Yizhou Liu

Determination of Relative Configuration from Residual Chemical Shift Anisotropy

Determination of relative configuration is frequently a rate-limiting step in the characterization of small organic molecules. Solution NMR-based nuclear Overhauser effect and scalar J-coupling constants can provide useful spatial information but often fail when stereocenters are separated by more than 4-5 Å. Residual dipolar couplings (RDCs) can provide a means of assigning relative configuration without limits of distance between stereocenters. However, sensitivity limits their application. Chemical shift is the most readily measured NMR parameter, and partial molecular alignment can reveal the anisotropic component of the chemical shift tensor, manifested as residual chemical shift anisotropy (RCSA). Hence, (13)C RCSAs provide information on the relative orientations of specific structural moieties including nonprotonated carbons and can be used for stereochemical assignment. Herein, we present two robust and sensitive methods to accurately measure and apply (13)C RCSAs for stereochemical assignment. The complementary techniques are demonstrated with five molecules representing differing structural classes.

Homodimericin A: A Complex Hexacyclic Fungal Metabolite

Microbes sense and respond to their environment with small molecules, and discovering these molecules and identifying their functions informs chemistry, biology, and medicine. As part of a study of molecular exchanges between termite-associated actinobacteria and pathogenic fungi, we uncovered a remarkable fungal metabolite, homodimericin A, which is strongly upregulated by the bacterial metabolite bafilomycin C1. Homodimericin A is a hexacyclic polyketide with a carbon backbone containing eight contiguous stereogenic carbons in a C20 hexacyclic core. Only half of its carbon atoms have an attached hydrogen, which presented a significant challenge for NMR-based structural analysis. In spite of its microbial production and rich stereochemistry, homodimericin A occurs naturally as a racemic mixture. A plausible nonenzymatic reaction cascade leading from two identical achiral monomers to homodimericin A is presented, and homodimericin A’s formation by this path, a six-electron oxidation, could be a response to oxidative stress triggered by bafilomycin C1.

Unequivocal determination of complex molecular structures using anisotropic NMR measurements

Picking structures out of a lineup Pharmaceutical research relies critically on determining the correct structures of numerous complex molecules. When well-ordered crystals are not available for x-ray analysis, nuclear magnetic resonance (NMR) spectroscopy is the most common structure-elucidation method. However, sometimes it is hard to distinguish isomers with similar spectra. Liu et al. showcase a protocol that combines computer modeling with anisotropic NMR data acquired using gel-aligned samples. Because of its uniform sensitivity to relative bond orientations across the whole molecular framework, the method overcomes common pitfalls that can lead to invalid structure assignments. Science, this issue p. eaam5349 A nuclear magnetic resonance method applied to aligned molecules helps to elucidate their complex structures. INTRODUCTION Single-crystal x-ray diffraction studies represent the gold standard for unequivocal establishment of molecular structure and configuration. For molecules that will not crystallize or that form poorly-diffracting crystals, alternative methods must be used. Crystalline sponges and atomic force microscopy are techniques with increasing potential, although nuclear magnetic resonance (NMR) spectroscopy methods provide the primary viable alternative means to determine molecular structures. However, misinterpretation of NMR data—as a result of poor data quality, inappropriate experiment selection, or investigator bias—has led to burgeoning numbers of structure revision reports. Clearly, the development of a method to more effectively use NMR data and simultaneously quell reports of incorrect structures would be highly beneficial. RATIONALE Combining computer-assisted structure elucidation (CASE) algorithms and density functional theory (DFT) calculations with measured anisotropic NMR parameters, specifically residual dipolar coupling (RDC), and residual chemical shift anisotropy (RCSA) holds strong promise as an effective alternative means of assigning three-dimensional (3D) molecular structures. Anisotropic NMR data provide a spatial view of the relative orientations between bonds (RDCs) and chemical shielding tensors (RCSAs), regardless of the separation between the bonds and atoms, respectively. Hence, these data are sensitive reporters of global structural validity. The combination of DFT calculations and anisotropic NMR data represents an orthogonal approach to conventional NMR data interpretation that is not subject to the interpretational biases of human investigators and, as such, mitigates the risk of incorrect structure assignments. RESULTS Anisotropic NMR data can be used directly to evaluate the validity of investigator-proposed structures or can be combined with a CASE program in conjunction with DFT calculations for both structural proposal and validation. The RDC data are typically used to structurally define C-H bond vectors, whereas the RCSA data report on the chemical shift tensors of both protonated and nonprotonated carbons, the latter only accessible by long-range RDC data that are difficult to measure and interpret. These data are used to evaluate a given structure proposal on the basis of the agreement between the experimentally measured data and theoretical values calculated for the corresponding 3D DFT models. When structures generated by a CASE program are being considered, the method only requires a multidimensional NMR data set of sufficient quality and sophistication to allow the CASE program to generate a set of proposals that contains the correct structure of the molecule. The molecules being studied should also be amenable to modern DFT calculations for 3D model building. The CASE program output is sorted on the basis of cumulative error between experimental and calculated 13C data for the ensemble of structures generated, and the best-fitting molecules are subsequently subjected to DFT calculation for analysis. Results obtained using the proposed method demonstrate its applicability to a diverse range of complex molecules, each of which challenged the investigators originally reporting the structures. CONCLUSION The technique described here represents a potential paradigm shift from conventional NMR data interpretation and can provide an unequivocal and unbiased confirmation of interatomic connectivity and relative configuration for organic and natural product structures. The principle of residual dipolar coupling (RDC)–based model differentiation is shown using aquatolide as an example. The revised structure of aquatolide is shown on the top left, with the originally reported structure shown on the bottom left. The selected C-H bond vectors in the two structures have different orientations, as is evident after translating them to the same origin in the middle diagrams. Theoretical RDC values associated with these vectors can be calculated for each model on the basis of the experimentally determined alignment tensor. Correlation data are shown for only the four highlighted CH groups, although the alignment tensor was actually determined using all available data. The originally proposed (incorrect) structure clearly shows poorer agreement between the calculated and experimental data. Assignment of complex molecular structures from nuclear magnetic resonance (NMR) data can be prone to interpretational mistakes. Residual dipolar couplings and residual chemical shift anisotropy provide a spatial view of the relative orientations between bonds and chemical shielding tensors, respectively, regardless of separation. Consequently, these data constitute a reliable reporter of global structural validity. Anisotropic NMR parameters can be used to evaluate investigators’ structure proposals or structures generated by computer-assisted structure elucidation. Application of the method to several complex structure assignment problems shows promising results that signal a potential paradigm shift from conventional NMR data interpretation, which may be of particular utility for compounds not amenable to x-ray crystallography.

Observation of potentially troublesome (2) JCC correlations in 1,1-ADEQUATE spectra.

Despite the tremendous usage of HMBC to establish long‐range 1H–13C and 1H–15N heteronuclear correlations, an inherent drawback of the experiment is the indeterminate nature of the nJXH correlations afforded by the experiment. A priori there is no reliable way of determining whether a given nJCH correlation is, for example, via two‐, three‐, or sometimes even four‐bonds. This limitation of the HMBC experiment spurred the development of the ADEQUATE family of NMR experiments that rely on, in the case of 1,1‐ADEQUATE, an out‐and‐back transfer of magnetization via the 1JCC homonuclear coupling constant, which is significantly larger than nJCC (where n = 2–4) couplings in most cases. Hence, the 1,1‐ADEQUATE experiment has generally been assumed to unequivocally provide the equivalent of 2JCH correlations. The recent development of the 1,1‐ and 1,n‐HD‐ADEQUATE experiments that can provide homodecoupling for certain 1JCC and nJCC correlations has increased the sensitivity of the ADEQUATE experiments significantly and can allow acquisition of these data in a fraction of the time required for the original iterations of this pulse sequence. With these gains in sensitivity, however, there occasionally come unanticipated consequences. We have observed that the collapse of proton multiplets, in addition to providing better s/n for the desired 1JCC correlations can facilitate the observation of typically weaker 2JCC correlations across intervening carbonyl resonances in 1,1‐HD‐ADEQUATE spectra. Several examples are shown, with the results supported by the measurement of the 2JCC coupling constants in question using J‐modulated‐HD‐ADEQUATE and DFT calculations. Copyright

Using Electron Paramagnetic Resonance Spectroscopy To Facilitate Problem Solving in Pharmaceutical Research and Development

As new chemical methodologies driven by single-electron chemistry emerge, process and analytical chemists must develop approaches to rapidly solve problems in this nontraditional arena. Electron paramagnetic resonance spectroscopy has been long known as a preferred technique for the study of paramagnetic species. However, it is only recently finding application in contemporary pharmaceutical development, both to study reactions and to track the presence of undesired impurities. Several case studies are presented here to illustrate its utility in modern pharmaceutical development efforts.

Selecting the Most Appropriate NMR Experiment to Access Weak and/or Very Long-Range Heteronuclear Correlations.

Heteronuclear long-range NMR experiments are well established as essential NMR techniques for the structure elucidation of unknown natural products and small molecules. It is generally accepted that the absence of a given (n)JXH correlation in an HMBC or HSQMBC spectrum would automatically place the proton at least four bonds away from the carbon in question. This assumption can, however, be misleading in the case of a mismatch between the actual coupling constant and the delay used to optimize the experiment, which can lead to structural misassignments. Another scenario arises when an investigator, for whatever reason, needs to have access to very long-range correlations to confirm or refute a structure. In such cases, a conventional HMBC experiment will most likely fail to provide the requisite correlation, regardless of the delay optimization. Two recent methods for visualizing extremely weak or very long-range connectivities are the LR-HSQMBC and the HSQMBC-TOCSY experiments. Although they are intended to provide similar structural information, they utilize different transfer mechanisms, which differentiates the experiments, making each better suited for specific classes of compounds. In this report we have sought to examine the considerations implicit in choosing the best experiment to access weak or very long-range correlations for different types of molecules.

Mechanistic insight into oxidized N,N-dimethylacetamide as a source of formaldehyde related derivatives

A hemiaminal derivative identified during preparation of the penultimate of ceftolozane 1, mandated a thorough investigation of the source of the impurity. N,N-Dimethylacetamide (DMAc) subjected to oxidation was found to be the culprit generating oxidized products that serve as sources of the hemiaminal derivative. Identification of DMAc as a source of formaldehyde derivatives and the mechanism of DMAc oxidation are elaborated.

A Practical Strategy for the Accurate Measurement of Residual Dipolar Couplings in Strongly Aligned Small Molecules

Accurate measurement of residual dipolar couplings (RDCs) requires an appropriate degree of alignment in order to optimize data quality. An overly weak alignment yields very small anisotropic data that are susceptible to measurement errors, whereas an overly strong alignment introduces extensive anisotropic effects that severely degrade spectral quality. The ideal alignment amplitude also depends on the specific pulse sequence used for the coupling measurement. In this work, we introduce a practical strategy for the accurate measurement of one-bond 13C-1H RDCs up to a range of ca. -300 to +300 Hz, corresponding to an alignment that is an order of magnitude stronger than typically employed for small molecule structural elucidation. This strong alignment was generated in the mesophase of the commercially available poly-γ-(benzyl-L-glutamate) polymer. The total coupling was measured by the simple and well-studied heteronuclear two-dimensional J-resolved experiment, which performs well in the presence of strong anisotropic effects. In order to unequivocally determine the sign of the total coupling and resolve ambiguities in assigning total couplings in the CH2 group, coupling measurements were conducted at an isotropic condition plus two anisotropic conditions of different alignment amplitudes. Most RDCs could be readily extracted from these measurements whereas more complicated spectral effects resulting from strong homonuclear coupling could be interpreted either theoretically or by simulation. Importantly, measurement of these very large RDCs actually offers significantly improved data quality and utility for the structure determination of small organic molecules.

Trichophycins B–F, Chlorovinylidene-Containing Polyketides Isolated from a Cyanobacterial Bloom

NMR-guided isolation (based on 1D 1H and 13C NMR resonances consistent with a chlorovinylidene moiety) resulted in the characterization of five new highly functionalized polyketides, trichophycins B-F (1-5), and one nonchlorinated metabolite tricholactone (6) from a collection of Trichodesmium bloom material from the Gulf of Mexico. The planar structures of 1-6 were determined using 1D and 2D NMR spectroscopy, mass spectrometry, and complementary spectroscopic procedures. Absolute configuration analysis of 1 and 2 were carried out by 1H NMR analysis of diastereomeric Mosher esters in addition to ECD spectroscopy, J-based configuration analysis, and DFT calculations. The absolute configurations of 3-6 were proposed on the basis of comparative analysis of 13C NMR chemical shifts, relative configurations, and optical rotation values to compounds 1 and 2. Compounds 1-5 represent new additions to the trichophycin family and are hallmarked by a chlorovinylidene moiety. These new trichophycins and tricholactone (1-6) feature intriguing variations with respect to putative biosynthetic starting units, halogenation, and terminations, and trichophycin E (4) features a rare alkynyl bromide functionality. The phenyl-containing trichophycins showed low cytotoxicity to neuro-2A cells, while the alkyne-containing trichophycins showed no toxicity.

Chapter 2:Assigning Molecular Configuration by Nuclear Magnetic Resonance

In this chapter, we have subdivided the important topic of assigning molecular configuration into two broad categories: assigning relative configuration and assigning absolute configuration. The former is more extensively covered since it lends itself more readily to nuclear magnetic resonance (NMR) methods and is probably more familiar to many readers. The latter category is treated more briefly and reflects the narrower focus of NMR-based work in that area, as well as the relative difficulty of assigning absolute configuration. This chapter is not intended to be a rigorous treatise on methods for stereochemical analysis, but rather is intended to provide the interested reader with a pathway into the extensive literature associated with this area of NMR investigation that can be explored in more depth as necessary to solve a given problem.