Metal-ion complexes of functionalised 1,10-Phenanthrolines as hydrolytic synzymes

J.G.J. Weijnen

Research output: Thesisinternal PhD, WU


<p>In this thesis metal-ion complexes of functionalised 1,10-phenanthroline derivatives have been studied as model systems for hydrolytic metallo-enzymes. Amphiphilic metallo- complexes incorporated into micelles or vesicles and water-soluble complexes in pure aqueous buffer solutions, have been found catalytically active in the hydrolysis of activated (chiral) carboxylic and phosphate esters. The effect of changing the ligand structure and the metal ion on the activity and enantioselectivity of the complexes has been investigated.<p>After a general introduction and a description of the aim and contents of the thesis in chapter 1, an overview is given in chapter 2 of the catalytic roles that metal ions perform in hydrolytic reactions and of the application of micelles and vesicles as biomimetic systems, with emphasis on functionalised metallo-aggregates.<p>Mixed micellar systems containing Zn <sup>II</SUP>and Cu <sup>II</SUP>complexes of lipophilic ligands with the 1,10-phenanthroline or pyridine group as chelating moiety and a pendant Nτ-alkylated imidazole group (ligands 1 and 3, chapter 3), are efficient catalysts in the hydrolysis of <em>p</em> -nitrophenyl picolinate (PNPP) and diphenyl <em>p</em> -nitrophenyl phosphate (DPPNPP). The lipophilic 1,10-phenanthroline ligands 1 and 2 have a higher affinity for metal ions than the pyridine ligand 3. In the presence of one equivalent of metal ions, almost all 1,10-phenanthroline ligand sites are occupied by MU, whereas for the pyridine ligand this amounts to only about 50%. Kinetic studies of the hydrolysis of PNPP strongly indicate that catalysis proceeds by preliminary formation of a reactive ternary complex composed of metal ion, ligand, and substrate. These synzymes operate via a metal-hydroxide-ion catalysed mechanism and exhibit turn-over behaviour while retaining their full catalytic activity.<p>The catalytic role of a hydroxymethyl group covalently bound to the 1,10-phenanthroline ligand in the vicinity of the reaction centre and the effect of incorporation of the ligand into micelles are discussed in chapter 4. The Zn <sup>II</SUP>complex of the lipophilic ligand bearing the hydroxymethyl group (ligand 1) is 25 times more active in the hydrolysis of PNPP than the lipophilic metallo-complex lacking this group (ligand 3). Under turn-over conditions, the hydroxymethyl-containing metallo-catalyst displays a kinetically biphasic behaviour, characteristic for an acylation-deacylation mechanism. The acylation of the hydroxymethyl group is 133 times faster than the deacylation step. Lipophilic 1,10-phenanthroline derivatives in mixed micelles are able to form only 1 : 1 complexes with bivalent metal ions, whereas the water-soluble ligand 2 can both form 1 : 1 and 2: 1 (ligand : M <sup>II</SUP>) complexes. Only 1 : 1 complexes appear to be catalytically active.<p>Metal-ion complexes of a lipophilic 1,10-phenanthroline ligand containing the <em></em> (S)-2-(hydroxymethyl)pyrrolidine function at the α-position (ligand 1, chapter 5) are highly active and enantioselective in the hydrolysis of <em>p</em> -nitrophenyl esters of N-protected phenylalanine. The direction and magnitude of enantioselective catalysis are remarkably dependent on the nature of the metal ion and the co-surfactant. The Co <sup>II</SUP>complex in Brij 35 micelles exhibits the highest degree of enantioselectivity: <em>k</em><sup>D</SUP><sub>a,obs</sub> / <em>k</em><sup>L</SUP><sub>a,obs</sub> = 15 .3 toward the substrate D(L)-C <sub>12</sub> -Phe-PNP. In mixed micellar systems composed of the Zn <sup>II</SUP>complex and Brij 35 as co- surfactant, hydrolysis of the D-enantiomer predominates over that of the L-enantiomer ( <em>k</em><sup>D</SUP><sub>a,obs</sub> / <em>k</em><sup>L</SUP><sub>a,obs</sub> = 2 .4), whereas with CTABr as the co-surfactant an <em></em> inversion of enantioselectivity is observed ( <em>k</em><sup>D</SUP><sub>a,obs</sub> / <em>k</em><sup>L</SUP><sub>a,obs</sub> = 0 .54). Enantioselective hydrolysis is an important effect of the hydrophobic interaction between substrate and catalyst, since water-soluble ligands containing a (S)-2-(hydroxymethyl)pyrrolidine group (ligands 5, 6, and 7) are less active and less stereoselective. Moreover, lipophilic 1,10-phenanthroline ligands with chiral ephedrine functions (ligands <em></em> 3 and 4) show a lower activity and stereoselectivity.<p>Metal-ion complexes of functionalised 1,10-phenanthroline ligands having two long alkyl chains and a nucleophilic hydroxymethyl (ligand 1, chapter 6), (S)-2-(hydroxymethyl)pyrrolidine (ligand 2), or ephedrine group (ligands 3 and 4) at the a-position, incorporated in C <sub>18</sub> C <sub>12</sub> vesicles, are catalytically active toward PNPP and show activity and enantioselectivity toward p-nitrophenyl esters of N-protected leucine as the substrate. In mixed metallo-vesicles, the amphiphilic ligand is anchored in the core of the bilayer membrane by the alkyl chains, whereas the chelated headgroup protrudes into the aqueous interface. The metallo-complexes appear to be active in both the exo- and endovesicular side of the bilayer and the fluidity of the vesicle membrane has no influence on <em></em> the enantioselectivity.<p>The catalytic activity of metal-ion complexes of 1,10-phenanthroline with two long alkyl chains at the 2 and 9 positions (C <sub>12</sub> Phen) in Brij 35 micelles toward various phosphate triesters, diesters, and monoesters is described in chapter 7. In the presence of Co <sup>II</SUP>and Zn <sup>II</SUP>complexes the rate of hydrolysis of DPPNPP is increased by factors of 600 and 240, respectively. The metallo-complexes exhibit turn-over behaviour without loss of activity. Saturation kinetics provide evidence for preliminary formation of ligand-M <sup>II</SUP>-phosphate ester complexes, which decay to products. Kinetic studies indicate that phosphate triesters containing a metal-ion binding site in the leaving group are hydrolysed by the same mechanism as DPPNPP.<p>In chapter 8 an outline is given of the synthesis and enzymatic resolution of mono <em>alanine a</em> mides containing pyridine (5a) or 1,10-phenanthroline (5b) side chains and of bis-alanine amides with 1,4-phenyl (10a) or 2,9-(1,10-phenanthroline) (10b) linker moieties. Resolution of the bis-alanine derivatives using aminopeptidase from <em>Pseudomonas putida</em> requires an excess amount of enzyme due to substrate and product inhibition. The large amount of biocatalyst which, is necessary for the reaction, strongly hampers the work-up procedure, preventing the isolation of satisfactory amounts of enantiomerically pure product. In the cases of the mono-alanine derivatives, enzymatic resolution is successful and treatment of the racemic amino acid amides with the aminopeptidase yields the L-amino acid and the unchanged D-amino acid amide, which can easily be separated.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • van der Plas, H.C., Promotor
  • Engbersen, J.F.J., Promotor, External person
Award date29 Nov 1993
Place of PublicationS.l.
Print ISBNs9789054851820
Publication statusPublished - 1993


  • metal ions
  • organomineral complexes
  • phenanthrene
  • hydrolases


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