Kinetics of BNPP hydrolysis

To avoid the aforementioned negative effects on peptide folding and DNA binding, the BNPP hydrolysis studies were performed at the most suitable pH of 7.8. On the one hand, this value is most frequently used for zinc-finger DNA binding studies found in the literature and, on the other hand, it allows the comparison of the obtained production data with already reported findings of similar phenolate-based building blocks. Prior to the experiments, the building blocks (2.5 mM) and ZnCl2 (5.1 mM for 13 and 15, 2.6 mM for 17 and 18) were dissolved in a buffer solution (600 L, 25 mM HEPES, I = 0.1) and the pH was adjusted to 7.74. All mixtures were incubated at 37 °C over night. Subsequently, the mixtures were transferred into a stirrable cuvette and 600 L of a solution of BNPP (2.5 mM) in acetonitrile was added. Upon the addition of BNPP, the hydrolysis reaction was initiated and its progress was monitored by means of UV-vis spectroscopy in a time-dependent manner. The

concentrations of the obtained cleavage products were calculated using LAMBERT-BEER law (Equation 2.1) with the molar extinction coefficient of the p-nitrophenolate (412 nm = 14.340 M^-1 cm^-1) species.^[75] The possibility of non-hydrolytic BNPP decay under the applied conditions was examined beforehand by measuring a sample without the active metal complexes. It was found that over the contemplated time, the auto decay of BNPP was negligible. This made a correction of the recorded spectra redundant.

(A = absorbance, = molar extinction coefficient, c = concentration and l = path length)

The data obtained from the spectroscopic measurements were processed in Equation 2.1 and the calculated concentrations were mathematically treated under the assumption of a pseudo-first-order rate law. This was appropriate because the hydrolytically active metal complex was used in excess and can therefore be regarded as constant over the contemplated reaction time what leads to Equation 2.2.

−𝑑𝑐(𝐵𝑁𝑃𝑃)

𝑑𝑡 = 𝑑𝑐(ℎ𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑖𝑠 𝑝𝑟𝑜𝑑𝑢𝑐𝑡)

𝑑𝑡 = 𝑘[𝑐𝑜𝑚𝑝𝑙𝑒𝑥][𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒] = 𝑘_𝑜𝑏𝑠[𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒] (2.2)

The equation can be further facilitated under the assumption that only values for initial rates smaller than 6% were used (initial slope method). This proceeding was also appropriate with respect to the formation of exactly one p-nitrophenol species and one p-nitrophenylphosphate species per hydrolyzed BNPP molecule. The latter can subsequently release a second p-nitrophenol moiety, which would negatively affect the concentration determination to provide false rate constants. By only using the initial values, the second release is assumed to be rather low and can therefore be neglected. Consequently, the substrate concentration can be seen as constant (Equation 2.3).

𝑘_𝑜𝑏𝑠=𝑑𝑐(ℎ𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑖𝑠 𝑝𝑟𝑜𝑑𝑢𝑐𝑡)

[𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒]₀ 𝑑𝑡 (2.3)

The observed rate constants kobs for each of the examined complexes were obtained by plotting the natural logarithm of the hydrolysis product concentration against the time. The slope of the thus obtained straight line equals the corresponding kobs (Equation 2.4).

ln(ℎ𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑖𝑠 𝑝𝑟𝑜𝑑𝑢𝑐𝑡) = ln[𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒]₀− 𝑘_𝑜𝑏𝑠∙ 𝑡 (2.4)

The determined kobs values for the examined building blocks are summarized in Table 2.1.

𝐴 = 𝜀 ∙ 𝑐 ∙ 𝑙 (2.1)

Building Block kobs [s^-1] (pH 7.74)

13 1.05 x 10^-5

17 1.84 x 10^-6

15 1.18 x 10^-5

18 2.21 x 10^-6

Table 2.1 Determined kobs values for the BNPP cleavage reaction of the mononuclear (17 and 18) and dinuclear (13 and 15) building blocks.

It becomes apparent that the overall hydrolysis abilities of the mononuclear complexes were lower compared to their dinuclear analogues. In fact, under the applied conditions the determined kobs values were approximately 5.5-fold decreased when compared to the dinuclear complexes. Considering that the aforementioned studies already revealed poor hydrolysis capacities for mononuclear complexes due to the absence of a cooperative effect with a second metal ion, this was not surprising. Thus, substrate activation and stabilization of the transition state were much less pronounced making an effective hydrolysis difficult.

However, the dinuclear building blocks 13 and 15 showed an increased BNPP cleavage ability under the applied conditions with comparable kobs values for the both structurally related compounds (Table 2.1). The experiments clearly demonstrated, that the building blocks fulfill the two major criteria for phosphodiester hydrolysis. Firstly, they were able to activate the BNPP substrate upon binding of the dinuclear metal complex to the phosphate group. Secondly, the hydrolytically active hydroxide species was provided by the metal site that finally triggered the cleavage and released the p-nitrophenol species. This was further demonstrated by studying the turnover numbers (TON) for the dinuclear complexes (50 nM) with BNPP (25 M) under similar conditions as mentioned above (Figure 2.4). A plot of the time against the absorbance at 412 nm revealed almost comparable TONs for both building blocks. This indicates a similar behavior in terms of substrate cleavage and product release over the contemplated reaction time.

It is to note, that the here presented values were produced with an activated DNA model-substrate and the applied pH was limited to 7.74. For a more profound analysis of the individual building blocks, the experiments also need to include a pH and concentration dependent characterization for the determination of a second-order rate constant for the BNPP hydrolysis. This was neglected with respect to the restrictions during the work with peptide incorporated building blocks and natural DNA, which narrow the range of applicability. Thus, the results with natural DNA might differ due to various other factors that come into play. These include, for instance, the accessibility of the phosphodiester backbone of the DNA by the zinc-finger-incorporated building block when bound to the latter. Whereas the individual building blocks and the BNPP substrate can freely move in solution guaranteeing enhanced substrate binding and subsequent activation, the DNA-bound zinc finger is much more restricted in its movement. Moreover, the substrate release, as demonstrated by the TONs, is limited due to the fixation of the zinc finger to exactly one dsDNA moiety. Consequently, the obtained findings provide a good initial suggestion for a possible cleavage of natural DNA by the building blocks. However, the actual hydrolysis ability towards natural DNA is discussed in detail in section 2.7.

(a) (b)

(c)

Figure 2.4 UV-vis spectra of the BNPP hydrolysis with 13 (a) and 15 (b) taken at different time points after complex addition. (c) Plot of the time against the p-nitrophenolate band (412 nm) for 15 (black squares) and 13 (red dots).

2.4 Incorporation of the dinuclear building blocks into the sequence of Zf3