Isotopically-labelled precursors

Pohnert G, Jung V (2003) Intracellular compartmentation in the biosynthesis of caulerpenyne study on intact macroalgae using stable-isotope-labeled precursors. Org Lett 5 5091-5093 Potin P, Bouarab K, Salaun JP, Pohnert G, Kloareg B (2002) Biotic interactions of marine algae. Curr Opin Plant Biol 5 308-317... 

In the determination of the sequence of intermediates of a specific pathway, one single probe, the use of isotopically labelled precursors, has proven of pivotal importance. With the appropriate safeguards, which we shall consider below, the administration of a compound isotopically labelled in a recognizable pattern and the subsequent isolation of a labelled metabolite in which the distribution of label can be recognized as occurring without randomization constitutes de facto evidence of a precursorial relationship. 

Tracer studies with isotopically labeled precursors have shown that both in mammalian tissues and in microorgan-isms, deoxyribonucleotides are formed from corresponding ribonucleotides by replacement of the 2 —OH group with hydrogen. 

It should be stressed that it is a prerequisite of successful flavor precursor studies that the contribution of the odorant under investigation to a food flavor or off-flavor has been established. Sometimes the structure of a precursor can be assumed on the basis of structural elements in the odorant. In such cases, additions of the respective isotope-labelled precursor to the food system is commonly used to elucidate the precursor and to clarify reaction pathways governing the formation of the odorant. This method has been frequently applied, especially, in studies on the enzymatic generation of odor-active aldehydes (e.g., (Z)-3-hexenal in tea leaves) or alcohols (e.g., l-octen-3-oI in mushrooms) [cf. reviews in 84, 85] as well as lactones [86] from unsaturated fatty acids. 

The majority of recent biosynthetic studies on bisbenzylisoquinoline alkaloids has been done by Bhakuni and co-workers, using feeding of isotopically labeled precursors to alkaloid-producing plants this work has been reviewed (339,340). In this manner, several of these alkaloids were shown to be derived by coupling of coclaurine or V-methylcoclaurine units the thalicarpine family of alkaloids, however, is formed from two (S)-reticuline (430a) units (148,339). 

The biosynthesis of the cucujolides was examined in the merchant grain beetle, O. mercator Fauvel, and the rusty grain beetle, C. ferrugineus (Stephens), using radio- and stable isotope-labeled precursors (Vanderwel et al., 1990,1992b). 

Inspired by the partly exotic structures of antibiotic sugars, a number of chemists and biochemists have tried to unravel their biosynthesis. As in other biosynthetic studies, labelling patterns obtained with isotopically labelled precursors dominated the earlier phases of this work, and this approach is still being used. Only recently has it been possible in some cases to study the reactions at the enzymic level. 

Dissection of the chemical structure of jamaicamides A-C led to the speculation that these metabolites derive from a mixture of polyketides (nine acetate units), amino acids (t-Ala and p-Ala), and the S-methyl group of methionine. To map out the biosynthetic subunits of these molecules, isotopically labeled precursors were supplied to I. majuscula JHB, and the labeling patterns discerned by NMR spectroscopy (Figure 6.12). From these experiments, insights were gained into the biochemical transformations that produce the jamaicamides, especially the mechanism of formation of the vinyl chloride group [157]. 

Sorensen and Ranganayakulu (1970) studied the 1,3,4,4-tetramethyl cyclo-hexenyl cation [274] and using isotopically labelled precursors observed a degenerate rearrangement. The reaction (177) which equilibrated the C(3)-and C(4)-methyl groups was suggested to involve a cyclopropylcarbinyl cationic intermediate or transition state. 

Feeding experiments with isotopically labeled precursors have shown that many NR fungal polyketides are formed by the use of advanced starter units. In the classic case of norsolorinic acid 7 biosynthesis, it has long been known that hexanoate forms the starter unit. Differential specific incorporation of acetate into the early and late positions in compounds such as citrinin 3 have been used to argue that these compounds may have been formed by more than one PKS so that one PKS makes an advanced starter unit, which is passed to a second PKS for additional extension. 

Today, despite recent developments in combinatorial and other chemosynthetic strategies, flmgi and streptomycetes remain the most prolific sources of new candidate drugs and agrochemicals. Both phyla elaborate bicyclic, tricyclic and tetracyclic fused ring polyketides, however, a preliminary survey of isotopically labelled precursor incorporation studies has revealed a consistent difference in the modes of cyclisation by which their characteristic polybenzenoid metabolites are formed. These and subsequent observations (yide infra) provide the basis for a novel biosynthetic classification of microbial fused ring polyketides. 

The common stable isotopes used in biosynthetic studies are H, N, and 0. Stable isotope-labeled precursors have replaced radiolabeled precursors in many biosynthetic studies for the following reasons 1) no appropriate radiolabeled isotope is available (e.g., N and O) 2) the detection methods frequently permit location of the label in the product directly and 3) radiocontamination and safety issues are reduced. The negative aspects of stable isotope studies... 

These additional data not only substantiate the generality of AT-less PKS for polyketide biosyndiesis but also enable us to speculate if AT-less PKS clusters could be predicted according to the structures of polyketide natural products. Reduced (also known as complex) polyketide natural products often carry alkyl branches on their carbon backbones. Feeding experiments with isotope-labeled precursors clearly established that the alkyl branches are typically derived from the acyl CoA extender units, such as methylmalonyl CoA, ethylmalonyl CoA, or methoxymalonyl ACP for the resultant methyl, ethyl, or methoxy branches, respectively. Noniterative type 1 PKSs account tisfactorily for their ability to... 

A biosynthetic pathway of the fungal metabolite chaetoglobosin A (155) " from Chaetmium globosum and Ch. suhhafme was extensively investigated by the incorporation experiments with isotopically labeled precursors... 

This approach assumes some knowledge, or at least some presumption, of the general biosynthetic pathway by which the metabolite is formed. It is usually possible to make a good guess at the biosynthetic origins of a molecule, particularly when biosynthetic information is available on a structurally similar molecule, and these theories can be tested by the use of isotopically labeled precursors. It also relies on the fact that the biosynthetic enzymes are not so specific as to exclude all but the natural precursor. Fortunately, the enzymes of secondary metabolism tend to be less specific than those of primary metabolism. 

As can be concluded from the above outline, access to carotenoids isotopically labelled with and at predetermined positions is an essential part of this strategy. Labelled carotenoids can be prepared in two ways, namely by biosynthesis or by chemical synthesis. For the preparation of specifically labelled carotenoids biosynthesis is not suitable the labelled carotenoids are obtained by growing bacteria or yeasts on media containing simple isotopically labelled precursors such as sodium acetate [19]. The position of labelling can often not be predetermined and multiple labelling often occurs. Another major disadvantage is that isotopic dilution occurs for studies of carotenoid-protein complexes, carotenoids labelled at specific positions with high isotopic enrichment (preferably >99%) are needed. 

As for the valine moiety, in the case of penicillins itsC-1 (i.e. penicillin C-3) has D configuration ((291)). The existence of LLL-ACV and the isotope-labelled precursors suggest that the primary form is in the L configuration the isomerization takes place after formation of the tripeptide. 

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