2D TOCSY

Gradient-enhanced 2D TOCSY spectrum of 10 mMof sucrose in D..,0 is shown in figure 7.26. The clean spectrum obtainable without any noise and without the nece.ssity of any phase cycling illustrates the power of this new technique in modern NMR spectroscopy. 

The stereochemistry and conformation of di-Schiff bases containing calix[4]arene unit U(IV) complexes have been deduced on the basis of the 2D TOCSY and T-ROESY measurements.116... 

Fig. 17.9 The H assignment of the unlabeled pheromone 2-see-butyl-4,5-dihydrothiazole, bound to [U-13C, 15N]-labeled mouse major urinary protein I, was accomplished by comparing the 2D TOCSY spectrum of free ligand (A) to the 13C-fil-tered TOCSY spectrum of the complex (B). Reproduced with permission from Ref. [37]. Copyright 1999 American Chemical Society.

NMR. The 300 MHz iR-spectrum of V-2 in D2O is shown in figure 5a. The three multiplets between 1.4 and 2.05 ppm, the two double doublets between 3.4 and 3.7 ppm, and the multiplet between 3.6 and 3.8 ppm comply well with a hydroxylysine substituent at a ring nitrogen (fig. 5b, table 2). No definite conclusion as to the total number of protons can be drawn from the spectrum, since impurities seem to be present, for example singlets at 2.2, 2.05 and 1.95 ppm. The compound V-2 was purified further by ion exchange- and adsorption chromatography and a 500 MHz ID iH- (fig. 6b) and a 2D TOCSY-spectrum (in D2O) were run. Also here, the main spin system present is the hydroxylysine residue (fig. 5b). 

The principle of multiple selective excitation has been incorporated into a few ID and 2D experiments, the schemes of which are shown below (fig. 1). Depending on the experiment, either a DANTE pulse train (ID TOCSY [2]), frequency selective 180° pulses (ID NOE [3], ID INADEQUATE [4], ID C/H COSY [5] and 2D TOCSY-COSY [6]) or frequency selective 90° pulses (2D HMBC [11]) are applied to selectively perturb and uniquely label selected spins. Besides the DANTE pulse , composed itself of a series of non-selective rectangular pulses, Gaussian-shaped 180° and... 

Fig. 1. Pulse sequences modified for multiple selective excitation. I ID TOCSY, II het-eronuclear ID NOE, III ID INADEQUATE, IVa heteronuclear ID COSY (optimized to detect Jch), IVb heteronuclear ID COSY (optimized to detect "Jch), V 2D TOCSY-COSY, Via 2D HMBC (designed to detect heteronuclear long-range couplings "Jch only), VIh 2D HMBC (extended pulse sequence to detect both heteronuclear long-range "Jch and...

TOPHAT-shaped 90° pulses are used in other cases as the best compromise with respect to the excitation profile, the phase homogeneity and length. Depending on the type of the detected spin-spin interaction - being either scalar or dipolar coupling - each selected spin is initially perturbed only once (ID TOCSY, ID INADEQUATE, ID C/H COSY, 2D TOCSY-COSY and 2D HMBC), or for several times (ID NOE). With each of the selected spins initially perturbed only once the inherently smaller transient NOEs would be detected in the latter case, whereas with the multiple excitation of a selected spin within the NOE build-up period the stronger steady-state NOEs are more or less approximated. 

Fig. 7. Normal COSY spectrum (expansion of the ring-protons) of the trisaccharide 1 dissolved in CDCI3 (d). Individual COSY subspectra A, B and C ((a), (b) and (c)) of the three monomer units, obtained with the optimized 2D TOCSY-COSY experiment V. The three anomeric protons were used to selectively perturb the three spin systems. They are assigned and their connectivities to protons 2A, 2B and 2C, respectively, are indicated. (Continued...

The pioneering work in this field, a two-dimensional relayed-NOE experiment proposed by Wagner [7], was quickly followed by the appearance of several related NMR techniques [8-17]. Application of isotropic mixing during the J-transfer period yielded the 2D TOCSY-NOESY [11, 15] and NOESY-TOCSY [12, 14] experiments. When spin-lock sequences were applied to both J and NOE-transfers, the 2D TOCSY-ROESY and ROESY-TOCSY experiments [10, 16, 17] emerged. 

The pulse sequence for ID TOCSY is a ID modification of the original TOCSY experiment [2] introduced by Braunschweiler and Ernst. The TOCSY experiment was also referred to as HOHAHA (which stands for HOmonuclear HArtman-HAhn) by Bax and Davis [3]. The ID TOCSY experiment was proposed by Bax and co-workers [4, 5], and by Kessler et al. [6]. The essential features of the pulse sequence involve the use of selective excitation of a desired resonance, followed by a homonu-clear Hartman-Hahn (or isotropic) mixing period [2, 7]. That is, the unit -Pnonsei - in the 2D TOCSY pulse sequence is replaced by Fsei -where P stands for a pulse (or pulses), ti is the evolution period in the 2D experiment and r is a fixed delay. 

By 2D TOCSY NMR spectroscopy (TOCSY - total correlated spectroscopy), the structure of a biosynthetic intermediate of PQQ was shown to be 3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic acid 15, not its constitutional isomer 16 <2004JA3452>. This result shows that the last enzyme on the biosynthetic pathway of PQQ facilitates a pyrrole ring closure and an unprecedented eight-electron oxidation of 15. 

Encapsulation of herbicides within anionic clays was readily identified by the loss of HRMAS NMR signal associated with immobilization of the molecules between clay layers.103 The application of HRMAS to soil samples has been shown to provide important results on the interaction of herbicide and other organic components with the soil matrix, using ID lH HRMAS, selective TOCSY and 2D TOCSY experiments.104 Significant advantages to the HRMAS approach are its reduced samples preparation needs, with no extraction, pre-treatment or purification required. 

An advantage of 2D TOCSY is that the net coherence transfer produced can be arranged to create pure positive absorption spectra, including the diagonal peaks, rather than spectra with equal positive and negative intensities obtained with differential coherence transfer as in the COSY experiment. 

Fig. 3.23 The 2D spectrum of peracetylated glucose from a 2D TOCSY experiment. The same sample has been used and the expansion is the same as for the 2D phase-sensitive, DQ-filtered COSY spectrum (Fig. 3.21). Note the additional cross peaks obtained with the TOCSY experiment.

Load the raw data obtained for peracetylated glucose with the 2D TOCSY experiment D NMRDATA GLUCOSE 2D HH GHHTO 001001.SER and perform a 2D FT following the guidelines given above. Enter the Manual phase correction option in the Process pull-down menu and perform a phase correction in F2 and Ft according to the procedure outlined above. Try to phase all peaks to positive absorption and store the spectrum (... 001001. RR). 

Through the H- C COSY (HMQC) spectrum, aglycone protons are identified and - H COSY (better E-COSY) or 2D-TOCSY experiments allow to assign all protons in the molecule (except for methyl groups). 

An additional 2D TOCSY spectrum (Figure 6.12) measured for row 11 (stop at 3.3 min) shows that one of the two co-eluting compounds (2 and 5) is a nonphosphorus material (compound 5). 

Figure 6.12 2D TOCSY spectrum with 31P decoupling acquired at row 11 (stop at 3.3 min) spectral size, 4k x 160 data points 8 scans per increment 65 ms TOCSY mixing time...

We saw a 2D TOCSY spectrum in Figure 9.22 and compared it to a COSY spectrum in the TOCSY spectrum, we have more peaks because starting from any proton in the spin system we can see correlations to all other members of the spin system, not just to the protons connected by a single J coupling. We saw the same thing in a real example by comparing the COSY spectrum of 3-heptanone (Fig. 9.23) with the TOCSY spectrum of the same sample (Fig. 9.24). 

The 600 MHz 2D TOCSY spectrum of cholesterol (Chapter 8, Fig. 8.35) is shown in Figure 9.44. Note that all peaks (diagonal and crosspeaks) are positive (black) and in-phase. 

---