3 -,4 -2 -Deoxythymidine, synthesis

Inhibit Enzymes Many drugs are competitive inhibitors of key enzymes in pathways. The statin drugs (lovastatin, simvastatin), used to control blood cholesterol levels, competitively inhibit 3-hvdroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase in cholesterol biosynthesis. Methotrexate, an antineoplastic drug, competitively inhibits dihydrofolate reductase, depriving the cell of active folate needed for purine and deoxythymidine synthesis, thus interfering with DNA replication during S phase. 

Introduction and Rationale. DHFR is an ideal system to study for a number of reasons. The catalytic properties of DHFR are such that under normal physiologic conditions and with the NADPH cofactor bound, 7,8-dihydrofolate (DHF) is reduced to 5,6,7,8-tetrahydrofolate (THF) (7). Thus DHFR plays an important role in cell metabolism by maintaining a supply of THF. THF is used by the cell as both a cofactor and in substrate quantities in the synthesis of deoxythymidine. By inhibiting the production of THF, deoxythymidine synthesis is curtailed, nucleic add replication comes to a halt, and cell proliferation ceases. It is this biochemical cascade which supplies the pharmacological and chemotherapeutic applications of inhibitors to DHFR. 

Rupprath, C., Kopp, M., Hirtz, D. et al. (2007) An enzyme module system for in situ regeneration of deoxythymidine 5 -diphosphate (dTDP)-activated deoxy sugars. Advanced Synthesis Catalysis, 349, 1489-1496. 

In 1995, Horie et al. described a polymorphic tandem repeat found in the 5 -un-translated region of the thymidylate synthase gene [70]. Thymidylate synthase (TS TYMS) catalyzes the intracellular transfer of a methyl group to deoxyuridine-5-monophosphate (dUMP) to form deoxythymidine-5-monophosphate (dTMP), which is anabolized in cells to the triphosphate (dTTP). This pathway is the only de- novo source of thymidine, an essential precursor for DNA synthesis and repair. The methyl donor for this reaction is the folate cofactor 5,10-methylenetetrahydro-folate (CH2-THF) (Figure 24.4). 

Ribonucleotide reductase works on ribo-A, -U, -G, -C diphosphates to give the deoxynucleotide. The deoxyuridine, which is useless for RNA synthesis, is converted to deoxythymidine by the enzyme thymidylate synthase, which uses methylene tetrahydrofolate as a one-carbon donor. The odd thing here is that ribonucleotide reductase uses the UDP as a substrate to give the dUDP. This must then be hydrolyzed to the dUMP before thymidylate synthase will use it to make dTMP. Then the dTMP has to be kinased (phosphorylated) up to dTTP before DNA can be made. 

The anaemia in B deficiency is caused by an inability to produce sufficient of the methylating agent S-adenosyhnethionine. This is required by proliferating cells for methyl group transfer, needed for synthesis of the deoxythymidine nucleotide for DNA synthesis (see below and Chapter 20). This leads to failure of the development of the nucleus in the precursor cells for erythrocytes. The neuropathy, which affects peripheral nerves as well as those in the brain, is probably due to lack of methionine for methyl transfer to form choline from ethanolamine, which is required for synthesis of phosphoglycerides and sphingomyelin which are required for formation of myelin and cell membranes. Hence, the neuropathy results from a... 

Figure 20.12 (a) Details of reaction catalysed by thymidylate synthase. Methylene FH4 represents N N methylene tetrahydro-folate (see Figure 15.2). (b) Reactions in the pathways in which either CDP or UDP gives rise to deoxythymidine monophosphate. Note that two processes can be involved in synthesis of deoxyuridine monophosphate. It is not known if one process dominates, but in (c) it is assumed that the pathway from CDP dominates formation of dTTP. (c) k summary of the reactions required for synthesis of deoxyribonucleotides required for DNA replication. 

The enzyme tetrahydrofolate reductase, which is essential for the synthesises deoxythymidine monophosphate (dTMP) from deoxyuridine monophosphate, a process essential for DNA synthesis. This enzyme catalyses formation of methylene tetrahydrofate (CH3-FH4) a necessary co-substrate for synthesis of d-TMP catalysed by thymidylate synthase (See Figure 20.12(a) and p. 477). 

Fig. 1. Folate-cobalamin interaction in the synthesis of purines and pyrimidines and, therefore, of DNA. (1) In gastrointestinal mucosa cells (2) in the liver (3) in peripheral tissues. C, cobalamine DAC, desoxyadenosylcobalamine HC, hydroxy cobalamine MC, methylcobalamine F, folic acid MTHF, methyltetrahydrofolic acid THF, tetrahydrofolic acid DHF, dihydrofolic acid dUMP, deoxyuridinemonophosphate dTMP, deoxythymidine-monophosphate. (Adapted from Far-...

The TS mediates the conversion of 2-deoxyuridine monophosphate (dUMP) into deoxythymidine monophosphate (dTMP). This enzymatic methylation reaction is a key step in the synthesis of DNA and involves a ternary complex between the substrate, the enzyme and the co-factor [methylene tetrahydrofolic acid (CH2FAH4)] (Fig. 24) [8,80,81], This transformation represents the sole de novo source of dTMP, a building block for DNA synthesis and repair [82]. 

The other major class of antimalarials are the folate synthesis antagonists. There is a considerable difference in the drug sensitivity and affinity of dihydrofolate reductase enzyme (DHFR) between humans and the Plasmodium parasite. The parasite can therefore be eliminated successfully without excessive toxic effects to the human host. DHFR inhibitors block the reaction that transforms deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) at the end of the pyrimidine-synthetic pathway. This reaction, a methylation, requires N °-methylene-tetrahydrofolate as a carbon carrier, which is oxidized to dihydrofolate. If the dihydrofolate cannot then be reduced back to tetrahydrofolate (THF), this essential step in DNA synthesis will come to a standstill. 

Synthesis of the thymidine derivative relies on the older two-step insertion of fluorine. Thus, the hydroxyl group in 3 -deoxythymidine benzoate (51-1) is first converted to the mesylate (51-2). Reaction with potassium fluoride in hydrogen fluoride replaces the mesylate by fluorine (51-3). The fact that this reaction, as that above, proceeds with retention of the configuration mles out simple displacement as the mechanism for this transform. The presence of the methyl group at position 5 negates the need for the chlorination step. Saponification then affords the antiviral agent alovudine (51-4) [52]. 

The folic acid-dependent conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) carried out by thymidylate synthase is an absolute requirement for DNA synthesis. An unusually high demand for uracil (ura) by certain tumor cells suggested that such an inhibitor of this process could have tumor cell selectivity. 5-Fluorouracil (fl5ura) (80), along with 5-fluorocytosine (fl5cyt) (81) and 5-fluoroorotic acid (fl5oro) (82), were synthesized by Heidelberger in 1957 as part of a... 

Cells making DNA must also be able to make deoxythymidine triphosphate (dTTP). The key step in the synthesis of dTTP is the conversion of dUMP to dTMP via thymidylate synthase. The reaction requires a source of N5,Nw-methylene tetrahydrofolate (see Sec. 15.7, Fig. 15-19) to provide the methyl group. In this reaction, the tetrahydrofolate is oxidized to dihydrofolate. Dihydrofolate must be reduced to tetrahydrofolate via the enzyme dihydrofolate reductase so that more Af5,A,l0-methylene tetrahydrofolate can be made from serine in a reaction catalyzed by serine hydroxymethyltransferase. These three reactions, which are essential for the formation of dTMP, are shown below. 

Direct measurement of DNA and protein synthesis in experimental animals suggest that in zinc deficiency protein synthesis is adversely affected (, ). Our own data on human subjects in these experiments show that the total protein, total collagen, and RNA-DNA ratio Increased as a result of zinc supplementation. The activity of deoxythymidine kinase was not measurable during the zinc restriction phase but became 70% of normal level after supplementation with zinc for 3 months. Similar data have been published for experimental animals. Thus, our data show that deoxythymidine kinase in human subjects also is a zinc-dependent enzyme, and an adverse effect of zinc deficiency on this enzyme may be responsible for decreased protein synthesis. Our studies do not rule out an adverse effect of zinc deficiency on protein catabolism. Further studies are required to establish the effect of zinc restriction on protein catabolism. 

D. Synthesis and anti-HIV activity of some novel phosphorodiamidate derivatives of 3 -azido-3 -deoxythymidine (AZT). Antiviral Chem. Chemother., 1991, 2 35-39. 

Fig. 6 Methyl trap hypothesis 5,10-Methylenetetrahydrofolate is reduced to 5-methyltetiahy-drofolate in an irreversible reaction. When vitamin Bn is deficient, methyl groups are trapped as 5-methyltetrahydrofolate, resulting in decreased substrates for DNA synthesis and neural lipid methylation. MTHFR, methylenetetrahydrofolate reductase DHFR, dihydrofolate reductase MS, Methionine synthase TS, thymidylate synthase SAM, S-adenosyl-methionine dUMP, deoxyuridine 5 -monophosphate dTTP, deoxythymidine 5 -monophosphate...

The addition of lithium azide to ferr-butyldimethylsilyloxy-2-penten-4-olides during the synthesis of a 3 -azido-3 -deoxythymidine derivative66 and the diastereoselective addition of benzy-lamine to (R)-4-0-benzyl-4-hydroxy-2-penten-5-olide giving substituted /9-amino acid derivatives67 have also been reported. 

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