Thymidine isolation

Thymine was isolated from hydrolyzates of bovine thymus or spleen in 1893, several years before uracil, but it was not made synthetically until 1901. Unlike uracil, it comes not from ribonucleic but from deoxyribonucleic acids via thymidine (3-D-2 -deoxyribofuranosidothymine). 

The TK+/ fine was originally isolated as a spontaneously arising revertant clone from a UV-induced TK / clone. The parental TK+/+ cell and the heterozygote were then the only TK-competent mouse lymphoma cells that could be maintained in THMG medium (3 pg ml-1 thymidine, 5 pg ml-1 hypoxanthine, 0.1 pg ml-1 methotrexate and 7.5 pg ml-1 glycine) (Clive, 1987). Thus, like most established lines, these cells are remote from wild-type cells. The karyotype of the TK+/ —3.7.2C line has a modal chromosome number of 40 like wild-type, but has a variety of chromosomal rearrangements and centromeric heteromorphisms (Blazak et al., 1986). 

Figure 1 Continued on next page) Immunopotentiating reconstituted influenza virosomes (IRIV) induced antigen specific proliferation of CD4+CD45RO+ cells. (A) Peripheral blood mononuclear cells (PBMQ from healthy donors n=3) were cultured in the absence of stimuli (Neg), in the presence of IRIV (V), and in the presence of control liposomes (L) at the indicated dilutions. Proliferation was measured on day 6 of culture by H-thymidine incorporation. (B) Cord blood mononuclear cells from two donors were cultured in the absence of stimuli (Neg) or in the presence of phytohaemag-glutinin (PHA), concanavalin A (ConA), IRIV (V) or L at the indicated concentrations. Proliferation was measured on day 3 of culture for PHA and ConA cultures and on day 6 for IRIV and L stimulated cultures. (Q Purified CD4+ or CD8+ cells were cocultured with autologous irradiated PBMC in the absence of stimuli (Neg) and in the presence of IRIV (V) at the indicated concentrations. Proliferation was measured on day 6 of culture by H-thymidine incorporation. (D) Purified CD4/CD45RA+ cells and CD4/CD45RO-I-cells were isolated from PBMC of one healthy donor and cocultured with autologous irradiated PBMC in the presence of IRIV (V) or L at the indicated concentration. Proliferation was measured on day 6 of culture by H-thymidine incorporation. Source From Ref 6.

The FOX assay applied to a skatole oxidation product isolated by HPLC gave a positive result, supporting the contention that it is skatolyl hydroperoxide (40) . Mixtures of 183 and the eight diastereoisomeric hydroperoxides 184 and 185 derived from thymidine (42), as shown in equation 64, can be separated and detected by RP-HPLC with UVD at 229 nm. Each isomer is determined by applying the FOX assay using a capillary reactor heated at 60 °C to provide sufficient time for total oxidation of the Fe(ll) ions, followed by UVD at 596 mn . A commercial kit based on the FOX assay for hydroperoxide determination in plasma, serum and tissue homogenizates appears in Table 2. 

An almost complete description of both OH radical-mediated and one-electron oxidation reactions of the thymine moiety (3) of DNA and related model compounds is now possible on the basis of detailed studies of the final oxidation products and their radical precursors. Relevant information on the structure and redox properties of transient pyrimidine radicals is available from pulse radiolysis measurements that in most cases have involved the use of the redox titration technique. It may be noted that most of the rate constants implicating the formation and the fate of the latter radicals have been also assessed. This has been completed by the isolation and characterization of the main thymine and thymidine hydroperoxides that arise from the fate of the pyrimidine radicals in aerated aqueous solutions. Information is also available on the formation of thymine hydroperoxides as the result of initial addition of radiation-induced reductive species including H" atom and solvated electron. 

The most important pyrimidine derivatives are those upon which biological organisms depend. Cytosine 1018 and uracil 1019 are found in ribonucleic acid (RNA) in the form of their ribonucleotides, cytidine 1020 and uridine 1021, while in deoxyribonucleic acid (DNA), cytosine and thymine 1022 are found in the form of their 2 -deoxyribonucleotides, 2 -deoxycytidine 1023 and thymidine 1024. 5-Methylcytosine 1025 is also found to a small extent (c. 5%) in human DNA in the form of its 2 -deoxyriboside 1026, and 5-(hydroxymethyl)cytosine-2 -deoxyriboside 1027 has also been detected in smaller amounts <2005CBI1>. Many variants of cytosine and uracil can be found in RNA including orotic acid 1028 in the form of its ribonucleotide orotidine 1029. Other pyrimidine derivatives to have been isolated from various biological sources include 2 -deoxyuridine 1030, alloxan 1031, and toxopyrimidine (pyramine) 1032 (Figure 2). 

Oxidation of 5-substituted barbimric acids 258 with concomitant ring contraction has been shown to afford 2,4-oxazolidinediones 260 (Scheme 6.58). Similarly, examples of 5-aryl- and 5-heteroaryl-2,4-oxazolidinediones, for example, 231 and 233-240 (Table 6.8 and Fig. 6.19) have been prepared from alloxan hydrate 261. Thus, conversion of 261 to the dilauric acid intermediates 262 and reaction with sodium hydroxide gave the target compounds.Swiss chemists isolated 265 as a side product (12% yield) from the oxidation of the thymidine base in 263 during their preparation of 264 (Scheme 6.58). 

Thymidine 5 -(a-D-glucopyranosyl pyrophosphate) (4) and the analogous D-galactose derivative have been isolated from extracts of Pasteurella pseudo tuberculosis.102 The ester of thymidine 5 -pyro-phosphate with a-D-mannopyranose was found in an extract of Strep-tomyces griseus,14,103 and the occurrence of the D-ribosyl ester in the... 

The most common derivative of this group of sugar nucleotides is thymidine 5 -(/3-L-rhamnopyranosyl pyrophosphate) (27), isolated from extracts of bacteria15,106,107 and streptomycetes.14 The assignment... 

The less basic purines generate different adducts. Both a C-8 adduct 107 and an 0-6 adduct 108 are produced in the presence of I, while the exclusive product of the reaction of A with 75n and 75o is the unique benzene imine 109. ° These purines also exhibit lower selectivity for trapping of the nitre-nium ions (Table 3). The pyrimidine nucleosides thymidine (T), uridine (U), and cytosine (C) showed negligible reactivity with these two nitrenium ions. ° The selectivity ratios for T, U, and C given in Table 3 are upper limits based on the decrease in the yield of the hydrolysis products at high nucleoside concentration (ca. 50mM). ° Since no adducts were isolated it is not clear that these selectivities represent nucleophilic trapping by the pyrimidines. 

Resistance to acyclovir can develop in HSV or VZV through alteration in either the viral thymidine kinase or the DNA polymerase, and clinically resistant infections have been reported in immunocompromised hosts. Most clinical isolates are resistant on the basis of deficient thymidine kinase activity and thus are cross-resistant to valacyclovir, famciclovir, and ganciclovir. Agents such as foscarnet, cidofovir, and trifluridine do not require activation by viral thymidine kinase and thus have preserved activity against... 

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