PH-dependent Si-fluorescein hypochlorous acid fluorescent probe: Spirocycle ring-opening and excess hypochlorous acid-induced chlorination

Article abstract of DOI:10.1021/ja401426s

We report the synthesis and characterization of a fluorescent probe (Hypo-SiF) designed for the detection of hypochlorous acid (HOCl) using a silicon analogue of fluorescein (SiF). The probe is regulated in an off-on fashion by a highly selective thioether spirocyclic nonfluorescent structure that opens to form a mixture of fluorescent products in the presence of HOCl. Over a range of pH values, the probe reacts with a stoichiometric amount of HOCl, resulting in a mixture of two pH-dependent fluorescent species, a SiF disulfide product and a SiF sulfonate product. The unique colorimetric properties of the individual SiF fluorophores were utilized to perform simultaneous detection of HOCl and pH. When an excess of HOCl is present, the SiF fluorophores become chlorinated, via an intermediate halohydrin, resulting in a more pH independent and red-shifted fluorophore.

Full text of DOI:10.1021/ja401426s

Article
pubs.acs.org/JACS
pH-Dependent Si-Fluorescein Hypochlorous Acid Fluorescent Probe:
Spirocycle Ring-Opening and Excess Hypochlorous Acid-Induced
Chlorination
Quinn A. Best, Narsimha Sattenapally, Daniel J. Dyer, Colleen N. Scott, and Matthew E. McCarroll*
Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale, Illinois 62901, United States
S
* Supporting Information
ABSTRACT: We report the synthesis and characterization of a fluorescent
probe (Hypo-SiF) designed for the detection of hypochlorous acid (HOCl)
using a silicon analogue of fluorescein (SiF). The probe is regulated in an
“off−on” fashion by a highly selective thioether spirocyclic nonfluorescent
structure that opens to form a mixture of fluorescent products in the presence
of HOCl. Over a range of pH values, the probe reacts with a stoichiometric
amount of HOCl, resulting in a mixture of two pH-dependent fluorescent
species, a SiF disulfide product and a SiF sulfonate product. The unique colorimetric properties of the individual SiF fluorophores
were utilized to perform simultaneous detection of HOCl and pH. When an excess of HOCl is present, the SiF fluorophores
become chlorinated, via an intermediate halohydrin, resulting in a more pH independent and red-shifted fluorophore.
Scheme 1. Neutral and Anionic Forms of Fluorescein
Derivatives Tokyo Green and Tokyo Magenta
INTRODUCTION
■
Reactive oxygen species (ROS) are of scientific interest because
of their role in biology and their link to a number of diseases.
Among the known ROS associated with biological processes,
hypochlorous acid (HOCl) has received an increasing amount
of attention. Its role in animal immune systems, specifically
during phagocytosis, is well documented. However, abnormal
production of HOCl has been linked to a variety of diseases,
including cystic fibrosis,1 kidney disease,2 and certain cancers.3
Development of imaging techniques for HOCl is therefore of
great interest to better our understanding of its role in the
immune system and its link to the aforementioned diseases.
Fluorescence is widely considered to be an advantageous
imaging technique because of its high sensitivity and relatively
low technical costs.
A variety of fluorescent probes designed for specific detection
of HOCl have been reported. These probes have a HOCl
reactive moiety that modulates the fluorescence of the
fluorophore, examples of which have included BODIPY,4
fluorescein,5 rhodamine,6 and a silicon analogue of rhodamine
(SiR).7 The SiR fluorophores are an interesting new class of
fluorophores because their subtle difference in the heteroatom,
i.e., replacing oxygen with silicon, leads to a large red shift (90
nm) in the absorption and emission spectra relative to
rhodamine.7,8 Nagano et al. reported that this approach was
also shown to have a comparable red shift (90 nm) on a
fluorescein-based analogue.9 They reported the synthesis of a
silicon analogue of their famous Tokyo Green (TG) that was
named Tokyo Magenta (TM) because of the significant red
shift. In addition to the significant red shift of this fluorophore,
a dual-color acid/base property was reported. Like the TG
series, TM can exist in the neutral or the anionic form (Scheme
1). However, TM shows a more pronounced change in the
absorption spectra as the fluorophore goes from the neutral to
the anionic form (110 nm), which gives rise to a clearly
distinguishable change in color, i.e., from yellow to magenta.
On the basis of this colorimetric property of TM, we chose
to couple the silicon analogue of fluorescein (SiF) fluorescent
core with a spirocyclized moiety to control the fluorescence.
We used a spirothiophene moiety, which was shown to have a
selective response to HOCl in the rhodamine6 and SiR7
systems. Over a range of pH values, the probe reacts with a
stochiometric amount of HOCl, resulting in a mixture of two
pH-dependent fluorescent species, a SiF disulfide (8) product
and a SiF sulfonate (SiF-SO3H) product (Scheme 2). The
unique colorimetric properties of the individual SiF fluo-
rophores were utilized to perform simultaneous detection of
HOCl and pH. When an excess of HOCl is present, the SiF
fluorophores undergo chlorination, resulting in a red-shifted,
more pH independent fluorophore (Scheme 2) and allowing
for the detection of a larger concentration range (0−80 equiv)
of HOCl compared to similar probe systems.6a,7 Herein, we
Received: February 15, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ja401426s | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
TBDMS group furnished compound 7 in good yields.
Lithiation of (2-bromobenzyl)(tert-butyl)sulfane with n-butyl-
lithium (n-BuLi) formed the lithiated nucleophile, which was
used to react with compound 7. The alcohol species, resulting
from the nucleophilic attack, simultaneously underwent
dehydration during deprotection of the TBDMS group. Further
treatment of the mixture with trifluoroacetic acid (TFA)
removed the tert-butyl group, leading to the spirocyclized
product Hypo-SiF.
Scheme 2. Oxidative Ring-Opening of Hypo-SiF with
Stoichiometric and Excess Amounts of HOCl
HOCl Titrations and Probe Specificity. Phosphate buffer
solutions containing 5 μM Hypo-SiF and 0.5% dimethylforma-
mide (DMF) for dissolution were prepared in various pH (4−
9) solutions. These solutions, in the absence of HOCl, are
colorless and nonfluorescent because the probe maintains its
spirocyclic structure. At pH 7.4, the addition of HOCl results in
a magenta-colored solution. The UV−vis spectrum of this
solution shows a λabs at 586 nm (Figure 1A). Excitation at this
present our synthetic approach to a spirocyclized SiF probe and
its fluorescence and colorimetric response to HOCl and pH.
RESULTS AND DISCUSSION
Synthesis of HOCl Probe. The Hypo-SiF probe was
synthesized starting from 3-bromophenol (Scheme 3). The
■
a
Scheme 3. Synthesis of Hypo-SiF
a
Reagents and conditions: (a) H2CO, hydrochloric acid (HCl)/
methanol, 50 °C; (b) tert-butyldimethylsilyl chloride ((TBDMS)Cl),
imidazole, DMF, rt; (c) n-BuLi, dimethylsilyl chloride, tetrahydrofuran
(THF), −78 °C to rt; (d) tetrabutylammonium fluoride (TBAF),
THF, rt; (e) DDQ, methanol, rt; (f) (TBDMS)Cl, imidazole, DMF, rt;
(g) 1-bromo-2-[(tert-butylsulfanyl)methyl]benzene, n-BuLi, THF,
−78 °C to rt; (h) TBAF, THF, dilute HCl, rt; (i) TFA, reflux.
Figure 1. Response of a 5 μM solution of Hypo-SiF with HOCl in pH
7.4 phosphate buffer solution and 0.5% DMF. (A) Absorption spectra
and (B) emission spectra with excitation at 570 nm (1 equiv of HOCl
added to a 5 μM concentration of the probe in different pH buffer
solutions). (C) Absorption spectra and (D) emission spectra with
excitation at 570 nm. (E) λ586 and λ475 absorption. (F) Specificity of
the probe under similar conditions with various ROS.
condensation of 3-bromophenol with formaldehyde in an acidic
medium yielded 2, which was then protected with the tert-
butyldimethylsilyl (TBDMS) protecting group on the phenolic
oxygens to give compound 3. The next step was lithiation of
compound 3 to provide a nucleophilic species that was trapped
with dimethyldichlorosilane, forming the cyclized product 4,
which after deprotection of the TBDMS groups gave
compound 5. Compound 5 was then oxidized to afford the
ketone intermediate 6. Unfortunately, oxidation of the
protected cyclized product with 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (DDQ) gave a very low yield, so it was necessary
to remove the TBDMS group before the oxidation step.
Reprotection of the phenolic alcohols on compound 6 with the
wavelength results in a strong fluorescence band centered
around 606 nm (Figure 1B). The intensities of these bands
increase linearly with additional amounts of HOCl, leading to a
stoichiometric amount. When 1 equiv of HOCl is added to
acidic solutions containing Hypo-SiF, a pH-dependent trend in
the UV−vis and fluorescence response is observed. Specifically,
the λ586 band begins to decrease, while a new relatively broad
band at 475 nm increases as the solutions containing HOCl
become more acidic (Figure 1C). The corresponding
fluorescence spectra of these solutions show that the probe’s
fluorescence intensity decreases under increasingly acidic
conditions (Figure 1D). The anomalously low response from
B
dx.doi.org/10.1021/ja401426s | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
pH 9 indicates that the probe is responsive toward HOCl and
not the hypochlorite anion, which has a pKa = 7.54. The ratio
between the λ586 and the λ475 bands remains constant as the
concentration of HOCl increases to 1 equiv. Therefore, the
fluorescence and UV−vis intensities and the ratio between the
λ586 and the λ475 bands can be used to indicate the
concentration of HOCl and the pH of the solution
simultaneously (Figure 1E). When the probe was tested against
other biologically relevant ROS, it was found to have a high
degree of specificity for HOCl. Hydrogen peroxide (H2O2),
superoxide (O2•−), nitric oxide (NO•), and peroxynitrite
(ONOO−) were all found to have little or no enhancement
in the absorbance and fluorescence of the probe (Figure 1F).
The selectivity observed in this system is believed to be due to
the higher oxidative strength of HOCl compared to other ROS
and the stability of the thioether probe, which is consistent with
similar probe systems.6a,7
Isolation and Characterization of SiF Chromophores.
On the basis of the complex response that was observed, we
decided to isolate the highly fluorescent products to identify the
species involved in the ratiometric response. This was achieved
by oxidizing Hypo-SiF with HOCl on a bulk scale at pH 7.4,
followed by immediate extraction with EtOAc, which allowed
for an 86% recovery by mass of a brightly colored mixture.
Upon further separation of the extracted fraction by
chromatography, it was shown to contain two compounds,
SiF sulfonate species (SiF-SO3H) and a SiF disulfide (8), as
shown Scheme 4. Both species are consistent with previous
studies on the oxidation of thiols by HOCl.6a,7,10
Figure 2. Photophysical properties of 5 μM SiF-SO3H (A, C) and 5
μM 8 (B, D) buffer solutions with 0.5% DMF. Fluorescence spectra
were excited at 570 nm. Titration curves (E) are from λmax at 586 nm.
however, the individual phenolic protons appear to have similar
pKa values, i.e., pKa ≈ 9.3 (Figure 2E).
The fluorescence spectra of 8 and SiF-SO3H show a similar
pH dependence, which correlates well with their absorption
spectra (Figure 2C,D).
Scheme 4. Oxidation of Hypo-SiF in pH 7.4 Buffer Solution
a
Forming SiF-SO3H and Disulfide 8
In addition to differences in the pH-dependent photophysical
properties of the two fluorescent species, the fluorescent
intensity was also notably lower for the disulfide species. The
sulfonate product SiF-SO3H displays a bright fluorescence with
a quantum yield of 18.5%, whereas the disulfide shows a lower
quantum yield and molar absorptivity (Table 1). The relatively
Table 1. Photochemical Properties of Hypo-SiF, Compound
a
8, and SiF-SO3H
a
λabs
molar exctinction coeff
λFl
quantum yield
Percent composition determined by NMR.
b
(nm)
(cm−1M−1
nd
)
(nm)
(ΦFl)
Hypo-
SiF
nd
nd
nd
We then investigated the pH-dependent photophysical
properties of the isolated species. Individually, these chromo-
phores’ spectra (Figure 2 A−D) resemble the response
observed from the Hypo-SiF probe (Figure 2 A−D). Both
compounds have λ586 and λ475 absorption bands that vary
depending on the pH (Figure 2 A,B). However, differences in
the phenolic proton pKa between the two chromophores were
observed.
The pH dependence of the absorption spectra from 8 was
found to differ slightly from that of SiF-SO3H. The absorption
spectra of 8 show a more sudden decrease in the 586 nm band
accompanied by an increase in the 475 nm band as the pH is
lowered from 10 to 7.4 (Figure 2B). These differences in the
spectral response of 8 relative to SiF-SO3H indicate that SiF-
SO3H has a lower pKa than 8. From the UV−vis pH titration
curve of SiF-SO3H, a single inflection point is observed
corresponding to a pKa ≈ 6.6 for phenolic proton (Figure 2E),
defined as half the maximum. Compound 8 is a diprotic system;
8
586
586
69000
606
606
0.0061
0.185
SiF-
102000
SO3H
a
The photochemical properties of Hypo-SiF, compound 8, and SiF-
b
SO3H were measured in 0.1 M NaOH/H2O solution. Determined by
using an average from rhodamine B and rhodamine 6G in ethanol as
the standard reference.
low fluorescence from 8 may be due to quenching from
intramolecular fluorescence resonance energy transfer (FRET),
which has been reported in a variety of other homodimers.11
Colorimetric Detection of HOCl and pH. The pH-
dependent response of Hypo-SiF is potentially useful in
environmental analysis of HOCl and could also benefit from
the colorimetric response provided by Hypo-SiF at different pH
values. For example, maintenance of a swimming pool requires
one to monitor HOCl and pH levels. Commercially available
colorimetric indicator solutions for pH and HOCl are typically
C
dx.doi.org/10.1021/ja401426s | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
sold together as a kit for proper pool maintenance.
Orthotolidine provides varying shades of yellow, allowing for
detection of 0.5−10 ppm HOCl in solution, while a separate
solution with phenol red displays a colorimetric response
indicating the pH. However, the probe Hypo-SiF provides a
colorimetric response, indicating both pH and HOCl
concentration, by generating a distinct color for different pH
values with HOCl (Figure 3). The varying degrees of these
mostly absorption around the λ475 band and a low amount of
fluorescence from the λ606 band. However, around 5 equiv, the
absorption λ586 band and the fluorescence λ606 band begin to
red shift and grow in intensity. By 40 equiv of HOCl, the peaks
have reached full intensity and have shifted to 612 and 624 nm
for the absorption and fluorescence spectra, respectively
(Figure 5). A similar response was also found at pH values of
Figure 5. Absorbance (A) and fluorescence (B) titrations of Hypo-SiF
with excess HOCl in pH 6.2. Fluorescence spectra were excited at 590
nm.
6.8 and 5 (Supporting Information). These results indicate that
the probe can show a different response toward HOCl
depending on the relative concentrations of the probe and
HOCl. If knowing the pH and concentration of HOCl in a
solution is desired, then maintaining a higher concentration of
the probe should be applied. If a relatively pH insensitive
fluorescence response is desired, then a lower concentration of
the probe can be applied. Furthermore, the chlorination effect
on the probe allows for the detection of a larger concentration
range (0−80 equiv) of HOCl compared to similar probe
systems.6a,7
To further characterize this system, we isolated the
fluorescent products generated when an excess of HOCl was
used by adding 80 equiv of HOCl to Hypo-SiF in a pH 7.4
buffer solution. The products were extracted using methylene
chloride after the solution was acidified to pH 3. The extracted
products were found to have the same λ612 band in the
absorption spectrum as that observed from excess HOCl
titrations. The NMR of the resulting crude product was
complex and difficult to interpret. Purification was similarly
problematic, and no meaningful separation was achieved.
However, liquid chromatography−mass spectrometry (LC−
MS) was helpful in characterizing the products in hand (Figure
6). A mixture of various chlorinated forms of the disulfide was
prominent in the LC−MS chromatogram. Molecular ions
Figure 3. Colorimetric response of Hypo-SiF to increasing
concentrations of HOCl at pH 5 (A), pH 7.4 (B), and pH 9 (C).
Simultaneous colorimetric detection for different concentrations of
HOCl and pH values was obtained by adding 800 μL from a 0.5 μM
stock solution containing Hypo-SiF in DMF to a 10 mL buffer
solution containing HOCl. Color from the isolated species 8 and SiF-
HySO3H in either pH 9 or pH 5 buffer solution (D).
distinct colors can be used to determine relative levels of HOCl
by the “naked eye” (Figure 3A−C). The multicolor response at
different pH values is due to the presence of the two main
products, 8 and SiF-SO3H, which differ in color (Figure 3D).
At low pH, both compounds are orange. However, at high pH,
SiF-SO3H appears magenta, while compound 8 has a darker
purple color. The combination of these colors as the pH
changes leads to different shades of magenta, purple, and
orange.
HOCl-Induced Chlorination. The response of Hypo-SiF
toward 1 equiv of HOCl produces a linear and well-defined
ratiometric absorption spectrum. However, as HOCl is titrated
above a stoichiometric amount, a different response is observed.
At pH 7.4, aliquots between 10 and 80 equiv of HOCl cause
the λ586 band to begin to red shift stepwise. This bathochromic
shift was also observed in the fluorescence spectra until finally
λ612 and λ624 bands were observed for the absorption and
fluorescence spectra, respectively (Figure 4).
When an excess amount of HOCl is added under more acidic
conditions, a larger change in the absorption and fluorescence is
observed. At pH 6.2, the response from 1 equiv of HOCl shows
Figure 4. Bathochromic shift in UV−vis (A) and fluorescence (B)
with additions of 1, 20, 40, and 80 equiv of HOCl to a 5 μM solution
of Hypo-SiF in pH 7.4 buffer solutions.
Figure 6. LC−MS chromatogram of the isolated mixture after
treatment of Hypo-SiF with 80 equiv of HOCl.
D
dx.doi.org/10.1021/ja401426s | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
corresponding to m/z of chlorinated isomers containing two,
three, four, and five chlorine atoms on the xanthene core were
detected. The exact structure and location of the chlorine atoms
could not be determined. Additionally, a tetrachloro-SiF
fluorophore containing a sulfonate moiety was also identified
in the LC−MS chromatogram. Chlorohydrins were also found
and are believed to be intermediates leading to the SiF-
fluorophore product.
the buffer has on the reaction. In the absence of phosphate
buffer, the probe was found to only produce the λ586 species.
The reaction kinetics for the oxidation at the sulfur center
and the chlorination of the Si-xanthene core were explored by
monitoring the fluorescence (Figure 9). The addition of 1 equiv
Although direct analysis of the individual products was not
possible because of difficulties in purification, the mixture’s pH-
dependent properties were investigated. The mixture charac-
terized to contain the chlorinated species (ChloroMix) was
titrated to examine its pH dependence (Figure 7). We found
Figure 9. Kinetic study for the reaction of 5 μM solutions of Hypo-SiF
with aliquots containing 1 and 50 equiv of HOCl. The fluorescence
intensity was monitored at 608 nm.
of HOCl results in a rapid rise in fluorescence intensity at pH
corresponding to the formation of a mixture of SiF-SO3H and
8. The rise in fluorescence occurs rapidly and remains stable
until additional HOCl is added. Upon addition of 50 equiv of
HOCl, a relatively slower rise in fluorescence is observed,
where maximum fluorescence is achieved over the course of
∼80 s. It is interesting to note that when excess HOCl is added
to Hypo-SiF, a similar fluorescence intensity is obtained for pH
7.4 and 6.6. Therefore, under these conditions the fluorescence
intensities of the chlorinated SiF products are less dependent
on pH compared to those of SiF-SO3H and 8. These results
also indicate that both the oxidative ring-opening and the
HOCl-induced chlorination take place on a relatively fast time
scale, making both processes suitable for various applications.
The reaction mechanism is presumably similar to that of
previous studies on the HOCl-induced chlorination of
fluorescein.12
Figure 7. pH-dependent absorbance spectra of ChloroMix (A) and
titration curve of λmax for ChloroMix, 8, and SiF-SO3H (B).
that ChloroMix is fluorescent at lower pH, whereas compound
8 and SiF-SO3H are relatively nonfluorescent under acidic
conditions. We believe this result is due to the increased acidity
of the phenolic alcohol caused by the electronegative chlorine
groups on the fluorophore. This correlates well with previous
studies on the pH dependence of chlorinated forms of
fluorescein, where the addition of chlorine to the xanthene
core is known to lower the pKa of the phenolic proton.12
Progress toward the formation of the new red-shifted
fluorescent species can be monitored through the shift in λmax
(Figure 8). The reaction proceeds more readily under acidic
Detection of Myeloperoxidase Activity. An exciting
potential application of this probe system is determination of
ROS in a biological system. However, the primary biological
application of this probe is in fluorescence imaging of the
cellular environment, where the colorimetric properties may
not be relevant. Therefore, we opted to evaluate a scenario
where the probe responds to an excess amount of HOCl to see
if we get the expected, more fluorescent, chlorinated product.
We examined the response of Hypo-SiF to the biological agent
myeloperoxidase (MPO). Neutrophils utilize the enzyme
(MPO) to generate HOCl as a defensive response to a
multitude of pathogens. In the presence of H2O2, MPO actively
converts Cl− to produce HOCl over a broad pH range. We
prepared solutions containing MPO (extracted from human
neutrophils), sodium chloride, and the fluorescent probe.
Aliquots of H2O2 were added, and the fluorescence was
measured. Leading to 2 equiv of H2O2, the probe forms the
characteristic fluorescent λ606 band (Figure 10A) from a
mixture of 8 and SiF-SO3H, where a relatively low fluorescence
is observed at lower pH compared with pH 7.4 (Figure 10B).
However, when excess H2O2 is added, the fluorescence λmax
begins to shift and the fluorescence intensity increase signifies
the formation of the more pH independent chlorinated
products. By 40 equiv of H2O2, a bright relatively stable
Figure 8. Change in λmax from the response of 5 μM solutions of
Hypo-SiF with increasing concentrations of HOCl for solutions in
water or buffered solutions at pH 9, 7.4, 6.8, and 6.2.
conditions relative to neutral or basic conditions. By 30 equiv of
HOCl, solutions buffered at pH 6.2 and 6.8 have fully shifted to
the λ612 band, whereas pH 7.4 requires approximately 90 equiv
of HOCl before the λ612 species are observed. Under basic
conditions (pH 9), the λ586 species are not observed and only a
red-shifted λmax at 595 nm is observed. We tested the effect that
E
dx.doi.org/10.1021/ja401426s | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
AUTHOR INFORMATION
Corresponding Author
mmccarroll@chem.siu.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Institutes of Health
(Grant NIGMS-1R15GM080721) and the National Science
Foundation (Grant CHE-0719185). Additional support was
provided by Southern Illinois University in the form of startup
(C.N.S.). This manuscript is dedicated to the memory of
Professor Daniel J. Dyer.
REFERENCES
■
(1) Perez-Vilar, J.; Boucher, R. C. Free Radical Biol. Med. 2004, 37,
1564.
(2) Rao, R. Front. Biosci. 2008, 13, 7210.
(3) (a) Guengoer, N.; Knaapen, A. M.; Munnia, A.; Peluso, M.;
Haenen, G. R.; Chiu, R. K.; Godschalk, R. W. L.; van Schooten, F. J.
Mutagenesis 2010, 25, 149. (b) Suzuki, T. Genes Environ. 2006, 28, 48.
(4) (a) Sun, Z. N.; Liu, F. Q.; Chen, Y.; Tam, P. K. H.; Yang, D. Org.
Lett. 2008, 10, 2171−2174. (b) Kim, T.; Park, S.; Choi, Y.; Kim, Y.
Chem.Asian J. 2011, 6, 1358−1361.
Figure 10. A 2.5 mL solution containing 50 nM MPO, 1 μM Hypo-
SiF, and 150 mM NaCl was prepared at various pH values. These
solutions were then titrated with aliquots of H2O2 and allowed to stir
for 5 min before the fluorescence was measured. (A) Fluorescence
λmax. (B) Fluorescence intensity at the respective λmax
.
(5) (a) Cheng, X.; Jia, H.; Long, T.; Feng, J.; Qin, J.; Li, Z. Chem.
Commun. 2011, 47, 11978−11980. (b) Shepherd, J.; Hilderbrand, S.
A.; Waterman, P.; Heinecke, J. W.; Weissleder, R.; Libby, P. Chem.
Biol. 2007, 14, 1221−1231.
fluorescence is observed at each pH. Interestingly, subtle
differences between the rates of HOCl-induced chlorination of
the fluorophore were observed at the different pH values. By
monitoring the shift in the fluorescence λmax, the degree of
chlorination on the SiF fluorophore can be assumed (Figure
10B). At lower pH values (5−6.2), a more chlorinated product
is observed with λ624 and λ622 for pH 5 and 6.2, respectively.
Under more alkaline conditions (pH 6.8−7.4), a less
chlorinated product is observed. On the basis of a comparison
of the MPO results with those from the HOCl titrations
(Figure 8), a likely explanation for this observation is that MPO
is more active in a more acidic environment. This observation
correlates well with previous studies on the pH-dependent
behavior of MPO.13,14
(6) (a) Kenmoku, S.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem.
Soc. 2007, 129, 7313. (b) Yang, Y.-K.; Cho, H. J.; Lee, J.; Shin, I.; Tae,
J. Org. Lett. 2009, 11, 859.
(7) Koide, Y.; Urano, Y.; Hanaoka, K.; Terai, T.; Nagano, T. J. Am.
Chem. Soc. 2011, 133, 5680.
(8) (a) Fu, M.; Xiao, Y.; Qian, X.; Zhao, D.; Xu, Y. Chem. Commun.
2008, 44, 1780. (b) Koide, Y.; Urano, Y.; Hanaoka, K.; Terai, T.;
Nagano, T. ACS Chem. Biol. 2011, 6, 600−608.
(9) Egawa, T.; Koide, Y.; Hanaoka, K.; Komatsu, T.; Terai, T.;
Nagano, T. Chem. Commun. 2011, 47, 4162.
(10) Hawkins, C. L.; Pattison, D. I.; Davies, M. J. Amino Acids 2003,
25, 259.
(11) (a) Piggott, A. M.; Karuso, P. Anal. Chem. 2007, 79, 8769.
(b) Ogawa, M.; Kosaka, N.; Choyke, P. L.; Kobayashi, H. ACS Chem.
Biol. 2009, 4, 535.
(12) Hurst, J. K.; Albrich, J. M.; Green, T. R.; Rosen, H.; Klebanoff,
S. J. Biol. Chem. 1984, 259, 4812.
(13) Andrews, P. C.; Krinsky, N. I. J. Biol. Chem. 1982, 257, 13240−
13245.
(14) Furtmuller, P. G.; Burner, U.; Obinger, C. Biochemistry 1998, 37,
17923−17930.
CONCLUSIONS
■
We have shown that the fluorescent properties of a silicon
analogue of fluorescein can be regulated by a spirocyclic
structure. The probe Hypo-SiF displays a distinct “off−on”
fluorescence-based response when reacted with HOCl. The
HOCl reaction with the probe generates a mixture of two
dominant fluorescent species, SiF-SO3H and 8, that were
characterized at various pH values. When an excess of HOCl is
present, the SiF fluorophores undergo chlorination, via an
intermediate halohydrin, resulting in a red-shifted more pH
independent fluorophore. The fluorescence from HOCl-
induced chlorination of the SiF fluorophore was utilized to
monitor the activity of MPO at low pH. Biological imaging
using Hypo-SiF is ongoing and will be reported in a future
paper.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details, synthetic procedures, and compound
characterization. This material is available free of charge via the
Internet at http://pubs.acs.org.
F
dx.doi.org/10.1021/ja401426s | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX