1H and 13C NMR spectra of N-substituted morpholines

(1)H and (13)C NMR data for N-substituted morpholines 1-20 were measured using 1D (DEPT, 1D NOE difference) and 2D NMR spectroscopic methods including (1)H-(1)H COSY, long-range (1)H-(1)H COSY, NOESY, gHMBC and gHMQC experiments. At room temperature the (1)H NMR spectra of protonated compounds 2 and 9 show the chair conformation for the morpholine ring. Spin-spin coupling constants were deduced from the resolution-enhanced proton spectra.     

Filtered backprojection for the reconstruction of a high-resolution (4,2)D CH3-NH NOESY spectrum on a 29 kDa protein

Projection-reconstruction (PR) NMR enables rapid collection of multidimensional NMR data. NOESY represents a particularly difficult challenge for currently existing reconstruction algorithms, as it requires the quantitative reconstruction of an unknown number of peaks, at full sensitivity. We have demonstrated the successful application of PR-NMR to NOESY by determining the 4D methyl/amide NOESY spectrum of a 29 kDa protein, human carbonic anhydrase II, from 2D projections, using filtered backprojection for reconstruction. Compared with a 3D control spectrum, all expected peaks were faithfully reconstructed, with correct volumes and with no artifacts. Filtered backprojection thus provides a way to obtain high-resolution 4D NOESY data in the time required for conventional 3D data collection.     

HOBS methods for enhancing resolution and sensitivity in small DNA oligonucleotide NMR studies

¹H NMR spectra from biopolymers give chemical shifts classified according to proton type and often suffer from signal degeneracy. Data from nucleic acids are particularly prone to this failing. Recent developments in proton broadband decoupling techniques with the promise of enhanced resolution at full sensitivity have allowed us to investigate the application of homonuclear band‐selective (HOBS) decoupling to the study of small synthetic DNA molecules and to compare these with results from classical and pure shift techniques. Improved signal resolution at full sensitivity in both HOBS‐1D ¹H and HOBS‐2D [¹H, ¹H] NOESY NMR data is reported for three example small DNA molecules. Comparisons of ¹H T₁ and integrals of signals from HOBS‐1D ¹H and HOBS‐2D [¹H, ¹H] NOESY NMR data with those of standard data collection methods are also reported. The results show that homonuclear HOBS‐NOESY data are useful for data assignment purposes and have some merit for quantification purposes. In general, we show that resolution and sensitivity enhancement of ¹H NMR data for small DNA samples may be achieved without recourse to higher magnetic field strength at full sensitivity in a band‐selected manner. Copyright © 2014 John Wiley & Sons, Ltd.     

Application of MEKC and monolithic CEC for the analysis of bioactive naphthoquinones in Eleutherine americana

Two microscale separation techniques for the analysis of bioactive naphthoquinones in Eleutherine americana were developed and validated. By MEKC four compounds (eleuthoside B, isoeleutherin, eleutherol and eleutherinoside A) could be determined in plant extracts using an aqueous electrolyte solution composed of 25 mM sodium tetraborate, 50 mM sodium cholate and 20% THF. CEC on a polymeric methacrylate‐based monolith with strong cationic properties showed promising results, as it additionally enabled the separation of two enantiomers, eleutherin and isoeleutherin. The mobile phase for CEC experiments comprised 3 mM ammonium formate in a mixture of ACN and water. At an applied voltage of ?25 kV, all five markers were baseline separated in less than 12 min. Both methods were successfully validated for linearity (MEKC: R²≥0.999; CEC: R²≥0.997), sensitivity (MEKC: LOD=4–5 μg/mL; CEC: LOD=2–8 μg/mL), accuracy (MEKC: 96.5–102.7% recovery; CEC: 97.1–103.5% recovery) and precision (MEKC: σ_rel≤2.43%; CEC: σ_rel≤2.21%). The quantitative analysis of naphthoquinone derivatives in several E. americana samples showed that both methods are suitable for practical applications, because the results were well comparable to those obtained by established techniques such as HPLC.     

Complete 1H and 13C NMR assignments of two phytosterols from roots of Piper nigrum

Using modern NMR techniques, including DQF-COSY, NOESY, HETCOR and HMBC, the full assignments of all 1H and 13C chemical shifts were made for stigmastane-3,6-dione and stigmast-4-ene-3,6-dione, isolated from the roots of Piper nigrum (Piperaceae). Their stereochemistry was resolved by a combination of 2D NOESY data and analysis of coupling constants. The two compounds were isolated from the genus Piper for the first time.     

Water-soluble constituents of the heartwood of Streblus asper

A new lignan, (7'R,8'R)-threo-strebluslignanol-2-O-beta-D-glucopyranoside, along with 8 known compounds (2-9) were isolated from the water-soluble part of the MeOH extract of the heartwood of Streblus asper. Their structures were elucidated through various spectroscopic methods, including 1D NMR (1H NMR, 13C NMR), 2D NMR (HMQC, HMBC, and NOESY), and HRMS. The stereochemistry at the chiral centers was determined using the CD spectrum, as well as analyses of coupling constants and optical rotation data. In the preliminary bioassay, the isolated compounds did not show anti-HBV activities in vitro using the HBV transfected HepG2.2.15 cell line.     

Formation of an Inclusion Complex of a New Transition Metal Ligand in β-Cyclodextrin

The inclusion complex of a new transition metal ligand, 2,4,9-trithia-tricyclo[3.3.1.1^3,7]decane-7-carboxylic acid (2,4,9-trithia-adamantane-7-carboxylic acid, TPCOOH) in β-cyclodextrin was studied by ¹H NMR, 2D NOESY NMR spectroscopy, host-induced CD spectroscopy, and tandem mass spectrometry. ¹H NMR, MS–MS and NOESY data show that the TPCOOH guest forms a 1:1 inclusion complex with the host β-cyclodextrin. The NOESY experiments also show that TPCOOH is oriented in the complex with the thioketal end preferentially located at the larger opening of β-cyclodextrin. The orientation of the guest in the host molecule is also confirmed by the induced CD of the ligand, which shows a positive Cotton effect. An association constant of 660±20?M^?1 was determined by ¹H NMR titration for the complex at room temperature in D₂O.     

1H NMR study of the inclusion of Costa-type organocobalt(III) complexes in cyclodextrins

The inclusion behavior between Costa-type complexes and cyclodextrins (CDs) was studied by 1H NMR in aqueous solution. The results indicated that 1:1 inclusion complex was formed, in which the alkyl group of the guest was included in the cavity of CDs. The stability constants of the inclusion complexes were determined by the quantitative 1H NMR method. The effects on stability constants were discussed when various host and guest compounds were used.     

Interaction between PEO-PPO-PEO copolymers and a hexapeptide in aqueous solutions

Interaction between PEO-PPO-PEO copolymers and a hexapeptide, growth hormone releasing peptide-6 (GHRP-6), was investigated by NMR to study the potential use of the copolymers in peptide drug delivery. (1)H NMR and nuclear Overhauser effect spectroscopy (NOESY) measurements determined that PO methyl protons interacted with methyl protons of the Ala moiety, aromatic protons of the Trp moiety, and some of the Phe aromatic protons. The Lys moiety and part of the Phe moiety entered the hydrophilic EO environment via hydrogen bonding. PEO-PPO-PEO copolymers and the peptide formed a complex in 1:1 stoichiometry. Binding constants between copolymers and GHRP-6 were determined, and it was indicated that the copolymers containing more EO and PO units will lead to greater affinity with the peptide. Isothermal titration calorimetry (ITC) measurements confirmed the results of NMR experiments. This study indicates that PEO-PPO-PEO copolymers have great potential in delivering peptide drugs.     

Unambiguous structural characterization of hydantoin reaction products using 2D HMBC NMR spectroscopy

Data from two-dimensional (2D) NMR experiments were used to identify the reaction products resulting from the opening of pyroglutamates with isocyanates or thioisocyanates. The reaction has the potential to produce compounds that would have very similar one-dimensional proton ((1)H) or carbon-13 ((13)C) NMR spectra. Careful analysis of (1)H--(1)H COSY, (1)H--(1)H NOESY, and HMBC data, including chemical shifts and coupling constants, were used to distinguish correctly between carbamoyl-2-pyrrolidinone, hydantoin, and perhydro-1,3-diazepine-2,4-dione type structures that could result from this reaction. This work describes their preparation and subsequent identification using 2D NMR spectroscopy, and includes complete (13)C assignments of the reaction products. The 2D NMR techniques and analysis described here can be applied successfully to other synthetic reactions with the potential to produce isomeric products.     

Conformational study of a guaiacyl beta-O-4 lignin model compound by NMR. Examination of intramolecular hydrogen bonding interactions and conformational flexibility in solution

Intramolecular H-bonding interactions were investigated in solution for the threo and erythro diastereomeric forms of a guaiacyl beta-O-4 lignin model compound by using the NMR data obtained from hydroxyl protons. Temperature coefficients of the chemical shifts (ddelta/dT) and coupling constants (3J(HCOH)) were measured in aprotic and protic solutions: DMSO-d6, acetone-d6 and acetone-d6-water. The NMR parameters do not support the existence of strong and persistent intramolecular H-bonds that could participate in the stabilization of the guaiacyl beta-O-4 structure in solution, but instead indicate that intermolecular H-bonds to solvent predominate. 1D NOE experiments nevertheless revealed the presence of a direct chemical exchange between the hydroxyl protons, suggesting the possible existence of weak and transient intramolecular H-bonding interactions. The conformational flexibility of the threo structure was also investigated in acetone solution from the measurement of long-range 1H, 1H and 1H, 13C coupling constants and from NOESY experiments. The NMR data are not consistent with any single conformation, indicating that different conformers co-exist in solution. The experimental results support the conformational flexibility predicted by molecular dynamics simulations performed in a previous study. Finally, both experimental and theoretical approaches indicate that weak intramolecular H-bonds can exist transiently in solution, breaking and reforming as the beta-O-4 molecule undergoes conformational interconversion, but cannot be invoked as possible means of conferring rigidity to the beta-O-4 structure.     

Dynamic NMR and ab initio studies of exchange between rotamers of derivatives of octahydrofuro[3,4-f]isoquinoline-7(1H)-carboxylate and tetrahydro-2,5,6(1H)-isoquinolinetricarboxylate

The 1H and 13C NMR spectra of methyl-8-(2-furyl)-5-methyl-1,3-dioxo-3,3a,4,6,8,9,9a,9b-octahydrofuro[3,4-f]isoquinoline-7(1H)-carboxylate (1) and trimethyl 8-methyl-3-phenyl-3,4,4a,7-tetrahydro-2,5,6(1H)-isoquinolinetricarboxylate (2) at room temperature displayed doubling of the majority of signals, suggesting the presence of two rotational conformers (rotamers) in a ratio approximately 1:1.2 (in a mixture of CDCl3 and C6D6), approximately 1:1 (in CD2Cl2) and approximately 1:1.4 (in C6D6). On the basis of the temperature-dependent 1H NMR spectra of 1 and 2, the barrier to interconversion of the rotamers was calculated to be approximately 16 kcal mol(-1). The average chemical shifts and spin-spin coupling constants were analyzed for the resolution-enhanced 300 MHz 1H NMR spectrum of 1 at 333 K in CDCl3 solution. From analysis of the spin-spin coupling constants, it is proposed that the nitrogen-containing ring is in a chair conformation with C-2-H equatorial. Low- and room-temperature NOESY experiments in conjunction with theoretical ab initio calculations supported the hypothesis that the two rotamers interchange via rotation about the C-N bond. Spectral assignments of all proton and carbon resonances were made on the basis of one- and two-dimensional NMR experiments (DEPT, DQCOSY, NOESY, HETCOR and gHMBC).     

Crown ether-based cryptand/tropylium cation inclusion complexes

Host–guest complexation between crown ether-based cryptand hosts and a carbonium ion, tropylium hexafluorophosphate was studied. ¹H NMR, NOESY NMR, and electrospray ionization mass spectrometry were employed to characterize these inclusion complexes. The contrast tests of ¹H NMR and association constants indicated that cryptands are much better hosts for tropylium hexafluorophosphate than the corresponding simple crown ethers. C–H?O hydrogen bonding, face-to-face π-stacking interactions, and charge-transfer interactions are thought to be the main driving forces for the formation of these host–guest complexes. These multiple non-covalent interactions may jointly contribute to the complex formation and considerably reinforce the complex stability. Moreover, the complexation between dibenzo-24-crown-8-based cryptand 4 and tropylium hexafluorophosphate 7 can be reversibly controlled by adding KPF₆ and then DB18C6 in 1:1 acetonitrile/chloroform, providing a new cation-responsive host–guest recognition motif for supramolecular chemistry.     

Improved resolution in two-dimensional 1H NMR spectra of peptides by band-selective, homonuclear decoupling during both the evolution and acquisition periods: application to characterization of the binding of peptides by heparin

Two-dimensional 1H NMR experiments that achieve band-selective, homonuclear decoupling in both the indirectly detected F1 and directly detected F2 dimensions were used to assign the highly overlapped 1H NMR spectrum of the peptide Ac-SRGKARVRAKVKDQTK-NH2, both free in solution and bound to heparin. Band-selective, homonuclear decoupling during the evolution period was achieved using a double pulsed field gradient spin-echo (DPFGSE) with semi-selective shaped pulses; band-selective, homonuclear decoupling during the acquisition period was achieved by time-shared semi-selective shaped pulse decoupling. Regular TOCSY, ROESY and NOESY spectra and TOCSY, ROESY and NOESY spectra measured with band-selective, homonuclear decoupling in the evolution (F1) dimension (BASHD-TOCSY, ROESY and NOESY spectra) and with band-selective, homonuclear decoupling in both the F1 and F2 dimensions (D-(or Double)-BASHD-TOCSY, ROESY and NOESY spectra) are reported and compared for the peptide and its heparin complex. Complete assignment of the 1H-NMR spectra of the free and heparin-complexed peptide was achieved with the high resolution of the D-BASHD-TOCSY, ROESY and NOESY spectra. Characterization of the heparin-complexed peptide is of interest because of the ability of the peptide to neutralize the anticoagulant activity of heparin.     

A new flavane acid from the fruits of Illicium verum

A new compound, illiciumflavane acid (1), along with 13 known compounds (2–14), were isolated from the fruits of Illicium verum Hook. F. Their structures were elucidated through various spectroscopic methods, including 1D NMR (¹H NMR, ¹³C NMR), 2D NMR (HMQC, HMBC and NOESY) and HRMS. The stereochemistry at the chiral centres was determined using CD spectrum as well as analyses of coupling constants and optical rotation data. Cytotoxicity evaluation of four compounds showed that illiciumflavane acid and (E)-1,2-bis(4-methoxyphenyl)ethene exhibited potential against A549 activities with IC₅₀ values of 4.63 μM and 9.17 μM, respectively.     

Eremophilane esters of Robinsonecio gerberifolius and their rearranged products. Study of the coupling constants (2)J(H, H), (3)J(H, H) and (4)J(H, H)

In the course of the basic hydrolysis of four eremophilane esters isolated from Robinsonecio gerberifolius, some rearrangements, eliminations, and additions occurred. Five compounds were obtained, three of them not previously described. Additionally, a new sesquiterpene was produced by autooxidation of compound 1. The (1)H and (13)C NMR spectra of these compounds were completely assigned by utilization of HMQC, HMBC, COSY, DEPT, and NOESY techniques. The long-range coupling constants of the peroxide 10 are reported, and all its coupling constants (2)J(H, H), (3)J(H, H), and (4)J(H, H) are calculated at the B3LYP/6-31G(d,p) level of theory. Their magnitude is explained in terms of electronic delocalization and the additivity of stereoelectronic effects.     

Structure, stability, and interconversion barriers of the rotamers of cis-[Pt(II)Cl(2)(quinoline)2] and cis-[Pt(II)Cl(2)(3-bromoquinoline)(quinoline)] from X-ray crystallography, NMR spectroscopy and molecular mechanics evidence

Reported are the preparations of cis-[PtCl(2)(quinoline)(2)] and cis-[PtCl(2)(3-bromoquinoline)(quinoline)] and an investigation of the stabilities and interconversion of the rotamer forms of these complexes. Both head-to-head (HTH) and head-to-tail (HTT) rotamer forms are found in the crystal structure of cis-[PtCl(2)(quinoline)(2)]. The NOESY NMR spectrum of cis-[PtCl(2)(quinoline)(2)] in dmf-d(7) at 300 K is consistent with conformational exchange brought about by rotation about the Pt-N(quinoline) bonds. H.H nonbonded distances between H atoms of the two different quinoline ligands were determined from NOESY data, and these distances are in accord with those observed in the crystal structure and derived from molecular mechanics models. cis-[PtCl(2)(3-bromoquinoline)(quinoline)] was prepared to alleviate the symmetry-imposed absence of inter-ring H2/H2 and H8/H8 NOESY cross-peaks for cis-[PtCl(2)(quinoline)(2)]. Molecular mechanics calculations on the complexes show the HTT rotamers to be 1-2 kJ mol(-)(1) more stable than the HTH forms, consistent with the (1)H spectra where the intensities of resonances for the two forms are approximately equal. Variable-temperature (1)H NMR spectra of cis-[PtCl(2)(quinoline)(2)] in dmf-d(7) indicate a rotational energy barrier of 82 +/- 4 kJ mol(-)(1). Variable-temperature (1)H NMR spectra indicate that the Br substituent on the quinoline ring does not affect the energy barrier to interconversion between the HTT and HTH forms (79 +/- 5 kJ mol(-)(1)). The steric contribution to the rotation barrier was calculated using molecular mechanics calculations and was found to be approximately 40 kJ mol(-)(1), pointing to a possible need for an electronic component to be included in future models.     

Amphidinolide X,a novel 16-membered macrodiolide from dinoflagellate Amphidinium sp

A novel cytotoxic 16-membered macrodiolide, amphidinolide X (1), has been isolated from a marine dinoflagellate Amphidinium sp. (strain Y-42). The gross structure of 1 was elucidated on the basis of spectroscopic data including one-bond and long-range (13)C-(13)C correlations. The relative and absolute stereochemistries were determined by combined analyses of NOESY data and (1)H-(1)H and (1)H-(13)C coupling constants of 1 and NMR data of the degradation products. Amphidinolide X (1) is the first macrodiolide consisting of polyketide-derived diacid and diol units from natural sources. The biosynthetic origins of 1 were investigated by means of feeding experiments with (13)C-labeled acetates.     

1H and 13C NMR structure determination of a new stereoisomer of isoreserpiline pseudoindoxyl from Rauvolfia grandiflora

An NMR study of a new stereoisomer of isoreserpiline pseudoindoxyl alkaloid is described. The complete ¹H and ¹³C NMR assignments of the compound were carried out using COSY, HMQC and HMBC. Stereochemistry at carbons C‐2, C‐3, C‐15, C‐20 and C‐19 was established using values of ¹H chemical shifts, coupling constants and NOESY experiments. Copyright © 2003 John Wiley & Sons, Ltd.     

Analysis of cyanobacterial toxins (anatoxin-a, cylindrospermopsin, microcystin-LR) by capillary electrophoresis

Capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) were applied to the simultaneous separation of cyanobacterial toxins (anatoxin-a, microcystin-LR, cylindrospermopsin). The analytical performance data of both methods, optimized for the three toxins, were similar with a precision of migration times smaller than 0.8 RSD% and a detection limit in the range of 1-4 microg/mL, using spectrophotometric detection at 230 nm. Both methods were applied to an analysis of cyanotoxins in water bloom samples and crude cyanobacterial extracts. The results obtained indicate that, for complex matrices, the sequential application of CZE and MEKC is necessary. It is recommended to use both CE techniques for the analysis of the same sample in order to confirm the results by an orthogonal approach.