them as chiral catalysts and by applying standard methodology

Transcription

1 Most biocatalysts can be used in a straightforward manner by regarding them as chiral catalysts and by applying standard methodology some pecial techniques have been developed in order to broaden their range of applications biocatalysts in nonaqueous media rather than in water can lead to the gain of some significant advantages 'fixation' of the enzyme by immobilization may be necessary, and the use of membrane technology may be advantageous

2 Water is a poor solvent for nearly all reactions in preparative organic chemistry most organic compounds are insoluble in this medium the removal of water is tedious and expensive due to its high boiling point high heat of vaporization Side-reactions such as hydrolysis, racemization, polymerization and decomposition are often facilitated in the presence of water. completely anhydrous solvents are incapable of supporting enzymatic activity because some water is always necessary for catalysis how much water is required to retain catalytic activity is enzyme-dependent a-chymotrypsin needs only 50 molecules l of water per enzyme molecule l to remain catalytically active.this is much less than is needed to form a monolayer of water around the enzyme

3 The water present in a biological system can be separated into several physically distinct categories. Whereas the majority of the water (>98%) serves as a true solvent ('bulk water'), a small fraction of it is tightly bound to the enzyme's surface ('bound water'). The physical state t of bound water is clearly l distinct t from the bulk water and it should be regarded as a crucial integral part of the enzyme's structure rather than as adventitious residual solvent. Bound water is also often referred to as 'structural water'. For enzymatic catalysis in organic media it should be possible to replace For enzymatic catalysis in organic media, it should be possible to replace the bulk water by an organic solvent without significant alteration of the enzyme's environment, as long as the structural water remains unaffected

4 Biocatalytic transformations performed in organic media offer the following advantages: The overall yields of processes performed in organic media are usually better due to the omission of an extractive step during work-up. The loss-causing formation of emulsions can be avoided and the recovery of product(s) is facilitated by the use of low-boiling organic solvents. Nonpolar substrates are transformed at better rates due to their increased solubility. An organic medium is a hostile environment for living cells, microbial contamination is negligible. This is particularly important for reactions on an industrial scale, where maintaining sterility may be a serious problem.

5 Deactivation and/or inhibition of the enzyme caused by lipophilic substrates t and/or products is minimized since their solubility in the organic medium leads to a reduced local concentration at the enzyme's surface. Many side-reactions such as hydrolysis of labile groups (e.g. epoxides, acid anhydrides), polymerization of quinones, racemization of cyanohydrins or acyl-migration are water-dependent and are therefore largely suppressed in an organic medium. -Immobilization of enzymes is not neccessary because they may be recovered by simple filtration after the reaction due to their insolubility in organic solvents. -Many of the reactions which are responsible for the denaturation of enzymes are hydrolytic y reactions and therefore require water, -Due to the conformational change of the enzyme during the formation of the enzyme-substrate complex (the 'induced-fit'), numerous hydrogen bonds are broken. It is often possible to control some of the enzyme's catalytic properties such as the substrate specificity the chemo, regio- and enantioselectivity by variation of the solvent. -The most important advantage, however, is the possibility of shifting thermodynamic equilibria to favor synthesis over hydrolysis. -Hydrolases (lipases and proteases) esters, polyesters, lactones, amides and peptides can be synthesized in a chemo-, regio-and enantioselective manner.

6 The solvent systems which have commonly been used for enzyme catalyzed reactions containing organic media can be classified into three different categories. Enzyme Dissolved in a Monophasic Aqueous-Organic Solution Enzyme Dissolved in a Biphasic Aqueous-Organic Solution Enzyme Suspended in a Monophasic Organic Solution

7 Enzyme Dissolved in a Monophasic Aqueous-Organic Solution The enzyme, the substrate and/or product are dissolved in a monophasic solution consisting of water and a water-miscible organic cosolvent dimethyl sulfoxide, (DMSO) dimethyl formamide, (DMF) tetrahydrofuran, (THF) dioxane, acetone one of the lower alcohols, e.g. methanol or tert-butanol As a rule of thumb, most water-miscible solvents can be applied in concentrations up to ~ 10% of the total volume, only in some rare enzyme/solvent combinations even 50-70% of cosolvent may be used If the proportion of the organic solvent exceeds a certain threshold, the essential p p g bound water is stripped from the enzyme's surface leading to deactivation

8 Reaction systems consisting of two macroscopic phases, namely the aqueous phase containing the dissolved enzyme, and a second phase of a nonpolar organic solvent (preferably lipophilic and of high molecular weight) such as hydrocarbons, ethers or chlorinated hydrocarbons, may be advantageous to achieve a spatial separation of the biocatalyst from the organic phase The biocatalyst is in a favorable aqueous environment and not in direct contact with the organic solvent, where most of the substrate/product is located the limited concentrations of organic material in the aqueous phase may circumvent inhibition phenomena the removal of product from the enzyme surface drives the reaction towards completion. In such biphasic systems the enzymatic reaction proceeds only in the aqueous phase, a sufficient mass transfer of the reactant(s) to and product(s) from the catalyst and between the two phases is necessary shaking or stirring represents a crucial parameter in such systems.

9 The number of phase distributions encountered in a given reaction depends on the number of reactants and products (A, B, C, D) which are involved in the transformation Each distribution, measured as the partition coefficient, is dependent on the solubilities of substrate(s) and product(s) in the two phases and this represents a potential rate-limiting factor.

10 Replacing all of the bulk water (which accounts for >98%) by a waterimmiscible organic solvent leads to a suspension of the solid enzyme in a monophasic organic solution The biocatalyst seems to be 'dry' on a macroscopic level, it must have the necessary residual bound water to remain catalytically active. In order to tune a biocatalytic reaction in a monophasic organic solvent system, the following parameters should be considered: ph Effects. In organic solutions that lack a distinct aqueous phase, ph cannot be measured easily the ionization state of the enzyme which is a function of the ph, determines its conformation and hence its properties such as activity and selectivity. Since the ionization state of the charged groups of a protein does not change when placed in an organic solvent and thus remains 'frozen', it is important to employ solid enzymes that have been recovered by lyophilization or precipitation from a buffer at their ph optimum

11 Enzyme state. Adsorption of enzymes onto the surface of a macroscopic (inorganic or organic) carrier material generates a better distribution of the biocatalyst and generally gives significantly enhanced reaction rates, in some cases up to one order of magnitude. Celite, silica gel or an organic nonionic support (e.g. XAD-8, Accurel [71]) may be used as the carrier Choice of solvent. a measure for the 'compatibility' of an organic solvent with high enzyme activity, many parameters a describing the hydrophobicity ob of the solvent, such as the Hildebrandt solubility parameter (δ), the dielectric constant (ε), and the dipole moment (µ), have been proposed The most reliable results were obtained by using the logarithm of the partition coefficient (log P) of a given solvent between l-octanol and water

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15 Effects of additives Polyalcohols such as carbohydrates, sugar alcohols or glycerol are well known to stabilize proteins as well as inactive proteins (bovine serum albumin) and polymers which have a certain structural resemblance to that of water (e.g. polyethylene glycol, polyvinyl alcohol). -Small polar organic molecules (e.g. N,N-dimethyl formamide and formamide) are known to enhance reaction rates by acting as 'molecular lubricants'. The addition of salts (LiCI, NaCI, KCI) or weak organic bases (e.g. triethylamine, pyridine ) may improve reaction rates and selectivities via formation of salt-pairs of substrate and/or product, which shift equilibria into the desired direction.

16 The following basic rules should be considered for the application of solid ('dry') enzymes in organic media having a low water content: -Hydrophobic solvents are more compatible than hydrophilic ones (log P of the organic solvent should be greater than -1.5). -The water layer bound to the enzyme must be maintained; this is accomplished by using water-saturated organic solvents or, alternatively, via control of the water activity. -The 'micro-ph' must be that of the ph-optimum of the enzyme in water, a prerequisite it that t is fulfilled if the protein was isolated from an aqueous solution at the ph-optimum. Stirring, shaking or sonication is necessary in order to maximize diffusion of substrate to the catalyst's surface. The addition of enzyme stabilizing agents may improve the stability of the solid -The addition of enzyme-stabilizing agents may improve the stability of the solid enzyme preparation significantly.

17 Due to the fact that the lipophilic solvent (log P >1.5) is unable to accommodate the water which is gradually produced during the course of the reaction, it is collected at the hydrophilic enzyme surface. As a consequence, the water gradually forms a discrete aqueous phase which encompasses the enzyme, finally separating substrate and enzyme from each other by a polar interface, which is difficult to penetrate t for lipophilic substrate/product t t molecules. l Thus, the rate slows down and the reaction may cease before reaching the desired extent of conversion. -Firstly, removal of water from the system (e.g. by evaporation, azeotropic distillation or chemical drying via addition of molecular sieves or waterscavenging inorganic salts. -Secondly, avoiding the formation of water by employing an acyl-transfer step rather than an esterification reaction

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19 Acyl Transfer Trans-or interesterifications, which do not release water during the course of the reaction, are usually easier to perform the water content of the reaction medi um (more accurately the 'water activity', aw), which is a crucial parameter for retaining the enzyme's activity, remains constant In order to avoid the undesired depletion of the optical purity of (predominantly) the remaining substrate during an enzymatic resolution under reversible reaction conditions, two tricks can be applied to shift the equilibrium of the reaction -Use of an excess of acyl donor; this may be expensive and not always compatible with the aim of maintaining high enzyme activity, but it may be helpful in some cases. -A better solution, however, is the use of special acyl donors which ensure a more or less irreversible type of reaction. The reversibility of transesterification reactions is caused by the comparable nucleophilicity of the attacking nucleophile (Nu 1) and the leaving group of the acyl donor (Nu 2 ), both of which h compete for the acyl-enzyme intermediate t in the forward and the reverse reaction

20 Acyl Transfer

21 Acyl Transfer

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23 Acid anhydrides Another useful method of achieving completely irreversible acyl-transfer reactions is the use of acid anhydrides The selectivities achieved are usually high and the reaction rates are about the same as with enol esters. One of the advantages of this technique is that no aldehydic by-products are formed and the enzyme is not acylated under the conditions employed, making its reuse possible

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25 Separation of E/Z-stereoisomers. Stereoisomeric mixtures of the allylic terpene alcohols geraniol and nerol, which are used as additives to flavor and fragrance preparations, were separated by selective acylation with an acid anhydride using porcine pancreatic lipase (PPL) as catalyst. Depending on the acyl donor employed, the slightly less hindered geraniol was more quickly acylated to give geranyl acetate leaving nerol unreacted. Acetic anhydride proved to be unsuitable, giving a low yield and poor selectivity, but longer-chain acid anhydrides were used successfully. Desymmetrization of prochiral and meso-diols. Chiral 1,3-propanediol derivatives are considered to be useful building blocks for p p g the preparation of enantiomerically pure bioactive compounds such as phospholipids, platelet activating factor (PAP), PAF-antagonists and renin inhibitors. A simple access to these synthons starts from 2-substituted 1,3propanediols. Depending on the substituent (R) in position 2, (R)-or (S)- monoesters were obtained in excellent optical purities using Pseudomonas sp. lipase (PSL). The last three entries demonstrate an enhancement in selectivity which may be obtained when the reaction temperature is lowered.

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31 Peptide Synthesis

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37 Review questions: What are the advantages of organic solvents? Is water needed for these reactions and how much? Strategies to control the amount of water? Peptide synthesis Transesterification? Acyl transfer?