Spectral tuning of azobenzene photoswitches for biological applications

Article abstract of DOI:10.1002/anie.200805013

Longer switching wavelengths and good photochemical yields and stabilities of the cis isomers in reducing aqueous environments are achieved by introducing 2,2 -aminoalkyl substituents into 4,4 - diamido-substituted azobenzenes. The products are thus suita

Full text of DOI:10.1002/anie.200805013

Communications
DOI: 10.1002/anie.200805013
Photoswitches
Spectral Tuning of Azobenzene Photoswitches for Biological
Applications**
Oleg Sadovski, Andrew A. Beharry, Fuzhong Zhang, and G. Andrew Woolley*
Photoisomerization of azobenzene has been used in diverse
fields to enable photocontrol of molecular processes. In
biology, the introduction of azobenzene photoswitches has led
to the development of photocontrolled ion channels and
enzymes, powerful tools for studying living systems.[1–4]
Currently, a limited range of switching wavelengths in the
UV region and/or sensitivity to the reducing intracellular
environment limits broad applicability of these photoswitches
for in vivo applications.[5,6] We report a series of azobenzene
derivatives in which longer switching wavelengths (up to
530 nm) are combined with good photochemical yields and
stabilities of the cis isomers. These derivatives can be used for
photocontrol of biomolecular structures in intracellular
environments.
A large number of azobenzene derivatives are known in
which enhanced electron-donating nature of ring substituents
increases both the wavelength of absorption of the trans
isomer and the rate of thermal back-isomerization from cis to
trans.[7,8] This phenomena has been attributed to similarities
between the electronic excited state of the trans isomer and
the thermal transition state for back-relaxation, that is, that
both species have substantial dipolar character.[7,9] The
dipolar nature of the transition state also results in strong
solvent sensitivity of the half-life for thermal relaxation. For
instance, the half-life of cis-4-diethylamino-4’-nitroazoben-
zene changes from 1 ms in DMSO to 100 s in cyclohexane.[9]
Of course, photoswitches intended for use in a biological
application must operate in water. In general, azobenzene
photoswitches that absorb at long wavelengths in water relax
very quickly back to the trans state so that only vanishingly
small amounts of the cis isomer can be produced under low-
power steady-state illumination.
which switches were used to control conformational changes
in peptides and proteins, as well as gating behavior in ion
channels.[4,10] The trans-to-cis isomerization of this unit
produces a change in mean end-to-end distance of about
4 ꢀ that can lead to effective control of biomolecules linked
through the 4,4’-amido arms. To preserve the conformational
change associated with this unit, we introduced electron-
donating amino substituents in the 2,2’-positions. Despite the
very large number of azo dyes known, only a limited variety of
ortho-amino-substituted azobenzenes has been previously
reported.[11–13] The key step in the synthesis was the use of
silver oxide in acetone for formation of the azo group
(Scheme 1). A wide variety of other oxidizing agents and
solvents was tried without success. The effectiveness of AgO
and the requirement for acetone as solvent in this case
indicate participation of a solvent-stabilized radical inter-
mediate.[14]
Here we report the synthesis of a series of ortho-amino-
substituted azobenzene derivatives in which long switching
wavelengths are combined with relatively slow thermal
relaxation rates and high cis-state yields. As a result, these
molecules can be used as effective long wavelength photo-
switches to drive conformational photocontrol in biochemical
systems.
Scheme 1. Synthesis of 4,4’-diacetamido azobenzenes bearing amino
substituents in the 2,2’-positions.
The 4,4’-diamido-substituted azobenzene unit (structure 5
with R = H) has served as a useful core in several studies in
Compared with the parent compound, 4,4’-diamido-sub-
stituted azobenzene (lmax = 370 nm), these derivatives exhibit
substantial redshifts (Figure 1). A number of factors are
expected to affect the electron-donating ability of the 2,2’
substituents. The presence of a six-membered ring (5c, 5e, 5 f)
introduces steric interactions that lead to loss of sp2 character
on the N atom, whereas a five-membered ring (5d), or no ring
at all (5a, 5b) produces enhanced N delocalization.[15,16] In
addition, compounds 5e and 5 f have heteroatoms in the 2,2’
[*] Dr. O. Sadovski, A. A. Beharry, F. Zhang, Prof. G. A. Woolley
Department of Chemistry, University of Toronto
80 St. George St., Toronto, M5S3H6 (Canada)
Fax: (+1)416-978-8775
E-mail: awoolley@chem.utoronto.ca
[**] We would like to thank NSERC for financial support of this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200805013.
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Figure 1. UV/Vis spectra of the trans isomers of 5a–f in a mixed
aqueous solvent (acetonitrile/water 7/3 with 1 mm sodium phosphate
buffer, pH 7.0) in which all compounds are soluble.
Figure 2. A) CD spectra of the JRK-W peptide intramolecularly cross-
linked via Cys residues with the chloroacetyl derivative of 5 f. Dark-
adapted (blue), irradiated (red), and calculated 100% cis spectra
(dashed) indicate increased helix content on trans-to-cis isomerization.
Models of the disordered peptide with a trans cross-linker (B) and the
helical cis-linked species (C). The ortho substituents are shown in
space-filling form.
substituents that are expected to decrease the basicity of the
N atoms.[17] In general the expected relative electron-donating
ability of the 2,2’ substituents correlates with the wavelength
of maximal absorption; species with the most electron
donating pyrrolidino substituents (5d) exhibit the longest
wavelength absorption.
resonance stabilization of dipolar transition states is
decreased.[8]
Inclusion of polar groups (5e, 5 f) on the ortho substitu-
ents distal to their attachment points to the azo chromophore
retains the wavelength and switching properties but greatly
enhances water solubility. For this reason, we chose the N-
methylpiperazino derivative 5 f for reaction with a test
peptide in aqueous solution. The peptide JRK-W, a model
helix-forming peptide that has been studied extensively,[19]
was intramolecularly cross-linked with the chloroacetyl
derivative of 5 f, as shown in Figure 2. As expected, based
on previous work with the parent cross-linker[19] the trans
isomer destabilized the JRK-W helix. Irradiation with a 400-
nm LED produced about 50% cis isomer and a substantial
increase in helix content as judged by circular dichroism
spectrometry (Figure 2). Since thermal reversion generates
about 100% trans isomer, production of 50% cis isomer
under low-power irradiation constitutes a large change in the
population of this cis isomer.[20] Attachment of the photo-
switch to the peptide slowed the relaxation rate even further.
The half-life for thermal relaxation of this photoswitchable
peptide was about 40 min at 48C, and about 5 min at 208C.
A variety of pathways for the in vivo modification of azo
compounds that could lead to loss of photoswitching are
known.[21] The detailed structure and route of administration
of an azo compound can determine whether it is metabolized
quickly or not at all.[21] In general, the electron-rich nature of
these photoswitches makes them less susceptible to reduction
(e.g., by intracellular thiols) than azobenzene photoswitches
studied previously.[6] Compound 5 f was incubated in 10 mm
reduced glutathione/5 mm reduced DTT (dithiothreitol) for
24 h at 378C, an environment more reducing than typically
found in the eukaryotic cytosol,[22] and no change in UV/Vis
absorption or switching kinetics was observed (see Support-
ing Information).
Despite the long wavelength absorption of the trans
isomers, these compounds relax from cis to trans on time-
scales of seconds to minutes, and substantial fractions of cis
isomers can be produced by low-power LED illumination
(Table 1). A previously reported photoswitch that absorbs in
this wavelength range relaxes with a time constant of about
10 ms under comparable conditions.[18]
Table 1: Properties of photoswitches 5a–f.
Photoswitch
lmax [nm][a]
e
[a] [mÀ1 cmÀ1
]
t1/2[a] [s]
t1/2[b] [s]
5a
5b
5c
5d
5e
5 f
470
488
445
513/537
435
13900
13000
10180
–
9610
10440
3.3Æ0.3
0.8Æ0.1
6.0Æ0.2
0.7Æ0.1
302Æ4
–
–
–
–
8.1Æ0.2
437
207Æ10
27Æ1
[a] In 70% acetonitrile/water, 1 mm sodium phosphate buffer, pH 7.0,
208C. [b] In 10 mm sodium phosphate buffer, pH 7.0, 208C.
There are a number of possible origins of the relatively
slow thermal relaxation rates. Possibly, the ortho substituents
selectively stabilize the cis isomer relative to the transition
state for thermal relaxation as compared to analogous para-
substituted analogues. Examining molecular models of cis
isomers shows that the ortho substituents can pack closely
around the azo group and may form a local hydrophobic cage
that stabilizes the structure (Figure 2C). Such a local hydro-
phobic environment around the azo group would also tend to
destabilize isomerization transition states that involve
buildup of negative charge on the azo nitrogen atoms.[7] The
ortho substituents may thus mimic the effects of solvents on
relaxation rates. In addition, ortho substituents are expected
to lead to twisting of the rings about the azo group so that
By combining long-wavelength switching with good
photochemical yields and stabilities of the cis isomers in
reducing aqueous environments, this series of azobenzene
photoswitches thus significantly expands the possibilities for
directed conformational control in biological settings.
Angew. Chem. Int. Ed. 2009, 48, 1484 –1486
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Communications
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Experimental Section
Compounds 3a–f, 4a–f, and 5a–f were prepared as outlined in
Scheme 1. Representative reaction procedures: 3 f: A solution of 3’-
chloro-4’-nitroacetanilide (2; 5 g, 23 mmol) in 1-methylpiperazine
(25 mL) was heated at 1008C with stirring for 12 h and then diluted
with water (150 mL). The solid was collected and used for the next
step without additional purification. Yield: 5.24 g (81%).
4 f: Zn dust (3 g) was added to 3 f (1 g, 3.6 mmol) in 28%
ammonium hydroxide (100 mL) over 1 min. The mixture was stirred
at room temperature (not higher than 358C) for 30 min, concentrated
under reduced pressure to 30% of its initial volume, and filtered. The
aqueous solution was washed with chloroform, and the organic layer
collected and dried with Na2SO4. The chloroform was evaporated to
give the product 4 f (0.7 g, 78%), which could be used directly in the
next step.
5 f: Freshly prepared, dry AgO (1.04 g, 8.4 mmol) was added to a
solution of 4 f (0.7 g, 2.8 mmol) in dry acetone (25 mL) at room
temperature with vigorous stirring. After stirring in the dark for 1 d, a
fresh portion of AgO (1.04 g) was added, stirring was continued for
another 1–4 d, and the presence of the initial amine was monitored by
thin-layer chromatography. The solid was collected by filtration,
washed with methanol, and the filtrates were combined and
evaporated. The product was further purified by column chromatog-
raphy on silica gel (MeOH/EtOAc 1/1; Rf = 0.1) to give 0.17 g (24%)
of a fine orange solid. Full experimental details of characterization of
compounds 3a–f, 4a–f, and 5a–f and the chloroacetyl derivative of 5 f
can be found in the Supporting Information. Peptide synthesis and
cross-linking were performed as described previously.[10] UV/Vis and
CD spectra were obtained and analyzed as detailed in the Supporting
Information.
[10] D. C. Burns, F. Zhang, G. A. Woolley, Nat. Protoc. 2007, 2, 251 –
258.
[11] H. Bisle, H. Rau, Chem. Phys. Lett. 1975, 31, 264 – 266.
[12] H. Mustroph, Dyes Pigm. 1991, 15, 129 – 137.
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[18] L. Chi, O. Sadovski, G. A. Woolley, Bioconjugate Chem. 2006,
17, 670 – 676.
[19] D. C. Burns, D. G. Flint, J. R. Kumita, H. J. Feldman, L. Serrano,
Z. Zhang, O. S. Smart, G. A. Woolley, Biochemistry 2004, 43,
15329 – 15338.
Received: October 14, 2008
Revised: November 20, 2008
Published online: January 15, 2009
[20] D. A. James, D. C. Burns, G. A. Woolley, Protein Eng. 2001, 14,
983 – 991.
[21] M. A. Brown, S. C. DeVito, Crit. Rev. Environ. Sci. Technol.
1993, 23, 249 – 324.
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Keywords: azo compounds · isomerization · peptides ·
photoswitches
.
[1] C. Renner, L. Moroder, ChemBioChem 2006, 7, 868 – 878.
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