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. 

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) 

O3’-tert.-Butyldimethylsilylthymidine (5) (6 g, 1.0 equiv.) was dissolved in pyridine (50 ml). Trichloroacetyl chloride 

## Analytical HPLC Methods for Synthetic Oligonucleotides 

## EXAMPLES 

## General Procedures 

## Example 1 

(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). 

Synthesis of 03 ’-tert. -butyidimethy lsily 1-O5’-ethoxy› dicarbonylthymidine, EtOX-dT-Si (6b) 

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). 

Synthesis of Oxalic Acid Benzyl Ester Chloride 

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. 

Synthesis of O5’-benzyloxydicarbonyl-O3’-tert.-bu› tyidimethylsilylthymidine, BnOX-dT-Si (6c) 

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). 

Synthesis of Oxalic Acid Cycclohexyl Ester Chloride 

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. 

