Snake venom, phosphodiesterase

FIGURE 11.31 Snake venom phosphodiesterase and spleen phosphodiesterase are exonncleases that degrade polynncleotides from opposite ends. 

Snake venom phosphodiesterase Both a Starts at 3 -end, 5 -NMP products... 

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. 

Using phosphotriester methods, dinucleoside (3 - 50-monophosphates containing 6-methyl-2,-deoxyuridine at the 3 - or 5 -end have been prepared.44 N.m.r. spectroscopy indicates that this nucleoside possesses the syn conformation in these compounds, and, on treatment with snake venom phosphodiesterase, d(m6UpT) is degraded, while d(Apm6U) is not, indicating that this enzyme, a 3 -exonuclease, requires the anti conformation to be present in the substrate. Two modified nucleo-side-5 -monophosphates, (20) and (21), which are resistant to 5 -nucleotidase, have been isolated from tRNA snake venom hydrolysates.45 A synthesis of (20) has been reported.46... 

The question of enzyme specificity for irradiated polynucleotides is taken up in more detail in the recent review of Johns.11 The specificities of four enzymes, spleen phosphodiesterase, snake venom phosphodiesterase, pancreatic ribonuclease, and pancreatic deoxyribonuclease are discussed. 

The existence of photoreversible, but not of heat-reversible, absorbance change in irradiated poly dI dC was taken to prove that the photoproducts are entirely dimers (in contrast to those in poly C irradiations where the product is almost entirely the hydrate82a). It was possible to detect dimers of uracil as well as those of cytosine, by means of the much slower photoreversal of uracil dimers. In the acid hydrolysates of irradiated dl-dC, both uracil dimers and uracil could be identified. Enzymatic hydrolysis (snake venom phosphodiesterase) does not split pyrimidine dimers, and the products of such hydrolysis of irradiated tritium-labeled poly dl dC contained trinucleotides shown by radioactivity to contain cytosine dimers. Thymine dimers were formed in the photolysis of the poly dA dT, and were detected and assayed by the same methods. The yield of thymine dimers in irradiated poly... 

When the Tg lesions is opened by ammonolysis, the resulting product (ureidoisobutyric acid) inhibits snake venom phosphodiesterase, A exonuclease and the Klenow (exo ) fragment (Matray et al. 1995 see also Greenberg and Matray 1997). It is, however, removed by E. coli Fpg and Nth proteins (Jurado et al. 1998). 

SVPDE/AP snake venom phosphodiesterase and alkaline phosphatase t-BOC tm-butoxycarbonyl (group)... 

Snake Venom Phosphodiesterase An exonuclease is an enzyme that sequentially cleaves nucleotides from the end of a polynucleotide strand. Snake venom phosphodiesterase, which hydrolyzes nucleotides from the 3 end of any oligonucleotide with a free 3 -hydroxyl group, cleaves between the 3 hydroxyl of the ribose or deoxyribose and the phosphoryl group of the next nucleotide. It acts on single-stranded DNA or RNA and has no base specificity. This enzyme was used in sequence... 

Iqbal et al. studied the phosphodiesterase inhibitory effect of durantins A (33), B (15), and C (16), isolated from Duranta repens (70). Compounds 16 and 33, along with compound 9, showed moderate to strong inhibitory activity against snake venom phosphodiesterase 1, using cysteine and EDTA as positive controls, while 15 showed weak activity. 

The mutagenic ribonucleotide rPTP (87) has been used in an in vitro retroviral replication model. After four rounds of replication the mutation frequency was raised to 3.8 x 10 per nucleotide, with C U and U C mutations observed. It was suggested that such an analogue could induce mutations in a retroviral target beyond its error threshold. The tricyclic cytosine analogues phenoxazine and 9-(2-aminoethoxy)-phenoxazine (g-clamp, see (141)) have been incorporated into ODNs to study their effect with exonuclease. It was found that a single substitution at the 3 -terminus afforded complete protection of the ODN with snake venom phosphodiesterase. ... 

Monomers [180]-15a-d were successfully used for the synthesis of several stereodefined oligo(nucleoside [180]phosphorothioate)s. Their stereochemistry and isotopic enrichment were confirmed by a combined enzymatic-mass spectrometry method employing snake venom phosphodiesterase or calf spleen nuclease, and MALDI TOF mass spectrometry. 

Contrary to information that LNA oligonucleotides are resistant to digestion with nucleases [67], we have observed that diastereomer 45a of LNA dinucleoside phosphorothioate (presumably RP), obtained from/asf-44, was readily digested by snake venom phosphodiesterase. 

On treatment of guanosine 2, 3 -monophosphate with ethanol, propanol, or glycerol in the presence of RNase Ni, the corresponding 3 -guanylyl esters are formed, in modest yield. In the last case, subsequent digestion with snake venom phosphodiesterase affords a mixture of d- and L-glycerol 3-phosphates in 1 2 ratio. 

Stec applied the above-described oxathiaphospholane approach to synthesise stereoselectively the phosphorothioates of the locked nucleic acids (84a) and (84b) from (83a) and (83b), respectively (Scheme 7). The oxathiaphospholane ring opening condensation reaction proceeded in acetonitrile in high yield and with 96% stereoselectivity. One of the two diastereomers thus prepared was found to be readily digested by snake venom phosphodiesterase, an enzyme known to be an Kp-specific nuclease. However, neither diastereoisomer was hydrolysed by nuclease PI, an enzyme known to preferentially hydrolyse phosphorothioate linkages of Sp-configuration. 

The reaction of 5 -amino-5 -deoxyadenosine with trimetaphosphate affords the 5 -Af-triphosphate (23). When (23) is employed as substrate with glucose in the hexokinase-catalysed reaction, the 5 -AT-diphosphate (24) is obtained the latter is cleaved by snake venom phosphodiesterase to the 5 -phosphoramidate, and hydrolyses in acid to the amino-nucleoside. It does not appear to be polymerized by polynucleotide phosphorylase. In this context it is noteworthy that uridine 5 -5-thiopyrophosphate (25) is a competitive inhibitor for polynucleotide phosphorylase from E. coli, but not a substrate, and that the 5 -S-thiotriphosphates (26) and (27) show neither substrate nor inhibitory properties for RNA polymerase or DNA polymerase I, respectively. However, (23) can be polymerized using the latter enzyme, showing that the introduction of a 5 -heteroatom does not completely exclude these modified nucleotides as substrates for the polymerizing enzymes. 

Nucleotides in aqueous solution can be alkylated at the phosphate (and in some cases the nucleoside also) by the action of l-oxidopyridin-2-yldiazo-methane (33). - The protecting group may be removed from the phosphate with snake venom phosphodiesterase, or generally by acetic anhydride treatment, followed by ammonia. Phosphoramidates have been described previously as phosphate-protecting groups, and if 2-naphthylamine is used as its anilidate for this purpose, organic solvent extraction (as above) is possible. A variation on this theme is to use dianilidophosphochloridate (34) as a... 

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