Phosphorothioates phosphorylation

The use of triphenylphosphine and 2,2 -bipyridyl disulphide in oxidation-reduction condensations has been extended to the phosphorylation of alcohols and phosphates, and to the preparation of 5 -(2-pyridyl) phosphorothioates (60) which have been used for the synthesis of pyrophosphates (see Chapter 6, Section 1). 

In the case of La3 + - and Zn2+-catalyzed methanolysis of the phosphorothioate esters the observed / ig values of —0.87 and —0.74 also signify an associative mechanism with some departure of the leaving group, but it is difficult to assign the extent of the bond cleavage since the /ieq value is not known for the phosphoryl transfer between thiol and oxygen nucleophiles. 

F. Hollfelder, D. Herschlag, The Nature of the Transition State for Enzyme-Catalyzed Phosphoryl Transfer. Hydrolysis of O-Aryl Phosphorothioates by Alkaline Phosphatase , Biochemistry 1995, 34, 12255-12264. 

The OPPs inhibit acetylcholinesterase (AChE) by phosphorylating the esteratic site of the enzyme. As a result of AChE inhibition, ACh accumulates and binds to muscarinic and nicotinic receptors throughout the nervous system. Transformation of OPPs in the organisms takes place by conversion of the phosphorothioate (P=S) group to oxon (P=0) analogs. These oxo compounds are of concern because they are the activated forms of the OPPs, with a considerably stronger inhibition of acetylcholinesterase activity (27). 

Using two enzymes, a mammalian adenylate cyclase and myosin ATPase, as examples the application of phosphorothioate analogues to the study of the mechanism of nucleotidyl and phosphoryl transfer will be described. 

Different OP compounds have structural similarities within classes. The phosphorus compounds have the characteristic phosphoryl bond, P=0. Most OP compounds have a phosphoryl bond or a thiophosphoryl bond (P=S). All OP compounds are esters of phosphorus with varying combinations of oxygen, carbon, sulfur, and nitrogen attached. These are classified as (1) phosphates (2) phos-phonates (3) phosphorothioates (4) phosphorodithioates (5) phosphorothiolates and (6) phosphoramidates. Further, the OP compounds are categorized as (1) aliphatic (2) phenyl and (3) heterocyclic derivatives. The aliphatic are carbon chainlike in structure. TEPP, which was used in agriculture for the first time in 1946, is a member of this group. Others include malathion, trichlorfon, monocrotophos, dimethoate, oxydemetonmethyl, dimethoate, dicrotophos, disulfoton, dichlorvos, mevinphos, methamidophos, and acephate. 

The first chiral phosphates to be used for stereochemical analyses were chiral phosphorothioates, which were used to determine the stereochemical courses of ribonuclease, UDP-glucose pyrophosphorylase, adenylate kinase and several other kinases and synthetases. The chiral phosphorothioates either had sulfur in place of an oxygen at an otherwise prochiral center of a phosphodiester or phosphoanhydride or stereospecifically placed sulfur and 180 (or nO) in a terminal phosphoryl group. The syntheses and configurational analyses of the most important of these compounds are outlined in the following. 

A convenient synthesis of (,Sp)-ATPaS is the enzymatic phosphorylation of AMPS, adenosine 5 -phosphorothioate by the adenylate kinase-pyruvate kinase system according to Equation [23] ... 

The chiral y-[l80]phosphorothioate of ATP, (Pp)-ATPyS, yl80(/3, y)lsO, was synthesized by the procedure outlined in Fig. 8 [25]. (Sp)-ADPaS, alsO(a,/3)l80 was prepared by stereospecific phosphorylation of AMPaS, al802 using the adenylate and pyruvate kinase system followed by dephosphorylation with glucose and hexokinase. This was the starting material for the synthesis, with the chiral a-... 

Fig. 14. Orientations of enzymatic phosphorylations of chiral [lsO]phosphorothioates.. Shown are the orientations of phosphorylation of AMPS by adenylate kinase and ATP and the phosphorylation of ADP/3S by pyruvate kinase and phosphoenolpyruvatc (PEP) and by acetate kinase and acetvl P...

Fig. 15. Configurational analysis of chiral [ lsO]phosphorothioates by stereospecific phosphorylation.

Many ribozymes and proteins that catalyze phosphoryl-transfer reactions use a mechanism employing two metal ions, and early on group II introns were hypothesized to use a similar mechanism (17). However, evidence for the existence of two metal ions in the catalytic core was only found very recently (12, 13, 18). A direct Mg + coordination of the pro-Sv oxygen of the first nucleotide of the catalytic triad is evident based on phosphorothioate substitution experiments (18). Recently, an intact group II was successfully crystallized for the first time (12). This structure confirms the metal contact from the first nucleotide of the catalytic triad and shows additional contacts to this metal ion from the second nucleotide of the catalytic triad and from the first nucleotide upstream of the bulge (Fig. 4a). This nucleotide is additionally coordinated to the second Mg + ion in the core. The distance between the two Mg + ions in the crystal stmcture is 3.9 A, which is in agreement with the proposed two-metal-ion mechanism (17). 

A quantum mechanical method has been used to study the TSs of the uridine phosphorylation reaction and an acid-catalysed Sn2 is the main mode of reaction. The ribonucleotide sulfur analogue 2 -deoxy-2 -thiouridine 3 -(p-nitrophenyl phosphate) (295) undergoes transphosphorylation to give 2, 3 -cychc phosphorothioate (296) followed by hydrolysis to give 2 -deoxy-2 -thiouridine 2 -phosphorothioate (297)... 

A difficulty is that the phosphorus centers in DNA are not chiral, because two of the groups bound to the phosphorus atom are simple oxygen atoms, identical with each other. This difficulty can be circumvented by preparing DNA molecules that contain chiral phosphoryl groups, made by replacing one oxygen atom with sulfur (called a phosphorothioate). Let us consider EcoKV endonuclease. This enzyme cleaves the phosphodiester bond between the T... 

Table 2 lists phosphatases for which the stereochemical outcome has been determined by using chiral phosphomonoester substrates. In such experiments the phosphoryl group is made chiral using lfiO, 170, and lsO, or, a phosphorothioate substrate is used that has a sulfur atom and two isotopes of oxygen in the nonbridging positions. Summaries of results for additional enzymes that catalyze phosphoryl transfer are available in other reviews.20,76,77... 

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