Journal of Planta 1983, Vol.47, pp.131—140,© Hippokrates Verlag GmbH Medicinal .PlantResearchmediCa Chemistry, Occurrence and Biosynthesis of C-Glycosyl Compounds in Plants Gerhard Franz, Michael Grun1 Lehrstuhl für Pharmazeutische Biologie, Universität Regensburg, Federal Republic of Germany Received: August 2, 1982 Introduction Therapid development of natural product chemi- stry has led to the isolation of a wide varieLty of secon- dary metabolites, which, in many cases, was shown to be of glycosidic nature. In particular our knowledge of the chemistry, occurrence and biogenesis of glyco- Flavone Xanthone sides has grown substantially during the last two de- cades [1, 2]. Most of this effort has been directed to- ward the large group of the 0-glycosides as well as the S- and N-glycosides. Far less effort has been given to the C-glycosides due to analytical difficulties. C-Glycosides are a special type of glycoside since the aglycone is directly attached to carbon 1 of a pyra- nose ring of a sugar while 0-glycosides possess a he- miacetal linkage. It is more correct to regard them as C-glycosyls or anhydropolyols as proposed by Chromone Anthrone BANDYUKOVA and YUGIN [3]. Since the original term C-glycoside is well established, it should be maintai- ned. One of the main characteristics of C-glycosides is COOH resistance towards acid hydrolysis. Even after pro- longed acid treatment the sugar residue, which is at- tached by a carbon-carbon-bond, remains attached HOOH to the aglycone. Cleavage of the C-C-linkage is achie- ved only under drastic conditions. When oxidizing reagents are used partial degradation of the sugar re- Gallic acid sidue is likely [4]. Classification of C-glycosides is usually based on Fig. 1. Basic structures of C-glycoside aglycones. the aglycone [5,6].Five aromatic ring systems are known to occur C-glycosylated (Fig. 1). The largest group, the C-glycosyl flavonoids, has been intensive-also exist. Application of such analytical tools as UV, ly studied by many authors and was the subject of twoJR. NMR and mass spectroscopy as well as optical recent review articles [3, 7]. Xanthone C-glycosidesmeasurements has resulted in the identification of appear to be biogenetically related to C-glycosyl fla-large numbers of such compounds in recent years. vonoid compounds. Another group, C-glycosylated Interestingly, C-glycosides are not restricted to chromone derivatives, are relatively rare. Aromaticplants. Carminic acid is an anthraquinone C-glycosi- three-ring systems, notably the anthrones, are C-lin-de from the insect Dactylopius coccus (references gi- ked to glucose. C-Glycosyl derivatives of gallic acidven in [3]). Kidamycin and toromycin are antibiotics produced by Streptomyces sp. and represent a new Present address: Institute of Biological Chemistry, Washing- type of polycyclic microbial metabolite with unusual ton State University, Pullmann, Washington 99164-6340, USA. C-glycosyl moieties [8, 9]. Recently, C-conjugated 132 Franz, GrOn compounds with glucuronic acid have been found inits specific substitution pattern. The same aglycone mammals, which are probably analogous to C-glyco-may possess a second C-linked sugar or 0-linked su- sylated compounds in plants. C-Glucuronides ofgar residues attached either to phenolic OH-groups drugs in man [10] and other mammals [11, 12] repre-or alcoholic OH-groups of the C-linked sugar. In the sent a novel type of drug metabolite. latter case, C,O-diglycosides and the C,O-triglycosi- As regards the biosynthetic pathway which leads todes are formed. It is interesting to note that in most the formation of the carbon-carbon bond in C-glyco-cases D-glucose serves as C-linked sugar but in the sides, very little is known. In some cases it is thoughtcase of flavonoid derivatives the only C-glycosyl com- that the attachment of the sugar moiety takes placepounds currently known contain other C-linked su- during an early stage of the aglycone formation butgar residues, i.e. galactose, rhamnose, xylose and ar- definite proof is still missing and cannot be generali-abinose [3]. A wide variety of 0-linked sugars may be zed for all groups of the C-glycosides. present as well. As for the aglycone moiety five types The aim of this article is to present a short review ofof flavonoid structures have been demonstrated, i.e., the principal structural features and occurrence offlavones, flavonols, flavanones, isoflavones and di- the different classes of C-glycosides and further, tohydrochalcones [7]. No anthocyanidines have been discuss the few biosynthetic findings of the C-glycosylidentified as C-glycosides. The most widespread attachment which have been published so far. aglycone structures which have been reported in lite- rature and which show different glycosidic variations are apigenin and luteolin [3]. More than 30 derivati- C-GlycosylFlavonoids ves of these aglycones have been reported so far. Chemistryof C-glycosyl Flavonoids Occurrence of C-Glycosyl Flavonoids As it has been pointed out, this group of C-glycosi- The review of ALSTON [13] on C-glycosyl flavonoids des has been the subject of a still increasing numbermentions about 20 Angiosperm families where these of reports, which mainly deal with the occurrencecompounds occur. There are no reports of C-glycosyl and structural investigation of these compounds. Sin-flavonoids in Gymnosperms and only a few of mosses ce they have been the subject of several review artic- and algae. Recently BANDYUKOVA and YUGIN [3] listed les [3, 6, 7, 13, 14] only the main features are given54 Angiosperm families with a varying number of here. species that contain C-glycosyl flavones in significant At the present over one hundred C-glycosyl fla-quantity. Gymnosperms and several lower plants al- vonoids have been shown to exist in the plant king-so possess these "unusual" flavonoid derivatives. dom. The model compound and one of the first to beOne can conclude that the C-glycosyl flavonoids are isolated was vitexin (8-C-3-D-glucopyranosylapige-widespread among plants, but with restricted chemo- nm) (Fig. 2), the structure of which was establishedtaxonomic significance as CHOPIN and BOUILLANT by NMR spectroscopy by HOROWITZ and GENTJLINIhave pointed out [7]. The most abi2ndant examples [15]. The 6-C-glycosyl isomer of vitexin, named iso-seem to be vitexin, isovitexin and their derivatives. vitexin (saponaretin), may be formed by an intramo- lecular rearrangement [16]. Biosynthesis of C-Glycosyl Flavonoids Most types of substituted flavonoid aglycones oc- OH HO cur as the O-glycoside with different sugars attached to alcoholic or phenolic groups. Biosynthesis of "nor- CH2OH mal" flavonoids and their glycosylated derivatives in- HO 0 volves three acetate units (ring A) and phenylalanine (ring B and the C3 segment) as shown by in vivo and in vitro tracer studies (see reviews by HAHLBROCK and GRISEBACH [17], HAHLBROCK [18]). In the case of C- HO1OS II glycosyl flavonoid aglycones, participation of phenyl- HO 0 alanine and p-coumaric acid in ring B and C forma- tion has been established [19, 20]. Vitexn The mechanism of flavonoid 0-glycosylation, Fig. 2. Vitexin as a representative C-glycosyl flavonoid. whereby the sugar moiety of a nucleotide sugar is transferred via more or less specific transferases to the aglycone acceptor is well understood and is the A free ortho-position to one or two phenolic hy-subject of recent reviews [2, 21]. The second type of droxyl groups appears to be a common feature that isglycoside formation, i.e., the biosynthesis of the C- prerequisite for the formation of a C-glycosidic linka-glycosidic linkage, has never been explained in de- ge in flavonoids. Therefore, the glycosyl residue istail. A few reports, however, give some indication linked to carbon 6 or 8 of ring A (Fig. 1) according toabout the biosynthetic stage of the aglycone at which C-Glycosyl Compounds in Plants 133 the C-glycosidic linkage might be formed. WALLACE Radioactive phenylalanine is rapidly incorpora- et al. [22]feddifferent radioactive precursors such asted into C-glycosyl flavones of the primary leaf of oat the flavone aglycones, apigenin and luteolin, to Spi-plants (A vena sativa, Poaceae). Two vitexin-derived rodela sp. or Lemna sp. (Lemnaceae) and detected0-rhamnosides and one isovitexin 0-arabinoside are radioactivity on 0-glycosylated and 0-methylatedformed and show a slow turnover during the experi- flavones. Radioactivity was not incorporated in fla-mental period of ten days, thus indicating that the C- vones containing C-glycosyl linkages, indicating thatglycosides are not necessarily metabolic end products 0-glycosylation of the flavones occurs readily in vi-[26, 27]. However, MARGNA and VAJNJARV [28] have vo, but C-glycosylation does not occur under condi-demonstrated that amongst the maj or flavonoids of tions in which both compounds are known to be syn-buckwheat seedlings (Fagopyrum esculentum, Faba- thesized in the plant. WALLACE and GRISEBACH [23], ceae), rutin and the C-glycosyl flavonoids do not on the other hand, showed that C-glycosylation wasundergo appreciable turnover during a seven-day only possible at the flavanone level using in vitro cul-period. tured Spirodela polyrhiza (Lemnaceae) clones. Genetic control of flavonoid biosynthesis and Again Wallace [24] stated that formation of the C-mainly 0-glycosylation has been extensively studied. glycosidic bond is always prior to 0-glycosylation.Vitexin and isovitexin glycosylation in Melandrium Pre-existing 0-glycosyl flavonoids are not C-glycosy-sp. and Silene sp. (Caryophyllaceae) are the subject lated. C-Glycosyl flavonoids, however, might be 0-of a series of publications (see review by HOSEL [21] glycosylated in a later step of biosynthesis (Fig. 3).and [29, 30, 31, 32]). Nothing, however, is shown for INouE and FUJITA [25] and Fujita and Inoue [20] supp- the control of the C-glycosylation process. orted these findings by feeding possible 14C-labeled Besides these in vivo studies concerning the forma- precursors to Pueraria lobata (Fabaceae) and Swertiation of C-glycosyl flavonoids essentially nothing is japonica (Gentianaceae) plants.