PL157777B1 - Method of obtaining oligonucleotides - Google Patents

Abstract

1. The invention concerns a method of producing oligonucleotides of general formula 1, where R2 indicates OH group or ethoxy group, Y indicates atom of oxygen, m=1, n indicates number 0-200, and B indicates rest of adenine, guanine or cytosine of respective formulae 2, 3 or 4, where R1 indicates atom of hydrogen or B indicates rest of thymine of formula 5, however all B do not indicate thymine, by conducted in presence of coupling agent condensation in positions 5' and 3' of consecutive nucleosides, such as derivatives of adenosine, guanosine, cytidine and thymidine, from which nucleotides the first is bounded by covalence with solid carrier, oxidation of product of each condensation and removal of group securing function 5'-hydroxyl and splitting off of produced digonucleotide from solid carrier, characterised in that as derivatives of adenosine, guanosine and cytidine are used nucleotides of formula 6 or 7, if the derivative is the first nucleotide, in which formulae Z indicates atom of hydrogen, and for formula 6 dimethoxytriphenylomethyl group, R2 indicates 2-cyanomethoxyl or ethoxy group, Ra indicates diisopropylamine, dimethylamine or morpholine group, S indicates solid carrier of extended chain, M=0, and B' indicates rest of adenine, guanine or cytosine of respective formulae 2, 3 or 4, where R1 indicates isopropoxyacetylene group, and group blocking 5'-hydroxyl function is removed from the last nucleotide by means of acidic hydrolysis, prior to splitting off of product from carrier or following splitting it off and cleansing.<IMAGE>

Description

REPUBLIC

POLAND

The Patent Office of the Republic of Poland ® PATENT DESCRIPTION® PL® 157777 ® BI

Application number: 273723 z-λ (£ 9 IntCl ⁵ :

C07H 21/02

Application daU: CZ ^ TELUIA

OGOLKA

Method for producing oligonucleotides

Application announced:

January 22, 1990 BUP 02/90

The holder of the patent:

Polish Academy of Sciences Research Center

Molecular and Macromolecular, Łódź, PL

The grant of the patent was announced on: 07/31/1992 WUP 07/92

Inventors:

Bogdan Uznański, Lodz, PL Andrzej Wilk, Lodz, PL Andrzej Grajkowski, Lodz, PL Wojciech J. Stec, Lodz, PL

PL 157777 BI

A method for producing oligonucleotides of general formula I, wherein R ₂ is an OH group or a group

57J ethoxy, Y is oxygen, ni = 1, n is 0-200, and B is an adenine, guanine or cytosine residue of the formulas 2, 3 or 4, respectively, where Ri is hydrogen or B is a thymine residue of formula 5 except that all B are not thymine, by condensation carried out in the presence of a coupling agent at the 5 'and 3' positions of consecutive nucleosides such as adenosine, guanosine, cytidine and thymidine derivatives, the first of which is covalently linked solid support, oxidation of the product of each condensation and removal of the 5'-hydroxyl protecting group and cleavage of the resulting digonucleotide from the solid support, characterized in that nucleosides of formula 6 or formula 7 are used as derivatives of adenosine, guanosine and cytidine, if this derivative is the first nucleoside in which Z is a hydrogen atom and for formula 6 a dimethoxytriphenylmethyl group, R2 is a 2-cyanoethoxy or ethoxy group, R3 is Significance diisopropylamino group, dimethylamino or morpholino group, S is a solid support with an extended chain, m = 0, and B 'is a residue of adenine, guanine or cytosine ⁰ formulas respectively to 2, 3 or 4, wherein Ri is izopropoksyacetylową, wherein the group the blocking 5 '- hydroxyl function is removed by the acid hydrolysis of the last 2 nucleotides, before cleavage of the product from the support or after it has been cleaved and purified.

pattern 2

MODEL 3

H-N-Ri

AND

PATTERN i

2-0

MODEL 5

MODEL 6

2- oA B

V?

AXIS

W&R 7

Method for producing oligonucleotides

Claims (6)

Patent Claims 1. ³ gtacagtagaagatc ⁵ A method for the production of oligonucleotides of general formula I, wherein R2 is OH or ethoxy, Y is oxygen, m = 1, n is 0-200, and B is an adenine, guanine or cytosine residue of the formulas 2, respectively, 3 or 4, in which R1 is hydrogen or B is a thymine residue of formula 5, except that all B are not thymine, by condensation carried out in the presence of a coupling agent at the 5 'and 3' positions of consecutive nucleosides such as adenosine derivatives, guanosine, cytidine and thymidine, the first of which is covalently bound to a solid support, oxidation of the product of each condensation and removal of the 5'-hydroxyl protecting group and cleavage of the produced digonucleotide from the solid support, characterized in that as derivatives of adenosine, guanosine and cytidine the nucleosides of formula 6 or formula 7 are used when this derivative is the first nucleoside, in which formulas Z is hydrogen and for formula 6 a dimethoxytriphenylmethyl, R ₂ is 2-cyjanoetoksylową or ethoxy, R3 is diisopropylamino, dimethylamino or morpholino group, S is a solid support with an extended chain, m = 0, and B 'is a residue of adenine, guanine or cytosine of formulas 2 3 or 4, in which R1 is an isopropoxyacetyl group, wherein the 5'-hydroxyl blocking group is removed from the last nucleotide by acid hydrolysis, prior to cleavage of the product from the support or after it has been cleaved and purified. 2. ^ TTCTACTGTACCTAG ⁵ 2. The method according to p. The method of claim 1, characterized in that in the step of cleaving the oligonucleotide from the support, all amino groups are simultaneously deblocked and the 2-cyanoethyl group is removed. 3. ^ TTGGGGCTCACT ⁵ Process for the production of oligonucleotides of general formula I, wherein R2 is a trifluoroethoxy group, Y is an oxygen atom, m = 1, n is 0-200, and B is an adenine, guanine or cytosine residue of the formulas 2, 3 or 4, respectively wherein R1 is hydrogen or B is a thymine residue of formula 5 with the proviso that all B are not thymine, characterized in that the nucleoside of formula 7 in which Z is hydrogen, S is a chain extended solid support, and B 'is an adenine, guanine or cytosine residue of formulas 2, 3 or 4, respectively, wherein R 1 is isopropoxyacetyl, or B' is a thymine residue of formula 5, is condensed in the presence of a coupling agent with a nucleoside of formula 6, wherein Z is a group protecting a 5'-hydroxy function, such as a dimethoxytriphenylmethyl group, R2 is a trifluoroethoxy group, R3 is a diisopropylamino, diethylamino or morpholino group, m = O, and B 'is as defined above, The condensation angle is oxidized with iodine or peroxides, and after deprotection by acid hydrolysis of the 5'-hydroxyl group, another nucleoside of formula 6 is condensed, in which all symbols have the above meaning, the obtained oligonucleotide is cleaved from the support and purified, and the function-blocking group is The 5'-hydroxyl is removed from the last nucleotide before the product is cleaved from the support or after it is purified. 4. 3GTTCGGACATCG ⁵ 4. The method according to p. The process of claim 3, wherein in the step of cleaving the oligonucleotide from the support, groups that block amino functions are simultaneously removed. The subject of the invention is a process for the preparation of oligonucleotides of general formula I, in which R2 is an OH group, an ethoxy group or a trifluoroethoxy group, Y is an oxygen atom, m = 1, n is 0-200, and B is an adenine, guanine or cytosine residue of Formulas 2,3 or 4, respectively, wherein R 1 is hydrogen or B is a thymine residue of formula 5 with the proviso that all B are not thymine. Oligonucleotides of formula I, in which R2 represents a hydroxyl group, and the remaining symbols have the meaning given above, are fragments of genes encoding protein biosynthesis, foreign to the pool of natural proteins of the biosynthetic cell. Oligonucleotides of the formula 1, wherein 157 777 3 R2 is ethoxy or trifluoroethoxy are used in studies of the interaction of oligonucleotides with proteins. Among the oligonucleotides according to the invention, the oligonucleotides of the formula I in which R2 is a trifluoroethoxy group have not been prepared so far. A known method of synthesizing oligonucleotides of formula I, in which R2 represents a hydroxyl or ethoxy group, and the remaining symbols have the above meanings, on a solid support, is that in a support-bound nucleoside of formula 7, in which Z is a group protecting a function hydroxy, preferably a dimethoxytriphenylmethyl group, S is a chain extended solid support, and B 'is a thymine residue of formula 5 or an adenine, guanine or cytosine residue of the formulas 2,3 or 4, respectively, in which Ri is a benzoyl or isobutyryl group, the group is deprotected 5'-hydroxyl and condensed with a nucleoside phosphite of formula 6, where Z and B 'are as defined above, m = 0, R2 is 2-cyanoethoxy, ethoxy or trifluoroethoxy and R3 is diisopropylamino, diethylamino or morpholino in the presence of a coupling agent to give a compound of formula 8, wherein S, Z, B 'are as defined above, R2 is 2-cyanoethoxy, ethoxy or trifluoroethoxy, m = 0. Optionally unreacted compound of formula 7, where Z is hydrogen and B ', S and R are as defined above, the 5'-hydroxy group is blocked to prevent formation wrong sequences. The obtained compound of formula 8 is oxidized to the compound of formula 8, where m = 1, Y is oxygen, and the remaining symbols are as defined above, the 5'-hydroxyl group is deprotected by acidic hydrolysis and the next nucleoside phosphite of formula 6 is attached. , in the presence of a coupling agent, repeating the first cycle of reactions until the desired oligonucleotide is formed. In the presented known method, after obtaining the oligonucleotide of the desired sequence, it is cleaved from the solid support by treatment with a concentrated aqueous ammonia solution for 2 hours. Under these conditions, the 2-cyanoethyl group is removed. In the next step, groups which block the amine function, i.e. the benzoyl or isobutyryl group, are removed by heating the synthesis product in concentrated aqueous ammonia solution for 16 hours at 50-60 ° C or by keeping it at room temperature for 48 hours. The use of elevated temperature makes it necessary to use sealed vessels that meet the requirements for working with biologically active materials and at the same time are resistant to increased pressure. Moreover, the difference in the rate at which the benzoyl and / or isobutyryl groups in adenine, guanine and cytosine are hydrolyzed results in oligonucleotides with incompletely deblocked amino groups, which adversely affects the yield and purity of the product. The severe temperature and time conditions in which the amino function protecting groups are removed prevent the preparation of compounds of formula I in which R2 is trifluoroethoxy, while compounds of formula I in which R2 is ethoxy are obtained in a low yield. Contrary to the known process, the method according to the invention allows the oligonucleotide to be cleaved from the solid support in one step, simultaneously with complete and at the same time selective deprotection of functional groups, under less drastic conditions of temperature and time, i.e. by treatment with concentrated ammonia for only 2 hours, in room temperature. The method according to the invention provides a high efficiency of a single chain extension reaction, exceeding 99.6%, and the obtained oligonucleotides are characterized by higher purity than before, which allows for obtaining pure gene fragments with the known methods of high pressure chromatography and / or electrophoresis. It has now been found that the yield and purity of the synthesized oligonucleotide depends on the protection of the amino groups. The method according to the invention is characterized in that in the known process of condensing consecutive nucleosides at the 5 'and 3' positions in the coupling agent, with subsequent oxidation of the product of each condensation, new nucleoside derivatives such as adenosine, guanosine and cytidine of the formulas 6 and 7 are used. wherein B 'is a group of the formula 2,3 or 4, in which R 1 is isopropoxyacetyl and the other symbols are as defined above, with Z representing a hydrogen atom for the compound of formula 7. 157 777 The method according to the invention consists in that the nucleoside of formula 7 in which B 'is an adenine, guanine or cytosine residue of the formulas 2,3 or 4, in which Ri is an isopropoxyacetyl group or B' is a thymine residue of the formula 5, Z is a hydrogen atom, and S is as defined above, is subjected to condensation, in the presence of a coupling agent, with a nucleoside of formula 6, in which B 'is an adenine, guanine or cytosine residue of the formulas 2, 3 or 4, in which Ri is an isopropoxyacetyl group, or B 'is a thymine residue of formula 5, m = 0, Z is a group protecting a hydroxyl function, preferably a dimethoxytriphenylmethyl group, R2 is a 2-cyanoethoxyl, ethoxy or trifluoroethoxy group, and R3 is as defined above to give a compound of formula 8, where Z represents a group protecting a hydroxyl function, and the other symbols have the meaning given above, this compound is oxidized to the compound of formula 8 where m = 1, Y represents an oxygen atom, and the other symbols are as defined above, then the 5'-hydroxy group is deprotected by acid hydrolysis and the next nuceloside of formula 6 is attached in the presence of a coupling agent, repeating the first reaction cycle until the desired oligonucleotide is formed. After obtaining the oligonucleotide with the desired sequence, it is cleaved from the solid support and purified. The group that blocks the 5'-hydroxy function is removed from the last nucleotide either before cleavage of the product from the solid support or after it has been cleaved from the support and purified. It depends on the method of cleaning. If the product is purified, for example, by high pressure liquid chromatography, then the 5'-hydroxy function is usually deblocked after purification. If, on the other hand, the purification is carried out by electrophoresis, the group that blocks the 5'-hydroxyl function is generally removed before cleavage of the oligonucleotide from the solid support. In the process according to the invention, unlike the known method, simultaneously with the liberation of the oligonucleotide from the solid support, all isopropoxyacetyl groups protecting the amine functions are deblocked and the 2-cyanoethyl group is removed to give the compound of formula 1 in which R2 is OH. When R2 is ethoxy or a trifluoroethoxy group, these groups are retained during the deblocking of the amino groups and the release of the product from the carrier. Cleavage of the oligonucleotide with simultaneous removal of said blocking groups is preferably carried out by treatment with a 25% ammonia solution for 2 hours at room temperature. In the method of the invention, the formation of the internucleotide bond is catalyzed by a weak acid coupling agent, preferably tetrazole. The oxidation of the internucleotide phosphite group in the compound of formula 8 is carried out with an aqueous-organic iodine solution or with organic peroxides such as e.g. t-butyl hydroperoxide. Cleavage of the protecting group for the 5'-hydroxy function is carried out with strong acid, preferably dichloroacetic acid. The carrier used is an inorganic or organic material with controlled granulation and porosity, preferably glass with controlled granulation and porosity. A properly blocked nucleoside is covalently bound to the carrier by an organic chain consisting of carbon, nitrogen and oxygen atoms. An example of a nucleoside bonded to a solid controlled-porosity (500Å) glass support with an amine chain extended is shown in Formula 9, where CPG is a controlled porosity glass, Z is a dimethoxytriphenylmethyl group, and B 'is a group of formula 2, 3, 4 or Wherein Ri is isopropoxyacetyl. A method for the preparation of novel nucleoside derivatives such as adenosine, guanosine and cytidine derivatives with amine functions blocked with an isopropoxyacetyl group is described in patent application No. P 273 722 published in BUP 8/90. As already mentioned, the method according to the invention ensures the complete removal of the amino function blocking groups together with the 2-cyanoethyl group during the cleavage of the product from the solid support, so that the obtained crude oligonucleotides are of higher purity than before, which allows for the preparation of high pressure chromatography and / or or electrophoresis, with higher yields, of pure oligonucleotide fragments used for gene synthesis. The oligonucleotides according to the invention have been used, for example, for the synthesis of a gene encoding the biosynthesis of a cytotoxic TNF protein. The process of the invention is illustrated by the following Examples, of which Examples 1-8 relate to the preparation of starting materials. Example I A. N6-isopropoxyacetyl-2'-deoxyadenosine. 50 mmoles of 2'-deoxyadenosine were dried under high vacuum in an oil bath at 50 ° C for 6 hours, then suspended in 80 ml of anhydrous pyridine and 250 mmoles of trimethylchlorosilane were added with stirring at room temperature. After 2 hours, 75 mmol of isopropoxyacetic anhydride was added and stirred for 2 hours. The precipitate of pyridine hydrochloride was filtered off, washed with 20 ml of benzene, the filtrates were combined and concentrated to about 40 ml, then 50 ml of dioxane and 30 ml of water were added. Stirred for 2 hours. The reaction mixture was concentrated to dryness. The oil obtained was purified on a gel column (Merck 60) using the eluent: chloroform / methanol 99: 1 - 90:10. The actual fractions were concentrated to dryness. The product was crystallized from chloroform, mp 134-135 ° C, Rf - 0.52 (CH 3 CN: H ₂ O 85:15), 76% yield, elemental analysis consistent with theoretical. B. 5'-dimethoxytriphenylmethyl-N6-isopropoxyacetyl-2'-deoxyadenosine. The compound obtained in step A (2 mmol) was dried by evaporation of anhydrous pyridine (5 ml) and suspended in 10 ml of anhydrous pyridine. 4,4'-Dimethoxytriphenylmethyl chloride (2.2 mmol) was added. The reaction mixture was stirred at room temperature for 5 hours. 2 ml of methanol was added and concentrated to dryness. The crude product was purified on a Merck 60H gel column, eluting with chloroform / methanol 99: 1 containing 0.1% pyridine, Rf-0.68 (CHCDCH3OH 90: 10 / HPTLC. Elemental analysis consistent with theory. Yield 76%) . C. N6-isopropoxyacetyl-5'-dimethoxytriphenylmethyl-2'-deoxyadenosine-3'-O- (0-2-cyanoethyl) -N, N-diisopropylphosphoramidite. The compound obtained in step B (1 mmol) and 1-H-tetrazole (1 mmol) were dried by evaporation of anhydrous pyridine (3 ml) and for 2 hours under high vacuum. Dissolved in anhydrous methylene chloride (3 mL) and 0-2 cyanoethyl N'-tetraisopropyl diamidophosphite (1.1 mmol) was added with stirring. Stirring was continued for 1 hour at room temperature. The reaction mixture was concentrated to dryness, purified on a Merck 60 gel column, eluting with benzene / methyl chloride / triethylamine 8: 1: 1. The product was obtained in a yield of 80%. Rf = 0.75 and 0.62 (benzene / CH ₂ Cl ₂₎ (CH3CH2 / 3N) (60:16:16) δ ppm ^ P NMR 149.73, 149.46 (CD 3 CN) 85% H3PO4. Example II A. N ₂ -isopropoxyacetyl-2'-deoxyguanosine. Proceeding analogously to Example IA, except that after adding dioxane and water, it was stirred for 12 hours, and the product was crystallized from the dioxane / ethyl ether 1: 1 mixture, the title compound was obtained with a yield of 81%, melting point> 200 ° C (with decomposition ); Rf = 0.50 (CH 3 CN · H 2 O 85:15); elemental analysis consistent with theoretical. B. 5'-dimethoxytriphenylmethyl-N2-isopropoxyacetyl-2'-deoxyguanosine. By proceeding analogously to example 1B, the product was obtained in 71% yield, Rt = 0.53 (CHCl3 · CH3OH 90:10) HPTLC. Elemental analysis consistent with theoretical. C. N2-isoporpoxyacetyl-5'-dimethoxytriphenylmethyl-2'-deoxyguanosine-3'-O- (0-2-cyanoethyl) -N, N-diisopropylphosphoramidite. By proceeding analogously to example IC, the product was obtained in 82% yield, Rf = 0.37 (benzene / methyl chloride / triethylamine 60:16:16), δ ppm ³¹ P NMR 149.86, 149.46 (CD3CN) 85% H3PO4. Example III A. N4-isopropoxyacetyl-2'-deoxycytidine. By proceeding analogously to Example 1, except that the crystallization was carried out in carbon tetrachloride, the product was obtained with a yield of 87%, Rf-0.59 (CH3CN: H2O 85:15); mp 157-158 ° C. Elemental analysis consistent with theoretical. 157 777 B. 5'-dimethoxytriphenylmethyl-N ₄ -isopropoxyacetyl-2'-deoxycytidine. The product was obtained in a yield of 80%, Rf-0.75 (CHCl2: CH3OH 90:10) by HPTLC by proceeding analogously to example 1B. Elemental analysis consistent with theoretical. C. N ₄ -isoporpoxyacetyl-5'-dimethoxytriphenylmethyl-2'-deoxycytidine-3'-O- (0-2-cyanoethyl) -N, N-diisopropylphosphoramidite. By proceeding analogously to Example 1 C, the product was obtained in 80% yield, Rt - 0.62 (methylene chloride / triethylamine 84:16), <ppm ^ P NMR '149.66, 149.52 (CD3CN) 85% H3PO4. Example IV A. 5'-dimethoxytriphenylthymidine. Proceeding analogously to the example IB. the product was obtained in 82% yield, Rf-0.65 (CHCl2 CHaOH 90:10) HPTLC. B. 5'-dimethoxytriphenylthymidine-3'-O- (0-2-cyanoethyl) -N, N-diisopropylphosphoramidite. By proceeding analogously to example IC, the product was obtained in 81% yield, Ri-0.68 (benzene / methylene chloride / triethylamine 60:16:16), <ppm 3 ¹ P NMR 148.5, 148.1 (CH2Cl2). Example 5 5'-dimethoxytriphenylmethyl-N6-isopropoxyacetyl-2'-deoxyadenosine bound to a solid support. 10 mmol of the compound obtained in Example 1B, 10 mmol of succinic anhydride and 10 mmol of dimethylaminopyridine were dried under high vacuum for 2 hours, 10 ml of anhydrous pyridine were added and stirred for 12 hours at room temperature. Concentrated to dryness, the product was purified on a gel column (Merck 60), eluted with chloroform: methanol 100: 5. 26 mmol of product, 28 mmol of 4-nitrophenol and 31 mmol of dicyclohexylurea were dried for 2 hours under high vacuum. Under anhydrous conditions, 3 ml of methylene chloride was added, stirred for 1 hour. The product in 1 ml of triethylamine was added to 1 g of a controlled porosity glass with a chain consisting of carbon, nitrogen and oxygen, stirred for 20 hours, filtered and washed with methanol and pyridine. The product was obtained with a content of 35 µmol per 1 g of solid support. Example VI. 5'-dimethoxytriphenylmethyl-2'-deoxyguanosine bound to a solid support. By analogy with Example B, the product was obtained with a content of 41.3 µmol per 1 g of solid support. Example VII. 5'-dimethoxytriphenylmethyl-2'-deoxycytidine bound to a solid support. By analogy with Example 5, the product was obtained with a content of 39.8 µmol per 1 g of solid support. Example VIII. 5'-dimethoxytriphenylmethylthymidine bound to a solid support. The product was obtained analogously to Example 5 with a content of 33.8 µmol per 1 g of solid support. Example IX. Generation of the fragment with the sequence ³ GACTACCACA ⁵ . This fragment is prepared according to the scheme where the following abbreviations are used: A-adenosine, G-guanosine, T-thymidine, C-cytidine, IPA-isopropoxyacetyl group, DMT-dimethoxytriphenylmethyl group, S-solid support, X-group -CH2CH2CN, with - diisopropylamine group. The cycle of one nucleoside attachment is as follows. 5 ml of 2% dichloroacetic acid in methylene chloride was passed through a 110 µl column protected on both sides with filters, containing 0.5 µmol of nucleoside bound to a solid support for 1 minute, washed with acetonitrile (5 ml) and dried. under argon and then under high vacuum. 50 µl of a 0.2 molar solution of the appropriate nucleoside in acetonitrile and 60 µl of a 0.5 molar solution of tetrazole in acetonitrile were added to the column thus prepared. The reaction was allowed to proceed for 2.5 minutes then washed with acetonitrile (5 ml) and dried under argon. Then 0.5 ml of A + B (1: 1) solution was passed through the column for 1 minute (A - 20% solution of methoxyacetic anhydride in tetrahydrofuran, B - 9 g of dimethylaminopyridine, 30 ml of 2,6-lutidine and 70 ml of tetrahydrofuran) . The column was washed with acetonitrile (5 ml) and dried under argon. 0.5 ml of a solution of iodine, water, 2,6-lutidine and tetra157 777 hydrofuran (2.6 g, 20 ml, 40 ml, 40 ml) was passed through the column for 0.5 minutes. The column was washed with acetonitrile (5 ml) and dried under argon. A solution of dichloroacetic acid in methylene chloride (3% by volume) was then passed through to unlock the 5'-hydroxyl function. The above cycle was repeated until a fragment with the sequence ³ GACTACCACA ^{5 was} obtained. After removal of the protecting group for the 5'-hydroxyl function of the last nucleotide, 1 ml of 25% aqueous ammonia was passed through the column for 2 hours. The product was concentrated to dryness and purified by 20% polyacrylamide gel electrophoresis. Elution from the gel was carried out using 0.4 ml of a solution with the following composition: 0.5 M CH3COONH4, 10 mM MgCl2, 10 mM sodium ethylenediaminetetraacetate, 0.02% NaN3, for 12 hours, and then separated from the salt on a preparative RP C18 gel ( 55-105pm) in a water-acetonitrophyl gradient. The product fraction was concentrated to dryness. Example 10 The procedure was analogous to that in Example IX, except that the generated fragment with the sequence given in Example IX was cleaved from the resin before the 5'-hydroxyl protecting group was removed from the last nucleotide. Cleavage from the bed was carried out by passing 1 ml of 25% aqueous ammonia solution for 2 hours. Then the product was concentrated to dryness and purified by high pressure liquid chromatography on RP, μ gel. Bondapak C18 on a water-acetonitrile gradient. The product fraction was concentrated to dryness. The 5'-hydroxy fraction was deprotected in acetic acid - water (4: 1 v / v) 0.5 ml solution for 45 minutes at room temperature. The product was then concentrated to dryness and purified once more by high pressure liquid chromatography under the same conditions. The following fragments were obtained in an analogous manner to that described in Example 9: 5. 3GGTACAACATC ⁵ 6. 3GTTTGGGAGTTC ⁵ 7. ^{3 5} gactccccgtcg 8. ³ acgtcgacggg ⁵ 9. ³ gagtcgaactc3 10. ³ CCAAACGATGT3 11. 3TGTACCCGATGT3 12. 3CCGAACAGTGA3 13. 3GCCCCAAGATC3 14. 3GGTTCAGCCACTGCA3 15. ³ GGAGGGCATTGGCCCGGC3 16. ³ GCTCCACGCCATTGGCCA3 17. ³ CACCAGCTGGTTATCTCTCA3 18. 3ACCAGCTGGTGGTAC3 19. ³ GCGTGGAGCTGAGATA? 20. ³ aatgccctcctggccaatg ⁵ 21. ³ GTGGCTGAACCGCCGGGCC3 22. ³ catggtagtctcccggacatgg ⁵ 23. 3AGTAGATGAGGGTCCAGGAGAAG3 24. ³ TTCCCGGTTCCGACGGGCAGCT5 25. ³ GCCCGTCGGAACCGGGAACTTCT ⁵ 26. 3CCTGGACCCTCAT3 27. ³ CTACTCCATGTCCGGGAGACTAC5 28. ³ gggtacacgaggagtgggtgtg ⁵ 29. ³ GTAGTCGGCGTAGCGGCAGAGGA3 30. ^ TGGTCTGGTTCC ⁵ 31. 3AGTTGGAGGAGAGACGTCAG5 32. ³ CGTCTCTCCTCCAACTGGAACCA3 33. ³ GACCATCCTCTGCCGCTACGCC3 34. 3CCCACTCCTCGTGTACCCAGCT5 35. 3TTCTCGGGGACGGTCTCCCTCTG3 36. ³ GGGTCTCCCCCGACTCCGGTTCG5 37. ³ GGACCATACTCGG3 38. ³ GTAGATAGACC'CTCCCCAGAA3 39. ³ GGTCGACCTCTTCCCACTGGCTG5 40. 5AGTCGCGACTIT ⁵ 41 3AGTTAGCCGGGCTGATAGATC ⁵ 42. 3ATTCAGCCCGGCTAACTAAAGTC5 43. ³ GCGACTCAGCCAGTGGGAAGA3 44. ³ GGTCGACCTTCTGG5 45. ³ GGAGGGTCTATCTACCCGAGTA5 46. ^ TGGTCCCGAACCGGAGTCGGGG ⁵ 47. ³ GAGACCCCAGA3 48. ³ GGGAGACCGTCCCCGAGAACTAG3 49. 3TGAAAGGGCTCAGACCCG ⁵ 50. ³ TCCAGATGAAACCCTAGTAACGGGACACTATCCTAC. ⁵ 51. ³ GATAGTGTCCCGTTACTA3 52. ³ GGGTTTCATCTGGACGGGTCTGAGCCGTATCAGATC5 157 777 '“θ''ο-ομγ ο w ο _ο ' i * _Λ Ρ · Ο 'Ό-ΟΜΤ _e * η Λ ^Μ £ ** (§ ί'?, ** θ '- ®0 ^{/ β} ' 0-Ρ-0 'Ό-Ρ-0 Ο-ΟΜΤ ^ θ' Ό-Ρ-0 ΟΗ Ο ¹ χ Ο Λ ρ-ο Ο-ΟΜΤ 9 Α? · * ~ Ϊ́Αα Μ ϊ Ο Ο-Ρ-0 Ο-Ρ-0 O-DMT ή Ó ο ο, ι •; χ »ν X SCHEME - / ΡΛ 6? 'α _ρ , ο AND Ο AND X ΙΡΑ Α ⁰ ΙΡΑ C ο ΙΡΑ Α ο JPA JPA ΙΡΑ BPA JPA A ο C ο Α • 0'Ό ·, Ο 'X, ο) β'0 *, 0 'Ό'Ο' ΌΑρ, Ο 'Ό ^ ρ.Ο'Ό ^ ρ.Ο' - _0Η 'χρχ' Ο