SEMUT editing of inadequate 13C NMR spectra

JOURNAL

OF MAGNETIC

RESONANCE

59, 332-337

(1984)

SEMUT Editing of INADEQUATE 13CNMR Spectra* OLE W. S~~RENSEN,~ULLA B. S~RENSEN,$ AND HANS J. JAKOBSEN# tLaboratorium Switzerland,

fir Physikalische and *Department

Chemie. Eidgeniissische of Chemistry, University Received

April

Technische of Aarhus,

Hochschule, CH-8092 Zurich, DK-8000 Aarhus C, Denmark

5, 1984

With the increasing complexity of molecules being subjected to structural elucidation by NMR spectroscopy the development of methods for spectrum simplification becomes more and more important. A particularly useful and routinely feasible experimental technique is the decomposition of a proton-decoupled 13CNMR spectrum into four subspectra, one for each proton multiplicity (C, CH, CH2, and CH3). For molecules with a small spread in one-bond 13C-iH coupling constants (‘Jcn) the methods of SEMUT (1) and DEPT (2) are capable of doing this job with acceptable accuracy (1). However, for an unequivocal spectral assignment of molecules or mixtures of molecules with large i&n ranges it is necessary to incorporate a purging sandwich, yielding the SEMUT GL and DEPT GL pulse sequences (3, 4). Another powerful method in NMR structural analysis is the INADEQUATE experiment (5) where pairs of 13C-13C doublets are observed via a double-quantum filter. From this experiment carbon-carbon connectivities may be deduced (6, 7). The present communication evaluates the usefulness of incorporating spectral editing techniques into the INADEQUATE experiment, proposes pulse sequences for this purpose, and demonstrates experimentally the additional information which may be obtained from such procedures, The first question to answer is why any NMR pulse sequence should be made more complicated by including an editing step. Obviously, the proton multiplicities for the 13C resonances are normally already known from a SEMUT or DEPT experiment and at first sight it does not seem to make much sense to confirm the assignment in a second experiment designed to give other spectral information. However, multiplicity assignment is not the only asset of subspectral editing. In one-dimensional INADEQUATE spectra it is not uncommon that different 13C-13C subsystems overlap and thereby render the assignment and measurement of 13C-13C coupling constants difficult and ambiguous. We propose editing extensions for the INADEQUATE sequence which, in addition to 13C-13C couplings, simultaneously provide information on the total proton multiplicity for pairs of 13C-13C coupled nuclei. This considerably facilitates the assignment of one-bond and longrange carbon-carbon links and thereby gives unambiguous information about the molecular framework. Two papers ( 7, 8) have already suggested the insertion of SEFI techniques (9) after the double-quantum filter in the INADEQUATE experiment. However, this usually * Presented 0022-2364184

at the 25th ENC,

Wilmington,

$3.00

Copyright 0 1984 by Academic Prt8.s. Inc. All rights of reproduction in any form reserved.

Delaware, 332

April

8-12,

1984.

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leads to phase distortions because of incomplete refocusing of the antiphase ‘sC-‘3C doublet created by the filter. For the two-dimensional experiment described in Ref. (7) this is not a problem, but in 1D spectra it is necessary to obtain clean phases. A modified sequence which circumvents this problem is shown in Fig. la. It is based on SEMUT (1, 3) and includes a z filter (10) to purge the phase anomalies. As for all the other pulse sequences discussed in this paper, and shown in Fig. 1, the editing procedure has the SEMUT level of accuracy (I). To achieve the GL level of accuracy a purging sandwich can be inserted in a manner very similar to that demonstrated in Ref. (3). The sequence also possessesthe symmetry required for uniform excitation of the 13C-13C double quantum coherences (11). Thus no compromise need be made in setting the 7c delay. Another possibility for SEMUT editing of INADEQUATE spectra, the general technique being dubbed SEMINA, is given by the pulse sequence shown in Fig. 1b.

0, t1

90::

4

1%

180'

90;90°

TC

TC

-T

T-

=3

180'

J, tc 2

8, 'H

'cl

t3

90::

b)

180 4r 2

'SC

90:

4

90:90°

180'

90::

90'

8,~

180'

J,

32

2

'H

t1 90;

'XC

180'

TC

13C

4

4J

2 2

180=' TC

T

90; tc 2

t2 180'

t2

=2 90'

'2

(3~~

0

=3 180' TC

T

FIG. 1. Pulse sequences for the SEMINA experiment, i.e., SEMUT editing of INADEQUATE “C NMR spectra. For sequences (a) and (b) the condition TC 2 2(7, + 7)) must be fulfilled, similarly for sequence (d) TV > 2(~* + TV).Otherwise two 180’ I3C refocusing pulses are needed in the relevant 7c delay for these sequences.

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This sequence has a SEMUT editing step placed before the doublequantum filter and may easily be modified to include polarization transfer and a DEFT editing step. The procedure outlined in Fig. 1b amplitude modulates the amount of double-quantum coherence created by the second 90” 13C pulse. For the fragment H:: A A -A the modulation

HE B

factor is given by a{ -cos e}n + b{ -cos e>m

111

where CIand b are determined by the one-bond 13C-‘H coupling constants according to (3) a = {sin(nJc.d,~d sin(J~,d4A73)}n b = {sin(?rJc,u,7,) sin(JceHB73)}m.

PI

With this sequence (Fig. 1b) editing is complicated and it is considered useful only for suppression of 13CH,-‘3CH m pairs with n + m being odd. This is achieved by setting 0 = 0” which makes expression [l] close to zero. The suppression occurs because the second 90” I3C pulse in this case generates zero-quantum coherence which gets eliminated by the double-quantum phase cycle. A more powerful variant of the SEMINA experiment is outlined in Fig. 1c. Carboncarbon double-quantum coherence (2QC) is created under the condition of proton decoupling, but instead of immediate reconversion to observable single-quantum coherence (1QC) the 2QC precesses for a period 27, before reconversion. During this period the decoupler is switched off and a SEMUT sequence is inserted. It turns out that the 2QC for fragment [A] behaves in the same way as the 1QC of a hypothetical CH,,, group would do in the SEMUT experiment. The general response for CA and CB of fragment [A] to the pulse sequence of Fig. lc is given by Z(CH,-CH,)

= sin(?rJcc7c){cos(~~,71) c0s(?rJ,7~) + sin(?rJ,r,) sin(?r.ZIT3)cos 0)’ X [co~(?rJ~~~)~os(9rJ~7~)+ sin(?r&J

sin(7r.Z273)cos 131~ [3]

whereJI = Jc.,H~ + Ju,,, JZ = JGSG + JC*HB. When the products of cosine factors are made to vanish by judicious setting of the 7, and 73 delays (3) it follows that the B flip-angle dependence for a CH,-CH, fragment is {cos e}“+“‘. Then appropriate linear combinations of experiments obtained with different B hip angles can decompose the INADEQUATE spectrum into seven subspectra (n + m = 0, 1, . . . , 6), each corresponding to a specific number n + m. These subspectra may simultaneously include spectra from one-bond as well as long-range 13C-13C two-spin systems. A complete editing generating seven INADEQUATE subspectra would, however, be accompanied by a considerable loss in sensitivity (4) and is not recommended for general applications. On the other hand the Fig. lc sequence may often be useful to suppress the resonances corresponding to a certain value for n + m or to generate two subspectra according to n + m being odd or even. The latter option is especially powerful since it ideally does not degrade the sensitivity of the INADEQUATE ex-

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periment (neglecting relaxation effects and long-range 13C-‘H couplings), and it has been chosen as an illustrative example in the present paper. This version of the sequence is performed by dividing the total time devoted to an INADEQUATE experiment into two subexperiments using fl= 0” and 0 = 180”, respectively. For this version 7’ and 73 can be chosen equal. The 19= 0” experiment gives a spectrum identical to the normal antiphase INADEQUATE spectrum whereas the 0 = 180” spectrum appears with inversion of all antiphase ‘3CH,-‘3CH,,, doublets for 12+ m being odd. Thus, addition and subtraction of the two spectra generate two subspectra with the 13C-13C doublets separated according to n + m being even and odd, respectively. 13C NMR SEMINA experiments using this variant of Fig. lc for SEMUT editing of INADEQUATE spectra were performed on a Varian XL-300 spectrometer at 75.45 MHz. A 64- or 128~step phase cycle with suppression of parent signals in alternate scans was used for the INADEQUATE part of the sequence. The 90” 13C and ‘H pulse widths were 19.5 and 34.0 ps, respectively. As an example Fig. 2 illustrates the edited n + m even and odd 13C SEMINA spectra obtained for a sample of 1,3dibromobutane. The SEMINA spectra in Fig. 2 were recorded with the 7c delay simultaneously optimized for the one- and three-bond 13C-13C couplings, 7c = (2n + 1)(2Jc&‘; i.e., 7c = 12 1.6 ms (see Table 1). Thus both the one- and three-bond ‘3CH,-‘3CH, satellite spectra are decomposed into the n + m even and odd subspectra in the same experiment. Less intense ‘3C-‘3C satellite spectra arising from the small two-bond couplings are also observed in the spectra of Fig. 2. The assignment of these resonances as two-bond 13C-13C satellites, and not residual parent signals, follows from the observed editing of all long-range satellite spectra into the n + m odd subspectrum and was also confirmed from separate experiments tuned for *Jcc. The results for the edited 13C-13C satellite spectra in Fig. 2 are summarized in Table 1 and are in full agreement with the proton multiplicities for 1,3-dibromobutane. Although the Fig. lc pulse sequence in principle can be applied to separate a conventional INADEQUATE 13C spectrum into a total of seven independent subspectra, it has the limitation that a CH2-CH2 system, for example, cannot be distinguished from a CH3-CH system (see, e.g., Fig. 2). Such ambiguities can occur in the three cases n + m = 2, 3, and 4. A pulse sequence which resolves these problems is outlined in Fig. Id. The 8’ pulse acts on the 13C-13C 2QC and fragment [A] again obtains a {cos 0}“+“’ flip-angle dependence by this editing pulse. In the following delay 272 the trapped proton multiple-quantum coherence must not be allowed to precess freely since the second editing pulse e2 may otherwise reconvert part of it to observable magnetization. A central 180” pulse takes care of this. At the time of this pulse the 13C-13C 2QC is transferred to 1QC which then undergoes a SEMUT sequence. Ideally this gives the following flip-angle dependences for the C, and CB resonances: CA:

{COS

e,)*+y--C0s

e,y

[4al

cB:

{COS

e,y+y--C0s

e,y.

[4bl

Therefore, from Eqs. [4a] and [4b] it is clear that the limitations mentioned above for the Fig. 1c sequence are absent in the experiment outlined in Fig. Id. Furthermore,

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CH3-CHBr-CH2-CH,Br AB

CD

-

d) 1’

FIG. 2. SEMINA ‘)C NMR spectra for 1,3dibromobutane (50% v/v in C&) obtained using the pulse sequence in Fig. lc. The two subexperiments in (a) and (b) were obtained using rc = 12 1.6 ms (see text), and 0 = 0” (a) and 0 = 180” (b), respectively. Spectrum (a) corresponds to the normal INADEQUATE spectrum. Edited SEMINA subspectra for ail CH,-CH, fragments are shown in (c) and (d). (c) The n + M even subspectrum obtained by addition of spectra (a) and (b). (d) The n + WI odd subspectrum obtained as the difference between (a) and (b). All spectra are recorded on the same absolute intensity scale. Experiments were performed on a Varian XL-300 spectrometer at 75.45 MHz.

for sensitivity reasons, a particularly useful version (slightly modified) of the Fig. Id sequence is to combine four experiments obtained using 8, = 0, 180” and & = 0, 180”. This version allows a subediting of the n + M even and odd subspectra, as obtained in Fig. 2, and with the sensitivity of the INADEQUATE experiment being preserved apart from losses caused by relaxation effects and long-range 13C-‘H coupling constants. A more detailed analysis and evaluation of the pulse sequences presented in this communication will be given elsewhere.

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1

‘3C-13C Coupling Constants’ Determined from n + rn Even and Odd SEMINA Subspectra for the CH,-CH, Fragments in 1,3-Dibromobutane (Fig. 2) CHr-CHBr-CHr-CH2Br

n+m

AB

Even

‘JAB ‘JBC

36.6 ” In hertz.

+O.l

’ JC.0 37.0

37.1

Odd

CD

=JAC

lJBD

0.70

1.45

‘JAI> 3.70

Hz

In conclusion, a SEMINA pulse sequence, with the same sensitivity as the conventional INADEQUATE experiment, is recommended for separation of r3CH,13CH, satellite spectra into two subspectra according to n + m being even or odd. This considerably simplifies spectra and assignments in cases of overlapping 13C-13C satellites. Combined with the information from an ordinary SEMUT GL or DEPT GL edited 13Cspectrum this SEMINA experiment is useful in constructing the carbon skeleton for molecular frameworks. Furthermore, a pulse sequence yielding simplifications beyond the 12+ m even/odd level is also proposed. The SEMINA experiments have clear advantages over the recently reported INADEQUATE-SEMI technique (8) both with respect to information content and experimental performance. ACKNOWLEDGMENTS This research was supported by the Danish Natural Science Research Council (J. Nos. 5 1I-15041 and 1l-3933). The use of the facilities at the University of Aarhus NMR Laboratory sponsored by the Danish Research Councils (SNF and STVF) and Carlsbergfondet is acknowledged. The support of a scholar stipend to U.B.S. by Carlsbergfondet is also acknowledged. O.W.S. thanks Professor R. R. Ernst for support and encouragement. REFERENCES 1. H. BILDS~E, S. D~NSTRUP, H. 2. D. M. D~DDRELL, D. T. PEGG,

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