transfer spectroscopy

JOURNAL

OF MAGNETIC

RESONANCE

63, 225-229

(1985)

Refocusing and Suppressionof Neighbors in Heteronuclear Relay/Transfer Spectroscopy PHILIP H. BOLTON* Department

of Chemistry,

Wesleyan Received

University, February

Middletown,

Connecticut

06457

11, 1985

The utility of NMR has been considerably enhanced by the introduction of methods which allow the tracing out of molecular backbones. The determination of carbon-carbon connectivities via the double-quantum INADEQUATE experiment is straightforward though of low sensitivity since only those sites with adjacent carbon- 13 nuclei are detected (1-5). Heteronuclear relay-transfer spectroscopy utilizes the transfer of information from remote protons to neighbor protons to heteronucleus with the neighbor protons being directly coupled to the heteronucleus and the remote protons being only coupled to the neighbor protons (6, 7). The adjacent carbon sites can be identified since they share neighbor and remote protons (7). The relay-transfer approach also gives the assignments of the proton signals. The heteronuclear relay-transfer experiment utilizes all of the carbon- 13 sites and hence has much higher sensitivity than INADEQUATE. Since the original demonstration of heteronuclear relay-transfer spectroscopy a number of variants have been introduced. These include a simplified phase ‘cycle (8, 9), the use of DEPT, rather than INEPT, based proton-proton transfer (II), the use of a proton-heteronucleus-proton transfer scheme (IO), proton detection of the relay-transfer data (I 7), a low-pass J-filter technique to discriminate between neighbor and remote-proton signals (12) and the use of proton double-quantum coherence as the correlation frequency (13). These latter two methods address a basic problem of relay-transfer spectroscopy which is the discrimination between the signals arising from neighbor and remote protons. It is not uncommon for protons at adjacent sites to have degenerate or overlapping resonances. In such situations it can be difficult or impossible to discern the actual connectivity pattern. The low-pass filter approach discriminates against the neighbor signals on the basis of their large scalar coupling to the carbons. The use of proton double-quantum coherence allows unambiguous determination of the connectivities since the sums of neighbor- and remote-proton frequencies are obtained. While both of these methods are functional other approaches are worthy of consideration. An alternative method is to utilize semiselective refocusing (14-16) of the neighbor protons at the middle of the evolution time. Since the neighbor protons are * Alfred

P. Sloan

Fellow. 225

0022-2364185

$3.00

Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.

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refocused the precession of the remote protons will be effectively decoupled from the scalar coupling to the neighbor protons. This feature necessitates the addition of a delay time to allow the remote protons to become out-of-phase with respect to the neighbor-remote scalar coupling so that efficient transfer from remote to neighbor proton can be made. Refocusing of the neighbor protons will give rise to neighbor proton signals which correspond to the proton-decoupled heteronuclear J spectrum of these protons (15). These signals are of no interest in relay-transfer spectroscopy and can be eliminated by the simple expedient of phase cycling the semiselective refocusing so that all signals arising from refocusing are canceled out as previously described for the case of semiselective refocusing in heteronuclear chemical-shift-correlation spectroscopy (16). A pulse sequence which combines semiselective refocusing of the neighbor protons with relay-transfer spectroscopy is ‘H: ‘T:

-r,,,/2-900($,)-r”-DCPL

90”(~,)-f,/2-SSR-~,/2-~,/2-180~(+_y~r,/2-90~(x>-7,/2-r’/2-.180~(+y)-r’/2-180”-

-SSR-

-90”($,)-r”-ACQ

with SSR the pulse sequence element ‘H: 13c.

90°(~3)-7-180”(~,)-7-900(~5), -

180” -

The pulse sequence is analogous to those demonstrated previously for relay-transfer spectroscopy and incorporates consolidated refocusing (9) during the second mixing time. The phase cycling of the pulses is given in Table 1 and includes four steps for “quadrature” detection in both dimensions (9, ZO), four steps for the phase cycling of the semiselective refocusing (17), and two steps each of the two 180” pulses which are cycled +y to reduce artifacts due to rnissetting these pulses. Thus, the entire phase cycle is 64 steps. Before examining some experimental results it is of interest to consider just what signals are expected. For those remote protons which are at a terminus, that is only

TABLE

I

Phase Cycle for Pulse Sequence”

’ Every fourth step $J,, &I~,and & are all incremented by 90”. E&y 16th step the proton 180” pulse in the first mixing time is incremented 180” and every 32nd step the phase of the proton 180” in the second mixing time is incremented by 180”.

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coupled to neighbor protons, the signals will be singlets. For those interior remote protons which are coupled to protons in addition to the neighbor protons the multiplet form will arise solely from the coupling to the non-neighbor protons. Thus, the data from a semiselective refocusing relay-transfer experiment supplements that of a conventional relay experiment by clearly identifying the remote-proton signals as well as allowing identification of terminal and interior remote protons. Results on 1-propanol are shown in Fig. 1 in the contour map mode of display. The data shows that the use of semiselective refocusing eliminates the neighbor signals. The assignments of the carbon and proton signals from the data is simple with the H, proton being remote to only Cz, the H: proton is remote to both C1 and C,, and H3 is only remote to Cz. A satisfactory protocol for a previously uninvestigated sample could be based on acquiring only a proton-carbon-l 3 chemical-shift-correlation map in addition to a semiselective refocused relay-transfer map. The first of these experiments would give the neighbor signal and the latter the remote signals. To more closely examine the effect of neighbor refocusing, selected slices from the propanol data are shown in Fig. 2. The H3 signal associated with C2 appears as a relatively sharp singlet and the H2 signal associated with C3 appears as a broad line in the refocused data. The resolution of the data, about 6 Hz, is not sufficient to resolve the triplet multiplet of the Hz. The data does show a sharpening of the remote-proton signals when semiselective refocusing is used due to the reduction in the multiplicity of the remote protons. The method demonstrated here allows for discrimination between neighbor and remote protons in relay-transfer spectroscopy as well as reducing the multiplicity of

I

700

Droton,Fl

500

300

100

I

700 Hl

I

500

I

,

,

300 H2

I

t

100 H3

FIG. 1. The spectra shown are for I-propanol in *Hz0 at 22’ obtained using a Varian XL-200 and the pulse sequence described in the text. The contour map on the left was obtained using the pulse sequence without refocusing of the neighbor protons and the one on the right was obtained with use of refocusing of the neighbor protons. The delay ‘T, was 35 ms and the delay ‘Twas 3.6 ms. The delays T’ and 7” were 3.2 and 3 ms, respectively, and are the usual delays to allow the protons and carbon-13 nuclei to become out-of-phase with respect to the heteronuclear coupling. The decoupler phase was cycled (18) to remove any decoupler-induced artifacts. The experiment consisted of one phase cycle, 64 acquisitions, for each of 128 increments of the evolution time. The proton spectral width was 800 Hz and the carbon-13 spectral width was 3200 Hz. The data were pseudo-Gaussian shaped in both dimensions (19). The two sets of data were obtained under analogous conditions and are presented at the same intensity and the same contour heights.

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228

I\

c3

c2

L-i

‘II

350

200

50

350

200

50 HZ

FIG. 2. The spectra shown are slices taken from the data shown in Fig. 1. The spectra on top were obtained with refocusing of the neighbor protons and those on the bottom without. The spectra on the left are those associated with the C, site and those on the right with the Cz site. The intensities of the spectra obtained with and without refocusing are directly comparable as are the linewidths because the data were obtained on the same sample in subsequent experiments.

the remote protons. This reduces the demand on spectral resolution as well as allowing ready identification of the remote signals. The sensitivity of the experiment is somewhat decreased by the reliance on two, rather than one, proton mixing periods. This decrease is typically more than offset by the enhanced signal-to-noise due to the reduced multiplicity of the remote protons. For the example shown there is a 10-40s increase in signal-to-noise when semiselective refocusing is used. It is noted that neighbor refocusing can also be incorporated into experiments utilizing proton detection. ACKNOWLEDGMENTS I thank the Alfred P. Sloan Foundation for a fellowship and the National Science Foundation Grant PCM-83 14322 which provided partial support for this project.

for

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