Hydroxylation of benzylic and allylic sites by plant cultured suspension cells

Article abstract of DOI:10.1016/S0040-4039(00)02132-8

A novel transformation of α-substituted benzylcyanides by hydroxylation of benzyl sites using cultured cells of cotton, Gossypium hirsutum, producing alkano- or benzophenones via presumably α-cyanohydrin is demonstrated. Also regioselective hydroxylation of 4S-(-)-perillyl alcohol and 3S-(-)-citronellol by cultured suspension cells of Catharanthus roseus is reported.

Full text of DOI:10.1016/S0040-4039(00)02132-8

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 909–911
Hydroxylation of benzylic and allylic sites by plant cultured
suspension cells
Hiroki Hamada,a,* Toshinori Tanaka,a Tsutomu Furuya,a Hiroki Takahatab and Hideo Nemotob
aDepartment of Applied Science, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan
bFaculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani 2630, Toyama 930-0194, Japan
Received 2 October 2000; revised 7 November 2000; accepted 17 November 2000
Abstract—A novel transformation of a-substituted benzylcyanides by hydroxylation of benzyl sites using cultured cells of cotton,
Gossypium hirsutum, producing alkano- or benzophenones via presumably a-cyanohydrin is demonstrated. Also regioselective
hydroxylation of 4S-(−)-perillyl alcohol and 3S-(−)-citronellol by cultured suspension cells of Catharanthus roseus is reported.
© 2001 Elsevier Science Ltd. All rights reserved.
Many investigators have studied biotransformation of
organic compounds by plant cultured suspension cells,
reporting selective and/or specific conversion ability
including enantioselective oxidation, stereoselective
reduction, regioselective hydroxylation, enantioselective
glycosylation, esterification, methylation, and isomer-
ization.1,2 In practice, the ability of plant cultured cells
to transform foreign substrates into potentially useful
substances is of great interest, as products may be
formed which are difficult to prepare by synthetic
chemical methods. Hitherto we have studied the
hydroxylation ability for foreign substrates such as
testosterone,3 carvone,4 geraniol,4 and piperitone5 by
plant cultured suspension cells. Our interest is now
focused on hydroxylation of a-positions of nitriles due
to the high synthetic utility of a-cyanohydrins,6 and at
allylic position of 4S-(−)-perillyl alcohol and 3S-(−)-cit-
ronellol. We now report a novel transformation of
a-substituted benzylcyanides by hydroxylation of ben-
zyl sites by the plant cultured suspension cells of cotton,
Gossypium hirsutum, into alkano- or benzophenones via
presumably an a-cyanohydrin intermediate and regiose-
lective hydroxylation of allylic positions by cultured
suspension cells of periwinkle, Catharanthus roseus.
out in a manner similar to that reported in Refs. 3–5.
Just prior to use for this work, a part of the callus
tissues (fresh weight 50 g) was transplanted to freshly
prepared Murashige and Skoog’s medium (100 mL in a
300 mL conical flask, pH 5.8) containing 2 ppm of
2,4-dichlorophenoxyacetic acid and 3% sucrose and
grown with continuous shaking for 1 week at 25°C in
the light (2000 lux). The substrate (20 mg/flask) was
added to the suspension cultures and the cultures were
incubated at 25°C for 3–5 days on a rotary shaker (120
rpm) in the light.
The incubation mixture was filtered and the culture
medium was extracted with EtOAc. Then, transforma-
tion products were isolated by preparative TLC and
1
were identified using H and 13C NMR, GC/MS, and
IR. We began with the hydroxylation of 1-(4-methoxy-
benzo)cyclobutenecarbonitrile (1) with cultured suspen-
sion cells of cotton, which produced the benzoketone 2
in 23.1% yield instead of the predicted cyanohydrin 3 of
1. Interestingly, ketone 2 was known as a key interme-
diate in the synthesis of 14a-hydroxyestrone.7 In order
to obtain 2 via 3, so far, attempts to hydroxylyze 1
Table 1. Biotransformation of benzylcyanides
The cotton and periwinkle cultured suspension cells
used were prepared as described in our previous papers
and feeding and incubation experiments were carried
Substrate
Product
Yielda (%)
1
4
5
6
2
7
8
9
23.1
37.5
9.1
Keywords: regioselective hydroxylation; a-cyanohydrin; (−)-perillyl
alcohol; cotton; Catharanthus roseus; biocatalyst.
* Corresponding author. Fax: +81-86-256-8468; e-mail: hamada@
das.ous.ac.jp
58.8
a Weight (%) of product relative to the substrate administered.
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)02132-8

910
H. Hamada et al. / Tetrahedron Letters 42 (2001) 909–911
using chemical methods remain unsuccessful. Accord-
ingly, the synthesis of 2 required a four-step sequence
from 1 via another route.8
In the case of the biotransformation of 13 after 24 h
incubation 13 was converted to 8- and 10-hydroxy
citronellol (15 and 16) in 13.0 and 50.0% yield, respec-
Therefore, the above procedure resulted in a new one-
step synthesis of 2. In a similar manner, the a-alkyl
substituted benzylcyanides 4 and 5 were converted into
the corresponding ketones 7 and 8, respectively (Table
1). As shown in Table 1, the yield of 8 was very low in
the case of the biotransformation of 5 and substrate 5
was recovered unchanged from the medium. Although
it was known that the hydroxylation of diphenylace-
tively. These results indicate that the cultured suspen-
sion cells of C. roseus regioselectively hydroxylate the
allylic position of 4S-(−)-perillyl alcohol and 3S-(−)-cit-
ronellol in good yields.
In summary, it was found that the cultured suspension
cells of cotton selectively converted benzylnitriles to the
corresponding ketones in moderate yields. This proce-
dure is a simple operation and is environmentally
friendly. Also, the cultured suspension cells of C. roseus
have the ability of regioselective hydroxylation. Further-
more, substrate specificity and generality of this reaction
are in progress and we are now using plant cultured
suspension cells in organic synthesis as biocatalysts.
tonitrile
6 with molybdenum peroxide MoO2-Py-
HMPA (MoOPH) using lithium diisopropylamide as a
base provided a mixture of a cyanohydrin 10, ben-
zophenone (9), and tetraphenylsuccinonitrile (11),9
treatment of 6 with cotton cultured suspension cells
interestingly gave only benzophenone (9) (Table 1).
We also studied the biotransformation of 4S-(−)-peril-
lyl alcohol (12) and 3S-(−)-citronellol (13) by cultured
suspension cells of C. roseus and the results are shown
in Table 2. After 48 h incubation 12 was converted to
10-hydroxy perillyl alcohol (14) in 72.0% yield.
Table 2. Biotransformation of monoterpens
Substrate
Product
Yielda (%)
12
13
14
15
16
72.0
13.0
50.0
a Weight (%) of product relative to the substrate administered.
Acknowledgements
The authors are grateful to Professor Margaret Essen-
berg, Oklahoma State University, for a gift of cotton
callus.
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