Use of 45° pulse pair as a filter for pure-phase two-dimensional NMR spectroscopy

Conventionally two-dimensional NMR spectra are recorded in the absolute-intensity mode (1-4). It has recently been demonstrated that absorption-mode 2D spectra have much higher resolution and are the preferred mode of presentation, especially for 2D spectra of biomolecules (5-7). Indeed, any experimental scheme which yields phasemixed lineshapes is subject to modification to yield pure-phase spectra, even at the expense of intensity and anomalous multiplet structure (8-10). For this purpose two types of filters are already known: the z filter (9, 20) and the purging pulse (8, 10). In this note, we propose a 45” pulse pair as a filter for obtaining pure-phase 2D spectra, mainly for experiments in which the above filters do not yield pure-phase spectra.

Coherence transfer via longitudinal spin order generalized pulse pair filtering for pure phase two-dimensional NMR spectroscopy

A generalized pulse pair has been suggested in which the longitudinal spin order is retained and the transverse components cancelled by random variation of the interval between pulses, in successive applications of the two-dimensional NMR algorithm. This method leads to pure phases and has been exploited to provide a simpler scheme for two-spin filtering and for pure phase spectroscopy in multiple-quantum-filtered two-dimensional NMR experiments.

NMR Spectroscopy of Multi-domain Proteins : Immunoglobulin-like Domains of Human Filamin A

Proteins are complex biomacromolecules playing fundamental roles in the physiological processes of all living organisms. They function as structural units, enzymes, transporters, process regulators, and signal transducers. Defects in protein functions often derive from genetic mutations altering the protein structure, and impairment of essential protein functions manifests itself as pathological conditions. Proteins operate through interactions, and all protein functions depend on protein structure. In order to understand biological mechanisms at the molecular level, one has to know the structures of the proteins involved. This thesis covers structural and functional characterization of human filamins. Filamins are actin-binding and -bundling proteins that have numerous interaction partners. In addition to their actin-organizing functions, filamins are also known to have roles in cell adhesion and locomotion, and to participate in the logistics of cell membrane receptors, and in the coordination of intracellular signaling pathways. Filamin mutations in humans induce severe pathological conditions affecting the brain, bones, limbs, and the cardiovascular system. Filamins are large modular proteins composed of an N-terminal actin-binding domain and 24 consecutive immunoglobulin-like domains (IgFLNs). Nuclear magnetic resonance (NMR) spectroscopy is a versatile method of gaining insight into protein structure, dynamics and interactions. NMR spectroscopy was employed in this thesis to study the atomic structure and interaction mechanisms of C-terminal IgFLNs, which are known to house the majority of the filamin interaction sites. The structures of IgFLN single-domains 17 and 23 and IgFLN domain pairs 16-17 and 18-19 were determined using NMR spectroscopy. The structures of domain pairs 16 17 and 18 19 both revealed novel domain domain interaction modes of IgFLNs. NMR titrations were employed to characterize the interactions of filamins with glycoprotein Ibα, FilGAP, integrin β7 and dopamine receptors. Domain packing of IgFLN domain sextet 16 21 was further characterized using residual dipolar couplings and NMR relaxation analysis. This thesis demonstrates the versatility and potential of NMR spectroscopy in structural and functional studies of multi-domain proteins.

Interactions of aromatic mannosyl disulfide derivatives with Concanavalin A: synthesis, thermodynamic and NMR spectroscopy studies

α-d-Mannopyranosyl units were attached to an aromatic scaffold through disulfide linkages to obtain mono- to trivalent glycosylated ligands for lectin binding studies. Isothermal titration calorimetric (ITC) measurements indicated that binding affinities of these derivatives to Concanavalin A (Con A) were comparable to or slightly higher than that of methyl α-d-mannopyranoside (Ka values in the range of 104 M−1). The stoichiometries of the lectin-ligand complexes were in agreement with the formal valencies (1–3) of the respective ligands indicating cross-linking in interactions with the di- and trivalent derivatives. Multivalency effects could not, however, be observed with the latter. These ligands were shown to bind to the carbohydrate binding site of Con A using saturation transfer difference (STD) NMR competition experiments.

Structural assignment of monothiocarbonohydrazones by 1H NMR spectroscopy

Analysis of the 1H NMR spectra of several monothiocarbonohydrazones, some of them synthesized for the first time, shows that they exist as two structural isomers. Whereas, in general, the derivatives of aromatic aldehydes conform to a linear structure, the aliphatic carbonyl derivatives conform to heterocyclic or linear structures, depending on the size of the substituent groups. This dual behaviour is explained in terms of extended conjugation and steric hindrance.

HR-MAS NMR spectroscopy shows regional metabolite variation in bovine articular cartilage

Mechanical stress is an important external factor effecting the development and maintenance of articular cartilage. The metabolite profile of diseased cartilage has been well studied but there is limited information about the variation in metabolite profile of healthy cartilage. With the importance of load in maintaining healthy cartilage, regional differences in metabolite profile associated with differences in load may provide information on how load contributes to the maintenance of healthy cartilage. HR-MAS NMR spectroscopy allows the assessment of tissue samples without modification and was used for assessing the difference in metabolic profile between the load bearing and non-load bearing regions of the bovine articular cartilage. In this preliminary study, we examined cartilage from tibia and femur of four knee joints. Sixteen pairs of 1D-NOESY spectra were acquired. Principle component analysis (PCA) identified chemical shifts responsible for variance. SBASE (AMIX) and the Human Metabolome Database were used in conjunction with previous reported cartilage data for identifying metabolites associated with the PCA results. The major contributors to load-related differences in metabolite profile were N-acetyl groups, lactate and phosphocholine peaks. Integrals of these regions were further analysed using a Student's t-test. In load bearing cartilage regions. N-acetyl groups and phosphocholine were found at significantly higher concentration (p < 0.05 and p < 0.005, respectively) in both femur and tibia, while lactate was reduced in load bearing cartilage (p < 0.005). The results of this pilot HR-MAS NMR study demonstrate its ability to provide useful metabolite information for healthy cartilage.

Two-dimensional NMR spectroscopy of connected transitions by linear combination of flip angle dependent cosy: An alternative scheme for E.COSY

An alternative pulse scheme which simplifies and improves the recently proposed P.E.COSY experiment is suggested for the retention of connected or unconnected transitions in a coupled spin system. An important feature of the proposed pulse scheme is the improved phase characteristics of the diagonal peaks. A comparison of various experiments designed for this purpose, namely COSY-45, E.COSY, P.E.COSY and the present scheme (A.E.COSY), is also presented. The suppression of unconnected transitions and the measurement of scalar coupling constants and their relative signs are illustrated from A.E.COSY spectra of 2,3-dibromopropionic acid and 2-(2-thienyl)pyridine.

On the models for deuterium long-range isotope effects in13C NMR spectroscopy

The long-range deuterium isotope effects on13C nuclear shielding are physically not yet completely understood. Two existing models for explaining these effects, vibrational and substituent, are compared here. The vibrational model is based on the Born-Oppenheimer approximation, but it can explain only one-bond deuterium effects. To the contrary, the substituent model may explain many long-range isotope effects, but it is controversial due to the assumption of some distinct electronic properties of isotopes. We explain how long-range deuterium isotope effects may be rationalized by the subtle electronic changes induced by isotope substitution, which does not violate the Born-Oppenheimer approximation.

1, 2-Bis (diphenylphosphino) etane bridged dinuclear copper (I) complexes: Investigations of solid state and solution structures by CP/MAS31P NMR spectroscopy, X-ray crystallography, IR spectroscopy and solution 31P NMR and 63Cu NMR spectroscopy

Three different complexes of copper (I) with bridging 1, 2-bis(diphenylphosphino)ethane (dppe), namely [Cu2 (mu-dppe) (CH3CN)6] (ClO4)2 (1), [Cu2 (mu-dppe)2 (CH3 CN)2] (ClO4)2 (2), and [Cu2 (mu-dppe) (dppe)2 (CH3CN)2] (ClO4)2 (3) have been prepared. The structure of [Cu2 (mu-dppe) (dPPe)2 (CH3CH)2] (ClO4)2 has been determined by X-ray crystallography. It crystallizes in the space group PT with a=12.984(6) angstrom, b=13.180(6) angstrom, c=14.001(3) angstrom, alpha=105.23(3), beta=105.60(2), gamma=112.53 (4), V=1944 (3) angstrom3, and Z=1. The structure was refined by least-squares method with R=0.0365; R(w)=0.0451 for 6321 reflections with F0 greater-than-or-equal-to 3 sigma (F0). The CP/MAS P-31 and IR spectra of the complexes have been analysed in the light of available crystallographic data. IR spectroscopy is particularly helpful in identifying the presence of chelating dppe. P-31 chemical shifts observed in solid state are very different from those observed in solution, and change significantly with slight changes in structure. In solution, complex 1 remains undissociated but complexes 2 and 3 undergo extensive dissociation. With a combination of room temperature H-1, Cu-63, and variable temperature P-31 NMR spectra, it is possible to understand the various processes occurring in solution.

Chiral polyhydroxylated tetrahydrothiophene derivatives: novel synthesis and structural elucidation by X-ray crystallography, NMR spectroscopy and molecular mechanics calculations

The dideoxygenation reaction of 1,3;4,6-di-O-alkylidene-2,5-di-S-methylthiocarbonyl-D-mannitol derivatives under Barton-McCombie reaction conditions gave the hexahydrodipyranothiophenes 4 and 7 instead of the expected 2,5-dideoxy products. Structural and conformational information on these novel derivatives has been obtained by NMR spectroscopy, single-crystal X-ray crystallography and molecular mechanics calculations.

Conformation of polymerizable diacetylenic lecithin, DC8,9PC, by 1H-NMR spectroscopy

Diacetylenic phospholipid, 1,2 bis-(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8,9PC), forms helices and tubules in addition to liposomes. The diacetylenic moiety responsible for the transformation is probed by 2-D NMR correlated spectroscopy. Chemical shift assignments and the analysis of 2D-COSY measurements were done on the lipid in chloroform-d solution. Based on this analysis, a model for the lipid is proposed. The geometry of the headgroup, glycerol backbone and acyl chains up to three methylenes from glycerol backbone [-(CH2)(3)-] is similar to that of dipalmitoyl phosphatidylcholine. The estimated torsional angle for methylene groups adjacent to diacetylenic moieties suggested an overall tilt of the diacetylenic lipid molecule from the bilayer axis of 25-30 degrees. This tilt could be negative or positive depending on the handedness of the resultant microstructures.

AM(1-x)Al(x)O(3-x) (A=Na or K; M=Nb or Ta): New anion-deficient Perovskite oxides exhibiting oxide ion conduction

Anion-deficient perovskite oxides of the formula AM(1-x)Al(x)O(3-x) (A = Na or K; M = Nb or Ta) have been prepared for 0 < x less than or equal to 0.5. Diffraction experiments reveal that while the potassium compounds adopt orthorhombic/cubic perovskite structures similar to the parent KNbO3/KTaO3, the sodium compound, NaNb0.5Al0.5O2.5, possesses a brownmillerite/LaSr-CuAlO5-like superstructure. Al-27 NMR spectra show an exclusive tetrahedral oxygen coordination for AI(III) in Na-Nb0.5Al0.5O2.5 (I) and both tetrahedral and octahedral coordination for Al(III) in KNb0.5Al0.5O2.5 (II). The results suggest a long-range and short-range ordering of oxide ion vacancies in I and II respectively. Electrical conductivity measurements show a significant oxide ion conduction for KNb1-xAlxO3-x, with the conductivity increasing with x up to x = 0.5. The differences in the Arrhenius plots of the ionic conductivity of I and II have been rationalized in terms of the long-range and short-range ordering of oxide ion vacancies in the anion-deficient perovskite oxides.