Enzymatic one-pot synthesis

Figure 10.15 Enzymatic one-pot synthesis of D-tagatose 1,6-bisphosphate based on the stereoselective TagA m coli.

Tricand de la Goutte, J Khan, JA Vulfson, EN. Identification of novel polyphenol oxidase inhibitors by enzymatic one-pot synthesis and deconvolution of combinatorial Mhidiiies. Biotechnology and Bioengineering, 2001 75, 93-99. 

W. D. Fessner and O. Eyrisch, One-pot synthesis of tagatose 1,6-bisphosphate by diastereoselec-tive enzymatic aldol addition, Angew. Chem. lnt. Ed. Engl. 31 56 (1992). 

Sequential one-pot synthesis Reactions occur sequentially Variable reaction conditions (e.g., pH, T°C) No isolation of intermediate products Regioselective enzymatic protection and chemical acetylation 15... 

Nakano, H., Shizuma, M., Murakami, H., Kiryu, T., and Kiso, T. 2005. One-pot synthesis of glycosyl poly(arbutin) by enzymatic glycosylation followed by polymerization with peroxidase. J. Mol. Catal. B Enzym., 33,1-8. 

S., and Martinelle, M. (2009) Enzymatic one-pot route to telechelic polypentadecalactone epoxide synthesis, UV curing, and characterization. Biomacromolecules,... 

CMP-N-acetylneuraminic acid (CMP-NeuAc). CMP-N-acetylneuraminic acid has been prepared enzymatically on small scales (> 0.5 mmol) from CTP and NeuAc, under catalysis by CMP-NeuAc synthetase (EC 2.7.7.43) [131l An improvement in this procedure, involving in situ production of CTP from CMP under adenylate kinase and pyruvate kinase catalysis, is suitable for multigram-scale synthesis11321. Adenylate kinase catalyzes the equilibration of CTP and CMP to CDP, which is subsequently phosphorylated by pyruvate kinase to provide CTP. A one-pot synthesis of CMP-NeuAc based on this procedure involves the in situ synthesis of NeuAc from N-acetylmannosamine and pyruvate, catalyzed by sialic acid aldolase (Fig. 11.3-12)[10S1. Chemical syntheses of CMP-NeuAc have also been reported11421. 

Unfortunately, this process cannot be performed in a more elegant and more efficient one-pot synthesis. On the one hand, the pH optima for the three enzymes are not compatible with each other, and on the other, lactate dehydrogenase is air sensitive. In addition to this, glycolate oxidase also catalyzes the reverse reaction under aerobic conditions, thus lowering the ee-value. Therefore, the reaction mixture is filtered (glycolate oxidase can be reused) and, after pH adjustment, the second enzymatic transformation is performed. Table 16.2-8 shows some results of this procedure. 

Heise, Palmans, de Geus, Villarroya and their collaborators (17,41,42) have been working on a chemoenzymatic cascade synthesis to prepare block copolymers. They combine enzymatic ring-opening polymerization (eROP) and atom transfer radical polymerization (ATRP). The synthesis of block copolymers was successful in two consecutive steps, i.e., eROP followed by ATRP. In the one-pot approach, block copolymers could be obtained by sequential addition of the ATRP catalyst, but side reactions were observed when all components were present from at the onset of reactions. A successful one-pot synthesis was achieved by conducting the reaction in supercritical carbon dioxide. 

Hilker et al (44) combined dynamic kinetic resolution with enzymatic polycondensation reactions to synthesize chiral polyesters from dimethyl adipate and racemic secondary diols. The concept offered an efficient route for the one-pot synthesis of chiral polymers from racemic monomers. Palmans at al (18,43) generalized the approach to Iterative Tandem Catalysis (ITC), in which chain growth during polymerization was effected by two or more intrinsically different catalytic processes that were compatible and complementary. 

We investigated the chemoenzymatic synthesis of block copolymers combining eROP and ATRP using a bifunctional initiator. A detailed analysis of the reaction conditions revealed that a high block copolymer yield can be realized under optimized reaction conditions. Side reactions, such as the formation of PCL homopolymer, in the enzymatic polymerization of CL could be minimized to < 5 % by an optimized enzyme (hying procedure. Moreover, the structure of the bifunctional initiator was foimd to play a major role in the initiation behavior and hence, the yield of PCL macroinitiator. Block copolymers were obtained in a consecutive ATRP. Detailed analysis of the obtained polymer confirmed the presence of predominantly block copolymer structures. Optimization of the one-pot procedure proved more difficult. While the eROP was compatible with the ATRP catalyst, incompatibility with MMA as an ATRP monomer led to side-reactions. A successfiil one-pot synthesis could only be achieved by sequential addition of the ATRP components or partly with inert monomers such as /-butyl methacrylate. One-pot block copolymer synthesis was successful, however, in supercritical carbon dioxide. Side reactions such as those observed in organic solvents were not apparent. 

One-pot synthesis focuses on the combination of multiple catalytic steps in one reaction vessel under the same reaction conditions. Optimization of the reaction conditions to suit all involved catalysts is crucial to reach shorter reactions times than for the sequential reaction type. Again, here the combination of chemical steps and enzymatic steps in one-pot is possible. 

Schoevaart, R., van Rantwijk, F., and Sheldon, R.A. (2000) A four-step enzymatic cascade for the one-pot synthesis of non-natural carbohydrates from glycerol. J. Org. Chem., 65, 6940-6943. 

Scheme 19.2 One-pot synthesis of D-mannitol based on combination of enzymatic isomerization of D-glucose and heterogeneous hydrogenation.

Scheme 19.22 One-pot synthesis of a poly(e-caprolactone)-type polyester based on combination of an initial amine-initiated lactone opening and subsequent enzymatic polymerization.

Scheme 19.27 One-pot synthesis of a cyclic malonic acid monoester derivative based on combination of metathesis and enzymatic ester hydrolysis.

Further studies by the same research group led to the sequential enzymatic reduction of diketone 40 to the one-pot synthesis of two stereoisomers of the corresponding diols 42a and 42b, in high chemical yields followed by excellent selectivities (Scheme 12.21) [56]. 

More recently, Heise and coworkers have shown that DKR can be combined with enzymatic polymerization for the synthesis of chiral polyesters from racemic secondary diols in one pot [34] (Figure 4.12). 

A tandem enzymatic aldol-intramolecular Homer-Wadsworth-Emmons reaction has been used in the synthesis of a cyclitol.310 The key steps are illustrated in Scheme 8.33. The phosphonate aldehyde was condensed with dihydroxyacetone phosphate (DHAP) in water with FDP aldolase to give the aldol adduct, which cyclizes with an intramolecular Horner-Wadsworth-Emmons reaction to give the cyclo-pentene product. The one-pot reaction takes place in aqueous solution at slightly acidic (pH 6.1-6.8) conditions. The aqueous Wittig-type reaction has also been investigated in DNA-templated synthesis.311... 

A classical approach to driving the unfavorable equilibrium of an enzymatic process is to couple it to another, irreversible enzymatic process. Griengl and coworkers have applied this concept to asymmetric synthesis of 1,2-amino alcohols with a threonine aldolase [24] (Figure 6.7). While the equilibrium in threonine aldolase reactions typically does not favor the synthetic direction, and the bond formation leads to nearly equal amounts of two diastereomers, coupling the aldolase reaction with a selective tyrosine decarboxylase leads to irreversible formation of aryl amino alcohols in reasonable enantiomeric excess via a dynamic kinetic asymmetric transformation. A one-pot, two-enzyme asymmetric synthesis of amino alcohols, including noradrenaline and octopamine, from readily available starting materials was developed [25]. 

Krauueer, M., Hummel, W. and Groeger, H. (2007) Enantioselective one-pot two-step synthesis of hydrophobic allylic alcohols in aqueous medium through the combination of a Wittig reaction and an enzymatic ketone reduction. European Journal of Organic Chemistry, (31), 5175—5179. 

An impressive one-pot six-step enzymatic synthesis of riboflavine from glucose on the laboratory scale has been reported with an overall yield of 35-50%. Six different enzymes are involved in the various synthesis steps, while two other enzymes take care for the in situ cofactor regenerations [12]. This example again shows that many more multi-enzyme cascade conversions will be developed in the near future, as a much greater variety of enzymes in sufficient amounts for organic synthetic purposes will become available through rapid developments in genomics and proteomics. 

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