4. Results
4.1.2 Kinetics and Biological Investigations
In order to determine how fast the three fucose derivatives react in the DAinv reaction, kinetic investigations were performed. The assay was established previously in our lab by Andrea Niederwieser[94] and is based on the characteristic absorption maximum of tetrazines at around 520 nm (Figure 11). As neither products nor other DAinv reactants have considerable absorption at this wavelength, the absorption at 522 nm can be measured and correlated to the tetrazine concentration using the Lambert-Beer law. Monitoring the reaction over time enables the calculation of second order rate constants. As solvent, acetic acid with a pH of 4.8 was used. The mild acidic conditions guarantee tetrazine stability and thus avoid tetrazine decomposition which also results in a decreasing absorbance at 522 nm.
4. Results 27
Figure 11: Principle of kinetic investigations. (A) Tetrazine 22 reacts with terminal alkene 9, 14 or 21 yielding different isomers and tautomers of DAinv products. (B) Decreasing UV/Vis absorbance of tetrazine 22 upon
reaction.
In orienting experiments the second-order rate constants (k2) of four alkenoles (allyl alcohol, butenol, pentenol, and hexenol) were determined. Therefore equimolar amounts of alkenoles and tetrazine 22 were mixed in a cuvette and the decreasing tetrazine absorbance at 522 nm was observed by UV/Vis spectroscopy (Figure 11B). Curve fitting delivered the rate constants which are depicted in Table 2. As expected, the DAinv reactivity increased with growing chain length which corresponds to a higher HOMO energy of the dienophile. For allyl alcohol that has a small distance between the electron withdrawing oxygen and the double bond the reactivity is very low (k2 = 0.002 M-1s-1). By prolonging the chain length to butenol this reactivity is increased by a factor of 5.5. When elongating the chain further a decreasing impact on reactivity was observed: k2
increased by factors of 3.1 and 2.4 when going to pentenol and hexenol.
Table 2: DAinv second order rate constants k2 of alkenoles, fucose derivatives and mannosamine derivatives with tetrazine 22. even less reactive than allyl alcohol. This difference might be due to a steric effect as the alkene is close to the ring and the axial hydroxyl group might sterically hinder the reaction. When comparing the longer chain lengths there is only a small difference between the alkenole and the corresponding fucose derivative. Fuc6Vin (14) with a rate constant of k2 = 0.009 M-1s-1 reacts in
Absorption
Wavelength [nm]
DAinv reaction
28 4. Results
the same range as butenol (k2 = 0.011 M-1s-1) and both, k2 of pentenol and Fuc6All (21) increase by a factor of about 3. In Figure 12 the decreasing tetrazine concentration over time of the three fucose derivatives is shown and reactivity differences can be seen.
Figure 12: Decreasing tetrazine 22 concentrations over time for reaction with fucose derivatives 9, 14 and 21.
During the compound design we hypothesized the dependence of reactivity and incorporation efficiency where we claimed adverse effects for the two parameters. Having the second order rate constants of the alkenols and fucose derivatives we can conclude, that the DAinv reactivity indeed increases with a growing chain length. To get more insights into this hypothesis, especially regarding the incorporation efficiency, we teamed up in our group and worked together on a project with mannosamine derivatives. Even though many experiments were performed by others I will depict the results for a complete overview. To get started, Anne-Katrin Späte and Sophie Schöllkopf synthesized a series of alkene mannosamine derivatives 23-26 differing in their chain lengths (Figure 13).[105] Measuring second order rate constants of free sugars again showed an increasing reactivity with a growing chain length (Table 2).
Figure 13: Mannosamine derivatives 23-26 with different chain lengths.
To study the suitability of the mannosamine derivatives 23-26 for MGE regarding cell surface sialic acids, microscopy experiments were performed by Anne-Katrin Späte.[105] HEK293T cells were cultured in the presence of sugars 23-26, respectively for 48 h. Incorporated, alkene modified sialic acids were reacted with Tz-biotin (27) (1 mM, 6 hours, 37°C) and labeled with streptavidin-AlexaFluor®(AF)647. Confocal fluorescence microscopy showed cell membrane staining for all four mannosamine derivatives 23-26, while no staining in the negative control was
4. Results 29
detected (Figure 14A). Only little membrane staining was observed for Ac4ManNAloc (23), which can be explained by its slow reactivity in the DAinv reaction. Interestingly, the staining intensity for the other derivatives increased from the long chain Ac4ManNHeoc (26) to the shorter Ac4ManNBeoc (24) in contrast to the reactivity. This indicates better incorporation efficiency for the short chain length derivative. With Ac4ManNBeoc (24) we found a mannosamine derivative with the perfect balance between reactivity and incorporation efficiency.[105]
Figure 14: (A) MGE for visualizing cell surface sialic acids. HEK293T cells were grown with DMSO or with 100 µM Ac4ManNAloc (23), Ac4ManNBeoc (24), Ac4ManNPeoc (25), and Ac4ManNHeoc (26), respectively,
for 48 h. Cells were labeled with Tz-biotin (27) (1 mM, 6 h, 37°C) followed by streptavidin-AF647 (20 min, 37°C). Nuclei were stained with Hoechst33342. Scale bar: 30 µm. (B) Structure of tetrazine-biotin (27). (C)
DMB-labeling reaction.
To finally verify incorporation efficiencies, Jeremias Dold applied a technique to label and quantify incorporated sialic acids, called DMB-labeling, in his master’s thesis.[153] To this end he cleaved off sialic acids using 3 M acetic acid (80°C, 90 min) and labeled them with 1,2-diamino-4,5-methylendioxybenzene (DMB (28), Figure 14C). DMB (28) is selective for α-keto acids and fluorescent upon reaction. Thus fluorescent readout upon RP-HPLC was performed. The retention times of incorporated unnatural sialic acid derivatives were compared with previously synthesized chemical standards. Using this method Jeremias Dold determined incorporation ratios, compared to natural sialic acids, of 50%, 13% and 6% for Ac4ManNAloc (23), Ac4ManNBeoc (24), and Ac4ManNPeoc (25), respectively. For Ac4ManNHeoc (26) no incorporation could be detected.[153] These results again showed better incorporation for short residues and thus perfectly fit to our microscopy data and support our hypothesis that incorporation efficiency decreases with a growing chain length while reactivity increases.
30 4. Results
Knowing that with reactivity and incorporation efficiency there are adverse effects for successful application in MGE we expect good incorporation for Ac4Fuc6CH2 (1), which however is not possible to react in a DAinv reaction. For good reacting fucose derivatives with longer chain lengths we needed to test if they are incorporated. Thus, Ac4Fuc6Vin (2) and Ac4Fuc6All (3) are promising sugars for the use in MGE whereas Ac4Fuc6CH2 (1) is not suited for biological applications in combination with the DAinv reaction.
Before MGE experiments with fucose derivatives were performed, cytotoxicity was tested using an AlamarBlue assay.[154] This assay is based on the conversion of blue resazurin to red resorufin by living cells. Comparing the absorbance of treated to untreated cells shows cell viability. Thus, HEK293T cells were grown for two days with different concentrations of unnatural carbohydrates before incubating them with resazurin for 90 minutes. Applying sugar concentrations of 0.03 –
survivability. Higher DMSO concentrations rapidly decrease the number of cells (Figure 15B). The literature-known fucose derivatives Ac4Fuc6Alk (5) and Ac4Fuc6Az (4) were also tested. The alkyne derivative 5 is in the same range as the alkene derivatives 1-3 and the azido fucose 4 is more toxic with an IC50 value of 200 µM (Figure 15B). This is in agreement with the literature where Ac4Fuc6Az (4) was reported to be toxic.[137, 138]
Figure 15: AlamarBlue assay. Dose-response curves of HEK293T cells incubated with increasing sugar concentrations. (A) Two independent measurements, each with four replicates were performed. (B) One or
two independent measurements, each with four replicates were performed.
Finally MGE experiments were performed to see if the fucose derivatives 2 and 3 are suited for cell surface fucose labeling. HEK293T or HeLa S3 cells were incubated with 200 or 500 µM Ac4Fuc6Vin (2) or Ac4Fuc6All (3) for 24 or 48 hours followed by labeling with 1 mM Tz-biotin 27
4. Results 31
for 6 hours at 37°C, which are the same conditions as previously used for terminal alkenes[94]. No reproducible cell membrane staining could be detected for all conditions. In addition, soluble fucosylated proteins were investigated using Western blot analysis. For these experiments HeLa S3 cells were treated with 200 or 500 µM Ac4Fuc6All (3). The cells were lysed and the lysate was reacted with Tz-biotin 27 (concentrations of 150 – 250 µM) for 3 – 4 hours. The samples were blotted and in the resulting Western blot, no differences between the fucose treated and the DMSO treated cells could be detected. Thus, neither by microscopy nor by Western blot analysis incorporation of sugars 2 and 3 could be detected. This can have two reasons: Either the fucose derivatives are not accepted by the biosynthetic machinery and thus not metabolized or the incorporated saccharide does not react sufficiently with the tetrazine. If the reason is insufficient enzyme acceptance, the synthesis of GDP-fucose derivatives or the use of CHO Lec13 cells might improve incorporation. Both approaches are depicted in more detail later. If the incorporation cannot be detected because of insufficient reactivity, faster reacting reporter groups can be applied. Especially, as fucose is less frequent on the cell membrane than sialic acids and probably shielded by other glycans, a faster reaction might help to reach the detection limit.
Terminal alkenes react slowly in the DAinv reaction compared to strained alkenes.[100] Thus the cyclopropene as the smallest strained alkene is a possible, fast reacting reporter.