JOURNAL

OF MAGNETIC

RESONANCE

84,648-653

1989)

SimultaneousTransfer of Saturation and Overhauser Enhancementin Difference NMR Spectroscopy

DANIEL KOST
Department of Chemistry, Ben Gurion University of the Negev, Beer Stieva 84120, Israel

Received February2, 1989;revised May 16, 1989

Double-resonance experiments have long been known and used in various methods (I). Saturation transfer and nuclear Overhauser enhancement are both well developed and documented methods for the observation of exchange phenomena, in one and two dimensions (2). NOE has been used extensively for the study of conformations of molecules in solution, while the use of saturation transfer experiments has not been as widespread (3). In 1D spectroscopy, both methods benefit from using difference measurements to enhance sensitivity and resolution.

1
SCHEME 1

In the course of a study, to be published elsewhere, on the structure and dynamic stereochemistry of a bicyclic compound ( I ) , we utilized difference NOE spectroscopy in order to assign exchanging spectral lines. The present paper reports what appears to be the first observation of signals which are due simultaneously to two different transfer mechanisms: NOE transfer by chemical exchange, and indirect NOE, generated by a nucleus which has been saturated by saturation transfer through exchange. Compound 1 is a bicyclic molecule composed of a nine-membered ring bridged by means of a two-carbon bridge to form a six-membered ring. A molecular model shows that the nine-membered ring can be inverted such that the six-membered ring is moved from the vicinity of one face of the aromatic plane to the other (Scheme 1) . This process is accompanied by topomerization (4) of diastereotopic proton pairs. At room temperature the proton NMR spectrum of 1 features sharp doublets for each
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of the ring protons labeled H,-H,, (Fig. 1) . The doublets are labeled l-8 in Fig. 1. When the temperature is raised, line broadening occurs over a large temperature range, until eventually a fast exchange spectrum is obtained, featuring four doublets for all of the eight ring protons (Fig. 1). The gradual passage of eight doublets into four involves extensive overlap and broadening of signals and makes it nearly impossible to identify with reasonable certainty the pairs of doublets which coalesce into each of the doublets observed at high temperature. In order to make this assignment we measured difference NOE and saturation transfer spectra at two temperatures, 300 and 323 K (Fig. 2, Table 1) . Difference NOE spectra were taken on a Bruker WP-200~SY spectrometer, at 200 MHz, using the multiplet irradiation method (5, 6). Each doublet (except the poorly resolved doublet 7) underwent six irradiation cycles, 0.4 s alternately at each site, for a total irradiation time of 4.8 s prior to application of the observation pulse. Fiftyfour decibels of attenuation was applied to the 0.2 W decoupler. It is evident from Fig. 2 and Table 1 that hardly any saturation transfer is found at 300 K. The only significant signals apparent in the difference spectra at 300 K are those due to NOES, and they enable straightforward identification of all geminal proton pairs (Table 2 ). The assignment of geminal protons has also been confirmed by a 2D shift correlation (COSY ) experiment. Two additional NOES at 300 K, other than those representing geminal connectivities, are found in Table 1: doublet 8 is connected to the aromatic protons at 7.3 ppm, and doublets 5 and 6 are mutually connected through a NOE. These NOES facilitate the following spectral assignments: The first effect is evidence that all four low-field doublets correspond to the benzylic protons H,-Hd; the other permits assignment of doublets 5 and 6 to protons Hi, and H,, which are substantially near each other. All spectral assignments are related to the proton labels on the left hand structure in Scheme 1. At 300 K the exchange is too slow to permit saturation transfer during irradiation. At 323 K, however, the exchange process is sufficiently rapid to cause formation of substantial saturation transfer signals, which are readily distinguished from NOE signals by their negative sign. Examination of difference NOE spectra at 323 K (Fig. 2) reveals the appearance of new signals. Thus when the doublet labeled 4, for instance, is irradiated for several seconds at low decoupler powler prior to the observation pulse and acquisition, four doublets are found in the resulting difference spectrum, corresponding to the chemical shifts of doublets l-4 (Fig. 2a). Two of those correspond to the signals obtained at 300 K (Fig. 2b), namely a negative (saturation) signal at the irradiation site 4 and a positive doublet at site 2 due to Overhauser enhancement. In addition, two new doublets appear, positive and negative, respectively, at sites 1 and 3. The negative doublet 3 is a result of saturation transfer from the irradiated proton, caused by chemical exchange, and hence provides evidence that sites 3 and 4 are mutually interchanged by the ring inversion process. The positive doublet at site 1 is totally absent from the difference spectrum at 300 K and must therefore also be associated with the temperature-dependent exchange process. It actually arises simultaneously from two d@rent magnetization transfers: In the first one, the initial Overhauser enhancement of doublet 2 is further transferred by exchange to 1. The positive sign of doublet 1 is opposite to that expected for ex-

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FIG. 1. The 200 MHz H NMR spectra of 1. Lower trace: Spectrum in CDC& solution of 300 K (the reference spectrum used for the difference NOE measurements). Inset shows detail between 3.9 and 4.4 ppm, and the AB quartet labeled 6, 7, which cannot be shown in the full spectrum due to overcrowding. Upper trace: Spectrum taken at 460 K in &nitrobenzene solution, near the fast exchange limit. The four doublets are labeled according to the low temperature pans of doublets which have coalesced into each one.

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FIG. 2. Difference NOE and saturation transfer spectra for 1. The following are the irradiated doublet and the temperature, respectively, for each trace: (a) 4, 323 K, (b) 4, 300 K; (c) 3, 323 K, (d) 3, 300 K; (e) 1,323 K;(f) 1,300 K.

change signals, because the exchange now is not with a saturated nucleus (i.e., one in which the excited level is populated in excess to the Boltzmann equilibrium distribution), but rather with one in which the population of the ground level is enhanced. The other process contributing to the positive intensity at doublet 1 is an indirect NOE generated by its geminal doublet 3, which is partially saturated through

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Percentages of Signal Enhancement in a Difference NOE and Saturation Transfer Experimentfor 1 Signal measured Signal measured Irradiated signal 1 2 3 4 -18.8 25.9 27.0 6.7 1 2 -18.2 9.1 23.6 14.9 3 23.0 24.7 6.8 -10.6 4 10.0 24.7 24.6 -17.9 Irradiated signal 5 6 8 5 6.7 5.8 14.5 14.0 6 3.9 6.5 -3.5 -6.5 7 -5.2 8 13.2 20.2 -2.9 -6.4 Arom. 7.3 ppm

5.8 4.3

Note. Upper figures: 300 K, lower figures: 323 K. Labels are as in Fig. 1. a Doublet 7 was insufficiently resolved to permit selective irradiation.

saturation transfer from signal 4. Analogous behavior is found for irradiation at other sites, as represented in the corresponding difference spectra (Fig. 2 ) . The immediate consequence arising from the observation of signals which are due to both NOE and NOE transfer is that the intensity of these signals does not con-espond in a simple manner to either the rate of exchange or the extent of dipolar relaxation. As a result it is obvious that the intensity of such signals should not be used either to calculate exchange rates or to calculate NOES for the derivation of geometrical parameters. On the other hand, the NOE and saturation transfer experiments described here have led to unequivocal assignment of exchanging doublets: the 300 K measurement serves to identify pairs of geminal protons (in conjunction with a COSY spectrum), as well as important spatial relationships, and the 323 K spectra show saturation transfer and readily lead to the assignments of interchanged doublets, as shown in Table 2. Individual proton assignments are based, in addition to the geminal and exchange connectivities established so far, on chemical shifts: Examination of a model shows that Hi, is located near the normal to the plane of the aromatic ring system, in a
TABLE 2 Geminal and Exchange Connections and Proton Assignments in the NMR Spectrum of 1 Doublet Geminal Exchange Proton assignment 1 3 2 Hh 2 4 I H, 3 1 4 4 2 3 Hf 5 8 7 Hb 6 7 8 7 6 5 & 8 5 6 H,

Note. Doublet labels are as in Fig. 1. Proton labels are as in the left hand structure 1 in Scheme 1. Connections are to be read vertically: Doublet 1 forms a geminal pair with 3, exchanges with 2, and corresponds to Hi,.

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strongly shielding region. This permits assignment of the high-field doublet 1 to proton Hh. The assignments of the rest of the protons H,-H, follow directly from this assignment with the aid of the known exchange and geminal pairs (Table 2). The low-field doublets H,-Hd are also individually assigned on the basis of their chemical shifts. It has already been established that doublets 5 and 6 belong to the inner protons, Hb and H,. Hence 7 and 8 correspond to the outer protons. Of these two, the one nearer to the anisotropic influence of the carbonyl group, H,, resonates at lower field, i.e., at the doublet site 8. This leads to the complete spectral assignment as listed in Table 2.
ACKNOWLEDGMENTS The author thanks Professor S. Bittner and Mrs. H. Strul for providing compound 1. Special thanks are due to the Kreitman family for a generous donation toward the purchase of the NMR spectrometer. REFERENCES I. 2. 3. 4. 5. 6. R.A.HOFFMANANDS.FORSEN, Prog. NMRSpectrosc. 1, 15(1966). J.K.M.SANDERSAND J.D.MERsH, Prog. NMRSpectrosc. 15,353(1982). J.BoYD,K. M. BRINDLE,I.D.CAMPBELL,ANDG. K. RADDA,J. Mugn.Reson.60, G.BINSCH,E.L.ELIEL,ANDH.KESSLER, Angew. Chem.Int.Ed 10,570( 1971). D. NEUHAUS, J. Mugn. Reson. 53,109 ( 1983). M. KINNS AND J. K. M. SANDERS, J. Magn. Reson. 56,5 18 ( 1984).

149(1984).