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

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 A. Basso, P. Braiuca, C. Ebert, L. Gardossi, P. Linda J. Chem. Technol. Biotechnol. 2006, 81, 1626-40 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 PEGA 1900 (Copolymer Acrylamide/PEG) used in Enzymatic Solid Phase synthesis of peptides and resolution of racemates.

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

6. Soluble Polymeric Supports Advantages 1- Easy monitoring of the support functional groups by common analytical techniques (e.g. 1 H 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 D. E. Bergbreiter Chem. Rev. 2002, 102, 3345-3384 POLYSTYRENES - Soluble in non polar organic solvents - Insoluble in MeOH - Good loading capacity POLYETHYLENE GLYCOLS - Soluble in water and most organic solvents - Insoluble in diethyl ether - Low loading capacity

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

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

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

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

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

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

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

15. Enzymatic Hydrolysis of (R,S)-Methyl mandelate Concept A R = OMe, OEt, OPr,n, Opr,iso, OBut,n, NH 2, NHPr,n, NHPr,iso S. Rocchietti et al. Enzyme Microb. Technol. 2002, 31, 88-93 From E. Coli on activated agarose gel

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

17. Efficient Polymer Functionalization: 1 H NMR and IR Primary OH Ester carbonyl IR: KBr, diffuse reflectance, polymer powder A B C D A+C B D A B+C A B C 1 H NMR: CDCl 3, solution

18. Efficient Control of Polydispersity RAFT reagent Reversible Addition-Fragmentation Chain Transfer (RAFT) Polymerization Functionalization “as usual” + Achieved control of Polydispersity:<1.2 Achieved control of Degree of polymerization (50 to 500) C. Barner-Kowollik, S. Perrier, J. Polym. Sci. A 2008, 46, 5715-5723

19. Copolymer/Substrate Solubility Tests SolventRatio (%)Solubility MeCN100+ MeCN/H 2 O50/50-/+ DMF100+++ DMF/H 2 O70/30++ DME100+++ DME/H 2 O70/30++ DMSO100+ DMSO/H 2 O50/50-/+ SolventRatio (%)Solubility MeCN100- DMF100++ DMA100+++ DMA/H 2 080/20++ DMA/H 2 020/80+ DMSO100-/+ THF100+++ THF/H 2 060/40+ 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 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% CH 3 CN T = 25°C 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 R S Acids Esters

23. Preliminary Data Results Immobilized PGA = 100UImmobilized PGA = 200U Same Hydrolysis Conditions in Aqueous Buffer 80 / DMA 20 (R,S)-Mandelate - Copolymer Hydrolysis Rate (  mol/min) 0.04 Conversion (30h) 41% ee% 18% E 1.61 (R,S)-Methyl mandelate Free Hydrolysis Rate (  mol/min) 0.73 Conversion (5h) 43% ee% 21% E 1.77

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 - Difficult to precipitate - Low yield - Impurities 2 -Copolymerization n m