Fluorescence and mass spectrometry studies of the interaction between naproxen and synthetic pseudopeptidic models in organic media

Article abstract of DOI:10.1016/j.tet.2009.07.031

Time-resolved/steady-state fluorescence and mass spectrometry measurements have shown the preferential binding of a non-steroidal anti-inflammatory drug (NSAID) like naproxen 4 to a synthetic pseudopeptidic receptor built using Phe (9), i.e., bearing an a

Full text of DOI:10.1016/j.tet.2009.07.031

Tetrahedron 65 (2009) 7801–7808
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Fluorescence and mass spectrometry studies of the interaction between naproxen
and synthetic pseudopeptidic models in organic media
,
a
a,
*
M. Isabel Burguete a, Ghinwa Fawaz b, Francisco Galindo a , M. Angeles Izquierdo , Santiago V. Luis
,
*
´
b
b,
*
´
Jean Martınez , Xavier J. Salom-Roig
a
´
´
´
´
Departamento de Quımica Inorganica y Organica, Universitat Jaume I/CSIC Av. Sos Baynat, s/n, 12071 Castellon, Spain
b Institut de Biomole´cules Max Mousseron (IBMM), UMR 5247, Synthe`ses Ste´re´ose´lectives, Place Euge`ne Bataillon, Universite´ Montpellier II, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Time-resolved/steady-state fluorescence and mass spectrometry measurements have shown the pref-
erential binding of a non-steroidal anti-inflammatory drug (NSAID) like naproxen 4 to a synthetic
pseudopeptidic receptor built using Phe (9), i.e., bearing an aromatic ring, compared to another model
synthesized using Lys (8), i.e., lacking such aromatic ring but with a basic binding site. The quenching of
the emission of naproxen by models 8 and 9 has been measured in solvents of different nature and
analyzed by means of the Stern–Volmer methodology. In non-polar solvent (dichloromethane) the
fluorescence of 4 is quenched to a higher extent by 8 than by 9 but in polar medium (methanol) the
Received 12 May 2009
Received in revised form 8 July 2009
Accepted 9 July 2009
Available online 14 July 2009
opposite occurs. The result in methanol is compatible with the existence of
p–p stacking interactions
between the aromatic rings of naproxen and the aromatic ring of 9. In order to proof this model, mass
spectrometry measurements have confirmed the higher stability of the complex formed by 4 and 9 over
the related one formed with 8. The observed phenomenon could help to understand the importance of
aromaticity in the interactions between NSAIDs and more complex biological macromolecules like
misfolded proteins, involved in the development of Alzheimer’s disease and other neuropathologies.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Neuropathologies are important health problems for modern
societies. Quite a number of them are linked to protein misfolding.
One of the paradigmatic examples is the Alzheimer’s disease (AD),1-4
which is characterized by deposits of b-amyloid (Ab) peptides in the
brain. Despite the enormous efforts made to elucidate the mecha-
nism of the genesis of the AD, there are still open questions that are
a challenge for a broad array of scientific disciplines.5–10 Much effort
is currently being carried out to design new molecules capable to
interrupt the self-assembly process. But the development of new
b
-blockers is hampered by the lack of a fundamental knowledge
about the mode of action of such ligands. Studies using molecules
targeting directly -amyloid derivatives have provided some clues
b
about the molecular mode of interaction between exogenous li-
gands and misfolded proteins.11–15 Thus, different binding sites have
been identified through the use of distinct imaging tracers like
Congo Red (1), Thioflavin T (2) and FDDP (3) (Chart 1).16–20 In this
Chart 1. Some molecules capable to bind amyloid aggregates.
regard, non-steroidal anti-inflammatory drugs (NSAID) like nap-
roxen (4) and ibuprofen (5) have been found to displace the hy-
drophobic probe 3 from the plaques of AD brain specimens,21 which
suggests hydrophobic rather than ionic interactions as driving forces
for the stacking of such NSAIDs to fibrils, also supporting the anti-
amyloidogenic properties of NSAIDs.22–25
* Corresponding authors. Tel.: þ34 964728239; fax: þ34 964728214.
E-mail addresses: francisco.galindo@uji.es (F. Galindo), luiss@uji.es (S.V. Luis),
salom-roig@univ-montp2.fr (X.J. Salom-Roig).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.07.031

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M.I. Burguete et al. / Tetrahedron 65 (2009) 7801–7808
The above-cited studies use complex chemical systems (amyloid
checked that the Trp subunit by itself was capable to quench the
emission of 4, by means of control experiments with tert-butyloxy-
carbonyl tryptophan (BOC-Trp, 6) and carbobenzyloxy tryptophan
(Cbz-Trp, 7), as will be shown later.
fibrils, synthetic polypeptides, etc.) and indirect measurements like
displacement assays. In order to understand more precisely the
intermolecular interactions between NSAIDs and pathogenic fibrils
it would be useful to simplify the peptide system under study by
means of models mimicking specific features of fibrils or poly-
peptides. One of the key issues to study in order to approach this
challenging problem is to disclose the chemical nature of the in-
2.1. Synthesis
Synthetic compounds 8 and 9 were prepared by coupling of
ethylendiamine to both amino acids forming each dyad (Trp and Lys,
or Trp and Phe) in a sequential manner, as indicated in Scheme 1.
Thus, starting from the Boc-protected aminoacid (Lys or Phe), in the
presence of PyBOP, HOBt and DIEA in anhydrous DMF,26–28 in-
termediates 10 or 11 were obtained completely free of the diacety-
lated C2 symmetry derivative. Successive coupling of the remaining
free amine end with BocTrp yielded model compound 9 and in-
termediate 12. For the latter, selective deprotection of the Cbz group
by means of H2/Pd–C led to model compound 8. All the products
were characterized unambiguously by means of 1H and 13C NMR,
ESI-MS, FABMS and HRMS (see Experimental section). This synthetic
approach constitutes a variation over the series of C2 symmetric
pseudopeptides reported by our group.29–32
teractions (H-bonding, p-stacking, ion–ion, ion–dipole, etc.) taking
place when a certain NSAIDs interacts with a certain misfolded
protein or, even better, with shorter peptides prone to aggregation
(for instance the well known Lys-Leu-Val-Phe-Phe). Two extreme
models can be considered for such interactions between peptides
and 2-arylpropionic NSAIDs: one in which ionic forces predominate
(through salt bridges between carboxylates of the drug and cationic
residues of the peptide receptor, like Lys) and another in which
solvophobic forces control the binding between NSAIDs and the
peptidic system. However, to discern clearly both contributions
studying a complex peptidic-NSAID assembly in a natural aqueous
environment is extremely difficult, and simplified models are
needed.
The objective of the present study is to demonstrate the im-
portance of
p-stacking interactions in the supramolecular recog-
2.2. Fluorescence measurements
nition of naproxen by synthetic receptors of pseudopeptidic nature.
For such purpose two models have been constructed, differing in
the nature of one building block. Whereas one receptor has been
made using Lys, and hence is carrying a basic residue, the other one
has been made using Phe, and thus contains an aromatic ring. From
the analysis of the static and dynamic quenching of the fluores-
cence of 4 it is demonstrated that the neutral aromatic derivative
binds naproxen in polar medium (methanol) stronger that the basic
synthetic model. Confirmatory evidence by mass spectrometry has
also been obtained. The aim of this study is not to emulate the
behaviour of misfolded peptides leading to pathological states,
which is a challenging issue, but to approach the phenomenon of
aromatic–aromatic interactions by using fluorescence techniques,
especially in the time-resolved mode, and under controlled con-
ditions (organic media).
The fluorescence of naproxen was measured in both polar
(methanol, MeOH) and apolar (dichloromethane, DCM) solvents.
The extent of the intermolecular quenching of the fluorescence of 4
by pseudopeptides 8 and 9 can be correlated with the extent of the
binding and hence, with the relative importance of the aromatic (9)
and the ionic (8) contributions for such interaction. In all the cases,
the corresponding control experiments with 6 and 7 were carried
out. The pseudopeptides here employed allowed reproducible
measurements in both methanol and dichloromethane, which al-
lows obtaining a more complete picture of the supramolecular
binding. Quenching experiments were attempted with commercial
dipeptides (TrpLys, TrpPhe) in water but the results were hardly
reproducible, most likely due to protonation processes leading to
several species coexisting in solution. On the other hand, the utili-
zation of buffers resulted in the introduction of additional variables,
adding complexity to the analysis. Therefore, we decided to study
compounds 8 and 9 in model solvents such as MeOH and DCM.
A simple mathematical model describing the fluorescence of
a molecule participating in an intermolecular quenching process is
that in which the probe molecule is quenched as a result of (a)
ground-state complexation or (b) collision during the lifetime of
the excited probe.
2. Results and discussion
In order to design the pseudopeptides capable to distinguish
between ionic and aromatic interactions, it was decided to syn-
thesize two dyads comprised by two amino acids, one amino acid
capable to perturb the fluorescence of naproxen, and another
amino acid with basic or aromatic features. Hence dyads 8 and 9
(Chart 2), containing Trp (as quencher of 4 likely via electron-
transfer or exciplex formation) linked by ethylenediamine to either
Lys (in 8) or Phe (in 9), were envisaged as potential receptors for
naproxen, enhancing the ionic or aromatic–aromatic interactions,
respectively. Prior to synthesize the mentioned receptors, it was
I0=I ¼ 1 þ K1½Qꢀ
so
¼ 1 þ K2½Qꢀ
(1)
(2)
=
s
If the quenching occurs only as a consequence of the collision of
the chemical species during the lifetime of the excited probe (dy-
namic quenching), the situation can be analyzed by means of the
Stern–Volmer relationships: Eq. 1 (where I0 and I are the fluores-
cence intensities of the fluorescent probe in the absence and in the
presence of quencher (Q), respectively) and Eq. 2 (in which so and s
are the lifetimes of the probe in the absence and in the presence of
Q, respectively).33 If the quenching is purely dynamic then I0/I¼so
/s,
and hence K1¼K2. A completely different situation occurs when the
quenching occurs only as a result of a ground-state complexation.
Then, the measured lifetimes are not affected by the quencher
concentration, in such a way that so
association constant in the ground state (static quenching). In many
/
s
¼1, K2¼0, and K1 reflects the
Chart 2. Model compounds (6, 7) and synthetic pseudopeptides (8, 9).
instances both situations coexist, and specific equations have been

M.I. Burguete et al. / Tetrahedron 65 (2009) 7801–7808
7803
Scheme 1. Synthetic steps for the chemical preparation of receptors 8 and 9.
developed to distinguish the dynamic and static contributions.33–38
However, it is not always possible to apply accurately such models,
and then the data can be analyzed by simplified Eqs. 1 and 2, K2
affording the dynamic quenching and K1 reflecting a combination of
static and dynamic processes. The closer K1/K2 ratio to unity, the
lower the complexation in the ground state would be. Although this
kind of analysis is estimative, it can provide, along with additional
techniques, an indication of the degree of complexation in systems
where supramolecular binding constants cannot be obtained in
other way because of low association constants, low sensitivity of
the employed techniques, etc.
We have applied Eqs.1 and 2 to fit the experimental quenching of
the emission of 4 by 8 and 9, to ascertain which mode of quenching
predominates (dynamic or static) and to know indirectly, which
quencher binds 4 in a higher extent. Thus, K1 and K2 for 8 and 9 were
extracted by fitting the experimental data of steady-state (I0/I) and
The analysis of the 16 quenching experiments was performed by
means of the Stern–Volmer equations to yield the fittings shown in
Figure 3. The constants associated to these analyses are reported in
Table 1.
The examination of data in Table 1 allows several conclusions.
Compounds 6 and 7 provide the reference values for the analysis.
The fact that both 6 and 7 present similar K1 and K2 in methanol
means that the fluorescence quenching of 4 by such compounds is
practically dynamic (ratio K1/K2w1.0–1.1). The same compounds in
a non-polar solvent afforded some degree of association, with K1/
K2w1.4–1.5. On the other hand, the peptidomimetic compounds
containing Lys and Phe displayed higher values of the K1/K2 ratio
than 6 and 7 in both solvents, indicating stronger binding, espe-
cially for 8 in DCM (6.9) and for 9 in MeOH (4.2). The binding af-
finity of the Lys containing model 8 in DCM is not surprising
providing that the carboxylic acid and the amine of the side chain
would form a strong salt bridge. However, compound 9 with the
aromatic side chain of Phe displays elevated values of K1/K2 in both
DCM (3.2) and MeOH (4.2). In fact, among the values in methanol,
the ratio K1/K2 for 9 is remarkably high when compared to the rest
of values in the same solvent. Indeed, this can be appreciated
graphically in Figure 3c for the fitting with the higher slope. Ad-
ditionally, the ratio for 8 in methanol results remarkably lower (1.5)
than for 9 (4.2), which also supports the higher association of
naproxen with 9 in polar solvent. This suggests that in MeOH
a strong participation of the aromatic rings in the intermolecular
association with the NSAID.
time-resolved (so/s) emissions to Eqs. 1 and 2. The reference com-
pounds containing only the indole moiety (6 and 7) were used as
controls.
Naproxen is a fluorescent drug emitting at 352 nm when excited
at 331 nm both in MeOH and DCM.39,40 Steady-state fluorescence
spectra of 4 in both solvents can be seen in Figure 1. The lifetimes of
naproxen in these solvents in the absence of quencher resulted
5.5 ns (DCM) and 9.3 ns (MeOH), in close agreement with the lit-
erature data.41,42
Controlled amounts of quenchers 6–9 were added to solutions
of pure naproxen in either DCM or MeOH, and the steady-state
emission and fluorescence lifetimes were recorded. For illustrative
purposes, the static and dynamic data for 4 quenched by reference
6 in methanol are shown in Figure 2.
2.3. Mass spectrometry
Since the Trp chromophore is a potential fluorescent element,
the wavelength at 331 nm was selected to excite selectively the
naproxen.43 Additionally, in order to prevent inner filter effects, all
the measurements were done in the front-face configuration of the
equipment. As it can be seen in Figure 2 for 6 and 4, the fluores-
cence decays of 4 resulted monoexponential even in the presence
of ca. 0.15 M of Boc-Trp, which indicates the absence of emission
from the quencher under the excitation conditions.
The fluorescence measurements provide an array of data that
analyzed by means of the Stern–Volmer methodology suggest the
higher stability of the complex formed by 9 and naproxen in MeOH.
Mass spectrometry measurements provided additional experi-
mental evidence supporting this model. The study of supramolec-
ular complexes in the gas phase is, in fact, the method of choice
when the objective is to suppress any secondary effects caused
by solvent in the complexation process (solvation/desolvation

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M.I. Burguete et al. / Tetrahedron 65 (2009) 7801–7808
Figure 2. Representative quenching of the fluorescence of 4 by 6 in MeOH: (a) steady-
state lexc¼325 nm, lem¼350 nm)
lexc¼331 nm) and (b) time-resolved
measurements.
(
(
a direct estimation of the gas-phase stability.49 Thus, the ion at m/z
827.4, corresponding to the complex of naproxen with 8, required
0.167 eV to reduce its intensity 50%, whereas the ion m/z 846.4 (for
9) needed 0.175 eV to experience the same dissociation. As can be
seen from the data of Figure 6, the curve for the complex between 9
and 4 reflects unequivocally the higher stability of this species,
when compared to the one formed between 8 and 4, in all the
collision energy range.
This result, combined with the fluorescence measurements,
confirms the preferential binding of naproxen to the receptor
containing an aromatic ring (from Phe) over the one with the amine
residue (from Lys). The supramolecular complex formed between
naproxen and models 9 should be stabilized by a combination of
Figure 1. (a) Steady-state fluorescence spectra (lexc¼331 nm) and (b) time-resolved
fluorescence decays (lexc¼325 nm, lem¼350 nm) of 4 in DCM (red) and MeOH (black).
changes, etc.). As it has been shown in the literature, the compar-
ative study of complexes in the gas phase affords precise in-
formation on the relationships between the structure of the model
and the ligand compound and the stability of such complexes,
without potential interferences by the surrounding environment.
Mass spectrometry has been used frequently as analytical tool in
such conditions,44 along with other techniques.45
In the case of naproxen and compounds 8 and 9 the electrospray
ionization-mass spectrometry (ESI-MS) experiments afforded,
firstly, the unambiguous evidence of the existence of the postulated
complexes. Mixtures of 8 or 9 in the presence of 4 in MeOH yielded
peaks at m/z 827.4 and m/z 846.4, which is compatible with
[4þ8þNa]þ and [4þ9þNa]þ species, respectively (Fig. 4).
weak interactions, not only by means of
p-stacking, since the oc-
currence of
p-stacking interactions in systems without conforma-
tional restrictions is very rare.50–55
In order to get a quantitative estimation of the strength of the
binding, collision induced dissociation (CID) experiments were per-
formed.46–48 Those experiments confirmed the higher stability of
complex formed between 4 and the Phe-contaning compound 9.
Upon CID conditions, the supramolecular singly charged [4þ(8 or
9)þNa]þ cation readily expels the ligand 4 leading to the [(8 or
9)þNa]þ cation (Fig. 5).
3. Conclusions
Extrapolation of the conclusions here presented to the behaviour
of systems with higher complexity like amyloid peptide must be
done with caution since this study has been done with model
compounds and under model conditions. However, we must remark
the necessity of studies in controlled conditions, where the possi-
bility of experimental artifacts is minimal, in order to progress in the
basic knowledge of fundamental questions.39 For instance, in water
under physiological conditions 4 should be mostly unprotonated
A plot of the intensity percentage of a certain ion versus the
collision energy allows a quantitative estimation of the gas-phase
stability of the complex. We choosed the collision energy in the
centre-of-mass frame necessary for 50% dissociation (ECM50) as

M.I. Burguete et al. / Tetrahedron 65 (2009) 7801–7808
7805
Figure 3. (a,b) Fluorescence quenching of 4 by 6 (squares) and 7 (up triangles). Steady-state (black) and time-resolved measurements (white). (c, d) Fluorescence quenching of 4 by
8 (circles) and 9 (down triangles). Steady-state (black) and time-resolved measurements (white).
Table 1
Stern–Volmer constants corresponding to the fluorescence quenching of 4 by
compounds 6–9 in polar and apolar solvents
Solvent
MeOH
Compound
K1 (Mꢁ1
)
K2 (Mꢁ1
)
K1/K2
6
7
8
9
12 ꢂ 1
17 ꢂ 1
12 ꢂ 1
38 ꢂ 2
12 ꢂ 1
15 ꢂ 1
8 ꢂ 1
1.0
1.1
1.5
4.2
9 ꢂ 1
DCM
6
7
8
9
29 ꢂ 1
27 ꢂ 2
19 ꢂ 2
20 ꢂ 1
41 ꢂ 9
20 ꢂ 2
1.5
1.4
6.9
3.2
282 ꢂ 30
63 ꢂ 4
due to its acidic pKa, which would be considered for future studies
with water-soluble pseudopeptides. Nevertheless, under model
conditions the higher binding affinity of 9 towards 4 (both in MeOH
and in the gas phase) could indirectly support the occurrence of p–p
stacking interactions also between the aromatic rings in more
complex systems, as it has been demonstrated to occur between
aromatic b-blockers of fibrilar aggregation. In fact, hydrophobicity
of some peptidic structures has been demonstrated to be a key
factor to explain their aggregation properties and, consequently,
aromatic inhibitors have been designed to stop the self-assembly
process.11–15,56–63 Gosh and co-workers have designed a poly-
peptide enriched with aromatic residues, capable to inhibit effi-
Figure 4. ESI-MS spectra: (a) 4 and 8 in MeOH (top: simulated, bottom: experimental);
(b) 4 and 9 in MeOH (top: simulated, bottom: experimental).
ciently the b
-amyloid fibrillization.64 On the other hand, Gazit and
co-workers have demonstrated the importance of aromaticity of
some polyphenols (rather than their antioxidant properties) to ex-
plain their anti-aggregation properties.65 Moreover, although the
presence of aromatic moieties is not a requisite for amyloid as-
sembly, it influences markedly in the kinetics of fibril formation.66
In summary, time-resolved/steady-state fluorescence and mass
spectrometry measurements have demonstrated the preferential
supramolecular binding of naproxen to a synthetic receptor built
with Phe, as compared to another model synthesized with Lys.
4. Experimental section
4.1. Materials
All chemicals were used directly as obtained from commercial
sources: (S)-(þ)-2-(6-Methoxy-2-naphthyl)-propionic acid (nap-
roxen, 98 %, Fluka); Boc-Lys(Z)-OH (Propeptide); Boc-Phe-OH, Boc-
Trp-OH, PyBOP, HOBt were purchased from Senn Chemicals;

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M.I. Burguete et al. / Tetrahedron 65 (2009) 7801–7808
Figure 5. Representative CID spectra in the Elab¼2–8 eV range for the mass-selected [4þ8þNa]þ (left) and [4þ9þNa]þ (right).
spectrometer was calibrated in the positive- and negative-ion ESI
mode. The samples were dissolved in H2O/CH3CN (50/50 v/v). For
the CID experiments a Quattro LC (QhQ quadrupole–hexapole–
quadrupole) mass spectrometer with an orthogonal Z-spray-elec-
trospray interface (Waters) was used. Sample solutions (ca.
1ꢃ10ꢁ4 M) in methanol were introduced through a fused-silica
capillary to the ESI source via a syringe pump at a flow rate of 10 mL/
min. The drying gas as well as nebulizing gas was nitrogen at a flow
of 400 L/h and 70 L/h, respectively. The temperature of the source
block was set to 80 ꢄC and the interface to 120 ꢄC. The capillary
voltage was set at 3.5 kV in the positive scan mode and the cone
voltage was adjusted (typically Uc 15 V) to control the extent of
fragmentation in the source region. The chemical composition of
each peak obtained in the full scan mode was assigned by com-
parison of the isotope experimental and theoretical patterns using
the MassLynx 4.1 program.
FAB mass spectra and HRMS (High Resolution Mass Spectrum)
were recorded on a JEOL JMS DX300-SX 102 in positive mode using
NBA (3-nitrobenzylalcohol) or GT (mixture of glycerol/thioglycerol
50/50 v/v) as matrix.
Figure 6. Collision induced dissociation performed on complexes formed by 4 with 8
(white squares) and 4 with 9 (black squares).
Collision induced dissociation (CID) experiments were per-
formed with argon at various collision energies, in the range of
Elab¼0–15 eV. The collision gas pressure was maintained at ap-
proximately 4ꢃ10ꢁ4 mbar. The most intense precursor peak of in-
terest was mass-selected with Q1, interacted with argon in the
hexapole cell while scanning Q2 to monitor the ionic fragments.
The resolution setting in Q1 (isolation width 3 u) and Q2 was low, in
order to obtain a good signal-to-noise ratio. For a qualitative
analysis of the energy-dependent CID experiments, the laboratory
collision energies (Elab) were converted to the centre-of-mass
frame, ECM¼m/(mþM)Elab, where m and M stand for the masses of
the collision gas and the ionic species, respectively. For the break-
down profiles representation, signal intensities were obtained from
the average of 40 scans and measuring the area of the fragmenta-
tion peaks. These graphs were represented taking into account the
ethylenediamine, DIEA, DMF (spectroscopy grade) were purchased
from Aldrich; methanol (spectroscopy grade, Scharlau). Dichloro-
methane was distilled over CaH2 previous use.
4.2. Instrumentation
1H and 13C NMR analyses were performed with a Bruker Avance
AM300 MHz and are reported in parts per million and calibrated
using residual undeuterated solvents as the internal reference. Data
are reported as: br¼broad, s¼singlet, d¼doublet, t¼triplet,
q¼quartet, m¼multiplet; coupling constant(s) in hertz, integration.
Mass spectra (electrospray ionization mode, ESIMS) were
recorded on a Platform II (Micromass) quadrupole mass spec-
trometer fitted with an electrospray interface. The mass
relative abundance of the precursor and product peaks of each
P
compound (Iprecursor
or Iproduct ion/Iprecursor ionþ Iproduct
)
ion
ion

M.I. Burguete et al. / Tetrahedron 65 (2009) 7801–7808
7807
against ECM. We selected the value of the collision energy required
for 50% reduction of the precursor ion (ECM50%) as a qualitative
measure of the gas-phase stability.
Melting points were measured on a Bu¨chi Melting Point 510
apparatus and are uncorrected.
4.3.2. N1-(2-Aminoethyl)-N6-[(benzyloxy)carbonyl]-N2-(tert-
butoxycarbonyl)- -lysinamide (11)
L
1,2-Ethanediamine (7.0 mL, 0.105 mol), 3.7 mL (21.0 mmol) of
DIEA, 4.0 g (10.5 mmol) of Boc-(Z)-lysine, 2.84 g (21.0 mmol) of
HOBt and 5.47 (10.5 mmol) of PyBOP in 150 ml DMF yielded 1.32 g
Analytical high performance liquid chromatography (HPLC) was
performed on a Waters Millenium 717 equipped with Autosampler,
with a variable wavelength diode detector using a CHROMOLITH
RP18 column (50ꢃ4.6 mm), flow 5 mL/min, linear gradient CH3CN
in water 0–100% (þ0.1% TFA) in 4.5 min.
(30%) of 11 as a colourless oil. 1H NMR (300 MHz; CDCl3)
d 7.34–7.25
(m, 5H), 7.04 (br, 1H), 5.50 (br, 1H), 5.25 (br, 1H), 5.07 (s, 2H), 4.07–
4.01 (m, 1H), 3.32–3.13 (m, 4H), 2.79 (t, 2H, J¼6 Hz), 2.33 (br, 2H),
1.85–1.30 (m, 6H), 1.37 (s, 9H); 13C NMR (300 MHz; CDCl3)
d 173.1,
157.1, 156.3, 136.9, 128.9, 128.4, 80.4, 77.6, 70.0, 53.4, 33.2, 32.4, 29.7,
28.7, 26.2, 25.5, 22.9; ESIMS m/z 423.3 [MþH]þ; FAB(þ) m/z 423.4
[MþH]þ; HRMS calcd for C21HN4O5 [MþH]þ: 423.2607, found
423.2589.
UV–vis absorption measurements were made using a Hewlett-
Packard 8453 spectrophotometer.
Steady-state fluorescence spectra were recorded in a Spex Flu-
orog 3-11 equipped with a 450 W xenon lamp. Time-resolved
fluorescence measurements were done with the technique of time
correlated single photon counting (TCSPC) in an IBH-5000U. Sam-
ples were excited with a hydrogen nanosecond flash lamp. Data
were fitted to the appropriate exponential model after deconvo-
lution of the instrument response function by an iterative decon-
volution technique, using the IBH DAS6 fluorescence decay analysis
software, where reduced c2 (values between 0.90–1.19) and
weighted residuals serve as parameters for goodness of fit.40 All the
samples were measured in aerated conditions.
Fluorescence was measured in the absence and in the presence
of quencher. For the fluorescence titrations, 4 mL of naproxen so-
lution (2–25)ꢃ10ꢁ5 M were placed in a fluorescence cell equipped
with a Teflon stopper and a magnetic stirrer. The solution was ti-
trated with successive additions of the quencher. The final con-
centrations of quenchers were 0.15 M (MeOH) or 0.01 M (DCM).
After each titration the fluorescence spectra and the lifetimes were
recorded. For the steady-state measurements, the excitation
wavelength was set at 331 nm and the maximum value at 350 nm
was taken as a measure of fluorescence intensity. For the time-re-
solved measurements, samples were excited at 325 nm and emis-
sion was monitored at 350 nm.
4.4. General procedure for the synthesis of asymmetric
diacylated diamines 9 and 12
The monoacylated diamine (1 equiv) was dissolved in DMF. To
the resultant solution were added DIEA (2.2 equiv) followed by the
Boc-protected tryptophan (1.1 equiv), 1-hydroxybenzotriazole
(HOBt, 2.2 equiv) and PyBOP (1.1 equiv). The reaction was allowed
to proceed at room temperature until judged complete by HPLC,
typically 24–30 h. DMF was then removed by evaporation under
reduced pressure and the resulting residue was suspended in ethyl
acetate and treated with an aqueous saturated solution of NaHCO3.
Phases were separated and the aqueous one extracted with ethyl
acetate. The organic layers were dried over magnesium sulfate and
rotary evaporated to produce a crude yellow oil, which was purified
by column chromatography (silica, CH2Cl2/MeOH 4:1).
4.4.1. N-(tert-Butoxycarbonyl)-N-(2-ethyl)-L-tryptophanamide (9)
Compound 10 (0.60 g, 1.95 mmol), 0.75 mL (4.29 mol) of DIEA,
0.65 g (2.15 mmol) of Boc-tryptophan, 0.58 (4.29 mmol) of HOBt
and 1.12 g (2.15 mmol) of PyBOP in 22 mL DMF yielded 0.61 g (53%)
of 9 as a white solid. Mp 178–180 ꢄC; 1H NMR (300 MHz; DMSO-d6)
d
10.84 (br s,1H), 7.99 (br s, 2H), 7.62 (d, 2H, J¼9.0 Hz), 7.37–6.98 (m,
9H), 6.90 (d, 1H, J¼9.0 Hz), 6.75 (d, 1H, J¼6 Hz), 4.18–4.11 (m, 2H),
4.3. General procedure for the synthesis of monoacylated
diamines 10 and 11
3.13–2.74 (m, 8H), 1.35 (s, 9H), 1.34 (s, 9H); 13C NMR (300 MHz;
DMSO-d6)
d 173.3, 172.9, 156.4, 139.4, 137.2, 130.4, 129.2, 128.6,
127.3, 124.8, 122.0, 119.7, 119.3, 112.4, 111.5, 79.2, 57.0, 56.4, 39.5,
38.9, 29.3, 29.1; ESIMS m/z 594.1 [MþH]þ; FAB(þ) m/z 594.4
[MþH]þ; HRMS calcd for C32H44N5O6 [MþH]þ: 594.3292, found
594.3285.
Ehtylenediamine (10 equiv) was suspended in DMF. Diisopro-
pylethylamine (2 equiv) was added, followed by the appropriate
Boc-protected aminoacid (1 equiv), 1-hydroxybenzotriazole (HOBt,
2 equiv) and PyBOP (1 equiv). The reaction was allowed to proceed
at room temperature until judged complete by HPLC, typically 24–
30 h. DMF was then removed by evaporation under reduced pres-
sure and the resultant residue was suspended in ethyl acetate and
treated with an aqueous saturated solution of NaHCO3. Phases were
separated and the aqueous one extracted with ethyl acetate. The
organic layers were dried over magnesium sulfate and rotary
evaporated to produce a crude yellow oil which was purified by
column chromatography (silica, CH2Cl2/MeOH 4:1).
4.4.2. N-(2-{[N6-[(Benzyloxy)carbonyl]-N2-(tert-butoxy-
carbonyl)-
carbonyl)-
L
-lysyl]amino}ethyl)-N-(tert-butoxy-
-tryptophanamide (12)
L
Compound 11 (0.93 g, 2.20 mmol), 0.84 mL (4.84 mol) of DIEA,
0.74 g (2.42 mmol) of Boc-tryptophan, 0.65 (4.84 mmol) of HOBt
and 1.26 g (2.42 mmol) of PyBOP in 40 mL DMF yielded 1.05 g (67%)
of 12 as a white solid. Mp 157–160 ꢄC; 1H NMR (300 MHz; CDCl3)
d
9.16 (br, 1H), 7.68 (d, 1H, J¼6 Hz), 7.41–7.04 (m, 9H), 6.38 (br, 1H),
5.75 (br, 1H), 5.26 (br, 1H), 5.17–5.15 (m, 1H), 5.10 (s, 2H), 5.06–5.04
(m, 1H), 4.44 (br, 1H), 3.85–3.83 (m, 1H), 3.35–2.99 (m, 8H), 1.82–
1.60 (m, 2H), 1.50–1.10 (m, 4H), 1.46 (s, 9H), 1.43 (s, 9H); 13C NMR
4.3.1. N1-(2-Aminoethyl)-N-(tert-butoxycarbonyl)-
L-
phenylalaninamide (10)
1,2-Ethanediamine (5 mL, 0.076 mol), 2.6 mL (15.1 mmol) of
DIEA, 2 g (7.55 mmol) of Boc-phenylalanine, 2.04 g (15.1 mmol) of
HOBt and 3.93 g (7.55 mmol) of PyBOP in 50 mL DMF yielded 0.72 g
(31%) of 10 as a white solid. Mp >260 ꢄC; 1H NMR (300 MHz; CDCl3)
(300 MHz; CDCl3) d 173.1, 172.6, 157.1, 156.2, 155.8, 136,9, 136.6,
128.9, 128.5, 128.4, 127.8, 123.6, 122.6, 120.0, 119.4, 111,9, 111.1, 80.6,
80.4, 77.6, 67.1, 55.8, 54.5, 40.9, 39.8, 39.4, 32.6, 29.8, 29.2, 28.7, 28.7,
22.8; ESIMS m/z 709.5 [MþH]þ; FAB(þ) m/z 709.6 [MþH]þ; HRMS
calcd for C37H53N6O8 [MþH]þ: 709.3925, found 709.3906.
d
8.15–8.11 (m, 1H), 7.47 (br, 2H), 7.34–7.21 (m, 5H), 7.01 (d, 2H,
J¼6 Hz), 4.16–4.13 (m, 1H), 3.34–3.27 (m, 2H), 3.03 (dd, 1H,
J0¼15 Hz, J00¼3 Hz), 2.84–2.76 (m, 3H), 1.34 (s, 9H); 13C NMR
4.4.3. N-(tert-Butoxycarbonyl)-N-(2-{[N2-(tert-butoxycarbonyl)-
lysyl]amino}ethyl)- -(tryptophanamide) (8)
L-
(300 MHz; DMSO-d6)
d
172.3, 155.2, 137.9, 129.1, 128.0, 126.2, 78.3,
L
55.7, 38.7, 37.4, 36.7, 28.0; ESIMS m/z 308.3 [MþH]þ; FAB(þ) m/z
308.3 [MþH]þ; HRMS calcd for C21HN4O5 [MþH]þ: 308.1974, found
308.1976.
A solution of 0.91 g of the Z-protected amine derivative 12 was
prepared in 60 mL of methanol degassed with argon. Under a pos-
itive argon flow, 92 mg of Pd-black catalyst was slowly added to the

7808
M.I. Burguete et al. / Tetrahedron 65 (2009) 7801–7808
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d 9.54 (br,
´
1H), 7.75–7.72 (br, 1H), 7.45 (d, 1H, J¼9 Hz), 7.26–7.07 (m, 4H), 5.35
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3.37–3.14 (m, 4H), 2.95–2.78 (m, 2H), 2.52 (m, 2H), 1.86 (br, 2H),
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Acknowledgements
Financial support from the Spanish MICINN (CTQ2006-15672-
C05-02, CTQ2008-02907-E/BQU), GVA (projects GV/2007/277,
ARVIV/2007/079, ARVIV/2007/081), Fundacio Caixa Castello-UJI
(projects P1$1B2004-13, P1$1A2007-05) is acknowledged. F.G.
thanks the support from MICINN (Ramon y Cajal Program). Tech-
´
´
´
40. Burguete, M. I.; Izquierdo, M. A.; Galindo, F.; Luis, S. V. Chem. Phys. Lett. 2008,
460, 503–506.
nical support by SCIC/UJI (Cristian Vicent) is also acknowledged.
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Supplementary data
1H, 13C NMR and ESI-MS spectra of synthesized compounds.
Supplementary data associated with this article can be found in the
online version, at doi:10.1016/j.tet.2009.07.031.
45. Fricke, H.; Gerlach, A.; Unterberg, C.; Wehner, M.; Schrader, T.; Gerhards, M.
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