Resolution Nuclear Magnetic Resonance Spectroscopy Research Articles

Synthesis of some novel halogenated 2,4‐diaminoquinazolines

AbstractThree new 2,4‐diaminoquinazolines, the 5,6‐difluoro, 6,7‐difluoro and 7,8‐difluoro isomers were prepared by the reaction of the requisite trifluorobenzonitrile and guanidine carbonate. Surprisingly, 2,3,6‐trifluorobenzonitriles gave 2,4‐diamino‐5,6‐difluoroquinazoline exclusively as determined by high resolution nuclear magnetic resonance spectroscopy. On the other hand, 3‐amino‐2,6‐difluorobenzonitrile on reaction with guanidine carbonate yielded only 5‐fluoro‐2,4,8‐triaminoquinazoline. This compound was subsequently converted to 8‐chloro‐2,4‐diamino‐5‐fluoroquinazoline using the Sandmeyer procedure. The nitration of 2,4‐diamino‐8‐fluoroquinazoline occurred exclusively at position six yielding 2,4‐diamino‐8‐fluoro‐6‐nitroquinazoline, which upon reduction with stannous chloride afforded 8‐fluoro‐2,4,6‐triaminoquinazoline. In a similar fashion 7‐fluoro‐2,4‐diaminoquinazoline underwent nitration at position six and was then reduced to give 7‐fluoro‐2,4,6‐triaminoquinazoline. Finally, both of these triaminoquinazolines were converted to the 6‐chloro derivatives under Sandmeyer conditions to yield 6‐chloro‐2,4‐diamino‐8‐fluoroquinazoline and 6‐chloro‐2,4‐diamino‐7‐fluoroquinazoline, respectively.

High resolution proton magnetic resonance spectroscopy of cyst fluids from patients with polycystic kidney disease

High field 1H-NMR spectra of fluid collected from the cysts of six renal transplant recipients with autosomal dominant polycystic kidney disease (ADPKD) have been measured and teh majro metabolite signals assigned. Quantitative NMR measurements have revealed a combination of unusual biochemical features of teh cystic fluids that shows them to be distinct from both blood plasma and urine. Isoleucine, lysine, threonine and valine were present at mM concentrations, in cyst fluid and in some cases levels up to 2 orders of magnitude higher than normal plasma or urine were recorded. Mean glucose concentrations in the cyst fluids ranged from 3.4–9.6 mM and a number of organic acids and bases, including acetate, lactate, succinate, creatinine and dimethylamine were also present at high concentration and in different ratios to those found in either plasma or urine. The majority of cyst fluid examined also contained significant quantities of glycoproteins with characteristic 1H-NMR signals from N-acetyl groups of amino-sugar and sialic acid side chains which had a high degree of molecular mobility (as indicated by their relatively long T 2 relaxation times, >120 ms. High levels of ethanol (0.5–12.6 mM/l) were found in all fluid samples from the six transplanted patients (confirmed by conventional analysis). In general there was little variation in the 1H-NMR spectral patterns of either the intra- or interpatient cyst fluids, although the contribution of the protein macromolecules to individual spectra was lower in a few cysts. This constancy of biochemical composition probably reflects the chronic nature of the accumulation of cyst fluid and a long turnover of the cystic fluid components which was the effect of averaging composition. These findings suggest that the dynamic composition of cyst fluid from ADPKD patients is unique among the other body fluids and that the unusual composition may be related to epithelial polarity reversed of the cystic epithelium which could also contribute ot the growth of the cysts.

Characterization of acylated steryl glycosides fromEuryale ferox by nuclear magnetic resonance spectroscopy

High resolution nuclear magnetic resonance spectroscopy has been used to identify the main acylated steryl glycosides isolated from Euryale ferox as 24-ethylcholest-5-en-3β-O-β-D-pyranoglucosyl palmitate and 24-ethylcholesta-5,22E-dien-3β-O-β-D-pyranoglucosyl palmitate. The application of this technique to the identification of acylated steryl glycosides within complex mixtures is described.

Porosity development in deep sandstones, Taranaki Basin, New Zealand

Reservoir quality in the Taranaki Basin is controlled by both depositional facies and diagenesis. High initial porosities and permeabilities in manyfluvial and littoral sandstones directed fluid flow such that these facies underwater widespread cementation by silica, carbonate and authigenic clays, and later dissolution of carbonates. Current commercial production of hydrocarbons in mainly from secondary, dissolution porosity in sandstones at depths of 1750–3600 m. Recent studies suggest that oil may not be released from terrestrial source rocks in western New Zealand until depths of 5.5 km or more. Knowledge of porosity development at such depths is therefore essential for understanding migration pathways and the controls on hydrocarbon accumulation. Organic species and/or carbon dioxide evolved from kerogen before liquid hydrocarbons are important to the formation of secondary porosity in sandstones by carbonate dissolution. Further, hydrocarbon migration should follow soon after the evolution of these, in order that re-cementation does not occur. High resolution nuclear magnetic resonance (NMR) spectroscopy shows that in western New Zealand basins, the evolution of solvents continues to at least 4 km and may persist almost until the expulsion of liquid hydrocarbons.

High Resolution Nuclear Magnetic Resonance Spectroscopy of Adsorbed Molecules

High Resolution Nuclear Magnetic Resonance Spectroscopy of Adsorbed Molecules

The structures of oligosaccharides excreted by sheep with swainsonine toxicosis.

Eleven oligosaccharides were purified form the urine of sheep with swainsonine toxicosis induced by the feeding of Astragalus lentiginosus. Oligosaccharides were extracted by charcoal adsorption, chromatographed on Bio-Gel P-2, and partially fractionated by preparative-layer chromatography. Separation into individual compounds was completed by semi-preparative high pressure liquid chromatography. Structures were determined by a combination of high pressure liquid chromatography and exo- and endo- glycosidase action, methanolysis followed by gas-liquid chromatography, methylation analysis, and high resolution nuclear magnetic resonance spectroscopy. Two homologous series of oligosaccharides were identified: (a) alpha-D-Manp-(1----6)-beta-D-Manp-(1----4)-D-GlcpNAc, alpha-D-Manp(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp+ ++-(1----4)-D-GlcpNAc, alpha-D-Manp-(1----2)-alpha-D-Manp(1----3)-[alpha-D-Manp+ ++-(1----6)]-beta-D-Manp-(1----4)-D-GlcpNAc, and alpha-D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Manp+ ++-(1----3)-[alpha- D-Manp-(1----6)]-beta-D-Manp-(1----4)-D-GlcpNAc (minor series); (b) alpha-D-Manp-(1----6)-beta-D-Manp-(1----4)-beta-D-GlcpNAc- (1----4)-D-GlcpNAc, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-beta-D-GlcpNAc-(1----4)-D-GlcpNAc, alpha-D-Manp(1----3)-alpha-D-Manp-(1----6)-beta-D-Manp -(1----4)-beta-D-GlcpNAc- (1----4)-D-GlcpNAc, alpha-D-Manp-(1----6)-alpha-D-Manp-(1----6)-beta-D-Manp++ +-(1----4)-beta-D-GlcpNAc - (1----4)-D-GlcpNAc, alpha-D-Manp-(1----3)-alpha-D-Manp-(1----6)-[alpha-D-Manp -(1----3)]-beta-D- Manp-(1----4)-beta-D-GlcpNAc-(1----4)-D-GlcpNAc, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-alpha-D-Man p-(1----6)-beta-D- Manp-(1----4)-beta-D-GlcpNAc-(1----4)-D-GlcpNAc, and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-alpha-D-Man p-(1----6)- [alpha-D-Manp-(1----3)]-beta-D-Manp-(1----4)-beta-D-GlcpNAc- (1----4)-D- GlcpNAc (major series).

Preparation and characterization of thermoplastic polymers from hydroxyalkyl methacrylates

Soluble polymers have been produced from technical-grade 2-hydroxyethyl methacrylate (HEMA) and hydroxypropyl methacrylate (HPHA) using a combination of vacuum distillation and solvent extractions to purify the monomers prior to solution polymerization. These extractions, particularly of HEMA with hexane and corn oil, were found to reduce the level of ethylene glycol dimethacrylate to < 1%, according to gas chromatographic analysis. Homopolymers of HEMA and HPMA and copolymers of HEMA with methyl methacrylate were prepared with a wide range of molecular weights and characterized by viscometry in dimethyl formamide, infrared spectroscopy including deuterium exchange experiments, and high resolution proton and carbon nuclear magnetic resonance (NMR) spectroscopy. The latter was used to determine copolymer composition and tacticity distribution.

High resolution proton magnetic resonance spectroscopy of human brain and liver

Water-suppressed and slice-selective proton spectra of live human brain exhibited several resonances that were tentatively assigned to metabolites such as N-acetylaspartate, glutamate, phosphocreatine and creatine, choline derivatives, and taurine. In the liver spectrum of a healthy volunteer, the major resonance was tentatively assigned to a fatty acyl methylene and the minor resonances to protons in carnitine, taurine, glutamate, and glutamine. In the spectrum of a cancerous liver, resonances in addition to those present in the normal liver were seen. Protein degradation in the liver with cancer was indicated by resonances from urea and from the ring protons in tryptophan, tyrosine, and phenylalanine. Furthermore, increased nucleic acid synthesis was indicated by resonances from nucleotide protons.

Chromatographic purification and identification of polar metabolites of benazolin-ethyl from soybean.

The metabolism of benazolin-ethyl (4-chloro-2-oxobenzothiazolin-3-ylacetic acid ethyl ester), a post emergence herbicide, has been studied in soybean using (14C)-phenyl labelled compound. Preliminary studies were performed on excised soybean leaves. Following hydrolysis of the ethyl ester to benazolin acid (4-chloro-2-oxobenzothiazolin-3-ylacetic acid), extensive metabolism to polar conjugates was observed. The polar fraction from a Bligh-Dyer extraction was purified by solvent partitioning, preparative TLC and reverse phase HPLC with ion suppression. The two major metabolites were characterised by fast atom bombardment mass spectrometry with accurate mass determination as an aspartate conjugate and a malonyl-beta-glucose ester of benazolin acid. Subsequent experiments were performed by spraying intact plants at growth stage V4. The major polar metabolite isolated one month after treatment was identified as the aspartate conjugate by mass spectrometry and high resolution nuclear magnetic resonance spectroscopy.

NMR studies on angiotensin II: Histidine and phenylalanine ring stacking and biological activity

The conformations of angiotensin II and the antagonist [Sar 1, Ile 8]angiotensin II in dimethylsulfoxide have been examined by high resolution proton magnetic resonance spectroscopy at 400MHz. The chemical shifts for the aromatic protons of the phenylalanine residue in angiotensin II are consistent with shielding and restricted rotation for this side-chain. The chemical shifts for the histidine C 2 and C 4 protons in angiotensin II also indicate shielding, whereas these same protons in the antagonist [Sar 1, Ile 8]angiotensin II do not demonstrate this shielding influence. These findings suggest a stacking interaction for the histidine and phenylalanine side-chains in angiotensin II which is important for activating angiotensin receptors.

On the stereoselective aminoacylation of RNA

Aminoacylation of the “internal” 2′-hydroxyl groups of poly(A) with the imidazolide of racemic DL-leucine resulted in the formation of an enantiomeric excess of the D-ester, whereas aminoacylation with the imidazolide of racemic N-3,5-dinitrobenzoyl-DL-leucine gave an enantiomeric excess of the L-ester. Comparison of these results with those obtained earlier for alanine shows that the larger side chain tends to favor the formation of the D-ester. High resolution proton magnetic resonance spectroscopy was performed on the D- and L-alanyl “internal” 2′-esters of 3′,5′-ApA. The resonance of the side chain methyl group of the D-ester is upfield from that of the L-ester, and the separation of the peaks increases with a decrease in temperature.

Rhodopsin's protein and carbohydrate structure: Selected aspects

A topographic model for rhodopsin has been constructed based upon evaluation of rhodopsin's sequence by a secondary structure prediction algorithm as well as chemical and enzymatic modification of rhodopsin in the membrane [Hargrave et al. (1983) Biophys. Struct. Mech. 9, 235–244]. The non-uniform distribution of several amino acids in the primary structure and within the topographic model is discussed. The seven predicted helices were evaluated and each helix was found to have one surface which is much more hydrophobic than the other. Stereoscopic views of a three dimensional model with a functional color-coding scheme incorporating these features are presented. The amino acid sequence of rhodopsin has been compared to other proteins in the Dayhoff Protein Data Bank. No obvious relationship to any other protein sequenced was found. High resolution proton magnetic resonance spectroscopy was used to reinvestigate the structure and relative proportions of rhodopsin's major and minor oligosaccharide chains. One major (Man 3GlcNAc 3) and two minor (Man 4GlcNAc 3 and Man 5GlcNAc 3) were observed.

Deletion mapping of the aerobactin gene complex of plasmid CoIV

With the possible exception of certain strains of lactic acid bacteria, iron is known to be an essential element for all species of microbes and for all higher organisms. Within the cell, iron protein catalysts participate in some of the reactions most fundamental to life, including aerobic and anaerobic energy metabolism, nitrogen fixation, photosynthesis, and generation of the deoxyribotides required for synthesis of DNA. Iron is abundant in the environment but may be insoluble or in some other way unavailable. Mechanisms for assimilation of iron have been evolved by all species but, because of the plasticity and diversity of microbial metabolism, those in unicellular organisms are the most readily studied. The particular system of microbial iron acquisition investigated in this laboratory is labelled high-affinity and consists of low molecular weight, virtually Fe(III) specific ligands, termed siderophores, and the matching membrane receptors for the complexed Fe(III) ion. This system, which is coordinately expressed under iron starvation, has been detected in virtually all aerobic and facultative anaerobic microorganisms carefully examined for its presence [1]. In general, siderophores can be relegated to one of two chemical classes, viz., hydrozamic acids and catechols. The number of siderophores characterized to date, some by crystallography and/or high resolution nuclear magnetic resonance spectroscopy, must now number several score. The complete systems, comprised of siderophore and specific receptor, is the converse is true, possibly because of the aromatic character of enterobactin and its propensity to adhere to proteins such as albumin.

Estimation of the distance between the two binding sites of ovotransferrin

Ovotransferrin (conabulmin, M.wt. 76,000) has been found in large quantities in egg white. Its physiological function is not known with certainty. Because of its large affinity for iron, it is an effective antimicrobial agent which could be important in the protection of the developing chick embryo [1]. In the presence of carbonate, or a difunctional anion, metal ions bind transferrin at two specific sites [2]. The binding of metal ions to the second specific site takes after the first site is filled, i.e. a sequential rather than a random binding process takes place [3]. In this communication the results of the addition of Gd(III) ion in the presence of malonate, using high resolution nuclear magnetic resonance spectroscopy, are reported. To 1.23 m M solution of protein containing 12.3 m M malonate, kept at pH 6.00 ± 0.02 by the presence of 50 m M trideuteroacetate buffer, one and two equivalents of Gd(III) per protein, dissolved in D 2O, were added. In each case, the spectrum was recorded using a Bruker-270 MHz spectrometer (Fig. 1). The spectrum of the protein solution containing one equivalent of Gd(III) shows that all histidine C 2-H resonances have been broadened. In fact the signals of the six histidines which were assigned to be involved in the binding sites [3] have nearly disappeared, indicating that these resonances broaden by at least 80 Hz which could make them undistinguishable from noise. On addition of the two equivalents, ▪ all the histidine C2-H resonances have disappeared, Fig. 1. Theoretical consideration of the line broadening by Gd(III): The line-width of the NMR signals is given by, ▪ where Δ v is the line-width (in Hz) at the half height and T 2M is the transverse relaxation time of the protein. In the presence of Gd(III) ions T 2M is given by the Solomon-Bloembergen equation [4]: ▪ where τ c is the correlation time which modulates the interaction, and r is the distance between the proton and the paramagnetic centre. Assuming τ c is the rotational tumbling time of the macromolecule (τ R, then from Stockes law, ▪ where M is the molecular weight V is the partial specific volume, η is the viscosity of the solvent, T is the temperature and R is the gas constant. Using the above equation, a value of τ R = 2.2 × 10 −8 s −1 could be obtained for ovotransferrin. At 270 MHz eqn. (1) reduces to ▪ giving r = 21 Å for an increase of 80 Hz in the line-width due to the presence of Gd(III). This is the upper limit of the distance between the first binding site of ovotransferrin and the C2-H protons in the second binding site. Thus we conclude from the broadening of the spectrum of the whole protein, when it contains two equivalents of Gd(III), that all the C2-H protons of histidines in ovotransferrin lie in a sphere of radius not exceeding 31.5 Å.

High resolution nuclear magnetic resonance spectroscopy of bile salts: individual proton assignments for sodium cholate in aqueous solution at 400 MHz.

The 400 MHz 1H-nuclear magnetic resonance spectrum of sodium cholate in dilute aqueous solution has been successfully resolved using a combination of decoupling, partial relaxation, and decoupled partial relaxation techniques. The individual carbon resonances in the 13C-NMR spectrum of sodium cholate have also have assigned. Assignments of individual methylene protons were made by consideration of the molecular structure of sodium cholate and the expected couplings and 1H-nuclear Overhauser enhancement experiments. Verification of the assignments of the methine protons was made by application of single frequency 1H-decoupled 13C-NMR. Variation of pH* from 6.0 to 11.0 did not alter the individual chemical shifts except for those between 2.12 delta and 2.30 delta, originating from the protons on the C23 position adjacent to the ionizable carboxyl group. The chemical shifts of the proton resonances were independent of concentration below 5 mM. Above 10 mM (micellar region), the proton chemical shifts were altered slightly and some band broadening occurred. These data are consistent with the formation of small micellar aggregates (up to N = 4) of cholate molecules.

Studies of static and dynamic aspects of spatial protein structures by high resolution nuclear magnetic resonance spectroscopy

The potential of high resolution nuclear magnetic resonance (NMR) for many-parameter characterization of biopolymer conformation has long been recognized. Currently new NMR techniques for use in this field are in full development. In the present paper a survey of fundamental aspects of NMR studies of protein conformation is used as a basis for discussion of recently developed one-dimensional and two-dimensional NMR experiments for studies of proteins.

Isolation of alpha-melanotropin and N, O-diacetylserine1-alpha-melanotropin from porcine pituitary extracts.

In the course of isolation of alpha-melanotropin (alpha-MSH) from porcine pituitary extracts, a peptide with identical amino acid composition but slightly less polar properties compared to alpha-MSH was detected and isolated by partition chromatography. The new peptide was identified as N, O-diacetyl-Ser1-alpha-MSH by high resolution nuclear magnetic resonance spectroscopy. N, O-diacetyl-Ser1-alpha-MSH was readily converted to alpha-MSH by incubation at pH 9.5. The O-acetyl analog of alpha-MSH was found to be as active as alpha-MSH in stimulating lipolysis in isolated rabbit adipocytes.

Review lecture: Nuclear magnetic resonance spectroscopy in two frequency dimensions

A spectrum is normally thought of in terms of a graph of absorption intensity as a function of frequency, and in this form it has served us well for many years. A recent development extends this idea into a second frequency dimension, the spectrum now being represented by a surface in three-dimensional space. The examples are in the field of high resolution nuclear magnetic resonance (n.m.r.) spectroscopy, although the principle is rather more general, being based on the two-dimensional Fourier transformation of a transient response that is a function of two independent time variables t 1 and t 2 . By arranging for different experimental conditions to prevail during t 1 and t 2 , it is possible to separate different n.m.r. parameters, for example chemical shifts and spin coupling constants, into the two frequency dimensions. There is also an important element of correlation involved, since during t 2 the nuclei ‘remember’ their past history during t 1 ,and this has been used to correlate proton and carbon-13 chemical shifts.

NMR study of the interaction of plastocyanin with chromium(III) analogues of inorganic electron transfer reagents

High resolution nuclear magnetic resonance spectroscopy has been used to examine the interaction of plastocyanins from French bean ( Phaseolus vulgaris) and cucumber ( Cucumis sativus) with three complexes—potassium hexacyanochromate(III), hexamminechromium(III) nitrate and tris(1,10-phenanthroline)-chromium(III) perchlorate—which are analogues of inorganic electron transfer reagents. The results indicate a high degree of specificity in the binding of these complexes and two binding sites on the protein are identified. One binding site is situated close to the copper atom and is clearly suited to outer sphere electron transfer through one of the histidine ligands. The other binding site is more distant from the copper atom and this mechanism cannot be operative. Electron transfer via hydrophobic channels or electron tunneling are possible mechanisms of electron transfer.

Electron transfer reagent binding sites on plastocyanin

Electron transfer is an essential process in all biological systems. The kinetics of electron transfer reactions between small inorganic complexes and a variety of metalloproteins have been extensively studied1–5. Recent studies on the electron transfer reactions of plastocyanin, an essential component of the photosynthetic electron transport chain, have shown6–9 that inorganic redox reagents such as the hexacyanoferrate(III) ion (Fe(CN)63−6) and the tris(1,10-phenanthroline)cobalt(III) ion (Co(1,10-phen)33+) form discrete complexes with the protein before the electron transfer step. Here, we have used high resolution nuclear magnetic resonance spectroscopy (NMR) to study the interaction of plastocyanins from French bean (Phaseolus vulgaris) and cucumber (Cucumis sativm) with the chromium(III) analogues of these redox reagents, K3Cr(CN)6 and Cr(1,10-phen)3 (ClO4)3. The binding of these complexes to plastocyanin is highly specific and we identify two binding sites, separated by about 15 A, which are apparently determined primarily by electrostatic interactions. Two distinct electron transfer pathways to and from the copper atom are indicated.