Synthesis of Kaempferol-3-0-(3",4'' -di-O-a-L-rhamnopyranosy^-^-D-galactopyranoside and its Comparison with the Natural Ascaside Isolated from Astragalus caucasicus Ingrid Riess-Maurer, Hildebert Wagner* Institut für Pharmazeutische Arzneimittellehre der Universität München, Karlstraße 29, D-8000 München 2 and András Lipták Institute of Biochemistry, L. Kossuth University, H-4010 Debrecen, P.O. Box 55, Hungary Z. Naturforsch. 36b, 257-261 (1981); received October 21, 1980 Trisaccharide, Synthesis, Synthetic Kaempferol Trioside The trisaccharide, 3,4-di-0-(a-L-rhamnopyranosyl)-D-galactose was synthesized using benzyl 2,6-di-0-benzyl-/?-D-galactopyranoside as starting compound. Its acetobromo derivative was coupled with 4',7-di-O-benzyl-kaempferol. The structure of the synthetic kaempferol trioside was characterised by different spectroscopic methods (UV, IR, XH, 13C NMR and MS). Chromatographic and mass spectrometric comparison of the synthetic product with ascaside precluded their identity. Introduction Some fiavonol triosides with branched structures While the synthesis of naturally occuring fla- [6, 7] have been isolated, but the ascaside is the first vonoid mono- and diglycosides are well documented representative among those which contain two and their synthesis is a routine process [1], the rhamnoses and one galactose. The ascaside has an chemical preparation of flavonoid glycosides, con- unique structure which prompted us to work out a taining a trisaccharide as sugar component seems to synthetic route for its preparation. It is the first be more difficult and only few examples of such synthesis of a flavonoid trioside with a branched syntheses may be found in the literature [2, 4]. sugar moiety. There seems to be a need for their syntheses, since the structure elucidation of such compounds by Result and Discussion means of spectroscopic analysis, lacking distinct The key step in the ascaside synthesis is the model compounds, proved to be equivocal [3]. preparation of its trisaccharide part. Generally, the Our results on the field of the structure elucida- synthesis of ohgosaccharides requires two main tion of the xanthorhamnin series showed clearly, operations: i) correct blocking to obtain the required that the distinction between the different structure aglycone, and ii) construction of the glycosidic 13 isomers by C NMR or MS methods is only possible linkage. In some cases the suitable protected ag- if there are synthetic models for the assignment on lycones are the bottle neck of the whole synthesis. hand [4]. An unequivocal synthesis of ascaside required a Here we wish to report on the synthesis of suitable protected galactopyranoside derivative for ascaside, a fiavonol trioside isolated from Astragalus the preparation of the trisaccharide component. For caucasicus [5], of which the structure was postulated with the aid of chemical degradation and spectros- this reason we chose the recently prepared benzyl copic investigations (UV, IR and XH NMR) to be a 2,6-di-0-benzyl-/5-D-galactopyranoside [8] (1) which kaempferol- 3 - 0- (3 " ,4 di - 0 - a-L-rhamnopyranosyl)- contains easily removable groups in all three posi- /3-D-galactopyranoside. tions. This aglycone was rhamnosylated by a-aceto- bromo-L-rhamnose in a modified Helferich procedure using a benzene-nitromethane (1:1) solvent system and Hg(CN)2 as acid acceptor to give compound 2. The high yield of the couphng (76%) and relatively Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung This work has been digitalized and published in 2013 by Verlag Zeitschrift in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der für Naturforschung in cooperation with the Max Planck Society for the Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Advancement of Science under a Creative Commons Attribution Creative Commons Namensnennung 4.0 Lizenz. 4.0 International License. short reaction time showed, that there was not any pounds, one a kaempferol-, the other one an iso- limited reactivity of the axial C4-OH group, which rhamnetin- or tamarixetin-derivative. was observed in the glycosylation reactions of 2,6- The analysis of the mass spectrum of the per- disubstituted galactopyranosyl derivatives [9, 10]. methylated isolated flavonoids showed the following Perhaps the high yield of our reaction may be characteristic fragments: m/e 189 for a rhamnose explained by the fact, that the reactivity of C4-OH trimethyl ether, accompanied with two secondary is increased by the glycosylation of C3-OH. fragments m/e 157 and 125. A fragment of a tetra- The structure of compound 2 was verified by its methyl hexopyranosyl moiety could not be detected. 13C NMR spectrum. The chemical shifts for the The fragment M-205 = m/e 689 may be assigned to anomeric carbons (102.9, 98.6 and 97.6 ppm) and a permethylated kaempferol-(0-rhamnosyl)-0-ga- their 1JCH values (160, 174 and 176 Hz) supported lactoside structure. The m/e 719 is assignable to the the suggested anomeric configurations; the two permethylated quercetin analogue. rhamnopyranosyl units have a-configuration. Com- The fragments m/e 517 and 547 (A -f- S) belong to pound 2 was readily hydrogenolysed over palladium the kaempferol and quercetin-monomethyl ether- on carbon and acetylated to give the crystalline dimethyl galactoside skeletons, respectively. These nonaacetyl-trisaccharide (3). The XH and 13C NMR facts seem to support the presence of a branched spectra of 3 showed the presence of both anomers of oligosaccharide moiety in ascaside. The absence of the reducing end in the galactopyranose unit in a a m/e 362 (T+ S2) fragment rules out a linear carbo- ratio of <z//5 = 5/3. Deacetylation of 3 using the hydrate chain. The spectrum of a peracetylated Zemplen method resulted in the trisaccharide (4) as derivative of the mixture verifies the absence of a an amorphous powder. linear structure, too. Taking into account these Treatment of 3 with HBr in acetic acid afforded fragments and the published data [5] (enzymatic the crystalline a-acetobromo derivative (5) in good degradation, diisopropylidene derivative) it may be yield. Condensation of the acetobromo-trisaccharide assumed, that the isolated substance is a mixture of 5 with 4',7-di-O-benzylkaempferol [11] (6) in a kaempferol-3-0-(di-rhamnosyl)-0-galactoside and quinoline in the presence of silver carbonate and its tamarixitin or isorhamnetine analogue. The Drierite resulted in the crystalline 7 - after separa- bond-types are most hkely a 3",6"- or a 4",6"-di-0- tion on silica gel column using a toluene-methanol, rhamnosyl-galactoside. 8:2 solvent system as eluent The XH NMR and UV spectra of 7 were in good accordance which the proposed structure, indicating that the glycosyla- tion took place in the position 3 of 6. The /S-anomer 8n0—-r-CHjOBn configuration of the new glycosidic linkage was suggested by the value of the optical rotation. Deacetylation of 7 resulted in crystalline 8, which after catalytic debenzylation gave the kaempferol- 2: R == OBn; R' = H; R" = Bn; R'" = Ac 3-0-(3",4"-,di-0-a-L-rhamnopyranosyl)-/?-D-galac- 3a: R = H; R' = OAc; R" = R'" = Ac 3/3: R = OAc; R' = H; R" = R'" = Ac topyranoside (9). Conventional acetylation of 9 gave 4a: R = R" = R'" = H; R' = OH the crystalline peracetylated kaempferoltrioside 4/5: R = OH; R' = R" = R'" = H (10) which also proved to be homogeneous. Though 9 5: R = H; R' = Br; R" = R'" = Ac was not crystalline, its purity was verified by TLC R' in different solvent systems and on different ad- sorbents. UV, IR, MS, *H and 13C NMR investiga- tions confirmed the proposed structure. The /?-glycosidic structure between the aglycone and the 6 OR" trisaccharide part is indicated by the presence of a 7: R = Bn; R' = H; R" = Ac low field signal at 101.3 ppm. 8: R = Bn; R' = R" = H 9: R = R' = R" = H The TLC comparison of the synthesized compound 10: R = R' = R" = Ac 9 with the supplied natural product showed clearly Bn = Benzyl that the latter is a mixture of at least two com- Ac = Acetyl Table. Assignment of the 13C NMR chemical shifts and 1 Jen data. Compound Galactosyl Rhamnosyl Rhamnosyl 1 23456 1 23456 1 23456 1» 102.5 79.3 73.6 69.4 74.6 69.1 (156) 2a 102.9 77.1 79.5 75.2 73.5 68.3 98.7 68.9 70.0 71.1 67.2 17.4 97.6 68.9 69.5 71.1 67.1 17.4 (160) (174) (176) 3* 93.2 69.2 75.6 75.2 69.2 62.11 98.3 69.9 69.6 71.2 67.5 98.7 69.9 69.5 71.2 67.3 97.9 73.2 78.4 75.4 74.4 62.1J 17.5 17.5 4b 93.2 69.6 76.8 76.1 71.7 61.91 101.5 70.8 71.1 72.7 70.0 17.5 102.3 71.0 71.1 72.8 70.2 17.6 97.2 73.1 80.0 75.7 75.5 61.9/ 9C* 101.3 71.6 78.2 73.3 75.6 60.4 100.7 70.3 70.8 71.9 68.9 17.9 101.8 70.5 70.9 72.0 68.9 17.8 * Chemical shift data for kaempferol skeleton of 9: 156.6 (C-2); 133.1 (C-3); 177.5 (C-4); 161.2 (C-5); 98.8 (C-6); 164.3 (C-7); 93.7 (C-8); 156.7 (C-9); 104.0 (C-10); 120.8 (C-l'); 130.9 (C-2',6'); 115.2 (C-3',5'); 160.1 (C-4'). 1«^CH coupling constants are given in brackets [Hz].