Biotransformation with Growing Cells

Use a biological total synthesis by fermentation or multi step biotransformation (with growing cells, resting cells, multi-enzyme systems, genetically or metabolically engineered biocatalysts, etc). 

Stereoselective biotransformation with growing cells, resting free or immobilized cells, or isolated enzymes has been demonstrated to be a useful preparative method for the synthesis of centrochiral optically active organosilicon compounds [1-3]. In continuation of our own studies in this field, we have investigated stereoselective microbial transformations of rac-1-(4-fluorophenyl)-l-methyl-l-sila-2-cyclohexanone (rac-1) and rac-(Si5,CR/SiR,C5)-2-acetoxy-l-(4-fluorophenyl)-l-methyl-1-silacyclohexane [rac-(Si5,C/ /Si/f,C5)-3a]. We report here on (i) the synthesis of rac-1 and rac- SiS,CR/SiR,CS)-3a, (ii) the diastereoselective microbial reduction of rac-1 [— (Si5,C/ )-2a, (SiR,C5)-2a], and (iii) the enantioselective microbial hydrolysis of rac-(SiS,CR/SiR,CS)-3a [- (SiR,C5)-2a],... 

Enantioselective reduction of the prochiral cyclic acylsilane 42 with growing cells of the yeast Kloeckera corticis (ATCC 20109) yielded the optically active reduction product (R)-43 (Scheme 8)53. On a preparative scale, the l-silacyclohexan-2-ol (R)-43 was isolated in 60% yield with an enantiomeric purity of 92% ee. Repeated recrystallization of the biotransformation product from n-hexane raised the enantiomeric purity to 99% ee. 

Intact microbial cells have also been used as biocatalyst for this particular type of bioconversion. Incubation of the racemic 2-acetoxy-l-silacyclohexane rac-(SiS,CR/SiR,CS)-81 with growing cells of the yeast Pichia pijperi (ATCC 20127) yielded the optically active l-silacyclohexan-2-ol (Si/t,CS)-70 (Scheme 16)66,67. Under preparative conditions, this biotransformation product was isolated as an almost enantiomerically pure compound (enantiomeric purity >96% ee) in ca 80% yield [relative to (Si/ ,CS )-81 in the racemic substrate]. 

Transformations with growing cells The reaction medium is inoculated with the microorganism and the substrate is added at the beginning or at a suitable phase of the growth. As growth of the organism and the biotransformation occur simultaneously, this is also a very simple method, provided that the expertise and facilities for microbiological work are available. 

All biotransformations mentioned above represent enantioselective reductions. In contrast, the reduction of the cyclic chiral acylsilane vac-225 with growing cells of Kloeckera corticis (ATCC 20109) into a 1 1 mixture of (S,R)-226 and (jR,S)-226 is an example of a diastereoselective conversion (yield of reduction product 95%, diaster-eomeric excess 90% de)19,283. 

In some cases it is more attractive to use whole microbial cells, rather than isolated enzymes, as biocatalysts. This is the case in many oxidative biotransformations where cofactor regeneration is required and/or the enzyme has low stability outside the cell. By performing the reaction as a fermentation, i.e. with growing microbial cells, the cofactor is continuously regenerated from the energy source, e.g. glucose. 

Initial scale-up of microbial biotransformation is conveniently run with multiple flasks without extensive reaction optimization. A typical flask fermentation is performed at 28 °C, 250 rpm with 100 mL culture in a 500 mL Erlenmeyer flask, although other settings will work fine too. Three parameters need to be investigated before scale-up the time for adding the substrate, the optimal substrate concentration and the time course of product formation. Optimization of other factors, such as medium composition and pH, growing cells versus resting cells [74], is helpful, if the timeline allows and if there is a sufficient amount of the substrate to support the screening. 

Analogous enantioselective biotransformations have been achieved with the silicon compounds 217 and 219, which were transformed into the respective optically active products (S)-218 (yield 90%, enantiomeric purity 84% ee) and (S)-220 (yield 80%, enantiomeric purity 76% ee) using growing cells of Kloeckera corticis (ATCC 20109)278,279. Particularly remarkable is the conversion of the hydridosilane 219 which could be performed without a noticeable degree of hydrolytic cleavage of the Si-H bond (incubation conditions pH 5.5, 27 °C). 

Whole-cell biotransformations frequently showed insufficient stereoselectivities and/or undesired side reactions because of competing enzymatic activities present in the cells. These side reactions can modify the substrates and/or products. Furthermore, whole-cell biotransformations are limited due to the intrinsic need to grow biomass, which generates its own metabolites that are not related to the biotransformation reactions and, therefore, which need to be removed during the downstream process. Both the cells themselves and the unrelated metabolites produced are impurities that need to be removed after the biotransformation reaction. With isolated enzymes, there are no organism and unrelated metabolites to remove after the biotransformation processes. 

Many reported biotransformations are initially only demonstrated on a very small scale, the substrates or products may be subject to competing reactions if other enzymes are present (this can be a serious issue in whole-cell biocatalysis), or the desired enzyme is insufficiently active or produced in low levels. For many biotransformations a little care and attention is needed in the growth of the microbe to achieve the desired results. Production of a specific enzyme from a microbe can often be increased by growing the cells in the presence of a very small concentration (typically micromolar) of an inducer. The inducer could be a natural enzyme substrate, a substrate mimic or a molecule which is in some way associated with a substrate s availability or role in metabolism. This process is called induction and represents a genetic switch which cells use to respond... 

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