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E. Fischer: “Um ein Bild zu gebrauchen, will ich sagen, Enzymes are Pure Chemistry Ulf Hanefeld Catalysis, An Integrated Approach Schiermonnikoog November 29 – December 04, 2009 dass Enzym und Glucosid wie Schloss und Schlüssel zu einander passen müssen, um eine chemische Wirkung auf einander ausüben zu können. Diese Vorstellung hat jedenfalls an Wahrscheinlichkeit und an Werth für die stereochemische Forschung gewonnen, nachdem die Erscheinung selbst aus dem biologischen auf das rein chemische Gebiet verlegt ist.” Ber. Dtsch. Chem. Ges., 1894, 27, 2985-2993. Emil Fischer To use a picture I would like to say, that enzyme and glycoside have to match each other like lock and key in order to potentially have a chemical effect on each other. This concept has become more likely and has gained value for the investigation for stereochemical research, now that it is part of the field of pure chemistry rather than biology. Ber. Dtsch. Chem. Ges., 1894, 27, 2985-2993. The first stereoselective synthesis M. North, Tetrahedron: Asymmetry, 2003, 14, 147-176. J. -M. Brunel, I. P. Holmes, Angew. Chem., Int. Ed. 2004, 43, 2752–2778. J. Holt, U. Hanefeld, Curr. Org. Syn. 2009, 6, 15-37. Prunus amygdalus Hydroxynitrile lyase ~61 000 Da 78x52x46 Å • highly R-selective • readily available • Prunus amygdalus = Almond • R-mandelonitrile is natural substrate L. Rosenthaler, Biochem. Z. 1908, 14, 238-253. 1 • highly S-selective • readily available • dimer in solution • acetone cyanohydrin is natural substrate Prunus amygdalus • highly R-selective Hydroxynitrile lyase • readily available ~61 000 Da 78x52x46 Å Hevea brasiliensis Hydroxynitrile lyase (HbHNL) 29 229 Da, 30x38x48 Å Results for one HNL cannot be directly compared with those for other HNL’s. Hevea brasiliensis Hydroxynitrile lyase (HbHNL) C1347H2066O384N326S8; 29 229 Da 30x38x48 Å Enzyme in synthesis Chemical Amount Price (S)-(-)-/(R)-(+)1,1’Bi(2-naphthol) 10 g 121.60 € TMSCN 25 g 96.80 € KCN 25 g 19.50 € PaHNL 1000 U 131.50 € MeHNL 1 ml (3000 U) 103.00 € PaHNL-CLEA/ MeHNL-CLEA 50 mg 125.00 € It has to be taken into account that they are structurally not related!!! • highly S-selective • readily available • dimer in solution Which conclusions can be drawn? • • • • Did the enzymes make or break a bond? Are enzymes difficult to obtain? Do they cost too much? In which unit (gram, mol, etc.) are enzymes used? • Why are they actually so huge? J. Holt, U. Hanefeld, Curr. Org. Syn. 2009, 6, 15-37. What types of catalysts are there? • Heterogeneous catalysts • Homogeneous catalysts • Enzymes (homogeneous or heterogeneous) What do they have in common? • All work as catalysts • All follow the rules of kinetics (Michaelis and Menten, Lindeman-Hinshelwood, Eley-Rideal mechanism and LangmuirHinshelwood, Arrhenius) • For many reactions either of the three types of catalysts can be applied • Each type of catalyst has advantages and disadvantages 2 What is different? What is different? • Homogeneous catalysts and enzymes have one active site • Heterogeneous catalysts have different active sites on the surface, at the edges and corners • Diffusion, adsorption, and particle size are very important parameters in heterogeneous catalysis but are rather unimportant for homogeneous catalysts and enzymes • Enzymes are huge, homogeneous catalysts are much smaller (H+) • Enzymes are used in units U and these have to be measured beforehand • Homogeneous catalysts are highly defined and are added in moles • Heterogeneous catalysts have different active sites and are added in grams Classification of Enzymes Which type of enzyme is used? EC numberClass Reaction 1 2 3 Oxidoreductases Transferases Hydrolases 4 5 6 Lyases Isomerases Ligases (synthetases) Electron transfer Group-transfer Hydrolysis (transfer of functional groups to water) Double bond additions Shuffling groups within a molecule Formation of C-C, C-S, C-O, etc. by condensations at the expense of ATP Why are enzymes so huge? • Degrading enzymes, such as lipases, esterases and proteases but also alcohol dehydrogenase and lyases have the advantage that they are selective for one functional group but not selective for a specific substrate • Synthetic enzymes (anabolism) tend to be very substrate specific, are therefore less flexible and can not be used so widely Why are enzymes so huge? No substrate bound Hevea brasiliensis Hydroxynitrile lyase (HbHNL) C1347H2066O384N326S8; 29 229 Da 30x38x48 Å Transition state bound When the transition state is reached the enzyme is much more ordered, more hydrogen bonds exist, more stable beta sheets and alpha helixes 3 Benefit in enthalpy (ΔΔH#) of some enzymecatalysed reactions relative to the reactions in free solution Enzymes align substrates D. H. Williams, E. Stephens and M. Zhou, Chem. Commun, 2003, 1973–1976. Enzyme Rate Acceleration ΔΔH#, kJ mol-1 (s-1) due to ΔΔH# Chorismate dismutase Chymotrypsin Staphylococcal nuclease Bacterial α-glucosidase Urease Yeast OMP decarboxylase 233 266 263 280 293 2143 106 1012 1011 1014 1016 1025 • Enzymes are huge, the scaffold fixes the substrate and aligns it with the binding sites of the enzyme • Due to the perfect alignment the acid base catalysis that occurs has its charges much more distributed than in normal acid base catalysis Enzymes align substrates Enzymes align substrates • The enzyme basically encompasses the substrates • This is only possible due to its size • The aligned substrate can now react with the relative weak acids and bases in the enzyme • What is the entropy here and in the homogeneous reaction? H. M. Weiss, J. Chem. Edu. 2007, 84, 440-442. Mechanism of a D-2-deoxyribose-5-phosphate aldolase. Intramolecular versus intermolecular Classification of Enzymes EC numberClass Reaction 1 2 3 Oxidoreductases Transferases Hydrolases 4 5 6 Lyases Isomerases Ligases (synthetases) Electron transfer Group-transfer Hydrolysis (transfer of functional groups to water) Double bond additions Shuffling groups within a molecule Formation of C-C, C-S, C-O, etc. by condensations at the expense of ATP Entropically favoured by fixing conformation 4 What is a (Serine) Hydrolase? • Serine hydrolases are enzymes that hydrolyse an ester, thioester or amide bond • Hydrolases are enzymes that hydrolyse other C-O or C-N or C-S bonds • Serine Hydrolases have a serine in their active site • Hydrolases catalyse hydrolysis reactions (enantio)selectively under mild conditions How to deprotonate? How does this Hydrolase work? • Serine acts as a nucleophile that attacks the substrate • It is the alcohol group of the serine that does the trick • This alcohol needs to be deprotonated to obtain an alcoholate => the nucleophile • How is this achieved? Chymotrypsin • Localised charge • Strong base necessary • Charge delocalisation => weak base O Asp O His H N N H O Ser O O Chymotrypsin Catalysis Asp102 His57 Ser195 Step 2, Formation of first Michaelis Complex Catalytic “triad’’ Step 1 Substrate diffuses into active site Positioning group (hydrophobic) 5 Step 3, Formation of Tetrahedral Intermediate Catalytic “triad’’ Step 4, Release of first product (amine) Catalytic “triad’’ “Oxy-anion hole” Acyl-enzyme “TI1” Product-1 Step 5, Water approaches (2nd substrate) Catalytic “triad’’ Step 6, Formation of Tetrahedral Intermediate Catalytic “triad’’ Acyl-enzyme Oxy-anion hole “TI2” Substrate-2 Nucleophilic addition of water Step 7, Formation of Michaelis Complex Step 8, Release of second product Catalytic “triad’’ Catalytic “triad’’ Repulsion Dissociation of acid Product-2 6 Catalytic triad: Charge relay Bi-bi Ping-pong kinetics Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 The tetrahedral intermediate Attack of the nucleophile Bi-bi Ping-pong kinetics Is this really reversible? J. H. Kastle, A. S. Loevenhart, Am. Chem. J. 1900, 24, 491-525. 7 Attack of the nucleophile Difference between ester and amide Why use a hydrolase? What are hydrolases used for? • Hydrolysis with acids and bases often require harsh reaction conditions • A lot of waste is generated • Lipases, esterases and proteases catalyse hydrolysis reactions (enantio)selectively under mild conditions • All this makes enzymatic catalysis very clean and green • As degrading enzymes they are selective for a functional group but not for a single molecule • Hydrolases are virtually never used for what they were evolved for: hydrolysis • Lipases, esterases and proteases catalyse hydrolysis reactions enantioselectively under mild conditions • Hydrolases are therefore used to obtain enantiopure compounds from racemates Rule of Kazlauskas Rule of Kazlauskas Secondary alcohols Secondary alcohols and primary amines R. J. Kazlauskas, A. N. E. Weissfloch, A. T. Rappaport, L. A. Cuccia, J. Org. Chem., 1991, 56, 2656-2665. C. K. Savile, R. J. Kazlauskas, J. Am. Chem. Soc., 2005, 127, 12228-12229. R. J. Kazlauskas, A. N. E. Weissfloch, A. T. Rappaport, L. A. Cuccia, J. Org. Chem., 1991, 56, 2656-2665. C. K. Savile, R. J. Kazlauskas, J. Am. Chem. Soc., 2005, 127, 12228-12229. 8 Rule of Kazlauskas What about tert. alcohols? primary alcohols • Very few enzymes accept these bulky substrates • Few examples and even less enantioselective examples • Limited scope but very interesting Burkholderia cepacia lipase (formerly called Pseudomonas cepacia lipase, PCL) preferably catalyses the hydrolysis/acylation of only one of the depicted enantiomers of the chiral primary alcohol (ester). The selectivity is low if oxygen is bound to the chiral carbon. L: largest substituent, M: medium sized substituent. A. N. E. Weissfloch, R. J. Kazlauskas, J. Org. Chem., 1995, 60, 6959-6969. Chymotrypsin Catalysis Asp102 His57 Ser195 What about chiral acids? What about chiral acids? Step 1 Substrate diffuses into active site H L COOH M Preferred enantiomer in Candida rugosa lipase (CRL)-catalysed hydrolysis and esterification reactions of chiral acids. L: largest substituent, M: medium sized substituent. Positioning group (hydrophobic) Differences between the enzymes? M. C. R. Franssen, H. Jongejan, H. Kooijman, A. L. Spek, N. L. F.L. Camacho Mondril, P. M. A. C. Boavida dos Santos, A. de Groot, Tetrahedron Asymmetry, 1996, 7, 497-510. Interfacial activation • Lipases are commonly interfacially activated. Their natural function is the hydrolysis of fat. They can be used in water and apolar organic solvents (MeOH, EtOH, DMSO tend to be bad) CMC = critical micellar concentration K. Faber, Biotransformations in Organic Chemistry, 5th edition, Springer, 2004. 9 Lid of lipase from Thermomyces lanuginosus U. Hanefeld, L. Gardossi and E. Magner Chem. Soc. Rev. 2009, 38, 453–468. Slight difference in catalytic triad catalytic triade His O N N H H Glu O oxyanion Ser O hole -RCOOR' +RCOOR' -RCOOH +RCOOH H His N H O Glu N H O O N H His H O N N R O Ser O oxyanion hole +R'OH -H2O O R O O Glu hole O Ser oxyanion O Glu N H O Ser +H2O R' His R O -R'OH O oxyanion hole Difference between ester and amide CAL B surface A. Basso, P. Braiuca, S. Cantone, C. Ebert, P. Linda, P. Spizzo,P. Caimi, U. Hanefeld, Giuliano Degrassi and L. Gardossi, Adv. Synth. Catal. 2007, 349, 877 – 886. Differences between the enzymes? • Esterases show no interfacial activation, they were evolved to hydrolyse esters and they tend to be sensitive to organic solvents. Mostly used for hydrolysis reaction in water (+ polar solvent). • Proteases were evolved to hydrolyse proteins. Since esters are less stable than proteins they can easily hydrolyse them. Many are stable in apolar organic solvents. No interfacial activation How to select the right hydrolase? • Hydrolases are degrading enzymes, therefore they are not substrate specific • They are selective for esters, amides and thioesters • Lipases and esterases were evolved to hydrolyse esters • Proteases were evolved to hydrolyse proteins • Although we understand a lot and can predict much the best approach is: 10 Reactions in water Summary • Almost hydrolysis only – but if amides need to be synthesised this can be done in water. • Either high buffer concentration or pH-stat (automatic burette). • Often low concentrations. • Often difficult separations – water is difficult to remove. • Serine Hydrolases can hydrolyse and synthesize esters and amides • Water, alcohols and amines can be nucleophiles • They are enantioselective • For secondary alcohols enzymes for both enantiomers exist • For primary alcohols stereo differentiation is also possible • Chiral acids can also be resolved Reactions in Organic Solvents Kinetic resolution A: in water • Substrates dissolve better • Higher substrate concentration • Enzyme does not dissolve → easy work up • Different reactions become possible • Hydrolases can be used to make esters • Hydrolases can be used to make amides • First described in 1913 (S)-Y +YH R C R' X C enzyme-catalysed (S)-Y acylation, fast B: in dry organic solvent R' C not catalysed, very slow XH X = O, NH; Y = C or heteroatom enzyme-catalysed (S)-Y acylation, fast C C Xacyl R' R XH not catalysed, very slow R (R)-Y C Xacyl R' Dynamic kinetic resolution R C Xacyl +YH R X = O, NH (R)-Y C R XH R' R XH R (R)-Y R (R)-Y XH R' R' dynamic racemisation C not catalysed, very slow Xacyl R' R' R (R)-Y C (S)-Y C R' R XH R' dynamic +YH C R (R)-Y Dynamic kinetic resolution R R enzyme-catalysed Xacyl (S)-Y hydrolysis, fast R' M. Bourquelot, M. Bridel, Ann. Chim. Phys. 1913, 145. (S)-Y X = O, NH; Y = C or heteroatom R C R' C R' Xacyl X (S)-Y C enzyme-catalysed (S)-Y acylation, fast R' C X = O, NH R (R)-Y C R' Xacyl R' dynamic racemisation dynamic +YH R XH R XH not catalysed, very slow (R)-Y C Xacyl R' Kinetic resolutions give only 50 % yield!!! 11 Dynamic kinetic resolution R (S)-Y +YH R C R' C R XH enzyme-catalysed (S)-Y acylation, fast R' C X = O, NH R +YH (R)-Y C Xacyl R' dynamic racemisation dynamic X Desymmetrisation Hydrolases desymmetrise symmetrical compounds. R XH not catalysed, very slow (R)-Y R' C Xacyl R' The “meso-trick” U. Hanefeld, Org. Biomol. Chem. 2003, 1, 2405 – 2415. Organic solvents • • • • Apolar, aprotic (alkanes, petrol ether) Polar, aprotic (DMSO, DMF) Protic (Alcohols) Water miscible (DMSO, DMF, THF, dioxane) • Water immiscible (toluene, many ethers, alkanes, esters) Degree of polarity solvent log p DMSO Dioxane DMF Methanol Actone Ethylacetate Diethylether Diisopropylether Toluene Hexane Octane -1.3 -1.1 -1.0 -0.76 -0.23 0.68 0.85 1.9 2.5 3.5 4.5 Degree of polarity • When working with enzymes log P is used • Log P is the log of the partition coefficient of a solvent between 1-octanol and water log P H2O miscible -2.5 to 0 yes Reaction in water with a little solvent (10-50 %), otherwise enzyme † 0 to 1.5 partially often bad for enzymes, only with very stable enzymes 1.5 to 2.0 low Works often but not always, unpredictable >2.0 no Enzymes are very stable in these solvents Effect on enzyme Organic solvents versus water • In water the enzyme is dissolved • In a polar protic (methanol) or polar aprotic (DMSO) solvent the enzyme can be dissolved • In a water immiscible solvent the enzyme does not dissolve • Does an enzyme need to move? • How does an enzyme deactivate? 12 Why are enzymes so huge? Benefit in enthalpy (ΔΔH#) of some enzymecatalysed reactions relative to the reactions in free solution D. H. Williams, E. Stephens and M. Zhou, Chem. Commun, 2003, 1973–1976. No substrate bound Transition state bound When the transition state is reached the enzyme is much more ordered, more hydrogen bonds exist, more stable beta sheets and alpha helixes Enzyme Rate Acceleration ΔΔH#, kJ mol-1 (s-1) due to ΔΔH# Chorismate dismutase Chymotrypsin Staphylococcal nuclease Bacterial α-glucosidase Urease Yeast OMP decarboxylase 233 266 263 280 293 2143 106 1012 1011 1014 1016 1025 Enzymes need to move a little to accept the substrate and to facilitate the transition state. Water acts as lubricant Organic solvents versus water Organic solvents versus water How does an enzyme deactivate? Hydrolysis of the peptide chain Unfolding Asparagine and Glutamine amide might hydrolyse • All of this is caused or aided by water • So in organic solvent the enzyme should be more stable • But the enzyme has to stay flexible • What about the pH? Enzymes have pH optima in water • In one phase + organic solvent nothing changes • In two phase systems there is a water layer, nothing changes • Only organic solvent (one phase) – there is no pH. • • • • Organic solvents versus water Organic solvents and water • Only organic solvent (one phase) – there is no pH. • An organic buffer pair can be used, but inconvenient • Approach of choice: make the enzyme preparation at the ideal pH of the enyzme. Lyophilise (freeze-dry) or immobilise at pH optimum • Enzyme has memory effect, since it is rigid in org sol is stays in optimal conformation. • The enzyme needs some flexibility • Most of the water is bulk water and if it is gone it does not matter • But a little water needs to be on the enzyme as structural water or molecular lubricant • Really dry enzymes tend to be less active • But too much water causes enzymes to lump together – unless they are immobilised (think of diffusion) 13 Organic solvents and water Reactions in organic solvents • How much water does the enzyme need? • If water saturated solvent is used enzymes tend to work well but it can cause many side reactions • Control water concentration i.e. water acitivity aw • Salt pairs can be used for this • Not all hydrolases are stable in dry organic solvents. Lipases that were evolved for hydrophobic substrates perform best, esterases worst. • Apolar solvents are better than water miscible ones • Immobilisation can help • Small amounts of water might be added, too • Additives might help aw Salt pair CaCl2 xH2O / 2 H2O 0.037 Na2HPO4/Na2HPO4•2H2O 0.16 Na2HPO4•2H2O/Na2HPO4•7H2O 0.57 Na2HPO4•7H2O/Na2HPO4•12H2O 0.80 Reactions in organic solvents How to synthesise an ester • If all the parameters discussed are taken care of enzymes can display similar activity in organic solvent as in water • Reactions performed: esterification, transesterification, amide synthesis U. Hanefeld, Org. Biomol. Chem. 2003, 1, 2405 – 2415. How to synthesise an ester U. Hanefeld, Org. Biomol. Chem. 2003, 1, 2405 – 2415. How to synthesise an ester U. Hanefeld, Org. Biomol. Chem. 2003, 1, 2405 – 2415. 14 Dynamic kinetic resolution H. Pellissier, Tetrahedron 2008, 64, 1563-1601 How to synthesise an amide Dynamic kinetic resolution H. Pellissier, Tetrahedron 2008, 64, 1563-1601 How to synthesise an amide U. Hanefeld, Org. Biomol. Chem. 2003, 1, 2405 – 2415. U. Hanefeld, Org. Biomol. Chem. 2003, 1, 2405 – 2415. Industrial application U. Hanefeld, Org. Biomol. Chem. 2003, 1, 2405 – 2415. Review: F. van Rantwijk, R.A. Sheldon, Tetrahedron 2004, 60, 501 dkr Y.K. Choi, M.J. Kim, Y. Ahn and M.J. Kim, Org. Lett., 2001, 3, 4099-4101. 15 Conclusions dkr Martijn A. J. Veld, Karl Hult, Anja R. A. Palmans, E. W. Meijer, Eur. J. Org. Chem. 2007, 5416–5421. kr for chiral intermediates AcO HO AcO + NH Burkholdia cepacia lipase (formerly called Pseudomonas cepacia lipase) NH O O racemate O Cl OMe Cl ee > 99 % OH O OMe + Cl O Cl NH O O CCL V. H. M. Elferink, J. G. T. Kierkels, M. Kloosterman, J. H. Roskam (Stamicarbon B.V.), EP 369553, 1990. N captopril OH OH O O DSM Andeno process Z. Liu, R. Weis, A. Glieder, Food Technol. Biotechnol., 2004, 42, 237–249. OH OH O kr for chiral intermediates COOH lactonase O O R. N. Patel, J. Howell, R. Chidambaram, S. Benoit and J. Kant, Tetrahedron: Asymmetry, 2003, 14, 3673–3677. AcO + NH O • Hydrolases are versatile catalysts for the clean and green synthesis of enantiopure compounds • Synthetic dynamic kinetic resolutions enable hydrolase-“catalysed” bond syntheses • Given the great promiscuity of the hydrolases, in particular the lipases, new nucleophiles other than water can be introduced • Reverse reaction in Organic Solvent possible + COOH HO HO D-pantoic acid rac-pantolactone O N J. Ogawa, S. Shimizu, Curr. Opin. Biotechnol., 2002, 13, 367–375. OH Vitamin B5 H.-J. Gais, C. Griebel, H. Buschmann, Tetrahedron: Asymmetry, 2000, 11, 917–928. chemical racemisation O O N N H O N O Bacillus lentus protease OEt N O N N H O O human rhinovirus protease inhibitors OH O + O N 5 % DBU, rt, 1 h C. A. Martinez, D. R. Yazbeck, J. Tao, Tetrahedron, 2004, 60, 759–764. O N N H O OEt O Clean-up dkr for chiral compounds O S CF3 in situ racemisation with trioctylamine O S CF3 O COOH Candida rugosa lipase O O Desymmetrisation C.-Y. Chen, Y.-C. Cheng, S.-W. Tsai, J. Chem. Technol. Biotechnol., 2002, 77, 699-705. S-fenoprofen OEt OEt in situ racemisation with NaOH O O COOH Candida rugosa lipase O O H. Fazlena, A. H. Kamaruddin, M. M. D. Zulkali, Bioprocess Biosyst. Eng., 2006, 28, 227–233. S-ibuprofen S S O O Pseudomonas cepacia lipase O O N NC N NC SPr NC SPr O SPr O O O O N NC OH O trimethylamine O N O N NC N NC roxifiban J. A. Pesti, J. Yin, L.-H. Zhang, L. Anzalone, J. Am. Chem. Soc., 2001, 123, 11075-11076. J. A. Pesti, J. Yin, L.-H. Zhang, L. Anzalone, R. E. Waltermire, P. Ma, E. Gorko, P. N. Confalone, J. Fortunak, C. Silverman, J. Blackwell, J. C. Chung, M. D. Hrytsak, M. Cooke, L. Powell, C. Ray, Org. Proc. Res. Dev., 2004, 8, 22-27. R. N. Patel, A. Banerjee, L. Chu, D. Brozozowski, V. Nanduri, L. J. Szarka, J. Am. Oil Chem. Soc., 1998, 75, 1473-1482. R. Öhrlein, G. Baisch, Adv. Synth. Catal., 2003, 345, 713 – 715. 16 “meso” trick O O AcO O Pseudomonas fluorenscens lipase (PFL) OAc O AcO yield = 98 % ee > 98 % OH meso O O O PFL OAc AcO HO O OAc yield = 79 % ee = 96 % meso C. Bonini, R. Racioppi, L. Viggiani, G. Righi, L. Rossi, Tetrahedron: Asymmetry, 1993, 4, 793-805. Z.-F. Xie, H. Suemune, K. Sakai, Tetrahedron: Asymmetry, 1993, 4, 973-980. U. Zutter, H. Iding, P. Spurr, and B. Wirz, J. Org. Chem. 2008, 73, 4895–4902. 17