Bioorthogonal Fluorescence Turn‐On Labeling Based on Bicyclononyne−Tetrazine Cycloaddition Reactions that Form Pyridazine Products

Abstract Fluorogenic bioorthogonal reactions enable visualization of biomolecules with excellent signal‐to‐noise ratio. A bicyclononyne−tetrazine ligation that produces fluorescent pyridazine products has been developed. In stark contrast to previous approaches, the formation of the dye is an inherent result of the chemical reaction and no additional fluorophores are needed in the reagents. The crucial structural elements that determine dye formation are electron‐donating groups present in the starting tetrazine unit. The newly formed pyridazine fluorophores show interesting photophysical properties the fluorescence intensity increase in the reaction can reach an excellent 900‐fold. Model imaging experiments demonstrate the application potential of this new fluorogenic bioorthogonal reaction.


General information
The chemicals were obtained from commercial suppliers and were used without further purification. Reactions with air-and moisture-sensitive reactants were performed in anhydrous solvents under nitrogen or argon atmosphere. Column chromatography was carried out on silica gel 60A (particle size: 40-60 μm) from Acros Organics. Mixtures of solvents are each stated as volume fractions. For flash column chromatography a CombiFlash ® Rf+ from Teledyne ISCO was used. Thin-layer chromatography was performed on aluminum sheets from Merck (silica gel 60 F254, 20 × 20 cm). Chromatograms were visualized by UV light (λ = 254 nm/ 366 nm) or by staining with KMnO 4 solution. 1 H-and 13 C-NMR spectra were measured on a Bruker Avance III™ HD 400 MHz NMR system equipped with Prodigy cryo-probe. Chemical shifts δ are quoted in ppm in relation to the chemical shift of the residual non-deuterated solvent peak (CDCl 3 : δ( 1 H) = 7.26, δ( 13 C) = 77.16; DMSO-d 6 : δ( 1 H) = 2.50, δ( 13 C) = 39.52). High-resolution mass spectra were recorded on an Agilent 5975C MSD Quadrupol, Q-Tof micro from Waters or LTQ Orbitrap XL from Thermo Fisher Scientific. HPLC-MS measurements were performed on an LCMS-2020 system from Shimadzu equipped with a Luna® C18(2) column (3µm, 100A, 100 × 4.6 mm). UV/VIS spectroscopy was performed on a Cary 60 UV/Vis spectrophotometer from Agilent Technologies. Data from kinetic experiments were processed using OriginPro 9.1 software. Fluorescence measurements were performed on a FluoroMax 4 spectrofluorometer (Jobin Yvon, Horiba) from Perkin Elmer equipped with a 450 W xenon lamp and a single cuvette reader using the dye quinine sulfate (solution in 0.5 M H 2 SO 4 ) as standard for determination of fluorescence quantum yields.

Synthetic procedures
The synthesis of tetrazines 1a [1] and 1b,c [2] has already been published in our earlier publications.

S5
To a solution of tetrazine 1a (30.0 mg, 0.0970 mmol) in CH 3 CN (3 mL) was added bicyclononyne (29.1 mg, 0.194 mmol). The reaction mixture was stirred for 18 h at room temperature until the starting materials disappeared (verified by TLC in DCM/EtOAc 1:1 or HPLC-MS, see Figure S1). The crude product was concentrated in vacuo and purified by flash column chromatography (0→40% EtOAc in DCM) to obtain an isomeric mixture of click product 2a (44 mg). Separation by preparative TLC (eluting with DCM/EtOAc 4:1 + 5% MeOH and elution of the major product from the silica with DCM/MeOH 4:1) provided the main isomer of 2a as yellow solid (27 mg, 64%).
As the occurrence of rotamers complicated the analysis of the cyclooctene moiety of 2a at room temperature, the NMR measurements were performed in DMSO-d 6 at 80 °C.

Photophysical properties of the click products of BCN with tetrazines 1a-1e
Conditions for the following absorption and emission measurements: A 1 mM solution of the respective tetrazine in DMSO was mixed with a 50 mM solution of BCN in the indicated solvent (CH 3 CN, CH 3 CN/H 2 O 1:1, CHCl 3 , acetone, dioxane, iPrOH or MeOH) and further diluted with the corresponding solvent to give a final tetrazine concentration of 500 µM using 10 equiv of BCN. The reaction mixtures were incubated at room temperature in the dark for 19 h and then measured by HPLC-MS ( Figure S2) to verify the formation of the corresponding click products.

S6
HPLC-MS measurements were performed on a Luna® C18(2) column (3µm, 100A, 100 x 4.6 mm) using a linear gradient of CH 3 CN + 0.05% HCOOH (5→95% in 9 min) in H 2 O + 0.05% HCOOH at a flow rate of 1.0 mL/min. All low-resolution masses found during these measurements are summarized in Table S1. These stock solutions were further diluted to a tetrazine concentration of 25 µM for absorption measurements and to a concentration of 2.5 µM for emission measurements.

Determination of second-order rate constants
Second-order rate constants of the reactions of bicyclononyne (BCN) with 1,2,4,5-tetrazines 1a and 1d were determined by following the decay in the concentration of the starting tetrazine over time.
The rate constants were calculated from the pseudo first order rate constants of the concentration decrease measured at different BCN concentrations (10 equiv, 12.5 equiv and 15 equiv) by UV/VIS spectroscopy. The measurements were performed in CH 3 CN/H 2 O 1:1 at room temperature using a final tetrazine concentration of 12.5 µM. All measurements were performed at least three times.

S19
Conditions: A 50 µM solution of the respective tetrazine in CH 3 CN/H 2 O 1:1 containing 5% of DMSO was mixed with a 500 µM solution of BCN in CH 3 CN/H 2 O 1:1. The mixture was further diluted with CH 3 CN/H 2 O 1:1 to give a final tetrazine concentration of 12.5 µM and was immediately measured on the UV/VIS spectrophotometer. The time-dependent measurements were performed at the corresponding absorption maxima of the tetrazines used, which were determined by UV/VIS spectroscopy before the measurement (Table S5). The measured intensity of the absorption was plotted against time. Fitting the curves with single exponential equation (y = y 0 + Ae -k/t ) provided the observed rate constants. Finally, the observed rate constants were plotted against the concentration of BCN in order to obtain the second order rate constants (Table S5) from the slope of the resulting plot.

Fluorogenic cell labeling
HeLa cells fixed with methanol were rehydrated in 0.05% Tween in PBS and incubated with 100 µM BCN-NHS for 1 hour. The cells were then washed three-times with 0.05% Tween in PBS, incubated with 20 µM tetrazines for 4 hours and the nucleus was stained using commercially available DRAQ5. The cells were again washed three-times with 0.05% Tween in PBS and inspected on confocal microscope.

Two-color fluorogenic labeling of segregated bilayer TG beads
The segregated bilayer Tentagel beads were prepared by following literature procedure. [7] Briefly: Tentagel NH 2 resin (25 mg, 130 µm beads) was swollen in ddH 2 O (2 mL) for 20 hours. After decantation the beads were briefly centrifuged, access water was pipetted off and the beads were briefly washed with DCM/Et 2 O = 55/45 mixture (2 mL We have also prepared Tentagel beads modified only with BCN or TCO moiety respectively by the reaction of TG NH 2 beads with the BCN/TCO active esters (1 equiv.) in DMF using DIPEA (2 equiv.) as the base. The BCN-NHS active ester is commercially available. TCO-NHS ester used in this experiment was prepared as previously described. [1] Structures of the NHS esters are shown below. Figure S19. Fluorogenic labeling of TG beads modified with BCN and TCO dienophiles. A) Fluorescent stereomicroscope images of TG beads modified with BCN-NHS ester after addition of tetrazine 1a. B) Fluorescent stereomicroscope images of TG beads modified with TCO-NHS ester after addition of tetrazine 1a. C) Fluorescent confocal microscope images of segregated TG beads modified with BCN in the outer layer and TCO in the inner part after reaction with tetrazine 1a. The images were captured by gray-scale camera and are in pseudocolors.
Toxicity studies 2x10 4 HeLa (or U2OS) cells were seeded on glass bottom 96 well plate (Cellvis) one day prior experiment. The cells were incubated with various concentrations of tetrazines 1e-1e for 24 hours. The tetrazines showed no toxicity up to 50 µM concentration. Compound 1d showed toxicity at 100 µM, the highest concentration tested. The toxicity was determined using XTT assay or by using crystal violet ( Figure S20). The experiments were performed in triplicate.
Briefly: Cells were cultivated for 24h with indicated concentrations of compounds. After 24h 50 µL of XTT(+PMS) was added to cell medium. Absorbance (difference 450-620 nm) was measured 1h after addition of XTT.
XTT (Thermo) was dissolved in DMEM (high glucose no serum) to give concentration 1mg/ml, PMS (phenmetrazine sulfate, Sigma) was dissolved in PBS to final concentration 0.383 mg/ml (1.25mM) solutions were combined in ratio 50:1 and added to cell culture medium. Total absorbane was calculated as absorbance measured at 450 nm subtracted by absorbance value measured at 620 nm.
Crystal violet: Cells grown on 96 well plate were fixed with 100µl of 100% methanol for 10 minutes, rinsed 2x with water and incubated with 50 µl of 0.1% crystal violet solution. After 15 minutes of incubation crystal violet was removed, cells were washed 2x with water. Crystal violet adsorbed on cells was dissolved in 50 µl of 100% methanol. Absorbance was measured at 595 nm using plate reader. Figure S20. Toxicity of the tetrazines determined by XTT or crystal violet on HeLa or U2OS cells. Viability was calculated as % of control (DMSO).

Time-lapse labeling of BCN-and TCO-modified TG beads
TG beads modified with BCN or TCO (prepared as described above) were reacted with tetrazines 1a, 1d and 1e as follows: To small portion of the beads (ca. 5 mg) was added CH 3 CN/H 2 O = 1/1 mixture (50 µL) and solution of the tetrazine in DMSO (10 µL of 1 mM stock). After 15 min, the reactions were inspected under UV-hand held lamp (365 nm) to confirm formation of the fluorophores. The beads were then washed twice with DMF, twice with CH 3 CN/PBS= 1/1 mixture and finally were incubated at 37°C in 75 µL CH 3 CN/PBS = 1/1 mixture for 24 hours. Small portion of the beads was pipetted off and the beads were inspected under fluorescence stereomicroscope at different time points (Leica M205 fluorescent stereomicroscope equipped with pE-300 white LED light source and DFC3000 G grayscale camera). Set-up on the microscope was as follows: UV: Ex. 350 nm, Em. 420 nm (long pass), intensity: 30%, gain: 8, exposure: 1.25 s. GFP: 480 nm, Em. 510 nm (long pass), intensity: 20%, gain: 8, exposure: 1.25 s. RFP: 546 nm, Em. 590 nm (long pass), intensity: 50%, gain: 8, exposure: 1.25 s. The results are shown in Figure S21 below.