Abstract:The synthesis of two selectively labeled isotopomers of L‐phenylalanine, (Phe), using chemical and enzymatic methods is reported. The [3R‐3H]‐L‐Phe isotopomer has been obtained from [3‐3H]cinnamic acid prepared from benzaldehyde and malonic acid using tritiated water as a source of radioactive label, and by addition of ammonia in the presence of enzyme PAL. The [3S‐3H]‐L‐Phe isotopomer has been synthesized by addition of ammonia to cinnamic acid in a buffered medium containing PAL and tritiated water. Copyrigh… Show more
“…The isotopomer [2‐ 14 C]‐ l ‐Phe, 2 , was obtained by the, partly similar, combined chemical and enzymatic methods using [2‐ 14 C]‐malonic acid as a source of label, Scheme . Also, the isotopomers [(3 R )‐ 3 H]‐ l ‐Phe, 3 , Scheme , and [(3 S )‐ 3 H]‐ l ‐Phe, 4 , Scheme , were synthesized by the chemical and enzymatic procedures . The doubly labeled [(3 S )‐ 2 H/ 3 H]‐ l ‐Phe, 5 , was obtained according to the Scheme by addition of ammonia to cinnamic acid carried out in fully deuteriated incubation medium containing tritiated water.…”
Aromatic amino acids such as l-phenylalanine, l-tryptophan, 3',4'-dihydroxy-l-phenylalanine (l-DOPA), and their derivatives 3',4'-dihydroxyphenylacelaldehyde (DOPAL) and 3',4'-dihydroxyphenylethanol (DOPET), play an essential role in human metabolic processes. Incorrect or slow biotransformation of these compounds leads to some metabolic dysfunctions and in some cases to some neurodegenerative diseases. Therefore, studies of the biotransformation mechanisms of these metabolites draw biochemists' and medical researchers' attention. This study investigates the mechanisms of biotransformation of the aforementioned compounds using kinetic (KIE) and solvent (SIE) isotope effect methods. The overview presents the results and the numerical values of KIE and SIE methods, obtained in the study of biotransformation of l-phenylalanine, 5'-chloro-l-tryptophan, and l-DOPA, catalyzed by the enzymes from the lyases group (phenylalanine ammonia lyase, tryptophan indole-lyase, and tyrosine decarboxylase). Deuterium KIE was also determined during the deamination of 2'-chloro-l-phenylalanine in the presence of the enzyme l-phenylalanine dehydrogenase, as well as in the conversion of DOPAL into DOPET catalyzed by the enzyme alcohol dehydrogenase. The values of KIE and SIE have been determined using a noncompetitive spectrophotometric and a competitive (combined with internal radioactivity standard) radiometric methods.
“…The isotopomer [2‐ 14 C]‐ l ‐Phe, 2 , was obtained by the, partly similar, combined chemical and enzymatic methods using [2‐ 14 C]‐malonic acid as a source of label, Scheme . Also, the isotopomers [(3 R )‐ 3 H]‐ l ‐Phe, 3 , Scheme , and [(3 S )‐ 3 H]‐ l ‐Phe, 4 , Scheme , were synthesized by the chemical and enzymatic procedures . The doubly labeled [(3 S )‐ 2 H/ 3 H]‐ l ‐Phe, 5 , was obtained according to the Scheme by addition of ammonia to cinnamic acid carried out in fully deuteriated incubation medium containing tritiated water.…”
Aromatic amino acids such as l-phenylalanine, l-tryptophan, 3',4'-dihydroxy-l-phenylalanine (l-DOPA), and their derivatives 3',4'-dihydroxyphenylacelaldehyde (DOPAL) and 3',4'-dihydroxyphenylethanol (DOPET), play an essential role in human metabolic processes. Incorrect or slow biotransformation of these compounds leads to some metabolic dysfunctions and in some cases to some neurodegenerative diseases. Therefore, studies of the biotransformation mechanisms of these metabolites draw biochemists' and medical researchers' attention. This study investigates the mechanisms of biotransformation of the aforementioned compounds using kinetic (KIE) and solvent (SIE) isotope effect methods. The overview presents the results and the numerical values of KIE and SIE methods, obtained in the study of biotransformation of l-phenylalanine, 5'-chloro-l-tryptophan, and l-DOPA, catalyzed by the enzymes from the lyases group (phenylalanine ammonia lyase, tryptophan indole-lyase, and tyrosine decarboxylase). Deuterium KIE was also determined during the deamination of 2'-chloro-l-phenylalanine in the presence of the enzyme l-phenylalanine dehydrogenase, as well as in the conversion of DOPAL into DOPET catalyzed by the enzyme alcohol dehydrogenase. The values of KIE and SIE have been determined using a noncompetitive spectrophotometric and a competitive (combined with internal radioactivity standard) radiometric methods.
“…3. Addition of ammonia to cinnamic acid, catalyzed by PAL, was carried out in the buffer containing tritiated water, HTO, leading to formation of ( 1a ) [25, 26]. Fig.…”
Section: Synthesismentioning
confidence: 99%
“…The tritiated benzaldehyde ( 4a ) was condensed with malonic acid ( 6 ) in pyridine solvent using catalytic amounts of piperidine giving [3- 3 H]-cinnamic acid ( 2a ). Compound 2a then was converted into ( 1d ) by addition of ammonia catalyzed by PAL [25] (Fig. 4).…”
Section: Synthesismentioning
confidence: 99%
“…[( 3R )- 3 H]- l Tyr ( 14f ) was synthesized by enzymatic hydroxylation of [( 3R )- 3 H]- l phenylalanine ( 1d ) obtained earlier [25] catalyzed by l -phenylalanine 4′-monooxygenase (Fig. 12).…”
This review compiles the combined chemical and enzymatic synthesis of aromatic l-amino acids (l-phenylalanine, l-tyrosine, l-DOPA, l-tryptophan, and their derivatives and precursors) specifically labeled with carbon and hydrogen isotopes, which were elaborated in our research group by the past 20 years. These compounds could be then employed to characterize the mechanisms of enzymatic reactions via kinetic and solvent isotope effects methods.
“…Final chemical conversion produced [ 14 C 4 ]everolimus.Concentration of precursor: The variation of the concentration may have a significant influence on features of the fermentation process, in particular the on incorporation (Inc), the dilution factor (Dil) and the mass yield of the metabolite 24. ADME studies of everolimus (51b), a rapamycin derivative with immunosuppressive activity, suggested that it was metabolically cleaved at two sites, indicated by hashed lines in the structure.…”
Biotransformations are selective chemical modifications of molecules by biological entities ranging from isolated enzymes (cell-free systems) to intact organisms 1 . In particular cases they are exploited by the chemical and pharmaceutical industries for production of chemicals; this approach can be especially advantageous for structurally complex compounds 2 . Preparative biotransformations have been used on a laboratory scale by radiochemists to prepare isotopically labeled compounds 3 necessary for drug development and other applications in the life sciences. Their synthetic use for isotope labeling includes:. selective modifications of individual functional groups; . synthesis of structurally complex molecules from less complex isotopically labeled precursors; . synthesis of key intermediates for reconstitution approaches to labeled compounds (see Chapter 10).Enzymes are proteins able to catalyze reactions with very high regio-and/or stereoselectivities under mild conditions. Those enzymes combining low substrate specificity (broad applicability) with high product selectivity are of optimal suitability for organic synthesis. Further increase of structural variety and stability by protein-and mediumengineering 4 has extended their synthetic applicability significantly and made them important tools in organic synthesis. The broad scope of their synthetic use is documented in numerous papers, reviews and textbooks 5 . Although successful in particular applications, biotransformations are less common in isotopic labeling.As the complexity of the biological system varies from highly purified enzymes to whole cell-containing systems, so do some of the practical dimensions of the experiments vary. Therefore this chapter is organized accordingly.In one-step biotransformations particularly, isolated enzymes have distinctive advantages, since they can be used simply, almost like any chemical reagent, and -importantly for isotopic labeling -they do not result in any isotope dilution. Since a large number of enzymes are commercially available, many types of reactions can be carried out.Examples of the use of isolated enzymes in isotopic labeling include N-or O-acylations, hydrolyses, redox reactions and group transfer reactions. Most of them aim for applications such as racemate resolution, catalysis of reactions and synthesis of enantiomerically pure molecules.
Optical Resolutions via DerivativesDespite the availability of several chemical strategies for diastereo-and enantioselective syntheses in isotopic labeling (see Chapter 11), kinetic racemate resolution remains a major application of enzymes in the chemistry of isotopically labeled compounds. Depending on the molecular entity, different possibilities have been described for achieving enantioselective differentiation through either derivatization (e.g. N-acyl formation and cleavage, O-acyl formation and cleavage) or selective degradation (e.g. redox reactions).
N-Acyl Formation: Enzymatically catalyzed N-acylation of racemates is used for preparative synthesis of ...
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