WO1989009780A1

WO1989009780A1 - Site-specific tritium-labeled oligodeoxynucleotides

Info

Publication number: WO1989009780A1
Application number: PCT/US1989/001535
Authority: WO
Inventors: Frank Charles Richardson; Thomas Richard Skopek
Original assignee: Chemical Industry Institute Of Toxicology
Priority date: 1988-04-15
Filing date: 1989-04-12
Publication date: 1989-10-19

Abstract

Nucleoside phosphoramidites have been synthesized and subsequently utilized in the preparation of tritium-labeled nucleotides at predetermined sites of polycnucleotides or oligonucleotides. Purine and pyrimidine nucleotides labeled in specific positions with tritium are protected at the reactive sites and then condensed in a predetermined sequence to yield tritium-labeled polynucleotides. The tritium-labeled oligonucleotides may be used to determine the effects of altering the primary DNA structure on the formation and repair of DNA adducts generated by chemical carcinogens and anticancer drugs.

Description

Site-specific Tritium-Labeled Oligodeoxynucleotides BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to the formation of new nucleotides and oligonudeotides. In particular, this invention relates to the formation of protected tritium- labeled nucleotide phosphoramidites and oligonudeotides with tritium located at predetermined nucleotide sites.

Description of the Related Art

Radioactively tagged nucleotides. Numerous investigators have incorporated radioactive labels into the nucleotides and other cell components of growing cells by growing the cells in media containing isotopically labeled compounds. See, for example, Oldham (U.S. Patent No. 3,854,240). The disclosure of this reference and of all other cited publications and patents is incorporated in full by reference herein. Most work using radioactively labeled nucleotides has involved use of nonspecific nucleotide labels such as in a uniformly labeled oligonucleotide to determine the presence of the particular oligonucleotide in organisms, tissues or cells. Thus, Stabinsky (U.S. Patent No. 4,652,639) manufactured structural genes tagged with (¹²⁵I) having up to 200 base pairs to enable the final product to be used as a labeled reagent. The patent of Wechter (U.S. Patent No. 3,520,872) provides a process for labeling specific positions in purine and pyrimidine-containing compounds. According to this method an oligonucleotide containing purines and pyrimidines is heated with deuterium or tritium oxide at a selected pH followed with treating with water or other proton-donating solvent so that the easily exchangeable hydrogen atoms are replaced by deuterium or tritium atoms. The result is that the oligonucleotide contains either (a) purines labeled at the C-8 position and pyrimidines labeled at the C-5 and/or C-6 positions or (b) only C-6 labeled pyrimidines. There is no selective labeling of only certain purines or pyrimidines in this patent.

Stepwise oligonucleotide synthesis methods. A variety of stepwise oligonucleotide synthesis methods employ repetition of 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 oligonudeotides by (1) reaction of a nucleoside with a phosphine derivative; (2) reaction 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'-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/RNA synthesizing machines. Manual techniques generally employ solid phase techniques wherein solid supports, having a particular desired

SUBSTITUTESHEET 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 oligonudeotides 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'- hydroxyl 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 500 angstrom 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

SUBSTITUTESHEET 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 for

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°C 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 methods of labeling particular single nucleotides within oligonudeotides. It is therefore an object of this invention to provide a means for synthesizing oligonudeotides that comprise a specific labeled nucleotide at a particular oligonucleotide site.

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

It is a further object of this invention to provide particular novel labeled nucleotide phorphoramidite intermediates.

It is another object of this invention to provide a series of oligonudeotides having the same nucleotide composition but having a radioactive label at a different nucleotide site in each oligonucleotide.

It is a further object of this invention to provide labeled oligonudeotides that may be used to examine the differential effect of various agents on different nucleotide locations within the oligonucleotide.

SUMMARY OF THE INVENTION

The novel phosphoramidite compositions of this invention differ from compositions previously reported in that they comprise a tritium label at the l'-C and 2'-C position on the ribose or deoxyribose moiety. The labeled oligonudeotides possess nucleotide sequences where the tritium-labeled nucleotide is positioned for study in biological applications. The nucleotide moieties that may be utilized in either tritium-labeled RNA or DNA sequences include: guanosine or deoxyguanosine, adenosine or deoxyadenosine, thymidine or deoxythymidine, cytidine or deoxycytidine, uridine or deoxyuridine. The tritium-labeled positions in the above nucleotides may include replaceable hydrogen atoms of the structures shown in Figures 1 and 1A, where the letter "T" has been substituted on a ring system.

The tritium-labeled nucleotide was obtained from Amersham (Arlington Heights, IL) . In the Examples below, 1' ,2' [³H]deox guanosine (tritium labels on the sugar moiety) was used to synthesize oligonudeotides. It might also be possible to place a ³H label on a purine or pyrimldine ring. Thus the C-8 hydrogen of purines could be replaced.

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 tritium label positions in representative nucleotides that may be used in the method of the invention. Figure 1A shows tritium label positions in another nucleotide that may be used in the method of the invention.

Figure 2 is a diagram of the protected nucleotide used in oligonucleotide synthesis.

Figure 3 shows steps and products of Examples I-IV.

DETAILED DESCRIPTION OF THE INVENTION AND

PREFERRED EMBODIMENTS THEREOF

The present invention generally comprises radioactively- labeled phosphoramidites and polynucleotides and methods of synthesis and use thereof. The synthesis steps to obtain the labeled phosphoramidites that are described in detail in Examples I-IV below are shown in Figure 3. A preferred embodiment of this invention comprises tritium-labeled polydeoxyribonucleotides. Four such oligonudeotides were synthesized with 1' ,2*-[³H]deoxyguanosine as illustrated in the following sequence: 5 ' -CC-G-T-G-G-G-ATATGGGCTG- 3 ' a b e d

where A is a deoxyadenosine residue; C is a deoxycytidine residue; G is a deoxyguanosine residue; T is a deoxythymidine residue; and a, b, c, and d are possible single sites of a tritium-labeled residue.

The tritium-labeled starting nucleotide, l',2'-[³H] deoxyguanosine, 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 where ac is an acyl group such as isobutyryl; ak is an alkyl group such as trityl; and ph is a phosphoramidite group.

In particular, the method employed to obtain the compounds of the invention from the tritium-labeled nucleotide triphosphate comprises the following steps:

(1) The hydroxyl groups on the deoxyribose 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 the N- isobutyryl-[³H]-nucleoside; (4) The nucleoside is tritylated (alkylation of a single hydroxyl group) at the 5 '-position of the deoxyribose 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 hydroxyl group is allowed to react with 2-cyanoethyl-N,N- diisopropylaminochlorophosphine in the presence of N,N,N-diisopropylethylamine to yield 5'-DMTr-N- isobutyryl-[ ³H]-2 ' -deoxyribonucleotide-3 '-[ (2- cyanoethyl)-(N,N-diisopropyl) ]-phosphoramidite; and

(6) A manual or automatic DNA synthesizing technique for sequential addition of nucleotides is used to add the nucleotide moiety of the nucleoside phosphite 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 (Applied Biosystems, Inc., Foster City,

CA) or other suitable equipment or procedures where the individual phosphoramidites derived from nucleoside or deoxynucleoside residues, for example, adenosine, cytidine, guanosine, uridine, thymidine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, and tritium-labeled nucleoside or deoxynucleoside (deoxyguanosine in the preferred embodiment of the invention) , are allowed to condense in a prescribed order. The resultant oligonudeotides can be used, for example to determine the effects of chemical carcinogens or anti-cancer drugs on the primary DNA/RNA structure.

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

Acylation. 1' ,2'-[³H]Deoxyguanosine (dG) was acylated as summarized below using a modification of the method of Jones,

1984. dG (280 mg) was first suspended in 7.5 ml of anhydrous pyridine and cooled to 4°C. Trimethylchlorosilane (520 ul) was added and the mixture stirred for 30 minutes and maintained at 4°C. Isobutyric anhydride (680 ul) was then added and the mixture stirred for 2 hours at room temperature. The reaction was cooled to 4°C and 2 ml of water added, followed 15 minutes later by addition of 1.8 ml of concentrated ammonium hydroxide. The sample was rotary evaporated to crystals and then a volume of 1-2 ml of water was added and the solution was extracted with 5 volumes of ether 2-3 times. The remaining aqueous phase was evaporated on a Savant Speed Vac (Savant Instruments, Inc., Farmingdale, NY) for 24 hours.

It was necessary to scale down the original method of Jones by a factor of greater than tenfol . This scaled down synthesis resulted in a product not pure enough for subsequent reactions. Therefore, the following additional purification steps were performed to enable significant increases in product yield. In the wash step, the resulting powder was washed with ice cold water in a glass sinter funnel to remove excess isobutyric salts and then dried on the Speed Vac. The white powder (220 mg) was dissolved in methanol (5 ml) and loaded onto a liquid chromatographic column containing 30 cc of silica gel (Merck Keiselgel 60, 20-200 uM, EM Reagents, Darmstadt, Germany) and eluted with 16% methanol/methylene chloride (95% of the tritium counts came out in the first 70 ml). The acylated product, 1 ' ,2' -[ ³H]-N²-isobutyryl- deoxyguanosine (Product 1), was then rotary evaporated to dryness, transferred to a brown bottle, sealed with a rubber septum, and placed under vacuum for 48 hours to assure dryness.

EXAMPLE II

Tritylation. Product 1 (200 mg) was tritylated at the 5'-position using the method described by Jones, 1984. Anhydrous pyridine (7 ml), 258 mg of dried 4,4'- dimethoxytrityl hydrochloride (DMTr-Cl, American Bionetics, Emeryville,CA) , 2-3 mg of dried 4-dimethylaminopyridine (DMAP) and 83 ul of distilled triethylamine (TEA) were added to the container containing Product 1. The reaction mixture was allowed to stand at room temperature for one hour to allow the reaction to come to completion. DMTr-Cl (129 mg) and TEA (41 ul) were added and the reaction was completed after one hour. The product (Product 2) was purified on silica gel using 8% methanol/methylene chloride, dried to an oil on the rotary evaporator, and then placed under vacuum for 16-24 hours. The yield was 400 mg of Product 2.

EXAMPLE III

Phosphitylation. Product 2 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 400 mg of dried Product 2 was added 3 ml of tetrahydrofuran, 430 ul of N,N- diisopropylethylamine, and 185 ul of chloro-2-cyanoethyl-N,N- diisopropylaminophosphine (American Bionetics). After 30 minutes the reaction was monitored by HPLC for production of phosphoramidite (Product 3). The phosphoramidite was filtered on a glass sinter funnel to remove salts, dried on a rotary evaporator, dissolved in 3 ml ethyl acetate and precipitated by pipetting into 10 volumes of hexanes at-78°C. Product 3 was redissolved in 2 ml acetonitrile, filtered through a 2 um filter into a brown vial and dried under vacuum for 24 hours.

Other nucleotides may be used in the procedure employed above to prepare acylated-alkoxylated-phosphitylated derivatives analogous to Product 3. Examples of other representative tritium-labeled starting nucleotides that may be used are shown in Figures 1 and 1A. EXAMPLE IV

Oligonucleotide synthesis and Isolation. Product 3 was redissolved in anhydrous acetonitrile (3.5 ml) and placed on the Applied Biosystems 381A DNA Synthesizer. Four separate oligonudeotides were synthesized with a l',2'[³H]- deoxyguanosine (dG) located at only one of the deoxyguanosine sites as indicated with "a, b, c, or d" below:

5'-C-C-G^a-T-G-G-G-A-T-A-T-G-G-G-C-T-G-3'

5'-C-C-G-T-G^b-G-G-A-T-A-T-G-G-G-C-T-G-3^• 5'-C-C-G-T-G-G^c-G-A-T-A-T-G-G-G-C-T-G-3'

5'-C-C-G-T-G-G-G^α-A-T-A-T-G-G-G-C-T-G-3

This oligonucleotide sequence is a region of the hypoxanthine-guanine phosphoribosyl transferase gene where position "d" is a site frequently mutated by methylnitrosourea (MNU) as compared to positions "a", "b", or "c"_ Oligodeoxyribonucleotide synthesis, performed on the Applied Biosystems 381A DNA Synthesizer, had an overall coupling yield of 83% for the entire oligomer with a 90% coupling efficiency when the labeled deoxyguanosine phosphoramidite^was inserted. The specific activities of the isolated four oligo ers were 10 uCi/mmol. Enzymatic digestion of each of the oligomers to nucleosides followed by HPLC separation and scintillation counting demonstrated that 95% of the radioactivity was located in the expected deoxyguanosine peak. No other major radioactivity peaks were observed. These results indicated that ³H-exchange was minimal during oligonucleotide synthesis and ammonia deprotection. Thus, the tritium-labeled nucleotide is well suited for identifying biochemical reactions in such constructed oligonudeotides. Preparation of a series of oligonudeotides that are identical except that a particular nucleotide (e.g., deoxyguanosine) is labeled at a different site in each oligonucleotide, allows the study of the differential effects of various treatments (exposure to mutagens, various temperature and pH regimes , etc. ) on the same nucleotide located at different sites in the same oligonucleotide.

Best Mode for Carrying Out the Invention. Preferably, the radioactively labeled polynucleotides of the invention comprise tritium-labeled polydeoxyribonucleotides. The nucleotides of the invention are synthesized by the following steps: (l) acylation of a labeled nucleoside 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 labeled third phosphoramidite product; and (4) insertion of the third phosphoramidite product in an oligonucleotide at a predetermined site using a sequential oligonucleotide synthesis technique.

Industrial Applicability. The invention provides a way to incorporate a radioactive label at a particular point in an oligonucleotide. Such an oligonucleotide enables study of the^' effect of specific treatments, such as exposure to various chemicals, as well as enabling the specifically labeled reagent to be used to study cellular chemical phenomena.

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.

Claims

THE CLAIMSWhat Is Claimed Is:

1. A method of synthesizing an oligonucleotide having a particular labeled nucleotide at a particular oligonucleotide site, comprising the steps of:

(a) acylation of labeled nucleoside to form a first product;

(b) tritylation of the first product to form a second product; (c) phosphitylation of the second product to form a labeled third phosphoramidite product; and (d) insertion of the third phosphoramidite product in an oligonucleotide at the particular oligonucleotide site using a sequential oligonucleotide synthesis technique.

2. A method of synthesizing an oligonucleotide having a particular labeled nucleotide at a particular oligonucleotide site according to claim 1, wherein the labeled nucleotide comprises tritium labeling at the 1' and 2' sites on the ribose or deoxyribose ring of the nucleotide.

3. A method of synthesizing an oligonucleotide having a particular labeled nucleotide at a particular oligonucleotide site according to claim 2, wherein the nucleotide is deoxyguanosine.

4. A labeled phosphoramidite compound formed by the method of claim 1.

5. An oligonucleotide formed by the method of claim 1.