Chemical Analysis of the Photo-Click Reaction Using its Fluorogenic

To gain insights into the optical parameters of the reaction, preparative scale photo-click reactions of the tetrazoles with acrylamide, which was known to be highly reactive^[163], were performed. To this end, tetrazoles 41 and 42, respectively, were dissolved in ethanol, mixed with 1.5 equivalents acrylamide and irradiated at 302 nm with a hand held UV-lamp for 120 minutes (Scheme 14). The reaction was monitored via TLC and a fluorescent spot, visible with 366 nm UV-light, appeared upon reaction. The product crystallized in ethanol and could be filtered off. The filtrate showed remaining fluorescent product in TLC which was thus purified by flash column chromatography.

The combined products yielded 65% pyrazoline 47 or 69% 48.

48 4. Results

Scheme 14: (A) Synthesis of pyrazoline 47. (B) Structure of pyrazoline 48 which was synthesized in the same way.

In order to know the optical properties for the reaction and the detection of the compounds, UV-spectra were measured from all tetrazole- and pyrazoline-derivatives (41, 42, 45 – 48) using 10 µM solutions in DMSO. The results show, that tetrazole 41 has an absorption maximum at 280 nm and tetrazole 42 at 290 nm (Figure 26A). The spectra of the tetrazole-biotin derivatives are similar to the corresponding tetrazole spectra and all tetrazole-derivatives show absorbance at 302 nm which is thus a good wavelength for irradiation. The pyrazolines 47 and 48 have the absorbance maximum around 360 nm which is therefore the excitation wavelength of choice for a fluorescent readout. During his bachelor’s thesis, Raphael Fahrner synthesized pyrazolines with the terminal alkene but-3-en-1-yl isopropylcarbamate.^[193] The spectra of these compounds are similar to the ones measured of 47 and 48. Therefore it is likely that most pyrazolines deriving from these tetrazoles can be excited at 360 nm.

For fluorescence spectra 100 µM solutions in DMSO were used. The compounds were irradiated at 360 nm and emission was measured from 400 to 600 nm. As depicted in Figure 26B the tetrazoles show no signal (identical blue and green line) while the pyrazolines have nice fluorescence. Pyrazoline 47 has its maximum at 445 nm and its fluorescence intensity is roughly doubled of the one of 48. This observation is in accordance to the literature were an increased quantum yield is reported.^[182] The fluorescence maximum of pyrazoline 48 is at 485 nm.

4. Results 49

Figure 26: (A) Absorption-spectra of tetrazole and pyrazoline derivatives. All compounds were measured in solutions of 10 µM in DMSO with solvent correction. (B) Fluorescence spectra of tetrazoles and pyrazolines

(100 µM, DMSO). Samples were excited at 360 nm.

Knowing these fluorogenic properties we could screen different reaction parameters in a 96-well format using a plate reader. For this assay a tetrazole and an alkene were dissolved in ethanol and mixed (Figure 27). For reaction, the plate was irradiated at 302 nm with a hand-held UV lamp which was placed directly on top of the 96-well plate. Using this setup different tetrazole concentrations (0 – 1 mM) were measured with acrylamide as alkene. 100 µM tetrazole were found to be sufficient but signal increased with a higher concentration. Thus 400 µM or 1 mM was used for most experiments. When varying the acrylamide concentration, the signal also increased with a higher concentration (Figure 28A, 0 – 400 µM). Increasing the concentration from 50 to 100 µM raised the fluorescent signal with a factor of 1.8. When further doubling the concentration fluorescence increased 1.4-times and with 400 µM only little signal increase (1.1 times) was observed, compared to 200 µM. A possible explanation could be that the tetrazole was also used in 400 µM. Thus, there is a 1:1 ratio of tetrazole and acrylamide. If side reactions occur there might be not enough nitrile imine which can react with the acrylamide. Therefore, in most experiments an excess of tetrazole was used.

50 4. Results

Figure 27: Principle of the plate reader assay.

Another parameter that was varied is the irradiation time. The same mixture of 1 mM tetrazole and 200 µM acrylamide was irradiated for 2, 5, 10 and 30 min, respectively (Figure 28B). For reaction with tetrazole 41 the fluorescence intensity increased from 2 to 10 minutes; reaction with tetrazole 42 showed the best signal after 5 min irradiation. Longer irradiation times decreased the signal which is likely to be due to oxidation of the pyrazolines. These corresponding pyrazoles are not fluorescent anymore and could be detected via UV-detecion using LC-MS analysis. Therefore irradiation times from 5 to 10 minutes are sufficient and best suited for fluorescent readout.

Figure 28: (A) Increasing acrylamide concentrations. 400 µM tetrazole 42 and different concentrations of acrylamide in ethanol were irradiated for 5 min with 302 nm. Fluorescence was detected at 485 nm upon excitation at 360 nm. Two replicates were performed. (B) Varying irradiation times. Mixtures of 1 mM tetrazole 41 (left) or 42 (right) and 200 µM acrylamide in ethanol were irradiated for 2 – 30 min with 302 nm.

Fluorescence was detected at 445 nm (41) or 485 nm (42) upon excitation at 360 nm. Black bars: no alkene;

grey bars: with acrylamide. Two replicates were performed.

Besides varying different concentrations and irradiation times, different solvents were investigated. Reaction in acetonitrile or DMSO was similar to ethanol and water with 25%

acetonitrile could also be used as solvent when using the tetrazole-biotin derivatives. These aqueous conditions were used to see if there are differences in reactivity when using phosphate buffered saline (PBS), which is rich in NaCl, instead of water. This was of interest as we need PBS for cell experiments but it was reported that chloride ions influence the reaction as they can also react with the nitrile imine.^[169] Thus tetrazole-biotin derivatives 45 or 46 were mixed with

4. Results 51

acrylamide in water or PBS with 25% acetonitrile and irradiated for 5 minutes (302 nm). As shown in Figure 29 no difference between water and PBS was observed. This is relevant for the application in biology and in accordance with other publications where PBS was used as solvent.[162, 165, 182]

Figure 29: Impact of chloride ions on the reaction. Mixtures of tetrazole-biotin derivative 45 or 46 (1 mM) and acrylamide (100 µM) were irradiated (5 min, 302 nm). Compounds were dissolved in water or PBS, each with 25% acetonitrile. Fluorescence was detected at 445 nm (tetrazole 45) or 485 nm (tetrazole 46) upon

excitation at 360 nm. Two replicates were performed.