Highly selective pyrophosphate sensor
AssigneeThe University of Akron
InventorsYi Pang, Weihua Chen
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Abstract
A dinuclear zinc complex PPi sensor is provided, that may be represented by the formula where X is hydrogen, —COOEt, —CH═CH2, —OR, or —NR2, wherein X is a monovalent organic group having from 1 to about 20 carbon atoms. A method of preparing the dinuclear zinc complex PPi sensor is also provided. Methods for detecting PPi in an aqueous solution and methods for pyrosequencing by using the dinuclear zinc complex are described.
Claims10
A dinuclear zinc complex PPi sensor represented by the formula
- having two Zn2+ centers and a phenoxide oxygen, wherein X is selected from the group consisting of hydrogen, —COOEt, —CH═CH2, —OR, and —NR2, wherein R is a monovalent organic group having from 1 to about 20 carbon atoms, and wherein one of the Zn2+ centers is bound to the phenoxide oxygen atom.
A method of preparing a dinuclear zinc complex PPi sensor, the method comprising the steps of:
- (1) reacting 2-(2′-hydroxyphenyl)-benzoxazole (HBO) or a derivative of HBO with 1-3 hexamethylenetetramine (HMTA) via a dual-Duff reaction, to form a dialdehyde intermediate that may be represented by the formula
- wherein X is selected from the group consisting of hydrogen, —COOEt, —CH═CH2, —OR, and —NR2, and wherein R is a monovalent organic group having from 1 to about 20 carbon atoms;
- then
- (2) combining the dialdehyde, sodium triacetoxyborohydride [NaBH(OAc)3], and a bis(2-pyridylmethyl)amino compound in a reductive amination reaction, to form a compound containing two bis(2-pyridylmethyl)amino groups, said compound being represented by the formula
- then
- (3) combining the product of the reductive amination reaction with zinc nitrate Zn(NO3)2 to form a dinuclear Zn2+ complex, that may be represented by the formula
- wherein one of the Zn2+ cations is bonded to a phenol oxygen, removing the phenolic proton and turning off the luminescence from the excited state intramolecular proton transfer (ESIPT).
A method of detecting PPi in an aqueous, the method comprising the steps of providing an aqueous solution containing a di-nuclear Zn complex PPi sensor capable of reacting with PPi, said PPi sensor having a structure represented by the formula.
- wherein X is selected from the group consisting of hydrogen, —COOEt, —CH═CH2, —OR, and —NR2, wherein R is a monovalent organic group having from 1 to about 20 carbon atoms, and wherein any PPi present in the aqueous solution will combine with said dinuclear Zn complex, thereby creating a measureable fluorescent signal.
A method of pyrosequencing is provided, the method comprising the steps of (1) providing a DNA template having a base at a target position and an extension primer;
- (2) subjecting the DNA template and extension primer to a polymerase reaction in the presence of (a) a deoxynucleotide or dideoxynucleotide, and (b) a dinuclear zinc complex PPi sensor represented by the formula
- X is selected from the group consisting of hydrogen, —COOEt, —CH═CH2, —OR, and —NR2, wherein R is a monovalent organic group having from 1 to about 20 carbon atoms, wherein said nucleotide will become incorporated onto the DNA template and release PPi if the nucleotide is complementary to the base in the target position, and wherein said released PPi combines with said dinuclear zinc complex and a fluorescent emission is produced.
Dependencies are inferred from the claim text.