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.

1. Obtaining Multi-step Correlations via Covariance
Processing 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. Abstract
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.
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 datasets10
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 observe multi-step long-range correlations in
COSY spectra acquired with modest numbers of increments of the evolution period, t1.
Generally, the observation of small, long-range homonuclear couplings in a COSY
spectrum requires either the acquisition of a spectrum with large numbers of increments
of the evolution period or a delay of the start of the evolution period. Covariance
processing of COSY or GCOSY spectra with more modest numbers of increments of the
evolution period, t1, can, however, provide spectra with resolution in both dimensions
defined by the resolution achieved in the directly acquired F2 frequency domain.13 In
those cases where remote protons are both coupled to a common partner, multi-step or
RCOSY-type correlations are observed linking the remote protons, e.g. A→B and B→C
giving rise to an A→C correlation. When protons are coupled to resonances with
overlapping 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. Covariance processing of a 2D FT NMR spectrum represented by the real N1 x N2
matrix, 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 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 t1 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. Superposition of the COSY and the covariance processed spectrum allows
facile determination of which are new responses based on the absence of overlap in the
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
4

5. has a prominent response at the chemical shift of H12 (4.26 ppm) when the F1 slice at the
1
H shift of H15a (2.36 ppm) is examined. The 600 MHz 1H reference spectrum is shown
in Figure 2A. The extracted F1 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 F1 slice from the covariance processed GCOSY spectrum
at the 1H shift of H15a is shown in trace D. Multi-step (RCOSY-type) 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 a multi-step correlation response 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 multi-step correlation
responses that arise when the two protons in question have a common coupling partner in
the 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, 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 F1 slices from the conventionally processed
COSY 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 traces. This
overlap leads to the artifact correlation observed at the 1H chemical shift of H18b in the
F1 slice corresponding to H13 shown in trace D. In similar fashion, other responses
shown in Figure 1B have been identified as artifact responses.
5

6. While some of the unsymmetrical indirect covariance processed spectra studied
thus far are amenable to artifact identification via algorithmic analysis through the use of
covariance spectra of one of the co-processed spectra14, 15 or by other methods16 at
present this is not possible for covariance processed COSY spectra. We are exploring the
possibility of algorithmic artifact identification but these efforts have thus far not yielded
a viable method.
Figure 4 shows extracted F2 slices for the H13 resonances from the conventional
and covariance processed GCOSY spectra shown in traces 4A and 4B, respectively. The
corresponding F2 slice of the covariance processed spectrum shown in Figure 1B is
presented as trace 4C; the corresponding trace from a zTOCSY spectrum acquired with
an 80 ms mixing time is shown in trace 4D; and finally, a segment of the 600 MHz high
resolution reference spectrum of strychnine is shown in trace 4E. All of the correlations
observed in the conventionally processed COSY spectrum are observed following
covariance processing as well as several multi-step correlation responses that are not
observed in the conventionally processed spectrum. Several undesired artifact responses
are also observed (trace 4C). Correlations observed in the covariance processed data
compare favorably with the correlations observed in the F1 slice taken from the zTOCSY
spectrum acquired with an 80 ms mixing time and shown in trace 4D except that most of
the correlation responses in the F1 trace from the covariance processed data are observed
with higher response intensity than the corresponding responses in the trace from the
zTOCSY spectrum.
6

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 or
RCOSY-type correlations as illustrated using strychnine (1) as a model compound. The
covariance processing algorithm, unfortunately, can also give rise to artifact responses as
shown and discussed with reference to Figures 2 and 3 when protons are coupled to other
protons with overlapping responses in the proton spectrum. While covariance processing
of a COSY or GCOSY spectrum will not replace the acquisition of long-range
homonuclear correlation spectra, this approach can provide access to multi-step or
RCOSY-type correlation responses if care is taken to ascertain, as shown in Figures 2 and
3, that the observed responses are not artifacts arising due to unfortuitous overlap. We
are working to develop an algorithmic method to identify artifact responses that would
make the process less subject to human interpretational error,
7

8. 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.
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. 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. increment in 3 h. The zTOCSY data were processed by linear prediction in the
second frequency domain to 1024 points prior to Fourier transformation.
10

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. 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. 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. 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. 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. A
4.0 3.5 3.0 2.5 2.0 1.5 1.0
Chemical Shift (ppm)
H18a H11a
B H17
H18b
4.0 3.5 3.0 2.5 2.0 1.5 1.0
Chemical Shift (ppm)
H8
C
H13
H12
4.0 3.5 3.0 2.5 2.0 1.5 1.0
Chemical Shift (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 Shift (ppm)
Figure 3.
16

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. 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 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
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 Shift (ppm)
Figure 4.
18

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