Optimized pH-responsive cyanine fluorochromes for detection of acidic environments

Article abstract of DOI:10.1039/b703764c

Modulation of pH-responsive cyanine dye pK_a values via heteroatom substitution allows for design of fluorescent reporters that are tuned for potential imaging of biologically relevant acidic environments. The Royal Society of Chemistry.

Full text of DOI:10.1039/b703764c

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Optimized pH-responsive cyanine fluorochromes for detection of acidic
environments{
Scott A. Hilderbrand* and Ralph Weissleder
Received (in Cambridge, UK) 13th March 2007, Accepted 2nd April 2007
First published as an Advance Article on the web 20th April 2007
DOI: 10.1039/b703764c
pH-responsive fluorochromes that are optimized for visualization
of biological acidic environments.
Modulation of pH-responsive cyanine dye pKa values via
heteroatom substitution allows for design of fluorescent
reporters that are tuned for potential imaging of biologically
relevant acidic environments.
The traditional synthesis of cyanine dyes involves the base-
catalyzed condensation of indolium salts with malonaldehyde
dianilide or gluconaldehyde dianilide in pyridine.16,17 Under these
conditions, the nitrogen atoms of the indolenine derivatives, used
in the synthesis of pH-sensitive cyanines, are deprotonated. This
decreases the acidity of the indolenine 2-methyl hydrogens and
deactivates them for reaction with malonaldehydes. We have
found that dye condensation can be effected under mildly acidic
conditions. The pH sensitive fluorochromes can be prepared
without the need for any base catalyst using commercially
available substituted malonaldehydes (Scheme 1). The reaction
of two equivalents of 2,3,3-trimethylindolenine-5-sulfonic acid,
which is readily prepared from 4-hydrazinobenzenesulfonic acid,
with one equivalent of the desired malonaldehyde in methanol
generates the crude pH sensitive dyes as dark blue solutions. The
desired pH-responsive dyes can also be synthesized from the
corresponding malonaldehyde dianilide, however in lower yields.
Purification by reverse-phase C18 chromatography followed by
cation exchange chromatography affords the pure fluorochromes
as bright blue solids after lyophilization. Using this synthetic route,
it is possible to prepare cyanine derivatives with different electron
withdrawing substituents on the polymethine backbone of the
dyes, which allows for modulation of the fluorophore pKa.
The emergence of near-infrared (NIR) fluorescence imaging
techniques using fluorescent probes has spurred the need for the
design of new environment-responsive dyes to visualize biological
processes in vivo. For efficient in vivo imaging, dyes that absorb
and emit in the far-red or NIR are necessary due to the increased
optical transparency and lower tissue autofluorescence in this
regime.1 A variety of NIR probes for interrogation of enzyme
activity,2,3 nitric oxide,4 zinc,5 and proton concentrations6–9 have
been reported. Imaging local pH is a promising target for the
development of new probes. Acidic environments are known to be
associated with solid tumors,10 cystic fibrosis,11 asthma,12 and a
variety of renal conditions. For example, intratumoral pH was
recently reported to range from 6.3 to 7.0,10,13 and lung airway
pHs as low as 5.2 have been recorded in asthmatics.12 Acid
responsive probes may also find application for cellular imaging of
acidic intracellular vesicles, such as endosomes, lysosomes, and
phagosomes, where pH can range from 4.5 to 6.5.14 Existing far-
red or NIR pH-responsive dyes are not optimized for in vivo
imaging of acidic environments due to their poor optical
properties, low aqueous solubility, or sub-optimal pKa values,
often greater than 7.0,7,9 that are not matched for visualization of
physiologically relevant acidic conditions. For example, a dye with
a pKa of 7.4 would be 50% activated at pH 7.4, this translates into
high background fluroescence and a two-fold maximum signal
increase as the pH of the local environment is lowered. There is
therefore a need to design novel bright, water-soluble, pH-sensitive
fluorochromes that are tuned for imaging acidic pH in vivo.
Cyanine dyes are a well known platform, which may be
modified to confer pH responsive properties.15 If one or both of
the indole nitrogen atoms on a cyanine dye are not alkylated, the
dye becomes sensitive to pH (Fig. 1). In acidic environments,
protonation of the indole nitrogen atoms gives rise to long-
wavelength absorption and strong fluorescence. Deprotonation of
the nitrogen atoms results in a blue-shifted absorption band and
little or no observable fluorescence emission. In this report, we
detail the synthesis and characterization of new water-soluble
Center for Molecular Imaging Research, Massachusetts General
Hospital, Harvard Medical School, Charlestown, Massachusetts, 02129,
USA. E-mail: Scott_Hilderbrand@hms.harvard.edu;
Fax: +1 617 726 5708; Tel: +1 617 726 5788
{ Electronic supplementary information (ESI) available: Detailed synthetic
procedures and characterization for all compounds. See DOI: 10.1039/
b703764c
Fig. 1 The core structure of a pH insensitive pentamethine cyanine dye
(a) and pH dependent equilibrium of the pH-responsive cyanine dyes (b).
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Fig. 3 Fluorescence-based pH titrations of 1 (triangles) and 2 (squares)
at various pH values in 50 mM phosphate–citric acid buffer. The pKa
values for 1 and 2 are 6.3 and 5.7, respectively. Excitation is at 590 nm and
the fluorescence intensity is integrated from 620 to 850 nm.
Scheme 1 Synthesis of the pH-responsive dyes.
Table 1 Optical properties of the pH-responsive fluorochromes in
aqueous media
b
in water.18 Following the expected heavy-atom effects, the
fluorescence quantum yields for the dyes show a sequential
decrease as the size of the substituent heteroatom increases in the
order H . Cl . Br. The deprotonated conjugate bases of the dyes
have absorption maxima at 480, 487 and 489 nm and weak
fluorescence emission at 648, 643 and 637 nm for 1, 2 and 3,
respectively.
Dye
labsa/nm
lema/nm
ea/M21 cm21
Ha (%)
pKa
1
2
3
a
638
642
639
664
663
659
141 000
167 000
146 000
12.9
6.4
3.5
6.3
5.7
5.7
b
Measured in 50 mM glycine buffer pH 3.0. Determined by pH
titration in 50 mM phosphate–citric acid buffer.
The addition of electron-withdrawing halogen substituents on
the pentamethine core of the dyes results in depression of the
indole nitrogen pKa values. Unsubstituted dye 1 has a pKa of 6.3;
whereas the chloride and bromide substituted derivatives have pKa
readings of 5.7. As a result of the low pKa values for dyes 2 and 3
at pH 7.4 less than 2% of the dye molecules are in their protonated,
fluorescent form. Dye 1 only displays minimal background
fluorescence at physiological pH, showing 7% activation (Fig. 2).
As with other pH sensitive fluorophores, these dyes can be used to
determine a wide range of pH values, but have maximum
sensitivity approximately ¡0.9 pH units from the pKa of the dye,
which corresponds to 10–90% protonation (Fig. 3). Thus, dye 1
has optimal sensitivity between pH 5.4 and 7.2, and is well suited
for potential visualization of the acidic environments associated
with tumors, which range from pH 6.3 to 7.0.10 Fluorochromes 2
and 3 with a more acidic pKa of 5.7 show strong fluorescence
response between pH 4.8 and 6.6, indicating these dyes are well
matched for imaging acidic intracellular vesicles or renal diseases
with pH values between 4.5 and 6.5.14
The pH-responsive dyes prepared in this report (Table 1) are
water-soluble and show strong long-wavelength fluorescence
emission in acidic media (Fig. 2). The two sulfonate groups on
the fluorochromes are also sufficient to prevent the formation of
dye–dye aggregates up to concentrations of at least 5 mM (Fig. S7,
ESI{), enabling their use in aqueous environments without the
need of organic co-solvents. Typical fluorescence reflectance
imaging (FRI) instruments only require concentrations in the
nanomolar to low micromolar range for visualization of NIR
fluorochromes. All three dyes have similar absorption and
emission properties (Table 1). When protonated, the dyes show
strong absorption near 640 nm with extinction coefficients greater
than 140 000 M21 cm21, and display strong fluorescence emission
near 660 nm. The quantum yields H of the dyes are 12.9, 6.4 and
3.5% for 1, 2 and 3, respectively (Table 1). These values compare
favorably with indocyanine green, a clinically approved non-pH
sensitive NIR imaging agent, which has a quantum yield of 2.7%
To demonstrate the potential utility of these pH-responsive
dyes, preliminary experiments using biological samples were
undertaken. Disorders in the urinary tract, such as renal tubular
acidosis,19 are characterized by altered urine pH. In order to image
the urinary tract in vivo, one must be able to differentiate between
urine, which is slightly acidic, and blood. Fig. 4 clearly
demonstrates that dye 2, which has low background fluorescence
emission at physiological pH, can distinguish between samples of
mouse urine and blood by fluorescence reflectance imaging.
In summary, new long-wavelength pH-responsive cyanine dyes
were prepared by a simple synthetic route. These fluorophores
show an increase in emission as the pH of their local environment
decreases. The pKa values for the dyes can be modulated by
variation of the heteroatom substituents on the polymethine-
conjugated system of the molecules. The dyes are water-soluble,
Fig. 2 Absorption spectrum of 2 in 50 mM phosphate–citric acid buffer
at pH 9.0 (solid line). Absorption (dashed line) and emission (dotted line)
spectra of 2 in 50 mM phosphate–citric acid buffer at pH 3.5.
2748 | Chem. Commun., 2007, 2747–2749
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Notes and references
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Fig. 4 Phantoms containing 1 mM dye 2 in: (A) PBS, pH 7.40; (B) mouse
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have large extinction coefficients, good quantum yields, and show
no tendency to aggregate in aqueous solution. The fluorochromes
prepared in this work have potential application for in vivo
imaging of pH in the renal and pulmonary systems where specific
targeting is not necessary for probe delivery. Additional function-
alization of these fluorochromes with carboxylic acid groups will
allow for conjugation of the pH sensitive dyes to biomolecules and
ultimately may enable targeting to specific biological tissues of
interest.
This research was supported in part by NIH grants CA86355
(P50) and HL080731 (U01).
This journal is ß The Royal Society of Chemistry 2007
Chem. Commun., 2007, 2747–2749 | 2749