A two-photon activatable amino acid linker for the induction of fluorescence

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General Information
All starting materials were obtained from commercially available sources and used without further purification.Activated zinc was bought from Rieke Metals Inc. ATTO565-NHS ester was obtained from ATTO-TEC GmbH.The hydrogel kit 3D Life PVA-PEG was obtained from Cellendes.
Flash chromatography was performed with silica gel 60 from Machery Nagel with a particle size of 40 -63 µm.The typical solvents were cyclohexane (CH) and ethyl acetate (EE).For TLC analysis aluminum plates coated with silica gel 60 F 254 from Merck were used.Compounds were visualized with 254 nm.

1.
2. Elemental analysis was conducted with a Vario Micro Cube from Elementar Analysesysteme GmbH.

Time
The elements Carbon, Hydrogen and Nitrogen were analyzed.
One-photon excitation was performed with a custom built setup containing LEDs from Roithner Lasertechnik.365 nm with 250 mW power was used.The one-photon actinometry was conducted with a cuvette holder, a 365 nm LED, and a LED controller from Thorlabs.Irradiation time was controlled by an external trigger.
For fluorescence measurements a TECAN plate reader infinite M200 PRO in fluorescence top reading mode was used.Excitation wavelength was 530 nm (bandwidth 9 nm) and the emission was recorded from 560 -850 nm (bandwidth 20 nm).
For the two-photon setup see section "Two-photon excitation".
For activation with 405 nm and 568 nm and imaging see section "One-photon widefield and superresolution imaging".

3-Nitrodibenzofuran-2-carbaldehyde
12.0 g (32.5 mmol) of 4-(2-Iodophenoxy)-2-nitrobenzaldehyde were dissolved in 150 mL of toluene.10.9 mL (195 mmol) of Ethylene glycol and 1.0 g of p-toluene sulfonic acid monohydrate (5.3 mmol) were added.The reaction was purged with argon and stirred at 120 °C.Reaction progress was monitored by TLC.After 20 h, the reaction was cooled to room temperature, diluted with 100 mL of CH 2 Cl 2 and extracted with 50 mL of NaHCO 3 .The aqueous phase was washed three times with 50 mL of CH 2 Cl 2 .The combined organic extracts were dried with anhydrous MgSO 4 , evaporated to dryness and purified by column chromatography (CH:EE 5:1).8.2 g (61.0%) of the intermediate product were isolated.
8.1 g (19.6 mmol) of the intermediate product were dissolved in 40 mL of dimethylacetamide.12.8 g (39.2 mmol) of Cs 2 CO 3 , 1.0 g (4.5 mmol) of Pd(OAc) 2 and 1 mL of H 2 O were added.The reaction was stirred at 80 °C.The progress of the reaction was followed by NMR.The reaction was complete after one week.After filtration over a Celite® pad, the filtrate was diluted with 80 mL of EE, three times washed with 80 mL of H 2 O and once with 80 mL of brine.The organic extract was dried with anhydrous MgSO 4 and the solvents evaporated to yield 5.4 g (96.6%) of the product 2-(1,3-dioxolan-2-yl)-3-nitrodibenzofuran, which was used in the next step without further purification.
5.3 g (18.6 mmol) of 2-(1,3-dioxolan-2-yl)-3-nitrodibenzofuran were dissolved in 50 mL of THF.After addition of 40 mL of 1M aqueous HCl, the reaction was stirred at room temperature.Reaction progress was monitored by TLC.After 48 h the reaction was extracted with 100 mL of ethyl acetate three times.The combined organic extracts were dried with anhydrous MgSO 4 and evaporated to dryness.The crude product was crystallized from CHCl 3 by adding n-hexane to yield 2.8 g (63%) of the pure product.The flask was purged with argon and cooled to 0 °C.58.4 mL of a solution of 2.5 g of activated zinc in 100 mL of THF (22.4 mmol) were added dropwise.The reaction was allowed to warm in 1 h to room temperature to form the Reformatzki reagent.In a second dried flask 2.7 g (11.2 mmol) of 3nitrodibenzofuran-2-carbaldehyde were dissolved in 50 mL of dry THF.This solution was also purged with argon and cooled to 0 °C.The Reformatzki reagent was slowly added to this solution via syringe.
The reaction was allowed to stir for 1 h at 0 °C.TLC control showed complete conversion of the starting material.100 mL of 1M HCl were added and the mixture was extracted three times with 100 mL of ethyl acetate.The combined organic extracts were dried with anhydrous MgSO 4 and purified by column chromatography (gradient from CH:EE 10:1 to CH:EE 1:1) to yield 3.9 g (98%) of the product.3.8 g (10.6 mmol) of tert-Butyl 3-hydroxy-3-(3-nitrodibenzofuran-2-yl)propanoate were placed together with 4.9 g (15.9 mmol) of Fmoc-β-alanine, 12.2 g (63.8 mmol) of EDC and 390 mg (3.2 mmol) of DMAP in a 250 mL flask.The flask was purged with Ar.The reagents were dissolved in 100 mL of CH 2 Cl 2 and stirred under protection of light.TLC showed complete conversion of starting material after 18 h, so the reaction was poured into ice water.The organic phase was separated and the aqueous phase extracted with EE twice.The combined organic extracts were washed with brine, dried with anhydrous MgSO 4 and evaporated to dryness.The crude product was purified by column chromatography (gradient from CH:EE 10:1 to 1:1) to yield 4.2 g (61%) of the desired product.

3-(3-(Fmoc)amino)propanoyloxy)-3-(3-nitrodibenzofuran-2-yl)propanoate
propanoate were dissolved in a mixture of 30 mL of CH 2 Cl 2 and 5 mL of trifluoroacetic acid.The reaction progress was monitored by TLC.The reaction was complete after 3 h at room temperature.The solvents were evaporated and the residue coevaporated with 10 mL of toluene three times to yield the final product in 3.5 g (93%) yield.

Peptide synthesis
Procedure A: 1 eq. of carboxylic acid was dissolved in dry THF (for 0.5 mmol 40 mL).1.1 eq.HOBt, 1.1 eq.EDC, 2 eq. of DIPEA and 1.1 eq. of the amine were added.The reaction was stirred at room temperature for 16 h.The solvent was evaporated and the residue dissolved in a mixture of 100 mL EE and 100 mL H 2 O.The organic phase was separated and washed with 100 mL H 2 O twice, three times with 60 mL 10% aqueous Na 2 CO 3 and 50 mL brine once.The organic phase was dried with MgSO 4 and evaporated to dryness.
Procedure B: The Fmoc protected starting material was dissolved in a mixture of 4 mL DMF with 1 mL piperidine.The reaction was stirred for 30 minutes and evaporated to dryness.
The product was purified by HPLC method 1.
HPLC: two diastereomers eluted at 17.5 and 17.9 min in quantitative HPLC (big column with flow of 4 mL/min).In analytical HPLC the retention time shifted to 20.4 and 20.7 min.

S5
The crude product was purified by column chromatography with a gradient of methanol in CH 2 Cl 2 from 1% to 5%.The isolated product did no longer possess an Fmoc group.
Yield: Starting with 400 mg of S1, the product yield over the two steps was 140 mg (21%).

Disulfide cleavage
Peptide 2 The disulfide containing peptide was dissolved in 50 mM TCEP, 100 mM Tris buffer pH 7.4.If solubility problems were observed small amounts of acetonitrile were added.After deprotection for 16 h the reaction was purified by HPLC method 2.
13 μL of the iron(III) oxalate solution were irradiated with 365 nm (184 mW) in a 1 cm cuvette.Irradiation time increased from 1 s, 2 s, 3 s, 5 s to 7 s.All experiments were repeated three times.After irradiation, each sample was vortexed in a 2 mL Eppendorf tube.Afterwards 4 μL of the irradiated ferrioxalate solution were mixed with 4 μL phenanthroline solution and 42 μL of buffer B. After waiting for 30 minutes, the absorption at 509 nm was measured (Fig. S2).As long as the results show a linear behavior (Fig. S2), the amount of generated Fe(II) can be determined from the increase in the absorption and the slope of the calibration curve (Fig. S1).

𝑉
The numbers of photons emitted by the LED per second can be calculated from the amount of Fe(II) and the quantum yield of iron(III) oxalate.
The amount of photons absorbed by the new compound is calculated from Lambert Beer's law.: quantum yield of the new molecule.

Determination of fluorescence quantum yield
For the determination of fluorescence quantum yields, peptide 1 was diluted directly in HBS buffer (20 mM HEPES, 150 mM NaCl, pH 7.5) and ATTO565 NHS ester (ATTO-TEC) was diluted in HBS from a 1 mM stock solution in absolute DMSO (≥ 99.5% (GC)).As reference dye we used Rhodamine 101 (Radiant Dyes) in absolute ethanol.The concentrations of the dye solutions were adjusted not to exceed an absorption of 0.1 in the S 0 -S 1 maximum and to matching absorbances at 540 nm, which was chosen as excitation wavelength for all dye constructs.Absorption and fluorescence spectra were recorded on a V-650 UV/Vis photometer (Jasco) and on a FP-8500 fluorimeter (Jasco) by keeping the spectrometer settings constant.The measurements were carried out at 25 °C by using a 3x3 mm quartz cuvette (Hellma Analytics).We calculated the fluorescence quantum yields according to C. Würth, M. Grabolle, J. Pauli, M. Spieles and U. Resch-Genger, Nat.Prot.2013, 8, 1535 with the following equations: For the reference dye Rhodamine 101 we assumed a fluorescence quantum yield of 91.5% in ethanol (C.Würth, M. Grabolle, J. Pauli, M. Spieles and U. Resch-Genger, Nat.Prot.2013, 8, 1535).We obtained quantum yields of 2.8% for caged peptide 1, 61.8% for uncaged peptide 1 and 78.6% for free ATTO565 NHS ester in HBS buffer.Note that the absorption spectrum of peptide 1 is blue-shifted of about 10 nm upon photoactivation, which results in an increased absorbance at the excitation wavelength.This yields a much higher fluorescence increase (81 fold) compared to the quantum yield increase (22 fold), for which the concentration of the uncaged compound was decreased to obtain a similar absorbance at the excitation wavelength compared to the caged sample.

Two-photon excitation
For two-photon irradiation, peptide 2 was immobilized in a commercially available maleimide containing hydrogel (Cellendes, see section "General Information").1.5 μL water, 0.63 μL CB buffer pH 5.5, 1.25 μL Mal PVA, 1 μL peptide (118 pmol in HBS buffer pH 7.5), and 1.87 μL PEG-Linker were mixed in a chambered cover glass according to manufactures instruction.The hydrogel was covered with 200 μL of HBS buffer pH 7.5.Irradiation was performed with an LSM 780 Axio Observer NLO with a C-Apochromat 63x/1.20 W Korr M27 objective from Zeiss.Two-photon excitation was performed with a Coherent Ultra II laser at 780 nm during simultaneous fluorescence excitation with a second laser at 561 nm.
The increase in the fluorescence was measured with 100 scans at 6 mW (Fig. S4).

Antibody labeling
The unlabeled F(ab') 2 fragment of an IgG (H + L) goat anti-mouse antibody (A10534, Life Technologies) was diluted to a concentration of 1 mg/mL in 50 µL NaHCO 3 buffer (0.1 M) at pH 8.3 and vortexed, while adding 0.1 µL of a freshly prepared solution of the dye NHS ester in DMF (2 mM).The reaction mixture was slightly stirred for 30 min.The labeled protein was purified by size exclusion chromatography using an Illustra NAP-5 column (Sephadex G-25, DNA-free, GE Healthcare) according to the protocol of the supplier.PBS was used as column equilibration buffer and elusion buffer, respectively.The average number of fluorophores per antibody was determined to 0.5 by absorption spectroscopy on a Cary 100 UV-Vis spectrometer (Agilent).
Cell culture and staining U2OS cells were cultured in Dulbecco's modified Eagle medium/nutrient mixture F-12 (DMEM/F-12) (3.15 g/L (+)D-glucose, Gibco, Life Technologies) supplemented with 1% Gluta MAX (Gibco, Life Technologies) and 10% fetal bovine serum (South America, Gibco, Life Technologies) at 37 °C in 5% CO 2 atmosphere.Cells were grown on 8-well chambered coverslips (170 µm thickness, Sarstedt; 1.5 x 10 4 cells per well) the day before fixation.Cell fixation and staining of microtubules was performed referring to a protocol from A. Desai (2012): Fluorescence procedures for the actin and tubulin cytoskeleton in fixed cells.http://mitchison.med.harvard.edu/protocols/general/Fluorescence%20Procedures%20for%20the%20Actin%20and%20Tubulin%20Cytoskeleton%20in%20Fixed%20Cells.pdfCells were washed shortly with PBS and extracted for 1 min with a buffer containing 80 mM PIPES, 1 mM MgCl 2 , 5 mM EGTA and 0.5% (v/v) Triton X-100 (pH adjusted to 6.8 with 6 M KOH).Glutaraldehyde was added from a 25% (v/v) stock solution (Grade I, Sigma) to final concentration of 0.5% (v/v) and incubated for 10 min.The solution was removed and cells were quenched with 0.1% (w/v) NaBH 4 in PBS for 7 min, followed by thorough washing with PBS.The cells were treated with a blocking buffer consisting of 2% (w/v) bovine serum albumin and 0.1% (v/v) Triton X-100 in PBS for 10 min, followed by incubation for 40 min with the primary mouse anti-β-tubulin antibody (Invitrogen) diluted to 1 µg/mL in the same buffer.After washing several times with a solution of PBS containing 0.1% (v/v) Triton X-100, the self-labeled secondary goat anti-mouse F(ab') 2 fragment diluted to 1 µg/mL in blocking buffer was added for 30 min, followed by another washing step with 0.1% (v/v) Triton X-100 in PBS and pure PBS.After post-fixation with 3.7% (v/v) formaldehyde in PBS, cells were rinsed with PBS and stored in PBS supplemented with 0.1% (w/v) NaN 3 at 4 °C.

Super-resolution imaging
One-photon activation and super-resolution imaging were performed on a custom-built widefield microscope setup suitable for single-molecule detection.A detailed description of the setup was published in F. Fricke et al., Histochem.Cell.Biol., 2014, 142, 91.Briefly, a 405 nm diode laser (CUBE 405-100C, Coherent) and a 568 nm laser (Saphire 568 LP, Coherent) were coupled into an IX-71 inverted microscope (Olympus) equipped with a 100x oil-immersion objective lens (PLAPO 100x TIRFM, NA ≥ 1.45, Olympus).An additional mirror was inserted in the excitation path for illumination of the sample in total internal reflection mode.The respective laser intensities were adjusted via an acousto-optic tunable filter (AA Optics).The sample was placed on a nosepiece stage (IX2-NPS, Olympus) to reduce thermal drift.Excitation and emission beam were separated with a quad-band dichroic mirror (HC Quad 410/504/582/669, AHF Analysentechnik) and a bandpass filter (ET 605/52, AHF Analysentechnik) in the detection channel.The fluorescence signal was collected on a 512 x 512 pixel chip of an electron multiplying charge-coupled device camera (iXon3, Andor).The image pixel size was adjusted to 160 nm using appropriate lenses.
One-photon activation was performed with a short 405 nm laser pulse (100 W/cm 2 ), while the sample was continuously illuminated with 568 nm (80 W/cm 2 ).500 frames were recorded with a frame rate of 33 Hz and an EM gain of 100.
Procedure C: Per mg starting material 100 µl CH 2 Cl 2 , 80 µl trifluoroacetic acid and 20 µl H 2 O were added.The reaction was stirred at room temperature for 16 h.The solvents were evaporated and the residue coevaporated with 200 µl toluene three times.with 715 mg of the new linker molecule, 940 mg (95%) of product were isolated.

Fig. S1 :
Fig. S1: Calibration curve for ferrioxalate actinometry.The absorption at 509 nm is recorded in dependence of an increasing concentration of Fe(II) ions in a solution with phenanthroline.

Fig. S2 :
Fig. S2:The absorption of ferrioxalate actinometry at 509 nm with 184 mW showed linear behavior with increasing irradiation time.

::
Increase of the absorption in dependence of the   concentration of Fe(II), slope from calibration curve (Fig. S1, Factor to correct the dilution of the sample (4 µL diluted to  50 µL; 0.08).: Measured volume (13 µL).

:
Amount of photons emitted by the LED at 365 nm.

:
Time of the irradiation (5 s).A solution of 492 μL 45 μM peptide 1 in HBS buffer pH 7.5 was mixed with 23 μL of internal standard.13 μL of this solution were irradiated for 0, 2, 5, 8, 10, 12, 15, 20, 25, 50 and 60 minutes using the same light source as in the previous experiments.After irradiation the samples were analyzed by HPLC (method 1, internal standard at 3.94 min, 1 at 27.52 min).The decrease of the peptide was measured by integration of the corresponding peak in the HPLC chromatogram.The values were referenced to the internal standard.The experiments were repeated thrice.The initial slope of the photoreaction was determined by exponential fit of the obtained data.

Fig. S3 :
Fig. S3: Photolysis of peptide 1.The initial slope of the decay was calculated by an exponential fit.

𝐸 365 =:𝐴
365 *  *  = -log   = 1 - = 1 -10 - 365 = 0.158 : Absorbance at 365 nm. 365 Coefficient of the photolabile group at 365 nm (The quantum yield of the new linker is then calculated from the amount of photons absorbed by the sample and the calculated slope of the photoreaction. The index represents the respective sample and the  index the standard. : fluorescence quantum yield Φ  : absorption factor  : absorbance at the excitation wavelength (  )   : integral emitted photon flux  : background-subtracted and corrected   fluorescence spectrum : emission wavelength   : refractive index of the solvent

Fig S4 :
Fig S4: Fluorescence increase during two-photon irradiation with 780 nm.Saturation indicates that all molecules in the area of the hydrogel were activated.
For super-resolution imaging, the UV laser was switched off and the intensity of the 568 nm laser line was increased continuously to a value of 230 W/cm 2 during the measurement.Image sequences of 10000 frames were recorded in PBS buffer with a frame rate of 33 Hz and an EM gain of 200.For localization of single emitters and reconstruction of the super-resolution image, the data was processed with the open-source software rapidSTORM V2.21, described in detail in S. Wolter, A. Löschberger, T. Holm, S. Aufmkolk, M.-C.Dabauvalle, S. van de Linde and M. Sauer, Nat.Meth., 2012, 9, 1040.

Fig S5 :
Fig S5:Widefield images of the microtubule network of an U2OS cell stained via immunofluorescence with the photoactivatable peptide 2 antibody conjugate: a) before activation with 405 nm, b) after activation with a short 405 nm puls.Images were averaged out of 50 frames without further image processing.Note that imaging in the microscope at a wavelength of 568 nm does lead to some photoactivation of the fluorophore ATTO565.

Fig S6 :
Fig S6: Widefield images of an U2OS cell stained via immunofluorescence against β-tubulin using the fluorogenic peptide 2 antibody conjugate.The sample was continuously illuminated with 568 nm.Images are selected from an image sequence at different time points: a) before activation, showing background fluorescence because of the non-zero quantum yield of the caged fluorophore; b) -d) consecutive activation and bleaching of single molecules, showing bright fluorescent spots (highlighted with orange arrows) that are successfully detected with rapidSTORM; e) blinking of residual ATTO565 conjugates.No further image processing was applied.