Phase HSQC-TOCSY

Haliclonacyclamine E (13) and arenosclerins A (14), B (15), and C (16) have been isolated from the marine sponge Arenosclera brasiliensis, endemic in Brazil. Crude extracts of this sponge displayed potent cytotoxic and antibiotic activities, and were subjected to fractionation by sihca-gel flash chromatography, medium pressure chromatography on a SiOH cyanopropyl-bonded column, and reversed-phase Cis column chromatography to give compounds 13-16 [18]. The structure elucidation was based on spectroscopic analysis, including HRFABMS, COSY, HSQC, HSQC-TOCSY, and HMBC NMR... 

Check it in 2D WIN-NMR C/H-COSY (HSQC-TOCSY, phase sensitive)... 

Beshah s work proceeded in four stages. Stage 1 involved analysis of the DEPT NMR spectra to identify S (25-40 ppm) and T (70-75 ppm) carbon resonances. The signal near 20 ppm was identified as a methyl resonance (from acetate). Stage 2 involved collection of an HSQC spectmm (Figure 29(a)) so that one-bond H-C correlations could be identified. He did not attempt to interpret this spectmm, but only used it to distinguish between the one-bond and multiple-bond correlations in the HSQC-TOCSY spectmm (Figure 29(b)). In modern versions of the HSQC-TOCSY sequence, spectra are produced with phases of the one-bond crosspeaks inverted relative to those TOCSY crosspeaks, and only one spectmm is needed (see Section 2.06.4.1.4). 

Fig. 8.2. Some of the most common 2D pulse sequences that can be employed using a proper choice of parameters to record 2D spectra of paramagnetic molecules (A) NOESY, (B) ROESY, (C) COSY, (D) ISECR COSY, (E) zero-quantum (double quantum) COSY, (F) TOCSY, (G) HMQC, (H) HSQC. Sequences (A), (B) and (F) are also used to obtain EXSY spectra. SL indicates a soft spin-lock sequence, while MLEV17 indicates a train of spin-locking hard pulses that optimizes the development of J/j coupling. In the reverse heteronuclear experiment (G) the upper and lower levels refer to H and heteronucleus, respectively. The phase cycles are not indicated. For clarity of discussion, all initial pulses can be thought to be applied along the y axis, in such a way that the coherence after the first 90° pulse is always along x. ...

For the purpose of adding a simple model to the following discussion. Figs. 5.1 and 5.2 present an overview of the covariance transformation types 1—4 on a selected set of matrices. The arrays used are of very small dimensions as compared to experimentally recorded NMR data. Still they reflect the essential features of 2D NMR spectra such as a H-H COSY or a H—TOCSY with a short mixing time, cf Fig. 5.1A, and an H-C HSQC, cf. Fig. 5.IB. The values of the matrix elements were arbitrarily chosen and indicate a signal or a correlation at that position. The relative signs should be interpreted as signal phases, cf. Fig. 5.IE as an example for a multiphcity-edited HSQC. 

Procedures have been published in detail elsewhere. Briefly, human skin was obtained at autopsy from individuals with diabetes and chronic renal failure or ESRD. After removal of the epidermis, a total of 75 g wet-weight skin tissue was defatted (2 1 chloroform-methanol) and extracted (1 M sodium chloride, 0.5 M acetic acid, Img/ml pepsin in 0.5 M acetic acid) which yielded 12 g of insoluble collagen. This collagen was digested sequentially with collagenase, peptidase, pronase, and aminopeptidase M LW-1 was purified by reverse-phase CIS HPLC and subjected to UV-fluorescence spectrometry, IH-NMR, 1H-13C HSQC, IH-IH TOCSY and mass spectrometry as described HPLC methods are described in Figs. 1-2. 

---