A two-photon fluorescent lipid raft probe derived from dicyanostilbene and similar to cholesterol’s structure

Article abstract of DOI:10.1007/s00706-021-02826-3

Lipid raft is a microdomain being rich in cholesterol and sphingolipid and closely related to neurodegenerative diseases such as Alzheimer’s disease and prion disease. Hence the study on the physiological mechanism of lipid raft is helpful to reveal its biological role and its correlations with diseases. Two-photon fluorescence probe for lipid raft is a sharp tool to achieve this goal. A dicyanocarbazolylstilbene-derived two-photon fluorescence probe for lipid raft (DLR) was developed. Both DLR and lipid raft belong to D-π-A (donor-π-acceptor; π: C = C) type rigid planar molecule, and contain four fused carbon rings (one five-membered ring and three six-membered ring), and bear a long carbon chain (C₈H₁₇). The excellent comparability between the rigid plane of the probe molecule and the rigid lipid raft could significantly enhance the specific affinity to lipid rafts. DLR’s maximum emission wavelength increased with medium polarity while its fluorescence intensity (FI) augmented with viscosity, and FI in DPPC (dipalmitoylphosphatidylcholine) was 20 times higher than that in DOPC (dioleoylphosphatidylcholine). DLR’s fluorescence lifetime in DPPC was more than 2.2 times longer than that in DOPC. These indicate that DLR can distinguish DPPC from DOPC. Φδs of DLR in DPPC and DOPC were 1350 GM and 67 GM, respectively. DLR can image lipid raft distribution in cells and tissues. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00706-021-02826-3

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-021-02826-3
ORIGINAL PAPER
A two‑photon fuorescent lipid raft probe derived
from dicyanostilbene and similar to cholesterol’s structure
Chibao Huang1,3 · Yuzhu Yang2 · Youfeng Li1 · Guoling Lv1
Received: 26 February 2021 / Accepted: 12 July 2021
© Springer-Verlag GmbH Austria, part of Springer Nature 2021
Abstract
Lipid raft is a microdomain being rich in cholesterol and sphingolipid and closely related to neurodegenerative diseases
such as Alzheimer’s disease and prion disease. Hence the study on the physiological mechanism of lipid raft is helpful to
reveal its biological role and its correlations with diseases. Two-photon fuorescence probe for lipid raft is a sharp tool to
achieve this goal. A dicyanocarbazolylstilbene-derived two-photon fuorescence probe for lipid raft (DLR) was developed.
Both DLR and lipid raft belong to D-π-A (donor-π-acceptor; π: C=C) type rigid planar molecule, and contain four fused
carbon rings (one fve-membered ring and three six-membered ring), and bear a long carbon chain (C8H17). The excellent
comparability between the rigid plane of the probe molecule and the rigid lipid raft could signifcantly enhance the specifc
afnity to lipid rafts. DLR’s maximum emission wavelength increased with medium polarity while its fuorescence intensity
(FI) augmented with viscosity, and FI in DPPC (dipalmitoylphosphatidylcholine) was 20 times higher than that in DOPC
(dioleoylphosphatidylcholine). DLR’s fuorescence lifetime in DPPC was more than 2.2 times longer than that in DOPC.
These indicate that DLR can distinguish DPPC from DOPC. Φδs of DLR in DPPC and DOPC were 1350 GM and 67 GM,
respectively. DLR can image lipid raft distribution in cells and tissues.
Graphic abstract
Keywords Lipid raft · Dicyanostilbene · Carbazole · Two-photon fuorescence · Cholesterol
Introduction
A lipid raft (10–100 nm membrane patches) is the plasma
membrane which has rigid compartments (liquid-ordered
* Chibao Huang
phase, lo) and is enriched with glycosphingolipids and cho-
lesterol [1, 2], and foating in the sea of glycerophospholipids
(liquid-disordered phase, ld) [3–7]. Its structural characteristics
and components are suitable for interactions between proteins
and conformation conversion. Therefore, lipid raft involves in
many cellular processes, such as signal transduction, pathogen
invasion, cholesterol homeostasis, neurodegenerative diseases
(Alzheimer’s disease and prion disease, etc.) and angiogenesis
huangchibao@163.com
1
School of Information Engineering, Zunyi Normal
University, Zunyi 563002, Guizhou, China
2
Shan County Lanbo Biotechnology Co., Ltd, Heze 274300,
China
3
Henry Fok School of Biology and Agriculture, Shaoguan
University, Shaoguan 512005, China
Vol.:(0123456789)
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C. Huang et al.
[3–8], and so on. Glycerophospholipids consists of polar heads
(hydrophilic ends) combined with phosphate groups and two
nonpolar tails (fatty acid chains, lipophilic ends). Sphingomye-
lin is a derivative of sphingosine. These two kinds of lipids have
strong polarity. On the other hand, cholesterol has extremely
weak polarity, and a high viscosity due to its characteristics of
four carbon rings leading to a strong molecular plane rigidity
and a long carbon chain containing eight carbon atoms. In addi-
tion, cholesterol belongs to a push–pull architecture [9] with a
donor-π-acceptor (D-π-A; D: electron donor; A: electron accep-
tor; π: conjugation bridge, C=C) group, where hydroxyl group
act as donor and long carbon chain act as acceptor. Cholesterol
is the main component of lipid raft, so its structure and proper-
ties represent that of lipid raft. It was not hard to understand
that the polarity of the raft is much smaller than the ocean of
glycerophospholipids, while its viscosity is relatively large.
With such properties as NIR (near infrared) photons
as excitation source, avoidance of photodamage and pho-
tobleaching, fxed target excitation, high transverse and lon-
gitudinal resolutions, small absorption coefcient of light in
tissue, and lower tissue auto-fuorescence, etc. [10, 11], two-
photon (TP) fuorescence (TPF) imaging is superior to one-
photon fuorescence imaging, and might be a powerful tool
for life science studies [12, 13]. A two-photon microscope
(TPM) [10, 11] excited by two near-infrared photons, can
achieve real-time dynamic three-dimensional (3D) imaging
of microscopic objects, and provide sharp tools for biologi-
cal research of lipid rafts [12–18].
benzene ring was replaced by the fused-ring carbazole
moiety to enhance the rigidity of molecular plane: one
was to improve the two-photon absorption cross-section
(δTPA) of the probe molecule to emit stronger fluores-
cence, which greatly heighten the sensitivity; the other
was to reduce the effect of water solution polarity (or
cell fluid polarity) upon the fluorescence of the probe
[19, 21], and to focus the probe on the response to vis-
cosity, namely lipid raft, so as to enhance the accuracy
of the detection; another was to boost the affinity and
efficacy of the probe with lipid raft rooting from the com-
parability between the rigidity of molecular plane for the
probe and the rigid lipid raft with rich glycosphingolip-
ids and cholesterol. The matching and similarity in the
structure of probe molecule and cholesterol which both
have four carbon rings (one five-membered ring and three
six-membered rings) contributed to their highly specific
recognition. Nitrogen atom on carbazolyl group and/or
cyano group of probe molecule ensured the preferential
interaction of the probe with lipid raft [22]. In the light
of these four points, on the basis of TPF matrix DCS [19,
21, 23–26], a TPF “on–off” probe for lipid raft (DLR) was
developed (Fig. 1) in this study.
Φδ of DLR was the largest in the reported TPF probes
for lipid raft. The larger the δΦ is, the higher the sensitivity,
accuracy, imaging clarity, and resolution are. DLR has a
broad range of solvent solvatochromism varing from 422 nm
in cyclohexane to 581 nm in DMSO. This feature greatly
improves the sensitivity of DLR to the polarity/viscosity
of the medium, and drastically enlarges the sensitivity and
accuracy for the specifc recognition of lipid rafts. Further-
more, the lipophilic long carbon chain (C8H17) of DLR and
the excellent comparability between the rigid plane of the
probe molecule and the rigid lipid raft with rich glycosphin-
golipids and cholesterol can signifcantly enhance the afnity
for lipid rafts.
An efficient bioimaging TPF probe, it should have a
large two-photon action cross-section (δΦ). However, the
δΦ values of previously reported TPF lipid raft probes were
relatively small, for example CL2 [18], its δΦ in DPPC
(dipalmitoylphosphatidylcholine) was only 80 GM (1 GM ≡
10–50 cm4 s / photon). Such small δΦ inevitably and greatly
afected the clarity and resolution of the imaging, as well as
the sensitivity and accuracy of the detection. Therefore, the
developments of TPF lipid raft probes with large δΦ have
become the focus of research in this feld.
We recently reported two solvatochromic probes (D-π-
A, C = C) derived from TP fluorophore DCS (4-methyl-
2,5-dicyano-4′-aminostilbene). Our probe exhibits a wide
range of solvatochromism and a large δΦ [19], therefore,
becoming the preferred candidate for lipid raft imaging.
Based on the molecular simulation calculation, Ai-Min
Ren had theoretically proved that, as a fluorophore, DCS
was able to lead to a significant increase in δΦ [20]. On
this basis, we modified the molecular structure of the
probe by introducing a lipophilic long carbon chain
(C8H17), which is similar to that of eight carbon atoms
in cholesterol with the purpose of augmenting the com-
patibility of the probe with the cytoplasmic membrane
(biliphytic molecular layer), and improving the matching
between probe molecule and lipid raft. In addition, the
Results and discussion
Design and synthesis of (E)‑2‑methyl‑5‑[2‑(9‑octyl‑
9H‑carbazol‑3‑yl)vinyl]terephthalonitrile (DLR)
1,4-Dibromo-2,5-dimethylbenzene (2) [27],
2 , 5 - d i m e t h y l t e r e p h t h a l o n i t r i l e ( 3 ) [ 2 7 ] ,
Fig. 1 Molecular structures of DLR
1 3

A two‑photon fuorescent lipid raft probe derived from dicyanostilbene and similar to…
2-(bromomethyl)-5-methylterephthalonitrile (4) [26],
and diethyl 2,5-dicyano-4-methylphenylphospho-
nate (5) [26] were synthesized according to literature
procedures. The dicyanocarbazolylstilbene-derived
DLR was obtained in 68% yield by the reaction of
9-octyl-9H-carbazole-3-carbaldehyde (8) with 5 in the pres-
ence of sodium hydride (NaH) (Scheme 1).
Efect of solvent polarity on emission spectrum
The maximum absorption wavelength of DLR hardly var-
ies with the solvent (Supporting Information, SI, Fig.
S1), while the maximum emission wavelength (λEM) of
DLR was positively correlated with the polarity of the
solvent (λEM (cHex) = 423 nm, cHex: cyclohexane; λEM
(DMSO) = 581 nm) (Fig. 2a, Table S1 (SI)), increasing
with the π* of solvent polarity empirical parameter [28]. In
cHex, there existed only an emission band at 400–480 nm in
the localized excitation state (LE). In DMF and DMSO, the
emission intensity at 400–480 nm in the delocalized excita-
tion state (DE) was much less than that at 480 nm, while in
THF (tetrahydrofuran), LE peak did not appeared.
Scheme 1
Br
CuCN, DMF
reflux, 24 h
Br2 , CH2Cl2
0~20 °C, 24 h
83~97%
75~93%
Br
2
1
CN
CN
NBS, CCl4
80 °C, 3 h
Br
56~68%
NC
NC
3
4
The emission intensity of DLR gradually decreased in
order of DPPC, Raft Mix and DOPC (20:12.8:1), and there
was a slight red shift (Fig. 2b). The emission intensity in
DPPC was 20 times higher than that in DOPC because the
viscosity of DPPC was much larger than that of DOPC. The
strong comparability between the rigid plane of the probe
molecule and DPPC with rich dipalmitoylphosphatidylcho-
line lipid could signifcantly enhance the afnity of DLR
for DPPC. DPPC and DOPC are similar to lipid rafts (lo)
and glycerophospholipids (ld), respectively, implying that the
fuorescence intensity of DLR in lo should be much greater
than that in ld. Therefore, DLR can be used for detection of
lipid rafts.
CN
P(OEt)3
reflux, 24 h
O
P(OEt)2
83~95%
NC
5
CH3(CH2)7Br
DMSO
N
N
H
(CH2)7CH3
6
7
CHO
POCl3
DMF
5
N
NaH, THF
(CH2)7CH3
Relationship between two‑photon action
cross section of DLR and its two‑photon excitation
wavelength
8
CN
H3C(H2C)7
N
The determination results of Φδs according to published
method [29, 30] were shown in Fig. 3, S2 (SI), and Table S1
(SI). Φδ decreased in DPPC, Raft Mix and DOPC order
(1350 (DPPC), 975 (Raft Mix), and 67 GM (DOPC)). These
CH3
NC
DLR
Fig. 2 Normalized (a) and
relative (b) emission spectra
of DLR in various solvent,
arranged in the order of increas-
ing π* value (cHex, THF, DMF,
DMSO=0.00, 0.58, 0.88,
1.00), as empirical parameter of
solvent polarity
(a)
(b)
1 3

C. Huang et al.
values are much larger than Φδ value of CL2 in DPPC [18],
and were the largest among reported TPF lipid raft probes
[14–18]. Maximum two-photon excitation wavelengths
(λEXTP) in DPPC, Raft Mix and DOPC were 780, 790, and
800 nm, respectively. It can be seen that Φδ increased upon
the increase of medium viscosity (Fig. S2, SI), and Φδ in
DPPC was more than 20 times more than that in DOPC.
Thus DLR was enough to distinguished between lo (similar
to DPPC) and ld (similar to DOPC). Due to the more easy
intramolecular torsion of excited molecules in less viscosity
media, the molecular coplanarity was weakened, thereupon
Φδ naturally decreased.
Time‑resolved fuorescence spectra
Time-resolved fluorescence spectra (TRFs) were deter-
mined according to the methods [31], as shown in Fig. 4;
Table S2 (SI). The fuorescence lifetime of DLR in DPPC
or cells (3.3–4.5 ns) was much larger than that in DOPC
(1.7–1.9 ns). The decay time constant of LE state in the
DOPC was 65 ps, while DE state 21 ps, and the former was
more than three times that of the latter. These data indicated
that the fuorescence of DLR in DOPC was very weak, and
the strength rapidly attenuated. Therefore, whether it was
fuorescence, or fuorescence lifetime, both decreased upon
the increase of medium polarity. Apparently, the TRFs in
cells was very similar to that in Raft Mix. This implied that
the fuorescence of DLR can clearly distinguish between
the gel phase (lipid raft lo) and the fuid phase (glycerophos-
phatide ld), because the afnity of DLR to the gel phase was
much greater than that to the fuid phase, and the fuores-
cence in polar medium was weaker.
Fig. 3 Two-photon action spectra of DLR (c=1 μmol dm−3) in DPPC
(■), raft mixture (●), and DOPC (▲) (lipid/DLR=100/1). These
data measured at 25 0.5 ℃
Fig. 4 Time resolved fuores-
(a)
(b)
cence of DLR in (a) DPPC, (b)
DOPC/sphingomyelin/choles-
terol (1:1:1), (c) DOPC, and (d)
cell (mouse fbroblast)
(c)
(d)
1 3

A two‑photon fuorescent lipid raft probe derived from dicyanostilbene and similar to…
signifcantly weakened after the addition of MβCD to tis-
Two‑photon fuorescence imaging
sues (Fig. 6b), indicating that most lipid rafts were destroyed
by MβCD. Subsequently, upon the addition of cholesterol
to the tissues whose inherent lipid rafts had been previously
destroyed, fuorescence was enhanced (Fig. 6c), demonstrat-
ing that DLR was able to image the distribution of lipid rafts
in tissue.
Of GUVs (Giant unilamellar vesicles) (DPPC, Raft Mix or
DOPC) labeled with DLR TPM images were obtained at
excitation wavelength 790 nm (Fig. S3, SI), and showed a
weak fuorescence region in the vertical direction. According
to the chart, the fuorescence intensities of DLR decreased in
order of DPPC, Raft Mix and DOPC, especially its fuores-
cence intensity in DOPC was rather weak, indicating a nega-
tive correlation between the fuorescence intensity and the
liposome polarity. DLR was more compatible with DPPC,
i.e., easier to combine with lo.
Conclusion
The dicyanocarbazolylstilbene-based two-photon fuorescent
lipid raft probe DLR showed many excellent properties such
as two-photon absorption, specifc afnity of lipid raft, and
bioimaging. Φδ of DLR was as high as 1350 GM in DPPC,
and it had a broad range of solvent solvatochromism [from
422 nm (cyclohexane) to 581 nm (DMSO)]. Good photo-
stability and no cytotoxicity were observed. The emission
intensity of DLR in DPPC was 20 times higher than that in
DOPC, and the fuorescence lifetime in DPPC was more
than 2 times longer than that in DOPC. DLR could well
recognize lipid rafts and image the distribution of lipid rafts
in cells and tissues. The excellent performances of the probe
were owing to the property of high two-photon absorption
of the dicyanostilbene. Furthermore, the large plane con-
jugation system of the carbazole group also makes a great
contribution to the two-photon absorption of the probe. The
excellent comparability between the rigid plane of the probe
molecule and the rigid lipid raft with rich glycosphingolip-
ids and cholesterol could signifcantly enhance the specifc
afnity to lipid rafts. This attributes to fve aspects: frstly,
probe molecule and lipid raft both belong to rigid planar
molecule, both of which contain four carbon rings, one of
which is the fve-membered ring and the other three are all
the six-membered ring, almost all of which are fused-ring;
secondly, they are both D-π-A (π: C = C) type molecule;
thirdly, they both contain a long carbon chain with eight
carbon atoms; fourthly, probe molecule contains the nitro-
gen atom on carbazolyl group and/or cyano group that can
preferentially interact with lipid raft; ffthly, they are both
very weakly polar molecules. These strategies provide a rare
To further verify and understand the feasibility of DLR
for the detection of lipid rafts and the corresponding appli-
cation performances, DLR was applied to cell and tissue
for imaging studies. The culture of mouse fbroblasts [18]
and the preparation of mouse brain tissue slices [26] were
followed according to the literature. The results of mouse
fbroblast imaging were shown in Fig. 4. Prior to the addi-
tion of MβCD (methyl-β-cyclodextrin), strong fuorescence
was seen, and the cellular regions with higher lipid raft con-
tent displayed greater fuorescence intensity (Fig. 5a). After
the addition of MβCD to the cells, the fuorescence became
rather faint, and cellular regions previously with relatively
strong fuorescence were not even visible (Fig. 5b), which is
due to the fact that MβCD is a lipid raft-destroying reagent
[18, 32], thus destroying the lipid rafts in cells, and lipid
raft content signifcantly decreased, and the correspond-
ing fuorescence was greatly weakened. After the treatment
with cholesterol (50 μmol dm−3), the fuorescence intensity
almost returned to the state before the addition of MβCD
(Fig. 5c). These results are sufcient to demonstrate that
DLR can be used to detect lipid rafts in the cytoplasmic
membrane, and that DLR has a good specifc recognition
of lipid rafts.
The results of tissue imaging were shown in Fig. 6, which
was similar to cell imaging. When rat brain tissue sections
were incubated with DLR, strong fuorescence was emitted
(Fig. 6a), and the fuorescence intensities of diferent tis-
sue regions varied enormously, refecting the non-uniform
distribution of lipid raft concentration. Fluorescence was
Fig. 5 Pseudo colored TPM
images of DLR-labeled
(1.0 μmol dm−3) mouse fbro-
blast before (a) and after (b)
treatment with 10 mM MβCD
for 30 min at 37 ℃. c TPM
image of cholesterol depleted
mouse fbroblast after repletion
with 50 μmol dm−3 cholesterol
for 1 h at 37 ℃. Scale bars,
20 μm
1 3

C. Huang et al.
Fig. 6 Pseudo colored TPM images of a mouse brain tissue slice
stained with DLR (5.0 μmol dm−3) before (a) and after (b) treatment
with 10 mM MβCD for 30 min at 37 ℃. c TPM image of cholesterol
depleted mouse fbroblast after repletion with 50 μmol dm−3 cho-
lesterol for 1 h at 37 ℃. The TPEF images were collected at 500–
600 nm upon excitation at 790 nm with fs pulses
experience and reference for the developments of new lipid
raft probes, and enrich the design theory of two-photon fuo-
rescent probes.
pH-dependent fuorescence studies were performed accord-
ing to the literature [35].
9‑Octyl‑9H‑carbazole (7, C20H25N) Carbazole (6, 5.0 g,
30.0 mmol), 3.0 g potassium hydroxide (53.6 mmol), and 30
cm3 anhydrous dimethyl sulfoxide was stirred and heated to
80–85 ℃. To this solution, 4 cm3 1-bromooctane was added
dropwise. The reaction mixture was stirred for 3 h at 80–85
℃, then the mixture was cooled to room temperature, fol-
lowed the addition of 100 cm3 distilled water, fltered, dried
over MgSO4, and concentrated in vacuo. The crude product
was recrystallized with anhydrous ethanol. Yield 5.7 g (68%)
of white needle crystal. M.p.: 68–70 ℃; IR (KBr): v=1608,
1595, 1493 (CH=CH) cm−1; 1H NMR (CDCl3): δ=8.07 (d,
J=7.6 Hz, 2H), 7.43 (t, J=7.6 Hz, 2H), 7.35 (d, J=8.0 Hz,
2H), 7.20 (t, J=7.6 Hz, 2H), 4.22 (t, J=7.2 Hz, 2H, NCH2),
1.18 (m, 2H, N–C–CH2), 1.21 (m, 10H, CH2), 0.85 (t,
J=6.4 Hz, 3H, CH3) ppm; 13C NMR (CDCl3): δ=140.59,
125.71, 122.99, 120.48, 118.84, 108.80, 77.52, 77.20, 76.88,
43.19, 31.97, 29.55, 29.35, 29.11, 27.47, 22.78, 14.25 ppm;
HRMS (EI): m/z=279.1987 (calcd for C20H25N 279.1987).
Experimental
NMR spectra were recorded on a VARIAN INOVA
400 MHz NMR spectrometer. Mass spectral determinations
were made on a ESI-Q-TOF mass spectrometer (Micromass,
UK). High resolution mass spectra measurements were per-
formed on a GC-TOF mass spectrometer (Micromass, UK).
Fluorescence measurements were performed on a PTI-C-
700 Felix and Time-Master system. Fluorescence quantum
yields were measured using standard methods [33] on air-
equilibrated samples at room temperature. Quinine bisulfate
in 0.05 mol dm−3 H2SO4 (Φ = 0.546) was used as a refer-
ence [33]. TPEF (two-photon-excited fuorescence) action
cross-section spectra were measured according to the experi-
mental protocol established by Xu and Webb [34], using a
mode-locked Ti/sapphire laser that delivers~ 80 fs pulses
at 76 MHz. Fluorescein (10–4 mol dm−3 in 0.1 mol dm−3
NaOH), whose TPEF action cross-sections are well-known
[34], served as the reference. The quadratic dependence of
the fuorescence intensity on the excitation intensity was ver-
ifed for each data point, indicating that the measurements
were carried out in intensity regimes in which saturation or
photodegradation does not occur. The measurements were
performed at room temperature on air-equilibrated solutions
(10–5 mol dm−3). The experimental uncertainty on the abso-
lute action cross-sections determined by this method has
been estimated to be 20% [34]. Absorption spectra were
measured on a HP-8453 spectrophotometer. Solvents were
generally dried and distilled prior to use. Reactions were
monitored by thin-layer chromatography on Merck silica gel
60 F254 precoated aluminum sheets. Column chromatogra-
phy: Merck silica gel Si 60 (40–63 μm, 230–400 mesh). The
9 ‑ O c t y l ‑ 9 H ‑ c a r b a z o l e ‑ 3 ‑ c a r b a l d e hyd e ( 8 ,
C21H25NO) Anhydrous DMF (6 cm3) was placed in a 100
cm3 fask and was cooled in ice bath. Then 2.2 g of phos-
phorus oxychloride (POCl3, 14.5 mmol) was added dropwise
with stirring. After 30 min, 4.0 g of compound 7 (14.3 mmol)
was added to the fask. The solution was heated at 90 °C
for 2 h. The reaction mixture was then cooled, poured over
crushed ice in a beaker, and neutralized to pH 6–8 by drop-
wise addition of saturated sodium acetate solution. The
mixture was extracted with ethyl acetate. The extracts were
washed with water, dried with magnesium sulfate, and then
concentrated. The residue was purifed by column chroma-
tography to yield 4.2 g (95%) of 8 as a white crystal. M.p.:
126–128 °C; IR (KBr): v = 1605, 1593, 1492 (CH = CH),
1643 (CO) cm−1; 1H NMR (CDCl3): δ=10.06 (s, 1H), 8.56
(s, 1H), 8.11 (d, J=8 Hz, 1H), 7.91 (d, J=8.4 Hz, 1H), 7.51
1 3

A two‑photon fuorescent lipid raft probe derived from dicyanostilbene and similar to…
Funding This work was supported by the Guizhou Province High-level
(t, J=8 Hz, 1H), 7.41 (d, J=8.4 Hz, 2H), 7.28 (t, J=7.6 Hz,
1H), 4.26 (t, J=7.6 Hz, 2H), 1.84 (m, 2H), 1.28 (m, 10H),
0.85 (t, J=6.4 Hz, 3H) ppm; 13C NMR (CDCl3): δ=191.93,
144.23, 141.34, 128.67, 127.29, 126.88, 124.13, 123.22,
123.17, 120.90, 120.46, 109.59, 109.11, 43.572, 31.96,
29.51, 29.33, 29.10, 27.44, 22.80, 14.27 ppm; HRMS (EI):
m/z=307.1938 (calcd for C21H25NO 307.1936).
Innovative Talents Training Project—Hundred Talents Program (No.
[2016]5683), Science and Technology Project in Guizhou Province
(No. [2020]4Y001), the Natural Science Research Project of Guizhou
Department of Education (Nos. KY[2019]068), the National Natural
Science Foundation of China (No. 21562050), the Special Fund Project
of the Construction of the Eighth Batch of Scientifc and Technologi-
cal Innovation Talent Team in Guizhou Province [No. (2015)4007],
Guizhou Science and Technology Fund Project (No. J[2015]2146),
the Key Project of Education Department of Guizhou Province (No.
KY[2014]296).
(E)‑2‑Methyl‑5‑[2‑(9‑octyl‑9H‑carbazol‑3‑yl)vinyl]‑
terephthalonitrile (DLR, C31H31N3) Aldehyde 8 (446 mg,
1 mmol) and 30 mg NaH (1.25 mmol) were dissolved in 10
cm3 of tetrahydrofuran (THF), and the solution was cooled
to 0 °C. To this solution, 292 mg of phosphonate 5 (1 mmol)
in 10 cm3 of THF was added dropwise, and the reaction
mixture was stirred for 24 h at 0 °C. Water was added to
the reaction mixture, and the product was extracted with
ethyl acetate. The organic layer was dried with over anhy-
drous MgSO4, and concentrated in vacuo. The crude product
was separated by column chromatography with a gradient
of hexane in dichloromethane (20–0%) and ethyl acetate
in dichloromethane (0–20%). Yield 303 mg (0.68 mmol,
68%). The resulting solid was recrystallized from acetone to
give yellow powder. M.p.: 235–237 ℃; IR (KBr): v=1602,
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1592, 1492 (CH = CH), 1653 (C = C), 2225 (CN) cm−1
;
10. Juvekar V, Lim CS, Lee DJ, Song DH, Noh CK, Kang H, Shin SJ,
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1H NMR (CDCl3): δ = 8.26 (s, 1H), 8.14 (d, J = 7.2 Hz,
1H), 8.03 (s, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.56 (s, 1H),
7.49 (t, J = 7.2 Hz, 2H), 7.44 (d, J = 11.6 Hz, 1H), 7.39
(d, J = 10.4 Hz, 1H), 7.32 (d, J = 16.4 Hz, 1H, CH = CH),
7.26 (d, J = 16.4 Hz, 1H, CH = CH), 4.30 (t, J = 7.2 Hz,
2H, NCH2), 2.56 (s, 3H, CH3), 1.88 (m, 2H), 1.36 (m, 2H,
NCH2CH2), 1.32 (m, 10H, (CH2)5), 0.86 (t, J=6.8 Hz, 3H,
CH2CH3) ppm; 13C NMR (CDCl3): δ = 141.12, 140.47,
139.84, 139.58, 136.22, 134.49, 129.05, 126.72, 126.37,
125.14, 123.51, 121.42, 120.76, 120.27, 119.65, 118.92,
117.58, 117.08, 114.23, 109.38, 43.48, 31.97, 29.54, 29.35,
29.18, 27.48, 23.70, 22.78, 20.12, 14.25 ppm; HRMS (EI):
m/z=445.2507 (calcd for C31H31N3 445.2518).
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Including DPPC + DLR, DOPC + DLR and DOPC/sphin-
gomyelin/cholesterol (1:1:1, mol %, Raft Mix, Also known
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stock solution with chloroform/methanol (9:1, v/v), general
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of lipid to the probe DLR was 200:1.
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