Chapter 4 - Separation procedures for complicated mixtures of sea cucumber triterpene glycosides with isolation of individual glycosides, their comparison with HPLC/MS metabolomic approach, and biosynthetic interpretation of the obtained structural data

Abstract

Sea cucumber glycosides differ from each other in the position and number of double bonds, the presence and position of a lactone, and other functional groups in aglycones as well as by the composition and sequence of sugar residues in carbohydrate chains, which can be modified in different manners such as sulfation or methylation. That is a reason why they form very complicated and almost inseparable mixtures in the animal producers. This chapter discusses the methodology of separation of glycoside fractions from the sea cucumbers developed during last 5 years. The analysis of results, obtained by LC-ESI MS metabolomic approach and by direct isolation of individual glycosides using extensive HPLC procedures on semipreparative columns, revealed that results are comparable and complement each other. The applying of isolation procedures allows to detect all the principal carbohydrate chain versions, even without of application of metabolomic approach, and moreover to establish complete chemical structures of the compounds investigated. In any case, some new variants of carbohydrate chains found by LC-ESI MS approach were confirmed in the glycosides isolated by preparative methods. As a result of using of separation methodology followed by the structure elucidation by physico-chemical methods, a few hundred triterpene glycosides have been isolated from the sea cucumbers of the orders Holoturiida, Synallactida, Elasipodida, and Dendrochirotida. Among them there are some minor compounds, so-called “hot metabolites,” or the glycosides, having unique chemical structures have been found. The combination of effective separation of complex mixtures of the glycosides and their structure elucidation makes it possible to analyze the biogenetic relationships of the compounds that finally lead to the construction of metabolic network illustrating the biosynthetic pathways of the glycosides. The analysis of the set of different carbohydrate chains allowed to suppose their biosynthesis as sequential attachment of monosaccharides to different positions of forming chain. The sulfation may occur at different stages of elongation of carbohydrate chain. The analysis of the aglycones set permits to deduce three main directions of biosynthesis leading to the formation of holostane and nonholostane–type aglycones as well as the aglycones having shortened side chains. The “mosaic” type of biosynthesis was established, which implies the aglycones and carbohydrate chains are biosynthesized simultaneously and independently from each other and different stages can be shifted in time relative to each other that leads to the formation of the same products by different pathways. Although large-scale HPLC isolation of individual glycosides takes a lot of hard work and time, it provides a possibility to get more structural information using NMR and can be considered as one of the approaches of studying on triterpene glycosides biosynthesis in the sea cucumbers.