Driving and photo-regulation of myosin–actin motors at molecular and macroscopic levels by photo-responsive high energy molecules

Halley M. Menezes ab, Md. Jahirul Islam ab, Masayuki Takahashi c and Nobuyuki Tamaoki *ab
aResearch Institute for Electronic Science, Hokkaido University, Kita 20, Nishi 10, Kita-Ku, Sapporo, Hokkaido, 001-0020, Japan. E-mail: tamaoki@es.hokudai.ac.jp
bGraduate School of Life Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan
cFaculty of Science, Hokkaido University, Kita 10, Nishi 8, Kita-Ku, Sapporo, Hokkaido 060-0810, Japan

Received 26th May 2017 , Accepted 3rd September 2017

First published on 13th September 2017


We employed an azobenzene based non-nucleoside triphosphate, AzoTP, in a myosin–actin motile system and demonstrated its efficiency as an energy molecule to drive and photo-regulate the myosin–actin motile function at the macroscopic level along with an in vitro motility assay. The AzoTP in its trans state induced shortening of a glycerinated muscle fibre whilst a cis isomer had no significant effect. Direct photoirradiation of a cis-AzoTP infused muscle fibre induced shortening triggered by a locally photo-generated trans-AzoTP in the muscle fibre. Furthermore, we designed and synthesized three new derivatives of AzoTPs that served as substrates for myosin by driving and photo-regulating the myosin–actin motile function at the molecular as well as the macroscopic level with varied efficiencies.


Introduction

The energy of nucleotide hydrolysis fuels biomolecular motors to lug a myriad of cargos through the cytoplasm and performs a range of cellular tasks.1,2 A purine nucleotide, adenosine triphosphate (ATP) is hydrolysed by cytoskeleton motors such as myosin, kinesin and dynein and the energy generated is converted into mechanical work with high efficiency by undergoing conformational changes.3–5 Harnessing these robust and versatile molecular motors for nanotechnology involves dynamic control over their motile properties including the velocity, direction of motion, processivity and on/off switching.6–8 Light impelled modulation of the ATP function is one of the pronounced approaches towards achieving the motor control.9–11 Uncaging of inactive caged ATPs by photo-irradiation (UV) to switch ON the motility from the OFF state is a remarkable study in this direction.9 However, the irreversibility of this system paved way for the development of high-efficiency reversible ATP analogues to control the motility. In the recent past, our group has reported photo-sensitive ATP analogue fuels for reversible control of the motility through illumination with two different wavelengths of light.12,13 An azobenzene based non-nucleoside triphosphate, AzoTP, in its trans state could drive a kinesin motor, and conversely, the cis isomer was unable to drive kinesin, thus facilitating the photo-control of the gliding velocity of microtubules on immobilized kinesin between the trans and cis states of the AzoTP.

In vitro motility assays provide an insight into the functioning of motor proteins on a molecular level where the motile interaction of only two isolated proteins under biochemical conditions is studied. Contrary to this, in a physiological macroscopic system motor proteins work collectively in large numbers along with other cellular components or enzymes, thus increasing the number of interacting molecules.14–17 In our present study, we explore the potential of AzoTPs to photo-control such a complex macroscopic system of molecular motors, hence extending its applicability over different scales. Myosin II, a muscle protein, is a convenient candidate for our study since one of its chief tasks, muscle contraction, is studied extensively via an in vitro motility assay as well as muscle fibre shortening.18–20 Myosin II ATPase translocates along an actin filament and the ATP dependent cyclic sliding interaction between them powers muscle contraction as well as movement.21,22 To substantiate this concept, glycerol extracted muscle preparation akin to living muscles has been developed and evolved over the years to carry out a number of muscle contraction regulation studies.23 Three-dimensional orderly arrays of myofilaments and the presence of actin-associated proteins like troponin and tropomyosin render the glycerinated muscle fibre system more complex than an in vitro motility system which involves isolated myosin and actin proteins without an orderly array. Ca2+ triggered regulation of glycerinated skeletal muscle fibre contractility is studied extensively.24,25 However, regulating the contraction by photoisomerizing the substrate locally in a muscle fibre by direct irradiation has not been studied copiously. Recently, Christian Hoppmann et al. reported the photo-control of living skeletal muscle fibre shortening in which a photo-switchable peptide ligand inhibited electrically stimulated fibre shortening in the cis state whilst the trans state had no effect on shortening.26

Herein, we report AzoTP triggered driving of myosin and photo-regulation of a myosin based macroscopic motile system in a glycerinated skeletal muscle fibre by direct photo-irradiation of the muscle fibre. The cis form of the AzoTP fails to initiate significant shortening; following irradiation with 510 nm light, the muscle fibre shortens remarkably in response to the photo-induced trans state. Furthermore, we synthesized AzoTP derivatives and employed them in a myosin–actin motile system to investigate the correlation between the structure of the substrate and its ability to perform as a photo-responsive energy molecule. We surveyed the efficiency of the newly synthesized three AzoTP derivatives to reversibly photo-control the in vitro actin filament gliding velocity as well as the shortening of the glycerinated muscle fibre. Amongst the newly synthesized AzoTP derivatives, the AzoTP with an ether group bridging the azobenzene and triphosphate moieties performed as a higher efficiency substrate for myosin motors.

Results and discussion

Synthesis and photoisomerization of AzoTP derivatives

AzoTP derivatives were synthesized by modifying the bridging group between azobenzene and triphosphate and by substitution on the azobenzene moiety of the previously reported parent AzoTP (4a). Substituting the amide linkage with ether and ethyl linkages resulted in azoethoxyTP (4b) and azoethylTP (4d), respectively, while the substitution of methyl groups at meta and para on the azobenzene moiety resulted in dimethylAzoTP (4c) (Fig. 1). These modifications in the parent AzoTP were done to probe the critical significance of the amide linkage in the functioning of an AzoTP as an energy molecule and for exploring the possibility of a further efficient azobenzene based photochromic non-nucleoside triphosphate.
image file: c7ob01293d-f1.tif
Fig. 1 (a) Structures of azobenzene based non-nucleoside triphosphates, AzoTP (4a), azoethoxyTP (4b), dimethylAzoTP (4c) and azoethylTP (4d). (b) Reversible photo-isomerization of 4a.

The reversible photoisomerization of these AzoTPs was confirmed by consecutive irradiation with 365 nm UV light and 436 nm visible light (Fig. S1). At the UV photo stationary state (PSS) the AzoTPs attain their cis-rich state which is reversed at visible PSS resulting in a thermodynamically stable trans-rich state. Our previous study of 4a in a kinesin–microtubule motile system suggested that the trans isomer of 4a was an efficient energy molecule that triggered the faster velocity of microtubules by driving a kinesin motor whereas the cis isomer was inefficient to drive kinesin. Table 1 shows the ratios of the cis[thin space (1/6-em)]:[thin space (1/6-em)]trans isomers of 4a, 4b, 4c and 4d at PSS induced by 365 nm and 436 nm light irradiation, determined by 1H NMR (Fig. S2). The thermal isomerization from cis to trans was evaluated by observing the changes in the absorption spectra when kept in the dark at room temperature. About 2% of the trans isomer was recovered after 3 h dismissing the possibility of thermal-back reactions during our experiments.

Table 1 Ratio of cis and trans isomers at the UV and Visible photo stationary state (PSS)
Non-nucleoside triphosphate UV PSS Visible PSS
cis trans cis trans
4a 92% 8% 38% 62%
4b 93% 7% 50% 50%
4c 93% 7% 35% 65%
4d 87% 13% 25% 75%


Reversible photo-control of the in vitro motility of a myosin–actin motile system

To assess the generalizability of 4a to drive and control cytoskeletal motor systems, we employed 4a and its derivatives to a myosin–actin motile system which is different from our previously reported kinesin–microtubule system. HMM (heavy meromyosin) is a soluble fragment of myosin consisting of two heads (containing a nucleotide binding site and an actin binding site) and a part of a myosin rod, and hence it was used as an ATPase motor in our in vitro motility assay experiments. All the four AzoTPs tested, served as substrates for myosin by driving the HMM induced gliding motility of F-actin on a HMM immobilized glass surface, of which 4a and 4b functioned at concentrations as low as 10 μM. The average velocity of F-actin triggered by 4a, 4b, 4c and 4d is 1.25 μm s−1, 1.50 μm s−1, 1.24 μm s−1 and 0.73 μm s−1, respectively, at saturated concentrations of 0.5 mM for 4a, 4c and 4d and 0.25 mM for 4b. Photo-induced reversible control of the gliding velocity of F-actin prompted by the photo-isomerization of AzoTPs was observed as the flow cell of motility solution was irradiated with 365 nm and 436 nm light alternately to the PSS. The velocity decreased remarkably after irradiation with 365 nm light for 5 s corresponding to the cis-rich state, and the subsequent irradiation with 436 nm for 20 s recovered the velocity, comparable to that of the initial velocity before irradiation. This phenomenon of reversible switching between the faster and slower velocities could be repeated over many cycles as represented in Fig. 2. We carried out in situ photo-regulation of the F-actin velocity by repeated alternating irradiation of a flow cell with 365 nm UV and 510 nm visible light for 3 s and 5 s, respectively, captured in a 5 min long video (ESI-Mv01). Speeding actin filaments slowed down following UV irradiation at a time interval and these slowly moving filaments recovered their speed after irradiation with 510 nm light. The magnitude of distance traveled by selected actin filaments facilitated by consecutive UV and Visible light irradiation at different time intervals is depicted in Fig. 3.
image file: c7ob01293d-f2.tif
Fig. 2 Repeatability of the complete and reversible photoregulation of the F-actin gliding velocity induced by 4b (azoethoxyTP) at a saturated concentration (0.25 mM). (BI: before irradiation; UV: after irradiation with 365 nm light; Vis: after irradiation with 436 nm light). Error bars: standard deviation for 10 actin filaments.

image file: c7ob01293d-f3.tif
Fig. 3 Fluorescence images of F-actin motility driven by trans and cis states of 4a (40 μM) by in situ photo-regulation experiments with alternating irradiation at 365 nm (3 s) and 510 nm (5 s). The distance traveled by two actin filaments is indicated in lines by tracking the path of filament heads; red circles denote the position of actin filament heads before irradiation (trans state), green and red lines denote the distance traveled by F-actin after UV (365 nm) and Vis (510 nm) irradiations, respectively.

The change in velocity between the two photoisomerized states of AzoTP molecules is 54%, 79%, 81% and 80% for 4a, 4b, 4c and 4d, respectively, at saturated concentrations. However, at 0.1 mM concentration the magnitude of switching is higher for all AzoTPs as the difference in the velocity is about 87–90%. The gliding velocities fuelled by AzoTPs in their trans state (black solid circles) and cis-rich state (blue solid circles) with respect to the range of the concentrations are shown in Fig. 4.


image file: c7ob01293d-f4.tif
Fig. 4 Gliding velocity of actin filaments as a function of AzoTP concentration. (Black solid circles: velocities before irradiation; black line: curve fitting using the Michaelis–Menten equation; blue circles: velocities after irradiating at 365 nm; red line: theoretical curve derived from the black line for the remaining trans in the cis-rich state.) Error bars: standard deviations for 10 actin filaments.

The concentration dependent velocity of actin filaments obeys the Michaelis–Menten equation and the obtained apparent Km (Kapp) values indicate that the apparent binding affinity of AzoTPs for myosin (1/Kapp = 9.9 mM−1) is twenty five times higher than that for a kinesin motor (1/Kapp = 0.4 mM−1) (as obtained in our previous work). The maximum gliding velocities (Vmax) of F-actin induced by 4a, 4b, 4c and 4d are 1.5 μm s−1, 1.9 μm s−1, 1.7 μm s−1 and 1.0 μm s−1, respectively, which are 53%, 68%, 59% and 35% of that of ATP (Vmax = 2.9 μm s−1) at saturated concentrations. Our previous report on AzoTPs explained that the microtubule velocity driven by the cis-rich state of AzoTPs is due to the remaining trans at cis-rich PSS, which corresponds to 8%. In our current study too, we plotted a theoretical curve corresponding to the remaining trans of all the four AzoTP molecules at their respective cis-rich PSS to validate this explanation. Fig. 4 shows the Michaelis–Menten plot for the concentration dependent velocity of all the four AzoTPs and theoretical curve (red lines) for the remaining trans amounting to 8%, 7%, 7% and 13% (Table 1) in the cis-rich state of 4a, 4b, 4c and 4d, respectively. These observations insinuate that AzoTPs in their cis state intrinsically have no ability to function as energy molecules to drive the motor proteins.

Although the Kapp of 4a and 4b is almost threefold less than 4c, the Vmax of these three AzoTPs is comparable, since all three Kapp values were in the submillimolar range. However, 4d has considerably lower Vmax, though its Kapp is in the same range as that of 4a, 4b and 4c. These results imply that the substrates 4b and 4a bind to the myosin motor with higher affinity than that of 4c and 4d. Structural studies of chicken skeletal myosin subfragment 1 revealed that the nucleotide binding pocket is formed by the residues from the N-terminal, central, and C-terminal sections of the myosin motor head domain.27,28 The adenine binding pocket is formed by amino acid residues Phe129–Tyr135 and Glu187–Lys191 contributed by the N-terminal segment as seen in the X-ray structural studies of a Dictyostelium discoideum myosin II complex (S1dC·MgADP·BeFx).29 Despite the presence of several water molecules with the potential for hydrogen bonding, very few specific interactions were seen between the adenine base and myosin heavy chain except the hydrogen bond formation between N6 of adenine and the side chain of Tyr135. This is in line with the observations that myosin utilizes a wide range of nucleotides and organic triphosphates,30,31 thus substantiating the functionality of our AzoTP molecules as substrates for myosin. Also the ribose moiety which bridges the adenine and triphosphate moieties forms very few interactions with the protein, enabling the myosin to utilize nucleotides and organic triphosphates with the ribose ring replaced by a variety of functional groups. However, ribose ring oxygen, O4′ forms a hydrogen bond with the Asn127 sidechain.29 Comparing the chemical structures of our AzoTP molecules with that of ATP suggests that the linker groups (ether, amide and ethyl) might fulfil the role of ribose and azobenzene takes adenine's place. This presumption provides an insight into the varied binding affinities of our four AzoTP substrates as well as their cis and trans isomers. The higher binding affinity of 4b (1/Kapp = 11.0 mM−1) could be attributed to the ether group bridging the triphosphate moiety and azobenzene, where the oxygen of ether forms a hydrogen bond with the side chain of Asn127, analogous to the ribose oxygen of ATP.29 Similarly the carbonyl oxygen of the amide group in 4a could participate in the hydrogen bonding by acting as a hydrogen acceptor. The absence of any potential hydrogen bond forming atoms in the bridging ethyl group elicits the lower binding affinity (1/Kapp = 5.7 mM−1) of 4d than 4a and 4b. Although there is carbonyl oxygen in 4c with the potential for hydrogen bond formation, yet the binding affinity (1/Kapp = 3.7 mM−1) is almost three fold lower than 4a and 4b. We assume that this weaker binding affinity is caused by the bulkiness of the substrate due to the presence of two methyl substituents on the azobenzene moiety which might sterically interfere with the binding of the substrate and myosin. AzoTPs in their cis state show no intrinsic ability as substrates for myosin unlike in their active trans state. It is well established that the isomerization of azobenzene from trans to cis changes the geometry from flat to bent or round shape resulting in bulkiness. We surmise that the inability of the cis isomer to bind in the nucleotide binding pocket could be ascribed to its bulkiness. In addition, the adenine binding site is relatively hydrophobic,32,33 and thus the hydrophilic cis isomer isn't favoured.

Photo-induced regulation of a macroscopic motile system of a myosin motor

As an approach to probe the efficiency of AzoTPs at the macroscopic level to drive and control the motile functions, we conducted simple glycerinated muscle fibre shortening experiments where the extent of shortening was assessed by the naked eye. The fibres used were of ∼0.5 mm in thickness and 7–8 mm in length. First we tested our parent AzoTP molecule 4a for macroscopic studies. 4a in its trans state induced the shortening of the muscle fibre accounting for 40–45% shortening of the muscle fibre's initial length. The pre-generated cis isomer of 4a didn't induce any significant shortening, thus affirming the poor activity of 4a in its cis-form as evidenced in our molecular in vitro motility experiments (Fig. 5a, ESI-Mv02). When the cis isomer infused muscle fibre was irradiated with 510 nm light for 10 s, a remarkable shortening of about 40% of its initial length was observed, thus confirming the efficiency of 4a to drive and photocontrol the myosin motor function in the macroscopic system (Fig. 5b, ESI-Mv04). To corroborate that the shortening was caused by the photoisomerization of 4a and not by the thermal energy of illumination, we irradiated the muscle fibre infused in buffer solution without 4a at 510 nm, which exhibited no shortening (ESI-Mv03).
image file: c7ob01293d-f5.tif
Fig. 5 Non-nucleoside triphosphate 4a induces and photo-controls the shortening of the glycerinated muscle fibre. (a) Buffer solution with trans-4a (3 mM) induces shortening while cis-4a has no significant effect on shortening. (b) The cis-4a infused muscle fibre shortens after irradiation with 510 nm light, no significant change in length in the non-irradiated fibre. Red arrows point at the two edges of the fibre. The scale seen in the photographs is 1 mm.

Further, the ability of all the three newly synthesized AzoTPs to initiate and photo-control the shortening was investigated. Fig. 6 represents the percentage change in the muscle fibre length with respect to time, induced by all the four AzoTPs at 3 mM (total). The shortening of the muscle fibre with respect to time increases in the order of 4a4b > 4c > 4d, where the substrates 4a and 4b induce the shortening swiftly with an evidently larger magnitude of length change than 4c and 4d over the time range. Similar to 4a, the cis-form of the energy molecules 4b, 4c and 4d was unable to induce any significant shortening and 510 nm light irradiation of cis infused muscle fibres induces the shortening except for 4d. Experiments involving the 4a concentration dependent shortening rate showed that there is no significant change in the muscle length below 0.5 mM of trans-4a (Fig. S3). This explains why no significant shortening was observed at cis-rich state of 4a at 3 mM (total) despite the presence of 8% (0.25 mM) of the remaining trans. The order of performance of the four AzoTP substrates (4a4b > 4c > 4d) in the macroscopic system is consistent with the Kapp values obtained by the molecular level studies. Unlike the molecular in vitro motility system, the glycerinated muscle fibre system involves an ordered array of myofibrils which could furnish steric hindrance, but the AzoTPs efficiently replicated the photo-regulation of the motor function in the glycerinated muscle fibre as well. AzoTP energy molecules photo-regulate the muscle fibre shortening by photoisomerizing to the active trans state from the inactive cis state locally in the muscle fibre by direct photo-irradiation.


image file: c7ob01293d-f6.tif
Fig. 6 Muscle fibre length change (L(t=0)L(t)/L(t=0)) with respect to the time for AzoTPs (3 mM) 4a, 4b, 4c and 4d inducing fibre shortening. Black, red, green, blue and pink circles represent the % length change induced by ATPs, 4a, 4b, 4c and 4d, respectively; the lines are best-fit through the data trend. Error bars: standard deviation for 4 fibres.

Conclusion

In a step towards employing our AzoTP energy molecules to regulate macroscopic systems, we have demonstrated the photo-regulated initiation of shortening in a glycerinated skeletal muscle fibre induced by photoisomerization. Direct irradiation with visible light initiated the shortening in the inactive cis-AzoTP infused muscle fibre by locally photo-generated trans-AzoTPs. The trans form of AzoTPs initiates the contraction and shortens the muscle fibre to almost half of its initial length, whilst cis isomer's contribution is insignificant. Also, we demonstrated the functionality of 4a as an efficient substrate for a myosin motor in addition to kinesin as reported previously, thus implying its generalizability for cytoskeletal motors. The newly designed and synthesized three AzoTPs serve as the substrates for myosin motors and photoregulate the motility at the molecular as well as the macroscopic level. All the four AzoTP molecules drive the myosin triggered in vitro gliding velocity of F-actin with 50 to 60% efficiency of that obtained with ATP and photo-control the velocity between fast/slow states by undergoing photoisomerization. Our demonstration of the photocontrol of a macroscopic glycerinated muscle fibre system is a promising indication towards regulating the much complex intact in vivo systems and organisms by utilizing photoresponsive energy molecules. The regulation of complex biological systems could benefit the investigation of disorders and targeted drug delivery. The efficient functionality of our AzoTP molecules in myosin–actin and kinesin–microtubule motile systems at the molecular as well as the macroscopic level makes them interesting photoswitches which could find potential in various biomolecular tasks.

Experimental

Instrumentation

1H, 13C and 31P NMR spectra were recorded using an ECX-400 (400 MHz) spectrometer (JEOL). Analysis of the AzoTPs was carried out in a Shimadzu reversed-phase (RP) HPLC system. An EYELA FDU-2200 lyophilisation system was used for freeze-drying. Electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) was performed using a JMS-T100CS instrument (JEOL) operated in the negative-ion mode. High-resolution mass spectrometry was performed on a Thermo Scientific Exactive mass spectrometer with Electrospray Ionization (ESI). Column chromatography was performed using silica gel 60 N (neutral, 60–120 μm, Kanto chemicals). Thin layer chromatography (TLC) was carried out on precoated silica gel 60 F254 aluminium sheets (Merck). UV-Vis absorption spectra were recorded using an Agilent 8453 single-beam spectrophotometer and a Shimadzu UV-1800 absorption spectrophotometer. A mercury lamp (Ushio) with band pass filters for 436 and a Hamamatsu LED Controller (model C11924-101) for 365 nm light was used for photoisomerization and in vitro motility experiments. A Hayasaka LED Controller (model CS_LED 3W_510) for 510 nm light was used for photoregulation of in situ motility and muscle fibre shortening experiments. An inverted fluorescence optical microscope (Olympus IX71) equipped with a UPlan F1 100×/1.30 oil C1 objective lens (Olympus) was used for the motility experiments in conjunction with appropriate filters (640 nm excitation filter). An EMCCD digital camera (Andor Solis Technology, model DL-604M-0EM-H1) was used to record videos.

Chemicals

All chemical and biochemical reagents were purchased from commercial sources (Tokyo Chemical Industry; Watanabe Chemical Industries; Wako Pure Chemical Industries; Dojindo Molecular Technologies) and used without purification.

Protein preparation

All experiments were performed in compliance with the relevant laws and institutional (Hokkaido University, Japan) guidelines. Skeletal muscle myosin was prepared from chicken pectoralis muscles according to the literature and was stored at −20 °C in 50% glycerol from which the HMM was prepared on the next day.34 HMM was produced by α-chymotrypsin digestion of myosin for 10 min at 25 °C, followed by dialysis and aliquots were quickly frozen in liquid N2 and stored at −80 °C.34 F-actin was prepared from rabbit skeletal muscle as described by Pardee and Spudich35 and then labelled with a phalloidin-CF™ 633 dye conjugate (Biotium) for performing in vitro motility assays. The concentration of proteins was determined by measuring the absorption at 280 nm with extinction coefficients of 0.63 and 1.1 for HMM and actin, respectively. The purity of proteins was confirmed by SDS-PAGE.

In vitro motility assay

Flow cell preparation and motility assay techniques were performed according to the literature.18,19,36 The gliding velocities were measured using ImageJ plugin MTrackJ.37 The average of velocity of 10 filaments was determined in each experiment. All the assays were performed at 23.5 °C. The motility solution consisted of 100 mM HEPES (pH 7.4), 25 mM NaCl, 4 mM MgCl2, 1 mM EGTA, 10 mM DTT, 0.5 mg ml−1 BSA and 20 mM glucose, 20 μg ml−1 catalase, 0.1 mg ml−1 glucose oxidase. The calculated ironic strength was 38 mM. The flow cell was directly irradiated with 365 nm (LED) light for 5 s and with 436 nm (Hg lamp) for 20 s alternately.

Muscle fibre preparation and shortening experiments

Chicken skeletal muscle stripes were prepared by a glycerol extraction method and stored at −20 °C in 50% glycerol-buffer.20,38 The stripes were transferred to a 20% glycerol-buffer solution 20 min before conducting the experiment and then transferred to the buffer solution without ATP/AzoTP 3 min before starting the experiment. The buffer consisted of 1.37 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4, pH 7.4. The muscle stripe was teased into ∼0.5 mm thick and 7 mm–8 mm long fibres which were then mounted on a glass slide and the buffer solution containing AzoTPs (4 μL) was added. The changes in the length were observed by the naked eye and analysed by video recording of the shortening process using a Canon digital camera. The length change vs. time was measured by dividing the net change in the length of the fibre with respect to its initial length at a time interval by the initial length (L(t=0)L(t)/L(t=0)). For the photo-regulation experiments, the muscle fibres mounted on the slide were directly irradiated with 510 nm (LED) light after adding the buffer solution containing cis-rich state AzoTP substrates.

Synthesis of new AzoTP derivatives

Photochromic non-nucleoside triphosphate 4a was synthesized as described in our previous report.13 The general synthetic route for AzoTP molecules involves two major steps: (1) synthesis of hydroxyl attached functional group tethered azobenzene; (2) phosphorylation of this functionalized azobenzene to obtain azobenzene based triphosphates. The new AzoTP derivatives, 4b and 4d, were synthesized by functionalizing azobenzene with ethoxy and ethyl groups; likewise 4c was synthesized by functionalizing dimethyl substituted azobenzene with the amide group as represented in Scheme 1. A detailed synthetic scheme of all the three derivatives is presented in the ESI.
image file: c7ob01293d-s1.tif
Scheme 1 General synthetic route for AzoTP molecules. Reaction conditions; (i) di-tert-butyl-N,N-diisopropylphosphoramidite, 1H-tetrazole, dry THF, Ar atmosphere, RT, 6 h and then mCPBA, 0 °C, 1 h followed by RT, 40 min; (ii) trifluoroacetic acid, dry CH2Cl2, Ar atmosphere, RT, 6 h; (iii) tributylamine, carbonyldiimidazole, pyrophosphate, dry DMF, Ar atmosphere, RT, overnight.
Synthesis of 1b. Azobenzene (1.0 g, 5.49 mmol), Pd(OAc)2 (0.123 g, 0.549 mmol), PhI(OAc)2 (3.536 g, 10.98 mmol) and AcOH (6.3 ml, 109.8 mmol) were all added to ethylene glycol (22 ml) in a RB flask and heated at 80 °C for 24 h. AcOH was removed by rotary evaporation and the reaction mixture was partitioned between organic (EtOAc) and aqueous layers. The organic phase was dried over MgSO4 and concentrated in a rotary evaporator and the purified compound was obtained through column chromatography (SiO2, hexane/EtOAc 7[thin space (1/6-em)]:[thin space (1/6-em)]3). Dark red crystals of 1b were obtained (1.03 g, 77%). Rf = 0.33 (hexane/EtOAc 7[thin space (1/6-em)]:[thin space (1/6-em)]3). Mp: 74.5–76 °C; 1H NMR [400 MHz, CDCl3]: δ 7.88 (dd, J1 = 8.3 Hz, J2 = 2.7 Hz, 2H), 7.69 (dd, J1 = 8.1 Hz, J2 = 1.7 Hz, 1H), 7.43–7.52 (m, 4H), 7.18–7.1 (m, 2H), 4.34 (t, J = 4.4 Hz, 2H), 3.96 (q, J = 6.4 Hz, 2H), 3.09 (t, J = 6.5 Hz, 1H). 13C NMR [100.5 MHz, CDCl3] δ = 156.10, 153.00, 143.42, 132.64, 131.18, 129.26, 122.97, 122.24, 118.00, 116.76, 72.55, 61.19. HRMS (ESI, m/z) calculated for C14H16N2O2Na [M + Na]+: 265.09475; found: 265.09473 (observed error of −0.07 ppm is within the range of instrumental error of ±5.00 ppm).

1c was synthesized via three steps via5 and 6 (Scheme 2).


image file: c7ob01293d-s2.tif
Scheme 2 Synthetic route for 1c. Reaction conditions: (i) acetoxyacetyl chloride, triethylamine, DCM, RT, 3 h; (ii) K2CO3, MeOH, RT, overnight.
Synthesis of 5. m-CPBA (9.40 g, 60.02 mmol) was added to a solution of 3,4-dimethylaniline (3.64 g, 30 mmol) in EtOAc (300 mL) at 0 °C in an ice bath. The reaction mixture was stirred for 3 h, extracted with EtOAc and saturated NaHCO3 and then washed with water. The organic phase was dried with MgSO4 and then the volume was reduced using a rotovap. When the volume reached approx. 150 mL, the evaporation was stopped and the solution was degassed with dry N2 for 15 min. 1,2-Phenylenediamine (3.2 g, 30 mmol) and acetic acid (1 mL) were added to this solution under an N2-atmosphere. The reaction mixture was stirred at 50 °C for 89 h. After 89 h the reaction mixture was extracted with water and EtOAc. The organic part was dried with MgSO4 and concentrated in a rotovap. The residue was purified by silica gel column chromatography. The compound was eluted with hexane/EtOAc, 9[thin space (1/6-em)]:[thin space (1/6-em)]1 to afford dark red crystals of 5 (2.70 g, 40%). Rf = 0.30 (hexane/EtOAc 9[thin space (1/6-em)]:[thin space (1/6-em)]1). Mp: 128–130 °C. 1H NMR [400 MHz, CDCl3] δ 7.80 (dd, J = 8.1, 1.5 Hz, 1H), 7.63–7.58 (m, 2H), 7.25–7.17 (m, 2H), 6.81 (ddd, J = 8.2, 7.1, 1.3 Hz, 1H), 6.76 (dd, J = 8.2, 1.1 Hz, 1H), 5.83 (br, 2H), 2.35 (s, 3H), 2.33 (s, 3H). 13C NMR [100.5 MHz, CDCl3] δ 151.42, 142.99, 139.27, 137.46, 137.26, 131.94, 130.34, 127.27, 123.14, 120.10, 117.49, 117.06, 20.03, 19.92. HRMS (ESI, m/z) calculated for C14H16N3 [M + H]+: 226.13387; found: 226.13383 (observed error of −0.19 ppm is within the range of instrumental error of ±5.00 ppm).
Synthesis of 6. Triethylamine (2.54 mL, 18.15 mmol) was added to a solution of 5 (2.70 g, 12.10 mmol) in DCM (90 mL) at 0 °C in an ice bath. While stirring, acetoxyacetyl chloride (1.95 mL, 18.15 mmol) was added dropwise to the reaction mixture at 0 °C in an ice bath. Then the reaction mixture was kept in an ice bath for 15 min followed by 3 h at room temperature. The reaction mixture was extracted with DCM and water. The organic part was dried with MgSO4 and all the solvents were removed using a rotovap. The residue was purified by washing with hexane several times and with DCM twice, and finally dried under vacuum to afford 3.51 g (89%) of an orange solid of 6. Rf = 0.1 (hexane/EtOAc 9[thin space (1/6-em)]:[thin space (1/6-em)]1). Mp 148–149 °C. 1H NMR (400 MHz, DMSO-D6) δ 10.23 (s, 1H), 8.32 (dd, J = 8.3, 1.1 Hz, 1H), 7.82–7.71 (m, 3H), 7.56–7.52 (m, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.28–7.24 (m, 1H), 4.81 (s, 2H), 2.35 (s, 3H), 2.33 (s, 3H), 2.15 (s, 3H). 13C NMR [100.5 MHz, DMSO-D6] δ 169.74, 165.85, 150.57, 141.03, 140.70, 137.55, 136.00, 132.25, 130.35, 124.41, 124.20, 121.79, 120.47, 116.42, 63.00, 20.53, 19.54, 19.45. HRMS (ESI, m/z) calculated for C18H19N3O3Na [M + Na]+: 348.13186; found: 348.13154 (observed error of −0.93 ppm is within the range of instrumental error of ±5.00 ppm).
Synthesis of 1c. K2CO3 (2.79 g, 20.0 mmol) was added to a solution of 6 (3.48 g, 10.69 mmol) in MeOH/DMSO (40 mL + 10 mL) at room temperature. The reaction mixture was stirred overnight. Then the reaction mixture was partitioned between EtOAc and water. The EtOAc part was dried with MgSO4 and all the solvents were removed using a rotovap. The residue was washed with hexane several times and with DCM twice and finally dried under vacuum to afford an orange solid of compound 1c (2.91 g, 96%). Rf = 0.13 (hexane/EtOAc 7[thin space (1/6-em)]:[thin space (1/6-em)]3). Mp 170–172 °C. 1H NMR (400 MHz, DMSO-D6) δ 11.10 (s, 1H), 8.64 (dd, J = 8.4, 1.1 Hz, 1H), 7.82–7.77 (m, 2H), 7.71 (dd, J = 8.0, 2.0 Hz, 1H), 7.58–7.53 (m, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.27–7.23 (m, 1H), 6.40 (s, 1H), 4.09 (s, 2H), 2.34 (s, 3H), 2.33 (s, 3H). 13C NMR [100.5 MHz, DMSO-D6] δ 170.99, 150.42, 141.22, 138.78, 137.73, 135.46, 132.77, 130.53, 123.65, 123.26, 120.67, 119.42, 118.95, 61.98, 19.59, 19.56. HRMS (ESI, m/z) calculated for C16H17N3O2Na [M + Na]+: 306.12130; found: 306.12127 (observed error of −0.09 ppm is within the range of instrumental error of ±5.00 ppm).
Synthesis of 1d. A solution of 2-(2-aminophenyl)ethanol (4.65 g, 33.90 mmol) in toluene (200 mL) was degassed under a stream of N2 for 15 min and then nitrosobenzene (3.63 g, 33.89 mmol) and acetic acid (0.8 mL) were added under an Ar-atmosphere. The reaction mixture was stirred at 60 °C for 72 h. The solvent of the reaction mixture was evaporated in a rotovap and partitioned between water and CH2Cl2 (DCM). The DCM part was dried (with MgSO4) and concentrated in a rotovap. The residue was purified by column chromatography (SiO2, hexane/EtOAc 6[thin space (1/6-em)]:[thin space (1/6-em)]4) to afford a dark red viscous liquid of 1d (5.75 g, 75%). Rf = 0.30 (hexane/EtOAc 7[thin space (1/6-em)]:[thin space (1/6-em)]3). 1H NMR [400 MHz, CDCl3]: δ 7.91–7.88 (m, 2H), 7.71 (dd, J1 = 8.0 Hz, J2 = 1.0 Hz, 1H), 7.55–7.45 (m, 3H), 7.44–7.39 (m, 2H), 7.37–7.33 (m, 1H), 3.96 (dd, J1 = 12.2 Hz, J2 = 6.4 Hz, 2H), 3.40 (t, J = 6.4 Hz, 2H), 2.02 (t, J = 5.6 Hz, 1H). 13C NMR [100.5 MHz, CDCl3] δ 152.72, 150.55, 138.70, 131.34, 131.23, 131.08, 129.14, 127.29, 122.94, 115.72, 63.87, 35.09. HRMS (ESI, m/z) calculated for C14H14N2ONa [M + Na]+: 249.09983; found: 249.09957 (observed error of −1.06 ppm is within the range of instrumental error of ±5.00 ppm).

General synthetic procedure for phosphorylation (step 2)

Monophosphate formation. 1H-Tetrazole (3 eq.) was added to a solution of 1a–d (1 eq.) and di-tert-butyl-N,N-diisopropylphosphoramidite (1.3 eq.) in dry THF. This reaction mixture was stirred for 6 h at room temperature. A solution of mCPBA (65%, 1.7 eq.) in dry CH2Cl2 was added and stirred for 1 h in an ice bath followed by stirring at room temperature for 25 min. Saturated aqueous NaHCO3 was added and the mixture was further stirred for 40 min. The reaction mixture was extracted in organic (EtOAc) and aqueous (NaCl) solutions. The organic phase was separated, dried over MgSO4, concentrated in a rotary evaporator, and the purified tert-butyl protected monophosphates 2a–d were obtained by column chromatography. Trifluoroacetic acid (16 eq.) was added to the solution of this protected monophosphate (1 eq.) in dry CH2Cl2 and stirred for 6 h at room temperature followed by solvent evaporation. For the complete removal of CF3COOH, the procedure of addition of MeOH and evaporation was repeated thrice followed by CH2Cl2 washing. The obtained residue of monophosphates (3a–d) was vacuum dried and dissolved in water by adjusting the pH to 7.5 using 1 M NaOH. This solution was eluted through a DEAE Sephadex A-25 column with 0.5 M triethylammonium hydrogencarbonate solution at 4 °C to convert the monophosphate into its triethylammonium salt. Triethylammonium hydrogencarbonate was removed by evaporation with EtOH several times.

2b: Reddish orange viscous liquid. Yield = 0.15 g (14%). Rf = 0.10 (hexane/EtOAc 7[thin space (1/6-em)]:[thin space (1/6-em)]3). 1H NMR (400 MHz, CDCl3): δ 8.6 (br, 1H), 7.91 (d, J = 8.0 Hz, 2H), 7.65 (d, J = 8.0 Hz, 1H), 7.52–7.40 (m, 4H), 7.12 (d, J = 8.2 Hz 1H), 7.05 (t, J = 7.6 Hz, 1H), 4.43–4.37 (m, 4H), 1.46 (s, 18 H). 13C NMR [100.5 MHz, CDCl3] δ 156.22, 153.07, 142.88, 132.38, 130.89, 129.05, 123.12, 121.64, 117.13, 115.34, 82.66 (d, J = 7.4 Hz), 68.99 (d, J = 8.5 Hz), 64.95 (d, J = 6.0 Hz), 29.88 (d, J = 4.3 Hz). HRMS (ESI, m/z) calculated for C22H31N2O5PNa [M + Na]+: 457.18628; found: 457.18634 (observed error of 0.13 ppm is within the range of instrumental error of ±5.00 ppm).

2c: Orange solid. Yield = 1.54 g (42%). Rf = 0.25 (hexane/EtOAc 7[thin space (1/6-em)]:[thin space (1/6-em)]3). Mp: 132–133 °C. 1H NMR (400 MHz, CDCl3): δ = 10.71 (s, 1H), 8.69 (dd, J1 = 8.4 Hz, J2 = 1.2 Hz, 1H), 7.87–7.80 (m, 3H), 7.49–7.45 (m, 1H), 7.31 (d, J = 7.8 Hz, 1H), 7.23–7.18 (m, 1H), 4.60 (d, J = 7.0 Hz, 2H), 2.41 (s, 3H), 2.35 (s, 3H), 1.46 (s, 18H). 13C NMR [100.5 MHz, CDCl3] δ 166.16 (d, J = 8.9 Hz), 150.95, 141.13, 139.82, 137.72, 135.50, 132.30, 130.64, 125.38, 124.21, 120.27, 120.30, 118.56, 83.64 (d, J = 7.1 Hz), 65.72 (d, J = 6.7 Hz), 29.92 (d, J = 4.2 Hz), 20.04, 19.90. HRMS (ESI, m/z) calculated for C24H34N3O5PNa [M + Na]+: 498.21283; found: 498.21237 (observed error of −0.92 ppm is within the range of instrumental error of ±5.00 ppm).

2d: Reddish orange viscous liquid. Yield = 1.3 g (38%). Rf = 0.19 (hexane/EtOAc 7[thin space (1/6-em)]:[thin space (1/6-em)]3). 1H NMR (400 MHz, CDCl3): δ = 7.93–7.90 (m, 2H), 7.70 (d, J = 7.6 Hz 1H), 7.54–7.39 (m, 3H), 7.43–7.39 (m, 2H), 7.36–7.31 (m, 1H), 4.23 (q, J = 7.1 Hz, 2H), 3.53 (t, J = 7.2 Hz, 2H), 1.40 (s, 18H). 13C NMR [100.5 MHz, CDCl3] δ 152.82, 150.45, 137.49, 131.52, 131.16, 131.02, 129.08, 127.52, 123.03, 115.46, 82.09 (d, J = 7.3 Hz), 67.55 (d, J = 6.7 Hz), 32.73 (d, J = 8.0 Hz), 29.77 (d, J = 4.2 Hz). HRMS (ESI, m/z) calculated for C22H31N2O4PNa [M + Na]+: 441.19137; found: 441.19107 (observed error of −0.67 ppm is within the range of instrumental error of ±5.00 ppm).

3b: Reddish orange semisolid. Yield = 0.11 g (96%). 1H NMR (400 MHz, CD3OD): δ = 9.27 (br, 1H), 7.91 (d, J = 6.9 Hz, 2H), 7.64 (d, J = 8.0 Hz, 1H), 7.55–7.45 (m, 4H), 7.26 (d, J = 8.3 Hz, 1H), 7.06 (t, J = 8.2 Hz, 1H), 4.43 (t, J = 5.1 Hz, 2H), 4.39–4.37 (m, 2H). 13C NMR [100.5 MHz, CD3OD] δ 157.72, 154.39, 143.83, 133.81, 132.08, 130.21, 123.98, 122.57, 117.84, 116.57, 70.48 (d, J = 7.7 Hz), 66.06 (d, J = 5.4 Hz). HRMS (ESI, m/z) calculated for C14H14N2O5P [M − H]: 321.06458; found: 321.06483 (observed error of 0.77 ppm is within the range of instrumental error of ±5.00 ppm).

3c in its triethylammonium salt: Orange solid. Yield = 0.77 g (94%). Mp: 139–141 °C. 1H NMR (CD3OD, 400 MHz): δ = 8.58 (dd, J = 8.3, 1.1 Hz, 1H), 7.90–7.82 (m, 3H), 7.49–7.46 (m, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.24–7.19 (m, 1H), 4.54 (d, J = 6.1 Hz, 2H), 3.17 (q, J = 7.3 Hz, 6H), 2.41 (s, 3H), 2.35 (s, 3H), 1.28 (t, J = 7.3 Hz, 9H). 13C NMR [100.5 MHz, CD3OD] δ 169.94 (d, J = 9.1 Hz), 152.27, 142.33, 141.41, 138.92, 137.07, 133.06, 131.83, 127.02, 125.30, 121.50, 120.88, 118.24, 65.71 (d, J = 4.7 Hz), 47.71, 19.94, 19.86, 9.16. HRMS (ESI, m/z) calculated for C16H17N3O5P [M − C6H17N(TEA)]: 362.09113; found: 362.09140 (observed error of 0.74 ppm is within the range of instrumental error of ±5.00 ppm).

3d in its triethylammonium salt: Reddish orange solid. Yield = 0.59 g (51%). Mp: 130–132 °C. 1H NMR (CD3OD, 400 MHz): δ = 7.94–7.92 (m, 2H), 7.67 (dd, J = 8.1, 1.2 Hz, 1H), 7.56–7.49 (m, 4H), 7.43 (td, J = 7.4, 1.3 Hz, 1H), 7.32–7.29 (m, 1H), 4.14 (dd, J = 14.0, 7.3 Hz, 2H), 3.51 (t, J = 7.4 Hz, 2H), 3.16 (q, J = 7.3 Hz, 6H), 1.28 (t, J = 7.3 Hz, 9H). 13C NMR [100.5 MHz, CD3OD] δ 154.31, 151.69, 139.79, 132.72, 132.41, 132.24, 130.31, 128.36, 124.00, 116.18, 67.24, 59.54, 34.02, 8.12. HRMS (ESI, m/z) calculated for C14H14N2O4P [M − C6H16N(TEA)]: 305.06967; found: 305.06991 (observed error of 0.13 ppm is within the range of instrumental error of ±5.00 ppm).

Triphosphate formation. The triethylammonium salt of monophosphates, 3b, 3c and 3d (1 eq.), was converted into its tributylammonium salt through the addition of tributylamine (3.3 eq.) in dry MeOH. Triethylamine and MeOH were removed through rotary evaporation. The tributylammonium salt was dissolved in dry DMF, a solution of 1,1′-carbonyldiimidazole (6.3 eq.) in dry DMF was added under an Ar atmosphere with stirring and then kept at room temperature for 16 h for the reaction to proceed. An excess of 1,1′-carbonyldiimidazole was removed by the addition of dry MeOH (0.25 eq.) and stirring for 1 h. This solution was then added dropwise with stirring to a solution of the tributylammonium salt of pyrophosphate in dry DMF. After reacting overnight at room temperature, the mixture was cooled to 0 °C in an ice bath. Cold water (4 °C) was added with stirring and the pH was brought to 7.5 using 1 M NaOH. The reaction mixture was extracted with ether and H2O; the aqueous phase was evaporated with EtOH at 30 °C and dried. The residue was dissolved in 0.2 M triethylammonium hydrogencarbonate, applied to a DEAE-Sephadex A-25 column (2.5 × 30 cm, 20 g) and eluted with a linear gradient (0.2–1.0 M; total volume: 1 L) of triethylammonium hydrogencarbonate at 4 °C. The product eluted in the range of 0.67–0.86 M was collected and evaporated with EtOH several times to remove triethylammonium hydrogencarbonate. The obtained residue of the product was converted into its sodium salt using 1 M NaI in acetone and freeze dried.

4b as sodium salt: Reddish orange solid. Yield = 0.05 g (44%). Mp 161–163 °C (color changes from reddish orange to dark brown at 110 °C). 1H NMR (D2O, 400 MHz): δ = 7.27 (d, J = 6.6 Hz, 2H), 7.06–7.02 (m, 5H), 6.86 (d, J = 7.0 Hz, 1H), 6.69 (t, J = 6.6 Hz, 1H), 4.56 (m, 2H), 4.48 (m, 2H). 13C NMR [100 MHz, D2O (CD3OD)] δ 156.22, 153.33, 142.86, 134.54, 132.67, 130.48, 123.58, 122.92, 118.18, 116.19, 70.16 (d, J = 7.9 Hz), 65.71 (d, J = 5.2 Hz). 31P NMR [160 MHz, D2O (H3PO4)] δ −10.89 to −11.20 (m, 2P), −23.03 to −23.19 (m, 1P). HRMS (ESI, m/z) calculated for C14H14N2O11Na4P3 [M + H]+: 570.93957; found: 570.93993 (observed error of 0.62 ppm is within the range of instrumental error of ±5.00 ppm). RP-HPLC [column – CN-80Ts, 4.6 × 250 mm (TOSOH); eluent – CH3CN/0.1 M aq. NaPi (pH 6.5); flow rate – 0.5 mL min−1] retention time = 55.01.

4c as sodium salt: Orange solid. Yield = 0.46 g (95%). Mp 168–170 °C (color changes from orange to dark brown at 118 °C). 1H NMR (D2O, 400 MHz): δ = 7.95 (d, J = 7.1 Hz, 1H), 7.76–7.68 (m, 3H), 7.63–7.59 (m, 1H), 7.45–7.40 (m, 2H), 4.69 (d, J = 7.4 Hz, 2H), 2.39 (s, 3H), 2.36 (s, 3H). 13C NMR [100 MHz, D2O (CD3OD)] δ = 170.93 (d, J = 9.6 Hz), 151.35, 143.78, 143.16, 139.35, 134.64, 133.11, 131.58, 127.47, 125.56, 124.48, 120.54, 118.18, 65.73 (d, J = 5.6 Hz), 20.03, 19.92. 31P NMR [160 MHz, D2O (H3PO4)] δ −10.90 (d, 1P, J = 18.6 Hz), −12.48 (d, 1P, J = 19.3 Hz), −23.06 (t, 1P, J = 17.8 Hz). HRMS (ESI, m/z) calculated for C16H17N3O11Na4P3 [M + H]+: 611.96612; found: 611.96757 (observed error of 2.36 ppm is within the range of instrumental error of ±5.00 ppm). RP-HPLC [column – CN-80Ts, 4.6 × 250 mm (TOSOH); eluent – CH3CN/0.1 M aq. NaPi (pH 6.5); flow rate – 0.5 mL min−1] retention time = 76.68.

4d as sodium salt: Dark orange solid. Yield = 0.181 g (64%). Mp 153–155 °C (color changes from reddish orange to dark brown at 110 °C). 1H NMR (D2O, 400 MHz): δ = 1H NMR (400 MHz, D2O) δ 7.96–7.93 (m, 2H), 7.64–7.59 (m, 3H), 7.56–7.52 (m, 2H), 7.44–7.41 (m, 1H), 4.24 (q, J = 8.0 Hz, 2H), 3.48 (t, J = 6.9 Hz, 2H). 13C NMR [100 MHz, D2O (CD3OD)] δ = 153.31, 151.51, 138.15, 132.69, 132.60, 130.51, 128.75, 123.67, 116.61, 68.12 (d, J = 6.1 Hz), 32.82 (d, J = 6.8 Hz). 31P NMR [160 MHz, D2O (H3PO4)] δ −10.71 to −11.19 (m, 2P), −23.28 (t, 1P, J = 19.6 Hz). HRMS (ESI, m/z) calculated for C14H14N2O10Na4P3 [M + H]+: 554.94466; found: 554.94501 (observed error of 0.63 ppm is within the range of instrumental error of ±5.00 ppm). RP-HPLC [column – CN-80Ts, 4.6 × 250 mm (TOSOH); eluent – CH3CN/0.1 M aq. NaPi (pH 6.5); flow rate – 0.5 mL min−1] retention time = 73.09.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

Authors thank the Research Foundation for Opto-Science and Technology for financial support.

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Footnote

Electronic supplementary information (ESI) available. See DOI: 10.1039/c7ob01293d

This journal is © The Royal Society of Chemistry 2017