Mark A. Findeis

Chapter 26. Enzymic Methods in Organic Synthesis

Publisher Summary This chapter discusses the procedures that use partially or highly purified enzymes to catalyze organic reactions potentially useful in medicinal chemistry. A widespread interest in asymmetric synthesis has focused attention on the demonstrated utility of enzymic catalysis in producing chiral fragments. In fact, the major hindrance to the widespread use of enzymic catalysis is the residual unfamiliarity of many classically trained synthetic chemists in the techniques of enzyme isolation, manipulation, and assay. More than two thousand enzymes are known and several hundred can be obtained commercially. Given sufficient demand, many enzymes can be produced in quantity by recombinant DNA techniques. Enzymes used in synthetic applications are commonly immobilized in or on insoluble materials because immobilization enhances their stability and allows their recovery and reuse. Enzyme stabilization is a concern before, during, and after immobilization. Immobilization often greatly increases the stability of enzymes. Reasons for this stabilization are not clearly understood. Enzyme deactivation during the immobilization process is often troublesome. Enzymic phosphorylation with coupled in situ adenosine triphosphate (ATP) regeneration has been used to prepare glucose-6-phosphate, sn-glycerol-3-phosphate, creatine-phosphate, and 5-phosphoribosyl-1-pyrophosphate. Nicotinamide cofactor dependent oxidoreductases have been applied in chiral synthesis. Enantioselectivity is often only moderate, but in certain cases excellent results have been obtained. Enzymic synthetic methods will see increased use in research and industry. Immunology, neurobiology, endocrinology, molecular genetics, membrane biology, and plant and insect biology are areas that are becoming more molecular in scope.

Protein synthesis in cell-free reticulocyte lysates on multi-hour incubation

Cell-free protein synthesis in rabbit reticulocyte lysate translation mixtures has been studied during multi-hour incubations. In an impaired lysate obtained from cells stored at 0°C before lysis, and showing a low initial rate of synthesis, translation could be stimulated by a factor of 4 by including RNase inhibitor and additional ATP and GTP. In translation mixtures prepared from normal lysates, protein synthesis could be improved by ∼50% by the addition of excess GTP. The observed increases in protein synthesis were obtained by improved maintenance of the initial rate of synthesis.