Snake venom, phosphodiesterases from

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

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... 

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. 

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. 

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... 

A uridine phosphate (5), obtained by treatment of uridine 5 -(a-D-glucopyranosyl pyrophosphate) with ammonia, imdergoes hydrazinolysis to D-ribose 5-phosphate (2) and 3-pyrazolone, which establishes the structure of (S) as uridine 5 -phosphate. - (Hydrazinolysis of uridine to pyrazolone, with the liberation of the sugar moiety, had been described, and has served as a useful tool in the determination of the position of attachment of the sugar moiety to the aglycon. ) The 5 -phosphates of adenosine, guanosine, cytidine, and uridine were obtained by enzymic hydrolysis of ribonucleic acid with phosphodiesterases from snake venom and from other sources (such as Streptomyces aureu ). 

Fig. 8. Terrapin bioprofiling pattern (adapted from ref. 47). Al, human gluthathione-5-transferase R8, rat glutathione-S-tranferase SI, schistosome glu-tathione-5-transferase HF2, housefly glutathione-i -transferase DAO, porcine D-amino acid oxidase BCh, equine butyryl cholinesterase Pap, papain PDE, snake venom phosphodiesterase I.

A new method for the preparation of 5 -amino-5 -deox)mucleotides has been described, based on the reaction between phosphorus triesters and phenyl azide. Treatment of protected 5 -azido-5 -deoxynucleosides with trimethyl or triphenyl phosphite leads, after removal of the nucleoside protecting groups and one of the esterifying groups from the phosphoryl residue, to the phosphoramidates (9). Snake venom phosphodiesterase hydrolyses (9) to 5 -aminonucleosides. 

When the product obtained from this oxidation of (41) is subsequently hydrolysed by snake venom phosphodiesterase in P OJHaO, a process in which the configuration at phosphorus is retained, and the chirality of the resulting 2 -deoxy-thymidine 5 -[ 0, 0, 0]phosphate analysed by cyclization to the 3, 5 -mono-phosphate, methylation and examination by P n.m.r., it is seen that the replacement of sulphur in (41) by proceeds with inversion at phosphorus, giving (43). Similarly, oxidation of (42) affords (44). Methylation of the potassium 18-crown-6 salt of (43) with methyl iodide gives the (i p)(45) and (5 p)(46) diastereoisomers of the methyl phosphotriester, and (45) gives a signal at higher field in the P n.m.r. spectrum than (46). The results show that little, if any,... 

The length of the polymer has been reported to vary from three to several hundred units of ADP-ribose 84) and may depend on experimental conditions. Number of chains and chain length can be estimated by isolation and quantification of the products (5 -AMP and iso-ADP-ribose) of the snake venom phosphodiesterase digestion. The number of 5 -AMP molecules represents the number of polymer chains. Average chain length can be estimated utilizing the relationship ... 

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