- Email: [email protected]
Broadband inversion of 1JCC responses in 1,n-ADEQUATE spectra
Journal of Magnetic Resonance 236 (2013) 126–133
Contents lists available at ScienceDirect
Journal of Magnetic Resonance journal homepage: www.elsevier.com/locate/jmr
Broadband inversion of 1JCC responses in 1,n-ADEQUATE spectra q Mikhail Reibarkh a,⇑, R. Thomas Williamson a, Gary E. Martin b,⇑, Wolfgang Bermel c a
Merck Research Laboratories, Discovery and Preclinical Sciences, Process and Analytical Chemistry, Structure Elucidation Group, Rahway, NJ 07065, USA Merck Research Laboratories, Discovery and Preclinical Sciences, Process and Analytical Chemistry, Structure Elucidation Group, Summit, NJ 07901, USA c BrukerBioSpin GmbH, Silberstreifen, 76287 Rheinstetten, Germany b
a r t i c l e
i n f o
Article history: Received 6 June 2013 Revised 27 July 2013 Available online 9 August 2013 Keywords: 1,n-ADEQUATE 1,1-ADEQUATE Pulse sequence Broadband inversion Strychnine
a b s t r a c t Establishing the carbon skeleton of a molecule greatly facilitates the process of structure elucidation, both manual and computer-assisted. Recent advances in the family of ADEQUATE experiments demonstrated their potential in this regard. 1,1-ADEQUATE, which provides direct 13C–13C correlation via 1JCC, and 1,n-ADEQUATE, which typically yields 3JCC and 1JCC correlations, are more sensitive and more widely applicable experiments than INADEQUATE and PANACEA. A recently reported modified pulse sequence that semi-selectively inverts 1JCC correlations in 1,n-ADEQUATE spectra provided a significant improvement, allowing 1JCC and nJCC correlations to be discerned in the same spectrum. However, the reported experiment requires a careful matching of the amplitude transfer function with 1JCC coupling constants in order to achieve the inversion, and even then some 1JCC correlations could still have positive intensity due to the oscillatory nature of the transfer function. Both shortcomings limit the practicality of the method. We now report a new, dual-optimized inverted 1JCC 1,n-ADEQUATE experiment, which provides more uniform inversion of 1JCC correlations across the range of 29–82 Hz. Unlike the original method, the dual optimization experiment does not require fine-tuning for the molecule’s 1JCC coupling constant values. Even more usefully, the dual-optimized version provides up to two-fold improvement in signalto-noise for some long-range correlations. Using modern, cryogenically-cooled probes, the experiment can be successfully applied to samples of 1 mg under favorable circumstances. The improvements afforded by dual optimization inverted 1JCC 1,n-ADEQUATE experiment make it a useful and practical tool for NMR structure elucidation and should facilitate the implementation and utilization of the experiment. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction An intrinsic property of NMR spectroscopy is the reliance on the modulations of various phenomena. Modulations of various types are an integral aspect of many 2D and nD NMR experiments. Some modulations are beneficial, enhancing response intensity while others can be problematic, leading to artifacts and/or to reduced correlation intensity or even lost responses. Problems due to modulations are long-standing. Early work in the area of heteronucleus-detected long-range heteronuclear chemical shift correlation experiments investigated the problem of fast modulations resulting from the relatively large 1JCH coupling constants, which were subsequently decoupled in modified variants of the experiment [1–3]. Modulation problems have not been circumvented with the advent of proton-detected long-range het-
q Presented at the 18th ISMAR Meeting, Rio de Janeiro, Brazil, May 19–24, 2013, ‘‘Broadband inversion of 1JCC correlations in 1,n-ADEQUATE spectra’’, paper OP118. ⇑ Corresponding authors. E-mail addresses: [email protected] (M. Reibarkh), gary.martin2@ merck.com (G.E. Martin).
1090-7807/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmr.2013.07.016
eronuclear shift correlation experiments. The widely used HMBC experiment provides a good example in this regard. HMBC optimization has generally been a compromise, biasing an experiment toward either smaller or larger heteronuclear couplings [4,5]. These exigencies have prompted several groups to develop modified experiments to allow the observation of a broader range of longrange heteronuclear couplings. The first such experiment was ACCORD-HMBC [6,7], followed by IMPEACH-MBC [8], CIGAR-HMBC [9], and others [10–12]. A detailed discussion of HMBC and experimental variants has been the subject of a number of reviews [13– 18]. Amplitude modulation of response intensity must also be taken into account when optimizing delays in experiments such as 1,1ADEQUATE [19,20] and inverted 1JCC 1,n-ADEQUATE [21]. In the former, correlations between adjacent carbons via 1JCC couplings may be lost as a consequence of the mismatch between the experimental optimization and the actual 1JCC coupling [19,20]. We have recently shown that modulation of magnetization as a function of the magnitude of direct and long-range 13C–13C couplings must also be taken into consideration in experiments such as inverted 1JCC 1,n-ADEQUATE [21,22]. The central premise
127
M. Reibarkh et al. / Journal of Magnetic Resonance 236 (2013) 126–133
of the inverted 1JCC 1,n-ADEQUATE pulse sequence is that by inverting the 1JCC correlations, which tend to unavoidably ‘‘leak’’ into a 1,n-ADEQUATE spectrum, they can be readily identified without having to resort to other experiments such as conventional 1,1ADEQUATE. Multiple-bond carbon–carbon correlations (nJCC) will always have positive phase in the inverted 1JCC 1,n-ADEQUATE experiment, readily differentiating them from 1JCC correlations when the latter are inverted [21,22]. A single spectrum that differentiates 1JCC from nJCC correlations has tremendous implications for structure elucidation. It would be particularly valuable as input for CASE (Computer-Assisted Structure Elucidation) programs where having unambiguous connectivity information drastically reduces both the number of structures generated and the corresponding computational time, as well as increasing the reliability of final structure(s) [23,24]. The amplitude transfer function that controls response intensity is governed by both the 1JCC and nJCC optimizations chosen. As a consequence of the oscillatory nature of the transfer function, 1JCC correlations can have unintended, positive response intensity in spectra acquired using single optimization [21,22] as predicted by the curve shown in Fig. 1. Aberrantly phased 1 JCC correlations are obviously highly undesirable as they complicate the data interpretation and could lead to errors in the elucidation of unknown structures due to the mistaken attribution of a one-bond correlation as an n-bond correlation. One approach to this problem is to attempt to optimize the amplitude transfer function so that all 1JCC couplings are inverted. However, this solution is both impractical and decidedly non-universal. Inverting a broad range of 1JCC couplings is quite difficult, since no single amplitude transfer function can achieve this objective due to the variability of 1JCC couplings, which are found to range from 32 to 72 Hz for strychnine [22,25]. Here, we report a dual optimization approach based on mutual cancellation of unwanted oscillations. We show that a dual-optimized, inverted 1 JCC 1,n-ADEQUATE experiment affords a much broader and more uniform inversion of 1JCC correlations in 1,n-ADEQUATE spectra than can be achieved using a single-optimized setup of the experiment. Additionally, it is demonstrated that the intensity of the nJCC correlations is improved relative to the previous variant of the experiment. 2. Optimization vs. 1JCC modulation Recently, a modified 1,n-ADEQUATE pulse sequence was reported that utilizes asymmetric delays to modify the oscillatory
Fig. 1. Amplitude transfer function for an inverted 1JCC 1,n-ADEQUATE [21] experiment optimized for 1JCC = 40 and nJCC = 7 Hz. The C22–C21 correlation in the spectrum of strychnine (1), which has a one-bond carbon–carbon coupling constant of 71.8 Hz, would be expected to exhibit a positive correlation and does (see Fig. 5C) [22,25].
behavior of the 1JCC and nJCC couplings, allowing the former to be semi-selectively inverted [21]. The experiment is not uniformly selective in that an inopportune 1JCC coupling may yield a positive response. For example, the 71.8 Hz C22–C21 coupling of strychnine (1) [25] yields a positive correlation when the experiment is optimized, for 1JCC = 40 and nJCC = 7 Hz [22], both commonly utilized optimizations for carbon–carbon coupling constants in 1,1- and 1,n-ADEQUATE experiments, respectively [20]. The amplitude transfer curve for the 1JCC correlations using the 1JCC/nJCC optimizations just noted is shown in Fig. 1. The other ‘‘islands’’ of positive intensity from 32–35 and 58–63 Hz could also yield unintended, positively phased 1JCC correlations for some aliphatic and aromatic carbon resonances (see Structure 1). Positive 1JCC correlations that could be mistaken for an nJCC correlation in the spectrum of an unknown represent a disadvantage and somewhat decrease the utility of the inverted 1JCC 1,n-ADEQUATE experiment. This problem can be partially circumvented by a judicious choice of 1JCC and nJCC optimization. Carefully optimizing the experiment, however, presupposes that one has a knowledge of the 1JCC coupling constants in the molecule being studied, which will only rarely be the case. Generally, an investigator will not have J-modulated ADEQUATE [25–30] data at his disposal and quantum mechanical calculation of the coupling constants with sufficient accuracy is impractical, requiring a significant computational effort [31–33] as well as prior knowledge of the actual structure before setting up every experiment. Furthermore, this approach is by no means a universal solution since no single choice of 1JCC and nJCC optima will likely achieve inversion of the full range of 1 JCC constants relevant for a given molecule (Supplementary material, Fig. S2). To the contrary, an unfortunate choice of 1JCC and nJCC optimization may lead to a spectrum where the majority of one-bond correlations are not inverted. Such is the case in the inverted 1JCC 1,n-ADEQUATE spectrum of strychnine optimized for 1JCC = 50 and nJCC = 5 Hz. While the choice of both optima is logical and quite reasonable, it results in a spectrum with very few inverted aliphatic one-bond correlations. Closer inspection of the amplitude transfer curve (see Fig. S3) confirms that most of strychnine’s aliphatic 1JCC couplings lie in the regions where the transfer curve is positive [25,30]. These considerations prompted us to explore further modifications of the pulse sequence to afford a broader range of inversion of the 1JCC correlations in 1,n-ADEQUATE spectra. Strategically, we wanted to improve the technique in two ways. First, it is highly desirable to have all 1JCC correlations either negative or, failing that, absent from the spectrum. When negative, 1JCC correlations are
Structure 1.
128
M. Reibarkh et al. / Journal of Magnetic Resonance 236 (2013) 126–133
readily identified and provide useful structural information. If absent, they cannot be mistaken for a nJCC long-range correlation in the 1,n-ADEQUATE spectrum of an unknown. The second objective is to have a simple and universal experimental setup that does not necessitate adjusting the optimization for every sample. These attributes improve the viability of the method and should facilitate the implementation and utilization of the experiment. All of the issues enumerated above are a consequence of the oscillatory nature of the 1JCC modulation. The amplitude transfer function is essentially a slowly oscillating sine wave (governed, in part, by the 1JCC optimization), modulated by a rapidly oscillating sine wave (conversely governed, in part, by nJCC optimization), as summarized mathematically in the following section. These attributes provide an avenue for exploitation: the sum of two transfer functions can be used to cancel out the fast oscillations while preserving the slow wave, which determines the 1JCC inversion. Judicious choices of pairs of optima can lead to amplitude transfer curves that, when superimposed and summed, lead to much broader inversion of 1JCC correlations.
3. Amplitude transfer functions of different versions of the ADEQUATE experiment In the ‘‘traditional’’ 1,1- and 1,n-ADEQUATE experiments, both JCC and 1JCC carbon–carbon couplings evolve according to sin(J p D) after the initial H,C-INEPT transfer. Being an outand-back experiment, the same transfer amplitudes apply for the delay prior to the reverse INEPT transfer. Thus, the overall transfer amplitude is defined by sin2(J p D). Hence, the transfer functions afford nJCC and 1JCC cross peaks that are always positive and therefore preclude discrimination between nJCC and 1JCC. In fact, the only parameter defining whether the experiment is 1,1- and 1,n- is the delay used to maximize the transfer function at particular value of J (typically 40–60 Hz for 1JCC and 5–7 Hz for nJCC) [20]. Because the 1JCC coupling constant is considerably larger than the nJCC coupling, the corresponding sine term for 1JCC oscillates much faster than the sine term for nJCC. This difference makes it possible to modulate the amplitude transfer function to keep it negative for 1JCC correlations, but still positive for nJCC correlations. Such modulation was achieved by making the pulse sequence asymmetric, that is, using slightly different delay settings before and after t1 evolution [21,22]. The approach yielded the 1JCC-edited 1,n-ADEQUATE experiment [21,22], which is referred to hereafter as single-optimization 1Jcc-edited 1,n-ADEQUATE in the manuscript. In this work, we report a further refinement of the asymmetric delay approach, which provides a more uniform and more robust inversion of 1JCC correlations, while improving the signal-to-noise (S/N) ratios for nJCC correlation. The amplitude transfer function of the modified dual optimization experiment is essentially a normalized sum of two differently optimized single-optimization transfer functions. The fundamental principle governing all ADEQUATE experiments described above (1,1-, 1,n-, single-optimization 1Jcc-edited, and dual-optimized 1Jcc-edited) is identical: H,C-INEPT transfer, followed by JCC evolution, followed by a reverse C,H-INEPT transfer. The difference between the experiments is in the modulation during the evolution, which is achieved by different delay optimizations. While 1,1- and 1,n-ADEQUATE experiments have symmetrical delays, 1JCC-edited experiments employ asymmetrical delays. The mathematical description of the amplitude transfer modulations as a function of JCC coupling constant follows: n
(1) 1,1-ADEQUATE: f(J) = sin2(J p D). D is usually optimized to match J of 40–60 Hz. (2) 1,n-ADEQUATE: f(J) = sin2(J p D). D is usually optimized to match J of 5–7 Hz.
Fig. 2. Calculated amplitude transfer curves governing the response intensity of an inverted 1JCC 1,n-ADEQUATE experiment modified for dual optimization of the carbon–carbon delays (blue). The two optimizations were 1JCC = 57 with nJCC = 9.5 Hz (red), and 1JCC = 64 with nJCC = 8 Hz (green). (A) This panel shows the response intensity of the 1JCC correlations as a function of the optimization. The blue trace shows the calculated, summed, and normalized response intensity across the optimization range from 30 to 90 Hz. One-bond correlations are inverted across the range from 29 to 82 Hz, which encompasses most commonly encountered onebond carbon–carbon [34,35]. (B) This panel shows the response intensity for the nJCC correlations as a function of the optimization across the range from 0 to 16 Hz. The blue trace shows the summed and normalized long-range carbon–carbon response intensity. This dual optimization pair gives usable response intensity (>0.5) across the range from approximately 4 to 12 Hz, which encompasses most structurally useful nJCC in the authors’ experience [20].
(3) Single-optimization 1Jcc-edited 1,n-ADEQUATE: f(J) = sin(J p ((2 m + 1)/(2 1Jopt) + d)) sin(J p ((2 m 1)/(2 1Jopt) + d)). Here, m is the truncated integer of 1Jopt/(2 nJopt). 1Jopt and nJopt are the optimization values for 1JCC and nJCC, respectively. d Is the sum of all additional delays in the pulse sequence (primarily due to the length of shaped pulses). (4) Dual optimization 1Jcc-edited 1,n-ADEQUATE: f(J) = 0.5 [sin(J p ((2 mA + 1)/(2 1JA)+d)) sin(J p ((2 mA 1)/(2 1JA) + d)) + sin(J p ((2 mB + 1)/(2 1JB) + d)) sin(J p ((2 mB 1)/(2 1JB) + d))].
M. Reibarkh et al. / Journal of Magnetic Resonance 236 (2013) 126–133
Here, mA is the truncated integer of 1JA/(2 nJA) and mB is the truncated integer of 1JB/(2 nJB). 1JA/nJA and 1JB/nJB are the two pairs of optimization values for 1JCC and nJCC, respectively. An example of the dual optimization scheme is shown in Fig. 2 in which two amplitude transfer curves, one optimized for 1JCC = 57 and nJCC = 9.5 Hz (red) and a second optimized for 1JCC = 64 and n JCC = 8 Hz (green) give the result shown by the blue curve when transfer function amplitudes are summed and normalized. Fig. 2A shows all three functions across the range of the most commonly encountered 1JCC coupling constants. One-bond carbon–carbon correlations are effectively inverted across the range from 29–82 Hz and 89–96 Hz. With the exception of cyclopropyl, cyclobutyl, and acetylenic carbon–carbon bonds, there are relatively few other structural moieties encountered in natural products or pharmaceuticals with one-bond carbon–carbon coupling constants outside of this range [34]. The corresponding amplitude transfer functions for the long-range nJCC couplings are shown in Fig. 2B; again, the blue trace corresponds to the summed and normalized amplitude for the two differently optimized transfer functions used. The useful range of nJCC couplings is 4–12 Hz (normalized intensity >0.5) with all couplings from 0 to 15 Hz having positive intensity. The range of long-range couplings with intensities of >0.5 incorporates the normally useful continuum of long-range carbon–carbon correlations [20]. The pulse sequence implemented to exploit dual optimization of the amplitude transfer functions shown in Fig. 3 is schematically indistinguishable from that previously reported [21]. The modified, dual optimization experiment performs n transients, typically set to a multiple of the 16-step phase cycle, for the first set of optimization parameters and then adds n transients for the second optimization before incrementing the gradients, phases, and delays (the Bruker TopSpin 3.1 pulse sequence code is reproduced in the Supplementary material). 4. Dual optimization inverted 1JCC 1,n-ADEQUATE The experimental results obtained using the inverted 1JCC 1,n-ADEQUATE experiment modified for dual optimization of the one-bond and long-range carbon–carbon coupling constant-based delays are shown in Fig. 4. The experiment was optimized using 57/9.5 and 64/8 Hz for the 1JCC/nJCC optimization pairs, the amplitude transfer curves for which are shown in Fig. 2. Positive nJCC correlations are plotted in black; inverted 1JCC correlations are plotted in red. As will be noted by inspection of the spectrum shown in Fig. 4, the problematic, positively phased C22–C21 correlation in the original version of the experiment [22] (1JCC/nJCC optimization
129
of 40/7 Hz) has been successfully inverted in the modified version of the experiment, as expected from the transfer curve (see also Supplemental Table 1). A comparison of the problematic C21–C22 response [22] is shown by the slices extracted from the 2D spectra in Fig. 5. The proton reference spectrum of strychnine (1) is shown in Fig. 5A. The F1 slice for the C21–C22 correlation from the dual-optimized experiment is shown in Fig. 5B while the corresponding slice from the original single-optimization inverted 1JCC spectrum optimized for 1JCC/nJCC = 40/7 Hz is shown in Fig. 5C. The traces in panels 5B and 5C were extracted at the 13C shift of the C21 quaternary carbon. Note that consistent with the amplitude curve shown in Fig. 1, the C21–C22 correlation with a coupling constant of 71.8 Hz has strong positive intensity rather than being inverted as intended in panel 5C. As intended, the C21–C22 correlation observed at the H22 proton chemical shift is inverted in the slice shown in Fig. 5B extracted from the dual-optimized spectrum shown in Fig. 4. The response intensity is consistent with the blue normalized summation curve shown in Fig. 2A. It is interesting to compare the relative S/N ratio of various responses in the slices shown in Fig. 5B and C. The most obvious difference is of course seen for the H22–C21 correlation which has a S/N ratio of 9.2:1 in the dual-optimized experiment (panel 5B) in contrast to a +34.2:1 S/N ratio in the single-optimized experiment (panel 5C). The change in phase and reduction in signal intensity for the C22–C21 correlation is consistent with what would be predicted from the amplitude transfer curves shown in Figs. 1 and 2A. The S/N for the C16–C21 correlation increased from +33.6:1 for this correlation in the single-optimization spectrum to +53.5:1 in the dual optimization experiment. In a similar fashion, the C13–C21 correlation intensity also increased significantly from +4.7:1 to +8.3:1 in going from single to dual optimization. The intensity of the C8–C21 correlation also increased, albeit only slightly from +3.6:1 to +4.1:1. As noted above, there is the expectation that the intensity of the 1 JCC correlations will be attenuated somewhat in the dual optimization experiment vs. the single optimization experiment. In the case of the 40/7 Hz single-optimized experiment, the C14–C21 correlation has the greatest intensity of 63.0:1. In contrast, the C14– C21 correlation is significantly attenuated in the dual-optimized experiment with a S/N ratio of 23.4:1. The same is true for the C20–C21 correlation that decreased from 42.5:1 to 9.3:1 for the correlation observed at the F2 shift of the H20a resonance and from 58.5:1 to 10.2:1 for the correlation observed at the F2 shift of the H20b resonance.
Fig. 3. Pulse sequence for dual optimization inverted 1JCC correlated 1,n-ADEQUATE. The duration of the D5 and D3 delays, were modified as described in the text. Narrow and wide bars indicate 90° and 180° pulses, respectively, with the exception of the 120° pulse that is specifically labeled. Unless otherwise indicated, phases were: U1 = 0000 2222, U2 = 0000 0000 2222 2222; U3 = 0022; U4 = 1133; UR = 0220 2002 2002 0220. Gradient ratios were: Gz1 = +78.4%; Gz2 = +77.4%; Gz3 = 59%. The Bruker TopSpin 3.1 pulse sequence code is contained in the supplemental material.
130
M. Reibarkh et al. / Journal of Magnetic Resonance 236 (2013) 126–133
Fig. 4. Dual-optimized inverted 1JCC 1,n-ADEQUATE spectrum of strychnine. The data were acquired as 3072 200 real points, with 128 transients accumulated for each set of optima. The experiment was dual-optimized for 1JCC = 57 and nJCC = 9.5 Hz for the first optimization and 1JCC = 64 and nJCC = 8 Hz for the second optimization. The acquisition time for the spectrum was 48 h 42 min. The data were linear predicted to 4096 points in F2 and to 512 points in F1 followed by zero-filling prior to the second Fourier transform to afford the 2 K 2 K spectrum shown. Data were subjected to sin2 apodization phase-shifted 90° prior to both Fourier transforms. Experimentally, the order in which the optima are entered into the pulse sequence and acquired is of no consequence.
Beneficial improvements in the intensity of the long-range correlations in the dual-optimized experiment are also highlighted in Fig. 6, which compares the correlations from the aromatic proton region to the aliphatic carbon region as an example. The trace shown in Fig. 6B was extracted from the spectrum shown in Fig. 4 at the 13C shift for C8/C16 (60.18 and 60.08 ppm, respectively); the trace shown in Fig. 6C was extracted at the 13C shift for the C8/C16 resonances in the 40/7 Hz single-optimized experiment [21,22]. Generally, the intensity of the nJCC correlations in the dual-optimized spectrum increased by 30–100% compared to the corresponding correlations in the 1JCC/nJCC = 40/7 Hz single-optimized spectrum [21,22]. The 1JCC correlations all have the intended negative phase and, as expected, somewhat reduced response intensity as predicted by the amplitude transfer curve shown in Fig. 2A. A comparison of the aliphatic region of the dual-optimized spectrum (panel A) and the corresponding region single-optimized spectrum (panel B) is shown in Supplemental Fig. S1. Both spectra were identically plotted with a 6% threshold. It can be clearly seen that there are correlations present in panel A that are absent in the single-optimized spectrum shown in panel B or below the 6% threshold. It should also be noted that correlations visible in both spectra are generally more intense in the dual-optimized spectrum shown in panel A.
5. Improved sensitivity for long-range correlations in dualoptimized inverted 1JCC 1,n-ADEQUATE As is evident from a comparison of the amplitude transfer functions in Figs. 1 and 2A, one-bond correlation intensities are expected to be somewhat lower for the dual-optimized inverted 1 JCC 1,n-ADEQUATE spectrum compared to the single optimum
experiment. Experimental observations support this premise, with 1D slices in Figs. 5 and 6 providing a clear illustration. However, one should keep in mind that the amplitude transfer function shown in Fig. 2A is essentially an attenuation function for 1JCC correlations that can tolerate some degree of attenuation. Here, the worst case scenario would be a missed one-bond correlation due to low intensity, which is still acceptable, and much better than an aberrant, positively phased 1JCC correlation, such as C22–C21 correlation in the 40/7 Hz single-optimized experiment [22]. More interestingly, nJCC correlations demonstrate improved sensitivity in dual-optimized inverted 1JCC 1,n-ADEQUATE spectra as shown in 1D slices (Figs. 5 and 6). For 2D data, improved sensitivity can translate into the detection of weaker nJCC correlations observed in the dual-optimized spectrum than is possible in the single optimum spectrum, as shown in Supplemental Fig. S1. Alternately, the improved sensitivity may allow the practical investigation of smaller samples. A plausible reason for the improved nJCC correlation S/N is the reduction of relaxation-related signal losses due to the choice of larger nJCC and 1JCC optima in comparison with the single-optimized experiment. Under favorable circumstances, it is possible to apply variants of the experiments described in this report to samples as small as 1 mg [36]. The experiment does, however, make significant demands on instrument performance and molecules with long T1 relaxation times may require the use of interpulse delays considerably longer than the 3 s delay normally used for ADEQUATE experiments. Improving nJCC correlation sensitivity at the cost of lower 1JCC correlation sensitivity is a viable trade-off for several reasons. First, not only do one-bond correlations generally have greater intrinsic intensity and can thereby tolerate some degree of attenuation due to the transfer function, but missing an occasional one-bond correlation is an acceptable scenario for the purposes of structure elucidation. Long-range correlations, in contrast, are weaker and more
M. Reibarkh et al. / Journal of Magnetic Resonance 236 (2013) 126–133
131
Fig. 5. Comparative slices extracted from the single- and dual-optimized inverted 1JCC 1,n-ADEQUATE and spectra of strychnine (1). (A) Proton reference spectrum of strychnine. (B) Slice extracted at the 13C F1 shift of C21 of the dual-optimized experiment showing negative intensity for the C22–C21 correlation. Correlations for the C14– C21 and C20–C21 correlations are also inverted as expected. The experiment was dual-optimized for 1JCC = 57 and nJCC = 9.5 Hz for the first optimization and 1JCC = 64 and n JCC = 8 Hz for the second optimization. (C) Slice extracted at the 13C F1 shift of C21 showing undesirable positive intensity for the C22–C21 correlation. The positive intensity is consistent with the result predicted for a 71.8 Hz C21–C22 coupling in Fig. 1 [25,30]. Correlations for C14-C21 and C20–C21 are inverted as desired. The experiment was single-optimized for 1JCC = 40 and nJCC = 7 Hz [21,22]. Acquisition times were essentially identical for the two spectra (panel B = 48 h 42 min; panel C = 48 h 25 min) from which the slices shown in Panels B and C were taken. The number of total transients and t1 increments accumulated were identical. The traces shown in panels B and C are scaled relative to one another based on the intensity of the H16 correlation in panel B. Representative ‘‘noise’’ was defined by the 4.5–5.0 ppm region of each trace enclosed within the dashed box.
difficult to observe, but have considerable value for structure elucidation. Since the majority of long-range correlations observed in inverted 1JCC 1,n-ADEQUATE spectra are 3JCC correlations (equivalent to 4JCH HMBC correlations) [22], they are especially valuable for structure elucidation of proton-deficient molecules, where a single such correlation could ultimately prove or disprove a structure [36,37]. Thus the dual-optimized inverted 1JCC 1,n-ADEQUATE experiment provides an additional benefit of higher sensitivity for long-range correlations. 6. Alternative choices of optima for dual-optimized inverted 1JCC 1,n-ADEQUATE Developing an experiment with optima that uniformly invert JCC correlations over a practical range of 13C–13C coupling constants while keeping nJCC correlations positively phased was the
1
primary objective of the present study. While the pair of optima discussed, 57/9.5 Hz and 64/8 Hz, perhaps best fits this goal, it is important to recognize that there are other pairs of optima that also generally satisfy the objective, albeit with differing degrees of success. Several such pairs of optima have been identified computationally and have also been examined experimentally. These pairings are described in detail in the Supplementary material. None of these pairings are better overall than the dual optimization parameters reported here. However, they may provide some advantage in specific cases, particularly for detecting small nJCC correlations although this would be at a cost of relaxation-related signal losses and potentially non-uniform 1JCC inversion. Another notable observation is that in order to achieve the best mutual cancellation of unwanted oscillations in dual optimization experiment one has to explicitly account for the evolution of components of magnetization during long shaped pulses. The
132
M. Reibarkh et al. / Journal of Magnetic Resonance 236 (2013) 126–133
Fig. 6. Comparison of slices at the F1 shift of C8/C16 (60.18 and 60.08 ppm, respectively) showing nJCC correlations from the aromatic proton region to aliphatic carbon region of C8/C16 of strychnine (1) in the single- and dual-optimized inverted 1JCC 1,n-ADEQUATE spectra. (A) Proton reference spectrum. (B) Trace from dual-optimized spectrum acquired with optima of 1JCC = 57 and nJCC = 9.5 Hz for the first pair and 1JCC = 64 and nJCC = 8 Hz for the second pair. (C) Corresponding trace from the 1JCC 1,n-ADEQUATE spectrum optimized for 1JCC = 40 and nJCC = 7 Hz [21,22]. Correlations shown are from C4 to C8 and from C1 to C8 and C16. Measured S/N ratios for the peaks are shown in the figure. The defined noise region was 300 Hz in width from 7.3 to 7.8 ppm. Correlations in the dual-optimized experiment have a 30–100% higher S/N than the corresponding resonances of the single optimum experiment. Further, the 4JCC correlation from C2 to either C8 or C16 is clearly above the noise level in the dual-optimized experiment whereas the S/N of this correlation in the single optimum experiment could lead an investigator to question whether or not this is a legitimate correlation.
corresponding parameter was described previously [22] and was set to be 1.5 ms for all dual-optimized experiments reported here. 7. Experimental All NMR experiments described in this report were performed on a Bruker AVANCE III 600 MHz three channel NMR spectrometer equipped with a 1H/13C/15N three channel TXI 1.7 mm MicroCryoProbe™. All data were recorded using a 4.5 mg sample of strychnine (Fluka, Pestanal™ grade) dissolved in 40 lL of deuteriochloroform (CIL) in a conical bottom vial. The resulting solution was transferred to a 1.7 mm NMR tube (Bruker) using a 24G Teflon™ needle attached to a Hamilton gas-tight syringe. The single optima inverted 1JCC 1,n-ADEQUATE data reported were acquired using the pulse sequence previously reported [21]. The dual optimization experiments used the modified pulse sequence shown in Fig. 3 with the pairs of optima specified. All data were acquired as 3072 real points in F2 and with 200 increments of the t1 evolution period. For the 40/7 Hz single optimum experiment, 256 transients were accumulated per t1. For the dual optimization experiment shown in Fig. 4, 128 transients were accumulated for each pair of optima at each t1 increment. The
approach used gave a total of 256 transients per t1 increment in both experiments with acquisition times of 48 h 25 min and 48 h 42 min, respectively. Data were linear predicted to 4096 points in F2; data were linear predicted to 512 points in F1 and zero-filled to afford 2 K points in the second frequency domain after processing using an using an echo/anti-echo protocol. The possible pairs of optima for the dual-optimized amplitude transfer function have been explored computationally using exhaustive search approach. A script was written that scanned both 1JCC optima from 25 to 95 Hz and nJCC optima from 2 to 16 Hz with the increment of 0.5 Hz. Several pairs of optima identified by this approach were then verified and explored experimentally. Spectral slices extracted from individual spectra for comparison in Figs. 5 and 6 were scaled and plotted identically. Contour plot segments for comparison in Fig. S1 were plotted with a 6% threshold and maximum intensity of 95%. 8. Conclusions The dual-optimized, inverted 1JCC 1,n-ADEQUATE experiment has been demonstrated to have multiple advantages over the
M. Reibarkh et al. / Journal of Magnetic Resonance 236 (2013) 126–133
single-optimized version reported previously [21,22]. Dual optimization with optima at 57/9.5 Hz and 64/8 Hz provides uniform inversion of one-bond carbon–carbon correlations across a practical range of 1JCC couplings from 29 to 82 Hz, while maintaining usable positive intensity for long-range correlations across a useful range of nJCC couplings (>0.5 from 4 to 12 Hz). The dual optimization protocol described also provides a convenient and robust experimental setup, eliminating the need for sample-specific parameter optimization choices. Improvements in long-range correlation sensitivity have been observed for the dual-optimized experiment. Yet another advantage provided by the reported dual optimization is the potential reduction of relaxation-related signal losses due to the choice of larger nJCC and 1JCC optima. Improvements that have been discussed make dual-optimized, inverted 1JCC 1,n-ADEQUATE experiment a robust and universal method for mapping out the carbon skeleton of organic molecules, while reliably differentiating one-bond and long-range carbon– carbon correlations. Spectra that are well digitized in F1 can be acquired overnight with a 4.5 mg sample of strychnine in a 1.7 mm MicroCryoProbe; the technique reported is also practical for routine molecular structure elucidation applications when longer acquisition times are possible [36]. Smaller samples, or those with long T1 relaxation times will take correspondingly longer to acquire. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jmr.2013.07.016. References [1] A.S. Zektzer, B.K. John, R.N. Castle, G.E. Martin, Decoupling modulations due to one-bond heteronuclear spin-couplings in long-range heteronuclear chemical shift correlation spectra, J. Magn. Reson. 72 (1987) 556–561. [2] M. Salazar, A.S. Zektzer, G.E. Martin, Modulation of methylene carbon response intensity in long-range heteronuclear 2D-NMR chemical shift correlation spectra, Magn. Reson. Chem. 26 (1988) 24–27. [3] M. Salazar, A.S. Zektzer, G.E. Martin, Elimination of direct responses and onebond modulations in long-range heteronuclear chemical shift correlation spectra through the use of low-pass J-filters, Magn. Reson. Chem. 26 (1988) 28–32. [4] W.F. Reynolds, R.G. Enriquez, Choosing the best pulse sequences, acquisition parameters, post acquisition processing strategies and probes for natural product structure elucidation by NMR spectroscopy, J. Nat. Prod. 65 (2002) 221–244. [5] R.C. Breton, W.F. Reynolds, Using NMR to identify and characterize natural products, Nat. Prod. Rep. 30 (2013) 501–524. [6] R. Wagner, S. Berger, ACCORD-HMBC: a superior technique for structural elucidation, Magn. Reson. Chem. 36 (1998) S44–S46. [7] G.E. Martin, C.E. Hadden, R.C. Crouch, V.V. Krishnamurthy, ACCORD-HMBC: advantages and disadvantages of static versus accordion excitation, Magn. Reson. Chem. 37 (1999) 517–528. [8] G.E. Martin, C.E. Hadden, V.V. Krishnamurthy, Improved performance accordion heteronuclear multiple-bond correlation spectroscopy – IMPEACHMBC, J. Magn. Reson. 140 (1999) 274–280. [9] C.E. Hadden, G.E. Martin, V.V. Krishnamurthy, Constant time inverse-detection gradient accordion rescaled heteronuclear multiple bond correlation spectroscopy: CIGAR-HMBC, Magn. Reson. Chem. 38 (2000) 143–147. [10] R. Berger, C. Schorn, P. Biger, BIRD-HMBC: improved detection of heteronuclear long-range couplings, Magn. Reson. Chem. 38 (2000) 964–967. [11] A. Meissner, O.W. Sørensen, Economizing spectrometer time and broadband excitation in small-molecule heteronuclear NMR correlation spectroscopy. Broadband HMBC, Magn. Reson. Chem. 38 (2000) 981–984.
133
[12] R. Berger, C. Schorn, P. Bigler, HMSC: simultaneously detected heteronuclear shift correlation through multiple and single bonds, J. Magn. Reson. 148 (2001) 88–94. [13] G.E. Martin, Qualitative and quantitative exploitation of heteronuclear coupling constants, in: G.A. Webb (Ed.), Ann Rep. NMR Spectrosc., vol. 46, Academic Press, London, 2002, pp. 37–100. [14] W.F. Reynolds, Heteronuclear Multiple Bond Correlation (HMBC) spectra, Encycl. NMR Spectrosc. (2011), http://dx.doi.org/10.1002/ 9780470034590.emrstm1176. [15] W. Schoefberger, J. Schlagnitweit, N.L. Müller, Recent developments in heteronuclear multiple-bond correlation experiments, in: G.A. Webb (Ed.), Ann. Rep. NMR Spectrosc., vol. 72, Academic Press, London, 2011, pp. 1–60. [16] J. Furrer, Recent developments in HMBC studies, in: G.A. Webb (Ed.), Ann. Rep. NMR Spectrosc, vol. 74, Elsevier, London, 2011, pp. 294–354. [17] J. Furrer, A comprehensive discussion of HMBC pulse sequences: 1. The classical HMBC, Concepts Magn. Reson. 40A (2012) 101–127. [18] J. Furrer, A comprehensive discussion of HMBC pulse sequences. 2. Some useful variants, Concepts Magn. Reson. 40A (2012) 146–169. [19] G.E. Martin, B.D. Hilton, K.A. Blinov, HSQC-1,1-ADEQUATE and HSQC-1,nADEQUATE: enhanced methods for establishing adjacent and long-range 13 C–13C connectivity networks, J. Nat. Prod. 74 (2011) 2400–2407. [20] G.E. Martin, Using 1,1- and 1,n-ADEQUATE 2D NMR data in structure elucidation protocols, in: G.A. Webb (Ed.), Ann. Rep. NMR Spectrosc., vol. 74, Elsevier, London, 2011, pp. 215–291. [21] G.E. Martin, R.T. Williamson, P.G. Dormer, W. Bermel, Inversion of 1JCC correlations in 1,n-ADEQUATE spectra, Magn. Reson. Chem. 50 (2012) 563– 568. [22] G.E. Martin, K.A. Blinov, M. Reibarkh, R.T. Williamson, 1JCC-edited HSQC-1,nADEQUATE: a new paradigm for simultaneous direct and long-range carbon– carbon correlation, Magn. Reson. Chem. 50 (2012) 722–728. [23] M.E. Elyashberg, A.J. Williams, G.E. Martin, Computer-assisted structure elucidation, in: J. Feeney, L. Sutcliff (Eds.), Prog. NMR Spectrosc., vol. 53, Pergammon Press, London, 2008, pp. 1–104. [24] S.F. Cheatham, M. Kline, R.R. Sasaki, K.A. Blinov, M.E. Elyashberg, S.G. Molodtsov, Enhanced automated structure elucidation by inclusion of twobond specific data, Magn. Reson. Chem. 48 (2010) 571–574. [25] R.T. Williamson, A.V. Buevich, G.E. Martin, Experimental and theoretical investigation of 1JCC and nJCC coupling constants in strychnine, Org. Lett. 14 (2012) 5098–5101. [26] B. Reif, M. Köck, R. Kerssebaum, J. Schleucher, C. Griesinger, Determination of 1 2 J, J, and 3J carbon–carbon coupling constants at natural abundance, J. Magn. Reson. 112 (1996) 295–301. [27] K.E. Kövér, P. Forgó, J-modulated ADEQUATE (JM-ADEQUATE) experiment for accurate measurement of carbon–carbon coupling constants, J. Magn. Reson. 166 (2004) 47–52. [28] C.M. Thiele, W. Bermel, J-modulated ADEQUATE experiments using different kinds of refocusing pulses, Magn. Reson. Chem. 45 (2007) 889–894. [29] C.M. Thiele, W. Bermel, Speeding up the measurement of one-bond scalar (1J) and residual dipolar couplings (1D) by using non-uniform sampling (NUS), J. Magn. Reson. 216 (2012) 134–143. [30] G. Bifulco, R. Riccio, G.E. Martin, A.V. Buevich, R.T. Williamson, Quantum chemical calculations of 1JCC coupling constants for the stereochemical determination of organic compounds, Org. Lett. 15 (2013) 654–657. [31] T. Helgaker, M. Jaszun´ski, K. Ruud, Ab initio methods for the calculation of NMR shielding and indirect spin–spin coupling constants, Chem. Rev. 99 (1999) 293–352. [32] G. Bifulco, C. Bassarello, R. Riccio, L. Gomez-Paloma, Quantum mechanical calculations of NMR J coupling values in the determination of relative configuration in organic compounds, Org. Lett. 6 (2004) 1025–1028. [33] T. Helgaker, M. Jaszun´ski, M. Pecul, The quantum-mechanical calculation of NMR indirect spin–spin coupling constants, Prog. Nucl. Magn. Reson. Spectrosc. 53 (2008) 249–268. [34] K. Kamien´ska-Trela, One-bond 13C–13C spin–spin coupling constants, in: G.A. Webb (Ed.), Ann. Rep. NMR Spectrosc., vol. 30, Academic Press, London, 1993, pp. 131–230. [35] J.B. Stothers, Carbon-13 NMR Spectroscopy, Academic Press, New York, 1972. pp. 370–375. [36] G.E. Martin, A.V. Buevich, M. Reibarkh, S.B. Singh, J.G. Ondeyka, R.T. Williamson, Coniothyrione: anatomy of a structure revision, Magn. Reson. Chem. 51 (2013) 383–389. [37] T.F. Molinski, Microscale methodology for structure elucidation of natural products, Curr. Opin. Biotechnol. 21 (2010) 819–826.
Contents lists available at ScienceDirect
Journal of Magnetic Resonance journal homepage: www.elsevier.com/locate/jmr
Broadband inversion of 1JCC responses in 1,n-ADEQUATE spectra q Mikhail Reibarkh a,⇑, R. Thomas Williamson a, Gary E. Martin b,⇑, Wolfgang Bermel c a
Merck Research Laboratories, Discovery and Preclinical Sciences, Process and Analytical Chemistry, Structure Elucidation Group, Rahway, NJ 07065, USA Merck Research Laboratories, Discovery and Preclinical Sciences, Process and Analytical Chemistry, Structure Elucidation Group, Summit, NJ 07901, USA c BrukerBioSpin GmbH, Silberstreifen, 76287 Rheinstetten, Germany b
a r t i c l e
i n f o
Article history: Received 6 June 2013 Revised 27 July 2013 Available online 9 August 2013 Keywords: 1,n-ADEQUATE 1,1-ADEQUATE Pulse sequence Broadband inversion Strychnine
a b s t r a c t Establishing the carbon skeleton of a molecule greatly facilitates the process of structure elucidation, both manual and computer-assisted. Recent advances in the family of ADEQUATE experiments demonstrated their potential in this regard. 1,1-ADEQUATE, which provides direct 13C–13C correlation via 1JCC, and 1,n-ADEQUATE, which typically yields 3JCC and 1JCC correlations, are more sensitive and more widely applicable experiments than INADEQUATE and PANACEA. A recently reported modified pulse sequence that semi-selectively inverts 1JCC correlations in 1,n-ADEQUATE spectra provided a significant improvement, allowing 1JCC and nJCC correlations to be discerned in the same spectrum. However, the reported experiment requires a careful matching of the amplitude transfer function with 1JCC coupling constants in order to achieve the inversion, and even then some 1JCC correlations could still have positive intensity due to the oscillatory nature of the transfer function. Both shortcomings limit the practicality of the method. We now report a new, dual-optimized inverted 1JCC 1,n-ADEQUATE experiment, which provides more uniform inversion of 1JCC correlations across the range of 29–82 Hz. Unlike the original method, the dual optimization experiment does not require fine-tuning for the molecule’s 1JCC coupling constant values. Even more usefully, the dual-optimized version provides up to two-fold improvement in signalto-noise for some long-range correlations. Using modern, cryogenically-cooled probes, the experiment can be successfully applied to samples of 1 mg under favorable circumstances. The improvements afforded by dual optimization inverted 1JCC 1,n-ADEQUATE experiment make it a useful and practical tool for NMR structure elucidation and should facilitate the implementation and utilization of the experiment. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction An intrinsic property of NMR spectroscopy is the reliance on the modulations of various phenomena. Modulations of various types are an integral aspect of many 2D and nD NMR experiments. Some modulations are beneficial, enhancing response intensity while others can be problematic, leading to artifacts and/or to reduced correlation intensity or even lost responses. Problems due to modulations are long-standing. Early work in the area of heteronucleus-detected long-range heteronuclear chemical shift correlation experiments investigated the problem of fast modulations resulting from the relatively large 1JCH coupling constants, which were subsequently decoupled in modified variants of the experiment [1–3]. Modulation problems have not been circumvented with the advent of proton-detected long-range het-
q Presented at the 18th ISMAR Meeting, Rio de Janeiro, Brazil, May 19–24, 2013, ‘‘Broadband inversion of 1JCC correlations in 1,n-ADEQUATE spectra’’, paper OP118. ⇑ Corresponding authors. E-mail addresses: [email protected] (M. Reibarkh), gary.martin2@ merck.com (G.E. Martin).
1090-7807/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmr.2013.07.016
eronuclear shift correlation experiments. The widely used HMBC experiment provides a good example in this regard. HMBC optimization has generally been a compromise, biasing an experiment toward either smaller or larger heteronuclear couplings [4,5]. These exigencies have prompted several groups to develop modified experiments to allow the observation of a broader range of longrange heteronuclear couplings. The first such experiment was ACCORD-HMBC [6,7], followed by IMPEACH-MBC [8], CIGAR-HMBC [9], and others [10–12]. A detailed discussion of HMBC and experimental variants has been the subject of a number of reviews [13– 18]. Amplitude modulation of response intensity must also be taken into account when optimizing delays in experiments such as 1,1ADEQUATE [19,20] and inverted 1JCC 1,n-ADEQUATE [21]. In the former, correlations between adjacent carbons via 1JCC couplings may be lost as a consequence of the mismatch between the experimental optimization and the actual 1JCC coupling [19,20]. We have recently shown that modulation of magnetization as a function of the magnitude of direct and long-range 13C–13C couplings must also be taken into consideration in experiments such as inverted 1JCC 1,n-ADEQUATE [21,22]. The central premise
127
M. Reibarkh et al. / Journal of Magnetic Resonance 236 (2013) 126–133
of the inverted 1JCC 1,n-ADEQUATE pulse sequence is that by inverting the 1JCC correlations, which tend to unavoidably ‘‘leak’’ into a 1,n-ADEQUATE spectrum, they can be readily identified without having to resort to other experiments such as conventional 1,1ADEQUATE. Multiple-bond carbon–carbon correlations (nJCC) will always have positive phase in the inverted 1JCC 1,n-ADEQUATE experiment, readily differentiating them from 1JCC correlations when the latter are inverted [21,22]. A single spectrum that differentiates 1JCC from nJCC correlations has tremendous implications for structure elucidation. It would be particularly valuable as input for CASE (Computer-Assisted Structure Elucidation) programs where having unambiguous connectivity information drastically reduces both the number of structures generated and the corresponding computational time, as well as increasing the reliability of final structure(s) [23,24]. The amplitude transfer function that controls response intensity is governed by both the 1JCC and nJCC optimizations chosen. As a consequence of the oscillatory nature of the transfer function, 1JCC correlations can have unintended, positive response intensity in spectra acquired using single optimization [21,22] as predicted by the curve shown in Fig. 1. Aberrantly phased 1 JCC correlations are obviously highly undesirable as they complicate the data interpretation and could lead to errors in the elucidation of unknown structures due to the mistaken attribution of a one-bond correlation as an n-bond correlation. One approach to this problem is to attempt to optimize the amplitude transfer function so that all 1JCC couplings are inverted. However, this solution is both impractical and decidedly non-universal. Inverting a broad range of 1JCC couplings is quite difficult, since no single amplitude transfer function can achieve this objective due to the variability of 1JCC couplings, which are found to range from 32 to 72 Hz for strychnine [22,25]. Here, we report a dual optimization approach based on mutual cancellation of unwanted oscillations. We show that a dual-optimized, inverted 1 JCC 1,n-ADEQUATE experiment affords a much broader and more uniform inversion of 1JCC correlations in 1,n-ADEQUATE spectra than can be achieved using a single-optimized setup of the experiment. Additionally, it is demonstrated that the intensity of the nJCC correlations is improved relative to the previous variant of the experiment. 2. Optimization vs. 1JCC modulation Recently, a modified 1,n-ADEQUATE pulse sequence was reported that utilizes asymmetric delays to modify the oscillatory
Fig. 1. Amplitude transfer function for an inverted 1JCC 1,n-ADEQUATE [21] experiment optimized for 1JCC = 40 and nJCC = 7 Hz. The C22–C21 correlation in the spectrum of strychnine (1), which has a one-bond carbon–carbon coupling constant of 71.8 Hz, would be expected to exhibit a positive correlation and does (see Fig. 5C) [22,25].
behavior of the 1JCC and nJCC couplings, allowing the former to be semi-selectively inverted [21]. The experiment is not uniformly selective in that an inopportune 1JCC coupling may yield a positive response. For example, the 71.8 Hz C22–C21 coupling of strychnine (1) [25] yields a positive correlation when the experiment is optimized, for 1JCC = 40 and nJCC = 7 Hz [22], both commonly utilized optimizations for carbon–carbon coupling constants in 1,1- and 1,n-ADEQUATE experiments, respectively [20]. The amplitude transfer curve for the 1JCC correlations using the 1JCC/nJCC optimizations just noted is shown in Fig. 1. The other ‘‘islands’’ of positive intensity from 32–35 and 58–63 Hz could also yield unintended, positively phased 1JCC correlations for some aliphatic and aromatic carbon resonances (see Structure 1). Positive 1JCC correlations that could be mistaken for an nJCC correlation in the spectrum of an unknown represent a disadvantage and somewhat decrease the utility of the inverted 1JCC 1,n-ADEQUATE experiment. This problem can be partially circumvented by a judicious choice of 1JCC and nJCC optimization. Carefully optimizing the experiment, however, presupposes that one has a knowledge of the 1JCC coupling constants in the molecule being studied, which will only rarely be the case. Generally, an investigator will not have J-modulated ADEQUATE [25–30] data at his disposal and quantum mechanical calculation of the coupling constants with sufficient accuracy is impractical, requiring a significant computational effort [31–33] as well as prior knowledge of the actual structure before setting up every experiment. Furthermore, this approach is by no means a universal solution since no single choice of 1JCC and nJCC optima will likely achieve inversion of the full range of 1 JCC constants relevant for a given molecule (Supplementary material, Fig. S2). To the contrary, an unfortunate choice of 1JCC and nJCC optimization may lead to a spectrum where the majority of one-bond correlations are not inverted. Such is the case in the inverted 1JCC 1,n-ADEQUATE spectrum of strychnine optimized for 1JCC = 50 and nJCC = 5 Hz. While the choice of both optima is logical and quite reasonable, it results in a spectrum with very few inverted aliphatic one-bond correlations. Closer inspection of the amplitude transfer curve (see Fig. S3) confirms that most of strychnine’s aliphatic 1JCC couplings lie in the regions where the transfer curve is positive [25,30]. These considerations prompted us to explore further modifications of the pulse sequence to afford a broader range of inversion of the 1JCC correlations in 1,n-ADEQUATE spectra. Strategically, we wanted to improve the technique in two ways. First, it is highly desirable to have all 1JCC correlations either negative or, failing that, absent from the spectrum. When negative, 1JCC correlations are
Structure 1.
128
M. Reibarkh et al. / Journal of Magnetic Resonance 236 (2013) 126–133
readily identified and provide useful structural information. If absent, they cannot be mistaken for a nJCC long-range correlation in the 1,n-ADEQUATE spectrum of an unknown. The second objective is to have a simple and universal experimental setup that does not necessitate adjusting the optimization for every sample. These attributes improve the viability of the method and should facilitate the implementation and utilization of the experiment. All of the issues enumerated above are a consequence of the oscillatory nature of the 1JCC modulation. The amplitude transfer function is essentially a slowly oscillating sine wave (governed, in part, by the 1JCC optimization), modulated by a rapidly oscillating sine wave (conversely governed, in part, by nJCC optimization), as summarized mathematically in the following section. These attributes provide an avenue for exploitation: the sum of two transfer functions can be used to cancel out the fast oscillations while preserving the slow wave, which determines the 1JCC inversion. Judicious choices of pairs of optima can lead to amplitude transfer curves that, when superimposed and summed, lead to much broader inversion of 1JCC correlations.
3. Amplitude transfer functions of different versions of the ADEQUATE experiment In the ‘‘traditional’’ 1,1- and 1,n-ADEQUATE experiments, both JCC and 1JCC carbon–carbon couplings evolve according to sin(J p D) after the initial H,C-INEPT transfer. Being an outand-back experiment, the same transfer amplitudes apply for the delay prior to the reverse INEPT transfer. Thus, the overall transfer amplitude is defined by sin2(J p D). Hence, the transfer functions afford nJCC and 1JCC cross peaks that are always positive and therefore preclude discrimination between nJCC and 1JCC. In fact, the only parameter defining whether the experiment is 1,1- and 1,n- is the delay used to maximize the transfer function at particular value of J (typically 40–60 Hz for 1JCC and 5–7 Hz for nJCC) [20]. Because the 1JCC coupling constant is considerably larger than the nJCC coupling, the corresponding sine term for 1JCC oscillates much faster than the sine term for nJCC. This difference makes it possible to modulate the amplitude transfer function to keep it negative for 1JCC correlations, but still positive for nJCC correlations. Such modulation was achieved by making the pulse sequence asymmetric, that is, using slightly different delay settings before and after t1 evolution [21,22]. The approach yielded the 1JCC-edited 1,n-ADEQUATE experiment [21,22], which is referred to hereafter as single-optimization 1Jcc-edited 1,n-ADEQUATE in the manuscript. In this work, we report a further refinement of the asymmetric delay approach, which provides a more uniform and more robust inversion of 1JCC correlations, while improving the signal-to-noise (S/N) ratios for nJCC correlation. The amplitude transfer function of the modified dual optimization experiment is essentially a normalized sum of two differently optimized single-optimization transfer functions. The fundamental principle governing all ADEQUATE experiments described above (1,1-, 1,n-, single-optimization 1Jcc-edited, and dual-optimized 1Jcc-edited) is identical: H,C-INEPT transfer, followed by JCC evolution, followed by a reverse C,H-INEPT transfer. The difference between the experiments is in the modulation during the evolution, which is achieved by different delay optimizations. While 1,1- and 1,n-ADEQUATE experiments have symmetrical delays, 1JCC-edited experiments employ asymmetrical delays. The mathematical description of the amplitude transfer modulations as a function of JCC coupling constant follows: n
(1) 1,1-ADEQUATE: f(J) = sin2(J p D). D is usually optimized to match J of 40–60 Hz. (2) 1,n-ADEQUATE: f(J) = sin2(J p D). D is usually optimized to match J of 5–7 Hz.
Fig. 2. Calculated amplitude transfer curves governing the response intensity of an inverted 1JCC 1,n-ADEQUATE experiment modified for dual optimization of the carbon–carbon delays (blue). The two optimizations were 1JCC = 57 with nJCC = 9.5 Hz (red), and 1JCC = 64 with nJCC = 8 Hz (green). (A) This panel shows the response intensity of the 1JCC correlations as a function of the optimization. The blue trace shows the calculated, summed, and normalized response intensity across the optimization range from 30 to 90 Hz. One-bond correlations are inverted across the range from 29 to 82 Hz, which encompasses most commonly encountered onebond carbon–carbon [34,35]. (B) This panel shows the response intensity for the nJCC correlations as a function of the optimization across the range from 0 to 16 Hz. The blue trace shows the summed and normalized long-range carbon–carbon response intensity. This dual optimization pair gives usable response intensity (>0.5) across the range from approximately 4 to 12 Hz, which encompasses most structurally useful nJCC in the authors’ experience [20].
(3) Single-optimization 1Jcc-edited 1,n-ADEQUATE: f(J) = sin(J p ((2 m + 1)/(2 1Jopt) + d)) sin(J p ((2 m 1)/(2 1Jopt) + d)). Here, m is the truncated integer of 1Jopt/(2 nJopt). 1Jopt and nJopt are the optimization values for 1JCC and nJCC, respectively. d Is the sum of all additional delays in the pulse sequence (primarily due to the length of shaped pulses). (4) Dual optimization 1Jcc-edited 1,n-ADEQUATE: f(J) = 0.5 [sin(J p ((2 mA + 1)/(2 1JA)+d)) sin(J p ((2 mA 1)/(2 1JA) + d)) + sin(J p ((2 mB + 1)/(2 1JB) + d)) sin(J p ((2 mB 1)/(2 1JB) + d))].
M. Reibarkh et al. / Journal of Magnetic Resonance 236 (2013) 126–133
Here, mA is the truncated integer of 1JA/(2 nJA) and mB is the truncated integer of 1JB/(2 nJB). 1JA/nJA and 1JB/nJB are the two pairs of optimization values for 1JCC and nJCC, respectively. An example of the dual optimization scheme is shown in Fig. 2 in which two amplitude transfer curves, one optimized for 1JCC = 57 and nJCC = 9.5 Hz (red) and a second optimized for 1JCC = 64 and n JCC = 8 Hz (green) give the result shown by the blue curve when transfer function amplitudes are summed and normalized. Fig. 2A shows all three functions across the range of the most commonly encountered 1JCC coupling constants. One-bond carbon–carbon correlations are effectively inverted across the range from 29–82 Hz and 89–96 Hz. With the exception of cyclopropyl, cyclobutyl, and acetylenic carbon–carbon bonds, there are relatively few other structural moieties encountered in natural products or pharmaceuticals with one-bond carbon–carbon coupling constants outside of this range [34]. The corresponding amplitude transfer functions for the long-range nJCC couplings are shown in Fig. 2B; again, the blue trace corresponds to the summed and normalized amplitude for the two differently optimized transfer functions used. The useful range of nJCC couplings is 4–12 Hz (normalized intensity >0.5) with all couplings from 0 to 15 Hz having positive intensity. The range of long-range couplings with intensities of >0.5 incorporates the normally useful continuum of long-range carbon–carbon correlations [20]. The pulse sequence implemented to exploit dual optimization of the amplitude transfer functions shown in Fig. 3 is schematically indistinguishable from that previously reported [21]. The modified, dual optimization experiment performs n transients, typically set to a multiple of the 16-step phase cycle, for the first set of optimization parameters and then adds n transients for the second optimization before incrementing the gradients, phases, and delays (the Bruker TopSpin 3.1 pulse sequence code is reproduced in the Supplementary material). 4. Dual optimization inverted 1JCC 1,n-ADEQUATE The experimental results obtained using the inverted 1JCC 1,n-ADEQUATE experiment modified for dual optimization of the one-bond and long-range carbon–carbon coupling constant-based delays are shown in Fig. 4. The experiment was optimized using 57/9.5 and 64/8 Hz for the 1JCC/nJCC optimization pairs, the amplitude transfer curves for which are shown in Fig. 2. Positive nJCC correlations are plotted in black; inverted 1JCC correlations are plotted in red. As will be noted by inspection of the spectrum shown in Fig. 4, the problematic, positively phased C22–C21 correlation in the original version of the experiment [22] (1JCC/nJCC optimization
129
of 40/7 Hz) has been successfully inverted in the modified version of the experiment, as expected from the transfer curve (see also Supplemental Table 1). A comparison of the problematic C21–C22 response [22] is shown by the slices extracted from the 2D spectra in Fig. 5. The proton reference spectrum of strychnine (1) is shown in Fig. 5A. The F1 slice for the C21–C22 correlation from the dual-optimized experiment is shown in Fig. 5B while the corresponding slice from the original single-optimization inverted 1JCC spectrum optimized for 1JCC/nJCC = 40/7 Hz is shown in Fig. 5C. The traces in panels 5B and 5C were extracted at the 13C shift of the C21 quaternary carbon. Note that consistent with the amplitude curve shown in Fig. 1, the C21–C22 correlation with a coupling constant of 71.8 Hz has strong positive intensity rather than being inverted as intended in panel 5C. As intended, the C21–C22 correlation observed at the H22 proton chemical shift is inverted in the slice shown in Fig. 5B extracted from the dual-optimized spectrum shown in Fig. 4. The response intensity is consistent with the blue normalized summation curve shown in Fig. 2A. It is interesting to compare the relative S/N ratio of various responses in the slices shown in Fig. 5B and C. The most obvious difference is of course seen for the H22–C21 correlation which has a S/N ratio of 9.2:1 in the dual-optimized experiment (panel 5B) in contrast to a +34.2:1 S/N ratio in the single-optimized experiment (panel 5C). The change in phase and reduction in signal intensity for the C22–C21 correlation is consistent with what would be predicted from the amplitude transfer curves shown in Figs. 1 and 2A. The S/N for the C16–C21 correlation increased from +33.6:1 for this correlation in the single-optimization spectrum to +53.5:1 in the dual optimization experiment. In a similar fashion, the C13–C21 correlation intensity also increased significantly from +4.7:1 to +8.3:1 in going from single to dual optimization. The intensity of the C8–C21 correlation also increased, albeit only slightly from +3.6:1 to +4.1:1. As noted above, there is the expectation that the intensity of the 1 JCC correlations will be attenuated somewhat in the dual optimization experiment vs. the single optimization experiment. In the case of the 40/7 Hz single-optimized experiment, the C14–C21 correlation has the greatest intensity of 63.0:1. In contrast, the C14– C21 correlation is significantly attenuated in the dual-optimized experiment with a S/N ratio of 23.4:1. The same is true for the C20–C21 correlation that decreased from 42.5:1 to 9.3:1 for the correlation observed at the F2 shift of the H20a resonance and from 58.5:1 to 10.2:1 for the correlation observed at the F2 shift of the H20b resonance.
Fig. 3. Pulse sequence for dual optimization inverted 1JCC correlated 1,n-ADEQUATE. The duration of the D5 and D3 delays, were modified as described in the text. Narrow and wide bars indicate 90° and 180° pulses, respectively, with the exception of the 120° pulse that is specifically labeled. Unless otherwise indicated, phases were: U1 = 0000 2222, U2 = 0000 0000 2222 2222; U3 = 0022; U4 = 1133; UR = 0220 2002 2002 0220. Gradient ratios were: Gz1 = +78.4%; Gz2 = +77.4%; Gz3 = 59%. The Bruker TopSpin 3.1 pulse sequence code is contained in the supplemental material.
130
M. Reibarkh et al. / Journal of Magnetic Resonance 236 (2013) 126–133
Fig. 4. Dual-optimized inverted 1JCC 1,n-ADEQUATE spectrum of strychnine. The data were acquired as 3072 200 real points, with 128 transients accumulated for each set of optima. The experiment was dual-optimized for 1JCC = 57 and nJCC = 9.5 Hz for the first optimization and 1JCC = 64 and nJCC = 8 Hz for the second optimization. The acquisition time for the spectrum was 48 h 42 min. The data were linear predicted to 4096 points in F2 and to 512 points in F1 followed by zero-filling prior to the second Fourier transform to afford the 2 K 2 K spectrum shown. Data were subjected to sin2 apodization phase-shifted 90° prior to both Fourier transforms. Experimentally, the order in which the optima are entered into the pulse sequence and acquired is of no consequence.
Beneficial improvements in the intensity of the long-range correlations in the dual-optimized experiment are also highlighted in Fig. 6, which compares the correlations from the aromatic proton region to the aliphatic carbon region as an example. The trace shown in Fig. 6B was extracted from the spectrum shown in Fig. 4 at the 13C shift for C8/C16 (60.18 and 60.08 ppm, respectively); the trace shown in Fig. 6C was extracted at the 13C shift for the C8/C16 resonances in the 40/7 Hz single-optimized experiment [21,22]. Generally, the intensity of the nJCC correlations in the dual-optimized spectrum increased by 30–100% compared to the corresponding correlations in the 1JCC/nJCC = 40/7 Hz single-optimized spectrum [21,22]. The 1JCC correlations all have the intended negative phase and, as expected, somewhat reduced response intensity as predicted by the amplitude transfer curve shown in Fig. 2A. A comparison of the aliphatic region of the dual-optimized spectrum (panel A) and the corresponding region single-optimized spectrum (panel B) is shown in Supplemental Fig. S1. Both spectra were identically plotted with a 6% threshold. It can be clearly seen that there are correlations present in panel A that are absent in the single-optimized spectrum shown in panel B or below the 6% threshold. It should also be noted that correlations visible in both spectra are generally more intense in the dual-optimized spectrum shown in panel A.
5. Improved sensitivity for long-range correlations in dualoptimized inverted 1JCC 1,n-ADEQUATE As is evident from a comparison of the amplitude transfer functions in Figs. 1 and 2A, one-bond correlation intensities are expected to be somewhat lower for the dual-optimized inverted 1 JCC 1,n-ADEQUATE spectrum compared to the single optimum
experiment. Experimental observations support this premise, with 1D slices in Figs. 5 and 6 providing a clear illustration. However, one should keep in mind that the amplitude transfer function shown in Fig. 2A is essentially an attenuation function for 1JCC correlations that can tolerate some degree of attenuation. Here, the worst case scenario would be a missed one-bond correlation due to low intensity, which is still acceptable, and much better than an aberrant, positively phased 1JCC correlation, such as C22–C21 correlation in the 40/7 Hz single-optimized experiment [22]. More interestingly, nJCC correlations demonstrate improved sensitivity in dual-optimized inverted 1JCC 1,n-ADEQUATE spectra as shown in 1D slices (Figs. 5 and 6). For 2D data, improved sensitivity can translate into the detection of weaker nJCC correlations observed in the dual-optimized spectrum than is possible in the single optimum spectrum, as shown in Supplemental Fig. S1. Alternately, the improved sensitivity may allow the practical investigation of smaller samples. A plausible reason for the improved nJCC correlation S/N is the reduction of relaxation-related signal losses due to the choice of larger nJCC and 1JCC optima in comparison with the single-optimized experiment. Under favorable circumstances, it is possible to apply variants of the experiments described in this report to samples as small as 1 mg [36]. The experiment does, however, make significant demands on instrument performance and molecules with long T1 relaxation times may require the use of interpulse delays considerably longer than the 3 s delay normally used for ADEQUATE experiments. Improving nJCC correlation sensitivity at the cost of lower 1JCC correlation sensitivity is a viable trade-off for several reasons. First, not only do one-bond correlations generally have greater intrinsic intensity and can thereby tolerate some degree of attenuation due to the transfer function, but missing an occasional one-bond correlation is an acceptable scenario for the purposes of structure elucidation. Long-range correlations, in contrast, are weaker and more
M. Reibarkh et al. / Journal of Magnetic Resonance 236 (2013) 126–133
131
Fig. 5. Comparative slices extracted from the single- and dual-optimized inverted 1JCC 1,n-ADEQUATE and spectra of strychnine (1). (A) Proton reference spectrum of strychnine. (B) Slice extracted at the 13C F1 shift of C21 of the dual-optimized experiment showing negative intensity for the C22–C21 correlation. Correlations for the C14– C21 and C20–C21 correlations are also inverted as expected. The experiment was dual-optimized for 1JCC = 57 and nJCC = 9.5 Hz for the first optimization and 1JCC = 64 and n JCC = 8 Hz for the second optimization. (C) Slice extracted at the 13C F1 shift of C21 showing undesirable positive intensity for the C22–C21 correlation. The positive intensity is consistent with the result predicted for a 71.8 Hz C21–C22 coupling in Fig. 1 [25,30]. Correlations for C14-C21 and C20–C21 are inverted as desired. The experiment was single-optimized for 1JCC = 40 and nJCC = 7 Hz [21,22]. Acquisition times were essentially identical for the two spectra (panel B = 48 h 42 min; panel C = 48 h 25 min) from which the slices shown in Panels B and C were taken. The number of total transients and t1 increments accumulated were identical. The traces shown in panels B and C are scaled relative to one another based on the intensity of the H16 correlation in panel B. Representative ‘‘noise’’ was defined by the 4.5–5.0 ppm region of each trace enclosed within the dashed box.
difficult to observe, but have considerable value for structure elucidation. Since the majority of long-range correlations observed in inverted 1JCC 1,n-ADEQUATE spectra are 3JCC correlations (equivalent to 4JCH HMBC correlations) [22], they are especially valuable for structure elucidation of proton-deficient molecules, where a single such correlation could ultimately prove or disprove a structure [36,37]. Thus the dual-optimized inverted 1JCC 1,n-ADEQUATE experiment provides an additional benefit of higher sensitivity for long-range correlations. 6. Alternative choices of optima for dual-optimized inverted 1JCC 1,n-ADEQUATE Developing an experiment with optima that uniformly invert JCC correlations over a practical range of 13C–13C coupling constants while keeping nJCC correlations positively phased was the
1
primary objective of the present study. While the pair of optima discussed, 57/9.5 Hz and 64/8 Hz, perhaps best fits this goal, it is important to recognize that there are other pairs of optima that also generally satisfy the objective, albeit with differing degrees of success. Several such pairs of optima have been identified computationally and have also been examined experimentally. These pairings are described in detail in the Supplementary material. None of these pairings are better overall than the dual optimization parameters reported here. However, they may provide some advantage in specific cases, particularly for detecting small nJCC correlations although this would be at a cost of relaxation-related signal losses and potentially non-uniform 1JCC inversion. Another notable observation is that in order to achieve the best mutual cancellation of unwanted oscillations in dual optimization experiment one has to explicitly account for the evolution of components of magnetization during long shaped pulses. The
132
M. Reibarkh et al. / Journal of Magnetic Resonance 236 (2013) 126–133
Fig. 6. Comparison of slices at the F1 shift of C8/C16 (60.18 and 60.08 ppm, respectively) showing nJCC correlations from the aromatic proton region to aliphatic carbon region of C8/C16 of strychnine (1) in the single- and dual-optimized inverted 1JCC 1,n-ADEQUATE spectra. (A) Proton reference spectrum. (B) Trace from dual-optimized spectrum acquired with optima of 1JCC = 57 and nJCC = 9.5 Hz for the first pair and 1JCC = 64 and nJCC = 8 Hz for the second pair. (C) Corresponding trace from the 1JCC 1,n-ADEQUATE spectrum optimized for 1JCC = 40 and nJCC = 7 Hz [21,22]. Correlations shown are from C4 to C8 and from C1 to C8 and C16. Measured S/N ratios for the peaks are shown in the figure. The defined noise region was 300 Hz in width from 7.3 to 7.8 ppm. Correlations in the dual-optimized experiment have a 30–100% higher S/N than the corresponding resonances of the single optimum experiment. Further, the 4JCC correlation from C2 to either C8 or C16 is clearly above the noise level in the dual-optimized experiment whereas the S/N of this correlation in the single optimum experiment could lead an investigator to question whether or not this is a legitimate correlation.
corresponding parameter was described previously [22] and was set to be 1.5 ms for all dual-optimized experiments reported here. 7. Experimental All NMR experiments described in this report were performed on a Bruker AVANCE III 600 MHz three channel NMR spectrometer equipped with a 1H/13C/15N three channel TXI 1.7 mm MicroCryoProbe™. All data were recorded using a 4.5 mg sample of strychnine (Fluka, Pestanal™ grade) dissolved in 40 lL of deuteriochloroform (CIL) in a conical bottom vial. The resulting solution was transferred to a 1.7 mm NMR tube (Bruker) using a 24G Teflon™ needle attached to a Hamilton gas-tight syringe. The single optima inverted 1JCC 1,n-ADEQUATE data reported were acquired using the pulse sequence previously reported [21]. The dual optimization experiments used the modified pulse sequence shown in Fig. 3 with the pairs of optima specified. All data were acquired as 3072 real points in F2 and with 200 increments of the t1 evolution period. For the 40/7 Hz single optimum experiment, 256 transients were accumulated per t1. For the dual optimization experiment shown in Fig. 4, 128 transients were accumulated for each pair of optima at each t1 increment. The
approach used gave a total of 256 transients per t1 increment in both experiments with acquisition times of 48 h 25 min and 48 h 42 min, respectively. Data were linear predicted to 4096 points in F2; data were linear predicted to 512 points in F1 and zero-filled to afford 2 K points in the second frequency domain after processing using an using an echo/anti-echo protocol. The possible pairs of optima for the dual-optimized amplitude transfer function have been explored computationally using exhaustive search approach. A script was written that scanned both 1JCC optima from 25 to 95 Hz and nJCC optima from 2 to 16 Hz with the increment of 0.5 Hz. Several pairs of optima identified by this approach were then verified and explored experimentally. Spectral slices extracted from individual spectra for comparison in Figs. 5 and 6 were scaled and plotted identically. Contour plot segments for comparison in Fig. S1 were plotted with a 6% threshold and maximum intensity of 95%. 8. Conclusions The dual-optimized, inverted 1JCC 1,n-ADEQUATE experiment has been demonstrated to have multiple advantages over the
M. Reibarkh et al. / Journal of Magnetic Resonance 236 (2013) 126–133
single-optimized version reported previously [21,22]. Dual optimization with optima at 57/9.5 Hz and 64/8 Hz provides uniform inversion of one-bond carbon–carbon correlations across a practical range of 1JCC couplings from 29 to 82 Hz, while maintaining usable positive intensity for long-range correlations across a useful range of nJCC couplings (>0.5 from 4 to 12 Hz). The dual optimization protocol described also provides a convenient and robust experimental setup, eliminating the need for sample-specific parameter optimization choices. Improvements in long-range correlation sensitivity have been observed for the dual-optimized experiment. Yet another advantage provided by the reported dual optimization is the potential reduction of relaxation-related signal losses due to the choice of larger nJCC and 1JCC optima. Improvements that have been discussed make dual-optimized, inverted 1JCC 1,n-ADEQUATE experiment a robust and universal method for mapping out the carbon skeleton of organic molecules, while reliably differentiating one-bond and long-range carbon– carbon correlations. Spectra that are well digitized in F1 can be acquired overnight with a 4.5 mg sample of strychnine in a 1.7 mm MicroCryoProbe; the technique reported is also practical for routine molecular structure elucidation applications when longer acquisition times are possible [36]. Smaller samples, or those with long T1 relaxation times will take correspondingly longer to acquire. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jmr.2013.07.016. References [1] A.S. Zektzer, B.K. John, R.N. Castle, G.E. Martin, Decoupling modulations due to one-bond heteronuclear spin-couplings in long-range heteronuclear chemical shift correlation spectra, J. Magn. Reson. 72 (1987) 556–561. [2] M. Salazar, A.S. Zektzer, G.E. Martin, Modulation of methylene carbon response intensity in long-range heteronuclear 2D-NMR chemical shift correlation spectra, Magn. Reson. Chem. 26 (1988) 24–27. [3] M. Salazar, A.S. Zektzer, G.E. Martin, Elimination of direct responses and onebond modulations in long-range heteronuclear chemical shift correlation spectra through the use of low-pass J-filters, Magn. Reson. Chem. 26 (1988) 28–32. [4] W.F. Reynolds, R.G. Enriquez, Choosing the best pulse sequences, acquisition parameters, post acquisition processing strategies and probes for natural product structure elucidation by NMR spectroscopy, J. Nat. Prod. 65 (2002) 221–244. [5] R.C. Breton, W.F. Reynolds, Using NMR to identify and characterize natural products, Nat. Prod. Rep. 30 (2013) 501–524. [6] R. Wagner, S. Berger, ACCORD-HMBC: a superior technique for structural elucidation, Magn. Reson. Chem. 36 (1998) S44–S46. [7] G.E. Martin, C.E. Hadden, R.C. Crouch, V.V. Krishnamurthy, ACCORD-HMBC: advantages and disadvantages of static versus accordion excitation, Magn. Reson. Chem. 37 (1999) 517–528. [8] G.E. Martin, C.E. Hadden, V.V. Krishnamurthy, Improved performance accordion heteronuclear multiple-bond correlation spectroscopy – IMPEACHMBC, J. Magn. Reson. 140 (1999) 274–280. [9] C.E. Hadden, G.E. Martin, V.V. Krishnamurthy, Constant time inverse-detection gradient accordion rescaled heteronuclear multiple bond correlation spectroscopy: CIGAR-HMBC, Magn. Reson. Chem. 38 (2000) 143–147. [10] R. Berger, C. Schorn, P. Biger, BIRD-HMBC: improved detection of heteronuclear long-range couplings, Magn. Reson. Chem. 38 (2000) 964–967. [11] A. Meissner, O.W. Sørensen, Economizing spectrometer time and broadband excitation in small-molecule heteronuclear NMR correlation spectroscopy. Broadband HMBC, Magn. Reson. Chem. 38 (2000) 981–984.
133
[12] R. Berger, C. Schorn, P. Bigler, HMSC: simultaneously detected heteronuclear shift correlation through multiple and single bonds, J. Magn. Reson. 148 (2001) 88–94. [13] G.E. Martin, Qualitative and quantitative exploitation of heteronuclear coupling constants, in: G.A. Webb (Ed.), Ann Rep. NMR Spectrosc., vol. 46, Academic Press, London, 2002, pp. 37–100. [14] W.F. Reynolds, Heteronuclear Multiple Bond Correlation (HMBC) spectra, Encycl. NMR Spectrosc. (2011), http://dx.doi.org/10.1002/ 9780470034590.emrstm1176. [15] W. Schoefberger, J. Schlagnitweit, N.L. Müller, Recent developments in heteronuclear multiple-bond correlation experiments, in: G.A. Webb (Ed.), Ann. Rep. NMR Spectrosc., vol. 72, Academic Press, London, 2011, pp. 1–60. [16] J. Furrer, Recent developments in HMBC studies, in: G.A. Webb (Ed.), Ann. Rep. NMR Spectrosc, vol. 74, Elsevier, London, 2011, pp. 294–354. [17] J. Furrer, A comprehensive discussion of HMBC pulse sequences: 1. The classical HMBC, Concepts Magn. Reson. 40A (2012) 101–127. [18] J. Furrer, A comprehensive discussion of HMBC pulse sequences. 2. Some useful variants, Concepts Magn. Reson. 40A (2012) 146–169. [19] G.E. Martin, B.D. Hilton, K.A. Blinov, HSQC-1,1-ADEQUATE and HSQC-1,nADEQUATE: enhanced methods for establishing adjacent and long-range 13 C–13C connectivity networks, J. Nat. Prod. 74 (2011) 2400–2407. [20] G.E. Martin, Using 1,1- and 1,n-ADEQUATE 2D NMR data in structure elucidation protocols, in: G.A. Webb (Ed.), Ann. Rep. NMR Spectrosc., vol. 74, Elsevier, London, 2011, pp. 215–291. [21] G.E. Martin, R.T. Williamson, P.G. Dormer, W. Bermel, Inversion of 1JCC correlations in 1,n-ADEQUATE spectra, Magn. Reson. Chem. 50 (2012) 563– 568. [22] G.E. Martin, K.A. Blinov, M. Reibarkh, R.T. Williamson, 1JCC-edited HSQC-1,nADEQUATE: a new paradigm for simultaneous direct and long-range carbon– carbon correlation, Magn. Reson. Chem. 50 (2012) 722–728. [23] M.E. Elyashberg, A.J. Williams, G.E. Martin, Computer-assisted structure elucidation, in: J. Feeney, L. Sutcliff (Eds.), Prog. NMR Spectrosc., vol. 53, Pergammon Press, London, 2008, pp. 1–104. [24] S.F. Cheatham, M. Kline, R.R. Sasaki, K.A. Blinov, M.E. Elyashberg, S.G. Molodtsov, Enhanced automated structure elucidation by inclusion of twobond specific data, Magn. Reson. Chem. 48 (2010) 571–574. [25] R.T. Williamson, A.V. Buevich, G.E. Martin, Experimental and theoretical investigation of 1JCC and nJCC coupling constants in strychnine, Org. Lett. 14 (2012) 5098–5101. [26] B. Reif, M. Köck, R. Kerssebaum, J. Schleucher, C. Griesinger, Determination of 1 2 J, J, and 3J carbon–carbon coupling constants at natural abundance, J. Magn. Reson. 112 (1996) 295–301. [27] K.E. Kövér, P. Forgó, J-modulated ADEQUATE (JM-ADEQUATE) experiment for accurate measurement of carbon–carbon coupling constants, J. Magn. Reson. 166 (2004) 47–52. [28] C.M. Thiele, W. Bermel, J-modulated ADEQUATE experiments using different kinds of refocusing pulses, Magn. Reson. Chem. 45 (2007) 889–894. [29] C.M. Thiele, W. Bermel, Speeding up the measurement of one-bond scalar (1J) and residual dipolar couplings (1D) by using non-uniform sampling (NUS), J. Magn. Reson. 216 (2012) 134–143. [30] G. Bifulco, R. Riccio, G.E. Martin, A.V. Buevich, R.T. Williamson, Quantum chemical calculations of 1JCC coupling constants for the stereochemical determination of organic compounds, Org. Lett. 15 (2013) 654–657. [31] T. Helgaker, M. Jaszun´ski, K. Ruud, Ab initio methods for the calculation of NMR shielding and indirect spin–spin coupling constants, Chem. Rev. 99 (1999) 293–352. [32] G. Bifulco, C. Bassarello, R. Riccio, L. Gomez-Paloma, Quantum mechanical calculations of NMR J coupling values in the determination of relative configuration in organic compounds, Org. Lett. 6 (2004) 1025–1028. [33] T. Helgaker, M. Jaszun´ski, M. Pecul, The quantum-mechanical calculation of NMR indirect spin–spin coupling constants, Prog. Nucl. Magn. Reson. Spectrosc. 53 (2008) 249–268. [34] K. Kamien´ska-Trela, One-bond 13C–13C spin–spin coupling constants, in: G.A. Webb (Ed.), Ann. Rep. NMR Spectrosc., vol. 30, Academic Press, London, 1993, pp. 131–230. [35] J.B. Stothers, Carbon-13 NMR Spectroscopy, Academic Press, New York, 1972. pp. 370–375. [36] G.E. Martin, A.V. Buevich, M. Reibarkh, S.B. Singh, J.G. Ondeyka, R.T. Williamson, Coniothyrione: anatomy of a structure revision, Magn. Reson. Chem. 51 (2013) 383–389. [37] T.F. Molinski, Microscale methodology for structure elucidation of natural products, Curr. Opin. Biotechnol. 21 (2010) 819–826.