Thymidine nucleotides synthesis

With the aid of cytosine permease, flucytosine reaches the fungal cell where it is converted by cytosine deaminase into 5-fluorouracil [51-21-8]. Cytosine deaminase is not present in the host, which explains the low toxicity of 5-FC. 5-Fluorouracil is then phosphorylated and incorporated into RNA and may also be converted into 5-fluorodeoxyuridine monophosphate, which is a potent and specific inhibitor of thymidylate synthetase. As a result, no more thymidine nucleotides are formed, which in turn leads to a disturbance of the DNA-synthesis. These effects produce an inhibition of the protein synthesis and cell repHcation (1,23,24). 5-Fluorouracil caimot be used as an antimycotic. It is poorly absorbed by the fungus to begin with and is also toxic for mammalian cells. 

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). 

Shoji A, Hasegawa T, Kuwahara M et al (2007) Chemico-enzymatic synthesis of a new fluorescent-labeled DNA by PCR with a thymidine nucleotide analogue bearing an acridone derivative. Bioorg Med Chem Lett 17 776-779... 

These one-carbon groups, which are required for the synthesis of purines, thymidine nucleotides and for the interconversion some amino acids, are attached to THF at nitrogen-5 (N5), nitrogen-10 (N10) or both N5and N10. Active forms of folate are derived metabolically from THF so a deficiency of the parent compound will affect a number of pathways which use any form of THF. 

In Fig. 1 various targets of some important cytostatic agents are depicted. Their main mechanisms of action can be briefly summarized as follows. Pentostatin blocks purine nucleotides by inhibiting adenosine deaminase. 6-Mercaptopurine and 6-thioguanine inhibit purine ring biosynthesis and they inhibit nucleotide interconversions. Methotrexate by inhibiting dihydrofolate reduction blocks thymidine monophosphate and purine synthesis. 5-Fluorouracil also blocks thymidine monophosphate synthesis. Dactinomycin, daunorubicin, doxorubicin and mitoxantrone intercalate with DNA and inhibit RNA synthesis. L-asparaginase deaminates... 

The inability to absorb Vitamin B12 occms in pernicious anemia. In pernicious anemia intrinsic factor is missing. The anemia results from impaired DNA synthesis due to a block in purine and thymidine biosynthesis. The block in nucleotide biosynthesis is a consequence of the effect of vitamin B12 on folate metabolism. When vitamin B-12 is deficient essentially all of the folate becomes trapped as the N -methyltetrahydrofolate derivative as a result of the loss of functional methionine synthase. This trapping prevents the synthesis of other tetrahydrofolate derivatives. required for the purine and thymidine nucleotide biosynthesis pathways. 

Synthesis of Thymidine nucleotides first requires deoxyribonucleotide synthesis. The enzyme responsible for this step is Ribonucleotide Reductase. This enzyme acts on oxynucleotides in their diphosphate form. Thioredoxin, a small protein, is oxidized as the 2 hydroxyl group on the ribose ring is reduced. Oxidized Thioredoxin (S-S) is then reduced by FADH2 and NADPH. The products are the respective deoxynucleotide diphosphates which are further phosphorylated and then used for DNA synthesis. 

Inhibition of the Reductase affects folate metabolism leading to decreased glycine formation from serine and decreased purine synthesis which requires CH3-THF. To facilitate normal cells folinic acid (Leucovorin) is given along with methotrexate. This acid aids normal cells by its conversion to the coenzyme of Thymidyiate S)mthetase, thus bypassing the block. Since the thymidine nucleotide requirements of rapidly proliferating cells are much greater than for quiescent cells folinic acid cannot meet the demands of the cancer cells. 

The brain needs the influx of nucleosides because the brain is deficient in de novo nucleotide synthesis (102). Purine and pyrimidine nucleosides are necessary for the synthesis of DNA and RNA, but nucleosides also influence many other biological processes. In addition, nucleosides play an important role in the treatment of diseases, such as cardiac diseases, brain cancers, and infections [parasitic and viral (103)]. Nucleosides are hydrophilic compounds, and the influx and efflux of these compounds is therefore mediated by a number of distinct transporters (104). Nucleoside transporters are membrane-fixed transporters and are classified by their transport mechanisms (e = equilibrative, c = concentrative), their sensitivity to the transport inhibitor nitrobenzylmercaptopurine riboside (NBMPR s = sensitive, i = insensitive), and their substrates. Presently, there are two equilibrative transporters (ENTs es and ei) and six concentrative nucleoside transporters [CNTs cif (concentrative, NBMPR insensitive, broad specificity Nl), cit (concentrative, NBMPR insensitive, common permeant thymidine N2), cib (concentrative, NBMPR insensitive, broad specificity N3), cib (concentrative, MBMPR insensitive, broad specificity N4), cs (concentrative, NBMPR sensitive N5), and csg (concentrative, NBMPR sensitive, accepts guanosine as permeant N6) (104)]. The equilibrative es and ei nucleoside transporters are widely expressed in mammalian cells and are present at cultured endothelial cells and brain capillaries (105). In these cells, the expression of concentrative transporter cit (N2) was demonstrated also. In other parts of the rat brain, ei and es nucleoside transport systems have... 

UMP is the parent compound in the synthesis of cytidine and deoxycytidine phosphates and thymidine nucleotides (which are deoxyribonucleotides). 

What are called antifolate drugs pertain in general to blocking the biosynthesis of purines and pyrimidines, the heterocyclic bases used in the further synthesis of DNA and RNA, where folic acid is required as a coenzyme (or vitamin) for the enzyme dihydrofolate reductase. The previously mentioned compound called methotrexate or amethopterin (4-amino-A °-methyl folic acid), being a structural analog of folate or folic acid, locks up the enzyme dihydrofolate reductase, which in turn blocks the synthesis of a thymidine nucleotide necessary for cell division. 

Cytosolic thymidine kinase salvages exogenous thymidine extremely efficiently. Experiments with radiolabeled precursors show that dTTP derived from salvage synthesis is usually incorporated into DNA in preference to thymidine nucleotides generated by de novo synthesis. 

To avoid incorporating uridines into DNA, cells have developed a rather a simple mechanism. The enzyme, dUXPase, converts dUTP (a substrate for incorporation into DNA) to dUMP (not a substrate for incorporation into DNA), and also provides a route to synthesis of thymidine nucleotides because the dUMP, in turn, is converted first to dTMP then to dTTP (Figure 22.17). 

The synthesis of deoxyuridine, cytidine, deoxycytidine and thymidine nucleotides from UMP (Fig. 6.13) involves three reactions CTP synthetase, ribonucleotide reductase, and thymidylate synthase (80). The first enzyme converts UTP into CTP and the second catalyzes the conversion of CDP, UDP, ADP and GDP into their respective deoxyribonucleotides. The last enzyme, thymidylate synthase, catalyzes the reductive methylation of deoxyUMP at the C-5 position giving deoxyTMP. The human enzyme has been extensively studied as it is a target enzyme in cancer chemotherapy. Besides these three enzymes, two other enzymes are involved in pyrimidine nucleotide synthesis and interconversion. DeoxyCMP deaminase converts deoxyCMP into deoxyUMP and deoxyUTP triphosphatase converts deoxyUTP into deoxyUMP. Giardia lamblia, and Trichomonas vaginalis lack both ribonucleotide reductase and thymidylate synthase and... 

The effect of acronycine (30) on the growth of cultured cells and of nucleoside uptake and incorporation into nucleic acids by the cells has been studied. It was found that, although acronycine does not inhibit nucleic acid synthesis, it does inhibit the accumulation of extracellular uridine and thymidine nucleotides in the precursor pool. Oral administration of dubamine to mice shows no apparent toxic elTects. ... 

Mechanism of action Fluorouracil is an analogue of uracil. After entry into cells it undergoes conversion to active metabolites fluorodeoxyuridine monophosphate (FdUMP), fluorodeoxyuridine triphosphate (FdUTP) and fluorouridine triphosphate (FUTP). FdUMP directly inhibits thymidy-late synthetase, reducing the availability of thymidine nucleotides, which are required for DNA synthesis, until new enzyme can be synthesized. FUTP is incorporated into RNA and causes impaired RNA processing and functioning, which disrupts cellular metabolism and viability [71 ]. 

The principle of selection is to place these cells into a selective medium in which the de novo pathway of nucleotide synthesis is inhibited. The key to this is the compound, aminopterin which is an analogue of folic acid and a specific inhibitor of dihydrofolate reductase, an essential enzyme for the formation of tetrahydrofolate (FH4) required as a coenzyme of the de novo purine nucleotide synthesis pathway. Tetrahydrofolate is also required for the formation of thymidine. However, if hypoxanthine and thymidine are provided in the culture media of HGPRT+ cells they will be able to grow normally. On the other hand HGPRT" cells would have no means of synthesizing purine nucleotides and consequently would be unable to grow. 

Human tissues can synthesize purines and pyrimidines from amphibolic intermediates. Ingested nucleic acids and nucleotides, which therefore are dietarily nonessential, are degraded in the intestinal tract to mononucleotides, which may be absorbed or converted to purine and pyrimidine bases. The purine bases are then oxidized to uric acid, which may be absorbed and excreted in the urine. While little or no dietary purine or pyrimidine is incorporated into tissue nucleic acids, injected compounds are incorporated. The incorporation of injected [ H] thymidine into newly synthesized DNA thus is used to measure the rate of DNA synthesis. 

Unmodified siRNA in this context is restricted to the 19 base pairing nucleotides of siRNA duplexes. Most siRNAs are synthesized with two nucleotide 3 overhangs. The last or the last two nucleotides are often desoxy-thymidines in order to increase the resistance of the siRNAs against exonucleases and because of technical reasons in RNA synthesis. siRNAs modified with 3 desoxy-thymidines are classified as unmodified in the context of this chapter. 

The pyrimidine antagonists inhibit the biosynthesis of pyrimidine nucleotides or interfere with vital cellular functions, such as the synthesis or function of nucleic acids. The analogues of deoxycytidine and thymidine that are used are inhibitors of DNA synthesis while 5-fluorouracil (5-FU) an analogue of uracil, is an inhibitor of both RNA function and of the synthesis of thymidylate (see Fig. 2). PALA (N-phosphonoacetyl-L-aspartate), an inhibitor of as-... 

Several cases of synthesis of a-D-galactosyl nucleotides from a-D-galactopyranosyl phosphate with bacterial enzymes have been reported. These included formation of UDP-Gal through reaction with uridine 5 -triphosphate22 or UDP-Glc,14,44 49 and of dTDP-Gal through interaction with thymidine 5 -triphosphate.50... 

---