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.

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