Venom esters

Lecithins and related phospholipids usually contain a saturated fatty acid in the C-l position but an unsaturated acid, which may contain from one to four double bonds, at C-2. Arachidonic acid is often present here. Hydrolysis of the ester linkage at C-2 yields a l-acyl-3-phosphoglycerol, better known as a Iysophosphatidylcholine. The name comes from the powerful detergent action of these substances which leads to lysis of cells. Some snake venoms contain phospholipases that form Iysophosphatidylcholine. Lysophosphatidic acid (l-acyl-glycerol-3-phosphate) is both an intermediate in phospholipid biosynthesis (Chapter 21) and also a signaling molecule released into the bloodstream by activated platelets.15... 

The phosphoric acid esters of diacyl glycerides, phospholipids, are important constituents of cellular membranes. Lecithins (phosphatidyl cholines) from egg white or soybeans are often added to foods as emulsifying agents or to modify flow characteristics and viscosity. Phospholipids have very low vapor pressures and decompose at elevated temperatures. The strategy for analysis involves preliminary isolation of the class, for example by TLC, followed by enzymatic hydrolysis, derivatization of the hydrolysis products, and then GC of the volatile derivatives. A number of phospholipases are known which are highly specific for particular positions on phospholipids. Phospholipase A2, usually isolated from snake venom, selectively hydrolyzes the 2-acyl ester linkage. The positions of attack for phospholipases A, C, and D are summarized on Figure 9.7 (24). Appropriate use of phospholipases followed by GC can thus be used to determine the composition of phospholipids. 

Tetrafluoroethene has been used as a 2-carbon difluoroacetic acid equivalent, methodology developed by Normanl and co-workers, in the synthesis of inhibitors of Cobra venom phospholipase A2.13 Conversion of the allyl alcohols into the 2,2-difluoropent-4-enoic acids 32 is performed in one pot. The crude acids 32 are then converted into the methyl esters 33. Although esters 33 can be obtained directly from the acyl fluorides 29, as originally described by Normant and co-workers,10 a two-step procedure facilitates the workup after the Claisen rearrangement. 

V. Other Venom Enzymes That Hydrolyze Phosphate Esters. . 328... 

Venom has long been known to be a good source of several enzymes that hydrolyze esters of phosphoric acid. It is not possible to discuss venom exonuclease without mentioning other enzymes of this group. An effort will be made, however, to limit the discussion of other phosphatases to the bare essentials and key references. The surveys of different species of snake with respect to these enzymes are fairly numerous (1-9) and allow several conclusions to be drawn. 

Other substrates for spleen exonuclease are the p-nitrophenyl esters of nucleoside-3 -phosphates and bis(p-nitrophenyl) phosphate, which is split only very slowly. These substrates are also split by enzymes having quite different natural substrates (Table I) (80-87). In fact, not only phosphodiesterases, in a broad sense, such as acid DNase, micrococcal nuclease, spleen and venom exonucleases, and cyclic phosphodiesterase but also enzymes such as nucleoside phosphoacyl hydrolase and nucleoside polyphosphatase split these substrates. As pointed out by Spahr and Gesteland (86), this may be explained by the fact that these substrates are not true diesters but rather mixed phosphoanhydrides because of the acidic character of the phenolic OH. It is evident that the use of the synthetic substrates, advocated by Razzell (3) as specific substrates for exonucleases, may be very misleading. Table II shows the distinctive characters of three spleen enzymes active on bis(p-nitrophenyl) phosphate which are present in the crude extracts from which acid exonuclease is prepared. 

In the biochemical method, the enzyme phospholipase A2, isolated from Naja naja snake venom can attack the native alkenylacylglycerophosphocho-line and liberate completely the esterified fatty acid and the alkenyl(lyso)glyc-erophosphocholine. On the basis of the stereospecific mode of attack of this enzyme on the 2-acyl ester position of sn-3 phosphoglycerides, it can be concluded that the naturally occurring alkenylacylglycerophosphocholine possessed the sn-3 stereochemical configuration. 

The enzyme phospholipase A2 (PLA2, EC 3.1.1.4) is widely distributed among various species in the animal kingdom, notably in the pancreatic tissues of mammals and venoms from snakes and bees It specifically catalyzes the hydrolysis of the acyl-ester bond at the sn-2 position of l,2-diacyl-3-.w-phosphoglycerides in the presence of Ca (Dennis, 1983)... 

The venom of poisonous snakes contains a class of enzymes known as phospholipases. These enzymes catalyze the breakdown of phospholipids, triglycerides in which one fatty acid has been replaced by a phosphate group. The venom of the eastern dia-mondback rattlesnake contains a phospholipase that hydrolyzes the ester bond at the middle carbon of phospholipids, if the larger of the two breakdown products of this reaction gets into the bloodstream, it dissolves the membranes of red blood cells, causing them to rupture. A bite from the eastern diamondback can lead to death if not treated immediately. 

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. 

The racemic ( )-3TC 232 and other pyrimidine and pnrine analogues were resolved by varions methods (such as separation on HPLC with a chiral column, enzyme-catalyzed hydrolysis of its 5 -0-butanoyl ester derivative. or enzy matic resolution of its monophosphate derivative with a nucleotidase derived from Crotalus atrox venom ). Resolution of racemic thia sugar precursors lias also been described. 

The syntheses of dithymidylyl-3, 5 -phosphorofluoridate and phosphorothiof-luoridate (87) and (88) have been described. These involved the fluorinolysis of the P-Se bond in the bis-dimethoxytrityl selenomethyl esters (89) and (90). Compounds (87) and (88) were reported to be hydrolytically unstable, with no inhibitory activity on the snake venom and spleen phosphodiesterases and alkaline phosphatases. Finally, neither was considered as a highly toxic dinuc-leotide. ... 

Nucleoside Pyrophosphates. - 2.3.1 Nucleoside Diphosphate Analogues. A large variety of esters with different nucleoside and alkyl moieties (llla-j) have been synthesised in small amounts using different combinations of nucleoside triphosphate, alcohols and snake venom pyrophosphatase. Potato tube pyrophosphatase was also reported as being a possible practical biocatalyst to synthesise such nucleotide pyrophosphate-O-alkyl esters, but using more stringent reaction requirements than that of the snake venom enzyme. ... 

When 5 -0-tritylthymidine-3 -phosphate is treated with excess tri-isopropyl benzenesulphonylchloride (TPS) and thymidine, and then deprotected, the trinucleoside monophosphate (7a) is obtained. The 5-bromo- and 5-fluoro-deoxyuridine analogues (7b) and (7c) are prepared similarly. All are resistant to snake venom and spleen phosphodiesterases, and hydrolyse too slowly under physiological conditions for the cytotoxic moiety to be effective. When protected UpU is treated with bis-(4-nitrophenyl) phosphorochloridate, and subsequently with an amine or amino-acid ester, the dinucleoside phosphor-amidates (8) are formed. Although the compounds investigated split the P—N bond under the conditions required for protecting-group removal, the method has potential for the preparation of easily fissionable neutral phospho-triesters. 

We have also studied the stereochemical outcomes of the reactions catalyzed by the phosphodiesterase from snake venom (99) and from bovine spleen (101). The former enzyme catalyzes the hydrolysis of esters of 5 -nucleotides (free 3 -hydroxyl group), and the latter enzyme catalyzes the hydrolysis of esters of 3 -nucleotides (free 5 -hydroxyl group). No structural information of mechanistic consequence is available for these enzymes. 

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