JOURNAL
OF MAGNETIC
99,42 f-425 ( 1992)
RESONANCE
Accurate Measurement of Homonuclear Coupling Constants Using JHH-TOCSY WIELAND
Institut
ftir
Organische
WILLKER
Chemie,
Received
March
AND
Universitiit
3 1,
DIETER
Bremen,
LEIBFRITZ
D-2800
Bremen
*
33, German)
1992; revised May 27, 1992
The homonuclear JHH coupling constant is one of the most important NMR parameters. In complex molecules with quite long relaxation times, E.COSY (1) in combination with the DISCO procedure (2) seems to be the best homonuclear method for quantifying J couplings. Other homonuclear methods are z-filtered COSY (3)) J-6 spectroscopy (4)) and selective excitation (5). These methods fail, however, in molecules with short spin-spin relaxation times where the linewidth is in the range of coupling constants. For these situations, some triple-resonance methods have been proposed recently (6-8), which can be used for 15N- and “Ccuenriched peptides and proteins. Their pulse sequences include a heteronuclear coherence transfer from “N to 13C. Additionally the magnetization transfer using a 13C spin lock works well for completely 13C-enriched molecules (9). In these spectra, the signal is split in Fi by the ‘JCH coupling constant, whereas the 3J(HN-H~) coupling constant can be extracted from the displacement in F2. From these methods, precisely estimated coupling constants are obtained, and they are, in distinction to E.COSY, independent of the linewidth. Until now no similar method has been proposed that could be used for molecules at natural abundance. For this purpose, we have developed two new pulse sequences shown in Figs. la and 1b, called JHH-TOCSY. Sequence 1a is a H,H correlation and sequence 1b is a H,X correlation. As for the methods mentioned above, these sequences provide the JHH splitting for XH groups only, whereas XH2 groups show no splitting. For overlapping XH and XH2 groups, and for spectral simplification, XH selection may be used as shown in Fig. 1c. Figure 1d shows the 30 extension of sequence 1a with and without XH selection. JHH-TOCSY is well suited to molecules at natural abundance or for 13C- or “N-monolabeled compounds. In comparison with the triple-resonance methods, the sequences are much shorter, because there is no need for the long 1 /( 2 JCN) delay. The evolution of the magnetization after 2, is as follows (only the relevant operators are shown); cf. Fig. la: * To whom correspondence should be addressed.
421
0022-2364192 $5.00 Copyright 0 1992 by Academic Press. Inc. All tights of reproduction in any form reserved.
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FIG. I. Pulse sequences for obtaining homonuclear coupling constants at natural abundance (JHHTOCSY ). (a) H,H-JHH-TOCSY, (b) H,X-JHH-TOCSY, (c) XH selection, (d) 3D version (without and with XH selection). Pulses marked with an asterisk (*) have their phases incremented by TPPI. Thick bars indicate 180” pulses. A = I /4J(X,H); BIRD pulses are used to suppress “C proton magnetization (10); p, = +x-x,p2 = +xtx -x-x,(oj = +y+y-y-y, cp(Rec.)= +x,-.x-x+x.
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The first part of the signal amplitude is in-phase in tI (cos rJIstl) and antiphase in t2 (sin rJ12t2), while the second part is antiphase in tI (sin rJIStl) and in-phase in t2 (cos ?rJ12t2). The superposition of these two amplitudes results in an antiphase structure of the signal. A schematic drawing of this superposition is given in Fig. 2a. For the H,C correlation (Fig. 1b) the amplitude is given by 12+os f&,cos n-J&,cos S2t2cos aJ,2t2 IzY sin &.t,sin 7rJlstl sin Q2t2sin rJIZtZ. Here the first part of the signal amplitude is in-phase in both dimensions (cos T Jlst 1, cos rJ12t2), while the second part is antiphase in both dimensions (sin rJIstl, sin rJ,2t2). The superposition gives an in-phase signal. This is shown in Fig. 2b. Instead of the 180” X pulse in the middle of the spin lock, one may use two 90” pulses alternately, one at the beginning (90; ) and one at the end (900,) of the MLEV cycle, which turns the S magnetization to z and back to x. If XH selection (Fig. lc) is used, the XH2 groups evolve in the second delay to
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FIG. 2. Schematic representation which shows the origin of the E.COSY-type patterns. (a) The pattern obtained with sequence 1a and (b) that obtained with sequence 1b.
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I,,12,S,, while the XH groups remain unchanged. The Ilx12& term is canceled by phase cycling (+x for the X pulse) and additionally by the trim pulse. Figure 3 shows the application of sequence 1a (with CH selection) to D-glucose. As can be seen from the rows, the two signal elements are antiphase to each other. If the relaxation times are long enough, an additional refocusing delay can be inserted prior to t, to obtain in-phase signals. Figure 4 compares a cross peak from the alkaloid angustifoline obtained with sequence la (Fig. 4a) with the same obtained with sequence lb (Fig. 4b). One disadvantage of the proposed sequences is that the coupling in CH2-CHZ elements is not accessible. Furthermore the two magnetization components after the spin lock could have different relaxation rates, which would lead to different intensities, resulting in incomplete signal elimination. For this reason, small artifacts can be seen in the rows of Fig. 3. We think that JHH-TOCSY is a useful method for measuring homonuclear coupling constants, including their sign. The possibility of obtaining a H,H and a H,C spectrum is useful for unraveling overlapping peaks in one of the spectra. The 3D extension of sequence la is especially suitable for large peptides and proteins.
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FIG. 3. Cross peaks of H 1 (cy and p) of D-ghcose obtained with sequence la (with C,H selection) The measurements were carried out on a Bruker AM 360 MHz spectrometer using 320 increments in I, with 64 scans per increment and a mixing time of 20 ms. The rows were zero-filled from 4K to 32K at a sweep width of 1000 Hz.
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0
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FIG. 4. Comparison ofthe cross peaks Hl I (2.88 ppm; “C, 56.8 ppm) with H14a (2.39 ppm) and H14b (2.23 ppm) of angustifoline. (a) Obtained with sequence la (with C,H selection) and (b) obtained with sequence 1b. The mixing time was 60 ms and the sweep width was 2500 Hz.
ACKNOWLEDGMENT We thank the Fonds der Chemischen Industrie for financial support. REFERENCES 1. C. GRIESINGER, 2. H. OSCHKINAT 3. H. OSCHKINAT,
0. W. SORENSEN, AND R. R. ERNST, J. Magn. Reson. 75,474 AND R. FREEMAN, .I Magn. Reson. 60, 164 ( 1984). A. PASTORE, P. PFICNDLER, AND G. BODENHAUSEN, J. Magn.
7. 8. 9. 10.
1987).
Reson.
(1976). 70, 106 ( 1986). G. T. MONTELIONE AND G. WAGNER, J. Am. Chem. Sot. 111,5414 ( 1989). 0. W. SORENSEN, J. Magn. Reson. 90,433 ( 1990). G. WAGNER, P. SCHMIEDER, AND V. THANABAL, J. Magn. Reson. 93,436 ( 199 1). G. GEMMECKER AND S. W. FESIK, J. Magn. Reson. 95,208 ( 199 1). A. BAX AND S. SLJBRAMANIAN, J. Magn. Reson. 67, 565 (1986).
4. W. P. AUE, J. KARHAN, AND R. R. ERNST, J. Chem. Phys. 64,4226 5. H. KESSLER, H. OSCHKINAT, AND C. GRIESINGER, J. Magn. Resort 6.
(
69,559
(
1986).