Monitoring penetratin interactions with lipid membranes and cell internalization using a new hydration-sensitive fluorescent probe

Article abstract of DOI:10.1039/c4ob01242a

A new fluorescent label N-[4′-(dimethylamino)-3-hydroxyflavone-7-yl]- N-methyl-β-alanine (7AF) was synthesized. Due to two electron donor groups at the opposite ends of the chromophore, an excited state intramolecular proton transfer (ESIPT) resulting in a dual emission was observed even in highly polar media and its fluorescence quantum yield was found to be remarkably high in a broad range of solvents including water. As a consequence, this label exhibits a remarkable sensitivity to the hydration of its environment, which is observed as a color switch between the emission of the ESIPT product (T* form) and that of the normal N* form. The 7AF label was coupled to the N-terminus of penetratin, a cell penetrating peptide, in order to study its interactions with lipid membranes and internalization inside the cells. As expected, the binding of penetratin to lipid membranes resulted in a dramatic switch in the relative intensity of its two emission bands as compared to its emission in buffer. Our studies with different lipid compositions confirmed the preference of penetratin to lipid membranes of the liquid disordered phase. After incubation of low concentrations of labeled penetratin with living cells, ratiometric imaging revealed, in addition to membrane-bound species, a significant fraction of free peptide in cytosol showing the characteristic emission from aqueous medium. At higher concentrations of penetratin, mainly peptides bound to cell membrane structures were observed. These observations confirmed the ability of penetratin to enter the cytosol by direct translocation through the cell plasma membrane, in addition to the classical entry by endocytosis. The present probe constitutes thus a powerful tool to study the interaction of peptides with living cells and their internalization mechanisms. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob01242a

Organic &
Biomolecular Chemistry
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PAPER
Monitoring penetratin interactions with lipid
membranes and cell internalization using a new
hydration-sensitive fluorescent probe†
Cite this: Org. Biomol. Chem., 2014,
12, 7036
Oleksandr M. Zamotaiev,a,b Viktoriia Y. Postupalenko,a Volodymyr V. Shvadchak,a
Vasyl G. Pivovarenko,*a,b Andrey S. Klymchenko*a and Yves Mélya
A new fluorescent label N-[4’-(dimethylamino)-3-hydroxyflavone-7-yl]-N-methyl-β-alanine (7AF) was
synthesized. Due to two electron donor groups at the opposite ends of the chromophore, an excited
state intramolecular proton transfer (ESIPT) resulting in a dual emission was observed even in highly polar
media and its fluorescence quantum yield was found to be remarkably high in a broad range of solvents
including water. As a consequence, this label exhibits a remarkable sensitivity to the hydration of its
environment, which is observed as a color switch between the emission of the ESIPT product (T* form)
and that of the normal N* form. The 7AF label was coupled to the N-terminus of penetratin, a cell pene-
trating peptide, in order to study its interactions with lipid membranes and internalization inside the cells.
As expected, the binding of penetratin to lipid membranes resulted in a dramatic switch in the relative
intensity of its two emission bands as compared to its emission in buffer. Our studies with different lipid
compositions confirmed the preference of penetratin to lipid membranes of the liquid disordered phase.
After incubation of low concentrations of labeled penetratin with living cells, ratiometric imaging revealed,
in addition to membrane-bound species, a significant fraction of free peptide in cytosol showing the
characteristic emission from aqueous medium. At higher concentrations of penetratin, mainly peptides
bound to cell membrane structures were observed. These observations confirmed the ability of penetratin
to enter the cytosol by direct translocation through the cell plasma membrane, in addition to the classical
entry by endocytosis. The present probe constitutes thus a powerful tool to study the interaction of
peptides with living cells and their internalization mechanisms.
Received 15th June 2014,
Accepted 15th July 2014
DOI: 10.1039/c4ob01242a
www.rsc.org/obc
(polarity, hydration, viscosity, etc.),6–9 appears as a promising
and simple alternative. These probes should sensitively report
Introduction
Despite the ever growing development of fluorescent probes on changes in the environment of a given biomolecule from
and instrumental techniques for tracking the localization of the highly polar environment of the cytoplasm to the apolar
biomolecules in living cells,1–3 it remains a challenge to environment in cellular lipid compartments.10
monitor directly their molecular environment and interactions
Most of the polarity sensors developed in the last decade
with their cellular targets.4,5 Förster resonance energy transfer for peptide labeling were based on popular solvatochromic
(FRET), being the most commonly used approach, is limited fluorescent dyes undergoing intramolecular charge transfer,
because it requires double labeling. In this respect, the use of namely Prodan derivatives,11–13 dimethylaminophthalimide,14
environment-sensitive probes, which change their emission and others.15–17 They were applied to investigate protein–
color or intensity as a function of their environment properties protein
interactions
and
protein
conformational
transitions.11–14,16,18,19 Though effective enough in the men-
tioned studies, these probes show limited spectroscopic pro-
perties and sensitivity to binding events. Most of them are
limited to applications in a relatively non-polar environment,
because in polar media, especially in water, their fluorescence
is quenched15 or is poorly sensitive to the environment.
3-Hydroxyflavones (3HFs) are fluorescent dyes exhibiting
strong sensitivity of their dual emission to the environment as
a result of an excited-state intramolecular proton transfer
aLaboratoire de Biophotonique et Pharmacologie, UMR 7213 du CNRS, Université de
Strasbourg, Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch, France.
E-mail: andrey.klymchenko@unistra.fr; Fax: +33 368 85 43 13;
Tel: +33 368 85 42 55
bOrganic Chemistry Chair, Chemistry Faculty, Taras Shevchenko National University
of Kyiv, 01601 Kyiv, Ukraine. E-mail: pvg_org@ukr.net; Fax: +38044-2898391;
Tel: +38044-2393312
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4ob01242a
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strong potential for applications as a vector for the intracellu-
lar delivery of hydrophilic biomolecules and drugs.43,44 The
labeled peptide was used to sensitively monitor its binding to
lipid vesicles of various compositions and lipid phases. More-
over, the new hydration-sensitive label allowed us to monitor
the internalization of penetratin into cells and distinguish for
the first time two of its internalization pathways by ratiometric
imaging.
Results and discussion
Label synthesis and characterization
Scheme 1 ESIPT reaction scheme in 3HF dyes. Structure of the 7AF
The synthesis of the new label (7AF) was performed in five
steps, through the preparation of substituted 4-amino-2-hydro-
xyacetophenone (3). It was converted into 3-hydroxyflavone (4)
by condensation with 4-N,N-dimethylaminobenzaldehyde and
a subsequent oxidative heterocyclization (Scheme 2). Then, the
target product 7AF was obtained through carboxyethylation of
the 7-amino group by a Michael reaction (see Materials and
methods and ESI†).
label and the sequence of penetratin.
(ESIPT) reaction20 (Scheme 1). The ratio of emission of the
normal (N*) to the photo-tautomer (T*) species provides a con-
centration-independent response to the environment polarity
and H-bonding interactions.21–26 Both the mentioned pro-
cesses inhibit the ESIPT reaction and thus decrease the relative
intensity of the T* band. Modification of the structure of 3HFs
gives the possibility of sensing separately the hydration27
(H-bond donating property28–30) and H-bond accepting prop-
erty27,30 of the surroundings. 2-Furyl-3-hydroxychromones31,32
and 4′-methoxy-3HFs26,27 present optimal two-band ratio-
metric response to the environment in polar media, which was
successfully used to study peptide–peptide and peptide–
nucleic acid interactions. However, their application for
microscopy techniques is limited due to their absorption in
the UV region. On the other hand, 4′-(dialkylamino)-3HFs
exhibit absorption and emission in the visible spectral region,
but due to much higher dipole moments,33 their dual emis-
sion is optimally sensing low- and middle-polar media, while
in water their T* band is poorly resolved. They were success-
fully applied to the study of model and cellular mem-
branes6,34,35 and protein–lipid interactions,24,36 but they are
strongly quenched in aqueous media. Recently, 4′-(dialkyl-
amino)-3HFs bearing an electron donating amino group in
7-position were shown to exhibit higher emission of the T*
form with better resolution of N* and T* bands,37 giving thus
the possibility of ratiometric sensing in highly polar media.
Therefore, we hypothesized that 7-substituted 4′-(dialkyl-
amino)-3HFs could be a promising environment-sensitive
label that combines absorption in the visible region with sen-
sitivity in polar (aqueous) media.
Spectroscopic properties
The absorption spectrum of 7AF in different solvents consists
of one band centered at about 400 nm, similar to its parent
4′-(dialkylamino)-3HF analogues.15,23,25,34,35 In organic sol-
vents, 7AF shows two well-separated emission bands corres-
ponding to the normal (N*) and tautomer (T*) forms of the
dye24 (Fig. 1A, S1A and Table S1†). The position of the N* emis-
sion band strongly shifts to the red upon an increase in
solvent polarity (Table S1†), like for other 4′-dialkylamino-
flavones.6,23,35,45,46 Remarkably, the relative intensity of the N*
band in aprotic solvents is very small even for highly polar sol-
vents, which is a clear difference with the parent 4′-dialkyl-
amino-3HFs (Table S1,† Fig. 1A and ref. 6,23,35,45). In protic
solvents, the N* band relative intensity increases drastically
and in water it becomes dominant (Fig. 1A). Thus, from polar
aprotic DMF to polar water solvent, 7AF undergoes a complete
switch from the red-shifted emission of the T* form to the
blue-shifted emission of the N* form (Fig. 1B). This is a clear
advantage in terms of environment-sensitivity compared to
other 4′-(dialkylamino)-3HF, which showed a relatively strong
To validate our hypothesis, we synthesized the N-[4′-(di-
methylamino)-3-hydroxyflavone-7-yl]-N-methyl-β-alanine (7AF)
probe and characterized its spectroscopic properties. The 7AF
probe was found to be highly sensitive to the local concen-
tration of water and could thus be used to quantify it. More-
over, we coupled 7AF to the N-terminus of the cell penetrating
peptide (CPP) penetratin, a cationic peptide containing 16
amino acids, 6 of which being lipophilic. Penetratin binds
lipid membranes, preferentially to their fluid phases,38 and
enters into cells by different mechanisms,38–42 showing a
Scheme 2 Synthesis of the 7AF label: (i) NaH, CH3I, THF; (ii) AcCl, AlCl3;
(iii) MeONa, DMF, p-Me2N–C6H4–CHO, then MeONa, H2O2; (iv) AcOH,
acrylic acid.
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to preferential solvation effects.27 Importantly, the 7AF emis-
sion is not sensitive to pH in the range of 6–9 (pKa = 10.5,
Fig. S2†) and to ionic strength in aqueous solutions
(Fig. S1B†). The IN*/IT* ratio is also poorly sensitive to basic sol-
vents, since the intensity of the N* form is negligible in DMF
(Fig. 1A). All mentioned features allow the application of the
log(IN*/IT*) fluorescence parameter for estimating the local
27
water concentration CL or, alternatively, the partial volume
WA of water in the probe surroundings (relative local water
access), defined as WA = 100% CL/55.5.
In contrast to most solvatochromic fluorophores including
3HFs, the 7AF label shows rather high fluorescence quantum
yields (11–44%) in all types of solvents, including water. More-
over, emission of N* and T* bands is better separated than for
other dialkylamino derivatives of 3HFs (Fig. 1A).24 Thus, in
line with our initial aim, the new label shows both absorption
in the visible region and highly sensitive dual emission, which
allows quantitative estimation of the probe hydration.
Applications of 7AF for investigating penetratin–membrane
interactions
Fig. 1 (A) Fluorescence spectra of the 7AF label in water, methanol,
ethanol, tetrahydrofuran and N,N-dimethylformamide, normalized at
their maxima. Excitation wavelength was at the absorption maximum.
(B) Fluorescence spectra of 7AF in mixtures of DMF and water. Inset:
plot of the logarithm of fluorescence intensity ratio log(IN*/IT*) versus
total concentration of water in the mixture. The solid line represents the
fit with the equation: log(IN*/IT*) = 0.0377Cwater − 0.975 (R2 = 0.97).
The penetratin peptide labeled by 7AF at its N-terminus
(Scheme 1) was prepared by solid-phase synthesis. In aqueous
buffer, the labeled peptide shows a single emission band close
to that of the label alone, but with a slightly higher fluo-
rescence quantum yield (Fig. 2A, Table 1). The shoulder of the
T*-band emission at 597 nm (Fig. 2A) is more pronounced
than for the free label, pointing to a slight decrease in
N* band emission in polar aprotic solvents.24 In line with pre- hydration due to the proximity of the peptide backbone.
vious reports,27 this dramatic effect of protic solvents is likely
Next, we investigated the interaction of the labeled penetra-
due to the inhibition of the ESIPT reaction by H-bonding of tin with model lipid membranes. Penetratin is known to
the dye with the solvent. Moreover, measurements of the ratio induce aggregation of zwitterionic lipid vesicles in a large
of the emission band intensities (IN*/IT*) for the 7AF probe in range of lipid to peptide (L/P) ratios.39 This makes problematic
DMF–water binary mixtures show the possibility to quantify the application of intensiometric probes for investigating
the water concentration. Indeed, the logarithm of IN*/IT* peptide–membrane interactions. The ratiometric response of
changes linearly with the total concentration of water (Fig. 1B the 7AF probe overcomes this problem, since the ratiometric
inset, Fig. S3†). Significant deviations from the linearity are signal, having internal calibration, is independent of the total
observed only at high water content (≥70%) that we attribute fluorescence intensity.
Fig. 2 Fluorescence spectra of 7AF-labeled penetratin alone and in the presence of lipid vesicles: (A) normalized spectra of the 7AF label and 7AF-
labeled penetratin in 20 mM phosphate buffer, 150 mM NaCl (pH = 7.4) at 20 °C. Deconvolution of their fluorescence spectra into the N* and T*
forms. (B) Fluorescence spectra of 7AF-labeled penetratin in the presence of DOPC LUVs at different lipid/peptide (L/P) molar ratios. (C) Changes of
the N*/T* ratio and corresponding local hydration of 7AF–penetratin bound to lipid vesicles at different L/P ratios.
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Table 1 Spectroscopic parameters and hydration of 7AF-labeled pene-
tratin in buffer and in the presence of lipid vesiclesa
Media
λABS λN*
λT*
IN*/IT* QY (%) WA (%)
7AF in buffer (25 °C)b 411
534 593 13
535 597 6.4
497 584 0.66
514 583 0.52
497 586 2.2
506 584 0.54
490 585 1.7
496 583 1.1
533 583 2.8
513 583 0.92
11
14
40
41
23
40
31
50
18
46
100
85
39
34
40
33
55
44
58
44
Buffer (25 °C)
DOPC (25 °C)
DOPC : Ch (6 : 4)
DPPC (25 °C)
414
411
413
411
—
DPPC (45 °C)
SM (25 °C)
SM (50 °C)
417
—
SM : Ch (6 : 4, 25 °C)
SM : Ch (6 : 4, 50 °C)
—
—
a λABS – position of absorption maxima (nm); λN* and λT* – positions of
the fluorescence maxima of N* and T* forms, respectively. IN*/IT*
–
ratio of integral intensities of the two emission bands (errors are 2%).
The presented fluorescence data were obtained from deconvolution of
the fluorescence spectra, as shown in Fig. 2A. QY (%), fluorescence
quantum yield (errors are 5%), measured using FE in EtOH (QY =
51%51) as a reference; WA – calculated water access (%): WA = 100% CW
/
55.5 mol L−1 where the concentration of water CW was obtained from
the calibration curve in Fig. 1B. Lipid/peptide ratio is 1000. b Data for
the 7AF probe alone.
Fluorescence spectra of penetratin in the presence of lipid
vesicles depend strongly on the L/P ratio (Fig. 2B). At low L/P
ratio (0–20), the spectra are close to that in buffer, indicating a
large fraction of free peptide. However, we cannot exclude the
presence of penetratin aggregates47 at the membrane surface,
but they are likely poorly fluorescent. Starting from a L/P ratio
of 40 (Fig. 2B and S4†), which corresponds to a dense mem-
brane surface covering (up to 1 peptide per 5 nm2),39,48 the
shape of the spectra changes drastically, so that the fluo-
rescence intensity grows and the IN*/IT* ratio drops. These
changes suggest a shift of the label from water to the poorly
hydrated lipid membranes, confirming the efficient binding of
the peptide to lipid membranes. Low IN*/IT* ratios were also
observed for other lipid compositions presenting the fluid
phase, such as DOPC : Ch, DPPC (45 °C) and SM : Ch (45 °C) at
L/P ratios >100. In contrast, for lipid vesicles presenting the gel
phase (DPPC and SM at 25 °C) or the liquid ordered phase
(SM : Ch at 25 °C), the IN*/IT* ratio and the shape of the spectra
are closer to those in aqueous buffer, even for high L/P ratios.
Therefore, the binding of penetratin to these lipid phases
appears much less efficient, so that a large fraction of the
peptide is in the free form with possibly some poorly fluo-
rescent aggregated species at the lipid membranes. The
observed higher affinity of penetratin for fluid phases is in line
with previous reports.38 Using the IN*/IT* ratio of the integral
band intensities, we estimated the decrease in water access
from 85% for the peptide in buffer to 33–50% in lipid mem-
branes of the fluid phase. These last values correspond to the
water access values of the interfacial region of the lipid bilayer,
determined with other methods.49,50
Fig. 3 Temperature dependence of the emission spectra of 7AF–pene-
tratin in the presence of lipid vesicles. (A) Fluorescence spectra of 7AF–
penetratin in the presence of DPPC LUVs at various temperatures.
(B) Temperature-dependence of the local hydration and N*/T* ratio of
7AF–penetratin in the presence of DPPC or SM-cholesterol (6 : 4) LUVs.
The lipid/peptide ratio is 250 in all cases.
rescence spectra show a large fraction of free peptide, character-
ized by a high IN*/IT* ratio. Further increase in the temperature
close to the phase transition (41 °C) leads to a sharp decrease in
the N*/T* ratio accompanied by an increase in the total fluo-
rescence intensity. This shows that the gel to fluid phase tran-
sition results in efficient binding and insertion of penetratin
into the lipid membranes. Remarkably, with lipid vesicles pre-
senting the liquid ordered phase (SM : Ch), the observed temp-
erature-dependent decrease in the N*/T* ratio was continuous
without a sharp phase transition (Fig. 3B), in line with the rela-
tively broad temperature range for its phase transition. Thus
our data indicate that penetratin avoids the liquid ordered
phase similarly to the gel phase.
Monitoring interactions with living cells
Understanding the mechanisms of internalization of cell pene-
trating peptides is of key importance for the development of
new efficient delivery systems.52 Due to strong sensitivity of 7AF
label to hydration and its high fluorescence quantum yield in
both aqueous and lipid media, we used 7AF–penetratin to
monitor by fluorescence microscopy internalization of the
peptide into the living cells. Two mechanisms of penetratin
entry into cells are described, namely endocytosis and direct
translocation of the peptide through the plasma membrane.40
Since endocytosis proceeds through binding of the peptide to
the plasma membrane, we expect this pathway to be associated
The effect of the phase preference can be further seen in
the temperature-dependence of the emission spectra of 7AF–
penetratin in the presence of LUVs composed of DPPC (Fig. 3A
and B). Below the phase transition temperature, the fluo-
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with low N*/T* ratios. In contrast, translocation is thought to the ratiometric images. This high ratio is in line with the high
drive the peptide into the cytosol, so that this pathway should N*/T* ratio observed in aqueous media, indicating a high level
be associated with high N*/T* ratios due to the aqueous nature of probe hydration. This diffuse fluorescence in the cytosol is
of the cytosol. In addition, as the quantum yield of the probe consistent with penetratin peptides that have been interna-
is comparable in aqueous and lipid media (Table 1), we lized by the translocation mechanism. The second population
should be able to visualize the two pathways simultaneously. is associated with cell plasma membrane and intracellular
Since the balance between the two mechanisms was reported dots, and is characterized by a low B/R intensity ratio, in line
to depend on the extracellular concentration of the peptide, we with the low N*/T* ratio observed on model membranes. This
monitored the internalization of 7AF-labeled penetratin at second population could be ascribed to penetratin molecules
2 µM and 10 µM, where translocation and endocytosis are, that are endocytosed by binding first to the plasma membrane
respectively, the major modes of internalization.53 Moreover, and then being internalized in early endosomes formed by
the images were recorded at different incubation times (5, 10 invaginations of the cell plasma membranes. After 30 min of
and 30 min), to take into account that penetratin internaliz- incubation, all penetratin molecules were found to exhibit low
ation is time dependent.53
B/R ratio values, suggesting their association with intracellular
At 2 µM concentration, the rather slow internalization of lipid compartments (Fig. 4D). This redistribution of the pep-
penetratin allowed us during the first minutes to clearly dis- tides likely results from the peptide partition coefficient that
tinguish two populations of 7AF-labeled penetratin molecules favors the lipid over the aqueous phase.
(Fig. 4A–D). The first and most important population is
At 10 µM concentration, penetratin internalizes rapidly, so
observed as a diffuse fluorescence in the cytosol, and is charac- that within 10 min most of the fluorescence is observed inside
terized by a high intensity ratio of blue/red (B/R) channels in the cells (Fig. 4E), including some fraction in the nucleus. In
the cytoplasm, the labeled penetratin molecules are associated
with intracellular dots and exhibit mainly low B/R ratios, in
full line with an accumulation of penetratin in endosomal
compartments, and thus, with endocytosis being the major
internalization pathway. Rare areas with high B/R ratios were
also observed, suggesting that a minor population of penetra-
tin may enter through translocation, but the signal associated
with this population is probably hidden by the signal of the
major population in the lipid compartments. In the nucleus,
penetratin is located in a highly hydrated medium (high B/R
values), with preferential binding to nucleoli. Thus, penetratin
after entering into the cytosol could cross the nuclear mem-
brane and then, due to its numerous positively charged amino
acids, it could bind to the negatively charged RNAs concen-
trated in the nucleoli. At 30 min, the images were very similar
to those at 10 min, suggesting that no major redistribution of
the penetratin molecules occurs in this time frame (Fig. 4F).
Taken together, our data confirm the two different internal-
ization modes of penetratin and their dependence on the
external concentration of penetratin. Due to the dependence of
its N*/T* ratio on the hydration of its environment and its
comparable quantum yields in hydrated and non-hydrated
media, this probe provides the possibility for the first time to
distinguish the two internalization pathways by imaging
techniques.
Conclusions
A fluorescent label based on 3-hydroxyflavone bearing electron
donor groups at 4′ and 7-positions (7AF) was synthesized. The
new label showed selective response to the local concentration
of water by the intensity ratio of the normal (N*) form to the
ESIPT product (T*) form and good quantum yields both in
Fig. 4 Fluorescence microscopy imaging of HeLa cells stained with
2 μM (A–D) and 10 μM (E–F) of 7AF–penetratin after 5 (A, C), 10 (B, E)
and 30 (D, F) min of incubation. The color of each pixel represents the
value of the intensity ratio of blue/red channels, while the pixel intensity
water and lipids. It was grafted to the N-terminus of the cell
penetrating peptide penetratin in order to monitor its local
corresponds to the total intensity at both channels. The two-photon
excitation wavelength was at 830 nm.
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hydration, which is expected to change after peptide inter- Yield 73%. 1H-NMR (300 MHz, CDCl3): δ 1.90 (s, 3H, NCOCH3),
action with lipid membranes. It was found that the probe 3.25 (s, 3H, NCH3), 3.82 (s, 3H, OCH3), 6.72 (t, 1H, J = 1.9 Hz,
hydration estimated from its dual emission showed a signifi- ArH), 6.77 (dd, 1H, J = 8.1 Hz, J = 1.9 Hz, ArH), 6.87 (dd, 1H, J =
cant decrease after binding to lipid membranes and the 8.1 Hz, J = 1.9 Hz, ArH), 7.31 (t, 1H, J = 8.1 Hz, ArH).
affinity of the peptide to lipid membranes depended strongly
N-(4-Acetyl-3-hydroxyphenyl)-N-methylacetamide (3). To a
on their phase state. Then, we monitored the internalization stirred ice-cooled solution of m-methoxy-N-methylacetanilide
of the labeled penetratin into living cells with the aim to (7.77 g, 0.043 mol) in 80 mL of dry CH2Cl2 under an Ar atmos-
image its intracellular distribution as well as to distinguish the phere, 7.8 mL (0.11 mol) of acetyl chloride was added. After
free peptide species from those bound to cell membranes. 5 minutes of stirring, 16.8 g (0.126 mol) of AlCl3 was added in
Using ratiometric imaging, we evidenced that at low penetratin small portions for 40 min. After that, a slightly red solution
concentrations, a significant fraction of the peptide is loca- was stirred for 15 min at room temperature and then boiled
lized in the cytosol without binding to membrane com- for 6 h on an oil bath. The reaction was monitored using TLC
ponents, suggesting that the peptide is mainly internalized (EtOAc–heptane, 80 : 20). Then the reaction mixture was
through direct translocation into the cytosol. At high concen- poured onto ice-water, the organic layer was separated and
trations, endocytosis becomes the major internalization mode, washed twice with water, dried with MgSO4 and evaporated.
so that the labeled peptides are mainly associated with the The obtained yellow oil was purified by repeated recrystalliza-
plasma membrane and endosomes, as indicated by the charac- tion from heptane. Yield 1.73 g (19.4%), white crystals.
teristic fluorescence intensity ratio of the label corresponding 1H-NMR (300 MHz, CDCl3): δ 2.01 (s, 3H, NCOCH3), 2.65 (s,
to the lipid environment. Thus, the new label can reveal the 3H, ArCOCH3), 3.29 (s, 3H, NCH3), 6.77 (dd, 1H, ArH, J =
internalization mechanisms of a cell penetrating peptide into 8.4 Hz, J = 2.2 Hz), 6.82 (d, 1H, ArH, J = 2.2 Hz), 7.79 (d, 1H,
the living cells.
ArH, J = 8.4 Hz), 12.38 (s, 1H, ArOH).
2-[4-(Dimethylamino)phenyl]-3-hydroxy-7-(methylamino)-4H-
chromen-4-one (4). 0.5 g (2.41 mmol) of compound (3) and
0.4 g (2.68 mmol) of 4-dimethylaminobenzaldehyde were dis-
solved in 6 mL of dry DMF under an argon atmosphere.
391 mg (7.23 mmol) of sodium methoxide were added to the
reaction mixture under stirring at rt. A red solution was stirred
for 6 h, and then diluted with 40 mL of dry ethanol. To the
obtained solution, 1.96 g (36.15 mmol) of NaOMe and 3 mL of
30% H2O2 were subsequently added. The mixture was gently
boiled for 5 min, and then left for cooling. In 15 min, the
obtained yellow solution was poured into 150 mL of water,
Materials and methods
Reagents and solvents
All the reagents were purchased from Sigma-Aldrich Chemical
Company. Solvents for synthesis were of reagent quality and
were appropriately dried if necessary. For absorption and
fluorescence studies, the solvents were of spectroscopic grade.
Synthesis
N-(3-Methoxyphenyl)acetamide (1). To an ice-cooled stirred acidified to pH 7.5 with 10% HCl and filtered. Then, the dried
solution of m-methoxyaniline (10 mL, 0.089 mol) in 100 mL of precipitate was washed with hot methanol. All stages of syn-
dry toluene, acetyl chloride (6.71 mL, 0.0979 mol) was added thesis were monitored by TLC (CH2Cl2–MeOH, 95 : 5). As a
1
dropwise for 20 min. Then the mixture was refluxed for 3 h. result, 150 mg (20%) of yellow powder was obtained. H-NMR
The reaction was monitored using TLC (EtOAc–heptane, (300 MHz, CDCl3): δ 2.97 (s, 3H, NHCH3), 3.08 (s, 6H, NMe2),
80 : 20). After the reaction was complete, the mixture was evap- 6.52 (d, 1H, ArH, J = 1.9 Hz), 6.63 (dd, 1H, ArH, J = 9.1 Hz, J =
orated in vacuo. The obtained oil crystallized after adding 1.9 Hz), 6.88 (d, 2H, ArH, J = 9.1 Hz), 7.98 (d, 1H, ArH, J =
20 mL of heptane. The gray precipitate was filtered and 9.1 Hz), 8.16 (d, 2H, ArH, J = 9.1 Hz).
washed with heptane. Yield 12.9 g (87.8%). 1H-NMR (300 MHz,
N-2-[4-(Dimethylamino)phenyl]-3-hydroxy-4-oxo-4H-
CDCl3): δ 2.09 (s, 3H, COCH3), 3.72 (s, 3H, OCH3), 6.59 (dd, chromen-7-yl-N-methyl-β-alanine (7AF). 50 mg of amine (4)
1H, J = 2 Hz, J = 8.3 Hz, ArH), 6.89 (d, 1H, J = 2 Hz, J = 8.3 Hz, were dissolved in 2 mL of anhydrous acetic acid. To the
ArH), 7.13 (t, 1H, J = 8.3 Hz, ArH), 7.20 (t, 1H, J = 2 Hz, ArH), obtained solution 0.5 mL of acrylic acid was added. The
7.24 (br. s, 1H, NH).
mixture was refluxed under an Ar atmosphere for 4 h. The reac-
N-(3-Methoxyphenyl)-N-methylacetamide (2). N-(3-Methoxy- tion was monitored by TLC (CH2Cl2–MeOH, 92 : 8). Then the
phenyl)acetamide (10 g, 0.06 mol) in dry THF (140 mL) was mixture was cooled and evaporated in vacuo. To the obtained
added to NaH (60%, 7 g; 0.175 mol) which was washed twice brown oil 15 mL of water was added. A yellow precipitate was
with n-hexane before use at 0 °C. 40 min later, methyl iodide collected, and washed twice with water. The crude product was
(28 mL) was added and the mixture was stirred at room temp- purified by preparative TLC (CH2Cl2–MeOH, 85 : 15). Yield
erature for 14 h. The reaction was monitored by TLC (CH2Cl2– 10 mg (16.2%). 1H-NMR (300 MHz, DMSO-d6): δ 2.50 (t, 2H, J =
MeOH, 95 : 5). After evaporation of the solvent in vacuo, the 7 Hz, CH2), 3.00 (s, 6H, NMe2), 3.03 (s, 3H, NMe), 3.71 (t, 2H,
residue was dissolved in methylene chloride (100 mL), filtered, J = 7 Hz, NCH2), 6.72 (d, 1H, J = 2.5 Hz, ArH), 6.82 (d, 2H, J =
washed twice with water and dried over MgSO4. After evapor- 9.3 Hz, ArH), 6.84 (dd, 1H, J = 9 Hz, J = 2,5 Hz, ArH), 7.82
ation of the solvent, 7.91 g of yellow oil was obtained. The sub- (d, 1H, J = 9 Hz, ArH), 8.07 (d, 2H, J = 9.3 Hz, ArH), 8.72 (br. s,
stance was pure enough for its usage in the next step. 1H, OH).
This journal is © The Royal Society of Chemistry 2014
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Organic & Biomolecular Chemistry
Peptide synthesis, labeling, and purification
laser (Malvern Instruments). The measured diameter of all
LUVs was 110 10 nm. The vesicles were used within 5 days of
preparation (stored at 4 °C). For vesicles composed of more
than one type of lipid, all given percentages are based on rela-
tive molar composition.
Peptides were synthesized by solid phase peptide synthesis on
a 433A synthesizer (ABI, Foster City, CA). The synthesis was
performed on a 0.1 mmol scale using standard side-chain pro-
tected fluorenylmethoxycarbonyl (Fmoc)-amino acids and the
HBTU/HOBt coupling protocol. LL-HMP resin (ABI, 0.44 mmol
g−1 reactive group concentrations) was used as a solid support.
At the end of the synthesis, peptidyl-resin was isolated and
washed twice with NMP.
Instrumentation
Proton NMR spectra were recorded on a 300 MHz Bruker
spectrometer and mass spectra were recorded on a Mariner
System 5155 mass spectrometer using the electrospray ioniza-
tion (ESI) method. All column chromatography experiments
were performed on silica gel (Merck, Kieselgel 60H, Art 7736).
Absorption and fluorescence spectra were recorded on a Cary
400 spectrophotometer (Varian) and a FluoroMax 3.0 spectro-
fluorimeter (Jobin Yvon, Horiba), respectively. For fluorescence
studies, the dyes were used at 0.5 to 1 µM concentrations. All
measurements were performed in 20 mM phosphate buffer,
containing 150 mM NaCl (pH = 7.4) at 20 °C. The excitation
wavelength was 400 nm for labeled peptides and 408 nm for
the free 7AF label. All the spectra were corrected for Raman
scattering and background fluorescence measured before the
addition of the labelled peptide. Fluorescence quantum yields
were determined using 3-hydroxy-4′-diethylaminoflavone as a
reference (quantum yield, φ = 51%51). For the experiments in
water, 10 mM phosphate buffer, containing 30 mM NaCl
(pH 7.0), was used systematically.
Two equivalents (0.15 mmol) of the label were dissolved in
1 mL of NMP mixed with two eq. of HBTU/HOBt coupling
solution (in DMF) and added to Fmoc-deprotected peptidyl-
resin (0.075 mmol) swelled in 1 mL of NMP. After a few
minutes of shaking, five eq. of DIEA solution were added.
Then, the reaction mixture was shaken overnight at 40 °C.
Resin was filtered and washed with NMP, methanol and DCM.
Cleavage and deprotection of the peptidylresin were per-
formed for 2 h using a 10 mL trifluoroacetic acid (TFA) solu-
tion containing water (5%, v/v), TIS (iPr3SiH, 2.5%, v/v),
phenol (1%, w/v), thioanisole (5%, v/v) and ethanedithiol
(2.5%, v/v). The solution was concentrated in vacuo and the
peptide was precipitated using ice-cold diethyl ether and then
pelleted by centrifugation. The pellet was washed with diethyl
ether and dried. The peptide was solubilized with aqueous
TFA (0.05%, v/v). HPLC purification was carried out on a C8
column (uptisphere 300A, 5 µm; 250 × 10, Interchim, France)
in a water–acetonitrile mixture containing 0.05% TFA with a
linear gradient (20–60% of acetonitrile for 30 min) and moni-
tored at 210 nm (detection of non-labeled peptide) and
370 nm (detection of labeled peptide). The molecular mass
obtained by ion spray mass spectrometry was 2610.4 for
7AF–penetratin (in agreement with the expected theoretical
mass of 2610.7). Prior to use, the peptide was dissolved in dis-
tilled water, aliquoted and stored at −20 °C. The concentration
of the labelled peptide was determined from the label absor-
Deconvolution of spectra into two bands and calculations of
polarity and hydration parameters
Deconvolution of the fluorescence spectra into N* and T*
bands was performed using the Siano software kindly provided
by Prof. A.O. Doroshenko (Kharkiv, Ukraine), as previously
described.54 Then, the fluorescence parameters (cited in
Table 1 or applied in figure drawings) were derived from the
separated N* and T* bands. The local water concentration of
the probe environment was calculated using the equation C =
[log(IN*/IT*) + 0.975]/0.0377 (Fig. 1B, inset, Fig. S3†). Then, this
concentration was converted into the water access coefficient
WA by the equation WA (%) = 100[H2O]L/55.5.
bance at 400 nm using ε = 25 000 M−1 cm−1
.
Preparation of liposomes
All lipidic components were purchased in lyophilized form or
in CHCl3 solution from Avanti Polar Lipids (Birmingham,
Fluorescence microscopy experiments
USA). Stock solutions of 2 mM were prepared in CHCl3 and Fluorescence microscopy experiments were performed using a
stored in glass vials at −20 °C. Aliquots of the appropriate home-built two-photon laser scanning setup based on an
amounts of stock solutions were mixed in glass flasks. The Olympus IX70 inverted microscope with an Olympus 60×1.2
CHCl3 was removed under vacuum for at least 1 h, and then NA water immersion objective.55 Two-photon excitation was
lipids were rehydrated in 25 mM phosphate buffer, pH 6.5, provided by a titanium-sapphire laser (Tsunami, Spectra
150 mM NaCl, to a final concentration of 1 mM lipid in the Physics), and photons were detected with Avalanche Photo-
form of multi-lamellar vesicles (MLVs). Large unilamellar vesi- diodes (APD SPCM-AQR-14-FC, Perkin-Elmer) connected to a
cles (LUVs) were prepared by extrusion through polycarbonate counter/timer PCI board (PCI6602, National Instrument).
films (Nucleopore) in a handheld extruder (Avanti Polar Imaging was carried out using two fast galvo-mirrors in the
Lipids). The MLV suspension was first reduced in size by 7 pas- descanned fluorescence collection mode. Sizes of the images
sages through a large pore size (1 µm) filter. The suspension were 70 × 70 μm, resolution 512 × 512 pixel. Images corres-
was then passed 10 times through a filter with 0.1 µm pore ponding to the blue and red channels were recorded simul-
size. The LUVs were prepared at temperatures 5–15 °C above taneously using a dichroic mirror (Beamsplitter 585 DCXR)
the transition points. The size of all vesicles was determined and two band-pass filters (Brightline HC 520/20 and HQ
by DLS using a Zetasizer Nano ZS equipped with a 633 nm 585/40). The images were processed with a home-made
7042 | Org. Biomol. Chem., 2014, 12, 7036–7044
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Organic & Biomolecular Chemistry
Paper
program under LabView that generates a ratiometric image by
dividing the image of the blue channel by that of the red
channel. For each pixel, a pseudocolor scale is used for coding
the ratio, while the intensity is defined by the integrated inten-
8 G. S. Loving, M. Sainlos and B. Imperiali, Trends Biotech-
nol., 2010, 28, 73–83.
9 A. R. Katritzky and T. Narindoshvili, Org. Biomol. Chem.,
2009, 7, 627–634.
sity recorded for both channels at the corresponding pixel.56 10 V. Y. Postupalenko, O. M. Zamotaiev, V. V. Shvadchak,
The sensitivity of the two detectors was calibrated using solu-
tions of the 7AF dye in DMF and EtOH.
A. V. Strizhak, V. G. Pivovarenko, A. S. Klymchenko and
Y. Mely, Bioconjugate Chem., 2013, 24, 1998–2007.
11 B. E. Cohen, T. B. McAnaney, E. S. Park, Y. N. Jan,
S. G. Boxer and L. Y. Jan, Science, 2002, 296, 1700–1703.
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Commun., 2002, 1912–1913.
13 M. E. Vazquez, M. Nitz, J. Stehn, M. B. Yaffe and
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16 B. E. Cohen, A. Pralle, X. Yao, G. Swaminath, C. S. Gandhi,
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Cell preparation and staining
HeLa cells were cultured in Dulbecco’s modified Eagle
medium (DMEM, high glucose, Gibco-Invitrogen) sup-
plemented with 10% (v/v) fetal bovine serum (FBS, Lonza), 1%
antibiotic solution (penicillin–streptomycin, Gibco-Invitrogen)
in a humidified incubator with a 5% CO2 atmosphere at 37 °C.
A cell concentration of 5–10 × 104 cells mL−1 was maintained
by removal of a portion of the culture and replacing with fresh
medium 3 times per week. For microscopy studies, cells were
seeded onto a chambered coverglass (IBiDi) at a density of
5 × 104 cells per IBiDi. After washing the cells with Opti-MEM,
a solution of 7AF–penetratin diluted in 1 mL of Opti-MEM was
added to the cells to final concentrations of 2 μM and 10 μM
and immediately measured.
17 M. E. Vazquez, J. B. Blanco and B. Imperiali, J. Am. Chem.
Soc., 2005, 127, 1300–1306.
18 P. Venkatraman, T. T. Nguyen, M. Sainlos, O. Bilsel,
S. Chitta, B. Imperiali and L. J. Stern, Nat. Chem. Biol.,
2007, 3, 222–228.
19 K. G. Harikumar, D. I. Pinon, W. S. Wessels,
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Abbreviations
LUVs Large unilamellar vesicles
DOPC 1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine
DPPC 1,2-Dipalmitoyl-sn-glycero-3-phosphatidylcholine
20 P. K. Sengupta and M. Kasha, Chem. Phys. Lett., 1979, 68,
382–385.
Ch
Cholesterol
21 A. S. Klymchenko, Y. Mely, A. P. Demchenko and
G. Duportail, Biochim. Biophys. Acta, 2004, 1665, 6–19.
22 G. M’Baye, Y. Mely, G. Duportail and A. S. Klymchenko,
Biophys. J., 2008, 95, 1217–1225.
SM
Brain sphingomyelin
Acknowledgements
23 S. Oncul, A. S. Klymchenko, O. A. Kucherak,
A. P. Demchenko, S. Martin, M. Dontenwill, Y. Arntz,
P. Didier, G. Duportail and Y. Mely, Biochim. Biophys. Acta,
2010, 1798, 1436–1443.
O.M.Z, V.Y.P., V.G.P., and this work were supported by ANR
Fluoaant, ANR Femtostack, ANR Fluometadn, ANR Cellmem-
probes, CNRS, and Université de Strasbourg.
24 V. Y. Postupalenko, V. V. Shvadchak, G. Duportail,
V. G. Pivovarenko, A. S. Klymchenko and Y. Mely, Biochim.
Biophys. Acta, 2011, 1808, 424–432.
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