Current Organic Chemistry - Volume 10, Issue 5, 2006

In this issue of Current Organic Chemistry new developments and applications of several analytical methods in life science, protein chemistry and natural product chemistry are documented. The article of Hirata and Fujii focuses on the direct detection of reactive oxygen radicals generated from xenobiotics, metal ions and drugs by in vivo detection with ESR spectroscopy and imaging. In their review, Griffith and Kaltashov provide a summary of numerous developments in mass spectrometry in the analysis of hemoglobins. Studies on all levels of protein structure and assembly, and interactions with other biomolecules are presented. Their clinical importance in the analysis of the hemoglobin mutations and posttranslational and chemical modifications is discussed. The article of Malliavin summarizes the methods published since 1997 for liquid-NMR of proteins. Methods for structure determination, spectral assignment and structure mobility are reviewed. The structural biology of antimicrobial peptides by solution and solid state NMR spectroscopy using phosphatylglycerol micelles as bacterial membrane-mimetic models is outlined by Wang. A picture is obtained regarding how antimicrobial peptides exert their perturbation activity on bacterial membranes. The relevance of the membrane-bound structures of these peptides to guide peptide engineering in order to improve the therapeutic effect is stressed. The applications of a wide range of separation techniques, spectroscopic and mass spectrometric methods for the determination of alkannins and shikonins are presented by Papageorgiou et al. As these natural products are susceptible for to several transformations, validated analytical methods are crucial for their use in pharmaceuticals and cosmetics. The editors thank all the authors for their efforts in preparing these interesting articles.

This review article describes recently developed technologies in electron paramagnetic resonance (EPR) spectroscopy and imaging. Automatic control techniques used for a continuous-wave (CW) EPR spectrometer are discussed. These techniques can solve problems created by the motion of animals. Recent developments with time-domain EPR spectroscopy are also reported. Time-domain EPR spectroscopy is a technically challenging method because of the very short relaxation time that free radicals have in biological tissue. EPR imaging techniques are also reviewed, which are able to visualize free radicals in animal subjects non-invasively. Current status and future trends in the development of instruments for EPR spectroscopy and imaging are also presented, especially for biomedical applications. An important and powerful application of in vivo EPR spectroscopy and imaging is the detection of free radicals generated in biological specimens, which are so-called bioradicals. This article reviews these bioradicals, such as reactive oxygen species (ROS), free radicals generated from xenobiotics, metal ions, and common drugs, and it especially focuses on the direct-detection of bioradicals, rather than indirect detection. Drug-induced reaction mechanisms with hydrazine-based drugs, carcinogenic nitroso compounds, and prescription drugs for patients with hypertension (nifedipine) are discussed in detail based on in vivo studies with small animals. Metal-related reactions in vivo are also discussed with irons, chromate, and manganese.

Hemoglobins are dioxygen transport proteins, which are universally present in higher vertebrates as a tetrameric protein. Recently there has been much interest in mutations and posttranslational modifications to hemoglobins as these may be directly implicated in disease states like thalassemia and diabetes mellitius. The analysis of covalent adducts to hemoglobin provides information on the extent of exposure to many carcinogenic compounds. Studies of the assembly of hemoglobins from mammalian sources are being pursued for their clinical use as blood transfusion products. Over the past several years, numerous developments in mass spectrometry (MS) methods and instrumentation have revolutionized the analysis of proteins from their primary sequences to the quaternary structures of large oligomeric complexes. This review provides a summary of the uses of mass spectrometry in the analysis of hemoglobins, in particular, tetrameric hemoglobins. It presents some results of novel mass spectrometric studies on hemoglobins on all levels of protein structure and assembly, and interaction with other biomolecules. Full and partial sequencing by MS in the analysis of hemoglobin mutations, posttranslational modifications and chemical modifications; analyses of hemoglobin structures, dynamics and assembly; and analyses of hemoglobin interactions with other biomolecules of clinical importance are discussed. The review will conclude with a discussion of the utility of MS techniques in hemoglobin analyses, where the field is headed, and possible areas for improvement.

Nuclear Magnetic Resonance (NMR) became during the two last decades an important method for biomolecular structure determination. NMR permits to study biomolecules in solution and gives access to the molecular flexibility at atomic level on a complete structure: in that respect, it is occupying a unique place in structural biology. During the first years of its development, NMR was trying to meet the requirements previously defined in X-ray crystallography. But, NMR then started to determine its own criteria for the definition of a structure. Indeed, the atomic coordinates of an NMR structure are calculated using restraints on geometrical parameters (angles and distances) of the structure, which are only indirectly related to atom positions: in that respect, NMR and X-ray crystallography are very different. The indirect relation between the NMR measurements and the molecular structure and dynamics makes critical the precision and the interpretation of the NMR parameters and the development of quantitative analysis methods. The methods published since 1997 for liquid-NMR of proteins are reviewed here. First, methods for structure determination are presented, as well as methods for spectral assignment and for structure quality assessment. Second, the quantitative analysis of structure mobility is reviewed.

Antimicrobial peptides are key components of innate immunity of all life forms. Understanding the structureactivity relationship of these peptides is essential for developing them into novel therapeutics that substitutes traditional antibiotics. NMR spectroscopy can provide insights into membrane-targeting antimicrobial peptides from a variety of angles. First, three-dimensional structures of antimicrobial peptides can be determined by solution NMR using short-chain phosphatidylglycerol micelles as a novel bacterial membrane-mimetic model. Natural abundance 15N and 13C chemical shifts of short peptides offer a practical approach for the refinement of distance-based structures. Isotope labeling will allow structures of antimicrobial peptides with longer or difficult sequences to be determined in lipid micelles or bicelles and further refined by residual dipolar couplings. The in-plane or transmembrane orientation of antimicrobial peptides in lipid bilayers can be determined by solid-state NMR. Second, the impact of antimicrobial peptides on the structure and dynamics of lipid bilayers can be probed by 31P and 2H NMR spectroscopy. Third, intermolecular nuclear Overhauser effects (NOE) provide direct evidence for the location of the peptides in the membranes and are key restraints for establishing the structure of peptide-lipid complexes. Peptide-lipid NOE patterns also reflect the penetration depth of peptides in membranes. A deeper penetration is required for a basic peptide to exert its membrane perturbation potential on acidic membranes. The combination of solution and solid-state NMR depicts a more complete picture how antimicrobial peptides perturb bacterial membranes. The membrane-bound structures of antimicrobial peptides can be harnessed to guide peptide engineering to improve the therapeutic index.

Isohexenylnaphthazarins (IHN), commonly known as Alkannins and Shikonins (A/S), are lipophilic red pigments. They are found in the underground parts, mainly roots, of at least a hundred and fifty species that belong to the genus Alkanna, Lithospermum, Echium, Onosma, Anchusa and Cynoglossum of the Boraginaceae family. The chiral pair A/S are potent pharmaceutical substances with a well-established and wide spectrum of wound healing, antimicrobial, anti-inflammatory, antioxidant, anticancer, radical scavenging and antithrombotic biological activity. The last years there has been extensive scientific research in many areas throughout the disciplines of chemistry and biology and more specifically in cancer chemotherapy and a number of papers have appeared in the literature. Significant research has been conducted on A/S effectiveness on several tumors and on their mechanism of anticancer action. A/S and their derivatives are susceptible to several transformations, such as photochemical decomposition, thermal degradation and polymerization. The stability of these substances during processing and storage is crucial to their use in pharmaceuticals and cosmetics, since polymerization of A/S results in a reduction in their antimicrobial activity, decrease in concentration of the active monomeric ones and to limited applications, due to loss of deep red colour and a decrease in solubility. Therefore, the determination of the impurities, degradation products or byproducts with the use of several analytical techniques, is of great importance. Additionally, the identification, qualitative and quantitative determination of A/S and their derivatives in raw materials for pharmaceuticals, such as natural products, samples prepared either by plant tissue cultures, or synthetically or by hydrolysis of naturally occurring IHN esters, is crucial for their use in pharmaceuticals. A large number of analytical techniques have been applied for the analysis and study of alkannin, shikonin and their derivatives. Chromatographic techniques used include TLC densitometry, Size Exclusion Chromatography, HPLC and chiral HPLC. The detection and identification techniques include: UV/Vis, IR, FTIR, 1H 1D and 2D NMR, 13C-NMR, mass spectrometry, FAB-MS, MALDI-MS, circular dichroism and indirect atomic absorption. Polarography, voltammetry, differential pulse voltammetry and even a photoacoustic technique for transdermal adsorption measurements have been utilized for qualitative and quantitative determinations of A/S and their derivatives. In the present study, all the above mentioned analytical methods on alkannins and shikonins are reviewed.