Isolation of erinacine P, a new parental metabolite of cyathane- xylosides, from Hericium erinaceum and its biomimetic conversion into erinacines A and B

Article abstract of DOI:10.1016/S0040-4039(00)00601-8

A new cyathane-xyloside, erinacine P, was isolated from mycelia of basidiomycetous Hericium erinaceum YB4-6237 in a shaken culture. Since its aglycon closely resembles typical cyathane diterpenoids such as cyathins and cyathatriols in its substitution pattern, this glycoside seems to be an important metabolite in the biosynthesis of erinacines and striatins. In fact, according to our biogenesis studies, erinacine P can be successfully converted chemically into erinacine B and, further, to erinacine A under mild conditions. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00601-8

Tetrahedron Letters 41 (2000) 4389±4393
Isolation of erinacine P, a new parental metabolite of
cyathane-xylosides, from Hericium erinaceum and its
biomimetic conversion into erinacines A and B
a
a,
b
Hiromichi Kenmoku, Takeshi Sassa * and Nobuo Kato
aDepartment of Bioresources, Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan
bInstitute of Advanced Material Study, Kyushu University, Kasuga, Fukuoka 816-8580, Japan
Received 10 March 2000; revised 24 March 2000; accepted 7 April 2000
Abstract
A new cyathane-xyloside, erinacine P, was isolated from mycelia of basidiomycetous Hericium erinaceum
YB4-6237 in a shaken culture. Since its aglycon closely resembles typical cyathane diterpenoids such as
cyathins and cyathatriols in its substitution pattern, this glycoside seems to be an important metabolite in
the biosynthesis of erinacines and striatins. In fact, according to our biogenesis studies, erinacine P can be
successfully converted chemically into erinacine B and, further, to erinacine A under mild conditions.
#
2000 Elsevier Science Ltd. All rights reserved.
Keywords: biomimetic reactions; biosynthesis; glycosides; terpenes and terpenoids.
Cyathane-xylosides of erinacines1 and striatins are currently attracting much attention
�3
4
because of their unique biological activities. Erinacines are known to have a potent stimulating
5
1�3
activity for nerve growth factor-synthesis and agonistic activity towards the k opioid receptor.
Striatins have leishmanicidal activity. In the course of our studies to isolate cDNAs encoding
6
7,8
novel fungal diterpene cyclases, we attempted the isolation of the cyathane hydrocarbon from
y
Hericium erinaceum YB4-6237, the erinacine-producing basidiomycete. During the investigation,
we succeeded in isolating a new cyathane-xyloside named erinacine P (1) from the mycelial extract
of this basidiomycete. Here, we report the structural elucidation of 1 and its in vitro biomimetic
conversions into erinacine A (2) and erinacine B (3) (Fig. 1).
The brownish aqueous residue from a dipping extract of wet mycelia of H. erinaceum YB4-
3
6
medium {5.0% glucose, 0.5% peptone, 1.0% Pharmamedia (Traders Protein Co.) and 0.5%
237 with acetone obtained from 32 culture ¯asks, each containing 100 cm of the culture
1
*
y
Corresponding author. Tel: +81-235-28-2862; fax: +81-235-28-2862; e-mail: tsassa@tds1.tr.yamagata-u.ac.jp
We have already isolated a cyathane hydrocarbon and the results will be reported in the near future.
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00601-8

4
390
Figure 1.
{
NaCl in deionized water}, was extracted with EtOAc at pH 9. The residue of the organic extract
was separated by silica gel ¯ash chromatography using mixtures of CHCl and EtOH (20:1±5:1)
3
to a€ord a new metabolite 1 (155 mg) as the major diterpene-glycoside together with the known
erinacines, A (2, 2.9 mg), B (3, 23 mg), and C (3.4 mg).1
2
5
The most polar metabolite, 1, was obtained as a pale yellow amorphous solid {‰ꢀŠ ^52.4
D
^
1
(
(
(
c 0.23, MeOH); ꢁ
(®lm) 3384, 1743, and 1693 cm ; l
(MeOH) 206 (" 7500) and 228
max
max
+
" 6900)}. Its molecular formula was determined as C H O from the (M+Na) ion peak
27
40
8
515.2629, Á+0.8 mmu) in HR-FABMS. The molecular formula and the NMR data listed in
Table 1 as well as its IR and UV data determine 1 to be a mono-acetate of a cyathane-xyloside.
The di€erence between 1 and erinacine B (3) is the substitution pattern on the C-ring. The former
has an additional acetoxy group at C11. The methine proton at this position appears at ꢂ 5.88 and
is coupled to the C10 methylene protons as evidenced in the H±H COSY spectrum. It has a
correlation peak with the carbonyl carbon (ꢂ 170.2) of the acetyl group in HMBC measurement.
The double bond in the C-ring is located at the C12±C13 position. The ole®nic proton at ꢂ 6.89
appears as a sharp doublet, which is coupled to the C14 oxymethine proton at ꢂ 4.43. The
Table 1
NMR data for erinacine P (1) in CDCl
3
at +50 C; 600 MHz for H and 150 MHz for 13C
ꢀ
a
1
{
ꢀ
The basidiomycete in a shaken culture was grown at 25 C in this medium for 18 days. The wet weight of the obtained
mycelia was ca. 157 g.

4
391
stereochemistry of C11 could be assigned unambiguously as depicted, since a clear NOE
x
enhancement was observed on the C5 methine proton upon irradiation of the C11 methine.
Thus, the structure of 1 was elucidated to be an erinacine-like xyloside of which the aglycon has
the typical substitution pattern of cyathane diterpenoids exempli®ed by 11-O-acetylcyathatriol
9
(4) shown in Scheme 1.
Scheme 1. A proposed biogenesis for the erinacine family. Reagents and conditions for in vitro conversions: (i) Et
3
N (3
, rt
equiv.)±LiBr (0.5 equiv.), THF, rt (40 h) (3, 72%); (ii) DABCO (2 equiv.)±LiBr (0.5 equiv. then 2.5 equiv.), THF-d
8
ꢀ
(
22 h) then 50 C (65 h) (2, 88%)
{
The characteristic structural features of the aglycon in 1 led us to propose a biogenesis that
links cyathins to erinacines as shown in Scheme 1. First, the typically oxygenated cyathane
0
diterpenoid such as 4 may form erinacine P (1). The 1,4-addition of the 2 -hydroxy group onto
the b-position of the a,b-unsaturated aldehyde followed by the elimination of the acetate would
form erinacine B (3). Erinacine A (2) may also be derived from 1 via an intermediary conjugated
diene 5 (path A). Alternatively, 5 may be formed from 3 by either the 1,4-addition±elimination
with participation of a nucleophile (path B) or a simple dienolate formation followed by the
0
elimination of the 2 -hydroxy group (path C). For the ®nal positional isomerization of the diene
moiety from 5 to 2, a [1,5]-sigmatropic hydrogen shift is more plausible than a simple protonation±
9
deprotonation process since the latter requires an unfavorable protonation at the conjugation
0
terminus of the a,b-unsaturated aldehyde moiety. Similarly, when the 3 -position of the xylose is
oxidized to a ketone prior to or during these transformations, a C±C bond between C13 and C2
0
3
4
may be formed, leading to erinacines E±G and/or striatins. Thus, in this proposed biogenesis, 1 can
be regarded as the parental metabolite of the cyathane-xyloside family. In fact, from a preliminary
analysis on the time-dependent formation of the metabolites, it was clear that 1 is formed in
advance of 2 and 3.
To support the above-mentioned biogenesis, we investigated chemical conversions of 1 to 2 and 3.
jj
In a treatment of a THF solution of 1 with Et N in the presence of LiBr, the formation of 3 was
3
x
{
The NOEs were measured in a mixture of CDCl
2
3
:CD
3
OD (10:1) to avoid oligomeric contributions of the substrate.
Although erinacine D has a similar substitution pattern, its stereochemistry at C-11 (b-OEt) is opposite to that of
9
,10
11,12
the known cyathins
jj
and scabronines.
Without LiBr, the reaction does not proceed smoothly. For use of lithium halide-tertiary amine, see Ref. 13 and Ref.
4.
1

4
392
actually realized even at ambient temperature. The absolute stereo-structure of 1 was unambiguously
con®rmed, since 3 obtained here as a main product in 72% yield is identical with the natural
2
D
6
26
D
substance including optical rotation {‰ꢀŠ ^123.0 (c 0.20, MeOH); natural, ‰ꢀŠ ^118.5 (c 0.20,
#
MeOH)}. Similarly, 1 was treated with 1,4-diazabicyclo[2.2.2]octane (DABCO, 2 equiv.) and LiBr
0.5 equiv.) in THF-d . Again, the predominant formation of 3 at an early stage of the reaction
(
was observed by H NMR analysis. After 22 h, another two sets of signals, which are ascribable
8
1
*
*
to 5 and 2, were detected. These results indicate that the biogenesis in Scheme 1 is quite plaus-
ible and the reaction proceeds not through path A but through paths B or C. The mixture was
warmed at 50 C for 65 h with additional LiBr (2 equiv.) in order to complete the reaction. During
this period, the formation of 2 occurred without an increase in the amount of 5, showing that the
ꢀ
[
1,5]-hydrogen shift proceeds smoothly taking advantage of the formation of the fully conjugated
triene-system in 2. Eventually, 2 was isolated as the sole product from the reaction mixture. Since
it is well known that DABCO acts as a nucleophile and forms an intermediary adduct in the
Baylis±Hillman reaction,15 it is plausible that this chemical conversion takes place via path B.
Consequently, the accomplishment of the chemical conversions of 1 into 2 and 3 under mild
yy
conditions supports the proposed biogenesis shown in Scheme 1.
Acknowledgements
We are grateful to Professor Shuji Kanemasa, Kyushu University, for his helpful discussion
and encouragement to use the lithium halide±tertiary amine combination in chemical conversions
of 1. We also thank Messrs. Eiichi Kimura and Takashi Shigihara, Edible Fungi Institute of
Kinox Co. Ltd, for providing H. erinaceum YB4-6237 and Mrs. Kaori Yamamura, a student of
Yamagata University, for her technical assistance.
References
1
2
3
. Kawagishi, H.; Shimada, A.; Shirai, R.; Okamoto, K.; Ojima, F.; Sakamoto, H.; Ishiguro, Y.; Furukawa, S.
Tetrahedron Lett. 1994, 35, 1569±1572.
. Kawagishi, H.; Shimada, A.; Shizuki, K.; Mori, H.; Okamoto, K.; Sakamoto, H.; Furukawa, S. Heterocycl.
Commun. 1996, 2, 51±54.
. Kawagishi, H.; Shimada, A.; Hosokawa, S.; Mori, H.; Sakamoto, H.; Ishiguro, Y.; Sakemi, S.; Bordner, J.;
Kojima, N.; Furukawa, S. Tetrahedron Lett. 1996, 37, 7399±7402.
. Hecht, H.-J.; Ho¯e, G.; Steglich, W.; Anke, T.; Oberwinkler, F. J. Chem. Soc., Chem. Commun. 1978, 665±666.
È
4
5
. Saito, T.; Aoki, F.; Hirai, H.; Inagaki, T.; Matsunaga, Y.; Sakakibara, T.; Sakemi, S.; Suzuki, Y.; Watanabe, S.;
Suga, O.; Sujaku, T.; Smogowicz, A. A.; Truesdell, S. J.; Wong, J. W.; Nagahisa, A.; Kojima, Y.; Kojima, N.
J. Antibiot. 1998, 51, 983±990.
6. Inchausti, A.; Yalu€, G.; Rojas de Arias, A.; Torres, S.; Ferreira, M. E.; Nakayama, H.; Schinini, A.; Lorenzen,
K.; Anke, T.; Fournet, A. Phytotherapy Res. 1997, 11, 193±197.
7. Kawaide, H.; Imai, R.; Sassa, T.; Kamiya, Y. J. Biol. Chem. 1997, 272, 21706±21712.
#
1
The absolute value of optical rotation of 3 di€ers greatly from the reported one (^34.9). The reason for this di€er-
ence is uncertain at this moment.
**
1
8
The diagnostic H signals for 5 are: ꢂ (THF-d ) 6.21, 6.31(each dm, J=12.2 Hz, 10/11-H), 6.82 (d, J=8.1 Hz, 13-H),
and 9.40 (s, 15-H).
yy
Added in proof: We noticed in a review that the planar structure of erinacine P (1) has been reported as herical
isolated from Hericium ramosum; see, Lorenzen, K.; Anke, T. Curr. Org. Chem. 1998, 2, 329±364.

4
393
8
9
. Kawaide, H.; Sassa, T.; Kamiya, Y. J. Biol. Chem. 2000, 275, 2276±2280.
. Ayer, W. A.; Lee, S. P. Can. J. Chem. 1979, 57, 3332±3337.
1
1
1
1
0. Ayer, W. A.; Browne, L. M.; Ferna
and references cited therein.
Â
ndez, S.; Ward, D. E.; Yoshida, T. Rev. Latinoamer. Quim. 1978, 9, 177±184,
1. Ohta, T.; Kita, T.; Kobayashi, N.; Obara, Y.; Nakahata, N.; Ohizumi, Y.; Takaya, Y.; Oshima, Y. Tetrahedron
Lett. 1998, 39, 6229±6232.
2. Kita, T.; Takaya, Y.; Oshima, Y.; Ohta, T.; Aizawa, K.; Hirano, T.; Inakuma, T. Tetrahedron 1998, 54, 11877±
1
1886.
3. Blanchette, M. A.; Choy, W.; Davis, J. T.; Essenfeld, A. P.; Masamune, S.; Roush, W. R.; Sakai, T. Tetrahedron
Lett. 1984, 25, 2183±2186.
1
1
4. Tsuge, O.; Kanemasa, S.; Yoshioka, M. J. Org. Chem. 1988, 53, 1384±1391.
5. Ciganek, E. Org. React. 1997, 51, 201±350