Method for preparing oligonucleotides

Abstract

A solution phase synthesis method for preparing an oligonucleotide, wherein at least some of the reagents are solid supported. The method suitable for large-scale synthesis comprises coupling a protected compound with a nucleotide derivative having a protection group in the presence of a solid supported activator to give an elongated oligonucleotide with a P(III)-internucleotide bond; optionally processing the elongated oligonucleotide by capping by reaction with a solid supported capping agent and/or by oxidizing or sulfurizing by reaction of the oligonucleotide with a solid supported oxidizing or sulfurization reagent; and removing the protection group. The coupling may include reacting a 3′-protected compound of formula: with a nucleotide derivative having a 5′-protection group, or reacting a 5′-protected compound of formula with a nucleotide derivative having a 3′-protection group.

1 . A method for preparing an oligonucleotide comprising the steps of
 a) providing a 3′-protected compound having the formula:   
     
       
         
         
             
             
         
       
       wherein
 B is a heterocyclic base, 
 R 2  is H, a protected 2′-hydroxyl group, F, a protected amino group, an O-alkyl group, an O-substituted alkyl, a substituted alkylamino or a C4′-O2′ methylene linkage, 
 R 3  is OR′ 3 , NHR″ 3 , NR″ 3 R′″ 3 , a 3′-protected nucleotide, or a 3′-protected oligonucleotide, 
 R′3 is a hydroxyl protecting group, 
 R′ 3 , R′″ 3  are independently an amine protecting group; 
 
       b) reacting said compound with a nucleotide derivative having a 5′-protection group in the presence of a solid supported activator to give an elongated oligonucleotide with a P(III)-internucleotide bond; 
       c) optionally processing the elongated oligonucleotide with a P(III)-internucleotide bond by either or both of steps cl) and c2) in any sequence:
 cl) capping by reacting with a solid supported capping agent; 
 c2) oxidizing or sulfurizing by reacting the oligonucleotide with a solid supported oxidizing reagent; 
 
       d) removing the 5′-protection group by treatment with a solid supported agent or removing the 5′-protection group with a removal agent followed by addition of a solid supported scavenger; and 
       e) repeating steps a) to d) at least once. 
     
   
   
       2 . The method of  claim 1 , wherein the nucleotide derivative having a 5′-protection group of step b) has the following formula: 
     
       
         
         
             
             
         
       
       wherein
 X is a P(III)-function, 
 B is a heterocyclic base, 
 R 2  is H, a protected 2′-hydroxyl group, F, a protected amino group, an O-alkyl group, an O-substituted alkyl, a substituted alkylamino or a C4′-O2′ methylene linkage, 
 R 5  is a hydroxyl protecting group, a 5′-protected nucleotide, or a 5′-protected oligonucleotide. 
 
     
   
   
       3 . A method for preparing an oligonucleotide comprising the steps of
 a) providing a 5′-protected compound having the formula:   
     
       
         
         
             
             
         
       
       wherein
 B is a heterocyclic base, 
 R 2  is H, a protected 2′-hydroxyl group, F, a protected amino group, an O-alkyl group, an O-substituted alkyl, a substituted alkylamino or a C4′-O2′ methylene linkage, 
 R 3  is OH, NH 2 , R 5  is a hydroxyl protecting group, a 5′-protected nucleotide or a 5′-protected oligonucleotide; 
 
       b) reacting said compound with a nucleotide derivative having a 3′-protection group in the presence of a solid supported activator to give an elongated oligonucleotide with a P(III)-internucleotide bond; 
       c) optionally processing the elongated oligonucleotide with a P(III)-internucleotide bond by either or both of steps cl) and c2) in any sequence;
 cl) capping by reacting with a solid supported capping agent; 
 c2) oxidizing or sulfurizing by reacting the oligonucleotide with a solid supported oxidizing reagent; 
 
       d) removing the 3′-protection group by treatment with a solid supported agent or removing the 3′-protection group with a removal agent followed by addition of a solid supported scavenger; and e) repeating steps a) to d) at least once. 
     
   
   
       4 . The method of  claim 3 , wherein the nucleotide derivative having a 3′-protection group has the following formula: 
     
       
         
         
             
             
         
       
       wherein
 X is a P(III)-function, 
 B is a heterocyclic base, 
 R 2  is H, a protected 2′-hydroxyl group, F, a protected amino group, an O-alkyl group, an O-substituted alkyl, a substituted alkylamino or a C4′-O2′ methylene linkage, 
 R 3  is OR′ 3 , NHR″ 3 , NR″ 3 R′″ 3 , a 3′-protected nucleotide or a 3′-protected oligonucleotide, 
 R′3 is a hydroxyl protecting group, 
 R″ 3 , R′″ 3  are independently an amine protecting group, 
 R′3 is a hydroxyl protecting group, a 3′-protected nucleotide or a 3′-protected oligonucleotide. 
 
     
   
   
       5 . The method of  claim 1 , wherein the nucleotide derivative of step b) is a phosphoramidite or a H-phosphonate. 
   
   
       6 . The method of  claim 1  wherein the solid supported activator of step b) is selected from the group consisting of a solid support bearing a pyridinium salt, a cation exchange solid support with an optionally substituted pyridinium salt, a cation exchange solid support with an optionally substituted imidazolium salt, a solid support bearing an optionally substituted azole (imidazol, triazole, tetrazole), a salt of a weak base anion exchange resin with a strong acid, a weak cation exchange resin (carboxylic) in its protonated form, a solid support bearing an optionally substituted phenol, a solid support bearing a carboxylic acid chloride/bromide, a sulfonic acid chloride/bromide, a chloroformate, a bromoformate, a chlorosulfite, a bromosulfite, a phosphorochloridate, a phosphorbromidate and a solid support bound carbodiimide. 
   
   
       7 . The method of  claim 1 , wherein the solid supported oxidizing reagent is selected from the group consisting of solid supported periodate, permanganate, osmium tetroxide, dichromate, hydroperoxide, substituted alkylamine oxide, percarboxylic acid and persulfonic acid. 
   
   
       8 . The method of  claim 1 , wherein the solid supported oxidizing reagent is a solid supported sulfurization reagent. 
   
   
       9 . The method of  claim 8 , wherein the solid supported sulfurization reagent is selected from the group consisting of a solid supported tetrathionate, a solid supported alkyl or aryl sulfonyl disulfide, a solid supported optionally substituted dibenzoyl tetrasulfide, a solid supported bis(alkyloxythiocarbonyl)tetrasulfide, a solid supported optionally substituted phenylacetyl disulfide, a solid supported N-[(alkyl or aryl)sulfanyl]alkyl or aryl substituted succinimide, and a solid supported (2-pyridinyldithio)alkyl or aryl. 
   
   
       10 . The method of  claim 1 , wherein the solid supported capping agent is a solid supported activated acid, preferably a carboxylic acid chloride, carboxylic acid bromide, azolide, substituted azolide, anhydride or chloroformate or phosphorochloridate, or a solid supported phosphoramidite, or a solid supported H-phosphonate monoester. 
   
   
       11 . The method of  claim 1 , wherein the 5′-protection is a dimethoxytrityl group (DMTr) or a monomethoxytrityl group (MMTr) and the solid supported agent of step d) is a cationic ion exchanger resin in the H +  form or solid supported ceric ammonium nitrate. 
   
   
       12 . A method for preparing an oligonucleotide comprising the steps of
 a) providing a compound having the formula:   
     
       
         
         
             
             
         
       
       wherein
 B is a heterocyclic base, 
 R 2  is H, a protected 2′-hydroxyl group, F, a protected amino group, an O-alkyl group, an O-substituted alkyl, a substituted alkylamino or a C4′-O2′ methylene linkage, 
 R 3  is OR′ 3 , NHR″ 3 , NR″ 3 R′″ 3 , a 3′-protected nucleotide or a 3′-protected oligonucleotide; and R 5  is a P(III)-function, 
 R′ 3  is a hydroxyl protecting group, 
 R″ 3 , R′″ 3  are independently an amine protecting group, or 
 R 5  is a hydroxyl protecting group, a protected nucleotide or a protected oligonucleotide and R 3  is a P(III)-function; 
 
       b) reacting said compound with a nucleotide derivative having a 3′ or 5′-free OH— group in the presence of a solid supported activator to give an elongated oligonucleotide with a P(III)-internucleotide bond; 
       c) optionally processing the elongated oligonucleotide with a P(III)-internucleotide bond by either or both of steps cl) and c2) in any sequence:
 cl) capping by reacting with a solid supported capping agent; 
 c2) oxidizing or sulfurizing by reacting the oligonucleotide with a solid supported oxidizing reagent; 
 
       d) removing the 3′ or 5′-protection group by treatment with a solid supported agent or removing the 5′-protection group with a removal agent followed by addition of a solid supported scavenger; and 
       e) repeating steps a) to d) at least once. 
     
   
   
       13 . The method of  claim 3 , wherein the nucleotide derivative of step b) is a phosphoramidite or a H-phosphonate. 
   
   
       14 . The method of  claim 3  wherein the solid supported activator of step b) is selected from the group consisting of a solid support bearing a pyridinium salt, a cation exchange solid support with an optionally substituted pyridinium, a cation exchange solid support with an optionally substituted imidazolium salt, a solid support bearing an optionally substituted azole (imidazol, triazole, tetrazole), a salt of a weak base anion exchange resin with a strong acid, a weak cation exchange resin (carboxylic) in its protonated form, a solid support bearing an optionally substituted phenol, a solid support bearing a carboxylic acid chloride/bromide, a sulfonic acid chloride/bromide, a chloroformate, a bromoformate, a chlorosulfite, a bromosulfite, a phosphorochloridate, a phosphorbromidate and a solid support bound carbodiimide. 
   
   
       15 . The method of  claim 3 , wherein the solid supported oxidizing reagent is selected from the group consisting of solid supported periodates, permanganates, osmium tetroxides, dichromates, hydroperoxides, substituted alkylamine oxides, percarboxylic acid, and persulfonic acid. 
   
   
       16 . The method of  claim 3 , wherein the solid supported oxidizing reagent is a solid supported sulfurization reagent. 
   
   
       17 . The method of  claim 16 , wherein the solid supported sulfurization reagent is selected from the group consisting of a solid supported tetrathionate, a solid supported alkyl or aryl sulfonyl disulfide, a solid supported optionally substituted dibenzoyl tetrasulfide, a solid supported bis(alkyloxythiocarbonyl)tetrasulfide, a solid supported optionally substituted phenylacetyl disulfide, a solid supported N-[(alkyl or aryl)sulfanyl]alkyl or aryl substituted succinimide and a solid supported (2-pyridinyldithio)alkyl or aryl. 
   
   
       18 . The method of  claim 3 , wherein the solid supported capping agent is a solid supported activated acid, preferably a carboxylic acid chloride, carboxylic acid bromide, azolide, substituted azolide, anhydride or chloroformate or phosphorochloridate, or a solid supported phosphoramidite, or a solid supported H-phosphonate monoester. 
   
   
       19 . The method of  claim 3 , wherein the 3′-protection is a silyl group and the solid supported agent of step d) is a solid-supported ammonium fluoride, or the 3′-protection is levuiinic acid, and the solid supported agent of step d) is a solid supported hydrazine or a solid supported hydrazinium. 
   
   
       20 . A method for sulfurization of an oligonucleotide with a P(III) internucleotide bond comprising the step of oxidizing with a solid supported sulfurization agent consisting of solid supported amine and a tetrathionate having the formula S 4 O 6   −2  or a cyanoethylthiosulfate having the formula (NC—CH 2 —CH 2 —S—SO 3   − ).