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COSY representation of two-dimensional homonuclear double-quantum spectra
JOURNAL
OF MAGNETIC
RESONANCE
66,
153- 156 ( 1986)
COSY Representationof Two-DimensionalHomonuclear Double-Quantum Spectra ERIK R. P. ZUIDERWEG Abbott Laboratories, Abbott Park, North Chicago, Illinois 60064 Received May 30, 1985
Many different 2D experiments are available for the analysis of J-coupling networks in liquid phase NMR spectra, such as correlated spectroscopy (COSY) (I, 2), doublequantum-filtered COSY (3), relayed coherence transfer (4, 5), and doublequantum spectroscopy (2, 6-8). Complicated spectra consisting of a superposition of many isolated networks, such as *H NMR spectra of proteins, are best analyzed by comparing the results of several different kinds of experiments. The 2D doublequantum (DQ) experiment is especially versatile for establishing J connectivities between resonances with a small chemical-shift difference, i.e., multiplets which would show cross peaks close to the diagonal in COSY experiments. Furthermore, information with respect to remotely coupled multiplets can be extracted from those spectra (6, 7). However, a major drawback of the DQ experiment is its twodimensional representation, which makes comparisons of complicated DQ spectra with COSY type spectra a tedious task. Recently, a modification of the basic DQ experiment which results in a COSY representation of the direct connectivity patterns has been suggested (9). In that experiment data acquisition commences at a time t, after the detection pulse, causing a mixing of the t, and t2 acquisition times analogous to what occurs in spin-echo correlated spectroscopy (SECSY) (10). The pulse sequence requires an anti-echo suppression phase cycle which causes phase twisted lineshapes. Therefore, the results of this elegant experiment can be displayed only in the absolute-value mode, which is undesirable for crowded spectra of larger biomacromolecules. Pure absorption phase DQ spectra can be obtained with a symmetrical excitation/ observation pulse sandwich terminated by a purge pulse (8). The experiment allows for the editing of direct and remote connectivities due to phase differences of the respective cross peaks, and when combined with preparation time averaging, for uniform doublequantum coherence excitation (8). The results of this superior experiment can easily be cast in the COSY representation, without affecting the experimental setup, in the following way. Prior to the tI - wI Fourier transformation the interferograms are zero filled to obtain in the final frequency-domain spectrum a number of points in w, twice as large as in ~2. After the transformation, the points i of the wI slices n are left-shifted to the positions j according to
(j, n) = (i - n, n). 153
0022-2364186 $3.00 Copyright 0 1986 by Academic Press, Inc. AU rights of reprod~on in my form -cd.
154
NOTES
In the original unshifted spectrum, direct double-quantum connectivities between two resonances at frequencies &?Aand QB, occur at (w,, w2) coordinates (QA + QB, %I) and (I?=4+ OB, SIB). When the data is processed as described here, the coordinates of these cross peaks are changed to (flB, !&I) and (M, QB), respectively, i.e., to the coordinates of COSY cross peaks. Remote connectivities, e.g., at coordinates &?A + QB, W) in the original spectrum, will be shifted to positions (SL.4+ QB - QC, K), which would fall into the window of the transformed matrix when fL4 + ilB - QC > 0. Other remote connectivities not satisfying this relation would be lost upon the transformation. However, such connectivities can still be analyzed in the original 1(
8.U
680
4.0 2,o 0.0 PPM FIG. 1. Pure absorption phase.500 MHz 2D ‘H-‘H double quantum spectrum (8) of 20 mkfvancomycin in DMSO-d,, 25°C. 5 12 accumulations of 40 transients each were recorded. Only positive levels arc plotted.
155
NOTES
spectrum. If desired, a standard symmetrization procedure can be applied to the transformed data set to reduce tl noise. In the phase sensitive DQ spectrum, remote connectivities are opposite in phase with respect to the direct connectivities (8). Therefore, no additional spectral editing effect is to be expected from the symmetrization of such spectra. However, if the data in the original matrix is in the absolute-value mode, the symmetrization will discard all remote connectivities, facilitating the spectral interpretation. Figure 1 shows a pure absorption phase ‘H- ‘H double quantum spectrum of vancomycin, a glycopeptide antibiotic (MW 1500). The spectrum was recorded on a General Electric GN500 500 MHz spectrometer, equipped with a Nicolet 293C pulse programmer, and interfaced to a Nicolet 1280 computer. The symmetric doublequantum coherence excitation/detection scheme (8) 90"-T-90"~t,-90"-7-45"+, in which the symbols have their usual meaning, with the minimal eight-step phase cycle was employed. One value was used for the antiphase preparation and refocusing period 7 (40 ms). Data processing was carried out on a VAX 1l/780 computer using commercially available software (I 1) which was slightly modified to accommodate the file sizes needed for the present application. The area between the two diagonal lines in Fig. 1 contains all direct doublequantum connectivities. This area corresponds to the square COSY representation of the same spectrum, obtained by column shifting as described above (Fig. 2). As is seen, the appearance of Fig. 2 is as of a COSY spectrum without a diagonal. However, J-coupling patterns are different than in COSY spectra: in w, , due to the absence of J coupling
8’. 0
6’. 0
4’. 0
2’. 0
0’. 0
PPM
FIG. 2. COSY representationof the spectrumof Fig. 1 as obtained by manipulation of the frequencydomain data setasdescribedin the text.
156
NOTES
between the active spins participating the double quantum coherence; in w2, due to the shifting of the columns and the absence of antiphase patterns within the multiplets. For larger molecules these differences, except the difference in phase, will be negligible due to the natural line width of the resonances. Thus, the obtained pattern of direct connectivities in the reformatted DQ spectrum may directly be overlaid with COSY, DQ-filtered COSY, relayed coherence transfer, and nuclear Overhauser enhancement (NOESY) (12) spectra. Remote connectivities, however, are best analyzed in the original, unshifted, DQ spectrum. ACKNOWLEDGMENT I thank Dr. G. Wagner (ETH-Zurich, Switzerland) for the discussions in which the idea of casting DQ spectra in a COSY representation arose. REFERENCES 1. W. P. AUE, E. BARTHOLDI, AND R. R. ERNST, J. Chem. Phys. 64,2229 (1976). 2. A. BAX, “Two Dimensional Nuclear Magnetic Resonance in Liquids,” Delhi Univ. 1982. 3. M. RANCYE, 0.
W. SORENSEN,
G. BODENHAUSEN,
G. WAGNER,
R. R. ERNST,
Press, Dordrecht,
AND K. W-RICH,
Biochem. Biophys. Res. Commun. 117,479 (1983). 4. P. H. BOLTDN AND G. BODENHAUSEN, Chem. Phys. L&t. 89, 139 (1982). 5. G. WAGNER, J. Magn: Reson. 55, 151 (1983). 6. L. BRAUNSCHWEILER, G. BODENHAUSEN, AND R. R. ERNST, Mol. Phys. 48,535 (1983). 7. G. WAGNER AND E. R. P. ZUIDERWEG, Biochem. Biophys. Res. Commun. 113,854 (1983). 8. M. RANCE,
0. W. WRENSEN,
W. LEUPIN,
H. KOGLER,
K. W~THRI~H,
AND R. R. ERNST,
J. Mugfz.
Reson. 61,67 ( 1984). 9. G. BODENHAUSEN, H. KOGLER, AND R. R. ERNST, J. Magn. Reson. 58,370 (I 984). 10. K. NAGAYAMA, K. WUTHRICH, AND R. R. ERNST, Biochem. Biophys. Res. Commun. 90,305 II. D. R. HARE, FTNMR Program, University of Washington, Seattle. 12. J. JEENER, B. H. MEIER, P. BACHMAN, AND R. R. ERNST, J. Chem. Phys. 71,4546 (1979).
(1979).
OF MAGNETIC
RESONANCE
66,
153- 156 ( 1986)
COSY Representationof Two-DimensionalHomonuclear Double-Quantum Spectra ERIK R. P. ZUIDERWEG Abbott Laboratories, Abbott Park, North Chicago, Illinois 60064 Received May 30, 1985
Many different 2D experiments are available for the analysis of J-coupling networks in liquid phase NMR spectra, such as correlated spectroscopy (COSY) (I, 2), doublequantum-filtered COSY (3), relayed coherence transfer (4, 5), and doublequantum spectroscopy (2, 6-8). Complicated spectra consisting of a superposition of many isolated networks, such as *H NMR spectra of proteins, are best analyzed by comparing the results of several different kinds of experiments. The 2D doublequantum (DQ) experiment is especially versatile for establishing J connectivities between resonances with a small chemical-shift difference, i.e., multiplets which would show cross peaks close to the diagonal in COSY experiments. Furthermore, information with respect to remotely coupled multiplets can be extracted from those spectra (6, 7). However, a major drawback of the DQ experiment is its twodimensional representation, which makes comparisons of complicated DQ spectra with COSY type spectra a tedious task. Recently, a modification of the basic DQ experiment which results in a COSY representation of the direct connectivity patterns has been suggested (9). In that experiment data acquisition commences at a time t, after the detection pulse, causing a mixing of the t, and t2 acquisition times analogous to what occurs in spin-echo correlated spectroscopy (SECSY) (10). The pulse sequence requires an anti-echo suppression phase cycle which causes phase twisted lineshapes. Therefore, the results of this elegant experiment can be displayed only in the absolute-value mode, which is undesirable for crowded spectra of larger biomacromolecules. Pure absorption phase DQ spectra can be obtained with a symmetrical excitation/ observation pulse sandwich terminated by a purge pulse (8). The experiment allows for the editing of direct and remote connectivities due to phase differences of the respective cross peaks, and when combined with preparation time averaging, for uniform doublequantum coherence excitation (8). The results of this superior experiment can easily be cast in the COSY representation, without affecting the experimental setup, in the following way. Prior to the tI - wI Fourier transformation the interferograms are zero filled to obtain in the final frequency-domain spectrum a number of points in w, twice as large as in ~2. After the transformation, the points i of the wI slices n are left-shifted to the positions j according to
(j, n) = (i - n, n). 153
0022-2364186 $3.00 Copyright 0 1986 by Academic Press, Inc. AU rights of reprod~on in my form -cd.
154
NOTES
In the original unshifted spectrum, direct double-quantum connectivities between two resonances at frequencies &?Aand QB, occur at (w,, w2) coordinates (QA + QB, %I) and (I?=4+ OB, SIB). When the data is processed as described here, the coordinates of these cross peaks are changed to (flB, !&I) and (M, QB), respectively, i.e., to the coordinates of COSY cross peaks. Remote connectivities, e.g., at coordinates &?A + QB, W) in the original spectrum, will be shifted to positions (SL.4+ QB - QC, K), which would fall into the window of the transformed matrix when fL4 + ilB - QC > 0. Other remote connectivities not satisfying this relation would be lost upon the transformation. However, such connectivities can still be analyzed in the original 1(
8.U
680
4.0 2,o 0.0 PPM FIG. 1. Pure absorption phase.500 MHz 2D ‘H-‘H double quantum spectrum (8) of 20 mkfvancomycin in DMSO-d,, 25°C. 5 12 accumulations of 40 transients each were recorded. Only positive levels arc plotted.
155
NOTES
spectrum. If desired, a standard symmetrization procedure can be applied to the transformed data set to reduce tl noise. In the phase sensitive DQ spectrum, remote connectivities are opposite in phase with respect to the direct connectivities (8). Therefore, no additional spectral editing effect is to be expected from the symmetrization of such spectra. However, if the data in the original matrix is in the absolute-value mode, the symmetrization will discard all remote connectivities, facilitating the spectral interpretation. Figure 1 shows a pure absorption phase ‘H- ‘H double quantum spectrum of vancomycin, a glycopeptide antibiotic (MW 1500). The spectrum was recorded on a General Electric GN500 500 MHz spectrometer, equipped with a Nicolet 293C pulse programmer, and interfaced to a Nicolet 1280 computer. The symmetric doublequantum coherence excitation/detection scheme (8) 90"-T-90"~t,-90"-7-45"+, in which the symbols have their usual meaning, with the minimal eight-step phase cycle was employed. One value was used for the antiphase preparation and refocusing period 7 (40 ms). Data processing was carried out on a VAX 1l/780 computer using commercially available software (I 1) which was slightly modified to accommodate the file sizes needed for the present application. The area between the two diagonal lines in Fig. 1 contains all direct doublequantum connectivities. This area corresponds to the square COSY representation of the same spectrum, obtained by column shifting as described above (Fig. 2). As is seen, the appearance of Fig. 2 is as of a COSY spectrum without a diagonal. However, J-coupling patterns are different than in COSY spectra: in w, , due to the absence of J coupling
8’. 0
6’. 0
4’. 0
2’. 0
0’. 0
PPM
FIG. 2. COSY representationof the spectrumof Fig. 1 as obtained by manipulation of the frequencydomain data setasdescribedin the text.
156
NOTES
between the active spins participating the double quantum coherence; in w2, due to the shifting of the columns and the absence of antiphase patterns within the multiplets. For larger molecules these differences, except the difference in phase, will be negligible due to the natural line width of the resonances. Thus, the obtained pattern of direct connectivities in the reformatted DQ spectrum may directly be overlaid with COSY, DQ-filtered COSY, relayed coherence transfer, and nuclear Overhauser enhancement (NOESY) (12) spectra. Remote connectivities, however, are best analyzed in the original, unshifted, DQ spectrum. ACKNOWLEDGMENT I thank Dr. G. Wagner (ETH-Zurich, Switzerland) for the discussions in which the idea of casting DQ spectra in a COSY representation arose. REFERENCES 1. W. P. AUE, E. BARTHOLDI, AND R. R. ERNST, J. Chem. Phys. 64,2229 (1976). 2. A. BAX, “Two Dimensional Nuclear Magnetic Resonance in Liquids,” Delhi Univ. 1982. 3. M. RANCYE, 0.
W. SORENSEN,
G. BODENHAUSEN,
G. WAGNER,
R. R. ERNST,
Press, Dordrecht,
AND K. W-RICH,
Biochem. Biophys. Res. Commun. 117,479 (1983). 4. P. H. BOLTDN AND G. BODENHAUSEN, Chem. Phys. L&t. 89, 139 (1982). 5. G. WAGNER, J. Magn: Reson. 55, 151 (1983). 6. L. BRAUNSCHWEILER, G. BODENHAUSEN, AND R. R. ERNST, Mol. Phys. 48,535 (1983). 7. G. WAGNER AND E. R. P. ZUIDERWEG, Biochem. Biophys. Res. Commun. 113,854 (1983). 8. M. RANCE,
0. W. WRENSEN,
W. LEUPIN,
H. KOGLER,
K. W~THRI~H,
AND R. R. ERNST,
J. Mugfz.
Reson. 61,67 ( 1984). 9. G. BODENHAUSEN, H. KOGLER, AND R. R. ERNST, J. Magn. Reson. 58,370 (I 984). 10. K. NAGAYAMA, K. WUTHRICH, AND R. R. ERNST, Biochem. Biophys. Res. Commun. 90,305 II. D. R. HARE, FTNMR Program, University of Washington, Seattle. 12. J. JEENER, B. H. MEIER, P. BACHMAN, AND R. R. ERNST, J. Chem. Phys. 71,4546 (1979).
(1979).