Combined Chemical-Enzymatic Syntheses

Synthetic oligonucleotides may be used as primers and be elongated stepwise with the aid of polynucleotide phosphorylase (PNPase) and nucleoside diphosphates. 

PCR can also be used to modify DNA sequences using primers differing at one or several positions from the target sequence. This is possible because PCR does not require perfect complementarity of a primer to the sequence flanking the target. Since all of the PCR products contain the primer sequence, an insertion or deletion can thus be incorporated into the product by modifying a primer. It is also possible to add new sequences to the 5 -ends of the primers. Modified or additional genetic information may thus be multiplied and transported. 

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The manipulation of DNA has been revolutionized by a combination of enzymatic synthesis and solid-state chemical synthesis ( glue technology ). Two enzyme types are involved in DNA synthesis. 

Similar procedures could be used for the efficient synthesis of F-labeled sugars which are useful as tracers (28). The combined chemical/enzymatic approach also provides new routes to a number of potentially useful antiviral and antibacterial fluorosugars and derivatives (29,30) ... 

For combined chemical and enzymatic synthesis, see Sabesan, S. Paulson, J. 

Several enzymatic procedures have been developed for the synthesis of carbohydrates from acyclic precursors. Aldolases appear to be useful catalysts for the construction of sugars through asymmeteric C-C bond formation. 2-deoxy-KDO, 2-deoxy-2-fluoro-KDO, 9-0-acetyl sialic acid and several unusual sugars were prepared by a combined chemical and enzymatic approach. Alcohol dehydrogenases and lipases have been used in the preparation of chiral furans, hydroxyaldehydes, and glycerol acetonide which are useful as building blocks in carbohydrate synthesis. 

Another important technique was based on the observation that synthetic trinucleotides induced the binding to ribosomes of tRNA molecules that were "charged" with their specific amino acids 38/39 For example, the trinucleotides UpUpU and ApApA stimulated the binding to ribosomes of 14C-labeled phenylalanyl-tRNA and lysyl-tRNA, respectively. The corresponding dinucleotides had no effect, an observation that not only verified the two codons but also provided direct evidence for the triplet nature of the genetic code. Another powerful approach was the use of artificial RNA polymers, synthesized by combined chemical and enzymatic approaches.40 For example, the polynucleotide CUCUCUCUCU led to the synthesis by ribosomes of a regular alternating polypeptide of leucine and serine. 

Fig. 1. In vitro selection scheme, showing an outline of the steps involved in the in vitro selection of functional aptamers. Sequences from an RNA pool created by combined chemical and enzymatic synthesis are partitioned according to their abilities to perform a desired task. Unfit RNAs are discarded, and the RNAs that are fit to do the task are reverse transcribed, PCR amplified, and regenerated through in vitro transcription. Multiple cycles of selection and amplification should result in the selective enrichment ofthe fittest species.

For this goal, more fundamental mechanistic studies of redox processes at BDD electrodes will be necessary. With chemically and mechanically more stable BDD/support combinations, preparative organic synthesis and enzymatic applications will conquer the field of research and will bring chemical innovation in a short time to application. 

Wang LX, Tang M, Suzuki T, Kitajima K, Inoue Y, Inoue S, Fan JQ, Lee YC. Combined chemical and enzymatic synthesis of a C-glycopeptide and its inhibitory activity toward glycoamidases. J. Am. Chem. Soc. 1997 119 11137-11146. 

Asano et al. [205] used a combination of enzymatic and chemical reactions to synthesize eight possible mono-j8-D-glucosides of validoxylamine A. In another synthesis by Ogawa and coworkers [206], treatment of validoxylamine A or its per-O-benzylated derivative with NBS under different conditions resulted in the formation of mixtures of keto- and aminocyclitol derivatives (e.g., 369 and 370). These compounds were used to prepare the dimeric aminocyclitol 371, which is a potent trehalose inhibitor (compounds 369-371). 

Although, in principal it is possible to perform totally enzymatic synthesis of peptides, in practice combined chemical and enzymatic steps are preferred. For the classical enzymochemical synthesis of polypeptides and even small proteins, the optimum approach is usually synthesis of fragments using the SPPS methodology... 

The biologically active monosaccharide 3-deoxy-D-ura6//io-heptulosonic acid 7-phosphate (8 DAMP) is an important intermediate in the biosynthesis of aromatic amino acids in plants (the shikimate pathway). As shown in Scheme 2, this compound has been produced in a combined chemical and enzymatic synthesis from racemic V-acetylaspartate 3-semialdehyde (4) and DHAP (1). The four-step synthesis proceeds in an overall yield of 13% (37% for the aldolase reaction). The enzymatic step generates the required, enantiomerically pure, syn aldol adduct compound (5). In view of the broad range of substrates tolerated by FDP aldolase, this method may be applicable to the production of analogs of DAMP. 

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