Asymmetric epoxidation of digeranyl by cultured cells of Nicotiana tabacum

Article abstract of DOI:10.1002/jlcr.681

Asymmetric epoxidation of digeranyl, which is a squalene analog, with cultured cells of Nicotiana tabacum was investigated. Feeding of [8-³H]-digeranyl into the cultured cells of N. tabacum resulted in the formation of (3S)-2,3-epoxydigeranyl and 6,7-epoxydigeranyl. It was found that the epoxidation of digeranyl with N. tabacum was highly stereoselective. Copyright

Full text of DOI:10.1002/jlcr.681

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 401–409.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.681
Research Article
Asymmetric epoxidation of digeranyl by
cultured cells of Nicotiana tabacum
Osamu Nakagawa, Kei Shimoda, Sunsuke Izumi and Toshifumi
Hirata*
Department of Mathematical and Life Sciences, Graduate School of Science,
Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan
Summary
Asymmetric epoxidation of digeranyl, which is a squalene analog, with
cultured cells of Nicotiana tabacum was investigated. Feeding of [8-3H]-
digeranyl into the cultured cells of N. tabacum resulted in the formation of
(3S)-2,3-epoxydigeranyl and 6,7-epoxydigeranyl. It was found that the
epoxidation of digeranyl with N. tabacum was highly stereoselective.
Copyright # 2003 John Wiley & Sons, Ltd.
Key Words: Asymmetric epoxidation; Biotransformation; Digeranyl; Cultured
cells of Nicotiana tabacum
Introduction
The epoxy group plays an important role in organic synthesis,l–4 with
the development of asymmetric epoxidation being particularly note-
worthy.5–8 However, many asymmetric epoxidation reactions can only
be performed under restricted conditions, e.g. by using organometallic
reagents, acting under rigorous exclusion of air, and act only when
specific substituents are present in the substrates.
*Correspondence to: Professor T. Hirata, Department of Mathematical and Life Sciences,
Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi- Hiroshima
739-8526, Japan. E-mail: thirata@sci.hiroshima-u.ac.jp
Copyright # 2003 John Wiley & Sons, Ltd.
Received August 2002
Revised October 2002
Accepted October 2002

402
O. NAKAGAWA ET AL.
Recently, biological transformations has enabled the accomplishment
of asymmetric epoxidations.9 Such studies revealed that several
bacteria, such as Pseudomonas and Corynebacterium, transformed
stereoselectively the unsaturated hydrocarbons into the corresponding
epoxides.10–14 We also found that plant cultured cells, such as Nicotiana
tabacum and Catharanthus roseus, have the ability to introduce
enantioselectively an epoxy group into monoterpenes.15–17 Asymmetric
epoxidation of squalene into (3S)-2,3-epoxysqualene is one of the most
fascinating reactions, because epoxysqualene is a key precursor for the
syntheses of steroids and triterpenoids.18 If such a reaction can be
adapted to squalene analogs, it may be extremely useful from the
viewpoint of formation of many cyclic compounds.
Here we have investigated the epoxidation of digeranyl (1), which is a
C20 squalene analog, with the cultured cells of N. tabacum.
Results and discussion
Biotransformation of digeranyl with cultured cells of N. tabacum
[8-3 H]-Digeranyl (1) was administered to the cultured suspension cells
of N. tabacum, and the cultures were incubated at 258C for 2 days. Two
products were found by TLC autoradiography and HPLC analyses of
the ether extract of the reaction mixture in 15.4 and 13.6% yields. These
products were identified as 2,3-epoxydigeranyl (2) and 6,7-epoxydiger-
anyl (3), respectively, by comparison of their TLC and GLC with the
synthetic specimens. Identification of these products was confirmed by
GC-MS analyses of the products in the incubation of non-labeled
digeranyl with the cultured cells of N. tabacum. The absolute
configuration and enantiomeric purity of the 2,3-epoxydigeranyl
obtained by the biotransformation were determined by comparison of
the chiral GLC with those of synthetic (3S)-2,3-epoxydigeranyl and
(RS)-2,3-epoxydigeranyl. The stereochemistry of the 2,3-epoxydigeranyl
was shown to be S in >99% ee (Scheme 1).
Thus, it was found that the cultured cells of N. tabacum are capable of
transforming digeranyl into 2,3-epoxy- and 6,7-epoxydigeranyls stereo-
selectively.
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 401–409

ASYMMETRIC EPOXIDATION OF DIGERANYL
403
Scheme 1. Biotransformation of digeranyl by cultured cells of N. tabacum
Scheme 2. Reagents and conditions: (a) AC2O, Pyridine, DMAP, rt; (b) PYDZ
Ligand, K2OsO4, K3Fe(CN)6, K2CO3, Methanesulfonamide, t-BuOH:H2O,
4 8C; (c) 2,2-dimethoxypropane, PPTS,CH2Cl2, rt ; (d) Pd(PPh3)4, TsNa-
4 H2O, THF, rt; (e) n-BuLi, THF, 0 8C; (f) PdCl2(dppp), LiHBEt3, THF, 0 8C;
(g) TsOH, MeOH, rt; (h) MsCl, TEA, CH2Cl2 À40 8C; (i) K2CO3, MeOH, rt
Synthesis of authentic (3S)-2,3-epoxydigeranyl
(3S)-2,3-Epoxydigeranyl (2a) was synthesized from geraniol (4) as
shown in Scheme 2. The asymmetric dihydroxylation of geranyl acetate
(5) using PYDZ ligand,19 K2OsO4, K2CO3 and K3Fe(CN)6 in 1:l
t-BuOH/H2O gave a diol 6 in 70% yield. Protection of the diol 6
followed by the sulfonylation of the resultant 7 with Pd(PPh3)4, TsNa-
4H2O in THF: MeOH furnished geranyl p-tolylsulfone (8). The product
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 401–409

404
O. NAKAGAWA ET AL.
8 was lithiated, followed by addition of geranyl bromide (9), which was
prepared using N-bromosuccinimide and dimethyl sulfide in CH2Cl2, to
give 10 in 60% yield. 10 was reduced with PdCl2(dppp) and LiHBEt3, in
THF to give 11 in 79% yield. 11 was deprotected to give a diol 12
with TsOH in MeOH. The resulting diol 12 was converted to
(3S)-2,3-epoxydigeranyl (2a) by sulfonylation of the secondary alcohol
with methanesulfonyl chloride and pyridine followed by ring closure
with K2CO3 in MeOH in 25% yield over two steps.
Thus, the synthesis of (3S)-2,3-epoxydigeranyl (2a) from geraniol (4)
was achieved by asymmetric dihydroxylation, coupling of geranyl
bromide (9) with geranyl p-tolylsulfone derivative (8), and reductive
desulfonylation, as key steps.
Experimental
General: Analytical TLC was performed on precoated TLC plates
(Merck 60 F254 Column chromatography was conducted using silica-gel
1
(Merck Silica gel 60, and Wakogel C-300 HG). H and 13C NMR
spectra were obtained using a JEOL JNM-LA500 spectrometer using
tetramethylsilane as an internal standard. Mass spectra (MS) were
obtained on JEOL JMS-SX102A and Hewlett Packard MSD-5971A
mass spectrometers.
Substrates: The preparation of [8-3H]-digeranyl (1) was assayed by
the synthetic method of [8-2H]-digeranyl reported in our previous
paper.20 To a solution of 8-p-tolylsulfonated digeranyl (500 mg,
0.583 mmol), prepared from geranyl p-tolylsulfone and geranyl chloride,
and 5 mol% 1,3-bis(diphenylphosphino)propane palladium(II) chloride
complex [PdCl2(dppp)] in THF (40 ml), LiB3HEt3 (1.17 mmol in 2.3 ml
THF; 1.48 GBq) was added at 08C under a N2 atmosphere. After
stirring at 08C for 1 h, 3 M NaOH and KCN (40 mg) was added to the
reaction mixture, and then the mixture was extracted with hexane to
give the crude product. The latter was subjected to column chromato-
graphy on silica gel and eluted with hexane to give [8-3H]-digeranyl (1)
(145 mg, 670 MBq, 90%).
Feeding of [8-3H]-digeranyl (1) with the cultured cells of N. tabacum:
The suspension cells of N. tabacum were prepared as reported
previously.21 To the flask containing the suspension cells (about 40 g
in 100 ml of Murashige and Skoog’s medium21), [8-3H]-digeranyl (1)
(58 mg; 131 kBq) was administered and incubated at 258C for 2 days on a
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 401–409

ASYMMETRIC EPOXIDATION OF DIGERANYL
405
rotary shaker. After incubation, the cells were collected by filtration and
extracted with ether. The ether layer was evaporated and then subjected
to short-column chromatography on silica gel with ether to give a crude
product (36 kBq)
The crude product was traced by TLC autoradiography. A charged
TLC was developed with hexane:EtOAc=5:1 (v/v) as eluent and then
analyzed using Fuji.BAS2000 imaging analyzer. The Rf values of the
radioactive spots were 0.51, 0.57, and 0.65, which were identical
with those of authentic 2,3-epoxydigeranyl, 6,7-epoxydigeranyl,
and digeranyl, respectively. A part of the crude product was sub-
jected to preparative HPLC (Inertsil SIL 5pm; 4.6 Â 250 mm) using
hexane:2-propanol=l000:l (v/v) as eluent and the radioactivities
of the isolated fractions were measured by liquid scintillation counting.
Three radioactive fractions eluted at Rt 10 (12 Bq; 71%), 16 (2.6 Bq;
15.4%), and 19 min (2.3 Bq; 13.6%) were identical with those of
authentic digeranyl, 6,7-epoxydigeranyl, and 2,3-epoxydigeranyl,
respectively.
Feeding of digeranyl (1) with the cultured cells of N. tabacum: To the
flask containing the suspension cells (about 40 g in 100 ml of MS
medium), non-labeled digeranyl (25 mg) was administered and the
mixture was incubated at 258C for 2 days on a rotary shaker. After
incubation, the cells were collected by filtration and extracted with
ether. The ether layer was evaporated and then subjected to short-
column chromatography on silica gel with ether to give a crude product
(2 mg). Two products were identified by the comparison of their
GLC [OV-17 on Chromosorb WAW-DMCS; 80–100 mesh; column
temperature., 100–2508C (108C/min, initial. time 5 min); inject
temparature., 2508C] and GC-MS analysis with those of authentic
2,3- and 6,7-epoxydigeranyl. Retention times for synthetic 2,3- and 6,
7-epoxydigeranyl in the GLC were 18.4 and 18.9 min, respectively.
GC-MS analyses of the peaks with retention times 18.4 and 18.9 min in
the administered sample with N. tabacum showed identical fragmenta-
tion patterns to synthetic 2,3- and 6,7-epoxydigeranyl, respectively.
The absolute configuration and enantiomeric purity of the 2,3-epoxy
product were determined by comparison of their chiral GLC (CP
cyclodextrin b236M-19 column; column temperature, 2008C; inject
temperature, 2208C) of incubated products with those of synthetic
samples, (3S)-2,3-epoxydigeranyl and racemic 2,3-epoxydigeranyl [Rt
values of (3S)- and (3R)-epoxydigeranyl: 17.3 and 17.5 min,
respectively].
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 401–409

406
O. NAKAGAWA ET AL.
Syntheses of racemic 2,3- and 6,7-epoxydigeranyl: Digeranyl (500 mg,
1.82 mmol) was dissolved in 52 ml of dry CH2C12. m-Chloroperbenzoic
acid (377 mg, 2.18 mmol) was added to the stirred reaction mixture at
08C. After stirring at room temperature for 12 h, aqueous Na2S203 and
NaHCO3 were added at 08C. The reaction mixture was extracted with
ether to give a crude product, which was purified by column
chromatography on silica-gel with hexane-ether (95:5) to give 2,3-
epoxydigeranyl (2) (4.1 mg, 7%) [EI-MS m/z 290 (M+), 275, 166, 153,
1
135, 109, 95, 69; H NMR (CDC13) d=5.14 (m, 3H), 2.70 (t, lH,
J=6.2 Hz), 2.07 (m, 10H), 1.68 (s, 3H), 1.64 (m, 2H), 1.62 (s, 3H), 1.60
(s, 6H), 1.30 (s, 3H) and 1.26 (s, 3H); 13C NMR (CDC13) d=135.2,
134.2, 131.3, 125.0, 124.4, 124.1, 64.2, 58.3, 39.7, 36.3, 28.3, 28.2, 27.5,
26.8, 25.7, 24.9, 18.7, 17.7 and 16.0] and 6,7-epoxydigeranyl (3) (9.4 mg,
1
16%) [EI-MS m/z 290 (M+); H NMR (CDC13) d=5.14 (m, 3H), 2.71
(t, lH, J=6.3 Hz), 2.07 (m, 8H), 1.68 (s, 6H), 1.64 (m, 2H), 1.61 (s, 9H),
1.51 (m, lH), 1.42 (m, 1H) and 1.25 (s, 3H); 13C NMR (CDC13)
d=135.9, 131.8, 131.4, 124.2, 123.7, 123.3, 63.3, 60.7, 39.7, 38.9, 29.0,
26.7, 25.7, 24.8, 23.9, 17.6, 16.5, 16.0 and 15.3].
Synthesis of (3S)-2,3-epoxydigeranyl. (6R)-6,7-Dihydroxygeranyl
acetate (6): Following the method described,23 a mixture of DHQD-
PYDZ ligand19 (190 mg, 0.26 mmol), K2OsO4 (20 mg, 0.05 mmol),
K2Fe(CN)6 (25 g, 76.5 mmol), K2CO3 (10.6 g, 76.5 mmol), methanesul-
fonamide (2.42 g, 25.5 mmol), geranyl acetate (5 g, 25.5 mmol) and
300 ml of 1:l t-BuOH-H2O was stirred for 12 h at 48C. Sodium sulfite
(30 g) was added, and the mixture was concentrated under reduced
pressure. The mixture was extracted with CH2Cl2, washed with 2 M
KOH and dried over Na2SO4. The crude product was purified by
column chromatography on silica gel using ethyl acetate–hexane (2:1) as
1
eluent to give a product 6 (2.6 g, 70%): EI-MS m/z 231 (MH+); H-
NMR (CDCl3) d=5.33 (t, lH, J=7.0 Hz), 4.53 (d, 2H, J=7.0 Hz), 3.27
(dd, lH, J=10.5, 2.0 Hz), 2.27 (m, lH), 2.04 (m, 1H), 2.00 (s, 3H), 1.66
(s, 3H), 1.55 (m, 1H), 1.39 (m, 1H), 1.15 (s, 3H) and 1.10 (s, 3H);
13C-NMR (CDCl3) d=171.2, 142.0, 118.5, 77.9, 72.9, 61.3, 36.5, 29.4,
26.3, 23.1, 20.9 and 16.3.
(5R)-3-Methyl-5(2,2,5,5-tetramethyl-[1,3]dioxolan-4-yl)-pent-2-enyl
acetate (7): To the suspension of 6 (4.8 g, 20.8 mmol) in 20 ml of
CH2C12, 2,2-dimethoxypropane (4.8 g, 41.7 mmol) and pyridinium
p-toluenesulfonate (10.3 g, 41.7 mmol) were added at 08C under a N2
atmosphere. After stirring at room temperature for 1 h, the reaction
mixture was treated with Et3N and concentrated. The crude product
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 401–409

ASYMMETRIC EPOXIDATION OF DIGERANYL
407
was purified by column chromatography on silica-gel with hexane-ethyl
acetate (2 : 1) to give a product 7 (5.3 g, 94%): EI-MS m/z 270 (M+);
1H-NMR (CDCl3) d=5.39 (bt, lH), 4.59 (d, 2H), 3.65 (dd, lH, J=9.7,
3.4 Hz), 2.28 (m, lH), 2.10 (m, lH), 2.04 (s, 3H), 1.73 (s, 3H), 1.64 (m,
3H), 1.51 (m, 3H), 1.40 (s, 3H), 1.31 (s, 3H), 1.24 (s, 3H) and 1.09 (s,
3H); 13C-NMR (CDCl3) d=170.4, 141.0, 118.6, 106.2, 82.4, 79.7, 60.9,
36.3, 28.3, 27.1, 26.5, 25.7, 22.7, 20.6 and 16.2.
(5R)-2,2,4,4-Tetramethyl-5-[3-methyl-5-(toluene-4-sulfonyl)-pent-3-
enyl-[1, 3]dioxolane (8): To the solution of sodium p-tolylsulfonate
tetrahydrate (5.8 g, 23.2 mmol) in THF (43 ml) and MeOH (14 ml) was
added acetate 7 (5.7 g, 21.1 mmol) and 5 mol% Pd(PPh3)4 in THF
(10 ml) under a N2 atmosphere. After stirring at room temperature
overnight, aqueous KCN was added dropwise at 08C to deactivate the
palladium catalyst. The reaction mixture was extracted with ether to
give a crude product, which was purified by column chromatography on
silica gel using hexane-ethyl acetate (4:1) as eluent to give a product 8
1
(5.3 g, 68%): FAB-MS m/z 367.8 (MH+); H-NMR (CDCl3) d=7.73
(d, 2H, J=8.2 Hz), 7.32 (d, 2H, J=8.2 Hz), 5.24 (t, lH, J=7.9 Hz), 3.80
(d, 2H, J=7.9 Hz), 3.63 (dd, lH, J=9.7, 3.0 Hz), 2.44 (s, 3H), 2.25 (m,
lH), 2.06 (m, lH), 1.55 (m, lH), 1.41 (s,3H), 1.40 (m, lH), 1.39 (s, 3H),
1.32 (s, 3H), 1.24 (s, 3H) and 1.08 (s, 3H); 13C-NMR (CDC13) d=145.0,
143.9, 135.6, 129.1, 127.9, 110.5, 106.0, 82.1, 79.5, 55.5, 36.3, 28.1, 27.0,
26.4, 25.6, 22.5, 21.1 and 15.8.
2,2,4,4-Tetramethyl-5-[3,8,12-trimethyl-5-(toluene-4-sulfonyl)-
trideca-3,7, 11-trienyl-[1,3]dioxolane (10): To the suspension of 8
(5.3 g, 14.5 mmol) in 20 ml of THF, 1.5 M n-BuLi (9.1 ml, 17.4 mmol) in
THF and geranyl bromide (9) (3.0 g, 17.4 mmol) in THF (10 ml) were
added dropwise at 08C under a N2 atmosphere. After stirring at 08C for
10 min, the reaction mixture was treated with 20 ml of 10% NH4C1 and
extracted with ether to give a crude product, which was purified by
column chromatography on silica-gel with hexane-ethyl acetate (4:1) to
give a product 10 (4.3 g, 60%).
2,2,4,4-Tetramethyl-5-[3,8,12-trimethyltrideca-3,7,11-trienyl-[1,3]
dioxolane (11): To the solution of 10 (2.6 g, 5.2 mmol) and 5 mol%
PdC12(dppp) in THF (173 ml), LiHBEt3, (1.2 ml, 10.2 mmol) was added
at 08C under N2 atmosphere. After stirring at 08C for 12 h, 3 M NaOH
and KCN (50 mg) was added to the reaction mixture, and then the
mixture was extracted with ether to give a crude product. The crude
product was subjected to column chromatography on silica-gel and
eluted with hexane-ethyl acetate (5:l) to give 11 (1.4 g, 79%): EI-MS m/z
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 401–409

408
O. NAKAGAWA ET AL.
1
348 (M+); H NMR (CDC13) d=5.13 (m, 3H), 3.67 (dd, lH, J=9.3,
3.5 Hz), 2.12 (m, lH), 1.68 (s, 3H), 1.62 (s, 3H), 1.60 (s, 6H), 1.48 (m,
2H), 1.42 (s, 3H), 1.33 (s, 3H), 1.24 (s, 3H) and 1.10 (s, 3H).
(3R)-Dihydroxydigeranyl (12): To the solution of 11 (23 mg,
66 mmol) in MeOH (2 ml) was added TsOH (500 mg, 79.2 mmol). After
stirring at room temperature for 3 h, 20 ml of 5% NaHCO3 was added,
and washed with brine. The reaction mixture was extracted with ether to
give a crude product, which was purified by column chromatography on
silica gel using hexane–ethyl acetate (3 : 1) as eluent to give a product 12
1
(25 mg, quant): EI-MS m/z 308 (M+); H-NMR (CDC13) d=5.13 (m,
3H), 3.34 (dd, lH, J=10.5, 1.7 Hz), 2.25 (m, lH), 2.02 (m, 9H), 1.68 (s,
3H), 1.62 (s, 3H), 1.60 (s, 6H), 1.55 (m, lH), 1.41 (m, lH), 1.19 (s, 3H)
and 1.15 (s, 3H); 13C-NMR (CDCl3) d=135.1, 134.9, 131.1, 124.9,
124.3, 124.1, 78.2, 73.0, 39.7, 36.8, 29.8, 28.2, 28.1, 26.7, 26.3, 25.6, 23.1,
17.6, 16.0 and 15.9.
(3S)-2,3-epoxydigeranyl (2a): A solution of 12 (63 mg, 0.20 mmol)
and pyridine (0.03 ml, 0.40 mmol) in 3 ml of CH2Cl2 was treated at
À408C with methanesulfonyl chloride (34 mg, 0.30 mmol). After 30 min,
the mixture was warmed to room temperature and stirred for 3 h. An
additional 0.3 ml pyridine was added, and the mixture was stirred for
8 h. The mixture was poured into a suspension of 1 g of K2CO3 in 5 ml
of MeOH and stirred for 6 h at room temperature. The mixture was
concentrated, diluted with water, extracted with ether, washed with
10% CuSO4 and brine. The crude product was purified by column
chromatography on silica gel using hexane–ether (95:5) as eluent to give
a product 2a (14.8 mg, 25%): EI-MS m/z 290 (M+), 275, 166, 153, 135,
1
109, 95, 69; H NMR (CDC13) d=5.14 (m, 3H), 2.70 (t, lH, J =
6.2 Hz), 2.07 (m, 10H), 1.68 (s, 3H), 1.64 (m, 2H), 1.62 (s, 3H), 1.60 (s,
6H), 1.30 (s, 3H) and 1.26 (s, 3H); 13C NMR (CDCl3) d=135.2, 134.2,
131.3, 125.0, 124.4, 124.1, 64.2, 58.3, 39.7, 36.3, 28.3, 28.2, 27.5, 26.8,
25.7, 24.9, 18.7, 17.7 and 16.0.
Acknowledgements
The authors thank Professor Tohru Kojima and Dr Mikio Yashiki,
School of Medicine, Hiroshima University for expertly carrying out the
1
EI-MS measurements. The H and 13C NMR and FAB-MS measure-
ments were run at the Instrument Center for Chemical Analysis,
Hiroshima University. The authors also thank the Radioisotope Center
of Hiroshima University for the experiment using the radioisotopes.
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 401–409

ASYMMETRIC EPOXIDATION OF DIGERANYL
409
References
1. Bartok M, Lang KL. In The Chemistry of Ethers, Crown Ethers, Hydroxyl
Groups and Their Sulphur Analoges, Supplement E, Vol 2, Patai, S. (ed.),
Wiley: New York, 1980; 609.
2. Chong JM, Sharpless KB. J Org Chem 1985; 50: 1569.
3. Smith JG, Synthesis 1984; 629 .
4. Rao AS, Paknikar SK, Kirtane JG. Tetrahedron 1983; 39: 2323.
5. Katsuki T, Sharpless KB. J Am Chem Soc 1980; 102: 5874.
6. Zhang W, Loebach JL, Wilson SR, Jacobsen EN. J Am Chem Soc 1990;
112: 2801.
7. Julia S, Masana J, Vega JC. Angew Chem Int Ed Engl 1980; 19: 929.
8. Yang D, Yip YC, Tang MW, Wong MK, Zheng JH, Cheung KK. J Am
Chem Soc 1996; 118: 491.
9. Buhler M, Schindler J. In Biotechnology, Vol 6a, Rehm H-J, Reed G (eds).
Verlag Chemie: Berlin, 1984; 329.
10. May SW, Schwartz RD. J Am Chem Soc 1974; 96: 4031.
11 Ohta H, Tetsukawa H. J Chem Soc Chem Commun 1978; 849.
12. Smet MJ, Witholt B, Wynberg H. J Org Chem, 1981; 46: 3128.
13. Smet MJ, Kingma J, Wynberg H, Witholt B. Enzyme Microb Technol
1983; 5: 352 .
14. Habets-Crutzen AQH, Carlier SJN, Bont JAM, et al. Enzyme Microb
Technol 1985; 7: 17
15. Hirata T, Izumi S, Kido T, Shimoi Y, Ikeda Y. Plant Tissue Culture Lett
1993; 10: 89.
16. Hirata T, Ikeda Y, Izumi S, Shimoda K, Hamada H, Kawamura T
Phytochemistry 1994; 37: 401.
17. Hirata T, Izumi M, Ogura M, Yawata T, Tetrahedron 1998; 54:15993.
18. Abe I, Prestwich PD. In Comprehensive Natural Products Chemistry, Vol 2
Barton SD, Nakanishi, K (eds). Elsevier: Amsterdam, 1999, 267.
19. Corey EJ, Noe MC, Sarshar S. J Am Chem Soc 1993; 115: 3828.
20. Nakagawa O, Shimoda K, Izumi S, Hirata T. J Label Compd. Radiopharm
2000; 43: 1301.
21. Lee YS, Hirata T, Suga T. J Chem Soc Perkin Trans 1983; 1:2475.
22 Murashige T, Skoog F. Physiol Plant 1962; 15: 473.
23. Corey EJ, Noe MC, Shieh WC. Tetahedron Lett 1993; 34: 5995.
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 401–409