Natural Abundance 13C NMR Spectra Research Articles

Dynamic Nuclear Polarization NMR Spectroscopy Allows High-Throughput Characterization of Microporous Organic Polymers

Dynamic nuclear polarization (DNP) solid-state NMR was used to obtain natural abundance (13)C and (15)N CP MAS NMR spectra of microporous organic polymers with excellent signal-to-noise ratio, allowing for unprecedented details in the molecular structure to be determined for these complex polymer networks. Sensitivity enhancements larger than 10 were obtained with bis-nitroxide radical at 14.1 T and low temperature (∼105 K). This DNP MAS NMR approach allows efficient, high-throughput characterization of libraries of porous polymers prepared by combinatorial chemistry methods.

Fast enantioselective amino acid quantitative 13C NMR determination by a praseodymium chiral shift reagent

The fast quantitative determination of enantiomers of amino acids by the use of a praseodymium-based water-soluble chiral shift reagent, utilizing natural abundance 13C NMR spectra, is described.

Natural-abundance 13C nuclear-magnetic-resonance study of toxin II from Anemonia sulcata.

Natural-abundance 13C NMR spectra (at 15.04 MHz) of the polypeptide toxin II from the sea anemone Anemonia sulcata have been analysed and compared with corresponding spectra reported recently for a closely related polypeptide anthopleurin A. The spectra contain many resolved one-carbon and two-carbon resonances from carbonyl, aromatic and methyl carbons, many of which have been assigned to individual carbons in the molecule on the basis of their chemical shifts, including their pH dependence, and by comparison with the 13C NMR spectrum of anthopleurin A. Analysis of the effects of pH on the spectrum yields estimates for the pKa values of a number of functional groups in the molecule, as follows: side-chain carboxylates of the two aspartic acid residues 2 and 3.1; COOH-terminal carboxylic acid, 3.5; imidazolium moieties of the two histidine residues, 6.7 and 7.6 NH2-terminal ammonium, 8. The similarity between the pKa values of these functional groups in toxin II and those of corresponding groups in anthopleurin A, together with the close agreement between chemical shifts of conserved carbons, indicates that many local interactions are nearly identical in the two molecules, and thus supports the thesis that their overall conformations in solution are similar. However, the local interactions involving one of the aspartic acid residues are altered in toxin II. Together with other data, this leads to a proposal for the site in these two molecules which is responsible for their cardiac stimulatory activity.

β-Poly( l -malate) production by Physarum polycephalum

β-Poly(l-malate) (PMLA) production in Physarum polycephalum has been followed by using d-[1-13C]glucose and Ca13CO3. Nuclear magnetic resonance studies of PMLA showed that the 13C label from [1-13C]glucose was incorporated in the presence of CaCO3 into positions C-3 (-CH2-) and C-4 (-CO-) of the l-malate repeating unit of PMLA. The 13C label from Ca13CO3 was incorporated into position C-4 and indicated that not only the endogenous CO2 but also the exogenous CO2 from CaCO3 served significantly as a carbon source for PMLA production. In the absence of CaCO3, the 13C labeling pattern of PMLA from d-[1-13C]glucose was almost indistinguishable from that for the natural abundance 13C-NMR spectrum of the polymer. These results indicated that l-malate used for PMLA production is synthesized either via carboxylation of pyruvate and reduction of oxaloacetate in the presence of CaCO3 or via the oxidative tricarboxylic acid (TCA) cycle in the absence of CaCO3. Avidin strongly inhibited the formation of l-malate via carboxylation; the 13C labeling pattern of PMLA in the presence of CaCO3 was almost identical with that for the natural abundance spectrum when avidin was added, indicating that l-malate utilized for PMLA production was supplied under this condition by the oxidative TCA cycle.

NMR parameters of cobalt and of the directly bonded organic carbons obtained from solution 59Co and 13C NMR spectra of cobaloximes

The one bond cobalt–carbon scalar coupling constants of the cobaloximes [RCo(DH)2PPh3] ((DH)2=bisdimethylglyoximate) (R=Me, n-Pr, CH2Cl) and [MeCo(DH)2L] (L=D2O, N-MeIm, py, P(OMe)3, P(OEt)3) were obtained by the joint use of 59Co and natural abundance 13C NMR spectra in solution. The 1J(Co, C) values of the methyl derivatives show a clear dependence on the axial ligand L. They increase in the order P(OMe)3<P(OEt)3⩽PPh3<py⩽N-MeIm<D2O and reflect changes in the cobalt–carbon binding. Since the reduced constants 1K(Co, C) are about one-half of the 1K(Rh, C) and the directly bonded carbon and phosphorus atoms are notably less shielded than in the corresponding rhodium compounds, important relativistic effects can be hypothesised in the latter. The correlation times for molecular reorientation (τc) of [MeCo(DH)2PPh3] and of [MeCo(DH)2py] were determined at various temperatures from the line-width at half-height of the 59Co NMR signal as its relaxation is inherently quadrupolar. The rotational diffusion exhibits a notable dependence on the solvent, revealing specific interactions with it.

13C Solid-State NMR of Gramicidin A in a Lipid Membrane

The natural-abundance 13C NMR spectrum of gramicidin A in a lipid membrane was acquired under magic-angle spinning conditions. With fast sample spinning (15 kHz) at ∼65°C the peaks from several of the aliphatic, β-, α-, aromatic, and carbonyl carbons in the peptide could be resolved. The resolution in the 13C spectrum was superior that observed with 1H NMR under similar conditions. The 13C linewidths were in the range 30–100 Hz, except for the α- and β-carbons, the widths of which were ∼350 Hz. The β-sheet-like local structure of gramicidin A was observed as an upfield shift of the gramicidin α and carbonyl resonances. Under slow sample spinning (500 Hz), the intensity of the spinning sidebands from 13C in the backbone carbonyls was used to determine the residual chemical shift tensor. As expected, the elements of the residual chemical shift tensor were consistent with the single-stranded, right-handed β 6.3 helix structure proposed for gramicidin A in lipid membranes.

Quantitation of metabolic compartmentation in hyperammonemic brain by natural abundance 13C-NMR detection of 13C-15N coupling patterns and isotopic shifts.

In the present study, the removal of cerebral ammonia by glutamine synthetase (GS) and by reductive amination of 2-oxoglutarate by glutamate dehydrogenase in the presence of an amino donor group, was determined in hyperammonemic rabbit brains. The 15N enrichments of brain metabolite alpha-amino and amide positions of glutamine, glutamate, and alanine were determined by the indirect detection of 15N-labeled compounds of the 13C-15N spin coupling patterns of natural abundance 13C-NMR spectra. The 13C-NMR spectra of brain extracts were obtained from rabbits infused with 15NH4Cl with or without intraperitoneal infusion of the GS inhibitor, L-methionine DL-sulfoximine, in a reasonable acquisition time period. When 15NH4Cl was infused, [5-15N]glutamine and [2-15N]glutamine concentrations reached 5.2 mumol/100 mg protein and 3.6 mumol/100 mg protein, respectively, which indicates the relatively high activity of reductive amination of 2-oxoglutarate in the glutamate dehydrogenase reaction. The low concentration of [2-15N]glutamate, which is about 30% of that of [2-15N]glutamine obtained in this study, suggests that very little glutamine serves as a precursor of neuronal glutamate. When GS was inhibited by L-methionine DL-sulfoximine, a flux of 15NH4+ via the residual activity of GS was accompanied by an apparent increase of [2-15N]glutamate and [15N]alanine concentrations (2.9 mumol/100 mg protein and 1.8 mumol/100 mg protein, respectively). These findings and those obtained from 13C-13C isotopomer analysis (Lapidot and Gopher, 1994b) suggest that astrocytic 2-oxoglutarate is partially utilized (together with an amino group donor) as a precursor for neuronal glutamate in the hyperammonemic brain when GS is inhibited. This process can partly replace GS activity in metabolizing ammonia in the hyperammonemic rabbit brain.

Identification and localization of two distinct microenvironments for the diacylglycerol component of lipophorin particles by 13C NMR

13C nuclear magnetic resonance spectroscopy of lipoproteins, isolated from the insect Manduca sexta, has been employed to probe the microenvironment of diacylglycerol (DG), their major neutral lipid component. Natural abundance 13C NMR spectra of high density lipophorin exhibited several well-separated resonances derived from its lipid moiety, including those for the carbonyl carbon atoms of phospholipid and DG fatty acyl chains in the region of 175-180 ppm. To verify the assignment of the DG acyl chain carbonyl carbon resonances, di[1-13C]oleoylglycerol high density lipophorin was isolated after instilling a bolus of tri[1-13C]oleoylglycerol into the midgut of larvae fed a fat-free diet. 13C NMR spectra of the isolated lipoprotein revealed a specific and dramatic enrichment of resonances at 175.5 ppm. Expansion of this region revealed two resonances separated by 0.08 ppm. These were assigned as 1,2- and 1,3- isomers of DG, the latter presumably arising from spontaneous acyl chain migration of 1,2-DG following lipoprotein isolation. On the basis of compositional and structural analysis of this lipoprotein, it is postulated that these DG species are localized predominantly in the hydrophobic core of the particle. By contrast, natural abundance 13C NMR spectra of the DG-rich, low density lipophorin (LDLp) subspecies revealed two additional resonances, separated by 0.2 ppm, that were tentatively assigned as 1,2- and 1,3-DG present at the surface of the particle. The verify this assignment, experiments employing phospholipase C, to convert lipophorin surface associated phospholipid into DG, were performed.(ABSTRACT TRUNCATED AT 250 WORDS)

The Conformations of 1-Thiacyclooctan-3-One Dynamic Nuclear Magnetic Resonance and Force-Field Calculation

The 1H and natural-abundance 13C-NMR spectra of 1-thiacyclooctan-3-one (1) have been measured from 25 to -100°C. Coalescence is observed in the 1H-NMR spectra of (1) at about -80°C, and attributed to ring inversion in a boat-chair conformation, which is the predominant conformation of (1). The free energy of activation (DG¹) for this process is 9.2±0.2 kcal/mol. The 13C-NMR spectra of (1) is temperature independent.

13C-NMR of the deoxyribose sugars in four DNA oligonucleotide duplexes: assignment and structural features.

Natural-abundance 13C-NMR spectra have been obtained for four self-complementary DNA oligonucleotides: [d(TAGCGCTA)]2, [d(GGTATACC)]2, [d(CG)3]2, and [d(TCGCG)]2; this paper focuses on the deoxyribose resonances. Assignments were made by a combination of the two-dimensional proton-detected heteronuclear correlation experiment and comparison of 1D spectra, accounting for 31P coupling, base composition, and similarities in chemical shift versus temperature profiles (delta vs T). Large shielding and deshielding of the sugar resonances (between 2.0 and -1.9 ppm) are observed upon thermal dissociation of the duplex. The shapes of the delta vs T profiles correlate strongly with the purine/pyrimidine nature of the base attached at C1' in these duplexes that have a substantial fraction of residues within alternating purine-pyrimidine sequences. The correlation is primarily associated with changes in the equilibrium distribution of furanose pseudorotational states that may arise in part from the relief of interstrand purine-purine steric clashes.

13C-NMR relaxation in three DNA oligonucleotide duplexes: model-free analysis of internal and overall motion.

Natural-abundance 13C-NMR spectra of [d(TCGCG)] (1), [d(CGCGCG)]2 (2), and [d(GGTATACC)]2 (3) were measured at 90.6 MHz to obtain 13C-1H NOEs and T1 relaxation times; relaxation data were also measured at 125.7 MHz for 1 and 2 and at 62.9 MHz for 1. Analysis of the relaxation data was performed in the context of the "model-free" approach of Lipari and Szabo [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559], leading to the following conclusions: (i) Optimized values for the overall correlation times of 0.9 ns for 1 and 1.4 ns for 2 are close to those predicted by light-scattering results on similar molecules [Eimer et al. (1990) Biochemistry 29, 799-811]. (ii) For the nonterminal residues, the "order parameter", S2, is around 0.8 for the protonated base carbons and 0.6 for the sugar carbons, indicating less spatial restriction on the sugar carbons (in the model-free approach, the order parameter is 1 for a rigid body and 0 for a system with completely unrestricted internal motion). (iii) The order parameters for the terminal residues vary over a wide range with the smallest values around 0.2-0.3 for the HO-13C5' and the 13C3'-OH; rational trends are seen in the variation of S2 with chain position in the terminal residues. (iv) The analysis shows that the order parameters are accurate within 15%. (v) The "effective internal correlation time", tau e, is very short for the sugar carbons (30-300 ps) and less well-defined, but probably also short, for the bases. (vi) The analysis indicates that most of the relaxation in DNA is accounted for by S2 and the tau e is so short that a good approximation to any relaxation property, P (e.g., T1, T2, 13C-1H NOE, 1H-1H cross-relaxation rate), is P = S2Prigid, where Prigid is the value for the property in a system without internal motion (the analysis assumes the same isotropic overall motion for both the rigid and flexible bodies).

Localized 13C NMR spectroscopy of myo-inositol in the human brain in vivo.

Natural abundance 13C NMR spectra obtained from 144-cm3 volumes in the human brain contained well-resolved resonances of myo-inositol after 60 min of data accumulation. A mean concentration of 7.2 +/- 0.5 mumol/g (+/- SE, n = 7) was calculated from the comparison with phantoms. 13C NMR spectroscopy thus provides a complementary role in the quantitation of metabolites also observed in the crowded 1H spectrum.

Definitive assignment of the 13C NMR signals of the indole C‐3 and C‐3a carbons of the ajmaline, picraline and sarpagine alkaloids

AbstractComparison of a 1H coupled natural abundance 13C NMR spectrum of 1‐oxo‐1,2,3,4‐tetrahydrocarbazole with a single‐frequency experiment in which the hydrogens three bonds away from C‐3 (indole numbering) were irradiated allowed the unambiguous distinction of the C‐3 and C‐3a signals. The results can be extrapolated for the assignment of the same signals in the ajmaline, picraline and sarpagine alkaloids, which also contain a carbonyl group as the substituent at the C‐2 position of the indole skeleton. The effect of this carbonyl group on the chemical shifts of the benzenoid carbons is also discussed.

13C NMR of the bases of three DNA oligonucleotide duplexes: assignment methods and structural features.

Natural abundance 13C NMR spectra of three DNA oligomers have been obtained. Most of the base resonances are well resolved from one another. A combination of two independent methods was used in making assignments: a one-dimensional spectral comparison method and a two-dimensional proton-detected 1H-13C correlated experiment for the protonated carbons. There are large shielding changes (between 1.62 and -1.40 ppm) upon thermal dissociation of the duplex. The shapes of the chemical shift vs temperature curves are largely independent of sequence. The base carbon resonance frequencies are sensitive to hydrogen bonding, base stacking, sugar conformation, and changes in the glycosyl torsion angle.

ChemInform Abstract: 13C NMR Spectra of α,α′‐Tetracyanosubstituted Platinacyclobutanes.

Abstract The natural abundance 13C NMR spectra of some bis-(triphenylphosphino)tetracyano substituted platinacyclobutanes are described. Coupling constants and chemical shifts are compared with the available literature data of similar compounds. A correlation is found between C Pt C, P Pt P angles and J(PtCα) coupling constants. The influence on NMR parameters of the nature of the ancillary ligands, the ring size and substitution on α carbons, is discussed.

Differentiation of human tumors from nonmalignant tissue by natural‐abundance 13C NMR spectroscopy

High-quality, high-resolution, proton-decoupled natural-abundance 13C NMR spectra have been obtained in vitro at 100.6 MHz from unprocessed human pathology specimens of tumors and adjacent nonneoplastic control tissues from lung, colon, and prostate. In these preliminary studies, specific molecular parameters were identified from the spectra that distinguished neoplastic from nonneoplastic tissue of a given organ in all sites studied. The NMR results were congruent with data derived from histochemical and biochemical examinations of the tissues and with previous studies using non-NMR methods. In particular, a comparison of the spectra of prostatic adenocarcinoma with that of adjacent hyperplastic tissue revealed the following differences: The tumors contained (1) larger amounts of triacylglycerols, (2) smaller amounts of citrate, and (3) acidic mucins. These transformation-associated deviations from the normally high amounts of citrate and low amounts of lipids in the prostate are consistent with an alteration in either the concentration or the activity of ATP-citrate lyase in the tumors. The 13C NMR spectra of colonic adenocarcinoma tissue showed that this tumor type contained (1) smaller signals from triacylglycerols, (2) larger signals from phospholipids and lactate, and (3) decreased lipid fatty acyl chain saturation, when compared to spectra from adjacent normal colon. Colloid carcinoma, another variant of colonic carcinoma, showed prominent 13C resonances from glycoproteins, which were absent from the spectra of normal colon, and from spectra from the more common pattern of colonic adenocarcinoma. Smaller 13C NMR signals from mucins and other proteins, and the presence of triacylglycerol signals distinguished poorly differentiated lung carcinoma and from nonmalignant lung tissue. These results indicate that natural-abundance 13C NMR spectroscopy may constitute a unique, nondestructive method, for the simultaneous measurement of a large number of tissue metabolites and structural components of significance to the study and diagnosis of a wide range of human tumors.

High-resolution 13C NMR study of pressure effects on the main phase transition in L-alpha-dipalmitoyl phosphatidylcholine vesicles.

The effects of pressure on the liquid-crystalline to gel transition in vesicles of L-alpha-dipalmitoyl phosphatidylcholine were investigated by high-resolution proton-decoupled natural-abundance 13C NMR spectroscopy. The linewidths of several 13C resonances, including the choline methyl groups, carbonyl carbons, and choline methylene groups and the palmitoyl methyl groups are reported as a function of pressure at 52.7 degrees C. These preliminary NMR experiments clearly demonstrate that high-pressure, high-resolution proton-decoupled natural-abundance 13C NMR spectra are a promising tool to study the phase-transition behavior and the dynamics of model membrane systems.

13C NMR spectra of α,α′-tetracyanosubstituted platinacyclobutanes

The natural abundance 13C NMR spectra of some bis-(triphenylphosphino)tetracyano substituted platinacyclobutanes are described. Coupling constants and chemical shifts are compared with the available literature data of similar compounds. A correlation is found between C Pt C, P Pt P angles and J(PtCα) coupling constants. The influence on NMR parameters of the nature of the ancillary ligands, the ring size and substitution on α carbons, is discussed.

Detection of human muscle glycogen by natural abundance 13C NMR.

Natural abundance 13C nuclear magnetic resonance spectroscopy was used to detect signals from glycogen in the human gastrocnemius muscle. The reproducibility of the measurement was demonstrated, and the ability to detect dynamic changes was confirmed by measuring a decrease in muscle glycogen levels after exercise and its subsequent repletion. Single frequency gated 1H decoupling was used to obtain decoupled natural abundance 13C NMR spectra of the C-1 position of muscle glycogen.

Natural abundance 13C-NMR study of paramagnetic horse heart ferricytochrome c cyanide complex: Assignment of hyperfine shifted heme methyl carbon resonances

Hyperfine shifted heme methyl carbon resonances of paramagnetic horse heart ferricytochrome c cyanide complex (Cyt-c(CN)) have been observed for the first time in the natural abundance 13C-NMR spectrum and assigned using 1H- 13C heteronuclear chemical shift correlated spectroscopy ( 1H- 13C COSY). Individual heme methyl carbon NMR signal assignment permits a direct comparison between the hyperfine shifts of heme methyl carbon and attached methyl proton resonances which provides a useful information on the delocalization mechanism of the unpaired spin from the π-conjugated system of porphyrin ring into the peripheral methyl side chains.