Thymidine 5’- , enzymic

BVdU is degraded by thymidine phosphorylase more rapidly than the natural substrate, thymidine. This rapid enzymic degradation may present a problem in its clinical use. Moreover, herpes vimses develop resistance to BVdU, apparendy because of mutant vimses that have lower thymidine kinase activity. G. D. Seade has dropped further development of BVdU because of increased animal tumor incidence induced by prolonged dosing (1). 

An amount of enzyme preparation equivalent to 900 mg of wet cells was made up to 25 ml with the above potassium phosphate buffer solution. 150 mg (1.15 mmol) of 5-fluorouracil and 1.0 gram of thymidine (4.12 mmol) were dissolved in 15 ml of the above potassium phosphate buffer solution. The mixture was incubated at 37°C for 18 hours. After this time, enzyme action was stopped by the addition of four volumes of acetone and one volume of peroxide-free diethyl ether. The precipitated solids were removed by filtration, and the filtrate was evaporated under nitrogen at reduced pressure until substantially all volatile organic solvent had been removed. About 20 ml of aqueous solution, essentially free of organic solvent, remained. This solution was diluted to 100 ml with distilled water. 

Methotrexate (MTX, chemical structure shown in Fig. 1.) competitively inhibits the dehyrofolate reductase, an enzyme that plays an essential role in purine synthesis. The dehydrofolate reductase regenerates reduced folates when thymidine monophosphate is formed from deoxyuridine monophosphate. Without reduced folates cells are unable to synthesize thymine. Administration of N-5 tetrahydrofolate or N-5 formyl-tetrahydrofolate (folinic acid) can bypass this block and rescue cells from methotrexate activity by serving as antidote. 

Ralitrexed is a folate analog with greater selectivity. It easily crosses the cell membrane and undergoes polyglutamation. Within tissues, ralitrexed may be stored up to 29 days. It directly inhibits thymidylate synthase, the key enzyme for synthesizing thymidine triphosphate (TTP). The drug has been described to induce apoptosis in tumor cells. Ralitrexed is used for the treatment of colon carcinomas. 

Methotrexate belongs to the class of antimetabolites. As a derivative of folic acid it inhibits the enzyme dihydrofolate reductase resulting in a decreased production of thymidine and purine bases essential for RNA and DNA synthesis. This interruption of the cellular metabolism and mitosis leads to cell death. 

All NRTIs, as exemplified for AZT (Fig. 7), act in a similar fashion following their uptake by the cells, they are phosphorylated successively to their 5 -monophosphate, 5 -diphosphate, and 5 -triphosphate form (De Clercq 2002). Unlike the first phosphorylation step in the metabolic pathway of the acyclic guanosine analogues (see above), which is carried out by a virus-encoded enzyme (thymidine kinase), the first as well as the subsequent phosphorylations of the 2, 3 -dideoxynucleosides are carried out by cellular enzymes, that is, a 2 -deoxynucleoside (e.g., dThd) kinase, a 2 -deoxynucleotide (e.g., dTMP) kinase, and a (2 -deoxy)nucleoside 5 -diphosphate (NDP) kinase. 

Deoxy-5 -fluorothymidine (838) was prepared by Langen and Kowol-jj. 796,797 fpQjyj 5 -0-tosyl precursor by treatment with fluoride. Compound 838 cannot be phosphorylated enzymically owing to the lack of OH-5, but it inhibits the growth of carcinoma cells. This was explained as follows the thymidine 5 -monophosphate (thymidylate) kinase in carcinoma cells, catalyzing the transformation of thymidine 5 -monophosphate into the diphosphate, is inhibited by 838, thus preventing the synthesis of... 

While mammahan cells reutilize few free pyrimidines, salvage reactions convert the ribonucleosides uridine and cytidine and the deoxyribonucleosides thymidine and deoxycytidine to their respective nucleotides. ATP-dependent phosphoryltransferases (kinases) catalyze the phosphorylation of the nucleoside diphosphates 2 "-de-oxycytidine, 2 -deoxyguanosine, and 2 -deoxyadenosine to their corresponding nucleoside triphosphates. In addition, orotate phosphoribosyltransferase (reaction 5, Figure 34-7), an enzyme of pyrimidine nucleotide synthesis, salvages orotic acid by converting it to orotidine monophosphate (OMP). 

The methylation of deoxyuridine monophosphate (dUMP) to thymidine monophosphate (TMP), catalyzed by thymidylate synthase, is essential for the synthesis of DNA. The one-carbon fragment of methy-lene-tetrahydrofolate is reduced to a methyl group with release of dihydrofolate, which is then reduced back to tetrahydrofolate by dihydrofolate reductase. Thymidylate synthase and dihydrofolate reductase are especially active in tissues with a high rate of cell division. Methotrexate, an analog of 10-methyl-tetrahydrofolate, inhibits dihydrofolate reductase and has been exploited as an anticancer drug. The dihydrofolate reductases of some bacteria and parasites differ from the human enzyme inhibitors of these enzymes can be used as antibacterial drugs, eg, trimethoprim, and anti-malarial drugs, eg, pyrimethamine. 

Human immunodeficiency virus (HIV) is a retrovirus, i.e. its RNA is converted in human cells by the en me reverse transcriptase to DNA which is incorporated into the human genome and is responsible for producing new HIV particles. Zidovudine (azidothymidine, AZT Fig. 5.22F) is a stmctural analogue of thymidine (Fig. 5.22A) and is used to treat AIDS patients. Zidovudine is converted in both infected and uninfected cells to the mono-, di- and eventually triphosphate derivatives. Zidovudine triphosphate, the aetive form, is a potent inhibitor of HIV replication, being mistaken for thymidine by reverse transeriptase. Premature ehain termination of viral DNA ensues. However, AZT is relatively toxic because, as pointed out above, it is converted to the triphosphate by eellular enzymes and is thus also aetivated in uninfected cells. 

Acyclovir (acycloguanosine. Fig. 5.221) is a novel type of nucleoside analogue which becomes achvated only in herpes-infected host cells by a herpes-specific enzyme, thymidine kinase. This enzyme inihates conversion of acyclovir initially to a monophosphate and then to the antiviral triphosphate which inhibits viral DNA polymerase. The host cell polymerase is not inhibited to the same extent, and the antiviral triphosphate is not produced in uninfected cells. Ganciclovir (Fig. 5.22J) is up to 100... 

The cell fusion mixture is transferred to a culture medium containing hypoxanthine, aminopterin and thymidine (HAT medium). Unflised myeloma cells are unable to grow as they lack HGPRT. Unflised normal spleen cells can grow but their proliferahon is limited and they eventually die out. The hybridoma cell can proliferate in the HAT medium as the normal spleen cell supplies the enzyme which enables the hybridoma to utilize extracellular hypoxanthine. 

Phosphoramidate analogues of dideoxyribonucleoside phosphates (26) and trideoxyribonucleoside phosphates are acid labile and can be hydrolysed enzymically. Snake venom phosphodiesterase cleaves (26) to thymidine and 5 -deoxy-5 -aminothymidine (27 R = H). The latter presumably arises by spontaneous decomposition of the phosphoramidate (27 R = PO3H2) and P—O fission must have occurred during the initial hydrolysis. With acid or spleen phosphodiesterase, (26) gave Tp and (27 R = H), i.e. P—N fission occurred. 

Fluorouracil (5-FU) acts as a false pyrimidine, inhibiting the formation of the DNA base thymidine.26,35 The main mechanism by which it accomplishes this is by inhibiting the enzyme thymidylate synthase, the rate-limiting step in thymidine formation. 5-FU first must be metabolized to its active metabolite (F-dUMP). Additionally, metabolites of 5-FU may incorporate into RNA, inhibiting its synthesis. 

DHFR has been the object of intense research for the last few decades. The enzyme catalyses the NADPH-dependent reduction of 7,8-dihydrofolate to 5,6,7,8 tetrahydrofolate, a chemical which participates in the thymidilate synthesis cycle. Thus, the enzyme is crucial in the synthesis of thymidine monophosphate as well as in various one-carbon unit transfer reactions. 

Variations The poly(A) tailing kit (Ambion) produces a mRNA population with varying lengths of poly(A) tails, controlled by altering poly(A) polymerase concentrations and incubation times. An alternate method to incorporate a poly(A) tail is to clone a defined stretch of adenosines/ thymidines into the > UTR of the template pDNA. To allow transcripts to finish on an adenosine, the insert should be followed by a restriction site for an enzyme that cleaves 5 of the last antisense strand thymidine, such as Nsi I. In this way, the poly (A) tail can be incorporated directly into the... 

---