Synthesis and Functional Assessment of a Novel Fatty Acid Probe, ω-Ethynyl Eicosapentaenoic Acid Analog, to Analyze the in Vivo Behavior of Eicosapentaenoic Acid

Article abstract of DOI:10.1021/acs.bioconjchem.7b00235

Eicosapentaenoic acid (EPA) is an ω-3 polyunsaturated fatty acid that plays various beneficial roles in organisms from bacteria to humans. Although its beneficial physiological functions are well-recognized, a molecular probe that enables the monitoring o

Full text of DOI:10.1021/acs.bioconjchem.7b00235

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Article
Synthesis and functional assessment of a novel fatty
acid probe, #-ethynyl eicosapentaenoic acid analog, to
analyze the in vivo behavior of eicosapentaenoic acid
Tomohisa Tokunaga, Bunta Watanabe, Sho Sato, Jun Kawamoto, and Tatsuo Kurihara
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00235 • Publication Date (Web): 06 Jul 2017
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Title
Synthesis and functional assessment of a novel fatty acid probe, ω-ethynyl eicosapentaenoic
acid analog, to analyze the in vivo behavior of eicosapentaenoic acid
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Tomohisa Tokunaga, Bunta Watanabe*, Sho Sato, Jun Kawamoto, Tatsuo Kurihara*
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611ꢀ0011, Japan
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*Corresponding authors
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B. W.: Tel: +81ꢀ774ꢀ38ꢀ3233, Fax: +81ꢀ774ꢀ38ꢀ3229, Eꢀmail: bunta@scl.kyotoꢀu.ac.jp
T. K.: Tel: +81ꢀ774ꢀ38ꢀ4710, Fax: +81ꢀ774ꢀ38ꢀ3248, Eꢀmail: kurihara@scl.kyotoꢀu.ac.jp
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Running Title: Novel probe to analyze eicosapentaenoic acid behavior in vivo
Key words: ωꢀ3 polyunsaturated fatty acid, eicosapentaenoic acid, ωꢀethynyl fatty acid probe
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Abstract
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Eicosapentaenoic acid (EPA) is an ωꢀ3 polyunsaturated fatty acid that plays various
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beneficial roles in organisms from bacteria to humans. Although its beneficial physiological
functions are wellꢀrecognized, a molecular probe that enables the monitoring of its in vivo behavior
without abolishing its native functions has not yet been developed. Here, we designed and
synthesized an ωꢀethynyl EPA analog (eEPA) as a tool for analyzing the in vivo behavior and function
of EPA. eEPA has an ωꢀethynyl group tag in place of the ωꢀmethyl group of EPA. An ethynyl group
has a characteristic Raman signal and can be visualized by Raman scattering microscopy. Moreover,
this group can specifically react in situ with azide compounds, such as those with fluorescent group,
via click chemistry. In this study, we first synthesized eEPA efficiently based on the following
wellꢀknown strategies. In order to introduce four CꢀC double bonds, a coupling reaction between
terminal acetylene and propargylic halide or tosylate was employed, and then, by simultaneous and
stereoselective partial hydrogenation with Pꢀ2 nickel, the triple bonds were converted to cis double
bonds. One double bond and an ωꢀterminal CꢀC triple bond were introduced by Wittig reaction with
a phosphonium salt harboring an ethynyl group. Then, we evaluated the in vivo function of the
resulting probe by using an EPAꢀproducing bacterium, Shewanella livingstonensis Ac10. This
coldꢀadapted bacterium inducibly produces EPA at low temperatures, and the EPAꢀdeficient mutant
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(∆EPA) shows growth retardation and abnormal morphology at low temperatures. When eEPA was
exogenously supplemented to ∆EPA, eEPA was incorporated into the membrane phospholipids as an
acyl chain, and the amount of eEPA was about 5% of the total fatty acids in the membrane, which is
comparable to the amount of EPA in the membrane of the parent strain. Notably, by supplementation
with eEPA, the growth retardation and abnormal morphology of ∆EPA were almost completely
suppressed. These results indicated that eEPA mimics EPA well and is useful for analyzing the in vivo
behavior of EPA.
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Introduction
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Eicosapentaenoic acid is an ωꢀ3 polyunsaturated fatty acid with 20 carbons and 5
nonconjugated cis double bonds. In various organisms from bacteria to humans, EPA exists as an acyl
chain of membrane phospholipids.1,2 Phospholipids containing polyunsaturated fatty acids, such as
EPA and docosahexaenoic acid (DHA), alter the physicochemical properties of membranes such as
elasticity, permeability, and fluidity, and modulate the function, localization, and activity of various
membrane proteins.3,4 EPA also plays important functions as a precursor of lipid mediators. In
humans, EPA and its related fatty acids, such as DHA, are converted to antiꢀinflammatory lipid
mediators that have beneficial effects on human health.5–7 These fatty acids play important roles in
the nervous system8 and signal transduction9. Moreover, supplementation of EPA and DHA
possesses antiꢀcancer effects10 and decrease the risk of cardiovascular disease11. In bacteria, EPA also
plays important physiological roles. It is involved in bacterial adaptation to various conditions such
as cold12, highꢀhydrostatic13, and oxidative environments14. De novo synthesis of EPA is observed in
many bacteria that live in cold and highꢀhydrostatic environments such as the polar regions and in
deep sea.15,16 We previously revealed that Shewanella livingstonensis Ac10, a coldꢀadapted
Gramꢀnegative bacterium isolated from Antarctic seawater, inducibly produces EPAꢀcontaining
phospholipids at 4 °C that support cell division, membrane organization, and membrane protein
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folding at low temperatures.12,17
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Although the physiological importance of EPA is wellꢀrecognized, information on its in
vivo behavior, particularly in biological membranes, is very limited. This is partly due to the lack of
tools for investigating EPA behavior. For example, it is currently difficult to visualize the distribution
and localization of EPA in vivo. For this purpose, visualizable EPA analogs will be very useful.
However, EPA analogs that retain the in vivo function of EPA and can be easily detected by standard
laboratory techniques have not yet been developed. Fluorescent tags are useful for visualizing the
localization of biological molecules, particularly of proteins. In the field of lipid biochemistry,
fluorescent tags such as nitrobenzoxadiazole (NBD) are often used.18,19 However, the relative
bulkiness of fluorescent tags compared with the size of lipid molecules generally affects the
biochemical properties of lipids. Therefore, the images obtained by using lipid molecules with
fluorescent tags may not reflect the native behavior of the corresponding lipid molecules.
In this study, to overcome these problems, we designed and synthesized an ethynylꢀtagged
EPA analog named eEPA, which has a CꢀC triple bond at the omega end of EPA (Fig. 1). Because this
functional group is much less bulky than fluorescent groups, eEPA may retain the in vivo function of
EPA. The ethynyl group has a characteristic Raman signal that enables the visualization of
ethynylꢀtagged compounds by Raman microscopy.20,21 In addition, the ethynyl group can specifically
react with azide compounds by a 1,3ꢀdipolar cycloaddition between azide and ethynyl groups
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affording a 1,2,3ꢀtriazole ring, so called “click chemistry”. Various applications of this reaction to
biological science have been reported.22 In situ labeling with azideꢀcontaining molecules has been
widely used for visualization and affinityꢀtag labeling of ethynylꢀtagged molecules.23,24
In order to evaluate the in vivo function of eEPA, the EPAꢀproducing bacterium S.
livingstonensis Ac10 and its EPAꢀdeficient mutant (∆EPA) are useful. We previously found that the
∆EPA strain shows growth retardation and abnormal morphology at 4 °C. Exogenous
supplementation of EPA suppresses these phenotypes of ∆EPA, whereas supplementation of oleic
acid does not12. Thus, the in vivo function of eEPA can be evaluated by supplementing the ∆EPA
strain with eEPA. Here, we report that eEPA newly synthesized in this study retained the in vivo
function of EPA and is therefore expected to be useful as a probe to understand the physiological
functions of EPA.
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Figure 1. Structures of EPA and eEPA
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Results and Discussion
Synthesis of eEPA
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The retrosynthetic analysis of eEPA is illustrated in Scheme 1. The wellꢀknown strategy
that has been successfully employed in the synthesis of PUFA analogs25–37 was adopted in this study.
The terminal ethynyl group of eEPA is introduced by a Wittig reaction of aldehyde 138 using
phosphonium salt 2.39,40 The fifth double bond between C17 and C18 is concomitantly built. The
four cis double bonds of the key intermediate 1 are constructed by selective hydrogenation of tetrayne
3. The skipped polyyne structure of 3 is assembled by coupling of the C1ꢀC9 fragment 441 and the
C10ꢀC17 fragment 5. These skipped diynes come from commercially available or easily accessible
materials. The convergent synthetic strategy outlined in Scheme 1 should have the advantage with
respect to overall yield and number of steps over the previously reported linear synthesis of
compound 1,38 utilizing continuous Wittig reactions. Especially, coupling reactions between terminal
acetylene and propargylic halide or tosylate in carbonꢀcarbon bond formation steps, and a
stereoselective partial reduction of carbonꢀcarbon triple bond42 in a simultaneous hydrogenation of a
resultant skipped polyyne should make the synthesis of 1 and eEPA convenient with excellent yield.
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Scheme 1. Retrosynthetic analysis of eEPA
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4
The synthesis of the C1ꢀC9 fragment 4 is illustrated in Scheme 2. Methyl hexꢀ5ꢀynoate (6)
and 3ꢀbromoꢀ1ꢀ(trimethylsilyl)ꢀ1ꢀpropyne (7), both commercially available, were coupled by skipped
diyne synthesis improved by Caruso and Spinella43 to give diyne 8,44,45 without purification, with
93% yield. The trimethylsilyl group of crude 8 was removed by an (nꢀBu)4NF/acetic acid system46 to
give the C1ꢀC9 fragment 441 with 87% yield after chromatographic purification. As mentioned in a
previous study,46 the addition of acetic acid was necessary to avoid a rearrangement of the terminal
triple bond to an allene. Compound 4 was highly unstable and decomposed during storage even at
–20 ºC under argon atmosphere.
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O
Br
+
TMS
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MeO
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CuI, NaI, Cs2CO3
DMF, rt, 16 h
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93% (without purification)
O
MeO
TMS
8
(nꢀBu)4NF, AcOH
THF, rt, 2 h
87%
O
MeO
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Scheme 2. Synthesis of the C1-C9 fragment 4
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5
The synthesis of the C10ꢀC17 fragment 5 is illustrated in Scheme 3. Monotosylate 10,47,48
prepared from 2ꢀbutyneꢀ1,4ꢀdiol (9), was coupled with terminal acetylene 1249,50 to give diyne 13,
without purification, with 68% yield. Compound 12 was synthesized by the reaction between an
organoaluminum reagent prepared from 3ꢀbromoꢀ1ꢀpropyne (11) and trimethyl orthoformate with
78% yield after distillation. In the previous syntheses of 12, the organoaluminum nucleophile was
generated in boiling solvents in the presence of catalytic HgCl2, and the yields were 50% (in
toluene)49 to 69% (in Et2O)50, respectively. Lately, Gao et al. reported that various organoaluminum
species could be prepared from propargylic bromides and aluminum powder in the presence of
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catalytic PbCl2 in tetrahydrofuran at 0 ºC.51 By applying the improved conditions, compound 12 was
prepared with 78% yield in this study The use of diethyl ether as coꢀsolvent was found to be required
for successful isolation and purification of compound 12 though it was still difficult to separate
tetrahydrofuran completely. Subsequent conversion of the terminal hydroxyl group of crude 13 to a
bromine atom gave the C10ꢀC17 fragment 5 with 51% yield after chromatographic purification.
Compound 5 was also highly unstable.
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OH
Br
HO
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1. Al, PbCl2
THF / Et2O
rt ~ 40 ºC, 5 h
2. (MeO)3CH
–78 ºC, 4 h
78%
pꢀTsCl, Et3N
Me3N•HCl
MeCN, 0 ºC, 1 h
53%
OMe
OTs
+
HO
OMe
10
12
CuI, NaI, Cs2CO3
DMF, rt, 17 h
68% (without purification)
OMe
OMe
HO
13
CBr4, PPh3
CH2Cl2, rt, 1 h
51%
OMe
OMe
Br
5
8
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Scheme 3. Synthesis of the C10-C17 fragment 5
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Now, with both the C1ꢀC9 fragment 4 and the C10ꢀC17 fragment 5 in hand, we coupled
them under the same reaction conditions as those employed for the preparation of skipped diynes 4
and 5 to obtain tetrayne 3, without purification, with 99% yield (Scheme 4). Compound 3 was
immediately used in the subsequent step without purification since it was prone to decomposition
during chromatographic purification.
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Stereoselective reduction of carbonꢀcarbon triple bonds to cis double bonds was
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extensively studied, and Pꢀ2 nickel52 is regarded as the most reliable and efficient catalyst for partial
hydrogenation of (poly)alkynic compounds to (poly)ꢀ(Z)ꢀolefins.42 Therefore, semiꢀhydrogenation
of the four triple bonds of compound 3 to cis double bonds was performed with Pꢀ2 nickel, prepared
from Ni(OAc)2•4H2O and NaBH4. According to the conditions developed by Balas and
coꢀworkers,46 the triple bonds of crude 3 were smoothly reduced as expected, and desired tetraene 14
was obtained with 65% yield after chromatographic purification. Acidic hydrolysis of the terminal
dimethyl acetal of 14 gave aldehyde 1, without purification, with 98% yield. Since aldehyde 1 is
oxidized easily, it is recommended to store it at the stage of dimethyl acetal 14. Compound 14 could
be stored without decomposition for 8 months in a refrigerator under argon atmosphere. Following
the Wittig reaction of crude aldehyde 1 with phosphonium ylide, prepared from phosphonium salt
239,40 and nꢀBuLi, pentaene 15 was obtained with 73% after chromatographic purification. Since the
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Wittig reactions between the above nonꢀstabilized ylide and aldehydes were reported to produce
Zꢀolefins38,53,54, the geometry of the newly formed double bond between C17 and C18 of pentaene 15
was considered to be Z. When sodium hexamethyldisilazide was used as a base in the generation of
the ylide in the reaction, the yield of the Wittig reaction was dramatically decreased. Finally, basic
hydrolysis of methyl ester of 15 gave the target eEPA, without purification, with 97% yield. Since
degradation of eEPA during chromatographic purification was observed, we used the product for the
following experiments without further purification.
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O
OMe
OMe
+
Br
MeO
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5
CuI, NaI, Cs2CO3
DMF, rt, 16 h
99% (without purification)
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OMe
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MeO
OMe
3
H2, Ni(OAc)2•4H2O, NaBH4
ethylenediamine
EtOH, rt, 8.5 h
65%
O
OMe
OMe
MeO
14
CF3CO2H, H2O
CH2Cl2, rt, 4 h
98% (without purification)
O
CHO
MeO
1
nꢀBuLi
THF, rt, 2 h
73%
BrPh3P
2
O
RO
15 : R = Me
LiOH, THF / H2O, rt, 17 h
97% (without purification)
cEPA: R = H
1
2
Scheme 4. Synthesis of eEPA
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Bacterial uptake of exogenous eEPA
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Because the ethynyl tag is a very small functional group with only two carbon atoms, and is
much smaller than the fluorescent tag, biomolecules that are tagged with the ethynyl group may retain
their native biological functions. To examine whether eEPA retains a specific function of EPA in
living cells, we tested whether this analog is incorporated into the EPAꢀdeficient mutant of S.
livingstonensis Ac10 (∆EPA) and compensates for the loss of EPA. This unique and remarkable
system enables the in vivo functional evaluation of a particular fatty acid of interest as a substitute for
EPA.
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We first analyzed the phospholipid compositions of the parent cells and the ∆EPA cells
grown with exogenous supplementation of EPA and eEPA at 4 °C. The phospholipids were extracted
and analyzed by electrospray ionizationꢀmass spectrometry (ESIꢀMS), and the mass spectra in the
range of m/z 690ꢀ800, where EPAꢀcontaining phospholipids were detected, are shown in Fig. 2. EPAꢀ
and eEPAꢀcontaining phosphatidylethanolamines (PEs) and phosphatidylglycerols (PGs) detected in
these samples by electrospray ionizationꢀtandem mass spectrometry are listed in Table 1. In all these
samples, phospholipids were mainly composed of PE and PG, containing various fatty acyl groups
such as 16:1, 15:0, 13:0, and 16:0 (data not shown), as demonstrated previously for the parent strain
and ∆EPA.12,55 In the parent strain, 16:1ꢀEPAꢀPE, 18:1ꢀEPAꢀPE, and 16:1ꢀEPAꢀPG were detected as
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major EPAꢀcontaining phospholipids (Fig. 2A, Table 1). EPAꢀcontaining phospholipids were not
found in ∆EPA (Fig. 2B). In the ∆EPA cells grown in the presence of EPA, 16:1ꢀEPAꢀPE,
16:0ꢀEPAꢀPE, 15:0ꢀEPAꢀPE, and 16:0ꢀEPAꢀPG were the major EPAꢀcontaining phospholipids (Fig.
2C, Table 1). In ∆EPA grown with exogenous eEPA supplementation, eEPAꢀcontaining PEs and PGs
were detected (Fig. 2D, Table 1). Among these, 16:1ꢀeEPAꢀPE, 16:0ꢀeEPAꢀPE, and 15:0ꢀeEPAꢀPE
were more abundant than other eEPAꢀcontaining phospholipids. As shown in Fig. 2 and Table 1, the
molecular weights of the eEPAꢀcontaining phospholipids are greater than the natural EPAꢀcontaining
counterparts by ten m/z units because of the structural modification of eEPA from EPA [+ ethylidene
group (CH) – three hydrogen atoms = 10].
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Table 1. EPA- or eEPA-containing phospholipids found in S. livingstonensis Ac10
WT*
∆EPA+EPA
∆EPA+eEPA
Acylꢀchain
composition
PE or PG
PE
m/z
N.D.**
708.5
722.7
734.6
736.6
748.6
N.D.
762.6
765.6
N.D.
779.3
N.D.
m/z
N.D.
m/z
13:0/EPA or eEPA
14:0/EPA or eEPA
15:0/EPA or eEPA
16:1/EPA or eEPA
16:0/EPA or eEPA
17:1/EPA or eEPA
17:0/EPA or eEPA
18:1/EPA or eEPA
16:1/EPA or eEPA
16:0/EPA or eEPA
17:1/EPA or eEPA
17:0/EPA or eEPA
18:1/EPA or eEPA
704.7
718.5
732.7
744.6
746.5
N.D.
N.D.
N.D.
775.4
777.3
N.D.
N.D.
N.D.
PE
PE
PE
PE
PE
PE
PE
PG
PG
PG
PG
PG
708.4
722.4
734.6
736.5
748.6
750.2
762.3
765.6
767.6
N.D.
781.5
793.5
793.5
12
13
* Parent strain. ** Not detected.
EPAꢀ and eEPAꢀcontaining phospholipids were analyzed by electrospray ionizationꢀtandem mass
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Figure 2. Phospholipid analysis of S. livingstonensis Ac10 grown with or without exogenous
EPA/eEPA supplementation
ESIꢀMS analysis of the phospholipid extracts of the parent strain (A), ∆EPA (B), and ∆EPA grown
with EPA (C) or eEPA (D). The spectra in the range of m/z 690ꢀ800 are shown. The peaks
corresponding to phospholipids are indicated by their m/z value, and those corresponding to EPAꢀ or
eEPAꢀcontaining phospholipids are shown in red and blue, respectively.
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Table 2. Acyl-chain compositions of the membrane phospholipids of S. livingstonensis Ac10 and
the EPA-deficient mutant grown with EPA and eEPA
Acyl chain
WT*
WT + EPA
WT + eEPA
ꢀEPA
ꢀEPA + EPA
ꢀEPA + eEPA
(%)
13:0
14:0
15:0
16:1
16:0
18:1
EPA
eEPA
2.5 ± 0.2
3.1 ± 0.2
12.6 ± 0.6
58.8 ± 0.7
12.8 ± 1.1
2.3 ± 0.3
8.0 ± 1.4
N.D.
3.7 ± 1.9
2.3 ± 1.0
17.9 ± 8.5
18.3 ± 6.0
14.8 ± 7.5
2.1 ± 1.3
24.7 ± 6.2
N.D.
22.6 ± 1.5
4.4 ± 0.5
41.9 ± 1.6
16.7 ± 3.6
9.2 ± 2.0
6.5 ± 6.5
0.4 ± 0.3
3.3 ± 1.4
7.5 ± 0.4
2.1 ± 0.1
7.6 ± 0.4
2.2 ± 0.0
29.0 ± 1.9
29.4 ± 3.6
13.3 ±1.1
2.4 ± 0.3
16.2 ± 1.2
N.D.
27.0 ± 4.7
4.5 ± 0.1
37.8 ± 1.1
18.8 ± 2.0
5.4 ± 1.5
1.2 ± 0.2
N.D.
25.1 ± 1.4
50.0 ± 1.6
11.2 ± 0.6
4.0 ± 0.3
N.D. **
N.D.
5.3 ± 2.7
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Percentages in the total fatty acids are shown. * Parent strain. ** Not detected. Each experiment was
performed three times, except for WT + EPA, with independently extracted fatty acids, and average
values ± SD are shown. The experiment for WT + EPA was carried out twice.
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We next analyzed the compositions of the fatty acids linked to the phospholipids. Fatty
acid methyl esters were prepared from the phospholipid extracts, which do not contain free fatty acids,
and quantitatively analyzed by gas chromatographyꢀmass spectrometry (GCꢀMS) (Table 2). In the
parent strain, the major fatty acids were 16:1, 16:0, and 15:0. The amount of EPA was 8% of the total
fatty acids. In ∆EPA, the major fatty acids were 16:1, 15:0, and 16:0. In the presence of exogenous
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EPA, the major fatty acids of ∆EPA were 16:1, 15:0, 16:0, and EPA. In the presence of eEPA, the
major fatty acids of this strain were 15:0, 13:0, and 16:1. The reason for the increase of 13:0, a short
chainꢀlength fatty acyl group, is unclear, but this might represent a counteraction to the incorporation
of eEPA, which has a longer chain length than EPA. The amount of eEPA was 5.3% of the total fatty
acids and was similar to the relative amount of endogenous EPA in the parent strain grown without
eEPA supplementation. These results indicate that exogenously supplemented eEPA served as the
substrate for eEPAꢀcontaining phospholipid production, probably after conversion into the CoA ester,
which is the substrate of 1ꢀacylꢀsnꢀglycerolꢀ3ꢀphosphate acyltransferase, responsible for
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incorporation of an acyl chain into phospholipids.56 The structural modification of the ωꢀterminal of
EPA to generate eEPA seems to be marginal for the enzyme in the substrateꢀrecognition mechanism.
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Biological function of eEPA
When EPA and eEPA were supplemented to the ∆EPA strain, these fatty acids were
incorporated into the membrane phospholipids as their acyl chains. To test whether eEPA retains
physiological functions of EPA, the growth and morphology of ∆EPA with eEPA supplementation
were characterized.
Without exogenous EPA supplementation, the ∆EPA strain showed growth retardation at 4
°C due to defects in cell division (Fig. 3B). The doubling times of the parent strain and ∆EPA were
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8.0 h and 13.5 h, respectively. The growth of the parent strain was not significantly affected by
supplementation with EPA or eEPA (Fig. 3A). In contrast, the growth of the ∆EPA strain was
improved by supplementation with EPA or eEPA (Fig. 3B). The growth rate of ∆EPA in
eEPAꢀcontaining medium was similar to its growth rate in EPAꢀcontaining medium: the doubling
times of ∆EPA under these conditions were 10.3 h and 9.9 h, respectively.
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3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
4.0
3.5
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2.5
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1.5
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0
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Time (h)
Time (h)
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Figure 3. Growth of S. livingstonensis Ac10 with or without exogenous EPA/eEPA
supplementation
4
(A) The growth curves of the parent strain in LB medium (filled circles), EPAꢀcontaining LB medium
(filled diamonds), and eEPAꢀcontaining LB medium (open diamonds) at 4 °C. (B) The growth curves
of ∆EPA in LB medium (filled triangles), EPAꢀcontaining LB medium (filled squares), and
eEPAꢀcontaining LB medium (open squares) at 4 °C. As a control, the growth of the parent strain in
LB medium (filled circle) at 4 °C is shown. The error bars indicate the standard deviation from three
independent experiments.
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We next analyzed the morphology of the parent strain and ∆EPA with and without
supplementation of EPA or eEPA. The parent strain formed rodꢀshaped cells, and the cell length of
about 80% of the cells was approximately 2−4 ꢁm (Figs. 4A and E), and the average cell length was
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2.9 ꢁm. For ∆EPA without supplementation of EPA or eEPA, about 70% of cells formed filamentous
cells with lengths of more than 4 ꢁm (Fig. 4B and E). The average cell length of the mutant was 12
ꢁm. We previously showed that the filamentous cells of ∆EPA contain multiple nucleoids, indicating
that EPA is important for cell division in a step after DNA replication.12 When EPA or eEPA was
added to the medium, the cell length of ∆EPA became significantly shorter. The average length of
∆EPA under these conditions was 3.9 ꢁm (Figs. 4C, D, and E). These data indicated that eEPA can
fulfill the physiological function of EPA in this strain, at least for cell division.
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0
Length (ꢁm)
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Figure 4. Morphology of S. livingstonensis Ac10 grown with or without exogenous EPA/eEPA
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Microscopic images of the parent strain (A) and ∆EPA (B, C, and D) are shown. The cells were
grown in LB medium (A and B), EPAꢀsupplemented LB medium (C), and eEPAꢀsupplemented LB
medium (D) at 4 °C. The bar indicates 2 ꢁm. The quantified cell size distributions of the parent strain
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grown in LB medium (white) and ∆EPA grown in LB medium (black), EPAꢀsupplemented LB
medium (dark gray), and eEPAꢀsupplemented LB medium (light gray) are shown in (E).
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Significance and prospects
5
PUFAs such as EPA and DHAplay important physiological roles in various organisms from
bacteria to humans. To understand the mechanisms underlying their function, it is crucial to reveal
the in vivo behavior of these molecules, including their subcellular localization. To analyze the
subcellular localization of EPAꢀcontaining phospholipids, we previously synthesized fluorescent
analogs of EPAꢀcontaining phospholipids. Although we succeeded in identifying polyunsaturated
hydrocarbon chainꢀdependent localization of phospholipids at the cell division site of S.
livingstonensis Ac10 using these analogs, they did not complement for the loss of EPA in this
bacterium. In contrast, eEPA developed in this study suppressed the growth defects caused by the
loss of EPA. Thus, our eEPA probe mimics native EPA well and is expected to provide information
on the behavior of native EPA in vivo. Similar to other probes harboring an ethynyl group, eEPA may
be visualized directly by Raman microscopy and indirectly by labeling with a fluorescent compound
containing an azide group by a click chemistry reaction.20,23,57 eEPA would also be useful for
identifying EPAꢀcontaining molecules, such as EPAꢀmodified proteins, by affinity purification58 of
eEPAꢀcontaining molecules.
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It should be noted that the ethynyl group is not totally inert in a biological system under
particular conditions: it was reported that the ethynyl group introduced into various steroids reacts
with human liver microsomal cytochrome P450.59 Thus it is desirable to verify that eEPA retains its
original structure in vivo and does not disturb normal cellular functions when it is used in various
organisms, although metabolic conversion of eEPA and its deleterious effects on cellular functions
were not observed in the bacterial strain we used in this study.
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The convergent synthetic route established in this study could synthesize compound 1, the
key intermediate for synthesis of various ωꢀmodified EPA analogs, in half the reaction steps
comparing to the previous method.38 We believe that our approach for the synthesis of eEPA is easily
applicable to the synthesis of various ωꢀmodified EPAs such as ωꢀazidoEPA, ωꢀaminoEPA, and
ωꢀdeuteriumꢀlabeled EPA. These ωꢀmodified EPAs would have various applications for studying
physiological functions of EPA. For example, ωꢀazidoEPA and deuteriumꢀlabeled EPA would be
useful for in situ imaging of EPA and mass spectrometric identification of EPA metabolites,
respectively. Thus, it is expected that eEPA, as well as these EPA analogs, remarkably contribute to
the progress of functional analysis of EPA.
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Experimental Procedures
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Synthesis of ω-ethynyl eicosapentaenoic acid analog, eEPA
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According to the retrosynthetic analysis (Scheme 1), we synthesized eEPA as shown in
Schemes 2ꢀ4. Full experimental procedures are described in the Supporting Information.
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Bacterial strains and growth conditions
8
Strains used in this study are listed in Table 3. An EPAꢀproducing bacterium, S.
livingstonensis Ac10, and the EPAꢀdeficient mutant of this strain (∆EPA), in which one of the
EPAꢀbiosynthesis genes, orf5, was disrupted, were used in this study. The strains cultured on solid
LB medium in the presence of 50 ꢁg/mL rifampicin at 18 °C were aerobically precultured in 5 mL
liquid LB medium (pH 7.0) for 24 h at 18 °C. The precultured cells were used as a seed culture for the
following supplement assays. For the ∆EPA strain, 30 ꢁg/mL kanamycin was added.
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Table 3. Strains used in this study
Strain
Shewanella livingstonensis Ac10 Parent strain of the EPAꢀdeficient mutant12
EPAꢀdeficient mutant of S. livingstonensis Ac10, orf5::Kmr
Description
∆EPA
12
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17
Growth and morphology in the presence of eEPA supplementation
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EPA or eEPA was dissolved in ethanol (20 ꢁL) and supplemented to 5 mL of LB liquid
media at a final concentration of 0.13 mM, and S. livingstonensis Ac10 and its EPAꢀdeficient mutant
were grown in these media. BioPhoto recorder TVS062CA (ADVANTEC Toyo, Tokyo, Japan) was
used to monitor the growth rate of the cells at 4 °C and 70 rpm. The cells were cultivated at 4 °C until
the optical density at 600 nm reached about 1.0. The microscopic images of the cells were observed
with a BX40F4 trinocular microscope (OLYMPUS, Tokyo, Japan) equipped with the digital
microscope system, Moticam 2500 (Shimadzu Rika, Tokyo, Japan). Cell length was measured using
the imageꢀanalyzing software, Image J ver. 1.6.0 (https://imagej.nih.gov/ij/).
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Analysis of phospholipid composition by ESI-MS
The cells were grown in 5 mL LB medium containing 0.13 mM of EPA or eEPA at 4 °C and
harvested by centrifugation when the optical density at 600 nm was approximately 1.0. After
centrifugation, the collected samples were frozen in liquid nitrogen and freezeꢀdried by being placed
in a lyophilizer, FreeZone 2.5 plus (Labconco, Kansas City, MO). Thereafter, phospholipids were
extracted by the BlighꢀDyer method60 and analyzed by ESIꢀMS with a tripleꢀquadrupole Sciex API
3000 liquid chromatographyꢀtandem mass spectrometry system (Applied Biosystems, Foster City,
CA) equipped with an IonSpray ion source in the negative mode. The fatty acyl residues in each
molecular species were analyzed in the precursor ion scan mode.
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Analysis of fatty acyl chain composition of phospholipids by GC-MS
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The cells were grown in 5 mL LB medium containing 0.13 mM EPA or eEPA at 4 °C.
Phospholipids extracted from freezeꢀdried cells by the BlighꢀDyer method60 were methylꢀesterified
with 0.5 mL of 10% HCl in methanol. Fatty acid methyl esters were extracted with 0.25 mL CH2Cl2
and 1 mL nꢀhexane.61 As authentic compounds, methylꢀesterified EPA and eEPA were prepared from
EPA and eEPA, respectively. The methylꢀesterified samples were analyzed with a Clarus 680 gas
chromatograph coupled with a Clarus SQ 8 C mass spectrometer (PerkinꢀElmer, Wellesley, MA)
equipped with an ULBON HRꢀ1 capillary column (Shinwa Chemical Industries, Ltd., Kyoto, Japan).
Individual compounds were identified by their mass spectra. The GCꢀMS analysis showed that the
esterification procedure did not cause significant isomerization and decomposition of authentic EPA
and eEPA, indicating that these compounds are stable enough under this condition.
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Acknowledgments
This work was supported in part by ICR Grants for Promoting Integrated Research from Institute for
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Chemical Research, Kyoto University (to JK and BW), GrantꢀinꢀAid for Challenging Exploratory
Research 26660065 (to JK), GrantsꢀinꢀAid for Scientific Research (B) from JSPS (24380047 and
15H04474 to TK), Grants from the Japan Foundation for Applied Enzymology and NAGASE
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Bioconjugate Chemistry
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Science Technology Foundation (to TK), and a GrantꢀinꢀAid for JSPS Fellows JP15J09839 to TT.
Some experimental measurements were carried out using the JEOL JNMꢀECA600 spectrometer and
the JOEL JMSꢀ700 mass spectrometer in the Joint Usage/Research Center (JURC) at the Institute for
Chemical Research, Kyoto University.
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Supporting Information
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Experimental procedures for eEPA synthesis.
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