Transaminase approach

A significant drawback of the transaminase approach is the need for a stoichio... 

Baltzer and co-workers have utilized a synthetic peptide scaffold to incorporate features of transaminase enzymes [ 13]. Using this approach, they have attempted to achieve the selective and tight binding of a pyridoxal phosphate coenzyme observed in transaminase enzymes. A synthetic helix-turn-helix peptide known to dimerize into a four-helix bundle was chosen as the platform for design [5]. 

The liver is also the principal metabolic center for hydrophobic amino acids, and hence changes in plasma concentrations or metabolism of these molecules is a good measure of the functional capacity of the liver. Two of the commonly used aromatic amino acids are phenylalanine and tyrosine, which are primarily metabolized by cytosolic enzymes in the liver [1,114-117]. Hydroxylation of phenylalanine to tyrosine by phenylalanine hydroxylase is very efficient by the liver first pass effect. In normal functioning liver, conversion of tyrosine to 4-hy-droxyphenylpyruvate by tyrosine transaminase and subsequent biotransformation to homogentisic acidby 4-hydroxyphenylpyruvic acid dioxygenase liberates CO2 from the C-1 position of the parent amino acid (Fig. 5) [1,118]. Thus, the C-1 position of phenylalanine or tyrosine is typically labeled with and the expired C02 is proportional to the metabolic activity of liver cytosolic enzymes, which corresponds to functional hepatic reserve. Oral or intravenous administration of the amino acids is possible [115]. This method is amenable to the continuous hepatic function measurement approach by monitoring changes in the spectral properties of tyrosine pre- and post-administration of the marker. 

Although the utility of transaminases has been widely examined, one such limitation is the fact that the equilibrium constant for the reaction is near unity. Therefore, a shift in this equilibrium is necessary for the reaction to be synthetically useful. A number of approaches to shift the equilibrium can be found in the literature.53 124135 Another method to shift the equilibrium is a modification of that previously described. Aspartate, when used as the amino donor, is converted into oxaloacetate (32) (Scheme 19.21). Because 32 is unstable, it decomposes to pyruvate (33) and thus favors product formation. However, because pyruvate is itself an a-keto acid, it must be removed, or it will serve as a substrate and be transaminated into alanine, which could potentially cause downstream processing problems. This is accomplished by including the alsS gene encoding for the enzyme acetolactate synthase (E.C. 4.1.3.18), which condenses two moles of pyruvate to form (S)-aceto-lactate (34). The (S)-acetolactate undergoes decarboxylation either spontaneously or by the enzyme acetolactate decarboxylase (E.C. 4.1.1.5) to the final by-product, UU-acetoin (35), which is meta-bolically inert. This process, for example, can be used for the production of both l- and d-2-aminobutyrate (36 and 37, respectively) (Scheme 19.21).8132 136 137... 

Unfortunately, no useful therapeutic drug has been designed by this approach so far. Inhibiting the transaminase enzyme has no medicinal use since the enzyme is crucial to mammalian biochemistry and inhibiting it would be toxic to the host. The main use for suicide substrates has been in labelling specific enzymes. The substrates can be labelled with radioactivity and reacted with their target enzyme in order to locate the enzyme in tissue preparations. 

Another example is provided by r>-tert-leucine (4) (Scheme 10), where an asymmetric approach cannot be used. This unnatural amino acid is one of the few that cannot be made by our current o-amino acid transaminase. While this enzymatic method is being researched, small amounts of material have been prepared by a resolution method [24]. 

Examples of uimatural L-amino acids that can be accessed by transaminase methodology are L-2-aminobutyric acid, i.-homophcnylalaninc, and L-lert-leu-cine the latter is not accessible by an asymmetric hydrogenation approach because of the absence of a P-hydrogen. 

Researchers at Celgene developed both (R) and (S) selective transaminases that were active on a range of aliphatic and aromatic ketones and amines [25, 55 57]. Two approaches were employed based upon kinetic resolution, which has been discussed above, and asymmetric synthesis. The asymmetric synthesis approach starts with a... 

Another approach involves the addition of lactate dehydrogenase to the reaction that reduces the pyruvate to lactate. In addition, the use of an ion exchange resin allows in situ product recovery of the product amine that reduces product inhibition of the transaminase. 

After the initial discovery that 0-fluoromethylene-substituted amines (e.g., 184, Table 1) were potent, mechanism-based inhibitors of monoamine oxidase (MAO) (41), the concept was successfully broadened to include most of the common amine oxidases (Table 1). This approach was also used to design inhibitors of y-aminobutyric acid transaminase both the a- and 0- substituted amino acids 189 and 190 were found to inactivate this enzyme. Recently, applica-tion of this concept to the design of inhibitors of S-adenosyl-homocysteine hydrolase (SAH) has led to the discovery of very potent inhibitors of this enzyme (e.g., 176, Table 1). 

A different approach led to the development of vigabatrin (Sabril). Its ability selectively and irreversibly to inhibit GABA-transaminase from metabolically inactive GABA (as a suicide inhibitor) makes it an effective agent (Fig. 12-5), particularly in complex partial seizures. 

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