Synthesis of photoactivatable azido-acyl caged oxazine fluorophores for live-cell imaging

Article abstract of DOI:10.1039/c6cc04882j

We report the design and synthesis of a photoactivatable azido-acyl oxazine fluorophore. Photoactivation is achieved cleanly and rapidly with UV light, producing a single fluorescent oxazine photoproduct. We demonstrate the utility of azido-acyl caged oxazines for protein specific labeling in living mammalian cells using the TMP-tag technology.

Full text of DOI:10.1039/c6cc04882j

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SynthesisꢀofꢀPhotoactivatableꢀAzido-acylꢀCagedꢀOxazineꢀ
FluorophoresꢀforꢀLive-cellꢀImagingꢀ
Receivedꢀ00thꢀJanuaryꢀ20xx,ꢀ
Acceptedꢀ00thꢀJanuaryꢀ20xxꢀ
a,b
a,c
a,b
AndrewꢀV.ꢀAnzalone ,ꢀZhixingꢀChen ꢀandꢀVirginiaꢀW.ꢀCornish ꢀ
DOI:ꢀ10.1039/x0xx00000xꢀ
www.rsc.org/ꢀ
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4
We report the design and synthesis of a photoactivatable azido- techniques. Oxazines also undergo photoswitching , which has been
1
5
acyl oxazine fluorophore. Photoactivation is achieved cleanly exploited for super-resolution dSTORM imaging . However, one
and rapidly with UV light, producing a single fluorescent drawback for oxazines relates to their unreliable behavior within the
living cell context, either due to low permeability or non-specific
oxazine photoproduct. We demonstrate the utility of azido-acyl
caged oxazines for protein specific labeling in living mammalian
cells using the TMP-tag technology.ꢀ
1
6
staining
leading to high background. Also, to date, the
derivatization of oxazine scaffolds has not been extensively
explored, potentially because of synthetic challenges. Therefore,
Fluorescence-based methods have emerged as powerful tools to probe development would be accelerated by modernized synthetic
study basic molecular processes within living cells. With the methods that enable the diversification of the scaffold with new
continuing development of new microscopy techniques comes the chemical features, either to improve cellular behavior or to install
1
,2
demand for brighter, multifunctional fluorescent probes . Great useful functionality.
strides have been made over the past several decades in devising
3
–8
Previously, other fluorophore scaffolds have been derivatized to
chemical tools for protein specific labeling
and intracellular
1
7–19
9
overcome challenges of solubility and cell-permeability
, and
molecular sensing using fluorescent reporters. Likewise, there has
been significant progress in the development of single molecule
2
0
brightness ,
and
to
introduce
functionality
such as
2
1–23
1
0,11
photoactivatability
. Representative examples include fluorescein
super-resolution
imaging
technologies
for
localizing
diacetate, which is cell permeable and cleaved by intracellular
biomolecules in cells with unprecedented precision. For live-cell
imaging, these methods require probes with high overall brightness
and photostability, preferably with absorbance and emission of red
or near-IR light, cellular permeability, and amenable cellular
2
4
esterases
to generate
a
fluorescent product, and the
2
5
photoactivatable fluorophores NVOC
2
-Rhodamine
(Fig. 1). While NVOC is commonly used as
photocleavable group, it incorporates unwanted bulk and
hydrophobicity into the probe. Also, in the case of NVOC
and azido-
2
6
DCDHF
a
1
properties (i.e. devoid of non-specific staining) . Several other useful
1
2
13
2
-
features, such as fluorogenicity and photoactivatability , are also
exploited in various applications. Thus, while the microscopy toolkit
expands to meet new challenges in the imaging arena, so too should
our repertoire of small molecule fluorophores in order to maximize
the potential of emerging technologies.
Rhodamine, full uncaging of the fluorophore requires two
photochemical cleavage events. Azido-DCDHF contains a smaller
photoactivation motif that minimally perturbs its overall structure
and requires just a single uncaging event; however, the photoreaction
gives rise to a mixture of products.
Oxazines comprise a class of live-cell fluorescent imaging
reagents exemplified by the popular commercial dye Atto 655. Their
remarkable photostability, notably higher in comparison to the
cyanine fluorophores, and emission in the red/far-red region of the
visible spectrum, notably redder than the rhodamine dye class, make
these fluorophores well-suited to cellular and single molecule-based
Here, we report the design and synthesis of new oxazine
derivatives and demonstrate feasibility for their use in live-cell
imaging. First, we have addressed the synthetic chemistry challenges
by employing recent advances in transition metal catalysis to
synthesize acyl oxazine intermediates, providing an avenue for facile
functionalization of the oxazine scaffold. Second, we prepared
azido-acyl oxazines and demonstrated that they undergo rapid and
clean photoconversion to the oxazine fluorophores through cleavage
of the acyl moiety accompanied by the concomitant release of
molecular nitrogen. Lastly, using the TMP chemical tagging
technology, we show that azido-acyl oxazines are useful reagents for
live mammalian cell imaging, especially when compared to the
native uncaged oxazine fluorophores.
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This synthetic strategy was initiated by synthesisVieowf AtrhtiecledOianlrinyel
ether 5 according to our previously reportedD sOt rI a: t1 e0 g. 1y0 ,3 u9 t/ iCl i6z Ci nC g0 4a 8 k8 e2 yJ
copper(I) promoted reaction between aminophenol
3
and
iodoindoline 4 (Scheme S1). Dibromination of 5 was achieved using
N-bromosuccinimide, proceeding exclusively with the desired
regioselectivity to afford 6 in high yield. Then, a second key
2
9
copper(I) promoted reaction was implemented to construct the
oxazine core, where the initial coupling between the amide or
carbamate and 6 is followed by a second intramolecular coupling
resulting in cyclized acyl oxazine products 7a-e. Importantly, this
reaction occurs in the presence of non-protected primary anilines,
which are orthogonal under the conditions employed.
Coupling/cyclization products bearing various acyl substituents
were prepared in good to excellent yields, and several of these
derivatives became colorful upon exposure to UV light. Of note, acyl
oxazines can be deprotected under the appropriate conditions to
yield leuco dyes, which upon exposure to air spontaneously oxidize
to furnish the fluorescent oxazines. Alternatively, further chemical
modification of the fluorophore can be carried out prior to removal
of the acyl substitutent. Thus, this approach provides a general route
to the construction of the oxazine core allowing for functionalization
of the scaffold before deprotection of the fluorophore’s sensitive
conjugated cationic system.
Fig. 1 Strategies for caging fluorescent dyes. Approaches include
trapping molecules in non-fluorescent states with enzymatically
cleavable or photocleavable capping groups. Some pro-fluorescent
molecules are intrinsically photoactivatable.
In order to apply the approaches displayed in Figure 1 to the
oxazine class of fluorophores, we chose to build upon the report that
acyl oxazines undergo UV-induced cleavage of the N-acyl bond to
2
7
generate oxazine dyes . However, because acyl oxazines uncage
relatively slowly, the extent of UV exposure required for efficient
photoactivation is incompatible with living cells. Moreover,
competitive Photo-Fries rearrangement products are obtained in
addition to the desired oxazines. Inspired by azido-DCDHF, we set
out to synthesize azido-acyl oxazines (Fig. 1, PA-oxazine 1a) in an
attempt to enhance photoactivation. Because these caged oxazines
would exist in the same oxidation state as their fluorophore products,
the net reaction does not require oxidation and therefore would likely
result in fewer side reactions and undesirable byproducts. We
anticipated that the release of molecular nitrogen would provide a
strong thermodynamic driving force for irreversibility of acyl bond
cleavage, favoring the forward reaction and resulting in fewer radical
recombination byproducts.
Previously, acyl oxazines have been prepared from their parent
oxazine fluorophores by in situ reduction of the oxazine to its leuco
form in the presence of an acylating agent (Scheme 1, Prior Works).
However, this procedure is limited in its compatibility with other
functionality, and is circuitous from a synthetic standpoint. To
circumvent these challenges, we sought to take advantage of our
Scheme 1 Synthesis of azido-acyl oxazines via acyl oxazine
intermediates. Prior synthesis of acyl oxazines was achieved through
the oxazine dye, whereas our approach directly synthesizes acyl
oxazines from diaryl ether intermediates. N,N’-DMED = N,N’-
dimethylethylenediamine. NBS = N-bromosuccinimide.
2
8
previously reported diaryl ether scaffold to prepare acyl oxazines
directly without passing through the oxazine dye. Given the
From acyl oxazines, synthesis of the target azido-acyl oxazines
electronically activated nature of the diaryl ether system, we 1a and 1b was achieved by diazotransfer from imidazole sulfonyl
reasoned that bromination followed by coupling with primary azide.ꢀAs anticipated, azido-acyl oxazines were extremely sensitive
amides or carbamates would furnish acyl oxazine products that could to UV light and readily converted to fluorescent photoproducts after
then be carried forward for facile derivitization (Scheme 1).
brief exposures (Fig. 2). In the UV-Vis absorbance and fluorescence
2
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traces of representative compound 1b, increasing exposure to UV
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In comparison to the acyl oxazines 7a and 7b, the VcioewrrAerstipcloenOdnilninge
light leads to an absorbance band that steadily arises in the visible azido-acyl oxazine derivatives 1a and 1 Db O Iu: n1 0d .e1 r0g3 o9 / Cm6 oC rCe 04r 8a 8p 2i dJ
region with a λmax of 612 nm as well as a fluorescence band with uncaging to the fluorophore in response to UV light, with rate
λ
max centered at 642 nm. Closer inspection of the UV region within accelerations of 20-30 fold (Fig. 2c). To assess whether the
Figure 2b displays several isosbestic points for the photochemical photoactivatable oxazines were viable reagents for live-cell imaging,
we first tested the azido-acetyl oxazine derivative 1a for
permeability and photoactivatability in living cells. HeLa cells
incubated with 2 µM of 1a were irradiated with 365 nm light for 1s,
process (Fig. S1, ESI), indicative of a clean reaction devoid of
accumulating intermediates or byproducts.
The absorbance and fluorescence traces of the photoproducts resulting in a dramatic increase in the fluorescence signal (Fig. S4,
overlay nearly identically with those of the independently ESI). This demonstrated that the photoactivatable oxazine is cell
synthesized oxazine 2, which is the expected product of the permeable and capable of undergoing photoactivation within the
photoreaction (Fig. S2, ESI). Additionally, HPLC analysis of the cellular environment.
crude photoreaction indicated formation of just a single major
product, identified as the anticipated oxazine by co-elution with the
Given these promising results, we then tested the trimethoprim
independently synthesized authentic marker 2 (Fig. S3, ESI). (TMP) conjugate of the photoactivatable oxazine for labeling E. coli
Together, these results demonstrate that the photoreaction proceeds dihydrofolate reductase (eDHFR) in HEK293T cells (Fig. 3). TMP
cleanly to a single oxazine product 2 generated by cleavage of the binds to eDHFR with nanomolar affinity, allowing specific labeling
acyl group and release of molecular nitrogen.
of a protein of interest (POI) when it is fused to eDHFR by staining
with TMP-fluorophore conjugates. We prepared the TMP conjugate
of the photoactivatable acetyl-azido oxazine (TMP-1a) as well as
the native oxazine fluorophore (TMP-2) as a point of comparison
(
Fig. 3a). Labeling of nuclear localized H2B-eDHFR with TMP-1a
followed by a brief UV excitation resulted in bright fluorescent
labeling of the target protein (Fig. 3b). This signal co-localizes well
with co-transfected H2B-EGFP. Interestingly, when TMP-2 was
imaged, very weak staining was observed, likely on account of poor
cell permeability. We also observed clean labeling of a plasma
membrane (PM) localized eDHFR with the photoactivatable TMP-
1
a, whereas TMP-2 showed only weak staining of the intracellular
DHFR (Fig. 3c). Of note, we have observed similarly poor cellular
behavior with other oxazine analogs (data not shown). These results
indicate that the caged azido-acetyl oxazine is a superior reagent for
live-cell applications when compared to its corresponding oxazine
fluorophore.
In conclusion, we present a new synthetic route to the synthesis
and derivatization of oxazine dyes and demonstrate the utility of this
approach through the design and synthesis of a photoactivatable
azido-acyl motif for oxazine fluorophores. We demonstrate that
azido-acyl oxazines undergo rapid and clean photoconversion to a
single major fluorescent product, and further apply these probes for
live-cell imaging. Importantly, caged oxazines were found to be
useful alternatives to native oxazine dyes on account of their
improved staining specificity. The comprehensive photophysical
characterizations of this dye are currently ongoing and will be
reported in due course. This new class of reagents should expand our
toolkit for probing molecular mechanisms of biology in living cells.
This work was supported by the US National Institutes of Health
Fig. 2
The azido-acyl oxazine uncaging photoreaction. (a) by grant R01 GM090126 to V.W.C. Support for A.V.A. was
Photochemical reaction of azido-acyl oxazines. (b) Absorbance and provided by NIH fellowship F30 CA174357.
fluorescence spectra (excitation = 594 nm) of 1b as a function of
total exposure to 254 nm light. (c) Comparison of photoactivation
rates for the azido-acyl oxazines 1a and 1b vs. the corresponding
acyl oxazines 7a and 7b in 100% ethanol.
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Fig. 3 Protein-specific labeling in HEK293T cells with TMP-oxazine conjugates. (a) Structures of the photoactivatable TMP-1a and the
oxazine fluorophore TMP-2. (b) Labeling of a nuclear localized H2B-eDHFR fusion and (c) a plasma membrane (PM) localized eDHFR in
HEK293T cells, co-tranfected with H2B-EGFP. Scale bar = 50 µm. Photoactivation conducted with 365nm light.
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