MAGNETIC RESONANCE IN CHEMISTRY, VOL. 30, 662-665 (1992)

High-Resolution Proton-Detected 2D HEHAHA Correlation Experiments with Spin-Echo Discrimination of Long-Range Correlations

Andrew Gibbs and Gareth A. Mor&* Department of Chemistry, University of Manchester, Oxford Road, Manchester M13 9PL, UK

Recent work has shown that broadband heteronuclear HartmanwHahn coherence transfer is an efficient alterna- tive to pulsed coherence transfer methods. A new technique for heteronuclear long-range correlation between ' 3C and protons, making use of proton detection and an extended mixing period, is described, which can have sensitivity and resoiution advantages over current techniques. Efficient regiospecific correlation is shown in a model system, menthol, and the use of a modulated spin-echo for the simultaneous determination of one-bond and long-range correlations is demonstrated.

KEY WORIX HEHAHA HOHAHA TOCSY Long-range correlation 2D NMR Structure elucidation

The possibility of performing a heteronuclear Hartmann-Hahn experiment (HEHAHA or hetero- TOCSY) for isotropic liquids has recently been demon- strated by a number of g r~ups . ' -~ Hartmann-Hahn transfer has the advantage of yielding in-phase magne- tization, and allows easy production of pure-phase absorption mode spectra, with a corresponding increase in signal-to-noise ratio and/or resolution compared with the more usual SQ/MQ e x p e r i m e n t ~ ~ . ~ where anti- phase magnetization transfer is used. In conjunction with proton-detection methods, 2D spectra with high resolution in the proton (fi) domain can thus be obtained, showing undistorted 'H-'H multiplet struc- ture on cross-peaks. This paper describes one particular application of proton-detected HEHAHA to long-range optimized 2D correlation experiments; it includes the use of a spin+cho pulse unit to provide selective inver- sion of the long-range cross-peaks, allowing discrimi- nation between one-bond and long-range signals in a single experiment. Such 'broadband' correlation experi- ments, involving the sharing of coherence amongst whole spin systems, are particulariy valuable in the structure elucidation of total unknowns, since they allow the regioselective identification of elements in a complex structure by identifying ' 3C signals with common patterns of proton coupling relationships.

Heteronuclear coherence transfer to distant nuclei may be achieved simply by lengthening the duration of the HEHAHA mixing p e r i ~ d , ~ allowing evolution via t he small long-range couplings, "J(C,H). An advantage of long-range correlation using HEHAHA compared with normal methods such as HMBC is the potential for transfer to occur through sequences of couplings, showing the linkages amongst members of extended spin systems, in a manner analogous to that of the TOCSY or HOHAHA experiment."*' ' Additionally, the HEHAHA experiment can also act as a HOHAHA experiment, allowing transfer via homonuclear coup-

* Author lo whom correspondence should be addressed

lings. For long-range experiments this opens up further correlation pathways, as in addition to direct C-H transfer via "J(C,H), multistep transfer is now posible via an initial one-bond heteronuclear transfer 'J(C,H) followed by one or more homonuclear transfers via "J(H,H). Such 'indirect' correlation pathways may often be favoured over the direct coupling route, as the initial step is very fast (occurring via the large one-bond coupling), and while the homonuclear scalar couplings are of the same order of magnitude as long-range het- eronuclear couplings, transfer occurs at twice the rate (on a timescale of l/J(C,H) for heteronuclear transfer, and 1/[2J(H,H)] for homonuclear transfer). The poten- tia1 for some long-range correiations to be formed very quickly (via hetero-hornonuclear transfer) allows long- range information to be obtained even with relatively short mixing times, important for samples with rather short T,s.

Long-range correlation spectra obtained using HEHAHA transfer thus typically show a wide range of correlations. As with existing techniques, it is desirable to distinguish between correlations arising from one- bond transfer and those from long-range transfer. This is typically achieved using a J-ñlter" to suppress one- bond signals in long-range correlation experiments. This method could be used with HEHAHA (requiring the phase-sensitive version of the J-filter), but the alter- native method proposed here is the use of the spin-echo unit -z-l8O(I,S)-z- [where z = 1/2'J(C,H)] to label long-range signals by selective inversion, as for example used recently in the proton relayed HSQC experi- ment.I3 This allows both one-bond and long-range spectra to be obtained in a single experiment.

The spin-echo HEHAHA experiment was imple- mented using a siightly modified Varian XL-300 spec- trometer; the pulse sequence is shown in Fig. 1 . Machine code reprogramming of the acquisition pro- cessor was needed for the WALTZ modulated HEHAHA transfer step, but this would not be neces- sary on a more modern instrument. Initial enhancement of carbon magnetization was by the nuclear Overhauser

0749-1 58 1/92/070662-O4 $07.00 Q 1992 by John Wiley & Sons, Ud .

Received 7 February 1992 Accepted 12 March 1992

PROTON-DETECTED 2D HEHAHA CORRELATION 663

1 - 1owpower - 180

@2

* - - * t c)c)

Di ti I m , , T T

Figure 1. Pulse sequence for spin-echo long-range ‘H detected HEHAHA I,,, is approximately l/”J(C,H), c = O S/’J(C,H) Phase cycling is shown in Table 1 The long x and y pulses were of 1 O and 15 ms, respectively

effect rather than polarization transfer, as for the 13C-’H system used a similar enhancement is obtained (a maximum factor of 3 rather than 4 Sor INEPT, DEPT or HEHAHA), along with easy suppression of unwanted proton magnetization at the start of the experiment. This greatly reduces the problems of stabil- ity and dynamic range associated with inverse mode experiments, a factor especially significant on older spectrometers. WALTZ mixing was used for the

Table 1. Phase cycling details for the sequence in Fig. 1

@, 0,2, + O, 1 (for hypercomplex 2D)

$2 0 @3 3 @, 0123 @, 0123+0,2,

Hartmann-Hahn transfer (other modulation sequences are posible*), following which a z-filterI5 allowed sup- pression of any out-of-phase coherence. The spin-echo unit was implemented using 90,-240,-90, composite pulses to minimize resonance offset effects. Broadband 13C decoupling could be used during acquisition if desired, but was not used here; thus splittings inf2 by J(C,H) remain, in addition to those by J(H,H).

Figure 2 shows a 2D HEHAHA correlation spectrum of a sample of menthol (1) in deuteriochloroform, using a mixing time of 40 ms to obtain long-range corre- lations (no spin-echo used). This shows the good proton resolution and low t , noise obtainable using this tech- nique. The extent of correlation is almost complete, al1

* The choice of modulation cequence is discussed in detail in a recent paper by Ernst et al.’4

I 1 I I

4 0 3 0 2 0 1 0 ’H PPm f2

Figure 2. Long-range HEHAHA spectrum of menthol obtained using the sequence of Fig 1 without the spin-echo unit, and with WALTZ mixing of 40 ms This spectrurn shows the low leve1 of r , noise, the high proton resolution and the pure phase obtainable using this techniaue

664 A. GIBBS AND G. A. MORRIS

Figure 3. Carbon ( f , ) trace along f2=3.4 ppm, showing the long-range couplings of ring carbons to proton H-1 of menthol. No decoupling was used during t , , resulting in splitting byJ(C,H); carbons C-2, C-6 and C-5 al1 show large one-bond Couplings (the long-range coupiings on C-4 and C-3 are not resolved), showing that these cross-peaks arose by the combined heterehornonuclear pathway.

ring proton signals showing correlations to al1 ring carbons and so forth. Note the different multiplet pat- terns seen for different long-range correlations (e.g. to H-1 at 3.4 ppm in f2); each multiplet consists of the normal proton multiplet split further by the appropriate long-range proton-carbon coupling. This allows the easy and specific determination of long-range proton- 13C coupling constants. The importance of the hetero- homo transfer route may be confirmed by comparing the observed heteronuclear coupling of signals before mixing (i.e. infi when no t , decoupling is used) to the final coupling in fi (with no t, decoupling). Figure 3 shows a n j i trace extracted from such a 2D experiment, showing five long-range correlations (the one-bond cross-peak is split inf, either side of this trace). Three of these cross-peaks show a large me-bond coupling (the much smaller long-range coupling is not resolved on the other two signals), and hence were transferred by the 'J(C,H)-"J(H,H) pathway.

1 1 I 1

4 3 2 '2 ' ' H PPm

Figure 5. Proton ( f,) traces showing correlations to carbon C-6 of menthol, obtained (A) without spin-echo (extracted from Fig. 2) and ( 6 ) with spin-echo (extracted from Fig. 4). showing inver- sion of long-range signals.

OH

8 CH3

9 CH3

1

The use of a spin-echo to invert selectively long- range signals is shown in the 2D contour plot of Fig. 4.

"1 fl 401

..<

aaJ¿c Y 4Bbs

6o 1 70 a w .. ' *'

1 l 1 I I 1 l , 4.0 3.5 3.0 2 5 2.0 1.5 1 .o 0.5

f2 1~ ppm

Figure 4. Long-range HEHAHA spectrum of menthol using the spin-echo rnodification (full sequence of Fig. 1) with WALTZ mixing of 40 ms and 5 = 4 ms. Negative peaks (long-range correlations) are shown as filled contourc. positive peaks (one-bond correiations) are shown as open contours; selective inversion has been achieved with minimum degradation of phase purity.

PROTON-DETECTED 2D H E H A H A CORRELATION 66 5

Positive contours (one-bond correlations) are shown open and negative contours (long-range correlations) are shown filled; here the residual J(C,H) spiitting inJ; confirms the selectivity of the method. Further detail is provided in Fig. 5, showing proton traces from C-6 of menthol acquired with and without the spin-echo iabel- ling pulse unit.

In conclusion, this technique illustrates one extension of the basic HEHAHA sequence which may prove useful in structure determination owing to the wide range of correlation information obtainable and the provision to observe directly the nature of cross-peaks without measuring separate one-bond and long-range spectra. The use of a spin-cho with very complex spectra increases the possibility of signal Ioss due to cancellation from overlapping one-bond and long-range

signals, and J-filter methods may be preferable. The good proton resoiution provides an aid to assignment and also offers the potential for measuring long-range coupling constants directly from the spectrum [this would obviously require no t , decoupling, and also the use of a J-filter before the mixing period to ensure that only direct 'J(C,H) transfer occurs.]

Acknowledgements

The XL-300 spectrorneter used in this work was purchased with the aid of grants from the Science and Engineering Research Council, the University of Manchester and the University of Manchester lnstitute of Science and Technology. A. G. thanks Glaxo Group Research, Greenford, for generous financia] support during the course of this work.

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