Multienzymatic synthesis

Scheme 20. Multienzymatic synthesis of D-tagatose 1,6-bisphosphate and its differentiation from the /rucfo-configurated side product...

Multistep enzymatic transformations can be done with combinations of compatible aldolases, glycosyltransferases and recycling systems. A practical and successful example for this concept is the multienzymatic synthesis of the sialyl Lewisx tetrasaccharide (sialyl Le ... 

Lopez-Gallego, F. and Schmidt-Dannert, C. (2010) Multienzymatic synthesis. Curr. Opin. Chem. Biol, 14, 174-183. 

Monti, D., Ferrandi, E.E., Zanellato, I., Hua, L, Polentini, F., Carrea, G., and Riva, S. (2009) One-pot multienzymatic synthesis of 12-ketoursodeoxycholic acid subtle cofactor specificities rule the reaction equilibria of five biocatalysts working in a row. Adv. Synth. Catal.,... 

P., Gambera, G., Kubac, D., and Martftikova, L. (2010) A novel chemo-multienzymatic synthesis of bioactive cydophellitol and epi-cydophellitol in both enantiopure forms. Tetrahedron Asymmetry, 21, 2448 2454. 

On the other hand, a-transaminases have been used extensively in the production of amino acids through kinetic resolution and asymmetric synthesis. While many studies rely on the use of an excess of cosubstrate to drive the reaction to completion, some multienzymatic approaches have been developed as well. As an example, aspartate has been used as an amino donor in a multienzymatic synthesis of L-2-aminobutyrate from L-threonine (Scheme 4.8). ° The rather complex multistep sequence started with the in situ formation of 2-ketobutyrate from L-threonine catalysed by threonine deaminase (ThrDA) from E. coli. A tyrosine transaminase (lyrAT) from E. coli converted 2-ketobutyrate and L-aspartie acid to L-2-aminobutyrate and oxaloacetate, which spontaneously decarboiq lated to give pyruvate. Since the... 

Scheme 11.4 Concurrent oxidation and reduction reactions in the stereoinversion of sec-alcohols (a) and in the one-pot multienzymatic synthesis of 12-ketoursodeoxycholic acid (b).

Another interesting example of a redox neutral cascade has been proposed for the multienzymatic synthesis of (JJ)-3-fluorolactic acid together with the resolution of racemic 3-fluoroalanine (Scheme 11.5b) [13]. Optically enriched (S)-3-fluoroalanine (88% ee) was recovered unreacted after the enantioselective oxidative deamination of the racemic substrate catalyzed by the L-alanine dehydrogenase (i-AlaDH) from Bacillus subtilis. This oxidative reaction, which is thermodynamically unfavorable, was driven by the coupled reduction reaction of the intermediate 3-fluoropyruvate catalyzed by rabbit muscle i-lactate dehydrogenase (L-LDH). Since both enzymes are NADH dependent, this coupled... 

Figure 13.35 Multienzymatic synthesis of S)-3-(l-aminoethyl)phenyl ethyl(methyl)carbamate, an intermediate of (S)-rivastigmine employing LDH for coproduct removal.

Rhodococcus opacus 71D a-Aminonitriles (R)- or (S)-a-Amino acids" Multienzymatic synthesis of enantiopure a-amino acids [111,112]... 

Apparently, all DHAP aldolases are highly specific for the donor component 22 for mechanistic reasons [29]. For synthetic applications, two equivalents of 22 are conveniently generated in situ from commercial fructose 1,6-bisphosphate 23 by the combined action of FruA and triose phosphate isomerase (EC 5.3.1.1) [93,101]. The reverse, synthetic reaction can be utilized to prepare ketose bisphosphates, as has been demonstrated by an expeditious multienzymatic synthesis of the (3S,4S) all-cis-configurated D-tagatose 1,6-bisphosphate 24 (Fig. 13) from dihydroxyacetone 27, including a cofactor-dependent phosphorylation, by employing the purified TagA from E. coli (Fig. 13) [95,96]. 

Flgure10.23 Sialyl Lewis -related selectin inhibitorandfluorogenicscreening compound for transketolase prepared using enzymatic aldolization, and multienzymatic oxidation-aldolization strategy for the synthesis of bicyclic higher carbon sugars. 

Figure 10.38 Multienzymatic scheme for the stereoselective synthesis oftwo equivalents ofxylulose 5-phosphate from fructose 1,6-bisphosphate.

The chemoenzymatic strategies described heretofore are based on DHAP-depen-dent aldolases. One of the main drawbacks of DHAP aldolases is their strict specificity toward DHAP and a negligible activity with the unphosphorylated DHA analog (29) (Figure 19.8). The chemical synthesis of DHAP involves several steps in an approximately 70% overall yield. [37] Alternatively, multienzymatic methods coupled with the aldol reaction have also been described [38]. However, drawbacks such as the different and often incompatible optimal reaction conditions of... 

Figure 1.8 shows the synthesis of fatty acids. This complex process is catalysed by the multienzymatic complex, fatty acid synthetase. This enzyme uses as substrates acetyl-coA and malonyl-coA to produce palmitic acid. Afterwards, palmitic acid, a saturated fatty acid of 16 carbon atoms, can be used to produce other fatty acids (Ratledge and Evans 1989). Fatty acids with more carbon units, such as estearic acid, are obtained by elongation of palmitic acid. 

Multienzymatic approaches of special interest for co-factor-dependent systems with recycling enzymes and multistep one-pot enzymatic synthesis FDH, kinases, glycosyltransferases... 

For a long time, kinetic resolution of alcohols via enantioselective oxidation or via acyl transfer employing, for example, lipases along with dynamic kinetic resolution have been the biocatalytic methods of choice for the preparation of chiral alcohols. In recent years, however, impressive progress has been made in the use of alcohol dehydrogenases (ADHs) and ketor-eductases (KREDs) for the asymmetric synthesis of alcohols by stereoselective reduction of the corresponding ketones. Furthermore, recent remarkable multienzymatic systems have been successfully applied to the deracemisation of alcohols via stereoinversion based on an enantioselective oxidation followed by an asymmetric reduction. 

Synthesis of poly(catechol) was demonstrated by multienzymatic processes (Fig. 1) [13]. Aromatic compounds were converted to catechol derivatives by the catalytic action of toluene dioxygenase and toluene cis-dihydrodiol dehydrogenase, followed by the peroxidase-catalyzed polymerization to give the polymer with molecular weight of several thousands. 

In this chapter, the focus is on in vitro enzyme catalysis for vinyl polymerization. To the best of our knowledge, prior to the work of Derango et al. (1992) there is a single short report showing the formation of low molecular weight vinyl polymers when studied in a suspension of Escherichia coli in the presence of methyl methacrylate [15,16]. Unhke polyaromatics, vinyl polymerization offers better control of polymer characteristics, as has been demonstrated with ternary systems (enzyme, oxidant, and initiator such as b-diketone). The number of different vinyl monomer chemistries investigated for susceptibility toward enzymatic polymerization (1-12) is fewer than reported aromatics, as is the extent of literature covering these types of syntheses. In addition, the discovery of multienzymatic approaches for the synthesis of antioxidant-functionalized vinyl polymers provides new impetus for the use of enzymatic methods related to vinyl polymers. 

However, when biocatalysts showing a sufficient cofactor specificity are not naturally available, possible undesired interferences between the cofactor regeneration systems can be circumvented by different approaches still maintaining the one-pot fashion of the multienzymatic process. For example, in the biocatalyzed synthesis of 12-ketoursodeoxycholic acid, the performances of the investigated cascade system, in which five enzymes were involved in concurrent oxidation and reduction reactions at different sites of the starting substrate cholic acid, were significantly improved by simple compartmentalization of the oxidative and reductive enzymes in two membrane reactors (Scheme 11.4b) [11]. 

A great effort has also been made for the development of multienzymatic cascade processes finalized to the preparation of enantiopure epoxides, important chiral synthones for the synthesis of drugs and biologically active compounds [2]. 

The main problem of the co-TA-catalyzed transamination is the generally unfavorable equilibrium that lies on the substrate side, especially when alanine is used as amino donor. Several methods have been successfully established to overcome this issue, for instance, coproduct removal [e.g., with lactate dehydrogenase (LDH)] or amino donor recycling [e.g., with alanine dehydrogenase (AlaDH)] [142-145]. The nicotinamide cofactor consumed in this reductive step is regenerated by an additional recycling system. These multienzymatic systems based on co-TAs were applied to the synthesis of biologically active compounds. 

Liu, L., et al.. One-step synthesis of 12-ketoursodeoxycholic add from dehydrocholic acid using a multienzymatic system. Appl. Microbiol. Biotechnol., 2013.97(2) 633-639. 

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