Nucleophilic attacks phosphate esters

DNA is not susceptible to alkaline hydrolysis. On the other hand, RNA is alkali labile and is readily hydrolyzed by dilute sodium hydroxide. Cleavage is random in RNA, and the ultimate products are a mixture of nucleoside 2 - and 3 -monophosphates. These products provide a clue to the reaction mechanism (Figure 11.29). Abstraction of the 2 -OH hydrogen by hydroxyl anion leaves a 2 -0 that carries out a nucleophilic attack on the phosphorus atom of the phosphate moiety, resulting in cleavage of the 5 -phosphodiester bond and formation of a cyclic 2, 3 -phosphate. This cyclic 2, 3 -phosphodiester is unstable and decomposes randomly to either a 2 - or 3 -phosphate ester. DNA has no 2 -OH therefore DNA is alkali stable. 

The mechanism of phosphate ester hydrolysis by hydroxide is shown in Figure 1 for a phosphodiester substrate. A SN2 mechanism with a trigonal-bipyramidal transition state is generally accepted for the uncatalyzed cleavage of phosphodiesters and phosphotriesters by nucleophilic attack at phosphorus. In uncatalyzed phosphate monoester hydrolysis, a SN1 mechanism with formation of a (POj) intermediate competes with the SN2 mechanism. For alkyl phosphates, nucleophilic attack at the carbon atom is also relevant. In contrast, all enzymatic cleavage reactions of mono-, di-, and triesters seem to follow an SN2... 

The nucleotide cyclic AMP (3, 5 -cyclic adenosine monophosphate, cAMP) is a cyclic phosphate ester of particular biochemical significance. It is formed from the triester ATP by the action of the enzyme adenylate cyclase, via nucleophilic attack of the ribose 3 -hydroxyl onto the nearest P=0 group, displacing diphosphate as leaving group. It is subsequently inactivated by hydrolysis to 5 -AMP through the action of a phosphodiesterase enzyme. 

ATP synthesis is achieved by ADP acting as the nucleophile towards this mixed anhydride, attacking the P=0 bond, with the carboxylate being the leaving group. Note that this reaction is favoured, whereas the alternative possibility involving hydrolysis of the phosphate ester does not occur. This is precisely what we would predict knowing the different reactivities of anhydrides and esters (see Section 7.8). This direct synthesis of ATP by a process in which... 

The half-life of Fe2(OH)2" at room temperature is a few seconds. An improved model for the kinetics of dissociation of this dinuclear cation recognizes significan articipation by Fe2(OH)3 + at higher pHs, thus clearing up earlier slight anomalies in this area. Phosphate ester hydrolysis at the di-iron center of uteroferrin has now been shown to involve nucleophilic attack by bridging hydroxide (as proposed but not conclusively demonstrated for several M—OH—M-containing catalytic species) rather than by hydroxide bonded to just one Fe. ... 

The two-step mechanism of phosphate ester hydrolysis by the (Znn)2-containing alkaline phosphatase (AP) (7) is thus somewhat mimicked by 24. The phosphoryl intermediate 25 is generated by nucleophilic attack of the alkoxide moiety in 24b at BNP" and is hydrolyzed by the intramolecular Zn11—OH" species in 25b. Thus, the attack at the BNP... 

Nucleophilic attack by an alcohol on the y phosphate (Pig. 13-10a) displaces ADP and produces a new phosphate ester. Studies with 180-labeled reactants have shown that the bridge oxygen in the new compound is derived from the alcohol, not from ATP the group transferred from ATP is a phosphoiyl (—POf-), not a phosphate (—OPOf-). Phosphoiyl group transfer from ATP to glutamate (Fig. 13-8) or to glucose... 

Cobalt-coordinated amide nucleophiles have also been observed to attack coordinated phosphate esters (equation 38),148 disulfides (Scheme 47)149 150 and nitriles (Scheme 48).151>152 Chelated amides can also be formed by intramolecular attack of cobalt hydroxides.153... 

The results of studies concerning reactions between nucleophiles and trimethyl phosphate and trimethyl phosphite should be mentioned briefly. In both esters the nucleophiles may attack the phosphorus centre to react in this way via pentacoordinate and tetrahedral type species, respectively, to products. Various nucleophiles, such as F-, CD30 , DNO , OH and NH2 have been allowed to react with trimethyl phosphate, but only OH and NH have been found to attack the phosphorus centre to a minor extent. The most prominent process is nucleophilic attack on one of the carbon atoms to generate by an SN2 reaction the phosphate diester anion as... 

However, most nucleophiles attack 5-oxazolones at the carbonyl group and the products are derivatives of a-amino acids formed by acyl-oxygen fission. Thus the action of alcohols, thiols, ammonia and amines leads, respectively, to esters, thioesters and amides orthophosphate anion gives acyl phosphates (Scheme 18). The use of a-amino acids in this reaction results in the establishment of a peptide link. Cysteine is acylated at the nitrogen atom in preference to the sulfur atom. Enzymes, e.g. a-chymotrypsin and papain, also readily combine with both saturated and unsaturated azlactones. A useful reagent for the introduction of an a-methylalanine residue is compound (202). Both the trifluoroacetamido and ester groups in the product are hydrolyzed by alkali to give a dipeptide. The alkaline hydrolyzate may be converted into the benzyloxycarbonyl derivative, which forms a new oxazolone on dehydration. Reaction with an ester of an amino acid then yields a protected tripeptide (equation 45). 

The saturated chain is added first, at Cl of glyceryl phosphate. The reagent is a thiol ester called acyl coenzyme A, whose full structure you will see in the next chapter. This reaction occurs by simple nucleophilic attack on the carbonyl group of the thiol ester followed by loss of the better leaving group, the thiolate anion. Then the process is repeated at the second OH group where an unsaturated fatty acid, perhaps oleic acid, is added by the same mechanism. 

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