Title of Invention	An Improved processes for the preparation of an improved support for the synthesis of oligodeoxyribo and oligorbonucleotides
Abstract	The process provides a polymer support containing an organic molecule having a cis-diol group where one hydroxyl group is attached via a covalent linkage and the other hydroxyl group is protected by an acid liable group. The process also relates to the cleavage of oligodeoxyribo and oligoribonucleotides from the universal support and simultaneously removal of protecting groups from exocyclic amino functionalities of nucleic bases.

Title of Invention

An Improved processes for the preparation of an improved support for the synthesis of oligodeoxyribo and oligorbonucleotides

Abstract

The process provides a polymer support containing an organic molecule having a cis-diol group where one hydroxyl group is attached via a covalent linkage and the other hydroxyl group is protected by an acid liable group. The process also relates to the cleavage of oligodeoxyribo and oligoribonucleotides from the universal support and simultaneously removal of protecting groups from exocyclic amino functionalities of nucleic bases.

Full Text	The present invention relates to a process for the preparation of an improved support for the synthesis of oligodeoxyribonucleotides and oligoribonucleotides. The polymer support containing an organic molecule having a cis-diol group where one hydroxyl group is attached via a covalent linkage and the other hydroxyl group is protected by an acid labile group. The process also rexates to the cleavage of oligo-deoxyribo- and oligoribonucleotides from the. universal support and simultaneously removal of protecting groups from exocyclic amino functionalities of nucleic bases and (2-cyanoethyl from internucleo-tidic phosphate functions. The improved universal polymer support of formula II accompanying the specifications wherein P represents a polymer support bearing aminoalkyl or hydroxyl functionalities and R stands for an organic substance bearing a pair of cis hydroxyl groups where one of the hydroxyl function is coupled to aminoalkyl or hydroxyl function via a succinate linkage. The other hydroxyl function of the organic substance was protected with monomethoxytrityl or 4,4'-dimethoxytrityl chloride. The support was employed for the synthesis of oligodeoxyribo- and oligoribonucleotides in an automated DNA synthesizer following phosphoramidite chemistry. The cleavage of oligonucleotide from the support and other protecting groups were removed in analogous way to the standard method. The known protecting groups of formula I, shown in the drawing accompanying the specification represented by figure I, employed, viz., conventional (set I) and labile (set II -IV) for exocyclic amino protection in nucleosides, are described below. Set I (Conventional Protecting Group) B = Adenine; R = Benzoyl (Bz) B = Cytosine; R = Benzoyl ( Bz) or acetyl (Ac) B = Guanine; R = Isobutyryl (Ibu) Set II(Labile Protecting Group) B = Adenine; R = Phenoxyacetyl (Pac) B = Cytosine; R = Isobutyryl (Ibu) or Acetyl(Ac) B = Guanine; R = Phenoxyacetyl (Pac) Set III(Labile Protecting Group) B = Adenine; R = Dimethylformamidine (DMF) B = Cytosine; R = Dimethylformamidine (DMF) B = Guanine; R = Dimethylformamidine (DMF) Set IV(Labile Protecting Group) B = Adenine; R = p-tert-Butylphenoxyacetyl B = Cytosine; R = p-tert-Butylphenoxyacetyl B = Guanine; R = p-tert-Butylphenoxyacetyl Synthetic oligonucleotides (oligodeoxyribo- and oligoribonucleo-tide) have become indispensable tools in modern biological sciences. The protocols for chemical synthesis and modifications of these molecules have been simplified to an extent that even a non-chemist, the actual user can synthesize these molecules without much difficulties. However, the support functionalization has been one of the time consuming tasks. Moreover, one still requires to use at least eight different (four for oligodeoxyribo- and four for oligoribonucleotides) supports, each bearing a separate nucleoside corresponding to the 3'-terminus of the desired oligomer. This number may even be more when 3'-terminus of the desired oligomer contains nucleotide other than the normal one. Though the time of functionalization of polymer supports required for oligonucleotide synthesis has been reduced considerably in the recent publications (Kumar et al., Nucleosides and Nucleotides, 15, 1996, 879), one still needs at least eight different supports for all kinds of oligonucleotides. Some attempts have been made to develop universal supports to eliminate the use of eight different supports. Gough et al. (Gough et al., Tet. Lett. 24, 1983, 5321) have proposed the use of a single universal support that consisted of controlled pore glass derivatized with 2'(3')-O-benzoyluridine 5'-0-succinyl residue and demonstrated its applications for the synthesis of oligodeoxyribo- and oligoribonucleotides via phosphotriester or the phosphite approach. The cleavage of oligodeoxyribo- and oligoribonu- o cleotides was effected by the use of aq. ammonia for 24h at 60-65 C o and aq. ammonia-pyridine (2:1) for 16h at 50 C, respectively in sealed vials. The universal support contains a nucleosidic material which does not get incorporated in the oligonucleotide chain and goes waste. The other serious limitations of this support are that the deprotection time is considerably longer in comparison to the time required in the standard protocol and the functinalization of the support involves multistep (4 steps) synthesis and purification procedure. In a recent publication, they (Gough et al., Tet. Lett., 36, 1995, 27) have further modified the functinalization of the universal support by the use of an adapter, 2'(3')-0-benzoyluridine-5'-0-(cya-noethyl- N,N-diisopropylphosphoramidite) and coupled it to a standard T-support, following phosphoramidite chemistry. The functionalized support was subsequently used for the synthesis of oligonucleotides. The deprotection of oligodeoxyribo- and oligoribonucleotides was achieved by the treatment of the support with 25 ml aq. ammonia for o 48h at 65 C in a pressure vial and 25 ml aq. ammonia-pyridine mix- o tare (4:1, v/v) for 24h at 50 C in a pressure vial, respectively. However, the universal support has still the following serious limita- tions: The support contains two nucleosidic units (expensive material) which do not get incorporated in the oligonucleotide chain and wasted. The adapter used for the functionalization of universal support involves multistep synthesis and purification strategy and yield of the final product is low, making the support functionalization a time consuming (2-3d), tedious and expensive task. Moreover, the time required for the deprotection of oligonucleotides is exhorbitantly high, almost three times to the time required in currently followed synthesis and deprotection protocol. In fact the time required for the deprotection of oligonucleotides is so much that one can afford to prepare all the eight different supports even in lesser time. In an elegant study Gough and co-workers described that an oligodeoxynucleotide having 3'-terminal protection with a ribonucleo-side linked by its 2'- or 3'-hydroxyl group to a 3'-phosphate is removed as its 2',3'-cyclic phosphate by lead cation. In this process oligodeoxynucleotide gets removed with 3'-hydroxyl function. This concept led Gough and co-workers developing two universal supports. Both of the universal, supports contain uridine as the terminal ribonucleoside providing a pair of cis-diol groups. Because of the rigid rotation of these hydroxy1 groups, oligodeoxyribo- and oligori-bonucleotides take considerable time (48h) for the cleavage from the universal supports. This problem might be taken care of if uridine, a source of cis diol groups is replaced by an organic molecule providing a pair of cis diol groups with free rotation. The free rotation of cis diol groups will help in rapid formation of cyclic phosphate in the presence of lead cation or alkaline conditions and hence will lead in rapid deprotection of oligodeoxyribo- and oligoribonucleotides from the universal support. Therefore, there is a need to develop a universal support suitable for the synthesis of oligodeoxyribo- and oligoribonucleotides and compatible to the existing methods of synthesis and deprotection (cleavage of oligodeoxyribo- and oligoribonucleotides from the universal support and simultaneously removal of protecting groups, labile or conventional from exocyclic amino functionalities of nucleic bases and (2-cyanoethyl from internucleotidic phosphate functions) of all possible oligodeoxyribo- and oligoribonucleotides. The main objective of the present invention is therefore to provide an improved process for the preparation of an improved universal polymer support for the synthesis of oligodeoxyribo- and oligoribonucleotides. Another objective of the present invention is to provide a process for the preparation of an improved universal polymer support devoid of any nucleosidic material, suitable for the synthesis of all possible oligodeoxyribo- and oligoribonucleotides Yet another objective of the present invention is to provide a process for the preparation of an improved universal polymer support compatible to the existing methodologies of synthesis and deprotection (cleavage of oligodeoxyribo- and oligoribonucleotides from the universal support and simultaneously removal of protecting groups, labile or conventional from exocyclic amino functionalities of nucleic bases and (2-cyanoethyl from internucleotidic phosphate functions) of all possible oligodeoxyribo- and oligoribonucleotides. Still another objective of the present invention is to provide a process for the preparation of an improved universal polymer support employing commonly available reagents. Accordingly, the present invention provides a process for the preparation of an improved support for the synthesis of oligodeoxyribonucleotides and oligoribonucleotides which comprises: (i) treating an organic molecule (5 mmol) having a pair of cis hydroxyl groups selected from a group a organic molecule comprising butane -2,3-diol, 1,4-anhydro-erythritol, D-ribose with 4, 4' - dimethoxytrityl chloride ( 2 mmol) for a period of 5 h and isolating a monosubstituted tritylated derivative from the above reaction mixture by stirring said reaction mixture with 5% aqueous sodium hydrogen carbonate for 10 min followed by concentrating the reaction mixture to get a syrupy liquid , dissolving the obtained syrupy liquid in ethyl acetate to get an organic layer and getting the said monosubstituted derivative by concentrating and drying the obtained organic layer, (ii) treating the monosubstituted derivative obtained in step (i) with one equivalent of a homobifunctional alkanoic acid chloride as defined herein , for a period period of 1 h , and transferring the mixture to a polymer support such as herein described having a hydroxyl or aminoalkyl functionalities, (iii) treating the polymer support obtained in step (ii) with dry alkanol as defined herein , followed by washing with dry alkanol and dialkyl ether, to get the desired improved polymer support represented by figure II of the drawing accompanying the specification wherein DMTr represents 4,4-dimethyl group, R represents an organic molecule bearing a pair of cis hydroxyl group as defined above and P represents a polymer support bearing aminoalkyl or hydroxyalkyl functions. The organic molecule having a pair of cis hydroxyl groups (both are at secondary positions) used for making monosubstituted tritylated derivative may be selected from butane-2,3-diol, 1,4-anhydro-erythritol, D-ribose and the like. The homobifunctional alkanoic acid chloride used in step (ii) may be selected from succinyl chloride, oxalyl chloride and the like. The polymer support employed in step (ii) may be selected from controlled pore glass (CPG) with variable pore size and linker arm, silica gel (porous or non-porous), cross-linked polystyrene having hydroxyl or aminoalkyl functions and the like. The alkanol used in step (iii) for capping purpose may be selected from methanol, ethanol and the like. The improved polymer support is then used for the synthesis of oligonucleotides (deoxyribo- and ribo-) which comprises (i) synthesizing oligodeoxyribo- and ribonucleotides on the polymer support proposed by the process following phosphoramidite chemistry using conventional or labile protecting groups for nucleic bases, (ii) treating the resultant oligodeoxynucleotide and oligoribo-nucleotides embedded in the support with aq. ammonia solution or aq alkylamine-ammonia mixture (1:1) at the temperature range o of ambient to 65 C. (iii)concentrating the resultant reaction mixture and dissolving the solid residue in water and subjecting to gel filtration to remove low m.w. impurities, (iv)concentrating the eluent obtained in step (iv) under vacuum concentrator and subjecting to high performance liquid chromatography (HPLC) to obtain the fully deprotected oligodeoxyribo- and ribonucleotides. The 2/-deoxyribonucleosidephosphoramidite and ribonucleosidephos-phoramidite used for the synthesis of oligodeoxyribo- and ribonucleotides in step (iv) carrying conventional protecting groups may be selected from benzoyl for adenine and cytosine and isobutyryl or acetyl for guanine or labile protecting groups may be selected from phenoxyacetyl for adenine and guanine and isobutyryl or acetyl for cytosine; dimethylformamidine (DMF) for adenine, guanine and cytosine; p-tert-butylphenoxyacetyl- for adenine, guanine and cytosine and the like for exocyclic amino functions of nucleic bases. The cleavage of oligodeoxyribo- and oligoribonucleotides from the universal support and simultaneously removal of protecting groups, labile or conventional from exocyclic amino functionalities of nucleic bases and (-cyanoethyl from internucleotidic phosphate functions) of all possible oligodeoxyribo- and oligoribonucleotides may be achieved in step (v) by the treatment with ag. ammonia or aq. ammonia-methylamine at the temperature renge to ambient to o 65 C (for oligodeoxyribonucleotides) and aq. ammonia-pyridine at o 50 C or aq. ammonia-methylamine at the temperature range of o ambient to 50 C for oligoribonucleotides. The following examples concerning the use of universal support for the synthesis of oligodeoxynucleotides and oligoribonucleotides and the cleavage of oligodeoxyribo- and oligoribonucleotides from the universal support and simultaneously removal of protecting groups (conventional and labile) from exocyclic amino functionalities of nucleic bases and (2-cyanoethyl from internucleotidic phosphate functions are provided to illustrate the invention and should not be construed to limit the scope of the invention. Example 1 The preparation of universal support comprises of two steps which are described below. In a 25 ml round bottomed flask, dl-butan-2,3-diol (5 mmol) was dried by coevaporation with pyridine (15 ml) and finally suspended in anhydrous pyridine (15 ml). 4,4'-Dimethoxytrityl chloride (2 mmol) was added and the reaction mixture was left for stirring at room, temperature. After 5h, 5% aq. sodium hydrogen carbonate (2 ml) was added and stirred for further 10 min. Then the reaction mixture was concentrated on a rotary evaporator and the syrupy material so obtained was redissolved in ethyl acetate (70 ml) and washed with aq. sodium hydrogen carbonate solution (2 x 25 ml) and water (1 x 25 ml), respectively. The organic layer was collected, dried over anhydrous sodium sulfate, concentrated and dried on an evaporator under reduced pressure to obtain, 2(3)-(4,4'-dimethaxytri-tyl)-butan-3(2)-diol, in 90% yield and fully characterized by its NMR, IR and MS spectra. In the second step, 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-diol was attached to the long chain aminoalkylated controlled pore glass (LCAA-CPG) by following procedure. Succinoyl chloride (6.3 (1, 0.05 mmol) was added to a solution of triazole (19.8 mg, 0.28 mmol) in an anhydrous mixture of pyridine (500(1) and acetonitrile (1.0 ml) taken in a septum sealed vial. To the solution was injected 2(3)~(4,4'-dimethoxytrityl)-butan-3(2)-diol (0.05 mmol) in acetonitrile (1 ml). The reaction mixture was agitated at room temperature for lh and then LCAA-CPG (200 mg) added and agitation continued at room temperature. After 15 min., the contents of the vial were transferred to sintered disc glass funnel, excess reagent washed off, residual triazolosucci-nyl groups on the polymer support were capped with anhydrous methanol (1 ml). After washing with dry acetonitrile (3x5 ml), residual amino groups were blocked following standard procedure (Matteucci, M.D. and Caruthers, M.H., J.Am.Chem.Soc., 103 (1981) 3185). The polymer support so obtained was used for oligonucleotide synthesis. The synthesized oligomers from the support and protecting groups from nucleic bases and phosphate groups were removed by the treatment of ag. ammonia for o 16h at 60 C. The fully deprotected oligomers were compared with oligomers synthesized on standard supports on HPLC, FPLC (Fast protein liguid chematography) and by biological activity. Example 2 In a 25 ml round bottomed flask, d-butan-2,3-diol (5 mmol) was dried by coevaporation with pyridine (15 ml) and finally suspended in anhydrous pyridine (15 ml). 4,4'-Dimethoxytrityl chloride (2 mmol) was added and the reaction mixture was left for stirring at room temperature. After 5h, 5% ag . sodium hydrogen carbonate (2 ml) was added and stirred for further 10 min. Then the reaction mixture was concentrated on a rotary evaporator and the syrupy material so obtained was redissolved in ethyl acetate (70 ml) and washed with ag. sodium hydrogen carbonate solution (2 x 25 ml) and water (1 x 25 ml), respectively. The organic layer was collected, dried over anhydrous sodium sulfate, concentrated and dried on an evaporator under reduced pressure to obtain, 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-diol, in 85% yield and fully characterized by its NMR, IR and MS spectra. The compound, 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-diol, (0.1 mmol) was reacted with succinic anhydride (0.2 mmol) in dichloroethane (4 ml) in the presence of 4-dimethyIaminopyridine (50 umol) and a o tertiary base, triethylamine (0.1 mmol) at 50 C for 20 min. After stipulated time (20 min), the reaction mixture was diluted with dichloroethane (25 ml) and washed with 5% cold ag. citric acid (2x 15 ml) and water (2x 10 ml), respectively. The organic phase was collected and concentrated to obtain 2-(4,4'-dimethoxytrityloxy)-l-methylpro-pyl-succinate in 93% yield. 2-(4,4'dimethoxytrityloxy)-1-methylpropyl- succinate (0.05 mmol) and 4-dimethylaminopyridine (10 umol) were dissolved in pyridine (2 ml) and added LCAA-CPG (200 mg) followed by dicyclohexylcarbodiimide (0.075 mmol) under anhydrous conditions. The resulting mixture was shaken occasionally at room temperature for 16h. Then the mixture was transferred to a sintered disc glass funnel and washed off the excess reagents with pyridine (1 x 10 ml), methanol (3 x 15 ml) and diethylether (2 x 10 ml). The washed support was dried under vacuum and capped the residual amino groups on the polymer support following standard procedure. The resulting functionalized support was used oligonucleotide synthesis using labile protecting groups containing nucleoside phosphoramidite. The cleavage of oligomer chain from the support and removal of protecting groups were achieved o by aq. ammonia (29%) treatment for 6h at 60 C. The fully deprotected oligomers were desalted and analyzed on HPLC and FPLC. Then the oligomers were compared with the standard oligomers synthesized on the commercially available conventional polymer supports. They were found to be identical in all respect, viz., retention times on HPLC as well as UV pattern. Example 3 In a 25 ml round bottomed flask, l-butan-2,3-diol (5 mmol) was dried by coevaporation with pyridine (15 ml) and finally suspended in anhydrous pyridine (15 ml). 4,4'-Dimethoxytrityl chloride (2 mmol) was added and the reaction mixture was left for stirring at room temperature. After 5h, 5% aq. sodium hydrogen carbonate (2 ml) was added and stirred for further 10 min. Then the reaction mixture was concentrated on a rotary evaporator and the syrupy material so obtained was redissolved in ethyl acetate (70 ml) and washed with. aq. sodium hydrogen carbonate solution (2 x 25 ml) and water (1 x 25 ml), respectively. The organic layer was collected, dried over anhydrous sodium sulfate, concentrated and dried on an evaporator under reduced pressure to obtain, 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-diol, in 88% yield and fully characterized fay its NMR, IR and MS spectra. In the next reaction, 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-dial, (0.1 mmol) was reacted with succinic anhydride (0.2 mmol) in dichloroethane (4 ml) in the presence of 4-dimethylaminopyridine o (0.05 mmol) and a tertiary basts, triethylamine (0.1 mmol) at 50 C for 20 min. After stipulated time, the reaction mixture was diluted with dichloroethane (25 ml) and washed, with 5% cold aq. citric acid (2 x 15 ml) and water (2 x 10 ml), respectively. The organic phase was collected and concentrated to obtain 2-(4,4'-dimethoxytrityloxy)-1-methyl- propylsuccinate in 93% yield. To the mixture of 2-(4,4'-dimethoxytrityloxy)-1- methylpropylsucci-nate (0.05 mmol), 2,2'-dithiobis(5-nitropyridine) (DTNP) (0.05 mmol) and 4-dimethylaminopyridine (0.05 mmol) dissolved in acetonitrile : dichloroethane (3:1, v/v, 2 ml) was added a solution of triphenylphosphine (0.05 mmol) in acetonitrile (0.5 ml) followed by addition of LCAA-CPG (250 mg) . The resulting mixture was shaken occasionally at room temperature for 3 0 min. Then the mixture was transferred to a sintered disc glass funnel and washed off the excess reagents with acetonitrile (1 x 10 ml), dichloroethane (3 x 10 ml), methanol (3 x 15 ml) and diethylether (2 x 10 ml). The washed support was dried under vacuum and blocked the residual amino groups on the polymer support with acetic anhydride. The resulting functionalized support was used oligoribonucleotide synthesis. The cleavage of oligomer from the support and removal of protecting groups from nucleic bases and internucleotidic phosphates were carried out by methylamine solution (2 ml) treatment at room temperature for 90 min. The deprotected oligoribonucleotides were analyzed on HPLC and compared with the oligoribonucleotide synthesized and deprotected from a conventional polymer support. Both the oligomers were found to be identical with respect to their retention times on HPLC. Example 4 In a 25 ml round bottomed flask, d-butan-2,3-diol (5 mmol) was dried by coevaporation with pyridine (15 ml) and finally suspended in anhydrous pyridine (15 ml). 4,4'-Dimethoxytrityl chloride (2 mmol) was added and the reaction mixture was left for stirring at room temperature. After 5h, 5% aq. sodium hydrogen carbonate (2 ml) was added and stirred for further 10 min. Then the reaction mixture was concentrated on a rotary evaporator and the syrupy material so obtained was redissolved in ethyl acetate (70 ml) and washed with aq. sodium hydrogen carbonate solution (2 x 25 ml) and water (1 x 25 ml), respectively. The organic layer was collected, dried over anhydrous sodium sulfate, concentrated and dried on an evaporator under reduced pressure to obtain, 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-diol, in 85% yield and fully characterized by its NMR, IR and MS spectra. The next step was carried out with slight modification. LCAA-CPG (500 mg) was activated with 3% trichloroacetic acid (20 ml) in dich-loromethane for 3h at room temperature followed by washings with dichloroethane (2 x 50 ml), triethylamine : diisopropylethylamine (1 : If v/v, 50 ml) and diethylether (1 x 25 ml). Then the support was dried in a vacuum desiccator. To the suspension of LCAA-CPG (250 mg) in dry dichloroethane (3 ml) was added succinic anhydride (0.25 mmol), triethylamine (0.05 mmol) and 4-dimethylaminopyridine (0.025 mmol) for o 5h at 55 C. Then it was filtered and excess of reagents were removed with dichloroethane (3 x 20 ml), methanol (2 x 20 ml) and diethylether (2 x 15 ml) followed by drying under vacuum. 2(3)-(4,4'-dimethoxytri-tyl)-butan-3(2)-diol (0.05 mmol) was mixed with 4-dimethylaminopyridine (0.05 mmol), l-( 3-dimethylaminopropyl) ethylcarbodiimi.de (0.25 mmol) and succinylated LCAA-CPG (200 mg) in pyridine (2 ml) and triethylamine (30 mmol). The flask was tightly sealed and shaken at room temperature for 16h. Pentafluorophenol (0.05 mmol) was added and shaking was continued for another 16h. Then piperidine (500 (mol) was added, shaken for 5 min and filtered off the support. It was washed with dichloromethane (2 x 20 ml) and diethylether (2 x 10 ml). After drying under vacuum, the residual amino groups were capped with acetic anhydride using standard procedure. Finally the support so obtained was used for synthesis of oligonucleotides. Cleavage from the support of oligonucleotides and removal of protecting groups were carried out by using aq. ammonia - methylamine (1:1, v/v, 2 ml) for 75 min at room temperature. The fully deprotected oligomers were desalted and analyzed on HPLC followed by comparison with standard oligomers. They were found to be identical in all respect (HPLC elution and UV pattern ) . Example 5 In a 25 ml round bottomed flask, dl-butan-2,3-diol (5 mmol) was dried by coevaporation with pyridine (15 ml) and finally suspended in anhydrous dichloroethane (15 ml). 4,4'-Dimethoxytrityl chloride (2 mmol) was added and the reaction mixture was left for stirring at room temperature. After 5h, 5% aq. sodium hydrogen carbonate (2 ml) was added and stirred for further 10 min. Then the reaction mixture was washed with aq. sodium hydrogen carbonate solution (2 x 25 ml) and water (1x25 ml), respectively. The organic layer was collected, dried over anhydrous sodium sulfate, concentrated and dried on an evaporator under reduced pressure to obtain, 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-diol, in 87% yield and fully characterized by its NMR, IR and MS spectra. After accomplishing the first step, the second step was carried out as follows. Oxalyl chloride (8.7 (1, 0.1 mmol) was added to a solution of triazole (35 mg, 0.5 mmol) in an anhydrous mixture of pyridine (1 ml) and acetonitrile (5.0 ml) taken in a septum sealed vial. To the solution was added 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-diol (0.1 mmol) in acetonitrile (3 ml). The reaction mixture was agitated at room temperature for Ih and then LCAA-CPG (300 mg) added and agitation continued at room temperature. After 15 min., the contents of the vial were transferred to sintered disc glass funnel, excess reagent washed off, residual triazolosuccinyl groups on the polymer support were capped with anhydrous methanol (5 mi). After washing with dry acetonitrile (3 x 25 ml), residual amino groups were blocked following standard procedure. The polymer support so obtained was used directly for oligonucleotide synthesis using labile protecting groups containing phosphoramidites. The synthesized oligomers were cleaved from the support and protecting groups from nucleic bases and o phosphate groups removed by using aq. ammonia for 2h at 60 C. The fully deprotected oligomers were compared with standard oligomers synthesized on conventional supports and found to be identical with respect to their retention tim.es on HPLC and FPLC. Example 5 In the first step, dl-butan-2,3-diol (5 mmol) was dried by coe-vaporation with pyridine (15 ml) and finally suspended in anhydrous acetonitrile (15 ml). 4,4'-Dimethoxytrityl chloride (2 mmol) was added and the reaction mixture was left for stirring at room temperature. After 5h, 5% aq. sodium hydrogen carbonate (2 ml) was added and stirred for further 10 min. Then the reaction mixture was concentrated on a rotary evaporator and the syrupy material so obtained was redissolved in ethyl acetate (70 ml) and washed with aq. sodium hydrogen carbonate solution (2 x 25 ml) and water (1 x 25 ml), respectively. The organic layer was collected, dried over anhydrous sodium sulfate, concentrated and dried on an evaporator under reduced pressure to obtain, 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-diol, in 83% yield and fully characterized by its NMR, IR and MS spectra. The second step was carried out as follows. Oxalyl chloride (8.7 (1, 0.1 mmol) was added to a solution of triazole (35 mg, 0.5 mmol) in an anhydrous mixture of pyridine (1 ml) and acetonitrile (5.0 ml) taken in a septum sealed vial. To the solution was added 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-dioI (0.1 mmol) in acetonitrile (3 ml). The reaction mixture was agitated at room temperature for Ih and then LCAA-CPG (300 mg) added and agitation continued at room temperature. After 15 min., the contents of the vial were transferred to sintered disc glass funnel, excess reagent washed off, residual triazolosucci-nyl groups on the polymer support were capped with anhydrous methanol (5 ml). After washing with dry acetonitrile (3 x 25 ml), residual amino groups were blocked following standard procedure. The polymer support so obtained was used directly for oligoribonucleotide synthe- sis. The synthesized oligoribomers were cleaved from the support and protecting groups from nucleic bases and phosphate groups removed by using methylamine solution (2 ml) for 90 min at room temperature. The fully deprotected oligoribomers were compared with the standard oligoribomers and found to be identical in all respects, viz., UV pattern, retention times on HPLC and FPLC. Example 6 In the first step, l-butan-2,3-diol (5 mmol) was dried by coe-vaporation with pyridine (15 ml) and finally suspended in anhydrous acetonitrile (15 ml). 4,4'-Dimethoxytrityl chloride (2 mmol) was added and the reaction mixture was left for stirring at room temperature. After 5h, 5% ag. sodium hydrogen carbonate (2 ml) was added and stirred for further 10 min. Then the reaction mixture was concentrated on a rotary evaporator and the syrupy material so obtained was redissolved in ethyl acetate (70 ml) and washed with ag. sodium hydrogen carbonate solution (2 x 25 ml) and water (1 x 25 ml), respectively. The organic layer was collected, dried over anhydrous sodium sulfate, concentrated and dried on an evaporator under reduced pressure to obtain, 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-diol, in 86% yield and fully characterized by its NMR, IR and MS spectra. After accomplishing the first step, succinoyl chloride (8.7 (1, 0.1 mmol) was added to a solution of triazole (35 mg, 0.5 mmol) in an anhydrous mixture of pyridine (1 ml) and acetonitrile (5.0 ml) taken in a septum sealed vial. To the solution was added 2(3)-(4,4'-dimethoxytrityl )-butan-3 ( 2 )-diol (0.1 mmol) in acetonitrile (3 ml). The reaction mixture was agitated at room temperature for in and then LCAA-CPG (300 mg) added and agitation continued at room temperature. After 15 min., the contents of the vial were transferred to sintered disc glass funnel, excess reagent washed off, residual triazolosucci-nyl groups on the polymer support were capped with anhydrous methanol (5 ml). After washing with dry acetonitrile (3 x 25 ml), residual amino groups were blocked following standard procedure. The polymer support so obtained was used directly for oligonucleotide synthesis using labile protecting groups containing nucleoside phosphoramidite. The synthesized oligomers were cleaved from the support and protecting groups from nucleic bases and phosphate groups were removed by methy-lamine-aq. ammonia solution (1:1, v/v, 2 ml) for 75 min at room temperature. The fully deprotected oligomers were compared with standard oligomers with respect to their UV pattern, retention times on HPLC and FPLC. Advantages of the invention: 1. The process is rapid and economical. 2. The improved universal support can be applied for both the synthesis of oligodeoxyribonucleotides and oligoribonucleotides, thus avoids the use of eight polymer supports. 3. Cleavage of oligonucleotides from the support and removal of protecting groups are achieved, in single step treatment. 4. Oligonucleotides cleaved from the support compare well with the standard oligomers in respect to retention times on FPLC and HPLC and biological properties (Polymerase chain reaction and sequencing) . We Claim: 1. A process for the preparation of an improved support useful for the synthesis of oligodeoxyribonucleotides and oligoribonucleotides which comprises: (i) treating an organic molecule (5 mmol) having a pair of cis hydroxyl groups , selected from a group comprising butane -2,3-diol, 1,4-anhydro-erythritol, D-ribose with 4, 4' - dimethoxytrityl chloride ( 2 mmol) for a period of 5 h and isolating a monosubstituted tritylated derivative from the above reaction mixture by stirring said reaction mixture with 5% aqueous sodium hydrogen carbonate for 10 min followed by concentrating the reaction mixture to get a syrupy liquid , dissolving the obtained syrupy liquid in ethyl acetate to get an organic layer and getting the said monosubstituted derivative by concentrating and drying the obtained organic layer, (ii) treating the monosubstituted derivative obtained in step (i) with one equivalent of a homobifunctional alkanoic acid chloride as defined herein , for a period period of 1 h , and transferring the mixture to a polymer support such as herein described having a hydroxyl or aminoalkyl functionalities, (iii) treating the polymer support obtained in step (ii) with dry alkanol as defined herein , followed by washing with dry alkanol and dialkyl ether, to get the desired improved polymer support represented by figure II of the drawing accompanying the specification wherein DMTr represents 4,4-dimethyl group, R represents an organic molecule bearing a pair of cis hydroxyl group as defined above and P represents a polymer support bearing aminoalkyl or hydroxyalkyl functions. 2. A process as claimed in claims 1 wherein the homobifunctional alkanoic acid chloride used in step (ii) is selected from succinoyl chloride, oxalyl chloride. 3. A process as claimed in claims 1 to 2 wherein the polymer support employed in step (ii) is selected from controlled pore glass (CPG) with variable pore size and linker arm, silica gel ( porous or non-porous ), cross - linked polystyrene having hydroxyl or aminoalkyl functions. 4. An improved process as claimed in claims 1-3 wherein the alkanol used in step (iii) is selected from methanol, ethanol. 5. A process for the preparation of an improved support useful for the synthesis of oligodeoxyribo and oligoribonucleotides substantially as herein described with reference to the examples.

Full Text

The present invention relates to a process for the preparation of an improved support for the synthesis of oligodeoxyribonucleotides and oligoribonucleotides. The polymer support containing an organic molecule having a cis-diol group where one hydroxyl group is attached via a covalent linkage and the other hydroxyl group is protected by an acid labile group. The process also rexates to the cleavage of oligo-deoxyribo- and oligoribonucleotides from the. universal support and simultaneously removal of protecting groups from exocyclic amino functionalities of nucleic bases and (2-cyanoethyl from internucleo-tidic phosphate functions. The improved universal polymer support of formula II accompanying the specifications wherein P represents a polymer support bearing aminoalkyl or hydroxyl functionalities and R stands for an organic substance bearing a pair of cis hydroxyl groups where one of the hydroxyl function is coupled to aminoalkyl or hydroxyl function via a succinate linkage. The other hydroxyl function of the organic substance was protected with monomethoxytrityl or 4,4'-dimethoxytrityl chloride. The support was employed for the synthesis of oligodeoxyribo- and oligoribonucleotides in an automated DNA synthesizer following phosphoramidite chemistry. The cleavage of oligonucleotide from the support and other protecting groups were removed in analogous way to the standard method.
The known protecting groups of formula I, shown in the drawing accompanying the specification represented by figure I, employed, viz., conventional (set I) and labile (set II -IV) for exocyclic amino protection in nucleosides, are described below.
Set I (Conventional Protecting Group)
B = Adenine; R = Benzoyl (Bz)
B = Cytosine; R = Benzoyl ( Bz) or acetyl (Ac)

B = Guanine; R = Isobutyryl (Ibu) Set II(Labile Protecting Group)
B = Adenine; R = Phenoxyacetyl (Pac) B = Cytosine; R = Isobutyryl (Ibu) or Acetyl(Ac) B = Guanine; R = Phenoxyacetyl (Pac) Set III(Labile Protecting Group)
B = Adenine; R = Dimethylformamidine (DMF) B = Cytosine; R = Dimethylformamidine (DMF) B = Guanine; R = Dimethylformamidine (DMF) Set IV(Labile Protecting Group)
B = Adenine; R = p-tert-Butylphenoxyacetyl B = Cytosine; R = p-tert-Butylphenoxyacetyl B = Guanine; R = p-tert-Butylphenoxyacetyl Synthetic oligonucleotides (oligodeoxyribo- and oligoribonucleo-tide) have become indispensable tools in modern biological sciences. The protocols for chemical synthesis and modifications of these molecules have been simplified to an extent that even a non-chemist, the actual user can synthesize these molecules without much difficulties. However, the support functionalization has been one of the time consuming tasks. Moreover, one still requires to use at least eight different (four for oligodeoxyribo- and four for oligoribonucleotides) supports, each bearing a separate nucleoside corresponding to the 3'-terminus of the desired oligomer. This number may even be more when 3'-terminus of the desired oligomer contains nucleotide other than the normal one. Though the time of functionalization of polymer supports required for oligonucleotide synthesis has been reduced considerably in the recent publications (Kumar et al., Nucleosides and Nucleotides,

15, 1996, 879), one still needs at least eight different supports for all kinds of oligonucleotides. Some attempts have been made to develop universal supports to eliminate the use of eight different supports. Gough et al. (Gough et al., Tet. Lett. 24, 1983, 5321) have proposed the use of a single universal support that consisted of controlled pore glass derivatized with 2'(3')-O-benzoyluridine 5'-0-succinyl residue and demonstrated its applications for the synthesis of oligodeoxyribo- and oligoribonucleotides via phosphotriester or the
phosphite approach. The cleavage of oligodeoxyribo- and oligoribonu-
o cleotides was effected by the use of aq. ammonia for 24h at 60-65 C
o and aq. ammonia-pyridine (2:1) for 16h at 50 C, respectively in sealed
vials. The universal support contains a nucleosidic material which does not get incorporated in the oligonucleotide chain and goes waste. The other serious limitations of this support are that the deprotection time is considerably longer in comparison to the time required in the standard protocol and the functinalization of the support involves multistep (4 steps) synthesis and purification procedure. In a recent publication, they (Gough et al., Tet. Lett., 36, 1995, 27) have further modified the functinalization of the universal support by the use of an adapter, 2'(3')-0-benzoyluridine-5'-0-(cya-noethyl- N,N-diisopropylphosphoramidite) and coupled it to a standard T-support, following phosphoramidite chemistry. The functionalized support was subsequently used for the synthesis of oligonucleotides. The deprotection of oligodeoxyribo- and oligoribonucleotides was
achieved by the treatment of the support with 25 ml aq. ammonia for
o 48h at 65 C in a pressure vial and 25 ml aq. ammonia-pyridine mix-
o tare (4:1, v/v) for 24h at 50 C in a pressure vial, respectively.
However, the universal support has still the following serious limita-

tions: The support contains two nucleosidic units (expensive material) which do not get incorporated in the oligonucleotide chain and wasted. The adapter used for the functionalization of universal support involves multistep synthesis and purification strategy and yield of the final product is low, making the support functionalization a time consuming (2-3d), tedious and expensive task. Moreover, the time required for the deprotection of oligonucleotides is exhorbitantly high, almost three times to the time required in currently followed synthesis and deprotection protocol. In fact the time required for the deprotection of oligonucleotides is so much that one can afford to prepare all the eight different supports even in lesser time. In an elegant study Gough and co-workers described that an oligodeoxynucleotide having 3'-terminal protection with a ribonucleo-side linked by its 2'- or 3'-hydroxyl group to a 3'-phosphate is removed as its 2',3'-cyclic phosphate by lead cation. In this process oligodeoxynucleotide gets removed with 3'-hydroxyl function. This concept led Gough and co-workers developing two universal supports. Both of the universal, supports contain uridine as the terminal ribonucleoside providing a pair of cis-diol groups. Because of the rigid rotation of these hydroxy1 groups, oligodeoxyribo- and oligori-bonucleotides take considerable time (48h) for the cleavage from the universal supports. This problem might be taken care of if uridine, a source of cis diol groups is replaced by an organic molecule providing a pair of cis diol groups with free rotation. The free rotation of cis diol groups will help in rapid formation of cyclic phosphate in the presence of lead cation or alkaline conditions and hence will lead in rapid deprotection of oligodeoxyribo- and oligoribonucleotides from

the universal support.
Therefore, there is a need to develop a universal support suitable for the synthesis of oligodeoxyribo- and oligoribonucleotides and compatible to the existing methods of synthesis and deprotection (cleavage of oligodeoxyribo- and oligoribonucleotides from the universal support and simultaneously removal of protecting groups, labile or conventional from exocyclic amino functionalities of nucleic bases and (2-cyanoethyl from internucleotidic phosphate functions) of all possible oligodeoxyribo- and oligoribonucleotides.
The main objective of the present invention is therefore to provide an improved process for the preparation of an improved universal polymer support for the synthesis of oligodeoxyribo- and oligoribonucleotides.
Another objective of the present invention is to provide a process for the preparation of an improved universal polymer support devoid of any nucleosidic material, suitable for the synthesis of all possible oligodeoxyribo- and oligoribonucleotides
Yet another objective of the present invention is to provide a process for the preparation of an improved universal polymer support compatible to the existing methodologies of synthesis and deprotection (cleavage of oligodeoxyribo- and oligoribonucleotides from the universal support and simultaneously removal of protecting groups, labile or conventional from exocyclic amino functionalities of nucleic bases and (2-cyanoethyl from internucleotidic phosphate functions) of all possible oligodeoxyribo- and oligoribonucleotides.
Still another objective of the present invention is to provide a process for the preparation of an improved universal polymer support

employing commonly available reagents.
Accordingly, the present invention provides a process for the preparation of
an improved support for the synthesis of oligodeoxyribonucleotides and
oligoribonucleotides which comprises:
(i) treating an organic molecule (5 mmol) having a pair of cis hydroxyl groups selected from a group a organic molecule comprising butane -2,3-diol, 1,4-anhydro-erythritol, D-ribose with 4, 4' - dimethoxytrityl chloride ( 2 mmol) for a period of 5 h and isolating a monosubstituted tritylated derivative from the above reaction mixture by stirring said reaction mixture with 5% aqueous sodium hydrogen carbonate for 10 min followed by concentrating the reaction mixture to get a syrupy liquid , dissolving the obtained syrupy liquid in ethyl acetate to get an organic layer and getting the said monosubstituted derivative by concentrating and drying the obtained organic layer,
(ii) treating the monosubstituted derivative obtained in step (i) with one equivalent of a homobifunctional alkanoic acid chloride as defined herein , for a period period of 1 h , and transferring the mixture to a polymer support such as herein described having a hydroxyl or aminoalkyl functionalities,
(iii) treating the polymer support obtained in step (ii) with dry alkanol as defined herein , followed by washing with dry alkanol and dialkyl ether, to get the desired improved polymer support represented by figure II of the drawing accompanying the specification wherein DMTr represents 4,4-dimethyl group, R represents an organic molecule bearing a pair of cis hydroxyl group as defined above and P represents a polymer support bearing aminoalkyl or hydroxyalkyl functions.
The organic molecule having a pair of cis hydroxyl groups (both are at
secondary positions) used for making monosubstituted tritylated derivative may be
selected from butane-2,3-diol, 1,4-anhydro-erythritol, D-ribose and the like.
The homobifunctional alkanoic acid chloride used in step (ii) may be selected
from succinyl chloride, oxalyl chloride and the like.

The polymer support employed in step (ii) may be selected from controlled pore glass (CPG) with variable pore size and linker arm, silica gel (porous or non-porous), cross-linked polystyrene having hydroxyl or aminoalkyl functions and the like.
The alkanol used in step (iii) for capping purpose may be selected from methanol, ethanol and the like.
The improved polymer support is then used for the synthesis of oligonucleotides (deoxyribo- and ribo-) which comprises (i) synthesizing oligodeoxyribo- and ribonucleotides on the polymer support proposed by the process following phosphoramidite chemistry using conventional or labile protecting groups for nucleic bases,
(ii) treating the resultant oligodeoxynucleotide and oligoribo-nucleotides embedded in the support with aq. ammonia solution
or aq alkylamine-ammonia mixture (1:1) at the temperature range
o of ambient to 65 C.
(iii)concentrating the resultant reaction mixture and dissolving the solid residue in water and subjecting to gel filtration to remove low m.w. impurities,
(iv)concentrating the eluent obtained in step (iv) under vacuum concentrator and subjecting to high performance liquid chromatography (HPLC) to obtain the fully deprotected oligodeoxyribo- and ribonucleotides.
The 2/-deoxyribonucleosidephosphoramidite and ribonucleosidephos-phoramidite used for the synthesis of oligodeoxyribo- and ribonucleotides in step (iv) carrying conventional protecting groups may be selected from benzoyl for adenine and cytosine and isobutyryl or

acetyl for guanine or labile protecting groups may be selected from phenoxyacetyl for adenine and guanine and isobutyryl or acetyl for cytosine; dimethylformamidine (DMF) for adenine, guanine and cytosine; p-tert-butylphenoxyacetyl- for adenine, guanine and cytosine and the like for exocyclic amino functions of nucleic bases.
The cleavage of oligodeoxyribo- and oligoribonucleotides from the universal support and simultaneously removal of protecting groups, labile or conventional from exocyclic amino functionalities of nucleic bases and (-cyanoethyl from internucleotidic phosphate functions) of all possible oligodeoxyribo- and oligoribonucleotides may be achieved in step (v) by the treatment with ag. ammonia or
aq. ammonia-methylamine at the temperature renge to ambient to
o 65 C (for oligodeoxyribonucleotides) and aq. ammonia-pyridine at
o 50 C or aq. ammonia-methylamine at the temperature range of
o ambient to 50 C for oligoribonucleotides.
The following examples concerning the use of universal support for the synthesis of oligodeoxynucleotides and oligoribonucleotides and the cleavage of oligodeoxyribo- and oligoribonucleotides from the universal support and simultaneously removal of protecting groups (conventional and labile) from exocyclic amino functionalities of nucleic bases and (2-cyanoethyl from internucleotidic phosphate functions are provided to illustrate the invention and should not be construed to limit the scope of the invention. Example 1
The preparation of universal support comprises of two steps which are described below. In a 25 ml round bottomed flask, dl-butan-2,3-diol (5 mmol) was dried by coevaporation with pyridine (15 ml) and finally suspended in anhydrous pyridine (15 ml). 4,4'-Dimethoxytrityl

chloride (2 mmol) was added and the reaction mixture was left for stirring at room, temperature. After 5h, 5% aq. sodium hydrogen carbonate (2 ml) was added and stirred for further 10 min. Then the reaction mixture was concentrated on a rotary evaporator and the syrupy material so obtained was redissolved in ethyl acetate (70 ml) and washed with aq. sodium hydrogen carbonate solution (2 x 25 ml) and water (1 x 25 ml), respectively. The organic layer was collected, dried over anhydrous sodium sulfate, concentrated and dried on an evaporator under reduced pressure to obtain, 2(3)-(4,4'-dimethaxytri-tyl)-butan-3(2)-diol, in 90% yield and fully characterized by its NMR, IR and MS spectra.
In the second step, 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-diol was attached to the long chain aminoalkylated controlled pore glass (LCAA-CPG) by following procedure. Succinoyl chloride (6.3 (1, 0.05 mmol) was added to a solution of triazole (19.8 mg, 0.28 mmol) in an anhydrous mixture of pyridine (500(1) and acetonitrile (1.0 ml) taken in a septum sealed vial. To the solution was injected 2(3)~(4,4'-dimethoxytrityl)-butan-3(2)-diol (0.05 mmol) in acetonitrile (1 ml). The reaction mixture was agitated at room temperature for lh and then LCAA-CPG (200 mg) added and agitation continued at room temperature. After 15 min., the contents of the vial were transferred to sintered disc glass funnel, excess reagent washed off, residual triazolosucci-nyl groups on the polymer support were capped with anhydrous methanol (1 ml). After washing with dry acetonitrile (3x5 ml), residual amino groups were blocked following standard procedure (Matteucci, M.D. and Caruthers, M.H., J.Am.Chem.Soc., 103 (1981) 3185). The polymer support so obtained was used for oligonucleotide synthesis. The synthesized

oligomers from the support and protecting groups from nucleic bases
and phosphate groups were removed by the treatment of ag. ammonia for
o 16h at 60 C. The fully deprotected oligomers were compared with
oligomers synthesized on standard supports on HPLC, FPLC (Fast
protein liguid chematography) and by biological activity.
Example 2
In a 25 ml round bottomed flask, d-butan-2,3-diol (5 mmol) was dried by coevaporation with pyridine (15 ml) and finally suspended in anhydrous pyridine (15 ml). 4,4'-Dimethoxytrityl chloride (2 mmol) was added and the reaction mixture was left for stirring at room temperature. After 5h, 5% ag . sodium hydrogen carbonate (2 ml) was added and stirred for further 10 min. Then the reaction mixture was concentrated on a rotary evaporator and the syrupy material so obtained was redissolved in ethyl acetate (70 ml) and washed with ag. sodium hydrogen carbonate solution (2 x 25 ml) and water (1 x 25 ml), respectively. The organic layer was collected, dried over anhydrous sodium sulfate, concentrated and dried on an evaporator under reduced pressure to obtain, 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-diol, in 85% yield and fully characterized by its NMR, IR and MS spectra.
The compound, 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-diol, (0.1
mmol) was reacted with succinic anhydride (0.2 mmol) in dichloroethane
(4 ml) in the presence of 4-dimethyIaminopyridine (50 umol) and a
o
tertiary base, triethylamine (0.1 mmol) at 50 C for 20 min. After
stipulated time (20 min), the reaction mixture was diluted with dichloroethane (25 ml) and washed with 5% cold ag. citric acid (2x 15 ml) and water (2x 10 ml), respectively. The organic phase was collected and concentrated to obtain 2-(4,4'-dimethoxytrityloxy)-l-methylpro-pyl-succinate in 93% yield. 2-(4,4'dimethoxytrityloxy)-1-methylpropyl-

succinate (0.05 mmol) and 4-dimethylaminopyridine (10 umol) were
dissolved in pyridine (2 ml) and added LCAA-CPG (200 mg) followed by
dicyclohexylcarbodiimide (0.075 mmol) under anhydrous conditions. The
resulting mixture was shaken occasionally at room temperature for 16h.
Then the mixture was transferred to a sintered disc glass funnel and
washed off the excess reagents with pyridine (1 x 10 ml), methanol (3
x 15 ml) and diethylether (2 x 10 ml). The washed support was dried
under vacuum and capped the residual amino groups on the polymer
support following standard procedure. The resulting functionalized
support was used oligonucleotide synthesis using labile protecting
groups containing nucleoside phosphoramidite. The cleavage of oligomer
chain from the support and removal of protecting groups were achieved
o by aq. ammonia (29%) treatment for 6h at 60 C. The fully deprotected
oligomers were desalted and analyzed on HPLC and FPLC. Then the oligomers were compared with the standard oligomers synthesized on the commercially available conventional polymer supports. They were found to be identical in all respect, viz., retention times on HPLC as well as UV pattern. Example 3
In a 25 ml round bottomed flask, l-butan-2,3-diol (5 mmol) was dried by coevaporation with pyridine (15 ml) and finally suspended in anhydrous pyridine (15 ml). 4,4'-Dimethoxytrityl chloride (2 mmol) was added and the reaction mixture was left for stirring at room temperature. After 5h, 5% aq. sodium hydrogen carbonate (2 ml) was added and stirred for further 10 min. Then the reaction mixture was concentrated on a rotary evaporator and the syrupy material so obtained was redissolved in ethyl acetate (70 ml) and washed with. aq. sodium

hydrogen carbonate solution (2 x 25 ml) and water (1 x 25 ml), respectively. The organic layer was collected, dried over anhydrous sodium sulfate, concentrated and dried on an evaporator under reduced pressure to obtain, 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-diol, in 88% yield and fully characterized fay its NMR, IR and MS spectra.
In the next reaction, 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-dial, (0.1 mmol) was reacted with succinic anhydride (0.2 mmol) in
dichloroethane (4 ml) in the presence of 4-dimethylaminopyridine
o (0.05 mmol) and a tertiary basts, triethylamine (0.1 mmol) at 50 C
for 20 min. After stipulated time, the reaction mixture was diluted with dichloroethane (25 ml) and washed, with 5% cold aq. citric acid (2 x 15 ml) and water (2 x 10 ml), respectively. The organic phase was collected and concentrated to obtain 2-(4,4'-dimethoxytrityloxy)-1-methyl- propylsuccinate in 93% yield. To the mixture of 2-(4,4'-dimethoxytrityloxy)-1- methylpropylsucci-nate (0.05 mmol), 2,2'-dithiobis(5-nitropyridine) (DTNP) (0.05 mmol) and 4-dimethylaminopyridine (0.05 mmol) dissolved in acetonitrile : dichloroethane (3:1, v/v, 2 ml) was added a solution of triphenylphosphine (0.05 mmol) in acetonitrile (0.5 ml) followed by addition of LCAA-CPG (250 mg) . The resulting mixture was shaken occasionally at room temperature for 3 0 min. Then the mixture was transferred to a sintered disc glass funnel and washed off the excess reagents with acetonitrile (1 x 10 ml), dichloroethane (3 x 10 ml), methanol (3 x 15 ml) and diethylether (2 x 10 ml). The washed support was dried under vacuum and blocked the residual amino groups on the polymer support with acetic anhydride. The resulting functionalized support was used oligoribonucleotide synthesis. The cleavage of oligomer from the

support and removal of protecting groups from nucleic bases and internucleotidic phosphates were carried out by methylamine solution (2 ml) treatment at room temperature for 90 min. The deprotected oligoribonucleotides were analyzed on HPLC and compared with the oligoribonucleotide synthesized and deprotected from a conventional polymer support. Both the oligomers were found to be identical with respect to their retention times on HPLC. Example 4
In a 25 ml round bottomed flask, d-butan-2,3-diol (5 mmol) was dried by coevaporation with pyridine (15 ml) and finally suspended in anhydrous pyridine (15 ml). 4,4'-Dimethoxytrityl chloride (2 mmol) was added and the reaction mixture was left for stirring at room temperature. After 5h, 5% aq. sodium hydrogen carbonate (2 ml) was added and stirred for further 10 min. Then the reaction mixture was concentrated on a rotary evaporator and the syrupy material so obtained was redissolved in ethyl acetate (70 ml) and washed with aq. sodium hydrogen carbonate solution (2 x 25 ml) and water (1 x 25 ml), respectively. The organic layer was collected, dried over anhydrous sodium sulfate, concentrated and dried on an evaporator under reduced pressure to obtain, 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-diol, in 85% yield and fully characterized by its NMR, IR and MS spectra.
The next step was carried out with slight modification. LCAA-CPG (500 mg) was activated with 3% trichloroacetic acid (20 ml) in dich-loromethane for 3h at room temperature followed by washings with dichloroethane (2 x 50 ml), triethylamine : diisopropylethylamine (1 : If v/v, 50 ml) and diethylether (1 x 25 ml). Then the support was

dried in a vacuum desiccator. To the suspension of LCAA-CPG (250 mg)
in dry dichloroethane (3 ml) was added succinic anhydride (0.25 mmol),
triethylamine (0.05 mmol) and 4-dimethylaminopyridine (0.025 mmol) for
o 5h at 55 C. Then it was filtered and excess of reagents were removed
with dichloroethane (3 x 20 ml), methanol (2 x 20 ml) and diethylether (2 x 15 ml) followed by drying under vacuum. 2(3)-(4,4'-dimethoxytri-tyl)-butan-3(2)-diol (0.05 mmol) was mixed with 4-dimethylaminopyridine (0.05 mmol), l-( 3-dimethylaminopropyl) ethylcarbodiimi.de (0.25 mmol) and succinylated LCAA-CPG (200 mg) in pyridine (2 ml) and triethylamine (30 mmol). The flask was tightly sealed and shaken at room temperature for 16h. Pentafluorophenol (0.05 mmol) was added and shaking was continued for another 16h. Then piperidine (500 (mol) was added, shaken for 5 min and filtered off the support. It was washed with dichloromethane (2 x 20 ml) and diethylether (2 x 10 ml). After drying under vacuum, the residual amino groups were capped with acetic anhydride using standard procedure. Finally the support so obtained was used for synthesis of oligonucleotides. Cleavage from the support of oligonucleotides and removal of protecting groups were carried out by using aq. ammonia - methylamine (1:1, v/v, 2 ml) for 75 min at room temperature. The fully deprotected oligomers were desalted and analyzed on HPLC followed by comparison with standard oligomers. They were found to be identical in all respect (HPLC elution and UV pattern ) . Example 5
In a 25 ml round bottomed flask, dl-butan-2,3-diol (5 mmol) was dried by coevaporation with pyridine (15 ml) and finally suspended in anhydrous dichloroethane (15 ml). 4,4'-Dimethoxytrityl chloride (2 mmol) was added and the reaction mixture was left for stirring at room

temperature. After 5h, 5% aq. sodium hydrogen carbonate (2 ml) was added and stirred for further 10 min. Then the reaction mixture was washed with aq. sodium hydrogen carbonate solution (2 x 25 ml) and water (1x25 ml), respectively. The organic layer was collected, dried over anhydrous sodium sulfate, concentrated and dried on an evaporator under reduced pressure to obtain, 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-diol, in 87% yield and fully characterized by its NMR, IR and MS spectra.
After accomplishing the first step, the second step was carried out as follows. Oxalyl chloride (8.7 (1, 0.1 mmol) was added to a solution of triazole (35 mg, 0.5 mmol) in an anhydrous mixture of pyridine (1 ml) and acetonitrile (5.0 ml) taken in a septum sealed vial. To the solution was added 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-diol (0.1 mmol) in acetonitrile (3 ml). The reaction mixture was agitated at room temperature for Ih and then LCAA-CPG (300 mg) added and agitation continued at room temperature. After 15 min., the contents of the vial were transferred to sintered disc glass funnel, excess reagent washed off, residual triazolosuccinyl groups on the polymer support were capped with anhydrous methanol (5 mi). After washing with dry acetonitrile (3 x 25 ml), residual amino groups were blocked following standard procedure. The polymer support so obtained was used directly for oligonucleotide synthesis using labile protecting groups containing phosphoramidites. The synthesized oligomers were
cleaved from the support and protecting groups from nucleic bases and
o phosphate groups removed by using aq. ammonia for 2h at 60 C. The
fully deprotected oligomers were compared with standard oligomers
synthesized on conventional supports and found to be identical with

respect to their retention tim.es on HPLC and FPLC. Example 5
In the first step, dl-butan-2,3-diol (5 mmol) was dried by coe-vaporation with pyridine (15 ml) and finally suspended in anhydrous acetonitrile (15 ml). 4,4'-Dimethoxytrityl chloride (2 mmol) was added and the reaction mixture was left for stirring at room temperature. After 5h, 5% aq. sodium hydrogen carbonate (2 ml) was added and stirred for further 10 min. Then the reaction mixture was concentrated on a rotary evaporator and the syrupy material so obtained was redissolved in ethyl acetate (70 ml) and washed with aq. sodium hydrogen carbonate solution (2 x 25 ml) and water (1 x 25 ml), respectively. The organic layer was collected, dried over anhydrous sodium sulfate, concentrated and dried on an evaporator under reduced pressure to obtain, 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-diol, in 83% yield and fully characterized by its NMR, IR and MS spectra.
The second step was carried out as follows. Oxalyl chloride (8.7 (1, 0.1 mmol) was added to a solution of triazole (35 mg, 0.5 mmol) in an anhydrous mixture of pyridine (1 ml) and acetonitrile (5.0 ml) taken in a septum sealed vial. To the solution was added 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-dioI (0.1 mmol) in acetonitrile (3 ml). The reaction mixture was agitated at room temperature for Ih and then LCAA-CPG (300 mg) added and agitation continued at room temperature. After 15 min., the contents of the vial were transferred to sintered disc glass funnel, excess reagent washed off, residual triazolosucci-nyl groups on the polymer support were capped with anhydrous methanol (5 ml). After washing with dry acetonitrile (3 x 25 ml), residual amino groups were blocked following standard procedure. The polymer support so obtained was used directly for oligoribonucleotide synthe-

sis. The synthesized oligoribomers were cleaved from the support and protecting groups from nucleic bases and phosphate groups removed by using methylamine solution (2 ml) for 90 min at room temperature. The fully deprotected oligoribomers were compared with the standard oligoribomers and found to be identical in all respects, viz., UV pattern, retention times on HPLC and FPLC. Example 6
In the first step, l-butan-2,3-diol (5 mmol) was dried by coe-vaporation with pyridine (15 ml) and finally suspended in anhydrous acetonitrile (15 ml). 4,4'-Dimethoxytrityl chloride (2 mmol) was added and the reaction mixture was left for stirring at room temperature. After 5h, 5% ag. sodium hydrogen carbonate (2 ml) was added and stirred for further 10 min. Then the reaction mixture was concentrated on a rotary evaporator and the syrupy material so obtained was redissolved in ethyl acetate (70 ml) and washed with ag. sodium hydrogen carbonate solution (2 x 25 ml) and water (1 x 25 ml), respectively. The organic layer was collected, dried over anhydrous sodium sulfate, concentrated and dried on an evaporator under reduced pressure to obtain, 2(3)-(4,4'-dimethoxytrityl)-butan-3(2)-diol, in 86% yield and fully characterized by its NMR, IR and MS spectra.
After accomplishing the first step, succinoyl chloride (8.7 (1, 0.1 mmol) was added to a solution of triazole (35 mg, 0.5 mmol) in an anhydrous mixture of pyridine (1 ml) and acetonitrile (5.0 ml) taken in a septum sealed vial. To the solution was added 2(3)-(4,4'-dimethoxytrityl )-butan-3 ( 2 )-diol (0.1 mmol) in acetonitrile (3 ml). The reaction mixture was agitated at room temperature for in and then LCAA-CPG (300 mg) added and agitation continued at room temperature.

After 15 min., the contents of the vial were transferred to sintered disc glass funnel, excess reagent washed off, residual triazolosucci-nyl groups on the polymer support were capped with anhydrous methanol (5 ml). After washing with dry acetonitrile (3 x 25 ml), residual amino groups were blocked following standard procedure. The polymer support so obtained was used directly for oligonucleotide synthesis using labile protecting groups containing nucleoside phosphoramidite. The synthesized oligomers were cleaved from the support and protecting groups from nucleic bases and phosphate groups were removed by methy-lamine-aq. ammonia solution (1:1, v/v, 2 ml) for 75 min at room temperature. The fully deprotected oligomers were compared with standard oligomers with respect to their UV pattern, retention times on HPLC and FPLC.
Advantages of the invention:
1. The process is rapid and economical.
2. The improved universal support can be applied for both the synthesis of oligodeoxyribonucleotides and oligoribonucleotides, thus avoids the use of eight polymer supports.
3. Cleavage of oligonucleotides from the support and removal of protecting groups are achieved, in single step treatment.
4. Oligonucleotides cleaved from the support compare well with the standard oligomers in respect to retention times on FPLC and HPLC
and biological properties (Polymerase chain reaction and sequencing) .

We Claim:
1. A process for the preparation of an improved support useful for the synthesis of oligodeoxyribonucleotides and oligoribonucleotides which comprises:
(i) treating an organic molecule (5 mmol) having a pair of cis hydroxyl groups , selected from a group comprising butane -2,3-diol, 1,4-anhydro-erythritol, D-ribose with 4, 4' - dimethoxytrityl chloride ( 2 mmol) for a period of 5 h and isolating a monosubstituted tritylated derivative from the above reaction mixture by stirring said reaction mixture with 5% aqueous sodium hydrogen carbonate for 10 min followed by concentrating the reaction mixture to get a syrupy liquid , dissolving the obtained syrupy liquid in ethyl acetate to get an organic layer and getting the said monosubstituted derivative by concentrating and drying the obtained organic layer,
(ii) treating the monosubstituted derivative obtained in step (i) with one equivalent of a homobifunctional alkanoic acid chloride as defined herein , for a period period of 1 h , and transferring the mixture to a polymer support such as herein described having a hydroxyl or aminoalkyl functionalities,
(iii) treating the polymer support obtained in step (ii) with dry alkanol as defined herein , followed by washing with dry alkanol and dialkyl ether, to get the desired improved polymer support represented by figure II of the drawing accompanying the specification wherein DMTr represents 4,4-dimethyl group, R represents an organic molecule bearing a pair of cis hydroxyl group as defined above and P represents a polymer support bearing aminoalkyl or hydroxyalkyl functions.

2. A process as claimed in claims 1 wherein the homobifunctional alkanoic acid chloride used in step (ii) is selected from succinoyl chloride, oxalyl chloride.
3. A process as claimed in claims 1 to 2 wherein the polymer support employed in step (ii) is selected from controlled pore glass (CPG) with variable pore size and linker arm, silica gel ( porous or non-porous ), cross - linked polystyrene having hydroxyl or aminoalkyl functions.
4. An improved process as claimed in claims 1-3 wherein the alkanol used in step (iii) is selected from methanol, ethanol.
5. A process for the preparation of an improved support useful for the synthesis of oligodeoxyribo and oligoribonucleotides substantially as herein described with reference to the examples.

Documents:

2338-del-1996-abstract.pdf

2338-del-1996-claims.pdf

2338-del-1996-complete specification (granted).pdf

2338-del-1996-correspondence-others.pdf

2338-del-1996-correspondence-po.pdf

2338-del-1996-description (complete).pdf

2338-del-1996-drawings.pdf

2338-del-1996-form-1.pdf

2338-del-1996-form-10.pdf

2338-del-1996-form-19.pdf

2338-del-1996-form-2.pdf

2338-del-1996-form-3.pdf

2338-del-1996-form-4.pdf

2338-del-1996-form-60.pdf

2338-del-1996-petition-138.pdf

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Patent Number

228278

Indian Patent Application Number

2338/DEL/1996

PG Journal Number

38/2008

Publication Date

19-Sep-2008

Grant Date

24-Nov-2006

Date of Filing

29-Oct-1996

Name of Patentee

Council of Scientific and Industrial Research,

Applicant Address

Rafi Marg,New Delhi-110001,India

Inventors:

#	Inventor's Name	Inventor's Address
1	Pradeep Kumar	Centre for Biochemical Technology, Mall Road, India.
2	Kailash Chand Gupta	Centre for Biochemical Technology, Mall Road

PCT International Classification Number

C08L71/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA