Isolation, structural elucidation and synthesis of a novel antioxidative pseudo-di-peptide, Hanasanagin, and its biogenetic precursor from the Isaria japonica mushroom

Article abstract of DOI:10.1016/j.tet.2009.06.069

The entomogenous 'Hanasanagitake' mushroom, Isaria japonica, is used as a folk medicine and as a traditional health food choice in Japan. A search for naturally occurring antioxidative compounds from the mushroom led to the isolation of a novel pseudo-di-

Full text of DOI:10.1016/j.tet.2009.06.069

Tetrahedron 65 (2009) 6822–6827
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Isolation, structural elucidation and synthesis of a novel antioxidative
pseudo-di-peptide, Hanasanagin, and its biogenetic precursor
from the Isaria japonica mushroom
,
,
Akira Sakakura a y, Kazufumi Shioya a y, Hirotaka Katsuzaki a, Takashi Komiya a,
Toshikatsu Imamura c, Yasuo Aizono b, Kunio Imai a
,
*
a Graduate School of Bioresources, Mie University, Kurimamachiyacho, Tsu 514-8507, Japan
b Faculty of Agriculture, Kobe University, Nada-ku, Kobe 657-8501, Japan
c Aseptic Sericulture System Laboratory, Minami-ku, Kyoto 601-8203, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 May 2009
Received in revised form 18 June 2009
Accepted 19 June 2009
Available online 24 June 2009
The entomogenous ‘Hanasanagitake’ mushroom, Isaria japonica, is used as a folk medicine and as a tra-
ditional health food choice in Japan. A search for naturally occurring antioxidative compounds from the
mushroom led to the isolation of a novel pseudo-di-peptide, named Hanasanagin, and a possible bio-
genetic precursor. The structures of the pseudo-di-peptides were determined as (R)-3,4-diguanidino-
butanoyl-(S)-DOPA and (R)-3,4-diguanidinobutanoyl-(S)-tyrosine by spectral analysis, chemical synthesis
and enzymatic conversion.
Keywords:
Hanasanagin
Ó 2009 Elsevier Ltd. All rights reserved.
Antioxidant
Pseudo-di-peptide
(R)-3,4-Diguanidinobutanoyl-(S)-DOPA
1. Introduction
with those of synthetic 1 and 2 confirmed their structures to be
(R)-3,4-diguanidinobutanoyl-(S)-DOPA and (R)-3,4-diguanidino-
The entomogenous ‘Hanasanagitake’ mushroom, Isaria japon-
ica, is a traditionally used folk medicine (‘Tochukaso’) for peren-
nial youth in Japan.1 Using the free radical scavenging test2 and
the superoxide dismutase (SOD)-like activity experiment3,4 to
identify antioxidative compounds from the mushroom, an active
novel pseudo-di-peptide 1 was recently isolated.5 We named the
compound ‘Hanasanagin’ and determined the chemical structure
to be 3,4-diguanidinobutanoyl-DOPA by spectral analysis and
chemical conversion.5 In this report, a pseudo-di-peptide 2, 3,4-
diguanidinobutanoyl-tyrosine, is also isolated from the mush-
room. We speculate that this pseudo-di-peptide is a biogenetic
precursor of 1 because of its structural resemblance to Hanasa-
nagin and from the enzymatic transformation of 2 to 1 by
a mushroom tyrosinase. To confirm the structures of the two
pseudo-di-peptides and to determine their configurations, the
diastereomeric mixture of 2 and chiral 2 were chemically syn-
thesized and 2 was enzymatically transformed to 1. Comparison of
the spectral data and the HPLC chromatograms of isolated 1 and 2
butanoyl-(S)-tyrosine, respectively.
This paper describes the isolation, structure elucidation and
synthesis of Hanasanagin 1 and its biogenetic precursor 2.
2. Results
2.1. Isolation of Hanasanagin 1 and the pseudo-di-peptide 2
The fruiting body of the Hanasanagitake mushroom cultivated
on silkworm pupae was collected and extracted with 60% ethanol.
After removing the ethanol from the extract, the aqueous residue
was separated by reversed-phase (RP) column chromatography
using a Diaion HP-20 column. Hanasanagin 1 and the pseudo-di-
peptide 2 were eluted from the column with 5% MeOH after
washing with water. The analytical RP-HPLC chromatogram of the
fraction is shown in Figure 1. Hanasanagin 1 eluted at 20.9 min and
the pseudo-di-peptide 2 at 22.3 min. Preparative HPLC of the
fraction afforded pure 1 and 2.
2.2. Structural analysis
* Corresponding author. Tel.: þ81 59 231 9610; fax: þ81 59 231 2986.
E-mail address: imai@bio.mie-u.ac.jp (K. Imai).
These authors contributed equally to this work.
Hanasanagin 1 and the pseudo-di-peptide 2 showed the pro-
tonated molecular ions at m/z¼382 and 366 in the MALDI-TOF-MS
y
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.06.069

A. Sakakura et al. / Tetrahedron 65 (2009) 6822–6827
6823
analysis. The molecular formulae of hanasanagin 1 and the pseudo-
di-peptide 2 were analyzed as C15H23N7O5 and C15H23N7O4 on the
bases of their HR-FABMS spectra (positive, in glycerin, m/z¼
382.1819,
D
ꢀ1.9 mmu and m/z¼366.1882,
D
ꢀ0.7 mmu), re-
spectively. The difference between the molecular formulae was one
oxygen atom, indicating that 1 was an oxygenated product of 2. The
UV spectra of the two compounds (lmax¼282 and 276 nm) sug-
gested the presence of phenol moieties. The 1H NMR, 13C NMR,
1H–1H COSY and HMBC spectra in D2O of the two species are
summarized in Table 1. The 1H–13C correlations were assigned by an
HSQC analysis.
The detailed spectral analysis of 15 confirmed that 1 was 3,4-
diguanidinobutanoyl-DOPA. The structure of 1 is shown in Figure 2
and the important 1H–1H and HMBC correlations are indicated by
bold lines and arrows, respectively.
6NH2
NH
NH2
6
HN
H
HN
NH
H
H
H
H
1
H
4
4
H
H
Nα COOH
Nα COOH
H6
2
2
H
H
HN
1
3
3
H6
HN
NH
O
H
NH
O
H
β
β
H
H
5
4
5
4
5
1
1
5
NH2
NH2
2
2
H
OH
H
OH
3
3
10
20
Retention time (min)
40
30
0
OH
H
Hanasanagin, 1
(3,4-diguanidinobutanoyl-DOPA)
pseudo-di-peptide, 2
(3,4-diguanidinobutanoyl-tyrosine)
Figure 1. RP-HPLC chromatogram of the 5% MeOH fraction.
Figure 2. Structures of Hanasanagin 1 and the pseudo-di-peptide 2.
Table 1
NMR spectral data of Hanasanagin 1 and the pseudo-di-peptide 2 in D2O.
Hanasanagin 1 (3,4-diguanidinobutanoyl-DOPA)
Residue
DOPA
Position
13C
1H
1H–1H COSY
HMBC (1H /13C)
C]O
a
b
176.8
55.8
37.2
4.47 (1H, dd, J¼5.0, 10.0 Hz)
a 2.66 (1H, dd, J¼10.0, 14.0 Hz)
b 3.01a (1H, m)
b
a
a
-Ha, Hb
CO,
CO,
CO,
b-C, C-1 (DOPA), C-1 (But)
a-C, C-1, C-2, C-6 (DOPA)
a-C, C-1, C-2, C-6 (DOPA)
-H,
-H,
b
b
-Hb
-Ha
1
2
3
4
5
6
130.6
117.6
144.6
143.6
116.9
122.3
6.67 (1H, d, J¼2.0 Hz)
H-6
b-C, C-3, C-4, C-6 (DOPA)
6.72 (1H, d, J¼8.0 Hz)
H-6
H-2, H-5
C-1, C-3 (DOPA)
-C, C-2, C-4 (DOPA)
6.58 (1H, dd, J¼2.0, 8.0 Hz)
b
But
1
2
172.0
38.1
a 2.35 (1H, dd, J¼6.6, 15.5 Hz)
b 2.41 (1H, dd, J¼8.0, 15.5 Hz)
3.84 (1H, br s)
Hb-2, H-3
Ha-2, H-3
Ha-2, Hb-2, H-4
H-3
C-1, C-3, C-4 (But)
C-1, C-3, C-4 (But)
C-1, C-2, C-4, C-5 (But)
C-2, C-3, C-6 (But)
3
4
5
6
49.8
44.8
157.5
157.8
3.01a (2H, m)
Pseudo-di-peptide 2 (3,4-diguanidinobutanoyl-tyrosine)
Residue
Tyr
Position
13C
1H
1H–1H COSY
HMBC (1H /13C)
C]O
a
b
175.8
55.1
36.7
4.52 (1H, dd, J¼5.0, 9.5 Hz)
a 2.74 (1H, dd, J¼9.5, 14.0 Hz)
b 3.07 (1H, dd, J¼5.0, 14.0 Hz)
b
a
a
-Ha, Hb
CO,
CO,
CO,
b-C, C-1 (Tyr), C-1 (But)
a-C, C-1, C-2/C-6 (Tyr)
a-C, C-1, C-2/C-6 (Tyr)
-H,
-H,
b
b
-Hb
-Ha
1
129.3
131.3
116.2
155.3
2/6
3/5
4
7.02 (2H, d, J¼8.5 Hz)
6.70 (2H, d, J¼8.5Hz)
H-3/H-5
H-2/H-6
b-C,C-1, C-3/ C-5, C-4 (Tyr)
C-1, C-2/C-6, C-4(Tyr)
But
1
2
172.2
38.1
a 2.36 (1H, dd, J¼6.0, 15.5 Hz)
b 2.41 (1H, dd, J¼7.5, 15.5 Hz)
3.84 (1H, m)
Hb-2, H-3
Ha-2, H-3
Ha-2, Hb-2, H-4
H-3
C-1, C-3, C-4 (But)
C-1, C-3, C-4 (But)
C-1, C-2, C-4, C-5 (But)
C-2, C-3, C-6 (But)
3
4
5
6
49.8
44.9
157.5
157.8
3.01 (2H, m)
But¼diguanidinobutanoyl moiety; DOPA¼dihydroxyphenylalanine moiety; Tyr¼tyrosine moiety.
a
Overlapped CH2 and CH signals were separately observed and assigned in the spectrum measured in D2O/pyridine-d5.

6824
A. Sakakura et al. / Tetrahedron 65 (2009) 6822–6827
As shown inTable 1, the signals in the 1H and 13C NMR spectra of 2
resembled the signals observed in the spectra recorded on 1. In
particular, the chemical shifts of the 1H and 13C signals of the buta-
noyl moieties were almost superimposable. This result suggests that
Starting from (R)-3 or racemic 3, (R)-3,4-diguanidinobutanoyl-
(S)-tyrosine, (R,S)-2 or the mixture of the diastereomers, (R,S)-2 and
(S,S)-2, was obtained. The diastereomeric mixture was separated
into two peaks (tR 9.5 peak and tR 10.6 peak) by RP-HPLC analysis
2 has the same butanoyl partial structure as 1. The carbonyl,
a
- and
b
-
using a Develosil ODS HG-5 column (4.6 mmꢁ150 mm, 5
mm, iso-
carbons and proton signals of the tyrosine moiety of 2 appeared at
similar chemical shifts to the corresponding signals of the DOPA
moiety of 1. However, differences were observed in the aromatic
regions of the spectra. Only two 2H aromatic proton signals (6.70 and
7.02 ppm), which were coupled by 8.5 Hz, were observed in the 1H
NMR spectrum of 2, whereas 1 showed three 1H signals at 6.58, 6.67
and 6.72 ppm. In the spectrum of 1, all of the six aromatic carbon
signals were identified individually, however; the spectrum of 2
showed four aromatic carbon signals (two 1C signals at 129.3 and
155.3 ppm and two 2C signals at 116.2 and 131.3 ppm). Among the
aromatic carbon signals of 2, only the one at 155.3 ppmwas assigned
to an oxygenated aromatic carbon. This assignment was based on the
chemical shift of the signal. In contrast, based on the NMR spectrum
of 1, two carbon atoms were oxygenated (143.6 and 144.6 ppm).
These data suggest that the aromatic group of 2 was a p-hydrox-
yphenyl group andthat 2 contained a tyrosine moiety rather than the
DOPA moiety of Hanasanagin 1. Thus, the structure of 2 was deduced
to be 3,4-diguanidinobutanoyl-tyrosine. The structure is shown in
Figure 2 and the important 1H–1H COSY and HMBC correlations of 2
are indicated by bold lines and arrows, respectively. All of these
correlations agreed with the deduced structure.
cratic elution with 10% acetonitrile containing 0.1% TFA,
0.5 mL minꢀ1) and the individual fractions were isolated (Fig. 3).
A:
t
= 9.5 min
R
B:
t
= 10.6 min
R
C:
(R,S)- 2
D:
natural 2
2.3. Chemical synthesis of 3,4-diguanidinobutanoyl-
tyrosine 2
To confirm the structures of 1 and 2 and to determine their
configurations, (R)-3,4-diguanidinobutanoyl-(S)-tyrosine, (R,S)-2
and the diastereomeric mixture of (R,S)-2 and (S,S)-2 were syn-
thesized, and (R,S)-2 was enzymatically converted to (R,S)-1
(Scheme 1).
5
10
retention time (min)
15
Figure 3. Comparison of the retention times of the diastereomers of diguanidinobu-
tanoyl-tyrosine 2 (A and B) isolated from the synthetic mixture with those of synthetic
(R,S)-2 (C) and natural 2 (D).
NHBoc
NHBoc
NH2
H
N
H
N
H2N COOMe
H
COOMe
OH
COOMe
a
b
c
+
H
H
H
H
H2N
H
O
HN
HN
O
O
Boc
Boc
O-t-Bu
O-t-Bu
OH
(R)-3
(R,S)-4
(R,S)-5
NBoc
NH
H2N NH
NH
BocHN NH
H2N NH
H
N
H
N
H
N
COOMe
d, e
COOH
COOH
mushroom tyrosinase
H
H
H
H
H
H
O
HN
HN
HN
O
O
in phosphate-ascorbatre
buffer containing borate
BocN NHBoc
HN NH2
HN NH2
OH
OH
OH
OH
(R,S)-6
(R,S)-2
Hanasanagin 1
(R,S)-1
Scheme 1. Synthesis of (R)-3,4-diguanidinobutanoyl-(S)-tyrosine 2 and enzymatic conversion of 2 to (R)-3,4-diguanidinobutanoyl-(S)-DOPA, Hanasanagin 1: (a) EDC, Et3N, DMF;
(b) TFA; (c) N,N0-di-Boc-S-methylisothiourea, 4-dimethylaminopyridine, Et3N, DMF; (d) aq NaOH, PrOH; (e) cleavage cocktail (82% TFA, 5% H2O, 5% thioanisole, 3% ethanedithiol,
2% ethyl methyl sulfide and 3% phenol).
3,4-Di-(Boc-amino)-butanoic acid3was condensed with (S)-O-tert-
butyltyrosine methyl ester to give the protected 3,4-diaminobu-
tanoyltyrosine 4. The protecting groups, Boc and tert-butyl, were then
removed by acid hydrolysis to afford 5. The freed amino groups of 5
were guanidinated with an excess of N,N0-di-Boc-S-methylisothiourea6
to give the protected 3,4-diguanidinobutanoyl-tyrosine 6. De-pro-
tection of 6 by hydrolysis with NaOH followed by treatment with the
acidic cleavage cocktail afforded the pseudo-di-peptide 2.
These fractions were identified as the stereoisomers of 3,4-
diguanidinobutanoyl-tyrosine by spectrometric analyses. The
spectral data of the tR 9.5 peak are shown in the Experimental
section. The spectral data and the retention time in the RP-HPLC
analysis of the tR 10.6 peak were the same as those of the synthetic
(R,S)-2. Accordingly, the tR 10.6 peak must have an (R,S) configu-
ration, and the tR 9.5 peak was concluded to have an (S,S) con-
figuration. As the retention time in the RP-HPLC analysis (Fig. 3)

A. Sakakura et al. / Tetrahedron 65 (2009) 6822–6827
6825
and spectral data of natural 2 were identical to those of synthetic
(R,S)-2 and the tR 10.6 peak, the stereochemistry of natural 2
should be (R,S) or (S,R).
To determine the configuration of natural 2, a CD spectrum of this
compound was acquired and compared with the CD spectrum of
synthetic (R,S)-2. The spectra were essentially superimposable
(Fig. 4A) and the configuration of natural 2 was concluded to be (R,S).
separation afforded pure 1 with a yield of 63%. The structure of
enzymatically derived 1 was confirmed by comparing the RP-HPLC
chromatogram and spectral data with those of natural 1.
The stereochemistry of natural Hanasanagin was determined to
be (R)-3,4-diguanidinobutanoyl-(S)-DOPA by comparison of the CD
spectrum of this compound with that of enzymatically derived
(R,S)-1 (Fig. 4B).
B
A
25
20
15
10
5
20
natural 2
15
natural Hanasanagin 1
enzymatically
derived (R,S)-1
synthetic (R,S)-2
10
5
0
250
300
λ (nm)
-5
0
250
300
-10
-15
λ (nm)
-5
Figure 4. CD spectra of (A): synthetic (R,S)-2 and natural pseudo-di-peptide 2, and (B): enzymatically derived (R,S)-1 and natural Hanasanagin 1.
2.4. Enzymatic conversion of 2 to Hanasanagin 1
2.5. Antioxidant activity
The enzymatic conversion of (R,S)-2 to Hanasanagin 1 was car-
ried out (Scheme 1). The substrate was treated with the mushroom
tyrosinase at 25 ꢂC in a 100 mM phosphate-ascorbate buffer, pH 7.0,
containing 20 mM borate.7 RP-HPLC analysis of the reaction mix-
ture (Fig. 5) revealed that the substrate disappeared in 100 min and
that 2 was converted to Hanasanagin, 1. Preparative RP-HPLC
The free radical scavenging activity of Hanasanagin was
evaluated against the stable 1,1-diphenyl-2-picrylhydrazyl
(DPPH) radical,2 and the SOD-like activity was measured using
the ESR spin trapping method according to Yes¸ilada et al. and
Sekine et al.3,4 The DPPH radical scavenging activity, EC50, was
8.1
m
M and was observed to be 2.6 times greater than the activity
mol.
of ascorbic acid. The SOD-like activity was 80 SOD unit/
m
This activity was 1.9 times stronger than the activity of ascorbic
acid.5
In contrast to Hanasanagin 1, the pseudo-di-peptide 2 showed
0 min
no antioxidant activity.
3. Discussion
10 min
In the present study, one novel antioxidant was isolated from
the entomogenous Hanasanagitake mushroom, and was named
Hanasanagin. The structure of Hanasanagin was determined by
spectral analyses, chemical synthesis and enzymatic conversion.
Hanasanagin was determined to be a new pseudo-di-peptide
composed of a unique (R)-3,4-diguanidinobutanoic acid and (S)-
DOPA. The DOPA moiety containing a catechol group is expected to
be the origin of the antioxidant activity because many natural
products displaying this moiety show such activity.3,4,8,9 Reactive
oxygen species are suspected to cause the peroxidative disinte-
gration of cells which is implicated in various pathological pro-
cesses and are involved in the pathogenesis of diseases such as the
initiation of cancer and the aging process. The antioxidant activity
of Hanasanagin from the Hanasanagitake mushroom may con-
tribute to reducing the reactive oxygen species, thus justifying the
use of this mushroom as a popular folk medicine and as a tradi-
tional health food choice.
100 min
after HPLC
purification
natural
Hanasanagin
15
20
25
30
retention time (min)
The modification of bioactive peptides has recently attracted
attention because such changes can add new favorable character-
istics to bioactive compounds such as the enhancement of
Figure 5. Time course change of the RP-HPLC chromatogram of the enzymatic reaction
mixture and comparison of the chromatogram of enzymatically derived Hanasanagin 1
after HPLC purification with that of natural Hanasanagin 1.

6826
A. Sakakura et al. / Tetrahedron 65 (2009) 6822–6827
activity10 and permeability.11,12 Hanasanagin is a small pseudo-di-
peptide that should be suitable for modification and as an anti-
oxidant additive to other bioactive peptides. Attempts are currently
underway to use Hanasanagin as a peptidic modifier of other
bioactive peptides, such as the diapauses hormone and Yamamarin
of insects.
In the course of the isolation of Hanasanagin 1, a new pseudo-
di-peptide 2, structurally related to 1, was obtained. This di-pep-
tide was easily converted to Hanasanagin 1 by the enzymatic
oxidation catalyzed by the commercial mushroom tyrosinase to
afford a good yield. As such, 2 is speculated to be a biogenetic
precursor of 1. The easy enzymatic conversion of 2 to 1 offers
a convenient method to obtain antioxidant 1 from inactive 2. This
is particularly attractive because the yield of 2 from the mushroom
was 20-fold higher than 1.
reaction, the mixture was evaporated to dryness to afford 290 mg
of crude (R,S)-5. Without further purification, 190 mg of crude
(R,S)-5 was mixed with 470 mg of N,N0-di-Boc-S-methylisothiourea,
4-dimethylaminopyridine (8 mg in 50 mL of DMF) and 270 mL of
Et3N in 4 mL of DMF. After stirring for 50 h at rt, the reaction
mixture was concentrated in vacuo. The remaining solution was
acidified with 10% citric acid and diluted with water. The diluted
solution was purified by Chromatorex ODS column chromatogra-
phy. As the acetonitrile fraction from the column contained the
desired (R,S)-6 and N,N0-di-Boc-S-methylisothiourea, the fraction
was purified by silica gel column chromatography (Wako gel C-200,
Wako Pure Chemical Industries Co.) using a mixture of hexane and
EtOAc (1:1) as the eluant. The yield of (R,S)-6 was 29 mg (11% from
R,S-4). 12 mg of (R,S)-6 was dissolved in a mixture of 2 mL of 2-
PrOH and 2 mL of 1 N NaOH. After stirring for 3 h, the reaction
mixture was neutralized with 10% citric acid and diluted with
80 mL of water. The diluted solution was loaded onto the Chro-
matorex ODS column and the column was washed with water and
50% CH3CN. The material was then eluted with CH3CN, 50% 2-PrOH
and 2-PrOH and the fractions containing the desired product were
collected and evaporated to dryness. The residue was treated with
1 mL of the cleavage cocktail composed of 82% TFA, 5% H2O, 5%
thioanisole, 3% ethanedithiol, 2% ethyl methyl sulfide and 3%
phenol. After a reaction period of 3 h, the mixture was evaporated
to dryness and the residue was washed with a mixture of hexane
and ether (1:1). The remaining precipitate was purified by RP-HPLC
to afford 4.1 mg (73% from (R,S)-6) of pure (R,S)-2. Spectral data
were identical to the natural 3,4-diguanidinobutanoyl-tyrosine
(Table 1).
4. Experimental
4.1. General information
Reagents and solvents were purchased from Wako Pure Chem-
ical Industries Co., Tokyo Chemical Industry Co. and Nacalai Tesque
Co., and were used without further purification. The mushroom
tyrosinase and SOD were obtained from Sigma Chemical Co. Mass
spectra were obtained with a Kompact Discovery MALDI-TOF-MS
system (Shimadzu Co.), and HRFABMS spectra were obtained with
a JMS-MS 700v mass spectrometer (JEOL Co.). NMR, CD and ESR
spectra were measured using a JNM-
a500 spectrometer (JEOL Co),
a J-720 spectropolarimeter (JASCO Co.) and a JES-RE1X spectrom-
eter (JEOL Co.), respectively. HPLC was performed using a Gulliver
HPLC system (JASCO Co.)
When the synthesis was started from the racemic 3,4-di-(Boc-
amino)-butanoic acid 3, a diastereomeric mixture of (R,S)-2 and
(S,S)-2 was obtained. The mixture was separated into two peaks
(tR 9.5 peak and tR 10.6 peak in Fig. 3) by the preparative RP-
HPLC.
4.2. Isolation of Hanasanagin 1 and the biogenetic precursor 2
The fruiting body of the Hanasanagitake mushroom (96 g) cul-
tivated on about 370 silkworm pupae was extracted with 400 mL of
60% ethanol. The extraction was repeated 4 times using 200 mL of
60% EtOH. After removing the ethanol from the collected extract,
the residue was separated by RP chromatography using a Diaion
HP-20 column (Mitsubishi Chemical Co.). The material was eluted
from the column with 5% MeOH after washing with water. Ana-
lytical RP-HPLC of the 5% MeOH fraction (column: Mightysil PR-
4.3.1. tR 10.6 Peak ((R)-3,4-diguanidinobutanoyl-(S)-tyrosine)
Spectral data of tR 10.6 peak were identical to those recorded on
synthesized (R)-3,4-diguanidinobutanoyl-(S)-tyrosine and natural
3,4-diguanidinobutanoyl-tyrosine (Table 1).
4.3.2. tR 9.5 Peak ((S)-3,4-diguanidinobutanoyl-(S)-tyrosine)
1H NMR (500 MHz, D2O):
d
2.27 (1H, dd, J¼8.9, 15.6 Hz), 2.44
18GP, 5
m
m, 20 mmꢁ250 mm, Kanto Chemical Co., gradient elution
(1H, dd, J¼4.6, 15.6 Hz), 2.70 (1H, dd, J¼10.1, 14.3 Hz), 3.00 (1H, dd,
J¼8.7, 14.3 Hz), 3.07 (1H, dd, J¼4.9, 14.3 Hz), 3.18 (1H, dd, J¼4.6,
14.3 Hz), 3.85 (1H, m), 4.52 (1H, dd, J¼4.6, 10.1 Hz), 6.67 (2H, d,
from 0.1% TFA to CH3CN containing 0.1% TFA, over 60 min, flow rate:
0.5 mL minꢀ1) is shown in Figure 1. Hanasanagin 1 and the pseudo-
di-peptide 2 were isolated by preparative RP-HPLC (column:
J¼8.4 Hz), 6.99 (2H, d, J¼8.4 Hz). 13C NMR (125 MHz, D2O):
d 36.8,
Mightysil PR-18GP, 20 mmꢁ250 mm, 5
m
m, gradient elution from
38.2, 44.9, 49.7, 55.0, 116.1, 129.3, 131.1, 155.1, 157.4, 157.8, 172.2,
0.1% TFA to 70% CH3CN containing 0.1% TFA, over 120 min, flow rate:
175.9. MALDI-TOF MS (DHBA): m/z¼366 ([MþH]þ).
4 mL minꢀ1).
The yields of 1 and 2 were 2 and 36 mg from 96 g of the
mushroom, respectively. The spectral data of 1 and 2 are presented
in the text and Table 1.
4.4. Enzymatic conversion of 2 to 1
The enzymatic conversion was carried out according to the
procedure reported by Taylor.7 The substrate 2 (30.1 mg) was dis-
solved in 30 mL of 100 mM phosphate-ascorbate buffer, pH 7,
4.3. Synthesis of (R)-3,4-diguanidinobutanoyl-(S)-tyrosine 2
containing 20 mM borate. To the solution, 500 mg of mushroom
A typical procedure for synthesis of (R,S)-2 is as follows. (R)-3,4-
Di-(Boc-amino)-butanoic acid (R)-3 (400 mg), 510 mg of (S)-O-tert-
butyltyrosine methyl ester hydrochloride, 540 mL of Et3N and
tyrosinase (2590 U mgꢀ1) dissolved in 1 mL of the buffer was added
and the reaction was carried out at 25 ꢂC. The time course of the
reaction was traced by RP-HPLC (column: Mightysil RP-18,
340 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
hydrochloride) were mixed in 30 mL of DMF and stirred for 30 h at
rt. The reaction mixture was acidified with 10% citric acid and
diluted with water. The diluted solution was separated by ODS
column chromatography (Chromatorex ODS, Fuji Silysia Co.). The
acetonitrile fraction from the column containing the desired
product was evaporated to afford 290 mg of (R,S)-4 (37% yield). The
obtained (R,S)-4 was treated with 13 mL of 80% TFA. After 4 h
4.6 mmꢁ150 mm, 5
mm, gradient elution from 0.1% TFA to aceto-
nitrile containing 0.1% TFA, over 60 min, 0.5 mL minꢀ1, Fig. 5). After
100 min, the reaction mixture was acidified to pH 2 by adding 1%
TFA and it was loaded onto Diaion HP20 column (15 mmꢁ200 mm)
equilibrated with 0.1% TFA. The column was eluted with 50%
2-PrOH containing 0.1% TFA after washing with 0.1% TFA. The 50%
2-PrOH fraction was subjected to RP-HPLC purification using
Mightysil RP-18 GP column (20 mmꢁ250 mm, 5
mm, gradient

A. Sakakura et al. / Tetrahedron 65 (2009) 6822–6827
6827
elution from 0.1% TFA to 50% CH3CN containing 0.1% TFA, over
120 min, flow rate: 4 mL minꢀ1). Pure Hanasanagin was obtained
with a 63% yield (19.7 mg). Its spectral data were identical with
those of natural 1.
Chemical Co.) in the same buffer. The solution was mixed well, and
after 40 s, the ESR spectrum was acquired.
Acknowledgements
This work was financially supported by the Research for the
Future Program of the Japan Society for the Promotion of Science
(JSPS-RFTF99L01203) and by the Research and Development
program for New Bio-industry Initiatives.
4.5. DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging
activity
The DPPH radical scavenging activity was measured
according to the procedure reported by Ahn et al.2 The sample
References and notes
was dissolved in 50% ethanol (200
a mixture of 100 L of water and 80
sample solution, 20 L of DPPH (300 M in ethanol) was added
m
L) and was diluted with
m
m
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1. Imamura, T.; Aizono, Y.; Mizuno, M. Japan Patent 2002-272267, 2002.
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The procedure reported by Yes¸ilada et al.3 and Sekine et al.4 was
used to measure the SOD-like activity. The sample solution (100
30 L of 0.92 M 5,5-dimethyl-1-pyrroline-N-oxide (Dojindo Co.) in
a 100 mM phosphate buffer, pH 7.8, and 100 L of xanthine oxidase
(0.4 U mLꢀ1 in the buffer, Roche Co.) were added to a mixture of
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m
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