Bioflavour ’87: Analysis, Biochemistry, Biotechnology. Proceedings of the International Conference Würzburg, Federal Republic of Germany, September 29–30, 1987 9783110867121, 9783110112047

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Bioflavour '87

Bioflavour '87 Analysis • Biochemistry Biotechnology Proceedings of the International Conference Wurzburg, Federal Republic of Germany September 29-30,1987 Editor Peter Schreier

W G DE

Walter de Gruyter • Berlin • New York 1988

Editor Peter Schreier, Prof. Dr. rer. nat. Universität Würzburg Lehrstuhl für Lebensmittelchemie A m Hubland D-8700 Würzburg Federal Republic of Germany

Library of Congress Cataloging in Publication Data

Bioflavour '87 : analysis, biochemistry, biotechnology : proceedings of the international conference, Wiirzburg, Federal Republic of Germany, September 29 - 30,1987 / editor, Peter Schreier. p. cm: Bibliography: p. Includes index. ISBN 0-89925-290-7 (U.S.) 1. Flavor—Congresses. 2. Biochemistry—Congresses. 3. Microbial metabolism—Congresses. 4. Plants—Analysis-Congresses. I. Schreier, Peter, 1942- . QP801.F45B56 1988 664'.06~dcl9

CIP-Kurztitelaufnahme

der Deutschen

Bibliothek

Bioflavour : [Bioflavour eighty-seven] Bioflavour '87 : analysis, biochemistry, biotechnology ; proceedings of the internat, conference, Würzburg, Fed. Republic of Germany, September 29 - 30,1987 / ed. Peter Schreier. - Berlin ; New York : de Gruyter, 1988 ISBN 3-11-011204-3 NE: Schreier, Peter [Hrsg.]; AST

Copyright © 1988 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. N o part of this book may be reproduced in any form - by photoprint, microfilm or any other means - nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike G m b H , Berlin. Binding: Liideritz & Bauer G m b H , Berlin. - Printed in Germany.

PREFACE

Many important food aromas originate via biochemical pathways.

These path-

ways comprise microbial reactions, endogenous and exogenous enzymic action, and plant metabolism. In the past, flavour research concentrated on characterizing

the

important

chemicals in foods responsible for their specific

aroma. Less information is therefore available on the biogeneration of flavours,

although the first publications on the biochemical pathways of food

flavours e.g.,

go back to the late sixties.

In contrast with the research done,

on the development of analytical techniques or non-enzymatic flavour

formation,

this work did not appreciably influence industrial development.

At present, however,

a

renaissance of studies of natural flavours, inclu-

ding their biogeneration, can be observed. A number of factors appear to be responsible for the renewed interest in

- consumers

have

come

bioflavour research, e.g.,

to reject more and more artificial flavours and to

demand natural ones; - bioflavour formation may provide possibilities for producing industrially important

secondary metabolites which are not available by

conventional

- advances in genetic research and genetic engineering permit

speculations

procedures;

to explore bioflavour production. Regardless chemistry of

the

of

whether this trend towards biological

will last long or not, development

BIOFLAVOUR '87,

(i)

are

worthy

both of

flavour research and

the scientific and applied aspects study.

Therefore,

it is the aim of

to review representative aspects of natural flavours,

stimulate new ideas, awaken

interest between the disciplines involved

and

bring about a cross-fertilization of the different approaches; (ii) to provide those working in specialized fields an opportunity to

see

their work

in a broader perspective; (iii) to discuss the relevance and limitations of the

various approaches;

and industrial fields:

workers;

(iv) to bring together the views of both academic and

(v) to

assess

the

needs in

the following

VI - Analytical Techniques - Biochemistry/Biomimetic

Studies

- Plant Cell/Tissue Cultures - Microbiology - Bioorganic Chemistry/Use of Enzymes.

This

book is intended to provide an overview of the developments in

these

different fields of flavour research. It is a interdisciplinary work intended for both chemists and biologists.

Würzburg, January

1988

Peter

Schreier

ACKNOWLEDGEMENTS

The Editor would like to express his gratitude to DOHLER GmbH, Darmstadt DRAGOCO GmbH, Holzminden HAARMANN & REIMER GmbH, Holzminden RAPS-STIFTUNG, Kulmbach QUEST-NAARDEN, Naarden-Bussum SILESIA KG, Neuss WILD GmbH, Heidelberg-Eppelheim for financial support.

The Editor is grateful to all contributors for their ready collaboration in the preparation of this book to the publishers of this book, Walter de Gruyter & Co., for their guidance and assistance and, in particular, to the members of his staff who acted as registrators, operators, technicians and in many other functions and helped create a friendly working atmosphere during the symposium.

to

CONTENTS

PREFACE ACKNOWLEDGEMENTS

V VII

BIOFLAVOUR - A CRITICAL VIEW

F. Drawert Bioflavour - what does it mean ?

ANALYTICAL TECHNIQUES

V. Schurig Enantiomer separation by complexation gas chromatography applications in chiral analysis of pheromones and flavours

35

A. Mosandl, C. Gunther, M. Gessner, W. Deger, G. Singer and G. Heusinger Structure and stereoanalysis of chiral flavour substances

55

K.H. Engel Investigation of chiral compounds in biological systems by chromatographic micromethods

75

G. Krammer, 0. Frohlich and P. Schreier Chirality evaluation of 1,4-deca- and 1,4-dodecanolide in strawberry

89

U. Ravid, E. Putievsky and M. Bassat Determination of the enantiomeric composition of natural and nature-identical semiochemicals by H-NMR "polarimetry"

97

G. Lange and W. Schultze Studies on terpenoid and non-terpenoid esters using chemical ionization mass spectrometry in GC/MS coupling

105

G. Lange and W. Schultze Differentiation of isopulegol isomers by chemical ionization mass spectrometry

X S. Nitz, H. Kollmannsberger and F. Drawert Analysis of flavours by means of combined cryogenic headspace enrichment and multidimensional GC

123

F. van Wassenhove, P. Dirinck and N. Schamp Analysis of the key components of celery by two dimensional capillary gas chromatography

137

C.Salles, H. Essaied, P. Chalier, J.C. Jallageas and J. Crouzet Evidence and characterization of glycosidically bound volatile components in fruits

145

G. Versini, A. Dalla Serra, M. Dell'Eva, A. Scienza and A. Rapp Evidence of some glycosidically bound new monoterpenes and norisoprenoids in grapes

161

BIOCHEMISTRY/BIOMIMETIC STUDIES F. Karp and R. Croteau Role of hydroxylases in monoterpene biosynthesis

173

W. Boland Odoriferous polyene hydrocarbons from marine and terrestrial plants

199

R. Tressl, J. Heidlas, W. Albrecht and K.H. Engel Biogenesis of chiral hydroxyacid esters

221

S. Tanaka, T. Yamaura and M. Tabata Localization and photoregulation of monoterpenoid biosynthesis in thyme seedlings

237

C.R. Enzell and I. Wahlberg Cembranoid derived tobacco flavour constituents

243

P. Winterhalter and P. Schreier Studies on C^-norisoprenoid precursors

255

T.T. Nguyen, M.M. Palcic and D. Hadziyev Eçzymatic formation of nucleotides and the flavour enhancer 5 -GMP during vegetable processing

275

XI

K. Knobloch, A. Pauli, B. Iberl, N. Weis and H. Weigand Mode of action of essential oil components on whole cells of bacteria and fungi in plate tests

287

PLANT CELL/TISSUE CULTURES B.V. Charlwood, J.T. Brown, C.Moustou, G.S. Morris and K.A. Charlwood The accumulation of isoprenoid flavour compounds in plant cell cultures

303

F. Constabel Cytodifferentiation

315

H. Sugisawa, K. Miwa, T. Matsuo and H. Tamura Volatile compounds produced from the cultured cells of thyme {Thymus vu.i.ganJ-6,L.)

327

J.W. Gramshaw, C.M. Cotton and L.V. Evans Production of flavour volatiles by callus and suspension cultures of tarragon (Ajvtemiàia cLnacunciilLLi )

341

F. Cormier and C.B. Do Selection of monoterpene producing Mentha

pj-pesiita

cell lines

I. Koch-Heitzmann and W. Schultze Compilation of volatile compounds found in plant cell cultures

357

365

MICROBIOLOGY W.R. Abraham, H.A. Arfmann, B. Stumpf, P. Washausen and K. Kieslich Microbial transformations of some terpenoids and natural compounds

399

R.G. Berger, F. Drawert and S. Hädrich Microbial sources of flavour components

415

P. Brunerie, I. Benda, G. Bock and P. Schreier Bioconversion of monoterpene alcohols and citral by BotsujLL* c-tnejiea

435

XII

A. Rapp and H. Mandery Influence of BotnyLu, c-in£yue.a on the monoterpene fraction of wine aroma

^45

L. Janssens, H.L. de Pooter, E.J. Vandamme and N.M. Schamp Biosynthesis of esters by Qe.otAJ.chum. peiu.cjJJ.aium

453

A. Latrasse, P. Dameron, M. Hassani and T. Staron An ester producing microorganism: QeotnJ.chum

candidum

(Staron)

465

K.G. Gupta and R. Singhal Electrochemical conversion of biologically produced diacetyl to acetaldehyde via 2,3-butanediole

473

BIOORGANIC CHEMISTRY/USE OF ENZYMES M.P. Schneider and K. Laumen Enzymes in organic synthesis - chiral building blocks from racemic and prochiral substrates

483

C. Triantaphylides, G. Langrand, H. Millet, M.S. Rangheard and G. Buono On the use of lipase specificity. Application to flavour chemistry

531

D. Gerlach, S. Schneider, T. Göllner, K.S. Kim and P. Schreier Screening of lipases for enantiomer resolution of secondary alcohols by esterification in organic medium

543

C. Fuganti and S. Servi Enzyme mediated synthesis of pheromones

555

Author index

571

Subject index

573

BIOFLAVOUR - A CRITICAL VIEW

BIOFLAVOR - WHAT DOES IT MEAN ?

F. Drawert Institut für Lebensmitteltechnologie und Analytische Chemie der TU München D-8050 Freising 12, FRG

Introduct ion Bioflavor - obviously a creation of Peter Schreier - means I presume natural and naturally produced flavors. As shall be illustrated,keywords such as bio-technology,

bio-process

technique, bio-activity, and the corresponding methods are addressed. Within the field of biotechnology, food technology is the oldest and economically most important part. About 220 billion $ of an estimated current sales of 250 billion $ worldwide account for the food and luxury goods industries. Up to now, the generation of volatile flavors amounts to a very small portion of these sales if we exclude the biotechnological production of ethanol and acetic acid. At present,

about

90 % of the industrially produced flavors and fragrances are of synthetic origin, the rest is mainly derived from agricultural sources (1). Numerous indications, for example also the coining of the term "bioflavor", which in some respects draws flavors nearer to biotechnology, point to a changing situation. This development has manifold reasons

: The in-

dustrial demand for flavors fo'r the production of aromatypical, aroma-intensive foods, convenient foods, extrusion foods is constantly increasing, in the same way as the consumers preference for natural food constituents. In view of the frequent correlation of stereochemical

structure of the

Bioflavour '87 © 1988 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

4 moleculs

and

a higher

standard

impulses

come from the recent

technique Scope of Which

sensory or g e n e r a l l y of p u r i t y

and cell b i o l o g y

physiological

is called

developments

(namely

activities

for. M o r e o v e r ,

new

in b i o p r o c e s s

the rDNA

methods).

Bioflavor

are the' a d v a n t a g e s

production

of flavors

attributed

to a

biotechnological

?

1) The p r o d u c t s may p o s s e s s the

legal

status of a natural

compound, 2) the h i g h

substrate

guarantees 3) optimized products

and

a defined

reaction

and to constant

pest

influences

are not p o s s i b l e

following

division

s y n t h e s i s will callus and

climate,

according to

among

especially

for example

processes.

Bioprocess

for the o p t i m i z a t i o n to achieve

space.

aged

with

and for

multiphase for

is

certain

requirement

downstreaming

production.

b i o a c t i v i t y with

important,

technique

bioreactors

is a n e c e s s a r y

precur-

mycophyta,

and

bioprocess

a cost-effective

includes

are

and

in immobilized

of p r o d u c t s

de-novo-

cells

tissue c u l t u r e s ,

and h o m o g e n a t e s

in o p t i m i z i n g control

and

Plant

them b a c t e r i a

of b i o f l a v o r s

essential,

the

1):

tissues,

In the field

also

described

cultures,

stored

as e n z y m e s ,

operations

is well

the b r o a d e s t

as m i c r o o r g a n i s m s ,

bioflavor

aqueous

drawbacks

box w i t h b i o t r a n s f o r m a t i o n

occupy

suspension

sor a t m o s p h e r e

systems.

or ecological

in

conditions,

such as u n f a v o r a b l e

economical

(Fig.

The " b i o t e c h n o l o g y "

as well

under mild

neglected.

In m y o p i n i o n b i o f l a v o r

just

uniform

productivity,

reactions, which

infestations,

can be

enzymes

lead to c o m p l e x ,

solution by chemical m e a n s , proceed 5) adverse external

of

stereochemistry,

reaction c o n d i t i o n s

4) m u l t i p l e - s t e p

specifity

The

physiological

term

5

BIOFLAVOR

BIO TECHNOLOGY

BIO PROCESS TECHNIQUE

BIO ACTIVITY

METHODS

BIO REACTORS

PHYSIOLOGICAL ACTIVITY

INSTRUMENTAL METHODS

BIO PROCESS CONTROL

SAR (STRUCTURE ACTIVITY RELATIONSHIP)

CAPILLARY - GC

BIO - TRANSFORMATION DE - NOVO - SYNTHESIS PLANT C E L L S

MICROORG.

CALLUS SUSPENSION CULTURES

BACTERIA (STARTER CULTURES)

AGED TISSUE CULTURES

MYCOPHYTA (YEASTS,MOLDS)

PRECURSOR ATMOSPHERE ( P A - ) STORAGE

ENZYMES | IMMOBILIZED E.

I HOMOGENATES

MULTIPHASE SYSTEMS

Fig.

1

The term

DOWN STREAMING

GC - MS GC - IR SNIFFING - GC

BIO SENSORS

BIOLOGICAL METHODS

bioflavor.

and SAR. Both b i o p r o c e s s

activity

are connected w i t h the detection

MULTIDIMEN SIONAL - GC

technique

instrumental m e t h o d s ,

of trace c o m p o u n d s ,

and

bioactivity

for example

and w i t h

for

biological

methods.

T h e pr imary problem catalyst".

For the s y n t h e s i s of v o l a t i l e

inexhaustible enzymes

is the choice of the most

reservoir

isolated

potential

and their

immanent

a

of selected

The m a j o r i t y

flavors

and plant

an

of the v a r i o u s

types of

"bioalmost

cells

out of them has to be c o n s i d e r e d .

synthetic number

of m i c r o b i a l

suited

and

The

biocatalysts

l i m i t a t i o n s will be d e m o n s t r a t e d

using

examples.

of natural

flavors

other parts of field plants. The

is

obtained from

fruits

totipotent cells of

or

callus

6 and s u s p e n s i o n c u l t u r e s , w h i c h are d e r i v e d from these m a y be u s e d

in b i o p r o c e s s e s .

of f l a v o r s are o b s e r v e d

Furthermore,

in a g e d fruit tissues and in

d i f f e r e n t i a t e d p a r t s of p l a n t s under i n c u b a t i o n or storage. H o m o g e n a t e s flavors via catabolic

pathways.

C a l l u s and S u s p e n s i o n

Cultures

plants,

de-novo-syntheses intact

suitable c o n d i t i o n s

of p l a n t cells

of

form

It is k n o w n since long that c a l l u s and s u s p e n s i o n

cultures,

d e s p i t e their p r o n o u n c e d p r i m a r y m e t a b o l i s m , posses a considerable potential

for the f o r m a t i o n of

secondary metabolites.

plant-typical

The f o r m a t i o n of a r o m a - a c t i v e

p o u n d s , h o w e v e r , f r e q u e n t l y fails to a p p e a r . a c c e p t e d that this

is due to the lack of

It is

generally

morphological

storage s t r u c t u r e s , a l t h o u g h e x a m p l e s d e m o n s t r a t i n g opposite

can be found

in l i t e r a t u r e

c h a i n to occur

single

pathway

steps of the

in an a c t i v e form. T h e r e f o r e ,

synthetic

suspension

c u l t u r e s are o f t e n able to p e r f o r m h i g h l y s p e c i f i c formations

Co-factor

the

(2,3).

The lack of end p r o d u c t s of a c e r t a i n m e t a b o l i c does, of c o u r s e , not e x c l u d e

com-

biotrans-

(4) .

dependent,

specific transformations

in s u s p e n s i o n c u l t u r e s

of

terpenes

of a r o m a t i c p l a n t s p r o c e e d

with

h i g h yields and o f t e n w i t h o u t w o r t h m e n t i o n i n g a m o u n t s s i d e p r o d u c t s . Not only o x i d a t i o n s

or r e d u c t i o n s ,

as

in Fig.2, but also n u m e r o u s other types of r e a c t i o n s been described major component

of

shown have

(4). The a l l y l i c o x i d a t i o n of v a l e n c e n e , in c i t r u s oils,

impact of g r a p e f r u i t

leads to n o o t k a t o n e ,

flavor with considerable market

(5). The a v a i l a b i l i t y of n a t u r a l During biotransformation

nootkatone

experiments

d e c r e a s e of the c o n c e n t r a t i o n

is

of the e x o g e n o u s

an value

limited.

a sometimes

a

rapid

terpenoid

Conversion

CR

Salvia

rate

0

off.

78 % 3 h Campher

Borneol

Melissa

off. 99,5 %

2 h

Citrus

sp.

VUY66*

6 h

Nootkatone

Valencene Fig. 2

Biotransformation cells.

of t e r p e n e s u s i n g

compound without a corresponding products

is o b s e r v e d .

f o r m a t i o n of

res of grape l a b e l l e d c a r o t i n o i d s

of

In s u s p e n s i o n

and s t e r o i d s w e r e

after a p p l i c a t i o n of l a b e l l e d m o n o t e r p e n e s is the

plant

volatile

In these cases the m e t a b o l i s m

terpenes may follow different pathways.

pathway

in v i t r o

(6). A

i n t r o d u c t i o n of a c a r b o x y f u n c t i o n

cultu-

found

second followed

by the ^ - o x i d a t i v e d e g r a d a t i o n of the acid, as o b s e r v e d the c o n v e r s i o n of e x o g e n o u s g e r a n i o l

to g e r a n i c acid

s u s p e n s i o n c u l t u r e s of a p p l e . C o n c u r r e n t W a r b u r g (Fig.3) s h o w e d a s t i m u l a t e d cell

respiration

cell c u l t u r e s up to c o n c e n t r a t i o n s geraniol convert to

measurements

in d i f f e r e n t

in the range of 1.5 mmol

per L (7). A t h i r d p a t h w a y

of t e r p e n e g l y c o s i d e s .

results

in the

S u s p e n s i o n c u l t u r e s of

formation

peppermint

e x o g e n o u s m e n t h o l w i t h h i g h c o n v e r s i o n rates

70 %) into m e n t h o l

glucosides

for

in

(Fig.4). A

striking

(up

8

Fig. 3

Cell r e s p i r a t i o n in C . l i m o n cells in the of e x o g e n o u s g e r a n i o l ; bars indicate the in three p a r a l l e l e x p e r i m e n t s .

result

is the high g l y c o s i d a t i o n

growing

c u l t u r e s . The m a x i m u m

is reached

alcohols

or p h e n o l i c

Some of the resulting

i.e., they have to be sorted

These

examples

show that

on h o m e o s t a t i c m e c h a n i s m s of substrates genous

in young,

concentration

on the 4th day of c u l t i v a t i o n .

also aliphatic silated.

activity

into the

cultivated

Except

rapidly glucoside

terpenols

can be

glyco-

are bitter

tasting,

bioflavors.

plant

cells

to e l i m i n a t e peak

and to avoid p r o b a b l y

of the

compounds

glycosides

presence deviation

toxic

can

switch

concentrations

effects

of

exo-

compounds. A further basic way for the removal

undesired m e t a b o l i t e s

from the cell or its s u r r o u n d i n g

of is

9

Conversion of Menthol

(%)i 70

o.

60

50

•O

L

Fig. 4

"ft~

4

3

5

7 (days)

6

C o n v e r s i o n of m e n t h o l to m e n t h o l g l u c o s i d e s u s p e n s i o n c u l t u r e s of M e n t h a sp.

the c o n j u g a t i o n w i t h l i p o p h i l i c m o i e t i e s to yield metabolites. Many

suspension cultures eliminate

acyl m o i e t i e s v i a the gas p h a s e by a l k y l a t i o n . c u l t u r e s of apple the f o r m a t i o n of m e t h y l

in

volatile

exogenous In

suspension

and ethyl

d o m i n a t e s ; u p o n a d d i t i o n of s u i t a b l e acids such as acid the c o r r e s p o n d i n g (Fig.5).

4- and 5 - o l i d e s are also

esters hexanoic

formed

In apple f r u i t s o n l y 4 - h e x a n o l i d e has b e e n

in t r a c e s .

It w a s s h o w n by s e v e r a l

a u t h o r s that

detected

in v i t r o

p l a n t cells are able to form o t h e r m e t a b o l i t e s

than

those

f o u n d in the intact p l a n t f r o m w h i c h t h e y w e r e

derived.

As d e m o n s t r a t e d w i t h the m e n t i o n e d apple c u l t u r e , the

possi-

b i l i t y e x i s t s to s t i m u l a t e the f o r m a t i o n of

species-untypical

metabolites with flavor-characteristics

"overflow"

via

mechanisms. In terms of an i n t e n s i v e f o r m a t i o n of f l a v o r s

it is

not only to a c c u m u l a t e the p r i m a r y p r e c u r s o r s , but circumvent

important, to

or e l i m i n a t e those r e g u l a t o r y m e c h a n i s m s ,

which

10

•C00H

ROH .urh

\

HOH

Hydroxylation

/ \

/ \ Fig.

5

R : Me

R : Et

37 %

4 %

48 %

Proposed m e t a b o l i s m apple cells.

of h e x a n o i c

modulate

secondary m e t a b o l i t e

choosing

the a p p r o p r i a t e

flavors

are formed

of h e t e r o t r o p h i c obtained w i t h a pronounced

acid

formation

culture

%

in

cells

effect

towards

light

could

factor.

of R u t a g r a v e o l e n s

constituents

: in dark

cultures

the root-typical

dominate,

in light

cultures

ketones

Another metabolic

and esters

difference between

and p h o t o m i x o t r o p h i c of a r o m a - a c t i v e

(Fig.6,

cell

cultures

C^-compounds

clevage of u n s a t u r a t e d

of

show volatile

irregular

leaftypical

(2,3)).

photoheterotrophic

refers to the

from the

C1R-fatty

exposition hence

Results

of light on the p r o p o r t i o n s the

natural

(rue)

monoterpenes aliphatic

by

c o n d i t i o n s . Many

role as an inducing

suspensions

cultivated

downwards

in green parts of plants. The

plant

play on important

11

acids

formation

1ipoxygenase-catalyzed (8). Incubation

of

11

Compound (pg kg fr.w. - 1 )

Ruta

graveolens

heterotrophic

Geijerene

mixotrophic

480

160

1040

230

2-Undecanone

80

1660

2-Tridecanone

20

210

Acetic acid n-nonyl ester 2-undecyl ester

20 50

230

Pregeijerene

3-Methylbutyric acid n-octyl ester n-nonyl ester 2-undecyl ester

Fig. 6


. I I 200 m/e

0"*••'' i vVi '>'!" T 100

155

100-1

100-1

200

150 137

137

155

50-

50-

81

81 1 00

)„ T

150

200

'

100

'

I

150

I ' '

200

Figure 2: Isobutane CI mass spectra of a) isopulegol b) isoc) neo- d) neoisoisopulegol. Ion source temperature 140°C One may suggest that the double bond of the isopropenyl group plays the essential role causing these pronounced steric effects. An intramolecular hydrogen bond may be formed between

118

the isopropenyl and the hydroxy1 group. This may yield differently stabilized protonated molecular ions (MH)+ according to the conformation and the spatial distance of the functional groups involving loss of B^O to a different extent in the respective isomer. This assumption can be supported by the fragmentation behaviour of the respective menthols which do not have the double bond and do not show this effect (3). Latter compounds do not even form an (MH)+ ion species. The effect of intramolecular hydrogen bonding in CI is described for various types of compounds and various functional groups (4). Among them it is discussed for norbornenols where only one isomer allows interaction between the double bond and the hydroxy1 group producing significant (MH)+ peaks (5). A fundamental parameter which may exercise great effects on the fragmentation behaviour in CI is the ion source temperature. Figure 3 depicts the isobutane CI spectra of the isopulegols measured at a source temperature of 200°C. As has to be expected, the intensities of the dehydration product m/e 137 are increased, thus yielding the (MH)+ as the base peak only for isopulegol. However, the stereospecifity is still contained. Reliable identification and differentiation of these isomers is possible as well at this source temperature. In addition, these differences in the spectral patterns clearly indicate that caution is required when comparing CI spectra. The fragmentation behaviour of the isopulegols is completely changed when methane or ammonia is employed for ionization. Figure 4a shows the methane CI spectrum of isopulegol. Abundant fragmentation products are observed according to the higher energy transfer in the ionization process. The protonated molecular ion (MH)+ is very small. There were only slight differences in the spectra of the four isomers. These differences, however, are too small to convincingly distinguish the isomers.

119

[MH]+

I nt.

50-

155

|MH-HjO]+

100-1

137 1 00-

155

137

50-

81

100

150

'

11 1

137

1 00-i

'

el ,

200 m/e

..i^ 100

.,1, , ,.,, i |i i 150

200

137

10 0-1 155

50-

50-

81

155

1 00

u

150

200

100

1 50

200

Figure 3: Isobutane CI mass spectra of a) isopulegol b) iso- c) neo- d) neoisoisopulegol. Ion source temperature 200°C The ammonia CI spectrum of isopulegol is depicted in Figure 4b, Ammonia which is a less energetic reagent gas does not protonate the isopulegols. It produces the (M+18)+ (= (M+NH4)+) and (M+35)+ (= (M+^H.^)^ adducts as the quasimolecular ions. They are the only significant peaks. The spectra of all four isomers are virtually identical and do not reflect steric differences. Variation of the ammonia pressure and the ion source temperature caused only little variations in the spectral pattern which do not allow to distinguish the isomers.

120

Int.

81

172

1 00-1

100-

|MH-HjO]+ 137

50-

50-

95

[M + 3 5] "

[mh]+ 109

JL

„Li 100

155 -i

150

200 m/e

0-

100

[M + NH,-1B]+ 154 'I ' 'i-r-r-* I

189

150

200

Figure 4: a) Methane CI mass spectrum of isopulegol b) Ammonia CI mass spectrum of isopulegol Source temperature 140°C; reagent gas pressure 0.35 mbar

Conclusion The preceding examples illustrate most impressively the influence of different reagent gases on CI spectra. It is most important to choose the proper reagent gas producing the desired effects to exploit the advantage of CI. The ' stereochemistry cannot be inferred from the methane or ammonia CI spectra. Only the isobutane CI spectra which provide pronounced differences can be used as a stereochemical probe to distinguish the isomers.

Experimental The EI mass spectra were run on a Hewlett-Packard GC/MS system (GC: 5890A; MSD 5970B; Data system 59970A). MS conditions: Ion source temperature: 200°C; electron energy: 70 eV; scan rate: 2.5 scans/sec.; the spectra are averaged

121

across the maxima of the GC peaks. GC conditions: The GC was a Hewlett-Packard 5890A, equipped with a 30m x 0.25mm i.d. DB-WAX (chemical bonded, cross linked) capillary column (J & W Scientific), film thickness 0.25 p.m. Carrier gas was Helium with a flow rate of 1 ml/min. The temperature program was 6 min. at 60°C, then raised to 150°C (3°C/min.). Temperature of the transfer

line was 200°C.

The CI mass spectra were obtained with a 8200/SS300 GC/MS/DS system (Finnigan MAT), ion source 140°C/200°C, accelerating voltage 3 kV; electron energy 70 to 100 eV; emission current 0.05 inA; scan

rate 0.7 sec/dec. The reagent gases were iso-

butane (2.5), methane (4.5) and ammonia (2.8) obtained from Messer Griesheim (Frankfurt, FRG). The nominal partial reagent r

gas pressure was 0.30 to 0.35 mbar. The scan range was from m/e 70 to m/e 300. The CI spectra are average spectra taken from five spectra across the maxima of the GC peaks. The gas chromatograph was a Varian 3700, equipped with an open split interface (temperature of the transfer line was 200°C). The samples were introduced through a 50m x 0.32mm i.d. WCOT fused silica CpWAX 57 CB (chemical bonded) capillary column, film thickness 0.22 |im (Chrompack) . The temperature program was 5 minutes at 60°C, then heated at

3°C/min. The carrier gas

was Helium, flow o.9 ml/min. The isopulegol sample (containing the four isomers) was obtained from Roth (Karlsruhe, FRG).

Acknowledgement Financial support of these studies by the "Sebastian-KneippStiftung" and by the "Fonds der Chemischen Industrie" to Prof.Franz-C.Czygan

(Wurzburg) is gratefully acknowledged.

122

References 1. Harrison,A.G., 1983. Chemical Ionization Mass Spectrometry. CRC Press, Boca Raton. 2. Lange,G., Schultze,W., 1987. Flavour and Fragrance Journal (in press). 3. Lange,G., Schultze,W., (in preparation). 4. Mandelbaum,A., 1983. Mass Spectr.Rev. 2, 223. 5. Jalonen,J., Taskinen,J., 1985. J.Chem.Soc. Perkin Trans.II, 1833.

ANALYSIS OF FLAVOURS BY MEANS OF COMBINED CRYOGENIC HEADSPACE ENRICHMENT AND MULTIDIMENSIONAL GC

S. Nitz, H. Kollmannsberger, F. Drawert Institut für Lebensmitteltechnologie und Analytische Chemie der Technischen Universität München 8050 Freising-Weihenstephan, FRG

Introduction In flavour research there is an increasing interest in the analysis of trace organic compounds in complex samples. However, gas chromatographic analysis of the volatile components of natural products (fruits, herbs, spices, etc.) requires sample preparation procedures suited for GC such as solvent extraction, steam distillation, etc. These procedures are often difficult and tedious to carry out on small amounts of sample, especially in the case of materials with high pigment or fat content. Additionally contamination of sample with solvent impurities during extraction is often unavoidable. These shortcomings can be eliminated by using headspace analysis. The sensitivity of this method can be considerably increased by combining continuous gas extraction with the concentration of substances before their admission to the chromatographic column. Combination with multidimensional gas chromatography (MDGC) noticeably improves the analysis of flavours in complex mixtures. A system for on-line cryogenic headspace enrichment and simultaneous multidimensional analysis is described and examples of applications to demonstrate the potentials and limitations of this method of analysis are given.

Bioflavour '87 © 1988 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

124 •ü O) G

-H XI

e o

u

e

1 w G Q) U •H h c H (U 0 (0 ra T3 rfl 1 si

a (Ö u

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o M

Si O

en ifl U ri

C O

•H w C 0)

(U si -p

E •H

4-1 o

•H •P — i1

ai

2

Si

-p

G a) o

en •rH

tu

0

125

System description and operation The schematic of the system used for enrichment of headspace volatiles and multidimensional analysis is shown in Fig. 1. Extraction of volatile components is achieved by stripping the headspace vial (8 ml capacity) with a constant helium flow (30 ml/min) followed by cryogenic concentration in a cooled (-130°C) trap, packed with 5% OV-lOl on chromosorb W. Total purging volume can be adjusted by time-programming solenoid valve SV3. During this sampling period breakthrough of substances into the first column is avoided by actuating SVlf thus maintaining the preselected column pressure (adjustment with PM 4 ) and establishing a minor gas-flow through a restriction capillary into the aforementioned trap (Fig.2). The sampling period is interrupted by closing SV2 and SV3. The gas-flow in the trap is reversed by actuating SV^. and enriched volatiles are introduced into column 1 by thermal desorption (Fig.3). After admittance into the column the original column pressure is reestablished by operating SVX again. The peakgroup of interest is transferred into column 2 by means of the valveless Live-T switching device (1). After final separation the substances are directed to the MS and/or sniffing mask (2). UN,

Fig.2: Gas- and substanceflow during sampling

Fig.3: Gas- and substance-flow during thermal desorption

126

Instrumentation Gas chromatograph Siemens Sichromat 2, dual oven GC, equipped with 2 FID~s, Sniffing device, "Total Transfer" (3,4) and cryogenic headspace enrichment system. An all-glass modification of the sampling system was found to be necessary since with the original device artifact formation was observed. Split ratio: 1:2.5 (60°C) Columns 1. SE-54 fused silica capillary column, 25m x 0.53 mm ID, 1.5 um film thickness, carrier gas: He, flow: 5ml/min (60°C), temp, progr.: 65°C-5°C/min-250°C. 2. CW20M glass capillary column, 25m x 0.3 mm ID, 0.3 um film thickness, carrier gas: He, flow: l,5ml/min (60°C), temp, progr.:depending on application The same GC-conditions were used for liquid injection. Mass Spectrometer Finnigan 4021 (quadrupole), equipped with Incos data system for registration and processing of mass spectral data.

Applications The following applications were selected to demonstrate the possibilities, advantages and limitations of the described system. Special attention was focused on its suitability for direct analysis of small amounts of biological material. 1. Analysis of volatiles of parsley leaves and oils A chromatogram of a commercial available oil obtained by liquid injection is shown in Fig. 4. The analysis of the same oil by means of cryogenic headspace enrichment (Fig.5) points

127

out a good qualitative agreement between both injection techniques in the retention range of monoterpenes (Compounds 1-10 in Tab.

1). Higher

boiling

components

like

(e.g. germacrene D) as well as phenolics

sesquiterpenes

(e.g. myristicin,

elemicin and apiole) cannot be stripped in comparable amounts under these analytical conditions

(Sampling at 40°C). These

substances have to be analyzed at higher temperatures. The relative amounts of the main components in the oil determined by means of the two aforementioned

sampling techniques are

listed in Tab.l. Table 1:

Major Components in a commercially available Essential Oil of Parsley

Nr.

Compound

Retention Index SE54

1 2 3 4 5 6 7 8 9 10 11 12 13 14

a-pinene ß-pinene myrcene a-phellandrene limonene ß-phellandrene p-cymene terpinolene a,p-dimethylstyrene p-1,3,8-menthatriene germacrene D myristicin elemicin apiole

Concentration (%) Liquid Injection Headspace

933 975 991 1003 1029 1029 1024 1086 1087 1109 1473 1516 1556 1677

8.4 5.9 8.2 0.7 6.2 1.9 0.5 1.9 5.9 30.6 0.8 10.2 0.8 6.6

10. 1 7. 5 9. 8 0. 8 7. 3 2. 3 0. 5 2. 3 7. 3 48. 2 0. 2 0. 3 -

The amount of essential oil in plain- or crisped-leaved parsley is in the order of 0.02-0.03 % (w/w) . Nearly 300 mg of fresh leaves are necessary to get similar amounts of volatiles in the headspace vial

(compared

to direct

headspace

analysis of 0.5 mg of oil; see Fig.5 and 6a). The chromatogram shown in Fig.6a corresponds to harvested leaves stored at 25°C in a closed headspace vial during 4 hours. It can be observed, that storage is accompanied by a marked increase of compounds 15-19, which are not resolved on column 1.

Headspace chromatograms on column 1, sampling: 1' at 40°C

1 3 5-7 8,9

J_L

Cut from ootumn 1

JL Fig. 6a: 300mg parsley leaves; 6b: Cut of peak group 5-7 into column 2

12

IiJIl

1

Cut to column 2

J 5-7

AJ UUmuU^UjjuJ. 1 2 3 57 8,9

. Jl. A 1 . 12

"

Fig. 5: 0.5mg parsley oil

M

ixji. LJJ^juAJII

Fig. 4: 1% parsley oil in ether, 1 ul liquid injection

129

After performing an appropriate cut - into column 2 they could be

properly

separated

due

to

the

polarity

change

of

the

column. Besides methanol, acetaldehyde, ethanol, methyl- and ethyl-acetate simultaneous "sniffing-MS" monitoring revealed the presence of methane-thiol and dimethyl-sulfide. The advantage of the combination of cryogenic headspace enrichment with MDGC (HR-(GC)2) becomes apparent if we consider the peak group 5-7 for example. Although p-cymene can be resolved using different temperature parameters on column 1, limonene and fi-phellandrene generally overlap on unpolar columns (5) . They can be very well resolved on a PEG-column (Fig.6B), but this type of stationary phase is not suitable for the complete analysis of parsley oil. Only the on-line combination of both types of stationary phases, using the Live-T switching

device,

allows

a rapid

and

comprehensive

analysis of the oil components with one single instrument. 2.Analysis of changes in the volatile composition of marjoram due to technological processing It is well established that sabinene, both sabinene-hydrates and cis-sabinenehydrate-acetate,

with a relative amount of

more than 70%, are the major components in fresh marjoram leaves (6,7,8). Especially the latter compound easily undergoes rearrangement reactions caused by thermal treatment or acid catalysis, mainly leading to 4-terpineol,

and a-ter-

pinene. Therefore artifact formation is to be expected during technological processing of marjoram, and its degree depends on the conditions applied. The changes in volatile composition due to drying or production of essential oil (by steam distillation) are illustrated in Fig.7-9. While the carefully dried leaves contain nearly the original monoterpene composition (Fig.7), a remarkable increase in 4terpineol and other artifacts

(compounds

1,4,5,6,7,9,11,12)

is observed in the case of overheating and steam distillation (Fig.8,9; Tab.2).

130

Headspace chromatograms on column 1, sampling:1' at 40°C

Fig. 7: 20 mg marjoram leaves I carefully dried

Fig. 8: 20 mg marjoram leaves II overheated during drying

w

JLJL

Fig. 9: 0.2mg commercial marjoram oil obtained by steam distillation.

This application clearly demonstrates the potentials of the analysis system: a) Fast analysis with minute sample amounts (20 mg leaves) b) No clean-up procedure is necessary and contaminations with solvent impurities are avoided. c) The combination with MDGC enables a reliable identification since 2 different retention indices and additionally mass spectral and sensory data are available.

131

Table 2:

Major Components in dried Leaves and distilled Oil of Marjoram

Nr.

Compound

Retention Index SE54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

a-pinene sabinene myrcene a-terpinene p-cymene limonene/B-phellandrene J^-terpinene E-sabinenehydrate terpinolene Z-sabinenehydrate 4-terpineol a-terpineol Z-sabinenehydrate-acetate longifolene caryophyllene bicyclo-germacrene

933 973 991 1016 1024 1029 1060 1066 1086 1094 1174 1186 1251 1393 1409 1487

Concentration (%) Leaves I Leaves II Oil 0. 3 6. 5 1.6 1.1 0.8 2. 9 1.7 4.9 0. 5 33.7 1.3 0.8 32. 0

0.5 7.7 2.6 5.9 8.1 4.9 8.3 9.8 1.8 24.4 12.8 1.0 4.0 -

-

1.3 0. 3

1.0 0.1

3.2 2.8 1.1 9.6 7.7 12.1 14.4 1.8 3.5 7.2 16.7 3.6 2.1 2.4 1.5 0.4

3.Analysis of secondary aroma formation in garlic Intact samples of garlic have no distinctive flavour characteristics. Immediately on cutting there is a rapid development of flavour due to the conversion of S-alk(en)yl-L-cysteine sulfoxides to dialk(en)yl-thiosulfinates by the enzyme alliinase (9). The reductive cleavage of these unstable intermediates leads to the formation of numerous volatiles (see Tab.3). This biological material was selected in order to show that the analysis of dynamic systems (e.g. secondary aroma formation, enzymatic conversions, biotransformations, thermal induced reactions, cooxidations, etc.) is straightforward with the analytical configuration described in this paper. The chromatograms obtained immediately after sample preparation and after one hour incubation at 40oC are shown in Fig. 10 and 11. The identification of low boiling volatiles was achieved by cutting the unresolved peak- group 1-8 into column 2, coupled to the mass-spectrometer (Fig.12).

132

Fig. 10: Immediately after Fig- 11: Incubation 1 hour sample preparation at 40°C Headspace (1 min, 40°C) of a Garlic Slice (20mg)on column 1

Cut from column 1

Fig. 12: Cut on column 2 (60°C isotherm)

Va.

J^ft

IH

i-JU

J

Fig. 13: Artifacts due to heating (column 1)

As can be seen from Fig. 11, di-2-propenyl-disulfide is the major constituent of garlic headspace, responsible for the typical flavour of fresh garlic. The analysis of garlic solvent extracts by means of liquid injection however leads to the formation of major amounts of 3-vinyl-l,2-dithi-5-ene and 3-vinyl-l,2-dithi-4-ene, due to dehydration of diallyl-thiosulfinate, present in the extract, as described by Brodnitz et al (10). This artifact formation can be induced by heating the ether extractives of one fresh crushed garlic clove for

133

15 minutes at 150°C. The headspace analysis revealed that in this case both vinyl-dithienes, a dimethyl-thiophene and 1,2dithio-cyclopentene are the major artifacts formed (Fig.13), possessing a typical odour of fried garlic. Table 3: 1 2 3 4 5 6 7 8

Identified Volatilies in Garlic Headspace

methane-thiol acetaldehyde propanal propane-thiol 2-propene-thiol ethanol 2-propenyl,methyl-sulfide propanol

9 10 11 12 13 14 15

di-2-propenyl-sulfide 2-propenyl,propyl-sulfide 2-propenyl,methyl-disulfide di-2-propenyl-disulfide 2-propenyl,propyl-disulfide 2-propenyl,1-propenyldisulfide di-2-propenyl-trisulfide

4.Analysis of a "peasy off-flavour" in coffee beans Coffe defects derived from moulds, overfermentation, insects or other influential factors often impair the flavour of the roasted end product considerably. Certain african coffee beans with an off-flavour, known among experts as "peasy", were investigated (11). In this case the major advantages of the system are exploited to a large extent: Analysis of trace volatiles in a complex matrix without clean-up procedures, small sample amounts (only 3 coffee beans), adjustable enrichment effect due to continuous gas extraction conditions and cryofocussing, heartcutting of off-flavour related peaks by means of MDGC and identification of the off-flavour responsible compound by simultaneous Sniffing-MS. Fig.l4a,b,c summarizes the aforementioned analytical steps. The enriched coffee headspace and the heart-cuts at the retention times corresponding to the elution of the "peasy" compounds is shown in Fig.14a. The chromatograms of the cuts from column 1 on column 2 for 3 normal and 3 defective coffee beans is shown in Fig.14b and Fig.14c respectively. Whereas 2-methoxy-3-isobutyl-pyrazine is present in nearly equal amounts in both samples, 2-methoxy-3-isopropyl-pyrazine is

134

markedly increased (10-fold) in the defective beans, and has to be considered as responsible for the off-flavour. Overloading of the first column in this case is of secondary importance, since the main separation is performed on column 2 with a preselected cut. Cuts from column 1

Sample: 3 coffee beans (5 min/ 80°C) Chromatogram on column 1

»off-flavour«

A/aJ

normal

T ~

i

Cuts to column 2

1: 2-methoxy-, 3-isopropyl-pyrazine 2: 2-methoxy-, 3-lsobutyl-pyrazine

Fig. 14:

A/Vjl

(odour: »peasy«) (odour: »bellpepper«)

Headspace of Coffee Beans (Column 1=CW20M, 2=SE54)

Conclusions The applications shown so far clearly demonstrate the advantages and possibilities of the .analytical system described in this paper. Nevertheless, some limitations have to be considered in this context. Although not observed during the analysis of the aforementioned applications, artifact formation during thermal desorption after enrichment of labile volatiles cannot be excluded in all cases. Additional traps or an

135

appropriate trap material are necessary for the analysis of samples containing higher water amounts. Research on these topics is in progress.

Acknowledgement We are grateful to the Deutsche Forschungsgemeinschaft financial support.

for

References 1. Schomburg , G., F. Weeke. 1982. Chromatographia 16, 87 2. Nitz, S., F. Drawert. 1986. Chromatographia 22, 51 3. Nitz, S. 1985. In: Topics in Flavour Research (R.G. Berger, S. Nitz, P. Schreier, eds.). Eichhorn, Germany 8051 Marzling, Hangenham 25, p.43 4. Nitz, S., E. Jülich. 1984. In: Analysis of Volatiles (P. Schreier, ed.). Walter de Gruyter & Co, Berlin - New York, p.151 5. Nykänen, I. 1986. Z. Lebensm. Unters. Forsch. 183, 172 6. Fischer, N., S. Nitz, F. Drawert. 1987. J. Agric. Food Chem., submitted 7. Fischer, N., S. Nitz, F. Drawert. 1987. Flavour and Fragrance J., in press 8. Fischer, N. 1986. Ph. D. Thesis, T.U.München, FRG 9. Whitaker, J. R. 1976. Adv. Food Res. 22, 73 10. Brodnitz, M. H., J. V. Pascale, L. Van Derslice. 1971. J. Agric. Food Chem. 19, 273 11. Becker, R., B. Döhla, S. Nitz, O. G. Vitzthum. 1987. In: Proceedings of the XII International Conference on Coffee Science. Montreux

ANALYSIS OF THE KEY COMPONENTS OF CELERY BY TWO DIMENSIONAL CAPILLARY GAS CHROMATOGRAPHY

F. Van Wassenhove, P. Dirinck and N. Schamp Laboratory of Organic Chemistry, Faculty of Agricultural Sciences, State University of Gent, Coupure Links 653, B-9000 Gent, Belgium

The characteristic odour of celery (Apium graveolens L.) is due to a series of phthalide derivates which have only slightly different structures.

The separation of phthalides in the

complex matrix of the flavour compounds of celery is very difficult.

Even with the modern capillary columns, which have

great separation power, it is not possible to obtain an unequivocal separation of the key celery components.

Two dimensional

capillary gas chromatography gives a solution to this problem. The phthalides in the essential oil, obtained from Apium graveolens L. var. dulce by two essence recovery methods, were separated by two dimensional capillary gas chromatography and identified by mass spectrometry.

The following phthalides were

identified : butylhexahydrophthalide, Z-butylidenephthalide, butyl-3a,4,5,7a-tetrahydrophthalide tylideen-4,5-dihydrophthalide

(cnidilide), E- and Z-bu-

(E- and Z-ligustilide), butyl-

phthalide, cis- and trans-butyl-3a,4,5,6-tetrahydrophthalide (cis- and trans-neocnidilide) and butyl-4,5-dihydrophthalide (sedanenolide = senkyunolide) .

Introduction The celery plant, particularly the seed, has been the subject of many investigations, probably because of its past medical importance.

Apium graveolens L. has been applied against

Bioflavour '87 © 1988 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

138

tumours (1), gout, rheumatism, bronchial catarrh (2) and, mainly the roots, have been used as a diuretic (3).

At pre-

sent, no important medical value is ascribed to celery.

Today

celery is used for its unique texture and appetizing flavour. The use of the celery plant as a vegetable in food blends, soups and other processed products has greatly increased the interest in the nature of its chemical constituents. Gold and Wilson (4,5,6) were the first to investigate the volatile flavour substances of the vegetable celery.

Among the

twenty-four components described by these authors several phthalides were identified.

Important work on the isolation and

identification of phthalides has since then been performed by Fehr (7,8) and Gijbels and co-workers (9,10,11).

A number of

phthalides are important flavour components of celery and are frequently used as flavouring agents in the food industry

(12).

The isolation and separation of phthalides from celery still remains difficult. This work reports on a fast method which allows separation of the phthalides, isolated by Likens-Nickerson and soxhlet extraction, in one analysis using two dimensional capillary gas chromatography with columns of different polarity.

The key

components are identified, after preparative gas chromatography and GC-MS analysis, by direct comparison of their mass spectra with published spectra

(13,14,15).

Isolation of celery oil Specimens of freshly harvested Apium graveolens L. var. dulce were obtained from the Agricultural Research Station in BeitemRoeselare.

The celery volatiles were recovered by two diffe-

rent methods : a. Likens-Nickerson extraction : a 400 gram sample of the stems of Apium graveolens L. var. dulce was chopped and, after addition of 2 1 distilled water into a 3 1 flask (A),

139

submitted to a simultaneous distillation-extraction procedure using a Likens-Nickerson extraction apparatus.

The

organic solvent flask (B) was filled with 3 ml dichloromethane.

After filling the demixing-return arms with dichlo-

romethane and water (ratio 4/1) the dichloromethane reflux was started by heating B in a oilbath at 80°C.

After 5 min

steam was generated by electrically heating of flask A. After 10 h the steam distillation-extraction was stopped. Due to the small amount of dichloromethane no further enrichment of the volatiles was required, b. Soxhlet extraction : a 50 gram sample of the stems of Apium graveolens L. var. dulce was chopped and the volatile compounds were isolated from the celery by soxhlet extraction with 200 ml diethylether.

After 4 h the extraction was

stopped and the extraction liquid was dried over Na2S0^ and concentrated under reduced pressure to a small volume (+ 1 ml) taking care to avoid loosing volatile celery compounds. As identical levels of the sum of the phthalides were obtained with the described methods both isolation procedures can be used for quantitative determination of key components in celery.

Two dimensional capillary gas chromatography Gas-liquid chromatography was performed with the aid of the MUSIC (Multiple Switching Intellegent Controller) instrument (Chrompack NV) built into a Varian 3700 gas chromatograph equipped with 2 flame ionisation detectors (FID).

The celery

extracts were analysed on a wide bore fused silica "pre" column of 30 m x 0.53 mm i.d. coated with OV1 (0,8 nm film thickness).

The column was temperature programmed from 20°C

to 220°C (2°/min) with a helium carrier flowrate of 4 ral/min. On this apolar phase the phthalides elute in one group without sufficient resolution (Rt=73 minutes).

After 73 min the

phthalide peaks are trapped into a CO- cooled fused silica

140

capillary during 10 minutes and the content of the trap was re-injected and analysed on a narrow bore fused silica "analytical" column of 25 m x 0,25 mm i.d. coated with cyanopropylsilicone (0,21 nm film thickness) (CP-Sil-88, Chrompack NV). The analytical column was temperature programmed from 160°-220°C (2°/min) with a helium pressure of 1.1 bar.

Preparative gas chromatography Preparative gas chromatography was performed on a Varian 1420 equipped with a micro katharometer.

The separation was done

on a packed column, 2 m x 2 mm i.d., glass, filled with 10% Carbowax 20 M on chromosorb W (60-80 mesh).

The column was

temperature programmed from 130°C to 220°C (6°/min) with a hydrogen gas flowrate of 20 ml/min.

The group of phthalides

to be isolated was trapped into an ici-cooled glass tube.

Gas chromatography - Mass spectrometry The GC-MS apparatus consisted of a Hewlett-Packard 5890 gas chromatograph directly coupled to a Hewlett-Packard 59 70 A series mass selective detector. Conditions were as follows : column : fused silica 25 m x 0,25 mm i.d. coated with cyanopropylsilicone (film thickness 0,21 urn) (identical as analytical column used by two dimensional capillary gas chromatography) ; temperature programming : 100-200°C (2°/min); carrier gas : helium 1 ml/min; ion source temperature 220°C; electron energy : 70 eV; accelerating voltage : 250 V.

Mass spectral identification was based on published spectra.

Results and Discussion Typical gas chromatograms of Likens-Nickerson and Soxhlet

141

Figure 1 : GC-MS analysis of celery essential oil isolated by Likens-Nickerson extraction. Normal alkanes, used as internal standard, are indicated on the chromatogram by their carbon numbers. l=3-methyl-4-ethylhexane, 2=a^thujene, 3=a-pinene, 4=camphene, 5=sabinene, 6=g-pinene, 7=myrcene, 8=p.cymene, 9=limonene, 10=ocimene-x, ll=ocimene-y, 12=y-terpinene, 13=allo-ocimene, 14=n-pentylcyclohexadiene, 15=l-terpinene-4-ol, 16=6-caryophyllene, 17=a-humulene, 18=8-selinene, 19=a-selinene, 20=butvlphthalide, 21=PHTHALIDE REGION, 22=unknown

73 83 minutes 0 Figure 2 : GC-MS analysis of celery essential oil obtained by soxhlet extraction. Normal alkanes, used as internal standards, are indicated on the chromatogram by their carbon numbers. l=3-methyl-4-ethylhexane, 2=ci-pinene, 3=ß-pinene, 4=myrcene, 5=limonene, 6=ocimene X, 7=Y-terpinene, 8=terpinolene, 9=npentylcyclohexadiene, 10=butylphthalide, 11=PHTHALIDE REGION, 12=unknown, 13=linoleic acid

142

1

butylhexahydrophthalide

2

Z-butylidenephthalide

3

cnidilide

4

Z-ligustilide

5

butylphthalide

6

trans-neocnidilide

7

cis-neocnidilide

8

senkyunolide

9

E-ligustilide

2 3 4

^jl 0

minutes

Figure 3 : "Phthalide cut" of celery essential oil, obtained by Likens-Nickerson extraction.

extracts from the stems of Apium graveolens L. var. dulce analysed on the "pre" column are presented in figure 1 and 2. Comparison of these figures showes that the soxhlet extraction is less efficient for the isolation of terpenes.

Both GC-MS

analyses show the presence of a badly resolved phthalide region. Gas chromatograms obtained by a "phthalide cut" from both ex-

143

I

V-vJ

_ 73

0 1

butylhexahydrophthalide

2

Z-butylidenephthalide

3

cnidilide

4

Z-ligustilide

5

butylphthalide

6

trans-neocnidilide

7

cis-neocnidilide

8

senkyunolide

9

E-ligustilide 0

83 minutes

26 minutes

Figure 4 : "Phthalide cut" of celery essential oil, obtained by soxhlet extraction. tracts are shown in figure 3 and 4.

After cold trapping of

the unresolved phthalide group and re-injection on the analytical column (heart cut) a clear separation of the phthalides without disturbance of the other peaks is obtained. The compounds identified by GC-MS analysis are indicated in figure 3 and 4.

Nine phthalides were found with trans-neocni-

dilide, senkyunolide and butylphthalide as main representatives. As a conclusion we may state that two dimensional capillary gas chromatography is a convenient procedure for determination of the key components in celery and may contribute to the objective measurement of the quality of the celery flavour.

144

Acknowledgement The authors thank the "Instituut ter bevordering van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw" for financial support and ir. G. Vulsteke (Onderzoeks- en Voorlichtingscentrum voor Land- en Tuinbouw, Beitem-Roeselare (Belgium)) for providing the celery plants.

References 1. Hartwell, J.L. 1971, Iloydia, 3_4, 310. 2. Thellung, A. 1925, Illustrierte Flora von Mitteleuropa, Band V, Tel 2. 3. Limpinuntana, C., P. Chaiars.1977, Varasarn Paesachasarthara 4, 10. 4. Gold, H.J., C.W. Wilson. 1961, Proc. Fla. St. Hort. Soc. 74, 291. 5. Gold, H.J., C.W. Wilson. 1963, J. Food Sei. 2^, 484. 6. Gold, H.J., C.W. Wilson. 1963, J. Org. Chem. 28^, 985. 7. Fehr, D. 1979, Pharmazie _34, 658. 8. Fehr, D. 1981, Pharmazie 36,

374.

9. Gijbels, M.J.M., J.J.C. Scheffer, A. Baerheim Svendsen. 1982, Planta Medica £4, 207. 10. Gijbels, M.J.M., F.C. Fischer, J.J.C. Scheffer, A. Baerheim Svendsen.1985, Fitoterapia J56, 17. 11. Gijbels, M.J.M., F.C. Fischer, J.J.C. Scheffer, A. Baerheim Svendsen.1983, Sei. Pharm. 51, 414. 12. Arctander, St. 1969, Perfume and Flavor Chemicals, Arctander St. (Publ.), Montclair, New Jersey, U.S.A. 13. Yamagishi, T. , H. Kaneshima. 1977, Yakugaku Zasshi 9J7, 237. 14. Bjeldanes, L.F., I.S. Kim. 1977, J. Org. Chem. 4_2, 2333. 15. Gijbels, M.J.M., Thesis University of Leiden, The Netherlands (1983).

EVIDENCE AND CHARACTERIZATION OF GLYCOSIDICALLY BOUND VOLATILE COMPONENTS IN FRUITS

C. S a l l e s , H. Essaied, P. C h a l i e r , J.C. Jallageas and J. Crouzet Centre de Génie et Technologie Alimentaires, Laboratoire de Biochimie Appliquée, Université des Sciences et Techniques du Languedoc, 34060 Montp e l l i e r Cedex, France

Introduction Many years ago, the presence of bound, nonvolatile terpene compounds in muscat grapes has been supposed (1). Later, Bayonove and Cordonnier (2) have shown that v o l a t i l e terpenes were released during acid or enzymatic h y d r o l y s i s of muscat j u i c e . Recently, monoterpene glycosides were i s o l a t e d by reversed phase l i q u i d chromatography (3,4) from Muscat of Alexandria. By acid h y d r o l y s i s of these compounds the aglycones g e r a n i o l ,

linalool,

nerol and a-terpineol and the sugar moieties glucose, 13-rutinose, 6 - 0 - a L-arabinofuranosyl-B-D-glucopyranose were i d e n t i f i e d . In a d d i t i o n , Williams et a l . (5,6) have shown that terpene compounds were also produced by chemi c a l rearrangement of nonvolatile d i o l s and t r i o l s during heat treatment in weak a c i d i c medium. F i n a l l y , the presence of g l y c o s i d i c a l l y bound polyo l s was demonostrated (3,7). Furthermore, in grapes cv. Muscat of Alexand r i a and R i e s l i n g (8) B-D-glucopyranosides, 13-rutinosides and 6 - 0 - a - L arabinofuranosyl-6-D-glucopyranosides of 2-phenylethanol and benzyl a l c o hol were found. All these bound v o l a t i l e components were a l s o detected in other grape c u l t i v a r s

(9,10) as well as in other f r u i t s , such as passion

f r u i t (11), papaya (12), or plant products, such as rose flowers (13), tea shoots (14), N i c o t i a n a s p .

(15).

Some r e s u l t s obtained during preparation of apricot puree (16) and during heat treatment of apricot or mango j u i c e

(17,18) suggested the occurrence

of bound terpene compounds in these f r u i t s . In a p r i c o t , an increase in the concentrations of furanoid l i n a l o o l oxides, nerol oxide and a-terpineol

Bioflavour '87 © 1988 Walter de Gruyter & Co., Berlin • New York - Fainted in Germany

146 was found during f r u i t processing or during heat treatment of the j u i c e . The increase in the concentration of a-terpineol observed during heating of mango juice could not be s o l e l y explained by the production of t h i s compound through chemical rearrangement and oxidation of terpenes, such as myrcene, limonene or B-pinene; thus the presence of g l y c o s i d i c a l l y bound a-terpineol was supposed. In the present work evidence of g l y c o s i d i c a l l y bound v o l a t i l e compounds i n apricot and mango f r u i t s using a rapid a n a l y t i c a l technique and degradat i v e methods i s given. On the other hand, the use of non-degradati ve methods, such as HPLC and s o f t i o n i s a t i o n modes in mass spectrometry for the characterization of these compounds i n grapes, apricot and mango i s d i s cussed.

Results and D i s c u s s i o n The r e s u l t s obtained f o r free and p o t e n t i a l l y v o l a t i l e terpene compounds in various f r u i t s

(cf. 10, 11, 16-20) using the rapid a n a l y t i c a l

technique

described by D i m i t r i a d i s and Williams (19) are outlined in Table 1. Table 1

Contents of free (FVT) and p o t e n t i a l l y v o l a t i l e terpenes (PVT)

in various f r u i t s using v a n i l l i n - s u l f u r i c acid reagent (19)

Fruit

Free v o l a t i l e terpenes

(mg/kg) a ^

Potentially volatile terpenes

(mg/kg) a ^

PVT FVT

Grapes (Muscat of Alexandria)

1.35

6.27

4.6

Passion f r u i t

1.94

6.29

3.2

Roussillon)

0.99

5.40

5.5

Mango

0.13

0.40

3.1

Apricot (Rouge du

expressed as l i n a l o o l

147

The f i n d i n g s obtained f o r grapes agreed with those of D i m i t r i a d i s and Williams (19) described for the cv. Muscat and Alexandria. As to apricot and mango the r e s u l t s showed that p o t e n t i a l l y v o l a t i l e terpenes might be present in these f r u i t s in g l y c o s i d i c a l l y bound form or as polyols as previously postulated (16-18). The values obtained for free and p o t e n t i a l l y v o l a t i l e terpenes were of the same order of magnitude for apricot as for grapes or passion f r u i t . For mango the contents of each of the two forms was lower, but t h e i r r a t i o was the same as found for passion f r u i t ; t h i s value was i n d i c a t i v e of a flavour potential in mango. However, the values shown in Table 1 must be considered approximative, since they were obtained using only l i n a l o o l for the q u a n t i f i c a t i o n .

V o l a t i l e compounds i s o l a t e d a f t e r a c i d i c h y d r o l y s i s of heterosidic extracts of f r u i t j u i c e s The v o l a t i l e compounds i d e n t i f i e d a f t e r a c i d i c h y d r o l y s i s of a crude heteros i d i c extract obtained by chromatography of j u i c e s on a C^g reversed phase column are represented i n Table 2. These r e s u l t s - for grapes in agreement with data previously obtained (4, 8, 10) - suggested the presence of g l y c o s i d i c a l l y bound terpenes and aromatic alcohols in mango and apricot

fruits.

However, some rearrangements of free monoterpene alcohols liberated by cleavage of the g l y c o s i d i c bound v o l a t i l e s have to be considered under the a c i d i c conditions used. Therefore, v o l a t i l e compounds produced by enzymatic h y d r o l y s i s of the heterosidic extract had to be studied, too.

V o l a t i l e compounds i s o l a t e d a f t e r enzymatic h y d r o l y s i s of heterosidic ext r a c t s of f r u i t j u i c e s The g l y c o s i d i c a l l y bound v o l a t i l e compounds i s o l a t e d from apricot and p u r i fied according to S a l l e s et a l . (21) were d i s s o l v e d in a few ml of phosphate-citrate buffer 0.1 M pH 5.0 und hydrolyzed at 40°C during d i f f e r e n t times (12 to 15 h) in the presence of almond B-glucosidase (5 mg per ml) and Pectinol VR (7 mg per ml) for the monoglucoside f r a c t i o n and pectinol VR (7 mg per ml) for the d i g l y c o s i d e f r a c t i o n . Each enzymatic preparation

148

Table 2

V o l a t i l e compounds i d e n t i f i e d a f t e r a c i d i c h y d r o l y s i s of hetero-

s i d i c f r a c t i o n s i s o l a t e d from grape, mango and apricot

(semi-quantitative

estimation: + = low; ++++ = high)

Grape (Muscat

V o l a t i l e compounds

Mango

of Alexandria) t r a n s - L i n a l o o l oxide, furanoid eis-

Linalool oxide, furanoid

Apricot (Rouge du R o u s s i l l o n )

++

+++

++

++

++

++

Linalool

+++

+

++++

a-Terpineol

++

+++

++++

Nerol

+

+

Citronel lol

+

Geraniol

++

Benzyl alcohol

+

2-Phenylethanol

+

Table 3

+ +++

+

+

Amounts (pg/1 f r u i t j u i c e ) a ^ of v o l a t i l e compounds i d e n t i f i e d

after enzymatic h y d r o l y s i s

(almond 13-glucosidase and Pectinol VR) of the

monoglucoside f r a c t i o n i s o l a t e d from apricot

V o l a t i l e compounds

ß- Gl ucosidase

Pectinol VR

(40°C over 12 h)

(40°C over 12 h)

Linalool oxides Li nalool a-Terpineol Citronellol

420

54

1039

81

331

125

66

-

Nerol

515

76

Geraniol

298

48

Benzyl alcohol

552

74

2-Phenylethanol

460

27

Internal standard: 1-octanol

149

Table 4

Amounts (pg/1 f r u i t j u i c e ) 3 ) of v o l a t i l e compounds i d e n t i f i e d

a f t e r enzymatic h y d r o l y s i s (Pectinol VR) of the d i g l y c o s i d e f r a c t i o n i s o lated from apricot

Pectinol VR

V o l a t i l e compounds 40°C over 12 h

40°C over 15 h

Linalool

11

13.5

a-Terpineol

17

22.5

Citronel l o i Nerol Geraniol Benzyl alcohol 2-Phenylethanol

5

-

10

20

547

625

48.5

58

20

25

Internal standard: 1-octanol exhibited the same a c t i v i t y towards p-nitrophenyl-13-D-glucoside. The res u l t s obtained for the monoglucoside and d i g l y c o s i d e f r a c t i o n s are o u t lined in Tables 3 and 4, r e s p e c t i v e l y . These r e s u l t s showed that in a p r i cot the monoglucosides were present in higher q u a n t i t i e s than the d i g l y c o sides ; contrary f i n d i n g s were observed in grapes. The presence of monoglucosides in apricot was attested by the higher s p e c i f i c i t y of 6-glucosidase in r e l a t i o n to Pectinol VR. S i m i l a r r e s u l t s were obtained during a k i n e t i c study of h y d r o l y s i s of neryl-6-D-glucoside by these two enzymes (Fig. 1). After 12 h the substrate was completely hydrolyzed in the prescence of B - g l u c o s i d a s e , whereas only 50% of nerol were released in the presence of Pectinol VR. The more important g l y c o s i d i c a l l y bound v o l a t i l e compounds in apricot were l i n a l y l , n e r y l , benzyl and phenylethyl glucosides and a geranyl

diglyco-

s i d e . The l i n a l o o l oxides i d e n t i f i e d among the v o l a t i l e s i s o l a t e d from the monoglucoside f r a c t i o n of apricot may have been produced through three d i f ferent ways, ( i ) h y d r o l y s i s of a g l y c o s i d i c a l l y bound l i n a l o o l oxide, h y d r o l y s i s of 3 , 7 - d i m e t h y l - o c t - 1 - e n e - 3 , 6 , 7 - t r i o l

(ii)

(5) in free or bound form

150 hydrolysis percent •

O

O

100

50-

0 6

Fig. 1 dase

12

18

24

Time

in

hours

K i n e t i c o f h y d r o l y s i s o f neryl B-D-glucoside by almond B - g l u c o s i -

o—o

and Pectinol VR

•—•

and ( i i i ) o x i d a t i o n of l i n a l o o l present i n large q u a n t i t i e s i n the hydrol y s a t e during the e x t r a c t i o n and concentration steps. As Pectinol i s known to possess g l y c o s i d i c a c i t i v i t y - i t has been used by several authors enzymatic h y d r o l y s i s of dissaccharide glycosides ( 7 , 10) - i t s

for

specificity

towards the d i f f e r e n t s t r u c t u r e s present i n natural products was questionable. Indeed, a f t e r 12 h at 40°C the h y d r o l y s i s o f a p r i c o t d i g l y c o s i d e s and monoglucosides or s y n t h e t i c neryl B-D-glucoside was not achieved. Under these conditions other ways studying g l y c o s i d i c a l l y bound v o l a t i l e compounds had to be explored. Among these, HPLC and s o f t i o n i z a t i o n techniques i n mass spectroscopy may be c a r r i e d out w i t h o u t any h y d r o l y s i s or d e r i v a t i z a t i o n of the compounds under study.

Separation o f g l y c o s i d i c a l l y bound v o l a t i l e components by HPLC on Cyclobond I

Cyclobond I i s a B - c y c l o d e x t r i n chemically bound to s i l i c a g e l ; the separ a t i o n s on t h i s s t a t i o n a r y phase are based on i t s a b i l i t y to form select i v e i n c l u s i o n complexes w i t h guest molecules. A p r e l i m i n a r y study (21) using authentic h e t e r o s i d i c compounds synthesized by condensation o f t e r -

151 pene or aromatic alcohols with a-acetobromoglucose or a-acetobromorutinose (22-24) in anhydrous ethyl ether in the presence of AggO (25) showed the p o s s i b l i t y of separation of these compounds on t h i s phase. The r e s u l t s obtained for g l y c o s i d i c a l l y bound v o l a t i l e s i s o l a t e d from grapes and apricot and p u r i f i e d according to S a l l e s et a l . (21) are outlined in F i g s . 2 and 3. In Muscat of Alexandria grapes, the presence of disaccharide glycos i d e s , i . e . neryl and geranyl B - D - r u t i n o s i d e , and of benzyl-B-D-qlucopyranoside, previously i d e n t i f i e d by Williams et a l . (4, 8) was suggested by HPLC without any h y d r o l y s i s or d e r i v a t i z a t i o n . Other compounds separated by HPLC on Cyclobond I remained unidentified due to the lack of corresponding reference compounds. In contrast to these r e s u l t s , monoglucoside compounds were the main cons t i t u e n t s found i n p u r i f i e d f r a c t i o n i s o l a t e d from apricot j u i c e . Based on the retention times, benzyl, neryl and l i n a l y l glucosides were suggested; t h i s r e s u l t was in good agreement with that obtained by enzymatic hydrolysis. As benzyl B-D-glucopyranoside has been previously i d e n t i f i e d in an aqueous extract of apricot kernels (26) i t may be assumed that t h i s compound occurs n a t u r a l l y i n the f r u i t . On the other hand, the presence of important q u a n t i t i e s of B-D-glucosides of nerol and l i n a l o o l in the absence of measurable q u a n t i t i e s of the corresponding disaccharide d e r i v a t i v e s was in f a vour of the existence of B-D-glucosides as natural products i n apricot fruits.

Mass spectrometry of monoterpene glycosides During the l a s t years s o f t i o n i z a t i o n methods have been used for mass spectrometry studies of n o n v o l a t i l e , polar and thermolabile compounds without preliminary d e r i v a t i z a t i o n . Field Desorption (FD), then Fast Atom Bombardment (FAB) and Desorption/Chemical

I o n i z a t i o n (DCI) was employed to

determine the structures of a number of g l y c o s i d i c compounds

(27-30).

Neryl and geranyl B-D-glucoside and geranyl B - D - r u t i n o s i d e were analyzed

152

Fig. 2 Separation of g l y c o s i d i c a l l y bound v o l a t i l e compounds of grapes cv. Muscat of Alexandria (diglycoside f r a c t i o n ) on Cyclobond I column (25x 0.46 cm) using an a c e t o n i t r i l e - w a t e r gradient (10/90 to 22.5/77.5, V/V) at 1 ml/min. Detection at 210 nm. 1 : benzyl B - D - r u t i n o s i d e , 2 : neryl B-Dr u t i n o s i d e , 3 : geranyl B-D-rutinoside

Fig. 3 Separation of g l y c o s i d i c a l l y bound v o l a t i l e compounds of apricot cv. Rouge du R o u s s i l l o n (whole p u r i f i e d f r a c t i o n ) on Cyclobond I column (25 x 0.46 cm) using an a c e t o n i t r i l e - w a t e r gradient (10/90 to 22.5/77.5, V/V) at 1 ml/min. Detection at 210 nm. 1 : benzyl B - D - g l u c o s i d e , 2 : neryl B - D - g l u c o s i d e , 3 : l i n a l y l B-D-glucoside

153

by these techniques. The FDMS spectrum of neryl B - D - g l u c o s i d e is shown in Fig. 4. Only the molecular ion at m/z 316 was observed using this ionization mode, thus relatively little information may be obtained with this method.

316

1

(

200

Fig. 4

300

FDMS spectrum of neryl B - D - g l u c o s i d e

In the negative ion FAB spectra of geranyl 6-D-rutinoside (Fig. 5) and of geranyl B-D-glucoside (Fig. 6) ions at m/z 179 attributed to the glucose moiety for the two compounds and at m/z 163 attributed to the rhamnose moiety of the rutinose were found besides molecular ions (M-H)

at m/z 315 and

461. Under these conditions, as previously stated (30) for steroid and flavonoid glycosides, negative ion FAB spectra indicated the molecular weight and some information concerning the saccharide moety of the studied molecules. More important information was obtained from DCI spectra in positive mode (Fig. 7 and 8) of geranyl B - D - g l u c o s i d e and geranyl B - D - r u t i n o s i d e . As to the first compound, the quasi-molecular ion (M+NH^)+ at m/z 334 and ions corresponding to the aglycone (A)

m/z 137 and (A.NH^)

m/z 154 and to the

glucose moiety (gluc.NH3)+ m/z 180 and (gluOH+NH^)4" m/z 198 were found. In the case of the disaccharide derivative besides the quasi-molecular ion (M+NH^)+ at m/z 480 and the ions corresponding to the aglycone at m/z 137 and 154, ions characteristic of the nature of the glucose moiety and of the saccharide sequence appeared; the ions at m/z 326 and 344 were character-

154

100-t 461 80-

60-

205 163

Jill 100

Fig. 5

150

200

244

250

305

337 373

300

514

dio

350

400

450

Negative ion FAB mass spectrum of geranyl

500

543

550

575

600

B-D-rutinoside

3

«ft 44, 457 475 —.4 523 331340?361 1 374387 407 l I 374 387Am 407ig Kg"* of berry -: undetected

(b)

26 11 2. 7 2 40 383 1 .4 10 36 6.5 12. 5 3.1 0.7 6. 5 67 87 114 325 196 635

Clonal 77 •(f)

(b)

17 1. 2 0. 3 1 1. 3 7. 5 0. 3 0.3 2. 1 0. 3 0. 6 0. 2 0. 6 0. 5 4. 2 9 16. 5 15 44 A6

Clonal 130 SMA

Clonal 116

(f)

(b)

(f)

(b)

7. 5 0. 4 1. 6 0. 4 3. 9 0. 9 0. 3 2. 6 14 29

1. 9 0. 3 4 0. 4 0. 7 0. 3 0. 2 7 13.5 22

54 4 3 0.2 1.2 0.5 0.7 2.2 9 28

2.0 0.3 17 0.3 0.3 0.2 0.7 2.8 6 12.7

95

90

0. 8

1.4

3

0. 8

0.8

1.5

181

36

-

-

-

-

-

-

70

54

8

20

7

14

9

21

84

334

32

104

15

72

11

62

535

976 36 424 218 77

107

474 52 1004 602 480 525 243

85 -

400 70 640 329 222

83

-

208 40 313 353 93

-

650 550

-

421 622

-

730 732

167 Table 1 refers to certain Chardonnay clonal grapes from Trentino: The relatively high contents of a^ and b have to be pointed out especially in bound forms and even in clones defined as "neutral", such as the 130 SMA and 116 (F). Usually the amount of J) was larger than that of a^ and the quantity of c and e larger than that of d. In Table 2 the results obtained from different white and red, aromatic and non-aromatic varieties are outlined. With these findings, the results shown in Table 1 were confirmed. Table 3

Distribution of free (f) and bound (b) compounds in different

parts of the berry in a Chardonnay grape (Trentino, height 500 m a.s.l.; harvested on 10/2/1986)

compounds

Juice + pulp (f)

tr.-furan llnalool oxide * eis furan llnalool oxide llnalool cH -terpineol tr.-pyran llnalool oxide cis-pyran llnalool oxide citronellol nerol geraniol tr.-geranic acid 2,6-dimethylocta-3 ,7-die ne-2,6-diol 2,6-dimethylocta-l ,7-dle ne-3,6-dlol 2,6-dimethyloct-7-i en-1,,6diol (E)-2 ,6-dime thy locta-2,,7diene-1,6-dlol (Z)-2,6-dlmethylocta-2 ,, 7dlene-1,6-diol nor-lsoprenic diketone (d) 3-oxo-«-ionol (e) benzyl alcohol 2-phenylethanol jig Kg~l of berry undetected

(b)

skin

% in skin

(f)

(b)

(f)

(b) 81 62 85

C free forms £ bound forms

4. 7 0. 3 34 O. 3 15. 5 11 1. 6 1. 3 4. 3 4. 3

1.0 0.3 3.6 0.1 0.4 0.1 0.1 0.2 0.5 5.2

6 1. 2 41. 5 0. 6 16 8. 5 0. 9 4. 8 24 64

4. 3 0. 5 20. 5 0. 1 1. 5 1. 2 0. 1 0. 8 2. 9 8

57 80 55 67 51 44 36 79 85 94

80 85 61

2. 0 1. 9 3. 1 4 16. 5 15 13 6. 1 8. 3 5. 2

18

0.7

23. 5

0. 8

57

53

28

0. 2

42

25

12

5. 5 28

0.6 4

4

15

65

79

4. 2

31

51

71

1 .8

81

73 40 68 66 72

12.5

41

24

16 26.5 47.5 81 31

101

-

291 126

-

52

39

-

-

79 92

-

562 576

42. 5 17. 5 101 121 78.5

-

66 82

2. 1 -

4. 2 6. 4

168 Table 3 shows the d i s t r i b u t i o n of free and bound compounds between berry s k i n and pulp of grapes from a 130 SMA Chardonnay vineyard located at a height of 500 s . a . l . : as outlined in Table 3, compounds a, j) and c - in bound and free forms - s u b s t a n t i a l l y predominanted in the s k i n

(prevailing

the free state over the bound one). This r e s u l t does not correspond to the f i n d i n g s reported for the same clonal type in Table 1. The r e s u l t s were obtained from the l a s t years and w i l l be v e r i f i e d in t h i s year using again the four Chardonnay clones cited (32,33). Obviously, there i s a remarkable analogy of development among the two norisoprenoid substances d and e, and ( Z ) - 2 , 6 - d i m e t h y l - o c t a - 2 , 7 - d i e n e - 1 , 6 - d i o l

- free and

bound - while the diol a showed a d i f f e r e n t behaviour. Thus i t seems to be u n l i k e l y that the s y n t h e s i s of a and t> d i r e c t l y correlates to that of linalool.

Acknowledgement

The authors would l i k e to thank D.J. Cerrato for the E n g l i s h t r a n s l a t i o n .

References 1.

Cordonnier,

R., C. Bayonove. 1974. C.R. Acad. S c i . , Ser. D 278, 3387.

2.

Di Stefano, R. 1982. Vigne Vini 9, 45.

3.

Williams, P. J. , C.R. S t r a u s s , B. Wilson, R.A. Massy-Westropp. 1982. Phytochemistry 21_, 2013.

4.

Williams, P . J . , C.R. S t r a u s s , B. Wilson, R.A. Massy-Westropp. 1982. J. Chromatogr. 2_35, 471 .

5.

Bayonove, C., Z. Gunata, R. Cordonnier. 1984. B u l l . O . I . V . 643-644, 741.

6.

W i l l i a m s , P . J . , C.R. S t r a u s s , B. Wilson, R.A. Massy-Westropp. 1982. J. Agric. Food Chem. 30, 1219.

7.

S t r a u s s , C.R., P.J. W i l l i a m s , B. Wilson, E. D i m i t r i a d i s . 1984. In: Flavour Research of A l c o h o l i c Beverages (L. Nykanen and P. Lehtonen eds). Foundation for Biotechnological and I n d u s t r i a l Fermentation re-

169 search, H e l s i n k i , p. 51. 8.

Grossmann, M., A. Rapp, W. Rieth. 1987. Dtsch. Lebensmittel-Rdsch. 83, 7. ~

9.

Williams, P . J . , C.R. S t r a u s s , B. Wilson, E. D i m i t r i a d i s . 1985. I n : Topics in Flavour Research (R.G. Berger, S . Nitz and P. S c h r e i e r , e d s . ) H. Eichhorn, Marzling, p. 335.

10. Paisarnrat, S . , C. Ambid. 1985. In: Topics in Flavour Research (R.G. Berger, S. Nitz and P. S c h r e i e r , eds.) H. Eichhorn, Marzling, p. 321. 11. Di Stefano, R., L. Corino, P.D. Bosia. 1983. Riv. V i t . Enol. 36, 245. 12. Wilson, B., C.R. S t r a u s s , P.J. Williams. 1984. J. A g r i c . Food Chem. 32, 919. 13. Williams, P . J . , C.R. S t r a u s s , B. Wilson, E. D i m i t r i a d i s . 1985. I n : Progress in Flavour Research (J. Adda, ed.) E l s e v i e r , Amsterdam, p. 349. 14. S t r a u s s , C.R., B. Wilson, R. Anderson, P.J. Williams. 1987. Am. J. Enol. V i t i c . 38, 23. 15. Gunata, Z . , C. Bayonove, R. Baumes, R. Cordonnier. 1985. J. S c i . Food A g r i c . 36, 857. 16. Wilson, B., C.R. S t r a u s s , P.J. Williams. 1986. Am. J. Enol. V i t i c . 37, 107. ' /. Simpson, R.F. 1977. Chem. Ind. ]_, 37. 18. Simpson, R . F . , G.C. M i l l e r . 1983. Vi t i s 22, 51. 19. Di Stefano, R., M. Castino. 1983. Riv. V i t . Enol. 36, 245. 20. Gunata, Z., C. Bayonove, R. Baumes, R. Cordonnier. 1986. Am. J. Enol. V i t i c . 37, 112. 21. Rapp, A., M. Güntert, H. Ullemeier. 1985. Z. Lebensm. 180, 109.

Unters.-Forsch.

22. Scienza, A., G. V e r s i n i , L. V a l e n t i , G. Romano: 196. I n : Comptes Rendus IV e Symposium International sur la Sélection Clonale de la Vigne (R. Bovey and M. Magnenat, e d s . ) . Stat. Fed. Rech. Agron., Wädenswil. p. 91. 23. Gunata, Z., C. Bayonove, R. Baumes, R. Cordonnier. 1985. J. Chromatogr. 331, 83. 24. Rapp, A . , H. Mandery, H. Niebergall. 1986. V i t i s 25, 79. 25. S t r a u s s , C.R., B. Wilson, P.J. Williams. 1987. In: 6th A u s t r a l . Wine Ind. Techn. Confer. (Proceed.), Adelaide, p. 117.

170 26. Schul te-El te, K.H., M. Gadola, G. Ohloff. 1973. Helv. Chim. Acta 56, 2028. 27. Aasen, A.J., B. Kimland, C.R. Enzell, 1971. Acta Chem. Scand. 2 5 , 1481. 28. Aasen, A.J., B. Kimland, C.R. Enzell. 1973. Acta Chem. Scand. 27, 2107. 29. Kodama, H., T. Fujimori, K. Kato. 1981. Agric. Biol. Chem. 45, 941. 30. Strauss, C.R., E. Dimitriadis, B. Wilson, P.J. Williams: 1986. J. Agric. Food Chem. 34, 145. 31. Dimitriadis, E., C.R. Strauss, B. Wilson, P.J. Williams. 1985. Phytochemistry 24, 767. 32. Scienza, A., G. Versini. 1987. In: The Aroma Components of Grapes and Wines (Symposium Proceed.), S. Michele all'Adige, (in press) 33. Versini, G., A. Rapp, A. Scienza, A. Dalla Serra, M. Dell'Eva. 1987. In: The Aroma Components of Grapes and Wines. (Symposium Proceed.), S. Michela all'Adige, (in press)

BIOCHEMISTRY/BIOMIMETIC

STUDIES

ROLE OF HYDROXYLASES IN MONOTERPENE BIOSYNTHESIS

F. Karp and R. Croteau Institute of Biological Chemistry Washington State University Pullman, Washington 99164-6340

Introduction Most of the several hundred naturally occurring monoterpenes are oxygenated compounds (e.g., camphor, carvone, menthol) (1,2) that are of particular significance because of the flavor and aroma properties they impart to the essential oils. The committed step in the biosynthesis of most monoterpenes is the cyclization of geranyl pyrophosphate, yet relatively few of these reactions result in the formation of oxygenated products, most of the parent cyclic compounds being simple olefins (3). These initial olefinic products are oxygenated in subsequent steps which generally involve cytochrome P450 systems and utilize molecular oxygen. In many higher organisms similar oxygenation reactions are often involved in the detoxification of xenobiotics or the biosynthesis of physiologically important compounds such as hormones (4). There have been few studies of cytochrome P450-dependent oxygenations of lower terpenoids in higher plants, but notable exceptions include the hydroxylation of the diterpene olefin kaurene en route to the gibberellin family of plant hormones (5) and the C-8 hydroxylation of the monoterpenols geraniol or nerol en route to iridoids and loganin, which becomes the terpenoid portion of the indole alkaloids (6,7). In this paper we describe the cytochrome

Bioflavour '87 © 1988 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

174

P450 system, provide an overview of monoterpene hydroxylations in microbes, insects and mammals, and report on recent studies of monoterpene hydroxylations in higher plants which have led to the elucidation of a general metabolic strategy for the formation of oxygenated monoterpenes of the essential oils.

Cytochrome P450 Systems Cytochrome P450 mixed function oxidase systems consist of a distinct group of protoheme-containing proteins, a flavoprotein reductase, and sometimes a nonheme iron-sulfur protein or lipid component. Unlike most cytochromes which are simple electron carriers, cytochrome P450 enzymes catalyze numerous oxygenation reactions, most notably hydroxylations, utilizing molecular oxygen and electrons derived from NADPH or HADH, which are delivered by the reductase component(s) of the system. The cytochrome functions as the oxygen-activating terminal electron acceptor and substrate-binding moiety of the complex. The term P450 resulted from the observation that the reduced hemoprotein binds CO and that in this inactive form gives rise to a prominent absorbtion at 450 nm (8). Photoreversal of CO binding, and consequent relief of inhibition of oxygenation function, also has a photoaction spectrum maximum at 450 nm (9), and this photoreversible inhibition has become a standard means for demonstrating the participation of cytochrome P450 in a given reaction. Cytochrome P450 systems are widely distributed in nature, from primitive bacteria (10,11) and yeast (12) to higher plants (13-15) and all types of animals (16-19). The systems vary considerably in form, from the microsomal type in which both cytochrome and flavoprotein reductase are membranous and the reductase contains one mole each of FAD and FMN for the electron transfer from NADPH (20), to bacterial types in

175

which both protein components are soluble, with the reductase containing a single FAD prosthetic group and NADH as the usual electron source (21). Bacterial and certain mammalian systems, also employ an additional iron-sulfur protein (e.g., putidaredoxin, adrenodoxin) which serves to transfer reducing equivalents from the reductase to the cytochrome (21,22). Substrate specificity varies enormously depending on the function of the cytochrome system. The catalytic cyclic (Fig. 1) begins with the binding of the substrate to the oxidized form of the cytochrome. One electron from a reduced pyridine nucleotide is transferred via the reductase component(s) to reduce the substrate-cytochrome complex, to which a molecule of dioxygen binds and a second electron is transferred. One oxygen atom is then lost (as water) while the remaining oxygen atom is bound to the substrate which is then released in hydroxylated form with concomitant recycling to the oxidized cytochrome in preparation for another cycle (23). ROH

Figure 1.

The cytochrome P450 catalytic cycle.

176

Monoterpene Hydroxylation by Microbes A broad range of microorganisms can utilize monoterpenes as a sole carbon and energy source (24), and the hydroxylation of these compounds by such organisms most often represents an early step in catabolism. Since the monoterpenes are adventitious substrates for these organisms, it is not surprising that these catabolic enzymes are under inductive or derepression control. Several bacterial cytochrome P450 systems involved in the catabolism of monoterpenes (camphor, linalool, 4i_-cymene) have been well-documented, and circumstantial evidence suggests the existence of many others. Cytochrome P450cam from Pseudomonas putida catalyzes the hydroxylation of (+)-camphor to 5-exo-hydroxycamphor (Fig. 2) and is the most thoroughly studied of these systems (25). It is characterized by rapid inducibility by a narrow range of analogs, relatively strict substrate specificity and high turnover number (19,25,26). This system yielded the first detailed spectral data on the cytochrome and has provided much of the basis for our present understanding of electron transfer, reductive cleavage of dioxygen, substrate activation and oxygen insertion (27), including the stereochemical details (28,29). This was the first system to be dissected into its components parts and to afford a pure, crystallized cytochrome (30). Genes coding for the hydroxylase proteins have been located on an extrachromosomal, transmissible plasmid, and the P450cam gene has recently been cloned and sequenced (31,32). A soil diptheroid (Rhodococcus) converts camphor to 6-endo-hydroxycamphor (Fig. 2) as the first step in catabolism, and after several subsequent transformations the pseudomonad and diptheroid degradative pathways converge (33). Cytochrome P450cym has been from isolated from another strain

177

of P. putida capable of growth on _p_-cymene (Fig. 2) . This cytochrome catalyzes the C-7 hydroxylation of the aromatic monoterpene to cuminyl alcohol as the first step of the degradative sequence which ultimately involves hydroxylation on the ring. Three pathways for the degradation of limonene by the same organism have been described (Fig. 2), two of which are allylic oxidations at C-6 and C-7 to yield carveol and perillyl alcohol, respectively, and the third is an epoxidation at C-l - C-2 which ultimately gives rise to the 1,2-diol and related products (34-38). The latter pathway (attack at C-l - C-2) appears to operate in several fungi as well (39). Several microorganisms are capable of growth on bicyclic olefins such as the pinenes and camphene since they are able to carry out prototropic rearrangements to limonene or borneol/isoborneol (and thus camphor) from which the aforementioned pathways apply (34,40,41).

10' Tr Jl-Cymene

Figure 2.

Monoterpene hydroxylase substrates.

178

The metabolism of linalool and related acyclic monoterpenes by Pseudomonas incognita has been well-documented (42-44), and two distinct substrate-inducible pathways appear to be involved in the formation of 8-hydroxy and 10-hydroxylinalool (Fig. 2). The responsible cytochrome P4501in systems have been examined in some detail, and they differ in subcellular location, spectral properties and other characteristics (43,45-47). Geraniol and nerol (Fig. 2) also undergo 10-hydroxylation and C-2 - C-3 double bond epoxidation in this organism; however, subsequent steps in these catabolic pathways differ from that of 10-hydroxylinalool. Bacillus cereus, isolated from the gut of Ips beetles, catalyzes the C-4 hydroxylation of a-pinene to trans-verbenol (major product) and cis-verbenol (Fig. 2) (48) , and is presumed to play a role in pheromone production. Aspergillis niger also produces cis-verbenol from a-pinene and transforms B-pinene by C-3 hydroxylation to pinocarveol (Fig. 2) among other products (24). The involvement of cytochrome P450 in the metabolism of pinenes by these microbes has yet to be confirmed.

Monoterpene Hydroxylation by Insects Cytochrome P450 systems in insects are involved in the metabolism of xenobiotics, such as pesticides, and of hostplant allelochemicals, and in the production of hormones and pheromones. The ability of dietary monoterpenes to induce high levels of cytochrome P450 and associated hydroxylase activities is particularly notable (49-51). Although many monoterpenoid-type inducers are subsequently metabolized (49,51,52), the more general effect of such cytochrome induction appears to be the conferring of resistance to poisoning by other, often diverse, xenobiotics (53), which

179

may have important ecological consequences (54) as well as significance in pest management (55). Some host-plant monoterpene olefins are transformed by insect cytochrome systems (or by those of symbiotic microbes) into oxygenated species with specific pheromone functions. Thus, the production of aggregation pheromones by numerous species of bark beetles involves the conversion of a-pinene by C-4 hydroxylation to cis- and trans-verbenol and by C-10 hydroxylation to myrtenol, and of B-pinene by C-3 hydroxylation to trans-pinocarveol (Fig. 2) (56-58). The particular product mixture varies with the species of beetle and host-tree chemistry, but ultimately depends upon the enantioselectivity, regiochemistry and stereochemistry of the responsible cytochrome P450 system(s) (57,58). The acyclic olefin myrcene is transformed by C-5 hydroxylation in several beetle species to ipsdienol (Fig. 2) which may also function as an aggregation pheromone (59). Most transformations of this type appear to involve direct allylic hydroxylation; yet, epoxidation followed by various allylic rearrangements may also play a role, as in the conversions of a-pinene oxide (60). Insect microsomal cytochrome P450 systems have been amply demonstrated (49,51,61); yet, in relatively few instances have specific inhibitors been employed or substrate binding spectra obtained to confirm the involvement of the cytochrome in monoterpene metabolism (49,51,53,60).

Monoterpene Hydroxylation by Mammals Considering the widespread occurrence of monoterpenes in the mammalian diet, surprisingly little work has been published on monoterpene metabolism. Since a major function of monoterpene oxygenation in mammals appears to be conversion to water-soluble excretion products, many early studies involved

180

the analysis of urinary metabolites. In rats and rabbits, myrcene (Fig. 2) is converted to a mixture of 1,2- and 3,10-diols via hydration of the corresponding epoxides (62,63). The isopropylidene function is unaltered, as are the allylic methyl groups, in contrast to the metabolism of the olefin by microbes (64). o-Pinene and B-pinene afford, by C-4, C-10 and C-3 hydroxylation respectively, the verbenols, myrtenol and pinocarveol (60,63,65,66), much like insect systems, although the regio- and stereochemistries may differ. Prototropic ring-opening also occurs (63,65), much like with microbial systems (34,67). Camphene, in rabbits, gives rise to a variety of hydroxylated metabolites (68) and in many animals limonene is converted to perillyl alcohol (C-7 hydroxylation) and carveols (C-6 hydroxylation) (69,70), in addition to 1,2- and 8,9-epoxides and the corresponding glycols (71), and metabolites bearing a hydroxyl group at C-l as a result of allylic rearrangement of the 1,2-epoxide (70). 41-Cymene yields _p.-cymen-9-ol and _p_-cymen-8-ol (Fig. 2) as urinary metabolites in the rabbit (63), but only C-7 hydroxylation (cuminyl alcohol) is observed in the koala and brushtail possum (72). The choice of the latter two species for metabolic studies is not capricious since both animals can efficiently survive solely on the monoterpene-rich leaves of Eucalyptus and excrete large quantities of monoterpenoid glucuronides. The metabolism of oxygenated monoterpenes has been approached in a manner similar to that for olefins, with an emphasis on detoxification and excretory mechanisms. In dogs, rabbits and rats, (+)-camphor (Fig. 2) is oxidized to 5-endo-hydroxycamphor with lesser amounts of the 5-exo- and 3-endo-isomers (73,74). This contrasts with the pseudomonad pathway in which 5-exo-hydroxycamphor is the primary product (25,75). In mammalian systems a broad spectrum of structurally diverse lipophilic substances are oxidized by cytochrome P450.

181

Multiple forms of the hemoprotein element having

different

but overlapping substrate specificities are known, and the complement of isozymes present in any particular organ varies widely due to physiological and genetic factors

(19,76). In

vitro experiments on monoterpene metabolism, mainly in liver and lung systems, have demonstrated the involvement of cytochrome P450, binding phenomena, inducibility of the cytochrome and oxygenase activity, and, occasionally, the destruction of the system by certain terpenoid compounds. Although myrcene does not induce cytochrome P450 in rat liver microsomes, the olefin does exhibit a typical cytochrome P450 type I binding spectrum (indicative of a shift in the spin state of the bound heme), and oxygenation is prevented by classical cytochrome P450 inhibitors

(77). Conversely,

a-pinene is an inducer of two types of rat liver cytochromes (P450 and P451) w h i c h hydroxylate the olefin at different rates and produce different product mixtures

(60,65). A

reconstituted cytochrome P4501m2 system from induced rabbit liver microsomes converts 8-pinene to the corresponding oxide (and to myrtenol by allylic rearrangement) and to pinocarveol by direct allylic hydroxylation at C-3 (66). A similar cytochrome system was employed to study binding

interactions

w i t h ji.-cymene (78) , w h i c h is hydroxylated at the isopropyl group and secondarily at the methyl substituent but which does not appear to undergo ring hydroxylation in mammals (79). Camphor, a-terpineol and isobornyl acetate all give rise to typical type I binding spectra and competitively inhibit cytochrome P450-dependent activities, but only isobornyl acetate was reported as a potent inducer of the cytochrome (74,80). Pulegone and carvone likewise yield typical binding spectra, and both result in the loss of cytochrome function. Carvone is responsible for the conversion of cytochrome P450 to the inactive P420 form, whereas incubation w i t h pulegone results in the loss of the heme function

(81).

182

The effectiveness of (+)-limonene in solubilizing accumulated cholesterol has led to considerable interest in the metabolism of this monoterpene olefin (82). Limonene does induce the production cytochrome P450 in mammals (83) and gives rise to a typical type 1 binding spectrum with liver microsomal systems (84). As noted earlier in the discussion of urinary metabolites, the cytochrome-dependent oxygenation of this olefin yields a variety of products derived from direct hydroxylation or via epoxides, the precise mixture being dependent on the particular mammalian species. Many of these transformations have now been demonstrated in microsomal preparations. The acyclic alcohols geraniol, nerol and linalool often serve as inducers of the cytochrome (77) and as hydroxylase and/or epoxidase substrates in liver and lung microsomal systems (77,84). The primary site of hydroxylation on geraniol and related compounds is the allylic C-8 methyl (Fig. 2), which is thus similar to the specificity observed in higher plants in the conversion of geraniol to loganin and related metabolites (6,7). Epoxidation of the 2,3- and 6,7-double bonds of geraniol and nerol has also been observed in liver which has been induced with phenobarbital (85). Unequivocal evidence for the involvement of cytochrome P450 in the C-8 hydroxylation of nerol and geraniol was provided with rabbit liver microsomes and with the reconstituted cytochrome P4501m2 system. In this case (84,85), the hydroxylase was inhibited by CO and other specific antagonists. Binding spectra showed a type 1 response for nerol but modified type 2 for geraniol suggesting interaction of the substrate hydroxyl function and heme moiety in this instance. A survey of the reactions of monoterpenes catalyzed by mammalian cytochrome P450 systems allows few generalizations, with the exception that the presence of a tri-substituted double bond tends to strongly promote allylic hydroxylation, whereas di-substituted double

183

bonds and eoco-methylenes foster epoxidation (71,86).

Monoterpene Hydroxylation by Plants The previous sections have amply demonstrated the involvement of cytochrome P450 in monoterpene hydroxylation throughout the major phyla, and made brief mention of such reaction types in higher plants which are, in fact, the major source of monoterpenes in nature. Gymnosperms produce primarily monoterpene olefins and the Angiosperms are dominated by oxygenated derivatives. Both types of monoterpenes arise, for the most part, via the conversion of the universal precursor geranyl pyrophosphate to olefinic products. In Angiosperms, these olefinic parent compounds undergo an impressive array of secondary transformations to produce oxygenated metabolites. Great difficulties have been encountered in studying hydroxylase systems and their cytochrome components in plants because of the rigorous extraction techniques imposed by the presence of a rigid cell wall and ubiquitous inhibitory phenolics and resins, as well as photosensitizing pigments. Additionally, subcellular separations are often compromised by the inability to isolate "pure" fractions free of adsorbed pigments and not contaminated by other organelle or membrane fragments (87). Fortunately, the enzymatic machinery for monoterpene biosynthesis and the storage reservoir for these compounds are sequestered in epidermal glandular structures (modified trichomes) in many herbaceous species (88). This unique feature has allowed the development of a highly selective, rapid extraction procedure for the isolation of gland contents (without disrupting the underlying plant tissue). The surface abrasion technique (89) , when coupled to refinements in capillary GLC-MS analysis for sub-nanomole

184

levels of metabolites (without need for radiolabeled substrates), has permitted the examination of a wide range of cytochrome P450-dependent monoterpene hydroxylase systems. Early in vivo studies in this area were directed toward the establishment of the relevant metabolic pathways for the conversion of parent olefins to their oxygenated derivatives. Thus, tracer studies with several plant species were employed to deduce that (+)-sabinene was transformed to (+)-transsabinol and thence to (+)-3-thujone and (-)-3-isothujone (Fig. 3) (90). Similar types of experiments with peppermint established that (-)-limonene, via (-)-trans-isopiperitenol, was the precursor of menthone and menthol, and that this same olefin gave rise to (-)-carvone, via (-)-trans-carveol, in spearmint (91) (Fig. 4).

c pyroi

(-)-3-lsothujone

Figure 3.

Biosynthesis and in©ta.bo 1 ism of (+) — sabinsnc.

185 x

-

X

Ó-4 -Ó4 p

"

x

x

—

-CK I tu c a) G O e •h

IH O

ew

o Cd 4-1 a) 0 X)

G

efl to •H CO a) 4->

A G ^

w O CO

•H

a) bC

186

Evidence for the role of cytochrome P450 in sabinene hydroxylation came with the development of a suitable cell-free enzyme system from sage (Salvia officinalis) leaf epidermis from which endogenous terpenoids were removed by repeated treatment with polystyrene beads. The hydroxylase was located in the light membranes (microsomes), showed an absolute dependence on molecular oxygen, preferred NADPH as reductant, was photoreversibly inhibited by CO, gave typical CO-difference and substrate binding spectra, and exhibited sensitivity to classical cytochrome P450 antagonists (15). In most characteristics, the plant microsomal system was similar to eucaryotic microsomal cytochrome P450 systems, and unlike bacterial and mitochondrial forms. A series of detergents was examined and sodium cholate was found to be most suitable in terms of efficiency of solubilization and hydroxylase stability. On removal of detergent, the solubilized hydroxylase system is easily maintained and yields superior difference and substrate binding spectra. As indicated above, limonene plays a pivotal role as progenitor of a broad range of oxygenated p-menthane monoterpenes. This olefin is of particular interest because of its widespread occurrence and because allylic hydroxylation could, in theory, yield three well-known metabolites (carveol,isopiperitenol, perillyl alcohol) which, however, rarely co-occur in the essential oils. Epidermis extracts from peppermint (Mentha piperita) have yielded very active microsomal preparations which convert limonene to trans-isopiperitenol (Fig. 4). This allylic hydroxylation is oxygen and NADPH-dependent, photoreversibly inhibited by CO, and fulfills other criteria indicative of the involvement of cytochrome P450 (see below). Both limonene antipodes give trans-isopiperitenol as product, but the system shows a strong preference for the (-)-isomer. Moreover, limonene is attacked specifically at C-3, and no other oxygenated derivatives (epoxides, diols, etc.) can be

187

detected as products. Importantly, the isomeric olefin terpinolene is completely unreactive as a substrate, confirming earlier in vivo observations that this olefin was not a precursor of oxygenated terpenes in mint (91). All subsequent steps leading from (-)-trans-isopiperitenol to the menthol stereoisomers (Fig. 4) have been demonstrated in cell-free extracts from peppermint and each of the soluble enzymes involved in this pathway has been characterized (91-93). It is notable that the general metabolic strategy employed in the menthol pathway is the same as that employed in the conversion of sabinene to thujone and isothujone; that is, an allylic hydroxylation followed by oxidation to the a,8-unsaturated carbonyl compound and then conjugate reduction (94) . In the case of menthol synthesis in peppermint, two such conjugate reductions occur and the carbonyl function is ultimately reduced.

Both of the aforementioned

pathways provide excellent examples of enzymes which exploit the inherent chemical reactivity of their substrates. Spearmint (Mentha spicata) produces an oil containing primarily (-)-carvone, presumed to be derived from (-)-limonene via (-)-trans-carveol (95), and microsomal preparations from leaf epidermis catalyze the cytochrome P450 dependent hydroxylation of (-)-limonene to (-)-trans-carveol as the sole product (no other oxygenated metabolites are detectable) (Fig. 4). (-)-Limonene is the naturally occurring enantiomer in spearmint. Interestingly, (+)-limonene is converted by this system (at slower rates) to only (+)-cis-carveol, the isomer typical of caraway and dill, which leads to (+)-carvone. Green perilla (Perilla frutescens var. crispa) accumulates perillyl aldehyde in the essential oil, and this product is presumed to be synthesized in the large resin-containing glands on the lower leaf surface. Extracts of these glands afford a membranous cytochrome P450 system that converts limonene to (-)-perillyl alcohol as the exclusive

188

oxygenated product (Fig. 4). The enzyme system hydroxylates both (+)- and (-)-limonene and exhibits little enantioselectivity. Hyssop (Hyssopus officinalis) produces an essential oil containing isopinocamphone and pinocamphone, with lesser amounts of myrtenol, as well as the olefins a-pinene and B-pinene which are thought to be the initial cyclic products in this species and the precursors of the oxygenated derivatives. Microsomal preparations from the oil glands of hyssop efficiently convert (-)-B-pinene to (+)-trans-pinocarveol, and it is presumed, but not yet demonstrated, that pinocarveol is oxidized to pinocarvone and the conjugated double bond then reduced to yield pinocamphone and isopinocamphone (Fig. 5) by a pathway analogous to that for the metabolism of sabinene (Fig. 3). Most dehydrogenases and reductases are operationally soluble enzymes, requiring a different experimental approach than that employed for the membranous hydroxylase systems. A minor product of B-pinene metabolism in hyssop is myrtenol, suggestive of the allylic rearrangement of B-pinene oxide as observed in mammalian systems (66); however, B-pinene oxide could not be detected as a metabolite of B-pinene with the hyssop microsome system. On the other hand, (-)-a-pinene was very efficiently transformed to myrtenol by this preparation (Fig. 5). Both the allylic hydroxylation of B-pinene to trans-pinocarveol and of a-pinene to myrtenol (no verbenol was observed) are cytochrome P450-dependent, but the number of cytochrome species responsible for these two reactions is at present uncertain because the microsomal system has yet to be solubilized and reconstituted from the purified enzyme components. The many similarities of the olefin hydroxylases described above allows many properties of these systems to be described together. Normal activity for these microsomal preparations

189

Figure 5.

Metabolism of pinenes.

ranges from 0.24 to 0.76 nmol product/min-mg protein with activity optimum at about pH 7.4. The requirement for molecular oxygen in catalysis is absolute, and H 2 02 and organic hydroperoxides fail to support the reactions tested. NADPH is the preferred reductant in all cases, with NADH only 10-30% as efficient. Cytochrome C effectively competes in a typical manner for reducing equivalents transferred from the reductase component of the systems. The cytochrome reductase is thought to contain equimolar amounts of FAD and FtlN. These prosthetic groups are unusually prone to dissociate from plant-derived microsomal systems, thus micromolar flavin supplements often improve reaction yields by 10-40%. Progressive inhibition of hydroxylation activity by increasing levels of CO is seen in all cases, which is one of the most characteristic features of cytochrome P450-dependent reaction types. C0/0 2 binding is competitive and at a C0/0 2 ratio of 9:1, 60-90% inhibition of hydroxylation is observed in the dark. Irradiation of the reaction mixture with blue light (450 nm) relieves CO-dependent inhibition.

190

Response to specific inhibitors has provided further supporting evidence for the involvement of cytochrome P450 in olefin hydroxylation by higher plants; however, four of the classical cytochrome P450 inhibitors (metyrapone, SKF-525a BHT and ellipticine) are relatively ineffective with the olefin hydroxylases (estimated I 5 0 > 1 mM), probably because of the very high substrate and oxygen binding affinities of these plant-derived enzymes. Substituted imidazoles, which are potent antimycotic agents, act as heterocyclic ligands which bind to both the oxidized and reduced forms of the cytochrome and compete with both substrate and oxygen, and they have provided very useful probes of the olefin hydroxylases. Thus, imidazole is a potent inhibitor of sabinene hydroxylase, miconazole inhibits spearmint-derived limonene C-6 hydroxylase, and clotrimazole inhibits all of the olefin hydroxylases at micromolar concentrations. Ancymidol, a powerful plant growth retardant, acts by inhibition of the cytochrome P450-dependent oxidation of the diterpene olefin kaurene and its derivatives en route to gibberellins, but this agent is completely without effect on monoterpene olefin hydroxylation. Direct spectral evidence for the presence of microsomal cytochrome P450 is difficult to obtain in preparations from whole leaf extracts because of the presence of interfering pigments, and suitable spectra of the sabinene hydroxylase from sage were obtained only after the system was solubilized with sodium cholate (15). Microsomes prepared from oil gland extracts contain minimal interfering materials since the leaf mesophyll cells remain largely intact by this procedure and thus release little chlorophyll or bulk protein. Such preparations exhibit typical type 1 substrate binding spectra, and provide maxima at 450 nm (or slightly higher) upon reduction with NADPH or dithionite. Calculations based on dithionite-reduced, CO difference spectra indicate yields

191

of from 0.83 to 1.64 nmol of cytochrome P450 per mg of microsomal protein, and reaction yields of 0.42 to 1.14 nmol product/min per nmol of cytochrome, figures which compare quite favorably with several plant (7,87), insect (49,51), and animal (19) microsomal systems. Many cytochrome P450 systems are inducible, resulting in higher cytochrome titers and enhanced oxygenase activity.

In

higher plants, induction has been accomplished by wounding and other stresses (96) and by exposure to xenobiotics or substrates of the oxygenase system (97). Exposure of sage plants to sabinene, and to several known herbicide-type inducers, failed to evoke any response. Etiolated sage seedlings grown in complete darkness are both capable of monoterpene biosynthesis and of producing oil glands. Exposure of these seedlings to 15 minutes of white light produces a three-fold increase in sabinene hydroxylase within 24 hours, and results in a notable increase in oxygenated monoterpenes in the oil. Similar light induction of cytochrome P450-dependent activities has been observed in other species of dark germinated seedlings (98,99). Perhaps the most unusual feature of the monoterpene olefin hydroxylases is their extreme substrate selectivity and the absolute regio- and stereospecificity in product formation. The search for reaction products in addition to these specified earlier has been exhaustive and uniformly negative, and over a dozen alternate olefin substrates have been tested with each system without success. The mutually exclusive, highly specific reactions observed with this set of monoterpene hydroxylases are in sharp contrast to many similar reactions in mammalian and microbial systems where the primary function is detoxification or carbon/energy metabolism. Rather, monoterpene hydroxylation in plants appears to be highly directed, much like monoterpene metabolism in

192

insects for the purpose of pheromone production; yet, such well-defined ecological or metabolic functions for the oxygenated monoterpenes in plants are not clear. The question of function notwithstanding, it is clear that these highly specific enzymes are the key catalysts w h i c h initiate a complex set of reactions (the allylic oxidation - conjugate reduction pathway) for the conversion of progenitor monoterpene olefins to their numerous oxygenated derivatives. Indeed, the allylic

hydroxylation-oxidation-conjugate

reduction pathway, and its simple variants, may account for well over half of all the monoterpenes of the essential oils. Based on the specificities of the hydroxylases examined thus far, it w o u l d appear that a large number of highly

selective

enzymes must be dedicated to monoterpene m e t a b o l i s m in higher plants.

Acknowledgement The research by the authors was supported by NSF grant no. DMB-8507121 and D O E grant no. DE-AT06-82ER12027.

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1035.

ODORIFEROUS POLYENE HYDROCARBONS FROM MARINE AND TERRESTRIAL PLANTS

Wilhelm Boland Institut für Organische Chemie der Universität Richard-Willstätter Allee, D-7500 Karlsruhe, FRG

Since the original work of R.E. Moore on odoriferous compounds from two species of Hawaiian Dictyopteris (1) considerable progress has been made in this field. To the smell of the ocean contribute mainly the numerous sulfur or halogen compounds, but also isoprenoid and nonisoprenoid

hydrocarbons

(2). Among the latter, a group of fatty acid derived C g and Cj^ olefins has gained particular interest, since most of them also act as powerful pheromones and/or release factors for male gametes of marine brown algae (3,4). Their occurrence, however, is not restricted to the marine environment. dealing with C ^

Reports

hydrocarbons as e.g. aroma components of ter-

restrial fruits and plants increase quickly. Many other polyenes of various chain lengths are found in algae and terrestrial plants as well. Some are released into the biosphere, and ecobiological functions may be therefore assumed. Special methods of enrichment, structure elucidation, determination of absolute configurations and enantiomeric purities as well as reliable biological activity tests had to be developed to obtain deeper insight into their mode of action, and ecobiological

significance.

Bioflavour '87 © 1988 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

biochemistry

200 1. Isolation and Structure Determination 1.1 Cg and C.^ hydrocarbons Like many other bioactive compounds these pheromone hydrocarbons are produced in very minute quantities. A successful enrichment takes advantage of their high volatility using the "Closed-Loop-Stripping"

technique (5) and living cultures. If

adequate conditions (illumination, temperature etc.) are maintained, the stripping period can be extended to several weeks and leads to sufficient accumulation of the trace organics. c Enrichments up to 10 fold are possible.

Figure 1. Extraction apparatus (4)

E = extraction F = charcoal

vessel

filter

P = pump S = sample all parts are commercially available. After desorbtion from the charcoal traps the samples can be processed with any highly sensitive analytical method like GC, GC/MS, GC/FTIR or HPLC. Best separations of the isomeric

C^

hydrocarbons (cf. Table la) are achieved by GC on unpolar coatings (SE 30, 0V 101; 50m x 0.32mm) or combined columns of low and moderate polarity (SE 30 + OV 17; 25m x 0.32mm, each). The Kov&ts index of all of the compounds has been reported and may

201

Table la

Sexual pheromones of brown algae (3,4)

cylopropanes

cyclopentenes

\\

cycloheptadienes

//

//

undecapolyenes

octatriene

Figure 2

Mass fragmentation of C ^ H ^

m/z =66

W]

hydrocarbons

W-]

m/z =82

202 be used for rapid identification. The mass spectra (70 eV) of the trienoic cyclopropanes, cyclopentenes and cycloheptadienes exhibit characteristic even numbered fragments (m/z = 66 and m/z = 82) which become dominant, if low voltage (17 eV) is applied. They are of considerable value for structural

assign-

ments and for the determination of the position of a stable isotope label (6). A plausible pathway for their formation is outlined in Figure 2. Irrespective of the high enrichment factor the collected amount of pheromones and related material usually does not exceed the lower ug or upper ng level and prevents the application of NMR-techniques. Furthermore, complex bouquets rather than single compounds are often observed

(7,8,9).

Thermal lability of compounds, easily recognized by comparative GC with cold and hot injection systems is indicative for Cope rearrangements of cyclopropane- or sigmatropic

hydrogen

shifts of 1,3Z,5Z-triene moieties (9). Microhydrogenations with H2 or

and microchemical operations with solid suppor-

ted reagents like N a B H 4 , LiAlH^ and

pyridiniumchlorochromate

(10) are other valuable tools for functional group and GC/MS analysis. If new structures emerge, biosynthetic ties and synthetic approaches have the highest

plausibili-

effectiveness.

Related compounds without known biological activity and divergent configuration (8) are also found. Most of them were observed as byproducts or trace components (7,8) in the pheromone blends of brown algae (Table lb). Cg and C ^

olefins

iso-

lated from terrestrial plants or sweet water sources are summarized in Table lc.

203

204 1.2

Long chain 1-alkenes

Two highly unsaturated and unbranched

C

21-5

and

C

21-6 Poly~

olefins were first isolated from the Australian phaeophyte Fucus vesiculosus (21). Later it was shown that these two compounds are particularly abundant in all brown algae. The chain length ( n - C 1 5 to

n

~c2i^

and

de

9ree

unsaturation

(monoenes

to hexaenes) of this type of oddnumbered 1-alkenes may vary considerably

(22,23).

Their odor ist characterized as 'seaweed' or 'algae'. While the fruity, sometimes flowery note of the C ^

hydrocarbons is

an intrinsic property, the odor quality of the C 2 1 penta- and hexaenes is due to some products of oxidative degradation. Besides the olfactory less satisfactory 5-hexenal, the unsaturated aldehydes (3Z,6Z)-3,6,11-dodecatrienal, 12-tridecatrienal and

(2E,4Z,7Z)-2,4,7,-

(3Z,6Z,9Z)-3,6,9,14-pentadecatetraena1

mainly contribute to the typical 'seaweed' or 'algae' odor (24). The biological function of these polyenes and whether or not, or to which extend, they are released into the marine environment, either intact or degradated, is not known. The same type of 1-alkenes, usually represented by the lower homologues in the range of C^g to C^y is found in the roots and germinating seeds of numerous terrestrial plants (12). In defense secretions of arthropods 1-alkenes (usually monoenes)

205 of various chain lengths (n-C g to n - C l g ) act as 'biosolvents' (25). Some oddnumbered vinylic acetylenes seem to fit into the same general scheme (26).

Their structures followed from UV, IR, MS and

1

Ozonolysis (22) and chemical ionization with N 0

H-NMR data.

+

(13) have

been used to determine the position of double bonds. A number of these polyenes has been synthesized for structural

confir-

mation, either de novo, or from appropriate fatty acid precursors (21,27).

13

C NMR data are available (27). Even numbered

hydrocarbons are also known, but usually only in traces (13). Occasional overlap between o(- and B-oxidation may be responsible for the odd/even chain lengths observed. The function and biochemistry of the compounds is unknown.

1.3 Optical purity and absolute configuration of C.... cyclopolyenes. The alicyclic C ^

pheromones have one or two asymmetric car-

bon(s) and may be formed as pure enantiomers or as enantiomeric mixtures in order to achieve species specific

interaction.

While the absolute configuration of two cycloheptadienes

and

two cyclopropanes from Hawaiian Dictyopteris spp. followed

206 from oxidative degradation (2), none of the pheromone is productive enough to allow for such wasteful

sources

approaches.

Chromatographic separations of the enantiomers are more sensitive and have been developed recently. Using cellulose

triace-

tate(benzoate) as chiral stationary phases, the absolute configuration and enantiomer excess of a cyclopropane- (28) and a cyclopentene pheromone (29) was determined. To establish the relative retention of the enantiomers, synthetic (+)- or ( - ) enantiomers are required. Due to the high UV absorption of the diene moiety (£ ca. 30 000) ng quantities are sufficient for analysis. Figure 3

ANALYTICAL

Separation of (j-)-hormosirene by inclusion chromatography on cellulose tribenzoate (28)

CONDITIONS:

Stationary phase: cellulose tribenzoate, 8 0 / 2 0 v/v. F l o w :

25 c m x 0.46 c m . E l u e n t :

methanol/water,

1.2 m l / m i n : D e t e k t i o n : UV, 247 nm. Sep. factor: (< = 1.40

207 Enantiomeric mixtures have been previously demonstrated to occur in insects. Table 2 shows the same to be true for the plant kingdom. The biological significance of the findings for the chemical communication of gametes of brown algae remains to be established. Table 2

Absolute configuration and enantiomer excess of hormosirene from various brown algae (28)

Species

Locality

major enantiomer

enantiomeric excess (e.e)

[%]

EUrvillaea potatorum

Sorrento (Victoria, Australia)

(-) —1R,2 R

51.7 + 1 3

Hormosira banksii

Flinders (Victoria, Australia)

(-)-lR,2R

82.8 + 2 3

Xiphophora chondrophylla

Flinders (Victoria, Australia)

(—)—1R,2R

82.0 + 2 7

(—)—1R,2R

72.3 + 3 5

(-) —1R,2R

71.2 + 1 0

( + )—1 S, 2 S

83.3 + 0 1

Hobart (Tasmania)

Xiphophora gladiata

DLctyopteris membranaceae

Villefranche (French Mediterr. coast) Halifax

Haplospora qlobosa

2.0 Stereo- and Enantiospecific

Syntheses

2.1 C 1:1 Hydrocarbons Stereospecific with two major

syntheses of the C ^

hydrocarbons are faced

problems:

1. Correct assembly of the alicyclic backbone; approaches being 2. Stereospecific

enantiospecific

preferred.

introduction of the side chains; Z/Z, E/Z,

Z/E and E/E combination must be optionally

possible.

208 A particularly versatile synthesis of C 1:L hydrocarbons utilizes lactones as starting materials. They are accessible for each ring size and may be prepared as pure enantiomers by enzymatic or chemical approaches (30,31). Reductive with diisobutylaluminium

olefination

hydride and an appropriate Wittig

re-

agent introduces the first substituent (32). Oxidation and a second Wittig reaction complete the sequence. Proper choice of bases, e.g. N a N ( S i ( C H g ) 3 ) 2 the phosphonium

(for Z-olefins), and of ligands in

salts like tricyclohexyl versus triphenyl

(for

E-olefins) can be used to exert complete control of stereochemistry

(33).

Figure 4

Synthesis of algal

pheromones

a ) e n z y m a t i c a p p r o a c h e s to chiral

PLE = pig

lactones

liver esterase, HLADH = h o r s e liver a l c o h o l

b) lactones as synthons for algal

dehydrogenase

pheromones x : _ru

Rjpcc

-

209 Chirality may be also introduced at a later stage of synthesis, since many alicyclic aldehydes are rather good substrates of horse liver alcohol dehydrogenase (HLADH). Such reactions are conducted with kinetic control, and after 50% conversion the products are extracted and separated by LC into the enantiomeric alcohols and aldehydes. Aldehydes having S-configuration are preferentially reduced and lead to alcohols with the same absolute stereochemistry. Depending on the structure of the substrates moderate to high optical purities have been reported (34). Figure 5

Kinetically controlled reduction of alicyclic aldehydes by HLADH (30)

rac

y

HLADH NAD* EtOH

p H 7.0

a

H

UR.fR) (unstable to

(-MIJ.ISI separation)

s'

OL

M-I33.4U »Irmene

731 e . e .

The stereocontrolled synthesis of conjugated polyenes has been achieved by a combination of acetylenic- and Wittig chemistry. Treatment of 2E-alkenals, or 2-alkynals with acetylenic phosporanes results in E,Z-pairs which are separated by LC on silica. Stereospecific reduction of the acetylenic bonds

210 with Zn(Cu/Ag) in aqueous methanol gives polyens with control of configuration

Figure 6

complete

(35).

Stereocontrolled

synthesis of polyene

hydrocarbons

J

"GIFFORDENE"

2.1 Methylene interrupted

hydrocarbons

Due to their close configurational

relationship

fatty acid precursors, such compounds are directly

to the accessible

from fatty acids by oxidative degradation with Pb(0-Ac) 4

(27).

Chain elongation of e.g. arachidonic aldehyde to give a C 2 1 pentaene by Wittig reaction has been also employed (21). De novo syntheses followed the usual protocol of the acetylenic approach to fatty acids

(24,27).

3. Biosynthetic Aspects 3.1 Biosynthesis of C ^

hydrocarbons

The biosynthesis of the

hydrocarbons has been studied

detail using the composite Senecio 2 and isotopically

labelled ( H and

isatideus as a model

in

system

3 H ) dodecapolyenoic

acids

(6). Leaves and stems of this plant produce large amounts of ectocarpene accompanied by minor quantities of butylcyclohep-

211

tadiene and 1,3,5,8-undecatetraenes (13). Tracer studies with

3

double bond labelled

H-dodeca-3Z,6Z,9Z-trienoic acid showed

this fatty acid as the natural precursor to the C ^

hydrocar-

bons. Administration of unnatural fatty acids like undeca- or trideca-3Z,6Z,9Z-trienoic

acids, having the double bonds in

the sane positions as the natural precursor, leads to artificial metabolites which can be analyzed by GC/MS without being superimposed by natural products (Figure 7a). Analysis of the fragments according to Figure 2 allows for localization of the 2

H

label. Summarizing the results of the administration 2

with

experiments

H labelled precursors, the biosynthesis of all C ^

drocarbons from dodeca-3Z,6Z,9Z-trienoic,

or

hy-

dodeca-3Z,6Z-di-

enoic acid follows a common pathway characterized by the topics below: 1. Fatty acid precursors are attacked at their methylene groups, either at C(5) or C(8). Only a single hydrogen is removed. 2. Attack at C(5) produces linear hydrocarbons (e.g.

1,3E,5Z-

undecatrienes; involvement of C(8) always leads to cyclopolyolefins. 3. The activated intermediate, being a radical, a cation or a covalently modified species, induces cyclization or rearrangement by neighbour group participation of double bonds. 4. After cyclization or rearrangement (and a second oxidation, if a radical is involved) the positive charge is in lî-position to the C00~ group. The system collapses into the two neutral fragments R-CH=CH„ and C0„ (Figure 8).

212

a

cT*

.li Jiiiii. uni., ii.ii

100l%]

>.

V H

a

c (

t> c

*

so-

i

/

^

8

li > H V t

H I K B9 J Ui J* J M

Figure 7

'I ' ' I 150

100

' 2 ÌÓ

M S a n a l y s i s of a r t i f i c i a l

metabolites 2 (70 eV) s p e c t r a of d e u t e r a t e d m e t a b o l i t e s , a ) n a s s s p e c t r u m of a H p e n 2 t e n y l - c y c l o h e p t a d i e n e o b t a i n e d a f t e r a d m i n i s t r a t i o n of (8- H 2 ) - t r i d e c a - 3 Z , 6 Z , 9 Z - t r i e n o i c Electron impact

a c i d to S e n e c i o i s a t i d e u s . T h e r e l e v a n t 2

s p e c t r u m of l l - p h e n y l ( 2 - H ) u n d e c e n e

f r a g m e n t s are i n d e x e d ; cf. F i g u r e 2.

b) n a s s 2

from i n c u b a t i o n e x p e r i m e n t s w i t h 1 2 - p h e n y l ( 2 - H 2 ) d o -

decanoic acid and germinating Carthamus

seeds.

213

Figure 8

Biosynthesis of alicy clic pheromones

Figure 8 illustrates the formation of C 1;L hydrocarbons by a radical attack onto C(8) of the precursor acid. The sequence of cyclization and oxidation may be reversed. Intermediates are likely, but have not been observed as yet. The biosynthesis of the cycloheptadienes probably

in-

volves an 'abiotic' Cope rearrangement of a thermolabile cyclopropane

(2).

Besides the cyclization between e.g. C(8) and C(4) yielding cyclopropanes and cyclopentenes, other combinations are possible. Attack at C(5) followed by direct cyclization with C(2) results in a recently discovered

4-(lE-hexenyl)cyclopentene

(8), and an attack at C(ll) in combination with a cyclization at C(3) yields 'aucantene' which has been found in the mediterranean phaeophyte Cutleria multifida (36) (Figure 9). Due to the multitude of attack- and cyclization sites the biosynthesis of the cycloheptadienes remains an open question. Two

214

undistinguishable pathways (by feeding studies) are possible. The route via a thermolabile cyclopropane is outlined in Figure 8, but a direct ring closure between C(8) and C(2) should be not

ignored.

Figure 9

Biosynthesis of other ring systems.

An 'abiotic' consecutive reaction clearly works in the biosynthesis of 'giffordene', the odoriferous principle of Giffordia mitchellae (9): 3,6,9-dodecatrienoic acid is first converted into 1,3Z,5Z,8Z-undecatriene as depicted in Figure 10. The compound is unstable at room temperature; it undergoes an antarafacial 1.7-sigmatropic hydrogen shift which results in the highly unfavourable 2Z,4Z,6E,8Z geometry of

'giffordene'

(35). The stereochemical course and the details of the enzymatic reactions have yet to be established.

215

Figure 10

3.2

Biosynthesis of

'giffordene'.

Biosynthesis of long chain 1-alkenes

The biosynthesis of the long chain 1-alkenes shows striking similarities to the biosynthesis of C ^

polyolefins. In order

to study the mechanistic aspects of the vinyl group

formation,

germinating seeds of C a r t h a m u s t i n c t o r i u s were chosen as a readily available model system because of their high production of 1,8,11,14-heptadecatetraene,

1,8,11-heptadecatriene

and 1-pentadecene, respectively (37). Their origin from

lino-

lenic-, linoleic or palmitic acid was readily evidenced by ad3 ministration of the

H labelled substrates. However,

insight into their biosynthesis following the feeding

further strategy

as discussed before (cf. 3.1.) remained unsuccessful; mainly because of the very low abundance of the molecular ion of such polyenes (usually below 0.1 %) (27). Using specifically 2 led

label-

H 12-phenyldodecanoic acids as metabolic probes providing

excellent analytical and mass spectroscopic properties (38) (Figure 7b), the following observations have been made:

216 *

Deuterium at C(2) is not attacked.

*

Only one deuterium from C(3) is removed.

*

Deuterium

at C(4) is not attacked.

These findings rule out previous hypotheses involving

inter-

mediates of K- or (1-oxidation (39) during 1-alkene formation. To test for a direct insertion of oxygen at C(3) followed by heterolytic

fragmentation

into an 1-alkene, water and C O 2 p

(40), racemic 3-hydroxy-2-( H 2 )-12-phenyldodecanoic

acid was

administered. Indeed, the expected 11-phenylundecene was formed, but in striking contrast to the aliphatic precursor, the reaction proceeded with loss of one deuterium

from C(2). A

lower homologue, 9-phenylnonene was also found. Thus, li-hydroxycarboxy1ic

acids are probably first channeled

into the

usual catabolic and anabolic pathways, before the 1-alkene is liberated from a saturated n-alkyl fatty acid Figure 11

precursor.

1-Alkenes from 3-hydroxy acids D

R

COOH

it • 2 [H] - HDO

D 1-( 2 H 2 )-11-phenylundecene (M+ = 232)

HO

HD 2 C0-SCOA

•COOH

»

R^"5^

9-phenylnonene (M+ • 204)

217

The results demonstrate that the biosynthesis of vinyl groups cannot be adequately described by heterolytic

fragmen-

tation of 3-hydroxy fatty acids or by decarboxylation of e.g. 3-oxo fatty acids (39). However, striking similarities to the formation of algal pheromones with regard to abstraction of a single hydrogen and loss of C 0 2 are observed. In both cases removal of a hydrogen from C(3) or C(8), respectively, bearing a racemic

H label shows a strong isotope effect (k^/k^ ca. 9-

10) pointing to an attack at this site. Figure 12 outlines mechanistic alternatives which are framed by the

experimental

results.

f^

R-ch^-C^1" ¿H

>

COj +

R-CHsCHj


* X - A-

154

Geranial (E)-3,7-Dimethyl-2,6octadienal Y 7-7-A>

152

C

10H16°

00470-82--06 10-71

05392-40-•05 02-41

334 30

logT x -logT M

(1)

logTN-logT.M M,N: Known Compound

I: Kovats Index of Compound N or M

X: Unknown Compound

T: Scan Number of Compound M,N or X

Known data ....

Compound X

R.T.

M

decanal, I M 1188, T M 1355 N undecanal, I N 1290, T^ 1748 Scan Number

K.I. from(l)

K.I. from ref.

A (carveol)

1409

1204

1202

B (nerol)

1452

1216

1212

C (carvone)

1478

1223

1225

D (geraniol)

1540

1239

1243

E (bornyl acetate)

1682

1275

1277

F i g . 2 Conversion system i n t o Kovats Index from .GC/MS scan number From the p r a c t i c a l

point of view of GC/MS a n a l y s i s , the measurement of the

KI value based on the scan number was e f f i c i e n t peaks (Fig.

2).

f o r the i d e n t i f i c a t i o n of

335 Identification of volatiles from Callus tissues 1). Callus culture. Callus culture and suspension culture of thyme (Thymus vulgaris L.) were prepared according to the method described in previous papers (1). Callus tissues of thyme were induced from the explants of the leaves on three kinds of media, Murashige & Skoog, Gamborg, and Nitsch & Nitsch, at 25°C for 8 weeks in a light intensity of 2000 lux. The medium contained 3.0 g of sucrose and 0.9 g of Difco Bactoaga and various combinations of the growth regulators, naphthaleneacetic acid (NAA) and kinetin, indoleacetic acid (IAA) and kinetin, and 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin as described in a previous paper (1). Callus tissues were transferred to new Murashige & Skoog agar media containing various combinations of growth regulators as mentioned above every 30 days when rapid growth was observed. 2.) Identification of volatiles. In the study of volatiles from callus tissues or suspension cells, sample preparation for GC and GC/'MS represents a special problem because the growth of cultured cells in vitro may be slow and the productivity of volatiles may be low. SDE or Simultaneous Distillation Absorption (SDA) (2) were adapted to the separation of volatiles from callus tissues at 0.01% to 0.001% level of fresh weight. SDE was used for large samples, SDA for small ones. Callus tissues (105 g fresh weight) were harvested from the 9th subcultures at 30 days after transference. Volatiles were separated by SDE and analyzed by GC/MS. The identification of compounds was carried out with our data base for identification as mentioned above. Fig. 3 shows a total ion monitoring (TIM) chromatogram of volatiles from the callus. As shown in Table 5, fifty seven components including 21 aldehydes, 7 alcohols, 4 ketones, 8 terpenes and others were identified. A remarkable feature of the essential oil obtained from callus was the presence of a variety of C15-C17 aldehydes and sesquiterpenes. Small amounts of thymol were also found. Geranylacetone, Bchamigrene, nerolidol, 6-guaiene, cuparene, y-cuprenene, which were not detected in the intact plant were found in this callus. At present, it is assumed that thymol in thyme is biosynthesized by aromatization of yterpinene leading to p-cymene followed by hydroxylation; thus, it has been reported that p-cycmene might be the precursor of oxygenated derivatives (6). However, y-terpinene and p-cymene were not found among the volatiles from

336

Fig. 3

Total ion monitoring chromatogram of v o l a t i l e s from the c a l l u s . GC

c o n d i t i o n s , c f . Tables 1 and 2

callus t i s s u e s . The quantitative a n a l y s i s of components in the o i l obtained from c a l l u s was carried out by using undecane as internal standard. Unfortunately, the y i e l d of the o i l from

a l i u s was very low, corresponding to 1/500-1/1000 of

that of the entire plant. Production of v o l a t i l e s under various culture conditions The effects of various constituents of culture media on c a l l u s

induction

and productivity of v o l a t i l e s were studied using f i v e kinds of basal media, Murashige & Skoog, Gamborg, Nitsch & N i t s c h , White, and Linsmaire & Skoog. Furthermore, preliminary examinations on the effections of growth regulators and precursors on the production of v o l a t i l e s were performed. S a t i s factory r e s u l t s on c a l l u s induction were obtained with NAA (5 ppm) and k i n e t i n (10 ppm) i n Murashige & Skoog medium.

337 Table 5. Volatiles identified in thyme callus Peak No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61

Kovats Index Found Ref.

Compound acetaldehyde ethanol biacetyl ethyl acetate chloroform * benzene pentanal 3-pentanone 1-pentanol hexanal furfural (E)-2-hexenal benzaldehyde 2,4-heptadienal 2-pentylfuran (2E,4E)-2,4-heptadienal benzyl alcohol phenyl acetaldehyde 3,5-octadien-2-one 1-octanol (3E,5E)-3,5-octadien-2-one nonanal 2,4-dichlorophenol decanal (2E,4E)-2,4-nonadienal (E)-2-decenal lH-indole 2,4-decadienal thymol 4-vinylguaiacol (2E,4E)-2,4-decadienal tridecane (E)-2-undecenal ¿-el emene tetradecane geranylacetone ^-chamigrene /3-guaiene 2,6-di-tert-butyl-p-cresol tridecanal cupa rene v-cuprenene nerolidol tridecanol tetradecanal hexadecane tetradecanol pentadecanal pentadecanol hexadecanal octadecane diisobutyl phthalate * heptadecanal methyl hexadecanoate dibutyl phthalate * hexadecanoic acid ethyl hexadecanoate ethyl 1 inoleate ethyl linolenate ethyl oleate ethyl octadecanoate

* : Artifact.

363 500 558 583 599 657 671 677 742 778 809 830 934 -

983 984 1010 1015 1040 1054 1064 1087 1147 1188 1191 1240 1255 -

*

+ : Could not calculate.

1269 1282 1290 1300 1339 1394 1400 1432 1480 1499 1495 1495 1506 1530 1550 -

1589 1600 1661 1691 1760 1791 1800 1823 1892 1909 1915 1946 1982 2144 2145 2150 2180

*

+ * +

656 671 676 751 777 805 830 934 970 979 985 1008 1014 1044 1057 1069 1087 1155 1188 1191 1242 1260 1268 1268 1286 1290 1300 1345 1394 1404 1432 1481 1493 1495 1496 1507 1530 1551 1563 1597 1600 1663 1707 1763 1796 1798 1821 1900 1903 1919 1945 1975 2136 2141 2145 2174

338 Additionally, the changes in the total amount of oil and the quantity of C14-C17 aldehydes in the subculture (8th generation) were investigated. On the 20th, 25th, 30th, and 35th day after transference volatiles were separated by SDE and analyzed by GLC. From the data obtained it was concluded that the rates of growth and essential oil production reached a plateau after 30 days; the rate of oil production increased distinctly from the 25th to 30th day. The changes in the quantities of C15-C17 aldehydes in the oil are shown in Table 6. The fraction of C15 aldehydes including an unsaturated compound comprised 50% of the total volatiles. The callus grown with IAA (1 ppm) and kinetin (10 ppm) in Murashige & Skoog medium produced volatiles containing mainly C17 aldehydes, i.e. a mixture of unsaturated aldehydes. The productivity of volatiles was increased by the addition of 2,4-D (10 ppm) and kinetin (10 ppm) to Murashige & Skoog medium, 5 times more than that in the combination NAA (1 ppm) and kinetin (1 ppm).

Table 6.

Effects of growth regulators and precursors on the volatile products

IAA 1 ppm Kinetin 1 1 Total Oil *

109..3 pg

10 ppm

661..0 pg

2,4-D

1 ppm 1

170,.1 yg

Thymol

0,.2 %

1..1 %

0..3 %

/3-Chamigrene

0..2

t

t

(3-Guaiene

5..2

13..8

4..3

Nerolidol Cjg aldehyde

10 ppm 10

MVA

554,.3 pg

1242,.5 jjg

0,.4 %

0..3 %

44,.0

11,.0

1..4

t

0,.3

1..1

5,.4

0,.8

0,.5

50..4

47..7

25..8

27,.9

9,.6

17..4

6..8

5.6

1,.0

4,.2

Heptadecene C

16, C17 Aldehydes

MVA: Mevalonic acid

*

1 mg/10 ml;

: in 100 g of fresh weight.

t: trace, less than 0.1 %

339 The influence of s u g a r s , amino acids or mevalonate on v o l a t i l e production was also examined. The precursor was added to the 8th generation c a l l u s on the 20th day a f t e r transference, and the c a l l u s was incubated for 10 days. After addition of mevalonic acid (1.09 mg/10 ml) the amount of v o l a t i l e s increased 12 times more than that of the reference sample (IAA 1 ppm, k i n e t i n 1 ppm). The amount of C15 aldehydes among v o l a t i l e s decreased dominantly. I t i s known that d i f f e r e n t i a t i o n or t i s s u e o r g a n i s a t i o n i s an important factor which influences the synthesis of secondary metabolites (7). In some cases of c e l l c u l t u r e s , environmental factors such as medium c o n s t i t u ents and growth regulators can show a marked influence on the production of certain secondary metabolites (7). In the present study, i t was possible to obtain 2-5 times as much v o l t i l e s without extending the c u l t i v a t i o n period. In p a r t i c u l a r , the fact that p r o d u c t i v i t y of a c y c l i c sesquiterpenes or the f r a c t i o n of C15 aldehyde including an unsaturated compound was affected by use of a growth regulator i s of great i n t e r e s t . Guaiene and other s e s q u i terpenes which were not found in the intact plant were also produced; t h e i r p r o d u c t i v i t i e s were increased by the influence of certain medium c o n s t i t u ents or growth r e g u l a t o r s . However, the y i e l d of v o l a t i l e s was s t i l l

too

low compared with the amounts occurring in the intact plant.

Sensory evaluation of the v o l a t i l e s from thyme c a l l u s The sensory evaluation of v o l a t i l e s from the c a l l u s was examined by GCs n i f f i n g as mentioned above. The aroma q u a l i t y of the c a l l u s speaking in ordinary language,

volatiles,

was judged to resemble to that of overripe

sweet o r i e n t a l persimmon f r u i t (Diospyros kaki L . ) , completely lacking any s p i c y odor.

Acknowledgement This work was supported by a grant from the Yamasaki Spice Research and Promotion Foundation, Tokyo, Japan.

340 References 1.

Nabeta, K., H. Sugisawa. 1983. in: Instrumental A n a l y s i s of Foods (G. Charalambous and G. I n g l e t t , e d s . ) . Academic P r e s s , New York. p. 65.

2.

Sugisawa, H., C. Chen, K. Nabeta. 1984. I n : A n a l y s i s of V o l a t i l e s , Methods. Application (P. Schreier ed.). Walter de Gruyter, B e r l i n , p. 357.

3.

Aeree, T . E . , J. Barnard. 1984. In: A n a l y s i s of V o l a t i l e s , Methods. Application (P. Schreier ed.). Walter de Gruyter, B e r l i n , p. 251.

4.

Sugisawa, H. 1986. Application of Data Base in Flavour Research, Symposium (Tokyo). Abstracts, p. 1.

5.

T s u g i t a , T. 1986. Application of Data Base in Flavour Research, Symposium (Tokyo). A b s t r a c t s , p. 9.

6.

Poulose, A. J . , R. Croteau. 1978. Arch. Biochem. Biophys. j_91> 400.

7.

Butcher, D.N. 1977. In: Plant C e l l , Tissue and Organ Culture (J. Reinert and V. P. S. Bajai e d s . ) . Springer Verlag, B e r l i n , p. 668.

PRODUCTION

OF

FLAVOUR

TARRAGON (A/itemUla

VOLATILES

BY CALLUS AND

SUSPENSION

CULTURES

OF

dn.acun.cu.luò)

J.W. Gramshaw, C.M. Cotton and L.V. Evans Procter Department of Food Science and Department of Plant University of Leeds, Leeds, England, LS2 9JT

Sciences,

The

Introduction

Tarragon, A/vieml^la

produces, upon steam distillation, a cha-

cLn.acun.culu»

racteristic essential oil which is prized as a flavouring in savoury applications such as the production of pizzas. herb,

The characteristic aroma of

which is broadly reproduced in the derived essential oil,

due to the presence of 4-methoxyallylbenzene, and

methyl chavicol, allylanisol

estragol with its aroma reminiscent of fennel.

predominant

the

is mainly

Methyl chavicol is the

volatile and is clearly the character impact compound of

both

herb and essential oil although the rather harsh nature of this compound is softened in each flavouring by minor components.

Two

broad

divisions of the herb (and its essential oil)

French tarragon (A. tarragon

(A.

cLiacunculoidcs,

),

recognised.

a

The two species

also be differentiated by the fact that Russian tarragon can

propagated vegetatively.

Russian

differences in quality apparently being

reflection of variation in the content of methyl chavicol. may

are

has a flavour superior to that of

dnacunculus)

only

be

In practice however, these distinctions are some-

what blurred and plants are encountered bearing characteristics between the two extremes. tends

to

To some degree at least, this may be because French tarragon

lose its ability to produce relatively large amounts

of

methyl

chavicol with the passage of time.

The

components of Oil of Tarragon have been studied much less

extensively

than those of many other essential oils; recorded components (Table I) fall mainly into the categories of terpenoid and phenylpropanoid compounds, thus

Bioflavour'87 © 1988 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

342 indicating the ability of the plant to produce secondary metabolites by the mevalonate pathway and by the acetate-shikimate route.

Table I.

Recorded Constituents of Oil of Tarragon (1-3)

limonene

ß-pinene

methyl chavicol

cis-ocimene

phellandrene ?

anethole

trans-ocimene

linalool

eugenol

allo-cis-ocimene

geraniol

eugenol methyl ether

allo-trans-ocimene

linalool oxide ?

A-methoxycinnamaldehyde

a-pinene

camphor

butanoic acid

"C9-aldehyde"

In

view of the prized status of Oil of Tarragon and the potential problems

attending its production, plant

source,

which stem from the long-term instability of the

it was of considerable interest to attempt to

maintain suspension cultures of A. diacaacuiui

produce

and

and to induce these cultures

to produce compounds recognised as important constituents of Tarragon Oil.

Results and discussion

Callus production

Plants

of A. d/iacunculuA

were grown in a greenhouse under long-day

condi-

tions and tissue material taken only from individuals which produced methyl chavicol

freely.

Harvested leaves were surface sterilized before explants

were taken and placed on an agar medium under sterile conditions.

The pre-

sence of both an auxin, a-naphtalene acetic acid (NAA), and a cytokin, benzyl aminopurine (BAP), was necessary for callus explants usually responding within 7 days.

growth to occur, succesful

343 It

proved

necessary to move each callus to fresh medium at

approximately proximately

28 days to maintain viability. 2 months,

intervals

of

Upon reaching the age of ap-

certain of the callus cultures became brown due

to

the production of phenolic compounds and the subsequent conversion of these into

melanoidin pigments; occurred,

melanoidins also discoulered the

When

this

and,

after repeated selection,

agar

healthy tissue was carefully selected at

medium.

subculture

a number of stable healthy callus

"lines"

were established. A callus producing a detectable amount of methyl chavicol was then used to initiate suspension cultures.

Suspension cultures

Suspension conical more

cultures were produced and maintained in continuously

flasks.

agitated

Successful suspension cultures were those taken from

the

friable of the callus cultures and at a time when the callus line had

been established for approximately 12 months.

Each culture contained small

aggregates of cells in addition to single cells; the hormone composition of the medium being one factor which influenced the balance between single and aggregated cells.

Subculturing was again essential and was required at in-

tervals of approximately 3 weeks to maintain viability.

Cultures

took

explants

had been taken that derived material was available to

many

months

to stabilize and it was

18-24

months

influence of environmental factors on the production of flavour

degree

growth

of selection will necessarily have occurred on the

and survival.

less

a cer-

basis

of

The selection process may be responsible in part for

the limited production of volatiles, cultures

the

volatiles.

Because of long periods required for establishing a stable culture, tain

after

study

particularly methyl chavicol, by cell

and some effort is presently being given to the use

of

younger,

well established cultures as experimental subjects for the production

of methyl chavicol and related compounds.

A second possible reason for the

reluctance of cultures to produce volatile compounds is that, in A. cuiuA grown as a plant,

those secondary metabolites

essential oil collect in glands.

d/iacun.-

which constitute

the

No such device is available to suspension

344 cultures into

which must either retain the metabolites in the cells or

excrete

the medium with the possibilities of further metabolization or an in-

hibitory effect occurring.

Isolation and analysis of volatile compounds Volatiles were isolated from plant (i.e.

leaf) tissue, from callus tissue,

and from suspension cultures (cells and media taken together).

Throughout,

a need existed for water free of all volatile impurities. As was discovered in an earlier investigation (4), treatment with adsorbents failed to remove all such impurities from glass-distilled water. method

was

based

on

the use of the

Thus,

Likens-Nickerson

distillation-solvent extraction procedure,

since the isolation concurrent

steam

the same technique was used

to

purify water. An acceptable level of purification was achieved only by carrying out the process on a relatively large-scale (2 1) for a period in excess of 24 h. Even so, traces of a number of phtalate esters were sometimes encountered in extracts; however these contaminants were very late to elute from

the

water

GC column and did not overlap any areas

of

interest.

Purified

was used not only for the isolation of volatiles but also for prepa-

ration of those batches of media to be used to give material for analysis.

Following

extraction using Likens-Nickerson procedure and concentration of

the extracts, volatile compounds were identified by linked GC-MS. An internal standard, extraction

dodecan-2-one,

was employed as a check on the efficiency of

and as a method of correlating the levels of

individual

vola-

tiles produced under different conditions.

Influence

of environmental factors on production of volatile compounds

by

A, d/iacunca£u/> in suspension culture The influence of plant hormones on the production of volatile compounds was studied

with particular regard to phenylpropanoid compounds related to me-

thyl chavicol.

Combinations of several different concentrations of NAA and

BAP were used and the effect of each of these upon the pattern of volatiles

345 produced

and the levels at which individual compounds occurred were

moni-

tored .

Tables

II and III record the phenylpropanoid compounds produced in culture

media

and

present 10 and 25 days after a subculture had

Table

II shows the effect of changing NAA concentration with that

held constant at 2 mg-1 against The

been

performed. of

a constant concentration of NAA of 2 mg-1 ^ is given in Table III.

two

tables

structure,

but

also

record the occurrence

of

a

compound

almost certainly a metabolic product of NAA,

prominent component in many extracts.

of

unknown

which was

MS studies indicate a molecular

mula CigH-^C^ and a naphtalene related skeleton for this compound. and

BAP

whilst the effect of changes in BAP concentration

a

for-

Figs. 1

2 are representations of total ion chromatograms of extracts from sus-

pension cultures rich in methyl chavicol and chavicol, respectively, whilst Fig. 3 shows the occurrence of C ^ H j ^ C ^ .

In

addition to analysis of volatile compounds,

cultures were assessed for

growth (trachid count) (5) and total soluble phenols in the cells tion

(extrac-

with 50 % methanol followed by colour development with the Folin-Cio-

calteau reagent) (6). Tables

A brief summary of these results is included in

as a guide to the condition of the cultures;

full details will

the be

given elsewhere.

Perhaps

the most striking result of the study to date is the reluctance of

suspension cultures to produce methyl chavicol,

the major

phenylpropanoid

produced being the parent phenol, chavicol (4-allylphenol). Methyl chavicol was

noted

growth

in appreciable amounts only when little NAA was present in

medium

and during the early period of culture.

Results

from

the the

growth of suspension cultures under conditions of low BAP concentration are incomplete

but,

since moderate levels of BAP permit production of

chavicol in trace amounts,

it is to be anticipated that rather more of the

compound will be observed in future experiments. allyl-2-methoxyphenol),

a

methyl

Production of eugenol (4-

minor component of tarragon also

methyl eugenol (3,4-dimethoxyallylbenzene),

occurred

which was more abundant

IV) than eugenol in tarragon plant tissue used as source material, detected in suspension cultures.

but

(Table was not

346

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347

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348

INTERNAL SUNOMO

ÄTHYL CKAVICOC CUGCMl DIAVtCOL

JIUJL

JJ 1^:31

if-.i'A

Fig. 1 Total ion chromatogram (0-47 rain) of an extract of. A. dracunculus suspension culture at 10 days (NAA at 0.001 rag-1 ; BAP at 0.5 m g - 1 ~ 1 ) .

Fig. 2 Total ion chromatogram of an extract of A. dracunculus culture at 10 days (NAA at 1.0 m g - l _ 1 ; BAP at 0.5 m g - ! " 1 ) .

suspension

349 Table IV. Phenylpropanoid components of A. diacu.n.culuA

leaf tissue (arbitry

units)

methyl chavicol

2580

106

eugenol methyl ether eugenol

24

chavicol

25

It may well be that the selection, during

the

which must have occurred to some extent

establishment of the cultures,

has led to a markedly

ability to carry out the methylation step in the biosynthetic

The

reduction

in methylating ability as cultures age is

interest in view of the reputation, has,

mentioned above,

reduced

pathway.

of

considerable

which A.

d/iacuncuiuA

as a plant, of losing flavour quality, presumably due to reduced pro-

duction of methyl chavicol, with time.

Clearly,

the

methoxylated conditions.

suspension

cultures

phenylpropanoids However,

when

respect to the allyl group,

this

connection, ring

that

an

eugenol

ability was

to noted

produce

other

under

some

the methoxy group is in the para position with it apparently becomes more difficult to

out the biosynthetic methylation step.

benzene

have

since

carry

It is perhaps relevant to note,

a methoxyl group para to second substituent

occurs in nature much less frequently than a

free

in

in a

hydroxyl

group in the same orientation.

The

production

of chavicol and eugenol depends markedly on the levels

hormones present in the medium.

Inspection of Table II (Day 10) shows that

the yield of chavicol increases with increasing NAA concentration to a maximum at 1 mg.l ^ (Fig. minent

of

reach

2) before falling. Eugenol reaches a less pro-

maximum at a concentration of 0.1 mg.l ^ before falling away rather

more rapidly than chavicol.

Of particular interest is the finding that, as

the culture period is extended to 25 days,

the concentration of the phenyl

propanoids decreases very sharply, possibly because the compounds have b'een

350 metabolized further. It is tempting to enquire whether they have become incorporated into the lignin biosynthetic pathway; an alternative

explanation

is that the compounds have become substrates for the phenol oxidase also

produced

by the cultures.

system

Further studies are necessary to

clarify

this point.

The

results

incomplete appear,

relating and

to

changes

in

BAP

concentration

so it is not possible to draw firm

are,

as

yet,

conclusions.

It

does

however, that increases in BAP concentration favour the production

of chavicol and that loss of this compound as the culture ages, occurs only at the highest

Tables

concentration.

II and III reveal no correlation between the production of

propanoid

phenyl-

compounds and either rapidity of culture growth or production of

soluble phenols in the cells.

Production of volatiles by young callus tissue

In view of the reduced ability to produce methyl chavicol noted between intact plant and suspension culture, production of phenylpropanoids in callus tissue soon after its induction was studied. levels

Table V shows the pattern and

of methyl chavicol and related compounds produced under a series of

hormone

regimes.

In some sets,

total volatiles are

callus and the agar medium upon which it was grown, whilst

in

recorded

(i.e.

the

were extracted as one)

other callus tissue and the supporting medium

were

separately

treated.

A

particular difficulty encountered in this part of the study was the fact

that agar retains, tile

certainly methyl chavicol and probably the other

phenylpropanoid compounds,

vola-

very strongly under the extraction condi-

tions. Recovery of methyl chavicol added to an agar plate was very low even when an amount much in excess of that produced by callus cultures was used. The results given in Table V should therefore be viewed with this in the

mind;

values given are certainly much lower than the true one and non-detec-

tion does not necessarily mean that a compound was absent.

Current experi-

351

8

I

I

I

I

I

I

'

I

I

I I «H

8

•H

©

o

1

M

M

ÎN

ÎN

I

I

I

I

I

I

3 U AJ

8 S

Ü •ÂS

tt 'S

, 8

Êt

u

9

S 5

S I 3

•

ÚÍ 1

J s *

U H «M H S -H >14J Ç

. J 8

11 giî

• s i l l

C 0

H c o

2 2

H

M e

(1S.2RM-)[a] p = -13 ° (c 1 ,CHC I3 ) Fig. 33:

N M R 93:7

V.2. Chiral alcohols

The enantioselective hydrolysis of esters in which the chiral or prochiral information resides in the alcohol moiety ean be used equally well for the preparation of chiral alcohols and diols.

507 The enantioselective preparation of optically active glycidol and glycidyl esters (Fig. 34) using procine pancreatic lipases (PPL) is noteworthy (27).

0

PPL

Fig. 34: Enzymatic preparation of (R)-Glycidyl-esters

The method epoxidation blocks with preparation and natural

is complementary to the use of the Sharpless and provides access to important chiral building glycerol substructures, intermediates for the of 3-blockers and many other biologically active products.

A large number of secondary alcohols, important chiral auxiliaries both for analytical and synthetic applications can be prepared by resolution of their esters using a lipase from Pseudomonas sp (28) (Fig. 35). A central building block for the synthesis of prostaglandins (Fig. 36) can be prepared

508

Fig. 35: Enantiomerically pure secondary alcohols by enzymatic resolution of their racemic esters

C02H

CO2H

Fig. 36: Synthetic routes ot prostaglandins

509 by enantioselctive hydrolysis of the corresponding meso-1,3diester (Fig. 37). While the (R)-ester-function is hydrolized using PLE, PPL and microbial ester hydrolases display a preference for the (S)-functionality, making both enantiomers accessible enzymatically (29, 30).

OAc

OAc

Fig. 37: A chiral building block for prostaglandins enzymatic preparation Chemoselective functional group manipulations, including protection and deprotection sequences can also provide access to both enantiomeric series of target molecules (Fig. 38).

(B)-fi

A

eol-Z

eol-fl

Fig. 38: Chemoenzymatic routes to both enantiomeric series of target molecules

510

Also diesters of acyclic and cyclic primary diols can be converted into the optically active monoesters using ester hydrolases. Chiral building blocks with glycerol substructures are usually prepared from carbohydrates (mannitol ,ascorbic acid etc.) or aminoacids (L-serine) (31). For the first time we were able to convert achiral glycerol itself into chiral building blocks of that kind using a chemo-enzymatic approach. The diacetate of 2-0-benzylglycerol was thus enzymatically hydrolized to provide the (R)-monoacetate in high chemical yield and optical purity (Fig. 39) (32).

R0

R=Ph3C=p-Tos=PhjBi/Si

OR R=Ac

HO

OAc

IBJ-

(R)-

THPO

OH

HO

OCPh

Fig. 39: An enzymatic route for the transformation of achiral glycerol derivatives into chiral building blocks with glycerol substructures

511

Although the initially o b t a i n e d p r o d u c t was not o p t i c a l l y p u r e , t h e c o r r e s p o n d i n g t o s y l a t e w a s u p g r a d e d t o > 97 % e.e by s i m p l e r e c r y s t a l l i s a t i o n . O b v i o u s l y , s t a r t i n g f r o m a n a c h i r a l s u b s t r a t e , b o t h e n a n t i o m e r i c s e r i e s of c o m p o u n d s are a g a i n a c c e s s i b l e by s e l e c t i v e f u n c t i o n a l g r o u p m a n i p u l a t i o n s including protection-deprotection-sequences. In a s i m i l i a r a p p r o a c h the d i a c e t a t e s of c y c l i c m e s o - d i m e t h a nols c a n be c o n v e r t e d i n t o the c h i r a l m o n o a c e t a t e s u s i n g e s t e r h y d r o l a s e s (Fig. 40) (33).

Fig.

40:

C l e a r l y , b o t h e n a n t i o m e r i c s e r i e s of p r o d u c t s c a n be o b t a i n e d by this c o m b i n a t i o n of e n z y m a t i c a n d c h e m i c a l m e t h o d s . The e n a n t i o m e r i c p u r i t i e s of the t h u s o b t a i n e d p r o d u c t s are remarkably h i g h . N e x t to P P L a m i c r o b i a l l i p a s e h a s p r o v e n p a r t i c u l a r l y u s e f u l in t h i s c a s e (Fig. 41) (28). The u s e f u l n e s s a n d f l e x i b i l i t y of c h e m o e n z y m a t i c a p p r o a c h e s to c h i r a l b u i l d i n g b l o c k s is n i c e l y d e m o n s t r a t e d in Fig. 42, s u m m a r i z i n g the d e s c r i b e d c h e m o e n z y m a t i c a p p r o a c h e s to e n a n tiomerically pure cyclic lactones.

512

oa 1

lAc OAc

or°Ac

PPL

1 % e.e.

Yiekfe:>90%

PPL

L-119

72

88

t=cssr

88

94

(XX

86

99

OCoh

78

50

ocr

99

97

c¿fc 2s.

21

2s.

Fig. 41:

2f

HO OH

Acylation

B

11) lipase

HLADH (NAD)

O

AcylÒ ÓAcyl12) raid Oj^ q >\

J1) PLE

O

12) re duct . 2C / c0V2Me Me0

Fig. 42: Chemoenzymatic routes to enantiomerically pure lactones

513

V.3. Esterhydrolases in organic solvents

As described earlier, ester hydrolases can be employed both for the hydrolysis of esters and their synthesis by a) esterification; b) alcoholysis While there are many enzymes which do not tolerate organic solvents, lipases have been shown to be remarkably stable in this environment, even under almost anhydrous conditions. These are obviously required for esterification reactions, since only under these conditions the equilibrium can be shifted towards the ester formation. In an aqueous environment the hydrolysis of esters is strongly favoured. Lipase RC0 2 H + R'OH

RCO 2 R'+ H 2 O

Several examples from the literature (34-36) are summarized below (Fig. 43).

R,CH-CO,H

Hal (•)-

R,0H

.

•

nBuOH

nPrCOjCHjCCIj

(i)

(il-Menthol • CH3ICH,)10CO,H

Candida cyl

IBI-RRÇHCO^U

Hal 80*/, >95%ee

BQ=

» |R|-nPrC0jR2 60-75% 70-90% ee

•

(SI-R,-ÇHCOjH

Hd 90% 60-9SV.ee

. (S)-R,0H 50-60% 90%ee

0 Candida cyl ^ n.^^.o-c-ICHj^CHj • W-Menlhel 45% 95%ee

80%ee

Fig. 43: Enantioselective ester synthesis using lipases

514

Alcoholyses (transesterifications) do not involve any free water. They are, at least in our view, to be preferred over the direct esterification for the synthesis of esters. It should be noted that the reversibility of these reactions must, in principle, have also stereochemical consequences. As already pointed out much earlier, enzymes are able to differentiate between a) enantiomers; b) enantiotopic groups. Clearly, as summarized below (Fig. 44,45) opposite enantiomers are becoming accessible by a simple change of reaction conditions.

OAc ^ X ^ Hydrolysis » Ph

(i)

P h

OH

OH

(R) •

i+ s)Ph

OAc

OAc

A

A

IS)

IR)

Ph

Alcoholysis EtOAc

AcO

OH IS)

HO

OAc (R)

Fig. 44: Hydrolysis and alcoholysisstereochemical consequences

la)

Bzl Alcoholysis

Hydrolysis OAc OAc

Ph

Ph

Bzl

Bzl

OH ^ ^

(b) OH

OH

515

HYDROLYSIS




Ac E

>

Ac E 2

1 Y

X E

recently

The of

(Y):

>

Ac Y 1

+

E

>

Ac Y 2

+

E

The acceptor compound (Y) is supposed to be in large concentration excess, which

allows

a

good enzyme recycling.

In terms of

initial

rates

the

Michaelian kinetic analysis leads to: v

Ac X 1

/v = a *Ac X/Ac X Ac X D 1 2 2 with

In

that

case

or a

D

Log(Ac X/Ac Xo) = Or *Log(Ac X/Ac Xo) 1 1 D 2 2

= (V/K) /(V/K) Ac X Ac X 1 2

the separation factor a

is also

characteristic

of

the

system but depends on the nature of the leaving group (X). Extension of this analysis to a mixture of donors (Ac X) allows a comparison i of the enzymatic specificity towards acyl transfer reactions, taking the faster-reacting compound as a reference. Consequently for a given mixture, a specificity spectrum of an enzyme towards various acyl groups can be easily obtained and drawn from a test reaction (13). This is useful for a selection of an enzyme for a given reaction.

However,

in opposition to nucleophile

separations, reaction modeling is not easy to realize for acyl separation.

535 Table 1 :

| 1 | 1 1 1 1 | | | 1

Competitive factors for Laurate Transfer Catalyzed by rugosa Lipase on/from Menthol Diastereoisomers.

Couple of compounds

Candida

| Alcohols ( OC.) | Lauric esters (CK^)!

(-)/(+) menthol (-)/(+) isomenthol (-)/(+) neomenthol (+)/(") neoisomenthol

1 | | | 1 1 | | 1

^ (-)menthol/(-)isomenthol^ (-)menthol/(-)neomenthol (-)menthol/(+)neoisomenthol

50 30 40 60

50

1 1 1 1 1 1 1 1 1

2.1 30 2.9

1 1 | | 1 | | | 1

n-dn-d. n.d. 2.1 n-d. n.d.

Reaction carried out on racemic mixtures- the reactivities of the others enantiomers were neglected for the calculations. n.d. : not determined. (-)Menthol Separation from Thymol Hvdrogenation Mixture. The different ways to prepare (-)menthol have been recently reviewed (14). The

well-known

after

Haarmann-Reimer process (15) starts from thymol

hydrogenation a mixture of

(±)menthol

(ca.

55%),

to

give

(±)neomenthol

(ca. 29%), (±)isomenthol (ca. 14%) and (±)neoisomenthol (ca. 2%). Biochemical the

separations of (-)menthol from this mixture were

assayed

acetates with various microbial hydrolases (16,17) and achieved

Rhodotorula mucilaginosa

(17).

on with

We have examined the direct separation of

(-)menthol from the whole mixture using lipase specificity.

The

reaction

in

organic medium is convenient and allows the use of high concentrations

of

substrate and the possibility of

these

conditions

an

enzyme

re-use.

Furthermore,

enhancement of enantioselectivity was

under

observed

on

(±)menthol resolution using Candida rugosa lipase (18).

In

the

following we have examined the potentiality of this enzyme

crude preparation (lipase MY,

in

a

Meito Sangyo Co.) for (-)menthol separation

from the previous diastereoisomeric mixture. First the best acyl group was choosen for the resolution of (±)menthol. reaction rate and enzyme stability, to shorter fatty acids.

In terms of enantioselectivity,

lauric acid ( a

= 50) was preferred A Phenyl valeric acid was also shown to be powerful

(19,12), but was not tested in this work.

536 The various separative constants

a

for laurate transfer were then deter-

mined

for couples of enantiomers or diastereoisomers of the mixture

Table

1).

(see

The enzyme is strongly selective in this series for R absolute

configuration of alcohols (20).

values for enantiomeric separaA tions are greater than 30. (see Table 1). When couples of diastereoisomers are

in competition,

All a

(-)menthol is preferred but the selectivity

towards

(-)isomenthol and (+)neoisomenthol is low (2.1 and 2.9 respectively). From the

values

taken

given in table 1,

(-)menthol

a selectivity scale has been

as a reference :

(-)menthol

100;

established

(-)iso-menthol

48;

(+)neoisomenthol 35; (-)neomenthol 3.3; (+)menthol 2.0; (+)isomenthol 1.6; (-)neoisomenthol 0.6; (+)neomenthol < 0 . 1 . If

thymol

hydrogénation

mixture is now considered as

substrate

for

a

direct synthesis reaction,

(±)neoisomenthol can be omitted with regard to

its low concentration (ca.

2%). It is expected that mainly (-)menthol and

(-)isomenthol would be first transformed in esters. This was observed from a

kinetic experiment which is given in figure 1.

of

ca.

35%

consumed. (see

was

reached,

(-)Neomenthol,

these two (+)menthol

compounds

When a conversion ratio were

almost

and to a less extend

completly

(+)isomenthol

Table 2) became substrates of the reaction which occurred then at

lower rate. lauric

Figure 1 : Lipase MY catalyzed lauric esters synthesis from thymol hydrogénation mixture of menthol (M.) diastereoisomers. Esters composition as a function of conversion ratio. Conditions : lauric acid and menthols : 0.25 M in heptane saturated with water; Lipase MY 5% w/v; T = 40°C

conversion

r a t i o (%)

a

537 Table 2 :

Composition of the Recovered Products from the Two Enzymatic Steps.

1 1 | | Compounds 1 1 1 1 | menthol | | isomenthol | | neomenthol | | | neoisomenthol 1 1 . . . 1| | Conversion ratio | | of each step | 1 1 *

,

.

1 st S„t e 2 n d Step | I 1 P 1 alcools | 1 laurgjes *l (%) | (%) | (ee (%)) 1 (ee (%)) 1 1 1 | (-) 91 82.9 | (-) >98. 92.4 | 7.6 | I (-) 86 12.7 | (-) >98. | n.d. 2.5 | 1 | n.d. 1.9 | 1 1 1 1 1 1

initial conditions

(%) (±) (±) (±) (±)

Successive

55.0 12.8 28.9 3.3

1

28

'

%

40. %

1 1

1

j 1

**

molar ratio percent. enantiomeric excess percent. See figure 1 and 2 for experimental conditions, n.d. : not determined.

Modeling the

of

reaction evolution was realized from the

thermodynamic conditions at equilibrium.

separate

experiment on (-)menthol,

mixture,

and

ot values and

from

These were determined in

the faster-reacting compound

it is assumed with a good approximation that the

of

a the

different

diastereoisomers were giving the same final equilibrium (6% free

alcohols

at equilibrium for 0.25 M alcohols and lauric acid). The simulated kinetic separations

in the mixture are given within experimental data in figure 2

for (±)menthol resolution on one side (figure 2A) and (-)menthol - (-)isomenthol

competition

perfectly

on the other (figure

the experimental results.

(-)menthol

cannot

2B).

Theoretical

curves

It is clear from these results

be separated directly from its

diastereoisomers

fit that with

this lipase. For this reason, we have examined the following strategy were two

successive enzymatic reactions carried out with this lipase have been

combined

with a separative distillation step of the

enriched

(-)menthyl

ester. Menthol

Lipase catalyzed

isomers

ester synthesis

mixture The

first

>

enriched in (-)menthol

lipase catalyzed > (-)menthol ester solvolysis

reactional step has been previously

(-)menthyl obtained

Ester

analyzed.

and (-)isomenthyl laurates (82.9 and 12.7 %,

A

mixture

of

respectively) is

when the reaction is stopped at 28 % conversion (see

table

2).

538

A

.oooe*oo

.190

CONVERSION »«TIO

CONVERS1 ON sono

B

CONVERSION Figure 2. Simulation : ester synthesis (A), (B); ester solvolysis (C) , (D). Theoretical curves and experimental results. A and C : (i)menthol resolution; enantiomeric excess as a function of conversion ratio of the two substrates ( Qf = 50; A : AcXe=AcYe= .2585 ; C : Xe= .558 , Ye= .0261). B and D : (-)menthol-(-)isomenthol separation; molar ratio as a function of conversion ratio of the two substrates (Ot = 2.1; B: AcXe= .2585; AcYe= .058, D: Xe=.583; Ye= .089). Ester synthesis conditions: see Figure 1. Solvolysis conditions : butyric esters 0.75 M; isopentanol 1 M in heptane saturated with water; lipase MY 5% w/v; T = 40°C

Modeling

of

the

solvolytic

second step is given in figure

menthyl laurate and 1 M isopentanol,

equilibrium conditions :

2

(0.75 70 %

M

free

menthol). The theoretical predictions have been verified (see figure (2)). (-)Menthol is obtained with high enantiomeric excess (figure 2C).

However

it is not possible to eliminate completly (-)isomenthol (figure 2D).

A preparative experiment was carried out under these conditions (see table 2). After two reactional steps 38% of the initial (-)menthol was recovered in

a

results

product which contains 92.4% of (-)menthol

(see

table

2).

are interesting but the purity of the product is not good

for industrial applications.

These enough

539 The

strategy

reactional

which

and

appropriate

was

developed is simple and limits

physical

specificity

enzymatic step.

separation could

be

steps. used,

Other

for

the

lipases

instance

in

number with the

of the

second

In this context quantification of a values is simple and

reaction modeling is powerful for further studies. Ethyl Butvrate from Butter Fat. Production

of

ethyl esters from butter fat by Candida rugosa lipase

has

been already investigated for a flavorant use (21). Hydrolyzed or unhydrolyzed

substrates

have

been

interesterification reaction,

tested

in

a

direct

respectively.

ester

synthesis

We have re-investigated

or the

latter reaction in organic media. A strategy based upon lipase specificity has

been developed in order to optimize butyryl transfer on ethanol.

lipases were tested for their specificity towards fatty acids in a interesterification

reaction

and

then

checked

in

the

The

simple

butter

fat

ethanolyse.

A mixture of ethyl esters of fatty acids and an acceptor alcohol (such propanol,

as

butanol, or pentanol...) was used to study the competitive acyl

transfer

reaction.

the ot values

Initial rate measurements allow the quantification of

towards

the

faster-reacting

substrate.

spectrum for this acyl transfer reaction is obtained. Mucor miehi

lipases (trade names :

A

specificity

Candida rugosa

lipase MY from MEITO SANGYO

Co.

and and

Lipozyme from NOVO Ind., respectively) have been studied and their spectra are described in Figure 3.

M. miehi lipase is a non specific enzyme but is more active on long

chain

fatty acid esters. In the opposite, Candida rugosa lipase is shown to have a

good

specificity

for

butyrate in the mixture

and

should

be

most

convenient for the preparation of ethyl butyrate from butter fat. Ethanolyse

of butter fat (which contains ca. 3.5% weight of butyric acid)

catalyzed by the two lipases was then studied.

In both cases optimization

of

according

the

experimental

experimental

conditions was realized

design (22).

to

a

computed

The kinetic results are described in Figure 4.

540 V a (%)

Lipases:DMy

® M.miehi

100-

JZl

XI

Chain length Figure 3 : Lipase specificity for fatty acids intersterification reaction (see text).

esters (M.)

Figure 4 : Ethanolyse of butter fat by Candida rueosa (lipase MY) ( ) and Mucor miehi (Lipozyme) ( ) lipases. Ethyl esters (C. to and ethyl butyrate formation as a function of time under optimized conditions.

0.5.

time (h.)

As expected from the previous test reaction, butyrate mixture

in of

molar ratio, reaction).

the triglycerides from butter

lipase MY is specific of the fat.

Using

ethyl esters with ethyl butyrate as the main ca.

this

lipase,

component

a

(50%

30% weight) is obtained under kinetic control (24 hours

541 Lipozyme mixture

is a more active catalyst but gives under the same conditions of ethyl esters with a lower content of ethyl butyrate

(ca.

a 10%

molar ratio). With

these

result the previous assumption based

on

lipase

specificity

towards acids was verified. This strategy allows the selection of the most convenient

lipase

for preparative purposes and can be extended to

other

substrates.

Acknowledgements This work was supported by different grants from : MIR (n° 83.V.0062) and ANVAR

(n°

X84.ll.1040) for menthol separation;

Byconversions"

and

from the GS

"Aromes

MERO-ROUSSELOT-SATIA for the preparation of

et

natural

ethyl butyrate.

REFERENCES 1.

Deleuze, H., G. Langrand, H. Millet, J. Baratti, G. Buono, C. Triantaphylides, C.. 1987. Biochim. Biophys. Acta, 911. 117.

2.

Langrand, G., J. Baratti, G. Buono, C. Triantaphylides. submitted to Biocatalysis.

3.

Desnuelle, P. 1972. In: The Enzymes, 3rd ed., (P.D. Acad. Press, 2> 575.

4.

Verger, R., G.H. De Haas. 1976. Ann. Rev. Biophys. 5, 77.

5.

Verger R. 1980. In: Methods in Enzym., (D.L. Purich Ed.), 64, 340.

6.

See for instance Ladner, W.E., G.M. Whitesides, 1984, J. Amer. Chem. Soc., 106, 7250., M. Schneider. In: Bioflavour 87.

7.

Lilly, M.D., J.M. Woodley. 1985. In: Studies inOrganic Chemistry 22: Biocatalysts in Organic Synthesis. (J. Tramper, H.C. Van der Plass, P.Linko Eds.)., 179.

8.

Klibanov, A. M.. 1987. Pharm. Technol., 32

Boyer Ed.),

542 9.

Marlot, C., G. Langrand, C. Triantaphylides, J. Baratti. 1985. Biotechnol. Lett., 7, 647.

10. Gancet, C., C. Guignard. 1986. Revue Française des Corps Gras, 423. 11. Zaks, A., A.M. Klibanov. 1985, Proc. Natl. Acad. Sci., 82, 3192. 12. Chen, C.S., Wu, S.H., Girdaukas, G. and Sih, C.J.. 1987. J. Amer. Chem. Soc., 109, 2812. 13. Unpublished data of the laboratory. 14. Garnero, J.. 1982. Labo. Pharma. Prob. Technol., 30, 413. 15. Davis, J.C.. 1978. Process. Technol. Chem. Eng., 22/5/1978, 62. 16. Aviron-Violet, P. 1980. VIII Intern. Congress on Essential Oils, 488. 17. Yamaguchi, Y., A. Komatsu, T. Moroe. 1976. J. Agric. Chem. Soc. Jpn., 50, p. 475, p. 501, p. 619. 18. Langrand, G., J. Baratti, G. Buono, C. Triantaphylides. 1986. Tetrahedron Lett., 27, 29. 19. Koshiro, S., K. Sonomoto, A. Tanaka, S. Fukui. 1985. J. Biotechnol., 2,47. 20. Langrand, G., M. Secchi, G. Buono, J. Baratti, C. Triantaphylides. 1985. Tetrahedron Lett., 26, 1857. 21. Kanisawa, T. 1983. Nippon Shokukin. Kogyo. Gakkai, 30, 572. 22. Mathieu, D., and R. Phan Tan Lu ., Logiciel NEMROD LPRAI Université Aix-Marseille III, 13297 Marseille cedex 13.

SCREENING

OF

LIPASES FOR ENANTIOMER RESOLUTION OF SECONDARY

ALCOHOLS

BY

ESTERIFICATION IN ORGANIC MEDIUM

D. Gerlach, S. Schneider, T. Göllner, K.S. Kim and P. Schreier Lehrstuhl für Lebensmittelchemie, Würzburg, FRG

Universität Würzburg, Am Hubland, D-8700

Introduction The

use of enzymes for the resolution of chiral molecules is a well estab-

lished

process (1,2).

utilize used

For apolar alcohols and esters

lipase suspended in organic medium (3,4).

to resolve,

e.g.,

convenient

systems

This technique has been

acyclic secondary alcohols (5,6),

epoxy alcohols

(7), ct-substituted cyclohexanols (A), d,l-menthol (8), (i)-sulcatol (9) and various nearly still

glycerol derivatives (10). all

the

Since a systematic

study

commercially available technical lipase

lacking,

the

considering

preparations

present work was carried out to screen

a

is

series

of

lipases from various sources for enantiomer resolution of acyclic aliphatic secondary alcohols (C6-C9).

From the potential reaction conditions, lipase

catalyzed ester synthesis in organic medium was chosen (4).

Results and Discussion The

following commercial lipase preparations were used:

(AòpejigiUuA Darmstadt,

aigcui', FRG);

80000 (Rkizopuò dida

c.yLLnd/iace.a\

(Fluco/i miehe.i\ PPL I (MKC,

Rohm,

Darmstadt,

S 12000 (Rhizopuò

aruihizu.ò\

FRG);

curnhlzuò',

2212 D and 2212 F

2212 E (ftucox. mieJxe.1',

Röhm,

Rapidase, Seclin, France); S

Gist-Brocades, Delft, The Netherlands); MY

(Can-

Meito Sangyo, Japan/Welding, Hamburg, FRG); Piccantase A Rapidase,

Seclin, France); porcine pancreas lipases (PPL):

Hannover, FRG); PPL II (Sigma, Darmstadt, FRG); PPL III (Roth,

Karlsruhe, FRG); PPL 7023 C (Röhm, Darmstadt, FRG).

Bioflavour '87 © 1988 Walter de Gruyter & Co., Berlin • New York - Printed In Germany

544 First of all, the lipases were characterized by means of their protein

and

water content, enzymatic activities (Tab. 1), protein patterns (Fig. 1) and isoelectric points (pi) (Fig. to

1; Tab. 2). Protein contents ranged from 3.5

62.5 % and most of the lipases exhibited a number of "side-activities",

such as, in particular, phosphatase, phosphoamidase as well as various protease were

and glycosidase activities. detected

by

ultrathin-layer

Analogously,

complex protein

patterns

isoelectric focusing (UDIEF) (Fig. 1).

Different pi values were determined for the lipases under study (Tab. 2).

MP

1

2

3

4

5

6

7

8

9

10

11

MP

lig. 1 UDIEF of commercial lipase preparations: protein patterns and isoelectric points (pi, marked by arrows), pi determinations were carried out by enzyme specific print technique on ultrathin-layer agar gels (11) (substrate: triacylglycerisjes C 2 - C ^ M P = marker groteins; 1 = 2212 D; 2 = 2212 F; 3 = 2212 E; '4 = wheat germ lipase ; = 7051 L ; 6 = S 80000; 7 = S 12000; 8 = MY; 9 = PPL 7023 C; 10 = PPL II; 11 = PPL I (* not further studied in this work).

545 Tab. 1 Screening of enzymatic activities in commercial lipase tions using the Api Zym test kit Activity

a

b

c

d

Alcal. phosphatase Esterase (C4) Esterase/Lipase (C8) Lipase (C14) Aminopeptidase (leu) Aminopeptidase (val) Aminopeptidase (cys) Trypsine Chymotrypsine Acid, phosphatase Phosphoamidase a-Galactosiuase ß-Galactosidase ß-Glucuronidase a-Glucosidase ß-Glucosidase ß-Glucosaminidase cvMannosidase ct-Fucosidase

1 2 3 1 2

4 2 3 1 5 +

2 3 4 5 5

-

4 2 4 3 2 1

-

-

-

-

-

-

-

-

5 5 5 5 5 5 5 5

3 3

1

1 2 4 3 1

2 3 4 2 1

1 2 4 4 5

1 3 3 2 2

-

-

-

-

-

-

-

-

-

-

3 4

1 1

-

5 5 3

-

-

-

-

-

-

k

-

1

+ 1

i

1 2 5 3 +

-

-

h

2 4 5 3 3 + +

-

-

g

4 3 5 4 5 2 1

5 5 5 5 3 5 5 5 1

-

Lipase f e

prepara-

-

-

-

-

-

-

-

-

-

-

+

5 2 1 1

4 2 3 4

1 4 4

4 2 1 1

-

-

-

-

-

-

-

-

-

-

-

1 5

-

-

1

-

-

3 + 1 1

3 1 1 +

4 + 1 2

3 + 1 1

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

1

1

-

-

-

-

-

-

1 1

-

1

a 2212 D; b 2212 E; c 2212 F; d S 12000; e S 80000; f MY; g Piccantase A; h PPL I; i PPL II; k PPL 7023 C; 1 PPL III. - = no activity; 1 to 5 = range of activity (low to strong); + = between - and 1.

Tab. 2 Isoelectric preparations

points (pi) of lipase activity in commercial

Enzyme

pi

2212 D 3/7 472 2212 E 3.9 2212 F 3.7 4.2 S 12000 4.4 5.4 S 80000 4.4 5.4 MY 4.2 4.8 Piccantase A 3.5 PPL I 4.2 5.3 PPL II 4.2 5.3 PPL 7023 C 4.3 Wheat germ lipase* 7051 L * 3.5 4.0 * not further studied in this work

lipase

8.3 5.6 5.6

5.9 5.9 6.0

6.5 6.5

5.9

6.7

5.8 5.8 5.7

7.7 7.7

7.2 7.2

8.2

546 Enzyme

(0.5 g and 1.0 g lipase preparation) catalyzed esterifications were

carried

out with

each of 2.5 mM racemic secondary alcohol (C6 to C9)

and

dodecanoic acid in 10 ml heptane. Different temperatures (4°, 25°,40°, 60°, 70°,90°C) and reaction times (6 to 216 h) were used. zyme

After cooling the en-

was filtered off and the volume adjusted to 50 ml with diethyl ether.

Ester formation was determined by quantitative capillary gas chromatography (HRGC) (standard: 2-octyl hexanoate). Use of preparative TLC led to quantitative

separation

of

pure

alcohol and ester; the ester was subjected to

LiAlH^ reduction. Control of enantioselectivity was carried out by HRGC after derivatization with S-(-)-l-phenylethyl isocyanate (12,13). The recovered enzyme could be reused after being dried; II

revealed

loss of activity of approximately

analysis performed

w i t h PPL

25 % in the second experi-

ment, but constant enantioselectivity was observed.

Starting

with

octanol,

first

the

study

of all,

of lipase catalyzed

of

the rate of ester formation was determined

different reaction times at 40°C.

Fig. 2 Lipase catalyzed acid in heptane at 40 C.

esterification

As outlined in Fig.

2,

yields

esterification of R,S-2-octanol with

R,S-2after ranging

dodecanoic

547

• 90° C , 48h

a

5-7

4,9

S 80000

1

2.n S

60* C,

96h

3, 7

0. I 12000

Fig. 3 Lipase catalyzed esterification of R,S-2-heptanol acid in heptane at 60°C (96 h) and 90°C (48 h).

with

dodecanoic

ester f ornat i on < V.) 80

70 60

S i gna 0 , 5g S i gna 1g

50

L ipase C

40

MKC

30 20 10

40»C, 96h

70»C, 48h reaction conditions

90°C,48h

Fig. 4 Lipase catalyzed esterif ication of P., S-2-neptanol with acid in heptane: Influence of temperature on ester formation.

dodecanoic

548 from 1 % to observed

67 % were obtained.

The highest rates of ester formation were

with lipase MY and PPL lipases. Similar results confirming

findings were found using R,S-2-heptanol (Figs. 3 and 4). Candida ce.a

lipase (MY) has been already described as catalyst

rification

suitable

in organic medium with high yields (3,4,5,6,8,14).

these c.ylindn_a-

for estePPL lipases

have been also found useful catalysts for the formation of various enantiomer products by enzymatic hydrolysis in organic solvent

From the lipases shown in Figs. 2 - 4 ,

(7,10,15,16).

the PPL preparations (leading to the

highest

ester formation rates) were further studied with R,S-2-octanol de-

pending

on temperature

and reaction time;

additionally, their enantiose-

lectivity was investigated. In spite of fast and high ester formation catalyzed by MY lipase,

this enzyme was not further studied because of its low

enantioselectivity. Due to the low ester formation rates observed with fungal lipases these enzymes were also excluded from complementary studies. As shown from Figs. 5 and 6, in which the results obtained at different

• S • •

tempe-

40»C, 9Gh 70° C, 481 90°C, 48li 4°C. I44h

Z 70« C, 241.

S igna (0,5q> I ipases

Lipase C

Fig. 5 Lipase (PPL) catalyzed esterification of R,S-2-octanol with dodecanoic acid in heptane: Influence of temperature and reaction time on ester formation

549 rature and reaction times are outlined, ester

the

PPL lipases showed both

formation and enantioselectivity. At 90°C in heptane,

only

high

partial

deactivation of lipases took place. The high stability of enzymes in waterfree medium has been recently pointed out by Zaks and Klibanov (17).

• 40° C, 96h

a

70° C , 4 8 h

0

9 0 ° C , 4 8h

•

4°C .

-

144h

70° C ,

?4h

dodecanoic Fig. 6 Lipase catalyzed esterification of R,S-2-octanol with acid in heptane: Influence of temperature and reaction time on enantioselectivity.

Using

R,S-2-octanol and

PPL 7023 C the ester formation

controlled at 70°C over 70 h. tion

As shown from Fig.

7,

was

continuously

maximal ester forma-

was reached at 54 % corresponding to an ee value of 75 % (R) (ester).

Maximal ee value (84 % (R)) was determined at 45 % ester formation.

As

to the experiments with PPL lipases,

different temperature optima were

found in studies carried out with PPL II and PPL 7023 C at 40°C (96 h) 70°C

(48 h) for enantioresolution of acyclic aliphatic secondary C6 to

alcohols. ester

As

shown from Figs.

formation

at 70°C,

8 and 9,

whereas at 40°C

use of PPL 7023 C led to - except

2-octanol

-

and C9

higher higher

550

Fig. 7 Ester formation (70°C) continuously controlled over 70 h using R,S2-octanol/dodecanoic acid and PPL 7023 C.

rates ponding

of esters were obtained using PPL II. In Figs. 10 and 11 the corresee

values determined are outlined.

As shown from the graphs, ex-

cept 2-hexanol (with PPL 7023 C), in all other cases enantioselectivity very similar. ester formation

Lower

values

were obtained in the reactions,

rate surpassed the 50 % value

was

in which the

(cf. Figs. 8 and 9).

551

Fig. 8 Lipase catalyzed esterification of dodecanoic acid with different secondary alcohols in heptane at 40 C (96 h).

2-hexanoI

2-heptanoI

I• 2-octanoI

2 -nonanoI

Fig. 9 Lipase catalyzed esterification of dodecanoic acid with different secondary alcohols in heptane at 70 C (48 h).

552 enantloneric excess ('/.) 911 -

i hexanol

2-heptanol

2-octanol

2-nonanot

Fig. 10 Enantioselectivity of lipase catalyzed esterification of dodecanoic acid with different secondary alcohols in heptane at 40 C (96 h).

enant iwipr Io

Fig. 11 Enantioselectivity of lipase catalyzed esterification of dodecanoic acid with different secondary alcohols in heptane at 70 C (48 h).

553 Concluding

it has to be pointed out that the optimal ester formation

rate

has to be determined depending on lipase and alcohol used. The optimal rate of ester production is the better the higher the talysis is

enantioselectivity of ca-

(theoretical value: 50 % at absolute enantioselectivity

of the

enzyme).

Acknowledgements

The assistance of Mrs. C. Missel and U. Keil is gratefully acknowledged. D. Gerlach thanks for the support of a DECHEMA Graduate Fellowship in the program

"Biotechnology" of the Bundesministerium für Forschung und

Technolo-

gie, Bonn.

References

1.

Jones, J.B. 1986. Enzymes in organic synthesis. 3403.

Tetrahedron 42_,

3351-

2.

Klibanov, A.M.1986. Enzymes that work in organic solvents. Chemtech. p. 354-359.

3.

Cambou B. and A.M. Klibanov.1984. Comparison of different strategies for the lipase-catalyzed preparative resolution of racemic acids and alcohols: Asymmetric hydrolysis,esterification and transesterification. Biotechnol. Bioeng. 26, 1449-1454.

4.

Langrand, G., M. Secchi, G. Buono, J. Baratti and C. Triantaphylides. Lipase catalyzed ester formation in organic solvents. 1985. Tetrahedron Lett. 26, 1857-1860.

5.

Cambou, B. and A.M. Klibanov.1984. Preparative production of optically active esters and alcohols using esterase-catalyzed stereospecific transesterification in organic media. J. Am. Chem. Soc. 106, 26872692.

6.

Kirchner, G., M.P. Scollar and A.M. Klibanov.1985. Resolution of racemic mixtures via lipase catalysis in organic solvents.J. Am. Chem. Soc. 107, 7072-7076.

7.

Ladner, W.E. and G.M. Whitesides.1984. Lipase-catalyzed hydrolysis as a route to esters of chiral epoxy alcohols. J. Am. Chem. Soc. 106, 72507252.

8.

Koshiro, S., K. Sonomoto, A. Tanaka and S. Fukui.1985. esterification of d,l-menthol by polyurethane-entrapped organic solvent. J. Biotechnol. _2, 47-57.

Stereoselective lipase in

554 9.

Stokes, T.M. and A.C. Oehlschlager. 1987. Enzyme reactions in apolar solvents: The resolution of (i)sulcatol with porcine pancreas lipase. Tetrahedron Lett. 28, 2091-2094.

10. Kerscher, V. and W. Kreiser. 1987. Enantiomerenreine Glycerin-Derivate durch enzymatische Hydrolyse prochiraler Ester. Tetrahedron Lett. 28, 531-534. 11. Hofelmann, M., R. Kittsteiner-Eberle and P. Schreier. 1983. Ultrathinlayer agar gels: a novel print technique for ultrathin-layer isoelectric focusing of enzymes. Anal. Biochem. 128, 217-222. 12. Gerlach, D., C. Missel and P. Schreier. 1987. Screening of lipases for enantioresolution of R,S-2-octanol by esterification in organic medium .Z. Lebensm. Unters. u. Forsch. in press. 13. Pereira, W., V.A. Bacon, W. Patton, B. Halpern and G.E. Pollock. 1970. The use of (R)-(+)-(1)-phenylethyl isocyanate in the optical analysis of asymmetric secondary alcohols by gas chromatography. Anal. Letters 3, 23-28. 14. Langrand, G. , J. Baratti, G. Buono and C.Triantaphylides.1986. Lipase catalyzed reactions and strategy for alcohol resolution. Tetrahedron Lett. 27, 29-32. 15. Ramos Tombo, G.M., H.P. Schar, X. Fernandez I Busquets and 0. Ghisalba. 1986. Synthesis of both enantiomeric forms of 2-substituted 1,3-propandiol monoacetate starting from a common prochiral precursor, using enzymatic transformations in aqueous and organic media. Tetrahedron Lett. 27, 5707-5710. 16. Laumen, K. und M.P. Schneider. 1986. A facile chemoenzymatic route to J. optically pure building blocks for cyclopentanoid natural products. Chem. Soc., Chem. Commun. pp. 1298-1299. 17. Zaks, A. and A.M. Klibanov. 1984. Enzymatic catalysis in organic at 100°C. Science 224, 1249-1251.

media

ENZYME MEDIATED SYNTHESIS OF PHEROMONES

Claudio Fuganti and Stefano Servi

Dipartimento di Chimica . Piazza L. d a Vinci 32

Politecnico di Milano.

20133 Milano, Italy.

Introduction

The

approach

to enantiomerically pure

synthetic

products

based on the chemical optical resolution of racemic material at

some

stage of the sequence is of limited value

in

the

field of insect pheromones, since the compounds are invariably

volatile

Accordingly, of

this

oils

lacking of

suitable

functionalities. 1

several preparations of representative members

class of compounds in optically active

relied

on

active

substances w h i c h are members of the so called 2

of

the use,

chirality", i.e.

as starting

materials,

the whole of easily

have

form

of

optically "pool

available

chiral

materials produced by nature including, among others, carbohydrates, the

amino acids, hydroxy acids and terpenes. However,

present composition of the "pool of chirality"

from beeing satisfactory,

is

far

the major limitation arising from

the fact that most of the components are really available in only one enantiomeric form, types

of

R,R'CHX,

chirality where

the

type

X= oxygen or nitrogen functions being

par-

ticularly abundant,

is

and, furthermore, the choice of

rather poor,

those

of

whereas those of the type R,R',R"CH and

Bioflavour '87 © 1988 Walter d e Gruyter & Co., Berlin • New York - Printed in Germany

556

R,R',R1'C(OR'''), mones,

quite frequent amongst the insect

phero-

occur rather rarely. Consequently, there is an inte-

rest in expanding the composition of the "pool of chirality" and new chiral products are expected to arise from the enzymic transformations of non conventional substrates. context, matic

In this

the baker's yeast mediated transformations of aro-

a, 3-unsaturated aldehydes,

Scheme

appeared

proceeding according

likely to be quite 3 4 synthetic point of view. '

fruitful

from

to the

OH

(1)

(2)

R = C6H5; 2-furyl

-15-25 %

(3) -85-75 t

R'- H, Me, Br Scheme I The

conversion

containing

of products (1) into the methyl diols

(2),

two additional carbon atoms with respect to

the

starting aldehydes and two additional chiral centres of

the

type R,R'CHOH, of

a multienzymatic process involving two distinct chemical

operations: dehyde position in

can be considered as the overall consequence

an

pyruvate

(i) Addition of a C^ unit equivalent of acetal-

onto the si face of the carbonyl carbon of

the

a -

unsaturated aldehydes forms (R) a-hydroxy ketones, acyloin-type condensation formally

paralleling

decarboxylase catalyzed synthesis of acetoin

pyruvate and acetaldehyde, intermediates

the from

and (ii) Reduction of the latter

by hydrogen addition onto the re face of

the

557

carbonyl

gives rise to the diols actually isolated (Scheme

2).

OH +

•h2"

"CHjCHO"

R1

OH

(2)

(1)

R = C6H5; 2-furyl R'= H; Me; Br Scheme 2 Under

suitable

ketones the

experimental

conditions

(R)

a-hydroxy

can be obtained as sole transformation products 5

aldehydes by the actively fermenting yeast.

some

tolerance

by

reaction of Scheme aldehydes

and

concerned.

the

the enzyme system(s) as

as

There

involved

is

in

the

the structure of the aromatic

substituents R' in the a -position

are

However, acetaldehyde seems to be the only alde-

hyde accepted as second terminus of the reaction. of

of

The

lack

incorporation of aldehydes such as a-ethylcinnamaldehyde 2,

and propionaldehyde into the type of diols of Scheme likely

to be due to the inability of these materials to

accepted part

as

substrates by the condensing enzyme(s)

of Scheme 2),

derived

from

since the synthetic a-hydroxy

the above aldehydes,

other carbonyl compounds, 4 yeast.

is

as well as

be

(first ketones

those

from

are stereospecifically reduced by

There is however, a dramatic difference in the yield

of conversion and in the type of products between the methyl ketones (4), and (9).

(5) and (6) and the higher homologues (7), (8)

Whereas (4),

(5) and (6) afforded

70-80% of a

558 nearly (14)

e q u i m o l e c u l a r m i x t u r e s of a n d (2S, 3S)

hydroxy ketones (16),

(17)

(11),

(7),

and

(13) a n d (15),

(10),

almost

exclusive

transformation

yield.

(10)

R=R1-R2=H;

(5) R=Me; R*=R2-H

(11)

R=R2=R3=H;

(6) R=R =H; R!=Me

(12)

R=Me;

R!=R =H;

R3=0H

R2=Me

(13)

R=Me;

R2=R3-H:

R^OH

U

R3=0H

2

R=RL-H;

and

respectively, a -

(t) R=Rj=R2=H

(7)

(12)

(8) a n d (9) gave rise to e r y t h r o d i o l s

(18) as

p r o d u c t s , in 15-20%

(2S, 3R)

2

R3=0H R!=0H 2

(8) R=Et; r!=R =H

(1U)

R=R =H;

R

(9) R=H; R!=R2=Me

(15)

R=R2=H;

r!=0H;

2

ME;

2

RJ=R2=H;

R3=Me

(16)

R=r!=H; R =Me;

R3=0H

(17)

R=Et:

R3=0H

1

(18) R=H; R =R =Me; R3=0H The diols

(10) a n d (12),

a-methyl cinnamaldehyde, yeast,

and

2

o b t a i n e d from c i n n a m a l d e h y d e r e s p e c t i v e l y , in f e r m e n t i n g

and

baker's

those a c c e s s i b l e from the r a c e m i c ot-hydroxy k e -

tones b y s t e r e o s p e c i f i c y e a s t r e d u c t i o n c a n be c o n s i d e r e d as carbohydrate-like, non carbohydrate derived chiral

synthons,

as

into

indicated

b y the c o n v e r s i o n of

(10) a n d (12)

d e o x y sugars of the L - s e r i e s L - a m i c e t o s e , L-mycarose

L-olivomycose and

. The s y n t h e t i c s i g n i f i c a n c e of these m a t e r i a l s

e n h a n c e d from the fact that from the c o r e s p o n d i n g lidene

the

d e r i v a t i v e s it is p o s s i b l e to extrude b y

the c h i r a l C

and C

carbonyl compounds

(19)-(27).

is

isopropyozonolysis 4

559

R

R'

0

0

R

R

(19) R=R1=R2=H

(20) R=R1=R2=H

(21) R=Me; RJ=R2=H

(22) R=Me; RJ=R2=H (2«> R=RXH; R2=Me

(23) R=R*=H; R2=Me (25) R=R2=H; RJ=Me (26) R=Et; RX=R2=H (27) R=H: R1=R2=Me Results

The latter materials seem particularly useful for the preparation of molecules containing in their framework relativelyfew carbon atoms chiral due to oxygen substitution. Thus,

the

diol (12),

(3S,4S) methyl ketone (21),

prepared from the

was used as precursor of (-)-frontalin (32), the

pheromone of Dendroctonus Frontalis bark beetle, possesseing a

chiral

dioxabicyclo can

be

centre

of

the

type

R,R',R"C(OR''')•

[3.2.1] octane system of

viewed

The

(-)-frontalin

as formed by internal ketalization

(32)

of

the

dihydroxyketone (31). This material resulted accessible from (21)

through the key intermediacy of the C

adduct

(28),

obtained

as the sole product upon reacting 4-methyl-pent-47 enylmagnesium bromide with (21) in THF . Subsequently, the two

chiral centres of (21) which assisted the formation

the

quaternary carbon of (28) were

Indeed, val

oxidatively

of

destroyed.

product (28) by O-benzylation, acid catalyzed remo-

of the isopropylidene protecting group,

and NaBH 4 reduction of the intermediate

HIO^ oxidation

aldehyde,

afforded

560

the C

material (29),

y

which

yielding, on ozonolysis the C

o

(-)-frontalin (32) was obtained

ketone

(30),

from

in

27%

yield

from (28) after hydrogenolysis and spontaneous cycli-

zation.

/

>

(21)

(29) (50) (31) At

R=CH2Ph: X=CH2 R=CH2Pn; X=0 R=H; X=0

(32)

variance w i t h the methyl ketone (21),

which

showed

a

precise anti facial selectivity in the addition of the above saturated the hyde

Grignard reagent,

the erythro aldehyde (19)

cyclohexylidene analogs of (19) and of the threo (20)

add saturated Grignard reagent w i t h poor

and alde-

steric

£ control . bromide

Indeed,

the latter materials w i t h ethylmagnesium

afford syn and anti adducts in 6:4 and

respectively.

The

major

diastereoisomers,

8:2

ratios

separated

by

column chromatography, were converted [ (i) NaH, DMF, C H CH CI, (ii) CH.COOH/H O, (iii) HICK , THF J1 into (2R) 2o b Z o Z 4 benzyloxy butyraldehyde (33) and into the enantiomer (34).

561

OH(\

OHC OCHjPh

(33) R=Me (41) R^CgHign (49) R=(CH2)2 CH=CH2

(34)

R=Me

(42)

R=CgHign

(50)

R=(CH2)2ch=ch2

OH

OH

OR

OR (35)

X=

J ; R=CH 2 Ph

/O" (36) x=: ; R=CH2Pn N 0-l

v OCH2Ph (37)

The

C^

X=

J;

V)

R=CH 2 PH

chiral aldehyde (33),

(38)

X=^]:R=CH2Ph

on reaction with the C^

turated Grignard reagent ClMgCH CH 2 CH 2 COCH^CH^OCH 3 ,

affords

the C g syn and anti adducts (35) and (36) in 6:4 ratio. latter

mixture,

on acid hydrolysis and debenzylation

10% Pd/C) gives rise to (+)-exobrevicomin (39) and

sa-

to

The ( H 2' (-)-

endobrevicomin (40), isolated by preparative gaschromatography. Similarly, the (2S) enantiomer (34), via (37) and (38), yields (-)-exo and (+)-endobrevicomin.

562

OR

OR

R1 i

(i(3) (44)

R

R=H; Rl=0H; R2=CH2Pn R=0H; Rl=H; R2=CH2Pn

T

R=0H; R1=H; R2