Impact of a conformationally restricted receptor on the BF2 chelated azadipyrromethene fluorosensing platform

Article abstract of DOI:10.1039/b513878g

Conformational control of the receptor-fluorophore orientation of BF ₂ chelated azadipyrromethene sensors reveals two photophysically different modes of analyte triggered fluorescence switching both of which exhibit large off-on fluorescence in

Full text of DOI:10.1039/b513878g

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Impact of a conformationally restricted receptor on the BF2 chelated
azadipyrromethene fluorosensing platform{
John Killoran and Donal F. O’Shea*
Received (in Cambridge, UK) 30th September 2005, Accepted 27th January 2006
First published as an Advance Article on the web 27th February 2006
DOI: 10.1039/b513878g
the system.7 The electronic separation of receptor and fluorophore
Conformational control of the receptor–fluorophore orienta-
can also be accomplished by a virtual spacer (C0), though this
approach is significantly less exploited (Fig. 2A).8 In this approach,
the receptor is covalently linked directly to the fluorophore but
their electronic systems remain disconnected as a result of a forced
orthogonal conformation between the receptor and fluorophore. If
a near orthogonal conformation exists, the electron system of the
receptor remains decoupled from that of the fluorophore due to
restricted p-orbital overlap. If this pre-orientation does not exist
then an integrated, or intrinsic, receptor–fluorophore is generated
in which there is no electronic separation of receptor and
fluorophore (Fig. 2B). In this case switching is modulated by an
internal charge transfer (ICT) process with integrated sensors often
having responses both in the ground state and the excited state
during substrate recognition.9
tion of BF2 chelated azadipyrromethene sensors reveals two
photophysically different modes of analyte triggered fluores-
cence switching both of which exhibit large off–on fluorescence
intensity responses to the light input–output of the sensors in
the visible red spectral region.
The development of new molecular fluorescent sensor platforms
for in vivo and in vitro analysis has emerged as an actively investi-
gated research field in recent years. In particular, fluorophores
which absorb and emit in the visible red and near-infrared (NIR)
spectral regions are much sought after for application in areas such
as tumour imaging, drug discovery and assay development.1 The
use of this spectral region is ideally suited to these applications due
to minimal endogenous chromophore absorption and autofluo-
rescence. Examples of chromophore classes which have been used
to address this spectral region are the cyanines,2 squaraines,3
modified BODIPY dyes4 and N-phenyl-1,8,-naphthalimides.5 The
BF2 chelated tetraarylazadipyrromethenes 1 have been recently
reported as a new class of chromophore with high absorption
extinction coefficients (70–80 000 M21 cm21) and fluorescence
quantum yields (0.23–0.36) between 650 and 750 nm (Fig. 1).6 We
are currently advancing the application of this class to visible red
and NIR molecular fluorescent sensors.
To determine the sensing potential and impact of non-restricted
and restricted aniline receptors, we chose to synthesize and examine
the photophysical properties of 1a and 1b (Fig. 1). We anticipated
that both would have the potential to be off–on pH responsive
sensors and, as anilino nitrogens (as part of a more structurally
elaborate receptor) often control the recognition response to metal
ions such as Ca2+ and Mg2+, we anticipated that examination of 1a
and 1b would delineate the design criteria for future receptors.10
We have employed a modular building block approach to the
synthesis of our target compounds by the condensation of 2,4-
diaryl-5-nitroso-pyrroles with 2,4-diarylpyrroles (Scheme 1).11 We
have previously described efficient routes to 2,4-diarylpyrroles and
their a-nitroso derivatives.11,12 The generation of the tetraarylaza-
dipyrromethene 4a was achieved by the condensation of 2-nitroso-
3,5-diphenylpyrrole 3a with the receptor functionalised 2,4-
diarylpyrrole 2a. Tetraarylazadipyrromethene 4b was formed from
the reaction of 2,4-diphenylpyrrole 2b with the 2-nitroso-3,5-
diarylpyrrole3b.ThefinalBF2-chelationsteptoyieldsensors1aand
1b was achieved by the room temperature reaction of 3a or 3b with
borontrifluoride diethyletherate with base in dichloromethane.
In order to obtain aqueous solutions of 1a,b for spectroscopic
analysis the emulsifier Cremophor EL (CrEL) was used. CrEL is
The design of fluorescent probes typically adopts a modular
receptor–spacer–fluorophore approach. The function of the
receptor is to detect the targeted analyte, whereas the role of the
fluorophore is to quantify and relay this detection. The purpose of
the spacer unit is to join the fluorophore to the receptor whilst
keeping the ground state electronic systems of the receptor and the
fluorophore disconnected. The most commonly used spacer is a
saturated methylene unit (C1 spacer), which effectively decouples
Fig. 1 BF2 chelated tetraarylazadipyrromethenes.
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: Experimental
procedures and analytical data, for 1a and 1b. Photophysical organic
solvent study of 1b. X-Ray crystallographic data of 1a and 1b. See DOI:
10.1039/b513878g
Fig. 2 A; Schematic of a fluorescent sensor with a virtual spacer.
Rectangle = fluorophore; oval = orthogonal receptor for substrate S; S =
substrate. B; Schematic of an intergral fluorescent sensor. Rectangle =
fluorophore; circle = co-planar receptor for substrate S; S = substrate.
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Chem. Commun., 2006, 1503–1505 | 1503

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Fig. 4 pH responsive absorbance spectra using a 1 6 1025 M solution
of 1a in water–CrEL. Red trace pH = y0.2, green trace pH = 2.5. INaCl
=
150 mmol L21. Inset shows the sigmoidal plot predicting an apparent pKa
value of less than 1.0 at 25 uC.
Scheme 1 Synthetic routes to 1a and 1b. (i) Acetic anhydride, acetic acid,
100 uC, 1 h. (ii) BF3?OEt2, DIEA, CH2Cl2, rt, 6 h.
single band appeared at 662 nm (Fig. 4, red trace). Plotting the
disappearance of the 750 nm band against pH gave a similar
sigmoidal response to the fluorescence titration (Fig. 4, inset).
The UV-visible spectral characteristics are indicative of a co-
planar receptor–fluorophore orientation in 1a. The molecular
structure of 1a, obtained by X-ray crystallography, confirmed a
small torsion angle about the receptor–fluorophore bond of only
18.5u (Fig. 5).{
We expected that the introduction of methyl groups on the
anilino ring of 1b would exert a steric influence on the
conformation about the aryl–fluorophore bond. Excitation of 1b
at 630 nm in aqueous media gave a very weak emission when
unprotonated but showed a strong acid induced fluorescence
enhancement with a l maximum at 673 nm and a fluorescence
intensity enhancement in excess of 250 fold (Fig. 6). A plot of pH
versus fluorescence provided a sigmoidal curve, which calculated
an apparent pKa of 2.0 (Fig. 6, inset). This higher value when
compared to 1a would indicate that some disconnection of the
sensor sub-units had been achieved.
frequently exploited in vivo as a delivery agent for drugs with poor
water solubility and we have found it suitable for the cellular
delivery of a range of analogues of 1.6b
We first examined the excited state response of aqueous
formulated solutions of 1a to varying acidic conditions. We were
pleased to observe virtually complete quenching of the fluorescence
in the pH range where the amine receptor remained unprotonated
(Fig. 3, green trace). Upon protonation, the fluorescence spectrum
showed a strong proton induced fluorescence enhancement with a
wavelength of maximum fluorescence at 683 nm (Fig. 3, red trace).
A sigmoidal plot of pH versus fluorescence intensity predicted an
apparent pKa of less than 1.0 (Fig. 3, inset).13 Encouragingly, the
enhancement of fluorescence intensity was greater than 250 fold
between the sensor off and on positions. We anticipated the lack of
a disconnecting modality between the receptor and fluorophore in
1a would facilitate an ICT between the receptor and the
fluorophore sub-units resulting in quenching of fluorescence at
higher pH values, but that upon protonation the ICT would be
suppressed and a strong fluorescence established.
This was corroborated by examination of the absorbance
properties during analyte detection. The visible red absorbance
spectrum of unprotonated 1b revealed that it consisted of a single
band with a wavelength maximum of 639 nm (Fig. 7, green trace).
During acid titration minor changes were recorded with the l
maximumundergoingasmallbathochromicshiftto651nmandthe
bandwidth decreasing. Overall the variations were minor especially
when compared to 1a and provide a powerful demonstration of the
effects of sub-unit pre-orientation in fluorescence sensor design. A
singlecrystalX-raystructurerevealedthatthetorsionanglebetween
the receptor and fluorophore had increased to 53.4(7)uas a result of
the methyl substituents (Fig. 8).§
A consequence of exploiting ICT formation as an excited state
quenching mechanism is the reorganisation of the ground state
properties of 1a upon analyte recognition. The absorbance
spectrum of unprotonated 1a has two bands in the red spectral
region centred at 618 and 750 nm (Fig. 4, green trace). At pH values
less than 2, upon protonation of the receptor, the absorbance bands
at 618 and 750 nm progressively reduced in intensity and a new
Fig. 3 pH responsive fluorescence spectra of 1a. Red trace pH = y0.2,
M
green trace pH = 2.5. Excitation at 630 nm, slit widths 5 nm, 1 6 1026
in water–CrEL. INaCl = 150 mmol L21. Inset shows the sigmoidal plot
predicting an apparent pKa value less than 1.0 at 25 uC.
Fig. 5 X-Ray crystal structure of 1a. Torsion angle (Q) C24–C25–C27–
C28 (marked by arrows) = 18.5(9)u.
1504 | Chem. Commun., 2006, 1503–1505
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changes would allow for the tailoring of sensor properties to an
application requirement. In addition the described synthetic route
would readily allow for analogues functionalised with other
substrate specific receptors to be generated.
This work was supported by the Program for Research in
Third-Level Institutions administered by the HEA. We thank Dr
D. Rai of the CSCB Mass Spectrometry Centre and Dr H.
Mueller-Bunz of the UCD Crystallographic Centre for analyses.
Notes and references
¯
{ Crystal data for compound 1a: C34H27BN4F2, M = 540.41, triclinic, P1
3
Fig. 6 pH responsive fluorescence spectra of 1b. Red trace pH = 1.0,
green trace pH = 4.0. Excitation at 630 nm, slit widths 5 nm, 1 6 1026
˚
˚
(#2), a = 10.755(3), b = 10.824(3), c = 12.163(4) A, V = 1288.9(7) A , m(Mo-
Ka) = 0.710 mm21, T = 113(2) K, Z = 2, Dc = 1.392 Mg m23, F(000) =
564, independent reflections 4977 (Rint = 0.0530). Final R for reflections
with I > 2s(I) R1 = 0.0944, wR2 = 0.2168, for all data R1 = 0.1443, wR2 =
0.2358. CCDC 285002.
M
in water–CrEL. INaCl = 150 mmol L21. Insert shows the sigmoidal plot
predicting an apparent pKa value of 2.0 at 25 uC.
§ Crystal data for compound 1b: C36H31BN4F2, M = 68.46, monoclinic,
As the effect of microenvironment polarity on excited state
sensing mechanisms is well established,14 we have examined the
emission sensitivity of 1a,b to polarity changes by determining the
spectral properties in four solvents of varying dipolarity. We were
pleased to observe that emissions from unprotonated 1a,b in
DMF, THF, 1,4-dioxane and cyclohexane were very weak and in
each case large fluorescence enhancements were recorded upon
addition of trifluoroacetic acid (ESI{).
In conclusion, we have outlined the flexible modular synthesis of
a new class of visible red fluorosensors based upon either an
integrated or virtual spacer design. Sensor performance is excellent
with large off–on fluorescence intensity responses and low
microenvironment polarity effects. The ability to control the
analyte induced photophysical responses by subtle conformational
3
˚
˚
C2, a = 42.555(7), b = 5.3195(9), c = 12.781(2) A, V = 2814.3(8) A , m(Mo-
Ka) = 0.710 mm21, T = 293(2) K, Z = 4, Dc = 1.342 Mg m23, F(000) =
1192, independent reflections 3777 (Rint = 0.0602). Final R for reflections
with I > 2s(I) R1 = 0.0509, wR2 = 0.0981, for all data R1 = 0.0854, wR2 =
0.1109. CCDC 285001. For crystallographic data in CIF or other electronic
format see DOI: 10.1039/b513878g
1 (a) V. Ntziachristos, J. Ripoll, L. V. Wang and R. Weissleder, Nat.
Biotechnol., 2005, 23, 313; (b) R. Weissleder, Nat. Rev., 2002, 2, 1; (c)
M. Rudin and R. Weissleder, Nat. Rev., 2003, 2, 123; P. Mitchell, Nat.
Biotechnol., 2001, 19, 1013.
2 (a) X. Peng, F. Song, B. Lu, Y. Wang, W. Zhou, J. Fan and Y. Gao,
J. Am. Chem. Soc., 2005, 127, 4170; (b) B. Ozmen and B. U. Akkaya,
Tetrahedron Lett., 2000, 41, 9185.
3 (a) B. Arunkumar and A. Ajayaghosh, Chem. Commun., 2005, 599; (b)
W. Pham, R. Weissleder and C.-H. Tung, Angew. Chem., Int. Ed., 2002,
41, 3659; (c) U. Oguz and B. U. Akkaya, J. Org. Chem., 1998, 63, 6059.
4 (a) Z. Shen, H. Ro¨hr, K. Rurack, H. Uno, M. Spieles, B. Schulz,
G. Reck and N. Ono, Chem.–Eur. J., 2004, 10, 4853; (b) K. Rurack,
M. Kollmannsberger and J. Daub, Angew. Chem., Int. Ed., 2001, 40,
385.
5 K. Rurack, U. Resch-Genger, J. L. Bricks and M. Spieles, Chem.
Commun., 2000, 2103.
6 (a) J. Killoran, L. Allen, J. F. Gallagher, W. M. Gallagher and
D. F. O’Shea, Chem. Commun., 2002, 1862; (b) A. Gorman, J. Killoran,
C. O’Shea, T. Kenna, W. M. Gallagher and D. F. O’Shea, J. Am.
Chem. Soc., 2004, 126, 10619; (c) W. M. Gallagher, L. T. Allen,
C. O’Shea, T. Kenna, M. Hall, J. Killoran and D. F. O’Shea, Br. J.
Cancer, 2005, 92, 1702; (d) S. O. McDonnell, M. J. Hall, L. T. Allen,
A. Byrne, W. M. Gallagher and D. F. O’Shea, J. Am. Chem. Soc., 2005,
127, 16360.
7 A. P. de Silva, H. Q. N. Gunaratne, T. Gunnlaugsson, A. J. M. Huxley,
C. P. McCoy, J. T. Rademacher and T. B. Rice, Chem. Rev., 1997, 97,
1515.
8 (a) K. Rurack and U. Resch-Genger, Chem. Soc. Rev., 2002, 31, 116; (b)
M. Maus and K. Rurack, New J. Chem., 2000, 24, 677; (c)
M. Kollmannsberger, T. Gareis, S. Heinl, J. Breu and J. Daub,
Angew. Chem., Int. Ed. Engl., 1997, 36, 1333.
Fig. 7 pH responsive absorbance spectra using a 1 6 1025 M solution
of 1b in water–CrEL. Red trace pH = 1.0, green trace pH = 4.0. INaCl
150 mmol L21
=
.
9 (a) J. F. Callan, A. P. de Silva, J. Ferguson, A. J. M. Huxley and
A. M. O’Brien, Tetrahedron, 2004, 60, 11125; (b) K. Rurack, A. Danel,
K. Rotkiewicz, D. Grabka, M. Spieles and W. Rettig, Org. Lett., 2002,
4, 4647.
10 J. H. Hartley, T. D. James and C. J. Ward, J. Chem. Soc., Perkin Trans.
1, 2000, 3155.
11 M. J. Hall, S. O. McDonnell, J. Killoran and D. F. O’Shea, J. Org.
Chem., 2005, 70, 5571.
12 J. Killoran, J. F. Gallagher, P. V. Murphy and D. F. O’Shea, New J.
Chem., 2005, 29, 1258.
13 pKa values in a micellar microenvironment are often lower than might
be anticipated. M. S. Fernandez and P. Fromherz, J. Phys. Chem., 1977,
81, 1755.
14 (a) B. Bag and P. K. Bharadwaj, J. Phys. Chem. B, 2005, 109, 4377; (b)
R. A. Bissell, A. P. de Silva, W. T. M. L. Fernando,
S. T. Patumathavithana and T. K. S. D. Samarasinghe, Tetrahedron
Lett., 1991, 32, 425.
Fig. 8 X-Ray crystal structure of 1b. Torsion angle (Q) C19–C18–C21–
C26 (marked by arrows) = 53.4(7)u.
This journal is ß The Royal Society of Chemistry 2006
Chem. Commun., 2006, 1503–1505 | 1505