1. SEPARATION BY ENZYMATICALLY CATALYZED REACTIONS Chapter 10

2. r-1-Phenylethylacetate r,s-1- Phenylethanol Vinylacetate Catalyst: Novozym 435 (Lipase) s-1-Phenylethanol Carbon dioxide, supercritical Carbon dioxide + r-1- Phenylethylacetate s-1-Phenylethanol Carbon dioxide Reactive separation

3. Enzyme Catalysis Kinetics: Influence of Substrates and Enzymes Michealis-Menten-mechanism Reaction of 2 nd order with an equilibrium in front. Free enzyme and substrate are in equilibrium with the enzyme-substrate-complex. [E]Enzyme concentration, [S]Substrate concentration, [ES] Concentration of enzyme-substrate-complex, [P]Product concentration.

4. Enzymes in supercritical carbon dioxide The use of enzymes in non-aqueous media has several reasons: Hydrolysis is avoided, Solubility of organic molecules is better, resulting in higher yields, In supercritical carbon dioxide the removal of reactands is possible during the reaction.

5. Randolph investigated the phospholipase (EC 3.1.3.1) catalyzed hydrolysis of Di-Natrium-p-nitrophenylphosphate in supercritical CO 2. Enzymes in supercritical carbon dioxide: Example Hydrolysis of Di-Natrium-p-nitrophenylphosphate in supercritical CO 2 (p = 10 MPa, T = 308 K).

6. Influence of water on stability and activity For most enzymes: activity increases with increasing water content. part of the water is fixed to the protein by hydrogen bonding as a water shell. enzymes can be active without water in organic media. additional amount of water should be kept low. conformation of the enzyme seems not to depend on acidity.

7. Comparison of contact rate of several enzymes in aqueous solution and of alcohol dehydrogenase in scCO 2 Activity in water and in sc-CO 2

8. Hrnjez et al.: regio specific and stereo specific activity of the lipase- catalyzed esterification of chiral dioles with anhydrous butyric acid. Lipase-catalyzed esterification of chiral dioles with anhydrous butyric acid. (p = 3.5 - 20 MPa, T = 40 °C). Specific enzymes and substrates

9. Lipases Lipases split fats into fatty acids and glycerol Activation of lipases by the surface between hydrophobic and hydrophilic medium.

10. Enrichment of enantiomers by lipase catalysis Enantio selective synthesis catalyzed by enzymes is due to the faster reaction of one enantiomer. Scheme of a catalyzed enantio-selective transesterification

11. Enantioselectivity k cat /K M apparent equilibrium constant for the 2 nd order reaction at infinitesimal substrate concentration, k cat and K M represent the turnover number and the Michaelis-Menten constant. If the Michaelis-Menten-constant is equal for both substrates: Indices A and B stand for the enantiomers.

12. For irreversible reactions the enantioselectivity E can be derived from yield (U) and the enantiomeric excess (ee). UYield [%], ee Produkt Enantiomeric excess of products [%], ee Substrat Enantiomeric exc. of substrate [%], c Prodkukt Product concentration [mol / l], c Substrat Substrate concentration [mol / l], c R Concentration R-enantiomer [mol / l], c S Concentration S-enantiomer [mol / l], EEnantioselectivity. Enantioselectivity

13. Change of enantiomeric excess with product formation. Enantioselectivity E =

14. The highest enantiomeric excess is achieved (with high enantioselectivity) at 50 %. Enantioselectivity E

15. If the reaction proceeds, ester and alcohol react reversibly into the initial substrates (educts). k cat and k r are the rate constants of forward and backward reaction. Enantioselectivity

16. Variation of enantiomeric excess of the remaining substrate with increasing K (K = 0; 0,1; 0,5; 1; 5) for E = 100. Enantioselectivity

17. Variation of enantiomeric excess with increasing K (K = 0; 0,1; 0,5; 1; 5) for E = 100. With the formation of products, enantiomeric excess drops rapidly. Enantioselectivity

18. Separation by enymatically catalyzed reactions: Examples and Experiments

19. Enrichment by enzyme catalyzed interesterification in supercritical carbon dioxide Racemic mixtures of Ibuprofen a. epi-Methyljasmonate

20. Reaction scheme Lipase:Novozym 435 (strain of Candida antarctica), 1-2 % w/w H 2 O Solvent:Supercritical CO 2, Hexane Conditions:100 - 200 bar, 40 - 60 °C

21. High pressure cell P: up to 4000 bar T: 150 °C. 0.1 mmol ester, 0.4 mmol alcohol 15 mg immobilized lipase Test cell

22. Conversion of Racemic Ibuprofenmethylester With Alcohols in n-Hexane p = 100 bar, T = 50 °C; Catalyst: Lipase Novozym 435 Enantiomeric Excess: Ibuprofen product educt

23. Enantiomer separation by chiral Gas chromatography Competitive Process: Chromatography

24. Influence of various reaction partners

25. Interesterification of ibuprofen methylester with ethanol (T = 50 °C) Enantiomeric excess: Ibuprofen

26. Reaction rate independent of Enzyme water content Influence of water

27. Enrichment of (+)-epi-Methyljasmonate in SC-CO 2

28.  Below 10 % percent yield the enzyme catalyzed reaction is irreversible.  (R)-Ibuprofenmethylester is preferably converted.  The enantiomeric excess increases with decreasing pressure and decreasing temperature.  The enzyme specificity is enhanced by introducing polar functional groups into the acyl acceptor.  Optimum water content is 1-2 % w/w.  Addition of water to Novozym 435 does not accelerate the reaction rate.  (+)-epi-Methyljasmonate is preferably converted.  The enzyme specificity in hexane is similar compared to that in supercritical carbon dioxide. Some conclusions

29. Lipase-catalysed kinetic resolution of racemates at temperatures from 40°C to 160°C in supercritical CO 2 The enzyme: Novozym 435, EC 3.1.1.3 from Candida antarctica B, 7000 PLU/g (activity expressed in propyl laurate units base on a batch synthesis assay), water content 1-2% w/w, Example: Phenylethanol

30. Reaction Scheme

31. s-1-Phenylethylacetate r,s-1- Phenylethanol Vinylacetate Catalyst: Novozym 435 (Lipase) s-1-Phenylethanol Carbon dioxide, supercritical Carbon dioxide + r-1- Phenylethylacetate s-1-Phenylethanol Carbon dioxide Reactive separation

32. Structure of the enzyme Lipase Arctica candida

33. Conversion of (R,S)-1-phenylethanol at 95°C ( ) and 136°C (  ) for phenylethanol esterification at 15 MPa. Reaction time [h] Yield rel. To racemate [%] Separation of r,s-1-phenylethanol

34. 1-Phenylethanol - vinylacetate - CO 2 Novozym 435 Initial substrate concentration: 0,5 M Reference: 1g immob. Enzyme, 100 o C Effect of temperature on reaction rate

35. Comparison of reaction rate

36. Pressure dependence of the enzyme activity at 60°C of 1-phenylethanol reaction. Pressure dependence Pressure [MPa] Yield racemate conversion mmol mg -1 h -1

37. Enantiomeric excess ee [%] of 1-phenylethanol ( ) and ibuprofen (  ) reaction respectively enantiomeric ratio E of ibuprofen (  ). Enantiomeric excess

38. Yield of the reaction of 1-phenylethanol with vinyl acetate in n-hexane in dependence on substrate quantity. Solvent: 4 ml Reaction in n-hexane

39. Initial reaction rate in dependence on substrate quantity. Non linear fit to Michaelis-Menten eq. Substrate quantity Reacted quantity Reaction in n-hexane

40. Eadie-Hofstee-diagram Reaction in n-hexane

41. Reactor types Stirred tank reactor Fixed bed tubular reactor Cycle pump Fixed bed CO 2 Substrate

42. Reaction in n-hexane Comparison of stirred tank and tubular fixed bed reactor tubular fixed bed reactor stirred tank reactor

43. 1: CO 2 -feed, 2: CO 2 -cooler, 3: CO 2 -pump, 4: inlet valve, 5: outlet valve, 6: spindle press, 7: reactor valve, 8: reactor pressure gauge, 9: injection valve, 10: view cell, 11: sample valve, 12: stirrer Flow scheme of batch apparatus

44. Yield Ibuprofen Esterification in sc-CO 2 Time Yield

45. Enantiomeric excess (ee) vs yield Yield

46. Temperature dependence of reaction rate

47. Temperature dependence of enantioselectivity

48. Esterification of FAEE Novozym 435 in 2-propanol

49. Transesterification with glycerol in n-hexane Enzyme: Lipase Novozym 435

50. Transesterification with glycerol in n-hexane (no enzyme)