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Gradient-Selected NOESY—A Fourfold Reduction of the Measurement Time for the NOESY Experiment
JOURNAL OF MAGNETIC RESONANCE, ARTICLE NO.
Series A 123, 119–121 (1996)
0222
Gradient-Selected NOESY—A Fourfold Reduction of the Measurement Time for the NOESY Experiment RONALD WAGNER
AND
STEFAN BERGER *
Department of Chemistry, Philipps University Marburg, Hans-Meerwein-Strasse, D-35032 Marburg, Germany Received July 29, 1996
The NOESY experiment (1, 2) is one of the fundamental and most often performed 2D NMR experiments for applications in organic chemistry and in biomolecular research. In the latter application, overnight and even longer measurements are often required due to the low concentration of proteins or oligonucleotides. A NOESY experiment for application in organic chemistry to determine stereochemical assignments typically runs about two hours, depending on the relaxation and mixing time (3). This rather long time is not due to the sensitivity of current NMR instruments but because an eight-step phase cycle (4) is required to suppress unwanted coherences, such as double-quantum contributions, COSY-like signals, and axial peaks. In recent years, the other standard sequences used in structural elucidation for organic chemistry, such as COSY (5) or HMQC (6) have experienced a significant reduction in experiment time with the introduction of pulsed field gradients (7). Today a gradient-selected COSY experiment or a HMQC-type inverse C,H correlation of typically 20–50 mg of organic material and a molecular weight on the order of 500 Da can be performed in 5 to 10 minutes (3). Compared with this, the corresponding NOESY measurement time is inconvenient, especially since it is desirable to perform more than one NOESY experiment using different mixing times. Within this context, Ko¨ck and Griesinger recently published a FAST NOESY method (8), reducing the relaxation waiting time. However, their procedure requires an extra measurement of these relaxation times and special data processing so that this method does not seem to have gained wide application in service laboratories. In this Communication, we wish to demonstrate that by an extremely easy modification of the standard experiment, the usual eight-step phase cycle of the experiment can effectively be reduced to two steps leading to the desired fourfold reduction in experiment time. We replace the NOESY mixing time tm simply by a weak pulsed field gradient, which acts during the entire exchange period as shown in Fig. 1. For a two-spin system, it can be shown with the productoperator formalism (9) that the second 907 pulse of the NOESY sequence creates the desired frequency-labeled z magnetization, but in addition double- and zero-quantum coherences and antiphase coherences leading to COSY-type signals. Despite the
zero-quantum coherences, these can be efficiently suppressed by the pulsed field gradient in the mixing time, whereas the field gradient will not affect the frequency-labeled z magnetization which is the working principle of the NOESY scheme. In addition, axial peaks which are caused by spins which have relaxed during t1 and which are made transverse again by the second 907 pulse are also suppressed. The idea of placing a pulsed field gradient within the mixing period seems to be straightforward and has indeed been used in more complicated 3D sequences (10) and in selective 1D versions of NOE difference spectroscopy (11). It was even mentioned before the introduction of self-shielded gradient coils in the first demonstration of the EXSY experiment (12) and applied for the HOESY sequence (13). Surprisingly, to the best of our knowledge, it has not been mentioned or demonstrated for the basic homonuclear 2D NOESY sequence, probably because the developers of modern NMR need many transients for the usual protein work. Best results were achieved when the gradient pulse was on during the entire mixing time. This is probably due to the integrated gradient strength which can be reached and to the suppression of stray-field effects during the mixing time (14). In principle, our method could be performed using no phase cycle at all; however, in practice, it seems to be advantageous to use a two-step phase cycle, similar to many other gradient-selected experiments (3, 7). As an example, we show in Fig. 2a a standard NOESY spectrum of a 10% solution of strychnine
* To whom correspondence should be addressed. 119
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1064-1858/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Pulse sequence for the gs-NOESY method. w1 , w2 , w3 , phases for the 907 1H NMR RF pulses; w4 , receiver phase; t1 , time increment for the indirect dimension; tm , NOE mixing time; gz , pulsed field gradient.
in CDCl3 using the eight-step phase cycle, a relaxation time of 2 s, and a mixing time of 250 ms requiring an experiment time of 83 min. Figure 2b gives as comparison the result of the gradient-selected method requiring one-fourth of the experimental time, which is identical except for some reduction in signal-to-noise. This reduction is somewhat counterbalanced by the slightly higher receiver gain possible with the gradient
method. Contrary to these measurements, a standard NOESY sequence under the same conditions, using only a two-step phase cycle for axial peak suppression, revealed the known COSY-type signal breakthroughs. These artifacts may become smaller toward longer mixing times; with the gradient method, however, one can be sure that these unwanted signals will be suppressed. Our new method will not be necessary for those
FIG. 2. (a) Expansion of the aliphatic region of a standard 2D NOESY measurement on 10% strychnine ( 1) in CDCl3 , using an eight-step phase cycle, 2K data points in F2 , 256 data points in F1 , recorded with a spectral width of 10 ppm (Bruker AMX-500 NMR spectrometer). (b) Same conditions as in (a), recorded with the gs-NOESY method of Fig. 1 with a pulsed field gradient of 250 ms length and strength of approximately 0.01 T/m.
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FIG. 2—Continued
who must accumulate NOESY spectra with many scans, but in summary we feel that the gs-NOESY method is a significant step toward improving the throughput for NOESY spectra in service laboratories for organic chemistry. ACKNOWLEDGMENTS This work has been supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.
REFERENCES 1. J. Jeener, B. H. Meier, P. Bachmann, and R. R. Ernst, J. Chem. Phys. 71, 4546 (1979). 2. D. Neuhaus and M. Williamson, ‘‘The Nuclear Overhauser Effect in Structural and Conformational Analysis,’’ VCH, Weinheim, 1989. 3. S. Braun, H.-O. Kalinowski, and S. Berger, ‘‘100 and More Basic NMR Experiments,’’ VCH, Weinheim, 1996.
AID
JMRA 0982
/
6j13$$$382
10-17-96 15:47:35
4. G. Bodenhausen, H. Kogler, and R. R. Ernst, J. Magn. Reson. 58, 370 (1984). 5. R. E. Hurd, J. Magn. Reson. 87, 422 (1990). 6. R. E. Hurd and B. K. John, J. Magn. Reson. 91, 648 (1991). 7. J. Keeler, R. T. Clowes, A. L. Davis, and E. D. Laue, Methods Enzymol. 239, 145 (1994). 8. M. Ko¨ck and C. Griesinger, Angew. Chem. 106, 338 (1994). 9. O. W. Sørensen, G. W. Eich, M. H. Levitt, G. Bodenhausen, and R. R. Ernst, Prog. NMR Spectrosc. 16, 163 (1983). 10. A. Majumdar and E. R. P. Zuiderweg, J. Magn. Reson. B 102, 242 (1993); D. R. Muhandiram, N. A. Farrow, G. Y. Xu, S. H. Smallcombe, and L. E. Kay, J. Magn. Reson. B 102, 317 (1993); J. Lee, J. Fejzo, and G. Wagner, J. Magn. Reson. B 102, 322 (1993). 11. J. Stonehouse, P. Adell, J. Keeler, and A. J. Shaka, J. Am. Chem. Soc. 116, 6037 (1994); K. Stott, J. Stonehouse, J. Keeler, T.-L. Hwang, and A. J. Shaka, J. Am. Chem. Soc. 117, 4199 (1995). 12. J. Jeener, B. H. Meier, P. Bachmann, and R. R. Ernst, J. Chem. Phys. 71, 4546 (1979). 13. P. L. Rinaldi, J. Am. Chem. Soc. 105, 5167 (1983). 14. A. Sodickson, W. E. Maas, and D. G. Cory, J. Magn. Reson. B 110, 298 (1996).
magal
AP: Mag Res, Series A
Series A 123, 119–121 (1996)
0222
Gradient-Selected NOESY—A Fourfold Reduction of the Measurement Time for the NOESY Experiment RONALD WAGNER
AND
STEFAN BERGER *
Department of Chemistry, Philipps University Marburg, Hans-Meerwein-Strasse, D-35032 Marburg, Germany Received July 29, 1996
The NOESY experiment (1, 2) is one of the fundamental and most often performed 2D NMR experiments for applications in organic chemistry and in biomolecular research. In the latter application, overnight and even longer measurements are often required due to the low concentration of proteins or oligonucleotides. A NOESY experiment for application in organic chemistry to determine stereochemical assignments typically runs about two hours, depending on the relaxation and mixing time (3). This rather long time is not due to the sensitivity of current NMR instruments but because an eight-step phase cycle (4) is required to suppress unwanted coherences, such as double-quantum contributions, COSY-like signals, and axial peaks. In recent years, the other standard sequences used in structural elucidation for organic chemistry, such as COSY (5) or HMQC (6) have experienced a significant reduction in experiment time with the introduction of pulsed field gradients (7). Today a gradient-selected COSY experiment or a HMQC-type inverse C,H correlation of typically 20–50 mg of organic material and a molecular weight on the order of 500 Da can be performed in 5 to 10 minutes (3). Compared with this, the corresponding NOESY measurement time is inconvenient, especially since it is desirable to perform more than one NOESY experiment using different mixing times. Within this context, Ko¨ck and Griesinger recently published a FAST NOESY method (8), reducing the relaxation waiting time. However, their procedure requires an extra measurement of these relaxation times and special data processing so that this method does not seem to have gained wide application in service laboratories. In this Communication, we wish to demonstrate that by an extremely easy modification of the standard experiment, the usual eight-step phase cycle of the experiment can effectively be reduced to two steps leading to the desired fourfold reduction in experiment time. We replace the NOESY mixing time tm simply by a weak pulsed field gradient, which acts during the entire exchange period as shown in Fig. 1. For a two-spin system, it can be shown with the productoperator formalism (9) that the second 907 pulse of the NOESY sequence creates the desired frequency-labeled z magnetization, but in addition double- and zero-quantum coherences and antiphase coherences leading to COSY-type signals. Despite the
zero-quantum coherences, these can be efficiently suppressed by the pulsed field gradient in the mixing time, whereas the field gradient will not affect the frequency-labeled z magnetization which is the working principle of the NOESY scheme. In addition, axial peaks which are caused by spins which have relaxed during t1 and which are made transverse again by the second 907 pulse are also suppressed. The idea of placing a pulsed field gradient within the mixing period seems to be straightforward and has indeed been used in more complicated 3D sequences (10) and in selective 1D versions of NOE difference spectroscopy (11). It was even mentioned before the introduction of self-shielded gradient coils in the first demonstration of the EXSY experiment (12) and applied for the HOESY sequence (13). Surprisingly, to the best of our knowledge, it has not been mentioned or demonstrated for the basic homonuclear 2D NOESY sequence, probably because the developers of modern NMR need many transients for the usual protein work. Best results were achieved when the gradient pulse was on during the entire mixing time. This is probably due to the integrated gradient strength which can be reached and to the suppression of stray-field effects during the mixing time (14). In principle, our method could be performed using no phase cycle at all; however, in practice, it seems to be advantageous to use a two-step phase cycle, similar to many other gradient-selected experiments (3, 7). As an example, we show in Fig. 2a a standard NOESY spectrum of a 10% solution of strychnine
* To whom correspondence should be addressed. 119
AID
JMRA 0982
/
6j13$$$381
10-17-96 15:47:35
1064-1858/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
magal
AP: Mag Res, Series A
120
COMMUNICATIONS
FIG. 1. Pulse sequence for the gs-NOESY method. w1 , w2 , w3 , phases for the 907 1H NMR RF pulses; w4 , receiver phase; t1 , time increment for the indirect dimension; tm , NOE mixing time; gz , pulsed field gradient.
in CDCl3 using the eight-step phase cycle, a relaxation time of 2 s, and a mixing time of 250 ms requiring an experiment time of 83 min. Figure 2b gives as comparison the result of the gradient-selected method requiring one-fourth of the experimental time, which is identical except for some reduction in signal-to-noise. This reduction is somewhat counterbalanced by the slightly higher receiver gain possible with the gradient
method. Contrary to these measurements, a standard NOESY sequence under the same conditions, using only a two-step phase cycle for axial peak suppression, revealed the known COSY-type signal breakthroughs. These artifacts may become smaller toward longer mixing times; with the gradient method, however, one can be sure that these unwanted signals will be suppressed. Our new method will not be necessary for those
FIG. 2. (a) Expansion of the aliphatic region of a standard 2D NOESY measurement on 10% strychnine ( 1) in CDCl3 , using an eight-step phase cycle, 2K data points in F2 , 256 data points in F1 , recorded with a spectral width of 10 ppm (Bruker AMX-500 NMR spectrometer). (b) Same conditions as in (a), recorded with the gs-NOESY method of Fig. 1 with a pulsed field gradient of 250 ms length and strength of approximately 0.01 T/m.
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/
6j13$$$382
10-17-96 15:47:35
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AP: Mag Res, Series A
121
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FIG. 2—Continued
who must accumulate NOESY spectra with many scans, but in summary we feel that the gs-NOESY method is a significant step toward improving the throughput for NOESY spectra in service laboratories for organic chemistry. ACKNOWLEDGMENTS This work has been supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.
REFERENCES 1. J. Jeener, B. H. Meier, P. Bachmann, and R. R. Ernst, J. Chem. Phys. 71, 4546 (1979). 2. D. Neuhaus and M. Williamson, ‘‘The Nuclear Overhauser Effect in Structural and Conformational Analysis,’’ VCH, Weinheim, 1989. 3. S. Braun, H.-O. Kalinowski, and S. Berger, ‘‘100 and More Basic NMR Experiments,’’ VCH, Weinheim, 1996.
AID
JMRA 0982
/
6j13$$$382
10-17-96 15:47:35
4. G. Bodenhausen, H. Kogler, and R. R. Ernst, J. Magn. Reson. 58, 370 (1984). 5. R. E. Hurd, J. Magn. Reson. 87, 422 (1990). 6. R. E. Hurd and B. K. John, J. Magn. Reson. 91, 648 (1991). 7. J. Keeler, R. T. Clowes, A. L. Davis, and E. D. Laue, Methods Enzymol. 239, 145 (1994). 8. M. Ko¨ck and C. Griesinger, Angew. Chem. 106, 338 (1994). 9. O. W. Sørensen, G. W. Eich, M. H. Levitt, G. Bodenhausen, and R. R. Ernst, Prog. NMR Spectrosc. 16, 163 (1983). 10. A. Majumdar and E. R. P. Zuiderweg, J. Magn. Reson. B 102, 242 (1993); D. R. Muhandiram, N. A. Farrow, G. Y. Xu, S. H. Smallcombe, and L. E. Kay, J. Magn. Reson. B 102, 317 (1993); J. Lee, J. Fejzo, and G. Wagner, J. Magn. Reson. B 102, 322 (1993). 11. J. Stonehouse, P. Adell, J. Keeler, and A. J. Shaka, J. Am. Chem. Soc. 116, 6037 (1994); K. Stott, J. Stonehouse, J. Keeler, T.-L. Hwang, and A. J. Shaka, J. Am. Chem. Soc. 117, 4199 (1995). 12. J. Jeener, B. H. Meier, P. Bachmann, and R. R. Ernst, J. Chem. Phys. 71, 4546 (1979). 13. P. L. Rinaldi, J. Am. Chem. Soc. 105, 5167 (1983). 14. A. Sodickson, W. E. Maas, and D. G. Cory, J. Magn. Reson. B 110, 298 (1996).
magal
AP: Mag Res, Series A