Method for preparing oligonucleotides

Abstract

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 R2 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′methylen linkage R3 is OR′3, NHR″3, NR″3R′″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-proctection 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 c1) and c2) in any sequence c1) capping preferably by reacting with a solid supported capping agent c2) oxidizing preferably by reacting the oligonucleotide with a solid supported oxidizing reagent d) removing the 5′-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′methylen 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′-proctection 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 c1) and c2) in any sequence 
 c1) capping preferably by reacting with a solid supported capping agent  
 c2) oxidizing preferably 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.    e) repeating steps a) to d) at least once.    
   
   
       2 . The method of  claim 1 , wherein the nucleotide derivative having a 5′-proctection 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′methylen 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′methylen 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′-proctection 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 c1) and c2) in any sequence    c1) capping, preferably by reacting with a solid supported capping agent    c2) oxidizing, preferably 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.    e) repeating steps a) to d) at least once.    
   
   
       4 . The method of  claim 3 , wherein the nucleotide derivative having a 3′-proctection 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′methylen linkage  
       R 3 ═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, 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 periodates, permanganates, osmium tetroxides, dichromates, hydroperoxides, substituted alkylamine oxides, percarboxylic acid and persulfonic acid.  
   
   
       8 . The method of  claim 1 , wherein the oxidizing is a sulfurization.  
   
   
       9 . The method of  claim 8 , wherein the solid supported oxidizing 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(akyloxythio-carbonyl)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 an cationic ion exchanger resin in the H +  form or solid supported ceric ammonium nitrate.  
   
   
       12 . The method of  claim 1 , wherein the 3′-protection is a silyl group and the solid supported agent of step d) is an anionic ion exchanger resin in the F-form or the 3′-proctection is levulinic acid and the solid supported agent of step d) is a solid supported hydrazine or a solid supported hydrazinium.  
   
   
       13 . (canceled)  
   
   
       14 . 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′methylen linkage and    R 3  is OR′ 3 , NHR″ 3 , NR″ 3 R′″ 3 ,    a protected nucleotide or a 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(II)-internucleotide bond    c) optionally processing the elongated oligonucleotide with a P(III)-internucleotide bond by either or both of steps c1) and c2) in any sequence    c1) capping by reacting with a solid supported capping agent    c2) oxidizing 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.    e) repeating steps a) to d) at least once.    
   
   
       15 . The method of  claim 3 , wherein the nucleotide derivative of step b) is a phosphoramidite or a H-phosphonate.  
   
   
       16 . 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.  
   
   
       17 . 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.  
   
   
       18 . The method of  claim 3 , wherein the oxidizing is a sulfurization.  
   
   
       19 . The method of  claim 18 , wherein the solid supported oxidizing 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(akyloxythio-carbonyl)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.  
   
   
       20 . 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.  
   
   
       21 . The method of  claim 3 , wherein the 5′-protection is a dimethoxytrityl group (DMTr) or a monomethoxytrityl group (MMTr) and the solid supported agent of step d) is an cationic ion exchanger resin in the H +  form or solid supported ceric ammonium nitrate.  
   
   
       22 . The method of  claim 3 , wherein the 3′-protection is a silyl group and the solid supported agent of step d) is an anionic ion exchanger resin in the F-form or the 3′-proctection is levulinic acid and the solid supported agent of step d) is a solid supported hydrazine or a solid supported hydrazinium.  
   
   
       23 . 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 a solid supported amine and a tetrathionate having the formula S 4 O 6  or a cyanoethylthiosulfate (NC—CH 2 —CH 2 —S—SO 3   − ).