Phosphohistidine analogs

A phosphoryltriazole of the formula:

• or a salt thereof wherein
• X and Y are independently —OR9, —NR9R10, SR9, COOR9, CONR9R10 or H, an NHJ or COQ;
• J is an amino protecting group;
• Q is a carboxylic acid protecting group;
• Z is CH, N or SiR11;
• R11 is an alkyl group or H;
• E and G are independently a chemical bond or a linker group selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, cycloalkenylalkyl, heterocyclic, heterocyclic alkyl, (O—CH2—CH2)n, or (CH2)q where one of the CH2 groups is replaced by O,
• n is 1-100;
• q is 1-6;
• R9 and R10 are independently hydrogen, alkyl, alkenyl, cycloalkyl, aryl, aryl alkyl, heterocyclic or heterocyclic alkyl;
• one of R and R1 is H and the other is
• and R2, R3, R4, R5, R6, R7, R8 are independently alkyl, alkenyl, cycloalkyl, aryl, cycloalkylalkyl, aryl alkyl, heterocyclic, heterocyclicalkyl or a protecting group.

A hapten comprised of a polypeptide comprised of a phosphoryltriazole fragment of the formula:

• wherein X1 and Y1 are independently a bond, —O—,
• or —S— or
• a salt thereof wherein
• X and Y are independently —OR9, —NR9R10, SR9, COOR9, CONR9R10 or
• Z is CH, N or SiR11;
• R11 is an alkyl group or H,
• E and G are independently a chemical bond or a linker group selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, cycloalkenyl alkyl, heterocyclic, heterocyclic alkyl, (O—CH2—CH2)n, or (CH2)q wherein one of the CH2 groups is replaced by O,
• n is 100;
• q is 1-6;
• R9 and R10 are independently hydrogen, alkyl, alkenyl, cycloalkyl, aryl, arylalkyl, heterocyclic or heterocyclic alkyl;
• one of R and R1 is H and the other is
• and R2, R3, R4, R5, R6, R7, R8 are independently alkyl, alkenyl, cycloalkyl, aryl, cycloalkylalkyl, aryl alkyl, heterocyclic, or heterocyclicalkyl.

A method for detecting a phosphopolypeptide containing at least one phosphorylated histidine in a biological sample, said method comprising the steps:

• (a) isolating an antibody to said phosphopolypeptide wherein at least one phosphorylated histidine residue is replaced by a phosphoryltriazolylalanine fragment of the formula
• or a salt thereof wherein
• X and Y are independently OR9, NR9R10, SR9, COOR9, CONR9R10 or H,
• Z is CH, N or SiR11;
• R11 a is an alkyl group or H;
• E and G are independently a chemical bond or a linker group selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, heterocyclic, heterocyclic alkyl, or (O—CH2—CH2)n, or (CH2)q where one of the CH2 groups is replaced by O:
• n is 1-100;
• q is 1-6;
• R9 and R10 are independently hydrogen, alkyl, alkenyl, alkynyl, cyclo alkyl, aryl, aryl alkyl, heterocyclic or heterocyclicalkyl;
• one of R and R1 is H and the other is
• and R2, R3, R4, R5, R6, R7, R8 are independently alkyl, alkenyl, cycloalkyl, aryl, cycloalkylalkyl, arylalkyl, heterocyclic or, heterocyclicalkyl;
• said antibody recognizing a phosphorylated polypeptide containing said phospuhonryltriazolylalanine fragment and recognizing a polypeptide having a phosphorylated histidine thereon but not recognizing a non-phosphorylated polypeptide or a phosphorylated polypeptide which contains no phosphorylated histidine but contains phosphorylation on at least one amino acid that is not histidine;
• (b) contacting a biological sample suspected of containing phosphorylated histidine with said antibody under conditions suitable for formation of a complex between said antibody and said phosphorylated polypeptide and
• (c) detecting the presence of said complex in said sample, wherein the presence of said complex indicates the presence of said phosphorylated polypeptide wherein at least one of said histidine residues is phosphorylated.