Substrate engineering

In parallel to the diethyl ester 9 the corresponding dimethyl ester was also tested as an alternative substrate for the enzymatic reaction [28], Compared with 9, however, the enzymatic hydrolysis slowed down to only 44% conversion after 74 hours and afforded the desired monoacid (R)-2b in merely 94% ee along with 6.7% (GC) of the undesired monoacid (corresponding to 16). Consequently, this approach was no longer considered. 

On the laboratory-scale the reaction products were easily separated by means of conventional extraction with ethyl acetate at neutral and acidic pH. Phase separation in the neutral extraction steps occurred almost instantly. Only in the first of the acidic extraction steps did the aqueous phase remain partly turbid and cloudy. The handling of this problem on the larger scale is described in Sections 6.3.2.2 and 6.3.3.3. 

Subtle modification of the substrate in order to change the enantioselectivity of a reaction (substrate engineering) is one of the simplest methods for obtaining changes in optical purity. For the oxidative KR of sec-alcohols using Rhodococcus ruher DSM 44541, it was found that the introduction of C=C bonds into the side chain improved the enantioselectivity of the reaction [29]. For example, 

Hohnquist et al. have reported a new approach for creating enantioselective hydrolytic en2ymes that they term engineered substrate-assisted catalysis (SAC) 

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Figure 6.5 Substrate engineering with protecting groups improves diastereoselectivity of KDGA O O RhaD y ...

Fechter, M.H., Gruber, K., Avi, M., Skranc, W., Schuster, C., Pbchlauer, P., Klepp, K.O. and Griengl, H., Stereoselective biocatalytic synthesis of (S)-2-hydroxy-2-methylbutyric acid via substrate engineering by using thio-disguised precursors and oxynitrilase catalysis. Chem. Eur. J., 2007,13, 3369. 

Chapter 8 describes the application of hydroxyl nitrile lyases to the synthesis of new chiral cyanohydrins and a-hydroxy acids and includes new approaches to the transformation of difficult aldehyde and ketone substrates using substrate engineering and immobilization techniques. 

Kim EJ, Perreira M, Thomas CJ, Hanover JA. An O-GlcNAcase-specific inhibitor and substrate engineered by the extension of the N-acetyl moiety. J. Am. Chem. Soc. 2006 128 4234-4235. 

Similar chiral alkinols have been used manifold in the synthesis of pharmaceuticals and biologically active compounds. Further development in the field of substrate engineering and chemoenzymatic synthesis might enable additional access to general chiral building blocks like compound 17. 

Figure 2 Metabolic substrate engineering as a strategy for modulating cell surface oligosaccharide structure. Modified metabolic substrates can intercept a biosynthetic pathway in two ways the pathway might be inhibited, leading to truncated structures on the cell surface, or the modified substrate might be incorporated into oligosaccharides in place of the normal substrate.

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