Microbial mannosidation of bioactive chlorogentisyl alcohol by the marine-derived fungus Chrysosporium synchronum

Article abstract of DOI:10.1248/cpb.59.499

The biological transformation of the biologically active chlorogentisyl alcohol (1), isolated from the marinederived fungus Aspergillus sp., was studied. Preparative-scale fermentation of chlorogentisyl alcohol with marine-derived fungus Chrysosporium synchronum resulted in the isolation of a new glycosidic metabolite, 1-O-(α-D-mannopyranosyl)chlorogentisyl alcohol (2). The stereostructure of the new metabolite obtained was assigned on the basis of detailed spectroscopic data analyses, chemical reaction, and chemical synthesis. Compounds 1 and 2 exhibited significant radical-scavenging activity against 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) with IC₅₀ values of 1.0 and 4.7 μM, respectively. The compounds 1 and 2 were more active than the positive control, L-ascorbic acid (IC₅₀, 20.0 μM).

Full text of DOI:10.1248/cpb.59.499

April 2011
Note
Chem. Pharm. Bull. 59(4) 499—501 (2011)
499
Microbial Mannosidation of Bioactive Chlorogentisyl Alcohol by the
Marine-Derived Fungus Chrysosporium synchronum
Keumja YUN,a Chinni Mahesh KONDEMPUDI,a Hong Dae CHOI,b Jung Sook KANG,c and
,a
*
Byeng Wha SON
a Department of Chemistry, Pukyong National University; Busan 608–737, Korea: b Department of Chemistry, Dongeui
University; Busan 614–714, Korea: and c College of Dentistry, Pusan National University; Yangsan, Gyeongnam 626–770,
Korea. Received November 25, 2010; accepted January 6, 2011; published online January 7, 2011
The biological transformation of the biologically active chlorogentisyl alcohol (1), isolated from the marine-
derived fungus Aspergillus sp., was studied. Preparative-scale fermentation of chlorogentisyl alcohol with ma-
rine-derived fungus Chrysosporium synchronum resulted in the isolation of a new glycosidic metabolite, 1-O-(a-
D-mannopyranosyl)chlorogentisyl alcohol (2). The stereostructure of the new metabolite obtained was assigned
on the basis of detailed spectroscopic data analyses, chemical reaction, and chemical synthesis. Compounds 1
and 2 exhibited significant radical-scavenging activity against 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) with
IC50 values of 1.0 and 4.7 mM, respectively. The compounds 1 and 2 were more active than the positive control, L-
ascorbic acid (IC50, 20.0 mM).
Key words marine-derived fungus; Chrysosporium synchronum; microbial mannosidation; 1-O-(a-D-mannopyranosyl)chloro-
gentisyl alcohol
The aim of our program is to explore the biological trans- new glycoside, 1-O-(a-D-mannopyranosyl)chlorogentisyl al-
formation of bioactive metabolites produced by microorgan- cohol (2) (3.2 mg). The metabolite 2, [a]D ꢁ3.70 (cꢂ0.3,
isms isolated from marine habitats. As part of this program, MeOH), was isolated as a colorless oil. A molecular formula
we identified the marine-derived ascomycete strains and of C13H17ClO8 was established by high resolution electro-
actinomycete strains that regioselectively oxidize bioactive spray ionization mass spectrometry (HR-ESI-MS) and 13C-
natural products to new and more active compounds: from NMR methods. The IR spectrum of 2 suggested the presence
terreusinone to the unsymmetrical alcohol derivative, ter- of hydroxyl (3400 cmꢀ1) and glycosidic (1018 cmꢀ1) groups.
reusinol, by Streptomyces sp.,1) from 6-n-pentyl-a-pyrone to The 1H- and 13C-NMR data of 2 closely resembled those of 1
two oxidized metabolites, 6-n-(4-oxopentyl)-a-pyrone and 6- except for the appearance of additional proton signals corre-
n-[(S)-1-hydroxypentyl]-a-pyrone, by Streptomyces sp.,2) sponding to the five sp3 oxymethines and one oxymethylene.
from cyclonerodiol to three metabolites, 10(Z)-, 10(E)-cy- An extensive analysis of the distortionless enhancement by
clonerotriol, and cyclonerodiol mannopyranoside, by the polarization transfer (DEPT) and 2D-NMR spectra, correla-
genus of Streptomyces and Penicillium,3) from geraniol to its tion spectroscopy (COSY), heteronuclear multiple quantum
7-oxidized metabolite, 1,7-dihydroxy-3,7-dimethyl-(E)-oct- correlation (HMQC), and heteronuclear multiple bond coher-
2-ene, by Hypocrea sp.,4) and from (ꢀ)-mellein to (3R,4S)-4- ence (HMBC), and nuclear Overhauser effect spectroscopy
hydroxymellein by Stappia sp.5) In our continuing study of (NOESY) experiments, suggested that 2 was a glycoside
applications of microbial transformations,6) we screened 50 comprising 1 as an aglycone and one sugar unit. The hexopy-
cultures for their ability to biotransform chlorogentisyl alco- ranose unit was presumed to be mannose due to the anti di-
hol (1). Chlorogentisyl alcohol was isolated from the marine axial orientations of H-3ꢃ/H-4ꢃ (JH3ꢃ–H4ꢃꢂ9.1 Hz) and H-4ꢃ/H-
algicolous fungus Aspergillus sp.7) and was found to induce 5ꢃ (JH4ꢃ–H5ꢃꢂ9.5 Hz) and the syn equatorial and axial orienta-
apoptosis in HeLa cells.8) The marine-derived fungus tion of H-2ꢃ/H-3ꢃ (JH2ꢃ–H3ꢃꢂ3.2 Hz) in the 1H-NMR
Chrysosporium synchronum was selected as the biotrans- (CD3OD). Acid hydrolysis of 2 furnished 1 and methyl man-
forming target strain.
nose,10) and the latter was identified on the basis of GC and
Preliminary small-scale fermentations (100 ml) of C. GC-MS analyses of the trimethylsilyl derivative. Comparison
synchronum, with the addition of monosaccharides (e.g., of the 13C-NMR data of 1 and 2, and the key HMBC correla-
glucose, galactose, mannose, rhamnose, or ribose) directly to tion from H-1ꢃ to C-1 were critical in establishing the posi-
the culture medium, respectively, did not provide promising tion of mannose as attached at the 1-OH of 1. The stereo-
result except for mannose. TLC analyses of the broth extracts chemistry of the anomeric position of the sugar moiety was
confirmed that mannose only stimulated mannoside produc- assigned as the a-configuration on the basis of the anomeric
1
tion relative to a control culture.
carbon signal at d 100.9 (d, C-1ꢃ) having a JC–H value of
Biotransformation of chlorogentisyl alcohol was carried 175 Hz.11) This value was deduced from DEPT nondecou-
out using the marine-derived fungus C. synchronum accord- pling measurements. The absolute configuration of the man-
ing to a two-stage fermentation protocol.9) The fermentations nose unit was determined to be D from measurement of the
of 1 (20 mg) with C. synchronum were harvested after two optical rotation. Obtained from acid hydrolysis of 2 with 9%
weeks, and the filtered broths were extracted with EtOAc to aq. HCl, the specific rotation of mannose, [a]D20 ꢁ28.8
afford crude extracts. The extract was purified by repeated (cꢂ0.03, H2O), indicated the presence of D-mannose in 2
silica gel flash chromatography (n-hexane in ethyl acetate) [ref: [a]D20 ꢁ29.3].12) These results led us to conclude that the
and HPLC (octadesyl silica (ODS), MeOH–H2O) to yield a structure of compound 2 was 1-O-(a-D-annopyranosyl)-
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: sonbw@pknu.ac.kr

500
Vol. 59, No. 4
chlorogentisyl alcohol (Fig. 1). To clarify the structure of 2, against epidermal growth factor receptor (EGFR),20,21) and
we synthesized 2 from 1 and 1-O-(2,3,4,6-tetra-O-acetyl-a- the results will be reported in due course.
D-mannopyranosyl)trichloroacetimide. D-(ꢁ)-Mannose was
acetylated by acetic anhydride in pyridine, followed by selec-
tive deacetylation of 1-acetyl group by piperidine to give
2,3,4,6-tetra-O-acetyl-D-mannopyranoside.13) Treatment with
trichloroacetonitrile of the C-1-unprotected mannopyranose,
2,3,4,6-tetra-O-acetyl-D-mannopyranoside, afforded 1-O-
(2,3,4,6-tetra-O-acetyl-D-mannopyranosyl)trichloroace-
timide.14,15) Glycosidation of 1 with the above imidate in the
presence of boron trifluoro-etherate16) provided 1-O-(2,3,4,6-
tetra-O-acetyl-a-D-mannopyranosyl)chlorogentisyl alcohols
(2a) in 25% yield as a major product as well as two minor
products, 4-O-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)-
chlorogentisyl alcohol (2b) and 7-O-(2,3,4,6-tetra-O-acetyl-
a-D-mannopyranosyl)chlorogentisyl alcohol (2c), in trace
amounts (Fig. 2). The a-configuration at C-1ꢃ of 2a was sub-
Experimental
General Optical rotation was determined on a Perkin Elmer model 341
polarimeter. UV/visible spectra were measured on a Hitachi U-2001 UV/Vis
spectrometer. IR spectra were recorded on a Bruker Fourier transform (FT)-
IR model IFS-88 spectrometer. 1H- (400 MHz) and 13C-NMR (100 MHz)
spectra were obtained on a JEOL JNM-ECP 400 NMR spectrometer, using
tetramethylsilane (TMS) or solvent peaks [DMSO-d6: 1H (d 2.50) and 13C (d
39.5)] as reference standard. LC-MS and MS spectra were obtained on API
2000 (Applied Bio System, U.S.A.) and IT-TOF (Shimadzu, Japan) spec-
trometer, respectively. HPLC was performed on a Young Lin ACME HPLC
system using
a
reversed-phase analytical column (Gemini C18,
4.6ꢄ250 mm, 5 mm) with UV detection. Incubations of microorganisms and
biotransformations were performed on an Incubator Shaker JS-FS-2500
(Johnsam Co., Inchon, Korea).
Fungal Isolation The fungal strain was isolated from the surface of edi-
ble brown alga Sargassum ringgoldium (Korean name: KeunIp Mojaban)
collected at Yokji Island of GyeongNam, Korea in 2009, and identified to be
C. synchronum (EMBL/AM943023.1) on the basis of morphology and 18S
stantiated by the small value of the coupling constant [d 5.49 ribosomal RNA (rRNA) analysis (SolGent Co., Ltd., Daejeon, Korea), iden-
tity of 98%. A voucher specimen is deposited at Pukyong National Univer-
(1H, br s, H-1ꢃ)] of the anomeric proton. Finally, the product
2a was subjected to alkaline hydrolysis (NaOMe) to afford 2.
sity with the code BAac049.
Biotransformation of 1 A two-stage fermentation protocol9) was used
The spectroscopic data for the synthetic compound were
for preparative scale formation of the metabolite of 1. The modified medium
contained soytone (0.1%), soluble starch (1.0%), mannose (0.1%), and sea-
1
identical to those of compound 2 in [a]D, ESI-MS, and H-
water (100%), and it was autoclaved at 121 °C for 15 min. Preparative incu-
bation was conducted in 1 l of sterile medium held in 3 l culture flask that
was incubated on a rotary shaker (130 rpm) at 29 °C for 1 week. A 10% in-
oculum derived from one week old stage I culture was used to initiate stage
II culture, which was incubated for 24 h more under the same condition be-
fore receiving 20 mg of 1 in 0.75 ml of N,N-dimethyl formamide (DMF),
and incubation was continued at 29 °C for two weeks in the same manner to
that described above. Substrate control consisted of sterile medium and sub-
strate incubated under the same conditions but without microorganism.
Also, culture control was composed of fermentation blanks in which the mi-
croorganism was grown under identical condition but without the addition of
substrate. After two weeks of incubation, each control was harvested and an-
alyzed by TLC. The culture was filtered through cheesecloth, and the filtrate
was extracted with EtOAc. The organic layer was dried over anhydrous
Na2SO4, filtered through sintered glass, and vacuum-concentrated to yield a
crude extract (95 mg).
NMR. Although many examples of microbial glucosidation
have been reported,17—19) microbial mannosidation is very
rare.3) Thus, to the best of our knowledge, compound 2 is the
second example of microbial mannosidation by the marine
isolate of the microorganism. Compounds 1 and 2 exhibited
significant radical scavenging activity against 1,1-diphenyl-
2-picrylhydrazyl radical (DPPH), with IC50 values of 1.0 and
4.7 mM, respectively. They were more active than the positive
control, L-ascorbic acid (IC50, 20.0 mM). We plan to inten-
sively examine compounds 1 and 2 for inhibitory activity
Isolation of the Metabolite (2) The crude extract (95 mg) was sub-
jected to silica gel flash column chromatography. Elution was performed
with n-hexane–EtOAc (stepwise, 0—100% EtOAc) to yield three fractions.
Fractions 2 and 3 were separated by medium-pressure liquid chromatogra-
phy (MPLC) (ODS) using a H2O–MeOH gradient elution to afford crude 1
and 2, respectively. These were further purified by HPLC (Gemini C18,
4.6ꢄ250 mm, 5 mm) utilizing a 30 min gradient program of 50 to 100%
MeOH in H2O to furnish the substrate, 1 (8.5 mg), and the metabolite (2)
(3.2 mg), respectively.
Fig. 1. Microbial Mannosidation of Chlorogentisyl Alcohol (1) and the
Structure of Metabolite, 1-O-(a-D-Mannopyranosyl)chlorogentisyl Alcohol
(2)
1-O-(a-D-Mannopyranosyl)chlorogentisyl alcohol (2): A colorless oil;
[a]D20 ꢁ3.70 (cꢂ0.3, MeOH); UV (MeOH) lmax (log e) 275 (3.3), 207 (0.6)
1
nm; IR (KBr) nmax 3400, 2924, 1627, 1241, 1018 cmꢁ1; H-NMR (CD3OD,
400 MHz) d: 7.03 (2H, s, H-3, 5), 4.64 (2H, s, H2-7), 5.37 (1H, d, Jꢂ1.5 Hz,
H-1ꢃ), 3.98 (1H, dd, Jꢂ3.2, 1.5 Hz, H-2ꢃ), 3.87 (1H, dd, Jꢂ9.1, 3.2Hz, H-
3ꢃ), 3.74—3.76 (3H, m, H-4ꢃ, 6ꢃ), 3.60 (1H, dt, Jꢂ9.5, 4.0 Hz, H-5ꢃ); 13C-
NMR (CD 3OD, 100 MHz) d: 150.9 (s, C-1), 132.0 (s, C-2), 117.6 (d, C-3),
146.7 (s, C-4), 116.3 (d, C-5), 121.6 (s, C-6), 60.9 (t, C-7), 100.9 (d, C-1ꢃ),
71.9 (d, C-2ꢃ), 72.2 (d, C-3ꢃ), 68.1 (d, C-4ꢃ), 75.3 (d, C-5ꢃ), 62.2 (t, C-6ꢃ);
low resolution (LR)-ESI-MS: m/z 335 [M (35Cl)ꢀH] and 337 [M*
(37Cl)ꢀH] (m/z 335 : 337ꢂca. 3 : 1); HR-ESI-MS: m/z 335.0538 [MꢀH]
(Calcd for C13H16O8Cl, 335.0539) (ꢀ0.30 ppm).
Acid Hydrolysis of 2, and Sugar Analysis A solution of 2 (0.5 mg) in
9% dry methanolic HCl (1.0 ml) was stirred at 80 °C for 1 h (N2 atmo-
sphere). The reaction mixture was neutralized with Ag2CO3 and filtrated.
The residue, obtained by removal of the solvent, was dissolved in pyridine
(0.5 ml) and treated with bis(trimethylsilyl)trifluoroacetamide (BSTFA)
(0.5 ml) at rt for 30 min. Solvent was removed by a nitrogen stream and the
residue dissolved in n-hexane was used for GC-MS analysis [(DB-1MS col-
umn, 60 mꢄ0.32 mm); N2 as a carrier gas at 0.7 ml/min; the program rate:
80—260 °C at 5 °C/min] showing peak at tR 16.60 min, which corresponded
to those of TMS/Me derivative of mannose (m/z 540 [M]ꢁ).
Fig. 2. Synthesis of 1-O-(a-D-Mannopyranosyl)chlorogentisyl Alcohol (2)
from Chlorogensyl Alcohol (1)

April 2011
501
Determination of the Stereochemistry of the Mannopyranose A so-
lution of 2 (1.0 mg) in 9% aq. HCl (1.0 ml) was stirred at 80 °C for 1 h. The
reaction mixture was treated as described above, and the residue thus ob-
tained was partitioned into H2O and CH2Cl2 to give the organic phase and
the aqueous phase. The aqueous portion was applied to a Diaion HP-20 col-
umn (Mitsubishi Kasei, Japan, 20 ml of resin, 10ꢄ250 mm). The column
was eluted with distilled water (100 ml) and MeOH (100 ml), successively.
The elute with MeOH was concentrated to afford mannose (0.3 mg). The ro-
tation recorded for mannose isolated in this study was [a]D ꢁ28.8 (cꢂ0.03,
H2O), which showed D-mannose in 2 [ref: [a]D20 ꢁ29.3].12)
tute. The second Brain Korea 21 graduate fellowship grant to students and a
post-doctor (CMK) is gratefully acknowledged (09B2519).
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Synthesis of 2 Molecular sieves 4A (60 mg) and 1-O-(2,3,4,6-tetra-O-
acetyl-D-mannopyranosyl)trichloroacetimide (38 mg, 0.08 mmol) were
added to a solution of 1 (15 mg, 0.08 mmol) in dry CH2Cl2 (7 ml) at rt, then
the mixture was treated with boron trifluoro-etherate (1% solution in dry
CH2Cl2, 0.5 ml) at ꢀ70 °C for 2 h. The reaction mixture was poured into ice
water, and the whole was extracted with CH2Cl2. The organic phase was
washed with aq. saturated NaHCO3 and brine, then dried over MgSO4. Re-
moval of the solvent under reduced pressure gave a product that was purified
by column chromatography (SiO2, n-hexane–EtOAcꢂ5 : 1) to afford not
only 1-O-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)chlorogentisyl alco-
hol (2a) (9.0 mg, 0.02 mmol, 25%) as the major product, but also the minor
products, 4-O-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)chlorogentisyl
alcohol (2b) and 7-O-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)chloro-
gentisyl alcohol (2c) in trace amounts. A solution of 2a (9 mg, 0.02 mmol) in
MeOH (2 ml) was treated with 28% NaOMe (1 drop) at rt for 4 h. The reac-
tion mixture was neutralized with Dowex [50Wꢄ8 (Hꢁ form)], and the resin
was removed by filtration. Removal of the solvent from the filtrate under re-
duced pressure gave a product (10 mg) that was purified by reversed-phase
ODS-A gel (YMC Co., Japan) flash column chromatography using MeOH,
followed by HPLC (ODS, MeOH–H2Oꢂ5 : 1) to furnish 2 (4.8 mg,
1
0.01 mmol, 50%) (Fig. 2). The spectroscopic data ([a]D, ESI-MS, and H-
NMR) for the synthetic compound were virtually identical to those of the
natural product (2).
Radical Scavenging Activity against DPPH7) Samples (1, 2, and posi-
tive control, L-ascorbic acid) to be tested were dissolved in MeOH and the
solution (160 ml) was dispensed into wells of a 96-well microtiter tray. Forty
microliters of the DPPH solution in MeOH (1.5ꢄ10ꢀ4 M) was added to each
well. The mixture was shaken and left to stand for 30 min, and the ab-
sorbance of the resulting solution was measured at 520 nm with microplate
reader (Packard Co., U.S.A., Spectra CountTM). The scavenging activity on
DPPH radical was expressed as IC50, which is the concentration of the tested
compound required to give a 50% decrease of the absorbance from that of
the blank solution [consisting of MeOH (160 ml) and DPPH solution
(40 ml)].
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Acknowledgment This research was supported by the National Re-
search Foundation of Korea Grant funded by the Korean Government
(MOEHRD, Basic Research Promotion Fund) (KRF-2008-314-F00048).
Mass spectral data were kindly provided by the Korea Basic Science Insti-
(2003).
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