WO1989009778A1 - Oligonucleotide containing 6-thioguanine at a predetermined site - Google Patents

Abstract

A 6-thioguanine containing phosphoramidite has been synthesized and subsequently utilized in the preparation of an oligonucleotide containing 6-thiodeoxyguanosine. The method includes steps of acylation, alkylation and phosphitylation of 6-thiodeoxyguanosine and incorporation of the resulting phosphoramidite into an oligonucleotide at a specific site. The new oligonucleotide may be used to study the effects of 6-thioguanine on replication, and on alkylation of 6-thioguanine by anticancer agents, and the possible repair of those alkylations.

Description

OLIGONUCLEOTIDE CONTAINING 6-THIOGUANINE AT A PREDETERMINED SITE

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to the formation of new phosphoramidites and oligonucleotides. In particular, this invention relates to the formation of a 6-thioguanine phosphoramidite intermediate and oligonucleotides containing 6-thioguanine inserted at particular oligonucleotide sites.

Description of the Related Art

6-Thioguanine. 6-Thioguanine (6TG) is a drug that is a thiol analog of guanine, having a sulfur substituted for the oxygen bound to the number 6 carbon in guanine. 6TG has been shown to be taken up by cells and incorporated into their DNA (Bodell, 1985). The disclosure of this reference and of all other cited publications and patents is incorporated in full by reference herein. Administ ation of the deoxyribonucleoside, 6- thiodeoxyguanine (6TdG), causes an arrest in growth after a lag period of one cell cycle (Barranco, 1971). 6TG is taken up by the cells and is converted by the purine salvage pathway into 6TdG, which is then incorporated into DNA during replication. This incorporation leads to DNA strand breaks, which may be associated with glycosylase activity that generates apurinic sites, or with a long- patch repair system (Christie, 1984). 6TG is commonly used in laboratories as a screen for cells lacking the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT). Cells deficient in HGPRT are unable to convert 6TG to 6TdG, so they do not incorporate the abnormal purine into the replicating DNA. Studies by Schwartz (1984) showed that HL-60 human acute promyelocytic cells show approximately 500 times the sensitivity to 6TG as did an HGPRT-negative subclone. 6TG is currently used in . humans for the treatment of some lymphomas (Kovach, 1986).

Stepwise oligonucleotide synthesis methods. A variety of stepwise oligonucleo i e synthesis methods employ repetition of the following steps including (a) blocking or protection of nucleotide sites in which no chemical moiety additions are desired; (b) exposure to the next desired nucleotide; and (c) removal of excess nucleotide and blocking agent. Thus, Kaufman (U.S. Patent No. 3,850,749) utilizes such a sequential synthesis with acyl nucleoside diphosphates added to an oligonucleotide. Patents of Caruthers (U.S. Patents No. 4,415,732, 4,458,066 and

4,668,777) include use of trityl groups to block unreacted hydroxyl groups, formation of nucleoside phosphoramidite compounds that are activated by acidic compounds to permit reaction with a further nucleotide to yield a polynucleotide.

The patent of Koster (U.S. Patent No. 4,725,677) provides for the preparation of oligonucleotides by (1) reaction of a nucleoside with a phosphine derivative; (2) reaction of the first product with a nucleoside bonded to a polymeric carrier; (3) oxidation of the carrier bound compound to form phosphotriester groups; (4) blocking of free primary 5'-hydroxy groups; (5) elimination of a protective group from the terminal 5^l-hydroxy group of the developing oligomer; (6) subsequent repetition to form the desired oligonucleotide; and (7) elimination of the protective groups as appropriate.

Automated oligonucleotide synthesis. Oligonucleotide synthesis of particular nucleotide sequences may be accomplished either manually or with DNA synthesizing machines. Manual techniques generally employ solid phase techniques wherein solid supports, having a particular desired 3 '-end nucleotide attached, are used to add nucleotides of the desired identity one by one. In both the manual and machine techniques, certain similar protection and deprotection steps are used to ensure addition of the appropriate nucleotide to the oligonucleotide being synthesized. Certain manual liquid phase DNA synthesis techniques are also used when large quantities of DNA are to be synthesized. See Li, 1987. DNA synthesizing machines have made the preparation of oligonucleotides having known nucleotide percent compositions and sequences a rapid and reproducible technique. For example, the Applied Biosystems Model 381A DNA Synthesizer uses as the chemistry of choice, the phosphoramidite method of oligonucleotide synthesis because of the inherently high coupling efficiencies and the stabilities of the starting materials. As in the manual solid phase technique discussed above, the growing nucleotide chain is attached to a solid support derivatized with the nucleoside which is to be the 3'-hydroxy1 end of the desired oligonucleotide product. The 5'-hydroxyl is blocked with a dimethoxytrityl group. In this particular synthesizer, the support used for DNA synthesis is Controlled Pore Glass (CPG), a porous, non-swelling particle of 125-177 microns diameter and having 500A pores. Because of the attachment to the solid phase, excess reagents present in the liquid phase may be removed by filtration prior to addition of the next reagents without the need for purification steps between base additions. The manufacturer's procedure of DNA synthesis on the Applied Biosystems machine involves the following steps:

(1) Treatment of the derivatized solid support in a reaction vessel with acid to remove the trityl group and free the 5'-hydroxyl for the addition of the nucleotide;

(2) Activation of a phosphoramidite derivative of the next nucleoside, which is blocked at the 5*hydroxyl end with the dimethoxytrityl group, with a weak acid, tetrazole, resulting in protonation of the amide-nitrogen of the phosphoramidite so that it is susceptible to nucleophilic attack;

(3) Addition of the activated phosphoramidite derivative to the reaction vessel;

(4) Capping or termination of any unreacted nucleotide chains by an acetylation process comprising the addition of acetic anhydride and 4-dimethylaminopyridine to minimize the length of impurities and facilitate purification of the desired oligonucleotide product; (5) Oxidation of the phosphite in the internucleotide linkage to phosphate using iodine as the oxidizing agent and water as the oxygen donor;

( 6 ) Removal of the dimethoxytrityl group from the nucleotide chain and repeat of the above steps until the oligonucleotide is of the desired length;

(7) Removal of the methyl groups on the phosphates when present with a thiophenol treatment (unnecessary with 2-cyanoethyl phosphoramidites) ; (8) Cleavage of the oligonucleotide chain from the solid support by treatment with ammonium hydroxide; and

(9) Treatment of the crude DNA solution in ammonium hydroxide at 55^ for 5-12 hours to remove the protecting groups on the exocyclic amines of the bases.

Detailed descriptions of the above-discussed DNA synthesizing procedures and others may be found in the user's manuals of the appropriate DNA synthesizers, in Biosystems Report, vol. 1, no. 1 (1984) (available from

Applied Biosystems) , and in the literature cited in these manuals .

See also Garegg, 1986.

The manual and automatic oligonucleotide synthesis techniques that have been developed do not comprise the addition of compounds such as 6-thioguanine to oligonucleotides at particular sites. It is therefore an object of this invention to provide a means for synthesizing oligonucleotides that comprise 6TG.

It is a further object of this invention to provide particular novel nucleotide phosphoramidites.

It is a further object of this invention to provide particular novel oligonucleotides.

It is another object of this invention to provide a DNA octamer having 6TG. It is a further object of this invention to provide oligonucleotides containing 6TG that may be used to examine the differential effect of various agents on oligonucleotides containing 6TG.

SUMMARY OF THE INVENTION

The novel phosphoramidite composition of this invention differs from compositions previously reported in that it comprises a 6TG derivative. The oligonucleotides possess nucleotide sequences where the 6TG is positioned for study in biological applications. Other aspects and features of the invention will be more fully apparent from the following disclosure and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the synthesis steps of the 6TdG- phosphoramidite.

Figure 2 is a diagram of the protected nucleotide. DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

The present invention generally comprises 6TdG- containing phorphoramidites and polynucleotides and methods of synthesis and use thereof. A preferred embodiment of the oligonucleotide of this invention comprises:

5'-CSACTCAT-3' where A is a deoxyadenosine residue; C is a deoxycytidine residue; S is a 6-thiodeoxyguanosine residue; and T is a deoxythymidine residue.

The starting nucleotide analog, 5 ' -( 4 , ' - dimethoxytrityl-N-isobutyryl-6-thio-2'-deoxyguanosine 3'- [ (2-cyanoethyl)-(N,N-diisopropyl) ]phosphoramidite (TdGPA) was synthesized from 6TdG. The 6TdG was protected by replacement of an active hydrogen atom of the pendant amino group on the purine base and hydrogen atoms of the hydroxyl groups on the deoxyribose nucleus as depicted in Figure 2 and discussed below and in the Examples.

Thus, the method employed to obtain the compounds of the invention from the 6TdG comprises the following steps: (1) The hydroxyl groups on the ribose nucleus are converted to trimethylsilyl ethers to allow for subsequent acylation to occur only at the amino position; (2) Acylation is carried out with isobutyric anhydride; (3) Trimethylsilyl groups are removed by the addition of concentrated ammonium hydroxide to yield N²- isobutyryl-6-thiodeoxyguanosine (N²-IBu6TdG) (4) The nucleotide is tritylated (alkylation of a single hydroxyl group) at the 5'-position of the ribose methylol group with 4,4'-dimethoxytrityl hydrochloride (DMTr-Cl) in the presence of a 4- dimethylaminopyridine (DMAP) catalyst and triethylamine (TEA);

(5) The remaining unreacted deoxyribose 3 '-hydroxyl group is allowed to react with chloro-N,N- diisopropylamino-2-cyanoethoxyphosphine in the presence of N,N,N-diisopropylethylamine to yield 5'-(4,4'-dimethoxytrityl-N²-isobutyryl-6-thio-2 '- deoxyguanosine 3 ' -[ (2-cyanoethyl)-(N,N- diisopropyl) ]-phosphoramidite (TdGPA); and

(6) A manual or automatic DNA synthesizing technique for sequential addition of nucleotides is used to add the TdGPA to the oligonucleotide in the desired position or positions. The actual synthesis of the desired polynucleotide sequence from selected nucleotides may be carried out by utilizing a DNA Synthesizer, such as the Applied Biosystems 381A DNA Synthesizer (Foster City, CA) or other suitable equipment or procedures. The individual phosphoramidite derived from nucleoside or deoxynucleoside residues, for example, adenosine, cytidine, guanosine, uridine, thymidine, and 6-thioguanosine, as well as the corresponding 2 ' -deoxyribonucleosides ( 6TdG in the preferred embodiment of the invention), are allowed to condense in a prescribed order. The resultant oligonucleotides can be used, for example to determine the effects of 6TG on replication as well as in studies on the alkylation of 6TG by anticancer agents, and the possible repair of those alkylations. The features and advantages of the present invention will be more clearly understood by reference to the following examples, which are not to be construed as limiting the invention. EXAMPLES

EXAMPLE I

Isobutylation. 6-Thiodeoxyguanine (6TdG) obtained from the National Cancer Institute (Bethesda, Maryland) (2.8 g or approximately 10 mmoles) was lyophilized overnight and then dissolved in 100 ml of anhydrous pyridine and cooled to 4°C. While the reaction mixture was stirred, 6.5 ml of trimethylchlorosilane (Aldrich) was slowly added and the mixture stirred for 30 minutes^• Isobutyric anhydride (8.5 ml) was then added and the mixture removed from the ice bath and stirred for 2 hours. The reaction was then cooled to 4°C and 20 ml of water added, followed 15 minutes later by addition of 20 ml of 2N ammonium hydroxide. The procedure up to this point is essentially the same as that of Jones, 1984, while the remaining steps in this procedural subsection are additions to and changes in the Jones procedure.

The sample was rotary evaporated to a volume of approximately 30 ml. When a precipitate began to form, it was allowed to settle. The supernatant pyridine was pipetted off and saved. The precipitate was washed six times with diethyl ether, using twice the volume of the precipitate for each wash volume. The ether wash was pooled with the the supernatant pyridine and dried on a rotary evaporator.

The residue from the rotary evaporation was resuspended and extracted several times with ethanol. The ethanol wash was dried on a rotary evaporator, leaving a yellow residue (N²-IBu6TdG) . For ease of handling, the remaining procedures employed a smaller aliquot of about 300 mg of this yellow residue that included excess isobutyrate salts. This was resuspended and washed twice with 200 volumes of water. After discarding the wash water, the residue was dried in a Savant Speed Vac. (Savant Instruments, Inc., Farmingdale, NY) for 24 hours. The dried residue was then dissolved in a minimal volume of methanol (preferably less than 10 ml). The methanol solution was pipetted onto a silica gel column (Merck Kieselgel 60 20-200 uM (EM Reagents, Darmstadt, Germany) of approximately 30 ml volume that had been allowed to settle in a 60 cc plastic syringe. The column was eluted with a mobile phase of 9% methanol in dichloromethane. Fractions of 7 ml were collected and checked for N²-IBu6TdG by spotting opposite an NMR-verified standard on a thin layer chromatography silica plate (J.T. Baker, Phillipsburg, NJ) run with 9% methanol/dichloromethane. The silica plates were inspected with an ultraviolet lamp. Because pyridine has a high mobility in this system, N²-IBu6TdG migrates to about 40% of the solvent front, while unreacted 6TdG has minimal mobility in this system. The fractions containing N²-IBu6TdG were pooled and 90% of the N²-IBu6TdG retained in fractions 3-8, although traces of the product persisted into fraction 24 or further. The N -IBu6TdG-containing fractions were then combined and the solvent removed on a rotary evaporator. An oily N²-IBu6TdG residue remained.

EXAMPLE II

Dimethoxytritylation of the 5' position. An aliquot of 200 mg of the N₂-IBu6TdG oil in a brown glass vial, that had been lyophilized overnight, was tritylated at the 5'- position using the method described by Jones, 1984. Anhydrous pyridine (4 ml), 200 mg of dried 4,4'- dimethoxytrityl chloride (DMTr-Cl, American Bionetics, Emeryville, CA) , 4 mg 4-dimethylaminopyridine, and 100 ul of distilled triethylamine were added to the container. The reaction mixture was stirred for one hour at room temperature. Additional dry DMTr-Cl (100 mg) was added and the reaction was stirred for one hour. Methanol (4 ml) and then water (4 ml) were added and the product was extracted three times with 40 ml diethyl ether. The ether wash was dried to an oil on a rotary evaporator, followed by addition of 50 ml ether, and final drying on a rotary evaporator. The ether addition and drying procedures were repeated, and the product was washed with dichloromethane and dried again. This step was to remove traces of pyridine. The oil was pipetted on to a silica gel column, and the column was eluted with 8% methanol in dichloromethane. Three-ml fractions were collected and checked for 5 ' -dimethoxytrityl-N²-isobutyryl-6- thiodeoxyguanine (5'DMT-N -IBu6TdG) by spotting thin layer chromatography silica plates run with 8% ethanol/dichloromethane and inspected under a ultraviolet lamp. The 5^•DMT-N²-IBu6TdG runs just behind the pyridine spot and ahead of the unreacted N²-IBu6TdG. The identification of the product was confirmed with a drop of 10% H₂SO₄, which causes the dimethoxytrityl groups to exhibit a bright orange color. Unreacted dimethoxytrityl chloride runs close to the solvent front, and faster than pyridine. The fractions containing 5'DMT-N²-IBu6TdG were pooled and dried to an oil on the rotary evaporator.

EXAMPLE III

Phosphitylation. 5'DMT-N²-IBu6TdG was phosphitylated using the method of Sinha et al. , 1984. This method is also disclosed by Koster (U.S. Patent No. 4,725,677). To 290 mg of 5'DMT-N²-IBu6TdG in a brown glass vial and that had been lyophilized overnight was added 2.1 ml of anhydrous tetrahydrofuran to dissolve the 5'DMT-N²-IBu6TdG. N,N- diisopropylethylamine (300 ul), and 250 ul of chloro- N,N-diisopropylamino-2-cyanoethyoxyphosphine (American Bionetics) were added. After 40 minutes of stirring the solution was filtered over a medium grade glass filter. The filter and residue were washed with 10-15 ml tetrahydrofuran and the filtrate dried to an oil on a rotary evaporator. The product was dissolved in 25 ml ethyl acetate and extracted twice with cold 10% (w/v) Na₂Cθ3. The ethyl acetate was dehydrated by stirring it over approximately 1 g anhydrous Na₂S0₄ for fifteen minutes. More Na₂S0₄ was added if the Na₂S0₄ appeared sticky in the reaction solution. The ethyl acetate was filtered through a coarse grade glass filter. The filter retaining the Na S0₄ was rinsed with about 20 ml ethyl acetate. The combined ethyl acetate filtrates were dried to an oil on a rotary evaporator. The oil was then redissolved in approximately 2 ml ethyl acetate and the solution pipetted into 30 ml hexane in a glass separation tube at -78°C forming a yellow semisolid precipitate. The hexane/ethyl acetate was pipetted off the product. The yellow colored precipitate was redissolved in 2 ml acetonitrile at room temperature, filtered through a 2 urn nylon filter and lyophilized overnight. This product was the 6-thiodeoxyguanosine phosphoramidite, 6TdGPA.

EXAMPLE IV

Oligonucleotide nucleotide synthesis and isolation. The 6TdGPA was redissolved in anhydrous acetonitrile (3.5 ml) and placed on the Applied Biosystems 381A DNA synthesizer. A DNA octamer was synthesized with a 6- thiodeoxyguanosine (6TdG) located at the site of the octamer indicated with "S": 5'-CSACTCAT-3' .

Oligodeoxyribonucleotide synthesis was performed on the Applied Biosystems 381A DNA Synthesizer with an overall coupling yield of 80% for the entire oligomer and an 86% coupling efficiency when the 6-thiodeoxyguanosin8 was inserted.

After cleavage of the oligonucleotide from the solid support by 2 N ammonium hydroxide (2 ul for 40 minutes at room temperature) and removal of the acyl groups by heating to 55°C for 12 hours, a 100 ul aliquot of the oligonucleotide was heated to 70°C for 30 minutes in 1 ml 0.1N HC1. This acid hydrolysis released the purine bases. THe purine base composition was analyzed by HPLC using a Whatman Partisil SCX 25 cm column (Whatman International Ltd., Maidenstone, England) eluted with 90% 75 mM ammonium formate, 10% methanol at a flow rate of 1.2 ml/min. The eluant was inspected for UV absorbance at a wavelength of 254 nm, and the two major absorbance peaks were found by comparison with standards to correspond to 6TG and adenine.

Best Mode for Carrying out the Invention. The invention comprises an oligonucleotide containing 6- thioguanosine and a preferred method of synthesis of this oligonucleotide: (1) acylation of 6-thiodeoxyguanosine to form a first product; (2) tritylation of the first product to form a second product; (3) phosphitylation of the second product to form a deoxyguanosine phosphoramidite product; and (4) insertion of the deoxyguanosine from the phosphoramidite product into an oligonucleotide at a predetermined site using a sequential oligonucleotide synthesis technique.

Industrial Applicability> The method of the invention provides a way to synthesize an oligonucleotide containing a thiol analog of guanine at a preselected site sot hat the effect of various parameters on the oligonucleotide with the incorporated analog may be studied. This is important because of the general effect that the analog, 6- thioguanine, has been shown to have on cells, cell growth and DNA replication.

While the invention has been described with reference to specific embodiments thereof, it will be appreciated that numerous variations, modifications, and embodiments are possible, and accordingly, all such variations. modifications, and embodiments are to be regarded as being within the spirit and scope of the invention.

LITERATURE CITED

Barranco, S.C. and R.M. Humphrey. 1971. The effects of beta-2'-deoxythioguanosine on survival and progression in mammalian cells. Cancer Research 31:583-586.

Bodell, W.J., W.F. Morgan, J. Rasmussen, M.E. Williams and D.F. Deen. 1985. Potentiation of l,3-bis(2-chloroethyl)-l- nitrosourea (BCNU)-induced cytotoxicity in 9L cells by pretreatement with 6-thioguanine. Biochem. Pharmacol. 34(4) :515-520.

Christie, N.T., S. Drake, R.E. Meyn and J.A. Nelson. 1984. 6-thioguanine-induced DNA damage as a determinant of cytotoxicity in cultured CHinese hamster ovary cells. Cancer Research 44:3665-3671.

Garegg, P.J., I. Lindh, T. Regberg, J. Stawinski, and R. Stromberg. 1986. Nucleoside H-phosphonates. III. Chemical sythesis of oligodeoxyribonucleotides by the hydrogenphosphonate approach. Tetrahedron Letters 27:4051- 4054.

Jones, R.A. 1984. Preparation of Protected Deoxyribonucleosides . Oligonucleotide Synthesis—a Practical Aproach, chapter 2, IRL Press, Washington, D.C.

Kovach, J.S., J. Rubin, E.T. Creagan, A.J. Schutt, L.K. Kvols and P.A. Svingen. 1986. Cancer Res. 46(11) :5959-5962.

Li, B.F.L., C.B. Reese and P.F. Swarm. 1987. Synthesis and characterization of oligode.oxynucleotides containing 4-0- methylthymine. Biochemistry 26(4) :1086-1093.

Schwartz, E.L., O.C. Blair and A.C. Sartorelli. 1984. Cell cycle events associated with the termination of proliferation by cytotoxic and differentiation-inducing actions of 6-thioguanine on HL-60 cells. Cancer Res. 44(9) :3907-3910.

Sinha, N.D., J. Biernat, J. McManus and H. Koster. 1984. Polymer support oligonucleotide synthesis XVIII: use of beta- cyanoethyl-N,N-dialkylamino-/N-morpholino phosphoramidite of deosynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product. Nucleic Acids Research 12:4539-4557.

Claims

THE CLAIMSWhat Is Claimed Is:

1. A method of synthesizing an oligonucleotide containing 6-thiodeoxyguanosine, comprising the steps of: (a) acylation of 6-thiodeoxyguanosine to form N²- isobutyryl-6-thiodeoxyguanosine; (b) tritylation of N²-isobutyryl-6-thiodeoxyguanosine to form 5" -dimethoxytrityl-N²-isobutyryl-6- thiodeoxyguanosine; (c) phosphitylation of 5' -(4,4' -dimethoxytrityl) -N²- isobutyryl-6-thiodeoxyguanosine to form 5 ' - ( 4 , 4 ' - dimethoxytrityl ) -N²-isobutyryl-6-thio-2 ' - deoxyguanos i e 3 ' - [ ( 2-cyanoeth l ) - , N- diisopropyl ) ] -phosphoramidite ; and (d) using 5' -(4,4 '-dimethoxytrityl )-N²-isobutyryl-6- thio-2 ' -deoxyguanosine 3'-[ ( 2-cyanoethyl )-N,N- diisopropyl) ] -phosphoramidite to insert 6- thiodeoxyguanosine into an oligonucloeotide at a particular base location using a sequential oligonucleotide synthesis technique.

2. A method of synthesizing an oligonucleotide according to claim 1, wherein the oligonucleotide formed that contains 6-thiodeoxyguanosine comprises 5 ' -CSACTCAT-3 ' , wherein A is a deoxyadenosine residue, C is a deoxycytidine residue, S is a 6-thiodeoxyguanosine residue, and T is a deoxythymidine residue.

3. A phosphoramidite compound formed by the method of claim 1, comprising 5 '-( 4 , 4 * -dimethoxytrityl) -N²- isobutyryl-6-thio-2' -deoxyguanosine 3 '-[ (2-cyanoethyl) -N,N- diisopropyl) ] -phosphoramidite.

4. An oligonucleotide containing 6-thiodeoxyguanosine.

5. An oligonucleotide according to claim 4, wherein the oligonucleotide was formed by the method of claim 1.