Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 17 © 1995 Elsevier Science B.V. All rights reserved. 451

Chemistry and Biosynthesis of Natural Diels-Alder Type Adducts from Moraceous Plants Taro Nomura, Yoshio Hano and Shinichi Ueda

Introducliion

[4 + 2]Cycloaddition, well known as the Diels-Alder reaction^

is a widely-used reaction in the synthesis of organic compounds

including natural products. On the other hand, many possible

biosynthetic Diels-Alder constructions from natural sources have

been reported [1 - 25]. Such constructions in natural are mostly

stereo- and regiospecific, suggesting the pivotal step of the

cycloaddition reaction in the biosynthesis to be enzymatic.

However, the evidence for a biological Diels-Alder reaction is

rare.

Mulberry tree is a rich source of intermolecular Diels-Alder

type adducts comprising two molecules of isoprenylphenols [26].

This article describes the chemistry of the Diels-Alder type

adducts from moraceous plants and the biosynthesis of the adducts

in Worus alha. cell cultures.

1. Mulberry Diels-Alder type adducts

Mulberry tree, a moraceous plant of the genus Morus, has been

widely cultivated in Japan and China in order to serve its leaves

for sillcworms. On the other hand, the root bar]c of the tree has

been used as a Chinese crude drug so called "Sang-Bai-Pi", for an

antiphlogistic, a diuretic, and an expectorant. A few pharma­

cological studies on the mulberry tree demonstrated a hypotensive

effect of the extract in rodents. Considering these reports, it

was suggested the active principle to be a mixture of many phenolic

components. Kuwanon G (1) is the first isolation of the active

substance exhibiting the hypotensive action from the Japanese Morus root baric.

Kuwanon G (1) [27], molecular formula C40H36OH, was assumed to

be a biogenetic intermolecular Diels-Alder type construction formed

from dehydrolcuwanon C (2) and a chalcone derivative (3) based on

the NMR spectroscopic studies (Figure 1). An evidence of the

452

HO

Figure 1

250**C, Z 0^-

Figure 2

regiochemistry for 1 was obtained by the following results: Diels-

Alder reaction of trans-chalcone and 2-methyl-3-phenyl-l,3-buta-

diene gave two cycloproducts, one of which is the all-trans type

adduct (4) in relative configuration among three substituents on

the methylcyclohexene ring and another is the cis-trans type adduct

(5) in relative configuration (Figure 2). The X-ray analyses of

these cycloproducts revealed that the regioselectivity in the [4 +

2] reaction was coincided with that estimated in the case of

kuwanon G (1). A pyrolysis of kuwanon G octamethyl ether (la)

afforded tra/3s-2,2',4^4'-methoxychalcone (6) and dehydro-kuwanon C

tetramethyl ether (2a) (Figure 3). Subsequently, the two

fragmentation products 6 and 2a gave, when heated in toluene at 160

®C for 61 h in a sealed tube, two [4 + 2]cycloadducts (Figure 4).

One of the adducts was identified with (±)-la and the other was a

453

HjCO

H3CO

HjCO

OCH3

OCH,

HjCO^^^v^^OCH,

H3CO

l a CH,0 O

6

Figure 3 Pyrolysis of kuwanon G octamethyl ether (la)

HsCO

H3CO

OCH9

HsCO^^^^Ss^OCHj

HjCO.

H3CO,

H3CO.

OCHs

OCH3

Figure 4 Diets-Aider reaction of the pyrolysis products 2a and 6

454

12: 3"-Hp 13: 3".Ha

OH O .'•vvi-°

X

10: 3".Ha 11: 3"-Hp

14: 3".Ha 15: 3"-Hp

F i g u r e 5 Typical Diels-Alder type adducts from Moms root bark

cis-trans type cycloadduct (7a) as was observed in the case of the

synthesis of the model compounds described above. The structure of

kuwanon G (1) has thus been established as depicted in Figure 1

[28]. Kuwanon G (1) is optically active ([a]D - 534°) and

considered to be formed through an enzymatic Diels-Alder reaction

of a chalcone derivative as a dienophile and dehydrokuwanon C as a

diene (Figure 1).

About forty kinds of optically active Diels-Alder type adducts,

which are presumably formed from a dehydroprenylphenol and a

chalcone derivative, have been isolated from Japanese Morns root

bark and Chinese crude drug ''Sang-Bai-Pi" [26]. The mulberry

Diels-Alder type adducts may be divided into the following four

types on the basis of the phenol nuclei; a) adducts of a chalcone

and a dehydroprenylflavonoid {e.g. (1), kuwanon H (8) [29] and

sanggenon C (9) [30]}, b) adducts of a chalcone and a

dehydroprenylchalcone {e.g. kuwanons I (10) [31] and J (11) [32]},

c) adducts of a chalcone and a dehydroprenyl-2-arylbenzofuran {e.g.

mulberrofurans C (12) [33] and J (13) [34]}, and d) adducts of a

chalcone and a dehydroprenylstilbenes {e.g. kuwanons X (14) [34]

and Y (15) [35]} (Figure 5). It is very interesting that a pair of

455

isomers, such as 10 and 11 and others, coexist in the Morus root

bark as in the case of the Diels-Alder reaction of a trans-chalcone

and a butadiene derivative giving rise to a pair of isomers, all-

trans and cis-trans type adducts.

On the other hand, callus tissues of Morus alba L. induced from

the seedlings or the leaves were cultured under specified

conditions and subjected to selection over nine years, giving rise

to cell lines producing characteristic Diels-Alder type adducts at

high levels [32]. Eight Diels-Alder type adducts, kuwanons J (11)

[32, 36], Q (16) [36], R (17) [36], V (18) [36], mulberrofurans E

(19) [37], T (20) [38], chalcomoracin (21) [32, 39], and kuwanol E

(22) [38], have been isolated from the callus tissues along with

morachalcones A (23) [36, 39], B (24) [36], and moracin C (25)

[36, 40] (Figure 6). Assuming that compounds (23), (24), and (25)

are a dienophile or an equivalent of a diene, kuwanon J (11) and

kuwanon V (18) are composed of two molecules of 23 and 24,

respectively. While, both kuwanons Q (16) and R (17) are formed

from 23 and 24. Similarly, chalcomoracin (21) is composed of 23

and 25 and mulberrofuran E (19) is formed from 24 and 25. It is

noteworthy that all combinations of these monomers, 23, 24, and

25, could be isolated from the M. alba callus tissues.

OH

11: RisR2=OH 16: Ri sOH Ri^H 17: R,sH R2= OH 18: Rt^RjssH

19 ! RirrRjsH/ 2 0 : R i = - - l ^ R2=OH

21: RisH R2-OH

22

F i g u r e 6 Phenolic components of Morus alba cell cultures

456

2. Other optically active Diels-Alder type adducts from

moraceous plants

The compounds structurally similar to the mulberry Diels-Alder

type adduct have also been isolated from the other species of the

plants of the family Moraceae. Brosimones A (26) [41], B (27) [14]

and D (28) [14] have been isolated from Brosimopsis oblongifolia, a

Brazilian moraceous plant (Figure 7). They are expected to be

formed through the saune way as the mulberry Diels-Alder type

adducts. Of these compounds, compound (26) is a unique adduct

which may be formed through an intramolecular [4+2] cycloaddition

reaction of the isoprenyl portion and the a, p-double bond of the

chalcone slceleton of compound (28) (Figure 8). Artonins C (29)

[42], D (30) [42] and I (31) [43] isolated from Artocarpus heterophyllus, an Indonesian moraceous plant, can also be regarded

OH

HO

OH 26 27 28

F i g u r e 7 Optically active Diels-Alder type adducts from Brosimopsis oblongifolia

HO-%^ OH

28 26

F i g u r e 8 A hypothesis of the formation of 26 from 28 through Diels-Alder reaction

457

30:R=

F i g u r e 9 Diels-Aider type adducts from Indonesian and Argentine moraceous plants

ny I MO MO

OH

HO^...^s^Oj,0^...;s^CHO TO' HO^...'fe^O

37

F i g u r e 10 Ketalized compounds from moraceous plants

458

as typical Diels-Alder type adducts of a chalcone derivative and a

dehydroprenylphenol (Figure 9). Mulberrofuran U (32) [44] has been

isolated from an Argentine moraceous plant Morns insignis (Figure

9).

Ketalized Diels-Alder type adducts^ mulberrofurans F (33) [45]^

G (34) [45], K (35) [46] and Icuwanol A (36) [47], have been

isolated from the Moras root barJc (Figure 10). Compounds (33) and

(34) are presumably formed through an intramolecular )cetalization

reaction of chalcomoracin (21) and mulberrofuran C (12),

respectively. Indeed, compounds (33) and (34) could be derived

stereospecifically from the original adducts 21 and 12,

respectively, under acidic conditions (Figure 11) [45]. Soroceal

(37) [20], soroceins A (38) [20] and B (39) [20], structurally

similar to the above described Jcetalized compounds, have been

isolated from Sorocea bonplandii, a Brazilian moraceous plant (Fig­

ure 10). These compounds are also considered to be modifications

of original adduct through an intramolecular Icetalization reaction.

H^

-H2O

12: R=H y

21:R= . ^ ^

34: R=H y

33:R=:

F i g u r e 11 The formation of the ketalized compounds from the original adducts under acidic conditions

40 D*

F i g u r e 12 Colouration mechanism of40 under acidic conditions

459

Mulberrofuran I (40) [48] has been isolated as a red pigment

from the root bark of the mulberry tree. The compound (40) colours

red under acidic conditions* The ^H-NMR spectrum of 40 in the

presence of deutelized trifluoroacetic acid revealed that only the

proton at C-2" position was exchangeable for deutelium. This

result suggested that the colouration mechanism of 40 is depicted

as in Figure 12 [26]. Compound (40) is considered to be one of the

modifications derived from mulberrofuran C (12). As shown in

Figure 13, the two ways of oxidative cyclizations of a hemilcetal

intermediate derived from 12 may afford Icetalized compound (34) and

the red pigment (40), respectively. Aromatized compound albanol B

(41) [49], presumably derived from 34 through a dehydrogenation,

has been isolated from Indian Morns alba L. However, this compound

is not optically active, indicating that the sole chriral center at

C-8" position is racemic [50]. Albanol B pentamethyl ether (41a)

formed from mulberrofuran G pentamethyl ether (34a) through the

dehydrogenation reaction with DDQ is optically active ([ctJo + US'*)

[50]. This fact indicated albanol B (41) to be an artefact formed

during the isolation procedures. As a result, it was concluded

that compound (41) is derived from mulberrofuran I (40), a red

pigment, through an auto-oxidation in the presence of an acid or

Lewis acid-li]ce catalyst such as Si02 (Figure 14) [50]. Aromatized

40 H2O, -H2

Figure 13 The formations of 34 and 40 through two ways (a and b) of oxidative cyclizations of a hemilcetal intermediate derived from 12

460

OCHg

H3CO OCH3

DDQ

-2H,

H3CO

34a

OCH3

41a (optically active)

41: R = H (racemate)

42: R = O H (racemate)

HO

-H2O2

HO^^^s^v^OH

OH

40: R=H 43 : R=OH

Figure 14

compound mulberrofuran P (42) [51] has also been isolated from

Japanese Morus root bark as a blue pigment. Considering the result

that aromatized compound such as 41 is an artefact from a red

pigment, compound (42) seems to be an artefact from the

corresponding red pigment. Indeed, the corresponding compound

mulberrofuran S (43) [50] has been isolated from the same source as

a red pigment (Figure 14). Many other substances regarded as

modifications biosynthetically derived from the original Diels-

Alder type adduct such as mulberrofuran C (12) have been isolated

from Japanese Morus root bark [26].

461

3. Absolute Configuration of Mulberry Diels-Alder Type

Adducts

As described above^ the stereochemistries of the mulberry Diels-

Alder type adducts could be divided into the following two groups;

one is an all-trans in relative configuration and the other is a

cis-^trans configuration. All-trans type adduct may correspond to

an exo-addition product in the Diels-Alder reaction of a chalcone

and a dehydroprenylphenol, whereas a cis-trans type adduct

corresponds to an endo-addition product in the reaction.

Absolute stereochemistries of the mulberry Diels-Alder type

adducts were confirmed by the circular dichroism (CD) spectroscopic

evidence and by X-ray analysis [26, 52, 53].

12a: R= < 13a: R= <

Ae

+ 2 0 -

+ 1 0 -

0"

- 1 0 -

-20-^

1 / '* \ / / *

r^ i \

' 1 t

r r—

1

—1 r

• 1 •? 1 — • ±z j

Ae

+ 5 -

0

- 5 -

\ : 12a

k,-/ : 13a

I , , , , 1 250 300 350 400 nm

F i g u r e 15 CD spectra of 12,13,12a, and 13a

250 300 350 400 nm

462

Both CD spectra of a pair of isomers, mulberrofurans C (12) and

J ( 1 3 ) , showed a large magnitude of Ae values more than those of

the other adducts [53]. On the other hand, the CD spectra of the

reduced compounds (12a) and (13a), which are formed from 12 and

13, respectively, by LiAlH4 reduction, are mirror images of each

other in the Jt - Ji* region due to the 2-arylbenzofuran chromophore

(Figure 15) [53]. This indicates that the stereochemistries of 12

and 13 at the C-3" chiral center bearing the 2-arylbenzofuran

chromophore are antipodal to each other. As descrived above,

mulberrofuran G (34) could be derived from 12 under acidic

conditions. Monobromomulberrofuran G pentamethyl ether (34b)

prepared from 34a by the treatment with NBS was converted to an

aromatized compound (34c) through dehydrogenation by DDQ (Figure

16 ) . The X-ray crystallographic analysis of 34c revealed that the

absolute configuration of the sole chiral center at C-8" is R [53].

OCHa

H3CO OCHa

OCH3

H3CO

NBS 34b DDQ R=Br — ^ g y;. 0CH3

OCH3

34a: R=H 34c (8"./?)

Figure 16

OH

12:3"S,4"/?,5"5

OH

13 : 3"/?, 4"/?, 5"S

Figure 17 Absolute stereochemistries of 12 and 13

463

i)OH

O H ii)(CH3)2S02

OH O 44

(3"1?, 4"i?, 5"S)

H3CO,

HaCO

iy)P'BtBzC\

45: R=H 45a: R^CHa

Figure 18

46: RisrRjsH 46a: Ri^H Rjsp-BrBz 46b: Ri=R2sp-BrBz

As the relative configuration of the four chiral centers in 34 has

been confirmed by the X-ray analysis of its pentamethyl ether (34a)

[49], the absolute configuration of 34 was specified as 3"S, 4"K,

5"S, B"R [53]. Hence, the absolute stereochemistry of 12 was also

determined to be 3"S, 4",JR, 5"S [53]. Since the stereochemistry of

13 at the C-3" position is antipodal to that of 12, the absolute

configuration of 13 was expressed as 3"R, A"R, 5"5 [53]. The

absolute stereochemistries of a pair of isomers, all-trans type and

ciS'-trans type adducts, has thus been determined as shown in Figure

17.

On the other hand, the absolute configuration of kuwanon L (44)

[54], an all-trans type adduct, was confirmed by the following

result: Alkali degradation followed by methylation of 44 gave its

pentcunethyl ether (45a) by way of 45. Treatment of 45a with OSO4

gave a cis-diol product (46), which was converted to the mono-p-

bromobenzoate (46a) and the di-p-bromobenzoate (46b) (Figure 18).

The CD spectrum of 46b exhibited a positive Cotton effect owing to

a typical exciton coupling between the two p-bromozenzoyl

chromophores [52]. Thus the absolute configuration of the three

chiral centers of the methylcyclohexene ring of 44 has been

specified as 3"R, 4t"R, 5"S [52]. This stereochemistry was in

accordance with that of an all-trans type adduct, mulberrofuran J

(13).

Optical rotations ([a]p) of typical mulberry Diels-Alder type

adducts are summarized in Table 1. The all-trans type adducts

exhibit negative optical rotation, while the cis-trans type adducts

exhibit positive values. Considering the absolute stereochemi-

464

Table 1 Optical rotation values ([a]^)

all-trans type adducts cis-trans type adducts

mulberrofuran J (13) -341®

kuwanon X (14) -322*

kuwanon I (10) -454*

kuwanon G (1) -534*

kuwanon H (8) -536*

kuwanon L (44) -277*

mulberrofuran C (12) +153**

kuwanon Y (15) +172**

kuwanon J (11) +85**

kuwanon Q (16) +160**

kuwanon R (17) + 56**

kuwanon V (18) +145**

k s A s ^ OH OH O

chalcone

OH

* 7--M

dehydroprenyl-phenol

jto-addition

L C -> 0

e/i^o-addition

HO

T 11 4" 13" OH 0 p

M

aW'trans 3"/?, 4"i?, 5"5

HO

OH 0 1 M

cis-trans 3"5,4"/?,5"5

F i g u r e 19 Absolute stereochemistries of the mulberry Diels-Alder type adducts

stries of a pair of isomers, mulberrofurans C (12) and J (13), the

chirality at the C-3" position influences the sign of the rotation.

Namely, the absolute configuration of the all-tra/is type adducts is

the same as that of 13, while that of the cis-trans type adducts is

the same as that of 12. The absolute stereochemistries of three

chiral centers in the methylcyclohexene ring of the all-trans type

465

adducts have thus been determined as Z^'R, 4"!?, 5"S, whereas those of the cis-trans type adducts as 3"S, ^"R, 5"S (Figure 19) [53].

4. Biosyniihesis of the mulberry Dlels-Alder type adducts

4.1. Biosynthesis of kuwanon J (11) and chalcomoracin (21)

As described in section 2, some cell strains of Morus alba callus tissues induced from the seedlings or the leaves have a high

productivity of the mulberry Diels-Alder type adducts. The yields

of major adducts chalcomoracin (21) and kuwanon J (11) by the cell

strains are about 100 - 1000 times more than those of the intact

plant [32]. The biosynthesis of the mulberry Diels-Alder type

adducts has been studied with the aid of the excellent cell

strains.

Administration of [1-^^C]-^ [2-l3c]-, or [1^2-l3c2]acetates to

the Morus alba cell cultures revealed the early stages of

biosynthesis of 11 and 21^ in which both adducts are originated

from two molecules of cinnamoylpolylcetide skeletons (Figure 20)

[55]. The chalcone skeleton of both adducts 11 and 21 has been

considered to be formed through deoxygenation at C-5 of the

cinnamoylpolyketide skeleton [56], followed by the Claisen-type

condensation at C-4 and C-9 of the skeleton (Figure 21). On the

other hand, the 2-arylbenzofuran skeleton of 21 is formed through

an aldol-type condensation at C-3 and C-8 of the cinnamoyl­

polyketide skeleton, followed by decarboxylation (Figure 21). The

other interesting result was obtained with respect to the

biosynthesis of the isoprenyl units of 21. Administration of i c-

lal elled acetate to the cell cultures resulted in the highly ^^C-

enriched aromatic carl)ons of 21 (about 18 % ) , while two isoprenyl

units of 21 were lal elled to a lesser extent (about 0.4 % ) . In

addition, on the basis of 13^.13^ spin-spin coupling, the labelling

of [ 2-l^c]acetate takes place in the contiguous carbons at the

starter acetate unit with regard to the mevalonate biosynthesis

(Figure 20b). On the contrary, [l-^^C]acetate was not incorporated

into the isoprenyl units of 21 [55], unlike the incorporation into

the cinnamoylpolyketide skeletons. These findings suggest the

participation of tricarboxylic acid (TCA) cycle to the biosynthesis

of the isoprenyl units of 21. In the experiment of [2-i3c]acetate,

the contiguous ^^C atoms can be derived from the two methyl groups of the intact acetates administered by way of at least two passages

through the TCA cycle, while in the case of [l-^^C]acetate the i C

atom was removed as carbon dioxide. Accordingly, the acetate

466

HO

HO

^A^OH

OH I

?a

11

(a)

• • CH3COSC0A

21

(b)

F i g u r e 2 0 ^^C-Labelling patterns of 11 and 21 from "C-labelied acetates

• • CHjCOGNa C2X3 "*• C o A S ^

O

SCoA x-'^^v^OH „ SCoA ,X5^^'

OH

CoAS

chalcone skeleton

OH

2-arylbenzofuran skeleton

F i g u r e 2 1 Biosyntheses of the chalcone and 2-arylbenzofuran skeletons in Moras alba cell cultures

467

CH3COSC0A

HOOC'V''°°"

/

"^COOH

•12 HOOC^^O

•^^COOH • / 2

• ^ ^COOH

1: HO ^COOH T C A cycle

• / 2 HOOC^^OH

ji/2

• /2

H O O C ^

O^COOH

/

«\1 • / 2

HOOC

CO2

•/2 HOOC

COOH • / 2 r COOH

• / 2

• / 2 HOOC^^O

•^^COOH • / 2

h • / 2 CO2

m/2 HOOC^ ^OH Tr •/2

• / 2 ^ HOOC^^O

•/2CH3

h • / 2 CO2

• / 2 1/2 CH3COSC0A

Figure 22

incorporated into the isoprenyl units of 21 was not the intact

acetate administered, but [1,2-^^02]acetate reorganized from the

methyl group of the intact acetate through the TCA cycle (Figure

22) [55]. This hypothesis was reinforced by the administration

experiment with (2-1^0]acetate in a pulsed manner (three times,

every 12 h) [57]. In this case, however, the ^^c-labelling at the

isoprenyl units of the resulting 21 was observed not only at the

starter acetate carbons, but also at the third acetate carbons at

C-7" and C-25", which showed i^c-^^c coupling with the adjoining

carbons at C-1" and C-23", respectively (Figure 23). In spite of

the laclc of ^^C-laljelling at the second acetate, the third acetate

carbon was apparently enriched here with ^^C. This fact can be

explained in terms of the isomerization of the two 3,3-

dimethylallyl groups via 3-methyl-l,3-butadienyl groups (Figure

23). Namely, the i^C-labelling at the third acetate carbon can be

attributable to the transfer of the i^C-labelling from the cis-

methyl carbon to the trans-methyl carbon involving an isomerization

of a cisoid and a transoid of a diene (Figure 23). This finding

468

7- ^ 2"

HO Y / \ \J

HO

f i — i T ^ Ho jii^ JL JL.<^

3^^^^,X*s.jS!^

X^o°" JIL,OH

1 I T 22" 23-T 21" 1^ OH •

24"

21

-K

,-Hr

11^1

V ^ '

^

F i g u r e 2 3 ^^C-Labelling pattern of 21 by pulse-administration experiment with [2."CJacetete

gave a confirmative evidence on the formation of the diene

structure at the isoprenyl portion for the Diels-Alder type

cycloaddition reaction. It is interesting that the isomerization

takes place not only at the isoprenyl unit participating in the

Diels-Alder type cycloaddition reaction, but also at the other

isoprenyl unit, which did not participate in the reaction (Figure

23). Thus the administration experiment with i^c.^^belled acetate

to the M. alba cell cultures revealed that the Diels-Alder type

adducts 11 and 21 are presumably biosynthesized through the [4 -«-

2]cycloaddition reaction between two molecules each of

cinnamoylpolyketide-derived skeleton and mevalonate. Final

confirmation of the biosynthesis of the mulberry Diels-Alder type

adducts was performed by the administration experiment with O-

methylchalcone derivatives, as modified precursors, to the M. alba

cell cultures [58]. 0-Methylated chalcones or 0-methylated Diels-

Alder type adducts have not been detected in the cell cultures.

Administration of 0-methylated chalcone (47) to the cell cultures

yielded the metabolites (48), (49), (50), (51), and (52) (Figure

24) [58]. The conversion of the precursory chalcone (47) into (48)

indicated that isoprenylation takes place after aromatization of

the chalcone skeleton derived through the Claisen-type condensation

of the cinnamoylpolyketide. The metabolites (49), (50), (51), and

(52) revealed that the precursory chalcone (47) was incorporated

469

OCHa

YV^o^^^c

50 : R1-CH3 R2=H R3SOH 5 1 : Ri=CHj R2=R3=H 5 2 : Ri=:R2=CH3 RjsOH

HaCO

Figure 24

H3C0

HaCq

HaCO

Figure 25

470

intact into the Diels-Alder type adducts, as a diene or a

dienophile. An analogous experiment with synthesized (48), one of

the metabolites from (47) in the cell cultures, yielded the same

Diels-Alder type metabolites (49), (50), (51), and (52), as in the

case of the experiment employing (47) (Figure 24) [58].

Subsequently, administration of tri-0-methylated chalcone (53) to

the cell cultures afforded the Diels-Alder type metabolite (54)

(Figure 25) [58], in which the precursory chalcone (53) was

incorporated as a dienophile. These results strongly indicate that

dehydrogenation at the isoprenyl portion of (48) followed by a [4 +

2]cycloaddition reaction with the a, P-double bond of another

molecule of isoprenylchalcone leads to the formation of the Diels-

Alder type metabolites. Furthermore, the Diels-Alder type metabo­

lites from the precursory chalcones (47), (48), and (53) are all

optically active, having the same stereochemistries as those of

kuwanon J (11) and chalcomoracin (21). This fact revealed the [4 +

2]cycloaddition step to be enzymatic. Administration of 0-

methylated precursory chalcone to the M. alba cell cultures has

thus demonstrated that optically active mulberry Diels-Alder type

adducts such as 11 and 21 are biosynthesized through an enzymatic

intermolecular [4 + 2]cycloaddition reaction.

Another interesting result concerning the enzyme system of the

M, alba cell cultures was obtained at the structure determination

experiment of artonin I (31) [43], which has been isolated from an

Indonesian moraceous plant Artocarpus heterophyllus, as described

in section 2. Artonin I (31) was regarded as a typical Diels-Alder

type adduct formed from a chalcone and a flavone, artocarpesin (55)

" N ^

HO y h=\

HO

HO^^jTs^O^

JLoH OH '

artonin I (31)

[ ^ " " " 1 k J 1 OH 1

F i g u r e 2 6 Bioconversion of 55 to 31 using Morus alba cell cultures

471

which co-occurs with 31. Administration of 55 into the M» alba

cell cultures yielded the metabolite (31) in high yield. The

structure of artonin I has thus been established as formula 31.

This finding also corroborates the biosynthetic pathway

simultaneously (Figure 26) [43]. This is the first example of

determining the structure of a target substance with the aid of an

enzyme system of cell cultures of other plant.

4.2. Shlkimate pathway and Isoprenoid biosynthesis in

Morus alba cell cultures

As described above, administration experiment with i^C-labelled

acetates revealed that both the mulberry chalcone and 2-

arylbenzofuran skeletons originate from cinnamoylpolyketide. In

order to confirm the biosynthesis of the cinnamoyl moiety derived

from shikimate via aromatic amino acid, phenylalanine or tyrosine,

further experiments administering phenylalanine and tyrosine to the

M. alba cell cultures were carried out [59]. In the administration

experiment with L-[3-i3C]phenylalanine to the cell cultures, a high

i^C-enrichment (about 33 %) from the amino acid was observed at the

11 21

Figure 2 7 '^C-Labeling from L-[3-"C]phenylalanine and L-[3->^C]tyrosine

472

C-3 and C-5" positions of chalcomoracin (21) as well as at the C-P

and C-5" positions of kuwanon J (11) (Figure 27). This finding

indicates that both the cinnamoyl moieties in the chalcone and 2-

arylbenzofuran moieties originate from L-phenylalanine. An

analogous experiment with L-[ 3-^^C]tyrosine also showed the same

^^C-labelling pattern as in the case of L-[3-^3c]phenylalanine

(Figure 27). The ^^C-incorporation was about 18 %. Both L-

phenylalanine and L-tyrosine, intermediates on the shikimate

pathway, are thus precursory to the mulberry chalcone and 2-

arylbenzofuran skeletons. Transformation of L-phenylalanine into

L-tyrosine by direct hydroxylation has been well established in

mammal cells [60]. An enzyme system isolated from spinach leaves

also caused the transformation [61]. In higher plants, however,

direct conversion of L-phenylalanine into L-tyrosine has been rare

[62]. It has also been reported that the overlapping of the

secondary metabolites from L-phenylalanine with those of L-tyrosine

is restricted, because of the presence of two independent metabolic

pathways for these aromatic amino acids. L-Phenylalanine has been

known to be converted to traus-cinnamate, which, in turn, is

subjected to 4-hydroxylation resulting in the formation of p-

coumarate through the action of cinnamate 4-hydroxylase in higher

plant [64]. In Morus alba cell cultures, it was suggested that both

O

r^COOH

X 6 phenyl pyruvate

prephenate

\ u COOH

NH2

r^COOH

6

OH

4-OH-phenylpyruvate

L-phenylalanine

NH2

I^COOH

Q • OH

L-tyrosine

COOH

cinnamate

[O]

COOH

OH /7-coumarate

F i g u r e 2 8 The shikimate pathway leading to p-coumarate from L-phenylalanine and L-tyrosine

473

L-phenylalanine and L-tyrosine are converted into p-coumarate,

which are conclusively incorporated into the chalcone and 2-

arylbenzofuran skeletons (Figure 28) [59].

On the other hand, in the experiment with ^^c.^abelled acetates,

the acetate incorporated into the isoprenyl units of chalcomoracin

(21) was reconstructed acetate from the methyl group of exogenous

acetate through the TCA cycle, as described in section 4.I. On the

basis of this novel finding, further studies with respect to the

biosynthesis of the isoprenyl unit of 21 were carried out by

administering dI-[ 2-1 0 Jmevalonate or L-t2-^^C]leucine, the

candidates for isoprenoid precursor, to the M» alba cell cultures

[57], In both experiments, no ^^C-incorporation was observed at the

isoprenyl carbons of 21. In the case of L-[2-^3c]leucine, however,

polyketide-derived aromatic carbons were enriched with ^ C in the

same labelling pattern as in the case of [l- c] acetate with

different degrees of ^^C-enrichment. This result indicated that L-

[2-1^0]leucine was catabolized to [l-^^cjacetyl CoA via 3-hydroxy-3-

^—2^1 • COOH

K I ? ^ \ HjCpHO

H,0 . HMG-CoA

CO2

CH3COSC0A / TCA \

'^ I cycle ) CH3COSC0A CH3

HO.

OH 0

rr°" OH

HO

li 1 V rS ^ ^ O H

Figure 29 Fate of L-leucine in Morus alba cell cultures

474

methylglutaryl CoA (HMG CoA) followed by the participation of the

polyketidG biosynthesis (Figure 29) [57]. Such a fate for L-

leucine has been reported in the case of sesquiterpene paniculide

biosynthesis in Andrographis paniculata tissue cultures [64].

Morus alba cell cultures also yield P-sitosterol (56) [32],

which is a good target for the examination of isoprenoid

biosynthesis from isoprenyl precursors in the cell cultures. The

^^C-labelling of 56 in the experiments with [l-^^C]-, [2'^^C]-, or

[1,2-^^02]acetates was in accordance with Ruziclca's biogenetic rule

[57], as was verified in the case of 56 in Rabdosia japonica tissue

cultures (Figure 30) [65]. Accordingly, the exogenous acetates

were incorporated intact into the isoprenyl units of 56. dl-[2-

^3c]Mevalonate was also incorporated into the expected positions of

56 (Figure 31), in spite of non-incorporation of this precursor

into the isoprenyl unit of 21 [57]. This result indicated that

non-incorporation of the precursor into the isoprenyl units of 21

was not due to the impermeability across the cell walls. Thus the

incorporation manner of the precursors, including acetate, into the

isoprenyl units of 21 is different from that observed in 56. It is

most lilcely that at least two independent isoprenoid biosynthetic

pathways, that for sterols and that for isoprenylphenols, operate

in the Morus alba cell cultures.

Figure 30 "C-LabcIIIng pattern of 56 from [1-* C]- (•), [2- C]- (•),

and [l,2-'X2]acetates {i

475

F i g u r e 3 1 ^^C-Labelling pattern of 56 fk*om [2-^^C]mevalonate

5. Conclusion

Optically active Diels-Alder type adducts isolated from Morus sp. were found to be biosynthesized through an enzymatic

intermolecular [4 + 2]cycloaddition reaction between an isoprenyl

portion of an isoprenylphenol as the diene and an a, ^-double bond

of a chalcone as the dienophile. This is the first example

demonstrating a biological intermolecular Diels-Alder reaction.

Although (-)-flavoskylin [2] produced by Penicillium sp., ageliferins [17] isolated from an Okinawan marine sponge, and the

sesquiterpene-monoterpene adducts [18] isolated from Artemisia AerJba-alba have also been considered to be formed via biosynthetic

intermolecular Diels-Alder reaction, the biological reaction has

not yet been demonstrated. On the other hand, a few examples of

biological intramolecular Diels-Alder reactions have been

unambiguously found. Betaenone B and solanapyrone A, phytotoxins

from Phoma tetae and Altenaria solani, respectively, were found to

be formed through intramolecular Diels-Alder reaction [7, 12, 15].

In the biosynthetic studies of these phytotoxins, specific

cytochrome P450 inhibitors have been used effectively for the

identification of the putative precursors. (+)-Brevianamides A and

B, mycotoxins from Penicillium brevicompactum, were also found to

be biosynthesized through the pathway involving intramolecular

Diels-Alder reaction, on the basis of experiments administering

supposed precursors labelled with H or ^ C [66].

It is noteworthy that enzymes catalysing a Diels-Alder reaction

actually occur in biological systems.

476

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