Processes and reagents for oligonucleotide synthesis and purification

Abstract

The present invention relates to processes and reagents for oligonucleotide synthesis and purification. One aspect of the present invention relates to compounds useful for activating phosphoramidites in oligonucleotide synthesis. Another aspect of the present invention relates to a method of preparing oligonucleotides via the phosphoramidite method using an activator of the invention. Another aspect of the present invention relates to sulfur-transfer agents. In a preferred embodiment, the sulfur-transfer agent is a 3-amino-1,2,4-dithiazolidine-5-one. Another aspect of the present invention relates to a method of preparing a phosphorothioate by treating a phosphite with a sulfur-transfer reagent of the invention. In a preferred embodiment, the sulfur-transfer agent is a 3-amino-1,2,4-dithiazolidine-5-one. Another aspect of the present invention relates to compounds that scavenge acrylonitrile produced during the deprotection of phosphate groups bearing ethylnitrile protecting groups. In a preferred embodiment, the acrylonitrile scavenger is a polymer-bound thiol. Another aspect of the present invention relates to agents used to oxidize a phosphite to a phosphate. In a preferred embodiment, the oxidizing agent is sodium chlorite, chloroamine, or pyridine-N-oxide. Another aspect of the present invention relates to methods of purifying an oligonucleotide by annealing a first single-stranded oligonucleotide and second single-stranded oligonucleotide to form a double-stranded oligonucleotide; and subjecting the double-stranded oligonucleotide to chromatographic purification. In a preferred embodiment, the chromatographic purification is high-performance liquid chromatography.

1 . A method of forming a phosphite compound, comprising the steps of: 
 admixing a phosphoramidite, alcohol, and activating agent to form a phosphite compound, wherein said activating agent is selected from the group consisting of                          wherein    X is C(R 6 ) or N;    R 1 , R 2 , R 3 , and R 6  each independently represent H, —NO 2 , —CN, —CF 3 , —SO 2 R 8 , —SR 8 , halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, alkoxyl, —OR 7 , —N(R 7 ) 2 , —N(R 7 )C(O)R 8 , —C(O)R 7 , or —CO 2 R 8 ; or an instance of R 1  and R 6 , R 1  and R 2 , or R 2  and R 3  can be taken together for form a 4-8 member ring containing 0-4 heteratoms selected from the group consisting of O, N and S;    R 4  is absent or represents independently for each occurrence —(C(R 9 ) 2 ) n CH 3 .Y;    R 5  is H or —(C(R 9 ) 2 ) n CH 3 ;    R 7  represents independently for each occurrence H, alkyl, aryl, or aralkyl;    R 8  represents independently for each occurrence alkyl, aryl, or aralkyl;    R 9  represents independently for each occurrence H or alkyl;    n represents independently for each occurrence 0 to 15 inclusive; and    Y represents independently for each occurrence halogen or R 8 CO 2   − ;                          wherein    R 1  and R 3  each represent independently H, —NO 2 , —CN, —CF 3 , —SO 2 R 6 , —SR 6 , halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, —N(R 5 )C(O)R 6 , —C(O)R 5 , or —CO 2 R 6 ;    R 2  is absent or represents independently for each occurrence —(C(R 7 ) 2 ) n CH 3 .Y;    R 4  is H or —(C(R 7 ) 2 ) n CH 3 ;    R 5  represents independently for each occurrence H, alkyl, aryl, or aralkyl;    R 6  represents independently for each occurrence alkyl aryl, or aralkyl;    R 7  represents independently for each occurrence H or alkyl;    n represents independently for each occurrence 0 to 15 inclusive; and    Y represents independently for each occurrence halogen or R 6 CO 2   − ;                          wherein    R 1  and R 2  each represent independently H, —NO 2 , —CN, —CF 3 , —SO 2 R 6 , —SR 6 , halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, —N(R 5 )C(O)R 6 , —C(O)R 5 , or —CO 2 R 6 ;    R 3  is absent or represents independently for each occurrence —(C(R 7 ) 2 ) n CH 3 .Y;    R 4  is H or —(C(R 7 ) 2 ) n CH 3 ;    R 5  represents independently for each occurrence H, alkyl, aryl, or aralkyl;    R 6  represents independently for each occurrence alkyl, aryl, or aralkyl;    R 7  represents independently for each occurrence H or alkyl;    n represents independently for each occurrence 0 to 15 inclusive; and    Y represents independently for each occurrence halogen or R 6 CO 2   − ;                          wherein    R 1  is H, —SR 5 , alkyl, aryl, —N(R 4 ) 2 , —(C(R 4 ) 2 ) m CO 2 R 5 , —NO 2 , —CN, —CF 3 , —SO 2 R 5 , —SR 5 , halogen, alkenyl, alkynyl, aralkyl, —N(R 4 )C(O)R 5 , —C(O)R 4 , or —CO 2 R 5 ;    R 2  is absent or represents independently for each occurrence —(C(R 6 ) 2 ) n CH 3 .Y;    R 3  is H or —(C(R 6 ) 2 ) n CH 3 ;    R 4  represents independently for each occurrence H, alkyl, aryl, or aralkyl;    R 5  represents independently for each occurrence alkyl, aryl, or aralkyl;    R 6  represents independently for each occurrence H or alkyl;    n represents independently for each occurrence 0 to 15 inclusive;    m is 1, 2, 3, 4, 5, 6, 7, or 8; and    Y represents independently for each occurrence halogen or R 5 CO 2   − ; and                          wherein    R 1 , R 3 , and R 4  each represent independently H, —NO 2 , —CN, —CF 3 , —SO 2 R 7 , —SR 7 , halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, —N(R 6 )C(O)R 5 , —C(O)R 6 , or —CO 2 R 7 ;    R 2  is absent or represents independently for each occurrence —(C(R 8 ) 2 ) n CH 3 .Y;    R 5  is H or —(C(R 8 ) 2 ) n CH 3 ;    R 6  represents independently for each occurrence H alkyl aryl, or aralkyl;    R 7  represents independently for each occurrence alkyl, aryl, or aralkyl;    R 8  represents independently for each occurrence H or alkyl;    n represents independently for each occurrence 0 to 15 inclusive; and    Y represents independently for each occurrence halogen or R 7 CO 2   − .    
     
     
         2 . The method of  claim 1 , wherein said phosphoramidite is a 3′-nucleoside phosphoramidite, 3′-nucleotide phosphoramidite, or 3′-oligonucleotide phosphoramidite.  
     
     
         3 . The method of  claim 1 , wherein said phosphoramidite is represented by formula A:  
       
         
           
           
               
               
           
         
         wherein  
         R 1  is alkyl, aryl, aralkyl, or —Si(R 5 ) 3 ; wherein said alkyl, aryl, and aralkyl group is optionally substituted with —CN, —NO 2 , —CF 3 , halogen, —O 2 CR 5 , or —OSO 2 R 5 ;  
         R 2  is optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, or alkenyl;  
         R 3  and R 4  each represent independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or aralkyl; or R 3  and R 4  taken together form a 3-8 member ring; and  
         R 5  is alkyl, cycloalkyl, heterocycloalkyl, aryl, or aralkyl.  
       
     
     
         4 . The method of  claim 3 , wherein R 1  is —CH 2 CH 2 CN.  
     
     
         5 . The method of  claim 3 , wherein R 2  is an optionally substituted heterocycloalkyl.  
     
     
         6 . The method of  claim 3 , wherein R 2  is an optionally substituted ribose.  
     
     
         7 . The method of  claim 3 , wherein R 2  is an optionally substituted deoxyribose.  
     
     
         8 . The method of  claim 3 , wherein R 2  is a nucleoside or nucleotide.  
     
     
         9 . The method of  claim 3 , wherein R 3  and R 4  are alkyl.  
     
     
         10 . The method of  claim 1 , wherein said alcohol is an optionally substituted ribose.  
     
     
         11 . The method of  claim 1 , wherein said alcohol is an optionally substituted deoxyribose.  
     
     
         12 . The method of  claim 1 , wherein said alcohol is a nucleoside, nucleotide, or oligonucleotide.  
     
     
         13 . The method of  claim 1 , wherein said alcohol is represented by R 5 —OH, wherein R 5  is optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, alkenyl, or —(C(R 6 ) 2 ) p heterocycloalkyl; R 6  is H or alkyl; and p is 1, 2, 3, 4, 5, 6, 7, or 8.  
     
     
         14 . The method of  claim 13 , wherein R 5  is —(C(R 6 ) 2 ) p heterocycloalkyl.  
     
     
         15 . The method of  claim 1 , further comprising the step of admixing a proton-shuttle compound to the mixture comprising said phosphoramidite, said alcohol, and said activating agent, wherein the pKa of said proton-shuttle compound is greater than the pKa of said activating agent, and the pKa of said proton-shuttle compound is less than the pKa of said phosphoramidite.  
     
     
         16 . The method of  claim 15 , wherein said proton-shuttle compound is a primary, secondary, or tertiary amine.  
     
     
         17 . The method of  claim 15 , wherein said proton-shuttle compound is represented by N(R 7 )(R 8 )R 9 , wherein R 7 , R 8 , and R 9  each represent independently for each occurrence H, alkyl, cycloalkyl, aryl, aralkyl, alkenyl; or R 7  and R 8  taken together form a 3-8 membered ring; and R 9  is H, alkyl, cycloalkyl, aryl, or aralkyl.  
     
     
         18 . A compound represented by formula E:  
       
         
           
           
               
               
           
         
         wherein  
         X is O or S;  
         R 1  is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;  
         R 2  is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, —C(O)N(R 3 )R 4 , —C(S)N(R 3 )R 4 , —C(S)N(R 3 ) 2 , —C(S)OR 4 , —CO 2 R 4 , —C(O)R 4 , or —C(S)R 4 ;  
         R 3  is H or alkyl; and  
         R 4  is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl.  
       
     
     
         19 . The compound of  claim 18 , wherein X is O.  
     
     
         20 . The compound of  claim 18 , wherein R 2  is H, alkyl, or cycloalkyl.  
     
     
         21 . The compound of  claim 18 , wherein R 2  is aryl or aralkyl.  
     
     
         22 . The compound of  claim 18 , wherein R 2  is —C(O)N(R 3 )R 4 , —C(S)N(R 3 )R 4 , —C(S)N(R 3 ) 2 , —C(S)OR 4 , —CO 2 R 4 , —C(O)R 4 , or —C(S)R 4 .  
     
     
         23 . The compound of  claim 18 , wherein R 3  is H.  
     
     
         24 . The compound of  claim 18 , wherein R 4  is alkyl or aryl.  
     
     
         25 . The compound of  claim 18 , wherein X is O, and R 2  is H.  
     
     
         26 . The compound formed by a process, comprising the steps of: 
 admixing about 1 equivalent of chlorocarbonyl sulfenyl chloride, about 1 equivalent of thiourea, and about 1 equivalent of triethylamine in a container cooled with a ice-bath at about 0° C. under an atmosphere of argon, stirring the resultant mixture for about 6 hours, filtering said mixture, concentrating said mixture to give a residue, and recystallizing said residue from dichloromethane-hexanes to give the compound.    
     
     
         27 . A method of removing an ethylcyanide protecting group, comprising the steps of: 
 admixing a phosphate compound bearing a ethylcyanide group with a base in the presence acrylonitrile scavenger, wherein said acrylonitrile scavenger is a polymer-bound thiol, 4-n-heptylphenylmethanethiol, alkane thiol having at least 10 carbon atoms, heteroarylthiol, the sodium salt of an alkyl thiol,                          wherein R 1  is alkyl; and R 2  is —SH, or —CH 2 SH.    
     
     
         28 . The method of  claim 27 , wherein said acrylonitrile scavenger is  
       
         
           
           
               
               
           
         
       
     
     
         29 . The method of  claim 27 , wherein said phosphate compound is an oligonucleotide.  
     
     
         30 . The method of  claim 27 , wherein said phosphate compound is an oligonucleotide containing at least one phosphorothioate group.  
     
     
         31 . The method of  claim 27 , wherein said phosphate compound is an oligomer of ribonucleotides.  
     
     
         32 . The method of  claim 27 , wherein said phosphate is represented by formula G:  
       
         
           
           
               
               
           
         
         wherein  
         R 1  is optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, or alkenyl;  
         R 2  is optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, alkenyl, or —(C(R 3 ) 2 ) p heterocycloalkyl;  
         R 3  is H or alkyl; and  
         p is 1, 2, 3, 4, 5, 6, 7, or 8.  
       
     
     
         33 . The method of  claim 32 , wherein R 1  is an optionally substituted heterocycloalkyl.  
     
     
         34 . The method of  claim 32 , wherein R 1  is an optionally substituted ribose.  
     
     
         35 . The method of  claim 32 , wherein R 1  is an optionally substituted deoxyribose.  
     
     
         36 . The method of  claim 32 , wherein R 1  is a nucleoside, nucleotide, or oligonucleotide.  
     
     
         37 . The method of  claim 32 , wherein R 1  is  
       
         
           
           
               
               
           
         
       
       wherein R′ 1  represents independently for each occurrence alkyl, aryl, aralkyl, or —Si(R 4 ) 3 ; wherein said alkyl, aryl, and aralkyl group is optionally substituted with —CN, —NO 2 , —CF 3 , or halogen; R 4  is alkyl, aryl, or aralkyl; and n 1  is 1 to 50 inclusive.  
     
     
         38 . The method of  claim 37 , wherein n 1  is 1 to 25 inclusive.  
     
     
         39 . The method of  claim 37 , wherein n 1  is 1 to 15 inclusive.  
     
     
         40 . The method of  claim 37 , wherein n 1  is 1 to 10 inclusive.  
     
     
         41 . The method of  claim 37 , wherein n 1  is 1 to 5 inclusive.  
     
     
         42 . A method of removing an amide protecting group from an oligonucleotide, comprising the steps of: 
 admixing an oligonucleotide bearing an amide protecting group with a polyamine, PEHA, PEG-NH 2 , Short PEG-NH 2 , cycloalkyl amine, hydroxycycloalkyl amine, hydroxyamine, K 2 CO 3 /MeOH microwave, thioalkylamine, thiolated amine, β-amino-ethyl-sulfonic acid, or the sodium sulfate of β-amino-ethyl-sulfonic acid.    
     
     
         43 . The method of  claim 42 , wherein said oligonucleotide is an oligomer of ribonucleotides.  
     
     
         44 . A method of purifying an oligonucleotide, comprising the steps of: 
 annealing a first oligonucleotide with a second oligonucleotide to form a double-stranded oligonucleotide, subjecting said double-stranded oligonucleotide to chromatographic purification.    
     
     
         45 . The method of  claim 44 , wherein said chromatographic purification is liquid chromatography.  
     
     
         46 . The method of  claim 44 , wherein said chromatographic purification is high-performance liquid chromatography.  
     
     
         47 . The method of  claim 44 , wherein said first oligonucleotide is an oligomer of ribonucleotides.  
     
     
         48 . The method of  claim 44 , wherein said second oligonucleotide is an oligomer of ribonucleotides.  
     
     
         49 . The method of  claim 44 , wherein said first oligonucleotide is an oligomer of ribonucleotides, and said second oligonucleotide is an oligomer of ribonucleotides.  
     
     
         50 . A method of preparing an oligonucleotide comprising a dinucleoside unit, comprising the steps of: 
 synthesizing a dinucleoside group via solution-phase chemistry, attaching said dinucleoside group to a solid support to form a primer, adding additional nucleotides to said primer using solid-phase synthesis techniques.    
     
     
         51 . The method of  claim 50 , wherein each nucleoside residue of said dinucleoside group is independently a natural or unnatural nucleoside.  
     
     
         52 . The method of  claim 50 , wherein said dinucleoside group comprises two nucleoside residues each independently comprising a sugar and a nucleobase, wherein said sugar is a D-ribose or D-deoxyribose, and said nucleobase is natural or unnatural.  
     
     
         53 . The method of  claim 50 , wherein said dinucleoside group comprises two nucleoside residues each independently comprising a sugar and a nucleobase, wherein said sugar is a L-ribose or L-deoxyribose, and said nucleobase is natural or unnatural.  
     
     
         54 . The method of  claim 50 , wherein said dinucleoside group comprises two thymidine residues.  
     
     
         55 . The method of  claim 50 , wherein said dinucleoside group comprises two deoxythymidine residues.  
     
     
         56 . The method of  claim 50 , wherein said dinucleoside group comprises two 2′-modified 5-methyl uridine or uridine residues, wherein the 2′-modifications are 2′-OTBDMS, 2′-OMe, 2′-F, 2′-O—CH2-CH2-O-Me, 2′-O-alkylaminoderivatives.  
     
     
         57 . The method of  claim 50 , wherein said dinucleoside group comprises a phosphorothioate linkage, phosphorodithioate linkage, alkyl phosphonate linkage, or boranophosphate linkage.  
     
     
         58 . The method of  claim 50 , wherein said dinucleoside group comprises a phosphorothioate linkage, alkyl phosphonate linkage, or boranophosphate linkage; and said dinucleoside group is a single stereoisomer at the phosphorus atom.  
     
     
         59 . The method of  claim 50 , wherein the linkage between the nucleoside residues of said dinucleoside group is a 3′-5′ linkage.  
     
     
         60 . The method of  claim 50 , wherein the linkage between the nucleoside residues of said dinucleoside group is a 2′-5′ linkage.  
     
     
         61 . The method of  claim 50 , wherein said dinucleoside group comprises two nucleoside residues each independently comprising a sugar and a nucleobase, wherein said sugar is a D-ribose or D-deoxyribose, and said nucleobase is natural or unnatural; and the linkage between the nucleoside residues of said dinucleotide group is unnatural and non-phosphate.  
     
     
         62 . The method of  claim 50 , wherein said dinucleoside group comprises two nucleoside residues each independently comprising a sugar and a nucleobase, wherein said sugar is a L-ribose or L-deoxyribose, and said nucleobase is natural or unnatural; and the linkage between the nucleoside residues of said dinucleotide group is MMI, amide linkage, or guanidinium linkage.