DQF-COSY sequence

Better coherence pathway selection is achieved by cycling more than one of the pulses in the sequence. For a DQF-COSY sequence, we could set N to 2, 2, and 4 for the three 90° pulses, thus making the last pulse the most selective so that we can allow both Ap = — 3 and Ap = 1 while blocking the NOESY Ap = —1. So we need to cycle the first two pulses with 180° phase shifts (360°/2) and the final pulse through a 90° phase shift (360°/4). To do all of these phase shifts independently will require 16 scans (2x2x4) because it requires two steps to sample all possible 180° phase shifts and four steps to sample all possible 90° phase shifts. Because these must be sampled independently, the number of scans required... 

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. 

Note that both pathways with p = 1 during the spin echo and with p = 2 during tx are retained. There are a number of possible phase cycles for this experiment and, not surprisingly, they are essentially the same as those for DQF COSY. If we regard the first three pulses as a unit, then they are required to achieve the overall transformation Ap = 2, which is the same as that for the first two pulses in the DQF COSY sequence. Thus the same cycle can be used with these three pulses going 0123 and the receiver going 0202. Alternatively the final pulse can be cycled 0123 with the receiver going 0 3 2 1, as in section 9.5.5.4. 

If neither PR nor PFG is used, radiation damping may bring about serious problems in water suppression. Because the water magnetization is not attenuated by hard pulses, radiation damping would make the [1, -1] sequence, the inversion-recovery sequence and the DQF-COSY sequence useless, as far as water suppression is concerned. For the simple... 

In 2006, Milosavljevic and co-workers64 reported a study of the complete 4H and 13C NMR assignment of a new triterpenoid saponin, leucantho-side-A (13), from Cephalaria leucantha L. In the course of determining the structure and assigning the spectra, the authors made extensive use of the normal ensemble of 2D NMR experiments in use for the characterization of natural product structures HSQC, HMBC, DQF-COSY, TOCSY, and NOESY. The authors supplemented the aforementioned list of experiments with 2D /-resolved, DINE-(Double INEPT-Edited)-HSQC, and INADEQUATE spectra. The authors made no mention of the use of the connectivity information derived from the 1,1-ADEQUATE spectrum in the assembly of the triterpene nucleus of the molecule but reported extensive tabulations of the 1,1-ADEQUATE correlations that were used to sequence and assign the saccharide resonances of the tri- and di-saccharide sub-units, 14 and 15, respectively, linked to the triterpene nucleus. 

The structures of the compounds were elucidated by a combination of NMR techniques (lH-, 13C-, and 13C-DEPT NMR) and chemical transformation, enzymatic degradation, and as well as mass spectrometry, which gives information on the saccharide sequence. A more recent approach consists of an extensive use of high-resolution 2D NMR techniques, such as homonuclear and heteronuclear correlated spectroscopy (DQF-COSY, HOHAHA, HSQC, HMBC) and NOE spectroscopy (NOESY, ROESY), which now play the most important role in the structural elucidation of intact glycosides. These techniques are very sensitive and non destructive and allow easy recovery of the intact compounds for subsequent biological testing. 

FIGURE 5.10 Pulse sequence for Double Quantum Filtered H—Tf COSY (DQF -COSY). 0 s are 90° pulses and 8 is a fixed delay of the order of a few microseconds. 

The sensitivity of the DQF COSY experiment could be increased by employing magnetic field gradient filtration instead of the phase cycling required in the above sequence. The authors obtained usable DQF COSY spectra from equimolar (or 0.37 M) aqueous solutions of siloxanes in 12 hours (10 mm NMR tubes at 59.595 MHz), illustrating the... 

As before, we use the table to adjust the phase according to the reference axis for each scan. Now we see that the 2IaIb terms alternate sign and cancel as we move from first scan, first term to second scan, second term to third scan, first term and finally to fourth scan, second term. Likewise, the 2IbIa terms alternate sign and cancel as we move down. So the ZQC, which exists between the second and third pulses of the DQF-COSY pulse sequence (Fig. 10.28) does not contribute anything to the observed FID after four scans. Just for completeness, we can show that all of the other terms present at the end of the 90S-fi-90j sequence are also destroyed by the phase cycle... 

We saw with the gradient DQF-COSY experiment that the relatively long gradients ( 1 ms) allow chemical shift evolution that will produce large chemical-shift dependent phase errors in the final spectrum. In the sequence of Figure 11.40, the gradient placed in the second half of the t period will set a minimum value for t of twice the gradient time (and... 

Using DQF-COSY and TOCSY we can link all of the protons within a single spin system, which corresponds to a single amino acid residue. We can classify each spin system as a pattern of chemical shifts unique to one amino acid or as a member of a class AMX or five spin. In order to get sequence-specific assignments, however, we have to have some way to correlate protons in one residue to protons in the next residue in the sequence. For unlabeled proteins this is done by NOE interactions certain protons in one residue are constrained by the peptide bond to be close in space to certain protons in the next residue. These NOE correlations are called sequential or z, i + 1 because they correlate a proton in residue z with a proton in the next residue in the sequence, residue z + 1. Specifically, we expect to see NOE correlations between Ha of residue z and Hn of residue z + 1 (Fig. 12.15) and sometimes between the protons of residue z and the Hn of the next residue. Because the DQF-COSY and TOCSY spectra correlate protons within a residue, we can move from... 

The NOESY spectrum (Fig. 12.21, right) gives the sequential connectivity, the proof that the Ser residue on the right side is followed in the primary sequence by the Val residue on the left side. On the vertical Hn = Val line we see the intraresidue crosspeaks to the Ha and Up of valine (which also appear in the TOCSY spectrum), but there is a new crosspeak that connects the of the Ser spin system (in F ) with the Hn of the Val spin system (in /Y). This crosspeak lines up with the intraresidue crosspeak on the serine Hn line (Hn = Ser in F2 and = Ser in F ) that is in the exact position of a crosspeak in the DQF-COSY... 

COSY can be combined with a z filter (see Section 9.5) into z-COSY, which is occasionally used to produce absorption phase spectra. A 90°, T, 90° sequence replaces the second 90° pulse in the usual COSY sequence, just as in DQF-COSY (see Fig. 12.6a). In this case, however, pulse 2 rotates in-phase magnetization to the z axis, where it is stored for the short r period and restored to the xy plane by pulse 3. Suitable phase cycling or imposition of a pulsed field gradient eliminates the coherences remaining in the xy plane during r, so that the final... 

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... 

Previous sections have already made the case for acquiring COSY data such that it may be presented in the phase-sensitive mode. The pure-absorption lineshapes associated with this provide the highest possible resolution and allow one to extract information from the fine-structure within crosspeak multiplets. However, it was also pointed out that the basic COSY-90 sequence suffers from one serious drawback in that diagonal peaks possess dispersion-mode lineshapes when crosspeaks are phased into pure absorption-mode. The broad tails associated with these can mask crosspeaks that fall close to the diagonal, so there is potential for useful information to be lost. The presence of dispersive contributions to the diagonal may be (largely) overcome by the use of the double-quantum filtered variant of COSY [37], and for this reason DQF-COSY is the experiment of choice for recording phase-sensitive COSY data. 

Figure 5.72. The INADEQUATE sequence and the corresponding eoherence transfer pathway. The experiment selects double-quantum coherence during the evolution period with a suitable phase cycle (analogous to the selection in the DQF-COSY experiment. Section 5.6.2). In doing so it rejects all contributions from all uncoupled spins.

The basic components of the INADEQUATE phase cycle comprise doublequantum filtration and fi quadrature detection. The filtration may be achieved as for the DQF-COSY experiment described previously, that is, all pulses involved in the DQ excitation (those prior to ti in this case) are stepped x, y, —X, —y with receiver inversion on each step (an equivalent scheme found in spectrometer pulse sequences is to step the ftnal 90° pulse x, y, —x, —y as the receiver steps in the opposite sense x, —y, —x, y, other possibilities also exist). This simple scheme may not be sufficient to fully suppress singlet contributions, which appear along fi = 0 as axial peaks and are distinct from genuine C-C correlations. Extension with the EXORCYCLE sequence (Section 7.2.2) on the 180° pulse together with CYCLOPS (Section 3.2.5) may improve this. Cleaner suppression could also be achieved by the use of pulsed field gradients, which for sensitivity reasons requires a gradient probe optimised for C observation. 

---