Enzymatic synthesis methods

Different types of antitemplates can be and, presumably, will be, synthesized by employing other chemical reactions that are sufficiently mild and selective for the modification of polynucleotides. Another approach to this problem is, of course, to synthesize an appropriately modified nucleoside triphosphate and utilize it as one of the substrates in a DNA or RNA polymerizing system, in the presence of the specific template for which an antitemplate is to be made. The enzymatic synthesis method has some obvious advantages however, the inhibitory activity of the product (antitemplate) may limit its applications. 

There are stUl other opportunities in using elegant enzymatic synthesis methods, for instance, the hydrolysis ofN-acetyl-(D)-amino acids from racemic mixtures by (L)-acylase cleavage, by dynamic kinetic resolution, or by employing a race-mase and a hydantoinase. 

The examples given in this chapter demonstrate that enzymatic synthesis methods are superior to chemical interesterifications for the production of... 

In addition to their in vivo function, these cofactors have important in vitro functions as co-factors in enzymatic synthesis. Because these co-factors are too expensive to be used in equimolar amounts, many methods have been developed for their regeneration. These include chemical (66), biological (67) and electrochemical (68) methods. En2ymatic regeneration has found particular utihty in this appHcation (69). 

Fig. 12. Enzymatic synthesis of polyester macromonomer and telechelics by terminator method...

Enzymatic Synthesis.—The total synthesis by chemical and enzymatic methods of the structural gene for the precursor of a tyrosine suppressor tRNA from E. coli, a DNA duplex that is 126 nucleotides long, has been described. This, Khorana s Meisterwerk to date, represents the highest state of the art, and proceeds by methods which are already famous, and the touchstone of those who wish to follow.135... 

This section describes the synthesis of oxazolidine esters used as polymer hardeners that cannot be synthesized using chemical catalysis, the synthesis of polyurethane polymers with methods that avoid the use of isocyanates and the enzymatic synthesis of polyesters with low molecular weight dispersity. 

The polymerization of 1 mentioned above should be compared with the enzymatic synthesis of chitin reported by Kobayashi and coworkers, in which an oxazoline derivative of A, A -diacetylchitobiose, the repeating unit of chitin was polymerized in the presence of chitinase enzyme via ring-opening addition process to give an artihcial chitin (Scheme 6) [5]. The method using an enzyme, however, may not enable synthesis of nonnatural-type aminopolysaccharide because the reaction catalyzed by chitinase enzyme is limited to the formation of (1 4)-P-glycosidic linkage. 

In 1990, Triantaphylidds and coworkers reported on the preparative enzymatic synthesis of hnoleic acid (135) hydroperoxide 24a using soybean lipoxygenase-1. In this dioxygenation asymmetry is induced by the catalyst, the enzyme. The reaction was later used by Dussault and also by Baba and coworkers as key step in the preparation of more complex peroxides. The enzyme is a non-heme iron dioxygenase which catalyzes the incorporation of dioxygen into polyunsaturated fatty acids to yield E,Z conjugated diene hydroperoxides 24a-d. With this enzymatic method, the hydroperoxide 24a could... 

For the purpose of synthesizing flavor peptides or proteins in large scale, we developed "protein recombination method" and "enzymatic synthesis using chemically modified enzyme". "Protein recombination method" was applied to the synthesis of C-terminal portion of p-casein and its analog. Chymotrypsin was chemically modified by Z-DSP in aqueous solution. It was stable for organic solvents. Using this modified enzyme, we succeeded in the synfiiesis of Inverted-Aspartame-Type Sweetener "Ac-Phe-Lys-OH" in one step. 

Today, it is well-known that peptides or proteins exhibit various kinds of taste. Our group has been researching on the relationship between taste and structure of peptides, BPIa (Bitter peptide la, Arg-Gly-Pro-Pro-Phe-Ile-Val) (7 as a bitter peptide, Om-p-Ala-HCl (OBA), Om-Tau-HCl as salty peptides(2j, and "Inverted-Aspartame-Type Sweetener" (Ac-Phe-Lys-OH) as a sweet peptide(5). The relationship between taste and chemical structure was partly made clear. Since commercial demand for these flavor peptides is increasing, we need to develop new synthetic methods which can prepare these peptides in large scale. We developed the following two methods (1) protein recombination method as a chemical method, (2) enzymatic synthesis using chemically modified enzyme as a biochemical method. 

Flavor notes and products of lipid oxidation in cooked meats, 85,86/ Flavor peptides convenient synthesis, 149-156 enzymatic synthesis, 151,154-156 protein recombination synthetic method, 149... 

Cooper, J.R. Nishino, K. Enzymatic synthesis of thiamin triphosphate. Methods EnzymoL, 122, 24-29 (1986)... 

Smaller peptides may be joined to form longer ones.298 Also useful is enzymatic synthesis. Proteinhydrolyzing enzymes under appropriate conditions will form peptide linkages, for example, joining together oligopeptides.302 303 Other new methods have been devised to join unprotected peptides.304 305... 

For example, a functional 17-bp gene for the 53-residue human epidermal growth factor was synthesized by joining ten oligonucleotides of lengths 11-59 bp.575 DNA is often synthesized enzymatically using methods described in Chapter 26. Cloned sequences of synthetic DNA can also be transcribed to produce polyribonucleotides of any desired sequence.583 New nonenzymatic methods for RNA synthesis have also been devised.583-587... 

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