Cualidades de los espacios por su percepción.

The first aim was to optimize the reaction time of the azido-protein with DIBODY. Therefore, ELP90-N3 and DIBODY were mixed in PBS/DMSO at room temperature, and samples were taken at different time points. The alkyne- containing ELP90 was subsequently labeled by the addition of a large excess of azidocoumarin (17, Figure 2). The labeling process is schematically shown in Scheme 6. In addition, all remaining DIBODY was quenched as well in this latter step. In the same experiment, the orthogonality of DIBODY was also investigated by treating non-modified ELP90 with DIBODY and azidocoumarin 17.

Figure 2. Dyes used for protein modification.

The results from these experiments are shown in Figure 3; panel A shows the fluorescence image, panel B shows the coomassie stain. Figure 3A shows that already after 5 minutes labeling is obtained. Prolonged reaction times seem to increase labeling only slightly. The lack of fluorescence in lane 7 of Figure 3A shows that labeling is selective for azide-functionalized protein.

Figure 3. SDS-PAGE gel analysis of ELP-90-N3 reacted with 1 for different amounts of time,

followed by N3-coumarin (17), visualized by fluorescence imaging (A) and coomassie stain (B).

Lane 1: 5 min; Lane 2: 10 min; Lane 3: 20 min; Lane 4: 30 min; Lane 5: 60 min; Lane 6: 120 min; Lane 7: ELP-90, 120 min.

However, when these experiments were repeated, the negative control experiment showed higher fluorescent labeling than obtained for ELP90-N3. To investigate whether this was caused by aspecificity of 1, ELP90 and azido- functionalized ELP90 were reacted with 1 or 13 followed by azidocoumarin 17. Figure 4 shows that labeling of ELP90-N3 with DIBODY or DIBAC2 resulted in fluorescently labeled ELP (Figure 4A, lanes 1 and 3 respectively). However, fluorescence for the negative control experiments (lanes 2 and 4) was in both cases higher than observed for ELP90-N3.

Figure 4. SDS-PAGE gel analysis of ELP90-N3 and ELP90 reacted with 1 or 13, followed by N3-

coumarin (17), visualized by fluorescence imaging (A) and coomassie staining (B). Lane 1: ELP90- N3 with 1; Lane 2: ELP90 with 1;Lane 3: ELP90-N3 with 13; Lane 4: ELP90 with 13.

It was already shown in different labeling studies that DIBAC reacts selectively with azide-functionalized proteins (see Chapters 4 and 7). Since the results as shown in Figure 4 were not in line with this observation, it was tested whether the aspecific signal originated from the azidocoumarin. To this end, fluorescein was equipped with an azide functionality, yielding azidofluorescein (18, Figure 2). ELP90 and ELP90-N3 were first treated with DIBODY for one hour, followed by labeling with azidofluorescein 18. Now, as can be observed in Figure 5A, ELP-N3 (lane 2) gave a clear fluorescent signal. Fortunately, ELP (lane 1) showed hardly any fluorescence, which was reproducible through several experiments. This indicated that azidocoumarin 17 caused problems with aspecific labeling, and not the DIBODY.

Figure 5. SDS-PAGE gel analysis of ELP90-N3 and ELP90 reacted with 1, followed by N3-

fluorescein, visualized by fluorescence imaging (A) and coomassie staining (B). Lane 1: ELP90; Lane 2: ELP90-N3.

Now that labeling of ELP-N3 using DIBODY was reproducible and selective, labeling of 147CalB-N3 was performed using the exact same procedure. Wildtype CalB (Figure 6A, lane 1) showed no fluorescent labeling. On the other hand, 147CalB-N3 was successfully fluorescently labeled (Figure 6A, lane 2). Surprisingly, not only labeled CalB was observed, but also multimer formation in fluorescence imaging (Figure 6A) and by coomassie stain (Figure 6B).

164 Dibenzoazacyclooctynes: Synthesis and Bioconjugation

Figure 6. SDS-PAGE gel analysis of CalB and CalB-AHA reacted with 1, followed by N3-

fluorescein (18), visualized by fluorescence imaging (A) and coomassie stain (B). Lane 1: CalB; Lane 2: CalB-AHA.

Multimer formation can be explained by reaction of 147CalB-N3 with a cyclooctyne-functionalized 147CalB-N3. However, Hosoya et al. showed that dimerization does not occur.20 Also, it was previously shown that dimer formation of 147CalB-N3 was troublesome.19 Therefore, it was considered that aggregation had occurred due to the high DMSO content in the reaction mixture (50%), as DMSO-mediated aggregation was already previously published.27

To investigate whether DMSO content and concentration would influence multimer formation and/or aggregation, labeling was performed at three different concentrations (1, 0.5 and 0.1 mg/mL) and at three different DMSO percentages (50, 25 and 10%). The results of this investigation are displayed in Figure 7. It shows that multimer formation increased upon increasing concentration independent of the DMSO content (compare for example lanes 2, 5, and 8 in Figure 7B). Also the DMSO content does influence dimer formation, which can be observed by comparing lanes 8 to 10 in Figure 7B. At 1 mg/mL and 50% DMSO content (lane 8), almost no 147CalB-N3 was observed, and only dimer and trimer were detected. When ligation was performed with either 25 or 10% DMSO at identical concentration, the main signal corresponded to fluorescein-labeled 147CalB-N3.

Figure 7. SDS-PAGE gel analysis of 147CalB-N3 labeled with 20 equiv of 1, followed by 50 equiv

N3-fluorescein at different concentrations and varying DMSO percentages, visualized by

fluorescence imaging (A) and coomassie staining (B). Lane 1: 147CalB-N3; Lane 2: 0.1 mg/mL,

50% DMSO; Lane 3: 0.1 mg/mL, 25% DMSO; Lane 4: 0.1 mg/mL, 10% DMSO; Lane 5: 0.5 mg/mL, 50% DMSO; Lane 6: 0.5 mg/mL, 25% DMSO; Lane 7: 0.5 mg/mL, 10% DMSO; Lane 8: 1 mg/mL, 50% DMSO; Lane 9: 1 mg/mL, 25% DMSO; Lane 10: 1 mg/mL, 10% DMSO.

Also, fluorescent labeling was influenced by concentration and DMSO content. As can be observed in Figure 7A, with 50% DMSO hardly any protein labeling was observed. A clear fluorescent signal was detected when 25 or 10% of DMSO was

used. The successful and quantitative labeling of 147CalB-N3 with azidofluorescein was confirmed by mass spectrometry (Figure 8), showing mono- and difunctionalized 147CalB-N3 (34996 and 35804 respectively) and 147CalB, containing methionine instead of azidohomoalanine (34213). The occurrence of 147CalB without azide can be explained by incorporation of methionine instead of azidohomoalanine during the production process, which is a common observation using this technique.28

Figure 8. Mass spectrometry analysis of fluorescein-labeled 147CalB-N3 showing the mass of

147CalB without azide (34213), 147CalB-N3 labeled with one fluorescein (34996), and 147CalB-N3

with two fluorescein groups (35804).

6.4 A hybrid catalyst via the dibenzocyclooctadiyne approach:

N

-catalysts

Now that labeling of 147CalB-N3 with DIBODY (1) was shown to be quantitative, the next step was the preparation of an azido-functionalized pincer complex. Amine-functionalized pincer ligand 7 was successfully converted to azide- functionalized pincer ligand 19 via a diazonium salt. However, complexation of 19

with Pd (forming 20) was unsuccessful.