Cu(II) immobilized on mesoporous organosilica as an efficient and reusable nanocatalyst for one‐pot Biginelli reaction under solvent‐free conditions

Cu(II) immobilized on mesoporous organosilica nanoparticles (Cu²⁺@MSNs‐(CO₂^?)₂) has been synthesized, as a inorganic–organic nanohybrid catalyst, through a post‐grafting approach. Its characterization is carried out by Fourier transform infrared spectroscopy (FT‐IR), X‐ray diffraction (XRD), Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), Energy dispersive X‐ray (EDX), Thermogravimetric/differential thermal analyses (TGA‐DTA), and Nitrogen adsorption–desorption analysis. Cu²⁺@MSNs‐(CO₂^?)₂ exhibits high catalytic activity in the Biginelli reaction for the synthesis of a diverse range of 3, 4‐dihydropyrimidin‐2(1H)‐ones, under mild conditions. The anchored Cu(II) could not leach out from the surface of the mesoporous catalyst during the reaction and it has been reused several times without appreciable loss in its catalytic activity.     

Terminal Hydride Species in [FeFe]‐Hydrogenases Are Vibrationally Coupled to the Active Site Environment

A combination of nuclear resonance vibrational spectroscopy (NRVS), FTIR spectroscopy, and DFT calculations was used to observe and characterize Fe?H/D bending modes in CrHydA1 [FeFe]‐hydrogenase Cys‐to‐Ser variant C169S. Mutagenesis of cysteine to serine at position 169 changes the functional group adjacent to the H‐cluster from a ‐SH to ‐OH, thus altering the proton transfer pathway. The catalytic activity of C169S is significantly reduced compared to that of native CrHydA1, presumably owing to less efficient proton transfer to the H‐cluster. This mutation enabled effective capture of a hydride/deuteride intermediate and facilitated direct detection of the Fe?H/D normal modes. We observed a significant shift to higher frequency in an Fe?H bending mode of the C169S variant, as compared to previous findings with reconstituted native and oxadithiolate (ODT)‐substituted CrHydA1. On the basis of DFT calculations, we propose that this shift is caused by the stronger interaction of the ‐OH group of C169S with the bridgehead ‐NH‐ moiety of the active site, as compared to that of the ‐SH group of C169 in the native enzyme.     

Purification of angiotensin‐converting enzyme from human plasma and investigation of the effect of some active ingredients isolated from Nigella sativa L. extract on the enzyme activity

In the present study, one‐step purification of angiotensin‐converting enzyme (ACE, peptidyldipeptidase A, EC 3.4.15.1), responsible for regulation of blood pressure, was achieved using affinity chromatography from human plasma. The enzyme was purified 12,860‐fold with a specific activtiy of 5080 EU/mg protein. Optimum temperature and pH were determined for the enzyme as 35–40°C and pH 7.4–7.5, respectively. The purity of ACE was determined by SDS–PAGE and the enzyme showed two bands at 60 and 70 kDa on the gel. The native molecular weight of ACE was found to be 260 kDa by gel filtration chromatography, demonstrating that the enzyme has a heterodimeric structure. Natural fatty acids of Nigella sativa (Ranunculaceae) were isolated by means of column chromatography. The structures of these compounds were determined using NMR and GC‐MS. The results showed that high concentrations of linoleic, oleic and palmitic acids were isolated from the plant. The effect of six fractions (Fr 1–6) on ACE activity was examined. Fraction 3 increased the ACE activity while the other fractions decreased the enzyme activity. The concentrations of the fractions inhibiting the half‐maximum activity of the enzyme were calculated as 1.597 mg/mL for Fr 1, 0.053 mg/mL for Fr 2, 0.527 mg/mL for Fr 4, 0.044 mg/mL for Fr 5 and 0.136 mg/mL for Fr 6 using a Lineweaver–Burk graph.     

Enzyme Catalysis Induced Polymer Growth in Nanochannels: A New Approach to Regulate Ion Transport and to Study Enzyme Kinetics in Nanospace

Method that could regulate the ion transport in nanochannel in an efficient and rapid manner is still a challenge. Here, we introduced enzyme‐catalysis‐induced polymer growth in nanochannels to develop a new method to regulate the ion transport and evaluate the enzyme catalysis kinetics in nano‐space. As a model enzyme, Horseradish peroxidase (HRP) was immobilized in the nanochannels through a volume‐controlled‐drying method. In the presence of H₂O₂, HRP catalyzed o‐phenylenediamine (o‐PD) to trigger its polymer growth, in turn blocked the ion transport and led to the decrease of the ion current. Taking advantages of the high efficiency of enzyme catalysis and the nano‐confinement of nanochannels, the system readily achieved blocking ratios of ion current even reaching 99.6 % of the initial. Based on above concept, we developed a new method to evaluate the enzyme catalysis kinetics in nano‐confined space. By comparing with those in free state in solution and absorbed on planar surface, HRP confined in nanochannels presented similar apparent Michaelis constant (K_m) values for the substrate H₂O₂ but much higher K_m values for the substrate o‐PD, due to the steric hindrance and diffusion suppression. The enzyme‐catalysis‐induced polymerization in nanochannels might lead to new concept for the nano‐blocking/switching and provide a new platform for single molecule analysis and detection.     

Revised mechanism of carboxylation of ribulose‐1,5‐biphosphate by rubisco from large scale quantum chemical calculations

Here, we describe a computational approach for studying enzymes that catalyze complex multi‐step reactions and apply it to Ribulose 1,5‐bisphosphate carboxylase–oxygenase (Rubisco), the enzyme that fixes atmospheric carbon dioxide within photosynthesis. In the 5‐step carboxylase reaction, the substrate Ribulose‐1,5‐bisphosphate (RuBP) first binds Rubisco and undergoes enolization before binding the second substrate, CO₂. Hydration of the RuBP.CO₂ complex is followed by C C bond scission and stereospecific protonation. However, details of the roles and protonation states of active‐site residues, and sources of protons and water, remain highly speculative. Large‐scale computations on active‐site models provide a means to better understand this complex chemical mechanism. The computational protocol comprises a combination of hybrid semi‐empirical quantum mechanics and molecular mechanics within constrained molecular dynamics simulations, together with constrained gradient minimization calculations using density functional theory. Alternative pathways for hydration of the RuBP.CO₂ complex and associated active‐site protonation networks and proton and water sources were investigated. The main findings from analysis of the resulting energetics advocate major revision to existing mechanisms such that: hydration takes place anti to the CO₂; both hydration and C C bond scission require early protonation of CO₂ in the RuBP.CO₂ complex; C C bond scission and stereospecific protonation reactions are concerted and, effectively, there is only one stable intermediate, the C3‐gemdiolate complex. Our main conclusions for interpreting enzyme kinetic results are that the gemdiolate may represent the elusive Michaelis–Menten‐like complex corresponding to the empirical K_m (=K_c) with turnover to product via bond scission concerted with stereospecific protonation consistent with the observed catalytic rate. © 2018 Wiley Periodicals, Inc.     

Quick screening of true tyrosinase inhibitors from natural products using tyrosinase‐immobilized magnetic nanoparticles and a magnetic microplate

Tyrosinase inhibitors from natural products have applications in the pharmaceutical, food, and cosmetic industries because of the functions of tyrosinase in skin disorders and in the enzymatic browning of fruits. Current in vitro inhibitor screening assays are based on the inhibition of the oxidation of l ‐3,4‐dihydroxyphenylalanine (l ‐DOPA) mediated by a mushroom tyrosinase. However, in these assays, a tyrosinase inhibitor or an antioxidant could inhibit dopaquinone formation. In this study, we aimed to eliminate this ambiguity by using a microplate assay integrating tyrosinase‐immobilized magnetic nanoparticles (TYR‐MNPs) and a homemade magnetic microplate for high‐throughput screening. After incubating extracts of natural products with TYR‐MNPs, the magnetic nanoparticles are attracted to the bottoms of wells, the extracts are rinsed, and TYR‐MNPs react with l ‐DOPA. This method can be used to screen compounds that interact with the active sites of the enzyme, or copper chelators that bind more strongly than tyrosinase to copper ions, distinguishing them from antioxidants or tyrosinase substrates. Integration with the homemade magnetic microplate enables high‐throughput inhibitor screening. Aloe vera flowers are crop by‐products, and litchi flowers fall after the blossom. Our work demonstrated that these flowers have tyrosinase inhibitory effects, thus increasing their value.     

A Mechanism‐Based Approach to Screening Metagenomic Libraries for Discovery of Unconventional Glycosidases

Functional metagenomics has opened new opportunities for enzyme discovery. To exploit the full potential of this new tool, the design of selective screens is essential, especially when searching for rare enzymes. To identify novel glycosidases that employ cleavage strategies other than the conventional Koshland mechanisms, a suitable screen was needed. Focusing on the unsaturated glucuronidases (UGLs), it was found that use of simple aryl glycoside substrates did not allow sufficient discrimination against β‐glucuronidases, which are widespread in bacteria. While conventional glycosidases cannot generally hydrolyze thioglycosides efficiently, UGLs follow a distinct mechanism that allows them to do so. Thus, fluorogenic thioglycoside substrates featuring thiol‐based self‐immolative linkers were synthesized and assessed as selective substrates. The generality of the approach was validated with another family of unconventional glycosidases, the GH4 enzymes. Finally, the utility of these substrates was tested by screening a small metagenomic library.     

三嗪噁二唑基吡唑衍生物抑酶活性的QSAR模型

基于拓扑化学理论,原子类型电拓扑态指数(Mk)被用于表征18种三嗪噁二唑基吡唑衍生物的化学微环境。采用最佳变量子集回归方法,分别建立上述化合物对蛋白酪氨酸磷酸酯酶1B(PTP1B)、细胞分裂周期25磷酸酯酶B(Cdc25B)的抑酶活性(P_t、C_d)与Mk的定量构效关系(QSAR)模型。它们的最佳三元QSAR模型的判定系数(R~2)依次为0.896、0.828,逐一剔除法交叉验证相关系数(R_(cv)~2)依次为0.830、0.688。经R_(cv)~2、VIF、FT、AC等检验,该模型具有良好的稳健性及预测能力。经训练集验证,上述模型均具有良好的外部预测能力。模型显示,影响Pt、Cd的因素既有不同的结构基团(-CH_3、-O-、-NH_2和芳环中-N=),也有相同的因素(芳环中-C=)。