Flemming H. Larsen

Mannose-binding lectin and its genetic variants

Mannose-binding lectin (MBL) is a collagen-like serum protein that mediates activation of the complement system and is of importance for host defence. Common variant alleles situated both in the promoter and structural region of the human MBL gene (MBL2) influence the stability and the serum concentration of the protein. Epidemiological studies have suggested that genetically determined variation in MBL serum concentration influences the susceptibility to and the course of different types of infections, autoimmune, metabolic and cardiovascular diseases, but this is still a subject of debate. The fact that these genetic variations are very frequent indicates a dual role for MBL in host defence. In this survey, we summarize the current molecular understanding of human MBL genetics.

HIGH-FIELD QCPMG-MAS NMR OF HALF-INTEGER QUADRUPOLAR NUCLEI WITH LARGE QUADRUPOLE COUPLINGS

The quadrupole Carr-Purcell-Meiboom-Gill NMR experiment using magic-angle spinning (QCPMG-MAS) is analysed as a means of determining quadrupolar coupling and anisotropic chemical shielding tensors for half-integer (I > 1/2) quadrupolar nuclei with large quadrupole coupling constants (C Q). This is accomplished by numerical simulations and 87Rb NMR experiments wih Rb2SO4 and Rb2CrO4 using different magnetic fields. It is demonstrated that (i) QCPMG-MAS experiments typically provide a sensitivity gain by more than an order of magnitude relative to quadrupolar-echo MAS experiments, (ii) non-secular second-order terms do not affect the spin evolution appreciably, and (iii) the effect of finite RF pulses needs to be considered when 2ω2 Q/(ω0ωRF) > 0.1, where ωQ = 2πC Q/(4I(2I—1)), ωRF is the RF amplitude, and ω0 the Larmor frequency. Using numerical simulations and iterative fitting the magnitudes and relative orientation of 87Rb quadrupolar coupling and chemical shielding tensors for Rb2SO4 and Rb2CrO4 have b...

Molecular dynamics from 2H Quadrupolar Carr–Purcell–Meiboom–Gill solid-state NMR spectroscopy

Abstract The 2 H quadrupolar Carr–Purcell–Meiboom–Gill (QCPMG) NMR experiment is proposed as a convenient method to obtain detailed information about molecular dynamics in solids. Compared to the quadrupolar-echo (QE) experiment QCPMG offers two advantages. First, a sensitivity enhancement by about an order of magnitude is achieved by splitting the QE spectrum into spin-echo sidebands. Second, the lineshape of the individual sidebands provides detailed information about the molecular dynamics and increases the dynamic range by two orders of magnitude. The 2 H QCPMG method is demonstrated experimentally and numerically using the two-fold flip process in dimethyl sulfone.

Residue Specific Hydration of Primary Cell Wall Potato Pectin Identified by Solid-State 13C Single-Pulse MAS and CP/MAS NMR Spectroscopy

Hydration of rhamnogalacturonan-I (RG-I) derived from potato cell wall was analyzed by (13)C single-pulse (SP) magic-angle-spinning (MAS) and (13)C cross-polarization (CP) MAS nuclear magnetic resonance (NMR) and supported by (2)H SP/MAS NMR experiments. The study shows that the arabinan side chains hydrate more readily than the galactan side chains and suggests that the overall hydration properties can be controlled by modifying the ratio of these side chains. Enzymatic modification of native (NA) RG-I provided samples with reduced content of arabinan (sample DA), galactan (sample DG), or both side chains (sample DB). Results of these samples suggested that hydration properties were determined by the length and character of the side chains. NA and DA exhibited similar hydration characteristics, whereas DG and DB were difficult to hydrate because of the less hydrophilic properties of the rhamnose-galacturonic acid (Rha-GalA) backbone in RG-I. Potential food ingredient uses of RG-I by tailoring of its structure are discussed.

Direct quantification of M/G ratio from 13C CP-MAS NMR spectra of alginate powders by multivariate curve resolution

Multivariate curve resolution (MCR) was applied to (13)C cross-polarisation (CP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of non-depolymerised alginate powders obtained from brown seaweed plus a pure mannuronate sample isolated from Pseudomonas fluorescens for estimation of the mannuronic acid/guluronic acid ratio (M/G ratio). An excellent MCR model with a correlation coefficient of r(2)=0.99 was established between the estimated M/G ratios and the M/G ratios obtained from the traditional (1)H solution state NMR method. The new method allows for successful determination of the M/G ratio independent of the calcium content (at least up to 2.4%, which was the upper limit in this study) with a root mean square error of prediction of 0.05. It is thus concluded that (13)C CP-MAS NMR in combination with multivariate curve resolution is a reliable, convenient (no sample preparation is required) and relatively rapid method for M/G ratio determinations of alginates and it may serve as a good alternative to the chemical techniques traditionally used.

Starch phosphorylation--maltosidic restrains upon 3'- and 6'-phosphorylation investigated by chemical synthesis, molecular dynamics and NMR spectroscopy.

Phosphorylation is the only known in vivo substitution of starch, yet no structural evidence has been provided to explain its implications of the amylosidic backbone and its stimulating effects on starch degradation in plants. In this study, we provide evidence for a major influence on the glucosidic bond in starch specifically induced by the 3‐O‐phosphate. Two phosphorylated maltose model compounds were synthesized and subjected to combined molecular dynamics (MD) studies and 950 MHz NMR studies. The two phosphorylated disaccharides represent the two possible phosphorylation sites observed in natural starches, namely maltose phosphorylated at the 3′‐ and 6′‐position (maltose‐3′‐O‐phosphate and maltose‐6′‐O‐phosphate). When compared with maltose, both of the maltose‐phosphates exhibit a restricted conformational space of the α(1→4) glycosidic linkage. When maltose is phosphorylated in the 3′‐position, MD and NMR show that the glucosidic space is seriously restricted to one narrow potential energy well which is strongly offset from the global potential energy well of maltose and almost 50°degrees from the Φ angle of the α‐maltose crystal structure. The driving force is primarily steric, but the configuration of the structural waters is also significantly altered. Both the favored conformation of the maltose‐3′‐phosphate and the maltose‐6′‐phosphate align well into the 6‐fold double helical structure of amylopectin when the effects on the glucosidic bond are not taken into account. However, the restrained geometry of the glucosidic linkage of maltose‐3′‐phosphate cannot be accommodated in the helical structure, suggesting a major local disturbing effect, if present in the starch granule semi‐crystalline lattice.

Formation Mechanism of Coamorphous Drug–Amino Acid Mixtures

Two coamorphous drug-amino acid systems, indomethacin-tryptophan (Ind-Trp) and furosemide-tryptophan (Fur-Trp), were analyzed toward their ease of amorphization and mechanism of coamorphization during ball milling. The two mixtures were compared to the corresponding amorphization of the pure drug without amino acid. Powder blends at a 1:1 molar ratio were milled for varying times, and their physicochemical properties were investigated using XRPD, (13)C solid state NMR (ssNMR), and DSC. Comilling the drug with the amino acid reduced the milling time required to obtain an amorphous powder from more than 90 min in the case of the pure drugs to 30 min for the coamorphous powders. Amorphization was observed as reductions in XRPD reflections and was additionally quantified based on normalized principal component analysis (PCA) scores of the ssNMR spectra. Furthermore, the evolution in the glass temperature (Tg) of the coamorphous systems over time indicated complete coamorphization after 30 min of milling. Based on the DSC data it was possible to identify the formation mechanism of the two coamorphous systems. The Tg position of the samples suggested that coamorphous Ind-Trp was formed by the amino acid being dissolved in the amorphous drug, whereas coamorphous Fur-Trp was formed by the drug being dissolved in the amorphous amino acid.

Exploring the solid-form landscape of pharmaceutical hydrates: transformation pathways of the sodium naproxen anhydrate-hydrate system.

ABSTRACTPurposeTo understand the transformation pathways amongst anhydrate/hydrate solid forms of sodium naproxen and to highlight the importance of a polymorphic dihydrate within this context.MethodsMulti-temperature dynamic vapour sorption (DVS) analysis combined with variable-humidity X-ray powder diffraction (XRPD) to establish the transformation pathways as a function of temperature and humidity. XRPD and thermogravimetric analysis (TGA) to characterise bulk samples. Monitoring of in-situ dehydration using solid-state 13C CP/MAS spectroscopy.ResultsAt 25°C, anhydrous sodium naproxen (AH) transforms directly to one dihydrate polymorph (DH-II). At 50°C, AH transforms stepwise to a monohydrate (MH) then to the other dihydrate polymorph (DH-I). DH-II transforms to a tetrahydrate (TH) more readily than DH-I transforms to TH. Both dihydrate polymorphs transform to the same MH.ConclusionsThe properties of the polymorphic dihydrate control the transformation pathways of sodium naproxen.

High throughput prediction of chylomicron triglycerides in human plasma by nuclear magnetic resonance and chemometrics

BackgroundThe lipid content of the chylomicrons is a key biomarker and risk factor of cardiovascular diseases and for the understanding of obesity. A high throughput determination of chylomicrons in human blood plasma is outlined.MethodsThe new method, which uses a combination of Nuclear Magnetic Resonance (NMR) analysis and multivariate calibration analysis (chemometrics), is based on a correlation analysis towards the established standard method (ultracentrifugation and colorimetric test kit) and enables extraordinarily fast, inexpensive, and robust prediction of triglyceride (TG) content in chylomicrons. It is the position and shape of the complex lipid methylene resonance band that determines the chylomicron TG status and this information is extracted by the multivariate regression method.ResultsThe resulting method is a relatively simple multivariate model that facilitates parsimonious and accurate prediction of chylomicron lipids from NMR spectra of blood. The chemometric model predicts the chylomicron TG content with a correlation coefficient (R) of 0.96 when plotted against density gradient ultracentrifugation data.ConclusionsThe new rapid method facilitates large scale clinical and nutritional trials with inclusion of diagnostics of chylomicron status and thus creates new opportunities for research in lifestyle diseases and obesity.

Structure and hydration of the amylopectin trisaccharide building blocks—Synthesis, NMR, and molecular dynamics

To gain insight into the molecular details and hydration of amylopectin, the five constituting trisaccharides have been chemically synthesized as their methyl alpha-glycosides. All five trisaccharides were subjected to 950 MHz NMR spectroscopy for complete assignment and nanosecond molecular dynamics trajectories were calculated to study the structure and dynamics of the trisaccharides in aqueous solution. Systematic analysis of the simulation data revealed several examples of bridging water molecules playing an important role in the stabilization of specific amylopectin conformations, which was also supported by the experimental NMR data such as interresidue NOEs and heteronuclear scalar couplings between nuclei from neighboring residues. Although alpha-maltotriose, alpha-iso-maltotriose, alpha-panose and alpha-isopanose are relatively well characterized structures, the study also includes one less characterized trisaccharide with the structure alphaGlcp(1-->4)alphaGlcp(1-->6)alphaGlcp. This trisaccharide, tentatively labelled alpha-forkose, is located at the branch point of amylopectin, forking the amylopectin into two strands that align into double-helical segments. The results show that the conformation of alpha-forkose takes a natural bend form which fits well into the structure of the double-helical segment of amylopectin. As the only trisaccharide in this study the structure of alpha-forkose is not significantly influenced by the hydration. In contrast, alpha-isopanose takes a restricted, but rather extended form due to an exceptionally strong localized water density. The two homo-linkage oligomers, alpha-maltotriose and alpha-iso-maltotriose, showed to be the most extended and the most flexible trimers, respectively, providing regular structure for crystalline domains and maximum linker flexibility for amorphous domains.