US 8,138,330 B2  32  31  The present invention can also be applied for the synthesis  of deoxyribooligo-nucleotides or ribooligonucleotides with  modifications in their nucleoside sugar or phosphate groups.  For instance, 2’-O-alkyl modified, e.g. 2’-O-methyl modified  oligonucleotides, or 2’-fluoro modified oligonucleotides can  be prepared through the use of respective 2’-0 alkyl or  2’-deoxy-2’-fluoro phosphoramidites with 5’-acyl groups, and  phosphorothioate deoxyribonucleosides can be prepared  through the use of a thioating reagent in the phosphite oxida› tion step of the synthesis cycles with 5’-acyl phosphoramid- ites. The invention is, however, not limited to the above modi› fications, but can be applied to the preparation of other  oligonucleotides modified in their sugar or phosphate groups  known to those skilled in the art and reviewed by Micklefield  (2001) Current Medicinal Chemistry 8:1157-1179.  In conclusion, the application of 5’-acyl phosphoramidites  (2) and (3) in the solid phase synthesis of oligonucleotides  with phosphoramidites represents a versatile method for the  synthesis of oligonucleotides which overcomes several limi› tations of the prior art. The cleavage of the front end protec› tive group is irreversible and fast in this method and the  by-products, which are primary amides or esters, are unreac› tive to the oligonucleotide product. Furthermore, the depro› tection reagents for the cleavage of the acyl groups are easy to  prepare and inexpensive. The application oflarge volumes of  the front end deprotection reagents as in the conventional  synthesis with 5’-DMT protected phosphoramidites is not  required, which reduces the cost of the synthesis and also  reduces the generation of hazardous chemical waste. The  methods of the invention improve the quality of the prepared  oligonucleotides due to irreversibility of the front end depro- tection step in the synthesis cycles, which reduces the forma› tion of deletion sequence side products, due to the avoidance  of repeated exposure of the growing oligonucleotide chain on  the support to strong acids, which prevents the formation of  depurination side products during chain assembly, and due to  the avoidance of premature loss of front end protective groups  during coupling reactions, which prevents the formation of  insertion sequence side products.  Method A (anion exchange): Dionex DNAPac PAl00 col› unm ( 4x250 mm), eluted with a linear gradient from 20% B to  80% B in 30 min. followed by a linear gradient from 80% B  to 100% B in 3 min. with a flow rate of 1.0 ml/min., detection  at A=260 nm, buffer A 25 mM Tris base/I mM EDTA, pH 8,  buffer B 25 mM Tris base/I mM EDTA/1 M NaCl, pH 8.  Method B (reversed phase): Waters XTerra MS C18 column  (2.5  µm, 4.6x50 mm), eluted with a linear gradient from 0%  to 67% Bin 40 min. with a flow rate of0.5 ml/min., detection  at A=260 nm, buffer A 100 mM triethylammonium acetate  (TEAA), pH 7 containing 5% acetonitrile, buffer B 100 mM  triethylammonium acetate (TEAA), pH 7 containing 30%  acetonitrile.  O3’-tert.-Butyldimethylsilylthymidine (5) (6 g, 1.0 equiv.)  was dissolved in pyridine (50 ml). Trichloroacetyl chloride  Synthesis of 5’-acyl-3 ’-tert. -butyidimethy lsily lthy› midines (6a-f)  Synthesis of 03 ’-tert. -butyidimethylsily 1-O5’-trichlo› roacety lthymidine, TCA-dT-Si (6a)  A precooled solution ofoxalyl chloride (316.8 g, 2.5 mo!)  in dichloromethane ( 400 ml) was added to a cooled solution  (ice bath) of cyclohexanol (50.0 g, 0.5 mo!) in dichlo› romethane (100 ml). The mixture was stirred at 0°  C. for 30  min, then allowed to warm to ambient temperature. The reac› tion was continued overnight. The mixture was concentrated  on a rotary evaporator and the residue was purified by frac- tionated distillation under reduced pressure. The product was  collected as the main fraction (68.5 g, 72%). bp 59°  C./1.6  mbar; nmo 1.462.  03’-tert.-Butyldimethylsilylthymidine (5)  (20  g,  1.0  equiv.) was dissolved in pyridine (100 ml). Commercially  available oxalic acid ethyl ester chloride (1.1 equiv.) was  added with cooling (ice bath) and the reaction was continued  at ambient temperature for 1 hour. Ethanol ( 4 ml) was added  and the reaction mixture was evaporated to a syrupy residue.  The residue was dissolved in dichloromethane (150 ml) and  the resultant solution was extracted with 5% sodium hydro› gencarbonate solution (3 times 150 ml). The organic phase  was dried over sodium sulfate and evaporated to a foam. The  foam was coevaporated with THF and dissolved in dichlo› romethane (65 ml). The solution was added to a mixture of  hexanes (750 ml) and diethylether (85 ml) with stirring at  ambient temperature. The resulting precipitate was collected  by filtration. Yield of pure (99.8%) EtOX-dT-Si (6b) (22 g,  86% yield).  Oxalyl chloride (17 .8 g, 5 equiv.) was dissolved in dichlo› romethane (25 ml). Benzyl alcohol (3.0 g, 1 equiv.) was added  dropwise as a solution in dichloromethane (10 ml) with stir› ring at ambient temperature. The mixture was stirred for 1  hour at ambient temperature and concentrated to a viscous  residue by evaporation to afford the crude product which was  used in subsequent reactions without further purification.  O3’-tert.-Butyldimethylsilylthymidine (5) (5 g, 1.0 equiv.)  was dissolved in pyridine (20 ml). Oxalic acid benzyl ester  chloride (1.3 equiv.) was added with cooling (ice bath) and the  reaction was continued at ambient temperature for 1 hour.  Ethanol ( 0. 5 ml) was added and the mixture was evaporated to  a syrupy residue. The residue was dissolved in dichlo- romethane and the resultant solution was extracted with 5%  sodium hydrogencarbonate solution (3 times). The organic  phase was dried over sodium sulfate and evaporated to a  foam. Purification by colunm chromatography on silica gel  gave pure (98.7%) BnOX-dT-Si (6c) (5.9 g, 81 % yield).  (1.1 equiv.) was added with cooling (ice bath) and the reaction  was continued at ambient temperature for 1 hour. The mixture  was concentrated to a syrupy residue by evaporation. The  residue was dissolved in dichloromethane (100 ml) and the  resulting solution was extracted with water (3 times 100 ml).  The organic phase was dried over sodium sulfate and evapo› rated to a foam. Purification by colunm chromatography on  silica gel gave pure (98.9%) TCA-dT-Si (6a) (5.6 g, 66%  yield).  EXAMPLES  General Procedures  Synthesis of 03 ’-tert. -butyidimethy lsily 1-O5’-ethoxy› dicarbonylthymidine, EtOX-dT-Si (6b)  Synthesis of Oxalic Acid Benzyl Ester Chloride  Synthesis of O5’-benzyloxydicarbonyl-O3’-tert.-bu› tyidimethylsilylthymidine, BnOX-dT-Si (6c)  Synthesis of Oxalic Acid Cycclohexyl Ester Chloride  Analytical HPLC Methods for Synthetic  Oligonucleotides  Example 1 