Medium engineering

The enantioselectivity of biocatalytic reactions is normally expressed as the enantiomeric ratio or the E value [la], a biochemical constant intrinsic to each enzyme that, contrary to enantiomeric excess, is independent of the extent of conversion. In an enzymatic resolution of a racemic substrate, the E value can be considered equal to the ratio of the rates of reaction for the two enantiomers, when the conversion is close to zero. More precisely, the value is defined as the ratio between the specificity constants (k st/Ku) for tho two enantiomers and can be obtained by determination of the k -at and Km of a given enzyme for the two individual enantiomers. 

However, considering practical limitations, that is, the availability of optically pure enantiomers, E values are more commonly determined on racemates by evaluating the enantiomeric excess values as a function of the extent of conversion in batch reactions. For irreversible reactions, the E value can be calculated from Equation 1 (when the enantiomeric excess ofthe product is known) or from Equation 2 (when the enantiomeric excess ofthe substrate is knovm) [la]. For reversible reactions, which may be the case in enzymatic resolution carried out in organic solvents (especially at extents of conversion higher than 40%), Equations 3 or 4, in which the reaction equilibrium constant has been introduced, should be used [lb]. 

For obtaining both the product and the remaining substrate in high enantiomeric excess in one reaction step, the E-value needs to be high, usually around or more than 100. 

Where cis conversion of substrate, eCp and ee are enantiomeric excesses of product (P) and remaining substrate (S), K= (1 — Ceq)/Cgq, and Ceq = conversion at equilibrium. 

Owing to the logarithmic relationship between E and AAG, a small increase in AAG produces a dramatic change in E. For instance, when AAG is increased by only 1 kcal mol approximately, the enantiomeric excess of the product is enhanced from 80 to 95% [2]. 

For instance, the almost exclusive specificity of proteases for L-configurated amino acid derivatives may be destroyed when reactions are carried out in organic solvents [346]. This makes them useful for the synthesis of peptides containing noimatural o-amino acids, which are usually not substrates for proteases. 

The influence of organic solvents on enzyme enantioselectivity is not limited to the group of proteases, but has also been observed with lipases, and is a general phenomenon [347-351]. As a rule of thumb, the stereochemical preference of an enzyme for one specific enantiomer usually remains the same, although its selectivity may vary significantly depending on the solvent. In rare cases, however, it was possible to even invert an enzyme s enantioselectivity [352-354]. 

The whole set of data available so far demonstrates that the nature of the organic solvent and its water content exerts a strong influence on the catalytic properties of an enzyme. However, no general rationale, which would allow the prediction of 

---

The value that is added during light-and medium-engineering work is larger, and this usually means that the economic constraint on the choice of materials is less severe - a far greater proportion of the cost of the structure is that associated with labour or with production and fabrication. Stainless steels, most alumiruum alloys and most polymers cost between UK 500 and UK 5000 (US 750 and US 7500) per... 

The specificity of enzyme reactions can be altered by varying the solvent system. For example, the addition of water-miscible organic co-solvents may improve the selectivity of hydrolase enzymes. Medium engineering is also important for synthetic reactions performed in pure organic solvents. In such cases, the selectivity of the reaction may depend on the organic solvent used. In non-aqueous solvents, hydrolytic enzymes catalyse the reverse reaction, ie the synthesis of esters and amides. The problem here is the low activity (catalytic power) of many hydrolases in organic solvents, and the unpredictable effects of the amount of water and type of solvent on the rate and selectivity. 

The term medium engineering , that is the possibility to affect enzyme selectivity simply by changing the solvent in which the reaction is carried out, was coined by Klibanov, who indicated it as an alternative or an integration to protein engineering [5aj. Indeed, several authors have confirmed that the enantio-, prochiral-, and even regioselectivity of enzymes can be influenced, sometimes very remarkably, by the nature of the organic solvent used. 

Before discussing the medium engineering phenomenon and its synthetic relevance in details, it is useful to offer a brief overview of the fundamentals of biocatalysis in organic media. 

The experimental evidences that medium engineering might represent an efficient method to modify or improve enzyme selectivity (alternative to protein engineering and to the time-consuming search for new catalysts) were immediately matched by the search for a sound rationale of this phenomenon. The different hypotheses formulated to try to rationalize the effects of the solvent on enzymatic enantioselectivity can be grouped into three different classes. The first hypothesis suggests that... 

The aspects of medium engineering summarized so far were a hot topic in biocatalysis research during the 1980s and 1990s [5]. Nowadays, all of them constitute a well-established methodology that is successfully employed by chemists in synthetic applications, both in academia and industry. In turn, the main research interests of medium engineering have moved toward the use of ionic liquids as reaction media and the employment of additives. 

As shown in this chapter, by focusing on the modulation of enzyme selectivity by medium engineering, quite simple modifications of the solvent composition can really have significant effects on the performances of the biocatalysts. The main drawback remains the lack of reliable predictive models. Despite the significant research efforts (particularly in the last decade), it is likely that a reasonable foresight of the enantioselective outcome of an enzymatic transformation will continue to be based solely on a careful analysis of the increasingly numerous literature reports. 

Efficient biocatalysis in neat organic solvent depends on the careful choice of the method of dehydrated enzyme preparation and solvent used. Optimization of these factors towards a given transformation is often known as catalyst formulation and solvent, or medium, engineering respectively, both of which will be briefly discussed below. Catalyst engineering which also provides a powerful method of improving activity and stability, is discussed in Chapter 2. 

In order to get good results in a kinetic resolution, E must be high, preferably well above 30 [66]. However, in organic media the enantioselectivity of an enzyme can depend strongly on parameters such as temperature and solvent, so medium engineering is often a fast and highly effective tool to increase E. 

Laane, C. (1987) Medium-Engineering for Bio-Organic Synthesis. Biocatalysis, 1, 17-22. 

Since then, a number of propositions to modify (if possible, to enhance) the enantioselectivity of enzymes (mostly hydrolases) by medium engineering have been put forward [20, 51, 60, 64—69], providing a challenging mix of scientifically and practically interesting questions. 

Despite the fact that solvent effects on enzyme enantioselectivity appear to resist our efforts to rationalize their outcome using commonly accepted solvent descriptors, the effects are certainly there. An impressive example is provided in a report on the successful resolution of ds/trans-( 1 R,5 R)-bicyclo[3.2.0]hept-6-ylidene-acetate ethyl esters, intermediates in the synthesis of GABA (y-aminobutyric acid) analogs, by the Pfizer Bio transformations and Global R D groups (Scheme 2.2) [136]. From a screening protocol, CaLB was identified as a reactive catalyst for the hydrolysis of the racemic mixture of / //-os lor enantiomers with approximately equal activity for the ds- and tmns-isomers and a rather modest (E = 2.7) preference for the /Z-(lR,5R)-enantiomers. Application of medium engineering resulted in a phenomenal increase in the enantioselectivity (addition of 40% acetone, E > 200), while the ds- and trans-isomers were still converted at an almost equal rate. 

Solvent as a Parameter for Reaction Optimization ( Medium Engineering )... 

---