Ligand-observed in-tube NMR in natural products research: A review on enzymatic biotransformations,protein–ligand interactions,and in-cell NMR spectroscopy

Natural product-observed NMR methods have considerably expanded the potentialities for in-tube NMR monitoring of complex enzymatic biotransformations and investigation of protein-natural product interactions even in living cells. We review, herein, the significant advantages of ligand-observed in-situ NMR monitoring of enzymatic biotransformations without restoring to laborious and time-consuming chromatographic methods. Emphasis will be given to the potentialities of the use of the NMR bioreactor: (i) to investigate through saturation transfer difference (STD), the capacity of natural products to serve as enzyme substrates, (ii) to monitor multiple biotransformation products of natural products with the use of immobilized enzymes and (iii) to investigate interactions of biotransformed products with protein targets. The use of STD and its variants, transfer effect Noes for PHArmacophore Mapping (INPHARMA) NMR, in conjunction with computational methods, can provide excellent tools in investigating competitive binding modes even in proteins with multiple binding sites. The method has been successfully applied in the study of unsaturated free fatty acids (UFFAs)-serum albumin complexes in which the location and conformational states of UFFAs could not be determined accurately, despite numerous X-ray structural studies, due to conformational averaging. This combined method, thus, may find promising applications in the field of protein-natural product recognition research. The emerging concept of in-cell NMR and recent applications will be discussed since they can provide atomic level insights into natural product-protein interactions in living cells without the need of isotope labelled techniques.     

Photosensitized oxidation of membrane lipids: reaction pathways, cytotoxic effects, and cytoprotective mechanisms.

Unsaturated lipids in cell membranes, including phospholipids and cholesterol, are well-known targets of oxidative modification, which can be induced by a variety of stresses, including ultraviolet A (UVA)- and visible light-induced photodynamic stress. Photodynamic lipid peroxidation has been associated with pathological conditions such as skin phototoxicity and carcinogenesis, as well as therapeutic treatments such as antitumor photodynamic therapy (PDT). Lipid hydroperoxides (LOOHs), including cholesterol hydroperoxides (ChOOHs), are important non-radical intermediates of the peroxidative process which can (i) serve as in situ reporters of type I vs. type II chemistry; (ii) undergo one-electron or two-electron reductive turnover which determines whether peroxidative injury is respectively intensified or suppressed; and (iii) mediate signaling cascades which either fortify antioxidant defenses of cells or evoke apoptotic death if oxidative pressure is too great. The purpose of this article is to review current understanding of photodynamic (UVA- or visible light-induced) lipid peroxidation with a special focus on LOOH generation and reactivity. Future goals in this area, many of which depend on continued development of state-of-the-art analytical techniques, will also be discussed.     

REACTIVITY OF PHOTOCHEMICALLY-GENERATED LIPID HYDROPEROXIDES IN CELL MEMBRANES WITH GLUTATHIONE PEROXIDASE

The ability of glutathione peroxidase (Gpx) to catalyze the reductive inactivation of photochemically-generated lipid hydroperoxides (LOOHs) was investigated, using hematoporphyrin derivative (HPD) as a photosensitizing agent and erythrocyte ghosts as membrane targets. Glutathione peroxidase was reactive toward photoperoxidized membranes only after their exposure to phospholipase A2 (PLA2). Iodometrically-determined LOOH values were typically 30-40% greater than values measured by enzymatic assay using Gpx and glutathione reductase. A consistent result was obtained when photooxidized membranes were treated with PLA2 and GSH/Gpx followed by iodometric assay, viz. persistence of approximately 40% of the starting LOOH. Whereas photooxidized egg phosphatidylcholine liposomes underwent total LOOH loss when incubated with PLA2 and GSH/Gpx, no net loss was observed with photooxidized cholesterol/dimyristoyl-phosphatidylcholine liposomes. The results suggest that cholesterol hydroperoxides in ghost membranes account for the Gpx-resistant fraction of LOOHs.     

环糊精在手性药物分离分析研究中的应用

环糊精由于自身特殊的手性环境和结构特征,已广泛运用于手性化合物的对映体拆分及分析。对近年来环糊精在各种色谱、质谱及核磁等分析方法中的应用进行了归纳总结,综述了环糊精及其衍生物在手性药物的分离分析方面的研究进展。     

Kinetics of oxidation of iron(II) ions with lipid hydroperoxides

One of the pathways for excessive production of free radicals is the decomposition of lipid hydroperoxides catalyzed by iron. A number of hydroperoxides of unsaturated fatty acids (LOOH), some prepared in our laboratory and others extracted from biological materials, were used to determine the rate constants of Fe²⁺ oxidation by measuring the formation rate of Fe³⁺ ions in the presence of simple unidentate ligands, chloride, and thiocyanate as the [FeCl]²⁺ and [FeNCS]²⁺ complexes, in a deoxygenated dichloromethane:methanol (2:1, v/v) mixture. The rates of Fe²⁺ oxidation with prepared LOOHs via the [FeNCS]²⁺ complex were approximately the same-the average second-order reaction rate constant was 1390 ± 340 dm³ mol⁻¹ s⁻¹; the rate constants of LOOHs from different biological materials were in the same range. The rates measured as the [FeNCS]²⁺ complex were somewhat higher than the rates measured as the [FeCl]²⁺ complex, indicating that ligands could interact in the transition state, thus affecting the disruption of the intermediate complex. Since there were no significant differences in the activation thermodynamic parameters for reactions within the reaction series of studied hydroperoxides, it was assumed that the oxidation proceeded by an inner sphere mechanism, considering that the breakdown of the successor inner sphere complex with the homolytic cleavage of peroxide bonds of hydroperoxides forming reactive alkoxyl radicals was the rate-limiting step. Based on this research, an indirect spectrophotometric method for quantitative determination of LOOH was reestimated. The microprocedure proposed for the lipid hydroperoxide assay could be applied to follow the early stages of lipid peroxidation processes in real biological samples.     

Review: Advances in the Determination of Peroxides by Optical and Spectroscopic Methods

This review focuses on the recent advances made in the field of organic peroxide analysis using spectroscopic methods. In this area, optical techniques and mass spectroscopy play a dominant role. Emphasis has been given to peroxides of great practical significance, such as lipid hydroperoxides, antimalarial peroxides, peroxide-based explosives, and industrially relevant peroxides. The discussion includes current challenges and trends in each of these groups. The underlying chemical principles of the described methods are briefly discussed. All the advances are categorized by branches of spectroscopy, and example applications are outlined. The scope of this paper is restricted to the most significant advances published between 2003 and mid-2013.     

Photosensitized lipid peroxidation and enzyme inactivation by membrane-bound merocyanine 540: reaction mechanisms in the absence and presence of ascorbate

The lipophilic photosensitizing dye merocyanine 540 (MC540) is being studied intensively as an antitumor and antiviral agent. Since plasma membranes are believed to be the principal cellular targets of MC540-mediated photodamage, we have studied membrane damage in a well characterized test system, the human erythrocyte ghost. When irradiated with white light, MC540-sensitized ghosts accumulated lipid hydroperoxides (LOOHs derived from phospholipids and cholesterol) at a rate dependent on initial dye concentration. Neither desferrioxamine nor butylated hydroxytoluene inhibited LOOH formation, suggesting that Type I (iron-mediated free radical) chemistry is not important. By contrast, azide inhibited the reaction in a dose-dependent fashion, implicating a Type II (singlet oxygen, 1O2) mechanism. Stern-Volmer analysis of the data gave a 1O2 quenching constant approximately 50 times lower than that determined for an extramembranous target, lactate dehydrogenase (the latter value agreeing with literature values). This suggests that 1O2 reacts primarily at its membrane sites of origin and that azide has limited access to these sites. Using [14C]cholesterol-labeled membranes and HPLC with radiodetection, we identified 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide as the major cholesterol photoproduct, thereby confirming 1O2 intermediacy. Irradiation of MC540-sensitized membranes in the presence of added iron and ascorbate resulted in a large burst of lipid peroxidation, as shown by thiobarbituric acid reactivity and appearance of 7-hydroperoxycholesterol and 7-hydroxycholesterol as major oxidation products. Amplification of MC540-initiated lipid peroxidation by iron/ascorbate (attributed to light-independent reduction of nascent photoperoxides, with ensuing free radical chain reactions) could prove useful in augmenting MC540's phototherapeutic effects.     

Infrared methods for high throughput screening of metabolites: food and medical applications

Chemical and physiological properties are related to individual or bioactive compounds such as essential oils, terpenoids, flavonoids, volatile compounds and other chemicals which are present in natural products in low concentrations (e.g. ppm or ppb). For many years, classical separation, chromatographic and spectrometric techniques such as high performance liquid chromatography (HPLC), gas chromatography (GC), liquid chromatography (LC) and mass spectrometry (MS) have been used for the elucidation of isolated compounds from different matrices. Hence, the use of standard separation, chromatographic and spectrometric methods was found useful in chemical and both plant and animal physiology studies, for fingerprinting and comparing natural and synthetic samples, as well as to identify single active compounds. It has been generally accepted that a single analytical technique will not provide sufficient visualization of the metabolome, hence holistic techniques are needed for comprehensive analysis. In the last 40 years near infrared (NIR) spectroscopy became one of the most attractive and used methods of analyzing agricultural related products and plant materials which provide simultaneous, rapid and non-destructive quantitation of major. This technique has been reported to determine other minor compounds in plant materials such as volatile compounds and elements. The aim of this short review is to describe some recent applications of NIR spectroscopy combined with multivariate data analysis for high throughput screening of metabolites with an emphasis on food and medical applications.     

Determination of chemical warfare agents and related compounds in environmental samples by solid-phase microextraction with gas chromatography

Solid phase microextraction (SPME) is an increasingly common method of sample isolation and enhancement. SPME is a convenient and simple sample preparation technique for chromatographic analysis and a useful alternative to liquid-liquid extraction and solid phase extraction. SPME is speed and simply method, which has been widely used in environmental analysis because it is a rather safe method when dealing with highly toxic chemicals. A combination of SPME and gas chromatography (GC) permits both the qualitative and quantitative analysis of toxic industrial compounds, pesticides and chemical warfare agents (CWAs), including their degradation products, in air, water and soil samples. This work presents a combination of SPME and GC methods with various types of detectors in the analysis of CWAs and their degradation products in air, water, soil and other matrices. The combination of SPME and GC methods allows for low detection limits depending on the analyte, matrix and detection system. Commercially available fibers have been mainly used to extract CWAs in headspace analysis. However, attempts have been made to introduce new fiber coatings that are characterized by higher selectivities towards different analytes of interest. Environmental decomposition of CWAs leads to the formation of more hydrophilic products. These compounds may be isolated from samples using SPME and analyzed using GC however, they must often be derivatized first to produce good chromatography. In these cases, one must ensure that the SPME method also meets the same needs. Otherwise, it is helpful to use derivatization methods. SPME may also be used with fieldportable mass spectrometry (MS) and GC-MS instruments for chemical defense applications, including field sampling and analysis. SPME fibers can be taken into contaminated areas to directly sample air, headspaces above solutions, soils and water.     

New look inside human breast ducts with Raman imaging. Raman candidates as diagnostic markers for breast cancer prognosis: Mammaglobin,palmitic acid and sphingomyelin

Looking inside the human body fascinated mankind for thousands of years. Current diagnostic and therapy methods are often limited by inadequate sensitivity, specificity and spatial resolution. Raman imaging may bring revolution in monitoring of disease and treatment. The main advantage of Raman imaging is that it gives spatial information about various chemical constituents in defined cellular organelles in contrast to conventional methods (liquid chromatography/mass spectrometry, NMR, HPLC) that rely on bulk or fractionated analyses of extracted components. We demonstrated how Raman imaging can drive the progress on breast cancer just unimaginable a few years ago. We looked inside human breast ducts answering fundamental questions about location and distribution of various biochemical components inside the lumen, epithelial cells of the duct and the stroma around the duct during cancer development. We have identified Raman candidates as diagnostic markers for breast cancer prognosis: carotenoids, mammaglobin, palmitic acid and sphingomyelin as key molecular targets in ductal breast cancer in situ, and propose the molecular mechanisms linking oncogenes with lipid programming.     

19F-Labeled Probes for Recognition-Enabled Chromatographic 19F NMR

The NMR technique is among the most powerful analytical methods for molecular structural elucidation, process monitoring, and mechanistic investigations; however, the direct analysis of complex real-world samples is often hampered by crowded NMR spectra that are difficult to interpret. The combination of fluorine chemistry and supramolecular interactions leads to a unique detection method named recognition-enabled chromatographic (REC) ¹⁹F NMR, where interactions between analytes and ¹⁹F-labeled probes are transduced into chromatogram-like ¹⁹F NMR signals of discrete chemical shifts. In this account, we summarize our endeavor to develop novel ¹⁹F-labeled probes tailored for separation-free multicomponent analysis. The strategies to achieve chiral discrimination, sensitivity enhancement, and automated analyte identification will be covered. The account will also provide a detailed discussion of the underlying principles for the design of molecular probes for REC ¹⁹F NMR where appropriate.     

Novel contribution of chromatography in the development and analyses of metformin hydrochloride in biological and environmental samples

Globally, metformin hydrochloride (HCl) is the most commonly used antidiabetic drug and provides safe medication because of low risk of the side effects (lactic acidosis). Therefore, various researches have been established in pharmacodynamics and pharmacokinetic studies using various analytical applications such as calorimetry, spectrophotometry, and chromatography. But the chromatographic techniques are most widely used methods for the analysis of metformin HCl. This review discussed the different chromatographic methods used for the analysis of metformin HCl and its sample preparation for the isolation in different biological and environmental samples. Moreover, the mechanism for the fragmentation of ion products in LC–MS/MS is indicated for high throughput of the methods involved in the determination of metformin HCl in various sample matrices. The advancement in the chromatographic modalities provides the wide range of assays discussed herein.     

Chromatography in authenticity and traceability tests of vegetable oils and dairy products: a review

The new applications of various chromatographic techniques such as gas-liquid chromatography, high-performance liquid chromatography and electrophoretic methods employed for the analysis in macro- and micro-components in vegetable oils and dairy products are compiled and critically evaluated. The employment of these methods for authenticity tests and traceability is discussed.     

The Existing Methods and Novel Approaches in Mycotoxins’ Detection

Mycotoxins represent a wide range of secondary, naturally occurring and practically unavoidable fungal metabolites. They contaminate various agricultural commodities like cereals, maize, peanuts, fruits, and feed at any stage in pre- or post-harvest conditions. Consumption of mycotoxin-contaminated food and feed can cause acute or chronic toxicity in human and animals. The risk that is posed to public health have prompted the need to develop methods of analysis and detection of mycotoxins in food products. Mycotoxins wide range of structural diversity, high chemical stability, and low concentrations in tested samples require robust, effective, and comprehensible detection methods. This review summarizes current methods, such as chromatographic and immunochemical techniques, as well as novel, alternative approaches like biosensors, electronic noses, or molecularly imprinted polymers that have been successfully applied in detection and identification of various mycotoxins in food commodities. In order to highlight the significance of sampling and sample treatment in the analytical process, these steps have been comprehensively described.     

NMR examination of fossil fuels Part I: solid samples

Nuclear Magnetic Resonance (NMR) spectroscopy¹ is one of the most valuable experimental methods available for the analysis of the chemical structure and composition of fuel products. This article describes applications of NMR in the analysis of solid fuels. Part II will describe applications of NMR in the liquid phase.²     

Stationary phases with special structural properties for high-throughput separation techniques: preparation, characterization and applications

Stationary phases with specific structural properties for high-throughput liquid chromatographic (LC) techniques are described. Special attention was paid to phases with special structural properties, mainly containing internal functional group (e.g. amide). Such materials are generally called "embedded phases". There are phases created in amidation process of aminopropylated silica gel, especially phases based on biological compounds, like phospholipids and cholesterol, which are called immobilized artificial membranes (IAM's). The synthesis and applications of polar embedded amide LC stationary phases were also reviewed. Methods of characterization of synthesized packing materials were presented, with general focusing on spectroscopic measurements like (13C and 29Si CP/MAS NMR and FT-IR), elemental and thermal analysis as well as chromatographic quantitative structure-retention relationships (QSRR) and extended chemometric tests. The potential applications of various dedicated stationary phases in a high-throughput LC screening procedures were also presented.     

色谱技术在肝素结构分析中的研究进展

肝素（heparin, Hp）是目前临床应用最为广泛的抗凝剂,是由重复二糖单元组成的多硫酸化酸性直链多糖。低分子量肝素（LMWHs）是以肝素为原料,经过化学或酶降解获得的相对分子质量相对较小的肝素衍生物,相对肝素,它们的出血副作用和免疫原性更小,皮下注射时生物利用度更高。肝素及低分子量肝素具有一系列结构特点,如相对分子质量偏大且有一定分布,多种糖残基同时存在,硫酸酯位置和数量呈现多样化,以及不同工艺产生的特殊残基的种类和含量不一等。该类药物结构的复杂性对分析方法提出了巨大的挑战,也限制了其质量控制提升、工艺优化、临床用药安全和新适应证拓展等。该文以色谱分析方法为中心,从结构分析的不同角度,包括单糖、二糖、寡糖、多糖的识别、组成分析和不同层次,系统地梳理和阐述近年来肝素类药物在色谱分析方法上的进展,并对这些方法的应用范畴、创新性、局限性等进行总结。该文将为肝素类药物的结构分析、质量控制提供较系统的方法学参考,为更多新方法开发提供思路,为更深入地研究肝素类药物结构、拓展其应用提供有力支撑。     

Formation constant and stoichiometry of the acetonitrile:18-crown-6 complex by nuclear magnetic resonance, raman and infrared spectroscopy

Crown ethers are increasingly used in a variety of chemical applications. While crown ether complexes with alkali-metal cations have been extensively studied, relatively little is known about their complexes with neutral molecules to form so-called host: guest complexes. The use of NMR is reported for the determination of the formation constant (2.1 +/- 0.1) for the acetonitrile: 18-crown-6 complex. The suitability of Raman spectroscopy for studies of the solid-phase complex is demonstrated and limitations in the use of infrared spectroscopy are discussed.     

Advancement in analysis of Salviae miltiorrhizae Radix et Rhizoma (Danshen)

This review summarizes the recent advances in the chemical analysis of Danshen and its finished products, including the introduction of the identified bioactive components, analytical methods for quantitative determination of target analytes and fingerprinting authentication, quality criteria of Danshen crude herb and its preparations, as well as the pharmacokinetic and pharmacodynamic studies on the active components of Danshen and its finished products. Danshen contains mainly two types of constituents, the hydrophilic depsides and lipophilic diterpenoidal quinones and both of them are responsible for the pharmacological activities of Danshen. In order to monitor simultaneously both types of components which have different physicochemical properties, numerous analytical methods have been reported using various chromatographic and spectrophotometric technologies. In this review, 110 papers on analysis of Danshen are discussed, various analytical methods and their chromatographic conditions are briefly described and their advantages/disadvantages are compared. For obtaining a quick, accurate and applicable analytical approach for quality evaluation and establishing a harmonized criteria of Danshen and its finished products, the authors’ suggestion and opinions are given, including the reasonable selection of marker compounds with high concentration and commercial availability, a simple sample preparation procedure with high recoveries of both the hydrophilic phenols and lipophilic tanshinones, and an optimized chromatographic condition with ideal resolutions of all the target components. The chemical degradation and transformation of the predominant constituent salvianolic acid B in Danshen during processing and manufacturing are also emphasized in order to assure the quality consistency of Danshen containing products.