Stereochemistry of hydroxy-bearing benzolactones: Isolation and structural determination of chrysoarticulins A-C from a marine-derived fungus Chrysosporium articulatum

Article abstract of DOI:10.1016/j.tetlet.2013.04.006

Three new benzolactone metabolites, chrysoarticulins A-C (1-3), were isolated from the culture broth of Chrysosporium articulatum, a marine-derived fungus collected from an unidentified dictyoceratid sponge collected off the coast of Gagu-do, Korea. Structurally, these novel compounds were determined to be benzolactones possessing a branched side chain based on combined spectroscopic analyses. The absolute configurations of the asymmetric carbon centers were determined by extensive chemical and spectroscopic analyses that provided more insights into the stereochemistry of hydroxy-bearing benzolactones. The new compounds exhibited weak cytotoxicity against the K562 and A549 cell lines, and 3 was more active than the other compounds. Compound 3 was also moderately active against sortase A, a bacterial transpeptidase.

Full text of DOI:10.1016/j.tetlet.2013.04.006

Tetrahedron Letters 54 (2013) 3111–3115
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Stereochemistry of hydroxy-bearing benzolactones: isolation and structural
determination of chrysoarticulins A–C from a marine-derived fungus
Chrysosporium articulatum
Ju-eun Jeon a, Elin Julianti a,b, Hana Oh a, Wanki Park c, Dong-Chan Oh a, Ki-Bong Oh c, , Jongheon Shin a,
⇑
⇑
a Natural Products Research Institute, College of Pharmacy, Seoul National University, San 56-1, Sillim, Gwanak, Seoul 151-742, Republic of Korea
b School of Pharmacy, Bandung Institute of Technology, Jl. Ganesha 10, Bandung 40132, Indonesia
c Department of Agricultural Biotechnology, College of Agriculture and Life Science, Seoul National University, San 56-1, Sillim, Gwanak, Seoul 151-921, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Three new benzolactone metabolites, chrysoarticulins A–C (1–3), were isolated from the culture broth of
Chrysosporium articulatum, a marine-derived fungus collected from an unidentified dictyoceratid sponge
collected off the coast of Gagu-do, Korea. Structurally, these novel compounds were determined to be
benzolactones possessing a branched side chain based on combined spectroscopic analyses. The absolute
configurations of the asymmetric carbon centers were determined by extensive chemical and spectro-
scopic analyses that provided more insights into the stereochemistry of hydroxy-bearing benzolactones.
The new compounds exhibited weak cytotoxicity against the K562 and A549 cell lines, and 3 was more
active than the other compounds. Compound 3 was also moderately active against sortase A, a bacterial
transpeptidase.
Received 27 February 2013
Revised 28 March 2013
Accepted 2 April 2013
Available online 13 April 2013
Keywords:
Chrysoarticulin
Benzolactone
Marine fungus
Chrysosporium articulatum
Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
OH
O
OH
Introduction
O
1
3
3
1
15
R
2
7
2
15
O
4
5
4
5
Marine microorganisms, in particular actinomycete bacteria
and fungi, produce a wide variety of biologically active and struc-
turally unique metabolites.1–5 Hundreds of novel compounds are
isolated from these organisms annually, and they have become
important sources of novel natural products.6 In our search for bio-
active compounds from marine fungi, we isolated a strain of Chry-
sosporium articulatum7 from an unidentified sponge collected off
the coast of Korea whose organic extract was found to contain sec-
ondary metabolites in an LC–MS analysis. The present report de-
tails the isolation and structural characterization of
chrysoarticulins A–C (1–3), benzolactone metabolites possessing
branched side chains. During this process, comprehensive chemical
and spectroscopic approaches were used to determine the absolute
stereochemistry of these hydroxy-bearing benzolactones.
O
9
10
13
8
13
11
12
7
8
6
6
10
H
H
9
OH
14
14
11
HO
12
1
2
R = CH3
R = CH2OH
3
the Laboratory of Natural Product and Structure Determination, Col-
lege of Pharmacy, Seoul National University. The isolate was cul-
tured on slants of YPG agar media (5 g yeast extract, 5 g peptone,
10 g glucose, and 18 g agar in 1 L artificial seawater) at 25 °C for
10 days. Spore solutions prepared from the slants (107 spores/mL)
were inoculated into 250 mL Erlenmeyer flasks, 100 flasks in total,
each containing 100 mL of YPG media (5 g yeast extract, 5 g pep-
tone, and 10 g glucose in 1 L artificial seawater). The flask cultures
were incubated at 28 °C on a rotary shaker at 150 rpm for 3 weeks.
The mycelia and culture broth of C. articulatum were separated
by filtration, and the broth (10 L) was extracted with EtOAc
(10 L ꢀ 3). The solvent was evaporated to obtain an organic extract
(790 mg). The extract was fractionated by C18 reversed-phase vac-
uum flash chromatography using a sequential mixture of MeOH
and H2O as eluents (five fractions eluted with a H2O–MeOH gradi-
ent from 80:20 to 0:100) and finally MeOH–CH2Cl2 (50:50). The
Results and discussion
The fungal strain C. articulatum was isolated from an unidenti-
fied marine sponge collected from Gagu-do, Korea, in October
2008. The isolate (strain number F085) was deposited at
⇑
Corresponding authors. Tel.: +82 2 880 2484; fax: +82 2 762 8322 (J.S.); tel.: +82
2 880 4646; fax: +82 2 873 3112 (K.-B.O).
E-mail addresses: ohkibong@snu.ac.kr (K.-B. Oh), shinj@snu.ac.kr (J. Shin).
0040-4039/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.04.006

3112
J. Jeon et al. / Tetrahedron Letters 54 (2013) 3111–3115
fraction (130 mg) eluted with H2O–MeOH (20:80) was separated
by reversed-phase HPLC (YMC ODS-A column, 10 ꢀ 250 mm;
H2O–MeOH, 26:74, 2 mL/min, detected at 254 nm) to afford 1
(15.1 mg) and 3 (6.7 mg). The fraction (70 mg) eluted with H2O–
MeOH (40:60) was separated by reversed-phase HPLC (H2O–
MeOH, gradient (50:50 to 0:100 in 40 min, then 0:100 in 5 min),
detected at 254 nm) to yield compound 2 (3.6 mg).
OH
O
O
OH
Figure 1. Selected gHMBC (arrows) and COSY (bold lines) correlations of compound
1.
The molecular formula of chrysoarticulin A (1) was deduced to
be C15H20O4 by HRFABMS analysis.8 The 13C NMR spectra of this
compound showed signals corresponding to
a carbonyl (dC
The molecular formula of a related compound, chrysoarticulin B
(2), was deduced to be C15H20O5 by HRFABMS analysis.9 The NMR
data for this compound were very similar to those for 1, with the
replacement of a methyl with an oxymethylene (dH 4.66, 4.64; dC
59.6) being the most noticeable difference. These spectroscopic dif-
ferences were readily identified as the result of the oxidation of the
C-15 methyl to a hydroxymethyl group by combined 2D NMR anal-
yses, including the HMBC correlations of the protons and carbons
at the oxymethylene and those at the neighboring C-3 and C-5.
Thus, chrysoarticulin B (2) was determined to be a benzylic oxidant
of 1.
The molecular formula of chrysoarticulin C (3) was determined
to be C15H20O4, identical to 1, by HRFABMS analysis.10 The NMR
spectra of this compound were also similar to those of 1. However,
detailed examination of the 1H and 13C NMR data revealed that the
signals for the C-8 and C-9 oxymethines were shifted noticeably (1:
dC 90.3 and 63.4, dH 4.47 and 4.93, 3: dC 85.8 and 78.1, dH 5.58 and
3.72) (Table 1), suggesting a change in the functional groups at
these positions. A combination of 1H COSY and gHSQC analyses
of 3 showed the same aliphatic proton spin systems as found in
1, adequately assigning the signals of the carbons and protons at
these positions (C-8: dC 85.8, dH 5.58, C-9: dC 78.1, dH 3.72). The sig-
nificant shifts of the protons relative to those of 1 suggested a
change of the lactone from a six-membered ring to the correspond-
ing five-membered ring. This difference was confirmed by the key
carbon-proton correlations at H-8/C-1, C-2, C-7 and H-9/C-7, C-8 in
the gHMBC data. Thus, chrysoarticulin C (3) was determined to be
a new benzolactone metabolite.
170.7), six aromatics or olefinics (dC 159.6–108.1), two oxymeth-
ines (dC 90.3 and 63.4), and six upfield (dC 38.3–10.9) carbons (Ta-
ble 1). The downfield shifts of the six carbon signals in the region of
dC 159.6–108.1 were indicative of a substituted benzene ring based
on the six degrees of unsaturation inherent in the molecular for-
mula. Thus, compound 1 must possess an additional ring structure.
Given this information, the structure of 1 was determined by a
combination of 2D NMR experiments. A partial structure accom-
modating the oxymethines and upfield NMR signals was readily
defined to be a 1,2-dioxy-3-methyl-pentyl chain by combined 1H
COSY and gHMBC analyses (Fig. 1). In addition, long-range pro-
ton–proton couplings of the aromatic proton at dH 7.32 with the
methyl protons at dH 2.32 and 2.22 in the 1H COSY data indicated
that the benzylic methyls were meta to each other, and this
arrangement was confirmed by the gHMBC data (Fig. 1). An addi-
tional long-range correlation of the methyl proton at dH 2.32 placed
the quaternary aromatic carbon at dC 135.1 at the neighboring C-7
position, which further coupled with the oxymethine protons at dH
4.93 and 4.47, thus connecting the pentyl chain and the aromatic
ring at this position. A long-range coupling between the other
methyl proton at dH 2.22 and the downfield carbon at dC 159.6
placed a hydroxyl group at C-3, ortho to this benzylic methyl group.
The aromatic carbon at dC 108.1 was determined to be located at C-
2 based on its upfield carbon chemical shift and its long-range cou-
pling with the H-8 benzylic proton at dH 4.93. Finally, the remain-
ing ester carbon at dC 170.7 located at C-1, the only available
position, was determined to form a six-membered lactone by its
long-range coupling with the H-9 oxymethine at dH 4.47 in the
gHMBC data. Thus, chrysoarticulin A (1) was determined to be a
new isocoumarin metabolite possessing a sec-butyl side chain.
The absolute configurations of 1 at the asymmetric centers at C-
8, C-9, and C-10 were assigned based on the results of combined
spectroscopic and chemical experiments described below together
with the results of congener 3.
Both compounds 1 and 3 possessed the same asymmetric cen-
ters at C-8, C-9, and C-10. A previously published study showed
that the configurations of similar lactone compounds can be as-
signed using data from diverse spectroscopic and crystallographic
methods.11–17 Among these spectroscopic methods, one widely
used approach is the analysis of the magnitude of the proton cou-
Table 1
13C NMR (ppm, mult) and 1H NMR (d, mult (J in Hz)) assignments for compounds 1–3a
Position
1
2
3
dC
dH
dC
dH
dC
dH
1
2
3
4
5
6
7
8
170.7, C
108.1, C
159.6, C
128.1, C
140.8, CH
127.5, C
135.1, C
63.4, CH
90.3, CH
38.3, CH
26.6, CH2
170.4, C
109.1, C
158.4, C
131.1, C
138.0, CH
127.6, C
136.4, C
63.3, CH
90.2, CH
38.2, CH
26.4, CH2
173.7, C
112.2, C
154.3, C
125.3, C
141.0, CH
126.9, C
145.8, C
85.8, CH
78.1, CH
38.2, CH
25.1, CH2
7.32, s
7.55, s
7.24, s
4.93, d (1.2)
4.95, d (1.3)
4.47, dd (9.6, 1.3)
1.42, m
1.64, m
1.29, m
0.89, t (7.1)
0.87, d (6.1)
2.36, s
4.66, d (17.7)
4.64, d (17.7)
5.58, d (5.0)
9
10
11
4.47, dd (9.6, 1.2)
1.43, dddq (9.6, 8.3, 3.5, 6.8)
1.65, ddq (13.6, 3.5, 7.5)
1.29, ddq (13.6, 8.3, 7.5)
0.89, t (7.5)
0.88, d (6.8)
2.32, s
2.22, s
3.72, dd (5.0, 5.0)
1.57, dddq (9.3, 5.0, 3.2, 6.9)
1.66, ddq (15.1, 3.2, 7.5)
1.22, ddq (15.1, 9.3, 7.5)
0.87, t (7.5)
0.80, d (6.9)
2.31, s
2.22, s
12
13
14
15
10.9, CH3
15.8, CH3
17.2, CH3
15.7, CH3
10.7, CH3
15.7, CH3
17.2, CH3
59.6, CH2
12.0, CH3
16.8, CH3
18.6, CH3
15.0, CH3
a
Data were obtained in MeOH-d4 solutions at 150 and 600 MHz (1), and 125 and 500 MHz (2 and 3).

J. Jeon et al. / Tetrahedron Letters 54 (2013) 3111–3115
3113
pling constant between oxymethines as a key indicator of their ori-
entation (typically, cis 6 2 Hz, trans P 4 Hz).11–15 The coupling
constant is usually combined with NOE data to assign the relative
configurations of the lactone moiety. In our data for compound 1,
the small coupling constant (J8,9 = 1.2 Hz) was indicative of the
cis orientation between H-8 and H-9. However, our initial NOESY
experiment showed conspicuous cross-peaks at H-8/H-13 that
should not be observed for an ordinary chair-type conformation.
Furthermore, in the ORTEP drawing of a crystal structure in the lit-
erature, the lactone ring appeared to have a half-chair conforma-
tion.14 These findings prompted us to thoroughly investigate the
stereochemistry of these compounds.
OH
O
OR
O
-0.047
-0.031
O
+0.030
+0.014
O
+0.144
-0.005
0
-0.093
+0.219
+0.246
-0.084
+0.001
+0.013
-0.023
-0.099
OR +0.007
-0.112
RO
+0.166
+0.074
+0.238
1S : R = (
S
)-MTPA
)-MTPA
3S : R = ( )-MTPA
3R
: R = (R)-MTPA
S
1R
: R = (
R
Figure 3.
3R).
DdS-R values for MTPA esters of compounds 1 (1S and 1R) and 3 (3S and
First, the relative configurations of 1 were investigated by pro-
ton coupling constant analysis. The large coupling constant
(J9,10 = 9.6 Hz) revealed an anti-orientation between these protons,
as suggested by Murata and coworkers.18 In contrast, the small
coupling constant (J8,9 = 1.2 Hz) suggested a gauche conformation
between H-8 and H-9. A molecular modeling study indicated that
this conformation could be interpreted as either the cis- or trans-
orientation depending on the conformation of the lactone ring.
That is, these protons could be cis-oriented in the half chair-type
conformation or trans-orientation in a boat-type conformation.
The NOESY data showed a conspicuous cross-peak at H-8/H-9, sup-
porting this interpretation (Fig. 2). Additional cross-peaks at H-8/
H-13 and H-9/H-13 also supported the anti-conformation between
H-9 and H-10. The absolute configuration at the C-8 oxymethine
was determined to be S by the Mosher method (Fig. 3).19,20 There-
fore, the absolute configuration was either 8S, 9R, and 10R or 8S, 9S,
and 10S depending on the conformation of the lactone ring, but
distinguishing these two conformations required additional data.
In contrast, the NOESY data of 3 showed cross-peaks at H-8/H-9
and H-9/H-13, as observed for 1. Although the mid-range coupling
constants (J8,9 = J9,10 = 5.0 Hz) were attributed to the five-mem-
bered lactone ring, these NOESY data were in good agreement with
the biosynthetic relationships between these compounds, in which
3 is formed from 1 by the attack of the lactone carbonyl by the C-8
hydroxyl group, resulting in ring opening. Therefore, the absolute
configurations of these two compounds must be identical. Because
the results of the Mosher analysis indicated that C-9 of 3 has the R
configuration (Fig. 3), the absolute configuration was assigned to
be 8S, 9R, and 10R for both 1 and 3.
files of these compounds differ significantly, and this difference
could be attributed to differences in either the sizes of the lactone
rings or the configurations of the asymmetric centers (Fig. 4). Sub-
sequently, both compounds were chemically converted by basic
hydrolysis (Scheme 1).21 The formation of ring-opened carboxylic
acids, as, was detected by HPLC-ESI-MS analysis (YMC ODS-A col-
umn, 5
lm, 4.6 ꢀ 250 mm, H2O–MeOH gradient (70:30 to 0:100
in 30 min, v/v), 0.7 ml/min flow rate, UV detector, 254 nm,
13.8 min, m/z 281 [M-H]ꢁ). However, these unstable intermediates
readily re-cyclized to form the five-membered lactone compound
3, as a result. The 1H NMR data, optical rotations, and CD profiles
of these lactone compounds were virtually identical to those of 3.
Moreover, MTPA esterification product of hydrolysate of 1 at C-9
yielded the same product as that derived from 3. Thus, the absolute
configurations of these compounds were unambiguously assigned.
Our results clearly demonstrate that the spectroscopic features
of hydroxy-bearing benzolactones are significantly influenced by
the conformation of the lactone moiety. Accordingly, the configu-
rations cannot be determined by simple proton–proton coupling
constants and NOE analysis alone. A more comprehensive ap-
proach combining diverse chemical and spectroscopic methods is
required to determine the stereochemistry of hydroxy-bearing
benzolactones.
Isocoumarins and structurally related benzolactones are widely
distributed among fungi. However, to the best of our knowledge,
the carbon framework of chrysoarticulins is unique. Compounds
structurally similar to chrysoarticulins may include melleins,11–
13,15
The stereochemical relationship between 1 and 3 was further
studied by both spectroscopic and chemical methods. The CD pro-
pestaphthalides,16 sclerolide,22 and sclerotinins23 as well as
the synthetic and biosynthetic intermediates of ajudazols,14
psymberin (irciniastatin A),24 and ascochitine.25
However, chrysoarticulins differ structurally from these com-
pounds because chrysoarticulins possess the combination of a 2-
hydroxy-3,5-dimethyl benzolactone and a 3-methyl-pentyl chain.
In our measurement of bioactivities, these compounds were
weakly active against the K562 and A549 cancer cell lines.
O
HO
HO
O
H
H3C
H
H9
8
H
10
11
CH3
H
14
CH3
CH3
13
1
O
HO
H3C
O
H
H
H
OH
8
11
9
10
CH3
CH3
14
H
H
H3C
13
3
Figure 4. Experimental CD spectra of compounds 1 (solid line), hydrolysate of 1
(dotted line), and 3 (bold line).
Figure 2. Selected NOESY correlations of compounds 1 and 3.

3114
J. Jeon et al. / Tetrahedron Letters 54 (2013) 3111–3115
OH
OH
O
OH
O
CO2H
3
1
15
2
7
a
OH
O
4
5
O
9
10
13
11
12
8
6
H
H
OH
OH
14
HO
a
1
3
OH
OH
OH
O
O
CO2H
OH
3
15
2
1
b
4
5
O
O
13
8
7
6
9
H
H
10
OH
14
11
HO
HO
12
a
3
3
Scheme 1. Reagents and conditions: (a) 1 M aqueous KOH, 0 °C, 30 min; (b) 1 M aqueous KOH, 0 °C, 22 h
26–28
Table 2
Results of bioactivity tests
10.1016/j.tetlet.2013.04.006. References
plementary data.
are cited in the Sup-
Compound
LC50
(l
M)
IC50
Sortase A
(lM)
K562
A549
ICL
References and notes
1
2
3
164.0
63.0
25.4
4.8
147.3
63.2
34.5
2.8
>300
>300
95.1
166.7
>300
236.4
1. Fenical, W. Chem. Rev. 1993, 93, 1673–1683.
2. Bugni, T. S.; Ireland, C. M. Nat. Prod. Rep. 2004, 21, 143–163.
3. Fenical, W.; Jensen, P. R. Nat. Chem. Biol. 2006, 2, 666–673.
4. Cragg, G. M.; Grothaus, P. G.; Newman, D. J. Chem. Rev. 2009, 109, 3012–3043.
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Doxorubicin
p-HMBa
3-NPb
104.4
12.6
a
para-hydroxymercuribenzoic acid.
b
7. The fungal strain (F085) was identified using standard molecular biological
protocols including DNA amplification and sequencing of the ITS region.
Genomic DNA extraction was performed using Intron’s i-genomic BYF DNA
Extraction Mini Kit according to the manufacturer’s protocol. The nucleotide
sequence of F085 has been deposited in the GenBank database under the
accession number JF901807. The 18S rDNA sequence of this strain showed
100% identity with Chrysosporium articulatum (GenBank accession number
AJ007841).
3-Nitropropionic acid.
Although compound 3 was more active than the six-membered
congeners 1 and 2, its cytotoxicity was far weaker than that of
doxorubicin. Compound 3 moderately inhibited sortase A, a key
enzyme involved in the adhesion and invasion of Gram-positive
bacteria. The inhibition of isocitrate lyase (ICL), another key en-
zyme in microbial biosynthesis, was also very weak Table 2). These
8. Chrysoarticulin A (1): brown, amorphous solid, ½a D25
ꢂ
+56 (c 0.40, MeOH); UV
(MeOH) kmax (log e) 212 (4.16), 250 (3.48), 328 (3.42) nm; IR (ZnSe) mmax 3349
(br), 2925, 1745, 1464 cmꢁ1 1H and 13C NMR data, see Table 1; HRFABMS m/z
;
265.1442 [M+H]+ (calcd for C15H21O4, 265.1440).
9. Chrysoarticulin B (2): brown, amorphous solid, ½a D25
ꢂ
+43 (c 0.25, MeOH); UV
(MeOH) kmax (log e) 212 (4.38), 250 (3.62), 328 (3.49) nm; IR (ZnSe) mmax 3344
compounds were also inactive (MIC > 100
pathogenic bacteria and fungi.
lg/mL) against diverse
(br), 2925, 1730, 1455 cmꢁ1 1H and 13C NMR data, see Table 1; HRFABMS m/z
;
281.1386 [M+H]+ (calcd for C15H21O5, 281.1389).
10. Chrysoarticulin C (3): brown, amorphous solid, ½a D25
ꢂ
+28 (c 0.25, MeOH); UV
In summary, we isolated three new benzolactone metabolites,
chrysoarticulins A–C (1-3), from the marine-derived fungus C.
articulatum. Their structures and stereochemistry were determined
using diverse spectroscopic and chemical approaches. Compounds
1-3 possess a unique branched side chain and have moderate or no
bioactivity.
(MeOH) kmax (log e) 210 (4.46), 276 (3.42), 312 (3.53) nm; IR (ZnSe) mmax 3423
(br), 2929, 1730, 1461 cmꢁ1
;
1H and 13C NMR data, see Table 1; HRFABMS m/z
265.1443 [M+H]+ (calcd for C15H21O4, 265.1440).
11. Aldridge, D. C.; Galt, S.; Giles, D.; Turner, W. B. J. Chem. Soc. 1971, 1623–1627.
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Aust, H.-J.; Draeger, S.; Schulz, B.; Antus, S. Phytochemistry 1997, 45, 313–320.
14. Hobson, S. J.; Parkin, A.; Marquez, R. Org. Lett. 2008, 10, 2813–2816.
15. Okuno, T.; Oikawa, S.; Goto, T.; Sawai, K.; Shirahama, H.; Matsumoto, T. Agric.
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Acknowledgments
We are grateful to the Basic Science Research Institute in Daegu,
Korea, for providing the mass data and the National Center for In-
ter-University Research Facilities, Seoul National University, for
providing the NMR data. This study was supported by grants from
the National Research Foundation of Korea (NRF) funded by the
Korean government (MEST, Ministry of Education, Science and
Technology) (M1A5A1-2010-0020429 and 2009-0083533).
16. Ding, G.; Liu, S.; Guo, L.; Zhou, Y.; Che, Y. J. Nat. Prod. 2008, 71, 615–618.
17. Tayone, W. C.; Honma, M.; Kanamaru, S.; Noguchi, S.; Tanaka, K.; Nehira, T.;
Hashimoto, M. J. Nat. Prod. 2011, 74, 425–429.
18. Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.; Tachibana, K. J. Org.
Chem. 1999, 64, 866–876.
19. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113,
4092–4096.
20. The esterification of compounds 1, hydrolysate of 1, and 3 was performed as
follows. A total of 30
containing 1.6, 0.5, and 1.2 mg of the alcoholic compound in 300
l
L of (ꢁ)-(R)-MTPA chloride was added to a solution
lL of dry
pyridine. The mixture was allowed to stand under N2 at room temperature for
2 h. After the consumption of the starting material was confirmed by thin-layer
chromatography, 1 mL of H2O–MeOH (10:90) was added. The solvent was
removed under vacuum, and the residue was separated by reversed-phase
HPLC (YMC-ODS column, 4.6 ꢀ 250 mm; H2O–MeOH, 15:85) to give 1.0, 0.4,
and 1.1 mg of (S)-MTPA esters (1S, hydrolysate of 1S, and 3S). The
corresponding (R)-MTPA esters (1R and 3R) were also obtained from the
same esterification reactions with (+)-(S)-MTPA chloride. 1H NMR (MeOH-d4,
Supplementary data
Supplementary data (1H and 13C NMR spectra of compounds 1–
3, and specific information of general experimental procedure, bio-
logical assays, and hydrolysates’ features) associated with this arti-
cle can be found, in the online version, at http://dx.doi.org/

J. Jeon et al. / Tetrahedron Letters 54 (2013) 3111–3115
3115
400 MHz) of 1S: dH 7.412ꢁ7.322 (5H, m, H-MTPA-Ar), 7.391 (1H, s, H-5), 6.382
(1H, d, J = 1.8 Hz, H-8), 4.642 (1H, dd, J = 9.7, 1.8 Hz, H-9), 3.530 (3H, s, H-
MTPA-OMe), 2.221 (3H, s, H-14), 2.219 (3H, s, H-15), 1.708 (1H, m, H-11a),
1.500 (1H, m, H-10), 1.318 (1H, m, H-11b), 0.978 (3H, d, J = 6.7 Hz, H-13), 0.891
(3H, t, J = 7.4 Hz, H-12); LR-FABMS m/z 481.3, [M+H]+ (calcd for C25H28F3O6,
481.2). 1H NMR (MeOH-d4, 400 MHz) of 1R: dH 7.413ꢁ7.347 (5H, m, H-MTPA-
Ar), 7.396 (1H, s, H-5), 6.466 (1H, d, J = 1.4 Hz, H-8), 4.498 (1H, dd, J = 9.6,
1.4 Hz, H-9), 3.389 (3H, s, H-MTPA-OMe), 2.320 (3H, s, H-14), 2.250 (3H, s, H-
15), 1.678 (1H, m, H-11a), 1.499 (1H, m, H-10), 1.304 (1H, m, H-11b), 0.965
(3H, d, J = 6.7 Hz, H-13), 0.884 (3H, t, J = 7.5 Hz, H-12); LR-FABMS m/z 481.3,
[M+H]+ (calcd for C25H28F3O6, 481.2). 1H NMR (MeOH-d4, 400 MHz) of 3S: dH
7.766ꢁ7.382 (10H, m, H-MTPA-Ar), 7.510 (1H, s, H-5), 5.882 (1H, d, J = 2.7 Hz,
H-8), 5.494 (1H, dd, J = 5.7, 2.7 Hz, H-9), 3.548 (3H, s, H-14), 3.339 (6H, s, H-
MTPA-OMe), 2.484 (3H, s, H-15), 1.663 (1H, m, H-11a), 1.610 (1H, m, H-10),
1.145 (1H, m, H-11b), 0.822 (3H, t, J = 7.5 Hz, H-12), 0.786 (3H, d, J = 6.9 Hz, H-
13); LR-ESIMS m/z 719.0, [M+Na]+ (calcd for C35H34F6O8Na, 719.2). 1H NMR
(MeOH-d4, 400 MHz) of 3R: dH 7.751–7.435 (10H, m, H-MTPA-Ar), 7.510 (1H,
s, H-5), 5.975 (1H, br s, H-8), 5.517 (1H, dd, J = 6.1, 2.1 Hz, H-9), 3.660 (3H, s, H-
14), 3.339 (6H, s, H-MTPA-OMe), 2.532 (3H, s, H-15), 1.588 (1H, m, H-11a),
1.364 (1H, m, H-10), 0.907 (1H, m, H-11b), 0.656 (3H, t, J = 7.5 Hz, H-12), 0.567
(3H, d, J = 6.9 Hz, H-13); LR-ESIMS m/z 719.0, [M+Na]+ (calcd for C35H34F6O8Na,
719.2). 1H NMR (MeOH-d4, 400 MHz) data of hydrolysate of 1S were
superimposed with 3S.
21. The hydrolysis of compounds 1 and 3 was performed as follows. An aliquot of 1
(0.8 mg) and 1 M aqueous KOH (0.5 mL) was stirred at 0 °C for 30 min. Then,
the reaction mixture was neutralized by adding 1 M aqueous HCl (0.5 mL).
After cooling, the solution was concentrated under a stream of N2, and then the
residue was purified by reversed-phase HPLC (YMC-ODS column,
4.6 ꢀ 250 mm; H2O–MeOH, 30:70) to obtain 3. A solution of 3 (0.7 mg) was
prepared in a similar manner (reaction time, 22 h). Detailed spectroscopic data
of corresponding hydrolysates are available in Supplementary data (S8).
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