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The microbiological transformation of some isoatisene diterpenoids into isoatisagibberellins and isoatisenolides by Gibberella fujikuroi
Phytochemiwy.
Vol. 26. No. 9. pp. 2521-2524.
1987. 0
Printedin GreatBritain.
0031~9422187 S3.00 + 0.00 I987 Pergamon JournalsLtd.
THE MICROBIOLOGICAL TRANSFORMATION OF SOME ISOATISENE DITERPENOIDS INTO ISOATISAGIBBERELLINS AND ISOATISENOLIDES BY GZBBERELLA FUJZKUROZ BRAULIOM. FRAGA and RICARDOGUILLERMO lnstituto de Productos Naturalcs Organ&s, C.S.I.C., La Laguna. Tencrife, Canary Islands, Spain (Revised received 12 February
Key Word Idex-Gibberella
1987)
jiijikuroi; gibbcrellins; kaurenolida; ent-atis-15cm; isoatisagiblxrcllins; isoatis-
cnolides; gummifcrolic acid.
Abstract-The microbiological transformations of ent-‘la-hydroxyatis-lSen-19-oic acid into isoatisagibberellins A, 2 and A , , , and of ent-l9-hydroxy-atis-6,lZ-diene into 7/?-hydroxyisoatisenolide and 7fi,18dihydroxyisoatisenolide have been demonstrated using Gibberellafujikuroi. The substrates incubated were chemically obtained from gummiferolic acid.
INTRODUCTION
The microbiological transformation of ent-7ahydroxyatis-Sen-19-oic acid (2) gave isoatisagibberellin The gibberellins are plant hormones that are biosynthetiAL2 (8) and isoatisagibbereliin A,, (12), in the acid tally derived from ent-kaur-16ene (1). Although some fraction. No metabolites were isolated from the neutral diterpenes structurally related to 1 are known, such as entfraction. The high resolution mass spectrum of isokaur-1Sene (isokaurene), ent-phyllocladene (13/L?atisagibberellin A, 2 dimethyl ester (10) was in accordance kaurene), ent-beyer-I S-ene, enl-atis- 16-ene. ent-atis-l5with the molecular formula C12H3204, and its ‘H NMR ene (isoatisene), and ent-trachylobane, the gibberellins spectrum contained three C-methyl signals, two singlets at which have been isolated so far belong to the ent-kaur-Id 60.78 and 1.08 for C-20 and C-18 methyls, respectively, ene series [l]. However, nothing is known about and one doublet at 1.73, assigned to the C-17 methyl. The gibberellin biosynthesis, which might preclude the natural characteristic gibberellin double doublets of H-5 and H-6 occurrence of analogues of gibberellins with other appeared at 2.04 and 3.25 resptively, with values similar skeletons, that show different arrangements of rings C and to those observed for the dimethyl ester ofatisagibberellin D. We have shown that the fungus Gibberellafujikuroican A, z [2]. The two methoxyls appeared at 3.66 and 3.74 and metabolize substrates with the atisane [2], trachylobane the vinylic proton as a broad singlet at 5.77. The mass [3,43, beyerene [S, 61, and isokaurane 173 skeletons to spectrum was very similar to those of gibberellin Al2 give atisa-, trachyloba-, beyer- and iso-gibberellins. Other dimethyl ester [13] and atisagibberellin A,, dimethyl authors have also published the results of the incubation ester [Z], with the molecular ion at m/z 360 and fragments of trachylobane and atisane derivatives by a mutant of this at m/z 328,300 and 285. fungus [8,9]. To complete these studies, we describe in The structure of the second gibberellin analogue obthis work the microbiological transformation by ‘wildtained in the methylated acid fraction was determined as type’ Giibbereh fujikuroi of the synthetic isoatisene isoatisagibberellin Al, methyl ester, on the basis of the derivatives, ent-7a-hydroxyatis-15en-19-oic acid (2) and following considerations: the molecular formula was ent-19-hydroxyatis-6-,lSdiene (a), to give isoestablished as CzlH,sOd by high resolution mass atisagibberellins and isoatisenolides, respectively. Both spectrometry. Its ‘H NMR spectrum showed signals of an compounds, 2 and 6, were synthetically prepared from angular methyl (6 I.1 1, s), of a methyl on a double bond gummiferolic acid (9, an atisane diteqxme isolated from (1.74, d, J = 1.5 Hz), ofa methoxyl(3.74, s)and of the H-5 Margotia gummifera [lo]. and H-6 hydrogens (2.29 and 2.44) d, J = 1I Hz). The methylene protons at C-20 also appear in this spectrum as RESULTS AND DISCUSSION a doublet at 64.1 I (J = I2 Hz) and a double doublet at The fermentations were carried out in the presence of 4.57 (J = I2 and 1.5 Hz). The second coupling constant of AM0 1618, an inhibitor of the biosynthesis of ent-kaurthis last hydrogen probably originates from a long-range 16-ene (I), in order to suppress the formation of the coupling with the H-la. The mass spectrum contains ions metabolites derived from this diterpene, and to facilitate similar to those obtained for GA15 methyl ester at the study of the product formed [ 11,121. The incubations m/z 344,326,3 12 and 284 [ 133. A third isoatisagibberellin were harvested after six days of growth, and the broth and was a minor compound in one of the fractions that mycelium extracts were mixed, and separated into acid contained isoatisagibberellin A,S, and it was tentatively and neutral fractions. The acid fraction was treated with identified as isoatisagibberellin AZ* on the evidence of the diazomethane and purified by chromatography of its high and low resolution mass spectrum of this fraction. methyl esters. It has been previously noted C2.93 that ent-atis-16-ene 2521
2522
8. M.
FRAGA
and
3 R’=
8 R’= H, R2=Me 9 R’=H.R’=CHO 10
R’ -
b
11
R’ = Me. R’ - CHO
R. GUILL~RMO
H, R2=
Ang
4
R’ * Me, R2 = Ang
5
R’-Me,
6 7
R = CHIOH R -
C02Me
Ra=H
u
R-H
14 R = H
w
R-Me
15
R = OH
= Me
: +I
& I
H
.. ..
R
16 R = COaMe 17 R - CH20H
. .._ Cl
2 R’ = R’ - H 18 R’ - H, R’ - Ang 19 R’ - Me, R2 - H
diterpenes were only metabolized for a few steps along the gibberellin biosynthetic pathway, and atisagibberellins oxidized at C-20 were not obtained. In contrast we have now obtained isoatisagibberellins with oxygen at this carbon, indicating that a small variation in the atisene D ring, can affect the substrate specificity of the enzyme(s) responsible for these oxidations. On the other hand, no C9 analogues of’gibberellin were isolated in the present work from the feedings of isoatisene derivatives, as also occurred in the incubation of enr-atis-16-ene diterpenes cz93. The incubation of ear-19-hydroxyatis-6,lSdiene (6) gave 7/?-hydroxyisoatisenolide (14)and 7fi-18dihydroxyisotisenolide (15) in the neutral fraction. The acid fraction did not contain gibberellin metabolites. This result is as expected because the A6double bond in 6, leads the metabolism to the kaurenolide biosynthetic pathway [14]. The less polar of these compounds, the 7/?hydroxyisoatisenolide (14), showed in its ‘H NMR spectrum signals of the three methyl groups, of the olefinic proton at C-15 and of the resonances originating from the hydrogens at C-6 and C-7 (64.74 and 4.11, I,, 6 = 7 Hx,
J
r 4 Hz). These last signals are similar to those 06!&ved in the kaurenolides [15]. The more polar hydroxy-lactone showed a ‘H NMR spectrum similar to that of 14, where the signal of one of the angular methyl groups has been substituted by a broad singlet at 63.71 (2H) of a hydroxymethyl group. By comparison with the spectrum of 7/?,18dihydroxykaurenolide, this alcoholic function was assigned to C-18, and the structure established as 7fiJ8dihydroxyisoatisenolide (15). The substrates incubated were synthesized in the following way. Hydrolysis of the methyl ester of gummiferolic acid (4) gave the alcohol 5 [IO], which was treated with ptoluenesulphonic acid in benzene to give the isomer&d compound 19. Reaction of 19 with thionyl chloride in pyridine gave the dehydrated product 7, contaminated with the chlorine derivative 16. Treatment of this mixture with lithium aluminium hydride afforded the alcohols 6 and 17, which were separated by silica gel chromatography. The hydroxy acid 2 was obtained by isomerization of gummiferolic acid (3), and then by hydrolysis of the product obtained (1s).
2524 7. Fraga, B. M., Hcmanda,
8. 9. 10. 11.
B. M. FRAGAand R. GUILURMO
M. G., Rodriguez, M. D., Diat, C. E., Gonzalez, P. and Hanson, J. R. (1987) Phyrochetirry 26, 1931. Bearder, J. R, MacMillan, J. and Matsuo, A. (1979) J. Chem. Sot. Chem. Comm. 650. Beale, M. H., Bearder, J. R., MacMillan, J., Matsuo, A. and Phinncy, B. 0. (1983) Phytochemistry 22, 875. Pinar, M.. Rodriguez_ B. and Alemany, A. (1978) Phytochemistry 17, 1636. Barnes, M. F., Light, E. N. and Lang, A. (1969)Planta 88,172.
12. Cross, B. E and Myers, P. L. (1969) Phytochemistry g, 79. 13. Binks, R, MacMillan, 1. and Pryce, R. J. (1969) Phytochemistry 8, 271. 14. Beale, M. H., Bearder, J. R., Down, G. H., Hutchison, M., MacMillan, J. and Phinncy, B. 0. (1982) Phytochemistry 21, 1279. 15. Hanson, J. R., Hawker, J. and White, A. F. (1966) Terra/w&on 22, 1701. 16. Hanson. J. R, Hawker, J. and White, A. F. (1972) J. Chem. Sot. Perkin Trans. 1, 1892.
Vol. 26. No. 9. pp. 2521-2524.
1987. 0
Printedin GreatBritain.
0031~9422187 S3.00 + 0.00 I987 Pergamon JournalsLtd.
THE MICROBIOLOGICAL TRANSFORMATION OF SOME ISOATISENE DITERPENOIDS INTO ISOATISAGIBBERELLINS AND ISOATISENOLIDES BY GZBBERELLA FUJZKUROZ BRAULIOM. FRAGA and RICARDOGUILLERMO lnstituto de Productos Naturalcs Organ&s, C.S.I.C., La Laguna. Tencrife, Canary Islands, Spain (Revised received 12 February
Key Word Idex-Gibberella
1987)
jiijikuroi; gibbcrellins; kaurenolida; ent-atis-15cm; isoatisagiblxrcllins; isoatis-
cnolides; gummifcrolic acid.
Abstract-The microbiological transformations of ent-‘la-hydroxyatis-lSen-19-oic acid into isoatisagibberellins A, 2 and A , , , and of ent-l9-hydroxy-atis-6,lZ-diene into 7/?-hydroxyisoatisenolide and 7fi,18dihydroxyisoatisenolide have been demonstrated using Gibberellafujikuroi. The substrates incubated were chemically obtained from gummiferolic acid.
INTRODUCTION
The microbiological transformation of ent-7ahydroxyatis-Sen-19-oic acid (2) gave isoatisagibberellin The gibberellins are plant hormones that are biosynthetiAL2 (8) and isoatisagibbereliin A,, (12), in the acid tally derived from ent-kaur-16ene (1). Although some fraction. No metabolites were isolated from the neutral diterpenes structurally related to 1 are known, such as entfraction. The high resolution mass spectrum of isokaur-1Sene (isokaurene), ent-phyllocladene (13/L?atisagibberellin A, 2 dimethyl ester (10) was in accordance kaurene), ent-beyer-I S-ene, enl-atis- 16-ene. ent-atis-l5with the molecular formula C12H3204, and its ‘H NMR ene (isoatisene), and ent-trachylobane, the gibberellins spectrum contained three C-methyl signals, two singlets at which have been isolated so far belong to the ent-kaur-Id 60.78 and 1.08 for C-20 and C-18 methyls, respectively, ene series [l]. However, nothing is known about and one doublet at 1.73, assigned to the C-17 methyl. The gibberellin biosynthesis, which might preclude the natural characteristic gibberellin double doublets of H-5 and H-6 occurrence of analogues of gibberellins with other appeared at 2.04 and 3.25 resptively, with values similar skeletons, that show different arrangements of rings C and to those observed for the dimethyl ester ofatisagibberellin D. We have shown that the fungus Gibberellafujikuroican A, z [2]. The two methoxyls appeared at 3.66 and 3.74 and metabolize substrates with the atisane [2], trachylobane the vinylic proton as a broad singlet at 5.77. The mass [3,43, beyerene [S, 61, and isokaurane 173 skeletons to spectrum was very similar to those of gibberellin Al2 give atisa-, trachyloba-, beyer- and iso-gibberellins. Other dimethyl ester [13] and atisagibberellin A,, dimethyl authors have also published the results of the incubation ester [Z], with the molecular ion at m/z 360 and fragments of trachylobane and atisane derivatives by a mutant of this at m/z 328,300 and 285. fungus [8,9]. To complete these studies, we describe in The structure of the second gibberellin analogue obthis work the microbiological transformation by ‘wildtained in the methylated acid fraction was determined as type’ Giibbereh fujikuroi of the synthetic isoatisene isoatisagibberellin Al, methyl ester, on the basis of the derivatives, ent-7a-hydroxyatis-15en-19-oic acid (2) and following considerations: the molecular formula was ent-19-hydroxyatis-6-,lSdiene (a), to give isoestablished as CzlH,sOd by high resolution mass atisagibberellins and isoatisenolides, respectively. Both spectrometry. Its ‘H NMR spectrum showed signals of an compounds, 2 and 6, were synthetically prepared from angular methyl (6 I.1 1, s), of a methyl on a double bond gummiferolic acid (9, an atisane diteqxme isolated from (1.74, d, J = 1.5 Hz), ofa methoxyl(3.74, s)and of the H-5 Margotia gummifera [lo]. and H-6 hydrogens (2.29 and 2.44) d, J = 1I Hz). The methylene protons at C-20 also appear in this spectrum as RESULTS AND DISCUSSION a doublet at 64.1 I (J = I2 Hz) and a double doublet at The fermentations were carried out in the presence of 4.57 (J = I2 and 1.5 Hz). The second coupling constant of AM0 1618, an inhibitor of the biosynthesis of ent-kaurthis last hydrogen probably originates from a long-range 16-ene (I), in order to suppress the formation of the coupling with the H-la. The mass spectrum contains ions metabolites derived from this diterpene, and to facilitate similar to those obtained for GA15 methyl ester at the study of the product formed [ 11,121. The incubations m/z 344,326,3 12 and 284 [ 133. A third isoatisagibberellin were harvested after six days of growth, and the broth and was a minor compound in one of the fractions that mycelium extracts were mixed, and separated into acid contained isoatisagibberellin A,S, and it was tentatively and neutral fractions. The acid fraction was treated with identified as isoatisagibberellin AZ* on the evidence of the diazomethane and purified by chromatography of its high and low resolution mass spectrum of this fraction. methyl esters. It has been previously noted C2.93 that ent-atis-16-ene 2521
2522
8. M.
FRAGA
and
3 R’=
8 R’= H, R2=Me 9 R’=H.R’=CHO 10
R’ -
b
11
R’ = Me. R’ - CHO
R. GUILL~RMO
H, R2=
Ang
4
R’ * Me, R2 = Ang
5
R’-Me,
6 7
R = CHIOH R -
C02Me
Ra=H
u
R-H
14 R = H
w
R-Me
15
R = OH
= Me
: +I
& I
H
.. ..
R
16 R = COaMe 17 R - CH20H
. .._ Cl
2 R’ = R’ - H 18 R’ - H, R’ - Ang 19 R’ - Me, R2 - H
diterpenes were only metabolized for a few steps along the gibberellin biosynthetic pathway, and atisagibberellins oxidized at C-20 were not obtained. In contrast we have now obtained isoatisagibberellins with oxygen at this carbon, indicating that a small variation in the atisene D ring, can affect the substrate specificity of the enzyme(s) responsible for these oxidations. On the other hand, no C9 analogues of’gibberellin were isolated in the present work from the feedings of isoatisene derivatives, as also occurred in the incubation of enr-atis-16-ene diterpenes cz93. The incubation of ear-19-hydroxyatis-6,lSdiene (6) gave 7/?-hydroxyisoatisenolide (14)and 7fi-18dihydroxyisotisenolide (15) in the neutral fraction. The acid fraction did not contain gibberellin metabolites. This result is as expected because the A6double bond in 6, leads the metabolism to the kaurenolide biosynthetic pathway [14]. The less polar of these compounds, the 7/?hydroxyisoatisenolide (14), showed in its ‘H NMR spectrum signals of the three methyl groups, of the olefinic proton at C-15 and of the resonances originating from the hydrogens at C-6 and C-7 (64.74 and 4.11, I,, 6 = 7 Hx,
J
r 4 Hz). These last signals are similar to those 06!&ved in the kaurenolides [15]. The more polar hydroxy-lactone showed a ‘H NMR spectrum similar to that of 14, where the signal of one of the angular methyl groups has been substituted by a broad singlet at 63.71 (2H) of a hydroxymethyl group. By comparison with the spectrum of 7/?,18dihydroxykaurenolide, this alcoholic function was assigned to C-18, and the structure established as 7fiJ8dihydroxyisoatisenolide (15). The substrates incubated were synthesized in the following way. Hydrolysis of the methyl ester of gummiferolic acid (4) gave the alcohol 5 [IO], which was treated with ptoluenesulphonic acid in benzene to give the isomer&d compound 19. Reaction of 19 with thionyl chloride in pyridine gave the dehydrated product 7, contaminated with the chlorine derivative 16. Treatment of this mixture with lithium aluminium hydride afforded the alcohols 6 and 17, which were separated by silica gel chromatography. The hydroxy acid 2 was obtained by isomerization of gummiferolic acid (3), and then by hydrolysis of the product obtained (1s).
2524 7. Fraga, B. M., Hcmanda,
8. 9. 10. 11.
B. M. FRAGAand R. GUILURMO
M. G., Rodriguez, M. D., Diat, C. E., Gonzalez, P. and Hanson, J. R. (1987) Phyrochetirry 26, 1931. Bearder, J. R, MacMillan, J. and Matsuo, A. (1979) J. Chem. Sot. Chem. Comm. 650. Beale, M. H., Bearder, J. R., MacMillan, J., Matsuo, A. and Phinncy, B. 0. (1983) Phytochemistry 22, 875. Pinar, M.. Rodriguez_ B. and Alemany, A. (1978) Phytochemistry 17, 1636. Barnes, M. F., Light, E. N. and Lang, A. (1969)Planta 88,172.
12. Cross, B. E and Myers, P. L. (1969) Phytochemistry g, 79. 13. Binks, R, MacMillan, 1. and Pryce, R. J. (1969) Phytochemistry 8, 271. 14. Beale, M. H., Bearder, J. R., Down, G. H., Hutchison, M., MacMillan, J. and Phinncy, B. 0. (1982) Phytochemistry 21, 1279. 15. Hanson, J. R., Hawker, J. and White, A. F. (1966) Terra/w&on 22, 1701. 16. Hanson. J. R, Hawker, J. and White, A. F. (1972) J. Chem. Sot. Perkin Trans. 1, 1892.