WO1992010505A1

WO1992010505A1 - Methods for labelling oligonucleotides

Info

Publication number: WO1992010505A1
Application number: PCT/US1991/009247
Authority: WO
Inventors: Clifford M. Chan
Original assignee: Abbott Laboratories
Priority date: 1990-12-11
Filing date: 1991-12-11
Publication date: 1992-06-25
Also published as: CA2098187A1; EP0562014A4; AU9158391A; JPH05509110A; EP0562014A1; KR930703340A

Abstract

A method for labelling the 5' end of a nucleotide synthesized on a solid support involves reacting the free 5' hydroxyl with a reactant member selected to activate the 5' terminus; reacting the activated 5' terminus with a marker molecule containing a nucleophilic group; and cleaving the labelled nucleotide from the solid phase. The invention also relates to nucleotides labelled at their 5' end by the above process, wherein the nucleophilic group of the marker moiety is covalently attached directly to the 5' carbon; and to nucleotide intermediates having activated 5' termini.

Description

METHODS FOR LABELLING OLIGONUCLEOTIDES This invention relates to the covalent coupling of detectable marker molecules into nucleic acid segments referred to as oligonucleotides. More specifically, it relates to 5' end labelling of oligonucleotides wherein each oligonucleotide contains only one marker molecule.

BACKGROUND Labelled oligonucleotides find utility in a number of applications, including DNA sequencing, diagnostic detection or quantitation and forensic science.

Typically, the labelled oligonucleotide is allowed to hybridize or anneal with nucleic acid present in the sample and the presence or absence of label is detected following separation steps. Many mechanisms and schemes have been used to introduce labels into oligonucleotides. For a comprehensive review of these methods see Goodchild, - Bioconjugate Chemistry, 1(3) .165-187 (1990). According to Goodchild, past researchers have labelled oligonucleotides at both internal and terminal locations; by enzymatic and chemical synthetic means; and utilizing a single or many marker molecules per oligonucleotide. Methods involving incorporation of marker moieties at internal locations in the oligonucleotides are generally less preferable due to their less predictable hybridization behavior; end- labelled oligonucleotides are preferred, particularly for automated detection systems. Similarly, methods for incorporating multiple label moieties into an oligonucleotide are less preferred for stoichiometric reasons. Labelling methods which randomly insert radiolabelled or haptenated bases into the oligonucleotide produce a labelled probe which is more difficult to quantitate. The correlation of signal generated to the amount of probe present is not straightforward.

For some applications, it may not be critical that the labelled oligonucleotides are poorly characterized in terms of exact positioning and number of label molecules.

However, for automated diagnostic detection and quantitation, it is desirable that each oligonucleotide have a single marker moiety at an end location. Methods for placing a single marker, or hapten capable of reacting with an antibody or other specific binding member, at a terminal position on an oligonucleotide have been described in the literature.

Most commonly, a linker member containing a primary amine or other nucleophilic group is attached at an available hydroxyl to enable conjugation to one of numerous electrophilic detectable markers. Alternatively, terminal deoxynucleotidyl transferase, ligase and phosphoramidite chemistry have been used to attach direct labels or reactive linkers to oligonucleotides. For example, Ke pe, et al. , Nucleic Acids Research 13(1) :45 (1985) describe biotinylation of the 5' end of synthesized oligonucleotides. In this scheme, an aminoethanol derivative of biotin is condensed with a polymer supported derivatized nucleotide. After the reaction and deprotection, a phosphodiester bond connects the animoethanol-biotin to the nucleoside at the 5' hydroxyl.

Smith, et al.. Nucleic Acids Research 13(7):2399- 2412 (1985) describe a 5' labeling technique utilizing a

5' amino deoxythymidine nucleotide. The 5' amino group is first protected and a phosphoramidite is prepared, which is then used as a final step to add the aminated base to the 5' hydroxyl of the synthesized chain. After cleavage and deprotection, the amino group can be reacted with a number of different electrophilic fluorophores to generate a 5' labelled oligonucleotide.

Coull, et al., Tetrahedron Letters 27(34) .3991-3994 (1986) describe a novel phosphoramidite reagent which introduces a protected primary aliphatic amino group into the 5' terminus of an oligonucleotide under automated synthesis. Following deprotection and cleavage from the support, the amino group can be reacted with a number of electrophilic labels to label the 5' end. Bischoff, et al., Anal. Biochem. 164:336-344 (1987) describe functionalization of the amino groups produced according to Coull's method above. For example, reaction of the 5' primary amine with succinic anhydride gives the

5' carboxylic acid. Similarly, reaction with dithiobis(succinimidylpropionate) followed by treatment with dithioerythritol produced 5' thiolated oligonucleotides. The various functional groups expand the range of possible reactions at the 5' end of these oligonucleotides.

Connolly, Nucleic Acids Research 15(7) :3131-3139 (1987) describes his attempt to prepare protected amino phosphoramidite reagents capable of producing a protected amino group at the 5' position on an oligonucleotide.

Connolly attempts to use different protecting groups selected from dimethoxytrityl and monomethoxytrityl, which can be removed under different conditions than previous protecting groups.

Thuong, et al.. Tetrahedron Letters 29(46) .5905-5908

(1988) describe a method for 5' end labeling a supported oligonucleotide with acridine using phosphoramidite chemistry. Although this method has certain advantages, a separate phosphoramidite reagent must be prepared for each desired label or marker. This adds process steps and expense to the procedure. In addition, the phospho ester bond and the spacer arm of the phosphoramidite reagent remain attached to the oligonucleotide between the sugar residue and the acridine hapten. Each of the above methods have been used to incorporate detectable marker compounds into end positions of oligonucleotides. However, each has drawbacks that make it less than desirable for completely automated synthesis of commercial quantities of oligonucleotides.

For example, reaction of the oligonucleotide with a label moiety after removal from the support requires additional handling. In addition, excess reagent must be used to drive the reaction. This may result in undesirable side reactions and the reaction is usually not completed. Therefore, additional steps of purification (eg. chromatography) are often required, further encumbering the synthesis process and resulting in low yields of labelled oligonucleotides. Accordingly, the present invention seeks to overcome the problems associated with prior methods, and to provide a method for end labelling of oligonucleotides which is amenable to automation and versatile enough to be useful for the addition of any one of many different markers, without the need to prepare specialized reagents for each marker. The invention also eliminates the spacer arms between the 5' carbon of the sugar residue and the marker molecule. SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a method for labeling the 5' end of a nucleotide synthesized on a solid support so as to have a free hydroxyl at the 5' terminal carbon of the nucleotide sugar residue, the method comprising: a. reacting the 5' terminus of the nucleotide sugar residue with a reactant member to produce an activated 5' terminus having a leaving group attached thereto; b. reacting the activated 5' terminus with a marker molecule containing a nucleophilic group to displace the leaving group and covalently attach the marker molecule to the 5' carbon of the nucleotide sugar residue; and c. cleaving the labeled nucleotide from the solid phase.

Preferably, the reactant member is a sulfonyl chloride and the nucleophilic group is selected from the group consisting of amines, thiols and alcohols. The most preferred nucleophilic group is amine. Preferred marker moieties are haptens.

In another aspect, the invention relates to nucleotides labeled at their 5' end by the above process.

Nucleotides according to the invention have the following general structure: B - Sugar-CH - N ' -MM

I ²

Rent wherein B represents a base selected from the group consisting of adenine, cytosine, guanine, thymine, uracil, and analog derivatives thereof; Sugar represents ribose, deoxyribose or analogs thereof; CH. is the methylene group at the 5' terminus of the sugar residue; MM represents a marker molecule; N' represents a nucleophilic group of the marker molecule; and Rem is attached to the sugar residue at its 3' carbon and represents OH, a phosphodiester linkage to a second nucleotide, or a spacer connected to a solid support.

In yet another embodiment the invention relates to intermediates useful for labelling the 5' end of an oligonucleotide. Intermediates have the following general structure: B - - LG

wherein B represents a base selected from the group

- consisting of adenine, cytosine, guanine, thymine, uracil, and analog derivatives thereof; Sugar represents ribose, deoxyribose or analogs thereof; CH_ is the methylene group at the 5' terminus of the sugar residue; LG represents a leaving group which can be displaced by a nucleophile; and Rem is attached to the sugar residue at its 3' carbon and represents OH, a phosphodiester linkage to a second nucleotide, or a spacer connected to a solid support. Exemplary leaving groups include aromatic or aliphatic sulfonates, eg. tosylates or esylates.

DETAILED DESCRIPTION As mentioned, the invention is a novel method for labelling the 5' end of an oligonucleotide synthesized on a solid support. Conceptually, the method comprises removing the 5' hydroxyl and activating the 5' carbon with a reactant member capable of exchanging the hydroxyl for a good leaving group; followed by reaction of the activated 5' terminus with a nucleophilic group of a marker molecule.

Many methods for synthesis of oligonucleotides on solid supports have been described in the literature. For example, Goodchild, supra, describes a diester approach; a triester approach; a trivalent phosphate approach, often referred to as the phosphoramidite approach; and, finally, a pentavalent phosphorous approach known as the hydrogen phosphonate approach. It is believed that any of these methods for synthesizing an oligonucleotide may be used in conjunction with the present invention, so long as the 5' hydroxyl group of the sugar of the terminal nucleoside remains available for activation. Presently preferred methods include the phosphoramidite method and the H- phosphonate method. As with the method of synthesis, the particular solid support employed with the invention is not critical.

Essentially the only requirement of the solid support is that it have a functionalized or derivatized group available for reacting with the 3' terminus of a nucleotide. Such solid supports include those which are already derivatized with the first nucleotide, and those which are merely functionalized for the incorporation of the first nucleotide in the chain. Typical solid supports include controlled pore glass (CPG), silica gel and polymer-based resins.

CPG supports are commercially available from Clontech (Palo Alto, CA) and CPG, Inc. (Fairfield, NJ) both with and without the first nucleotide attached. Functionalized solid supports are available with short chain and long chain amino groups as the functional group. The long chain aminated CPG supports are presently preferred.

The reactant member is a reagent capable of exchanging the hydroxyl at the 5' carbon for a "good leaving group". Good leaving groups are well known to those of skill in the art. They are characterized by their electron density which renders them easily displaced by a nucleophile in a substitution reaction generally known as SN2. Reactant members must satisfy two criteria: they must include an "R" group which comprises a "good leaving group"; and they must be sufficiently reactive to displace the 5' hydroxyl and exchange the leaving group in its place. Exemplary and preferred reactant members are described below.

An "activated 5' terminus" refers to the 5^r carbon of the sugar residue to which is covalently attached a "good leaving group" instead of the usual hydroxyl group. It is well known to those of skill in the art that good leaving groups comprise the salts of strong acids. Illustrative leaving groups and activated termini are discussed below. A preferred reactant member is an aromatic or aliphatic sulfonyl halide having the general structure:

R - SO. - X where R is an aromatic or aliphatic group and X is a halide, preferably Cl or Br. The term "aromatic" refers to substituted or unsubstituted ring structures generally

^•known to those skilled in the art to fall into this class, including those compounds which fulfill the Huckel (4n+2) τ~ electrons rule. Particularly preferred are single ring derivatives of benzene having substitutions which enhance the "leaving" ability of the group. Certain substituents in the para-position are generally recognized as fulfilling this function.

Preferred aromatic sulfonyl halide reactant members include p-toluenesulfonyl chloride (toεyl chloride) p- bromobenzenesulfonyl chloride (brosyl chloride) and p- nitrobenzenesulfonyl chloride (nosyl chloride). The R group of the sulfonyl halide reactant members may also be aliphatic. The term "aliphatic" includes groups which may be straight or branched, preferably straight; and ideally no longer than eight backbone atoms total. Alkyl chains having less than about 8 carbons, preferably less than 4, are preferred aliphatic groups for sulfonyl halide reactant members. Both aliphatic and alkyl R groups may include substitutions provided they do not interfere with the criteria of the reactant member. Certain substitutions are useful to enhance the reactant members ability to serve as a leaving group. Exemplary aliphatic reactant members are methanesulfonyl chloride, trifluoromethane sulfonyl chloride, ethanesulfonyl chloride,^' 2,2,2 trifluoroethanesulfonyl chloride, propanesulfonyl chloride, and ammonioalkane sulfonyl halides.

Although the recited reactant members have largely been sulfonyl chlorides, it will be recognized by one of ordinary skill in the art that other halides may also be used. For example, it is contemplated that sulfonic acid bromides will function as reactant members in the present invention, when reaction conditions are appropriately modified.

It will also be recognized by those skilled in the art that reactant members equivalent to the sulfonyl halides include any reactant member which bears a suitable "leaving group" and can be made to displace the 5' hydroxyl. It is also possible to utilize a combination of two or more reagents as reactant members. For example, one can convert the 5' hydroxyl to a bromide leaving group using carbontetrahalides and triphenylphosphine.

It will be further recognized that the preferred reactant members, when reacted with a hydroxyl group under appropriate conditions, produce the activated termini shown in Table 1.

Table 1.

Reactant Member Activated 5 ' Termini p-toluenesulfonyl chloride tosylate p-bromobenzensulfonyl ^chloride brosylate p-nitrobenzenesulfonyl chloride nosylate methanesulfonyl chloride mesylate trifluoromethanesulfonyl chloride triflate 2,2,2 trifluoroethane¬ sulfonyl chloride tresylate ammonioalkanesulfonyl halide betalates

The reaction conditions under which aromatic and/or aliphatic sulfonyl halides will react with the 5' hydroxyl to form the activated 5' terminus are generally described in the literature. For example. See March, J. , Advanced Organic Chemistry 3rd Ed (1985) pp310-320. It is within the ability of one of ordinary skill in the art to modify the reaction conditions as necessary for any particular reactant member. Although applicant does not wish to be limited to any particular theory or mechanism of action, it is presumed that the activated 5' terminus undergoes nucleophilic displacement by the nucleophilic group of the marker molecule. It is thus contemplated that other strong leaving groups are the equivalent of the sulfonyl halides discussed above.

The marker molecule employed in the labelling step can be selected from a wide variety of such compounds having nucleophilic groups. The term "marker molecule" is intended to include at least the following 3 classes of molecules:

(1) molecules that are capable of directly providing a detectable signal such as radioactive molecules and chemiluminescent molecules;

(2) molecules which give a detectable signal upon the addition of an external stimulus, such as fluorophores (stimulus is incident radiation); and

(3) molecules, sometimes referred to as "hooks", which are one member of a pair of specific binding partners, such as haptens (bind to antibodies) and biotin (binds to avidin or antibody) .

Of course, many other examples of each type of marker molecule are known to those of skill in the art. Virtually any such molecule may be employed with the present invention, provided it has, or can be modified to contain, a nucleophilic group sterically available for reaction with the activated 5' terminus of the oligonucleotide.

It is presently preferred to utilize marker molecules which fall into the third category; and particularly preferred to utilize small molecular weight haptens. Exemplary haptens are generally known to those skilled in the art and include, without limitation, biotin, iminobiotin, fluorescein, dansyl, acridine, dinitrophenol, dibenzofuran, luminols (eg. N-4-aminobutyl-N-ethyl isoluminol - ABEI). Other useful marker molecules include fluorophores, such as fluorescein and rhodamine; and chemilumiphores, such as acridine and luminols.

Nucleophilic groups useful in the invention include amines, thiols and alcohols, although other nucleophilic groups may be deemed equivalent. Generally, the stronger the nucleophile, the weaker the leaving group may be in terms of its "leaving" ability. Thus, stronger nucleophilic groups would generally be preferred over weaker ones, so that a greater range of leaving groups and reactant members may be employed. However, for reasons of stability and ease of synthesis, the preferred nucleophilic group for the present invention is amines. Primary nucleophilic groups are typically preferred over secondary or tertiary groups. The nucleophilic group found most commonly and most readily available in a plurality of marker moieties is an amino group, preferably a primary amino group.

The reaction conditions under which the nucleophilic group of the marker molecule will react with the activated 5' terminus are well known to those of ordinary skill in the art. Because this reaction is believed to be a nucleophilic displacement, standard textbooks (See for example March, J., supra) give the reaction conditions under which the nucleophilic group will react with the activated 5' terminus to displace the leaving group and covalently bond the marker molecule directly to the 5' carbon of the sugar residue. Variations on these reaction conditions are well within the ability of the routine practitioner. Once the marker molecule has been covalently bonded to the oligonucleotide, the labelled oligonucleotide is cleaved from the solid support. Once again, methods for cleaving the oligonucleotide from the solid support are known to those of ordinary skill in the art. For example, the Applied Biosystems 380B synthesizer generally employs concentrated ammonium hydroxide to separate the oligonucleotide from the support. Other available methods include strong sodium hydroxide and ethanolic 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU - Aldrich Chemical, Milwaukee, WI) . Labelled oligonucleotides prepared according to this invention have utility in a number of diagnostic applications. For example, Whitely, et al., U.S. 4,883,750 describe a diagnostic method in which a diagnostic probe and a contiguous probe are laid down on a target strand and are ligated together. The ligated product is then detected. Incorporation of a marker molecule at the 5 ' end of the probe whose 3' end undergoes ligation would permit rapid detection of the ligated product.

Another diagnostic method is disclosed in EP-A-320 308 and is known as ligase chain reaction (LCR) . In this method, two pairs of probes are utilized, one member of each pair being hybridizable with the other member and with adjacent sections of the target molecule. Using a ligase enzyme, the adjacent probes are covalently bonded together. By repeated cycles of hybridization, ligation and separation, the number of molecules of ligated product increases rapidly if the target is present to serve as a template for adjacent hybridization.

The following examples serve to illustrate the invention further. However, the invention is limited only by the claims which are appended hereto. EXAMPLE 1: Preparation of a 5' Labelled Oligonucleotide Using p-Toluenesulfonyl Chloride

An oligodeoxynucleotide was synthesized using phosphoramidite chemistry on an ABI 380A automated DNA synthesizer according to standard instrument procedure. A 1 /xmole scale protocol, modified to increase reaction time, was used to synthesize a 16 mer. For this experiment the sequence was regularly alternating bases, although it is believed that the specific sequence is immaterial to the invention. The synthesis was programmed with 5' dimethoxytrityl protection off ("DMT-off", see ABI 380A operator manual) while the protected oligonucleotide remained attached to the control pore glass.

The column was removed from the synthesizer. A 10 ml syringe filled with 5 ml of 0.1M solution p-

^• toluenesulfonyl chloride (Aldrich Chemical, Milwaukee, WI) freshly prepared in dry pyridine was attached to one end of the column via a luer tip. An empty syringe was mounted on the opposite end to receive the eluate. The 0.1M p-toluenesulfonyl chloride solution was carefully passed through the column five times, followed by incubation at 55°C for an additional 24 hours. The excess p-toluenesulfonyl chloride solution was removed and the column was washed with 10 ml of dry acetonitrile. Next, 2 ml of 0.1M N-4-Aminobutyl-N-ethyl isoluminol (ABEI), a hapten and chemilumiphore, in dry dimethylformamide (DMF) was loaded into a 5 ml syringe with luer tip (ABEI, a hapten, and DMF are available from

Aldrich Chemical, Milwaukee, WI). This syringe was mounted on one end of the column. Another syringe was filled with 2 ml of triethylamine solution and mounted on the other end of the column. The solutions were slowly mixed by passing through the column. The mixture was incubated at 55°C for 24 hours, the excess reagent was removed and the column was washed with 20 ml acetonitrile. The 5' labelled DNA was removed from the CPG with the treatment of concentrated NH 4OH for 1 hour under standard

Applied Biosystem Inc. protocols (ABI 380A DNA synthesizer user manual). The 5' labelled oligonucleotide was heated at 55°C overnight, to remove the protecting group from the protected bases. Evaporation of NH₄0H gave crude 5' labelled oligonucleotides.

EXAMPLE 2: Preparation of a 5' Labelled Oligonucleotide Using Methanesulfonyl Chloride An oligodeoxynucleotide was synthesized using phosphoramidite chemistry on an ABI 380A automated DNA synthesizer according to standard instrument procedure. A 1 /.mole scale protocol, modified to increase the reaction time, was used to synthesize a second 16 mer. For this experiment the sequence was a single base repeated although it is believed that the specific sequence is immaterial to the invention. The synthesis was programmed with 5' dimethoxytrityl protection off ( "DMT- off", see ABI 380A operator manual) while the protected oligonucleotide remained attached to the control pore glass.

The column was removed from the synthesizer. A 10 ml syringe filled with 5 ml of 0.1M solution methanesulfonyl chloride (Aldrich Chemical, Milwaukee, WI) freshly prepared in dry pyridine was attached to one end of the column via a luer tip. An empty syringe was mounted on the opposite end to receive the eluate.

The 0.1M methanesulfonyl chloride solution was carefully passed through the column several times, followed by incubation at 25°C for an additional 24 hours. The excess methanesulfonyl chloride solution was removed and the column was washed with 10 ml of dry acetonitrile.

Next, 2 ml of 0.1M N-4-Aminobutyl-N-ethyl isoluminol

- (ABEI), a hapten and chemilumiphore, in dry dimethylformamide (DMF) was loaded into a 5 ml syringe with luer tip (ABEI, a hapten, and DMF are available from

Aldrich Chemical, Milwaukee, WI). This syringe was mounted on one end of the column. Another syringe was filled with 2 ml of triethylamine solution and mounted on the other end of the column. The solutions were slowly mixed by passing through the column. The mixture was incubated at 25°C for 24 hours, the excess reagent was removed and the column was washed with 20 ml acetonitrile.

The 5' labelled DNA was removed from the CPG with the treatment of concentrated NH 4OH for 1 hour under standard. Applied Biosystem Inc. protocols (ABI 380A DNA synthesizer user manual). The 5' labelled oligonucleotide was heated at 55°C overnight to remove the protecting groups from the protected bases. Evaporation of NH₄0H gave crude 5' labelled oligonucleotides.

EXAMPLE 3: Evaluation of ABEI Labelled Oligonucleotide

A. By gel separation: The crude 5' ABEI-labelled oligonucleotide from both Examples 1 and 2 were separately redissolved in 0.3M NaOAc followed by 70% ethanol precipitation at 4°C. The DNA pellet was isolated from the mother liquid and evaporated to dryness. The 5' ABEI- labelled DNA was analyzed by electrophoresis in 15% polyacrylamide, 7M urea gel. In both cases, the electrophoretogram showed the hapten labelled DNA migrated as a single blue fluorescent band closer to the origin relative to the unmodified, control oligonucleotide.

B. By UV/VIS spectra: The 5' ABEI-labelled oligonucleotide band and the control oligonucleotide band were extracted from the polyacrylamide gel and assayed with UV/VIS spectroscopy. In both cases, the spectra indicated an absorption peak at 327nm (for ABEI) along with the absorption peak at 260nm (characteristic of DNA).

C. By chemiluminescence: One μL of the 5' ABEI labelled oligonucleotide from Example 1 was added to a TD * (Abbott Laboratories, Abbott Park, IL) cuvette containing 100 μL of distilled water. Then 100 μl> of 0.3 mg/ml Vanadium IV catalyst was added and the cuvette was placed inside a chemiluminometer (Abbott Laboratories, IL; although any commercial chemiluminometer will suffice). 200 μL of 3% hydrogen peroxide in 0.25 N NaOH solution was injected into the cuvette and the emitted light was measured for 5 seconds immediately after the injection. As a control, the experiment was repeated with non- labelled oligonucleotides and no light was emitted. This evaluation procedure can also be repeated with the labelled oligonucleotides of Example 2; or with oligonucleotides labelled with any chemiluminescent marker molecule.

EXAMPLE 4: Preparation of a 5' Labelled Oligonucleotide Using p-Toluenesulfonyl Chloride

Example 1 is repeated except aminomethylfluorescein (AMF: See U.S. Patent 4,510,251, Abbott Laboratories) is used instead of the ABEI reagent. For this example the conditions are the same as in Example 1, but for the marker reagent. Evaluation of this labelled oligonucleotide is done using electrophoresis and/or UV/VIS spectroscopy. The AMF labelled oligonucleotide will have an absorption peak at about 495 nm, in addition to the characteristic DNA peak at 260 nm.

EXAMPLE 5: Preparation of a 5' Labelled Oligonucleotide Using p-Nitrobenzenesulfonyl Chloride

Example 2 is repeated except that 0.1M p- nitrobenzenesulfonyl chloride is used in place of the 0.1M methanesulfonyl chloride. Other conditions and steps remain the same except the reaction times may take a little longer at 25°. Performing the reactions at a higher temperature should decrease the reaction times.

EXAMPLE 6: Preparation of a 5' Labelled Oligonucleotide via a 5^r-chloro leaving group

Example 2 is repeated except a solution consisting of carbontetrachloride and triphenylphosphine in acetonitrile, each 0.1M, is used in place of 0.1M methanesulfonyl chloride. The result is a 5'-chloro activated derivative which is reacted with a nucleophilic marker molecule under conditions as in example 2 to label the oligonucleotide. EXAMPLE 7: Preparation of a 5' Labelled Oligonucleotide via a 5'-bromo leaving group

Example 2 is repeated except a solution consisting of carbontetrabromide and triphenylphosphine in acetonitrile, each 0.1M, is used in place of 0.1M methanesulfonyl chloride. The result is a 5'-bromo activated derivative which is reacted with a nucleophilic marker molecule under conditions as in example 2 to label the oligonucleotide.

EXAMPLE 8: Preparation of a 5' Labelled Oligonucleotide via a 5' activation with Mitsunobu reagent

Example 2 is repeated except the syringe at a first end of the column contains a solution (0.1M, 5ml) consisting of triphenylphosphine and 3-hydroxy- dibenzofuran (a hapten) in acetonitrile. The syringe attached to the other end of the column contains a solution (0.1M, 5ml) of diethylazodicarboxylate in acetonitrile. The two solutions are passed alternately through the column to acheive adequate mixing and the column is incubated at 25°C for 24 hours. The excess reagent is removed and the column is washed with acetonitrile (20ml). The result is a dibenzofuran-labeled oligonucleotide (at the 5' end) and removal of the labeled oligonucleotide proceeds as in example 2.

Claims

What is claimed is:

1. A method for labeling the 5' end of a nucleotide synthesized on a solid support so as to have a free hydroxyl at the 5' terminal carbon of the nucleotide sugar residue, the method comprising: a. reacting the 5' terminus of the nucleotide sugar residue with a reactant member to produce an activated 5' terminus having a leaving group attached thereto; b. reacting the activated 5' terminus with a marker molecule containing a nucleophilic group to displace the leaving group and covalently attach the marker molecule to the 5' carbon of the nucleotide sugar residue; and c. cleaving the labeled nucleotide from the solid phase.

2. The method of claim 1 wherein the reactant member of step a is. an aromatic sulfonyl halide.

3. The method of claim 1 wherein the reactant member of step a is an aliphatic sulfonyl halide.

4. The method of claim 3 wherein the reactant member is an alkylsulfonyl chloride.

5. The method of claim 1 wherein the reactant member of step a is a sulfonyl chloride.

6. The method of claim 1 wherein the nucleophilic group of step b is an amine.

7. A nucleotide labeled at its 5' end having the general structure:

B - Sugar-CH₂ - N'-MM Rem wherein B represents a base selected from the group consisting of adenine, cytosine, guanine, thymine, uracil, and analog derivatives thereof; Sugar represents ribose, deoxyribose or analogs thereof; CH_∑ is the methylene group at the 5' terminus of the sugar residue; MM represents a marker molecule; N' represents a nucleophilic group of the marker molecule; and Rem is attached to the sugar residue at its 3' carbon and represents OH, a phosphodiester linkage to a second nucleotide, or a spacer connected to a solid support.

8. The nucleotide of claim 7 wherein N' is selected from the group consisting of nitrogen, sulfur and oxygen.

9. The nucleotide of claim 7 wherein MM is selected from the group consisting of fluorophores, chemilumiphores and haptens.

10. The nucleotide of claim 7 wherein Rem comprises a phosphodiester linkage to a second nucleotide which, in turn, is connected to one or more additional nucleotides and wherein the 3' terminal nucleotide is attached to a solid support.

11. A nucleotide intermediate having an activated 5' terminus, said intermediate having the following general structure:

B - Sugar-CH_ - LG Rem wherein B represents a base selected from the group consisting of adenine, cytosine, guanine, thymine, uracil, and analog derivatives thereof; Sugar represents ribose, deoxyribose or analogs thereof; CH. is the methylene group at the 5' terminus of the sugar residue; LG represents a leaving group which can be displaced by a nucleophile; and Rem is attached to the sugar residue at its 3' carbon and represents OH, a phosphodiester linkage to a second nucleotide, or a spacer connected to a solid support.

12. The nucleotide intermediate of claim 11 wherein LG has the general structure:

—S0₂-R wherein R represents an aromatic or aliphatic group.

13. The nucleotide intermediate of claim 12 wherein LG is selected from the group consisting of tosylate, brosylate and nosylate.

14. The nucleotide intermediate of claim 12 wherein R is an aliphatic group comprising a substituted or unsubstituted alkyl chain having less than 4 backbone carbons.