RAMPED-UP NMR: multiplexed NMR-based screening for drug discovery

The crucial step in drug discovery is the identification of a lead compound from a vast chemical library by any number of screening techniques. NMR-based screening has the advantage of directly detecting binding of a compound to the target. The spectra resulting from these screens can also be very complex and difficult to analyze, making this an inefficient process. We present here a method, RAMPED-UP NMR, (Rapid Analysis and Multiplexing of Experimentally Discriminated Uniquely Labeled Proteins using NMR) which generates simple spectra which are easy to interpret and allows several proteins to be screened simultaneously. In this method, the proteins to be screened are uniquely labeled with one amino acid type. There are several benefits derived from this unique labeling strategy: the spectra are greatly simplified, resonances that are most likely to be affected by binding are the only ones observed, and peaks that yield little or no information upon binding are eliminated, allowing the analysis of multiple proteins easily and simultaneously. We demonstrate the ability of three different proteins to be analyzed simultaneously for binding to two different ligands. This method will have significant impact in the use of NMR spectroscopy for both the lead generation and lead optimization phases of drug discovery by its ability to increase screening throughput and the ability to examine selectivity. To the best of our knowledge, this is the first time in any format that multiple proteins can be screened in one tube.     

The absolute sign of J coupling constants determined using the order matrix calculation

A novel methodology using the order matrix calculation to determine the absolute sign of spin-spin couplings based on the structure of organic compounds is presented. The sign of the residual dipolar coupling (RDC) depends on the sign of corresponding scalar spin-spin coupling constant and the sign of the RDC has a dramatic influence on the order matrix calculation. Therefore, the sign of the spin-spin coupling constant can be obtained by an order matrix calculation through the corresponding RDC. Six types of spin-spin coupling constants, including 2J(H,H), 1J(C,F), 2J(C,F), 3J(C,F), 2J(F,H) and 3J(F,H), were obtained simultaneously. Except for 3J(C,F) where the measured RDCs have very small magnitudes, the signs were determined unambiguously.     

Synthesis,characterization and antioxidant,antibacterial activity Zn2+, Cu2+, Ni2+ and Co2+, complexes of ligand [2-(thiophen-2-yl)-1-(thiophen-2-ylmethyl)-1H-benzo[d]imidazole]

New metal complexes of (Zn(II), Co(II), Ni(II) and Cu(II)) based on the ligand 2-(thiophen-2-yl)-1-(thiophen-2-ylmethyl)-1H-benzo[d]imidazole] were synthesized, whose structures were determined with the different spectroscopic techniques ¹H NMR,¹³C NMR, FT-IR, UV–Visible and by mass spectrometry. The thermal analysis was performed by TG-DTA. The antioxidant activity of the ligand and its complexes was evaluated by DPPH (2,2-diphenyl-1-picrylhydrazyl) method, in comparison with the synthetic antioxidant, ascorbic acid. The results obtained showed that the antioxidant activity of the ligand and its complexes is moderate and that the copper complex has a high activity that exceeds that of ascorbic acid. Antimicrobial activity of the ligand and its metal complexes was studied against two Gram-positive bacteria: Bacillus subtilis ILP1428B, Staphylococcus aureus CIP543154 and two Gram-negative bacteria: Pseudomonas aeruginosa ATCC27653, Escherichia coli CIP5412 (American Type Culture Collection)the activity data show that the metal complexes are more potent than the free ligand.     

固相萃取-毛细管柱气相色谱-质谱测定土壤和蔬菜中三唑酮残留量

提出固相萃取分离,毛细管柱气相色谱和质谱联用法测定土壤及蔬菜中三唑酮的残留量.试样用乙腈提取,所得提取液在80℃吹氮至近干,并加正己烷2 mL溶解残渣.此溶液流经填充酸性氧化铝的固相萃取小柱,用正己烷-丙酮(2+1)混合溶剂淋洗小柱,使土壤样品中存在的硫磺与需测定的三唑酮分离.用正己烷一丙酮(1+3)混合溶剂将三唑酮从小柱上解吸洗下,所得洗出液蒸发浓缩,用正己烷定容为5.0 mL供GC-MS分析用.测定中用三唑酮标准溶液制备浓度范围在0.006～1.44 mg·L-1之间的标准曲线.方法的检出限(3S/N)为0.001 mg·kg-1.在3个浓度水平上对方法的回收及精密度作了试验,测得其回收率及相对标准偏差(n=5)依次在84%～92 9,5及1.93%～4.04%之间.     

Differential non-destructive image current detection in a fourier transform quadrupole ion trap

Dual-detector differential non-destructive Fourier transform detection in a quadrupole ion trap is shown to improve signal intensity and reduce noise compared with spectra recorded using a single detector. A larger area detector in each end-cap electrode is machined to fit its hyperbolic shape and so minimize field imperfections on the z-axis. Argon, acetophenone and bromobenzene spectra were recorded to allow a comparison between single- and dual-detector (differential) modes of detection and to demonstrate the improvement achieved with differential detection. Copyright 1999 John Wiley & Sons, Ltd.     

Determination of Methanol Increment in Mobile Phase Consisting of Methanol and Water by On—line UV Spectrometry in Reversed Phase Liquid Chromatography

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乙基曙红碘翁盐的染料再生循环光敏体系

本文设计了染料循环再生的光敏反应体系, 通过对占吨碘翁盐电子反应机制的深入研究, 提出了占吨染料在光敏反应过程中的染料再生循环, 它依次由三个反应过程组成: 在光作用下, 通过电子转移反应, 产生染料氧化褪色体(I); 在H给体存在下, 通过H转移反应, 使产物(I)转化为染料酸式隐色体(II); 在碱作用下, (II)发生去质子反应, 染料再生回复到染料的起始结构。其中光、H给体和碱的作用是再生循环的三个要素, 而控制染料酸式隐色体生成比例是染料再生效率的关键。由乙基曙红碘翁盐/三乙胺组成的染料再生体系的染料敏化效率比单一体系高4倍左右, 同时使活性自由基的数量增殖, 这种体系在实际应用方面具有重要意义。     

The effect of relaxation on the epitope mapping by saturation transfer difference NMR

The effect of longitudinal relaxation of ligand protons on saturation transfer difference (STD) was investigated by using a known binding system, dihydrofolate reductase and trimethoprim. The results indicate that T1 relaxation of ligand protons has a severe interference on the epitope map derived from a STD measurement. When the T1s of individual ligand protons are distinctly different, STD experiments may not give an accurate epitope map for the ligand-target interactions. Measuring the relaxation times prior to mapping is strongly advised. A saturation time shorter than T1s is suggested for improving the potential epitope map. Reduction in temperature was seen to enhance the saturation efficiency in small to medium size targets.     

Variation of molecular alignment as a means of resolving orientational ambiguities in protein structures from dipolar couplings

Residual dipolar couplings for pairs of proximate magnetic nuclei in macromolecules can easily be measured using high-resolution NMR methods when the molecules are dissolved in dilute liquid crystalline media. The resulting couplings can in principle be used to constrain the relative orientation of molecular fragments in macromolecular systems to build a complete structure. However, determination of relative fragment orientations based on a single set of residual dipolar couplings is inherently hindered by the multi-valued nature of the angular dependence of the dipolar interaction. Even with unlimited dipolar data, this gives rise to a fourfold degeneracy in fragment orientations. In this Communication, we demonstrate a procedure based on an order tensor analysis that completely removes this degeneracy by combining residual dipolar coupling measurements from two alignment media. Application is demonstrated on (15)N-(1)H residual dipolar coupling data acquired on the protein zinc rubredoxin from Clostridium pasteurianum dissolved in two different bicelle media.