Multienzyme one-pot

The aforementioned enzymatic reaction conditions were utilized to construct the sLe -containing glycopeptides using the multienzyme one-pot, three-step strategy (Scheme 43). Substrates 32 and 33 were incubated with GalT and SiaT in the presence of UDP-Gal and CMP-NeuAc. Subsequently, FucT and GDP-Fuc were added and incubated for another 48 h. After purification, the truncated PSGL-1 glycopeptides with or without sulfation, 36 and 37, were obtained with 43 and 25% yield, respectively. 

Scheme 43 Multienzyme one-pot synthesis of unsulfated and sulfated glycopeptides.

On the other hand, Kren and Thiem have developed a sequential multienzyme one-pot system with cofactor regeneration in order to prepare rather complicated hetero-oligoglycosides such as the sialylated antigen T-epitope... 

Franke, D., Machajewski, T., Hsu, C.-C. and Wong, C.-H. (2003) One-pot synthesis of L-fructose using coupled multienzyme systems based on rhamnulose-1-phosphate aldolase. The Journal of Organic Chemistry, 68 (17), 6828-6831. 

In summary, the concept of multienzyme reactions with integrated cofactor regeneration has been shown to be useful for sequential synthesis of rather complicated heterooligosaccharides. This conception opens up new perspectives for the synthesis of glycosides having up to three or four different glycosyl units in one-pot reactions. 

The 3-deoxy-D-ara6mo-2-heptulosonic acid 7-phosphate (DAHP) synthetase (EC 4.1.2.15) is an enzyme involved in the shikimic pathway of aromatic amino acids biosynthesis in bacteria and plants, where catalyzes the construction of 3-deoxy-D-ara6/ o-2-heptulosonic acid 7-phosphate from phosphoenolpyruvate and D-erythrose 4-phosphate [6]. Although 3-deoxy-D-ara6/H0-2-heptulosonic acid 7-phosphate (DAHP) synthetase has not been widely investigated it has been employed for the DAHP synthesis on preparative scale from D-fructose in multienzyme system [68], This one-pot synthesis was subsequently even more simplified by the results of further studies which indicated that it was more efficient and economical to use the whole cells containing a DAHP synthetase plasmid [69]. 

Multienzyme modular assemblies such as PKSs and NRPSs have flexible swinging tethers which channel covalently bound intermediates between successive active sites. Swinging arms plus specific protein-protein interactions offer mechanisms for the transfer of substrates between modules and offer concepts for the development of one-pot multi-reaction biocatalytic processes. 

K. et al. (2013) Statistical experimental design guided optimization of a one-pot biphasic multienzyme total synthesis of amorpha-4,11-diene. PLoS One, 8, e79650. 

L., Yu, H., Ding, L., Malekan, H., and Chen, X. (2012) Efficient one-pot multienzyme synthesis of UDP-sugars using a promiscuous UDP-sugar pyrophosphorylase from Bifidobacterium longum (BLUSP). Chem. 

Perhaps the first decision to be made in process development is the difficult decision of whether the enzymes to be used should be used in an integrated format. Such a question does not arise with conventional single biocatalytic steps but is highly important in multienzyme processes. One of the key criteria here is whether the enzymes can be operated together without compromise to any of the individual enzyme s activity or stability. An interaction matrix (see Section 10.6) can be used to assist such decision making. In cases where the cost of one or more of the enzyme(s) is not critical, it will be possible to combine in a one-pot operation. In other cases, where the cost of an individual enzyme becomes critical, then it may be necessary to separate the catalysts, such that each can operate under optimal conditions. Likewise, selection of the biocatalyst format (immobilized enzyme, whole cell, cell-free extract, soluble enzyme, or combinations thereof) in combination with the basic reactor type (packed bed, stirred tank, or combinations thereof) and biocatalyst recovery (mesh, microfiltration, ultrafiltration, or combinations thereof) will determine the structure of the process flowsheet and therefore is an early consideration in the development of any bioprocess. The criterion for selection of the final type of biocatalyst and reactor combination is primarily economic and may best be evaluated by the four metrics in common use to assess the economic feasibility of biocatalytic processes [29] ... 

In another context, another remarkable multienzyme cocktail was used for the one-pot synthesis of precorrin-5, starting from 8-aminolevulinic acid. In this transformation, up to eight different enzymes were used, including 5-aminolevulinic acid dehydratase to form porphobilinogen followed by porphobilinogen deaminase, uroporphyringen III synthase, uroporphyringen m methyltransferase, precorrin-2 methyltransferase, precorrin-3 oxidase, pre-corrin-3 hydrojq lactone methyltransferase, and precorrin methyltransfer-ase. The desired enantiopure precorrin-5 was achieved in 30% overall yield, as shown in Scheme 4.24. 

The total synthesis of sialosides by using the chemoenzymatic approach is as follows [74]. Sialic acid itself can be synthesized from ManNAc, mannose, or their derivatives by sialic acid aldolase enzyme through aldol condensation reaction. If ManNAc is chemically or enzymatically modified at C2, C4—C6 positions, sialic acid has structural modifications at C5, C7-C9 positions, respectively. The sialic acids are subsequently activated by a CMP-siahc acid synthetase to form a CMP-sialic acid, which is the donor used by sialyltransferases. Because CMP-sialic acid is tmstable, the CMP-Neu5Ac synthetase is valuable for the preparative enzymatic synthesis of sialosides. In the last steps, the CMP-sialic acid is transferred to galactose or GalNAc terminated glycosides by sialyltransferases to form structurally defined sialosides. Examples are that Chen and co-workers have recently developed a one-pot multienzyme system for the efficient synthesis of a-sialosides (Table 2) [12,76,79]. In this system, recombinant E. coli K-12 sialic acid aldolase catalyzed the synthesis of sialic acid precursors for... 

Table 2 Example of one-pot multienzyme approach for the s)mthesis of sialosides with versatile structural modification... 

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