Polar dyes

An oligonucleotide conjugate comprising an oligonucleotide covalently bonded with a dye having an arsonate group, wherein the oligonucleotide conjugate is represented by:

• wherein,
• v is 0, 1, 2, 3, 4, 5 or 6;
• Y represents atoms necessary to form one or two fused aromatic rings having 6 atoms in each ring, each ring atom is the same or different and independently ═CR46—, ═N+R47— or ═N—;
• W represents atoms necessary to form one or two fused aromatic rings having 6 atoms in each ring, each ring atom is the same or different and independently ═CR46—, ═N+R47— or ═N—;
• Z is —O—, —S—, —Se—, —C(R48)2— or —NR49—;
• R20 and R23 are the same or different and independently alkyl, aralkyl or substituted alkyl substituted with hydroxy, cyano, nitro amino sulfonyl, phosphonyl, —As(═O)(OR6)(OR7), —As(OR6)2(OR7)2, —COOH, alkoxy, monoalkylamino dialkylamino or trialkylamonium or L-oligonucleotide;
• R52 and R53 are the same or different and independently hydrogen, alkyl, alkyl substituted hydroxy, —COOH, halogen, cyano, nitro, amine, alkylamino, dialkylamino, sulfonyl, phosphonyl, —As(═O)(OR6)(OR7), As(OR6)2(OR7)2 or L-oligonucleotide;
• R21 and each R22 are the same or different and independently hydrogen, halogen, alkoxy, hydroxy, mercapto, aryloxy, heterocycle, iminium ion, L-oligonucleotide, or two adjacent R21 and R22 or any two adjacent R22 form a 4, 5 or 6 member carbocycle, optionally substituted with alkyl, halogen or oxo;
• or two R22 that are one carbon away from each other, together with the carbons to which they are attached, form a 4, 5 or 6 member carbocycle, optionally substituted with alkyl, halogen, alkylthio, arylthio, alkoxy, aryloxy, oxo (═O), ═S or) ═C(R50)2;
• each R46 and R47 is the same or different and independently hydrogen, alkyl, aryl, halogen, cyano, nitro, amine, alkylamino, dialkylamino, sulfonyl, phosphonyl, —As(═O)(OR6)(OR7), As(OR6)2(OR7)2 or L-oligonucleotide;
• each R48 and R49 is the same or different and independently hydrogen or alkyl optionally substituted with hydroxy, —COOH, halogen, cyano, nitro, amine, alkylamino, dialkylamino, sulfonyl, phosphonyl, —As(═O)(OR6)(OR7), —As(OR6)2(OR7)2 or L-oligonucleotide;
• each R50 is the same or different and independently hydrogen, alkyl, cyano, —C(O)NHR48;
• R6 and R7 are the same or different and independently H, alkyl, aryl, or heteroaryl;
• or R6 and R7 are linked together to form a 5 or 6-member heterocycle comprising the arsenic and the two oxygen atoms to which R6 and R7 are attached, wherein —R6—R7— is —[C(R8)2]w— (w is 2 or 3), wherein each R8 is the same or different and independently hydrogen, hydroxy, cyano, —OC(O)-alkyl, —OC(O)-aryl, alkyl, aryl, alkylcarbonyl, arylcarbonyl, —CH2CN, —CH2SO2Me, —CH2SO2Ph, or two R8 of adjacent carbons together with the carbons to which they are attached form an aryl or heteroaryl, or two R8 of the same carbon form an oxo;
• and
• L is a single covalent bond or a covalent linkage having 1-30 non-hydrogen atoms selected from the group consisting of C, N, O and S and is composed of any combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds and carbon-sulfur bonds;
• provided, at least one of the R20, R23 R46, R47, R48, R49, R52 and R53 is —As(═O)(OR6)(OR7) or —As(OR6)2(OR7)2.

A method of preparing an oligonucleotide conjugate in an automated synthesis, using a dye reagent of Formula (V),

• wherein,
• v is 0, 1, 2, 3, 4, 5 or 6;
• Y represents atoms necessary to form one or two fused aromatic rings having 6 atoms in each ring, each ring atom is the same or different and independently ═CR46—, ═N+R47— or ═N—;
• W represents atoms necessary to form one or two fused aromatic rings having 6 atoms in each ring, each ring atom is the same or different and independently ═CR46—, ═N+R47— or ═N—;
• Z is —O—, —S—, —Se—, —C(R48)2— or —NR49—;
• R20 and R23 are the same or different and independently alkyl, aralkyl or substituted alkyl substituted with hydroxy, cyano, nitro, amino, sulfonyl, phosphonyl, —As(═O)(OR6)(OR7), —As(OR6)2(OR7)2, —COOH, alkoxy, monoalkylamino, dialkylamino or trialkylamonium or L-Rx;
• R52 and R53 are the same or different and independently hydrogen, alkyl, alkyl substituted hydroxy, —COOH, halogen, cyano, nitro, amine, alkylamino, dialkylamino, sulfonyl, phosphonyl,—As(═O)(OR6)(OR7), AS(OR6)2(OR7)2or L-Rx;
• R21 and each R22 are the same or different and independently hydrogen, halogen, alkoxy, hydroxy, mercapto, aryloxy, heterocycle, iminium ion ,L-Rx, or two adjacent R21 and R22 or any two adjacent R22 form a 4, 5 or 6 member carbocycle, optionally substituted with alkyl, halogen or oxo;
• or two R22 that are one carbon away from each other, together with the carbons to which they are attached, form a 4, 5 or 6 member carbocycle, optionally substituted with alkyl, halogen, alkylthio, arylthio, alkoxy, aryloxy, oxo (═O), ═S or ═C(R50)2;
• each R46 and R47 is the same or different and independently hydrogen, alkyl, aryl, halogen, cyano, nitro, amine, alkylamino, dialkylamino, sulfonyl, phosphonyl, —As(═O)(OR6)(OR7), As(OR6)2(OR7)2or L-Rx;
• each R48 and R49 is the same or different and independently hydrogen or alkyl optionally substituted with hydroxy, —COOH, halogen, cyano, nitro, amine, alkylamino, dialkylamino, sulfonyl, phosphonyl, —As(═O)(OR6)(OR7), —As(OR6)2(OR7)2 or L-Rx;
• each R50 is the same or different and independently hydrogen, alkyl, cyano, —C(O)NHR48 ;
• R6 and R7 are the same or different and independently H, alkyl, aryl, or heteroaryl;
• or
• R6 and R7 are linked together to form a 5 or 6-member heterocycle comprising the arsenic and the two oxygen atoms to which R6 and R7 are attached, wherein —R6—R7— is —[C(R8)2]w— (w is 2 or 3), wherein each R8 is the same or different and independently hydrogen, hydroxy, cyano, —OC(O)-alkyl, —OC(O)-aryl, alkyl, aryl, alkylcarbonyl, arylcarbonyl, —CH2CN, —CH2SO2Me, —CH2SO2Ph, or two R8 of adjacent carbons together with the carbons to which they are attached form an aryl or heteroaryl, or two R8 of the same carbon form an oxo;
• L is a single covalent bond or a covalent linkage having 1-30 non-hydrogen atoms selected from the group consisting of C, N, O and S and is composed of any combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds and carbon-sulfur bonds;
• provided, at least one of the R20, R23 R46, R47, R48, R49, R52 and R53 is —As(═O)(OR6)(OR7) or —As(OR6)2(OR7)2,
• wherein Rx is a phosphoramidite group, the method comprising:
• performing automated synthesis of oligonucleotide using phosphoramidite method to provide a dye-labeled oligonucleotide;
• deprotecting —As(═O)(OR6)(OR7) or —As(OR6)2(OR7)2 of the dye-labeled oligonucleotide to provide a dye-labeled oligonucleotide having negatively charged polar group selected from —AsO32- and —AsO3H−;
• and
• isolating the dye-labeled oligonucleotide from the reaction medium.