Structure Determination Using HSQC and HMBC

6 STRUCTURE DETERMINATION USING HSQC AND HMBC 11.6.1 Testosterone Metabolites 

HMBC is a powerful tool for locating the position of a functional group within a known carbon skeleton. Oxidation of testosterone with the enzyme cytochrome P-450 (Fig. 11.29) leads to a number of hydroxylation (C-H - C-OH) and di-hydroxylation products. One 

H-7 would be five bonds away, so an HMBC crosspeak would be impossible. Note that the corresponding crosspeak between H-17 and C-15 (TQ = 3.48 ppm, F = 70 ppm, upper right side) is not observed this is because the H-C17-C16-C15 dihedral angle is different from the C17-C16-C15-H dihedral angle, leading to a small 37ch for the former and a large 3/ch for the latter. HMBC does not exhibit the same symmetry as the COSY spectrum because the relationships are not equivalent. 

HMBC Observed No HMBC Am = 5.8 HMBC Observed No HMBC 

INVERSE HETERONUCLEAR 2D EXPERIMENTS HSQC, HMQC, AND HMBC 

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ATpairs, and is the basis of many so-called double-resonance experiments used for the structural determination of proteins and of other biological macromolecules, as we shall see later. A variation on the HSQC experiment is the heteronuclear multiple bond correlation (HMBC) experiment. This is a sensitive technique that maybe used to identify heteronuclear and spin-spin coupled nuclei. 

LC/NMR in various combinations with LC/UV-DAD, LC/MS, LC/MSMS, LC/IR, and/or LC/CD has been used in many applications related to the online identification of natural products. In this field, the challenge for hyphenated techniques is important since often the characterization of completely unknown molecules is required in very complex biological matrices. In this case, LC hyphenated techniques are used for the chemical evaluation of biologically active fractions or extracts and for dereplication purposes. As full structure assignment is often needed, all online spectroscopic data are taken into consideration. Most applications are performed in the stop-flow mode and extensive 2D NMR correlation experiments are measured. For unknown online determination the need for data is often mandatory. This type of information can be deduced from HSQC and HMBC indirect measurements and very recently it has been demonstrated that even direct measurements were possible in a crude plant extract. For this application the LC peak of interest was preconcentrated by trapping on SPE and the measurement was performed on a cryogenic flow... 

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 HMBC experiment correlates long-range (two to three bond) H C pairs (Fig. 12.11) three is used to determine the C chemical shifts and structural connectivity of quaternary and carbonyl carbons. Quaternary and carbonyl carbons do not have directly bonded hydrogens and as a result do not have a cross peak in the 2D H- C HSQC spectrum. Fig. 12.11 shows cross peaks for the correlation between hydrogens 10 and 13 with carbonyl carbon 12, and hydrogen 20 with carbonyl carbon 19. Also shown in the HMBC spectrum is the correlation between hydrogens 23 and 25 with quaternary carbon 21. Despite a lack of correlations to spiro-carbon 14, the overwhelming body of evidence from interpretations of multiple NMR... 

The structures of most benzophenones have been determined by 1D ( H, l3C, and DEPT) and 2D (COSY, HSQC, HMBC, and NOESY) NMR experiments. The majority of benzophenone NMR spectra have been recorded in CDCI3 and CD3OD. Benzene-c4, mixtures of benzene-<4 and CDCI3 [75], or pyridine-ds have also been used [88,93]. The aforementioned solvents were used to resolve overlapping signals of studied compounds. Deuterated TFA (0.1%) has also been used to increase the rate of keto-enol interconversion in benzophenones. We now turn our attention to the structural elucidation of xanthochymol (138). 

For metabolite identification, 8 male Cij CD(SD) rats (7 weeks old) were treated orally with [ " C-phenylJ-l at 300 mg/1cg b.w./day for 2 days. Urine and feces were collected for 3 days after the first dose. Feces were not used following isolation. Urine was lyophilized and fractionated by solvent extractions (hexane, ethyl acetate, etc.). Eight metabolites were isolated from extracts by preparative TLC and preparative HPLC. Their chemical structures were determined by NMR ( H, H-H COSY, HSQC, HMBC, etc.) and MS (ESI, El) spectroanalyses. Their chemical structures are shown in Figure 3 (8, 9, 10, 11,13,14,15 and 16). 

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