Green light triggered [2+2] cycloaddition of halochromic styrylquinoxaline—controlling photoreactivity by pH

Photochemical reactions are a powerful tool in (bio)materials design due to the spatial and temporal control light can provide. To extend their applications in biological setting, the use of low-energy, long wavelength light with high penetration propertiesis required. Further regulation of the photochemical process by additional stimuli, such as pH, will open the door for construction of highly regulated systems in nanotechnology- and biology-driven applications. Here we report the green light induced [2+2] cycloaddition of a halochromic system based on a styrylquinoxaline moiety, which allows for its photo-reactivity to be switched on and off by adjusting the pH of the system. Critically, the [2+2] photocycloaddition can be activated by green light (λ up to 550 nm), which is the longest wavelength employed to date in catalyst-free photocycloadditions in solution. Importantly, the pH-dependence of the photo-reactivity was mapped by constant photon action plots. The action plots further indicate that the choice of solvent strongly impacts the system’s photo-reactivity. Indeed, higher conversion and longer activation wavelengths were observed in water compared to acetonitrile under identical reaction conditions. The wider applicability of the system was demonstrated in the crosslinking of an 8-arm PEG to form hydrogels (ca. 1 cm in thickness) with a range of mechanical properties and pH responsiveness, highlighting the potential of the system in materials science.


SEC-ESI-MS
Spectra were recorded on a Q Exactive Plus (Orbitrap) mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with a HESI II probe. The instrument was calibrated in the m/z range 74-1822 using premixed calibration solutions (Thermo Scientific) and for the high mass mode in the m/z range of 600-8000 using ammonium hexafluorophosphate solution. A constant spray voltage of 3.5 kV, a dimensionless sheath gas and a dimensionless auxiliary gas flow rate of 10 and 0 were applied, respectively. The capillary temperature was set to 320 °C, the S-lens RF level was set to 150 and the aux gas heater temperature was set to 125 °C. The Q Exactive was coupled to an UltiMate 3000 UHPLC System (Dionex, Sunnyvale, CA, USA) consisting of a pump (LPG 3400SD), autosampler (WPS 3000TSL), and a temperature-controlled column department (TCC 3000). Separation was performed on two mixed bed size exclusion chromatography columns (Agilent, Mesopore 250 × 4.6 mm, particle diameter 3 µm) with a precolumn (Mesopore 50 × 7.5 mm) operating at 30 °C. THF at a flow rate of 0.30 mL·min -1 was used as eluent. The mass spectrometer was coupled to the column in parallel to an UV-detector (VWD 3400, Dionex), and a RI-detector (RefractoMax520, ERC, Japan) in a setup described earlier. [1] 0.27 mL·min -1 of the eluent were directed through the UV-and RI-detector and 30 µL·min -1 were infused into the electrospray source after post-column addition of a 50 µM solution of sodium iodide in methanol at 20 µL·min -1 by a micro-flow HPLC syringe pump (Teledyne ISCO, Model 100DM). A 200 µL aliquot of a polymer solution with a concentration of 2 mg·mL -1 was injected into the SEC system.

DMAC-SEC
The SEC measurements were conducted on a

1 H NMR Measurements
NMR spectra were recorded on a Bruker Avance III 400 MHz or 600 MHz with a 5 mm broadband auto-tunable probe with Z-gradients at 293 K. Chemical shifts are reported as δ in parts per million (ppm) and referenced to the chemical shift of the residual solvent resonances (CDCl3 δ = 7.26 ppm), couplings are shown as s: singlet, d: doublet, t: triplet, m: multiplet. Polymer samples were prepared at a concentration of 10 mg mL -1 . In most spectra traces of water appears as a broad singlet at around 1.5-2.5 ppm. NMR spectra were processed using MestReNova software.

Mass Spectrometry
Test compounds were infused directly into the MS via a kdScientific infusion pump at a static flow rate of 650 µL/h. MS setup was as followed: Agilent 6220 TOF MS system (Santa Clara, CA, USA) with a multimode dual nebuliser ESI/APCI source. The MS was operated in positive or negative mode using the following conditions: nebulizer pressure 35 psi, gas flowrate 8 L/min, gas temperature 300°C, capillary voltage 2500/-2500 V, fragmentor 150 and skimmer 65 V. The instrument was operated in the extended dynamic range mode with data collected in m/z range 100-3200.

UV-VIS Spectroscopy
UV/vis spectra were recorded on a Shimadzu UV-2700 spectrophotometer equipped with a CPS-100 electronic temperature control cell positioner. Samples were prepared in THF and measured in Hellma Analytics quartz high precision cells with a path length of 10 mm at ambient temperature.

Rheology
Rheological experiments were studied using an Anton Paar Physica rheometer with a plate-plate configuration. The lower plate is made of quartz and the upper plate is made of stainless steel with a diameter of 15 mm. A liquid light guild, which was connected to the WheeLED light source, was equipped below the quartz plate. In a typical experiment, 50 µL of a solution of P2 was placed on the lower plate and the upper plate was brought to a measurement gap of 0.2 mm. A layer of paraffin oil was applied on the edge of the stainless-steel plate to prevent dehydration of hydrogel and the test was started by applying a 1% strain with the frequency of 0.1 Hz on the sample.

Experiments using LED lamps
Light irradiation for polymer crosslinking was performed using a Mightex's WheeLED wavelengthswitchable LED for λ = 405-505 nm (built-in filters). The intensity of light irradiance on the gel sample was tuned to the desired intensity (20 mW cm -2 ) using a RM-12 radiometer (Opsytec) with a sensor VISBG 400-570 nm.

Experiments using laser irradiation
The incident light used for laser experiments was a Coherent Opolette 355 tunable OPO operated at 410-500 nm with a full width half maximum of 7 ns and a repetition rate of 20 Hz. The emitted pulse, which has a flat-top spatial profile, was expanded to 6mm diameter using focusing lenses and directed upwards using a prism. The beam was then centered on a glass laser vial which is positioned in a 6mm diameter slot in a temperature-controlled sample holder. The energy transmitted through the sample holder was measured using a Coherent Energy Max PC power meter.
Supplementary Figure 12. Experimental setup for tunable laser experiments.

Dimerization of PEG-SQ
PEG-SQ (1 mg) was dissolved in 0.1 mL of Acetonitrile or water in laser vials, the vials were crimped airtight and degassed for 5 min applying Argon. Each solution was irradiated at λ = 410-550 nm with the same number of photons.

Control over the constant number of photons [2]
The number of photons np ([np] = mol) that a monochromatic laser pulse contains can be calculated by application of the Planck-Einstein relation from the energy of the pulse Epulse, the incident wavelength λ, Planck's constant h and the speed of light c: If the absorption of the glass vial and the extent of reflection and scattering at the vial at the respectively relevant wavelength is known, a target energy value can be calculated that must be reached during the above described measurement to guarantee that the desired number of photons penetrates the sample solution during the subsequent irradiation. The wavelength dependent transmittance of the glass vials was determined experimentally using the above setup. Three glass vials were randomly selected as calibration vials. For varying wavelengths and in each case at a constant power output of the laser the energy was measured both with and without the calibration vials fitted into the sample holder. The top parts of these vials were cut off to minimize errors in the procedure, since only the bottom and sides of the glass vials would contribute significantly to the reduction of the photon flux that enters the solution.
The measured energy per pulse without a calibration vial in the sample holder is denoted as E0 and the measured energy per pulse with a calibration vial in the sample holder as En. The transmittance was calculated as the ratio of En to E0. The average transmittance over the measurements of the three vials (Tλ) was plotted together with the respective error (compare7): The target energy per pulse E0 can be calculated directly from the wavelength λ, the number of pulses , the transmittance of the glass vial at the respective wavelength λ and the desired total photon count p : By controlling the target E0 at the respective wavelength, the number of photons that penetrate each sample solution of one set of experiments as described in the following subsections was guaranteed to be identical despite irradiation at different wavelengths.

Supplementary Table 1.
Transmittance of the bottom of the glass vials used in this study. The transmittance values shown and used here were obtained analogously to a method reported previously. [3] The glass vials were cut at a height of 3 mm. Thus, the number of photons delivered into the sample solution can be determined more precisely, than in initial attempts to estimate the number of photons. [4] The values are in agreement to the previously found transmittance at 285 nm, which was determined with the same glass vials as used here. [5] l / nm

Calculation of conversions for action plot
The conversion of the dimerization reactions was calculated from the SEC data (Supplementary Figure  4). Initially, the integral of the peaks was calculated by applying Gaussian formulation for the peak fitting. Then the below equation was used to obtain the conversion as percentage where A1 is the area under first peak (PEG-SQ) and A2 is the area under second peak ((PEG-SQ)2).

Hydrogel swelling
To study the effect of pH on hydrogel swelling, a polymer solution (c = 7.5 mM, 200 µL) in a 5 mL sealed vial was irradiated with light at 455 nm for 3 h. The resultant solid gel was extracted from the vial and placed in excess PBS solution pH 7.4. The weight of the hydrogel was monitored until no further change in the weight was observed. The pH of the solution was subsequently adjusted using HCl 1 M and NaOH 1 M solution. At each set pH, the weight of the hydrogel (wt) was recorded when no further change in the weight was observed. The swelling ratio was calculated by: Where w0 is the weight of as-prepared hydrogel. The experiment was done in triplicate.

Cell culture study
Cell culture was carried out using commercial mouse fibroblasts L929 (NCTC clone 929, ATCC® CCL-1™). Cells were cultured on tissue culture flasks as per manufacturers' instructions, then trypsinised with Tryple Express to detach from the culture surfaces. The cells were centrifuged for 3 min at 0.3 g and the supernatant discarded. For cell culture studies, fibroblasts were resuspended in PEG-(SQ)8 solution (c = 5 mM) to achieve a cell density of 5 × 10 6 cells per mL. The solution was agitated gently to allow the cells to distribute throughout the solution and pipetted into tissue culture inserts. The inserts were exposed to green light (λ = 505 nm, I = 20 mW cm -2 ) irradiation for 30 minutes and cell culture media (Dulbecco's Modified Eagle Medium) were added. Triplicates were prepared. The cell-laden hydrogel samples were rinsed twice with culture media and maintained at 37 °C and 5% CO2. At day 1 cell culture media were exchanged once. To assess cell viability, gels were removed from tissue culture inserts after day 1 and day 3 in culture, washed in PBS and stained using Live/Dead® Viability/Cytotoxicity Kit for mammalian cells (Invitrogen) following the manufacturer's recommended protocol.

Synthesis of 8-arm PEG-NH2
8-arm PEG20k (5 g, 0.25 mmol) was dissolved in CH2Cl2 (20 mL). Triethylamine (1.2 g, 12 mmol) was added and the solution was cooled on an ice bath. Methansulfonyl chloride (1.14 g, 10 mmol) was added dropwise over 30 min and the solution was allowed to warm to room temperature. The solution was stirred for 2 h, filtered and precipitated into diethyl ether (200 mL) to give white powder that was used directly in the next step.
The above product was dissolved in DMF (10 mL) and sodium azide (1.3 g, 20 mmol) was added and the solution was stirred at 80 °C for 10 h. DMF was concentrated in vacuo and the residue was taken up in dichloromethane (50 mL), and filtered. The filtrate was washed with water (100 mL x2), brine (100 mL), dried (MgSO4), concentrated in vacuo to ca. 10 mL and precipitated into diethyl ether (200 mL) to give white polymer that was used directly in the next step.
The above product was dissolved in methanol (50 mL) and triphenylphosphine (2.62 g, 10 mmol) was added. The resultant solution was heated at 80 °C under refluxing condition overnight. The solution was then concentrated in vacuo and water (50 mL) was added. The mixture was then extracted with diethyl ether (50 mL x 3) and the organic phase was discarded. The aqueous phase was then extracted with dichloromethane (50 mL x2). The combined organic phases were dried (MgSO4), concentrated in vacuo to ca. 10 mL and precipitated into diethyl ether (200 mL) to give product as white powder (total yield: 3.8 g, ca. 76%). 1