monophosphate, CDI… N,N’-carbonyldiimidazole.
For modification in position 6, precursor 17 was substituted with propargylamine under reflux conditions yielding modified adenosine 77 after crystallisation. In parallel, commercially available adenosine (78) was brominated in position 8 to obtain 8-bromoadenosine (79), which was further substituted with propargylamine to give derivative 80 after column chromatography.[57] Both compounds were then phosphorylated[113-114] and the respective monophosphates 81 and 82 were obtained. Finally, coupling with pre-activated
β-Substrate Scope of PAR Synthesising Enzymes
nicotinamide monophosphate gave compounds 2 and 4 as previously.[115] Of note, repeated freeze-drying of monophosphates 81 and 82 cause them to be insoluble in DMF. Thus, they should either be directly used after one round of lyophilisation or dissolved and evaporated from methanol and triethylamine (1:1) prior to reaction in DMF.
6.2 Biochemical Evaluation of NAD
+Analogues 1-4
Having all four derivatives in hands, their substrate properties were investigated in ADP-ribosylation assays with histone H1.2 as acceptor (Figure 31) and in ARTD automodification (Figure 32).
The respective assay conditions for ARTD2, ARTD5 and ARTD6 were found to be similar.
Only reaction temperatures were decreased to 30 °C and reaction times had to be elongated to 1 h, 4 h and 2 h respectively. After the times indicated, copper-catalysed click reactions were performed with a fluorophore-containing azide. The fluorescence was detected and compared to the Coomassie Blue stained gel as before (Figure 13). Every analogue was as well tested in a 1:1 mixture with natural NAD+ and all gels contain control reactions with natural substrate. For a better comparison, ARTD1 ADP-ribosylation assays were also performed. Moreover, all four analogues have never been tested in parallel before.
Comparing Figure 31A and Figure 32A, ARTD1 behaves similar in auto- and trans(ADP-ribos)ylation. As known from previous work,[58, 152] the enzyme was not able to process 7- and 8-modified NADs 3 and 4 (Figure 31, lanes 9 to 14 and Figure 32, lanes 7 to 10). The 6-modified derivative 2 forms in both assays only little amounts of 6-modified PAR in the absence of natural substrate (Figure 31, lane 7 and Figure 32, lane 5), when compared in parallel with compound 1. This is surprising, because 6-alkyne-NAD+ is the most frequently applied compound in literature and it is already quite hampered in PAR formation. Nevertheless, strong signal is detected in a mixture containing NAD+ (Figure 31, lane 8 and Figure 32, lane 6). Application of compound 1 results in the strongest signal and is competitive towards natural substrate (Figure 31, lanes 4 to 5 and Figure 32, lanes 3 to 4). In summary, 2-modified analogue 1 is the most efficiently used substrate. This is in line with the results obtained in chapters 1 and 5.
As expected from the structural similarity between ARTD1 and ARTD2, the outcome of the ARTD2 ADP-ribosylation assays (Figure 31B and Figure 32B) is comparable. Again, NADs 3 and 4 are not accepted as substrates substrate (Figure 31, lanes 9 to 10 and Figure 32, lanes 7 to 10), whereas 1 and 2 are (Figure 31, lanes 3 to 8 and Figure 32, lanes 3 to 6). As before, compound 1 is better metabolised than 2.
Also in ARTD5 and ARTD6 ADP-ribosylation, analogues 3 and 4 were not used as substrates (Figure 31C and D, lanes 9 to 10 and Figure 32C and D, lanes 7 to 10). In contrast, compounds 1 and 2 were both used by both enzymes for PAR formation, even in the absence of natural NAD+. In case of ARTD5, derivative 1 seems to be slightly better incorporated than 2 in histone ADP-ribosylation, whereas in case of ARTD6 both are equally used as substrates.
Figure 31. SDS PAGE analyses of ADP-ribosylation of histone H1.2 with ARTD1 (A), ARTD2 (B), ARTD5 (C) and ARTD6 (D) using NAD+ analogues 1-4. Left panel shows Coomassie Blue staining; right panel shows Cy3 fluorescence. Total concentration of NADs was 1 mM. Controls were performed using either natural substrate (lane 1 and 2) or no enzyme (lane 1, 3, 6, 9 and 12).
Substrate Scope of PAR Synthesising Enzymes
Figure 32. SDS PAGE analyses of auto(ADP-ribos)ylation of ARTD1 (A), ARTD2 (B), ARTD5 (C) and ARTD6 (D) using NAD+ analogues 1-4. Left panel shows Coomassie Blue staining; right panel shows Cy3 fluorescence. Total concentration of NADs was 1 mM. Controls were performed using either natural substrate (lane 2) or loading the same amount of ARTD1 (lane 1).
6.3 Expanding towards Dye-Modified NAD
+Analogues 11 and 12
The alkyne-tag induces only small alterations to the NAD+ scaffold. As a next step, bulkier dye-modified NAD+ analogues were investigated in these assays. As all enzymes only accepted 2- and 6-modified analogues, compounds 11 and 12 were already available for this purpose. As shown in chapter 5, direct modification with fluorophores offers the opportunity to study enzyme activity in real-time, saving the additional labelling step.
For a better comparison, ARTD1 catalysed ADP-ribosylation was again performed in parallel. Results are depicted in Figure 33 and Figure 34.
As already described in chapter 5.3, ARTD1 incorporated analogue 11 in a competitive manner and compound 12 could be used for polymer elongation in mixture with natural substrate (Figure 33A and Figure 34A). ARTD2 (Figure 33B and Figure 34B) behaves again like ARTD1, but small fluorescent signal is already obtained in histone ADP-ribosylation but not in automodification using analogue 12 in the absence of natural NAD+ (Figure 33B, lane 7 vs Figure 34B, lane 5). This indicates that ARTD2 can better cope with modifications in position 2 than ARTD1.
The tankyrase ARTD5 showed decreased incorporation of the bigger analogues. During automodifcation, both compounds failed to form detectable, fluorescent PAR chains (Figure 34C). In general, it should be stated that ARTD5 showed less activity in automodification compared to the other ARTDs, making the judgement more difficult. In the histone assay, analogue 12 was slightly incorporated as seen by fluorescent heterogeneous polymer in the absence of natural substrate and increased polymer in the presence of NAD+ (Figure 33C, lanes 7 and 8). Although, compound 11 shows also fluorescent chains in the presence of NAD+, it cannot be concluded that 11 is used for polymer elongation, because of the weak signal compared to the strong unspecific signal resulting from histone background.
In case of ARTD6, both analogues were equally used for the ADP-ribosylation of histone (Figure 33D) and automodification (Figure 34D).
In conclusion, all enzymes except ARTD5 used analogue 11 for ADP-ribosylation, whereas compound 12 is differently accepted among the enzymes and in dependency on the PARylation substrate. Thus, modification of adenine position 2 can be applied for the investigation of DNA-dependent ARTDs and modification of adenine position 6 is suitable for the tankyrases. Moreover, 2-modified analogues can be furthermore used to distinguish ARTD5 from ARTD6 activity.
Substrate Scope of PAR Synthesising Enzymes
Figure 33. SDS PAGE analyses of ADP-ribosylation of histone H1.2 with ARTD1 (A), ARTD2 (B), ARTD5 (C) and ARTD6 (D) using NAD+ analogues 11 and 12. Left panel shows Coomassie Blue staining; right panel shows Cy3 fluorescence. Total concentration of NADs was 1 mM. Controls were performed using either natural substrate (lane 1 and 2) or no enzyme (lane 1, 3, 6, 9 and 12).
Figure 34. SDS PAGE analyses of auto(ADP-ribos)ylation of ARTD1 (A), ARTD2 (B), ARTD5 (C) and ARTD6 (D) using NAD+ analogues 11 and 12. Left panel shows Coomassie Blue staining; right panel shows Cy3 fluorescence. Total concentration of NADs was 1 mM. Controls were performed using either natural substrate (lane 2) or loading the same amount of ARTD1 (lane 1).
Summary and Discussion
7 Summary and Discussion
Within this PhD project, a range of twelve NAD+ analogues (1-12) was applied for the investigation of ADP-ribosylation processes in vitro and in cellula. Out of these, seven derivatives (6-12) were described for the first time to the best of the author’s knowledge.
These compounds have been synthesised and modified at different sites of the adenine base with varying types of reporter groups to find analogues, that enable the cellular detection of poly(ribose) with the help of fluorescence microscopy. By testing them in ADP-ribosylation assays, new insights into the substrate scopes of ARTDs were gained. The best-performing ones pave the way for the design of new experiments for the study of ADP-ribosylation processes and should thus contribute to the understanding of the highly dynamic PAR metabolism.
In the focus of this project were the most abundant enzyme ARTD1 and its involvement in DNA repair processes. A comprehensive understanding of its substrate scope was obtained.
Whereas already alkyne-tags in adenine position 7 and 8 disturb the enzyme’s ability to process these analogues (3 and 4), small perturbations such as the propargyl group in 6 (2) are tolerated to a certain extent. Bulkier substitutions such as present in derivatives 7, 8 and 12 cannot be processed by ARTD1 any longer. As these analogues are nevertheless processed in a mixture with natural NAD+, it is assumed that ARTD1 exhibits different constraints during the attachment of first ADP-ribose units to the acceptor protein and the elongation of already existing chains. Most effectively used are 2-modified NADs 1, 6 and 7, and even long and bulky modifications such as in 9, 10 and 11 are accepted. When applied in mixtures with natural NAD+, these compounds were found to be compatible and incorporated to a similar extent. As a conclusion, 2-modified NADs should be the first choice for studying ARTD1 catalysed ADP-ribosylation.
After testing these analogues, all were found to cause staining of involved proteins at least to a certain extent, which might be explained by either electrostatic attraction between the negatively charged NAD+ and positively charged proteins and/or to non-enzymatic addition of ADP-ribose at lysine residues[158] via Schiff base formation. Consequently, meaningful controls must be included in these kinds of experiments to ensure that the signals obtained result indeed from enzyme activity rather than from side product formation.
In chapter 4, the different possibilities to apply bioorthogonal chemistry for the conjugation of fluorescence dyes were explored. The NAD+ scaffold was modified with bioorthogonal reporter groups such as alkyne, azide, alkene, cyclooctyne or cyclopropene and to label modified PAR chains with CuAAC, SPAAC or DAinv chemistry. After successful optimisation of the labelling conditions in vitro, the applicability for the best performing ones was expanded towards cellular systems. For the first time, it was shown that these reporter-tagged analogues are metabolised in cells upon DNA damage by ARTD1 and PAR was readily detected with the help of fluorescence microscopy. The bioorthogonal design of NAD+ analogues 9 and 10 enabled furthermore the simultaneous and parallel detection of PAR, which offers the opportunity of multi-channel read-outs as desired for instance in time-dependent studies or pulse-chase-experiments.
In the course of this project and also reported by others,[92] it was found to be challenging to achieve ‘real’ bioorthogonality for applications inside of cells. Thus, careful examination of labelling conditions and appropriate controls are essential to distinguish signal from background.
In chapter 5, NAD+ was directly modified with a fluorophore to enable optical real-time imaging of ADP-ribosylation. Despite its bulky modification, the NAD+ analogue 11 proved to be a competitive and efficiently used substrate of ARTD1 in vitro as well as in cellula. This probe allowed to directly monitor the turnover of PAR on the level of the polymer as both, the formation and degradation of PAR, was imaged upon photoinduced DNA damage in a living cell. In combination with FLIM-FRET microscopy, compound 11 provides a powerful tool for detecting protein-specific PARylation in cells as shown by proof-of-principle with the covalent ARTD1 automodification or the non-covalent binding of macroH2A to PAR after DNA damage. This approach thus provides the basis for future experiments, where both covalent and non-covalent interactions with PAR can be studied for selected proteins. This could be extremely useful for the identification and characterisation of different PAR synthesising and degrading enzymes as well as of hitherto unknown protein targets, which has not been possible so far in the context of intact cells.[169]
Despite achieving the central aim of this project, the method to deliver NAD+ into the cell still needs optimisation for a more convenient applicability and cellular studies lasting longer than one hour. When planning further FLIM measurements, one must consider that the site of the eGFP label at the protein of interest is very crucial for successful FRET and thus can be the limiting factor for further applications.
Chapter 6 deals with the question, whether the NAD+ analogues can be applied for the study of other PARylating enzymes such as ARTD2, ARTD5 or ARTD6. It was found that NAD+ analogues modified with alkyne groups in adenine position 2 and 6 are used by all these enzymes to a certain extent. DNA-dependent ARTDs can incorporate 2-modified analogues best as also sterically demanding compounds are incorporated. On the other hand, the tankyrases prefer 6-modified derivatives. Because ARTD5 and ARTD6 exhibit different constraints for metabolising bulky 2-modified analogues, this behaviour can be used to discriminate their activity.
Although the in vitro experiments look very promising, these findings need to be approved in more complex systems such as a cellular environment. Because ARTD-specific inhibitors are not-well established by now, the presented FLIM-FRET approach can help to clarify their substrate properties also in a protein-specific manner.
Outlook and Future Research Directions
8 Outlook and Future Research Directions
In summary, the NAD+ analogues described in here have proven their potential for deciphering important questions in PAR research by offering the opportunity to monitor the polymer in real-time and in its natural environment with the help of fluorescence microscopy.
Although all compounds synthesised in here were obtained in reasonable yields, the NAD+ synthesis route offers still some possibilities for optimisations. In case of compounds 9, 10 and 11, the introduction of a different linker could be beneficial to avoid the formation of an unreactive side product during TFA deprotection. For instance, diaminoalkyl spacers resulting in compound 83 would be a possible option as they are known to be stable under the chosen deprotection conditions. Moreover, the reporter groups in this project were mostly introduced on the level of adenosine or adenosine monophosphate. Using a suitable protection group and mild cleavage conditions, the functionalisation at the level of NAD+ would become possible and thus creating a more flexible synthesis route and easier access to derivatives (Scheme 17).