Correlation NMR Spectroscopy Research Articles

Microbial Hydroxylation and Methylation of Genistein by Streptomycetes

Streptomyces griseus (ATCC 10137) and Streptomyces catenulae (ATCC 23893) were used to convert genistein5,7,4‘-trihydroxyisoflavone (1)a major isoflavone found in soybeans, into five metabolites, four of which were previously unknown. These were 5,7,3‘,4‘-tetrahydroxy-8-methylisoflavone (3), 5,7,3‘,4‘-tetrahydroxy-8-methylisoflavanone (4), 5,7,4‘-trihydroxy-3‘-methoxyisoflavone (5), 5,7,8,3‘,4‘-pentahydroxyisoflavone (7), and the known compound, 5,7-dihydroxy-3‘,4‘-dimethoxyisoflavone (6). Identities of these metabolites were established by HRFABMS and 1D and 2D NMR correlation spectroscopy.

Elucidation of the structure of an alanine-lacking core tetrasaccharide trisphosphate from the lipopolysaccharide of Pseudomonas aeruginosa mutant H4.

Lipopolysaccharide (LPS) of Pseudomonas aeruginosa rough mutant H4 was isolated by hot water/phenol extraction followed by a modified phenol/chloroform/petroleum ether procedure. Upon SDS/PAGE, the LPS showed a strong major band corresponding to the expected rough-type LPS. Additional faint high molecular-mass bands revealed that the O-chain was present, indicating that the H4 mutant is genetically unstable. Mild acid hydrolysis of the LPS removed lipid A and released a phosphorylated core oligosaccharide that was purified by gel-permeation chromatography and high-performance anion-exchange liquid chromatography. The oligosaccharide contained two residues of L-glycero-D-manno-heptose (Hep) and one residue each of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and GalNAc. Upon matrix-assisted laser desorption/ionization mass spectroscopy in the negative ion mode, the main fraction expressed a peak for the molecular ion [M-H]- at m/z 1106.41, which was compatible with a carbamoylated, trisphosphorylated tetrasaccharide. The structure was further investigated using one- and two-dimensional homonuclear and heteronuclear correlated NMR spectroscopy at pD 3 and, after borohydride reduction, at pD 9. The NMR data of the two phosphorylated tetrasaccharides recorded at different pD allowed determination of the positions of the three phosphate (P) groups and the carbamoyl group (Cm) thus establishing the following structure of the core oligosaccharide: [equation: see text] Two unusual structural features in the core oligosaccharide of P. aeruginosa were identified for the first time, i.e. the replacement of an amide-linked alanyl group in GalN with an acetyl group and the phosphorylation at position 6 of HepII.

Structural features and anti-complementary activity of some heteroxylan polysaccharide fractions from the seeds of Plantago major L.

Polysaccharides were isolated from the seeds of Plantago major L. by water extraction and fractionated by ion exchange and size exclusion chromatography. Methods such as methanolysis and GC, methylation analysis and GC-MS, weak acid hydrolysis, enzyme hydrolysis, 13C, APT, 1H and 2D heteronuclear (1H–13C) chemical shift correlated NMR spectroscopy have revealed that the polysaccharides isolated are heteroxylans which consist of a 1,4-linked (β-D-Xylp backbone with short side chains attached to position 2 in some 1,4-linked β-D-Xylp residues and to position 3 in other 1,4-linked β-D-Xylp residues. The side chains consist of β-D-Xylp, α-L-Araf, α-L-Araf 1→3 β-D-Xylp and α-D-Glcp A1→3 α-L-Araf.1,4-linked α-D-GalpA residues were also detected in addition to small amounts of 1,2,4-linked Rhap and 1,3- 1,6- and 1,3,6-linked Galp. The crude extract and some of the fractions obtained had potent anti-complementary activity.

Spin Diffusion with 13C Selection and Detection for the Characterization of Morphology in Labeled Polymer Blends with MAS NMR

A pulse sequence has been implemented for the determination of domain sizes in 13C enriched organic materials with two-dimensional high-resolution magic angle spinning (MAS) NMR correlation spectroscopy. The method integrates the high resolution of 13C MAS for selection and detection with an efficient polarization transfer across domain boundaries via strong 1H dipolar interactions. A one-dimensional version with spectral editing allows for improved efficiency with respect to measurement time. With this MAS NMR and labeling approach heterogeneity at length scales from 1 to 100 nm can be determined for systems without long-range order. As an example, proton spin diffusion times up to 500 ms were used to study the morphology of a phase-separated semi-interpenetrating network of poly(styrene-co-acrylonitrile) and cross-linked poly(styrene-co-maleic anhydride). From the spin diffusion behavior a characteristic domain diameter d = 63 ± 4 nm was calculated.

Solvolysis of Caryophyllen-8β-yl Derivatives: Biomimetic Rearrangement−Cyclization to 12-Nor-8α-presilphiperfolan-9β-ol

The solvolyses of caryophyllen-8β-yl p-nitrobenzoate (14-OpNB) and 15-norcaryophyllen-8β-yl tosylate (15-OTs) were investigated as potential model reactions for the biogenesis of the tricyclic presilphiperfolanol sesquiterpenes. Buffered solvolysis of 14-OpNB in 60% aqueous acetone at 125 °C afforded caryophyllene (3) as major product, accompanied by small amounts of caryophyllen-8β-ol (14-OH) and 5,8-cyclocaryophyllen-4α-ol (16). In contrast, 15-OTs underwent a stereospecific rearrangement−cyclization to 12-nor-8α-presilphiperfolan-9β-ol (17) upon solvolysis in 60% aqueous acetone at 75 °C. The structure and stereochemistry of this trans,cis,trans-tricyclo[6.2.1.05,11]undecane derivative were established by NMR correlation spectroscopy and X-ray crystallography. Two different mechanisms (paths A and B) for the conversion of 15-OTs to 17 by initial 1,2-migration of either the external or internal cyclobutane ring bonds (C10 and C1) followed by π−σ cyclization onto the trans double bond are discussed.

Spatial Ordering of Organic and Inorganic Charge Centers in As-Made High-Silica Zeolites Determined by Multidimensional {1H} →2H CPMAS NMR Correlation Spectroscopy

Evidence is presented for the spatial ordering of the organic and inorganic charge centers in the as-made tectosilicates nonasil, SSZ-23, and ZSM-12. This result is based on two-dimensional 2H−{1H} NMR correlation spectroscopy experiments, which show correlation between the silanol protons of the defect site and the N-methyl groups of the structure-directing agent. The two-dimensional NMR spectra for all samples suggest that the deuterons of the methyl groups adjacent to the organic charge center are preferentially ordered relative to the defect site. Experiments performed on a sample synthesized in D2O are consistent with the interpretation of the two-dimensional NMR experiments. The results also suggest that Coloumbic organic−inorganic forces influence the location of the charge-compensating defect site in all-silica zeolites. Moreover, we suggest an additional role for certain cationic structure-directing agents in zeolite synthesis, controlling the location of T+3 atoms in the zeolite framework.

Longitudinal and Transverse 1H−15N Dipolar/15N Chemical Shift Anisotropy Relaxation Interference: Unambiguous Determination of Rotational Diffusion Tensors and Chemical Exchange Effects in Biological Macromolecules

High-resolution proton-detected heteronuclear correlation NMR spectroscopy allows the measurement of 15N spin relaxation rates at multiple sites throughout a biological macromolecule. The rate constants are determined by stochastic internal motions on time scales of picoseconds to nanoseconds, overall molecular rotational diffusion on time scales of nanoseconds, and chemical exchange rates on time scales of microseconds to milliseconds. A new method has been developed for distinguishing the contributions of chemical exchange from the contributions due to anisotropic rotational diffusion by measuring both longitudinal and transverse interference between the 1H−15N dipolar and 15N chemical shift anisotropy interactions. The spectroscopic experiment for measuring the longitudinal cross-correlation rate constant for 1H−15N dipolar/15N chemical shift anisotropy interference is based on the approach for measuring the transverse cross-correlation rate constant (Tjandra, N.; Szabo, A.; Bax, A. J. Am. Chem. Soc. 1...

Facile synthesis of a bis‐linked dioxocyclam porphyrin

Reaction of the 5,15-di-(2-alkylamidophenyl)-etioporphyrin 3 (a,a-atropisomer) with the 'self- trans-diprotected' dioxocyclam 4 leads easily, in only two steps, to a new symmetrical dioxocyclam porphyrin 5 in an overall 41% yield. This ligand is a precursor of models of cytochromes. All the compounds have been fully characterized by elemental analysis, mass spectrometry and UV-vis, 1 H NMR, 13 C NMR and 1 H- 13 C heteronuclear correlation NMR spectroscopies. X-ray structural data are presented for the Zn(II) porphyrin 6 confirming that the porphyrin and the dioxocyclam adopt cofacial orientations.# 1998 John Wiley & Sons, Ltd.

Assignment and Measurement of Deuterium Quadrupolar Couplings in Liquid Crystals by Deuterium−Carbon NMR Correlation Spectroscopy

The performance of deuterium−carbon correlation (DECOR) spectroscopy for the assignment and measurement of deuterium quadrupolar couplings in fully deuterated liquid crystalline phases is analyzed. The experiment relies on a broadband polarization transfer step from deuterium to carbons. The efficiency of this step is demonstrated experimentally together with a brief study of cross-polarization dynamics. We show that resolution can be improved by removing the substantial contributions from heteronuclear dipolar couplings in a refocused version of the experiment. Quantification of the quadrupolar couplings is illustrated by the application to the thermotropic liquid crystal 5CB. A complete assignment is obtained by using both short-range and long-range correlations, allowing the individual measurement of all the aromatic couplings that are overlapping in the one-dimensional deuterium spectrum. The heteronuclear C−D couplings are shown to be measurable by direct inspection of the 2D spectra. By way of demonstration, we show that the experiment allows the measurement of quadrupolar couplings as a function of temperature.

Synthesis and characterization of a novel series of bis-linked diaza-18-crown-6 porphyrins

The synthesis and physicochemical characterization of a novel family of ‘crowned-porphyrins’ is presented. The compounds uniquely possess a 5,15-di(2-alkylamidophenyl)etioporphyrin bis-linked to the nitrogens on opposite sides of a 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane moiety. The size of the cavity in between the porphyrin and the diaza-18-crown-6 ring has been regulated by varying the length of the alkylamido linking units. The structures of 5 and 7 bear diacetamido linkers whereas 6 is linked via dipropionamido groups. The synthetic pathway presented is generalized such that a wide variety of useful porphyrin-based bis-macrocycles can be prepared. Products have been fully characterized by elemental analysis, mass spectrometry, spectrophotometry, 1H NMR, 13C NMR and 1H–13C heteronuclear correlation NMR spectroscopies. X-Ray structural data are presented for the free-base porphyrin 6 and the ZnII porphyrin 7. The structural data confirm that the porphyrins and their diaza-18-crown-6 substituents adopt cofacial orientations. The size of the cavity between the macrocycles, while demonstrated to accommodate small molecules as shown by the binding of H2O as an axial ligand to the central ZnII ion in the crystal structure of 7, will probably not accommodate large ligands or substrates. Moreover, these compounds should allow sequential metallations to yield heterobimetallic species. Therefore, they are ideally suited for study as model systems for biologically important heme-dependent phenomena such as xenon-129 binding to myoglobin or the mimicking of processes related to the structure and function of cytochrome c oxidase.

The nature and fate of natural resins in the geosphere—VIII. NMR and Py–GC–MS characterization of soluble labdanoid polymers, isolated from Holocene class I resins

Soluble polylabdanoids isolated by sequential solvent extraction have been characterized by liquid-state 13C- and 1H NMR and 13C- 1H HMQC (heteronuclear correlation) NMR spectroscopy in addition to solid-state NMR and Py–GC–MS techniques. Two Holocene resins originating from Santander, Colombia and Mombasa, Kenya were analyzed. Soluble polymers were isolated by extraction with a 1:1 (v/v) methylene chloride–methanol mixture following sequential extractions with methylene chloride and methanol. The molecular weight of polymer extracts was shown by GPC analyses to exceed that of non-polymeric occluded terpenoids. Py–GC–MS, solid-state 13C CP/MAS and 13C cross-polarization/depolarization NMR spectroscopy results indicated that chemical compositions of soluble polymers isolated from immature resins are highly representative of the structure of corresponding insoluble polymers, i.e. polylabdatrienes. These data provide evidence for cross-linking or cyclization of side-chain olefinic carbons during or shortly after polymerization. Generally, the characterization of soluble resin polymers by liquid-state NMR spectroscopy has proven to be an excellent means for investigating the maturation mechanism of polylabdanoid resinites, and has potential for furthering the application of Class I resinites as geothermal indicators.

Jenisseensosides C and D, biologically active acylated triterpene saponins from Silene jenisseensis

We previously reported the isolation and structure elucidation of a new trans- p-methoxycinnamoyl triterpene-saponin along with its cis- p-methoxycinnamoyl isomer as an inseparable mixture from the roots of Silene jenisseensis. In a continuing study on this plant, two additional new acylated triterpene-saponins were obtained as an inseparable mixture. Their structures have been established by chemical means and spectroscopic methods including 1D and 2D homonuclear and heteronuclear correlation NMR spectroscopy as 3- O-[β- d-galactopyranosyl-(1 → 2)-β- d-glucuronopyranosyl]-28- O-[{α- l-rhamnopyranosyl-(1 → 2)}- {4- O- trans- p-methoxycinnamoyl}-β- d-fucopyranosyl] quillaic acid and its cis-isomer, respectively. They showed a significant enhancement of the granulocyte phagocytosis in vitro. © 1997 Elsevier Science Ltd. All rights reserved

Multiple-quantum magic-angle spinning NMR with cross-polarization: Spectral editing of high-resolution spectra of quadrupolar nuclei

An experiment is presented that combines the multiple-quantum magic-angle spinning (MQMAS) technique with cross-polarization (CP). As a preliminary test of this new method, we measured and compared the 27Al 3QMAS and 19F → 27A1 CP 3QMAS spectra of a fluorinated AlPO 4 aluminophosphate. Complete discrimination between the fluorinated and nonfluorinated Al sites was easily achieved, which demonstrates the usefulness of CP MQMAS for spectral editing. Future applications of this experiment will include other spin pairs and heteronuclear correlation NMR spectroscopy.

Deprotonation of the carbaborane nido-7-NH2But-7-CB10 H12: crystal structures of nido-7-NH2But-7-CB10 H12 and [NEt3(CH2Ph)][ nido-7-NHBut-7-CB10H12

The crystal structure of nido-7-NH 2 Bu t -7-CB 10 H 12 1 has been determined by X-ray diffraction. The compound crystallises in the orthorhombic space group Pbca [a = 10.507(5), b = 13.805(6), c = 18.093(9) A]. The nido-icosahedral structure was established with two endo-B–H–B and two NH 2 hydrogen atoms located in Fourier-difference maps and refined. The structure determination is also supported by 11 B-{ 1 H}– 11 B-{ 1 H} correlation NMR spectroscopy. A molecular orbital calculation and frontier density analysis of 1 indicated that deprotonation should initially occur at the exo-nitrogen atom. Treatment of 1 with 1 equivalent of LiBu n , followed by addition of [NEt 3 (CH 2 Ph)]Cl, gave [NEt 3 (CH 2 Ph)][nido-7-NHBu t -7-CB 10 H 12 ] 2 as the only product and single crystals were grown for an X-ray diffraction study. The salt crystallises in the monoclinic space group P2 1 /n [a = 10.651(2), b = 11.652(2), c = 20.926(3) A, β = 101.77(2)°]. The 1 H, 13 C-{ 1 H} and 11 B-{ 1 H} NMR spectra of 1 and 2 have also been recorded.

Backbone dynamics of oxidized and reduced D. vulgaris flavodoxin in solution.

Recombinant Desulfovibrio vulgaris flavodoxin was produced in Escherichia coli. A complete backbone NMR assignment for the two-electron reduced protein revealed significant changes of chemical shift values compared to the oxidized protein, in particular for the flavine mononucleotide (FMN)-binding site. A comparison of homo- and heteronuclear NOESY spectra for the two redox states led to the assumption that reduction is not accompanied by significant changes of the global fold of the protein. The backbone dynamics of both the oxidized and reduced forms of D. vulgaris flavodoxin were investigated using two-dimensional 15N-1H correlation NMR spectroscopy. T1, T2 and NOE data are obtained for 95% of the backbone amide groups in both redox states. These values were analysed in terms of the 'model-free' approach introduced by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546-4559, 4559-4570]. A comparison of the two redox states indicates that in the reduced species significantly more flexibility occurs in the two loop regions enclosing FMN. Also, a higher amplitude of local motion could be found for the N(3)H group of FMN bound to the reduced protein compared to the oxidized state.

Bacterial overexpression, isotope enrichment, and NMR analysis of the N-terminal domain of human apolipoprotein E

The nucleotide sequence encoding the N-terminal domain (residues 1-183) of human apolipoprotein E3 (apoE3) was cloned into the pET expression vector and introduced into Escherichia coli. Induction of protein expression with isopropyl beta-D-thiogalactopyranoside resulted in production of recombinant apoE3(1-183). Immunoblot analysis revealed that recombinant protein was present in both the cell pellet and cell culture supernatant. Analysis revealed that a significant portion of the rApoE3(1-183) in the cell pellet still possessed the bacterial N-terminal pel B leader sequence, encoded by plasmid DNA directly upstream of the apoE3(1-183) coding sequence. By contrast, this hydrophobic leader sequence had been removed from recombinant protein specifically accumulating in the culture medium. This behavior is novel for bacterial expression of apolipoprotein E and its truncated variants and permits efficient overexpression of the recombinant protein (> 100 mg/L cell culture). Recombinant apoE3(1-183) was isolated by a combination of heparin-Sepharose chromatography and reverse-phase HPLC. Electrospray mass spectrometry provided a mass of 21 191 daltons, corresponding directly to that expected from the known sequence. Circular dichroism spectroscopy revealed that the recombinant protein possesses significant amounts of alpha-helical secondary structure. The lipid binding ability of rApoE3(1-183) was evaluated using an in vitro lipoprotein binding assay. It was observed that recombinant apoE3(1-183) protected human low density lipoprotein (LDL) from lipid accumulation induced particle aggregation, indicating that it is capable of associating with lipoprotein surfaces. In addition, rApoE3(1-183) forms disk complexes with the model phospholipid dimyristoylphosphatidylcholine. In competition experiments, it was observed that rApoE3(1-183) phospholipid disks compete with 125I-LDL for binding to the apoB/E receptor on human skin fibroblasts to an extent similar to that observed for intact rApoE3. Taken together, these data show that recombinant apoE3(1-183) is fully functional as an apolipoprotein and receptor ligand. Given the high expression level and its known existence as a monomer in solution, we evaluated the potential for application of NMR spectroscopy to study the structure-function relationship of rApoE3(1-183). Bacteria were cultured in media supplemented with 15NH4Cl or [15N]glycine and the isotopically labeled recombinant apoE3(1-183) was analyzed by heteronuclear single quantum correlation NMR spectroscopy. The data revealed that rApoE3(1-183) is an excellent candidate for solution structure studies by NMR, including conformational adaptations associated with lipid association.

Core mutants of the immunoglobulin binding domain of streptococcal protein G: Stability and structural integrity

A library of core mutants of the GB1 domain of streptococcal protein G was created, and the structure and stability of selected members was assessed by 1H- 15N heteronuclear correlation NMR spectroscopy and fluorescence. All mutants comprised changes in β-sheet residues, with sidechains at positions 5 (Leu), 7 (Leu), 52 (Phe) and 54 (Val) forming the β-sheet side of the sheet-helix core interface. A solvent exposed position Ile-6 was chosen as a control. Randomization of bases at codon positions 1 and 3 with thymine at position 2 introduces five possible hydrophobic amino acids, namely Leu, Val, Ile, Phe, and Met. The distribution of encoded amino acids at all five positions is approximately as expected theoretically and indicates that no major bias was introduced towards particular residues. The overall structural integrity of several mutants, as assessed by NMR, ranges from very close to wild type to fully unfolded. Interestingly, the stability of the mutants is not strictly correlated with the number of changes or residue volume.

Stereochemical Course and Reaction Products of the Action of β‐Xylosidase from Thermoanaerobacterium saccharolyticum Strain B6A‐RI

Beta-Xylosidases are grouped in families 39 and 43 of a general classification of glycosyl hydrolases based on amino acid sequence similarities. The Beta-xylosidase from Butyrivibrio fibrisolvens, which belongs to family 43, has been shown to operate by a molecular mechanism which results in the inversion of the anomeric configuration. Thermoanaerobacterium saccharolyticum B6A-RI Beta-xylosidase which belongs to family 39 was purified as a recombinant enzyme from Escherichia coli. The stereochemistry of the hydrolysis of p-nitrophenyl Beta-D-xylopyranoside was followed by 1H NMR. The spectrum recorded after 2 h hydrolysis showed a large signal centred at 4.47 ppm (J approximately 10 Hz) assignable to H1 of free Beta-xylose with a small amount of alpha-xylose (5.05 ppm, J approximately 3 Hz) attributable to mutarotation. This result indicates that T. saccharolyticum Beta-xylosidase operates with overall retention of the anomeric configuration. This result, with the lack of sequence similarity between the two families of Beta-xylosidases, suggests that these two families have major differences in their active-site geometries. Consistent with its retaining mechanism, Beta-xylosidase of T. saccharolyticum B6A-RI also displayed transglycosylating activity:reverse-phase HPLC showed approximately 30% conversion of p-nitrophenyl Beta-D-xylopyranoside into a number of higher nitrophenyl oligosaccharides after 5 min incubation with the enzyme. The structure of the most abundant oligosaccharides could be determined by total correlation spectroscopy NMR and showed that the enzyme can build Beta-1,4, Beta-1,3- and Beta-1,2-linked xylo-oligosaccharides.

Multiple-Frequency Decoupling in Selective Correlation Spectroscopy

New variants are presented of the selectively inverted soft correlation NMR spectroscopy using multiple-frequency decoupling. Tests were made using the std. Me trans-2-phenylcyclopropanecarboxylate. This method can det. arbitrary coupling consts. with great accuracy. [on SciFinder (R)]

Improved Sensitivity in Selective NMR Correlation Spectroscopy and Applications to the Determination of Scalar Couplings in Peptides and Proteins

A new method, providing a significant improvement in sensitivity, is presented for obtaining selective two-dimensional NMR correlation (COSY) spectra. The method is demonstrated on a small molecule having nonlinear optical properties and is further demonstrated by the precise determination of scalar coupling constants in the peptide gramicidin and in a 57 amino acid fragment of the nucleocapsid of the hepatitis C virus. In these systems we observe sensitivity improvements of between a factor of 2 and 6. In the case of the protein it was not practical to record the selective COSY spectra using conventional techniques. The method makes use of the recently introduced concept of “linear” selective pulses which provide a simple way of obtaining pure-phase band selective excitation with an arbitrary shape for the response. In a selective 2D COSY experiment the relative sensitivity improvement provided by these pulses is cubed with respect to a 1D experiment. The resulting pulse sequences are highly unusual in that they require a modulation of the pulse shape and pulse length as the indirect detection period is increased.