- Email: [email protected]
Two-dimensional correlation spectroscopy with heteronuclear relay
JOURNAL
OF MAGNETIC
RESONANCE
56, 163-166 (1984)
Two-Dimensional Correlation Spectroscopy with Heteronuclear Relay M. A. DELSUC, Laboratoire
de Rbonance
E. GUITTET, MagnBique
N. TROTIN, AND J. Y. LALLEMAND Nucliaire,
CNRS-ICSN,
91190
Gif-sur-
Yvette,
France
ReceivedAugust 8, 1983 Several recent two-dimensional NMR experiments, particularly those using multiplequantum spectroscopy (1) and relay coherence transfer (RELAY) (2), have shown that it is possible to obtain much more specific information than by use of the classical one-dimensional approach. The second of these techniques takes advantage of spy nuclei to elucidate the coupling network and to establish resonance assignments and is based on a transfer of magnetization from one nucleus to another using an intermediate spin. Both types of relay experiments which have already been reported involve an initial homonuclear magnetization transfer (proton-proton) and differ only in that the coherence is relayed to a third spin of either the same (proton, homonuclear) or a different kind (carbon- 13, heteronuclear). We now describe a variation of these relay techniques in which the coherence is first transferred to a heteronuclear spin and is then relayed and detected on a third spin (proton in the present application). The need for such a sequence arose in the course of an oligonucleotide study where extensive overlap of phosphorus resonances precluded the application of classical approaches (such as heteronuclear shift correlated spectroscopy (3) or low-power SFORD (4)). In effect, the problem was to correlate groups of protons which share a common coupled partner but are not themselves coupled to one another (Fig. 1). The technique is based, as in the relays already reported, on the merging of two distinct coherence transfer processes and can be thought of as a ‘H-X heteronuclear shift correlation followed by a X-‘H coherence transfer which brings the information back into the proton spectrum. The resulting pulse sequence is set out in Fig. 2. The amplitude of the relayed cross-peaks can be shown to be proportional to the transfer coefficient: a = sin (7rJILTm) sin (7r&Tm)
ill and the time T,,, is chosen to maximise this transfer coefficient. The phase sequence is designed to eliminate undesirable signals. More elaborate phase sequences can be derived from the basic scheme shown in Table 1, in order to compensate for pulse or phase imperfections. The experiment is demonstrated with a spectrum of diethyl phosphite obtained on a Broker WM 400 equipped with an ASPECT 2000 computer. Minor modifications 163
0022-2364/84 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
164
COMMUNICATIONS J,,=O
FIG. 1. Spin connectivities as revealed by the HERPECS experiment. I and S are homonuclear spins, L is heteronuclear.
of the spectrometer had to be introduced to allow the phase-alternated 3’P pulses. At present the “P pulses are 300 psec, a value that could prove prohibitively long for many applications but should be capable of improvement by use of a booster pulse amplifier. A 128 X 1024 data matrix was acquired and 16 experiments were performed for each I, value. In the present case the heteronuclear coupling values are very different for the hydride (695 Hz) and for the methylene protons (9 Hz), hence a compromise had to be found for the proper choice for T,,, . The value of T, for the experiment of Fig. 3 is 55 msec which corresponds to a transfer coefficient of 0.65. No attempt has been made at present to optimize this value. The spectrum in Fig. 3 shows the correlation of the methylene and the hydride resonances through their common 31P partner. Additional correlations are present in the spectrum that actually correspond to either autocorrelation or residual COSY peaks.
FIG. 2. Pulse schemes of the heteronuclear shift correlation (a), heteronuclear coherence transfer (b), and HERPECS (c) experiments.
165
COMMUNICATIONS TABLE
1
BASIC PHASE SEQUENCE FOR HERPECS EXPERIMENT
Y Y Y Y
x
Y Y
-X -X
-Y -Y
X -x X
X
-X
To rule out any homonuclear correlation due to long-range homonuclear couplings, a blank experiment with the same experimental parameters but with the 31Ptransmitter turned off was performed that showed the absence of such correlations. We are currently applying this technique to oligonucleotides, where the individual
H-P I
CH,
CHz I
0
0 H-;/O-cHrCH, ‘O-CH,-CH,
FI
0
.
Q
0
5
ppm
F2
5
0
FIG. 3. The HERPECS spectrum of diethylphosphite obtained in 45 min from 500 mg of the compound in CDQ, with quadrature detection in t2 and pseudoquadrature detection in I,. The “P carrier is on resonance and the 180” refocusing pulses have been omitted.
166
COMMUNICATIONS
proton subspectra for each ribose are easily identified and isolated by correlated spectroscopy experiments (5). The position in the sequence would be best unraveled by establishing the mutual couplings of selected protons from different riboses to one single 3’P nucleus from the phosphorylated backbone. Water solvent, solubility restriction, and small couplings introduce additional difficulties. We consider that this technique should prove fruitful in elucidating coupling networks and establishing spin connectivities when the strong overlap of heteronuclear resonances hampers the use of more classical approaches. Although this technique should be prescribed in pathological situations, we propose to nickname this HEteronucleus Relayed Proton Correlated Spectroscopy as HERPECS. REFERENCES
1. G. BODENHALJSEN, Prog. NMR Spectrosc. 4, 137 (198 1). 2. P. H. BOLTON, J. Magn. Reson. 48, 336 (1982); P. H. BOLTON AND G. BODENHAUSEN, Chem. Phys. Lett. 89, I39 (1982); A. BAX, J. Magn. Reson. 53, 149 (1983); G. EICH, G. BODENHAUSEN, AND R. R. ERNST, J. Am. Chem. Sot. 104, 1304 (1982). 3. A. A. MAUDSLEY AND R. R. ERNST, Chem. Phys. Lett. 50, 368 (1977). 4. J. C. BELOEIL, C. LE COCQ, V. MICHON, AND J. Y. LALLEMAND, Tetrahedron 37, 1943 (198 1). 5. A. PARDI, R. WALKER, H. RAPOPORT, G. WIDER, AND K. WOTHRICH, J. Am. Chem. Sot. 105, 1652 (1983); P. P. LANKHORST, G. WILLE, J. VAN BOOM, C. ALTONA, AND C. A. G. MAASNOOT, Nucleic Acids Rex 9, 2839 (1983).
OF MAGNETIC
RESONANCE
56, 163-166 (1984)
Two-Dimensional Correlation Spectroscopy with Heteronuclear Relay M. A. DELSUC, Laboratoire
de Rbonance
E. GUITTET, MagnBique
N. TROTIN, AND J. Y. LALLEMAND Nucliaire,
CNRS-ICSN,
91190
Gif-sur-
Yvette,
France
ReceivedAugust 8, 1983 Several recent two-dimensional NMR experiments, particularly those using multiplequantum spectroscopy (1) and relay coherence transfer (RELAY) (2), have shown that it is possible to obtain much more specific information than by use of the classical one-dimensional approach. The second of these techniques takes advantage of spy nuclei to elucidate the coupling network and to establish resonance assignments and is based on a transfer of magnetization from one nucleus to another using an intermediate spin. Both types of relay experiments which have already been reported involve an initial homonuclear magnetization transfer (proton-proton) and differ only in that the coherence is relayed to a third spin of either the same (proton, homonuclear) or a different kind (carbon- 13, heteronuclear). We now describe a variation of these relay techniques in which the coherence is first transferred to a heteronuclear spin and is then relayed and detected on a third spin (proton in the present application). The need for such a sequence arose in the course of an oligonucleotide study where extensive overlap of phosphorus resonances precluded the application of classical approaches (such as heteronuclear shift correlated spectroscopy (3) or low-power SFORD (4)). In effect, the problem was to correlate groups of protons which share a common coupled partner but are not themselves coupled to one another (Fig. 1). The technique is based, as in the relays already reported, on the merging of two distinct coherence transfer processes and can be thought of as a ‘H-X heteronuclear shift correlation followed by a X-‘H coherence transfer which brings the information back into the proton spectrum. The resulting pulse sequence is set out in Fig. 2. The amplitude of the relayed cross-peaks can be shown to be proportional to the transfer coefficient: a = sin (7rJILTm) sin (7r&Tm)
ill and the time T,,, is chosen to maximise this transfer coefficient. The phase sequence is designed to eliminate undesirable signals. More elaborate phase sequences can be derived from the basic scheme shown in Table 1, in order to compensate for pulse or phase imperfections. The experiment is demonstrated with a spectrum of diethyl phosphite obtained on a Broker WM 400 equipped with an ASPECT 2000 computer. Minor modifications 163
0022-2364/84 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
164
COMMUNICATIONS J,,=O
FIG. 1. Spin connectivities as revealed by the HERPECS experiment. I and S are homonuclear spins, L is heteronuclear.
of the spectrometer had to be introduced to allow the phase-alternated 3’P pulses. At present the “P pulses are 300 psec, a value that could prove prohibitively long for many applications but should be capable of improvement by use of a booster pulse amplifier. A 128 X 1024 data matrix was acquired and 16 experiments were performed for each I, value. In the present case the heteronuclear coupling values are very different for the hydride (695 Hz) and for the methylene protons (9 Hz), hence a compromise had to be found for the proper choice for T,,, . The value of T, for the experiment of Fig. 3 is 55 msec which corresponds to a transfer coefficient of 0.65. No attempt has been made at present to optimize this value. The spectrum in Fig. 3 shows the correlation of the methylene and the hydride resonances through their common 31P partner. Additional correlations are present in the spectrum that actually correspond to either autocorrelation or residual COSY peaks.
FIG. 2. Pulse schemes of the heteronuclear shift correlation (a), heteronuclear coherence transfer (b), and HERPECS (c) experiments.
165
COMMUNICATIONS TABLE
1
BASIC PHASE SEQUENCE FOR HERPECS EXPERIMENT
Y Y Y Y
x
Y Y
-X -X
-Y -Y
X -x X
X
-X
To rule out any homonuclear correlation due to long-range homonuclear couplings, a blank experiment with the same experimental parameters but with the 31Ptransmitter turned off was performed that showed the absence of such correlations. We are currently applying this technique to oligonucleotides, where the individual
H-P I
CH,
CHz I
0
0 H-;/O-cHrCH, ‘O-CH,-CH,
FI
0
.
Q
0
5
ppm
F2
5
0
FIG. 3. The HERPECS spectrum of diethylphosphite obtained in 45 min from 500 mg of the compound in CDQ, with quadrature detection in t2 and pseudoquadrature detection in I,. The “P carrier is on resonance and the 180” refocusing pulses have been omitted.
166
COMMUNICATIONS
proton subspectra for each ribose are easily identified and isolated by correlated spectroscopy experiments (5). The position in the sequence would be best unraveled by establishing the mutual couplings of selected protons from different riboses to one single 3’P nucleus from the phosphorylated backbone. Water solvent, solubility restriction, and small couplings introduce additional difficulties. We consider that this technique should prove fruitful in elucidating coupling networks and establishing spin connectivities when the strong overlap of heteronuclear resonances hampers the use of more classical approaches. Although this technique should be prescribed in pathological situations, we propose to nickname this HEteronucleus Relayed Proton Correlated Spectroscopy as HERPECS. REFERENCES
1. G. BODENHALJSEN, Prog. NMR Spectrosc. 4, 137 (198 1). 2. P. H. BOLTON, J. Magn. Reson. 48, 336 (1982); P. H. BOLTON AND G. BODENHAUSEN, Chem. Phys. Lett. 89, I39 (1982); A. BAX, J. Magn. Reson. 53, 149 (1983); G. EICH, G. BODENHAUSEN, AND R. R. ERNST, J. Am. Chem. Sot. 104, 1304 (1982). 3. A. A. MAUDSLEY AND R. R. ERNST, Chem. Phys. Lett. 50, 368 (1977). 4. J. C. BELOEIL, C. LE COCQ, V. MICHON, AND J. Y. LALLEMAND, Tetrahedron 37, 1943 (198 1). 5. A. PARDI, R. WALKER, H. RAPOPORT, G. WIDER, AND K. WOTHRICH, J. Am. Chem. Sot. 105, 1652 (1983); P. P. LANKHORST, G. WILLE, J. VAN BOOM, C. ALTONA, AND C. A. G. MAASNOOT, Nucleic Acids Rex 9, 2839 (1983).