Stereoselective oxidation at C-4 of flavans by the endophytic fungus Diaporthe sp. isolated from a tea plant

Article abstract of DOI:10.1248/cpb.53.1565

The microbial transformation of five flavans (1-5) by endophytic fungi isolated from the tea plant Camellia sinensis was investigated. It was found that the endophytic filamentous fungus Diaporthe sp. oxidized stereoselectively at C-4 position of (+)-catechin (1) and (-)-epicatechin (2) to give the correspondent 3,4-cis-dihydroxyflavan derivatives (6, 10), respectively. (-)-Epicatechin 3-O-gallate (3) and (-)-epigallocatechin 3-O-gallate (4) were also oxidized by the fungus into 3,4-dihydroxyflavan derivatives (10, 12) via (-)-epicatechin (2) and (-)-epigallocatechin (11), respectively. Meanwhile, (-)-gallocatechin 3-O-gallate (5), (-)-catechin (ent-1) and (+)-epicatechin (ent-2), which possess a 2S-phenyl substitution, resisted the biotransformation.

Full text of DOI:10.1248/cpb.53.1565

December 2005
Chem. Pharm. Bull. 53(12) 1565—1569 (2005)
1565
Stereoselective Oxidation at C-4 of Flavans by the Endophytic Fungus
Diaporthe sp. Isolated from a Tea Plant1)
Andria AGUSTA,a Shoji MAEHARA,a Kazuyoshi OHASHI,a Partomuan SIMANJUNTAK,b and
,a
*
Hirotaka SHIBUYA
a Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University; Sanzo, 1 Gakuen-cho, Fukuyama, Hiroshima
729–0292, Japan: and b Research Centre for Biotechnology, Indonesian Institute of Sciences; Jalan Raya Bogor Km 46,
Cibinong, Bogor 16911, Indonesia. Received July 25, 2005; accepted August 19, 2005
The microbial transformation of five flavans (1—5) by endophytic fungi isolated from the tea plant Camellia
sinensis was investigated. It was found that the endophytic filamentous fungus Diaporthe sp. oxidized stereoselec-
tively at C-4 position of (ꢀ)-catechin (1) and (ꢁ)-epicatechin (2) to give the correspondent 3,4-cis-dihydroxyfla-
van derivatives (6, 10), respectively. (ꢁ)-Epicatechin 3-O-gallate (3) and (ꢁ)-epigallocatechin 3-O-gallate (4) were
also oxidized by the fungus into 3,4-dihydroxyflavan derivatives (10, 12) via (ꢁ)-epicatechin (2) and (ꢁ)-epigallo-
catechin (11), respectively. Meanwhile, (ꢁ)-gallocatechin 3-O-gallate (5), (ꢁ)-catechin (ent-1) and (ꢀ)-epicate-
chin (ent-2), which possess a 2S-phenyl substitution, resisted the biotransformation.
Key words microbial transformation; flavan; endophyte; Diaporthe sp.; tea plant
Endophytic microbes living inside plants2) may have a ca-
pacity to transform the chemical constituents of the plants to
yield their chemical derivatives. Flavans, [e.g. (ꢀ)-catechin
(1), (ꢁ)-epicatechin (2), (ꢁ)-epicatechin 3-O-gallate (3),
(ꢁ)-epigallocatechin 3-O-gallate (4) and (ꢁ)-gallocatechin
3-O-gallate (5)], have been isolated from tea leaves3) and are
well known as free-radical scavengers.4) In order to shed light
on the availability of endophytic microbes, we attempted the
microbial transformation of five typical flavans (1—5, shown
in Fig. 1) by endophytic fungi isolated from the tea plant
Camellia sinensis (L.) O.K. (Theaceae), which is parasitized
with Scurrula atropurpurea (BL.) DANS. (Loranthaceae).5,6)
A total of 35 filamentous endophytic fungi was obtained
from the young stems of Camellia sinensis collected in the
Puncak area, West Java, Indonesia, through the same proce-
dure as described in our previous paper.7) The fungi obtained
were classified into 6 species by morphological considera-
tions and RAPD analyses.
By screening tests in several liquid cultivation mediums, it
was found that one filamentous fungus among them trans-
formed (ꢀ)-catechin (1) into the chemical derivative.1) The
fungus was inoculated in glucose–yeast extract–peptone
medium (glucose 20 g, yeast extract 1.0 g, peptone 5.0 g,
K2HPO4 0.5 g, MgSO4 0.5 g, FeSO4·7H2O 0.01 g, CaCO3
1.0 g and tap water 1 l, at pH 6.44) and then cultivated for
5 d under shaking at 90 rpm at 27 °C. A MeOH solution
of (ꢀ)-catechin (1) was then added to the medium and
shaken for one more day. The whole medium was extracted
with EtOAc and evaporated to give a product. The product
was purified by SiO2 column chromatography (eluted with
CHCl3 : MeOH : H2Oꢂ65 : 35 : 10 lower phase) and subse-
quent reverse-phase HPLC (with H2O) to afford a biotrans-
formed product (6, 45%) in addition to the recovered (ꢀ)-
catechin (1, 8.5%).
The IR spectrum of the product (6), C15H14O7, [a]D ꢀ5.5°
(EtOH), showed absorption bands due to hydroxyl and aro-
1
matic groups. The H-NMR spectrum led us to belive that 6
was the known substance (ꢀ)-2,3-trans-3,4-cis-3,4,5,7,3ꢃ,4ꢃ-
hexahydroxyflavan (leucocyanidin). However, since the
physicochemical data in the literature8,9) was insufficient for
comparison with 6, we attempted the following chemical de-
rivatization according to a previously reported procedure,10)
as shown in Fig. 2.
(ꢀ)-(2R,3R)-3,5,7,3ꢃ,4ꢃ-Pentahydroxyflavanone (taxifolin,
7) was converted by CH2N2 methylation followed by NaBH4
reduction into two 3,4-dihydroxyflavan derivatives (8, 9),
whose stereostructures were clarified by the NOE observa-
tion between 2b-H and 4b-H in 9. The methylated product of
6 was identical with 8, including the optical rotation. From
the above findings, the biotransformed product (6) was deter-
mined as (ꢀ)-(2R,3S,4S)-3,4,5,7,3ꢃ,4ꢃ-hexahydroxyflavan.
Next, we conducted rDNA analysis for the 18S, ITS1, 5.8S
and ITS2 regions of the fungus that exhibited biotransforma-
tion activity against (ꢀ)-catechin (1). After consideration of
the phylogenetic relationships for the ITS1, 5.8S, and ITS2
rDNA regions using the databases,11) we directly compared
the rDNA base sequences of the endophytic fungus and Dia-
porthe phaseolorum var. sojae (IFO 6709). The results
Fig. 1
∗ To whom correspondence should be addressed. e-mail: shibuya@fupharm.fukuyama-u.ac.jp
© 2005 Pharmaceutical Society of Japan

1566
Vol. 53, No. 12
Fig. 2
Table 1. Comparison 18S-ITS1-5.8S-ITS2 rDNA Sequence of the Endophytic Fungus with Diaporthe phaseolorum var. sojae
18S rDNA
(bp)
Similarity
(%)
ITS1
(bp)
Similarity 5.8S rDNA Similarity
ITS2
(bp)
Similarity Total similarity
(%)
(bp)
(%)
(%)
(%)
The endophytic fungus
Diaporthe phaseolorum
Insert
1734
1736
177
179
2
7
0
153
153
0
0
0
156
158
2
7
0
99.5
93.2
100.0
90.4
98.3
6
18
0
2
4
0
Difference
Gap
showed that the similarity for the 18S region is 99.5% with
D. phaseolorum, 93.8% for the ITS1, 100% for the 5.8S, and
93.0% for the ITS2, shown in Table 1. These findings indi-
cated that the endophytic fungus may be a species closely re-
lated to Diaporthe phaseolorum var. sojae.
In contrast, exposure of the endophytic fungus to (ꢁ)-gal-
locatechin 3-O-gallate (5) did not yield a product oxidized at
C-4, but only afforded hydrolyzed (ꢁ)-gallocatechin (13,
75%). Since the resistance to biooxidation at the C-4 position
was thought to be due to a 2S-configuration function, we next
examined the biotransformation of (ꢁ)-catechin (ent-1) and
(ꢀ)-epicatechin (ent-2), which also have a 2S-phenyl substi-
tution. As expected, ent-1 and -2 strongly resisted the biooxi-
dation (Fig. 4).
In conclusion, the endophytic fungus Diaporthe sp. iso-
lated from the tea plant Camellia sinensis, stereoselectively
oxidizes the C-4 carbon of flavans possessing a 2R-substitu-
tion from the same direction to the configuration of 3-hy-
droxyl function. This evidence agrees with the hypothesis
that endophytic microbes exhibit the ability to transform the
chemical constituents of the plants.
Finally, to determine the factors for this microbial oxida-
tion, the following cultivations were examined. When the
cultivation reaction was carried out under an environment of
nitrogen gas, the oxidation did not proceed, but restarted to
afford the oxidized products (6, 10, 12) after the rebubbling
of oxygen gas. The filtrate of the fungus cultivation medium
did not transform the flavans (1—4). These findings show
that the biooxidation occurs in an endoenzyme manner using
molecular oxygen.
The transformation of (ꢁ)-epicatechin (2) by the above-
mentioned endophytic fungus Diaporthe sp. gave a product
(10, 39%) together with unchanged 2 (2.4%). The IR and UV
spectra of 10, C15H14O7, [a]D ꢁ8.4° (EtOH), showed similar
absorptions to those of 6 transformed from (ꢀ)-catechin (1).
1
The H- and 13C-NMR spectra both showed a characteristic
three consecutive methine-proton system (d 5.00, br s, 2-H; d
3.88, dd, Jꢂ1.2, 2.2 Hz, 3-H; d 4.75, d, Jꢂ2.2 Hz, 4-H). Fur-
thermore, the NOE was observed between 2b-H and 4b-H in
1
the H-NMR spectrum. From these findings, the chemical
structure of 10 was elucidated as a new compound, (ꢁ)-
(2R,3R,4R)-3,4,5,7,3ꢃ,4ꢃ-hexahydroxyflavan.
(ꢁ)-Epicatechin 3-O-gallate (3) was also biotransformed
into the same product 10 (53%) via (ꢁ)-epicatechin (2,
13%), which was obtained as another product.
The biotransformation of (ꢁ)-epigallocatechin 3-O-gallate
(4), a major chemical constituent of tea leaves, by the fungus
gave a product (12, 43%) together with (ꢁ)-epigallocatechin
(11, 19%). The IR and UV spectra of 12, C15H14O8, [a]D
ꢁ16.9°, showed similar absorption patterns to those of 10,
the product from (ꢁ)-epicatechin (2) and (ꢁ)-epicatechin 3-
1
O-gallate (3). The H- and 13C-NMR spectra of 12 also
Experimental
Optical rotations were measured using a JASCO DIP-360 digital po-
larimeter and a cell length of 50 mm. FAB-MS and high resolution FAB-MS
were taken on a JMS SX-102 A mass spectrometer. IR spectra were run on a
Shimadzu FT-IR 8500 spectrophotometer and UV spectra on a Hitachi U-
3500 spectrometer. 1H- and 13C-NMR spectra were recorded on a JEOL
showed similar signal patterns to those of 10 except for a sig-
nal pattern due to a phenyl moiety; d 6.61 (2H, s, 2ꢃ-, 6ꢃ-H)
1
in H-NMR, dC 131.1 (1ꢃ-C), 106.9 (2ꢃ-, 6ꢃ-C), 146.2 (3ꢃ-,
5ꢃ-C), 132.9 (4ꢃ-C) in the 13C-NMR. Furthermore, the NOE
was observed between 2b-H and 4b-H. These findings
prove that the chemical structure of 12 is a new compound,
(ꢁ)-(2R,3R,4R)-3,4,5,7,3ꢃ,4ꢃ,5ꢃ-heptahydroxyflavan (leuco-
delphinidin) (Fig. 3).
1
JNM-Lambda 500 spectrometer operating at 500 MHz and 125 MHz for H
and 13C nuclei, respectively. Chemical shifts are given in d scale (ppm) rela-
tive to tetramethylsilane (dꢂ0) as an internal standard, and coupling con-
stants are given in hertz. PCR amplification was carried out with an ABI
GeneAmp PCR System 9700 amplifier. DNA sequencing was performed

December 2005
1567
Table 2. Primers Used for PCR and rDNA Sequencing
Primer
Sequence (5ꢃ–3ꢃ)
Application
Location
18STscFw
TAA GCC ATG CAA GTC TAA GT
18S amplification
18S sequencing
18S rDNA
18SFt1
18SFt2
18SFt3
18SFt5
18SFt6
18SFt7
18SFt8
18SRt2
18SRt4
18SRt5
18SRt6
18SRt7
18SRt8
18STscRev
ACG GGT AAC GGA GGG TTA GG
CTG GCT GGC CGG TCT GCC TC
GCA TTC GCC AAG GAT GTT TT
ATC CCT AGT AAG CGC AAG TC
TCC AAA CTC GAT CAT TTA GAG
CAG ACA CAA CTA GGA TTG AVA G
GGA ATC CCT AGT AAG CGC AAG T
AGA ACA TCT AAG GGC ATC AC
TCC CTC CGC TGG TTC ACC AAC GG
CAA TTG TTC CTC GTT AGG GG
CAT CGC CGG CAC AAG GCC AT
CCT TGG ATG TAG TAG CCG TTT C
ACG GGC TAT TTA GCA GGT TA
TCC CTC CGC TGG TTC ACC AAC GG
18S sequencing
18S sequencing
18S sequencing
18S sequencing
18S sequencing
18S sequencing
18S sequencing
18S sequencing
18S sequencing
18S sequencing
18S sequencing
18S sequencing
18S sequencing
18S amplification
18S sequencing
18S rDNA
18S rDNA
18S rDNA
18S rDNA
18S rDNA
18S rDNA
18S rDNA
18S rDNA
18S rDNA
18S rDNA
18S rDNA
18S rDNA
18S rDNA
18S rDNA
FG18SF10
TCT GTG ATG CCC TTA GAT GTT TG
ITS1-5.8S-ITS2 amplification
ITS1-5.8S-ITS2 sequencing
ITS1-5.8S-ITS2 sequencing
ITS1-5.8S-ITS2 sequencing
ITS1-5.8S-ITS2 sequencing
ITS1-5.8S-ITS2 sequencing
18S rDNA
5.8SFt1
5.8SRt1
ITS-TscRev
CAC GGT AGT GGT TCG GTC CG
GCC TGG CTT GGT GAT GGC AC
CAC ACA GGC TTG GTG TCC GAC C
5.8S rDNA
5.8S rDNA
28S rDNA
Fig. 3
MgSO4, 0.01 g of FeSO4·7H2O, 1.0 g of CaCO3 and 1 l of tap-water, at pH
6.44.
DNA extraction, PCR amplification, DNA sequencing, and phylogenetic
analysis were carried out using the primers shown in Table 2, through the
same procedures as described in our previous paper.7)
Plant Material The young stems of the tea plant Camellia sinensis (L.)
O.K. (Theaceae) were collected in the Puncak area, West Java, Indonesia, in
August 2003 and identified at the Herbarium Bogoriense, the Research Cen-
tre for Biology, Indonesian Institute of Sciences, Indonesia.
Fig. 4
Isolation of the Endophytic Fungi from the Tea Plant The young
stems were cut into pieces (ca. 1 cm in length) and washed with tap water
for 10 min. The pieces were treated with 70% aq. EtOH for 1 min, 5.3% aq.
sodium hypochlorite for 5 min, and 75% aq. EtOH for 0.5 min, and then cut
into two pieces. The cut stems were placed on corn malt meal agar contain-
ing chloramphenicol (0.05 mg/ml) in a Petri dish and incubated at 27 °C.
After 3 d, individual colonies were transferred onto PDA in a Petri dish and
then incubated again at 27 °C for several days with periodic checks for the
purity to obtain the 35 endophytic filamentous fungi and 9 yeasts.
with an ABI PRISM 3100 Genetic Analyzer. HPLC was carried out with a
TOSOH PD-8020. Column chromatography was carried out using Kieselgel
60 (230—400 mesh, Merck). TLC on Kieselgel 60 F254 (Merck) was used to
ascertain the purity of the compounds. The spots were visualized by spray-
ing with 5% vanillin in concentrated H2SO4. All microbial transformations
were carried out in glucose–yeast extract–peptone medium containing 20 g
of glucose, 1.0 g of yeast extract, 5.0 g of peptone, 0.5 g of K2HPO4, 0.5 g of

1568
RAPD Analysis of the Endophytic Fungi Random amplified polymor-
Vol. 53, No. 12
(ꢁ)-(2R,3R,4R)-3,4,5,7,3ꢃ,4ꢃ-Hexahydroxyflavan (10): A white powder,
[a]D ꢁ8.4° (cꢂ0.77, in EtOH at 26 °C). IR (KBr) cmꢁ1: 3250, 1628. UV
(EtOH) nm (e): 279 (4200). H-NMR (acetone-d6) d: 3.88 (1H, dd, Jꢂ1.2,
phic DNA (RAPD) analyses of the extracted DNA from 35 endophytic fila-
mentous fungi were performed in a mixture of 50 ng of the DNA template,
5 ml of a primer [either primer-1 (5ꢃ-GTAGACCCGT-3ꢃ) or primer-2 (5ꢃ-
CCCGTCAGCA-3ꢃ)], Ready-To-Go RAPD Analysis Beads (Amersham
Biosciences Corp.) and sterile distilled water to a final volume of 25 ml. The
whole mixture was subjected to thermal cycling programmed for 5 min at
95 °C followed by 45 cycles of 1 min at 95 °C, 1 min at 36 °C, and 2 min at
72 °C. The RAPD products were then loaded on 2.3% agarose gel at 150
volts and 140 mA for 240 min and then stained in ethidium bromide for
20 min. The electrophoresis patterns of the amplified products were visual-
ized under UV light.
Microbial Transformation of (ꢀ)-Catechin (1) by the Endophytic
Fungus Diaporthe sp. The endophytic fungus Diaporthe sp. was inocu-
lated into the glucose–yeast extract–peptone medium (200 ml) and cultivated
for 5 d under shaking at 90 rpm at 27 °C. A solution of (ꢀ)-catechin (1,
20 mg) in MeOH (20 ml) was added to the cultivation medium and shaking
continued for 1 d. The reaction mixture was filtrated to remove the fungus
bodies. The filtrate was extracted with EtOAc and concentrated under re-
duced pressure to give a product (45 mg), which was purified by silica gel
column chromatography (SiO2 10 g, CHCl3 : MeOH : H2Oꢂ65 : 35 : 10 lower
phase) and HPLC (Capcell Pak Phenyl SG-120, H2O) to afford (ꢀ)-
(2R,3S,4S)-3,4,5,7,3ꢃ,4ꢃ-hexahydroxyflavan (6, 9.5 mg, 45%)8,9) and the re-
covered (ꢀ)-catechin (1, 1.7 mg, 8.5%).
1
2.2 Hz, 3-H), 4.75 (1H, d, Jꢂ2.2 Hz, 4-H), 5.00 (1H, br s, 2-H), 5.92 (1H, d,
Jꢂ2.1 Hz, 8-H), 6.03 (1H, d, Jꢂ2.1 Hz, 6-H), 6.80 (1H, d, Jꢂ7.9 Hz, 5ꢃ-H),
6.85 (1H, dd, Jꢂ1.8, 7.9 Hz, 6ꢃ-H), 7.08 (1H, d, Jꢂ1.8 Hz, 2ꢃ-H). 13C-NMR
(acetone-d6) d: 64.6 (4-C), 72.3 (3-C), 75.8 (2-C), 95.4 (8-C), 96.3 (6-C),
103.6 (4a-C), 115.5, 115.6 (2ꢃ-C, 5ꢃ-C), 119.4 (6ꢃ-C), 131.9 (1ꢃ-C), 145.4,
145.5 (3ꢃ-C, 4ꢃ-C), 157.6 (8a-C), 159.2 (5-C), 159.3 (7-C). FAB-MS m/z:
307 [MꢀH]ꢀ. High-resolution FAB-MS m/z: Calcd for C15H15O7: 307.0818
[MꢀH]ꢀ. Found 307.0819.
Microbial Transformation of (ꢁ)-Epicatechin 3-O-Gallate (3) by the
Endophytic Fungus Diaporthe sp. The endophytic fungus Diaporthe sp.
was inoculated into glucose–yeast extract–peptone medium (200 ml) and
shaken at 90 rpm at 27 °C for 5 d. A solution of (ꢁ)-epicatechin 3-O-gallate
(3, 20 mg) in MeOH (20 ml) was added to the medium and shaking contin-
ued for 1 d. After filtration to remove the fungus bodies, the filtrate was ex-
tracted with EtOAc. The EtOAc extract was concentrated under reduced
pressure to give a product (51 mg), which was purified by silica gel column
chromatography (SiO2 10 g, CHCl3 : MeOH : H2Oꢂ65 : 35 : 10 lower phase)
followed by HPLC (Capcell Pak Phenyl SG-120, H2O) to afford (ꢁ)-
(2R,3R,4R)-3,4,5,7,3ꢃ,4ꢃ-hexahydroxyflavan (10, 7.2 mg, 53%) and (ꢁ)-epi-
catechin (2, 1.7 mg, 13%).
Microbial Transformation of (ꢁ)-Epigallocatechin 3-O-Gallate (4) by
the Endophytic Fungus Diaporthe sp. The endophytic fungus Diaporthe
sp. was inoculated in glucose–yeast extract–peptone medium (200 ml) and
cultivated for 5 d under shaking at 90 rpm at 27 °C. A solution of (ꢁ)-epigal-
locatechin 3-O-gallate (4, 20 mg) in MeOH (20 ml) was added to the
medium and then shaken for one more day. The reaction mixture was fil-
trated and the filtrate was extracted with EtOAc. The EtOAc layer was con-
centrated under reduced pressure to give a product (48 mg), which was puri-
fied by silica gel column chromatography (SiO2 10 g, CHCl3 : MeOH : H2Oꢂ
65 : 35 : 10 lower phase) and HPLC (Capcell Pak Phenyl SG-120, H2O) to af-
ford (ꢁ)-(2R,3R,4R)-3,4,5,7,3ꢃ,4ꢃ,5ꢃ-heptahydroxyflavan (12, 6.2 mg, 43%)
and (ꢁ)-epigallocatechin (11, 2.6 mg, 19%).
(ꢁ)-(2R,3R,4R)-3,4,5,7,3ꢃ,4ꢃ,5ꢃ-Heptahydroxyflavan (12): A white pow-
der, [a]D ꢁ16.9° (cꢂ0.69, in EtOH at 24 °C). IR (KBr) cmꢁ1: 3300, 1628.
UV (EtOH) nm (e): 279 (4200). 1H-NMR (acetone-d6) d: 3.90 (1H, dd,
Jꢂ1.2, 3.1 Hz, 3-H), 4.76 (1H, d, Jꢂ3.1 Hz, 4-H), 4.95 (1H, br s, 2-H), 5.93
(1H, d, Jꢂ2.4 Hz, 8-H), 6.03 (1H, d, Jꢂ2.4 Hz, 6-H), 6.61 (2H, s, 2ꢃ-, 6ꢃ-H).
13C-NMR (acetone-d6) d: 64.5 (4-C), 72.3 (3-C), 75.7 (2-C), 95.4 (8-C),
96.3 (6-C), 103.6 (4a-C), 106.9 (2ꢃ-C, 6ꢃ-C), 131.1 (1ꢃ-C), 132.9 (4ꢃ-C),
146.2 (3ꢃ-C, 5ꢃ-C), 157.5 (8a-C), 159.2 (5-C, 7-C). FAB-MS m/z: 323
[MꢀH]ꢀ. High-resolution FAB-MS m/z: Calcd for C15H15O8: 323.0767
[MꢀH]ꢀ. Found 323.0737.
Microbial Transformation of (ꢁ)-Gallocatechin 3-O-Gallate (5) by the
Endophytic Fungus Diaporthe sp. The endophytic fungus Diaporthe sp.
was inoculated into glucose–yeast extract–peptone medium (200 ml). After
cultivation for 5 d under shaking at 90 rpm at 27 °C, a solution of (ꢁ)-gallo-
catechin 3-O-gallate (5, 20 mg) in MeOH (20 ml) was added to the medium
and shaking continued for 1 d. The reaction mixture was filtrated and the fil-
trate was extracted with EtOAc. The EtOAc layer was evaporated in vacuo to
give a product (60 mg), which was purified by silica gel column chromatog-
raphy (SiO2 10 g, CHCl3 : MeOH : H2Oꢂ65 : 35 : 10 lower phase) and HPLC
(Capcell Pak Phenyl SG-120, H2O) to give (ꢁ)-gallocatechin (13, 10.1 mg,
75%).
Treatment of (ꢁ)-Catechin (ent-1) and (ꢀ)-Epicatechin (ent-2) by the
Endophytic Fungus Diaporthe sp. After cultivation of the endophytic
fungus Diaporthe sp. in glucose–yeast extract–peptone medium (200 ml) for
5 d under shaking at 90 rpm at 27 °C, each solution of (ꢁ)-catechin (ent-1,
20 mg) and (ꢀ)-epicatechin (ent-2, 20 mg) in MeOH (20 ml) was individu-
ally added to the medium and shaking continued for 1 d. After filtration of
each reaction mixture, the filtrates were extracted with EtOAc and concen-
trated under reduced pressure to give products (43 mg from ent-1, 48 mg
from ent-2), which were purified by silica gel column chromatography (SiO2
10 g, CHCl3 : MeOH : H2Oꢂ65 : 35 : 10 lower phase) to recover (ꢁ)-catechin
(ent-1, 16 mg, 80%) and (ꢀ)-epicatechin (ent-2, 17 mg, 85%), respectively.
The physicochemical properties of (ꢀ)-(2R,3S,4S)-3,4,5,7,3ꢃ,4ꢃ-hexahy-
1
droxyflavan (6) are given here, since Kristiansen8,9) reported only H-NMR
and UV spectra for 6.
(ꢀ)-(2R,3S,4S)-3,4,5,7,3ꢃ,4ꢃ-Hexahydroxyflavan (6): A white powder,
[a]D ꢀ5.5° (cꢂ0.55, in EtOH at 25 °C). IR (KBr) cmꢁ1: 3300, 1612. UV
(EtOH) nm (e): 280 (4,000). 1H-NMR (acetone-d6: 3.85 (1H, dd, Jꢂ3.7,
9.5 Hz, 3-H), 4.84 (1H, d, Jꢂ9.5 Hz, 2-H), 4.87 (1H, d, Jꢂ3.7 Hz, 4-H), 5.65
(1H, d, Jꢂ2.1 Hz, 8-H), 6.02 (1H, d, Jꢂ2.1 Hz, 6-H), 6.80 (1H, dd, Jꢂ2.2,
7.9 Hz, 6ꢃ-H), 6.81 (1H, d, Jꢂ7.9 Hz, 5ꢃ-H), 6.94 (1H, d, Jꢂ2.2 Hz, 2ꢃ-H).
13C-NMR (acetone-d6) d: 62.8 (4-C), 71.4 (3-C), 77.7 (2-C), 95.2 (8-C),
96.4 (6-C), 103.9 (4a-C), 115.7 (2ꢃ-C, 5ꢃ-C), 120.5 (6ꢃ-C), 131.8 (1ꢃ-C),
145.6 (4ꢃ-C), 145.8 (3ꢃ-C), 157.1 (8a-C), 158.9 (5-C), 159.8 (7-C). FAB-MS
m/z: 307 [MꢀH]ꢀ. High-resolution FAB-MS m/z: Calcd for C15H15O7:
307.0818 [MꢀH]ꢀ. Found 307.0810.
Chemical Transformation of (ꢀ)-(2R,3S)-3,5,7,3ꢂ,4ꢂ-Pentahydroxyfla-
vanone (7) into 8 and 9 A solution of (ꢀ)-(2R,3S)-3,5,7,3ꢃ,4ꢃ-pentahy-
droxyflavanone (7, 80 mg, Wako Pure Chemical Industries Ltd.) in MeOH
(5.0 ml) was treated with ethereal diazomethane for 8 h at 0 °C. The reaction
mixture was worked up in a usual manner to give a product (105 mg), which
was purified by silica gel column chromatography (SiO2 15 g, benzene : ace-
toneꢂ8 : 1) to afford (ꢀ)-3-hydroxy-5,7,3ꢃ,4ꢃ-tetramethoxyflavanone (44
mg, 46%). NaBH4 (3.1 mg) was added to a solution of (ꢀ)-3-hydroxy-
5,7,3ꢃ,4ꢃ-tetramethoxyflavanone (10 mg) in MeOH (3.0 ml) at ꢁ20 °C and
the mixture was stirred for 1.5 h at ꢁ20 °C. The reaction mixture was
poured into H2O and extracted with CHCl3. The CHCl3 extract was washed
with brine and dried over Na2SO4. Removal of the solvent gave a product
(12 mg), which was separated by silica gel column chromatography (SiO2
1 g, benzene : acetoneꢂ5 : 1) to afford (ꢀ)-(2R,3S,4S)-3,4-dihydroxy-
5,7,3ꢃ,4ꢃ-tetramethoxyflavan10) (8, 2.9 mg, 29%) and (ꢀ)-(2R,3S,4R)-3,4-di-
hydroxy-5,7,3ꢃ,4ꢃ-tetramethoxyflavan10) (9, 7.0 mg, 68%).
Methylation of (ꢀ)-(2R,3S,4S)-3,4,5,7,3ꢂ,4ꢂ-Hexahydroxyflavan (6)
(ꢀ)-(2R,3S,4S)-3,4,5,7,3ꢃ,4ꢃ-Hexahydroxyflavan (6, 24 mg) was dissolved in
MeOH (1.0 ml) and treated with ethereal diazomethane for 8 h at ꢁ20 °C.
The reaction mixture was worked up in a usual manner to give a product
(35 mg), which was purified by silica gel column chromatography (SiO2
10 g, benzene : acetoneꢂ5 : 1) to afford (2R,3S,4S)-3,4-dihydroxy-5,7,3ꢃ,4ꢃ-
tetramethoxyflavan (8, 12 mg, 42%), which was identified with 8 prepared
from 7.
Microbial Transformation of (ꢁ)-Epicatechin (2) by the Endophytic
Fungus Diaporthe sp. After cultivation of the endophytic fungus Dia-
porthe sp. in glucose–yeast extract–peptone medium (200 ml) for 5 d under
shaking at 90 rpm at 27 °C, a solution of (ꢁ)-epicatechin (2, 20 mg) in
MeOH (20 ml) was added to the cultivation medium and shaken for one
more day. The reaction mixture was filtrated and the filtrate was extracted
with EtOAc. The EtOAc layer was concentrated under reduced pressure to
give a product (48 mg), which was purified by silica gel column chromatog-
raphy (SiO2 10 g, CHCl3 : MeOH : H2Oꢂ65 : 35 : 10 lower phase) and HPLC
(Capcell Pak Phenyl SG-120, H2O) to afford 10 (8.2 mg, 39%) and the re-
covered (ꢁ)-epicatechin (2, 0.48 mg, 2.4%).
Acknowledgement This work was supported by the “High-Tech Re-
search Center” Project for Private Universities: matching fund subsidy from
the Minister of Education, Culture, Sports, Science and Technology, 2004—
2008.

December 2005
1569
References and Notes
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