Efficient synthesis of tetrazole derivatives of cytisine using the azido-Ugi reaction

The azido-Ugi reaction with natural alkaloid cytisine was investigated. It was demonstrated that the reaction could be performed with various carbonyls (both aldehydes and ketones) and isocyanides. The transformation proceeded under mild conditions in methanol using TMSN3 as a source of hydrazoic acid to give target tetrazole derivatives of cytisine in up to 98% yield. The diastereoselectivity of this reaction was studied using both aliphatic and aromatic aldehydes. A family of tetrazole derived cytisine compounds was prepared. Selective deprotection of tetrazoles was elaborated to synthesize the corresponding NH-tetrazoles.     

离子液体吸附剂对木犀草素的富集及CARS变量筛选的近红外光谱分析方法

该文以咪唑型离子液体作为原料制备吸附剂富集稀溶液中的木犀草素,利用竞争性自适应权重(CARS)变量筛选的方法建立了一种快速测定木犀草素的近红外光谱分析方法。考察了吸附剂用量、pH值、振荡时间对吸附效果的影响,并探究了吸附剂的吸附能力;富集木犀草素的吸附剂经近红外漫反射光谱检测,采用CARS变量筛选的方法结合偏最小二乘回归(PLS)建立了木犀草素的定量校正模型。结果表明,吸附剂用量为0.15 g、pH值为7、振荡时间为20 min的最佳条件下,吸附率达90.9%,且该吸附符合Langmuir等温吸附模型,最大吸附量为7.1 mg/g。近红外光谱建模中,与未经CARS变量筛选处理作为对照,对比发现经CARS变量筛选的方法结果更优,并采用连续小波变换(CWT)的光谱预处理进行验证,结果表明经CWT处理后,预测残差(RPD)值增大,说明了模型的可靠性。该方法可有效富集稀溶液中的木犀草素,采用CARS变量筛选结合CWT光谱预处理的近红外光谱方法可实现对稀溶液中木犀草素的灵敏、快捷检测。     

Characterization of fluorescence of ANS-tear lipocalin complex: evidence for multiple-binding modes

ANS is widely used as a probe for locating binding sites of proteins and studying structural changes under various external conditions. However, the nature of ANS-binding sites in proteins and the accompanying changes in fluorescence properties are controversial. We examined the steady-state and time-resolved fluorescence of the ANS-protein complexes for tear lipocalin (TL) and its mutants in order to discern the origin of lifetime components via analysis that included the multiexponential decay and the model-free maximum entropy methods. Fluorescence lifetimes of ANS-TL complexes can be grouped into two species, 14.01-17.42 ns and 2.72-4.37 ns. The log-normal analyses of fluorescence spectral shapes reveal the heterogeneous nature of both long- and short-lifetime species. The constructed time-resolved emission, amplitude (TRES) and area normalized (TRANES), and decay-associated spectra are consistent with a model that includes heterogeneous modes of ANS binding with two separate lifetime components. The two lifetime components are not derived from solvent relaxation, but rather may represent different binding modes.     

A Simple Model-Free Method for Direct Assessment of Fluorescent Ligand Binding by Linear Spectral Summation

Fluorescent tagged ligands are commonly used to determine binding to proteins. However, bound and free ligand concentrations are not directly determined. Instead the response in a fluorescent ligand titration experiment is considered to be proportional to the extent of binding and, therefore, the maximum value of binding is scaled to the total protein concentration. Here, a simple model-free method is presented to be performed in two steps. In the first step, normalized bound and free spectra of the ligand are determined. In the second step, these spectra are used to fit composite spectra as the sum of individual components or linear spectral summation. Using linear spectral summation, free and bound 1-Anilinonaphthalene-8-Sulfonic Acid (ANS) fluorescent ligand concentrations are directly calculated to determine ANS binding to tear lipocalin (TL), an archetypical ligand binding protein. Error analysis shows that the parameters that determine bound and free ligand concentrations were recovered with high certainty. The linear spectral summation method is feasible when fluorescence intensity is accompanied by a spectral shift upon protein binding. Computer simulations of the experiments of ANS binding to TL indicate that the method is feasible when the fluorescence spectral shift between bound and free forms of the ligand is just 8 nm. Ligands tagged with environmentally sensitive fluorescent dyes, e.g., dansyl chromophore, are particularly suitable for this method.

Theoretical modelling of physiologically stretched vessel in magnetisable stent assisted magnetic drug targetingapplication

The magnetisable stent assisted magnetic targeted drug delivery system in a physiologically stretched vessel is considered theoretically. The changes in the mechanical behaviour of the vessel are analysed under the influence of mechanical forces generated by blood pressure. In this 2D mathematical model a ferromagnetic, coiled wire stent is implanted to aid collection of magnetic drug carrier particles in an elastic tube, which has similar mechanical properties to the blood vessel. A cyclic mechanical force is applied to the elastic tube to mimic the mechanical stress and strain of both the stent and vessel while in the body due to pulsatile blood circulation. The magnetic dipole–dipole and hydrodynamic interactions for multiple particles are included and agglomeration of particles is also modelled. The resulting collection efficiency of the mathematical model shows that the system performance can decrease by as much as 10% due to the effects of the pulsatile blood circulation.     

Flow of power-law fluids over a moving wedge surface with wall mass injection

The boundary layer problem of a power-law fluid flow with fluid injection on a wedge whose surface is moving with a constant velocity in the opposite direction to that of the uniform mainstream is analyzed. The free stream velocity, the injection velocity at the surface, moving velocity of the wedge surface, the wedge angle and the power law index of non-Newtonian fluid are assumed variables. The fourth order Runge–Kutta method modified by Gill is used to solve the non-dimensional boundary layer equations for non-Newtonian flow field. Without fluid injection, for every angle of wedge β, a limiting value for velocity ratio λ _cr (velocity of the wedge surface/velocity of the uniform flow) is found for each power-law index n. The value of λ _cr increases with the increasing wedge angle β. The value of wedge angle also restricts the physical characteristics of the fluid to be used. The effects of the different parameters on velocity profile and on skin friction are studied and the drag reduction is discussed. In case of C = 2.5 and velocity ratio λ = 0.2 for wedge angle β = 0.5 with the fluid with power law-index n = 0.5, 48.8% drag reduction is obtained.     

Gelatin-Immobilised Poly(hydroxyethyl methacrylate) Cryogel for Affinity Purification of Fibronectin

In this work, fibronectin purification from human plasma with the gelatin-immobilised poly(hydroxyethyl methacrylate) (PHEMA) cryogel has been evaluated. The PHEMA cryogel was prepared by cryo-polymerisation which proceeds in an aqueous solution of monomer frozen inside a plastic syringe. The PHEMA cryogel contained interconnected macrochannels of 10–200 μm in diameter. Gelatin molecules were covalently immobilised onto the PHEMA cryogel via carbodiimide activation. The gelatin-immobilised PHEMA cryogel was used to purify fibronectin from human plasma. Fibronectin adsorption from human plasma on the PHEMA cryogel was 0.30 mg/ml, while much higher adsorption values, up to 38 mg/ml, was obtained with the gelatin-immobilised PHEMA cryogel. The fibronectin adsorption capacity of the gelatin-immobilised PHEMA cryogel did not change with an increase in the flow rate of plasma. Up to 92 % of the adsorbed fibronectin was eluted using 2 M urea containing 1 M NaCl as elution agent. The adsorption–elution cycle was repeated ten times using the same PHEMA cryogel. No remarkable decrease was detected in the adsorption capacity of the gelatin-immobilised PHEMA cryogel.     

Purification of Alcohol Dehydrogenase from Saccharomyces cerevisiae Using Magnetic Dye-Ligand Affinity Nanostructures

Reactive Green 19 was covalently immobilized onto magnetic nanostructures for purification of alcohol dehydrogenase from Saccharomyces cerevisiae. The Reactive Green 19 immobilized magnetic nanostructures were characterized by Fourier transform infrared spectroscopy, electron spin resonance, atomic force microscope, and energy dispersive X-ray analysis. Particle size of nanostructures was found to be roughly 70 nm. Alcohol dehydrogenase adsorption experiments were investigated under different conditions in batch system (i.e., medium pH, alcohol dehydrogenase concentration, temperature, and ionic strength). Maximum alcohol dehydrogenase adsorption capacity was found to be 176.09 mg/g polymer while nonspecific alcohol dehydrogenase adsorption onto plain magnetic nanostructures was negligible (19.4 mg/g polymer). Alcohol dehydrogenase molecules were desorbed by using 1.0 M NaCl with 98.4 % recovery. Alcohol dehydrogenase from S. cerevisiae was purified 45.63-fold in single step with dye-immobilized magnetic nanostructures, and purity of alcohol dehydrogenase was shown by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Hemoglobin binding from human blood hemolysate with poly(glycidyl methacrylate) beads

Metal-chelating affinity beads have attracted increasing interest in recent years for protein purification. In this study, iminodiacetic acid (IDA) was covalently attached to the poly(glycidyl methacrylate) [PGMA] beads (1.6 μm in diameter). Cu(2+) ions were chelated via IDA groups on PGMA beads for affinity binding of hemoglobin (Hb) from human blood hemolysate. The PGMA beads were characterized by scanning electron microscopy (SEM). The PGMA-Cu(2+) beads (628 μmol/g) were used in the Hb binding-elution studies. The effects of Hb concentration, pH and temperature on the binding efficiency of PGMA-Cu(2+) beads were performed in a batch system. Non-specific binding of Hb to PGMA beads in the absence of Cu(2+) ions was very low (0.39 mg/g). The maximum Hb binding was 130.3 mg/g. The equilibrium Hb binding increased with increasing temperature. The negative change in Gibbs free energy (ΔG°<0) indicated that the binding of Hb on the PGMA-Cu(2+) beads was a thermodynamically favorable process. The ΔS and ΔH values were 102.2 J/mol K and -2.02 kJ/mol, respectively. Significant amount of the bound Hb (up to 95.8%) was eluted in the elution medium containing 1.0 M NaCl in 1 h. The binding followed Langmuir isotherm model with monolayer binding capacity of 80.3-135.7 mg/g. Consecutive binding-elution experiments showed that the PGMA-Cu(2+) beads can be reused almost without any loss in the Hb binding capacity. To test the efficiency of Hb depletion from blood hemolysate, eluted portion was analyzed by fast protein liquid chromatography. The depletion efficiency for Hb was above 97.5%. This study determined that the PGMA-Cu(2+) beads had a superior binding capacity for Hb compared to the other carriers within this study.