7. COMPONENTE DE INGENIARÍA AGRONÓMICA
7.8. Plan de manejo de nutrición mineral
2.4.1 Hydrolysis and Ligand Exchange
As seen in other examples, the aqueous behaviour of half-sandwich complexes is often relevant to their activity.59,65,66,172 Complexes containing a monodentate halide often exhibit ligand exchange in aqueous conditions to form an aqua species. The subsequent complex offers more lability at the monodentate coordination site, allowing DNA binding within the cell as a potential mechanism for activity.
Figure 2.4 1H-NMR spectra (D
2O : MeOD-d4 9:1, 298 K, 400 MHz) of complex Ru1PP4 (A) in D2O, (B)
0.1 M NaCl solution and (C) addition of AgNO3 and filtration of AgCl.
To test the properties of the pyridylphosphinate complexes under aqueous conditions, complex Ru1PP4 was dissolved in a D2O : CD3OD (9 : 1) mix and 1H-NMR was used to determine the extent of ligand exchange by monitoring the p-cymene protons (5.4-6.0 ppm). Equilibrium was reached between the chloride (Ru1PP4) and aqua (Ru1PP4.1) species in solution (Figure 2.4A) within 5 min and remained stable at room temperature over 24 h. To mimic extracellular chloride
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concentration, the complex was dissolved in 100 mM NaCl solution (Figure 2.4B) to provide chloride bound species (Ru1PP4) as the major adduct and confirmed the chemical shift of the
p-cymene protons of the chloride species in D2O. To remove the chloride ligand and force the formation of the aqua species (Ru1PP4.1), AgNO3 was dissolved in solution, followed by filtration of the AgCl salt through a 0.2 μm pore filter to provide only the aqua species in solution (Figure 2.4C). Having identified the respective chloride (Ru1PP4) and aqua (Ru1PP4.1) diastereotopic proton peaks, the equilibrium reached in D2O after 24 h (Figure 2.4A) displays approximately 62% of the aqua and 38% of the chloride species. These NMR experiments suggest the chloride complex would be the major species in the extracellular environment (chloride concentration of approximately 100 mM) while once inside the cell, the lower chloride concentration (approximately 20 mM) would encourage the formation of the aqua species. The analogous Ru-iodide complex also undergoes rapid hydrolysis in D2O however 60% of the iodide species remains intact, implying an increased stability of the Ru-I bond compared to Ru-Cl.
2.4.2 Variable pH Conditions
Alongside hydrolysis, other physiological factors such as pH can play a significant role in the activity of potential metal complexes as anticancer agents.173 While generally pH varies depending on cell location and function, rapid proliferation of malignant cells causes a reduction in oxygen supply to tumour areas which become more hypoxic (acidic).173 This microenvironment can directly affect the structure and hence overall efficacy of a drug candidate. The stability of the aqua species is desirable within the cellular environment, with the pH influencing deprotonation of the bound water molecule, forming the hydroxyl ligand (Figure 2.5). The hydroxyl ligand is much less labile than water and could prevent binding of the metal centre to nucleobase.173 Due to the fast rate of hydrolysis observed for the pyridylphosphinate complexes, the effect of pH on the aquated species was investigated. Calculation of pKa values of the water bound molecule in the pyridylphosphinate complexes was therefore of interest to determine the likelihood of DNA binding and how the pKa varies depending on the coordinated ligands and the metal centre.
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NMR spectroscopy is a common methodology used for calculating pKa. The chemical shifts of NMR-active nuclei (e.g. 1H and 13C) depend on the chemical environment. Alteration of pH can cause deprotonation or protonation of acidic or basic sites which can be monitored by an adjacent atom’s chemical shift.174 In this case, the deprotonation of the water bound molecule was monitored by peaks H6 -adjacent to the pyridine nitrogen, the phosphorus peak and the arene protons of the selected complexes. To reduce discrepancies, dioxane (1H-NMR) and triphenylphosphine (31P-NMR) were used as reference compounds. To a solution of pyridylphosphinate complex (D2O : MeOH-d6 9:1), AgNO3 (2.5 equivalents) was added to form the aqua adduct and the AgCl salt was removed by filtration. The selected peak shifts in the 1H and 31P- NMR spectra were measured at intervals in the pH range 2.0-14.0; adjusted using 0.1 M NaOD and DCl solutions. The initial calculation of the raw data was a correction of chemical shift observed (δpeak - δreference) and was plotted as a function of pH. Using an established method (equations noted in Appendix 2) the pKa was extracted from the inflection point of the resulting sigmoidal curve (Figure 2.6) for all complexes (Table 2.5) - calculated as an average of the three nuclei monitored.
Figure 2.6 Measurement of the pKa of aqua complex Ru1PP5.1 by monitoring the 31P-NMR spectrum
(D2O : MeOH-d6 9:1, 298 K, 162 MHz) as a function of pH.
The pKa values for all the aqua complexes tested were higher than physiological pH (7.4) indicating the complexes would be present in their aqua form and not the less reactive hydroxo species in this environment. By comparing the pKa values of the different aqua complexes (Table 2.5), some trends become apparent. The electron-donating group on the pyridyl ligand appears to promote a higher pKa of the water bound molecule. This is shown by the increase in values in the order
Ru1PP5.1<Ru1PP3.1<Ru1PP7.1 considering 4-Me, 3-Me and 4-OCD3 pyridyl substituents (R), respectively. The highest pKa of the Ru(II) centred complexes is that of Ru1PP7.1 with the 4-OCD3 pyridyl substituent. The significant increase in pKa could be attributed to the more electron donating nature of the 4-OCD3 group, which creates a more electron rich metal centre requiring a
43.0 44.0 45.0 46.0 47.0 48.0 2.0 4.0 6.0 8.0 10.0 δ (31 P ) / p p m pH
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higher pH to deprotonate the coordinated H2O, compared to the poorer electron donating alkyl substituents of complexes Ru1PP3.1 and Ru1PP5.1.
Table 2.5 pKa values for selected aqua complexes (D2O : MeOD-d6 9:1, 298 K). pKa* values were measured
by monitoring changes in 1H-NMR and 31P-NMR spectra and converted to pK
a using the equation pKa =
0.929pKa* + 0.42.175
Complex R R1 {M(R2)} pK
a
Ru1PP3.1 3-Me Ph {Ru(p-cymene)} 9.34 ± 0.04 Ru1PP4.1 3-Me Me {Ru(p-cymene)} 9.18 ± 0.19 Ru1PP5.1 4-Me Ph {Ru(p-cymene)} 7.76 ± 0.13 Ru1PP7.1 4-OCD3 Ph {Ru(p-cymene)} 10.08 ± 0.05
Ir1PP6.1 4-Me Me {Ir(Cp*)} 9.31 ± 0.07 RhPP6.1 4-Me Me {Rh(Cp*)} 10.95 ± 0.04
The phosphorus R1 substituent has little effect on the pK
a as shown in complexes Ru1PP3.1 and
Ru1PP4.1 although the electron withdrawing inductive effect of the phenyl group could have influenced the pKa, this effect is clearly negligible. The significance of the metal centre is illustrated when comparing the complexes Ir1PP6.1 and RhPP6.1. The water bound molecule of the rhodium(III) aqua complex is less acidic than the iridium(III) analogue, reflecting the increased metal-oxygen bond length of the heavier congener176; a trend also observed in analogous complexes of ruthenium(II) and osmium(II).177
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Figure 2.7 1H-NMR spectra showing the deprotonation of Ru1PP6 to form the hydroxyl species Ru1PP6.1
with irreversible formation of the [(p-cymene)2Ru2(OH)3]+ dimer above pH 11.0.
While the pKa values suggest relative stability of the aqua complexes at physiological pH, at strongly basic conditions the formation of a new species was observed. The 1H-NMR spectrum of the ruthenium complexes (Figure 2.7) showed an increased intensity of two doublets in the arene-bound proton region (5.0 – 6.0 ppm) indicating a loss of stereogenic centre within the new species. Subsequent reduction of the pyridylphosphinate complex peaks was accompanied by an increase in free ligand peaks showing dissociation of the pyridylphosphinate ligand. Upon lowering the pH of the sample, only partial regeneration of the original complexes was observed. The new species was consistently formed for each of the ruthenium complexes at elevated pH (>11.0) and was identified as the hydroxyl-bridged dimer (D1, Figure 2.7). The formation of the dimeric ruthenium species (D1) has been previously noted for other half-sandwich complexes under strongly basic conditions and is known to be non-cytotoxic.178,179