Synthesis and optical properties of water-soluble biperylene-based dendrimers

Article abstract of DOI:10.1039/c4cc01742k

We report the synthesis and photophysical properties of three biperylene-based dendrimers, which show red fluorescence in water. A fluorescence microscopy study demonstrated uptake of biperylene-based dendrimers in living cells. Our results indicate that these biperylene-based dendrimers are promising candidates in fluorescence imaging applications with the potential as therapeutic carriers. the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc01742k

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Synthesis and optical properties of water-soluble
biperylene-based dendrimers†
Pin Shao,‡a Ningyang Jia,‡ab Shaojuan Zhangac and Mingfeng Bai*ade
Cite this: Chem. Commun., 2014,
50, 5648
Received 7th March 2014,
Accepted 2nd April 2014
DOI: 10.1039/c4cc01742k
www.rsc.org/chemcomm
We report the synthesis and photophysical properties of three highly fluorescent in water.14 Water-soluble dendrons are efficient
biperylene-based dendrimers, which show red fluorescence in water. building blocks to increase hydrophilicity and reduce intermolecular
A fluorescence microscopy study demonstrated uptake of biperylene- stacking. In addition, dendrimers with surface functional groups are
based dendrimers in living cells. Our results indicate that these attractive biomaterials that allow for targeting capability, imaging
biperylene-based dendrimers are promising candidates in fluores- applications and drug delivery.16 Inspired by these findings, we set out
cence imaging applications with the potential as therapeutic carriers. to develop a dendrimeric biperylene dye with the potential for
biomedical imaging and therapy applications.
Rylene derivatives are fluorophores well-known for their exceptional
In our previous study, we developed a dendrimeric quaterrylene-
photochemical stability and high fluorescence quantum yields.1,2 As diimide dye by covalently attaching polyamide dendrons to the bay
such, they have become attractive materials for various applications regions of a quaterrylenediimide structure.17 We demonstrated that
such as in solar cells,3 laser dyes,4 organic light-emitting field-effect such a strategy was effective in increasing hydrophilicity, reducing
transistors,5 and optical switches.6 In recent years, some rylene aggregation and introducing functionality. Using a similar strategy,
derivatives have been developed as versatile fluorescent probes for here we developed water-soluble and functional biperylene dyes with
biological imaging.7–10 Rylene derivatives also have potential as four polyamide-based dendrons (Scheme 1). Compared to quaterry-
photosensitizers in photodynamic therapy.11 However, the rigid aro- lenediimide, biperylene has the advantage of reduced aggregation
matic structures of rylene derivatives make them highly hydrophobic due to the fact that the two perylene units connected by a C–C bond
and prone to aggregation, especially in aqueous solutions, leading to are twisted instead of being in the same plane. We report herein the
fluorescence quenching.12 To overcome this challenge, several strate- synthesis and photophysical properties of three biperylene-based
gies have been developed. One method is to attach charged groups, dendrimers, BiPI-G0, BiPI-G1 and BiPI-G2, which possess four, twelve
such as sulfonic acid and quaternized amine groups, to the bay region and thirty-six carboxylic acid groups on the surface, respectively. To
of the rylene rings. The charges on the rylene structure inhibited the the best of our knowledge, these are the first water-soluble, functional
intermolecular aggregation due to the electrostatic repulsion forces.13 and dendrimeric biperylene dyes. To demonstrate the potential of
Another strategy is to attach bulky substituents to rylene structures to these dendrimers in biomedical imaging, we imaged the uptake of
shield p–p stacking.14,15 For example, Wu¨rthner et al. introduced the dendrimers in living cells using fluorescence microscopy.
dendronized polyglycerols in the end imide positions and successfully Together, these new biperylene-based dendrimers hold great promise
developed polyglycerol-dendronized perylene bisimide dyes which are in biomedical applications.
BiPI-G0 was synthesized by following the pathway shown in
Scheme S1 (ESI†). The key precursor 3 was prepared by selective
nucleophilic substitution of the two bromine atoms of 2 using
potassium carbonate as the base. Due to the undesired substitution
a Molecular Imaging Laboratory, Department of Radiology, University of Pittsburgh,
Pittsburgh, PA 15219, USA. E-mail: baim@upmc.edu; Fax: +1-412-624-2598;
Tel: +1-412-624-2565
reaction on the 9-bromine atom of perylene 2, the reaction yield was
moderate (44%). Biperylene 4 was synthesized by the Yamamoto
homocoupling reaction of 3, and the subsequent deprotecting reac-
tion of t-butyl groups using trifluoroacetic acid afforded BiPI-G0 in a
high yield (95%). BiPI-G0 was then used as the core structure to
synthesize the two generations of dendrimers BiPI-G1 and BiPI-G2.
Dendrons 5 and 7 were conjugated to the four carboxylic acid groups
of BiPI-G0 using HBTU/HOBt as the coupling reagent to yield 6 and 8,
b Department of Radiology, Eastern Hepatobiliary Surgery Hospital,
Second Military Medical University, Shanghai, 200438, P. R. China
c Department of Diagnostic Radiology, the First Hospital of Medical School,
Xi’an Jiaotong University, Xi’an, Shaanxi 710061, P. R. China
d University of Pittsburgh Cancer Institute, Pittsburgh, PA 15232, USA
e Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15261, USA
† Electronic supplementary information (ESI) available: Synthesis and character-
ization. See DOI: 10.1039/c4cc01742k
‡ P. Shao and N. Jia equally contributed to this work.
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Fig. 1 The normalized concentration-dependent absorption and emission
(lex = 530 nm) spectra of (a) BiPI-G0, (b) BiPI-G1 and (c) BiPI-G2 in water.
The insets show the extinction coefficients at different concentrations.
Scheme 1 The structures of BiPI dendrimers.
BiPI-G0, BiPI-G1 and BiPI-G2 showed broad fluorescence from 600 to
800 nm, with peaks at 663, 661 and 646 nm, respectively. Similar to
the concentration-dependent absorption results, the fluorescence
intensity increased gradually with increasing concentrations from
0.5 to 10 mM (Fig. S1, ESI†) and the emission band profiles remained
almost the same at the concentrations studied.
To further verify the inhibited dye aggregation due to the twisted
configuration of BiPI dyes, we studied temperature-dependent absorp-
tion and emission profiles. Similar to the concentration-dependent
results, the absorption and emission spectra profiles of all three dyes
showed an insignificant change when temperature was increased from
20 1C to 70 1C (Fig. 2), indicating that no significant aggregation
existed. We also found that the fluorescence peaks of BiPI-G0 and BiPI-
G1 showed slight blue shift as well as a decrease in intensity (Fig. 2 and
Fig. S2, ESI†), while the fluorescence intensity of BiPI-G2 decreased
dramatically when temperature was increased (Fig. S2, ESI†), which is
likely due to the significantly increased nonradiative decays of the
highly flexible system of BiPI-G2 at high temperatures.18,19
When dissolved in water at a concentration of 1 mM, BiPI-G0,
BiPI-G1 and BiPI-G2 exhibited broad and structureless absorption
bands in the range of 450 to 650 nm (Fig. 3) and detectable fluores-
cence with quantum yields of 0.57%, 0.85% and 1.24%, respectively
(Table 1). The fluorescence intensity of the three biperylene dyes in
water increased as the size of the dendron enlarged, although the
dendritic effect was not as large as that observed in perylene bisimide
dyes.14 There are two possible reasons: (1) nonradiative decays (such
as intramolecular rotation and vibration) other than intermolecular
aggregation are critical in the fluorescence quenching and (2) fluores-
cence of the hydrophobic biperylene core is quenched by water. It has
been previously reported that polar solvents could cause fluorescence
respectively. It is noteworthy that the initial attempt of chromato-
graphic purification of 6 and 8 using silica gel failed due to the
difficulty of eluting the products off the column. Therefore, thin layer
chromatography using large silica gel plates was employed to purify 6
and 8. Because of the loss during purification, 6 and 8 were obtained
in low yields (51% and 30%, respectively). At last, the termini of 6 and
8 were deprotected using trifluoroacetic acid resulting in BiPI-G1 and
BiPI-G2 with 12 and 36 carboxylic acid groups, respectively. The
complete deprotection was confirmed by the disappearance of the
t-butyl protons at around 1.4 ppm in the 1H NMR spectra.
Next, we studied the spectroscopic properties of BiPI dendrimers.
As expected, the twisted structure of biperylene can efficiently inhibit
intermolecular aggregation. Fig. 1 shows the normalized absorption
spectral change of BiPI dendrimers at different concentrations
ranging from 0.5 to 10 mM in water. All three dendrimers showed
strong broad absorption bands in the region of 450–650 nm in water,
with the maximum absorption at 535, 540 and 545 nm for BiPI-G0,
BiPI-G1 and BiPI-G2, respectively. With an increase in concentration,
all three dyes showed increased absorption intensity (Fig. S1, ESI†),
whereas the band profiles of the dendrimers, including shape,
position and molar extinction coefficients, showed a negligible
change as demonstrated in Fig. 1. It has been reported that perylene
dyes showed distinct absorption curves at different concentrations
due to aggregation.14 These results indicate that BiPI-G0, BiPI-G1 and
BiPI-G2 exist mainly as monomeric dye molecules in water in the
concentration range from 0.5 to 10 mM.
The concentration-dependent fluorescence behavior of these
biperylene dendrimers was also investigated. As illustrated in Fig. 1,
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Table 1 Photophysical properties of BiPI dendrimers in different solvents
at a concentration of 1 mM
Solvent
CH3CN
Sample labs/nm e/104 cmÀ1 MÀ1 em/nm ja/%
l
BiPI-G0 524
BiPI-G1 528
BiPI-G2 536
4.62
5.25
6.20
633
634
656
3.99
5.42
3.18
DMSO
Water
BiPI-G0 532
BiPI-G1 531
BiPI-G2 534
7.14
7.65
7.31
607
628
611
0.85
0.98
1.19
BiPI-G0 535
BiPI-G1 540
BiPI-G2 545
4.05
4.49
4.45
663
661
646
0.57
0.85
1.24
Water, 10 wt% P123 BiPI-G0 530
BiPI-G1 536
7.66
7.53
7.59
597
602
607
24.38
11.63
1.58
BiPI-G2 545
Water, 0.5 wt% CTAB BiPI-G0 537
BiPI-G1 538
5.89
7.55
7.74
633
624
607
2.78
3.08
29.52
BiPI-G2 543
a
Rhodamine B was used as the standard (j = 65% in ethanol, ref. 26).
Fig. 2 The normalized temperature-dependent absorption and emission
(lex = 530 nm) spectra of (a) BiPI-G0, (b) BiPI-G1 and (c) BiPI-G2 in water at
a concentration of 10 mM.
two surfactants, Pluronic P123 and cetyltrimethylammonium bro-
mide (CTAB), to coat the BiPI dyes. Researchers have used Pluronic
P123 and CTAB to enhance fluorescence of perylene and terrylene
dyes.10,22,23 In our study, we found that Pluronic P123 and CTAB were
able to enhance the fluorescence of BiPI dendrimers in aqueous
solutions. As shown in Fig. 3, all three BiPI dendrimers exhibited
enhanced, narrowed and structural absorption bands in Pluronic
P123 and CTAB solutions. The absorption change is due to restricted
movements of BiPI molecules by the copolymer Pluronic P123 and
quaternized amine CTAB micelles. When the BiPI dyes were incorpo-
rated into the hydrophobic center of micelles, the intramolecular
movements such as rotation and vibration were significantly inhibited
and the quenching effect of water was avoided. Therefore, their
fluorescence quantum yields increased dramatically. When treated
with 10 wt% P123, the fluorescence quantum yield of BiPI-G0
increased 43-fold from 0.57% to 24.38% and that of BiPI-G1 increased
13-fold from 0.85% to 11.63%. No significant increase of quantum
yield was observed when BiPI-G2 was treated with P123. In contrast,
CTAB appeared to be more effective on large dye molecules. Treat-
ment of CTAB did not show significant effect on the quantum yields
of BiPI-G0 and BiPI-G1, but greatly increased the quantum yield of
BiPI-G2 by 18-fold from 1.24% to 29.52%. The improved fluorescence
performance indicated that the hydrophobic environment and the
confined dye configuration in micelles efficiently reduced the non-
radioactive decay and water quenching of the BiPI dyes. Besides the
fluorescence intensity change, the fluorescence peaks of these dyes
showed a significant blue shift in the presence of Pluronic P123 or
CTAB (from 663 to 597 nm for BiPI-G0 with P123; from 661 to 602 nm
for BiPI-G1 with P123; and from 646 to 607 nm for BiPI-G2 with
CTAB). Generally, two kinds of excited states contribute to the
fluorescence of symmetrical biaryl dyes, including the locally excited
(LE) state and the charge transfer (CT) state. The contribution from
the CT state typically increases in polar solvents;24,25 therefore, the
increase of fluorescence from biperylene dyes is mainly due to the
increased contribution from the LE state that relates to the non-polar
quenching of the hydrophobic core.20,21 Nevertheless, the fluores-
cence quantum yield increased over two-fold from G0 to G2 (0.57%
for BiPI-G0 and 1.24% for BiPI-G2). Importantly, the higher generation
of BiPI dendrimers has improved hydrophilicity and drug loading
capability, due to the increased number of surface carboxylic acid
groups and larger volume available for drug encapsulation. To
suppress intramolecular movements and water quenching, we used
Fig. 3 The UV-vis absorption (solid) and emission spectra (dash) of (a)
BiPI-G0, (b) BiPI-G1 and (c) BiPI-G2 in water (black), in water in the presence of
0.5 wt%/wt CTAB (red), and in water in the presence of 10 wt%/wt Pluronic P123
(green) at a concentration of 1 mM (lex = 500 nm).
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concentration-dependent and an incubation time-dependent man-
ner, with stronger fluorescence signals observed at higher concentra-
tions and longer incubation time. BiPI-G1 and BiPI-G2 also showed
concentration-dependent cell uptake, whereas the incubation time
did not have much effect on the dendrimer uptake after 5 minutes
(Fig. S5, ESI†).
In conclusion, we have developed three polyamide-based
biperylene dendrimers with four, twelve and thirty-six carboxylic acid
termini. We found that the twisted configuration of the biperylene
dyes efficiently suppressed the dye aggregation. The dendrimeric
biperylene dyes showed red fluorescence in water and the fluores-
cence quantum yields increased with an increase of the dendron size.
Moreover, these biperylene dendrimers showed low cytotoxicity and
strong fluorescence signals in living cells. Additionally, the prospect
that targeting, signaling and therapeutic molecules can be attached to
or incorporated into these dendrimers offers BiPI dyes great promise
in imaging and therapeutic applications.
Fig. 4 Fluorescence microscopy images of BiPI-G0, BiPI-G1 and BiPI-G2.
WT-DBT cells were incubated with BiPI-G0, BiPI-G1 and BiPI-G2 at a concen-
tration of 5 mM and 37 1C for 3 h before imaging. Upper: fluorescence images.
Lower: differential interference contrast (DIC) images. Scale bars: 20 mm.
environment of inner micelles. Importantly, increased contribution
We thank Dr Nephi Stella at the University of Washington for
from the LE state causes a blue shift,22 which matches our providing WT-DBT cells. This work was financially supported by the
observations.
NIH Grant # R21CA174541 (PI: Bai) and the startup fund was
To evaluate the cytotoxicity of BiPI dyes, we used the provided by the Department of Radiology, University of Pittsburgh.
hemocytometer-based trypan blue dye exclusion method, as we A grant from Shanghai Science and Technology (12DZ1940606,
previously described.17 Wild type astrocytoma delayed brain tumor 12ZR1439900) and a grant from Shanghai Municipal Health Bureau
(WT-DBT) cells were treated with indicated concentrations (0, 1, 5 and (20124195) supported Dr N. Jia to conduct this work.
10 mM) of BiPI-G0, BiPI-G1 and BiPI-G2 for 24 h before evaluating the
cytotoxicity. As shown in Fig. S3 (ESI†), BiPI-G0, BiPI-G1 and BiPI-G2
exhibited comparably low cytotoxicity in WT-DBT cells, when indo-
cyanine green (ICG), the clinically approved near infrared fluorescent
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