Thymidylic acid synthesis

Folates carry one-carbon groups in transfer reactions required for purine and thymidylic acid synthesis. Dihydrofolate reductase is the enzyme responsible for supplying reduced folates intracellularly for thymidylate and purine synthesis. 

Deficiency of folate or vitamin Bn can cause hematological changes similar to hereditary orotic aciduria. Folate is directly involved in thymidylic acid synthesis and indirectly involved in vitamin Bn synthesis. Orotic aciduria without the characteristic hematological abnormalities occurs in disorders of the urea cycle that lead to accumulation of carbamoyl phosphate in mitochondria (e.g., ornithine transcarbamoylase deficiency see Chapter 17). The carbamoyl phosphate exits from the mitochondria and augments cytosolic pyrimidine biosynthesis. Treatment with allopurinol or 6-azauridine also produces orotic aciduria as a result of inhibition of orotidine-5 phosphate decarboxylase by their metabolic products. 

Folate vitamins are essential cofactors in DNA synthesis. They carry one-carbon groups in transfer reactions that are required for purine and thymidylic acid synthesis, and in turn for formation of DNA and for cell division. Natural folates circulating in the blood have a single glutamic acid group, but within cells they are converted to polyglutamates, which are more efficient cofactors and which are preferentially retained inside the cells. ... 

Aminouracil produces a block in the mitotic cycle of various plants. For example, cessation of mitosis occurred in Vida faba roots incubated 24 h with this compound [392—394]. Depending on different experimental conditions, thymidine or thymidylic acid may or may not alleviate these effects [392—394]. It was concluded that 5-aminouracil depressed the rate of DNA synthesis, which led to an accumulation of cells in the S phase. After removal of the agent, DNA synthesis resumed. Similar results have been observed with Allium cepa and Haplopappus gracilis [395, 396]. Inhibition of guanosine incorporation into RNA of meristematic cells in Vida faba by 5-aminouracil was also reported [397]. 

A short time later, these authors reported an elegant adaptation of this work for the synthesis of multisubstrate analogues (170a-c) of the proposed intermediate in the thymidylate synthase-mediated synthesis of thymidylic acid, as... 

Fluorouracil [flure oh YOOR a sil] (5-FU), a pyrimidine analog, has a stable fluorine atom in place of a hydrogen atom at position 5 of the uracil ring. The fluorine interferes with the conversion of deoxyuridylic acid to thymidylic acid, thus depriving the cell of one of the essential precursors for DNA synthesis. 

In 1981, Beaucage and Caruthers4 extended the domain of phosphorus(lll) chemistry with the development of the phosphoramidite approach to oligonucleotide synthesis (see reviews in section 1.9.5.) which, in its later variants, is unsurpassed in its speed and efficiency in both solid and solution phase. An early synthesis of thymidylic acid derivative 1.7 illustrates the phosphoramidite approach [Scheme 7.1]. The sequence begins with the conversion of the 3 -hy-droxyl of nucleoside 1.1 to the phosphoramidite 13 by reaction with chloro-... 

Dihydrofolate reductase (DR) is showm near the top of Figure 9.6, This enzyme catalyzes the reduction of Hjfolate to H folate. DR is part of the cycle of reactions in the synthesis of thymidylic acid. The enzyme also catalyzes the reduction of folic acid to dihydrofolic acid and then to EclrahydrofoJic acid, DR is the target of the anti cancer drug methotrexate (MTX), MTX exerts its toxic effects more nn rapidly... 

Fluorouracil is a lluorinated pyrimidine, which is converted intraceUularly to the active form, fluorodeoxyuridine monophosphate, which inhibits thymidylate synthetase and hence reduces the production of thymidylic acid, the deox)Tibonucleotide of thymine (5-methyluracil), a DNA pyrimidine base, blocking DNA synthesis. In addition, intracellular conversion to 5-fluorouridine monophosphate results in incorporation of the activated antimetaboUte into RNA and consequent RNA dysfunction. 

Purines, pyrimidines and their nucleosides and nucleoside triphosphates are synthesized in the cytoplasm. At this stage the antifolate drugs (sulphonamides and dihydrofolate reductase inhibitors) act by interfering with the synthesis and recycling of the co-factor dihydrofolic acid (DHF). Thymidylic acid (2-deoxy-thymidine monophosphate, dTMP) is an essential nucleotide precursor of DNA synthesis. It is produced by the enzyme thymidylate synthetase by transfer of a methyl group from tetrahydrofolic acid (THF) to the uracil base on uridylic acid (2-deoxyuridine monophosphate, dUMP) (Fig. 12.5). THF is converted to DHF in this process and must be reverted to THF by the enzyme dihydrofolate reductase (DHFR) before... 

The antifungal agent 5-fluorocytosine also interferes with these early stages of DNA synthesis. Through conversion to the nucleoside triphosphate it subsequently blocks thymidylic acid production through inhibition of the enzyme thymidylate synthetase (Fig. 12.6). 

Fig. 12.5 Conversion of uridylic acid to thymidylic acid by the enzyme thymidylate synthetase, a vital early stage in the synthesis of DNA.

Synthesis of thymidylic acid (TMP). Fluorodeoxyuridylate inhibits conversion of dUMP to TMP, and methotrexate inhibits regeneration of the tetrahydrofolate coenzyme. 

In vitamin B12 or folate deficiency anemia, megaloblastosis results from interference in fohc acid-and vitamin B -interdependent nucleic acid synthesis in the immature erythrocyte. The rate of RNA and cytoplasm production exceeds the rate of DNA production. The maturation process is retarded, resulting in immature large RBCs (macrocytosis). Synthesis of the RNA and DNA necessary for cell division depends on a series of reactions catalyzed by vitamin B12 and folic acid, as they have a role in the conversion of midine to thymidine. As shown in Fig. 99-4, dietary folates are absorbed in this process and converted (A) to 5-methyl tetrahydrofolate, which is then converted via a Bi2-dependent reaction (B) to tetrahydrofolate (C). After gaining a carbon, tetrahydrofolate is converted to a folate cofactor (D), 5,10-methyl-tetrahydrofolate, used by thymidylate synthetase (E) in the... 

In 1968, my graduate student Charles Walsh and I addressed the following question What are the pyrimidine sources for nucleic acid synthesis by Plasmodium lophurae We found the parasite synthesized pyrimidines de novo (Walsh and Sherman, 1968b). The evidence for a de novo synthesis was the presence of the key enzymes, thymidylate synthetase and oroti-dine-5-monophosphate pyrophosphorylase, as well as the demonstration of the incorporation of 14C-bicarbonate into cytosine, uracil and thymine. Finding a de novo pathway for the synthesis of pyrimidines by the malaria parasite would, in the next three decades, provide a biochemical basis for the mechanism of action of anti-folate anti-malarials as well as contributing to an understanding of the unique properties of the malaria parasite mitochondrion. 

With the normal substrate the next step would involve a hydride shift to the methylene of the folate and a proton abstraction from C-5, leading to thymidylic acid. Here, however, removal of an F from C-5 is impossible. The result is an irreversible blockade of the enzyme by a ternary covalent complex (Fig. 4-13). The synthesis of thymidylic acid (Fig. 

Fluorouracil (5-FU) interferes with DNA synthesis by blocking the methylation of deoxyuridylic acid to thymidylic acid. Topical formulations (carac, efudex, fluoroplex) are used in multiple actinic keratoses, actinic cheilitis, Bowen s disease, and superficial basal cell carcinomas not amenable to other treatments. 

One of the most well-known examples of effective replacement of hydrogen with fluorine is observed in the antineoplastic drug 5-fluorouracil (Figure 2.1). This compound is metabolized in vivo to 5-fluoro-2 -deoxyuridylic acid (5-fluoro-dUMP), which is the active drug that covalently binds to thymidylate synthase, the enzyme responsible for the essential conversion in DNA synthesis of uridylic acid to thymidylic acid. 

Cytidylic acid, thymidylic acid, and uridylic acid (UMP) are compounds in which the sugar moiety of each of the related nucleosides described above is phosphorylated at the 5 position.The sugar moiety combined with the uracil base of the uridyHc acid used for RNA formation is D-ribose, whereas the sugar combined with the thymine of thymidylic acid used in DNA formation is D-2-deoxyribose. 2 -Deoxycytidyhc acid is used in RNA synthesis, whereas cytidyHc acid is used in the formation of DNA. 

N °-Methylene-FH4 acts as a reducing agent as well as a source of one- carbon units in the synthesis of thymidylic acid (see Pyrimidine biosynthesis). 

The megaloblast differs from the normoblast in its increased amount of cytoplasmic RNA. The DNA and the nuclear RNA are apparently unchanged, and therefore the thymine-uracil ratio is decreased. From such observations, it was concluded that the megalo-blasts are red blood cell precursors whose interphase is abnormally prolonged because of interference with DNA synthesis. On the basis of the alteration of thymine-uracil ratios, it was assumed that the metabolic block would be located on the passage from deoxyuri-dylic acid to thymidylic acid. Such conclusions are unwarranted at present because the role of vitamin B12 in the methylation of thymidine is not established. Furthermore, it is not certain that cytoplasmic RNA acts as a reservoir for DNA as the above hypothesis implies. 

Bromoacetamidophenyl derivatives of thymidylic acid have been used to elucidate the structure of the substrate-binding site of Staphylococcus aureus nuclease. Similarly, bromoacetamidophenyl derivatives of the initiation codon, A-U-G, and the termination codon, U-G-A, have been employed as affinity probes of the ribosomal codon binding sites. The synthesis of these chemically reactive oligonucleotides combines chemical and enzymic methods as outlined for the synthesis of 5 -[4-(bromo-[2-i C] acetamido) phenylphospho ] adenylyl- (3 -5 ) -uridiylyl- (3 -6 ) guano-sine ( A-U-G) in Fig. 1. 

Cell proliferation may be decreased by using various structural analogues to inhibit nucleic acid synthesis. Amethopterin (methotrexate) closely resembles folic acid (page 165) in structure and prevents the formation of the purine precursors of DNA, RNA and ATP. 6-Mercaptopurine is a purine analogue which interferes with the pathway in which the phosphoribosyl precursors of the purine nucleotides are formed. 5-Fluorouracilis a competitive inhibitor of the conversion of uridylic acid (dUMP) into thymidylic acid (dTMP). 

Nakahara, Y., and Ogawa, T. (1983) Chemical synthesis of nucleotide oligomers convergent synthesis of fully protected SOmer of thymidylic acid. Nucl Acids Symp. Ser. 12, 59-62. 

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