Strong coupling effects and their suppression in two-dimensional heteronuclear NOE experiments

JOURNAL

OF MAGNETIC

RESONANCE

74,397-405

(1987)

Strong Coupling Effects and Their Suppressionin Two-Dimensional Heteronuclear NOE Experiments KATALIN

E. K~v~R*

AND GY. BATTA~

*Biogal Pharmaceutical Works, H-4032 Debrecen. Hungary, and TDepatiment of Organic Chemistry, L. Kossuth University, H-4010 Debrecen, Hungary Received September 22, 1986; revised March 16, 1987 Strong coupling effectsin two-dimensional heteronuclear NOE spectroscopy are analyzed experimentally. A simple method is proposed for suppression of spurious “ghost” peaks resulting from second-order effects. The disadvantage of this method is that it decreases the sensitivity. 0 1987 Academic PISS, Inc.

Two-dimensional heteronuclear NOE spectroscopy is promising in studies of heteronuclear dipole-dipole interactions and chemical exchange processes of biological macromolecules (1-7). Cross peaks occurring in 2D heteronuclear (‘H, i3C) NOE spectra can provide unique connectivities between quatemary carbons and their nearest neighbor protons, rendering the assignment of quatemary carbons unambiguous. Furthermore, the well-known distance (rnc) dependence of the intensity of cross peaks between isolated pairs of nuclei allows determination of internuclear distances (6). However, in these earlier reports (1-7) the effect of strongly coupled spin systems is neglected. In this paper we demonstrate that strong coupling effects may lead to considerable confusion in evaluation of 2D heteronuclear NOE experiments due to the appearance of spurious ghost peaks in 2D NOE spectra. Moreover, distortions in cross-peak intensities may complicate the interpretation of peak intensities in terms of molecular structure and render the first-order approach in evaluation of proton-carbon distances useless. Furthermore, a new and simple procedure is proposed for suppression of peaks resulting from strong coupling. This method does not require a special, sophisticated spectrometer. We have examined these second-order effects in the case of oligosaccharides which often suffer from strong J couplings in their 13C satellite spectra. It has been found that such effects are not unique in the 2D heteronuclear NOE spectroscopy of oligosaccharides. The simplest spin system showing strong couplings between protons and carbon is an ABX three spin system (A, B = ‘H; X = 13C) in which JAB # 0 and the chemical397

0022-2364187 $3.00 Copyri%lt Q 1987 by Academic FVes, Inc. AU tights of repmdwtion in any form resewed

398

KiiVfiR T/2(PHl)

‘H

( 4

13c Dl/

AND BATTA

Wp~3) t1

1 z mix I

BB

TI(PH2) ;

I

FIG. 1. Pulse sequence for the 13C-{‘H} heteronuclear 2D NOE experiment. The phases of the pulses and the receiver are cycled to obtain the pure absorption phase in four quadrants (4). PHI xy-x-y-X-yxy PH2 y PH3 0 Y --x -Y), (Y --x -Y x)2 PH4 x PH5 x4 -x4 y4 -y4 with CYCLOPS rotation

shift difference between the protons A and B, Au AB, is nearly equal to half of the scalar coupling constant, JAx (Eq. [ 11) AvAe = 1JAx . [II If Eq. [ l] is valid for a spin system then one of the satellite signals and the transitions of the proton on the adjacent carbon are nearly degenerate, and consequently the zero-order wavefunctions of protons A and B are mixed. Therefore the effect of the carbon 180” pulse in the middle of the evolution time t, in the pulse sequence (Fig. 1) will be to mix the A and B magnetizations similarly to the. heteronuclear chemical-shift correlation experiments (8, 9). As a consequence, besides the one-bond cross peaks, there are other intense positive peaks in the 2D heteronuclear NOE spectrum. This means that the magnetization has been transferred not only from the protons directly bonded to the carbons but also from the protons strongly coupled to the directly bonded protons. Theoretical analysis of such effects requires a density matrix approach which will not be presented here. However, an experimental demonstration of these second-order effects is provided by the examples of suitable model compounds methyL2,3,4,6tetraO-acetyl-cr-Dglucopyranosid (1) (Fig. 2) and &i-Me-&CD (2) (Fig. 3). Figure 4 shows selected traces of the twodimensional pure absorption phase heteronuclear NOE (4) spectrum of 1 in DMSO-& (Bruker WP 200 SY, 7,ix = 1 s). As is expected, cross peaks appear between all directly bound proton-carbon pairs of nuclei (H 1-C 1, H2-C2, H3-C3, H4-C4, and H5-C5) due to the heteronuclear dipolar coupling. However, there are intense signals from protons adjacent to the site from which signal is expected, too. For example, the row of carbon C2 contains a signal at the chemical shift of H3, the spectrum of carbon C3 shows peaks at protons H2 as AcO,

Fk. 2. Methyl-2,3,4,6-tetra-O-acetyl+Bglucopyranosid

(1).

STRONG COUPLING

IN 2D NOE EXPERIMENTS

399

Me0

RG. 3. Tri-Me-&CD

(2) heptakis-2,3,6-tri-0-methyl-cyclomaltohexaose.

well as H4, and the row of carbon C4 contains an extra peak at H3. It can be observed that these peaks are symmetrically positioned (e.g., H3-C4, H4-C3). To decide whether these signals originate from heteronuclear dipolar interactions, we have performed some model calculations using the known standard heteronuclear distances for sugar skeleton. These calculations show that cross peaks of comparable intensities with cross peaks due to one-bond interactions cannot result from intraunit dipolar interactions because of the geometrical restrictions. However, the chemicalshift differences for H3-H4 and H2-H3 pairs of proton nuclei satisfy the corresponding equalities given by Eq. [I] supporting our hypothesis that these extra signals are due to strong-coupling effects. To give a further verification of the presence of strong coupling in the spin system of 1 we repeated the experiment using 7,,,ix = 0. In this case only carbons involved in an ABX spin system can be observed in the corresponding 2D spectrum (Fig. 5).

C-5

5.5

5.0

4.5

4.0

3.5 pm

l3c. 4. Selected traces of the phase-sensitive 2D heteronuclear NOE spectrum of 1 obtained in an overnight experiment (about 200 mg 1 dissolved in 1.5 ml DMSO-& T = 3 10 K). A waiting time of 2 s was allowed between each pulse sequence. The 2D map is composed of 32 X 4K data points. The chemical-shift differences: A$yHy = 7 1Hz; A&‘“y = 94 Hz.

KijVeR AND BATTA

400

5.5

5.0

4.5

LO

3.5

ppm

FIG.5. Crosssectionsof the 2D heteronuclearmap of 1 obtained with T,ix = 0. (Further experimental parametersarethe sameasbefore.)

Carbons Cl and C5 of 1 will not give observable signals because incoherent magnetization transfer between proton and carbon nuclei is precluded when 7mix = 0. The anomalous phase behavior of signals can be explained by the insertion of an extra

c-5

C-4 c-3

c-2 C-l

5.5

5.0

4.5

4.0

3.5

wm

FIG.6. Selectedtracesof the phase-sensitive 2D heteronuclearNOE spectrumof 1 obtained on a Bruker Wh4 250. The chemical-shiftdifferences:AvH3,H4 = 88 Hz; AYIQ~,= 117Hz.

STRONG

COUPLING

IN 2D NOE EXPERIMENTS

H-5

H-l I 50

I 4.0

H-2

H-4 H-3

1 3.0 ppm

FIG. 7. Cross sections of the 2D heteronuclear NOE spectrum of 2 (500 mg 2 dissolved in 1.5 ml CDC& , T = 3 10 K). A waiting time of 2 s was allowed between each pulse sequence. A fixed mixing time of 0.5 s was used before the final 13Cpulse.

907-

H5jC5

6543-

HJ/CC

FIG. 8. The changes of the cross-peak intensities due to one-bond ‘H/13C dipole-dipole (H5/C5) and strong-coupling effect (H3/C4) in 1 as a function of proton pulse length ((Y).

interaction

402

KijVfiR

AND BATTA

5.0

4.5

H3

5.5

4.0

Pm nc. 9. Selected traces of the phase-sensitive 2D heteronuclear NOE spectrum of 1 obtained with the modified pulse sequence. A carbon 180” pulse has been inserted in the middle of the mixing period. A mixing time of 0.66 s was used and a waiting time of 2 s was applied between each pulse sequence. The negative cross peaks result from strong coupling.

delay, about 5 ms, after the read carbon 90” pulse to rephase the carbon magnetization components for broadband decoupling. Figure 6 shows the selected rows of 2D ( ‘H-13C) NOE spectrum of 1 obtained at a higher magnetic field (Bruker WM 250). It has been found that in the rows of carbon C2 and C3 the spurious peaks H2-C3 and H3-C2 disappear, demonstrating that one can avoid the second-order effects by increasing the strength of the magnetic field. Of course, it may happen for a particular group of spins that even the higher field causes Eq. [l] to be satisfied. A more direct way of circumventing this problem might be preferable; e.g., the decoupling of 13C from protons during tl can reduce these effects, as has already been suggested in heteronuclear correlation spectroscopy (8,9). However, using this approach one should be careful, as 13C decoupling can eliminate strong coupling in the satellite spectra, but it certainly does not eliminate second-order effects in the parent proton spectrum. The main inconvenience with these procedures is that the necessary facilities are available only in a minority of NMR laboratories. For this reason, a simple method

STRONG

, 55

COUPLING

IN 2D NOE EXPERIMENTS

1

1

50

4.5

Go

403

Ppm

10. Selected cross Sections of 2D ‘kZ-{ ‘H} NOE spectrum of 1 obtained under the same experimental conditions as in the previous experiment except that proton 60” pulse simultaneously is applied with the carbon inversion pulse during the mixing period. FIG.

which can be carried out on the majority of spectrometers would be desirable. A method which results in the suppression of ghost peaks would be especially important in those cases when the intensities of NOE and ghost peaks are nearly identical. This situation has been found in the case of &i-Me-&CD (2). Figure 7 shows the selected traces of 2D heteronuclear NOE spectrum of 2. It can be observed that the intensities of cross peaks in the row of carbon C3 are nearly the same. There is no simple way to decide which one is the NOE and which is the ghost peak using the conventional pulse sequence. Application of nonselective pulses during the mixing period in homonuclear 2D NOE experiment is straightforward for J-peak suppression (ID). In the case of a heteronuclear experiment the heteronuclear spin system provides new experimental possibilities for spin manipulations; e.g., it makes possible asymmetric spin preparation. We have studied the effect of nonselective proton and carbon pulses inserted in the middle of the mixing period on the intensities of NOE and ghost peaks. A series of

404

K&‘fiR

AND BATTA

C-l 1

I

50

4.0

I

30 ppm

FIG. 11. Cross sections of 2D heteronuclear NOE spectrum of 2. Ghost peaks are eliminated using the modified pulse sequence with (Y = 60”. A mixing time of 0.17 s was applied for magnetization transfer.

experiments have been carried out where the length of the proton pulse, applied simultaneously with the carbon inversion pulse, has been incremented by 30” from 0’ to 180’ in each experiment. Figure 8 illustrates the intensity changes of direct (H5-C5) and ghost (H3-C4) peaks of 1 as a function of proton pulse length (a). If (Y is in the range 0” to 60”, signals resulting from NOE and strong-coupling effects are opposite to each other in the phase-sensitive representation. By increasing the proton

FIG. 12. Pulse sequence of heteronuclear 2D NOE-experiment involved in the middle of the evolution period tl .

where the bilinear rotation pulses are

STRONG

COUPLING

IN 2D NOE EXPERIMENTS

405

pulse length an intensity enhancement of the direct NOE peak (HSC5) can be observed while ghost peaks show a phase alternation with a zero-crossing at (Y = 60’. In the range of 60” to 180” for (Y,all signals have the same phase, and consequently there is no simple way to distinguish them. Figure 9 shows the selected rows of 1 when carbon inversion pulse has been applied only in the middle of the mixing period. NOE and ghost peaks can be easily distinguished on their phase behavior. Figure 10 demonstrates suppression of ghost peaks when (Y = 60”. The efficiency of our procedure is also illustrated for tri-Me-P-CD (2) (Fig. 11). From the above experimental results it is clear that strong coupling in the satellite spectrum is capable of producing intensive spurious cross peaks in 2D heteronuclear NOE spectra as well. If such signals are misinterpreted it may lead to considerable confusion in evaluation of these experiments. However, our simple procedure proposed for suppression of these additional signals may be an effective solution to this problem. The main advantage of our method is the simplicity. It should be mentioned that the only disadvantage of our procedure is that it decreases the sensitivity by reducing the intensity of direct peaks (Fig. 8). However, the effective suppression of ghost peaks compensates for the loss in sensitivity. Furthermore, the unambiguous differentiation of NOE and ghost peaks enables the use of this experiment for assignment of carbon signals. These ghost peaks give the same information as the relay peaks in the 2D heteronuclear relay experiment (II). As an alternative suppression of ghost peaks, we have studied the effect of the bilinear rotation pulses (12-14) involved in the basic pulse sequence in the middle of the evolution period (Fig. 12). We have found that the pulse sequence eliminates the spurious signals arising from strong couplings. Direct (one-bond) NOE peaks, where the corresponding spins are the members of a strongly coupled spin system, show additional splittings, as has already been observed in heteronuclear chemical-shift correlation experiments (15). However, when using this method one should be careful because of the appearance of additional and sometimes intense, misleading signals. REFERENCES RINALDI, J. Am. Chem. Sot. 105,5167 (1983). AND G. C. LEW, J. Am. Chem. Sot. 105,6694 (1983). AND G. C. LEW, J. Am. Chem. Sot. 106,6533 (1984). KC)VBR AND GY. BATTA, J. Magn. Reson. 69, 5 19 (1986). KOVBR AND GY. BATTA, J. Magn. Reson. 69,344 (1986). KC)&R, GY. BAJA, AND Z. MADI, J. Mugn. Reson. 69,538 (1986). K0ti~ AND GY. BATTA, J. Chem. Sot. Chem. Commun., 647 1 (1986). 8. P. H. BOLTON, J. Magn. Reson. 51, 134 (1983). 9. G. A. MORRIS AND K. J. SMITH, J. Magn. Reson. 65,506 (1985). 10. S. MACURA, K. WOTHRICH, AND R. R. ERNST, J. Magn. Reson. 47,351 (1982). Il. P. H. B~LTON AND G. BODENHAUSEN, Chem. Phys. Lett. 89, 139 (1982). 12. J. R. GARBOW, D. P. WEITEKAMP, AND A. PINES, Chem. Phys. Lett. 93, 504 (1982). 13. A. BAX, J. Magn. Reson. 52,330 (1983). 14. V. RUTAR, J. Am. Chem. Sot. 105,4095 (1983). 15. V. RUTAR, T. C. WONG, AND W. Guo, J. Magn. Reson. 64,8 (1985). 1. 2. 3. 4. 5. 6. 7.

P. C. C. K. K. K. K.

L. Yu Yu E. E. E. E.