Oligonucleotide labeling reactants and their use

Abstract

The invention relates to a novel labeling reactant of formula (I) suitable for labeling an oligonucleotide wherein: R is a temporary protecting group. A is either a phosphorylating moiety or a solid support tethered to a bridge point Z via a linker arm E. E′ is a linker arm between G and Z. G is a bivalent aromatic structure, tethered to two iminodiacetic acid ester groups N(COOR′″) 2 or G is a structure selected from a group consisting of  or G is a protected functional group. The invention further concerns a method for direct attachment of a conjugate group to an oligonucleotide structure enabling the attachment of a desired number of these groups during chain assembly. The method comprises a Mitsonobu alkylation.

1 - 13 . (Canceled).  
     
     
         14 . A labeling reactant of formula (I) suitable for labeling an oligonucleotide  
       
         
           
           
               
               
           
         
       
       wherein 
 R is a protecting group or is not present;  
 A is eithera phosphorylating moiety  
                     
  where 
 L is O, S, or is not present  
 L′ is H, L′″CH 2 CH 2 CN or L′″r, where Ar is phenyl or its substituted derivative, where the substituent is nitro or chlorine, and L′″ is O or S;  
 L″ is O − , S − , Cl, N(i-Pr) 2 ; or  
 
 A is a solid support tethered to Z via a linker arm, which is formed of one to ten moieties, each moiety being selected from a group consisting of phenylene, alkylene containing 1-12 carbon atoms, ethynediyl, ether, thioether, amide, carbonyl, ester, disulfide, diaza, and tertiary amine;  
 Z is a bridge point and is formed from  
                     
  or trivalent derivatives, substituted or unsubstituted, of cyclohexane, cyclohexene, cyclohexadiene, phenyl, cyclopentane, cyclopentene, cyclopentadiene, cyclobutane, cyclobutene, cyclobutadiene, aziridine, diaziridine, oxetane, thietaneazete, azetidine, 1,2-dihydro-1,2-diazete, 1,2-diazetidine, furan, tetrahydrofuran, thiophene, 2,5-dihydrothiophene, thiolane, selenophene, pyrrole, pyrrolidine, phosphole, 1,3-dioxolane, 1,2-dithiole, 1,2-thiolane, 1,3-dithiole, 1,3-dithiolane, oxazole, 4,5-dihydrooxazole, isoxazole, 4,5-dihydoisozaole, 2,3-dihydroisoxazole, thiazole, isothiazole, imidazole, imidazolidine, pyrazole, 4,5-dihydropyrazole, pyrazolidine, triazole, pyran, pyran-2-one, 3,4-dihydro-2H-pyran, tetrahydropyran, 4H-pyran, pyran-4-one, pyridine, pyridone, piperidine, phosphabenzene, 1,4-dioxin, 1,4-dithiin, 1,4-oxathiin, oxazine, 1,3-oxazinone, morpholine, 1,3-dioxane, 1,3-dithiane, pyridazine, pyrimidine, pyrazine, piperazine, 1,2,4-triazine, 1,3,5-triazine, 1,3,5-triaza-cyclohexane-2,4,6-trione;  
 where 
 R″ is H or X′X″, where  
 X′ is —O—, —S—, —N—, ON— or —NH— and X″ is a protection group or  
 X′ is —O— and X″ is alkyl or alkoxyalkyl;  
 X is H, alkyl, alkynyl, allyl, Cl, Br, I, F, S, O, NHCOCH(CH 3 ) 2 , NHCOCH 3 , NHCOPh, SPh 3 , OCOCH 3  or OCOPh;  
 
 E is a linker arm between R and Z and is formed of one to ten moieties, each moiety being selected from a group consisting of phenylene, alkylene containing 1-12 carbon atoms, ethynediyl, ether, thioether, amide, carbonyl, ester, disulfide, diaza, and tertiary amine, or not present;  
 E′ is a linker arm between G and Z, and is formed of one to ten moieties, each moiety being selected from the group consisting of phenylene, alkylene containing 1-12 carbon atoms, ethynediyl, ether, thioether, amide, carbonyl, ester, disulfide, diaza, and tertiary amine, or is not present;  
 G is a bivalent aromatic structure, tethered to two iminodiacetic acid ester groups N(CH 2 COOR′″) 2  where 
 R′″ is an alkyl of 1 to 4 carbon atoms, allyl, ethyltrimethylsilyl, phenyl or benzyl, which phenyl or benzyl is substituted or unsubstituted, and  
 
 said bivalent aromatic structure is capable of absorbing light or energy and transferring the excitation energy to a lanthanide ion after the solid phase synthesis made labeling reactant has been released from the used solid support, deprotected and converted to a lanthanide chelate, or  
 G is a structure selected from a group consisting of  
                     
  where 
 R′″ is an alkyl of 1 to 4 carbon atoms, allyl, ethyltrimethylsilyl, phenyl or benzyl, which phenyl or benzyl is substituted or unsubstituted, and  
 one of the hydrogen atoms is substituted with E′, or G is a protected functional group, where the functional group is amino, aminooxy, carboxyl, thiol, and the protecting group is phthaloyl, trityl, 2-(4-nitrophenylsulfonyl)ethoxycarbonyl, fluorenylmethyloxycarbonyl, benzyloxycarbonyl, trifluoroacetyl or t-butoxycarbonyl for amino and aminooxy, alkyl for carbonyl and alkyl or trityl for thiol, provided that bridge point Z is selected from a group consisting of  
                     
 
 
     
     
         15 . The labeling reactant of  claim 14 , wherein R is a member of the group consisting of 4,4′dimethoxytrityl, 4-methoxytrityl, trityl, and (9-phenyl)xanthen-9-yl.  
     
     
         16 . The labeling reactant of  claim 14 , wherein X″ is a member of the group consisting of t-butyldimethylsilyl-, tetrahydropyranyl, 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl-, 1-[2-chloro-4-methyl)phenyl]-4-metoxypiperidin-4-yl-, 4-methoxytetrahydropyran-4-yl-, phthaloyl-, acetyl, pivaloyl-, benzoyl-, 4-methylbenzoyl, benzyl-, and trityl.  
     
     
         17 . The labeling reactant of  claim 14 , wherein G is a protected functional group.  
     
     
         18 . The labeling reactant of  claim 17 , wherein said protected functional group is selected from the group consisting of amino, carboxyl, aminooxy and thiol.  
     
     
         19 . The labeling reactant of  claim 14 , wherein G is an organic dye.  
     
     
         20 . The labeling reactant of  claim 19 , wherein said organic dye is selected from the group consisting of dabsyl, dansyl, fluorescein, rhodamine and tetramethyl-6-carboxyrhodamine (TAMRA).  
     
     
         21 . The labeling reactant of  claim 14 , wherein the temporary protecting group R is 4,4′-dimethoxytrityl.  
     
     
         22 . The labeling reactant of  claim 14 , wherein said reactant is a nucleotide and the sugar of the nucleotide is 2-deoxyribose or 3-deoxyribose.  
     
     
         23 . The labeling reactant of  claim 22 , wherein X′ is hydroxyl.  
     
     
         24 . The labeling reactant of  claim 23 , wherein the permanent protection group X″ of X′ is selected from the group consisting of t-butyldimethylsilyl, tetrahydropyranyl, 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl-, 1-[2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl- and 4-methoxytetrahydropyran-4-yl-.  
     
     
         25 . The labeling reactant of  claim 22 , wherein X″ is an alkyl or alkoxyalkyl.  
     
     
         26 . The labeling reactant of  claim 25 , wherein X″ is selected from the group consisting of methyl, methoxymethyl and ethoxymethyl.  
     
     
         27 . The labeling reactant of  claim 14 , wherein G is a bivalent aromatic structure.  
     
     
         28 . The labeling reactant of  claim 27 , wherein G is selected from the group consisting of carbostyryl,  
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         29 . The labeling reactant of  claim 14 , wherein said reactant is non-luminescent and G is selected from a group consisting of  
       
         
           
           
               
               
           
         
       
       and wherein 
 R′″ is an alkyl of 1 to 4 carbon atoms, allyl, ethyltrimethylsilyl, phenyl or benzyl, which phenyl or benzyl can be substituted or unsubstituted, and one of the hydrogen atoms is substituted with E′.  
 
     
     
         30 . The labeling reactant of  claim 29 , wherein R′″ is selected from the group consisting of methyl, ethyl and allyl.  
     
     
         31 . The labeling reactant of  claim 14  wherein A is a solid support.  
     
     
         32 . The labeling reactant of  claim 31 , where A is 5′-O-(4,4′-dimethoxytrityl)-3′-O-succinyl-N3-{tetramethyl-2,2′,2″,2′″-{6,6′-[4′-hydroxyethoxyethoxyphenylethynyl]pyridine-2,6-diyl}bis-(methylenenitrilo)tetrakis(acetato)}thymidine long chain alkylamine controlled pore glass.  
     
     
         33 . A method for a direct attachment of a conjugate group to an oligonucleotide structure enabling the attachment of a desired number of these groups during chain assembly wherein it comprises a Mitsunobu alkylation of a compound of formula (II)  
       R-Z  (II)  
       wherein 
 R is a protecting group;  
 Z′ is an acidic bridge point selected from a group consisting of  
                     
  where 
 R″ is H or X′X″, where X′ is —O—, —S—, —N—, ON— or —NH— and X″ is a protection group;  
 X is H, alkyl, alkynyl, allyl, Cl, Br, I, F, S, O, NHCOCH(CH 3 ) 2 , NHCOCH 3 , NHCOPh, SPh 3 , OCOCH 3  or OCOPh;  
 
 and pK a  of said acidic bridge point is <14;  
 with a compound of formula (III)  
 G-E″  (III)  
  wherein  
 E″ is an arm with a primary aliphatic OH group at the end, which arm is formed of one to ten moieties, each moiety being selected from a group consisting of phenylene, alkylene containing 1-12 carbon atoms, ethynediyl, ether, thioether, amide, carbonyl, ester, disulfide, diaza, and tertiary amine;  
 G is a bivalent aromatic structure, tethered to two iminodiacetic acid ester groups N(CH 2 COOR′″) 2 , where 
 R′″ is an alkyl of 1 to 4 carbon atoms, allyl, ethyltrimethylsilyl, phenyl or benzyl, which phenyl or benzyl can be substituted or unsubstituted and  
 
 said bivalent aromatic structure is capable of absorbing light or energy and transferring the excitation energy to a lanthanide ion after the solid phase synthesis made labeling reactant has been released from the used solid support, deprotected and converted to a lanthanide chelate, or  
 G is a structure selected from a group consisting of  
                     
  where 
 R′″ is an alkyl of 1 to 4 carbon atoms, allyl, ethyltrimethylsilyl, phenyl or benzyl, which phenyl or benzyl can be substituted or unsubstituted, and one of the hydrogen atoms is substituted with E′, or  
 
 G is a protected functional group, where the functional group is amino, aminooxy, carboxyl, thiol, and the protecting group is pthaloyl, trityl, 2-(4-nitrophenyl-sulfonyl)ethoxycarbonyl, fluorenylmethyloxycarbonyl, benzyloxycarbonyl or t-butoxycarbonyl for amino and aminooxy, alkyl for carbonyl and alkyl or trityl for thiol, or  
 G is not present; and  
 the functional groups of E′ and G, excluding said primary aliphatic OH group, are protected;  
 to produce a compound of formula (IV)  
                     
  wherein G and R of compound (IV) are as defined above;  
 E′″ is a linker arm between G and Z, and is formed of one to ten moieties, each moiety being selected from a group consisting of phenylene, alkylene containing 1-12 carbon atoms, ethynediyl, ether, thioether, amide, carbonyl, ester, disulfide, diaza, and tertiary amine; and  
 Z″ is a bridge point selected from a group consisting of  
                     
  where 
 R″ is H or X′X″, where X′ is —O—, —S—, —N—, ON— or —NH— and X″ is a protection group;  
 
 X is H, alkyl, alkynyl, allyl, Cl, Br, I, F, S, O, NHCOCH(CH 3 ) 2 , NHCOCH 3 , NHCOPh, SPh 3 , OCOCH 3  or OCOPh.  
 
     
     
         34 . The method of  claim 33 , wherein X″ is a member selected from the group consisting of t-butyldimethylsilyl-, tetrahydropyranyl, 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl-, 1-[2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl-, 4-methoxytetrahydropyran-4-yl-, pthaloyl-, acetyl, pivaloyl-, benzoyl-, 4-methylbenzoyl, benzyl-, trityl and alkyl.