,while only M1 and G1 were detected in HIM-supplemented system. The CLintfor M1 in HLM and HIM were 104. 3,and57. 6 µL·min~(-1)·mg~(-1),respectively,while those for G1 were 543. 3,and 75. 9 µL·min~(-1)·mg~(-1),respectively. Furthermore,reaction phenotyping was performed to identify the main contributors to psoralidin metabolism after incubation of psoralidin with NADPH-supplemented twelve CYP isozymes( or UDPGA-supplemented twelve UGT enzymes),respectively. The results showed that CYP1 A1( 39. 5 µL·min~(-1)·mg~(-1)),CYP2 C8( 88. 0 µL·min~(-1)·mg~(-1)),CYP2 C19( 166. 7 µL·min~(-1)·mg~(-1)),and CYP2 D6( 9. 1 µL·min~(-1)·mg~(-1)) were identified as the main CYP isoforms for M1,whereas CYP2 C19( 42. 0 µL·min~(-1)·mg~(-1)) participated more in producing M2. In addition,UGT1 A1( 1 184. 4 µL·min~(-1)·mg~(-1)),UGT1 A7( 922. 8 µL·min~(-1)·mg~(-1)),UGT1 A8( 133. 0 µL·min~(-1)·mg~(-1)),UGT1 A9( 348. 6 µL·min~(-1)·mg~(-1)) and UGT2 B7( 118. 7 µL·min~(-1)·mg~(-1)) played important roles in the generation of G1,while UGT1 A9( 111. 3 µL·min~(-1)·mg~(-1)) was regarded as the key UGT isozyme for G2. Moreover,different concentrations of psoralidin were incubated with monkey liver microsomes( MkLM),rat liver microsomes( RLM),mice liver microsomes( MLM),dog liver microsomes( DLM) and mini-pig liver microsomes( MpLM),respectively. The obtained CLintwere used to evaluate the species differences.Phase Ⅰ metabolism and glucuronidation of psoralidinby liver microsomes showed significant species differences. In general,psoralidin underwent efficient hepatic and intestinal metabolisms. CYP1 A1,CYP2 C8,CYP2 C19,CYP2 D6 and UGT1 A1,UGT1 A7,UGT1 A8,UGT1 A9,UGT2 B7 were identified as the main contributors responsible for phase Ⅰ metabolism and glucuronidation,respectively. Rat and mini-pig were considered as the appropriate model animals to investigate phase Ⅰ metabolism and glucuronidation,respectively.

Outcome of scleral buckling with or without gas tamponade for recurrent retinal detachment in post-vitrectomy eyes.

BACKGROUND: Treatment of recurrent retinal detachment (re-RD) following vitrectomy (post-gas/air tamponade and post-silicone oil removal) is challenging. Previously reported treatment is commonly revision pars plana vitrectomy (PPV) combined with tamponade, which is invasive and a burden both economically and emotionally when compared with scleral buckling (SB). The purpose of this study is to report anatomical and functional outcomes of SB with or without gas tamponade in eyes with recurrent retinal detachment (re-RD) that previously underwent PPV at least once. METHODS: We retrospectively reviewed the medical records of 14 patients (14 eyes) who underwent PPV at least once and were treated with SB after re-RD. Preoperative characteristics, intraoperative complications, and postoperative data were assessed. The final anatomical and functional outcomes were analyzed. RESULTS: The original PPV was performed for primary rhegmatogenous retinal detachment in 11 eyes, macular hole retinal detachment in 2 eyes, and myopic foveoschisis in 1 eye. Previously, 3 eyes underwent one PPV with gas tamponade, and the remaining 11 (79%) eyes underwent 2-5 operations. Seven eyes underwent the procedure with gas injection. At the last follow-up, 13 eyes achieved total retinal attachment and 1 eye had re-RD. The postoperative intraocular pressure was within the normal range, except in 1 eye (6 mmHg). The finest postoperative best-corrected visual acuity (BCVA) was 20/25. There was a significant improvement in BCVA from 20/160 ± 20/63 at baseline to 20/80 ± 20/50 at the last visit in the 13 successfully treated eyes (P = 0.025). CONCLUSIONS: SB can be effective for re-RD after PPV in specific cases.

[Clinical manifestations and gene mutation analysis of children with noncompaction of the ventricular myocardium: an analysis of 6 cases].

This article summarizes and analyzes the clinical features and gene mutation characteristics of children with noncompaction of the ventricular myocardium (NVM). For the 6 children with NVM (4 boys and 2 girls), the age of onset ranged from 3 months to 12 years. Of the 6 children, 5 had arrhythmia, 3 had cardiac insufficiency, 1 had poor mental state, and 1 had chest distress and sighing. NVM-related gene mutations were detected in 4 children, among whom 2 had MYH7 gene mutation, 1 had PRDM16 gene mutation, and 1 had mutations in the ACTN2 and TNNT2 genes. Four children had improvement in cardiac function. The two children with no significant improvement in cardiac function had a younger age of onset, a greater reduction in systolic function on echocardiography, and greater increases in myocardial enzyme and N-terminal pro-brain natriuretic peptide. It is concluded that for children with the initial symptoms of chest distress, sighing, arrhythmia, enlarged heart shadow, and increased myocardial enzyme, echocardiography and cardiac magnetic resonance are recommended for the diagnosis of NVM. NVM can have various genetic mutations.

Efficient production of an ezetimibe intermediate using carbonyl reductase coupled with glucose dehydrogenase.

Ezetimibe is a top-selling hypolipidemic drug for the treatment of cardiovascular diseases. Biosynthesis of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one ((S)-ET-5) using carbonyl reductase has shown advantages including high catalytic efficiency, excellent stereoselectivity, mild reaction conditions, and environmental friendness, and was considered as the key step for ezetimibe production. The regeneration efficiency of the cofactor, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) is one of the main restricted factor. Recombinant Escherichia coli strain (smCR125) coexpressing carbonyl reductase (CR125) and glucose dehydrogenase were successfully constructed and applied for the production of (S)-ET-5 for the first time. Without extra addition of the coenzyme NADPH, the yield of 99.8% and the enantiomeric excess (e.e.) of 99.9% were achieved under ET-4 concentration of 200 g/L. Using a substrate fed-batch strategy, under the optimal conditions, the substrate ET-4 concentration was increased to 250 g/L with the yield of 98.9% and the e.e. of 99.9% after 12 hr reaction. The space-time yield of 494.5 g L-1 d-1 and the space-time yield per gram biocatalyst of 24.7 g L-1 d-1 g-1 DCW were achieved, which were higher than ever reported for the biosynthesis of the ezetimibe intermediate.

Deciphering the Relationship Between the Trough Concentration of Posaconazole and Its Efficacy and Safety in Chinese Patients With Hematological Disorders.

Posaconazole (PCZ) is effective in preventing and salvage treatment invasive fungal infections in patients with hematologic disorders. However, PCZ displays highly variable individual pharmacokinetics affecting its efficacy and safety. To investigate the correlation between PCZ concentration and efficacy and safety, the following key influencing factors were explored. A total of 285 trough plasma concentrations (Cmin) of 81 Chinese patients receiving PCZ oral suspension for prophylaxis or treatment of invasive fungal infections were collected in this study. The relationships between Cmin values and clinical response and hepatotoxicity were investigated as well as the incidence of clinical response under different Cmin values of PCZ with a logistic regression model. The concentration of PCZ showed remarkable differences among patients with haematologic disorders. PCZ Cmin values of 0.76 and 1.0 µg/mL were both associated with an over 80% probability of successful response to prophylaxis and treatment of fungal infections, respectively. No association between Cmin values and hepatotoxicity was noted (P > 0.05). Gender, albumin, and co-administration of proton pump inhibitor (PPI) were identified as independent factors influencing PCZ Cmin by multiple linear regression analysis. Furthermore, patients' C-reactive protein (CRP), albumin, and co-administration of PPI exhibited significant effects on the therapeutic window of patients receiving PCZ for prophylaxis. The plasma concentration is closely associated with therapeutic efficacy of PCZ. It is necessary to adjust the dosing regimens based on PCZ Cmin to obtain an optimal therapeutic response.

Creation of a robust and R-selective ω-amine transaminase for the asymmetric synthesis of sitagliptin intermediate on a kilogram scale.

The creation of an R-selective ω-amine transaminase (ω-ATA) as biocatalyst is crucial for the asymmetric amination of prochiral ketones to produce sitagliptin intermediates because rare ω-ATAs are R-selective in nature and most of them suffer from poor stability and low activity toward bulky prochiral ketones. Here, the gene of an R-selective ω-ATA was cloned from Arthrobacter cumminsii ZJUT212 (AcATA) and expressed in Escherichia coli. The best variants (M1 + M122H and M1+T134 G) were obtained using a semi-rational protein design after screening. These variants not only exhibited improved activity and substrate affinity but also enhanced stability in aqueous phase containing 20 % dimethyl sulfoxide. The conversion of asymmetric amination on 50 g/L pro-sitagliptin ketone PTfpB (1-[1-piperidinyl]-4-[2,4,5-trifluorophenyl]-1,3-butanedione) achieved 92 %, with an extremely high e.e. of >99 %, using 2 gDCW/L E. coli cells harboring M1 + M122H as biocatalyst. In the kilogram-scale experiment, approximately 40 kg of (R)-APTfpB (e.e. >99 %) was produced within 30 h when 50 kg PTfpB was used as the substrate. Furthermore, the space-time yield reached ≈32 g/(L·d).