Heteronuclear J spectroscopy

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Heteronuclear J Spectroscopy Most applications of heteronuclear two-dimensional NMR spectroscopy (1) have been concerned with the correlation of chemical shifts of unlike nuclei (2-7). The same two-dimensional method offers a unique way to measure proton-proton couplings indirectly (2, 8, 9). This approach may be advantageous in complex systems where the ordinary proton spectrum is too crowded for analysis, or obscured by resonances due to the solvent and to impurities. The indirect measurement of homonuclear proton couplings in ribose rings in nucleotides provides a new tool for the conformational analysis of such systems (8, 9). If the measurement of the coupling constants is the primary object of the experiment, the chemical shift of the protons is often of little interest, while the resolution of the multiplet structure is of paramount importance. Since heteronuclear two-dimensional spectra are normally obtained with carbon-13 or phosphorus-31 probes designed for lo-mm sample tubes, the homogeneity of the static field is invariably unfavorable for the resolution of proton-proton interactions. This problem may be alleviated by a new twodimensional Fourier transform technique which combines features of spin-echo (10) or J spectroscopy (11, 12) with heteronuclear NMR (I) to enhance the resolution of the proton spectrum. In the normal heteronuclear experiment (I), two 90” pulses are applied to the protons, separated by an evolution period tl, and followed by a 90” phosphorus-31 observation pulse with subsequent signal acquisition. In the modified experiment, a pair of 180” pulses are applied in synchronism to both proton and phosphorus resonances at the midpoint of the evolution period. As a result, both the proton chemical shifts and the dephasing associated with the imperfect homogeneity of the static field are canceled at the end of the evolution period, just before the transfer of the magnetization from the proton to the phosphorus transitions. On the other hand, the precession of the proton magnetization due to either heteronuclear or homonuclear couplings is not refocused, and the echoes are modulated by JPH and JHH as a function of tl. It is assumed that the weak coupling approximation applies to the homonuclear interactions. At first sight, the cancellation of the proton shifts appears to complicate the two-dimensional experiment, since the signals will appear at the origin of the F1 frequency domain. In cellular phosphates, the 3JPocH coupling to the nearest proton is typically 5 to 10 Hz, leading to complicated overlapping multiplets in the presence of homonuclear proton couplings, which are of the same order of magnitude. If these multiplets “pile up” at the origin of the Fl domain, complex aliasing problems arise, particularly when a real, single-sided Fourier transformation is used. (In amplitudemodulated spectra, a real transform is preferable since it produces two-dimensional absorption lines rather than phase-twisted lineshapes (13).) The remedy to this confusion is the introduction of an apparent chemical shift, which can be achieved by 175

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advancing the phase of the proton echo by 90” in each increment of the evolution time tr. In this study, the rf phase of the initial 90” pulse was shifted together with the phase of the refocusing pulse. A similar approach has been employed before to introduce a tl modulation when the basic pulse sequence yields unmodulated signals (14), and has recently found applications in multiple-quantum spin-echo spectroscopy (15, 16). In heteronuclear spectra, the phase cycling method shifts all resonances by AFI = (4AtJ’ corresponding to one-half of the spectral width in the FI domain. Thus the proton transmitter can be set in the middle of the proton spectrum without introducing aliasing problems, and the technique does not interfere with ordinary quadrature detection, which is routinely used in the F2 or phosphorus dimension. The phase-cycling scheme represents a simple alternative to the “quadruple phase detection” approach recently proposed by Ernst and coworkers (7, 17) since it does not require additional software for a hypercomplex Fourier transform. To eliminate the unmodulated component in the phosphorus spectra, the phase of the last 90” proton pulse is alternated while the phosphorus free-induction decays are added and subtracted in alternate transients (1). The heteronuclear J spectrum of trimethylphosphate, shown in part in Fig. 1, consists of a decaplet in the F2 or phosphorus dimension and a doublet in the FI or proton domain. This doublet appears precisely centered at FI = 20 Hz, corresponding to the artificial chemical shift due to the phase-cycling procedure. The evolution period was incremented in 200 steps of 12.5 msec each. The FI linewidth is 0.30 Hz, which is quite narrow for a proton spectrum at 270 MHz in a lo-mm sample tube, though spin-diffusion effects (18) cannot be excluded. The basic rules governing the signal amplitudes are not modified by the use of refocusing pulses. The normal

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FIG. 1. Selected sections taken from a two-dimensional heteronuclear J spectrum of trimethylphosphate, obtained at a phosphorus resonance frequency of 109.3 MHz with proton pulses generated by gating the 270-MHz decoupler. Only 4 of the 10 phosphorus resonances are shown in the vertical F2 domain. The proton doublets appearing in the horizontal Fl dimension have linewidths that are limited only by transverse relaxation and spin diffusion.

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binomial intensities of the decaplet are enhanced by a factor proportional to the algebraic frequency difference between a given line in the multiplet and the center of the multiplet. The resulting intensity distribution in the F2 domain follows an unusual pattern: +l, +7, +20, +28,+14,

-14, -28, -20, -7, -1.

Only the four central resonances in the F2 domain are shown in Fig. 1. Clearly, this experiment contains no novel information, since the magnitude of JPH can be obtained directly from the ordinary phosphorus spectrum. In 2’-guanosine monophosphate (2’GMP), the heteronuclear J spectrum provides an accurate measure of both heteronuclear and homonuclear couplings between the I’, 2’, and 3’ protons in the ribose ring (8). These scalar coupling constants provide insight into the conformation of the nucleotide through Karplus-type relationships, and have been the object of extensive research (19). In Fig. 2, the experimental heteronuclear J spectrum of 2’GMP (middle) shows a good agreement with the stick spectrum (top). The spectrum was obtained by subtracting two sections taken from a two-dimensional plot at the Fz frequencies of the phosphorus doublet (9). Eight

H FIG. 2. subspectra form of a displaced connectivity well with refocusing spectrum

IOHz

The heteronuclear J spectrum of 2’ guanosine monophosphate is made up of the two-proton belonging to the +$ and -$polarizations of the phosphorus spin (8). Each subspectrum has the triplet because of the accidental degeneracy J nl.nI. = JHZ.HB. = 5.35 Hz. The two triplets are by Jprr2. = 7.6 Hz and appear with opposite signs because of progressive and regressive relationships (9). The experimental J spectrum (middle) has linewidths of 1.1 Hz and agrees the stick spectrum (top), but the conventional heteronuclear spectrum, obtained without (bottom) is broadened by the inhomogeneity of the static field across the lo-mm sample. Each required about 2 hours.

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transients were averaged for each of the 200 tl increments. Only half of the lOO-Hz spectral width is shown in the Fl domain, which has been filtered by a 0.25-Hz Lorentzian broadening. The lOO-mM solution in *II20 with 1 A4 NaC104 was passed through a Chelex column and paramagnetic impurities were masked by the addition of ethylenediaminetetraacetate (19) prior to the adjustment of the pH to 7, yet the natural linewidth is too broad to resolve any difference between the two homonuclear couplings. For comparison, the lower trace in Fig. 2 has been obtained under identical conditions except for the fact that the 180” pulses were skipped. This experiment is similar to the original heteronuclear method (I), although the phase-cycling was retained to shift the resonance away from the origin of the FI domain. (The proton transmitter frequency was placed near the chemical shift of the 2’proton at 5.09 ppm (21).) The lines are clearly much broader because of the inhomogeneity, in spite of careful shimming, and the interpretation of the multiplet in terms of the two triplets shown in the stick spectrum is no longer straightforward. In systems with strong coupling among the protons, the proton magnetization vectors will be mixed by the proton 180” pulse. This leads to additional frequencies in the Fl domain and alters the intensity rules of the individual proton resonances (ZZ), while the strength of the coupling also affects the transfer of the magnetization (9). In systems of the ABX type like 2’3’-cyclic guanosine monophosphate (9), the cancellation of the proton chemical shifts leads to the superposition of the multiplets due to the A and B protons, and it appears that J spectroscopy offers no advantage in these systems. J spectroscopy and related techniques have often been neglected, as the gain in resolution does not always justify the time and effort required. In the present case, however, if one assumes that the proton couplings cannot be extracted from the conventional proton spectrum, thus rendering the indirect approach essential, the use of refocusing pulses does not substantially lengthen the experiment, although the maximum value of the evolution period may have to be increased for the best resolution. Since it is no longer necessary to pay attention to the exact position of the proton carrier frequency, heteronuclear J spectroscopy is particularly simple to operate. The pulse sequence described in this Communication bears a superficial resemblance to the Morris-Freeman method for Insensitive Nuclei Enhanced by Polarization Transfer (23), but the type of information sought is quite distinct in the two experiments. ACKNOWLEDGMENTS Helpful discussions with Drs. D. J. Ruben and P. H. Bolton experiments were performed at the NMR facility for Biomolecular and supported by Grant RR00995 from the Division of Research National Science Foundation under Contract C-670.

are gratefully acknowledged. The Research located in this laboratory, Resources of the NIH and by the

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Francis Bitter National Magnet Laboratory Massachusetts Institute of Technology Cambridge, Massachusetts 02139 Received January 8, 1980