Anisotropic NMR Research Articles

Characterization by Empirical and Computational Methods of Dictyospiromide, an Intriguing Antioxidant Alkaloid from the Marine Alga Dictyota coriacea.

The challenging structural motif of dictyospiromide (1), a spirosuccinimide alkaloid with antioxidant properties that are associated with activation of the Nrf2/ARE signaling pathway, was assigned using contemporary NMR experiments complemented with anisotropic NMR, chiroptical, and computational methodologies. Anisotropic NMR parameters provided critical orthogonal verification of the configuration of the difficult to assign spiro carbon and the other stereogenic centers in1.

Determination of the Absolute Configurations and Sensory Properties of the Enantiomers of a Homologous Series (C6-C10) of 2-Mercapto-4-alkanones.

The enantiomers of a homologous series (C6-C10) of 2-mercapto-4-alkanones were obtained by lipase-catalyzed kinetic resolution of the corresponding racemic 2-acetylthio-4-alkanones. Their configurations were assigned via vibrational circular dichroism and 1H NMR anisotropy based methods. Odor thresholds and odor qualities were determined by capillary gas chromatography-olfactometry using chiral stationary phases. There were minima of the odor thresholds for the chain lengths C7 and C8. Except for chain length C8, the enantiomers of the other homologues showed similar odor thresholds. The odor qualities ranged from pungent (C5) to mushroom (C9 and C10) and were similar to those known for the corresponding 1-alken-3-ones with one less C atom. In contrast to their positional isomers (4-mercapto-2-alkanones), the investigated 2-mercapto-4-alkanones do not meet the requirements of a "tropical olfactophore" (i.e., compounds possessing a 1,3-oxygen-sulfur functionality and specific arrangements of the substituents).

Configuration determination by residual dipolar couplings: accessing the full conformational space by molecular dynamics with tensorial constraints.

Residual dipolar couplings (RDCs) and other residual anisotropic NMR parameters provide valuable structural information of high quality and quantity, bringing detailed structural models of flexible molecules in solution in reach. The corresponding data interpretation so far is directly or indirectly based on the concept of a molecular alignment tensor, which, however, is ill-defined for flexible molecules. The concept is typically applied to a single or a small set of lowest energy structures, ignoring the effect of vibrational averaging. Here, we introduce an entirely different approach based on time averaged molecular dynamics with dipolar couplings as tensorial orientational restraints that can be used to solve structural problems in molecules of any size without the need of introducing an explicit molecular alignment tensor into the computation. RDC restraints are represented by their full 3D interaction tensor in the laboratory frame, for which pseudo forces are calculated using a secular dipolar Hamiltonian as the target. The resulting rotational averaging of each individual tensorial restraint leads to structural ensembles that best fulfil the experimental data. Using one-bond RDCs, the approach has been implemented in the force field procedures of the program COSMOS and extensively tested. A concise theoretical introduction, including the special treatment of force fields for stable and fast MD simulations, as well as applications regarding configurational analyses of small to medium-sized organic molecules with different degrees of flexibility, is given. The observed results are discussed in detail.

PBLG as a versatile liquid crystalline medium for anisotropic NMR data acquisition.

Anisotropic NMR of small molecules has thus far utilized different solvent systems and alignment media to analyze molecules of different polarities. The value of utilizing PBLG liquid crystalline phase in a co-solvent system, expanding its range as a versatile alignment medium, is shown with three molecules of different polarities: strychnine, parthenolide, and sucrose.

Application of anisotropic NMR parameters to the confirmation of molecular structure.

The use of anisotropic NMR data, such as residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs), has emerged as a powerful technique for structural characterization of organic small molecules. RDCs typically report the relative orientations of different 1H-13C bonds; RCSAs report the relative orientations of different carbon chemical shielding tensors and hence are more useful for proton-deficient molecules. This information is complementary to that obtained from conventional NMR data such as J couplings, isotropic chemical shifts, and nuclear Overhauser effects (NOEs)/rotational frame nuclear Overhauser effects (ROEs). Obtaining anisotropic NMR data requires the creation of an anisotropic sample environment through an alignment medium. Here, we focus on the use of compressed or stretched polymeric gels as two different but fundamentally equivalent methods for introducing sample anisotropy. Protocols are provided for the synthesis of the chloroform-compatible poly(methyl methacrylate) and dimethyl sulfoxide (DMSO)-compatible poly(2-hydroxyethyl methacrylate) gels and sample setup with a preparation time of 2-3 d. The bond-specific RDC data and the atom-specific RCSA data are extracted as changes in 1H-13C couplings and 13C chemical shifts, respectively, between two measurements under different alignment conditions, with a total experimental time of 0.5-4 d. NMR data acquisition and important considerations are described in detail. We also provide step-by-step procedures for the density functional theory (DFT) calculations involved and data analysis using the commercial software MSpin. We use three example compounds, namely cryptospirolepine (505 Da), retrorsine (351 Da), and estrone (270 Da), to demonstrate some important aspects of the workflow, such as input data preparation, handling of structural flexibility, and RCSA data correction when necessary.

Poly-γ-p-Biphenylmethyl-Glutamate as Enantiodifferentiating Alignment Medium for NMR Spectroscopy with Temperature-Tunable Properties.

The use of anisotropic NMR parameters-especially residual dipolar couplings (RDCs)-offers access to additional structural information and, therefore, alignment media required for this approach are under a continuous development. Here, we present poly-γ-p-biphenylmethyl-glutamate (PBPMG) as a new versatile enantiodifferentiating alignment medium. The thermoresponsive properties of this polymer allowed for RDC measurements of more than one orientation within the same sample at different temperatures. Moreover, the outstanding enantiodifferentiation along with excellent spectral quality even offered the opportunity to differentiate enantiomers in mixtures.

A New Method of Assessing Lipid Mixtures by 31P Magic-Angle Spinning NMR

A variety of lipids that differ by their chains and headgroups are found in biomembranes. In addition to studying the overall membrane phase, determination of the structure, dynamics, and headgroup conformation of individual lipids in the mixture would be of great interest. We have thus developed, to our knowledge, a new approach using solid-state 31P NMR, magic-angle spinning, and chemical-shift anisotropy (CSA) recoupling, using an altered version of the recoupling of chemical shift anisotropy (ROCSA) pulse sequence, here penned PROCSA. The resulting two-dimensional spectra allowed the simultaneous measurement of the isotropic chemical shift and CSA of each lipid headgroup, thus providing a valuable measure of its dynamics and structure. PROCSA was applied to mixtures of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) in various relative proportions, to mimic bacterial membranes and assess the respective roles of lipids in shaping these bilayers. The results were interpreted in terms of membrane topology, lipid propensity to adopt various phases or conformations, and lipid-lipid miscibility. Our results showed that PG dictates the lipid behavior when present in a proportion of 20 mol % or more. A small proportion of PG is thus able to impose a bilayer structure to the hexagonal phase forming PE. We discuss the requirement for lipids, such as PE, to be able to adopt non-bilayer phases in a membrane.

Correction to "Detection of Curvature-Radius-Dependent Interfacial pH/Polarity for Amphiphilic Self-Assemblies: Positive versus Negative Curvature".

It is possible that a defined curvature at the membrane interface controls its pH/polarity to exhibit specific bioactivity. By utilizing an interface-interacting spiro-rhodamine pH probe and the Schiff base polarity probe, we have shown that the pH deviation from the bulk phase to the interface (ΔpH)/interfacial dielectric constant (κ(i)) for amphiphilic self-assemblies can be regulated by the curvature geometry (positive/negative) and its radius. According to 1H NMR and fluorescence anisotropy investigations, the probes selectively interact with an anionic interfacial Stern layer. The ΔpH/κ(i) values for the Stern layer are estimated by UV–vis absorption and fluorescence studies. For the anionic sodium bis-2-ethylhexyl-sulfosuccinate (AOT) inverted micellar (IM) negative interface, the highly restricted water and proton penetration into the Stern layer owing to tight surfactant packing or a reduced water-exposed headgroup area may be responsible for the much lower ΔpH ≈ −0.45 and κ(i) ≈ 28 in comparison ...

Rotational Diffusion of Membrane Proteins Probed by Anisotropic T2and T1ρ NMR Relaxation in Aligned Lipid Bilayers

Rotational Diffusion of Membrane Proteins Probed by Anisotropic T2and T1ρ NMR Relaxation in Aligned Lipid Bilayers

Computer-Assisted 3D Structure Elucidation (CASE-3D) of Natural Products Combining Isotropic and Anisotropic NMR Parameters.

A computer-assisted structural elucidation (CASE-3D) strategy based on the use of isotropic and/or anisotropic NMR data is proposed to elucidate relative configuration and preferred conformation in complex natural products. The methodology involves the selection of conformational models through the use of the Akaike Information Criterion and scoring of the different configurations. As illustrative examples, the methodology furnished the correct configuration of the already known compounds artemisinin (1) and homodimericin A (2). Revised structures (5 and 6), including their absolute configuration, for the recently reported curcusones I (3) and J (4) are proposed.

Protein Rotational Dynamics in Aligned Lipid Membranes Probed by Anisotropic T1ρ NMR Relaxation

A membrane-bound form of Pf1 coat protein reconstituted in magnetically aligned DMPC/DHPC bicelles was used as a molecular probe to quantify for the viscosity of the lipid membrane interior by measuring the uniaxial rotational diffusion coefficient of the protein. Orientationally dependent 15N NMR relaxation times in the rotating frame, or T1ρ, were determined by fitting individually the decay of the resolved NMR peaks corresponding to the transmembrane helix of Pf1 coat protein as a function of the spin-lock time incorporated into the 2D SAMPI4 pulse sequence. The T1ρ relaxation mechanism was modeled by uniaxial rotational diffusion on a cone, which yields a linear correlation with respect to the bond factor sin4θB, where θB is the angle that the NH bond forms with respect to the axis of rotation. Importantly, the bond factors can be independently measured from the dipolar couplings in the separated local-field SAMPI4 spectra. From this dependence, the value of the diffusion coefficient D|| = 8.0 × 105 s−1 was inferred from linear regression of the experimental T1ρ data even without any spectroscopic assignment. Alternatively, a close value of D|| = 7.7 × 105 s−1 was obtained by fitting the T1ρ relaxation data for the assigned NMR peaks of the transmembrane domain of Pf1 to a wavelike pattern as a function of residue number. The method illustrates the use of single-helix transmembrane peptides as molecular probes to assess the dynamic parameters of biological membranes by NMR relaxation in oriented lipid bilayers.

Detection of Curvature-Radius-Dependent Interfacial pH/Polarity for Amphiphilic Self-Assemblies: Positive versus Negative Curvature.

It is possible that a defined curvature at the membrane interface controls its pH/polarity to exhibit specific bioactivity. By utilizing an interface-interacting spiro-rhodamine pH probe and the Schiff base polarity probe, we have shown that the pH deviation from the bulk phase to the interface (ΔpH)/interfacial dielectric constant (κ(i)) for amphiphilic self-assemblies can be regulated by the curvature geometry (positive/negative) and its radius. According to 1H NMR and fluorescence anisotropy investigations, the probes selectively interact with an anionic interfacial Stern layer. The ΔpH/κ(i) values for the Stern layer are estimated by UV-vis absorption and fluorescence studies. For the anionic sodium bis-2-ethylhexyl-sulfosuccinate (AOT) inverted micellar (IM) negative interface, the highly restricted water and proton penetration into the Stern layer owing to tight surfactant packing or a reduced water-exposed headgroup area may be responsible for the much lower ΔpH ≈ -0.45 and κ(i) ≈ 28 in comparison to ∼-2.35 and ∼44, respectively, for the anionic sodium dodecyl sulfate (SDS) micellar positive interface with a close similar Stern layer. With increasing AOT IM water-pool radius (1.7-9.5 nm) or [water]/[AOT] ratio ( w0) (8.0-43.0), the ΔpH and κ(i) increase maximally up to ∼-1.22 and ∼45, respectively, due to a greater water-exposed headgroup area. However, the unchanged ΔpH ≈ -0.65 and κ(i) ≈ 53.0 within radii ∼3.5-8.0 nm for the positive interface of a mixed Triton X-100 (TX-100)/SDS (4:1) micelle justify its packing flexibility. Interestingly, the continuously increasing ΔpH trend for IM up to its largest possible water-pool radius of ∼9.5 nm may rationalize the increase in ΔpH (∼-1.4 to -1.6) with the change in the curvature radii (∼15 to 50 nm) for sodium 1,2-dimyristoyl- sn-glycero-3-phosphorylglycerol (DMPG)/1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) (2:1) large unilamellar vesicles (LUV) owing to its negative interface. Whereas, similar to the micellar positive interface, the unchanged ΔpH at the positive LUV interface was confirmed by fluorescence microscopic studies with giant unilamellar vesicles of identical lipids composition. The present study offers a unique and simple method of monitoring the curvature-radius-dependent interfacial pH/polarity for biologically related membranes.

Polyaspartates as Thermoresponsive Enantiodifferentiating Helically Chiral Alignment Media for Anisotropic NMR Spectroscopy.

Lyotropic liquid crystalline phases of poly-β-phenethylaspartates are presented as new helically chiral enantiodifferentiating alignment media with thermoresponsive properties. In addition to displaying the highest enantiodifferentiation observed for homopolypeptides, the alignment media undergo a temperature induced helix reversal without perturbing the nematic phase. This offers the opportunity to measure residual dipolar couplings (RDCs) in high and low temperature helix conformation (P- and M-helix) in one polymer. Thus different mean orientations of a chiral analyte can be determined within the same sample. Furthermore, we investigated whether the resulting media are diastereomeric and whether we are able to obtain information about the alignment process.

Biphasic Liquid Crystal and the Simultaneous Measurement of Isotropic and Anisotropic Parameters by Spatially Resolved NMR Spectroscopy.

Residual dipolar couplings and other anisotropic NMR parameters are powerful tools for molecular structure elucidation when conventional techniques do not suffice. With current liquid crystalline preparations it is necessary to prepare two samples to extract isotropic and anisotropic data from spectra and to derive the residual dipolar couplings. Here, we present the preparation, measurement, and interpretation of a novel biphasic liquid crystalline phase where a single sample can be used to generate both isotropic and anisotropic data. First, we introduce the synthesis of the chiral polymer leading to the biphasic liquid crystal. Second, we present two approaches to measure spatially selective CLIP-HSQC spectra. From these spectra, we extracted the couplings, performed an assignment of diastereotopic protons, and achieved the enantiomeric discrimination of isopinocampheol as a well-studied test molecule.

Nuclear magnetic and nuclear quadrupole resonance parameters of β-carboline derivatives calculated using density functional theory

A density functional theory (DFT) calculations using B3LYP/6-311++G(d,p) method were carried out to investigate the relative stability of the molecules of β-carboline derivatives such as harmaline, harmine, harmalol, harmane and norharmane. Calculated nuclear quadrupole resonance (NQR) parameters were used to determine the 14N nuclear quadrupole coupling constant χ, asymmetry parameter η and EFG tensor (q zz ). For better understanding of the electronic structure of β-carboline derivatives, natural bond orbital (NBO) analysis, isotropic and anisotropic NMR chemical shieldings were calculated for 14N nuclei using GIAO method for the optimized structures. The NBO analysis shows that pyrrole ring nitrogen (N9) atom has greater tendency than pyridine ring nitrogen (N2) atom to participate in resonance interactions and aromaticity development in the all of these structures. The NMR and NQR parameters were studied in order to find the correlations between electronic structure and the structural stability of the studied molecules.

Complementarity of DFT Calculations, NMR Anisotropy, and ECD for the Configurational Analysis of Brevipolides K-O from Hyptis brevipes.

Brevipolides K-O (1-5), five new cytotoxic 6-(6'-cinnamoyloxy-2',5'-epoxy-1'-hydroxyheptyl)-5,6-dihydro-2H-pyran-2-ones (IC50 values against six cancer cell lines, 1.7-10 μM), were purified by recycling HPLC from Hyptis brevipes. The structures, containing a distinctive tetrahydrofuran ring, were established by comprehensive quantum mechanical calculations and experimental spectroscopic analysis of their NMR and ECD data. Detailed analysis of the experimental NMR 1H-1H vicinal coupling constants in comparison with the corresponding DFT-calculated values at the B3LYP/DGDZVP level confirmed the absolute configuration of 3 and revealed its conformational preferences, which were further strengthened by NOESY correlations. NMR anisotropy experiments by the application of Mosher's ester methodology and chemical correlations were also used to conclude that this novel brevipolide series (1-5) share the same absolute configuration corresponding to C-6(R), C-1'(S), C-2'(R), C-5'(S), and C-6'(S).

Relaxation-Assisted Separation of Overlapping Patterns in Ultra-Wideline NMR Spectra.

Efficient acquisition of high-quality ultra-wideline (UW) solid-state NMR powder patterns in short experimental time frames is challenging. UW NMR powder patterns often possess inherently low signal-to-noise (S/N) and usually overlap for samples containing two or more magnetically distinct nuclides, which obscures spectral features and drastically lowers the spectral resolution. Currently, there is no reliable method for resolving overlapping powder patterns originating from unreceptive nuclei affected by large anisotropic NMR interactions. Herein, we discuss new methods for resolving individual UW NMR spectra associated with magnetically distinct nuclei by exploiting their different relaxation characteristics using 2D relaxation-assisted separation (RAS) experiments. These experiments use a non-negative Tikhonov fitting (NNTF) routine to process high-quality T1 and T2eff relaxation data sets to produce high-resolution, 2D spin-relaxation correlation spectra for both spin-1/2 and quadrupolar nuclei in organic and organometallic solids under static (i.e., stationary) conditions. It is found that (i) T2eff RAS data sets can be acquired in a fraction of the time required for analogous T1 RAS data sets, because a time-incremented 2D data set is not required for the former, and (ii) Tikhonov regularization is superior to conventional non-negative least-squares fitting, as it more reliably and robustly results in cleaner separation of patterns based on relaxation time constants.

Reinvestigation of the Absolute Configurations of Chiral β-Mercaptoalkanones Using Vibrational Circular Dichroism and 1H NMR Analysis.

The absolute configurations of chiral β-mercaptoalkanones were previously assigned on the basis of the 1H NMR anisotropy method using (S)-2-methoxy-2-(1-naphthyl)propionic acid ((S)-MαNP) as the chiral auxiliary. This study presents a reinvestigation of the configurations of 4-mercapto-2-pentanone 1, 4-mercapto-2-heptanone 2, and 2-mercapto-4-heptanone 3. Enantiomers of 1, 2, and 3 were obtained by lipase-catalyzed hydrolyses of the respective acetylthioalkanones. Upon derivatization with (S)-MαNP, the configurations of the reaction products were deduced based on the order of the HPLC elution of the diastereoisomeric thioesters, assuming that the sector rule previously developed for secondary alcohols is also valid for thiols. In addition, the configurations were experimentally determined by vibrational circular dichroism (VCD) and 1H NMR analyses after esterification with (R)-hydratropic acid (HTA) and 2-methoxy-2-phenylacetic acid (MPA). The assignments of the configurations using VCD and NMR analyses of HTA- and MPA-thioesters were in agreement. However, they were opposite to those deduced for (S)-MαNP thioesters via the sector rule. Consequently, the formerly assigned configurations of β-mercaptoalkanones deduced via investigation of (S)-MαNP-derivatives have to be revised.

Mechanical Behavior of Polymer Gels for RDCs and RCSAs Collection: NMR Imaging Study of Buckling Phenomena.

Anisotropic NMR parameters, such as residual dipolar couplings (RDCs), residual chemical shift anisotropies (RCSAs) and residual quadrupolar couplings (RQCs or ΔνQ ), appear in solution-state NMR when the molecules under study are subjected to a degree of order. The tunable alignment by reversible compression/relaxation of gels (PMMA and p-HEMA) is an easy, user-friendly, and very affordable method to measure them. When using this method, a fraction of isotropic NMR signals is observed in the NMR spectra, even at a maximum degree of compression. To explain the origin of these isotropic signals we decided to investigate their physical location inside the NMR tube using deuterium 1D imaging and MRI micro-imaging experiments. It was observed that after a certain degree of compression the gels start to buckle and they generate pockets of isotropic solvent, which are never eliminated. The amount of buckling depends on the amount of cross-linker and the length of the gel.

Elucidation of the Enantiodiscrimination Properties of a Nonracemic Chiral Alignment Medium through Gel-based Capillary Electrochromatography: Separation of the Mefloquine Stereoisomers.

Enantiodiscrimination and enantioseparation are two highly important processes in chemistry, often performed by using NMR spectroscopy and chromatography. For a better understanding of the mechanistic details, the same system should be studied by both methods. In addition, isotropic and anisotropic NMR parameters should be obtained, the latter using alignment media so that residual dipolar couplings and chemical‐shift anisotropies can be measured. Consequently, a chiral alignment medium was used for the first time in chiral gel‐based capillary electrochromatography with the four stereoisomers of the antimalaria drug mefloquine as test compounds. Chromatographic data verify that enantiodiscrimination obtained with this alignment gel is caused by differences in the equilibrium constants related to associate formation. Hence, the chromatographic separation provides physicochemical data that form a basis for the understanding and optimization of alignment processes, and vice versa.