Synthesis of one double bond-inserted retinal analogs and their binding experiments with opsins: Preparation of novel red-shifted channelrhodopsin variants

Article abstract of DOI:10.1248/cpb.c19-01005

In optogenetics, red-shifted channelrhodopsins (ChRs) are eagerly sought. We prepared six kinds of new chromophores with one double bond inserted into the polyene side chain of retinal (A1) or 3,4-didehy-droretinal (A2), and examined their binding efficie

Full text of DOI:10.1248/cpb.c19-01005

Vol. 68, No. 3
Chem. Pharm. Bull. 68, 265–272 (2020)
265
Regular Article
Synthesis of One Double Bond-Inserted Retinal Analogs and Their
Binding Experiments with Opsins: Preparation of Novel Red-Shifted
Channelrhodopsin Variants
Takashi Okitsu,a Yumiko Yamano,a Yi-Chung Shen,b Toshikazu Sasaki,b Yuka Kobayashi,a
Shoko Morisawa,a Takahiro Yamashita,b Yasushi Imamoto,b Yoshinori Shichida,c and
,a
Akimori Wada*
a Laboratory of Organic Chemistry for Life Science, Kobe Pharmaceutical University; Kobe 658–8558, Japan:
b Department of Biophysics, Graduate School of Science, Kyoto University; Kyoto 606–8502, Japan: and
c Research Organization of Science and Technology, Ritsumeikan University; Kusatsu, Shiga 525–8577, Japan.
Received November 14, 2019; accepted December 18, 2019
In optogenetics, red-shifted channelrhodopsins (ChRs) are eagerly sought. We prepared six kinds of
new chromophores with one double bond inserted into the polyene side chain of retinal (A1) or 3,4-didehy-
droretinal (A2), and examined their binding efficiency with opsins (ReaChR and ChrimsonR). All analogs
bound with opsins to afford new ChRs. Among them, A2-10ex (an extra double bond is inserted at the C10–
C11 position of A2) showed the greatest red-shift in the absorption spectrum of ChrimsonR, with a maxi-
mum absorbance at 654nm (67nm red-shifted from that of A1-ChrimsonR). Moreover, a long-wavelength
spectral boundary of A2-10ex-ChrimsonR was extended to 756nm, which reached into the far-red region
(710–850 nm).
Key words channelrhodopsin; retinal analog; chromophore; red-shift; optogenetics
togenetic tools, which were controlled by currently unused
Introduction
Channelrhodopsin-1 (ChR1, 1) was discovered from Chlam- wave-length light, but also deeply penetrate the targeted neu-
ydomonas reinhardtii by Nagel et al. in 2002.1) It is a member ral tissues without the influence of other tissue components
of the microbial rhodopsin and is a light-gated cation channel. such as water and blood (scattering and absorption light).
ChR1 consists of the chromophore (all-trans-retinal, 2a) and
One method to create red-shifted ChR is to introduce muta-
a seven-transmembrane helix apoprotein (opsin). The retinal tions of the amino acids of opsin and several such achieve-
covalently binds to the ε-amino group of lysine residue 296 ments have been reported.10–12) Another approach has been to
(K296) through a protonated Schiff base (PSB). Upon absorp- replace retinal with another red-shifted chromophore having a
tion of light, photoisomerization of retinal from the all-trans system of extended conjugation.13–15) In connection with our
to the 13-cis forms occurs with a conformational structure study on development of new red-shifted ChRs,16) very re-
change of the opsin, and then ChR1 activate the membrane cently we designed and prepared new chromophores in which
with opening the channel and ion transportation (depolariza- a one double bond was inserted into the polyene side chain of
tion). Since the first finding of ChR1, plenty of similar chan- all-trans-retinal (A1, 2a) and 3,4-didehydroretinal (A2, 2b) at
nelrhodopsins (ChRs) have been discovered from archaea, C6–C7, C10–C11 or C14–C15 positions (nomenclature based
bacteria and eukaryotes.2)
on retinal numbering), which were named A1-6ex (3a), A2-
Recently, a new technique for treatment of cellular per- 6ex (3b), A1-10ex (4a), A2-10ex (4b), A1-14ex (5a) and A2-
turbation tools called optogenetics has been developed.3–6) 14ex (5b) (Fig. 1). All analogs except for 3a were incorporated
Optogenetics is a technology for controlling specific neural into C1C2, which is one of the most widely used ChR vari-
activities by light irradiation that arises from the depolariza- ants in optogenetics (an absorption maximum at 477nm),17)
tion of ChRs-expressing neural cells. This technique is less and among the newly created ChRs, A2-10ex-C1C2 showed
invasive compared to the traditional electrode method that the longest absorption maximum at 510nm.18) These results
affects other cells. Therefore, optogenetics are deemed to be indicated that polyene chain-elongated retinals could be in-
a useful tool to clarify the neural circuits and the mechanism corporated into the retinal-binding pocket of C1C2. However,
of various neural diseases.7,8) ChRs now used in optogenetics the red-shift value of absorption maximum of A2-10ex-C1C2
are mostly sensitive to blue-green light (430–550nm) and bear was not enough compared to the ideal red-shifted ChRs,
several limitations, such as low expression levels, ability of whose absorption maxima are required in the near-infrared
depolarization, kinetics (response of excitation and desensiti- region (750-1500nm). We anticipated that it might be possible
zation), spectra response and ion selectivity due to their bio- to produce the even more red-shifted new ChRs by changing
logical and physical properties. To overcome these problems, opsins for binding. As other opsins, we chose ReaChR11) and
several approaches for the development of new ChRs have ChrimsonR19) because their absorption maxima were 61nm
been achieved.9) Among them, to create the red-shifted ChRs and 110nm red-shifted, respectively, compared to that of
that are sensitive to long-wavelength light is highly attracted C1C2. Although the data of A2 analogs (3b, 4b and 5b) were
because the red-shifted ChRs can not only provide new op- already reported in the literature,20) we describe here a full ac-
*To whom correspondence should be addressed. e-mail: a-wada@kobepharma-u.ac.jp
© 2020 The Pharmaceutical Society of Japan

266
Chem. Pharm. Bull.
Vol. 68, No. 3 (2020)
count of the synthesis of six kinds of chromophores and their ford the corresponding C2- or C5-elongated conjugate esters
binding experiments with other opsins.
in a highly E-stereoselective manner. In the second reduction
step, the resultant conjugate ester would be converted to the
allyl alcohol by using lithium aluminum hydride (LiAlH4)
Chemistry
With the exception of 5a,21) our designed one double bond or diisobutylaluminum hydride (DIBAL). The third oxida-
elongated retinal analogs 3–5 had been unknown. We there- tion step from allyl alcohol to conjugate aldehyde would be
fore planned a systematic synthetic route for 3–5 from the achieved by using manganese dioxide (MnO2). If we appropri-
known aldehydes: a 3-step sequence of Horner–Wadsworth– ately chose C2- or C5-phosphonate in this 3-step sequence, re-
Emmons reaction/reduction/oxidation (Chart 1). The first step peating this 3-step sequence would ultimately provide access
of the Horner–Wadsworth–Emmons reaction with aldehyde to the desired retinal analogs 3–5. This methodology contains
using triethyl phosphonoacetate (C2-phosphonate) or triethyl a quite traditional polyene construction, but we believed this
3-methyl-4-phosphonocrotonate (C5-phosphonate) would af- protocol would be a practical, scalable, and robust procedure.
A1-6ex (3a) was prepared from commercially available
β-cyclocitral (6a) by a C2-/C5-/C5-elongated sequence (Chart
2). In the Horner–Wadsworth–Emmons reaction of C2-
phosphonate, the E-isomer was obtained exclusively. On the
contrary, in the case of C5-phoshonate, the newly produced
conjugate ester was obtained as a stereoisomeric mixture with
a terminal double bond (E:Z=9:1 to 18:1), and it is well
known that N,N-dimethylpropyleneurea (DMPU) plays an
important role for this high stereoselectivity.22) As the separa-
tion of stereoisomers was difficult, the stereoisomeric mixture
was used for the next reaction, and the all-E isomer was easily
isolated after conversion to the aldehyde by column chroma-
tography. In a similar fashion, A2-6ex (3b) was prepared from
safranal (6b).
A1-10ex (4a) and A2-10ex (4b) were prepared from
β-ionylideneacetaldehyde
(9a)23)
and
3,4-didehydro-β-
ionylideneacetaldehyde (9b)24) by C2-/C5-elongated sequence
(Chart 3).
Fig. 1. Channelrhodopsin-1, Retinal and Its Analogs
Chart 1. Three-Step Sequence for Conjugate Aldehyde Construction
Reagents and conditions: (a) triethyl phosphonoacetate, n-BuLi or NaH, THF, 0°C; (b) LiAlH4, Et2O, 0°C (for 6a, 7a, 8a) or DIBAL, THF, −78°C (for 6b, 7b, 8b); (c)
MnO2, Et2O, room temperature (r.t.); (d) triethyl 3-methyl-4-phosphonocrotonate, n-BuLi, DMPU, THF, −78°C to r.t.
Chart 2. Synthesis of A1-6ex (3a) and A2-6ex (3b)

Vol. 68, No. 3 (2020)
Chem. Pharm. Bull.
267
Reagents and conditions: (a) triethyl phosphonoacetate, NaN(TMS)2 or NaH, THF, 0°C; (b) LiAlH4, Et2O, 0°C (for 9a, 10a) or DIBAL, THF, −78°C (for 9b, 10b); (c)
MnO2, Et2O, r.t.; (d) triethyl 3-methyl-4-phosphonocrotonate, n-BuLi, DMPU, THF, −78°C to r.t.
Chart 3. Synthesis of A1-10ex (4a) and A2-10ex (4b)
Reagents and conditions: (a) triethyl phosphonoacetate, NaN(TMS)2 or NaH, THF, 0°C; (b) LiAlH4, Et2O, 0°C (for 2a) or DIBAL, THF, −78°C (for 2b); (c) MnO2, Et2O,
r.t.
Chart 4. Synthesis of A1-14ex (5a) and A2-14ex (5b)
Although A1-14ex (5a) was
a known compound as logs. In the case of A1 analogs having the same conjugated
14′-apo-β-carotenal, which was produced by the oxidative system of A2, absorption maxima of all new ChRs (A1-6ex-
degradation of β,β-carotene,21) we prepared 5a by the C2- ChrimsonR [625nm] and A1-10ex-ChrimsonR [636nm],
elongated sequence from retinal (2a).25) In a similar fashion, A1-14ex-ChrimsonR [597nm]) were the same or of shorter
A2-14ex (5b) was prepared in 54% overall yield from 3,4-di- wavelengths than that of A2-ChrimsonR (636nm). When A2
dehydroretinal (2b)24) (Chart 4).
analogs were used as the chromophore, the λmax of A2-14ex-
ChrimsonR (634nm) was shorter than that of A2-ChrimsonR;
on the other side, the λmax of A2-6ex-ChrimsonR (651nm)
Binding Experiment with Opsins
With the new chromophores in hand, we next examined and A2-10ex-ChrimsonR (654nm) were slightly red-shifted
their binding efficiency with opsins of ReaChR and Chrim- (15 and 18nm) compared to that of A2-ChrimsonR. The red-
sonR. All analogs bound to both opsins to form new ChRs. shift values relative to A1-ChrimsonR (native ChrimsonR,
The spectra from ReaChR and ChrimsonR are shown in Fig. 587nm) were moved 64 and 67nm, respectively. Furthermore,
2 and 3, respectively, where the absorption spectra were nor- the width expansion of the absorption spectra in A2-6ex-
malized to absorbance 1.0 at 278nm. These data were sum- ChrimsonR and A2-10ex-ChrimsonR were more remarkable
marized in Tables 1, 2.
than for A2-14ex-ChrimsonR, with the long-wavelength spec-
Among the ReaChR analogs, the A2-ReaChR formed from tral boundaries being extended to 746nm (shift of 102nm)
2b, having an extra double bond in the cyclohexene ring and 756nm (shift of 112nm), respectively. In both cases, these
compared to retinal, showed λmax at 580nm with a 42nm positions were over 700nm and reached into the far-red region
red-shift compared to the A1-ReaChR (native ReaChR, λmax (710–850 nm).
at 538nm). In using A1 analogs having the same conjugated
The capability of a new ChR to absorb the visible light
system of A2, the absorption maxima of three new ChRs was estimated by the value of relative absorbance (Absmax
/
(A1-6ex-ReaChR [570nm], A1-10ex-ReaChR [569nm] and Abs278nm). While the relative absorbance depends on the bind-
A1-14ex-ReaChR [546nm]) were shorter than that of A2- ing efficiency of the analogs, it would be affected by the dif-
ReaChR. When A2-analogs, possessing a π-electron system ference in the extinction coefficients and/or stability of formed
elongated by one more double bond than A2, were used as pigments.18) All analogs seemed to bind with opsin smoothly
chromophore, the different results were obtained according to to afford new ReaChRs (Table 1). In contrast, in the case of
the chromophore. Thus, the A2-14ex-ReaChR showed λmax at ChrimsonR, a remarkable difference was observed. Thus,
579nm, which was almost the same as A2-ReaChR, whereas A2 and its analogs gave values similar to retinal, whereas the
A2-6ex-ReaChR and A2-10ex-ReaChR exhibited λmax at value of A1 analogs were significantly decreased (Table 2).
607nm and 596nm, respectively, which are longer than that of These results suggested that the binding pocket in ChrimsonR
A2-ReaChR and their red-shift values were 69nm and 58nm was not suitable for the A1 analogs due to their steric hin-
relative to the A1-ReaChR.
drance. Such steric hindrances might be reduced in the case
In addition, the shape of absorption spectra broadened of A2 analogs by the introduction of a double bond into the
the band width. As a result, the long-wavelength spectral cyclohexene ring. To clarify these observations, the interac-
boundary13) of A2-6ex-ReaChR and A2-10ex-ReaChR were tions between chromophore and amino acid residues of opsin
extended to 672nm (shift of 84nm) and 673nm (shift of are now in progress using computational docking simulations.
85nm), respectively. These red-shift values were greater than
that of A2-14ex-ReaChR (68nm). It is noteworthy that the po-
sition of inserted one double bond was very important in caus-
ing the effective red-shift in the absorption spectra.
Conclusion
We designed and prepared six new kinds of chromophores,
with one double bond inserted into the polyene side chain of
Similar tendencies were also observed in ChrimsonR ana- retinal (A1) or 3,4-didehydroretinal (A2) for producing the

268
Chem. Pharm. Bull.
Vol. 68, No. 3 (2020)
Fig. 3. UV-Vis Absorption Spectra of ChrimsonRs
Fig. 2. UV-Vis Absorption Spectra of ReaChRs
Bearing (A) A1 or its analogs and (B) A2 or its analogs. With A1 or A2 (black,
1), 6ex (red, 2), 10ex (green, 3) and 14ex (blue, 4). The contribution of ChrimsonR
regenerated by the endogenous A1 in HEK293 cells was subtracted. All the absorp-
tion spectra were normalized to 1.0 at 278nm.
Bearing (A) A1 or its analogs and (B) A2 or its analogs. With A1 or A2 (black,
1), 6ex (red, 2), 10ex (green, 3) and 14ex (blue, 4). The contribution of ReaChR
regenerated by the endogenous A1 in HEK293 cells was subtracted. All the absorp-
tion spectra were normalized to 1.0 at 278nm.
Table 1. Absorption Characteristics of ReaChR Aanalogs
A1-Analog
A2-Analog
A1
6ex
10ex
14ex
546
A2
580
6ex
607
10ex
14ex
579
λmax (nm)
538
570
569
596
Shift (Δ nm)a)
1/e λmax (nm)
Shift (Δ nm)a)
Relative absorbanceb)
—
32
632
44
31
635
47
8
620
32
42
634
46
69
672
84
58
673
85
41
656
68
588
—
0.35
0.20
0.21
0.20
0.28
0.20
0.24
0.21
a) Shift from A1-ReaChR. b) The values of Absmax/Abs278nm
.
Table 2. Absorption Characteristics of ChrimsonR Analogs
A1-Analog
A2-Analog
A1
6ex
10ex
14ex
597
A2
636
6ex
651
10ex
14ex
634
λmax (nm)
587
625
636
654
Shift (Δ nm)a)
1/e λmax (nm)
Shift (Δ nm)a)
Relative absorbanceb)
—
—
38
700
56
49
711
67
10
660
16
49
703
59
64
746
102
67
756
112
47
702
58
644
0.26
0.08
0.20
0.12
0.26
0.23
0.24
0.28
a) Shift from A1-ChrimsonR. b) The values of Absmax/Abs278nm
.
red-shifted ChRs. All chromophores bound with ReaChR promising for further development of a new red-shifted ChR.
and ChrimsonR to afford the new red-shifted ChRs. Further- Photochemical behaviors of these new ChRs are in progress
more, the absorption spectra of all new ChRs were broadened and will be published elsewhere.
towards the long-wavelength side and remarkable red-shifts
were observed. Among the new ChRs, A2-10ex-ChrimsonR
exhibited the longest red-shift effect in its absorption spec-
Experimental
UV-vis spectra were recorded on a JASCO Ubest-55 or
trum. Therefore, A2-10ex chromophore seemed to be the most JASCO V-650 instrument. IR spectra were measured on a

Vol. 68, No. 3 (2020)
Chem. Pharm. Bull.
269
PerkinElmer, Inc. FT-IR spectrometer, model Paragon 1000 ture was filtered through Celite with Et2O, and evaporated in
or Horiba FT-IR spectrometer, model FT-720. NMR spectra vacuo. The resultant allyl alcohol was used for the next MnO2
were obtained on a Varian Mercury-300, a VXR-500 or a oxidation step without further purification.
Brucker Avance III 600 superconducting FT-NMR spectrom-
To a solution of ally alcohol in Et2O (0.04M) was added
eter. Chemical shifts (δ) are reported in ppm relative to tetra- MnO2 (10 times weight relative to ester) at r.t. and was stirred
methylsilane as an internal reference (CDCl3: δ=0ppm for for 14–18h. The resulting mixture was filtered through Celite
1H) or residual solvent signals (CDCl3: δ=77ppm for 13C). with Et2O, and concentrated in vacuo. The residue was puri-
Mass spectra were taken on a Thermo Fisher Scientific Exac- fied by flash column chromatography on silica gel to afford
tive spectrometer. Column chromatography was performed the aldehyde as pure E stereoisomer.
using Kanto Silica Gel 60N (spherical, neutral). All reagents
were used as obtained commercially unless otherwise noted.
(E)-3-(2,6,6-Trimethylcyclohexen-1-yl)-2-propen-1-al
(7a) According to GP1, ethyl (E)-3-(2,6,6-trimethylcyclo-
General Procedure for the Synthesis of C2-Elongeted hexen-1-yl)-2-propenate (1.37g, 94%) was obtained from
Conjugate Ester by Horner–Wadsworth–Emmons Reaction β-cyclocitral (6a: 1.00g, 6.57mmol), triethyl phosphonoacetate
(GP1) To a stirred solution of triethyl phosphonoacetate (C2- (7.36g, 32.9mmol) and n-BuLi (20.5mL, 1.57M in hexane,
phosphonate, 2.5–5.0eq.) in dry tetrahydrofuran (THF) (0.2M/ 32.2mmol). Eluent: hexane/EtOAc=9:1. According to GP3,
phosphonate) was added a solution of NaN(TMS)2 (2.5–5.0eq.) 7a (428mg, 37%, in 3 steps from 6a) was obtained from ethyl
or n-BuLi (2.5–5.0eq.) at 0°C, and the mixture was stirred at (E)-3-(2,6,6-trimethylcyclohexen-1-yl)-2-propenoate
(1.37g,
the same temperature for 15min. To this mixture a solution 6.18mmol), LiAlH4 (469mg, 12.4mmol) and MnO2 (85% pu-
of aldehyde (1eq.) in THF (1M/aldehyde) was added, and the rity, 14.0g, 137mmol). Eluent: hexane/Et2O=4:1. Spectral
reaction mixture was stirred at room temperature (r.t.) until data were identical with those of the literature.26)
the reaction was completed. The reaction was quenched with a
7a: Pale yellow oil. IR (CHCl3) νmax cm−1: 2928, 1676, 1605.
saturated aqueous solution of NH4Cl, and then extracted with 1H-NMR (300MHz, CDCl3) δ: 9.51 (1H, d, J=7.8Hz), 7.26
ethyl acetate. The combined extracts were washed with brine, (1H, d, J=16.2Hz), 6.17 (1H, dd, J=16.2, 7.8Hz), 2.10 (2H,
dried over Na2SO4, and concentrated in vacuo. The residue t, J=6.0Hz), 1.79 (3H, s), 1.70–1.53 (2H, m), 1.53–1.47 (2H,
was purified by flash chromatography on silica gel to afford m), 1.09 (6H, s). 13C-NMR (75MHz, CDCl3) δ: 194.95, 152.99,
the one double bond elongated conjugate ester. In the case of 139.80, 136.25, 132.79, 40.17, 34.26, 34.24, 28.90, 22.02, 18.94.
A2 analogs, sodium hydride (NaH) (1.5eq., 60% suspension in
mineral oil) was used as a base instead of NaN(TMS)2.
(E)-3-(2,6,6-Trimethylcyclohexa-1,3-dien-1-yl)-2-propen-
1-al (7b) According to GP1, ethyl (E)-3-(2,6,6-trimethylcy-
General Procedure for the Synthesis of C5-Elongeted clohexa-1,3-dien-1-yl)-2-propenate (11.9g, 87%) was obtained
Conjugate Ester by Horner–Wadsworth–Emmons Reaction from safranal (6b: 9.33mL, 60.0mmol), triethyl phosphono-
(GP2) To a stirred solution of triethyl 3-methyl-4-phospho- acetate (20.2g, 90.0mmol) and NaH (60%, 3.60g, 90.0mmol).
nocrotonate (C5-phosphonate, 3.0eq.) and DMPU (4eq.) in Eluent: hexane/Et2O=20:1. According to GP3, 7b (3.74g,
dry THF (0.2M/aldehyde), n-BuLi (3.0eq.) was added at 0°C. 35%, in 3 steps from 6b) was obtained from ethyl (E)-
The resulting mixture was stirred at the same temperature for 3-(2,6,6-trimethylcyclohexa-1,3-dien-1-yl)-2-propenate (11.9g,
20min and then cooled to −78°C. To this mixture a solution 53.8mmol), LiAlH4 (5.11g, 135mmol) and MnO2 (85% purity,
of aldehyde (1eq.) in THF (1M/aldehyde) was slowly added, 119g, 1.16mol). Eluent: hexane/Et2O=10:1
and the reaction mixture was allowed to come to r.t. and
7b: Yellow solid. IR (CHCl3) νmax cm−1: 2962, 1670, 1607.
then stirred for overnight. The reaction was quenched with a 1H-NMR (300MHz, CDCl3) δ: 9.54 (1H, d, J=7.8Hz), 7.26
saturated aqueous solution of NH4Cl, and then extracted with (1H, d, J=16.2Hz), 6.26 (1H, dd, J=16.2, 7.8Hz), 5.99–5.89
ethyl acetate. The combined extracts were washed with brine, (2H, m), 2.15 (2H, d, J=3.3Hz), 1.94 (3H, s), 1.11 (6H, s).
dried over Na2SO4, and concentrated in vacuo. The residue 13C-NMR (75MHz, CDCl3) δ: 194.35, 151.23, 135.69, 135.09,
was purified by flash chromatography on silica gel to afford 131.62, 129.68, 129.64, 40.16, 34.01, 26.53, 20.41. High
the two double bonds elongated conjugate ester as a stereoiso- resolution electrospray ionization (HR-ESI) MS Calcd for
meric mixture (E:Z=9:1 to 18:1). The mixture was used for C12H17O [M+H]+: 177.1274. Found: 177.1273.
the next reaction without further separation of stereoisomers.
(2E,4E,6E)-3-Methyl-7-(2,6,6-trimethylcyclohexen-1-yl)-
General Procedure for the Conversion from Ester to 2,4,6-heptatrien-1-al (8a) According to GP2, ethyl (E)-
Aldehyde (GP3) In the case of A1-analogs, to a stirred sus- 3-methyl-7-(2,6,6-trimethylcyclohexen-1-yl)-2,4,6-heptatrieno-
pension of LiAlH4 (2.0eq.) in dry Et2O (0.2M/aldehyde) was ate (628mg, 90%) was obtained from 7a (428mg, 2.40mmol),
added a solution of ester (1eq.) in Et2O (0.2M/ester) at 0°C, triethyl 3-methyl-4-phosphonocrotonate (1.77g, 7.20mmol),
and the resulting mixture was stirred at the same temperature n-BuLi (4.50mL, 1.57M in hexane, 7.07mmol) and DMPU
for 1h. To this mixture aqueous silica gel (H2O/SiO2 =1:5) (1.10mL, 9.59mmol). Eluent: hexane/EtOAc=9:1. Ac-
was added and the resulting mixture was stirred at r.t. for 1h. cording to GP3, 8a (360mg, 58%, in 3 steps from 7a) was
The reaction mixture was filtered through Celite with Et2O, obtained from ethyl (2E,4E,6E)-3-methyl-7-(2,6,6-trimeth-
dried over Na2SO4, and concentrated in vacuo. The resultant ylcyclohexen-1-yl)-2,4,6-heptatrienoate (628mg, 2.18mmol),
allyl alcohol was used for the next MnO2 oxidation step with- LiAlH4 (165mg, 4.35mmol) and MnO2 (85% purity, 6.30g,
out further purification.
61.6mmol). Eluent: hexane/Et2O=4:1. This compound is
1
In the case of A2-analogs, to a solution of ester (1eq.) in known and main signals of H-NMR were reported in the
THF (0.1M/ester) was added DIBAL (3.5eq., 1.0M in hexane) literature.27)
dropwise at −78°C, and the mixture was stirred at the same
8a: Pale yellow oil. IR (CHCl3) νmax cm−1: 2932, 1656, 1594.
temperature for 2h. The mixture was quenched with H2O/ 1H-NMR (300MHz, CDCl3) δ: 10.10 (1H, d, J=8.1Hz), 6.82
SiO2, and was stirred at r.t. for 30min. The resulting mix- (1H, dd, J=15.6, 10.5Hz), 6.46 (1H, d, J=15.6Hz), 6.31 (1H,

270
Chem. Pharm. Bull.
Vol. 68, No. 3 (2020)
d, J=15.6Hz), 6.26 (1H, dd, J=15.6, 10.5Hz), 5.95 (1H, d, 3-methyl-4-phosphonocrotonate (3.75g, 12.0mmol), n-BuLi
J=8.1Hz), 2.31 (3H, s), 2.05 (2H, t, J=6.3Hz), 1.75 (3H, s), (7.59mL, 1.58M in hexane, 12.0mmol) and DMPU (1.93mL,
1.64–1.56 (2H, m), 1.52–1.44 (2H, m), 1.05 (6H, s). 13C-NMR 16.0mmol). Eluent: hexane/EtOAc=30:1. According to
(75MHz, CDCl3) δ: 191.16, 154.91, 137.53, 137.25, 133.17, GP3, 3b (1.32g, 54%, in 3 steps from 8b) was obtained from
132.56, 132.40, 128.95, 39.71, 34.15, 33.39, 28.90, 21.79, 19.05, ethyl
(2E,4E,6E,8E,10E)-3,7-dimethyl-11-(2,6,6-trimethyl-
13.00.
cyclohexa-1,3-dien-1-yl)-2,4,6,8,10-undecapentaenoate (2.99g,
(2E,4E,6E)-3-Methyl-7-(2,6,6-trimethylcyclohexa-1,3- 8.49mmol), DIBAL (29.7mL, 1.0M in hexane, 29.7mmol)
dien-1-yl)-2,4,6-heptatrien-1-al (8b) According to GP2, and MnO2 (85% purity, 29.9g, 292mmol). Eluent: hexane/
ethyl
dien-1-yl)-2,4,6-heptatrienoate (7.98g, 95%) was obtained
from 7b (5.20g, 29.5mmol), triethyl 3-methyl-4- phospho- en-1-yl)-2,4,6-heptatrien-1-al (10a) According to GP1,
nocrotonate (11.6g, 44.2mmol), n-BuLi (28.0mL, 1.58M in ethyl
(2E,4E,6E)-5-methyl-7-(2,6,6-trimethylcyclohexen-
hexane, 44.2mmol) and DMPU (7.11mL, 59.0mmol). Eluent: 1-yl)-2,4,6- heptatrienoate (1.20g, 99%) was obtaind
hexane/EtOAc=30:1.
from β-ionylideneacetaldehyde23) (9a: 916mg, 4.20mmol),
(2E,4E,6E)-3-methyl-7-(2,6,6-trimethylcyclohexa-1,3- Et2O=5:1. Spectral data were shown in the literature.20)
(2E,4E,6E)-5-Methyl-7-(2,6,6-trimethylcyclohex-
According to GP3, 8b (4.72g, 66%, in 3 steps from 7b) was NaN(TMS)2 (10.5mL, 1.0M in THF, 10.5mmol) and tri-
obtained from ethyl (2E,4E,6E)-3-methyl-7-(2,6,6-trimethylcy- ethyl phosphonoacetate (2.40g, 10.5mmol). Eluent: hexane/
clohexa-1,3-dien-1-yl)-2,4,6-heptatrienoate (7.98g, 27.9mmol), Et2O=8:1. According to GP3, 10a (586mg, 62%, in 3 steps
DIBAL (100mL, 1.0M in hexane, 100mmol) and MnO2 (85% from 9a) was obtained from ethyl (2E,4E,6E)-5-methyl-
purity, 79.8g, 780mmol). Eluent: hexane/Et2O=4:1.
7-(2,6,6-trimethylcyclohexen-1-yl)-2,4,6-heptatrienoate (1.09g,
8b: Yellow solid. IR (CHCl3) νmax cm−1: 3013, 1651, 1592. 3.78mmol), LiAlH4 (287mg, 7.57mmol) and MnO2 (85%
1H-NMR (300MHz, CDCl3) δ: 10.11 (1H, d, J=8.1Hz), 6.83 purity, 11.0g, 107mmol). Eluent: hexane/Et2O=4:1. Spectra
(1H, dd, J=15.6, 9.9Hz), 6.48 (1H, d, J=15.6Hz), 6.36 (1H, data were identical with those of the literature.26)
dd, J=15.6, 9.9Hz), 6.35 (1H, d, J=15.6Hz), 5.96 (1H, d,
10a: Pale yellow oil. IR (CHCl3) νmax cm−1: 2932, 1663,
1
J=8.1Hz), 5.87 (1H, d, J=9.6Hz), 5.79 (1H, dt, J=9.6, 1590. H-NMR (300MHz, CDCl3) δ: 9.61 (1H, d, J=8.1Hz),
4.2Hz), 2.31 (s, 3H), 2.10 (2H, dd, J=4.2, 0.9Hz), 1.90 (3H, 7.53 (1H, dd, J=15.0, 12.0Hz), 6.50 (1H, d, J=15.9Hz), 6.29
s), 1.06 (6H, s). 13C-NMR (75MHz, CDCl3) δ: 191.00, 154.66, (1H, d, J=12.0Hz), 6.20 (1H, d, J=15.9Hz), 6.15 (1H, dd,
137.48, 137.42, 135.86, 133.45, 131.81, 129.84, 129.51, 128.96, J=15.0, 8.1Hz), 2.09 (3H, s), 2.04 (2H, t, J=6.0Hz), 1.72
126.43, 39.92, 33.86, 26.71, 20.39, 12.92. HR-ESIMS Calcd for (3H, s), 1.69–1.58 (2H, m), 1.51–1.42 (2H, m), 1.04 (6H, s).
C17H23O [M+H]+: 243.1743. Found: 243.1745.
13C-NMR (75MHz, CDCl3) δ: 193.95, 148.29, 147.01, 137.60,
(2E,4E,6E,8E,10E)-3,7-Dimethyl-11-(2,6,6-trimethyl- 136.64, 132.73, 131.82, 130.95, 128.65, 39.80, 34.48, 33.43,
cyclohexen-1-yl)-2,4,6,8,10-undecapentaen-1-al (A1-6ex, 29.18, 22.01, 19.34, 13.37.
3a) According to GP2, ethyl (2E,4E,6E,8E,10E)-3,7- (2E,4E,6E)-5-Methyl-7-(2,6,6-trimethylcyclohexa-1,3-
dimethyl-11-(2,6,6-trimethylcyclohexen-1-yl)-2,4,6,8,10-un- dien-1-yl)-2,4,6-heptatrien-1-al (10b) According to GP1,
decapentaenoate (459mg, 93%) was obtained from 8a ethyl (2E,4E,6E)-5-methyl-7-(2,6,6-trimethylcyclohexa-1,3-
(360mg, 1.39mmol), triethyl 3-methyl-4-phosphonocrotonate dien-1-yl)-2,4,6-heptatrienoate (1.74g, 79%) was obtained from
(1.02g, 4.16mmol), n-BuLi (2.60mL, 1.57M in hexane, 9b24) (1.66g, 7.69mmol), triethyl phosphonoacetate (2.59g,
4.08mmol) and DMPU (0.630mL, 5.23mmol). Eluent: hexane/ 11.5mmol) and NaH (60%, 0.46g, 11.5mmol). Eluent: hex-
EtOAc =9:1. According to GP3, 3a (99.0mg, 21%, in 3 steps ane/Et2O=15:1. According to GP3, 10b (0.914g, 49%, in 3
from 8a) was obtained from ethyl (2E,4E,6E,8E,10E)-3,7- steps from 9b) was obtained from ethyl (2E,4E,6E)-5-methyl-
dimethyl-11-(2,6,6-trimethylcyclohexen-1-yl)-2,4,6,8,10-un- 7-(2,6,6-trimethylcyclohexa-1,3-dien-1-yl)-2,4,6-heptatrieno-
decapentaenoate (358mg, 1.01mmol), LiAlH4 (77.0mg, ate (1.74g, 6.07mmol), DIBAL (21.2mL, 1.0M in hexane,
2.02mmol) and MnO2 (85% purity, 3.61g, 35.3mmol). Eluent: 21.2mmol) and MnO2 (85% purity, 17.4g, 170mmol). Eluent:
hexane/Et2O=4:1.
3a: Orange solid. UV-vis (EtOH) λmax 403nm (ε=54300).
hexane/Et2O=8:1.
10b: Yellow solid. IR (CHCl3) νmax cm−1: 3018, 1667, 1609.
nm. IR (CHCl3) νmax cm−1: 2931, 1651, 1602, 1561. H-NMR 1H-NMR (300MHz, CDCl3) δ: 9.62 (1H, d, J=8.1Hz), 7.55
(300MHz, CDCl3) δ: 10.10 (1H, d, J=8.4Hz), 7.12 (1H,dd, (1H, dd, J=15.0, 11.7Hz), 6.51 (1H, d, J=16.2Hz), 6.34
J=15.0, 11.4Hz), 6.50 (1H, dd, J=15.0, 9.9Hz), 6.37 (1H, d, (1H, d, J=16.2Hz), 6.33 (1H, d, J=11.7Hz), 6.18 (1H, dd,
J=15.0Hz), 6.29 (2H, d, J=15.0Hz), 6.22 (1H, d, J=11.4Hz), J=15.0, 8.1Hz), 5.87 (1H, d, J=9.6Hz), 5.79 (1H, dt, J=9.6,
6.19 (1H, dd, J=15.3, 9.9Hz), 5.98 (1H, d, J=8.1Hz), 2.33 4.5Hz), 2.12–2.08 (5H, m), 1.89 (3H, s), 1.05 (6H, s). 13C-NMR
(3H, s), 2.03 (2H, t, J=6.0Hz), 2.02 (3H, s), 1.74 (3H, s), (75MHz, CDCl3) δ: 193.60, 147.83, 146.53, 137.78, 135.67,
1.64–1.58 (2H, m), 148–1.45 (2H, m), 1.04 (6H, s). 13C-NMR 131.18, 130.76, 129.76, 128.79, 127.80, 126.20, 39.86, 33.98,
(75MHz, CDCl3) δ: 191.06, 154.70, 141.15, 137.47, 134.86, 26.71, 20.32, 12.99. HR-ESIMS Calcd for C17H23O [M+H]+:
134.71, 133.52, 133.38, 132.44, 132.19, 131.04, 130.24, 129.07, 243.1743. Found: 243.1741.
1
39.72, 34.17, 33.30, 28.92, 21.79, 19.15, 13.08. HR-ESIMS
(2E,4E,6E,8E,10E)-3,9-Dimethyl-11-(2,6,6-trimethyl-
cyclohexen-1-yl)-2,4,6,8,10-undecapentaen-1-al (A1-10ex,
Calcd for C22H31O [M+H]+: 311.2369. Found: 311.2368.
(2E,4E,6E, 8E,10E)-3,7-Dimethyl-11-(2,6,6 -tri- 4a) According to GP2, ethyl (2E,4E,6E,8E,10E)-3,9-
methylcyclohexa-1, 3-dien-1-yl)-2,4,6,8,10-undeca- dimethyl-11-(2,6,6-trimethylcyclohexen-1-yl)-2,4,6,8,10-unde-
pentaen-1-al (A2-6ex, 3b) According to GP2, ethyl capentaenoate (820mg, 96%) was obtaind from 10a (586mg,
(2E,4E,6E,8E,10E)-3,7-dimethyl-11-(2,6,6-trimethyl- 2.40mmol), triethyl 3-methyl-4-phosphonocrotonate (1.77g,
cyclohexa-1,3-dien-1-yl)-2,4,6,8,10-undecapentaenoate (2.99g, 7.19mmol), n-BuLi (4.50mL, 1.57M in hexane, 7.07mmol)
quant.) was obtained from 8b (1.94g, 8.00mmol), triethyl and DMPU (1.10mL, 9.59mmol). Eluent: hexane/Et2O=8:1.

Vol. 68, No. 3 (2020)
Chem. Pharm. Bull.
271
According to GP3, 4a (235mg, 36%, in 3 steps from 10a) was 14ex, 5b) According to GP1, ethyl (2E,4E,6E,8E,10E)-5,9-
obtained from ethyl (2E,4E,6E,8E,10E)-3,9-dimethyl-11-(2,6,6- dimethyl-11-(2,6,6-trimethylcyclo-hexa-1,3-dien-1-yl)-
trimethyl-cyclohexen-1-yl)-2,4,6,8,10-undecapentaenoate 2,4,6,8,10-undecapentaenoate (115mg, 85%) was obtained
(709mg, 2.00mmol), LiAlH4 (152mg, 4.01mmol) and MnO2 from 3,4-didehydroretinal22) (2b: 109mg, 0.387mmol), tri-
(85% purity, 7.10g, 69.4mmol). Eluent: hexane/Et2O=4:1.
ethyl phosphonoacetate (130mg, 0.581mmol) and NaH
4a: Orange solid. UV-vis (EtOH) λmax 403nm (ε=54000). (60%, 23.2mg, 0.581mmol). Eluent: hexane/Et2O=20:1.
IR (CHCl3) νmax cm−1: 2926, 1651, 1599,1561. 1H-NMR According to GP3, 5b (64.1mg, 54%, in 3 steps from 2b)
(300MHz, CDCl3) δ: 10.10 (1H, d, J=8.1Hz), 6.99–6.78 (2H, was obtained from ethyl (2E,4E,6E,8E,10E)-5,9-dimethyl-
m), 6.35 (1H, d, J=15.0Hz), 6.34 (1H, dd, J=15.0, 10.5Hz), 11-(2,6,6-trimethyl-cyclohexa-1,3-dien-1-yl)-2,4,6,8,10-undeca-
6.27 (1H, d, J=15.6Hz), 6.15 (1H, d, J=10.5Hz), 6.14 (1H, pentaenoate (115mg, 0.327mmol), DIBAL (1.31mL, 1.0M in
d, J=15.6Hz), 5.96 (1H, d, J=8.1Hz), 2.30 (3H, s), 2.02 (2H, hexane, 1.31mmol) and MnO2 (85% purity,1.15g, 11.2mmol).
t, J=6.3Hz), 1.99 (3H, s), 1.72 (3H, s), 1.63–1.54 (2H, m), Eluent: hexane/Et2O=5:1. Spectral data were shown in the
1.50–1.42 (2H, m), 1.03 (6H, s). 13C-NMR (75MHz, CDCl3) δ: literature.20)
191.09, 154.56, 139.51, 137.70, 137.23, 136.92, 134.46, 134.23,
Preparation of ChR Analogs and Spectroscopy In this
131.59, 130.18, 129.68, 129.14, 128.81, 39.59, 34.26, 33.13, study, two ChR variants, ReaCh (NCBI accession number,
28.96, 21.76, 19.19, 12.98, 12.79. HR-ESIMS Calcd for C22H31O KF448069)11) and ChrimsonR (Chrimson (KF992060) K176R
[M+H]+:311.2369. Found: 311.2369.
mutant)19) were used. These ChR variants were expressed by
(2E,4E,6E,8E,10E)-3,9-Dimethyl-11-(2,6,6-trimethyl- HEK293 system as reported previously.18) Briefly, the cDNA of
cyclohexa-1,3-dien-1-yl)-2,4,6,8,10-undecapentaen-1-al (A2- these ChR variants were inserted into the mammalian expres-
10ex, 4b) According to GP2, ethyl (2E,4E,6E,8E,10E)-3,9-di- sion vector pCAGGS with the epitope sequence of anti-bovine
methyl-11-(2,6,6-trimethylcyclohexa-1,3-dien-1-yl)-2,4,6,8,10- rhodopsin monoclonal antibody rho1D4 (ETSQVAPA) on the
undecapentaenoate (714mg, quant.) was obtained from 10b C-terminus. HEK293 cells transfected with the plasmid bear-
(450mg, 1.86mmol), triethyl 3-methyl-4-phosphonocrotonate ing the ChR gene were collected after incubation for 2d, and
(746mg, 2.79mmol), n-BuLi (1.74mL, 1.58M in hexane, suspended in Buffer P (50mM N-(2-hydroxyethyl)piperazine-
2.79mmol) and DMPU (0.448mL, 3.71mmol). Eluent: hexane/ N′-2-ethanesulfonic acid (HEPES) and 140mM NaCl, pH
EtOAc =30:1. According to GP3, 4b (127mg, 22%, in 3 steps 6.5). Retinal or analog dissolved in ethanol was added to the
from 10b) was obtained from ethyl (2E,4E,6E,8E,10E)-3,9-di- sample at final concentration of 20µM. After overnight incu-
methyl-11-(2,6,6-trimethylcyclohexa-1,3-dien-1-yl)-2,4,6,8,10- bation at 4°C, the regenerated pigments were extracted by 1%
undecapentaenoate (714mg, 2.02mmol), DIBAL (7.08mL, n-dodecyl-β-D-maltoside (DDM) in Buffer P, and purified by
1.0M in hexane, 7.08mmol) and MnO2 (85% purity, 7.14g, rho1D4-antibody conjugated agarose column. The final solvent
69.8mmol). Eluent: hexane/Et2O=5:1. Spectral data were of the sample was Buffer P with 0.02% DDM at pH 6.5. All
shown in the literature.20)
(2E,4E,6E,8E,10E)-5,9-Dimethyl-11-(2,6,6-trimethyl-
the procedures above were performed under deep-red light.
UV-visible absorption spectra of pigments were measured
cyclohexen-1-yl)-2,4,6,8,10-undecapentaen-1-al
(A1-14ex, by UV-visible spectrophotometer (Shimadzu UV-2400), which
5a) According to GP1, ethyl (2E,4E,6E,8E,10E)-5,9- is equipped with a water circulation system to the cell holder
dimethyl-11-(2,6,6-trimethylcyclohexen-1-yl)-2,4,6,8,10-unde- to keep the sample at 4°C. HEK293 cells produce A1,29) which
capentaenoate (565mg, 96%) was obtaind from retinal25) generates approx. 10% (ReaChR) or approx. 50% (Chrim-
(2a: 477mg, 1.59mmol), triethyl phosphonoacetate (1.07g, sonR) of fully regenerated pigment with excess A1. To show
4.47mmol) and NaN(TMS)2 (4.47mL, 1.0M in THF, the pure spectra of new ChRs, the contribution of ChR regen-
4.47mmol). Eluent: hexane/EtOAc=9:1.
erated by the endogenous A1 was subtracted from the sample
According to GP3, 5a (82.0mg, 19%, in 3 steps from 2a) spectra, and the absorption spectra were normalized to absor-
was obtained from ethyl (2E,4E,6E,8E,10E)-5,9-dimethyl- bance 1.0 at 278nm.
11-(2,6,6-trimethylcyclohexen-1-yl)-2,4,6,8,10-undecapentae-
noate (466mg, 1.26mmol), LiAlH4 (96.0mg, 2.52mmol)
Acknowledgments This work was supported in part by
and MnO2 (85% purity, 4.60g, 45.0mmol). Eluent: hexane/ JSPS KAKENHI Grant No 26450146 (to AW), The Science
Et2O=4:1. Spectra data were identical with those of the lit- Research Promotion Fund from The Promotion and Mutual
erature.28)
Aid Corporation for Private Schools of Japan and The Kobe
5a: Orange solid. UV-vis (EtOH) λmax 405nm (ε=48000). Pharmaceutical Collaboration Fund.
IR (CHCl3) νmax cm−1: 2926, 1667, 1603, 1559. 1H-NMR
(600MHz, CDCl3) δ: 9.61 (1H, d, J=7.8Hz), 7.51 (1H,
Conflict of Interest The authors declare no conflict of
dd, J=15.0, 12.0Hz), 6.92 (1H, dd, J=14.4, 11.4Hz), 6.38 interest.
(1H, d, J=15.0Hz), 6.35 (1H, d, J=12.0Hz), 6.29 (1H, d,
J=16.2Hz), 6.18 (1H, dd, J=12.6, 7.8Hz), 6.17 (1H, d,
Supplementary Materials The online version of this ar-
J=12.6Hz), 6.15 (1H, d, J=16.2Hz), 2.11 (3H, s), 2.04 (2H, ticle contains supplementary materials.
t, J=6.0Hz), 2.01 (3H, s), 1.72 (3H, s), 1.65–1.58 (2H, m),
1.49–1.45 (2H, m), 1.04 (6H, s). 13C-NMR (150MHz, CDCl3)
References
1) Nagel G., Ollig D., Fuhrmann M., Kateriya S., Musti A. M., Bam-
δ: 193.56, 147.64, 146.68, 139.45, 137.75, 137.32, 135.51, 130.77,
130.15, 129.96, 129.89, 128.72, 128.48, 39.64, 34.29, 33.15,
28.98, 21.77, 19.22, 13.29, 12.93.
(2E,4E,6E,8E,10E)-5,9-Dimethyl-11-(2,6,6-trimethyl-
cyclohexa-1,3-dien-1-yl)-2,4,6,8,10-undecapentaen-1-al (A2-
berg E., Hegemann P., Science, 296, 2395–2398 (2002).
2) Ernst O. P., Lodowski D. T., Elstner M., Hegemann P., Brown L. S.,
Kandori H., Chem. Rev., 114, 126–163 (2014).
3) Deisseroth K., Feng G., Majewska A. K., Miesenböck G., Ting A.,

272
Chem. Pharm. Bull.
Vol. 68, No. 3 (2020)
Schnitzer M. J., J. Neurosci., 26, 10380–10386 (2006).
moto Y., Shichida Y., Wada A., Chem. Pharm. Bull., 65, 356–358
(2017).
17) Wang H., Sugiyama Y., Hikima T., Sugano E., Tomita H., Takahashi
T., Ishizaki T., Yawo H., J. Biol. Chem., 284, 5685–5696 (2009).
4) Deisseroth K., Nat. Methods, 8, 26–29 (2011).
5) Yizhar O., Fenno L. E., Davidson T. J., Mogri M., Deisseroth K.,
Neuron, 71, 9–34 (2011).
6) Zhang F., Vierock J., Yizhar O., Fenno L. E., Tsunoda S., Kianian- 18) Shen Y. C., Sasaki T., Matsuyama T., Yamashita T., Shichida Y.,
momeni A., Prigge M., Berndt A., Cushman J., Polle J., Magnuson
J., Hegemann P., Deisseroth K., Cell, 147, 1446–1457 (2011).
7) Bi A., Cui J., Ma Y.-P., Olshevskaya E., Pu M., Dizhoor A. M., Pan
Z.-H., Neuron, 50, 23–33 (2006).
Okitsu T., Yamano Y., Wada A., Ishizuka T., Yawo H., Imamoto Y.,
Biochemistry, 57, 5544–5556 (2018).
19) Klapoetke N. C., Murata Y., Kim S. S., et al., Nat. Methods, 11,
338–346 (2014).
8) Kravitz A. V., Freeze B. S., Parker P. R. L., Kay K., Thwin M. T.,
Deisseroth K., Kreitzer A. C., Nature (London), 466, 622–626 (2010).
9) Lin J. Y., Exp. Physiol., 96, 19–25 (2011).
10) Prigge M., Schneider F., Tsunoda S. P., Shilyansky C., Wietek J.,
Deisseroth K., Hegemann P., J. Biol. Chem., 287, 31804–31812 (2012).
11) Lin J. Y., Knutsen P. M., Muller A., Kleinfeld D., Tsien R. Y., Nat.
Neurosci., 16, 1499–1508 (2013).
20) Takayama R., Kaneko A., Okitsu T., Tsunoda S. P., Shimono K.,
Mizuno M., Kojima K., Tsukamoto T., Kandori H., Mizutani Y.,
Wada A., Sudo Y., J. Phys. Chem. Lett, 9, 2857–2862 (2018).
21) Eroglu A., Harrison E. H., J. Lipid Res., 54, 1719–1730 (2013).
22) Bennani Y. L., J. Org. Chem., 61, 3542–3544 (1996).
23) Wada A., Hiraishi S., Takamura N., Date T., Aoe K., Ito M., J. Org.
Chem., 62, 4343–4348 (1997).
12) Zhang F., Prigge M., Beyrière F., Tsunoda S. P., Mattis J., Yizhar
24) Wada A., Wang F., Ito M., Chem. Pharm. Bull., 56, 112–114 (2008).
O., Hegemann P., Deisseroth K., Nat. Neurosci., 11, 631–633 (2008). 25) Hruszkewycz D. P., Cavanaugh K. R., Takamura K. T., Wayman L.
13) Sineshchekov O. A., Govorunova E. G., Wang J., Spudich J. L., Bio-
chemistry, 51, 4499–4506 (2012).
14) AzimiHashemi N., Erbguth K., Vogt A., Riemensperger T., Rauch
E., Woodmansee D., Nagpal J., Brauner M., Sheves M., Fiala A.,
Kattner L., Trauner D., Hegemann P., Gottschalk A., Liewald J. F.,
Nat. Commun., 5, 5810 (2014).
15) Ganapathy S., Bécheau O., Venselaar H., Frölich S., van der Steen J.
B., Chen Q., Radwan S., Lugtenburg J., Hellingwerf K. J., de Groot
H. J. M., de Grip W. J., Biochem. J., 467, 333–343 (2015).
16) Okitsu T., Matsuyama T., Yamashita T., Ishizuka T., Yawo H., Ima-
M., Curley R. W. Jr., Synthesis, 2011, 2205–2207 (2011).
26) Bassolino G., Sovdat T., Liebel M., Schnedermann C., Odell B.,
Claridge T. D. W., Kukura P., Fletcher S. P., J. Am. Chem. Soc., 136,
2650–2658 (2014).
27) Colmenares L. U., Liu R. S. H., Tetrahedron, 47, 3711–3718 (1991).
28) Domínguez M., Pequerul R., Alvarez R., Giménez-Dejoz J., Birta
E., Porté S., Rűhl R., Parés X., Farrés J., de Lera A. R., Tetrahe-
dron, 74, 2567–2574 (2018).
29) Brueggemann L. I., Sullivan J. M., J. Gen. Physiol., 119, 593–612
(2002).