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]OWMI of Natural Prochis Vof. 5 3 , No. 2 , p p . 441-450, Mar-Apr 1990
MICROBIAL TRANSFORMATION OF TETRACYCLIC DITERPENES: CONVERSION OF ENT-KAURENONES BY CURVULARZA A N D RHZZOPUS STRAINS ANDRES
GARCIA-GRANADOS,~ ANTONIO W T I N E Z ,
ANTONIO ORTIZ,
Departamento de Quimica Organira MARIA ESTHER ONORATO,
Amifisisy Detewninacih de Estrucruras, Swicios Timicas and JOSE MARIAARIW
Departamto de Mimbiofogia, F m f r a d de Cimias, Uniwrsi&d de G r a d , I8071 G r a d , Spain Assmcr.-We have biotransformed several mt-kaur-15-enones and mt-iaur- 16enones with Cunvkatia lunata and Rhizopus nigricans cultures. Incubation ofmt- 18-acetoxykaur15-ene-3,7dione with C. lunata led to its 17-hydroxy derivative, and with R . nigricans to 17hydroxy and 13-hydroxy derivatives. Incubation of ent- 18-acetoxykaur-15-en-3-0ne with C . lunata also gave the 17-hydroxyderivative, while incubation with R . nipcans produced 13-hydroxy- and 3,13dihydroxyderivatives.Moreover, biotransformation of mt- 18-acetoxykaur- 16ene-3,7-dione with C . lunata gave its (16R), 17-epoxy, (16R), 17- and ( l a ) , 17dihydroxy, and (7Shhydroxy derivatives. The structures of the metabolites have been determined by spectroscopy and chemical correlations.
As a continuation of our systematic studies on microbial transformations of terpenes (I+), we now report the bioconversion ofent-kaur- 15-en-7-one and -3,7-dione by Curvukzria lunata and Rhizopus nigricans to determine the possible influence of the ketone group at C-3. Furthermore, we have biotransformed one ent-kaur- 16-ene-3,7-dione with C. lunata cultures. These data together with previous results (1,2,4) help to discover the relationship between the structure of ent-kaurane substrates and the site at which the microorganism acts. MATERIAL AND METHODS PHVSIC~LANALYSES.-M~'Swere determined on a Kofler apparatus and are uncorrected. 'H-nmr spectra were measured at 300 MHz (CDCI,, 6 CHCI, = 7.25) in a Bruker AM-300. '3C-nmr spectra were determined at 74.75 MHz also in CDCI, (6CHCI, = 77.100). Assignments were made with the aid of distonionless enhancement by polarization transfer (DEPT), using a "flip angle" of 135". Ir spectra were recorded on an Ft-ir Nicolet 20SX spectrometer. Mass spectra were measured at 70 eV in a Hewlett-Packard 5988 A spectrometer. Elemental analyses were measured in a Perkin-Elmer 24OC analyzer. T h e rotatory powers were measured on a Perkin-Elmer 240 polarimeter at 20".
ISOUTIONOF STARTINGmmruhL.-The mt- 18-acetoxy-kaur-16-ene-3f3,7adiol (linear01 111) and mt- 18-acetoxykaur-15-ene-3@,7adiol (isolinearol [Z]) were isolated from Sideritic funkiana Willk. (Iabiatae) (9), and the ent-7a-acetoxykaur-15-en-18-01 (siderol [31) was isolated from Sideritis pusifka (Lange) Pau ssp. pavoVirens (Rouy) Malagarriga (Labiatae) (lo), and Sideritis arbvrescm Salzw. ex Bentham ssp. paulii (Pau) P.W. Ball ex Heywood (Labiatae) (1 1).
SYNTHESIS OF SUBSTRATEs.-LiflearO1 [l](2 g) was dissolved in Me2C0 (60 ml) and oxidized with Jones reagent (12). After cc, 1.8 g of mt-18-acetoxykaur-16-ene-3,7dione[4) was obtained (4). Isolinearol [Z)(0.6 g) was dissolved in Me2C0 (25 ml) and oxidized with Jonesreagent. After cc, 480 mg of ent-18-acetoxykaur-l5-ene-3,7dione[f was isolated as a gum: l a l -27' ~ (c= 1, CHCI,); ir Y max (neat) cm-' 1744, 1697, 1376, 1219, 1039, 998; 'H q r (6) 5.49 ( l H , s, H-15), 4.15 and 3.75 (2H, AB system,J= 11.3 Hz, H2-18), 1.97 (3H, s, A&), 1.72 (3H, d , J = 1.6 Hz, Me-17), 1.32 and 1.00 (3H, s,Me-19andMe-20); '3CnmrseeTable l;msdz(%)[M7+358(78), 298(18), 285(19), 273(11), 255 (20), 227 (7), 201 (7), 199 (lo), 316 (17). Found C 73.4, H 8.6; C22H3004 requires C 73.71, H 8.43%. Siderol 131(1 g) was saponified with MeOH-H,O (70:30) (30 ml) and KOH (1.5 g) for 18 h at room
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Journal of Natural Products
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Mar-Apr 19901 Garcia-Granadoset al. : Conversionofent-Kaurenones
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temperature. After cc, 860 mg ofent-kaur- 15-ene-7a, 18diol (sideridiol161)(13)was obtained. Product 6 (800 mg) was acetylated with pyridine-Ac,O (50:20) for 2 h a t 0'. After cc, 675 mg ofmt- 18-acetoxykaur15-en-7a-01 (sideripol [q) (14) was isolated: mp 83-85"(from hexane/CHCI3);[a]D +4O(c= 1, CHCI,); ir Y rnax (KBr) cm-' 3504, 1739, 1243, 1039; 'H nmr (6) 5.48 ( l H , s, H-15), 4.00 and 3.44 (2H, AB system,]= 11.1 Hz, H,-18), 3.54(1H, dd, J 1 = J 2 = 2 . 8 H z , H-7), 2.02(3H, s, A d ) , 1.67 (3H, d, ]= 1.6 Hz, Me-17), 1.02 and 0.76 (3H each, s, Me-19 and Me-20); I3C nmr seeTable 1; ms m/z(%) [MI+ 346 (14), 286 (7), 255 (1 l), 164 (16), 149 (9), 122 (26), 43 (100). Product 7 (600 mg) was dissolved in Me,CO (25 ml) and oxidized with Jones reagent. After cc 490 mg ofent-18-acetoxykaur-15-en-7-0ne181 wasobtainedasagum: [a]D -7O(c=O.5, CHCI,); irvmax(neat)cm-' 1739, 1700, 1380, 1336, 1238, 810, 755; 'H nmr (6) 5.44 ( l H , s, H-15), 3.67 and 3.61 (2H, AB system,]= 11.4 Hz, H,-18), 1.97 (3H, s,AcO), 1.67(3H,d,]= 1.6Hz,Me-17), 1.15and0.80(3Heach,Me-19andMe-20); 13Cnmrsee Table l;msrn/z(%)[M]+ 344(100), 329(7), 316(15), 301(20), 284(15), 269(56), 241(25), 201(13). Found C 76.3, H 9.5; C,,H,,O, requires C 76.70, H 9.36%. ORGANISMS.
