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Obtaining RCOSY-type Correlations via Covariance Processing of GCOSY Spectra
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Obtaining RCOSY-type Correlations via Covariance Processing of GCOSY Spectra

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Observing small, long-range homonuclear coupling pathways in COSY or GCOSY spectra generally requires the time-consuming acquisition of spectra with large numbers of increments of the evolution …

Observing small, long-range homonuclear coupling pathways in COSY or GCOSY spectra generally requires the time-consuming acquisition of spectra with large numbers of increments of the evolution period, t1. Covariance processing of spectra acquired with modest numbers of t1 increments, however, allows the observation of long-range coupling correlations with considerable instrument time savings. In this work results obtained from covariance processed GCOSY spectra are fully analyzed and compared to normally processed GCOSY and 80 ms zTOCSY spectra. RCOSY-type correlations are observed when remote protons both exhibit correlations to the same coupling partner. Artifact correlations are observed when protons couple to different protons that overlap or partially overlap.

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  • 1. Obtaining RCOSY-type Correlations via Covariance Processing of GCOSY Spectra Gary E. Martin* and Bruce D. Hilton Rapid Structure Characterization Laboratory Pharmaceutical Sciences Schering-Plough Research Institute Summit, New Jersey 07901 Kirill A. Blinov Advanced Chemistry Development Moscow Department Moscow 117513 Russian Federation and Antony J. Williams ChemZoo, Inc. Wake Forest, NC 27587
  • 2. Abstract Observing small, long-range homonuclear coupling pathways in COSY or GCOSY spectra generally requires the time-consuming acquisition of spectra with large numbers of increments of the evolution period, t1. Covariance processing of spectra acquired with modest numbers of t 1 increments, however, allows the observation of long-range coupling correlations with considerable instrument time savings. In this work results obtained from covariance processed GCOSY spectra are fully analyzed and compared to normally processed GCOSY and 80 ms zTOCSY spectra. RCOSY-type correlations are observed when remote protons both exhibit correlations to the same coupling partner. Artifact correlations are observed when protons couple to different protons that overlap or partially overlap. 2
  • 3. Sir: We have recently reported the use of unsymmetrical indirect covariance NMR processing methods to provide convenient access to hyphenated 2D NMR correlation data1-3 and access to experimentally inaccessible 13C-15N heteronuclear shift correlation plots.4-7 It is important to recall, however, that covariance NMR processing methods can also be advantageously applied to individual 2D NMR spectra.8,9 Brüschweiler and co- workers have demonstrated the acquisition of 2D NMR spectra with minimal datasets 10 as well as the use of covariance processing methods with TOCSY spectra to extract individual component spectra from a mixture. 11,12 We now report the application of covariance NMR processing methods to access RCOSY-type long-range correlations in GCOSY spectra acquired with modest numbers of increments of the evolution period, t1. Generally, the observation of small, long-range homonuclear couplings in GCOSY spectra requires the acquisition of spectra with large numbers of increments of the evolution period and this can be time-consuming. Covariance processing of COSY or GCOSY spectra with more modest numbers of increments of the evolution period, t 1, can however provide spectra with resolution in both dimensions defined by the resolution achieved in the directly acquired F2 frequency domain.13 As a consequence of the improved F1 resolution achievable through covariance processing, weaker long-range homonuclear correlation responses that are normally only observed with high digital resolution in the F1 frequency domain can be observed. In those cases where remote protons are both coupled to a common partner, RCOSY-type correlations are observed linking the remote protons as a beneficial “artifact” of the covariance processing method. 3
  • 4. When protons are coupled to different resonances with overlapping proton multiplets, undesired artifact responses can also be observed. Covariance processing of a 2D FT NMR spectrum represented by the real N1 x N2 matrix, F, affords a symmetric matrix, C: C = (FT ∙ F)1/2 1 where the superscript T refers to the transposed matrix and the square root denotes the matrix square root. It should also be noted that the resolution in both dimensions is determined by the resolution of matrix F in the F2 dimension8,13 Thus, subjecting the GCOSY spectrum of strychnine (1) shown in Figure 1A (1K points in F2 after the first FT; 128 increments of t 1 linear predicted to 256 points and then zero-filled to 1K points prior to the second FT processing step) to covariance processing affords the result shown in Figure 1B. Even by casual comparison of the two contour plots it is obvious that there is improved resolution in the F1 frequency domain as well as a significant difference in the information content after covariance processing relative to the starting, conventionally processed COSY spectrum. The threshold levels of both plots are identical. There are numerous responses defined by black or red boxes in Figure 1B. These responses are two types of artifacts from the covariance processing to which the data were subjected. The analysis of the responses in the covariance processed data warrants comment. Superimposition of the GCOSY and the covariance processed spectrum allows facile determination of which are new responses based on the absence of overlap in the 4
  • 5. two spectra. Once a given response has been identified as new in the covariance processed data, slices can be extracted from the conventional GCOSY spectra at the 1H shifts of the two resonances involved. For example, the covariance processed spectrum has a prominent response at the chemical shift of H12 (4.26 ppm) when the vertical slice at the 1H shift of H15a (2.36 ppm) is examined. The 600 MHz 1H reference spectrum is shown in Figure 2A. The extracted vertical slices from the conventionally processed GCOSY spectrum at the 1H chemical shifts of H15a and H12 are shown as traces B and C, respectively, in Figure 2. The slice from the covariance processed GCOSY spectrum at the 1H shift of H15a is shown in trace D. RCOSY responses are denoted with black boxed assignments; artifact responses are denoted by red boxed assignments. Note that both resonances have a common coupling partner in H14 (black hatched box) in traces B and C. The common coupling partner in this case gives rise to the response at the H12 chemical shift affording an RCOSY-type of cross peak in the covariance processed spectrum shown in Figure 1B (black boxed response) and trace 2D. All of the black boxed responses shown in Figure 1B correspond to RCOSY type responses that arise when the two protons in question have a common coupling partner in the conventional GCOSY spectrum. In contrast, other types of response overlap during covariance processing are non- beneficial giving rise to the artifact responses that are boxed in red. As an example, the H13 resonance (1.27 ppm) exhibits a cross peak at the 1H chemical shift of the H18b resonance (2.86 ppm). Once again extracting vertical slices from the conventionally processed GCOSY spectrum affords the traces shown in panels B and C, respectively, in Figure 3. In this case, there is an overlap of the H18a and H11a resonances in the two 5
  • 6. traces. This overlap leads to the artifact correlation observed at the 1H chemical shift of H18b in the vertical slice corresponding to H13 shown in trace D. In similar fashion, the other responses shown in Figure 1B have been identified as artifact responses. Figure 4 shows extracted slices for the H13 resonances from the conventional and covariance processed GCOSY spectra shown in traces 4A and 4B, respectively. The corresponding segment of the 600 MHz high resolution reference spectrum of strychnine (trace 4C) and the corresponding trace from a zTOCSY spectrum acquired with an 80 ms mixing time (trace 4D). All of the correlations observed in the conventionally processed GCOSY spectrum are observed following covariance processing as well as several RCOSY-type correlations that are not observed in the conventionally processed spectrum as well as several undesired artifact responses. Correlations observed in the covariance processed data compare favorably with the correlations observed in the slices taken from the zTOCSY spectrum acquired with an 80 ms mixing time except for the fact that most of the correlation responses in the trace from the covariance processed data are observed with higher intensity than the corresponding responses in the trace from the zTOCSY spectrum. 18 N 17 H 20 16 15 H 8 14 N 13 22 H H 12 23 O 11 O H 6
  • 7. 1 Covariance processing of COSY or GCOSY spectra can be used to advantage to access RCOSY-type and weak long-range correlations as illustrated for strychnine (1) in this report. Data can be acquired with modest digitization in the second frequency domain, e.g. 128 increments for the spectrum shown in Figure 1A for which the data were acquired in ~30 min.14 Covariance processing affords a data matrix in which the resolution in the second frequency domain, F1, is defined by the resolution in F2 of the starting data matrix. To acquire a spectrum with comparable digital resolution in F 1 would require 1024 increments of the evolution period that would require ~6 h of spectrometer. The data shown in the 80 msec zTOCSY traces used to validate the results obtained from the covariance processing were acquired with 512 increments of the evolution time in 3 h 6 min. REFERENCES 1. Blinov, K. A.; Larin, N. I.; Williams, A. J.; Mills, K. A.; Martin, G. E. J. Heterocycl. Chem. 2006; 43: 163. 2. Martin, G. E.; Hilton, B. D.; Irish, P. A.; Blinov, K. A.; Williams, A. J. J. Nat. Prod. 2007; 70: 1393. 3. Blinov, K. A.; Williams, A. J.; Hilton, B. D.; Irish, P. A.; Martin, G. E. Magn. Reson. Chem., 2007; 45: 544. 7
  • 8. 4. Martin, G. E.; Hilton, B. D.; Irish, P. A.; Blinov, K. A.; Williams, A. J. Magn. Reson. Chem., 2007; 45: 624. 5. Martin, G. E.; Hilton, B. D.; Blinov, K. A.; Williams, A. J. Magn. Reson. Chem. 2007; 45: 883. 6. Martin, G. E.; Hilton, B. D.; Irish, P. A.; Blinov, K. A.; Williams, A. J. J. Heterocycl. Chem. 2007; 44: 1219. 7. Martin, G. E.; Hilton, B. D.; Blinov, K. A.; Williams, A. J. J. Nat. Prod. 2007; 70: 1966. 8. Brüschweiler, R.; Zhang, F. J. Chem. Phys. 2004; 120: 5253. 9. Schoefberger, W; Smrečki, V.; Vikić-Topić, D;Müller, N. Magn. Reson. Chem. 2007; 45:583. 10. Chen, Y.; Zhang, W.; Bermel, W.; Brüscheiler, R. J. Am. Chem. Soc. 2006; 128: 15564. 11. Zhang, F.; Brüscheiler, R. Chem. Phys. Chem. 2004; 5: 794. 12. Zhang, F.; Dossey, A. T.; Zachariah, C.; Edison, A. S.; Bruschweiler, R. Anal. Chem. 2007; 79: 7748. 13. Trbovic, N.; Smirnov, S.; Zhang, F.; Brüschweiler, R. J. Magn. Reson. 2004; 171: 277. 14. All NMR data shown were recorded using a sample of 2 mg of strychnine dissolved in ~200 µL CDCl3 (Cambridge Isotope Laboratories) in a 3 mm NMR tube (Wilmad). Data were acquired using a Varian three channel NMR spectrometer operating at a 1H observation frequency of 599.75 MHz and equipped with a 5 mm cold probe operating at an rf coil temperature of 20 K. The 8
  • 9. sample temperature was regulated at 26o C. GCOSY data for the spectrum shown in Figure 1A were acquired as 128 x 2K points with 16 transients/t 1 increment in 30 min to insure a completely flat noise floor in the 2D spectrum. The data were processed by linear prediction to 256 points and zero-filling to 1K points prior to the second Fourier transform. The 80 ms zTOCSY data used for comparison purposes were acquired as 512 x 2K points with 16 transients/t 1 increment in 3 h 6 min. The zTOCSY data were processed by linear prediction in the second frequency domain to 1024 points prior to Fourier transformation. 9
  • 10. A 1.5 2.0 2.5 F1 Chemic al Shift (ppm) 3.0 3.5 4.0 4.5 5.0 5.5 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 F2 Chemical Shif t (ppm) Figure 1A. 10
  • 11. B 1.5 2.0 2.5 F1 Chemical Shift (ppm) 3.0 3.5 4.0 4.5 RCOSY 5.0 Peak overlap artifact 5.5 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 F2 Chemical Shif t (ppm) Figure 1B. 11
  • 12. Figure 1. A.) GCOSY spectrum of a 2 mg sample of strychnine dissolved in ~200 µL CDCl3 recorded as 128 x 2K points in approximately 30 min.14 The data were linear predicted to 256 points and zero-filled to 1K points in F1 prior to the second Fourier transform. B.) Result obtained from covariance processing of the GCOSY spectrum shown in Figure 1A. Even a cursory comparison of the two spectra reveals that there are considerably more responses contained in the covariance processed spectrum. Analysis of the covariance processed spectrum reveals numerous RCOSY-type responses (black boxed responses) as well as a similar number of undesired artifact responses (red boxed responses). Responses with no labeling correspond to responses that would normally appear in the GCOSY spectrum. 12
  • 13. A 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shif t (ppm) B H15b H16 H14 H15a 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shif t (ppm) C H14 H11b H12 H13 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shif t (ppm) H12 D H14, H11a H11b H15a H16 H17 H13 H20a 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shif t (ppm) Figure 2. 13
  • 14. Figure 2. A.) 1H reference spectrum of strychnine recorded at 600 MHz. B.) Vertical slice taken through the GCOSY spectrum shown in Figure 1A at the 1H shift of the H15a resonance. C.) Vertical slice taken through the GCOSY spectrum shown in Figure 1A at the 1H shift of H12. As will be noted from the black hatched boxed region, both the H15a and H12 resonances have H14 as a common coupling partner. This commonality in their coupling pathways gives rise to the RCOSY-type response between H15a and H12 that is observed in the H15a vertical slice from the covariance processed spectrum shown in Figure 1B. D.) Vertical slice at the 1H shift of H15a in the covariance processed spectrum shown in Figure 1B. The artifact response is labeled in red and boxed; The RCOSY-type response is black boxed; normal COSY responses are labeled in black. 14
  • 15. A 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shif t (ppm) H18a H11a B H17 H18b 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shif t (ppm) H8 C H13 H12 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shif t (ppm) H13 H16 H8 D H12 H11a H18b H15a H17a/b H11b 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shif t (ppm) Figure 3. 15
  • 16. Figure 3. A.) 1H reference spectrum of strychnine recorded at 600 MHz. B.) Vertical slice taken through the GCOSY spectrum shown in Figure 1A at the 1H shift of the H18b resonance. C.) Vertical slice taken through the GCOSY spectrum shown in Figure 1A at the 1H shift of H13. As will be noted from the red hatched boxed region, the H18b resonance has a correlation to H18a and H13 shows a correlation to the H11a resonance. The responses to H18a and H11a are partially overlapped, which gives rise to the artifact response to H18b at the 1H chemical shift of H13 in the covariance processed spectrum shown in Figure 1B. D.) Vertical slice at the 1H shift of H13 in the covariance processed spectrum shown in Figure 1B. Artifact responses are labeled in red and boxed; RCOSY-type responses are black boxed; normal COSY responses are labeled in black. 16
  • 17. 8 A 14 13 12 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shif t (ppm) B 8 13 12 14 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shif t (ppm) C 14 11a 13 11b 8 15a 17a/b 12 16 20a 18b 22 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shif t (ppm) D 13 8 11a 12 14 11b 15b 15a 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shif t (ppm) 11a 20b 16 8 14 23a 23b 17a/b 11b E 18b 20a 15b 13 22 15a 12 18a 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shif t (ppm) Figure 4. 17
  • 18. Figure 4. A.) Slice taken at the 1H shift of the H13 resonance from the conventionally processed GCOSY spectrum of strychnine (1) shown in Figure 1A. Slice take at the 1H shift of the H13 resonance of a GCOSY spectrum (not shown) acquired with 1024 increments of the evolution time, t 1. C.) Slice taken at the 1 H shift of the H13 resonance from the covariance processed GCOSY spectrum shown in Figure 1B. D.) Slice taken at the 1H shift of the H13 resonance of a zTOCSY spectrum (not shown) of strychnine (1) acquired with an 80 ms mixing time. E.) Segment of the high resolution 600 MHz reference spectrum of strychnine shown for comparison. 18