Fluorescent macrocyclic probes with pendant functional groups as markers of acidic organelles within live cells

Article abstract of DOI:10.1039/c3ob41773e

A new family of acidity sensitive fluorescent macrocycles has been synthesized and fully characterized. Their photophysical properties including emission quantum yield and fluorescence lifetime have been determined. The acid-base properties of the new molecules can be tuned by the incorporation of pendant functional groups. The nature of such functional groups (carboxylic acid or ester) influences dramatically the pK_a of the probes, two compounds of which exhibit low values. Preliminary intracellular studies using confocal microscopy together with emission spectra of the probes from the cellular environment have shown that the synthesized fluorescent macrocycles mark the acidic organelles of RAW 264.7 macrophage cells.

Full text of DOI:10.1039/c3ob41773e

Organic &
Biomolecular Chemistry
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PAPER
Fluorescent macrocyclic probes with pendant
functional groups as markers of acidic organelles
within live cells†
Cite this: Org. Biomol. Chem., 2014,
12, 823
Prashant D. Wadhavane,a M. Ángeles Izquierdo,a Dennis Lutters,a
M. Isabel Burguete,a María J. Marín,b David A. Russell,b Francisco Galindo*a and
Santiago V. Luis*a
A new family of acidity sensitive fluorescent macrocycles has been synthesized and fully characterized.
Their photophysical properties including emission quantum yield and fluorescence lifetime have been
determined. The acid–base properties of the new molecules can be tuned by the incorporation of
pendant functional groups. The nature of such functional groups (carboxylic acid or ester) influences dra-
matically the pKa of the probes, two compounds of which exhibit low values. Preliminary intracellular
studies using confocal microscopy together with emission spectra of the probes from the cellular
environment have shown that the synthesized fluorescent macrocycles mark the acidic organelles of RAW
264.7 macrophage cells.
Received 30th August 2013,
Accepted 13th November 2013
DOI: 10.1039/c3ob41773e
www.rsc.org/obc
A number of strategies have been followed in order to
develop fluorescent pH probes for bioimaging of cellular com-
Introduction
Fluorescence imaging of intracellular species is a field in con- ponents, in particular for acidic environments.13–18 Substi-
tinuous development.1–3 The number of molecules able to fluo- tution at the fluorophore core with electron withdrawing
resce selectively in the presence of specific species of interest substituents such as chloride and fluoride has been developed
such as cations, anions, radicals and neutral molecules has to lower the pKa values of well-established near-neutral pH
grown considerably over the last decades.4–9 Protons are probes such as fluorescein and other xanthene derivatives.19,20
perhaps the simplest analyte to target, but despite their simpli- This strategy involves significant modifications of the synthetic
city the role that pH plays in the biological realm is of para- procedures and, sometimes, unpredictable results of the final
mount importance.10 The concentration of protons determines pKa values of the resulting compounds. In addition, the substi-
the correct function of the cellular machinery, while un- tution pattern of the fluorophore can often modify the absorp-
balanced pH is linked to many diseased states. For instance, tion and emission properties, requiring a modification of the
Alzheimer’s disease, lysosomal storage diseases, renal tubular experimental protocols established for the imaging of the
acidosis, diabetes, inflammation and cancer are disorders in parent compounds.
which cellular pH lies far from the normal levels.11 In many
A different strategy for designing fluorescent pH indicators
cases, the endosomal–lysosomal pathway (working at acidic for acidic organelles is based on the synthetic modification
pH, between 4 and 6) is malfunctioning, which implies a of anthrylmethylamines.21–26 The steric hindrance of the bulky
defective degradation of waste material, or an inadequate work and hydrophobic anthracene moiety contributes to the lower
of proton pumps in the membranes of the acidic organelles.12
basicity of this family of pH sensors as compared to other
amines containing smaller aromatic subunits or alkyl substitu-
ents. This characteristic makes them suitable for the measure-
ment of pH in acidic environments and sets the basis for the
development of, for example, the commercial LysoSensors
DND-192 and DND-167. We have followed a similar strategy to
develop a family of anthracene derivatives of pseudopeptidic
nature.27,28 Structural modifications of 9,10-disubstituted
anthracenes resulted in compounds exhibiting identical
absorption and emission spectral parameters except for the
fluorescent quantum yield, which is higher at acidic pH and
aUniversitat Jaume I, Departamento de Química Inorgánica y Orgánica, Av. Sos
Baynat, s/n, E-12071 Castelló de la Plana, Spain. E-mail: francisco.galindo@uji.es,
luiss@uji.es; Tel: +34 964 72 8236
bSchool of Chemistry, University of East Anglia, Norwich, Norfolk NR4 7TJ, UK.
Tel: +44 (0)1603 593012
†Electronic supplementary information (ESI) available: 1H and 13C NMR and
mass spectra of compounds 1–4; absorption and emission spectra of com-
pounds 1–4; fluorescence decay traces of compounds 1–4; distribution and fluo-
rescence emission spectra of compounds 1 and 2 in RAW 264.7 macrophage
cells. See DOI: 10.1039/c3ob41773e
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depends on the chemical nature of the substituents. Biological
experiments with RAW 264.7 macrophage cells and human
monocytes demonstrated that the anthracene derivatives of
pseudopeptidic nature could be used to study lysosomal struc-
tures within living cells by means of confocal fluorescence
microscopy and flow cytometry.27,28 The work here presented
reports on a further development of such a set of pseudopepti-
dic probes. The new compounds here described bear an
additional pendant arm consisting of an acid or an ester
group, which significantly modifies their acid–base properties,
providing pH probes displaying lower pKa values than related
systems, and also enables future post-functionalization and
modification of their solubility properties. The introduction of
such functionalities is not trivial from the synthetic perspec-
tive. The synthesized anthracene derivatives have been chemi-
Scheme 1 Synthesis of the anthracenophanes functionalized within
cally and photophysically characterized in solution and have the methylene bridge (1–4). Key: (i) DCC, N-hydroxysuccinimide, THF, rt,
6
h; (ii) NH2(CH2)2NH(CH2)2NH2, DME, 45 °C (18 h), rt (6 h); (iii)
BrCH2CO2Me, K2CO3, dry DMF, 50 °C, 24 h; (iv) H2/Pd, dry THF, 50 °C,
h; (v) 9,10-bis-(bromomethyl)-anthracene, K2CO3, CH3CN, reflux,
been successfully tested as markers of acidic organelles in
RAW 264.7 macrophage cells.
4
24 h; and (vi) LiOH, 2 : 1 ( v/v) H2O–THF (2 : 1), rt, 24 h.
Results and discussion
Synthesis
its substituents, on the basicity and solubility properties of the
compounds; and (2) to evaluate their intracellular performance
as fluorescent markers for acidic organelles in living cells
using confocal fluorescence microscopy. Further, the new syn-
thetic strategy herein reported allows the successful introduc-
tion of additional functional groups in the polymethylene
bridge and sets the basis for the future development of new
series of derivatives via anchoring other moieties or
fluorophores.29
Different pseudopeptidic macrocyclic structures containing an
anthracene moiety have been reported previously by our
group.27,28 An important example is the valine derivative 5
(Chart 1), which has been used as the reference molecule for
this work. Variations in the macrocyclic architecture, such as
the ring size or the nature of the side chain, have useful effects
in the analytical performance of the macrocycles as fluorescent
probes. Thus, it is possible to smoothly change the pKa of the
probes by 0.1–0.2 units, only by adding, for example, some
strain to the macrocycle. This small effect could also be
achieved by changing the hydrophobic character of the com-
pound by varying the amino acid side chain. This fine tuning
ability is of great interest in the area of sensor development
since chemical changes performed with other probes can lead
to drastic variations of the fluorescent properties (for instance
in the fluorescein family of pH probes).19,20 Taking the fine
tuning ability into account, macrocycles 1–4 (Chart 1) were
designed and synthesized with a double objective: (1) to study
the effect of a third nitrogen atom in the macrocyclic ring, and
Following a standard methodology developed in our group,
compounds 1–4 were synthesized starting from the corres-
ponding Cbz-protected amino acid (valine (Val) or phenyl-
alanine (Phe)) which was activated at the C end by means of
N-hydroxysuccinimide/N,N′-dicyclohexylcarbodiimide (DCC) and
further reacted with diethylenediamine to afford intermediates
8 and 16 (see Scheme 1). The central nitrogen of the bridge
was then derivatized with methyl bromoacetate to afford inter-
mediates 9 and 17 with good yields (75% and 76%, respect-
ively). After deprotection of the Cbz groups the diamines 10
and 18 were obtained. These amines were reacted with 9,10-
bis-(bromomethyl)-anthracene to afford the macrocyclic com-
pounds 1 and 3 in 60–70% yield. Although moderate, these
yields can be considered as an excellent result considering
that no high dilution methods were employed. As we have pre-
viously studied,30 the reason for such efficient macrocycliza-
tion could reside in the preorganized conformation of the
reactive intermediates favoured by intramolecular hydrogen
bonding and solvophobic effects, as well as by a kinetic tem-
plate effect associated to the presence of halide anions.31 In
the synthesis reported here, the presence of an alkylated nitro-
gen atom in the methylene bridge seems not to have a negative
effect on the preorganization needed for the macrocyclization.
Once the macrocycles with an ester pendant arm were
obtained, the next step was the hydrolysis of these compounds
with LiOH to yield the corresponding carboxylates. Isolation of
Chart 1 New anthracenophanes functionalized within the methylene
bridge (1–4) and reference compound 5.
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the compounds 2 and 4 required the utilization of exchange
resins with a strong acidic character and final elution with an
ammonia solution.32 The valine derivative 2 was obtained in a
higher yield than the phenylalanine derivative 4, probably due
to the lower solubility of the more hydrophobic derivative 4 in
the aqueous solvents needed for the ion exchange purification.
At the end of the synthesis, several hundreds of milligrams of
each macrocycle 1–4 were ready for chemical and photo-
physical characterization.
Table 1 Photophysical features of compounds 1–4 and reference
compound 5
Probea
λabs b (nm)
λem b,c (nm)
ϕmax
ϕmin
τ
b,e (ns)
b
d
1
2
3
359, 378, 400
359, 378, 398
359, 378, 400
359, 378, 400
358, 376, 397
405, 426, 450
404, 426, 451
405, 427, 451
405, 427, 453
405, 427, 453
0.75
0.73
0.77
0.78
0.77
0.017
0.010
0.031
0.020
0.009
13.3
13.0
13.7
13.3
13.0
4
5f
a Probe concentration (2 µM) in H2O (0.2% DMSO). b Measured at pH
1.7. c λexc = 374 nm. d Measured at pH 8. e λexc = 372 nm, λem = 420 nm.
f Data obtained from the literature.28
Characterization
Compounds 1–4 were characterized by means of Nuclear Mag-
netic Resonance spectroscopy (1H and 13C NMR), Electro-Spray
Ionization Mass Spectrometry (ESI-MS) and Fourier Transform
Infrared Spectroscopy (FTIR) confirming the structures
depicted in Chart 1. As described previously for other related
systems, the 1H NMR signals corresponding to the CH2 at posi-
tions 9 and 10 of the anthracene moiety appear as two well
defined doublets in the region located between 4.5 and 5 ppm,
while the signal for the amide protons appear at 6.8 ppm for 1,
2 and 4; and 6.6 ppm for 3 (see ESI Fig. S4, S5, S9 and S10† for
compounds 1–4, respectively). These features constitute a
characteristic landmark of this family of pseudopeptidic
anthracene derivatives and indicate that the essential struc-
tural features are not affected by the introduction of a functio-
nalized third nitrogen atom in the central spacer.
The UV-visible spectra of the macrocycles display the
typical absorption of 9,10-disubstituted anthracene derivatives
below 400 nm (see ESI Fig. S11A†). Thus, the presence of the
pendant arms does not affect this feature; hence all of the
spectra are almost identical and also match that of the refer-
ence compound 5.
The fluorescence of the new macrocycles was recorded as a
function of the acidity of the medium. Hence, pH titrations
were conducted affording intensity variations as expected,
i.e. strong emission at acidic values and low fluorescence in
the neutral-alkaline environment (Fig. 1A). This phenomenon
has been interpreted by the classical photoinduced-electron
transfer (PET) mechanism extensively described in the
literature.22–26,33–35 PET involves excitation of the anthracene
moiety and fast quenching of the excited state by the neigh-
bouring amines in their unprotonated form (in neutral-
alkaline media). Protonation of the amines results in the dis-
ruption of the PET and hence fluorescence enhancement in
acidic media.
One of the striking features of the macrocycles described
herein is the difference that exists between the on (fluorescent)
and off (quenched) states, which is not commonly reported in
the literature of pH sensors.36–41 The fluorescence quantum
yields at pH 1.7 are in the range 0.73–0.78 whereas at pH 8.0
these values are substantially reduced to 0.010–0.031 (Table 1).
This feature is ideal for obtaining microscopy images in which
a high contrast between the bright (lower pH) and dark (high
pH) areas of organelles within cells is desirable. The fluo-
rescence emission spectra of the macrocycles 1–4 also exhibit
the characteristic pattern of 9,10-disubstituted anthracene
derivatives as shown in Fig. S11B.†
Time resolved fluorescence spectroscopy has also been
used to characterize the singlet excited state of the new probes,
providing emission lifetimes ranging between 13.0 and 13.7 ns
(Table 1 and Fig. S12†). These values are in agreement with
the one previously reported for the reference compound 5,
which indicates that the attachment of functionalities has no
effect on the emission lifetime of the compounds.
From the pH fluorescence titrations, the intensity vs. pH
profiles (Fig. 1B) were fitted to eqn (1) to calculate the corres-
ponding pKa values related to fluorescence quenching
(Table 2). It should be noted that these pKa values are related
to the first deprotonation of one ammonium group located in
proximity to the anthracene moiety, in the fully protonated
macrocycle (polyammonium species). Once one of such amine
is free, the electron pair located at that position is able to
undergo PET and so quench the emission of the probe. Fig. 1B
shows that there are two types of macrocycles according to the
position of the titration curve: (1) those with the ester group
as the pending functionality, with calculated pKa values
ca. 3.0–3.2; and (2) those probes with a carboxylic pending
group, with calculated pKa ca. 4.4–4.8. For comparison, the
curve of the reference compound 5 (pKa 5.06) has also been
plotted in Fig. 1B.
ðImax ꢀ IÞ
log
¼ pH ꢀ pKa
ð1Þ
ðImin ꢀ IÞ
Fig. 1 (A) Fluorescence emission spectra of compound 3 as a function
of the pH. (B) Fluorescence titration curves of compounds 1–4 and
reference compound 5. Experimental conditions: probe concentration
(2 µM) in aqueous solution (0.2% DMSO); λexc = 374 nm.
Compounds 1 and 3 differ from the parent compound 5 in
the number of amines of the macrocycle (considering that the
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Table 2 Calculated pKa values for compounds 1–5 using the fluo-
rescence titration curves and eqn (1)
Compound
pKa
1
2
3
3.21
4.83
3.04
4.45
5.06
4
5a
a Data obtained from the literature.28
ester group is playing a negligible effect on the acid–base equi-
libria). Hence, the differences between the calculated pKa
values (5.06 vs. 3.0–3.2) can be rationalized by considering that
the new compounds are triamines whereas the reference is a
diamine. It is well established in polyamine chemistry that the
higher the number of amines in a system, the greater the
difficulty to fully protonate the compound due to electrostatic
repulsion between positive charges. This is particularly true
when macrocyclic polyamines are considered, as the lack
of conformational freedom makes the relaxation of this
Coulombic repulsion difficult.42
The hydrolysis of the ester group in 1 and 3 to yield mole-
cules 2 and 4 has a dramatic effect on the pKa of the resulting
compounds. Thus, valine derivative 2 displays a pKa of 4.83 in
contrast with the pKa of the related valine ester 1 (pKa 3.21).
The same effect was observed in 4 (pKa 4.45) vs. 3 (pKa 3.04).
This effect is easily accounted for by charge neutralization due
to the formation of a zwitterion (carboxylate-ammonium) in
the central part of the bridge. This neutralization would
render the pKa of the macrocycles with the acidic functionality
more similar to the pKa of the reference compound 5 (pKa
5.06) than to the pKa of the probes 1 and 3, with the ester
pendant group, i.e. behaving as a diamine rather than as tri-
amines, in agreement with the behaviour reported in the
literature for other compounds containing carboxylate or
methylenephosphonate groups attached to the amino groups
of polyazamacrocycles.43
Fig. 2 Distribution and colocalization experiments with probes 1 and 2
and LysoSensor Green DND-189 in RAW 264.7 macrophage cells. (A)
Fluorescence image of the compound 1, collected with a confocal laser
scanning microscope in the blue channel. (B) Fluorescence image of the
DND-189 probe collected with a confocal laser scanning microscope in
the green channel. (C) Composite images of blue, green and DIC chan-
nels for compound 1. (D) Fluorescence image of the compound 2, col-
lected with a confocal laser scanning microscope in the blue channel.
(E) Fluorescence image of the DND-189 probe collected with a confocal
laser scanning microscope in the green channel. (F) Composite images
of blue, green and DIC channels for compound 2.
ESI†). These experiments confirm that probes 1 and 2 act as
markers of the acidic organelles within RAW 264.7 macrophage
cells.
A final experiment was performed in order to confirm that
the fluorescence emission observed in the blue channel corre-
sponds to the anthracene fluorophore and not to autofluore-
scence from the components of the cellular environment.
Autofluorescence of the cells can lead to significant emission
when the cells are excited with UV light. To discriminate the
autofluorescence of the cells from the fluorescence due to the
molecular probe, the fluorescence emission spectra of the
areas inside the cell with higher fluorescence intensity were
recorded. The shape and position of the spectrum recorded
within the cell was compared to the spectrum of 2 recorded in
solution. This strategy has been recently applied to study the
nature of the intracellular reaction products of 1,2-diamino-
anthraquinone (a fluorescent probe for nitric oxide).45 Also, the
measurement of intracellular spectra has been of great utility
to calculate the pH of localized areas within Chinese hamster
ovary (CHO) cells using a gold nanoparticle based fluorescent
sensor.46,47 Fig. 3A–C show a RAW 264.7 macrophage cell incu-
bated with 2 that displays areas of strong fluorescence emis-
sion when excited with a 364 nm UV laser. The fluorescence
emission spectra of three areas within the bright spot reveal
clearly the anthracenic origin of the emission (Fig. 3D, areas
1–3). Control experiments were performed recording spectra of
darker zones (see Fig. 3D, areas 4–6). More examples of
fluorescence emission spectra recorded from within RAW
264.7 macrophage cells incubated with probe 2 are given in
Fig. S15.† The same study was carried out for probe 1 and
analogous results were obtained (see ESI Fig. S16 and S17†).
Cellular imaging using confocal microscopy
Once the basic photophysical and analytical features of the
new compounds were established, their potential use as cell
imaging probes was studied. Intracellular studies with
compounds 1 and 2 were performed. Thus, a culture of RAW
264.7 macrophage cells was incubated with these compounds
and simultaneously with the marker of acidic organelles Lyso-
Sensor Green DND-189. The tested molecules were readily
uptaken by the cells, possibly via a plasma membrane carrier,
with consideration of their polyamine nature, as previous
examples described in the literature.44 The images in Fig. 2
show that the fluorescence from the blue channel (corres-
ponding to the emission of the anthracene, Fig. 2A and 2D)
and from the green channel (fluorescence due to the LysoSen-
sor Green DND-189, Fig. 2B and 2E) appear to co-locate. The
same pattern was observed repeatedly in a number of cells (see
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for 13C). Chemical shifts for all the compounds are reported in
ppm (δ).
Mass spectrometry. Mass spectra were recorded on a hybrid
QTOF I (quadrupole–hexapole–TOP) mass spectrometer with
an orthogonal Z-spray-electrospray interface. Nitrogen was
used as the desolvation and the nebulizing gas at a flow of
700 L h−1 and 20 L h−1, respectively. The temperature of the
source block was set to 120 °C and the desolvation tempera-
ture to 150 °C. A capillary voltage of 3.5 and 3.3 kV was used in
the positive and negative scan mode, respectively. The cone
voltage was typically set to 20 V to control the extent of frag-
mentation of the identified ions. Sample solutions were
infused via a syringe pump directly connected to the SI source
at a flow rate of 10 L min−1. The observed isotopic pattern of
each intermediate matched perfectly to the theoretical isotope
pattern calculated from their elemental composition equipped
with an electrospray ionization source and a triple-quadrupole
analyzer.
Fig. 3 Intracellular emission spectra of probe 2. (A) DIC image was col-
lected with a confocal laser scanning microscope in the DIC mode. (B)
Fluorescence image of 2 was collected with a confocal laser scanning
microscope in the blue channel. (C) Composite image of blue and DIC
channels. (D) Fluorescence emission spectra of compound 2 inside the
cells selecting different areas within the cells.
UV-Vis experiments. UV-visible absorption measurements
were made using a UV-visible spectrophotometer. All samples
were measured in aerated conditions unless otherwise stated.
Steady-state fluorescence spectroscopy. Steady-state fluo-
rescence spectra were recorded with a Spex Fluorog 3-11
equipped with a 450 W xenon lamp. Fluorescence spectra were
Conclusions
Four new fluorescent macrocycles with high structural com- recorded in the front face mode. The samples were measured
in aerated conditions, unless otherwise stated.
Time-resolved fluorescence spectroscopy. Time-resolved
plexity have been synthesized, characterized and their photo-
physical properties, including emission quantum yields and
fluorescence lifetimes, have been determined. The sensitivity fluorescence measurements were performed using the tech-
nique of time correlated single photon counting (TCSPC) in an
IBH-5000U. The samples were excited using a 372 nm
towards pH has been studied, with calculation of the pKa
values of the new probes. Preliminary intracellular studies
using RAW 264.7 macrophage cells have demonstrated that the NanoLED with a FWHM of 1.3 ns at repetition rate of 100 kHz.
Data were fitted to the appropriate exponential model after
deconvolution of the instrument response function by an itera-
new macrocycles can be used as markers of acidic organelles,
as demonstrated by colocalization with a known lysosomal
probe. Additionally, the fluorescence emission spectra, tive deconvolution technique, where reduced χ2 and weighted
residuals serve as parameters for the goodness of fit. The
samples were measured in aerated conditions, unless other-
recorded directly from within the live cells, have confirmed the
identity of the probe responsible for the fluorescence. Of
important significance is the fact that the introduction of an wise stated.
Sample preparation for photophysical characterization.
ester group as a pendant arm far from the fluorescent moiety
has led to a dramatic shift of the apparent pKa of the probe
towards acidic pH.
Stock solutions of compounds 1–4 (1 mM) were prepared in
DMSO. The stock solutions were diluted in H2O to a final con-
centration of 2 µM. The pH of the diluted solutions was
adjusted by adding aliquots of HCl and NaOH at different
concentrations.
Fluorescence quantum yield measurements. Fluorescence
quantum yields (ϕs) of compounds 1–4 in water, upon exci-
tation at 372 nm, were reported relative to 5 (ϕr = 0.77).28 The
experiments were performed using optically matched solutions
and the quantum yields were calculated using eqn (2),
Experimental section
Chemicals and starting materials
The solvents and reagents were used as received, without
further purification. 9,10-Bis(bromomethyl)-anthracene was
synthesized using a procedure described in the literature.27
Compounds 7 and 15 were synthesized using a procedure deve-
loped in our group and were obtained with similar yields.30
2
ArFs ηs
AsFr ηr
ϕs ¼ ϕr
ð2Þ
2
Methods and techniques
where As and Ar are the absorbance of the sample and refer-
NMR spectroscopy. 1H and 13C NMR spectra were recorded ence solutions, respectively, at the same excitation wavelength;
on a 500 MHz spectrometer (500 MHz for 1H and 125 MHz for Fs and Fr are the corresponding relative integrated fluorescence
13C) or a 300 MHz spectrometer (300 MHz for H and 75 MHz intensities of the sample and the reference; and ηs and ηr are
1
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the refractive indexes of the solvent used to measure the between 378 and 549 nm exciting the sample with a 364 nm
sample and the reference.
UV laser. The fluorescence emission intensity was measured in
spectral bands each of 10.8 nm width. Images and spectra
were processed using the Zeiss LSM Image Browser Version
Intracellular studies
Imaging medium. Imaging medium was prepared in 4.2.0.121 software.
analytical reagent grade water containing sodium chloride
Imaging of RAW 264.7 macrophage cells loaded with 1 or 2
(120 mM), potassium chloride (5 mM), calcium chloride and LysoSensor Green DND-189. Imaging of RAW 264.7 macro-
(2 mM), magnesium chloride (1 mM), sodium dihydrogen phage cells loaded with both the compounds 1 or 2 and the
phosphate (1 mM), sodium hydrogen carbonate (1 mM) and LysoSensor Green DND-189 was achieved by exciting the cells
4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES, at two different wavelengths. As previously described, the cells
25 mM). The pH of the imaging medium was adjusted to pH were excited with a 364 nm UV laser to collect the emission
7.2. The imaging medium was supplemented with bovine from 1 or 2. The fluorescence emission was collected between
serum albumin (1 mg mL−1), basal medium Eagle amino acid 383 and 447 nm (blue channel). To collect the fluorescence
(2%), glutamine (2 mM) and glucose (11 mM). The imaging emission due to the LysoSensor Green DND-189, the cells were
medium was sterilized by filtration through a Millex® GP excited with a 488 nm argon-ion laser and the fluorescence
syringe driven filter unit (0.22 µm) prior to use.
emission was collected between 505 and 550 nm (green
Cell culture. The mouse macrophage cell line RAW 264.7 channel). DIC images were collected simultaneously using a
was cultured in Dulbecco’s Modified Eagle’s Medium (1×) con- 488 nm argon-ion laser.
taining 4.5 g L−1 D-glucose (DMEM) supplemented with 1%
Synthesis and characterization of organic compounds
L-glutamine (200 mM), 1% penicillin–streptomycin (100 U
mL−1 and 100 µg mL−1, respectively) and 10% fetal calf serum.
Synthesis of 8. To a clear solution of the N-hydroxysuccini-
The cells were grown at 37 °C in a 7.5% CO2 atmosphere in mide ester of N-Cbz-L-valine 7 (30.0 g, 86.12 mmol) in
Nunc Easy flasks with porous caps. Subcultures (1 : 3) were anhydrous dimethoxyethane (DME, 250 mL) at 0 °C, diethyl-
made every 3 days by dislodging the cells from the flask enetriamine (4.46 g, 43.06 mmol) dissolved in dry DME (20 mL)
surface using a cell scraper. For imaging, RAW 264.7 macro- was added in a dropwise manner. The reaction mixture was
phage cells were cultured on 18 mm diameter glass coverslips stirred at room temperature for 18 h and then was heated to
a day prior to performing the imaging experiments. Cells were 40–50 °C for 6 h. The white solid formed was filtered and
harvested from the Nunc Easy flask using a cell scraper and washed with cold water and cold methanol (23.27 g, 0.04 mol,
placed on a sterile coverslip in each well of a 6-well Nunc 94% yield): mp 175–176 °C; [α]2D5 = 11.39° (c 0.1, CHCl3); IR
1
multidish. The cells were cultured in supplemented DMEM at (ATR) 3285, 2957, 1685, 1649, 1535 cm−1; H NMR (500 MHz,
37 °C in a 7.5% CO2 atmosphere overnight.
DMSO-d6) δ 7.83 (s, 2H), 7.38–7.19 (m, 12H), 5.03 (s, 4H), 3.79
Incubation of RAW 264.7 macrophage cells with 1 or 2. The (m, 2H), 3.10 (dd, 4H, J = 12.6, 6.1 Hz), 2.51 (m, 4H), 1.92 (m,
RAW 264.7 macrophage cells were loaded with 1 (50 µM, 0.5% 2H), 0.83 (d, 12H, J = 6.1 Hz); 13C NMR (75 MHz, DMSO-d6)
DMSO) or 2 (50 µM, 0.5% DMSO) and incubated at 37 °C in a δ 174.9, 171.8, 156.8, 137.8, 128.9, 128.3, 66.1, 61.1, 48.9, 30.9,
7.5% CO2 environment for 20 min. Stock solutions of com- 25.7, 19.9, 18.9; HRMS (ESI-TOF)+ (m/z) calcd for C30H43N5O6
pounds 1 and 2 (10 mM in DMSO) were used to load the cells.
Incubation of RAW 264.7 macrophage cells with 1 or 2 and H2O: C, 61.3; H, 7.7; N, 11.9. Found: C, 61.4; H, 7.8; N, 11.9.
LysoSensor Green DND-189. After incubating the RAW Synthesis of 9. A mixture of compound (10 g,
(M + H+) 570.3292, found 570.3296. Anal. calcd for C30H43N5O6
8
264.7 macrophage cells with compound 1 or 2 for 20 min, 17.55 mmol), anhydrous K2CO3 (24.25 g, 175.5 mmol) and
LysoSensor Green DND-189 (2 µM, 0.2% DMSO) was added to methyl bromoacetate (2.68 g, 19.30 mmol) was placed in a
the cells. The cells were incubated at 37 °C in a 7.5% CO2 flask containing dry dimethylformamide (DMF, 10 mL) and
atmosphere for 5 min.
heated to 50 °C for 24 h under a nitrogen atmosphere. The
Imaging of RAW 264.7 macrophage cells loaded with 1 or reaction was monitored by TLC. After complete consumption
2. For imaging, 18 mm coverslips containing the RAW of the starting material, distilled water was added (30 mL).
264.7 macrophage cells were placed in a Ludin chamber (Life This solution was extracted by using ethyl acetate (EtOAc,
Imaging Service, Olten, Switzerland) which was securely tigh- 30 mL, ×3). The organic phase was dried over anhydrous
tened. The cells on the coverslip were washed three times with MgSO4 and evaporated under vacuum. The product was puri-
the imaging medium and the Ludin chamber was mounted on fied by silica flash chromatography using MeOH–CH2Cl2
a heating stage at 37 °C in a Carl Zeiss LSM510 META confocal (1 : 50) to give the white solid 9 (8.19 g, 12.80 mmol, 75%
laser scanning microscope. The images were acquired with a yield): mp 156–158 °C; [α]2D5 = −10.45° (c 0.1, CHCl3); IR (ATR)
Plan-Neofluar ×40, a 1.3NA oil immersion objective. The cells 3300, 3064, 3031, 2949, 2869, 2360, 1736, 1685, 1644,
were excited with a 364 nm UV laser and the fluorescence 1532 cm−1 1H NMR (500 MHz, DMSO-d6) δ 7.77 (s, 2H),
;
emission was collected above 385 nm. Differential interference 7.39–7.24 (m, 10H), 7.17 (d, 2H, J = 8.8 Hz), 5.00 (m, 4H), 3.78
contrast (DIC) images were collected simultaneously using a (t, 2H, J = 7.9 Hz), 3.58 (s, 3H), 3.37 (d, 2H), 3.10 (m, 4H), 2.63
488 nm argon-ion laser. The fluorescence emission spectra (m, 4H), 1.91 (m, 4H), 0.82 (m, 12H); 13C NMR (126 MHz,
of the samples were obtained using a lambda scan mode CDCl3) δ 173.73, 171.50, 139.76, 129.31, 128.59, 127.47, 67.53,
828 | Org. Biomol. Chem., 2014, 12, 823–831
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55.69, 54.98, 53.56, 51.53, 37.45, 31.24, 19.49, 17.96; HRMS exchange resin and deionized H2O. The eluents used for the
(ESI-TOF)+ (m/z) calcd for C33H47N5O8 (M + H+) 642.3503, resin column purification were: H2O (100 mL), 1 : 1 THF–H2O
found 642.3508. Anal. calcd for C33H47N5O8: C, 61.8; H, 7.4; (100 mL), 5% NH3 in H2O and then, 10% NH3 in H2O. The
N, 10.9. Found: C, 61.7; H, 7.5; N, 10.9.
compound was isolated in the range of 5–10% NH3 in H2O
Synthesis of 10. To a clear solution of compound 9 (2.00 g, solutions. Then the water was evaporated under vacuum to
3.11 mmol) in dry tetrahydrofuran (THF), 10 mol% of the cata- yield the yellowish solid 2 (0.103 g, 0.182 mmol, 35% yield):
lyst (5 wt% Pd on activated carbon) was added. The reaction mp 105–107 °C; [α]2D5 = −85.25 (c 0.1, CHCl3); IR (ATR) 3428,
mixture was stirred under an atmosphere of H2 (H2 balloon) at 3149, 2965, 2871, 1651, 1568 cm−1; 1H NMR (500 MHz, DMSO-
50 °C for 4 h. The reaction was monitored by TLC, and after d6) δ 8.44 (m, 4H), 7.52 (m, 4H), 6.76 (s, 2H), 4.88 (d, 2H, J =
complete consumption of the starting material the catalyst was 13.9 Hz), 4.60 (d, 2H, J = 13.7 Hz), 2.98 (m, 2H, J = 4.6 Hz), 2.84
filtered off through a Celite® bed and washed with THF. The (s, 2H), 2.00 (m, 6H), 1.80 (m, 2H), 0.94 (d, 6H, J = 6.9 Hz), 0.74
crude product was purified by column chromatography using (d, 6H, J = 6.9 Hz); 13C NMR (126 MHz, DMSO-d6) δ 173.2,
MeOH–CH2Cl2 (1 : 15) as an eluent, to which NH3 (20 µL) was 132.8, 129.9, 125.8, 125.6, 68.8, 53.2, 52.7, 45.2, 37.1, 31.2,
also added at every addition of the eluent throughout the 19.9, 18.3; HRMS (ESI-TOF)+ (m/z) calcd for C32H43N5O4
column. Evaporation of the solvent under vacuum yielded the (M
+
H+) 562.3294, found 562.3298. Anal. calcd for
green oil 10 (0.462 g, 1.24 mmol, 80% yield): [α]2D5 = 6.26° C32H43N5O4·H2O: C, 66.3; H, 7.8; N, 12.0. Found: C, 66.3; H,
(c 0.1, CHCl3); IR (ATR) 3289, 3073, 2963, 2869, 1736, 1640, 7.7; N, 12.2.
1
1526 cm−1; H NMR (500 MHz, CDCl3) δ 7.50 (s, 2H), 3.26 (m,
Synthesis of 16. To a clear solution of the N-hydroxysuccini-
4H), 3.09 (m, 2H), 2.66 (m, 4H), 2.10 (m, 2H), 1.59 (m, 5H), mide ester of N-Cbz-L-phenyl 15 (30.0 g, 86.12 mmol) in anhy-
0.87 (d, 6H, J = 6.8 Hz), 0.74 (d, 6H, J = 6.8 Hz); 13C NMR drous DME (250 mL) at 0 °C, diethylenetriamine (4.46 g,
(75 MHz, CDCl3) δ 174.3, 171.8, 68.2, 54.8, 54.1, 51.0, 43.4, 43.06 mmol) dissolved in dry DME (20 mL) was added in a
36.9, 31.2, 19.6, 17.9, 15.2; HRMS (ESI-TOF)+ (m/z) calcd for dropwise manner. The reaction mixture was stirred at room
C17H35N5O4 (M + H+) 374.2767, found 374.2762. Anal. calcd temperature for 18 h and then was heated to 40–50 °C for 6 h.
for C17H35N5O4: C, 49.9; H, 9.6; N, 17.1. Found: C, 49.9; H, 9.7; The white solid was filtered and washed with cold distilled
N, 17.0.
Synthesis of 1. A mixture of compound 10 (0.50 g, 40.05 mmol, yield 93%): mp 172–173 °C; IR (ATR): 3304, 1685,
1.33 mmol), anhydrous K2CO3 (1.85 g, 13.38 mmol) and 9,10- 1654, 1531, 1496, 1455 cm−1 1H NMR (500 MHz, DMSO-d6)
water and cold methanol to obtain a white solid (26.63 g,
;
bis(bromomethyl)-anthracene (0.54 g, 1.47 mmol) in dry δ 7.91 (s, 2H), 7.47 (d, 2H, J = 8.2 Hz), 7.12–7.37 (m, 20H), 4.95
CH3CN (175 mL) was stirred till reflux for 24 h under a nitro- (m, 4H), 4.22 (m, 2H), 3.12 (m, 4H), 2.98 (dd, 2H, J = 4.2 Hz; J =
gen atmosphere. The reaction was monitored by TLC. After 13.3 Hz), 2.77 (dd, 2H, J = 10.2 Hz; J = 12.9 Hz), 2.58 (s, 4H);
complete consumption of the starting material, the reaction 13C NMR (125 MHz, DMSO-d6) δ 172.8, 171.2, 155.8, 138.0,
mixture was filtered and the solvent was evaporated under 136.9, 129.2, 128.2, 127.9, 127.6, 127.4, 126.2, 65.2, 56.2, 48.0,
vacuum. The crude product was purified by silica flash chrom- 37.7, 25.2; HRMS (ESI-TOF)+ (m/z) calcd for C38H43N5O6
atography using MeOH–CH2Cl2 (1 : 25) as the eluent to yield (M
+
H+) 666.3292, found 666.3301. Anal. calcd for
the yellowish solid compound 1 (0.542 g, 0.942 mmol, 70% C38H43N5O6·H2O: C, 66.7; H, 6.6; N, 10.2. Found: C, 66.5; H,
yield): mp 94–95 °C; [α]2D5 = −51.30° (c 0.1, CHCl3), IR (ATR). 6.8; N, 10.2.
3329, 2950, 2920, 2861, 1746, 1661, 1590 cm−1
;
1H NMR
Synthesis of 17. To a stirred mixture of 16 (10.0 g,
(500 MHz, DMSO-d6) δ 8.57 (m, 4H), 7.65 (m, 4H), 6.87 (s, 2H), 15.0 mmol) and anhydrous K2CO3 in dry DMF (25 mL),
5.01 (d, 2H, J = 13.9 Hz), 4.72 (d, 2H, J = 13.8 Hz), 3.55 (s, 3H), methyl-2-bromoacetate (3.44 g, 22.5 mmol) was added and the
3.11 (m, 4H), 2.70 (s, 2H), 2.19–1.99 (m, 6H), 1.92 (m, 2H), 1.06 reaction mixture was stirred at 50 °C for 24 h. The reaction pro-
(d, 6H, J = 6.3 Hz), 0.89 (d, 6H, J = 6.9 Hz); 13C NMR (126 MHz, gress was followed by TLC. After complete consumption of the
CDCl3) δ 173.2, 171.7, 132.9, 129.9, 125.8, 125.6, 68.7, 52.9, starting material, distilled cold water (120 mL) was added and
52.6, 51.2, 45.1, 36.8, 31.2, 19.8, 18.2; HRMS (ESI-TOF)+ (m/z) the reaction mixture extracted with EtOAc (75 mL, ×3). The
calcd for C33H45N5O4 (M + H+) 576.3550, found 576.3557. Anal. organic phases were dried over anhydrous MgSO4, and the
calcd for C33H45N5O4: C, 66.8; H, 7.9; N, 12.2. Found: C, 66.6; solvent was evaporated under vacuum. The crude product was
H, 7.7; N, 12.2.
purified by silica flash chromatography using MeOH–CH2Cl2
Synthesis of 2. To a clear solution of 1 (0.3 g, 0.52 mmol) in (1 : 40) as the eluent to yield the white solid compound (8.40 g,
6 mL THF–H2O (2 : 1), LiOH (0.087 g, 3.64 mmol) was added 11.40 mmol, 76% yield): mp 156–158 °C; [α]2D5 = −16.84° (c 0.1,
and the reaction was stirred at room temperature for 24 h. The CHCl3); IR (ATR) 3305, 1698, 1685, 1643, 1533, 1455 cm−1
;
reaction was monitored by TLC and, after complete consump- 1H-NMR (500 MHz, DMSO-d6) δ 7.87 (s, 2H), 7.45 (d, 2H, J =
tion of the starting material, the reaction mixture was made 8.7 Hz), 7.11–7.36 (m, 20H), 4.97 (d, 2H, J = 12.7 Hz), 4.92 (d,
acidic (pH 6) with 1 : 1 HBr–H2O. This solution was added to 2H, J = 12.8 Hz), 4.23 (m, 2H), 3.61 (s, 3H), 3.40 (s, 2H), 3.12
the washed DOWEX® ion exchange resin (4 g), and then left (m, 4H), 2.99 (dd, 2H, J = 4.4 Hz; 13.9 Hz), 2.77 (dd, 2H, J =
for 18 h at room temperature. The solvent of this mixture was 10.4 Hz; 13.4 Hz), 2.61 (m, 4H); 13C-NMR (125 MHz, CDCl3)
evaporated under vacuum, and then the resulting solid was δ 172.0, 171.9, 156.3, 137.1, 136.3, 129.3, 128.4, 127.8, 127.7,
added to a column containing 20 g washed DOWEX® ion 126.6, 66.7, 56.4, 53.9, 53.4, 51.4, 38.3, 37.6; HRMS (ESI-TOF)+
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(m/z) calcd for C41H47N5O8 (M + H+) 738.3503, found 738.3505 DOWEX® ion exchange resin and deionized H2O. The eluents
(M + H+). Anal. calcd for C41H47N5O8: C, 66.7; H, 6.4; N, 9.5. used for the resin column purification were: 100 mL H2O, 1 : 1
Found: C, 66.9; H, 6.7; N, 9.5.
THF–H2O 100 mL, 5% NH3 in H2O and then, 10% NH3 in
Synthesis of 18. To a stirred clear solution of 17 (1.0 g, H2O. From this resin column purification the compound was
1.4 mmol) in dry THF, 10 mol% of the catalyst (5 wt% Pd on isolated in the range of 5–10% NH3 in H2O solutions. Then
activated carbon) was added. The reaction mixture was stirred the water was evaporated under vacuum to yield the yellowish
under H2 pressure (H2 balloon) at 50 °C for 4 h. The reaction solid 4 (0.124 g, 0.189 mmol, 21% yield): mp 107–110 °C;
was monitored by using TLC. After complete consumption of [α]2D5 = −47.15° (c = 0.1, CHCl3); IR (ATR) 3269, 3027, 1669,
the starting material the catalyst was filtered off through 1539 cm−1 1H NMR (500 MHz, DMSO-d6) δ 8.34 (d, J = 8.6 Hz,
Celite® and washed with dry THF. The crude product was puri- 2H), 8.18 (d, J = 9.4 Hz, 2H), 7.51 (m, 4H), 7.37 (s, 8H), 7.29 (s,
fied by silica flash chromatography using MeOH–CH2Cl2 2H), 6.84 (s, 2H), 4.54 (m, 4H), 3.10 (m, 2H), 2.80 (s, 2H), 2.67
(1 : 15) as an eluent, to which 20 µL NH3 were also added at (dd, J = 13.5, 9.3 Hz, 3H), 2.08 (m, 4H), 1.87 (m, 2H); 13C NMR
every addition of the eluent throughout the column purifi- (125 MHz, DMSO-d6) δ 173.2, 139.1, 132.4, 129.9, 129.8, 128.8,
cation to give the sticky greenish oil 18 (0.401 g, 0.854 mmol, 126.9, 125.8, 125.7, 125.4, 64.05, 53.2, 44.6, 39.1, 37.5; HRMS
61% yield): [α]2D5 = −48.14° (c 0.1, CHCl3); IR (ATR) 3296, 3086, (ESI-TOF)+ (m/z) calcd for C40H43N5O4 (M + H+) 658.3393,
3026, 2946, 2848, 1734, 1644, 1521, 1454 cm−1 1H NMR found 658.3397 (M + H+). Anal. calcd for C40H43N5O4·H2O: C,
;
(500 MHz,DMSO-d6) δ 7.80 (s, 2H), 7.26 (m, 4H), 7.20 (m, 6H), 71.1; H, 6.7; N, 10.4. Found: C, 71.0; H, 6.9; N, 10.2.
3.60 (s, 3H), 3.37 (m, 4H), 3.09 (m, 4H), 2.93 (m, 2H), 2.55–2.66
(m, 6H), 1.90 (s, 2H); 13C NMR (75 MHz, DMSO-d6) δ 173.9,
171.6, 138.7, 129.5, 128.3, 127.7, 126.4, 125.7, 56.3, 54.4, 53.04,
Acknowledgements
50.9, 40.94, 36.9; HRMS (ESI-TOF)+ (m/z) calcd for C25H35N5O4
(M
C25H35N5O4·2H2O: C, 59.4; H, 7.8; N, 13.8. Found: C, 59.4; H, C03-01) and UJI (project P1·1B-2012-41) are acknowledged.
7.7; N, 13.5. P.D.W. thanks the financial support from GV (Grisolia Fellow-
+
H+) 470.2767, found 470.2760. Anal. calcd for Financial support from the Spanish MINECO (CTQ2012-38543-
Synthesis of 3. Compound 18 (0.80 g, 1.7 mmol), anhydrous ship). M.J.M. acknowledges the School of Chemistry (Univer-
K2CO3 (2.36 g, 17.0 mmol) and 9,10-bis-(bromomethyl)-anthra- sity of East Anglia, Norwich, UK) for a studentship. The
cene (0.68 g, 1.7 mmol) were placed in a flask containing dry authors are grateful to Dr Jelena Gavrilovic (School of Biologi-
CH3CN (150 mL) and the mixture was refluxed for 24 h under cal Sciences, University of East Anglia, Norwich, UK) for the
a nitrogen atmosphere. The reaction was filtered and the kind gift of the RAW 264.7 mouse macrophage cells used in
solvent evaporated under vacuum. The product was purified by this work.
flash silica chromatography using MeOH–CH2Cl2 (1 : 20) as an
eluent to yield a yellow solid (0.708 g, 1.054 mmol, 62% yield):
mp 77–78 °C; [α]2D5 = −171.34° (c 0.1, CHCl3); IR (ATR) 3335,
Notes and references
2943, 1734, 1657, 1509, 1437 cm−1 1H NMR (500 MHz,
;
CD3CN) δ 8.39 (d, 2H, J = 8.4 Hz), 8.12 (d, 2H, J = 8.4 Hz), 7.50
(m, 4H), 7.43 (m, 4H), 7.36 (m, 6H), 6.68 (s, 2H), 4.63 (d, 2H,
J = 13.8 Hz), 4.53 (d, 2H, J = 13.8 Hz), 3.55 (s, 3H), 3.47 (dd,
2H, J = 9.1 Hz; J = 4.6 Hz), 3.18 (dd, 2H, J = 13.9 Hz; J = 4.6 Hz),
3.01 (s, 2H), 2.70 (dd, 2H, J = 13.9 Hz; J = 9.1 Hz), 2.56 (m, 2H),
2.14–2.21 (m, 2H), 2.01–2.09 (m, 2H), 1.89–1.95 (m, 2H); 13C
NMR (500 MHz, CD3CN,) δ 173.1, 171.5, 138.4, 131.9, 129.8,
129.8, 129.4, 128.6, 126.7, 125.6, 125.5, 125.1, 125.0, 64.1, 52.8,
51.6, 50.7, 44.5, 39.0, 37.2; HRMS (ESI-TOF)+ (m/z) calcd for
C41H45N5O4 (M + H+) 672.3550, found 672.3555. Anal. calcd for
C41H45N5O4: C, 73.3; H, 6.8; N, 10.4. Found: C, 73.0; H, 6.9; N,
10.2.
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