Compositions and methods to co-localize luminophores with luminescent proteins

A compound of formula (XII)

• 172
• wherein
• R7 is H, alkyl, heteroalkyl, aryl, or —CH2—C6H4OR14;
• R8 is H, alkyl, heteroalkyl, or aryl;
• R9 is H, alkyl, heteroalkyl, aryl, or —C6H4OR15;
• R10 is —H, —CH3, or —CH(CH3)2;
• and
• R11, R14, and R15 are independently enzyme-removable groups;
• and
• wherein the concentration of the compound in a mixture comprising F12 medium and 10% fetal bovine serum at 22° C. is reduced by less than 50% after 45 minutes.

A compound of formula (XII)

• 173
• wherein
• R7 is H, alkyl, heteroalkyl, aryl, or —CH2—C6H4OR14;
• R8 is H, alkyl, heteroalkyl, or aryl;
• R9 is H, alkyl, heteroalkyl, aryl, or —C6H4OR15;
• R10 is —H, —CH3, or —CH(CH3)2;
• and
• R11, R14, and R15 are independently enzyme-removable groups;
• and
• wherein the removal of at least one enzyme-removable group provides a parent compound;
• and
• wherein the time necessary for the concentration of the compound in a mixture comprising F12 medium and 10% fetal bovine serum at 22° C. to be reduced by 50% is greater than the time necessary for the concentration of the parent compound in a mixture comprising F12 medium and 10% fetal bovine serum at 22° C. to be reduced by 50%.

A compound of formula (XIII) or (XIV)

• 174
• wherein
• R7 is H, alkyl, heteroalkyl, aryl, or —CH2—C6H4OR 14;
• R8 is H, alkyl, heteroalkyl, or aryl;
• R12 and R13 are independently —H, —OH, alkyl, heteroalkyl, aryl, or —OR16;
• n is 0, 1, or 2;
• and
• R11, R14, and R16 are independently enzyme-removable groups.

A protected luminophore, which is

• a modified coelenterazine;
• wherein the enol group has been converted to an ester or an ether comprising an enzyme-removable group;
• the removal of said enzyme-removable group providing a parent coelenterazine;
• and
• wherein the time necessary for the concentration of the modified coelenterazine in a mixture comprising F12 medium and 10% fetal bovine serum at 22° C. to be reduced by 50% is greater than the time necessary for the concentration of the parent coelenterazine in a mixture comprising F12 medium and 10% fetal bovine serum at 22° C. to be reduced by 50%.

A kit, comprising:

• a protected luminophore;
• and
• a deprotecting enzyme;
• wherein the luminophore and the deprotecting enzyme are in separate containers.