New production of a monoterpene glycoside, 1-O-(α-D-mannopyranosyl) geraniol, by the marine-derived fungus Thielavia hyalocarpa

Full text of DOI:10.1002/bkcs.10451

Note
DOI: 10.1002/bkcs.10451
BULLETIN OF THE
K. Yun et al.
KOREAN CHEMICAL SOCIETY
NewProductionofaMonoterpeneGlycoside, 1-O-(α-D-Mannopyranosyl)
geraniol, by the Marine-derived Fungus Thielavia hyalocarpa
*
Keumja Yun, Chinni Mahesh Kondempudi, Alain Simplice Leutou, and Byeng Wha Son
Department of Chemistry, Pukyong National University, Busan 608-737, Korea. *E-mail: sonbw@pknu.ac.kr
Received April 7, 2015, Accepted May 27, 2015, Published online August 25, 2015
Keywords: Microbial transformation, 1-O-(α-D-Mannopyranosyl)geraniol, Marine-derived fungus,
Thielavia hyalocarpa
The application of microbial transformation to natural pro-
ducts has been shown to be a powerful tool for the generation
72.5 (CH), 74.5 (CH), and 100.2 (CH), indicative of the pres-
8,10
ence of an α-mannopyranoside.
1
,2
of new, active, and less toxic derivatives. We are currently
exploring the microbial transformation of bioactive natural
products by marine-derived microorganisms. We have identi-
fied marine-derived bacteria and fungi that regioselectively
oxidize bioactive natural products to new, more bioactive
The α configuration of the mannose moiety was further con-
firmed by the coupling constant of the anomeric proton at 4.77
(d, J
0
0
= 1.5 Hz), as well as the anomeric carbon signal at
H1 –H2
0
1
11
δ 100.2 (d, C-1 ) with a J
DEPT nondecoupling measurements.
value of 175 Hz, deduced from
C–H
3–9
compounds.
In this study, many growing cultures were
The absolute configuration of the mannose moiety was
found to be D (vide infra). Acid hydrolysis of 2 with 9%
aq. HCl yielded 1 and mannose (Supporting Information,
initially screened for their abilities to catalyze interesting
biotransformation reactions with geraniol (1) as substrate.
A culture of the mudflat-derived Thielavia hyalocarpa was
able to metabolize and transform compound 1 to a more polar
metabolite. Therefore, this strain was selected for preparative-
scale fermentation of 1.
Examination of the biotransformation-culture broth led to
the isolation of a metabolite (2) (Figure 1).
High-resolution electrospray ionization mass spectrometry
20
p. 8). The specific rotation of mannose (½αꢀ = + 28:8) indi-
D
20
12
cated a D-configuration (reference: ½αꢀ = + 29:3).
D
Based on these results, the structure of compound 2 is 1-O-
α-D-mannopyranosyl)geraniol (Figure 1).
In order to clarify the structure of 2, we synthesized 2 from
(
1
and 1-O-(2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl)tri-
chloroacetimide in the same manner as described for the syn8-
(
HR-ESI-MS) analysis of 2 suggested a quasi-molecular for-
thesis of 1-O-(α-D-mannopyranosyl)chlorogentisyl alcohol
+
mula of C H O Na ([M + Na] = 339.1718, Δ = 0 mDa)
16 28 6
(
[
Scheme S1 and Supporting Information p. 8). Values of
α] , ESI-MS, and H-NMR for the synthetic compound were
(Figure S4, Supporting Information), which was in accord-
1
D
ance with the structural information provided by the
identical to those of compound 2.
1
3
C-NMR spectrum (Figure S2, Supporting Information).
The IR spectrum of 2 exhibited strong absorptions at 3372
Although some examples of microbial glucosidation have
13–15
5,8
been reported,
microbial mannosidation is very rare.
−1
and1062 cm indicativeofhydroxylandglycosidicfunction-
Geraniol (1) is an important component of the essential oil
alities, respectively, suggesting that 2 was a glycoside of 1.
16
from the rhizome of Alpinia galangal, and is also a biosyn-
1
13
Detailed analyses of 1-D NMR ( H- and C-NMR, DEPT)
thetic precursor of many types of acyclic and cyclic monoter-
2
of 2 revealed the presence of two sp quaternary carbons,
17
penoids. Furthermore, geraniol (1) is an important flavor
2
3
3
two sp methines, five sp oxymethines, two sp oxymethy-
component of some grape juices and wines, for which flavor
development is important.
The production of useful flavor compounds using a marine-
derived fungus is a potentially important application of micro-
bial technology.
3
lenes, two sp methylenes, and three olefinic methyls
(Table 1).
COSY spectral data suggested the presence of partial struc-
tures consisting of two sequential methylene groups with
,1,2-trisubstituted vinyl group, 1-oxy-3,3-disubstituted
1
allylic group, and sequentially oxygenated five methines
and one methylene. Connectivities of those partial structures
and quaternary carbons were determined by analyses of
HMBC spectral data. The HMBC correlations of the olefinic
protons H-2 with C-10 and H-6 with C-8 and C-9 established
Experimental
General. The instruments used to obtain the physical data
were the same as those described in our previous paper.
9
the geraniol moiety, and further HMBC correlation of the oxy-
Isolation of the Marine-derived Fungus Thielavia hyalo-
carpa. The fungal strain, T. hyalocarpa, was isolated from
the mudflat collected at Suncheon Bay, Korea, and identified
based on 18S rRNA analyses (SolGent Co., Ltd., Daejeon,
0
methylene proton H -1 with C-1 aided the assignment of
2
1
3
the glycosidic linkage. The C-NMR spectrum showed
carbon resonances at δ 62.8 (CH ), 68.5 (CH), 72.2 (CH),
c
2
Correction added on 01 October 2015, after first online publication: ISSN (Print) has been corrected.
Bull. Korean Chem. Soc. 2015, Vol. 36, 2391–2393
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BULLETIN OF THE
ISSN (Print) 0253-2964 | (Online) 1229-5949
KOREAN CHEMICAL SOCIETY
Korea), with an identity of 99%. Avoucherspecimen is depos-
ited at Pukyong National University (code MSA018).
Biotransformation of 1. Organisms were cultivated in two
stages in the modified SWS medium composed of soytone
control was composed of fermentation blanks in which the
microorganism was grown under identical condition but with-
out the addition of substrate. After 2 weeks of incubation, each
control was harvested and analyzed by TLC. The culture was
filtered through cheesecloth, and the filtrate was extracted
with EtOAc. The organic layer was dried over anhydrous
(
(
0.1%), soluble starch (1.0%), mannose (0.1%), and seawater
100%). The media were sterilized in an autoclave at 121 C
ꢁ
for 15 min. Cultures were grown in 3 L culture flasks contain-
ing one-third of their volumes of culture medium. Incubations
were conducted at 29 C on a rotary shaker (130 rpm) for 1
week. Stage I cultures were inoculated from fresh
T. hyalocarpa slants and were grown as described. A 10%
Na SO , filtered through sintered glass, and vacuum-concen-
2 4
trated to yield a crude extract (78 mg).
ꢁ
Isolation of the metabolite (2). The extract (78 mg) was sub-
jected to silica gel flash column chromatography eluting with
n-hexane-EtOAc (stepwise, 0–100% EtOAc). From the 80%
EtOAc/n-hexane and 100% EtOAc fractions, semipure
1and2wereobtainedbyreversephaseC MPLCelutingwith
(v/v) inoculum derived from a 24-h-old, first-stage culture
was used to initiate the second-stage culture, which was
incubated as described. After 24 h, compound 1 dissolved in
N,N-dimethyl formamide (DMF) (30 mg/mL) was added to
second-stage cultures to a final concentration of 0.5 mg/mL.
The progress of microbial transformation reactions was mon-
itored by thin-layer chromatography (TLC). Substrate control
consisted of sterile medium and substrate incubated under the
same conditions but without microorganism. Also, culture
18
50% methanol/water, respectively. Final purification by
repeated C18 HPLC (Gemini C18, 4.6 × 250 mm, 5 μm) with
a 30 min gradient program of 50–100% MeOH in H O pro-
2
vided the substrate (1) (5.0 mg) and the metabolite (2) (11.5
mg), respectively.
1-O-(α-D-Mannopyranosyl)geraniol (2): Colorless oil;
2
D
0
½
αꢀ = + 45:6 (c 0.5, MeOH); UV (MeOH) λ
(log ε)
3372, 1667, 1448,
max
2
74 (3.4), 225 (0.8) nm; IR (neat) ν
379, 1124, 1062, 1029, 976 cm ; H-NMR (CD OD,
400 MHz) and
Table 1); LR-ESI-MS: m/z 339 [M + Na] ; HR-ESI-MS:
max
−1
1
1
3
OH
13
OH
O
7
C-NMR (CD OD, 100 MHz) (see
3
4
'
+
HO
HO
1'
+
5
m/z 339.1778 [M + Na] (calcd. for C H O , 339.1778)
16 28 6
O
1
(Δ = 0 mDa).
Thielavia hyalocarpa
3
HO
1
Acknowledgment. Thisresearch wassupportedbyBasicSci-
ence Research Program through the National Research Foun-
dation of Korea (NRF) funded by the Ministry of Education
(2014R1A1A2053912).
1
0
Figure 1. Microbial mannosidation of geraniol (1) to the metabolite
-O-(α-D-mannopyranosyl)geraniol (2).
1
a
b
Table 1. NMR spectroscopic data for geraniol (1) and 1-O-(α-D-mannopyranosyl)geraniol (2).
Geraniol (1)
1-O-(α-D-mannopyranosyl)geraniol (2)
(mult., J in Hz) (mult.)
4.05 (dd, 12.0, 7.5) 64.2 (t)
.18 (dd, 12.0, 6.5)
Carbon position
1
δ
H
(mult., J in Hz)
δ
C
(mult.)
δ
H
δ
C
4.13 (d, 7.0)
59.2 (t)
4
2
3
4
5
6
7
8
9
5.39 (d, 7.0)
123.5 (d)
139.2 (s)
39.6 (t)
5.31 (dd, 7.5, 6.5)
121.3 (d)
142.0 (s)
40.7 (t)
2.02 ( t, 7.0)
2.09 (dt, 8.0, 7.0)
5.09 (t, 7.0)
2.04 ( t-like, 6.5)
2.10 (dt-like, 7.0, 6.5)
5.09 (t, 7.0)
26.4 (t)
27.4 (t)
124.0 (d)
131.6 (s)
17.6 (q)
25.6 (q)
16.2 (q)
125.0 (d)
132.5 (s)
17.7 (q)
25.9 (q)
16.5 (q)
100.2 (d)
72.2 (d)
72.5 (d)
68.5 (d)
74.5 (d)
62.8 (t)
1.60 (s)
1.67 (s)
1.66 (s)
1.60 (s)
1.67 (s)
1.69 (s)
4.77 (d, 1.5)
3.75 (dd, 3.0, 1.5)
3.68 (dd, 10.0, 3.0)
3.60 (dd, 10.0, 9.0)
3.51 (ddd, 9.0, 5.5, 2.0)
3.71 (dd, 11.8, 5.5)
1
1
2
3
4
5
6
0
0
0
0
0
0
0
3
.82 (dd, 11.8, 2.0)
a
b
1
13
Recorded in DMSO-d
6
at 400 MHz ( H) and 100 MHz ( C).
1
13
3
Recorded in CD OD at 400 MHz ( H) and 100 MHz ( C).
Bull. Korean Chem. Soc. 2015, Vol. 36, 2391–2393
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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BULLETIN OF THE
Note
KOREAN CHEMICAL SOCIETY
1
13
Supporting Information. H-NMR, C-NMR, and LR- and
HR-ESI-MS spectra of 2, Scheme S1 for synthesis of 2 from 1,
and Experimental.
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0. J. B. Stothers, Carbon-13 NMR Spectroscopy, Academic Press,
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Bull. Korean Chem. Soc. 2015, Vol. 36, 2391–2393
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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