Practical Aspects of Carbon-13 Double Quantum NMR
We want to address an erroneous view that has recently been advanced on 13C double quantum NMR. A recent publication (I) introduced a modified version (I) of the original double quantum NMR experiments; this new version claims to offer an attractive increase in sensitivity by a factor of three, or a decrease in the size of the required data matrix by a factor of four. To avoid disappointment among spectroscopists we wish to make it clear that this modified version offers no gain in sensitivity whatsoever in comparison with the other double quantum experiments (2-6), provided they are properly optimized. It is shown here that the sensitivity of this modified experiment can be even slightly lower. The claim of a reduction of the size of the required data matrix is also shown to be incorrect. In the original two-dimensional double quantum “C experiments, the acquisition time t2 starts immediately after the final “read pulse” (Fig. 1), and the time during which the double quantum coherence evolves is labeled ti. The detected 13C-13C satellites (7) are modulated in amplitude as a function of tl with the double quantum frequencies (2). In the quadrature-Fi-detection version of this experiment (3), results are combined with those of a second experiment which contains a composite 45: pulse (8) in the evolution period. This allows the sign of the modulation frequency to be determined, which simplifies the interpretation of the results. A simpler and more sensitive alternative to obtain quadrature F, detection has been proposed more recently (6); an increase of the flip angle of the final “read pulse” to 135” provides directly satellite signals which are modulated in phase as a function oft,. In either case, data acquisition is started after the final read pulse and the coherence transfer echo (9) is detected (4, 6). The time domain signal of a carbon A, directly coupled to carbon X, is then given by sA(tl, t2) = c exp[-i(nA + &)t,] exp[i& + d&2]
HSQC-1,1-ADEQUATE and HSQC-1,n-ADEQUATE: enhanced methods for establishing adjacent and long-range 13C-13C connectivity networks.
1H-13C GHSQC and GHMBC spectra are irrefutably among the most valuable 2D NMR experiments for the establishment of unknown chemical structures. However, the indeterminate nature of the length of the long-range coupling(s) observed via the (n)J(CH)-optimized delay of the GHMBC experiment can complicate the interpretation of the data when dealing with novel chemical structures. A priori there is no way to differentiate 2J(CH) from (n)J(CH) correlations, where n ≥ 3. Access to high-field spectrometers with cryogenic NMR probes brings 1,1- and 1,n-ADEQUATE experiments into range for modest samples. Subjecting ADEQUATE spectra to covariance processing with high sensitivity experiments such as multiplicity-edited GHSQC affords a diagonally symmetric 13C-13C correlation spectrum in which correlation data are observed with the apparent sensitivity of the GHSQC spectrum. HSQC-1,1-ADEQUATE covariance spectra derived by co-processing of GHSQC and 1,1-ADEQUATE spectra allow the carbon skeleton of molecules to be conveniently traced. HSQC-1,n-ADEQUATE spectra provide enhanced access to correlations equivalent to 4J(CH) correlations in a GHMBC spectrum. When these data are used to supplement GHMBC data, a powerfully synergistic set of heteronuclear correlations are available. The methods discussed are illustrated using retrorsine (1) as a model compound.
Two-dimensional J-resolved 1H n.m.r. spectroscopy for studies of biological macromolecules.
The technique of two-dimensional J-resolved 1H n.m.r. spectroscopy has been extended to handle the very wide spectra of proteins and other macromolecules at 360 MHz. The potential of the method to resolve and assign individual spin multiplets in the complex spectra encountered in structural studies of biopolymers is illustrated with some experiments with amino acids and with a protein, the basic pancreatic trypsin inhibitor.