A near-IR uncaging strategy based on cyanine photochemistry

Article abstract of DOI:10.1021/ja5065203

The development of photocaging groups activated by near-IR light would enable new approaches for basic research and allow for spatial and temporal control of drug delivery. Here we report a near-IR light-initiated uncaging reaction sequence based on readily synthesized C4'-dialkylamine-substituted heptamethine cyanines. Phenol-containing small molecules are uncaged through sequential release of the C4'-amine and intramolecular cyclization. The release sequence is initiated by a previously unexploited photochemical reaction of the cyanine fluorophore scaffold. The uncaging process is compatible with biological milieu and is initiated with low intensity 690 nm light. We show that cell viability can be inhibited through light-dependent release of the estrogen receptor antagonist, 4-hydroxycyclofen. In addition, through uncaging of the same compound, gene expression is controlled with near-IR light in a ligand-dependent CreER^T/LoxP-reporter cell line derived from transgenic mice. These studies provide a chemical foundation that we expect will enable specific delivery of small molecules using cytocompatible, tissue penetrant near-IR light.

Full text of DOI:10.1021/ja5065203

Article
pubs.acs.org/JACS
A Near-IR Uncaging Strategy Based on Cyanine Photochemistry
Alexander P. Gorka,†,§ Roger R. Nani,†,§ Jianjian Zhu,‡ Susan Mackem,‡ and Martin J. Schnermann*,†
†Chemical Biology Laboratory, National Cancer Institute, 376 Boyles Street, Frederick, Maryland 21702, United States
‡Cancer and Developmental Biology Laboratory, Center for Cancer Research, National Cancer Institute-Frederick, Frederick,
Maryland 21702, United States
S
* Supporting Information
ABSTRACT: The development of photocaging groups
activated by near-IR light would enable new approaches for
basic research and allow for spatial and temporal control of
drug delivery. Here we report a near-IR light-initiated uncaging
reaction sequence based on readily synthesized C4′-dialkyl-
amine-substituted heptamethine cyanines. Phenol-containing
small molecules are uncaged through sequential release of the
C4′-amine and intramolecular cyclization. The release
sequence is initiated by a previously unexploited photo-
chemical reaction of the cyanine fluorophore scaffold. The
uncaging process is compatible with biological milieu and is initiated with low intensity 690 nm light. We show that cell viability
can be inhibited through light-dependent release of the estrogen receptor antagonist, 4-hydroxycyclofen. In addition, through
uncaging of the same compound, gene expression is controlled with near-IR light in a ligand-dependent CreERT/LoxP-reporter
cell line derived from transgenic mice. These studies provide a chemical foundation that we expect will enable specific delivery of
small molecules using cytocompatible, tissue penetrant near-IR light.
which cleaves the olefinic caging group. An inherent
consequence of this design is an intact photosensitizer after
uncaging with resulting sensitizer-dependent phototoxicity
effects.7b Very recent advances from Lawrence and co-workers
using contact-quenching induced scission of a Co−C bond are
also quite promising.8 While these methods are proving quite
useful, the full potential of near-IR uncaging has almost
certainly not yet been realized. Essential to the development of
this area is the identification of promising candidate photo-
chemical reactions.
We speculated that the intrinsic photochemistry of the
heptamethine cyanine fluorophore scaffold might serve
admirably in this context. Heptamethine cyanines are used in
numerous fluorescence-based biomedical applications, and find
broad application as conjugated labels for in vivo imaging.9,10
One example, indocyanine green, is a clinical diagnostic agent
with a long history of well-tolerated human use.11 Although
useful, cyanine fluorophores are prone to light-dependent
decomposition or photobleaching. The chemical basis of
cyanine photobleaching has been shown to be a regioselective
photooxidative polyene cleavage reaction through numerous
mechanistic studies.12−21 While historically a liability, this
reactivity is exploited here as the central component of a near-
IR uncaging strategy.
INTRODUCTION
■
Chemical reactions that proceed efficiently in complex
biological settings underpin many biomedical methods. Photo-
removable protecting groups, most notably those based on o-
nitroaryl ring systems, that control or “cage” the activity of
small molecules represent one such class of reactions. Since
seminal studies over 35 years ago with photocaged ATP, these
approaches have found application in diverse fields ranging
from cell biology to materials science.1 Nevertheless, the
general requirement of UV or blue light is a significant
limitation due to associated toxicity and poor tissue
penetration. By contrast, light between 650 and 900 nm,
often referred to as the near-IR window, is cytocompatible and
has significant tissue penetration.2 Moreover, the application of
near-IR light has been broadly validated through numerous
optical methods, including in vivo fluorescence imaging. For
these reasons, expanding the repertoire of uncaging reactions to
include approaches initiated by near-IR light is an important
objective.
The substantial challenge in identifying near-IR photocages
centers on translating modest photonic energy into bond
cleavage and small molecule release. Key considerations include
removal with readily achievable light intensity, straightforward
synthesis, and biological compatibility. Most existing near-IR
uncaging relies on two-photon excitation, which requires pulsed
laser sources with limited release in only the small focal
volume.3−5 A previous single-photon near-IR uncaging
approach, suggested by Breslow, employs a photosensitizer
and an electron rich olefin attached to the payload.6,7
Irradiation with red or near-IR light forms singlet oxygen,
Here we report a near-IR (690 nm) light-initiated uncaging
approach based on the C4′-dialkylamine-substituted variant of
the heptamethine cyanine fluorophore scaffold (1). The
Received: June 28, 2014
Published: September 11, 2014
This article not subject to U.S. Copyright.
Published 2014 by the American Chemical
Society
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Scheme 1. Synthesis of 6−13
Figure 1. Uncaging reaction sequence of C4′-dialkylamine-substituted
heptamethine cyanines.
reaction sequence, shown in Figure 1, entails photooxidative
cleavage of 1 at the C1−C1′ and C2′−C3′ bonds to afford 2
and 3, which both then hydrolyze (C4′−N) and cyclize to
liberate the previously caged molecule. The increased hydro-
lytic susceptibility of 2 and 3, relative to 1, was predicted by
considering that altered π-conjugation would increase the
electrophilic reactivity of the key C4′−N bond (i.e., through
increased iminium character). Here it is shown that these
readily synthesized cyanine derivatives liberate phenol-contain-
ing small molecules upon irradiation with 690 nm light. This
method is applied to alter gene expression through ligand-
dependent genetic recombination and, in a separate demon-
stration, to release cytotoxic concentrations of a therapeutic
agent.
cyanine intermediate formed from N,N′-dimethylethylenedi-
amine and 5. We also prepared N-methylethanolamine-
substituted cyanines, 12 and 13, which are designed to release
only N-methylethanolamine. Cyanine 12 proved useful to
evaluate the amine release reaction by NMR and 13 served as a
negative control in the cellular studies. These two molecules
were prepared by heating 4 or 5 and N-methylethanolamine in
MeCN or DMF, respectively. Of note, cyanines 6−11 exhibit
optical properties similar to that of other N-substituted
heptamethine cyanines, including useful quantum yield of
fluorescent emission (Supporting Information Table S1).
Uncaging Analysis. With cyanines 6−11, the phenol
release reaction was investigated under a range of conditions
and concentrations. UV−vis analysis indicated that these
compounds do not aggregate at micromolar concentrations
under a variety of aqueous conditions, including those shown in
Figure 2. Irradiation of a 50 μM solution of 8 in pH 7.5 HEPES
buffer with 1 mW/cm2 690 ( 20) nm light from a commercial
LED source provided an increase in the characteristic
nitrophenolate absorbance (400 nm), with concomitant
reduction of the cyanine absorbance (680 nm) (Figure 2B).
Consistent with the accumulation of intermediate species that
convert to the final phenolate product, the timing for
disappearance of the cyanine absorbance signal and appearance
of the 4-nitrophenolate signal diverge significantly, with half-
lives (t1/2) of 8.5 and 40 min, respectively. Indicating that
uncaging can occur in complex biological mixtures, similar
absorbance profiles were seen in Dulbecco’s Modified Eagle’s
RESULTS AND DISCUSSION
■
Design and Synthesis. To test this approach, a series of
dialkylamine-substituted cyanines were synthesized (Scheme
1).22 N-Methyl carbamates 6−11 were designed to unveil
phenols through sequential amine release/intramolecular
cyclization (Figure 2A).23 Cyanines 6−10 serve as mechanistic
probes through release of optically silent free phenol, the
fluorophore 4-methylumbelliferone, and the absorbance
reporter 4-nitrophenol. We also prepared 11, which is designed
to release 4-hydroxycyclofen, a bioactive small molecule. The
indoline nitrogen of the cyanine was substituted with either n-
propyl or 4-butanesulfonate substituents, the latter being more
well tolerated by biological systems.24 Compounds 6−10 were
accessed through a protocol comprising initial conjugation of
N,N′-dimethylethylenediamine to commercially available IR-
780 (4) and IR-783 (5), followed by addition of a
chloroformate to the unpurified diamine cyanine intermediate.
The 4-hydroxycyclofen conjugate 11 was prepared by adding
the corresponding mixed nitrophenyl carbonate to the same
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solution of 12 at 690 nm for 24 h at 25 °C in 1:1 D2O:d4-
methanol provided substantial conversion (66−70%) to N-
1
methylethanolamine (14) (Scheme 2), as determined by H
Scheme 2. Products Observed upon 690 nm Irradiation of
a
12
a
Yield is based on an NMR internal standard and was run in triplicate
to provide the range shown.
NMR. As seen in previous studies, oxindole 15 and aldehyde 16
were also generated.20,21 Compound 12 is stable in the dark
under these conditions with greater than 95% remaining after
14 days, indicating the light dependence of this reactivity.
The proposed release sequence is shown in Scheme 3 with
compound 10. Photooxidative cleavage occurs through initial
formation of dioxetane intermediates 17 and 18, which
thermally decompose to form carbonyl products 19 and 21,
respectively. Subsequent hydrolysis of both of these inter-
mediates provides 25, which undergoes rapid intramolecular
cyclization to provide the now uncaged phenolate 26. To
provide support for this proposal, we have carried out a series
of mass spectrometry experiments. Spectral signals for all
intermediates shown in Scheme 3 are observed by high-
resolution mass spectrometry (HRMS) of an irradiated solution
of 10 in H2O. Notably, signals consistent with dioxetanes 17
and 18 are obtained, an observation that has also been made
elsewhere.20,21 Singlet oxygen (1O2), generated through energy
transfer from the triplet excited state of the cyanine, is generally
assumed to be the major reactive oxygen species involved in the
photooxidative cleavage,12−21 although light-generated super-
Figure 2. (A) General scheme for phenolate release. (B) Absorbance
traces at 400 nm (blue) and 680 nm (red) with (solid line) or without
(dashed line) 1 mW/cm2 690 nm irradiation of a 50 μM solution of 8
(50 mM HEPES, pH 7.5 with 5% DMSO). (C) As (B), but with 3
mW/cm2 690 nm irradiation in DMEM (buffered with 50 mM
HEPES, pH 7.5, 0.1% DMSO) with 10% FBS. (D) As (B), but with
intermittent irradiation. (E) Fluorescence traces (360 nm ex., 460 nm
em.) with (solid line) or without (dashed line) 1 mW/cm2 690 nm
irradiation of a 50 nM solution of 7 (50 mM HEPES, pH 7.5, 5%
DMSO). All data shown are the average of three independent
experiments with the standard deviation ≤5% in all cases.
Medium (DMEM) containing 10% fetal bovine serum (FBS),
albeit requiring a somewhat higher light intensity of 3 mW/cm2
to achieve a similar release rate (t1/2 for release = 34 min)
(Figure 2C). To examine the effect of intermittent irradiation,
compound 8 was irradiated for 10 min, maintained in the dark
for 50 min, and irradiated for another 10 min (Figure 2D). The
profile of the cyanine absorption decrease correlates directly
with irradiation, while the nitrophenolate signal increases in
interim periods. This result suggests that only irradiation
sufficient to disrupt the cyanine absorption is required, and that
the subsequent steps that culminate in release occur in the
absence of irradiation. Fluorescence was used to examine the
release reaction at lower concentrations. Irradiation of a 50 nM
solution of 7, or the corresponding butanesulfonate-substituted
10, led to appearance of 4-methylumbelliferone fluorescence
(Figure 2E and Supporting Information Figure S1). We also
subjected control compounds 6 and 9, which release only
phenol, to the conditions above. Verifying that the signals
discussed above derive from release of the absorbance and
fluorescence reporters, irradiation of 6 and 9 demonstrated that
the byproducts of photolysis only minimally absorb at 400 nm
and are not fluorescent at the excitation/emission wavelengths
of 4-methylumbelliferone (Supporting Information Figure S2).
Our approach is based on the prediction that phenol release
is preceded by cleavage of the C4′ secondary amine.
Accordingly, we examined the C4′-amine release step in
isolation with a simpler N-methylethanolamine-substituted
cyanine, which cannot undergo a subsequent cyclization step.
Continuous irradiation (690 nm, 5 mW/cm2) of a 1 mM
−
oxide (O2 ) has been suggested to be a competing, albeit likely
minor, O2 source.15,18,19 We note that methods, such as
chromophore-assisted light inactivation (CALI), using the
highly localized nature of singlet oxygen generated by
photosensitization find broad use for a variety of biological
applications.25
We sought to provide further support for the notion that the
photooxidation intermediates accumulate during irradiation and
then undergo subsequent light-independent hydrolysis. Mass
spectral ion counts of starting material 10, the indistinguishable
dioxetanes, 17/18, the photooxidative cleavage products, 19
and 21, and released 4-methylumbelliferone, 26, were measured
at various time points. As shown in Figure 3, 10 is consumed
after 30 min of irradiation with concomitant increase in 17/18,
19, and 21. During the ensuing incubation at 37 °C without
irradiation, these signals decrease as the signal corresponding to
26 increases. These results, as well as the mass spectral
characterization of the direct products of the hydrolysis
reaction, 23−25, are noteworthy because, unlike the photo-
oxidative cleavage step, the hydrolysis step has only indirect
precedent, and then not under neutral aqueous conditions.26,27
It is plausible that hydrolysis is particularly facile in this case as
a consequence of the electron-withdrawing α and γ carbonyls.
Further characterization of 23 and 24, for example through
chromatographic isolation and spectral analysis, has not been
possible to date.
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Scheme 3. Proposed Intermediates in Uncaging Sequence of 10
analog of 4-hydroxytamoxifen that exhibits similarly potent
estrogen receptor antagonist/agonist activity.28 We first
measured the yield of light-dependent release in biological
media. A 10 μM solution of 11 in DMEM with 10% FBS was
irradiated using a standard set of conditions also employed in
the experiments below (690 nm, 10 mW/cm2, 30 min, 25 °C),
followed by 37 °C incubation. Conversion exceeded 40% 1 h
after the irradiation and approached 60% after 4 h, as
determined by HPLC. The unirradiated control showed
minimal release (less than 2%) under otherwise identical
conditions (Supporting Information Figure S3).
Tamoxifen and its analogues can exhibit significant
cytotoxicity, particularly against breast cancer cell lines that
overexpress the estrogen receptor.29 With MCF-7 cells,
irradiation of 11 recapitulated the IC50 of 4-hydroxycyclofen
(9.4 μM for irradiated 11 vs 10 μM for 4-hydroxycyclofen,
Figure 4). By contrast, unirradiated 11 exhibited an IC50 of 150
μM. We also established that the majority of observed
cytotoxicity derived from released 4-hydroxycyclofen and not
the cyanine irradiation process or byproducts from cyanine
uncaging. Cyanine 13, which should only release N-
methylethanolamine, exhibited an IC50 of >200 μM upon
identical irradiation, and an IC50 of >200 μM in the absence of
irradiation. This result is consistent with previous observations
suggesting that heptamethine cyanine fluorophores are
generally only weakly cytotoxic, even upon protracted
irradiation.24 The findings presented here, as well as these
previous observations, appear to suggest the products of the
cyanine photodecomposition process, 19−24, are relatively
well-tolerated, despite the presence of potentially reactive
carbonyl groups. As expected with this low energy light, no
phototoxicity was observed with irradiation alone.
Figure 3. Relative spectral ion counts of 10, 17/18, 19, 21, and 26
over time upon exposing a 20 μM solution of 10 in H2O to 10 mW/
cm2 690 nm light for 30 min at rt, and at 37 °C for an additional 60
min without irradiation. Ion counts were determined at each time
point relative to an internal standard (phenylalanine).
Cellular Studies. We next examined if this technique could
be used to control cellular function using near-IR light. The
cyanine caged form of 4-hydroxycyclofen, 11, was used to
illustrate two objectives where near-IR uncaging could be of
significant benefit: release of a pharmacological agent and
regulation of gene expression. In the case of the former, the
goal is selective drug delivery to distinct tissue, and, in the
latter, to obtain fine spatial control of expression in cellular
subpopulations. 4-Hydroxycyclofen is a readily synthesized
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Figure 4. Light-dependent (690 nm, 30 min, 10 mW/cm2)
cytotoxicity of 11 and 13 against MCF-7 cells. Error bars represent
standard deviation of at least three independent experiments for the
percent change in cell viability, relative to control, as measured by
MTT assay.
Among various strategies that use light to achieve precise
regulation of gene expression, a number of studies over the past
15 years have used UV light-mediated uncaging of small
molecules in combination with inducible gene expression
systems.30,31 The distinct advantages of near-IR light might
prove beneficial as these techniques progress in complex
biological settings and organismal contexts. To pursue this, we
used a CreER/LoxP-reporter approach in which recombination
is initiated by tamoxifen and its analogues, such as 4-
hydroxycyclofen.28,32−34 Small molecule binding to the
CreER chimera promotes nuclear translocation and site-specific
recombination at LoxP sites. We employed a Rosa26-driven
dual mT/mG reporter in transgenic mouse embryonic
fibroblast (MEFs) cells.35 Here, a ligand-dependent Rosa26-
CreERT excises a LoxP flanked fluorescent tdTomato reporter
(mT), enabling expression of a second downstream locus,
Enhanced Green Fluorescent Protein (EGFP, mG), now under
Rosa promoter control (Figure 5A).36 We chose this approach
because it allows EGFP expression to be used as an easily
assessed readout of 4-hydroxycyclofen uncaging.
Irradiation of 11 (1 μM) followed by 48 h incubation
significantly increased the proportion of EGFP positive cells, as
measured by flow cytometry (Figure 5B). The persistent
tdTomato fluorescence after uncaging likely reflects the long
half-life of this fluorescent protein.34 As shown in Figure 5C,
EGFP expression was similar to the 1 μM 4-hydroxycyclofen
control and, critically, only background expression levels were
seen without irradiation. We also verified these EGFP
expression patterns using confocal microscopy (Figure 5D).
We sought to examine the cellular uptake of 11, noting that
other disulfonated heptamethine cyanines show intracellular
staining, presumably via an endosomal uptake pathway.24b,37,38
In doing so, we were able to take advantage of the fluorescent
properties of the cyanine scaffold. After preincubating the cells
for 2 h in DMEM and washing, significant intracellular signal
derived from cyanine fluorescence was observed using confocal
microscopy (Figure 5E). Similar punctate intracellular signal
was also observed in another mammalian cell line (HeLa), and
this colocalized with LysoTracker staining (Supporting
Information Figure S4). Irradiating the preincubated/washed
transgenic MEF cells led to meaningful EGFP expression, albeit
to a slightly lower level than at the same concentration when
media is maintained throughout (1 μM), suggesting that
uncaging occurs, at least in part, within endosomal compart-
ments. Indeed, greater EGFP expression was obtained after
preincubation and media exchange with a higher concentration
of 11 (5 μM). Further studies to define the uptake mechanism
Figure 5. (A) Schematic of light-dependent EGFP expression. (B and
C) Flow cytometry of light-dependent EGFP expression in MEFs
obtained from Rosa26CreERT;Rosa26mT/mG embryos (n = 3). (D)
Confocal microscopy images of live MEF cells treated with 11 (1 μM)
and Hoechst 33342. The cells in panels 1−3 were irradiated, and the
cells in 4−6 were not. From the left, emission (1 and 4) from Hoescht
(nuclear staining), emission (2 and 5) from EGFP, and overlay (3 and
6) are shown. (E) Confocal microscopy images of live MEF cells
treated with 11 (1 μM) and Hoechst 33342. From the left, emission
(1) from Hoescht (nuclear staining), emission (2) from 11, and
overlay (3) are shown.
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and to determine and optimize the location of uncaging are
ongoing.
light source, Dr. Stephen Lockett, Ms. Kim Peifley, and the
Optical Microscopy and Analysis Laboratory (Advanced
Technology Program, Frederick National Laboratory for
Cancer Research) for their invaluable expertise and assistance
in obtaining confocal fluorescence images, and Ms. Kathleen
Noer, Ms. Roberta Matthai, and the Frederick CCR Flow
Cytometry Core (Cancer and Inflammation Program, NCI-
Frederick) for help with flow cytometry analysis. This work was
supported by the Intramural Research Program of the National
Institutes of Health, Center for Cancer Research, and the
National Cancer Institute, National Institutes of Health.
CONCLUSION
■
In summary, we have developed a near-IR light-initiated
reaction sequence of dialkylamine-substituted heptamethine
cyanines that can be used to uncage small molecules. This
technique involves easily synthesized compounds, functions
over a wide concentration range (nM to mM), and uses easily
attainable light intensity. This method was applied both to
inhibit cell survival and to alter gene expression. Mechanisti-
cally, cleavage from the cyanine scaffold occurs through two
discrete stages. In the first stage, which is dependent on light,
photooxidative C−C cleavage renders the C−N bond
susceptible to hydrolysis by altering the structure, and
consequently the reactivity, of the cyanine polyene. The second
stage, light-independent C−N bond hydrolysis, provides a
liberated secondary amine. Here the secondary amine under-
goes an intramolecular cyclization to release a phenol, though
this approach could likely also be applied to the direct uncaging
of biologically active secondary amines. The cleavage kinetics
achieved here are similar to existing single-photon photo-
sensitizer-based uncaging methods, despite using much lower
intensity light.7a Moreover, unlike these methods, the
compounds used here display minimal intrinsic phototoxicity,
which will allow for biological responses to be confidently
assigned to drug release. We note that it is plausible that
alteration of the cyanine scaffold will yield significant
improvements, and such efforts are underway.
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ASSOCIATED CONTENT
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Corresponding Author
martin.schnermann@nih.gov
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ACKNOWLEDGMENTS
■
We thank members of the Chemical Biology Laboratory for
helpful comments and suggestions. We thank Dr. Joseph Barchi
for NMR assistance and Dr. James Kelley for mass
spectrometric analysis. We also gratefully acknowledge Dr.
Hisataka Kobayashi for advice and assistance with the LED
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