Natural Abundance, Single-Scan 13C-13C-based Structural

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Natural Abundance, Single-Scan 13C-13C-based Structural Elucidations by Dissolution DNP NMR Martins Otikovs, Gregory L Olsen, Eriks Kupce, and Lucio Frydman J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b12216 • Publication Date (Web): 16 Jan 2019 Downloaded from http://pubs.acs.org on January 16, 2019

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Journal of the American Chemical Society

Natural Abundance, Single-Scan 13C-13C-based Structural Elucidations by Dissolution DNP NMR Martins Otikovsa, Gregory L. Olsena, Ēriks Kupčeb, Lucio Frydmana,* aDepartment bBruker

of Chemical and Biological Physics, Weizmann Institute of Science, Rehovot 7610001, Israel

UK Limited, Banner Lane, Coventry CV4 9GH, UK

Supporting Information Placeholder ABSTRACT: While 13C-based INADEQUATE experiments offer an

attractive alternative for establishing molecular structures, they suffer from low sensitivities arising from the scarcity of spin pairs present at natural abundance. Herein we demonstrate that dissolution dynamic nuclear polarization (dDNP) provides sufficient sensitivity to acquire 1D 13C INADEQUATE spectra in a single scan and at natural abundance. Moreover, if steps are adopted to endow sub-Hertz precision to these measurements, they allow one to measure carbon-carbon J couplings over both one and multiple bonds for each chemical site. As these JCCcouplings are usually sufficiently distinct to enable univocal pairing of the nuclei involved, essentially the same information as in 2D INADEQUATE can be obtained. The feasibility of the method is demonstrated for a range of compounds, including natural products such as α-pinene, menthol and limonene. Features and extensions of this approach are briefly discussed.

The Incredible Natural Abundance DoublE QUAntum Transfer Experiment (INADEQUATE) is one of the most attractive tools available for NMR-based structural analyses. INADEQUATE leverages the strictly pair-wise carbon-carbon information that arises at natural isotopic abundance, to provide a direct picture of interatomic connectivities within a molecule1. At the same time, the analytical applications of INADEQUATE have been challenged by the method’s low sensitivity: the same reliance on natural abundance 13C-13C J-couplings (JCCs) that endows INADEQUATE with its simplicity implies that its signals derive from only ≈0.01% of all carbons present in the sample. By endowing NMR with unprecedented enhancements arising from both cryogenic operation and from transferring polarization from electrons to nuclei, Dynamic Nuclear Polarization (DNP)2 can provide an answer to this challenge. This has been recently shown with carbon-carbon correlations at natural abundance under magic-angle-spinning3–6. DNP measurements in solutions are also feasible, thanks to the advent of rapid dissolution procedures7–10. The ensuing dDNP approach relies on suddenly melting the polarization-enhanced, cryogenic pellet using an aliquot of super-heated solvent, followed by the rapid transfer of the hyperpolarized liquid into an NMR tube for a conventional solution-state observation. While this could, in principle, offset the sensitivity penalties associated with the INADEQUATE scheme, the measurement of carbon-carbon couplings in hyperpolarized solutions will still be challenged by a number of

factors that were absent in the solid state counterpart. For instance the latter were based on multi-scan, hours-long experiments, whereas dDNP is largely restricted to a single-scan acquisition. Moreover, unless combined with ultrafast procedures11,12, the NMR-invisible double-quantum states underlying INADEQUATE will demand the use of multi-scan 2D approaches for their characterization. On the other hand, the fact that both one- and multiple-bond JCC couplings are highly structure-sensitive parameters, provides an alternative avenue for establishing structural networks via 1D versions of INADEQUATE13–18. This route relies on measuring the multiple JCC couplings affecting every individual 13C site; if accurately characterized and sufficiently distinct to enable univocal 13C-13C pairings, this allows one to extract –in the absence of spectral overlap– the kind of information contained in a 2D INADEQUATE counterpart, but in a 1D fashion. To make such double-quantumfiltered measurements compatible with the single-scan nature of dDNP, they will therefore require sub-Hz line shapes, as well as a nearly complete suppression of the intense signals associated with isolated 13C nuclei that would otherwise obscure small (≤5Hz) but important multiple-bond JCC information. Herein we show that these requirements can be satisfied by dDNP, enabling single-scan structural measurements at natural abundance for a range of natural products. The targets of this study included both self-glassing liquids and room temperature solids; for the former DNP samples were prepared by dissolving 20 mM BDPA (SigmaAldrich) into ≈100150 µL of the neat compound; for the latter 20mM BDPA was dissolved in ≈200 µL made from 1:1 mixtures with toluene to ensure the formation of a glassy matrix. In all cases samples were hyperpolarized by irradiating the electrons in the comixed BDPA with 100 mW of microwaves for ca. 3 hours at 1.2 K in an Oxford Instruments Hypersense® at a frequency of 94.085 GHz, corresponding to the sum of the electron and 13C Larmor frequencies. For the NMR measurements samples were dissolved in 3 mL of natural abundance superheated (138 ˚C) methanol at 9 bar, pushed between the polarizing and NMR magnets in under 3 seconds through a plastic line by 5.7 bar of He gas, and injected in a 5 mm NMR sample tube placed in a 500 MHz Varian iNova® spectrometer equipped with a broadband HX probe (Fig. 1). After this transfer a delay of ca. 5 s was introduced prior to the execution of an NMR pulse sequence in order to achieve optimized lineshapes, and prevent magnetization losses during subsequent gradient-based coherence manipulations. All post-

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dissolution sample volumes were 500 μL and final sample concentrations were in the 100-250 mM range. A tuning sample was used for pre-shimming and pre-tuning before performing each dissolution. The pulse sequence used in these measurements was a slightly modified proposal of the original INADEQUATE pulse sequence19, tailored to the single-shot nature of dDNP NMR (Fig. 1b). Modifications included replacing 180° refocusing by a composite

Figure 1. Schematic representation of the experimental setup (left) and pulse sequence (right) used to test 1D 13C dDNP INADEQUATE at natural abundance. A 1:2 Gz gradient (1 and 2 ms at 16 G/cm respectively) was used to choose the coherence transfer pathways indicated in the lower panel by the continuous lines; FID signals were acquired over 1 second.

90𝑜𝑦 ― 180𝑜𝑥 ― 90𝑜𝑦 pulse20, selecting only one of the two doubleto-single quantum conversion pathways to better suppress resonances originating from uncoupled 13C nuclei by strong coherence selection gradients, and maximizing the 2Q1Q conversion efficiency by the application of a 120° read-out pulse21,22. Representative experimental results for the analyzed compounds are shown in Fig. 2, with a more detailed analysis of selected sites presented in Fig. 3. In all cases multiple antiphase doublets originating in the distinct one- and multiple-bond JCC couplings affecting each 13C site are evident. One-bond splittings range from ≈35-75 Hz depending on the hybridization and bonding of the carbon site in question23, while two- and threebond splittings in the ≈3-10 Hz range24 are also discernible. The ca. 10-12% nuclear polarization levels provided post-dissolution by the DNP setup employed allowed us to acquire, in a single scan, INADEQUATE spectra possessing similar sensitivities as ≈24h runs on neat compounds under thermal polarization conditions (see Fig. S1 for details). These substantial gains materialized for all spin pairs, despite the fact that the doublequantum pumping delay T/2 was set solely for optimizing intraaliphatic one-bond transfers (JCC = 40 Hz)23. Quality line shapes with half-widths ≤1Hz were in all cases achieved, allowing us to resolve the multiple similar couplings associated with a given carbon site, and discern the bonding patterns among chemically inequivalent neighbors based on the coincidence of specific JCC values –even when these are very similar. Notice for instance the method’s ability to distinguish among all coupling partners within toluene’s aromatic ring, despite the fact that the carbons’ identical hybridization requires for this a 1JCC accuracy of ≈0.1 Hz (Fig. 3A). The quality of these double-quantum-filtered line shapes also enabled measuring longer-range, multiple-bond connectivities, requiring a nearly perfect suppression of the central 13C signals (Fig. 3A and B). A feature evidenced by these experiments concerns asymmetrical doublet lines –both in terms of their intensities and of their positions around the respective isotropic chemical shifts.

Consider for instance the splittings arising between C2 and C3 or C3 and C4 carbons for toluene (Fig. 3A), or between C2 and C3 carbons in t-butyl acrylate (Fig. 3C). The center-of-mass displacements can be ascribed to isotope effects; by comparing dDNP, thermal and simulated spectra for the isotropic chemical shifts and JCC couplings in question (Fig. S1), we conclude that second-order coupling effects25 are the dominant mechanism behind these doublet intensity asymmetries. Differences in populations between lower and higher energy levels of the passive spins in these hyperpolarized experiments or crosscorrelated relaxation effects that could generate such asymmetries for these 1H-decoupled sp- and sp2-hybridized sites26–28 do not seem to contribute significantly to these effects. Note that for such strongly-coupled spin systems a good enhancement is also observed, despite the fact that the optimal mixing time is a complex function of both the difference of resonance frequencies between the two sites and their JCC couplings29. Another peculiarity of these experiments concerns the presence of signals in several scans acquired consecutively during the course of dDNP injections (Figs. S2-S3). The nominal pulse angles shown in Figure 1 should leave no signals observable for the second (and subsequent) INADEQUATE scans. Still some signals such as the C2 and C3 doublets in limonene are present beyond the first spectrum, while doublets between other sites (e.g., C2 and C6, C2 and C9, C3 and C7 Fig. S2) have disappeared. Figure S3 shows a similarly long survival of the C3 and C5, C4 and C5 doublets in α-pinene. This could originate from distinct relaxation times for the same carbon in distinct isotopomers, or from off-resonance effects; quantitation analyses, however, end up pointing to variations in the JCC couplings –resulting in magnetization being brought back to +/-z by the last read pulse in the sequence remaining available for subsequent scans– as the signals’ source. This could serve as an additional filter to detect small couplings where suppression of the main carbon-13 peak may not be perfect in the first scan due to its high intensity. It could also be the starting point of a combined INADEQUATE/single-quantum acquisition whereby leftover “afterglow” z-magnetizations35 are used to gain additional information –particularly information related to the bonded 1H sites, of the kind revealed by HSQC or HMBC correlations36,37. Figures 2 and 3 reveal that sufficiently intense signals arise in these single-shot INADEQUATE analyses to study the more rapidly relaxing protonated (13CHn)n=1-3 carbons, including all of the methylene and methyl groups (e.g., C9 and C10 of limonene). This is in agreement with other studies in the field, which demonstrated that rapid transfers enable one to apply dDNP to small-molecule and natural products research30–34. The success of this experiment hinges on achieving high quality post-dissolution spectra, with complete single-13C resonance suppression, peak widths ≤1 Hz and center peak accuracies ≤0.1Hz. This high spectral quality requires strict avoidance of post-dissolution bubbles and/or of fluctuations in the injected volume, so as to enable an optimal pre-shimming and tuning on a separate sample prior to injection. Such conditions can be achieved using low-viscosity solvents, and/or pressurized fast-dissolution systems30,38. The uniqueness of JCC values can then enable univocal pairing of different 13C peaks, and provide valuable stereochemical and conformational insight capable of defining a chemical’s structure16. One could also consider selecting zerorather than double-quantum coherences as part of the 13C-13C filtering process, and thereby reduce the effects of turbulences


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Journal of the American Chemical Society associated with the sudden transfer and the signal losses associated with the coherence selection gradients. Although attractive in theory, these experiments (not shown) resulted in suboptimal suppression of the ≈200x times more intense resonances arising from uncoupled 13C sites. In terms of its generality, the main limitation of the method herein described stems from its need to dissolve a sufficiently high amounts of the compound to be analyzed in the DNP matrix, to withstand the ca. 10-fold dilution experienced upon dissolution. Notwithstanding this requirement, the quantities demanded by these dDNP

studies are ca. an order of magnitude lower than the ones required to acquire a comparable 1D thermal INADEQUATE spectrum over a day-long acquisition (Fig. S1). Extensions of the approach described herein that are being explored include applications to other nuclei with relatively low natural abundance (e.g., 29Si), characterization of other challenging heteronuclear systems at natural abundance (naturally-occurring 13C-15N pairs), dissolution protocols capable of reducing dDNP’s dilution effects, and using cryogenically cooled probes optimized for carbon detection.

Figure 2. 1D 13C INADEQUATE spectra acquired in one scan for dDNP-enhanced solutions of: (A) toluene, (B) t-butyl acrylate, (C) limonene, (D) α-pinene and (E) menthol at natural abundance. For clarity the traces are displayed zoomed for every site, with the center value indicated in ppm and a 50 Hz marker. For menthol (E) one of the diastereotopic carbons (C9) overlaps with methyl carbon of toluene (21.0 ppm) and is not distinguishable (corresponding trace not shown). Spectra were obtained after zero-filling and Fourier transformation only; no window function was applied in order to better illustrate the experiments’ sensitivity and resolution. See Supporting Information Fig. S4 for full plots of these 13C NMR traces.

Full 13C NMR traces for all dDNP-enhanced 1D INADEQUATE data.

ASSOCIATED CONTENT Supporting Information Supporting Information for this article is available free of charge on the ACS Publications website at DOI: xxxxx/yyyyyyy. Thermal equilibrium and simulated 1D INADEQUATE spectra for toluene and t-butylacrylate. dDNP NMR traces acquired consecutively during the course of dDNP injections for a variety of compounds.

AUTHOR INFORMATION Corresponding Author *[email protected]



ACKNOWLEDGMENT Financial support from the Kimmel Institute for Magnetic Resonance (Weizmann Institute), the Israel Science Foundation

(ISF 965/18) and the EU Horizon 2020 program (Marie Sklodowska-Curie Grant 642773) are gratefully acknowledged.

Figure 3. Assignment of selected doublets observed in the single-scan, natural-abundance, dDNP-enhanced 1D 13C INADEQUATE spectra of: (A) toluene, (B) α-pinene and (C) t-butyl acrylate. Carbon labels in bold correspond to the site originating the base frequency (axes in ppm). In the case of α-pinene (B), exact one-bond coupling constants for overlapping doublets have been reported39: 27.3 (J35), 27.5 (J36), and 28.2 (J45) and 27.9 (J46) Hz –hence the single doublet observed for C3-C5,C6 and the broadening of the C4-C5,C6 doublet. sample preparation., Angew Chem Int Ed Engl. 51 (2012) 11766–11769. doi:10.1002/anie.201206102.

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Natural Abundance, Single-Scan 13C-13C-based Structural

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