Selective 2D DEPT heteronuclear shift correlation spectroscopy

JOURNAL

OF MAGNETIC

RESONANCE

59, 124- 13 1 ( 1984)

Selective 2D DEPT Heteronuclear Shift Correlation Spectroscopy* T. T. NAKASHIMA, BOBAN K. JOHN,

AND

R. E. D. MCCLUNG

Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada Received

December

6, 1983

Selective 2D DEIT heteronuclear shift correlation maps for cholesterol in CDCls are reported and demonstrate the selectivity and utility of these experiments. Improvements in the basic 2D DEPT sequence to effect quadrature detection and proton-proton decoupling in thefi domain are reported. An implementation in which the 2D data files for proton flip angles 0 = 30, 90, and 150” are acquired and used to construct selective CH, CH2, and CH, maps is shown to be twice as efficient as the previous implementation where separate experiments were performed to construct each selective 2D map.

INTRODUCTION Two-dimensional (2D) heteronuclear shift correlation experiments (I, 2) are used in 13C NMR spectroscopy to identify the chemical shift(s) of the proton(s) which are directly bonded to each of the 13Cnuclei in the molecule. With effective 13Cdecoupling in thefi domain (‘H spectral frequencies) and ‘H decoupling in the f2 domain (13C frequencies), ‘H-13C shift correlation experiments provide a powerful technique for the assignment of the ‘H and 13C spectra of large molecules (3). Recently, it has been suggested that the DEPT pulse sequence (4) can be used to produce selective magnetization transfer in heteronuclear shift correlation experiments (5, 6) and in other 2D experiments (7). The basis of the selectivity of magnetization transfer using DEPT is the fact that the intensities of the 13C{ ‘H} signals for CH, CH2, and CH3 fragments have characteristic dependence on the flip angle 0 of the last ‘H pulse in the sequence (4), and the demonstration that an appropriate linear combination of FIDs collected using different values of 0 gives a 13C spectrum w hi ch is dominated by resonances from one particular type of CH, fragment. In a preliminary report (8), we have demonstrated that 2D heteronuclear correlation maps with only CH, CH2, or CH3 peaks present can be obtained with the 2D DEPT sequence shown in Fig. 1A. This implementation of the 2D DEPT experiment involved the collection of a series of FIDs, each of which is a particular linear combination of FIDs for the different values of 0 required to selectively enhance resonances from the desired CH, fragments [FID(90”) selects CH resonances, FID(45”) - FID( 135”) selects CHP resonances, and FID(30”) + FID(l50’) - FID(90”) selects CH3 resonances]. The data collection process was carried out with a microprogram in which the pulse sequences for the different 19values were arranged consecutively, and separated by memory negation * Research supported under operating Grant 0022-2364184

in part by the Natural A5887 and Infrastructure

$3.00

Copyright 0 1984 by Academic Pres, Inc. All rights of reproduction in any form reserved.

Sciences and Grant Al593. 124

Engineering

Research

Council

of Canada

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125

if required, so that the accumulated FID for each ti value is the required linear combination of FIDs for different 0. This approach was facilitated by recognizing that the CH3 selective linear combination of the FIDs for 6’ values of 30, 90, 150’ is simpler than the CH3 selective linear combination for 13values of 45, 90, 135” commonly used in DEPT experiments (4). This incorporation of the linear combinatorics into the data collection process avoids manipulation of large 2D data sets (except in the final Fourier transformation step), but makes unnecessary demands on spectrometer time. Recently, broadband homonuclear decoupling in the fi (proton) domain of 2D heteronuclear correlation spectroscopy has been demonstrated (9). This proton-proton decoupling is achieved by including a bilinear rotation pulse sequence (10) at the center of the t, evolution period. This bilinear rotation sequence selectively protonproton decouples the resonances of the protons, which are directly bonded to 13C atoms, during the t1 period. In this paper, we report the results of an investigation of the basic features and the selectivity of 2D DEPT heteronuclear correlation experiments on cholesterol whose 13C NMR spectrum has already been assigned (II). Sensitivity and resolution enhancement by the incorporation of proton-proton decoupling in thefi domain, and the use of appropriate cycling of the phases of the pulses in the 2D DEPT sequence to effect quadrature detection in fi are also described. An implementation of the 2D DEPT experiment which decreases the time required for data collection by a factor of 2 over our initial approach (8) is reported. In this implementation, the full 2D data sets are collected for each of the three 8 values, 30,90, and 150”, and the selective linear combinations can be constructed and transformed at a data station to produce the selective 2D correlation maps for CH, CH2, and CH3 groups. EXPERIMENTAL

Selective 2D DEPT experiments have been performed on Bruker WH-200, AM300, and WH-400 spectrometers, each equipped with an Aspect 2000 computer. The manipulation and transformation of the 2D data sets acquired with the WH-200 and WH-400 spectrometers were performed on an Aspect 2000 data station. Unfortunately the disk drives on the AM-300 system and the data station are different so data acquired with the AM-300 was worked up on the computer associated with that spectrometer. All spectra shown in the figures were obtained from a saturated solution of cholesterol (13C in natural abundance) in CDC13 in a 10 mm tube using the Bruker AM-300 spectrometer with transmitters operating at 300.13 MHz (‘H) and 75.48 MHz (13C). The 90” pulse lengths were 3 1.5 ps (‘H) and 13 ps (13C), and a relaxation delay of 3 s separated successive scans. Sixteen scans were accumulated for each value of t,, and the total time required for the acquisition of the full 2D data sets for 0 = 30, 90, and 150’ was about 2.7 h. The 2D data sets consisted of 1K FIDs for 64 values of tl , and covered frequency ranges of 1080 Hz and 5000 Hz in thefi (‘H) and X (i3C) domains. The ‘H frequency range excluded the vinyl proton and the range used for 13C excluded the resonances from C-5 and C-6. The data sets were zero-filled to 256 points in thef; domain, and were apodized with exponential weighting functions corresponding to line broadenings of 4.2 and 10 Hz inf; andfi, respectively. These line broadenings are equal to the digital (Hz/pt) resolutions.

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CLUNG

DISCUSSION

The pulse sequence for the 2D DEFT experiment is shown in Fig. 1A. The proton magnetizations are put onto the y’ axis of the rotating frame by the proton 90”, pulse and are allowed to precess at their characteristic frequencies for a period t,/2. The 180” carbon pulse relabels the magnetizations of protons coupled to a 13C nucleus so that the 13C-‘H coupling is refocussed during the second t,/2 precession period (3). At this point, the proton magnetizations are labeled with the frequencies in the “carbon-decoupled” proton spectrum. These magnetizations are allowed to precess for a further time T = 1/(2.&u) so that the magnetization components for a proton or group of protons directly bonded to a 13C nucleus will acquire a phase difference of 180”. At this point, a carbon 90” pulse transfers the proton magnetizations into zero- and double-quantum coherences which are allowed to precess for 7 = 1/(2&n) before the proton 8 pulse transfers the multiple-quantum coherences into 13C perpendicular magnetization. During the last 7 = 1/(2&u) precession, the components of the 13C multiplets come into phase with each other and acquisition with broadband proton decoupling is then performed. The 180” proton and carbon pulses, which are applied simultaneously with the 90” carbon and 0 proton pulses, respectively, serve to refocus any phase differences which arise in the T = 1/(2&n) precession periods due to resonance offsets. The efficiency and selectivity of the transfer of magnetization

A

90x

decoupling

‘H

FIG. I. (A) The pulse sequence for the 2D DEPT heteronuclear correlation experiment. The value of t, is incremented in equal intervals and r is set to l/U% (0.00388 s in the cholesterol study). 2D data sets were collected for 0 values of 30, 90, and 150”. The phases of the final ‘H pulse (13,) and the “C 90” pulse (90+) were cycled as shown in order to suppress the normal ‘% Boltzmann signal and to enable quadrature detection in the f, domain. The receiver phase is held constant (CP). (B) To remove ‘H-‘H couplings between weakly coupled protons in thefi domain, the bilinear rotation pulse sequence shown replaces the 180” 13C pulse at the center of the evolution period.

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from the protons to the 13C nuclei is determined by the flip angle B of the final proton pulse in the DEPT sequence (4). The intensities of the i’C{ ‘H} signals for CH, CH2, and CH3 groups are proportional to sin 8, sin 28, and sin B + sin 30, respectively. In the standard DEPT experiment (4), one collects FIDs for 8 = 45, 90, and 135”, accumulating twice as many transients for 6 = 90” as for the other values of 8, and generates subspectra which selectively show the CH, CH2, or CH3 resonances by transformation of FID(90’), FID( 135”) - FID(45”), or FID(45”) + FID( 135”) - 0.707 FID(90”). We have found it more convenient to collect data at 8 = 30, 90, and 150”, accumulating the same number of transients at each value of 0, since the selective linear combinations FID(90”), FID(150”) - FID(30’), and FID(30”) + FID( 150”) - FID(90”) are easier to incorporate into the microprograms used to collect (8) or to manipulate the data. The use of 0 = 30 and 150’ rather than B = 45 and 135” reduces the intensities of the CH2 peaks by about 30%, and increases the intensities of the CH3 peaks by about 20%. The phases of the 90” carbon pulse and the proton B pulse are cycled as shown in Fig. 1 in order to suppress the normal Boltzmann carbon signals and to allow quadrature detection in fi . This phase cycling was designed by computer simulation of the 2D DEPT sequence using the density-matrix simulation scheme described earlier (12). In our previous work (8), we incorporated the linear combinatorics necessary for construction of selective 2D correlation maps into the data acquisition microprograms so that no manipulation of large 2D data sets was required. This approach is inefficient if all of the selective CH, maps are required in that, for example, the FIDs collected for 0 = 90” to generate the selective CH correlation map are not used in the construction of the selective CH3 map where more FIDs for 0 = 90” are also acquired. We have overcome this deficiency by collecting the full 2D data sets for 8 = 30, 90, and 150’ in one data acquisition microprogram,’ and using appropriate microprograms’ to manipulate these large data files and construct the selective linear combinations required for the CH2 and CH3 maps. This implementation of the 2D DEPT experiment reduces the data acquisition time by about 50%. The selective 2D DEPT correlation maps obtained for cholesterol in CDC13 are shown in Fig. 2. The correlation map in Fig. 2A contains peaks from all CH, fragments and was obtained by transforming the 2D DEPT data set acquired with B = 30”. All maps are power spectra so that separate absorption and dispersion components were not computed in the Fourier transformations. With the exception of two small peaks due to CH3 groups which appear on the map for CH2 groups (Fig. 2C), the separation of the resonances into the selected CH, CH2, and CH3 correlation maps is excellent. There are three regions in which CH and CH2 resonances are in close proximity in both ‘H and 13C frequencies: (a) the resonances due to carbons C-20 and C-22 at ’ We were able to write a microprogram to collect all three of the 2D data sets using the pulser board version FP8 10505 of Bruker NMR software, but not with the DISNMRP.R30 version of the Bruker disk NMR software. With DISNMRP.R30, we found it necessary to collect the 0 = 30” data set with one microprogram and to collect the 19 = 90 and 150” data sets with a second one. 2 The addition and subtraction of the large 2D data sets was performed with microprograms described in the Bruker PP8 105 15 software documentation. The construction of the CH2 and CHx selected 2D data files from the 2D data sets for 0 = 30, 90, and 150” required about 6 min on the Aspect 2000 computer.

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JOHN, AND MC CLUNG

I” II FIG. 2. The ‘H-W shift correlation maps of cholesterol containing resonances from (A) all CH, CH2, and CHs groups obtained by processing the 0 = 30 2D data set [FID(30)]; (B) only CH fragments [FID(90)]; (C) only CHa groups [FID( 150) - FID(30)]; and (D) only CHJ fragments [FID(30) + FTD( 150) - FID(90)]. A 5000 Hz (horizontal) and 1080 Hz (vertical) window is plotted for each map corresponding to 13Cshifts ( fi, 8.2 to 74.4 ppm) and ‘H shifts (1;, 0.2 to 3.8 ppm), respectively. In A and C, a, b, and c indicate the regions of congestion which are discussed in the text. The asterisks in Fig. 2C indicate low-intensity peaks from CH, groups.

36 ppm; (b) the resonances due to carbons C-2, C-7, and C-8 at 32 ppm inf2; and (c) the resonances due to carbons C- 16 and C-25 at 28 ppm. The maximum separation in f2 of the resonances in each of these regions is about 0.5 ppm. The peaks due to CH and CH2 groups in these regions are cleanly separated in the selective CH and CH2 correlation maps in Figs. 2B and C. The patterns due to the CH2 groups at carbons C- 1, C-2, C-7, C- 15, C- 16, C-22, C-23, and C-24 are shown to have chemically nonequivalent protons. The increased resolution in region b (- 32 ppm) in the CH2 selected map (Fig. 2C) compared to the nonselective map (Fig. 2A) is significant and is brought about by the removal of the CH resonance due to C-8 which obscures the nonselective map. In the implementations of 2D DEPT correlation spectroscopy by

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Levitt et al. (5) and by Bendall and Pegg (6), spectral editing relied on the fact that, for 6 = 135”, the CH2 correlation peaks are 180” out of phase with the CH and CH3 peaks so that 2D maps with only CH + CH3 resonances or with only CH2 resonances could be obtained by selecting positive or negative contours of the phase sensitive transform of FID(l35’). This approach does not give such clean separation of the peaks for C-2, C-7, and C-8 in the positive and negative contours of the phase sensitive transform of FID( 150’) as we obtained in the selective CH and CH2 maps in Fig. 2. One may obtain selective proton spectra containing only the resonances for CH, CH2, or CH3 groups by projecting the selective 2D DEPT correlation maps onto the f, axis. In cholesterol, the ‘H spectrum at 300 MHz shows sharp peaks due to the methyl protons on a broad structured background, and isolated resonances for the vinyl proton (at C-6), the methylene protons on C-4, and the proton attached to C3. The selective proton spectrum for the methyl protons is very clean and shows only minor overlapping of resonances. The projections of the CH and CH;? maps onto thefi axis show significant overlapping of lines as one would anticipate from Figs. 2B and C. To get increased resolution in a particular selective 2D DEPT correlation map (e.g., the CH1 selected map for cholesterol), one can decrease the spectral widths for acquisition in bothfi and fi so that only the resonances of a particular group of CH, are enclosed. One can acquire the selected linear combination of FIDs using the narrower spectral widths, and the resonances due to the nonselected CH, fragments will be folded into the restricted spectral range but, since they are of much lower intensity, they should not produce significant interference. We have performed CH selective 2D DEPT experiments at 100.6 MHz (i3C) on bis[hydrotris(pyrazol-lyl)borato][3-(trifluoroacetyl)-d-camphorato]lutetium(III) in CDzClz solution with reduced sweep widths of 5000 Hz ( 13C) and 1000 Hz (‘H) enclosing only the pyrazole resonances of both carbon and proton spectra. The normal 13Cand ‘H spectra require spectral widths of 25,000 and 5000 Hz, respectively, so that all resonances are displayed without folding. In the 2D DEPT experiment with reduced spectral windows, the peaks from the CH;! and CH3 groups of the camphorate moiety were selectively suppressed and did not appear on the selective CH map. The peak from the bridgehead CH of the camphorate did fold into the restricted spectral range but did not interfere with the CH peaks of the pyrazole groups. The 2D DEPT experiment can be modified to incorporate proton-proton decoupling in f; by replacing the 180”, carbon pulse at the center of the preparation period with the bilinear rotation sandwich (10) shown in Fig. 1B in the same way that the normal ‘H-13C correlation experiment has been modified by Bax (9). In Fig. 3, some representative vertical cross sections of the selective CH and CH2 2D DEPT correlation maps for cholesterol obtained with the modified sequence are compared with the corresponding cross sections obtained without proton-proton decoupling in fi . The CH proton cross sections (e.g., Fig. 3A) are significantly sharper and more intense in the proton decoupled map because the CH protons are coupled to a number of protons at adjacent positions. The effect of proton-proton decoupling on the cross sections at the methylene centers at positions C-l (Fig. 3C) and C-22 (Fig. 3B) are not so pronounced because the two protons at each of these positions are chemically nonequivalent and form a strongly coupled AB spin system. The bilinear rotation

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JOHN,

AND

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FIG. 3. Representative vertical cross sections of the selective CH maps at frequencies fi corresponding to C-8 (A), C-22 (B), and C- 1 from the map without ‘H-‘H decoupling infi (pulse sequence Fig. map with decoupling infi (pulse sequence Fig. IB). Frequency scales and C).

(A) (C). IA), are

and CH2 (B and C) correlation In each case, the upper trace is and the lower trace is from the 25 Hz/div (A) and 50 Hz/div (B

sequence does not remove the geminal proton couplings in strongly coupled systems (IO), but does remove the couplings of this AB system to other protons in the molecule. In Fig. 3C, the geminal proton coupling is unresolved in the proton coupled cross section, but is partially resolved in the decoupled cross section. For the strongly coupled geminal pairs of protons, one finds that proton-proton decoupling produces a peak at approximately the average chemical shift of the geminal pair. The intensity of this central peak varies from one geminal pair to another, and is most intense when the chemical-shift difference for the geminal protons is smallest (Fig. 2B vs Fig. 2C). The origin of this peak and the application of the quadraplexed bilinear rotation sequence suggested by Garbow et al. (10) to effect proton-proton decoupling in the presence of strong AB coupling are currently under investigation. CONCLUSIONS

We have reported an implementation of the selective 2D DEPT heteronuclear correlation experiment which halves the time required for data acquisition of our earlier implementation (8). The selectivity and utility of selective correlation experiments has been demonstrated in a study of cholesterol. Modifications of the basic 2D DEPT experiment which produce quadrature detection and proton-proton decoupling in thefr domain are shown to enhance the resolution and sensitivity of this selective correlation experiment. ACKNOWLEDGMENTS We thank Professor camphorato]lutetium(III),

J. Takats for the sample of bis[hydrotris(pyrazol-l-yl)borato][3-(trifluoroacetyl)-dand Dr. E. Knaus and Dr. Y. Theriault of the Faculty of Pharmacy

for the use

SELECTIVE of the AM-300 Grant 2623.

spectrometer

which

DEPT

CORRELATION

was purchased

under

Alberta

131

SPECTROSCOPY Heritage

Foundation

for Medical

REFERENCES I. 2. 3. 4. 5. 6. 7. 8.

A. G. R. D. M. M. D. T. 9. A. 10. J. 11. J. 12. B.

A. MAUDSLEY AND R. R. ERNST, Chem. Phys. Lett. 50, 368 (1977). B~DENHAUSEN AND R. FREEMAN, J. Magn. Reson. 28, 471 (1977). FREEMAN AND G. A. MORRIS, J. Chem. Sot. Chem. Commun.. 684 (1978). M. D~DDRELL, D. T. PEGG, AND M. R. BENDALL, J. Mugn. Reson. 48, 323 (1982). H. LEVIS, 0. W. SC~RENSEN, AND R. R. ERNST, Chem. Phys. Lett. 94, 540 (1983). R. BENDALL AND D. T. PEGG, J. Mugn. Reson. 53, 144 (1983). T. Peck AND M. R. BENDALL, J. Mugn. Reson. 55, 114 (1983). T. NAKASHIMA, B. K. JOHN, AND R. E. D. MCCLUNG, J. Magn. Reson. 57, 149 (1984). BAX, .I. Mugn. Reson. 53, 5 17 (1983). R. GARBOW, D. P. WEITEKAMP, AND A. PINES, Chem. Phys. Let?. 93, 504 (1982). R. BLUNT AND J. B. STOTHERS, Org. Magn. Reson. 9,439 (1977). K. JOHN AND R. E. D. MCCLIJNG, J. Mugn. Reson. 58, 47 (1984).

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