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2-Hydroxymethylene-8-dimethylamino quinoline-(DMAQ) derived photosensitive probes were prepared and tested under 366 nm and 730 nm 150 fs pulsed (“two-photon”) irradiation conditions. Brief bursts of photolysis of 250 μM solution of the kainate derivative of 2f generated sufficient kainate in a small spot to excite large inward currents and somatic spiking in Purkinje neurons.
© 2016 Optical Society of America
1. Introduction
The photorelease of ligands from biologically inert precursors with a spatially defined pulse of light provides a tool frequently used to probe the function of highly organized biological systems [1, 2]. The photoactivation can be realized by near-UV light, usually in the 250-400 nm window, corresponding to one photon (OP) photolysis, or, by two-photon (TP) activation by using pulsed near-IR light. The TP excitation presents some advantages over OP photolysis, such as (i) the excitation volume is better defined (ca. 1 fL) and (ii) deeper penetration of the localized photolysis is possible in turbid tissues because scattered near-IR photons do not produce out of focus excitation, unlike the scattered near UV photons in OP photolysis [1, 2, 4].
As a part of a larger program on light-sensitive probes designed for molecular biology, physiology and neurosciences [5, 6], we were interested revisiting the dimethylamino quinoline (DMAQ) platform [3, 7] considered as one of the most efficient photosensitive probe for biological applications in terms of sensitivity, solubility and fragmentation kinetics. The systematic study on the substitution effect of the 8-dimethylamino quinoline (8-DMAQ), 1, realized by our group [8] has revealed that p-benzoyl group at the C5 position (2) enhances the TP sensitivity, giving significantly improved TP uncaging cross sections while retaining high aqueous solubility (5-benzoyl-8-DMAQ-OAc δu = 2.00 GM) (Fig. 1) [9]. Based on these results it appeared to us interesting to examine the substituent effect impacting the TP sensitivity of aryl-conjugated acceptor (EWG) and donor (D) derivatives of the 8-DMAQ acetate platform (Fig. 1).
2. Experimental details
Organic synthesis: Compounds 2a and 2b were prepared from the TBS-protected 5-bromo-8-DMAQ 3a and the corresponding aryl boronates, 4a and 4b, respectively under Suzuki - Miyaura conditions in the presence of Pd(PPh3)4 (10 mol%) and Na2CO3 (aq, 2 M), in DME at 80 °C (51% 5a; 56% 5b). For the preparation of 2c the 5-iodo-8-DMAQ 3b and the p-N,N-dimethylaniline pinacolboronate 4c was used [10]. The protected intermediates were transformed to the corresponding acetates by HF / pyridine treatment followed by acetylation under usual conditions (Fig. 2).
Fig. 2 Synthesis of 5-(p-cyanophenyl)-8-DMAQ-OAc (2a), 5-(p-methoxyphenyl)-8-DMAQ-OAc (2b), 5-(p-dimethylaminophenyl)-8-DMAQ-OAc (2c).
Compounds 2d and 2e were prepared by “inverse polarity” Suzuki-Miyamura coupling. The boronate ester 3c was prepared from bromine 3a and the bromobenzene 4d and the monobrominated triphenylamine 4e [11] were added respectively under similar conditions described above (Fig. 3). The deprotection of 5d and 5e followed by acetylation of the corresponding alcohols afforded 2d and 2e in 57% and 46% yields, respectively for the two steps.
Photonics: UV and NIR irradiations were realized in acetonitrile / TRIS buffer 1/1 (pH 7.4) solution (c = 0.1 mM). For the UV irradiation, an aliquot (1 mL) of this solution was irradiated at approximately 366 nm by using 8W Carl Roth lamp. One-photon quantum yields were determined by using Eq. (1) [3, 7]:
where ε(λexc) is the molar absorption coefficient of the compound at the excitation wavelength (M−1cm−1), t90% is the time at which 90% of the compound was converted, as determined by HPLC, and I0(λexc) is the photon flux at the excitation wavelength (einstein cm−2s−1) determined by ferrioxalate actinometry. Between each irradiation, a small aliquot (20 µL) of the solution was removed for analysis by reverse-phase HPLC using absorbance detection at 360 nm. Optical densities at 366 nm were kept around 0.1 so that inner-filtering of the irradiation and spatial gradients of concentrations could be neglected, and the progress curves were simple decaying exponentials. Dark hydrolysis rates were measured similarly except without illumination.Uncaging two photon cross-sections (δu) were calculated from the fractional conversion of the cage with exposures of 2 - 4 hours in a 45 microlitre quartz cuvette of 3 mm pathlength (Starna, UK). A MaiTai BB (Spectra-Physics) TiS, set up here for 730 nm at 150 fs pulse width at 80 MHz. The 3x expanded output was focused into the cuvette with a 32 mm focal length AR coated lens.The two-photon excitation volume was entirely contained within the cuvette volume, determined by calculation from the beam waist radius of 13 µm, Rayleigh length 950 µm and standardized by measuring the two photon induced fluorescence generated in the volume in 1 mM fluorescein (pH 11). The average power was set in the range 100 −150 mW, measured after the cuvette, avoiding heating. Initial cage concentrations were 0.1 mM. Samples were centrifuged to remove particles apparent in the transmitted beam. Non-irradiated controls were included in the assays. The two-photon uncaging cross section δu was calculated from the fractional conversion, exposure time, volume and beam parameters as described by Kiskin et al. (Eq. (2)) [14]:
where C = fractional conversion in time t; δu = TP uncaging cross-section (cm4s/photon); P = time averaged power (W); λ = wavelength (cm); h = Plancks constant; c = velocity of light in vacuo (cm/s); ω = waist radius (cm), T = pulse interval (s); τ = corrected pulse width (s, FWHM) assuming sech2 pulse shape; n = refractive index; VF = fractional irradiated volume; t = exposure time (s).The factor 1.17 takes account of the spatial and temporal distributions of instantaneous intensity squared for a Gaussian-Lorentzian profile, and two photons absorbed for each excitation [4, 14]. In most measurements the excitation volume was entirely contained within the cuvette, simplifying the calculation because geometric parameters of the spatial intensity distribution and excitation volume cancel, requiring only the sample volume VS (cm3) as a separate measurement (Eq. (3)) [4, 14]:Pulse widths were measured with a Carpe or Femtochrome 103 autocorrelator and corrected to sech2 profile by dividing by 1.76. The two photon excitation volume in the cuvette irradiations had a beam waist radius of 13 µm, giving intensities 350 fold lower than the same average power in a submicron focus of a microscope objective. The calculated diffusional exchange rate between the cuvette excitation volume and bulk cuvette solution was much faster than the photolysis rates observed at the irradiation intensities used and was not rate limiting. There was no evidence of heating. For reference the two-photon uncaging cross-section for L-glutamate release from the widely used MNI-caged glutamate determined in this way was 0.05 GM (10−50 cm4s/photon). The conversion of the product was assayed by HPLC by monitoring the remaining caged compound. HPLC analyses were carried out on Water device 600 with an inverse phase column X-Terra® MS C18 HPLC-MS analysis of irradiated samples confirmed the presence of the corresponding alcohol photoproducts in the samples.Slice preparation: Experiments were performed in vitro sagittal slices 220 µm thick cut from the cerebellum of 17-23 day old Sprague-Dawley male or female rats. Briefly, rats were killed by decapitation under general anaesthesia following inhalation of the volatile anesthetic isoflurane in accordance with the Directive 2010/63/UE and the cerebellum was quickly removed and cooled in ice-cold solution. After removal of the brain stem, the tissue was glued to the stage of a vibrotome (Leica VT1200S, Germany). Slices were kept in a vessel bubbled with 95% O2 / 5% CO2 at 34 °C for 1h and then allowed to cool down to room temperature. Slice preparation and recordings were made in a bicarbonate buffered solution containing in mM: 115 NaCl, 2.5 KCl, 1.3 NaH2PO4, 26 NaHCO3 and 25 glucose. For preparation of the slices, the solution contained 4 mM MgSO4 and 0.5 mM CaCl2; for recovery and recording, 1 mM MgSO4 and 2 mM CaCl2 respectively.
Recording: Recordings were made in 1 ml Hepes buffered saline of composition (mM) NaCl 145, KCl 4, Hepes 10, glucose 25, NaHCO3 2, CaCl2 2, MgSO4 1, pH 7.3 with NaOH. Whole cell patch clamp recordings were made from Purkinje neurons, identified by their size and location at the edge of the molecular and granule cell layers with an Axoclamp 200B amplifier (Molecular Devices, USA), digitised at 10 kHz after LP filtering at 2 kHz (Spike 2, P1401, Cambridge Electronic Design, UK). The internal solution contained in mM: 140 gluconate, 10 KCl, 10 Hepes, 0.1 EGTA, 4.6 MgCl2, 4 ATPNa2 and 0.4 GTPNa, pH adjusted to 7.3 with KOH and osmolarity to 300 mOsm/kg. When filled with internal solution recording pipettes had a resistance between 2.5 and 4.0 MΩ. Membrane currents were recorded at a pipette potential of –60 mV (not corrected for junction potential of approximately –12 mV pipette-bath). Data were analysed in Igor Pro (Wavemetrics, USA).
Ethical approval: Sprague Dawley rats were provided by Janvier (St Berthevin, France) and subsequently housed in agreement with the European Directive 2010/63/UE regarding the protection of animals used for experimental and other scientific purposes. Experimental procedures were approved by the Directorate of Paris Veterinary Services and by the ethical committee for animal experimentation of Paris Descartes.
3. Results and discussion
Acetate derivatives were only sparely soluble in TRIS buffer, all photophysical analysis were realized in a 1/1 mixture of acetonitrile/TRIS buffer (20 mM, pH 7.4) at roughly 0.1 mM concentration at 293 K. The UV absorptions of these compounds, measured in the same conditions, present qualitatively similar UV spectra. Compounds 2a-f have a first absorption maxima (λmax) located between 343 and 366 nm, with molar extinction coefficient (εmax) at the λmax between 2025 and 11370 M−1cm−1 (Fig. 4, Table 1). The UV spectra shown that compounds 2a-c and 2e have higher εmax values compared to the 5-phenyl (2d) and 5-benzoyl-8-DMAQ-OAc (2f), reflecting the influence of strong electron-withdrawing and donating groups on the OP absorption. Furthermore, the 5-dimethylaminophenyl (2c) and 5-diphenylaminophenyl-8-DMAQ-OAc (2e) are red-shifted, compared to the 5-benzoyl derivative (2f), with λmax at 365 and 366 nm and εmax = 5881 and 11370 M−1cm−1 respectively. Moreover, a hyperchromic effect of the nitrile (2a) and also, of the diphenylamino (2e) derivative can be observed that is probably the consequence of an important ICT within these compounds.
Fig. 4 UV absorption spectra of 5-benzoyl-8-DMAQ-OAc derivatives in acetonitrile/TRIS (20 mM) 1/1 at 293 K.
Table 1. Photophysical properties of chromophores 2a-f. a Measured at 366 nm; bQu values were determined according to Eq. (1).
Compounds 2a-f were photolyzed by UV irradiation, for 0.5 - 1 hours in the same conditions described previously [6, 8, 9]. The time course of UV photolysis and the conversion of the starting materials to the corresponding hydroxymethylenes over time was monitored by HPLC, and results are depicted in Fig. 5. No quantitative analysis of the photoreleased acetic acid was realized. With the exception of the dimethylaniline derivative 2c all derivative showed increased sensitivity and photolysis efficiency under UV irradiation compared to 5-benzoyl-8-DMAQ-Ac 2f with εQu between 724 and 2083 M−1.cm−1. While the 5-dimethylaminophenyl-derivative 2c was the less efficient in this series, the 5-diphenylaminophenyl-derivative (2e) revealed to be the most sensitive, with εQu = 2083 M−1cm−1 with a high molar extinction at 366 nm (ε366 = 11297 M−1cm−1), probably due to the synergistic effects of the extension of π-conjugation and the strong EDG, contributing to a more efficient ICT.
Fig. 5 OP photolysis of 5-benzoyl-8-DMAQ-OAc derivatives (2a-f) at 366 nm. The remaining fraction was determined by HPLC and the reported fittings correspond to an exponential decay.
Although the OP excitation of chromophores 2a, 2b, 2d and 2e was more efficient than the UV photolysis of the 5-benzoyl-8-DMAQ-OAc, 2f, their TP sensitivity appeared less relevant (Table 1): TP cross section values δu varied between 0.25 and 0.64 GM. This observation could be rationalized by a lower ICT within these platforms, or by the influence of these substituents on the interfering relaxation pathways (e.g. fluorescence, phosphorescence). Nevertheless, the trend is noteworthy: the introduction of a nitrile group to the 5-phenyl-8-DMAQ-OAc 2d allowed more than two fold enhancement of the δu value in 2a that can be probably attributed to the formation of an efficient push-pull system within an extended π-conjugated system. Moreover, similar δu value is obtained with 2e, reflecting the synergistic effects between an EDG and an extended π-system. Furthermore, with weaker EDG (2b and 2c), the TP cross-section is less efficient than the δu value for 2d, showing a lower ICT with respect to the 5-phenyl-8-DMAQ-OAc.
Physiological experiments: We have previously investigated several caged neuroactive amino acids for their ability to activate glutamate receptors after uncaging in cerebellar Purkinje neurons [13]. MNI-kainate was found to be effective at low concentration in OP photolysis, generating large currents in Purkinje neurons by activating both AMPA and kainate sub-types of glutamate receptor, suggesting the use of kainate as an agonist to produce photostimulation by activating glutamate receptors. 5-Benzoyl-8-DMAQ kainate was prepared and was found to be hydrolytically stable, with a half-life around 7 days. The TP uncaging cross-section was measured at 1 GM for kainate release. The caged kainate was dissolved in 154 mM NaCl buffered with 10 mM Hepes pH 7.0 to give a stock solution of 13 mM (estimated from the molar absorption coefficient 2025 M−1 cm−1 at 343 nm). 50 µL stocks were kept frozen at −20 °C and added to the bath solution to generate cage concentrations from 0.1 to 1 mM. The results showed that addition of 25 µl of the stock to 1 mL of physiological solution to produce 0.3 mM caged kainate resulted in no apparent changes of membrane conductance or excitability.
The photolysis laser spot (3 µm diameter) was applied to the bifurcation of the primary dendrite. Progressively increasing the photolysis by increasingly longer bursts of pulses of 0.2 ms duration at 100-1000 Hz generated inward currents up to 0.5 nA amplitude in cerebellar Purkinje neurons and dendritically evoked spikes at large amplitudes. The results indicate that 5-benzoyl-8-DMAQ-kainate has no large effect on excitability as judged by lack of effect on spontaneous synaptic activity or action potentials evoked by depolarisation. Brief bursts of photolysis of 250 μM solution generated sufficient kainate in a small spot to evoke large inward currents and somatic spiking in Purkinje neurons.
Acknowledgments
This research was supported by a Marie Curie Intra European Fellowship within the 7th European Community Framework Programme (FP7-PEOPLE-2013-IEF: 629675). We thank Tocris Biosciences for providing the protected kainic acid used in this study. Authors acknowledge also the generous financial support of the ANR-14-CE08-0013-01 grant.
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