Double-quantum filtered COSY experiment

The MQC intermediate state in coherence ( INEPT ) transfer can also be used to clean up the spectrum. In this case, we can apply a double-quantum filter (using either gradients or a phase cycle) to kill all coherences at the intermediate step that are not DQC. We will see the usefulness of this technique in the DQF (double-quantum filtered) COSY experiment (Chapter 10). As with the spoiler gradient applied to the 2IZSZ intermediate state, a doublequantum filter destroys any unwanted magnetization, leaving only DQC that can then be carried on to observable antiphase magnetization in the second step of INEPT transfer. 

Double Quantum Filtered COSY The double quantum filtered COSY experiment (DQF-COSY, Section 6-1 and Figure 6-16) is similar to COSY, with three 90° pulses in the sequence 90°-/i-90°-T-90°-f2 (acquire). The DQF-COSY experiment is performed in the phase-sensitive mode, but, unlike the situation in the phase-sensitive COSY experiment, in the DQF-COSY both diagonal and cross peaks can be phased as absorptive signals. This difference not... 

The application of three and more gradients is necessary if multiquantum states are to be suppressed as in the double-quantum filtered COSY experiment or if CT pathways in heteronuclear spin systems should be selected in sequences which include magnetization transfer steps. 

Figure 1. Pulse sequences of some typical 2D-NMR experiments. COSY = correlation SpectroscopY, DQFCOSY = Double Quantum Filtered COSY, RELAY = RELAYed Magnetization Spectroscopy, and NOESY = Nuclear Overhauser Effect SpectroscopY.

H/ H-double quantum filtered COSY Note that with this experiment both the diagonal and the cross peaks may be phased to pure absorption. Therefore it is best to select diagonal peaks at the extremes and in the center of the spectrum for phase adjustment. The cros.s peaks when correctly phased consist of positive and negative peaks, which are anti-phase with respect to the active, and are in-phase with respect to the passive coupling(s). 

A simple variant of the COSY experiment is COSY-35 (sometimes called COSY-45), in which the second 90° pulse is reduced from a 90° pulse to a 35° or 45° pulse (Fig. B.3). The result is that the fine structure of crosspeaks is simplified, with half the number of peaks within the crosspeak. This makes it much easier to sort out the coupling patterns in both dimensions and to measure couplings (active and passive) from the crosspeak fine structure. A more important variant of the COSY experiment is the DQF (double-quantum filtered)— COSY (Fig. B.4), which adds a short delay and a third 90° pulse. The INEPT transfer is divided into two steps antiphase I spin SQC to I,S DQC, and I,S DQC to antiphase S spin SQC. The filter enforces the DQC state during the short delay between the second and third pulses either by phase cycling or with gradients. DQF-COSY spectra have better phase characteristics and weaker diagonal peaks than a simple COSY, so this has become the standard COSY experiment. 

Today, a number of one- and two-dimensional NMR experiments are available for the detection of homonuclear Li, Li and Li, Li couplings. Aside from the COSY experiment, the double quantum filtered COSY (COSY-DQF), the TOCSY, and the ID and 2D INADEQUATE experiments [24] have been successfully employed. An attractive feature of all these experiments is their sensitivity for small scalar interactions which give rise to crosspeaks even if line splittings in the corresponding ID spectra are not resolved. This was first demonstrated with COSY experiments for a paramagnetic nickel complex [82] and for quadrupolar nuclei in the case of boron-11 [83]. 

The DQF-COSY sequence (Fig. 5.40) differs from the basic COSY experiment by the addition of a third pulse and the use of a modified phase-cycle or gradient sequence to provide the desired selection. Thus, following tj frequency labelling, the second 90° pulse generates multiple-quantum coherence which is not observed in the COSY-90 sequence since it remains invisible to the detector. This may, however, be reconverted into single-quantum coherence by the application of the third pulse, and hence subsequently detected. The required phase-cycle or gradient combination selects only signals that existed as double-quantum coherence between the last two pulses, whilst all other routes are cancelled, hence the term double-quantum filtered COSY. 

The homonudear 2-D NMR experiments that use I-coupling indude the correlation spectroscopy (COSY, and variants induding gradient-selected COSY or gCOSY, double-quantum filtered COSY or DQF-COSY) experiment, the total correlation spectroscopy (TOCSY) experiment, and the incredible natural abimdance double quantum transfer experiment (INADEQUATE) [3]. 

In some instances, we may find that examination of a cross peak close to the diagonal (5j is almost equal to 82) is difficult because of distortion of the cross peak due to intensity on the diagonal of the spectrum. The double-quantum filtered COSY (DQF-COSY) experiment fortunately provides a method for suppression of most of the signal intensity found on the diagonal. 

Double-quantum filtered COSY is now the most widely used version of the COSY experiment. It has two main advantages over its precursor diagonal and off-diagonal peaks can be phased to be absorptive simultaneously, and singlets are removed from the spectrum. The pulse sequence is... 

The first factor governing the sensitivity of the experiment is the nudeus detected these are shown in the second column of Table 4. As discussed in Section 2.06.2.1 for the same number of nuclei, the signals from are almost 2 orders of magnitude greater than those from In cases where limited sample is available, H-detected experiments are preferred. Most 2D-NMR experiments produce correlations between nudei, with the correlations indicating the presence of spedfic NMR interactions such as coupling. The most sensitive NMR experiments produce correlations between abundant spins, for example, between two H s. These are called homonudear correlation experiments. Examples of these are correlation spectroscopy (COSY), double quantum filtered COSY (DQ-COSY),... 

Suppression of the tme diagonal peaks by double-quantum filtering (DQF-COSY) may resolve such problems. Finally, quantitative measurements of the magnitude of the coupling constants is possible using the Z-COSY modification, These experiments ate restricted to systems of abundant spins such as H, and which have reasonably narrow linewidths. 

Figure 1.45 Coherence transfer pathways in 2D NMR experiments. (A) Pathways in homonuclear 2D correlation spectroscopy. The first 90° pulse excites singlequantum coherence of order p= . The second mixing pulse of angle /3 converts the coherence into detectable magnetization (p= —1). (Bra) Coherence transfer pathways in NOESY/2D exchange spectroscopy (B b) relayed COSY (B c) doublequantum spectroscopy (B d) 2D COSY with double-quantum filter (t = 0). The pathways shown in (B a,b, and d) involve a fixed mixing interval (t ). (Reprinted from G. Bodenhausen et al, J. Magn. Resonance, 58, 370, copyright 1984, Rights and Permission Department, Academic Press Inc., 6277 Sea Harbor Drive, Orlando, Florida 32887.)...

The data from H NMR studies of 63, which included double quantum filtered phase sensitive correlated spectroscopy (DQF-COSY) and rotating frame nuclear Overhauser effect spectroscopy (ROESY) experiments (Figure 12), are collected in Table 17. 

The double quantum filter eliminates or at least suppresses the strong signals from protons that do not experience J-coupling, e.g. the solvent signal, which would otherwise dominate the spectrum and possibly be a source of troublesome tl noise. Compared to a phase-sensitive but non-DQ-filtered COSY with pure absorption lineshapes for the cross peaks but mixed lineshapes for the diagonal peaks, the phase-sensitive, DQ-filtered COSY has pure absoiption lineshapes throughout. 

Double quantum filters can be used as a component of many different 2D, 3D, and 4D experiments. In some applications, COSY benefits from DQF, as we discuss in the next section. 

By allowing multiple-quantum coherence to process during the evolution period of a two-dimensional experiment, Drobny et al. were able to detect its effects indirectly. This idea subsequently blossomed into the new technique of filtration through double-quantum coherence. Multiple-quantum coherence of order n possesses an n-fold sensitivity to radiofrequency phase shifts, which permits separation from the normal single-quantum coherence. This concept inspired the popular new techniques of double-quantum filtered correlation spectroscopy (DQ-COSY) and the carbon-carbon backbone experiment (INADEQUATE), both designed to extract useful connectivity information from undesirable interfering signals. 

All proton NMR spectra were recorded on a Varian Unity 600 at 25 C. 6 to 10 mg of the disulfide linked c-Myc-Max heterodimeric LZ were dissolved in 0.5 mL of potassium phosphate buffer (50 mM, 10% DiO / 90% H2O and pH 4.7) containing 100 mM KCl and ImM 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS) to yield solutions ranging from 0.75 to 1.25 mM. Proton resonances were assigned from two-dimensional double quantum filtered correlation spectroscopy (DQF-COSY (21)), two-dimensional total correlation spectrocopy (TOCSY mixing time = 50 ms (22)) and two-dimensional nuclear Overhauser enhancement spectrocopy (NOESY mixing times = 150 and 200 ms (23)) experiments. Sequential assignment of the proton resonances was performed as described by Wuthrich (24). 

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