1. logo here… Biomimetic Modelling of Catechol Oxidase Activity Pule Molokoane Department of Chemistry, University of the Free State 10 th Inkaba yeAfrica/!Khure Africa (AEON) Conference/Workshop Lord Milner Hotel, Matjiesfontein - Karoo 29 September – 3 October 2014

2. Overview  Introduction  Synthesis  Aim  Characterization  Model  Reaction kinetics  Conclusion  Acknowledgements 2

3. Introduction  Catechol oxidase;  Belongs type III protein family of enzymes  Exclusively catalyses the oxidation of catechols to quinones  This enzyme is a binuclear metalloprotein, internuclear distance of 2.9 Å (met form)  Internuclear distance of 4.4 Å (deoxy form)  Generally copper enzymes are important for their role of acting as oxygen carriers in oxidation reactions of aromatic substrates 3 A. Majumder, S. Goswami, S. R. Batten, M. S. El Fallah, J. Ribas, S. Mitra, Inorg. Chim. Acta. 359 (2006) 2375.

4. Introduction C. Belle, K. Selmeczi, S. Torelli, J. –L. Pierre, C. R. Chimie. 10 (2007) 271. 4

5. Synthetic approach  Ligand Synthesis  Synthesis of Bis(pyridinonato)copper(II) Complexes 5

6. Aim  Emulate biological activity with simple synthetic molecules  Decrypt the biological code of reactivity, determine optimum conditions for a model system  Models would grant the opportunity to examine biological reactivity at a small-molecule level of detail through systematic and comparative studies as opposed to macromolecular level of detail (enzymes)  Development of similar (enzymes) chemical architectures for efficient catalysis 6

7. Characterization ( 1 H-proton) 7

8. Characterisation (NMR-HMQC) 8

9. Characterisation (NMR-HMBC) 9

10. Characterisation (X-ray Diffraction)  2-Ethyl-3-hydroxy-1-isopropylpyrid-4(H)-one ( EP(naltol)H) Cell Parameters a = 11.741(5) Å b = 13.355(5) Å c = 37.552(5) Å α = 90° β = 90° γ = 90° Space GroupOrthorhombic Crystal systemPbca Z24 10  Molecules A, B and C in colours red, blue and green respectively  r.m.s values;  A and B = 0.645 Å  B and C = 0.147 Å  A and C = 1.167 Å

11. Hydrogen bonding 11 D-H…A D-H( Å )H…A ( Å )D…A ( Å ) D-H…A ( ˚ ) O2B-H2B…O1B0.882.342.7648110 O2B-H2B…O1C (i) 0.881.862.6506149 O2A-H2A…O1A0.872.342.7580110 O2A-H2A…O1A (ii) 0.871.872.6484147 O2C-H2C…O1B (iii) 0.861.802.5871150 O2C-H2C…O1C0.862.382.7820109

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13. Insights from Structural Data  In the solid state all structures are in the ketone tautomeric form  In all the cases where a clear packing order was observed, dimers were formed due to weak O—H…O hydrogen interaction (≈ 2.5 Å with bond angles 120-180 ˚) which primarily stabilized the structure.  Most bond angles and bond lengths were comparable to the parent structures  Carbonyl length were slightly increased in the newly synthesized structures  The increase in C=O bond length was proportional to the electron donating capacity of the primary amines 13

14. Characterisation (X-ray Diffraction)  Bis(1-ethyl-3-hydroxy-2-methyl-4-pyridinonato)copper(II) [Cu(ME(naltol)) 2 ] 14  Molecules A and B in red and blue respectively  r.m.s value 0.0743 Å Cell Parameters a = 11.741(5) Å b = 13.355(5) Å c = 37.552(5) Å α = 89.65° β = 104.98° γ = 89.71° Space GroupTriclinic Crystal systemP-1 Z2

15. Hydrogen bonding and Packing 15 D-H…A d(D-H) ( Å )d(H…A) ( Å )d(D…A) ( Å ) ⊾ (D-H…A )( ˚ ) C4A-H4A…O1B (i) 0.932.393.201(3)146 C11A-H11B…O1B (ii) 0.972.503.355(3)146 C11A-H11B…O2B (iii) 0.972.543.302(3)136 C11B-H11C…O2A0.972.443.250(3)140 C11B-H11C…O2A (iv) 0.972.423.234(3)142

16. Model 16  Synthesize system of ligands with various electronic and steric properties (serving to tune the oxygen binding process)  Formation of Cu(II) nano-particles  Aerobic Pseudo-first order conditions

17. Reaction kinetics  3,5-di-tert-butyl-1.2-benzoquinone formation at 400 nm ( t(initial) = 540 min, ∆t = 60 min, t(final) = 1080 min)  All model complexes showed increasing limiting kinetics  The study was carried out at 25 °C  The model substrate and nano-particle prepared with dry oxygen saturated methanol 17 (a)

18. Reaction kinetics  Bis(1-ethyl-3-hydroxy-2-methyl-4-pyridinonato)copper(II) 18 [3,5-di-tert- butylcatechol] (mol.dm -3 ) [Cu(ME(naltol)) 2 ] (mol.dm -3 ) k obs (s -1 ) k 1 (M -1.s -1 ) k -1 (s -1 ) K (M -1 ) 0.0101.88 x 10 -5 7.33(1) x 10 -6 3.4(6) x 10 -4 3.6(4) x 10 -6 94(18) 0.0081.89 x 10 -5 5.99(7) x 10 -6 0.0061.87 x 10 -5 5.66(5) x 10 -6 0.0041.88 x 10 -5 5.52(7) x 10 -6 0.0021.89 x 10 -5 4.16(7) x 10 -6

19. Reaction kinetics  Bis(3-hydroxy-2-methyl-1-isopropyl-4-pyridinonato)copper(II) 19 [3,5-di-tert-butylcatechol] (mol.dm -3 ) [Cu(MP(naltol)) 2 ] (mol.dm -3 ) [O 2 ] (mol.dm -3 ) k obs (s -1 ) 0.0101 1.89 x 10 -5 5.59 x 10 -3 1.1(1) x 10 -6 0.0100 1.88 x 10 -5 6.14 x 10 -3 3.76(5) x 10 -6 0.0102 1.87 x 10 -5 7.33 x 10 -3 5.42(4) x 10 -6 0.0104 1.86 x 10 -5 9.14 x 10 -3 6.21(5) x 10 -6 0.0103 1.88 x 10 -5 9.25 x 10 -3 7.03(1) x 10 -6

20. Reaction kinetics  Comparison of the three complexes 20 Complexk 1 (M -1.s -1 )Colour [Cu(MP(naltol)) 2 ]4.8(9) x 10 -4 red [Cu(EP(naltol)) 2 ]8.7(7) x 10 -4 Blue [Cu(ME(naltol)) 2 ]3.4(6) x 10 -4 green

21. Comparative study ComplexRate constant (s -1 ) x 10 -4 [{Cu(L1)} 2 (l-CH 3 COO) 2 ] (1)7.72 [(CuL2)(CH 3 COO)] (2)6.08 [{Cu(CH 3 COO)} 2 (l-L3) 2 ] (3)6.95 [{Cu(L4)} 2 (l-(CH 3 COO) 2 ] (4)13.53 [Cu 3 (L5)(CH 3 COO) 3 ]. 3H 2 O (5) 6.78 21 V. K. Bhardwaj, N. Aliaga-Alcalde, M. Corbella, G. Hundal, Inorg. Chim. Acta. 363 (2010) 97.

22. Conclusion  In the solid state all ligands were in the ketone tautomeric form  All the synthesized compounds exhibited weak O—H…O hydrogen interactions  Most bond lengths and bond angles were comparable to the parent structures (C=O slightly longer)  The increase in C=O bond length was proportional to the electron donating capacity of the primary amines  Steric bulk, electron donating capacity and the strength of the hydrogen interaction were the most influential parameters  Activity of the enzyme >> nano-particles 22

23. Acknowledgements  Prof A Roodt and G Steyl  UFS Inorganic Chemistry group  Funds  Inkaba ye Africa Foundation  NRF  Sasol  Thrip  UFS Research Fund 23