Background of the Invention This invention relates to nucleotide analogs capable of being attached to oligonucleotides and labeled for use as nucleic acid hybridization probes. Such probes are used to detect DNA and, in some instances, RNA, characteristic of particular cells or microorganisms. Both isotopic and non-isotopic labeling methods have been used.

Summary of the Invention In general, the invention features a cytidine analog in which the cytosine base portion thereof has the formula

wherein Y is derived from a group capable of reacting with the N-hydroxy-succinimide ester of biotin amino caproic acid, Z is a protecting group capable of preventing Y from reacting with a phosphite, and X is a linking group connecting Y and N.

Preferably, Y is NH, Z is C-CP-., and X is

0 (CH ₂ ) ₃ .

As will be explained in more detail below, the cytidine analogs of the invention are attached to synthetic oligonucleotide probes, and the protective group Z removed to expose the reactive group Y, which is spaced from the cytidine analog by spacer X, to prevent steric hindrance. A reporter group is then attached to reactive group Y (which may change slightly in structure in the process of deprotection; e.g., NH becomes NH ₂ ) , thus labeling the probe.

The invention provides rapid, simple labeling which can be precisely controlled in degree and position of label.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Description of the Preferred Embodiments The drawings will first be described. Drawings Fig. 1 is the structure of a preferred labeled oligonucleotide of the invention.

Figs. 2-5 are the structures of the intermediates in the synthesis of said labeled oligonucleotide. Structure

The labeled oligonucleotides of the invention have the structure given in the Summary of the Invention, above. The substituents are now described in more detail. Reactive Group Y

Reactive group Y is derived from a group capable of reacting with the N-hydroxy-succinimide ester of biotin amino caproic acid. This does not mean that the reporter group need be biotin or a derivative thereof, but is simply a convenient way of defining the reactivity of Y necessary for its being labeled with any desired reporter group.

The presently most preferred reactive group Y is an amino group or derivative thereof. Other suitable reactive groups are S- and 0

C-O.

Linking Group X

Linking group X does not participate directly in the derivatization reaction but rather serves the function of preventing steric hindrance between the amino group of cytidine and reactive group Y. Consequently, it is the size of X, rather than its chemical composition, that is important. Generally, X therefore can include any of a wide range of chemical substituents, provided they are not more reactive than group Y. Examples of suitable linking groups X are

Protecting Group 3

Protecting group Z is any group which bonds to reactive group Y to prevent Y from reacting with a phosphite reagent. The protecting group Z must also be capable of being removed after the cytidine analog has been attached to the oligonucleotide, under conditions which do not substantially impair the hybridizing ability of the oligonucleotide or the reactivity of Y.

Where the reactive group Y is an amino group, suitable protecting groups are amides, e.g., N-Formyl, N-Acetyl, N-Chloroacetyl, N-Trichloroacetyl, N-Trifluoroacetyl, N-o-Nitrophenylacetyl, N-o-Nitrophenoxyacetyl, N-Acetoacetyl, N-3-Phenylproρionyl, N-3-(p-Hydroxyphenyl)propionyl,

4 -

N-2-Methyl-2-(o-nitrophenoxy)propionyl, N-2-Methyl-2- (o-phenylazophenoxy)propionyl, N-4-Chlorobutyryl, N-o-Nitrocinnamoyl, N-Picolinoyl,

N-(N'-Acetylmethionyl) , N-Benzoyl, N-Phthaloyl, and N-Dithiasuccinoyl.

Other suitable amino protecting groups are carbamates, e.g. Methyl Carbamate, 9-Fluorenylmethyl Carbamate, 2,2,2-Trichloroethyl Carbamate, 2-Trimethylsilylethyl Carbamate, 1,1-Dimethylpropynyl Carbamate, 1-Methyl-l-phenylethyl Carbamate, 1-Methyl-l- (4-biphenylyl)ethyl Carbamate, l-l-Dimethyl-2-haloethyl Carbamate, 1,1-Dimethyl- 2-cyanoethyl Carbamate, t-Butyl Carbamate, Cyclobutyl Carbamate, 1-Methylcγclobutyl Carbamate, 1-Adamantyl Carbamate, Vinyl Carbamate, Allyl Carbamate, Cinnamyl Carbamate, 8-Quinolyl Carbamate, N-Hydroxypiperidinyl Carbamate, 4,5-Diphenyl-3-oxazolin-2-one, Benzyl Carbamate, p-Nitrobenzyl Carbamate, 3,4-Dimethoxy- 6-nitrobenzyl Carbamate, 2,4-Dichlorobenzyl Carbamate, 5-Benzisoxazolylmethyl Carbamate, 9-Anthrylmethyl Carbamate, Diphenylmethyl Carbamate, Isonicotinyl Carbamate, S-Benzyl Carbamate, and N-(N'-Phenylaminothiocarbonyl) Derivatives.

Other suitable amino protecting groups include N-Allyl, N-Phenacyl, N-3-Acetoxypropyl, Quaternary Ammonium Salts, N-Methoxymethyl, N-Benzyloxymethyl, N-Pivaloyloxymethyl, N-Tetrahydropyranyl, N-2,4-Dinitrophenyl, N-Benzyl, N-o-Nitrobenzyl, N-Di(p-methoxyphenyl)methyl, N-Triphenylmethyl, N-(p-Methoxyphenyl)diphenylmethyl,

N-Diphenyl-4-pyridylmethyl, N-2-Picolyl N'-Oxide, N,N'-Isopropylidene, N-Benzylidene, N-p-Nitrobenzylidene, N-Salicylidene, N- (5,5-Dimethyl-

3-oxo-l-cyclohexenyl) , N-Nitro, N-Oxide, N-Diphenylphosphinyl, N-Dimethylthiophosphinyl, N-Benzenesulfenyl, N-o-Nitrobenzenesulfenyl, N-2,4,6-Trimethylbenzenesulfonyl, N-Toluenesulfonyl, N-Benzylsulfonyl, N-Trifluoromethylsulfonyl, and N-Phenacylsulfonyl.

Where reactive group Y is a thiol group, suitable protecting groups include S-Benzyl Thioether, S-p-Methoxybenzyl Thioether, S-p-Nitrobenzyl Thioether, S-4-Picolyl Thioether, S-2-Picolyl N-Oxide Thioether,

S-9-Anthrylmethyl Thioether, S-Diphenylmethyl Thioester, S-Di(p-methoxyphenyl)methyl Thioether, S-Triphenylmethyl Thioether, S-2,4-Dinitrophenyl Thioester, S-t-Butyl Thioether, S-Isobutoxymethyl Hemithioacetal, S-2-Tetrahydropyranyl Hemithioacetal, S-Acetamidomethyl Aminothioacetal, S-Cyanomethyl Thioether, S-2-Nitro-l-Phenylethyl Thioether, S-2,2-Bis(carboethoxy)ethyl Thioether, S-Benzoyl Derivative, S-(N-Ethylcarbamate) , and S-Ethyl Disulfide. where the reactive group Y is a carboxyl group, suitable protecting groups include Methyl Ester, Methoxymethyl Ester, Methylthiomethyl Ester, Tetrahydropyranyl Ester, Benzyloxymethyl Ester, Phenacyl Ester, N-Phthalimidomethyl Ester, 2,2,2-Trichloroethyl Ester, 2-Haloethyl Ester, 2-(p-Toluenesulfonyl)ethyl Ester, t-Butyl Ester, Cinnamyl Ester, Benzyl Ester, Triphenylmethyl Ester, Bis(o-nitrophenyl)methyl Ester, 9-Anthrylmethyl Ester, 2-(9,10-Dioxo)anthrylmethyl Ester, Piperonyl Ester, Trimethylsilyl Ester, t-Butyldimethylsilyl Ester, S-t-Butyl Ester, 2-Alkyl-l,3-oxazolines, N,N-Dimethylamide, N-7-Nitroindoylamide, Hydrazides, N-Phenylhydrazide, and N,N-Diisopropylhydrazide.

Sugar Moiety

For convenience and economy, it is preferred that the sugar moiety to which the cytosine base is bonded have the conventional 5-membered heterocyclic configuration, with oxygen and four carbon atoms constituting the ring. Strict adherence to this configuration is not important to the labeling or hybridization reactions, however, and various other substituents, e.g., S, NH, or CH ₂ , can be substituted for the ring oxygen if desired.

Similarly, the group bonded to Cj. of the sugar moiety is most conveniently a hydroxyl group in which the hydrogen is replaced by a protecting group which is capable of preventing the reaction of the hydroxyl with a phosphite reagent; this protecting group can be any conventional hydroxyl protecting group, e.g., 4,4'-dimethoxytrityl (DMT), ethers such as methyl ether, esters such as acetate esters, and cyclic acetals and ketals such as ethylidine. if desired, the group attached to C ₅ of the sugar moiety can be, rather than a protected hydroxyl group, a conventionally protected S group. Linkage to Oligonucleotide

The cytidine analog is attached to the oligonucleotide by means of a linking group substituted for the hydrogen in the hydroxyl group bonded to C of the sugar moiety. As mentioned above, the most

/ • _* preferred linking group is CH ₃ 0-P-N 0. Any other suitable phosphorus-containing group can be used, however, e.g., other phosphite groups, or phosphate groups, including groups containing more than one phosphorus atom.

Reporter Group

Following the attachment of the cytidine analog to the oligonucleotide and the deprotection of reactive group Y, Y is reacted with any suitable reporter group. The reporter group can be directly detectable, as in the case of fluorophores such as fluorescein, or it can be a group which confers binding specificity on the oligonucleotide to enable detection by a subsequent compound or series of compounds. One well-known example of the latter is the system using biotin, avidin, an enzyme, and a substrate for the enzyme which produces a detectable change when acted on by the enzyme. Synthesis

The synthesis of the labeled oligonucleotides of the invention is carried out generally as follows. The first step is to synthesize a cytidine analog having the formula given in the Summary of the Invention, above. The analog has, attached to the amino group at C4, a linking group X, a reactive group, or derivative thereof, Y, and a protective group Z. To construct the analog, X and Y are attached to the amino group of C4 of cytidine, and the reactive group Y is then protected with protective group Z.

The next step is to attach the cytidine analog to the 5* end of an oligonucleotide to be used as a hybridization probe. Following this step, additional nucleotides can be attached to the cytidine analog, including additional cytidine analogs, to give as many labeled positions in the oligonucleotide as desired, at precisely predetermined positions. This is conveniently done using commercially available DNA synthesizers, into which the cytidine analog has been loaded.

- 8 -

Finally, the cytidine analog is deprotected and a label attached at Y to yield a labeled oligonucleotide.

Referring now to Fig. 1, there is shown a labeled oligonucleotide in which X is (CH ₂ ) ₃ , Y is NH, and the detectable label is biotin, a well-known reporter compound whose affinity for avidin has previously rendered it useful in labeling DNA probes.

Figs. 2-5 show the synthetic steps in the construction of the labeled oligonucleotide of Fig. 1. Referring to Fig. 2, the addition of the linker (CH ₂ )3 and reactive group H ₂ is accomplished in a single step. This is followed by the addition to the NH ₂ of the protective group COCF... The next step is protection of the C5 hydroxyl with the protective group DMT. The remaining hydroxyl (C3) is then reacted with a phosphite reagent to provide a site for the attachment of the cytidine analog to the oligonucleotide.

Referring to Fig. 3, the cytidine analog is then reacted with the oligonucleotide. Referring to Fig. 4, the protective groups COCF ₃ and DMT are then removed and (Fig. 5) biotin added at the reactive NH ₂ via an acylation reaction.

In more detail, the illustrated synthesis was carried out according to the following steps. N-(3-aminopropyl) -2'-deoxycytidine (compound

(3) of Fig. 2) was first prepared, as follows. A solution of sodium bisulfite (2M) and 1,3-diaminopropane (1M) was prepared by the addition of 1,3-diaminopropane (18.38g, 0.25M, 20.8ml) to an ice cold solution of sodium bisulfite (52.3g, 0.5M) in distilled water (200ml) , the pH adjusted to 7 with concentrated HCl, and the volume made up to 250ml with distilled water.

2'-deoxycytidine monohydrate (5.0g, 20mM) was added and the reaction mixture warmed to 70°C and incubated overnight under argon. TLC in isopropanol: ammonia:water (60:15:5) gave two spots of equal intensity (Rf = 0.29, 0.19). The reaction mixture was diluted 10-fold with 0.1M TRIS-HC1 (30.28g, 0.25m, ph9) and the solution heated to 37°C. TLC as developed above gave one spot at Rf = 0.29 after one hour. The solution was concentrated to approximately 150ml and applied to a reversed phase C-18 column for desalting. The column was washed with two liters of distilled water and the product eluted with 10% acetonitrile in water to give compound (3) (2.0g) .

Next, compound (3) was suspended in a mixture of dichloromethane (50ml)/triethylamine (4ml) and cooled to 4°C under argon. Trifluoroacetic anhydride (5.4g, 25mmol) was added dropwise by syringe over a ten minute period. The reaction mixture was stirred at room temperature for one hour and then an excess of trifluoroacetic anhydride (5.4g, 25mmol) was added.

After one hour methanol (30ml) was added and the solvent was then removed invacuo yielding a yellow oil (approximately 15g) , which was purified by column chromatography on silica gel (Baker Flash grade) . Elution with dichloromethane:methanol (8:2) and evaporation of the solvent invacuo gave 4 N-(3-trifluoroacetylammopropyl) -2'-deoxycytidine

(compound (4) of Fig. 2) .

Compound (4) (lg, 2.6mmol) was co-evaporated with anhydrous pyridine (3 x 25ml) and dissolved in anhydrous pyridine (30ml). 4,4'-dimethoxytritylchloride

(0.97g, 2.8mmol) was added and the mixture stirred at room temperature for one hour. Methanol (30ml) was

added and then the reaction mixture was concentrated invacuo. The residue was partitioned between dichloromethane (100ml) and cold saturated sodium bicarbonate solution (100ml) . The aqueous layer was further extracted with dichloromethane (100ml) . The organic extracts were combined and dried using Na ₂ SO.. Evaporation of the solvent invacuo gave a yellow oil which was purified by column chromatography on silica gel (Baker Flash Grade) . Elution with dichloromethane/methanol/pyridine (950:49:1) and evaporation of the solvent invacuo gave 5 ^• -0-(4,4-dimethoxytrityl) - ⁴ N-(3-trifluoro- acetylaminopropyl)-2'-deoxycytidine (compound (5) of Fig. 2) . Compound (5) (1.3g, 1.9mmol) was dissolved in a mixture of chloroform (10ml, filtered through basic alumina) and diisopropylethylamine (1.3ml, 7.6mmol) under argon. To the ice cooled reaction mixture was added chloro-morpholino-methoxy-phosphine (0.29ml, 2.85mmol) , dropwise, by syringe over three minutes.

After 15 minutes at room temperature, the solution was transferred with 50ml ethylacetate into a 250ml separating funnel and extracted with cold, saturated sodium chloride solution (8 x 80ml) . The organic layer was dried ( a ₂ S0 ₄ ) and then concentrated invacuo to yield a buff colored foam. The foam was dissolved in toluene (15ml) and added dropwise to hexane (200ml) at

-80 ^Q C. The colorless precipitate, collected at room temperature and dried under a stream of argon, was 5'-0-(4,4-dimethoxyt ityl)-3'-methoxymorpholinophosphine- 4 N-(3-tπfluoro-acetylaminopropyl)-2'-deoxycytidine

(compound (6) of Fig. 2) .

The next step was the incorporation of compound (6) into synthetic DNA. Compound (6), dissolved in acetonitrile (80mg ml , 9.7 umolsml ) was used to functionalize oligonucleotides synthesised on an Applied Biosystems 380A DNA synthesiser, according to standard techniques. This resulted in functionalized oligonucleotide (7) of Fig. 3.

Following deprotection by treatment with thiophenol, ammonia and acetic acid to form the deprotected oligonucleotide (8) of Fig. 4, labeling with biotin was carried out as follows.

To oligonucleotide (8) (25 ug, 2.5 x 10 -9moles) dissolved in 0.1M sodium borate, pH 8.0, was added the N-hydroxysuccinimide ester of biotin amino

—8 caproic acid (23 m g, 5 x 10 moles) (Enzo Biochem) in dimethyl formamide (3 m l), and the reaction allowed to proceed for 4 hours. Residual biotin acylating reagent was removed from the biotinylated oligomer by chromotography on Sephadex G50. Further purification can be accomplished by reverse phase high performance liquid chromatography. Use

The oligonucleotides which have been labeled using the cytidine analogs of the invention are used in nucleic acid hybridization reactions to detect nucleic acid sequences characteristic of specific bacteria, viruses, or cells, according to conventional techniques, e.g., as described in Landes U.S. Pat. Appln. S.N. 529,044, filed September 2, 1983, assigned to the same assignee as the present application, hereby incorporated by reference.