Thymine synthesis

SV40 Promoter This is the promoter for expression of the DHFR gene. DHFR The enzyme dihydrofolate reductase (DHFR) is required for nucleotide (thymine) synthesis and cell growth and serves as a selection marker in mammalian cells. 

Decreased levels of folate coenzymes needed for various reactions of de novo purine synthesis and thymine synthesis produce shortages of deoxyribonudeotides and consequent impaired DNA synthesis in many tissues. 

Other useful targets for pharmaceutical agents are thymidylate synthase and dihydrofolate reductase, enzymes that provide the only cellular pathway for thymine synthesis (Fig. 22-49). One inhibitor that acts on thymidylate synthase, fluorouracil, is an important chemotherapeutic agent. Fluorouracil itself is not the enzyme inhibitor. In the cell, salvage pathways convert it to the deoxynucleoside monophosphate FdUMP, which binds to and inactivates the enzyme. Inhibition by FdUMP (Fig. 22-50) is a classic example of mechanism-based enzyme inactivation. Another prominent chemotherapeutic agent, methotrexate, is an inhibitor of dihydrofolate reductase. This folate analog acts as a competitive inhibitor the enzyme binds methotrexate with about 100 times higher affinity than dihydrofolate. Aminopterin is a related compound that acts similarly. 

The effects of cobalamin deficiency are most pronounced in rapidy dividing cells, such as the erythropoietic tissue of bone marrow and the mucosal cells of the intestine. Such tissues need both Die N5-N10-methylene and N10-formyl forms of tetrahydrofolate for Ihe synthesis of nucleotides required for DNA replication (see pp. 291, 301). However, in vitamin B12 deficiency, the N5-methyl form of tetrahydrofolate is not efficiently used. Because the methylated fonn cannot be converted directly to other forms of tetrahydrofolate, tie Ns-methyl form accumulates, whereas the levels of the other forms decrease. Thus, cobalamin deficiency is hypothesized to lead to a deficiency of the tetrahydrofolate forms needed in purine and thymine synthesis, resulting in the symptoms of megaloblastic anemia. 

Folic acid Tetrahydrofolate Transfer of one-carbon components thymine synthesis Anemia, neural-tube defects in development... 

Methotrexate inhibits dihydrofolate reductase, which catalyzes reduction of FH2 to FH4. When dUMP is converted to dTMP, A ,A1 methylene tetrahydrofolate is converted to dihydrofolate, which must be reduced to tetrahydrofolate in order for the production of thymine to continue. If the reductase is inhibited by methotrexate, thymine synthesis also is inhibited, thus preventing DNA synthesis. 

Endova, M., Masojidkova. M.. Budesinsky. M.. and Rosenberg, L, 3. 5 -O-Phosphonoalkylidene derivatives of l-(2-deoxy-P-D-//zrec>-pentofuranosyl)thymine. Synthesis and reactivity. Tetrahedron. 54, 11187, 1998. 

The unique aspects of thymine synthesis allow the design of antimetabolites that may have greater specificity in this process. Aminopterin and methotrexate have been used with some success. These are inhibitors of tetrahydrofolate formation where, at the correct dosage, the conversion of dUMP to dTMP appears to be more sensitive than many other reactions and is preferentially inhibited. This inhibits DNA formation and cell proliferation. Other... 

J. Lee, A highly stereoselective glycosylation of 2-(phenylselenyl)-2,3-dideoxy-ribose derivative with thymine Synthesis of 3 -deoxy-2, 3 -didehydrothymidine and 3 -deoxythmidine, J. Org. Chem. 55 1418 (1990). 

The mode of action has been a subject for research for a number of years. While it was originally thought that maleic hydrazide replaced uracil in the RNA sequence, it has been deterrnined that the molecule may be a pyrimidine or purine analogue and therefore base-pair formation is possible with uracil and thymine and there exists the probabiHty of base-pair formation with adenine however, if maleic hydrazide occurs in an in vivo system as the diketo species, then there remains the possibiHty of base-pairing with guanine (50). Whatever the mechanism, it is apparent that the inhibitory effects are the result of a shutdown of the de novo synthesis of protein. 

The best laboratory synthesis of thymine (947) is probably from 3-methylmalic acid (945) which gives 2-formylpropionic acid (946 R = H) in situ by decarboxylation and oxidation in fuming sulfuric acid prior to condensation with urea (46JA912) a similar method from ethyl 2-formylpropionate (946 R = Et) is also described (68IZV918). 

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. 

Deoxy-3 -fluorothymidine (813), a selective inhibitor of DNA synthesis, was prepared " in moderate yields from 3 -0-mesyl- or 3, 5 -di-O-mesyl-thymidine, through 2,3 -anhydro-1 -(2-deoxy- -D-t/2reopentofur-anosyl)thymine (808), by treatment with hydrogen fluoride (0.1% HF in l,4-dioxane-AlF3, 3.764 hf in DMF-AlFj, or 10% HF in DMF ),... 

Incorporation of a flavin electron donor and a thymine dimer acceptor into DNA double strands was achieved as depicted in Scheme 5 using a complex phosphoramidite/H-phosphonate/phosphoramidite DNA synthesis protocol. For the preparation of a flavin-base, which fits well into a DNA double strand structure, riboflavin was reacted with benzaldehyde-dimethylacetale to rigidify the ribityl-chain as a part of a 1,3-dioxane substructure [49]. The benzacetal-protected flavin was finally converted into the 5 -dimethoxytri-tyl-protected-3 -H-phosphonate ready for the incorporation into DNA using machine assisted DNA synthesis (Scheme 5a). For the cyclobutane pyrimidine dimer acceptor, a formacetal-linked thymine dimer phosphoramidite was prepared, which was found to be accessible in large quantities [50]. Both the flavin base and the formacetal-linked thymidine dimer, were finally incorporated into DNA strands like 7-12 (Scheme 5c). As depicted in... 

Scheme 5 a Flavin-H-phosphonate and formacetal-linked thymine dimer phospho-ramidite used for the synthesis of the flavin and dimer containing DNA-strands 7-12. b Representation of a reduced flavin- and formacetal-linked cyclobutane pyrimidine dimer containing DNA strand, which upon irradiation (hv) and electron transfer (ET) performs a cycloreversion (CR) of the dimer unit, c Depiction of the investigated oligonucleotides... 

Large doses of ultraviolet light can damage DNA. In humans this damage is confined to the skin, since, unlike x-rays, ultraviolet light is easily absorbed. The chemical lesion in this case is the formation of dimers between adjacent thymine residues on the same DNA strand. Unless corrected or removed, these dimers will stop DNA synthesis. 

An improved synthesis of nC-2 thymine 49, for use in PET scans, was made possible by an efficient and rapid synthesis of nC-phosgene, previously reported <02NMB345> by the same authors. nC is a particularly interesting challenge due to its very short half life (20 minutes) and the whole sequence and purification from the end of the bombardment took 16 minutes. The scale was necessarily small (0.2 mg) <06TL5321>. 

Pd(PPh3)4 and N,N -dimethyl-NfNf-propylene urea (DMPU). The product of this reaction 315 was a key intermediate in the synthesis of (Z)-olefinic RNA containing adenine and thymine as bases. 

Transformation of chiral nitrones into enantiomer enriched a-chiral N -hydroxylamines and their derivatives, has been successfully employed in the enantioselective synthesis of (+ )-(R)- and (—)-(S)-zileuton (216). An expeditious synthesis of thymine polyoxin C (347), based on the stereocontrolled addition of 2-lithiofuran (a masked carboxylate group) to the A-benzyl nitrone derived from methyl 2,3-O-isopropylidene-dialdo-D-ribofuranoside, is described (Scheme 2.151) (194). 

The reaction of silyl ketene acetal addition to nitrones has been used for the synthesis of optically active (2S,3S)-benzoyl- and /V- oc-phenyl isoserine (636a) of isoxazolidine nucleoside-analog of thymine polyoxine C(636b) and of... 

---