PET modulated fluorescent sensing from the BF2 chelated azadipyrromethene platform

Article abstract of DOI:10.1039/b514788c

A convergent building block synthesis has been applied to new off/on photoinduced electron transfer (PET) modulated fluorescent sensors which are based on a BF₂ chelated tetraarylazadipyrromethene platform and operate in the biomedically import

Full text of DOI:10.1039/b514788c

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www.rsc.org/obc | Organic & Biomolecular Chemistry
PET modulated fluorescent sensing from the BF2 chelated azadipyrromethene
platform†
Michael J. Hall, Lorcan T. Allen and Donal F. O’Shea*
Received 18th October 2005, Accepted 20th December 2005
First published as an Advance Article on the web 19th January 2006
DOI: 10.1039/b514788c
A convergent building block synthesis has been applied to new off/on photoinduced electron transfer
(PET) modulated fluorescent sensors which are based on a BF2 chelated tetraarylazadipyrromethene
platform and operate in the biomedically important red region of the visible spectrum. Incorporation of
diethylamine and morpholine receptors facilitates off/on microenvironment polarity and pH sensing.
Aqueous formulation and in vitro cellular imaging demonstrates their potential for intracellular sensing.
Introduction
The development of molecular fluorescent sensors for biologically
related applications is a very active research area.1 The use of
high sensitivity fluorescence detection has become a widely used
tool for probing the molecular processes of biological systems
in vitro,2 and the application of fluorescence detection to non-
Fig. 1 BF2 chelated tetraarylazadipyrromethene fluorochromes.
invasive in vivo optical imaging is currently emerging.3 The
majority of fluorescent molecular sensors have operational light
input/output wavelengths in the 300–550 nm wavelength range.
In the case of in vivo imaging applications, this spectral region
suffers strong interference from endogenous chromophores. In
addition, fluorochrome excitation with blue and green light can
be damaging to the cellular systems under observation. The most
efficient light penetration of biological tissue occurs in the lower
energy red and near-infrared (NIR) spectral regions.4 As such,
the development of new visible red and NIR off/on fluorescent
sensors would be of benefit to future imaging applications. Several
research groups have recently reported a number of visible red
molecular sensors based on cyanines, squaraines and rhodamines,
modified BODIPY dyes and N-phenyl-1,8-naphthalimides.5
Herein, we report a new class of fluorescent sensors based
upon the BF2 chelated tetraarylazadipyrromethene fluorophore 1
(Fig. 1) which employs a receptor–methylene spacer–fluorophore
architecture. We have recently reported that this class of compound
displays strong absorption and fluorescence in the 650–750 nm
region of the spectrum.6 For example, the parent tetraphenyl
analogue of 1 (Ar1–4 = Ph) has an absorption kmax at 650 nm (e =
79 000 dm−3 mol−1 cm−1) and emission at 672 nm (φF = 0.34)
in chloroform. These promising photophysical characteristics
suggest that these fluorophores would make an excellent platform
from which visible red sensors could be constructed.
the targeted substrate, while the fluorophore quantifies and
communicates this information to the observer. The spacer unit
covalently links the fluorophore to the receptor whilst keeping the
ground state electronic systems of the receptor and the fluorophore
disconnected (Fig. 2). This sensing technology can be applied to a
diverse set of substrates by the use of substrate specific receptors,
allowing analytes such as protons, cations, anions, carbohydrates
and peptides to be detected.8
Fig. 2 Schematic of a PET fluorescent sensor design. Rectangle =
fluorophore; CH2 = methylene spacer; circle = receptor for substrate;
S = substrate.
In order to examine the PET sensing properties of BF2 chelated
tetraarylazadipyrromethenes, we synthesised and examined the
photophysical properties of the benzylamine substituted ana-
logues 1a and 1b, which would have the potential to act as
off/on switching receptors for pH9 and microenvironment polarity
(Fig. 3).10
The synthesis of our target compounds utilised our previously
described modular approach, involving the synthesis and con-
densation of 2,4-diaryl-5-nitrosopyrroles with 2,4-diarylpyrroles.11
Generation of 1a and 1b was accomplished through the con-
densation of 2-nitroso-3,5-diphenylpyrrole 2 with the receptor
substituted pyrroles 3a,b to yield the tetraarylazadipyrromethenes
4a,b. BF2 chelation of 4a,b was achieved by reaction with boron-
trifluoride diethyletherate with diisopropylethylamine as the base
in dichloromethane at room temperature for 16 hours. Purification
The design of fluorescent sensors most commonly adopts a
receptor–spacer–fluorophore architecture with the fluorescence
switching properties controlled by a photoinduced electron trans-
fer (PET) mechanism.7 The role of the receptor is to detect
Centre for Synthesis and Chemical Biology, Conway Institute, School of
Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin
4, Ireland. E-mail: donal.f.oshea@ucd.ie; Tel: +353 (0) 17162425
† Electronic supplementary information (ESI) available: 1H and 13C NMR
spectra for 1a and 1b. UV–Visible and fluorescence solvent studies for 1a
and 1b. See DOI: 10.1039/b514788c
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Table 1 Spectral characteristics of 1a and 1b in nm
DMF
THF
Dioxane
Cyclohexane
1a
Absa
657
680
657
682
653
677
655
676
652
676
653
677
647
668
650
672
Flub
Abs/H+c
Flu/H+c
1b
Absa
656
681
657
684
654
676
656
677
652
675
654
678
647
669
652
672
Flub
Abs/H+c
Flu/H+c
Fig. 3 PET modulated fluorescence molecular sensors.
a Concentration of 1 × 10−5 M. b Concentration of 8 × 10−7 M, excitation
630 nm. c 5 lL of TFA added to a 3 mL sample.
of the final products was by chromatography on alumina, which
provided 1a,b as copper coloured solids in 88 and 91% yields
respectively (Scheme 1).
Our expectations for the sensing properties of 1a,b were that lit-
tle change in the ground state UV–visible spectral properties would
be observed on substrate recognition, however, large changes in the
fluorescence intensity would act as the sole signalling event. The
UV–visiblespectra of 1a infour solvents of decreasingdipolarity—
DMF, THF, dioxane and cyclohexane—are shown in Fig. 4.12 The
wavelengths of the absorption maxima for 1a show little solvent
dependency with only a 10 nm hypsochromic shift observed for
cyclohexane (647 nm) when compared to DMF (657 nm) (Table 1,
Fig. 4). Identical behaviour was recorded for 1b (Table 1, ESI†).
Similarly, only a 12 nm difference between the wavelengths of the
emission maxima in cyclohexane and DMF was observed for 1a
and 1b which mirrored the minor hypsochromic shifts observed
for non-polar solvents in the UV–visible spectra (Table 1).
In contrast to the invariance of the emission wavelength
maxima in various solvents, the emission intensity showed a
marked response to solvent polarity. The trend observed was
that fluorescence intensity increased as a function of decreasing
polarity along the series of DMF < THF < dioxane < cyclohexane
(Fig. 5). A ninefold enhancement in fluorescence intensity was
recorded when the extremes of DMF and cyclohexane were
compared. It can be interpreted that in more polar solvents,
little fluorescence is observed as the excited state quenching by
the PET process is efficient (sensor is off). Whereas in non-polar
solvents the PET process is an ineffective competing process for
fluorescence emission and the sensor is switched on. The switching
Fig. 4 UV–Visible spectra of 1a in DMF (blue), THF (yellow), 1,4-diox-
ane (black), cyclohexane (green) at 1 × 10−5 M.
Fig. 5 Fluorescence spectra of 1a in DMF (blue), THF (yellow),
1,4-dioxane (black) and cyclohexane (green). Concentration 8 × 10−7 M,
excitation 630 nm, slit widths 5 nm.
Scheme 1 Synthesis of 1a and 1b. Reagents and conditions: (i) acetic anhydride, acetic acid, 100 ◦C, 1 h. (ii) BF3·OEt2, DIEA, CH2Cl2, rt, 16 h.
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effect of microenvironment polarity on the PET mechanism has
been observed previously.10b,13
The one-dimensional switching of our sensors in response
to an acid substrate (i.e. no significant ground state change in
response to substrate recognition) is shown in Fig. 6. There is
virtually no variance in the peak shape or the wavelength of
maximum absorbance in the UV–visible spectra for protonated
1a in any of the four solvents examined (Table 1, ESI). There is a
marked response however to the acid analyte in the fluorescence
spectra. For example the non-protonated 1a is weakly emissive
in DMF solutions but upon protonation with aqueous HCl a
strong emission is observed at 682 nm. The increase in fluorescence
intensity was greater than eightfold from the off to the on state
(Fig. 6).
Fig. 7 Fluorescence titration of 1a in H2O/CrEL/phosphate buffered
saline (INaCl = 150 mmol L−1). Apparent pKa determined to be 6.9 at
25 ◦C. Red trace pH = 4.9, dark green trace pH = 9. Concentration 8 ×
10−7 M, excitation 630 nm, slit widths 5 nm.
Fig. 6 Overlaid UV–visible (1 × 10−5 M) and fluorescence (8 × 10−7 M,
excitation 630 nm, slit widths 5 nm) spectra of 1a in DMF (blue) and in
DMF–aq. HCl (red).
As would be expected, THF, dioxane and cyclohexane each gave
smaller fluorescence intensity enhancements upon the addition
of trifluoroacetic acid (ESI). A similar trend for off to on
fluorescence enhancement (3-fold) in response to acidic conditions
was observed for 1b in DMF (ESI).
Fig. 8 Individual cellular focal plane sections of 1a in a HeLa cell.
Cytoplasm – red color; central dark area is the nucleus. Numbers in the
left hand corner of each image indicate the cellular position at which the
image was recorded.
Each image represents the fluorescence intensity at a specific
focal plane of increasing depth through the cell. It can be seen that
fluorescence intensity increases as you transect the cell (from 0 to
3 lM), reaches a maximum near the centre of the cell (∼4 to 5
lM) and diminishes as the further edge of the cell is reached. In a
cellular context 1a has the potential to come into contact with a
variety of different pH ranges and subcellular microenvironment
polarities and as such the exact mode of intracellular switching is
still under investigation.
In order to obtain aqueous solutions suitable for in vitro
imaging, the emulsifier Cremophor EL (CrEL) was used. CrEL
is a non-ionic surfactant, frequently used in vivo as a delivery
agent for poorly water-soluble drugs such as Taxol.14 The aqueous
formulated spectroscopic properties of 1a,b were very similar to
those obtained in organic solvents. The fluorescence titration of
1a with aqueous HCl showed a significant increase in fluorescence
intensity upon sequential addition of acid aliquots, whereas a
more moderate increase was recorded for the morpholine receptor
analogue 1b. The titration data predicted apparent pKa values of
6.9 and 4.8, for 1a and 1b respectively, providing a useful window
of sensing under physiological conditions (Fig. 7, ESI). It should
be noted that pKa values of amines in a micellar microenvironment
are often lower than would be anticipated.15
To examine the potential of our molecular sensor class for
imaging in vitro, an aqueous formulated solution of 1a was
incubated with HeLa cells for 1 hour. The cells were washed of
surface bound material, slide mounted and imaged using confocal
laser scanning microscopy. Cellular localisation of emissive 1a was
examined through 16 focal plane sections of 0.48 lm apart through
a single cell.16 Analysis of the Z-stack plane views confirmed that
emission from 1a was exclusively in the cytoplasm (red) with no
fluorescence observed in the nucleus (dark area) (Fig. 8).
Conclusions
We have developed a new fluorophore template for PET based
sensing, using the receptor–methylene spacer–fluorophore ap-
proach from the boron-difluoro chelated azadipyrromethene plat-
form, with an optical advantage of sensing in the red region of the
spectrum. The flexible modular synthesis would readily allow for
modified derivatives to be produced to include a range of receptor
classes and to fine-tune any desired photophysical parameters. The
ability of 1a to localize and be readily detected in vitro gives an
indication of the potential of this fluorochrome class as markers
for specific cellular events in conjunction with other receptor units
and fine-tuning of the switching responses. Future advances in the
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field of fluorescent molecular sensing and imaging will provide
new tools to assist in the understanding of biological processes at
the molecular level.
as a copper colored solid (108 mg, 91%); mp 170–174 ◦C;
kmax(CHCl3)/nm 652 (e/dm−3 mol−1 cm−1 82 000); mmax(KBr
disk)/cm−1 3438, 1515; dH(300 MHz; CDCl3; Me4Si) 2.49 (4H,
t, J 4.5, N(CH2CH2)2), 3.55 (2H, s, CH2N), 3.74 (4H, t, J 4.5,
(CH2CH2)2O), 7.00 (1H, s), 7.01 (1H, s), 7.40–7.48 (11H, m),
8.02–8.06 (8H, m); dC(75 MHz; CDCl3; Me4Si) 53.7, 63.2, 67.0,
118.9, 119.1, 128.6, 129.3, 129.4, 129.5, 129.5, 129.6, 129.6, 130.9,
131.2, 131.6, 132.3, 139.7, 144.0, 144.1, 145.5, 145.7, 159.4, 159.7;
m/z (ES) 597.2634 (M + H+, C37H32BF2N4O requires 597.2637).
Experimental
THF was distilled under N2 over sodium wire and benzophenone.
Cyclohexane, dioxane and chloroform were distilled over K2CO3.
DMF was distilled under reduced pressure over K2CO3. Solutions
for the solvent studies were prepared from a stock solution of 1
(0.005 mmol in 10 mL THF). 1 mL was diluted into 25 mL of
either cyclohexane, DMF, dioxane or THF to provide a second
stock solution. For UV–visible spectra, 5 mL of the second
stock solution was diluted into 10 mL of the relevant solvent
to give samples for UV–visible measurements. A 3 mL sample
was removed and the UV–visible spectra recorded. 5 lL of
trifluoroacetic acid (TFA) was added and the UV–visible spectra
were recorded again. For fluorescence spectra 1 mL of the second
stock solution was diluted into 25 mL of the relevant solvent
to give samples for UV–visible measurements at 8 × 10−7 M. A
3 mL sample was removed and the fluorescence spectra were
recorded. 5 lL of TFA was added and the fluorescence spectra
were recorded again. Fluorescence measurements were recorded
with the following setting; excitation and emission slit widths of
5 nm used, excitation wavelength 630 nm.
Formulation of 1a for in vitro cellular imaging
Compound 1a (0.005 mmol) was dissolved THF (1 mL), Cre-
mophor EL (0.07 mL) and 1,2-propanediol (0.03 mL) were added
and the sample was placed in a sonic bath for 30 min. The THF
was removed under reduced pressure and the oily mixture was
dissolved in phosphate buffered saline (PBS) solution (10 mL) and
filtered through an Acrodisc 25 mm syringe filter (with 0.2 lm HT
Tuffryn membrane). The concentration was checked by diluting a
portion of the sample (1 mL to 25 mL) with PBS and UV–visible
spectral analysis.
Confocal laser scanning microscopy
Cells, grown on 8-well chamber slides (Nunc), were incubated in
the dark at 37 ◦C with 1 × 10−5 M 1a for 1 h. Prior to visualisation,
excess probe was washed off by rinsing in PBS 4 times and cells
were fixed in 3.7% formaldehyde–PBS. Cells were mounted as
above and image analysis was performed using an LSM510 META
confocal laser scanning microscope (Zeiss) equipped with a 40X
numerical aperture 1.0 objective, with a pinhole of 100 lm in
diameter being used to capture each image at a resolution of 512 ×
512 pixels. 1a was excited by a 543 nm helium neon laser.
Synthesis of [3-(4-diethylamino-methylphenyl)-5-phenylpyrrol-2-
ylidene]-(3,5-diphenyl-1H-pyrrol-2-yl)-amine BF2 chelate 1a
A
stirred solution of [3-(4-diethylamino-methylphenyl)-5-
phenylpyrrol-2-ylidene]-(3,5-diphenyl-1H-pyrrol-2-yl)-amine11
(107 mg, 0.2 mmol) in CH2Cl2 (50 mL) was treated with
borontrifluoride diethyletherate (350 lL, 2.8 mmol) and
diisopropylethylamine (350 lL, 2 mmol). The reaction was stirred
at room temperature for 16 h, washed with water (2 × 50 mL),
dried over Na2SO4, the solvent was removed under reduced
pressure and the resulting solid was purified by chromatography
on alumina (CH2Cl2–EtOAc, 4 : 1) to give the product 1a
as a copper colored solid (102 mg, 88%); mp 140–142 ◦C
(cyclohexane); kmax(CHCl3)/nm 653 (e/dm−3 mol−1 cm−1 82 000);
mmax(KBr disk)/cm−1 3446, 1513; dH(300 MHz; CDCl3; Me4Si) 1.08
(6H, t, J 7.1, N(CH2CH3)2), 2.57 (4H, q, J 7.1, N(CH2CH3)2),
3.63 (2H, s, CH2N), 7.00 (2H, s), 7.43–7.47 (11H, m), 8.01–8.06
(8H, m); dC(75 MHz; CDCl3; Me4Si) 11.8, 46.9, 57.4, 118.8, 119.0,
128.6, 128.6, 129.1, 129.2, 129.4, 129.4, 129.6, 130.8, 130.9, 131.6,
131.7, 132.4, 141.8, 143.9, 144.3, 145.4, 145.8, 159.1, 159.8; m/z
(ES) 583.2850 (M + H+. C37H34BF2N4 requires 583.2845).
Acknowledgements
This research was supported by the Program for Research in Third-
Level Institutions administered by the HEA. Thanks to Dr D. Rai
of the CSCB Mass Spectrometry Centre for analyses.
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