NO892042L - NON-Nucleotide Binding Reagents for Nucleotide Probes. - Google Patents

Description

Non-nucleotide binding reagents for nucleotide probes QField of invention

The present invention relates to non-nucleotide reagents which can conveniently allow single or multiple residues, such as markers or insertions, to bind to a nucleotide probe, at any specific preselected site(s) thereon.

Technological retrospectives

In clinical research and diagnosis, a known technique for determining the presence of a particular nucleotide sequence (the "target nucleotide sequence" or simply the "target sequence") in either RNA or DNA is to perform a nucleic acid hybridization experiment. In such an experiment, a nucleotide multimer probe (typically an oligonucleotide) is selected which has a nucleotide sequence complementary to at least part of the target sequence. The probe is typically labeled, i.e. it is equipped with an atom or group bound to it, the presence of which can be easily detected. When the labeled probe is exposed to a test sample presumed to contain the target nucleotide sequence, under hybridization conditions, the target will hybridize with any such labeled probe. Presence of the target sequence in the sample can be determined qualitatively or quantitatively usually by separating hybridized and non-hybridized probe, after which the amount of labeled probe that hybridized is determined, either by determining the presence of marker in the probe hybrids or by determining the amount of marker in non-hybridized probes.

Previously, radioactive markers were used. However, due to handling difficulties, non-isotopic markers were later developed. Such markers include those whose presence is determined either directly or indirectly. Examples of direct markers include chemiluminescence, phosphorescence, fluorescence or spectroscopically detectable markers. Examples of indirect markers include compounds such as biotin and various antigens that can be detected using proteins conjugated to a suitable detectable marker.

Certain previous methods for binding a marker to a nucleotide probe have typically been based on the binding of a simple marker to a nucleotide, after which one or more of these nucleotides are incorporated into the probe. Analogues of dUTP and UTP containing a biotin residue attached to the C-5 position of the pyrimidine ring have, for example, been synthesized chemically and incorporated enzymatically into polynucleotides (P.R. Langer et al., Proe. Nat. Acad. Sei., USA, vol. 78, p (6633, 1981)). Such biotin-labelled nucleotides can be incorporated into nucleic acid probes of biological or synthetic origin by means of enzymatic procedures. In addition, the 5'-allylamine precursors of the foregoing analogs are proposed for use in a similar manner, leading to the incorporation of nucleophilic amine residues in the probe, which can be attached to labels using commonly used methods. Other deoxynucleotide triphosphate analogs are incorporated enzymatically into probes. In particular, bromo-dUTP and iodo-dUTP have been incorporated and detected immunochemically (Boultwodd, J. et al., J. Pathol. vol 148, page 61, 1986). In addition, 4-thio-UTP (H. Eshaghpour et al., Nucl. Acids Res., vol. 7, page 1485, 1979), has been attached to the 3<1-> end of DNA fragments and then labeled in its nucleophilic sulfhydryl residue. A PCT application by Tchen (International Publication No. WO 86/00074; published Jan. 3/86) relates to a technique in which apparently random pyrimidine base nucleotides can be depyrimidated, and the resulting sugar rings opened so that an amine-bearing residue can be attached to that.

Chemical methods of labeling have also been proposed, as in

all essentially allow markers to bind randomly to any nucleotides in a nucleotide multimer. N-acetoxy-N-acetyl-aminofluorene has, for example, been linked to guanine residues by

nucleic acids and then detected by immunochemical techniques (P. Chen et al., Proe. Nat. Acad. Sei. USA, vol. 81, page 3466, 1984). Another such method, which provides a nucleophilic amine group to which a label can be attached, involves bisulfite-catalyzed transamination at the C-6 position of the cytosine residues of nucleic acid probes (R.P. Viscidi et al., J. Clin. Biol. vol. 23, page 311, 1986). Both the chemical and enzymatic methods described above suffer from the fact that either single nucleotides cannot be specifically labeled or the procedure modifies the excyclic amines on the nucleotide bases and thus interferes with hybridization.

Other techniques are discussed which allow attachment of only a single marker to the 5' or 3' end of a nucleotide multimer, typically an oligonucleotide. Such a technique is, for example, indicated at CF. Chu et al., Nucl. Acids res., vol. 11, page 6513, 1983 and by A. Chollet et al., nucl. Acids res., vol 13, page 1529, 1985. Similar terminal methods of labeling are discussed, which are better suited for label attachment as a final step in solid/phase oligonucleotide synthesis. For example see B.A. Connolly, Nucl,. Acids Res., vol. 13, page 4485, 1985; S. Agrawal et al. Nucl. Acids Res., vol. 14,

page 6227, 1986 and B.A. Connolly, Nucl. Acids Res., vol. 15, page 3131, 1987. However, these terminal labeling methods are limited in that they only allow attachment of a single label to the end of the nucleotide multimer.

Compounds have been proposed which can be used to introduce a primary amine-modified nucleotide residue into selected positions in a synthetic oligonucleotide under standard automated synthesis procedures. Such compounds include analogs of deoxythymidine, deoxyadenine, deoxyguanine and deoxytytidine (G.B. Dreyer et al., Proe. Nati. Acad. Sei., USA vol 82, page 968, 1985; J.L. Ruth, PCT application publication number WO 84/03285, published 30 August, 1984). Such compounds can theoretically allow labeled nucleotides to be placed at a number of locations along a sequence, thus allowing the use of multiple markers to increase detection sensitivity. However, it has been shown that the use of such labeled nucleotides in a probe can reduce the stability of a hybrid formed with a target sequence, particularly when multiple markers are present. Such reduced hybrid stability has been shown for nucleic acid probes of biological origin that have multiple biotin residues attached to either uridine, cytidine or adenine bases (R.P. Viscidi et al., J. Clin. Microbiol., vol. 23, page 311, 1986; G. Gebeyehu et al., Nucl. Acids Res., vol. 15 page 4513, 1987). Reduced hybrid stability has also been reported for synthetic oligonucleotides having multiple fluorescent labels attached to modified uridine residues (J. Haralambidis, et al., Nucl. Acids Res., vol. 15, page 4857, 1987). Such instability can also be demonstrated for synthetic oligonucleotides with biotin and fluorescent markers attached to the N-4 position in the cytidine bases. In order to place a marker or markers at any desired position(s) in the synthetic oligonucleotides, it will further be necessary to have a total of 8 different compounds (four for deoxyribonucleotide multimers, and four for ribonucleotide multimers).

In addition, derivatives of nucleotide binding phosphate groups are discussed, whose nucleophilic residue can be labeled after their incorporation into an oligonucleotide (R.L. Letsinger and M.E. Schott, J. Am. Chem. Soc. 1981, vol. 103, page 7394; Japanese patents by N. Sugimoto , with numbers 61 44,353; 61 57,595; 61 44,352, all published March 1986). However, such compounds which are based on nucleotide derivatives will exhibit at least some of the disadvantages discussed above for nucleotide-based derivatives. Furthermore, the binder examples discussed above will not be able to be used in current standard solid-phase synthesis techniques, without modifications to these techniques.

Definitions

As used in the description and claims herein, the following terms are defined as: nucleotide: a subunit of a nucleic acid consisting of a phosphate group, a sugar with 5 carbon atoms and a nitrogen-containing base. In RNA, the sugar with 5 carbon atoms is ribose. In DNA there is 2-deoxyribose. The term also includes analogues of such subunits.

nucleotide multimer: a chain of nucleotides attached by phosphodiester bonds, or analogues thereof.

olio<g>nucleotide: a nucleotide multimer having a length of about 10 to about 100 nucleotides, but which may be more than 100 nucleotides in length. They are usually thought to be synthesized from nucleotide monomers, but can also be obtained by enzymatic methods.

deoxyribonucleotide: an olionucleotide consisting of deoxyribonucleotide monomers.

polynucleotide: a nucleotide multimer generally having a length of about 100 nucleotides or more. These are usually of biological origin and are obtained by enzymatic methods.

nucleotide multimer probe: a nucleotide multimer with a nucleotide sequence complementary to a target nucleotide sequence contained in another nucleotide multimer, usually a polynucleotide. Typically, the probe is chosen to be exactly complementary to the corresponding base in the target sequence. In some cases, however, it may be appropriate or even desirable that one or more of the nucleotides in the probe is not complementary to the corresponding base in the target sequence. The sample is typically labeled.

non-nucleotide monomer unit: refers to a monomer unit that does not participate significantly in the hybridization of a polymer. Such monomer units must, for example, not take part in any significant hydrogen bonding with a nucleotide, and will exclude monomer units that have one of the five nucleotide bases or analogues thereof as a component.

nucleotide/non-nucleotide polymer: a polymer comprising nucleotide and/non-nucleotide monomer units. When used as a probe, it will typically be labeled.

Oleonucleotide/non-nucleotide multimer: a multimer of generally synthetic origin of less than 100 nucleotides, but which may contain more than 200 nucleotides and which contains one or more non-nucleotide monomer units.

monomer unit: A unit of either a nucleotide reagent or a non-nucleotide reagent according to the invention, where the reagent contributes to a polymer.

hybrid: the complex formed between two nucleotide multimers by means of Watson-Crick base pairing between the complementary bases.

Summary of the invention

The present invention provides a non-nucleotide reagent, with a non-nucleotide monomer unit that can be linked synthetically with specific nucleotide monomer units from nucleotide reagents, to produce a defined sequence polymer with a main chain comprising nucleotide and non-nucleotide monomer units. Said non-nucleotide reagent also has a ligand which is either a linker arm residue that participates in conjugation reactions as soon as the linker arm is deprotected or it can be a side arm to which a usable desired chemical residue is attached before initiation of the polymer synthesis. In general, the techniques for attaching residues to the linker arm are similar to the techniques for attaching labels to groups of proteins. However, modifications to such techniques may be necessary. Examples of useful chemical transformations include the reaction of alkylamines with active esters, active imines, aryl fluorides or isothiocyanates, and the reaction of thiols with maleimides, haloacetyls, etc. (for additional potential techniques see G.M. Means and R.E. Feeney, "Chemical Modification and Proteins ", Holden-Day Inc., 1971; R.E. Feeney, Int. J. Peptide Protein Res., vol. 29, 1987, pages 145-161). Suitable protecting groups that can be used to protect the functional group on the linker arm during the formation of a polymer are also similar to those used in protein chemistry (see, e.g., "The Peptides Analysis Synthesis, Biology," vol. 3, ed. E. Gross and J. Meienhofer Academic Press, 1971). Because of the chemical nature of the non-nucleotide reagent, it can be placed at any desired position within the backbone sequence. This makes it possible to give the polymer containing nucleotide monomers a variety of properties. These include: (1) attachment of specific chemical residues at any desired location in the polymer, such residues may include (but are not limited to) detectable markers, insertion agents, gelling agents, drugs, hormones, proteins, peptides, haptens, radical generators, nucleolytic agents, proteolytic agents, catalysts, receptor-binding substances and other binding substances of biological interest, and agents that modify DNA transport across a biological barrier (such as a membrane), and substances that change the solubility of a nucleotide multimer. This means that it is possible to place such markers and insertion agents in the vicinity of any desired nucleotide; (2) the ability to immobilize the indicated sequence to a solid support using its linker arm for binding to a chemical residue of said support to construct, for example, nucleotide affinity carriers; (3) the ability to attach multiple chemical residues to the polymer through the linker arms by incorporating multiple non-nucleotide monomer units into the polymers; (4) the ability to construct polymers that differ from naturally occurring polynucleotides in that they have altered activities with proteins and enzymes that act on polynucleotides. For example, placing the non-nucleotide monomer unit on the 3' terminal end of an otherwise pure polynucleotide will confer resistance to degradation by snake venom phosphodiesterase. Such non-nucleotide monomer units can form specific cleavage sites for other nucleases; (5) the ability to construct hybridization probes by dispersed placement of hybridizable nucleotide monomer units and non-nucleotide monomer units. For example, a mixed block synthesis of nucleotide and non-nucleotide monomers can be produced, whereby a defined sequence of nucleotide monomers is synthesized followed by a piece of one or more non-nucleotide monomer units followed by a second block of nucleotide monomers of defined sequence; (6) the ability to construct synthetic probes that simultaneously detect target nucleotide multimers that differ by one or more base pairs. This is accomplished by using the non-nucleotide reagent described herein to replace the nucleotides in the probe with non-nucleotide monomer units at locations where differences occur in the nucleotide sequence of the various target nucleotide multimers.

In a preferred embodiment of the invention, labeled hybridization probes are constructed with a defined sequence comprising nucleotide and non-nucleotide monomers. In another preferred embodiment of the invention, the non-nucleotide monomer units are used to connect two or more nucleotide multimers with defined sequence, and the non-nucleotide monomer units are chemically labeled for use in hybridization reactions.

In another preferred embodiment, the non-nucleotide reagent is structured in such a way that it is allowed to be added in a stepwise manner to give a mixed nucleotide/non-nucleotide polymer using one of the commonly used DNA synthesis methods. Such nucleotide and non-nucleotide reagents typically add in a stepwise fashion so that their corresponding monomer units are attached to a growing oligonucleotide chain that is covalently immobilized to a solid support. The first nucleotide is typically attached to the carrier through a cleavable ester bond before initiation of synthesis. Stepwise extension of the oligonucleotide chain is normally carried out in the 3' to 5' direction. For standard DNA and RNA synthesis methods, see for example "Synthesis and Applications of DNA and RNA" ed. SO. Narang, Academic Press, 1987, and M.J. Gait, "Oligonucleotide Synthesis", IRL Press, Wash. D.C. U.S.A., 1984. When the synthesis is complete, the polymer is cleaved from the support by hydrolysis of the ester bond as mentioned above and the nucleotide originally attached to the support becomes the 3' terminal end of the resulting oligomer. By analogy, an alternative way is to introduce a non-nucleotide monomer unit and simultaneously attach it to a DNA synthesis carrier before initiation of DNA synthesis. In a preferred embodiment, the non-nucleotide monomer unit is attached to a DNA synthesis carrier through an ester bond which is formed by using the free alcohol form of the non-nucleotide monomer.

Accordingly, the present invention provides a reagent for the preparation of polymers containing a mixture of nucleotide and non-nucleotide monomer units. Said non-nucleotide monomers further contain one or more protected linker arms or one or more linker arms conjugated to a desired chemical residue such as a marker or insertion agent.

Such a non-nucleotide monomer has two additional linking groups so that its stepwise inclusion into a polymer of nucleotide and non-nucleotide monomer units is permitted. A first of the aforementioned linking groups has the property that it can connect effectively to the terminal end of a growing chain of monomer units. The second of said linking groups is able to further extend, in a stepwise manner, the growing chain of mixed nucleotide and non-nucleotide monomers. This requires that the second linking group be inactivated while the first linking group is being linked, so that it does not significantly link at this time, but can then be activated so that it then links to the non-nucleotide monomer unit. The "inactivation" is preferably carried out with a protective group on the second coupling group, which can be removed to "activate" the second coupling group. However, it is within the scope of the invention that such "inactivation" and "activation" can be carried out by simply changing reaction conditions (for example pH, temperature, changing the concentration of certain other components in the reaction system) with other linking groups of a suitable chemical structure, which can also be used for inactivation and activation by such techniques. Said linking groups allow the proximal attachment of either nucleotide or non-nucleotide monomer units. In a preferred embodiment, said coupling groups operate through coupling and deprotection steps which are compatible with them according to standard DNA synthesis methods.

Such methods require that synthesis occur in a non-directional manner and that all cleavage and deprotection steps in connection with coupling occur under "non-adverse" conditions, that is, they do not adversely affect the polymer backbone and its sugar, base , linker arm and marker components, nor the monomer reagents to a significant extent. One skilled in the art can readily identify functionalities, coupling methods, deprotection procedures and cleavage conditions that meet these criteria (see, for example, Gait reference, supra).

The non-nucleotide monomer preferably has a main chain to whose ends linking groups are attached. The main chain is preferably an acyl group with from one to twenty carbon atoms in the chain, and preferably an acyl hydrocarbon chain with from one to twenty carbon atoms.

In one embodiment of the invention, the reagent is selected from both compounds I or II among:

Whether I or II is chosen, the described groups are as follows:

(i) R 2 = non-nucleotide backbone;

are first coupling groups in which:

X2= halogen or substituted amino

X4= halogen, amino or 0~

R3= alkyl, alkoxy or phenoxy

R5= alkyl, alkoxy or aryloxy or can be H,

but only if X4 = 0~

(iii) Ri~xi~is the protected second linking group in which X]_ = 0, S, NH or HN =N-

R]_ = the protecting group which can be deprotected under deprotection conditions for the linking group for

to obtain the second linking group H-X]_-;

(iv) n is an integer,

(v) R4-X3- is the ligand preferably selected from a functional residue or from a protected binding arm that can be deprotected under non-damaging conditions so that it is then able to bind with a functional residue (again under non-damaging conditions ). In the latter case, X3 is the binding arm and R4 is the protecting group.

More preferably, any of the indicated groups are as follows: X 2 = Cl or secondary amine (preferably selected from

dialkylamino and heterocyclic N-amines)

R3= methoxy, ethoxy, chlorophenoxy or p-cyanoethoxy X3: terminates with 0, S, NH or HN=N- and preferably has from 1 to 25 carbon atoms in the chain extending from R2

X4= Cl, secondary amine or 0~

X 2 is further preferably diisopropylamine, dimethylamine or morpholine. R-|_ is further preferably triphenylmethyl

(which includes derivatives thereof, typically dimethoxytriphenylmethyl)

and Xi is 0.

R2 preferably has a secondary carbon attached to -0-. This allows the use of a secondary alcohol during the synthesis, which has the advantage that a higher yield of the protected reagents is formed from the alcohol.

A method for producing a nucleotide/non-nucleotide polymer is also described. One such method comprises using a reagent with a non-nucleotide monomer unit and a liquid (as described above) bound thereto, and coupling the non-nucleotide monomer unit under non-adverse conditions to a first monomer unit, and to either one of a second additional monomer unit or a solid support. At least one of the above additional monomer units is a nucleotide monomer unit. Should another monomer unit from a non-nucleotide reagent be chosen, it is of course possible to then link this monomer unit to yet another non-nucleotide monomer unit, etc. The above-mentioned links are typically carried out through a first linker group and a protected second linker group, both of which are bound to the non-nucleotide monomer unit. Thus, the non-nucleotide monomer unit is first linked to a first nucleotide monomer unit through the first linking group, after which the second linking group is then deprotected to then link the non-nucleotide monomer unit to either another nucleotide or another non-nucleotide monomer unit reagent. The preferred chemical composition of the reagents used in such a method is described above.

Products obtained from the above reagents and methods are also described.

Polymeric probes are also described which comprise a plurality of nucleotide and non-nucleotide monomer units and at least one acridinium ester marker residue which will typically serve as a marker, bound to a corresponding monomer unit of the probe.

The acridinium ester moiety is preferably a chemiluminescent acridinium ester marker. There is also specified a method for producing such probes, comprising binding of at least one such acridinium ester part to a corresponding monomer unit of a polymer with a plurality of nucleotide monomer units.

Drawings

Embodiments of the invention will now be described with reference to the drawings, in which: Figure 1 shows the preparation of a reagent according to the invention; Figure 2 shows the preparation of another reagent according to the invention; and Figure 3 shows the production of yet another reagent according to the invention. Figures 4-8 show the preparation of further other reagents according to the invention.

Detailed description of embodiments of the invention.

The synthesis of three different "linker" reagents according to the invention will now be described in detail in subsequent examples 1-3. Example 4 in the following shows methods for the production of substituted nucleotides (especially oligonucleotides) according to the invention, which have the main chain of the reagent linked, at different specific preselected locations, on the oligonucleotide. Example 5 shows binding of a marker to a binding group in a substituted nucleotide according to the invention, while example 6 shows a further advantage that can be provided by substituted nucleotides according to the invention, namely resistance to hydrolysis catalyzed by a phosphordiesterase.

Example 1: Synthesis of 2-(3-aminopropyl)-1, 3-dihydroxypropane linker reagent.

The schematic representation for this synthesis is given in figure 1, and is indicated below. (a) Synthesis of 2-(nitrilepropyl)-diethylmalonate (1): The procedure used is an adaptation of the method according to R. Adams and R.M. Kamm, Organic Synthesis, vol. 1 page 250-251, Gilman&Blatt, eds.

Materials: Diethyl malonate, 3-bromopropionitrile, and sodium ethoxide (21% solution in ethanol) were obtained from Aldrich Chemical Company (Milwaukee, WI). Absolute ethanol (200 proof) was from the U.S. Industrial Chemicals.

Procedure: Sodium ethoxide (0.1 mol) was diluted with absolute ethanol to a final volume of 100 ml. A solution of diethyl malonate (0.1 mol) in 50 ml of absolute ethanol was added dropwise with stirring, the reaction apparatus being protected from moisture with a CaCl 2 drying tube. Stirring was continued for one hour at room temperature. A solution of 3-bromoproponitrile in 50 ml of absolute ethanol was then added dropwise with stirring and the mixture was stirred overnight at room temperature. The resulting solution was filtered to remove precipitated sodium bromide, concentrated and extracted into diethyl ether (50 mL). This solution was then extracted with water (50 mL), dried over anhydrous magnesium sulfate and concentrated to an oil. Thin-layer chromatography on silica plates with chloroform as mobile phase gave three spots after visualization with iodine vapor: Rf 0.58, 0.51 and 0.38, which were then identified as diethyl malonate, 2-(nitrilepropyl)-diethyl malonate, and 2.2 respectively -di-(nitrilepropyl)malonate. After several days in the cooler, crystals separated from the crude oil which was filtered off, dissolved in toluene (10 mL) and reprecipitated by addition of hexanes to give 3.28 g of white solid (12%); thin layer chromatography (as described above), Rf 0.38. The structure of this compound was confirmed by NMR (CDCl 3 ) to be 2,2-di-(nitrilepropyl)malonate (6): 61.30 (t, 6H), 2.26 (t, 4H), 2.47 ( t, 4H), 4.27 (q, 4H). The filtered oil was distilled in vacuo to give the title compound (1), (bp 99-103°C, 0.3 mm Hg) in 20% yield; <1>H NMR analysis in CDCl3: 51.24 (t, 6H ), 2.19 (q, 2H), 2.47 (t, 2H), 3.46 (t, 1H), 4.18 (q, 4H). (b) Synthesis of 2-(3-aminopropyl)-1,3-dihydroxypropane (2).

Materials: In addition to those given in part (1), lithium aluminum hydride (1.0 M solution in diethyl ether) was obtained from Aldrich Fine Chemicals, Milwaukee, Wisconsin.

Procedure: 2-(3-Nitrilepropyl)-diethyl malonate (1, 3.21 g, 15.1 mmol) in anhydrous diethyl ether (50 mL) was added dropwise to a stirred solution of lithium aluminum hydride (0.1 mol in 100 mL diethyl ether) under nitrogen. The resulting mixture was heated under reflux for two hours and then stirred at room temperature overnight. Then a 2.5 mM solution of sodium hydroxide in water (100 ml) was added slowly to suppress unreacted hydride. This mixture was stirred for two hours and the ether layer was decanted and discarded. (The product remains in the water layer). The white gelatinous solid was removed from the aqueous layer by centrifugation, washed with water and the aqueous supernatant and washings were combined and evaporated to a syrup under vacuum. Thin layer chromatography (Analtech reverse phase plates, water mobile phase, visualized with ninhydrin reagent) gave a major spot identified as the title compound (2), Rf 0.48, and a minor spot attributed to a condensation byproduct, Rf 0.29. The title compound (2) was purified by ion exchange chromatography (Dowex 50X8, 0.5 M HCl mobile phase)

■in a total dividend of 50%. 1 H NMR analyzes (D 2 O) : 61.36 (distinct quartet, 2H), 1.68 (m, 3H), 2.97 (t, 2H) 3.57 (t, 4H).

(C) Synthesis of 2-(3-N-trifluoroacetylaminopropyl) 1,3-dihydroxypropane (3): The procedure was adapted from the method

after R.F. Oldfinger in "Methods in Enzymology", vol. XI, page 317, incorporated herein by reference.

Materials: (In addition to those listed above) S-ethyltrifluorothioacetate was used; from Aldrich Fine Chemicals, Milwaukee, Wisconsin.

Procedure: 2-(3-aminopropyl)-1,3-dihydroxypropane (2), (3 mmol) was dissolved in water (25 mL). The pH of the solution was lowered to 9.5 by dropwise addition of 6 N HCl. Subsequent reactions were carried out in a hood: S-ethyl trifluorothioacetate (2 ml) was added dropwise to the vigorously stirred solution; The pH was maintained between 9.5 and 10.0 by dropwise addition of 6 N KOH. After thirty minutes, a further milliliter of S-ethyltrifluorothioacetate was added, maintaining the pH as described above. The mixture was stirred for an additional 45 minutes. Then the pH was adjusted to 7 using 6 N KOH and the mixture was concentrated to dryness by rotary evaporation under vacuum. The residue was vortexed with acetone (20 mL) and filtered to remove potassium acetate precipitate. The filtrate was concentrated to a syrup, redissolved in acetone (2 ml) and applied to a flash chromatography column containing 40 g of silica gel (average particle diameter 40 µm, from J.T. Baker Chemical Co.

(Phillipsburg, New Jersey, USA). The column was eluted with a 50:50 solution (v/v) of methylene chloride/acetone (500 ml) withdrawing 25 ml fractions. Fractions were analyzed for product content by placing 2 µl samples on silica gel plates, spraying with 10% piperidine in water, leaving the plates for 15 minutes, then drying them with a heat gun, and then treating them with ninhydrin reagent. Omission of the piperidine spray treatment prevented a colorimetric reaction with ninhydrin, confirming trifluoroacetylation on the primary amine. Using this procedure, product was found between fractions 13 and 18; these factions

colorless oil, Rf 0.4 (silica gel thin layer chromatography using the same solvent system and visualization method as described above). (d) Synthesis of 1-O-(dimethoxytrityl)-2-(3-N-trifluoroacetylaminopropyl-1,3-dihydroxypropane (4): Materials: (In addition to those listed above) Dimethoxytrityl chloride was obtained from Aldrich Fine Chemicals (Milwaukee, WI, USA). Methylene chloride was refluxed and distilled over CaH2 and stored over 4 Angstrom (4 A) molecular sieves. Pyridine was distilled over potassium hydroxide pellets and p-toluenesulfonate and stored under dry nitrogen.

Procedure: 2-(3-N-trifluroacetylaminopropyl)-1, 3-dihydroxypropane (3) (362 mg, 1.58 mmol) was dried with several evaporations of dry pyridine under reduced pressure and then further dried for several hours under complete vacuum . The residue was then dissolved in 10 ml of dry pyridine under dry nitrogen. Dimethoxytrityl chloride (401 mg, 1.18 mmol) in dry methylene chloride (1.5 mL) was added with stirring, and the resulting solution was stirred for one hour at room temperature. The solvent was removed under reduced pressure and the residue was dissolved in chloroform (50 mL). This solution was extracted three times with 5% sodium bicarbonate in water and then dried over anhydrous magnesium sulfate. The resulting solution was concentrated to an oil, redissolved in 2 ml of chloroform and fractionated by flash chromatography as described above, with the exception of using chloroform/ethyl acetate/pyridine (95:5:0.2 v/v /v) as the mobile phase. Fractions were analyzed by thin layer chromatography on silica plates using the same solvent system. Spots visualized with HCl vapor and with Rf values of 0.27, 0.87 and 0.93 were identified as the 1-dimethoxytrityl product (4), dimethoxytritanol and the 1,3-di-(dimethoxytrityl) by-product. The latter material can be hydrolyzed to the title compound (4) by shaking with a mixture of 4% dichloroacetic acid in methylene chloride saturated with water. The product (4) was isolated by evaporation of the solvent from the appropriate fractions and dried under complete vacuum, yielding a foam (370 mg, 44%). (e) Synthesis of 1-O-(dimethoxytrityl)-2-(3-N-trifluoroacetylaminopropyl)-3-O-(methyl-N,N-diisopropylphosphoramido)-1,3-dihydroxypropane (5): Materials: (I in addition to those indicated above). N,N-diisopropylethylamine and N,N-diisopropylmethylphosphoramide chloride were obtained from Aldrich Chemical Company (Milwaukee, WI, USA). Dimethylformamide was heated at reflux and distilled over CaH and stored over 4 A molecular sieves.

Preparation: 1-O-(dimethyloxytrityl)-2-(3-trifluoroacetylaminopropyl)-1,3-dihydroxypropane (4,300 mg, 0.56 mmol) dried with several evaporations of dry pyridine and dissolved in 10 mL of dry dimethylformamide. The following reaction was carried out under dry nitrogen: N,N-diisopropylethylamine (245 µl, 1.3 mmol) was added with stirring, followed by N,N-diisopropylmethylphosphoramide chloride (140 µl, 0.7 mmol). The reaction mixture was stirred for two hours. The mixture was then concentrated under reduced pressure and dissolved in methylene chloride (50 mL). This solution was extracted three times with 5% aqueous sodium bicarbonate, dried over anhydrous magnesium sulfate, and concentrated to an oil under reduced pressure. Conversion of the starting material (4) to the corresponding phosphoramidite (5)

31

was confirmed by P NMR (CDCl 3 , trimethyl phosphate, external standard): 6147.9. Purity was estimated at more than 70%.

Example 2: Synthesis of 2,2-di-(3-aminopropyl)-1,3-dihydroxypropari linker reagent.

This example comprises of a monomer for incorporation into the phosphodiester backbone of a synthetic oleonucleotide having two aminopropyl linkers for multiple label attachment. The schematic presentation of the synthesis is given in figure 2.

Materials: The materials are the same as those given in Example 1, except when specifically indicated otherwise. (a) Synthesis of 2,2-di-(3-aminopropyl)-1, 3-dihydroxypropane (7): The following procedure is a brief description of an adaptation of the method described above in Example 1 (b). 2,2-Di-(nitrilepropyl)-diethyl malonate (6) (2.00 g, 7.51 mmol), whose synthesis is described in Example 1(a) was dissolved in anhydrous diethyl ether (80 ml). The obtained solution was added dropwise to a stirred solution of lithium aluminum hydride (0.1 mol) in diethyl ether (100 ml). After fifteen minutes, the mixture was heated at reflux for two hours and then stirred at room temperature overnight. Production and extraction of the raw product was carried out as described above in example 1(b). A thin-layer chromatography, also described in example 1(b), gave a main spot (Rf approx. 0.2). (b) Synthesis of 2,2-di-(3-trifluoroacetylaminopropyl)-1 3-dihydroxypropane (8) : 2,2-di-(3-aminopropyl)-1,3-dihydroxypropane (7) (3.7 mmol ) was dissolved in water (25 mL) and the pH was then adjusted to about 10 with 6 NHCl. 1.0 ml of S-ethyl trifluorothioacetate was added with vigorous stirring, the pH was maintained between 9.5 and 10.0 by dropwise addition of 6 N KOH. Two additional 1.0 mL additions of S-ethyl trifluorothioacetate were added in the same manner at thirty minute intervals. The mixture was concentrated to an oil under vacuum and then dissolved in acetone (30 mL). Precipitated potassium acetate was removed by filtration. The product (8) was purified by silica gel flash chromatography as described in example 1(c) using methylene chloride/acetone (50:50 v/v) mobile phase. The purified material gave a single spot (Rf 0.7) by silica gel thin-layer chromatography using

same solvent system; visualization was carried out by treatment with piperidine followed by ninhydrin as in example 1(c). The yield was 510 mg (1.48 mmol). (c) Synthesis of 1-O-(dimethoxytrityl)-2,2-di(trifluoroacetylaminopropyl)-1,3-dihydroxypropane (9): 2,2-di-(3-trifluoroacetylaminopropyl)-1,3-dihydroxypropane (8 ) (510 mg, 1.48 mmol) was dried by several evaporations of dry pyridine under vacuum and then dissolved in 5 mL of dry pyridine under nitrogen. Dimethoxytrityl chloride (3.76 mg, 1.11 mmol) in dry methylene chloride (1.5 mL) was added under nitrogen with stirring, and the mixture was stirred for one hour. The mixture was concentrated under reduced pressure and dissolved in chloroform (50 mL). This solution was extracted three times with saturated aqueous sodium bicarbonate and once with saturated sodium chloride. The solution was then dried over anhydrous magnesium sulfate and concentrated to an oil under vacuum. The oil was then dissolved in 2 ml of chloroform and fractionated by silica gel flash chromatography as described above using chloroform/ethyl acetate/pyridine (80:20:0.2 v/v/v). The product (9) was identified by silica gel thin-layer chromatography using the same solvent system, visualized with HCl vapor (Rf 0.3); it was concentrated under reduced pressure and dried under complete vacuum to give a pale yellow foam (517 mg, 52%). (d) Synthesis of 1-O-(dimethoxytrityl)-2,2-di-(3 trifluoroacetylaminopropyl)-3-O-(methyl-N,N-diisopropylphosphoro-amido)-1,3-dihydroxypropane (10) : Procedure : 1-O-(dimethoxytrityl)-2,2-di-(trifluoroacetylaminopropyl)-1,3-dihydroxypropane (9) (136 mg, 0.2 mmol) was dried with three coevaporations of dry pyridine (3 mL) . The obtained residue was dissolved in dry methylene chloride (1.5 mL) under argon and N,N-diisopropylethylamine (175 µl, 1.0 mmol) was added with gentle stirring. N,N-diisopropylmethylphosphoramide chloride (80 u, 0.4 mmol) was then added, and the reaction mixture

no one was stirred for an hour. The resulting mixture was diluted with ethyl acetate/triethylamine (98.2) (50 mL) and extracted twice with saturated aqueous sodium bicarbonate (25 mL). The organic layer was dried over anhydrous MgSO4 and concentrated to an oil (240 mg). Conversion to phosphoramidite (10) was

31

confirmed by P NMR (CDCl 3 trimethoxyphosphate, external standard): 6145.5. Purity was estimated at more than 60

Example 3: Synthesis of a 3-amino-1,2-propanediol-based linker reagent.

The synthesis is shown schematically in figure 3, and is described below. (a) Synthesis of a 3-(trifluoroacetylamino)-1,2-propanediol (11): Materials: 3-amino-1,2-propanediol and S-ethyltrifluorothioacetate were obtained from Aldrich Chemical Co. (Milwaukee WI, USA) . Procedure: S-ethyl trifluorothioacetate (5.13 mL, 45 mmol) was added to a rapidly stirred mixture of 3-amino-1,2-propanediol (2.32 mL, 30 mmol) and ethyl acetate (5.0 mL). After several minutes, the mixture became homogeneous. After one hour, the reaction solution was shaken with petroleum ether (100 ml), an oil was obtained which was separated and concentrated under vacuum. This material was analyzed by means of thin-layer chromatography on silica plates using ethyl acetate/methylene chloride (2:1) as mobile phase. The plates were first visualized with ninhydrin reagent, which showed a mark of unreacted amine starting material at the starting point. The plates were then visualized by being sprayed with 10% aqueous piperidine, dried with a heat gun after 15 minutes and then treated with ninhydrin reagent. In the latter case, a main spot was visible (Rf 0.28) which was estimated to comprise more than 95% of the material. Purification of the material associated with the main spot (11) was achieved by thin layer chromatography on a preparative scale. (b) Synthesis of 3-(trifluoroacetylamino)-1-0-(dimethoxytrityl)-].,2-propanediol (12): The materials and general procedures are described above in Example 1 (d). 3-(Trifluoroacetylamino)-1,2-dihydroxypropane (11) (1.87 g, 10 mmol) was dried by three co-evaporations of dry pyridine (10 mL) under reduced pressure. The material was then dissolved in dry pyridine (10 mL). Then a solution of dimethoxytrityl chloride (3.73 g, 11 mmol) in dry pyridine (10 mL) was added dropwise with stirring under nitrogen. After approx. one hour, methanol (0.2 mL) was added. The resulting solution was diluted with ethyl acetate (80 mL) and extracted twice with saturated aqueous sodium bicarbonate (30 mL) and twice with water (20 mL). The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to give 6 g of crude oil. 1.1 g of the crude material was fractionated by silica gel flash chromatography as described above using methylene chloride/ethyl acetate/pyridine (10:1:0.01 v/v/v). Fractions were analyzed by thin-layer chromatography on silica gel plates with the same solvent system. Spots were visualized with HCl gases, and showed two minor components at Rf values of 0.94 and 0.87 and a major component at Rf 0.53 which was identified as the 1,2-di-(dimethoxytrityl) by-product, dimethoxytritanol and the calculated product (12). Fractions with an Rf of 0.53 determined by thin layer chromatography as described above were pooled and evaporated to give 0.72 g of (12), the structure of which was confirmed by <1>H NMR. Total yield of (12) was 80% based on the yield from the fast column. (c) synthesis of 3-(trifluoroacetylamino)-1-0-(dimethoxytrityl)-2-0-(methyl-N,N-diisopropylphosphoramido)-1,2-propanediol (13): The reagents for this synthesis are given in preceding in example 1.

3-(Trifluoroacetylamino)-1-O-(dimethoxytrityl-1,2-propanediol (12) (19 6 mg, 0.4 mmol) was dissolved in dry methylene chloride (1.5 mL) containing diisopropylethylamine (348 µl, 2 mmol ).

N,N-diisopropylmethylphosphoramide chloride (200 µl, 1 mmol) was added dropwise with stirring under argon. After one hour, ethyl acetate containing 1% triethylamine was added (50 mL), and the resulting solution was extracted three times with saturated aqueous sodium bicarbonate. The organic layer was dried over anhydrous magnesium sulfate and concentrated to an oil under reduced pressure. The purity of this material was estimated to

31

be greater than 95% by P NMR (CD3CN, trimethoxyphosphoric acid, external standard): 5147.5. Attempts to crystallize this material in toluene/hexane mixtures at -7 8°C were not successful, and the impure samples were therefore used directly for linker addition.

Example 3(a): Synthesis of 6-amino-1,2-hexanediol based linker reagent.

The synthesis is shown schematically in figure 4, and is described in what follows. (a) Synthesis of 1,2-(isopropylidine)-1,2,6-trihydroxyhexane (14): Materials: 1,2,6-trihydroxyhexane and 2,2-dimethoxypropane were obtained from Aldrich Chemical Co. (Milwaukee,

WI, USA).

Procedure: 1,2,6-trihydroxyhexane (1.00 g, 7.45 mmol), dry acetone (10 mL) and concentrated sulfuric acid (30 µL) were added to a 50 mL round-bottomed flask along with a magnet for magnetic stirring. The flask was purged with nitrogen and a rubber septum was attached to exclude moisture. Then 2,2-dimethoxypropane (3.00 mL, 24.4 mmol) was added slowly via syringe to the stirred solution over a period of 30 minutes. Stirring was continued for two hours. Anhydrous sodium carbonate (150 mg) was added to quench

the reaction, and the mixture was then stirred overnight.

Finally, the solution was filtered and concentrated under vacuum to a pale yellow syrup (1.6 g). This material was used in the next step without purification. (b) Synthesis of 1,2-(isopropylidine)-6-(p-toluenesulfonyl)-1,2,6-trihydroxyhexane (15): Materials: p-toluenesulfonyl chloride was obtained from Aldrich Chemical Co. (Milwaukee, WI, USA).

Procedure: The crude isopropylidinated material from the previous step (14, about 7.45 mmol) was dissolved in dry acetone (15 mL). Then p-toluenesulfonyl chloride (2.8 g, 14.9 mmol) and dry pyridine (5 ml) were added, and the contents were stirred for 3 hours at room temperature with exclusion of moisture. The solvent was then removed under vacuum and the residue was partitioned between ethylene chloride (25ml), dried over anhydrous magnesium sulfate, filtered and then concentrated under vacuum to give a crystalline solid (15, 2.8g). The crude product was purified by silica gel flash chromatography as described above using chloroform as mobile phase. Fractions were analyzed by thin layer chromatography on fluorescent silica gel plates using the same solvent. Spots were visualized under an ultraviolet lamp. Fractions containing product (R, 0.50) were collected and concentrated under vacuum to give an oil (15, 2.37 g) in a total yield of 96.9%. (c) Synthesis of 1,2-(isopropylidine)-6-azido-1,2-dihydroxyhexane (16): Procedure: The tosylate from the previous step (15, 2.37 g, 7.22 mmol) was dissolved in dry dimethylformamide (30 mL). Sodium azide (1.64 g, 25.2 mmol) was added along with a magnet for magnetic stirring, and a reflux condenser and CaCl 2 drying tube were attached. The mixture was then stirred in a water bath at 60-65°C for three hours. Stirring was continued overnight at room temperature. The precipitate was then removed by centrifugation, and the resulting solution was concentrated under vacuum to a final volume of approximately 5 mL. The concentrated solution was partitioned between chloroform (50 mL) and water (15 mL). The organic layer was further washed with water (15 mL), dried over anhydrous MgSO 4 , filtered and concentrated under vacuum to an amber oil (16). The crude product was then used in the next step without further purification.

(d) Synthesis of 6-amino-1,2-dihydroxyhexane (17):

Materials: A solution of lithium aluminum hydride

(1.9 M) in diethyl ether was obtained from Aldrich Chemical Company (Milwaukee, WI, USA).

Procedure: Anhydrous diethyl ether (10 mL) and a solution of lithium aluminum hydride (1.0 M) in diethyl ether (15 mL) were added to a 250 mL round-bottomed flask under an argon atmosphere. A solution of the impure azide from the previous step (16, ca.

7 mmol) in anhydrous diethyl ether (25 mL) was then added through an addition funnel with stirring under argon. After complete addition, the mixture was stirred under argon. After complete addition, the mixture was stirred under reflux for 90 minutes. The resulting slurry was diluted with diethyl ether (25 mL) and the following solutions were added with stirring in the order indicated: Water

(1 ml), 5 N NaOH (1 ml) and water (1 ml). The mixture was then filtered through a medium glass scinter. The filtrate was concentrated by distillation at room temperature followed by high vacuum to give a pale yellow oil. Then water (10.8 ml) and 88% formic acid (14.2 ml) were added. The resulting mixture was allowed to stand overnight at room temperature and was then heated to 70-75°C for 2 hours. The solution was concentrated under vacuum to a syrup, which was then dissolved in water (50 mL) and applied to a cation exchange column containing AG 50W-X8 resin (H form, 50 mL bed volume, Bio-Rad labs, Richmond, CA, USA ). The column was eluted with 1 N HCl. Fractions containing the amine product were visualized

by spot formation on silica gel TLC plates, spraying with ninhydrin reagent and heating as described above. Fractions containing the product were pooled and concentrated under vacuum to give a syrup, which was further co-evaporated with methanol to give pale yellow needles (17, as hydrochloride). (e) Synthesis of 6-N-(9-fluorenylmethoxycarbonyl)-amino-1,2-dihydroxypropane (18): Materials: 9-fluorenylmethylsuccinimidyl carbonate (Fmoc-NHS) was obtained from Bachem, Inc. (Torrance, CA. USA) . Procedure: 6-amino-1,2-dihydroxyhexane hydrochloride (17), in an amount consistent with the yield obtained in the previous step, was dissolved in water (10 mL) and adjusted with 5 N NaOH to a final pH of 8.7 . Sodium bicarbonate (588 mg, 7 mmol), Fmoc-NHS (2.76 g, 7 mmol) and acetone (10 mL) were added. The suspension was stirred overnight at room temperature, after which all the Fmoc-NHS had gone into solution. The reaction mixture was concentrated under vacuum to remove acetone. 1 N HCl (50 mL) and ethyl acetate (150 mL) were added and the mixture was transferred to a separatory funnel. The organic layer was separated and washed with 0.1 N HCl (50 mL) and then with water (2 x 50 mL). The organic layer was then dried over anhydrous MgSO 4 , filtered and concentrated to an oil. The product was purified by silica gel flash chromatography as described above using chloroform/acetone (50:50) as mobile phase. Fractions were analyzed by thin-layer chromatography on fluorescent silica gel plates using the same solvent system. Spots were visualized under an ultraviolet lamp. Fractions containing the product (R, 0.25) were collected and evaporated to give a white crystalline solid (18, 1.20 g). Overall yield was 45%, based on the amount of 1,2,6-trihydroxyhexane starting material. (f) Synthesis of 1-O-(dimethoxytrityl)-6-N-(fluorenylmethoxycarbonyl)-6-amino-1,2-dihydroxyhexane: Procedure: The product from the previous step (18, 0.5 g, 1.41 mmol ) was co-evaporated with dry pyridine (3 x 3 mL) and then dissolved in dry pyridine (8 mL) under argon. A solution of dimethoxytrityl chloride (0.5736 g, 1.69 mmol) in dry methylene chloride (2 mL) was added via syringe with stirring over a period of several minutes. Stirring was continued for 2 hours at room temperature, after which methanol (100 µl) was added to quench the reaction. The solvent was removed under vacuum and the residue was dissolved in chloroform (100 mL). This obtained solution was transferred to a separatory funnel and washed with saturated aqueous sodium bicarbonate (3 x 20 mL) followed by 5 M NaCl (20 mL). The organic layer was then dried over anhydrous MgSO 4 <filtered and concentrated to an oil under vacuum. The product was purified by silica gel flash chromatography as described above using a methylene chloride/ethyl acetate/triethylamine (95:5:0.5) solvent system. Fractions were analyzed by thin-layer chromatography on silica gel plates using the same solvent; spots were visualized by subjecting the plates to HC1 vapor. Fractions containing product (19, R, 0.35) were collected and evaporated under vacuum to a foam (910 mg, 100% of theoretical yield). (g) synthesis of 1-O-(dimethoxytrityl)-6-N-(fluorenylmethoxycarbonyl)-2-O-(methyl-N,N-diisopropylphosphoramido)-6-amino-1,2-dihydroxyhexane (20): Materials: The materials are described in the previous examples.

Procedure: N,N-diisopropylmethoxyphosphinyl chloride (102 µl, 0.513 mmol) was added dropwise to a stirred solution of 19 (225 mg, 0.34 mmol) and N,N-diisopropylethylamine (236 µl, 1.36 mmol) in dry methylene chloride (3 mL) under an argon atmosphere. After 90 minutes, the reaction mixture was diluted in ethyl acetate containing 2% triethylamine (50 mL) and washed with saturated aqueous sodium bicarbonate (2 x 25 mL). The organic layer was dried over anhydrous MgSO 4 , filtered and evaporated to dryness under vacuum. The residue was dissolved in toluene (2 ml) and added dropwise with rapid stirring to petroleum ether at -20°C. The resulting mixture was then stored at -20°C for 16 hours. It was then warmed to room temperature and the supernatant was decanted. The precipitated product (20) was then dried under vacuum: yield = 160 mg (58% yield). The purity of this material was demonstrated by thin-layer chromatography on silica gel plates using a methylene chloride/ethyl acetate/triethylamine (10:1:0.1) solvent system and visualized under ultraviolet light (R, 0.9, compared to an R, of 0 .25 for starting material).

Extended analogs of the linker reagent described in Example 3(a) were also formed. The structure of these analogues is shown in Figure 5 (21-24). The preparation of these analogues is described in the following examples.

Example 3(b): Synthesis of 3-N-(glycidyl)-amino-1,2-propanediol-based linker reagent (21).

The reaction core for this synthesis is shown in Figure 6.

(a) synthesis of 3-N-[B-(fluorenylmethoxycarbonyl)-glycidyl]-amino-1,2-propanediol (25).

Materials: N-(fluorenylmethoxycarbonyl)-glycine N-hydroxysuccinimide (Fmoc-glycine-NHS) was obtained from Barchem, Inc. (Torrance, CA, USA). Other reagents have been described previously.

Procedure: 3-Amino-1,2-propanediol (91 mg, 1 mmol) was added to a solution of Fmoc-glycine-NHS (394 mg, 1 mmol) in acetone (7 mL). To this solution was added a solution of sodium bicarbonate (84 mg, 1 mmol) in water (5 mL). The reaction mixture was stirred at room temperature for 16 hours. Thin layer chromatography using silica gel plates and a methylene chloride/methanol/acetic acid (20:2:0.1) solvent system showed that the reaction was complete. The product (25) appeared in the flask as a precipitate, which was filtered off and dried under vacuum over P2O5i for two days. The yield was 310 mg (84%). (b) Synthesis of 1-O-(dimethoxytrityl)-3-N-[N-(fluorenylmethoxycarbonyl)-glycidyl]-amino-1,2-propanediol (26).

Materials: The materials are described in the preceding examples.

Procedure: Compound 25 (185 mg, 0.5 mmol) was dried by co-evaporation with dry pyridine (3 x 3 mL). It was then dissolved in dry pyridine (3 mL) and a solution of dimethoxytrityl chloride (222 mg, 0.57 mmol) in a 1:1 mixture of methylene chloride/pyridine (4 mL) was added dropwise with stirring. Stirring was continued for 1.5 hours and the reaction was measured by silica gel thin layer chromatography using a methylene chloride/methanol (8:1) solvent system. The reaction was quenched by the addition of methanol (0.2 mL); stirring was continued for 10 minutes. Pyridine was evaporated under vacuum. The residue was dissolved in methylene chloride (150 mL) and washed with saturated aqueous sodium bicarbonate (2 x 50 mL) followed by water (50 mL). After drying over anhydrous MgSO 4 , the methylene chloride solution was evaporated to dryness under vacuum. The residue was purified by silica gel flash chromatography using a methylene chloride/ethyl acetate (11:5) solvent system containing 0.1% pyridine in accordance with the method described above. Fractions containing product were identified by silica gel thin-layer chromatography in , as described above. These fractions were combined and evaporated to dryness, yielding 250 mg of 26 (75% yield). (c) Synthesis of 1-O-(dimethoxytrityl)-2-O-(N,N-diisopropylamino-methoxyphosphinamido)-3-N-[N-(fluorenylmethoxycarbonyl)-glycidyl]-amino-1,2-propanediol (21).

Materials: The materials are described in previous examples.

Procedure: Compound 21 (235 mg, 0.35 mmol) was dried by co-evaporation with dry pyridine (2 x 3 mL). It was then dissolved in dry methylene chloride (2 mL) and N,N-diisopropylethylamine (244 µl, 1.4 mmol) was added. Then fine N,N-diisopropylaminochloromethoxyphos (105 µl, 0.53 mmol) was added dropwise with stirring under an argon atmosphere. The reaction was found to be complete after 20 minutes by silica gel thin layer chromatography using a methylene chloride/ethyl acetate/triethylamine (10:5:0.5) solvent system. The reaction mixture was then diluted in ethyl with saturated aqueous bicarbonate (2 x 25 mL). After drying over anhydrous MgSO 4 , the ethyl acetate layer was evaporated under vacuum. The residue was redissolved in ethyl acetate (3 mL) and poured into hexane (150 mL) at -25°C. The precipitate was filtered and dried under vacuum to give 210 mg of 21 (72%).<31>P-NMR (CDCl 3 , in ppm relative to trimethyl phosphate): 147.5 (d). The structure was also confirmed by ^-H-NMR analyses.

Example 3(c): Synthesis of 3-N-(4-aminobutyryl)-amino-1,2-propanediol and 3-N-(6-aminocaproyl)-amino-1,2-propanediol-based linker reagents (22, 23 ).

The steps in the synthesis which are unique to this example are given schematically in Figure 7 and are described below. (a) Synthesis of N-fluorenylmethoxycarbonyl-protected forms of 4-aminobutyric acid and 6-aminocaproic acid (N-Fmoc-4-aminobutyric acid, 27, and N-Fmoc-6-aminocaproic acid, 28).

Materials: 4-aminobutyric acid and 6-aminocaproic acid were obtained from Aldrich Chemical Co. Milwaukee, WI, USA). Fmoc-NHS was described in Example 3(a).

Procedure: These syntheses were carried out as described in

the method according to A. Paquet (Can. J. Chem., 1982, 60,

976). (b) Coupling of both N-Fmoc-4-aminobutyric acid and N-Fmoc-6-aminocaproic acid with 3-amino-1,2-propanediol.

Materials: Trimethylacetyl chloride was obtained from Aldirch Chemical Co. (Milwaukee, WI, USA). Other materials were described in previous examples.

Procedure: Both compounds 27 and 28 (1 mmol) were first dried by co-evaporation with pyridine (2 x 3 mL). The residue was then dissolved in a mixture of dry dimethylformamide (3 mL) and dry tetrahydrofuran (3 mL). The resulting solution was cooled in an ice bath and N,N-diisopropylethylamine (1 mmol) was added followed by the slow addition of trimethylacetyl chloride (1 mmol) with stirring. Stirring was continued in an ice bath for 45 minutes. Then a solution of 3-amino-1,2-propanediol (1.2 mmol) in dry dimethylformamide (3 ml) was added, and the resulting mixture was warmed to room temperature and stirred for 1 hour. The reaction was measured by silica gel thin layer chromatography using a methylene chloride/methanol/acetic acid (10:1:0.1) solvent system. Based on this analysis, the reaction was determined to have gone to about 90% completeness. The reaction mixture was then concentrated under vacuum, diluted with ethyl acetate (100 mL) transferred to a separatory funnel. The organic solution was washed with saturated aqueous sodium bicarbonate (2 x 50 mL) and water (50 mL). After drying over anhydrous MgSO 4 , the organic layer was evaporated to dryness. The product obtained was determined to have a purity of over 95% and was used in the subsequent steps without further purification. In those cases where purification was necessary, however, it was carried out by means of silica gel flash chromatography as described in the preceding examples, using a methylene chloride/methanol (40:1) solvent system. Purities of 28 and 29 were confirmed by<1>H-NMR analyses.

(c) 1-O-Dimethoxytritylation of 28 and 29:

The materials and procedure for this synthesis were as described in Example 3(b), part (b). The purity of these materials was confirmed by H-NMR. (d) Conversion of the compounds discussed in part (c) above, to the corresponding 2-O-(N,N-diisopropylmethyl)-phosphoramidites (22 and 23): The materials and procedure for this synthesis were as described in Example 3(b ), part (c). The purity of products 22 and 23 was confirmed by <31>P-NMR.

Example 3(d): Synthesis of a further extended analogue of a 3-N-(6-aminocaproyl)-amino-1,2-propanediol-based linker reagent.

The unique steps in this synthesis are indicated schematically in Figure 8 and are described below. (a) Synthesis of 1-O-(dimethoxytrityl)-3-N-6-amino-caproyl)-Amino-1,2-dihydroxypropane (31).

Materials: Compound 30 was prepared as described in Example 3(c), part (c).

Procedure: Compound 30 (0.89 g, 1.1 mmol) was subjected to ammonolysis with concentrated ammonium hydroxide (10 mL) and pyridine (10 mL) at room temperature overnight. Samples from the reaction were applied to silica gel TLC plates and treated with ninhydrin reagent to investigate deprotection of the primary amine. The reaction mixture was then dried under vacuum and the obtained residue (30) was used in the subsequent step without purification. (b) Coupling of compound 31 with compound 28.

Materials: Compound 28 was synthesized in accordance with the procedure described in Ex. 3(c) subsection (a).

Procedure: N-Fmoc-aminocaproic acid (28, 1.1 mmol) was reacted with triethylacetyl chloride (1.1 mmol) according to the procedure described in Ex. 3(c), subdivision (b). Then a solution of compound 30 (1.1 mmol) in dry dimethylformamide was added, again in accordance with the procedure in Ex. 3(c) subsection (b). The resulting adduct, 32, was purified by silica gel flash chromatography as described in the aforementioned examples using a chloroform/methanol (30:1) solvent system. The yield of the product was 250 mg

(23%). (c) Conversion of compound 32 to the corresponding 2-O-(N,N-diisopropylamine)-methoxyphosphoramidite (24).

Procedure: The method was essentially the same as that described in example 3(b), part (c). Thus, compound 32 (240 mg, 0.285 mmol) was reacted with N,N-diisopropylaminochloromethoxyphosphine (73 µl, 0.371 mmol) in dry methylene chloride (3 mL) containing N,N-diisopropylethylamine (198 µl, 1.14 mmol). The reaction was carried out by diluting with 2% triethylamine in ethyl acetate (50 ml) and extracting with saturated aqueous sodium bicarbonate (25 ml) and water (25 ml). The organic layer obtained was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was dissolved in several milliliters of ethyl acetate and precipitated in hexanes (150 ml) as described in the above example. The yield of 32 was 200 mg, the purity of which was confirmed by<1>H- and<32>P-NMR spectroscopy.

Example 4: Automated attachment of 2-(3-aminopropyl)-1,3-dihydroxypropane-based linker to synthetic oligonucleotides.

Attachment of the linker reagent described in Example 1, hereinafter referred to as "LI", to various synthetic oligonucleotides will now be described. (a) The "LI" reagent was attached to the 5' end of a deoxyoligonucleotide. A deoxynucleotide with the sequence "5'-GCTCGTTGCGGGACTTAACCCAACAT-3'" was synthesized on a controlled porous glass support with an Applied Biosystems, Inc., Model 3 80A DNA Synthesizer using standard phosphoramidite chemistry. The 5'-dimethoxytrityl group was removed and a solution of "LI" (0.1M) in dry acetonitrile was coupled twice using standard coupling cycles. Percent couplings at the first and second additions of "LI" were quantified relative to the amount of full-length deoxyoligonucleotide, by measuring the absorbance at 49 8 nm of dimethoxytrityl released at the end of each coupling cycle; as these values were determined to be 29 and 42% respectively. The 5'-(L1) and 5'-(LI)-(LI) oligonucleotides were purified by gel electrophoresis on a 20% polyacrylamide gel containing 7 M urea. The corresponding bands were visualized by UV shadowing and were estimated to migrate more slowly on the gel with spacing approximately 1.5 times that of the corresponding additional nucleotide spacing. These bands were excised and linker-modified deoxy-nucleotides were recovered and purified by standard methods. (b) The "LI" reagent was attached to the 3' end of a deoxyoligonucleotide. For this synthesis, a Teflon oxidizable solid support was used (Molecular Biosystems, Inc., San Diego, CA, USA, catalog #0SS-01). When a deoxyribonucleotide is cleaved from this support, the compound used during the first cycle of coupling remains at the 3'-end together with a 3'-terminal phosphate group. A solution of "LI" (0.2 M) in dry acetonitrile was coupled to this support using three cycles of coupling with standard phosphoramidite chemistry on an Applied Biosystems, Inc., Model 380A DNA Synthesizer. Next, a deoxyoligonucleotide sequence identical to that presented immediately above in part (a) was added using the same coupling chemistry. Initial percent couplings with the "LI" reagent were determined as described above to be 40%, 63%, and 63% for the first, second, and third couplings, respectively. After removal of the terminal dimethoxytrityl group from the obtained trimer, a deoxyoligonucleotide of the same sequence as in 4(a) was added using standard phosphoramidite chemistry. The carrier material was then removed from the synthesizer and treated with concentrated ammonium hydroxide at 55°C for 16 hours. The support was then washed three times with water and treated with 50 mM NaI0 4 in 20 mM sodium phosphate buffer (pH 7.4) for 2.5 hours at room temperature. Finally, the support was washed several times with water and treated with a 10% aqueous solution of n-propylamine at 55°C for 3 hours. The obtained solution was applied to a 20% polyacrylamide gel containing 7 M urea and electrophoresis was performed. Corresponding 3'-(LI)-(LI)-(LI) deoxyoligonucleotide was prepared as described above.

(c) "LI" was joined at the 3' end of a deoxynucleotide with the sequence

The synthesis method was the same as described in part (b), except that a Biosearch Model 8750 DNA Synthesizer was used.

(d) "LI" was linked at the 3' end of a deoxyoligonucleotide with the sequence

Again, the method described in part (b) was used with the exception that the automated part of the synthesis was performed on a Biosearch Model 8750 DNA Synthesizer. (e) Hybridization and melting temperatures (Tm) of synthetic deoxyoligonucleotide probes containing 2-(3-aminopropyl)-1,3-dihydroxypropane ("LI") linkers inserted between nucleotide bases.

Materials: Two L1 derivatives of a 33-mer deoxyoligonucleotide probe were synthesized by methods similar to those described above: "L1 insertion" has L1 inserted between nucleotide residues 21 and 22 (numbering from the 5' end);

"Ll replacement" provides LI between nucleotide residues 20 and 22 as a replacement for residue 21. Both probes have sequences complementary to ribosomal RNA from Clamydia trachomatis ("target rRNA"). The probes were labeled with 125j using a standard procedure developed by Gen-probe Inc., "hydroxyapatite" (HAP) was from Behring Diagnostic, Calbiochem Division, La Jolla, CA; sodium dodecyl sulfate (SDS), sodium phosphate (mono- and dibasic salts), and hydrochloric acid were graded reagents from Fisher Scientific Corp.; Betagel (scintillation liquid mixture) was from West Chem, San Diego, CA. All other materials were graded reagents. Manipulations were carried out in 1.5 ml polypropylene Eppendorf tubes with screw caps unless otherwise stated.

Hybridizations were carried out as follows: 48 µl 1 M sodium phosphate (pH 6.8), 10 µl 1% SDS (v/v), 10 µl 125x labeled probe (approximately 200,000 CPM), 29.5 µl water and either 25 µl rRNA solution (0.5 µg, "target") or 2.5 µl water ("control") was mixed and incubated at 60°C for one hour. 10 µl samples were then diluted with 1 ml of 0.12 M sodium phosphate (pH 6.8)/- 0.02% SDS/0.02% sodium azide and vortexed for 5 seconds. The diluted samples were incubated in a water bath heated from room temperature to 80°C; the samples were removed at set temperatures and stored on ice. The samples were then passed through a small column of hydroxyapatite equilibrated with 0.12 M sodium phosphate (pH 6.8)/0.02% SDS/0.02% sodium azide, and the eluents were counted by scintillation using standard procedures. (Percent counts that remained bound to the column corresponded to hybridized probe). The Tm values were calculated as the temperatures at which 50% of the initially formed hybrid was denatured thermally to single-chain species. Results:

The data indicate that both probes hybridize to the target rRNA as expected, with the Tm for the "inserted" probe being approximately three degrees higher than the Tm for the "replacement" probe.

Example 5: The ability of "LI" modified deoxyoligonucleotides to be labeled with biotin and fluorescein was demonstrated.

(a) 5'-(LI)- and 5'-(LI)-(LI)-modified deoxyoligonucleotides described above in Example 4(a)

Materials: Alpha-32p adenosine triphosphate was obtained from New England Nuclear (DuPont, Boston, MA, USA). Thermal deoxynucleotidyl transferase (TdT) and 5X tailing buffer were products of Bethesda Research Laboratories (Gaithersburg,

MD, USA).

20 pmol 51 -(LI)-(LI)-modified oleonucleotides were reacted with 16.5 pmol alpha-P-32 adenosine triphosphate (specific

activity 3000 Ci/mmol) and 40 units of TdT in 20 µl IX "tailing" buffer at 37°C for one hour. The obtained P-32 labeled oleonucleotides were purified on a Nensorb-20 (TM) column (New England Nuclear, DuPont, Corp., Boston MA, USA) according to the vendor's procedure, which is incorporated herein by reference.

(b) The P-32 labeled 5'-(LI)-(LI)-oleonucleotides were reacted with biotin-E-aminocaproic acid-N-hydroxysuccinimide ("Bio-X-NHS", Calbiochem-Behring Corp., San Diego, CA USA). Streptavidin-agarose was obtained from Bethesda Research Laboratories (Gaithersburg, MD, USA), and D(+) biotin was from Calbiochem-Behring Corp. (San Diego, CA, USA) . 1 pmol of each modified oleonucleotide described above was reacted with 2.5 mM Bio-X-NHS in 125 mM borate buffer (pH 9) containing 12.5% dimethylsulfoxide for 1.5 hours. Small samples of the resulting reaction mixtures were then tested for binding to streptavidin agarose in 50 mM sodium phosphate (pH 7.4)/2 mM EDTA/0.5 NaCl, either in the presence ("non-specifically bound") or absence of (" specifically bound") 0.2 mg/ml D(+) biotin. Bound material was quantified by scintillation counting:

Binding of biotin to these L1-modified oligomers was also confirmed by electrophoresing samples of the above reaction mixtures on a 20% polyacrylamide/7 M urea gel. Representative bands were visualized by autoradiography, which indicated almost quantitative conversion to biotinylated forms, which migrated more slowly than the non-biotinylated controls.

(c) 3'-Ll-modified deoxyoligonucleotides described in Example 4(c,d)

("%'-AAATAACGAACCCTTGCAGGTCCTTTCAACTTTGAT-L1-3'" and

"5'-CAGTCAAACTCTAGCCATTACCTGCTAAAGTCATTT-L1-3'") were labeled with fluorescein isothiocyanate and biotin-X-NHS, respectively. The oleonucleotides were first kinased with [gamma- 32 P] adenosine triphosphate using T4 polynucleotide kinase according to the procedure of Maxam and Gilbert (Proe. Nati. Acad. Sei. USA, 1977, vol. 74, page 560), which is incorporated herein as a reference.

The first modified oleonucleotide was reacted with fluorescein isothiocyanate (FITC, Sigma Chemical Co., St. Louis, MO, USA). 40 pmol of this oligomer was treated with 90 mM FITC in 0.1 M borate buffer (pH 9) containing 90% DMSO for 12 hours. The reaction mixture was then electrophoresed on a 20% polyacrylamide/7 M urea gel. Bands were visualized by autoradiography. The top band from each field was excised and FITC-labeled oligomers were prepared from the gel and purified.

A binding assay to an anti-FITC antibody derivatized solid support was used to confirm the attachment of fluorescein to the oleonucleotide described above. Anti-FITC magnetic microspheres were obtained from Advanced Magnetic, Inc.

32 (Cambridge, MA, USA), Cat. #4310. Samples of the purified P labeled FITC modified oleonucleotide were mixed with 0.5 ml of buffer solution (50 mM sodium phosphate, pH 7.4/2 mM EDTA/0.5 M NaCl) containing 20 µl of anti-FITC microbeads in the presence of ("no -specific binding") or absence of ("specific binding") 20 mM hydrolyzed FITC. After 1 hour, the beads were removed by magnetic separation and the supernatants were counted by Cerenkov radiation to determine the amount of bound material:

The second modified oligonucleotide was reacted with biotin-X-NHS. 40 pmol of this oligomer was treated with 10 mM bio-X-NHA in 0.1 M borate buffer (pH 9) containing 20% DMSO for 1 hour. The biotinylated oligomer obtained was purified by polyacrylamide gel electrophoresis as described above. The presence of biotin attached to this oligonucleotide was confirmed by analysis of binding on streptavidin-agarose as described earlier in this example:

Example 6: Resistance of a 3'-L1-modified deoxyoligonucleotide to hydrolysis catalyzed by a phosphordiesterase.

Materials: Phosphodiesterase from Crotalus durissus was obtained from Boehringer-Mannehim Biochemicals (Indianapolis IN, USA). This enzyme catalyzed exonucleolytic cleavage from the 3<1> end of an oligonucleotide. A synthetic oxyoligonucleotide with the sequence

"5'-AAATAACGAACCCTTGCAGGTCCTTTCAACTTTGAT-3'" was synthesized on an Applied Biosystems Model 380A DNA Synthesizer using standard phosphoramidite chemistry. A probe with

the same sequence, but with a 3'-L1 linker attached, was synthesized according to the procedure given in Example 4.

3 2

Both oligonucleotides were kinased with P according to the method mentioned in Example 5. Approximately 350,000 CPM of each labeled oligonucleotide was reacted in 10 µl buffer (0.1 M tris-HCl, pH 8.0/20.mM MgCl2) containing either 3 x 10~<5>or 3 X 10 units of phosphodiesterase. 1.5 µl samples were removed at time intervals of 5, 10, 15 and 30 minutes; the reaction was stopped by the addition of 3 µl of 0.1 N NaOH. Then 5 µl of 90% formamide containing bromophenyl blue and xylanol XCFF dyes were added to each sample, and electrophoresis was performed on the obtained samples on a 20% polyacrylamide/7M urea gel. The gel was then analyzed by autoradiography.

The 3'-LI modified oleonucleotide was found to be more than 95% resistant to phosphodiesterase-catalyzed hydrolysis after 30 minutes, with both enzyme concentrations tested. There was essentially no full-length unmodified oligonucleotide detectable on the gel, indicating that the enzyme normally cuts the oligomer without a 3'-LI group.

Example 7: Automated incorporation of 2,2-di-(3-aminopropyl)-1,3-dihydroxypropane-based linker reagent into a synthetic oleonucleotide.

The incorporation of the linker reagent described in Example 2, hereafter designated "L2" was inserted between the bases of a synthetic oligonucleotide, forming the sequence

"5'-CGTTACTCGGATGCCCAAAT(L2)ATCGCCACATTOG-3'". The method used was similar to that described in Example 1(a), except that a solution of "L2" (0.1 M in acetonitrile) was reacted in the 13th coupling cycle instead of in a final coupling cycle which described in Example 4(a)). The yield of coupling with "L2" was about 30%, as estimated from the amount of dimethoxytrityl liberated (see Ex. 4(a)).

Example 8: Automated incorporation of 3-amino-1,2-propanediol-based linker reagent into a synthetic oleonucleotide.

The incorporation of this linker, hereafter designated "L3", into a synthetic oligonucleotide with the sequence

"5'CCCGCACGTCCCTATT(L3)AATCATTACGATGG-3'" was carried out in accordance with the procedure given in Example (a). In this example, a solution of "L3" (0.3 M in dry acetonitrile) was reacted in the fifteenth coupling cycle; whereas the coupling yield in this step, estimated from dimethoxytrityl release (see Ex. 4(a)), was approximately 60%.

Example 8(a): Automated incorporation of linker reagents 20, 21, 22, 23 and 24 into synthetic oleonucleotides.

The incorporation of linker reagents 20-24, whose synthesis is described above in Examples 3(a)-3(d), was carried out as described above in Example 4, part (a). The corresponding linkers associated with these reagents are hereafter denoted respectively "L4",

"L5", "L6", "L7" and "L8". In a special case corresponding to the use of one of the above-mentioned reagents, a 0.12-0.2 M solution of the reagent in dry acetonitrile was thus inserted into

position #6 in an Applied Biosystems Model 380A DNA Synthesizer (Foster City, CA, USA). The incorporation of the reagent into an oligonucleotide polymer was achieved using a standard

phosphoramidite coupling method. A series of oligonucleotides were prepared, ranging in length from 17 to 35 bases, each of the linkers L4-L8 being inserted at different places in the sequences. The coupling yields associated with these reagents, as measured by trityl release at the end of the coupling cycle, were between 75 and 98%.

Example 9: Labeling of amine linker-arm probe with acridinium ester and subsequent purification.

A 25 mM stock solution of acridinium ester (for composition refer to I. Weeks et al., Clin. Chem. vol. 29, page 1474, 1983) was prepared in distilled DMSO. The desired amount of a polymer prepared in Examples 4, 7 or 8(a) was evaporated to dryness in a 1.5 ml conical polypropylene tube. The following liquid mixture was prepared by adding the following ingredients in the order given below:

3 µL H2O

1 yL 1 m HEPES (8.0)

4 µl DMSO (distilled)

2 µl 25 mM acridinium ester in DMSO (distilled)

The mixture was vortexed, centrifuged in a microcentrifuge for 2 seconds (to bring the contents to the bottom of the tube) and incubated at 37°C for 20 minutes. At this point, the following ingredients were added to the reaction liquid mixture in the order given below:

3.0 µl 25 mM acridinium ester in DMSO (distilled)

1.5 µl H 2 O

0.5 μl 1 M (HEPES (8.0)

The liquid mixture was vortexed again, centrifuged and incubated for a further 20 minutes at 37°C. Unreacted label was suppressed using a 5-fold excess of lysine, by adding 5 µl of 0.125 M lysine in 0.1 M HEPES (8.0), 50% DMSO and incubating for 5 min at room temperature.

At this point, the acridinium ester-labeled oligomer was purified using the following method. To 20 µl reaction mixture where the reaction had been interrupted, 30 µl 3 M NaOAc (5.0),

245 µl H 2 O and 5 µl glycogen added as a carrier (glycogen was pretreated to remove any nuclease activity). The sample was vortexed gently and 640 µl of absolute EtOH was added. The sample was vortexed gently and incubated on ice for 5 to 10 min, then centrifuged 5 min at

15,000 rpm in a microcentrifuge. The supernatant was carefully removed and the pellet was redissolved in 20 µl of 0.1 M NaOSc (5.0), 0.1% SDS. The sample was further purified by ion-exchange high-pressure liquid chromatography (HPLC) as follows: 20 µl of redissolved pellet was injected into a Nucleogen-DEAE 60-7 ion exchanger

HPLC column mounted in an IBM 9533 HPLC system. All the buffers used in this process were prepared with HPLC graded water, acetonitrile (CH3CN) and sodium acetate (NaOAc), and graded glacial acetic acid (HOAc) and LiCl. In addition, other buffers were filtered through nylon-66 filters with a pore size of 0.45 um before use. In the specific case of a nucleotide/non-nucleotide multimer with a total of 26 monomer units, of which only one was a non-nucleotide monomer unit, the following elution procedure was used. Buffer A was 2 mM NaOAc, pH 5.5, 20% CH 3 CN; buffer B was 20 mM NaOAC (pH 5.5), 20% CH 3 CN and 1 M LiCl. Elution was achieved with a linear gradient from 55% buffer A, 45% buffer B to 30% buffer A, 70% buffer B for 25 minutes at a flow rate of 1 ml/min. Absorbance at 260 nm was measured during the run, 0.5 ml fractions were collected in 1.5 ml conical polypropylene tubes. Immediately after the run, 5 µl of 10% SDS was added to each tube followed by Vortex mixing of each tube (this was done to ensure that the acrdinium ester labeled probe did not adhere to the tube wall). 0.5 µl sample was removed from fractions 21-42 and added to 200 µl water in a 12 x 75 mm tube (a separate pipette tip was used for each sample to avoid the carryover problem). Chemiluminescence for each sample was then determined in a Berthoid Clinilumat by automatic injection of 200 µl 0.25 N HNO3, 0.1% H2O2, followed after a 1 second dwell by 200 µl INNaOH and chemiluminescence was read for 10 seconds.

Fractions 29-33 were EtOH precipitated by adding 5 µl of glycogen to each, vortexing, adding 1 ml ETOH to each, vortexing, incubating 5-10 minutes on ice and centrifuging for 5 minutes at 15,000 rpm in a microcentrifuge. Each supernatant was carefully removed and each pellet was redissolved in 20 µl of 0.1 M NaOAc, pH 5, 0.1% SDS and these separate fractions were then pooled.

With the help of the above-mentioned examples, it has thus been shown that the reagents according to the present invention can be prepared, each having a non-nucleotide main chain, first and second linking groups and a ligand, all as previously described under "summary of the invention".

Particular reagent (5) has a non-nucleotide propyl main chain, to which is attached a first linking group of methyl-N,N-diisopropylphosphoramide,

a second linking group of 1-hydroxy protected with dimethoxytrityl (DMT), and a ligand in the form of the aminopropyl linker arm protected with trifluoroacetyl. Reagent (10) is the same as reagent (5) except that the former is provided with two identical ligands (in the form of two protected trifluoroacetylaminopropyl linker arms). Reagent (13) is also similar to reagent (5) with the exception that the non-nucleotide main chain is ethyl or a propyl, and the linker arm is shorter, being only trifluoroacetyl-protected methylamine rather than a similarly protected aminopropyl.

As shown above, a single reagent according to the invention can be used to provide a linker arm specifically at any preselected position(s) on a nucleotide multimer, without introducing unwanted nucleotides. As also described, by linking one backbone to a nucleotide and another backbone, (which in turn is then linked to another backbone, etc. to produce a chain), the reagents of the invention now allow the possibility that a series of adjacent ligands (eg labels or linker arms to which labels can be attached) are provided in a nucleotide multimer. Thus, multiple nearby markers can be bound to the probe, thereby increasing the sensitivity of a hybridization experiment using such a probe.

Furthermore, the use of one or a number of sequence-bound non-nucleotide main chains according to the invention can further serve to bridge two nucleotide sequences on the probe that are complementary to the corresponding sequences on a target nucleotide multimer, which can be linked by a simple different nucleotide or a different sequence in a test sample. Thus, the target nucleotide multimer can actually be a group of two or more nucleotide multimers consisting of common nucleotide sequences of interest, which are linked together by a single different nucleotide or different nucleotide sequences that are not of interest. Even when a single target sequence is of interest, it is possible to prepare the probe with a complementary sequence and with the non-nucleotide monomer unit carrying the marker group linked between any two nucleotides. In such a case, the probe hybridizes to the target nucleotide sequence in the normal manner except that the monomer unit carrying the marker group will conform in such a way that it does not interfere with the previous hybridization (ie, it will "bulge out" of the hybrid structure ). Such an arrangement can be particularly advantageous in situations where it is desirable to take advantage of insertion effects (as described by U. Asseline et al, Proe. Nati. Acad, Sei. USA, vol. 81, pages 3297-3301), for example, to increase probe specificity. The fact that non-nucleotide monomer units are used in the present invention of course considerably reduces interference of the type described above, compared to previous probes that use nucleotide monomer units.

As discussed in the "summary of the invention" above, compounds (4), (9) and (12) can be attached to a solid phase synthesis support through their primary hydroxyl groups (see Gait and Sarang for techniques adapted to such a purpose). The resulting derivatized supports, when used for further polymer synthesis, result in the attachment of a non-nucleotide monomer unit to the 3'-terminal end of the resulting nucleotide/non-nucleotide polymer.

It will be understood that a number of variations of the above described invention are possible. The ligand can, for example, actually be a marker or an insert which is provided on the reagents according to the invention before they are linked to a nucleotide of a nucleotide multimer. Furthermore, as described in "summary" above, various other protecting groups may be used than those specified in the examples above. However, the use of either the trifluoroacetyl or 9-fluorenylmethoxycarbonyl amino protecting group is particularly preferred, as it is cleaved to deprotect the amine under the same alkaline conditions used in known standard oligonucleotide syntheses, to deprotect exocyclic nucleotide amines (typically concentrated NH4OH at 50°C for 1 to 12 hours). Likewise, the use of dimethoxytrityl 5'-hydroxy protection, methyl or beta-cyanoethylphosphite O-protection, and N,N-diisopropyl as a phosphite leaving group during the coupling allows all reagents to be fully compatible with current standard oligonucleotide solid phase synthesis techniques , so that the necessity for other additional special steps is minimized.

Other variations and changes to the embodiments according to the invention defined above will be possible for the expert in the field. Accordingly, the present invention is not limited to the embodiments described in detail above.

Claims (50)

1. Non-nucleotide reagent suitable for the preparation of a nucleotide/non-nucleotide polymer, characterized in that it includes: (a) a non-nucleotide monomer unit with a ligand selected from a side arm with an associated chemical residue, and from a binding arm which can be activated under non-adverse conditions to be capable of binding with a chemical residue; (b) first and second non-nucleotide linking groups bound to the monomer unit, the first linking group being able to link the non-nucleotide monomer unit to a first further monomer unit, while the second linking group remains inactive so that it is substantially not capable of coupling, but which can then be activated under non-adverse conditions to link the non-nucleotide monomer unit to a second additional monomer unit, at least one of the first and second additional monomer units being a nucleotide monomer unit. 2. Reagent as stated in claim 1, characterized in that said ligand is selected from a marker, an intercalator insertion, a metal chelator, drugs, hormones, proteins, peptides, haptens, radical generators, nucleolytic agents, proteolytic agents, - catalysts, specific binding substances, agents that modify DNA transport and substances that alters nucleotide multimer solubility, and from a binding functional group which may be attached to any of the above chemical residues. 3. Non-nucleotide reagents suitable for the preparation of a nucleotide/non-nucleotide polymer, characterized in that it includes: a) a non-nucleotide monomer unit with a ligand selected from a side arm with an associated chemical residue and from a protected binding arm that can be deprotected under non-adverse conditions to become capable of binding with a chemical residue; b) a non-nucleotide first linker group and protected second linker group bound to the monomer unit, the first linker group being capable of linking the non-nucleotide monomer unit to a first further monomer unit while the second is substantially incapable of link, but which can then be deprotected under non-adverse conditions so that it is able to link the non-nucleotide monomer unit to a second additional monomer unit, at least one of the first and second monomer units being a nucleotide monomer unit. 4. Reagent as stated in claim 3, characterized in that said ligand is selected from a marker, an intercalator insertion, a metal chelator, drugs, hormones, proteins, peptides, haptens, radical generators, nucleolytic agents, proteolytic agents, catalysts, specific binding substances, agents that modify DNA transport and substances that change nucleotide multimer solubility, and from a binding functional group that can be attached to any of the above chemical residues. 5. Reagent as specified in claim 4, characterized in that the first and second linking groups are able to link the non-nucleotide monomer unit to a 5 <1-> hydroxyl group and a 3'-phosphate group in a monomer unit, respectively. 6. Reagent as specified in claim 5, characterized in that the non-nucleotide monomer unit has an acyclic main chain to whose respective ends the first and second linking groups are attached. 7. Reagent as stated in claim 5, characterized in that the non-nucleotide monomer unit has a main chain to whose respective ends the first and second linking groups are attached, the main chain having from 1 to 20 atoms. 8. Reagent as specified in claim 5, characterized in that the non-nucleotide monomer unit has a main chain to whose respective ends the coupling groups are attached, the main chain being an acyclic hydrocarbon chain with from 1 to 10 carbon atoms. 9. Reagent as specified in claim 5, characterized in that the non-nucleotide monomer unit has a main chain to whose respective ends the linking groups are attached and in which the linking arm is a chain of from 1 to 25 atoms extending from the main chain. 10. Reagent as stated in claim 2, characterized in that it is selected from one of the compounds with formula: wherein (a) R 2 = a main chain of the non-nucleotide monomer unit, are first coupling groups in which: X2 = halogen or substituted amine X4 = halogen, amino or0 ~ R 3 = alkyl, alkoxy or phenoxy R5 = alkyl, alkoxy or aryloxy or is H only if X4 = 0 (c) R^ - Xi - is the protected second linking group wherein: X1 = 0, S, NH or HN=N- R] _ = the protecting group which can be cleaved under linking group deprotection conditions to obtain the second linking group H-X] _-, (d) n is an integer, (e) X3 -R4 - is the ligand. 11. Reagent as stated in claim 10, characterized in that X2 = Cl or secondary amine, R2 = chlorophenoxy, methoxy, ethoxy or beta- cyanoethoxy, X4 = Cl, secondary amine or 0 <_> when R5 = H R5 = methoxy, ethoxy, monochlorophenoxy or betacyanethoxy, or is H only if X4 = 0~ 12. Reagent as stated in claim 11, characterized in that the ligand is a protected binding arm in which X3 is an alkylamine bound to R2 at C- and to R4 at N-, and in which R4 is selected from trifluoroacetyl and 9-fluoromethylmethoxycarbonyl. 13. Reagent as stated in claim 11, characterized in that R2 has a secondary carbon bound to -0-. 14. Reagent as stated in claim 10, characterized in that the secondary amino groups are selected from dialkylamine and heterocyclic N-amines. 15. Reagent as specified in claim 10, 11 or 14, characterized in that R4 is triphenylmethyl or an alkoxy derivative thereof. 16. Reagent as stated in claim 10, 11 or 14, characterized by a t = 0 and = triphenylmethyl or an alkoxy derivative thereof. 17. Reagent as specified in claim 10, 11 or 14, characterized in that X1 =O and R^ = dimethoxytriphenylmethyl. 18. Method for producing a substituted nucleotide from a reagent with: (a) a non-nucleotide monomer unit, and (b) a ligand bound to the non-nucleotide monomer unit and selected from a side arm with an attached chemical residue and from a binding arm to which a chemical residue can be attached under non-favorable conditions, characterized in that the non-nucleotide monomer unit is linked to a nucleotide -monomer unit and to one of a second monomer unit and a solid support under non-adverse conditions. 19. Method for producing a substituted nucleotide from a reagent with: (i) a non-nucleotide monomer unit with a ligand selected from a side arm with an attached chemical residue and from a protected binding arm to which a chemical residue can be attached, and (ii) a first linking group and a protected second linking group attached to the non-nucleotide monomer unit, characterized by , under non-adverse conditions, (a) first linking the non-nucleotide monomer unit to a first additional monomer unit through the first linking group; (b) then deprotection of the second linking group; (c) then linking the non-nucleotide monomer unit through the second linking group to a second additional monomer unit; wherein at least one of the first and second monomer units is a nucleotide monomer unit while the other is selected from a monomer unit and a solid support. 20. Method as stated in claim 18 or 19, characterized in that both the first and second further monomer units are nucleotide monomer units. 21. Method as stated in claim 18, characterized in that the first and second linking group can link the non-nucleotide monomer unit to a 5 <1-> hydroxyl group and a 3'-phosphate group in a nucleotide monomer unit, respectively. 22. Method as stated in claim 19, characterized in that the non-nucleotide monomer unit has an acyclic main chain to whose respective ends the first and second linking groups are attached. 23. Method as stated in claim 19, characterized in that the reagent is selected from one of the compounds with formula: wherein: (a) R 2 = a main chain of the non-nucleotide monomer unit, are first coupling groups in which: X2 = halogen or substituted amine X2 = halogen, amino or 0~ R3 = alkyl, alkoxy or phenoxy r <5> = alkyl, alkoxy or phenoxy or may be H only where X4 = 0~ (c) Ri~ Xi~ is the protected second linking group in which: X± = 0, S, NH or HN=N - R] _ = the protecting group which can be cleaved under linking group deprotection conditions to obtain the second linking group H-X^ -, (d) n is an integer (e) X3 -R4 is the ligand. 24. Method as stated in claim 23, characterized in that said ligand is selected from a marker, an intercalator insertion, a metal chelator, and from a binding functional group which can be bound to a marker, an intercalator insertion and a metal chelator. 25. Method as stated in claim 24, characterized in that X2 = Cl or secondary amine R3 = chlorophenoxy, methoxy, ethoxy or beta-cyanoethoxy X4 = Cl, secondary amine or 0~ when R5 = H R5 = methoxy, ethoxy, monochlorophenoxy, beta-cyanoethoxy or can be H only if X4 = 0". 26. Method as stated in claim 24, characterized in that the secondary amino groups in the reagent are selected from dialkylamine and heterocyclic N-amines. 27. Method as stated in claim 23 or 26, characterized by Ri in the reagent being triphenylmethyl or an alkoxy derivative thereof. 28. Method as stated in claim 23 or 26, characterized in that the reagent R] = dimethoxytriphenylmethyl. 29. Method as stated in claim 23 or 26, characterized in that the reagent has = 0 and Ri = dimethoxytriphenylmethyl. 30. Method as stated in claims 23 to 26, characterized in that it further comprises linking further monomer units to the linked nucleotide/non-nucleotide polymer, for the production of a polymer with a nucleotide sequence that is complementary to at least part of a target nucleotide sequence , but whose complementary nucleotide sequence has at least one non-nucleotide monomer unit placed therein, whereupon the nucleotide/non-nucleotide polymer sequence is hybridized with the target sequence. 31. Composition containing a single sequence nucleotide/non-nucleotide polymer as the majority of all nucleotide/non-nucleotide polymers present, the single nucleotide/non-nucleotide polymer comprising: (a) a non-nucleotide monomer unit having bound thereto a ligand selected from a side arm with an attached chemical residue and from a binding arm capable of binding to a chemical residue under non-adverse conditions; (b) a first nucleotide monomer unit linked to the non-nucleotide monomer unit, and (c) an additional monomer unit linked to the non-nucleotide monomer unit. 32. Composition as stated in claim 31, characterized in that said ligand is selected from a marker, an intercalator insert, a metal chelator and from a binding functional group which can be bound to the marker, the intercalator insert and a metal chelator. 33. Composition as stated in claim 31 or 32, characterized in that the further unit is a nucleotide monomer unit. 34. Composition as stated in claim 31 or 32, characterized in that the additional monomer unit is a non-nucleotide monomer unit which is linked to another non-nucleotide monomer unit. 35. Composition containing a nucleotide non-nucleotide polymer characterized in that it includes: (a) a non-nucleotide monomer unit having a ligand attached thereto selected from a side arm with an attached chemical residue attached to the main chain through a saturated binding group, and from a saturated binding arm which can be attached to a chemical residue under non-adverse conditions , (b) a first nucleotide monomer unit linked to the non-nucleotide monomer unit, and (c) an additional monomer unit linked to the non-nucleotide monomer unit. 36. Composition as stated in claim 35, characterized in that said ligand is selected from a marker, an intercalator insertion, a metal chelator and from a saturated binding arm which can be bound to either a marker or an intercalator. 37. Composition as stated in claim 36, characterized in that the ligand group is the saturated binding arm. 38. Composition as set forth in claim 34, characterized in that the non-nucleotide monomer unit has a backbone to whose ends the linking groups are bound and wherein the ligand comprises a marker bound to the backbone through the binding group selected from an alkylamine and alkylthio. 39. Composition as stated in claim 37, characterized in that the binding arm is selected from an alkylamine and an alkylthio. 40. Composition stated in claim 35, 37 or 39, characterized in that the non-nucleotide monomer unit has a main chain to whose ends the coupling groups are attached, the main chain being an acyclic hydrocarbon chain with from 1 to 20 carbon atoms. 41. Composition as stated in claim 35, characterized in that the marker is a chemiluminescent acridinium ester. 42. Composition as stated in claim 36, characterized in that the intercalator insertion is an acridinium master. 43. Composition as stated in claim 35, characterized in that the marker is selected from biotin, fluorescein, dinitrobenzene, rhodamine and Texas Red. 44. Composition as stated in claim 35, characterized in that the intercalator addition is selected from psoralen, acridine, acridine salts, acriflavins and ethidium. 45. Polymer probe, characterized in that it comprises a plurality of nucleotide monomer units and at least one acridinium ester residue bound to a corresponding monomer unit in the probe. 46. Probe as specified in claim 45, characterized by the acridinium ester marker is a kemiluminescent-acridinium ester marker. 47. Method for producing a probe, characterized in that at least one acridinium ester residue is bound to a corresponding monomer unit of a polymer with a plurality of nucleotide monomer units. 48. Method as stated in claim 47, characterized in that the acridinium ester marker is a chemiluminescent acridinium ester marker. 49. Reagent as stated in claim 10, 11, 24, 25, 31 or 35, characterized in that the ligand is a protected binding arm in which R4 is the binding arm and X3 is the protecting group. 50. Reagent as stated in claim 10, 11, 24, 25, 31 or 35, characterized in that the ligand is a protected binding arm in which X3 is an alkylamino binding arm bound to R 2 at a C- and to R4 at N-, and R4 is the protecting group.