Phosphate esters enzymic synthesis

Three types of synthases catalyze the addition of phosphoenolpyruvate (PEP) to aldoses or the corresponding terminal phosphate esters. By concurrent release of inorganic phosphate from the preformed enolate nucleophile, the additions are essentially irreversible. None of the enzymes are yet commercially available and little data are available oil the individual specificities for the aldehydic substrates. A bacterial NeuAc synthase (EC 4.1.3.19) has been used for the microscale synthesis of A -acetylncuraminic acid from Af-acetyl-D-mannosamine31 and its 9-azido analog from 2-acetamido-6-azido-2,6-dideoxy-D-mannose32. 

Furthermore, the GPO procedure can also be used for a preparative synthesis of the corresponding phosphorothioate (37), phosphoramidate (38), and methylene phosphonate (39) analogs of (25) (Figure 10.20) from suitable diol precursors [106] to be used as aldolase substrates [102]. In fact, such isosteric replacements of the phosphate ester oxygen were found to be tolerable by a number of class I and class II aldolases, and only some specific enzymes failed to accept the less polar phosphonate (39) [107]. Thus, sugar phosphonates (e.g. (71)/(72)) that mimic metabolic intermediates but are hydrolytically stable to phosphatase degradation can be rapidly synthesized (Figure 10.28). 

A practical, inexpensive one-step procedure was developed for the RhaD-catalyzed gram-scale synthesis of L-fructose. The requirement for DHAP as the donor substrate was circumvented by use of borate buffer, presumably by in situ formation of borate esters as a phosphate ester mimic. Racemic glyceraldehyde was also used, as the enzyme preferentially accepted the L-enantiomer as a substrate. The method can also be apphed to other products, including L-rhamnulose, and towards a two-step synthesis of L-iminocychtols. ... 

Considerable ingenuity was required in both the synthesis of these chiral compounds695 697 and the stereochemical analysis of the products formed from them by enzymes.698 700 In one experiment the phospho group was transferred from chiral phenyl phosphate to a diol acceptor using E. coli alkaline phosphatase as a catalyst (Eq. 12-36). In this reaction transfer of the phospho group occurred without inversion. The chirality of the product was determined as follows. It was cyclized by a nonenzymatic in-line displacement to give equimolar ratios of three isomeric cyclic diesters. These were methylated with diazomethane to a mixture of three pairs of diastereoisomers triesters. These dia-stereoisomers were separated and the chirality was determined by a sophisticated mass spectrometric analysis.692 A simpler analysis employs 31P NMR spectroscopy and is illustrated in Fig. 12-22. Since alkaline phosphatase is relatively nonspecific, most phosphate esters produced by the action of phosphotransferases can have their phospho groups transferred without inversion to 1,2-propanediol and the chirality can be determined by this method. 

It appears that phosphorus is related to the formation of starch in the plant. Thus Hanes has synthesized a linear polysaccharide from a-D-glucopyranosc 1-phosphate (Cori ester) through the action of potato phosphorylase. Dunlap and Beckmann and likewise Cori have found that the B-fraction activates this enzymic synthesis, while the A-frac-tion is inactive. It has not been established whether this effect is due to the branched character of the B-fraction or to the presence of phosphate in its structure. 

In 1952 G. M. Shmm, head of the British Research Council on the campus of the University of British Columbia, Vancouver, Canada, offered Khorana the opportunity to form his own research group on whatever topic he wished. His group became very successful in developing methods for synthesizing phosphate ester derivatives of nucleic acids, and in 1959 he and John G. Moffatt announced the synthesis of acetyl coenzyme A (acetyl CoA), a molecule essential to the biochemical processing of proteins fats and carbohydrates. Prior to this work, the coenzyme had to be extracted from yeast by a very laborious and expensive process, so this discovery led to Khorana s international recognition within the scientific community and he received many job offers as a result. He accepted the position of codirector of the Institute for Enzyme Research at the University of Wisconsin. 

Vitamin B6 has been shown to be essential in many biochemical reactions that occur in plants and animals. Although it may occur in any one of the three forms listed above, the compound usually acts as the phosphate ester, pyridoxine phosphate. Pyridoxine phosphate functions as a coenzyme in the transformation of amino acids, the building blocks from which proteins are made. A coenzyme is a chemical compound that works with an enzyme to catalyze some essential chemical reaction in the body. Pyridoxine phosphate appears to be necessary for the synthesis of proteins from amino acids as well as the metabolism of amino acids to produce energy needed for normal body functioning. 

Whereas enzymes synthesize DNA and RNA in a 5 to 3 direction, chemical oligonucleotide synthesis is carried out in the opposite, 3 to 5 direction using a solid phase approach. Solid phase polynucleotide synthesis requires stepwise creation of a phosphate ester intemucleotide linkage. Currently, phosphoramidite... 

From my laboratory and the laboratories of Ef Racker, Frank Dickens and Melvin Calvin, the years that followed witnessed a series of parallel and often highly synergistic discoveries on the nature of the pentose phosphate pathway and the path of carbon in photosynthesis. Andrew Benson and others in Calvin s laboratory, had shown that phosphate esters of ribulose and sedoheptulose were early products of CO2 fixation in photosynthesis,and the immediate precursor of phosphoglyceric acid, and therefore the primary CO2 acceptor, appeared to be ribulose diphosphate. The major problems became (1) to find the enzyme or enzymes that catalyzed the formation of phosphoglyceric acid from ribulose diphosphate and (2) to define the reactions leading to the synthesis of ribulose diphosphate from triose and hexose phosphates. 

Present data indicates that chain growth in both gramicidin S and the tyrocidines commences at the D-phenylalanine residue adjacent to proline and that the first step in the synthesis is the conversion of L-phenylalanine to D-phenyldanine. In the case of the tyrocidines, the synthesis then proceeds in order from the amino to the carboxyl terminus to form a linear decapeptide ending with a thiol ester linked leucine. The peptide then cyclises relatively slowly to the final product. Yamada and Kurahashi showed that the epimerisation of L-phenylalanine in the initial step did not require pyridoxal phosphate or FAD and they suggested that the reaction occurs via the thiol ester enzyme bound form (101), Figure 3.18. 

Recent investigations indicate that synthesis of sucrose in plants is not accomphshed in the same way as in P. saccharophila. Despite numerous attempts, no enzyme system that would combine D-glucose-1-phosphate and D-fructose to form sucrose and inorganic phosphate could be isolated from the tissues of higher plants. However, biochemical studies on various species of plants support the view that the synthesis of sucrose may involve chemical reactions in which phosphate esters of both n-glucose and D-fructose serve as substrates. It is also significant that the experimental evidence shows that aerobic metabofism is indispensable for the synthesis of sucrose in the plant. Possibly aerobic oxidations are essential to the phosphorylation of one of the substrates involved in the synthesis. 

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