Deoxythymidine triphosphate

Pharmacology Zidovudine is a synthetic nucleoside analog of the naturally occurring nucleoside thymidine. The active metabolite, zidovudine 5 -triphosphate (AztTP), inhibits the activity of the HIV reverse transcriptase by competing for utilization with the natural substrate deoxythymidine 5 -triphosphate (dTTP) and by its... 

Radiochemicals tritiated 2 -deoxythymidine 5 -triphosphate [me/fty/-3H]dTTP, 2 -deoxyadenosine 5 -triphosphate [2,8-3H]dATP, 2 -deoxyguanosine 5 -triphos-phate [8-3H]dGTP or 2 -deoxycytidine 5 -triphosphate [5-3H]dCTP. The radiospecificities of the deoxy nucleotides usually range between 15 and 30 Ci/mmol. They can be obtained from several companies such as Amersham (Buckinghamshire, UK), ICN (Costa Mesa, CA), NEN (DuPont Ltd., Hertfordshire, UK), Moravek Biochemicals (Brea, CA), and so forth. They can be used at 2 pCi/assay. 

A chemoenzymatic synthesis of the P-a-methyl 2 -deoxynucleoside triphosphates 122 has been described which involves reaction of the 5 -0-(methylpho-sphonyl)-N-protected nucleosides with pyrophosphate in the presence of CDI. Removal of the base protection by treatment with penicillin amidase gave compounds 122 leaving the labile a-methylphosphonate intact. A number of 2 -deoxythymidine 5 -triphosphate and 3 -azido-2, 3 -dideoxythymidine 5 -tripho-sphate analogues (123) containing a hydrophobic phosphonate group have also been synthesised and evaluated as substrates for several viral and mammalian polymerases. Some y-ester (124) and y-amide (125) derivatives of dTTP and 3 -azido-2, 3 -dideoxythymidine 5 -triphosphate (AZTTP) were also synthesized and studied. The y-phenylphosphonate triphosphate 126 and its conjugation to biotin and fluorescein labels has also been described. 

Select from the following those compounds that are precursors of 2 -deoxythymidine-5-triphosphate (dTTP) in mammals and place them in their correct biosynthetic order. 

Deoxy-maw o-oct-2-ulosonic acid D-form 8-Phosphate, D-329 2-Deoxy-crj t o-pentose D-form 5-Phosphate, D-345 3 -Deoxythymidine 5 -Triphosphate, D-380 3 -Deoxy-5 -tl imdylic add, 9CI, D-380 PO -Digu osine 5 -tetraphosphate, D-670 l,T-Di-my -inosityl phosphate, D-712... 

Many other nucleotide analogs, notably those of dTTP, have been developed as potential RTase inhibitors having pharmacological value. For example, 3 -azido-3 -deoxythymidine 5 -triphosphate (NjdTTP) is incorporated by AMV RTase (and HIV-1 RTase as well) into the T sites of DNA and terminates chain elongation in a dose-dependent manner (48). The N3dTTP, which is converted from 3 -azido-3 -deoxythymidine (AZT) by cellular kinases, is believed to be the active metabolite producing the potent inhibitory effect of AZT on HIV-1 replication. 

A chain-terminating dTTP analog, 3 -azido-3 -deoxythymidine 5 -triphosphate (N3dTTP), is also efficiently incorporated by MoLV RTase into the T sites of elongating DNA in a dose-dependent manner (25). 

Thymine Deoxythymidme Deoxythymidylic acid Deoxythymidine monophosphate (dTMP) Deoxythymidine diphosphate (dTDP) Deoxythymidine triphosphate (dTTP)... 

The DNA component deoxythymidine triphosphate (dTTP) is synthesized from UDP in several steps. The base thymine, which only occurs in DNA (see p. 80), is formed by meth-ylation of dUMP at the nucleoside monophosphate level. Thymidylate synthase and its helper enzyme dihydrofolate reductase are important target enzymes for cytostatic drugs (see p. 402). 

Trifluridine (Viroptic) is a fluorinated pyrimidine nucleoside that has in vitro activity against HSV-1 and HSV-2, vaccinia, and to a lesser extent, some adenoviruses. Activation of trifluridine requires its conversion to the 5 monophosphate form by cellular enzymes. Trifluridine monophosphate inhibits the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) by thymidylate synthetase. In addition, it competes with deoxythymidine triphosphate (dTTP) for incorporation by both viral and cellular DNA polymerases. Trifluridine-resistant mutants have been found to have alterations in thymidylate synthetase specificity. 

Rossi, M. Woodward, D.O. Enzymes of deoxythymidine triphosphate biosynthesis in Neurospora crassa mitochondria. J. BacterioL, 121, 640-647 (1975)... 

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. 

The mono-, di-, and tri-phosphorylates of d4T were analysed by ion-pair LC-MS after lysis of the PBMC cell in Tris/methanol and centrifugation [50]. The supernatant was injected into the LC system with a 150x2.1-mm-ID Cjg column (5 pm) and a mobile-phase gradient of 70 to 35% solvent A (10 mmol/1 DMHA and 3 mmol/1 ammonium formate adjusted at pH 11.5) in solvent B (50% acetonitrile in 20 mmol/1 DMHA and 6 mmol/1 ammonium formate). Negative-ion ESI-MS was performed in SRM mode. The method enabled the direct measurement of the chain terminator ratio (d4T-triphosphate/deoxythymidine-triphosphate). Subsequently, the same group [51] reported modifications of this method, including simplifications of the sample pretreatment, replacement of the LC column for another type, and reduction of the column inner diameter from 2 mm ID to 0.32 mm ID. This improved method was applied to the determination of the phosphorylates of d4T, 3TC, and ddl. The sample throughput is 200 samples per week. The determination of intracellular AZT-triphosphate in PBMC [52], and the validation of the method for the determination of the ddl and d4T triphosphates was reported separately [53]. 

Zidovudine (7) (previously azidothymidine, AZT) is a deoxythymidine analog that also requires anabolic phosphorylation for activation. It competitively inhibits deoxythymidine triphosphate for the RT. It also acts as a chain terminator in the synthesis of proviral DNA. It is active against HIV-1, HIV-2 and the... 

A variety of derivatives of 3 -azido-2, 3 -dideoxynucleosides have been prepared in order to study the mechanism of action, to improve the transport of the drug or to obtain more selective compounds. These include tritium labelled derivatives of AZT [64, 65], various derivatives of 3 -azido-3 -deoxythymidine triphosphate [66, 67] and quaternary salts and dihydropyridine derivatives of AZT [68]. 

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). 

Fig. 14.1 Cellular pathway of methotrexate. ABCBl, ABCCl-4, ABC transporters ADA, adenosine deaminase ADP, adenosine diphosphate AICAR, aminoimidazole carboxamide ribonucleotide AMP, adenosine monophosphate ATIC, AICAR transformylase ATP, adenosine triphosphate SjlO-CH -THF, 5,10-methylene tetrahydrofolate 5-CHj-THF, 5-methyl tetrahydro-folate DHFR, dihydrofolate reductase dTMP, deoxythymidine monophosphate dUMP, deoxy-uridine monophosphate FAICAR, 10-formyl AICAR FH, dihydrofolate FPGS, folylpolyglutamyl synthase GGH, y-glutamyl hydrolase IMP, inosine monophosphate MTHFR, methylene tetrahydrofolate reductase MTR, methyl tetrahydrofolate reductase MTX-PG, methotrexate polyglutamate RFCl, reduced folate carrier 1 TYMS, thymidylate synthase. Italicized genes have been targets of pharmacogenetic analyses in studies published so far. (Reproduced from ref. 73 by permission of John Wiley and Sons Inc.)...

Fig. 3. Metabolism of the fluoropyrimidines dTMP = deoxythymidine monophosphate, dUMP = deoxyuridine monophosphate, FdUDP = fluorodeoxyuridine diphosphate, FdUMP - fluoro-deoxyuridine monophosphate, FdUTP = fluorodeoxyuridine triphosphate, FU-DNA= fluorouracil-deoxyribonucleic acid, FUDP = fluorouracil diphosphate, FUMP = fluorouracil monophosphate, FU-RNA = fluorouracil-ribonucleic acid, FUTP = fluorouracil triphosphate.

Azido-3 -deoxythymidine, or AZT, is a nucleoside analog approved for use to treat AIDS. Its metabolites, the mono-, di- and triphosphate forms, accu-... 

Furman PA, Fyfe JA, St. Clair MH, Weinhold K, et al. 1986. Phosphorylation of 3-azido-3 deoxythymidine and selective interaction of the 5 triphosphate with human immunodeficiency virus reverse transcriptase. Proc Natl Acad Sci USA. 83 8333-8337. 

ADP adenosine diphosphate, ANP atrial natriuretic peptide, AP5DTPT(5 -adenosyl)P5-(5 -thymidyl)pentaphosphate, ATP adenosine triphosphate, AZTMP 3 -azido-3 -deoxythymidine monophosphate, dGMP deoxyguanosine monophosphate, P 4-(2-hydroxyethyl)-l-piperazine ethanesulfonic acid, HMD hymenialdisine, TMP thymidine monophosphate... 

In the past, various serendipitous discoveries have capitalized on the differential expression of enzymes by host and viral infected cells. For example, the prodrug Acyclovir, used widely for the treatment of herpes simplex and herpes zoster infections, is selectively activated through phosphorylation by viral thymidine kinase to acyclovir monophosphate which is then converted to the triphosphate, which inhibits DNA polymerase, by host cellular enzymes. Similarly several 2, 3 -dideoxynucleoside analogs such as Zidovudine (azidothymidine, AZT) and 2, 3 -didehydro-3 -deoxythymidine (D4T) have potent antiviral activity against human immunodeficiency vims (HIV). These compounds are selectively phosphoiylated intracellularly to the 5 -triphosphate derivatives which inhibit the viral reverse transcriptase. 

A-residue, G-residue etc. or more simply dA, dG (or A, G etc. where there is no possibility of confusion with the ribonucleotides, for example as in a DNA sequence) pdT etc. dTMP, dAMP etc. rATP, rCTP etc. A deoxyadenosine (deoxyguanosine) monophosphate either at one end, or within, a polynucleotide chain and linked to the adjacent nucleotide(s) by phosphodiester bond(s) deoxythymidine 5 -monophosphate the deoxynucleoside 3 -monophosphate the ribonudeoside triphosphates, adenosine triphosphate, cytidine triphosphate etc. 

However, equivalent potency of enzyme inhibition has been reported for the triphosphates of 3 -deoxythymidine and 3 -amino-3 -deoxythymidine despite the weak antiviral activity of the parent nucleosides (163) and (90) in vitro, both of which are poor substrates for phosphorylation [213, 214]. These observations suggest that the antiviral aetivity of 2, 3 -dideoxynucleo-sides may depend less on the relative inhibitory activities of the triphosphates against isolated RT, but rather reflects the affinity of the parent nucleosides for the cellular kinase enzymes effecting triphosphorylation. Nevertheless, other studies of RT inhibition by nucleoside triphosphates have demonstrated a defined SAR for modifications at the 3 -position [215], and it is likely that the azido substituent of AZT contributes both to the requisite substrate activity for the cellular phosphorylating enzymes, and the specificity of RT inhibition following conversion to the triphosphate. 

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