Heteronuclear relay transfer spectroscopy with proton detection

Heteronuclear relay transfer spectroscopy with proton detection

JOURNAL OF MAGNETIC 62, 143-146 (1985) RESONANCE COMMUNICATIONS Heteronuclear Relay Transfer Spectroscopywith Proton Detection PHILIP H. BOLTON* D...

239KB Sizes 0 Downloads 80 Views

JOURNAL

OF MAGNETIC

62, 143-146 (1985)

RESONANCE

COMMUNICATIONS Heteronuclear Relay Transfer Spectroscopywith Proton Detection PHILIP H. BOLTON* Department of Chemistry, Wesleyan University, Middletown, Connecticut 0645 7 Received November 8, 1984

Investigations of the conformations and structures of macromolecules in solution by proton NMR spectroscopy are often hindered by the extensive overlap of signals. One approach to overcoming the difficulties associated with spectral overlap is to use proton two-dimensional spectroscopy which also has limitations (I). The limitations arise from extensive overlap in the two-dimensional maps, the problems in making unambiguous assignments, and the need for good resolution in both dimensions. In addition, even when the situation is favorable for data acquisition the interpretation of the data can be tedious and time-consuming. In an effort to overcome some of these problems with proton spectroscopy the use of heteronuclear probes has been proposed (2, 3). In this sort of experiment the proton spectral information is transferred to a heteronucleus whose signal is both resolved and assignable. This approach can allow the determination of information about those protons, the neighbors, which are directly coupled to the heteronucleus, and about the remote protons which are coupled to the neighbors (4, 5). If the heteronucleus is in a strategic location, this methodology can lead to detailed coupling-constant and chemical-shift information (6). One limitation on the methods proposed to date using heteronuclear probes is that the sensitivity is rather poor. The use of proton observation can increase the sensitivity by a factor of (y proton/~heteronucleus)2 (I) and hence make the millimolar concentration range quite accessible. To combine the advantageous features of proton detection and relay transfer spectroscopy a new set of experiments has been devised. To obtain information via proton detection which is essentially equivalent to that obtained with heteronuclear detection, pulse sequence 1 is used: ‘H: 90°(~,)-~/2-180”(&)-~/2-t,/2-180’(&)-t,/2-t,-90”(~4)-acquisition IQ

-~~O’(C#Q)&:XY-x-y;

-9O’(+,)-

-90”(h)-

$*:XY-x-y;

lp,: XxXx;

64: xxxx

Every fourth step @2is incremented by 90” and 4s by 180”. Every sixteenth step & and 42 are incremented by 90”. Every sixty-fourth step & is incremented by 90”. The time 7 is set equal to 1/4J(CH) (3.125 ms for glucose) and tm is set according to 3JHH (32 ms). The acquisition phase is X. * Alfred P. Sloan Foundation Fellow. 143

0022-2364185 $3.00 Copyright Q 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.

144

COMMUNICATIONS

The first part of the pulse sequence is a heteronuclear zero-quantum chemicalshift correlation (7, 8). This procedure is followed by a mixing time to allow the proton magnetizations to end up out of phase with respect to both homonuclear and heteronuclear couplings (4, 5). The net result of this experiment is that signals will be obtained whose F2 frequencies are those of the remote and neighbor protons and whose F, frequency is that of the heteronucleus. This is an interchange of the frequency axes from heteronuclear detected relay transfer spectroscopy. The contour map in Fig. 1 was obtained using this pulse sequence with a sample of mutarotated glucose, anomers I and II, which contained 90% 13C at position 1. HOC H2

HOCH2

H!&$H

H:@ OH I

II

The contour map clearly gives the chemical shifts of the HI and H2 protons of both anomers. Figure 1 also compares the normal proton spectrum of the sample with absolute-value projections of the data from the contour map. The projections

1

I 6

I 5

4

PPM

FIG. 1. The contour map shown was obtained using a sample of mutarotated glucose, I and II, in *Hz0 at 4O’C using a Varian XL-200. The sample was 120 m&f in glucose in a 10 mm sample tube. The pulse sequence used is described in the text. The Fi frequencies, in hertz, are those of the 13Cnuclei and the F2 frequencies, in hertz, are those of the neighbor and remote protons. The data were collected using a 0.9 s acquisition time and a spectral width of 600 Hz with zero filling of the data to 2048 points. The experiment consisted of 64 increments of C, and a spectral width of 600 Hz in F, and the data were zero filled to 128 points. Pseudo-Gaussian line shaping was applied in both dimensions (10). The onedimensional conventional proton spectrum of glucose is shown (top) for comparison with absolute-value projections onto F2 of the same data shown in the contour map. In addition to the signals from H, and Hz a small signal from H3 is observed in the projection of the (Y anomer data which occurs due to the moderate coupling of the H2 and H3 protons.

145

COMMUNICATIONS

demonstrate the resolution of this approach which is primarily governed by the FZ acquisition time rather than FI . The heteronuclear couplings have not been suppressed from the proton dimension. In general, the presence of ‘JCH is an advantage with one of the 13C satellites being in the weak coupling limit (9). The heteronuclear coupling can be removed by including an additional delay time and applying a decoupling field to the 13C during proton acquisition. A variation on proton detection of relay transfer can be useful. Pulse sequence 2 is ‘H:

90’(&)-7/2-180’(,#+/2-

13c.

-180”(&)-

-tl-900(~4)-acquisition -90”(9,k90”(~3)-

The phase cycle and delay time are the same as for pulse sequence 1. This pulse sequence begins with a heteronuclear zero-quantum filter to selectively generate proton transverse magnetization. This is followed by an evolution time which ends with a proton pulse to transfer magnetization from neighbor to remote protons. This experiment is similar to the familiar 90”-ti-90”-acquisition approach used for proton chemical-shift-correlation spectroscopy (1) except that the zeroquantum filter is used in place of the first 90” pulse. The diagonal signals in the two-dimensional map will be from the neighbor protons and off-diagonal signals will have the F2 frequencies of the remote protons and the F, frequencies of the neighbor protons. A contour map obtained for the mutarotated glucose sample is shown in Fig. 2 along with absolute-value projections onto F2 of the same data. While there is less information present in this variant (the 13C chemical shifts are not present), there is a greater dispersion of the signals. The advantages of these experiments for utilizing heteronuclear probes demon-

H

FIG. 2. The contour map is of the same sample and conditions as described in Fig. 1. The pulse sequence used correlates the chemical shifts of neighbor and remote protons. The onedimensional spectra shown are the projections onto F2 of the data in the contour map.

146

COMMUNICATIONS

strated here are high sensitivity and high resolution. The sensitivity of the experiments is one-half that of conventional proton chemical-shift-correlation experiment and the resolution is essentially limited by the F2 acquisition time. These experiments make the investigation of millimolar, or lower, concentration solutions of 15N- or 13C-labeled molecules feasible. These experiments can also be applied to samples with only the natural abundance of “N or 13C but only the first version would be useful in such an application. When the heteronuclear coupling is comparable in magnitude to the proton-proton couplings the net efficiency of the experiments can be low (7). For those samples with only a few, isolated heteronuclei a onedimensional version of proton detected relay transfer can be used.’ Utilization of selective excitation of the neighbor protons could be adapted to allow the Overhauser effect to be the proton-proton transfer connection. ACKNOWLEDGMENTS This research was supported, in part, by Grant PCM-8314322 from the National Science Foundation and a fellowship from the Alfred P. Sloan Foundation. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il.

R. BENN AND H. GUNTHER, Angew. Chem. Int. Ed. Engl. 22, 350 (1983) is a recent review. G. BODENHAUSENAND R. FREEMAN, J. Magn. Reson. 28,471 (1977). A. A. MAUDSLEY AND R. R. ERNST, Chem. Phys. Lett. 50, 368 (1977). P.H. BOLTONANDG.BODENHAUSEN, Chem.Phys. Lett.84, 139(1982). P. H. BOLTON, J. Magn. Reson. 48, 336 (1982). P. H. BOLTON, “Biological Magnetic Resonance” (L. J. Berliner and J. Ruben, Eds.), Vol. VI, pp. l22, Plenum, New York, 1984. P. H. BOLTON, J. Mugn. Reson. 57, 427 (1984). A. BAX, R. H. GRIFFEY, AND B. L. HAWKINS, J. Magn. Reson. 55, 301 (1983). P. H. BOLTON, J. Mugn. Reson. 51, 134 (1983). A. BAX, R. FREEMAN, AND G. A. MORRIS, J. Magn. Reson. 43, 333 (1981). P. H. BOLTON, J. Am. Chem. Sot. 106,4299 (1984).

’ A one-dimensional experiment uses the first pulse sequence with t, = 0. This allows detection of ail neighbor and remote protons in an analogous fashion to a recently proposed homonuclear experiment (II) and is suitable for those samples not requiring the resolution of a two-dimensional experiment. The data will be. difficult to interpret if a proton is remote to more than one heteronucleus.