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Catalytic cleavage agents for phosphate esters hydrolysis
thesisposted on 16.01.2017, 22:57 by Tjioe, Linda
This thesis describes the synthesis of series of novel copper(II) complexes of bis(2-pyridylmethyl)amine (DPA) and 1,4,7-triazacyclononane (tacn) ligands with appended guanidinium pendants, and the study of their hydrolytic reactivity with two model phosphodiesters, bis(p-nitrophenyl)phosphate (BNPP), 2-hydroxypropyl-p-nitrophenyl phosphate (HPNPP), as well as supercoiled pBR 322 plasmid DNA. A comparative kinetic study indicated that the introduction of guanidinium groups, capable of acting as hydrogen-bond donors, significantly increases the hydrolytic activity of a number of metal complexes. The ligands were prepared by multi-step syntheses and isolated as their hydrochloride salts. The DPA derivatives bearing guanidinium pendants of varying lengths were made by reacting the corresponding N,N-bis(2-pyridylmethyl)alkane-α,ω-diamines with 1H-pyrazole-1-carboxamidine hydrochloride. Tacn-based ligand families, featuring alkyl/xylyl guanidinium pendants, were prepared following a common synthetic approach. This involved coupling of protected amino-bearing pendants to the free secondary amine group(s) of an appropriate N-protected tacn derivative, deprotection of the pendants, and subsequent conversion of the pendant amino groups to guanidinium groups via treatment with N,N′-Boc2-1H-pyrazole-1-carboxamidine. Global trifluoroacetic acid (TFA)-mediated Boc-deprotection yielded the final products. A family of alkyl-guanidine bridged bis(tacn) ligands was prepared following a synthetic strategy which required the formation of tacn carbamoyl thiourea derivatives and their EDC-mediated coupling to the corresponding Boc2tacn-alkylamines to give protected carbamoyl guanidines. Removal of the ethyl carbamate group under basic conditions, followed by Boc group removal using TFA, gave the target bis(tacn) guanidinium ligands. The perchlorate salts of copper(II)-DPA complexes were prepared by reacting the ligands with Cu(NO3)2•3H2O in aqueous solution, followed by cation exchange chromatography, using aqueous NaClO4 as the eluent. Single crystals of copper(II)-tacn complexes were isolated following addition of an equimolar amount of Cu2+ to the tacn-based ligands in aqueous solution, and adjustment of the pH to 9. X-ray crystallographic analyses of the copper(II)-DPA complexes revealed that the DPA moieties of the ligands coordinate to the distorted octahedral copper(II) centers in a meridional fashion, with a water/methanol occupying the fourth basal position and the perchlorate anions occupying the axial positions. In contrast, Cu(II)-tacn complexes with single alkyl/xylyl guanidinium pendants, exhibited distorted square pyramidal copper(II) geometries. Interestingly, for the complexes featuring ethylguanidinium and bis(ethylguanidinium) pendants, one secondary amine nitrogen from each guanidine pendant coordinated to the copper(II) center, forming very stable five-membered chelate ring(s). The copper(II)-tacn complex with a single ethylguanidinium pendant also incorporated a single µ-hydroxo bridge between two Cu(II)-ligand units. This bridge was found to facilitate strong antiferromagnetic coupling between the Cu(II) centers. Except for the Cu(II)-tacn complex with bis(ethylguanidinium) pendants, each copper(II) complex featured labile coordination sites, an important feature when designing biomimetic model compounds for the hydrolysis of phosphate esters. The copper(II) complexes were tested for their hydrolytic activity towards the well-established model phosphodiesters, BNPP and HPNPP. In some instances, the introduction of the positively charged guanidinium pendant resulted in enhancements in the rate of phosphate ester hydrolysis compared to the parent complex, [Cu(tacn)(OH2)2]2+. This appears to be due to a combination of: (i) the pendants reducing the extent of formation of inactive dihydroxo-bridged dimers, and (ii) the guanidinium groups enhancing substrate binding and/or stabilizing transition states involved in the cleavage process. The monoalkyl-linked guanidinium complexes were the most active of those investigated, with the most active being the copper(II) complex containing the butyl spacer (at pH 7.0, kobs values measured were 10.2 ( 0.9) x 10-6 s-1 for BNPP cleavage at 50 ºC, and 54.2 ( 2.0) x 10-6 s-1 for HPNPP cleavage at 37 ºC, corresponding to 6- and 10-times faster activity than [Cu(tacn)(OH2)2]2+). The mononuclear Cu(II) complex with two coordinated ethylguanidinium pendant arms exhibited no measurable cleavage activity towards the model substrates employed, indicating the requirement for labile ligand coordination sites. The Cu(II) complexes featuring xylyl-linked guanidinium pendant groups showed no significant enhancement in cleavage rates compared to [Cu(tacn)(OH2)2]2+, suggesting that the more rigid spacers within these complexes prohibited simultaneous interaction of the Cu(II) centers and guanidinium groups with the phosphodiester groups in BNPP and HPNPP. The binuclear copper(II) complexes with bridging guanidine groups were found to be less effective cleavage agents compared to their mononuclear counterparts. This finding is ascribed to intermolecular µ-hydroxo bridge formation between the two copper(II) centers inhibiting interactions with the model substrates employed. Assessment of plasmid pBR 322 DNA cleavage activities showed that all complexes exhibited significant nuclease activity, which was predominantly hydrolytic in nature. Kinetic analyses revealed the copper(II) complex featuring an ortho-xylyl guanidinium pendant as the most active cleavage agent (kobs = 2.7 x 10-4 s-1), representing a 3 x 107-fold rate enhancement over the uncatalyzed cleavage reaction. This remarkable cleavage activity is proposed to be due to the charged guanidinium pendant interacting with a neighboring phosphodiester group in the DNA backbone, rather than in concert with the Cu(II) center to activate the phosphodiester linkage undergoing cleavage.