Biotechnology and biosynthesis of quinones* Albert J.J. Van den Berg *Based on a lecture given at the 16th LOF Symposium, 27 October 1989, Utrecht, the Netherlands.
C
Introduction Cultured cells of higher plants may serve as sources of various known quinones [1 2]. But new quinones have also been produced in plant cell cultures, which are not biosynthesized or accumulated by intact plants, e.g. 7-methylphyscion (1) [3], 7-methyltorosachrysone (2) [3], echinone (3) [4], and dihydroshikonofuran (4) [5]. Research in the field of plant cell biotechnology aims at the production of useful metabolites (e.g. drugs and food additives) by plant cell cultures on an industrial scale. Successful commercialization has been achieved for the production of shikonin pigments (naphthoquinone derivatives) with plant cell cultures of Lithospermum erythrorhizon [6]. In the latter cultures m a x i m u m yields of 3.5 g/1 shikonin derivatives have been reported, whereas cell cultures of Morinda citrifolia were found to accumulate anthraquinones at levels of 2.5 g/1 [7]. Most cultures, however, still lack the capability to produce secondary metabolites (e.g. anthraquinones) in commercially feasible amounts. Empirical approaches to increase the productivity have been unsuccessful in most cases, and the examples mentioned
above are exceptions. Therefore, it is generally agreed that more fundamental research is needed to deepen our insight into biosynthetic processes. Pathways of biosynthesis have to be completely elucidated by determining each intermediate and by characterizing the enzymes involved in their formation. Nowadays, plant cell biotechnology and biosynthesis cannot be seen apart. Cultured cells r a t h e r t h a n intact plants are used in biosynthesis studies. Plant cell cultures can be grown for short growth cycles under controlled standard conditions. Precursors added to the culture medium are easily incorporated. Cell-free extracts and enzymes can be prepared more easily from cultured cells t h a n from intact plants. Thus, plant cell cultures may also serve as sources of enzymes catalyzing steps in quinone biosynthesis [8]. The next sections deal with quinones derived from acetate/malonate, iso-chorismate/o-succinylbenzoic acid, and p-hydroxybenzoic acid, respectively. A selection of recent literature is presented illustrating different aspects of quinone biosynthesis research.
0 II OCH3 0/C-CH3
Acetate/malonate-derived (polyketide) quinones In the biosynthesis of fatty acids and polyketides, a starter unit - usually acetyl-CoA - is extended with malonyl-CoA units. The biosynthesis of polyketides involves a hypothetical intermediate chain of alternating keto and methylene groups, whereas fatty acid precursors contain methylene groups only, since the malonyl keto group is reduced after each condensation [9 10]. In suspension cultures of Rhamnus purshiana, particularly the emodin content was significantly raised by a 12 h light/dark cycle, whereas levels of physcion were much less affected. However, when the cultures were continuously illuminated anthraquinone production was strongly suppressed [11]. The synthesis of the polyketide alternariol (benzocoumarin derivative) in the fungus Alternaria alternata was also inhibited by irradiation with light, whereas lipid synthesis increased under this condition [12].
HO 0 OH H 3 ~
CH30~ ~
y 0 i
~
O. "CH3
i
[ 1]
7-methylphyscion
echinone
OH
HO OH 0 C H 3 ~ ~ J ~ C
H
3
0
~
7-methyltorosachrysone
[3 ]
CH3 [2 ]
OH dihydroshikonofuran
[4 ]
Keywords Biosynthesis Quinones Technology
Dr. A.J.J. Van den Berg: Division of Pharmacognosy, Utrecht University, P.O. Box 80.082, 3508 TB Utrecht, the Netherlands.
74
Van den Berg AJJ. Biotechnology and biosynthesis of quinones. Pharm Weekbl [Sci] 1991;13(2):74-7.
Abstract Nowadays, it is generally agreed that intensive investigation of biosynthetic pathways is a prerequisite for attaining industrial-scale production of secondary metabolites (e.g. quinones) by plant cell cultures. Literature data are presented to illustrate different aspects of today's quinone biosynthesis research. Accepted 20 November 1990.
Pharmaceutisch Weekblad Scientific edition
13(2) 1991
The reductive steps in fatty acid synthesis require NADPH, and it has been suggested, that the NADPH/NADP ratio in fungi might be a factor regulating the incorporation of malonyl-CoA units into either fatty acids or polyketides [13]. Recent studies revealed, however, that the enzymatic NADPH-generating capacity in darkgrown mycelia of Alternaria alternata was not reduced in comparison with light-grown mycelia. Preliminary results indicate that enzymes directly involved in the biosynthesis of alternariol are not expressed in cultures grown with light [14]. A direct relationship between polyketide anthraquinone and fatty acid biosynthesis was demonstrated in mycelia of Cortinarius orichalceus by addition of cerulenin (5) [15]. Cerulenin inhibits the condensing enzyme (3-oxo-acyl-acyl carrier protein synthetase) catalyzing the con-
O
O
cerulenin
[5 ]
densation reactions in fatty acid biosynthesis [9 10]. Alkylation of the condensing enzyme, accompanied by the SN 2 type of epoxide-opening reaction, results in loss of enzyme activity [16]. By addition of cerulenin a decrease in fatty acid content of the mycelia, and decreased incorporation rates of 1-[14C] acetate into mycelial triacylglycerols were observed. Incorporation of 1-[14C] acetate into anthraquinones was also drastically diminished. Therefore, it can be concluded that condensation of activated acetate/ malonate units is a common initial step of both fatty acid and anthraquinone biosynthesis. Alternatively, two condensation reactions occur in two enzyme systems which are very similar and therefore both inhibitable by cerulenin [15]. In the biosynthesis of polyketide anthraquinones one acetyl-CoA is extended with seven
HO
OH
O
HO
O
OH
duction of anthraquinones and anthrones in Rhamnus purshiana cell cultures; acetate, however, exerted an inhibitory effect under similar conditions. It was suggested that cultivated Rhamnus purshiana cells or maybe even intact plants are not capable of converting acetyl-CoA into malonyl-CoA [18]. Callus cultures of Aloe saponaria, grown in the dark, accumulated besides known tetrahydroanthracene glucosides, also the glucoside of a new tetrahydroanthracene derivative, aloesaponol IV (6). Upon continuous illumination, however, tetrahydroanthracene glucosides originally present i n d a r k grown cultures, disappeared and were replaced by glucosides of anthraquinones (e.g. chrysophanol) [19]. In cell-free extracts from Aloe arborescens, it was demonstrated that the enzyme responsible for the C-glucosylation at C-10 of aloe emodinanthrone leading to aloin A and B (7), was specific for UDP-glucose as glucosyl donor and aloe-emodinanthrone as C-glucosyl acceptor [20]. The aloin actively biosynthesized by Aloe arborescens is aloin B, which is partially transformed into the diastereomer aloin A [21].
Is~>chorismate/r acid derived quinones In the biosynthesis of iso-chorismate-derived anthraquinones and naphthoquinones, o-succinylbenzoic acid (8) arises from iso-chorismic acid (9) and ~-ketoglutarate in the presence of thiamine pyrophosphate [22]. Cyclization of o-succinylbenzoic acid via its co-enzyme A derivative results in the formation of 1,4-dihydroxy-2-naphthoic acid (DHNA) (10). In the genera Galium, Morinda and Cinchona (Rubiaceae) anthraquinone biosynthesis proceeds via DHNA prenylated at C-3. In the genera Catalpa, Tabebuia (Bignoniaceae), and Streptocarpus (Gesneriaceae) quinones are biosynthesized by preny]ation at C-2 of 2-carboxy-2,3-dihydro-l,4naphthoquinone (11). Simultaneous administration of (2R)-l-[14C]catalponone (12) and (2S)-
HO
O
OH COOH COOH
CH3/
~
~
~
CH20H
CH20H H
aloesaponol IV
[6 ]
aloin A
aloin B
0 [7 ]
malonyl-CoA units. Acetyl-CoA is produced H O O C y OH through the glycolytic pathway, or is synthesized by plants from acetate and co-enzyme A. In most O~~/COOH organisms, malonyl-CoA is formed by carboxylation of acetyl-CoA, but in plants malonyl-CoA can also be synthesized from malonate and co-enzyme A. Addition of the basic precursor acetate to suspension cultures of Rheum palmatum resulted in an increased anthraquinone formation [17]. Exogenous malonate stimulated the pro- iso-chorismic acid [9 ] 13(2) 1991
Pharmaceutisch Weekblad Scientific edition
OSB
[8]
OH ~ / C O O H
OH DHNA
[ 10] 75
O
O
O
O
[ 11 ]
(2R)- catalponone
[ 12 ]
8-[3H]catalponone to the wood of Catalpa ovata showed that the biosynthesis of catalponone congeners proceeds through the 2R enantiomer [23]. From suspension cultures of Streptocarpus dunnii several naphthoquinones, prenylated at C-3 [e.g. dunnione (13)] were isolated [24]. These unusually prenylated metabolites arise via osuccinylbenzoic acid, DHNA, lawsone (14) and O-prenylated lawsone (lawsone 2-prenyl ether) (15). By the Claisen-type rearrangement of the latter compound, the key intermediate 2-hydroxy-3-(l',l'-dimethylallyl)-l,4-naphthoquinone (16) is formed [26]. In tissue cultures of Cinchona ledgeriana, anthraquinones were found to occur in the genus Cinchona for the first time [26]. In healthy bark of Cinchona ledgeriana, anthraquinones could not be detected. However, examination of bark samples of the same tree, infected with Phytophthora cinnamomi (a fungus pathogenic to Cinchona species), revealed the presence of anthraquinones [27]. Addition of sterilized mycelia of Phytophthora cinnamomi to Cinchona ledgeriana suspension cultures resulted in a ninefold increase of the anthraquinone content [28]. Photoautotrophic suspension cultures of Morinda lucida accumulated chlorophyll and lipoquinones (phylloquinone, plastoquinone, ubiquinone), whereas anthraquinone glycosides could not be detected. When the photosynthesizing plant cells were transferred to the dark, a strong increase in anthraquinone production coincided with a rapid disappearance of lipoquinones and chlorophyll, provided that saccharose was present. Quinones produced in heterotrophic cultures (i.e. anthraquinones) as well as photoautotrophic cultures [i.e. phylloquinone 0
dunnione
0
[13] 0
[161
76
lawsone
0
[ 14 ]
lawsone 2-prenyl ether 0
phylloquinone (vitamin kl)
[ 17 ]
(17)] are derived from iso-chorismate (9) via osuccinylbenzoic acid (8) and DHNA (10), the latter compound representing the branch point for both pathways leading to (secondary) anthraquinones or (primary) phylloquinone [29]. In suspension cultures of Morinda citrifolia anthraquinone synthesis was inhibited by L-tryptophan. The site of inhibition is probably located further down in the specific anthraquinone pathway, since addition of shikimic acid or o-succinylbenzoic acid did not restore pigment formation [30]. This study also demonstrates the possibility that certain secondary pathways (e.g. leading to anthraquinones) could be repressed by accumulation of primary metabolites, such as L-tryptophan.
Quinones derived from/>hydroxybenzoic acid In several Boraginaceous plants, quinones are derived from p-hydroxybenzoic acid (p-HBA) (18), which arises from shikimic acid via phenylCOOH
COOH
OH
OH
_.p- HBA
[18]
m- geranyl - p - HBA
OH
O
OH
0
D
[19]
m
OH
R1 =OH
i [21 ]
OH geranylquinol
[20]
shikonin alkannin RI= H
, R2 = H , R2=OH
alanine and cinnamic acid. Prenylation of phydroxybenzoic acid at C-3 results in m-geranylp-HBA (19). The latter compound is converted into geranylquinol (geranylhydroquinone) (20), which is the key intermediate in the biosynthesis of the stereoisomers shikonin and alkannin (21) [31]. Shikonin is produced by suspension cultures of Lithospermum erythrorhizon, whereas both [ 15 ] shikonin and alkannin are found to occur in Echium lycopsis callus [31 32]. Benzoquinones accumulating in Lithospermum cultures [i.e. echinofuran B, dihydroshikonofuran (4)] and Echium callus (i.e. echinofuran) may also be considered as metabolites of geranylquinol [4 5 33]. The formation of naphthoquinones in Lithospermum erythrorhizon as well as in Echium lycopsis cultures is almost completely inhibited by white or blue light. Recently, the influence of light on the activity of p-HBA-geranyltransferase, pHBA-O-glucosyltransferase, p-HBA-O-gluco-
Pharmaceutisch Weekblad Scientific edition
13(2) 1991
sidase a n d p h e n y l a l a n i n e a m m o n i a l y a s e was inv e s t i g a t e d i n s u s p e n s i o n c u l t u r e s of Lithosperm u m erythrorhizon i n r e l a t i o n to t h e f o r m a t i o n of s h i k o n i n a n d p-HBA-O-glucoside [34]. U p o n i l l u m i n a t i o n , p-HBA-O-glucoside w a s produced a n d s h i k o n i n w a s not. I l l u m i n a t i o n s t r o n g l y i n h i b i t e d p - H B A - g e r a n y l t r a n s f e r a s e activity, whereas p-HBA-O-glucosyltransferase activity was s t i m u l a t e d ; p-HBA-O-glucosidase a n d p h e n y l a l a n i n e a m m o n i a l y a s e were o n l y s l i g h t l y affected b y light. T h e r a t i o of a c t i v i t i e s ofp-HBAgeranyltransferase and p-HBA-O-glucosyltransferase m i g h t be one of t h e p r i n c i p l e s d e t e r m i n i n g w h e t h e r p - h y d r o x y b e n z o i c acid is c o n v e r t e d i n t o s h i k o n i n or i n t o its glucoside [34].
17 Dusek J, Sicha J, Duskova J. Influence on the production of anthracene derivatives in the tissue culture of Rheum palmatum by a modification of the cultivating medium, or potential precursors. Cesk Farm 1989;38:210-3. 18 Van den Berg AJJ, Radema MH, Labadie RP. Influence of acetate and malonate on the production of 1,8dihydroxyanthracene derivatives in suspension cultures of Rhamnus purshiana. Planta 1988;174:417-21. 19 Yagi A, Shoyama Y, Nishioka I. Formation of tetrahydroanthracene glucosides by callus tissue of Aloe saponaria. Phytochemistry 1983;22:1483-4. 20 Grfin M, Franz G. In vitro biosynthesis of the Cglycosidic bond in aloin. Planta 1981;152:562-4. 21 Gr(in M, Franz G. Untersuchungen zur Biosynthese der Aloine in Aloe arborescens Mill. Arch Pharm 1982; 315:231-41. 22 Simantiras M, Leistner E. Formation of o-succinylbenzoic acid from iso-chorismic acid in protein extracts from anthraquinone-producing plant cell suspension References cultures. Phytochemistry 1989;28:1381-2. 1 Thomson RH. Naturally occurring quinones. III. 23 Inouye H, Ueda S, Inoue K, Shiobara Y. (2R)Catalponone, a biosynthetic intermediate for prenylLondon: Chapman and Hall, 1987. naphthoquinone congeners of the wood of Catalpa 2 Van den Berg AJJ, Labadie RP. Quinones. In: Dey PM, ovata. Phytochemistry 1981;20:1707-10. Harborne JB, eds. Methods in plant biochemistry. 24 Inoue K, Ueda S, Nayeshiro H, Inouye H. Quinones Vol. 1. London: Academic Press, 1989:451-91. from Streptocarpus dunnii. Phytochemistry 1983;22: 3 Kitanaka S, Igarashi H, Takido M. Formation of pig737-41. ments by the tissue culture of Cassia occidentalis. 25 Inoue K, Ueda S, Nayeshiro H, Moritome N, Inouye H. Chem Pharm Bull 1985;33:971-4. Biosynthesis of naphthoquinones and anthraquinones 4 Inouye H, Matsumura H, Kawasaki M, Inoue K, in Streptocarpus dunnii cell cultures. Phytochemistry Tsukada M, Tabata M. Two quinones from callus cul1984;23:313-8. tures of Echium lycopsis. Phytochemistry 1981;20: 26 Mulder-Krieger Th, Verpoorte R, De Water A, Van 1701-5. Gessel M, Van Oeveren BCJA, Baerheim Svendsen A. 5 Yazaki K, Fukui H, Tabata M. Dihydroshikonofuran, Identification of the alkaloids and anthraquinones in an unusual metabolite of quinone biosynthesis in Cinchona ledgeriana callus cultures. Planta Med 1982; Lithospermum cell cultures. Chem Pharm Bull 1987; 46:19-24. 35:898-901. 6 Curtin ME. Harvesting profitable products from plant 27 Wijnsma R, Van Weerden IN, Verpoorte R, et al. Anthraquinones in Cinchona ledgeriana bark infected tissue culture. Biotechnology 1983;1:649-57. with Phytophthora cinnamomi. Planta Med 1986;52: 7 StSckigt J, Schfibel H. Naturstoffe aus pflanzlichen 211-2. Zellkulturen. Dtsch Apoth Ztg 1989;129:1187-92. 8 Khouri HE, Ibrahim RK. Purification and some 28 Wijnsma R, Go JTKA, Van Weerden IN, Harkes PAA, Verpoorte R, Baerheim Svendsen A. Anthraquinones properties of five anthraquinone-specific glucosylas phytoalexins in cell and tissue cultures of Cinchona transferases from Cinchona succirubra cell suspension spec. Plant Cell Rep 1985;4:241-4. culture. Phytochemistry 1987;26:2531-5. 9 Mann J. Secondary metabolism. Oxford: Oxford Uni- 29 Igbavboa U, Sieweke HJ, Leistner E, RSwer I, H(isemann W, Barz W. Alternative formation of versity Press, 1978:1-77. anthraquinones and lipoquinones in heterotrophic and 10 Vickery ML, Vickery B. Secondary plant metabolism. photoautotrophic cell suspension cultures of Morinda London: MacMillan Press, 1981:56-111. lucida Benth. Planta 1985;166:537-44. 11 Van den Berg AJJ, Radema MH, Labadie RP. Effects of light on anthraquinone production in Rhamnus 30 E1-Shagi H, Shulte U, Zenk MH. Specific inhibition of anthraquinone formation by amino compounds in Mopurshiana suspension cultures. Phytochemistry 1988; rinda cell cultures. Naturwissenschaften 1984;71:267. 27:415-7. 12 Hiiggblom P. Light effects on polyketide and lipid 31 Inouye H, Ueda S, Inoue K, Matsumura H. Biosynthesis of shikonin in callus cultures of Lithospermum metabolism in Alternaria alternata. Int Bot Congr erythrorhizon. Phytochemistry 1979;18:1301-8. Abstr 1987;17:174. 13 Mosbach K, B~ivertoft I. A comparative study on the 32 Fukui H, Tsukada M, Mizukami H, Tabata M. Formation of stereoisomeric mixtures of naphthoquinone biosynthesis of palmitic and orseUinic acids in Penicilderivatives in Echium lycopsis callus cultures. Phytolium baarnense. Acta Chem Scand 1971;25:1931-6. chemistry 1983;22:453-6. 14 Orvehed M, H~iggblom P, SSderh~ill K. Activity of NADPH-generatingpathways in relation to polyketide 33 Fukui H, Yoshikawa N, Tabata M. Induction of benzoquinone formation by activated carbon in Lithospersynthesis in the fungus Alternaria alternata. Exp mum erythrorhizon cell suspension cultures. PhytoMycol 1987;11:187-96. chemistry 1984;23:301-5. 15 Gstraunthaler GJA. The effect of cerulenin on fatty acid and anthraquinone biosynthesis in vegetative my- 34 Heide L, Nishioka N, Fukui H, Tabata M. Enzymatic regulation of shikonin biosynthesis in Lithospermum celia of Cortinarius orichalceus Fr. Biochim Biophys erythrorhizon cell cultures. Phytochemistry 1989;28: Acta 1983;750:424-7. 1873-7. 16 Omura S. Philosophy of new drug discovery. Microbiol Rev 1986;50:259-79.
13(2) 1991
P h a r m a c e u t i s c h W e e k b l a d Scientific e d i t i o n
77
C
Introduction Cultured cells of higher plants may serve as sources of various known quinones [1 2]. But new quinones have also been produced in plant cell cultures, which are not biosynthesized or accumulated by intact plants, e.g. 7-methylphyscion (1) [3], 7-methyltorosachrysone (2) [3], echinone (3) [4], and dihydroshikonofuran (4) [5]. Research in the field of plant cell biotechnology aims at the production of useful metabolites (e.g. drugs and food additives) by plant cell cultures on an industrial scale. Successful commercialization has been achieved for the production of shikonin pigments (naphthoquinone derivatives) with plant cell cultures of Lithospermum erythrorhizon [6]. In the latter cultures m a x i m u m yields of 3.5 g/1 shikonin derivatives have been reported, whereas cell cultures of Morinda citrifolia were found to accumulate anthraquinones at levels of 2.5 g/1 [7]. Most cultures, however, still lack the capability to produce secondary metabolites (e.g. anthraquinones) in commercially feasible amounts. Empirical approaches to increase the productivity have been unsuccessful in most cases, and the examples mentioned
above are exceptions. Therefore, it is generally agreed that more fundamental research is needed to deepen our insight into biosynthetic processes. Pathways of biosynthesis have to be completely elucidated by determining each intermediate and by characterizing the enzymes involved in their formation. Nowadays, plant cell biotechnology and biosynthesis cannot be seen apart. Cultured cells r a t h e r t h a n intact plants are used in biosynthesis studies. Plant cell cultures can be grown for short growth cycles under controlled standard conditions. Precursors added to the culture medium are easily incorporated. Cell-free extracts and enzymes can be prepared more easily from cultured cells t h a n from intact plants. Thus, plant cell cultures may also serve as sources of enzymes catalyzing steps in quinone biosynthesis [8]. The next sections deal with quinones derived from acetate/malonate, iso-chorismate/o-succinylbenzoic acid, and p-hydroxybenzoic acid, respectively. A selection of recent literature is presented illustrating different aspects of quinone biosynthesis research.
0 II OCH3 0/C-CH3
Acetate/malonate-derived (polyketide) quinones In the biosynthesis of fatty acids and polyketides, a starter unit - usually acetyl-CoA - is extended with malonyl-CoA units. The biosynthesis of polyketides involves a hypothetical intermediate chain of alternating keto and methylene groups, whereas fatty acid precursors contain methylene groups only, since the malonyl keto group is reduced after each condensation [9 10]. In suspension cultures of Rhamnus purshiana, particularly the emodin content was significantly raised by a 12 h light/dark cycle, whereas levels of physcion were much less affected. However, when the cultures were continuously illuminated anthraquinone production was strongly suppressed [11]. The synthesis of the polyketide alternariol (benzocoumarin derivative) in the fungus Alternaria alternata was also inhibited by irradiation with light, whereas lipid synthesis increased under this condition [12].
HO 0 OH H 3 ~
CH30~ ~
y 0 i
~
O. "CH3
i
[ 1]
7-methylphyscion
echinone
OH
HO OH 0 C H 3 ~ ~ J ~ C
H
3
0
~
7-methyltorosachrysone
[3 ]
CH3 [2 ]
OH dihydroshikonofuran
[4 ]
Keywords Biosynthesis Quinones Technology
Dr. A.J.J. Van den Berg: Division of Pharmacognosy, Utrecht University, P.O. Box 80.082, 3508 TB Utrecht, the Netherlands.
74
Van den Berg AJJ. Biotechnology and biosynthesis of quinones. Pharm Weekbl [Sci] 1991;13(2):74-7.
Abstract Nowadays, it is generally agreed that intensive investigation of biosynthetic pathways is a prerequisite for attaining industrial-scale production of secondary metabolites (e.g. quinones) by plant cell cultures. Literature data are presented to illustrate different aspects of today's quinone biosynthesis research. Accepted 20 November 1990.
Pharmaceutisch Weekblad Scientific edition
13(2) 1991
The reductive steps in fatty acid synthesis require NADPH, and it has been suggested, that the NADPH/NADP ratio in fungi might be a factor regulating the incorporation of malonyl-CoA units into either fatty acids or polyketides [13]. Recent studies revealed, however, that the enzymatic NADPH-generating capacity in darkgrown mycelia of Alternaria alternata was not reduced in comparison with light-grown mycelia. Preliminary results indicate that enzymes directly involved in the biosynthesis of alternariol are not expressed in cultures grown with light [14]. A direct relationship between polyketide anthraquinone and fatty acid biosynthesis was demonstrated in mycelia of Cortinarius orichalceus by addition of cerulenin (5) [15]. Cerulenin inhibits the condensing enzyme (3-oxo-acyl-acyl carrier protein synthetase) catalyzing the con-
O
O
cerulenin
[5 ]
densation reactions in fatty acid biosynthesis [9 10]. Alkylation of the condensing enzyme, accompanied by the SN 2 type of epoxide-opening reaction, results in loss of enzyme activity [16]. By addition of cerulenin a decrease in fatty acid content of the mycelia, and decreased incorporation rates of 1-[14C] acetate into mycelial triacylglycerols were observed. Incorporation of 1-[14C] acetate into anthraquinones was also drastically diminished. Therefore, it can be concluded that condensation of activated acetate/ malonate units is a common initial step of both fatty acid and anthraquinone biosynthesis. Alternatively, two condensation reactions occur in two enzyme systems which are very similar and therefore both inhibitable by cerulenin [15]. In the biosynthesis of polyketide anthraquinones one acetyl-CoA is extended with seven
HO
OH
O
HO
O
OH
duction of anthraquinones and anthrones in Rhamnus purshiana cell cultures; acetate, however, exerted an inhibitory effect under similar conditions. It was suggested that cultivated Rhamnus purshiana cells or maybe even intact plants are not capable of converting acetyl-CoA into malonyl-CoA [18]. Callus cultures of Aloe saponaria, grown in the dark, accumulated besides known tetrahydroanthracene glucosides, also the glucoside of a new tetrahydroanthracene derivative, aloesaponol IV (6). Upon continuous illumination, however, tetrahydroanthracene glucosides originally present i n d a r k grown cultures, disappeared and were replaced by glucosides of anthraquinones (e.g. chrysophanol) [19]. In cell-free extracts from Aloe arborescens, it was demonstrated that the enzyme responsible for the C-glucosylation at C-10 of aloe emodinanthrone leading to aloin A and B (7), was specific for UDP-glucose as glucosyl donor and aloe-emodinanthrone as C-glucosyl acceptor [20]. The aloin actively biosynthesized by Aloe arborescens is aloin B, which is partially transformed into the diastereomer aloin A [21].
Is~>chorismate/r acid derived quinones In the biosynthesis of iso-chorismate-derived anthraquinones and naphthoquinones, o-succinylbenzoic acid (8) arises from iso-chorismic acid (9) and ~-ketoglutarate in the presence of thiamine pyrophosphate [22]. Cyclization of o-succinylbenzoic acid via its co-enzyme A derivative results in the formation of 1,4-dihydroxy-2-naphthoic acid (DHNA) (10). In the genera Galium, Morinda and Cinchona (Rubiaceae) anthraquinone biosynthesis proceeds via DHNA prenylated at C-3. In the genera Catalpa, Tabebuia (Bignoniaceae), and Streptocarpus (Gesneriaceae) quinones are biosynthesized by preny]ation at C-2 of 2-carboxy-2,3-dihydro-l,4naphthoquinone (11). Simultaneous administration of (2R)-l-[14C]catalponone (12) and (2S)-
HO
O
OH COOH COOH
CH3/
~
~
~
CH20H
CH20H H
aloesaponol IV
[6 ]
aloin A
aloin B
0 [7 ]
malonyl-CoA units. Acetyl-CoA is produced H O O C y OH through the glycolytic pathway, or is synthesized by plants from acetate and co-enzyme A. In most O~~/COOH organisms, malonyl-CoA is formed by carboxylation of acetyl-CoA, but in plants malonyl-CoA can also be synthesized from malonate and co-enzyme A. Addition of the basic precursor acetate to suspension cultures of Rheum palmatum resulted in an increased anthraquinone formation [17]. Exogenous malonate stimulated the pro- iso-chorismic acid [9 ] 13(2) 1991
Pharmaceutisch Weekblad Scientific edition
OSB
[8]
OH ~ / C O O H
OH DHNA
[ 10] 75
O
O
O
O
[ 11 ]
(2R)- catalponone
[ 12 ]
8-[3H]catalponone to the wood of Catalpa ovata showed that the biosynthesis of catalponone congeners proceeds through the 2R enantiomer [23]. From suspension cultures of Streptocarpus dunnii several naphthoquinones, prenylated at C-3 [e.g. dunnione (13)] were isolated [24]. These unusually prenylated metabolites arise via osuccinylbenzoic acid, DHNA, lawsone (14) and O-prenylated lawsone (lawsone 2-prenyl ether) (15). By the Claisen-type rearrangement of the latter compound, the key intermediate 2-hydroxy-3-(l',l'-dimethylallyl)-l,4-naphthoquinone (16) is formed [26]. In tissue cultures of Cinchona ledgeriana, anthraquinones were found to occur in the genus Cinchona for the first time [26]. In healthy bark of Cinchona ledgeriana, anthraquinones could not be detected. However, examination of bark samples of the same tree, infected with Phytophthora cinnamomi (a fungus pathogenic to Cinchona species), revealed the presence of anthraquinones [27]. Addition of sterilized mycelia of Phytophthora cinnamomi to Cinchona ledgeriana suspension cultures resulted in a ninefold increase of the anthraquinone content [28]. Photoautotrophic suspension cultures of Morinda lucida accumulated chlorophyll and lipoquinones (phylloquinone, plastoquinone, ubiquinone), whereas anthraquinone glycosides could not be detected. When the photosynthesizing plant cells were transferred to the dark, a strong increase in anthraquinone production coincided with a rapid disappearance of lipoquinones and chlorophyll, provided that saccharose was present. Quinones produced in heterotrophic cultures (i.e. anthraquinones) as well as photoautotrophic cultures [i.e. phylloquinone 0
dunnione
0
[13] 0
[161
76
lawsone
0
[ 14 ]
lawsone 2-prenyl ether 0
phylloquinone (vitamin kl)
[ 17 ]
(17)] are derived from iso-chorismate (9) via osuccinylbenzoic acid (8) and DHNA (10), the latter compound representing the branch point for both pathways leading to (secondary) anthraquinones or (primary) phylloquinone [29]. In suspension cultures of Morinda citrifolia anthraquinone synthesis was inhibited by L-tryptophan. The site of inhibition is probably located further down in the specific anthraquinone pathway, since addition of shikimic acid or o-succinylbenzoic acid did not restore pigment formation [30]. This study also demonstrates the possibility that certain secondary pathways (e.g. leading to anthraquinones) could be repressed by accumulation of primary metabolites, such as L-tryptophan.
Quinones derived from/>hydroxybenzoic acid In several Boraginaceous plants, quinones are derived from p-hydroxybenzoic acid (p-HBA) (18), which arises from shikimic acid via phenylCOOH
COOH
OH
OH
_.p- HBA
[18]
m- geranyl - p - HBA
OH
O
OH
0
D
[19]
m
OH
R1 =OH
i [21 ]
OH geranylquinol
[20]
shikonin alkannin RI= H
, R2 = H , R2=OH
alanine and cinnamic acid. Prenylation of phydroxybenzoic acid at C-3 results in m-geranylp-HBA (19). The latter compound is converted into geranylquinol (geranylhydroquinone) (20), which is the key intermediate in the biosynthesis of the stereoisomers shikonin and alkannin (21) [31]. Shikonin is produced by suspension cultures of Lithospermum erythrorhizon, whereas both [ 15 ] shikonin and alkannin are found to occur in Echium lycopsis callus [31 32]. Benzoquinones accumulating in Lithospermum cultures [i.e. echinofuran B, dihydroshikonofuran (4)] and Echium callus (i.e. echinofuran) may also be considered as metabolites of geranylquinol [4 5 33]. The formation of naphthoquinones in Lithospermum erythrorhizon as well as in Echium lycopsis cultures is almost completely inhibited by white or blue light. Recently, the influence of light on the activity of p-HBA-geranyltransferase, pHBA-O-glucosyltransferase, p-HBA-O-gluco-
Pharmaceutisch Weekblad Scientific edition
13(2) 1991
sidase a n d p h e n y l a l a n i n e a m m o n i a l y a s e was inv e s t i g a t e d i n s u s p e n s i o n c u l t u r e s of Lithosperm u m erythrorhizon i n r e l a t i o n to t h e f o r m a t i o n of s h i k o n i n a n d p-HBA-O-glucoside [34]. U p o n i l l u m i n a t i o n , p-HBA-O-glucoside w a s produced a n d s h i k o n i n w a s not. I l l u m i n a t i o n s t r o n g l y i n h i b i t e d p - H B A - g e r a n y l t r a n s f e r a s e activity, whereas p-HBA-O-glucosyltransferase activity was s t i m u l a t e d ; p-HBA-O-glucosidase a n d p h e n y l a l a n i n e a m m o n i a l y a s e were o n l y s l i g h t l y affected b y light. T h e r a t i o of a c t i v i t i e s ofp-HBAgeranyltransferase and p-HBA-O-glucosyltransferase m i g h t be one of t h e p r i n c i p l e s d e t e r m i n i n g w h e t h e r p - h y d r o x y b e n z o i c acid is c o n v e r t e d i n t o s h i k o n i n or i n t o its glucoside [34].
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