6-N,N-dimethylamino-2,3-naphthalimide: A new environment-sensitive fluorescent probe in δ- and μ-selective opioid peptides

Article abstract of DOI:10.1021/jm060343t

A new environment-sensitive fluorophore, 6-N,N-(dimethylamino)-2,3- naphthalimide (6DMN) was introduced in the o-selective opioid peptide agonist H-Dmt-Tic-Glu-NH2 and in the μ-selective opioid peptide agonist endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2). Enviro

Full text of DOI:10.1021/jm060343t

J. Med. Chem. 2006, 49, 3653-3658
3653
6-N,N-Dimethylamino-2,3-naphthalimide: A New Environment-Sensitive Fluorescent Probe in
δ- and µ-Selective Opioid Peptides
M. Eugenio Va´zquez,† Juan B. Blanco,† Severo Salvadori,‡ Claudio Trapella,‡ Roberto Argazzi,§ Sharon D. Bryant,|
Yunden Jinsmaa,| Lawrence H. Lazarus,| Lucia Negri, Elisa Giannini, Roberta Lattanzi, Mariantonella Colucci, and
Gianfranco Balboni*,‡,#
Departamento de Qu´ımica Orga´nica y Unidad Asociada al CSIC, UniVersidad de Santiago de Compostela, 15782 Santiago de Compostela,
Spain, Department of Pharmaceutical Sciences and Biotechnology Center, UniVersity of Ferrara, I-44100 Ferrara, Italy, Department of
Chemistry, UniVersity of Ferrara, I-44100 Ferrara, Italy, Medicinal Chemistry Group, Laboratory of Pharmacology and Chemistry, National
Institute of EnVironmental Health Sciences, Research Triangle Park, North Carolina 27709, Department of Human Physiology and
Pharmacology “Vittorio Erspamer,” UniVersity La Sapienza, I-00185 Rome, Italy, and Department of Toxicology, UniVersity of Cagliari,
I-09124 Cagliari, Italy
ReceiVed March 24, 2006
A new environment-sensitive fluorophore, 6-N,N-(dimethylamino)-2,3-naphthalimide (6DMN) was introduced
in the δ-selective opioid peptide agonist H-Dmt-Tic-Glu-NH2 and in the µ-selective opioid peptide agonist
endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2). Environment-sensitive fluorophores are a special class of
chromophores that generally exhibit a low quantum yield in aqueous solution but become highly fluorescent
in nonpolar solvents or when bound to hydrophobic sites in proteins or membranes. New fluorescent
δ-selective irreversible antagonists (H-Dmt-Tic-Glu-NH-(CH2)5-CO-Dap(6DMN)-NH2 (1) and H-Dmt-Tic-
Glu-Dap(6DMN)-NH2 (2)) were identified as potential fluorescent probes showing good properties for use
in studies of distribution and internalization of δ receptors by confocal laser scanning microscopy.
Introduction
attached to a free carboxylic acid or an amino group on the
peptides in one of two ways: (i) to a side chain functional group
of a noncritical residue or (ii) by extending the peptide backbone
in a manner that has minimal influence on receptor binding.21
Other studies show that a nonpeptidic fluorescent probe, derived
from the naltrindole template for the δ-opioid receptor, is a
potent δ-opioid receptor antagonist in the mouse vas deferens
(MVD) (smooth muscle) assay and binds to the δ-opioid
receptor with relatively high affinity (Ki ) 1 nM) and selectiv-
ity.20 However, with the exception of the arylacetamide-derived
fluorescent ligands,22 none of these compounds have been
reported as molecular probes nor was their selectivity for any
of the major opioid receptor types (δ, µ, κ) studied.
Schiller et al. reported the synthesis of the µ-opioid peptide
[Dmt1]DALDA (H-Dmt-D-Arg-Phe-Lys-NH2) containing dansyl
or anthranoyl fluorophores,17,18 and subsequently, this group
published the synthesis of a fluorescent δ-opioid peptide
containing the environment-sensitive amino acid Aladan (H-
Tyr-Tic-Aladan-Phe-OH) (Ki (δ) ) 2.56 nM, selectivity µ/δ )
7930, Φ ) 0.150 in Tris-HCl buffer at pH 6.6).14 Furthermore,
our group reported the synthesis of a δ-selective tripeptide
containing fluorescein at the C-terminal (H-Dmt-Tic-Glu-NH-
(CH2)5-NH-C(dS)-NH-fluorescein (Ki (δ) ) 0.035 nM, selec-
tivity µ/δ ) 4370, Φ ) 0.227 in Tris-HCl buffer at pH 6.6). 23
Recently, Imperiali et al. published the synthesis of the new
environment-sensitive fluorophore 6-N,N-(dimethylamino)-2,3-
naphthalimide (6DMN) and the corresponding N-protected
amino acid Fmoc-R-amino-â-(6-N,N-dimethylamino naphthal-
imide) propanoic acid (Fmoc-Dap(6DMN)-OH).24 They dem-
onstrated that the corresponding model compound methyl
2-(6-(dimethylamino)-1,3-dioxo-1H-benzo[f]isoindol-2(3H)-yl)-
acetate (6DMN-Gly-OMe) had a red-shifted fluorescence in
polar protic environments, with a maximum emission intensity
that shifted from 491 nm in toluene to 592 nm in water, and
the fluorescence quantum yield (Φ) decreased more than 100-
fold from chloroform (0.225) to water (0.002). The 6DMN
Fluorescence spectroscopy has become one of the most
valuable tools for the development of new probes for biochemi-
cal research,1,2 being extensively used for monitoring ions,3
small molecules,4 and biological processes,5 such as protein
folding,6 protein-protein interactions,7 and phosphorylation
events.8 Whereas many fluorescence applications rely on the
use of intrinsic fluorophores, the development of new extrinsic
fluorophores remains an essential element for the design of new
fluorescent probes. Environment-sensitive fluorophores are a
special class of chromophores whose spectroscopic behavior
depends on the physicochemical properties of its immediate
environment.9 These molecules generally exhibit a low quantum
yield in aqueous solution but become highly fluorescent in
nonpolar solvents or when bound to hydrophobic sites in
proteins or membranes. Particularly useful are the solvatochro-
mic fluorophores that display sensitivity to the polarity of the
local environment, such as the 2-propionyl-6-dimethylaminon-
aphthalene (PRODAN),10 4-dimethylamino phthalimide (4-
DMAP),11 and 4-amino-1,8-naphthalimide derivatives.12 Prodan
and derivatives (Aladan, an alanine derivative of 6-(dimethyl-
amino)-2-acyl-naphthalene)13,14 still constitute the most widely
used environment-sensitive fluorophores, regardless of certain
limitations mainly resulting from the relatively intense fluores-
cence even in aqueous environments.
Peptide ligands for opioid receptors were previously labeled
with fluorescent functionalities, such as rhodamine,15 pyrene,16
dansyl,17,18 and fluorescein.19,20 These groups can be readily
* To whom correspondence should be addressed. Tel: +39-532-291-
275. Fax: +39-532-291-296. E-mail: gbalboni@unica.it; bbg@unife.it.
† Universidad de Santiago de Compostela.
‡ Department of Pharmaceutical Sciences and Biotechnology Center,
University of Ferrara.
§ Department of Chemistry, University of Ferrara.
| National Institute of Environmental Health Sciences.
University La Sapienza.
# University of Cagliari.
10.1021/jm060343t CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/17/2006

3654 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Table 1. Receptor Binding and Functional Bioactivity
Va´zquez et al.
a The Ki values (nM) were determined according to Chang and Prusoff37 as detailed in the Experimental Section. The mean ( SE with n repetitions in
parentheses is based on independent duplicate binding assays with five to eight peptide doses using several different synaptosomal preparations. b Agonist
activity was expressed as the IC50 values obtained from dose-response curves. These values represent the mean ( SE for at least five fresh tissue semples.
Deltorphin C and dermorphin were the internal standards for MVD (δ-opioid receptor bioactivity) and GPI (µ-opioid receptor bioactivity) tissue preparation,
respectively. c The Ke (equilibrium dissociation constant) values of opioid antagonists against the agonists (deltorphin C and dermorphin) were determined
by the method of Kosterlitz and Watt.40 d Data taken from Balboni, G. et al.23
fluorophore combines some of the advantageous fluorescence
properties of PRODAN with the extreme sensitivity to the local
polarity exhibited by the 4-aminophthalimide family of the
environment-sensitive fluorophores.11 Such environment-sensi-
tive molecules could be useful in confocal microscopy studies,
where flurescence will be higher for opioid-receptor interac-
tions and lower for nonspecific binding.
crude product (3) that was purified by preparative HPLC as
detailed in the Supporting Information.
Results and Discussion
In Vitro Opioid Activity Profile. Receptor binding and
functional bioactivity are reported in Table 1. In comparison to
the reference δ-selective fluorescent compound derivative,23
1
In this article, we evaluate the opportunities offered by this
new fluorescent amino acid, starting from the data we collected
with our reference compound H-Dmt-Tic-Glu-NH-(CH2)5-NH-
C(dS)-NH-fluorescein.23 We report the synthesis, biological
evaluation, and in situ visualization of δ-opioid receptors in
mouse neuroblastoma cells of µ- and δ-selective opioid peptides
derived from endomorphin-225 and Dmt-Tic pharmacophore,
respectively (Table 1).26
is characterized by the substitutions of 1,5-pentanediamine,
introduced to reduce the potential interference with the opioid
receptors, with 6-aminohexanoic acid (the same number of
methylene units) and fluorescein by the new fluorescent probe
Dap(6DMN). These modifications are detrimental for the
binding and selectivity of δ-opioid receptors. In fact, the affinity
for δ-opioid receptors decreases 4.5-fold, whereas that for
µ-opioid receptors increases 3.7-fold, yielding a loss of selectiv-
ity of about 17-fold. Unexpectedly, 2, the same analogue without
the pentamethylene spacer, decreased δ-affinity by 49-fold but
increased selectivity by 3.9-fold (Kiδ ) 7.8 nM, selectivity )
1010) relative to that of 1. Compounds 3-5 depict three attempts
to synthesize fluorescent derivatives selective for µ-opioid
receptors; only few examples are reported in the literature. In
fact, the last active but not selective µ peptides synthesized were
reported by Schiller et al.;17,18 the fluorescent analogues of
[Dmt1]DALDA were endowed with high µ affinity (Kiµ ranging
from 0.508 to 0.589 nM) but exhibited very low selectivities
(Kiδ/Kiµ ) 3.2-6.1). Compd 4 coincides with 1, where the
δ-selective tripeptide Dmt-Tic-Glu is substituted by the µ-selec-
tive tetrapeptide Tyr-Pro-Phe-Phe corresponding to the sequence
of endomorphin-2. This new analogue (4), despite its low µ
affinity (Kiµ ) 244.5 nM), is characterized by a selectivity (Kiδ/
Kiµ ) 24) better than that of other published µ-selective
Chemistry
The peptides, including the fluorescent-sensitive amino acid
(1, 2, 4, and 5), were synthesized using standard Fmoc solid-
phase peptide synthesis (SPPS), cleaved/deprotected from the
solid support, and purified by HPLC as detailed in the
Experimental Section. Compound 3, containing fluorescein, was
prepared by standard solution peptide synthesis as reported in
Scheme 1, Supporting Information. Boc-Tyr-Pro27 was con-
densed with H-Phe-Phe-OBzl28 via WSC/HOBt. After C-
terminal benzyl ester deprotection by catalytic hydrogenation
(Pd/C, 10%), N-Z-1,5-pentanediamine was condensed (WSC/
HOBt). The N-Z deprotection by catalytic hydrogenation (Pd/
C, 10%) gave the intermediate Boc-Tyr-Pro-Phe-Phe-NH-
(CH2)5-NH2 suitable for the reaction with fluorescein 5-isothio-
cyanate.23 Final N-Boc-deprotection with TFA gave the final

6-N,N-Dimethylamino-2,3-naphthalimide
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3655
selectivity, and peptides 4 and 5 failed to elicit opioid activity,
confirming the difficulty in obtaining biologically functional
fluorescent µ-ligands based on an endomorphin scaffold.
Fluorescence Detection. The visualization of δ-opioid recep-
tors in situ with our fluorescent probe was obtained by
incubating (15 min at 35 °C) the fluorescent compound (1) (20
nM) with the NG108-15 (mouse N18 neuroblastoma × rat C6
glioma) cell line, which expresses mouse δ-opioid receptors.
Figure 2 shows the fluorescent photomicrographs obtained using
1 in the absence (left panel) or in the presence (right panel) of
the nonspecific opioid-receptor antagonist naloxone (20 µM);
preincubation with naloxone (10 min) essentially abolished the
fluorescence bound to δ-opioid receptors. This illustrates the
advantage of using environment-sensitive fluorophores with low
Φ in the unbound state. This results in a lower background
signal from the free probe and, therefore, better sensitivity and
spatial resolution.
Fluorescence Quantum Yield Measurements. To probe the
environment affecting the sensitivity of the fluorescent peptides,
the emission spectra and the fluorescent quantum yields of 1
were recorded in solvents of different polarity: water (Tris-
HCl buffer at pH ) 6.6), acetonitrile, and dichloromethane.
Fluorescence quantum yields were calculated with respect to
quinine sulfate in 0.5 M H2SO4 as a standard (Φ ) 0.546).30
Steady-state fluorescence parameters of 1 and 2 are reported in
Table 2. Solutions of both the samples and the reference
compound were prepared by dilution in the appropriate solvent
from the primary solutions whose absorbance was below 0.2 at
the same excitation wavelength (350 nm). Fluorescent measure-
ments were taken for each solution with the same instrument
parameters, and the fluorescence spectra were corrected for
instrumental response before integration. The integrated cor-
rected emission spectra values were plotted against the absor-
bance data, and least-squares fit was used to fit the data to a
straight line. The slope of the best-fit line was assumed to be
proportional to the emission quantum yield. The yield for each
sample was calculated as detailed in the Experimental Section.
For samples in aqueous solution, the correction for refractive
index was assumed to be insignificant. The spectroscopic
behavior of 1 resembled that of the model compound 6DMN-
GlyOMe reported by Imperiali et al.;24 the lower energy
absorption maxima, around 380 nm, showed only a small
dependence on solvent polarity, whereas emission maxima
followed the trend observed for 6DMN-GlyOMe in CH3CN and
CH2Cl2. The emission maxima found in water at pH 6.6 for 1
and 2 (λem ) 467 and 464 nm, respectively) are significantly
blue shifted with respect to those expected on the basis of the
previously reported behavior of the fluorophore in protic solvents
and water at neutral pH. This could be attributed to the role of
protonation inside the compound, which might have a strong
influence on the electronic structure of the excited state.
Figure 1. Functional bioactivity of compounds 1 and 2 on MVD. The
inhibition of DEL C-induced twitch by (2) (10 and 100 nM) and (1)
(0.3 and 1 nM) were conducted as given in the Experimental Section
using MVD preparations.
compounds.29 However, 3 is comparable to the reference
compound, where the δ-selective tripeptide Dmt-Tic-Glu was
substituted by the µ-selective tetrapeptide Tyr-Pro-Phe-Phe. The
endomorphin-2 analogue (5) (H-Tyr-Pro-Dap(6DMN)-Phe-NH2)
is derived from a similar modification reported in the δ-selective
tetrapeptide TIPP, where Phe3 is substituted by the fluorescent
amino acid Aladan to yield [Aladan3]TIPP.14 Considering this
modification, we substituted Dap(6DMN) in the third position
of the endomorphin-2 sequence; however, neither of these two
compounds (4 and 5) had negligible affinity and selectivity for
µ- and δ-opioid receptors.
Compounds 1-5 were tested for functional bioactivity in
MVD and GPI preparations (Table 1). Interestingly, these
fluorescent derivatives (1 and 2) and the reference compound
in the MVD assay demonstrated nonequilibrium antagonist
activity (Figures 1 and 2). The log dose-response curves of
deltorphin C (δ agonist) in the presence of increasing concentra-
tions of 1 or 2 reduced the apparent efficacy and Hill slope
(deltorphin C ) 1.1; +0.3 nM (1) ) 0.7 and at +1 nM, (1) )
0.5; at +10 nM, (2) ) 0.9 and at +100 nM, (2) ) 0.6). Both
compounds bind tightly and dissociate very slowly in the tissue
preparation. The δ antagonism could not be reversed by washing
the tissue with a drug-free solution for over a 3 h period of
time. Moreover, the longer the compound remained in contact
with the tissue, the greater was the magnitude of the observed
δ antagonism. Compd 1, containing the pentamethylene spacer
between the opioid sequence and the fluorescent amino acid,
demonstrated a δ-irreversible antagonism starting from a
concentration of 0.3 nM, whereas the corresponding derivative,
lacking the pentamethylene spacer (2), exhibited an irreversible
δ-antagonist activity that began at a 30-fold higher concentration
(10 nM), confirming the importance of the spacer between the
opioid pharmacophore and the fluorophore.
Conclusion
The incorporation of the amino acid H-Dap(6DMN)-OH
containing the environment-sensitive fluorophore 6-N,N-(di-
methylamino)-2.3-naphthalimide (6DMN) into compounds con-
taining the δ-opioid antagonist pharmacophore Dmt-Tic yielded
selective probes (Kiδ/Kiµ ) 260 and 1000 relative to that of the
µ-opioid receptor), which bound as apparent irreversible an-
tagonists, to δ-opioid receptors in the membranes of NG108-
15 cells. The maintenance of the opioid binding properties and
fluorescence parameters indicated that the fluorophore had a
weak effect on δ-receptor interaction. Our fluorescent conjugates
1 and 2 could be applied as specific probes to study the in vitro
In the GPI assay, 1 and 2 are endowed with a weak agonist
or partial agonist activity, which is in agreement with their
affinity data and selectivity. Compd 3, projected as an analogue
of the reference compound modified to have potential µ-opioid

3656 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Va´zquez et al.
Figure 2. Microscopic visualization of compound 1 in NG108-15 cells. Left panel: NG108-15 cells with fluorescence compound. Right panel:
nonspecific binding obtained by preincubation with the opioid receptor antagonist naloxone.
Table 2. Steady State Fluorescence Parameters of Compounds 1 and 2a
systems: (A) 1-butanol/AcOH/H2O (3:1:1, v/v/v) and (B) CH2Cl2/
methanol/toluene (17:1:2), and ninhydrin (1%, Merck), fluores-
no.
λ
em (nm)
λ
em,corr (nm)
Φ
camine (Hoffman-La Roche), and chlorine reagents were used as
sprays. The melting points were determined on a Kofler apparatus
and are uncorrected. Optical rotations were determined at 10 mg/
mL in methanol with a Perkin-Elmer 241 polarimeter with a 10
cm water-jacketed cell.
Peptide synthesis used standard SPPS protocols on a 0.02 to 0.05
mmol scale using a 0.21 mmol/g loading PAL-PEG-PS solid
support. Amino acids were purchased as protected Fmoc amino
acids with the standard side chain protecting scheme: Fmoc-6-
aminohexanoic acid, Fmoc-Glu(OtBu)-OH, Fmoc-Tic-OH, Fmoc-
Phe-OH, Fmoc-Pro-OH. Boc-Tyr(tBu)-OH and Boc-Dmt-OH31
were used as N-terminal amino acids. The synthesis of Fmoc-Dap-
(6DMN)-OH fluorescent building block was made using previously
reported procedures.24
Amino acids were manually coupled in 4-fold excess using a
mixture of N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-
N-methylmethanaminium (HBTU) and 1-hidroxybenzotriazole
(HOBt) and N,N-diisopropylethylamine (DIEA) in DMF as activat-
ing agents. The coupling of Fmoc-Dap(6DMN)-OH was performed
using 2-(1H-7-aza-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate methanaminium (HATU) but otherwise using
the same protocol as the other amino acids. Each amino acid was
activated for two minutes in DMF before being added onto the
resin. Peptide-bond-forming couplings were conducted for 1 h and
monitored using the TNBS test.32 The deprotection of the temporal
Fmoc protecting group was performed by treating the resin with
20% piperidine in DMF solution for 15 min.
The peptides were cleaved from the resin, and side-chain
protecting groups were simultaneously removed by treatment with
the following cleavage mixture: 50 µL of dichloromethane, 25 µL
of triisopropyl silane, 25 µL of water, and 950 µL of TFA (1 mL
of mixture/50 mg of resin) for 1.5-2 h at room temperature. All
peptides were precipitated with diethyl ether (4 °C) and further
purified by reverse-phase HPLC.
reference
520a
464a
557b
534c
467a
522a
464a
571b
541c
467a
0.24 ( 0.01a
0.012 ( 0.001a
1
2
0.194 ( 0.01b
0.396 ( 0.01c
0.022 ( 0.001a
554b
531c
567b
538c
0.192 ( 0.01b
0.394 ( 0.01c
a Recorded in 50 mM Tris-HCl buffer (pH 6.6, 20 °C); λ ) 350 nm.
b Recorded in CH3CN. c Recorded in CH2Cl2.
localization of δ-opioid receptors in tissues, receptor internaliza-
tion, and the trafficking in live cells in real time by using
confocal microscopy.29 It is important to emphasize that
environment-sensitive fluorophores become highly fluorescent
in either nonpolar solvents or when bound to hydrophobic sites
in proteins or membranes. Therefore, there will be some
background signal resulting from the nonspecific binding of the
probes to lipid membranes that will have to be taken into
account. Such environment-sensitive molecules could be useful
in confocal microscopy studies, where flurescence will be higher
for interactions between the opioid ligand and its receptor and
lower for nonspecific binding.
The comparison between the environment-nonsensitive fluo-
rophore (reference compound) and the confocal microscopic
images obtained after treatment with the environment-sensitive
fluorophore 1 could be useful for various applications, including
studies on membrane interactions, binding to receptors, cellular
uptake and intracellular distribution, and tissue distribution.
Experimental Section
All peptide synthesis reagents and amino acid derivatives were
purchased from GL Biochem (Shanghai) and Novabiochem. C-
terminal amide peptides were synthesized on Fmoc-PAL-PEG-PS
resin from Applied Biosystems, and all other chemicals were
purchased from Aldrich or Fluka. High-performance liquid chro-
matography was performed using an Agilent 1100 series Liquid
Chromatograph Mass Spectrometer system. Analytical HPLC was
run using LiChrospher RP-18 (5 µm) 4.6 × 150 mm analytical
column from Merck. The purification of the peptides was performed
on a ZORBAX ODS C18, 94 × 250 mm reverse-phase column.
The standard gradient used for analytical and preparative HPLC
was 90:10 to 20:80 over 30 min (water/acetonitrile, 0.1% TFA).
Electrospray Ionization Mass Spectrometry (ESI/MS) was per-
formed with an Agilent 1100 Series LC/MSD model in positive
scan mode using direct injection of the purified peptide solution
into the MS. 1H NMR (δ) spectra were measured, when not
specified, in DMSO-d6 (Bruker AC-200 spectrometer), and the
peaks are parts per million downfield from tetramethylsilane
(internal standard). TLC was performed on precoated plates of silica
gel F254 (Merck, Darmstadt, Germany) using the following solvent
Deprotection. To 0.05 mmol of Fmoc-HN-(aa)n in solid support,
piperidine (3 mL, 20% in DMF) was added, and nitrogen was
passed through the mixture for 15 min. The resin was then filtered
and washed with DMF (3 × 3 mL × 3 min), and the TNBS test
was run with a small resin sample to confirm that deprotection was
successful.
Coupling. Fmoc-aa-OH (0.2 mmol) was dissolved in an HOBt/
HBTU solution (1 mL of 0.2 M HBTU, 0.2M HOBt in DMF), and
DIEA (1.5 mL of 0.195 M solution in DMF) was added. The
resulting mixture was activated for 2 min and then added over the
previously deprotected resin. Nitrogen was passed through the resin
suspension for 1 h, when the TNBS test of a small resin sample
was negative. The resin was then washed with DMF (3 × 3 mL ×
3 min) and subjected to the following deprotection/coupling cycles
in a similar way.
Coupling of Fmoc-Dap(6DMN)-OH. Fmoc-Dap(6DMN)-OH
(55 mg, 0.1 mmol) and HATU (38 mg, 0.1 mmol) were dissolved
in DMF (0.5 mL), and DIEA (0.75 mL of 0.195 M solution in
DMF) was added, the resulting mixture was activated for 2 min,

6-N,N-Dimethylamino-2,3-naphthalimide
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3657
and then added over the resin. Nitrogen was passed through the
resin suspension for 1 h, when the TNBS test of a small resin sample
was negative. The resin was then washed with DMF (3 × 3 mL ×
3 min) and recoupled with Fmoc-Dap(6DMN)-OH following the
same protocol described above. The resin was then subjected to
the following deprotection/coupling cycles using the HOBt/HBTU
coupling scheme.
to the bath and allowed to interact with tissue receptor sites for 5
min before adding the standard peptide. Competitive antagonist
activities were evaluated for their ability to shift the deltorphin C
(MVD) and dermorphin (GPI) log concentration-response curve
to the right; pA2 values were determined using the Schild Plot.39
IC50 (nM, mean ( SE) as well as the pA2 values were obtained
from at least six experiments conducted with fresh tissues.
Cleavage. The resin-bound peptide dried overnight (0.05 mmol)
was placed in a 50 mL flask, to which was added 5 mL of the
cleavage cocktail (250 µL of CH2Cl2, 125 µL of water, 125 µL of
TIS, and TFA to 5 mL), and the resulting mixture was shaken for
2 h, the resin was filtered, and the TFA filtrate was concentrated
under an argon current until a volume of 2 mL. Then it was added
to ice-cold ethyl ether (40 mL). After 5 min, the peptide was
centrifuged and washed again with 20 mL of ice-cold ether. The
solid residue was dried under Ar and redissolved in acetonitrile/
water 1:1 (2 mL), and purified by preparative reverse-phase HPLC.
The collected fractions were lyophilized and stored at -20 °C.
H-Dmt-Tic-Glu-NH-(CH2)5-(CdO)-Dap(6DMN)-NH2 (1). Rf(A)
0.72; HPLC K′ 7.91; mp 161-163 °C; [R]20D -7.8; MH+ 920; 1H
NMR (DMSO-d6) δ 1.29-1.57 (m, 6H), 2.06-3.20 (m, 24H),
3.95-5.16 (m, 8H), 6.29-8.42 (m, 11H).
Fluorescence Spectroscopy. Fluorescence quantum yields were
calculated with respect to quinine sulfate (Fluka) in 0.5 M H2SO4
as a standard (φ ) 0.546).30 Solutions of both the sample and the
reference were prepared by dilution with the appropriate solvent
of mother solutions whose absorbance was below 0.2 at the same
excitation wavelength (350 nm). Fluorescence measurements were
taken for each solution with the same instrument parameters, and
the fluorescence spectra were corrected for instrumental response
before integration. The integrated corrected emission spectra values
were plotted against the absorbance, and data were least-squares
fitted to a straight line. The slope of the best-fit line was assumed
to be proportional to the emission quantum yield. The yield for
each sample was calculated according to the formula
2
mS ηS
H-Dmt-Tic-Glu-Dap(6DMN)-NH2 (2). Rf(A) 0.65; HPLC K′
7.38; mp 157-159 °C; [R]20D -9.2; MH+ 807; 1H NMR (DMSO-
d6) δ 2.06-3.17 (m, 20H), 3.95-5.16 (m, 8H), 6.29-8.42 (m, 11H).
TFA‚H-Tyr-Pro-Phe-Phe-NH-(CH2)5-NH-(CdS)-NH-fluores-
cein (3). Rf(A) 0.66; HPLC K′ 7.26; mp 153-155 °C; [R]20D -15.1;
φS ) φR
( )
mR ηR
where φ is the emission quantum yield, m is the slope of the best-
fit line, and η is the refractive index of the solvent for the sample
(S) and the reference (R), respectively. Absorption spectra were
recorded with a Jasco V-570 UV-Vis-NIR spectrophotometer,
fluorescence spectra were obtained with a Jobin-Yvon FluoroMax-2
spectrofluorometer. To detect the fluorescent signal, a Nikon
Optiphot microscope (Hoechest filter EX 350 nm, EM 467 nm)
was used with a 40× magnification.
1
MH+ 1047; H NMR (DMSO-d6) δ 1.29-1.55 (m, 6H), 1.92-
2.34 (m, 4H), 2.92-3.29 (m, 8H), 3.41-3.51 (m, 4H), 3.95-4.92
(m, 4H), 6.11-7.28 (m, 23H).
H-Tyr-Pro-Phe-Phe-NH-(CH2)5-(CdO)-Dap(6DMN)-NH2 (4).
Rf(A) 0.74; HPLC K′ 7.96; mp 165-167 °C; [R]20 -13.8; MH+
D
1
995; H NMR (DMSO-d6) δ 1.29-1.57 (m, 6H), 1.92-2.34 (m,
6H), 2.85-3.20 (m, 14H), 3.41-3.51 (m, 2H), 3.95-5.16 (m, 7H),
6.68-8.37 (m, 19H).
Acknowledgment. This research was supported in part by
the University of Cagliari (PRIN 2004, assigned to G.B.),
University of Ferrara (PRIN 2004, assigned to S.S.), Italy, and
the Intramural Research Program of the NIH, and NIEHS. We
appreciate the professional services of the library staff of the
NIEHS. M.E.V. thanks the Human Science Frontier Program
Organization for their support with the Career development
Award (CDA0032/2005-C) and the Spanish Ministry of Educa-
tion and Science for the “Ramon y Cajal” contract. M.E.V. and
G.B. sincerely thank Prof. Barbara Imperiali (M. I. T.; U.S.A.)
for her support and useful advice in the development of this
project.
H-Tyr-Pro-Dap(6DMN)-Phe-NH2 (5). Rf(A) 0.69; HPLC K′
7.48; mp 160-162 °C; [R]20D -14.3; MH+ 735; 1H NMR (DMSO-
d6) δ 1.92-2.34 (m, 4H), 2.92-3.17 (m, 10H), 3.41-3.51 (m, 2H),
3.95-5.16 (m, 6H), 6.68-8.42 (m, 14H).
Elemental analysis of compounds 1-5 is reported in Supporting
Information, Table 3.
Competitive Receptor Binding Assays. These assays were
conducted as described in detail elsewhere using rat brain synap-
tosomes (P2 fraction).31,33-35 Membrane preparations were prein-
cubated to eliminate endogenous opioid peptides and stored at -80
°C in buffered 20% glycerol.34,36 Each analogue was analyzed in
duplicate using five to eight dosages of peptide and independent
repetitions with different synaptosomal preparations (n values are
listed in Table 1 in parentheses, and the results are listed as the
mean ( SE). An unlabeled peptide (2 µM) was used to determine
nonspecific binding in the presence of 1.9 nM [3H]deltorphin C
(45.0 Ci/mmol, Perkin-Elmer, Boston, MA; KD ) 1.4 nM) for
δ-opioid receptors, and for µ-opioid receptors, 3.5 nM [3H]DAMGO
(50.0 Ci/mmol, Amersham Biosciences, Buckinghamshire, U.K.;
KD ) 1.5 nM) was used. Glass fiber filters (Whatman GFC) were
soaked in 0.1% polyethylenimine to enhance the signal/noise ratio
of the bound radiolabeled synaptosome complex, and the filters
were washed thrice in ice-cold buffered BSA.34 The affinity
constants (Ki) were calculated according to Cheng and Prusoff.37
Functional Bioactivity in Isolated Organ Preparations. The
preparations of myenteric plexus-longitudinal muscle obtained from
the male guinea pig ileum (GPI, enriched in µ-opioid receptors)
and the preparations of MVD (containing δ-opioid receptors) were
used for field stimulation with bipolar rectangular pulses of
supramaximal voltage.38 Agonists were evaluated for their ability
to inhibit the electrically evoked twitch. The biological potency of
the compounds was compared against the activity of the µ-opioid
receptor agonist dermorphin in GPI and with MVD for the δ-opioid
receptor measured agonist deltorphin C. The results are expressed
as IC50 values obtained from dose-response curves (Prism,
GraphPad). To evaluate antagonism, the compounds were added
Supporting Information Available: Experimental details for
the synthesis of compound 3and references cited herein; elemental
analysis. This material is available free of charge via the Internet
at http://pubs.acs.org.
References
(1) Abbreviations: in addition to the IUPAC-IUB Commission on
Biochemical Nomenclature (J. Biol. Chem. 1985, 260, 14-42), this
article uses the following additional symbols and abbreviations: Ac,
acetyl; Bid, 1H-benzimidazol-2-yl; Boc, tert-butyloxycarbonyl; DAM-
GO, [D-Ala2,N-Me-Phe4, Gly-ol5] enkephalin; DEL C, deltorphin C,
(H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2); DER, dermorphin (H-Tyr-
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DMSO-d6, hexadeuteriodimethyl sulfoxide; Dmt, 2′,6′-dimethyl-L-
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HPLC, high performance liquid chromatography; MALDI-TOF,
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raphy; TNBS, 2,4,6-trinitrobenzene sulfonic acid; WSC ) EDC,
1-ethyl-3-[3′-dimethyl)aminopropyl]-carbodiimide hydrochloride; Z,
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