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Dario Pasini

Styrene-Based Copolymers as Soluble Platforms for the Biocatalytic Transformation of Organic Substrates with Immobilized Enzymes. Dario Pasini. Dipartimento di Chimica Organica Università degli Studi di Pavia. APIB-2009 Pavia, 3 rd June 200 9. Overview.

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Dario Pasini

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  1. Styrene-Based Copolymers as Soluble Platforms for the Biocatalytic Transformation of Organic Substrates with Immobilized Enzymes Dario Pasini Dipartimento di Chimica Organica Università degli Studi di Pavia APIB-2009 Pavia, 3rd June 2009

  2. Overview 1) Biocatalysis, Solid Phase Synthesis and Soluble Polymers 2) Soluble Polymer-Achiral Substrate / Immobilized Enzyme 3) Enzymatic Hydrolysis of (R,S)-Mandelate Copolymer 4) Conclusions and Outlook

  3. General concepts involved in the use of supported organic targets and their biocatalytic transformations

  4. Crosslinked Polymers-Substrate / Free Enzyme Concept B • Tentagel and Argogel resins (polyethylene glycol chains grafted onto classical polystyrene/divinylbenzene cores) • High swelling characteristics in aqueous solvents • Low loading capacity • Limited success in combination with biocatalysis • PEGA1900(Copolymer Acrylamide/PEG) used in Enzymatic Solid Phase synthesis of peptides and resolution of racemates. A. Basso, P. Braiuca, C. Ebert, L. Gardossi, P. Linda J. Chem. Technol. Biotechnol.2006, 81, 1626-40

  5. = Merrifield resin or Wang resins Biocatalysis and Solid Phase Synthesis Concept C versus Concept D Biocatalitically-Triggered Safety-Catch Linker X = O, NH, NR Only when is a soluble linear polymer (PEG = polyethyleneglycol) high yields of the product could be achieved (Concept D) ImmobilizedPGA - + U. Grether, H. Waldmann Chem. Eur. J.2001, 7, 959-971

  6. Soluble Polymeric Supports Advantages 1- Easy monitoring of the support functional groups by common analytical techniques (e.g. 1H NMR) 2-Reactivity similar to the solution, homogeneous phase 3 – Facile product/reagent separation by precipitation of the polymer in a non -solvent

  7. Soluble Polymeric Supports POLYSTYRENES POLYETHYLENE GLYCOLS - Soluble in water and most organic solvents - Insoluble in diethyl ether - Low loading capacity - Soluble in non polar organic solvents - Insoluble in MeOH - Good loading capacity D. E. Bergbreiter Chem. Rev.2002, 102, 3345-3384

  8. Soluble PS Copolymer-Substrate / Immobilized Enzyme Concept D Immobilized Enzyme + i) Enzymatic hydrolysis (PGA) ii) Filtration of the Enzyme Soluble PS Copolymer -Substrate iii) Recovery of the Copolymer by precipitation iv) Isolation of substrate from the solution D. Pasini, M. Filippini, I. Pianetti, M. Pregnolato Adv. Synth. Catal.2007, 349, 971-978

  9. Monomer and Polymer Synthesis 83-89% 70-90% Introduction of phenylacetic ester monomers and copolymerization with styrene at several loadings y x 60-80%

  10. Characterization by 1H NMR Spectroscopy Hm Hd He Hi Ha Hb Hf,g Hc Excellent agreement between feed and observed ratios of monomers

  11. Gel permeation chromatography Solvent Polymer Average Molecular Mass Mn (number average) Mw (weight average) PDI Polydispersity Index Values between 1.5 and 2.4 Mw / Mn

  12. Properties as Supports High Comonomer Loading (60:40) Bad precipitation in MeOH: centrifugation needed Low Comonomer Loading (93:7) Excellent precipitation in MeOH Sample Molecular Weight Distribution Mn = 11080 ; Mw = 18950 ; PD = 1.7 Good precipitation in MeOH Medium Comonomer Loading (80:20) Sample Molecular Weight Distribution Mn = 9080 ; Mw = 17630 ; PD = 1.9

  13. Copolymer Substrate Hydrolysis by PGA Immobilized on Eupergit (brown) Immobilized on Agarose (yellow) Hydrolysis Conditions -Temperature: 37°C -Mechanical stirring -Mixed solvent system (aqueous buffer 80/ DMF 20) Quantitative release First order kinetics D. Pasini, M. Filippini, I. Pianetti, M. Pregnolato, Adv. Synth. Catal., 2007, 349, 971– 978.

  14. (R)* (S,R)* (S,R)* Soluble Copolymer Soluble Copolymer Enantiomeric Resolution Strategy Immobilized enzyme Soluble Copolymer i)Enantioselective Enzymatic Cleavage ii) Immobilized Enzyme Recovery iii) Optically-Active Substrate and Soluble Copolymer Recovery Chemical Refunctionalization (S)* (R)* i) Chemical Cleavage ii) Soluble Copolymer Recovery (R)* Possible application to enantioselective resolution of racemic carboxylic acids?

  15. Enzymatic Hydrolysis of (R,S)-Methyl mandelate Concept A From E. Coli on activated agarose gel R = OMe, OEt, OPr,n, Opr,iso, OBut,n, NH2, NHPr,n, NHPr,iso S. Rocchietti et al. Enzyme Microb. Technol.2002, 31, 88-93

  16. Alternative Synthesis of Copolymer/Substrate 1 - Copolymerization n m 2 - Functionalization Good yields Good purity

  17. Efficient Polymer Functionalization: 1H NMR and IR A B+C B A C Primary OH 1H NMR: CDCl3, solution IR: KBr, diffuse reflectance, polymer powder D B C A A+C B D Ester carbonyl

  18. Efficient Control of Polydispersity RAFT reagent + Functionalization “as usual” Reversible Addition-Fragmentation Chain Transfer (RAFT) Polymerization Achieved control of Polydispersity:<1.2 Achieved control of Degree of polymerization (50 to 500) C. Barner-Kowollik, S. Perrier, J. Polym. Sci. A2008, 46, 5715-5723

  19. Copolymer/Substrate Solubility Tests Phenylacetate Copolymer (R,S)-Mandelate Copolymer DMF / Water Best Solvent DMA / Water Best Solvent

  20. Stability of Immobilized PGA in DMA/Water

  21. Enzymatic Hydrolysis of (R,S)-Mandelate Copolymer Hydrolysis Conditions -Temperature: 25°C -Mechanical stirring -Mixed solvent system (aqueous buffer 80/ DMA 20)

  22. Analytical Control Enantioselectivity monitoring HPLC: Merck Hitachi LaChrom L-7000 Column: REGIS (S,S) Whelko-O1; 4,6 x 250mm  = 220 nm Flow: 2 ml/min Method: 90% Hexane10 mM-10%Ammonium acetate 100 mM in Ethanol T = 25°C Conversion monitoring HPLC: Merck Hitachi LaChrom L-7000 Column: AGILENT ZORBAX C18; 4,6 x 250mm  = 220 nm Flow: 1 ml/min Method (Gradient elution): A: 98% phosphate buffer 10 mM pH 3,2 B: 2% CH3CN T = 25°C R S Acids Esters

  23. Preliminary Data Results Immobilized PGA = 100U Immobilized PGA = 200U Same Hydrolysis Conditions in Aqueous Buffer 80 / DMA 20

  24. Conclusions and Perspectives 1 – The use of Polystyrene Soluble Polymers as Tags for Substrates in combination with Immobilized Enzymes is feasible 2- In a biocatalytic reaction on a racemate, Enantioselectivity seems to be retained (more experiments needed to confirm preliminary data) 3- Work-up, recovery and refunctionalization of the Soluble Polymer need to be optimized

  25. Acknowledgments Dep. Organic Chemistry Prof. Dario Pasini Dr. Carmine Coluccini Dr. Claudio Cornaggia Michele Petenzi Dep. Pharmaceutical Chemistry Prof. Massimo Pregnolato Prof. Daniela Ubiali Dr. Teodora Bavaro Dr. Davide A. Cecchini Dr. Chiara Savarino Visit: www.unipv.it/labt

  26. Classical Synthesis of Copolymer/Substrate 1 –Functionalization of monomer m n 2 -Copolymerization - Difficult to precipitate - Low yield - Impurities

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