Unsymmetrical Indirect Covariance NMR or NMR Outside of the Box

Pharmaceutical Sciences

Unsymmetrical Indirect

Covariance NMR orNMR Outside of the Box!

G. E. Martin, K. A. Blinov, and A. J. Williams

Schering-Plough Research InstituteSummit, NJ 07901

ACD Laboratories, MoscowRussian Federation

ACD Laboratories, Toronto, Ontario, Canada

2Pharmaceutical Sciences

2D NMR experiments can be thought of as being comprised of “building blocks” that can have different functions.

The simplest of the hyphenated 2D NMR experiments is GHSQC-COSY in which protons are labeled with the 13C chemical shift of their directly bound carbon in the first phase of the experiment, followed by the establishment of proton-proton connectivities in the COSY experiment “tacked” on the back of the GHSQC segment. This approach serves to sort proton-proton connectivity by 13C chemical shift, which is useful for complex molecules.

Hyphenated 2D-NMRHyphenated 2D-NMR


GHSQC-COSY GHSQC-TOCSY

• GHSQC-TOCSY with Inverted Direct Responses (IDR-GHSQC-TOCSY)

• GHSQC-TOCSY with Suppressed Direct Responses (SDR-GHSQC-TOCSY)

• GHSQC-TOCSY – conventional experiment, all responses (+)

GHSQC-NOESY GHSQC-ROESY

Hyphenated 2D-NMR ExperimentsHyphenated 2D-NMR Experiments


From a sensitivity standpoint, depending on the author, estimates of the relative sensitivity of GHSQC-TOCSY place the experiment at about ½ the relative sensitivity of an GHMBC experiment. The inherently low sensitivity of GHSQC-TOCSY has prevented many workers from utilizing this otherwise very beneficial heteronuclear 2D NMR experiment, and hyphenated heteronuclear 2D NMR experiments in general.

GHSQC-COSY & -TOCSY GHSQC-COSY & -TOCSY


GHSQC-TOCSY

Why resort to an experiment like GHSQC-TOCSY if it has low sensitivity when there are perfectly good experiments like GCOSY and multiplicity-edited GHSQC around?

What happens in a GCOSY when you have multiple proton resonances overlapped and aren’t sure how to disentangle the resulting mess? GHSQC-TOCSY provides a useful alternative to the problem by sorting proton-proton connectivity in the second frequency domain as a function of the 13C shift of the directly bound carbon.


Conventional GCOSY & GHSQC Interpretation of the conventional GCOSY and GHSQC spectra

leads to ambiguity as to which of the carbons is the vicinal neighbor of the upfield heteronuclide pair in this example because of spectral overlap. There are three possible vicinal neighbor carbons shown in the boxed region of panel B.

135 130 125 120 115 110F1 Chemical Shift (ppm)

7.5

8.0

8.5

F2

Che

mic

al S

hift

(ppm

)

9.0 8.5 8.0 7.5F2 Chemical Shift (ppm)

A B

N

N

N N

CH3


IDR-GHSQC-TOCSY The IDR-GHSQC-TOCSY

spectrum shown in panel C resolves the ambiguity, the phase of the direct responses vs. the vicinally relayed response readily differentiating the responses and identifying the vicinal neighbor at ~130.8 ppm.


7.5

8.0

8.5

F2

Che

mic

al S

hift

(ppm

)



112

114

116

118

120

122

124

126

128

130

132

134

F1

Che

mic

al S

hift

(ppm

)

A B

C

N

N

N N

CH3


F. Zhang and R. Bruschweiler, J. Am. Chem. Soc., 126, 13180 (2004).

K. A. Blinov, N. I. Larin, M. P. Kvasha, A. Moser, A. J. Williams, and G. E. Martin, Magn. Reson. Chem., 43, 999 (2005).

K. A. Blinov, N. I. Larin, A. J. Williams, M. Zell, and G. E. Martin, Magn. Reson. Chem., 44, 107 (2006).

K. A. Blinov, N. I. Larin, A. J. Williams, K. A. Mills, and G. E. Martin, J. Heterocyclic Chem., 43, 163 (2006).

K. A. Blinov, A. J. Williams, B. D. Hilton, P. A. Irish, and G. E. Martin, Magn. Reson. Chem., 45 in press (2007).

G.E. Martin, P. A. Irish, B. D. Hilton, K. A. Blinov, and A. J. Williams, Magn. Reson. Chem., 45, in press (2007).

G.E. Martin, B.D. Hilton, P.A. Irish, K.A. Blinov, and A.J. Williams, J. Heterocyclic Chem., submitted (2007).

G.E. Martin, B.D. Hilton, P.A. Irish, K.A. Blinov, and A.J. Williams, J. Nat. Prod., submitted (2007).

This list does not include other papers by Bruschweiler and co-workers that deal with homonuclear covariance NMR methods.

Indirect Covariance SpectroscopyCurrent Published Literature


Provides an alternative presentation format in the case of GHSQC-TOCSY spectra analogous to an auto-correlated INADEQATE spectrum. Protonated-carbon to protonated carbon correlations are symmetrically positioned about a diagonal similar to responses in a COSY spectrum.

F. Zhang and R. Bruschweiler, J. Am. Chem. Soc., 126, 13180-13181 (2004).

Indirect Covariance Spectroscopy


Indirect Covariance Processing

Schematic representation of indirect covariance processing

Original matrix Covariance matrix

Row I

Row J

Row I

Co

lum

n J

Result of first multiplication of rows

Result of second multiplication of rows

Co

lum

n I

Row J



5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5F2 Chemical Shift (ppm)

35

40

45

50

55

60

65

70

75

80

85

90

F1

Ch

em

ica

l Sh

ift (

pp

m)

9 > 410 < 9

5 < 4

3 < 4

3 > 8b

8a < 3

2 < 3

2 > 6

5 > 6a

5 > 6b

6a 6b

5 < 6a/b

5 > 4

11 < 5

2 < 6a/b

2 > 3

9 < 4

3 > 4

7

8

O1

3

2

4

5

6

OH11

O12

9 OH10

2 mg sample in 180 µL d6-DMSO in3 mm NMR tube.

18 ms conventionalHSQC-TOCSY spectrum.

Data acquisition time 16 h.



90 85 80 75 70 65 60 55 50 45 40 35 30F2 Chemical Shift (ppm)

35

40

45

50

55

60

65

70

75

80

85

90

F1

Che

mic

al S

hift

(ppm

)

C2C5 C9 C4 C3

C6C8

7

8

O1

3

2

4

5

6

OH11

O12

9 OH10

C3-C2

C6-C2

C4-C5

C6-C5C3-C4

C8-C3

C4-C9

Covariance matrix

Row I

Co

lum

n J

Result of first multiplication of

rows

Result of second multiplication of rows

Co

lum

n I

Row J

Result from the IDC processing of the 18 ms HSQC-TOCSY spectrum.


Indirect Covariance Processing of IDR-GHSQC-TOCSY spectra

90 85 80 75 70 65 60 55 50 45 40 35 30F2 Chemical Shift (ppm)

35

40

45

50

55

60

65

70

75

80

85

90

F1

Che

mic

al S

hift

(ppm

)

C2C5 C9 C4 C3

C6C8

7

8

O1

3

2

4

5

6

OH11

O12

9 OH10

C3-C2

C6-C2

C4-C5

C6-C5C3-C4

C8-C3

C4-C9

C4-C6 Type I

Artifact response

5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5F2 Chemical Shift (ppm)

35

40

45

50

55

60

65

70

75

80

85

90

F1

Ch

em

ica

l Sh

ift (

pp

m)

9 > 410 < 9

5 < 4

3 < 4

3 > 8b

8a < 3

2 < 3

2 > 6

5 > 6a

5 > 6b

6a 6b

5 < 6a/b

5 > 4

11 < 5

2 < 6a/b

2 > 3

9 < 4

3 > 4

7

8

O1

3

2

4

5

6

OH11

O12

9 OH10

Phase information is retained in the processed result, butnot only in the usual sense of inverted direct responses!



1.9 1.8 1.7 1.6 1.5F2 Chemical Shift (p...

34

36

38

40

42

44

46

48

50

52

54

56

58

60

62

64

F1

Che

mic

al S

hift

(ppm

)

64 62 60 58 56 54 52 50 48 46 44 42 40 38 36 34F2 Chemical Shift (ppm)

34

36

38

40

42

44

46

48

50

52

54

56

58

60

62

64

F1

Che

mic

al S

hift

(ppm

)

7

8

O1

3

2

4

5

6

O12

9 OH10

OH11

C3-C4

C8-C3

C4-C9

C6-C9 Type II

Artifact Response

C4-C6 Type I

Artifact Response

C4-C9

C3-C4

C4-C6

As noted in the original work by Zhang and Bruschweiler,proton resonance overlap can give rise to artifact responses.

Type I artifacts are inverted (red).

Type II artifactsare indistinguishableon the basis of response phase.




9.1 9.0 8.9 8.8 8.7 8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8F2 Chemical Shift (ppm)

125

126

127

128

129

130

131

132

133

134

135

136

137

F1

Che

mic

al S

hift

(ppm

)

S

1514

N

6

5

H5H15 H6

H14

C6

C5

C15

C14

Consider the morecomplex example ofa polynuclear aromaticsystem with considerableoverlap in the protonspectrum even at 600 MHz.

H6 & H15 are completelyoverlapped and would beexpected to give rise to artifact responses: Type I – red solid lineType II – dashed black line



136.5 136.0 135.5 135.0 134.5 134.0 133.5 133.0 132.5 132.0 131.5 131.0 130.5 130.0F2 Chemical Shift (ppm)

129.5

130.0

130.5

131.0

131.5

132.0

132.5

133.0

133.5

134.0

134.5

135.0

135.5

136.0

F1

Ch

em

ica

l Sh

ift (

pp

m)

C15C10 C16C14

C17C12 C13

C1 C2C5 C3

C11

11-10

12-15 16-1514-15

17-16

12-14 5-14

13-1

12-13

13-2

1-2

12-2

12-1

2-15

15-3, 15-5

3-4

The indirect covariance processed result of the IDR-HSQC-TOCSY spectrum shown on the previous slide.

Type I artifacts are observed with negative phase (red);

Type II artifacts are denoted by dashed black lines. These also differ in integrated peak volume.

Projections of the 13C spectrum are shown flanking the F1

axis while a 13C spectrum is plotted along F2.



S

1514

1011

13

12

8N

6

516

174

312

134.9

134.7

134.4

129.8

134.5

125.4

144.4

135.3

136.3

134.6

131.4

132.5

129.7131.2

Complete analysis of theType I (red) and Type II(dashed black) artifactresponses observed in theindirect covariance processedresult from the IDR-HSQC-TOCSY spectrum of acomplex polynuclear aromatic.

Obviously the very long-rangecorrelations are artifact responses.

K.A. Blinov, N.I. Larin, M.P. Kvasha, A. Moser, A.J. Williams, andG.E. Martin, Magn. Reson. Chem., 43, 999 (2005).


Unsymmetrical Indirect Covariance Processing

Row I

Positive matrix (relayed responses)

Row J

Row I

Negative matrix (direct responses)

Row J

IDR-HSQC-TOCSY data matrix

Direct responsesRelayed or

TOCSY

responses

(positive)

Unsymmetrical indirectcovariance processingworks on a pair of data matrices. In the case of IDR-HSQC-TOCSY spectrathe data matrix is “decomposed”into a positive (relayed) andnegative (direct) response matrix as shown schematically.

K. A. Blinov, N. I. Larin, A. J. Williams, M. Zell, and G. E. Martin, Magn. Reson. Chem., 44, 107 (2006).


Unsymmetrical Indirect Covariance Processing m,n -

ADEQUATE

80 75 70 65 60 55 50 45 40 35 30 25F2 Chemical Shift (ppm)

25

30

35

40

45

50

55

60

65

70

75

80

F1

Che

mic

al S

hift

(ppm

)

C14

C11

C7

C8

C23

C12

C20* C15

Long-range carbon-carbon connectivities are shown for the C12 methine resonance. The sole artifact response observedinvolves C20.Red arrows denote mutually coupledresonant pairs; black arrows denote unidirectional correlations.

1413

12

8

N9

1110

1516

7

5

6

43

12

N19

17

18 20

O

2122

23

O24


Unsymmetrical Indirect Covariance Processing GHSQC-COSY

A more interesting possibility is found in the unsymmetrical indirect covariance co-processing of an HSQC spectrum and a COSY or TOCSY spectrum to produce the equivalent of an HSQC-COSY or HSQC-TOCSY spectrum.

Data matrices were acquired with identical F2 spectralwindows and digitization using the simple sesquiterpenelactone autumnolide as a model compound for the study.

K.A. Blinov, N.I. Larin, A.J. Williams, K.A. Mills, and G.E. Martin, J. Heterocyclic Chem., 43, 163-166 (2005).


Unsymmetrical Indirect Covariance Processing GHSQC-

COSY

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0F2 Chemical Shift (ppm)

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

F1

Che

mic

al S

hift

(ppm

)


16

24

32

40

48

56

64

72

80

88

96

104

112

120

128

136

F1

Che

mic

al S

hift

(ppm

)

O

O

CH2OHOH

O

Standard GCOSY and multiplicity-edited GHSQC spectra of a 2 mg sample of autumnolide that might be acquired to elucidate a structure. Acquisition times were 10 and 60 m, respectively.


Unsymmetrical Indirect Covariance ProcessingExtending the Boundaries


16

24

32

40

48

56

64

72

80

88

96

104

112

120

128

136

F1

Che

mic

al S

hift

(ppm

)

18 msec IDR-HSQC-TOCSYspectrum of autumnolideacquired in 16 h using a600 MHz spectrometer equipped with a 3 mmgradient indirect-detection NMR probe.

O

O

CH2OHOH

O


Unsymmetrical Indirect Covariance Processing GHSQC-COSY

Unsymmetrical indirect covariance processing of COSY and HSQC spectra affords a data matrix, equivalent to anHSQC-COSY spectrum.

Subjecting the calculated HSQC-COSY spectrum to indirect covariance processing, reduces the data to a presentation of 13C-13C direct connectivity information identical to what one would obtain by subjecting an HSQC-TOCSY spectrum to this manipulation as described previously by Zhang andBruschweiler.



COSY

8 7 6 5 4 3 2 1 0F1 Chemical Shift (ppm)

0

20

40

60

80

100

120

140

F2

Che

mic

al S

hift

(ppm

)

8 7 6 5 4 3 2 1 0F2 Chemical Shift (ppm)

0

20

40

60

80

100

120

140F

1 C

hem

ical

Shi

ft (p

pm)

HSQC-TOCSY, 18 ms mixing time16 h acquisition at 600 MHz using a 2 mg sample and 3 mm probe.

Unsymmetrical indirect covariancecalculated HSQC-COSY spectrum.Total instrument time ~70 m; 4 scalculation from the processed spectra.


150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10Chemical Shift (ppm)

1

2

Top trace – projection through F1 unsymmetrical indirect covariance processed HSQC-COSY spectrum. Instrument time ~70 m. Signal-to-noise = 77:1.

Bottom trace – projection through F1 of the 18 msec HSQC-TOCSY spectrum. Instrument time 16 h. Signal-to-noise = 8:1. Time to equivalent s/n… a week?

Unsymmetrical Indirect Covariance Processing – GHSQC-COSY



COSY


24

32

40

48

56

64

72

80

F1

Che

mic

al S

hift

(ppm

)


1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

F1

Che

mic

al S

hift

(ppm

)


24

32

40

48

56

64

72

80

F1

Che

mic

al S

hift

(ppm

)

N

N

OH

H H

H

O

H

Multiplicity-edited GHSQC

COSY

Unsymm. Indirect covariance processed GHSQC-COSY

Calculation of aGHSQC-COSY spectrum of strychnine from a multiplicity-edited GHSQC and a conven-tional GCOSY spectrum. Total data acquisition time was <<1 hr.

Total post processingtime was ~5 sec.



COSY Comparison plots ofA) 24 ms GHSQC-TOCSYspectrum of strychnine. Approx. 8 h. data acquisition.s/n = 40:1

B) GHSQC-COSY spectrumcalculated from a conven-tional GCOSY spectrum anda multiplicity-edited GHSQCspectrum. Data acquisition <<1 h.s/n = 144:1

Information content is duplicated; numerous valid responses are now visible above the threshold.

Plots have identical threshold levels.

5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ppm

24

32

40

48

56

64

72

80

F1 C

hem

ical

Shi

ft (p

pm)

5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0F2 Chemical Shift (ppm)

24

32

40

48

56

64

72

80

F1 C

hem

ical

Shi

ft (p

pm)

A

B



COSY Comparison F1 projections ofstrychnine GHSQC-COSY andGHSQC-TOCSY spectra.

A) GHSQC-COSY spectrumcalculated from a conven-tional GCOSY spectrum anda multiplicity-edited GHSQCspectrum. Data acquisition <<1 h.Post processing ~ 5 s.s/n = 144:1

B) 24 ms GHSQC-TOCSYspectrum of strychnine. Approx. 8 h. data acquisition.s/n = 40:1

80 75 70 65 60 55 50 45 40 35 30 25 20Chemical Shift (ppm)

80 75 70 65 60 55 50 45 40 35 30 25 20Chemical Shift (ppm)

B

A

40:1

144:1

Both spectra were subjected to magnitude calculation prior to F1 projection.


GHSQC-NOESY is another very low sensitivity hyphenated 2D-NMR experiment that receives relatively little use in the case of unlabeled small molecules.

Unsymmetrical indirect covariance processing offers the intriguing possibility of experimental access to GHSQC-NOESY data through the co-processing of much higher sensitivity GHSQC and NOESY spectra.

Unsymmetrical Indirect Covariance Spectroscopy – GHSQC-NOESY

G.E. Martin, P. A. Irish, B. D. Hilton, K.A. Blinov, and A.J. Williams,, Magn. Reson. Chem., 45, in press (2007).


A GHSQC-NOESY experiment was performed on a 2 mg sample of ibuprofen using a mixing time of 450 ms. The acquisition of a spectrum with usable s/n consumed 44 h of spectrometer time on a 500 MHz instrument equipped with a 3 mm gradient inverse triple resonance probe.

For purposes of unsymmetrical indirect covariance processing, a GHSQC spectrum was recorded in 30 m and a 450 ms NOESY spectrum was recorded in 3.75 h.

Unsymmetrical Indirect Covariance Spectroscopy – GHSQC-NOESY


Unsymmetrical Indirect Covariance ProcessingGHSQC-NOESY

7 6 5 4 3 2 1F2 Chemical Shift (ppm)

20

40

60

80

100

120

F1

Ch

em

ica

l Sh

ift (

pp

m)


20

40

60

80

100

120

F2

Ch

em

ica

l Sh

ift (

pp

m)

44 h GHSQC-NOESY with Unsymmetrical indirect 450 ms mixing time. covariance processed GHSQC and NOESY data.

4.25 h of spectrometer time.


Unsymmetrical Indirect Covariance ProcessingGHSQC-NOESY

7 6 5 4 3 2 1Chemical Shift (ppm)

-0.01

0

0.01

0.02

0.03

0.04

0.05

Nor

mal

ized

Inte

nsity

21:1 58:1 65:1

7 6 5 4 3 2 1Chemical Shift (ppm)

-0.01

0

0.01

0.02

0.03

0.04

0.05

Nor

mal

ized

Inte

nsity

19:1 25:1

27:1

Slices taken from the 44 h GHSQC-NOESY (left) and unsym-metrical indirect covariance calculated HSQC-NOESY spectraof ibuprofen (right). Slices were taken at the 13C shift of thesec –butyl methine resonance at ~22.5 ppm.


Unsymmetrical Indirect Covariance Processing13C-15N Heteronuclear Shift Correlation

The 13C-13C INADEQUATE experiment depends on thestatistical probability of two 13C atoms being in the same molecule, a 1:10,000 probability based on the ~1% relative natural abundance of 13C. The probability of adjacent 13C-13C is correspondingly lower.

Now consider the statistical probability of a 13C and a 15N anywhere in the molecular structure. Roughly a 1:27,000 probability based on 1.1% 13C and 0.37% 15N.

The net result of these probabilities is that we have the very low sensitivity 13C-13C INADEQUATE experiment but no 13C-15N analog, at least not at natural abundance.


Unsymmetrical Indirect Covariance Processing13C-15N Heteronuclear Shift Correlation -

Strychnine

4.0 3.5 3.0 2.5 2.0 1.5 1.0F2 Chemical Shift (ppm)

40

60

80

100

120

140

F1 C

hem

ical S

hift

(ppm

)

1H-15N GHMBC

65 60 55 50 45 40 35 30 25 20F1 Chemical Shift (ppm)

1.5

2.0

2.5

3.0

3.5

4.0

F2 C

hem

ical S

hift

(ppm

)

multiplicity-edited1H-13C GHSQC

13C

15N


40

60

80

100

120

140

F1 C

hem

ical S

hift

(ppm

)

13C-15N HSQC-HMBC

C16 C18 C20 C17 C14 C15

C11C13C8

N19

N9

B ↔ C

N

N

OH

H H

H

O

H



Strychnine

15N


40

60

80

100

120

140

F1

Ch

em

ica

l Sh

ift (

pp

m)

13C-15N HSQC-HMBC

C16 C18 C20 C17 C14 C15

C11C13C8

N19

N9

1413

12

8

N9

1110

1516

7

N19

17

18 20

O

2122

23

O

39.6

155.2

G.E. Martin, P.A. Irish, B.D. Hilton, K.A. Blinov, and A.J. Williams, Magn. Reson. Chem., 45, in press (2007).



Strychnine

15N


40

60

80

100

120

140

F1

Ch

em

ica

l S

hift

(pp

m)

13C-15N HSQC-HMBC

C16 C18 C20 C17 C14 C15

C11C13C8

N19

N9

1413

12

8

N9

1110

1516

7

N19

17

18 20

O

2122

23

O

39.6

155.2

Although a 13C-15N heteronuclear shift correlation experiment is infeasibleexperimentally, we can still calculate this correlation matrix using unsym-metrical indirect covariance processing methods.

1JCN correlations arise via 2JNH correlations in the 1H-15N GHMBC data. 2JCN and 3JCN correlations arise via 3JNH and 4JNH correlations, respectively. The multiplicity arises via the phase of the multiplicity-edited 1H-13C GHSQCAD spectrum direct response.



Eburnamonine

N2

7

14

133

16

8

N

15

6

O

5

17

20

12 19

9

18

21

11

10

H

(189.4) 173.6

34.5 (30.8)


0

1

2

3

4

5

6

7

8

F2

Ch

em

ica

l S

hift (p

pm

)


40

60

80

100

120

140

160

180

F1

Ch

em

ica

l S

hift (p

pm

)

8 7 6 5 4 3 2 1F2 Chemical Shift (ppm)

40

60

80

100

120

140

160

180

F1

Ch

em

ica

l S

hift (p

pm

)


Unsymmetrical indirect covariance processing provides quick access to what are often time-prohibitive hyphenated 2D-NMR data because of the inherently low sensitivity of many hyphenated 2D-NMR experiments.

Examples reported where comparison spectra have been recorded include:

GHSQC-COSYGHSQC-NOESY

Unsymmetrical Indirect Covariance Spectroscopy – Conclusions


Unsymmetrical indirect covariance methods may generate artifact responses due to resonance overlap – more work is needed to evaluate this aspect of the processing method and is on-going.

• We have reported the analysis of artifacts in indirect covariance processed GHSQC-TOCSY spectra.


• Further work needs to be done with GHSQC-COSY calculated by unsymmetrical indirect covariance processing methods to

examine the frequency of artifact responses.

• 13C-15N correlation spectra derived via unsymmetrical indirect covariance methods need to be examined to determine whether or not artifacts can occur and how prevalent they are.



Unsymmetrical indirect covariance processing can provide access to 13C-15N heteronuclear chemical shift correlation data via the co-processing of 1H-13C GHSQC and long-range 1H-15N (GHMBC, IMPEACH, etc.) spectra.

• The value of having access to 13C-15N chemical shift correlation information remains to be explored.

• Recent work of Kupče and Freeman also reported the derivation of 13C-15N heteronuclear shift correlation data using projection-reconstruction NMR methods.

E. Kupče and R. Freeman, Magn. Reson. Chem., 45, 103-105 (2007).



Acknowledgements

The authors would like to acknowledge Sr. Management of Schering-Plough

Research Institute, particularlyDrs. R. Imwinkelreid and J. B. Landis

for their support.

The authors would also like to acknowledgethe contributions of B.D. Hilton and

P.A. Irish to this work.

Technology

Unsymmetrical Indirect Covariance NMR or NMR Outside of the Box