Proof of the existence of an unstable amino acid: Pleurocybellaziridine in pleurocybella porrigens

Article abstract of DOI:10.1002/anie.201004646

Angel's wing mushroom, Pleurocybella porrigens, caused fatal acute encephalopathy in Japan in 2004. The structures of cytotoxic amino acids previously isolated from the mushroom motivated a study to prove the existence of an aziridine amino acid, pleurocy

Full text of DOI:10.1002/anie.201004646

Communications
DOI: 10.1002/anie.201004646
Natural Products
Proof of the Existence of an Unstable Amino Acid:
Pleurocybellaziridine in Pleurocybella porrigens**
Toshiyuki Wakimoto, Tomohiro Asakawa, Saeko Akahoshi, Tomohiro Suzuki, Kaoru Nagai,
Hirokazu Kawagishi,* and Toshiyuki Kan*
Angelꢀs wing mushroom, Pleurocybella porrigens (Pers.)
Singer, is distributed worldwide in temperate areas and has
been eaten throughout the world, especially in Japan. None-
theless, seventeen people who ate angelꢀs wing mushroom
died of acute encephalopathy in 2004 in Japan.[1–3] Epidemio-
logical investigation of the incident indicated that most
patients were undergoing hemodialysis treatment for chronic
renal failure and had digested the wild fruiting bodies before
the onset of neurological symptoms. Although the mechanism
of the acute encephalopathy has not yet been elucidated,
several chemical investigations have reported that vitamin D
analogues,[4] fatty acids,[5] and saccharides[6] are potential
causative agents of poisoning. Recently our group reported a
lectin[7] and cytotoxic amino acids,[8] six of which were novel.
The structural novelty and analogy of the amino acids is such
that each acid has the b-hydroxyvaline unit attached to
endogenous molecules (Scheme 1), which inspired us to
conclude the occurrence of an aziridine amino acid (1) as
the common precursor; one carbon atom of the aziridine ring
is substituted with a carboxy group and the other with geminal
Scheme 1. Structure of the cytotoxic compounds[8a] (top) and their
proposed precursor 1.
methyl groups (Scheme 1). Thus the instability of this
molecule could be demonstrated by nucleophilic attack by
endogenous molecules and successive ring opening. There-
fore, we hypothesized that the labile feature of the precursor
should be the reason why it could not be isolated from the
mushroom in the previous study.
[*] Dr. T. Wakimoto,[+] Dr. T. Asakawa, S. Akahoshi, Prof. Dr. T. Kan
School of Pharmaceutical Sciences, University of Shizuoka and
Global COE Program
52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan)
Fax: (+81)54-264-5745
To confirm the existence of the aziridine as a component
within the mushroom, we launched this unique project;
synthesis of the proposed molecule and its esters, and
isolation of the corresponding esters from the crude extract
of the mushroom after esterification.
Our synthesis plan is illustrated in Scheme 2. As a result of
the labile nature of 1, utilizing appropriate protection of the
nitrogen atom as well as the carboxylic group of 2 would play
a crucial step in the total synthesis. Although racemic 1 has
already been synthesized,[9] employing a Mitsunobu reaction
with 3 would be suitable for the preparation of an optically
active compound. Recently we have developed a highly
E-mail: kant@u-shizuoka-ken.ac.jp
Dr. T. Suzuki, Prof. Dr. H. Kawagishi
Department of Applied Biological Chemistry, Faculty of Agriculture,
Shizuoka University
836 Ohya, Suruga-ku, Shizuoka 422-8529 (Japan)
Fax: (+81)54-238-4885
E-mail: achkawa@ipc.shizuoka.ac.jp
Dr. K. Nagai
Department of Epigenetics Medicine, Interdisciplinary Graduate
School of Medicine and Engneering, University of Yamanashi
Yamanashi 409-3898 (Japan)
[+] Present address: Graduate School of Pharmaceutical Sciences, The
University of Tokyo, Tokyo 113-0033 (Japan)
[**] This work was partially supported by a Grant-in-Aid for Research and
Development Projects for Application in Promoting New Policy of
Agriculture Forestry and Fisheries from the Ministry of Agriculture,
Forestry and Fisheries (MAFF) (Japan) and a Grant-in-Aid for
Scientific Research on Priority from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) (Japan). We also
thank V. K. Deo (Shizuoka University) for valuable discussions, Dr.
Toru Ogata (National Rehabilitation Center for Persons with
Disabilities (Japan)), and Dr. Araceli Espinosa (UCLA) for providing
CG4-16 cells.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201004646.
Scheme 2. Retrosynthetic analysis of the aziridine amino acid 1.
1168
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2011, 50, 1168 –1170

efficient methodology for construction of secondary amines
using nitrobenzenesulfonamides (Ns strategy).[10] Since our
Mitsunobu reaction, mediated by the 2,4-dinitrobenzenesul-
fonyl (DNs) group, proceeded under extra mild reaction
conditions, the incorporation of DNs into 3 would enable the
total synthesis of labile compound 1.[11] The change in the
oxidation state of 4 into that of 3 could proceed by conversion
of the geminal dimethyl alcohol unit in ester 5 and subsequent
oxidation of 4. Garnerꢀs ester (5)[12] is readily obtained from
both enantiomeric forms of serine, which would serve as an
appropriate starting material.
Scheme 4. Reagent and conditions: a) Ph2CN2, CH2Cl2, 77%; b) HCl
gas, MeOH;[19] c) DNsCl, Na2CO3, THF/H2O (2:1), 59% (2 steps);
d) DIAD, Ph3P, toluene, 70%; e) nPrNH2, CH2Cl2, 08C–R.T., 90%;
f) H2, 5% Pd/C, MeOH, 67%. DIAD=diisopropyl azodicarboxylate,
Dpm=diphenylmethyl.
As shown in Scheme 3, we first synthesized methyl ester
derivative 10 to confirm the existence of 1 in the mushroomꢀs
extracts. After treatment of 5 with excess MeMgBr and acidic
proceeded smoothly to provide pleurocybellaziridine (1).
During the course of the isolation, pleurocybellaziridine (1)
fortunately crystallized in a mixture of CHCl3 and MeOH
(20:1) as colorless prisms.[18]
With the desired compounds 11 and 16 in hand, we turned
our attention to confirmation of the existence of 1 in P.
porrigens. Considering the instability of 1, we tried to isolate
11 or 16 from the extract of the mushroom after esterification.
The lyophilized fruiting bodies were suspended in MeOH.
The resulting extract was treated with CH2N2 or Ph2CN2, and
then the resulting mixture was purified by repeated silica gel
flash chromatography; the Rf values of the corresponding
synthetic materials (11 or 16) were used to identify the
components using TLC analysis. Although the methyl ester 11
showed partial decomposition during the separation steps and
when concentrated to dryness, we finally succeeded in
isolation of the esters. Their spectral data were identical
with those of the synthetic compounds, and were the first
confirmation of the existence of this labile amino acid,
pleurocybellaziridine (1), in a natural source.
Scheme 3. Reagent and conditions: a) MeMgBr, THF, ꢁ208C;
b) PPTS, MeOH, 96% (2 steps); c) TEMPO, PhI(OAc)2, NaClO2,
MeCN, Buffer pH 6.4, 96%; d) CH2N2, Et2O 87%; e) HCl gas, MeOH;
f) DNsCl, 2,6-lutidine, CH2Cl2, 72% (2 steps); g) DEAD, Ph3P, toluene,
83%; h) nPrNH2, CH2Cl2, 08C–R.T., 52%. Boc=tert-Butoxycarbonyl,
DEAD=diethyl azodicarboxylate, DNsCl=2,4-dinitrobenzenesulfonyl,
PPTS=pyridinium p-toluenesulfonate, TEMPO=2,2,6,6-tetramethyl-
piperidine-1-oxyl.
hydrolysis, the resulting diol 4 was subjected to TEMPO-
catalyzed oxidation[13] to give the carboxylic acid 6[14] without
loss of any functional group. After conversion of 6 into 7 by
treatment with CH2N2, the protecting group was exchanged
with the DNs group. Upon treatment of 9 with DEAD and
Interestingly, the content of 1 in the mushroom was
extraordinarily high; 23 mg of 16 was obtained from a MeOH
extract of the lyophilized fruiting bodies (4.0 g). Furthermore,
the absolute configuration of 16 that isolated from the
mushroom was determined by comparison of its specific
[15]
PPh3 in toluene at room temperature, the desired aziridi-
20
nation reaction proceeded smoothly to produce 10 in 83%
yield. Finally, removal of the DNs group in 10 was accom-
plished by treatment with n-propylamine to provide 11.[10]
Since hydrolysis of the methyl ester of 11 caused decom-
position of its aziridine ring, a diphenymethyl ester was
considered to be a more suitable protecting group for the
carboxy group in 1. This ester counterpart has the advantage
of not only facile incorporation and deprotection, but also
provided a quantitative analysis of 16 because of its strong
UV absorption. Furthermore, 16 seemed to be more stable
than methyl ester 11 because of the steric hindrance around
the aziridine ring.[16] After treatment of the N-Boc b-hydroxy
valine 6 with diphenyldiazomethane,[17] the Boc protecting
group in 12 was exchanged for a DNs group to provide the
cyclization precursor 14 (Scheme 4). Upon treatment of 14
with DIAD and PPh3, the desired cyclization reaction
proceeded smoothly to give 15 in 70% yield. During this
reaction, employment of DIAD gave a superior result
compared to that of DEAD. After removal of the DNs
group in 15, hydrogenolysis of the diphenylmethyl group of 16
rotation with that of the synthetic one: ½aꢀD (natural) = 25
20
(c = 0.50, CHCl3); ½aꢀD (synthetic) = 24 (c = 1.0, CHCl3). In
contrast, only trace amounts of 1 could be detected in the H2O
fraction prepared according to the previous report,[8a] and the
synthetic 1 gradually decomposed in D2O in an NMR tube
(see Figures S8 and S10 in the Supporting Information).
These results suggest that most of 1 decomposed during the
previous isolation procedure.[20]
Histological findings of the brain tissues affected by the
encephalopathy showed demyelinating symptoms.[21] It indi-
cates that toxic substance(s) in the mushroom damaged
oligodendrocytes which constitutes the myelin sheath in the
brain. Therefore, we examined the toxicity of both pleuro-
cybellaziridine (1) and 11 against rat CG4-16 oligodendrocyte
cells. Given the results of an MTT assay, 1 significantly
reduced the cell viability at concentrations of up to
10 mgmLꢁ1 (87 mm), but the methyl ester 11 showed only
weak toxicity at 30 mgmLꢁ1 (233 mm; Figure 1a). The cell
staining also revealed that most of the cells treated with
30 mgmLꢁ1 (260 mm) of 1 showed red fluorescence, thus
Angew. Chem. Int. Ed. 2011, 50, 1168 –1170
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
1169

Communications
Received: July 28, 2010
Revised: November 12, 2010
Published online: December 23, 2010
Keywords: amino acids · aziridines · cytotoxicity ·
.
natural products · structure elucidation
[1] T. Kuwabara, T. Arai, N. Honma, M. Nishizawa, Rinsho
Shinkeigaku 2005, 45, 239 – 245 (In Japanese with English
abstract).
[2] K. Obara, S. Okawa, M. Kobayashi, S. Watanabe, I. Toyoshima,
Rinsho Shinkeigaku 2005, 45, 253 – 256 (In Japanese with English
abstract).
[3] K. Obara, C. Wada, T. Yoshioka, K. Enomoto, S. Yagishita, I.
Toyoshima, Neuropathology 2008, 28, 151 – 156.
[4] H. Sasaki, H. Akiyama, Y. Yoshida, K. Kondo, Y. Amakura, Y.
Kasahara, T. Maitani, Biol. Pharm. Bull. 2006, 29, 2514 – 2528.
[5] T. Hasegawa, M. Ishibashi, T. Takata, F. Takano, T. Ohta, Chem.
Pharm. Bull. 2007, 55, 1748 – 1749.
[6] T. Tanaka, T. Hasegawa, T. Tatsuno, J. Date, Y. Ishigaki, Y.
Nakamura, N. Tomosugi, F. Takano, T. Ohta, J. Health Sci. 2009,
55, 373 – 379.
[7] T. Suzuki, Y. Amano, M. Fujita, Y. Kobayashi, H. Dohra, H.
Hirai, T. Murata, T. Usui, H. Kawagishi, Biosci. Biotechnol.
Biochem. 2009, 73, 702 – 709.
[8] a) T. Kawaguchi, T. Suzuki, Y. Kobayashi, S. Kodani, H. Hirai, K.
Nagai, H. Kawagishi, Tetrahedron 2010, 66, 504 – 507; b) Y.
Aoyagi, T. Sugahara, Phytochemistry 1988, 27, 3306 – 3307.
[9] a) M. P. Sammes, A Rahman, J. Chem. Soc. Perkin Trans. 1 1972,
344 – 346; b) T. Hiyama, H. Taguchi, H. Nozaki, Bull. Chem. Soc.
Jpn. 1974, 47, 2909 – 2910.
[10] For reviews on synthetic strategies involving the Ns group, see:
a) T. Kan, T. Fukuyama, J. Syn. Org. Chem. Jpn. 2001, 59, 779 –
789; b) T. Kan, T. Fukuyama, Chem. Commun. 2004, 353 – 360.
[11] T. Fukuyama, M. Cheung, C.-K. Jow, Y. Hidai, T. Kan,
Tetrahedron Lett. 1997, 38, 5831 – 5834.
[12] a) P. Garner, J. M. Park, Org. Synth., Coll. Vol. IX, Wiley, New
York, 1998, pp. 300 – 306; b) A. Dondoni, D. Perrone, Org.
Synth., Coll. Vol. X, Wiley, New York, 2004, pp. 320 – 326.
[13] M. Zhao, J. Li, E. Mano, Z. Song, D. M. Tschaen, E. J. J.
Grabowski, P. J. Reider, J. Org. Chem. 1999, 64, 2564 – 2566.
[14] J. E. Dettwiler, W. D. Lubell, J. Org. Chem. 2003, 68, 177 – 179.
[15] For a review of Mitsunobu reactions, see: a) O. Mitsunobu,
Synthesis 1981, 1 – 28; b) D. L. Hughes, Org. React. 1992, 42,
335 – 656.
Figure 1. Toxicity of pleurocybellaziridine (1) and its methyl ester (11)
for CG4-16 cells. CG4-16 cells treated with 1 or 11 were subjected to
an MTT assay (a) and calcein-AM/PI staining (b). a) The values are
represented as the mean of the relative percentage of surviving cells
and the standard error of the mean is shown (n=12; n: the number of
each experiment). Statistical analysis was performed by one-way
ANOVA and then a Tukey-Kramer post hoc test. The differences
between mean values were considered to be significant when p-values
were less than 0.01 (**p<0.01). CG4-16 cells were treated with 30
mgmL-1 of 1 or 11, and subjected to live/death staining. Upper panels
represent a typical staining pattern of live (calcein, green) and dead
(PI, red) cells. Lower panels represent nuclear staining with
Hoechst33342 for confirmation that the stained parts in upper panels
were actual cells, not materials other than cells.
[16] Diphenylmethyl ester of 1 was stable enough to allow diode
array detection (DAD) HPLC analysis: see Figure S3 in the
Supporting Information.
[17] L. I. Smith, K. L. Howard, Org. Synth., Coll. Vol. III, Wiley, New
York, 1995, pp. 351 – 355.
[18] Detailed experimental procedures and spectral data are de-
scribed in the Supporting Information.
[19] Z. Tozuka, H. Takasugi, T. Takaya, J. Antibiot. 1983, 36, 142 –
146.
[20] 1 could be stabilized by salt formation with a counter ion in the
natural environment.
representing the dead ones, whereas only a few cells showed
red fluorescence when treated with 30 mgmLꢁ1 of 11 (Fig-
ure 1b).[22] In addition, the adducts previously isolated from
the mushroom (Scheme 1) exhibited much less toxicity than 1
(see Figure S2 in the Supporting Information).[8a] Further-
more, 1 could be detected in the assay media during and after
the test (see Figures S3–S7 in the Supporting Information).
These data suggest that 1 maybe the cause of the demyelinat-
ing symptom and the carboxylic residue and the aziridine
skeleton are crucial for the cytotoxicity.
[21] N. Arai, H. Kawagishi, unpublished data.
[22] 1 induced not only significant toxicity but also molphological
changes in CG4-16 cells; see Figure S1 in the Supporting
Information.
In summary, we synthesized a labile and toxic aziridine
amino acid (1) and elucidated its presence in the angelꢀs wing
mushroom, Pleurocybella porrigens.
1170
www.angewandte.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2011, 50, 1168 –1170