JOURNAL
OF MAGNETIC
RESONANCE
75, 162- 166 ( 1987)
Proton Longitudinal Relaxation Times of 13CIsotopomers PETER BIGLER Institute of Organic Chemistry, University of Beme. Freiestrasse 3, 3012 Bern, Switzerland Received April 28, 1987
Modem pulse sequences designed either to detect carbon signals with improved sensitivity [INEPT (1) DEPT (2)] or to correlate carbon and proton chemical shifts via scalar spin-spin coupling [C-H COSY (3, 4), H-H-C RELAY (5, 6)] are based mainly on polarization transfer. The large population differences between energy levels of proton transitions are transferred in these sequences to the transitions of directly bound r3C nuclei. One of the factors influencing the overall sensitivity of these experiments is the length of the initial proton relaxation delay. To optimize this delay some knowledge of the relaxation rates of protons directly bound to 13C is essential. The present results demonstrate that these rates may strongly differ from the normally measured values for the corresponding 12C isotopomer. The neglect of this “isotope effect” may be responsible for the sometimes unexpected poor sensitivity in measured spectra. Simulation or computational optimization of such 13C experiments on the other hand need precise proton relaxation rates of 13C isotopomers. Figure 1 depicts a pulse sequence developed to measure relaxation rates of protons directly bound to r3C. In its basic version (nonselective mode) all proton magnetizations in a given molecule are inverted with a normal or composite 180” decoupler pulse. A slightly modified version (selective mode) with the 180” decoupler pulse replaced by 90:(H)-2’J&180,“(H), 180:(C)-2’J&-90,0(H), a so-called “bilinear rotation operator” (7), selectively inverts in a given molecule only z magnetization of protons directly bound to 13C. Due to the high dilution of i3C in natural abundance it leaves all the other proton z magnetizations in the same molecule unperturbed. This version which suppresses proton-proton cross-relaxation effects corresponds to the inversion-recovery experiment with selective 180” pulses applied to measure proton relaxation rates in 12Cisotopomers. Proton relaxation proceeds during a variable delay T. Several 90” I3C pulses may be applied in this part for cancellation of eventual proton-carbon cross-relaxation effects. In the second part of the sequence proton polarization is transferred to carbon via the well-known INEPT (I) sequence and the relaxation process is detected in the carbon domain under proton broadband decoupling. Compared to a direct observation of the relaxation process in the 13C-satellite ‘H spectra, by use of the double-quantum tilter technique for suppression of the intense center signal of the 12C isotopomer, as in 2D C-H shift correlation experiments in 0022-2364187 $3.00 Cowi& 0 1987 by Acarkaic Rcss, Inc. All rights of reproduction in any form z2ewcd.
162
163
NOTES
/ INEPT
selective ‘H
non selective
1 T-J \
I L--, 180
‘90
a0
180
13C
FIG. 1. Pulse sequence to detect relaxation rates of protons bound to “C starting with a nonselective or selective (dashed scheme) proton inversion pulse. D2, D3, D4 are set equal to f&, $&, and &JCH, respectively.
“reverse mode” (8) sensitivity is lost. Furthermore, only mean relaxation rates are accessible for nonequivalent methylene protons. With the normally well-resolved proton broadband decoupled carbon spectra, on the other hand, no overlapping problems arise either with poorly working doublequantum filters or with complex highly crowded proton spectra. Since this latter problem not only appears in proton spectra of 13C but also of 12C isotopomers the proposed method might be a good alternative in this case to determine indirectly relaxation rates of 12C isotopomers. According to the relation T;‘(H),sc = T;‘(H)I~
1 +- T;‘(C) N
DD [II
proton longitudinal relaxation rates of 13C and 12C isotopomers differ by an amount determined by the dipolar interaction between 13C and ‘H nuclei. This amount is given by the dipolar carbon relaxation rate TT’(C)~~ divided by the number of interacting protons. Due to the r& dependence of the carbon relaxation rate this contribution is negligible for protons not directly bound to r3C. Highly crowded proton spectra not only make the accurate detection of relaxation rates difficult but also prevent the application of selective 180” inversion pulses when cross-relaxation e&c& should be avoided. Application of the mod&d (selective mode) version of the pro@osed pulse sequence elegantly solves this problem for both 13Cand 12Cisotopomers. &cause of the above-mentioned high dilution of 13C spins, only one inversion-recovery experiment is necessary to get all relevant data, whereas several experiments must be performed in the conventional experiment depending on the number of protons to be selectively inverted.
To test its reliability, the proposed method was applied to a sample of 200 mg eugenol dissolved in 200 ~1 acetone d-6. Subsequently two inversion-recovery experiments for determination of carbon and proton ( ‘*C isotopomer) relaxation rates and a NOE experiment for the evaluation of the dipolar part of the carbon relaxation rate were carried out. To test the reproducibility of the results this queue of experiments was repeated three times. The corresponding experiments for the detection of proton TABLE 1 ‘H and “C Relaxation Rates Ti’ (s-l) of 13Cand ‘% Isotopomers of Eugenol Location in eugenol 3
1.14” 0.96 1.02
0.69b 0.55 0.58 0.61
1.82’ 1.67 1.54 1.79
1.83d 1.69 1.54 1.63
4
1.28 1.10 1.09
0.73 0.57 0.63 0.63
2.00 1.82 1.56 1.92
2.01 1.85 1.73 1.72
6
1.15 0.98 1.01
0.85 0.67 0.73 0.74
2.12 2.0 1.61 2.00
2.0 1.82 1.71 1.75
7
0.71 0.67 0.68
1.32 1.18 1.16 1.16
1.59 1.59 1.43 1.54
1.68 1.54 1.50 1.50
8
0.24 0.21 0.22
0.35 0.33 0.29 0.29
0.62 0.58 0.52 0.55
0.59 0.57 0.50 0.51
979
0.48 0.42 0.39
0.55 0.53 0.46 0.47
0.79 0.78 0.67 0.72
0.79 0.77 0.63 0.67
10
0.56 0.48 0.52
1.56 1.45 I .08 1.14
1.20 1.19 0.98 1.15
1.75 1.64 1.24 1.31
a Dipolar carbon relaxation rates measured with an inversion recovery and a { ‘H}13C NOE experiment. The relaxation &Lay Between subsequent scans was set to 60 sin Both experiments. Eight scans for each of 20 7 values varied Between 0.25 to 50 s were accumulated in the former, 64 scans in the latter experiment. b Broton relaxation rates of the ‘Zc isotopomer measured with inversion-recovery experiments with a nonselective (left column) and a selective (right column) 180” inversion pulse. The relaxation delay was set to 20 s. Eighteen 7 vafues in the range of 0.1 to 16 s were used; for each value eight scans were accumulated. c Proton relaxation rates of ‘C isotopomers measured with the experiment shown in Fig. 1 in the nonselective (let% column) and in the selective mode (right column). The relaxation delay was set to 20 s. Eighteen 7 values in the range of 0.1 to 16 s were used, for each value 16 scans were accumulated. d Proton relaxation rates of 13Cisotopomexs calculated from 13Cand ‘H (‘w isotopomers) relaxation rates according to IQ. [I].
I 6
165
NOTES 9
6
7
4
\S
3 I
*/‘O
/
I
OH SCHEME
1
relaxation rates with selective inversion of 2 magnetization were carried out only once. Relaxation rates were calculated using a three-parameter curve fit routine. The results are summarized in Table 1. According to Eq. [l] the experimentally determined T;‘(H)I~ and T;l(C)DD values were combined to calculate the proton relaxation rates of the 13C isotopomers of eugenol. These indirectly determined proton relaxation rates T;‘(H)!7 are compared
T,-‘(”),I’” s
-1
+ 0 *
C
n
+ A 0
2
H-3 H-4 H-6 H-7 H-8 H-9 H-10
/.+‘* /
/’ l
/. /
l-
I
0
I
I
I
1
2 T;‘(“);3ND, C
FIG. 2. Proton relaxation rates of the ‘% isotopomers of eugenol. The values 7’;‘(He measured directly with the puk sequence shown in Fig. I are compared with the values T;‘(Hxp calculated according to F.q. [ 11 from data obtained with normal inversion-recovery techniques.
NOTES
values, using the proposed pulse sequence in with the directly measured r;‘(C):: Fig. 2. Apart from the slight but significant deviation of the values for the methyl group, the reasons for which are not fully understood, good agreement between the two independently determined data sets can be achieved with both the nonselective and the selective (Table 1) variant of the experiment. From the data in Table 1, it is obvious that corresponding proton relaxation rates generally increase in going from 12C to 13C isotopomers. The relative amount of this increase is dependent on the proton type and decreases in the order CH > CH2 > CH3. This is reasonable since the contribution of the 13C nucleus to the overall proton relaxation rate of a CH3 group with strong dipole-dipole interactions among the methyl protons is of minor importance whereas it strongly dominates the relaxation behavior of the relatively isolated methine protons. Reduced values for relaxation rates are measured when experiments with selective inversion of proton magnetization are applied. The marked effects detected for methine protons reflect that proton-proton cross relaxation in these cases is effective and can be greatly suppressed. Cross-relaxation processes among equivalent protons of CH2 and CH3 groups, on the other hand, can not be suppressed, of course, but certainly dominate the overall cross-relaxation rates of these protons. This explains the fact that corresponding values for methylene and methyl protons measured with selective and nonselective proton magnetization inversion differ only slightly. REFERENCES
1. G. A. MORRIS, 2. 3. 4. 5. 6. 7. 8.
D. A. J. P. A. J. A.
J. Magn. Reson. 41, 185 ( 1980). M. D~DDRELL, D. T. PEGG, AND M. R. BENDALL, J. Magn. BAX, J. Magn. Reson. 53,5 17 (1983). A. WILDE AND P. H. BOLTON, J. Magn. Reson. 59, 343 (1984). H. BOLTON, J. Magn. Reson. 48,336 (1982). BAX, J. Magn. Reson. 53, 149 (1983). R. GARBOW, D. P. WEITEKAMP, AND A. PINES, Chem. Phys. BAX, J. Magn. Reson. 67,565 (1986).
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Left. 93,504 (1982).