Linalyl and bornyl disaccharide glycosides from Gardenia jasminoides flowers

Pergamon

LINALYL

0031-9422(94)EO232-H

AND BORNYL

Phymckmwy. Vol 37, No. 2 pp. 457459. 1994 Copytight Q 1994 tbck sdeacr Lid m0ced in Gmat Btitun. AlI ng~btrruemd 0031-94zm4 17.00+0.00

DISACCHARIDE GLYCOSIDES JASMZNOZDES FLOWERS*

FROM

GARDENIA

NAOHARU WATANABE,t RYUTA NAKAJIMA. SHUZO WATANABE, JAE-HAK MOON,$ JUNJI INAGAKI, KANZO SAKATA. AKIHITO

YAGI and KAZUO INA

Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya. Shizuoka 422, Japan; SUnited Graduate School of Agricultural Sciences, Gifu University (Shizuoka University), 836 Ohya, Shizuoka 422, Japan (Received 24 January

1994)

Key Word Index-Gardenia

jasminoides; linalyl6-0-a-t-arabinopyranosyl-/_?-D-glucopyranoside; bornyl 6-O-p-D-xylopyranosyl-/?-D-glucopyranoside; bornyl /?-primeveroside; monoterpcne alcohol glycoside; aroma precursor; flower fragrance.

Abstract-(R)-Linalyl 6-0-a-t_-arabinopyranosyl-/?-D-glucopyranoside and bomyl6-O-/?-D-xylopyranosyl+D-glucopyranoside were isolated as aroma precursors of linalool and bomeol from flower buds of Gardenia jasminoides, guided by enzymatic hydrolysis followed by GC and GC-MS analyses.

In the course of our studies focused on flower fragrance formation [I], we have confirmed its formation from glycosidic precursors by endogenous enzymatic hydrolysis just before flower opening. Linalyl 6-0-malonyl-B-Dglucopyranoside was isolated as one of the aroma precursors of linalool from flower buds of Jasminum sambac [2]. We have also reported that linalool and bomeol were liberated from the precursor solution prepared from flower buds by treatment with crude enzyme preparation obtained from flowers of Gardenia josminoides [l]. The highest activity of the crude enzyme preparation was at the anthesis stage of G. jasminoides [I]. The aroma precursors of linalool and bomeol were suggested to be their glycosides rather than their glucosides. We have now attempted to isolate the aroma precursors of linalool and borneol from G. josminoides flower buds. RESULTS AND DI!XXJSSlON

Isolation of linalool and borneol glycosides was guided by detection of linalool and bomeol by GC or GC-MS after enzymic hydrolysis of glycosidic fractions with a crude enzyme prepared from Gardenia flowers and naringinase (from Penicillium decumbens). Flower buds just before anthesis were extracted and chromatographed on an Amberlite XAD-2 column (H,O, EtOAc and MeOH) and a LH-20 column (50% MeOH), successively. Both linalool and bomed were liberated from the glycosidic

*Part

3 in the

series 'Studieson Aroma

of Flowers’.

For Part 2 see ref. 123.

tAuthor

to whom correspondence

Formation

Mechanism

should be addressed. 457

fraction D-3 by treatment with the crude enzyme preparation and naringinase. As naringinase showed higher activity in a shorter incubation period than the crude enzyme, naringinase was used for further purification steps. Repeated HPLC (four times) on ODS columns (20-80% MeOH) gave the aroma precursors, linalyl and bornyl glycosides (1 and 2). The molecular formula C,,H,,O,o of 1 was determined from its high resolution FAB-mass spectrum m/r 471.2211 (+0.5 mmu for C,,H,,O,,Na) together with other spectral data. In the r3C NMR spectrum (Table l), 1 showed I1 carbon signals including two anomeric carbons (699.6 and 104.6), besides those of a linalyl moiety. In the ‘H NMR spectrum, two anomeric protons at 64.32(H-l’,d,J=7.7 Hz)and4.30(H-l”,d,J=6.2 Hz) were observed. The 13C and ‘HNMR signals are in agreement with those of (S)-linalyl 6-O-a-t-arabinopyranosyl-B-D-glucopyranoside (3) isolated from Rudus idoeus by Pabst et al. [3], except the r3C NMR signal due to

N. WATANABE

458 Table

1. “CNMR

spectral (CD,OD.

data (6) of 100 MHz)

compounds

et al.

oses. Since t_-xylose does not occur naturally,

1-5

configuration

of the disaccharide

the absolute

moiety of 2 must be D.

Because no significant difference in the chemical shifts of

C

1 2 3 4 5 6 7 8 9 10 I’ 2 3’ 4 5’ 6

115.2 144.4 81.5 41.7 23.7 125.8 132.1 25.9 17.8 23.5 99.3 75.0 78. I 72.3 76.3 69.3” 104.8 71.7’ 74.1 69.2” 66.2

I ,, 2” 3”

4” 5”

4 3 (50 MHz) (C,D,N)

2

1

50.1 84.6 36.8 46.4 29.1 27.7 49.2 19.6 20.3 41.1 103.2 75.2 78.2’ 71.7 77.6’ 69.7 IO55 74.9 77.2 71.2 66.9

5

C-6’ between 2 and 5 was observed, the D-xylosyl moiety is linked to C-6’. On the basis of the above evidence, the structure

of 2 is concluded

115.7 144.4 81.5 42.7 23.6

49.9 86.1 38.2 45.4 28.6

_ _

125.7 132.1 25.6

27.2 47.6 18.9

--_

19.9 14.1 106.3 75.5 78.6 72.3 77.0 68.4 110.1 83.4 78.9 86.3 62.7

-

from G.jusminoides

-

and

17.7 23.3 99.6 75.2 78. I 12.3’ 76.4 69.4” 104.6 71.78 74.1 69.2” 66.3

-

to

be bornyl

xylopyranosyl-P-D-glucopyranoside,

a new

~-O-/?-Dbornyl

di-

saccharide glycoside. Several glucosides of monoterpene and aromatic

alcohols have been isolated from Rowers

C7.83, but no disaccharide

glycosides of these volatile

alcohols have been reported. Compounds

1 and 2 liberated

linalool

and borneol,

respectively, by the action of a crude enzyme preparation

102.7 75.3 78.2 71.6 78.2 70.0 105.6 74.8 76.9 71.2 66.9

1

flowers or naringinase. Compounds

2 are suggested to be transformed

into

volatile

linalool and borne01 during flower opening by the action of an endogenous enzyme system. EXPERIMENTAL

Preparution jasminoides

of crude

enzyme.

From

5OOg fresh G.

flowers (stage 5) [ 13, 50 g of Me&O

was obtained

by conventional

powder was solubilized

methods.

powder

The

Me&O

in Pi buffer (0.1 M, pH 7) for 3 hr

and centrifuged to yield the crude enzyme prepn (750 ml)

111. Enzymaric

‘.bChemical shifts may be interchangeable within the same column. Compound 3: Linalyl 6-0-a-L-arabinopyranosyl-/?-o-©ranoside [3]. Compound 4: bomyl6-O-a-L-arabinofuranosyl/3-D-glucopyranoside [S]. Compound 5 vomifolyl 6-0-fi-D-xylopyranosyl-/&II-glucopyranoside [63; signals due 10 sugar moiety were listed.

hydrolysis.

sample equivalent [l]

and crude

A mixt. of an aroma

to lo-30

enzyme

precursor

g of fr. flower buds (stage 3) (3.75 ml) in 10 ml of Pi buffer

prepn

(0.1 M, pH 7) was kept at 30” for 72 hr in the presence of

_ ‘). When naringinase (I mg. Sigma,

Na azide (500 pg ml from Penicillium

decumbens) was used, a shorter incuba-

tion (in 0.5 M citrate

buffer, pH

5) period

(17 hr) was

enough. To the reaction mixt. was added 0.5 ml of a Et,0 C-4 (Table from

1). Thechirality

1 was determined

cyclodex mined

moiety

pair of diastereomeric

chemical

shift difference

was previously

2, [ali

Compound

(+2.5

spectrum

with 21 carbon

molecular

formula of C21HJ60,0.

The presence of two anomeric = 7.7 Hz), 4.45 (d. 5=8.1 disaccharide

spec-

signals at

moiety

must

proton signals E64.51 (d. J that the

be in a pyranoside

form.

different from those of bornyl 6-0-a-

t_-arabinofuranosyl-/?-D-glucopyranoside glucopyranoside lysis, including

‘H-‘H

I). ‘H NMR

(D,O)

spin-decoupling experiments tranc-1,2-diaxial

Hz) in H-l’-H-5’

that the disaccharide

[S],

and

relationship

and H-I”-H-5”,

fused silica capillary

i.d.) and

GC-MS

(EI

70 eV)

analyses [ 11. Isolation

of linalyl

and bornyl

glycosides

(1 and 2).

was guided by the above enzymatic

hydrolysis

analyses. Flower

morning

from 5 to 20 June 1991. at Fujieda,

Japan) were extracted ice-cooling

with 80%

buds just

immediately

MeOH

Shizuoka,

(8 I x 3) under

after plucking.

Combined

tracts were coned in uacuo. A portion (equivalent an Amberlite and

XAD-2

successively

MeOH).

The EtOAc

column

eluted

(equilibrated

with

pentane,

and MeOH

ex-

to 690 g

subjected to Sephadex LH-20

CC (50% MeOH)

vol.). Fr.

(4.3 g from

purified

by HPLC

2.1 kg of flower

(Develosil

which linalyl

on 2.

successively purified by HPLC

indicating

consists of xylo- and glucopyran-

osil ODS-IO:

and bornyl

ODS-10:

bed

buds) was

4 20 x 250 mm)

MeOH.

Fr. Db-3,

in

glycosides were detected, was on ODS columns (Devel-

4 20 x 250 mm; YMC-Pack

x 250 mm; 40-100%

and

lo afford

alcohol glycoside fr. (fr. D-3: 0.7-0.9

D-3

on H,O

EtOAc

a monoterpene

ana(/

with

frs were combined then

with stepwise elution of 20-100%

6-O-t)-D-xylopyranosyl+D-

(5) [63 (Table

a sequential

(4)

it was

fr. wt) of the resulting aq. soln was chromatographed

glycoside of borneol.

Hz)] clearly indicated

closer to those of vomifolyl

=6.2-9.5

a

chemical shifts due to the saccharide moiety of

2 were significantly

clarified

and

103.2, besides nine signals at 678.2-66.9,

suggested that it was a disaccharide

(PEG-2OM

25 m x 0.25 mm

which

to give an aroma

before flower opening (stage 3 [ 11.2.7 kg, harvested in the

FAB-mass

In the ‘.‘C NMR carbon

for GC

after

work-up

followed by GC and GC-MS

signals. indicated

1) of 2, two anomeric

(20pgml-‘),

to conventional

concentrate

Isolation

[4].

mmu for C,,H,,O,,)

13C NMR

b 105.5 and

observed in a

- 24”. high resolution

spectrum m/r 449.2412

(Table

of

glycosides, (R)- and (S)-linalyl6-O-

p-D-xylopyranosyl-/?-D-ducopyranoside

subjected column,

6-0-a-L-arabinopyranosyl-b-D-

A similar

C-4 on the linalyl

13C NMR

(CP-

B-236M). Thus. the structure of t was deter-

to be (R)-linalyl

glucopyranoside.

trum

soln of Et octanoate

at C-3 of the linalool liberated to be R by chiral-GC

MeOH).

was finally subjected to HPLC

ODS-AM:

The partially (YMC-Pack

4 20

purified ODS-AM:

fr. 4

Glycosides from Gardenia jasminoides 10 x 250 mm; 50% MeOH; detected at UV 210 nm) to give linalyl glycoside (1, 30 mg) and bomyl glycoside (2, 3 mg) from 2 kg of the flower buds. Compound 1. [a];’ -24” (MeOH; c 1.0). FAB-MS (neg., NOBA) m/z 447 [M -HI-; (pas.. NOBA) m/r 471 [M +Na]+; HRFAB-MS (pas., NOBA) m/z 471.2211 (+OS mmu for C,,H,,O,oNa). ‘HNMR (400 MHz, D,O): 66.01 (H-2, dd, J= 17.6, 11.0 Hz), 5.29 (H-la, br d, .I= 17.6 Hz), 5.28 (H-lb, br d, J= 11.0 Hz), 5.22 (H-6, br t, J=7.0 Hz), 4.53 (H-l’, d, 3=8.1 Hz), 4.39 (H-l”, d, J = 7.3 Hz), 4.09 (H-6’a. br d, J = 11.4 Hz), 3.95 (H-4”. m), 3.93 (H-5”a. dd, J= 12.8,2.6 Hz), 3.81 (H-6’b, dd, J = 11.4. 4 Hz), 3.68 (H-3”. dd, J-9.5, 3.3 Hz), 3.66 (H-S’b, br d, J = 12.8 Hz), 3.60 (H-2”. dd, J=9.5, 7.3 Hz), 3.50-3.45 (H3’, H-Q’, H-5’, m), 3.23 (H-2’. dd, f= 8.8.8.1 Hz), 2.08-2.02 (H-5, m), 1.70 (H,-8, s). 1.69-1.66 (H-4, m), 1.63 (H,-9, s), 1.36 (H,-10, s). “C NMR (Table 1). Compound 2. [a];’ -42” (MeOH; ~0.3). FAB-MS (neg., NOBA), m/r 447 [M -H] -; (pas., NOBA) m/z 449 HRFAB-MS (pos., NOBA) m/z 449.2412 [M+H]+; (2.5 mmu for C,,H,,O,,). ‘HNMR (400 MHz, D,O): b4.51 (H-l”, d, J=7.7 Hz), 4.45 (H-l’, d, 5=8.1 Hz), 4.20 (H-2, br d, J=9.2 Hz), 4.12 (H-6’a, dd, J= 12.1, 2.2 HZ), 3.97 (H-S’b, dd, J = 11.7.5.5 Hz), 3.86 (H-4”. ddd, J = 10.6, 9.2, 5.5 Hz), 3.58 (H-S, ddd, J-9.5, 5.5, 2.2 Hz), 3.48 (H3”,t,J=9.2Hz),3.44(H-3’,dd,J=9.2,9.2Hz),3.42(H-4’, dd,J=9.5,9.2 Hz),3.30(H-S’a,dd,J= 11.7,10.6 Hz), 3.30 (H-2”, dd, J=9.2, 7.7 Hz), 3.28 (H-2’, dd, J=9.2, 8.1 Hz), 2.25 (H-3b, m), 1.90 (H-6a, m), 1.75 (H-Sb, m), 1.69 (H-4, t, J=4.4 Hz), 1.30(H-6b.m). 1.22(H-Sa,m), l.O7(H-3a.dd.J =13.2, 3.3 Hz), 0.90 (HJ-10, s), 0.87 (H,-8, H,-9, s). 13C NMR (Table 1). Determination o/ absolute stereochemistry of linalool. The chirality of linalooi liberated from 1 was determined

459

by GC using a chiral column of CP-cyclodex

B-236M

(50 m x 0.25 mm i.d.): temp. 60” (8 min) -2OO”, 1” min- ‘;

N,, 1.19 ml min- I. Et decanoate (25 pg) was used as int. standard. The RRp of linalool were 0.844 and 0.839 for (S)- and (R)-linalooi, respectively. authors thank Prof. A. Kobayashi (Ochanomizu University) for his assistance in determining the chirality of linalool. Acknowledgement-The

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