Nebraska Red: A phosphinate-based near-infrared fluorophore scaffold for chemical biology applications

Article abstract of DOI:10.1039/c6cc05717a

A series of novel phosphinate-based dyes displaying near-infrared fluorescence (NIR) are reported. These dyes exhibit remarkable photostability and brightness. The phosphinate functionality is leveraged as an additional reactive handle in order to tune ce

Full text of DOI:10.1039/c6cc05717a

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Nebraska Red: A Phosphinate-Based Near-Infrared Fluorophore
Scaffold for Chemical Biology Applications†
Received 00th January 20xx,
Accepted 00th January 20xx
a
a,b
a
a,b
a,
Xinqi Zhou, Rui Lai, Jon R. Beck, Hui Li, and Cliff I. Stains *
DOI: 10.1039/x0xx00000x
www.rsc.org/
A series of novel phosphinate-based dyes displaying near-infrared such as rhodamine is to replace the bridging oxygen atom with
fluorescence (NIR) are reported. These dyes exhibit remarkable other elements. Chalcogens like sulfur, selenium, and
1
photostability and brightness. The phosphinate functionality is tellurium along with group 14 elements like carbon, silicon,
2
13
1
4
leveraged as an additional reactive handle in order to tune cell germanium, and tin have been incorporated in this manner
permeability as well as provide a proof-of-principle for a self- and the resulting fluorophores all display red-shifted excitation
reporting small molecule delivery vehicle.
and emission spectra compared to the parent rhodamine
scaffold. However, this approach can also negatively impact
the brightness of the resulting fluorophore in certain cases
Fluorophores can be used as optical “paints” to visualize
otherwise invisible structures or processes. Due to this
property, fluorophores have been intensely studied for over 70
(Table S1†). In addition, the exploration of new chemical
handles in NIR scaffolds could enrich the tools available for
fundamental biomedical research. Thus, more work is needed
to identify robust and malleable NIR scaffolds for applications
in chemical biology.
1
-2
years. More recently, fluorophores that absorb and emit
3
light within the NIR window (650 nm - 900 nm) have attracted
attention for bioimaging applications since NIR wavelengths
have deeper tissue penetration and mitigate issues associated
4
with autofluorescence of natively occurring fluorophores. For
Inspired by the replacement of the bridging xanthene
oxygen of tetramethylrhodamine (TMR) with varying elements,
we envisioned a potential NIR scaffold in which the bridging
xanthene oxygen of TMR was replaced by the group 15
element phosphorous. We termed these dyes the Nebraska
Red (NR) series (Fig. 1). Remarkably, although phosphorous
translates to “light-bringer” in Greek, fluorophores containing
example, the cyanine and porphyrin dye series have found
5
applications in photochemistry, image-guided surgery, and
6
7
photodynamic therapy. Although powerful, these scaffolds
are hindered by poor chemical as well as photostability,
relatively large molecular weights, and low quantum
8
efficiencies. By comparison, fluorescein and rhodamine,
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this element have been relatively underexplored.
We
which are based on the xanthene ring system, have been well-
documented as bright, low molecular weight fluorophore
hypothesized that the use of a phosphinate functionality may
provide an additional reactive handle on the scaffold that
could be leveraged to produce changes in the spectral as well
as physical properties of the dye. As an initial test of our
hypothesis, we synthesized NR666 and its ethyl ester
9
templates. However, these dyes typically generate green to
orange fluorescence, which has prompted numerous efforts to
1
0
red-shift their emission wavelength. One of the most
common strategies employed in this area is to decrease the
energy gap between highest occupied molecular orbital
(HOMO) and lowest unoccupied molecular orbital (LUMO) by
increasing the π-conjugation of the fluorophore. However this
method typically results in a decrease in brightness (ε × φ) of
11
the resulting fluorophores. An alternative strategy for
modulating the emission wavelength of xanthene-based dyes
Fig. 1 Structures of Nebraska Red dye series.
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counterpart NR700 (Scheme 1). Investigation of the
photophysical properties of these two dyes showed that both
dyes absorbed and emitted in the NIR region in aqueous
solutions (Fig. 2a-c). Moreover, we observed a red-shift in both
the excitation and emission spectra of NR700 compared to
NR666. The molar extinction coefficients and quantum yields of
both dyes were determined and, in particular, NR666 was found
to be 1.3-fold brighter than the parent TMR scaffold (Table S1
1
1
†
). In addition, NR666 was stable to continuous irradiation
for 1 hr (Fig. 2d). Over the same time period we observed a
decrease in NR700 fluorescence, similar to the commonly used
Cy 5.5 dye (Fig. 2d). Further investigation indicated that the
decrease in fluorescence of NR700 was due to autohydrolysis of
the phosphinate ester, yielding NR666 (Fig. S1a, ESI
†).
Monitoring conversion of NR700 to NR666 yielded a first-order
-
1
rate of 0.026 min for hydrolysis (Fig. S1b, ESI
†). These
experiments demonstrate that the ether substituents on the
phosphinate of NR dyes can be used to modulate fluorescence
and could potentially be exploited as additional sites for
modification of the physical properties of these dyes.
Computational calculations showed that introduction of the
phosphinate functionality resulted in stabilization of dye
LUMO energies relative to the parent TMR scaffold, providing
a mechanism for the red-shift observed in these fluorophores
(Fig. S2†).
Encouraged by these initial results, we set out to identify
dyes with excitation and emissions above 700 nm by replacing
the dimethylaniline functionality of our initial NR dyes with a
julolidine substituent. Accordingly, we synthesized NR698 and
Fig. 2 Photophysical properties of the NR dye series. All experiments were performed
in PBS (10 mM, pH = 7.4 with 1% DMSO). (a) Absorption spectra. (b) Emission spectra.
its phosphinate ethyl ester counterpart, NR744 (detailed (c) Summary of optical parameters. (d) Photostability comparison with Cy 5.5.
synthetic procedures in the ESI
†). Satisfyingly, both these
derivatives displayed excitation and emission peaks at or
above 700 nm and maintained the photostability of the parent
xanthene scaffold (Fig. 2). Interestingly, we did not observe
spontaneous hydrolysis of NR744 during the timeframe of our
experiment. The resistance of NR744 to hydrolysis may be a
result of increased steric bulk and is currently under further
investigation. Nonetheless, these results demonstrate that the
phosphinate functionality can be utilized as a transposable
element in fluorophore design.
intensity, yielding mid-points of 0.5 and 1.8 pH units for NR666
and NR698, respectively. Based on these measurements, both
dyes can be used under physiological conditions with
essentially no inference due to changes in pH. Taken together,
the NR template displays remarkable photostability compared
1
8
to commonly employed NIR fluorophores, providing a robust
scaffold for the design of NIR dyes.
Building upon the observation that NR700 underwent
spontaneous hydrolysis to yield NR666, we asked whether
phosphinate ester substituents could be used to tune the
physical properties of NR dyes, such as cell permeability.
Accordingly, we incubated HeLa cells with either NR666 or
NR700. Gratifyingly, cell uptake was only observed for the NR700
derivative, indicating that phosphinate ester substituents can
be used to modulate the cell permeability of NR dyes (Fig. 3).
Our laboratory is currently pursuing this approach to afford a
suite of cell permeable and impermeable reagents for labelling
studies.
To investigate the potential sensitivity of phosphinate-
containing dyes to changes in pH, we determined the
fluorescence of NR666 and NR698 at varying pHs (Fig. S3, ESI
†).
Low pHs induced a red-shift in the excitation and emission
wavelength of both dyes as well as a decrease in fluorescence
Lastly, we sought to demonstrate a proof-of-principle for
generation of a self-reporting small molecule delivery reagent
using NR666 as a template. Given our laboratory’s interest in
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detecting biologically relevant signalling molecules,
we
chose to demonstrate the utility of the NR scaffold by
developing a reagent that would deliver a small molecule in
response to a biologically relevant stimulus. Reactive oxygen
species (ROS) and reactive nitrogen species (RNS) represent a
Scheme 1 Synthesis of NR666 and NR700
.
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Fig. 3 Cell permeability comparison between NR666 and NR700. (a) Living HeLa cells
were incubated with 5 µM dye for 15 min at 37 °C followed by washing with PBS (3x).
Scale bar: 25 µm. (b) The average fluorescence intensity of cells from panel a.
group of small molecules that are associated with numerous
2
1
human diseases. HOCl, a member of the ROS family, causes
inflammation at the site of tissue injury due to its strong
2
oxidative properties. Building upon previously reported
2
Scheme 2 Cleavage of NR-HOCl phosphinate ester upon reaction with HOCl.
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3-26
fluorescent sensors for HOCl,
we sought to construct a self-
we sought to determine whether the production of
endogenous HOCl by macrophages (RAW 264.7) stimulated
with lipopolysaccharide (LPS) and phorbol 12-myristate 13-
acetate (PMA) would be capable of triggering small molecule
release. These experiments yielded a clear increase in cellular
NR-HOCl fluorescence that was proportional to the amount of
stimuli applied, validating the potential to self-report on small
molecule release using endogenous HOCl as a stimulus (Fig. 4).
These results indicate the intriguing possibility of utilizing the
NR scaffold as a potential template for reaction-induced
release of small molecules capable of eliciting a biological
response. We are currently investigating this application in our
laboratory.
reporting small molecule delivery reagent triggered by HOCl.
Combining the selective reaction of spirocyclic thioethers with
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7-33
HOCl
and the NIR properties of the NR scaffold, we
envisioned a reagent that would remain intact and non-
fluorescent until reaction with HOCl. Importantly, we
anticipated that this reagent, termed NR-HOCl (detailed
synthetic procedures in the ESI
hydrolyze upon reaction with HOCl to yield ethanol and NR666
Scheme 2). Indeed, NR-HOCl was found to be non-fluorescent
†), would spontaneously
(
until exposure to HOCl, which yielded a product with excitation
and emission maxima at 705 and 730 nm, respectively
(
Scheme 2 and Fig. S4, ESI
HOCl compared to excess off-target ROS and RNS (Fig. S5, ESI
). Titration experiments revealed that the limit of detection
of NR-HOCl for HOCl is 3 nM (Fig. S6, ESI ), well below
physiologically relevant concentrations of HOCl (5 - 50 μM).
NR-HOCl also was capable of detecting HOCl across a wide
†). NR-HOCl was highly selective for
†
†
3
4
Conclusions
In summary, we have developed a novel NIR fluorophore
range of pHs (3 - 9) with a maximum increase in fluorescence scaffold that shows remarkable brightness and photostability
at pH 7 (Fig. S7, ESI ). As expected, incubation of NR-HOCl under physiological conditions. The generality of our design
†
after treatment with HOCl resulted in hydrolysis of its
phosphinate ester, providing a final product with excitation
and emission maxima at 673 and 690 nm, respectively (Fig. S4,
ESI
after 24 hrs in the absence of HOCl (Scheme 2 and Fig. S8, ESI
).
†). The phosphinate ester of NR-HOCl remained intact
†
Confident in the ability of NR-HOCl to selectively react with
HOCl and subsequently undergo phosphinate ester hydrolysis,
we next asked whether this reagent could function in the
context of living cells. As an initial trial, HeLa cells were
incubated with NR-HOCl and HOCl was subsequently added. A
distinct “off-on” signal was observed in presence of HOCl (Fig.
S9, ESI
†), demonstrating that NR-HOCl could self-report on
small molecule delivery in living cells in response to an
exogenous HOCl input. Importantly, no cellular toxicity was
observed from NR-HOCl alone at the concentrations used in
Fig. 4
(a) Confocal fluorescence microscopy imaging of living RAW 264.7 cells
incubated with 10 μM NR-HOCl and (I) without LPS or PMA, (II) with LPS (1 μg/mL) and
PMA (0.1 μg/mL), or (III) with LPS (1 μg/mL) and PMA (1 μg/mL) induction. Scale bar: 25
μm. (b) The average fluorescence intensity of cells from panel a.
these experiments (Fig. S10, ESI†). Bolstered by these results
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pallet via introduction of phosphinate functionalities into
existing fluorophores. We further demonstrated the chemical
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