Cleavage of Paraoxon

4 6 8 10 12 14 16

18 4

L¹ Zn²⁺

control

v0[10-10 M s-1 ]

pH

Figure 2.22: Initial rates in 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP) transesterification/hydrolysis of 4, L¹, Zn²⁺ and a control.

2.4.4. Cleavage of Paraoxon

Organophosphate triesters are highly toxic compounds, which have been used as insecticides and warfare agents. The degradation of these compounds is necessary to decrease their toxicity. Paraoxon is a phosphate triester that has been used as insecticide.

This substrate can be defanged by the cleavage of the P−O bond to form diethyl phosphate and p-nitrophenolate (Scheme 2.45).

The activity of 2, 4, 5a and 12 to cleave Paraoxon was investigated applying ³¹P NMR spectroscopy. The experiments were performed under different conditions as summarised in Table 2.24.

P O O

O O

NO₂

O

NO2

P O O

O

O

Paraoxon diethyl phosphate p-nitrophenolate

Scheme 2.45: Degradation reaction of Paraoxon resulting in diethyl phosphate and p-nitrophenolate.

Table 2.24: Overview of complexes and conditions during Paraoxon cleavage.

Complex pH independent pH dependent Equivalents Paraoxon

2 1.0, 2.0

4 − 1.0

5a 1.0

12 − 1.0

Hydrolytic experiments of 2, 5a and 12 were performed in d3-MeCN/D2O (9:1, v/v).

Equimolar amounts of each complex and Paraoxon were combined in a NMR tube and measured initially. The reaction was followed in time intervals of one hour. A signal at 7.29 ppm was observed in all spectra, which did not change in chemical shift for more than 31 hours. As a control, pure substrate was analysed under the same conditions revealing a signal at 7.29 ppm. These results ended up in the conclusion that the substrate was not hydrolysed under these conditions. A reason might be the acetate co-ligands in 5a and 12, which are tightly bound to the zinc atoms and do not allow coordination of the substrate. 2 does not bear any co-ligand, but also lacks a nucleophile that is required to mediate substrate cleavage.

In addition to pH independent experiments, compounds 2, 4 and 5a were studied towards hydrolytic activity within a pH range of 5 to 11 (buffers are listed in Table 2.23). NMR measurements were carried out in buffered H2O/d6-DMSO (8:2, v/v) to maintain a stable pH and good solubility of the complexes. Spectra were recorded initially after mixing equimolar amounts of complex and Paraoxon and followed at hourly intervals for eight hours, later in 12 hour intervals for three to five days. Between the measurements, the NMR tubes were stored at 40 °C (water bath) to promote the catalytic reaction. The signal

of pure Paraoxon was obtained at 6.85 ppm and did not change within the mentioned pH range. However, after three days of incubation, an additional signal appeared at 0.34 ppm at pH 10 and 11 probably due to background hydrolysis caused by the strongly basic conditions.

The experiments with 2 and 4 have been monitored for five days. It was surprising that addition of 2 and 4 did not influence the chemical shift at 6.85 ppm, which was the only signal obtained in the spectra. After three days, the signal at 0.34 ppm was also observed at high pH values in both experiments, which resulted from the background hydrolysis.

The formed diethyl phosphate did probably not coordinate to the complexes. If so, a chemical shift from 0.34 ppm would be reasonably expected. Using an excessive amount of substrate (ratio 1:2) did not reveal other signals than 6.85 ppm and 0.34 ppm.

A possible reason might be the coordination of the sulfonic acid buffers to the complexes that can inhibit the binding of the phosphate group. In addition, 5a did also not show activity in the hydrolysis of Paraoxon at different pH. This might be due to the bridging acetate group that inhibits the reaction, as discussed above.

To investigate the influence of the used buffer system, an additional experiment was performed in H2O/d6-DMSO (8:2, v/v). Equimolar amounts of 2, KO^tBu and Paraoxon were mixed and the spectra were recorded in regular intervals (12 hours). In addition to the resonance at 6.85 ppm (not hydrolysed substrate) a new signal was observed at 0.82 ppm after three days of incubation at 40 °C. This signal presumably resulted from the hydrolysed substrate (diethyl phosphate), which coordinates to the complex.

In addition to hydrolytic experiments, a suspension containing L¹, KO^tBu, Zn(SO₃CF₃)₂ and Paraoxon was stirred in the presence of acetonitrile (Scheme 2.46) until the solution became clear. ESI-MS analysis of the reaction solution showed fragments that matched the calculated masses of [Zn2H₋₁L¹(OP(O)(OEt)2)(SO3CF3)]⁺ at m/z = 931 and [Zn2H₋₁L¹(OP(O)(OEt)2)]²⁺ at m/z = 391. Signals in d3-MeCN were observed at 0.87 ppm (product) and 7.34 ppm (Paraoxon) in ³¹P NMR.

N space group P2₁/n with four molecules per unit cell. As shown in Figure 2.23, each zinc atom is found in trigonal bipyramidal environment (Zn1: τ₅ = 0.91 and Zn2: τ₅ = 0.88)^[53]

by four N-donor moieties of the ligand and one oxygen atom of the phosphate group. The zinc atoms and P1 are located within the pyrazolato plane. The oxygen atoms O1 and O2 are arranged above this plane (d(O1···pz) = 0.34(1) Å) and below (d(O2···pz) = 0.44(6) Å). The zinc atoms are separated by 4.1086(9) Å, which is comparable to 2 (d(Zn1···Zn2) = 4.1028(5) Å) and might explain the twisted arrangement of the phosphate. Selected bond lengths and angles for 18 are listed in Table 2.25.

Figure 2.23: Molecular structure of 18, hydrogen atoms and counter ions omitted for clarity.

Table 2.25: Selected bond lengths [Å] and angles [°] of 18.

Atoms Bond length Atoms Angle Atoms Angle Zn1-N1 2.021(5) N1-Zn1-N3 76.1(2) N2-Zn2-N8 76.5(2) Zn1-N3 2.378(5) N1-Zn1-N4 113.8(2) N2-Zn2-N9 118.9(2) Zn1-N4 2.041(6) N1-Zn1-N6 119.2(2) N2-Zn2-N11 112.5(2) Zn1-N6 2.026(6) N1-Zn1-O1 106.9(2) N2-Zn2-O2 106.9(2) Zn1-O1 1.991(5) N3-Zn1-N4 75.6(2) N8-Zn2-N9 75.8(2) Zn2-N2 2.005(5) N3-Zn1-N6 76.6(2) N8-Zn2-N11 75.9(2) Zn2-N8 2.429(5) N3-Zn1-O1 173.9(2) N8-Zn2-O2 171.7(2) Zn2-N9 2.023(6) N4-Zn1-N6 110.2(2) N9-Zn2-N11 111.8(2) Zn2-N11 2.031(6) N4-Zn1-O1 107.4(3) N9-Zn2-O2 96.0(2)

Zn2-O2 1.983(5) N6-Zn1-O1 97.4(2) N11-Zn2-O2 108.9(3) Zn1···Zn2 4.1086(9)

31P NMR analysis of 18 in d3-MeCN solution revealed one signal at 0.87 ppm. This confirmed the hypothesis that the signal at 0.82 ppm, which was observed in hydrolytic reactions without buffer, was the hydrolysed substrate (diethyl phosphate) that coordinated to the complex. In addition, it could be demonstrated that the used buffer inhibits the binding of the hydrolysed substrate to the complex as only the free (and not coordinated) diethyl phosphate and Paraoxon were present in the experiments in buffered solutions. Performing the hydrolytic experiments in other buffer systems, which do not interact with the complex, might increase the activity to cleave the P−O bond of Paraoxon.