Azide conjugatable and pH responsive near-infrared fluorescent imaging probes

Article abstract of DOI:10.1021/ol902140v

"Chemical Equation Presented" The synthesis and photophysical characteristics of a pH responsive near-Infrared fluorescence Imaging probe Is described. A key feature is the ability to conjugate the probe by an alkyne-azide cycloaddition reaction and its r

Full text of DOI:10.1021/ol902140v

ORGANIC
LETTERS
2009
Vol. 11, No. 23
5386-5389
Azide Conjugatable and pH Responsive
Near-Infrared Fluorescent Imaging
Probes
Julie Murtagh, Daniel O. Frimannsson, and Donal F. O’Shea*
Centre for Synthesis and Chemical Biology, School of Chemistry and Chemical
Biology, UniVersity College Dublin, Belfield, Dublin 4, Ireland
donal.f.oshea@ucd.ie
Received September 16, 2009
ABSTRACT
The synthesis and photophysical characteristics of a pH responsive near-infrared fluorescence imaging probe is described. A key feature is
the ability to conjugate the probe by an alkyne-azide cycloaddition reaction and its reversible response of fluorescence intensity across the
physiological pH range.
The use of high-sensitivity fluorescence imaging as a reporter
of in vitro and in vivo molecular processes of biological
systems is a powerful research tool.1 The recent technological
advances in noninvasive small animal fluorescence imaging
and the future prospect of human fluorescence imaging
applications has given rise to a resurgent interest in suitable
small molecule fluorophores.2 In the case of in vivo imaging
it is highly preferential to use fluorophores with absorption/
emission profiles in the far visible red or near-infrared (NIR)
spectral regions (0.7-1.4 µm), as at lower wavelengths,
strong interference from endogenous chromophores is prob-
lematic.3 These stringment spectral requirements have limited
such applications to a select few organic fluorophore classes
such as cyanines, squaraines, and seminapthofluorones.4-6
In conjunction with optimal photophysical characteristics,
two complementary approaches are often adopted to enhance
differentiation of the imaging target from background fluo-
rophore. The first, and more common, is the use of
conjugated fluorophores generated by the covalent attachment
(3) (a) Ntziachristos, V.; Ripoll, J.; Wang, L. V.; Weissleder, R. Nat.
Biotechnol. 2005, 23, 313. (b) Frangioni, J. V. Curr. Opin. Chem. Biol.
2003, 626
.
(4) For examples see: (a) Lin, Y.; Weissleder, R.; Tung, C.-H.
Bioconjugate Chem. 2002, 13, 605. (b) Citrin, D.; Scott, T.; Sproull, M.;
Menard, C.; Tofilon, P. J.; Camphausen, K. Int. J. Radiat. Oncol. Biol.
Phys. 2004, 58, 536. (c) Ballou, B.; Ernst, L. A.; Waggoner, A. S. Curr.
Med. Chem. 2005, 12, 795
.
(5) For examples see: Johnson, J. R.; Fu, N.; Arunkumar, E.; Leevy,
W. M.; Gammon, S. T.; Piwnica-Worms, D.; Smith, B. D. Angew. Chem.,
Int. Ed. 2007, 46, 5528
(6) Yang, Y.; Lowry, M.; Xu, X.; Escobedo, J. O.; Sibrian-Vazquez,
M.; Wong, L.; Schowalter, C. M.; Jensen, T. J.; Fronczek, F. R.; Warner,
.
(1) Rudin, M.; Weissleder, R. Nat. ReV. Drug DiscoVery 2003, 2, 123.
(2) De Grand, A. M.; Frangioni, J. V. Technol. Cancer Res. Treat. 2003,
2, 1.
I. M.; Strongin, R. M. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 8829
.
10.1021/ol902140v CCC: $40.75
Published on Web 11/02/2009
 2009 American Chemical Society

of a targeting (bio)-molecule to the fluorescent probe, which
facilitates a target-selective accumulation of fluorophore.7
An alternative approach is the modulation of the fluorescence
signal intensity (from low to high) in response to a specific
molecular recognition at the endogenous target.8,9 In spite
of the success of both strategies a combination of both
processes is rarely investigated. Herein, we outline our
strategy to achieve a prototype azide conjugatable and pH
responsive NIR fluorescent platform. The on/off fluorescence
switching operation would be governed by a straightforward
phenol/phenolate interconversion on the fluorophore with
conjugation to a molecular targeting motif via an alkyne-azide
cycloaddition. To date, there are few literature reports of NIR
pH responsive fluorophores in spite of their potential imaging
applications for disease states that can induce localized intra-
and extracellular pH changes such as cancers, renal failure,
and ischemia.10 Recently, pH responsive cyanine fluoro-
phores covalently attached to bacteriophage particles have
been reported.11
would be capable of conjugation via azide cycloaddition and
the intensity of fluorescence output would be controlled by
a phenol/phenolate interconversion.
The synthetic route commenced with an addition of
nitromethane to chalcone 3, which gave 1-(4-hydroxyphe-
nyl)-4-nitro-3-phenylbutan-1-one 4 in 73% yield (Scheme
1). Subsequent generation of the bis-phenol-substituted
Scheme 1. Synthesis of Fluorophore 2
We have recently developed the BF2-chelated tetraary-
lazadipyrromethene fluorophore class 1 with excellent ab-
sorption and fluorescence properties in the 650-750 nm
spectral region.12 For example, the tetraaryl analogue 1 has
an absorption λmax at 688 nm (ε ) 85 000 dm-3 mol-1 cm-1)
and emission at 716 nm (φF ) 0.36) in chloroform (Figure
1). These promising photophysical characteristics have
azadipyrromethene 5 was achieved by the reflux of 4 with
ammonium acetate in ethanol for 24 h. Filtration of the
precipitate from the crude reaction mixture gave the pure
product in 51% yield. Compound 5 was converted to its BF2-
chelated analogue 6 with BF3 diethyletherate and diisopro-
pylethylamine (DIEA) in dichloromethane for 24 h. An
Figure 1. BF2-chelated tetraarylazadipyrromethenes.
encouraged the adaption of this class to specific functions
such as fluorescent sensors13 and photodynamic therapeutic
agents.14 The synthetic strategy adopted for our current goal
was to develop a short route to the monoalkyne-monophenol-
substituted analogue 2 (Figure 1). It was anticipated that 2
(11) (a) Hilderbrand, S. A.; Kelly, K. A.; Niedre, M.; Weissleder, R.
Bioconjugate Chem. 2008, 19, 1635. (b) Zhang, Z.; Achilefu, S. Chem.
Commun. 2005, 5887.
(12) (a) Gorman, A.; Killoran, J.; O’Shea, C.; Kenna, T.; Gallagher,
W. M.; O’Shea, D. F. J. Am. Chem. Soc. 2004, 126, 10619. (b) Killoran,
J.; Allen, L.; Gallagher, J. F.; Gallagher, W. M.; O’Shea, D. F. Chem.
Commun. 2002, 1862.
(7) For examples see: (a) Citrin, D.; Lee, A. K.; Scott, T.; Sproull, M.;
Menard, C.; Tofilon, P. J.; Camphausen, K. Mol. Cancer Ther. 2004, 3,
481. (b) Petrovsky, A.; Schellenberger, E.; Josephson, L.; Weissleder, R.;
Bogdanov, A. Cancer Res. 2003, 8, 1936. (c) Ramjiawan, B.; Maiti, P.;
Aftanas, A.; Kaplan, H.; Fast, D.; Mantsch, H. H.; Jackson, M. Cancer
2000, 89, 1134.
(13) (a) Killoran, J.; McDonnell, S. O.; Gallagher, J. F.; O’Shea, D. F.
New J. Chem. 2008, 483. (b) Loudet, A.; Bandichhor, R.; Wu, L.; Burgess,
K. Tetrahedron 2008, 64, 3642. (c) Gawley, R. E.; Mao, H.; Mahbubul
Haque, M.; Thorne, J. B.; Pharr, J. S. J. Org. Chem. 2007, 72, 2187. (d)
Killoran, J.; O’Shea, D. F. Chem. Commun. 2006, 1503. (e) Hall, M. J.;
Allen, L. T.; O’Shea, D. F. Org. Biomol. Chem. 2006, 776. (f) McDonnell,
S. O.; O’Shea, D. F. Org. Lett. 2006, 8, 3493.
(8) Rao, J.; Dragulescu-Andrasi, A.; Yao, H. Curr. Opin. Biotechnol.
2007, 18, 17
.
(14) (a) McDonnell, S. O.; Hall, M. J.; Allen, L. T.; Byrne, A.; Gallagher,
W. M.; O’Shea, D. F. J. Am. Chem. Soc. 2005, 16360. (b) Gallagher, W. M.;
Allen, L. T.; O’Shea, C.; Kenna, T.; Hall, M.; Killoran, J.; O’Shea, D. F.
Br. J. Cancer 2005, 92, 1702. (c) Byrne, A. T.; O’Connor, A.; Hall, M.;
Murtagh, J.; O’Neill, K.; Curran, K.; Mongrain, K.; Rousseau, J. A.;
Lecomte, R.; McGee, S.; Callanan, J. J.; O’Shea, D. F.; Gallagher, W. M.
Br. J. Cancer 2009, 101, 1565.
(9) (a) Fonovic´, M.; Bogyo, M. Curr. Pharm. Des. 2007, 13, 253. (b)
Blum, G.; Mullins, S. R.; Keren, K.; Fonovic, M.; Jedeszko, C.; Rice, M. J.;
Sloane, B. F.; Bogyo, M. Nat. Chem. Biol. 2005, 1, 203
.
(10) (a) Gillies, R. J.; Raghunand, N.; Garcia-Martin, M. L.; Gatenby,
R. A. IEEE Eng. Med. Biol. Mag. 2004, 57. (b) Stubbs, M.; McSheehy,
P. M. J.; Griffiths, J. R.; Bashford, C. L. Mol. Med. Today 2000, 6, 15.
Org. Lett., Vol. 11, No. 23, 2009
5387

isolated yield of 69% was obtained following chroma-
tography on silica gel. The synthesis of our target
compound 2 required a desymmetrization step involving
alkylation of one of the two phenol groups.15 This
alkylation was carried out with 2.2 equiv of propargyl-
tosylate and NaH in THF for 4 h under reflux. It was found
that these specific conditions biased the distribution of
mono-2 and bis-alkylated 7 products toward the mono-
substituted derivative (Scheme 1). Exploiting the large
polarity difference between 2 and 7 allowed for facile
separation on silica gel chromatography and gave products
in isolated yields of 30% and 25%, respectively.
aqueous solutions showed a greater than 15-fold fluorescence
intensity differential between pH 6 and 8 with virtually
complete suppression of fluorescence signal at pH 9 (Figure
2). A sigmoidal plot of pH versus fluorescence intensity
predicted an apparent pKa of 6.9 (Figure 2, inset).16 As would
be expected for an ICT process, the UV-visible spectrum
of 2 was strongly influenced by pH. The absorption band at
700 nm was progressively reduced in intensity with increas-
ing pH and a new band appeared at 775 nm with an isosbestic
point at 740 nm indicative of the formation of a monodepro-
tonated species (SI).
The mild aqueous conditions required for azide-alkyne
cycloadditions offer distinct advantages when utilized for
bioconjugation reactions.17 To demonstrate functional group
tolerance of azide reactions with 2, three azides containing
amino, carboxy, and carbohydrate substituents were tested.
The optimized reaction conditions with 1-azido-1-deoxy-ꢀ-
D-galactopyranoside, 4-azidobutyric acid, and (2-azidoeth-
yl)carbamic acid tert-butyl ester were identified as CuSO4/
Cu/sodium ascorbate in THF/H2O (3:1) under reflux for 3 h
(Scheme 2). The copper salts were removed by aqueous
Spectral properties of 2 in organic solvents were very
similar to those of 1 with the absorption and emission
maxima of 2 in CHCl3 at 680 and 708 nm, respectively, with
a high fluorescence quantum yield (Φf) of 0.37 (Table 1,
Table 1. Absorption/Emission Properties of 2
entry
solvent
λmax abs (nm)a
λmax flu (nm)b
,
1
2
3
4
CHCl3
C7H8
CH3OH
H2O/CrEL
680
685
688
700
708c d
711
716
729
Scheme 2. Azide Cycloaddition Reactions
a 5 × 10-6 M. b 5 × 10-7 M. c Φf ) 0.37 (1 as standard). d Addition of
DBU resulted in a 75-fold reduction of fluorescence intensity (SI).
entry 1, SI). Spectral characteristics showed a slight depen-
dence upon solvent dipolarity with bathochromatic shifts of
8 nm for absorbance and emission maxima in methanol
(Table 1, entry 3, SI). Additionally, an aqueous solution
generated by formulation using Cremophor EL12a showed
further small bathochromic shifts (700/729 nm) when
compared to organic solvents (entry 4, SI).
In contrast to 1, the spectral properties of 2 displayed a
striking response across the physiological pH range (Figure
2). The excited state response of 2 at the λmax of 729 nm in
Figure 2. pH responsive fluorescence spectra of 2: red trace, pH
6.0; gray trace, pH 9.0. Excitation at 640 nm, slit widths 10 nm, 5
× 10-7 M in water/CrEL. INaCl ) 150 mmol/L. Inset: Sigmoidal
plot predicting a pKa value of 6.9.
extraction and cycloadducts 8a-c were isolated in good
yields of 68% to 88% following either recrystallization from
methanol or column chromatography.
5388
Org. Lett., Vol. 11, No. 23, 2009

We were pleased to observe that the UV-vis/fluorescence
spectra of 8a-c in methanol showed little difference from each
other or from the alkyne derivative 2 demonstrating that the
cycloaddition reaction had negligible effect on these spectral
characteristics (Figure 3). As a representative example, the pH
231 cells for 1 h followed by blue nuclear costaining with
4,6-diamidino-2-phenylindole (DAPI). Dual-color imaging
with confocal laser scanning microscopy (CLSM) showed a
distinct red emission from 8a localized to the cyctosol (SI).
Illustrative reVersible on/off switching of intracellular 8a
could be achieved by treating a population of dual-stained
cells with aqueous carbonate buffer of pH 8.0 or acidic buffer
of pH 6.6 (Figure 5, SI). Imaging of the same cell population
Figure 5. CLSM images of 8a (red) with fixed MDA-MB-231 cells
costained with nuclear stain DAPI (blue). Cell population imaged
following treatment with (a) pH 6.6 buffer and (b) pH 8.0 buffer.
Figure 3
.
Normalized UV-visible (5 × 10-6 M) and fluorescence
(5 × 10-7 M) spectra of 8a (λmax 687/716 nm; brown), 8b (λmax
687/716 nm; black), and 8c (λmax 687/716 nm; blue) in MeOH.
following addition of basic buffer showed almost complete
quenching of the red emission of 8a with the blue nuclear
DAPI emission still clearly visible. In contrast, following
the addition of pH 6.6 buffer to the cells the emission from
8a was re-established. The difference in averaged whole cell
red fluorescence taken from off-cells to on-cells was 6-fold.
In summary, an efficient synthesis, photophysical char-
acteristics, and intracellular demonstration of a new pH
responsive fluorochrome platform with emission at 730 nm
is outlined. The noninvasive in vivo imaging potential of
bioconjugated derivatives of 2 is currently under investigation
and will be described in due course.
responsive nature of the galactose conjugated derivative 8a was
examined and shown to have similar ground and excited state
responses as that of 2 with pKa of 6.9 (SI). Analysis of the
fluorescence intensity of 8a at five different pH values illustrated
how a significant bias toward higher fluorescence intensity at
low physiological pH regions was accomplished. For example,
comparison of the measured fluorescence intensity difference
from pH 6.1 to 7.2 was greater than 2-fold and the difference
between pH 6.6 and 8 was 6-fold (Figure 4).
Acknowledgment. Funding support from Science Foun-
dation Ireland is gratefully acknowledged. The authors thank
the CSCB Mass Spectrometry and NMR Centres.
Supporting Information Available: All experimental
procedures and spectra. This material is available free of
charge via the Internet at http://pubs.acs.org.
OL902140V
(15) For an alternative synthetic approach to unsymmetrical derivatives
see: Hall, M. J.; McDonnell, S. O.; Killoran, J.; O’Shea, D. F. J. Org. Chem.
2005, 70, 5571.
(16) pKa values in a micellar microenvironment are often lower than
anticipated.
Figure 4. Relative fluorescence intensity at 730 nm of 8a at pH
6.1, 6.6, 7.2, 7.6, and 8.0. Excitation at 640 nm, slit widths 20 nm,
(17) For recent examples see: (a) Laughlin, S. T.; Baskin, J. M.;
Amacher, S. L.; Bertozzi, C. R. Science 2008, 320, 664. (b) Gramlich,
P. M. E.; Warnche, S.; Gierlich, J.; Carell, T. Angew. Chem., Int. Ed. 2008,
47, 3442. (c) Mindt, T. L.; Struthers, H.; Brans, L.; Anguelov, T.;
Schweinsberg, C.; Maes, V.; Tourwe´, D.; Schibli, R. J. Am. Chem. Soc.
2006, 28, 15096. (d) Casas-Solvas, J. M.; Vargas-Berenguel, A.; Capita´n-
Vallvey, L. F.; Santoyo-Gonza´lez, F. Org. Lett. 2004, 3687.
5 × 10-7 M in water/CrEL. INaCl ) 150 mmol/L.
Demonstration of cell internalization and in vitro pH
response of 8a was achieved by incubation with MDA-MB-
Org. Lett., Vol. 11, No. 23, 2009
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