Facile synthesis of fluorescent distyrylnaphthalene derivatives for bioapplications

Article abstract of DOI:10.1039/c7nj00004a

A series of novel 2,6-bis(4-aminostyryl)naphthalene based derivatives were synthesized and their spectroscopic properties investigated. Due to their low cytotoxicity, suitable optical properties and potential affinity towards biological structures these d

Full text of DOI:10.1039/c7nj00004a

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ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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Facile Synthesis of Fluorescent Distyrylnaphthalene Derivatives
for Bioapplications†
Beata Łukasik,a# Justyna Milczarek,b# Roza Pawlowska,b Remigiusz Żurawiński*a and Arkadiusz
Chworos*b
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A
series of novel 2,6-bis(4-aminostyryl)naphthalene based properties, often suffer low brightness, sensitivity to
derivatives were synthesized and their spectroscopic properties photobleaching or narrow Stokes shift.12 In turn, FPs whose
investigated. Due to the low cytotoxicity, suitable optical photophysical properties are very similar to organic dyes may
properties and potential affinity towards biological structures undergo oligomerization and aggregation. These processes can
these derivatives proved promising dyes for application in lead to a loss of function or incorrect localization of proteins.
fluorescence-based bioimaging. Selective intercalation of tested Protein aggregates may also adversely influence the cell
compounds into cell membranes was revealed by fluorescence viability.13 QDs offer several advantages over organic dyes
microscopy.
concerning the narrow emission band of mostly symmetric
shape, higher molar absorption coefficient, long
a
a
Fluorescence bioimaging is one of the most widespread
methods in biosciences. This developing field of research uses
different types of fluorescent probes for understanding the
functions and mechanisms of various biological processes from
molecular level to cellular and tissue stages. Fluorescent
markers with molecular imaging functionality may also play an
important role as carriers or indicators in diagnostics and
therapy. Given this significance of bioimaging in life science
and biomedicine, the better fluorescent probes with excellent
photostability, high quantum efficiency, tunable absorption-
emission wavelength, biocompatibility, low cytotoxicity,
energy transfer capability and easily functionalized groups in
aqueous or biological environment are highly desirable by
scientists.1,2
fluorescence lifetime (typically 5-100 ns) and a high quantum
yield especially for NIR wavelength.12 However, the promise of
QD applications are limited by their significant toxicity due to
the presence of heavy metal components and generation of
free radicals during excitation.14
The promising fluorescent probes, which have received
considerable attention since the 1990s are conjugated
polyelectrolytes (CPEs) and their shorter analogues conjugated
oligoelectrolytes (COEs).15 CPEs and COEs are specialized
macromolecules with π-conjugated molecular frameworks and
polar pendant groups.16,17 Fluorescent markers based on this
class of compounds show good light-harvesting properties,
which are usually better than the properties of conventional
fluorescent probes (QDs, FPs or others organic dyes). Various
applications of COEs in visualization or detection of specific
peptides and proteins,18 cellular imaging,19,20 drug tracking or
gene delivery systems21,22 have been widely investigated.
Comparing these types of fluorescent probes, COEs seem to be
more attractive due to their controllable length and structural
precision.23 More attention has been paid to design the
photoactivatable dyes based on COEs for in vitro and in vivo
imaging of targeted cells or organelles.19,24 One of the key
examples of COE involved in direct cellular imaging are
compounds, which contain the phenylene-vinylene (PV-COEs)
frameworks. These distyrylbenzene and distyrylstilbene
derivatives are characterized by linear and non-linear optical
properties, which are valuable for uses in modern technology
including fluorescent bioimaging.20,25,26 PV-COEs were
successfully incorporated into bacteria,27 yeast20,26 and
mammalian cells.28,29 Their promising bioimaging applications
There is a large toolbox of fluorescent probes, which
includes small organic dyes,3 metal-ligand complexes,4-6
fluorescent proteins (FPs)7-9 and quantum dots (QDs).10,11
Unfortunately, all these types of chromophores have some
limitations that hamper their applicability in bioanalytics or
medicinal diagnostics. Thus, the most versatile organic dyes
which are highly diverse in terms of structures and optical
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Fig. 1 Structures of synthesized distyrylnaphthalene derivatives.
for staining the extracellular vesicles (exosomes), which take
part in intracellular signaling or adaptive immune responses,
were also screened.30
Scheme 2 Synthesis of distyrylnaphthalene derivatives.
iodohexyl)amino]styryl}naphthalene
(DSNN-I), which was
Recently, in the frame of our research program aimed at
the invention and development of the general methods of the
synthesis of novel staining agents for fluorescence based
bioimaging, the suitability of some PV-COEs as
fluorescentmarkers was investigated.28,30 In the course of our
studies we have compared the effectiveness of membrane
staining by PV-COEs dyes depending on their structural
differences in terms of length of conjugated core and ionic
functionality. We found that the four-ring distyrylstilbene
easily available in a four step reaction sequence starting from
dimethyl 2,6-naphthalenedicarboxylate (Scheme 1). Thus, the
reduction of both ethoxycarbonyl groups with lithium
aluminum hydride, followed by bromination of 2,6-
bis(hydroxymethyl)naphthalene (2) formed with phosphorus
tribromide afforded the corresponding dibromide 3. The
Michaelis-Arbuzov reaction of 3 with triethyl phosphite gave
bisphosphonate 4, which subsequently was subjected to the
Horner-Wittig olefination with aldehyde 5 in the presence of
sodium tert-butoxide to yield DSNN-I. Configuration of the
carbon-carbon double bond in DSNN-I was assigned based on
the vicinal proton coupling constant value of 15.9 Hz, which is
characteristic for trans stereoisomers.
derivative composed of π-delocalized phenylvinylene repeated
units and contains ammonium terminated pendant groups
bound at both extremities is the best candidate for
visualization of membrane-rich cellular compartments.31
Continuing our interest in this field, we herein report a simple
and effective synthesis of a series of distyrylnaphthalene
(DSNN) derivatives (Fig. 1), which belong to a new class of
molecules with potential for fluorescent imaging applications.
The structures of the synthesized DSNN derivatives were
designed to have the molecule lengths close to the thickness
of the lipid bilayer membranes and consist of the hydrophobic
core and the polr terminal groups. Optical properties of these
compounds, their cytotoxicity determined against the human
cervical cancer cell line (HeLa), and the preliminary results of
their utility as membrane-staining dyes for cell imaging are
also presented.
Having DSNN-I in hand, we focused on the synthesis of a
series of distyrylnaphthalene derivatives. The synthetic
pathway to these compounds is outlined in Scheme 2. To
evaluate the influence of the different functional terminal
groups on the cytotoxicity of these compounds against
mammalian cells and effectiveness of their incorporation into
mammalian lipid membranes, the ionic and polar DSNN
derivatives were synthesized. Two cationic derivatives DSNN-
NMe3+ and DSNN-Py+ were obtained in the reaction of DSNN-I
with excess of trimethylamine 33 wt.% solution in ethanol and
pyridine, respectively. Anionic derivative DSNN-POK was
synthesized in two steps involving the Michaelis-Arbuzov
reaction of DSNN-I with trimethyl phosphite, followed by
DSNN derivatives were prepared from 2,6-bis{4-[N,N-bis(6-
Scheme 1 Synthesis of DSNN-I.
2 | J. Name., 2012, 00, 1-3
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Table 1 Optical properties of distyrylnaphthalene derivatives.
Water
MeOH
DMSO
Compound
Stokes
shift [nm]
λabs
λem [nm]
Stokes
λabs [nm]
λem [nm]
Stokes
shift [nm]
100
λabs [nm]
λem [nm]
shift [nm]
100
90
+
DSNN-NMe3
DSNN-Py+
DSNN-P
DSNN-Mor
DSNN-POK
DSNN-NH2
405
408
416
409
363
406
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
403
404
407
406
405
405
503
494
504
505
523
492
421
422
421
423
394
422
521
525
523
523
503
517
103
102
100
109
97
99
118
87
95
partial hydrolysis of dimethoxyphosphoryl group in the of 492 nm to 523 nm. The bathochromic shifts (18-20 nm)
phosphonate DSNN-P formed carried out in the presence of were observed in both absorption and emission spectra when
o
excess of KOH in dioxane/water solution at 60 C for 24h. On changing from higher to lower polarity solvents (MeOH vs.
the other hand, the neutral distyrylnaphthalene derivative, DMSO) except for phosphonic acid salt DSNN-POK. Comparing
+
DSNN-Mor, containing a morpholinyl groups at the terminus of DSNN-NMe3 to the phenylene-vinylene analog DSSN+, the
the four side chains was obtained upon treatment of DSNN-I spectra of styrylnaphthalene derivative is blue shifted (421nm
+
with an excess of morpholine. The synthesis of DSNN-NH2 was vs. 435nm in DMSO). The Stokes shifts of DSNN-NMe3
,
DSSN+
accomplished in two steps. In the first, DSNN-I was reacted and shorter distyrylbenzene derivative DSBN+ in DMSO are 100
with sodium azide to give azide DSNN-N3
which was nm, 161 nm and 92 nm, respectively.26
subsequently subjected to the Staudinger reaction with To evaluate the cytotoxicity of the synthesized DSNN
triphenylphosphine to give the corresponding derivatives on human cells, live-cell experiments were
iminophosphorane, whose hydrolysis led to desired DSNN- performed using the HeLa cell line. Cytotoxicity was assessed
NH2 using a standard MTT assay based on the conversion of the 3-
To appraise the optical properties of the synthesized DSNN (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
,
.
derivatives we recorded their absorption and emission spectra salt into formazan by mitochondrial dehydrogenase, which is
in three solvents of different polarity (water, MeOH and active only in living cells. The intensity of formazan was
DMSO). Stokes shift, absorbance (λabs) and photoluminescence quantitatively measured in three independent experiments
(PL) (λem) maxima for these compounds are given in Table 1 performed in five repeats. We probed the influence of DSNN
(normalized UV-Vis and PL spectra are available in the ESI). derivatives at concentrations of 1, 5 and 10 ꢀM on the HeLa
Because for the investigated compounds the PL intensity in cell viability after 48h. The results of the cytotoxicity test
water was much lower than in other solvents, the results were indicate that DSNN derivatives exhibit negligible cytotoxicity
not normalized. All DSNN derivatives except DSNN-I (a starting effects at 1ꢀ
material for other derivatives) and DSNN-N3 (intermediate in concentration (5-10
M concentration (Fig. 2). Within the higher
M), only 10
DSNN-Me3N+ derivative
ꢀ
ꢀM
the synthesis of DSNN-NH2) were sufficiently water-soluble exhibits significant cytotoxicity, which manifests as a 26.4%
and low fluorescent emission in this solvent may be ascribed reduction in HeLa cell viability while non-ionic DSNN
to self-quenching caused by aggregate formation. In the derivatives DSNN-P and DSNN-Mor are completely safe for live
methanol and DMSO solutions, DSNN derivatives exhibit cells under these conditions.
absorbance maxima in the 363-416 nm range. Relatively small
After determining that DSNN derivatives could be added to
M,
observed in absorbance spectra. The emission λem values for all DSNN-NMe3 and DSNN-Mor were tested as fluorescence
chromophores measured in MeOH and DMSO was in the range probes. After 24 hours of incubation in humidified
atmosphere with 5% CO2 at 37 C the dyes were found to be
sensitivity to the polarity of the surrounding solvents was human cell lines without significant toxic effects at 1
ꢀ
+
a
°
+
cell permeable. For both DSNN-NMe3 and DSNN-Mor
emissions in HeLa cells can be observed in cell membranes
surrounding cell nuclei and lipid bilayer-rich organelles such as
the Golgi apparatus and endoplasmic reticulum, corresponding
to the green light emission (Fig. 3).
1uM
5uM
10uM
120%
100%
80%
60%
40%
20%
0%
Conclusions
Fig. 2 The viability of HeLa cells after 48 of incubation in the presence of DSNN
derivatives at various concentrations. Mean values ± SD for 3 independent experiments
are shown.
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+
Fig. 3 Fluorescence microscopy images of fixed HeLa cells incubated 24h with charged 1 µM DSNN-NMe3 (left two panels) and neutral 1 µM DSNN-Mor (right two panels),
collected at 20x magnification (left - phase contrast; right - B2A filter)
A series of 2,6-bis(4-aminostyryl)naphthalene derivatives with
11 B. A. Kairdolf, A. M. Smith, T. H. Stokes, M. D. Wang, A. N.
embedded terminal ionic/neutral polar groups were easily and
effectively synthesized starting from dimethyl 2,6-
naphthalenedicarboxylate. In contrast to ionic PV-COEs, the
synthesized DSNN derivatives with the uncharged polar amino
and azido substituents can easily be further bioconjugated
with peptides or click-functionalized nucleotides. The
cytotoxicity of the DSNN derivatives against the HeLa cell line
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compounds is a safe level for cells and also sufficient for
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+
localization of DSNN-NMe3 and DSNN-Mor in plasma
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We gratefully acknowledge financial support from the National
Science Centre of Poland (grant number NCN-
2012/05/B/ST5/00364).
4
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Synthesis of a novel type of distyrylnaphthalene derivatives and their application as
molecular fluorescent probes for bioimaging.