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Using pure shift HSQC to characterize microgram samples of drug metabolites
Tetrahedron Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Using pure shift HSQC to characterize microgram samples of drug metabolites Yong Liu a, Mitchell D. Green b, Rosemary Marques a, Tony Pereira b, Roy Helmy a, R. Thomas Williamson a, Wolfgang Bermel c, Gary E. Martin a,⇑ a b c
Merck Research Laboratories, Process & Analytical Chemistry, Rahway, NJ 07065, United States Merck Research Laboratories, Pharmacokinetics, Pharmacodynamics & Drug Metabolism, Rahway, NJ 07065, United States Bruker BioSpin GmbH, Silberstreifen, 76287 Rheinstetten, Germany
a r t i c l e
i n f o
Article history: Received 17 May 2014 Accepted 16 June 2014 Available online xxxx Keywords: HSQC—heteronuclear single quantum coherence Metabolite structure characterization Pure shift NMR 3-Hydroxy carbamazepine 3-Hydroxy amiodarone
a b s t r a c t Difficulties in isolating samples from complex biological matrices and sensitivity limitations have long stymied the utilization of heteronuclear 2D NMR for the characterization of drug metabolites. Small diameter cryogenic NMR probes have largely ameliorated sensitivity limitations and the recently reported pure shift HSQC 2D NMR pulse sequence offers a further and marked improvement in both resolution and sensitivity. Using a 7.4 lg sample of the commercially available metabolite 3-hydroxy carbamazepine dissolved in 30 lL of deuterated solvent and a 600 MHz NMR equipped with a 1.7 mm cryogenic NMR probe, it was possible to acquire high signal-to-noise pure shift HSQC data in just over 30 min. A conventional HSQC spectrum acquired with identical parameters had approximately half the signal-to-noise of the pure shift HSQC spectrum. Collapsing the vicinal homonuclear couplings in the pure shift HSQC spectrum also significantly improves resolution. A practical, real world application of the technique is illustrated with the chromatographically isolated metabolite 3-hydroxy amiodarone from incubation with CYP2J2 recombinant enzyme. High quality pure shift HSQC data were recorded in slightly over 14 h for a 3 lg sample of the metabolite. Ó 2014 Published by Elsevier Ltd.
Metabolite structure characterization is challenging on many levels. Hyphenated techniques such as LC/MS are primarily used to circumvent the difficulties inherent in isolating metabolites from complex, biological matrices. While mass spectrometric methods can localize the site of, for example, oxidative metabolism to a given ring or molecular fragment, in many cases the exact site of metabolism cannot be determined from MS data alone. NMR spectroscopy, in contrast, provides the means of unequivocally determining the exact site of metabolism albeit with far lower sensitivity than afforded by MS. Resorting to classical NMR techniques also mandates the isolation of the metabolite in sufficient quantity and purity to facilitate the acquisition of the necessary NMR data to determine the site of metabolism.1 The inherently low sensitivity of NMR techniques initially limited the use of this approach to 1H–1H homonuclear experiments for most metabolite characterization. There have, however, been technological advances on several fronts that have changed the scope of what is possible by NMR. First, there have been continuing developments in superconducting magnet technology allowing ⇑ Corresponding author. Tel.: +1 732 594 5398. E-mail address: [email protected] (G.E. Martin).
NMR experiments to be routinely performed at proton observation frequencies of 600 MHz and higher. Development of protondetected or ‘inverse’ NMR methods has greatly enhanced experimental access to heteronuclear shift correlation data.2,3 Finally, advances in probe technology, first with the development of small diameter probes beginning in the early 1990s, followed by the development of cryogenic NMR probes, and in 2006 the initial development of 1.7 mm ‘micro’ cryogenic probes have made it possible to acquire direct and even long-range 1H–13C heteronuclear shift correlation data on samples of a few micrograms of analytically pure material.4–8 Despite the cumulative impact of the advances delineated above, recent developments in the area of pure shift heteronuclear 2D NMR methods have the potential to further enhance experimental access to heteronuclear 2D NMR data for metabolite characterization. When dealing with severely limited sample quantities, the ability to fully or even partially collapse multiplet structures that is afforded by pure shift HSQC experiment very significantly increases both resolution and sensitivity. Multiple approaches to pure shift NMR methods are presently being explored and a review of the ongoing research in this area of NMR spectroscopy is beyond the scope of this Letter.9a–h In the
http://dx.doi.org/10.1016/j.tetlet.2014.06.067 0040-4039/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Liu, Y.; et al. Tetrahedron Lett. (2014), http://dx.doi.org/10.1016/j.tetlet.2014.06.067
2
Y. Liu et al. / Tetrahedron Letters xxx (2014) xxx–xxx
present study, we have utilized an adaption of the pure shift HSQC method recently described by Morris and co-workers9d that employs real-time acquisition with composite pulse decoupling applied interspersed with periodic intervals comprised of BIRD pulses and hard, non-selective 180° pulses. Although the BIRD operator used in pure shift HSQC experiments cannot collapse the geminal methylene proton couplings, it does effectively reduce the coupling pattern of anisochronous methylene protons to doublets since the contribution from remote protons is removed. To illustrate the benefit derived from using this approach for the acquisition of HSQC data on metabolite samples, a sample targeted to be 5 lg of commercially available 3-hydroxy carbamazepine (1) was first investigated as a model system. 3-Hydroxy carbamazepine was chosen as a model specifically because of the limited dispersion in both the proton and carbon spectra. NH2
O
3
OH
N 5
1
1 All NMR data were acquired using a Bruker AVANCE III 600 MHz three channel NMR spectrometer equipped with a 1.7 mm TCI MicroCryoProbe™. The sample for NMR was prepared by serial dilution in deuterated solvents using volumetric glassware (see Supplemental information). Based on prior experience, errors in concentration can be fairly large when working with small amounts of compounds that have been reconstituted from a dried sample in limited volumes of deuterated solvents.7 For this reason, a MS calibration curve was prepared using volumetric glassware and non-deuterated solvents, and the concentration of the solution in the 1.7 mm NMR tube was determined after completion of the NMR experiments. Using mass spectrometry to quantitate the analyte, the sample used to acquire the example spectra was found to contain 7.4 lg of 3-hydroxy carbamazepine (1) per 30 lL of solvent (see Supplemental information). The conventional HSQC spectrum was acquired using the standard Bruker TopSpin 3.1 pulse sequence code (hsqcedetgpprsisp.2) supplied by the spectrometer manufacturer. The pure shift HSQC spectrum employed the pulse sequence reset_hsqcsi_bbhd_2d.t1_pr (see Supplemental information) based on the experiment described by Morris and co-workers.9d Both 2D spectra were acquired with identical parameters to facilitate direct comparison other than those parameters specific to the pure shift pulse sequence. The data were acquired as 3072 points with 32 increments in the second dimension. The full proton spectral width of 11.00 ppm was employed with the transmitter centered on the water resonance at 3.341 ppm to allow presaturation of the water resonance during the 1.5 s interpulse delay between transients. The 25 ppm F1 spectral window was centered at 122.5 ppm to encompass the aromatic resonances of the 3-hydroxy carbamazepine. Both spectra were recorded using 32 transients/t1 increment, which gave acquisition times of 30 m 45 s and 33 m, respectively, for the conventional and pure shift HSQC spectra shown in Figure 1. Both spectra were identically processed by linear prediction to 4096 and 64 points in F2 and F1, respectively, followed by zero-filling in F1 to afford final spectra that were 4096 512 points. Sine bell squared apodization phase shifted by p/2 was applied prior to both Fourier transforms. The reset_hsqcsi_bbhd_2d.t1_pr pulse sequence employs explicit dwell mode acquisition, which allows data acquisition with interspersed BIRD pulses followed by a hard 180° pulse and was coded with a loop, L0, executed 11 times
Figure 1. Comparison of conventional HSQC (panel A) and pure shift HSQC spectra (panel B) of a 7.4 lg sample of 3-hydroxy carbamazepine (1) dissolved in 30 lL of DMSO-d6. Both spectra were acquired as 3072 points with 32 transient accumulated/t1 increment, giving acquisition times of 31 and 33 min, respectively. The proton reference spectrum is plotted in (panel C). Data were processed by linear prediction of the data to 4096 64 points followed by zero-filling in the indirect frequency domain to afford final spectra that were 4096 512 points. Sinebell apodization phase shifted p/2 was applied prior to both Fourier transforms. The two contour plots were prepared with an identical threshold.
(L0 = 1; see Supplemental information). For the pure shift data shown in Figure 1B, a setting of L0 = 8 was employed (block length 19.97 ms). Generally we have found that settings of L0 = 8 work well. Figure 1 shows a comparison of the aromatic region of a conventional HSQC spectrum (panel A) and a pure shift HSQC spectrum (panel B) of the 3-hydroxy carbamazepine (1) sample. The proton reference spectrum is shown in panel C. The acquisition times for the two experiments were 31 and 33 min, respectively. The difference in acquisition time is due to the BIRD and hard 180° pulses being applied during the acquisition of the pure shift data. Both spectra were acquired with presaturation of the water resonance in the spectrum. As expected, the pure shift spectrum shows dramatically better resolution than the conventional HSQC spectrum. The more telling comparison of the data is found in the evaluation of the F2 projections of the two spectra shown in Figure 2. The well-resolved singlet resonating at 6.86 ppm in the projection of the pure shift HSQC spectrum (Figure 2B) and the corresponding doublet in the conventional HSQC spectrum (Fig. 2A) were used for comparison. The region from 5.0 to 5.5 ppm was used to define representative ‘noise’ for the signal-to-noise (S/N) measurement. The measured S/N of the resonance at 6.86 ppm in the pure shift HSQC spectrum (Fig. 2B) was 13.1:1. In contrast, averaging both resonances of the corresponding doublet in the projection of the conventional HSQC spectrum (Fig. 2A) gave a S/N
Please cite this article in press as: Liu, Y.; et al. Tetrahedron Lett. (2014), http://dx.doi.org/10.1016/j.tetlet.2014.06.067
Y. Liu et al. / Tetrahedron Letters xxx (2014) xxx–xxx
3
The improvement in resolution afforded by the pure shift experiment is seen by comparing the resonances in the region of the projections downfield of 7.2 ppm. While the pure shift spectrum shows all five resonances in this crowded region of the proton spectrum as resolved singlets, in the conventional HSQC spectrum only one resonance is clearly resolved, one partially resolved, and the remaining three are overlapped. While developing and testing NMR experiments using model compounds is necessary, the practical value of any experiment is determined by its utility when dealing with real world samples. For this reason, we undertook the preparation and isolation of 3hydroxy amiodarone (2) metabolite from incubation using CYP2J2 recombinant enzyme (see Supplemental information). Structural characterization of isolated metabolites, as noted in the introduction, is complicated by the other components of the biological matrix used to prepare the metabolite and chromatographic artifacts associated with the isolation process. The pure shift HSQC spectrum of the isolated metabolite was acquired in 14 h as 3072 points with 80 increments in the indirect dimension with 288 transients accumulated/t1 increment. The data were processed using linear prediction in the indirect frequency domain to 192 points followed by zero-filling to afford the 4 K 1 K spectrum shown in Figure 3. A spectrum was acquired in 2 h 35 m using the same parameters with 64 transient accumulated/t1 increment that showed all of the resonances in the spectrum with the exception of the anisochronous 1-methylene resonances, which are the weakest resonances in the spectrum. Figure 2. The F2 projection of the conventional HSQC spectrum is shown in panel A; the F2 projection of the pure shift HSQC spectrum is shown in panel B; and the corresponding region of the proton reference spectrum is shown in panel C. The region of the two projections from 5.0 to 5.5 ppm was selected for representative ‘‘noise’’ for the measurement of the signal-to-noise (S/N) ratio. In panel A, the resolved doublet at 6.86 ppm and the corresponding singlet in the projection shown in panel B were selected for comparison as the best resolved resonances. Measuring and averaging both limbs of the doublet in the conventional HSQC spectrum shown in panel A gave a S/N measurement of 6.6:1. The measured S/N ratio for the corresponding singlet at 6.86 ppm from the pure shift HSQC spectrum was 13.1:1. It should also be noted that in the region of the projections downfield of 7.2 ppm, in the pure shift HSQC spectrum all five correlations are observed as resolved singlets whereas in the conventional HSQC spectrum, only one of the five resonances is fully resolved with a second partially resolved, and the other three resonances are overlapped.
ratio of 6.6:1, roughly half of that obtained in essentially the same time with the pure shift HSQC experiment.
Figure 4 shows the proton spectrum of the metabolite sample without water suppression in trace A. The 256 transient proton spectrum with the water resonance suppressed is shown in panel
Figure 3. Pure shift HSQC spectrum of a 3 lg sample of 3-hydroxy amiodarone prepared using from incubation using CYP2J2 recombinant enzyme. The spectrum was acquired in 14 h as 3072 points 80 increments in the indirect dimension points; 288 transients were accumulated/t1 increment. The 3-hydroxy methine resonance is enclosed in the green box. The anisochronous 1-methylene protons are the weakest resonances in the metabolite spectrum and are enclosed in the blue box.
Please cite this article in press as: Liu, Y.; et al. Tetrahedron Lett. (2014), http://dx.doi.org/10.1016/j.tetlet.2014.06.067
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Y. Liu et al. / Tetrahedron Letters xxx (2014) xxx–xxx
due to the collapse of multiplet structures, other than anisochronous geminal methylene resonances, despite the longer acquisition time used for the latter. Intense recent research efforts by a number of prominent NMR laboratories have led to the development of a robust pure shift HSQC experiment that can be employed as a powerful tool in the characterization of isolated drug metabolite samples. The improvement in both resolution and sensitivity is extremely beneficial given the typically small amount of metabolite or other mass-limited samples isolated for characterization by NMR methods. In most cases, sensitivity enhancement will be less than the factor of 2 observed for the vinyl resonance of 1 in this study. Enhancements in the range of 40–70% would be more typical.9h In cases where it is useful, a multiplicity-edited version of the pure shift HSQC experiment is also available. Supplementary data
Figure 4. (A) Proton spectrum of the 3 lg 3-hydroxy amiodarone (2) metabolite sample in DMSO-d6 without water suppression. The spectrum was acquired in 32 transients. (B) Proton spectrum of the metabolite sample acquired in 256 transients with water suppression. (C) Slice taken at the 13C chemical shift (65.1 ppm; predicted 67.0 ppm, ACD CNMR v14.0) of the 3-methine resonance. (D) Slice taken at the 13C chemical shift of the 1-methylene resonance (24.5 ppm; predicted 26.0 ppm, ACD CNMR v14.0).
B. The 3-methine proton resonance was located on the downfield flank of the water resonance but it was nevertheless still possible to acquire a pure shift HSQC spectrum with water suppression that gave excellent signal-to-noise for the 3-methine resonance. The slice from the HSQC spectrum taken at the 13C F1 chemical shift of the 3-methin is shown in panel C. The weak anisochronous 1methylene resonances are shown in panel D. Our experience with the pure shift HSQC experiment with sensitivity enhancement used in this work with metabolite samples in the range of 2–3 lg with a molecular weight in the range of 400 Da has shown that it is reasonable to expect to be able to acquire a spectrum with a satisfactory signal-to-noise ratio in approximately 10 h in comparison to an acquisition time in the range of 20–24 h using conventional HSQC with sensitivity enhancement. Furthermore, the pure shift spectrum will generally afford 40% better signal-to-noise than the conventional spectrum
Supplementary data (the procedures used for the preparation and quantitation of the metabolite samples by MS and NMR methods; Bruker TopSpin 3.1 pulse sequence code for the sensitivity improved pure shift HSQC experiment with water suppression; full page plots of the 3-hydroxy carbamazepine and 3-hydroxy amiodarone 2D spectra (both 14 h and 2 h 35 min); slice comparisons for both metabolites; and a plot of the 5 h COSY spectrum with water suppression of the 3-hydroxy amiodarone sample) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tetlet.2014.06.067. References and notes 1. Orbach, S. R.; Dalvie, D. K.; Walker, G. S. In Encyclopedia of Drug Metabolism; Lyubimov, A. V., Ed.; ; John Wiley & Sons: New York, 2012; Vol. 3, pp 121–175. 2. Breton, R. C.; Reynolds, W. F. Nat. Prod. Rep. 2013, 30, 501–524. 3. Reynolds, W. F.; Burns, D. C. In Ann. Rep. NMR Spectrosc.; Webb, G. A., Ed.; ; Elsevier: London, 2012; Vol. 76, pp 1–21. 4. Martin, G. E. In Ann Rep NMR Spectrosc.; Webb, G. A., Ed.; ; Elsevier: London, 2005; Vol. 56, pp 1–99. 5. Martin, G. E. In Handbook of Modern Magnetic Resonance; Webb, G. A., Ed.; ; Elsevier: London, 2006; Vol II, pp 1187–1194. 6. Molinski, T. F. Nat. Prod. Rep. 2010, 27, 321–329. 7. Hilton, B. D.; Martin, G. E. J. Nat. Prod. 2010, 73, 1465–1469. 8. Martin, G. E. In Encyclopedia of NMR; Harris, R. K., Wasylishen, R. A., Eds.; Wiley: New York, 2012. DOI:1002/-9780470034590.emrstm1300. 9. (a) Zangger, K.; Sterk, H. J. Magn. Reson. 1997, 124, 486–489; (b) Sakhaii, P.; Hasse, B.; Bermel, W. J. Magn. Reson. 2009, 199, 192–198; (c) Aguilar, J. A.; Nilsson, M.; Morris, G. A. Angew. Chem., Intl. Ed. 2011, 50, 9716–9717; (d) Paudel, L.; Adams, R. W.; Király, P.; Aguilar, J. A.; Foroozandeh, M.; Cliff, M. J.; Nilsson, M.; Sándor, P.; Waltho, J. P.; Morris, G. A. Angew. Chem., Intl. Ed. 2013, 52, 1–5; (e) Meyer, N. H.; Zangger, K. Angew. Chem., Intl. Ed. 2013, 52, 7143–7146; (f) Castañar, L.; Saurí, J.; Nolis, P.; Virgili, A.; Parella J. Magn. Reson. 2014, 238, 63– 69; (g) Timári, I.; Kaltschnee, L.; Kolmer, A.; Adams, R. W.; Nilsson, M.; Thiele, C. M.; Morris, G. A.; Köver, K. E. J. Magn. Reson. 2014. doi: org/10.10.1016/ j.jmr.2013.10.023 (in press); (h) Ying, J. R.; Joche, J.; Bax, A. J. Magn. Reson. 2014. doi: org/10.1016/j.jmr. 2013.11.006 (in press).
Please cite this article in press as: Liu, Y.; et al. Tetrahedron Lett. (2014), http://dx.doi.org/10.1016/j.tetlet.2014.06.067
Contents lists available at ScienceDirect
Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Using pure shift HSQC to characterize microgram samples of drug metabolites Yong Liu a, Mitchell D. Green b, Rosemary Marques a, Tony Pereira b, Roy Helmy a, R. Thomas Williamson a, Wolfgang Bermel c, Gary E. Martin a,⇑ a b c
Merck Research Laboratories, Process & Analytical Chemistry, Rahway, NJ 07065, United States Merck Research Laboratories, Pharmacokinetics, Pharmacodynamics & Drug Metabolism, Rahway, NJ 07065, United States Bruker BioSpin GmbH, Silberstreifen, 76287 Rheinstetten, Germany
a r t i c l e
i n f o
Article history: Received 17 May 2014 Accepted 16 June 2014 Available online xxxx Keywords: HSQC—heteronuclear single quantum coherence Metabolite structure characterization Pure shift NMR 3-Hydroxy carbamazepine 3-Hydroxy amiodarone
a b s t r a c t Difficulties in isolating samples from complex biological matrices and sensitivity limitations have long stymied the utilization of heteronuclear 2D NMR for the characterization of drug metabolites. Small diameter cryogenic NMR probes have largely ameliorated sensitivity limitations and the recently reported pure shift HSQC 2D NMR pulse sequence offers a further and marked improvement in both resolution and sensitivity. Using a 7.4 lg sample of the commercially available metabolite 3-hydroxy carbamazepine dissolved in 30 lL of deuterated solvent and a 600 MHz NMR equipped with a 1.7 mm cryogenic NMR probe, it was possible to acquire high signal-to-noise pure shift HSQC data in just over 30 min. A conventional HSQC spectrum acquired with identical parameters had approximately half the signal-to-noise of the pure shift HSQC spectrum. Collapsing the vicinal homonuclear couplings in the pure shift HSQC spectrum also significantly improves resolution. A practical, real world application of the technique is illustrated with the chromatographically isolated metabolite 3-hydroxy amiodarone from incubation with CYP2J2 recombinant enzyme. High quality pure shift HSQC data were recorded in slightly over 14 h for a 3 lg sample of the metabolite. Ó 2014 Published by Elsevier Ltd.
Metabolite structure characterization is challenging on many levels. Hyphenated techniques such as LC/MS are primarily used to circumvent the difficulties inherent in isolating metabolites from complex, biological matrices. While mass spectrometric methods can localize the site of, for example, oxidative metabolism to a given ring or molecular fragment, in many cases the exact site of metabolism cannot be determined from MS data alone. NMR spectroscopy, in contrast, provides the means of unequivocally determining the exact site of metabolism albeit with far lower sensitivity than afforded by MS. Resorting to classical NMR techniques also mandates the isolation of the metabolite in sufficient quantity and purity to facilitate the acquisition of the necessary NMR data to determine the site of metabolism.1 The inherently low sensitivity of NMR techniques initially limited the use of this approach to 1H–1H homonuclear experiments for most metabolite characterization. There have, however, been technological advances on several fronts that have changed the scope of what is possible by NMR. First, there have been continuing developments in superconducting magnet technology allowing ⇑ Corresponding author. Tel.: +1 732 594 5398. E-mail address: [email protected] (G.E. Martin).
NMR experiments to be routinely performed at proton observation frequencies of 600 MHz and higher. Development of protondetected or ‘inverse’ NMR methods has greatly enhanced experimental access to heteronuclear shift correlation data.2,3 Finally, advances in probe technology, first with the development of small diameter probes beginning in the early 1990s, followed by the development of cryogenic NMR probes, and in 2006 the initial development of 1.7 mm ‘micro’ cryogenic probes have made it possible to acquire direct and even long-range 1H–13C heteronuclear shift correlation data on samples of a few micrograms of analytically pure material.4–8 Despite the cumulative impact of the advances delineated above, recent developments in the area of pure shift heteronuclear 2D NMR methods have the potential to further enhance experimental access to heteronuclear 2D NMR data for metabolite characterization. When dealing with severely limited sample quantities, the ability to fully or even partially collapse multiplet structures that is afforded by pure shift HSQC experiment very significantly increases both resolution and sensitivity. Multiple approaches to pure shift NMR methods are presently being explored and a review of the ongoing research in this area of NMR spectroscopy is beyond the scope of this Letter.9a–h In the
http://dx.doi.org/10.1016/j.tetlet.2014.06.067 0040-4039/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Liu, Y.; et al. Tetrahedron Lett. (2014), http://dx.doi.org/10.1016/j.tetlet.2014.06.067
2
Y. Liu et al. / Tetrahedron Letters xxx (2014) xxx–xxx
present study, we have utilized an adaption of the pure shift HSQC method recently described by Morris and co-workers9d that employs real-time acquisition with composite pulse decoupling applied interspersed with periodic intervals comprised of BIRD pulses and hard, non-selective 180° pulses. Although the BIRD operator used in pure shift HSQC experiments cannot collapse the geminal methylene proton couplings, it does effectively reduce the coupling pattern of anisochronous methylene protons to doublets since the contribution from remote protons is removed. To illustrate the benefit derived from using this approach for the acquisition of HSQC data on metabolite samples, a sample targeted to be 5 lg of commercially available 3-hydroxy carbamazepine (1) was first investigated as a model system. 3-Hydroxy carbamazepine was chosen as a model specifically because of the limited dispersion in both the proton and carbon spectra. NH2
O
3
OH
N 5
1
1 All NMR data were acquired using a Bruker AVANCE III 600 MHz three channel NMR spectrometer equipped with a 1.7 mm TCI MicroCryoProbe™. The sample for NMR was prepared by serial dilution in deuterated solvents using volumetric glassware (see Supplemental information). Based on prior experience, errors in concentration can be fairly large when working with small amounts of compounds that have been reconstituted from a dried sample in limited volumes of deuterated solvents.7 For this reason, a MS calibration curve was prepared using volumetric glassware and non-deuterated solvents, and the concentration of the solution in the 1.7 mm NMR tube was determined after completion of the NMR experiments. Using mass spectrometry to quantitate the analyte, the sample used to acquire the example spectra was found to contain 7.4 lg of 3-hydroxy carbamazepine (1) per 30 lL of solvent (see Supplemental information). The conventional HSQC spectrum was acquired using the standard Bruker TopSpin 3.1 pulse sequence code (hsqcedetgpprsisp.2) supplied by the spectrometer manufacturer. The pure shift HSQC spectrum employed the pulse sequence reset_hsqcsi_bbhd_2d.t1_pr (see Supplemental information) based on the experiment described by Morris and co-workers.9d Both 2D spectra were acquired with identical parameters to facilitate direct comparison other than those parameters specific to the pure shift pulse sequence. The data were acquired as 3072 points with 32 increments in the second dimension. The full proton spectral width of 11.00 ppm was employed with the transmitter centered on the water resonance at 3.341 ppm to allow presaturation of the water resonance during the 1.5 s interpulse delay between transients. The 25 ppm F1 spectral window was centered at 122.5 ppm to encompass the aromatic resonances of the 3-hydroxy carbamazepine. Both spectra were recorded using 32 transients/t1 increment, which gave acquisition times of 30 m 45 s and 33 m, respectively, for the conventional and pure shift HSQC spectra shown in Figure 1. Both spectra were identically processed by linear prediction to 4096 and 64 points in F2 and F1, respectively, followed by zero-filling in F1 to afford final spectra that were 4096 512 points. Sine bell squared apodization phase shifted by p/2 was applied prior to both Fourier transforms. The reset_hsqcsi_bbhd_2d.t1_pr pulse sequence employs explicit dwell mode acquisition, which allows data acquisition with interspersed BIRD pulses followed by a hard 180° pulse and was coded with a loop, L0, executed 11 times
Figure 1. Comparison of conventional HSQC (panel A) and pure shift HSQC spectra (panel B) of a 7.4 lg sample of 3-hydroxy carbamazepine (1) dissolved in 30 lL of DMSO-d6. Both spectra were acquired as 3072 points with 32 transient accumulated/t1 increment, giving acquisition times of 31 and 33 min, respectively. The proton reference spectrum is plotted in (panel C). Data were processed by linear prediction of the data to 4096 64 points followed by zero-filling in the indirect frequency domain to afford final spectra that were 4096 512 points. Sinebell apodization phase shifted p/2 was applied prior to both Fourier transforms. The two contour plots were prepared with an identical threshold.
(L0 = 1; see Supplemental information). For the pure shift data shown in Figure 1B, a setting of L0 = 8 was employed (block length 19.97 ms). Generally we have found that settings of L0 = 8 work well. Figure 1 shows a comparison of the aromatic region of a conventional HSQC spectrum (panel A) and a pure shift HSQC spectrum (panel B) of the 3-hydroxy carbamazepine (1) sample. The proton reference spectrum is shown in panel C. The acquisition times for the two experiments were 31 and 33 min, respectively. The difference in acquisition time is due to the BIRD and hard 180° pulses being applied during the acquisition of the pure shift data. Both spectra were acquired with presaturation of the water resonance in the spectrum. As expected, the pure shift spectrum shows dramatically better resolution than the conventional HSQC spectrum. The more telling comparison of the data is found in the evaluation of the F2 projections of the two spectra shown in Figure 2. The well-resolved singlet resonating at 6.86 ppm in the projection of the pure shift HSQC spectrum (Figure 2B) and the corresponding doublet in the conventional HSQC spectrum (Fig. 2A) were used for comparison. The region from 5.0 to 5.5 ppm was used to define representative ‘noise’ for the signal-to-noise (S/N) measurement. The measured S/N of the resonance at 6.86 ppm in the pure shift HSQC spectrum (Fig. 2B) was 13.1:1. In contrast, averaging both resonances of the corresponding doublet in the projection of the conventional HSQC spectrum (Fig. 2A) gave a S/N
Please cite this article in press as: Liu, Y.; et al. Tetrahedron Lett. (2014), http://dx.doi.org/10.1016/j.tetlet.2014.06.067
Y. Liu et al. / Tetrahedron Letters xxx (2014) xxx–xxx
3
The improvement in resolution afforded by the pure shift experiment is seen by comparing the resonances in the region of the projections downfield of 7.2 ppm. While the pure shift spectrum shows all five resonances in this crowded region of the proton spectrum as resolved singlets, in the conventional HSQC spectrum only one resonance is clearly resolved, one partially resolved, and the remaining three are overlapped. While developing and testing NMR experiments using model compounds is necessary, the practical value of any experiment is determined by its utility when dealing with real world samples. For this reason, we undertook the preparation and isolation of 3hydroxy amiodarone (2) metabolite from incubation using CYP2J2 recombinant enzyme (see Supplemental information). Structural characterization of isolated metabolites, as noted in the introduction, is complicated by the other components of the biological matrix used to prepare the metabolite and chromatographic artifacts associated with the isolation process. The pure shift HSQC spectrum of the isolated metabolite was acquired in 14 h as 3072 points with 80 increments in the indirect dimension with 288 transients accumulated/t1 increment. The data were processed using linear prediction in the indirect frequency domain to 192 points followed by zero-filling to afford the 4 K 1 K spectrum shown in Figure 3. A spectrum was acquired in 2 h 35 m using the same parameters with 64 transient accumulated/t1 increment that showed all of the resonances in the spectrum with the exception of the anisochronous 1-methylene resonances, which are the weakest resonances in the spectrum. Figure 2. The F2 projection of the conventional HSQC spectrum is shown in panel A; the F2 projection of the pure shift HSQC spectrum is shown in panel B; and the corresponding region of the proton reference spectrum is shown in panel C. The region of the two projections from 5.0 to 5.5 ppm was selected for representative ‘‘noise’’ for the measurement of the signal-to-noise (S/N) ratio. In panel A, the resolved doublet at 6.86 ppm and the corresponding singlet in the projection shown in panel B were selected for comparison as the best resolved resonances. Measuring and averaging both limbs of the doublet in the conventional HSQC spectrum shown in panel A gave a S/N measurement of 6.6:1. The measured S/N ratio for the corresponding singlet at 6.86 ppm from the pure shift HSQC spectrum was 13.1:1. It should also be noted that in the region of the projections downfield of 7.2 ppm, in the pure shift HSQC spectrum all five correlations are observed as resolved singlets whereas in the conventional HSQC spectrum, only one of the five resonances is fully resolved with a second partially resolved, and the other three resonances are overlapped.
ratio of 6.6:1, roughly half of that obtained in essentially the same time with the pure shift HSQC experiment.
Figure 4 shows the proton spectrum of the metabolite sample without water suppression in trace A. The 256 transient proton spectrum with the water resonance suppressed is shown in panel
Figure 3. Pure shift HSQC spectrum of a 3 lg sample of 3-hydroxy amiodarone prepared using from incubation using CYP2J2 recombinant enzyme. The spectrum was acquired in 14 h as 3072 points 80 increments in the indirect dimension points; 288 transients were accumulated/t1 increment. The 3-hydroxy methine resonance is enclosed in the green box. The anisochronous 1-methylene protons are the weakest resonances in the metabolite spectrum and are enclosed in the blue box.
Please cite this article in press as: Liu, Y.; et al. Tetrahedron Lett. (2014), http://dx.doi.org/10.1016/j.tetlet.2014.06.067
4
Y. Liu et al. / Tetrahedron Letters xxx (2014) xxx–xxx
due to the collapse of multiplet structures, other than anisochronous geminal methylene resonances, despite the longer acquisition time used for the latter. Intense recent research efforts by a number of prominent NMR laboratories have led to the development of a robust pure shift HSQC experiment that can be employed as a powerful tool in the characterization of isolated drug metabolite samples. The improvement in both resolution and sensitivity is extremely beneficial given the typically small amount of metabolite or other mass-limited samples isolated for characterization by NMR methods. In most cases, sensitivity enhancement will be less than the factor of 2 observed for the vinyl resonance of 1 in this study. Enhancements in the range of 40–70% would be more typical.9h In cases where it is useful, a multiplicity-edited version of the pure shift HSQC experiment is also available. Supplementary data
Figure 4. (A) Proton spectrum of the 3 lg 3-hydroxy amiodarone (2) metabolite sample in DMSO-d6 without water suppression. The spectrum was acquired in 32 transients. (B) Proton spectrum of the metabolite sample acquired in 256 transients with water suppression. (C) Slice taken at the 13C chemical shift (65.1 ppm; predicted 67.0 ppm, ACD CNMR v14.0) of the 3-methine resonance. (D) Slice taken at the 13C chemical shift of the 1-methylene resonance (24.5 ppm; predicted 26.0 ppm, ACD CNMR v14.0).
B. The 3-methine proton resonance was located on the downfield flank of the water resonance but it was nevertheless still possible to acquire a pure shift HSQC spectrum with water suppression that gave excellent signal-to-noise for the 3-methine resonance. The slice from the HSQC spectrum taken at the 13C F1 chemical shift of the 3-methin is shown in panel C. The weak anisochronous 1methylene resonances are shown in panel D. Our experience with the pure shift HSQC experiment with sensitivity enhancement used in this work with metabolite samples in the range of 2–3 lg with a molecular weight in the range of 400 Da has shown that it is reasonable to expect to be able to acquire a spectrum with a satisfactory signal-to-noise ratio in approximately 10 h in comparison to an acquisition time in the range of 20–24 h using conventional HSQC with sensitivity enhancement. Furthermore, the pure shift spectrum will generally afford 40% better signal-to-noise than the conventional spectrum
Supplementary data (the procedures used for the preparation and quantitation of the metabolite samples by MS and NMR methods; Bruker TopSpin 3.1 pulse sequence code for the sensitivity improved pure shift HSQC experiment with water suppression; full page plots of the 3-hydroxy carbamazepine and 3-hydroxy amiodarone 2D spectra (both 14 h and 2 h 35 min); slice comparisons for both metabolites; and a plot of the 5 h COSY spectrum with water suppression of the 3-hydroxy amiodarone sample) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tetlet.2014.06.067. References and notes 1. Orbach, S. R.; Dalvie, D. K.; Walker, G. S. In Encyclopedia of Drug Metabolism; Lyubimov, A. V., Ed.; ; John Wiley & Sons: New York, 2012; Vol. 3, pp 121–175. 2. Breton, R. C.; Reynolds, W. F. Nat. Prod. Rep. 2013, 30, 501–524. 3. Reynolds, W. F.; Burns, D. C. In Ann. Rep. NMR Spectrosc.; Webb, G. A., Ed.; ; Elsevier: London, 2012; Vol. 76, pp 1–21. 4. Martin, G. E. In Ann Rep NMR Spectrosc.; Webb, G. A., Ed.; ; Elsevier: London, 2005; Vol. 56, pp 1–99. 5. Martin, G. E. In Handbook of Modern Magnetic Resonance; Webb, G. A., Ed.; ; Elsevier: London, 2006; Vol II, pp 1187–1194. 6. Molinski, T. F. Nat. Prod. Rep. 2010, 27, 321–329. 7. Hilton, B. D.; Martin, G. E. J. Nat. Prod. 2010, 73, 1465–1469. 8. Martin, G. E. In Encyclopedia of NMR; Harris, R. K., Wasylishen, R. A., Eds.; Wiley: New York, 2012. DOI:1002/-9780470034590.emrstm1300. 9. (a) Zangger, K.; Sterk, H. J. Magn. Reson. 1997, 124, 486–489; (b) Sakhaii, P.; Hasse, B.; Bermel, W. J. Magn. Reson. 2009, 199, 192–198; (c) Aguilar, J. A.; Nilsson, M.; Morris, G. A. Angew. Chem., Intl. Ed. 2011, 50, 9716–9717; (d) Paudel, L.; Adams, R. W.; Király, P.; Aguilar, J. A.; Foroozandeh, M.; Cliff, M. J.; Nilsson, M.; Sándor, P.; Waltho, J. P.; Morris, G. A. Angew. Chem., Intl. Ed. 2013, 52, 1–5; (e) Meyer, N. H.; Zangger, K. Angew. Chem., Intl. Ed. 2013, 52, 7143–7146; (f) Castañar, L.; Saurí, J.; Nolis, P.; Virgili, A.; Parella J. Magn. Reson. 2014, 238, 63– 69; (g) Timári, I.; Kaltschnee, L.; Kolmer, A.; Adams, R. W.; Nilsson, M.; Thiele, C. M.; Morris, G. A.; Köver, K. E. J. Magn. Reson. 2014. doi: org/10.10.1016/ j.jmr.2013.10.023 (in press); (h) Ying, J. R.; Joche, J.; Bax, A. J. Magn. Reson. 2014. doi: org/10.1016/j.jmr. 2013.11.006 (in press).
Please cite this article in press as: Liu, Y.; et al. Tetrahedron Lett. (2014), http://dx.doi.org/10.1016/j.tetlet.2014.06.067