Nucleic Acids in Chemistry and Biology

5 -O-nucleoside residue. The enzymatic reaction is completed by the regioselective ring opening of the
cyclic phosphate to give only a 3-nucleoside phosphate (Figure 3.51). By contrast, alkaline hydrolysis
leads to a mixture of 2- and 3- phosphates. These reactions exhibit overall retention of configuration at
the phosphorus centre. This is accounted for by the double inversion of stereochemistry that occurs in the
two successive ‘in-line’ displacement processes.
It must be emphasised that this remarkable reactivity appears to be exclusive to five-membered cyclic
phosphate esters and esters of 1,2-diols. This contrasts with the relative stability of esters of 1,3-diols and
6-ring cyclic phosphates. An important example is 3,5-cAMP, whose key role as the second messenger
in cell signalling is dependent on its kinetic stability to non-enzymatic hydrolysis.

3.2.2.4 Phosphate Monoesters.^87 The hydrolysis of monoalkyl phosphates at very low pH proceeds

viathe conjugate acid, and is similar in mechanism to that of triesters and neutral diesters (Table 3.1).
These esters are very resistant to hydrolysis under alkaline conditions where they exist as dianions (and
catalysis by hydroxide is never observed). The reaction of the dianion proceeds by P O cleavage and has
many of the characteristics of a dissociative process viaa hypothetical metaphosphateintermediate. A bet-
ter description invokes the idea of an ‘exploded’ transition state in which there is very weak bonding to the
incoming nucleophile and outgoing leaving group (i.e. a concerted reaction which is very dissociative in
character). The reaction is extremely sensitive to the pKaof the leaving group, and alkyl phosphate monoester
dianions are even more stable than the corresponding diesters. By contrast, the monoanion shows both an
unusually high relative reactivity towards hydrolysis and is very insensitive to the leaving group pKa. This
is explained by involving the minor tautomer (where the leaving group O carries the proton) as the react-
ive ionic form, which then hydrolyses through a similar transition state to the dianion (Figure 3.52). For

106 Chapter 3

O

P

O

O

OH

O P

O

OH

OH

OH

H 2 O

HO P

O

OH

O

OH O

P

O

O

OH

P

O

O OHOH

HO P

O

O OHOH

HO

Ψrot

endocyclic

cleavage

endocyclic

cleavage

exocyclic

cleavage

exocyclic

cleavage

Figure 3.50 Role of trigonal bipyramidal pseudorotation (crot) in^18 O isotope (O) incorporation into ethylene
phosphate

O

O

O

B

H

O

O

O

B

P O

O

OH

O

B

P O

O

O

O

O

OH

O

OH B

O

OH

O

B

P O

HO O

N

N

H

His^12

H

H

His^119

N

N N

N

H

His^119

H

O

H

N

N

H

H His^12

N

N

H

His^12

5'

5'

2'

3' 2'

RNaseA RNaseA

Figure 3.51 Ribonuclease A hydrolysis of RNA via 2,3-cyclic phosphate. Imidazoles (of His-12 and His-119
residues) act as a general acid and general base

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