O N Ph H Ph O O N Ph H Ph O HN Ph O CO 2 Bu H Ph HN Ph O CO 2 H H Ph lipase BuOH + (R)-1 (S)-1 (S)-2 3 (trace) Enhancement of Candida antarctica lipase B enantioselectivity and activity in organic solvents Marie-Claire Parker,* a † Stuart A. Brown, b Lindsey Robertson b and Nicholas J. Turner b a Department of Chemistry, Joseph Black Building, University of Glasgow, Glasgow, UK G12 8QQ b Edinburgh Centre for Protein Technology, Department of Chemistry, University of Edinburgh, King’s Buildings, West Mains Road, Edinburgh, UK EH9 3JJ The enantioselectivity and catalytic activity of Novozym 435 ® [Candida antarctica lipase B (CALB)] in organic solvents was found to dramatically increase upon the addition of a non-reactive organic base, such as Et 3 N, to the reaction system. It has been shown that the unusual microenvironment of enzymes in organic solvents can affect a number of parameters, including the degree of protein hydration, 1,2 secondary struc- ture, 3 the susceptibility of the protein to inactivation and variations in the ionisation state 4 of side-chain residues. Frequently, these differences have been shown to result in interesting changes in the enzymes, including reversal of substrate specificity and changes in stereoselectivity, although the underlying reasons remain poorly understood. It is commonly accepted that the best predictor of enzyme catalytic activity in low water organic media is thermodynamic water activity (a w ). 1 ‡ Over the past few years although much has been reported on enzyme enantioselectivity in organic media there are as yet no predictive rules available. Crude lipase preparations have proved to be simple and effective biocatalysts for kinetic resolutions, e.g. chiral carboxylic acids and alcohols. However, the low purity of these preparations (presence of other lipases and competing hydrolases) can, in specific reactions, lead to low and unpredictable enantioselective behaviour. This effect can be compounded when using organic solvents, due to the effect of different solvent properties on catalytic activity. The starting point for the work described herein was the lipase (Lipozyme ® Mucor miehei) catalysed dynamic resolution of 4-substituted oxazol-5(4H)-ones, a reaction we have pre- viously employed for the synthesis of enantiomerically pure (S)-L-tert-leucine. 5 It was previously found that the modest enantioselectivity in toluene (ca. 68% ee) could be enhanced (ca. 97% ee) by the addition of a catalytic amount of Et 3 N to the reaction; the role of Et 3 N is not to facilitate racemisation of the substrate. We decided to investigate this effect in more detail by using a commercially available immobilised lipase,§ Novozym 435 (Candida antarctica lipase B 6 (CALB), since a larger substrate range could be tested with this enzyme. The catalytic activity and enantioselectivity of the alcoholysis of (±)-2-phenyl- 4-benzyloxazol-5(4H)-one 1 using butan-1-ol as the nucleo- phile (Scheme 1) was monitored¶ under a range of reaction conditions, including controlled water activity. Hydration was controlled by equilibrating∑ enzyme and solvent with the appropriate saturated salt solution 7 of known thermodynamic water activity a w . Therefore a low a w system will be one in which the solvent is poorly hydrated and the enzyme, similarly, has a low level of hydration, and at high a w (e.g. 0.97) the solvent is near water saturation and the enzyme is fully hydrated (as would be found in an aqueous system). Table 1 shows the effect of hydration on the initial catalytic rate and enantiose- lectivity, in three different solvents, n-hexane, toluene and MeCN, either with or without Et 3 N.** It can immediately be seen that the lipase-catalysed reaction is very sensitive to water activity. The addition of a non-reactive organic base,†† Et 3 N, to the reaction enhances significantly both the enantioselectivity and catalytic activity of the enzyme. Even low levels of hydration, present in the more nonpolar solvents such as n-hexane and toluene, are detrimental to the overall catalytic performance of CALB. We find that generally for optimum yield and enantioselectivity, both the enzyme and solvent should be rigorously dried prior to addition of Et 3 N. We were interested to see if addition of Et 3 N to a reaction already in progress and of poor enantioselectivity, could reverse this effect. As can be seen from Fig. 1, the addition of Et 3 N after 140 min immediately results in enhanced catalytic rate and en- antioselectivity. In order to examine the generality of the effect of Et 3 N we investigated a second reaction, namely the CALB-catalysed Table1 Effect of water activity on initial catalytic rate a,b and enantiospecificity as a function of hydration, with and without Et 3 N No Et 3 N Et 3 N Solvent c a w Initial rate/nmol min 21 mg 21 Ee (%) Initial rate/nmol min 21 mg 21 Ee (%) n-hexane ~ 0 (anhydrous) 26 (± 1.5) 85 (± 3) 30 (± 1.5) 90 (± 3) n-hexane 0.69 4 (± 0.5) 55 (± 2) 20 (± 1) 87 (± 3) n-hexane 0.97 1.5 (± 0.15) 30 (± 5) 18 (± 0.9) 80 (± 5) toluene ~0 15 (± 0.8) 85 (± 4) 27 (± 1.5) 93 (± 3) toluene 0.22 3 61 (± 6) 17 (± 1) 95 (± 2) MeCN d ~0 15 > 99 10 97 (± 2) MeCN d 0.1 (0.5% v/v H 2 O) NR e — 5 (± 0.3) 90 (± 4) MeCN d 0.4 (2% v/v H 2 O) NR e — NR e — a Initial rate for (S)-butyl ester enantiomer 2. b Results reported are the average of three separate measurements. c Note ∑. d Ref. 8. e No reaction. Scheme 1 Chem. Commun., 1998 2247 Published on 01 January 1998. 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