Compounds, compositions and methods for generating chemiluminescence with phosphatase enzymes

A reagent composition which produces chemiluminescence in the presence of a phosphatase enzyme which comprises in an aqueous solution:

• a) a compound of formula I:
• which reacts with the phosphatase enzyme wherein Het is selected from the group consisting of:
• wherein * indicates the point of attachment of an enol phosphate group, wherein Z is selected from the group consisting of O and S atoms, wherein R6 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl and aralkyl groups, wherein each M is independently selected from H and a cationic center and wherein n is a number which satisfies electroneutrality;
• and
• b) a cationic aromatic compound in an amount effective to increase the chemiluminescence compared to that generated in the absence of the cationic aromatic compound.

A method for producing chemiluminescence which comprises reacting a phosphatase enzyme with

• a) at least one compound of formula I:
• wherein Het is selected from the group consisting of:
• wherein * indicates the point of attachment of an enol phosphate group, wherein Z is selected from the group consisting of O and S atoms, wherein R6 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl and aralkyl groups, wherein each M is independently selected from H and a cationic center and wherein n is a number which satisfies electroneutrality;
• and
• b) a cationic aromatic compound in an amount effective to increase the chemiluminescence compared to that generated in the absence of the cationic aromatic compound.

A method for detecting acid phosphatase and alkaline phosphatase in a sample suspected of containing both acid phosphatase and alkaline phosphatase by a chemiluminescent assay procedure which comprises:

• (a) reacting the sample with a reagent composition comprising at least one compound of formula I,
• wherein in the compound of formula I, Het is selected from the group consisting of:
• wherein * indicates the point of attachment of an enol phosphate group, wherein Z is selected from the group consisting of O and S atoms, wherein R6 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl and aralkyl groups, wherein each M is independently selected from H and a cationic center and wherein n is a number which satisfies electroneutrality, a cationic aromatic compound, an anionic surfactant in an amount effective to increase the speed with which maximum chemiluminescence intensity is reached and a non-ionic surfactant in an amount effective to increase the chemiluminescence;
• (b) detecting the amount or intensity of chemiluminescence during an initial period;
• (c) waiting a second period of time until the chemiluminescence has achieved a constant level;
• (d) detecting the amount or intensity of chemiluminescence during a third period;
• (e) relating the chemiluminescence in the initial time period to the amount of acid phosphatase;
• and
• (f) relating the chemiluminescence in the third time period to the amount of alkaline phosphatase.

A process for the preparation of a compound of formula I:

• wherein Het is selected from the group consisting of:
• wherein * indicates the point of attachment of an enol phosphate group, wherein Z is selected from the group consisting of O and S atoms, wherein R6 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl and aralkyl groups, wherein each M is independently selected from H and a cationic center and wherein n is a number which satisfies electroneutrality comprising the steps of:
• (a) reacting a heterocyclic ester or thioester compound VIII having the formula:
• wherein Het, Z and are as defined for compound I with a base to form an enolate of VIII;
• (b) reacting the enolate with a phosphorylating agent to form a protected enol phosphate TX having the formula:
• wherein Het, Z and R6 are as defined above and Y is a protecting group;
• and
• (c) deprotecting the enol phosphate to form the enol phosphate salt compound I by reacting IX with at least one deprotecting agent in the presence of a cationic species M if the cationic species is not a part of the deprotecting agent.