Metabolite structure determination

Owing to rapid development in analytical techniques, metabolite identification and structure elucidation have become possible even with trace levels of metabolites generated with in vitro or in vivo mammalian systems. However, the microbial bioreactor is still a valuable system for metabolite structure determination, especially when the metabolite of interest presents at a low level in in vitro or in vivo mammalian systems and the isolation from these matrices is hindered by the interference of other metabolites, the parent drug or endogenous compounds, or the structure determination requires appreciable amounts of samples due to structure complexity. 

In this chapter we provide a brief introduction to NMR theory and describe the most common experiments used for metabolite structure determination by NMR. Selected examples of metabolite structure elucidation are presented to illustrate relevant hardware, key parameters, and NMR methodology. Detailed explanations of NMR fundamentals have been previously reviewed in a series of books that are listed at the end of this chapter (Berger and Brunn, 2004 Breitmaier, 2002 Freeman, 1997 Friebolin, 2005 Homans, 2005 Keeler, 2005 Martin and Zektzer, 1988). 

Four general classes of NMR experiments are routinely used to analyze metabolites (1) ID NMR experiments (2) 2D NMR experiments (3) Solvent suppression methods and (4) Hyphenated NMR experiments. The ID and 2D NMR experiments are commonly used for metabolite structure determination. The various solvent suppression techniques (Gaggelli and Valensin, 1993 Hwang and Shaka, 1995 Smallcombe and Patt, 1995) are crucial for dilute metabolite samples where the solvent peak is the most intense peak in the NMR spectrum. These solvent suppression techniques can be incorporated as needed in both ID and 2D NMR experiments. Since their introduction in the 1990s, hyphenated NMR methods have become common tools in the identification of metabolites. These methods include LC-NMR (Albert, 1995 Spraul et al., 1993, 1994), LC-NMR-MS (Mass Spectrometry) (Shockcor et al., 1996) and LC/SPE (solid phase extraction)/NMR (Alexander et al., 2006 Bieri et al., 2006 Xu et al., 2005 Wilson et al., 2006). 

EXAMPLES OF METABOLITE STRUCTURE DETERMINATION FROM KNOWN BIOTRANSFORMATIONS... 

The Use of High-Accuracy Mass Measurements in Combination with LC-MS for the Structure Determination of Drug Metabolites... 

The biodegradation of branched-chain alkanol ethoxyethylates was carried out by the standard OECD confirmatory tests and the metabolites fractionated after solid-phase extraction. The structures of the metabolites were determined by electrospray mass spectrometry and this made it possible to derive a scheme for the partial degradation of the compounds (Di Corcia et al. 1998). 

Marine sponges are a source of an array of polycyclic diamine alkaloids of common biogenetic origin. This class of secondary metabolites has been the subject of four previous reviews [4-7]. Therefore, the present review will include literature reports previously not discussed, dealing with the isolation, structure determination, biological activities, and total synthesis of polycycUc diamine alkaloids isolated from marine sponges. This review will not include guanidine alkaloids [8,9] or the manzamine alkaloids [10,11], since these compounds have been recently reviewed elsewhere. Only polycycUc... 

Metabolite biosynthesis has demonstrated its utility in drug metabolite preparation and characterization, and contributed to drag discovery and development. Although metabolite biosynthesis is a prerequisite step for metabolite structure elucidation in many cases, it is complementary to chemical synthesis in large-scale metabolite preparations. The merits for using these techniques should be determined on a case-by-case fashion. New techniques, such as recombinant enzyme and microbial glucuronidation systems, would have a great impact on the field. 

Barbuch, R.J., Campanale, K., Hadden, C.E. et al. (2006) In vivo metabolism of [14C]ruboxistaurin in dogs, mice, and rats following oral administration and the structure determination of its metabolites by liquid chromatogra-phy/mass spectrometry and NMR spectrometry. Drug Metabolism and Disposition The Biological Fate of Chemicals, 34, 213-224. 

Lee HC, Walseth TF, Bratt GT, Hayes RN, Clapper DL 1989 Structural determination of a cyclic metabolite of NAD+ with intracellular Ca2+-mobilizing activity. J Biol Chem 264 1608-1615... 

The mechanisms of autoimmunity may also entail interaction with MHC structures determined by the HLA alleles. Individuals carrying certain HLA alleles have been shown to be predisposed to certain autoimmune diseases, which may account in part for the genetic variability of autoimmunity. In addition, metabolites of a particular drug may vary between individuals to confound the development of drug-induced autoimmunity. Dendritic cells, such as the Langerhans cells of the skin and B lymphocytes that function to present antigens to Th cells, express class-II... 

Purifications of the methanolic extract of adults and larvae of the New Guinean species Epilachna signatipennis led to the isolation and structure determination of three nitrogen-containing secondary metabolites choline (24), L-hypaphorine (25), and signatipennine (26) (Fig. 5). From a biosynthetic... 

G. Cassinelli, R. Corigli, P. Orezzi, G. Ventrella, A. Bedeschi, E. Perrone, D. Borghi, G. Franceschi, Structure Determination of the Primary Renal Metabolite of the Penem... 

Stadler M et at. New metabolites with nematicidal and antimicrobial activities from the ascomycete Lachnum papyraceum (Karst.) Karst. VII. Structure determination of brominated lachnumon and mycorrhizin A derivatives, J Antibiot 48 158-161, 1995. 

In the North Sea project, the results from primary screening for biological activity or new compounds guide the selection of strains for upscaling and finally isolation and structural elucidation. Since even modern methods for structure determination and an initial biomedical evaluation require 10 mg of every compound or more, a scale-up fermentation is necessary. With the aid of biotechni-cal methods, fermentation conditions have to be optimized to achieve maximum yield of metabolites, to increase the genetic stability of the producer or to improve other parameters. 

Ascosalipyrrolidinone A and B (116,117) were isolated and their structures determined by spectroscopic techniques. The two metabolites differ... 

HSCCC fractions containing known metabolites as determined by dereplication were eliminated from further study and only fractions with novel structures and high potency were designated "hits."... 

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