Complete relative stereochemistry of multiple stereocenters using only residual dipolar couplings

Residual dipolar couplings (RDCs), in combination with molecular order matrix calculations, were used to unambiguously determine the complete relative stereochemistry of an organic compound with five stereocenters. Three simple one-dimensional experiments were utilized for the measurements of (13)C-(1)H, (13)C-(19)F, (19)F-(1)H, and (1)H-(1)H RDCs. The order matrix calculation was performed on each chiral isomer independently. The fits were evaluated by the comparison of the root-mean-square deviation (rmsd) of calculated and measured RDCs. The order tensor simulations based on two different sets of RDC data collected with phage and bicelles are consistent. The resulting stereochemical assignments of the stereocenters obtained from using only RDCs are in perfect agreement with those obtained from the single-crystal X-ray structure. Six RDCs are found to be necessary to run the simulation, and seven are the minimum to get an acceptable result for the investigated compound. It was also shown that (13)C-(1)H and (1)H-(1)H RDCs, which are the easiest to measure, are also the most important and information-rich data for the order matrix calculation. The effect of each RDC on the calculation depends on the location of the corresponding vector in the structure. The direct RDC of a stereocenter is important to the configuration determination, but the configuration of stereocenters devoid of protons can also be obtained from analysis of nearby RDCs.     

Computer‐Assisted 3D Structure Elucidation of Natural Products using Residual Dipolar Couplings

An enhanced computer‐assisted procedure for the determination of the relative configuration of natural products, which starts from the molecular formula and uses a combination of conventional 1D and 2D NMR spectra, and residual dipolar couplings (RDCs), is reported. Having already the data acquired (1D/2D NMR and RDCs), the procedure begins with the determination of the molecular constitution using standard computer‐assisted structure elucidation (CASE) and is followed by fully automated determination of relative configuration through RDC analysis. In the case of moderately flexible molecules the simplest data‐explaining conformational model is selected by the use of the Akaike information criterion.     

Conformational analysis of a tetrasaccharide based on NMR spectroscopy and molecular dynamics simulations

The conformational preference of the human milk oligosaccharide lacto-N-neotetraose, beta-d-Galp-(1 --> 4)-beta-d-GlcpNAc-(1 --> 3)-beta-d-Galp-(1 --> 4)-d-Glcp, has been analyzed using (1)H,(1)H T-ROESY and (1)H,(13)C trans-glycosidic J coupling experiments in isotropic solution and (1)H,(13)C residual dipolar couplings (RDCs) obtained in lyotropic liquid crystalline media. Molecular dynamics simulations of the tetrasaccharide with explicit water as the solvent revealed that two conformational states are significantly populated at the psi glycosidic torsion angle, defined by C(anomeric)-O-C-H, of the (1 --> 3)-linkage. Calculation of order parameters, related to the molecular shape, were based on the inertia tensor and fitting of experimental RDCs to different conformational states showed that psi(+) > 0 degrees is the major and psi(-) < 0 degrees is the minor conformation in solution, in complete agreement with a two-state analysis based on the T-ROESY data. Attention was also given to the effect of salt (200 mM NaCl) in the anisotropic medium, which was a ternary mixture of n-octyl-penta(ethylene glycol), n-octanol, and D(2)O.     

16-fold degeneracy of peptide plane orientations from residual dipolar couplings: analytical treatment and implications for protein structure determination

Residual dipolar couplings (RDCs) measured for internally rigid molecular fragments provide important information about the relative orientations of these fragments. Dependent on the symmetry of the alignment tensor and the symmetry of the molecular fragment, however, there generally exist more than one solution for the fragment orientation consistent with the measured RDCs. Analytical solutions are presented that describe the complete set of orientations of internally rigid fragments that are consistent with multiple dipolar couplings measured in a single alignment medium that is rhombic. For the first time, it is shown that, for a planar fragment such as the peptide plane, there generally exist 16 different solutions with their analytical expressions presented explicitly. The presence of these solutions is shown to be highly relevant for standard structure determination protocols using RDCs to refine molecular structures. In particular, when using standard protein structure refinement with RDCs that were measured in a single alignment medium as constraints, it is found that often more than one of the peptide plane solutions is physically viable; i.e., despite being consistent with measured RDCs, the local backbone structure can be incorrect. On the basis of experimental and simulated examples, it is rationalized why protein structures that are refined against RDCs measured in a single medium can have lower resolution (precision) than one would expect on the basis of the experimental accuracy of the RDCs. Conditions are discussed under which the correct solution can be identified.     

Characterization of stereochemistry and molecular conformation using solid-state NMR tensors

A solid-state NMR technique is described for establishing stereochemistry using the natural product terrein as a model compound. This method involves comparison of experimental (13)C tensor principal values with ab initio computed values for all possible computer-generated stereoisomers. In terrein the relative stereochemistry is confirmed by NMR to be 2R*,3S with high statistical probability (>99.5%). The proposed approach also simultaneously verifies the molecular conformation of the two hydroxy groups in terrein established by X-ray data. It is sufficient to use only shift tensor values at carbons 2 and 3, the stereocenters, to characterize both the stereochemistry and molecular conformations. The solid-state NMR method appears to be especially useful for determining relative stereochemistry of compounds or their derivatives that are difficult to crystallize.     

Measurement of 1H-1H residual dipolar coupling constants for structural studies of medium size molecules.

Residual dipolar coupling constants (RDCs) are being increasingly applied to elucidate the configuration and conformation of small organic molecules, peptides and oligosaccharides. In this paper we describe a set of robust 1D NMR methods for accurate and precise measurement of proton-proton RDCs of small and medium size molecules. The performance of these techniques is not impeded by the presence of overlapping and broad (1)H multiplets that are typically observed for such molecules in weakly aligned media. The use of these techniques provides access to a large pool of proton-proton RDCs opening new avenues for the solution structure elucidation of medium size molecules by NMR. The techniques are illustrated on the determination of the alignment tensor of the reducing monosaccharide ring of cellobiose and the determination of the relative configuration of sodium cholate.     

Limited flexibility of lactose detected from residual dipolar couplings using molecular dynamics simulations and steric alignment methods

The conformational flexibility of lactose in solution has been investigated by residual dipolar couplings (RDCs). One-bond carbon-proton and proton-proton coupling constants have been measured in two oriented media and interpreted in combination with molecular dynamics simulations (MD). Two different approaches, known as PALES (Zweckstetter et al., J. Am. Chem. Soc. 2000, 122, 3791-3792) and TRAMITE (Azurmendi et al., J. Am. Chem. Soc. 2002, 124, 2426-2427), have been used to determine the alignment tensor from a shape-induced alignment model with the oriented medium. The steric alignment of the structures from several MD trajectories has provided ensemble averaged RDCs that have been compared with the experimental ones. The obtained results reveal the almost exclusive presence of a major low energy region defined as syn-phi/syn-psi (> 97%), for which sampling occurs in a dynamic manner. This result satisfactorily agrees with that determined by standard NOE-based methods.     

Long‐Range Residual Dipolar Couplings: A Tool for Determining the Configuration of Small Molecules

Together with NOE and J coupling, one‐bond residual dipolar coupling (RDC), which reports on the three‐dimensional orientation of an internuclear vector in the molecular frame, plays an important role in the conformation and configuration analysis of small molecules in solution by NMR spectroscopy. When the molecule has few C? H bonds, or too many bonds are in parallel, the available RDCs may not be sufficient to obtain the alignment tensor used for structure elucidation. Long‐range RDCs that connect nuclei over multiple bonds are normally not parallel to the single bonds and therefore complement one‐bond RDCs. Herein we present a method for extracting the long‐range RDC of a chosen proton or group of protons to all remotely connected carbon atoms, including non‐protonated carbon atoms. Alignment tensors fitted directly to the total long‐range couplings (T=J+D) enabled straightforward analysis of both the long‐range and one‐bond RDCs for strychnine.     

Side chain orientation from methyl 1H-1H residual dipolar couplings measured in highly deuterated proteins

High-level deuteration is a prerequisite for the study of high molecular weight systems using liquid-state NMR. Here, we present new experiments for the measurement of proton-proton dipolar couplings in CH(2)D methyl groups of (13)C labeled, highly deuterated (70-80%) proteins. (1)H-(1)H residual dipolar couplings (RDCs) have been measured in two alignment media for 57 out of 70 possible methyl containing residues in the 167-residue flavodoxin-like domain of the E. coli sulfite reductase. These data yield information on the orientation of the methyl symmetry axis with respect to the molecular alignment frame. The alignment tensor characteristics were obtained very accurately from a set of backbone RDCs measured on the same protein sample. To demonstrate that accurate structural information is obtained from these data, the measured methyl RDCs for Valine residues are analyzed in terms of chi(1) torsion angles and stereospecific assignment of the prochiral methyl groups. On the basis of the previously determined backbone solution structure of this protein, the methyl RDC data proved sufficient to determine the chi(1) torsion angles in seven out of nine valines, assuming a single-rotamer model. Methyl RDCs are complementary to other NMR data, for example, methyl-methyl NOE, to determine side chain conformation in high molecular weight systems.     

Multiple alignment tensors from a denatured protein

The structural content of the denatured state has yet to be fully characterized. In recent years, large residual dipolar couplings (RDCs) from denatured proteins have been observed under alignment conditions produced by bicelles and strained polyacrylamide gels. In this report, we describe efforts to extend our picture of the residual structure in denatured nuclease by measuring RDCs with multiple alignment tensors. Backbone amide 15N-1H RDCs were collected from 4 M urea for a total of eight RDC data sets. The RDCs were analyzed by singular value decomposition (SVD) to determine the number of independent alignment tensors present in the data. On the basis of the resultant singular values and propagated error estimates, it is clear that there are at least three independent alignment tensors. These three independent RDC datasets can be reconstituted as orthogonal linear combinations, (OLC)-RDC datasets, of the eight actually recorded. The first, second, and third OLC-RDC datasets are highly robust to the removal of any single experimental RDC dataset, establishing the presence of three independent alignment tensors, sampled well above the level of experimental uncertainty. The observation that the RDC data span three or more dimensions of the five-dimensional parameter space demonstrates that the ensemble average structure of denatured nuclease must be asymmetric with respect to these three orthogonal principal axes, which is not inconsistent with earlier work demonstrating that it has a nativelike topology.     

Determining the absolute configuration of two marine compounds using vibrational chiroptical spectroscopy

Chiroptical techniques are increasingly employed for assigning the absolute configuration of chiral molecules through comparison of experimental spectra with theoretical predictions. For assignment of natural products, electronic chiroptical spectroscopies such as electronic circular dichroism (ECD) are routinely applied. However, the sensitivity of electronic spectral parameters to experimental conditions and the theoretical methods employed can lead to incorrect assignments. Vibrational chiroptical methods (vibrational circular dichroism, VCD, and Raman optical activity, ROA) provide more reliable assignments, although they, in particular ROA, have been little explored for assignments of natural products. In this study, the ECD, VCD, and ROA chiroptical spectroscopies are evaluated for the assignment of the absolute configuration of a highly flexible natural compound with two stereocenters and an asymmetrically substituted double bond, the marine antibiotic Synoxazolidinone A (SynOxA), recently isolated from the sub-Arctic ascidian Synoicum pulmonaria. Conformationally averaged nuclear magnetic resonance (NMR), ECD, Raman, ROA, infrared (IR) and VCD spectral parameters are computed for the eight possible stereoisomers of SynOxA and compared to experimental results. In contrast to previously reported results, the stereochemical assignment of SynOxA based on ECD spectral bands is found to be unreliable. On the other hand, ROA spectra allow for a reliable determination of the configuration at the double bond and the ring stereocenter. However, ROA is not able to resolve the chlorine-substituted stereogenic center on the guanidinium side chain of SynOxA. Application of the third chiroptical method, VCD, indicates unique spectral features for all eight SynOxA isomers in the theoretical spectra. Although the experimental VCD is weak and restricted by the limited amount of sample, it allows for a tentative assignment of the elusive chlorine-substituted stereocenter. VCD chiroptical analysis of a SynOxA derivative with three stereocenters, SynOxC, results in the same absolute configuration as for SynOxA. Despite the experimental challenges, the results convincingly prove that the assignment of absolute configuration based on vibrational chiroptical methods is more reliable than for ECD.     

Highly populated turn conformations in natively unfolded tau protein identified from residual dipolar couplings and molecular simulation

Tau, a natively unstructured protein that regulates the organization of neuronal microtubules, is also found in high concentrations in neurofibrillary tangles of Alzheimer's disease and other neurodegenerative disorders. The conformational transition between these vastly different healthy and pathological forms remains poorly understood. We have measured residual dipolar couplings (RDCs), J-couplings, and nuclear Overhauser enhancement (NOE) in construct K18 of tau, containing all four repeat domains R1-R4. NHN RDCs were compared with prediction on the basis of a statistical model describing the intrinsic conformational sampling of unfolded proteins in solution. While local variation and relative amplitude of RDCs agrees with propensity-based prediction for most of the protein, homologous sequences in each repeat domain (DLKN, DLSN, DLSK, and DKFD in repeats R1-R4) show strong disagreement characterized by inversion of the sign of the central couplings. Accelerated molecular dynamic simulations (AMD) in explicit solvent revealed strong tendencies to form turns, identified as type I beta-turns for repeats R1-R3. Incorporation of the backbone dihedral sampling resulting from AMD into the statistical coil model closely reproduces experimental RDC values. These localized sequence-dependent conformational tendencies interrupt the propensity to sample more extended conformations in adjacent strands and are remarkably resistant to local environmental factors, as demonstrated by the persistence of the RDC signature even under harsh denaturing conditions (8 M urea). The role that this specific conformational behavior may play in the transition to the pathological form is discussed.     

Residual Dipolar-Coupling-Based Conformational Comparison of Noncovalent Ubiquitin Homodimer with Covalently Linked Diubiquitin

Although the conformation of the polymer chain of Ubiquitin (Ub) mainly depends on the type of isopeptide linkage connecting two Ub molecules, the non-covalent (noncovalent) interaction between two Ub molecules within the chain could also tune their conformational preference. Here, we studied the conformation of noncovalently formed Ub dimers in solution using residual dipolar couplings (RDCs). Comparing the RDC derived alignment tensor of the noncovalently formed dimer with the two most abundant (K11 and K48) covalent linked Ub dimers revealed that the conformation of K11 linked and noncovalent Ub dimers were similar. Between the various NMR and crystal structures of K11 linked Ub dimers, RDC tensor analysis showed that the structure of K11 linked dimer crystalized at neutral pH is similar to noncovalent dimer. Analogous to the experimental study, the comparison of predicted order matrix of various covalent Ub dimers with that of the experimentally determined order matrix of noncovalent Ub dimer also suggests that the conformation of K11 linked dimers crystalized at neutral pH is similar to the noncovalent dimer.     

Is Enantiomer Assignment Possible by NMR Spectroscopy Using Residual Dipolar Couplings from Chiral Nonracemic Alignment Media?—A Critical Assessment

Insensitive towards inversion: Can residual dipolar couplings (RDCs) and other anisotropic NMR observables be used to determine absolute configuration? A critical assessment of recent approaches is provided to determine the absolute configuration from RDCs.     

Catalytic Stereoselective Borylative Transannular Reactions

Medium‐sized carbocycles containing an α,β‐unsaturated ketone moiety as Michael acceptor site and a ketone moiety as internal electrophilic site are ideal substrates to conduct Cu(I)‐catalyzed conjugated borylation followed by electrophilic intramolecular trapping that results into a pioneer transannular borylative ring closing reaction. The relative configuration of three adjacent stereocenters is controlled, giving access to a single diastereoisomer for a wide range of substrates tested. Moreover, when a chiral ligand is incorporated, the reaction provides enantioenriched polycyclic products with up to 99 % ee.     

Structure elucidation of phenoxathiin-based thiacalix[4]arene conformations using NOE and RDC data

Phenoxathiin-based thiacalix[4]arene, obtained by the acid-catalysed rearrangement of the corresponding spirodienone derivative, was immobilized using the alkylation with chloroacetonitrile to yield three (out of four theoretically possible) stereoisomers. As the conformational outcome of the reaction could not be unambiguously assigned using standard NMR techniques, the method of Residual Dipolar Coupling constants (RDCs) was applied. The measuring of an anisotropic through-space dipole-dipole interactions in the lyotropic liquid crystalline alignment medium (PELG, poly-γ-ethyl-l-glutamate, and PBLG, poly-γ-benzyl-l-glutamate) enabled the assignment of the individual conformers. The usefulness of this approach was finally confirmed by the X-ray crystallography data.     

Mapping the potential energy landscape of intrinsically disordered proteins at amino Acid resolution

Intrinsically disordered regions are predicted to exist in a significant fraction of proteins encoded in eukaryotic genomes. The high levels of conformational plasticity of this class of proteins endows them with unique capacities to act in functional modes not achievable by folded proteins, but also places their molecular characterization beyond the reach of classical structural biology. New techniques are therefore required to understand the relationship between primary sequence and biological function in this class of proteins. Although dependences of some NMR parameters such as chemical shifts (CSs) or residual dipolar couplings (RDCs) on structural propensity are known, so that sampling regimes are often inferred from experimental observation, there is currently no framework that allows for a statistical mapping of the available Ramachandran space of each amino acid in terms of conformational propensity. In this study we develop such an approach, combining highly efficient conformational sampling with ensemble selection to map the backbone conformational sampling of IDPs on a residue specific level. By systematically analyzing the ability of NMR data to map the conformational landscape of disordered proteins, we identify combinations of RDCs and CSs that can be used to raise conformational degeneracies inherent to different data types, and apply these approaches to characterize the conformational behavior of two intrinsically disordered proteins, the K18 domain from Tau protein and N(TAIL) from measles virus nucleoprotein. In both cases, we identify the enhanced populations of turn and helical regions in key regions of the proteins, as well as contiguous strands that show clear and enhanced polyproline II sampling.     

Simultaneous assignment of all diastereotopic protons in strychnine using RDCs: PELG as alignment medium for organic molecules

The concept of using residual dipolar couplings (RDCs) for the structure determination of organic molecules is applied to the simultaneous assignment of all diastereotopic protons in strychnine. To use this important NMR parameter the molecule has to be aligned in the magnetic field. Here we present a new alignment medium for organic substrates. The optimization of the alignment properties of mixtures of poly-gamma-ethyl-L-glutamate (PELG) and CDCl(3) are described and the alignment properties of PELG at different concentrations are evaluated. A comparison of PELG with poly-gamma-benzyl-L-glutamate (PBLG) shows considerable differences in the magnitude of alignment for strychnine in the two alignment media. PELG induces a lower degree of order and makes the measurement of residual dipolar couplings (RDCs) in strychnine possible. All one-bond C-H RDCs of strychnine in PELG were determined by using 2D heteronuclear single quantum coherence (HSQC) spectroscopy. The strategy for the extraction of RDCs for methylene groups is described in detail. The RDCs and order parameters are used to assign pairs of diastereotopic protons. This methodology can distinguish not only one pair of diastereotopic protons but it can be used to assign all pairs of diastereotopic protons simultaneously. Two different calculation approaches to achieve this task are described in detail.