Most biocatalysts can be used in a straightforward manner by regardingMost 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 theirsome 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 y q gof some significant advantages

'fixation' of the enzyme by immobilization may be necessary,and the use of membrane technology may be advantageous

Water is a poor solvent for nearly all reactions in preparative organic chemistry

most organic compounds are insoluble in this mediummost organic compounds are insoluble in this medium

the removal of water is tedious and expensive due to its high boiling point g g phigh heat of vaporization

Side-reactions such as hydrolysis,racemization,polymerization and decompositionare 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 h t i d l 50 l l f t l la-chymotrypsin needs only 50 molecules of water per enzyme molecule

to remain catalytically active.This is much less than is needed to forma monolayer of water around the enzyme

•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').

Th h i l t t f b d t i l l di ti t f th b lk t d•The physical state of bound water is clearly distinct 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

Biocatalytic transformations performed in organic media offer the following advantages:

The overall yields of processes performed in organic media are usually betterThe overall yields of processes performed in organic media are usually betterdue 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)g y p ( )is facilitated by the use of low-boiling organic solvents.

Nonpolar substrates are transformed at better rates due to their increased solubility . p y

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.

D ti ti d/ i hibiti f th d b li hili b t t d/Deactivation and/or inhibition of the enzyme caused by lipophilic substrates 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• Many side-reactions such as hydrolysis of labile groups (e.g. epoxides, acid anhydrides),polymerization of quinones,racemization of cyanohydrinsracemization 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 byImmobilization 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 reactions and therefore require water,y y q ,-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 y ysubstrate 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.

The solvent systems which have commonly been used for enzymecatalyzed reactions containing organic media can be classified into threecatalyzed 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

Enzyme Dissolved in a MonophasicAqueous-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)tetrahydrofuran, (THF)dioxane, acetone one of the lower alcohols e g methanol or tert-butanolone 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 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 gbound water is stripped from the enzyme's surface leading to deactivation

Reaction systems consisting of two macroscopic phases, namely the aqueous phase containing the dissolved enzyme, and a second phase of a nonpolarorganic 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 aqueousIn 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.

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 p ( , , , )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.

Replacing all of the bulk water (which accounts for >98%) by a waterimmiscibleorganic solvent leads to a suspension of the solid enzyme in a monophasicorganic solvent leads to a suspension of the solid enzyme in a monophasicorganic solution

The biocatalyst seems to be 'dry' on a macroscopic level, it must have theThe 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, y p g y ,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 b ff t th i H tibuffer at their pH optimum

Enzyme state. Adsorption of enzymes onto the surface of a macroscopic (inorganic or organic) carrier material generates a better distribution of the biocatalyst and generallycarrier 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]) mayCelite, silica gel or an organic nonionic support (e.g. XAD-8, Accurel [71]) may be used as the carrier

Choice of solventChoice of solvent.

a measure for the 'compatibility' of an organic solvent with high enzyme activity, many parameters describing the hydrophobicity of the solvent, such as the a y pa a ete s desc b g t e yd op ob c ty o t e so e t, suc as t eHildebrandt 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

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.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' . y gThe 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.

The following basic rules should be considered for the application of solid ('dry') enzymes in organic media having a low water content: y g g

-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 i it th t i f lfill d if th t i i l t d f l ti t thprerequisite that 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 ofStirring, 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.

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

t t f li hili b t t / d t l l Th th t l d dpenetrate for lipophilic substrate/product molecules. 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-Firstly, removal of water from the system (e.g. by evaporation , azeotropic distillation or chemical drying via addition of molecular sieves or water-scavenging inorganic salts . Secondly avoiding the formation of water by employing an acyl transfer step rather-Secondly, avoiding the formation of water by employing an acyl-transfer step rather

than an esterification reaction

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'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) p p p y (p y)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

l d (N 2) b th f hi h t f th l i t di t i thacyl donor (Nu2), both of which compete for the acyl-enzyme intermediate in the forward and the reverse reaction

Acyl Transfer

Acyl TransferAcyl Transfer

Acid anhydrides Another useful method of achieving completely irreversible acyl-transfer reactions isthe use of acid anhydridesThe selectivities achieved are usually high and the reaction rates are about theThe selectivities achieved are usually high and the reaction rates are about thesame as with enol esters. One of the advantages of this technique is that noaldehydic by-products are formed and the enzyme is not acylated under theconditions employed making its reuse possibleconditions employed, making its reuse possible

Separation of E/Z stereoisomersSeparation 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 byare 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 nerolgeraniol was more quickly acylated to give geranyl acetate leaving nerolunreacted. 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 gthe preparation of enantiomerically pure bioactive compounds such as phospholipids, platelet activating factor (PAP), PAF-antagonists and renininhibitors. 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.

Peptide Synthesis

Review questions:Review questions:

What are the advantages of organic solvents?

Is water needed for these reactions and how much?Is water needed for these reactions and how much?

Strategies to control the amount of water?

Peptide synthesis

Transesterification?Transesterification?

Acyl transfer?