Harri Koskela

Atomic Structures of Two Novel Immunoglobulin-like Domain Pairs in the Actin Cross-linking Protein Filamin

Filamins are actin filament cross-linking proteins composed of an N-terminal actin-binding domain and 24 immunoglobulin-like domains (IgFLNs). Filamins interact with numerous proteins, including the cytoplasmic domains of plasma membrane signaling and cell adhesion receptors. Thereby filamins mechanically and functionally link the cell membrane to the cytoskeleton. Most of the interactions have been mapped to the C-terminal IgFLNs 16–24. Similarly, as with the previously known compact domain pair of IgFLNa20–21, the two-domain fragments IgFLNa16–17 and IgFLNa18–19 were more compact in small angle x-ray scattering analysis than would be expected for two independent domains. Solution state NMR structures revealed that the domain packing in IgFLNa18–19 resembles the structure of IgFLNa20–21. In both domain pairs the integrin-binding site is masked, although the details of the domain-domain interaction are partly distinct. The structure of IgFLNa16–17 revealed a new domain packing mode where the adhesion receptor binding site of domain 17 is not masked. Sequence comparison suggests that similar packing of three tandem filamin domain pairs is present throughout the animal kingdom, and we propose that this packing is involved in the regulation of filamin interactions through a mechanosensor mechanism.

Quantitative two-dimensional HSQC experiment for high magnetic field NMR spectrometers.

The finite RF power available on carbon channel in proton-carbon correlation experiments leads to non-uniform cross peak intensity response across carbon chemical shift range. Several classes of broadband pulses are available that alleviate this problem. Adiabatic pulses provide an excellent magnetization inversion over a large bandwidth, and very recently, novel phase-modulated pulses have been proposed that perform 90 degrees and 180 degrees magnetization rotations with good offset tolerance. Here, we present a study how these broadband pulses (adiabatic and phase-modulated) can improve quantitative application of the heteronuclear single quantum coherence (HSQC) experiment on high magnetic field strength NMR spectrometers. Theoretical and experimental examinations of the quantitative, offset-compensated, CPMG-adjusted HSQC (Q-OCCAHSQC) experiment are presented. The proposed experiment offers a formidable improvement to the offset performance; (13)C offset-dependent standard deviation of the peak intensity was below 6% in range of+/-20 kHz. This covers the carbon chemical shift range of 150 ppm, which contains the protonated carbons excluding the aldehydes, for 22.3 T NMR magnets. A demonstration of the quantitative analysis of a fasting blood plasma sample obtained from a healthy volunteer is given.

Chapter 1 Quantitative 2D NMR Studies

Abstract Nuclear magnetic resonance (NMR) spectroscopy is regarded as one of the most important analytical techniques in chemistry for characterization of molecular structure. In addition to the structural information, NMR spectroscopy also gives quantitative information about the sample constituent. The induced current in the coil can be regarded as linearly dependent on the concentration of the nucleus in the sample. Therefore the resonance integrals in a simple one-dimensional spectrum measured with the excitation–acquisition scheme offer a way to measure absolute amounts of the chemicals present in the sample. Recently, the need for quantitative analysis of highly complex samples has led to a situation where resonance overlap in one-dimensional spectra can compromise or even prevent accurate quantification of sample compounds. Two-dimensional NMR offers improved resolution of resonances, and therefore the use of two-dimensional NMR experiments in determination of sample constituent has gained interest in many fields of research where quantification of compounds in complex samples is needed. Concepts of the quantitative two-dimensional NMR and recent applications are discussed.

Use of NMR techniques for toxic organophosphorus compound profiling

This review presents with selected examples the versatility of nuclear magnetic resonance (NMR) spectroscopy in the analysis of toxic organophosphorus (OP) compounds, i.e. OP pesticides and chemical warfare agents (CWAs). Several NMR applications of biological importance, like studies on inhibition mechanism, metabolism, and exposure determination, are presented. The review also concerns with the environmental analysis of OP compounds by NMR spectroscopy. Residue analysis of environment and food samples as well as characterization of degradation in environment is discussed. Some of the NMR studies that have been done to support the Chemical Weapons Convention, i.e. the development of suitable CWA detoxification means and the method development of verification analysis for CWAs and their degradation products, are outlined.

Identification of gymnodimine D and presence of gymnodimine variants in the dinoflagellate Alexandrium ostenfeldii from the Baltic Sea

Gymnodimines are lipophilic toxins produced by the marine dinoflagellates Karenia selliformis and Alexandrium ostenfeldii. Currently four gymnodimine analogues are known and characterized. Here we describe a novel gymnodimine and a range of gymnodimine related compounds found in an A. ostenfeldii isolate from the northern Baltic Sea. Gymnodimine D (1) was extracted and purified from clonal cultures, and characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS), nuclear magnetic resonance (NMR) spectroscopy, and liquid chromatography-high resolution mass spectrometry (LC-HRMS) experiments. The structure of 1 is related to known gymnodimines (2-5) with a six-membered cyclic imine ring and several other fragments typical of gymnodimines. However, the carbon chain in the gymnodimine macrocyclic ring differs from the known gymnodimines in having two tetrahydrofuran rings in the macrocyclic ring.

NMR properties of conjugated linoleic acid (CLA) methyl ester hydroperoxides.

NMR data on lipid hydroperoxides is scarce. In this study, hydroperoxides were produced from methyl 9-cis,11-trans-octadecadienoate and from methyl 10-trans,12-cis-octadecadienoate by autoxidation in the presence of 20% of alpha-tocopherol. Ten different hydroperoxides were isolated from the autoxidation mixtures of the two conjugated linoleic acid (CLA) methyl esters by SPE and HPLC. The assignment of the 1H and 13C NMR spectra of these hydroperoxides was accomplished by 2D NMR experiments and by spectral simulations. Substitution of a hydroperoxyl group at the allylic position in CLA methyl esters induced a 53.93 ppm downfield shift on the hydroperoxyl-bearing carbon resonance. The effects on the olefinic alpha, beta, gamma, and delta carbon resonances were -3.45, +4.96, -1.22, and +4.42 ppm, respectively. Furthermore, the solvent effects of deuterochloroform, deuteroacetone, and deuterobenzene on the 13C resonances of the hydroperoxides suggest that deuterochloroform is the appropriate solvent for 13C NMR studies on mixtures of lipid hydroperoxides.

Structural Characterization of Chemical Warfare Agent Degradation Products in Decontamination Solutions with Proton Band-Selective 1H−31P NMR Spectroscopy

Decontamination solutions, which are usually composed of strong alkaline chemicals, are used for efficient detoxification of chemical warfare agents (CWAs). The analysis of CWA degradation products directly in decontamination solutions is challenging due to the nature of the matrix. Furthermore, occasionally an unforeseen degradation pathway can result in degradation products which could be eluded to in standard analyses. Here, we present the results of the application of proton band-selective (1)H-(31)P NMR spectroscopy, i.e., band-selective 1D (1)H-(31)P heteronuclear single quantum coherence (HSQC) and band-selective 2D (1)H-(31)P HSQC-total correlation spectroscopy (TOCSY), for ester side chain characterization of organophosphorus nerve agent degradation products in decontamination solutions. The viability of the approach is demonstrated with a test mixture of typical degradation products of nerve agents sarin, soman, and VX. The proton band-selective (1)H-(31)P NMR spectroscopy is also applied in characterization of unusual degradation products of VX in GDS 2000 solution.

Rapid Chemical Characterisation of Stilbenes in the Root Bark of Norway Spruce by Off‐line HPLC/DAD–NMR

INTRODUCTION Stilbenes are plant secondary metabolites that have shown promising and varied biological activities. Stilbenes are presently actively studied for the exploitation of this primary raw material resource, involving the concept of biorefining. Methods for the rapid discovery of new and known stilbene structures from various plant sources are thus keenly sought. OBJECTIVE To establish a simple and rapid technique of off-line HPLC with a diode-array detector (DAD) and NMR for the unambiguous structural elucidation of stilbene structures in the root bark of Norway spruce [Picea abies (L.) Karst.]. MATERIAL AND METHODS The stilbene containing fraction was extracted from the plant bark with an ethanol:water mixture (95:5, v/v) preceded by defatting of hydrophobic compounds with n-hexane using the accelerated solvent extraction technique. A portion of the ethanol-water soluble extract was hydrolysed with β-glucosidase to prepare stilbene aglycones. The extracts were further purified and enriched using a polymeric adsorbent. Stilbene-enriched extracts were directly characterised by off-line HPLC/DAD-NMR in conjunction with HPLC/DAD and HPLC/DAD with electrospray ionisation MS(n). RESULTS Trans-isorhapontin and trans-astringin were identified as the major, and trans-piceid as a minor, stilbene glucosides of the bark of roots of Picea abies. Not only stilbene glucosides but also the corresponding stilbene aglycones, such as trans-resveratrol, trans-piceatannol and trans-isorhapontigenin, were rapidly identified from the hydrolysed extract. The acquired heteronuclear single-quantum coherence and heteronuclear multiple bond correlation spectra were used to assign the complete carbon NMR chemical shifts of trans-isorhapontin and trans-astringin without the need of acquiring a (13)C-NMR spectrum. CONCLUSION The off-line HPLC/DAD-NMR method is expedient for the unambiguous identication of structurally similar stilbenes in plant extracts.

On-flow pulsed field gradient heteronuclear correlation spectrometry in off-line LC-SPE-NMR analysis of chemicals related to the chemical weapons convention.

Hyphenation of liquid chromatography with nuclear magnetic resonance spectroscopy (LC-NMR) is a useful technique in the analysis of complex samples. However, application of on-flow 1H NMR spectrometry during the LC-NMR analysis usually suffers from high intensity of eluent resonances. The poor dynamic range can be improved either with use of deuterated eluents or with various signal suppression schemes. Deuterated eluents are expensive, and peak-selective signal suppression schemes are often unsatisfactory when detection of chemicals at low concentration is needed. If the analytes have a common heteronucleus, on-flow pulsed field gradient heteronuclear correlation spectrometry can offer several benefits. The analytes can be monitored selectively, while the intense nondeuterated eluent and impurity background can be effectively eliminated. In our study, on-flow one-dimensional (1D) 1H-31P heteronuclear single quantum coherence (HSQC) spectrometry was utilized in the analysis of characteristic organophosphorus degradation products of nerve agents sarin and soman during chromatographic separation. These chemicals were not detectable by UV, so their retention times were monitored using on-flow 1D 1H-31P HSQC. This enabled application of LC-NMR combined with solid-phase extraction (LC-SPE-NMR) in analysis of these organophosphorus chemicals in an alkaline decontamination solution. The analytes were extracted from the SPE cartridges with deuterated eluent, and the off-line NMR analysis was performed using a mass-sensitive microcoil probe head. The used on-flow 1D 1H-31P HSQC approach offered a high dynamic range and good detection limit (ca. 10 microg/55 nmol) with a high sampling frequency (1 point per 2 s) in the acquired pseudo-two-dimensional spectrum. No significant impurity background was present in the off-line NMR samples, and identification of the extracted analytes was straightforward.

Application of a Microcoil Probe Head in NMR Analysis of Chemicals Related to the Chemical Weapons Convention

A 1.7-mm microcoil probe head was tested in the analysis of organophosphorus compounds related to the Chemical Weapons Convention. The microcoil probe head demonstrated a high mass sensitivity in the detection of traces of organophosphorus compounds in samples. Methylphosphonic acid, the common secondary degradation product of sarin, soman, and VX, was detected at level 50 ng (0.52 nmol) from a 30-microL water sample using proton-observed experiments. Direct phosphorus observation of methylphosphonic acid with (31)P{(1)H} NMR experiment was feasible at the 400-ng (4.17 nmol) level. Application of the microcoil probe head in the spiked sample analysis was studied with a test water sample containing 2-10 microg/mL of three organophosphorus compounds. High-quality (1)H NMR, (31)P{(1)H} NMR, 2D (1)H-(31)P fast-HMQC, and 2D TOCSY spectra were obtained in 3 h from the concentrated 1.7-mm NMR sample prepared from 1 mL of the water solution. Furthermore, a 2D (1)H-(13)C fast-HMQC spectrum with sufficient quality was possible to measure in 5 h. The microcoil probe head demonstrated a considerable sensitivity improvement and reduction of measurement times for the NMR spectroscopy in identification of chemicals related to the Chemical Weapons Convention.