Asymmetric Total Syntheses and Structure Revisions of Eurotiumide A and Eurotiumide B, and Their Evaluation as Natural Fluorescent Probes

Article abstract of DOI:10.1002/ejoc.201800535

Asymmetric total syntheses and structure revisions of dihydroisocoumarin-type natural products, eurotiumide A and eurotiumide B have been described. The key features of these total syntheses are the asymmetric Shi epoxidation, regio- and stereoselective e

Full text of DOI:10.1002/ejoc.201800535

A Journal of
Accepted Article
Title: Asymmetric Total Syntheses and Structure Revisions of
Eurotiumide A and Eurotiumide B, and Evaluation of their
Fluorescent Properties as Natural Probes
Authors: Atsushi Nakayama, Hideo Sato, Sangita Karanjit, Naoki
Hayashi, Masataka Oda, and Kosuke Namba
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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content of this Accepted Article.
To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201800535
Link to VoR: http://dx.doi.org/10.1002/ejoc.201800535
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10.1002/ejoc.201800535
European Journal of Organic Chemistry
COMMUNICATION
Asymmetric Total Syntheses and Structure Revisions of
Eurotiumide A and Eurotiumide B, and Evaluation of their
Fluorescent Properties as Natural Probes
Atsushi Nakayama*[a], Hideo Sato[a], Sangita Karanjit, Naoki Hayashi[b], Masataka Oda[b], and Kosuke
Namba*[a]
Abstract: Asymmetric total syntheses and structure revisions of
dihydroisocoumarin-type natural products, eurotiumide and
gastrointestinal illness, respectively. Eurotiumide B (2), which was
assigned cis configuration at H3/H4 positions by the same way as
1, exhibited potent antifouling activity toward the larval settlement
of the barnacle Balanus amphitrite (EC50 = 0.7–1.4 µg/mL) and
also displayed a high therapeutic ratio (LC50/EC50 = >68.5). These
results suggest that 1 and 2 might be good candidates for a potent
antimicrobial drug and an environment-friendly antifouling agent.
To date, there is no report on the total syntheses of 1 and 2, and
their mode of actions are still unknown. Herein we report the first
asymmetric total syntheses of the proposed structures of 1 and 2,
and clarified that their relative and absolute configurations on the
basis of the X-ray crystallographic analysis, and their revised
structures as 1’ and 2’. Moreover, we found that 1’ and 2’
displayed the highly fluorescence in several solvents and
demonstrated that it might be useful for studying the mode of
action of 1’ toward Bacillus cereus.
A
eurotiumide B have been described. The key features of these total
syntheses are the asymmetric Shi epoxidation, regio and stereo-
selective epoxide opening, C1 insertion/lactonization cascade
reaction for constructing 4-methoxyisochroman-1-one skeleton. We
confirmed the structures and configurations of eurotiumide A and B on
the basis of X-ray crystallographic analysis of the key intermediate,
and revealed that eurotiumide A and
B have cis and trans
configurations at H3/H4 positions, which are the opposite relationship
of the stereochemistry from the previous report, respectively. The
absolute configurations of them are also determined. These natural
products exhibited highly fluorescence in several solvents with large
Stokes shifts involving the excited-state intramolecular proton transfer
mechanism, which is supported by time-dependent density functional
theory. Eurotiumide A also emitted fluorescence in Bacillus cereus.
Marine-derived fungi are an important source of diverse
secondary metabolites possessing both unique structures and
biological activities, such as antitumor,[1] antibacterial,[2a,b]
antifouling,[3a-c] and other activities. These compounds are
considered as important drug candidates and have attracted
much attention in a variety of research fields. In 2014, Wang and
co-workers reported the isolation of dihydroisocoumarin
analogues as racemates, named eurotiumides, from a gorgonian-
derived fungus, Eurotium sp. XS-200900E6 (Figure 1).[4] Among
them, eurotiumide A (1), which was assigned trans configuration
at H3/H4 positions on the basis of coupling constants between H3
and H4, displayed potent antibacterial activities especially toward
Vibrio anguillarum and Vibrio parahaemolyticus (MIC = 0.39–0.78
µM), which are pathogens that cause vibriosis and
[a]
Dr. A. Nakayama, H. Sato, Dr. S. Karanjit, Prof. Dr. K. Namba
Graduate School of Pharmaceutical Sciences
Tokushima University
1-78 Shomachi, Tokushima
Tokushima 770-8505 (Japan)
E-mail: anakaya@tokushima-u.ac.jp
E-mail: namba@tokushima-u.ac.jp
http://www.tokushima-u.ac.jp/ph/faculty/labo/bot/
Dr. N. Hayashi, Prof. Dr. M. Oda
[b]
Department of Microbiology and Infection Control Sciences
Kyoto Pharmaceutical University
Figure 1. Structures of eurotiumide A and B.
Misasaginakauchi-cho, Yamashina-ku
Kyoto 607-8414 (Japan)
Supporting information for this article is given via a link at the end of
the document.
Our synthetic plan toward proposed structure of (–)-eurotiumide A
(1) and (+)-eurotiumide B (2), whose stereochemistry at C3-C4
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10.1002/ejoc.201800535
European Journal of Organic Chemistry
COMMUNICATION
positions as (3R,4S) and (3S,4S), is outlined in Scheme 1. The
difference between 1 and 2 is the stereochemistry at the C3
position. On the basis of this point, we considered that both 1 and
2 would be obtained from common intermediate 3, from which 2
would be readily synthesized by introducing the prenyl side chain
to 3. The late stage introduction of the side chain by Pd-catalyzed
cross coupling would make the structure-activity relationship
(SAR) studies easier. On the other hand, 1 would be accessible
by a similar cross coupling reaction from 4, which would be
obtained from 3 through inversion at the C3 position by sequential
hydrolysis and intramolecular Mitsunobu reactions. We
envisioned that the cis 4-methoxyisochroman-1-one skeleton,
which is the key framework of 2, could be constructed from
alcohol derivative 5 via Pd-catalyzed C1 insertion/lactonization
cascade reaction. Contiguous cis 3-hydroxy/4-methoxy groups
would be obtained from chiral epoxide 6 through regio and
stereoselective epoxide opening with MeOH, and 6 is expected to
be obtained from alkene 7 by the asymmetric Shi epoxidation.
Finally, 7 would be available by the Julia-Kocienski olefination
between aldehyde 8 and 1-phenyl-1-H-tetrazol-5-yl (PT) sulfone
9.
bromination
of
commercially
available 2,5-
dihydroxybenzaldehyde (10) and MOM protection of the two
phenolic hydroxyl groups (11) (Scheme 2(a)). PT-sulfone 9 was
obtained through the Mitsunobu reaction of 1-phenyl-1H-tetrazol-
5-yl thiol (PT-SH) with nhexanol (13) followed by oxidation of the
sulfide to sulfone (Scheme 2(b)). The Julia-Kocienski olefination
between aldehyde 12 and PT-sulfone 9 proceeded smoothly to
afford trans olefin 14 in 87% yield. Next, we examined the
asymmetric epoxidation of 14 by the application of Shi’s method.
[6] First, the epoxidation of trans alkene 14 with a catalytic amount
of chiral ketone 15 (30 mol%) was carried out under the general
conditions of the asymmetric Shi epoxidation. However, the
desired chiral epoxide 16 was obtained in only 37% yield. After
several optimizations, we found that the use of 1.0 equivalent of
15 increased the isolated yield to 76% and the enantiomeric
excess, which was determined by chiral HPLC analysis, was 86%.
Then, we conducted the regio and stereoselective epoxide
opening reaction with MeOH. Thus, treatment of epoxide 16 with
BF3-OEt2 in DCM with an excess amount of MeOH (10 equiv) at
–40 °C afforded desired product 17 in 94% yield as a single
isomer. Interestingly, we found that 17 preferred to form a racemic
crystal and the most of minor enantiomer of 17 was removed by
simple filtration after recrystalization.[7] The enantiomeric excess
of the resultant filtrate increased up to 94% ee. Moreover, its X-
ray
crystallographic
analysis
confirmed
the
relative
stereochemistry of 17.[8] We propose this high stereo selectivity
as illustrated in Scheme 2. Ring opening, which is assisted by the
lone pair of the oxygen atom at the ortho-position, occurs after
activation of the epoxide with BF3-OEt2. The conformation of the
opened intermediate should exist as TS1 expected from the allylic
1,3-strain effect, and then MeOH attacks the C4 benzylic position
from the opposite side of the bulkier npentyl group.[9]
Scheme 1. Retrosynthetic analysis of proposed structures of (–)-eurotiumide A
(1) and (+)-eurotiumide B (2).
We initially prepared the aldehyde derivative and PT-sulfone as
substrates of the Julia-Kocienski olefination (Scheme 2).
Aldehyde 12[5] was synthesized by a sequential operation of
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10.1002/ejoc.201800535
European Journal of Organic Chemistry
COMMUNICATION
Pd cat.
MOMO
RO
O
P ligand
Additive
CO source
Br
OH
C5H11
O
DMF
C5H11
T
MOMO
OMe
MOMO
R =
OMe
17
MOM (18), H (19
)
Pd cat[a]
Additive
(equiv)
CO
source
T
[°C]
Yield
[%]
18
Yield
[%]
19
Entry
P ligand[b]
Pd(PPh3)
–
nBu3N
(2.0)
CO gas
(1 atm)
1
2
3
4
5
80
80
0
0
0
Pd(OAc)2
Xantphos
CO gas
(1 atm)
–
8
Pd(OAc)2
Xantphos
KF
(2.5)
N-formyl-
80
27
60
76
5
saccharin[c]
Pd(OAc)2
Xantphos
KF
(5.0)
N-formyl-
80
5
saccharin[c]
Pd(OAc)2
Xantphos
KF
(5.0)
N-formyl-
115
19
saccharin[c]
[a] 20 mol%. [b] 30 mol%. [c] 1.5 equiv.
Having established the method for the construction of the 4-
methoxyisochroman-1-one skeleton, which is the key core of
eurotiumides, we turned our attention to completion of the total
Scheme 2. Syntheses of (a) aldehyde derivative 12, (b) PT sulfone 9, (c) lactone
precursor 17.
synthesis of
1 and 2 (Scheme 3). The Pd-catalyzed C1
insertion/lactonization cascade reaction under the optimum
conditions afforded the mixture of 18 and 19 with excellent
conversion, and the MOM group of the salicylic acid moiety of 18
was subsequently selectively removed by treatment with
magnesium bromide to afford 19 in a one-pot operation with 89%
overall yield.[11,12] After the introduction of bromine to the ortho-
position of the salicylic hydroxyl group, the phenolic hydroxyl
group was reprotected with MOM in one-pot. It should be noted
that bromination did not occur with MOM-protected 18. At this
stage, 20 was crystallized and its enantiomeric excess increased
up to 99%. The relative and absolute configuration of 20 were
confirmed by X-ray crystallographic analysis, and the
stereochemistry at C3-C4 positions was determined as
(3S,4S).[13] The Stille coupling reaction in the presence of
PdCl2(PPh3)2, CsF in DMF for the introduction of the prenyl group
proceeded smoothly, and subsequent addition of 6 M HCl aq
removed the two MOM groups to furnish the proposed structure
for (+)-2 in 55% yield in one-pot. Although we achieved the total
synthesis of the proposed structure of (+)-2, its all spectral data
did not match the reported (+)-2, instead matched the reported
(–)-1. It indicates that the assignment of the relative configurations
between C3-C4 positions was incorrect, and the correct
configuration at the C3 position is opposite from the previous
report. Toward the proposed structure of (–)-1, hydrolysis of 20
followed by intramolecular Mitsunobu reaction afforded the
corresponding trans 4-methoxyisochroman-1-one compound 21
in good yield. The similar sequential operation of 21 afforded the
proposed structure of (–)-1. We also compared the all spectral
data of our synthetic 1 with the data of the reported (–)-1, however,
our data were not identified with the reported (–)-1, and finally, the
data of ours matched the data of the reported (+)-2. On the basis
Having prepared the precursor for the C1 insertion/lactonization,
we turned our attention to construction of the cis 4-
methoxyisochroman-1-one skeleton (Table 1). First, the general
conditions used for Pd-catalyzed C1 insertion reaction under CO
atmosphere did not give any products (entry 1). However, on
screening phosphine ligands, we found that the use of Xantphos
gave the desired lactone 18 in 8% yield (entry 2). Recently,
Manabe et al. reported that N-formylsaccharin is an efficient CO
source and could be applied to Pd-catalyzed fluorocarbonylation
reactions of aryl halides in the presence of slight excess of
potassium fluoride (KF).[10a-c] Following their procedure, the
desired C1 insertion/lactonization cascade reaction proceeded to
afford lactone 18 in 27% yield along with lactone 19 (5%), in which
one MOM group was removed (entry 3). Encouraged by this result,
we investigated further optimization of the reaction conditions. To
our delight, increasing the amount of KF (5.0 equiv) significantly
improved the conversion and afforded cyclized products in 65%
combined yield (entry 4). The starting material was almost totally
consumed at 115 °C to give 18 and 19 in excellent yields (entry
5). This result displayed that the C1 insertion reaction by using N-
formylsaccharin is useful for natural product synthesis.
Table 1. C1 Insertion/lactonization cascade reaction for constructing
cis 4-methoxyisochroman-1-one skeleton.
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10.1002/ejoc.201800535
European Journal of Organic Chemistry
COMMUNICATION
of these results, we revised the structures of the isolated natural
eurotiumide A and eurotiumide B having contiguous cis and trans
H3/H4 configurations, and the correct structures of (–)-
eurotiumide A and eurotiumide B are 1’ and 2’ as displayed in
scheme 3, respectively.
support this observation (see the Supporting Information for
details). Moreover, although two peaks are observed in the
fluorescence spectrum in protic solvents such as methanol and
water, the emission intensity of the longer wavelength peak
substantially decreases in methanol and almost disappears in
water. These experimental results suggest that the ESIPT of 1’
occurs readily in aprotic solvents. In contrast, protic solvents such
as methanol and water facilitate the formation of intermolecular
hydrogen bonds with the phenolic oxygen or carbonyl group of the
lactone moiety preventing the ESIPT from occurring in these
solvents.
Finally, we evaluated the utility of 1’ as a fluorescent probe for
the investigation of the mode of action of antibiotics. Treatment of
incubated Bacillus cereus with 1’ revealed an MIC 6.25 μM of 1’,
and the fluorescence of 1’ from bacteria was observed. As shown
in figure 2 (c), the living bacteria treated with 1’ emitted blue
fluorescence upon excitation at 345 nm, whereas no fluorescence
was observed from the control treated with DMSO. On the other
hand, rapid discoloration of the fluorescence of 1’ was observed
from Staphylococcus aureus, which is treated with 1’ and
inhibited the growth in MIC 12.5 µM of 1’. This difference in the
speed of the discoloration of 1’ indicates a possibility of different
mode of actions or localization between each grampositive
bacteria.
OH
O
Pd(OAc)2, Xantphos
N-formylsaccharin
KF, DMF, 115 °C;
NBS
DMF
65 °C;
O
17
then
MOMCl, TBAI
DIPEA
C5H11
MgBr2, Benzene
rt
MOMO
OMe
89%
gram scale
85%
19
MOMO
Br
O
Br
O
3S
3S
4S
C5H11
4S
MOMO
OMe
20
(99% ee after recrystallization)
(X-ray analysis)
1) KOH aq, EtOH
2)
MOMO
Br
O
DEAD, PPh3
THF, rt
O
3R
4S
76%
(2 steps)
C5H11
MOMO
OMe
21
SnBu3
PdCl2(PPh3)2
CsF, DMF
80 °C;
SnBu3 then
then
6 M HCl aq
MeOH
6 M HCl aq
MeOH
PdCl2(PPh3)2
CsF, DMF
80 °C;
55%
49%
OH
O
OH
O
O
O
3R
3S
4S
4S
C5H11
C5H11
OH OMe
OH OMe
revised structure of
(–)-Eurotiumide A (1’)
revised structure of
(+)-Eurotiumide B (2’)
Scheme 3. Completion of total syntheses of (–)-eurotiumide A (1’) and (+)-
eurotiumide B (2’).
Interestingly 1’ and 2’ emitted high fluorescence in
dichloromethane after excitation at 365 nm, and thus, further
investigation of their fluorescent properties was conducted. Figure
2 displays the fluorescence (a) spectra and (b) properties of 1’ in
several solvents. The absorption maximum of 1’ is around 350 nm
in dichloromethane, acetone, dimethyl sulfoxide, methanol, and
water. Two peaks are observed in the fluorescence spectra at 486
nm/428 nm, 501 nm/439 nm, and 503 nm/439 nm in
dichloromethane, acetone, and dimethyl sulfoxide, respectively,
and the emission intensities of the longer wavelength peaks are
stronger than those at the shorter wavelength region.[14] These
observations suggest that the fluorescence of 1’ is derived from
the excited-state intramolecular proton transfer (ESIPT).[15] Time-
dependent density functional theory (TD-DFT) calculations
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10.1002/ejoc.201800535
European Journal of Organic Chemistry
COMMUNICATION
(a)
excited at 365 nm
Experimental Section
H
O
O
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.2018xxxxx.
O
C5H11
OH OMe
400
(Int)
Acknowledgements
H
O
O
200
This work was supported by JSPS KAKENHI Grant Nos.
17K08365 (AN), 16K01927 (KN), and 16H01156 (KN), and the
Kurita Water and Environment Foundation and Japan Ecology
Foundation. We are grateful to Dr. Hiroshi Imagawa (Tokushima
Bunri University) and Dr. Takashi Ooi (Tokushima University) for
having useful discussion about the X-ray crystallographic analysis.
O
C5H11
OH OMe
400
500
600
(nm)
H2O
(b)
solvent
DCM Acetone DMSO MeOH
λmax
Keywords: natural products• total synthesis • fluorescent probes
353
348
348
351
350
(nm)
abs
em
λmax
(nm)
442
–
486
428
501
439
503
439
485
433
[1]
[2]
D. –L. Zhao, C.-L. Shao, Q. Zhang, K.-L. Wang, F.-F. Guan, T. Shi, C.-Y.
Wang. J. Nat. Prod. 2015, 78, 2310–2314.
0.033
0.47
0.14
0.23
0.16
φF
a) E. Cohen, L. Koch, K. M. Thu, Y. Rahamim, Y. Aluma, M. Ilan, O.
Yarden, S. Carmeli. Bioorg. Med. Chem. 2011, 19, 6587–6593; b) J. Bao,
Y.-L. Sun, X.-Y. Zhang, Z. Han, H.-C. Gao, F. He, P.-Y. Qian, S.-H. Qi. J.
Antibiot. 2013, 66, 219–223.
color
(365 nm)
[3]
a) C.-L. Shao, H. X. Wu, C.-Y. Wang, Q. A. Liu, Y. Xu, M. Y. Wei, P.-Y.
Qian, Y. C. Gu, C.-J. Zheng, Z.-G. She. J. Nat. Prod. 2011, 74, 629–633;
b) C.-L. Shao, R.-F. Xu, C.-Y. Wang, P.-Y. Qian, K.-L. Wang, M.-Y. Wei.
Mar. Biotechnol. 2015, 17, 408–415.
(c)
Bright Field
Blue fluorescence
[4]
M. Chen, C.-H. Shao, K.-L. Wang, Y. Xu, Z. G. She, C.-Y. Wang.
Tetrahedron 2014, 70, 9132–9138.
(A)
(C)
(B)
[5]
[6]
[7]
A. Little, J. A. Porco Jr. Org. Lett. 2012, 14, 2862–2865.
Y. Tu, Z.-X. Wang, Y. Shi. J. Am. Chem. Soc. 1996, 118, 9806–9807.
Chiral 17, whose enantiomeric excess is quite high, was not crystallized
under several trials.
[8]
[9]
CCDC 1821023.
(D)
We also conducted the same reaction with the substrate which has
methyl group instead of MOM group and observed the same selectivity.
Thus, this selectivity does not come from the double inversion at C4
position involving the neighbouring effect of MOM group. Moreover,
increasing amount of MeOH accelerates SN2 reaction at C4 position and
gave the mixture of cis and trans products.
Figure 2. Fluorescent properties of eurotiumide A (1’). (a) Fluorescence spectra
of 1’ in several solvents. (b) Fluorescent properties of 1’. (c) Fluorescent labeling
of Bacillus cereus with 1’. (A) and (B) were treated with 1 µM DMSO. (C) and
(D) were treated with 1 µM of 1’.
[10] a) T. Ueda, H. Konishi, K. Manabe. Angew. Chem. Int. Ed. 2013, 52,
8611-8615; Angew. Chem. 2013, 125, 8773–8777; b) T. Ueda, H. Konishi,
K. Manabe. Org. Lett. 2013, 15, 5370–5373; c) H. Konishi, K. Manabe.
Synlett 2014, 25, 1971–1986.
[11] G. G. Haraldsson, J. E. Baldwin. Tetrahedron 1997, 53, 215–224.
[12] This reaction proceeded in 76% even in 9.8 g scale with racemic 17.
[13] CCDC 1821161.
In conclusion, we have achieved the first asymmetric total
syntheses of (–)-eurotiumide A (1’) and (+)-eurotiumide B (2’) in
longest linear 8 and 10 steps (totally 10 and 12 steps),
respectively. We have also revised the relative configurations of
H3/H4 of eurotiumide A and eurotiumide B. These syntheses
involve the construction of a cis 4-methoxyisochroman-1-one
skeleton by means of the asymmetric Shi epoxidation, region and
stereoselective epoxide opening, and subsequent Pd-catalyzed
C1 insertion/lactonization cascade reaction using N-
formylsaccharin. This strategy is applicable to the synthesis of
other related natural products that have a 4-oxoisochroman-1-one
architecture. We have also discovered the high fluorescence
property of 1’ and 2’, and observed large Stokes shifts in several
solvents. This fascinating property can be utilized for elucidating
the mode of action of antimicrobial activities of 1’ against several
bacteria by fluorescence imaging. Further investigations of the
mode of action are underway in our laboratory.
[14] Detail of the fluorescence properties of 2’ is disclosed in the Supporting
Information.
[15] G. J. Woolfe, M. Melzig, S. Schneider, F. C. Dorr. Chem. Phys. 1983, 77,
213–221.
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10.1002/ejoc.201800535
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COMMUNICATION
Entry for the Table of Contents
(Please choose one layout)
COMMUNICATION
Atsushi Nakayama*, Hideo Sato,
OH
O
OH O
Sangita Karanjit, Naoki Hayashi,
Masataka Oda, and Kosuke Namba*
O
3
O
3
4
4
OH OMe
OH OMe
(–)-Eurotiumide A
(+)-Eurotiumide B
(H3/H4 cis)
(H3/H4 trans)
revised
revised
in DCM
(365 nm)
Page No. – Page No.
proposed Eurotiumide A
proposed Eurotiumide B
(H3/H4 trans)
(H3/H4 cis)
Asymmetric Total Syntheses and
Structure Revisions of Eurotiumide A
and Eurotiumide B, and Evaluation of
their Fluorescent Properties as
Natural Probes
Asymmetric total
syntheses and
structure revisions of dihydroisocoumarin-type natural products, eurotiumide A and
eurotiumide B are disclosed. Key framework of these compounds was constructed
by Pd-catalyzed C1 insertion/lactonization cascade reaction by using N-
formylsaccharin as CO source. These compounds emitted highly fluorescence in
several solvents.
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