Phenylalanine as a Precursor

Phenylalanine as a Precursor.— Tropic acid is found in Nature as the add moiety of the ester alkaloids hyoscyamine and hyosdne. A large amount of information, some conflicting, has been published in the past decade on the origin of this relatively simple molecule. We will review these data critically in the hope that it will aid work on the currently unsolved problem of its biosynthesis. It was discovered in 1960 that the administration of [3- C]phenylalanine to intact Datura stramonium plants yielded tropic acid having essentially all its activity at C-2. Later workers confirmed this result in D. stramonium (intact plants) and D. metel (sterile root cultures). It was then established that the other carbons of the phenylalanine side-chain were used for the production of the side-chain of tropic acid. The pattern of labelling found in the tropic 

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The chemistry and nomenclature of these compounds are somewhat confusing, and are best understood by reference to the synthetic route used by plants to make ephedrine. All ephedra plants contain phenylalanine-derived alkaloids. Plants use phenylalanine as a precursor, but incorporate only seven of its carbon atoms. Phenylalanine is metabolized to benzoic acid, which is then acetylated and decarboxylated to form pyruvic acid. Transamination, results in the formation of forms (-)-cathinone. 

Erythrina berterona. However, more recently a hydroxylase has been isolated from spinach which catalyses the direct conversion of L-phenylalanine to L-tyrosine . Studies on the biosynthesis of selected alkaloids using isotopically labelled L-phenylalanine as a precursor also indicated that hydroxylation of L-phenylalanine to give L-tyrosine is possible in peyote and barley . In barley, for example, it was established that L-phenylalanine, L-tyrosine and tyramine (45) were precursors of the alkaloid hordenine (47) and the biosynthetic sequence (42 - 44 45 - 46 47) was inferred. 

Consider one small molecule, phenylalanine. It is an essential amino acid in our diet and is important in protein synthesis (a component of protein), as well as a precursor to tyrosine and neurotransmitters. Phenylalanine is one of several amino acids that are measured in a variety of clinical methods, which include immunoassay, fluorometry, high performance liquid chromatography (HPLC see Section 4.1.2) and most recently MS/MS (see Chapter 3). Historically, screening labs utilized immunoassays or fluorimetric analysis. Diagnostic metabolic labs used the amino acid analyzer, which was a form of HPLC. Most recently, the tandem mass spectrometer has been used extensively in screening labs to analyze amino acids or in diagnostic labs as a universal detector for GC and LC techniques. Why did MS/MS replace older technological systems The answer to this question lies in the power of mass spectrometer. 

The general scheme of the biosynthesis of catecholamines was first postulated in 1939 (29) and finally confirmed in 1964 (Fig. 2) (30). Although not shown in Figure 2, in some cases the amino acid phenylalanine [63-91-2] can serve as a precursor it is converted in the liver to (-)-tyrosine [60-18-4] by the enzyme phenylalanine hydroxylase. Four enzymes are involved in E formation in the adrenal medulla and certain neurons in the brain tyrosine hydroxylase, dopa decarboxylase (also referred to as L-aromatic amino acid decarboxylase), dopamine-P-hydroxylase, and phenylethanolamine iV-methyltransferase. Neurons that form DA as their transmitter lack the last two of these enzymes, and sympathetic neurons and other neurons in the central nervous system that form NE as a transmitter do not contain phenylethanolamine N-methyl-transferase. The component enzymes and their properties involved in the formation of catecholamines have been purified to homogeneity and their properties examined. The human genes for tyrosine hydroxylase, dopamine- 3-oxidase and dopa decarboxylase, have been cloned (31,32). It is anticipated that further studies on the molecular structure and expression of these enzymes should yield interesting information about their regulation and function. 

When [1-14C]phenylalanine was tested as a precursor, the entire activity of decinine and decodine was unequally divided between C-ll and C-3. The carbonyl carbon at C-ll [isolated as benzophenone oxime as a result of phenylation with the Grignard reagent, dehydration, and chromic acid... 

The finding that tryptophan-2-i4C (CII) may also serve as a precursor for tropic acid 94) labeled in the carboxyl group (XXIIId) seemed to be contradictory. However, it was interpreted later 93) by assuming tryptophan as a source for C02, which becomes incorporated into phenylpyruvic acid this acid is supposed to be formed from phenylalanine. However, this is an explanation but not a rigorous proof. 

Cinnamic acid (24), a metabolite of phenylalanine, has been linked with tropic acid biosynthesis via its epoxide on chemical grounds but neither of these compounds have previously been found to act as a tropic acid precursor. Results with cinnamoyltropine were similarly negative. " More recently examination of [2- C]cinnamic acid [as (24)] as a precursor for the acid moieties of hyoscyamine... 

Phenylacetic acid has been reported as a precursor of tropic acid (19)/" " but in a recent experiment it was found not to label hyoscyamine (20) or scopolamine (21)/ Nor was phenylalanine labelled which excludes incorporation of phenylacetic acid through this amino-acid. 

Isolation of the amino acids from such wastewaters might be of commercial value. For example, L-phenylalanine has been used as a precursor in the synthesis of a variety of industrial products, for example, the artificial sweetener Aspartame [117]. Purified amino acids can, on the other hand, be used as nutritional supplements in the diet of livestock [118], as weU as in human nutrition [119]. Therefore, there is potential for the extraction of amino acids from dairy and proteinaceous wastewaters for additional income of the producing industries. Application of LMs in treatment of such wastewaters could be of substantial benefit. 

The following were administered separately to P. cyclopium [carboxyl- and [ N]-anthranilic acid, phenylalanine with labels at positions 1,2, and 3, and also N-labelled phenylalanine and [methyl- C]methiomne. The results show an intact incorporation of all the atoms of phenylalanine and anthranilic acid into both (49) and (50), with L-phenylalanine preferred over the D-isomer. The iV-methyl group originates from the S-methyl group of methionine. The cyclic dipeptide formed from these two amino-acids is presumably an intermediate on the pathway to the alkaloids. As phenylalanine serves as a precursor for cyclopenol, the origin of the hydroxy-group is by meta-hydroxylation of phenylalanine. Further, m-tyrosine and tyrosine are only unspecific precursors. 

Incorporation of DL-[2- C]tyrosine was to give specific labelling of C-1, whilst the results with L-[C/- C]tyrosine confirmed the utilization of this ammo-acid as the source of a Cg unit for rings c and D. (Tyramine, dopa, and homogentisic acid were much less effective precursors " for securinine, and homogentisic acid label was randomized.) The results with phenylalanine exclude it as a precursor and provide yet another example of the non-equivalence of phenylalanine and tyrosine in biosynthesis in higher plants. 

The L-phenylalanine-L-proline lactam (137), which has been found to be present in C. purpurea cultures, serves as a precursor for ergotamine (133). This result mirrors the derivation of ergocryptine/ergocryptinine (134) from the L-leucyl-L-proline lactam (136).A pointer for ergotamine (133) biosynthesis is the clear implication of D-lysergyl-L-valine (138) in the biosynthesis 01(134)." However, lysergylalanine has been found to be a non-intact precursor of ergotamine. 

Under the action of phenylalanine ammonia lyase (EC 4.3.1.5) Phe is transformed to rrans- cinnamic acid. This plays an important role in plants as a precursor of numerous phenolic secondary products such as flav-onoids, lignin, etc. D-Phe is a component of grami-cidin S and tyrocidines. L-Phe tastes weakly bitter, D-Phe tastes sweet. 

However, even though this reaction proceeds in a very efficient way when an aromatic aldehyde is employed as the Michael donor, the use of aliphatic aldehydes is much more problematic due to the intrinsic instability of enoliz-able aldehydes in the basic media required in the reactions catalyzed by A-heterocyclic carbenes. In fact, pre-catalyst 119a performed poorly in this case, but this limitation was overcome with the use of bicyclic triazolium salt 120a derived from phenylalanine as catalyst precursor. This new A-heterocyclic... 

In the compound calophyllolide (123) from Calophyllum inophyllum (Clusiaceae or Hypericaceae), phenylalanine was incorporated into the 2,3,4-position and the aromatic ring attached at the C-4 position. Acetate served as a precursor for the aromatic ring with the phloroglucinol substitution. Compounds from the Fabaceae generally have a 6,7-oxygen-ated pattern rather than that of calophyllolide. The possibility of different pathways cannot be excluded. 

The biosynthesis of Lythraceae alkaloids is not well understood. Lysine serves as a precursor for part of the molecule all carbons except the carboxyl group of lysine are incorporated. Further, the lysine-derived portion enters the molecule via a symmetrical intermediate. Phenylalanine... 

Some of the possible pathways for the biosyntheses of the epinephrine bodies are shown in Fig. 6. That phenylalanine can serve as a precursor... 

A widespread alkaloid family is made up of phenylalanine-tyrosine derivatives. These amino acids are precursors of a variety of compounds alkaloids, flower pigments, phenylpropane acids, lignins, etc. In most cases, deamination is the first reaction, but sometimes decarboxylation, O-methylation, or A-methylation occurs. The resultant protoalkaloids can be transformed into an impressive number of alkaloids. In closely related orders, e.g., Ranales, Berberidales, Aristolochiales, and Rhoeadales, the commonly synthesized compound is norlaudanosoline, and it acts as a precursor for all alkaloids in these plants. In some orders such as Aristolochiales, the alkaloids can be converted into compounds that do not give an alkaloid-positive reaction. Another group of plants with tyrosine- and phenylalanine-derived alkaloids are the Amaryllidaceae and related taxa. 

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