COSY-TOCSY

Proton resonances for aU residues were assigned using a combination of COSY, TOCSY, HSQC, and HMBC experiments. The large values of y(NH/H-C(/9)) and the small values of J(H-C(a)/H-C(y9) were indicative of antiperiplanar and synclinal arrangements respectively, around those bonds. In addition, medium-range NOE connectivities H-C(/ )i/NH +i, H-C(a)i/NH, + i, NHi/NH +i were consistent... 

A detailed analysis of the proton high field NMR spectra of tomato juice and pulp has recently been acquired [15]. The combination of suitable selective and two-dimensional techniques (J-resolved, COSY, TOCSY, DOSY, etc.) was used for... 

Applications Useful 2D NMR experiments for identification of surfactants are homonuclear proton correlation (COSY, TOCSY) and heteronuclear proton-carbon correlation (HETCOR, HMQC) spectroscopy [200,201]. 2D NMR experiments employing proton detection can be performed in 5 to 20 min for surfactant solutions of more than 50 mM. Van Gorkum and Jensen [238] have described several 2D NMR techniques that are often used for identification and quantification of anionic surfactants. The resonance frequencies of spin-coupled nuclei are correlated and hence give detailed information on the structure of organic molecules. 

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 second Chilocorini species studied, Chilocorus cacti, contained several alkaloids closely related to the former. The structure of the heptacyclic chilo-corine A (19) was deduced from a series of NMR experiment (DEPT, HMQC, DQ-COSY, TOCSY) and from a comparison of its spectral properties with those of exochomine (18) from which it differ by having two C-C linkages between the two tricyclic partners. However, the configuration at the asymmetric center in the azaacenaphthylene ring was not established [33]. 

In many cases, the analytical tasks are simply to detect and quantify a specific known analyte. Examples include the detection and quantification of commonly used buffer components (e.g., Tris, acetate, citrate, MES, propylene glycol, etc.). These simple tasks can readily be accomplished by using a standard one-dimensional NMR method. In other situations, the analytical tasks may involve identifying unknown compounds. This type of task usually requires homonuclear and heteronuclear two-dimensional NMR experiments, such as COSY, TOCSY, NOESY, HSQC, HMBC, etc. The identification of unknown molecules may also require additional information from other analytical methods, such as mass spectrometry, UV-Vis spectroscopy, and IR spectroscopy.14... 

Figure 6.47 shows a decision scheme for the analysis of impurities by LC-NMR at 500 MHz. The weights indicate the amount of analyte required in the active volume of the flow cell to obtain good quality data from which reliable conclusions can be drawn. For a cryogenic flow probe with an active volume of 120 p,l, 0.3 [tg of analyte is required to give a one-dimensional proton spectmm in 2 h, approximately 2 ig for two-dimensional homonuclear spectra (COSY, TOCSY) and 25 ig for inverse correlation spectra (HMQC, HMBC). Cryogenic flow probes with lower active volumes (30 or 60 p,l) have higher mass sensitivity and therefore require less analyte to acquire spectra of the same quality. 

One-dimensional spectra obtained in these experiments can be compared to ID traces of nD NMR spectra but offering much better digital resolution and shorter acquisition times. On the negative side each trace needs to be acquired separately and thus, if several sites are to be inspected, a series of ID experiments must be performed. In practice, this exercise is preceded by careful inspection of standard two-dimensional COSY, TOCSY, NOESY or ROESY spectra and only the ambiguous assignments are tackled by combined ID techniques. 

Fig. 15. ID COSY-TOCSY spectra of the oligosaccharide 5 acquired using the pulse sequence of fig. 13(b) with the initial polarization transfer from overlapping anomeric protons of two terminal glucoses, (a) Partial H spectrum of 1 at 600 MHz after 8 scans. The summation (b) and subtraction (c) of two ID COSY-TOCSY spectra acquired using the following parameters for the original spectra (not shown) = 50 ms, to = 39 ms, Tr = 28 ms,...

Replacement of the TOCSY transfer in a ID COSY-TOCSY experiment by the NOESY step yields a ID COSY-NOESY sequence [38] (fig. 13(c)). The experiment is illustrated by the determination of NOEs from the H-7 proton of a terminal 3,6-dideoxy-4-C-(l-hydroxyethyl)-D-xylohexose (6)... 

The complex structure isolated from Scleranthus uncinatus, 5,7-dihydroxy-3 -meth-oxy-4 -acetoxyflavone-8-C-p-D-xylopyranoside-2 -0-glucoside, was determined by using ID NMR ( H, C, DEPT) and 2D NMR (H-COSY, TOCSY, HMQC, HMBC, NOESY) data sequence and linkage of the sugar chain and acylation site were confirmed by observation of inter-residue NOEs in the NOESY spectrum. 

Nuclear magnetic resonance (NMR) has proved to be a very useful tool for structural elucidation of natural products. Recent progress in the development of two-dimensional 1H- and 13C-NMR techniques has contributed to the unambiguously assignment of proton and carbon chemical shifts, in particular in complex molecules. The more used techniques include direct correlations through homonuclear (COSY, TOCSY, ROESY, NOESY) [62-65] and heteronuclear (HMQC, HMBC) [66. 67] couplings. 

Today, it is possible to make complete assignments of all proton and carbon atoms in the NMR spectra of most isolated anthocyanins. These assignments are normally based on chemical shifts (8) and coupling constants (J) observed in 1-D H and l3C NMR spectra (Fig. FI.4.2), combined with correlations observed as cross-peaks in various homo- and heteronu-clear 2-D NMR experiments (see below for details on COSY, TOCSY, HSQC, HMBC, NOESY, and ROESY). 

In natural products analysis, most frequently the stop-flow mode is chosen to acquire H spectra of the compounds of interest, or if further structural information is required to perform two-dimensional H NMR spectra, such as COSY, TOCSY, NOESY or ROESY. In many cases an on-flow NMR chromatogram (usually at flow rates between 0.3 and 1ml min-1) is recorded beforehand, either to screen for the presence of particular groups of compounds or to gain a general overview on the sample composition. (Heteronuclear LC-NMR experiments, such as HSQC and HMBC of a natural product, have been reported in the literature once [9] however, this was of a highly enriched fraction.) More recently, time-sliced stop-flow [14,16] and on-flow approaches at low flow rates [34,35] have been applied to natural product extracts in order to combine the advantages of both on-flow (a ready overview on the entire sample) and stop-flow (sufficient acquisition time for minor compounds) modes. 

In 1989, the first applications of multidimensional NMR were applied to humic substances (Buddrus et al., 1989).This study involved the application of 13C detected J-resolved (J-Res) spectroscopy. The study was successful in that it showed multidimensional NMR was applicable to the study of humic substances. However, in 1989 the lack of various modern experiments and the corresponding hardware (mainly probes fitted with pulse field gradients) made applying NMR to humic materials very challenging. In 1997, Simpson et al. demonstrated that the more sensitive inverse-detected NMR experiments were applicable to NOM (Simpson et al., 1997). In this manuscript COSY,TOCSY and HMQC were applied (Simpson et al.,... 

DEPT, H-H COSY, TOCSY, short range C-H chemical shift correlation and long range C-H chemical shift correlation experiments were performed on Bruker AMX 360 and AM400 instruments using acetone-d6 as solvent. C-H chemical shift correlation experiments were carried out with inverse detection. Standard pulse sequence programs provided with the instruments were used for all 2-D experiments. 

Although ID experiments are useful as a first step in the analysis of peptides, 2D NMR is really the workhorse technique of peptide NMR, with DQF-COSY, TOCSY, and NOESY being the main techniques used for homonuclear studies and HSQC for heteronuclear studies. 

Vulcanisate structures of BR crosslinked with cyclic disulfides was studied by NMR.36 Using high resolution MAS techniques of DEPT, COSY, TOCSY, and HETCOR, the resulting spectra showed that crosslinking gave an addition product to the double bond and not the allylic structure found in typical sulfur vulcanisations. 

The combined information from the l3C/DEPT, COSY, TOCSY, and HMQC enables us to assign all protons in the 3H spectrum except those that are exchanging rapidly (i.e., the carboxyl and free amino protons) and all of the non-quaternary carbon resonances in the 13C spectrum. There is no need to assign the rapidly exchanging protons, and the non-quaternary carbons will be assigned during the sequencing discussion. 

The elucidation of the primary structure of small organic molecules by tracing their carbon skeletons was, traditionally, the main focus of INADEQUATE experiments. It is not the first NMR experiment to be considered for such a task typically a standard set of NMR spectra, that is COSY, TOCSY, NOESY, HSQC and HMBC, are performed and analysed first. If ambiguities remain after inspection of standard spectra, the tracing of carbon-carbon connectivities is embarked on. Nevertheless, an example is presented below where carbon-carbon connectivities are included at an earlier stage in order to reduce the number of computer-generated structures compatible with the experimental data. 

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