Development of a high‐performance liquid chromatography procedure to identify known and detect novel C‐13 oligosaccharide moieties in diterpene glycosides from Stevia rebaudiana (Bertoni) Bertoni (Asteraceae): Structure elucidation of rebaudiosides V and W

As an aid for structure elucidation of new steviol glycosides, reversed‐phase C18 high‐performance liquid chromatography method was developed with several previously characterized diterpene glycosides, to identify known and detect novel aglycone‐C13 oligosaccharide moieties and indirectly identify C‐19 interlinkages. Elution order of several diterpene glycosides and their aglycone‐C13 oligosaccharide substituted with different sugar arrangements were also summarized. Comparison of the retention time of a product obtained after alkaline hydrolysis with the aglycone‐C‐13 portions of known compounds reported herein allowed us to deduce the exact positions of the sugars in the C‐13 oligosaccharide portion. The elution position of several steviol glycosides with an ent‐kaurene skeleton was helpful to describe an identification key. Two previously uncharacterized diterpene glycosides together with two known compounds were isolated from a commercial Stevia rebaudiana leaf extract. One was found to be 13‐[(2‐O‐β‐d‐xylopyranosyl‐3‐O‐β‐d‐glucopyranosyl‐β‐d‐glucopyranosyl)oxy]ent‐kaur‐16‐en‐19‐oic acid‐(2‐O‐β‐d‐glucopyranosyl‐β‐d‐glucopyranosyl) ester (rebaudioside V), whereas the other was determined to be 13‐[(2‐O‐β‐d‐xylopyranosyl‐3‐O‐β‐d‐glucopyranosyl‐β‐d‐glucopyranosyl)oxy]ent‐kaur‐16‐en‐19‐oic acid‐(2‐O‐α‐l‐rhamnopyranosyl‐3‐O‐β‐d‐glucopyranosyl‐β‐d‐glucopyranosyl) ester (rebaudioside W). Previously reported compounds were isolated in gram quantities and identified as rebaudioside J and rebaudioside H. In addition, a C‐19 sugar‐free derivative was also prepared from rebaudioside H to afford rebaudioside H<jats:sub/>1. Chemical structures were partially determined by the high‐performance liquid chromatography method and unambiguously characterized by using one‐dimensional and two‐dimensional nuclear magnetic resonance experiments.