Selective 1d Tocsy Using Dpfgse

A selective TOCSY experiment starts with putting the net magnetization of just one resonance in the x'-y' plane and locking it with the TOCSY mixing spin lock. After an appropriate mixing time, the spin lock field is turned off and we simply start acquiring the FID. These steps can be summarized as follows:

1. Preparation: Selectively excite the peak of interest to place its magnetization on the y axis.

2. Mixing: Apply the TOCSY mixing sequence (spin lock) on the y axis for a period of time between 30 and 85 ms.

3. Detection: Record the FID.

Any coherence that we observe in the FID must have come from the selected resonance by TOCSY transfer and therefore must belong to the same spin system. The best selective 90o pulse is DPFGSE, the same building block used as the front end of the selective NOE experiment: A 90o hard pulse followed by a gradient, and then a selective 180o pulse followed by another gradient of the same magnitude and sign as the first (Fig. 8.42). The first gradient twists all the sample magnetization and the shaped 180o pulse refocuses only the selected spins, whereas all other spins are unaffected. The second gradient "unwinds" the coherence helix of the selected spins and doubles the twist of all the other spins. At the end, we have only the selected net magnetization aligned on the y' axis and all other

magnetization is destroyed. The PFGSE sequence is repeated to reinforce the selectivity (not shown in Fig. 8.42), and then the spin-lock mixing sequence is started with phase on the j' axis. Unlike the NOE experiment, we do not need a 90o pulse at the end of the DPFGSE because we already have the selected resonance's net magnetization right where we want it: in the x'-y' plane. At the end of mixing we have positive, in-phase magnetization on y' for the selected spin and all other spins in the spin system that received the transferred magnetization. As it is already in the x'-y' plane, we just turn on the ADC and collect the FID data. The 1D proton spectrum will have in-phase peaks for the selected resonance and for any other resonance to which magnetization was transferred during the spin lock. The intensity of each peak (integral area) will depend on the efficiency of TOCSY transfer from the selected spins. A small coupling constant (e.g., a fixed gauche relationship for vicinal couplings or a long-range (>3 bond) coupling) will lead to a "bottleneck" in TOCSY transfer, reducing the intensity of the destination peak and the peaks of all other protons after it in a linear spin system.

8.12.1 Factors That Affect TOCSY Mixing Efficiency

In the real world, complete mixing throughout the spin system is not observed. Magnetization transfer is a stepwise process starting from the selected proton, so protons near the selected proton in the spin system usually give more intense signals, and protons farther away give weaker peaks in the 1D spectrum. The simplest case, where there are only two protons (Ha and Hb coupled with Jab) in the spin system, has been analyzed precisely. If we start with magnetization on Ha along the y' axis at the beginning of the TOCSY mixing sequence, the magnetization will oscillate between Ha and Hb:

where t is the mixing time (Fig. 8.43). Note that at time zero we have pure Iy, at time t = 1/(2Jab) we have pure Ib and none of the starting magnetization (100% transfer), and at time t = 1/Jab we are back to pure Iy (no transfer). So we can conclude that when magnetization hits the end of a spin system, it "bounces back" and we see oscillatory behavior as a function of mixing time. For a typical vicinal coupling (J = 7.0 Hz), a mixing

TOCSY mixing time

TOCSY mixing time time of 71 ms gives complete transfer. For a small coupling constant such as Jab = 2.0 Hz, you would get only 4% transfer and with Jab = 0.5 (long range) you would get 0.24% transfer. This is why small coupling constants cause a "bottleneck" in transfer of magnetization and most of the magnetization remains on the starting nucleus.

In long spin systems such as flexible chains of CH2 groups, magnetization transfer is more of a diffusion-like process. The signal is strongest on the starting spin and weaker as you move farther away along the chain. With longer mixing times, magnetization spreads farther along the chain. For example, with a mixing time of 70 ms it is usually possible to reach the s position of the lysine side chain (five jumps) starting with magnetization on the amide NH in a peptide or protein:

Short mixing times (e.g., 30 ms) lead to INEPT-type spectra (or COSY-type 2D spectra), where transfer is mostly limited to a single jump over one J coupling. Unlike INEPT and COSY, however, the transfer results in an in-phase rather than antiphase signal. This is a significant advantage as the peaks have the same shape and pattern as they do in a 1D spectrum.

Figure 8.44 shows two selective 1D TOCSY spectra of sucrose in D2O, with 70 ms of MLEV-17 mixing. Selecting the anomeric H-g1 (glucose) proton as a downfield "handle," we can see the spin system of the glucose unit, without any peaks from the fructose unit (Fig. 8.44, center). Because of the a-glycosidic linkage, H-g1 is in an equatorial position and has a small (3.8 Hz) coupling to H-g2. This "bottleneck" accounts for the inefficient transfer to H-g2 and H-g3. The triplet at 3.71 ppm is much larger than the distorted triplet

Figure 8.45

at 3.42 ppm, so we can assign the former to H-g3 and the latter to H-g4. No transfer is observed beyond H-g4 (three jumps). Selecting the H-f3 doublet (fructose unit: Fig. 8.44, bottom), we see the fructose spin system in a 1D proton spectrum, with none of the glucose peaks. The triplet at 3.99 must be H-f4 as we expect more complex splitting for H-f5 and H-f6. The multiplet at 3.83 ppm can be assigned to H-f5 (three couplings) and the tall peak at 3.77 ppm is H-f6. We can assign the two-proton singlet at 3.62 to H-f1, so the entire fructose system is assigned. In this way, we can "light up" one unit (residue) of a biological polymer and see only the peaks due to spins in that residue.

Figure 8.45 shows two selective 1D TOCSY spectra of cholesterol. Between the two spectra at the right-hand side is shown the very crowded and heavily overlapped upfield region of the 1H spectrum of cholesterol for comparison. Selecting the H3 multiplet at 3.54 ppm (Fig. 8.45, top), we can follow the spin system clockwise around the A ring to H2ax and H2eq and on to H1ax and H1eq (Fig. 8.46). The completely resolved H1ax peak is shown in the inset (Fig. 8.45, top); this peak is hopelessly overlapped in the normal 1H spectrum. The dt coupling pattern is due to large coupling constants to H1eq (geminal) and H2ax (axial-axial) and a small coupling to H2eq (axial-equatorial). We are now at a dead end because we run into the quaternary carbon, C10, at the A-B ring juncture. Moving counterclockwise around the A ring from H3 we see H4ax and H4eq, the peaks we selected in the 1D NOE experiment (Fig. 8.35), and then there is a jump through a long-range ("allylic") coupling to H6 (Fig. 8.46). The H6 peak is very small due to this TOCSY transfer "bottleneck" of a small long-range coupling. Then we see transfer from H6 to H7ax and H7eq. These peaks are small primarily because the peak they derive from, H6, is small.

Figure 8.46

Selecting the H6 peak at 5.35 ppm (Fig. 8.45, bottom) gives a relatively weak transfer to H7ax and H7eq, and these efficiently give magnetization to H8 (Fig. 8.47). The inset (Fig. 8.45, bottom) shows the resolved H7eq "doublet" peak, which has one large coupling (geminal coupling to H7ax) and a number of small couplings (to H8, H6, and H4ax). In the other direction (Fig. 8.47), we see weak transfer from H6 to H4ax and H4eq and from these we are just beginning to get transfer to H3 (Fig. 8.47). Note that technically cholesterol is a single spin system (if we count the allylic H4 to H6 coupling), but in reality magnetization does not spread indefinitely. The bottleneck (H4 to H6) confines magnetization to some extent to the A ring, but even without this the spread of magnetization seldom goes more than five jumps (J couplings) in the extreme. Eventually, we "run out of time" for magnetization transfer, and the spins farther away from the selected spin give very weak peaks or none at all. The peak assignments in Figure 8.45 are derived from 2D NMR analysis and are not obvious, based only on the 1D selective TOCSY experiment shown here. We can get some ideas of axial and equatorial from the number of large couplings ("doublet" vs. "triplet" patterns) and we might be able to sort it out completely using a short mixing time (tm — 30 ms) to see the direct "one-jump" relationships only.

Comparing Figure 8.45 (TOCSY) to Figure 8.36 (NOE), we can see that TOCSY transfer is very efficient. Compared to the selected peak, the transfer peaks are of comparable intensity. NOE transfer is very inefficient and, for small molecules, of opposite sign. In the NOE spectrum, the selected peak is negative and enormous compared to the very small (around 1%) positive transfer peaks. This is an important factor in the design of NMR


Figure 8.47


Figure 8.47

experiments: Through-space transfer is very inefficient, whereas J-coupling transfer is very efficient. TOCSY transfer can be so efficient (even 100%, see Fig. 8.43) that the selected peak may be small or even missing in the spectrum!

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