Fluorescence assays with improved sensitivity
View Patent ↗Latent fluorescent compounds, comprising a fluorescent molecule with one or more blocking groups attached and optionally one or more urea-containing groups are provided. The urea-containing group can be used to further attach one or more molecules of interest, such as proteins, peptides or nucleic acids. The blocking group(s) is released from the latent fluorescent compound by reaction with a trigger, forming the fluorescent molecule which can be detected. Also provided herein are methods of using latent fluorescent compounds to detect triggers.
1. A latent fluorescent compound comprising the following structure:
wherein F is a fluorophore having the structure:
each A is independently a C1-C12 straight chain or branched alkyl group or alkoxy group;
R is a C1-C12 straight chain or branched alkyl group or alkoxy group;
Y is selected from hydrogen, —PO 3 (R 1 ) 2 , straight-chain, branched or cyclic C1-C20 alkyl groups in which one or more of the carbon atoms are optionally substituted with non-hydrogen substituents and wherein one or more C, CH, or CH 2 moieties can be replaced with an O atom, a nitrogen atom, an NR 1 group, or a S atom; straight-chain, branched or cyclic C1-C20 alkenyl group which contains one or more double bonds in which one or more of the carbon atoms are optionally substituted with non-hydrogen substituents and wherein one or more C, CH, or CH 2 moieties can be replaced with an O atom, a —CO— group, a —OCO— group, a nitrogen atom, an NR 1 group, or a S atom; straight-chain, branched or cyclic C1-C20 alkynyl group which contains one or more triple bonds in which one or more of the carbon atoms are optionally substituted with non-hydrogen substituents and wherein one or more C, CH, or CH 2 moieties can be replaced with an O atom, a —CO— group, a —OCO— group, a nitrogen atom, an NR 1 group, or a S atom; and an optionally substituted aromatic or non-aromatic ring structure, wherein: R can optionally be substituted with one or more substituent groups selected from halogens; nitro groups; cyano groups; isocyano groups; thiocyano groups; isothiocyano groups; azide groups; —SO 2 groups; —OSO 3 H groups; one or more optionally substituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl groups; OR 1 ; —CO—OR 1 ; —O—CO—R 1 ; —N(R 1 ) 2 ; —CO—N(R 1 ) 2 ; —NR 1 —CO—OR 1 ; —SR 1 ; —SOR 1 ; —SO 2 —R 1 ; —SO 3 R 1 ; —SO 2 N(R 1 ) 2 ; —P(R 1 ) 2 ; —OPO 3 (R 1 ) 2 ; and —Si(R 1 ) 3 wherein each R 1 , independent of other R 1 in the substituent group can be a hydrogen, an optionally substituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2 groups therein can be replaced with an O atom, N atom, S atom or —NH group; an optionally substituted aromatic group, two or more R 1 groups can be linked together to form a ring which may contain one or more of the same or different heteroatoms; and R 1 can in turn be optionally substituted with one or more groups selected from the group consisting of halogens, nitro groups; cyano groups; isocyano groups; thiocyano groups; isothiocyano groups; azide groups; —SO 2 groups; —OSO 3 H groups; straight-chain, branched or cyclic alkyl, alkenyl or alkynyl groups; halogenated alkyl groups; hydroxyl groups; alkoxy groups; carboxylic acid and carboxylic ester groups; amine groups; carbamate groups, thiol groups, thioether and thioester groups; sulfoxide groups, sulfone groups; sulfide groups; sulfate and sulfate ester groups; sulfonate and sulfonate ester groups; sulfonamide groups, sulfonate ester groups; phosphine groups; phosphate and phosphate ester groups; phosphonate and phosphonate ester groups; and alkyl-substituted silyl groups.
2. The compound of claim 1 , wherein each A is independently a C1-C5 alkyl or alkoxy group.
3. The compound of claim 1 , wherein each A is a methyl group.
4. The compound of claim 1 having the structure:
wherein the B's may be the same or different and B is a blocking group having the structure:
where L is a direct bond, —NH—, or —O—;
Y is selected from the group consisting of:
hydrogen, —PO 3 (R 1 ) 2 ,
straight-chain, branched or cyclic C1-C20 alkyl groups in which one or more of the carbon atoms are optionally substituted with non-hydrogen substituents and wherein one or more C, CH, or CH 2 moieties can be replaced with an O atom, a nitrogen atom, an NR 1 group, or a S atom;
straight-chain, branched or cyclic C1-C20 alkenyl group which contains one or more double bonds in which one or more of the carbon atoms are optionally substituted with non-hydrogen substituents and wherein one or more C, CH, or CH 2 moieties can be replaced with an O atom, a —CO— group, a —OCO— group, a nitrogen atom, an NR 1 group, or a S atom;
straight-chain, branched or cyclic C1-C20 alkynyl group which contains one or more triple bonds in which one or more of the carbon atoms are optionally substituted with non-hydrogen substituents and wherein one or more C, CH, or CH 2 moieties can be replaced with an O atom, a —CO— group, a —OCO— group, a nitrogen atom, an NR 1 group, or a S atom; and
an optionally substituted aromatic or non-aromatic ring structure, wherein:
R can optionally be substituted with one or more substituent groups selected from halogens; nitro groups; cyano groups; isocyano groups; thiocyano groups; isothiocyano groups; azide groups; —SO 2 groups; —OSO 3 H groups; one or more optionally substituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl groups; OR 1 ; —CO—OR 1 ; —O—CO—R 1 ; —N(R 1 ) 2 ; —CO—N(R 1 ) 2 ; —NR 1 —CO—OR 1 ; —SR 1 ; —SOR 1 ; —SO 2 —R 1 ; —SO 3 R 1 ; —SO 2 N(R 1 ) 2 ; —P(R 1 ) 2 ; —OPO 3 (R 1 ) 2 ; and —Si(R 1 ) 3 wherein each R 1 , independent of other R 1 in the substituent group can be a hydrogen, an optionally substituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2 groups therein can be replaced with an O atom, N atom, S atom or —NH group; an optionally substituted aromatic group, two or more R 1 groups can be linked together to form a ring which may contain one or more of the same or different heteroatoms; and
R 1 can in turn be optionally substituted with one or more groups selected from the group consisting of halogens, nitro groups; cyano groups; isocyano groups; thiocyano groups; isothiocyano groups; azide groups; —SO 2 groups; —OSO 3 H groups; straight-chain, branched or cyclic alkyl, alkenyl or alkynyl groups; halogenated alkyl groups; hydroxyl groups; alkoxy groups; carboxylic acid and carboxylic ester groups; amine groups; carbamate groups, thiol groups, thioether and thioester groups; sulfoxide groups, sulfone groups; sulfide groups; sulfate and sulfate ester groups; sulfonate and sulfonate ester groups; sulfonamide groups, sulfonate ester groups; phosphine groups; phosphate and phosphate ester groups; phosphonate and phosphonate ester groups; and alkyl-substituted silyl groups;
each A is independently a C1-C12 straight chain or branched alkyl group or alkoxy group; and
R is a C1-C12 straight chain or branched alkyl group or alkoxy group.
5. The compound of claim 1 , having the following structure:
6. A latent fluorescent compound having the structure:
(B) a —F
where a is 1 or 2;
F is a fluorescent molecule having the structure
wherein each B is a blocking group having the structure:
where L is either a direct bond, —NH—, or —O—;
Y is selected from the group consisting of:
hydrogen, —PO 3 (R 1 ) 2 ,
straight-chain, branched or cyclic C1-C20 alkyl groups in which one or more of the carbon atoms are optionally substituted with non-hydrogen substituents and wherein one or more C, CH, or CH 2 moieties can be replaced with an O atom, a nitrogen atom, an NR 1 group, or a S atom;
straight-chain, branched or cyclic C1-C20 alkenyl group which contains one or more double bonds in which one or more of the carbon atoms are optionally substituted with non-hydrogen substituents and wherein one or more C, CH, or CH 2 moieties can be replaced with an O atom, a —CO— group, a —OCO— group, a nitrogen atom, an NR 1 group, or a S atom;
straight-chain, branched or cyclic C1-C20 alkynyl group which contains one or more triple bonds in which one or more of the carbon atoms are optionally substituted with non-hydrogen substituents and wherein one or more C, CH, or CH 2 moieties can be replaced with an O atom, a —CO— group, a —OCO— group, a nitrogen atom, an NR 1 group, or a S atom; and
an optionally substituted aromatic or non-aromatic ring structure, wherein:
R can optionally be substituted with one or more substituent groups selected from halogens; nitro groups; cyano groups; isocyano groups; thiocyano groups; isothiocyano groups; azide groups; —SO 2 groups; —OSO 3 H groups; one or more optionally substituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl groups; OR 1 ; —CO—OR 1 ; —O—CO—R 1 ; —N(R 1 ) 2 ; —CO—N(R 1 ) 2 ; —NR 1 —CO—OR 1 ; —SR 1 ; —SOR 1 ; —SO 2 —R 1 ; —SO 3 R 1 ; —SO 2 N(R 1 ) 2 ; —P(R 1 ) 2 ; —OPO 3 (R 1 ) 2 ; and —Si(R 1 ) 3 wherein each R 1 , independent of other R 1 in the substituent group can be a hydrogen, an optionally substituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2 groups therein can be replaced with an O atom, N atom, S atom or —NH group; an optionally substituted aromatic group, two or more R 1 groups can be linked together to form a ring which may contain one or more of the same or different heteroatoms; and
R 1 can in turn be optionally substituted with one or more groups selected from the group consisting of halogens, nitro groups; cyano groups; isocyano groups; thiocyano groups; isothiocyano groups; azide groups; —SO 2 groups; —OSO 3 H groups; straight-chain, branched or cyclic alkyl, alkenyl or alkynyl groups; halogenated alkyl groups; hydroxyl groups; alkoxy groups; carboxylic acid and carboxylic ester groups; amine groups; carbamate groups, thiol groups, thioether and thioester groups; sulfoxide groups, sulfone groups; sulfide groups; sulfate and sulfate ester groups; sulfonate and sulfonate ester groups; sulfonamide groups, sulfonate ester groups; phosphine groups; phosphate and phosphate ester groups; phosphonate and phosphonate ester groups; and alkyl-substituted silyl groups;
each A is independently a C1-C12 straight chain or branched alkyl group or alkoxy group;
R is a C1-C12 straight chain or branched alkyl group or alkoxy group.