Fluorescent dyes containing phosphorus or arsenic
The present invention provides a fluorescent dye that incorporates a pentavalent phosphorus or arsenic into its ring structure. Also provided is a fluorescence energy transfer system that comprises the above fluorescent dye and a second dye, where the second dye is capable of energy transfer with the fluorescent dye. Additionally provided is a kit for labeling a target molecule. In further embodiments, another kit for labeling a target molecule is provided. A target molecule labeled with the above-described fluorescent dye is also provided. In additional embodiments, methods of labeling a target molecule are provided.
1. A fluorescent dye comprising Structure I, II, III or IV:
wherein
R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are independently H, F, Cl, Br, I, CN, nitro, azido, hydroxyl, amino, hydrazino, (substituted) aryl, (substituted) aroxyl, alkenyl, alkynyl, alkyl, alkoxy, alkylamino, dialkylamino, arylamino, diarylamino, alkyl(aryl)amino, alkanoylamino, alkylthio, alkylcarbonyl, aryl carbonyl, alkylthiocarbonyl, arylthiocarbonyl, alkyloxycarbonyl, aroxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl, alkyl(aryl)aminocarbonyl, arylcarboxamido, or Q, the alkyl or alkoxy portions of which are saturated or unsaturated, linear or branched, and unsubstituted or substituted; wherein Q comprises a carboxyl group (CO 2 − ), a carbonyl halide, a carbonate ester or thioester (COSR 9 ) or amide (COER 9 ), a thiocarboxylic acid, a thiocarbonyl halide, a thiocarbonate ester or amide (CSER 9 ), a sulfonate acid or halide or ester (SO 2 ER 9 ), a sulfoxide (SOR 9 ), a sulfone (SO 2 CR 9 R 10 R 11 ), a sulfonamide (SO 2 NR 9 R 10 ), a phosphate (PO 4 = ), a phosphoryl halide, a phosphate monoester or monoamide (PO 3 − ER 9 ), a phosphate diester or diamide (PO 2 ER 9 ER 10 ), a phosphonate (PO 3 = ), a phosphonyl halide, a phosphonate monoester or monoamide (PO 2 − ER 9 ), a phosphonate diester or diamide (POER 9 ER 10 ), a thiophosphate (PSO 3 = ), a thiophosphate monoester or monoamide (PSO 2 − ER 9 ), a thiophosphate diester or diamide (PSOER 9 ER 10 ), a thiophosphonate (PSO 2 = ), a thiophosphonyl halide, a thiophosphonate monoester or monoamide (PSO − ER 9 ), a thiophosphonate diester or diamide (PSER 9 ER 10 ), a phosphonamide (PONR 9 R 10 NR 12 R 13 ), a phosphonamide thioanalogue (PSNR 9 R 10 NR 12 R 13 ), a phosphoramide (PONR 9 R 10 NR 11 NR 12 R 13 ), a phosphoramide thioanalogue (PSNR 9 R 10 NR 11 NR 12 R 13 ), a phosphoramidite (PO 2 R 12 NR 9 R 10 ) or a phosphoramidite thioanalogue (POSR 12 NR 9 R 10 ), where each E is independently N, NH, O or S;
R 1 in combination with R 2 , R 2 in combination with R 3 , R 3 in combination with R 4 , R 3 in combination with R 7 , R 4 in combination with R 7 , or R 4 in combination with R 5 can independently form a 5-10 member ring structure which is saturated, unsaturated or aromatic, unsubstituted or comprising a heteroatom, and/or further substituted;
R 9 , R 10 , R 11 , R 12 and R 13 are independently a hydrogen, a halogen, an amino group, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, or substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated, branched or linear, or substituted or unsubstituted, an aryl group wherein said aryl group is unsubstituted or substituted; wherein R 9 in combination with R 10 , R 12 in combination with R 13 , R 9 in combination with R 11 , R 9 in combination with R 12 , R 10 in combination with R 13 , or R 11 in combination with R 12 can independently form a 5-10 member ring;
X is O, OR 14 , S, SR 15 , CR 16 R 17 , NR 16 R 17 or N + R 16 R 17 ; Y is O, OR 14 , S, SR 15 , CR 18 R 19 , NR 18 R 19 or N + R 18 R 19 , wherein
R 14 , R 15 , R 16 , R 17 , R 18 and R 19 are independently H, alkyl, aryl, alkylcarbonyl, arylcarbonyl, or Q, in which the alkyl or aryl potions are unsubstituted or substituted, saturated or unsaturated, or
R 16 in combination with R 17 , or R 18 in combination with R 19 can independently form an unsubstituted or substituted 5-10 member ring structure, and R 16 in combination with R 1 , R 17 in combination with R 2 , R 18 in combination with R 5 , or R 19 in combination with R 6 , can independently form a 5- to 10-member ring structure that is saturated or unsaturated and substituted or unsubstituted;
Z is a pentavalent P or As;
A and B are substituents that link to Z through single or double bonds and are independently H, O, S, NH, F, Cl, Br, I, CN, nitro, azido, hydroxyl, amino, imino, hydrazino, (substituted) aryl, (substituted) aroxyl, alkenyl, alkynyl, alkyl, alkoxy, alkylamino, dialkylamino, arylamino, diarylamino, alkyl(aryl)amino, alkanoylamino, alkylthio, alkylcarbonyl, aryl carbonyl, alkylthiocarbonyl, arylthiocarbonyl, alkyloxycarbonyl, aroxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl, alkyl(aryl)aminocarbonyl, arylcarboxamido, or Q, the alkyl or alkoxy portions of which are saturated or unsaturated, linear or branched, and substituted or unsubstituted;
A in combination with B, A in combination with a ring formed by R 1 and R 2 , B in combination with a ring formed by R 4 and R 5 of structures I or II, A in combination with R 1 , B in combination with R 6 , A in combination with R 6 , or B in combination with R 7 , can independently form a 5- to 10-member ring structure that is saturated or unsaturated, and unsubstituted or substituted; and
D 1 , D 2 , D 3 and D 4 are either C or N, wherein only 1 or none of D 1 , D 2 , D 3 or D 4 are N.
2. The fluorescent dye of claim 1 , wherein
at least one of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 comprises an alkyl, alkoxy and/or aryl that is further substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide, or thioamide; and/or at least one of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 comprises an alkyl and/or alkoxy that is further substituted by Q; and/or
at least one of R 1 in combination with R 2 , R 2 in combination with R 3 , R 3 in combination with R 4 , R 3 in combination with R 7 , R 4 in combination with R 7 , or R 4 in combination with R 5 independently forms a 5-10 member ring structure that is further substituted with an alkyl, an aryl, an alkenyl, an alkynyl, an alkoxy, an aroxyl, a hydroxyl, an F, a Cl, a Br, an I, a CN, a nitro, an alkylsulfonyl, an arylsulfonyl, an alkylsulfinyl, an arylsulfinyl, a (thio)carbonyl, a (thio)carboxylic acid, a (thio)carboxylic acid ester, a nitro, an amino, a (thio)amide, an azido, a hydrazino, or a (thio)phosphonate where each alkyl group or alkoxy group is independently saturated or unsaturated, linear or branched, or substituted or unsubstituted, and each aryl group is unsubstituted or substituted and/or
at least one of A or B is an alkyl or alkoxy that is further substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide, thioamide, or Q; and/or
at least one of A in combination with B, A in combination with a ring formed by R 1 and R 2 , B in combination with a ring formed by R 4 and R 5 , A in combination with R 1 , B in combination with R 6 , A in combination with R 6 , or B in combination with R 7 , independently form a 5- to 10-member ring structure that is substituted with an alkyl, an aryl, an alkenyl, an alkynyl, an alkoxy, an aroxyl, a hydroxyl, an F, a Cl, a Br, an I, a CN, a nitro, a carbonyl, a thiocarbonyl, a thiocarboxylic acid, a thiocarboxylic acid ester, a nitro, an amino, a (thio)amide, an azido, a hydrazino, or Q, wherein the alkyl group herein is saturated or unsaturated, linear or branched, substituted or unsubstituted, an alkoxy group wherein the alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted.
3. The fluorescent dye of claim 2 , wherein
at least one of the 5-10 member ring structure formed by R 1 in combination with R 2 , R 2 in combination with R 3 , R 3 in combination with R 4 , or R 4 in combination with R 5 is substituted with an aryl substituted with an F, a Cl, a Br, an I, a CN, an OH, an alkyl, an alkenyl, an alkynyl, an alkoxy, an aryoxy, an alkylthio, an arylthio, a nitro, an azido, a hydrazino, a carboxyl, a thiocarboxyl, a carbonyl, a thiocarbonyl, a carboxylic acid ester, a thiocarboxylic acid ester, or an unsubstituted or substituted amino, amide, thioamide, or Q.
4. The fluorescent dye of claim 1 , wherein Z is P, A is ═O and B is —OH or —O − .
5. The fluorescent dye of claim 1 , comprising structure II or III and further comprising the 5-10 member ring structure independently formed by R 16 in combination with R 17 , and/or R 18 in combination with R 19 , further substituted with alkyl, alkenyl, alkynyl, aryl, alkoxy, F, Cl, Br, I, carboxylic acid or carboxylic acid ester, where the alkyl group is saturated or unsaturated, linear or branched, and substituted or unsubstituted.
6. The fluorescent dye of claim 5 , wherein the 5-10 member ring structure independently formed by R 16 in combination with R 17 , and/or R 18 in combination with R 19 comprises
an alkyl group that is further substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, nitro, azido, hydrazino, alkoxy, aryoxy, alkylthio, arylthio, thiocarboxyl, carbonyl, thiocarbonyl, thiocarboxylic acid ester, unsubstituted or substituted amino, amide, thioamide, or Q, and/or
an aryl group that is further substituted by F, Cl, Br, I, CN, OH, alkoxy, aryoxy, alkylthio, arylthio, nitro, azido, hydrazino, carboxyl, thiocarboxyl, carbonyl, thiocarbonyl, carboxylic acid ester, thiocarboxylic acid ester, unsubstituted or substituted amino, amide, thioamide, or Q.
7. The fluorescent dye of claim 1 , comprising
wherein
R 20 , R 21 , R 22 , R 23 and R 24 are independently H, F, Cl, Br, I, CN, nitro, azido, hydroxyl, amino, hydrazino, (substituted) aryl, (substituted) aroxyl, alkenyl, alkynyl, alkyl, alkoxy, alkylamino, dialkylamino, arylamino, diarylamino, alkyl(aryl)amino, alkanoylamino, alkylthio, alkylcarbonyl, aryl carbonyl, alkylthiocarbonyl, arylthiocarbonyl, alkyloxycarbonyl, aroxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl, alkyl(aryl)aminocarbonyl, arylcarboxamido, or Q, the alkyl or alkoxy portions of which are saturated or unsaturated, linear or branched, and substituted or unsubstituted.
8. The fluorescent dye of claim 7 , wherein at least one of R 20 , R 21 , R 22 , R 23 and R 24 is independently alkyl or alkoxy further substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide, thioamide, or Q.
9. The fluorescent dye of claim 7 , wherein either R 22 or R 24 is —COOH, —O(CH 2 ) n COOH or SO 3 H, wherein n=1-10.
10. The fluorescent dye of claim 1 , comprising
11. The fluorescent dye of claim 1 , further comprising a reactive group L.
12. The fluorescent dye of claim 11 , comprising
13. The fluorescent dye of claim 11 , comprising
wherein X and Y are
(a) NH 2 and NH, respectively;
(b) —NHCH 2 CH 3 and —NCH 2 CH 3 , respectively;
(c) —NHCH 3 and —NCH 3 , respectively;
(d) —N(CH 2 CH 3 ) 2 and —N + (CH 2 CH 3 ) 2 , respectively; or
(e) a tertiary amine and a quaternary ammonium in a ring system, respectively.
14. The fluorescent dye of claim 11 , comprising
15. The fluorescent dye of claim 1 , further comprising a member of a binding pair.
16. A fluorescence energy transfer system, comprising the fluorescent dye of claim 1 and a second dye wherein the second dye is capable of energy transfer with the fluorescent dye.
17. The fluorescent dye of claim 1 , bound to a target molecule.
18. A target molecule labeled with the fluorescent dye of claim 1 .
19. A method of labeling a target molecule, the method comprising contacting reactive group L of the fluorescent dye of claim 11 with the target molecule such that reactive group L reacts with the target molecule to form a covalent bond between reactive group L and the target molecule.
20. A method of labeling a target molecule, the method comprising contacting the fluorescent dye of claim 15 with the target molecule, wherein the target molecule comprises a second member of the binding pair.