(2R)-2-amino-6-hydroxy-4-hexynoic acid, and related amino acids in the fruiting bodies of Amanita miculifera

Article abstract of DOI:10.1016/S0031-9422(98)00177-0

(2R)-2-amino-6-hydroxy-4-hexynoic acid, (2RS)-2-amino-4,5-hexadienoic acid, (2S)-2-amino-4-hexynoic acid, and 2-amino-5-chloro-5-hexenoic acid were isolated and characterised from the fruit bodies of Amanita miculifera, with the first and the last mentioned amino acids being new natural products. Their structures were based on analysis of the results from elementary analysis, oxidation with KMnO₄, determination of optical rotations, ¹H NMR- spectra, and hydrogenation over Adams or Lindlar catalysts. (2RS)-2-Amino-6- hydroxy-4-hexynoic acid was synthesized and optically resolved by renal acylase to confirm its natural (2R)-configuration. The configuration at C-2 of 2-amino-4,5-hexadienoic acid was also assumed from its ORD spectrum, as well as that of its hydrogenation product. The optical rotation (2-amino-5- chloro-5-hexenoic acid) was not determined because it was isolated in trace amounts; it was also prepared from 2-amino-5-hexynoic acid, although in very low yield. A possible biosynthetic route leading to the non-protein amino acids in this fungus is presented.

Full text of DOI:10.1016/S0031-9422(98)00177-0

Phytochemistry\ Vol[ 38\ No[ 1\ pp[ 462Ð467\ 0887
Þ 0887 Elsevier Science Ltd[ All rights reserved
Printed in Great Britain
Pergamon
\
9920Ð8311:87:,Ðsee front matter
PII] S9920Ð8311"87#99066Ð9
"1R#!1!AMINO!5!HYDROXY!3!HEXYNOIC ACID\ AND RELATED
AMINO ACIDS IN THE FRUITING BODIES OF AMANITA MICULIFERA0
IN HONOUR OF PROFESSOR G[ H[ NEIL TOWERS 64TH BIRTHDAY
SHIN!ICHI HATANAKA\aꢀ YUKIO NIIMURA\b KUNIO TAKISHIMAc and JUNTA SUGIYAMAd
aDepartment of Biology\ Division of Natural Sciences\ International Christian University\ Osawa 2!09!1\ Mitaka!shi\
Tokyo 070\ Japan^ bRadioisotope Research Center\ Teikyo University School of Medicine\ Kaga 1!00!0\ Itabashi!ku\ Tokyo
062\ Japan^ cDepartment of Biochemistry I\ National Defense Medical College\ Tokorozawa\ Saitama 248\ Japan^
dInstitute of Molecular and Cellular Biosciences\ The University of Tokyo\ Yayoi 0!0!0\ Bunkyo!ku\ Tokyo 002\ Japan
"Received 2 October 0886^ received in revised form 01 January 0887^ accepted 19 February 0887#
Key Word Index*non!protein amino acids^ "1R#!1!amino!5!hydroxy!3!hexynoic acid^ D!amino
acid^ Amanita miculifera^ Amanitaceae^ fruiting bodies
Abstract*"1R#!1!amino!5!hydroxy!3!hexynoic acid\ "1RS#!1!amino!3\4!hexadienoic acid\ "1S#!1!amino!3!
hexynoic acid\ and 1!amino!4!chloro!4!hexenoic acid were isolated and characterised from the fruit bodies of
Amanita miculifera\ with the _rst and the last mentioned amino acids being new natural products[ Their
structures were based on analysis of the results from elementary analysis\ oxidation with KMnO3\ determination
of optical rotations\ 0H NMR!spectra\ and hydrogenation over Adams or Lindlar catalysts[ "1RS#!1!Amino!
5!hydroxy!3!hexynoic acid was synthesized and optically resolved by renal acylase to con_rm its natural "1R#!
con_guration[ The con_guration at C!1 of 1!amino!3\4!hexadienoic acid was also assumed from its ORD
spectrum\ as well as that of its hydrogenation product[ The optical rotation "1!amino!4!chloro!4!hexenoic
acid# was not determined because it was isolated in trace amounts^ it was also prepared from 1!amino!4!
hexynoic acid\ although in very low yield[ A possible biosynthetic route leading to the non!protein amino acids
in this fungus is presented[ Þ 0887 Elsevier Science Ltd[ All rights reserved
INTRODUCTION
acids were detected from its fruit bodies "Fig[ 0\ com!
pounds 0Ð3#[ Now we report details of their isolation
and structural elucidation\ giving special attention to
their optical properties[ Possible biosynthetic path!
ways and speci_c distribution pattern of non!protein
amino acids of this species are also discussed[
It has been known that the fruit bodies of the section
Roanokenses "Singer ð0ŁꢁLepidella^ ðBas\ 0858Ł ð1Ł#\
subgenus Lepidella of the genus Amanita contain vari!
ous non!protein amino acids of speci_c chemical
structure ð2Ł[ Interestingly several have unbranched
unsaturated carbon!chains\ i[e[\ unsaturated nor!
valine or norleucine\ and chlorine!containing amino
acids are also known[ Most recently\ "1S#!1!amino!
4!chloro!3!hydroxy!4!hexenoic acid was isolated and
characterized from Amanita `ymnopus Corner and
Bas ð3Ł\ and from an undescribed Amanita which also
belongs to the section Roanokenses ð4Ł[
RESULTS AND DISCUSSION
Although it is possible that the distribution pattern
of non!protein amino acids might re~ect phylogenetic
relationships among fungal species\ some knowledge
of new biosynthetic relationships is _rst required to
support this view[ Figure 1 shows a presumed biosyn!
thetic route leading to the formation of the speci_c
non!protein amino acids found in A[ miculifera[ 1!
Amino!3\4!hexadienoic acid 1 was _rst reported in
0857 from the fruiting bodies of Amanita solitaria
"Fr[# Secr[ sensu D[ E[ Stuntz ð6Ł "now regarded as A[
smithiana Bas in the section Roanokenses#[ Although
the isolate by Chilton et al[ was itself optically inactive\
it was concluded that it belongs to the L!series of
amino acids\ because its hydrogenation product was
optically similar to L!norleucine[ Since then this amino
In 0873\ Bas and one of us reported an undescribed
Amanita sp[ and named it Amanita miculifera Bas and
Hatanaka ð5Ł[ This species also belongs to the section
Roanokenses\ and several speci_c non!protein amino
0 Dedicated to Prof[ G[ H[ N[ Towers on the occasion of
his 64th birthday[ Part 14 in the series {{Biochemical studies
on nitrogen compounds of fungi||[ For Part 13\ see ref[ ð4Ł[
ꢀ Author to whom correspondence should be addressed[
462

463
SHIN!ICHI HATANAKA et al[
oxidized by L!amino acid oxidase[ Therefore it pos!
sibly occurs in many species in a partially racemized
form[ Regrettably its resolution form an isolate from
A[ miculifera by renal acylase\ was unsuccessful[
L!1!Amino!3!hexynoic acid 2 was isolated _rst
from the fruit bodies of Tricholomopsis rutilans "Fr[#
Sing[ ð00Ł\ and then detected in other Tricholomopsis
species ð01Ł[ Some years later it was also isolated from
A[ pseudoporphyria\ amongst other amino acids of
unsaturated norleucine!type ð09Ł[ The speci_c optical
rotations in water of this amino acid isolated from T[
rutilans\ A[ pseudoporphyria\ and A[ miculifera were
determined to be −43> "c 0[9#\ −28> "c 0[9#\ and −04>
"c 9[7#\ at room temperature\ respectively[ Therefore\
the optical purity of this amino acid again seems not
to be uniform[
We then reported earlier that species of the pter!
idophyte\ genus Asplenium\ sometimes contain pure D!
and sometimes partially racemized D!1!aminopimelic
acid and trans!2\3!dehydro!D!1!aminopimelic acid[
Furthermore the D] L ratio depends on the season\ as
well as on the plant species ð02\ 03Ł[
It is worth emphasizing that 3 exists in pure D!form
and its possible direct precursor\ 2\ occurs pre!
dominantly in the L!form[ Compound 1\ which is very
likely metabolically related\ is\ however\ a racemate[
We cannot yet explain\ what kind of chemical or enzy!
matic processes might account for these observations[
EXPERIMENTAL
Fun`us
The fruiting bodies of Amanita miculifera "479 g\
fresh weight# were collected in August 0864 in Nagano
Prefecture and stored in a refrigerator for one day
before extraction[ We should like to propose to name
this fungus {{Haikaburi!Tengu!Take|| "Ash!covered
Amanita#[
Fig[ 0[ Structures of the non!protein amino acids from Aman!
ita miculifera[
General
Evapn of solvents was carried out using a rotary
acid has been reported to be distributed sporadically evaporator on a water bath below 39>C[ For PC\
not only in the section Roanokenses\ but in other sec! Whatman No[ 49 and for cellulose chromatograpy\
tions such as A[ abrupta Peck ð7Ł\ and A[ `ymnopus thin layer and column {{Avicel|| "Funakoshi Phar!
ð3Ł "both in the Roanokenses section#\ as well as A[ maceutical Co[\ Ltd[ Tokyo\ Japan# were employed[
neoovoidea Hongo ð8Ł "section Amidellae#\ and in A[ The solvent systems used were as follows] n!BuOH!
pseudoporphyria Hongo ð09Ł "Phalloideae section#[ HOAc!H1O "52]09]16# "A#\ Phenol!H1O "14]7\ in NH2
The possible chemical reactivity and the rather wide vapor# "B#\ t!AmylOH!Pyridine!H1O "24]24]29# "C#\
distribution of this allenic amino acid\ suggests a role n!BuOH!methyl ethyl ketone!"conc[# NH3OH!H1O
as a central intermediate in the biosynthesis of many "04]8]3]1# "D# and t!AmylOH!methyl ethyl ketone!
speci_c\ in particular\ norleucine!type amino acids in "conc[# NH3OH!H1O "61]34]1]0# "E#[
fungi[
Infrared spectra were measured using a Hitachi IR
Interestingly 1 from this fungus gave no uniform spectrometer model 159!29\ whereas 0H NMR!spectra
optical rotation^ and the ðaŁ1D1!values in H1O of this were recorded on a Jeol JNM!PS!099 NMR spec!
acid from A[ neoovoidea and A[ pseudoporphyria were trometer\ 099 MHz\ in D1O with TPS as an
reported −13> "c 0[9# and −41[0> "c 9[7#\ respectively[ int[ standard "dꢁ9#\ and a Hitachi RMU5C mass
Furthermore\ although no optical rotation was spectrometer\ respectively[ Optical rotations were cal!
reported in the case of A[ abrupta\ it was completely culated from the results of the measurements of ORDs

A D!amino acid from Amanita miculifera
464
Fig[ 1[ Possible biosynthetic relationships among the non!protein amino acids in Amanita miculifera[
at 478 nm[ Melting points were determined in capil! acid fraction[ Although only one unusual spot near
laries and uncorrected[
valine was detected on a two dimensional PC
developed with the solvents A and B successively\
careful fractionation on a cellulose column with the
Extraction\ fractionation\ and puri_cation of the amino solvent A\ D\ or E revealed the existence of at least
acids
four unusual amino acids[ We designated them 0Ð3 in
the order of the Rf!values in the solvent E[ To obtain
On the day followin` collects on the fruitin` bodies\ each amino acid in crystalline form\ the amino acid
these were then extracted "4×# with 79) ethanol in a fraction was puri_ed repeatedly mainly using cellulose
homo`enizer[ The combined extract "3 l# was next chromotography with columns eluted with various
passed throu`h a column of Amberlite IR 019B "H¦# solvent systems "see above#[ When an ion exchanger
"099 ml#\ the resin was then washed successively tho! resin was used\ Dowex!0×3 "199Ð399 mesh# in acetate
roughly with 79) ethanol and water\ and the retained form was the most successful[ For the sepn of these
amino acids were displaced with 1N NH3OH "0 l#[ amino acids\ solvent system D with a cellulose column
Ammonia was removed by evapn\ giving the amino proved to be the most e}ective[

465
SHIN!ICHI HATANAKA et al[
amount of activated charcoal\ the crystals obtained
Isolation of the amino acids
were recrystallized three times from aq[ EtOH\ yield!
ing 49 mg of pure 2[ Elemental analysis\ Found] C\
45[76^ H\ 6[07^ N\ 09[83)[ Calcd for C5H8NO1] C\
45[57^ H\ 6[02^ N\ 00[91)[ mp[ 088>C "decomp[#[
ðaŁ1D1 −04> "H1O^ c 0[3\#\ −3> "2N HCl^ c 9[7\#[ Hydro!
genation over Adams and Lindlar catalysts revealed
that the isolate absorbed 0[75 and 0[92 equiv[ moles
of H1\ respectively\ with the only product identi_ed as
norleucine\ by TLC comparison using solvents sys!
tems A\ B\ and E[ Oxidation with KMnO3 yielded
Asp\ as before\ with its identi_cation con_rmed using
TLC comparison with solvent systems A\ B\ and E^
the colour of the reaction with ninhydrin was _rst
yellow\ then changed to brown with time\ and _nally
to normal violet[ Compound 3[ Following treatment
with a small amount of activated charcoal\ crude sam!
ple was recrystallized from aq[ EtOH three times[
Elemental analysis\ Found] C\ 49[00^ H\ 5[28^ N\
8[66)[ C5H8NO2 requires C\ 49[24^ H\ 5[23^ N\
8[68)[ mp[×044>C[ ðaŁ1D1 ¦25> "H1O^ c 0#\ ¦09> "2N
HCl^ c 9[4\#[ 0H NMR! "d#] 1[75 "1H\ m\ !CH1!#\ 2[72
"0H\ t\ Jꢁ4[4 Hz\ −CH=−#\ 3[05 "1H\ t\ Jꢁ1[9 Hz\
HOCH1!#[ MS] m:z\ ðM!H1OŁ¦\ 014^ ðM!CO1HŁ¦\ 87^
ð!CH"NH1#CO1HŁ¦\ 63[ IR!Spectra "cm−0#] 1159\
1129 "acetylene#[ On hydrogenation\ the pure sample
absorbed 1[97 and 0[97 equiv[ moles H1 over Adams
and Lindlar catalysts\ respectively[ Hydrogenation
product obtained with the former catalyst was puri_ed
and a few mg were isolated[ When heated in a sealed
tube\ at 019Ð029>C with 44) HI "ca[ 0 ml# and a small
amount of red phosphorus for 05 h\ norleucine was
the only ninhydrin!positive substance formed[ Re!
action of 3 with ninhydrin gave a brown product[
Compound 3 seemed to be a D!amino acid\ when
the Lutz and Jirgensons rule was applied[ It was\ there!
fore\ synthesized and resolved optically to con_rm the
natural occurrence of the D!isomer[
As a result of the _rst fractionation of the total
amino acids on a cellulose column "1[6×649 cm#
eluted using solvent system A\ several fractions were
obtained containing 0Ð3 in di}erent concn[
Rechromatography under nearly the same conditions
gave fractions I "1¦2#\ II "3#\ and III "0#\ respectively[
Fraction I was further successively separated by
cellulose column\ "1[6×54 cm chromatography# using
solvent system C\ then by
a cellulose column
"1[6×64 cm# with solvent system D\ and a cellulose
column "0[4×54 cm# with solvent system E\ respec!
tively[ As a result\ amino acids 1 "465 mg# and 2
"49 mg# were obtained nearly homogeneous[ From
Fraction II\ 3 "009 mg# was obtained following rech!
romatography with a cellulose column "1[6×64 cm#
with solvent system D[ Finally\ Fraction III
was placed on a cellulose column of 1[6×64 cm
and developed with solvent system D\ to yield 0
"07 mg#[
Properties of the puri_ed amino acids[
Compound 0 was recrystallized from aq[ EtOH[
Elemental analysis\ Found] C\ 33[08^ H\ 5[11^ N\ 7[43^
Cl\ 08[59)[ C5H09NO1Cl requires] C\ 33[94^ H\ 5[05^
N\ 7[45^ Cl\ 10[56)[ mp[×064>C "decomp[#[ 0H
NMR!spectra "d#] 1[0Ð1[3 "3H\ m\ !CH1!CH1!#\ 3[3
"0H\ t\ !CH!#\ 3[8\ 4[0 "1H\ CH1ꢁ#[ MS] m:z\ ðMÐ
ClŁ¦^ 017\ ðMÐCO1HŁ¦^ 007\ ð!CH"NH1#CO1HŁ¦^ 63[
Hydrogenation over Adams catalyst^ 1[09 equiv[ mole
H1 absorbed with norleucine as product\ with the
latter identi_ed by TLC comparison using solvent sys!
tem A and E with visualization violet using ninhydrin[
The yield of 0 was so small that its optical rotation
could not be determined[ Compound 1 was recrys!
tallized from aq[ EtOH[ Elementary analysis\ Found]
C\ 45\85^ H\ 6[16^ N\ 00[06)[ Calcd for C5H8NO1] C\
45\57^ H\ 6[02^ N\ 00[91)[ mp[×079>C "decomp[#[
Synthesis of amino acids 3 and 0
0H NMR!spectra "d#] 1[43 "1H\ m\ !CH1!#\ 2[65 "0H\
=
2[5[0[ Compound 3[ Sodium "1[2 g# was dissolved in
abs[ EtOH "27 ml# under re~ux[ Diethyl for!
q\ ÐCH =#\ 3[79Ð4[91 "2H\ m\ H1CꢁCꢁCHÐ#[ IR spec!
=
tra "cm−0#] 0844\ 734 "allene#[ Hydrogenation over mamidomalonate "19[2 g# in abs[ EtOH "49 ml# was
Adams catalyst] 0[71 equiv[ moles H1\ with the prod! then added and re~uxed further for 29 min[ 0!Chloro!
uct being norleucine as before\ as established by TLC 1!butyne!3!ol "09[4 g#\ which had been prepd accord!
comparisons using solvent systems A\ B\ C\ and E[ ing to Colonge and Poilane ð04Ł "09[4 g# was added
Oxidation with 0) KMnO3 in 09) H1SO3 at room dropwise and re~ux was a continued a further 4 h[
temp[ gave Asp\ which was identi_ed by TLC com! The ppt[ formed was removed by _ltration and the
parison using solvent systems A\ B and E with vis! _ltrate concd under reduced pressure[ The syrup was
ualization with ninhydrin\ which gave _rst a brown then dissolved in EtOH "039 ml#\ with NaOH solution
and then changed a violet color over time[ For deter! "19 g in 099 ml H1O# and the mixture re~uxed for
mination of its optical activity\ two solutions of 09 mg 0 h[ The reaction mixt[ was then concd to about half
and 4 mg of this amino acid in H1O and 2N HCl volume\ and its pH was adjusted to ca[ 1 by addition
respectively were prepared and both gave no optical of conc[ HCl under stirring in ice!water[ The mixture
rotation when prescribed at 478 nm[ The hydro! was then heated until re~ux began\ which was con!
genation product\ norleucine\ was also inactive tinued for 0 h[ The resulting mixture was then passed
between 249 and 599 nm in the equivalent concn[ in through 0 l Amberlite 019B "H¦#\ with the resin
H1O and HCl[ Compound 2[ After the _nal chro! washed with EtOH and H1O\ successively\ and the
matography prepn was further treated with a small amino acid eluted with 09 l NH3OH[ Because the prod!

A D!amino acid from Amanita miculifera
466
uct contained a small amount of Gly\ it was subjected the whole was heated until re~ux began\ this being
to further chromotagraphy using Dowex 0×3 "−OAc\ maintained for 0[4 h[ After cooling\ the bulk of EtOH
199Ð399 mesh# eluted with 9[994 N acetic acid[ Rel! was then removed by evaporation under reduced pres!
sure and the pH of the mixt[ was adjusted to 1 with
conc HCl "ca[ 14 ml#[ It was hydrolyzed by re~ux for
0[4 h[ The temperature was again raised until re~ux
began\ which was held for an additional 0[4 h[ The
solution was cooled\ and the crude product was sub!
jected to Amberlite 019B "H¦\ 499 ml# chro!
matography as before[ The ammonia eluate "4 l# was
evaporated with ca[ half of it fractionated using cellu!
lose column "2×65 cm\ solvent system A#[ Crude crys!
tals were obtained "44 mg# and recrystallized from aq[
EtOH[ Elemental analyses\ Found] C\ 45[35^ H\ 6[94^
N\ 09[89)[ C5H00NO1 requires] C\ 45\57^ H\ 6[03^ N\
evant fractions were combined and concentrated\
yielding 5[95 g crude crystals "31) yield#[ Recrys!
tallized from aq[ EtOH gave colourless sample[
Elemental analysis\ Found] C\ 49[14^ H\ 5\22^ N\
8[88)[ C5H8NO2 requires] C\ 49[23^ H\5[23^ N\
8[68)[ mp[×057>C "decomp[#[ 0H NMR! "d#1[75
=
"1H\ m\ !CH1!#\ 2[72 "0H\ t\ Jꢁ4[4 Hz\ ÐCH=Ð#\ 3[05
=
"1H\ t\ Jꢁ1[9 Hz\ HOCH1!#[ MS] m:z\ ðMŁ¦\ 032^ ðM!
H1OŁ¦\ 014^ ðM!CO1HŁ¦\ 87^ ð!CH"NH1#CO1HŁ¦\ 63[
The racemate "0[54 g# was next dissolved in ice!cold
1N NaOH "4[7 ml# and chloroacetyl chloride "0[0 ml#
and 1N NaOH "6 ml# were added to it alternatively
dropwise over 1 h while stirring[ Ten ml H1O was
added and the mixt[ was passed through a column of
Dowex!49 "H¦\ 49 ml#[ The eluate was concd to give
a partially crystallised syrup "1[5 g#[ Which was dis!
solved in water "099 ml#[ Co"CH2CO1#1 "25[1 mg# was
then added\ with the pH adjusted to 6[4 with 2N
NaOH[ Commercial renal acylase "19 mg# was added
and incubated at 26>C[ During the incubation a small
amount of acylase was additionally provided several
times\ and at each calculation\ the pH!value of the
reaction mixt[ was adjusted to 6[4 with 1N NaOH[
Following incubation for 13 h\ the mixt[ was treated
with a small amount of activated charcoal\ _ltered
with a glass _lter\ and the _ltrate was passed through
Amberlite 019B "H¦\ 79 ml#[ The water eluate and
washings were combined and the thick syrup was
hydrolyzed for 0 h in 49) ethanolic soln of NaOH
"019 ml#[ The hydrolysate was treated as usual with
Amberlite 019B "H¦#\ then subsequently puri_ed with
a column of Dowex 0×3 "−OAc\ 8×109 mm# and
9[94 N acetic acid[ Relevant fractions were combined
and concd\ to give crystals "85[2 mg# "D!Form#[
Recrystallized from EtOH!H1O[ Elemental analysis\
Found] C\ 49\03^ H\ 5[22^ N\ 8[47)[ C5H8NO2
requires] C\ 49[03^ H\ 5[22^ N\ 8[47)[ mp[×044>C
gradually decomp[ ðaŁ1D1ꢀ¦27> "H1O^ c 0#\ 6[9> "2N
HCl^ c 0#[ From the remaining soln as well as washings
from the former Amberlite!019B column\ the free L!
amino acid was also obtained "52[4 mg#[ Elemental
analysis\ Found] C\ 49[97^ H\ 5[30^ N\ 8[54)[ Calcd
for C5H8NO2]C\ 49[24^ H\ 5[23^ N\ 8[68)[
mp[×049>C gradually decomp[ ðaŁ1D1ꢀ−24> "H1O^ c
0#\ −09>"2N HCl^ c 9[4#[
0
00\ 91)[ The MS\ H NMR! and IR!spectra agreed
with those of the authentic DL!1!amino!4!hexynoic
acid[ This product "44 mg# was dissolved in 2N HCl
"09 ml# and re~uxed at 099Ð094>C\ following which
after cooling\ the solution was evapd under reduced
pressure\ with the resulting residue puri_ed on a cellu!
lose column "1[4×49 cm# using water!saturated t!
AmylOH[ Under these conditions\ _rst 1!amino!4!
chloro!3!hexenoic acid was eluted\ then 0\ and lastly
unchanged amino acid[ Yield of crude crystals] Com!
pound 0\ 3 mg "5)#[ The MS and IR of the natural
and synthetic product were superimposable\ respec!
tively\ and TLC comparison using with various sol!
vent systems were identical[
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2[5[1[ Compound 0[ Sodium "0[12 g# was dissolved
in abs[ EtOH "19 ml#\ with diethyl formamido!
malonate "09[6 g# in abs[ EtOH "16 ml# subsequently
added to soln and the mixture heated until re~ux
begun\ this being maintained for 29 min[ 0!Bromo!2!
butyne "6[0 g#\ prepared according to Schulte and
Reiss ð05Ł was added dropwise under stirring and
re~ux was continued for a further 06 hr[ After cooling\
the salt was removed by _ltration with the _ltrate
concd[\ re_ltered[ To the _nal _ltrate\ EtOH "56 ml# 00[ Hatanaka\ S[ I[ and Niimura\ Y[\ Phytochemistry\
and NaOH "09[6 g in 43 ml H1O# were added and 0861\ 00\ 2216[

467
SHIN!ICHI HATANAKA et al[
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Ã