Enzymatic synthesis 2-

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 

Mikhailov and J. Smrt, Coll. Czech. Chem. Comm., 1975, 40, 2353. 

When E. coli cells in log phase are subjected to simultaneous osmotic and temperature shock, they can be rendered permeable. If ribonucleoside triphosphates are incubated with H3 P04 and shocked cells, the label is introduced as the -phosphate. With ATP the y-phosphate also becomes labelled. The mechanism is thought to involve polynucleotide phosphorylase, and although the levels of labelling are not very high, the procedure may be synthetically useful. 

Kikuchi, K. Hirai, F. Hishinuma, and K. Sakaguchi, Biochim. Biophys. Acta, 1977, 476, 287. 

Large quantities of hybridization probes can be prepared from small amoimts of DNA by filter priming the DNA fragment is immobilized on a nylon filter using UV light and used as template to prepare radioactive probes in a random primed labelling reaction.309 probes are removed from the filter by heating, purified and used, while the filter-bound DNA may be re-used. 

The amplification by PCR of a DNA segment containing a functional RNA promoter together with an insert (probe sequence) under its control affords double stranded products which are suitable templates for transcription by DNA-dependent RNA polymerase.313 The RNA transcripts obtained are then useful for in situ RNA-RNA hybridization. By in vitro transcription of synthetic DNA templates, RNA hairpin sequences of 16-mer to 22-mer length containing homopolymer loops of 3 to 9 nucleotides, consisting of adenosine, cytidine or uridine residues have been prepared, and their thermal stabilities determined.3 The stability varied with loop 

Finally, synthesis of oligonucleotides by degradation Homologues of oligouridylate have been prepared by successive digestion of RNA with RNase Ti and RNase U2, followed by deamination, to afford oligouridylate sequences with 3 -terminal purine nucleotides, which were subjected to enzymic dephosphorylation, periodate oxidation and P-elimination of the terminal residue, and a second dephosphorylation step.325 xhis afforded (Up)nU species (n = 1 - 7), which were separated chromatographically. 

Gloagen, L. Vebret, L. Hoifmann, H. Morvan, Y. Planeke, J.-M. Wieruszeski, G. Strecker, XVlllInt. Carbohydr. Symp., 1996, Milan, Italy, AP075. 

Suzuki, N. Shibata, M. Suzuki, F. Saitoh, H. Oyamada, H. Kobayashi, S. Suzuki, Y. Okawa, J. Biol. Chem., 1997, 272, 16822-16828. 

Branefors-Helander, L. Kenne, B. Lindberg, K. Petersson, P. Unger, Carbohydr. Res., 1981, 97, 285-291. 

---

C, chemical synthesis Enz, enzymatic synthesis Ext, extraction E, fermentation. Ref. 219. 

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). 

Microemulsions or reverse micelles are composed of enzyme-containing, surfactant-stabiHzed aqueous microdroplets in a continuous organic phase. Such systems may be considered as a kind of immobilization in enzymatic synthesis reactions. 

Dica.rboxyIic AcidMonoesters. Enzymatic synthesis of monoesters of dicarboxyUc acids by hydrolysis of the corresponding diesters is a widely used and thoroughly studied reaction. It is catalyzed by a number of esterases. Upases, and proteases and is usually carried out in an aqueous buffer, pH 6—8 at room temperature. Organic cosolvents may be added to increase solubiUty of the substrates. The pH is maintained at a constant level by the addition of aqueous hydroxide. After one equivalent of base is consumed the monoesters are isolated by conventional means. 

Mono cylDiols. Enzymatic synthesis of chiral monoacyl diols can be carried out either by direct enzymatic acylation of prochiral diols or by hydrolysis of chemically synthesized dicarboxylates. 

Peptide Synthesis. The literature on the enzymatic synthesis of peptides is enormous (120—124). Here the basic principles that govern peptide synthesis are illustrated and recent trends in this area reviewed. 

Sephacryl S-500/S-1000 is an appropriate system to control the extent of enzymatic synthesis of nonbranched a( 1— 4)-linked glucans. The degree of... 

By comparing the dissociation constant of 6-azauracil and 6-aza-uridine with those or uracil and uridine, 6-azauridine is now considered to be 1-ribofuranosyl derivative (2-ribofuranosyl-3,5-dioxo-2,3,4,5-tetrahydro-l,2,4-triazine), The same was shown more exactly by comparing the UV and IR spectra and the dissociation constants of 6-azauridine with the two monomethyl derivatives of 6-aza-uracil," Enzymatic synthesis thus, proceeds, in the same way in natural bases and in their aza analogs. 

In the beginning of the investigation it was believed that enzymatic synthesis of j8-poly(L-malate) follows a route, which is similar to the synthesis of poly(j8-hydrox-ybutyrate) (for a review see [15]). However, enzymati-... 

It is well known that the 1-phosphates of the ketoses, L-fuculose (51) and L-rhamnulose (52) have considerable biochemical interest. Their chemical synthesis has not been described as far as is known to the writer, but the rate of acid hydrolysis of L-fuculose 1-phosphate, obtained by enzymatic synthesis, has been determined by Heath and Ghalambor (20) and that of L-rhamnulose 1-phosphate by H. Sawada (48) and by Chiu and Feingold (II). They found that the rate of... 

Figure A8.13 Two step enzymatic synthesis of L-alanine from fumaric acid.

Shoshani I, Boudou V, Pierra C et al (1999) Enzymatic synthesis of unlabeled and [(3-32P]-labeled p-L-2, 3 dideoxyadenosine-5 -triphosphate as a potent inhibitor of adenylyl cyclases and its use as reversible binding ligand. J Biol Chem 274 34735-34741... 

Scheme 7.5 Enzymatic synthesis of dipeptides on solid phase.

Also the impact of various reaction parameters on enzymatic synthesis of amide surfactants from ethanolamine and diethanolamine has been studied, although the possibilities of acyl migration are not investigated. However, it was found that the selectivity of the reaction depended on the solubility of the product in the solvent used, and that the choice of solvent was critical to obtain an efficient process [17]. 

Scheme 7.6 Solvent-free enzymatic synthesis of fatty alkanolamines.

Enzyme preparations from liver or microbial sources were reported to show rather high substrate specificity [76] for the natural phosphorylated acceptor d-(18) but, at much reduced reaction rates, offer a rather broad substrate tolerance for polar, short-chain aldehydes [77-79]. Simple aliphatic or aromatic aldehydes are not converted. Therefore, the aldolase from Escherichia coli has been mutated for improved acceptance of nonphosphorylated and enantiomeric substrates toward facilitated enzymatic syntheses ofboth d- and t-sugars [80,81]. High stereoselectivity of the wild-type enzyme has been utilized in the preparation of compounds (23) / (24) and in a two-step enzymatic synthesis of (22), the N-terminal amino acid portion of nikkomycin antibiotics (Figure 10.12) [82]. 

Figure 10.26 Short enzymatic synthesis of L-fucose and hydrophobic analogs, and of L-rhamnose, by aldolization-ketol isomerization, including kinetic resolution of racemic hydroxyaldehyde precursors.

Al-Mulla, E.A.J., Yunus, W.M.Z., Ibrahim, N.A., Abdul Rahman, MZ. 2010a. Enzymatic synthesis of fatty amides from palm olein. Journal of Oleo Science 59 59-64. 

De Luca, V. and Ibrahim, R. K. 1985a. Enzymatic synthesis of polymethylated flavonols of Chrys-osplenium americarmm. I. Partial purification and some properties of S-adenosyl-L-methionine flavonol 3-, 6-, 7-, and 4 -0-methyltransferases. Arch. Biochem. Biophys. 238 596-605. 

C. Oligo- and Poly-nucleotides.—The stepwise enzymatic synthesis of internucleotide bonds has been reviewed. A number of polynucleotides containing modified bases have been synthesised " in the past year from nucleoside triphosphates with the aid of a polymerase enzyme, and the enzymatic synthesis of oligodeoxyribonucleotides using terminal deoxynucleotidyl transferase has been studied. Primer-independent polynucleotide phosphorylase from Micrococcus luteus has been attached to cellulose after the latter has been activated with cyanogen bromide. The preparation of insolubilized enzyme has enabled large quantities of synthetic polynucleotides to be made. The soluble enzyme has been used to prepare various modified polycytidylic acids. ... 

Porter, J.W., Enzymatic synthesis of carotenes and related compounds. Pure Appl. Chem. 20, 449, 1969. 

Enzymes are generally classified into six groups. Table 1 shows typical polymers produced with catalysis by respective enzymes. The target macromolecules for the enzymatic polymerization have been polysaccharides, poly(amino acid)s, polyesters, polycarbonates, phenolic polymers, poly(aniline)s, vinyl polymers, etc. In the standpoint of potential industrial applications, this chapter deals with recent topics on enzymatic synthesis of polyesters and phenolic polymers by using enzymes as catalyst. 

In polyester synthesis via ring-opening polymerizations, metal catalysts are often used. For medical applications of polyesters, however, there has been concern about harmful effects of the metallic residues. Enzymatic synthesis of a metal-free polyester was demonstrated by the polymerization of l,4-dioxan-2-one using Candida antarctica lipase (lipase CA). Under appropriate reaction conditions, the high molecular weight polymer (molecular weight = 4.1 x 10" ) was obtained. 

Enzymatic synthesis of aliphatic polyesters was also achieved by the ringopening polymerization of cyclic diesters. Lactide was not enzymatically polymerized under mild reaction conditions however, poly(lacfic acid) with the molecular weight higher than 1 x 10" was formed using lipase BC as catalyst at higher temperatures (80-130°C). Protease (proteinase K) also induced the polymerization however, the catalytic activity was relatively low. 

Activated esters of halogenated alcohols, such as 2-chloroethanol, 2,2,2-trifluoroethanol, and 2,2,2-trichloroethanol, have been often used as substrate for enzymatic synthesis of esters, owing to an increase in the electrophilicity (reactivity) of the acyl carbonyl and avoid significant alcoholysis of the products by decreasing the nucleophilicity of the leaving alcohols. ... 

The enzymatic synthesis of polyesters from activated diesters was achieved under mild reaction conditions. The polymerization of bis(2,2,2-trichloroethyl) glutarate and 1,4-butanediol proceeded in the presence of PPL at room temperature in diethyl ether to produce the polyesters with molecular weight of 8.2 x 10. Vacuum was applied to shift the equilibrium forward by removal of the activated alcohol formed, leading to the production of high molecular weight polyesters. The polycondensation of bis(2,2,2-trifluoroethyl) sebacate and aliphatic diols took place using lipases BC, CR, MM, and PPL as catalyst in diphenyl ether. Under the... 

Enzymatic synthesis and biological properties of flavonoid polymers... 

---