US 8,138,330 B2  12  11  groups known to those skilled in the art, and may also contain  optional ether groups. The alkyl groups may also contain  additional amino groups which may be primary, secondary or  tertiary amino groups. Examples of primary amines as used  herein include, but are not limited to n-butylamine, tert.› butylamine, n-hexylamine, 2-methoxyethylamine, ethylene  diamine,  2-(N,N-diethylamino )ethylamine,  cyclohexy› lamine and the like. Examples of secondary amines as used  herein include, but are not limited to diethylamine, di-n› propylamine, 1,2-dimethylethylene diamine and the like. The  alky 1 groups R and R’ in secondary amines may or may not be  identical.  The term "solid support" as used herein refers to a polymer,  that is insoluble in the media employed in the reaction steps  performed to synthesize oligonucleotides, and is derivatized  to comprise hydroxyl groups. The polymer can be an inor› ganic polymer, including, but not limited to silica, alumina  and controlled pore glass (CPG), or an organic polymer,  including, but not limited to polystyrene, polyacrylamide,  polymethacrylate, polyvinylalcohol, or other synthetic poly› mers, carbohydrates such as cellulose and starch or other  polymeric carbohydrates, or other organic polymers and any  copolymers, composite materials or combination of the above  inorganic or organic materials. The hydroxy groups of a solid  support may be unprotected or protected, e.g. with a DMT› protective group, or with another hydroxyl group protective  group. During oligonucleotide synthesis a solid support is  treated with various reagents in several synthesis cycles to  achieve the stepwise elongation of a growing oligonucleotide  chain with individual nucleotide units. The nucleoside unit at  the end of the chain which is directly linked to the solid  support is termed "the first nucleoside" as used herein. The  first nucleoside is bound to the solid support via a linker  moiety, i.e. a diradical with covalent bonds to both the poly› mer of the solid support and the nucleoside. The linker stays  intact during the synthesis cycles performed to assemble the  oligonucleotide chain and is cleaved after the chain assembly  to liberate the oligonucleotide from the support.  The term "acidic reagent" as used herein refers to an acidic  solution. Acidic solutions in this context may be aqueous  solutions with a pH ofless than 7, e.g. the solution of a protic  acid in water or a buffer solution with a pH ofless than 7, or  non-aqueous solutions, e.g. the solution of an acid in an  organic solvent. Examples of acidic reagents include, but are  not limited to diluted mineral acids, e.g. 0.01 M hydrochloric  acid in water or 0.01 M sulfuric acid in water, mixtures of  carboxylic acids with water, e.g. 80% acetic acid in water or  40% formic acid in water, aqueous citrate buffers, e.g. citrate  buffer pH 3, aqueous acetate buffer, e.g. acetate buffer pH 4,  solutions of dichloroacetic acid in organic solvents, e.g. 3%  dichloroacetic acid in dichloromethane or toluene, solutions  of acetic acid in dichloromethane, e.g. 10% acetic acid in  dichloromethane, and the like.  The term "acid labile linker" as used herein refers to a  linker moiety which is susceptible to cleavage with an acidic  reagent.  The term "2’-protective group" as used herein refers to a  protective group applied to the 2’-hydroxy group of ribo› nucleosides. In particular, the term 2’-protective group refers  to the protective group R$_{2 }$for nucleoside phosphoramidites in  formulae (2) and (3). The cleavage of2’-protective groups is  orthogonal to the cleavage of front end protective groups in an  oligonucleotide synthesis, i.e. they stay intact during the syn- thesis cycles on the solid support and are collectively  removed after the chain assembly. Preferably, the 2’-protec- In a preferred embodiment of the invention the nucleobase  protective groups, if present, are cleavable after the assembly  of the respective oligonucleotides with an acidic reagent. In  another embodiment of the invention one or more of the  nucleobase protective groups is cleavable under neither  The present invention discloses novel nucleoside phos› phoramidites which are useful for the synthesis of oligonucle- otides. The novel phosphoramidites of the invention are rep› resented by formulae (2) and (3) wherein R$_{1 }$ denotes an acyl  group as defined above. The novel phosphoramidites are fur› ther characterized therein that the acyl group R$_{1 }$ is cleavable  in a synthesis cycle of the phosphoramidite mediated synthe- sis of oligonucleotides on solid supports with an organic base  such as a primary amine, a secondary amine, or a mixture  thereof.  Phosphoramidites which are applied in the coupling step of  an oligonucleotide synthesis cycle are typically monomer  phosphoramidites, i.e. they contain one nucleoside unit as  displayed in formulae (2) and (3).  It has nevertheless been  demonstrated that suitably constructed dimer phosphoramid› ites, i.e. phosphoramidites derived from dinucleotides as  described by Kumar et al. (1984) J. Org. Chem. 49:4905-12,  or trimeric phosphoramidites, i.e. phosphoramidites derived  from trinucleotides as described by Ono et al., Nucleic Acids  Res. 23, 4677-82 (1995), can be applied successfully in a  phosphoramidite mediated synthesis of oligonucleotides on a  solid support. The synthesis cycles for monomeric, dimeric,  trimeric and possibly higher oligomeric phosphoramidites  are nevertheless very similar to each other, if not identical,  provided the type of employed phosphoramidite as defined  through the nature of the groups Ri, R$_{2}$ ,  R$_{3}$ ,  R$_{3}$ ’  and R$_{4 }$ in (2)  and (3) is identical. Any feature of the present invention  described in this disclosure for the use of monomeric phos› phoramidites is therefore directly applicable to the use of  dimeric, trimeric or higher oligomeric phosphoramidites in  the synthesis of oligonucleotides on solid supports. The appli› cation of dimeric, trimeric or higher oligomeric phosphora- midites in the synthesis of oligonucleotides on solid supports  is consequently well within the breath and scope of the inven› tion.  tive groups are removed simultaneously with the cleavage of  the linker to the support and with the removal of nucleobase  protective groups.  The extension of an oligonucleotide chain during a phos- phoramidite mediated synthesis on a solid support is typically  pursued in the 3’ to 5’ direction with phosphoramidites (2).  During synthesis with (2) the front end protective group is  removed from the 5’-position and the subsequent coupling  occurs between a support-bound 5’-hydroxy group and a  3’-phosphoramidite group of (2). The extension of the oligo› nucleotide chain may alternatively be pursued in the 5’ to 3’  direction with phosphoramidites (3). During synthesis with  (3) the front end protective group is removed from the 3’-po› sition and the subsequent coupling occurs between a support- bound 3’-hydroxy group and a 5’-phosphoramidite group of  (3). The latter approach is exemplified in the synthesis of  oligodeoxynucleotides with 3’-DMT protected deoxynucleo› side 5’-phosphoramidites, as described by e.g. Robles et al.  (1995) Nucleic Acids Res. 23:4151-61 (1995). The synthesis  cycles for the chain elongation in 3’ to 5’ direction and the  chain elongation in 5’ to 3’ direction are nevertheless very  similar, if not identical for a given type of employed phos› phoramidites. Any feature of the present invention described  in this disclosure for the chain elongation in 3’ to 5’ direction  is therefore directly applicable to chain elongation in 5’ to 3’  direction and chain elongation in 5’ to 3’ direction is conse› quently well within the breath and scope of the invention. 