A bisazobenzene crosslinker that isomerizes with visible light

Article abstract of DOI:10.3762/bjoc.8.246

Background: Large conformational and functional changes of azobenzene-modified biomolecules require longer azobenzene derivatives that undergo large end-to-end distance changes upon photoisomerization. In addition, isomerization that occurs with visible r

Full text of DOI:10.3762/bjoc.8.246

A bisazobenzene crosslinker that
isomerizes with visible light
Subhas Samanta, Harris I. Qureshi and G. Andrew Woolley*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2012, 8, 2184–2190.
Department of Chemistry, University of Toronto, 80 St. George St.,
Toronto, ON, M5S 3H6, Canada; telephone: (416) 978-0675, fax:
(416) 978-8775
doi:10.3762/bjoc.8.246
Received: 24 August 2012
Accepted: 26 November 2012
Published: 14 December 2012
Email:
G. Andrew Woolley* - awoolley@chem.utoronto.ca
This article is part of the Thematic Series "Molecular switches and cages".
Guest Editor: D. Trauner
* Corresponding author
Keywords:
azobenzene; molecular switches; switch; photo-control; visible
© 2012 Samanta et al; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
Background: Large conformational and functional changes of azobenzene-modified biomolecules require longer azobenzene
derivatives that undergo large end-to-end distance changes upon photoisomerization. In addition, isomerization that occurs with
visible rather than UV irradiation is preferred for biological applications.
Results: We report the synthesis and characterization of a new crosslinker in which a central piperazine unit links two azobenzene
chromophores. Molecular modeling indicates that this crosslinker can undergo a large change in end-to-end distance upon
trans,trans to cis,cis isomerization. Photochemical characterization indicates that it does isomerize with visible light (violet to blue
wavelengths). However, the thermal relaxation rate of this crosslinker is rather high (τ½ ~ 1 s in aqueous buffer at neutral pH) so
that it is difficult to produce large fractions of the cis,cis-species without very bright light sources.
Conclusion: While cis-lifetimes may be longer when the crosslinker is attached to a biomolecule, it appears the para-piperazine
unit may be best suited for applications where rapid thermal relaxation is required.
Introduction
Azobenzene derivatives have been used for reversible manipu- achieved by using rigid crosslinkers that undergo a large change
lation of biological targets, including peptide and protein struc- in end-to-end distance upon photoisomerization [6]. Previously,
ture and function [1-12], enzyme activities [13-17], oligo- we reported the design and synthesis of the rigid bisazobenzene
nucleotide functions [18-20], and ion-channel activities [21-23]. crosslinker BPDBS, (4,4'-bis(4-(2-chloroacetamido)phenyl)-
A quantitative analysis of the effects of crosslinkers on protein diazenylbiphenyl-2,2'-disulfonate) (1, Scheme 1) [24]. This
folding led to the conclusion that photocontrol of folding is best photoswitch can produce a minimum ~5 Å and a maximum
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Scheme 1: Bisazobenzene derivatives.
~23 Å end-to-end distance change upon trans–cis isomeriza- pairs to extend the π-system of each independent azo unit. In
tion. It has a high absorption coefficient (45–60,000 M−1cm−1) this manner, independent photoswitching behavior of the two
and can produce up to ~80% trans–cis isomerization under units at wavelengths shifted towards the visible range was
favorable conditions. The suitability of this photoswitch for bio- expected to occur.
logical systems would be enhanced if the wavelengths required
for photoisomerization were longer, so that UV light was not Results and Discussion
required. UV light is highly scattered by biological samples and The p-diacetamido piperazine-linked bisazobenzene derivative
can be a toxic stimulus [25,26]. In an attempt to red-shift the 2 was prepared by a double diazo-coupling reaction between the
switching wavelength while maintaining a large end-to-end dis- donor 1,4-diphenylpiperazine (7) and the acceptor 4-acetamido-
tance change upon photoisomerization, we designed compound 3-sulfobenzenediazonium chloride (3), which was synthesized
2 (Scheme 1). The central piperazine unit in this structure was in situ by diazotization of sodium 2-acetamido-5-aminoben-
expected to disconnect the π-delocalization between the two azo zenesulfonate (4) using sodium nitrite with HCl (Scheme 2).
chromophores completely, while providing N-centered lone Compound 4 was prepared by hydrogenation with H2/Pd-C
Scheme 2: Synthetic route for the preparation of bisazobenzene derivative 2.
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from sodium 2-acetamido-5-nitrobenzenesulfonate (5), which coefficient for 2 was determined by 1H NMR coupled with
was obtained from commercially available sodium 2-amino-5- UV–vis absorption spectra, to be 66,400 in DMSO at 428 nm,
nitrobenzenesulfonate (6) by acetylation using acetic anhydride. such that on a molar basis, the photoswitch is intensely colored.
1,4-Diphenylpiperazine (7) was synthesized by a cross-coupling
reaction of bromobenzene with 1-phenylpiperazine in the pres- The spectra in aqueous solutions are pH dependent below
ence of t-BuOK.
pH 6.0 (Figure 2b). A band corresponding to the azonium
species [27] near 600 nm is evident at pH 5 and this does not
As with previous crosslinkers, attachment to a target peptide or appear to change upon irradiation. At lower pH values the
protein would involve replacement of the two terminal spectrum undergoes further changes with enhanced absorbance
acetamido units with chloro- or iodoacetamide units to enable from 530–700 nm consistent with the formation of a doubly
reaction with Cys residues [24]. The end-to-end distance protonated bisazonium species. This species is expected to
changes expected upon photoisomerization of the resulting be in equilibrium with the corresponding bisammonium species
crosslink were estimated by molecular dynamics methods. [27].
Figure 1 shows models of compound 2 in trans,trans, trans,cis,
and cis,cis-isomeric states. These simulations indicate that the Irradiation of 2 in DMSO and in acetonitrile caused only small
trans,trans and cis,cis-states give well-separated end-to-end dis- changes in the spectra indicating rapid thermal reversal.
tance changes. The trans,trans-isomer has a preferred end-to- However, switching in aqueous solutions was measurable
end distance of 32.8 Å but some flexibility in the piperazine (Figure 3). The half-life of the irradiated isomers of 2 in phos-
linkage permits distances between 25 and 35 Å. The cis,cis- phate buffer at 4 °C was measured to be 1.5 ± 0.3 s.
isomer permits distances between ~4 and 25 Å. The trans,cis-
isomers produce an intermediate range of distances between 7 Numerous cycles of photoisomerization could be carried out
and 30 Å, which overlaps with both trans,trans and cis,cis- without noticeable photobleaching (Figure 3b). The relatively
isomers. This rather large and overlapping range of distances rapid thermal relaxation, however, means that the overall trans
for the trans,cis-species is problematic from the point of view versus cis-isomer ratio is small, and the fraction of the cis,cis-
of photocontrol since a large light-driven change in the end-to- species is low. While cis lifetimes may be longer when the
end distribution will require very efficient photoisomerization crosslinker is attached to a biomolecule, it is likely that this
to the cis,cis-species. We were, therefore, interested in the crosslink will predominantly cycle between the trans,trans and
photochemical behavior of 2.
trans,cis-species under low irradiation intensities. These species
have overlapping end-to-end distances and would not be
Figure 2 shows UV–vis absorbance spectra of 2 in the dark- expected to drive significant conformational change in a single
adapted trans,trans-form in various solvents. As expected, the attached biomolecule [6]. However, high (e.g., pulsed) light in-
main absorbance band (π–π* transition) is red-shifted compared tensities may be expected to populate the cis,cis-state and may
to BPDBS with a maximum near 420 nm. The molar extinction permit pulsed-light-driven conformational changes. It is
Figure 1: (a) Histograms showing the relative populations of conformations having the sulfur-to-sulfur distances indicated. (b) Examples of
trans,trans, trans,cis and cis,cis conformations with the S–S distances indicated.
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Beilstein J. Org. Chem. 2012, 8, 2184–2190.
Figure 2: (a) Normalized UV–vis absorption spectra of 2 in DMSO, acetonitrile, MeOH and sodium phosphate buffer (pH 7.0). (b) UV–vis absorption
spectra of 2 (~10 μM) in aqueous buffer at different pHs (indicated).
Figure 3: (a) Photoswitching of 2 with violet light (407–410 nm) at different temperatures (4, 10 and 20 °C) in 25 mM sodium phosphate buffer at pH
8. (b) Multiple rounds of photoswitching of 2 with alternating violet light (violet bars) and dark adaption (white bars) in 25 mM phosphate buffer, pH 8,
at 10 °C.
possible that further substitution, particularly in positions ortho recorded either on Varian Vnmr-S 400 MHz, Varian Mercury
to the azo units may slow down the thermal relaxation rates and 400 MHz or Agilent DD2 500 MHz spectrometers. Silica gel of
make production of a large fraction of the cis,cis-species particle size 40–63 μm from Silicycle Chemical Division was
possible [28]. Alternatively, azobenzene-based switches with used for column chromatography.
rapid thermal relaxation times are preferred in materials-based
applications where they can lead to light-driven changes in Synthesis of 1,4-diphenylpiperazine (7) [34]: A mixture of
polymer properties. Bisazobenzene-based compounds analo- 1-phenylpiperazine (8, 2.0 g, 12.3 mmol), bromobenzene (1.9 g,
gous to 2 may be useful in this context [29-33].
12.1 mmol) and t-BuOK (2.7 g, 24.11 mmol) in anhydrous
DMSO (25 mL) was heated at 100 °C for 2 h. The reaction was
quenched with water and extracted with dichloromethane. The
Experimental
General aspects: All commercial materials (solvents, reagents combined organic layer was dried over anhydrous sodium
and substrates) were used as received. NMR spectra were sulfate and concentrated. The crude oil was subjected to silica-
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gel column chromatography to afford 1,4-diphenylpiperazine to a short silica gel column chromatography to isolate
(7, 1.2 g, yield 41%) as a colorless solid. 1H NMR (400 MHz, p-diacetamido piperazine-linked bisazobenzene 2 (108.0 mg,
CDCl3) δ 3.34 (s, 8H), 6.89 (t, J = 7.6 Hz, 2H), 6.99 (d, J = 8.0 yield 7%), which was further HPLC purified on a semiprepara-
Hz, 4H), 7.29 (t, J = 7.6 Hz, 4H) ppm.
tive SB-C8 column with a linear solvent gradient of 10–75%
acetonitrile/H2O (containing 0.1% trifluoroacetic acid) over a
Synthesis of sodium 2-acetamido-5-nitrobenzenesulfonate course of 25 min; the product was eluted at 54.5% acetonitrile.
(5): To sodium 2-amino-5-nitrobenzenesulfonate (6, 4.0 g, 1H NMR (400 MHz, DMSO-d6) δ 2.08 (s, 6H), 3.54 (s, 8H),
16.6 mmol) in acetic acid (40 mL) was added acetic anhydride 7.11 (d, J = 9.2 Hz, 4H), 7.77–7.81 (m, 6H), 8.09 (d, J = 2.4 Hz,
(2.6 g, 24.8 mmol) and the solution was heated under reflux for 2H), 8.45 (d, J = 8.8 Hz, 2H), 10.58 (br, NH) ppm; 13C NMR
4 h. After cooling to rt, the precipitate was filtered, washed with (125 MHz, DMSO-d6) δ 25.5, 47.0, 114.6, 119.5, 120.1, 124.8,
hot acetic acid and then ether, and dried under high vacuum. 125.6, 136.2, 137.1, 144.6, 147.1, 152.9, 168.3 ppm;
The crude product was resuspended in hot ethanol, filtered and HRMS–ESI (m/z): [M − H]− calcd for C32H31N8O8S2,
dried to obtain sodium 2-acetamido-5-nitrobenzenesulfonate (5, 719.1711; found, 719.1711.
4.3 g, yield 90%) as a colorless solid. 1H NMR (400 MHz,
DMSO-d6) δ 2.13 (s, 3H), 8.21 (dd, J1 = 9.2 Hz, J2 = 2.8 Hz, UV–vis spectra and photoisomerization: UV–vis absorption
1H), 8.43 (d, J = 2.8 Hz, 1H), 8.55 (d, J = 9.2 Hz, 1H), 10.66 spectra were obtained using either a Perkin-Elmer Lambda 25
(br s, NH) ppm; 13C NMR (100 MHz, DMSO-d6) δ 25.7, 120.2, spectrophotometer or using a diode array UV–vis spectropho-
123.1, 126.2, 135.9, 141.6, 141.8, 169.3 ppm; HRMS–ESI tometer (Ocean Optics Inc., USB4000) coupled to a tempera-
(m/z): [M − H]− calcd for C8H7N2O6S, 259.2174; found, ture-controlled cuvette holder (Quantum Northwest, Inc.
259.2172.
Spokane, WA, USA). The latter arrangement was used to
measure thermal relaxation rates, and steady-state spectra under
Synthesis of sodium 2-acetamido-5-aminobenzenesulfonate UV–vis illumination. For steady-state spectra, typically, a
(4) [35]: To a solution of sodium 2-acetamido-5-nitrobenzene- 5–10 µM solution of 2 in 25 mM sodium phosphate buffer
sulfonate (5, 4.2 g, 14.9 mmol) in water (100 mL) contained in (pH 8.0), contained in a 1 cm path length quartz cuvette in the
a double-necked round-bottom flask was added 10% Pd/C temperature-controlled cuvette holder, was irradiated (at 90o to
(0.3 g) under nitrogen gas atmosphere. A filled hydrogen the measuring beam) with LEDs emitting at 407–410 nm
balloon was then connected to one neck and the reaction vessel (~460 mW, LEDengin LZ1-10UA05) at 4, 10 and 20 °C for
was thoroughly degassed and purged with hydrogen gas. The 2 min until no further decrease in absorbance was observed, and
reaction mixture was vigorously stirred under hydrogen-balloon under this steady-state-irradiation condition the spectra were
pressure at rt for 14 h. The reaction mixture was filtered through recorded.
a celite pad, and the water was removed under vacuum. The
crude product was resuspended in hot ethanol, filtered and dried Rates of thermal reversion of the irradiated samples were
to obtain sodium 2-acetamido-5-aminobenzenesulfonate (4, measured at 4 °C by monitoring absorbance at 425 nm with
3.6 g, yield 97%) as a colorless solid. 1H NMR (400 MHz, time. All curves were fitted to mono-exponential decay kinetics.
DMSO-d6) δ 1.96 (s, 3H), 4.91 (br, NH2), 6.46 (dd, J1 = 8.4 Hz, The light used for the absorbance measurement was of suffi-
J2 = 2.8 Hz, 1H), 6.95 (d, J = 2.8 Hz, 1H), 7.88 (d, J = 8.4 Hz, ciently low intensity to cause negligible isomerization.
1H), 9.98 (br, NH) ppm.
To determine the molar extinction coefficient 1H NMR spectra
Synthesis of p-diacetamido piperazine-linked bisazobenzene, of a solution of 2 in DMSO-d6 containing a known concentra-
5,5'-((piperazine-1,4-diylbis(4,1-phenylene))bis(diazene-2,1- tion of 1,2-dichloroethane (DCE) as an internal reference was
diyl))bis(2-acetamidobenzenesulfonic acid) (2): An ice-cold recorded. The NMR solution was later diluted in DMSO and the
mixture of sodium 2-acetamido-5-aminobenzenesulfonate (4, UV–vis spectrum was recorded to calculate the molar extinc-
1.6 g, 6.3 mmol) and sodium nitrite (0.47 g, 6.8 mmol) in water tion coefficient.
(30 mL) was added dropwise to a mixture of concentrated
hydrochloric acid (2.8 mL) and ice (30.0 g). The resulting mix- Molecular modeling: Models of trans,trans, trans,cis and
ture was stirred at 0–5 °C for 1 h. To this diazonium solution cis,cis-crosslinkers were built by using HyperChem (v.8,
was added dropwise an ice-cold solution of 1,4-diphenylpiper- Hypercube Inc.) with the linker terminated with methyl groups
azine (7, 0.5 g, 2.1 mmol) in DMF (10 mL). After stirring for representing the β-carbon of Cys in the cross-linked peptide,
2 h at 0–5 °C, it was stirred at room temperature for 24 h. The and minimized by using the Amber99 force field. Restraints
reaction was made acidic (pH 1 to 0.5), filtered and the residue were added to the azo-bond for the trans,cis and cis,cis-isomers
was washed with cold water. The crude product was subjected (force constant 16). Molecular dynamics runs were performed
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Supporting Information
Supporting Information File 1
NMR spectra for compounds 2, 4, 5 and 7.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-8-246-S1.pdf]
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We are grateful to the Natural Science and Engineering
Research Council of Canada for funding.
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