The Structure of a New Dipeptide from the Mushroom Clitocybe acromelalga
K. Y am ano and H. Shirahama
D ep artm en t o f C hem istry, Faculty o f Science, H o k k aid o U niversity, S a p p o ro 060, Jap a n
Z. N aturforsch. 49c, 1 57-162 (1994); received D ecem ber 13, 1993
Clitocybe acromelalga, T oadstool, G A B A , D ipeptide, N -fy-am inobutyry^-L -glutam ic Acid
F ro m the poisonous m ushroom Clitocybe acromelalga, a new g lu tam ate-co n tain in g d ip ep ­
tide w as isolated. Its structure was deduced to be 7V-(y-am inobutyryl)-L-glutamic acid (10)
based on spectral d a ta and was confirm ed by synthesis.
Introduction
Clitocybe acromelalga (Japanese name: Dokusasako), found only in Japan, is a famous toadstool
that has a unique biological activity. The acciden­
tal ingestion o f this fungus causes violent pain, a
marked reddish edema on the hands and feet after
several days, and this pain continues for about a
m onth (M iura, 1936). These physiologically char­
acteristic properties prom pted us to study the toxic
constituent o f this mushroom. Although it was dif­
ficult to reproduce the symptoms in experimental
animals, on the basis of a lethal effect in mice acromelic acids A and B were isolated as the toxic prin­
ciples (K onno et al ., 1988). Acromelic acids are the
extraordinarily potent neuroexcitatory amino
acids. Because of their remarkable physiological
activity, they attract significant interest both phar­
macologically and physiologically. N ot only the
complete study of these acids (Ishida and Shinozaki, 1991; Shinozaki and Ishida, 1991) but also
investigation o f other toxins is expected. Further
separation o f the water extracts of this mushroom
led to isolation o f nine new compounds with struc­
tures 1 - 9 (Fig. 1) (Konno et al., 1982; Yamano
and Shiraham a, 1994; Hirayam a et al., 1989; K on­
no et al., 1984; Yamano et al., 1991; Yamano and
Shiraham a, 1992; Yamano et al., 1992; Yamano
and Shiraham a, 1993; Yamano et al., 1993; Y a­
m ano and Shiraham a, 1993). Amino acids 6 - 8 ex­
hibited a weakly depolarizing activity in the prepa­
ration o f the spinal cord of a newborn rat. New
pyridine nucleoside 1 and nucleotide 2 also showed
a lethal effect in mice on intraperitoneal injection.
R ep rin t requests to Prof. D r. H. S hiraham a.
Verlag der Zeitschrift für Naturforschung,
D -72072 Tübingen
0 9 3 9 -5 0 7 5 /9 4 /0 3 0 0 -0 1 5 7 $03.00/0
y-Aminobutyric acid (GABA), which plays an
im portant role in the central inhibitory process in
mammals (Elliott, 1965), was also found from this
fu n g u s. Its fu n c tio n is just o p p o s ite to that o f g lu ­
tamic acid (Glu) within the central nervous system.
The title m ushroom biosynthesizes such com ­
pounds with opposite activities as instrumental in
a n im a ls. Ar-(y -a m in o b u ty r y l-)-L -g lu ta m ic acid (10)
was also isolated in the course o f further investiga­
tion. Isolation of 10 is the first example of a natu­
ral product. We wish to describe the determination
of structure and synthesis of 10.
Results and Discussion
The water extracts were diluted with acetone
to give a copious residue which was dialyzed
against water. The dialyzate was further fraction­
ated by chrom atography and paper electropho­
resis (Scheme 1). Each step o f the fractionation
was carried out by m onitoring a lethal effect in
mice. A new com pound 10 was contained in the
poisonous fraction.
A new dipeptide 10, [a]^+ 18.0° (c 0.16, H 20 ),
showed a violet coloration with ninhydrin. The 'H
N M R spectrum (250 M Hz) o f 10 in D 20 indicated
the presence of glutamic acid and y-aminobutyric
acid (GABA) moieties (Table). The FAB MS spec­
trum exhibited a protonated ion peak at m /z 233
[M + H]+, corresponding to the m olecular formula
C 9H 170 5N 2. These spectral data implied structures
10-a, -y and -N as possible structures (Fig. 2).
Confirm ation of the structure for 10 was
achieved by synthesis (Scheme 2). Selective peptide
bond form ation is necessary for the synthesis of
10-a, and -y. To achieve this coupling reaction, a
commercially available y -b e n z y l-L -g lu ta m a te was
used for the starting material, whose Ar-protected
form 11 was coupled with methyl y-am inobutyrate
TFA salt (GABA-M e-TFA) assisted by diphenyl-
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K. Y am ano and H. Shirahama • The Structure o f a New Dipeptide from Clitocybe acromelalga
158
Fig 1.
H 0 2C
N ^O
O ^N
C 0 2H
K )-:
I J : ..
^ N ^ C O iH
"~COlH
^ N ^ C O iH
H
H
acromelic acid A
acromelic acid B
+H3N
+h 3n
NH
X ^
0 2C
"!C\
BO
I I
HO OH
R=H (1, d itidin e)
R = H 2P 0 3 (2)
co2
*
H
*• dithionein
NH2
L ° -J
n— n
1 1 Jl NMe3+
^ N
I ^ , C O ;H
£
T
N ^ C 0 2H
h 2n
3
s— C 0 2H
O '
v
N
H
u
^ C 0 2H
10
phosphoryl azide (DPPA) to yield an adduct 12 in
53% yield. It was deprotected to give 10-a in 84%
yield from 12. Further, a glutam ate derivative 11
was converted into a y-free glutam ate 13 in 94%
yield which produced 10-y (45% yield from 14) in
a way similar to that employed in the synthesis of
10-a. On the other hand, the synthesis o f 10-N was
achieved starting from dimethylglutamate 15
which was coupled with y-(Af-r-butyloxycarbonyl)aminobutyric acid (Boc-GABA) to provide
an adduct 16 in 50% yield. Removal of protective
groups yielded 10-jV in 91% yield from 16.
The N M R spectrum, [a]^ (+ 20.8°, c 0.16, H 20 )
and R/'value on cellulose TLC (R / 0.46, "B uO H -
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K. Yamano and H. Shirahama • The Structure o f a N ew D ipeptide from Clitocybe acromelalga
H C 0 2H - H 20 , 6 :1 :2 ) of synthetic 10-N were
completely coincident with those of the natural
compound.
Garyaev et al. (1991) discussed the excitatory
activity of some synthetic glutam ate-containing di­
peptides. They reported that the com pound correScheme 1.
fruit bodies (6 kg)
H20
extracts
acetone
precipitates
d ialysis
charcoal
2.5-5% aq. EtOH fr. (41 g)
IR-45 (HC02 form)
10-20% aq. H C 02H fr. (3.7 g)
PEP (pH 4.6, 600 V, 1.5 h)
0-+3 cm fr.
HPLC
(Shodex Sugar
SC-1821)
Rf 10-16 min
cellulose TLC
(" B u 0H -H C 02H-H20 , 6:1:2)
R / 0.45
(10, 3.2 mg)
Table. 'H N M R d a ta o f 10.
5 1.86
2.11
2.32
2.39
2.91
4.31
cf. G A B A (250 M H z, D 2Q)
(3 H ,m )
5 1.80 (2 H ,m )
(1 H, m)
2 .1 9 ( 2 H ,t, 7 = 7 .5 )
( 2 H ,t,7 = 7 .0 )
2.90 (2 H , t, 7 = 7.3)
( 2 H ,t, 7 = 7 .6 )
(2H , t, 7 = 7.6)
(1 H, dd, 7 = 5.5, 9.2)
H ,N
CO.H
10-y
10-a
Experimental
General procedure
IR spectra were measured on a JASCO model
IR-S spectrophotom eter with a NaCl cell. Mass
spectra were obtained on a JEOL model JM S-DX
300 spectrometer. Optical rotations were taken
with a JASCO model DIP-360 autom atic polarimeter. ’H N M R spectra were recorded in D 20 on
a Hitachi R-250 H (250 MHz) using 3-(trimethylsilyl)-1-propane-sulfonic acid sodium salt (DSS) as
an internal standard.
Fruiting bodies o f Clitocybe acromelalga were
collected in autum n in Nagaokashi, Niigata-ken,
Japan, and frozen upon collection, and stored at
- 2 0 °C.
Isolation o f 10
Frozen fruit bodies (6 kg) were extracted with
H 20 (7 1 x 3) at 4 °C overnight. The combined ex­
tracts were concentrated in vacuo to about 1 1. To
this turbid solution acetone (2.5 1) was added and
the mixture was allowed to stand at 4 °C over­
night. The supernatant was decanted and the low­
er m uddy layer was then evaporated. The copious
residue was dialyzed against H20 (3 1 x 4) at 4 °C
overnight. The combined dialyzate was evaporat­
ed and the residue (338 g) was applied to a column
of charcoal (300 g, packed in H 20 ). The column
was eluted stepwise with several concentrations of
aq. EtO H (H 20 , 2.5, 5, 10 and 30% aq. EtOH,
each 101). The 2 .5 -5 % aq. EtOH fraction was
collected and the solvent was removed in vacuo
(42 g). The residue (6 g x 7) was chrom atographed
on a column of weakly basic ion-exchange resin
(Amberlite IR-45, H C 0 2~ form) using H 20 H C 0 2H (H 20 , 5, 10 and 20% aq. H C 0 2H, each
6 1) as a solvent. The eluate with 10-20% aq.
H C 0 2H was concentrated in vacuo and a portion
10-JV
C O N H '^ ^ ^ C O zH
c o 2h
h 2ni ' ^ c o n h ^
sponding to 10 exhibited a seizure activity to convulsant potency of 11.4, relative to L-glutamate.
Mushroom material
dialyzate (338 g)
10
^ ^
c o 2h
h 2n
^
159
c o 2h
c o 2h
O
ki
N
H
C 0 2H
Fig. 2. Possible structures.
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160
K. Y am ano and H. Shirahama • The Structure o f a N ew Dipeptide from Clitocybe acromelalga
Schem e 2.
CO2B1I
GABA-Me-TFA
©
BocHN
0
COH
l.u2h
D PPA ’ N E t3
11
(53.2%)
B o c 2 , NEtj ,
dioxane-H,0
CO,Bn
h 2n
BocHN
C0NH/ ^
C02H
H,N
/ S 'C 0 2Me
CONH
10-a
12
1) CH2N2, MeOH
2) H2, Pd-C, EtOH
(2steps, 95.0%)
^ c o 2h
c o 2h
©
(N)
1) H2, Pd-C, EtOH
2) IN KOH, MeOH
3) TFA_________
(3steps, 84.4%)
COzBn
BocHN
f
C02Me t h f
(50.6%)
13
BocHN
^ — C 02Me
Boc-GABA
*A
DPPA, NEt3
Et3
THF
(49.5%)
TFA H2N ^ C 0 2Me
15
CONH^
''COiMe
GABA-Me-TFA
DPPA, NEt-i
N-----s.
>
f
J .
1
O '^ 'N
H
BocHN
CONH—
1) IN KOH. MeOH
2) TFA
(2steps, 88.7%) H2N
C 02Me
C02H
14
— C 02Me
10-y
BocHN
IN KOH
C02Me
16
MeOH
MeOH
tQ0 1®-'
(92.1%)
H2N
N-----v.
>
O
1
C 02H N---v.
f
1
N ^C02H
H
1?
TFA
(quant.)
v—
(^ - C
>
J
0 2H
2
I
uO
n ^ w
r n uH
N ^ ^C02
H
ION
cellulose TLC: "Bu0H -H C02H-H20 (6:1:2)
R/ value .
TFA ^
H2N ^
.
~"'C02Me
GABA-Me-TFA
BocHN
Boc-GABA
a:
0.49
r-
0.47
N:
0 .4 6
n a tu r a l:0 .4 6
o f the resultant paste (3.7 g) was subjected to
paper electrophoresis (46 * 20 cm, pH 4.6, 600 V,
1.5 h). An area of 0 ~ + 3 cm was cut off and the
strips were extracted with H 20 . The solvent was
then removed. The residue was separated by
HPLC (Shodex Sugar SC -1821: H ,0 , 52 °C, 1 ml/
min, R/ 10— 16 min). After removal of the solvent,
the residue was placed on cellulose TLC and devel­
oped by a mixed solvent ('IB u 0 H - H C 0 2H - H 20 ,
6:1 :2 ). A new dipeptide 10 was isolated from the
band at R /0 .4 5 (3.2 mg). 10: [aß6+ 18.0° (c 0.16,
H 20 ); IR vmax (nujol) cm -1: 3680-2400 and 1770—
1590; ]H N M R (250 MHz, D 20 ) 5 ppm: 1.86 (3H,
m), 2.11 (1H , m), 2.32 (2H , t, J = 7.0 Hz), 2.39
(2H , t, J = 7.6 Hz), 2.91 (2H , t, 7 = 7.6 Hz) and
4.31 (1H , dd, 7 = 5 .5 , 9.2 Hz); HR-FAB MS
found: m/z 233.1131 [M + H]+, calcd for
C 9H 170 5N 2: 233.1 138.
Synthesis o f 12
To a stirred solution of y-benzyl-L-glutamate
(300 mg, 1.26 mmol) in H :0-dioxane (2 ml - 1 ml)
was added triethylamine (154 mg, 1.52 mmol,
1 .2 equiv.) and subsequently a solution of di-/-
butyl dicarbonate (Boc 20 , 331 mg, 1.52 mmol,
1.2 equiv.) in dioxane (1 ml). The reaction mixture
was allowed to stand at room tem perature over­
night and then neutralized with 1 N HC1. This so­
lution was extracted with AcOEt (10 ml x 3). The
combined extracts were dried over anhydrous so­
dium sulfate and evaporated to yield the TV-pro­
tected form 11 (420 mg, quant.). To the mixture of
11 (100 mg, 0.30 mmol) and triethylamine
(66.0 mg, 2.2 equiv.) in T H F (1 ml) was added the
solution of GABA-M e-TFA (69.3 mg, 0.30 mmol,
1.0 equiv.) in T H F (0.5 ml) at 0 °C. To this mix­
ture was subsequently added diphenylphosphoryl
azide (DPPA, 82.0 mg, 0.30 mmol, 1.0 equiv.) in
T H F (1 ml) and allowed to stand at room temper­
ature overnight. W ater was added to the reaction
mixture which was extracted with AcOEt
(100 m ix 3). The combined extracts were dried
over anhydrous sodium sulfate and evaporated.
The residue was chrom atographed on silica gel
column (100 g, hexane-ether) to give an adduct 12
(69.0 mg, 53.2%). 12: IR vmax (neat) cm -1: 36803120, 1758-1620, 1580-1478, 1450, 1393, 1370,
1245, 1168, 870 and 757; >H N M R (250 MHz,
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K. Y am ano and H. Shirahama • The Structure o f a N ew D ipeptide from C litocybe acromelalga
CDCI 3) 8 ppm: 1.42 (9H , s), 1.76-1.98 (3H , m),
2.13 (1H , m), 2.31-2.61 (4H , m), 3.28 (2H , q,
7 = 6.5 Hz), 3.64 (3H , s), 4.10 (1H , m), 5.15 (2H ,
s), and 4.31 (1H, dd, 7 = 5 .5 , 9.2 Hz); EIM S
(probe) 70 eV m /z (rel. int.): 436 [M]+
(C 22H 320 7N 2) (1), 380 (2), 363 (2), 305 (3), 292 (17),
236 (15), 192 (61), 129 (6), 112 (10), 108 (8), 102
(5), 91 (100), 84 (30), 79 (7), 69 (5), 65 (5), 57 (84)
and 41 (23); HR-M S found: m /z 436.2221 [M]+,
calcd for C 22H 320 7N 2: 436.2210.
1 0 -a
The coupling product 12 (69.0 mg, 0.16 mmol)
was stirred with palladium carbon powder
(70.0 mg) in EtOH (3 ml) at room tem perature un­
der hydrogen gas for 1 h. This solution was filtered
through Celite and evaporated. The residue was
dissolved in MeOH (3 ml). To this stirred solution
was added 1 N KOH (0.8 ml, 0.8 mmol, 5.0
equiv.), and the mixture was allowed to stand at
room temperature overnight. After neutralization
of the reaction mixture with 1 N HC1, it was ex­
tracted with AcOEt (10 ml x 3). The combined ex­
tracts were evaporated. The residue was stirred in
TFA (1 ml) at room tem perature. After 45 min, to
the mixture was added H zO, and it was evaporat­
ed. Purification of the product was perform ed by
paper electrophoresis to yield 10 -a (31.0 mg,
3 steps 84.4%). 10-a: [a]2D5+ 1.3° (c 0.83, H 20 ); IR
vmax (nujol) cm ”1: 3740-2400 and 1775-1590; ’H
N M R (250 MHz, D 20 ) 5 ppm: 1.81 (2H , m), 2.14
(2H , dt, 7 = 7.0, 7.5 Hz), 2.41 (2H , t, 7 = 7.5 Hz),
2.50 (2H , t, 7 = 7.0 Hz), 3.26 (2H , t, 7 = 6.5 Hz)
and 3.98 (1 H, t, 7 = 7.0 Hz); HR-FAB MS found:
m /z 233.1134 [M + H]+, calcd for C 9H I7O sN 2:
233.1138.
Synthesis o f 14
The compound 11 (200 mg, 0.60 mmol) was dis­
solved in MeOH (10 ml). To this solution was add­
ed an ethereal solution of diazom ethane until the
yellow color was developed, and the solvent was
then removed. This resultant residue and palla­
dium carbon (200 mg) was stirred in EtOH at
room temperature under hydrogen gas for 1 h.
This reaction mixture was filtered through Celite.
The filtrate was evaporated to provide 13 (147 mg,
2 steps 95%). To the mixture o f 13 (100 mg,
0.38 mmol)
and
triethylamine
(86.0 mg,
0.85 mmol, 2.2 equiv.) in T H F (1 ml) was added
161
the solution o f GABA-M e-TFA (90.0 mg,
0.39 mmol, 1.0 equiv.) in TH F (1 ml) at 0 °C. To
this mixture was subsequently added DPPA
(106 mg, 0.39 mmol, 1.0 equiv.) in T H F (1 ml) and
allowed to stand at room tem perature overnight.
W ater was added to the reaction mixture which
was extracted with AcOEt (100 ml x 3). The com ­
bined extracts were dried over anhydrous sodium
sulfate and evaporated. The residue was chro­
m atographed on silica gel column (100 g, hexaneether) to give an adduct 14 (70.0 mg, 50.6%). 14:
IR vmax (neat) cm “1: 3680-3120, 1780-1610,
1580-1490, 1450, 1373, 1235 and 1170; >H N M R
(250 M Hz, CDCI 3) 5 ppm: 1.42 (9H , s), 1.78-2.00
(3 H, m), 2.17 (1H , m), 2.25 (2H , m), 2.38 (2H , t,
7 = 7.0 Hz), 3.30 (2H , dt, 7 = 6.5 Hz), 3.68 (3H , s),
3.74 (3H , s) and 4.27 (1H , m); EIM S (probe)
70 eV m /z (rel. int.): 360 [M]+ (C 16H 280 7N 2) (1),
304 ( 8), 301 (10), 287 ( 8), 273 (4), 260 (4), 245 (5),
213 (2), 211 (3), 201 (4), 187 (13), 184 (42), 172 ( 6),
159 (25), 152 (10), 144 (18), 142 (12), 127 (5), 124
(5), 118 (15), 116 (31), 101 (20), 86 (60), 84 (43), 73
( 8), 57 (100) and 41 (55); HR-M S found: m /z
360.1906 [M]+, calcd for C 16H 280 7N 2: 360.1896.
10-y
To the stirred solution of 14 (70.0 mg,
0.19 mmol) in MeOH (3 ml) was added 1 N KOH
(0.95 ml, 0.95 mmol, 5.0 equiv.), and the mixture
was allowed to stand at room tem perature over­
night. After neutralization of the reaction mixture
with 1 N HC1, it was extracted with AcOEt
(10 ml x 3). The combined extracts were evaporat­
ed. The residue was stirred in TFA (1 ml) at room
temperature. After 45 min, to the mixture was
added H 20 , and it was evaporated. Purification of
the product was performed by paper electropho­
resis to yield 10-y (40.0 mg, 2 steps 88.7%). 10-y:
[a]o + 2.8° (c 0.08, H 20 ); IR vmax (nujol) cm-1:
3660-2400 and 1730-1590; ‘H N M R (250 MHz,
D 70 ) 5 ppm: 1.78 (2H , m), 2.18 (2H , m), 2 .3 0 2.60 (4H , m), 3.19 (2H , t, 7 = 6 .5 Hz) and 4.05
(1H , t, 7 = 6 .5 Hz); HR-FAB MS found: m /z
233.1131
[M + H]+, calcd for C 9H 170 5N 2:
233.1 138.
Synthesis o f 16
To the mixture of Boc-GABA (1.14 g,
5.62 mmol) and triethylamine (1.70 g, 16.8 mmol,
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162
K. Y am ano and H. Shirahama • The Structure o f a N ew D ipeptide from Clitocybe acromelalga
3.0 equiv.) in T H F (10 ml) was added the solu­
tion of dim ethylglutamate TFA salt (1.63 g,
5.64 mmol, 1.0 equiv.) in TH F (15 ml) at 0 °C. To
this mixture was subsequently added DPPA
(1.55 g, 5.63 mmol, 1.0 equiv.) in TH F (10 ml) and
allowed to stand at room tem perature overnight.
W ater was added to the reaction mixture which
was extracted with AcOEt (100 ml x 3). The com­
bined extracts were dried over anhydrous sodium
sulfate and evaporated. The residue was chro­
m atographed on silica gel column (100 g, hexaneether) to give an adduct 16 (1.0 g, 49.5%). 16: IR
vmax (neat) cm “': 3620-3160, 1770-1630, 15651498, 1450, 1370, 1250 and 1172; 'H N M R
(250 M Hz, CDC13), 5 ppm: 1.42 (9H, s), 1.80 (2H,
m), 2.03 (1H , m), 2.14-2.34 (3H, m), 2.42 (2H,
m), 3.19 (2H , m), 3.67 (3H , s), 3.73 (3H , s) and
4.59 (1 H, m); EIMS (probe) 70 eV m /z (rel. int.):
360 [M]+ (C 16H 280 7N 2) (1), 304 (5), 303 (5), 287
(17), 273 (6), 259 (2), 242 (25), 227 (4), 217 (85),
201 (14), 185 (10), 176 (18), 158 (20), 144 (48), 131
(26), 116 (100), 98 (9), 86 (41), 84 (57), 69 (17), 57
(96) and 41 (53); HR-M S found: m/z 360.1874
[M]+, calcd for C ]6H 280 7N 2: 360.1896.
E lliott K. A. C. (1965), y-A m inobutyric acid and oth er
inhibitory substances. Brit. M ed. Bull. 21, 7 0 -7 5 .
G aryaev A. P., D um pis M. A., Poznjakova L. N ., Z aitzev Y. V. and Piotrivsky L. B. (1991), E xcitatory ac­
tion o f som e a sp artate- an d glutam ate-containing d i­
peptides after in tracereb ro v en tricu lar injection in
mice. E u ropean J. P harm acol. 197, 1 5 7 -160.
H irayam a F., K o n n o K ., S hirah am a H. an d M atsu m o to
T. (1989), 4-A m inopyridine-2, 3-dicarboxylic acid
from Clitocybe acromelalga. Phytochem istry 28,
1 133 -1 135. '
Ishida M . and Shinozaki H. (1991), N ovel k ain ate deriv­
atives: p o ten t depolarizing actions on spinal m o to ­
neurons and d orsal ro o t fibres in new born rats. Brit.
J. Pharm acol. 104, 8 7 3 -8 7 8 .
K on n o K., H ash im o to K ., O hfune Y., S h irah am a H.
and M atsu m o to T. (1988), A crom elic acids A and B.
P otent neuroex citato ry am ino acids isolated from Cli­
tocybe acromelalga. J. Am. Chem . Soc. 110, 4 8 0 7 4815.
K on n o K ., H ay an o K ., S h irah am a H ., Saito H. and
M atsu m o to T. (1982). Clitidine, a new toxic pyridine
nucleoside from Clitocybe acromelalga. T etra h edron
3 8 ,3 2 8 1 -3 2 8 4 .
K on n o K., Shirah am a H. and M atsu m o to T. (1984),
C lithioneine, an am ino acid betaine from Clitocybe
acromelalga. Phytochem istry 23, 1003-1006.
M iura O. (1936), Studies on the pharm acological effect
o f Clitocybe acromelalga, Ichim ura and Biological ef­
fect o f Clitocybe acromelalga, Ichim ura on a rooster.
T o h o k u J. Exp. M ed. 30, 1 5 0 -1 6 9 and 1 9 6 -2 0 2 .
10-W
To the stirred solution of 16 (90 mg, 0.25 mmol)
in M eOH (3 ml) was added 1 N KOH (1.25 ml,
1.25 mmol, 5.0 equiv.), and the mixture was al­
lowed to stand at room tem perature overnight.
After neutralization of the reaction mixture with
1 N HC1, it was extracted with AcOEt (10 ml * 3).
The combined extracts were evaporated to give 17
(76.4 mg, 92.1%). The com pound 17 (32 mg,
0.01 mmol) was stirred in TFA (1 ml) at room
tem perature. After 45 min, to the mixture was
added H 20 , and it was evaporated. Purification of
the product was performed by paper electropho­
resis to yield 10-A^ (28.6 mg, quant.). 10-N:
[a]2D6 + 20.8° (c 0.16, H 20 ); IR vmax (nujol) cm "1:
3680-2400 and 1770-1590; 'H N M R (250 MHz,
D ,0 ) 5 ppm: 1.86 (3 H ,m ), 2.11 (1 H, m), 2.32 (2H,
t, J = 7.0 Hz), 2.39 (2H , t, J = 7.6 Hz), 2.91 (2H, t,
J = 7.6 Hz) and 4.31 (1H , dd, .7=5.5, 9.2 Hz);
H R -FA B MS found: m /z 233.1121 [M + H]+, calcd
for C9H 170 5N 2: 233.1137.
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am in o acid, L-3-(2-carboxy-4-pyrrolyl)alanine from
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in term ed iate in the biogenesis o f acrom elic acids.
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T etra h ed ro n 49, 2 4 2 7 -2 4 3 6 .
Unauthenticated
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