1965]Phosphate Esters : Efect of Changing Ester Grot@ 6559 and not to any peculiarity of the cyclohexyl ester as such. This general view has been confirmed by an investigation of the rates of hydrolysis of a series of esters of 2-hydroxypropyl phosphate (I). The esters were prepared by reaction between the alkyl or aryl phosphate and 1,2-epoxypropane in aqueous solution at about pH 8. The rates and product CHzO 0 -* The first-order rate constant for hydrolysis of barium propane-1,2-diol cyclic phosphate in 0 . 1~-NaOH a t 30" can be calculated as very roughly 3 x sec.-l from the data of T. Ukita, K. Nazasawa, and M. Irie. Pharm. Bull. (Tokyo), 1957, 5, 127. t The equation of the line for alkaline hydrolysis of the acetates is log koH = -0.26 pK, + 5-0 (cf. ref. 5 ) .D.

Dodecaadenylic acid containing a single 2',5'-linkage at a defined position was formed by the coupling of two hexamers on a poly(U) template at 20. The rate of hydrolysis of this dodecamer was compared with that of a dodecamer that contained only the natural 3'-'-linkages. At 400, in 1 M aqueous ethylenediamine at pH 8 in the absence of poly(U), both dodecamers hydrolyzed at comparable rates, but the addition of two equivalents of poly(U) caused a 7-fold increase in the initial rate of hydrolysis of the oligomer containing the 2',5'-bond, and a 5-fold decrease in the initial rate of hydrolysis of the natural oligomer. When the oligomers are fully constrained in helical form, the ratio of the rates of cleavage of one 2',5'-bond to one 3',5'-bond under these conditions is probably about 900:1. The use of the 3',5'-bond, in combination with a right-handed helix, appears to have had a large selective advantage over the use of the 2',5'-bond for the storage of genetic information. Naturally occurring RNA appears to contain solely the 3',5'-internucleotide linkage. By contrast, successful attempts to demonstrate the template-directed non-enzymatic polymerization of ribonucleotides, which are of interest as models of prebiotic synthesis, have almost invariably resulted-in the production of a large excess of the unnatural 2',5'-isomer (1). Thus, coupling of adenosine 2',5'-cyclic phosphate (A>p) on a poly(U) template is catalyzed by aqueous ethylenediamine, but the resulting dimer contains about 97% of the 2',5'-linkage (2). It has been suggested (3) that these two observations may be related: that the constraint of the helix forces the formation of the 2',5'-bond from the cyclic phosphate, and also stabilizes a 3',5'-bond against hydrolysis in mildly basic solution. As a corollary, the 2',5'-bond is still labile when the constituent nucleotides are part of a right-handed helix, and thus storage of "genetic" information is better accomplished by use of the 3',5'-bond. Previous evidence for the stability of the 3',5'-bond in helical conformation comes from the isolation of oligo(purines) from the base-catalyzed partial hydrolysis of single-stranded RNA. Purine-rich regions have a higher helical content than pyrimidine-rich regions, and better resist alkaline hydrolysis (4, 5).We have made an unambiguous test of the ideas expressed above by measuring the rate of hydrolysis at pH 8 of two dodecaadenylates, one (I) that is entirely 3',5'-linked, the other (II) that contains a single 2',5'-bond at the central position. The measurements have been made both in the presence and in the absence of poly(U). The preparation of the synthetic dodecamer (II) could not be achieved by the Abbreviations: BAP, bacterial alkaline phosphatase; HPLC, high pressure liquid chromatography; A>p, adenosine 2',3'-cyclic phosphate; DP, degree of polymerization; percentages are given as % of total nucleoside units present; molar absorptivities are given per nucleoside unit. * corder. Baseline separation of (A)5A'p from (A)sA>p, and (A)ujA...

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3',5'-Linked hexa-adenylic acid with a 2',3'-cyclic phosphate terminus [(A5A less than p] couples on a polyuridylic acid template in the presence of ethylenediamine to form the dodecamer (24 percent) and octadecamer (5 percent). The bond produced is largely that of the 2',5' isomer.

Advantage was taken of the reversibility of the first step of ribonuclease-A action to synthesize the dinucleoside phosphorothioate Up(S)C from the crystalline isomer of uridine 2',3'-cyclic phosphorothioate [Up(S)] and cytidine. Cyclic phosphorothioate was then reformed from Up(S)C by a nonenzymic reaction known to proceed by an in-line mechanism. The geometry of the enzymic reaction was determined to be in-line by a comparison of the Up(S) product with the Up(S) originally used. By the principle of microscopic reversibility, the geometry of the first step in the action of ribonuclease-A is shown to be in-line.Bovine pancreatic ribonuclease-A catalyzes the hydrolysis of a ribonucleic acid or nucleotide ester by a two-step reaction. A transesterification in which the 2'-OH group attacks one of the phosphodiester bonds to form a 2',3'-cyclic phosphate is followed by the hydrolysis of this intermediate to yield a terminal 3'-phosphate (1). Each of these steps could proceed by either an in-line or an adjacent mechanism, depending on the relative geometry of the attacking and leaving groups in the displacement reaction (2). An adjacent mechanism requires the formation of a pentacoordinate intermediate, which must undergo pseudorotation before the product can be released.Since Rabin's original proposal in 1961 (3), several mechanisms have been suggested (4-8). Most of these can be classified as in-line or adjacent by our combining a knowledge of the mechanism of hydrolysis of phosphate esters with an examination of the groups claimed to participate in the catalysis. For example, in the first step, we would infer an adjacent mechanism if the same group acts first as a general base to deprotonate the 2'-OH and then as a general acid toward the leaving ester group (2). Nuclear magnetic resonance (NMR) and x-ray evidence have been presented to support an in-line mechanism (9), but this conclusion, being based on the mode of binding of dianionic inhibitors, is equivocal. Until now, the most pertinent evidence favoring an in-line mechanism for the first step has been x-ray diffraction studies on a complex of the enzyme with a phosphonate analog of the natural substrate uridylyl-adenosine (10).A conclusive determination of the geometry of the second step, in which the monoanionic diester uridine 2',3'-cyclic phosphorothioate was used as substrate, has been reported (11); the mechanism was in-line. Although the ring-opening step is often shown as the microscopic reverse of the ring closure, the two steps need not have the same mechanism, since water is obviously not equivalent to a nucleoside. We have, therefore, extended our work to provide a conclusive determination of the geometry of the first step of this reaction. EXPERIMENTALMaterials. Bovine pancreatic ribonuclease-A was obtained as a lyophilized, phosphate-free powder from Worthington Biochemical Corp. Cytidine was from Sigma Chemical Co. Uridine 2',3'-cyclic thiophosphate was prepared from 5'-Oacetyl uridine and trisimidazole phosphine sulfide by the meth...