A novel dipeptide, N-γ-glutamyl boletine, and a cyclic iminium toxin from the mushroom Tylopilus sp. (Boletaceae)

Article abstract of DOI:10.1016/S0040-4039(02)01495-8

N-γ-Glutamyl boletine and a toxin, 2-butyl-1-azacyclohexene iminium salt, were isolated from the mushroom Tylopilus sp. (Boltaceae). The absolute stereostructure of N-γ-glutamyl boletine was clarified based on spectroscopic analysis, acidic hydrolysis and total synthesis. N-γ-Glutamyl boletine exhibited moderate antibacterial activity. 2-Butyl-1-azacyclohexene iminium salt exhibited moderate acute toxicity against ddY mice. We proposed feasible biosynthetic pathway of piperidine alkaloids that the cyclic iminium compound might be biosynthesized from boletine, a new amino acid containing an δ-amino ketone moiety.

Full text of DOI:10.1016/S0040-4039(02)01495-8

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 6501–6504
A novel dipeptide, N-g-glutamyl boletine, and a cyclic iminium
toxin from the mushroom Tylopilus sp. (Boletaceae)
Reiko Watanabe, Masaki Kita and Daisuke Uemura*
Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa, Nagoya 464-8602, Japan
Received 29 June 2002; revised 19 July 2002; accepted 22 July 2002
Abstract—N-g-Glutamyl boletine and a toxin, 2-butyl-1-azacyclohexene iminium salt, were isolated from the mushroom Tylopilus
sp. (Boltaceae). The absolute stereostructure of N-g-glutamyl boletine was clarified based on spectroscopic analysis, acidic
hydrolysis and total synthesis. N-g-Glutamyl boletine exhibited moderate antibacterial activity. 2-Butyl-1-azacyclohexene iminium
salt exhibited moderate acute toxicity against ddY mice. We proposed feasible biosynthetic pathway of piperidine alkaloids that
the cyclic iminium compound might be biosynthesized from boletine, a new amino acid containing an d-amino ketone moiety.
© 2002 Elsevier Science Ltd. All rights reserved.
Various mushrooms have attracted a great deal of
attention as health foods in Japan. The chemical con-
stituents of mushrooms, especially the toxic varieties,
have been well studied and numerous bioactive com-
pounds have been reported. Nevertheless, food poison-
ing caused by the ingestion of mushrooms is still
common, since the fungi have not been well identified
compared to plants and animals. To prevent mushroom
poisoning and to assure the safety of the food supply,
we have searched for the bioactive constituents of a
large variety of mushrooms.
boletine (1), and an iminium compound 2, a toxic
principle, were obtained (Fig. 1). We report here the
isolation and structure determination of these
compounds.
The aqueous 80% EtOH extract of Tylopilus sp. was
partitioned between water and EtOAc. Guided by acute
toxicity against ddY mice, the water-soluble fraction
was subjected to fractionation using column chro-
matography (TSK G-3000S polystyrene gel, Sephadex
LH-20, ODS, and SiO2) and reversed-phase HPLC.
Final purification was achieved by reversed-phase
HPLC (MeOH/aqueous NaCl) to give N-g-glutamyl
boletine (1, 2.8×10−3% yield based on wet wt) and
2-butyl-1-azacyclohexene iminium salt (2, 7.1×10−4%
yield based on wet wt).2 2-Butyl-1-azacyclohexene
iminium salt (2) exhibited moderate acute toxicity
against ddY mice (LD99 25 mg/kg). Although N-g-glu-
tamyl boletine (1) did not show acute toxicity against
mice, it exhibited moderate antibacterial activity against
the marine bacteria Rhodospirillum salexigens SCRC
113 strain (9 mm, 1 mg/disk).
Recently, a new variety of mushroom Tylopilus sp.
(Boletaceae) was discovered at Mikawa, Aichi Prefec-
ture, and its aqueous ethanol extract exhibited acute
toxicity in mice. Since the Boletaceae are generally
considered to be edible, this finding could be unique.
Several bioactive compounds have been isolated from
Tylopilus sp., but the toxic constituent has not been
reported.1 Therefore, we focused on the chemical con-
stituents of this novel mushroom. Guided by acute
toxicity against mice, a novel dipeptide, N-g-glutamyl
Figure 1.
* Corresponding author. Tel./fax: +81-52-789-3654; e-mail: uemura@chem3.chem.nagoya-u.ac.jp
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)01495-8

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R. Watanabe et al. / Tetrahedron Letters 43 (2002) 6501–6504
The molecular formula of 1 was determined to be
C15H26N2O6 by ESI MS (m/z 331.1870, calcd for
C15H27N2O6 [M+H]+ 331.1869). The NMR data for 1
1
are summarized in Table 1. The H NMR, 13C NMR,
and HMQC spectra of 1 showed the presence of one
methyl carbon (lc 12.8), eight methylene carbons, two
methine carbons (lc 54.0, 54.5) connected to hetero
atoms, and four carbonyl carbons (lc 173.3, 174.1,
176.9, 213.7). A detailed analysis of the COSY spec-
trum of 1 allowed three partial structures, C-2 to C-5,
C-7 to C-10, and C-2% to C-4% (Fig. 2). The remaining
connectivities of 1 were clarified by the HMBC correla-
tions H-2/C-1 and C-5%, H-5/C-6, H-7/C-6, H-2%/C-1%,
and H-4%/C-5%. Thus, the gross structure of N-g-glu-
tamyl boletine was determined to be a dipeptide con-
sisting of glutamic acid and a new amino acid, which
was named boletine, as shown in 1.
Scheme 1. Hydrolysis of N-g-glutamyl boletine (1).
the carbon adjacent to the carboxyl group in 4 could
not be determined.
To confirm the absolute stereochemistry of N-g-glu-
tamyl boletine (1) and to obtain a greater supply for
further biological studies, both diastereomers, (2S, 2%S)-
and (2R, 2%S)-dipeptides 11 and 12, were synthesized
(Scheme 2). The synthesis of 1 started from commer-
The absolute stereostructure of 1 was elucidated as
follows. Acidic hydrolysis of 1 followed by separation
using reversed-phase HPLC gave glutamic acid (3) and
cyclic amino acid 4 (Scheme 1). The absolute configura-
tion of the glutamic acid (3) was determined to be L by
chiral HPLC analysis.3 The gross structure of cyclic
amino acid 4 was determined by analysis of COSY,
HMQC and HMBC spectra, but the stereochemistry of
cially available Boc- -Asp-OBn (5). Compound 5 was
L
converted into a mixed anhydride and then subjected to
reduction using sodium borohydride to give alcohol 6
in 69% yield.4 Dess–Martin oxidation of alcohol 6 gave
aldehyde 7 in 79% yield. The Horner–Wadsworth–
Emmons reaction of aldehyde 7 with b-ketophospho-
nate gave a,b-unsaturated ketone 8 in 77% yield.5,6 The
geometry of the olefin in 8 was confirmed to be E based
on the magnitude of the coupling constant between
vinyl protons (16.0 Hz). Acidic deprotection of the Boc
Table 1. NMR data for N-g-glutamyl boletine (1) in
CD3OD
group in 8 followed by condensation with Z-L-Glu-OBn
13Ca
1Hb
HMBCc
(9) gave dipeptide 10 in 53% yield. Finally, hydrogena-
tion of 10 using Pd(OH)2 led to deprotection of the Z
and Bn groups and reduction of the olefin to give (2S,
2%S)-dipeptide 11 in quantitative yield. Similarly, (2R,
Atom
1
2
3
4
5
6
7
8
176.9 s
54.0 d
31.4 t
20.3 t
41.0 t
213.7 s
41.7 t
26.8 t
22.4 t
12.8 q
173.3 s
54.5 d
27.0 t
H-2, 3
H-3, 4
H-2, 5
H-2, 3, 5
H-3
H-5, 7, 8
H-8, 9
H-7, 9, 10
H-7, 8, 10
H-8, 9
H-2%, 3%
H-3%, 4%
H-2%, 4%
4.23 m 1H
1.62 m 2H
1.84 m 2H
2.51 m 2H
2%S)-dipeptide 12 was synthesized from Boc-D-Asp-OBn
(ent-5) in 27% yield in six steps. Synthetic (2S, 2%S)-
dipeptide 11 was identical to natural 1 with respect to
TLC analysis and spectroscopic data (1H and 13C
NMR, and optical rotation).7 Thus, the absolute
stereostructure in N-g-glutamyl boletine (1) was shown
to be 2S and 2%S. Synthetic N-g-glutamyl boletine (11)
also exhibited moderate antibacterial activity against R.
salexigens (9 mm, 1 mg/disk) as with natural products.
It is interesting to note that 2R-isomer 12 did not show
any antibacterial activity.
2.46 t (6.7) 2H
1.51 tt (6.7, 6.9) 2H
1.31 tq (6.9, 6.9) 2H
0.92 t (6.9) 3H
9
10
1%
2%
3%a
3%b
4%
5%
3.64 m 1H
2.10 m 1H
2.23 m 1H
2.52 m 2H
32.5 t
174.1 s
H-2%, 3%
H-2, 3%, 4%
2-Butyl-1-azacyclohexene iminium salt (2) showed an
ion peak at m/z 140 [M+] in its FAB MS spectrum. The
a Recorded at 200 MHz. Multiplicity was based on the HMQC
spectrum.
1
NMR data for 2 are summarized in Table 2. The H
NMR, 13C NMR, and HMQC spectra of 2 showed the
presence of one methyl carbon (lc 12.6), seven methyl-
ene carbons, and one carbonyl carbon (lc 192.3). Analy-
sis of the COSY, HMQC, HMBC spectra of 2 allowed
us to construct its entire framework (Fig. 3). Treatment
b Recorded at 800 MHz. Coupling constants (Hz) are in parentheses.
c Based on the correlation from each carbon atom.
1
of 2 with triethylamine gave an imine, the H NMR
data of which coincided with those of synthetic 2-butyl-
1-azacyclohexene previously reported.8 Thus, the struc-
ture was confirmed to be as shown in Fig. 3.
The biosynthesis of piperidine alkaloids has been well
studied. Experiments using labeled precursors have
established that a large group of piperidine alkaloids
can be derived from lysine or acetic acid.9 For example,
Figure 2. Gross structure of N-g-glutamyl boletine (1).

R. Watanabe et al. / Tetrahedron Letters 43 (2002) 6501–6504
6503
Scheme 2. (a) N-methylmorpholine, isobutyl chloroformate, THF, 0°C, 15 min; then NaBH4, MeOH, 0°C, 1 h, 69%; (b)
Dess–Martin periodinane, CH2Cl2, 0°C, 35 min, 79%; (c) (MeO)2P(O)CH2C(O)nBu, Cs2CO3, MeCN, rt, 35 min, 77%; (d) TFA,
CH2Cl2, 0°C, 1.5 h, 79%; (e) 9, WSCD·HCl, HOBt, (i-Pr)2NEt, CH2Cl2, 0°C, 5 h, 53%; (f) H2, Pd(OH)2, EtOH, rt, 9 h, quant.
Table 2. NMR data for 2-butyl-1-azacyclohexene iminium
salt (2) in CD3OH
anabasine, sedamine, and pelletierine, have been shown
to arise from lysine. On the other hand, nigrifactin,
coniine, and g-conicerine can be derived from four to
six acetate units in linear combination in their biosyn-
thesis.10 The structure of 2-butyl-1-azacyclohexene
iminium salt (2) is quite similar to that of g-conicerine.
However, the difference between number of carbon
atoms in these compounds suggests that the biosynthe-
sis of 2 does not only involve acetate units.
Atom
13Cb
1Hc
HMBCd
2
3
45.1 t
20.0 t
18.0 t
3.66 m 2H
1.88 m 2H
1.84 m 1H
1.86 m 1H
2.70 m 2H
H-2, 3
H-3, 4
H-1, 2
4a
4b
5a
6
28.0 t
192.3 s
38.0 t
27.6 t
22.0 t
12.6 q
H-2, 3a
H-1, 3a, 7
H-7
H-6, 8, 9
H-7
7a
8
9
2.60 t (7.2) 2H
Scheme 3 shows the possible biosynthetic pathways of
compounds 1 and 2. N-g-Glutamyl boletine (1) might
be biosynthesized by the condensation of boletine (13)
1.64 tt (7.2, 7.2) 2H
1.42 tq (7.2, 7.2) 2H
0.98 t (7.2) 3H
10
H-8
and -glutamic acid. Since boletine (13) has a d-amino
L
ketone moiety, however, intramolecular cyclization may
easily occur to give a dehydrated form such as 4.
Decarboxylation of dehydrated boletine 4 may lead to
substituted azacyclohexene 2. However, reductive
decarboxylation followed by cyclization may also be
possible. Consequently, we assumed that both 1 and 2
were biosynthesized from the novel amino acid boletine
(13).
a Protons were almost not observed in CD3OD due to deuterium
exchange.
b Recorded at 200 MHz. Multiplicity was based on the HMQC
spectrum.
c Recorded at 800 MHz. Coupling constants (Hz) are in parentheses.
d Based on the correlation from each carbon atom.
In conclusion, N-g-glutamyl boletine (1) and 2-butyl-1-
azacyclohexene iminium salt (2) were isolated from the
mushroom Tylopilus sp. Compound 1 was clarified to
be a dipeptide consisting of
L-glutamic acid and
boletine, a novel amino acid, based on spectroscopic
analysis and acidic hydrolysis. The total synthesis of 11
and its 2R-isomer 12 elucidated the absolute stereo-
chemistry of 1. Both natural and synthetic N-g-glu-
tamyl boletine 1 and 11 showed moderate antibacterial
Figure 3. Gross structure of 2-butyl-1-azacyclohexene
iminium salt (2).

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R. Watanabe et al. / Tetrahedron Letters 43 (2002) 6501–6504
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2. Conditions for the isolation of N-g-glutamyl boletine (1)
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Develosil ODS-HG-5 (ƒ 20×250 mm); eluent, 30%
MeOH/70% H2O containing 0.1% NaCl; flow rate, 5.0
mL/min; detection at 215 nm. Desalting was achieved by
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Scheme 3. Possible biosynthetic pathways of compounds 1
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This work was supported in part by Grants-in-Aid for
Scientific Research (No. 11558079) and Scientific
Research on Priority Areas (A) (No. 12045235) from
the Ministry of Education, Culture, Sports, Science,
and Technology, Japan. We are indebted to Wako Pure
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Mycological Society of Japan) for supplying the mush-
room Tylopilus sp.
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