核磁共振波谱在药物发现中的应用

核磁共振波谱通过检测组成有机化合物分子的原子核跃迁而得到反映核性质的参数以及周围化学环境对这些参数的影响规律. 这些参数的相关内容包含了极其详尽的有机化合物分子结构和分子间相互作用的信息,并构成了核磁共振结构解析和生物靶分子-配体相互作用研究的理论基础. 在生物医药研发领域内,科研院所和公司企业的研发工作者们一直在努力探索利用核磁共振波谱监测生物靶分子-配体相互作用作为药物发现工具的潜能. 本文旨在针对核磁共振波谱在药物发现过程中活性化合物筛选的最新研究进展进行综述.     

Small crystals and small coils in variable-temperature single-crystal NMR

Time savings by a factor of between 20 and 30 in the acquisition of multinuclear single-crystal (SC) NMR spectra have been obtained for submillimeter-size (0.01 to 0.03 mm(3)) single crystals when compared to recent results for (31)P and (87)Rb SC NMR. This gain in sensitivity is achieved by optimizing the filling factor using the smallest possible rf coil (2.0 mm inner diameter) for the specific SC probe design. Furthermore, this small coil is particularly useful for variable-temperature SC NMR studies. A probe design for such studies is presented and demonstrated experimentally.     

Plate-based diversity subset screening generation 2: an improved paradigm for high-throughput screening of large compound files

High-throughput screening (HTS) is an effective method for lead and probe discovery that is widely used in industry and academia to identify novel chemical matter and to initiate the drug discovery process. However, HTS can be time consuming and costly and the use of subsets as an efficient alternative to screening entire compound collections has been investigated. Subsets may be selected on the basis of chemical diversity, molecular properties, biological activity diversity or biological target focus. Previously, we described a novel form of subset screening: plate-based diversity subset (PBDS) screening, in which the screening subset is constructed by plate selection (rather than individual compound cherry-picking), using algorithms that select for compound quality and chemical diversity on a plate basis. In this paper, we describe a second-generation approach to the construction of an updated subset: PBDS2, using both plate and individual compound selection, that has an improved coverage of the chemical space of the screening file, whilst only selecting the same number of plates for screening. We describe the validation of PBDS2 and its successful use in hit and lead discovery. PBDS2 screening became the default mode of singleton (one compound per well) HTS for lead discovery in Pfizer.     

A Probe Design for the Acquisition of Homonuclear, Heteronuclear, and Inverse Detected NMR Spectra from Multiple Samples

A new probe design is presented for obtaining homonuclear, heteronuclear, and inverse detected NMR spectra from more than one sample in the same total data acquisition time as for a single sample, thus increasing data acquisition efficiency. Specifically, a two-coil system, with each solenoidal coil impedance matched to 50 Omega at both proton and nitrogen frequencies, has been designed for operation at 11.7 T with an observe volume of 15 microL for each coil. Isolation between the two frequencies for each individual coil, and at each frequency between coils, was greater than 30 dB. Two-dimensional COSY and HMQC spectra were obtained with negligible NMR cross-talk between the two coils.     

基于片段的先导化合物发现中的核磁应用

基于片段的药物筛选与设计在过去10年开始出现并获得了重要的应用,数十种基于片段的药物已经进入临床测试期. 源于靶标蛋白和小分子片段本质上的弱相互作用,现代核磁技术在其中发挥着无可替代的作用. 该文简略介绍了核磁片段筛选的基本流程和重要概念,包括靶标蛋白的选择、片段库的设计、质量控制和重要的核磁筛选技术. 在后续的基于片段的先导化合物发现阶段,阐述了核磁新技术的基本理论框架,包括化学位移扰动、分子间NOE、残留偶极耦合和顺磁标记等方法,以及这些新技术在靶标/配基复合体结构研究中的实际应用,穿插演示了片段组装的基本思路和成功案例.     

Compensation of effect of field instability by reference deconvolution with phase reconstruction

A compensation method based on reference deconvolution is developed to obtain high-resolution NMR spectra under an unstable magnetic field. It is shown that the applicability of the original deconvolution method is limited for small fluctuation, and a process what may be called phase reconstruction is proposed to compensate large field fluctuation. We demonstrate the method using a probe with a coil that can generate a fluctuation field artificially. A high-resolution 1H NMR spectrum of ethylbenzene was obtained under the unstable field after compensation with this method.     

Design of small volume HX and triple-resonance probes for improved limits of detection in protein NMR experiments

Three- and four-frequency nuclear magnetic-resonance probes have been designed for the study of small amounts of protein. Both "HX" (1H, X, and 2H channels) and "triple-resonance" (1H, 15N, 13C, and 2H) probes were implemented using a single transmit/receive coil and multiple-frequency impedance matching circuits. The coil used was a six-turn solenoid with an observe volume of 15 microl. A variable pitch design was used to improve the B1 homogeneity of the coil. Two-dimensional HSQC spectra of approximately 1mM single labeled 15N- and double labeled 15N/13C-proteins were acquired in experimental times of approximately 2h. Triple-resonance capability of the small-volume triple-resonance probe was demonstrated by acquiring three-dimensional HNCO spectra from the same protein samples. In addition to enabling very small quantities of protein to be used, the extremely short pulse widths (1H = 4, 15N = 4, and 13C = 2 micros) of this particular design result in low power decoupling and wide-bandwidth coverage, an important factor for the ever-higher operating frequencies used for protein NMR studies.     

Reduced data acquisition time in multi-dimensional NMR spectroscopy using multiple-coil probes

A new hardware-based approach is presented to reduce data acquisition times in multi-dimensional NMR spectroscopy using a multiple-coil probe. Using a four-coil setup, two-dimensional COSY and TOCSY spectra were acquired in one-quarter the time of conventional spectra by simultaneous acquisition of different effective t1 evolution times for each coil. Data processing consists of simple phase-shifting and intensity normalization of the individual data sets, and results in spectra almost identical to those acquired in a conventional manner. This method can potentially be integrated with other new data acquisition and processing schemes for further increases in data acquisition speed.     

Design,Synthesis, Photophysical Properties,Biological Estimation and Molecular Docking Studies of Novel Schiff Base Derivatives as Potential Urease Inhibitors

A quinoline functionalized two novel fluorescent Schiff bases, N-(quinolin-2-ylmethylene) anthracen-1-amine (SB1) and 2-(quinolin-2-ylmethyleneamino) benzene thiol (SB2) were synthesized and confirmed by using ¹H NMR, IR and GC-MS techniques. The spectroscopic properties were examined by absorption spectroscopy and fluorescence spectroscopy. The absorption and fluorescence spectra of the probes (SB1 and SB2) were measured in a variety of solvents. Both the compounds were tested for urease inhibitory activity. The synthesized compound SB2 proved to be the most effective screening for enzyme inhibitory activity with IC₅₀?=?0.111 μM than SB1 (IC₅₀?=?0.287 μM). Molecular docking studies were performed to delineate the binding affinity and conformational positions of chemical compounds within the active region of the target protein. In-vitro analysis depicts the potency of SB1 in free radical scavenging as compared to the reference drug vitamin C.     

An easily constructed, tuning free, ultra-broadband probe for NMR

We have developed an easy to construct, non-resonant wideband NMR probe. The probe is of the saddle coil geometry and is designed such that the coil itself forms a transmission line. The probe thus requires no tuning or matching elements. We use the probe with a spectrometer whose duplexer circuitry employs a simple RF switch instead of the more common lambda/4 lines, so the entire probe and spectrometer perform in an essentially frequency-independent manner. Despite being designed with electro- and magnetostatic formulas, the probe performs well at frequencies up to 150 MHz and beyond. We expect that with additional design effort, the probe could be modified for use at significantly higher frequencies. Because our construction method relies on commercial circuit fabrication techniques, identical probes can be easily and accurately produced.     

A low-E magic angle spinning probe for biological solid state NMR at 750 MHz

Crossed-coil NMR probes are a useful tool for reducing sample heating for biological solid state NMR. In a crossed-coil probe, the higher frequency ¹H field, which is the primary source of sample heating in conventional probes, is produced by a separate low-inductance resonator. Because a smaller driving voltage is required, the electric field across the sample and the resultant heating is reduced. In this work we describe the development of a magic angle spinning (MAS) solid state NMR probe utilizing a dual resonator. This dual resonator approach, referred to as “low-E,” was originally developed to reduce heating in samples of mechanically aligned membranes. The study of inherently dilute systems, such as proteins in lipid bilayers, via MAS techniques requires large sample volumes at high field to obtain spectra with adequate signal-to-noise ratio under physiologically relevant conditions. With the low-E approach, we are able to obtain homogeneous and sufficiently strong radiofrequency fields for both ¹H and ¹³C frequencies in a 4 mm probe with a ¹H frequency of 750 MHz. The performance of the probe using windowless dipolar recoupling sequences is demonstrated on model compounds as well as membrane-embedded peptides.     

[ONSO]型Salen化合物的合成、单晶结构及金属离子识别

一些重金属离子即使在较低浓度时也会对环境、生物体产生毒性,所以研究痕量金属离子识别具有重大意义。荧光传感器由于具有选择性好、灵敏度高、成本低、实时响应等优点, 得到了广泛关注。以2,4-二叔丁基苯酚,3,5-二叔丁基水杨醛和邻氨基苯硫酚为原料合成了类Salen配体L1, 并用1H NMR,13C NMR,IR,元素分析及X射线单晶衍射等手段对其进行了表征。并通过自由挥发法得到了配体L1的单晶结构,实验表明L1是三斜晶、P-1空间群的一个空穴平面[ONSO]四配位环境。通过荧光光谱考察了类Salen配体与金属离子(Li+,Na+,K+,Cd2+,Cs+,Co2+,Cu2+,Hg2+,Mn2+,Ni2+,Zn2+,Ag+)的识别与配位性能。光谱滴定分析表明L1与Zn2+以1∶1的化学计量数配位。另外,L1与Zn2+结合后荧光显著增强,荧光检测限达到5.01×10-5 mol·L-1,而上述提到的其他常见金属离子不引起荧光光谱变化。结果表明L1是一个对检测Zn2+的选择性高,灵敏度强的荧光增强型探针。     

Design and construction of a versatile dual volume heteronuclear double resonance microcoil NMR probe

Improved NMR detection of mass limited samples can be obtained by taking advantage of the mass sensitivity of microcoil NMR, while throughput issues can be addressed using multiple, parallel sample detection coils. We present the design and construction of a double resonance 300-MHz dual volume microcoil NMR probe with thermally etched 440-nL detection volumes and fused silica transfer lines for high-throughput stopped-flow or flow-through sample analysis. Two orthogonal solenoidal detection coils and the novel use of shielded inductors allowed the construction of a probe with negligible radio-frequency cross talk. The probe was resonated at ¹H–²D (upper coil) and ¹H–¹³C (lower coil) frequencies such that it could perform 1D and 2D experiments with active locking frequency. The coils exhibited line widths of 0.8–1.1 Hz with good mass sensitivity for both ¹H and ¹³C NMR detection. ¹³C-directly detected ²D HETCOR spectra of 5% v/v ¹³C labeled acetic acid were obtained in less than 5 min. Demonstration of the probe characteristics as well as applications of the versatile two-coil double resonance probe are discussed.     

Spectral unraveling by space-selective Hadamard spectroscopy

Spectral unraveling by space-selective Hadamard spectroscopy (SUSHY) enables recording of NMR spectra of multiple samples loaded in multiple sample tubes in a modified spinner turbine and a regular 5mm liquids NMR probe equipped with a tri-axis pulsed field gradient coil. The individual spectrum from each sample is extracted by adding and subtracting data that are simultaneously obtained from all the tubes based on the principles of spatially resolved Hadamard spectroscopy. The well-known Hadamard spectroscopy has been applied for spatial selection and the method utilizes standard configuration of NMR instrument hardware. The SUSHY method can be easily incorporated in multi-dimensional multi-tube NMR experiments. This method combines the excitation multiplexing, natural advantage of FTNMR, and sample multiplexing and offers high-throughput by reducing the total experimental time by up to a factor of four in a 4-tube mode.     

Radiation damping in microcoil NMR probes

Radiation damping arises from the field induced in the receiver coil by large bulk magnetization and tends to selectively drive this magnetization back to equilibrium much faster than relaxation processes. The demand for increased sensitivity in mass-limited samples has led to the development of microcoil NMR probes that are capable of obtaining high quality NMR spectra with small sample volumes (nL-microL). Microcoil probes are optimized to increase sensitivity by increasing either the sample-to-coil ratio (filling factor) of the probe or quality factor of the detection coil. Though radiation damping effects have been studied in standard NMR probes, these effects have not been measured in the microcoil probes. Here a systematic evaluation of radiation damping effects in a microcoil NMR probe is presented and the results are compared with similar measurements in conventional large volume samples. These results show that radiation-damping effects in microcoil probe is much more pronounced than in 5 mm probes, and that it is critically important to optimize NMR experiments to minimize these effects. As microcoil probes provide better control of the bulk magnetization, with good RF and B0 inhomogeneity, in addition to negligible dipolar field effects due to nearly spherical sample volumes, these probes can be used exclusively to study the complex behavior of radiation damping.     

X-[1H, 19F] triple resonance with a X-[1H] CP MAS probe and characterisation of a 29Si-19F spin pair

An economic approach for implementing X-[1H,19F] double-decoupling MAS NMR experiments with a conventional X-[1H] dual-channel CP MAS probe is demonstrated. The parameters characterising the isolated 29Si-19F spin pair in an organosilicon compound R(3)SiF (R = 9-anthryl) are determined. In addition, we discuss the optimum choice of experimental parameters for determining all 29Si-19F spin-pair parameters from straightforward 29Si MAS NMR spectra with only 1H decoupling applied during acquisition.     

Ultra-broadband NMR probe: numerical and experimental study of transmission line NMR probe

We have reinvestigated a transmission line NMR probe first published by Lowe and co-workers in 1970s [Rev. Sci. Instrum. 45 (1974) 631; 48 (1977) 268] numerically and experimentally. The probe is expected to be ultra-broadband, thus might enable new types of solid-state NMR experiments. The NMR probe consists of a coil and capacitors which are connected to the coil at regular intervals. The circuit is the same as a cascaded LC low-pass filter, except there are nonzero mutual inductances between different coil sections. We evaluated the mutual inductances by Neumann's formula and calculated the electrical characteristics of the probe as a function of a carrier frequency. We found that they were almost the same as those of a cascaded LC low-pass filter, when the inductance L of a section was estimated from the inductance of the whole coil divided by the number of the sections, and if C was set to the capacitance in a section. For example, the characteristic impedance of a transmission line coil is given by Z=(L/C)(1/2). We also calculated the magnitude and the distribution of RF magnetic field inside the probe. The magnitude of RF field decreases when the carrier frequency is increased because the phase delay between neighboring sections is proportional to the carrier frequency. For cylindrical coils, the RF field is proportional to (pinu/2nu(d))(1/2)exp(-nu/nu(d)), where the decay frequency nu(d) is determined by the dimensions of the coil. The observed carrier frequency thus must be much smaller than the decay frequency. This condition restricts the size of transmission line coils. We made a cylindrical coil for a 1H NMR probe operating below 400 MHz. It had a diameter 2.3mm and a pitch 1.2mm. Five capacitors of 6pF were connected at every three turns. The RF field strength was 40 and 60 kHz at the input RF power 100 W by a calculation and by experiments, respectively. The calculations showed that the RF field inhomogeneity along the coil axis was caused by a standing wave of current, which arose from the reflections at the coil ends. The calculation showed that the homogeneity could be improved by decreasing the pitch near the both ends and making their impedance close to that at the center.     

Estimation and measurement of flat or solenoidal coil inductance for radiofrequency NMR coil design

The inductance of a radiofrequency coil determines its compatibility with a given NMR probe circuit. However, calculation (or estimation) of inductance for radiofrequency coils of dimensions suitable for use in an NMR probe is not trivial, particularly for flat-coils. A comparison of a number of formulae for calculation of inductance is presented through the use of a straightforward inductance measurement circuit. This technique relies upon instrumentation available in many NMR laboratories rather than upon more expensive and specialized instrumentation often utilized in the literature. Inductance estimation methods are suggested and validated for both flat-coils and solenoids. These have proven very useful for fabrication of a number of new coils in our laboratory for use in static solid-state NMR probes operating at (1)H frequencies of 300 and 600MHz. Solenoidal coils with very similar measured and estimated inductances having inner diameters from 1 to 5mm are directly compared as an example of the practical application of inductance estimation for interchange of coils within an existing solid-state NMR probe.     

NMR probe for the simultaneous acquisition of multiple samples.

A dual channel probe for the simultaneous acquisition of NMR data from multiple samples has been developed. This multiplex probe consists of two noninteracting sample coils that are each capable of detecting NMR signals at the same resonant frequency with good sensitivity and resolution. 13C free induction decays for the two samples, methanol (13C, 99%) and carbon tetrachloride (13C, 99%), were acquired simultaneously at 75.44 MHz using a single transmitter pulse and separate NMR receivers. S/N measurements are comparable to those observed using single coils. No evidence of cross talk is evident in the spectra even after considerable signal averaging. The probe demonstrates the feasibility of significant parallelism in NMR, which will be of interest in situations where high throughput analysis is desired.