Enzymatic syntheses multiple products

The anticoagulant fondaparinux, a synthetic analogue of the terminal fragment of heparin, is synthesized using multiple protection/deprotection steps that result in a route of up to 50 steps. There is, as yet, no enzymatic system that approaches the capability to make such a molecule." As this modified pentasaccharide is a natural product, it should, in theory, be accessible through a series of biotransformations, but we currently lack the biocatalytic tools to achieve more than a few steps and would stiU need to use some protection steps to avoid multiple products. Enzymatic synthesis in vivo depends largely on the levels and selectivities of glycosylating enzymes to achieve multistep reactions, a situation that has been mimicked in vitro for simpler systems." ... 

The enzymatic synthesis of DNA has been described using a circular DNA template. Multiple copies of the template are produced without the requirement for the heating and cooling cycles associated with PCR. The concatermeric products can be cleaved with enzymes to yield shorter multiple copies of oligonucleotides. An automated multiplex DNA synthesiser has been developed which can simultaneously and rapidly synthesise up to 96 different oligonucleotides. ... 

Synthesis of DNA. An enzymatic synthesis of polymers of deoxy-ribonucleotides has been described recently. Enzyme preparations from E. coli form acid-insoluble products that are digestible by DNAase. Study of the reaction was complicated by multiple requirements, but extensive purification has permitted the determination of some of the properties of the system. The enzyme is relatively inactive when a single nucleotide is used as a substrate, and appears to have a requirement for the simultaneous presence of four deoxynucleoside triphosphates, deoxy ATP, deoxy GTP, deoxy CTP, and deoxy TTP. Only the triphosphates, not the corresponding disphosphates, are active. In addition to nucleotides, a primer is required. DNA from various sources serves as primer Mg++ is also required. The products of the reaction are polymer and inorganic pyrophosphate. Reversibility has been indicated by experiments in which labeled inorganic pyrophosphate was incorporated into the nucleotides. 

JAs are derived from linolenic acid via an octadecanoid pathway consisting of several enzymatic steps (Figure 36). Multiple compartments in plant cells participate in JA synthesis. The early steps of this pathway occur in chloroplasts, where linolenic acid is converted to OPDA by means of the three enzymes lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC).867-869 Linolenic acid is oxygenated by 13-LOX producing a peroxidized fatty acid 13-hydroperoxylinolenic acid. The product is subsequently metabolized by AOS to an unstable compound allene oxide. Allene oxide is sequentially converted by AOC to produce OPDA. An alternative pathway from another trienoic fatty acid, hexadecatrienoic acid (16 3), is present in chloroplasts.870 In this pathway, dinor OPDA is produced instead of OPDA. OPDA and dinor OPDA are transported into the peroxisome. An ABC transporter involved in this transport was identified in... 

One of the most exciting areas in the field of multifunctional enzyme systems is the synthesis of a wide array of organic molecules by polyketide and nonribosomal protein synthetases. These enzymes are generally characterized by multiple subunits which themselves consist of individual domains with distinct enzymatic activities (Fig. 9.11a). The range of natural products synthesized by these mega-synthetases includes a considerable number of important antibiotics, antifungals, antitumor and cholesterol-lowering compounds, immunosuppressants, and siderophores. 

Modular PKS enzymes are responsible for the synthesis of a wide diversity of structures and seem to have more relaxed specificities in several of the enzymatic steps. Their enormous appeal for combinatorial purposes, though, derives from the presence of multiple modules that can be manipulated independently, allowing the production of rings of different sizes and with potential stereochemical variation at each PK carbon. The higher complexity of these pathways has somewhat hindered their exploitation, but recently, several have been fully characterized. Among them, by far the most studied modular multienzyme complex is 6-deoxyerythronolide B synthase (DEBS 240,266,267), which produces the 14-member macrolide 6-deoxyerythronolide B (10.70, Fig. 10.45). DEBS contains three large subunits each of which contains two PKS enzyme modules. Each module contains the minimal PKS enzyme vide supra) and either none (M3), one (ketoreductase KR Ml, M2, MS, and M6), or three (dehydratase DH-enoyl reductase ER-ketoreductase KR, M4) catalytic activities that produce a keto (M3), an hydroxy (Ml, M2, MS and M6), or an unsubstituted methylene (M4) on the last monomeric unit of the growing chain (Fig. 10.45). A final thioesterase (TE) activity catalyzes lactone formation with concomitant release of 10.70 from the multienzyme complex. Introduction of TE activity after an upstream module allows various reduced-size macrolides (10.71-10.73, Eig. 10.45) to be obtained. 

Figure 5 Chemoenzymatic approaches for the production of novel bioactive compounds. In this example, the enzymatic buildup of the linear precursor of daptomycin by its NRPSs (DptA, DptBC, and DptD) is substituted by solid-phase synthesis (a). By using the 4 Ppan transferase Sfp and the CoA-thioester of the linear peptide, the opo-enzyme PCP-TE and be modified, and after trans-esterification cyclized by the TE domain (b). Because the resulting ho/o-enzyme cannot be modified again, this is a single turnover reaction. Another strategy uses thiophenole-esters of the linear peptides to be cyclized (c). When these compounds are used, no PCP domain is necessary. The TE domain is readily acylated, and regiospecific and stereospecific cyclization toward daptomycin or, depending on the linear peptide provided, toward variants thereof occurs. Because the enzyme is not altered in any way after product release, this setup results in a multiple turnover.

Organisms in which resistance has increased most dramatically include enterococci, pneumococci, and Mycobacterium tuberculosis. Enterococci have been isolated with multiple resistance patterns. They may be resistant to /S-lactams (by virtue of -lactamase production, altered penicillin-binding proteins [PBPs], or both), vancomycin (via alterations in peptidoglycan synthesis), and high levels of aminoglycosides (via enzymatic degradation). 

Heterocydes are common motifs in natural products, which may occur as single, tandem, and multiple moieties within a given molecule (Scheme 8.5) [35-37]. These motifs often provide molecular interaction with nudeotide and protein targets. In the biosynthesis of NRPs, an oxazoline was usually formed from a dipeptide containing serine in the second position upon dehydration (Scheme 8.5) [38]. The syntheses of a thiazoline from cysteine and a 2-methyloxazoline from threonine follow a similar mechanism. These heterocycles can be further custom-made to provide thiazolidines/oxazolidines upon reduction or thiazoles/oxazoles upon oxidation. Enzymatic heterocyclization can be portable, as demonstrated in the synthesis of novel chiral heterocyclic carboxylic adds by hybrid enzymes [39]. 

The first type of cascade reaction is sequential synthesis which describes the usage of multiple consecutive catalytic steps for building up complex stractures. Each step may be performed under different reaction conditions however, no purification of the intermediate products is necessary. Herein chemical catalytic steps may be combined with enzymatically catalyzed ones or the cascade may be operated with enzymes solely. 

Sequential biocatalytic cascade reactions are characterized by the use of multiple enzymatic steps involving various biocatalysts. One cascade reaction can consist of an enzyme-module with several enzymes if substrate and inhibitor kinetics are compatible with these combinations. Sequential use of such enzyme-modules surpassing the work-up of intermediate products is the criterion for the idea of cascade reactions we address here on the one hand, the synthesis of nucleotide sugars and their derivatives, on the other hand, the synthesis of glycan epitopes with multiple GTs. 

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