3 :5 -cyclic phosphate hydrolysis

The turnover reaction of hydrolysis of 2, 3 -CMP could be made negligibly slow at temperatures below -60°C at pH 3-6 in 70% methanol, and below -35°C at pH 2.1. The rate of the catalytic reaction using crystalline enzyme was found to be 50-fold slower than that of dissolved enzyme for cyclic phosphate hydrolysis, and 200-fold slower for dinucleotide hydrolysis (presumably the greater reduction for the larger substrate reflects increased diffusional hindrance by the small solvent chan-... 

The first detailed proposal for the mechanism of action of ribonuclease was put forward by Mathias and Rabin and their colleagues (514) An original diagram from their paper is shown in Fig. 28 BIB, 516). It bears a remarkable similarity to the geometry of the active site as defined by the X-ray studies and shown in Fig. 23. For Step 1 the mechanism proposes (1) removal of the proton on the 2 -OH by an imidazole residue in the base form, (2) protonation of the 5 0 of the leaving nucleoside by the other imidazole in the acid form, and (3) attack by the 2 alkoxide on the phosphorus atom to yield the cyclic phosphate. Hydrolysis or alcoholysis of the cyclic phosphate requires the reverse of each of these steps. At the start of step 1, one histidine is in the acidic form and one in the basic form. At the start of step 2 the roles of the two histidine residues are reversed. 

D-Xylose 3-phosphate is formed from the 5-phenyl phosphate of 1,2-iso-propyhdene xylofuranose which by alkaline treatment gives a3,5-cyclic phosphate. Hydrolysis of the latter yields a mixture of the 3- and 5-phosphates. ... 

B. Solvolysis of Phosphoric Acid Derivatives.—Interest continues in neighbouring-group participation in the solvolysis of phosphate esters. As a potential model compound for investigating the mechanism of ribo-nuclease action, the phenyl hydrogen phosphate ester of c/j-3,4-tetrahydro-furandiol (24) has been the subject of a detailed study. Above (and probably also below) pH 4 hydrolysis gives solely the cyclic phosphate (25)... 

Isomers of D-apiofuranosyl 1-phosphate have been prepared by treating a mixture of jS-o-apio-D-furanosyl and /3-D-apio-L-furanosyl tetra-acetates with crystalline phosphoric acid. a-o-Apio-o- (59) and a-o-apio-L-furanosyl-1-phosphate (60) and their cyclic phosphates were separated by chromatography and identified by H n.m.r. o-Apiose is metabolized in parsley and Lemna minor with the possible formation of UDP-D-apiose. L. minor will convert UDP-a-glucuronic acid into a-o-apio-D-furanosyl-1,2-cyclic phosphate (61) but no evidence of UDP-o-apiose was found, although it is possible that (61) arose from the rapid hydrolysis of UDP-o-apiose. 

A similar reaction of methyl P-D-ribopyranoside (5) with P(NMe2)3 in dioxane gave 2,3,4-bicyclophosphite of P-methyl-D-ribopyranoside, 6a in 90% yield. It was sulfurated and selenated to give the corresponding thiophosphate 6b and selenophosphate 6c in 89 and 81% yields, respectively. Oxidation of 6a with hydrogen peroxide gave cyclic phosphate 6d (92%), whereas its hydrolysis in aqueous... 

An estimate of the rate enhancement associated with the intramolecular phosphorylation can be made by using isopropyl p-nitrophenyl methyl-phosphonate as a model for the covalent intermediate formed in the initial step of the reaction of cycloheptaamylose with bis (p-nitrophenyl) me thy 1-phosphonate. The first-order rate constant for the alkaline hydrolysis of isopropyl p-nitrophenyl methylphosphonate at pH 9.86 can be obtained from the data of van Hooidonk and Groos (1970) kun = 1.4 X 10-5 sec-1. This value may be compared with the maximal rate constant for the reaction of cycloheptaamylose with bis(p-nitrophenyl) methylphosphonate— k2 = 1.59 X 10-1 sec-1 at pH 9.86—which must be a minimal value for the rate of the intramolecular phosphorylation. This comparison implies a kinetic acceleration of at least 104 which is similar to rate enhancements associated with the formation of cyclic phosphates from nucleoside phosphate diesters. 

By hydrolysis under very mild alkaline conditions (with a boiling suspension of barium carbonate), ribonucleic acids have been shown to yield small quantities of cyclic phosphates as well as the normal nucleotides.96 These materials were identical electrophoretically with synthetic cyclic phosphates and were readily hydrolyzed to mixtures of 2- and 3-phosphates. Their formation in this way constitutes strong support for Brown and Todd s theory. The precise way in which the alkaline hydrolysis of the polynucleotide occurs has been studied using isotopically labeled water, and the results are in agreement202 with the scheme outlined above. 

Hydrolysis of ribonucleic acids by snake venom was found to yield inorganic phosphate, nucleosides, and pyrimidine ribonucleoside diphosphates.197 These diphosphates were shown by their behavior toward various enzymes to be mixtures of 2,5- and 3,5-diphosphates, and it therefore seems likely that they were formed through intermediate, cyclic phosphates. Thus, although this evidence confirms the existence of 2(or 3) — 5 linkages, it does not distinguish between the 2- and 3-positions. 

A novel synthesis of D-glucose 6-phosphate (in 55% yield) has been reported it involves alcoholysis of the cyclic phosphate of catechol with 1,2-O-isopropylidene-a-D-glucofuranose, followed by acid hydrolysis of the so-formed phosphoric diester.192 The reagent... 

The alcoholysis of the cyclic phosphate of catechol by alditols can lead, after acid hydrolysis of intermediate, cyclic phosphates, to the selective formation of phosphoric esters of the primary hydroxyl groups in the alditols. Thus, erythritol and D-mannitol afford, after chromatographic purification of the reaction products, their 1-phosphates in yields of 31 and 38%, respectively.217 The method was used to convert riboflavine into riboflavine 5 -phosphate.218 1-Deoxy-1-fluoro-L-glycerol has been converted into the 3-(dibenzyl phosphate) in 54% yield by selective reaction with dibenzyl phosphorochloridate. 219... 

Figure 6.8. Cyclic and non-cyclic inositol phosphates. Hydrolysis of phosphatidyl 4,5-bisphosphate (PIP2) by phospholipase C can generate cyclic and non-cyclic inositol phosphates.

Fig. 9.10. Partial metabolic scheme of tris(2-methylphenyl) phosphate (9.44) showing hydrolysis to the diester 9.45, the monoester, and phosphate. Also shown is a reaction of oxidation to the hydroxymethyl analogue 9.46, followed by cyclization to the toxic cyclic phosphate 9.47...

Ah initio calculations to map out the gas-phase activation free energy profiles of the reactions of trimethyl phosphate (TMP) (246) with three nucleophiles, HO, MeO and F have been carried out. The calculations revealed, inter alia, a novel activation free-energy pathway for HO attack on TMP in the gas phase in which initial addition at phosphorus is followed by pseudorotation and subsequent elimination with simultaneous intramolecular proton transfer. Ah initio calculations and continuum dielectric methods have been employed to map out the lowest activation free-energy profiles for the alkaline hydrolysis of a five-membered cyclic phosphate, methyl ethylene phosphate (247), its acyclic analogue, trimethyl phosphate (246), and its six-membered ring counterpart, methyl propylene phosphate (248). The rate-limiting step for the three reactions was found to be hydroxyl ion attack at the phosphorus atom of the triester. ... 

A series of diaquatetraaza cobalt(III) complexes accelerated the hydrolysis of adenylyl(3 -50adenosine (ApA) (304), an enhancement of 10 -fold being observed with the triethylenetetramine complex (303) at pH 7. The pentacoordinated intermediate (305), which is formed with the complex initially acting as an electrophilic catalyst, then suffers general acid catalysis by the coordination water on the Co(III) ion to yield the complexed 1,2-cyclic phosphate (306), the hydrolysis of which occurs via intracomplex nucleophilic attack by the metal-bound hydroxide ion on the phosphorus atom. Neomycin B (307) has also been shown to accelerate the phosphodiester hydrolysis of ApA (304) more effectively than a simple unstructured diamine. 

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