13C NMR spectral analysis of some isoquinoline alkaloids

The ¹³C NMR spectra of some isoquinoline and tetrahydroisoquinoline alkaloids and their corresponding N-methosalts and of the bisbenzylisoquinoline alkaloid isochondodendrine were recorded and the signals assigned. The substituent shielding effects and the ¹³C¹H long range couplings were analysed and utilized in the spectral interpretation.     

13C NMR spectral and conformational analysis of 8-O-4′ neolignans

The ¹³C NMR spectra of the erythro and threo forms of representative members of the 8-O-4′ type of neolignans were recorded and the signals assigned. Based on these assignments and on the comparison with previously reported ¹H NMR data, the most probable conformations for the above mentioned stereoisomers are suggested.     

Structures of chlorosomes and aggregated BChlc inChlorobium tepidum from solid state high resolution CP/MAS13C NMR

Cross polarization/magic angle spinning (CP/MAS)¹³C (solid state high resolution) NMR spectra were observed for chlorosomes and BChlc aggregates. Similarity of both kinds of spectra (except for some signals assignable to proteins and lipids in chlorosomes) indicates that BChlc's in chlorosomes are present just as in synthetic BChlc aggregates. Chemical shifts for C13¹ carbonyl and C3¹ hydroxylethyl carbons indicate hydrogen bonding between them. Comparison of solution and solid state¹³C NMR chemical shifts shows the five coordinated nature of BChlc aggregates. Some chemical shift differences were attributable to ring currents shifts. Their comparisons with calculated ring current shift values predicted structures for the aggregates. Cross polarization dynamics of the CP/MAS¹³C NMR signals explored dynamic and structural nature of the BChlc aggregates.     

13C NMR analysis of aporphine alkaloids

The ¹³C NMR spectra of some tertiary and quaternary aporphine alkaloids are recorded and the signals assigned. The substituent shielding effects together with the effects of N- and O-methylation, and the twisting of the biphenyl system, are analysed and utilized in the spectral interpretation.     

13C NMR and IR analyses of structure,aging and botanical origin of Dominican and Mexican ambers

¹³C NMR analysis of Oligocene amber from the Dominican Republic, Oligo-Miocene amber from Mexico and trunk resins from certain extant species of Hymenaea, suggests a polylabdane structure for all and gives insights into structural changes during aging. Additionally, IR spectra, some aspects of the ¹³C NMR spectra and paleobotanical data suggest a close relationship between Dominican amber and H. verrucosa. By contrast, spectral evidence shows that Mexican amber differs, not only from the former polymers, but from the H. courbaril polymer.     

Fungal macrolides: Structure determination and biosynthesis of achaetolide,a lactone from Achaetomium cristalliferum

The structure of a new ten-membered lactone, achaetolide, isolated from cultures of Achaetomium cristalliferum is deduced from its mass and NMR spectra and from the study ofsomederivatives. The ¹³C NMR spectra of achaetolide enriched with [1-¹³C], [2-¹³C] and [1, 2-¹³C] acetate established its formation from eight intact acetate units via a precursor octaketide chain.     

1H NMR Spectroscopy of pyrrolizidine alkaloids

The ¹H NMR spectra of three pyrrolizidine alkaloids of the macrocyclic diester type, retrorsine, seneciphylline and senecionine, plus their three N-oxides have been assigned. Previous ¹H NMR studies of these pyrrolizidine alkaloids have stressed the difficulties of spectral intrepretation. The results reported here will provide a useful resource for analysis of tertiary structure in these and related compounds.     

One simple step in the identification of the cofactors signals, one giant leap for the solution structure determination of multiheme proteins

Multiheme proteins play major roles in various biological systems. Structural information on these systems in solution is crucial to understand their functional mechanisms. However, the presence of numerous proton-containing groups in the heme cofactors and the magnetic properties of the heme iron, in particular in the oxidised state, complicates significantly the assignment of the NMR signals. Consequently, the multiheme proteins superfamily is extremely under-represented in structural databases, which constitutes a severe bottleneck in the elucidation of their structural-functional relationships. In this work, we present a strategy that simplifies the assignment of the NMR signals in multiheme proteins and, concomitantly, their solution structure determination, using the triheme cytochrome PpcA from the bacterium Geobacter sulfurreducens as a model. Cost-effective isotopic labeling was used to double label (¹³C/¹⁵N) the protein in its polypeptide chain, with the correct folding and heme post-translational modifications. The combined analysis of ¹H-¹³C HSQC NMR spectra obtained for labeled and unlabeled samples of PpcA allowed a straight discrimination between the heme cofactors and the polypeptide chain signals and their confident assignment. The results presented here will be the foundations to assist solution structure determination of multiheme proteins, which are still very scarce in the literature.     

The core of Ure2p prion fibrils is formed by the N-terminal segment in a parallel cross-β structure: evidence from solid-state NMR

Intracellular fibril formation by Ure2p produces the non-Mendelian genetic element [URE3] in Saccharomyces cerevisiae, making Ure2p a prion protein. We show that solid-state NMR spectra of full-length Ure2p fibrils, seeded with infectious prions from a specific [URE3] strain and labeled with uniformly ¹⁵N-¹³C-enriched Ile, include strong, sharp signals from Ile residues in the globular C-terminal domain (CTD) with both helical and nonhelical ¹³C chemical shifts. Treatment with proteinase K eliminates these CTD signals, leaving only nonhelical signals from the Gln-rich and Asn-rich N-terminal segment, which are also observed in the solid-state NMR spectra of Ile-labeled fibrils formed by residues 1-89 of Ure2p. Thus, the N-terminal segment, or “prion domain” (PD), forms the fibril core, while CTD units are located outside the core. We additionally show that, after proteinase K treatment, Ile-labeled Ure2p fibrils formed without prion seeding exhibit a broader set of solid-state NMR signals than do prion-seeded fibrils, consistent with the idea that structural variations within the PD core account for prion strains. Measurements of ¹³C-¹³C magnetic dipole-dipole couplings among ¹³C-labeled Ile carbonyl sites in full-length Ure2p fibrils support an in-register parallel β-sheet structure for the PD core of Ure2p fibrils. Finally, we show that a model in which CTD units are attached rigidly to the parallel β-sheet core is consistent with steric constraints.     

Lignans of Araucaria angustifolia and 13C NMR analysis of some phenyltetralin lignans

Secoisolariciresinol monomethyl ether and lariciresinol-4-methyl ether were isolated from the knots of dead trees of Araucaria angustifolia. On the basis of spectral evidence, the position of the OH group was located in these compounds. The ¹³C NMR spectra of the phenyltetralin lignans galbulin, galcatin, isogalcatin and cyclogalgravin have also been recorded and the signals assigned, based on the methyl shifts of cyclogalgravin.     

13C NMR spectral analysis of lignans from Araucaria angustifolia

Pinoresinol dimethyl ether, secoisolariciresinol, lariciresinol, isolariciresinol and isolariciresinol-4′-methyl ether were isolated from the knots of dead trees of Araucaria angustifolia. The ¹³C NMR spectra of these compounds, their methyl and acetyl derivatives, and the corresponding one of matairesinol, have been recorded and the signals assigned. On the basis of these assignments, the structure of the new monomethyl ether of isolariciresinol has been established.     

Detection and characterization of serine and threonine hydroxyl protons in Bacillus circulans xylanase by NMR spectroscopy

Hydroxyl protons on serine and threonine residues are not well characterized in protein structures determined by both NMR spectroscopy and X-ray crystallography. In the case of NMR spectroscopy, this is in large part because hydroxyl proton signals are usually hidden under crowded regions of ¹H-NMR spectra and remain undetected by conventional heteronuclear correlation approaches that rely on strong one-bond ¹H–¹⁵N or ¹H–¹³C couplings. However, by filtering against protons directly bonded to ¹³C or ¹⁵N nuclei, signals from slowly-exchanging hydroxyls can be observed in the ¹H-NMR spectrum of a uniformly ¹³C/¹⁵N-labeled protein. Here we demonstrate the use of a simple selective labeling scheme in combination with long-range heteronuclear scalar correlation experiments as an easy and relatively inexpensive way to detect and assign these hydroxyl proton signals. Using auxtrophic Escherichia coli strains, we produced Bacillus circulans xylanase (BcX) labeled with ¹³C/¹⁵N-serine or ¹³C/¹⁵N-threonine. Signals from two serine and three threonine hydroxyls in these protein samples were readily observed via ³J_C–OH couplings in long-range ¹³C-HSQC spectra. These scalar couplings (~5–7 Hz) were measured in a sample of uniformly ¹³C/¹⁵N-labeled BcX using a quantitative ¹³C/¹⁵N-filtered spin-echo difference experiment. In a similar approach, the threonine and serine hydroxyl hydrogen exchange kinetics were measured using a ¹³C/¹⁵N-filtered CLEANEX-PM pulse sequence. Collectively, these experiments provide insights into the structural and dynamic properties of several serine and threonine hydroxyls within this model protein.     

Characterization of uniformly and atom-specifically C-labeled heparin and heparan sulfate polysaccharide precursors using C NMR spectroscopy and ESI mass spectrometry

The biological actions of heparin and heparan sulfate, two structurally related glycosaminoglycans, depend on the organization of the complex heparanome. Due to the structural complexity of the heparanome, the sequence of variably sulfonated uronic acid and glucosamine residues is usually characterized by the analysis of smaller oligosaccharide and disaccharide fragments. Even characterization of smaller heparin and heparan sulfate oligosaccharide or disaccharide fragments using simple 1D ¹H NMR spectroscopy is often complicated by the extensive signal overlap. ¹³C NMR signals, on the other hand, overlap less and therefore, ¹³C NMR spectroscopy can greatly facilitate the structural elucidation of the complex heparanome and provide finer insights into the structural basis for biological functions. This is the first report of the preparation of anomeric carbon-specific ¹³C-labeled heparin and heparan sulfate precursors from the Escherichia coli K5 strain. Uniformly ¹³C- and ¹⁵N-labeled precursors were also produced and characterized by ¹³C NMR spectroscopy. Mass spectrometric analysis of enzymatically fragmented disaccharides revealed that anomeric carbon-specific labeling efforts resulted in a minor loss/scrambling of ¹³C in the precursor backbone, whereas uniform labeling efforts resulted in greater than 95% ¹³C isotope enrichment in the precursor backbone. These labeled precursors provided high-resolution NMR signals with great sensitivity and set the stage for studying the heparanome-proteome interactions.     

13C NMR des alcaloïdes des Strychnos: Les dérivés de l'harmane et de l'usambarensine

¹³C NMR spectra of some tertiary and quaternary indole alkaloids are recorded and the signals assigned. Graphic interpretation of off-resonance spectra and substituent shielding effects together with the effect of N_b-methylation are utilized in the spectral interpretation.     

The incorporation of ornithine-[2,3-13C2] into nicotine and nornicotine established by NMR

dl-Ornithine-[2,3-¹³C₂] was synthesized from acetate-[1-¹³C] and ethyl acetamidocyanoacetate-[2-¹³C]. This labelled material was mixed with dl-ornithine-[5-¹⁴C] and fed to Nicotiana glutinosa plants by the wick method. After 10 days the plants were harvested affording radioactive nicotine and nornicotine (0.14% and 0.051% specific incorporations, respectively). Even at these low specific incorporations an examination of their ¹³C NMR spectra established the incorporation of ornithine symmetrically into the pyrrolidine rings of these alkaloids. Satellites were observable at the signals due to C-2′, 3′, 4′ and 5′ positions, arising by the presence of contiguous carbons at C-2′, 3′ and C-4′, 5′.     

Secondary structural analysis of proteins based on 13C chemical shift assignments in unresolved solid-state NMR spectra enhanced by fragmented structure database

Magic-angle-spinning solid-state ¹³C NMR spectroscopy is useful for structural analysis of non-crystalline proteins. However, the signal assignments and structural analysis are often hampered by the signal overlaps primarily due to minor structural heterogeneities, especially for uniformly-¹³C,¹⁵N labeled samples. To overcome this problem, we present a method for assigning ¹³C chemical shifts and secondary structures from unresolved two-dimensional ¹³C–¹³C MAS NMR spectra by spectral fitting, named reconstruction of spectra using protein local structures (RESPLS). The spectral fitting was conducted using databases of protein fragmented structures related to ¹³C^α, ¹³C^β, and ¹³C′ chemical shifts and cross-peak intensities. The experimental ¹³C–¹³C inter- and intra-residue correlation spectra of uniformly isotope-labeled ubiquitin in the lyophilized state had a few broad peaks. The fitting analysis for these spectra provided sequence-specific C^α, C^β, and C′ chemical shifts with an accuracy of about 1.5 ppm, which enabled the assignment of the secondary structures with an accuracy of 79 %. The structural heterogeneity of the lyophilized ubiquitin is revealed from the results. Test of RESPLS analysis for simulated spectra of five different types of proteins indicated that the method allowed the secondary structure determination with accuracy of about 80 % for the 50–200 residue proteins. These results demonstrate that the RESPLS approach expands the applicability of the NMR to non-crystalline proteins exhibiting unresolved ¹³C NMR spectra, such as lyophilized proteins, amyloids, membrane proteins and proteins in living cells.     

Isolation and detailed 1H and 13C NMR structural assignment for three trachylobanes from Psiadia punctulata (Asteraceae) grown in Africa

The first isolation of a trachylobane from an African specimen of Psiadia punctulata (Asteraceae) is presented in this paper. A complete ¹H and ¹³C NMR spectral analysis of this compound and two other trachylobane diterpenes, previously isolated from the same plant, are also provided. The use of NMR techniques such as gCOSY, gHSQC, gHMBC and 2D-J-resolved, in combination with a software-assisted methodology, led to a complete and unequivocal assignment of ¹H and ¹³C signals. This was achieved together with the measurement of all homonuclear hydrogen coupling constants. The presented detail level of the assignment data has never been published before for trachylobanes. Furthermore, with all determined NMR experimental data from the spectra and to obtain a reliability assessment, signals were simulated in the FOMSC3 and NMR_MultSim software.     

Differences in lysine pKa values may be used to improve NMR signal dispersion in reductively methylated proteins

Reductive methylation of lysine residues in proteins offers a way to introduce ¹³C methyl groups into otherwise unlabeled molecules. The ¹³C methyl groups on lysines possess favorable relaxation properties that allow highly sensitive NMR signal detection. One of the major limitations in the use of reductive methylation in NMR is the signal overlap of ¹³C methyl groups in NMR spectra. Here we show that the uniform influence of the solvent on chemical shifts of exposed lysine methyl groups could be overcome by adjusting the pH of the buffering solution closer to the pKa of lysine side chains. Under these conditions, due to variable pKa values of individual lysine side chains in the protein of interest different levels of lysine protonation are observed. These differences are reflected in the chemical shift differences of methyl groups in reductively methylated lysines. We show that this approach is successful in four different proteins including Ca²⁺-bound Calmodulin, Lysozyme, Ca²⁺-bound Troponin C, and Glutathione S-Transferase. In all cases significant improvement in NMR spectral resolution of methyl signals in reductively methylated proteins was obtained. The increased spectral resolution helps with more precise characterization of protein structural rearrangements caused by ligand binding as shown by studying binding of Calmodulin antagonist trifluoperazine to Calmodulin. Thus, this approach may be used to increase resolution in NMR spectra of ¹³C methyl groups on lysine residues in reductively methylated proteins that enhances the accuracy of protein structural assessment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

13C and31P NMR spectroscopic studies on glucose metabolism inTribolium confusum

Nuclear magnetic resonance (NMR) technology was applied to study the glucose metabolism inTribolium confusum (Coleoptera).¹³C signals of D-(1-¹³C)glucose eaten by beetles were clearly detected in such metabolites of the glucose metabolism as glycogen, trehalose, triacylglycerol, alanine and proline by¹³C-NMR. After glucose feeding the³¹P-NMR spectra ofT. confusum showed the signal intensity increases in arginine-phosphate, sugar-phosphate and uridine diphosphoglucose. The results demonstrated the potential of NMR analysis for the study of glucose metabolism inT. confusum.     

Deuterium isotope effects in carbohydrates revisited. Cryoprobe studies of the anomerization and NH to ND deuterium isotope induced 13C NMR chemical shifts of acetamidodeoxy and aminodeoxy sugars

Complete ¹H and ¹³C NMR chemical shift assignments have been generated from a series of acetamidodeoxy and aminodeoxy sugar derivatives. For free sugars, the enhanced sensitivity of an NMR cryoprobe allowed simple 1D and 2D NMR spectra to be obtained from essentially single anomers, before significant mutarotation had occurred. The NMR assignments have been used to characterize deuterium isotope effects on ¹³C chemical shifts measured under conditions of slow NH to ND exchange in single solutions. Within a range of 0 to −0.138 ppm, β, γ, δ, and ζ deuterium isotope effects have been observed, thus providing additional reference data for assignment of the ¹³C NMR spectra of nitrogenous saccharides.