Synthesis of Flavonoid Sulfates. II. The Use of Aryl Sulfatase in the Synthesis of Flavonol-3-sulfates* Denis Barron** and Ragai K. Ibrahim Plant Biochemistry Laboratory, Department of Biology, Concordia University, 1455 De Maisonneuve Boulevard West, Montreal, Quebec, Canada H3G 1M8 Z. Naturforsch. 43c, 625-630 (1988); received February 23/May 17, 1988 Flavonol Sulfate Esters, Synthesis, 13C NMR, FAB-MS, UV Spectra The rates of aryl sulfatase hydrolysis of several 7-, 4'- and 3-sulfated flavonoids were compared and found to follow the order 7 or 4' >» 3. The complete resistance of the 3-sulfate ester to enzyme hydrolysis provided a unique and convenient method for the synthesis of a number of naturally occurring flavonol-3-sulfates from the corresponding higher sulfated analogs in quanti- tative yield. Introduction famic acid method [24] which gives rise to complex There has been an increasing number of reports in mixture of flavonoid sulfate isomers [25]. Although recent years on the natural occurrence of flavonoid flavonol-3-sulfates represent one of the most com- sulfates in the plant kingdom [2—21]. This consider- mon groups of naturally occurring sulfate esters [6, able interest seems to be related to their suggested 7], however, no method is yet available for the role in the detoxification of excess sulfate in response specific sulfation of position 3, due to its chelation to high sulfur environment [2], From structural point with the neighbouring carbonyl group. The fact that of view, the naturally occurring flavonoid sulfates are the 3-sulfate is the only group resistant to hydrolysis derivatives of common hydroxyflavones (Fig. 1) and with aryl sulfatase [14] allows the specific synthesis of hydroxyflavonols (Fig. 2) or their methyl ethers. The flavonol-3-sulfates from highly sulfated flavonoid-3- recent development of a novel sulfotransferase assay sulfates obtained by the DCC-TBAHS method. We [22] and the design of an original method for the wish to report here on the synthesis of a number of synthesis of specifically sulfated compounds [1] al- flavonol-3-sulfates and their identification by spec- lowed, for the first time, to demonstrate the enzyma- troscopic methods. tic synthesis of flavonoid sulfate esters [23]. The method used for their organic synthesis utilized N,N'-dicyclohexylcarbodiimide (DCC) plus tetra- Fig. 1. Structures of the flavone sulfate esters. butylammonium hydrogen sulfate (TBAHS) and performed stepwise sulfation of positions 7, 4' and 3 R2 of the flavonoid ring [1], It allowed the synthesis of a R3 number of naturally occurring flavone-7-sulfates, as well as polysulfated flavonol-3-sulfates. In addition, the DCC-mediated sulfation afforded one major product that was easily separated from by-products by gel filtration [1], and therefore, represents a sig- OH 0 nificant improvement of the previously described sul- Flavonoid compound R, R2 r3 * For Part I, see Ref. [1]. 1 Apigenin OH H OH ** Present address: Laboratoire Arômes, INRA, 17 rue la -7-sulfate OSO,K H OH Sully, F-21034 Dijon Cedex, France. lb -4'-sulfate OH H OSO^K Abbreviations: DCC, NN'-dicyclohexylcarbodiimide; lc -7,4'-disulfate OSO3K H OSO3K TBAHS, tetrabutylammonium hydrogen sulphate. 2 Luteolin OH OH OH Reprint requests to Prof. Dr. R. K. Ibrahim. 2 a -7-sulfate OSO,K OH OH 2b -4'-sulfate OH OH OSO,K Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen 2c -7,4'-disulfate OSO,K OH OSO,K 0341 - 0382/88/0900- 0625 $01.30/0 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. 626 D. Barron and R. K. Ibrahim • Synthesis of Flavonoid Sulfates. III. Fig. 2. Structures of the flavonol sulfate esters. Flavonoid compound R, R: R3 R4 R5 3 Kaempferol H OH H OH OH 3a -3-sulfate H OH H OH OSO,K 3b -3,7,4'-trisulfate H OSO,K H OSO,K OSO,K 4 Quercetin H OH OH OH OH 4a -3-sulfate H OH OH OH OSO,K 4b -7-sulfate H OSO3K OH OH OH 4c -4'-sulfate H OH OH OSO,K OH 4d -3,4'-disulfate H OH OH OSO,K OSO,K 4e -3,7-disulfate H OSO,K OH OH OSO,K 4f -7,4'-disulfate H OSO3K OH OSO,K OH 4g -3,7,4'-trisulfate H OSO,K OH OSO,K OSO,K 5 Tamarixetin H OH OH OCH, OH 5a -3-sulfate H OH OH OCH, OSO,K 5b -3,7-disulfate H OSO3K OH OCH3 OSO,K 6 Rhamnetin H OCH3 OH OH OH 6a -3-sulfate H OCH3 OH OH OSO,K 6b -3,4'-disulfate H OCH3 OH OSO,K OSO,K 7 Eupalitin OCH3 OCH, H OH OH 7a -3-sulfate OCH, OCH3 H OH OSO,K 7b -3,4'-disulfate OCH3 OCH, H OSO,K OSO,K 8 Eupatolitin OCH3 OCH, OH OH OH 8a -3-sulfate OCH3 OCH, OH OH OSO,K 8b -3,4'-disulfate OCH, OCH, OH OSO,K OSO,K 9 Veronicafolin OCH, OCH, OCH, OH OH 9a -3-sulfate OCH3 OCH3 OCH, OH OSO,K 9b -3,4'-disulfate OCH, OCH3 OCH, OSO,K OSO,K General methods Experimental Analytical HPLC was carried out on m-Bondapack Source of reference compounds and enzyme C18 column (300 x 3.9 mm) using Waters HPLC ap- Rhamnetin was obtained from Extrasynthese paratus equipped with two pumps (Model 510), a (Gernay, France). Tamarixetin-3,7-disulfate, apige- Rheodyne injector (Model 7125), an automated gra- nin-, luteolin-, and quercetin-7,4'-disulfates, as well dient controller (Model 680) and a UV detector as quercetin- and kaempferol-3,7,4'-trisulfates were (Model 441) for detection at 340 nm. The following synthesized according to [1]. Aryl sulfatase type H-l solvents were used for ion pairing chromatography: from Helix pomati was purchased from Sigma (St. A, 0.1 M aqueous tetrabutylammonium dihydrogen Louis, Mo.). phosphate; B, Me0H-H20-H0Ac (90:5:5, v/v/v). 'H 627 D. Barron and R. K. Ibrahim • Synthesis of Flavonoid Sulfates. III. NMR (299.9 MHz) and I3C NMR (75.4 MHz) spec- tamarixetin-3-sulfate (5a). Under similar conditions, tra were recorded using a Bruker spectrometer at the the 3-monosulfates of rhamnetin (6a), eupalitin Montreal Regional High Field NMR laboratory. For (7a), eupatolitin (8a) and veronicafolin (9a) were negative FAB-MS, a Kratos MS-50-TC-TA instru- prepared from their corresponding 3,4'-disulfate ment (6.7 kV gun; Xe beam, 2 mA; 9 kV source) esters. was used, after the sample had been dissolved in a The 3-sulfates of kaempferol (3a) and quercetin glycerol matrix. (4a) were synthesized from their respective 3,7,4'- trisulfate esters [1] (3 b) and 4g as described for tamarixetin-3-sulfate, except that double the amount Synthesis of flavonoids of aryl sulfatase was used. Eupalitin (7), eupatolitin (8) and veronicafolin (9) The 4'-sulfates of apigenin (lb), luteolin (2b) and were synthesized according to the method of Wagner quercetin (4 c) were obtained after aryl sulfatase et al. [26, 27] except that the benzyl protecting hydrolysis of their corresponding 7,4'-disulfates lc, groups were replaced with isopropyl groups. Iso- 2 c and 4f using the same conditions as described for propylation of p-hydroxybenzaldehyde, vanillin and tamarixetin-3,7-disulfate. Separation of the isomeric protocatechualdehyde was carried out as described 4'- and 7-sulfate esters of apigenin, luteolin and by Johnstone and Rose [28] and the isopropyloxy- quercetin was performed on Sephadex G-10 using a flavonols were obtained after transformation of their gradient of water and 20% aqueous MeOH, which corresponding chalcones [29], Deisopropylation and was followed by column chromatography on cellu- removal of the 5-methyl group were performed by lose using water in the case of quercetin isomers. refluxing the isopropyloxyflavonols in conc. HCl-gla- cial HOAc (1:1, v/v) for 2 h [26], After dilution with ice, the precipitates of eupalitin, eupatolitin and veronicafolin were purified by column chromatogra- Results and Discussion phy on cellulose using H20-Me2CO-n-butanol-diox- Tamarixetin-3,7-disulfate (5b) [1], after hydrolysis ane (80:10:5:5), v/v/v/v). with aryl sulfatase, yielded tamarixetin-3-sulfate (5a) Rhamnetin (6b), eupalitin (7b), eupatolitin (8b) [31] as the only product. Similarly, using double the and veronicafolin (9b) 3,4'-disulfates were syn- amount of enzyme, quercetin (4g) and kaempferol thesized by sulfation of the corresponding aglycones (3b) 3,7,4'-trisulfates [1] gave quercetin-3-sulfate using the DCC-TBAHS method [1]. The resulting (4 a) [32] and kaempferol-3-sulfate (3 a) [33], respec- TBA-salts were converted to their potassium salts tively. Rhamnetin (6), eupalitin (7), eupatolitin (8) using saturated K2C03 in MeOH. They were sepa- and veronicafolin (9), on sulfation with DCC + rated by centrifugation, dissolved in water and fur- TBAHS gave their respective 3,4'-disulfate esters ther purified on Sephadex G-10 using water as sol- 6b, 7b, 8b and 9b, and upon aryl sulfatase hydrolysis vent with 6 b and a gradient of 20—50% aqueous gave their corresponding 3-monosulfates 6a [32], 7a MeOH with 7b, 8b and 9b. Yields ranged from 65 [17], 8a [34] and 9a [34], The rates of enzymic hydro- to 70%. lysis of the 7- and 4'-sulfate groups were compared by subjecting a number of flavonoid 7,4'-disulfates to the same hydrolytic conditions as the respective Synthesis of flavonoid-3-sulfates monosulfates (i.e.