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Heteronuclear zero quantum spectroscopy as a conformational probe
JOURNAL
OF MAGNETIC
RESONANCE
52, 326-329
(1983)
Heteronuclear Zero Quantum Spectroscopy as a Conformational Probe PHILIP
H. BOLTON
Chemistry Department, Wesleyan University, Middletown, Connecticut 06457 Received
November
17, 1982; revised
December
27, 1982
Investigations of the conformations of molecules in liquids are often aided by the information present in the proton NMR spectrum of the sample. The proton chemical shifts and proton-proton couplings often contain sufficient information for determination of the structural features of interest. However, in many cases the proton spectrum is obscured by the signals from solvent or an enzyme or by overlap of signals from the molecule being studied. One approach to overcoming this problem is to use heteronuclear two-dimensional NMR to detect indirectly the proton spectral information (1, 2). The heteronuclear two-dimensional NMR method can be used to obtain proton spectra of sufficient quality to answer conformational questions (35). The technique has recently been extended to detection of protons which share a coupling partner with the heteronucleus but are not directly coupled to the heteronucleus (6, 7). Another approach to spying on protons is to use heteronuclear multiple quantum spectroscopy (8-10). Since indirect detection of protons is of use in examining the conformations and structures of molecules where conventional approaches fail, it was of interest to begin an investigation of the merits of multiple quantum spectroscopy relative to heteronuclear two-dimensional NMR. Zero quantum spectroscopy, in particular, appears to be quite promising. The linewidths obtained in the crucial domain containing the proton information are only weakly dependent on the field homogeneity. A more advantageous feature is that the proton spectra obtained via heteronuclear zero quantum spectroscopy correspond to the heteronuclear decoupled proton spectrum when only one proton is coupled to the heteronucleus. The pulse sequence used for the generation and detection of heteronuclear zero quantum coherence is shown in Fig. 1. The phases of the pulses are cycled so that the phosphorus-31 signals are modulated only by the zero quantum coherence as a function of tl. Unlike the situation for homonuclear zero quantum spectroscopy, phase cycling can discriminate between zero quantum coherence and longitudinal magnetization. The phase cycling modulates the phase of the detected signal, allowing “quadrature” detection to be used in both frequency domains. The frequency of the heteronuclear zero quantum signal, in the absence of protonproton coupling, is the difference between the proton Larmor frequency and the proton transmitter frequency minus the difference between the phosphorus-31 Lar0022-2364183
$3.00
Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.
326
327
COMMUNICATIONS
preparation tP
evolution
detection
‘1
‘2
FIG. 1. The pulse sequence used in the heteronuclear zero quantum NMR experiments. The preparation period has two proton 90” pulses and a single phosphorus-3 1 90” pulse. These three pulses are phase cycled together through the sequence x, y, -x, -y. The phase of the proton 180” pulse applied at the middle of the preparation period is cycled through y, -x, -y, x. After each pass through the four step phase cycle the phase of the proton refocusing pulse is incremented by 180”. At the end of the evolution period a 90” pulse with x phase is applied to the protons. All of the phosphorus transients for a given value of t, are added together. As is usual f, is arithmetically incremented and a second Fourier transformation of the data is performed. Only the heteronuclear zero quantum modulation of the phosphorus-31 signals is detected.
mor frequency and the phosphorus-3 1 transmitter frequency. Proton-proton coupling gives rise to splitting of the signal in the usual fashion. The heteronuclear zero quantum signal does not exhibit the characteristic “up-down” pattern found in heteronuclear two-dimensional NMR. The zero quantum signals associated with the two phosphorus-31 lines are of opposite algebraic sign. Thus, it is expected that the zero quantum signals will be particularly simple, corresponding directly to the phosphorus3 1 decoupled proton spectrum. Enhanced signal-to-noise can be gained by taking the difference between the zero quantum spectra associated with the two phosphorus-3 1 lines as is the case for heteronuclear two-dimensional NMR (4, 5). The field homogeneity dependence of the linewidths of the zero quantum signals can be analyzed by extension of a previous treatment of coherence transfer echoes (11). In a heteronuclear two-dimensional experiment the magnetization defocuses as proton magnetization during t, and a portion refocuses as phosphorus-31 magnetization during t2. Thus, a coherence transfer echo is formed when t2 = (~&y&r. In the heteronuclear zero quantum experiment the magnetization defocuses as zero quantum coherence and refocuses as phosphorus-3 1 magnetization. The refocusing time is (in - yr/yr)t, . As shown elsewhere (II, 12) the presence of coherence transfer echoes leads to a “sloping” pattern of the signals in the two-dimensional map. The slope for the zero quantum signals is (vu - yp/-yp), which is 60% of that of the heteronuclear two-dimensional experiment. The smaller the slope the narrower will be the linewidths along any given slice through the two-dimensional map. In the same magnetic field it is expected that the linewidths obtained via the zero quantum method will be 60% of those obtained via heteronuclear two-dimensional NMR when relaxation can be neglected. The field homogeneity dependence can be completely eliminated from both experiments by the RECYCLE technique (12). An experimental examination of the homogeneity dependence of the indirectly detected proton signals is shown in Fig. 2. It is seen that an approximately threefold decrease in the field homogeneity induces an increase of less than 20% in the linewidths of the proton signals. Examination of contour plots of data obtained in very inhomogeneous fields, not shown, confirmed the prediction that the slope of the signals is (mu - y&r) = 1S. The weak homogeneity dependence of the zero quantum
328
COMMUNICATIONS
FIG.2. Heteronuclear zero quantum spectra obtained from phosphothreonine in fields of different homogeneity. The spectra on the right are the normal proton coupled phosphorus-31 spectra and indicate the extent of the field inhomogeneity. The spectra on the left are the phase sensitive zero quantum spectra obtained in the same magnetic field. The field homogeneity was degraded by stopping the sample. Linewidth in the field of better homogeneity was 0.8 to 0.9 Hz and in the poor homogeneity field, 1.0 to 1.1 Hz.
signals indicates that high resolution data can be obtained from samples in moderately inhomogeneous environments. If zero quantum spectroscopy is to be used as a conformational probe, it must be shown that the data obtained can be related to proton chemical shifts and 3’ CMP
(5He
phasphothreonine
5H?.
H
FIG.3. Simulations of the zero quantum spectra of cytidine 3’-phosphate (3’CMP) and phosphothreonine compared with the phase sensitive experimental data. For 3’CMP it was assumed that the 3’ proton was coupled only to the 2’ and 4’ protons with coupling constants of 5.1 and 6.3 Hz. For phosphothreonine, Jh was 7.1 and Jaewas 6.8 Hz. The linewidths used in the simulations were 0.9 Hz. The experiments used 128 evolution time increments of 2.5 msec. The data were zero filled to 5 points per hertz in the proton dimension. All experiments were performed on a Varian XL200 spectrometer using 20 mM samples in 10 mm tubes.
329
COMMUNICATIONS
scalar couplings. Figure 3 compares simulations of the normal, phosphorus-31 decoupled, proton spectra of cytidine 3’-phosphate and phosphothreonine, CH,CH(OPO,)CH(NH)CO,H, with the zero quantum spectra. The correlation is particularly pleasing in its simplicity. The heteronuclear coupling is absent from the proton signals but is present in the phosphorus-31 dimension. The zero quantum spectra are easy to simulate and hence to analyze for the proton-proton couplings. The proton chemical shifts can be determined by knowing the chemical shift of the proton transmitter and the frequency of the phosphorus-31 signal relative to the phosphorus-3 1 transmitter. For those samples which have only one proton coupled to the heteronuclear spy the zero quantum experiment is an attractive alternative to heteronuclear two-dimensional NMR. The prime advantage of the zero quantum approach lies in the simplicity of the results and a lesser advantage is the weaker dependence of the linewidths on the field homogeneity. In actual practice the heteronuclear twodimensional experiment is about twice as sensitive as the zero quantum one. Thus it seems that the heteronuclear zero quantum experiment does have a place as yet another means of indirectly determining proton spectral information. Preliminary investigation of more complicated spin systems shows that the zero quantum approach will be clearly superior to heteronuclear two-dimensional NMR in those cases where more than one proton is coupled to the heteronuclear spy. ACKNOWLEDGMENTS This research Research Fund
was supported, of the American
in part, by the Camille Chemical Society.
and Henry
Dreyfus
Foundation
and the Petroleum
REFERENCES
1. A. A. MAUDSLEY 2. 3. 4. 5.
R. P. G. P.
6. 7. 8. 9. 10. II. 12.
P. P. G. A. L. A. P.
AND R. R. ERNST, Chem. Phys. Lett. 50, 368 (1977). FREEMAN AND G. A. MORRIS, Bull. Magn. Reson. 1, 5 (1979). H. BOLTON AND G. BODENHALJSEN, J. Am. Chem. Sot. 101, 1080 (1979). B~DENHAUSEN AND P. H. BOLTON, J. Magn. Reson. 39, 399 (I 980). H. BOLTON, “Biomolecular Stereodynamics” (R. H. Sarma, Ed.), Vol. II, p. 437, New York, 1981. H. BOLTON, J. Magn. Reson. 48, 336 (1982). H. B~LTON AND G. B~DENHAUSEN, Chem. Phys. Lett. 89, 139 (1982). B~DENHAUSEN, Progr. NMR Spectr. 14, 137 (198 1). WOUAUN AND R. R. ERNST, Chem. Phys. Lett. 52,407 (1977). MUELLER, J. Am. Chem. Sot. 101,4481 (1979). A. MAVDSLEY, A. WOKAUN, AND R. R. ERNST, Chem. Phys. Lett. 55, 9 (1978). H. BOLTON AND G. B~DENHAVSEN, J. Magn. Reson. 46, 306 (1982).
Adenine
Press,
OF MAGNETIC
RESONANCE
52, 326-329
(1983)
Heteronuclear Zero Quantum Spectroscopy as a Conformational Probe PHILIP
H. BOLTON
Chemistry Department, Wesleyan University, Middletown, Connecticut 06457 Received
November
17, 1982; revised
December
27, 1982
Investigations of the conformations of molecules in liquids are often aided by the information present in the proton NMR spectrum of the sample. The proton chemical shifts and proton-proton couplings often contain sufficient information for determination of the structural features of interest. However, in many cases the proton spectrum is obscured by the signals from solvent or an enzyme or by overlap of signals from the molecule being studied. One approach to overcoming this problem is to use heteronuclear two-dimensional NMR to detect indirectly the proton spectral information (1, 2). The heteronuclear two-dimensional NMR method can be used to obtain proton spectra of sufficient quality to answer conformational questions (35). The technique has recently been extended to detection of protons which share a coupling partner with the heteronucleus but are not directly coupled to the heteronucleus (6, 7). Another approach to spying on protons is to use heteronuclear multiple quantum spectroscopy (8-10). Since indirect detection of protons is of use in examining the conformations and structures of molecules where conventional approaches fail, it was of interest to begin an investigation of the merits of multiple quantum spectroscopy relative to heteronuclear two-dimensional NMR. Zero quantum spectroscopy, in particular, appears to be quite promising. The linewidths obtained in the crucial domain containing the proton information are only weakly dependent on the field homogeneity. A more advantageous feature is that the proton spectra obtained via heteronuclear zero quantum spectroscopy correspond to the heteronuclear decoupled proton spectrum when only one proton is coupled to the heteronucleus. The pulse sequence used for the generation and detection of heteronuclear zero quantum coherence is shown in Fig. 1. The phases of the pulses are cycled so that the phosphorus-31 signals are modulated only by the zero quantum coherence as a function of tl. Unlike the situation for homonuclear zero quantum spectroscopy, phase cycling can discriminate between zero quantum coherence and longitudinal magnetization. The phase cycling modulates the phase of the detected signal, allowing “quadrature” detection to be used in both frequency domains. The frequency of the heteronuclear zero quantum signal, in the absence of protonproton coupling, is the difference between the proton Larmor frequency and the proton transmitter frequency minus the difference between the phosphorus-31 Lar0022-2364183
$3.00
Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.
326
327
COMMUNICATIONS
preparation tP
evolution
detection
‘1
‘2
FIG. 1. The pulse sequence used in the heteronuclear zero quantum NMR experiments. The preparation period has two proton 90” pulses and a single phosphorus-3 1 90” pulse. These three pulses are phase cycled together through the sequence x, y, -x, -y. The phase of the proton 180” pulse applied at the middle of the preparation period is cycled through y, -x, -y, x. After each pass through the four step phase cycle the phase of the proton refocusing pulse is incremented by 180”. At the end of the evolution period a 90” pulse with x phase is applied to the protons. All of the phosphorus transients for a given value of t, are added together. As is usual f, is arithmetically incremented and a second Fourier transformation of the data is performed. Only the heteronuclear zero quantum modulation of the phosphorus-31 signals is detected.
mor frequency and the phosphorus-3 1 transmitter frequency. Proton-proton coupling gives rise to splitting of the signal in the usual fashion. The heteronuclear zero quantum signal does not exhibit the characteristic “up-down” pattern found in heteronuclear two-dimensional NMR. The zero quantum signals associated with the two phosphorus-31 lines are of opposite algebraic sign. Thus, it is expected that the zero quantum signals will be particularly simple, corresponding directly to the phosphorus3 1 decoupled proton spectrum. Enhanced signal-to-noise can be gained by taking the difference between the zero quantum spectra associated with the two phosphorus-3 1 lines as is the case for heteronuclear two-dimensional NMR (4, 5). The field homogeneity dependence of the linewidths of the zero quantum signals can be analyzed by extension of a previous treatment of coherence transfer echoes (11). In a heteronuclear two-dimensional experiment the magnetization defocuses as proton magnetization during t, and a portion refocuses as phosphorus-31 magnetization during t2. Thus, a coherence transfer echo is formed when t2 = (~&y&r. In the heteronuclear zero quantum experiment the magnetization defocuses as zero quantum coherence and refocuses as phosphorus-3 1 magnetization. The refocusing time is (in - yr/yr)t, . As shown elsewhere (II, 12) the presence of coherence transfer echoes leads to a “sloping” pattern of the signals in the two-dimensional map. The slope for the zero quantum signals is (vu - yp/-yp), which is 60% of that of the heteronuclear two-dimensional experiment. The smaller the slope the narrower will be the linewidths along any given slice through the two-dimensional map. In the same magnetic field it is expected that the linewidths obtained via the zero quantum method will be 60% of those obtained via heteronuclear two-dimensional NMR when relaxation can be neglected. The field homogeneity dependence can be completely eliminated from both experiments by the RECYCLE technique (12). An experimental examination of the homogeneity dependence of the indirectly detected proton signals is shown in Fig. 2. It is seen that an approximately threefold decrease in the field homogeneity induces an increase of less than 20% in the linewidths of the proton signals. Examination of contour plots of data obtained in very inhomogeneous fields, not shown, confirmed the prediction that the slope of the signals is (mu - y&r) = 1S. The weak homogeneity dependence of the zero quantum
328
COMMUNICATIONS
FIG.2. Heteronuclear zero quantum spectra obtained from phosphothreonine in fields of different homogeneity. The spectra on the right are the normal proton coupled phosphorus-31 spectra and indicate the extent of the field inhomogeneity. The spectra on the left are the phase sensitive zero quantum spectra obtained in the same magnetic field. The field homogeneity was degraded by stopping the sample. Linewidth in the field of better homogeneity was 0.8 to 0.9 Hz and in the poor homogeneity field, 1.0 to 1.1 Hz.
signals indicates that high resolution data can be obtained from samples in moderately inhomogeneous environments. If zero quantum spectroscopy is to be used as a conformational probe, it must be shown that the data obtained can be related to proton chemical shifts and 3’ CMP
(5He
phasphothreonine
5H?.
H
FIG.3. Simulations of the zero quantum spectra of cytidine 3’-phosphate (3’CMP) and phosphothreonine compared with the phase sensitive experimental data. For 3’CMP it was assumed that the 3’ proton was coupled only to the 2’ and 4’ protons with coupling constants of 5.1 and 6.3 Hz. For phosphothreonine, Jh was 7.1 and Jaewas 6.8 Hz. The linewidths used in the simulations were 0.9 Hz. The experiments used 128 evolution time increments of 2.5 msec. The data were zero filled to 5 points per hertz in the proton dimension. All experiments were performed on a Varian XL200 spectrometer using 20 mM samples in 10 mm tubes.
329
COMMUNICATIONS
scalar couplings. Figure 3 compares simulations of the normal, phosphorus-31 decoupled, proton spectra of cytidine 3’-phosphate and phosphothreonine, CH,CH(OPO,)CH(NH)CO,H, with the zero quantum spectra. The correlation is particularly pleasing in its simplicity. The heteronuclear coupling is absent from the proton signals but is present in the phosphorus-31 dimension. The zero quantum spectra are easy to simulate and hence to analyze for the proton-proton couplings. The proton chemical shifts can be determined by knowing the chemical shift of the proton transmitter and the frequency of the phosphorus-31 signal relative to the phosphorus-3 1 transmitter. For those samples which have only one proton coupled to the heteronuclear spy the zero quantum experiment is an attractive alternative to heteronuclear two-dimensional NMR. The prime advantage of the zero quantum approach lies in the simplicity of the results and a lesser advantage is the weaker dependence of the linewidths on the field homogeneity. In actual practice the heteronuclear twodimensional experiment is about twice as sensitive as the zero quantum one. Thus it seems that the heteronuclear zero quantum experiment does have a place as yet another means of indirectly determining proton spectral information. Preliminary investigation of more complicated spin systems shows that the zero quantum approach will be clearly superior to heteronuclear two-dimensional NMR in those cases where more than one proton is coupled to the heteronuclear spy. ACKNOWLEDGMENTS This research Research Fund
was supported, of the American
in part, by the Camille Chemical Society.
and Henry
Dreyfus
Foundation
and the Petroleum
REFERENCES
1. A. A. MAUDSLEY 2. 3. 4. 5.
R. P. G. P.
6. 7. 8. 9. 10. II. 12.
P. P. G. A. L. A. P.
AND R. R. ERNST, Chem. Phys. Lett. 50, 368 (1977). FREEMAN AND G. A. MORRIS, Bull. Magn. Reson. 1, 5 (1979). H. BOLTON AND G. BODENHALJSEN, J. Am. Chem. Sot. 101, 1080 (1979). B~DENHAUSEN AND P. H. BOLTON, J. Magn. Reson. 39, 399 (I 980). H. BOLTON, “Biomolecular Stereodynamics” (R. H. Sarma, Ed.), Vol. II, p. 437, New York, 1981. H. BOLTON, J. Magn. Reson. 48, 336 (1982). H. B~LTON AND G. B~DENHAUSEN, Chem. Phys. Lett. 89, 139 (1982). B~DENHAUSEN, Progr. NMR Spectr. 14, 137 (198 1). WOUAUN AND R. R. ERNST, Chem. Phys. Lett. 52,407 (1977). MUELLER, J. Am. Chem. Sot. 101,4481 (1979). A. MAVDSLEY, A. WOKAUN, AND R. R. ERNST, Chem. Phys. Lett. 55, 9 (1978). H. BOLTON AND G. B~DENHAVSEN, J. Magn. Reson. 46, 306 (1982).
Adenine
Press,