Fluorescent probe and NMR studies of the aggregation of bile salts in aqueous solution

The binding of the fluorescent probes 1-anilino-8-naphthalene sulfonate and dansyl cadaverine to the sodium salts of cholic, deoxycholic and dehydrocholic acids has been investigated. Enhanced probe solubilisation accompanies aggregation. Monitoring of fluorescence intensities as a function of bile salt concentration permits the detection of primary micelle formation, as well as secondary association. The transition concentrations obtained by fluorescence are in good agreement with values determined for the critical micelle concentrations, by other methods. Differences in the behaviour of cholate and deoxycholate have been noted. Fluorescence polarisation studies of 1,6-diphenyl-1,3,5-hexatriene solubilised in bile salt micelles suggest a higher microviscosity for the interior of the deoxycholate micelle as compared to cholate. 1H NMR studies of deoxycholate over the range 1–100 mg/ml suggest that micelle formation leads to a greater immobilisation of the C18 and C19 methyl groups as compared to the C21 methyl group. Well resolved 13C resonances are observed for all three steroids even at high concentration. Both fluorescence and NMR studies confirm that dehydrocholate does not aggregate.

Structural studies of the hen's egg lipovitellins. NMR and fluorescence investigations

The pH dependent reversible association-dissociation reaction of α- and β-lipovitellins from egg yolk has been studied by 1H NMR and fluorescence probe methods. Increased mobility of the choline methyl groups has been demonstrated on dissociation. The lipid methylene resonance of β-lipovitellin shows clear doublet character suggesting that the fatty acid chains exist in distinct environments. The high field component increases with temperature but is suppressed on treatment with pronase, suggesting a significant role for proteins in maintaining the differences in lipid environments. 1-Anilino-8-naphthalene sulfonate has been shown to bind less effectively to the monomeric lipovitellins. This is in agreement with earlier results suggesting that dissociation may be accompanied by increased hydration and conformational changes.

High-pressure NMR investigations of the protonic conductor (NH4)4Fe(CN)6.1.5H2O

The effect of high hydrostatic pressure up to 1.5 GPa on ionic motion in (NH4)4Fe(CN)6.1.5H2O has been studied by wide-line 1H NMR experiments performed in the temperature range from room temperature to 77 K. The experiments at room temperature have shown a large increase in the second moment at 0.45 GPa as a result of a pressure-induced phase transition. The temperature dependence study up to 0.425 GPa has shown a gradual increase in the values of activation energy and attempt frequency with increase in pressure. The activation volume for motion at 300 K has been estimated to be 6% of molar volume. Vacancy-assisted ionic jumps are concluded to be the mode of charge transport. Second moments estimated at 77 K show evidence for tunnelling reorientation of at least one of the two NH4+ groups in the compound.

Conformations of dehydrophenylalanine containing peptides: nmr studies of an acyclic hexapeptide with two delta Z-Phe residues

The conformation of an acyclic dehydrophenylalanine (delta Z-Phe) containing hexapeptide, Boc-Phe-delta Z-Phe-Val-Phe-delta Z-Phe-Val-OMe, has been investigated in CDCl3 and (CD3)2SO by 270-MHz 1H-nmr. Studies of NH group solvent accessibility and observation of interresidue nuclear Overhauser effects (NOEs) suggest a significant solvent-dependent conformational variability. In CDCl3, a population of folded helical conformations is supported by the inaccessibility to solvent of the NH groups of residues 3-6 and the detection of several NiH----Ni + 1H NOEs. Evidence is also obtained for conformational heterogeneity from the detection of some Ci alpha H----Ni + 1H NOEs characteristic of extended strands. In (CD3)2SO, the peptide largely favors an extended conformation, characterized by five solvent-exposed NH groups and successive Ci alpha H----Ni + 1H NOEs for the L-residues and Ci beta H----Ni + 1H NOEs for the delta Z-Phe residues. The results suggest that delta Z-Phe residues do not provide compelling conformational constraints.

Conformations of dehydrophenylalanine containing peptides. NMR studies on three tripeptides with a central dehydrophenylalanyl residue.

Three tripeptides containing a central Z-dehydrophenylalanine residue (Δz-Phe), Boc-L-Phe-Δz-Phe-X-OMe (X = L-Val 1, L-Leu 2 and X = L-Ala 3) have been synthesized and their solution conformations investigated by 270 MHz 1H NMR spectroscopy. In all three peptides, conformations involving the X residue NH in an intramolecular hydrogen bond were favoured in CDCl3 solutions. Studies of the nuclear Overhauser effect (NOE) provided support for a Type II β turn conformation in these peptides with Phe and Δz-Phe occupying the i + 1 and i + 2 positions, respectively. Significantly different conformations lacking any intramolecular hydrogen bonds were observed for peptide 1 in (CD3)2SO. NOE results were consistent with a significant population of molecules having semi-extended conformations (ø > 100°) at the Δz-Phe residue.

CD and NMR studies on the aggregation of amphotericin-B in solution

We report in this paper the aggregation properties of amphotericin-B (amp-B) in solution using CD and 1H-NMR techniques. Our results indicate that the preferred structure of amp-B in dimethylsulfoxide is a monomer at low concentrations (10−4M and below) and a stable dimer at higher concentrations (range 5 · 103 M to 10−2M). In a DMSO/ethanol mixture (1:1 (v/v)), the antibiotic is monomeric, irrespective of the concentration within the range studied. We propose a head-to-tail model based on NMR data. An understanding of the head-to-tail dimer, is, we believe important, particularly in view of the recent report wherein it is proposed that the drug inserts into bilayers as head-to-tail oligomers.

基于NMR的代谢组学方法对稀土生物效应的研究

近年来，随着高场傅里叶变换核磁共振波谱仪灵敏度的提高、分辨率的增加、化学位移的扩展、多脉冲实验技术的发展以及计算机技术的不断进步，使NMR技术在生物和医学领域的应用有了飞速的发展。而基于NMR技术结合模式识别的代谢组学方法更是在医学临床诊断、药物毒性、环境毒理学等领域得到广泛应用。 稀土由于其特殊的电子层结构在工业、农业、畜牧业和现代生物医学上具有非常广泛的应用。本论文工作采用基于核磁共振技术的代谢组学方法，分别对给药稀土钕、镨(农用稀土微肥常乐中两种主要成分)以及重稀土钆后大鼠的体液(尿液、血清)和组织(肝、肾)中代谢物的浓度变化进行了分析。结合生化指标数据和组织切片显微照片，系统的研究了稀土钕、镨和钆的急性生物效应。 运用高分辨1H NMR技术，检测分析了大鼠腹腔注射不同剂量硝酸钕(2，10，50 mg/kg体重，48 h)、氯化钆(10，50 mg/kg体重，168 h)和硝酸镨(2，10，50 mg/kg体重，168 h)后尿液、血清及肝肾组织中的代谢物如柠檬酸、肌酸、肌酸酐、二甲胺、二甲基甘氨酸、氮氧三甲胺(TMAO)、氨基酸、乳酸、琥珀酸、牛磺酸、胆碱、甘油三脂、糖元等的浓度、物种的变化。结合模式识别方法，并根据血中生化指标和组织光镜图，对稀土在体内与细胞、组织和器官的作用机理及急性毒性进行了有意义的探讨。结果表明：高剂量硝酸钕的引入可能使动物体内的代谢出现异常，动物肾脏和肝脏的特定部位(如肾小管、肾乳头、肝脏线粒体)受到选择性的损伤；氯化钆对大鼠造成损伤的靶器官是肝脏，同时肾脏功能也受到伤害，其主要表现为通过改变大鼠体内酶代谢而使大鼠肝脏中正常的能量代谢(糖代谢和脂肪代谢)和氨基酸代谢等受到扰乱，同时能使肾脏中维持渗透平衡的渗透质浓度降低；硝酸镨靶向器官为肝脏和肾脏，以肝脏为主，且呈现明显的剂量-反应关系，低、中剂量Pr(NO3)3对大鼠肝脏和肾脏的损伤在168 h内是可逆的。基于NMR的代谢组学方法也可用于其它化合物的毒性研究。

现代NMR技术在生物医学中的应用－－稀土对大鼠代谢产物的影响及磁共振造影剂性质的研究

近年来，随着脉冲傅里叶变换核磁共振波谱仪灵敏度的提高及计算机技术的进步，使NMR技术在生物领域的应用有了飞速的发展。论文应用NMR技术研究了稀土化合物（硝酸镧）对大鼠体内代谢产物的影响。对Wistar种大鼠按不同剂量给药（灌胃和腹腔注射两种方式）后的尿液和血清中的代谢物质的~1H NMR谱数据进行研究，并结合血清、尿液中生化指标的检测结果，指出稀土作用后大鼠肾脏的肾小管及肝脏线粒体均受到一定程度的损害，随稀土摄入量的增多，损害程度越严重。并提出代谢物中尿素、Suc、Kg、Cit、DMA、DMG、TMAO和氨基酸等可以作为肾脏受损伤的NMR markers，而乙醇、Lac和Tau可作为肝脏受损伤的NMR markers。实验中最小剂量为0.05 mg La(NO_3)_3/kg体重，仍有机体受损的NMR信号，说明稀土的安全剂量小于这一数值。在临床磁共振成像中，造影剂是医学诊断中的一种重要辅助试剂。本文采用NMR水弛豫分析法研究了GdDTPA及Gd(cycle-DTPA-1,2-PN)、Gd(DTPA－BIN)在水溶液和BSA溶液中的弛豫性质，为研究顺磁性金属配合物与蛋白质间相互作用情况提供了有效方法；对配体cycle-DTPA-1,2-pn、DTPA－BIN做了~1H NMR滴定研究，得出其质子解离过程和解离常数，并采用半经验量化计算方法计算了两种配体的质子化过程，得到了与~1H NMR滴定一致的结果。结果表明将NMR实验和量化计算方法结合，是考察复杂配体热力学稳定性的有效方法。

氨三乙酸稀土络合物配体交换反应的NMR研究

利用变温13C、1H－NMR研究了过量配体存在下，氨三乙酸稀土络合物Ln（NTA）2（Ln＝Ce，Pr和Nd）配体交换反应，在中性水溶液中，分子间的配体交换过程按如下机制进行：由溶液中自由NTA信号线宽分析了交换速率，确定了反应的活化能．结果表明，分子间配体交换反应的活化能与相应稀土络合物的热力学稳定性有一定关系.

混合物中稀土元素的NMR法测定

核磁共振检测混合物中的共存金属是新近发展起来的无机分析方法。Fedorov以EDTA和NTA为配体,通过观察络合物Ln(EDTA)和Ln(NTA)_2配体的~1H NMR谱线,率先开展了水溶液中共存稀土元素的同时间接测定研究,这种方法比分别直接检测各稀土核磁核共振信号的方法实用可靠,且方便易行。但采用~1H NMR谱检测水溶液中顺磁稀土离子时,强的溶剂水峰易掩蔽近邻的稀土配合物谱线;某些重稀土配合物的~1H谱线宽,相互重叠,难以用作分

聚甲基丙烯酸乙酯链结构核磁共振(NMR)研究

本文用~1H-NMR,~(13)C-NMR和化学位移相关谱(COSY)研究了聚甲基丙烯酸乙酯(PEMA)的链结构.虽然质子峰的重叠给谱的识别带来一些困难,但是利用H-H COSY和C-H COSY提供的信息讨论了PEMA的~1H谱和~(13)C谱中各谱峰的归属.归属β-CH_2的四元组立构序列仍有一些问题.最后计算了无规PEMA的五元组立构序列分布.

Use of NMR metabolomic plasma profiling methodologies to identify illicit growth-promoting administrations

Detection of growth-promoter use in animal production systems still proves to be an analytical challenge despite years of activity in the field. This study reports on the capability of NMR metabolomic profiling techniques to discriminate between plasma samples obtained from cattle treated with different groups of growth-promoting hormones (dexamethasone, prednisolone, oestradiol) based on recorded metabolite profiles. Two methods of NMR analysis were investigated—a Carr–Purcell–Meiboom–Gill (CPMG)-pulse sequence technique and a conventional 1H NMR method using pre-extracted plasma. Using the CPMG method, 17 distinct metabolites could be identified from the spectra. 1H NMR analysis of extracted plasma facilitated identification of 23 metabolites—six more than the alternative method and all within the aromatic region. Multivariate statistical analysis of acquired data from both forms of NMR analysis separated the plasma metabolite profiles into distinct sample cluster sets representative of the different animal study groups. Samples from both sets of corticosteroid-treated animals—dexamethasone and prednisolone—were found to be clustered relatively closely and had similar alterations to identified metabolite panels. Distinctive metabolite profiles, different from those observed within plasma from corticosteroid-treated animal plasma, were observed in oestradiol-treated animals and samples from these animals formed a cluster spatially isolated from control animal plasma samples. These findings suggest the potential use of NMR methodologies of plasma metabolite analysis as a high-throughput screening technique to aid detection of growth promoter use.

Development and application of NMR techniques to study water-in-CO2 microemulsions

Self-assembly is a phenomenon that occurs frequently throughout the universe. In this work, two self-assembling systems were studied: the formation of reverse micelles in isooctane and in supercritical CO2 (scCO2), and the formation of gels in organic solvents. The goal was the physicochemical study of these systems and the development of an NMR methodology to study them. In this work, AOT was used as a model molecule both to comprehensively study a widely researched system water/AOT/isooctane at different water concentrations and to assess its aggregation in supercritical carbon dioxide at different pressures. In order to do so an NMR methodology was devised, in which it was possible to accurately determine hydrodynamic radius of the micelle (in agreement with DLS measurements) using diffusion ordered spectroscopy (DOSY), the micellar stability and its dynamics. This was mostly assessed by 1H NMR relaxation studies, which allowed to determine correlation times and size of correlating water molecules, which are in agreement with the size of the shell that interacts with the micellar layer. The encapsulation of differently-sized carbohydrates was also studied and allowed to understand the dynamics and stability of the aggregates in such conditions. A W/CO2 microemulsion was prepared using AOT and water in scCO2, with ethanol as cosurfactant. The behaviour of the components of the system at different pressures was assessed and it is likely that above 130 bar reverse microemulsions were achieved. The homogeneity of the system was also determined by NMR. The formation of the gel network by two small molecular organogelators in toluene-d8 was studied by DOSY. A methodology using One-shot DOSY to perform the spectra was designed and applied with success. This yielded an understanding about the role of the solvent and gelator in the aggregation process, as an estimation of the time of gelation.

The power of NMR.: in two and three dimensions

Soon after its discovery in the 1950s, NMR had become an indispensable tool fr chemists. In the 1970s and 1980s, the power of the technique was extended from one dimension to two and even three dimensions, opening up exciting applkications in both chemistry and biochemistry. the success of one dimensional. high-resolution NMR stems from the unique insights that it can provide about molecular structure. The chemical shift of a nucleus gives invaluable information abut the chemical environment in which that nucleus is located, Coupling interactions between hydorgen nuclei, as revealed by characteristic splitting patterns inthe 1H-NMR spectrum, provide informaton about the loaction of one group of hydorgen atoms relative to others inthe molecule. And the nuclearf Overhauser effect (nOe) can shed light on molecular stereochemistry.

Application of on-line C30 RP-HPLC-NMR for the analysis of flavonoids from leaf extract of Maytenus aquifolium

The application of on-line C30-reversed-phase high-pressure liquid chromatography-nuclear magnetic resonance spectroscopy is described for the analysis of tetraglycosylated flavonoids in aqueous and hydroalcoholic extracts of the leaves of Maytenus aquifolium (Celastraceae). Triacontyl stationary phases showed adequate separation for on-line 1H-NMR measurements at 600 MHz and allowed the characterisation of these flavonoids by detection of both aromatic and anomeric proton signals. Copyright (C) 2000 John Wiley and Sons, Ltd.