Carbon Multiplicity Editing in Long-Range Heteronuclear Correlation

Article pubs.acs.org/jnp

Carbon Multiplicity Editing in Long-Range Heteronuclear Correlation NMR Experiments: A Valuable Tool for the Structure Elucidation of Natural Products Josep Saurí,† Michel Frédérich,‡ Alembert T. Tchinda,§ Teodor Parella,⊥ R. Thomas Williamson,† and Gary E. Martin*,† †

NMR Structure Elucidation, Process and Analytical Chemistry, Merck & Co. Inc., Rahway, New Jersey 07065, United States Laboratory of Pharmacognosy, Department of Pharmacy, CIRM, University of Liège, B36, 4000 Liège, Belgium § Center for Studies on Medicinal Plants and Traditional Medicine, Institute of Medical Research and Medicinal Plants Studies (IMPM), P.O. Box 6163, Yaoundé, Cameroon ⊥ Servei de Ressonància Magnètica Nuclear, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain

Downloaded by SUNY UPSTATE MEDICAL UNIV on August 27, 2015 | http://pubs.acs.org Publication Date (Web): August 25, 2015 | doi: 10.1021/acs.jnatprod.5b00447

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S Supporting Information *

ABSTRACT: A recently developed NMR method to simultaneously obtain both long-range heteronuclear correlations and carbon multiplicity information in a single experiment, ME-selHSQMBC, is demonstrated as a potentially useful technique for chemical shift assignment and structure elucidation of natural products presenting complicated NMR spectra. Carbon multiplicities, even for C/CH2 and odd for CH/CH3 resonances, can be distinguished directly from the relative positive/negative phase of cross-peaks. In addition, connectivity networks can be further extended by incorporating a TOCSY propagation step. Staurosporine (1) and sungucine (2) are utilized as model compounds to demonstrate these techniques.

with them in the form of unwanted “crosstalk” artifacts that need to be identified and taken into account since they might constitute false responses. Consequently, there is still not a routine and robust way to obtain carbon multiplicity information from long-range heteronuclear correlation experiments, although such information would be very helpful in elucidating the structure of natural products when carbon resonances are overlapped anywhere in the spectrum. Herein, we wish to demonstrate how the application of a recently developed multiplicity-edited long-range heteronuclear correlation NMR experiment8 provides carbon multiplicity information as a useful additional bonus for the structural elucidation of complex natural products. The compounds used as examples in this work were staurosporine (1)9a and sungucine (2).9b

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ne-bond heteronuclear correlation experiments, based on the HSQC pulse sequence,1 and long-range heteronuclear correlation experiments, typically based on HMBC2 or long-range optimized HSQC (HSQMBC)3 pulse sequences, are widely used NMR techniques for the elucidation of natural product structures. These experiments correlate protons and heteronuclei (13C or 15N) separated by one or n (where n = 2− 4) bonds, respectively. Since these methods first appeared, numerous interesting modifications have been incorporated and evaluated with the broad goal of maximizing the information that can be gleaned from a single NMR experiment.4 One obvious example is the multiplicity-edited HSQC experiment,5 which differentiates CH2 carbons from CH/CH3 by the relative phases of the cross-peaks. ME-HSQC optimizes spectrometer time because it avoids the need for recording a more timeconsuming strategy based on the sequential collection of a 2D HSQC and a 1D DEPT experiment. Insertion of a multiplicity-editing element in conventional HMBC/HSQMBC-like experiments is more challenging due to the mixed-phase character of the multiplets obtained in these spectra.6 Edited HMBC variants have been reported based on the acquisition of two different data sets that are further manipulated during processing to obtain separate subspectra, allowing C/CH2 and CH/CH3 resonances to be differentiated.7 However, these experiments have a serious drawback associated © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION In the HSQC pulse sequence, the 1JCH magnetization terms evolve during the classic INEPT period. To achieve efficient magnetization transfer from a given proton to its directly bound carbon, the delay must be optimized as a function of the 1JCH coupling constant, which is usually in the range 120−210 Hz, Received: May 19, 2015

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DOI: 10.1021/acs.jnatprod.5b00447 J. Nat. Prod. XXXX, XXX, XXX−XXX


Journal of Natural Products

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depending on the degree of saturation (aliphatic, olephinic, aromatic, or heteroaromatic). Typically, a compromise value is chosen for the one-bond coupling, 1JCH. Likewise, long-range correlation experiments (HSQMBC) are designed in such a way that nJCH (n > 1) components of magnetization evolve during the INEPT period, usually optimized in the range 5−10 Hz, again based on the nJCH coupling constant. The effects of the evolution of homonuclear proton−proton couplings (JHH) become very important in HSQMBC because they are on the same order of magnitude as the nJCH heteronuclear couplings. Consequently, the intensity and the phase of long-range crosspeaks are strongly affected by undesired JHH modulation(s), and hence correlations are usually displayed as magnitudecalculated data due to the mixed phase of the resulting crosspeaks. This is especially relevant in the HMBC experiment because multiple-quantum coherences also evolve during the variable evolution period.10 The difference of 1 order of magnitude between 1JCH and JHH coupling constant values allows the HSQC pulse sequence to be modified easily since JHH modulation effects can be largely neglected. Hence, a multiplicity-editing block allowing differentiation of C/CH2 carbons from CH/CH3 by visualizing their relative opposite phase is readily incorporated. The idea of obtaining the carbon multiplicity information has been adapted into other NMR experiments that are based on a 1JCH transfer mechanism, for instance as in HSQC-TOCSY, H2BC, or ADEQUATE.11 Beyond simple multiplicity editing, the inverted 1JCC‑1,n-ADEQUATE experiment could be considered a “J-edited” experiment,12 where 1JCC correlations are differentiated from nJCC correlations. Mixed-phase cross-peaks due to the JHH evolution makes the HMBC/HSQMBC experiments unsuited to modifications designed to obtain phase-edited (±) spectra based on resonance multiplicity. To minimize and remove J HH modulations, several approaches have been described in the literature.13 One simple and elegant approach used in HSQMBC-like sequences is based on the application of 1H frequency-selective 180° pulses during the INEPT transfer so that no JHH evolution takes place for the selectively excited proton(s) provided that the excited proton frequencies are not mutually coupled. Thus, pure in-phase cross-peaks with respect to JHH can be obtained. If a refocusing INEPT pulse sequence element is applied, in-phase multiplet patterns with respect to the active nJCH can be also obtained irrespective of the homonuclear coupling pattern and/or the complexity of the proton multiplet. Such an experiment is referred to as selHSQMBC.14 As demonstrated in a recent publication,8 these attributes allow the insertion of a multiplicity-editing element in the pulse sequence that affords a long-range

Figure 1. (A) Nine different ME-selHSQMBC experiments performed on staurosporine (1) superimposed after selective inversion of nine different proton frequencies. Correlations plotted in red represent negative relative phase (C/CH2), whereas black responses represent positive relative phase (CH/CH3). The large inset is shown in Figure 2. More details on the small insets are described in the text. (B) Expansion corresponding to H4 proton with the carbon 1D projection shown on the right. (C) Expansion corresponding to H6′ proton with the carbon 1D projection shown on the right. Direct correlations (panels B and C) arise due to the excitation of the 13C satellites of the proton being selected.

heteronuclear correlation experiment providing carbon multiplicity information. Interpretation of these spectra is very straightforward based on the simple visual analysis of the relative phase (±) of the cross-peaks, analogous to the interpretation of multiplicity-edited HSQC spectra. Thus, correlations associated with C/CH2 carbons will exhibit the opposite phase with respect to those originating from CH/CH3 resonances. Despite the fact that in principle only a single proton frequency can be selected per HSQMBC experiment, selective pulses that can be generated on a modern NMR spectrometer are completely compatible with band-selective excitation B




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Figure 2. Expansion of the boxed aromatic region of the spectra of staurosporine (1) shown in Figure 1.

sequential experiments using nine different single selective excitation pulses. The nine resulting spectra from these experiments were superimposed to obtain the correlation “map” shown in Figure 1. ME-selHSQMBC affords a remarkably clean spectrum where carbon multiplicity information is easily decoded by simple inspection of the relative up/down phases. A distinction between CH/CH3 and C/CH2 groups can sometimes be made on a chemical shift basis. For instance, rarely do CH3 signals appear downfield of 60 ppm or CH2 resonances downfield of 80 ppm, with the exception of exocyclic CH2 or −OCH2O− functional groups (see Figure 1C as an example). In addition, CH3 signals are usually quickly distinguished based on their stronger intensities in 1D and 2D spectra. Hence, the selHSQMBC experiment provides a powerful tool for the differentiation of quaternary carbons from CH groups in the aromatic region as well as for differentiating quaternary or CH2 carbons from CH groups in the aliphatic region. Despite the fact that CH resonances cannot be differentiated from CH3, or C from CH2, the latter can be identified by comparing the MEselHSQMBC spectrum to the conventional ME-HSQC spectrum, in which correlations associated with quaternary carbons are identified by their absence. An expanded view of the aromatic region of the spectrum shown in Figure 1 is provided in Figure 2 to illustrate how well the experiment works even in the presence of a crowded carbon spectral region, where the differentiation of protons long-range coupled to methine carbons from those that are long-range coupled to quaternary carbons might become relevant for elucidating an unknown structure. Taking into account that the ME-selHSQMBC experiment is typically optimized for 8 Hz, three-bond correlations are generally expected to predominate among responses from the aromatic protons. A close inspection reveals some important structural information. For instance, the H4 doublet resonating at 9.40 ppm shows three long-range cross-peaks: (a) a three-bond correlation to a methine carbon (positive response) located at 124.9 ppm (C2); (b) a threebond correlation to a quaternary carbon (negative response) located at 114.2 ppm (C4b); and (c) a three-bond correlation to another quaternary carbon (negative response) located at 136.4 ppm (C13a). Since the latter quaternary carbon is also long-range coupled to an aromatic proton (H2 resonating at 7.55 ppm), the quaternary carbon resonating at 114.2 ppm can

Figure 3. ME-selHSQMBC experiment after multiple-site excitation of different non-mutually coupled protons in the spectrum of sungucine (2)9b achieved by using shifted-laminar pulses (see Supporting Information for details). Direct response cross-peaks are highlighted in the insets.

provided that the excited protons are non-mutually coupled. Otherwise JHH modulation(s) could occur, leading to possible misinterpretation due to distorted cross-peaks. It is also possible to excite several non-mutually coupled protons located in different parts of the molecule by using shifted-laminar pulses to achieve multiple-frequency excitation (see Supporting Information for additional details). Even overlapped protons can be inverted, again provided that they are not mutually coupled. Using multiple resonance excitation(s), the experimental acquisition time can be drastically reduced. Figure 1 shows an example of the ME-selHSQMBC experiment performed on staurosporine (1).9a Selective inversion of nine different proton frequencies was achieved in C




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Figure 4. Top: ME-selHSQMBC experiment (A) after the selective inversion of the aliphatic H15/H15′ and H23/H23′ proton pairs of sungucine (2); (B) ME-selHSQMBC-TOCSY experiment after inversion of the same protons. Note the increased number of cross-peaks that are available for analysis in B. Bottom: Schematic representations of the performance of both experiments are shown in A′ and B′/B″, respectively. Arrow labeled 1 denotes the forward INEPT transfer, whereas arrow labeled 2 corresponds to the reverse INEPT transfer. Arrows labeled 3 identify the TOCSY transfers from H15 to H16 and H14. Correlations from the TOCSY transfer following reverse INEPT (2) will be observed from the transfer of magnetization from H15 to H16 and H14. See main text for additional details.

be identified as the C4b carbon, located near the central core of the molecule with no other long-range correlation to any other proton. In a similar fashion, the C7b quaternary carbon was identified as the carbon located in the central core of the molecule by examining at H8 doublet (8.06 ppm) correlations, which show the same pattern as H4: three cross-peaks, two of them associated with quaternary carbon correlations. Furthermore, since the H7 resonance (5.06 ppm) is longrange coupled to carbon C7b, the latter can be differentiated from carbon C4b. Similarly, due to a correlation peak arising from the H6′ (7.00 ppm) proton (see the small insets highlighting those correlations in Figure 1), C12b is differentiated from C12a. In addition C11a can be differentiated from C13a based on the correlation from H4 to C13a. Another example is shown in Figure 3 using the complex, dimeric Strychnos alkaloid sungucine (2).9b In this case, several non-mutually coupled protons located in different parts of the molecule were selectively excited by using shifted-laminar

Figure 5. (A) Correlations obtained from ME-selHSQMBC after selective inversion of the H15/H15′ and H23/H23′ protons. (B) Additional correlations obtained from the ME-selHSQMBC-TOCSY experiment. Note that additional correlations from H20/H20′ to C21 and C22 are obtained due to a 4JHH between H20/H20′ and H22/ H22′. For details, see main text.

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Figure 6. (A) 1D slice extracted at the quaternary C21′ carbon frequency from the ME-selHSQMBC experiment. Only long-range proton−carbon correlations from the selectively excited H15′ and H23′ protons are obtained. (B) 1D slice extracted at the same carbon frequency from the MEselHSQMBC-TOCSY experiment. Note that not only correlations from the H15′ and H23′ protons are obtained but also long-range proton− carbon correlations from the protons that have been reached by the TOCSY transfer. 1D slices are shown in red with the relative negative phase, as they display protons long-range coupled to the quaternary C21′ carbon. Also, both 1D slices are shown with the same scale in order to compare sensitivity. As expected, ME-selHSQMBC-TOCSY is somewhat less sensitive due to the fact that the TOCSY transfer makes the pulse sequence longer.

Therefore, additional H16−C7 and H14−C7 correlations can be obtained (red arrows). Note that the initial H15−C7 response will also be obtained from the same spectrum. A comparison of all long-range heteronuclear correlations obtained from each experiment performed on sungucine (2) is summarized in Figure 5. In (A), long-range proton−carbon correlations are obtained only from the initially excited H15/ H15′ and H23/H23′ resonances (highlighted with black and blue arrows, respectively). However, in (B), the subsequent TOCSY step transfers the magnetization from the remote proton(s) to their vicinal neighbor(s), providing a set of additional correlations. Correlations coming from the TOCSY transfer from the H15/15′ protons are denoted by the red arrows (must be paired with the black arrows from (A)), while those coming from the TOCSY transfer from H23/23′ protons are denoted by the green arrows (must be paired with the blue arrows from (A)). Another comparison of the performance of both experiments can be made by extracting a 1D slice from a given carbon frequency. For instance, Figure 6 shows a 1D slice taken at the carbon frequency of the carbon C21′ (resonating at 142.4 ppm) that highlights the increased number of correlations afforded by the ME-selHSQMBC-TOCSY experiment, which offers five additional correlations when compared to the corresponding ME-selHSQMBC experiment. Thus, ME-selHSQMBC-TOCSY permits the identification of the different spin systems of the molecule, in a manner analogous to the more familiar HSQC-TOCSY experiment. Another advantage is that the TOCSY module can provide long-range correlation cross-peaks even in congested areas with overlapped proton signals where the application of selective pulses is not feasible. In summary, it has been shown that the ME-selHSQMBC experiment has the potential to become an important tool for resonance assignment purposes and the elucidation of complex natural product structures. Long-range heteronuclear correlations and carbon multiplicity information are both obtained in a

pulses to achieve multiple-frequency excitation. Again, a very clean spectrum was obtained, facilitating the analysis of the carbon resonance multiplicity. Owing to better control over magnetization components due to the selective nature of the experiment and the in-phase properties of the multiplet line shapes obtained, a TOCSY transfer can be appended to the pulse sequence to extend the correlation information to a remote spin system.15 Consequently, much more information can be garnered from a single experiment in minimal experimental time. Figure 4 shows a side-by-side comparison of the performance of the MEselHSQMBC (panel A) and the related ME-selHSQMBCTOCSY experiment (panel B) after simultaneous selective inversion of the aliphatic H15/H15′ methylene and H23/H23′ methyl resonances of sungucine (2), which appear overlapped in the 1.6−1.9 ppm area. It is clearly seen that when the TOCSY transfer takes place, not only does the number of cross-peaks increase significantly but the carbon multiplicity information is also retained. The schemes (A′ and B′/B′′) at the bottom of Figure 4 summarize the performance of both experiments. To better understand the information afforded by the ME-selHSQMBCTOCSY with respect to the ME-selHSQMBC experiment, we will focus on the spin system constituted by the H15−C7 correlation response from sungucine (2). In ME-selHSQMBC (A′) only correlations due to long-range coupling constants between the selectively inverted H15 proton and the respective carbons are obtained (indicated by the black arrows 1 and 2) in an out-and-back process. Note that as we are specifically looking at the H15−C7 spin system, only this correlation is highlighted, but additional correlations from H15 to other longrange coupled carbons would also be obtained, as shown in the top left panel (A) of Figure 4. However, in ME-selHSQMBCTOCSY the information obtained from the initial out-and-back process (see B′, arrows 1 and 2) will be transferred to all those protons that belong to the same spin system via homonuclear coupling constants (see B′′ and gray arrows labeled as 3). E



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(2) Bax, A.; Summers, M. F. J. Am. Chem. Soc. 1986, 108, 2093−2094. (3) Williamson, R. T.; Marquez, B. L.; Gerwick, W. H.; Kover, K. E. Magn. Reson. Chem. 2000, 38, 265−273. (4) Castañar, L.; Parella, T. In Annual Reports in NMR Spectroscopy; Academic Press: San Diego, CA, 2015; Vol. 84, Chapter 4, pp 163− 232. (5) (a) Willker, W.; Leibfritz, D.; Kerssebaum, R.; Bermel, W. Magn. Reson. Chem. 1993, 31, 287−292. (b) Parella, T.; Belloc, J.; SánchezFerrando, F.; Virgili, A. Magn. Reson. Chem. 1998, 36, 715−719. (c) Hu, H.; Krishnamurthy, K. Magn. Reson. Chem. 2008, 46, 683−689. (6) Saurí, J.; Parella, T. Magn. Reson. Chem. 2013, 51, 509−516. (7) (a) Nyberg, N. T.; Sørensen, O. W. Magn. Reson. Chem. 2006, 44, 451−454. (b) Benie, A. J.; Sørensen, O. W. Magn. Reson. Chem. 2006, 44, 739−743. (8) Saurí, J.; Sistaré, E.; Williamson, R. T.; Martin, G. E.; Parella, T. J. Magn. Reson. 2015, 252, 170−175. (9) (a) Meksuriyen, D.; Cordell, G. J. Nat. Prod. 1988, 51, 884−892. (b) Lamotte, J.; Dupont, L.; Dideberg, O.; Kambu, K.; Angenot, L. Tetrahedron Lett. 1979, 20, 4227−4228. (10) (a) Furrer, J. Concepts Magn. Reson., Part A 2012, 40A, 101− 127. (b) Furrer, J. Concepts Magn. Reson., Part A 2012, 40A, 146−169. (c) Furrer, J. Conc. Magn. Reson. 2015, 43A, in press. doi: 10.1002/ cmr.a.21317. (11) (a) Parella, T.; Sanchez-Ferrando, F.; Virgili, A. J. Magn. Reson. 1997, 126, 274−277. (b) Nyberg, N. T.; Duus, J. Ø.; Sørensen, O. W. Magn. Reson. Chem. 2005, 43, 971−974. (c) Parella, T.; SanchezFerrando, F. J. Magn. Reson. 2004, 166, 123−128. (12) Reibarkh, M.; Williamson, R. T.; Martin, G. E.; Bermel, W. J. Magn. Reson. 2013, 236, 126−133. (13) Koskela, H.; Kilpeläinen, I.; Heikkinen, S. J. Magn. Reson. 2003, 164, 228−232. (14) Gil, S.; Espinosa, J. F.; Parella, T. J. Magn. Reson. 2011, 213, 145−150. (15) Saurí, J.; Espinosa, J. F.; Parella, T. Angew. Chem., Int. Ed. 2012, 51, 3919−3922. (16) Tchinda, A. T.; Tamze, V.; Ngono, A. R. N.; Ayimele, G. A.; Cao, M.; Angenot, L.; Frédérich, M. Phytochem. Lett. 2012, 5, 108− 113.


single NMR experiment. Still more spectral information can be garnered by inserting a TOCSY transfer at the end of the MEselHSQMBC sequence, as shown in Figure 4 for sungucine (2).9b

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EXPERIMENTAL SECTION

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ASSOCIATED CONTENT

General Experimental Procedures. All NMR data reported in this study were acquired using either a sample of 2.1 mg of staurosporine (1) or a 4.0 mg sample of sungucine (2). Staurosporine was purchased from LC Laboratories (Woburn, MA, USA). Sungucine was extracted and purified from Strychnos icaja from Cameroon, according to a previously published procedure.16 Both samples were dissolved in 35 μL of 99.96% DMSO-d6, which was then transferred to a 1.7 mm NMR tube. All of the NMR data were acquired using a 600 MHz Bruker three-channel AVANCE III NMR spectrometer equipped with a Bruker gradient triple resonance TXI 1.7 mm TCI 1H/13C−15N MicroCryoProbe. The nine different experiments shown in Figure 1 were acquired in 1 h 18 min each as 4096 data points with 8 scans accumulated for each of the 256 t1 increments accumulated. Spectral windows in F2 and F1 were set in all cases to 1802 and 24 150 Hz, respectively. All experiments were optimized for 8 Hz, and the duration and shape of the selective 180° 1H pulse were set accordingly to the required selectivity in each case (see Supporting Information for further discussion). Prior to Fourier transformation of the data, zero-filling to 1024 points in F1 and 8192 points in F2 and sine-squared apodization were applied in both dimensions. Figure 3 and Figure 4A data were acquired in 1 h 57 min as 4096 data points with 12 scans accumulated for each one of the 384 t1 increments. Spectral windows in F2 and F1 were set to 5411 and 27 169 Hz, respectively. The experiment was optimized for 8 Hz, and the duration of the Gaussian selective 180° 1H pulse was set to 10 ms. Prior to Fourier transformation of the data, zero-filling to 1024 points in F1 and 8192 points in F2 and sine-squared apodization were applied in both dimensions. Data shown in Figure 4B were acquired and processed in exactly the same way as those in Figure 4A. The only difference was the mixing time used for the TOCSY element, which was set to 60 ms. S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00447. Brief tutorial to perform multiple-site excitation using shifted-laminar pulses in TopSpin3.2 (Bruker). Pulse sequence scheme with full details of both MEselHSQMBC and ME-selHSQMBC-TOCSY experiments. Pulse program code for Bruker. Expansions of Figure 1A, Figure 3, and Figure 4B (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: (+1) 732.594.5398. Fax: (+1) 732.594.9456. E-mail: gary. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS T.P. gratefully acknowledges financial support from the MINECO (project no CTQ2012-32436). REFERENCES

(1) Bodenhausen, G.; Ruben, D. J. Chem. Phys. Lett. 1980, 69, 185− 189. F


Carbon Multiplicity Editing in Long-Range Heteronuclear Correlation

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