STREPTAVIDIN-COATED SOLID PHASES WITH A MEMBER OF A BINDING PAIR

Abstract

The present disclosure relates to a solid phase coated with (strept)avidin and having attached thereto, by way of biotin:(strept)avidin interaction, a biotinylated first member of a binding pair, wherein the attached first member is capable of binding to a second member of the binding pair, but is not capable of binding to biotin or to (strept)avidin, and wherein no member of the binding pair is capable of hybridizing with a naturally-occurring single-stranded nucleic acid. The solid phase is particularly useful in immunoassays with samples having high content in biotin or (strept)avidin-binding derivatives thereof. The present disclosure further provides uses, kits and methods, particularly for determination of an analyte in a sample.

Claims

1. A solid phase coated with (strept)avidin and having attached thereto, by way of <biotin:(strept)avidin> interaction, a biotinylated first member of a binding pair, wherein the attached first member is capable of binding to a second member of the binding pair, but is not capable of binding to biotin or to (strept)avidin, wherein the second member is capable of becoming bound by the first member when the second member is part of a conjugate comprising any of an analyte, an analyte analogon, and an analyte-specific capturing agent, and wherein no member of the binding pair is capable of hybridizing with a naturally-occurring single-stranded nucleic acid.

2. The solid phase of claim 1, obtainable by a method of claim 10.

3. The solid phase of claim 1, wherein the binding pair is selected from the group consisting of a first and a second oligonucleotide spiegelmer, each consisting of L-ribose- or L-2′-deoxyribose-containing nucleoside monomers, the first oligonucleotide spiegelmer being capable of hybridizing with the second oligonucleotide spiegelmer; a first and a second oligomer consisting of beta-L-LNA nucleoside monomers, the first oligomer being capable of hybridizing with the second oligomer; an antigen and an antigen-specific antibody; a hapten and a hapten-specific antibody; a ligand and a specific ligand-binding domain; an oligo- or polysaccharide and a lectin, the lectin being capable of specifically binding to the oligo- or polysaccharide; a histidine-tag and a metal-chelate complex comprising a metal ion selected from Zn.sup.2+, Ni.sup.2+, Co.sup.2+, and Cu.sup.2+, the metal-chelate complex being capable of binding to the histidine-tag; an indium chelate complex and the CHA255 antibody; a cucurbit[n]uril host residue and a guest residue capable of binding the host residue; and a first and a second protein dimerization domain, optionally in the presence of a dimerization inducing or enhancing agent.

4. The solid phase of claim 1, wherein the solid phase is selected from the group consisting of a microparticle, a microwell plate, a test tube, a cuvette, a membrane, a quartz crystal, a film, a filter paper, a disc and a chip.

5. The solid phase of claim 4, wherein the solid phase is a microparticle with a diameter from 0.05 μm to 200 μm.

6. The solid phase of claim 5, wherein the microparticle is a monodisperse paramagnetic bead.

7. The solid phase of claim 6, wherein the diameter of the bead is about 3 μm.

8. The solid phase of claim 1, wherein the solid phase is in contact with an aqueous liquid phase.

9. The solid phase of claim 8, wherein in the liquid phase contains a conjugate, the conjugate comprising a second member of the binding pair.

10. A method of preparing a solid phase having attached thereto a member of a binding pair, the method comprising the steps of (a) providing a solid phase coated with (strept)avidin; (b) selecting a binding pair with a first member and a second member; (c) providing the first member of the binding pair selected in step (b); (d) biotinylating the first member of step (c); (e) attaching the biotinylated first member obtained from step (d) to the solid phase of step (a) by contacting the biotinylated first member with the coated solid phase and incubating, thereby attaching the biotinylated first member to the solid phase by way of biotin-(strept)avidin interaction; wherein in step (b) the pair is selected such that without biotinylation the first and the second member of the binding pair are not capable of binding to streptavidin, in biotinylated form and non-covalently attached to the coated solid phase by way of a biotin:(strept)avidin bond the first member of the binding pair is capable of binding to the second member, in conjugated form and covalently attached to an analyte-specific capturing agent the second member of the binding pair is capable of binding to the biotinylated first member attached to the solid phase, and no member of the binding pair is capable of hybridizing with a naturally-occurring single-stranded nucleic acid; thereby obtaining the solid phase having attached thereto the member of the binding pair.

11. Use of a solid phase of claim 1 or of a solid phase obtained from the method of claim 10 in an assay to determine an analyte in a sample.

12. A kit for determining an analyte in a sample, the kit comprising (a) in a first container, and either (b) or (c) in a second container, wherein (a) is a solid phase having attached thereto a first member of a binding pair, wherein the solid phase is a solid phase of claim 1 or a solid phase obtained from the method of claim 10, (b) is a first conjugate, the conjugate comprising a second member of the binding pair coupled to an analyte-specific capturing agent, (c) is a second conjugate, the conjugate comprising a second member of the binding pair coupled to the analyte or an analogon of the analyte.

13. The kit of claim 12, the kit comprising (a) and (b), the kit further comprising a labeled analyte-specific detecting agent, wherein the detecting agent and (b) are in different containers, and wherein the analyte-specific capturing agent of (b) and the labeled analyte-specific detecting agent are capable of forming a sandwich complex with the analyte.

14. The kit of claim 12, the kit comprising (a) and (c), the kit further comprising a labeled analyte-specific detecting agent, wherein the detecting agent and (c) are in different containers, and wherein the analyte or analyte analogon comprised in the conjugate and the analyte in the sample are capable of being bound by the detecting agent.

15. A complex comprising (a) and either (b) or (c), wherein (a) is a solid phase having attached thereto a first member of a binding pair, wherein the solid phase is a solid phase of claim 1 or a solid phase obtained from the method of claim 10, (b) is a first conjugate, the conjugate comprising a second member of the binding pair coupled to an analyte-specific capturing agent, (c) is a second conjugate, the conjugate comprising a second member of the binding pair coupled to the analyte or an analogon of the analyte, wherein in the complex (a) is bound to (b) or (c), respectively, and wherein in the complex a first member of the binding pair is bound to a second member of the binding pair.

16. A complex of claim 15, obtainable by a method of claim 17.

17. A method to form a complex, the method comprising the step of contacting (a) with either (b) or (c), wherein (a) is a solid phase having attached thereto a first member of a binding pair, wherein the solid phase is a solid phase of claim 1 or a solid phase obtained from the method of claim 10, (b) is a first conjugate, the conjugate comprising a second member of the binding pair coupled to an analyte-specific capturing agent, (c) is a second conjugate, the conjugate comprising a second member of the binding pair coupled to the analyte or an analogon of the analyte, followed by the step of incubating (a) and either (b) or (c), respectively, thereby forming the complex, wherein in the complex (a) is bound to (b) or (c), respectively, and wherein a first member of the binding pair is bound to a second member of the binding pair.

18. A method to determine an analyte in a sample, the method comprising the steps of (a) providing the sample with the analyte; (b) providing a solid phase having attached thereto a first member of a binding pair, wherein the solid phase is a solid phase of claim 1 or a solid phase obtained from the method of claim 10; (c) providing a conjugate, the conjugate comprising a second member of the binding pair coupled to an analyte-specific capturing agent; (d) contacting, mixing and incubating the sample of (a) with conjugate of (c), thereby forming a complex, the complex comprising the analyte being captured by the analyte-specific capturing agent comprised in the conjugate; (e) immobilizing complex formed in step (d) by contacting and incubating complex with the solid phase of step (b), wherein the first member of the binding pair binds to the second member; (f) optionally washing immobilized complex obtained from step (e); (g) determining analyte comprised in immobilized complex; thereby determining the analyte in the sample.

19. The method of claim 18, wherein steps (d) and (e) are performed subsequently or simultaneously.

20. The method of claim 18, wherein step (c) additionally comprises providing a labeled analyte-specific detecting agent, the analyte is capable of being bound simultaneously by the capturing agent comprised in the conjugate and by the detecting agent, thereby being capable of forming a sandwich complex; step (d) comprises contacting, mixing and incubating the sample of (a) with the conjugate of (c) and additionally labeled analyte-specific detecting agent, thereby forming a complex, the complex comprising analyte being sandwiched between the capturing agent and the detecting agent, and step (g) is performed by determining label comprised in immobilized complex.

21. The method of claim 20, wherein prior to step (g) unbound labeled analyte-specific detecting agent is removed from immobilized complex.

22. A method to determine an analyte in a sample, the method comprising the steps of (a) providing the sample with the analyte; (b) providing a solid phase having attached thereto a first member of a binding pair, wherein the solid phase is a solid phase of claim 1 or a solid phase obtained from the method of claim 10; (c) providing a conjugate, the conjugate comprising a second member of the binding pair coupled to the analyte or an analogon of the analyte; (d) providing a labeled analyte-specific detecting agent, wherein the analyte or analyte analogon comprised in the conjugate of step (c) and the analyte in the sample are capable of being bound by the detecting agent; (e) contacting, mixing and incubating the sample of step (a) with conjugate of step (c) and detecting agent of step (d), thereby forming a first complex comprising the analyte and the detecting agent and a second complex comprising the conjugate and the detecting agent; (f) immobilizing second complex formed in step (e) by contacting and incubating complex with the solid phase of step (b), wherein the first member of the binding pair binds to the second member; (g) optionally washing the immobilized complex obtained from step; (h) determining label comprised in the immobilized complex obtained from step (f) or step (g); thereby determining the analyte in the sample.

23. The method of claim 22, wherein steps (e) and (f) are performed subsequently or simultaneously.

24. The method of claim 22, wherein prior to step (h) unbound labeled analyte-specific detecting agent is removed from immobilized complex.

25. The method of claim 22, wherein a predetermined amount of each of (c) and/or (d) is provided.

Description

DESCRIPTION OF THE FIGURES

[0291] FIG. 1 Synthesis scheme illustrating Example 1.

[0292] FIG. 2 Synthesis scheme illustrating Example 2.

[0293] FIG. 3 Synthesis scheme illustrating Example 3.

[0294] FIG. 4 Synthesis scheme illustrating Example 4.

[0295] FIG. 5 Synthesis scheme illustrating Example 5.

[0296] FIG. 6 A and B are illustrations of the results of Example 10.

[0297] FIG. 7 A and B are illustrations of the results of Example 12.

[0298] FIG. 8 Diagram illustrating the results of Example 13.

EXAMPLE 1

Synthesis of sugar intermediate 1,2-O-Diacetyl-3-O-benzyl-5-O-mesyl-4-C-(mesyloxymethyl)-L-ribose

[0299] This example illustrates the multi-step synthesis via several intermediate products as indicated in the following. FIG. 1 illustrates the synthesis scheme.

[0300] (i)

1,2:5,6-Di-O-isopropylidene-α-L-glucofuranose

[0301] 1,2:5,6-Di-O-isopropylidene-α-L-glucofuranose was synthesized according to Qu et al., Research on Chemical Intermediates 2014, 40 (4), 1557-1564.

[0302] To a stirred suspension of anhydrous L-glucose (50 g, 0.28 mol; available from Carbosynth) in anhydrous acetone (500 mL) pulverized anhydrous zinc chloride (40 g) was added followed by 1.5 mL of 85% phosphoric acid. This mixture was stirred for 30 h at room temperature, and the unreacted sugar was collected by filtration and washed with a small volume of acetone. Filtrate and washings were cooled and made slightly alkaline with 2.5 M sodium hydroxide. Insoluble inorganic material was removed by filtration and washed with acetone. The almost colorless filtrate and washings were concentrated under reduced pressure and the residue was diluted with water, extracted with dichloromethane, dried over magnesium sulfate, and concentrated under reduced pressure. Crystallization of the residue from hexane gave 1,2:5,6-di-O-isopropylidene-α-L-glucofuranose as colorless needles (51 g, 70%).

[0303] R.sub.f (ethyl acetate/hexane 1:1)=0.5.

[0304] .sup.1H NMR (CDCl.sub.3): δ 5.94 (d, 1H), 4.53 (d, 1H), 4.39-4.29 (m, 2H), 4.16 (dd, 1H), 4.06 (dd, 1H), 3.98 (dd, 1H), 2.65 (d, 1H), 1.49 (s, 3H), 1.44 (s, 3H), 1.36 (s, 3H), 1.31 (s, 3H).

[0305] (ii)

1,2:5,6-Di-O-isopropylidene-α-L-ribo-hexofuranose-3-ulose

1,2:5,6-Di-O-isopropylidene-α-L-allofuranose

[0306] 1,2:5,6-Di-O-isopropylidene-α-L-allofuranose was synthesized according to Hassan et al., Bioorganic Chemistry 2016, 65, 9-16.

[0307] To a 2 L, 3-necked flask equipped with a mechanical stirrer and a condenser connected at the top to a mineral oil bubbler, a solution of 1,2:5,6-di-O-isopropylidene-α-L-glucofuranose (100 g, 0.38 mol) in ethanol free chloroform (500 mL), potassium carbonate (16.2 g, 0.12 mol), potassium periodate (148.5 g, 0.65 mmol), benzyltriethylammonium chloride (0.9 g, 3.83 mmol) and activated ruthenium(IV) oxide hydrate (1.75 g) were added. The mixture was stirred for 1 h at 0° C., thereafter at room temperature overnight. The mixture was filtered over a Celite pad and the organic phase was separated and washed with water. The aqueous phase was washed with chloroform and the combined organic phases were dried over magnesium sulfate, evaporated and dried under reduced pressure to give 1,2:5,6-di-0-isopropylidene-α-L-ribo-hexofuranose-3-ulose (R.sub.f (ethyl acetate/hexane 3:1)=0.85). The residue was used in the next reaction step without further purification.

[0308] 1,2:5,6-Di-O-isopropylidene-α-L-ribo-hexofuranose-3-ulose was dissolved in ethanol/water 7:3 (600 mL) and treated with sodium borohydrate (8.73 g) portion wise at 0° C. The mixture turned colorless and was stirred for 3 h at 0° C. and thereafter for 1 h at room temperature. The solvent was concentrated to about 400 mL and another 400 mL of water was added to the mixture. Thereafter the mixture was concentrated to a volume of ca. 400 mL and extracted with dichloromethane. The organic phase was dried over magnesium sulfate and evaporated under reduced pressure to give 1,2:5,6-di-O-isopropylidine-α-L-allofuranose (60 g, 61%) as a white solid.

[0309] .sup.1H NMR (DMSO-d6): δ 5.66 (d, 1H), 5.05 (d, 1H), 4.45 (t, 1H), 4.23 (dt, 1H), 3.93 (dd, 1H), 3.83 (m, 2H), 3.74 (dd, 1H), 1.45 (s, 3H), 1.32 (s, 3H), 1.28 (s, 3H), 1.27 (s, 3H).

[0310] (iii)

3-O-Benzyl-1,2:5,6-di-O-isopropylidene-α-L-allofuranose

3-O-Benzyl-1,2-O-isopropylidene-α-L-allofuranose

[0311] 3-O-Benzyl-1,2-O-isopropylidene-α-L-allofuranose was synthesized according to Hassan et al., Bioorganic Chemistry 2016, 65, 9-16.

[0312] To 1,2:5,6-di-O-isopropylidine-α-L-allofuranose (60 g, 0.235 mol) in DMF (150 mL) benzyl bromide (29.2 mL, 0.245 mol) was added dropwise at 0° C. The reaction mixture was stirred overnight at room temperature. Water (100 mL) was added to the mixture and the product was allowed to crystalize overnight in a refrigerator. The crystals were filtered off, washed with water, dried under reduced pressure to give 3-O-benzyl-1,2:5,6-di-O-isopropylidene-α-L-allofuranose. The residue was dissolved in 70% acetic acid (390 mL) and stirred for 7 h at 36° C. The mixture was evaporated under reduced pressure to yield 3-O-benzyl-1,2-O-isopropylidene-α-L-allofuranose (62.8 g, 86%) as a clear viscous oil.

[0313] R.sub.f (ethyl acetate/hexane 1:1)=ca. 0.9.

[0314] .sup.1H NMR (CDCl3) δ 7.39-7.30 (m, 5H), 5.76 (d, 1H), 4.77 (d, 1H), 4.63-4.54 (m, 2H), 4.13-4.06 (dd, 1H), 4.01-3.91 (m, 2H), 3.68 (d, 2H), 2.88 (br s, 1H), 2.71 (br s, 1H), 1.54 (s, 3H), 1.33 (s, 3H).

[0315] (iv)

3-O-Benzyl-1,2-O-isopropylidene-α-L-ribo-pentodialdofuranose

3-O-Benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-L-ribose

[0316] A 2 L Erlenmeyer flask equipped with a magnetic stir bar was charged with silica gel (80.6 g, EM Science, catalog no. 9385-9) and dichloromethane (800 mL). An aqueous solution of sodium periodate (80 mL, 52 mmol, 0.65 M) was added dropwise over 5 min, and a white precipitate formed. A solution of 3-O-benzyl-1,2-O-isopropylidene-α-L-allofuranose (10.0 g, 32.3 mmol, 0.5 M) in dichloromethane (65 mL) was added in one portion to the Erlenmeyer flask. The mixture was stirred at room temperature for 1.5 h. Thereafter the reaction was diluted with water (275 mL), and the suspension was transferred to a 2 L separatory funnel. The aqueous and organic layers were separated, and the aqueous layer was extracted with dichloromethane. The combined organic layers were dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to yield a colorless oil that was dried under high vacuum for 12 h to afford 3-O-benzyl-1,2-O-isopropylidene-α-L-ribo-pentodialdofuranose (5.3 g, 59%).

[0317] Aqueous 37% formaldehyde (10.6 mL) and then 1 M sodium hydroxide (53 mL) were added at 0° C. to a stirred solution of crude 3-O-benzyl-1,2-O-isopropylidene-α-L-ribo-pentodialdofuranose (5.3 g, 19 mmol) in water (50 mL). The reaction mixture was then kept at room temperature for 4 days and concentrated under reduced pressure. The residue was extracted with dichloromethane, dried over magnesium sulfate and evaporated to dryness. The residue was purified by column chromatography (silica gel; toluene/diethylether) to give 3-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-L-ribose (4.3 g; 72%).

[0318] R.sub.f (ethyl acetate)=0.42.

[0319] .sup.1H NMR (CDCl.sub.3): δ 7.41-7.28 (m, 5H), 5.76 (d, 1H), 4.80 (d, 1H), 4.62 (dd, 1H), 4.52 (d, 1H), 4.21 (d, 1H), 3.90 (dd, 2H), 3.78 (dd, 1H), 3.55 (dd, 1H), 2.37 (t, 1H), 1.89 (dd, 1H), 1.63 (s, 3H), 1.33 (s, 3H).

[0320] (v)

3-O-Benzyl-1,2-O-isopropylidene-5-O-mesyl-4-C-(mesyloxymethyl)-α-L-ribose

[0321] A solution of 3-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-L-ribose (10 g, 32 mmol) in anhydrous pyridine (30 mL) was cooled in an ice bath, and mesyl chloride (7.5 mL, 96.5 mmol) was added. The mixture was stirred for 1 h at room temperature, diluted with diethyl ether (200 mL), and washed with water. The organic layer was dried over sodium sulfate, concentrated under reduced pressure, coevaporated with toluene (2×100 mL), and dried in vacuo to yield 3-O-benzyl-1,2-O-isopropylidene-5-O-mesyl-4-C-(mesyloxymethyl)-α-L-ribose as a white solid (15.0 g, 95%).

[0322] R.sub.f (hexane/ethyl acetate 15:85)=ca. 0.5.

[0323] .sup.1H NMR (CDCl.sub.3): δ 7.42-7.29 (m, 5H), 5.79 (d, 1H), 4.89 (d, 1H), 4.78 (d, 1H), 4.65 (dd, 1H), 4.58 (d, 1H), 4.42 (d, 1H), 4.33 (d, 1H), 4.19 (d, 1H), 4.14 (d, 1H), 3.08 (s, 3H), 2.98 (s, 3H), 1.69 (s, 3H), 1.34 (s, 3H).

[0324] (vi)

1,2-O-Diacetyl-3-O-benzyl-5-O-mesyl-4-C-(mesyloxymethyl)-L-ribose

[0325] Acetic anhydride (22.6 mL, 240 mmol) and concentrated sulfuric acid (23 μL) were added to a solution of 3-O-benzyl-1,2-O-isopropylidene-5-O-mesyl-4-C-(mesyloxymethyl)-α-L-ribose (15.0 g, 30.2 mmol) in acetic acid (230 mL), and the mixture was stirred overnight at room temperature. More concentrated sulfuric acid (4 μL) was added, and the reaction was continued for 24 h. Thereafter, water (150 mL) was added, and the mixture was stirred for 3 h and washed twice with dichloromethane. The organic layers was washed with saturated sodium hydrogen carbonate (4×200 mL), dried over sodium sulfate, and concentrated under reduced pressure to give 1,2-O-diacetyl-3-O-benzyl-5-O-mesyl-4-C-(mesyloxymethyl)-L-ribose as a colorless oil (14.5 g, 97%; two anomers of ratio ca. 1:5).

[0326] .sup.1H NMR (CDCl.sub.3): δ 7.42-7.24 (m, 5H), 6.17 (s, 1H), 5.37 (d, 1H), 4.62 (d, 1H), 4.52 (d, 1H), 4.50 (d, 1H), 4.42 (d, 1H), 4.38 (d, 1H), 4.30 (d, 1H), 4.19 (d, 1H), 3.02 (s, 3H), 3.01 (s, 3H), 2.14 (s, 3H), 2.10 (s, 3H).

EXAMPLE 2

[0327] β-L-LNA-T Phosphoramidite Compounds

[0328] This example illustrates the multi-step synthesis via several intermediate products as indicated in the following. FIG. 2 illustrates the synthesis scheme. Synthesis was basically performed analogously to Koshkin et al., Journal of Organic Chemistry 2001, 66 (25), 8504-8512.

[0329] (i)

1-(2-O-Acetyl-3-O-benzyl-5-O-mesyl-4-C-(mesyloxymethyl)-β-L-ribofuranosyl)thymine

[0330] N,O-Bis(trimethylsilyl)acetamide (33.7 mL, 136 mmol) was added to a mixture of 1,2-O-diacetyl-3-O-benzyl-5-O-mesyl-4-C-(mesyloxymethyl)-L-ribose (25 g, 49 mmol) and thymine (7.7 g, 61 mmol) in anhydrous acetonitril (120 mL). The reaction mixture was refluxed for 1 h, thereafter trimethylsilyl triflate (11.5 mL, 64 mmol) was added, and refluxing was continued further for 5 h. The solution was kept at room temperature overnight, thereafter diluted with dichloromethane (100 mL), and washed with saturated sodium hydrogen carbonate solution. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. Then the residue was purified by silica gel column chromatography (ethyl acetate) to result 1-(2-O-acetyl-3-O-benzyl-5-O-mesyl-4-C-(mesyloxymethyl)-β-L-ribofuranosyl)thymine (24.0 g, 85%) as a white solid.

[0331] .sup.1H NMR (CDCl.sub.3): δ 9.33 (s, 1H), 7.40-7.28 (m, 5H), 7.08 (d, 1H), 5.71 (d, 1H), 5.58 (dd, 1H), 4.70 (d, 1H), 4.60 (d, 1H), 4.55 (d, 1H), 4.53 (d, 1H), 4.38 (d, 1H), 4.34 (d, 1H), 4.32 (d, 1H), 3.02 (s, 3H), 3.00 (s, 3H), 2.11 (s, 3H), 1.92 (d, 3H).

[0332] MS (ESI): 576.6 Da confirmed.

[0333] (ii)

3′-O-Benzyl-5′-O-mesyl-β-L-LNA-T

[0334] To a solution of 1-(2-O-acetyl-3-O-benzyl-5-O-mesyl-4-C-(mesyloxymethyl)-β-L-ribofuranosyl)thymine (22 g, 38.2 mmol) in 1,4-dioxane/water 1:1 (100 mL) was added 2 M sodium hydroxide (100 mL). The mixture was stirred for 1 h at room temperature, diluted with saturated sodium hydrogen carbonate solution (100 mL), and extracted with dichloromethane. The aqueous phase was acidified by 10% hydrochloric acid and thereafter extracted with dichloromethane. The organic layer was dried over sodium sulfate, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (1-3% methanol in dichloromethane) to yield 3′-O-benzyl-5′-O-mesyl-3-L-LNA-T (16.1 g, 96%) as a white solid.

[0335] .sup.1H NMR (CDCl.sub.3): δ 9.24 (s, 1H), 7.41-7.22 (m, 6H), 5.68 (s, 1H), 4.66 (d, 1H), 4.61 (s, 1H), 4.59 (d, 1H), 4.56 (d, 1H), 4.52 (d, 1H), 4.08 (d, 1H), 3.93 (s, 1H), 3.87 (d, 1H), 3.08 (s, 3H), 1.93 (s, 3H).

[0336] MS (ESI): 438.5 Da confirmed.

[0337] (iii)

5′-O-Benzoyl-3′-O-benzyl-β-L-LNA-T

[0338] Sodium benzoate (9.9 g, 68.7 mmol) was added to a solution of 3′-O-benzyl-5′-O-mesyl-β-L-LNA-T (15.0 g, 34.2 mmol) in anhydrous N,N-dimethylformamide (400 mL). The mixture was stirred for 5 h at 100° C., cooled to room temperature and filtrated. N,N-dimethylformamide was evaporated under reduced pressure, and the residue was suspended in ethyl acetate (150 mL), washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated to dryness. Crystallization from ethanol yielded 5′-O-benzoyl-3′-O-benzyl-β-L-LNA-T (14.9 g, 94%) as a white solid.

[0339] R.sub.f(ethyl acetate)=0.78.

[0340] .sup.1H NMR (CDCl.sub.3): δ 8.78 (s, 1H), 7.94 (m, 2H), 7.61 (m, 1H), 7.45 (m, 2H), 7.30-7.20 (m, 6H), 5.63 (s, 1H), 4.83 (d, 1H), 4.73 (d, 1H), 4.66 (s, 1H), 4.56 (d, 1H), 4.52 (d, 1H), 4.18 (d, 1H), 3.97 (d, 1H), 3.91 (s, 1H), 1.58 (s, 3H).

[0341] MS (ESI): 464.5 Da confirmed.

[0342] (iv)

3′-O-Benzyl-β-L-LNA-T

[0343] Water (25 mL) and 2 M sodium hydroxide (100 mL) were added to a solution of 5′-O-benzoyl-3′-O-benzyl-β-L-LNA-T (14 g, 31.8 mmol) in 1,4-dioxane (100 mL). The reaction mixture was refluxed for 24 h, cooled to room temperature, and neutralized with acetic acid (12.5 mL). Saturated sodium hydrogen carbonate solution (100 mL) was added, and the mixture was washed with dichloromethane. Organic layer was dried over sodium sulfate, and concentrated under reduced pressure. Purification by silica gel column chromatography (1-3% methanol in dichloromethane) yielded 3′-O-benzyl-β-L-LNA-T (10.3 g, 90%) as a white solid.

[0344] R.sub.f (ethyl acetate)=0.51.

[0345] .sup.1H NMR (CDCl.sub.3): δ 9.28 (s, 1H), 7.45 (d, 1H), 7.38-7.22 (m, 5H), 5.66 (s, 1H), 4.67 (d, 1H), 4.56 (d, 1H), 4.54 (s, 1H), 4.05 (d, 1H), 4.01 (d, 1H), 3.96 (s, 1H), 3.95 (d, 1H), 3.83 (d, 1H), 1.88 (d, 3H).

[0346] MS (ESI): 360.4 Da confirmed.

[0347] (v)

[0348] β-L-LNA-T Nucleoside

[0349] A mixture of 3′-O-benzyl-β-L-LNA-T (10 g, 27.5 mmol), 20% palladium hydroxide on carbon (5 g), and ammonium formiate (5.3 g, 84.6 mmol) was suspended in methanol (70 mL). After refluxing the mixture for 10 min, the catalyst was filtered off and washed with methanol. The combined filtrates were concentrated to a white solid. Crystallization from 15% methanol in dichloromethane afforded 8-L-LNA-T (6.5 g, 87%) as a white solid.

[0350] R.sub.f(ethyl acetate)=0.11.

[0351] .sup.1H NMR (DMSO-d6): δ 11.32 (br s, 1H), 7.60 (d, 1H), 5.68 (d, 1H), 5.38 (s, 1H), 5.20 (t, 1H), 4.09 (s, 1H), 3.89 (d, 1H), 3.80 (d, 1H), 3.74 (d, 2H), 3.61 (d, 1H), 1.75 (d, 3H).

[0352] MS (ESI): 270.2 Da confirmed.

[0353] (vi)

5′-O-(4,4′-Dimethoxytrityl)-β-L-LNA-T

[0354] β-L-LNA-T nucleoside (5 g, 18.5 mmol) was coevaporated with anhydrous pyridine (50 mL) and redissolved in anhydrous pyridine (150 mL). 4,4′-dimethoxytrityl chloride (7.5 g, 22.1 mmol) and 4-(dimethylamino)pyridine (225 mg, 1.8 mmol) were added. The solution was stirred overnight at room temperature. After addition of methanol the reaction mixture was concentrated under reduced pressure. Thereafter the residue was dissolved in ethyl acetate (150 mL) and extracted with saturated sodium hydrogen carbonate solution. The organic layer was washed with brine (150 mL), dried over sodium sulfate and concentrated under reduced pressure to dryness. Purification by silica gel column chromatography (starting from 40% hexane in ethyl acetate) afforded 5′-O-(4,4′-dimethoxytrityl)-β-L-LNA-T (9.3 g, 88%) as an off-white solid.

[0355] R.sub.f(ethyl acetate)=0.60.

[0356] .sup.1H NMR (CDCl.sub.3): δ 9.88 (s br, 1H), 7.64 (d, 1H), 7.47-7.14 (m, 9H), 6.85 (dd, 4H), 5.56 (s, 1H), 4.53 (s, 1H), 4.31 (m, 1H), 4.00-3.75 (m, 9H), 3.50 (m, 2H), 1.65 (d, 3H).

[0357] MS (ESI): 572.6 Da confirmed.

[0358] (vii)

5′-O-(4,4′-Dimethoxytrityl)-β-L-LNA-T, 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite

[0359] 5′-O-dimethoxytrityl-β-L-LNA-T (8 g, 14 mmol) was dissolved in anhydrous dichloromethane (125 mL). Thereafter N,N-diisopropylethylamine (6.1 mL, 35 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (5.29 g, 22.4 mmol) were added. The solution was stirred for 3 h at room temperature, and then washed with saturated sodium hydrogen carbonate solution. The organic layer was dried over sodium sulfate and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (40% hexane in ethyl acetate) to yield 5′-O-(4,4′-dimethoxytrityl)-β-L-LNA-T, 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite (8.8 g, 81%) as a white solid.

[0360] .sup.31P NMR (CDCl.sub.3): δ 149.32, 149.18.

[0361] MS (ESI): 772.8 Da confirmed.

EXAMPLE 3

[0362] β-L-LNA-C Phosphoramidite Compounds

[0363] This example illustrates the multi-step synthesis via several intermediate products as indicated in the following. FIG. 3 illustrates the synthesis scheme.

[0364] (i)

5′-O-(4,4′-Dimethoxytrityl)-β-L-LNA-N4-benzoyl-5-methyl-C

[0365] 5′-O-(4,4′-Dimethoxytrityl)-β-L-LNA-T (13.5 g, 23.6 mmol) was dissolved in anhydrous acetonitrile. At 0° C. triethylamine (9.86 mL, 70.8 mmol) was added, followed by dropwise addition of trimethylsilyl chloride (7.48 mL, 59 mmol). The reaction was stirred for 1 h at 0° C. (reaction mixture A). In parallel, 1,2,4-triazole (24.4 g, 353.3 mmol) was dissolved in anhydrous acetonitrile (150 mL) and thereafter at 0° C. phosphoryl chloride (7.71 mL, 82.5 mmol) was added slowly. After stirring for 15 min at 0° C. triethylamine (59.15 mL, 424.4 mmol) was added. Stirring was continued at 0° C. for 50 min and thereafter reaction mixture A was added. The reaction mixture was stirred for 3 h at 0° C., and then extracted with saturated sodium hydrogen carbonate solution/dichloromethane. The organic phase was dried over sodium sulfate and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (20% hexane in ethyl acetate to 100% ethyl acetate) to yield the C4-1,2,4-triazolid nucleoside intermediate (15.2 g) as a white solid (LC-MS: 697.4 [M+H].sup.+). Thereafter, benzamide (15.67 g, 129.4 mmol) was suspended in dioxane (100 mL). To this suspension 60% sodium hydride (5.17 g, 129.4 mmol) was added. After 15 min stirring at room temperature the C4-1,2,4-triazolid nucleoside intermediate in dioxane (150 mL) was added. The reaction mixture was stirred for 2 h at room temperature, and thereafter extracted with 5% citric acid/ethyl acetate. The organic layer was washed with saturated sodium hydrogen carbonate solution and brine. Then the organic phase was dried over sodium sulfate and concentrated to dryness to yield 5′-O-(4,4′-dimethoxytrityl)-3′-O-trimethylsilyl-β-L-LNA-N6-benzoyl-5-methyl-C nucleoside R.sub.f (20% hexane in ethyl acetate)=0.87). This intermediate was dissolved in tetrahydrofurane (250 mL), and 1 M tetrabutylammonium fluoride in tetrahydrofurane (23.7 mL, 23.7 mmol) was added. After 15 min stirring at room temperature the solvent was evaporated. The residue was dissolved in dichloromethane and washed with saturated sodium hydrogen carbonate solution. The organic layer was dried over sodium sulfate and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (40% hexane in ethyl acetate) to yield 5′-O-(4,4′-dimethoxytrityl)-β-L-LNA-N4-benzoyl-5-methyl-C (14 g, 88% from 5′-O-dimethoxytrityl-β-L-LNA-T) as an off-white solid.

[0366] R.sub.f (20% hexane in ethyl acetate)=0.64.

[0367] .sup.1H NMR (CDCl.sub.3): δ 8.30 (d, 2H), 7.80 (s, 1H), 7.56-7.23 (m, 12H), 6.86 (dd, 4H), 5.66 (s, 1H), 4.46 (s, 1H), 4.29 (s, 1H), 3.90-3.79 (m, 8H), 3.60-3-47 (dd, 2H), 2.03 (s, 3H).

[0368] MS (ESI): 675.7 Da confirmed.

[0369] (ii)

5′-O-(4,4′-Dimethoxytrityl)-β-L-LNA-N4-benzoyl-5-methyl-C, 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite

[0370] 5′-O-(4,4′-dimethoxytrityl)-β-L-LNA-N4-benzoyl-5-methyl-C (4.6 g, 6.8 mmol) was dissolved in anhydrous dichloromethane (70 mL). Thereafter N,N-diisopropylethylamine (2.37 mL, 13.6 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (3.22 g, 13.6 mmol) were added. The solution was stirred for 1 h at room temperature, and then washed with 5-10% sodium hydrogen carbonate solution. The organic layer was dried over sodium sulfate and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (40% hexane in ethyl acetate) to yield 5′-O-(4,4′-dimethoxytrityl)-β-L-LNA-N4-benzoyl-5-methyl-C, 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite (5.0 g, 84%) as a white solid.

[0371] R.sub.f(ethyl acetate/hexane 3:2)=0.84.

[0372] .sup.31P NMR (CDCl.sub.3): δ 149.46, 149.42.

[0373] MS (ESI): 875.9 Da confirmed.

EXAMPLE 4

[0374] β-L-LNA-A Phosphoramidite Compounds

[0375] This example illustrates the multi-step synthesis via several intermediate products as indicated in the following. FIG. 4 illustrates the synthesis scheme. Synthesis was basically performed analogously to Koshkin et al., Journal of Organic Chemistry 2001, 66 (25), 8504-8512.

[0376] (i)

9-(2-O-Acetyl-3-O-benzyl-5-O-mesyl-4-C-(mesyloxymethyl)-β-L-ribofuranosyl)N6-benzoyladenine

[0377] To a suspension of 1,2-O-diacetyl-3-O-benzyl-5-O-mesyl-4-C-(mesyloxymethyl)-L-ribose (25 g, 49 mmol) and N6-benzoyladenine (14.06 g, 58.8 mmol) in anhydrous 1,2-dichloroethane (200 mL) was added N,O-bis(trimethylsilyl)acetamide (32 mL, 128.8 mmol). The mixture was refluxed for 1 h, thereafter trimethylsilyl triflate (17.7 mL, 98 mmol) was added at room temperature, and refluxing was continued for 48 h. Thereafter the reaction mixture was poured into ice-cold saturated sodium hydrogen carbonate solution (200 mL), stirred for 0.5 h, and filtrated. The phases were separated, and the organic phase was washed with saturated sodium hydrogen carbonate solution, dried over sodium sulfate, and concentrated to dryness under reduced pressure. Purification by silica gel column chromatography (1-2% methanol in dichloromethane gave 9-(2-O-acetyl-3-O-benzyl-5-O-mesyl-4-C-(mesyloxymethyl)-13-L-ribofuranosyl)N6-benzoyladenine (27.4 g, 81%) as an off-white solid.

[0378] .sup.1H NMR (CDCl.sub.3): δ 8.76 (s, 1H), 8.12 (s, 1H), 8.02 (m, 2H), 7.61 (m, 1H), 7.51 (m, 2H), 7.40-7.34 (m, 5H), 6.23 (d, 1H), 6.08 (dd, 1H), 5.12 (d, 1H), 4.68 (d, 1H), 4.67 (d, 1H), 4.64 (d, 1H), 4.44 (d, 1H), 4.39 (d, 1H), 4.36 (d, 1H), 3.03 (s, 3H), 2.87 (s, 3H), 2.13 (s, 3H).

[0379] MS (ESI): 689.7 Da confirmed.

[0380] (ii)

3′-O-Benzyl-5′-O-mesyl-β-L-LNA-N6-benzoyl-A

[0381] 9-(2-O-Acetyl-3-O-benzyl-5-O-mesyl-4-C-(mesyloxymethyl)-13-L-ribofuranosyl)N6-benzoyladenine (25 g, 36.3 mmol) was dissolved in a mixture of tetrahydrofurane (220 mL) and water (150 mL). Lithium hydroxide monohydrate (7.7 g, 183 mmol) was added, and the reaction mixture was stirred for 3.5 h at room temperature. The solution was neutralized with acetic acid (8.4 mL) to result a precipitate which was filtered off and washed with water to afford 3′-O-benzyl-5′-O-mesyl-β-L-LNA-N6-benzoyl-A (19.6 g, 98%) as a white solid.

[0382] .sup.1H NMR (CDCl.sub.3): δ 8.72 (s, 1H), 8.15 (s, 1H), 8.02 (m, 2H), 7.63-7.59 (m, 1H), 7.54-7.50 (m, 2H), 7.32-7.27 (m, 5H), 6.10 (s, 1H), 4.95 (s, 1H), 4.67 (d, 1H), 4.62 (d, 1H), 4.60 (d, 1H), 4.57 (d, 1H), 4.33 (s, 1H), 4.21 (d, 1H), 4.01 (d, 1H), 3.04 (s, 3H).

[0383] MS (ESI): 551.6 Da confirmed.

[0384] (iii)

N6,5′-O-Di-benzoyl-3′-O-benzyl-β-L-LNA-A

[0385] 3′-O-Benzyl-5′-O-mesyl-13-L-LNA-N6-benzoyl-A (18 g, 31.6 mmol) was dissolved in anhydrous N,N-dimethylformamide (700 mL). Thereafter sodium benzoate (8.45 g, 58.5 mmol) was added, and the mixture was stirred at 90° C. for 7 h, cooled to room temperature, filtrated, concentrated under reduced pressure, and coevaporated with acetonitrile. The residue was dissolved in dichloromethane (200 mL), washed with saturated sodium hydrogen carbonate solution and brine, dried over sodium sulfate, and concentrated to dryness under reduced pressure. Crystallization from water/ethanol 1:1 afforded N6,5′-O-di-benzoyl-3′-O-benzyl-13-L-LNA-A (15.5 g, 85%) as a white solid.

[0386] .sup.1H NMR (DMSO-d6): δ 11.2 (br s, 1H), 8.72 (s, 1H), 8.48 (s, 1H), 8.06 (m, 2H), 7.94 (m, 2H), 7.66 (m, 2H), 7.54 (m, 4H), 7.36-7.26 (m, 5H), 6.11 (s, 1H), 4.97 (s, 1H), 4.82 (s, 2H), 4.77 (s, 1H), 4.75 (d, 1H), 4.69 (d, 1H), 4.19 (d, 1H), 4.07 (d, 1H).

[0387] MS (ESI): 577.6 Da confirmed.

[0388] (iv)

3′-O-Benzyl-β-L-LNA-A

[0389] N6,5′-O-Di-benzoyl-3′-O-benzyl-β-L-LNA-A (15 g, 25.9 mmol) was suspended in a mixture of methanol (150 mL) and concentrated ammonia (200 mL). The solution was stirred for 2 days at room temperature. Thereafter methylamine (40%, 19.4 mL) was added, and the mixture was stirred overnight. The precipitate was filtered off, dried in vacuo, and crystallized from ethanol to afford 3′-O-benzyl-3-L-LNA-A (8.1 g, 85%) as a white solid.

[0390] .sup.1H NMR (DMSO-d6): δ 8.18 (s, 1H), 8.14 (s, 1H), 7.33-7.30 (m, 5H), 5.97 (s, 1H), 5.17 (t, 1H), 4.73 (s, 1H), 4.63 (s, 2H), 4.35 (s, 1H), 3.95 (d, 1H), 3.84-3.81 (m, 3H).

[0391] MS (ESI): 369.4 Da confirmed.

[0392] (v)

β-L-LNA-A Nucleoside

[0393] To a suspension of 3′-O-benzyl-3-L-LNA-A (7.4 g, 20.0 mmol) in ethanol (100 mL) were added 20% palladium hydroxide on carbon (2 g) and ammonium formiate (6.4 g, 100.8 mmol). The reaction mixture was refluxed for 3 h, and more ammonium formiate (2 g, 31.8 mmol) was added. After 2 h, the hot solution was filtrated through a Celite pad which was washed with boiling ethanol/water (200 mL). The combined filtrates were concentrated under reduced pressure to afford β-L-LNA-A nucleoside (5.5 g, 98%) as white crystals.

[0394] .sup.1H NMR (DMSO-d6): δ 8.22 (s, 1H), 8.15 (s, 1H), 7.30 (br s, 2H), 5.89 (s, 1H), 5.68 (d, 1H), 5.05 (t, 1H), 4.41 (s, 1H), 4.25 (d, 1H), 3.92 (d, 1H), 3.82 (m, 2H), 3.76 (d, 2H).

[0395] MS (ESI): 279.3 Da confirmed.

[0396] (vi)

β-L-LNA-N6-benzoyl-A

[0397] β-L-LNA-A nucleoside (4.8 g, 17.2 mmol) was coevaporated in anhydrous pyridine (50 mL), thereafter suspended in anhydrous pyridine (100 mL). Then trimethylsilyl chloride (11.6 mL, 91 mmol) was added. The reaction mixture was stirred for 1 h at room temperature. Thereafter benzoyl chloride (2.6 mL, 22.4 mmol) was added. The reaction mixture was stirred for 4 h at room temperature. Thereafter the reaction mixture was cooled to 0° C. Then water (20 mL) and concentrated ammonia (25 mL) were added. The ice bath was removed, and the reaction mixture was stirred for 1 h at room temperature, then concentrated under reduced pressure and extracted with dichloromethane/water. The organic layer was dried over sodium sulfate and concentrated to dryness under reduced pressure to give β-L-LNA-N6-benzoyl-A (6.2 g, 95%) as white solid.

[0398] .sup.1H NMR (methanol-d4): δ 8.73 (s, 1H), 8.57 (s, 1H), 8.11 (m, 2H), 7.69 (m, 1H), 7.59 (m, 2H), 6.16 (s, 1H), 4.67 (s, 1H), 4.42 (s, 1H), 4.12 (d, 1H), 4.01 (s, 2H), 3.95 (d, 1H).

[0399] MS (ESI): 383.4 Da confirmed.

[0400] (vii)

5′-O-(4,4′-Dimethoxytrityl)-β-L-LNA-N6-benzoyl-A

[0401] β-L-LNA-N6-benzoyl-A nucleoside (5 g, 13.0 mmol) was coevaporated with anhydrous pyridine (50 mL) and redissolved in anhydrous pyridine (150 mL). 4,4′-dimethoxytrityl chloride (5.3 g, 15.5 mmol) and 4-(dimethylamino)pyridine (0.16 g, 1.3 mmol) were added. The solution was stirred overnight at room temperature. After addition of methanol the reaction mixture was concentrated under reduced pressure. Thereafter the residue was dissolved in ethyl acetate (150 mL) and extracted with saturated sodium hydrogen carbonate. The organic layer was washed with brine, dried over sodium sulfate and concentrated under reduced pressure to dryness. Purification by silica gel column chromatography (starting from 20% hexane in ethyl acetate to ethyl acetate) afforded 5′-O-(4,4′-dimethoxytrityl)-β-L-LNA-N6-benzoyl-A (6.7 g, 75%) as an off-white solid.

[0402] .sup.1H NMR (DMSO-d6): δ 11.21 (s, 1H), 8.75 (s, 1H), 8.47 (s, 1H), 8.02 (d, 2H), 7.65-7.18 (m, 12H), 6.87 (dd, 4H), 6.12 (s, 1H), 5.75 (d, 1H), 4.57 (s, 1H), 4.42 (d, 1H), 3.98 (dd, 1H), 3.93 (dd, 1H), 3.70 (s, 6H), 3.54 (dd, 1H), 3.31 (dd, 1H).

[0403] MS (ESI): 685.7 Da confirmed

[0404] (viii)

5′-O-(4,4′-Dimethoxytrityl)-β-L-LNA-N6-benzoyl-A,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite

[0405] 5′-O-(4,4′-Dimethoxytrityl)-β-L-LNA-N6-benzoyl-A (6.5 g, 9.4 mmol) was dissolved in anhydrous dichloromethane (100 mL). Thereafter N,N-diisopropylethylamine (4.1 mL, 23.7 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (3.57 g, 15.1 mmol) were added. The solution was stirred for 3 h at room temperature, and then washed with saturated sodium hydrogen carbonate solution. The organic layer was dried over sodium sulfate and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (20% hexane in ethyl acetate) to yield 5′-O-(4,4′-dimethoxytrityl)-β-L-LNA-N6-benzoyl-A,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite (7.0 g, 84%).

[0406] .sup.31P NMR (CDCl.sub.3): δ 149.58, 149.43.

[0407] MS (ESI): 885.9 Da confirmed.

EXAMPLE 5

β-L-LNA-G Phosphoramidite Compounds

[0408] This example illustrates the multi-step synthesis via several intermediate products as indicated in the following. FIG. 5 illustrates the synthesis scheme. Synthesis was basically performed analogously to Koshkin et al., Journal of Organic Chemistry 2001, 66 (25), 8504-8512

[0409] (i)

9-(2-O-Acetyl-3-O-benzyl-5-O-mesyl-4-C-(mesyloxymethyl)-β-L-ribofuranosyl)N2-isobutyrylguanine

[0410] To a suspension of 1,2-O-diacetyl-3-O-benzyl-5-O-mesyl-4-C-(mesyloxymethyl)-L-ribose (25 g, 49 mmol) and N2-isobutyrylguanine (12.36 g, 55.9 mmol) in anhydrous 1,2-dichloroethane (200 mL) was added N,O-bis(trimethylsilyl)acetamide (40.5 mL, 164.9 mmol). The mixture was refluxed for 1 h, thereafter trimethylsilyl triflate (18.2 mL, 100.4 mmol) was added at room temperature, and refluxing was continued for 3.5 h. Thereafter the reaction was stirred overnight at room temperature. Then the reaction mixture was diluted with dichloromethane (200 mL), washed with saturated sodium hydrogen carbonate solution, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (1-2% methanol in dichloromethane) to afford 9-(2-O-acetyl-3-O-benzyl-5-O-mesyl-4-C-(mesyloxymethyl)-β-L-ribofuranosyl)N2-isobutyrylguanine (27.5 g, 83%) as a white solid.

[0411] .sup.1H NMR (CDCl.sub.3): δ 12.20 (br s, 1H), 9.34 (br s, 1H), 7.76 (s, 1H), 7.40-7.30 (m, 5H), 6.03 (d, 1H), 5.76 (dd, 1H), 5.08 (d, 1H), 4.91 (d, 1H), 4.67 (d, 1H), 4.61 (d, 2H), 4.49 (d, 1H), 4.39 (d, 1H), 4.32 (d, 1H), 3.14 (s, 3H), 3.02 (s, 3H), 2.70 (m, 1H), 2.09 (s, 3H) 1.24 (m, 6H).

[0412] MS (ESI): 671.7 Da confirmed.

[0413] (ii)

3′-O-Benzyl-5′-O-mesyl-β-L-LNA-N2-isobutyryl-G

[0414] 9-(2-O-Acetyl-3-O-benzyl-5-O-mesyl-4-C-(mesyloxymethyl)-13-L-ribofuranosyl)N2-isobutyrylguanine (25 g, 37.2 mmol) was dissolved in tetrahydrofurane (250 mL). Then 1 M sodium hydroxide (250 mL, 250 mmol) was added, and the reaction mixture was stirred for 1 h at 0° C. Thereafter the solution was neutralized with acetic acid (15 mL) and concentrated under reduced pressure. The concentrated reaction mixture was diluted with water and extracted with dichloromethane. The organic layer was dried over sodium sulfate and concentrated to dryness under reduced pressure to yield 3′-O-benzyl-5′-O-mesyl-13-L-LNA-N2-isobutyryl-G (14.7 g, 74%) as a white solid which was used without purification for next reaction step.

[0415] R.sub.f(5% methanol in dichloromethane)=0.57

[0416] .sup.1H NMR (CDCl.sub.3): δ 12.14 (br s, 1H), 9.51 (br s, 1H), 7.77 (s, 1H), 7.30-7.26 (m, 5H), 5.84 (s, 1H), 4.67 (d, 1H), 4.63 (d, 1H), 4.62 (s, 1H), 4.62 (d, 1H), 4.56 (d, 1H), 4.50 (s, 1H), 4.12 (d, 1H), 3.93 (d, 1H), 3.06 (s, 3H), 2.78 (m, 1H), 1.26 (m, 6H).

[0417] MS (ESI): 533.6 Da confirmed.

[0418] (iii)

5′-O-Benzoyl-3′-O-benzyl-β-L-LNA-N2-isobutyryl-G

3′-O-Benzyl-β-L-LNA-N2-isobutyryl-G

[0419] 3′-O-Benzyl-5′-O-mesyl-β-L-LNA-N2-isobutyryl-G (12.5 g, 23.4 mmol) was dissolved in anhydrous N,N-dimethylformamide (250 mL). Thereafter sodium benzoate (6.8 g, 47 mmol) was added, and the mixture was stirred at 90° C. overnight, then cooled to room temperature, filtrated and concentrated under reduced pressure. The residue was dissolved in ethyl acetate (200 mL), washed with saturated sodium hydrogen carbonate solution and brine, dried over sodium sulfate, and concentrated to dryness under reduced pressure to afford 5′-O-benzoyl-3′-O-benzyl-13-L-LNA-N2-isobutyryl-G (MS (ESI): calc. 559.6, found 560.1). The product was used for the next reaction step without further purification. 5′-O-Benzoyl-3′-O-benzyl-13-L-LNA-N2-isobutyryl-G was dissolved in ethanol/pyridine 8:1 (350 mL). To this solution 2 M sodium hydroxide (13.5 mL) was added, and the mixture was stirred for 30 min at room temperature. Thereafter acetic acid (21.5 mL) was added, and the reaction mixture was concentrated under reduced pressure. The residue was crystallized from water/ethanol 1:1 to afford 3′-O-benzyl-β-L-LNA-N2-isobutyryl-G (8.2 g, 77%) as a white solid.

[0420] R.sub.f (10% methanol in ethyl acetate)=0.75

[0421] .sup.1H NMR (DMSO-d.sub.6): δ 8.05 (s, 1H), 7.33-7.26 (m, 5H), 5.85 (s, 1H), 5.17 (t, 1H), 4.69 (s, 1H), 4.64 (s, 2H), 4.23 (s, 1H), 3.95 (d, 1H), 3.83 (m, 2H), 3.80 (d, 1H), 2.78 (m, 1H), 1.12 (m, 6H).

[0422] MS (ESI): 455.5 Da confirmed.

[0423] (vi)

β-L-LNA-N2-isobutyryl-G

[0424] 3′-O-Benzyl-β-L-LNA-N2-isobutyryl-G (8.2 g, 18.0 mmol) was dissolved in methanol (75 mL). Thereafter 20% palladium hydroxide on carbon (3 g) and formic acid (4.2 mL, 111.3 mmol) were added. The reaction mixture was refluxed for 5 h, cooled to room temperature and filtrated through a Celite pad. The filtrate was concentrated under reduced pressure to give β-L-LNA-N2-isobutyryl-G (6.2 g, 94%) as white solid.

[0425] R.sub.f(10% methanol in ethyl acetate)=ca. 0.3.

[0426] .sup.1H NMR (DMSO-d.sub.6): δ 7.80 (s, 1H), 5.51 (s, 1H), 5.44 (br s, 1H), 4.77 (br s, 1H), 4.12 (s, 1H), 3.88 (s, 1H), 3.65 (d, 1H), 3.53 (m, 2H), 3.47 (d, 1H), 2.50 (m, 1H), 0.84 (m, 6H).

[0427] MS (ESI): 365.3 Da confirmed.

[0428] (v)

5′-O-(4,4′-Dimethoxytrityl)-β-L-LNA-N2-isobutyryl-G

[0429] β-L-LNA-N2-isobutyryl-G nucleoside (5 g, 13.7 mmol) was coevaporated with anhydrous pyridine (50 mL) and redissolved in anhydrous pyridine (150 mL). 4,4′-dimethoxytrityl chloride (6.0 g, 17.8 mmol) and 4-(dimethylamino)pyridine (0.17 g, 1.4 mmol) were added. The solution was stirred overnight at room temperature. After addition of methanol the reaction mixture was concentrated under reduced pressure. Thereafter the residue was dissolved in ethyl acetate (150 mL) and washed with saturated sodium hydrogen carbonate solution and brine, dried over sodium sulfate and concentrated under reduced pressure to dryness. Purification by silica gel column chromatography (starting from 20% hexane in ethyl acetate to ethyl acetate) afforded 5′-O-(4,4′-dimethoxytrityl)-β-L-LNA-N2-isobutyryl-G (8.0 g, 87%) as an off-white solid.

[0430] R.sub.f(ethyl acetate)=0.39.

[0431] .sup.1H NMR (DMSO-d.sub.6): δ 12.09 (s, 1H), 11.78 (s, 1H), 8.00 (s, 1H), 7.39 (d, 2H), 7.30-7.24 (m, 7H), 6.88 (dd, 4H), 5.86 (s, 1H), 5.73 (d, 1H), 4.42 (s, 1H), 4.26 (d, 1H), 3.92 (dd, 1H), 3.88 (dd, 1H), 3.72 (s, 6H), 3.53 (d, 1H), 3.30 (d, 1H), 2.76 (m, 1H), 1.10 (d, 6H).

[0432] MS (ESI): 667.7 Da confirmed.

[0433] (vi)

5′-O-(4,4′-Dimethoxytrityl)-β-L-LNA-N2-isobutyryl-G,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite

[0434] 5′-O-(4,4′-dimethoxytrityl)-β-L-LNA-N2-isobutyryl-G (7.5 g, 11.2 mmol) was dissolved in anhydrous dichloromethane (100 mL). Thereafter N,N-diisopropylethylamine (4.8 mL, 28.0 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (4.24 g, 17.9 mmol) were added. The solution was stirred for 3 h at room temperature, and then washed with saturated sodium hydrogen carbonate solution. The organic layer was dried over sodium sulfate and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (40% hexane in ethyl acetate) to yield 5′-O-(4,4′-dimethoxytrityl)-β-L-LNA-N2-isobutyryl-G,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite (6.8 g, 70%).

[0435] R.sub.f(ethyl acetate)=0.66, 0.75.

[0436] .sup.31P NMR (acetonitrile-d3): δ 148.51, 148.12.

[0437] MS (ESI): 867.9 Da confirmed.

EXAMPLE 6

Synthesis of 5′-biotinylated 13-L-LNA oligonucleotides

[0438] 5′-Biotinylated 13-L-LNA oligonucleotides were synthesized in a 2×1 μmole scale synthesis on an ABI 394 DNA synthesizer using standard automated solid phase DNA synthesis procedure and applying phosphoramidite chemistry. Glen UnySupport PS (Glen Research cat no. 26-5040) and β-L-LNA phosphoramidites (from examples 2-5) as well as spacer phosphoramidite 18 (Glen Research cat. no. 10-1918) and 5′-biotin phosphoramidate (Glen Research cat. no. 10-5950) were used as building blocks. All phosphoramidites were applied at a concentration of 0.1 M in DNA grade acetonitrile. Standard DNA cycles with extended coupling time (180 sec), extended oxidation (45 sec) and detritylation time (85 sec) as well as standard synthesis reagents and solvents were used. 5′-Biotinylated oligonucleotides were synthesized DMToff. A standard cleavage program was applied for the cleavage of the LNA oligonucleotides from the support by concentrated ammonia, residual protecting groups were also cleaved by treatment with concentrated ammonia (8 h at 56° C.). Crude 5′-biotinylated β-L-LNA oligonucleotides were evaporated and purified by RP HPLC (column: PRP-1, 7 μm, 250×21.5 mm (Hamilton, part no. 79352)) using a 0.1 M triethylammonium acetate pH 7/acetonitrile gradient. Product fractions were combined and desalted by dialysis (MWCO 1000, SpectraPor 6, part no. 132638) against water. Finally, the LNA oligonucleotides were quantified and lyophilized.

[0439] Yields ranged from about 800 to 900 nmoles.

[0440] 5′-Biotinylated β-L-LNA oligonucleotides were analyzed by RP18 HPLC (Chromolith RP18e, Merck, part no. 1.02129.0001) using a 0.1 M triethylammonium acetate pH 7/acetonitrile gradient. Typical purities were >90%. Identity of 5′-biotinylated β-L-LNA oligonucleotides were confirmed by LC-MS analysis.

EXAMPLE 7

Synthesis of 5′-maleimide-modified β-L-LNA oligonucleotides

[0441] 5′-Maleimide-modified β-L-LNA oligonucleotides were synthesized in a 20 μmole scale synthesis on an Äkta Oligopilot plus 10 DNA synthesizer (GE Healthcare) using standard automated solid phase DNA synthesis procedure and applying phosphoramidite chemistry. Glen UnySupport PS (Glen Research cat no. 26-5040) and β-L-LNA phosphoramidites (from Examples 2-5) as well as spacer phosphoramidite 18 (Glen Research cat. no. 10-1918) and 5′-amino-modifier C6 phosphoramidite (Glen Research cat. no. 10-1906) were used as building blocks. All phosphoramidites were applied at a concentration of 0.15 M in DNA grade acetonitrile. Standard DNA cycles with extended coupling time (240 sec) and extended oxidation (45 sec) as well as standard synthesis reagents and solvents were used for the assembly of 5′-amino-modified β-L-LNA oligonucleotides which were synthesized MMTon. A standard cleavage program was applied for the cleavage of the LNA oligonucleotides from the support by concentrated ammonia, residual protecting groups were also cleaved by treatment with concentrated ammonia (8 h at 56° C.). Crude 5′-modified β-L-LNA oligonucleotides were evaporated and purified by RP HPLC (column: PRP-1, 12-20 μm, 250×30 mm (Hamilton, part no. 79352)) using a 0.1 M triethylammonium acetate pH 7/acetonitrile gradient. Product fractions were combined and desalted by dialysis (MWCO 1000, SpectraPor 6, part no. 132638) against water, thereby also cleaving MMT group of MMTon purified oligonucleotides. Finally, the 5′-amino-modified LNA oligonucleotides were quantified and lyophilized (typical yield: ca. 3.5 μmol). To synthesize 5′-maleimide modified β-L-LNA oligonucleotides 5′-amino-modified β-L-LNA oligonucleotides were dissolved in 0.1 M sodium borate buffer pH 7.5 (2.5 mL). After addition of acetonitrile (0.5 mL) and 6-maleimidohexanoic acid N-hydroxysuccinimide ester (15 mg; Sigma, cat. no. M9794) the reaction mixture was shaken for 0.5 h at room temperature, stopped with 80% acetic acid and desalted applying an Amicon ultra centrifugal filter device (MWCO 3000, Merck, cat no. UFC9003). The retentate was quantified and lyophilized to yield 5′-maleimide-modified β-L-LNA oligonucleotides.

[0442] Typical yields: ca. 2 μmoles.

[0443] 5′-Maleimide-modified β-L-LNA oligonucleotides were analyzed by RP18 HPLC (Chromolith RP18e, Merck part no. 1.02129.0001) using a 0.1 M triethylammonium acetate pH 7/acetonitrile gradient. Typical purities were >90%. Identity of LNA oligonucleotides were confirmed by LC-MS analysis.

EXAMPLE 8

Synthesis of Unmodified β-L-LNA Oligonucleotides

[0444] Unmodified β-L-LNA oligonucleotides were synthesized in a 1 μmole scale synthesis on an ABI 394 DNA synthesizer using standard automated solid phase DNA synthesis procedure and applying phosphoramidite chemistry. Glen UnySupport PS (Glen Research cat no. 26-5040) and β-L-LNA phosphoramidites (from Examples 2-5) were used as building blocks. All phosphoramidites were applied at a concentration of 0.1 M in DNA grade acetonitrile. Standard DNA cycles with extended coupling time (180 sec), extended oxidation (45 sec) and detritylation time (85 sec) and standard synthesis reagents and solvents were used for the assembly of the LNA oligonucleotides which were synthesized as 5′-DMTon oligonucleotides. Then, a standard cleavage program was applied for the cleavage of the LNA oligonucleotides from the support by conc. ammonia. Residual protecting groups were cleaved by treatment with conc. ammonia (8 h at 56° C.). Crude LNA oligonucleotides were evaporated and purified by RP HPLC (column: PRP-1, 7 μm, 250×21.5 mm (Hamilton, part no. 79352)) using a 0.1 M triethylammonium acetate pH 7/acetonitrile gradient. In few cases, difficult to purify LNA oligonucleotides were additionally purified by anion exchange HPLC chromatography under denaturing conditions (column: Source 15Q, GE Healthcare). Product fractions were combined and desalted by dialysis (MWCO 1000, SpectraPor 6, part no. 132638) against water, thereby also cleaving DMT group of DMTon purified oligonucleotides. Finally, the LNA oligonucleotides were quantified and lyophilized.

[0445] Yields ranged from about 100 to 400 nmoles.

[0446] LNA oligonucleotides were analyzed by RP18 HPLC (Chromolith RP18e, Merck part no. 1.02129.0001) using a 0.1 M triethylammonium acetate pH 7/acetonitrile gradient. Typical purities were ≥90%. Identity of LNA oligonucleotides were confirmed by LC-MS analysis.

EXAMPLE 9

Coupling of L-LNA Oligonucleotides to TSH-Specific F(Ab′).SUB.2.-Fragments (Thyrotropin-Specific Capturing Agent)

[0447] F(ab′).sub.2-Fragments were conjugated with LNA in a two step reaction. Firstly, thiol-groups were introduced into F(ab′).sub.2-fragments via conjugation with SATP (N-succinimidyl-S-acetythiopropionate) and deacetylation by hydroxylamine (see Greg T. Hermanson Bioconjugate Techniques, 3rd edition 2013). L-LNA-oligonucleotides of Seq ID NO:9 were then conjugated to free thiols via maleimiide chemistry. L-LNA labeled F(ab′).sub.2 fragments were purified via Superdex 200 size-exclusion and mono Q anion exchange chromatography to obtain conjugated F(ab′).sub.2 fragments of high purity, with each conjugate comprising a single L-LNA oligomer.

EXAMPLE 10

Use of Complementary L-LNA Oligonucleotides as a Binding Pair in a Roche Cobas® Elecsys® Analytical Electro-Chemiluminescence Immunoassay to Determine TSH (Thyrotropin) in Human Serum and Plasma

[0448] ECL (ElectroChemiLuminescence) is Roche's technology for immunoassay detection. Based on this technology and combined with specific and sensitive TSH immunoassays, Elecsys yielded reproducible results. The development of ECL immunoassays is based on the use of a ruthenium-complex and tripropylamine (TPA). The chemiluminescence reaction for the detection of the reaction complex is initiated by applying a voltage to the sample solution resulting in a precisely controlled reaction. ECL technology can accommodate many immunoassay principles and formats while providing advantageous performance.

[0449] Commercially available Cobas® Elecsys® TSH kits (No. 11731459, Roche Diagnostics GmbH, Mannheim, Germany) were modified. The reagent kit contains three bottles, one bottle containing a suspension of streptavidin-coated beads, one bottle containing the first reagent (R1) and one bottle containing the second reagent (R2). To the bottle containing the streptavidin-coated beads the L-LNA oligonucleotide of Seq ID NO:10 which was 5′-labeled with a biotin-(HEG).sub.4-moiety (HEG=hexaethylene glycol) was added at two different concentrations, either 0.2 nmol or 0.5 nmol L-LNA oligonucleotide per mg beads.

[0450] The R1 bottle contained the same components as the commercially available kit, except that the biotin-antiTSH antibody conjugate was replaced by an L-LNA antiTSH conjugate described above in Example 9 (concentration 2.5 μg/ml). The ingredients in the R2 bottle were identical as in the commercially available kit.

[0451] The above mentioned assay reagents were used to measure samples (calibrators and controls), in comparison with the commercially available assay. Measurements were performed on a Cobas® Elecsys® e411 analyzer. Results are depicted in FIGS. 6A and 6B.

[0452] Elecsys® TSH test characteristics, commercially available version, not modified

[0453] Test principle One-step sandwich assay

[0454] Sample material Serum [0455] Li-, Na-, NH.sub.4.sup.+-heparin plasma [0456] K.sub.3-EDTA, Na-citrate, NaF, K-oxalate plasma

[0457] Sample volume 50 μL

[0458] Detection limit 0.005 IU/mL

[0459] Functional sensitivity 0.014 IU/mL

[0460] Measuring range 0.005-100 IU/mL

[0461] FIG. 6A shows the comparison of signals (counts) measured with the commercially available assay Cobas® Elecsys® TSH kit (designated “TSH” in FIG. 6A) and modified kits containing 0.2 nmol/mg beads (designated “0.2 L-LNA” in FIG. 6A) or 0.5 nmol/mg beads Biotin-L-LNA oligonucleotides (designated “0.5 L-LNA” in FIG. 6A) in the bead reagent bottle. Samples measured were calibrator 1 (“Cal1”) and a level 1 control (“PCU1”). Reagent 1 (R1) and Reagent 2 (R2) bottle content as described above.

[0462] FIG. 6B shows the comparison of signals (counts) measured with the commercially available assay Cobas® Elecsys® TSH kit (designated “TSH” in FIG. 6B) and modified kits containing 0.2 nmol/mg beads (designated “0.2 L-LNA” in FIG. 6B) or 0.5 nmol/mg beads Biotin-L-LNA oligonucleotides (designated “0.5 L-LNA” in FIG. 6B) in the bead reagent bottle. Samples measured were calibrator 2 (“Cal2”) and a level 2 control (PCU2). Reagent 1 (R1) and Reagent 2 (R2) bottle content as described above.

[0463] Roche Product Id numbers were Elecsys® TSH 200 tests 11731459; TSH CalSet 4×1.3 mL 04738551; PreciControl Universal (PCU) 2×3 mL each 11731416; PreciControl TSH 4×2 mL 11776479; Diluent MultiAssay 2×16 mL 03609987.

EXAMPLE 11

Coupling of L-LNA Oligonucleotides to TSH-Specific F(Ab′).SUB.2.-Fragments (Thyrotropin-Specific Capturing Agent)

[0464] F(ab′).sub.2-Fragments were conjugated with LNA in a two step reaction. Firstly, thiol-groups were introduced into F(ab′)2-fragments via conjugation with SATP (N-succinimidyl-S-acetythiopropionate) and deacetylation by hydroxylamine (see Greg T. Hermanson Bioconjugate Techniques, 3rd edition 2013). L-LNA-oligonucleotides of Seq ID NO:9 were then conjugated to free thiols via maleimide chemistry. L-LNA labeled F(ab′).sub.2 fragments were purified via Superdex 200 size-exclusion and mono Q anion exchange chromatography to obtain conjugated F(ab′).sub.2 fragments of high purity, with each conjugate comprising a single L-LNA oligomer.

EXAMPLE 12

Use of Complementary L-LNA Oligonucleotides as a Binding Pair in a Roche Cobas® Elecsys® Analytical Electro-Chemiluminescence Immunoassay to Determine TSH (Thyrotropin) in Human Serum and Plasma

[0465] The Elecsys® Troponin T Immunoassay is an immunoassay for the in vitro quantitative determination of cardiac troponin T in Heparin, EDTA plasma and serum. The immunoassay is intended to aid in the diagnosis of myocardial infarction.

[0466] The electrochemiluminescence immunoassay “ECLIA” is intended for use on the Cobas® system analyzers.

[0467] Sample material: Serum, [0468] Li-, Na-Heparin-Plasma, [0469] K.sub.2- and K.sub.3-EDTA

[0470] Sample volume 50 μL

[0471] 10% CV precision: 13 ng/L (pg/mL)

[0472] Measuring range 3-10,000 pg/mL

[0473] Limit of detection: 5 ng/L (pg/mL)

[0474] Limit of blank: 3 ng/L (pg/mL)

[0475] Commercially available Cobas® Elecsys® TNThs kits (No. 05092744190, Roche Diagnostics GmbH, Mannheim, Germany) were modified. The reagent kit contains three bottles, one bottle containing a suspension of streptavidin-coated beads, one bottle containing the first reagent (R1) and one bottle containing the second reagent (R2). To the bottle containing the streptavidin-coated beads the L-LNA oligonucleotide of Seq ID NO:10 which was 5′-labeled with a biotin-(HEG).sub.4-moiety (HEG=hexaethylene glycol) was added at two different concentrations, either 0.2 nmol or 0.5 nmol L-LNA oligonucleotide per mg beads.

[0476] The R1 bottle contained the same components as the commercially available kit, except that the biotin-antiTNT antibody conjugate was replaced by an L-LNA antiTSH conjugate described above in Example 11 (concentration 2.5 μg/ml). The ingredients in the R2 bottle were identical as in the commercially available kit.

[0477] The above mentioned assay reagents were used to measure samples (calibrators and controls), in comparison with the commercially available assay. Measurements were performed on a Cobas® Elecsys® e411 analyzer. Results are depicted in FIGS. 7A and 7B.

[0478] FIG. 7A shows the comparison of signals (counts) measured with the commercially available assay Cobas® Elecsys® TNThs kit (designated “NT” in FIG. 7A) and modified kits containing 0.2 nmol/mg beads (designated “0.2 L-LNA” in FIG. 7A) or 0.5 nmol/mg beads Biotin-L-LNA oligonucleotides (designated “0.5 L-LNA” in FIG. 7A) in the bead reagent bottle. Samples measured were calibrator 1 (“Cal2”) and a dilution medium containing no analyte (“DilMa”). Reagent 1 (R1) and Reagent 2 (R2) bottle content as described above.

[0479] FIG. 7B shows the comparison of signals (counts) measured with the commercially available assay Cobas® Elecsys® TSHhs kit (designated “TNT” in FIG. 7B) and modified kits containing 0.2 nmol/mg beads (designated “0.2 L-LNA” in FIG. 7B) or 0.5 nmol/mg beads Biotin-L-LNA oligonucleotides (designated “0.5 L-LNA” in FIG. 7B) in the bead reagent bottle. Samples measured were calibrator 2 (“Cal2”). Reagent 1 (R1) and Reagent 2 (R2) bottle content as described above.

[0480] Roche Product Id numbers were Elecsys Troponin T high sensitive 200 Tests 05 092 744 190; ElecsysT Troponin T high sensitive (STAT) 100 Tests 05 092 728 190; CalSet Troponin T high sensitive ElecsysT 10 calibrations 05 092 752 190; CalSet Troponin T high sensitive (STAT) ElecsysT 10 calibrations 05 092 736 190; Diluent Universal ElecsysT 2×16 mL/2×36 mL 11 732 277 122/03 183 971 122.

EXAMPLE 13

Formation of L-LNA Oligonucleotide Binding Pairs is not Affected by Interference by Free Biotin

[0481] Original Elecsys® beads in a Troponin T hs Elecssys® assay (Id. 05092744190) were replaced by Elecsys beads coated with biotinylated LNA oligos (0.308 nMol L-LNA/ml beads). Additionally the biotinylated specifier Mab<TN-T>-Fab-Bi was replaced in the R1 bottle by a Fab conjugated containing a complementary L-LNA-oligomer at an engineered cysteine at a position Q195 conjugated by maleimide chemistry. This new Troponin T hs Elecsys® assay variant using LNA hybridization to immobilize Troponin T immune-complexes on the streptavidin-coated beads, and a conventional commercial Troponin T hs Elecsys assay (Id. 05092744190) were run in parallel on a cobas E170 device with Cal2 samples from Troponin T hs CalSet (Id. 05092752190) without and supplemented with D-Biotin at concentrations of 100, 250, 500, 1000 and 2000 ng/ml. See FIG. 8, dark bars indicating the results of the unmodified commercial assay, bright bars representing the measurements using the complementary pair of L-LNA oligonucleotides. In contrast to the original assay no significant signal loss could be observed in the new assay variant using LNA hybridization to immobilize Troponin T immune-complexes on streptavidin-coated Elecsys® beads.

[0482] Comparable results can be obtained using different binding pairs, e.g.

TABLE-US-00003 (Seq ID NO: 5) 5′ tgctcctg 3′ (Seq ID NO: 6) 5′ caggagca 3′, (Seq ID NO: 7) 5′ gcctgacg 3′ (Seq ID NO: 8) 5′ cgtcaggc 3′, (Seq ID NO: 9) 5′ tgctcctgt 3′ (Seq ID NO: 10) 5′ acaggagca 3′, (Seq ID NO: 11) 5′ gtgcgtct 3′ (Seq ID NO: 12) 5′ agacgcac 3′, (Seq ID NO: 13) 5′ gttggtgt 3′ (Seq ID NO: 14) 5′ acaccaac 3′ (Seq ID NO: 15) 5′ gttggtgtgttggtg 3′ (Seq ID NO: 16) 5′ caccaacacaccaac 3′ (Seq ID NO: 17) 5′ gttggtgtg 3′ (Seq ID NO: 18) 5′ cacaccaac 3′ (Seq ID NO: 19) 5′ ggaagagaa 3′ (Seq ID NO: 20) 5′ ttctatcc 3′.