Thymidine 5 - , hydrolysis

An isobutyl carbonate was prepared by reaction with isobutyl chloroformate (Pyr, 20°, 3 days, 73% yield), to protect the 5 -OH group in thymidine. It was cleaved by acidic hydrolysis (80% AcOH, reflux, 15 min, 88% yield). ... 

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. 

A polynucleoside with an unnatural polymeric backbone was synthesized by SBP-catalyzed oxidative polymerization of thymidine 5 -p-hydroxyphenylacetate. Chemoenzymafic synthesis of a new class of poly(amino acid), poly(tyrosine) containing no peptide bonds, was achieved by the peroxidase-catalyzed oxidative polymerization of tyrosine ethyl esters, followed by alkaline hydrolysis. Amphiphile higher alkyl ester derivatives were also polymerized in... 

Selective reaction at the ci.s-2,3-diol grouping of unprotected D-ribonucleosides has occasionally been observed. Treatment of D-ribonucleosides with tris(tetramethylammonium) trimetaphosphate in M sodium hydroxide for 4 days at room temperature led to a mixture of nucleoside 2 - and 3 -phosphates in yields of >70% no 5 -phosphate was detected.213 Reaction of ethyl (trichloromethyl)phos-phonate with nucleosides in N,N-dimethylformamide containing triethylamine, followed by basic hydrolysis of the reaction product, yielded 2 (3 )-phosphates in variable yields.214 The participation of the cis-diol grouping in the reaction was suggested by the failure of thymidine or 2, 3 -0-isopropylideneuridine to undergo reaction. 

Scheme 1.23 Enzyme-catalysed hydrolysis of thymidine-17 -monophosphate...

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. 

Conversion of the 3, 5 -diacetyl thymidine 652 (R = Me) to the chloro derivative 653 followed by reaction with sodium azide in anhydrous DMF gave 654, whose hydrolysis gave 655 (86JHC1401). The 2 -deoxy-2, 2 -difluoro analog of 655 was prepared (93EUP576230) (Scheme 132). 

The action of venom exonuclease is also blocked by thymine dimers produced as a result of UV irradiation of DNA at 280 nm. Setlow et al. (66) subjected irradiated DNA to exhaustive digestion by venom exonuclease. They isolated and identified the products of the reaction, which were composed of large amounts of all four 5 -mononucleotides and of small amounts of trinucleotides of the type d-pNpTpT where N was any of four common nucleosides and TpT was the irradiation-induced dimer of thymidine. These trinucleotides were totally resistant to further digestion with venom exonuclease but became partially susceptible after UV irradiation at 240 nm, known to reverse dimerization. The authors picture the action of venom exonuclease as proceeding linearly from the internal bond one base beyond the dimer. From there on conventional hydrolysis is resumed until the next block. This experiment touches upon one of the most pressing problems connected with venom exonuclease. Is the endonucleolytic activity an intrinsic property of the enzyme ... 

Murray and Friedrichs (80) have obtained a 5 -nucleotidase from a particulate fraction of Ehrlich ascites tumor cells using deoxycholate. The relative rates of hydrolysis of 5 -UMP, 5 -AMP, 5 -CMP, 5 -GMP, and 5 -IMP are 129, 100, 93, 83, and 79, respectively. Adenosine and thymidine triphosphate are competitive inhibitors of 5 -AMP hydrolysis... 

The same thymine dimer has been obtained indirectly from thymidylyl-(3, 5 )-thymidine,252 and has been isolated from DNA by hydrolysis after irradiation with UV light both in vivoZ5Z and in vitro.254 The identity of this dimer has recently been confirmed chemically.288 This dimerization between two adjacent thymine residues is thought to be one of the major factors responsible for the photochemical inactivation of transforming DNA. There are conflicting views as to whether the dimerization occurs within one strand or... 

Since it is well established that the P-chiral phosphorothio-ates serve as effective probes in mechanistic studies for the phos-phoryl group transfer enzymes /16/, we turned our attention to the application of diastereomeric 8 (B=Thy, Ar=pN02C6Hi -,/17f) to elucidate the mode of action of spleen phosphodiesterase (SPDE, EC 3.1.1. 18). This enzyme splits the phosphodiester bonds to yield nucleoside 3 -phosphates. In the case of it was expected that its SPDE-catalyzed hydrolysis in 180 H 2O medium leads to P-chiral thymidine 3 - 180 phosphorothioate. On the contrary to our expectation the main product of this reaction was thymidine cyclic 3 ,5 - Rp phosphorothioate (10) /6/. By treatment of 8 under the same conditions, but in the absence of the enzyme, no trace of J 0 was detected. ... 

A possibility would be monomeric thymidine 3 -nietaphosphorothioate, which in the active site allowing the rotation around C-0 bond would lead to the same preferred orientation being adopted whatever the stereochemistry of the starting diester and this would then lead to a single diastereomeric product P>p -10. If this explanation is correct and hydrolysis of thymidine 3 -p-nitrophenyl- 80 phos-phate would follow the same mechanistic course, its hydrolysis in 1170 H20 containing medium in the presence of SPDE should lead to formation of P-racemic thymidine 3 160,170,180 phosphate. The work on elucidation of this hypothesis is in progress. 

More difficulty was encountered with the pyrimidine deoxynucleosides. It was tacitly assumed that the sugar radical in thymidine and deoxy-cytidine, by analogy with the ribonucleosides, is attached to position Nl. The first significant experiment111 to shed some light upon the validity of this assumption was based upon the methylation studies of Levene and Tipson.108 Deoxyribonucleic acid from thymus was methylated with dimethyl sulfate plus alkali, and the product was degraded by strong-acid hydrolysis. One of the products obtained was 3-methylthymine (XI).111 A... 

A parallel experiment146 was performed with 5-deoxy-5-iodothymidine (XXX), a nucleoside derivative which was prepared146,146 by a series of reactions from thymidine. Treatment of XXX with silver acetate in acetonitrile yielded the 2, 5-anhydronucleoside (XXXI) in crystalline form. Alkaline hydrolysis of XXXI regenerated thymidine. An x-ray crystallo-... 

The 1,2-photodimer (194), of some biological significance, was obtained as the main product on irradiation of a frozen aqueous solution of thymine or DNA (followed by hydrolysis in the latter case).275 This dimerization between two adjacent thymine residues is considered to be the major change responsible for the photochemical inactivation of DNA dimerization appeared to occur within one strand (intrastrand).276 Upon irradiation of 1,3-dimethylthymine,277 1,3-dimethyluracil,216,278 uracil,279 and thymidine,280 in frozen aqueous solutions, a mixture of all four possible 1,2-dimerization products (i.e., cis and trans head-to-head, cis and trans head-to-tail) have been produced. A triplet state has recently been proposed as their precursor.216 It has also been shown281 that the trans head-to-head photodimer (195) of 2-quinolone, obtained in ethanol,282 is formed by condensation of the triplet state monomer with another unexcited molecule. 

A practical example of separation of a mixture of a/P-anomers of the mother liquor collected from the commercial-scale synthesis of P-thymidine is shown in Scheme 10.14. PSLC is used as a biocatalyst because this lipase has shown the desired opposite regioselectivity toward the hydrolysis of a- and P-nucleosides. The hydrolysis reaction takes place with excellent selectivity toward the 5 -p-... 

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