The first in vivo observation of (13)C-(15)N coupling in mammalian brain.

[5-(13)C,(15)N]Glutamine, with (1)J((13)C-(15)N) of 16 Hz, was observed in vivo in the brain of spontaneously breathing rats by (13)C MRS at 4.7 T. The brain [5-(13)C]glutamine peak consisted of the doublet from [5-(13)C,(15)N]glutamine and the center [5-(13)C,(14)N]glutamine peak, resulting in an apparent triplet with a separation of 8 Hz. The time course of formation of brain [5-(13)C,(15)N]glutamine was monitored in vivo with a time resolution of 20-35 min. This [5-(13)C,(15)N]glutamine was formed by glial uptake of released neurotransmitter [5-(13)C]glutamate and its reaction with (15)NH(3) catalyzed by the glia-specific glutamine synthetase. The neurotransmitter glutamate C5 was selectively (13)C-enriched by intravenous [2,5-(13)C]glucose infusion to (13)C-label whole-brain glutamate C5, followed by [(12)C]glucose infusion to chase (13)C from the small and rapidly turning-over glial glutamate pool, leaving (13)C mainly in the neurotransmitter [5-(13)C]glutamate pool, which is sequestered in vesicles until release. Hence, the observed [5-(13)C,(15)N]glutamine arises from a coupling between (13)C of neuronal origin and (15)N of glial origin. Measurement of the rate of brain [5-(13)C,(15)N]glutamine formation provides a novel noninvasive method of studying the kinetics of neurotransmitter uptake into glia in vivo, a process that is crucial for protecting the brain from glutamate excitotoxicity.     

The First in Vivo Observation of C–N Coupling in Mammalian Brain

[5-¹³C,¹⁵N]Glutamine, with ¹J(¹³C–¹⁵N) of 16 Hz, was observed in vivo in the brain of spontaneously breathing rats by ¹³C MRS at 4.7 T. The brain [5-¹³C]glutamine peak consisted of the doublet from [5-¹³C,¹⁵N]glutamine and the center [5-¹³C,¹⁴N]glutamine peak, resulting in an apparent triplet with a separation of 8 Hz. The time course of formation of brain [5-¹³C,¹⁵N]glutamine was monitored in vivo with a time resolution of 20–35 min. This [5-¹³C,¹⁵N]glutamine was formed by glial uptake of released neurotransmitter [5-¹³C]glutamate and its reaction with ¹⁵NH₃ catalyzed by the glia-specific glutamine synthetase. The neurotransmitter glutamate C5 was selectively¹³C-enriched by intravenous [2,5-¹³C]glucose infusion to ¹³C-label whole-brain glutamate C5, followed by [¹²C]glucose infusion to chase ¹³C from the small and rapidly turning-over glial glutamate pool, leaving ¹³C mainly in the neurotransmitter [5-¹³C]glutamate pool, which is sequestered in vesicles until release. Hence, the observed [5-¹³C,¹⁵N]glutamine arises from a coupling between ¹³C of neuronal origin and ¹⁵N of glial origin. Measurement of the rate of brain [5-¹³C,¹⁵N]glutamine formation provides a novel noninvasive method of studying the kinetics of neurotransmitter uptake into glia in vivo, a process that is crucial for protecting the brain from glutamate excitotoxicity.     

Novel peak assignments of in vivo (13)C MRS in human brain at 1.5 T

(13)C MRS studies at natural abundance and after intravenous 1-(13)C glucose infusion were performed on a 1.5-T clinical scanner in four subjects. Localization to the occipital cortex was achieved by a surface coil. In natural abundance spectra glucose C(3beta,5beta), myo-inositol, glutamate C(1,2,5), glutamine C(1,2,5), N-acetyl-aspartate C(1-4,C=O), creatine CH(2), CH(3), and C(C=N), taurine C(2,3), bicarbonate HCO(-)(3) were identified. After glucose infusion (13)C enrichment of glucose C(1alpha,1beta), glutamate C(1-4), glutamine C(1-4), aspartate C(2,3), N-acetyl-aspartate C(2,3), lactate C(3), alanine C(3), and HCO(-)(3) were observed. The observation of (13)C enrichment of resonances resonating at >150 ppm is an extension of previously published studies and will provide a more precise determination of metabolic rates and substrate decarboxylation in human brain.     

Observation of nitrogen-14, carbon-13 indirect spin-spin coupling in high-resolution 13C CP/MAS spectra of solids.

The 13C CP/MAS NMR spectrum of [(n-C3H7)4N][Cd(SCN)3], 1, indicates the presence of three non-equivalent thiocyanate ligands, in agreement with the results of a recent single-crystal X-ray diffraction study. Examination of the 13C MAS line shapes allows direct measurement of the indirect spin-spin coupling constants, 1J(14N, 13C) = 16 +/- 1 Hz and 2J(111/113Cd, 13C) = 75 +/- 5 Hz, for the unique N-bonded thiocyanate ligand. This is the first reported measurement of 1J(14N, 13C) and 2J(111/113Cd, 13C) in the solid state. Possible reasons for the failure to observe 1J(14N, 13C) values in previous high-resolution 13C CP/MAS NMR studies are summarized.     

13C NMR Chemical Shifts of Heterocycles: Empirical Substituent Effects in 5-Halomethylisoxazoles

Evaluation by empirically derived equations for the Substituent effect (α, β, γ, δ) on the ¹³C NMR chemical shifts for C-3, C-4. C-5 and halomethyl-substituent carbon (C-6) in isoxazoles 1-5 [where C-3 substituent (R¹) = H, alkyl or phenyl, C-4 Substituent (R²) = H, alkyl, and C-5 substituent (R³) = di-or trihalomethyl, methyl and H], taking as reference the compound la, is reported. From the calculated values for the α, β, γ, δ effects for each substituent it was possible to estimate the chemical shift of each carbon of the compounds 1–5. The ¹³ C chemical shifts of the C-3, C-4, C-5, C-6 of these compounds, can be estimated with good precision: 94% of the calculated chemical shifts are found to be within ±1.0ppm, and 100% are found to be within ±1.5ppm.     

Novel Peak Assignments of in VivoC MRS in Human Brain at 1.5 T

¹³C MRS studies at natural abundance and after intravenous 1-¹³C glucose infusion were performed on a 1.5-T clinical scanner in four subjects. Localization to the occipital cortex was achieved by a surface coil. In natural abundance spectra glucose C_3β,5β, myo-inositol, glutamate C_1,2,5, glutamine C_1,2,5, N-acetyl-aspartate C_1-4,C=O, creatine CH₂, CH₃, and C_C=N, taurine C_2,3, bicarbonate HCO⁻₃ were identified. After glucose infusion ¹³C enrichment of glucose C_1α,1β, glutamate C_1-4, glutamine C_1-4, aspartate C_2,3, N-acetyl-aspartate C_2,3, lactate C₃, alanine C₃, and HCO⁻₃ were observed. The observation of ¹³C enrichment of resonances resonating at >150 ppm is an extension of previously published studies and will provide a more precise determination of metabolic rates and substrate decarboxylation in human brain.     

Insight into the CSA tensors of nucleobase carbons in RNA polynucleotides from solution measurements of residual CSA: towards new long-range orientational constraints

Using residual chemical shift anisotropies (RCSAs) measured in a weakly aligned stem-loop RNA, we examined the carbon chemical shift anisotropy (CSA) tensors of nucleobase adenine C2, pyrimidine C5 and C6, and purine C8. The differences between the measured RCSAs and the values back-calculated using three nucleobase carbon CSA sets [D. Stueber, D.M. Grant, 13C and 15N chemical shift tensors in adenosine, guanosine dihydrate, 2'-deoxythymidine, and cytidine, J. Am. Chem. Soc. 124 (2002) 10539-10551; D. Sitkoff, D.A. Case, Theories of chemical shift anisotropies in proteins and nucleic acids, Prog. NMR Spectrosc. 32 (1998) 165-190; R. Fiala, J. Czernek, V. Sklenar, Transverse relaxation optimized triple-resonance NMR experiments for nucleic acids, J. Biomol. NMR 16 (2000) 291-302] reported previously for mononucleotides (1.4 Hz) is significantly smaller than the predicted RCSA range (-10-10 Hz) but remains larger than the RCSA measurement uncertainty (0.8 Hz). Fitting of the traceless principal CSA values to the measured RCSAs using a grid search procedure yields a cytosine C5 CSA magnitude (CSAa=(3/2.(delta11(2)+delta22(2)+delta33(2)))1/2=173+/-21 ppm), which is significantly higher than the reported mononucleotide values (131-138 ppm) and a guanine C8 CSAa (148+/-13 ppm) that is in very good agreement with the mononucleotide value reported by solid-state NMR [134 ppm, D. Stueber, D.M. Grant, 13C and (15)N chemical shift tensors in adenosine, guanosine dihydrate, 2'-deoxythymidine, and cytidine, J. Am. Chem. Soc. 124 (2002) 10539-10551]. Owing to a unique sensitivity to directions normal to the base plane, the RCSAs can be translated into useful long-range orientational constraints for RNA structure determination even after allowing for substantial uncertainty in the nucleobase carbon CSA tensors.     

In Vivo3D LocalizedC Spectroscopy Using Modified INEPT and DEPT

The 3D localized¹³C spectroscopy methods LINEPT and LODEPT, which are modifications of INEPT and DEPT, are proposed. As long as a¹³C inversion pulse (180-degree pulse) is applied at 1/(4J) before the proton echo time in LINEPT and a¹³C excitation pulse (90-degree pulse) is applied at 1/(2J) before the proton echo time in LODEPT, the proton echo time can be set to any value longer than 1/(2J) in LINEPT and longer than 1/Jin LODEPT. As a result, the proton and the¹³C pulses can be applied separately and these proton pulses can be made slice-selective pulses. These localization features of LINEPT and LODEPT were evaluated using a phantom consisting of a cylinder filled with ethanol placed inside another cylinder filled with oil, and localized ethanol spectra could be obtained.In vivo3D localized¹³C spectra from the brain of a monkey could be obtained using decoupled LINEPT, and glutamate C-4 appeared directly after the administration of glucose C-1, followed by the appearance of glutamate C-2, C-3 and glutamine C-2, C-3, C-4.     

Sensitivity enhancement, assignment, and distance measurement in 13C solid-state NMR spectroscopy for paramagnetic systems under fast magic angle spinning

Despite success of previous studies, high-resolution solid-state NMR (SSNMR) of paramagnetic systems has been still largely unexplored because of limited sensitivity/resolution and difficulty in assignment due to large paramagnetic shifts. Recently, we demonstrated that an approach using very-fast magic angle spinning (VFMAS; spinning speed 20kHz) enhances resolution/sensitivity in (13)C SSNMR for paramagnetic complexes [Y. Ishii, S. Chimon, N.P. Wickramasinghe, A new approach in 1D and 2D (13)C high resolution solid-state NMR spectroscopy of paramagnetic organometallic complexes by very fast magic-angle spinning, J. Am. Chem. Soc. 125 (2003) 3438-3439]. In this study, we present a new strategy for sensitivity enhancement, signal assignment, and distance measurement in (13)C SSNMR under VFMAS for unlabeled paramagnetic complexes using recoupling-based polarization transfer. As a robust alternative of cross-polarization (CP), rapid application of recoupling-based polarization transfer under VFMAS is proposed. In the present approach, a dipolar-based analog of INEPT (dipolar INEPT) methods is used for polarization transfer and a (13)C signal is observed under VFMAS without (1)H decoupling. The resulting low duty factor permits rapid signal accumulation without probe arcing at recycle times ( approximately 3 ms/scan) matched to short (1)H T(1) values of small paramagnetic systems ( approximately 1 ms). Experiments on Cu(dl-Ala)(2) showed that the fast repetition approach under VFMAS provided sensitivity enhancement by a factor of 8-66 for a given sample, compared with the (13)C MAS spectrum under moderate MAS at 5kHz. The applicability of this approach was also demonstrated for a more challenging system, Mn(acac)(3), for which (13)C and (1)H paramagnetic shift dispersions reach 1500 and 700 ppm, respectively. It was shown that effective-evolution-time dependence of transferred signals in dipolar INEPT permitted one to distinguish (13)CH, (13)CH(2), (13)CH(3), (13)CO2- groups in 1D experiments for Cu(DL-Ala)(2) and Cu(Gly)(2). Applications of this technique to 2D (13)C/(1)H correlation NMR under VFMAS yielded reliable assignments of (1)H resonances as well as (13)C resonances for Cu(DL-Ala)(2) and Mn(acac)(3). Quantitative analysis of cross-peak intensities in 2D (13)C/(1)H correlation NMR spectra of Cu(DL-Ala)(2) provided distance information between non-bonded (13)C-(1)H pairs in the paramagnetic system.     

Simultaneous measurement of neuronal and glial metabolism in rat brain in vivo using co-infusion of [1,6-C2]glucose and [1,2-C2]acetate

In this work the feasibility of measuring neuronal-glial metabolism in rat brain in vivo using co-infusion of [1,6-¹³C₂]glucose and [1,2-¹³C₂]acetate was investigated. Time courses of ¹³C spectra were measured in vivo while infusing both ¹³C-labeled substrates simultaneously. Individual ¹³C isotopomers (singlets and multiplets observed in ¹³C spectra) were quantified automatically using LCModel. The distinct ¹³C spectral pattern observed in glutamate and glutamine directly reflected the fact that glucose was metabolized primarily in the neuronal compartment and acetate in the glial compartment. Time courses of concentration of singly and multiply-labeled isotopomers of glutamate and glutamine were obtained with a temporal resolution of 11 min. Although dynamic metabolic modeling of these ¹³C isotopomer data will require further work and is not reported here, we expect that these new data will allow more precise determination of metabolic rates as is currently possible when using either glucose or acetate as the sole ¹³C-labeled substrate.     

17O and 13C NMR Studies of Isobenzopyrylium Salts

¹³C and ¹⁷O NMR chemical shifts for a series of isobenzopyrylium salts are reported. The oxygen signal range from 300 to 270 ppm as a double bonded carboxylic oxygen, From the ¹⁷O and ¹³C data valuable informations on the conjugative and substituent effects of isobenzopyrylium salts were obtained.     

Astrocytic energy metabolism and glutamate formation--relevance for 13C-NMR spectroscopy and importance of cytosolic/mitochondrial trafficking

Glutamate plays a double role in ¹³C-nuclear magnetic resonance (NMR) spectroscopic determination of glucose metabolism in the brain. Bidirectional exchange between initially unlabeled glutamate and labeled α-ketoglutarate, formed from pyruvate via pyruvate dehydrogenase (PDH), indicates the rate of energy metabolism in the tricarboxylic acid (V_TCA) cycle in neurons (V_{PDH, n}) and, with additional computation, also in astrocytes (V_{PDH, g}), as confirmed using the astrocyte-specific substrate [¹³C]acetate. Formation of new molecules of glutamate during increased glutamatergic activity occurs only in astrocytes by combined pyruvate carboxylase (V_PC) and astrocytic PDH activity. V_{PDH, g} accounts for ∼15% of total pyruvate metabolism in the brain cortex, and V_PC accounts for another ∼10%. Since both PDH-generated and PC-generated pyruvates are needed for glutamate synthesis, ∼20/25 (80%) of astrocytic pyruvate metabolism proceed via glutamate formation. Net transmitter glutamate [γ-aminobutyric acid (GABA)] formation requires transfer of newly synthesized α-ketoglutarate to the astrocytic cytosol, α-ketoglutarate transamination to glutamate, amidation to glutamine, glutamine transfer to neurons, its hydrolysis to glutamate and glutamate release (or GABA formation). Glutamate-glutamine cycling, measured as glutamine synthesis rate (V_cycle), also transfers previously released glutamate/GABA to neurons after an initial astrocytic accumulation and measures predominantly glutamate signaling. An empirically established ∼1/1 ratio between glucose metabolism and V_cycle may reflect glucose utilization associated with oxidation/reduction processes during glutamate production, which together with associated transamination processes are balanced by subsequent glutamate oxidation after cessation of increased signaling activity. Astrocytic glutamate formation and subsequent oxidative metabolism provide large amounts of adenosine triphosphate used for accumulation from extracellular clefts of neuronally released K⁺ and glutamate and for cytosolic Ca²⁺ homeostasis.     

Combination of two fat saturation pulses improves detectability of glucose signals in carbon-13 MR spectroscopy

In order to improve the fat suppression performance of in vivo (13)C-MRS operating at 3.0 Tesla, a phantom model study was conducted using a combination of two fat suppression techniques; a set of pulses for frequency (chemical shift) selective suppression (CHESS), and spatial saturation (SAT). By optimizing the slab thickness for SAT and the irradiation bandwidth for CHESS, the signals of the -(13)CH(3) peak at 49 ppm and the -(13)CH(2)- peak at 26 ppm simulating fat components were suppressed to 5% and 19%, respectively. Combination of these two fat suppression pulses achieved a 53% increase of the height ratio of the glucose C1β peak compared with the sum of all other peaks, indicating better sensitivity for glucose signal detection. This method will be applicable for in vivo (13)C-MRS by additional adjustment with the in vivo relaxation times of the metabolites.     

Determination of the Stereochemistry of Substituted 5-Methylenehydantoins and Thiohydantoins by 13C NMR and Homonuclear NOE Experiments

Basically the aim of this work is to define the accurate configuration of the exocyclic double bond of substituted 5-methylenehydantoins and thiohydantoins which have been conceived as potential Aldose Reductase inhibitors. A previsional survey based upon the chemical shifts analysis from ¹H and decoupled ¹³C NMR spectra discloses, for a part of the family of compounds, the assignment of the Zconfiguration for unsubstituted (2,3) and N-3 substituted (6,7,9) derivatives, and the E-configuration for the N-1 substituted (8,11) ones. The qualitative study with Homonuclear NOE (8,11) and the coupling constant measuring ^{3 J}C4-C=C-H6 from coupled ¹³C NMR (1–11), lead to the assignment of the accurate configuration of the whole family's compounds in agreement with the previsional study.     

NMR Studies of Crowded Diels–Alder Adducts of Phencyclone with N‐(2,6‐Dialkylphenyl)maleimides. Hindered Rotations and Magnetic Anisotropy

Abstract

Phencyclone, 1, reacted with N‐(2,6‐dimethylphenyl)maleimide, 2a; with N‐(2,6‐diethylphenyl)maleimide, 2b; and with N‐(2,6‐diisopropylphenyl)maleimide, 2c, respectively, to yield the corresponding Diels–Alder adducts, 3a–c. The adducts were extensively characterized by NMR (7 T) at ambient temperatures using one‐ and two‐dimensional (1D and 2D) proton and carbon‐13 techniques for assignments. Slow exchange limit (SEL) spectra were observed, demonstrating slow rotations on the NMR timescales, for the unsubstituted bridgehead phenyl groups [C(sp³)–C(aryl sp²) bond rotations] and for the 2,6‐dialkylphenyl groups [N(sp²)–C(aryl sp²) bond rotations]. Substantial magnetic anisotropic shifts were seen in the adducts. For example, in the N‐(2,6‐dialkylphenyl) moieties of the adducts, one of the alkyl groups is directed “into” the adduct cavity, toward the phenanthrenoid portion, and these “inner” alkyl proton NMR signals were shifted upfield. Thus, in CDCl₃, the “inner” methyl of adduct 3a exhibits a proton resonance at ?0.13 ppm, upfield of tetramethylsilane (TMS); the “inner” ethyl group signals from 3b appear at 0.026 ppm (CH₂, quartet), and ?0.21 ppm (CH₃, triplet); and the “inner” isopropyl group from 3c is seen at ?0.06 ppm (methine, approx. septet) and ?0.39 ppm (CH₃, doublet). Proton NMR of the crude N‐(2,6‐dialkylphenyl)maleamic acids (used as precursors of the maleimides, 2a–c) exhibited two sets of AB quartet signals, suggesting possible conformers from hindered rotation in the amide groups about the HN–C?O bonds.     

A 13C solid-state NMR investigation of the alkynyl carbon chemical shift tensors for 2-butyne-1,4-diol

The alkynyl carbon chemical shift (CS) tensors for 2-butyne-1,4-diol are reported, based on analyses of the carbon-13 NMR spectra of stationary-powder and slow magic-angle spinning (MAS) samples for which the alkynyl carbon nuclei are enriched in 13C. NMR spectra of slow MAS samples exhibit spinning-frequency-dependent fine structure typical of crystallographically equivalent but magnetically distinct nuclei. Simulated spectra of slow MAS samples of this two-spin system are particularly sensitive to the relative orientations of the CS tensors. In addition, the value of 1J(13C, 13C), +175 +/- 10 Hz, is determined by examination of the total NMR lineshape of slow MAS samples. The CS tensors are almost axially symmetric, delta11 = 158.9 +/- 1.0 ppm and delta22 = 155.7 +/- 1.0 ppm; the direction of greatest shielding is approximately along the alkynyl C-C bond, delta33 = -57.8 +/- 2.0 ppm. Both the magnitudes of the principal components of the CS tensors and their orientations are in agreement with those predicted from first-principles calculations at the HF and MP2 levels of theory. This study demonstrates the importance of examining the NMR spectra of homonuclear two-spin systems with and without MAS under a variety of conditions (e.g., two or more applied magnetic fields and slow MAS).     

Automated data processing of [1H-decoupled] 13C MR spectra acquired from human brain in vivo

In clinical 13C infusion studies, broadband excitation of 200 ppm of the human brain yields 13C MR spectra with a time resolution of 2-5 min and generates up to 2000 metabolite peaks over 2h. We describe a fast, automated, observer-independent technique for processing [1H-decoupled] 13C spectra. Quantified 13C spectroscopic signals, before and after the administration of [1-13C]glucose and/or [1-13C]acetate in human subjects are determined. Stepwise improvements of data processing are illustrated by examples of normal and pathological results. Variation in analysis of individual 13C resonances ranged between 2 and 14%. Using this method it is possible to reliably identify subtle metabolic effects of brain disease including Alzheimer's disease and epilepsy.     

1H and 13C NMR Spectra of Alditolyl Derivatives of 3-Hydrazino-5-Methyl[1,2,4]triazino[5,6-b]indole and Their Cyclized Products.

The ¹H and ¹³C NMR spectra of sugar (5-methyl [1, 2, 4]-triazino [5, 6-b] indol-3-yl) hydrazones (1), per-0-acetyl aldehydo sugar 1-acetyl-1-(5-methyl [1, 2, 4] triazino [5, 6-b]-indol-3-yl) hydrazones (2), l- (penta-0-acetyl-pentitol-1-yl)-10-methyl [l, 2, 4] triazolo [3′, 4′:3, 4] [l, 2, 4] triazino [5, 6-b]-indoles (3) have been investigated. The 2 D NMR (H, C COSY) spectrum of 2a has been studied.     

Solid State C NMR of 30-Crown-10 Ether and 30-Crown-10.4H2O

The ¹³C NMR solution spectra of 30-crown-10 ether and its tetrahydrate show only one resonance at all accessible temperatures. In contrast, the solid state ¹³C NMR spectrum of the 30-crown-10.4H₂O shows two resonances in the ratio of 4:1, separated by 1.2 ppm. In the case of 30-crown-10 itself, six resolvable ¹³C resonances in the ratio of 4:1:1:2:1:1 are observed in the solid with an overall chemical shift dispersion of 5 ppm. The remarkably different spectral behavior of these two systems in the solid state is discussed in terms of the torsional environments of the crystallographically unique carbons and the results of GIAO calculations of isotropic ¹³C shieldings for simpler model compounds. Results of dipolar dephased ¹³C CPMAS spectra indicate that 30-crown-10 does not undergo a large amplitude molecular motion, in contrast to earlier results for 18-crown-6. Only a small amount of residual intensity is found in the dipolar dephased spectrum of 30-crown-10.4H₂O, indicating that it also is relatively rigid in the solid.     

C, N CPMAS NMR and GIAO DFT calculations of stereoisomeric oxindole alkaloids from Cat's Claw (Uncaria tomentosa)

Oxindole alkaloids, isolated from the bark of Uncaria tomentosa [Willd. ex Schult.] Rubiaceae, are considered to be responsible for the biological activity of this herb. Five pentacyclic and two tetracyclic alkaloids were studied by solid-state NMR and theoretical GIAO DFT methods. The ¹³C and ¹⁵N CPMAS NMR spectra were recorded for mitraphylline, isomitraphylline, pteropodine (uncarine C), isopteropodine (uncarine E), speciophylline (uncarine D), rhynchophylline and isorhynchophylline. Theoretical GIAO DFT calculations of shielding constants provide arguments for identification of asymmetric centers and proper assignment of NMR spectra. These alkaloids are 7R/7S and 20R/20S stereoisomeric pairs. Based on the ¹³C CP MAS chemical shifts the 7S alkaloids (δ C3 70–71 ppm) can be easily and conveniently distinguished from 7R (δC3 74.5–74.9 ppm), also 20R (δC20 41.3–41.7 ppm) from the 20S (δC20 36.3–38.3 ppm). The epiallo-type isomer (3R, 20S) of speciophylline is characterized by a larger ¹⁵N MAS chemical shift of N4 (64.6 ppm) than the allo-type (3S, 20S) of isopteropodine (δN4 53.3 ppm). ¹⁵N MAS chemical shifts of N1–H in pentacyclic alkaloids are within 131.9–140.4 ppm.