Fluorescent peptides labeled with environment-sensitive 7-aminocoumarins and their interactions with lipid bilayer membranes and living cells

Article abstract of DOI:10.1246/bcsj.20120314

The photophysical properties of protected nonnatural amino acids BocL- Dap(7DEAC)OMe (7DEAC: 7-diethylaminocoumarin) and BocL- Dap(C343)OMe (C343: coumarin 343) were investigated to evaluate the suitability of these amino acids as fluorescent units in peptide-based fluorescent biosensors. The absorption and fluorescence spectra of BocL- Dap(7DEAC)OMe and BocL- Dap(C343)OMe exhibited significant red shifts with increasing solvent polarity. The fluorescence quantum yield and lifetime of BocL- Dap(7DEAC)OMe solutions decreased remarkably with increasing solvent polarity, whereas those of BocL- Dap(C343)OMe were slightly affected by the solvent polarity. Fluorescent peptides HDap( 7DEAC)LLAOMe (1), HDap( 7DEAC)KLAOMe (2), and HDap( 7DEAC)ELAOMe (3) labeled with environment-sensitive 7DEAC were synthesized to examine the interactions of these labeled peptides with lipid membranes and living cells. Neutral 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and anionic 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG) liposomes were used to investigate peptidemembrane interactions. Hydrophobic peptide 1 exhibited high affinities to both DMPC and DMPC/DMPG mixed membranes. Of the three peptides, cationic peptide 2 exhibited the strongest affinity to DMPC/DMPG membranes, whereas anionic peptide 3 showed a much lower affinity to DMPC/DMPG membranes. These results could be interpreted based on hydrophobic and electrostatic interactions between the peptides and membranes. Peptide 1 was efficiently internalized into HeLa cells, whereas peptides 2 and 3 showed a much lower intracellular delivery.

Full text of DOI:10.1246/bcsj.20120314

510
Bull. Chem. Soc. Jpn. Vol. 86, No. 4, 510-519 (2013)
© 2013 The Chemical Society of Japan
Fluorescent Peptides Labeled with Environment-Sensitive
7-Aminocoumarins and Their Interactions with
Lipid Bilayer Membranes and Living Cells
Tokiko Murase, Toshitada Yoshihara, Keiichi Yamada, and Seiji Tobita*
Department of Chemistry and Chemical Biology, Gunma University, Kiryu, Gunma 376-8515
Received November 13, 2012; E-mail: tobita@gunma-u.ac.jp
The photophysical properties of protected nonnatural amino acids Boc-L-Dap(7DEAC)-OMe (7DEAC: 7-di-
ethylaminocoumarin) and Boc-L-Dap(C343)-OMe (C343: coumarin 343) were investigated to evaluate the suitability of
these amino acids as fluorescent units in peptide-based fluorescent biosensors. The absorption and fluorescence spectra
of Boc-L-Dap(7DEAC)-OMe and Boc-L-Dap(C343)-OMe exhibited significant red shifts with increasing solvent
polarity. The fluorescence quantum yield and lifetime of Boc-L-Dap(7DEAC)-OMe solutions decreased remarkably with
increasing solvent polarity, whereas those of Boc-L-Dap(C343)-OMe were slightly affected by the solvent polarity.
Fluorescent peptides H-Dap(7DEAC)-LLA-OMe (1), H-Dap(7DEAC)-KLA-OMe (2), and H-Dap(7DEAC)-ELA-
OMe (3) labeled with environment-sensitive 7DEAC were synthesized to examine the interactions of these labeled
peptides with lipid membranes and living cells. Neutral 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and
anionic 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG) liposomes were used to investigate peptide-
membrane interactions. Hydrophobic peptide 1 exhibited high affinities to both DMPC and DMPC/DMPG mixed
membranes. Of the three peptides, cationic peptide 2 exhibited the strongest affinity to DMPC/DMPG membranes,
whereas anionic peptide 3 showed a much lower affinity to DMPC/DMPG membranes. These results could be interpreted
based on hydrophobic and electrostatic interactions between the peptides and membranes. Peptide 1 was efficiently
internalized into HeLa cells, whereas peptides 2 and 3 showed a much lower intracellular delivery.
Peptide-based fluorescent biosensors1,2 have been developed
cal water-sensitive fluorophores used in biological studies in-
clude 2-dimethylamino-6-propionylnaphthalene,22 nitrobenzo-
furazan,23 dimethylaminonaphthalenesulfonyl,24 N,N-dimethyl-
amino-1,8-naphthalimide,25 and coumarin derivatives.26 We
recently synthesized a small fluorescent amino acid 3-[2-cyano-
4-(dimethylamino)phenyl]alanine that is almost nonfluorescent
in water but whose fluorescence intensity and lifetime are sig-
nificantly enhanced when the molecule partitions to nonaqueous
environments.27 We have also developed a new fluorophore
8-methoxy-4-methyl-2H-benzo[g]-chromen-2-one that displays
the opposite behavior: it is almost nonfluorescent in nonpolar
aprotic media, but becomes highly emissive in polar protic
media such as water.28
To apply these environment-sensitive fluorophores to bio-
logical studies, it is essential to determine the photophysical
mechanism of the environment-sensitive fluorescence. This
study focuses on aminocoumarins. They are attractive fluoro-
phores due to their relatively long-wavelength absorption and
fluorescence spectra, high emission quantum yield, high photo-
stability, and excellent environment-sensitive emission proper-
ties.26,29 Synthesis of fluorescent coumarin-bearing amino acids
has been reported for biological imaging.30 Aminocoumarin-
labeled peptides with fluorogenic activity have also been syn-
thesized for fluorescence imaging of proteins.31
for investigating the interactions between peptides and bio-
logical membranes,3-6 for detecting ions7-10 and studying pro-
tein functions11-16 in living cells, and for various biomedical
applications.17-20 Fluorescent peptides are often imparted
with fluorogenic properties to visualize biologically targeted
substances or events in living organisms. One way to achieve
this is to synthesize fluorogenic peptides with excitation donor
and acceptor units or with a quenching unit having a fluo-
rescent group. The fluorescence intensity of these peptides is
enhanced through Förster resonance energy transfer or sup-
pression of electron-transfer quenching induced by structural
changes in the peptide in response to the detection of the
targeted substances or biological events. Another approach
involves using an environment-sensitive fluorophore that
exhibits distinct fluorescence properties (e.g., fluorescence
quantum yield, lifetime, and wavelength) when it binds with
targeted substances or when a certain biological event occurs.
This approach has been used to turn the fluorescence of labeled
peptides on and off.1,2,5,21 This latter approach has the advan-
tage that the labeled peptides are simple to synthesize and it is
particularly useful for investigating peptide-membrane inter-
actions in real-time.
Water-sensitive fluorophores that exhibit intense visible
fluorescence in nonaqueous environments but whose fluores-
cence is remarkably quenched in aqueous media are especially
useful for investigating protein functions in living cells and
interactions between peptides and biological membranes. Typi-
In this study, we synthesized fluorescent amino acids bearing
a fluorophore (7DEAC or C343) and evaluated their poten-
tial as fluorescence units for peptide-based fluorescent bio-
sensors by examining their photophysical properties. The two
Published on the web March 20, 2013; doi:10.1246/bcsj.20120314

T. Murase et al.
Bull. Chem. Soc. Jpn. Vol. 86, No. 4 (2013)
511
O
O
H
N
H
N
(a) in n–hexane
1
1
O
O
O
O
O
O
HN
HN
0.5
0.5
O
O
O
O
O
O
0
1
0
1
(b) in acetonitrile
N
N
(a)
(b)
0.5
0.5
Scheme 1. Structures of (a) Boc-L-Dap(7DEAC)-OMe and
(b) Boc-L-Dap(C343)-OMe.
0
1
0
1
fluorophores were incorporated into the side chain amino
group of Dap (L-2,3-diaminopropionic acid) via amide bond.
Furthermore, short fluorescent peptides H-Dap(7DEAC)-
LLA-OMe (1), H-Dap(7DEAC)-KLA-OMe (2), and H-Dap-
(7DEAC)-ELA-OMe (3) labeled with environment-sensitive
7DEAC were synthesized to examine the interactions of the
labeled peptides with lipid membranes and living cells. These
tetrapeptides consist of three different dipeptide units contain-
ing fluorescent L-Dap(7DEAC) residue at N-terminal (Dap-
(7DEAC)-X; X = L (Leu), K (Lys), and E (Glu)). In addition,
hydrophobic dipeptide ester moiety (LA-OMe) was incorpo-
rated to assist the entry of the labeled peptides into the lipid
membrane since the LA sequence is frequently used in the
model peptides of transmembrane helices32,33 and coiled-coil
peptides.31 Peptides 1-3 possess distinct charge and hydro-
phobicity. They are thus expected to exhibit different affinities
to biological membranes.
(c) in Tris–HCl buffer/MeOH = 9 : 1
0.5
0.5
0
0
300
400
500
600
Wavelength / nm
Figure 1. Absorption and fluorescence spectra of Boc-
L-Dap(7DEAC)-OMe (solid line) and Boc-L-Dap(C343)-
OMe (broken line) in (a) n-hexane, (b) MeCN, and (c) Tris-
HCl buffer/MeOH (9:1 v/v) at 20 °C. -exc: (a) 370 and
380 nm, (b) 390 and 400 nm, and (c) 400 and 420 nm for
Boc-L-Dap(7DEAC)-OMe and Boc-L-Dap(C343)-OMe,
respectively.
Results and Discussion
Photophysical Properties of Boc-L-Dap(7DEAC)-OMe
properties, namely Taft’s hydrogen-bonding ability (¡), the
dielectric constant (¾), and the orientation polarizability (ꢀf),
which is given by:
and Boc-L-Dap(C343)-OMe.
We synthesized protected
nonnatural amino acids Boc-L-Dap(7DEAC)-OMe and Boc-
L-Dap(C343)-OMe (Scheme 1) to evaluate their suitability as
environment-sensitive fluorescent units of peptide-based fluo-
rescent biosensors. Their photophysical properties were mea-
sured in various solvents that have different polarities and
hydrogen-bonding abilities to determine their environment-
sensitive properties. Figure 1 shows the absorption and fluores-
cence spectra of Boc-L-Dap(7DEAC)-OMe and Boc-L-Dap-
(C343)-OMe obtained in nonpolar solvent n-hexane, polar
solvents acetonitrile (MeCN), and Tris-HCl/MeOH (9:1, v/v)
at 20 °C. The absorption and fluorescence spectra of both com-
pounds exhibit similar shapes and shift remarkably to the red
when the solvent is changed from nonpolar n-hexane to polar
MeCN and Tris-HCl/MeOH. In these solvents the absorption
and fluorescence bands of Boc-L-Dap(C343)-OMe are shifted
to wavelengths that are 15-20 nm longer than those of Boc-
L-Dap(7DEAC)-OMe. The large red shifts in the absorption
spectra observed when the solvent was changed from a nonpolar
solvent to a polar solvent suggest that the dipole moment of the
¾ ꢀ 1
n2 ꢀ 1
ꢀf ¼ fð¾Þ ꢀ fðn2Þ ¼
ꢀ
ð1Þ
2¾ þ 1 2n2 þ 1
where fð¾Þ and fðn2Þ are respectively the high and low
frequency polarizabilities.34 At high solvent polarities, the
absorption and fluorescence spectra exhibit significant red
shifts on changing the solvent from n-hexane to Tris-HCl/
MeOH (9:1, v/v): 28 and 65 nm respectively for -
of Boc-L-Dap(7DEAC)-OMe and 31 and 66 nm respectively
abs
max
flu
max
and -
abs
flu
max
for -
and -
of Boc-L-Dap(C343)-OMe. The protic sol-
max
vents with hydrogen-donating ability increase the red shifts in
both the absorption and fluorescence spectra; this is probably
due to the hydrogen-bonding interaction with the carbonyl
oxygen in the coumarin unit. These findings indicate that Boc-
L-Dap(7DEAC)-OMe and Boc-L-Dap(C343)-OMe exhibit
intramolecular charge transfer from the diethyl amino group
to the carbonyl moieties in the first excited singlet (S1) state. In
fact, Lippert-Mataga plots suggest that the ground and S1 states
of Boc-L-Dap(7DEAC)-OMe and Boc-L-Dap(C343)-OMe
have relatively large dipole moment differences (®e ¹ ®g =
2.52 and 2.43 D, respectively)35 (see Figure S1 in Supporting
Information). In protic solvents, hydrogen-bonding interactions
molecules is enhanced by the electronic transition to the S1 state.
abs
max
Table 1 lists the absorption and fluorescence maxima (-
flu
ꢁ
and -max) and the Stokes shift (ꢀ¯) of Boc-L-Dap(7DEAC)-
OMe and Boc-L-Dap(C343)-OMe together with the solvent

512
Bull. Chem. Soc. Jpn. Vol. 86, No. 4 (2013)
Fluorescent Peptides Labeled with 7-Aminocoumarin
abs
flu
ꢁ
Table 1. Absorption Maximum (-max), Fluorescence Maximum (-max), and Stokes Shift (ꢀ¯) of Boc-L-Dap(7DEAC)-OMe and Boc-
L-Dap(C343)-OMe in Selected Solvents with Different Polarity and Proton-Donor Ability at 20 °C
Boc-L-Dap(7DEAC)-OMe
Boc-L-Dap(C343)-OMe
a)
b)
Solvents
¡
¾
¦fc)
abs
flu
abs
flu
3
¹1
3
¹1
ꢁ
ꢁ
-max/nm ꢀ¯/10 cm
-
max/nm
-
max/nm ꢀ¯/10 cm
-
max/nm
n-Hexane
0.00
0.00
0.00
0.00
®
1.89 0.000
3.08 0.0957
4.27 0.165
6.08 0.201
20.0 0.269
24.8 0.280
29.7 0.292
403
408
408
412
417
417
417
417
419
431
420
431
415
433
438
452
462
463
464
466
466
471
470
480
0.752
1.42
1.76
2.24
2.40
2.45
2.52
2.62
2.47
2.06
2.59
2.49
418
423
423
428
435
435
435
435
437
450
439
449
430
448
453
467
478
479
480
482
483
487
485
496
0.657
1.35
1.63
2.06
2.12
2.18
2.23
2.30
2.19
1.72
2.19
2.15
Dibutyl ether
Diethyl ether
Ethyl acetate
Butyl cyanide
Propyl cyanide
Ethyl cyanide
Acetonitrile
Ethanol
2,2,2-Trifluoroethanol
Methanol
®
®
0.19 36.6 0.305
0.83 25.3 0.290
1.51 27.7 0.316
0.93 33.0 0.309
Tris-HCl/MeOH (9:1, v/v) 1.05 75.4 0.320
a) Hydrogen-bond donor ability of solvent. b) Dielectric constant of solvent. c) Orientational polarizability of solvents.
Table 2. Fluorescence Quantum Yield (Φf ), Fluorescence Lifetime (¸f ), Fluorescence Rate Constant (kf ) and Nonradiative Rate Constant
(knr) of Boc-L-Dap(7DEAC)-OMe and Boc-L-Dap(C343)-OMe in Selected Solvents at 20 °C
Boc-L-Dap(7DEAC)-OMe
Boc-L-Dap(C343)-OMe
Solvents
¦f a)
Φfb)
¸
f
b)/ns
kf/108 s
knr/108 s
Φfb)
¸
f
b)/ns
kf/108 s
knr/108 s
¹1
¹1
¹1
¹1
n-Hexane
0.000
0.0957
0.165
0.201
0.269
0.280
0.292
0.305
0.290
0.316
0.309
0.320
0.86
0.95
0.96
0.76
0.24
0.16
0.10
0.065
0.085
0.037
0.034
0.018
2.6
2.7
2.9
2.5
0.87
0.55
0.36
0.23
0.27
0.15
0.13
0.072
3.3
3.5
3.3
3.0
2.8
2.9
2.8
2.8
3.1
2.5
2.6
2.5
0.54
0.19
0.14
0.96
8.7
15
25
41
34
64
74
136
0.80
0.93
0.94
0.97
0.85
0.95
0.90
0.94
0.93
0.95
0.95
0.89
2.9
3.0
3.3
3.4
3.5
3.6
3.7
3.8
3.7
4.3
3.9
4.1
2.8
3.1
2.8
2.9
2.4
2.6
2.4
2.5
2.5
2.2
2.4
2.2
0.69
0.23
0.18
0.088
0.43
0.14
0.27
0.16
0.19
0.12
0.13
0.27
Dibutyl ether
Diethyl ether
Ethyl acetate
Butyl cyanide
Propyl cyanide
Ethyl cyanide
Acetonitrile
Ethanol
2,2,2-Trifluoroethanol
Methanol
Tris-HCl/MeOH (9:1, v/v)
a) Orientational polarizability of solvents. b) Error is within 10%.
between proton-donating solvents and the carbonyl moieties of
aminocoumarins are enhanced in the S1 state that has charge-
transfer character. This may further stabilize the excited state.
The fluorescence decay profiles were measured by exciting
Boc-L-Dap(7DEAC)-OMe and Boc-L-Dap(C343)-OMe; they
exhibited single exponential decays in all the solvents. Table 2
lists the fluorescence quantum yield (Φf) and the fluorescence
lifetime (¸f) along with the radiative (kf) and nonradiative (knr)
rate constants, which were calculated from Φf and ¸f using the
following equations.
hydrogen-bonding interactions enhance the nonradiative rate.
Ramakrishna and Ghosh36 have reported similar photophysical
properties for a similar coumarin derivative, 7-diethylamino-
coumarin-3-carboxylic acid.
In contrast to the environment-sensitive relaxation properties
of excited Boc-L-Dap(7DEAC)-OMe, the fluorescence quan-
tum yield and lifetime of Boc-L-Dap(C343)-OMe are almost
independent of the solvent parameters, as shown in Table 2.
Specifically, the nonradiative transition rates are little affected
by changes in the solvent polarity and the hydrogen-bonding
ability, despite the remarkable shifts in the absorption and fluo-
rescence spectra with changing the solvent polarity. Moreover,
the fluorescence quantum yield is close to unity irrespective
of the solvent properties. The most remarkable difference
between the fluorophores in Boc-L-Dap(7DEAC)-OMe and
Boc-L-Dap(C343)-OMe is that the internal rotational motion
and out-of-bending vibrations of the amino group are structur-
ally restricted in C343, unlike in 7DEAC. This clearly suggests
that the structural changes in the diethylamino group on elec-
tronic excitation are responsible for the environment-sensitive
photophysical character of Boc-L-Dap(7DEAC)-OMe.
kf ¼ ꢀf=¸f; knr ¼ ð1 ꢀ ꢀfÞ=¸f
ð2Þ
Table 2 shows that the fluorescence quantum yield and fluo-
rescence lifetime of Boc-L-Dap(7DEAC)-OMe vary remark-
ably with the solvent properties: an increase in the solvent
polarity significantly reduces Φf and ¸f due to an enhanced
nonradiative rate. In particular, the most remarkable quenching
is observed in strongly proton-donating solvents such as 2,2,2-
trifluoroethanol and Tris-HCl/MeOH (9:1). The nonradia-
tive rate constant of Boc-L-Dap(7DEAC)-OMe in Tris-HCl/
MeOH (9:1) is of the order of 1010 s¹1, which suggests that

T. Murase et al.
Bull. Chem. Soc. Jpn. Vol. 86, No. 4 (2013)
513
(a)
(c)
1
0.5
0
1
0.5
0
Φ
Φ
0
0.1
0.2
0.3
0
0.1
0.2
0.3
Δf
Δf
4
3
2
1
0
6
4
2
0
(b)
(d)
τ
τ
0
0.1
0.2
0.3
0
0.1
0.2
0.3
Δf
Δf
Figure 2. Plots of fluorescence quantum yield (Φf ) and lifetime (¸f) of Boc-L-Dap(7DEAC)-OMe ((a) and (b)) and Boc-
L-Dap(C343)-OMe ((c) and (d)) against solvent parameter (¦f).
Figure 2 shows a plot of Φf and ¸f of Boc-L-Dap(7DEAC)-
OMe and Boc-L-Dap(C343)-OMe as a function of the sol-
vent polarity parameter ðꢀfÞ. Interestingly, the plots for Boc-
L-Dap(7DEAC)-OMe (Figures 2a and 2b) show that Φf and ¸f
increase gradually with increasing solvent polarity, whereas
in highly polar solvents with ꢀf larger than ca. 0.17, Φf and
¸f tend to decrease rapidly. In contrast, Φf and ¸f of Boc-
L-Dap(C343)-OMe (Figures 2c and 2d) are almost constant in
all the solvents used, although moderate increases in ¸f are
observed in strongly polar solvents. The drastic changes in
the solvent polarity dependences of Φf and ¸f, which were
observed only in Boc-L-Dap(7DEAC)-OMe, suggest that a
polarity-dependent nonradiative route opened in solvents with
ꢀf larger than ca. 0.17 due to conformational changes in the
diethylamino group in the excited state.26,36
The distinct photophysical properties of Boc-L-Dap-
(7DEAC)-OMe and Boc-L-Dap(C343)-OMe suggest that
7DEAC can be used as a fluorophore for developing an envi-
ronment-sensitive fluorescent probe that is water sensitive,
while C343 is suitable for use as a biological labeling agent
that does not respond to the surrounding environment.
Affinity of Coumarin-Labeled Peptides to Lipid Bilayer
Membranes. We synthesized three 7DEAC-labeled peptides
1-3 (Scheme 2) to examine the interactions between the
labeled peptides and lipid membranes through the environ-
ment-sensitive fluorescence character of 7DEAC. Coumarin-
labeled compounds 1-3 are tetrapeptides, which have a residue
with a distinct charge in the second position from the N-
terminus. Peptide 1 has a neutral amino acid leucine, while
peptides 2 and 3 respectively have a cationic lysine and anionic
glutamic acid as the second residue. As mentioned in the
previous section, 7DEAC has a very low fluorescence quantum
O
R
O
H
N
H2N
O
O
N
H
N
H
O
O
NH
O
O
N
R = CH2CH(CH3)2 (Leu) (1)
= (CH2)4NH2
(Lys) (2)
= (CH2)2COOH (Glu) (3)
Scheme 2. Structures of coumarin-labeled peptides 1-3.
yield in aqueous media, whereas it is much higher in non-
aqueous and less polar environments. Due to the environment-
sensitive character of 7DEAC, fluorescence enhancement is
expected to occur when the 7DEAC-labeled peptides interact
with lipid bilayers. In addition, the fluorescence spectra will
shift to shorter wavelengths when the peptides are partitioned
into hydrophobic and less polar sites in lipid bilayer mem-
branes from the highly polar aqueous phase.
To examine the affinity of coumarin-labeled peptides 1-3
with lipid bilayer membranes, we employed zwitterionic 1,2-
dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and ani-
onic 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]
(DMPG) membranes. Unilamellar vesicles composed of
DMPC or DMPC/DMPG (1:1) membranes were prepared at
35 °C. The fluorescent peptide concentration was adjusted to be
1 ¯M. Figure 3 shows the variation of the fluorescence spectra
of 1-3 on adding DMPC (0.2-5.0 mM) and DMPC/DMPG
(1:1) (0.02-1.0 mM) liposomes to a Tris-HCl buffer (pH 7.0) at

514 Bull. Chem. Soc. Jpn. Vol. 86, No. 4 (2013)
Fluorescent Peptides Labeled with 7-Aminocoumarin
150
300
200
100
0
(a) 1
(d) 1
[DMPC/DMPG]
= 0.02-1.0 mM
[DMPC] = 0.2-5.0 mM
100
50
0
400
500
500
500
600
400
500
500
500
600
600
600
40
20
0
300
200
100
(b) 2
(e) 2
0
400
600 400
40
20
0
(f) 3
(c) 3
40
20
0
400
400
600
Wavelength / nm
Figure 3. Fluorescence spectral changes of peptides (a) 1, (b) 2, and (c) 3 (1 ¯M) on addition of 0.2-5.0 mM DMPC liposomes,
and (d) 1, (e) 2, and (f) 3 (1 ¯M) on addition of 0.02-1.0 mM DMPC/DMPG (1:1) liposomes in Tris-HCl buffer (pH 7.0) at
35 °C. The broken line represents the fluorescence spectra of 1-3 in Tris-HCl buffer (pH 7.0) without addition of liposomes (-exc
:
380 nm).
35 °C. Figures 3a-3c show that the fluorescence intensity due
Iw þ Kp£L½LꢁIL
I ¼
ð4Þ
to the 7DEAC moiety in the peptide increases significantly
and the spectral peak shifts to the blue with increasing DMPC
concentration, demonstrating that peptides 1-3 are partitioned
inside the DMPC membrane. Similar changes in the fluo-
rescence spectra of 1-3 are also observed for DMPC/DMPG
(1:1) (0.02-1.0 mM) liposomes (Figures 3d-3f), although the
degree of enhancement depends on the type of membrane.
To quantify the extent of the interaction of the peptides with
the membrane model system, the partition coefficient (Kp)
between the lipid and aqueous phases is defined as
1 þ Kp£L½Lꢁ
where Iw and IL are respectively the maximum fluorescence
intensities obtained when all the fluorescent peptides are in the
water and in the membrane and £L is the molar volume of the
¹1 38
membrane (0.67 L mol
)
and [L] is the lipid concentration.
Peptide 1 was partitioned into the DMPC and DMPC/DMPG
(1:1) membranes with Kp = 4.9 («0.5) © 102 and 4.2 («0.4) ©
103, respectively. Cationic peptide 2 was more efficiently
incorporated into the DMPC/DMPG membrane with Kp = 1.4
(«0.1) © 104, although it seems to have a much lower affinity
to the DMPC membrane than to the DMPC/DMPG (1:1)
membrane. These findings indicate that the negative charge
of the DMPG membrane facilitates interactions between the
membrane and peptide 2, which has a positively charged lysine
residue. Peptide 3, which has a negative charge on the glutamic
acid residue, was not partitioned efficiently into either mem-
brane, probably due to repulsive interactions with DMPG.
These results indicate that hydrophobic and electrostatic inter-
nL=VL
Kp ¼
ð3Þ
nW=VW
where nL and nW are respectively the moles of peptide in lipid
and water phases and VL and VW are respectively the volumes
of lipid and water. From plots of the integrated fluorescence
intensity I of the peptides against the concentration of DMPC
or DMPC/DMPG (1:1) in Tris-HCl buffer (Figure 4), Kp be-
tween the lipid and aqueous phases was determined using:37

T. Murase et al.
Bull. Chem. Soc. Jpn. Vol. 86, No. 4 (2013)
515
5
0
(a)
8000
–5
103
(a)
(b)
(c)
102
101
100
4000
0
0
2
4
6
[DMPC] / mM
0
2
4
6
15000
10000
5000
0
5
0
(b)
–5
103
102
101
100
0
0.4
0.8
1.2
[DMPC/DMPG(1:1)] / mM
0
2
4
6
Figure 4. Fluorescence intensity changes of 1 ( ), 2 ( ),
and 3 ( ) (1 ¯M) vs. (a) DMPC and (b) DMPC/DMPG
concentrations in Tris-HCl buffer (pH 7.0) at 35 °C.
5
0
–5
103
actions play an important role in determining the affinity of
short peptides to lipid bilayer membranes.39
102
101
100
Fluorescence Decay Characteristics of Coumarin-
Labeled Peptides in Lipid Bilayer Membranes. Measuring
the fluorescence decay characteristics is a powerful approach
for directly monitoring the environment and dynamics around
a fluorescent peptide, which reflects interactions with lipid
bilayer membranes. Figures 5a-5c show the fluorescence decay
profiles of peptide 1 (1 ¯M) in Tris-HCl buffer and in the
presence of DMPC (1 mM) and DMPC/DMPG (1:1; 1 mM)
liposomes in Tris-HCl buffer at 35 °C, respectively. The fluo-
rescence decay curves were deconvoluted with the instrument
response function and analyzed as a sum of exponential terms:
X
0
5
10
Time / ns
Figure 5. Fluorescence decay profiles of 1 (1 ¯M) in
(a) Tris-HCl buffer (pH 7.0) and in Tris-HCl buffer under
the presence of (b) DMPC (1 mM) and (c) DMPC/DMPG
(1:1; 1 mM) at 35 °C. -exc: 266 nm and -mon: (a) 480,
(b) 461, and (c) 458 nm.
FðtÞ ¼
Ai expðꢀt=¸iÞ
ð5Þ
i
close to the lifetime observed in buffer solutions, which
suggests that this component originates from peptide 1 mole-
cules in the water phase (i.e., on the outside of the liposomes).
The proportion (12%) of the shortest lifetime component is
much lower than that (88%) of the longer lifetime components,
which are not observed for peptides in buffer solutions. These
results clearly show that most of peptide 1 is incorporated into
the liposome membrane, which is consistent with the relatively
large Kp value (4.9 («0.5) © 102) of 1 in DMPC membranes.
The observation of two long-lifetime components (805 ps and
1.92 ns) suggests that peptide 1 is localized in at least two
distinct sites with different polarities in DMPC liposome mem-
branes. It is conceivable from Table 2 that peptide 1 molecules,
which give the longest lifetime (1.92 ns) component with the
pre-exponential factor of 0.46, are localized in a site with its
polarity between ethyl acetate and n-butyl cyanide in liposome
membranes. In the same way, peptide 1 molecules, which give
where F(t) is the fluorescence intensity at time t, and ¸i and Ai
are respectively the lifetime and the pre-exponential factor of
the ith component. The average lifetime was calculated from
¸i and Ai using
n
X
Ai¸2i
i¼1
h¸i ¼
ð6Þ
n
X
Ai¸i
i¼1
The fluorescence decay profile of peptide 1 in Tris-HCl buffer
(Figure 5a) could be analyzed by a single exponential with a
lifetime of 62 ps, while much slower decay components were
observed in the presence of DMPC (1 mM) (Figure 5b) and the
decay curve was best fitted using three decay components with
lifetimes of 65 ps (A1 = 0.12), 805 ps (A2 = 0.42), and 1.92 ns
(A3 = 0.46). The shortest lifetime (65 ps) component is very

516
Bull. Chem. Soc. Jpn. Vol. 86, No. 4 (2013)
Fluorescent Peptides Labeled with 7-Aminocoumarin
5
0
5
0
–5
–5
103
103
102
101
100
(a)
(a)
(b)
(c)
102
101
100
0
2
4
6
0
0
0
2
2
2
4
4
4
6
6
6
5
0
5
0
–5
–5
103
103
(b)
102
101
100
102
101
100
0
2
4
6
5
0
5
0
–5
–5
103
(c)
103
102
101
100
102
101
100
0
5
10
Time / ns
Time / ns
Figure 6. Fluorescence decay profiles of 2 (1 ¯M) in
(a) Tris-HCl buffer (pH 7.0) and in Tris-HCl buffer under
the presence of (b) DMPC (1 mM) and (c) DMPC/DMPG
(1:1; 1 mM) at 35 °C. -exc: 266 nm and -mon: (a) 476,
(b) 472, and (c) 457 nm.
Figure 7. Fluorescence decay profiles of 3 (1 ¯M) in (a)
Tris-HCl buffer (pH 7.0) and in Tris-HCl buffer under the
presence of (b) DMPC (1 mM) and, (c) DMPC/DMPG
(1:1; 1 mM) at 35 °C. -exc: 266 nm and -mon: (a) 480, (b)
476, and (c) 468 nm.
the second lifetime (805 ps) component with the pre-exponen-
tial factor of 0.42, are considered to be localized in a site with
its polarity between butyl cyanide and propyl cyanide, i.e., a
more polar site with its location close to the head group of the
liposome membranes. In the presence of DMPC/DMPG (1:1)
liposomes, the shortest lifetime component almost disappeared
and the fluorescence profile could be analyzed in terms of two
exponential decays with lifetimes of 638 ps (A1 = 0.22) and
1.74 ns (A2 = 0.78). This indicates that almost all the peptide
molecules are bound to DMPC/DMPG (1:1) membranes and
hence the higher affinity to DMPC/DMPG (1:1) membranes is
probably due to electrostatic interactions between the proto-
nated N-terminus of the peptide and the anionic DMPG lipid in
DMPC/DMPG (1:1) membranes.
analysis together with the result of 1. The fluorescence decay
curves of 2 and 3 in Tris-HCl buffer were best analyzed by two
exponentials with lifetimes of 50-60 and 150-190 ps (Table 3).
Although the origin of the two exponential decay components
in Tris-HCl buffer is not obvious, one possible explanation is
given in terms of different conformers that can be involved in
labeled peptides in water.27 In the presence of DMPC, peptide 2
has a much slower decay component with a lifetime of 1.63 ns
(A2 = 0.45), in addition to the fast decay component with a
lifetime of 0.063 ns (A1 = 0.55). Here, the magnitude of the
pre-exponential factor is not quantitatively related to partition-
ing of the peptides between the membrane and the aqueous
phase due to the fluorescence spectral shift accompanying
partitioning of the peptides into the less polar membrane.
However, the above result suggests that nearly half of peptide 2
is bound to the DMPC membrane. In the presence of DMPC/
DMPG (1:1) membranes, the component with lifetimes of 50-
Figures 6 and 7 respectively show the fluorescence decay
profiles of peptides 2 and 3 in Tris-HCl buffer and in lipid
membranes. Table 3 lists the results of fluorescence decay

T. Murase et al.
Bull. Chem. Soc. Jpn. Vol. 86, No. 4 (2013)
517
Table 3. Fluorescence Lifetimes of 1-3 (1 ¯M) under the Presence of DMPC (1 mM) and DMPC/DMPG (1:1; 1 mM)
Membranes in Tris-HCl Buffer (pH 7.0) at 35 °C
2
Compounds
Lipid
-
mon/nm
¸/ns
A1
¸2/ns
A2
¸3/ns
A3
h¸i=ns
»
1
No Lipid
DMPC
DMPC/DMPG
No Lipid
DMPC
DMPC/DMPG
No Lipid
DMPC
480
461
458
476
472
457
480
476
468
0.062
0.065
0.638
0.052
0.063
0.88
0.056
0.053
0.062
1.00
0.12
0.22
0.76
0.55
0.34
0.70
0.75
0.39
0.062
1.23
1.03
1.05
1.02
1.13
0.99
1.13
1.20
1.02
1.01
0.805
1.74
0.155
1.63
0.42
0.78
0.24
0.45
0.66
0.30
0.25
0.61
1.92
0.46
1.50
0.077
0.77
2
3
2.07
1.67
0.182
0.244
1.37
0.094
0.102
0.86
DMPC/DMPG
60 ps almost disappeared and the proportion of the long
lifetime (2.07 ns) component increased in peptide 2, which is
in agreement with the large Kp value (1.4 («0.1) © 104) of 2 in
DMPC/DMPG membranes.
¹0.66, and 0.207, respectively. Goodwin et al. investigated
the relationship between the transcellular permeability coef-
ficients and log Po/w by using a series of dipeptides40 and found
that the cellular permeability increases with increasing log Po/w
,
In contrast to peptide 2, peptide 3 exhibits similar two-
component decays in Tris-HCl buffer and in the same buffer
containing DMPC liposomes. This shows that anionic peptide
3 has a very low affinity to the zwitterionic DMPC membrane.
The decay profile of peptide 3 in the presence of DMPC/
DMPG (1:1) membranes in Tris-HCl buffer was best fitted by
two exponentials with lifetimes of 62 ps (A1 = 0.39) and 1.37
ns (A2 = 0.61). The lifetime of the faster decay component
is close to that in Tris-HCl buffer. These findings show that
peptide 3 has a much lower affinity to DMPC/DMPG (1:1)
membranes that contain the negatively charged DMPG lipid
than peptides 1 and 2. The electrostatic repulsion between the
anionic carboxy group of 3 and the negatively charged DMPG
lipid in DMPC/DMPG (1:1) membranes is related to the lower
affinity of 3 to DMPC/DMPG (1:1) membranes.
Hydrophobicity of the LA sequence in 1-3 will promote
the incorporation of the labeled peptides into the hydrophobic
region of lipid bilayer membranes, resulting in the appearance
of the long lifetime components. On the other hand, the pro-
tonated amino group of the peptides will facilitate the elec-
trostatic and/or hydrogen-bonding interactions of the peptides
with the anionic head group of lipid membranes. The latter ionic
interactions might be related to the second lifetime (805 ps)
component observed for 1 and 2 in DMPC and/or DMPC/
DMPG membranes, because partial water penetration occurs
in the membrane interfacial region. In the case of the zwitter
ionic peptide 3, however, such ionic interactions would be
suppressed by the electrostatic repulsion due to the anionic
carboxy group.
Affinity of Peptides 1-3 to Living Cells. We then investi-
gated the interactions between peptides 1-3 and living cells
(HeLa cells). Figures 8a-8c show emission images of HeLa
cells that were loaded with peptides 1-3 (10 ¯M). Peptide 1
gives a much brighter image than peptides 2 and 3, showing
that peptide 1 has a much higher cellular uptake efficiency than
peptides 2 and 3. The logarithm of the water-octanol parti-
tion coefficient (log Po/w) of a substance loaded into the cell
medium is known to be correlated with the ability of the
substance to penetrate the plasma membrane of living cells40,41
and it is used as a measure of molecular hydrophobicity. The
log Po/w values of our peptides 1-3 were obtained as 1.735,
at least between log Po/w = 0 and 2. This is consistent with
our results, namely that 1 (log Po/w = 1.735) has a much higher
cellular uptake efficiency than 2 (log Po/w = ¹0.66) and 3
(log Po/w = 0.207).
We then verified the intracellular localization of peptide 1 in
living cells by performing a morphologic examination using
HeLa cells. Cellular localization of peptide 1 seemed to be
limited to the cytoplasm and not to the nucleus (Figures 8d-8f).
We examined color merging using the red lysosome-specific
probe Lysotracker, and observed purple between peptide 1 and
Lysotracker (Figure 8f). These observations suggest that pep-
tide 1 exhibits selective lysosome localization in the cytoplasm.
Conclusion
The protected nonnatural amino acids Boc-L-Dap(7DEAC)-
OMe and Boc-L-Dap(C343)-OMe were synthesized and their
photophysical properties were examined in a series of sol-
vents that have different polarities and hydrogen-bonding
abilities. The fluorescence quantum yield and lifetime of Boc-
L-Dap(7DEAC)-OMe decreased significantly from nonpolar
n-hexane (Φf = 0.86, ¸f = 2.6 ns) to polar Tris-HCl/MeOH
(9:1 v/v) mixed solvent (Φf = 0.018, ¸f = 0.072 ns), whereas
those of Boc-L-Dap(C343)-OMe were almost independent
of the solvent parameters. Short peptides H-Dap(7DEAC)-
LLA-OMe (1), H-Dap(7DEAC)-KLA-OMe (2), and H-Dap-
(7DEAC)-ELA-OMe (3) labeled with environment-sensitive
7DEAC were synthesized to explore the application of 7DEAC
to probe biological interactions. The fluorescence intensity
of 7DEAC-labeled peptides 1-3 significantly increased upon
binding to DMPC and DMPC/DMPG (1:1) liposomes in Tris-
HCl buffer, showing that the affinity of 1-3 to these mem-
branes depends on hydrophobic and electrostatic interactions.
Furthermore, the fluorescence decay profiles of peptides 1-3
varied with affinities of the peptides to lipid membranes. These
results indicate that the environment-sensitive fluorophore
7DEAC is useful for labeling peptides or proteins to monitor
biological interactions.
Experimental
General. Absorption and emission spectra were respec-
tively measured using a UV-vis spectrophotometer (Jasco,

518
Bull. Chem. Soc. Jpn. Vol. 86, No. 4 (2013)
(a) H-Dap(7DEAC)LLA-OMe (1)
Fluorescent Peptides Labeled with 7-Aminocoumarin
(c) H-Dap(7DEAC)ELA-OMe (3)
(b) H-Dap(7DEAC)KLA-OMe (2)
50 μm
(d) H-Dap(7DEAC)LLA-OMe (1)
(e) Lysotracker
(f) merge
20 μm
Figure 8. Upper: Luminescence images of HeLa cells treated with (a) 1, (b) 2, and (c) 3. Lower: Subcellular localization of the
peptide 1 in HeLa cells. Peptide 1 was added to the medium at a final concentration of 10 ¯M. Lysotracker (100 nM), which
selectively stains lysosome, was used as an organelle marker.
Ubest-V550) and a spectrofluorometer (Hitachi, F-4010). The
emission spectrum was corrected for spectral sensitivity.
Picosecond fluorescence lifetime measurements were perfor-
med using a femtosecond laser system that was based on
a mode-locked Ti:sapphire laser (Spectra-Physics, Tsunami;
center wavelength: 800 nm; pulse width: ca. 70 fs; repetition
rate: 82 MHz) pumped by a CW green laser (Spectra-Physics,
Millenia V; 532 nm, 4.5 W).42 The repetition frequency was
reduced to 4 MHz by using a pulse picker (Spectra-Physics,
3980) and the third harmonic (266 nm; FWHM: ca. 250 fs) was
used as the excitation source. The monitoring system consisted
of a microchannel plate photomultiplier tube (MCP-PMT;
Hamamatsu, R3809U-51) that was cooled to ¹20 °C and a
single-photon counting module (Becker and Hickl, SPC-530).
The fluorescence photon signal detected by the MCP-PMT
and the photon signal of the second harmonic (400 nm) of the
Ti:sapphire laser were used for the start and stop pulses of
a time-to-amplitude converter in this system. The instrument
response function had a half width of about 25 ps. The fluo-
rescence time profiles were analyzed by deconvoluting with the
instrument response function.
Fluorescence quantum yields of all the compounds in de-
aerated solution were measured using an absolute photolumi-
nescence quantum yield system (Hamamatsu, C9920-02)43 that
consisted of a Xe arc lamp, a monochromator, an integrating
sphere, a multichannel detector, and a personal computer.
Cellular Experiments. HeLa cells were purchased from
American Type Culture Collection and were cultured in
Dulbecco’s Modified Eagle Medium supplemented with 10%
fetal bovine serum, penicillin (50 units/mL), streptomycin (50
mg mL¹1) for 24 h at 37 °C in a 5% CO2 atmosphere. Peptides
1-3 were added to the medium at a final concentration of
10 ¯M and the cells were incubated for 2 h. The medium was
then removed and the cell layer was washed with phosphate-
buffered saline. Luminescence microscopy images of HeLa
cells treated with peptides 1-3 were obtained using an inverted
microscope (Olympus, IX71) equipped with a ©40 objective
lens or a ©100 oil-immersion objective lens and an electron
multiplying CCD camera (Evolve 512, Photometrics) driven
by MetaVue software. Samples were excited using a 100 W
mercury lamp and imaged using custom filter settings (blue
channel: excitation band path = 400-410 nm, emission band
path = 460-510 nm; red channel: excitation band path = 545-
580 nm, emission cut > 610 nm).
Materials. Solvents were of spectrograde quality if availa-
ble and were used without further purification except propyl
cyanide and ethyl cyanide which were purified by column
chromatography (activated carbon and alumina). Deionized
water was purified by using a Millipore (MILLI-Q-Labo).
Supporting Information
Synthesis of Boc-L-Dap(7DEAC)-OMe, Boc-L-Dap-
(C343)-OMe, H-Dap(7DEAC)-LLA-OMe (1), H-Dap-
(7DEAC)-KLA-OMe (2), and H-Dap(7DEAC)-ELA-OMe
(3). Lippert-Mataga plot. This material is available free of
charge on the Web at http://www.csj.jp/journals/bcsj/.
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