6-D-Glucopyranosyl Fatty Acid Esters from Brassica Napus Pollen

Phyrochemisrry. 19i8. Vol. Ii. pp.1187-1189. © Pergamon Press Ltd. Printed in England. 0031-9422/78,0701-1187502.00,0

6-D-GLUCOPYRANOSYL FATTY ACID ESTERS FROM BRASSICA NAPUS POLLEN

MICHAEL D. GROVE,* GAYLAND F. SPENCER,* PHILIP E. PFEFFER,t NAGABHUSHANAM MANDAVA,t J. DAVID WARTHEN, JR.t and JOSEPH F. WORLEyt * Northern Regional Research Center. Agricultural Research Service, U.S. Department ofAgriculture. Peoria, IL 61604: t Eastern Regional Research Center. Agricultural Research Service. U.S. Department of Agriculture. Philadelphia. PA 19118; t Beltsville Agricultural Research Center. Agricultural Research Service, U.S. Department of Agriculture. Beltsville. MD 20705. U.S.A.

(Revised received 6 January 1978)

Key Word Index-Brassica napus; Cruciferae: rape pollen: identification: glucosyl esters: fatty acids.

Abstract-6-D-Glucopyranosyl esters of palmitic, oleic, linoleic and linolenic acids were identified in Brassica napus (rape) pollen. These esters are inactive as plant growth promoters in the bean second-internode bioassay.

INTRODUCTION acids as oleic. linoleic and linolenic acids. The identitv of o-glucose in hydrolysates of esters subsequently In previous work, fractionation of Brassica nap tiS (rape) isolated by PLC was established by TLC, GLC and pollen gave a preparation called 'brassins' which exhibited colour reaction. novel plant growth-promoting activity, and which Individual glucosyl esters were separated by PLC contained a series of glucosyl esters of various fatty on AgN03-Si gel. Although the layers exhibited gray acids [1]. When bioassayed by the bean second-internode streaks indicating reduction of Ag ion, the reaction was method [2J, brassin preparations cause both elongation only partial. Because reduction of Ag ion was not and thickening of the internode. As the concentration of observed with synthetic 1- and 2-substituted glucosyl the active pr~lciple increases, swelling and curvature esters, an alternate site of fatty acid attachment in the of the stem are enhanced and often give rise to split natural material was indicated. Three bands were internodes. The esters \vere reported earlier [1 J to be removed which afforded the following major glucosyl I-glucosyl esters mainly on the basis of MS evidence esters in order of decreasing R (: palmitate, linoleate and obtained from a mixture. Individual esters were not linolenate. Alternatively. glucosyl esters could be sepa separated. and there was no correlation of biological rated by reversed phase HPLC on ,u-Bondapak C , 18 activity \vith any physical measurement [3]. As a con Esters were eluted in the order: linolenate, linoleate. tinuation of this research, we report here a series of new palmitate and oleate (contaminated with palmitate). 6-glucosyl esters from rape pollen which are distinct HPLC ofa biologically active fraction that also contained from the unidentified active principle, Esters identified as glucosyl esters resulted in elution of material that exhi 6-o-glucopyranosyl linolenate, palmitate, linoleate and bited brassin growth-promoting activity well before the oleate are inactive in the bean second-internode bioassay. elution of glucosyllinolenate. GC-MS of TMSi derivatives of natural glucosyl RESULTS AND DISCUSSION esters (see Experimental) gave ions for M + minus Me and/or one and two TMSiOH groups. In addition An iso-PrOH extract of H,O washed Brassica napus to acylium ions (RCO+) indicative of the fatty acid, (rape) pollen was fractionated by countercurrent distribu- ions corresponding to rearranged RCO,DMSi + were tion (CCD) and column chromatography on Si gel. also observed. The derivatized esters exhibited two The bean second-internode bioassay was used to follow GLC peaks of variable intensity representing the fractionation. Rechromatography of those fractions ex- anomeric mixture; the first peak due to the fl-anomer hibiting significant brassin-type activity afforded an (see below) was usually less intense. The MS of the first inactive major fraction which was eluted just prior G LC peak exhibited strong ions at m/e 217 (100 %) and to the growth-promoting material. The IR (Nujol) 204 (ca 50 %) while the second peak showed these spectrum ofthe inactive fraction showed ester absorption ions with the intensities reversed [m/e 217 (50 %), at 1730 cm - 1. Transesterification with NaGMe gave 204 (100 %)J. These ions represent three- and two Me esters which were analyzed by GLC on the basis carbon fragments from the carbohydrate ring [5]. of equivalent chain length. The following fatty acid In comparison, MS of synthetic TMSi-I-glucopyranosyl Me esters were characterized (percent composition palmitates showed the ion at m/e 217 three times stronger given in parentheses): 14: 0(0.6), 16: 0(39), 18: 0(0.8), than the 204 ion. 18: 1(3), 18:2(11.8), 18:3(44.8). Assignments were con- The 90 MHz PMR spectrum (CsDsN) of natural firmed by GC-MS which showed the appropriate glucosyl palmitate showed it to be an anomeric mixture M + for each ester. Double bond positions in unsaturated of 75 % C( (<5 5.88, J = 3.5 Hz): 25 % fl (<55.32, J = 7.0 Hz). esters were established by GC-MS of TMSi derivatives In contrast, anomeric protons for synthetic 1- and 2 of the alcohols obtained by per-hydroxylation with glucopyranosyl palmitates [6. 7J were observed as OsO4 [4]. These procedures identified the unsaturated follows: I-C(, <5 6.92 (J = 3.5 Hz): I-fl, <56.41 (J = 6.7 Hz): 1187 1188 Short Reports

2-y.:2-f3 (1:1 mixture), (j 6.10 (J=3.5Hz), (j 5.38 TLC, 95 ~io EtOH-toluene, 1: 3) just prior to the active material. (J = 8 Hz). The 220 MHz PMR spectrum of natural Combined inactive fractions from 6 kg pollen (230 mg) were glucosyl palmitate was compared with that of synthetic rechromatographed on Si gel (23 g) to give the glucosyl esters 6-y.-D-glucopyranosyl palmitate [8J. Except for dif (190mg). ferences due to anomeric composition, the spectra were Fatty acid analysis. A soln of glucosyl esters (40 mg) in identical. Substitution at C-6 was verified by the observa CHoClo-MeOH was treated with 0.5 M NOlO Me (2 ml) at 60' for 20 min. The soln was cooled, acidfied with 4 N HCl, diluted tion of the lovv field resonances of the C-6 methylene with HoO and extracted with hexane to give the Me esters: protons at (j 4.3 (Me CO-d ) in agreement with pre 2 6 IR (Cd.) 1740 cm- 1. no trans unsaturation. GLC analvsis viously reported values [9J. However, a detailed analysis was at 180' on a 3 m ~ 4 mrn column packed with 5% LAC-2 of the coupling constants could not be readily performed R446. GLC separation of TMSi derivatives of the alcohols due to the complexities given by the anomeric mixture obtained from OsO" per-hydroxylation of unsaturated Me and the degeneracy of the C-6 methylene proton reso esters [4J was on a 1.2 m x 4 mm column packed with 3 % nances. Heating the syntheticy.-anomer in C D N-D 0 OV-I programmed from 150 to 250' at 4'/min. s s 2 at 50' for 15 min gave a 60:40y.:f3-anomeric distribution Sugar identification. Samples of hexose esters from PLC that more closely resembled the natural material. (0.6 mg) in 2 N HCI (l ml) were heated under reflux for 1 hr. cooled, washed with hexane and evap. to dryness under red: Similarly, a sample consisting of at least 90 %y.-anomer pres. The residues were dissolved in H,O (30 pI) and applied was converted to a 50:50 mixture after 3 days in CsDsN to Lilly Tes-tape R urine sugar analysis paper to give an immedi at room temperature. GC-MS of the TMSi derivative ate positive reaction for D-glucose. The sugar migrated with of this mixture now showed a corresponding increase D-glucose on TLC using 0.1 NH 3B03-Si gel G (n-BuOH in the first GLC peak representing the f3-anomer and MeoCO-H,O: 4:5:1). GLC analvsis of the TMSi derivative exhibiting the intense mle 217 (100 ~~) ion. The mutarota on Ii 1.2 m'x 2 mm column pack~d with 3 ~!~ Dexsil 300 and tion of glucose in pyridine explains why 6-glucosyl programmed from 100 to 200' at 3'/min confirmed the presence palmitate was obtained as an anomeric mixture [1 OJ. of D-glucose. The PMR spectra of natural 6-glucosyl linoleate and Separation ofglucosyl esters. PLC: Solns of esters in MeOH CHCI3 (l :9) were applied to 0.5 mm thick layers of AgN03-Si Iinolenate likewise showed the predominant y.-anomeric gel G (5: 95) which were developed with EtOH-toluene (l: 3). protons at (j 5.88 (J = 3.8 Hz) and (j 5.87 (J = 3.5 Hz) The plates were dried in a stream of No and I em of each side respectively. The amount off3-anomer was notdetermined was sprayed with 0.2 % 2',7'-dichlorofluorescein in EtOH. because of proximity to the olefinic proton signals Three bands observed under long UV light were removed and centered at (j 5.49. extracted with MeOH-CHCI3 (l :9). Each extract was re· These esters are new natural products, but are closely chromatographed on a Si gel column (I g) with EtOH-toluene related to the steryl-f3-D-( 6-0-fattyacyl)-glucopyranosides (l :9). Glucosyl linolenate and linoleate were obtained as oils. which are well-known plant constituents [11 J. Although Combined samples of glucosyl palmitate were subjected to we were able to separate the brassin growth-promoting PLC on Si gel G. Material migrating 5.5-9 em from the origin was extracted and crystallized from EtOH :mp 129-132': mmp activity from the reported 6-glucosyl esters, we were \vith synthetic 6-y.-D-glucopyranosyl palmitate was not de unable to confirm by PMR the presence of 1-glucosyl pressed. HPLC: A Waters Associates ALC-201 instrument esters. The following synthetic glucosyl esters were equipped with two 0.33 m x 4 mm !D p-Bondapak C 8 nevertheless evaluated in the bean second-internode columns and a refractive index detector was used. The solve~t bioassay and all failed to exhibit brassin-type activity: was MeOH-HzO (3: 1) at 200 kgcm Z and 1ml/min. Typically, 1-0.- and I-f3-palmitates: l-cz-stearate; 2-cz- and 2-13 glucosyl esters were eluted with the following solvent vo1s: palmitates: 6-y.-palmitate, oleate, linoleate and linolenate. 18:3 (26-31 ml), 18:2 (37--40ml), 16:0 (44-52ml), 16:0 plus 18: I (53-60 ml). Synrhesis of 6-y.-D-glucopyranosyl palmitate. Dry ,B-D-glucose EXPERIMENTAL was allowed.to react with palmitoyl chloride [8]. The MeOH

Extraction and preliminary Factionation. Bee-collected CHCI3 (1 :9) soluble portion of the reaction mixture was Brassica napus L. pollen (70-90 % B. napus plus mixed pollens) chromatographed on Si gel with EtOH-toluene to remove some from the McRory Apiaries, Benito, Manitoba, Canada (500 g) 1-,B-D-glucopyranosyl palmitate which eluted prior to the was washed with H,O (1.21.. 11.. 11.1 and centrifuged. The 6-isomer. The major 6-:/-ester was crystallized from absolute residual pollen (ca 250 g) was' extracted twice at ro;m temp EtOH: mp 133-135' (lit mp 134-136'). Other 6-glucosyl for I hr with iso-PrOH (1400 ml. 1000 ml). The extracts were ester standards were similarly prepared. filtered through Celite and cone. under red. pres. to give 55-60 g. GC-MS of glucosyl esters. The esters were converted to Countercurrent distribution was carried out in four 250 ml TMSi esters by treatment with excess HMDS and TMCS in separatory funnels with CCl,,-MeOH-H10 (2:1:0.15) [12]. CsHsN for 1 hr at room temp. GLC separation was on a The extract was dissolved in 200 ml of upper and lower phase I m x 2 mm column packed with 3 % OV-l and programmed in funnel 1. Three remaining funnels contained 100 ml of from 200 to 350' at 4'/min. Effiuent from the GLC was directed upper phase. Fifteen 100 ml portions of 100ver phase were to a Dupont (CEC) 21-492-1 MS through a jet-type separator sequentially equilibrated with upper phases. Twelve portions with a transfer line temp. of 300'. Spectra were obtained \vith of lower phase were drawn off funnel 4 and discarded. Upper an ionizing energy of 70 eV and a source temp. of 250'. MS mie and lower phases in funnels 2--4 were combined and cone (reI. int.): 6-:/-D-glucopyranosyl linolenate: 728 [M '] (0.3), carefully (foams) under red. pres. to give co 4 g trom each 713 (0.2), 638 (2), 623 (0.3), 548 (l), 533 (0.3), 335 [RC02 DMSi+J 500 g of pollen (4),261 [RCO'] (41.2171461.204 (100). 191 (34). 6-fi-D-glucopy Column chromaTography. Combined CCD fractions from ranosyl hnolenate: 548 10.7), 533 (0.2), 335 (2),261 (3),217 (100), 204 (33),191 (12). 1 kg of pollen in MeOH-CHCI3 (5:95) were applied to Si 6-:/-D-glucopyranosyl linoleate: 730 [M +] (0.2), 715 (0.2), gel (l50 g, 70-230 mesh). Material eluted with MeOH-CHCI3 640 (I), 625 (0.3), 550 (1), 535 (0.3), 337 (3), 263 (3), 217 (49), (I :9) \vas combined on the basis of TLC analysis (CHCI 3 MeOH-HOAc, 90:10:1: sprayed with 3'10 eerie sulfate in 204 (100), 191 (50). 6-,B-D-glucopyranosyl linoleate: 640 (0.6), 62510.2),550 (0.8), 535 (0.3), 337 (3), 263 (6), 217 (l00). 204 (46), 3 N H 2 S04 and heated at 120'1. Fractions exhibiting significant activity in the bean second-internode bioassay were rechromato 191 (23). graphed on Si gel with absolute EtOH-toluene. A biologically 6-):-D-g1ucopyranosyl oleate: 717 [M -Me] (0.11, 642 (2). 627 (R (0.2),552 (1), 537 (U.2), 339 (31. 265 (4), 217 (51),204 (100),191 inactive major fraction was eluted with 10% EtOH f 0.4 on Short Reports 1189

(52). 6-{J-D-glucopyranosyl oleate: 642 (0.3), 627 (0.1), 552 (J), 2. Mitchell, J. W. and Livingston, G. A. (1968) Methods oj 537 (0.2), 339 (2), 265 (4), 217 (J 00), 204 (43), 191 (J8). Stlldyillg Plam H ormOlles alld Growth-Regulating Substances, 6-:t.-D-glucopyranosyl palmitate: 691 [M+-Me] (J), 616 p. 26. Agriculture Handbook No. 336. U.S. Government Printing Office. Washington. D.C. (0.7), 601 (J), 526 (J), 511 (J), 313 [RC02 DMSi+] (12), 239 [RCO~] (8), 217 (54), 204 (100),191 (44). 6-{J-D-glucopyranosyl .). Milbor;ow. B. V. and P;yce. R. J. (1973) Natllre 243, 46. palmitate: 691 (1),616 (J), 526 (1), 511 (1),313 (21), 239 (J7), 217 4. Murawski. U. and Egge. H. (1975) J. Chromotogr. Sci. 13. (100),204 (55),191 (39). 497. 1-:t.-D-glucopyranosyl palmitate: 691 (0.6),616 (0.3), 601 (2), 5. Dejongh, D. c., Radford, T., Hribar. J. H.. Hanessian. S.. 511 (3),313 (JO), 239 (18), 217 (99),204 (30), 191 (71). l-{J-D Bieber, M., Dawson, G. and Sweeley: C. C. (1969) J. Amer. glucopyranosyl palmitate: 691 (0.3),601 (0.4),511 (0.6), 313 (6), Cheln. Soc. 91, 1728. 239 (11), 217 (100), 204 (35), 191 (25). 6. Pfeffer, P. E., Rothman, E. S. and Moore, G. G. (1976) 2-:t.:2-{J-D-glucopyranosyl palmitate: 691 (0.7), 616 (0.3), J. Org. Chem. 41, 2925. 601 (J), 511 (1),313 (9), 239 (18), 217 (93), 204 (39),191 (71). 7. Pfeffer, P. E., Moore, G. G., Hoagland, P. D. and Rothman, E. S. (1977) Synthetic Methods Jor Carbohydrates (El Ackllowledgemems-We thank Dr. D. Weisleder for providing Khadem, H. S., ed.), p. 155, ACS Symposium Series, Vol. 39. American Chemical Societv. Washington. D.C. some of the 90 MHZ PMR data, M. V. Wakeman and D. W. 8. Reinefeld, E. and Korn, H. F. (1968) Sta;rke 20,181. Spaulding for technical assistance. The 220 MHZ PMR spectra 9. Moyer, B. G., Pfeffer, P. E., Moniot, J. L., Shamma. M. and were recorded at the Middle Atlantic Regional NMR facilitv which is supported by NIH grant RR 542 at the Universit)· Gustine, D. L. (1977) Phytochemistry 16, 375. of Pennsylvania. 10. Hill, A. S. and Shallenberger, R. S. (1969) Carbohydr. Res. II. 541. 11. Aneja. R. and Harries, P. C. (1974) Chem. Phrs. Lipids 12.351. REFERENCES 12. Hecker, E. and Schmidt, R. (1974) Fortschr. Chem. Org. I. Mandava, N. and Mitchell, J. W. (J972) Chem. Illd. 930. Natllrst. 31. 377.

Phyrochemisrry.i978. Vol. 17. pp. 1189-1190. {£1 Pergamon Press Ltd. Printed in England. 0031-9412/78:0701-1189 S02.00/0

NEUE EUDESMAN-DERIVATE AUS PLUCHEA FOETIDA*

FERDINAND BOHLMANN und PRADIP K. MAHANTA Institut fUr Organische Chemie der Technischen Universitiit Berlin, Straf3e des 17. Juni 135. D-I00 Berlin 12. Deutschland

(Eingegangen am 13 Januar 1978)

Key Word Index-Pluclzea Joetida: Compositae; new eudesmane derivatives.

Die bisher untersuchten Pillchea-Arten (Tribus Inuleae) meter. CHCI 3 • Die lufttrockenen zerkleinerten Pfianzenteile enthalten neben Thiophenacetylenverbindungen vor (Herbar Nr. R.M.K. 230) extrahierte man mit Et,O-Petrol aHem Eudesman-Derivate mit charakteristischem Sub (1 :2) und trennte die erhaltenen Extrakte zuniichst g-rob durch stitutionsmuster [1]. Auch die Wurzeln von Pillchea SC (Si gel, Akt.-St. II) und we iter durch mehrfache DC (Si gel, foetida (L.) DC. enthalten 1, 2 und 3, wlihrend die GF 254).50 g Wurzeln ergaben 13 mg 1 und 2 (ca I: I) und 8 mg 3, wiihrend 500 g oberirdische Teile 50 mg 9. Spuren von 2, oberirdischen Teile neben 2, 3, und 9 die ebenfalls bekannten Sesquiterpene 4 und 5 enthalten. Daneben isoliert man zwei weitere Verbindungen. bei denen es Tabelle I. 'H-NMR-Daten von 6. 7 und 8 (270 MHz,

J (Hz) 6: 2edf3 = 2{J.3f3 = 3: 5:t.,6:t. = 4: 5:t..6f3 = 13: 6:t..6fi * 146. Mitteil. in der Serie 'Naturlich vorkommende Terpen = 15: 6:1,12 = 2: 9:t.,9{J = 15: 7: 2:t..3{J = 2{J,3f! = 3: 5:t.,6 = Derivate': 145. Mitteil.: Bohlmann, F. und Le Van, N. (1978) 2.5: 9:t.,9{J = 17: 17,18 = 6.8: 2:t..3f3 = 2{J.3{J = 3: 5:t..6:1 = 3: Phytochemistry 17.1173. 6:t..6f3 = 13 (Acetonid s 1.49 und 1.38). 1190 Short Reports

MeC=C ~[C=C]2CH=CH2 Me[C=C]2~C=CCH=CH2

1[3] 2[3J

14 3 [1] 4 [lJ 5 [2J 6 R Epoxyang Ang Epoxyang H 12 R' H H Ac H

15 ~ 9

90mg 4, 140mg 3, 180mg 5, 14mg 7 (CH,Cl,-MeOH, 9:1) 589 578 546 436 nm J~4' und 18 mg 6 (CH,Cl,-MeOH, 9: 1). -- [x = - ---:-:-,._- -:-:-:-::_:_ (c = 1.4) + 104.6 + 109.0 + 213.8' Cuauhr;mon (6). Farblose Kristalle aus Ether, Schmp. 143'. IR (CHCI ). OH 3620, 3300; C=C-C=O 1670 cm-I. MS. 3 Anerkenmmg-Herrn Dr. R. M. King, Smithsonian Institution, MOo mle 252.1725 (IOO%) (ber. fUr C,sH2403 252.1725): Washington, danken wir fUr das Pflanzenmaterial, der Deuts -'CH 237 (I2): -H 0 234 (I9): 234 -'CH 219 (I7); 219 3 2 3 chen Forschungsgemeinschaft fUr die F6rderung dieser Arbeit. -H,O 201 (40): 219 - CO 191 (23). Die Daten entsprechen den en des durch Verseifung von 5 erhaltenen Diols [2]. 10 mg 6 in 2 ml Aceton versetzte man mit 10 mg p-Toluo1sulfonsaure. Nach 12 hr Stehen bei Raumtemp. wurde neutralisiert, in Ether LITERATUR aufgenommen und der Riickstand durch DC (Et,O-Petrol. 1: 1) gereinigt. Man erhielt 7 mg 8, farbloses 01. JR. C- C-C=O 1. Bohlmann, F. und Zdero. C. (I 976) Chem. Ber. 109,2653. 1690. 1610 cm - 1. 2. Nakanishi, K., Crouch, R., Miura, 1.. Dominguez, X., 4o:-Aceroxy-3x-(2-merhyl-2,3-epoxy-buryryloxy)- 11-hydroxy-6, Zamudio, A. und Villarreal, R. (I 974). J. Am. Chem. Soc. 96, 7-dehvdroeudesman-8-on (7). Farbloses 01. IR. OH 3500: CO,R, 609. OAc i750: C=C-C=O 1665 cm-I. MS. MOo mle 408 (0.3%): 3. Bohlmann, F., Burkhardt. T. und Zdero, C. (973) Nafllrally -H2=C=O 366 (4): -AcOH 348 (2): 348 -'CH3 333 (IOO). Occurring Acerylenes. Academic Press, London.

A NEW GLAUCOLIDE DERIVATIVE FROM ERLANGEA RElv.fIFOLIA* FERDINAND BOHLMANN and HORST CZERSON Institute of Organic Chemistry, Technical University Berlin, Strasse d. 17. Juni 135, D-1000 Berlin 12, W. Germany

(Received 22 November 1977)

Key Word Index-Erlallgea remifolia: Compositae: Vernonieae: glaucolide.

So far only two Erlangea species (Tribe Vernonieae, closely related to glaucolide A (6) [3]. Though the MS Subtr. Vernoniinae) have been investigated chemically, did not show a molecular ion, all the data indicated one containing unusual coumarin and chromone deriva that we were dealing with a glaucolide with an additional tives[1J, the other guaianolides [2]. We now have hydroxy group in the ester attached to C-8. The com investigated E. remifolia Willd. et Pope collected in parison of the 270 MHz NMR spectrum at 80' with Botswana. The roots contained the hydrocarbons 1 that of glaucolide A clearly showed that the new lactone and 2, while the aerial parts afforded besides germacrene had structure 4, which was supported by acetylation D (3) in relatively high concentration a single lactone, of the free hydroxyl (see Table 1). All assignments were established by extensive double resonance experi ments. The isolation of 4 and the occurrence of guaiano * Part 140 in the series "Naturally Occurring Terpene Deriva lides in Erlangea inyangana [2J may indicate- a close tives", for part 139 see: Bohlmann, F. and Zdero, C., Phyto relationship to the genus Vernonia. However, more chemistry (in press). species have to be investigated.