Tunable visible and near-IR photoactivation of light-responsive compounds by using fluorophores as light-capturing antennas

Article abstract of DOI:10.1002/anie.201308816

Although the corrin ring of vitamin B₁₂ is unable to efficiently absorb light beyond 550 nm, it is shown that commercially available fluorophores can be used as antennas to capture long-wavelength light to promote scission of the Co-C bond at wavelengths up to 800 nm. The ability to control the molecular properties of bioactive species with long visible and near-IR light has implications for drug delivery, nanotechnology, and the spatiotemporal control of cellular behavior. See it split: A palette of photoresponsive agents is described in which the desired activating wavelength is encoded with readily available fluorophores. Photomanipulation is feasible throughout the visible range into the near IR, and in a manner that provides orthogonal control over multiple species. This technology has been used to trigger 1) interorganelle trafficking, 2) disassembly of stress fibers, and 3) light-dose-dependent cell death. Copyright

Full text of DOI:10.1002/anie.201308816

Angewandte
Chemie
DOI: 10.1002/anie.201308816
Photoactivatable Agents Very Important Paper
Tunable Visible and Near-IR Photoactivation of Light-Responsive
Compounds by Using Fluorophores as Light-Capturing Antennas**
Thomas A. Shell,* Jennifer R. Shell, Zachary L. Rodgers, and David S. Lawrence*
Abstract: Although the corrin ring of vitamin B12 is unable to
efficiently absorb light beyond 550 nm, it is shown that
commercially available fluorophores can be used as antennas
to capture long-wavelength light to promote scission of the Coꢀ
C bond at wavelengths up to 800 nm. The ability to control the
molecular properties of bioactive species with long visible and
near-IR light has implications for drug delivery, nanotechnol-
ogy, and the spatiotemporal control of cellular behavior.
P
hotoresponsive species are used to manipulate intracellu-
lar biochemical pathways, light-sensitive nanoparticles are
employed to site-selectively deliver cytotoxic agents, and
photoinitiated hydrogels are applied as biomaterial replace-
[
1]
ments. However, the application of these molecular entities
is dependent upon short wavelengths of light (< 450 nm),
which inflict biological damage and are unable to take
[2]
advantage of the optical window of tissue (600–1000 nm).
Furthermore, the narrow wavelength–response range limits
the ability to design a family of photoactivatable species that
[
3]
can be orthogonally triggered at distinct wavelengths. We
recently reported expansion of the range of photoresponsive
[
4]
compounds to green light (approximately 560 nm), however
these compounds do not absorb light that falls within the
optical window of tissue. With these challenges in mind, we
report herein wavelength-tunable photoresponsive species
based on the vitamin B12 cobalamin ring framework. These
species are easily encoded, using commercially available
[
5]
fluorophores, to respond to specific wavelengths throughout
the visible spectrum and into the near IR.
Figure 1. Methylcobalamin (MeCbl) and cobalamin–fluorophore conju-
gates: Cbl-1 (TAMRA), Cbl-2 (acetyl), Cbl-3 (SulfoCy5), Cbl-4 (Atto725),
Cbl-5 (DyLight800), Cbl-6 (Alexa700), AdoCbl-1 (TAMRA), AdoCbl-2
A fluorescence increase is observed upon photolysis of
a
5-carboxytetramethylrhodamine (TAMRA)-substituted
(SulfoCy5), AdoCbl-3 (Atto725), and AdoCbl-4 (DyLight800).
cobalamin (Cbl-1) as a result of the ability of cobalamin to
quench the fluorescence of attached fluorophores
[
4,6]
(
Figure 1).
This effect is likely a consequence of contact
quencher, respectively, for energy transfer to transpire. Given
the fact that the cobalt–alkyl bond is weak (< 30 kcalmol ),
ꢀ
1
[7]
quenching between the fluorophore and the corrin ring
[6b]
system.
Contact quenching, unlike fluorescence energy
it occurred to us that fluorophores excited at wavelengths
beyond that absorbed by cobalamin (> 560 nm) could transfer
their excited state energy to the corrin ring and thus promote
CoꢀC bond scission (Figure 1). Indeed, the rate of photolytic
resonance transfer, does not require an overlap between the
emission and absorption wavelengths of the fluorophore and
ꢀ
3
ꢀ1
conversion of Cbl-1 ((8.0 ꢁ 0.4) ꢀ 10 s ) is twice that of its
[
*] Prof. T. A. Shell, Prof. J. R. Shell, Z. L. Rodgers, Prof. D. S. Lawrence
Department of Chemistry, Division of Chemical Biology and
Medicinal Chemistry, and the Department of Pharmacology
University of North Carolina,Chapel Hill, NC 27599 (USA)
E-mail: tshell@email.unc.edu
non-fluorophore-containing counterparts MeCbl and Cbl-2
ꢀ
3
ꢀ1
ꢀ3 ꢀ1
(
(3.8 ꢁ 0.2) ꢀ 10
s
and (4.2 ꢁ 0.2) ꢀ 10 s , respectively;
Figure 1, and Figures S1–S4 in the Supporting Information),
thus suggesting that excitation of an appended fluorophore
could play a role in promoting photocleavage of the CoꢀC
lawrencd@email.unc.edu
bond.
[
**] We thank the National Institutes of Health for financial support
Several fluorophores were appended to the alkylcobala-
min framework, all with excitation wavelengths longer than
that absorbed by the corrin ring (Cbl-2–Cbl-6). A number of
features are apparent from the results presented in Table 1.
(
RO1 CA79954).
Supporting information (including materials and methods) for this
article is available on the WWW under http://dx.doi.org/10.1002/
anie.201308816.
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875

.
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Table 1: Percentage photolysis of cobalamin–fluorophore conjugate upon illumination at 546 nm
5 min), 646 nm (5 min), 700/727 nm (20 min), and 777 nm (10 min). Percentage photolysis was
calculated using the maximum observed fluorescence increase as 100% photolysis (see Figures S8, S10,
S12, S14, S17, and S24).
Table 1. By contrast, since Cbl-3
and Cbl-5 are photochemically dis-
tinct at 646 nm and 777 nm
(
(Table 1, Figure S19), they can be
[
a]
Cobalamin Fluorophore
conjugate
lab/ex [nm]
Percent photolysis at applied wavelength [nm]
individually photomanipulated
without the need to resort to a spe-
cific illumination sequence (Fig-
ure S20). The more than 200 nm
bandwidth of the cobalamin–fluo-
rophore conjugates in Table 1 fur-
nishes a spectral range in which
specific compounds in a mixture can
be independently acted upon.
Indeed, since the dissociation
energy of the CoꢀC bond is esti-
[
b]
5
46
646
700/727
777
[
c]
[c]
c]
[c]
[c]
Cbl-1
Cbl-3
Cbl-4
Cbl-5
Cbl-6
Cbl-Bod
TAMRA
SulfoCy5
Atto725
DyLight800
Alexa700
Bodipy650
546
546, 646*
546, 646, 727*, 777
546, 727, 777*
546, 646, 700*
546, 646*
100ꢁ17
13ꢁ21
62ꢁ8
[
100ꢁ21
35ꢁ5
100ꢁ5
20ꢁ12
100ꢁ3
9ꢁ5
[
c]
22ꢁ12
56ꢁ3
100ꢁ12
[
c]
48ꢁ5
[
c]
[c]
48ꢁ12
100ꢁ12
[
a] Absorbance/excitation of the cobalamin–fluorophore conjugate where * is the major fluorophore
excitation band. [b] All compounds were photolyzed at 727 nm, except Cbl-6, which was photolyzed at
00 nm (3 min). [c]<5% photolyzed under the experimental conditions.
7
First, illumination at the corrin absorption wavelength
546 nm) completely converts all the derivatives to their
photolyzed products if sufficient light exposure is applied
25 min under the conditions described in the Supporting
Information). However, at a much shorter illumination time
5 min), Cbl-1 is photolyzed to a greater extent at 546 nm than
its counterparts, presumably as a result of the significant
(
(
(
ꢀ1
ꢀ1
TAMRA absorbance in this region (e = 90000 cm m ).
Second, all the compounds undergo photolysis at their
excitation wavelengths, even when those wavelengths are
far beyond the absorbance of the corrin ring. The cobalamin–
SulfoCy5 conjugate Cbl-3 (lex 650 nm, lem 660 nm), when
illuminated at (646 ꢁ 10) nm, is photocleaved to hydroxoco-
Figure 2. Selective photolysis of individual cobalamin conjugates from
a mixture by using serial illumination [(a) 777 nm!(b) 700 nm!(c)
6
46 nm!(d) 546 nm] sequentially photolyzes Cbl-5, Cbl-6, Cbl-3, and
balamin (B1 ) and SulfoCy5 products as assessed by UV/Vis
Cbl-1, respectively.
2a
spectroscopy (Figure S5) and LC–MS (Table S2, Scheme S7).
The Atto725 (Cbl-4, lex 730 nm, lem 750 nm) and DyLight800
ꢀ
1 [7]
(
Cbl-5, lex 775 nm, lem 794 nm) conjugates likewise undergo
mated to be less than 30 kcalmol , it may be feasible to
sensitize bond cleavage up to and beyond 1000 nm.
photolysis at wavelengths absorbed by the appended fluo-
rophores, namely (730 ꢁ 10) and (780 ꢁ 10) nm, respectively
Fluorophores appended to the ribose 5’-OH of alkylco-
balamins also promote photocleavage of the cobalt–alkyl
linkage (Figure 1, AdoCbl-1–AdoCbl-4). Our initial studies
employed coenzyme B12 (AdoCbl) derivatives since Schwartz
and Frey had previously identified the adenosine products of
photolysis. LC–MS confirmed that 546 nm illumination of
AdoCbl (no fluorophore) and AdoCbl-1 (TAMRA) gener-
ates the expected products (Tables S6, S7). Furthermore, both
AdoCbl and AdoCbl-1 are resistant to photolysis at wave-
lengths beyond 600 nm. In a fashion analogous to the
behavior displayed by the Cbl-3–Cbl-6 series, the fluoro-
(
Figures S6, S7, Tables S3 and S4). Third, all the cobalamin–
fluorophore conjugates are stable in the dark. Fourth,
exposure of these conjugates to wavelengths that they do
not absorb has no effect on their structural integrity. The
TAMRA derivative, Cbl-1, is stable at 646 nm and longer
[
8]
(
Table 1). Cbl-3, with an appended fluorophore that absorbs
6
46 nm light, is unaffected by 727 and 777 nm illumination.
The results in Table 1 suggest that it should be possible to
selectively photolyze specific compounds from a mixture of
cobalamin-substituted derivatives by employing the appro-
priate illumination sequence and/or wavelengths. Owing to
the slight photolysis observed for Cbl-4 at 777 nm, the latter
was replaced with the Alexa700-appended conjugate Cbl-6,
which lacks an absorption band at 777 nm. Only Cbl-5
photolysis occurs when illuminating a mixture of Cbl-5, Cbl-
phore-substituted
AdoCbl-2
(SulfoCy5),
AdoCbl-3
(Atto725), and AdoCbl-4 (DyLight800) derivatives produce
the anticipated adenosine products at the wavelengths
absorbed by their appended fluorophores (Tables S8, S10).
In short, the photolytic release of compounds attached to the
cobalt ion of cobalamin can be tuned in a predictable fashion
by using commercially available fluorophores appended to
either the ribose ring or the metal ligand.
6
, Cbl-3, and Cbl-1 at 777 nm (Figure 2a). Subsequent
exposure of the mixture to 700 nm elicits the conversion of
Cbl-6 to its photoproducts without affecting Cbl-3 or Cbl-
1
5
(Figure 2b). A final sequential illumination at 646 and
46 nm (Figures 2c, d) furnishes the stepwise photolysis of
Light-responsive small molecules are commonly prepared
by covalently modifying a functional group essential for
biological activity with a photocleavable moiety, whereas
genetically expressed light-responsive proteins are typically
designed by engineering a link between light-induced con-
[1]
Cbl-3 and Cbl-1, respectively. As expected, 546 nm illumina-
tion of a mixture of these Cbl derivatives failed to furnish
selective photolysis; a predictable result based on the data in
876
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[
9]
formational changes and biochemical activities. Cobalamins
offer a potentially attractive alternative since conjugates of
Bodipy650 from the quenching effect of the cobalamin.
Under these conditions, Bodipy650 is simultaneously trans-
ferred from endosomes to mitochondria, as assessed by using
the mitochondrial marker Mitotracker Green (Pearsonꢁs
coefficient: 0.81). This supports the notion that the endosomal
capture of cobalamin derivatives can be exploited as a means
to sequester cell-permeable bioactive agents from their
intracellular targets. As an aside, it should be feasible to
employ nonendosomal strategies for the sequestration and
[
10]
cobalamins appear to be retained by endosomes. There has
been a huge effort to create delivery strategies that avoid
these organelles since endosomally embedded bioactive
species are unable to interact with their intracellular targets.
This liability represents an opportunity for cobalamin con-
jugates: cell-permeable bioactive species can potentially be
endosomally sequestered, which should preclude their action
until released by light. With this in mind, the Bodipy650
derivative Cbl-Bod was prepared (Figure 3, Schemes S2–S4).
[12]
release of light-sensitive cobalamin derivatives as well.
We examined the use of light to control the activity of
a cAMP-dependent protein kinase (PKA) signaling pathway.
A cobalamin-appended analogue of cAMP, Cbl-cAMP, was
prepared (Figure 3, Scheme S10). cAMP-induced activation
of PKA is known to promote dramatic morphological changes
[
13]
in cells, including loss of stress fibers and cell rounding. In
the absence of illumination, Cbl-cAMP has no effect on the
morphology of REF52 cells. Upon illumination in the
presence of Cbl-cAMP, REF52 cells undergo the expected
loss of their stress fibers as well as cell shrinking and rounding
(Figure 5). In addition, an 8-substituted acetylated cAMP
Figure 3. The Bodipy650–, cAMP–, and doxorubicin–cobalamin conju-
gates Cbl-Bod, Cbl-cAMP, and Cbl-Dox, respectively.
Bodipy650 is cytotoxic through a mechanism based on the
[
11]
targeting of mitochondria.
However, the corresponding
Cbl-Bod conjugate, unlike Bodipy650, is endosomal as judged
by using an endosomal marker (Pearsonꢁs coefficient 0.89;
Figure 4). Furthermore, it remains endosomally sequestered
even after 5 h in the dark. Illumination of cells containing Cbl-
Bod at 650 nm furnishes a fluorescence increase ((230 ꢁ 6)%,
Figures S26, S27) similar to that observed in the spectrofluor-
ometer ((220 ꢁ 30)%), thus signaling liberation of the
Figure 5. Light-dependent Cbl-cAMP-mediated rearrangement of actin
cytoskeleton in rat embryonic fibroblasts (REF52 cells). REF52 cells
were exposed to Cbl-cAMP, illuminated at 530 nm, and stained with
DyLight488–phalloidin to visualize the actin stress fibers. a) No treat-
ment, media alone. b) CPT-cAMP as a positive control. c) Cbl-cAMP,
no illumination. d) Cbl-cAMP with illumination. Images are representa-
tive of 10–20 cells.
analogue (Ac-cAMP, Scheme S9) was prepared and used as
a control. Both Ac-cAMP and a commercially available cell-
permeable cAMP analogue [8-(4-chloro-phenylthio)-2’-O-
methyladenosine-3’,5’-cyclic monophosphate; CPT-cAMP]
are active in the absence of light, producing morphological
effects on REF52 cells analogous to those induced by Cbl-
cAMP upon illumination (Figure S28). These results demon-
strate that the cobalamin in Cbl-cAMP precludes cytosolic
penetration by an appended cAMP derivative (Ac-cAMP is
cell permeable) and that illumination releases cAMP from its
cobalamin constraint.
Figure 4. Red light induced translocation of Bodipy 650 in HeLa cells.
a) Cbl-Bod before photolysis. b) Rhodamine B-dextran endosomal
marker. c) Overlay of (a) and (b). d) Cbl-Bod after photolysis e) Mito-
Tracker Green mitochondria marker. (f) Overlay of (d) and (e).
There is significant interest in the use of light to site-
selectively deliver therapeutic agents in vivo. For example, in
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the arena of chemotherapy, strategies that limit cytotoxicity to
targeted regions offer the promise of reduced side effects and
the application of agents that are otherwise too toxic to use.
Doxorubicin highlights this need since this valuable anti-
cancer agent displays severe cardiotoxicity. We examined
the cytotoxicity of the cobalamin–doxorubicin conjugate Cbl-
Dox (Figure 3, Scheme S11) in HeLa cells, after illumination
for different periods of time (Figure 6). Light-only treatment
delivery, material fabrication, nanotechnology, and the spa-
tiotemporal control of cellular behavior.
Received: October 9, 2013
Published online: November 27, 2013
[14]
Keywords: cobalamins · drug delivery · energy transfer ·
.
fluorescence · photochemistry
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3
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878
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