1) Unsymmetrical indirect covariance processing can be used to derive 13C-15N heteronuclear chemical shift correlation spectra from the separate acquisition of 1H-13C GHSQC and 1H-15N IMPEACH spectra. 2) This provides long-range 13C-15N connectivity information that may assist in structural characterization, especially of nitrogen-containing compounds. 3) Artifacts from overlapped protons are avoided, and the specific proton correlated to 15N can be determined from 13C chemical shifts.

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 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 long-range
(GHMBC, IMPEACH-MBC, CIGAR-HMBC, etc.) heteronuclear chemical shift correlation spectra to establish 13C-15N
correlation pathways.
J. Heterocyclic Chem., 44, (2007).
Sir: range heteronuclear shift correlation data, for the
Long-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 describing
subject of several recent reviews.1-5 Interest, and the pulse sequences that allow the simultaneous acquisition of
growing importance of being able to access 1H-15N long- long-range 1H-13C and 1H-15N HMBC data.6,7

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 in
development of indirect covariance NMR methods, first the top left panel and corresponds to the A→B coherence
reported by Brüschweiler and co-workers.8-13 Insofar as transfer spectrum; the multiplicity-edited 1H-13C GHSQC
potential for small molecule applications, to the authors, spectrum is shown in the bottom right panel and can be
the most interesting report was that describing indirect considered the A→C coherence transfer experiment. The
covariance methods, which affords the capability of GHSQC spectrum has been transposed to reflect the
extracting carbon-carbon connectivity information from a eventual orientation of the 13C spectrum as the F2 axis in
GHSQC-TOCSY spectrum.11 In their report, the indirectly determined 13C-15N correlation spectrum
Brüschweiler and Zhang commented that proton shown in the top right panel, which corresponds to the
resonance overlap could lead to artifacts in the calculated B→C coherence transfer spectrum. In the 13C-15N
carbon-carbon correlation spectra although they did not correlation spectrum, 15N Chemical shift information is
explore or document that observation.11 We subsequently displayed in the F1 frequency domain while 13C chemical
reported the analysis of two types of artifacts observed in shift information is presented in the F2 frequency domain.
13
IDR-(Inverted Direct Response)-GHSQC-TOCSY spectra C-15N correlation responses in the spectrum shown in
with overlapped proton resonances, which, in turn, Figure 1 arise from transfer between 13C and 15N via the
n
prompted us to explore the elimination of these artifacts JNH correlation response in the 1H-15N IMPEACH
via a method that we have named unsymmetrical indirect spectrum, the 13C chemical shift information derives from
covariance.14 Subsequent work has shown that it is also the chemical shift of the carbon directly bound to the
possible to mathematically combine various discretely proton in question in the 1H-13C GHSQC spectrum. 2JNH
acquired 2D NMR spectra. The calculation of hyphenated long-range correlations lead to 13C-15N direct correlation
2D NMR experiments from discretely acquired 2D responses in the spectrum shown in Figure 1. 3JNH and
4
spectra is based on the premise that using coherence JNH long-range correlations in the 1H-15N GHMBC give
transfer experiments of the type A→B and A→C, that one rise to 13C-15N responsed corresponding to correlations via
can indirectly determine B→C. The first effort in this two- and three-bonds, respectively.
direction demonstrated the combination of 1H-13C
GHSQC and GHMBC spectra to afford the equivalent of
an m,n-ADEQUATE spectrum.15 Subsequent studies 9 6
have demonstrated the calculation of GHSQC-COSY16,17 10 8 7 5
and GHSQC-NOESY18 spectra from discretely acquired H
34.5 (30.8)
COSY, NOESY, and 1H-13C GHSQC 2D NMR spectra. 11 13 2
Recently, Kupče and Freeman7 have demonstrated the use N
of projection reconstruction techniques to establish 15N- 12 N 3 19
13
C correlations at natural abundance, using vitamin B-12
as a model compound for their study, also noting in (189.4) 173.6 14 16 18
parallel that indirect covariance methods can be used to 15 17
obtain homonuclear correlation spectra indirectly. Thus, O
we would now like to demonstrate specifically, that 20
unsymmetrical indirect covariance NMR methods can 21
also be used to derive 15N-13C connectivity information
1
from discretely acquired 1H-13C GHSQC and 1H-15N
HMBC 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-15N
hereafter) spectra were acquired using an ~5 mg sample
correlation spectrum shown in Figure 1 arise from
of (-) 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 from
using a Varian three channel spectrometer equipped with
methine carbons to nitrogen (or methyl carbons, although
a 3 mm gradient inverse detection probe. The 1H-13C
there are no methyl groups correlated to nitrogen in
GHSQC spectrum was recorded in 23 m as 1024 x 96 data
the1H-15N IMPEACH spectrum of (-)eburnamonine). The
points; the 1H-15N IMPEACH spectrum was recorded in
phase of the 13C resonance from the multiplicity-editing of
16.2 h as 1024 x 66 data points. The spectra were
responses in the GHSQC spectrum is carried forward into
acquired with identical proton spectral widths and the data
the 13C-15N HSQC-IMPEACH unsymmetrical indirect
were processed to yield data matrices that were identically
covariance processed spectrum shown in the top right
digitized 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 to
IMPEACH 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 both
IMPEACH long-range correlation spectrum shown in

3. 13
Jan-Feb 2007 C-15N Connectivity Networks via Unsymmetrical Indirect Covariance Processing
conventional and multiplicity-edited 1H-13C GHSQC
spectra when the data are acquired since these data can be
accumulated in a very reasonable period of time. For REFERENCES
weaker samples, the sensitivity loss associated with
multiplicity-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 Tools
protons, 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 have
1 [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, in
which 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 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 right
panel, transposed to reflect the orientation of the 13C chemical shift axis in the final 13C-15N correlation spectrum) and the
1
H-15N IMPEACH spectrum (top left panel). The spectral traces flanking the 13C-15N HSQC-IMPEACH spectrum are
projections 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 comprised
of 512 x 2048 points that were then subjected to unsymmetrical indirect covariance processing. Processing time was
approximately 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 weaker
correlations to the C11 and C12 aromatic carbons. Correlations are observed from the C3, C5, C6, C18, and C19
resonances to the N4 aliphatic nitrogen resonance. The C10 and C19 resonances are overlapped at ~44.7 ppm and
correlate to the N1 and N4, resonances, respectively, with no observed artifacts of any type. The phase of the responses
in the 13C-15N correlation spectrum arises from the multiplicity-editing of the GHSQC spectrum; methine and methyl
carbons give rise to correlation responses with positive intensity; methylene resonances are negatively phased.