Photophysics of novel coumarin-labeled depsipeptides in solution: Sensing interactions with SDS micelle via TICT model

Article abstract of DOI:10.1007/s00726-013-1483-3

N-Acylbenzotriazoles enable the synthesis (69-92 % yield) of blue to green fluorescent coumarin-labeled depsidipeptides 8a-f (quantum yields 0.004-0.97) and depsitripeptides 12a-d (quantum yields 0.02-0.96). Detailed photophysical studies of fluorescent c

Full text of DOI:10.1007/s00726-013-1483-3

Amino Acids (2013) 45:159–170
DOI 10.1007/s00726-013-1483-3
ORIGINAL ARTICLE
Photophysics of novel coumarin-labeled depsipeptides in solution:
sensing interactions with SDS micelle via TICT model
•
Suvendu Biswas Ilker Avan Akash K. Basak
•
•
•
Nader E. Abo-Dya Abdullah Asiri
•
Alan R. Katritzky
Received: 8 August 2012 / Accepted: 23 February 2013 / Published online: 4 April 2013
Ó Springer-Verlag Wien 2013
Abstract N-Acylbenzotriazoles enable the synthesis
(69–92 % yield) of blue to green fluorescent coumarin-
labeled depsidipeptides 8a–f (quantum yields 0.004–0.97)
and depsitripeptides 12a–d (quantum yields 0.02–0.96).
Detailed photophysical studies of fluorescent coumarin-
labeled depsipeptides 8a–f and 12a–d are reported for both
polar protic and polar aprotic solvents. 7-Methoxy and
7-diethylaminocoumarin-3-ylcarbonyl depsipeptides 8c,f
and 12d are highly solvent sensitive. These highly fluo-
rescent compounds could be useful for peptide assays.
Further photophysical studies of 7-diethylaminocoumarin-
labeled depsipeptides 8c,12d within the micellar microen-
vironment of SDS reflect their ability to bind with the
biological membrane, suggesting potential applications in
the fields of bio- and medicinal chemistry.
Keywords Coumarin ꢀ Peptidomimetics ꢀ Depsipeptides ꢀ
SDS ꢀ Micelle ꢀ TICT
Abbreviations
DCC N0,N0-dicyclohexylcarbodiimide
SDS
Sodium dodecyl sulfate
TICT Twisted intramolecular charge transfer
Introduction
Proteins and peptides labeled with fluorescent groups are
widely applied in biology, biotechnology and medicinal
chemistry for the detection and monitoring of physio-
chemical activity (Griffin et al. 1998; Chen et al. 2005;
Dragulescu-Andrasi and Rao 2007). Fluorescent peptides
have been used to label human v1b vasopressin or oxytocin
receptors selectively (Corbani et al. 2011) and used in the
construction of highly sensitive fluorescent tags for the
detection of vascular endothelial growth factor, a bio-
marker for angiogenesis (Suzuki and Yokoyama 2011).
Protein engineering has inserted position-specific, non-
natural amino acids into biosynthetic proteins (Hohsaka
et al. 2001) and peptide analogs containing non-natural
amino acids are broadly applied in structure–activity
studies. Incorporation of non-natural amino or hydroxy
acids into peptides and depsides expands the scope of
structural perturbation and can induce specific steric
properties (Scheike et al. 2007). Depsipeptides, containing
both amino acid units linked by amide bonds and
a-hydroxy acid units linked by ester bonds, are analogs of
peptides and differ significantly in hydrogen-bonding
capacity compared to natural peptides. Thus, the incorpo-
ration of hydroxy acids into a peptide chain is a useful tool
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-013-1483-3) contains supplementary
material, which is available to authorized users.
S. Biswas ꢀ I. Avan ꢀ A. K. Basak ꢀ N. E. Abo-Dya ꢀ
A. R. Katritzky (&)
Department of Chemistry, Center for Heterocyclic Compounds,
University of Florida, Gainesville, FL 32611-7200, USA
e-mail: katritzky@chem.ufl.edu
I. Avan
Department of Chemistry, Faculty of Science, Anadolu
University, 26470 Eskis¸ehir, Turkey
N. E. Abo-Dya
Department of Pharmaceutical Organic Chemistry, Faculty
of Pharmacy, Zagazig University, Zagazig, 44519, Egypt
A. Asiri ꢀ A. R. Katritzky
Department of Chemistry, King Abdulaziz University,
Jeddah 21589, Saudi Arabia
123

160
S. Biswas et al.
for the diversification of peptidomimetics and for gaining a
better understanding of their structural properties (Yang
and Gellman 1998; Gallo and Gellman 1993). Depsipep-
tides exhibit useful biological (antimicrobial, antifungal,
anti-inflammatory) and therapeutic activity (anticancer and
anti-HIV) (Ballard et al. 2002); didemnin B and dolastatin
10 have anticarcinogenic activities (SirDeshpande and
Toogood 1995; Kingston 2009). Cyclic depsipeptides such
as mirabamides E–H, callipeltins A and also quinoxapeptin
show promising inhibitory activities against HIV (Lu et al.
2011; Zampella et al. 1996; Boger et al. 1999). Valino-
mycin (Kuisle et al. 1999), a natural ionophore, can act as a
non-metallic isoforming agent in potassium selective
electrodes with the best K?/Na? selectivity of all K?
ionophores to date (Dudev and Lim 2009).
applicability in biological system. No literature report was
found on the synthesis and/or spectroscopical properties of
labeled depsipeptides. Our aim in this project was to syn-
thesize and explore the use of labeled depsipeptides, thus,
showing their potential utility in biological system. Herein,
we report the efficient synthesis and study of steady-state
absorption, fluorescence properties of coumarin-labeled
depsipeptides in polar protic and polar aprotic solvents as
well as in the organized confined media of SDS micelles.
Results and discussion
Materials and methods
Coumarins afford commercially important ‘‘blue–
green’’ fluorescent dyes which can be highly sensitive to
their environment, (Wagner 2009) possess good solubility
in many solvents with extended spectral ranges, high
emission quantum yields and photostability (Heiner et al.
2006; Zhou and Fahrni 2004). These compounds are used
to investigate ultrafast solvation dynamics and various
electron transfer processes. Fluorescent coumarin-labeled
peptides provide a sensitive and specific assay of matrix
metalloproteinases, cathepsin D and E activity in biological
samples (Knight et al. 1992; Yasuda et al. 1999). They are
hydrolyzed by leucine aminopeptidase and, hence, act as
inhibitors of clostridial aminopeptidase (Carmel et al.
1977).
Melting points were determined on a capillary point
apparatus equipped with a digital thermometer and are
uncorrected. NMR spectra were recorded in CDCl3 and
DMSO–d6 with TMS for 1H (300 MHz) and 13C (75 MHz)
as an internal reference. Elemental analyses were per-
formed on a Carlo Erba–EA 1108 Elemental Analyzer.
Mass spectrometry was done on Agilent 6210 TOF–MS
with electro spray ionization (ESI). CH2Cl2 was dried and
distilled over CaH2, whereas THF was used after distilla-
tion over Na-benzophenone. Unprotected amino acids L-
phenylalanine, L-methionine and N-(protected)-aminoacid
Boc-Gly-OH 1 were purchased from Sigma and used
without further purification. Boc-Gly-Bt 2, Hydroxycarb-
oxylic acids 3, Boc-protected depsidipeptide 4c and cou-
marinoylbenzotriazoles 7a–c were prepared by the reported
method (Avan et al. 2011; Katritzky et al. 2008a, b, 2010).
Unprotected depsidipeptides 5a,b and depsitripeptides
Sodium dodecyl sulfate (SDS) micelles are capable of
mimicking the tertiary interactions of protein-, lipid- and
aqueous-exposed helical surfaces and are used as mem-
brane mimetics to study the complex biological phenomena
(Tulumello and Deber 2009; Maciejewski et al. 2005).
They can solubilize proteins and stabilize the intramolec-
ular interactions and, hence, are important to biosensor
study (Bandyopadhyay and Saha 2008; Mishra et al. 2004).
These special properties of SDS are widely used to dissolve
and denature proteins, (Bhuyan 2010; Sun et al. 2012) to
characterize membrane protein non-native states (Dutta
et al. 2010) and to investigate solvation dynamics of cou-
marin dyes (Sarkar et al. 1996).
1
11a–c were characterized by H, 13C-NMR and used for
the coupling step without further purifications. Absorption
spectra were recorded on a Lambda–25 (Perkin Elmer) and
fluorescence spectra were recorded on FluoroMax–3 Job-
inYuon Horiba Spectrofluoremeter at 23 °C. Quantum
yield (U) was obtained by comparison of the integrated
area of the corrected emission spectra of standard sample
with solution of coumarin 30 in acetonitrile or stilbene in
methanol. The concentration of the standard was adjusted
to give the same absorbance, which is around 0.1 as the
sample at the excitation wavelength.
Although several literature papers concern general
methods and simple photophysical studies of coumarin-
labeled peptides and peptidomimetics (Avan et al. 2011;
Katritzky et al. 2008a, b, 2010); a report showing their
potential application inside the biological system should be
interesting and useful. In this connection, depsipeptides
show in contrast to their natural analogs (i) high affinity for
specific receptors, (ii) good metabolic stability towards
endogenous proteases, (iii) greater oral bioavailability and
(iv) longer duration of action. These features interested us
to explore the fields of labeled peptidomimetics and their
Preparation of unprotected depsidipeptides 5a–b
Free or protected optically pure L-a-dipeptides (dipeptides)
are useful building blocks for longer peptide analogs. The
functions and applications of dipeptides have been poorly
examined in the literature because of lack of an efficient
protocol for the synthesis of dipeptide compared with
proteins and amino acids (Yagasaki and Hashimoto
2008). Here, an efficient route to synthesize unprotected
123

Photophysics of novel coumarin-labeled depsipeptides in solution
161
O
O
O
O
OH
HO
OH
O
O
N
N
H
H
SOCl2/BtH
DCM
DCC
BtH,
R
3a,b
N
N
N
R1
R1
O
O
6a-c
O
Boc
OH
O
Boc
HCl
Bt
DCM
THF
DMAP,
R
7a-c
1
2
O
R
O
H
4N
R
+H3N
Cl-
O
N
OH
OH
Cl-
O
O
Boc
O
in dioxane
H3N
OH
O
O
O
O
O
5a-b
4a,b
=1-benzotriazolyl
N
H
OH
5a,b
R
O
Bt
=
R1
5a R CH2Ph
,
O
=
O
=
Et3N/MeCN/H2O
5b R
,
CH(CH3)2
8a-f
8a R1
R
=
CH2Ph
,
H;
Scheme 1 Preparation of depsidipeptides 5a,b
8b R1
R
R
CH2Ph
CH2Ph
=
CH(CH3)2
=
=
=
,
OMe;
8c R1 NEt2
=
,
;
depsipeptides is reported under mild reactionconditions. Boc-
protected amino acid 1 was coupled with 1H-benzotriazole
using DCC to obtain N-Boc(a-aminoacyl)benzotriazole 2,
which was further reacted with L-a-hydroxycarboxylic
acids 3a,b in THF in the presence of 4-dimethylamino-
pyridine (DMAP) as a base to obtain Boc-protected dep-
sidipeptides 4a,b. Without isolation, 4a,b were Boc-
deprotected by 4 N HCl solution in dry dioxane to yield
unprotected depsidipeptides 5a,b as hydrochloride salts
(Scheme 1). These free depsidipeptides were characterized
by 1H, 13C-NMR and reacted with coumarinoylbenzo-
triazoles 7a–c to prepare coumarin-labeled fluorescent
depsidipeptides 8a–f (Scheme 2).
8d R1
8e R1
R
=
,
H;
OMe;
=
=
R
=
,
CH(CH3)2
R1 NEt2
R
;
=
8f
,
CH(CH3)2
Scheme 2 Preparation of coumarin-labeled depsidipeptides 8a–f
7b,c to prepare coumarin-labeled fluorescent depsitripep-
tides (Scheme 4).
Preparation of N-coumarinoyl-labeled depsitripeptides
12a–d
N-Coumarinoyl-labeled depsitripeptides 12a–d were pre-
pared by the treatment of coumarinoylbenzotriazoles 7b,c
with unprotected depsitripeptides 11a–c in the presence of
triethylamine and MeCN/H2O (4:1 v/v) at 20 °C (Scheme 4).
The novel fluorescent compounds were characterized by 1H,
13C-NMR and elemental analysis.
Preparation of N-coumarinoyl-labeled depsidipeptides
8a–f
Coumarinoylbenzotriazoles 7a–c were prepared from the
corresponding coumarinoyl acids 6a–c by known benzo-
triazole methodology (Katritzky et al. 2008a, b, 2010). N-
Coumarinoyl-labeled depsidipeptides 8a–f were obtained
by treatment of coumarinoylbenzotriazoles 7a–c with various
unprotected depsipeptides 5a,b in the presence of triethyl-
amine and MeCN/H2O (3:1 v/v) at 20 °C (Scheme 2). Novel
fluorescent compounds were characterized by 1H, 13C-NMR
and elemental analysis.
Photophysical studies of coumarin-labeled
depsipeptides 8a–f and 12a–d
Due to a change in the dipole moments between the ground
and excited electronic states of the coumarin moiety, the
absorption and fluorescence maxima of coumarin-labeled
conjugates are sensitive to the solvent polarity and
H-bonding ability (Senthilkumar et al. 2004; Schimitschek
Preparation of unprotected depsitripeptides 11a–c
O
Boc-protected depsidipeptides 4a–c (4c were prepared by
literature method) (Avan et al. 2011) were coupled with
1H-benzotriazole using 1-ethyl-3-(3-dimethylaminopro-
pyl)carbodiimide (EDCI) to obtain N-Boc(a-amino-
acyl)benzotriazoles 9a,b, which were reacted with L-
phenylalanine and L-methionine in THF in the presence of
triethylamine to obtain the Boc-protected depsitripeptides
10a–c. Without isolation, 10a–c were Boc-deprotected by
4 N HCl solution in dry dioxane to yield the unprotected
depsitripeptides 11a–c as hydrochloride salts (Scheme 3).
The novel depsitripeptides 11a–c were characterized by
1H, 13C-NMR and coupled with coumarinoylbenzotriazoles
H2N
OH
O
R
O
R
H
N
R2
H
N
BtH, EDCI
DCM
OH
Bt
R
Boc
Boc
O
Boc
O
Et3N, MeCN
O
O
4a, c
9a-b
O
Cl-
H3N
O
O
R
O
O
O
H
H
N
H
N
4N HCl/
dioxane
N
OH
O
OH
R2
O
R2
11a-c
10a-c
11a, R = CH2CH(CH3)2; R2 = CH2Ph
11b, R = CH2CH(CH3)2; R2 = CH2CH2SCH3
11c, R = CH2Ph; R2= CH2CH2SCH3
Scheme 3 Preparation of depsitripeptides 11a–c
123

162
S. Biswas et al.
O
O
O
R
O
H
N
H3N
Cl-
+
N
O
OH
N
O
R2
N
R1
O
11a-c
7b-c
Et3N, MeCN
H2O
O
O
O
R2
O
N
H
R
OH
N
H
O
O
R1
O
12a-d
12a, R1 = OMe; R = CH2CH(CH3)2; R2 = CH2Ph
12b, R1= OMe; R = CH2CH(CH3)2; R2 = CH2CH2SCH3
12c, R1 = OMe; R = CH2Ph; R2 = CH2CH2SCH3
12d, R1= NEt2; R = CH2CH(CH3)2; R2= CH2Ph
Scheme 4 Preparation of coumarin-labeled depsitripeptides 12a–d
Fig. 1 Emission and absorption spectra (inset) of 8b, 8e, 12a–c in
PBS buffer at pH 7.4
et al. 1976; McCarthy and Blanchard 1993; Samanta and
Fessenden 2000). The spectroscopic properties of couma-
rins can be tuned by substituents at the positions 6 or 7,
which affect the energy of the excited states (Furuta et al.
1999; Eckardt et al. 2002). This special property prompted
researchers to use these fluorescent dyes as a probe to
investigate many physiochemical processes. Photophysical
properties of coumarin-labeled depsipeptides 8a–f and
12a–d were investigated in both polar protic and polar
aprotic solvents over a wide range of solvent polarity. To
simulate the physiological pH and gain, a better under-
standing of the solvent polarity effects inside a membrane-
like environment, photophysical properties of two specific
compounds 8c and 12d were studied within the micellar
microenvironment of SDS in phosphate buffered saline
(PBS) solution at pH 7.4.
Fig. 2 Emission and absorption spectra (inset) of 8c, 8f, 12d in PBS
buffer at pH 7.4
Absorbance, absorptivity, fluorescence and quantum yield
data for coumarin-labeled depsipeptides 8a–f and 12a–d
The absorption and emission spectra of 8a–f and 12a–d in
polar protic (methanol and PBS buffer) and polar aprotic
(dichloromethane) solvents are shown in Figs. 1, 2, 3, 4, 5,
6 and wavelengths of absorption maxima (kmaxabs), fluo-
rescence emission maxima (kmaxem), molar extinction
coefficients (e) and quantum yields (U) are listed in
Tables 1, 2, 3.
Electron-donating substituents, methoxy (–OMe) and
diethylamino (–NEt2) at position 7 of the coumarin skele-
ton cause a bathochromic shift of the fluorescence emission
maxima (kmaxem) and increase quantum yields. As
expected 7-methoxycoumarin-3-ylcarbonyl-labeled depsi-
peptides 8b, 8e, 12a–c showed high quantum yields in PBS
buffer (U = 0.20–0.24), in MeOH (U = 0.27–0.52) and in
CH2Cl2 (U = 0.26–0.57) relative to unsubstituted couma-
rin-labeled depsipeptides 8a and 8d (U = 0.004–0.006).
Fig. 3 Emission and absorption spectra (inset) of 8b, 8e, 12a–c in
MeOH
123

Photophysics of novel coumarin-labeled depsipeptides in solution
163
Table 1 Absorption and emission data in PBS buffer
Entry Comp.
no.
kmaxabs (nm)
k
maxem (nm) e (104cm-1M-1
)
U (quantum
yield)
1
2
8a
301
343
430
301
343
430
346
345
346
428
415.0
407.0
481.0
419.0
410.0
480.0
406.0
405.0
406.0
480.0
1.55
2.78
5.00
1.91
2.39
4.56
2.24
2.30
2.03
6.65
0.006
0.239
0.018
0.006
0.234
0.014
0.242
0.197
0.222
0.020
8b
3
8c
4
8d
5
8e
6
8f
7
12a
12b
12c
12d
8
9
10
The quantum yields of 7-methoxycoumarin-labeled depsi-
peptides are interpreted in terms of emission from an
intramolecular charge transfer (ICT) excited state (Taka-
date et al. 2000).
Fig. 4 Emission and absorption spectra (inset) of 8c, 8f, 12d in
MeOH
The methoxycoumarin-labeled depsipeptides showed
reductions in quantum yields with increased solvent
polarity, particularly in PBS buffer (Figs. 1, 3, 5). Inter-
estingly, the quantum yields of 7-N,N-diethylaminocou-
marin-3-ylcarbonyl containing 8c, 8f and 12d were
significantly higher (U = 0.85–0.97) in CH2Cl2 (polar
aprotic solvent) and sharply decreased in polar protic sol-
vents (U = 0.014–0.020 in buffer and U = 0.029–0.052 in
MeOH) (Figs. 2, 4, 6). In polar aprotic solvent (CH2Cl2),
diethylaminocoumarin-labeled depsipeptides fluoresce
from a highly emissive ICT excited state but in polar protic
solvent (PBS buffer, MeOH), rotation of the diethylamino
group of the ICT excited state leads to a twisted intramo-
lecular charge transfer (TICT) excited state from which
non-radiative decay to the ground state occurs (Scheme 5)
(Jones et al. 1985; Krishnamoorthy and Dogra 2000). The
polar solvent stabilizes charge in the twisted zwitterionic
TICT and consequently the interconversion of the
ICT ? TICT is facilitated by an increase of the solvent
polarity (Senthilkumar et al. 2004). Increasing solvent
polarity stabilizes the TICT exited state relatively to
ground state, which explains the bathochromic shift in
emission maxima (kmaxem) (Figs. 7, 8). The solvatochro-
mic shifts are directly proportional to the dipole moments
of the excited and ground state.
Fig. 5 Emission and absorption spectra (inset) of 8b, 8e, 12a–c in
DCM
Photophysical properties of 7-N, N-
diethylaminocoumarin-labeled depsipeptides 8c
and 12d in SDS micellar microenvironment
Potential applications of the coumarin-labeled depsipep-
tides were investigated by further photophysical study inside a
membrane-like system. From the earlier experiments it was
found that7-N,N-diethylaminocoumarin-labeled depsipeptides
Fig. 6 Emission and absorption spectra (inset) of 8c, 8f, 12d in DCM
123

164
S. Biswas et al.
Table 2 Absorption and emission data in MeOH
Entry Comp.
no.
kmaxabs (nm)
kmaxem (nm)
e
U (quantum
yield)
(104cm-1M-1
)
1
8a
291
411
403
469
410
404
467
403
404
404
468
1.62
1.87
4.80
1.52
2.51
4.22
2.41
3.29
3.67
4.09
0.004
0.516
0.029
0.005
0.451
0.049
0.413
0.315
0.274
0.052
2
8b
347.5
420
3
8c
4
8d
291
5
8e
346.5
420
6
8f
7
12a
12b
12c
12d
347
8
348.5
347
9
10
420
Fig. 7 Emission spectra of 8c in PBS buffer, MeOH and DCM
Table 3 Absorption and emission data in DCM
Entry Comp.
no
kmaxabs (nm)
kmaxem (nm)
e
U (quantum
yield)
(104cm-1M-1
)
1
8a
291
351
427
291
350
425
351
351
352
426
409
402
458
405
402
458
404
404
404
460
1.42
2.26
4.86
1.51
3.05
6.36
2.94
4.25
3.68
5.01
0.006
0.363
0.849
0.004
0.566
0.970
0.494
0.262
0.360
0.960
2
8b
3
8c
4
8d
5
8e
6
8f
7
12a
12b
12c
12d
8
9
10
Et
N
Et
N
O
O
O
O
O
O
Et
Et
R
Fig. 8 Emission spectra of 12d in PBS buffer, MeOH and DCM
R
Non-Emissive TICT
Emissive ICT
All data were taken with the SDS concentration
(6.0–200 mM) kept well above the critical micellar con-
centration (CMC) of SDS micelles in PBS buffer (Fuguet
et al. 2005), while the concentration of 8c and 12d were
very low (8.63–11.15 lM); according to Poisson statistics
this should allow not more than one labeled depsipeptide to
interact with each SDS micelle (Tachiya 1975). The
absorption spectra of 8c and 12d were unchanged at dif-
ferent SDS concentrations, but increasing SDS concentra-
tion resulted in a gradual increase in steady-state emission
intensity of both 8c and 12d (Figs. 9, 10).
Scheme 5 ICT ? TICT conversion
are highly sensitive to the solvent polarity compared to the
other coumarin-labeled depsipeptides. Two 7-N,N-diethyl-
amino coumarin-labeled compounds 8c and 12d were
chosen to study any change in the photophysical behavior
inside a membrane-like environment. Having observed
TICT for depsipeptides, our study was extended to a SDS
microenvironment, since the TICT state of fluorophores is
sensitive to the polarity, H-bonding ability and viscosity of
the solvent (Rettig 1986; Cazeau-Dubroca et al. 1989). The
steady-state absorption and fluorescence spectra of 7-N,N-
diethylaminocoumarin-labeled depsidipeptide 8c and dep-
sitripeptide 12d were recorded in PBS buffer solution at
physiological pH 7.4 with different concentration of SDS.
This result clearly indicates that there is interaction
between the 7-N,N-diethylaminocoumarin-labeled depsi-
peptides and SDS micelle. To get a better understanding of
the phenomena, the emission and absorption spectra of
both the compounds were recorded in PBS buffer solution
(SDS free condition) and in SDS solution made in PBS
123

Photophysics of novel coumarin-labeled depsipeptides in solution
165
Fig. 9 Emission and absorption spectra (inset) of 8c in PBS with
different SDS conc
Fig. 11 Binding of coumarin moiety to the Stern layer
Fig. 10 Emission and absorption spectra (inset) of 12d in PBS with
different SDS conc
Fig. 12 Blue shift in the emission spectra 8c and 12d in PBS, and
100 mM SDS solution
buffer keeping the same 50 nM concentration in both
cases. It was found that the quantum yields of 8c and 12d
were significantly higher (around four times) even in very
low 50 mM concentration of SDS solution (U = 0.073
and 0.093, respectively) than in SDS free PBS buffer
solution (U = 0.018 and 0.020, respectively). The con-
centration-dependent enhancement of fluorescence by
SDS can be explained by the diffusion of the 7-N,N-die-
thylaminocoumarin-labeled depsipeptides to the micellar
Stern layer. The hydrophobic depsipeptide chains of 8c
and 12d make them less soluble in a polar solvent such as
water, thereby; they prefer the hydrophobic inner core of
the micelle. The microenvironment around the 7-N,N-
diehtylaminocoumarin moiety, however, is polar and it
remains bound to Stern layer of the SDS micelle (Shirota
et al. 2004; Dhenadhayalan et al. 2011) (Fig. 11). This
hypothesis was further supported for both the compounds
8c and 12d by a k = 15 nm blue shift of the emission
maxima in SDS solution compared to PBS buffer solution
(Fig. 12).
Since, TICT requires twisting of the donor diethylamino
group (Scheme 5), an organized SDS Stern layer will
restrict this twisting motion and retard TICT (Bhattachar-
yya and Chowdhury 1993). Conversion of ICT to non-
emissive TICT state was restricted by simultaneous
decreased polarity and increased confinement at the Stern
layer of the SDS micelle resulting in an increased emission
intensity with increasing SDS concentration. This signifi-
cant change of the fluorescence property in the SDS
micelle could make these compounds useful to monitor the
reaction dynamics of a peptide-based drug inside a bio-
logical system.
123

166
S. Biswas et al.
150–152 °C; [a]D23 = –29.0 (c 1.0, CH3OH); 1H NMR
(DMSO–d6) d 3.08 (dd, J = 14.4, 8.1 Hz, 1H), 3.19 (dd,
J = 14.2, 4.2 Hz, 1H), 3.75–3.88 (m, 2H), 5.23–5.27 (m,
1H), 7.19–7.35 (m, 5H), 8.54 (br s, 3H); 13C NMR
(DMSO–d6) d 36.3, 73.9, 126.9, 128.4, 129.4, 136.1, 167.3,
169.7.
Conclusion
In this paper, we have documented the synthesis and
photophysical studies of novel coumarin-labeled depsi-
peptides. We believe that the present study complements
on dye-labeled natural peptides and facilitated application
of labeled peptidomimetics or depsipeptides. Variations of
quantum yields in different solvents are reported and
rationalized in terms of ICT–TICT excited states.
7-Methoxycoumarin-labeled depsipeptides are efficient for
probes, since they exhibit high quantum yield values.
Moreover, 7-diethylaminocoumarin-labeled depsipeptides,
with their unique TICT state in polar protic solvents, are
highly sensitive to solvent polarity, H-bonding ability and
organized nature of the solvent medium such as an SDS
micelle. Thus, 7-diethylaminocoumarin-labeled depsipep-
tides may be good candidates for the real-time monitoring
of physiological processes. Their drastic change in fluo-
rescence properties due to binding with the Stern layer of
SDS micelle suggests potential utility in investigating
reaction dynamics in biological membrane interfaces as
well as for the monitoring of drug delivery.
(S)–2–(1–carboxy–2–methylpropoxy)–2–oxoethanamini-
um chloride (5b) White microcrystals (79 %), mp
165–168 °C; [a]D23 = -35.0 (c 1.0, CH3OH); 1H NMR
(DMSO–d6) d 0.91–0.98 (m, 6H), 2.14–2.24 (m, 1H),
3.78–4.00 (m, 2H), 4.83 (d, J = 3.9 Hz, 1H), 8.60 (br s,
3H); 13C NMR (DMSO–d6) d 17.0, 18.6, 29.5, 77.5, 167.5,
169.9.
General preparation of N-coumarinoyl-labeled
depsidipeptides 8a–f
Hydrochloride salts of Gly-L-(O-Phe) 5a (0.04 g,
0.16 mmol, 1.2 equiv) or Gly-L-(O-Val) 5b (0.03 g,
0.16 mmol) and TEA (0.04 g, 0.32 mmol, 2.0 equiv) were
dissolved in minimum amount of cold water (2 mL).
Acetonitrile (6 mL) was added to this solution and cooled
to 10 °C. A solution of N-acylcoumarinoyl-Bt 7a–c
(0.04–0.06 g, 1.0 equiv) was added and stirred for 1 h at
25 °C. The reaction mixture was monitored with TLC
[EtOAc-Hexanes (1:2)]. After the completion of reaction,
solvent was evaporated. 4 N HCl solution (5 mL) was
added drop wise just to acidify the reaction mixture. The
precipitate was filtered and washed with 1 N HCl (5 mL)
and water (5 mL) to afford desired N-Coumarinoyl-labeled
depsidipeptides 8a–f (Note: for 8c and 8f acidification was
done carefully just to neutralize the solution).
Experimental section
General preparation of unprotected depsidipeptides
5a,b
4–Dimethylaminopyridine (1.2 mmol) was added to a
stirred solution of Boc–Gly–Bt 2 (1.0 mmol) and a–hy-
droxycarboxylic acid 3a,b (1.2 mmol) in dry THF
(5.0 mL) at 4 °C. The reaction mixture was stirred for 4 h
at room temperature until the reaction was completed by
TLC [EtOAc-hexanes (1:2)]. The solvent was evaporated
under reduced pressure, and the residue was taken into
EtOAc (10.0 mL), washed with saturated citric acid solu-
tion (3 9 5 mL) and brine (5 mL) and dried over MgSO4.
The solvent was evaporated under reduced pressure to
yield the crude product as oil. Without isolation, Boc-
deprotection was conducted by 4 N HCl solution in dry
dioxane (5.0 mL) for 2 h. Boc-protected depsidipeptides
4a (0.31 g, 1.0 mmol for 4a) or 4b (0.27 g, 1.0 mmol for
4b) was dissolved in dry dioxane (2.0 mL) and cooled to
0 °C. Then dry 4 N HCl in dioxane (3.0 mL) was added
into the solution through a syringe for 5 min at 0 °C. The
mixture was stirred for another 2 h at 0 °C. The solvent
was evaporated and the precipitate was washed with dry
diethyl ether to yield unprotected depsidipeptides 5a,b as
hydrochloride salts.
(S)–2–(2–(2–oxo–2H–chromene–3–carboxamido)acetoxy)
–3–phenylpropanoic acid (8a) White microcrystals
(86 %), mp 185–187 °C; [a]2D3 = -17.0 (c 1.0, CH3OH);
1H NMR (DMSO-d6) d 3.07 (dd, J = 14.6, 8.0 Hz, 1H),
3.14 (dd, J = 14.6, 4.7 Hz, 1H), 4.17 (d, J = 6.0 Hz, 2H),
5.14 (dd, J = 7.7, 4.5 Hz, 1H), 7.14–7.30 (m, 5H), 7.45 (t,
J = 7.3 Hz, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.77 (t,
J = 7.9 Hz, 1H), 8.00 (dd, J = 8.0, 1.4 Hz, 1H), 8.89 (s,
1H), 9.07 (t, J = 5.6 Hz, 1H), 13.22 (br s, 1H); 13C NMR
(DMSO-d6) d 36.5, 41.3, 73.1, 116.2, 118.1, 118.4, 125.3,
126.7, 128.2, 129.4, 130.5, 134.4, 136.2, 148.3, 154.0,
160.3, 161.4, 169.0, 170.2; Anal. Calcd for C21H17NO7: C,
63.80; H, 4.33, N, 3.54. Found: C, 63.51; H, 4.50; N, 3.51.
(S)–2–(2–(7–methoxy–2–oxo–2H–chromene–3–carboxamido)
acetoxy)–3–phenylpropanoic acid (8b) White micro-
crystals (89 %), mp 184–187 °C; [a]2D3 = -20.0 (c 1.0,
1
CH3OH); H NMR (DMSO–d6) d 3.00–3.18 (m, 2H), 3.91
(s, 3H), 4.16 (d, J = 5.7 Hz, 2H), 5.13 (dd, J = 7.7,
4.7 Hz, 1H), 7.02–7.14 (m, 2H), 7.15–7.29 (m, 5H), 7.93
(S)–2–(1–carboxy–2–phenylethoxy)–2–oxoethanaminium
chloride (5a) White microcrystals (54 %), mp
123

Photophysics of novel coumarin-labeled depsipeptides in solution
167
(d, J = 8.7 Hz, 1H), 8.84 (s, 1H), 9.01 (t, J = 5.4 Hz, 1H),
13.25 (br s, 1H);13C NMR (DMSO–d6) d 36.4, 41.2, 56.3,
73.1, 100.3, 112.1, 113.8, 114.0, 126.7, 128.2, 129.4,
131.8, 136.2, 148.5, 156.4, 160.7, 161.7, 164.7, 169.1,
170.2 Anal. Calcd for C22H19NO8: C, 62.12; H, 4.50, N,
3.29. Found: C, 61.97; H, 4.40; N, 3.24.
1H), 8.67 (s, 1H), 9.02 (t, J = 5.7 Hz, 1H), 13.07 (br s,
1H); 13C NMR (DMSO–d6) d 12.3,16.9, 18.5, 29.4, 41.1,
44.4, 76.6, 95.9, 107.5, 107.6, 110.2, 131.7, 148.1, 152.6,
157.3, 162.6, 169.5, 170.3, Anal. Calcd for C21H26N2O7: C,
60.28; H, 6.26, N, 6.69. Found: C, 59.73; H, 6.12, N, 7.45.
General preparation of unprotected depsidipeptides
(S)–2–(2–(7–(diethylamino)–2–oxo–2H–chromene–3–car-
boxamido)acetoxy)–3–phenylpropanoic acid (8c) Yellow
microcrystals (86 %), mp 191–193 °C; [a]2D3 = -39.0 (c
1.0, CH3OH); 1H NMR (DMSO–d6) d 1.14 (t, J = 6.9 Hz,
6H), 3.00–3.17 (m, 2H),3.48 (q, J = 6.9 Hz, 4H), 4.14 (d,
J = 5.7 Hz, 2H), 5.12 (dd, J = 7.4, 4.5 Hz, 1H), 6.63 (s,
1H), 6.81 (d, J = 8.7 Hz, 1H), 7.17–7.26 (m, 5H), 7.69 (d,
J = 9.0 Hz, 1H), 8.65 (s, 1H), 8.98 (t, J = 5.6 Hz, 1H),
13.22 (br s, 1H); 13C NMR (DMSO–d6) d 12.3, 36.4, 41.0,
44.4, 73.0, 95.8, 107.6, 108.5, 110.2, 126.7, 128.2, 129.4,
131.8, 136.2, 148.2, 152.6, 157.4, 161.6, 162.5, 169.3,
170.2; Anal. Calcd for C25H26N2O7: C, 64.37; H, 5.62, N,
6.01. Found: C, 64.21; H, 5.50; N, 6.08.
11a–c
A solution of EDCI (1.10 g, 5.80 mmol, 1.0 equiv.) was
added to a stirred solution of 4a, and 4c (1.0 equiv.) and
benzotriazole (0.69 g, 5.80 mmol) in CH2Cl2 (50 mL) at
0 °C. The reaction mixture was stirred for 16 h at room
temperature. Then reaction was quenched with water. The
reaction mixture was washed with 20 % citric acid solution
(3 9 10 mL), saturated Na2CO3 (3 9 15 mL), water
(2 9 10 mL) and brine (15 mL), dried over MgSO4 and
solvent was evaporated under reduced pressure to yield the
desired product 9a–b. L-Phenylalanine (0.50 g, 3.0 mmol,
1.5 equiv) or L-methionine (0.22 g, 1.5 mmol) and TEA
(300 mg, 3.0 mmol, 1.5 equiv) were dissolved in minimum
amount of cold water (5 mL). Acetonitrile (10 mL) was
added to the solution and cooled to 10 °C. A solution of
9a–b (1.0 equiv., 2.0 mmol) in acetonitrile (5 mL) was
added and stirred for 2 h at 25 °C. The reaction mixture
was monitored with TLC [EtOAc-Hexanes (1:2)]. After the
completion of the reaction, solvent was evaporated. The
residue was dissolved in CH2Cl2 (30 mL) and washed with
saturated citric acid solution (4 9 10 mL), water (10 mL)
and brine (10 mL), dried over MgSO4 and evaporated to
give 10a–c. Crude 10a–c (1.0 mmol) was dissolved in dry
dioxane (2 mL) and cooled to 0 °C. Then dry 4 N HCl in
dioxane (3 mL) was added into the solution through a
syringe for 5 min at 0 °C and stirred for another 2 h at
0 °C. The solvent was evaporated and the precipitate was
washed with dry diethyl ether to give the desired product
11a–c as white microcrystals.
(S)–3–methyl–2–(2–(2–oxo–2H–chromene–3–carboxa-
mido) acetoxy)butanoic acid (8d) White microcrystals
(69 %), mp 137–140 °C; [a]2D3 = -26.0 (c 1.0, CH3OH);
1H NMR (CDCl3) d 1.03 (d, J = 6.6 Hz, 3H), 1.06 (d,
J = 8.1 Hz, 3H), 2.26–2.40 (m, 1H), 4.28–4.46 (m, 2H),
5.04 (d, J = 3.9 Hz, 1H), 7.36–7.44 (m, 2H), 7.64–7.74
(m, 2H), 8.95 (s, 1H), 9.35 (t, J = 6.0 Hz, 1H); 13C NMR
(CDCl3) d 17.2, 19.0, 30.2, 42.0, 77.4, 116.9, 117.8, 118.7,
125.6, 130.3, 134.6, 149.4, 154.8, 161.4, 162.5, 169.1,
173.1; Anal. Calcd for C17H17NO7: C, 58.79; H, 4.93, N,
4.03. Found: C, 58.53; H, 4.87; N, 4.07.
(S)–2–(2–(7–methoxy–2–oxo–2H–chromene–3–carboxa-
mido) acetoxy)–3–methylbutanoic acid (8e) White
microcrystals (90 %), mp 182–185 °C; [a]2D3 = -24.0 (c
1.0, CH3OH); 1H NMR (DMSO–d6) d 0.91 (d, J = 6.6 Hz,
3H), 0.95 (d, J = 6.6 Hz, 3H), 2.11–2.22 (m, 1H),3.90 (s,
3H), 4.22 (d, J = 5.7 Hz, 2H),4.76 (d, J = 4.2 Hz, 1H),
7.02–7.07 (m, 1H), 7.11 (d, J = 2.4 Hz, 1H), 7.91 (dd,
J = 8.6, 2.9 Hz, 1H), 8.86 (d, J = 2.7 Hz, 1H), 9.06 (t,
J = 5.6 Hz, 1H), 13.11 (br s, 1H); 13C NMR (DMSO–d6) d
16.9, 18.6, 29.4, 41.2, 56.3, 76.7, 100.3, 112.1, 113.7,
114.0, 131.7, 148.5, 156.3, 160.7, 161.8, 164.7, 169.3;
170.3, Anal. Calcd for C18H19NO8: C, 57.29; H, 5.08, N,
3.71. Found: C, 57.20; H, 5.08; N, 3.63.
(S)–2–((S)–2–(2–aminoacetoxy)–4–methylpentanamido)–
3–phenylpropanoic acid hydrochloride (11a) White
microcrystals (42 %), mp 175–178 °C; [a]2D3 = -43.0 (c
1.0, CH3OH); 1H NMR (DMSO–d6) d 0.82 (d, J = 5.1 Hz,
3H) 0.84 (d, J = 4.5 Hz, 3H) 1.40–1.64 (m, 3H), 2.94 (dd,
J = 13.8, 9.0, 1H), 3.07 (dd, J = 13.7, 5.0 Hz, 1H),
3.70–3.90 (m, 2H), 4.40–4.56 (m, 1H), 5.04 (dd, J = 8.7,
3.5 Hz, 1H), 7.18–7.36 (m, 5H), 8.34–8.46 (br s, 3H), 8.52
(d, J = 7.2 Hz, 1H); 13C NMR (DMSO–d6) d 21.5, 23.1,
23.8, 24.5 36.5, 53.2, 73.0, 126.5, 128.2, 129.2, 137.5,
167.1, 168.9, 172.5.
(S)–2–(2–(7–(diethylamino)–2–oxo–2H–chromene–3–car-
boxamido)acetoxy)–3–methylbutanoic acid (8f) Yellow
microcrystals (92 %), mp 174–176 °C; [a]2D3 = -19.0 (c
1.0, CH3OH); 1H NMR (DMSO–d6) d 0.91 (d, J = 6.6 Hz,
3H), 0.95 (d, J = 6.6 Hz, 3H), 1.14 (t, J = 7.1 Hz, 6H),
2.12–2.19 (m, 1H), 3.48 (q, J = 7.0 Hz, 4H), 4.20 (d,
J = 6.0 Hz, 2H), 4.75 (d, J = 4.2 Hz, 1H), 6.61 (d,
J = 2.1 Hz, 1H), 6.79–6.83 (m, 1H), 7.69 (d, J = 9.0 Hz,
(S)–2–((S)–2–(2–aminoacetoxy)–4–methylpentanamido)–
4–(methylthio)butanoic acid hydrochloride (11b) White
microcrystals (30 %), mp 104–107 °C; [a]2D3 = -32.0 (c
1.0, CH3OH); 1H NMR (DMSO–d6) d 0.84 (d, J = 5.7 Hz,
123

168
S. Biswas et al.
3H); 0.86 (d, J = 6.3 Hz, 3H); 1.44–1.74 (m, 3H),
1.80–2.00 (m, 5H), 2.34–2.45 (m, 2H), 3.70–3.93 (m, 2H),
4.23–4.32 (m, 1H), 5.01 (dd, J = 9.5, 4.1 Hz, 1H),
8.40–8.62 (m, 4H); 13C NMR (DMSO–d6) d 14.7, 21.6,
23.2, 23.8, 29.8, 30.7, 50.8, 73.1, 167.2, 169.1, 172.9.
(S)–2–((S)–2–(2–(7–methoxy–2–oxo–2H–chromene–3–
carboxamido)acetoxy)–4–methylpentanamido)–4–(methyl-
thio)-butanoic acid (12b) White microcrystals (92 %),
mp 182–184 °C; [a]D23 = -17.0 (c 1.0, CH3OH); 1H NMR
(CDCl3) d 0.92 (d, J = 6 Hz, 3H), 0.94 (d, J = 6.3 Hz,
3H), 1.65–1.82 (m, 3H), 2.10 (s, 3H), 2.10–2.31(m, 2H),
2.55–2.61 (m, 2H), 3.92 (s, 3H), 4.17–4.35 (m, 2H),
4.60–4.69 (m, 1H), 5.35 (t, J = 6.5 Hz, 1H), 6.85 (d,
J = 2.4 Hz, 1H), 6.95 (dd, J = 8.7, 2.4 Hz, 1H), 7.50
(d, J = 7.8 Hz, 1H), 7.65 (d, J = 8.7 Hz, 1H), 8.83 (s,
1H), 9.38 (t, J = 5.6 Hz, 1H); 13C NMR (CDCl3) d
15.4, 21.6, 23.2, 24.6, 30.2, 30.8, 40.4, 42.4, 51.8, 56.1,
73.5, 100.3, 112.2, 113.3, 114.3, 131.5, 149.3, 156.9,
161.7, 163.8, 165.4, 168.3, 171.1, 173.7; HRMS,
[M ? Na]?: Calcd for [C28H30N2O9Na]?: 545.1564.
Found: 545.1562.
2–(((S)–1–(((S)–1–carboxy–3–(methylthio)propyl)amino)–
1–oxo–3–phenylpropan–2–yl)oxy)–2–oxoethanaminium
chloride (11c) White microcrystals (35 %), mp
180–182 °C; [a]D23 = -21.0 (c 1.0, CH3OH); 1H NMR
(DMSO–d6) d 1.80–2.02 (m, 2H), 2.04 (s, 3H), 2.32–2.48
(m, 2H), 2.99 (dd, J = 14.3, 8.9 Hz, 1H), 3.14 (dd,
J = 14.4, 3.6 Hz, 1H), 3.72 (d, J = 17.3, 1H), 3.89 (d,
J = 18.2, 1H), 4.31–4.39 (m, 1H), 5.29 (dd, J = 8.1,
3.6 Hz, 1H), 7.20–7.36 (m, 5H), 8.65 (d, J = 7.8 Hz, 1H);
13C NMR (DMSO–d6) d 14.6, 29.7, 30.6, 37.1, 50.9, 75.0,
126.7, 128.3, 129.4, 136.3, 167.1, 168.2, 172.9.
(S)–2–((S)–2–(2–(7–methoxy–2–oxo–2H–chromene–3–
carboxamido)acetoxy)–3–phenylpropanamido)–4–(methyl-
thio)butanoic acid (12c) White microcrystals (83 %), mp
170–172 °C; [a]D23 = -43.0 (c 1.0, CH3OH); 1H NMR
(CDCl3) d 2.03 (s, 3H), 2.02–2.25 (m, 4H), 3.14 (dd,
J = 14.7, 4.5 Hz, 1H), 3.24 (dd, J = 14.3, 5.2 Hz, 1H),
3.95 (s, 3H), 4.09 (dd, J = 17.4, 5.7 Hz, 1H), 4.32 (dd,
J = 17.4, 5.7 Hz, 1H), 4.52–4.62 (m, 1H), 5.55 (t,
J = 4.7 Hz, 1H), 6.80–7.14 (m, 7H), 7.39 (d, J = 7.8 Hz,
1H), 7.67 (d, J = 8.7 Hz, 1H), 8.69 (s, 1H), 9.39 (t,
J = 5.4 Hz, 1H); 13C NMR (CDCl3) d 15.4, 30.0, 30.6,
37.4, 42.7, 52.0, 56.4, 74.6, 100.6, 112.5, 113.5, 114.6,
127.0, 128.4, 130.0, 131.8, 135.5, 149.5, 157.1, 162.0,
164.3, 165.7, 167.7, 169.9, 173.7; Anal. Calcd for
C27H28N2O9S: C, 58.26; H, 5.07, N, 5.03. Found: C, 57.97;
H, 5.20; N, 4.60.
General preparation of N-coumarinoyl-labeled
depsitripeptides 12a–d
Hydrochloride salts of 11a–c (0.050-0.055 g, 0.11 mmol,
1.1 equiv.) and TEA (0.04 g, 0.22 mmol, 2.0 equiv.) were
dissolved in minimum amount of cold water (1 mL).
Acetonitrile (4 mL) was added to the solution and cooled
to 10 °C. A solution of N-coumarinoyl-Bt 7a–c (0.04–
0.05 g, 1.0 equiv.) was added and stirred for 1 h at 25 °C.
The reaction mixture was monitored with TLC [EtOAc-
Hexanes (1:2)]. After the completion of reaction, solvent
was evaporated. 4 N HCl solution (5 mL) was added drop
wise just to acidify the reaction mixture. The precipitated
was filtered and washed with 1 N HCl (5 mL) and water
(5 mL) to afford desired N-Coumarinoyl-labeled depsidi-
peptides 12a–d. (Note: for 12d acidification was done
carefully just to neutralize the solution, at neutralization
point thick precipitate was formed and it was filtered off).
The crude compound was recrystallized from EtOAc-
hexanes.
(S)–2–((S)–2–(2–(7–(diethylamino)–2–oxo–2H–chro-
mene–3–carboxamido)acetoxy)–4–methylpentanamido)–
3–phenylpropanoic acid (12d) Yellow microcrystals
(90 %), mp 162–165 °C; [a]2D3 = -25.0 (c 1.0, CH3OH);
1H NMR (CDCl3) d 0.83 (s, 3H), 0.91 (s, 3H), 1.23(t,
J = 6.9, 6H), 1.57 (s, 3H), 3.17(dd, J = 14.2, 8.4, 1H),
3.33 (dd, J = 14.2,4.5,1H) 3.44 (d, J = 6.9 4H), 4.11 (d,
J = 4.8 Hz, 2H), 4.72–4.85 (m, 1H), 5.25 (br s, 1H), 6.46
(s, 1H), 6.45 (d, J = 8.4 Hz, 1H), 7.10–7.28 (m, 6H),
7.35–7.45 (m, 2H), 8.56 (s, 1H), 9.37 (s, 1H); 13C NMR
(CDCl3) d 12.7, 21.9, 23.3, 24.7, 37.3, 40.5, 42.7, 45.4,
54.3, 73.6, 96.7, 108.5, 110.5, 127.1, 128.7, 129.5, 131.9,
136.6, 149.0, 153.3, 158.1, 162.8, 165.2, 168.7, 171.3,
172.8, HRMS, [M ? Na]?: Calcd for: [C31H37N2O8Na]?:
602.2473. Found: 602.2467.
(S)–2–((S)–2–(2–(7–methoxy–2–oxo–2H–chromene–3–
carboxamido)acetoxy)–4–methylpentanamido)–3–phenyl-
propanoic acid (12a) White microcrystals (88 %), mp
160–163 °C; [a]D23 = -22.0 (c 1.0, CH3OH); 1H NMR
(CDCl3)d0.86(d, J = 6.3 Hz, 3H),0.87(d, J = 6.0 Hz, 3H),
1.54–1.66 (m, 3H), 3.16 (dd, J = 13.8, 8.4 Hz, 1H), 3.32 (dd,
J = 14.3, 5.3 Hz, 1H), 3.92 (s, 3H), 4.16–4.20 (m, 2H),
4.76–4.88 (m, 1H), 5.27 (t, J = 6.5 Hz, 1H), 6.85 (d,
J = 2.4 Hz, 1H), 6.95 (dd, J = 8.7, 2.4 Hz, 1H), 7.14–7.30
(m, 6H), 7.59 (d, J = 8.7 Hz, 1H), 8.75 (s, 1H), 9.31 (t,
J = 5.9 Hz, 1H); 13C NMR (CDCl3) d 21.9, 23.3, 24.7, 37.3,
40.6, 42.5, 53.7, 56.3, 73.8, 100.5, 112.4, 113.6, 114.5, 127.2,
128.7, 129.5, 131.6, 136.4, 149.4, 157.0, 161.8, 163.7, 165.5,
168.5, 170.8, 173.4; HRMS, [M ? Na]?: Calcd for
[C28H30N2O9Na]?: 561.1844. Found: 561.1847.
Acknowledgments We thank Dr. C. D. Hall, Dr. S. Tala and Mr.
Z. Wang for helpful discussions. The authors would like to
acknowledge Dr. Schanze and Mr. Emir Yasun (University of Flor-
ida) for their support.
123

Photophysics of novel coumarin-labeled depsipeptides in solution
169
Conflict of interest The authors declare that they have no conflict
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