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Obtaining Multi-step Correlations via CovarianceProcessing of COSY/GCOSY Spectra: Opportunities and Artifacts             ...
AbstractSmall, long-range homonuclear coupling pathways in COSY or GCOSY spectra by theacquisition of spectra with large n...
Sir:       We have recently reported the use of unsymmetrical indirect covariance NMRprocessing methods to provide conveni...
Covariance processing of a 2D FT NMR spectrum represented by the real N1 x N2matrix, F, affords a symmetric matrix, C, acc...
has a prominent response at the chemical shift of H12 (4.26 ppm) when the F1 slice at the1    H shift of H15a (2.36 ppm) i...
While some of the unsymmetrical indirect covariance processed spectra studiedthus far are amenable to artifact identificat...
18                                                           N                                           17 H             ...
REFERENCES1.    Blinov, K. A.; Larin, N. I.; Williams, A. J.; Mills, K. A.; Martin, G. E. J.      Heterocycl. Chem. 2006; ...
12.   Zhang, F.; Dossey, A. T.; Zachariah, C.; Edison, A. S.; Bruschweiler, R. Anal.      Chem. 2007; 79: 7748.13.   Trbov...
increment in 3 h. The zTOCSY data were processed by linear prediction in thesecond frequency domain to 1024 points prior t...
A                                                                                         1.5                             ...
B                                                                                         1.5                             ...
Figure 1. A.) GCOSY spectrum of a 2 mg sample of strychnine dissolved in ~200 µL          CDCl3 recorded as 128 x 2K point...
A             4.0      3.5         3.0                   2.5          2.0         1.5          1.0                        ...
Figure 2. A.) 1H reference spectrum of strychnine recorded at 600 MHz. B.) F1 slice          taken through the GCOSY spect...
A              4.0             3.5      3.0                   2.5          2.0            1.5           1.0               ...
Figure 3. A.) 1H reference spectrum of strychnine recorded at 600 MHz. B.) F1 slice          taken through the COSY spectr...
8A                                                                        14                                              ...
Figure 4. A.) F2 slice taken at the 1H shift of the H13 resonance from the conventionally           processed GCOSY spectr...
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Obtaining multi step correlations via covariance processing of COSY and GCOSY spectra opportunities and artifacts

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Small, long-range homonuclear coupling pathways in COSY or GCOSY spectra by the acquisition of spectra with large numbers of increments of the evolution period, t1, than would normally be used. Alternatively, covariance processing of COSY-type spectra acquired with modest numbers of t1 increments, however, allows the observation of multi-stage correlations. In this work results obtained from covariance processed GCOSY spectra are fully analyzed and compared to normally processed COSY and 80 ms TOCSY spectra. Multi-stage or “RCOSY-type” correlations are observed when remote protons both exhibit correlations to the same coupling partner e.g. A→B and B→C gives rise to an A→C correlation. Artifact correlations are observed when protons couple to other protons that overlap or partially overlap.

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Transcript of "Obtaining multi step correlations via covariance processing of COSY and GCOSY spectra opportunities and artifacts"

  1. 1. Obtaining Multi-step Correlations via CovarianceProcessing of COSY/GCOSY Spectra: Opportunities and Artifacts 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. 2. AbstractSmall, long-range homonuclear coupling pathways in COSY or GCOSY spectra by theacquisition of spectra with large numbers of increments of the evolution period, t1, thanwould normally be used. Alternatively, covariance processing of COSY-type spectraacquired with modest numbers of t1 increments, however, allows the observation ofmulti-stage correlations. In this work results obtained from covariance processedGCOSY spectra are fully analyzed and compared to normally processed COSY and 80ms TOCSY spectra. Multi-stage or “RCOSY-type” correlations are observed whenremote protons both exhibit correlations to the same coupling partner e.g. A→B andB→C gives rise to an A→C correlation. Artifact correlations are observed when protonscouple to other protons that overlap or partially overlap. 2
  3. 3. Sir: We have recently reported the use of unsymmetrical indirect covariance NMRprocessing methods to provide convenient access to hyphenated 2D NMR correlationdata1-3 and access to experimentally inaccessible 13C-15N heteronuclear shift correlationplots.4-7 It is important to recall, however, that covariance NMR processing methods canalso be advantageously applied to individual 2D NMR spectra.8,9 Brüschweiler and co-workers have demonstrated the acquisition of 2D NMR spectra with minimal datasets10as well as the use of covariance processing methods with TOCSY spectra to extractindividual component spectra from a mixture.11,12 We now report the application ofcovariance NMR processing methods to observe multi-step long-range correlations inCOSY spectra acquired with modest numbers of increments of the evolution period, t1.Generally, the observation of small, long-range homonuclear couplings in a COSYspectrum requires either the acquisition of a spectrum with large numbers of incrementsof the evolution period or a delay of the start of the evolution period. Covarianceprocessing of COSY or GCOSY spectra with more modest numbers of increments of theevolution period, t1, can, however, provide spectra with resolution in both dimensionsdefined by the resolution achieved in the directly acquired F2 frequency domain.13 Inthose cases where remote protons are both coupled to a common partner, multi-step orRCOSY-type correlations are observed linking the remote protons, e.g. A→B and B→Cgiving rise to an A→C correlation. When protons are coupled to resonances withoverlapping proton multiplets, undesired artifact responses can also be observed,although this has not been discussed in the work of Brüschweiler and co-workers.11,12 3
  4. 4. Covariance processing of a 2D FT NMR spectrum represented by the real N1 x N2matrix, F, affords a symmetric matrix, C, according to [1]: C = FT ∙ F [1]where the FT refers to the transposed matrix. It should also be noted that the resolution inboth 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 F2after the first FT; 128 increments of t1 linear predicted to 256 points and then zero-filledto 1K points prior to the second FT processing step) to covariance processing affords theresult shown in Figure 1B. Even by casual comparison of the two contour plots it isobvious that there is improved resolution in the F1 frequency domain as well as asignificant difference in the information content after covariance processing relative tothe starting, conventionally processed COSY spectrum. The threshold levels of bothplots are identical. There are numerous responses defined by black or red boxes in Figure 1B. Theseresponses are two types of artifacts from the covariance processing to which the datawere subjected. The analysis of the responses in the covariance processed data warrantscomment. Superposition of the COSY and the covariance processed spectrum allowsfacile determination of which are new responses based on the absence of overlap in thetwo spectra. Once a given response has been identified as “new” in the covarianceprocessed data, slices can be extracted from the conventional GCOSY spectra at the 1Hshifts of the two resonances involved. For example, the covariance processed spectrum 4
  5. 5. has a prominent response at the chemical shift of H12 (4.26 ppm) when the F1 slice at the1 H shift of H15a (2.36 ppm) is examined. The 600 MHz 1H reference spectrum is shownin Figure 2A. The extracted F1 slices from the conventionally processed GCOSYspectrum at the 1H chemical shifts of H15a and H12 are shown as traces B and C,respectively, in Figure 2. The F1 slice from the covariance processed GCOSY spectrumat the 1H shift of H15a is shown in trace D. Multi-step (RCOSY-type) responses aredenoted with black boxed assignments; artifact responses are denoted by red boxedassignments. Note that both resonances have a common coupling partner in H14 (blackhatched box) in traces B and C. The common coupling partner in this case gives rise tothe response at the H12 chemical shift affording a multi-step correlation response in thecovariance processed spectrum shown in Figure 1B (black boxed response) and trace 2D.All of the black boxed responses shown in Figure 1B correspond to multi-step correlationresponses that arise when the two protons in question have a common coupling partner inthe conventional COSY or 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, theH13 resonance (1.27 ppm) exhibits a cross peak at the 1H chemical shift of the H18bresonance (2.86 ppm). Once again extracting F1 slices from the conventionally processedCOSY spectrum affords the traces shown in panels B and C, respectively, in Figure 3. Inthis case, there is an overlap of the H18a and H11a resonances in the two traces. Thisoverlap leads to the artifact correlation observed at the 1H chemical shift of H18b in theF1 slice corresponding to H13 shown in trace D. In similar fashion, other responsesshown in Figure 1B have been identified as artifact responses. 5
  6. 6. While some of the unsymmetrical indirect covariance processed spectra studiedthus far are amenable to artifact identification via algorithmic analysis through the use ofcovariance spectra of one of the co-processed spectra14, 15 or by other methods16 atpresent this is not possible for covariance processed COSY spectra. We are exploring thepossibility of algorithmic artifact identification but these efforts have thus far not yieldeda viable method. Figure 4 shows extracted F2 slices for the H13 resonances from the conventionaland covariance processed GCOSY spectra shown in traces 4A and 4B, respectively. Thecorresponding F2 slice of the covariance processed spectrum shown in Figure 1B ispresented as trace 4C; the corresponding trace from a zTOCSY spectrum acquired withan 80 ms mixing time is shown in trace 4D; and finally, a segment of the 600 MHz highresolution reference spectrum of strychnine is shown in trace 4E. All of the correlationsobserved in the conventionally processed COSY spectrum are observed followingcovariance processing as well as several multi-step correlation responses that are notobserved in the conventionally processed spectrum. Several undesired artifact responsesare also observed (trace 4C). Correlations observed in the covariance processed datacompare favorably with the correlations observed in the F1 slice taken from the zTOCSYspectrum acquired with an 80 ms mixing time and shown in trace 4D except that most ofthe correlation responses in the F1 trace from the covariance processed data are observedwith higher response intensity than the corresponding responses in the trace from thezTOCSY spectrum. 6
  7. 7. 18 N 17 H 20 16 15 H 8 14 N 13 22 H H 12 23 O 11 O H 1 Covariance processing of COSY or GCOSY spectra afford access to multi-step orRCOSY-type correlations as illustrated using strychnine (1) as a model compound. Thecovariance processing algorithm, unfortunately, can also give rise to artifact responses asshown and discussed with reference to Figures 2 and 3 when protons are coupled to otherprotons with overlapping responses in the proton spectrum. While covariance processingof a COSY or GCOSY spectrum will not replace the acquisition of long-rangehomonuclear correlation spectra, this approach can provide access to multi-step orRCOSY-type correlation responses if care is taken to ascertain, as shown in Figures 2 and3, that the observed responses are not artifacts arising due to unfortuitous overlap. Weare working to develop an algorithmic method to identify artifact responses that wouldmake the process less subject to human interpretational error, 7
  8. 8. REFERENCES1. 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: 544.4. 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. 8
  9. 9. 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. Martin, G. E.; Hilton, B. D.; Blinov, K. A.; Williams, A. J. Magn. Reson. Chem. 2008; 46:138.15. Martin, G. E.; Hilton, B. D.; Blinov, K. A.; Williams, A. J. J. Nat. Prod. 2007; 70: 1966.16. Blinov, K. A.; Larin, N. I.; Kvasha, M. P.; Moser, A.; Williams, A. J.; Martin, G. E. Magn. Reson. Chem. 2005; 43: 999.17. 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 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/t1 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 GCOSY spectrum acquired with 1024 increments of the evolution period that provided trace B in Figure 4 was acquired with 16 transients/t1 increment in 6 h. The 80 ms zTOCSY data used for comparison purposes were acquired as 512 x 2K points with 16 transients/t1 9
  10. 10. increment in 3 h. The zTOCSY data were processed by linear prediction in thesecond frequency domain to 1024 points prior to Fourier transformation. 10
  11. 11. A 1.5 2.0 2.5 F1 Chemical 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 Shift (ppm) Figure 1A. 11
  12. 12. 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 Shift (ppm) Figure 1B. 12
  13. 13. 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 multi-step correlation responses (black boxed responses) as well as a similar number of undesired artifact responses (red boxed responses) that arise due to resonance overlap. Responses with no labeling correspond to responses that would normally appear in the GCOSY spectrum. 13
  14. 14. A 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shift (ppm) B H15b H16 H14 H15a 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shift (ppm) C H14 H11b H12 H13 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shift (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 Shift (ppm)Figure 2. 14
  15. 15. Figure 2. A.) 1H reference spectrum of strychnine recorded at 600 MHz. B.) F1 slice taken through the GCOSY spectrum shown in Figure 1A at the 1H shift of the H15a resonance. C.) F1 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 multi-step or RCOSY-type correlation response between H15a and H12 (A→C) that is observed in the H15a F1 slice from the covariance processed spectrum shown in Figure 1B. D.) F1 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 multi-step correlation response is black boxed; normal COSY correlation responses are labeled in black. 15
  16. 16. A 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shift (ppm) H18a H11aB H17 H18b 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shift (ppm) H8C H13 H12 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shift (ppm) H13 H16 H8D H12 H11a H18b H15a H17a/b H11b 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shift (ppm)Figure 3. 16
  17. 17. Figure 3. A.) 1H reference spectrum of strychnine recorded at 600 MHz. B.) F1 slice taken through the COSY spectrum shown in Figure 1A at the 1H shift of the H18b resonance. C.) F1 slice taken through the COSY 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 1 H chemical shift of H13 in the covariance processed spectrum shown in Figure 1B. D.) F1 slice at the 1H shift of H13 in the covariance processed spectrum shown in Figure 1B. Artifact responses are labeled in red and boxed; multi-step or RCOSY-type correlation responses (A→C) are black boxed; normal COSY responses are labeled in black. 17
  18. 18. 8A 14 13 12 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shift (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 Shift (ppm)C 14 11a 13 8 11b 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 Shift (ppm)D 13 8 11a 12 14 11b 15a 15b 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shift (ppm) 11a 20b 16 8 14 23a 23b 17a/b 11bE 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 Shift (ppm)Figure 4. 18
  19. 19. Figure 4. A.) F2 slice taken at the 1H shift of the H13 resonance from the conventionally processed GCOSY spectrum of strychnine (1) shown in Figure 1A. B.) F2 slice taken at the 1H shift of the H13 resonance of a GCOSY spectrum (not shown) acquired with 1024 increments of the evolution time, t1. C.) F2 slice taken at the 1H shift of the H13 resonance from the covariance processed GCOSY spectrum shown in Figure 1B. D.) F2 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 600 MHz reference spectrum of strychnine shown for comparison. 19

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