Nuclear magnetic resonance carbon-13 enrichment

Generally, the most powerful method for stmctural elucidation of steroids is nuclear magnetic resonance (nmr) spectroscopy. There are several classical reviews on the one-dimensional (1-D) proton H-nmr spectroscopy of steroids (267). C-nmr, a technique used to observe individual carbons, is used for stmcture elucidation of steroids. In addition, C-nmr is used for biosynthesis experiments with C-enriched precursors (268). The availability of higher magnetic field instmments coupled with the arrival of 1-D and two-dimensional (2-D) techniques such as DEPT, COSY, NOESY, 2-D J-resolved, HOHAHA, etc, have provided powerful new tools for the stmctural elucidation of complex natural products including steroids (269). 

Example Isotopic enrichment is a standard means to enhance the response of an analyte in nuclear magnetic resonance (NMR). Such measures gain importance if extremely low solubility is combined with a large number of carbons, as is often the case with [60]fullerene compounds. [19] The molecular ion signals, IVT, of Qo with natural isotopic abundance and of C-enriched Cgo are shown below (Fig. 3.11 for EI-MS of [60]fullerenes cf. Refs. [20-22]). From these mass spectra, the enrichment can be determined by use of Eq. 3.1. For Qo of natural isotopic abundance we obtain Mrceo = 60 x 12.0108 u = 720.65 u. Applying Eq. 

Walker, T.E., Han, C.H., Kollman, V.H., London, R.E., Matwiyoff, N.A. (1982). 13C nuclear magnetic resonance studies of the biosynthesis by Microbacterium ammoniaphilum of L-gluta-mate selectively enriched with carbon-13. J. Biol. Chem. 257,1189-1195. 

As the glucose is metabolized, the metabolic derivatives become enriched with the isotopic carbon label and are detected using advanced technologies such as mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Importantly, incorporation of isotopes into molecules can alter reaction mechanisms including the rate of a reaction-this is termed isotopic substitution and forms the basis of the kinetic isotope effect. 

Incorporation studies of C-labeled precursors require extensive nuclear magnetic resonance (NMR) measurements. The signals of C-NMR-spectra of the analyzed metabolites must be assigned fully. The C-NMR spectra of the reference compound of natural abundance (1.1%) and of the labeled metabolite must be recorded in the same conditions. A comparison between the relative signal intensities of the two spectra indicates that the carbon atom is labeled and indicates the magnitude of the isotope enrichment. 

Mikita, M. A., Steelink, C., and Wershaw, R. L. (1981). Carbon-13 enriched nuclear magnetic resonance method for the determination of hydroxyl functionality in humic substances. Anal. Chem. S3, 1715-1717. 

Xie, Y., and Terashima, N. (1991) Selective carbon 13-enrichment of side chain carbons of ginkgo [Ginkgo bUoba] lignin traced by carbon 13 nuclear magnetic resonance. J. Japan Wood Res. Soc. 37(10), 935-941. 

Griffin, J. H., Alazard, R., Dibello, C., Sala, E., Mermet-Bouvier, R., and Cohen, P. (1975). FEBS Letters 50, 168. Carbon-13 Nuclear Magnetic Resonances Studies on (85% 1 3C-Enriched Gly9) Oxytocin. 

NMR spectra of (a) a soil humic acid (b) a groundwater fulvic acid (c) a mixture of C-methylated reference compounds (1) pentamethylquercetin (2) glycerol dimethyl ether (3) methyl benzoate (4) methyl pentanoate. Reprinted with permission from Mikita et al., Carbon-13 Enriched Nuclear Magnetic Resonance Method for the Determination of Hydroxyl Functionality in Humic Substances, Anal. Chem. 53, 1715-1717. Copyright 1981, American Chemical Society. 

Walker, T. E., R. E. London, T. W. Whaley, R. Barker, and N. A. Matwiyoff Carbon-13 Nuclear Magnetic Resonance Spectroscopy of (1- C) Enriched monosaccharides. Signal Assignments and orientation dependance of geminal and vicinal Carbon-Carbon and Carbon-Hydrogen Spin-Spin coupling constants. J. Amer. Chem. Soc. 98,5807 (1976) and references cited therein. 

Lin RC, Davis EJ (1974) Malic enzymes of rabbit heart mitochondria. J Biol Chem 249 3867-3875 London RE, Kollman VH, Matwiyoff NA (1975a) The quantitative analysis of carbon-carbon coupling in the C nuclear magnetic resonance spectra of molecules biosynthesized from " C enriched precusors. J Am Chem Soc 97 3565-3573 London RE, Matwiyoff NA, Kollman VH (1975b) Differential nuclear overhauser enhancement for i C siglet and multiplet resonances of a carboxyl carbon. J Magn Res 18 555-557... 

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