Photoswitching of ortho-substituted azonium ions by red light in whole blood

Article abstract of DOI:10.1002/anie.201306352

Red-light switches: Tetra-ortho-methoxy substituted aminoazobenzenes form azonium ions at neutral pH, isomerize to the cis form when illuminated with red light (635 nm), and relax thermally to the trans form on a timescale of seconds. Copyright

Full text of DOI:10.1002/anie.201306352

Angewandte
Chemie
DOI: 10.1002/anie.201306352
Photoisomerism
Photoswitching of ortho-Substituted Azonium Ions by Red Light in
Whole Blood**
Subhas Samanta, Amirhossein Babalhavaeji, Ming-xin Dong, and G. Andrew Woolley*
Photocontrol using red light is highly desirable for biological
applications, as red wavelengths are the only part of the
visible spectrum that can effectively penetrate tissue.[1] Efforts
to develop optogenetic and optochemical genetic methods
that are red-shifted range from exploring natural biodiversity
in the search for red-shifted opsins[2] to the conjugation of
chemical photoswitches to upconverting nanoparticles.[3] We
recently reported that azobenzenes with bulky polar sub-
stituents in all four positions ortho to the azo group could
undergo red-light-driven photoisomerization.[4] However, the
compounds require intense red light or long irradiation times
to reach the photostationary state because the absorption
coefficients for wavelengths of more than 600 nm are very
small.[4]
ions, such as protonated methyl orange (1), have pKa values
in the range of 1.5–3.5[5a–c,6] so that the azonium species is
hardly present at the neutral pH normally encountered
in vivo. Second, the cis-to-trans thermal isomerization rate
of azonium ions is fast, with cis half-lives in the ms range, so
that production of a significant concentration of the cis isomer
is difficult without very bright light sources.[7] The rapid
thermal isomerization of the cis azonium species is attributed
ꢀ
to the decreased double-bond character of the N N bond
owing to the contribution of resonance structure 1(d).[8]
We report herein that introduction of methoxy substitu-
ents to all four positions ortho to the azo group in an
aminoazobenzene derivative has a remarkable effect on the
photochemistry in that it enables photoswitching of the
related azonium ion at neutral pH with red light. We
synthesized the tetra-ortho-methoxy substituted aminoazo-
benzene derivative 2, a structure that permits two-point
attached to a thiol containing target biomolecule. Synthesis of
2 was initiated from 1-bromo-3,5-dimethoxybenzene, which
was transformed into 2,2’,6,6’-tetramethoxy-4,4’-dibromoazo-
benzene, as described previously.[4] Palladium-catalyzed two-
fold amination at the 4 and 4’ positions with tert-butylpiper-
azine-1-carboxylate led to di-tert-butyl-4,4’-(diazene-1,2-diyl-
bis(3,5-dimethoxy-4,1-phenylene))bis-piperazine-1-carboxyl-
ate (see the Supporting Information). Boc deprotection of
amines followed by chloroacetylation using chloroacetyl
chloride resulted in (2), which was then reacted with a test
peptide AB15 (Ac-WGCAEAAAREAAAREAACRQ-
NH2) having Cys residues at i and i + 15 positions.[9] Attach-
ment to a peptide ensures water solubility and prevents self-
association of the dye as well as mimicking the target
environment for the photoswitch. Azobenzene derivatives
attached as cross-linkers to peptides and proteins have been
used to drive conformational and functional changes in
a variety of targets.[1a,10]
Azonium ions formed by amino-substituted azobenzenes
(Scheme 1) are well-known species that have strong absorb-
ance in the red region of the spectrum.[5] Two features,
however, make typical azonium ions difficult to use as
photoswitches in a biological context. First, most azonium
Figure 1 shows UV/Vis spectra in the range pH 5–9 of 2
and 3 (the non-methoxy substituted counterpart[9]) in their
trans states after attachment to AB15. For the tetra-ortho-
Scheme 1. Neutral and protonated forms of methyl orange (1) and the
ortho-substituted (2) and unsubstituted (3) aminoazobenzenes studied
herein. Diprotonated species can also form (see Ref. [5a]).
methoxy-substituted compound 2, the azonium ion (lmax
=
560 nm) is produced with an apparent pKa of 7.2. The
corresponding non-ortho-substituted species 3 does not
become protonated in this pH range (Figure 1b). The
azonium ion of 3 is only seen below pH 3 (Supporting
Information, Figure S1).
Irradiation of the azonium peak of 2 cross-linked to AB15
at pH 7.5 with red light (635 nm, 80 mWcmꢀ2) produces
a marked photochromism (Figure 2a). The strong absorbance
of the azonium species (ca. 20000 Lmolꢀ1 cmꢀ1 at 600 nm,
pH 7.0; see the Supporting Information) results in rapid (ca.
1 s) production of the photostationary state (Figure 2b).
[*] Dr. S. Samanta, A. Babalhavaeji, Dr. M.-x. Dong, Prof. G. A. Woolley
Departement of Chemistry, University of Toronto
80 St. George St., Toronto, ON M5S 3H6 (Canada)
E-mail: awoolley@chem.utoronto.ca
[**] We are grateful to the NSERC and the NIH (R01 MH086379) for
financial support of this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201306352.
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Communications
2–3 ms. Thus, under comparable
conditions, the thermal recovery
rate of 2 is about 106 times slower.
The consequence of this dramati-
cally slowed thermal isomerization
rate is that substantial populations
of the thermally less-stable cis
species can be produced by red
LED illumination under physio-
logical conditions.
In an effort to understand the
remarkable behavior of 2, we car-
ried out DFT (B3LYP, 6-311 ++
G(d,p)) calculations to find mini-
mum energy structures (see the
Supporting Information). We used
piperidino analogues of 2 to sim-
plify the calculations. As shown in
Figure 3, the neutral species 2a is
non-planar (Figure 3a) with substan-
tial twisting of the rings, as was
observed in X-ray crystal structures
of other neutral tetra-ortho-methoxy
species reported earlier.[4] Calcula-
tions predict the azonium species 2-
(c/d), however, is planar (Figure 3b).
Planarity facilitates resonance stabili-
zation and H-bonding interactions as
diagrammed in Figure 3c. The pres-
ence of an H-bond as shown in Fig-
ure 3b was confirmed by an atoms-in-
molecules analysis[11](see the Support-
ing Information). Stabilization of azo-
nium species by a single ortho sub-
stituent capable of H-bonding has
been reported previously.[12] In 2,
however, these effects apparently
raise the pKa by about 4–5 pH units
(compared to 3), so that the azonium
species is the dominant species at
physiological pH values.
Figure 1. UV/Vis spectra of the trans isomers of a) 2 and b) 3 cross-linked to the peptide AB15 in
aqueous buffer at the indicated pH values (228C).
The red-light-induced photo-
chromism observed (Figure 2) implies
the trans azonium species can absorb
red light and isomerize to produce
a cis species that has a lower absorb-
ance coefficient. Figure 4 shows cal-
culated minimum energy structures
for the protonated and neutral cis
species (Figure 4a,b). Unlike the trans
Figure 2. a) UV/Vis spectra of 2 cross-linked to the peptide AB15: (c) dark-adapted, (a) with
red-light irradiation (pH 7.5, 228C). b) Photoisomerization under red light (indicated by the gray
bar) followed by thermal recovery in the dark (black bar). c) Multiple photoswitching cycles show
no evidence of photobleaching (pH 7.5, 228C).
After removing the red-light irradiation, the dark-state
spectrum recovers thermally in a monoexponential manner
with a half-life of about 10 s at pH 7.5 (Figure 2b). This
process can be repeated over many cycles with no apparent
photobleaching (Figure 2c).
The thermal isomerization of methyl orange (1) has been
studied in detail using laser flash photolysis techniques by
Barra, Sanchez, and de Rossi.[7] These authors observed half-
lives for thermal recovery by the azonium ion on the order of
azonium species, the proton on the azo moiety of the cis
azonium ion is too far from the ortho methoxy substituents to
form an effective H-bond, suggesting the pKa of the cis
azonium species is lower than the trans. DFT calculations also
indicate the proton affinity of the cis isomer is lower than the
trans (see the Supporting Information). Thus, photoisomeri-
zation to the cis azonium species may be accompanied by
conversion to the neutral cis state. Time-dependent DFT
calculations predict that both neutral and protonated cis
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Angewandte
Chemie
species have lower absorbance coefficients in the red region
than the trans azonium ion (see the Supporting Information).
A neutral cis isomer would be expected to undergo
thermal relaxation to the trans state much more slowly than
the cis azonium species.[7,8] We measured the rate of thermal
recovery as a function of pH (Figure 4c) and found a sigmoi-
dal dependence to the observed rate constant. This behavior
is consistent with the simple kinetic model shown in Figure 4d
in which protonation/deprotonation rates are fast compared
to rates of thermal relaxation and the neutral cis species
undergoes relaxation (kn) about 1000 times more slowly than
the cis azonium ion (kaz). From these data, the cis azonium ion
is estimated to have a pKa of 5.7, which is lower by 1.5 pH
units than the trans azonium ion. The million-fold decrease in
the rate of thermal relaxation of 2 compared to typical
azonium ions therefore appears to be due to two factors: 1) an
intrinsic effect of the tetra-ortho substitution pattern on the
thermal barrier; and 2) deprotonation accompanying conver-
sion into the cis isomer at physiological pH values.
Figure 3. Calculated minimum-energy structures of trans neutral (a)
and protonated (b) forms of a model compound representative of 2.
c) Resonance representations showing effects that stabilize the azo-
nium ion.
Red light has significantly higher penetration through
biological tissue than other parts of the visible spectrum,
which is primarily because wavelengths of more than about
600 nm can avoid absorbance by hemoglobin.[13] To test
directly the possibility of using 2 as a photoswitch for
controlling conformational changes and ultimately bioactivity
in vivo, we mixed 2-cross-linked peptide directly with whole
blood. The azonium absorption band extends beyond the
hemoglobin absorbance (Figure 5a). Irradiation of the
Figure 5. a) UV/Vis spectra of whole blood (g), only AB15 cross-
linked with 2 (c), and AB15 cross-linked with 2 (1 mm; d) in
whole blood. Each sample was diluted 1000-fold in Tris-buffered saline,
pH 7.4 before scanning (b) Thermal relaxation after red-light irradia-
tion (80 mWcmꢀ2, 635 nm) of blood only (g) and undiluted blood
containing AB15 (c) cross-linked with 2 (after incubation at room
temperature for 12 h).
sample at 635 nm produced photoswitching that was stable
for at least 12 h (Figure 5b).
Figure 4. Cis-protonated (a) and neutral (b) forms of a model com-
pound representative of 2. c) Observed rate constant for thermal
relaxation as a function of pH. Limiting half-lives are about 1 min for
the neutral species and about 100 ms for the cis azonium ion.
d) Kinetic model showing isomerization and protonation of 2 (R
represents the linkage to the peptide). The data in (c) are fit to the
following equation:
The tetra-ortho-methoxy substitution pattern could be
used to develop a class of azo compounds for which isomer-
ization can be triggered by long-wavelength light. Azonium
species are known with absorbance maxima of more than
660 nm[5b,d] and with further tuning of the pKa difference
between cis and trans isomers, azo compounds might be
developed for manipulating biomolecules in vivo with near-
ꢀ
ꢁ
ꢀ
ꢁ
10ꢀpK
10ꢀpH
cis
kobs ¼ kn
þ kaz
.
ð10ꢀpHÞþð10ꢀpK
Þ
ð10ꢀpHÞþð10ꢀpK
Þ
cis
cis
Angew. Chem. Int. Ed. 2013, 52, 14127 –14130
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Communications
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2010, 7, 827 – 829.
infrared light. As might be anticipated based on previous
work,[4] there is some sensitivity to reduction by high
concentrations of glutathione, however (the compound is
reduced by 10 mm GSH with a half-life of about 1.4 h; see the
Supporting Information), so that use may be restricted to
extracellular and/or oxidizing environments in vivo. For
example, blood-borne peptide hormones and growth factors
could be coupled to an azonium-based photoswitch and
manipulated non-invasively with a high degree of spatiotem-
poral control.
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Experimental Section
The peptide AB15 was prepared using standard Fmoc-based solid-
phase synthetic methods. Intramolecular cross-linking of cysteine
residues on AB15 with 2 (freshly prepared, see details in the
Supporting Information) was performed in 50% DMSO as follows: A
solution of 0.5 mm peptide (freshly purified) and 2 mm cross-linker 2
in 50 mm Tris buffer at pH 8 was stirred at 408C under a nitrogen
atmosphere for 16–20 h. The completion of the reaction was judged
by MALDI mass spectra. The reaction was dried under high vacuum,
and the cross-linked peptide was purified by reverse-phase HPLC on
a semipreparative RX-C8 column (Zorbax, 9.4 mm ID ꢀ 255 mm)
with a linear gradient of 10–65% acetonitrile/water (containing 0.1%
trifluoroacetic acid) over a period of 25 min. Cross-linked AB15 was
eluted at 47% acetonitrile. ESI-MS: m/z calcd for C114H173N38O34S2:
2683.9 [M+], observed: 2682.4.
Received: July 21, 2013
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Published online: November 8, 2013
Keywords: azobenzenes · blood · cis–trans isomerism ·
.
photoisomerization · photoswitches
[1] a) W. Szymanski, J. M. Beierle, H. A. Kistemaker, W. A.
Velema, B. L. Feringa, Chem. Rev. 2013, 113, 6114 – 6178; b) D.
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