Nucleoside heterocycle that binds to both thymidine and cytidine

Abstract

This application discloses nucleoside analogs that when incorporated into a oligonucleotide, forms a nucleobase pair with either thymidine or cytidine that are present in a complementary strand at the paired position. These analogs are called purine biversals. Such compounds and their associated processes have utility in processes that detect complementary oligonucleotides, in particular oligonucleotides from natural sources and in complex biological mixtures, where the sequence of the complementary oligonucleotide being detected is not known precisely. The invention also includes compositions of matter that are oligonucleotides that contain purine biversals of the instant invention, and duplexes of said oligonucleotides.

Claims

1. A compound having the structure ##STR00001## wherein X is either H or OH and Y is H, phosphate, diphosphates, or triphosphate.

2. The compound of claim 1, wherein X is H.

3. An oligonucleotide chain that contains within it a nucleotide having the structure, ##STR00002## wherein X is either H or OH and Y is either H, phosphate, diphosphates, or the point of attachment of the remainder of the oligonucleotide chain to said nucleotide.

4. The oligonucleotide chain of claim 3, wherein X is H.

5. A duplex between an oligonucleotide chain that contains within it a nucleotide unit having the structure ##STR00003## wherein X is either H or OH and Y is either H, phosphate, diphosphates, or the point of attachment of the remainder of the oligonucleotide chain to said nucleotide, and a substantially complementary oligonucleotide, wherein said duplex includes at least one Watson-Crick pair between said nucleotide unit in said oligonucleotide chain and a thymidine or a cytidine in said substantially complementary oligonucleotide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. The pyrimidine (left) [Lin, P. K., Brown, D. M. (1989) Synthesis and duplex stability of oligonucleotides containing cytosine-thymine analogues. Nucl Acids Res. 17, 10373-10383] and purine (right) biversals [Brown, D. M., Lin, P. K. T. (1991) Synthesis and duplex stability of oligonucleotides containing adenine-guanine analogs. Carbohydrate Res. 216, 129-139] presently known in the art.

(2) FIG. 2. The heterocycle of the instant invention, where are is the point of attachment of the heterocycle to a 2-deoxyribose, a ribose, or the oligonucleotide of the instant invention.

(3) FIG. 3. Synthetic route for creating the heterocycle to be attached to the carbohydrate. Attaching a side chain to the heterocycle-2-deoxyriboside.

(4) FIG. 4. Synthetic route for attaching the heterocycle to a carbohydrate derivative, and adding and iodine to it to prepare for the addition of a sidechain.

(5) FIG. 5. Synthetic route for addition of a sidechain and preparation of the sidechain for the formation of the tricyclic heterocycle-2-deoxyriboside.

(6) FIG. 6. Synthetic route for the formation of the tricyclic heterocycle-2-deoxyriboside and its transformation to give protected phosphoramidites suitable for incorporation into oligonucleotides using solid phase synthesis.

(7) FIG. 7. UV spectrum of protected species, with a .sub.max=291 nm

(8) FIG. 8. UV spectrum of unprotected species; .sub.max=301 nm (=5,050), 270 nm (=4,950), 237 nm (=23,700).

(9) FIG. 9. Two tautomeric forms and their proposed pairs. R is the point of attachment to the carbohydrate in the DNA or RNA oligonucleotide.

DETAILED DESCRIPTION OF THE INVENTION

(10) The specific invention covers ribonucleosides, 2-deoxyribonucleosides, and sugar analogues (for example threose, fluoro sugars, 2-O-methoxy sugars, and others known in the art) that have the heterocycle shown in FIG. 2 attached to the carbohydrate, instead of a standard heterocycle. This system has two tautomeric forms, one with hydrogen atoms placed so it is complementary in the Watson-Crick sense to thymine and its analogs, and the other that is complementary to cytosine and its analogs.

(11) In addition, this invention concerns oligonucleotides that contain this heterocyclic system, either embedded within the oligonucleotide chain, or at the 5-end of the oligonucleotide (where its 5-hydroxyl group can be free, or esterified with a monophosphate, diphosphate, or triphosphate group), or an oligonucleotide synthesized by solid phase phosphoramidites chemistry following procedures well-known in the art using phosphoramidites described in Example 1. Likewise, this invention covers processes that exploit these purine biversals as monophosphates, diphosphates, and triphosphates for enzymatic reactions, and processes that exploit oligonucleotides containing them, without limitation, in products and processes for binding to substantially complementary oligonucleotides, including those from natural sources, and especially where the precise sequence of the complementary oligonucleotide being detected is not known precisely.

(12) Further, the invention includes compositions of matter that are building blocks for oligonucleotides that contain these purine biversals,

(13) The presently preferred implementation are the structures shown in the Figures, prepared as described in Example 1. As I well known in the art, functionally equivalent species can be obtained by derivatization in the minor groove. The procedure in Example 1 can be modified by methods well known in the art to make the corresponding D-ribonucleoside, as well as the enantiomeric species.

Example 1

(14) The synthesis of a purine biversal as a 2-deoxyribonucleoside is described.

(15) Step 1. Creation of the heterocycle (FIG. 3). To a solution of 2,4-diamino-6-hydroxypyrimidine (1, 25.2 g) in DMF (480 mL) and water (80 mL) was added sodium acetate (27.2 g). The resulting yellow solution was stirred for 1 h. To the solution was added chloroacetaldehyde (50% solution, 25.4 mL) and the mixture was stirred at rt for 2 days. The volatiles were removed in vacuo and the residue was mixed with methanol (70 mL) and stored at rt overnight. The resulting solid was filtered. The solid was mixed with methanol (150 mL) and heated at 60 C. for 10 min and cooled to rt overnight. The resulting solid was filtered and dried. The yield of 2 was 15 g to 19 g.

(16) A mixture of 7-deazaguanine (14.2 g) and dimethylaniline (6 mL) in POCl.sub.3 (200 mL) was refluxed for 3 h (bath temp. 130 C.). After cooling to rt, volatiles were removed by distillation (bath temp 60 C.). The residue was mixed with water (2300 mL) and neutralized with ammonium hydroxide until complete precipitation of solid (pH4). The resulting precipitate was filtered and further purified by column chromatography (silica, MC:MeOH=10:1) to give 3.6 g (21.4 mmol, 23%) of solid (FIG. 3).

(17) Step 2. Creating the nucleoside analog in preparation for addition of a side chain (FIG. 4). To a mixture of powdered KOH (2.6 g) in acetonitrile (150 mL) was added TDA-1 (0.27 mL) and the mixture was stirred for 5 min. To this mixture was added the amino chloro 7-deazapurine (3, 3.6 g) and the mixture was stirred for 5 min; the chloro-sugar (4, 9.1 g) was then added to the mixture. Stirring was continued for another 40 min (reaction complete by TLC). The mixture was adsorbed on silica gel and eluted with MC/EA until all the product was eluted. The filtrate was evaporated with silica gel and purified on silica column (MC:EA=20:1 to 10:1) to give a white solid (9.0 g, 17.3 mmol, 80%) which contains small amount of impurities. It was used for the next reaction without further purification (The actual yield could be around 75%)

(18) A mixture of the starting material (9.6 g) and pivaloyl chloride (11.5 mL) in pyridine (95 mL) was stirred at rt for 2 h. TLC showed reaction was complete (Almost one spot, which is higher than starting material). Volatiles were removed by rotary evaporation and the residue was dissolved in dichloromethane/aq. HCl and extracted with dichloromethane. The organic layer was dried and purified on silica column to give a white solid (solvent is not easily removed by evaporation). It was dissolved in DMF (150 mL) and treated with N-iodosuccinimide (NIS, 6.2 g) and stirred at rt overnight. The mixture was mixed with water and extracted with dichloromethane. The organic layer (contained some DMF) was evaporated and purified on silica column.

(19) Rf=0.3 (MC:Hx:EA=1:1:0.2) After column chromatography, the appropriate fractions were combined and evaporated to give white solid. It was treated with ethyl acetate/hexane and the resulting solid was filtered and dried. The filtrate was evaporated and stored at rt overnight. This was treated with ethyl acetate/hexane and the resulting solid was filtered. The filtered solids were combined. The filtrate was analyzed by TLC to confirm the existence of the product. The total yield was around 9.7 g, 13.3 mmol, 72%.

(20) Step 3. Forming a tagged heterocycle and its manipulation (FIG. 5). To a mixture of the starting material (6, 15.8 g) and Pd(PPh.sub.3).sub.4 (4.7 g) in toluene (300 mL) was added allyl tributylstannane (16.2 mL) and the mixture was heated at 95 C. for 18 hours. After cooling to rt, the volatiles were removed by rotary evaporation and the residue was purified on silica column (MC to MC:EA) to give 7.7 g of white solid (7, 11.9 mmol, 55%).

(21) To a mixture of starting material (7, 7.7 g) and NMO (1.68 g, 1.2 eq) in acetone (390 mL) and water (65 mL) was added osmium tetroxide (4% in water, 1.5 mL, 0.02 eq) and the mixture was stirred at rt for 24 hours. The mixture was mixed with water and extracted with dichloromethane. Purification on silica column (Hx:EA=1:1 to EA 100%) gave white solid. It was dissolved in THF/H.sub.2O (300 mL/150 mL) and treated with sodium periodate. Stirring was continued for 30 min and mixed with water and extracted with dichloromethane. Purification on silica column (MC:Hx=1:1 to MC:Hx:EA=3:3:2) gave an intermediate as a white solid. This white solid was dissolved in MeOH/THF (150 mL: 150 mL) then treated with sodium borohydride. The mixture was stirred at rt for 20 min (reaction complete). The mixture was mixed with water and extracted with dichloromethane to give 8. Purification of 8 on silica column (MC:Hx:EA=1:1:2) gave 4.3 g (6.6 mmol, 55% for 3 steps from the allyl compound) of white solid. Mp; 163164 C.

(22) This compound was then mesylated (FIG. 5). To a solution of starting material (8, 4.3 g) in dichloromethane (100 mL) and pyridine (7 mL) was added methanesulfonyl chloride (3.5 mL). The mixture was stirred at rt for 2 hours. The mixture was washed with diluted HCl and purified on silica column, EA:Hx=1:2 to 1:1 (Rf=0.5, EA:Hx=1:1) to give a white solid. 4.7 g, 6.46 mmol, 97%. Mp; 6667 C.

(23) This was then prepared for cyclization. A mixture of starting material (9, 4.0 g, 5.5 mmol), hydroxylamine hydrochloride (3.82 g, 55 mmol) and trimethylamine (18 mL) in methanol (180 mL) and dioxane (180 mL) was stirred at 45 C. for 24 hrs. The mixture was mixed with water and organic solvents were removed by rotary evaporation. The mixture was extracted with ethyl acetate. The organic layer was evaporated and purified on silica column, EA:Hx=1:1 to give a white solid. 2.6 g, 3.6 mmol, 65%, mp; 82-83 C.

(24) Step 4. Cyclization and conversion to deprotected nucleoside and phosphoramidite suitable for solid phase DNA synthesis (FIG. 6). To a solution of starting material (2.6 g, 3.6 mmol) in DMF (150 mL) was added potassium tert-butoxide (605 mg, 5.4 mmol). The mixture was stirred at rt for 8 hours. The mixture was mixed with water and extracted with ethyl acetate. The organic layer was evaporated and purified on silica column, MC:EA=5:1 to give a white solid (1.3 g, 2.1 mmol, 58%). This material could not be obtained pure form and contained some impurities. It was dissolved in saturated ammonia in methanol (50 mL) and stirred at rt for 24 hours. Volatiles were removed by rotary evaporation and the residue was purified on silica column, EA to EA:MeOH=10:1 to give a white solid, mp; 5859 C., 490 mg, 1.25 mmol, 35%

(25) For deprotection of the heterocycle (FIG. 6), a solution of the starting material (11, 25 mg) in methanol (0.2 mL) was mixed with ammonium hydroxide (2 mL) and the mixture was heated at 55 C. overnight. Volatiles were removed by rotary evaporation and the residue was purified on silica column (EA to EA:MeOH=8:1) to give a white solid, mp; 113115 C., 13 mg, 0.042 mmol, 66%.

(26) For preparation of the phosphoramidite, to a mixture of the starting material (500 mg, 1.28 mmol) and imidazole (436 mg, 6.4 mmol) in dichloromethane (15 mL) was added TBDMSCl (578 mg, 3.83 mmol) and the mixture was stirred at rt for 6 hrs. (Extended reaction gave tri-TBDMS product) It was mixed with water and extracted with dichloromethane. The organic layer was dried and evaporated, purified on silica column gave di-TBDMS product. This was dissolved in dichloromethane (15 mL) and pyridine (0.6 mL) and treated with acetic anhydride (0.4 mL). The mixture was stirred at rt for 30 min. It was mixed with diluted HCl and extracted with dichloromethane. The organic layer was dried and evaporated, purified on silica column, EA:Hx=1:1 to give a white solid, mp; 9293 C., 466 mg, 0.70 mmol, 55%.

(27) A mixture of the starting material (420 mg, 0.634 mmol), TBAF (2.5 mL, 1M in THF) and acetic acid (0.25 mL) in THF (20 mL) was stirred at 55 C. for 6 hours. The mixture was mixed with water and THF was removed by rotary evaporation. The residue was extracted with dichloromethane and purified on silica column, EA:MeOH=10:1 to give a white solid. It was dissolved in dichloromethane (10 mL) and 4-methylmorpholine (0.2 mL) and treated with dimethoxytrityl chloride (230 mg). The mixture was stirred at rt for 20 min and poured into water and extracted with dichloromethane. The organic layer was dried and evaporated, purified on silica column, EA:Hx=2:1 to EA 100% to give a white solid, mp; 11012 C., 380 mg, 0.516 mmol, 81%.

(28) Finally, to a mixture of the starting material (14, 365 mg, 0.496 mmol) and 4-methylmorpholine (0.2 mL) was added 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.2 mL). The mixture was stirred at rt for 30 min and poured into water and extracted with dichloromethane. The organic layer was dried and evaporated, purified on neutral silica column, EA:Hx=1:1 to 2:1 to give a white solid (15) mp; 7374 C., 390 mg, 0.416 mmol, 84%.

(29) Developing Deprotection Conditions for the Biversal Purine Base in Oligonucleotides Synthesized by Solid Phase Phosphoramidite-Based Synthesis.

(30) To a solution (20 L) of protected nucleotide methanol was added ammonia solution (400 L) or potassium carbonate (0.05 M in methanol, 69 mg/10 mL, 400 L). The mixture was incubated in various conditions. Small portion (20 L) of the mixture was diluted with 25 mM TEAA (180 L) and analyzed by rp HPLC. The results are shown below.

(31) 1. Ammonia solution at room temperature

(32) TABLE-US-00001 Result (HPLC peak integration) Deprotected Protected base Temperature Time (11.7 min) (14.6 min) 1 NH.sub.4OH rt 1 day 468 292 2 NH.sub.4OH rt 3 days 725 111
Deprotection with ammonia solution at room temperature proceeded very slowly. Even after 3 days15% of starting material was left unchanged.
2. Ammonia solution at 55 C.

(33) TABLE-US-00002 Result (HPLC peak integration) Deprotected Protected base Temperature Time (11.7 min) (14.6 min) 3 NH.sub.4OH 55 C. 1 h 323 356 4 NH.sub.4OH 55 C. 2 h 462 221 5 NH.sub.4OH 55 C. 4 h 643 94 6 NH.sub.4OH 55 C. 6 h 545 43 7 NH.sub.4OH 55 C. 22 h 439 0

(34) Deprotection with ammonia solution at 55 C. proceeded much faster than the deprotection at room temperature. After 6 hours, 10% of starting material was left and the reaction was complete with overnight incubation. However, the amount of deprotected compound decreased with longer incubation without any noticeable side products. This implies the biversal purine decompose with long incubation in ammonia solution, but the decomposed products are not detectable by UV detection.

(35) 3. Potassium carbonate in methanol solution at 55 C.

(36) TABLE-US-00003 Result (HPLC peak integration) Deprotected Protected base Temperature Time (11.7 min) (14.6 min) 8 K.sub.2CO.sub.3 55 C. 1 h 716 166 9 K.sub.2CO.sub.3 55 C. 2 h 981 46 10 K.sub.2CO.sub.3 55 C. 4 h 1052 0 11 K.sub.2CO.sub.3 55 C. 6 h 1100 0 12 K.sub.2CO.sub.3 55 C. 18 h 913 0

(37) In 0.05 M potassium carbonate in methanol, the complete deprotection occurred in less than 4 hours. But, overnight incubation resulted decreased amount of deprotected compound. The optimum condition is 46 hours at 55 C.

(38) Ammonia treatment of the protected biversal purine nucleoside was slow (at room temperature) or decomposed (at 55 C.). However, the deprotection with potassium carbonate in methanol was complete in 4 hours and did not show decomposition. These conditions are applied to oligomer containing biversal purine.

(39) Stability of Duplexes Containing the Purine Biversal Base, Compared with the 6(N)Methoxydiamino Purine of Brown [Op. Cit.].

(40) T.sub.m was measured with a solution containing each oligo (1.0 M), NaCl (100 mM) and pH 7 buffer (20 mM sodium cacodylate). The heating and cooling cycle was run from 20 C. to 85 C. with 0.5 C./min gradient.

(41) Results;

(42) TABLE-US-00004 Base Base pair pair Sequence X:Y Tm X:Y Tm 2B- 5-GAG TCT CGA CAX AGX Biversal 63 F:C 60 Int TCC CAG AGG SEQ ID purine:C NO 1 Biversal 68 F:T 62 3-CTC AGA GCT GTY TCY purine:T AGG GTC TCC SEQ ID NO 2 Biversal 63 F:G 57 purine:G Biversal 59 F:A 58 purine:A G:C 72 G:T 60 A:C 55 A:T 67 2B- 5'-GAX TCT CGA CAG AGA Biversal 58 F:C 60 Ends TCC CAX AGG SEQ ID purine:C NO 3 Biversal 65 F:T 61 3-CTY AGA GCT GTC TCT purine:T AGG GTY TCC SEQ ID NO 4 Biversal 60 F:G 57 purine:G Biversal 59 F:A 57 purine:A G:C 69 G:T 57 A:C 56 A:T 64 3B 5-GAG TCT CGX CAG AGX Biversal 56 F:C 57 TCC CAX AGG SEQ ID purine:C NO 5 Biversal 65 F:T 59 3-CTC AGA GCY GTC TCY purine:T AGG GTY TCC SEQ ID NO 6 Biversal 58 F:G 48 purine:G Biversal 54 F:A 53 purine:A G:C 71 G:T 55 A:C 51 A:T 67 4B 5-GAX TCT CGX CAG AGX Biversal 46 F:C 52 TCC CAX AGG SEQ ID purine:C NO 7 Biversal 64 F:T 54 3-CTY AGA GCY GTC TCY purine:T AGG GTY TCC SEQ ID NO 8 Biversal No F:G 50 Purine:G melting Biversal 38? F:A 45 purine:A G:C 72 G:T 49 A:C No melting A:T 65