13                            C-15N Connectivity Networks via Unsymmetrical                                               ...
2                             G. Martin, B. Hilton, P. Irish, K. Blinov, and A. Williams.Considerable recent attention has...
13Jan-Feb 2007         C-15N Connectivity Networks via Unsymmetrical Indirect Covariance Processingconventional and multip...
4                                     G. Martin, B. Hilton, P. Irish, K. Blinov, and A. Williams.[23] A. Grube and M. Köck...
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13C-15N Connectivity networks via unsymmetrical indirect covariance processing of 1H-13C HSQC and 1H-15N IMPEACH spectra

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Long-range 1H-15N heteronuclear shift correlation methods at natural abundance to facilitate the elucidation of small molecule structures have assumed a role of growing importance over the past decade. Recently, there has also been a high level of interest in the exploration of indirect covariance NMR methods. From two coherence transfer experiments, A→B and A→C, it is possible to indirectly determine B⟷C. We have shown that unsymmetrical indirect covariance methods can be employed to indirectly determine several types of hyphenated 2D NMR data from higher sensitivity experiments. Examples include the calculation of hyphenated 2D NMR spectra such as 2D GHSQC-COSY and GHSQC-NOESY from the discrete component 2D NMR experiments. We now wish to report the further extension of unsymmetrical indirect covariance NMR methods for the combination of 1H-13C GHSQC and 1H-15N longrange (GHMBC, IMPEACH-MBC, CIGAR-HMBC, etc.) heteronuclear chemical shift correlation spectra to establish 13C-15N correlation pathways.

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13C-15N Connectivity networks via unsymmetrical indirect covariance processing of 1H-13C HSQC and 1H-15N IMPEACH spectra

  1. 1. 13 C-15N Connectivity Networks via Unsymmetrical 1 Indirect Covariance Processing of 1H-13C HSQC and 1 H-15N IMPEACH Spectra Gary E. Martin,* Bruce D. Hilton, and Patrick A. Irish Schering-Plough Research Institute Rapid Structure Characterization Laboratory Pharmaceutical Sciences, Summit, NJ 07901 Kirill A. Blinov Advanced Chemistry Development Moscow Department, Moscow 117513 Russian Federation and Antony J. Williams Advanced Chemistry Development Toronto, Ontario, M5C 1T4, Canada 9 6 10 8 7 5 F1 Chemical Shift (ppm) H 50 11 13 2 N 15 12 N 3 19 N 100 14 16 18 O 15 17 150 20 21 100 50 0 F2 Chemical Shift (ppm) 13 C Long-range 1H-15N heteronuclear shift correlation methods at natural abundance to facilitate the elucidation of smallmolecule structures have assumed a role of growing importance over the past decade. Recently, there has also been ahigh level of interest in the exploration of indirect covariance NMR methods. From two coherence transfer experiments,A→B and A→C, it is possible to indirectly determine B→C. We have shown that unsymmetrical indirect covariancemethods can be employed to indirectly determine several types of hyphenated 2D NMR data from higher sensitivityexperiments. Examples include the calculation of hyphenated 2D NMR spectra such as 2D GHSQC-COSY andGHSQC-NOESY from the discrete component 2D NMR experiments. We now wish to report the further extension ofunsymmetrical indirect covariance NMR methods for the combination of 1H-13C GHSQC and 1H-15N long-range(GHMBC, IMPEACH-MBC, CIGAR-HMBC, etc.) heteronuclear chemical shift correlation spectra to establish 13C-15Ncorrelation pathways.J. Heterocyclic Chem., 44, (2007).Sir: range heteronuclear shift correlation data, for theLong-range 1H-15N heteronuclear chemical shift characterization of natural products and pharmaceuticals,correlation methods at natural abundance have been the in particular, has fostered two recent reports describingsubject of several recent reviews.1-5 Interest, and the pulse sequences that allow the simultaneous acquisition ofgrowing importance of being able to access 1H-15N long- long-range 1H-13C and 1H-15N HMBC data.6,7
  2. 2. 2 G. Martin, B. Hilton, P. Irish, K. Blinov, and A. Williams.Considerable recent attention has also been focused on the Figure 1. The 1H-15N IMPEACH spectrum is shown indevelopment of indirect covariance NMR methods, first the top left panel and corresponds to the A→B coherencereported by Brüschweiler and co-workers.8-13 Insofar as transfer spectrum; the multiplicity-edited 1H-13C GHSQCpotential for small molecule applications, to the authors, spectrum is shown in the bottom right panel and can bethe most interesting report was that describing indirect considered the A→C coherence transfer experiment. Thecovariance methods, which affords the capability of GHSQC spectrum has been transposed to reflect theextracting carbon-carbon connectivity information from a eventual orientation of the 13C spectrum as the F2 axis inGHSQC-TOCSY spectrum.11 In their report, the indirectly determined 13C-15N correlation spectrumBrüschweiler and Zhang commented that proton shown in the top right panel, which corresponds to theresonance overlap could lead to artifacts in the calculated B→C coherence transfer spectrum. In the 13C-15Ncarbon-carbon correlation spectra although they did not correlation spectrum, 15N Chemical shift information isexplore or document that observation.11 We subsequently displayed in the F1 frequency domain while 13C chemicalreported the analysis of two types of artifacts observed in shift information is presented in the F2 frequency domain. 13IDR-(Inverted Direct Response)-GHSQC-TOCSY spectra C-15N correlation responses in the spectrum shown inwith overlapped proton resonances, which, in turn, Figure 1 arise from transfer between 13C and 15N via the nprompted us to explore the elimination of these artifacts JNH correlation response in the 1H-15N IMPEACHvia a method that we have named unsymmetrical indirect spectrum, the 13C chemical shift information derives fromcovariance.14 Subsequent work has shown that it is also the chemical shift of the carbon directly bound to thepossible to mathematically combine various discretely proton in question in the 1H-13C GHSQC spectrum. 2JNHacquired 2D NMR spectra. The calculation of hyphenated long-range correlations lead to 13C-15N direct correlation2D NMR experiments from discretely acquired 2D responses in the spectrum shown in Figure 1. 3JNH and 4spectra is based on the premise that using coherence JNH long-range correlations in the 1H-15N GHMBC givetransfer experiments of the type A→B and A→C, that one rise to 13C-15N responsed corresponding to correlations viacan indirectly determine B→C. The first effort in this two- and three-bonds, respectively.direction demonstrated the combination of 1H-13CGHSQC and GHMBC spectra to afford the equivalent ofan m,n-ADEQUATE spectrum.15 Subsequent studies 9 6have demonstrated the calculation of GHSQC-COSY16,17 10 8 7 5and GHSQC-NOESY18 spectra from discretely acquired H 34.5 (30.8)COSY, NOESY, and 1H-13C GHSQC 2D NMR spectra. 11 13 2Recently, Kupče and Freeman7 have demonstrated the use Nof projection reconstruction techniques to establish 15N- 12 N 3 1913 C correlations at natural abundance, using vitamin B-12as a model compound for their study, also noting in (189.4) 173.6 14 16 18parallel that indirect covariance methods can be used to 15 17obtain homonuclear correlation spectra indirectly. Thus, Owe would now like to demonstrate specifically, that 20unsymmetrical indirect covariance NMR methods can 21also be used to derive 15N-13C connectivity information 1from discretely acquired 1H-13C GHSQC and 1H-15NHMBC spectra. 13 C-15N Heteronuclear correlation responses observed For the present study, multiplicity-edited 1H-13C in Figure 1 are shown on the structure above.GHSQC and 1H-15N IMPEACH-MBC (IMPEACH Correlations plotted with red contours in the 13C-15Nhereafter) spectra were acquired using an ~5 mg sample correlation spectrum shown in Figure 1 arise fromof (-) eburnamonine (1) dissolved in ~180 μL CDCl3 in a correlations between methylene carbons and the nitrogen;3 mm NMR tube. The spectra were recorded at 600 MHz correlations plotted in black arise from correlations fromusing a Varian three channel spectrometer equipped with methine carbons to nitrogen (or methyl carbons, althougha 3 mm gradient inverse detection probe. The 1H-13C there are no methyl groups correlated to nitrogen inGHSQC spectrum was recorded in 23 m as 1024 x 96 data the1H-15N IMPEACH spectrum of (-)eburnamonine). Thepoints; the 1H-15N IMPEACH spectrum was recorded in phase of the 13C resonance from the multiplicity-editing of16.2 h as 1024 x 66 data points. The spectra were responses in the GHSQC spectrum is carried forward intoacquired with identical proton spectral widths and the data the 13C-15N HSQC-IMPEACH unsymmetrical indirectwere processed to yield data matrices that were identically covariance processed spectrum shown in the top rightdigitized with F1,F2 dimensions of 512 x 2048 points. panel in Figure 1. Presumably, methine and methylene The processed 1H-13C GHSQC and 1H-15N carbons with the same 13C chemical shift that correlate toIMPEACH spectra were subjected to unsymmetrical the same nitrogen could partially or completely cancel,indirect covariance processing to yield the 13C-15N HSQC- hence it may useful to consider the acquisition of bothIMPEACH long-range correlation spectrum shown in
  3. 3. 13Jan-Feb 2007 C-15N Connectivity Networks via Unsymmetrical Indirect Covariance Processingconventional and multiplicity-edited 1H-13C GHSQCspectra when the data are acquired since these data can beaccumulated in a very reasonable period of time. For REFERENCESweaker samples, the sensitivity loss associated withmultiplicity-editing (~20%) may obviate the acquisition of [1] G. E. Martin and C. E. Hadden, J. Nat. Prod., 2000,a multiplicity-edited GHSQC spectrum in any case. It is 65, 543.also interesting to note that in the 13C-15N HSQC- [2] R. Marek and A. Lyčka, Curr. Org. Chem., 2002,6,IMPEACH correlation spectrum shown in Figure 1, the 35.C10 and C19 resonances, which have identical 13C [3] G. E. Martin and A. J. Williams, “Long-Range 1H-chemical shifts (~44.7 ppm), correlate to N1 and N4, 15 N 2D NMR Methods,” in Ann. Rep. NMR Spectrosc.,respectively, but do not produce artifacts as seen in the vol. 55, G. A. Webb, Ed., Elsevier, Amsterdam, 200, pp.case of IDR-GHSQC-TOCSY spectra with overlapping 1-119.proton resonances as in our previous work. 14 An [4] P. Forgo, J. Homann, G. Dombi, and L. Máthé,interesting corollary arises in the case of overlapped “Advanced Multidimensional NMR Experiments as Toolsprotons, one of which is long-range correlated to a for Structure Determination of Amaryllidaceae Alk-nitrogen resonance. By calculating the 13C-15N aloids,” in Poisonous Plants and Related Toxins, T.correlation spectrum, as shown in Figure 1, the specific Acamovic, S. Steward and T. W. Pennycott, Eds.,proton (via the 1H/13C response in the GHSQC spectrum) Wallingford, UK, 2004, pp. 322-328.correlating to the 15N resonance could be determined via [5] G. E. Martin, M. Solntseva, and A. J. Williams,the 13C-15N correlation response. “Applications of 15N NMR in Alkaloid Chemistry,” in Modern Alkaloids, E. Fattorusso and O. Taglialatela- CONCLUSION Scafati, Eds., Wiley-VCH, 2007, in press. [6] M. Pérez-Trujillo, P. Nolis, and T. Parella, Org. Lett., In conclusion, 13C-15N HSQC-IMPEACH hetero- 2007, 9, 29.nuclear chemical shift correlation spectra can be derived [7] E. Kupče and R. Freeman, Magn. Reson. Chem.,through the unsymmetrical indirect covariance processing 2007, 45, 103.of 1H-13C GHSQC and 1H-15N long-range heteronuclear [8] R. Brüschweiler and F. Zhang, J. Chem. Phys., 2004,chemical shift correlation spectra (1H-15N IMPEACH in 120, 5253.the present example, but we have obtained the comparable [9] R. Brüschweiler, J. Chem. Phys., 2004, 121, 409.results by co-processing 1H-13C GHSQC with 1H-15N [10] F. Zhang and R. Brüschweiler, Chem. Phys. Chem.,IMPEACH19,20 and 1H-15N CIGAR-HMBC21 spectra). 2004, 5, 794.We propose the “labeling” convention 13C-15N HSQC- [11] F. Zhang, and R. Brüschweiler, J. Am. Chem. Soc.,HMBC, 13C-15N HSQC-IMPEACH, etc. to denote the 2004, 126, 13180.component 2D experiments used in the unsymmetrical [12] N. Trbovic, S. Smirnov, F. Zhang, and R.indirect covariance processing of the data. The utilization Brüschweiler, J. Magn Reson., 2004, 171, 177.of 13C-15N heteronuclear chemical shift connectivity [13] Y. Chen, F. Zhang, W. Bermel, and R. Brüschweiler,information may prove useful in the structural J. Am. Chem. Soc., 2006, 126, 15564.characterization of pharmaceuticals (>80% contain [14] K. A. Blinov, N. I. Larin, M. P. Kvasha, A. Moser,nitrogen in their structures), alkaloids, as well as in the A. J. Williams, and G. E. Martin, Magn. Reson. Chem.,characterization of other nitrogen-containing heterocyclic 2005, 43, 999.compounds. The availability of 13C-15N heteronuclear [15] K. A. Blinov, N. I. Larin, A. J. Williams, M. Zell,connectivity information may also prove useful in and G. E. Martin, Magn. Reson. Chem., 2006, 44, 107.computer-assisted structure elucidation studies, as have1 [16] K. A. Blinov, N. I. Larin, A. J. Williams, K. A. Mills, H-!5N long-range heteronuclear chemical shift correlation and G. E. Martin, J. Heterocyclic Chem., 2006, 43, 163.data.5,22,23 These data may also allow investigators to [17] G. E. Martin, K. A. Blinov, and A. J. Williams, J.differentiate between overlapped protons, one of which is Nat. Prod., 2007; submitted.long-range coupled to 15N, provided that the carbons [18] K. A. Blinov, A. J. Williams, B. D. Hilton, P. A.directly bound to the overlapped protons are resolved, Irish, and G. E. Martin, Magn. Reson. Chem., 2007, 45, inwhich is almost always the case. It will be quite press (2007).interesting to see what other applications for 13C-15N [19] C. E. Hadden, G. E. Martin, and V. V.heteronuclear chemical shift correlation data may arise. Krishnamurthy, J. Magn. Reson., 1999, 140, 274.We are currently engaged in the exploration of further [20] G. E. Martin and C. E. Hadden, Magn. Reson. Chem.,unsymmetrical indirect covariance processing 2000, 38, 251.possibilities; the results of that work forms the subject of [21] C. E. Hadden, G. E. Martin, and V. V. Krishna-forthcoming reports. murthy Magn. Reson. Chem., 2000, 38, 143. [22] M Köck, J. Junker, and T. Lindel Org. Lett.,1999, 1, 2041.
  4. 4. 4 G. Martin, B. Hilton, P. Irish, K. Blinov, and A. Williams.[23] A. Grube and M. Köck J. Nat. Prod., 2006 69, 1212. 40 40 60 60 F1 Chemical Shift (ppm) F1 Chemical Shift (ppm) 80 80 100 100 120 120 140 140 160 160 180 180 8 7 6 5 4 3 2 1 120 100 80 60 40 20 0 F2 Chemical Shift (ppm) F2 Chemical Shift (ppm) 0 1 2 F2 Chemical Shift (ppm) 3 4 5 6 7 8 120 100 80 60 40 20 0 F1 Chemical Shift (ppm)Figure 1. 13C-15N HSQC-IMPEACH (F2,F1) heteronuclear chemical shift correlation spectrum (upper right panel)derived by the unsymmetrical indirect covariance processing of discretely acquired 1H-13C GHSQCAD (bottom rightpanel, transposed to reflect the orientation of the 13C chemical shift axis in the final 13C-15N correlation spectrum) and the1 H-15N IMPEACH spectrum (top left panel). The spectral traces flanking the 13C-15N HSQC-IMPEACH spectrum areprojections through both frequency domains. The data were recorded at 600 MHz using an ~5 mg sample of (-)eburnamonine (1) in 180 μL CDCl3. The individual 2D NMR spectra were processed to yield a pair of spectra comprisedof 512 x 2048 points that were then subjected to unsymmetrical indirect covariance processing. Processing time wasapproximately 4 sec. The N1 and N4 resonances of (-) eburnamonine (1) are observed at 173.6 and 34.5 ppm,respectively. 15N Chemical shifts calculated using a neural network are shown parenthetically on the structure.Correlations are observed, as expected, between C15 and the N1 amide nitrogen resonance in addition to much weakercorrelations to the C11 and C12 aromatic carbons. Correlations are observed from the C3, C5, C6, C18, and C19resonances to the N4 aliphatic nitrogen resonance. The C10 and C19 resonances are overlapped at ~44.7 ppm andcorrelate to the N1 and N4, resonances, respectively, with no observed artifacts of any type. The phase of the responsesin the 13C-15N correlation spectrum arises from the multiplicity-editing of the GHSQC spectrum; methine and methylcarbons give rise to correlation responses with positive intensity; methylene resonances are negatively phased.

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