Competition STD NMR for the detection of high-affinity ligands and NMR-based screening

The reported competition STD NMR method combines saturation transfer difference (STD) NMR with competition binding experiments to allow the detection of high-affinity ligands that undergo slow chemical exchange on the NMR time-scale. With this technique, the presence of a competing high-affinity ligand in the compound mixture can be detected by the disappearance or reduction of the STD signals of a low-affinity indicator ligand. This is demonstrated on a BACE1 (beta-site amyloid precursor protein cleaving enzyme 1) protein-inhibitor system. This method can also be used to derive an approximate value, or a lower limit, for the dissociation constant of the potential ligand based on the reduction of the signal intensity of the STD indicator, which is illustrated on an HSA (human serum albumin) model system. This leads to important applications of the competition STD NMR method for lead discovery: it can be used (i) for compound library screening against a broad range of drug targets to identify both high- and low-affinity ligands and (ii) to rank order analogs rapidly and derive structure-activity relationships, which are used to optimize these NMR hits into viable drug leads.     

Structure‐Based Design of Inhibitors of the Aspartic Protease Endothiapepsin by Exploiting Dynamic Combinatorial Chemistry

Structure‐based design (SBD) can be used for the design and/or optimization of new inhibitors for a biological target. Whereas de novo SBD is rarely used, most reports on SBD are dealing with the optimization of an initial hit. Dynamic combinatorial chemistry (DCC) has emerged as a powerful strategy to identify bioactive ligands given that it enables the target to direct the synthesis of its strongest binder. We have designed a library of potential inhibitors (acylhydrazones) generated from five aldehydes and five hydrazides and used DCC to identify the best binder(s). After addition of the aspartic protease endothiapepsin, we characterized the protein‐bound library member(s) by saturation‐transfer difference NMR spectroscopy. Cocrystallization experiments validated the predicted binding mode of the two most potent inhibitors, thus demonstrating that the combination of de novo SBD and DCC constitutes an efficient starting point for hit identification and optimization.     

AlN靶材射频磁控溅射制备AlN薄膜及性质研究

以AlN作为靶材,使用射频磁控溅射法在Si（100）和玻璃衬底上,在纯氮气气氛条件下制备得到AlN薄膜,并研究了衬底温度对溥膜的结构,形貌和性质的影响．实验表明,衬底温度为370℃的条件下制备的AlN溥膜具有C轴择优取向,薄膜表面均匀、致密和平整,均方根粗糙度为4．83nm．随着基片温度的增加,溥膜的折射率增加,对应着薄膜从非晶态到晶态过程的演变．     

Application of WET sequence for the detection of the ligand signals resonating close to water

An efficient pulse sequence for observing the ligand signals resonating close to the water signal has been developed by incorporating the WET technique into the saturation transfer difference pulse sequence. Although several pulse sequences have been developed for observing a ligand binding with a protein receptor, the ligand signals resonating close to the water were undetectable owing to the interference of the huge water signal in the samples containing 95% ¹H₂O. On the point of sample preparation, it is preferable to avoid the solvent exchange in the protein samples. In the proposed pulse sequence, a WET sequence is incorporated for the selective suppression of the water resonance. The efficient water suppression and the clear observation of the bound ligand signals close to the water have been demonstrated using the lysozyme‐glucose complex. Copyright © 2009 John Wiley & Sons, Ltd.     

13C‐NMR detection of STD spectra

We have investigated the use of ¹³C for the detection of saturation transfer difference (STD) NMR spectra. By detecting the STD spectrum in the ¹³C channel it is possible to eliminate the residual water signal in the STD‐NMR spectrum. We have employed an INEPT transfer in order to shift the magnetization from the proton channel to ¹³C. As a sample system to check our method we have used human serum albumin and phenylalanine. We have shown that such a transfer can be accomplished and gives reasonable signal intensities. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of nucleotides binding to chromatography supports provided by nuclear magnetic resonance spectroscopy

The epitope mapping of nucleotides bound to three chromatography supports is accomplished using saturation transfer difference (STD)-NMR spectroscopy. This experiment involves subtracting a spectrum in which the support was selectively saturated from one recorded without support saturation. In the difference spectrum only the signals of the ligands that bind to the support and received saturation transfer remain. The nucleotide protons in closer contact with the support have more intense signals due to a more efficient transfer of saturation. We investigate the effects on the binding to the nucleotides by the introduction of a spacer arm between l-histidine and Sepharose. Our NMR experiments evidence a clear contribution of the spacer to the interaction with all the nucleotides, increasing the mobility of the amino acid and giving different STD responses. This enhanced mobility originates the reinforcement of the interactions with the sugar moiety and phosphate group of 5'-CMP and 5'-TMP or the base of 5'-GMP and 5'-UMP. Hence, with this study we show that by using STD NMR technique on chromatographic systems it is possible to provide a fast, robust and efficient way of screening the atoms involved in the binding to the supports.     

碲锌镉晶体晶格畸变的测定与分析

在高分辨X射线衍射仪上,利用不对称布拉格衍射STD技术与单晶摇摆曲线技术相结合的方法,对碲锌镉单晶材料的晶格畸变进行了分析测定.此方法实现了一次装样,多角度、多次测定晶体的摇摆曲线,从而利用多组实验数据完成多元线性回归方程组的求解,避开了一般应变测定方法中难以确定无应力状态下衍射角的问题.所测定的生长态碲锌镉晶片晶格畸变量为10-3～10-2数量级,该畸变量是碲锌镉晶片存在成份偏析、位错和空位等缺陷以及晶片受到的应力综合作用的结果.     

Bulk derivatization and cation exchange restricted access media-based trap-and-elute liquid chromatography–mass spectrometry method for determination of trace estrogens in serum

Estrone (E1), estradiols (α/β-E2), and estriol (E3) are four major metabolically active estrogens exerting strong biological activities at very low circulating concentrations. This paper reports a sensitive and efficient method with automated, on-line clean-up and detection to determine trace estrogens in a small volume of serum samples using liquid chromatography–electrospray ionization–tandem mass spectrometry directly, without off-line liquid–liquid or solid-phase extraction pretreatments. Serum aliquots (charcoal stripped fetal bovine serum, 100 μL) were spiked with four estrogen standards and their corresponding isotope-labeled internal standards, then bulk derivatized with 2-fluoro-1-methyl-pyridium p-toluenesulfonate (2-FMP) to establish the calibration curves and perform method validation. Calibration was established in the concentration ranges of 5–1000 pg mL⁻¹, and demonstrated good linearity of R² from 0.9944 to 0.9997 for the four derivatized estrogens. The lower detection limits obtained were 3–7 pg mL⁻¹. Good accuracy and precision in the range of 86–112% and 2.3–11.9%, respectively, were observed for the quality control (QC) samples at low, medium, and high concentration levels. The stability tests showed that the derivatized serum samples were stable 8 h after derivatization at room temperature and at least to 48 h if stored at −20 °C. The method was applied to measure trace estrogens in real human and bovine serum samples, and three of four estrogen compounds studied were observed and quantified.     

大型医用氧舱监测与控制系统

介绍大型医用氧舱的实时监控系统，从氧舱对系统提出的要求出发，讨论以ＳＴＤ工控机为核心的系统组成，软、硬件设计以及对象模型和控制方式。该系统投入运行表明已完全达到开发设计要求。