Recently, we found that acetyl resorufin (AR) is transformed to fluorescent resorufin through deacetylation by H 2 O 2 (perhydrolysis), and hence perhydrolysis of AR can be used as a fluorometric indicator reaction for glucose determination using only glucose oxidase (GOD). 1) In the fluorometric method with GOD and AR, glucose analysis is performed without interference by ascorbic acid, uric acid or bilirubin. These compounds interfere with colorimetric determination of serum components with indicator reactions based on H 2 O 2 -dependent oxidative coupling of two chromogens in the presence of peroxidase (POD), which are represented by so called Trinder's reactions of 4-aminoantipyrine with phenolic or anilinic derivatives. 2) Thus, it was demonstrated that the fluorometric method with GOD and AR offers a more reliable and accurate tool for determination of blood glucose than the POD-dependent colorimetric method with 4-aminoantipyrine and phenol. 3) However, there is a problem to be resolved in utilizing perhydrolysis of AR as a general fluorometric indicator reaction for determination of H 2 O 2 : although AR remains intact in CH 3 CN at room temperature for more than 6 months, 3) AR undergoes spontaneous hydrolysis when its CH 3 CN solution is mixed with pH 7.4 phosphate buffer (blank solution), which prevents the fluorometric method from being employed for the measurement of glucose over a concentration range of more than two orders of magnitude.The key to success in improving the measurement range in the fluorometric method seems to be molecular design conferring the ability to resist hydrolysis of AR. The susceptibility of esters to hydrolysis can be controlled by changing steric factors of acyl groups. To our knowledge, there have been no reports of the effects of acyl groups on perhydrolysis of esters, although perhydrolysis of esters or amides has been recognized as a useful tool for generation of peroxyacids in chemical bleaching processes. 4,5) Since the molecular sizes of HOO Ϫ and HO Ϫ are quite similar, steric effects in perhydrolysis and hydrolysis must be in the same order of magnitude. Accordingly, it seemed doubtful whether judicious choice of acyl groups in AR could shift the competition between perhydrolysis and hydrolysis in a manner favorable towards H 2 O 2 -based deacetylation. However, perhydrolysis of aryl acetates is known to be much faster than their hydrolysis, i.e., the nucleophilic reactivity of HOO Ϫ toward aryl esters is markedly higher than that of HO Ϫ , 6-9) which is referred to as the a-effect: nucleophilicity is enhanced when the atom adjacent to a nucleophilic site bears a lone pair of electrons. [10][11][12] AR is a type of aryl acetate, and hence the a-effect is likely to be operative in the H 2 O 2 -dependent deacetylation of AR, which allows the fluorometric determination of glucose with the generation of resorufin as an indicator reaction even though AR undergoes hydrolysis. Taking the a-effect into consideration, it was expected that establishing reaction conditions ...