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Biotransformation of exogenous substrates by plant cell cultures
Phytochemistry, Vol. 29, No. 8, pp. 2393-2406, 1990. Printed in Great Britain.
0
003l-9422/90 $3.00+ 0.00 1990 Pergamon Press plc
REVIEW ARTICLE NUMBER 55 BIOTRANSFORMATION OF EXOGENOUS PLANT CELL CULTURES TAKAYUKI Department
of
Chemistry, Faculty
SUGA
and
TOSHIFUMI
SUBSTRATES
BY
HIRATA
of Science, Hiroshima University, Higashisenda-machi, Naka-ku, Hiroshima 730, Japan
(Received29 January 1990) Key Word Index-Plant cell cultures; immobilized plant cells; biotransformation; stereospecificity; enantioselectivity; hydroxylation; oxido-reduction; hydrogenation; glycosyl conjugation; hydrolysis.
Abstract-This review outlines the progress during the last 10 years in the biotransformation of exogenous substrates administered to plant cell cultures. The reaction types, stereochemistry and mechanisms involved in the biotransformations are described in six tables covering 200 substrates and 30 plant species. The development of techniques using immobilized plant ceils are also delineated.
INTRODUCTION
The biochemical potential of plant cell cultures to produce specific secondary metabolites such as drugs, flavours, pigments and agrochemicals is of considerable interest in connection with their biotechnological utilization. However, it has been reported that formation and accumulation of some secondary metabolites does not normally occur in the cell cultures of higher plants [l-4]. There is evidence that such cultures retain an ability to transform specifically exogenous substrates administered to the cultured cells [S-7]. Therefore, plant cell cultures are considered to be useful for transforming cheap and plentiful substances into rare and expensive substances by using the cell culture as a bioreactor. Many studies have focused on the ability of plant cell cultures to transform exogenous substrates and the large amount of information on these biotransformations called for an update of previous reviews [S-11]. This article summarizes the advances in the biotransformation of exogenous substrates by plant cell cultures which have been reported during the last 10 years. Biotransformational capability of the plant cell cultures
The reaction type, stereospecificity, enantioselectivity, and mechanism involved in the biotransformations of the exogenous substrates by plant cell cultures are summarized according to classes of chemical reactions as follows: (i) hydroxylation, (ii) oxido-reduction between alcohols and ketones, (iii) reduction ofthe carbon-carbon double bond, (iv) glycosyl conjugation, (v) hydrolysis and (vi) miscellaneous reactions. The substrates, products, types of reaction and plant species participating in the biotransformations of exogenous substrates are summarized in Tables 1, 3, 5-8. Hydroxylation. Regio- and stereoselective introduction of oxygenated functions at the various positions of the molecule is one of the important categories in the bio-
transformation of exogenous substrates by plant cell cultures, because this offers great potential for the production of useful substances. Hydroxylations of exogenous substrates with the plant cell cultures are summarized in Table 1. It was reported that the suspension cultures of Nicotiana tabacum have the ability to hydroxylate the trans-methyl group in the isopropylidene moiety of linalool (l), dihydrolinalool (4) and their acetates (3 and 6) to give the corresponding Shydroxy derivatives [ 123. Such an ability of the cultured cells was also investigated with the monoterpenoids having terminal, endocyclic and exocyclic C-C double bonds, such as p-menth-1-en-8-01 (a-terpineol) (7) and its acetate (10) [ 13-161, c-4-p-menth-8(9)-en-r-l-01(/3-terpineol) (26) and its acetate (29) [13, 173 and 1-acetoxy-p-menth-4(8)-ene (y-terpinyl acetate) (33) [18], as substrates. The terpineols were hydroxylated at the carbon atoms allylic to the C-C double bond to yield the corresponding ally1 alcohols. On the other hand, terpinyl acetates were hydroxylated, not only at the allylic positions, but also at the C-C double bond to give glycols as the major products. All these hydroxylations were stereoselective; (i) the hydroxylation at C-4 of /I-terpineol(26) and its acetate (29) afforded only trans-homers (28 and 32, respectively), (ii) the hydroxylation of the endocyclic linkage of cr-terpinyl acetate (10) resulted in the predominant formation of a trans-diol (11) and (iii) the hydroxylation of y-terpinyl acetate (33) gave predominantly a diol(34) having the hydroxyl group trans to the 1-acetoxyl group. Thus cultured cells possess the ability to hydroxylate regio- and stereospecifically to the C-C double bond in the allylic position. The enantioselectivity in the hydroxylation by cultured ceils was tested using the enantiomers of a-terpineol (7) and its acetate (10) [14, 161 (Table 2). The hydroxylation at the 6-position of (4R)-a-terpineol (14) and its acetate (20) took place in preference to that of their (4S)-isomers. On the other hand, the hydroxylation at the ethylenic linkage of (4S)-a-terpinyl acetate (24) was in preference to that of its (4R)-isomer. Hence the cells discriminate
2393
Papaverine
*S and I denote
(48)
the use of suspension
cells and immobilized
Papaverinol(49)
Tryptamine
cells, respectively.
Hydroxylation
Hydroxylation Hydroxylation Hydroxylation
(37) (39)
(33)
(29)
(46) (47)
12fi-Hydroxydigitoxigenin 16/CHydroxydigitoxigenin S-Hydroxytryptamine
(lR,rlS)-p-Menth-3-one (1 R,4R)-p-Menth-3-one Solavetivone (41) Digitoxigenin (43)
I-Acetoxy-p-menth-4(8)-ene
r-I-Acetoxy-c-4-p-menth-8(9)-ene
(26)
(24)
(4S)-8-Acetoxy-p-menth-1-ene
c-4-p-Menth-8(9)-en-r-l-01
(20)
(17)
(4S)-p-Menth-l-en-8-01
(10)
(4R)-8-Acetoxy-p-menth-l-ene
(14)
(4R)-p-Menth-1-en-8-01
(RS)-8-Acetoxy-p-menth-1-ene
Hydroxylation Hydroxyiation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Glycol formation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Glycol formation Hydroxylation Glycol formation Hydroxylation Hydroxylation Hydroxylation Glycol formation Hydroxylation Hydroxylation Glycol formation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation
(3R)-8-Hydroxylinalool (2) (3R)-8-Hydroxyhnalool (2) (3S)-8-Hydroxydihydrolinalool (5) (3S)-8-Hydroxydihydrolinalool (5) p-Menth-I-ene-7,8-diol (8) f-4-p-Menth-1-ene-r-6,8-diol (9) 8-Acetoxy-c-4-p-menthane-r-l,t-2-diol (11) X-Acetoxy-t-4-p-menth-1-en-r-6-01 (12) 8-Acetoxy-p-menth-1-en-7-01 (13) (4S,6R)-p-Menth-I-ene-6,8-diol (15) (4R)-p-Menth-I-ene-7,8-dial (16) (4R,6S)-p-Menth-1-ene-6,8-dial (18) (4S)-p-Menth-I-ene-7,8-dial (19) (4S,6R)-8-Acetoxy-p-menth-1-en-6-01 (21) (lS,2&4R)-&Acetoxy-p-menthane-1,2-diol (22) (4R)-8-Acetoxy-p-menth-I-en-7-01 (23) (lR,2R,4S)-8-Acetoxy-p-menthane-1,2-diol (25) (4S)-p-Menth-1-ene-7,8-diol (19) c-4-p-Menth-8(9)-ene-r-l,lO-diol (27) 4-p-Menth-8(9)-ene-r-l,f-4-dial (28) r-l-Acetoxy-c-4-p-menthane-8,9-dial (30) r-l-Acetoxy-c-4-p-menth-8(9)-en-lo-01 (31) r-1-Acetoxy-c-4-p-menth-8(9)-en-t-4-01 (32) r-I-Acetoxy-p-menthane-t-4,8-diol (34) I-Acetoxy-p-menth-4(8)-en-9-01 (35) I-Acetoxy-p-menth-3-en-8-01 (36) (lR,4R)-4-Hydroxy-p-menth-3-one (38) (lR,4S)-4-Hydroxy-p-menth-3-one (40) Hydroxysolavetivone (42) IS-Hydroxydigitoxigenin (44) 5/I-Hydroxydigitoxigenin (45)
(3R)-Linalool (1) (3R)-Linalyl acetate (3) (3S)-Dihydrolinalool (4) (3S)-Dihydrolinalyl acetate (6) (RS)-p-Menth-l-en-S-o1 (7)
with plant cell cultures
Type of reaction
1. Hydroxylations
Products
Table
Substrates
-
S S S
S S S
S
S S S S S S I S S S I S S S
Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum
Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum
Nicotiana tabacum
Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Strophanthus gratus Strophanthus intermedius Strophanthus gratus Daucus carota Digitalis lanata Digitalis purpurea Peganum harmala Ochrosia elliptica Saponaria oficinalis Glycyrrhiza glabra
14, 15
S
Nicotiana tabacum
19 19 20 21,22 21,22 22 23 21 24 25, 26 27 28 29
18
14, 16
14, 16
14, 16
14, 16
12 12 12 12 13, 14
Ref.
tabacum tahacum tabacum tabacum tabacum
Nicotiana Nicotiana Nicotiana Nicotiana Nicotianu
Plant species
Explant used*
2
3 z
g
i3
Biotransformation of exogenous R
9‘,,
‘.‘,
P 7
R
5? 3
:”81: 10
2395
substrates
HO.,,
OH
8
4: R=OH
1: R=OH 3: R=OAc
19: R=OH
18
6: R=OAc
CHW ‘..,
OH
:,,
5
R
,\“ OH
R
Ok
5 7: R=OH IO: R=OAc
8: R=OH 13: R=OAc
25
‘11,
HO...
9: R=OH 12: R=OAc
CH,OH
26: R=OH
27: R=OH
29: R=OAc
31: R=OAc
R
‘,,,
d
6 2
R
d
OAc
“‘OH
14: R=OH u): R=OAc
Q
CYR
28: R=OH
33: R=H
32: R=OAc
35: R=OH
HO
R 3 IS: R=OH 21: R=OAc
16: R=OH 23; R=OAc
17: R=OH 24: R=OAc
37: R=H
36
38: R=OH
between the enantiomers of the substrates and hydroxylate one of the enantiomers selectively. The difference of enantioselectivity in the hydroxylation between the allylic position of the C-C double bond and the C-C double bond itself suggests that these hydroxylations are catalysed by different enzyme systems in the cells [30]. The process of glycol formation was investigated in the biotransformation of cc-terpinyl acetate (10) and y-terpinyl acetate (33) in the cultured cells of N. tabacum [31]. It was found that glycols were formed from epoxidation of the C-C double bond, followed by hydrolysis of the resulting epoxides. The presence of an epoxidase in the plant cell cultures was also demonstrated by the epoxidation of isopentenol with a cell-free system from the callus tissues of Jasminum officinale, though neither callus nor suspension cultures of the cells were able to form any epoxides [32]. The other types of hydroxylation, as well as the hydroxylation at the C-C double bond and its allylic position with the plant cultured cells, are listed in Table 1. Regio- and stereoselective hydroxylation at the C-l, C-5, C-12 and C-16 of digitoxigenin (43) were reported [21-241. Hydroxylation at the a-position to the carbonyl group was found in the biotransformation of the 3-0x0-pmenthanes, (lR,4S)- and (lR,4R)-p-menth-3-ones (37 and 39), with a suspension culture of N. tabacum; the hydroxyl group at the 4-position occupies the same spatial arrangement as that of the leaving methine proton [19]. Interestingly, such a hydroxylation did not occur in the biotransformation of 2-oxo-p-menthane derivatives by the same cultured cells [33].
P ‘,a
0
9
39: R=H
41: R=H
40: R=OH
42: R=OH
CYR
HO
43:R,=R2=R3=R4=H
48: R=H
44:R,=OH.R2=Rj=R4=H
49: R=OH
45:R,=R3=R4=H,RZ=OH 46:R,=R2=R4=H,R3=OH 47:R,=R2=R3=H.R4=OH
Oxido-reduction between alcohols and ketones. Oxidation of the hydroxyl group. Alcohols are converted to the
corresponding ketones by the palnt cell cultures, (Table 3). Conversion of mono- and bicyclic monoterpene alcohols by the cultured cells of N. tabacum is
2396
T. SUGA and T.
HIRATA
Table 2. Enantioselectivities in the biotransformation of wterpineol
(7) and its acetate
wTerpineo1 Reaction
4R
types
Hydroxylation C-6 position C-7 position Glycoi formation Hydrolysis
+++ +++
(7) 4s
(10)
cc-Terpinyl acetate 4R 4s
+ +++ -I-
+++ + ++ +
% (T
CHO
of the enantiomers (10)
+ + +++ +++
CH,W
9
‘33,
CH,Ctl
A.
52: R , =OH, Rz =H
!?I
Q 72
71
70
53:R,.R2=0 54:R,=H,Rz=OH RI “,R
Ri
2
0
2
b
Rz
“‘R,
& A
R, =OH,
55: R ,=OH. R2 =H
58:
RZ =H
56:R,,R2=0
59:R,.R2=0
57:R,=H,R2=OH
M):R,=H.R2=OH
61: R , =OH. R2 =H
73:R,.R2=0
75:R,.R2=0
78:R,,R2=0
62:R,.R2=0
74:R,=OH,Rz=H
76:R
,=H.R,=OH
79:R,=OH,Rz=H
77:R
,=OH,RZ=H
63: R , =OH. R z =H 65:R
64:R,.Rz=0
,=OH.R2=H
66: R ,.R2=0
80:R,.R2=0
83:R,.R,=O
85:R,.R,=O
81:R
84:R,=OH.RZ=H
86:R,=H,R2=OH
,=H,R2=OH
87: R , =OH, RZ =H
82:R,=OH.R2=H
1
CHO
67
ypCH” QHO 4
68
69
enantioselective; the cultured cells discriminate the enantiomers of p-menthan-2-01, bicyclo[2.2.l]heptan-2-01 and bicyclo[3.l.l]heptan-3-01 derivatives, and oxidize their hydroxyl group enantioselectively [33, 411. Transformation of (RS)-borne01 and (RS)-isoborneol with the cultured cells of N. tabacum gave (lR,4R)-camphor (56) and unchanged substrates, (lS,2R,4S)-borne01 or (lS,2S,4S)isoborneol, of which the optical purities were about 90-95% enantiomer excess (Table 4) [30, 411. Such a enantioselective oxidation is useful for the preparation of chiral compounds. Reduction of rhe carbonyl group. There are many reports of the reduction of ketones and aldehydes to the corresponding alcohols with plant cell cultures (Table 3). Reduction of 2- and 3-oxygenated p-menthanes, such as (1R,4R)- and (1S,4S)-dihydrocarvones (73 and 75), (1&4R)- and (lR,4S)-isodihydrocarvones (78 and 80), (1 R,4S)-menthone (83) and (1R,4R)- and (1S,4S)-
88:R,.R2=0,R,.R,=0
90:R,.R2=0
89.R,=R4=OH,R2=Rj=H
9l:R,=OH.R,=H
R; 92 93:
R,
M
R j =OH. R, =H
R2 =0
carvomenthones (53 and 85) with cultured cells of N. tabacum occur stereospecifically; the hydrogen attack in the reduction takes place preferentially from there-face of the carbonyl group to give the hydroxy compounds with
Nicotiana tabacum Dendrobium phalaenopsis Papavm somnifkum
Reduction of C=O
(78) (80)
a-Dicarvone (88) Androstenolone (90) Androstenedione (66) Codeinone (93)
Menthone (83) (lR,4R)-Carvomenthone (53) (lS,4S)-Carvomenthone (B5)
(lS,4R)-Isodihydrocarvone (lR,4S)-Isodihydrocarvone
tabacum tabacum Mentha sp. Nicotiana tabacum Nicotiana tabacum
Nicotiana Nicotiana
48 44
47 33,39 33, 39
46 46
46 46
Nicotiana tabacum Nicotiana tabacum
Ref. 34-36 34-36 34-36 34-36 37 38 39 39 40 40 41 41 42 33,42 33,42 33,42 43 44 44 45
Explant used*
Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Spirodela oligorrhiza Glycine max Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Dendrobium phalaenopsis Nicotiana tabacum Nicotiana tabacum Lavandula angustifolia
Oxido-reduction Oxido-reduction Oxido-reduction Oxido-reduction Oxidation of OH Oxidation of OH Oxidation of OH Oxidation of OH Oxidation of OH Oxidation of OH Oxidation of OH Oxidation of OH Oxidation of OH Oxidation of OH Oxidation of OH Oxidation of OH Oxidation of OH Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O
Cyclopentanone Cyclohexanone Cycloheptanone Cyclooctanone Acetophenone Geranial(51) (lR,4R)-Carvomenthone (53) (I R,4R)-Carvomenthone (53) (lR,4R)-Camphor (56) (1R,4R)-Camphor (56) (lR,4R)-Camphor (56) (lR,4R)-Camphor (56) (lS,2S,SR)-Isopinocamphone (59) (lS,2S,SR)-Isopinocamphone (59) (lS,2R,SS)-cis-Verbanone (62) (lR,2S,SR)-cis-Verbanone (64) Androstenedione (66) Ethyl 3-hydroxybutanoate Butyl 3-hydroxybutanoate Benzyl alcohol Cinnamyl alcohol Citronellol(68) Geraniol(50) Nero1 (70) Perillyl alcohol (72) (lR,2S,4R)-Neodihydrocarveol (74) (lS,2R,4S)-Neodihydrocarveol(76) (lS,2S,4S)-Dihydrocarveol (77) (lS,2S,4R)-Isodihydrocarveol (79) (lR,2R,4S)-Isodihydrocarveol(81) (lR,2S,4S)-Neoisodihydrocarveol (82) Neomenthol(B4) (lR,2&4R)-Neocarvomenthol(52) (lS,2R,4S)-Neocarvomenthol (86) (lS,2S,4S)-Carvomenthol (87) 6,6’-p-Menth-8-en-2-01 (89) l7-Hydroxy derivative (91) 17-Hydroxy derivative (92) Codeine (94)
Cyclopentanol Cyclohexanol Cycloheptanol Cyclooctanol l-Phenylethanol Geraniol(50) (lR,2S,4R)-Neocarvomenthol (52) (lR,2R,4R)-Carvomenthol(54) (1R,2&4R)-Borneo1 (55) (lR,2R,4R)-Isoborneol (57) (RS)-Borneo1 (RS)-Isobomeol (lS,2S,3S,SR)-Isopinocampheol (5B) (lS,2S,3R,SR)-Neoisopinocampheol (69) (lS,2R,4S,W)-Neoisoverbanol(61) (lR,2S,4R,5R)-Neoisoverbanol(63) Testosterone (65) Ethyl acetoacetate Butyl acetoacetate Benzaldehyde Cinnamylaldehyde Citronella1 (67) Geranial (51) Neral(69) Perilladehyde (71) (lR,4R)-Dihydrocarvone (73) (lS,4S)-Dihydrocarvone (75)
Plant species
Type of reaction
Products
Substrates
Table 3. Oxido-reduction between alcohols and ketones with plant cell cultures
T. SUGAand T. HIRATA
2398 Table
4. Biotransformation cultured
of (RS)-borne01 and (RS)-isoborneol suspension cells of N. tahncum (56) produced
Unchanged
Substrates
Ial I?’
Camphor
o.p.*
Crl A”
o.p.*
(RS)-Borneo] (RS)-Isoborneol
f40.7 +40.1
92.1 90.7
- 36.4 f32.5
95.5 94.8
*O.p. denotes
the S-chirality
at the position
bearing
optical
purity
the hydroxyl
(% enantiomer
with
the
alcohols
excess).
group
[33, 39, 461. Interestingly cultured cells discriminate between the 2and 3-oxygenated p-menthanes in their reductive conversions; p-menthan-2-ones are converted to their corresponding alcohols in good yield, but this is not the case with the p-menthan-3-ones [19]. In addition, the cells stereospecifically reduce (lR,4R)-2-oxo-p-menthanes, whereas the specificity is low in the case of the (4S)-epimer c331. Oxidation--reduction relationships. The conversion between cycloalkanols and the corresponding ketones in the cultured cells of N. tabacum is reversible [34] and the oxido-reductions are governed by an NAD’-dependent alcohol dehydrogenase [30, 361. The balance of the equilibrium depends on the carbon number in the carbocyclic ring of the cyclic compounds; the equilibrium tends to lie toward the side of the alcohol in the case of a six-membered cyclic compound, while it is mostly in the direction of the ketones for the five-, seven-, and eightmembered cyclic compounds [34]. The equilibrium constants of the oxido-reduction between the cycloalkanols and their corresponding cycloalkanones are correlated with the r3C NMR chemical shift values of the carbonyl carbon of the oxidation products [35, 361. A method for simulating of the time course of the oxidation of cycloalkanols and the reduction of cycloalkanones by cultured cells of N. tabacum was developed on the basis of the permeability constant of the substrates into the cultured cells and the 13C NMR chemical shift of the carbonyl carbon of the cycloalkanones [36, 401. It was found that the proportion (P) of the amount of product at the incubation time, t, to the initial amount of substrate in the oxidation and reduction by the cultured cells can be predicted by the following equations: P={l-exp(-4.82x 10-6t)}/{l+exp(-0.284&=o + 60.5)} for the oxidation and P= { 1-exp (-4.82 x 10-h t)}/{ 1 +exp (0.284 &=o- 60.5)) for the reduction, where a,,, denotes the 13CNMR chemical shift of the carbonyl carbon. Further, the method can be widely applied for the simulation of the time course in the oxidoreduction of the cyclic alcohols and ketones by other cultured cells (Fig. 1) [36]. This method is useful for predicting the equilibrium constant and the time course in the biotransformation, prior to the incubation. Hydrogenation of the carbon-carbon double bond. There are several reports of the reduction of a C-C double bond by a plant cell culture (Table 5). The cultured cells of N. tabacum reduce the C-C double bond adjacent to the carbonyl group of (4R)- and (4S)-carvones (95 and 96) whereas the cells do not attack the C-C double bond in the I-methylethenyl group [46]. The stereochemistry of the reduction of the endocyclic C-C double bond of (4R)carvone (95) was investigated with cultured cells of N.
Fig. 1. Time courses for the transformation of borne01 to camphor with the cultured cells of A’. tabacum (-a-), the enzyme preparation (--O-) and by simulation (- - - -).
tabacum and the enzyme preparation from the cultured cells: (i) the conjugated C--C double bond was regioselectively reduced; (ii) the reduction occurred stereospecifically by anti-addition of hydrogen from the si-face at C-l and the re-face at C-6 of carvone to give (lR,4R)dihydrocarvone (73); (iii) the hydrogen atoms participating in the enzymatic reduction at C-l and C-6 originate from the medium and the pro-4R hydrogen of NADH, respectively [54, 551. Such a stereospecific reduction occurs in the biotransformation of pulegone (97) and verbenone (98) [19, 33,42, 511. In addition, cultured cells discriminate the enantiomer of verbenone (98) and enantioselectively reduce the C-C double bond of only the (lS,SS)-enantiomer [33, 421. Glycosyl conjugation. Glycosyl conjugation is of special interest, because of the possibility of producing new cardenolides, as well as converting water-insoluble substances to water-soluble compounds. Two types of glycosyl conjugations are listed in Table 6. One involves the esterification between carboxylic acids and sugar moieties, whereas the other is ether formation (glycosidation) of alcohols and sugar moities. Esterifications of propionic acid derivatives and several sugars, such as glucose, xylose, inositol and sucrose, have been carried out by using root cultures of Panax ginseng and suspension cultures of N. tabacum, Dioscoreophyllum cummensii, Coffea arabica and Coronilla uaria [54-571. On the other hand, when the capacity for glucosylation of plant cell cultures is examined, it is clear that cultured cells have high activity for the glucosylation of phenolic substrates [57-601. Glucosylation of esculetin (110)by cultured cells is highest at the late stationary phase of the cell growth cycle and ca 10% of the added substrate is converted to 6O-/?-D-glucosylesculetin (111)in 24 hr [65]. On the other hand, higher activity is observed with salicyl alcohol (107)
(104)
(102)
*S and I denote suspension
Cathenamine
10-Oxocitronellol
cells and immobilized
cells, respectively.
(lS,2R,SS)-cis-Verbanone (62) lo-Hydroxycitronellol (100) 6,7-dihydro-lo-hydroxygeraniol (101) lo-Hydroxycitronellol (100) 6,7-Dihydro-lo-hydroxycitronellol(lO3) Ajamalicine (105)
Catharanthus roseus
I
S
Catharanthus roseus
of of of of of of
Reduction Reduction Reduction Reduction Reduction Reduction
(lS,SS)-Verbenone (98) lO-Hydroxygeraniol(99)
C=C C=C C=C C=C C=C C=C
of C=C and C=O
(&I)
Reduction
Neomenthol
46 50 46 50 46
S S S S S
53
52
51 19 51 19 33.42 52
50
S
I S I S S S
of C=C of C=C and C=O of C=C
Reduction Reduction Reduction
(lR,4S)-Isodihydrocarvone (80) (lR,2S,4S)-Neoisodihydrocarveol Isomenthone (39)
(%)
(4QCarvone
Ref.
Explant used*
Mentha sp. Nicotiana tabacum Mentha sp. Nicotiana tabacum Nicotiana tabacum Catharanthus roseus
of C=C and C=O
Reduction
(lR,2S,4R)-Neodihydrocarveol(74)
(97)
of C=C
Reduction
(73)
(lR,4R)-Dihydrocarvone
(95)
(4R)Xarvone
Pulegone
Medicago sativa Glycine max Vinca minor Catharanthus roseus Nicotiana tabacum Medicago sativa Nicotiana tabacum Medicaga sativa Nicotiana tabacum
of C=C
Reduction
Cyclohexanone
Cyclohex-Zen-l-one
(82)
Plant species
bonds with plant cell cultures
Type of reaction
of C-C double
Products
5. Reduction
Substrates
Table
L? B u z z E
r% B
acid
*R, S, and I denote
ceils and immobilized
(120)
cells, respectively.
Hesperitin 7-glucoside (124) Digitoxigenin p-o-glucoside (125) Purpureaglucoside (127)
Hesperitin (123) Digitoxigenin (43) Gigitoxin (126)
(115) (117)
suspension
Nicotinic acid-N-fl-D-glucopyranoside Prunin (122)
Nicotinic acid (119) Naringenin (121)
Isorhamnetin Umbelliferone
the use of root cultures,
Salicin (108) Isosalicin (109) 6-0-Glucosylesculetin (111) Quercetin 3-0-glucoside (113) Quercetin 3-0-diglucoside (114) Isorhamnetin 3-0-glucoside (116) 7-0-Glucosylumbelliferone (118)
(107)
Salicyl alcohol
Esculetin (110) Quercetin (112)
0-Glucosylphenol 0-Glucosylcathechol 0-Glucosylresorcinol 0-Glucosylhydroquinone 0-Glucosyl-o-nitrophenol 0-Glucosyl-m-nitrophenol 0-Glucosyl-p-nitrophenol 0-Glucosylpentanitrophenol
Phenol Cathechol Resorcinol Hydroquinone o-Nitrophenol m-Nitrophenol p-Nitrophenol Pentachlorophenol
acid acid
Glycosylation Glycosylation Glycosylation
Glycosylation Glycosylation
Glycosidation Glycosidation Glycosidation Glycosidation Glycosylation
Glycosylation
Glycosylation Glycosylation Glycosylation Glycosylation Glycosylation Glycosylation Glycosylation Glycosylation
Esterification Esterification Glvcosvlation Gl&o&ation Glycosylation
Esterification Esterification Esterification
(2RS)-2-O-(2-phenylpropionyI)-D-Glucoside (RS)-2-Phenylpropionic acid sucrose ester 2-(3-benzoylphenyl)Propionylglucoside 2-[2-(6-methoxy)naphthyl]Propionylglucoside 3-NitropropanyI-D-glucopyranose Geranyl acetate (106) (2R)-2-(4-O-B-D-gIucopyranosyIphenyI)Propionic p-0-fi-D-Glucosylbenzoic acid m-O-B-D-GlucoHylbenzoic acid
acid
(RS)-2-Phenylpropionic
(RS)-2-Phenylpropionic acid 2-(3knzoylphenyl)Propionic acid 2-[2-(6-methoxy)naphthyl]Propionic 3-Nitropropanoic acid Geraniol (SO) (RS)-2-(4-HydroxyphenyI)-propionic p-Hydroxybenzoic acid m-Hydroxybenzoic acid
Esterification Esterification Esterification Esterification Esterification
Type of reaction
with plant cell cultures
(RS)-2-Phenylpropionyl /l-o-glucoside (2RS)-2-O-(2-phenylpropionyI)-D-glucoside (2S)-2-Phenylpropionyl-6-0-8-D-xylosyl-B-D-glucoside myo-Inositol ester of (R)-2-phenylpropionic acid (RS)-2-Phenylpropionyl b-o-glucoside
conjugation
acid
6. Glycosyl
(RS)-2-Phenylpropionic
Table
Products
--_
Substrates
__..-._
ginseng
S S
Cannabis sativa Datura innoxia Perilla frutescens Catharanthus roseus Lithospermum erythrorhizon Petroselinum hortense Citrus paradisi Citrus limon Strophanthus amboensis Digitalis lanata
S S
Gardenia jasminoides Cannabis sativa
S I
S S
S S S S S S S S S
S S S S S
S R
S S
R
Explant used*
Coronilla varia Citrus sp. Panax ginseng Mallotus japonicus Datura innoxia Perilla frutescens Catharanthus roseus Lithospermum erythrorhizon Gardenia jasminoides Gardenia jasminoides Gardenia jasminoides Gardenia jasminoides Gardenia jasminoides Gardenia jasminoides Gardenia jasminoides Glycine max Triticum aestivum Gardenia jasminoides
Coffea arabica Panax ginseng
Nicotiana tahacum Dioscoreophyllum cummensii
Panax
Plant species
70 71, 72
67 68, 69
66 60
65 66
63, 64
61 61 61 61 61 61 61 62
58 59 51 60 60
56 57
55
54
Ref.
Biotransformation
6_
2401
of exogenous substrates
0
0
A
OR
OH
97
96
95
% ;I-;-r A0
Q+9
0
0 CC&
CH,m -0
0
OH H
CH&ti
0
H
4
OH
HOb OH 126: R=H t
100
99
CH,C+l
CH,Cii
t
127: R=Glc
CHpl
CH,Ctl
Y?
CH,Cti
CH,C+i
CHO
102
IO1
103
%~I
&w
CW~
107:R
,=RZ=H
108: R , =H, R 2 =Glc 106
;plJJP 1 lo: R=H Ill:
R=Glc
109: R , =Glc. R z =H
OH
117: R=H 112: R ,=R2=H 113:R
118: R=Glc
,=Glc.R,=H
114: R , =Glc-0.Glc,
R 2 =H
115: R ,=H. R *=CHg OH
116: R I=Glc. RZ=CHB
ticoo. GIG
CL,,, I19
121: R=H
120 OH
123: R=H 124: R=Glc
122: R=Glc
at the exponential phase of the growth cycle and about 70% of the alcohol administered is converted to the corresponding glucoside within four days [63,64]. Interestingly, a major product of glucosylation with the cultured cells of Gardenia jasminoides is salicin (lo@, although other culture strains derived from different plant species predominantly produce isosalicin (109) [63, 641. Furthermore, glycosyl conjugation of (RS)-2-phenylpropionic acid derivatives occur enantioselectively to give the C-2 chiral products [54, 571. Hydrolysis. The ability of cultured cells to hydrolyse the acetoxyl group has been widely investigated (Table 7). Enantioselective hydrolysis is of interest because the transformation is considered to be useful for the optical resolution of racemic acetates. The enantiomers (20 and 24) of or-terpinyl acetate tend to experience enantioselective hydrolysis in the cultured cells of N. tabacum [14,30] (Table 2). Therefore, the ability of the cultured cells for enantioselective hydrolysis was examined in the biotransformation of bornyl acetate (136) isobornyl acetate (139) and isopinocampheyl acetate (140). The enantiomers with the R-configuration at the carbon atom bearing the acetoxyl group are preferentially hydrolysed [70]. Enantioselective hydrolysis was also observed in the biotransformation of (RS)-1-phenylethyl acetate and its derivatives with cultured cells of Spirodela oligorrhiza in which the biotransformations gave only (R)-alcohols [73]. Other examples have been reported for the hydrolyses of the hydroxyimino group and an ether bond (Table 7); carvoximes (142 and 143) and dihydrocarvoxime (144) are hydrolysed to the corresponding ketones by cultured cells of N. tabncum [76] and thebaine (145) is hydrolysed to the corresponding alcohol by cultured cells of Papauer somniferum [77]. Miscellaneous. Many other types of biotransformation have been reported, such as isomerization, epoxidation, degradation and dehydrogenation (Table 8). Salvage synthesis of ajmalicine (150) with tryptamine and secologanin (151) by immobilized cells of Catharanthus roseus is an interesting example of the utilization ofcomplex multienzyme processes in plant cells [SO, 811. Biotransformation with immobilized plant cell cultures During the last 10 years, the techniques for immobilization of plant cells have progressed considerably [87-911. Many studies have been focused on de nova synthesis of useful substances by the immobilized plant cells [90], but only a few examples have been reported on the biotransformation of foreign substrates by use of the immobilized plant cells [27, 44, 49, 51, 53, 711. Immobilization of the plant cells offers some advantages for the biotransforma-
*S denotes
suspension
cells.
(IS,ZR,JS)-Bornyl acetate (137) (lR,ZR,4R)-lsobornyl acetate (139) ( I R,ZK,3R,SS)-Isopinocamphcyl acetate (4R)Karvoxime (142) (4Sj-Carvoxime (143) I I K,4R)-Dihydrocarvowime (144) Thebaine (145)
(140)
acetate (130) acetate (132) acetate (134)
(138) (58) (lR.2R,3R,5S)-IsopinocampheoI (4R)Xarvone (95) (4S)Carvone (96) (I R,4R)-Dihydrocarvone (73) Neopine (146)
(I R,2R,4R)-Isoborneoi
(1&2R,4S)-Borneo1
!rcms-2-Hydroxy-truns-dihydropinanol c,i.s-2-Hydroxy-trans-dihydropinanol rrans-2-Hydroxy-cis-dihydropinanol Borneo1
Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis
Hydrolysis Hydrolysis Hydrolysis Hydrolysis
Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis
of of of of of of of
of of of of
ester ester ester oxime oxime oxime ether
ester ester ester ester
of ester of ester of ester of ester of ester and oxidation of ester and oxidation of ester of ester
Type of reaction _____.-
with plant cell cultures
(131) (133) (135)
(141)
(R)-I-Phenylethanol (R)-1-(l-naphthyl)Ethanol (R)-I-(2-naphthyI)Ethanol (K)-2-Phenylbutanol Benzyl alcohol (3R)-8-Hydroxylinalool (2) Linalooi (3SWHydroxydihydrolinalool (5) (4S)-p-Menth-I-ene-7,8-diol (19) Menthol (129)
(RS)-l-Phenylethyl acetate (RS)-l-(I-naphthyl)EthyI acetate (RS)-I-(2-naphthyl)Ethyl acetate (KS)-2-Phenylbutanyl acetate Benzyl acetate (3R)-Linalyl acetate (3) LinaIyI acetate (3S)-Dihydrolinaiyl acetate (6) (4S)-R-acetoxy-p-menth-l-ene (24) Menthyl acetate (128)
trans-2-Hydroxy-truns-dihydropinanyl cis-2-Hydroxy-trans-dihydropinanyl rrcrns-2-Hydroxy-cis-dihydropinanyl Bornyl acetate (136)
Products
7. Hydrolyses
Substrates
Table ___
Spirodela oligorrhizu Spirodelu oligorrhizu Spirodela oligorrhizu Spirodelu oligorrhiza Spirodela oligorrhizu Nicotianu tubucum Lavandula angustijtiliu Nicotiana tabacum Nicotiana tabacum Spirodela oligorrhiza Epidendrum ochraceum Spirodela oligorrhiza Spirodela oligorrhiza Spirodela oligorrhiza Spirodelrr oligorrhiza LLloandula ungustifolia Nicotianu tabacum ,Vicotiunu tabacum Nicotiana tabucum Nicotianu tabucum Nlcotiana tabacum Nicntiunu tubucum Paprrr:er ,somn[ferum
Plant species
-
73 73 73 73 73 12 45 12 14, 16 74 43 74 74 74 74 45 75 75 75 76 76 76 77
S S S S S S S S s S S S S S s S
Ref. S S S S S S S
Explant used*
Z
$
Z
! > ;
ri
*S denotes suspension cells.
Anhydrovinblastine (162)
84.85 86
Silene alba Catharanthus roseus
S
82 20 27
S
Lonicera tatarica Lonicera morrowii Lonicera korolkowii Lonicera minutijlora Lo&era glaucescens Weigelia japonica Weigelia middendorfana Weigelia j7orida Hydrangea macrophylla Symphoricarpus orbiculatus Coptis japonica Phytophthora infestane Ochrosia elliptica
Dehydrogenation Oxidation Oxidation Oxidation Demethylation Demethylation Dehydration
S
Nicotiana tabacum
(S)-Berberine (157) Lubimin (158) Papaveraldine (159) Papaverine N-oxide 6-Demethylpapaverine (160) 4’-Demethylpapaverine (161) Vinblastine (163)
(10)
(S)-Tetrahydroberberine (1%) Solavetivone (41) Papaverine (48)
Loganin (155)
8-Acetoxy-p-menth-I-ene
20 21 80781 31
79 18
64
78
Ref.
S S S S
Isomerization Isomerization Isomerization and hydroxylation Isomerization and hydroxylation Isomerization and hydroxylation Hydroxylation Isomerization Salvage synthesis Epoxidation Epoxidation Epoxidation Epoxidation Oxidative degradation
Isosalicin (109) D-Glucose I-Acetoxy-p-menth-3-en-S-01 (36) r-I-Acetoxy-p-menth-S(9)-en-t-4-01 (32) r-l-Acetoxy-p-menth-8(9)-en-c-4-ol(147) Rishitin (148) 17/l-H-Digitoxigenin (149) Ajamalicine (105) r-1-Acetoxy-t-4,8-epoxy-p-menthane (151) r-1-Acetoxy-c-4,8_epoxy-p-menthane (152) 8-Acetoxy-r-l,c-2-epoxy-c-4-p-menthane (153) 8-Acetoxy-r-l,c-2-epoxy-t-4-p-menthane (154) Secologanin (150)
Salicin (108) L-Rhamnose I-Acetoxy-p-menth-4(S)-ene (33)
Explant used*
Solanum tuberosum Strophanthus grarus Catharanthus roseus Nicotiana tabacum
Euphorbia characias Nicotiana tabacum Catharanthus roseus Glycine max Gardenia jasminoides Duboisia myoporoides Nicotiana tabacum
Isomerization
Nero1 (70)
Geraniol (50)
Solavetivone (41) Digitoxigenin (43) Tryptamine plus secologanin (150) l-Acetoxy-p-menth-4(S)-ene (33)
Plant species
Type of reaction
Products
Substrates
Table 8. Miscellaneous reactions with plant cell cultures
m 2 3 & 0” 2 s 5’ u
T. SUGAand T.
2404
12X: R=Ac
130: R=Ac
132: u=i\c
129: R=H
131. R=ll
133, R=ll
HIRATA
AcO ._ cb 137: R=Ac
136
138: R=H 134: R=Ac
I60
13.5: R=H
101: R ,=CII1,
139
R, =Il
rNnj”,
140: R=Ac
AHn dH
142
141: R=H
143
K , =H, R 2 =CH,
CO,CH,
3
144 145: R=CH9
I63
146: R=H
(J4
149
ci
OHC
‘,,,
,Qc
151
a ‘,
‘,,
0
$iIY
150
OAC
152
0
OAC
153
H
155
tion of exogenous substrates [87-911: (i) the cells become resistant to shear damage by immobilization, (ii) the immobilized cells can be used repeatedly over a prolonged period, (iii) high concentrations of biomass are possible, thus giving high conversions of substrate, (iv) the method facilitates recovery of the cell mass and products and (v) sequential chemical treatments are possible. CONCLUSIONS
As can be seen from the examples given above, the plant cell cultures possess considerable biochemical ability to transform foreign substrates administered exogenously. The reaction types and stereochemistry in the biotransformation depends on the functional group in the substrates administered and the structural moieties in the vicinity of the functional group. Therefore, the biotransformations by plant cell cultures are considered to serve as important tools for the structural modification of molecules to give compounds possessing useful properties. Fundamental information, such as the reaction types, stereospecificity and enantioselectivity in the biotransformation of exogenous substrates, is essential for the development of the biotechnology for using higher plant cells. Further investigations, especially the development of methods to utilize the multi-reaction processes, will be necessary for the practical applications of biotransformations with plant cell cultures. REFERENCES
156
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Biotransformation
of exogenous substrates
2405
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0
003l-9422/90 $3.00+ 0.00 1990 Pergamon Press plc
REVIEW ARTICLE NUMBER 55 BIOTRANSFORMATION OF EXOGENOUS PLANT CELL CULTURES TAKAYUKI Department
of
Chemistry, Faculty
SUGA
and
TOSHIFUMI
SUBSTRATES
BY
HIRATA
of Science, Hiroshima University, Higashisenda-machi, Naka-ku, Hiroshima 730, Japan
(Received29 January 1990) Key Word Index-Plant cell cultures; immobilized plant cells; biotransformation; stereospecificity; enantioselectivity; hydroxylation; oxido-reduction; hydrogenation; glycosyl conjugation; hydrolysis.
Abstract-This review outlines the progress during the last 10 years in the biotransformation of exogenous substrates administered to plant cell cultures. The reaction types, stereochemistry and mechanisms involved in the biotransformations are described in six tables covering 200 substrates and 30 plant species. The development of techniques using immobilized plant ceils are also delineated.
INTRODUCTION
The biochemical potential of plant cell cultures to produce specific secondary metabolites such as drugs, flavours, pigments and agrochemicals is of considerable interest in connection with their biotechnological utilization. However, it has been reported that formation and accumulation of some secondary metabolites does not normally occur in the cell cultures of higher plants [l-4]. There is evidence that such cultures retain an ability to transform specifically exogenous substrates administered to the cultured cells [S-7]. Therefore, plant cell cultures are considered to be useful for transforming cheap and plentiful substances into rare and expensive substances by using the cell culture as a bioreactor. Many studies have focused on the ability of plant cell cultures to transform exogenous substrates and the large amount of information on these biotransformations called for an update of previous reviews [S-11]. This article summarizes the advances in the biotransformation of exogenous substrates by plant cell cultures which have been reported during the last 10 years. Biotransformational capability of the plant cell cultures
The reaction type, stereospecificity, enantioselectivity, and mechanism involved in the biotransformations of the exogenous substrates by plant cell cultures are summarized according to classes of chemical reactions as follows: (i) hydroxylation, (ii) oxido-reduction between alcohols and ketones, (iii) reduction ofthe carbon-carbon double bond, (iv) glycosyl conjugation, (v) hydrolysis and (vi) miscellaneous reactions. The substrates, products, types of reaction and plant species participating in the biotransformations of exogenous substrates are summarized in Tables 1, 3, 5-8. Hydroxylation. Regio- and stereoselective introduction of oxygenated functions at the various positions of the molecule is one of the important categories in the bio-
transformation of exogenous substrates by plant cell cultures, because this offers great potential for the production of useful substances. Hydroxylations of exogenous substrates with the plant cell cultures are summarized in Table 1. It was reported that the suspension cultures of Nicotiana tabacum have the ability to hydroxylate the trans-methyl group in the isopropylidene moiety of linalool (l), dihydrolinalool (4) and their acetates (3 and 6) to give the corresponding Shydroxy derivatives [ 123. Such an ability of the cultured cells was also investigated with the monoterpenoids having terminal, endocyclic and exocyclic C-C double bonds, such as p-menth-1-en-8-01 (a-terpineol) (7) and its acetate (10) [ 13-161, c-4-p-menth-8(9)-en-r-l-01(/3-terpineol) (26) and its acetate (29) [13, 173 and 1-acetoxy-p-menth-4(8)-ene (y-terpinyl acetate) (33) [18], as substrates. The terpineols were hydroxylated at the carbon atoms allylic to the C-C double bond to yield the corresponding ally1 alcohols. On the other hand, terpinyl acetates were hydroxylated, not only at the allylic positions, but also at the C-C double bond to give glycols as the major products. All these hydroxylations were stereoselective; (i) the hydroxylation at C-4 of /I-terpineol(26) and its acetate (29) afforded only trans-homers (28 and 32, respectively), (ii) the hydroxylation of the endocyclic linkage of cr-terpinyl acetate (10) resulted in the predominant formation of a trans-diol (11) and (iii) the hydroxylation of y-terpinyl acetate (33) gave predominantly a diol(34) having the hydroxyl group trans to the 1-acetoxyl group. Thus cultured cells possess the ability to hydroxylate regio- and stereospecifically to the C-C double bond in the allylic position. The enantioselectivity in the hydroxylation by cultured ceils was tested using the enantiomers of a-terpineol (7) and its acetate (10) [14, 161 (Table 2). The hydroxylation at the 6-position of (4R)-a-terpineol (14) and its acetate (20) took place in preference to that of their (4S)-isomers. On the other hand, the hydroxylation at the ethylenic linkage of (4S)-a-terpinyl acetate (24) was in preference to that of its (4R)-isomer. Hence the cells discriminate
2393
Papaverine
*S and I denote
(48)
the use of suspension
cells and immobilized
Papaverinol(49)
Tryptamine
cells, respectively.
Hydroxylation
Hydroxylation Hydroxylation Hydroxylation
(37) (39)
(33)
(29)
(46) (47)
12fi-Hydroxydigitoxigenin 16/CHydroxydigitoxigenin S-Hydroxytryptamine
(lR,rlS)-p-Menth-3-one (1 R,4R)-p-Menth-3-one Solavetivone (41) Digitoxigenin (43)
I-Acetoxy-p-menth-4(8)-ene
r-I-Acetoxy-c-4-p-menth-8(9)-ene
(26)
(24)
(4S)-8-Acetoxy-p-menth-1-ene
c-4-p-Menth-8(9)-en-r-l-01
(20)
(17)
(4S)-p-Menth-l-en-8-01
(10)
(4R)-8-Acetoxy-p-menth-l-ene
(14)
(4R)-p-Menth-1-en-8-01
(RS)-8-Acetoxy-p-menth-1-ene
Hydroxylation Hydroxyiation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Glycol formation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Glycol formation Hydroxylation Glycol formation Hydroxylation Hydroxylation Hydroxylation Glycol formation Hydroxylation Hydroxylation Glycol formation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation
(3R)-8-Hydroxylinalool (2) (3R)-8-Hydroxyhnalool (2) (3S)-8-Hydroxydihydrolinalool (5) (3S)-8-Hydroxydihydrolinalool (5) p-Menth-I-ene-7,8-diol (8) f-4-p-Menth-1-ene-r-6,8-diol (9) 8-Acetoxy-c-4-p-menthane-r-l,t-2-diol (11) X-Acetoxy-t-4-p-menth-1-en-r-6-01 (12) 8-Acetoxy-p-menth-1-en-7-01 (13) (4S,6R)-p-Menth-I-ene-6,8-diol (15) (4R)-p-Menth-I-ene-7,8-dial (16) (4R,6S)-p-Menth-1-ene-6,8-dial (18) (4S)-p-Menth-I-ene-7,8-dial (19) (4S,6R)-8-Acetoxy-p-menth-1-en-6-01 (21) (lS,2&4R)-&Acetoxy-p-menthane-1,2-diol (22) (4R)-8-Acetoxy-p-menth-I-en-7-01 (23) (lR,2R,4S)-8-Acetoxy-p-menthane-1,2-diol (25) (4S)-p-Menth-1-ene-7,8-diol (19) c-4-p-Menth-8(9)-ene-r-l,lO-diol (27) 4-p-Menth-8(9)-ene-r-l,f-4-dial (28) r-l-Acetoxy-c-4-p-menthane-8,9-dial (30) r-l-Acetoxy-c-4-p-menth-8(9)-en-lo-01 (31) r-1-Acetoxy-c-4-p-menth-8(9)-en-t-4-01 (32) r-I-Acetoxy-p-menthane-t-4,8-diol (34) I-Acetoxy-p-menth-4(8)-en-9-01 (35) I-Acetoxy-p-menth-3-en-8-01 (36) (lR,4R)-4-Hydroxy-p-menth-3-one (38) (lR,4S)-4-Hydroxy-p-menth-3-one (40) Hydroxysolavetivone (42) IS-Hydroxydigitoxigenin (44) 5/I-Hydroxydigitoxigenin (45)
(3R)-Linalool (1) (3R)-Linalyl acetate (3) (3S)-Dihydrolinalool (4) (3S)-Dihydrolinalyl acetate (6) (RS)-p-Menth-l-en-S-o1 (7)
with plant cell cultures
Type of reaction
1. Hydroxylations
Products
Table
Substrates
-
S S S
S S S
S
S S S S S S I S S S I S S S
Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum
Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum
Nicotiana tabacum
Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Strophanthus gratus Strophanthus intermedius Strophanthus gratus Daucus carota Digitalis lanata Digitalis purpurea Peganum harmala Ochrosia elliptica Saponaria oficinalis Glycyrrhiza glabra
14, 15
S
Nicotiana tabacum
19 19 20 21,22 21,22 22 23 21 24 25, 26 27 28 29
18
14, 16
14, 16
14, 16
14, 16
12 12 12 12 13, 14
Ref.
tabacum tahacum tabacum tabacum tabacum
Nicotiana Nicotiana Nicotiana Nicotiana Nicotianu
Plant species
Explant used*
2
3 z
g
i3
Biotransformation of exogenous R
9‘,,
‘.‘,
P 7
R
5? 3
:”81: 10
2395
substrates
HO.,,
OH
8
4: R=OH
1: R=OH 3: R=OAc
19: R=OH
18
6: R=OAc
CHW ‘..,
OH
:,,
5
R
,\“ OH
R
Ok
5 7: R=OH IO: R=OAc
8: R=OH 13: R=OAc
25
‘11,
HO...
9: R=OH 12: R=OAc
CH,OH
26: R=OH
27: R=OH
29: R=OAc
31: R=OAc
R
‘,,,
d
6 2
R
d
OAc
“‘OH
14: R=OH u): R=OAc
Q
CYR
28: R=OH
33: R=H
32: R=OAc
35: R=OH
HO
R 3 IS: R=OH 21: R=OAc
16: R=OH 23; R=OAc
17: R=OH 24: R=OAc
37: R=H
36
38: R=OH
between the enantiomers of the substrates and hydroxylate one of the enantiomers selectively. The difference of enantioselectivity in the hydroxylation between the allylic position of the C-C double bond and the C-C double bond itself suggests that these hydroxylations are catalysed by different enzyme systems in the cells [30]. The process of glycol formation was investigated in the biotransformation of cc-terpinyl acetate (10) and y-terpinyl acetate (33) in the cultured cells of N. tabacum [31]. It was found that glycols were formed from epoxidation of the C-C double bond, followed by hydrolysis of the resulting epoxides. The presence of an epoxidase in the plant cell cultures was also demonstrated by the epoxidation of isopentenol with a cell-free system from the callus tissues of Jasminum officinale, though neither callus nor suspension cultures of the cells were able to form any epoxides [32]. The other types of hydroxylation, as well as the hydroxylation at the C-C double bond and its allylic position with the plant cultured cells, are listed in Table 1. Regio- and stereoselective hydroxylation at the C-l, C-5, C-12 and C-16 of digitoxigenin (43) were reported [21-241. Hydroxylation at the a-position to the carbonyl group was found in the biotransformation of the 3-0x0-pmenthanes, (lR,4S)- and (lR,4R)-p-menth-3-ones (37 and 39), with a suspension culture of N. tabacum; the hydroxyl group at the 4-position occupies the same spatial arrangement as that of the leaving methine proton [19]. Interestingly, such a hydroxylation did not occur in the biotransformation of 2-oxo-p-menthane derivatives by the same cultured cells [33].
P ‘,a
0
9
39: R=H
41: R=H
40: R=OH
42: R=OH
CYR
HO
43:R,=R2=R3=R4=H
48: R=H
44:R,=OH.R2=Rj=R4=H
49: R=OH
45:R,=R3=R4=H,RZ=OH 46:R,=R2=R4=H,R3=OH 47:R,=R2=R3=H.R4=OH
Oxido-reduction between alcohols and ketones. Oxidation of the hydroxyl group. Alcohols are converted to the
corresponding ketones by the palnt cell cultures, (Table 3). Conversion of mono- and bicyclic monoterpene alcohols by the cultured cells of N. tabacum is
2396
T. SUGA and T.
HIRATA
Table 2. Enantioselectivities in the biotransformation of wterpineol
(7) and its acetate
wTerpineo1 Reaction
4R
types
Hydroxylation C-6 position C-7 position Glycoi formation Hydrolysis
+++ +++
(7) 4s
(10)
cc-Terpinyl acetate 4R 4s
+ +++ -I-
+++ + ++ +
% (T
CHO
of the enantiomers (10)
+ + +++ +++
CH,W
9
‘33,
CH,Ctl
A.
52: R , =OH, Rz =H
!?I
Q 72
71
70
53:R,.R2=0 54:R,=H,Rz=OH RI “,R
Ri
2
0
2
b
Rz
“‘R,
& A
R, =OH,
55: R ,=OH. R2 =H
58:
RZ =H
56:R,,R2=0
59:R,.R2=0
57:R,=H,R2=OH
M):R,=H.R2=OH
61: R , =OH. R2 =H
73:R,.R2=0
75:R,.R2=0
78:R,,R2=0
62:R,.R2=0
74:R,=OH,Rz=H
76:R
,=H.R,=OH
79:R,=OH,Rz=H
77:R
,=OH,RZ=H
63: R , =OH. R z =H 65:R
64:R,.Rz=0
,=OH.R2=H
66: R ,.R2=0
80:R,.R2=0
83:R,.R,=O
85:R,.R,=O
81:R
84:R,=OH.RZ=H
86:R,=H,R2=OH
,=H,R2=OH
87: R , =OH, RZ =H
82:R,=OH.R2=H
1
CHO
67
ypCH” QHO 4
68
69
enantioselective; the cultured cells discriminate the enantiomers of p-menthan-2-01, bicyclo[2.2.l]heptan-2-01 and bicyclo[3.l.l]heptan-3-01 derivatives, and oxidize their hydroxyl group enantioselectively [33, 411. Transformation of (RS)-borne01 and (RS)-isoborneol with the cultured cells of N. tabacum gave (lR,4R)-camphor (56) and unchanged substrates, (lS,2R,4S)-borne01 or (lS,2S,4S)isoborneol, of which the optical purities were about 90-95% enantiomer excess (Table 4) [30, 411. Such a enantioselective oxidation is useful for the preparation of chiral compounds. Reduction of rhe carbonyl group. There are many reports of the reduction of ketones and aldehydes to the corresponding alcohols with plant cell cultures (Table 3). Reduction of 2- and 3-oxygenated p-menthanes, such as (1R,4R)- and (1S,4S)-dihydrocarvones (73 and 75), (1&4R)- and (lR,4S)-isodihydrocarvones (78 and 80), (1 R,4S)-menthone (83) and (1R,4R)- and (1S,4S)-
88:R,.R2=0,R,.R,=0
90:R,.R2=0
89.R,=R4=OH,R2=Rj=H
9l:R,=OH.R,=H
R; 92 93:
R,
M
R j =OH. R, =H
R2 =0
carvomenthones (53 and 85) with cultured cells of N. tabacum occur stereospecifically; the hydrogen attack in the reduction takes place preferentially from there-face of the carbonyl group to give the hydroxy compounds with
Nicotiana tabacum Dendrobium phalaenopsis Papavm somnifkum
Reduction of C=O
(78) (80)
a-Dicarvone (88) Androstenolone (90) Androstenedione (66) Codeinone (93)
Menthone (83) (lR,4R)-Carvomenthone (53) (lS,4S)-Carvomenthone (B5)
(lS,4R)-Isodihydrocarvone (lR,4S)-Isodihydrocarvone
tabacum tabacum Mentha sp. Nicotiana tabacum Nicotiana tabacum
Nicotiana Nicotiana
48 44
47 33,39 33, 39
46 46
46 46
Nicotiana tabacum Nicotiana tabacum
Ref. 34-36 34-36 34-36 34-36 37 38 39 39 40 40 41 41 42 33,42 33,42 33,42 43 44 44 45
Explant used*
Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Spirodela oligorrhiza Glycine max Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Dendrobium phalaenopsis Nicotiana tabacum Nicotiana tabacum Lavandula angustifolia
Oxido-reduction Oxido-reduction Oxido-reduction Oxido-reduction Oxidation of OH Oxidation of OH Oxidation of OH Oxidation of OH Oxidation of OH Oxidation of OH Oxidation of OH Oxidation of OH Oxidation of OH Oxidation of OH Oxidation of OH Oxidation of OH Oxidation of OH Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O Reduction of C=O
Cyclopentanone Cyclohexanone Cycloheptanone Cyclooctanone Acetophenone Geranial(51) (lR,4R)-Carvomenthone (53) (I R,4R)-Carvomenthone (53) (lR,4R)-Camphor (56) (1R,4R)-Camphor (56) (lR,4R)-Camphor (56) (lR,4R)-Camphor (56) (lS,2S,SR)-Isopinocamphone (59) (lS,2S,SR)-Isopinocamphone (59) (lS,2R,SS)-cis-Verbanone (62) (lR,2S,SR)-cis-Verbanone (64) Androstenedione (66) Ethyl 3-hydroxybutanoate Butyl 3-hydroxybutanoate Benzyl alcohol Cinnamyl alcohol Citronellol(68) Geraniol(50) Nero1 (70) Perillyl alcohol (72) (lR,2S,4R)-Neodihydrocarveol (74) (lS,2R,4S)-Neodihydrocarveol(76) (lS,2S,4S)-Dihydrocarveol (77) (lS,2S,4R)-Isodihydrocarveol (79) (lR,2R,4S)-Isodihydrocarveol(81) (lR,2S,4S)-Neoisodihydrocarveol (82) Neomenthol(B4) (lR,2&4R)-Neocarvomenthol(52) (lS,2R,4S)-Neocarvomenthol (86) (lS,2S,4S)-Carvomenthol (87) 6,6’-p-Menth-8-en-2-01 (89) l7-Hydroxy derivative (91) 17-Hydroxy derivative (92) Codeine (94)
Cyclopentanol Cyclohexanol Cycloheptanol Cyclooctanol l-Phenylethanol Geraniol(50) (lR,2S,4R)-Neocarvomenthol (52) (lR,2R,4R)-Carvomenthol(54) (1R,2&4R)-Borneo1 (55) (lR,2R,4R)-Isoborneol (57) (RS)-Borneo1 (RS)-Isobomeol (lS,2S,3S,SR)-Isopinocampheol (5B) (lS,2S,3R,SR)-Neoisopinocampheol (69) (lS,2R,4S,W)-Neoisoverbanol(61) (lR,2S,4R,5R)-Neoisoverbanol(63) Testosterone (65) Ethyl acetoacetate Butyl acetoacetate Benzaldehyde Cinnamylaldehyde Citronella1 (67) Geranial (51) Neral(69) Perilladehyde (71) (lR,4R)-Dihydrocarvone (73) (lS,4S)-Dihydrocarvone (75)
Plant species
Type of reaction
Products
Substrates
Table 3. Oxido-reduction between alcohols and ketones with plant cell cultures
T. SUGAand T. HIRATA
2398 Table
4. Biotransformation cultured
of (RS)-borne01 and (RS)-isoborneol suspension cells of N. tahncum (56) produced
Unchanged
Substrates
Ial I?’
Camphor
o.p.*
Crl A”
o.p.*
(RS)-Borneo] (RS)-Isoborneol
f40.7 +40.1
92.1 90.7
- 36.4 f32.5
95.5 94.8
*O.p. denotes
the S-chirality
at the position
bearing
optical
purity
the hydroxyl
(% enantiomer
with
the
alcohols
excess).
group
[33, 39, 461. Interestingly cultured cells discriminate between the 2and 3-oxygenated p-menthanes in their reductive conversions; p-menthan-2-ones are converted to their corresponding alcohols in good yield, but this is not the case with the p-menthan-3-ones [19]. In addition, the cells stereospecifically reduce (lR,4R)-2-oxo-p-menthanes, whereas the specificity is low in the case of the (4S)-epimer c331. Oxidation--reduction relationships. The conversion between cycloalkanols and the corresponding ketones in the cultured cells of N. tabacum is reversible [34] and the oxido-reductions are governed by an NAD’-dependent alcohol dehydrogenase [30, 361. The balance of the equilibrium depends on the carbon number in the carbocyclic ring of the cyclic compounds; the equilibrium tends to lie toward the side of the alcohol in the case of a six-membered cyclic compound, while it is mostly in the direction of the ketones for the five-, seven-, and eightmembered cyclic compounds [34]. The equilibrium constants of the oxido-reduction between the cycloalkanols and their corresponding cycloalkanones are correlated with the r3C NMR chemical shift values of the carbonyl carbon of the oxidation products [35, 361. A method for simulating of the time course of the oxidation of cycloalkanols and the reduction of cycloalkanones by cultured cells of N. tabacum was developed on the basis of the permeability constant of the substrates into the cultured cells and the 13C NMR chemical shift of the carbonyl carbon of the cycloalkanones [36, 401. It was found that the proportion (P) of the amount of product at the incubation time, t, to the initial amount of substrate in the oxidation and reduction by the cultured cells can be predicted by the following equations: P={l-exp(-4.82x 10-6t)}/{l+exp(-0.284&=o + 60.5)} for the oxidation and P= { 1-exp (-4.82 x 10-h t)}/{ 1 +exp (0.284 &=o- 60.5)) for the reduction, where a,,, denotes the 13CNMR chemical shift of the carbonyl carbon. Further, the method can be widely applied for the simulation of the time course in the oxidoreduction of the cyclic alcohols and ketones by other cultured cells (Fig. 1) [36]. This method is useful for predicting the equilibrium constant and the time course in the biotransformation, prior to the incubation. Hydrogenation of the carbon-carbon double bond. There are several reports of the reduction of a C-C double bond by a plant cell culture (Table 5). The cultured cells of N. tabacum reduce the C-C double bond adjacent to the carbonyl group of (4R)- and (4S)-carvones (95 and 96) whereas the cells do not attack the C-C double bond in the I-methylethenyl group [46]. The stereochemistry of the reduction of the endocyclic C-C double bond of (4R)carvone (95) was investigated with cultured cells of N.
Fig. 1. Time courses for the transformation of borne01 to camphor with the cultured cells of A’. tabacum (-a-), the enzyme preparation (--O-) and by simulation (- - - -).
tabacum and the enzyme preparation from the cultured cells: (i) the conjugated C--C double bond was regioselectively reduced; (ii) the reduction occurred stereospecifically by anti-addition of hydrogen from the si-face at C-l and the re-face at C-6 of carvone to give (lR,4R)dihydrocarvone (73); (iii) the hydrogen atoms participating in the enzymatic reduction at C-l and C-6 originate from the medium and the pro-4R hydrogen of NADH, respectively [54, 551. Such a stereospecific reduction occurs in the biotransformation of pulegone (97) and verbenone (98) [19, 33,42, 511. In addition, cultured cells discriminate the enantiomer of verbenone (98) and enantioselectively reduce the C-C double bond of only the (lS,SS)-enantiomer [33, 421. Glycosyl conjugation. Glycosyl conjugation is of special interest, because of the possibility of producing new cardenolides, as well as converting water-insoluble substances to water-soluble compounds. Two types of glycosyl conjugations are listed in Table 6. One involves the esterification between carboxylic acids and sugar moieties, whereas the other is ether formation (glycosidation) of alcohols and sugar moities. Esterifications of propionic acid derivatives and several sugars, such as glucose, xylose, inositol and sucrose, have been carried out by using root cultures of Panax ginseng and suspension cultures of N. tabacum, Dioscoreophyllum cummensii, Coffea arabica and Coronilla uaria [54-571. On the other hand, when the capacity for glucosylation of plant cell cultures is examined, it is clear that cultured cells have high activity for the glucosylation of phenolic substrates [57-601. Glucosylation of esculetin (110)by cultured cells is highest at the late stationary phase of the cell growth cycle and ca 10% of the added substrate is converted to 6O-/?-D-glucosylesculetin (111)in 24 hr [65]. On the other hand, higher activity is observed with salicyl alcohol (107)
(104)
(102)
*S and I denote suspension
Cathenamine
10-Oxocitronellol
cells and immobilized
cells, respectively.
(lS,2R,SS)-cis-Verbanone (62) lo-Hydroxycitronellol (100) 6,7-dihydro-lo-hydroxygeraniol (101) lo-Hydroxycitronellol (100) 6,7-Dihydro-lo-hydroxycitronellol(lO3) Ajamalicine (105)
Catharanthus roseus
I
S
Catharanthus roseus
of of of of of of
Reduction Reduction Reduction Reduction Reduction Reduction
(lS,SS)-Verbenone (98) lO-Hydroxygeraniol(99)
C=C C=C C=C C=C C=C C=C
of C=C and C=O
(&I)
Reduction
Neomenthol
46 50 46 50 46
S S S S S
53
52
51 19 51 19 33.42 52
50
S
I S I S S S
of C=C of C=C and C=O of C=C
Reduction Reduction Reduction
(lR,4S)-Isodihydrocarvone (80) (lR,2S,4S)-Neoisodihydrocarveol Isomenthone (39)
(%)
(4QCarvone
Ref.
Explant used*
Mentha sp. Nicotiana tabacum Mentha sp. Nicotiana tabacum Nicotiana tabacum Catharanthus roseus
of C=C and C=O
Reduction
(lR,2S,4R)-Neodihydrocarveol(74)
(97)
of C=C
Reduction
(73)
(lR,4R)-Dihydrocarvone
(95)
(4R)Xarvone
Pulegone
Medicago sativa Glycine max Vinca minor Catharanthus roseus Nicotiana tabacum Medicago sativa Nicotiana tabacum Medicaga sativa Nicotiana tabacum
of C=C
Reduction
Cyclohexanone
Cyclohex-Zen-l-one
(82)
Plant species
bonds with plant cell cultures
Type of reaction
of C-C double
Products
5. Reduction
Substrates
Table
L? B u z z E
r% B
acid
*R, S, and I denote
ceils and immobilized
(120)
cells, respectively.
Hesperitin 7-glucoside (124) Digitoxigenin p-o-glucoside (125) Purpureaglucoside (127)
Hesperitin (123) Digitoxigenin (43) Gigitoxin (126)
(115) (117)
suspension
Nicotinic acid-N-fl-D-glucopyranoside Prunin (122)
Nicotinic acid (119) Naringenin (121)
Isorhamnetin Umbelliferone
the use of root cultures,
Salicin (108) Isosalicin (109) 6-0-Glucosylesculetin (111) Quercetin 3-0-glucoside (113) Quercetin 3-0-diglucoside (114) Isorhamnetin 3-0-glucoside (116) 7-0-Glucosylumbelliferone (118)
(107)
Salicyl alcohol
Esculetin (110) Quercetin (112)
0-Glucosylphenol 0-Glucosylcathechol 0-Glucosylresorcinol 0-Glucosylhydroquinone 0-Glucosyl-o-nitrophenol 0-Glucosyl-m-nitrophenol 0-Glucosyl-p-nitrophenol 0-Glucosylpentanitrophenol
Phenol Cathechol Resorcinol Hydroquinone o-Nitrophenol m-Nitrophenol p-Nitrophenol Pentachlorophenol
acid acid
Glycosylation Glycosylation Glycosylation
Glycosylation Glycosylation
Glycosidation Glycosidation Glycosidation Glycosidation Glycosylation
Glycosylation
Glycosylation Glycosylation Glycosylation Glycosylation Glycosylation Glycosylation Glycosylation Glycosylation
Esterification Esterification Glvcosvlation Gl&o&ation Glycosylation
Esterification Esterification Esterification
(2RS)-2-O-(2-phenylpropionyI)-D-Glucoside (RS)-2-Phenylpropionic acid sucrose ester 2-(3-benzoylphenyl)Propionylglucoside 2-[2-(6-methoxy)naphthyl]Propionylglucoside 3-NitropropanyI-D-glucopyranose Geranyl acetate (106) (2R)-2-(4-O-B-D-gIucopyranosyIphenyI)Propionic p-0-fi-D-Glucosylbenzoic acid m-O-B-D-GlucoHylbenzoic acid
acid
(RS)-2-Phenylpropionic
(RS)-2-Phenylpropionic acid 2-(3knzoylphenyl)Propionic acid 2-[2-(6-methoxy)naphthyl]Propionic 3-Nitropropanoic acid Geraniol (SO) (RS)-2-(4-HydroxyphenyI)-propionic p-Hydroxybenzoic acid m-Hydroxybenzoic acid
Esterification Esterification Esterification Esterification Esterification
Type of reaction
with plant cell cultures
(RS)-2-Phenylpropionyl /l-o-glucoside (2RS)-2-O-(2-phenylpropionyI)-D-glucoside (2S)-2-Phenylpropionyl-6-0-8-D-xylosyl-B-D-glucoside myo-Inositol ester of (R)-2-phenylpropionic acid (RS)-2-Phenylpropionyl b-o-glucoside
conjugation
acid
6. Glycosyl
(RS)-2-Phenylpropionic
Table
Products
--_
Substrates
__..-._
ginseng
S S
Cannabis sativa Datura innoxia Perilla frutescens Catharanthus roseus Lithospermum erythrorhizon Petroselinum hortense Citrus paradisi Citrus limon Strophanthus amboensis Digitalis lanata
S S
Gardenia jasminoides Cannabis sativa
S I
S S
S S S S S S S S S
S S S S S
S R
S S
R
Explant used*
Coronilla varia Citrus sp. Panax ginseng Mallotus japonicus Datura innoxia Perilla frutescens Catharanthus roseus Lithospermum erythrorhizon Gardenia jasminoides Gardenia jasminoides Gardenia jasminoides Gardenia jasminoides Gardenia jasminoides Gardenia jasminoides Gardenia jasminoides Glycine max Triticum aestivum Gardenia jasminoides
Coffea arabica Panax ginseng
Nicotiana tahacum Dioscoreophyllum cummensii
Panax
Plant species
70 71, 72
67 68, 69
66 60
65 66
63, 64
61 61 61 61 61 61 61 62
58 59 51 60 60
56 57
55
54
Ref.
Biotransformation
6_
2401
of exogenous substrates
0
0
A
OR
OH
97
96
95
% ;I-;-r A0
Q+9
0
0 CC&
CH,m -0
0
OH H
CH&ti
0
H
4
OH
HOb OH 126: R=H t
100
99
CH,C+l
CH,Cii
t
127: R=Glc
CHpl
CH,Ctl
Y?
CH,Cti
CH,C+i
CHO
102
IO1
103
%~I
&w
CW~
107:R
,=RZ=H
108: R , =H, R 2 =Glc 106
;plJJP 1 lo: R=H Ill:
R=Glc
109: R , =Glc. R z =H
OH
117: R=H 112: R ,=R2=H 113:R
118: R=Glc
,=Glc.R,=H
114: R , =Glc-0.Glc,
R 2 =H
115: R ,=H. R *=CHg OH
116: R I=Glc. RZ=CHB
ticoo. GIG
CL,,, I19
121: R=H
120 OH
123: R=H 124: R=Glc
122: R=Glc
at the exponential phase of the growth cycle and about 70% of the alcohol administered is converted to the corresponding glucoside within four days [63,64]. Interestingly, a major product of glucosylation with the cultured cells of Gardenia jasminoides is salicin (lo@, although other culture strains derived from different plant species predominantly produce isosalicin (109) [63, 641. Furthermore, glycosyl conjugation of (RS)-2-phenylpropionic acid derivatives occur enantioselectively to give the C-2 chiral products [54, 571. Hydrolysis. The ability of cultured cells to hydrolyse the acetoxyl group has been widely investigated (Table 7). Enantioselective hydrolysis is of interest because the transformation is considered to be useful for the optical resolution of racemic acetates. The enantiomers (20 and 24) of or-terpinyl acetate tend to experience enantioselective hydrolysis in the cultured cells of N. tabacum [14,30] (Table 2). Therefore, the ability of the cultured cells for enantioselective hydrolysis was examined in the biotransformation of bornyl acetate (136) isobornyl acetate (139) and isopinocampheyl acetate (140). The enantiomers with the R-configuration at the carbon atom bearing the acetoxyl group are preferentially hydrolysed [70]. Enantioselective hydrolysis was also observed in the biotransformation of (RS)-1-phenylethyl acetate and its derivatives with cultured cells of Spirodela oligorrhiza in which the biotransformations gave only (R)-alcohols [73]. Other examples have been reported for the hydrolyses of the hydroxyimino group and an ether bond (Table 7); carvoximes (142 and 143) and dihydrocarvoxime (144) are hydrolysed to the corresponding ketones by cultured cells of N. tabncum [76] and thebaine (145) is hydrolysed to the corresponding alcohol by cultured cells of Papauer somniferum [77]. Miscellaneous. Many other types of biotransformation have been reported, such as isomerization, epoxidation, degradation and dehydrogenation (Table 8). Salvage synthesis of ajmalicine (150) with tryptamine and secologanin (151) by immobilized cells of Catharanthus roseus is an interesting example of the utilization ofcomplex multienzyme processes in plant cells [SO, 811. Biotransformation with immobilized plant cell cultures During the last 10 years, the techniques for immobilization of plant cells have progressed considerably [87-911. Many studies have been focused on de nova synthesis of useful substances by the immobilized plant cells [90], but only a few examples have been reported on the biotransformation of foreign substrates by use of the immobilized plant cells [27, 44, 49, 51, 53, 711. Immobilization of the plant cells offers some advantages for the biotransforma-
*S denotes
suspension
cells.
(IS,ZR,JS)-Bornyl acetate (137) (lR,ZR,4R)-lsobornyl acetate (139) ( I R,ZK,3R,SS)-Isopinocamphcyl acetate (4R)Karvoxime (142) (4Sj-Carvoxime (143) I I K,4R)-Dihydrocarvowime (144) Thebaine (145)
(140)
acetate (130) acetate (132) acetate (134)
(138) (58) (lR.2R,3R,5S)-IsopinocampheoI (4R)Xarvone (95) (4S)Carvone (96) (I R,4R)-Dihydrocarvone (73) Neopine (146)
(I R,2R,4R)-Isoborneoi
(1&2R,4S)-Borneo1
!rcms-2-Hydroxy-truns-dihydropinanol c,i.s-2-Hydroxy-trans-dihydropinanol rrans-2-Hydroxy-cis-dihydropinanol Borneo1
Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis
Hydrolysis Hydrolysis Hydrolysis Hydrolysis
Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis
of of of of of of of
of of of of
ester ester ester oxime oxime oxime ether
ester ester ester ester
of ester of ester of ester of ester of ester and oxidation of ester and oxidation of ester of ester
Type of reaction _____.-
with plant cell cultures
(131) (133) (135)
(141)
(R)-I-Phenylethanol (R)-1-(l-naphthyl)Ethanol (R)-I-(2-naphthyI)Ethanol (K)-2-Phenylbutanol Benzyl alcohol (3R)-8-Hydroxylinalool (2) Linalooi (3SWHydroxydihydrolinalool (5) (4S)-p-Menth-I-ene-7,8-diol (19) Menthol (129)
(RS)-l-Phenylethyl acetate (RS)-l-(I-naphthyl)EthyI acetate (RS)-I-(2-naphthyl)Ethyl acetate (KS)-2-Phenylbutanyl acetate Benzyl acetate (3R)-Linalyl acetate (3) LinaIyI acetate (3S)-Dihydrolinaiyl acetate (6) (4S)-R-acetoxy-p-menth-l-ene (24) Menthyl acetate (128)
trans-2-Hydroxy-truns-dihydropinanyl cis-2-Hydroxy-trans-dihydropinanyl rrcrns-2-Hydroxy-cis-dihydropinanyl Bornyl acetate (136)
Products
7. Hydrolyses
Substrates
Table ___
Spirodela oligorrhizu Spirodelu oligorrhizu Spirodela oligorrhizu Spirodelu oligorrhiza Spirodela oligorrhizu Nicotianu tubucum Lavandula angustijtiliu Nicotiana tabacum Nicotiana tabacum Spirodela oligorrhiza Epidendrum ochraceum Spirodela oligorrhiza Spirodela oligorrhiza Spirodela oligorrhiza Spirodelrr oligorrhiza LLloandula ungustifolia Nicotianu tabacum ,Vicotiunu tabacum Nicotiana tabucum Nicotianu tabucum Nlcotiana tabacum Nicntiunu tubucum Paprrr:er ,somn[ferum
Plant species
-
73 73 73 73 73 12 45 12 14, 16 74 43 74 74 74 74 45 75 75 75 76 76 76 77
S S S S S S S S s S S S S S s S
Ref. S S S S S S S
Explant used*
Z
$
Z
! > ;
ri
*S denotes suspension cells.
Anhydrovinblastine (162)
84.85 86
Silene alba Catharanthus roseus
S
82 20 27
S
Lonicera tatarica Lonicera morrowii Lonicera korolkowii Lonicera minutijlora Lo&era glaucescens Weigelia japonica Weigelia middendorfana Weigelia j7orida Hydrangea macrophylla Symphoricarpus orbiculatus Coptis japonica Phytophthora infestane Ochrosia elliptica
Dehydrogenation Oxidation Oxidation Oxidation Demethylation Demethylation Dehydration
S
Nicotiana tabacum
(S)-Berberine (157) Lubimin (158) Papaveraldine (159) Papaverine N-oxide 6-Demethylpapaverine (160) 4’-Demethylpapaverine (161) Vinblastine (163)
(10)
(S)-Tetrahydroberberine (1%) Solavetivone (41) Papaverine (48)
Loganin (155)
8-Acetoxy-p-menth-I-ene
20 21 80781 31
79 18
64
78
Ref.
S S S S
Isomerization Isomerization Isomerization and hydroxylation Isomerization and hydroxylation Isomerization and hydroxylation Hydroxylation Isomerization Salvage synthesis Epoxidation Epoxidation Epoxidation Epoxidation Oxidative degradation
Isosalicin (109) D-Glucose I-Acetoxy-p-menth-3-en-S-01 (36) r-I-Acetoxy-p-menth-S(9)-en-t-4-01 (32) r-l-Acetoxy-p-menth-8(9)-en-c-4-ol(147) Rishitin (148) 17/l-H-Digitoxigenin (149) Ajamalicine (105) r-1-Acetoxy-t-4,8-epoxy-p-menthane (151) r-1-Acetoxy-c-4,8_epoxy-p-menthane (152) 8-Acetoxy-r-l,c-2-epoxy-c-4-p-menthane (153) 8-Acetoxy-r-l,c-2-epoxy-t-4-p-menthane (154) Secologanin (150)
Salicin (108) L-Rhamnose I-Acetoxy-p-menth-4(S)-ene (33)
Explant used*
Solanum tuberosum Strophanthus grarus Catharanthus roseus Nicotiana tabacum
Euphorbia characias Nicotiana tabacum Catharanthus roseus Glycine max Gardenia jasminoides Duboisia myoporoides Nicotiana tabacum
Isomerization
Nero1 (70)
Geraniol (50)
Solavetivone (41) Digitoxigenin (43) Tryptamine plus secologanin (150) l-Acetoxy-p-menth-4(S)-ene (33)
Plant species
Type of reaction
Products
Substrates
Table 8. Miscellaneous reactions with plant cell cultures
m 2 3 & 0” 2 s 5’ u
T. SUGAand T.
2404
12X: R=Ac
130: R=Ac
132: u=i\c
129: R=H
131. R=ll
133, R=ll
HIRATA
AcO ._ cb 137: R=Ac
136
138: R=H 134: R=Ac
I60
13.5: R=H
101: R ,=CII1,
139
R, =Il
rNnj”,
140: R=Ac
AHn dH
142
141: R=H
143
K , =H, R 2 =CH,
CO,CH,
3
144 145: R=CH9
I63
146: R=H
(J4
149
ci
OHC
‘,,,
,Qc
151
a ‘,
‘,,
0
$iIY
150
OAC
152
0
OAC
153
H
155
tion of exogenous substrates [87-911: (i) the cells become resistant to shear damage by immobilization, (ii) the immobilized cells can be used repeatedly over a prolonged period, (iii) high concentrations of biomass are possible, thus giving high conversions of substrate, (iv) the method facilitates recovery of the cell mass and products and (v) sequential chemical treatments are possible. CONCLUSIONS
As can be seen from the examples given above, the plant cell cultures possess considerable biochemical ability to transform foreign substrates administered exogenously. The reaction types and stereochemistry in the biotransformation depends on the functional group in the substrates administered and the structural moieties in the vicinity of the functional group. Therefore, the biotransformations by plant cell cultures are considered to serve as important tools for the structural modification of molecules to give compounds possessing useful properties. Fundamental information, such as the reaction types, stereospecificity and enantioselectivity in the biotransformation of exogenous substrates, is essential for the development of the biotechnology for using higher plant cells. Further investigations, especially the development of methods to utilize the multi-reaction processes, will be necessary for the practical applications of biotransformations with plant cell cultures. REFERENCES
156
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Biotransformation
of exogenous substrates
2405
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