Identification of 10-desoxyiridal as an intermediate in the biosynthesis of iridals

Article abstract of DOI:10.1016/S0031-9422(98)00639-6

The metabolism of iridals, unusual triterpenoids found in sword lilies, was studied in a cell culture of Iris pseudacorus Linn. The cells contain mainly spiroiridals, esterified with capric acid, which are hydrolyzed by esterases upon cell disruption. In ether extracts of these cell homogenates 10-desoxyiridal, a hitherto unknown precursor of the iridals, was identified by comparison with a semisynthetic standard. The compound was isolated and its structure verified by spectroscopic means. Upon incubation of the cells with 3-amino-1,2,4-triazole, a bleaching agent, the amounts of iridal and 10- desoxyiridal in the cells increase, whereas the level of the conjugated iridotrienes is diminished. This proves that 10-desoxyiridal plays a central role in iridal biosynthesis and that the conjugated triene moiety of the iridotrienes is formed by the action of a desaturase on iridal.

Full text of DOI:10.1016/S0031-9422(98)00639-6

\
PERGAMON
Phytochemistry 49 "0888# 884Ð0992
Identi_cation of 09!desoxyiridal as an intermediate in the
biosynthesis of iridals
Ingrid Ritzdorf\ Melanie Bartels\ Bianca Kerp\ Torsten Kasel\ Sabine Klonowski\
Franz!Josef Marnerꢀ
Institute of Biochemistry\ University of Cologne\ Zulpicher Stra)e 36\ 49563 Cologne\ Germany
Ã
Received 0 September 0887
Abstract
The metabolism of iridals\ unusual triterpenoids found in sword lilies\ was studied in a cell culture of Iris pseudacorus Linn[ The
cells contain mainly spiroiridals\ esteri_ed with capric acid\ which are hydrolyzed by esterases upon cell disruption[ In ether extracts
of these cell homogenates 09!desoxyiridal\ a hitherto unknown precursor of the iridals\ was identi_ed by comparison with a
semisynthetic standard[ The compound was isolated and its structure veri_ed by spectroscopic means[ Upon incubation of the cells
with 2!amino!0\1\3!triazole\ a bleaching agent\ the amounts of iridal and 09!desoxyiridal in the cells increase\ whereas the level of
the conjugated iridotrienes is diminished[ This proves that 09!desoxyiridal plays a central role in iridal biosynthesis and that the
conjugated triene moiety of the iridotrienes is formed by the action of a desaturase on iridal[ Þ 0888 Elsevier Science Ltd[ All rights
reserved[
Keywords] Iris pseudacorus Linn[^ Iridaceae^ Cell culture^ Biosynthesis^ Metabolism^ Triterpenoids^ Iridals^ Spiroiridals^ Iridal esters^ 09!Desoxyiridal
0[ Introduction
serves most likely as precursor of the other triterpenoids[
Thus\ it has been shown by incubation of an Iris pallida
cell culture with various precursors that the conjugated
triene moiety of the spiroiridals "e[g[ 2# is formed by the
action of a dehydrogenase on iridal 0 or 15!hydroxyiridal
6\ respectively "Scheme 1# "Marner\ Ritzdorf\ + Johnen\
0882#[ Although compound 6 in this study undoubtedly
was accepted by the dehydrogenase as a substrate\ it was
not possible to rule out that the formation of the 15!
hydroxytriene 8 also may proceed via dehydrogenation
of 0 and subsequent oxidation of the iridotriene 7 at C15
"Marner et al[\ 0882#[ We set out to get further insight
into these reaction sequences by incubating a cell culture
of I[ pseudacorus\ which synthesizes appreciable amounts
of spiroiridals\ with an appropriate enzyme inhibitor[
The addition of a dehydrogenase inhibitor should\ for
instance\ suppress the formation of the conjugated triene
and lead to the accumulation of the respective precursors[
We report here on the results of these studies[
The iridals "e[g[ 0Ð2# and 09!desoxyiridals "e[g[ 3# are
unusual monocyclic\ bicyclic or spirobicyclic triter!
penoids\ which are found in the lipid extracts of Iridaceae
"Marner\ 0886#[ Their biosynthesis undoubtedly is
initiated by cyclization of 1\2!epoxysqualene to a bicyclic
intermediate\ which subsequently is rearranged to give
the characteristic seco!ring A structure of these natural
products "Marner\ 0886#[ In the course of this reaction a
CH2 group is shifted from C5 to C00[0 For stereochemical
reasons we assume that 1\2!epoxysqualene is folded in a
chair!boat form during this process and the _rst product
to be expected is the aldehyde 4\ which may be converted
via several steps to 09!desoxyiridal 5 "Scheme 0#[ Both
compounds have so far not been detected in nature\ but
the latter is the most plausible educt for all iridals and
09!desoxyiridals known today "Marner\ 0886#[ Thus\ the
action of a monooxygenase should\ with retention of the
con_guration at C09\ give iridal 0\ whereas oxidation of
the side chain of 5 should result in the formation of 09!
desoxy!06!hydroxyiridal 3 or its homologues[ Iridal 0
1[ Results and discussion
It seemed probable that the inhibition of the dehydro!
genase as a key step in the iridotriene biosynthesis might
not only lead to the accumulation of 15!hydroxyiridal 6
ꢀCorresponding author[
0 The carbon skeleton of all compounds is numbered in analogy to
squalene[
9920!8311:88:, ! see front matter Þ 0888 Elsevier Science Ltd[ All rights reserved[
PII] S9920!8311 "87#99528!5

885
I[ Ritzdorf et al[:Phytochemistry 49 "0888# 884Ð0992
Structure 0[
or iridal 0 but also of one or more of its precursors\ which
are depicted in Scheme 0[ Of special interest was 09!
desoxyiridal 5 as a central intermediate in the proposed
biosynthetic pathway[ Since it was not at hand as a
natural product\ a semisynthetic access to this compound
was worked out\ which is outlined in Scheme 2[ 09!
Desoxy!06!hydroxyiridal 3\ which can be isolated from
extracts of I[ sibirica in reasonable amounts "Marner
+ Longerich\ 0880#\ served as starting material[ After
reduction of the aldehyde function and protection of the
two primary hydroxy groups as benzoates\ the secondary
OH in the terpenoid side chain was converted to the
bromide[ Subsequent reaction with LiAlH3 lead in one
step to removal of the protecting ester groups and
reduction of the halide[ Finally\ the allylic alcohol was
selectively oxidized with MnO1 and 09!desoxyiridal 5 was
obtained[ With the help of this synthetic standard we
tried to track down the substance in extracts of the cul!
tured cells[
Upon HPLC analysis of total lipid extracts of the I[
pseudacorus cells\ prepared by soaking freshly harvested
cells in CHCl2:MeOH "0]1 v:v# according to Bligh and
Dyer "0848#\ the three spiroiridals\ esteri_ed at C2 with
capric acid\ 2a "45)#\ 01a "00)# and 02a "16)# were
found as main products adding up to more than 89) of
the iridal content[ Side products are the capric acid esters
of iridal 0a "3)# and 05!hydroxyiridal 03 "1)#[ Isolation
of a small amount of the compounds by semipreparative
HPLC and enzymatic hydrolysis with Lipozym 09999L\
as described elsewhere "Marner et al[\ 0882#\ gave the free
spiroiridals and monocyclic iridals\ which were identi_ed
by comparison with authentic standards "Marner\
Karimi!Nejad\ Jaenicke\ + Wray\ 0889^ Littek + Marner\
0880#[ After transesteri_cation of the compounds with
CH2OH:HCl the fatty acid was identi_ed by GC:MS[
The retention behaviour of synthetic 5 was identical with
that of the spirotriene ester 02a and therefore no 09!
desoxyiridal could be detected in these extracts[ Also the
search for this compound in essential oils of several Iris
species was unsuccessful[ When the cultured cells of I[
pseudacorus are homogenated in a mortar or by ultra!
sound under addition of phosphate bu}er prior to extrac!
tion\ however\ the fatty acid esters of the iridals are
hydrolyzed by esterases present in the cell homogenate[
Reversed phase chromatography of an ether extract now
showed the unesteri_ed iridals and spiroiridals\ which
elute much earlier than their corresponding esters[ In
addition\ now small amounts of 09!desoxyiridal 5 were
detected[ To ensure its structure\ the substance was iso!
lated and characterized by NMR "0H\ 0202C\ DEPT\ H\H!
COSY and H\C long range#\ mass and UV spectroscopy[
The absolute con_guration was assured in analogy to 09!
desoxy!06!hydroxyiridal 3 "Marner + Longerich\ 0880#
and by NOE measurements[
The dehydrogenation of iridal 0 or 15!hydroxyiridal 6\
resulting in an all!E conjugated triene moiety\ closely
resembles the reactions catalyzed by phytoene desaturase
in the biosynthesis of carotenoids[ The inhibition of these
desaturase reactions in plants by {chlorosis inducing
herbicides| leads to the lack of cyclic carotenoids\ which
are used for the protection of chlorophylls against photo!
oxygenation[ Subsequently the chlorophylls are
destroyed\ as seen by the {bleaching| of the plant\ and
_nally the plants die[ It has been shown that 2!amino!

I[ Ritzdorf et al[:Phytochemistry 49 "0888# 884Ð0992
886
Scheme 0[ Proposed biosynthesis of iridals and 09!desoxyiridals[
0\1\3!triazole "amitrole# belongs to this group of reagents[
Thus\ the treatment of cell suspension cultures or ger!
minating seeds with this compound leads to the accumu!
lation of carotenoids with low degree of unsaturation\ e[g[
phytoene and phyto~uene "Burns\ Buchanan\ + Carter\
0860^ Hassal\ 0889#[ When 2!amino!0\1\3!triazole is
added to the culture medium of the I[ pseudacorus cells\ a
signi_cant change in the iridal composition was observed
after a few days[ As shown in Fig[ 0\ in comparison to
a blind\ in the treated culture within two weeks a 1!\
respectively\ 3!fold increase in the relative amounts of
iridal 0 and 09!desoxyiridal 5 was observed[ The per!
centage of 17!hydroxyspiroiridal 01 and 17!acetoxy!
spiroiridal 02 remained nearly constant\ whereas the rela!
tive amount of spiroiridal 2 decreased by a factor of
about 0[4 to less than 24)[ It is noteworthy to mention
that 09!desoxyiridal 5 is\ in contrary to all other iridals\
not esteri_ed\ as seen in Bligh and Dyer extracts of the
aminotriazole treated cultures[ In course of the incu!
bation period the growing HPLC peak of compound 5
can be observed\ which previously was hidden under!
neath the signal of 02a[
These results alone\ however\ are not signi_cant
enough for a sound statement\ if iridal 0 and 09!desox!
yiridal 5 really accumulate due to a desaturase inhibition[
Thus\ these changes in the relative composition of iridals

887
I[ Ritzdorf et al[:Phytochemistry 49 "0888# 884Ð0992
of the compounds present in course of the incubation
period[
The development of the fresh weight of the harvested
cells is depicted in Fig[ 1[ It is obvious that within the
_rst week of the aminotriazole incubation the treated and
untreated cells grow at the same rate[ From day 8 on the
control cells grow signi_cantly faster than the treated
culture\ which starts to die after 01 days[ In untreated
cultures of I[ pseudacorus a slowdown of the cell growth
is observed after two weeks "data not shown# due to an
overpopulation in the culture vessel[ It should be men!
tioned that the cells were transferred into fresh medium
after 6 days\ which in case of the treated culture again
contained the inhibitor in the same concentration as
before[ This certainly sped up the inhibition of important
enzymatic reactions\ thus leading to the slower growth
and _nally the cell death observed[ Fig[ 2 shows the
absolute amount of the triterpenoids within the cells in
relation to their dry weight\ which was determined after
freeze drying the homogenated cells[ Subsequently the
cell powder was resuspended in water and after extraction
with ether the unesteri_ed compounds could be analyzed
as before[ The amount of iridals and spiroiridals present
was determined by comparison with appropriate stan!
dards[ As seen in Fig[ 1 a steady decline in the contents
of the spirotrienes 2\ 01 and 02 can be observed in the
treated cell cultures\ whereas the values in the control
remain nearly constant[ In contrast\ the amount of the
monocyclic precursors increases within the _rst week
upon aminotriazole inhibition[ From day 8 on also a
drop in the quantity of these compounds is encountered
which may be due to the increase in inhibitor as men!
tioned above[ As for the trienes\ also the amount of 0 and
5 is unchanged within the blind throughout the culture
period[
Scheme 1[ Formation of the iridotrienes[
may also be observed\ when the total of iridals in the cell
culture decreases and when the spirotrienes 2\ 01 and 02
disappear at a faster rate than 0 and 5[ This may happen\
for instance\ when the cells die under the in~uence of
aminotriazole and the iridal biosynthesis ceases[ Since the
conjugated trienes are much more susceptible to oxidative
degradation than the less desaturated compounds 0 and
5 "Marner\ 0886#\ the ratio of the latter to the former
should increase in this situation[ Therefore\ it was essen!
tial to determine the cell growth and the absolute amount
These results undoubtedly point to an inhibition of the
desaturase\ which is responsible for the formation of the
Scheme 2[ Synthesis of 09!desoxyiridal 5[ Reagents] "a# NaBH3\ "b# PhCOCl\ pyridine\ "c# Ph2PBr1\ "d# LiAlH3 and "e# MnO1[

I[ Ritzdorf et al[:Phytochemistry 49 "0888# 884Ð0992
888
Fig[ 0[ Iridal composition ")# of an I[ pseudacorus cell culture upon incubation with 2!amino!0\1\3!triazole[ Top] spiroiridal 2\ 17!hydroxyspiroiridal
01 and 17!acetoxyspiroiridal 02[ Bottom] iridal 0 and 09!desoxyiridal 5[ Straight lines] probe and dashed lines] control[
conjugated triene[ Certainly\ iridal 0 is its natural sub!
strate and not 15!hydroxyiridal 6\ at least in the cell
strain of I[ pseudacorus used in these experiments\ since
otherwise the latter compound should have accumulated[
Also\ neither iridotriene 7 nor 15!hydroxytriene 8 could
be found in the treated cells[ Therefore\ it can be excluded
that aminotriazole prevents only the formation of the
spirobicyclic ring system\ which presumably is the prod!

0999
I[ Ritzdorf et al[:Phytochemistry 49 "0888# 884Ð0992
Structure 1[
uct of an intramolecular Prins reaction after oxidation of
C15 to an aldehyde "Marner\ 0886#[ It can be concluded
from the data shown in Figs 0 and 2 that the drop in the
levels of 17!hydroxyspiroiridal 01 and its acetate 02 is
less pronounced than that of their immediate precursor
2[ Therefore the oxidation of C17 and its acetylation are
not a}ected in the same way by the presence of ami!
notriazole as the desaturation and formation of the spi!
robicyclic triene moiety[ Since most of the other iridal
precursors\ outlined in Scheme 0\ lack a chromophore\ it
was not possible to detect them with the analytical
methods used[ It should\ however\ be possible to establish
their presence with other detectors\ e[g[ LC:MS[ Cor!
responding studies are underway and it will be interesting
to _nd out if it is possible to trace the biosynthetic path!
way of the iridals this way further back to epoxysqualene[
Fig[ 1[ Growth of an I[ pseudacorus cell culture "fresh weight of cells# upon incubation with 2!amino!0\1\3!triazole[

I[ Ritzdorf et al[:Phytochemistry 49 "0888# 884Ð0992
0990
Fig[ 2[ Absolute amount of iridals "mg:g dry weight# in I[ pseudacorus cells upon incubation with 2!amino!0\1\3!triazole[ Top] spiroiridal 2\ 17!
hydroxyspiroiridal 01 and 17!acetoxyspiroiridal 02[ Bottom] iridal 0 and 09!desoxyiridal 5[ Straight lines] probe and dashed lines] control[

0991
I[ Ritzdorf et al[:Phytochemistry 49 "0888# 884Ð0992
2[ Experimental
the diester 09 "119 mg\ 9[21 mmol#\ dissolved in 0 ml
benzene\ was added[ The reaction mixture was stirred for
1[4 h\ _ltrated and evaporated at RT[ The crude product
was dissolved in 4 ml THF and a soln of 799 mg LiAlH3
in 6 ml THF was added at −19>C within 3 h[ Sub!
sequently H1O "0 ml# and Et1O "04 ml# were stirred in
and the mixture was allowed to warm to RT[ The organic
phase was washed with satd NH3Cl soln\ dried "Na1SO3#
and evaptd[ The product was puri_ed by ~ash CC on
silica gel "pentane:Et1O 29]69 v:v# to give 14 mg "19)#
00[ EI!MS m:z "rel[ int[#] 333 ðM¦Ł "9[4#\ 315 ðM¦!H1OŁ
"3#\ 176 "2[4#\ 163 "1#\ 023 "28#\ 84 "47#\ 70 "43#\ 58 "099#^
0H NMR] d 4[04Ð3[84 "2H\ m\ H!03\ H07 and H!11#\ 3[01
"1H\ AB\ JABꢁ7[4 Hz\ H!0#\ 2[5 "1H\ t\ Jꢁ6 Hz\ H!2#\
1[59 "0H\ m\ H!5#\ 1[04Ð0[74 "xH#\ CH1\ CH#\ 0[71 "2H\
s\ H!14#\ 0[69 "2H\ s\ CH2#\ 0[47 "8H\ s\ 2 CH2#\ 9[89 "2H\
s\ H!15#\ 9[67 "2H\ d\ H!16#[ The compound contained
11) of the D06\07 isomer\ formed by allylic shift of the
D07\08 double bond during the LiAlH3 reduction[ This
is seen by signals at d 4[17 "1H\ m\ H06 and H07# and
1[48 "1H\ d\ H!05#[
2[0[ General
HPLC] Kontron model 199\ column] LiChrocart RP
07 "014 mm\ Merck#^ solvent] MeOH:H1O 6]2 "4 min#\
linear gradient to 099) MeOH "04 min#\ 099) MeOH
"19 min#^ Hewlett!Packard 0939A diode!array detector[
Flash CC] Merck Kieselgel 59 "39Ð52 mm#[ MS] Finnigan!
MAT 3409 GC:MS "EI] 69 eV#\ inlets] capillary column
OV0 "04 m\ 9[14 mm i[d[# and solid probe[ NMR] Bruker
AM 299 "0H] 299 MHz\ 02C] 64 MHz#\ solvent CDCl2[
UV] Hitachi U 1999\ solvent MeOH[ Ultrasound pro!
cessor] Dr[ Hieschler UP 399S[ Freeze drying] Leybold
Lyovac GT1[
2[1[ Compounds
The iridals used in this study as standards were isolated
from extracts of I[ foetidissima as described elsewhere
"Marner et al[\ 0889^ Littek
+
Marner\ 0880#[
5S\09R\00R!09!Desoxyiridal
5 was synthesized as
follows[
2[1[2[ 5S\09R\00R!09!Desoxyiridal\ 5[ 14 mg "9[945
mmol# of the diol 00 and 074 mg MnO1 "1[02 mmol# in 2
ml CH1Cl1 were shaken at RT for 05 h[ The soln was
_ltrated over 099 mg silica gel and evaptd in vacuo[ The
crude product was puri_ed by ~ash CC on silica gel "29)
Et1O# to yield 01 mg "37)# 5\ containing 11) of the
D06\07 isomer[ The spectral characterization can be seen
in Section 2[4[
2[1[0[ 5S\09R\00R\06j!0\2!Dibenzoyl!0!dihydro!06!hy!
droxy!09!desoxyiridal\ 09[ 359 mg "0 mmol#
5S\09R\00R\06j!09!desoxy!06!hydroxyiridal\ 3\ ob!
tained from extracts of I[ sibirica "Marner + Longerich\
0880#\ were dissolved in 49 ml MeOH[ 59 mg "0[5 mmol#
NaBH3 were added and the soln was stirred for 1 h at
RT[ After concentration in vacuo\ 49 ml satd NH3Cl soln
were added and the cloudy soln was extracted twice with
49 ml Et1O[ The organic phase was washed with H1O\
dried "Na1SO3# and evaporated to dryness[ The product
"314 mg\ 9[8 mmol#\ dissolved in 14 ml CH1Cl1 was cooled
to −39>C[ Pyridine "599 mg\ 6[5 mmol# and benzoyl
chloride "219 mg\ 1[2 mmol# were added and the soln was
stirred for 7 h[ After warming to RT the reaction mixture
was diluted with 19 ml CH1Cl1\ washed with H1O and
dried "Na1SO3#[ After evapn of the solvent the product
was puri_ed by ~ash CC on silica gel with pentane:Et1O
"79]19\ v:v# to yield 119 mg "25)# of the diester[ EI!MS
m:z "rel[ int[#] 549 ðM¦!H1OŁ "9[0#\ 417 "0[4#\ 348 "0#\ 393
"1#\ 283 "2#\ 094 "099#\ 58 "49#^ 0H NMR] d 7[91Ð6[81 "3H\
AB\ Phe!H#\ 6[41 "1H\ m\ Phe!H#\ 6[33Ð6[29 "3H\ AB\
Phe!H#\ 4[1Ð4[94 "2H\ m\ H!03\ H07 and H!11#\ 3[89 "1H\
d\ H!0#\ 3[31 "0H\ m\ H!06#\ 3[14 "1H\ m\ H!2#\ 1[57 "0H\
m\ H!5#\ 1[04Ð0[74 "xH#\ CH1\ CH#\ 0[71 "2H\ s\ H!14#\
0[55 "2H\ s\ CH2#\ 0[51 "2H\ s\ CH2#\ 0[59 "2H\ s\ CH2#\
0[43 "2H\ s\ CH2#\ 9[89 "2H\ s\ H!15#\ 9[79 "2H\ d\ H!16#[
2[2[ Cell culture
Callus cultures of I[ pseudacorus were prepared by plac!
ing sterile cuts of a seedling on agar plates "9[7) agar#
containing the medium described below[ After several
weeks of growth a suspension culture was established
from the calli[ The cells were raised in a Linsmaier!Skoo`
medium "Linsmaier + Skoog\ 0854# pH 5[9 with 2)
sucrose\ 0[1 mM thiamine hydrochloride\ 9[45 mM myo!
inositol\ 0 mM 1\3!dichlorophenoxyacetic acid and 0 mM
0!naphthaleneacetic acid[ The cultures were cultivated at
continuous light "299 lx# and 12>C on a gyratory shaker
"099 rpm#[ They were subcultured every 6 days by trans!
ferring 09 g of the old cells to 149 ml of fresh medium in
a 0 l Erlenmeyer ~ask[ During this time the fresh weight
of the cells doubled[
2[3[ Extraction and analysis of the iridals
For the extraction of the lipids according to Bligh and
Dyer "0848# the cells were _ltrated through a sieve and
extracted twice with CHCl2:MeOH "0]1\ v:v\ 49 ml#[ After
evaporation of the solvent the residue was partitioned
between Et1O "1×099 ml# and H1O "49 ml#\ the organic
phase was separated\ dried "Na1SO3# and the solvent was
2[1[1[ 5S\09R\00R!0!Dihydro!09!desoxyiridal\ 00[ 159 mg
"0 mmol# triphenylphosphine were dissolved in 1[4 ml
benzene and a soln of 059 mg "0 mmol# Br1 in 0[4 ml
benzene was added dropwise[ After 04 min at RT the
light yellow suspension of PPh2Br1 was cooled to 4>C and

I[ Ritzdorf et al[:Phytochemistry 49 "0888# 884Ð0992
0992
evaporated in vacuo[ A soln of the crude oil in MeOH
was analyzed by HPLC[
autoclaved medium in a 0 l Erlenmeyer ~ask and the
solution was added to 09 g of the cells[ The medium of
the control culture was mixed with 199 ml ethanol only[
For incubation periods longer than a week\ after 6 days
the cells were transferred into 499 ml fresh medium con!
taining 9[4 mM aminotriazole\ respectively\ the appro!
priate amount of ethanol[ After the appropriate
incubation time 09 g of the cell material was mixed with
09 ml bu}er soln "9[0 M KH1PO3\ 0 mM EDTA\ 0 mM
glutathione\ pH 5[1#\ treated with ultrasound and the
homogenate was freeze dried[ After determination of the
dry weight "the weight of the bu}er salts was subtracted#
the material was resuspended in 09 ml H1O and extracted
once with 69 ml and two times with 49 ml Et1O[ The
combined organic phases were dried "Na1SO3# and evaptd
in vacuo[ A 0) soln of the extract in MeOH was used
for the quantitative analysis of the iridals by RP HPLC[
For the determination of the relative composition the
response of the compounds at 143 nm was used\ since it
is known that the molar extinction coe.cient of the iri!
dals at this wavelength is equal "Marner + Kerp\ 0881#[
09!Desoxyiridal 5 and spiroiridal 2 served as standards
for the calculation of the absolute amount present[
2[4[ Isolation of 5S\09R\00R!09!desoxyiridal\ 5
49 g of freshly harvested cells were mixed with 49 ml
bu}er solution "9[0 M KH1PO3\ 0 mM EDTA\ 0 mM
gutathione\ pH 5[1# and 09 g quartz sand and ground in
a mortar[ The homogenate was pressed through a nylon
net "49 mm#\ allowing also the sand particles to pass[ The
_ltrate was discarded and the residue was stirred 3Ð5 h
with 099 ml Et1O\ transferred into a Waring blendor
and mixed for several minutes[ The organic phase was
separated and the aqueous phase mixed two more times
with Et1O in the blendor[ The combined ether phases
were dried and evaptd to give 074 mg of crude extract[
399 mg of the oil were separated on silica gel "09 g#
using a pentane:Et1O gradient[ The fraction eluting with
pentane:Et1O "19]79 v:v# was rechromatographed on sil!
ica gel with pentane:Et1O "14]64 v:v# to give 5 mg of pure
5[ UV lmax nm "o#] 143 "01499#^ EI!MS m:z "rel[ int[#] 331
ðM¦Ł "2#\ 313 ðM¦!H1OŁ "1#\ 194 "6[4#\ 098 "36#\ 84 "37#\
70 "42#\ 58 "099#\ 44 "34#^ 0H NMR] d 09[07 "0H\ s\ H!0#\
4[96 "0H\ m\ H!11#\ 4[94 "0H\ m\ H!07#\ 3[85 "0H\ t\ Jꢁ5
Hz\ H!03#\ 2[5 "1H\ t\ Jꢁ5[2 Hz\ H!2#\ 2[24 "0H\ dd\
Jꢁ6[6:5[8 Hz\ H!5#\ 1[52:1[04 "1H\ m\ H!7#\ 1[91 "1H\
m\ H!06#\ 0[82 "3H\ m\ H!05\ H!19#\ 0[77 "0H\ m\ H!09#\
0[68 "1H\ m\ H!02#\ 0[67 "2H\ s\ H!14#\ 0[54 "2H\ s\ H!
13#\ 0[52 "1H\ m\ H!4#\ 0[59:0[27 "1H\ m\ H!8#\ 0[46 "2H\
s\ H!29#\ 0[44 "2H\ s\ H!18#\ 0[38 "2H\ s\ H!17#\ 0[2 "1H\
m\ H!3#\ 0[07:0[98 "1H\ m\ H!01#\ 9[85 "2H\ s\ H!15#\ 9[79
"2H\ d\ Jꢁ5[7 Hz\ H!16#^ 02C NMR] 089[9 "d\ C!0#\ 052[2
"s\ C!6#\ 024[1 "s\ C!04#\ 023[8 "s\ C!08#\ 022[2 "s\ C!1#\
020[1 "s\ C!12#\ 013[3 "d\ C!03#\ 013[3 "d\ C!11#\ 013[1 "d\
C!07#\ 52[9 "t\ C!2#\ 32[2 "d\ C!5#\ 39[0 "s\ C!00#\ 28[6 "t\
C!05#\ 28\6 "t\ C!19#\ 24[6 "d\ C!09#\ 20[7 "t\ C!01#\ 20[4
"t\ C!3#\ 29[4 "t\ C!8#\ 16\3 "t\ C!7#\ 15\7 "t\ C!10#\ 15[5 "t\
C!06#\ 14[6 "q\ C!13#\ 13[1 "q\ C!15#\ 13[9 "t\ C!4#\ 10[0 "t\
C!02#\ 06[6 "q\ C!29#\ 05[9 "q\ C!18#\ 04[8 "q\ C!17#\ 04[1
"q\ C!16#\ 09[1 "q\ C!14#[
Acknowledgements
The authors wish to thank Dr[ H[ Schmickler "Institute
of Organic Chemistry\ University of Cologne# for record!
ing the NMR spectra[ Financial support by the Fonds
der chemischen Industrie\ Frankfurt\ is gratefully
acknowledged[
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