CalFluors: A Universal Motif for Fluorogenic Azide Probes across the Visible Spectrum

Article abstract of DOI:10.1021/jacs.5b02383

Fluorescent bioorthogonal smart probes across the visible spectrum will enable sensitive visualization of metabolically labeled molecules in biological systems. Here we present a unified design, based on the principle of photoinduced electron transfer, to access a panel of highly fluorogenic azide probes that are activated by conversion to the corresponding triazoles via click chemistry. Termed the CalFluors, these probes possess emission maxima that range from green to far red wavelengths, and enable sensitive biomolecule detection under no-wash conditions. We used the CalFluor probes to image various alkyne-labeled biomolecules (glycans, DNA, RNA, and proteins) in cells, developing zebrafish, and mouse brain tissue slices.

Full text of DOI:10.1021/jacs.5b02383

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Article
CalFluors: A Universal Motif for Fluorogenic
Azide Probes Across the Visible Spectrum
Peyton Shieh, Vivian T. Dien, Brendan J Beahm, Joseph
M. Castellano, Tony Wyss-Coray, and Carolyn R Bertozzi
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b02383 • Publication Date (Web): 22 Apr 2015
Downloaded from http://pubs.acs.org on May 3, 2015
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CalFluors: A Universal Motif for Fluorogenic Azide Probes Across the Visible Spectrum
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Peyton Shieh , Vivian T. Dien , Brendan J. Beahm , Joseph M. Castellano , Tony Wyss-Coray ,
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Carolyn R. Bertozzi
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Departments of Chemistry , Molecular and Cell Biology , and Howard Hughes Medical
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Institute , University of California, Berkeley, CA, 94720
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Department of Neurology and Neurological Sciences , Stanford University School of Medicine,
Stanford, CA, 94305
Abstract:
Fluorescent bioorthogonal smart probes across the visible spectrum will enable sensitive
visualization of metabolically labeled molecules in biological systems. Here we present a
unified design, based on the principle of photoinduced electron transfer (PeT), to access a panel
of highly fluorogenic azide probes that are activated by conversion to the corresponding triazoles
via click chemistry. Termed the CalFluors, these probes possess emission maxima that range
from green to far red wavelengths, and enable sensitive biomolecule detection under no-wash
conditions. We used the CalFluor probes to image various alkyne-labeled biomolecules
(glycans, DNA, RNA and proteins) in cells, developing zebrafish and mouse brain tissue slices.
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Introduction
Bioorthogonal smart probes enable the detection of labeled biomolecules in settings where it is
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difficult to remove excess probe. In a typical embodiment, the bonding changes accompanying
the bioorthogonal reaction lead to unquenching of a latent fluorophore. As an example, we
recently reported the design of fluorogenic azide probes that are activated by Cu-catalyzed or
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metal-free click chemistry with alkyne-labeled biomolecules. Building from the elegant work
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of Nagano and coworkers , we designed these molecules to be internally quenched via
photoinduced electron transfer (PeT) in the azide-functionalized form, and unquenched upon
conversion of the azide to a triazole (Fig 1A).
Key to these efforts was the identification of a pendent aryl ring system that imparts the desired
electronic effects on a given fluorophore scaffold. Using computational methods, we first
arrived at the azidonaphthyl group of fluorescein analog 1 (Fig. 1B), which afforded a 30-fold
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fluorescence enhancement upon triazole formation. However, the azidonaphthyl “switch” was
considerably less effective in other fluorophore structures. Instead, a Si-rhodamine analog was
more potently quenched by a pendent 3-azido 4,6-dimethoxybenzene moiety (compound 2, Fig.
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B) . Indeed, this switching group afforded a 5-fold higher fluorescence response than any other
aryl system screened.
In an effort to accelerate the design of activatable azide probes with various photophysical and
photochemical properties, we sought to identify a universal switch capable of PeT across a
variety of dye structures. Here we report that the 3-azido 4,6-dialkoxyaryl group possesses this
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capability. Incorporation of this group into various xanthene scaffolds afforded a palette of dyes
that emit at green to far red wavelengths. When functionalized with zwitterionic solubilizing
groups, these probes enabled robust and sensitive detection of alkyne-labeled biomolecules under
no-wash conditions and in a variety of settings, including live cells and tissue sections. We call
the probes Click Activated Luminogenic Fluorophores, or CalFluors.
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Figure 1: PeT-based fluorogenic azide probes activated by click chemistry. (A) General strategy.
(B) Structures of previously described fluorogenic azide probes.
Results and Discussion
In our recent study, we identified the 3-azido-4,6-dimethoxy benzene substituent as a fluorogenic
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switch that outperformed many other aryl substituents examined. To further understand the
electronic basis of this superior performance, we synthesized a panel of azide- and triazole-
functionalized aryl rings (3ꢀ7, Fig. 2A) and determined their oxidation potentials using cyclic
voltammetry (Fig. 2B). The measured oxidation potentials reflect the compounds’ propensity to
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donate an electron, which would result in fluorescence quenching via PeT. Consistent with
calculations, we found that all the triazoles had higher oxidation potentials than their parent
azides (Fig. 2C). Among the aryl azides, compound 3a was the most electron rich, underscoring
its potent quenching activity, and its change in oxidation potential upon conversion to triazole 3b
was the most dramatic (Fig. 2D). This large change is consistent with computational predictions
that an ortho-substituent forces the triazole to twist further out of plane and prevents donation
from the nitrogen lone pair into the aryl system (Fig. S1). We found that calculated EHOMO and
oxidation potential correlate (Fig. S2), though the modest fit suggests that more sophisticated
approaches towards predicting PeT efficiency may be necessary for future improvements in
computation-based probe design. Nevertheless, these results gave us confidence that dimethoxy
aryl ring 3 would efficiently switch fluorescence in a variety of fluorophore systems, regardless
of subtle differences in their electronics.
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Figure 2: Cyclic voltammetry analysis of substituted aryl systems. (A) Aryl azides (3aꢀ7a) and
triazoles (3bꢀ7b) synthesized and studied by cyclic voltammetry. (B) Cyclic voltammetry plots
of compounds 3ꢀ7. (C) Oxidation potentials of compounds 3ꢀ7. Note that all redox cycles were
completely or partially irreversible. Cyclic voltammetry was performed in acetonitrile containing
0.1 M NBu PF as an electrolyte using platinum and glassy carbon electrodes, with a silver
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reference standard. Ferrocene was added afterwards as an internal standard. Scans were
performed at 100 mV/s from 0 to 2 V.
To test this hypothesis, we expanded our panel of xanthene fluorophores containing the 3-azido
2,4-dimethoxy benzene substituent. We focused on fluorescein, tetramethylrhodamine
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rhodamine) and dimethylsilicon-substituted fluorescein (Si-fluorescein) scaffolds, which have
emission maxima of around 520 nm (green, compound 8), 580 nm (orange, compound 9) and
610 nm (red, compound 10), respectively (Fig. 3A). Following a general route, 3-bromo-4,6-
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dimethoxy aniline was protected, lithiated, and then added into various protected xanthones to
generate the corresponding amino-fluorescein, -rhodamine, and -Si-fluorescein derivatives
(Scheme S1). While the fluorescein and Si-fluorescein xanthones were readily prepared via
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literature procedures, we developed a new route to access the rhodamine xanthone from
commercially available Pyronin Y (see Supporting Information). These amine-functionalized
probes were converted to aryl azides by diazotization and displacement with sodium azide to
generate compounds 8a to 10a. The corresponding triazoles, 8b to 10b, were prepared under Cu-
catalyzed click conditions.
The fluorescence quantum yields of the azides and triazole products were measured in pH 7.4
phosphate-buffered saline. Gratifyingly, we found that their common dimethoxy aryl substituent
efficiently switched fluorescence for all fluorophores tested (Table 1). Notably, the fluorescein
and Si-fluorescein probes offered higher levels of fluorescence enhancement than any of our
previously reported fluorogenic azide probes. In particular, Si-fluorescein 10a, which has an
emission maximum beyond 600 nm, underwent >100-fold enhancement in fluorescence upon
click reaction. Rhodamine 9a, however, showed a significantly lower turn-on ratio (20-fold) due
to reduced quenching of the azide starting material. All of the probes were good substrates for
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the Cu-catalyzed click reaction, achieving complete conversion to the corresponding triazoles
within 10 min using micromolar concentrations of catalyst and reagents (Fig. S3).
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Figure 3: Structures of initial fluorophores prepared in this study. (A) Parent dimethoxy-
substituted fluorophores 8 to 10. (B) Oligoethylene-glycol functionalized fluorescein derivative
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Compound
λmax
λem
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Enhancement
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a
497 nm 516 nm
498 nm 520 nm
555 nm 577 nm
557 nm 580 nm
586 nm 604 nm
588 nma 604 nm
0.0059
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0.0178
0.351
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--
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a
654 nm 666 nm
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triazole
11a
665 nm 668 nm
48x
499 nm 519 nm
499 nm 520 nm
498 nm 520 nm
0.00589
0.743
0.00306
0.0747
0.0174
0.604
0.00250
0.473
0.0056
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--
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CalFluor 488
CalFluor 488 triazole 500 nm 521 nm
CalFluor 555
557 nm 577 nm
CalFluor 555 triazole 561 nm 583 nm
CalFluor 580
588 nm 611 nm
CalFluor 580 triazole 591 nm 609 nm
CalFluor 647
655 nm 678 nm
CalFluor 647 triazole 657 nm 674 nm
45x
Table 1: Photophysical properties of all fluorophores. Measurements were made in pH 7.4
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phosphate-buffered saline. Data from Ref. 4.
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A significant advantage of these probes is that, by replacing the methoxy groups with other
alkyloxy functionalities, their physical properties can be altered without perturbing the
electronics of the system. In previous work, we found that introducing oligoethylene glycol tails
significantly improved the water solubility of our probes while maintaining fluorescence
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enhancement. We synthesized the bis-oligoethylene glycol functionalized azidofluorescein
derivative 11a as well as its triazole derivative 11b (Fig. 3B, Scheme S2). This fluorescein
analogue maintained the enhancement in fluorescence of the parent fluorophore while showing
good water solubility, making it very well-suited for biological labeling experiments (Table 1,
also see Supporting Information).
The robustness of this route enabled us to access oligoethylene glycol functionalized rhodamine
and Si-fluorescein probes as well, however we found their aqueous solubility to be limiting for
biological labeling purposes. We were concerned that potential for nonspecific protein binding
would lead to spurious fluorescence turn-on, as documented by Nagano and coworkers in other
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systems. While were able to improve water solubility of the Si-rhodamine derivative by adding
sulfate esters at the termini of oligoethylene glycol chains, but this compound was still subject to
fluorescence enhancements due to protein and lipid binding (Fig. S4). As well, selective
sulfation would have been difficult to achieve for fluorescein-based probes that possess
competing phenolic hydroxyl groups.
In a search for alternative solubilizing groups, we were drawn to zwitterions, which are growing
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in popularity as anti-fouling agents in biomaterials applications. Indeed, zwitterionic coatings
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have been shown to outperform polyethylene glycols in many situations.
Additionally,
zwitterionic fluorophores have been recently demonstrated to have dramatically reduced serum
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binding compared to anionic counterparts. To generate fluorophores containing zwitterionic
tails, we were drawn to the sulfo-betaine scaffold, which can be generated by the reaction of
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tertiary amines with 1,3-propanesultone. The mild reaction conditions suggested that these
functionalities could be introduced at the end of our synthetic route, thereby minimizing
difficulties in product isolation. Accordingly, we prepared a panel of zwitterionic azide
fluorophores according to the route shown in Scheme S3. In keeping with tradition in this field,
we have given the azide fluorophore products the common names CalFluor 488, 555, 580 and
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All the CalFluors underwent significant fluorescence enhancement upon triazole formation at a
level that was at least as high as their dimethoxy substituted counterparts (Fig. 4B, Table 1).
CalFluor 488 and 580 showed higher levels of fluorescence enhancement compared to their
parent derivatives, potentially due to subtle electronic differences conferred by the oligoethylene
glycol tails. Additionally, the fluorescence of these probes remained virtually unaltered in
proteinaceous environments (e.g., 3% BSA or neat fetal bovine serum, Fig. S5) or in the
presence of detergents such as Triton X-100 and Tween-20 (Fig. S6), demonstrating how
effectively these zwitterionic tails minimize non-specific interactions.
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Figure 4: CalFluors and their fluorescence enhancements. (A) Structures of CalFluors 488, 555,
580, and 647. (B) Fluorescence enhancements of CalFluors during copper-catalyzed click
reactions. To a mixture of 2 µM fluorophore, 50 µM, 300 µM BTTAA ligand, and 2.5 mM
sodium ascorbate was added 100 µM alkyne and emission scans were taken every 30 seconds.
The first scan was taken immediately before addition of alkyne.
Our next goal was to evaluate the CalFluors’ performance in no-wash cell imaging experiments.
Using established methods, we metabolically labeled cell-surface glycoconjugates with
peracetylated N-pentynoyl mannosamine (Ac ManNAl), which introduces terminal alkyne
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groups into sialic acid residues (SiaNAl).
HEK 293T cells treated in this fashion were then
incubated, without fixation, with a cocktail containing 50 µM CuSO , 300 µM BTTAA, 5 mM
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sodium ascorbate and 10 µM CalFluor dye. After 15 min, cell surface glycans were robustly
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labeled with the given fluorophore, whereas control cells treated with peracetylated N-
acetylmannosamine (Ac ManNAc) showed no detectable fluorescence (Fig. 5A). The only
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background fluorescence was from free fluorophore in solution (Fig. S7, only observable at high
contrast). Similar results were achieved with CHO K1 cells (Fig. S8). Labeling with CalFluor
probes could be observed in real time and clear fluorescence over background was visible just
minutes into the reaction (Fig. S9). Notably, under the same reaction conditions, CalFluors
labeled cells far more intensely than the blue fluorogenic probe 3-azido-7-hydroxycoumarin (Fig.
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observation is consistent with studies by Finn and coworkers showing that electron-rich aryl
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azides undergo more rapid copper-catalyzed click reactions.
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Figure 5: No-wash labeling of cell-surface glycoproteins on HEK 293T cells. Cells were grown
with 50 µM Ac ManNAl or Ac ManNAc for 3 days, then subjected to click labeling with
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CalFluor probes. (A) Labeling glycoproteins on live cell surfaces. Cells were treated with 10 µM
azide probe, 50 µM CuSO , 300 µM BTTAA ligand, and 5 mM sodium ascorbate. The reaction
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was quenched with 1 mM BCS and the cells imaged without further wash steps. (B) Labeling
glycoproteins on fixed cells. Cells were fixed with 3% paraformaldehyde, then treated with 10
µM CalFluor probe, 1 mM CuSO , 100 µM TBTA ligand, and 2 mM sodium ascorbate. 0.1
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mg/mL BSA was added to prevent the TBTA from precipitating over the course of the reaction.
Scale bar = 10 µm.
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We also evaluated fixed (3% paraformaldehyde) Ac ManNAl-treated HEK 293T cells as
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BTTAA gave poor labeling, potentially due to sequestration of the copper catalyst by denatured
proteins. However, use of higher catalyst concentrations (100 µM TBTA, 1 mM CuSO , 2 mM
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sodium ascorbate) gave robust labeling with 10 µM CalFluor dyes (Fig. 5B). While some
background was apparent when using CalFluor 555 at this concentration, a single wash step was
sufficient to eliminate this background fluorescence (Fig. S12). Similar results were achieved
when performing the same experiments on fixed CHO K1 cells (Figs. S13, S14).
Under these copper-click conditions, the 3-azido-7-hydroxycoumarin probe performed
comparably to the CalFluors when labeling fixed cells (Fig. S15). By contrast, the sulfated
predecessor S1 or the oligoethylene glycol containing probe 11 gave higher background
fluorescence, underscoring the benefit of the zwitterionic tails in reducing non-specific
interactions. (Figs. S16, S17). The advantage of the fluorescence turn-on of CalFluors was clear
when comparing labeling by the non-fluorogenic AlexaFluor 647 alkyl azide. (Fig. S18). Even
with a washing step, background from unreacted fluorophore obscured any alkyne-dependent
signal.
We next tested our probes for visualization of glycans in vivo. Zebrafish have been powerful
model system for the study of development using optical methods due to their optical
transparency. Our lab has had a longstanding interest in imaging glycans during zebrafish
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development.
To test the utility of CalFluors in this system for such applications, we injected
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zebrafish embryos at the single-cell stage with SiaNAl. After either 24 or 36 hours post
fertilization (hpf), the embryos were bathed in a solution containing a CalFluor probe along with
copper catalyst. We observed bright alkyne-dependent fluorescence on cells of the enveloping
layer without washing (Fig. 6A). Fluorescence signal increased over time and maximized after
20 minutes. Importantly, in similar labeling experiments performed with the non-fluorogenic
AlexaFluor 594 alkyl azide probe or the blue-emitting 3-azido-7-hydroxycoumarin (Fig. 6B),
high levels of background fluorescence from unreacted probe or endogenous biomolecules,
respectively, obscured alkyne-dependent labeling.
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Figure 6: Visualizing sialic acids on live developing zebrafish with CalFluors. Zebrafish were
injected with 50 pmol SiaNAl at the one to four-cell stage and allowed to develop over time. (A)
Real-time labeling of sialic acids. After 24 hpf, zebrafish were incubated in a solution containing
1 µM CalFluor 580 and copper catalyst. Alkyne-dependent labeling was observable after 5
minutes, and appeared to saturate at 20 minutes. Scale bar = 500 µm. (B) Comparing no-wash
labeling performance by azide probes. After 36 hpf, the embryos were transferred to a solution
containing the fluorophore (1 µM for CalFluor 580 and AlexaFluor 594 alkyl azide, or 5 µM for
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-azido-7-hydroxycoumarin) and copper catalyst and imaged without washing after 20 minutes.
Only zebrafish labeled with CalFluor 580 show alkyne-dependent fluorescence signal. Scale bar
100 µm.
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Beyond glycans, the small size of linear alkynes has enabled its incorporation into a wide variety
of intracellular biomolecules. For example, linear alkynes have been used to label newly
synthesized DNA and RNA via 5-ethynyl deoxyuridine (EdU) and 5-ethynyl uridine (EU),
respectively. In particular, this click-chemistry based approach to visualizing newly synthesized
DNA/RNA remains superior to related methods such as BrdU labeling as a harsh denaturation
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step is not necessary.
Additionally, linear alkynes can be incorporated into newly synthesized
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proteins by the incorporation of homopropargylglycine (HPG) in methionine-starved cells.
We first tested the suitability of our probes for imaging EdU-labeled DNA. We treated HEK
93T cells with 10 µM EdU for 12 h, fixed and permeabilized the cells, then labeled them with
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our azide probes under copper-click conditions. We hoped that the use of fluorogenic azide
probes for EdU detection would streamline the visualization of newly synthesized DNA.
Gratifyingly, we saw robust labeling using all four of our probes without the need to wash away
excess reagent (Figs. 7A, 7B). Results were significantly better than those achieved using other
azide fluorophores under similar labeling conditions. (Figs. 7B, S19, and S20). We noticed that
the fluorescein-based probes (CalFluors 488 and 580) labeled intracellular DNA more weakly
than the related rhodamine-based probes (CalFluors 555 and 647, Figs. 6B). Similar results were
achieved when we labeled newly synthesized RNA by treatment of cells with 1 mM EU (Figs.
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S21-S23). This difference may be due to the pH sensitivity of our fluorescein-based dyes,
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especially when conjugated to the anionic oligonucleotides (Figs. S24, S25).
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Importantly, our palette of probes enabled two-color imaging with other well established probes,
such as with cells pre-labeled with the blue-emitting Hoescht 33342 nuclear stain (Fig. 7C). No-
wash labeling was also suitable for CHO K1 cells labeled with EdU and EU under identical
conditions, although specific signal was lower for all probes tested, likely due to poorer alkyne
incorporation (Figs. S26-S28). Beyond imaging mammalian cells in culture, EdU has also been
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used to label newly synthesized DNA in bacteria. E. coli were grown in the presence of EdU,
fixed, permeabilized, and labeled with CalFluor 647; robust alkyne-dependent labeling was
observed by flow cytometry, and significantly higher signal over background was achieved
compared to AlexaFluor 647 alkyl azide (Fig. S29). One of the more exciting applications of
EdU labeling is to visualize actively proliferating cells in vivo. Tissue slices were obtained from
the subventricular zone of mice injected with 150 mg/kg EdU two hours before perfusion.
Gratifyingly, CalFluor 647 were able to efficiently visualize EdU from these tissue sections with
excellent signal over background (Fig. 7D). Finally, all four CalFluor probes were suitable for
the robust detection of newly synthesized proteins containing HPG (Fig. S30).
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Figure 7: Visualizing EdU-labeled DNA in fixed and permeabilized samples using fluorogenic
azide probes. (A) No-wash labeling of EdU-labeled HEK 293T cells. Cells were treated with
EdU for 16 h, fixed and permeabilized, then treated with 10 µM CalFluor probe, 1 mM CuSO4,
100 µM TBTA ligand, 2 mM sodium ascorbate, and 0.1 mg/mL BSA and imaged without further
wash steps after 1 hour. Scale bar = 10 µm. (B) Quantification of normalized signal over
background for the four panels in (A), and comparison to labeling under identical conditions
using the non-fluorogenic AlexaFluor 647 alkyl azide (AF). (C) Two-color labeling using
Hoescht 33342 and CalFluor 555. After staining with Hoescht, the cells were incubated with a
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solution of 1 µM CalFluor probe and copper catalyst and imaged in real-time. Scale bar = 10 µm.
(D) Visualization of EdU-labeled newly proliferating cells in mouse brain slices with CalFluor
647. Scale bar = 100 µm.
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Conclusion
Here, we report a general platform to generate fluorogenic azide probes across the visible
spectrum. Our experiments clearly demonstrate the broad applicability of these optimized probes
for labeling a large panel of alkyne-functionalized biomolecules in both live and fixed cells, in
tissue and in vivo. Given the generality of PeT, we anticipate that this bis-zwitterionic dialkoxy
aryl azide motif will switch fluorescence in a wide variety of fluorophores beyond the xanthenes.
For example, other fluorophore scaffolds, such as BODIPY, cyanines, and pyrazolines, can all be
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efficiently switched via PeT.
Consistent with this prediction, a recent report from Wong and coworkers demonstrated that the
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BODIPY scaffold can also be used as a platform for PeT-based fluorogenic azide probes.
Beyond developing probes with higher photostability, using our optimized aryl ring in
conjunction with cyanine probes or modified Si-rhodamine probes may push emission maxima
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into the near-infrared. PeT has also been used to modulate other properties besides
fluorescence, such as the rate of singlet oxygen generation or luminescence from metal
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complexes.
We note that this transportable design element is reminiscent of the 1,2-
diaminobenzene-based trigger for nitric oxide sensing that has been integrated into myriad dye
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scaffolds.
In addition, the incorporation of zwitterionic sulfobetaine tails, which significantly
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outperformed both oligoethylene glycol or sulfated modifications in our hands, may be a general
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strategy for increasing the performance of PeT-based probes in complex environments. These
modifications prevent probe access to the interior of living cells, but the cytotoxicity of copper
catalysts likewise limits reactions with linear alkyne-functionalized biomolecules to fixed
samples or the live cell surface. Future work to overcome these shortcomings may benefit from
the modularity of our synthetic route towards a wide variety of fluorogenic azide probes. Beyond
the applications described here, CalFluor probes may find use in visualizing linear alkyne-tagged
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biomolecules in large tissue samples, where reagents are difficult to wash out.
Nonetheless, this current work, in combination with the established 3-azido-7-hydroxycoumarin
probe, completes the panel of fluorogenic azide probes with emission maxima across the visible
spectrum. With recent advances in increasing the efficiency of Cu-catalyzed click reactions,
these technologies will further enhance the ease and sensitivity of detecting linear alkyne-
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functionalized biomolecules in biological systems.
Associated Content:
Supporting Information.
Supplementary figures, synthetic procedures and experimental protocols are available in the
Supporting Information. This material is available free of charge via the Internet at
http://pubs.acs.org.
Author Information:
Corresponding Author
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Carolyn R. Bertozzi, crb@berkeley.edu
Acknowledgments:
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This work was funded by NIH grants GM058867 and AI051622 to C.R.B. We would like to
thank Prof. C. Chang (UC Berkeley) for the generous use of his potentiostat and Rachelle J.
Abbey (Stanford) for assistance with mouse experiments.
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