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Identification of unnatural phaseic acid as a metabolite derived from exogenously added (−)-abscisic acid in a maize cell suspension culture
Pergamon
Phymheminry,
Vol. 36. No. 3, pp 647450, 1994 Ehevier S&a Lid Printed in Greal Britain m-9422/94 17.00+0.00
IDENTIFICATION OF UNNATURAL PHASEIC ACID AS A METABOLITE DERIVED FROM EXOGENOUSLY ADDED (-)-ABSCISIC ACID IN A MAIZE CELL SUSPENSION CULTURE JOHN J. BALSEVICH,*SUZANNER. ABRAM&NANCY LAMB and WILFRIED A. KdNlGt Plant Biotechnology Institute, National Research Council of Canada, Organische
Chemie,
Universitit
(Received in revised form
Key Word Index-Zea gas chromatography.
Saskatoon,
Hamburg,
Saskatchewan,
2000 Hamburg
Canada
S7N OWP, tlnstitut
fiir
13, Germany
1 December 1993)
mays; Gramineae; cell culture; metabolism; abscisic acid; phaseic acid; chiral
Abstract-Unnatural phaseic acid (U-PA) was identified by chiral GC as a minor component of the metabolite mixture obtained from the medium of a maize cell suspension culture treated with unnatural (-)-(R)-abscisic acid (U-ABA). When U-[3’,5’,5’,7’,7’,7’]-hexadeutero-ABA was fed to the cell suspension, d6-PA was obtained, establishing its origin from the exogenously-added U-ABA. The major metabolite produced was identified as U-T-hydroxyabscisic acid (U7’-HOABA) [ds-7’-HOABA from I,-U-ABA]. Confirmation of the assignments was obtained by HPLC/LC/CFSIMS analysis of the metabolite mixtures. This result corroborates a previous report of the formation of U-PA from racemic ABA in avocado mesocarp tissue.
INTRODUCI’ION The plant hormone (+ )-(S)-abscisic acid [N-ABA: = natural ABA] (1) has been implicated in the regulation of a variety of developmental and stress-related responses of plants [l]. In many of the previous metabolic and physiological studies involving exogenously applied ABA, synthetic racemic material was used due to availability and lower cost. Unfortunately, the use of racemic material has complicated the analysis of results, both from physiological and metabolic standpoints. Recent work with individual enantiomers has indicated, e.g. that the main oxidative metabolic pathway for U-ABA (2) is to U-II’-HOABA (3), while the main oxidative metabolic pathway for N-ABA is via oxidation of C-8’ to N-PA (4) [2-41. These results had previously been rationalized [S, 63 by the fact that ABA is relatively symmetrical about the plane which intersects C-l’-C-4’ and contains the side chain. Alignment of the side chains, the C-l’ hydroxyl groups and the C-4 ketones of U- and N-ABA could, with some distortion of the ring and the C-l’ hydroxyl group in an axial orientation, result in C-7’ of U-ABA occupying a similar spatial position as C-8’ of N-ABA.
The C-7’ and C-8’ methyl groups of U- and N-ABA, respectively, could thus be perceived similarly in the reactive site of the enzyme, leading to the observed results. Although the main modes of oxidative metabolism of U- and N-ABA were at C-7’ and C-8’, respectively, a recent report has established that N-ABA does yield N-7’HOABA (5) as a minor metabolite in a bromegrass (Br0mu.s inermis) cell suspension cultures [fl. One might also expect that U-ABA should afford U-PA (6) as a metabolite and there has been one recent report where UPA was identified by chiral HPLC as a metabolite produced on feeding racemic ABA to avocado (Perseu americana) mesocarp tissue [8]. A much earlier report of the formation of dihydro PA and PA from U-ABA [9] was based largely on TLC mobilities and the assumption that U-ABA metabolism was the same as N-ABA metabolism. This report occurred prior to the identification and characterization of 7’-HOABA as the major oxidative metabolite of U-ABA. Here we report direct and conclusive evidence that exogenously added U-ABA can indeed be metabolized to afford U-PA, in our case by a maize cell suspension culture. RESULTS AND DECUS!3ION
*Author to whom correspondence should be addressed. Published as NRCC No. 37340. ZThe prefixes N- and U- have been used here to denote derivatives possessing the natural and unnatural stereochemistry at C-l’. A change in the sign of optical rotations is observed between ABA and PA which can be confusing; thus, natural ABA is the ( +) form, while natural PA is the (-) form.
A maize cell suspension culture (Zea muys L. cv Black Mexican Sweet) was treated with U-ABA (lo-50 PM) and after two-four days, acidic metabolites were isolated from the medium and subjected to analysis via liquid chromatography/continuous flow secondary ion mass spectrometry (LC/CFSIMS) using authentic samples as 647
J. J.
BALSEVICH
et al.
0 R
R
C-1
2 R-H 3 R-OH
1 R-H 5 R=ctl
+
AL I(+)
standards [7, lo-121. Three components were identified by co-chromatography (LC) with standards and comparison of their CFSIMS with those of the authentic materials as: ABA, 7’-HOABA and PA. Repetition of feedings indicated that results were reproducible although relative proportions of the components were variable. The metabolite mixture consisted predominantly of ABA (83390%) followed by 7’-HOABA (7-14%) and PA (2-4%). Recovery of the metabolites from the medium was ca 75595% by weight. To ensure that the products were derived from the exogenously applied U-ABA and not endogenous NABA, U-[3’,5’,5’,7’,7’,7’]-hexadeutero-ABA was prepared via base catalysed exchange reaction in D,O [13] and incubated with the maize cell suspension culture. The LC/CFSIMS analysis of the isolated metabolite mixture established the presence of &-ABA (m/z 271, [M + H] +, 97%; 253 [M + H - H,O]‘, lOO%), d,-7’-HOABA (m/z 286, [M + H] +, 90%; 268, [M + H - H,O] ’ , lOO%), and d,-PA (m/z 287, [M +H]‘, 52%; 269, [M+H-H,O]‘, 77%). The mass spectral experiments indicated that there was no significant scrambling of label or quantities of unlabelled products present. To establish that no racemization at C- 1’ had occurred, a metabolite mixture was methylated with diazomethane, and 7’-HOABA and ABA were confirmed as the Uenantiomers by chiral HPLC [7, 101. Chiral HPLC with our column was not useful for determining the conhguration of the PA produced; capillary GC of the methyl esters on a y-cyclodextrin column [ 143 however was useful, as racemic methyl phaseatc was readily resolvable (baseline separation by several min) [Fig. 11. U- and N-ABA methyl esters were also baseline resolved on this column, but 7’-HOABA (methyl ester) did not elute from the column. Co-injection of authentic standards (methyl esters) with the methylated metabolites unequivocally established that the PA produced possessed the U configuration, and also provided corroboration that only UABA was present in the metabolite mixture (Fig. 1). In
two
recent
studies
of
U-ABA
metabolism,
7’-
HOABA and ABA glucose ester were observed as major metabolites, but no mention of PA was made [2,4]. In the recent report [S] where U-PA was identified as a minor
C-1
(-1
9” \
Jw 0
\ coohie
(-)(+I (+) t-1
I
0
0
.o: \_* J3w
-lL 10 4 Min
20
Fig. I. Gas chromatograms of ABA and PA (methyl esters) obtained on a chiral column. Top: methylated metabolite mixture obtained from feeding U-ABA to a maize cell suspension doped with racemic ABA and racemic PA (methyl esters); middle: methylated metabolite mixture only; bottom: racemic ABA and PA (methyl esters). 7’-HOABA (methyl ester) does not elute on this column. U-ABA =( -) enantiomer and U-PA = (+) enantiomer.
product, racemic ABA was used as the additive, leaving open the possibility that the U-PA arose via racemization. In the early report [9], PA and dihydro PA were assumed to be the metabolites derived from U-ABA, but sufficient characterization was not performed and the claims were made prior to the identification and characterization of 7’-HOABA.
Unnatural phaseic acid as a metabolite of maize cells In conclusion, it was determined that U-ABA can indeed be metabolized, albeit via a minor pathway, to UPA, mirroring the results from the study which established that N-ABA is capable of being metabolized (via a minor pathway) to N-7’-HOABA [7], and corroborating the recent report of the formation of U-PA from racemic ABA [8]. Still to be determined is whether the oxidation of the 7’ and 8’ positions of ABA is performed by different enzymes. Finally, it should be noted that no racemization at C-l’ of U-ABA (or metabolites) was observed, suggesting that physiological responses induced by U-ABA are not due to its conversion to N-ABA. EXPERIMENTAL
Racemic ABA was purchased from Aldrich and converted to the methyl ester by treatment with ethereal diazomethane. N- and U-ABA were obtained by sepn of racemic ABA methyl ester as described in ref. [ 1S], except a prep. Chiralcel OD 1 x 50 cm coated silica column (Daicel, Los Angeles, CA) using isocratic hexane-isoPrOH (4: 1) as solvent was used. Hydrolysis of the methyl esters was achieved by saponification with I M NaOH in aq. MeOH (ca 25% MeOH) at room temp. for 4 hr. Synthetic U- and N-7’-HOABA were available from our ref. collection [7, lo]. N-PA was obtained from the biotransformations of N-ABA by cell suspensions [7, 123. U-PA was synthesized according to the method previously reported for racemic PA [lS], except that the oxoisophorone was reduced with Baker’s yeast to give (-)-(6R)-2,2,6-trimethyl-cyclohexane 1,4-dione [ 163. The methyl phaseate produced by this method gave ‘H NMR and mass spectra identical with lit. values [17] and mp 158-159”, [z]:: +28.7” (MeOH; c 1.91); lit. mp 158.5-159.5”. [alA + 33” (CHCI,; c 0.97) [17]. Saponification alforded U-PA, [a]h4 + 16.7” (MeOH; c 1.22); lit. [a];’ + 18.5” (MeOH; c 0.11) [17]. Hexadeutero U-ABA was prepd by modification of the procedure of Bonnafous et al. [13] as follows: d,deuteromethanol (3 ml, 99.8% D) was treated with sodium (0.2 g) followed by D,O (2 ml, 99.8% D) and UABA (100 mg). The resultant soln was stirred at room temp. for 3 days, made acidic by addition of tartaric acid (1.5 g) and H,O (50 ml). After extraction with EtOAc and drying of the combined organic extract (Na,SO,) the solvent was removed in uacuo yielding the U[3’,5’,5’,7’,7’,7’]-hexadeutero-ABA (100 mg) as a crystal-43.9” (CHCI,; c 0.114); ‘HNMR line solid. [a];’ (CDCI,): 67.79 (lH, d, 16 Hz), 6.15 (lH, d, 16 Hz), 5.74 (lH, s), 2.03 (3H, s), 1.09 (3H, s), 1.01 (3H, s); CFSIMS: m/z 272 (25%), 271 (M + + 1, lOO%), 270 (20%), 253 (65%). Analytical chiral LC was performed with a Chiralcel OD 0.46 x 25 cm column using hexane-iso-PrOH (6: 1) as described in refs [7, 123. UV detection at 262 nm was used. Gas chromatography was performed using a 10m fused silica capillary column with octakis (3-O-butyryl2,6-di-0-pentyl)-y-cyclodextrin (60% in polysiloxane OV 1701, w/w) at 180” with hydrogen (40 kPa) as carrier gas as described in ref. [14]. A Carlo Erba model 2101 gas PHY
36:3-I
649
chromatograph with split injector and flame ionization detector was employed. Samples were injected in CH,Cl,. A Merck-Hitachi Chromato-Integrator D-2500 served for integration of peak areas. LC/CFSIMS was performed as outlined in ref. [l 11, using a 0.32 x 150 mm Spherisorb 3 micron ODS-2 packed capillary column interfaced with a VG Analytical (Manchester, U.K.) 70-250 SEQ hybrid mass spectrometer equipped for continuous flow sims analysis. Gradient elution using a 2 solvent system consisting of A: 2% glycerol and 0.1% trifluoroacetic acid in H,O and B: 80% acetonitrile, 2% glycerol and 0.1% trifluoroacetic acid in H,O. The gradient elution used was 50% A to 10% A in 10 min which was held for a further 35 min. Suspension cultures of corn (Zea mays L. cv Black Mexican Sweet) were maintained at 22” in low light on a rotary shaker at 140 rpm in modified Murashige and Skoog medium as described previously [18]. Incubations were performed by adding solns of U-ABA (or d,-U-ABA) (0.1-0.5 mg) in 70% EtOH (20-30 pl) to the cell suspensions (typically 5-10 flasks with 60 ml flask - ‘, ca 2 g cells fr. wt flask- ‘) 24-30 hr after having been subcultured into fr. medium. Incubations were carried out at 28” for 24 days. Metabolites were isolated as follows: medium was sepd from cells by filtration and extracted with EtOAc (3 x ) [20 ml EtOAc per each 60 ml medium]. The combined organic portion was extracted with 5% NaHCO, (2 x ) and discarded. The combined aq. portion was acidified with 1 N HCI and extracted with EtOAc (3 x ). The combined organic extract was washed with H,O (1 x ), dried (Na,SO,) and coned in vacua to afford the metabolite mixt. (ca 75-95% wt recovery). For GC and chiral LC analyses, a portion of the metabolite mixt. was dissolved in EtOAc-MeOH (3: 1) and treated with an appropriate amount of ethereal diazomethane to afford the methyl esters. Acknowledgements-We wish to thank Mr Doug Olson and Mr Lawrence Hogge for running the LC/CFSIMS analyses, Mr Greg Bishop for technical help and for separation of racemic ABA methyl ester, MS Angela Shaw for the synthesis of U-Pa, Dr Garth Abrams and Mr Dennis Barton for advice and help in LC analyses, and Dr Adrian Cutler for supplying the maize cell suspension.
REFERENCES
1. Davies, W. J. and Jones, H. G. (1991) Abscisic Acid Physiology and Biochemistry. Bios Scientific Publishers, Oxford. 2. Mertens, R., Stiining, M. and Weiler, E. W. (1982) Naturwissen 69, 595. 3. Vaughan, G. T. and Milborrow, B. V. (1984) J. Exp. Botany 35, 110. 4. Zeevaart, J. A. D., Gage, D. A. and Creelman, R. A. (1990) in Plant Growth Substances 1988 (Pharis, R. P. and Rood, S. B., eds), p. 239. Springer, Berlin. 5. Boyer, G. L. and Zeevaart, J. A. D. (1986) Phytochemistry 25, 1103.
650
J. J. BALSEVICH
6. Loveys, B. and Milborrow, B. V. (1991) Phytochemistry 31, 67. 7. Hampson, C. R., Reaney, M. J. T., Abrams, G. D., Abrams, S. R. and Gusta, L. V. (1991) Phyrochemistry 31, 2645. 8. Okamoto, M. and Nakazawa, H. (21-26 July 1991) Abstract No. MO-Cl-P22 in 14th International Conjkrence on Plant Growth Suhstonces, p. 20. Amsterdam, Netherlands. 9. Sondheimer, E., Galson, E. C., Tinelli, E. and Walton, D. C. (1974) PIant Physiol. 54, 803. 10. Nelson, L. A. K., Shaw, A. C. and Abrams,S. R.(1991) Tetruhedron
47, 3259.
11. Hogge. L. R., Balsevich, J. J., Olson, J. H., Abrams, G. D. and Jacques, Spectr. 7. 6.
S. L. (1993) Rapid
Comm.
Mass
er al
12. Dunstan, D. I., Bock, C. A., Abrams, G. D. and Abrams, S. R. (1992) Phytochemistry 31, 1451. 13. Bonnafous, J.-C.. Fonzes, L. and Mousseron-Canet, M. (1971) Bull. Sot. Chim. France 4552. 14. Konig, W. A.. Krebber, R. and Mischnick, P. (1989) .I. High
Res. Chromarogr.
15. Abrams,
12, 732.
G. D., Abrams, S. R.. Nelson, L. A. K. and Gusta, L. V. (1990) Tetrahedron 46, 5543. 16. Lamb, N. and Abram% S. R. (1990) Can. J. Chem. 68, 1151. 17. Kitahara, T., Touhara, K., Watanabe, H. and Mori, K. (1989) Tetrahedron 45, 6387. 18. Ludwig, S. R., Somers, D. A.. Peterson. W. L., PohImann, B. F., Zarovitz, M. A., Gengenbach, B. G. and Messing, J. (1985) Theoret. Appl. Genet. 71, 344.
Phymheminry,
Vol. 36. No. 3, pp 647450, 1994 Ehevier S&a Lid Printed in Greal Britain m-9422/94 17.00+0.00
IDENTIFICATION OF UNNATURAL PHASEIC ACID AS A METABOLITE DERIVED FROM EXOGENOUSLY ADDED (-)-ABSCISIC ACID IN A MAIZE CELL SUSPENSION CULTURE JOHN J. BALSEVICH,*SUZANNER. ABRAM&NANCY LAMB and WILFRIED A. KdNlGt Plant Biotechnology Institute, National Research Council of Canada, Organische
Chemie,
Universitit
(Received in revised form
Key Word Index-Zea gas chromatography.
Saskatoon,
Hamburg,
Saskatchewan,
2000 Hamburg
Canada
S7N OWP, tlnstitut
fiir
13, Germany
1 December 1993)
mays; Gramineae; cell culture; metabolism; abscisic acid; phaseic acid; chiral
Abstract-Unnatural phaseic acid (U-PA) was identified by chiral GC as a minor component of the metabolite mixture obtained from the medium of a maize cell suspension culture treated with unnatural (-)-(R)-abscisic acid (U-ABA). When U-[3’,5’,5’,7’,7’,7’]-hexadeutero-ABA was fed to the cell suspension, d6-PA was obtained, establishing its origin from the exogenously-added U-ABA. The major metabolite produced was identified as U-T-hydroxyabscisic acid (U7’-HOABA) [ds-7’-HOABA from I,-U-ABA]. Confirmation of the assignments was obtained by HPLC/LC/CFSIMS analysis of the metabolite mixtures. This result corroborates a previous report of the formation of U-PA from racemic ABA in avocado mesocarp tissue.
INTRODUCI’ION The plant hormone (+ )-(S)-abscisic acid [N-ABA: = natural ABA] (1) has been implicated in the regulation of a variety of developmental and stress-related responses of plants [l]. In many of the previous metabolic and physiological studies involving exogenously applied ABA, synthetic racemic material was used due to availability and lower cost. Unfortunately, the use of racemic material has complicated the analysis of results, both from physiological and metabolic standpoints. Recent work with individual enantiomers has indicated, e.g. that the main oxidative metabolic pathway for U-ABA (2) is to U-II’-HOABA (3), while the main oxidative metabolic pathway for N-ABA is via oxidation of C-8’ to N-PA (4) [2-41. These results had previously been rationalized [S, 63 by the fact that ABA is relatively symmetrical about the plane which intersects C-l’-C-4’ and contains the side chain. Alignment of the side chains, the C-l’ hydroxyl groups and the C-4 ketones of U- and N-ABA could, with some distortion of the ring and the C-l’ hydroxyl group in an axial orientation, result in C-7’ of U-ABA occupying a similar spatial position as C-8’ of N-ABA.
The C-7’ and C-8’ methyl groups of U- and N-ABA, respectively, could thus be perceived similarly in the reactive site of the enzyme, leading to the observed results. Although the main modes of oxidative metabolism of U- and N-ABA were at C-7’ and C-8’, respectively, a recent report has established that N-ABA does yield N-7’HOABA (5) as a minor metabolite in a bromegrass (Br0mu.s inermis) cell suspension cultures [fl. One might also expect that U-ABA should afford U-PA (6) as a metabolite and there has been one recent report where UPA was identified by chiral HPLC as a metabolite produced on feeding racemic ABA to avocado (Perseu americana) mesocarp tissue [8]. A much earlier report of the formation of dihydro PA and PA from U-ABA [9] was based largely on TLC mobilities and the assumption that U-ABA metabolism was the same as N-ABA metabolism. This report occurred prior to the identification and characterization of 7’-HOABA as the major oxidative metabolite of U-ABA. Here we report direct and conclusive evidence that exogenously added U-ABA can indeed be metabolized to afford U-PA, in our case by a maize cell suspension culture. RESULTS AND DECUS!3ION
*Author to whom correspondence should be addressed. Published as NRCC No. 37340. ZThe prefixes N- and U- have been used here to denote derivatives possessing the natural and unnatural stereochemistry at C-l’. A change in the sign of optical rotations is observed between ABA and PA which can be confusing; thus, natural ABA is the ( +) form, while natural PA is the (-) form.
A maize cell suspension culture (Zea muys L. cv Black Mexican Sweet) was treated with U-ABA (lo-50 PM) and after two-four days, acidic metabolites were isolated from the medium and subjected to analysis via liquid chromatography/continuous flow secondary ion mass spectrometry (LC/CFSIMS) using authentic samples as 647
J. J.
BALSEVICH
et al.
0 R
R
C-1
2 R-H 3 R-OH
1 R-H 5 R=ctl
+
AL I(+)
standards [7, lo-121. Three components were identified by co-chromatography (LC) with standards and comparison of their CFSIMS with those of the authentic materials as: ABA, 7’-HOABA and PA. Repetition of feedings indicated that results were reproducible although relative proportions of the components were variable. The metabolite mixture consisted predominantly of ABA (83390%) followed by 7’-HOABA (7-14%) and PA (2-4%). Recovery of the metabolites from the medium was ca 75595% by weight. To ensure that the products were derived from the exogenously applied U-ABA and not endogenous NABA, U-[3’,5’,5’,7’,7’,7’]-hexadeutero-ABA was prepared via base catalysed exchange reaction in D,O [13] and incubated with the maize cell suspension culture. The LC/CFSIMS analysis of the isolated metabolite mixture established the presence of &-ABA (m/z 271, [M + H] +, 97%; 253 [M + H - H,O]‘, lOO%), d,-7’-HOABA (m/z 286, [M + H] +, 90%; 268, [M + H - H,O] ’ , lOO%), and d,-PA (m/z 287, [M +H]‘, 52%; 269, [M+H-H,O]‘, 77%). The mass spectral experiments indicated that there was no significant scrambling of label or quantities of unlabelled products present. To establish that no racemization at C- 1’ had occurred, a metabolite mixture was methylated with diazomethane, and 7’-HOABA and ABA were confirmed as the Uenantiomers by chiral HPLC [7, 101. Chiral HPLC with our column was not useful for determining the conhguration of the PA produced; capillary GC of the methyl esters on a y-cyclodextrin column [ 143 however was useful, as racemic methyl phaseatc was readily resolvable (baseline separation by several min) [Fig. 11. U- and N-ABA methyl esters were also baseline resolved on this column, but 7’-HOABA (methyl ester) did not elute from the column. Co-injection of authentic standards (methyl esters) with the methylated metabolites unequivocally established that the PA produced possessed the U configuration, and also provided corroboration that only UABA was present in the metabolite mixture (Fig. 1). In
two
recent
studies
of
U-ABA
metabolism,
7’-
HOABA and ABA glucose ester were observed as major metabolites, but no mention of PA was made [2,4]. In the recent report [S] where U-PA was identified as a minor
C-1
(-1
9” \
Jw 0
\ coohie
(-)(+I (+) t-1
I
0
0
.o: \_* J3w
-lL 10 4 Min
20
Fig. I. Gas chromatograms of ABA and PA (methyl esters) obtained on a chiral column. Top: methylated metabolite mixture obtained from feeding U-ABA to a maize cell suspension doped with racemic ABA and racemic PA (methyl esters); middle: methylated metabolite mixture only; bottom: racemic ABA and PA (methyl esters). 7’-HOABA (methyl ester) does not elute on this column. U-ABA =( -) enantiomer and U-PA = (+) enantiomer.
product, racemic ABA was used as the additive, leaving open the possibility that the U-PA arose via racemization. In the early report [9], PA and dihydro PA were assumed to be the metabolites derived from U-ABA, but sufficient characterization was not performed and the claims were made prior to the identification and characterization of 7’-HOABA.
Unnatural phaseic acid as a metabolite of maize cells In conclusion, it was determined that U-ABA can indeed be metabolized, albeit via a minor pathway, to UPA, mirroring the results from the study which established that N-ABA is capable of being metabolized (via a minor pathway) to N-7’-HOABA [7], and corroborating the recent report of the formation of U-PA from racemic ABA [8]. Still to be determined is whether the oxidation of the 7’ and 8’ positions of ABA is performed by different enzymes. Finally, it should be noted that no racemization at C-l’ of U-ABA (or metabolites) was observed, suggesting that physiological responses induced by U-ABA are not due to its conversion to N-ABA. EXPERIMENTAL
Racemic ABA was purchased from Aldrich and converted to the methyl ester by treatment with ethereal diazomethane. N- and U-ABA were obtained by sepn of racemic ABA methyl ester as described in ref. [ 1S], except a prep. Chiralcel OD 1 x 50 cm coated silica column (Daicel, Los Angeles, CA) using isocratic hexane-isoPrOH (4: 1) as solvent was used. Hydrolysis of the methyl esters was achieved by saponification with I M NaOH in aq. MeOH (ca 25% MeOH) at room temp. for 4 hr. Synthetic U- and N-7’-HOABA were available from our ref. collection [7, lo]. N-PA was obtained from the biotransformations of N-ABA by cell suspensions [7, 123. U-PA was synthesized according to the method previously reported for racemic PA [lS], except that the oxoisophorone was reduced with Baker’s yeast to give (-)-(6R)-2,2,6-trimethyl-cyclohexane 1,4-dione [ 163. The methyl phaseate produced by this method gave ‘H NMR and mass spectra identical with lit. values [17] and mp 158-159”, [z]:: +28.7” (MeOH; c 1.91); lit. mp 158.5-159.5”. [alA + 33” (CHCI,; c 0.97) [17]. Saponification alforded U-PA, [a]h4 + 16.7” (MeOH; c 1.22); lit. [a];’ + 18.5” (MeOH; c 0.11) [17]. Hexadeutero U-ABA was prepd by modification of the procedure of Bonnafous et al. [13] as follows: d,deuteromethanol (3 ml, 99.8% D) was treated with sodium (0.2 g) followed by D,O (2 ml, 99.8% D) and UABA (100 mg). The resultant soln was stirred at room temp. for 3 days, made acidic by addition of tartaric acid (1.5 g) and H,O (50 ml). After extraction with EtOAc and drying of the combined organic extract (Na,SO,) the solvent was removed in uacuo yielding the U[3’,5’,5’,7’,7’,7’]-hexadeutero-ABA (100 mg) as a crystal-43.9” (CHCI,; c 0.114); ‘HNMR line solid. [a];’ (CDCI,): 67.79 (lH, d, 16 Hz), 6.15 (lH, d, 16 Hz), 5.74 (lH, s), 2.03 (3H, s), 1.09 (3H, s), 1.01 (3H, s); CFSIMS: m/z 272 (25%), 271 (M + + 1, lOO%), 270 (20%), 253 (65%). Analytical chiral LC was performed with a Chiralcel OD 0.46 x 25 cm column using hexane-iso-PrOH (6: 1) as described in refs [7, 123. UV detection at 262 nm was used. Gas chromatography was performed using a 10m fused silica capillary column with octakis (3-O-butyryl2,6-di-0-pentyl)-y-cyclodextrin (60% in polysiloxane OV 1701, w/w) at 180” with hydrogen (40 kPa) as carrier gas as described in ref. [14]. A Carlo Erba model 2101 gas PHY
36:3-I
649
chromatograph with split injector and flame ionization detector was employed. Samples were injected in CH,Cl,. A Merck-Hitachi Chromato-Integrator D-2500 served for integration of peak areas. LC/CFSIMS was performed as outlined in ref. [l 11, using a 0.32 x 150 mm Spherisorb 3 micron ODS-2 packed capillary column interfaced with a VG Analytical (Manchester, U.K.) 70-250 SEQ hybrid mass spectrometer equipped for continuous flow sims analysis. Gradient elution using a 2 solvent system consisting of A: 2% glycerol and 0.1% trifluoroacetic acid in H,O and B: 80% acetonitrile, 2% glycerol and 0.1% trifluoroacetic acid in H,O. The gradient elution used was 50% A to 10% A in 10 min which was held for a further 35 min. Suspension cultures of corn (Zea mays L. cv Black Mexican Sweet) were maintained at 22” in low light on a rotary shaker at 140 rpm in modified Murashige and Skoog medium as described previously [18]. Incubations were performed by adding solns of U-ABA (or d,-U-ABA) (0.1-0.5 mg) in 70% EtOH (20-30 pl) to the cell suspensions (typically 5-10 flasks with 60 ml flask - ‘, ca 2 g cells fr. wt flask- ‘) 24-30 hr after having been subcultured into fr. medium. Incubations were carried out at 28” for 24 days. Metabolites were isolated as follows: medium was sepd from cells by filtration and extracted with EtOAc (3 x ) [20 ml EtOAc per each 60 ml medium]. The combined organic portion was extracted with 5% NaHCO, (2 x ) and discarded. The combined aq. portion was acidified with 1 N HCI and extracted with EtOAc (3 x ). The combined organic extract was washed with H,O (1 x ), dried (Na,SO,) and coned in vacua to afford the metabolite mixt. (ca 75-95% wt recovery). For GC and chiral LC analyses, a portion of the metabolite mixt. was dissolved in EtOAc-MeOH (3: 1) and treated with an appropriate amount of ethereal diazomethane to afford the methyl esters. Acknowledgements-We wish to thank Mr Doug Olson and Mr Lawrence Hogge for running the LC/CFSIMS analyses, Mr Greg Bishop for technical help and for separation of racemic ABA methyl ester, MS Angela Shaw for the synthesis of U-Pa, Dr Garth Abrams and Mr Dennis Barton for advice and help in LC analyses, and Dr Adrian Cutler for supplying the maize cell suspension.
REFERENCES
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