Bidirectional photocontrol of peptide conformation with a bridged azobenzene derivative

Article abstract of DOI:10.1002/anie.201202383

It goes both ways: A thiol-reactive cross-linker based on a bridged azobenzene derivative permits photoreversible control of peptide conformation on irradiation with violet (407a nm) and green (500-550a nm) light (see picture) through isomerization of the

Full text of DOI:10.1002/anie.201202383

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DOI: 10.1002/anie.201202383
Photoisomerization
Bidirectional Photocontrol of Peptide Conformation with a Bridged
Azobenzene Derivative**
Subhas Samanta, Chuanguang Qin, Alan J. Lough, and G. Andrew Woolley*
A major challenge in photocontrol is to combine a large
conformational change with complete bidirectional photo-
switching.[1] The azobenzene chromophore undergoes a large
conformational change upon isomerization but, because of
the overlap of the absorbance spectra of the cis and trans
isomers, photoswitching is incomplete.[2] Thermal isomeriza-
tion can fully repopulate the thermodynamically more stable
trans state, but this process may be too slow for many
applications. Increasing the thermal relaxation rate by
introducing appropriate ring substituents is possible,[3] but
means that one cannot maintain the cis isomer without
continual irradiation.
In 2009 Siewertsen et al. reported the remarkable photo-
chemistry of the bridged azobenzene derivative 1.[4] This
compound had been described 100 years earlier, but its
photochemical properties had apparently not been realized.[5]
The C2 bridge in 1 produces a highly twisted trans isomer that
is less stable than the cis isomer—opposite to the situation
with normal azobenzenes. Most importantly, from the point of
stability of the azo derivative to reduction by glutathione,
which is present intracellularly at concentrations between
1 and 10 mm.[7] We reasoned, based on our experience with
the substitution of azobenzene itself,[8] that p-acetamido
substitution (i.e. compound 2) might result in a suitable red-
shift and confer stability against reduction by glutathione.[9]
Moreover, p-acetamido substitution permits the synthesis of
a thiol-reactive cross-linker version (3) of the photoswitch
that is suitable for introduction into biomolecules such as
peptides and proteins.[10]
Scheme 1 shows the synthetic approach we used to
produce the p-acetamido-substituted C2-bridged azobenzene
2 and the thiol-reactive chloroacetamido derivative 3. A
view of photocontrol, the C2 bridge leads to an 85 nm
separation of the n-p* absorption bands of the cis and trans
isomers, so that there are wavelengths where only the trans
isomer absorbs. The separation of the n-p* transitions of cis
and trans isomers has also been seen recently with tetra-ortho-
methoxy-substituted azobenzene derivatives,[6] although in
this case the separation is only 35 nm.
The large separation of the n-p* transitions of C2-bridged
azobenzene 1 would be even more useful from the standpoint
of biomolecular photoswitching if the cis-to-trans switching
wavelength (where ecis/etrans is maximal) were slightly red-
shifted so that it was in the visible range rather than in the UV
region.[4] An additional concern for biological studies is the
Scheme 1. Synthesis of bridged azobenzene photoswitches 2 and 3.
a) PPh3, toluene, reflux, yield 96%; b) tBuOK, 3-nitrobenzaldehyde
THF, 08C–RT, 57%; c) H2, Pd/C, MeOH, yield 90%; d) Ac2O, Py, THF,
yield 78%; e) KNO3, AcOH/H2SO4 (conc.), 08C–RT, 31%;
f) Ba(OH)2·8H2O, Zn, EtOH, reflux; g) HgO, EtOH, reflux (overall
yield for two steps 8%); h) KOH, MeOH, reflux, 70%; i) chloroacetic
anhydride, Py, diethyl ether, 50%. Py=pyridine.
Wittig reaction was used to produce 1,2-bis(3-nitrophenyl)-
ethene (8) from (3-nitrobenzyl)triphenylphosphonium bro-
mide (9) and 3-nitrobenzaldehyde. The alkene and the two
nitro groups of 8 were reduced using H2-Pd/C to give 1,2-
bis(3-aminophenyl)ethane (7; see the Supporting Informa-
tion). This compound was acetylated (to give 6) and then
nitrated to give 1,2-bis(2-nitro-5-acetamidophenyl)ethane (5).
A two-step oxidation protocol, adapted from that of Paudler
and Zeiler,[11] using Ba(OH)2, Zn, and HgO led to para-
diacetamido-substituted C2-bridged azobenzene 2. Hydrolysis
of the two acetamido groups of 2 with KOH resulted in para-
diamino-substituted C2-bridged azobenzene 4, which was
treated with chloroacetic anhydride/pyridine to give para-
di(a-chloroacetamido)-substituted C2-bridged azobenzene 3.
[*] Dr. S. Samanta, Dr. A. J. Lough, Prof. G. A. Woolley
Dept. of Chemistry, University of Toronto
80 St. George Street, Toronto, ON M5S 3H6 (Canada)
E-mail: awoolley@chem.utoronto.ca
Prof. C. Qin
School of Life Science, Northwestern Polytechnical University
127 West Youyi Rd., Xi’an, Shaanxi, 710072 (China)
[**] We are grateful to the 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.201202383.
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Computational (B3LYP/cc-pVTZ) methods were used to
calculate the relative stability of the cis and trans isomers of 2,
and these calculations confirmed that, similar to the parent
unsubstituted C2-bridged compound 1, the cis isomer was the
more stable by approximately 8 kcalmolÀ1 (see the Support-
ing Information). An X-ray crystal structure of the cis isomer
confirmed the calculated structure (Figure 1). Time-depen-
in the concentration of the trans isomer of at least 400-fold
(see the Supporting Information) is possible. The large
separation of the n-p* bands of the cis and trans isomers
also results in a convenient visible color change of these
solutions from pale yellow for the cis to reddish purple for the
trans isomer.
The photoisomerization of azobenzene has been used to
control a wide variety of biomolecular targets (peptides,
proteins, and nucleic acids) in vitro[10,13] as well as in cell
extracts.[14] It has been applied to the photocontrol of proteins
in living cells and tissues[15] and to the photocontrol of ion
channels in vivo.[16] Photocontrol of the conformation of
helical peptides has been extensively investigated as a proto-
type for conformational control, where the mechanism of the
coupling of the azo isomerization to the peptide conforma-
tional response can be well described.[17,18] Peptides bearing
pairs of Cys residues can be intramolecularly cross-linked
with thiol-reactive azobenzene-based photoswitches such as 3
(Figure 2). When a peptide bearing a pair of Cys residues
Figure 1. a) UV/Vis spectra of the photostationary states (PSS)
obtained for 2 in DMSO. b) X-ray crystal structure of cis-2. c) Calcu-
lated (B3LYP/cc-pVTZ) structure of trans-2.
dent DFT calculations also supported our expectation that
the p-acetamido substituent would red-shift the wavelengths
of both the cis and trans n-p* transitions (see the Supporting
Information). Figure 1 shows the UV/Vis spectra of cis-2,
prepared in the dark, in DMSO where the n-p* transition is
approximately 10 nm red-shifted compared to that of the
parent bridged compound.[4] Irradiation of this species
produced the trans isomer, which exhibited an n-p* transition
maximum near 505 nm (Figure 1), approximately 15 nm red-
shifted compared to the parent compound and 90 nm red-
shifted compared to the cis form.
The trans isomer was most effectively produced by
irradiation at 407 nm, the wavelength where ecis/etrans was
maximal. This wavelength is conveniently available in many
low-cost violet light-emitting diodes (LEDs) and microscope
illuminators, and has been chosen in other studies as a target
wavelength for the design of photoswitches.[12] Irradiation at
407 nm produced a photostationary state that was 70:30 trans/
cis as judged by NMR spectroscopy (see the Supporting
Information). The extrapolated spectrum of the pure trans
isomer is also shown in Figure 1. Thermal relaxation from the
trans to the cis isomer occurred with a half-life of about 4.8 h
at 208C in DMSO (see the Supporting Information), a rate
similar to that of the parent bridged compound.[4] Impor-
tantly, the spectrum of the trans isomer has a long wavelength
tail where the cis isomer essentially does not absorb (see the
Supporting Information for the absorbance spectrum of
a very concentrated solution of the cis isomer at long
wavelengths). This feature means that irradiation with green
light (500–550 nm) completely (> 99.7%) repopulates the
more stable cis isomeric state. Thus, a photocontrolled change
Figure 2. Photoswitching of helical peptide conformation with the
bridged azobenzene derivative 3. a) CD spectra obtained with irradi-
ation conditions shown at 208C (5 mm Na phosphate buffer, pH 7.0).
b) Models showing FK-11 (AcWGEACAREAAAREAACRQ-NH2) cross-
linked with 3 in the cis (left) and trans (right) conformations. c) Multi-
ple rounds of photoswitching (monitored by the absorbance of the
trans isomer) can be carried out in the presence of 5 mm reduced
glutathione.
undergoes a helix–coil transition, the spacing between the Cys
thiol groups changes. The greatest degree of photocontrol
over the peptide conformational can be achieved when the
spacing between the Cys thiol residues in the helical state of
the peptide matches one isomer of the photoswitch but not
the other.[10,18]
To investigate optimal Cys spacings for the bridged
photoswitchable cross-linker 3 we performed molecular
dynamics simulations on the isolated cross-linker (see the
Supporting Information). These simulations indicated that
the mean end-to-end distances are somewhat shorter for both
isomers compared to their nonbridged counterparts,[19] but
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the change in the end-to-end distance is similar for the
bridged and nonbridged photoswitches. The simulations
indicated that the trans isomer has an end-to-end distance
that matches well with the thiol (S-S) spacing in a helical
peptide with Cys residues spaced i, i + 11 in the primary
sequence. This distance is too long to be spanned by the cis
nonhelical) conformational state for long periods of time,
then pulsed “on” (helical) with violet light and “off” again
with green light. Such photocontrolled biomolecules are
expected to find applications in diverse settings.[21]
isomer of 3. In the case of the bridged photoswitch, the cis Experimental Section
The peptide FK-11 (AcWGEACAREAAAREAACRQ-amide) was
isomer is the thermodynamically more stable state, thus the
nonhelical conformation is expected to be preferred—oppo-
site to the case for the nonbridged analogue.[20]
prepared by using standard 9-fluorenylmethoxycarbonyl (Fmoc)
based solid-phase synthetic methods. Intramolecular cross-linking of
cysteine residues in FK-11 with 3 (prepared as described in the
Supporting Information) was performed in 25% DMSO as follows: A
solution of 0.5 mm peptide (freshly purified) and 2 mm cross-linker 3
in 50 mm tris(hydroxymethyl)aminomethane (Tris) buffer at pH 8 was
stirred at 378C under a nitrogen atmosphere for 24 h. The completion
of the reaction was judged by ESI mass spectrometry. The reaction
was dried under high vacuum, and the cross-linked peptide was
purified by reverse-phase HPLC. FK11 cross-linked with 3 was eluted
at 46% acetonitrile/water and characterized by ESI-MS: m/z calcd
for C98H142N34O28S2: 2308.53 [M+]; found: 2308.71. Details of the UV/
Vis, CD, and NMR spectroscopy measurements are provided in the
Supporting Information.
We synthesized the peptide FK-11, known to be a helix-
forming peptide in aqueous solution,[20] and cross-linked it
with 3. Figure 2 shows the circular dichroism (CD) spectra for
the cross-linked peptide after irradiation at 407 nm and
518 nm. These spectra indicate that, as expected, irradiation
with violet light causes an increase in the helix content for FK-
11 cross-linked with 3, while irradiation with green light
completely reverses this conformational change (Figure 2).
The thermal relaxation from the trans to the cis isomer was
slower when the photoswitch was attached to the peptide (t1/2
= 8.3 h at 208C, compared to 4.8 h for 2 in DMSO; see the
Supporting Information). A CD signal is also seen at longer
wavelengths for the trans isomer (not for the cis isomer; see
the Supporting Information). This observation of induced CD
indicates that the n-p* transition in the trans isomer senses the
chiral environment of the peptide.
As with the parent bridged azobenzene derivative studied
by Siewertsen et al., the p-acetamido derivative was found to
be photostable, undergoing hundreds of cycles of irradiation
with violet and green light without noticeable decay, both as
a free compound in DMSO and when attached to the peptide
in aqueous solution (see the Supporting Information). We
then tested the redox stability of the p-acetamido-substituted
bridged azobenzene when attached to the FK-11 peptide by
exposing it to various concentrations of reduced glutathione
under different irradiation conditions. The cis isomer was
found to be completely stable to overnight incubation with
10 mm reduced glutathione (see the Supporting Information).
The trans isomer, however, underwent a bleaching process,
whose rate depended on the concentration of the reduced
glutathione (see the Supporting Information). The process
was slow enough, however, under typical physiological
conditions (t1/2 ꢀ 3 h at 258C, 5 mm GSH, pH 7.0) that
multiple cycles of photoswitching could be carried out
without a measurable decay of the signal (Figure 2). This
sensitivity of the trans isomer to reduction is likely a result of
its highly strained nature relative to the nonbridged analogue,
which is not reduced under comparable conditions.[9]
Received: March 27, 2012
Published online: May 29, 2012
Keywords: azo compounds · helical structures · peptides ·
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photocontrol · photoisomerization
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