RU2258708C2 - Analogues of l-ribo-cna - Google Patents

Abstract

FIELD: organic chemistry, biochemistry.

SUBSTANCE: invention relates to oligomer comprising at least one nucleoside analogue of L-ribo-CNA of the general formula (Ia)

wherein X represents -O-; B represents nitrogen base; P means radical position in an internucleoside linkage followed by monomer or 5'-terminal hydroxy-group; P* means an internucleoside linkage with precede monomer or 3'-terminal hydroxy-group; R^2* and R^4* mean in common biradical -(CH₂)_0-1-O-(CH₂)_1-3-(CH₂)_0-1-S-(CH₂)_1-3- or -(CH₂)_0-1-NR-(CH₂)_1-3- wherein R means hydrogen atom, alkyl or acyl; R^1*, R², R^3*, R⁵ and R^5* mean hydrogen atom. Also, invention proposes nucleoside analogues used in preparing oligomers. Proposed oligomers elicit the enhanced affinity to complementary nucleic acids and can be used as a tool in molecular-biological investigations and as antisense, antigen agents of agents activating genes.

EFFECT: valuable properties of analogues.

15 cl, 3 tbl, 4 dwg, 17 ex

Description

FIELD OF THE INVENTION

The present invention relates to the field of bicyclic nucleoside analogs in the L-rib configuration and to the synthesis of such nucleoside analogs that are used in the preparation of synthetic oligonucleotides capable of forming specific nucleotide base duplexes with complementary single-stranded and double-stranded nucleic acids. The invention also relates to the field of analogues of bicyclic nucleosides in the L-ribo configuration, which can be used as drugs and which can be included in oligonucleotides.

BACKGROUND OF THE INVENTION

Synthetic oligonucleotides are compounds that are widely used in various fields, such as molecular biology, as well as diagnostics and treatment using DNA-based drugs.

general description

For use in a wide range of different applications, the above, oligonucleotides must satisfy many different requirements. For example, for use as a medicament, the oligonucleotide must be able to penetrate the cell membrane, have good resistance to extra- and intracellular nucleases, and preferably have the ability to mobilize endogenous enzymes such as PHKseH. Other properties are also important in DNA-based diagnostics and in molecular biology, such as, for example, the ability of oligonucleotides to act as effective substrates for a wide range of different enzymes that affect natural nucleic acids, such as polymerases, kinases, ligases and phosphatases. However, the fundamental property of oligonucleotides, which underlies all variants of their use, is their ability to recognize and hybridize at a specific site in the sequence with complementary single-stranded nucleic acids using either Watson-Crick hydrogen bonds (AT and GC) or other hydrogen bonds such as described in the Hoogsteen model. Two important parameters are usually used to characterize the ability of a given oligonucleotide to hybridize: affinity and specificity. Affinity is a measure of the binding strength of an oligonucleotide to a complementary target sequence (expressed as thermal stability (T _m ) of a duplex). Each nucleotide pair in a duplex contributes to thermal stability and, thus, affinity increases with the size (number of nucleotide bases) of the oligonucleotide. Specificity is a measure of the ability of an oligonucleotide to distinguish between complete complementarity and an erroneous target sequence. In other words, specificity is a measure of the loss of affinity associated with the presence of erroneous pairs of nucleotide bases in a target molecule.

With a constant oligonucleotide size, specificity increases with an increase in the number of mismatches between the oligonucleotide and its target sequences (i.e., the percentage of errors increases). Conversely, specificity decreases when the size of the oligonucleotide increases with a constant number of errors (i.e., the percentage of mismatches decreases). In other words, an increase in the affinity of an oligonucleotide occurs due to the loss of specificity and vice versa.

In view of the deficiencies inherent in natural oligonucleotides, it is highly desirable to develop new approaches to increase the specificity and affinity of DNA-based drugs, diagnostic methods, and, in general, molecular biology methods using DNA.

Conformationally Limited Nucleosides

It is known that oligonucleotides undergo conformational transitions during hybridization with the target sequence from a relatively randomly folded structure of a single-chain state to an ordered structure of a duplex.

So, in recent years, in the search for analogues of oligonucleotides with improved hybridization properties in comparison with unmodified (2'-deoxy) oligonucleotides, the conformational restriction method was used. These include, for example, bicyclo [3.3.0] nucleosides with an additional C-3 ′, C-5′-ethano-bridge (M. Tarköy, M. Bolli, B. Schweizer and C. Leumann, Helv. Chem. Acta, 1993, 76, 481; Tarköy and C. Leumann, Angew. Chem., Int. Ed. Engl., 1993, 32, 1432; M. Egli, P. Lubini, M. Dobler and C. Leumann, J. Am. Chem. Soc., 1993, 115, 5855; M. Tarköy, M. Bolli and C. Leumann, Helv. Chem. Acta, 1994, 77, 716; M. Bolli and C. Leumann, Angew. Chem., Int. Ed. Engl., 1995, 34, 694; M. Bolli, P. Lubini and C. Leumann, Helv. Chem. Acta, 1995, 78, 2077; JC Litten, C. Epple and C. Leumann, Bioorg. Med. Chem. Lett., 1995, 5, 1231; JC Litten and C. Leumann, Helv. Chem. Acta, 1996, 79, 1129; M. Bolli, JC Litten, R. Schultz and C. Leumann, Chem. Biol ., 1996. 3, 197; M. Bolli, HU Trafelet and C. Leumann, Nucleic Acids Res., 1996, 24, 4660), bicarbocyclo [3.1.0] nucleosides with additional C-1 ', C-6' or C-6 ', C-4'-methane bridge (K.-N. Altmann, R. Kesselring, E. Francotte and G. Rihs, Tetrahedron Lett., 1994, 35 , 2331; K.-H. Altmann, R. Imwinkelried, R. Kesselring and G. Rihs, Tetrahedron Lett., 1994, 35, 7625; V.E. Marquez, M.A. Siddiqui, A. Ezzitouni, P. Russ, J. Wang, R.W. Wagner and M.D. Matteucci, J. Med. Chem., 1996, 39, 3739; A. Ezzitouni and V.E. Marquez, J. Chem. Soc., Perkin Trans. 1, 1997. 1073), bicyclo [3.3.0] - and [4.3.0] nucleosides containing an additional C-2 ', C-3'-dioxalane ring synthesized as a dimer with an unmodified nucleoside in which the additional ring represents a part of the internucleoside bond replacing the natural phosphodiester bond (RJ Jones, S. Swaminathan, JF Millagan, S. Wadwani, BS Froehler and M. Matteucci, J. Am. Chem. Soc., 1993, 115, 9816; J. Wang and MD Matteucci, Bioorg. Med. Chem. Lett., 1997, 7, 229), dimers containing a bicyclo [3.1.0] nucleoside with a C-2 ', C-3'-methane bridge as part of the internucleoside bonds of the amide and sulfonamide type (CGYannopoulus , WQZhou, P. Nower, D. Peoch, YS Sanghvi and G. Just, Synlett., 1997, 378), a nucleoside analog derived from bicyclo [3.3.0] glucose incorporated into the middle of the trimer via formacetal internucleoside bonds (CG Yannopoulus, WQ Zhou, P. Nower, D. Peoch, YS Sanghvi and G. Just, Synlett., 1997, 378) and bicyclic [4.3.0] and [3.3.0] nucleosides with additional C-2 ', C A 3'-linked six- and five-membered ring (R. Nielsen, N.M. Pfundheller, J. Wengel, Chem. Commun., 1997, 826; R. Nielsen, H.M. Pfundheller, J. Wengel, XII International Roundtable: Nucleosides, Nucleotides and Their Biological Applications; La Jolla, California, September 15-19, 1996; Poster PPI 43), which were synthesized and incorporated into oligodeoxynucleotides. Unfortunately, oligonucleotides including these analogs in most cases form less stable duplexes with complementary nucleic acids compared to unmodified oligonucleotides. In the case when there is a moderate increase in the stability of the duplex, such an increase is associated only with the DNA or RNA target, or is related to fully, but not partially modified oligonucleotides, or vice versa.

Evaluation of most of these analogues is also complicated by the lack of data on analogues with G, A and C nucleotide bases, as well as data on the specificity and method of hybridization. In many cases, the synthesis of these monomeric analogues is a very complex process, and in some cases, the synthesis of fully modified oligonucleotides is incompatible with commonly used standard methods of phosphoramidite chemistry.

Recently, there have been reports of oligomers comprising closed nucleic acids (ZNA, Locked Nucleic Acids, LNA) (Nielsen, P., Pfundheller, HM, Olsen, CE and Wengel, J., J. Chem. Soc., Perkin Trans. 1, 1997, 3423; Nielsen, P., Pfundheller, HM, Wengel, J., Chem. Commun., 1997, 9, 825; Christensen, NK, Petersen M., Nielsen, P., Jacobsen, JP and Wengel, J., J. Am. Chem. Soc., 1998, 220, 5458; Koshkin, AA and Wengel, J., J. Orq. Chem., 1998, 63, 2778; Obika, S., Morio, K.- I., Hari, Y. and Imanishi, T., Bioorg. Med. Chem. Lett., 1999, 515). It turned out that the inclusion of ZNA monomers containing a 2'-O, 4'-C-methylene bridge in the oligonucleotide sequence leads to an unprecedented improvement in the hybridization properties of such a modified oligonucleotide (Singh, SK, Nielsen, P., Koshkin, AA, Olsen, CE and Wengel, J., Chem. Commun., 1998, 455; Koshkin, A.K., Singh, SK, Nielsen, P., Rajwanshi, VK, Kumar, R., Meldgaard, M., Olsen, SE and Wengel , J., Tetrahedron, 1998, 54, 3607; Koshkin, AA, Rajwanshi, VK and Wengel, J., Tetrahedron Lett., 1998, 39, 4381; Singh, Sanjay, K. and Wengel, J., Chem. Commun. 1998, 1247; Kumar, R., Singh, SK, Koshkin, AA, Rajwanshi, VK, Meldgaard, M., and Wengel, J., Bioorg. Med. Chem. Lett., 1998, 8, 2219; Obika, S. et al. Tetrahedron Lett., 1997, 38, 8735; Obika, S. et al. Tetrahedron Lett., 1998, 39, 5401 ; Singh, SK, Kumar, R. and Wengel, J., J. Org. Chem., 1998, 63, 6078; Koshkin, A.A., Nielsen, P., Meldgaard, M., Rajwanshi, VK, Singh , SK and Wengel, J., J. Am. Chem. Soc., 1998, 120, 13252; Singh, SK, Kumar, R. and Wengei, J., J. Org. Chem., 1998, 63, 10035). Oligonucleotides, including the indicated LNA monomers and the corresponding 2'-thio-LNA analog, form duplexes with complementary DNA and RNA with thermal stability values that were not previously observed for bi- and tricyclic nucleosides in modified oligonucleotides (ΔТ _pl / modification = from +3 up to + 11 ° C), and also demonstrate improved selectivity.

In a number of works Seela et al. (Seela et al.) Studied xylo DNA (Figure 1, base = adenin-9-yl, cytosin-1-yl, guanine-9-yl or thymin-1-yl), including one or more 2'-deoxy β-D-xylofuranosyl nucleotide monomers (Rosemeyer, H .; Seela, F. Helv. Chem. Acta, 1991, 74, 748; Rosemeyer, H .; Krecmerova, M .; Seela, F. Helv. Chem. Acta, 1991, 74, 2054; Seela, F .; Wörner, Rosemeyer, H. Helv. Chem. Acta, 1994, 77, 883; Seela, F .; Heckel, M .; Rosemeyer, H. Helv. Chem. Acta, 1996 79, 1451; Rosemeyer, H .; Seela, F. Nucleosides Nucleotides, 1995, 14, 1041; Schoeppe, A., Hinz, H.-J., Rosemeyer, H., Seela, F. Eur. J. Biochem . 1996, 239, 33). Compared to the corresponding natural 2'-deoxy-β-D-ribofuranosyl oligonucleotides, xylo DNA is mainly characterized by a mirror secondary structure, entropically favoring the formation of a duplex, increasing stability against exonucleases and, in the case of oligonucleotides, including a small number of 2'-deoxy β-D-xylofuranosyl monomers, reducing thermal affinity for complementary DNA (Rosemeyer, H .; Seela, F. Helv. Chem. Acta, 1991, 74, 748; Rosemeyer, H .; Krecmerova, M .; Seela, F Helv. Chem. Acta, 1991, 74, 2054; Seela, F .; Wörner, Rosemeyer, H. Helv. Chem. Acta, 1994, 77, 883; Seela, F .; Heckel, M .; Rosemeyer, H. Helv. Chem. Acta, 1996, 79, 1451).

SUMMARY OF THE INVENTION

In light of the foregoing, and based on the known properties of ZNA monomers with a 2'-O, 4'-C-methylene bridge, it was decided to synthesize oligonucleotides comprising one or more 2'-O, 4'-C-methylene-α-L- ribofuranosyl nucleotide monomer (s). Computer simulation on α-L-ribo-ZNA monomers indicated the conformation of the S-type furanose ring. In this regard, the aim of this work is the synthesis of 2'-O, 4'-C-methylene-α-L-ribofuranosyl nucleotide monomer and the study of the thermal stability of oligonucleotides comprising this monomer. The results show that modified L-ribo-LNA can be used for high affinity recognition of complementary nucleic acids. If we take into account the invert stereochemistry of the molecule at the C-3'-end and C-4'-end, this fact is very surprising.

Thus, the authors of the present invention have proposed new analogues of nucleoside LNA (L-ribo-LNA) and oligonucleotides, which include L-ribo-LNA nucleoside analogues. Were synthesized new analogues of L-ribo-ZNA nucleosides, including thymine as the nucleic base, however, they can be easily synthesized also with four other nucleotide bases with the formation of a complete set of nucleoside analogues for inclusion in oligonucleotides.

The present invention relates to oligomers comprising at least one nucleoside analogue (hereinafter referred to as “L-ribo-ZNA”) of the general formula I

where X is selected from —O—, —S—, —N (R ^{N *} ) -, —C (R ⁶ R ^{6 *} ) -;

B is selected from hydrogen, hydroxy, optionally substituted C _1-4 alkoxy, optionally substituted C _1-4 alkyl, optionally substituted C _1-4 acyloxy, nucleotide bases, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups reporter groups and ligands;

P denotes the position of the radical in the internucleoside bond with respect to the subsequent monomer or 5'-terminal group, and such an internucleoside bond or 5'-terminal group optionally includes a substituent R ⁵ or an equally acceptable substituent R ⁵ *;

P * denotes an internucleoside bond with a preceding monomer or a 3'-terminal group;

R ² * and R ⁴ * are biradicals consisting of 1-4 groups / atoms selected from —C (R ^a R ^b ) -, —C (R ^a ) = C (R ^a ) -, —C (R ^a ) = N-, -O-, -Si (R ^a ) ₂ -, -S-, -SO ₂ -, -N (R ^a ) - and> C = Z, where Z is selected from -O-, -S - and —N (R ^a ) -, and R ³ and R ¹³ are each independently selected from hydrogen, optionally substituted C _1-12 alkyl, optionally substituted C _2-12 alkenyl, optionally substituted C _2-12 - alkynyl, hydroxy, C _1-12 alkoxy, C _2-12 alkenyloxy, carboxy, C _1-12 alkoxycarbonyl, C _1-12 alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, g heteroarylcarbonyl, amino, mono- and di (C _1-6 alkyl) amino, carbamoyl, mono and di (C _1-6 alkyl) aminocarbonyl, amino C _1-6 alkylaminocarbonyl, mono and di (C _{1 -6-} alkyl) amino-C _1-6 -alkylaminocarbonyl, C _1-6 -alkylcarbonyl-amino, carbamido, C _1-6 -alkanoyloxy, sulfono, C _1-6 -alkylsulfonyloxy, nitro, azido, sulfanyl, C _{1- 6} -alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted and where two geminal Override I R ^a and R ^b together may designate optionally substituted metilenolefin (= CH _2);

each of the substituents present R ¹ *, R ² , R ³ *, R ⁵ , R ⁵ *, R ⁶ and R ⁶ * are independently selected from hydrogen, optionally substituted C _1-12 alkyl, optionally substituted C _2-12 alkenyl optionally substituted C _2-12 alkynyl, hydroxy, C _1-12 alkoxy, C _2-12 alkenyloxy, carboxy, C _1-12 alkoxycarbonyl, C _1-12 alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di _(C1-6 alkyl) amino, carbamoyl, mono- and di (C _1-6 -alkyl) aminocarbonyl, amino-C _1-6 - lkilaminokarbonila, mono- and di (C _1-6 -alkyl) amino-C _1-6 -alkylaminocarbonyl, C _1-6 -alkylcarbonyl-amino, carbamido, C _1-6 -alkanoyloxy, sulphono, C _1-6 -alkilsulfoniloksi, nitro, azido, sulfanyl, C _1-6 alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands, where aryl and heteroaryl may optionally be substituted and where two geminal substituents together can represent oxo, thioxo, imino or optionally substituted methylene, or together they can form Vat spiro biradical consisting of an alkylene chain containing 1-5 carbon atoms which optionally contains inside and / or on the end of one / one or more heteroatoms / groups selected from -O-, -S- or - (NR ^N) -, where R ^{N is} selected from hydrogen and C _1-4 alkyl and where two adjacent (non-heminal) substituents may indicate an additional bond leading to the formation of a double bond; and R ^{N *} , if present, is selected from hydrogen and C _1-4 alkyl;

and to their basic salts and acid addition salts.

The present invention also relates to nucleoside analogues (L-ribo-ZNA) of the general formula II

where Deputy B is selected from nucleotide bases, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands;

X is selected from —O—, —S—, —N (R ^{N *} ) - and —C (R ⁶ R ^{6 *} ) -;

each of Q and Q ^{* is} independently selected from hydrogen, azido, halogen, cyano, nitro, hydroxy, Prot-O-, Act-O-, mercapto, Prot-S-, Act-S-, C _1-6 -alkylthio, amino, Prot-N (R ^H ) -, Act-N (R ^H ) -, mono- or di (C _1-6 -alkyl) amino, optionally substituted C _1-6 alkoxy, optionally substituted C _1-6 - alkyl, optionally substituted C _2-6 alkenyl, optionally substituted C _2-6 alkenyloxy, optionally substituted C _2-6 alkynyl, optionally substituted C _2-6 alkynyloxy, monophosphate, diphosphate, triphosphate, DNA intercalators, photochemically active groups thermochemically active g rup, chelating groups, reporter groups, ligands, carboxy, sulfono, hydroxymethyl, Prot-O-CH ₂ -, Act-O-CH ₂ -, aminomethyl, Prot-N (R ^H ) -CH ₂ -, Act-N ( R ^H ) —CH ₂ -, carboxymethyl, sulfonomethyl, where Prot is a protecting group for —OH, —SH and —NH (R ^H ) -, respectively, Act is an activating group for —OH, —SH and —NH (R ^H ), respectively, and R ^{H is} selected from hydrogen and C _1-6 alkyl; and R ² * and R ⁴ * together denote a biradical selected from -O-, - (CR * R *) _{r + s + l} -, - (CR * R *) _r -O- (CR * R *) _s -, - (CR * R *) _r -S- (CR * R *) _s -, - (CR * R *) _r -N (R *) - (CR * R *) _s -, -O- ( CR * R *) _{r + s} -O-, -S- (CR * R *) _{r + s} -O-, -O- (CR * R *) _{r + s} -S-, -N- (R * ) - (CR * R *) _{r + s} -O-, -O- (CR * R *) _{r + s} -N (R *) -, -S- (CR * R *) _{r + s} -S- , -N (R *) - (CR * R *) _{r + s} -N (R *) -, -N (R *) - (CR * R) _{r + s} -S- and -S- (CR * R *) _{r + s-} N (R *) -;

where each R * is independently selected from hydrogen, halogen, azido, cyano, nitro, hydroxy, mercapto, amino, mono- or di (C _1-6 alkyl) amino, optionally substituted C _1-6 alkoxy, optionally substituted C _{1 -6} -alkyl, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands, and / or two adjacent (non-heminal) R * can together denote a double bond and each r and s is 0-3, when provided that the sum r + s is 1-4;

each of the present substituents R ¹ *, R ² , R ³ *, R ⁵ , R ⁵ *, R ⁶ and R ⁶ * are independently selected from hydrogen, optionally substituted C _1-12 alkyl, optionally substituted C _2-12 alkenyl optionally substituted C _2-12 alkynyl, hydroxy, C _1-12 alkoxy, C _2-12 alkenyloxy, carboxy, C _1-12 alkoxycarbonyl, C _1-12 alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di (C _1-6 -alkyl) amino, carbamoyl, mono- and di (C _1-6 -alkyl) aminocarbonyl, amino-C _1-6 -al kilaminocarbonyl, mono- and di (C _1-6 -alkyl) amino-C _1-6 -alkylaminocarbonyl, C _1-6 -alkylcarbonyl-amino, carbamido, C _1-6 -alkanoyloxy, sulfono, C _1-6 -alkylsulfonyloxy, nitro, azido, sulfanyl, C _1-6 alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands, where aryl and heteroaryl may optionally be substituted and where two geminal substituents together can represent oxo, thioxo, imino or optionally substituted methylene, or together they can form be a spiro biradical consisting of an alkylene chain containing 1-5 carbon atoms which optionally contains inside and / or on the end of one / one or more heteroatoms / groups selected from -O-, -S- and - (NR ^H) -, where R ^{H is} selected from hydrogen and C _1-4 alkyl and where two adjacent (non-heminal) substituents may indicate an additional bond, leading to the formation of a double bond;

and R ^H *, if present and not included in the biradical, is selected from hydrogen and C _1-4 alkyl;

and their basic salts and acid addition salts;

provided that any chemical group (including any nucleotide base) that is reactive under the conditions prevailing in the synthesis of oligonucleotides is an optionally protected functional group.

The present invention also relates to the use of nucleoside analogues (L-ribo-ZNAs) for the preparation of oligomers and to the use of oligomers as well as nucleoside analogues (L-ribo-ZNAs) in diagnostics, molecular biological studies and therapy.

DETAILED DESCRIPTION OF THE INVENTION

In the context of the present description, the term "L-ribo-ZNA" (closed nucleoside analogues in the L-ribo configuration) refers to bicyclic nucleoside analogs in the L-ribo-configuration, which are either included in the oligomer according to the present invention (basic formula I), or represent a separate chemical species (general formula II). The term "monomeric L-ribo-ZNA" specifically refers to the latter case.

Oligomers and nucleoside analogues

As indicated above, the present invention relates, among other objects, to new oligomers (oligonucleotides) comprising one or more bicyclic nucleoside analogues in the L-ribo configuration (hereinafter referred to as “L-ribo-ZNA”).

Each of the possible L-ribo-ZNAs included in the oligomer (oligonucleotide) has the general formula I:

where X is selected from -O- (L-ribofuranose motif), -S-, -M (R ^{N *} ) -, -C (R ⁶ R ^{6 *} ) -, where R ⁶ , R ^{6 *} and R ^{N * are} defined below. Thus, L-ribo-ZNAs included in the oligomer contain a 5-membered ring as an essential part of the bicyclic structure.

Among the possible 5-membered rings, a very interesting situation is when X is —O—, —S— and —N (R ^{N *} ), and a situation where X is —O— is particularly interesting.

Substituent B may denote a group which, when the oligomer forms a complex with DNA or RNA, is capable of interacting (for example, via hydrogen bonds, or covalent bonds, or through electronic interaction) with DNA or RNA, especially with the nucleotide bases of DNA or RNA. Alternatively, substituent B may indicate a group that acts as a label or reporter, or substituent B may indicate a group (eg, hydrogen) that is expected to interact slightly with DNA or RNA, or the interaction will not occur at all. Thus, substituent B is preferably selected from hydrogen, hydroxy, optionally substituted C _1-4 alkoxy, optionally substituted C _1-4 alkyl, optionally substituted C _1-4 acyloxy, nucleotide bases, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands.

In the context of the present description, the term "nucleotide base" encompasses both natural nucleotide bases and non-natural nucleotide bases. It is obvious to every specialist with an average level of knowledge in this field that various nucleotide bases, which were previously considered unnatural, were subsequently found in nature. Thus, the term “nucleotide bases” includes not only known purine and pyrimidine heterocycles, but also analogues of heterocyclic compounds and their tautomers. Representative examples of nucleotide bases include adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurin, 8-oxo-N ^6- methyladenine, 7-deazaxanthin, 7-deazaguanine, N ⁴ , N ^4- ethanocytosine, N ⁶ , N ^6- ethano-2,6-diaminopurin, 5-methylcytosine, 5- (C ³ -C ⁶ ) alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine, and the "unnatural" nucleotide bases described in US Pat. 5,432,272 (Benner et al.). The term "nucleotide base" covers all of the above examples, as well as their analogues and tautomers. Particularly interesting nucleotide bases are adenine, guanine, thymine, cytosine and uracil, which are considered as natural nucleotide bases in the context of their therapeutic and diagnostic use in humans.

In the context of the present description, the term "DNA intercalator" means a group that can intercalate into a helix, duplex or triplex of DNA or RNA. Examples of the functional parts of DNA intercalators include acridines, anthracenes, quinones, such as anthraquinone, indole, quinoline, isoquinoline, dihydroquinones, anthracyclines, tetracyclines, methylene blue, anthracyclines, psoralenes, coumarins, ethidium halides, dynemycin, metal, 10-phenanthroline-copper, tris (4,7-diphenyl-1,10-phenanthroline) ruthenium-cobalt-enedines, such as calheamycin, porphyrins, distamycin, netropcin, viologen, daunomycin. Particularly interesting examples are acridines, quinones, such as anthraquinone, methylene blue, psoralen, coumarins, and ethidium halides.

In the context of the present description, the term "photochemically active groups" covers compounds capable of undergoing chemical reactions when irradiated with light. Illustrative examples of the functional groups of such compounds are quinones, especially 6-methyl-1,4-naphthoquinone, anthraquinone, naphthoquinone and 1,4-dimethyl-anthraquinone, diazirines, aromatic azides, benzophenones, psoralenes, diazo compounds and diazirino compounds.

In the context of the present description, the term "thermochemically reactive group" means a functional group that, when thermochemical induction is capable of forming a covalent bond with other groups. Illustrative examples of the functional parts of thermochemically active groups are carboxylic acids, esters of carboxylic acids, such as activated esters, carboxylic acid halides, such as fluorohydrides, acid chlorides, bromoanhydrides and iodohydrides, as well as carboxylic acid azides, carboxylic acid hydrides, sulfones sulfonic acid esters, sulfonic acid halides, semicarbazides, thiosemicarbazides, aldehydes, ketones, primary alcohols, secondary alcohols, tertiary alcohol , Phenols, alkyl halides, thiols, disulphides, primary amines, secondary amines, tertiary amines, hydrazines, epoxides, maleimides, and boronic acid derivatives.

In the context of the present description, the term "chelating group" means a molecule that includes more than one binding site and often connects to another molecule, another atom or other ion through more than one binding site at the same time. Examples of the functional parts of the chelating groups include iminodiacetic acid, nitrilotriacetic acid, ethylenediaminetetraacetic acid (EDTA), aminophosphonic acid, etc.

In the context of the present description, the term "reporter group" means a group that is detected either by itself or as part of a detectable series. Examples of the functional parts of the reporter groups include biotin, digoxygenin, fluorescent groups (groups that can absorb electromagnetic radiation, such as light or X-rays with a specific wavelength, followed by re-emission of absorbed energy in the form of radiation with a longer wavelength; illustrative examples include dansyl ( 5-dimethylamino) -1-naphthalenesulfonyl), DOXYL (N-oxyl-4,4-dimethyloxazolidine), PROXYL (N-oxyl-2,2,5,5-tetramethylpyrrolidine), TEMPO (N-oxyl-2,2, 6,6-tetramethylpiperidine), dinitrophenyl, acridines, coumarins, y3 and Сy5 (trademarks of Biological Detection Systems, Inc.), erythrosine, coumaric acid, umbelliferone, Texas Red (Roxamine), rhodamine, tetramethyl rhodamine, Rox (Rox), 7-nitrobenzo-2-oxa-1-diazole (BND), pyrene, fluorescein, europium, ruthenium, samarium and other rare earth metals), radioisotope labels, chemiluminescent labels (labels detected by light emission during a chemical reaction), spin labels (free radicals (for example, substituted organic nitroxides) or other paramagnetic probes (e.g. Cu ²⁺ , Mg ²⁺ ) that, when bound to bi the biological molecule makes it detectable using electron paramagnetic resonance spectroscopy), enzymes (such as peroxidases, alkaline phosphatases, β-galactosidases and glucose oxidases), antigens, antibodies, haptens (groups that can bind to the antibody, but which by themselves do not cause immune response, such as peptides and steroid hormones), carrier systems for penetration through the cell membrane, such as fatty acid residues, steroid groups (cholesterol), vitamin A, vitamin D, vitamin E, folic acid acid, peptides for specific receptors, endocytosis mediating groups, epidermal growth factor (EGF), bradykinin and platelet growth factor (PDGF). Particularly interesting examples include biotin, fluorescein, Texas red, rhodamine, dinitrophenyl, digoxygenin, ruthenium, europium, Cy5 and Cy3.

In the context of the present description, the term "ligand" means a group that can bind to something. Ligands may include functional groups such as aromatic groups (such as benzene, pyridine, naphthalene, anthracene and phenanthrene), heteroaromatic groups (such as thiophene, furan, tetrahydrofuran, pyridine, dioxane and pyrimidine), carboxylic acids, carboxylic acid esters, carboxylic acid halides, carboxylic acid azides, carboxylic acid hydrazides, sulfonic acids, sulfonic acid esters, sulfonic acid halides, semicarbazides, thiosemicarbazides, aldehydes, ketones, primary alcohols, secondary pirs, tertiary alcohols, phenols, alkyl halides, thiols, disulfides, primary amines, secondary amines, tertiary amines, hydrazines, epoxides, maleimides, C ₁ -C ₂₀ alkyl groups, optionally including one or more heteroatoms within a group or at its end, such as oxygen atoms, nitrogen atoms and / or sulfur atoms, optionally including aromatic or mono / polyunsaturated hydrocarbons, polyoxyethylene such as polyethylene glycol, oligo / polyamides such as poly-β-alanine, polyglycine, polylysine, peptides, oligo / polysaccharides, oligo / po lyphosphates, toxins, antibiotics, cell poisons and steroids, as well as “affinity ligands”, i.e. functional groups or biomolecules that have specific affinity for sites on certain proteins, antibodies, poly- and oligosaccharides and other biomolecules.

It is obvious to any person skilled in the art that the above specific examples of DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands correspond to the “active / functional” part of the groups under consideration. It is also clear to those skilled in the art that DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands are typically in the M-K form, where M denotes the "active / functional" part of the group in question, and K denotes a spacer through which the specified "active / functional" part is attached to the 5-membered ring. Thus, it should be understood that group B when B is selected from intercalators of DNA, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands, has the form M-K-, where M stands for "active / functional "Part of the DNA intercalator, photochemically active group, thermochemically active group, chelating group, reporter group and ligand, respectively, and where K denotes an optional spacer comprising 1-50 atoms, preferably 1-30 atoms, in particular 1-15 atoms, du 5-membered ring and the "active / functional" part of the molecule.

, в случае некоторых применений предпочтительно, чтобы длина была меньше 100

. Таким образом, спейсер включает цепь углеродных атомов, необязательно содержащую внутри или на конце один или более гетероатомов, таких как атомы кислорода, атомы азота и/или атомы серы. Таким образом, спейсер К может включать одну или более амидных, сложноэфирных, амино, эфирных и/или тиоэфирных функциональных групп и необязательно ароматические или моно/полиненасыщенные углеводороды, полиоксиэтилен, такой как полиэтиленгликоль, олиго/полиамиды, такие как поли-р-аланин, полиглицин, полилизин, и пептиды в целом, олигосахариды, олиго/полифосфаты. Кроме того, спейсер может состоять из объединенных единиц. Длина спейсера может варьировать, в зависимости от его желательного или необходимого положения и пространственной ориентации «активной/функциональной» части рассматриваемой группы относительно 5-членного кольца. В особенно интересных вариантах реализации настоящего изобретения спейсер включает химически расщепляемую группу. Примеры таких химически расщепляемых групп включают дисульфидные группы, расщепляемые в восстановительных условиях, пептидные фрагменты, расщепляемые пептидазами, и др.In the context of the present description, the term "spacer" means a photochemically and thermochemically inactive group that performs the function of spatial distance when two or more different groups of the above type are connected. Spacers are selected based on many characteristics, including hydrophobicity, hydrophilicity, the flexibility of their molecules and length (see, in particular, Hermanson et al., "Immobilized Affinity Ligand Techniques", Academic Press, San Diego, California (1992), p. 137 -ff). Basically, the spacer length is less than about 400

, in the case of some applications, it is preferable that the length is less than 100

. Thus, the spacer includes a chain of carbon atoms, optionally containing inside or at the end of one or more heteroatoms, such as oxygen atoms, nitrogen atoms and / or sulfur atoms. Thus, spacer K may include one or more amide, ester, amino, ester and / or thioether functional groups and optionally aromatic or mono / polyunsaturated hydrocarbons, polyoxyethylene such as polyethylene glycol, oligo / polyamides such as poly-p-alanine, polyglycine, polylysine, and peptides in general, oligosaccharides, oligo / polyphosphates. In addition, the spacer may consist of combined units. The length of the spacer may vary, depending on its desired or necessary position and spatial orientation of the "active / functional" part of the group in question relative to the 5-membered ring. In particularly interesting embodiments of the present invention, the spacer includes a chemically cleavable group. Examples of such chemically cleavable groups include disulfide groups cleaved under reducing conditions, peptide fragments cleaved by peptidases, and others.

In one embodiment of the present invention, K denotes a single bond, so that the "active / functional" part of the group in question is attached directly to the 5-membered ring.

In a preferred embodiment of the present invention, Deputy B in the basic formulas I and II is preferably selected from nucleotide bases, in particular from adenine, guanine, thymine, cytosine and uracil.

In the oligomers according to the present invention (formula I), P denotes the position of the radical in the internucleoside bond with the subsequent monomer or 5'-terminal group. The first option occurs when the considered L-ribo-ZNA is not a 5'-terminal "monomer", while the last option is applicable when the considered L-ribo-ZNA is a 5'-terminal "monomer" . It should be understood (which is also obvious from the definition of internucleoside linkage and the 5'-terminal group given below) that such an internucleoside linkage or 5'-terminal group may include a substituent R ⁵ (or an equally acceptable substituent R ^{5 *} ), thereby forming a double bond with R. group (5'-end refers to the position corresponding to the 5'-carbon atom in the ribose group of the nucleoside).

On the other hand, P ^* denotes the position of the radical in the internucleoside bond with the preceding monomer or a 3'-terminal group. Similarly, the first option refers to the case when the considered L-ribo-ZNA is not a 3'-terminal "monomer", while the latter option occurs when the considered L-ribo-ZNA is a 3'-terminal " monomer "(3'-end refers to the position corresponding to the 3'-carbon atom in the ribose group in the nucleoside).

In the context of the present description, the term "monomer" refers to natural nucleosides, unnatural nucleosides, PNA, ZNA, and also to L-ribo-ZNA. Thus, the term “downstream monomer” refers to an adjacent monomer in the 5 ′ end direction, and the term “upstream monomer” refers to an adjacent monomer in the 3 ′ end direction. Such subsequent and preceding monomers from the point of view of the position of the L-ribo-LNA monomer can be represented by natural nucleosides or unnatural nucleosides or even L-ribo-LNA monomers.

Therefore, in the context of the present description (as can be seen from the above definitions), the term "oligomer" means an oligonucleotide modified by the inclusion of one or more L-ribo-ZNAs.

The most important part of the present invention is the L-ribo configuration of the combined 5-membered ring, provided that R ^{2 *} and R ^{4 *} together represent the diradical forming a fused ring on the 5-membered ring.

In the groups comprising the biradical (s), Z is selected from —O—, —S—, and —N (R ^a ) -, and R ^a and R ^b are each independently selected from hydrogen optionally substituted with C _1-12 - alkyl, optionally substituted C _2-12 alkenyl, optionally substituted C _2-12 alkynyl, hydroxy, C _1-12 alkoxy, C _2-12 alkenyloxy, carboxy, C _1-12 alkoxycarbonyl, C _1-12 alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono and di (C _1-6 alkyl) amino, carbamoyl, mono and di (C _1-6 alkyl) aminocarbo yl, amino-C _1-6 -alkylaminocarbonyl, mono- and di (C _1-6 -alkyl) amino-C _1-6 -alkylaminocarbonyl, C _1-6 -alkylcarbonyl-amino, carbamido, C _1-6 alkanoyloxy, sulfono, C _1-6 alkylsulfonyloxy, nitro, azido, sulfanyl, C _1-6 alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands (the latter groups may include a spacer, defined for substituent B), where aryl and heteroaryl may be optionally substituted. In addition, the two geminal substituents R ^a and R ^b together may be optionally substituted methylene olefin (= CH ₂ optionally substituted at one or two positions with substituents defined for optional aryl substituents).

In the context of the present invention, that is, in the context of the description and claims, the orientation of the biradicals is such that the left side represents the substituent with the lowest number, and the right side displays the substituent with the largest number, so that when R ^{2 *} and R ^{4 *} together denote the biradical "-O-CH ₂ -", it should be understood that the oxygen atom corresponds to R ^{2 *} and the methylene group corresponds to R ^{4 *} .

In terms of the possibilities afforded by the structure of the biradical (s) in L-ribo-ZNA to be included in the oligomers of the present invention, it is believed that the biradicals made up by the pair (s) of non-heminal substituents are preferably selected from - (CR ^* R ^* ) _r - Y- (CR ^* R ^* ) _s -, - (CR ^* R ^* ) _r -Y- (CR ^* R ^* ) _s -Y-, -Y- (CR ^* R ^* ) _{r + s} -Y-, - Y- (CR ^* R ^* ) _r -Y- (CR ^* R ^* ) _s -, - (CR ^* R ^* ) _{r + s} -, -Y-, -YY-, where each Y is independently selected from -O- , -S-, -Si (R ^* ) ₂ -, -N (R ^* ) -,> С = O, -C (= O) -N (R ^* ) - and -N (R ^* ) -С ( = O) -, where each R ^* is independently selected from hydrogen, halogen, azido, cyano, nitro, hydroxy, mercapto, amino, mono- or di _(C1-6 alkyl) and ino optionally substituted C _1-6 -alkoxy, optionally substituted C _1-6 -alkyl, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, and / or two adjacent (negeminalnyh) R ^* may together denote a double bond and each r and s is 0-4, provided that the sum of r + s is 1-4. Of particular interest are situations in which each biradical is independently selected from -Y-, - (CR ^* R ^* ) _{r + s} -, - (CR ^* R ^* ) _r -Y- (CR ^* R ^* ) _s - and -Y - (CR ^* R ^* ) _{r + s} -Y-, where each r and s is 0-3, provided that the sum of r + s is 1-4.

Particularly interesting oligomers are those that satisfy the following criteria applicable to L-ribo-ZNA in oligomers: R ^{2 *} and R ^{4 *} together denote a biradical selected from —O—, —S—, —N (R ^* ) -, - (CR ^* R ^* ) _{r + s + l} -, - (CR ^* R ^* ) _r -O- (CR ^* R ^* ) _s -, - (CR ^* R ^* ) _r -S- (CR ^* R ^* ) _s -, - (CR ^* R ^* ) _r -N (R ^* ) - (CR ^* R ^* ) _s -, -O- (CR ^* R ^* ) _{r + s} -O-, -S- (CR ^* R ^* ) _{r + s} -O-, -O- (CR ^* R ^* ) _{r + s} -S-, -N (R ^* ) - (CR ^* R ^* ) _{r + s} -O-, -O- ( CR ^* R ^* ) _{r + s} -N (R ^* ) -, -S- (CR ^* R ^* ) _{r + s} -S-, -N (R ^* ) - (CR ^* R ^* ) _{r + s} -N (R ^* ) -, -N (R ^* ) - (CR ^* R ^* ) _{r + s} -S- and -S- (CR ^* R ^* ) _{r + s} -N (R ^* ) -, where each r and s = 0-3, with the proviso that the sum r + s is 1-4, and wherein R ^* is selected from hydrogen, hydroxy, optionally substituted C _1-6 -alk xi, optionally substituted C _1-6 -alkyl, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, and the remaining substituents R ^* are hydrogen.

In one preferred embodiment of the present invention, one R ^* group in the biradical of at least one ZNA is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands (the latter groups may include a spacer specific for the substituent IN).

In another preferred embodiment, one R ^* group in the biradical of at least one ZNA is selected from hydrogen, hydroxy, optionally substituted C _1-6 alkoxy, optionally substituted C _1-6 alkyl, DNA intercalators, photochemically active groups, and thermochemically active groups, chelating groups, reporter groups and ligands, and any remaining R ^* substituents are hydrogen.

Existing substituents R ^{1 *} , R ² , R ^{3 *} , R ⁵ , R ^{5 *} , R ⁶ and R ^{6 *} are independently selected from hydrogen, optionally substituted C _1-12 alkyl, optionally substituted C _2-12 alkenyl, optionally substituted C _2-12 alkynyl, hydroxy, C _1-12 alkoxy, C _2-12 alkenyloxy, carboxy, C _1-12 alkoxycarbonyl, C _1-12 alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di (C _1-6 alkyl) amino, carbamoyl, mono- and di (C _1-6 -alkyl) aminocarbonyl, amino-C _1-6 -alkilaminokarbo yl, mono- and di (C _1-6 -alkyl) amino-C _1-6 -alkylaminocarbonyl, C _1-6 -alkilkarbonilamino, carbamido, C _1-6 -alkanoyloxy, sulphono, C _1-6 -alkilsulfoniloksi, nitro, azido, sulfanyl, C _1-6 alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands (the latter groups may include a spacer specific for substituent B), where aryl and heteroaryl can be optionally substituted and where two geminal substituents together may represent oxo, thioxo, imino or optionally substituted methylene, or together they can form a spiro-biradical consisting of an alkylene chain of 1-5 carbon atoms, which may optionally contain one / one or more heteroatoms / groups selected from -O- inside and / or at the end , -S- and - (NR ^N ) -, where R ^{N is} selected from hydrogen and C _1-4 -alkyl and where two adjacent (non-heminal) substituents may represent an additional bond, leading to the formation of a double bond; and R ^{N *} , if present, is selected from hydrogen and C _1-4 alkyl.

Preferably, each of the substituents R ^{1 *} , R ² , R ^{3 *} , R ⁵ , R ^{5 *} , R ⁶ and R ^{6 *} in L-ribo-LNA, if present, is independently selected from hydrogen, optionally substituted with C _{1- 6} -alkyl, optionally substituted C _2-6 alkenyl, hydroxy, C _1-6 alkoxy, C _2-6 alkenyloxy, carboxy, C _1-6 alkoxycarbonyl, C _1-6 alkylcarbonyl, formyl, amino, mono - and di (C _1-6 -alkyl) amino, carbamoyl, mono- and di (C _1-6 -alkyl) aminocarbonyl, C _1-6 -alkylcarbonylamino, carbamido, azido, C _1-6 -alkanoyloxy, sulfono, sulfanyl , C _1-6 -alkylthio, DNA intercalators, photochemically active groups, thermo Chemically active groups, chelating groups, reporter groups, and ligands, and halogen, where two geminal substituents together may form an oxo, and R ^{N *,} when present, is selected from hydrogen and C _1-4 -alkyl.

In a preferred embodiment of the present invention, X is selected from —O—, —S— and —NR ^{N *} -, in particular —O—, and each of the substituents R ^{1 *} , R ² , R ^{3 *} , R ⁵ , R ^{5 *} , R ⁶ and R ^{6 *} in L-ribo-ZNA, if present, is hydrogen.

In an even more preferred embodiment of the invention, X is O, the substituents R ^{1 *} , R ² , R ³ , R ⁵ and R ^{5 *} are hydrogen, and R ^{2 *} and R ^{4 *} in L-ribo-ZNA included in the oligomer, together denote a biradical selected from —O—, - (CH ₂ ) _0-1 —O— (CH ₂ ) _1-3 -, - (CH ₂ ) _0-1 —S- (CH ₂ ) _1-3 -, - (CH ₂ ) _0-1 -N (R ^N ) - (CH ₂ ) _1-3 - and - (CH ₂ ) _2-4 -, in particular from -O-CH ₂ -, -S-CH ₂ - and —NR ^H —CH ₂ -. Basically, in accordance with the results obtained, it is preferable that the biradical containing R ^{2 *} and R ^{4 *} form a diatomic bridge, i.e. so that the radical forms a five-membered ring with a furanose ring (X = O).

In one preferred embodiment, the biradical is - (CH ₂ ) _2-4 .

In the case of these embodiments of the invention, it is preferable that the L-ribo-ZNA has / had the following general structure Ia:

As a separate aspect of the present invention is also considered a variant of formula Ia, in which B is in the "β-configuration".

The oligomers of the present invention typically include 1-10000 L-ribo-ZNAs of general formula I (or more detailed general formula Ia) and 0-10000 nucleosides selected from natural nucleosides and nucleoside analogues. The total number of nucleosides and L-ribo-ZNA (n) is at least 2, preferably at least 3, in particular at least 5, especially at least 7, and is, for example, in the range of 2-15000 preferably in the range of 2-100, such as 3-100, in particular in the range of 2-50, such as 3-50 or 5-50 or 7-50.

It was found that partially L-ribo-ZNA modified oligomers have the ability to strongly hybridize (with increased affinity) with DNA and RNA. Currently, it is believed that fully modified L-ribo-LNA oligomers and oligomers consisting of L-ribo-LNA monomers together with other nucleotide analogues in the L-ribo configuration lead to an increase in hybridization properties to comparable values.

In the context of the present description, the term "nucleoside" refers to a glycoside of a heterocyclic base. The term "nucleoside" is used in a broad sense and includes non-natural nucleosides, natural nucleosides, as well as other nucleoside analogues.

Illustrative examples of nucleosides include ribonucleosides containing a ribose group, as well as deoxyribonucleosides containing a deoxyribose group. Regarding the bases of such nucleosides, it should also be noted that they can be any natural base, for example adenine, guanine, cytosine, thymine and uracil, as well as any modified versions thereof or any possible non-natural bases.

As for the known nucleosides (natural and non-natural) and nucleoside analogues (including known bi- and tricyclic analogues), it is obvious from these definitions that the oligomer may include one or more L-ribo-ZNAs (which may be the same or different in terms of choice of substituent and from the point of view of biradical choice) and one or more nucleosides and / or nucleoside analogues. In the context of the present description, the term "oligonucleotide" refers to a sequential chain of nucleosides connected via internucleoside bonds, however, it should be understood that the nucleotide base in one or more nucleotide units (monomers) in the oligomer (oologonucleotide) can be modified using Deputy B, as defined above.

Oligomers can be linear, branched or cyclic. In the case of a branched oligomer, the branch points can be located in the nucleoside, in the internucleoside bond, or, in the case of the most interesting embodiment of the invention, in L-ribo-ZNA. It can be assumed that in the latter embodiment, the substituents R ² and R ³ * may represent a group P * showing an internucleoside bond with the preceding monomer, in particular R ² is the subsequent P *.

As indicated above, L-ribo-ZNA oligomers are linked to other monomers via an internucleoside bond. In the context of the present description, the term "internucleoside bond" means a bond consisting of 2-4, preferably 3, groups / atoms selected from -CH ₂ -, -O-, -S-, -NR ^H -,> C = O, > C = NR ^H ,> C = S, -Si (R``) <sub>2</sub> -, -SO-, -S (O) <sub>2</sub> -, -P (O) <sub>2</sub> -, -RO (BH <sub>3</sub> ) -, - P (O, S) -, -P (S) <sub>2</sub> -, -PO (R``) -, -PO (OCH ₃ ) - and -PO (NHR ^H ) -, where R ^{H is} selected from hydrogen and C _{1 -4-} alkyl, and R ″ is selected from C _1-6 -alkyl and phenyl. Illustrative examples of such internucleoside bonds are —CH ₂ —CH ₂ —CH ₂ -, —CH ₂ —CO — CH ₂ -, —CH ₂ —CHOH — CH ₂ -, —O — CH ₂ —O—, —O — CH ₂ —CH ₂ -, —O — CH ₂ —CH = (including R ⁵ , if used as a bond with a subsequent monomer), —CH ₂ —CH ₂ —O—, —NR ^H —CH ₂ —CH ₂ - , -CH ₂ -CH ₂ -NR ^H -, -CH ₂ -NR ^H -CH ₂ -, -O-CH ₂ -CH ₂ -NR ^H -, -NR ^H -CO-O-, -NR ^H -CO -NR ^H -, -NR ^H -CS-NR ^H -, -NR ^H -C - (= NR ^H ) -NR ^H -, -NR ^H -CO-CH ₂ -NR ^H -, -O-CO-O -, -O-CO-CH ₂ -O-, -O-CH ₂ -CO-O-, -CH ₂ -CO-NR ^H -, -O-CO-NR ^H -, -NR ^H -CO-CH ₂ -, -O-CH ₂ -CO-NR ^H -, -O-CH ₂ -CH ₂ -NR ^H -, -CH = NO-, -CH ₂ -NR ^H -O-, -CH ₂ -ON = (including R ⁵ , if used as a bond with a subsequent monomer), —CH ₂ —O — NR ^H -, —CO — NR ^H —CH ₂ -, —CH ₂ —NR ^H —O—, —CH ₂ - NR ^H —CO—, —O — NR ^H —CH ₂ -, -O-NR ^H -, -O-CH ₂ -S-, -S-CH ₂ -O-, -CH ₂ -CH ₂ -S-, -O-CH ₂ -CH ₂ -S-, - S-CH ₂ -CH = (including R ⁵ , if used as a bond with a subsequent monomer), -S-CH ₂ -CH ₂ -, -S-CH ₂ -CH ₂ -O-, -S-CH ₂ -CH ₂ -S-, -CH ₂ -S-CH ₂ -, -CH ₂ -SO-CH ₂ -, -CH ₂ -SO ₂ -CH ₂ -, -O-SO-O-, -OS (O ) ₂ -O-, -OS (O) ₂ -CH ₂ -, -OS (O) ₂ -NR ^H -, -NR ^H -S (O) ₂ -CH ₂ -, -OS (O) ₂ -CH ₂ -, -O-P (O) ₂ -O-, -O-P (O, S) -O-, -O-P (S) ₂ -O-, -S-P (O) ₂ -O -, -S-P (O, S) -O-, -S-P (S) ₂ -O-, O-P (O) ₂ -S-, -O-P (O, S) -S- , -O-P (S) ₂ -S-, -S-P (O) ₂ -S-, -SP (O, S) -S, -S-P (S) ₂ -S-, -O- ^{PO (R '') -O-,} -O- PO _(OCH3) -O-, -O-PO (OCH ₂ CH ₃₎ -O-, -O-PO (OCH ₂ CH ₂ SR) -O- , -O-PO (BH ₃ ) -O, -O-PO (NHR ^N ) -O-, -O-P (O) ₂ -NR ^H -, -NR ^H -P (O) ₂ -O-, —OP (O, NR ^H ) —O—, —CH ₂ —P (O) ₂ —O—, —O — P (O) ₂ —CH ₂ -, and O — Si (R ″) ₂ —O— ; among which —CH ₂ —CO — NR ^H -, —CH ₂ —NR ^H —O—, —S — CH ₂ —O—, —O — P (O) ₂ —O—, —O — P ( O, S) -O-, -O-P (S) ₂ -O-, -NR ^H -P (O) ₂ -O-, -OP (O, NR ^H ) -O-, -O-PO ( R ^″ ) —O—, —O — PO (CH ₃ ) —O— and —O — PO (NHR ^N ) —O—, where R ^{H is} selected from hydrogen and C _1-4 alkyl and R ^{H is} selected from C _1-6 alkyl and phenyl. Other illustrative examples are given by Mesmaeker et al., Current Opinion in Structural Biology 1995, 5, 343-355. The left side of the internucleoside bond is connected to the 5-membered ring as a substituent P *, while the right side of the bond is connected to the 5'-position of the preceding monomer.

From the above it is also clear that the P group can also denote a 5'-terminal group, in the case when the considered L-ribo-ZNA is a 5'-terminal monomer. Examples of such 5'-terminal groups include hydrogen, hydroxy, optionally substituted C _1-6 alkyl, optionally substituted C _1-6 alkoxy, optionally substituted C _1-6 alkylcarbonyloxy, optionally substituted aryloxy, monophosphate, diphosphate, triphosphate and - W-A ', where W is selected from -O-, -S- and -N (R ^H ) -, where R ^{H is} selected from hydrogen and C _1-6 alkyl, and where A' is selected from intercalators of DNA, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands (the latter groups may include a spacer defined A substituent B).

In the context of the present description and claims, the terms "monophosphate", "diphosphate" and "triphosphate" mean groups of the formulas -O-P (O) ₂ -O ^- , -O-P (O) ₂ -O-P (O) ₂ -O ^- and -O-P (O) ₂ -O-P (O) ₂ -O-P (O) ₂ -O ^- , respectively.

In a particularly interesting embodiment of the invention, group P denotes 5'-terminal groups selected from monophosphate, diphosphate and triphosphate. The triphosphate variant is particularly interesting as a substrate, such as enzymes, especially those acting on nucleic acids.

Similarly, the P ^* group may denote a 3'-terminal group in the case when the considered L-ribo-ZNA is a 3'-terminal monomer. Examples of such 3'-terminal groups include hydrogen, hydroxy, optionally substituted C _1-6 alkoxy, optionally substituted C _1-6 alkylcarbonyloxy, optionally substituted aryloxy and -W-A ', where W is selected from -O-, -S - and -N (R ^H ) -, where R ^{H is} selected from hydrogen and C _1-6 -alkyl, and where A 'is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands (when the latter groups may include a spacer defined for substituent B).

In a preferred embodiment, the oligomer has the following formula III:

Where

q is 1-50;

each of n (0), ..., n (q) is independently 0-10000;

each of m (1), ..., m (q) is independently 1-10000;

provided that the sum of n (0), ..., n (q) and m (1), ..., m (q) is 2-15000;

G represents a 5'-terminal group;

each Nu independently represents a nucleoside selected from natural nucleosides and nucleoside analogues;

each L-ribo-ZNA independently denotes a nucleoside analogue;

each L independently represents an internucleoside bond between two groups selected from Nu and L-ribo-ZNA, or L together with G * represents a 3'-terminal group; and each (L-ribo-ZNA) -L independently represents a nucleoside analog of the general formula I as defined above, or preferably the general formula Ia as defined above.

This option, as well as the whole invention, provides the possibility of incorporating L-ribo-LNAs with various nucleotide bases, in particular with nucleotide bases selected from thymine, cytosine and uracil, as well as with nucleotide bases selected from adenine and guanine. The oligomer may include, in one embodiment, at least one L-ribo-LNA, in which B (in formula I or Ia) is selected from the group consisting of adenine and guanine, and at least one L-ribo-LNA, in which B is selected from the group consisting of thymine, cytosine and uracil.

The present invention covers, in addition to the oligomers defined above, also monomeric L-ribo-ZNAs, used, for example, in the preparation of oligomers for the purpose of their further use as substrates, for example, nucleic acid polymerases, polynucleotide kinases, terminal transferases, and also as medicines (see below for more details). Monomeric L-ribo-LNAs (especially with regard to possible biradicals) correspond mainly to the structure of L-ribo-LNAs, defined as components of oligomers. However, with regard to the groups P and P ^* , the monomeric L-ribo-ZNAs are somewhat different from the case of occurrence as oligomer constituents, which will be explained later. In addition, monomeric L-ribo-LNAs may include protective functional groups, especially when monomeric L-ribo-LNAs are incorporated into oligomers by chemical synthesis.

The invention also relates to monomeric L-ribo-ZNA nucleosides of the general formula II:

where Deputy B is selected from nucleotide bases, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands; X is selected from —O—, —S—, —N (R ^{N *} ) - and —C (R ⁶ R ^{6 *} ) -, preferably from —O—, —S-, —N (R ^{N *} ) -;

each of Q and Q ^{* is} independently selected from hydrogen, azido, halogen, cyano, nitro, hydroxy, Prot-O-, Act-O-, mercapto, Prot-S-, Act-S-, C _{1-6 -alkylthio} , amino, Prot-N (R ^H ) -, Act-N (R ^H ) -, mono- or di (C _1-6 alkyl) amino, optionally substituted C _1-6 alkoxy, optionally substituted C _1-6 - alkyl, optionally substituted C _2-6 alkenyl, optionally substituted C _2-6 alkenyloxy, optionally substituted C _2-6 alkynyl, optionally substituted C _2-6 alkynyloxy, monophosphate, diphosphate, triphosphate, DNA intercalators, photochemically active groups thermochemically active RUPP, chelating groups, reporter groups, ligands, carboxy, sulphono, hydroxymethyl, Prot-O-CH ₂ -, Act-O-CH ₂ -, ^{aminomethyl, Prot-N (R H)} -CH ₂ -, Act-N ( R ^H ) —CH ₂ -, carboxymethyl, sulfonomethyl, where Prot is a protecting group for —OH, —SH and —NH (R ^H ), respectively. Act is an activating group for —OH, —SH and —NH (R ^H ), respectively, and R ^{H is} selected from hydrogen and C _1-6 alkyl;

R ^{2 *} and R ^{4 *} together denote a biradical selected from -O-, -S-, -N (R ^* ) -, - (CR * R *) _{r + s + 1} -, - (CR ^* R ^* ) _r -O- (CR ^* R ^* ) _s -, - (CR ^* R ^* ) _r -S- (CR ^* R ^* ) _s -, - (CR ^* R ^* ) _r -N (R ^* ) - (CR ^* R ^* ) _s -, -O- (CR ^* R ^* ) _{r + s} -O-, -S- (CR ^* R ^* ) _{r + s} -O-, -O- (CR ^* R ^* ) _{r + s} -S-, -N (R ^* ) - (CR ^* R ^* ) _{r + s} -O-, -O- (CR ^* R ^* ) _{r + s} -N (R ^* ) -, -S- (CR ^* R ^* ) _{r + s} -S-, -N (R ^* ) - (CR ^* R ^* ) _{r + s} -N (R ^* ) -, -N (R ^* ) - (CR ^* R ^* ) _{r + s} -S- and -S- (CR ^* R ^* ) _{r + s} -N (R ^* ) -, where R ^{* is as} defined above for oligomers; and each substituent from among R ^{1 *} , R ² , R ^{3 *} , R ⁵ and R ^{5 *} , which are not included in Q or Q ^* , is also defined above for oligomers.

Monomeric L-ribo-ZNAs also include their basic salts and acid addition salts.

In addition, it should be understood that any chemical group (including any nucleotide base) that is reactive under the conditions prevailing during the chemical synthesis of oligonucleotides, is an optionally functional group protected by methods known in the art. This position means that groups such as hydroxy, amino, carboxy, sulfono and mercapto groups, as well as nucleotide bases in monomeric L-ribo-ZNA, are optionally protected functional groups. Protection (and deprotection) is carried out using methods known to those skilled in the art (see, for example, Greene, T.W. and Wuts, PGM, "Protective Groups in Organic Synthesis", 2 ^nd ed., John Wiley, NY (1991) and MJ Gait, Oligonucleotide Synthesis, IRL Press, 1984).

Illustrative examples of hydroxy protecting groups are optionally substituted trityl (Tr), such as 4,4'-dimethoxytrityl (DMT), 4-monomethoxytrityl (MMT), and trityl, optionally substituted 9- (9-phenyl) xanthenyl (pixel), optionally substituted ethoxycarbonyloxy, p-phenylazophenyloxycarbonyloxy, tetrahydropyranyl (tgp), 9-fluorenylmethoxycarbonyl (Fmoc), methoxytetrahydropyranyl (mtgp), silyloxy such as trimethylsilyl (TMS), triisopropylsilyl (TIPSyl) trityl, trimethyl, trimethyl, trimethyl, trimethyl, trimethyl), phenyldimethylsilyl as well as eff benzyloxycarbonyl or substituted benzyloxycarbonyl such as 2-bromobenzyloxycarbonyl, tert-butyl ethers, alkyl ethers such as methyl ether, acetals (including two hydroxy groups), acyloxy such as acetyl or halogen-substituted acetyls, for example, chloroacetyl or fluoroacetyl, isobutyryl, pivaloyl, benzoyl and substituted benzoyl, methoxymethyl (MOM), benzyl esters or substituted benzyl esters such as 2,6-dichlorobenzyl (2,6-Cl ₂ Bzl). Alternatively, the hydroxy group may be protected by binding to a solid support, optionally via a linker.

Illustrative examples of protecting groups for amino groups include Fmoc (fluorenylmethoxycarbonyl), BOC (tert-butyloxycarbonyl), trifluoroacetyl, allyloxycarbonyl (alloc, AOC), benzyloxycarbonyl (Z, Cbz), substituted benzyloxycarbonyl, 2-chlorobenzyloxymethoxycarbonyl) (MMT), dimethoxytrityl (DMT), phthaloyl and 9- (9-phenyl) xanthenyl (pixel).

Illustrative examples of carboxy protecting groups include allyl esters, methyl esters, ethyl esters, 2-cyanoethyl esters, trimethylsilylethyl esters, benzyl esters (Obzl), 2-adamantyl esters (O-2-Ada), cyclohexyl esters (OsNech), 1,3-oxazolines, oxazolera, 1,3-oxazolidines, amides or hydrazides.

Illustrative examples of protecting groups for mercapto groups are trityl (Tr), acetamidomethyl (acm), trimethylacetamidomethyl (tacm), 2,4,6-trimethoxybenzyl (Tmob), tert-butylsulfenyl (StBu), 9-fluorenylmethyl (Fm), 3-nitro -2-pyridine sulfenyl (Npys) and 4-methylbenzyl (Meb).

In addition, it may be necessary or desirable to introduce a protective group for any nucleotide base included in the monomeric L-ribo-LNA, especially when the monomeric L-ribo-LNA is included in the oligomer according to the present invention. In the context of the present description, the term “protected nucleotide base” means that the nucleotide base in question carries a protecting group selected from groups known to those skilled in the art (see, for example, “Protocols for Oligonucleotides and Analogs”, vol. 20, (Sudhir Agrawal , ed.), Humana Press, 1993, Totowa, NJ; SL Beaucage and RPIyer, Tetrahedron, 1993, 49, 6123; SL Beaucage and RPIyer, Tetrahedron, 1992, 48, 2223; and E. Uhlmann and A. Peyman Chem. Rev. 90, 543.). Illustrative examples include benzoyl, isobutyryl, tert-butyl, tert-butyloxycarbonyl, 4-chlorobenzyloxycarbonyl, 9-fluorenylmethyl, 9-fluorenylmethyl-hydroxycarbonyl, 4-methoxybenzoyl, 4-methoxytriphenylmethyl, optionally substituted triazolo, p-toluenesol , isopropyl, optionally substituted amidines, optionally substituted trityl, phenoxyacetyl, optionally substituted acyl, pixel, tetrahydropyranyl, optionally substituted silyl ethers and 4-methoxybenzyloxycarbonyl. Other relevant examples are disclosed in the literature (Chapter 1 in "Protocols for oligonucleotide conjugates", Methods in Molecular Biology, vol. 26, (Sudhir Agrawal, ed.), Humana Press, 1993, Totowa, NJ. And SL Beaucage and RPIyer, Tetrahedron, 1992, 48, 2223).

In a preferred embodiment of the invention, group B for monomeric L-ribo-ZNA is preferably selected from nucleotide bases and protected nucleotide bases.

In one embodiment of the present invention, in the monomeric L-ribo-LNA of the present invention, one of Q and Q *, preferably Q *, is a group selected from Act-O-, Act-S-, Act-N (R ^H ) - , Act-O-CH ₂ -, Act-S-CH ₂ -, Act-N (R ^H ) -CH ₂ -, and the other of Q and Q ^* , preferably Q, represents a group selected from hydrogen, azido, halogen , cyano, nitro, hydroxy, Prot-O-, mercapto, Prot-S-, C _1-6 -alkylthio, amino, Prot-N (R ^H ) -, mono- or di (C _1-6 -alkyl) amino optionally substituted C _1-6 -alkoxy, optionally substituted C _1-6 alkyl, optionally substituted C _1-6 -alkenyl, optionally substituted C _1-6 -alkeniloksi optionally substituted C _2-6 -alkynyl, optionally substituted C _2-6 -alkiniloksi, monophosphate, diphosphate, triphosphate, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands , carboxy, sulfono, hydroxymethyl, Prot-O-CH ₂ -, aminomethyl, Prot-N (R ^H ) -CH ₂ -, carboxymethyl, sulfonomethyl, and R ^H are selected from hydrogen and C _1-6 alkyl.

In the above embodiment, the Prot group is a protecting group for —OH, —SH and —N (R ^H ), respectively. Such protecting groups are selected from the same groups as indicated above with respect to protecting groups for hydroxy, protecting groups for mercapto and protecting groups for amino, respectively, however, taking into account the need for a stable and reversible protecting group. However, it is preferred that any protecting group for —OH be selected from optionally substituted trityl such as dimethoxytrityl (DMT), monomethoxytrityl (MMT) and trityl, as well as 9- (9-phenyl) xanthenyl (pixel), optionally substituted, tetrahydropyranyl (tgp) (other suitable hydroxy protecting groups for phosphoramidite oligonucleotide synthesis are described in the literature: Agrawal, ed. "Protocols for Oligonucleotide Conjugates"; Methods in Molecular Biology, vol. 26, Humana Press, Totowa, NJ (1994) and Protocols for Oligonucleotides and Analogs, vol. 20, (Sudhir Agrawal, ed.), Humana Press, 1993, Totowa, NJ), or to be protected an acetal; and also that any protecting group for —SH be selected from trityl such as dimethoxytrityl (DMT), monomethoxytrityl (MMT), and trityl, as well as 9- (9-phenyl) xanthenyl (pixel) optionally substituted, tetrahydropyranyl (thp) (other suitable protecting groups for mercapto in phosphoramidite synthesis of oligonucleotides are also described in the literature (Agrawal, see above)); and that any protecting group for —NH (R ^H ) is selected from trityl, such as dimethoxytrityl (DMT), monomethoxytrityl (MMT) and trityl, as well as 9- (9-phenyl) xanthenyl (pixel), optionally substituted, tetrahydropyranyl (mpr) ) (other suitable protecting groups for the amino group in the phosphoramidite synthesis of oligonucleotides are also described in Agrawal (see above)).

In the aforementioned embodiment of the invention, as well as in the case of any monomeric L-ribo-LNA defined in the present description, Act denotes an activating group for —OH, —SH and —NH (R ^H ), respectively. Such activating groups are selected, for example, from optionally substituted O-phosphoramidite, optionally substituted O-phosphoroester, optionally substituted O-phosphodiester, optionally substituted H-phosphonate and optionally substituted O-phosphonate.

As used herein, the term “phosphoramidite” means a group of the formula —P (OR ^x ) —N (R ^Y ) ₂ , where R ^x is an optionally substituted alkyl group, for example methyl, 2-cyanoethyl or benzyl, and each R ^y is optionally substituted alkyl groups, for example ethyl or isopropyl, or the group —N (R ^y ) ₂ forms a morpholino group (—N (CH ₂ CH ₂ ) ₂ O). R ^{x is} preferably 2-cyanoethyl and two R ^{Y are} preferably identical and are isopropyl. Thus, a particularly suitable phosphoramidite is N, N-diisopropyl-O- (2-cyanoethyl) phosphoramidite.

It should be understood that the protecting groups used in the present invention for one monomeric L-ribo-LNA or several monomeric L-ribo-LNAs can be selected so that when said / indicated L-ribo-LNAs are included in the oligomer according to the present invention, either the simultaneous removal of protective groups, or the sequential removal of protective groups from functional groups. The latter situation opens up the possibility of regioselective administration of one or more “active / functional” groups, such as DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands, and such groups can be attached via a spacer as defined above.

In a preferred embodiment of the invention, Q is selected from hydrogen, azido, halogen, cyano, nitro, hydroxy, Prot-O-, mercapto, Prot-S-, C _1-6 -alkylthio, amino, Prot-N (R ^H ) -, mono- or di (C _1-6 alkyl) amino, optionally substituted C _1-6 alkoxy, optionally substituted C _1-6 alkyl, optionally substituted C _2-6 alkenyl, optionally substituted C _2-6 alkenyloxy, optionally substituted C _2-6 alkynyl, optionally substituted C _2-6 alkynyloxy, monophosphate, diphosphate, triphosphate, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands, carboxy, sulfono, hydroxymethyl, Prot-O-CH ₂ -, aminomethyl, Prot-N (R ^H ) -CH ₂ -, carboxymethyl, sulfonomethyl, where Prot denotes a protective group for - OH, —SH and —NH (R ^H ), respectively, and R ^H are selected from hydrogen and C _1-6 alkyl; and Q ^* are selected from hydrogen, azido, halogen, cyano, nitro, hydroxy, Act-O-, mercapto, Act-S-, C _1-6 -alkylthio, amino, Act-N (R ^H ) -, mono- or di (C _1-6 alkyl) amino, optionally substituted C _1-6 alkoxy, optionally substituted C _1-6 alkyl, optionally substituted C _2-6 alkenyl, optionally substituted C _2-6 alkenyloxy, optionally substituted C _2-6 -alkynyl, optionally substituted C _2-6 -alkiniloksi, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands, carboxy, sulphono, rD Act represents an activating group for -OH, -SH and -NH (R ^H), respectively, and R ^H is selected from hydrogen and C _2-6 -alkyl.

Monomeric L-ribo-ZNAs of the general formula II can, like the L-ribo-ZNAs included in the oligomers, have various stereoisomers. Thus, the stereochemical variants described above for L-ribo-LNAs included in oligomers are equally acceptable for the case of monomeric L-ribo-LNAs (however, it should be noted that P must then be replaced by Q).

In a preferred embodiment, monomeric ZNA has the general formula IIa:

where the substituents are defined above.

In addition, with regard to the determination of substituents, biradicals, R ^* and others, they include the same preferred options as were defined above for the oligomer according to the present invention, they are also applicable in the case of monomeric L-ribo-ZNAs.

In a particularly interesting embodiment, in the monomeric L-ribo-LNAs of the present invention, B is a nucleotide base, preferably a nucleotide base selected from thymine, cytosine, uracil, adenine and guanine (especially from adenine and guanine), X is —O—, R ^{2 *} and R ^{4 *} together denote a biradical selected from - (CH ₂ ) _0-1 -O- (CH ₂ ) _1-3 -, - (CH ₂ ) _0-1 -S- (CH ₂ ) _{1- 3} - and - (CH ₂ ) _0-1 -N (R ^N ) - (CH ₂ ) _1-3 -, in particular -O-CH ₂ -, -S-CH ₂ - and -R ^N -CH ₂ - where R ^{N is} selected from hydrogen and C _1-4 alkyl, Q is Prot-O-, Q * is Act-OH, and R ^{1 *} , R ² , R ³ *, R ⁵ and R ^{5 *} , each denotes water b. In this embodiment, R ^N may also be selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands.

In an even more interesting embodiment, in the monomeric L-ribo-LNAs of the present invention, B is a nucleotide base, preferably a nucleotide base selected from thymine, cytosine, uracil, adenine and guanine (especially adenine and guanine), X is —O -, R ^{2 *} and R ^{4 *} together denote a biradical selected from - (CH ₂ ) _0-1 -O- (CH ₂ ) _1-3 -, - (CH ₂ ) _0-1 -S- (CH ₂ ) _1-3 - and - (CH ₂ ) _0-1 -N (R ^N ) - (CH ₂ ) _1-3 -, in particular -O-CH ₂ -, -S-CH ₂ - and -R ^N -CH ₂ -, where R ^{N is} selected from hydrogen and C _1-4 alkyl, Q is selected from hydroxy, mercapto, C _1-6 alkylthio, amino, mono or di (C _1-6 -alkyl) amino, optionally substituted C _1-6 alkoxy, optionally substituted C _2-6 alkenyloxy, optionally substituted C _2-6 alkynyloxy, monophosphate, diphosphate and triphosphate, Q ^{* is} selected from hydrogen, azido, halogen, cyano, nitro, hydroxy, mercapto, C _1-6 alkylthio, amino, mono or di (C _1-6 alkyl) amino, optionally substituted C _1-6 alkoxy, optionally substituted C _1-6 alkyl, optionally substituted C _2-6 alkenyl, optionally substituted C _2-6 alkenyloxy, optionally substituted C _2-6 alkynyl and optionally substituted C _2-6 alk inyloxy, R ^{3 * is} selected from hydrogen, optionally substituted C _1-6 alkyl, optionally substituted C _2-6 alkenyl and optionally substituted C _2-6 alkynyl, and R ^{1 *} , R ² , R ⁵ and R ^{5 *} each denotes hydrogen. In the context of the present description, R ^N can also be selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands.

One object of the present invention relates to various derivatives of L-ribo-ZNA for inclusion in the oligomer in the solid phase and / or in the phase of the solution. Illustrative examples of monomers suitable for incorporating (1R, 3R, 4S, 7R) -7-hydroxy-1-hydroxymethyl-3- (thymin-1-yl) -2,5-dioxabicyclo [2.2.1] heptane, (1R, 3R, 4S, 7R) -7-hydroxy-1-hydroxymethyl-3- (cytosin-1-yl) -2,5-dioxabicyclo [2.2.1] heptane, (1R, 3R, 4S, 7R) -7-hydroxy -1-hydroxymethyl-3- (uracil-1-yl) -2,5-dioxabicyclo [2.2.1] heptane, (1R, 3R, 4S, 7R) -7-hydroxy-1-hydroxymethyl-3- (guanine- 1-yl) -2,5-dioxabicyclo [2.2.1] heptane and (1R, 3R, 4S, 7R) -7-hydroxy-1-hydroxymethyl-3- (adenin-1-yl) -2,5-dioxabicyclo [2.2.1] heptane using the phosphoramidite approach, the phosphate ester approach and the H-phosphonate approach are, with responsible, (1R, 3R, 4S, 7R) -7- (2-cyanoethoxy (diisopropylamino) phosphinoxy) -1- (4,4'-dimethoxytrityloxymethyl) -3- (thymin-1-yl) -2,5-dioxabicyclo [2.2.1] heptane, (1R, 3R, 4S, 7R) -7-hydroxy-1- (4,4'-dimethoxytrityloxymethyl) -3- (thymin-1-yl) -2,5-dioxabicyclo [2.2. 1] heptan-7-O- (2-chlorophenylphosphate) and (1R, 3R, 4S, 7R) -7-hydroxy-1- (4,4'-dimethoxytrityloxymethyl) -3- (thymin-1-yl) -2 , 5-dioxabicyclo [2.2.1] heptan-7-O- (N-phosphonate) and their 3- (cytosin-1-yl), 3- (uracil-1-yl), 3- (adenin-1-yl ) and 3- (guanin-1-yl) analogues, respectively. In addition, analogues in which methyleneoxy diradical monomers are substituted with methylentio, methyleneamino or 1,2-ethylene diradical will most likely also be particularly interesting embodiments of the present invention. It is assumed that the analogues of methylentio and methyleneamino will be applicable to the same extent as the analog of methyleneoxy, and in this regard, the specific reagents needed to include the indicated (1R, 3R, 4S, 7R) -7-hydroxy-1-hydroxymethyl-3- (thymin-1-yl) -2,5-dioxabicyclo [2.2.1] heptane, (1R, 3R, 4S, 7R) -7-hydroxy-1-hydroxymethyl-3- (cytosin-1-yl) -2, 5-dioxabicyclo [2.2.1] heptane, (1R, 3R, 4S, 7R) -7-hydroxy-1-hydroxymethyl-3- (uracil-1-yl) -2,5-dioxabicyclo [2.2.1] heptane, (1R, 3R, 4S, 7R) -7-hydroxy-1-hydroxymethyl-3- (guanin-1-yl) -2,5-dioxabicyclo [2.2.1] heptane and (1R, 3R, 4S, 7R) - 7-hydroxy-1-hydroxymethyl-3- (adenin-1-yl) -2,5-wild sabicyclo [2.2.1] heptane should also be considered as particularly interesting reactive monomers according to the present invention. Regarding methyleneamine analogs, it should be noted that the secondary amine may carry a substituent selected from optionally substituted C _1-6 alkyl, such as methyl and benzyl, optionally substituted C _1-6 alkylcarbonyl, such as trifluoroacetyl, optionally substituted arylcarbonyl and optionally substituted heteroarylcarbonyl .

Also, as a separate aspect of the present invention, an embodiment of formula II or IIa is interesting, wherein B is a “β-configuration”.

Preparation of Monomers

In a preferred embodiment, an α-L-ribo-ZNA containing a 2′-O, 4′-C-methylene bridge is synthesized using the following procedure: benzoylation of 4-C-hydroxymethyl-α-D-xylofuranose 1 (TFTam and B. Fraser-Ried, Can.J. Chem., 1979, 57, 2818) gives the di-O-benzoyl derivative 2, which is then converted to 1,2-di-O-acetylated furanose 3 by acetolysis using 80% acetic acid acid followed by acetylation. Using a modified Forbruggen technique (H. Vorbrüggen, K. Krolikiewicz and B. Bennua, Chem. Ber., 1981, 114, 1234; H. Vorbrüggen and G. Höfle, Chem. Ber., 1981, 114, 1256) thymine nucleoside in the β-configuration 4 are obtained in stereoselective mode by in situ silylation of thymine and binding with the participation of trimethylsilyl triflate. Treatment of compound 4 with sodium methoxide leads to deacetylation with the formation of a nucleoside triol 5. Protection with 4,4'-dimethoxytrityl followed by tosylation gives the 5'-O-4,4'-dimethoxytrityl-protected nucleoside derivative 7. Ring closure induced by the base, yields a bicyclic nucleoside derivative 6. Debenzylation results in a bicyclic nucleoside analog 9, which is transformed into phosphoramidite derivative 10 for oligonucleotide synthesis. The binding technique used in this example is only one of several possible approaches, which is obvious to specialists in this field.

As an alternative synthesis route, the procedure shown in FIG. 3 (Examples 12-14) can be used. In accordance with it, nucleoside 5 is trimesylated to obtain nucleoside 11, which can then be cyclized with NaOH / EtOH / H ₂ O. Under the conditions of the experiment, the remaining mesyloxy group is converted to a hydroxyl group to form nucleoside 12. Standard DMT protection shown in example 14 should lead to the formation of nucleoside 8, a suitable intermediate for the synthesis of phosphoramidite derivative of α-L-ribo-ZNA nucleoside 10 (figure 2).

The following examples are given to illustrate the procedures and examples of the present invention. The structures of the synthesized compounds were confirmed using 1D NMR.

The methods depicted in Schemes 1, 2, and 3 can similarly be used to synthesize α-L-ribo-ZNA nucleoside derivatives of other pyrimidine bases other than thymine, for example, uracil, cytosine, 5-substituted uracil, 5-substituted cytosine, as well as otherwise substituted pyrimidines. Alternatively, uracil derivatives can be converted to the corresponding cytosine derivatives and thymine derivatives can be converted to the corresponding 5-methylcytosine derivatives using known methods (Koshkin, AA, Singh, SK, Nielsen, P., Rajwanshi, VK, Kumar, R., Meldgaard, M., Oisen, CE, Wengel, J. Tetrahedron 1998, 54, 3607; Obika, S., Nanbu, D., Hari, Y., Andoh, J., Morio, K., Doi, T., Imanishi, T. Tetrahedron Lett. 1998, 39, 5401).

For the synthesis of α-L-ribo-LNA nucleoside derivatives of purine, many suitable synthesis techniques can be used. It should be noted that the term “α-side” in the context used below refers to the α-side of natural nucleoside RNA monomers, and the term “β-side” in the context used below refers to the β-side of natural RNA nucleoside monomers, and the terms “β- purine nucleoside "and β-pyrimidine nucleoside" mean that these nucleotide bases are located, as in the natural nucleoside monomers of RNA. As an example of possible routes for the synthesis of α-L-ribo-ZNA nucleoside derivatives of purine, one can point to a procedure involving cyclization of analogs in the arabino configuration (2'-OH group is located on the β-side of the furanose ring). These nucleosides can be obtained from the corresponding starting nucleosides in the arabino configuration via 5'-oxidation, aldol condensation and reduction.

For the purposes of the above desired cyclization, conditions must be prepared for the manipulations associated with the introduction of the protective group and the activation of the 5'-OH group (located on the β-side of the furanose ring). Alternatively, the procedure of 2'-oxidation of the 2'-OH group to 4'-C-hydroxymethyl derivatives of β-purine ribofuranosyl nucleosides (with the location of 2'-OH in them and 3'-OH groups on the α-side of the furanose ring and with 3'-OH located on the β-side of the furanose ring (or alternatively, on the α-side of the furanose ring) and with concomitant inversion at C3 '), followed by selective reduction ( using, for example, NaBH ₄ ). Then, to carry out the above desired cyclization, conditions should be prepared for the manipulations associated with the introduction of the protective group and activation of the 5'-OH group (located on the β-side of the furanose ring). Other procedures may be useful for inverting the configuration of the 2'-carbon atom in 4'-C-hydroxymethyl derivatives of β-purine ribofuranosyl nucleosides (when 2'-OH and 3'-OH groups are located on the α-side of the furanose ring and with a 3'-OH group located on the β-side of the furanose ring, or alternatively, on the α-side, when performing a converse inversion at the C3 'furanose ring), for example, the Mitsunobu reaction or nucleophilic substitution reactions of 2'-O-activated derivatives (e.g. , 2'-O-mesyl, 2'-O-tosyl and whether 2'-O-trifluoromethanesulfonyl derivatives) to O-nucleophiles such as acetate, benzoate, alkoxide, etc. Subsequent removal of the protective group to obtain the 5'-hydroxy-4'-C-hydroxymethyl derivative, activation in order to prepare for cyclization (for example, by mono- or dimesylation, mono- or ditosylation or mono- or dithrifluoromethanesulfonation), cyclization (after removal, if necessary, of the protective group from the 2'-OH group) and removal of the protective groups lead to the desired α-L-ribo nucleosides. It should be noted that the protection of purine nucleosides should preferably be carried out in the target monomers and that this procedure can be carried out during the selected synthesis route or, as a final step of the process, by trimethylsilylation to protect the amino group of purine bases and desilylation. The original 4'-C-hydroxymethyl derivatives of β-purine nucleosides can be obtained, within one embodiment of the invention, by coupling the furanose derivative 3 (FIG. 1) with suitably protected adenine or guanine derivatives in accordance with known Forbruggen binding methods ( Vorbrüggen type) (see, for example, nucleoside 4 synthesis, FIG. 1) (Koshkin, AA, Singh, SK, Nielsen, P., Rajwanshi, VK, Kumar, R., Meldgaard, M., Olsen, CE, Wengel , J. Tetrahedron 1998, 54, 3607).

It can be assumed that the inversion of the above configuration can be carried out on the basis of natural β-purine ribofuranosyl nucleosides (when the 2'-OH group is located on the α-side of the furanose ring and the 3'-OH group on the β-side of the furanose ring, or, alternatively, on the α-side, with concomitant inversion at the C3 ′ furanose ring) with the introduction of an additional 4′-C-hydroxymethyl group, using further known procedures, in particular the ones mentioned above. It can be expected that either enzymatic or chemical transglycosylation reactions carried out on the basis of the corresponding derivatives of protected pyrimidine nucleosides, or β-pyrimidine furanosyl nucleosides in the arabino configuration, or 4'-C-hydroxymethyl-β-pyrimidine furanosyl nucleosides in the arabino configuration, or the already cyclized α-L-ribo-LNA pyrimidine nucleosides belong to possible routes for the synthesis of α-L-ribo-LNA derivatives of purine nucleosides. Alternatively, starting from furanose or hexose, 4-C-hydroxymethylation, configuration inversion at the 2-carbon atom and cyclization, or one of these procedures, or two of these procedures (which is necessary, depends on the starting material) are carried out. Then, before or after cyclization, in order to obtain nucleoside derivatives used in the synthesis of α-L-ribo-ZNA of pyrimidine or purine nucleosides, after necessary manipulations to protect and / or activate the OH group, binding is carried out with various bases (purines or pyrimidines , if necessary, protected). As another procedure that can be used to synthesize α-L-ribo-LNA of pyrimidine or purine nucleosides, one can indicate the possibility of the direct formation of the desired nucleotide bases, carried out in two or more chemical steps) from the corresponding furanosyl derivative, for example, furanosylamine.

In a preferred embodiment of the invention, the procedures described in Examples 15, 16 and 17 (FIG. 4) can be used to obtain purine α-L-3HK monomers, for example derivatives of adenine and guanine. Thus, sugar 3 can be combined with N-6-benzoyladenine to form nucleoside 13, which is subjected to selective deacetylation and then converted to the 2'-O-trifluoromethanesulfonyl derivative. The concomitant reaction with potassium acetate gives the 2'-O-acetyl derivative 14 with inversion at C2 '. After complete deacylation, subsequent removal of the protective group from the adenine group, selective mesylation of two primary hydroxyl groups and treatment with sodium hydroxide in a mixture of water: dioxane, an α-L-SHK derivative of an adenine nucleoside 15 is obtained. DMT protection of nucleoside 15 followed by debenzylation and 3 ' -O-phosphitylation (Koshkin, AA, Singh, SK, Nielsen, P., Rajwanshi, VK, Kumar, R., Meldgaard, M., Olsen, CE, Wengel, J. Tetrahedron 1998, 54, 3607) is one of the possible ways to obtain phosphoramidite derivative 16. Debenzylation 15 followed by sat The objective DMT protection of the primary hydroxyl group and 3'-O-phosphitylation indicate another variant of the formation of the phosphoramidite derivative 16.

All methods and procedures described above with respect to the synthesis of α-L-ribo-LNA of purine nucleosides are also acceptable as alternative methods for the synthesis of α-L-ribo-LNA of pyrimidine nucleosides.

The methods described above for the synthesis of α-L-ribo-LNA of pyrimidine and purine nucleosides lead quite naturally to the methods used for the synthesis of 2'-amino and 2'-thio derivatives of α-L-ribo-LNA nucleosides. As an example, we can point to the cyclization procedure through the action of the 2'-amino or 2'-thio group located on the β-side of the furanose ring on the correspondingly activated 5'-OH group, which must be carried out to form 2'-amino and 2'-thio derivatives of α-L-ribo-ZNA pyrimidine or purine nucleosides. Alternatively, cyclization through the action of the 5'-amino or 5'-thio group located on the P-side of the furanose ring on the suitably activated 2'-OH group located on the β-side of the furanose ring also leads to the formation of 2'-amino - and 2'-thio derivatives of α-L-ribo-ZNA of pyrimidine or purine nucleosides. As another acceptable method, one can indicate the participation in the formation of 2'-amino and 2'-thio derivatives of α-L-ZNA nucleosides of the cyclization process of appropriately activated, protected and spatially oriented derivatives, for example, 2'-O, 5'- O-dimesyl, 2'-O, 5'-O-ditosyl or 2'-O, 5'-O-difluoromethanesulfonyl nucleosides using amino or thio-containing nucleophiles (e.g., benzylamine and potassium thioacetate, respectively). Similarly, the effect of the 5'-OH group located on the β-side of the furanose ring on a suitably activated 2'-OH group located on the α-side of the furanose ring should lead to the formation of the parent α-L-ribo-ZNA pyrimidine or purine nucleosides.

It is expected that any method used to oligomerize α-L-ribo-LNA of pyrimidine nucleosides will also be successful in the case of α-L-ribo-LNA of purine nucleosides.

Alternatively, any known method used for automated synthesis or synthesis in the solution phase of oligonucleotides and analogs, for example, the phosphate ester method, the H-phosphonate method, or any variant of the phosphoramidite method used for oligomerization of pyrimidine nucleosides α-L-ribo-ZNA, will also be acceptable .

Obtaining oligomers

Linear, branched (M.GrotIi and BSSproat, J. Cheni. Soc., Chem. Commun., 1995, 495; RHE Hudson and MJ Damha, J. Am. Chem. Soc., 1993, 115, 2119; M. Von Büren, GV Petersen, K. Rasmussen, G. Brandenburg, J. Wengel and F. Kirpekar, Tetrahedron, 1995, 51, 8491) and ring (G. Prakash and ET Kool, J. Am. Chem. Soc., 1992 114, 3523) oligo and polynucleotides according to the present invention can be obtained using nucleic acid polymerization methods known to those skilled in the art of organic chemistry. Methods of phosphoramidite chemistry (SLBeaucage and RPIyer, Tetrahedron, 1993, 49, 6123; SL Beaucage and RPIyer. Tetrahedron, 1992, 48, 2223), as well as, for example, methods of H-phosphonate chemistry, phosphoric ether chemistry, or enzymatic synthesis methods. The standard binding conditions in the framework of the phosphoramidite approach were slightly changed due to the use of pyridine hydrochloride instead of 1H-tetrazole, since it is a more effective reagent for activating nucleoside phosphoramidites during oligonucleotide synthesis, which allows to prolong the binding period from 10 to 30 minutes.

After synthesis of the desired sequence, removal of the protective groups and separation from the solid support (separation from the solid support and removal of the protective groups is carried out using concentrated ammonia in methanol at room temperature for 12 hours) and subsequent purification with phase reversal using commercially available disposable cartridges ( which includes detritylation) get the finished oligomeric product. Alternatively, purification of L-ribo-ZNA oligonucleotides can be carried out using a single phase reversal HPLC system and / or by precipitation from ethanol or butanol. Capillary gel electrophoresis is used to confirm the purity and composition of the synthesized oligonucleotide analogues, however, phase reversal HPLC and MALDI-MS can also be used to confirm the purity and composition.

Basically, the present invention relates to the use of L-ribo-LNAs as defined herein for the purpose of producing L-ribo-LNA modified oligonucleotides. It should be understood that L-ribo-LNA modified nucleotides may include normal nucleosides (e.g., natural nucleosides, such as ribonucleosides and / or deoxyribonucleosides), as well as modified nucleosides that differ from certain nucleosides of the general formula II.

In addition, a solid support material that contains an optionally protected nucleotide base and optionally 5'-OH protected LNA immobilized on it is especially interesting as a material suitable for the synthesis of LNA modified oligonucleotides, in those cases when the LNA monomer is incorporated at 3'- the end. In this case, the solid support material is preferably CPG, for example, readily (commercially) available CPG material on which a 3'-functional, optionally launched nucleotide base and optionally 5'-OH protected ZNA are combined under the conditions specified by the supplier for this material. So, for example, the substrate BioGenex Universial CPG Support (BioGenex, USA) can be used. The 5'-OH protecting group may be, for example, a DMT group. The 3'-functional group should be selected taking into account the conditions applicable to the CPG material in question.

Applications

The present invention discloses a surprising discovery in which L-ribo-ZNA derivatives, when incorporated into partially modified oligonucleotides, reduce the affinity of these modified oligonucleotides for complementary DNA and RNA in comparison with unmodified oligonucleotides. However, when completely modified oligonucleotides are incorporated into fully L-ribo-LNAs, a sharp increase in hybridization properties is observed for complementary single-stranded DNA and single-stranded RNA. α-L-ribo-ZNA is a special variant of L-ribo-ZNA - in addition to the described properties, it has the ability to distinguish between hybrid RNA and DNA targets. Depending on the application, the use of fully modified L-ribo-ZNA oligonucleotides provides a promising opportunity to either significantly increase the affinity of a standard oligonucleotide without negatively affecting specificity (constant oligonucleotide size) or significantly increase specificity without negatively affecting affinity (decreasing oligonucleotide size) or carry out specific hybridization with the target RNA.

It is also believed that L-ribo-ZNA modified oligonucleotides, in addition to significantly enhanced hybridization abilities, demonstrate many useful physicochemical properties inherent in normal DNA and RNA oligonucleotides. This promising list includes excellent solubility, the reaction of LNA modified oligonucleotides to salts such as sodium chloride and tetramethylammonium chloride, which mimics the reaction of unmodified oligonucleotides, the ability of LNA modified oligonucleotides to act as primers for many polymerases, the ability of LNA modified oligonucleotides to act as a target primer amplification reactions using thermostable DNA polymerase, the ability of ZNA to modify of oligonucleotides act as a substrate for T4 polynucleotide kinase; the ability of biotinylated LNAs to capture sequence-specific PCR amplicons on a solid surface coated with streptavidin; the ability of immobilized LNAs of modified oligonucleotides to sequence-specific capture of amplicons and the very important ability of the oligonucleotide sequence-specific oligonucleotides to access oligonucleotides double-stranded DNA with chain invasion. In this regard, it is obvious to any specialist in this field that these new nucleoside analogs are an extremely useful tool for improving the overall technology of using oligonucleotides in the aspect of therapy, diagnosis and molecular biological research.

An object of the present invention is to provide monomeric L-ribo-ZNAs according to the present invention, which can be incorporated into oligonucleotides using procedures and equipment known to those skilled in the art of oligonucleotide synthesis.

Another object of the present invention is the production of fully or partially L-ribo-ZNA-modified nucleotides (oligomers) capable of hybridizing in a sequence-specific manner with complementary oligonucleotides to form either duplexes or triplexes with significantly higher affinity than the corresponding complexes formed by unmodified oligonucleotides.

Another object of the present invention is the use of fully modified L-ribo-ZNA oligonucleotides to achieve increased specificity of oligonucleotides without adversely affecting their affinity.

Another object of the present invention is to obtain fully or partially modified oligonucleotides, including L-ribo-ZNA, normal nucleosides and other nucleoside analogues.

Another object of the present invention is the use of high affinity of L-ribo-LNAs to create fully modified oligonucleotides with extremely high affinity, which are able to bind to their target sequences in the dsDNA molecule by "chain substitution".

Another object of the present invention is to obtain other classes of L-ribo-ZNAs, which, when incorporated into oligonucleotides, differ in affinity from their complementary nucleosides. This goal can be achieved, for example, by replacing the normal nucleotide bases G, A, T, C, and Y with derivatives having, for example, an altered picture of hydrogen bonds.

Another object of the present invention is to obtain L-ribo-ZNA modified oligonucleotides, which are characterized by greater resistance to nucleases than their unmodified partners.

Another object of the present invention is the production of L-ribo-ZNA modified oligonucleotides that, when hybridized, can distinguish between DNA and RNA targets. It was found when measuring T _PL that T _PL , α-L-ribo-ZNA in relation to complementary RNA oligonucleotides increases by 5.7 ° C due to the modification, compared with a value of only 2.7 ° C per modification, in relation to complementary DNA (as shown in example 11, table 3). α-L-ribo-ZNA oligonucleotides will thus have an increased affinity for RNA compared to DNA, which creates conditions under which α-L-ribo-ZNA will specifically hybridize with this RNA, but not with DNA having that same sequence of bases. The indicated ability to distinguish between RNA and DNA can be used in a variety of situations, described below.

Another object of the present invention is the production of L-ribo-LNA modified oligonucleotides that can mobilize PHKseH.

An additional object of the present invention is the production of L-ribo-ZNAs, which can function as substrates for DNA and RNA polymerases, thereby allowing analogues to either be included in the growing nucleic acid chain or act as chain terminators.

Another object of the present invention is to obtain L-ribo-ZNA, which can function as drugs. Many examples of nucleoside analogues used for therapeutic purposes are known, and similar derivatives of nucleoside analogues disclosed herein can be synthesized using procedures known in the literature (E. De Clercq, J. Med. Chem. 1995, 38, 2491; P. Herdewijn and E. De Clercq: Classical Antiviral Agents and Design of New Antiviral Agents. In: A Textbook of Drug Design and Development; Eds. P. Krogsgaard-Larsen, T. Liljefors and U. Madsen; Harwood Academic Publishers, Amsterdam 1996, p. 425; IKLarsen: Anticancer Agents. In: A Textbook of Drug Design and Development; Eds. P. Krogsgaard-Larsen, T. Liljefors and U. Madsen; Harwood Academic Publishers, Amsterdam, 1996, p. 460 )

Double-stranded RNA has been shown to have antiviral and tumor-suppressing activity (Sharp et al., Eur. J. Biochem. 230 (1): 97-103, 1995, Lengyel-P. Et al., Proc. Natl. Acad. Sci USA, 90 (13): 5893-5, 1993, and Laurent-Crawford et al., AIDS Res. Hum. Retroviruses, 8 (2): 285-90, 1992). It is likely that double-stranded LNAs can mimic the effect of therapeutically active double-stranded RNAs and, accordingly, such double-stranded LNAs have the potential for drug use.

In the context of the present description, the term "natural nucleic acid" refers to nucleic acids in the broadest sense, for example, to nucleic acids present in intact cells of any origin or viruses, or to nucleic acids released from such sources by chemical or physical methods, or nucleic acids obtained from such primary sources by amplification. The natural nucleic acid may be single-stranded, double-stranded or partially double-stranded and may be a relatively pure type or mixture of different nucleic acids. It can also be a component of a crude biological sample, including other nucleic acids and other cellular components. On the other hand, the term "synthetic nucleic acids" refers to any nucleic acid obtained by chemical synthesis.

The present invention also relates to the use of L-ribo-ZNA modified nucleotides in nucleic acid-based therapeutics, diagnostics, and molecular biology. L-ribo-ZNA modified oligonucleotides can be used to detect, identify, capture, characterize, quantify and fragment natural or synthetic nucleic acids and as agents that block translation and transcription in vivo and in vitro. In many cases, it may be of interest to attach various molecules to L-ribo-LNA modified oligonucleotides. These molecules can be attached to either end of the oligonucleotide, or they can be attached at one or more internal positions. Alternatively, they can be attached to the oligonucleotide via spacers attached to the 5 ′ or 3 ′ end. Representative groups of such molecules include DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands. Basically, all methods for labeling unmodified oligonucleotides in DNA and RNA using these molecules can also be used to label L-ribo-ZNA modified oligonucleotides. And similarly, all methods used to detect labeled oligonucleotides are generally applicable for appropriately labeled L-ribo-LNA modified oligonucleotides.

Thus, L-ribo-ZNA modified oligonucleotides can be used to label cells, so that these labels can distinguish or separate such cells from unlabeled cells.

Treatment

The term “chain substitution” refers to a method by which an oligonucleotide binds to its complementary target sequence in double-stranded DNA or RNA, such that another strand is displaced from said target strand.

In one aspect of the present invention, L-ribo-LNA modified nucleotides capable of "chain substitution" are used in the development of new pharmaceuticals based on the "antigenic" approach. In contrast to oligonucleotides capable of forming triple helices, such oligonucleotides used to “replace the chain” allow any sequence in dsDNA to be achievable as a target at any physiological values of ionic strength and pH.

The ability of oligonucleotides to "replace the chain" can also lead to success in the framework of the approach based on the use of the antisense sequence, in those cases where the target RNA sequence is unavailable due to the presence of intramolecular hydrogen bonds. Such intramolecular structures can be found in mRNA and can cause serious problems when trying to “turn off” the translation of mRNA as part of an antisense approach.

Other classes of cellular RNA, such as, for example, tRNA, rRNA, snRNA and mcRNA, include intramolecular structures that are important for their functioning. These classes of highly structured RNAs do not encode proteins, but rather (in the form of RNA / protein particles) are involved in a wide range of different cellular functions, such as mRNA splicing, polyadenylation, translation, editing, maintaining the integrity of chromosome ends, etc. Due to the high degree of structurization, which complicates or even hinders effective hybridization in the case of normal oligonucleotides, these RNA classes did not attract attention as possible targets for antisense sequences. However, in light of the new results obtained using the described α-L-ribo-LNAs, the recognition of such RNAs using α-L-ribo-LNAs has become possible, as will be shown below.

It is known that many antibiotics interact with the bacterial ribosome and, in this regard, inhibit translation. It is known that certain antibiotics (e.g., streptomycin, tetracycline, spectinomycin, edein, hygromycin and neomycin) bind to specific sites of bacterial 163 rRNA (Moazed D. and Noller H.F., Nature, 1987, 327 (6121), 389). Similarly, other antibiotics (e.g., chloramphenicol, erythromycin, carbomycin, and vernamycin B) interact with specific sites of bacterial 23S rRNA (Moazed D. and Noller H.F., Biochimie, 1987, 69 (8), 879). A similar situation is likely to occur in higher organisms (Spangler E.A. and Elackburn E.H., J. Biol. Chem., 1985, 260 (10), 6334).

In addition, it is known that PNA-PNA (PNA - peptide nucleic acids, PNA) are molecules that specifically interact with DNA according to the Watson-Crick base pairing rule, which they do with slightly increased thermal stability ( _Tm ) with respect to functional and accessible sites in ribosomal RNA and can inhibit translation into Escherichia coli (Good L. and Nielsen PE, Proc. Natl. Acad. Sci. USA, 1998, 95 (5), 2073), indicating that high affinity oligonucleotides that bind to certain rRNA sites can mimic the effect of antibiotics, binding to rRNA. Since LNAs bind to rRNA with an even higher T _PL value than PNA, it is likely that it is possible to obtain LNAs that will specifically bind to bacterial rRNA and inhibit translation in bacteria. As a development of this approach, it should be pointed out that it is possible to use small but important differences in rRNA sequences between higher organisms to construct LNA oligonucleotides that inhibit translation in one of them, but not in the other. One of the obvious applications of this approach can be the development of ZNKs that specifically inhibit translation in Plasmodium species (malaria parasites), Schistosoma species (causing schistosomiasis), various filarias (causing elephantiasis and River Blindness), cryogenic heads (causing anemia), and other pathogenic parasites .

The use of high affinity L-ribo-ZNA monomers should facilitate the construction of antisense probes with sufficient thermal stability in order to efficiently hybridize with such target RNAs. In this regard, in a preferred embodiment of the invention, L-ribo-ZNAs are used to give sufficient affinity to the oligonucleotide in order to facilitate hybridization with these classes of RNAs by qualitative and / or quantitative modulation of the functioning of the particles in which these RNAs are located.

L-ribo-ZNA-modified oligonucleotides intended for use as drugs within the antisense mechanism of action are being developed with a double purpose: to achieve high affinity and the ability to mobilize PHKseH. These goals can be achieved, for example, due to the presence of L-ribo-ZNA segments flanking the unmodified central DNA segment. In addition, the special ability of α-L-ribo-ZNA to distinguish between RNA and DNA can be used in various major therapeutic applications of the antisense approach due to the preferred nature of the activity of α-L-ribo-ZNA in relation to RNA. When creating α-L-ribo-LNA oligonucleotides specific to a particular RNA, nonspecific binding to DNA fragments whose nucleotide sequence is similar to the target RNA is avoided, which prevents stable association of α-L-ribo-LNA oligonucleotides with chromosomal DNA, which could change the structure of DNA and thereby induce mutations in the gene in question. Said alteration of DNA structure and associated mutations can cause undesirable toxic side effects.

Another embodiment of the present invention relates to the construction of ribozymes with increased specificity. Ribozymes are oligodeoxyribonucleotides and their analogues that combine RNAase activity with the ability to sequence-specific interaction with complementary target RNA. They are of great interest as molecules of therapeutic action, and it is likely that the attractive properties of α-L-ribo-ZNA oligonucleotides can be used to improve the ribozymes acting against specific RNAs.

Another embodiment of the present invention relates to L-ribo-ZNA oligonucleotides that specifically interact with cellular nucleoproteins that contain RNA as an integrated and indispensable component of the active protein, two examples of which are ribosomes and telomerase. The ability of α-L-ribo-ZNA oligonucleotides to inhibit telomerase can be used in a number of important applications.

Chromosomes of higher eukaryotes (including humans) are linear. The primary structure (DNA sequence) of the chromosomal ends was determined, and it turned out that the DNA sequences of all chromosomal ends, in each particular organism, consist of a simple repeating unit with a protruding single-stranded end. The end of the chromosome is called the telomere. In humans, telomeres contain long strands of double-stranded multiple repeats of the 5'-TTAGGG-3 'sequence (a sequence of one chain in the direction from the centromere toward the end of the chromosome). Since all DNA polymerases require the presence of a template strand and oligonucleotide primer to initiate the synthesis of a complementary strand of DNA, DNA polymerase alone is not able to replicate the far ends of the chromosome. This leads to progressive shortening of chromosomes during their replication. When considering the length of telomeres in normal somatic cells, it can be seen that the telomere length actually becomes shorter with each replication cycle until the telomere size reaches 5-15 TPO in length. When telomeres become so short, the cells stop dividing normally and gradually enter the aging phase. The only exceptions to this are stem cells. Stem cells are specialized cells that can continue to divide throughout the life of the body. Interestingly, stem cell telomeres remain long (10-15 mps). They remain so due to the activity of a special enzyme - telomerase. Telomerase is a unique enzyme that is able to specifically extend the protruding single-stranded end of the telomere, thereby allowing the telomere to maintain a stable length. Telomerase is a ribonucleoprotein enzyme, i.e. it is a protein that contains RNA and is dependent on RNA in its enzymatic activity. The structure of telomerase somewhat resembles reverse transcriptase - a viral protein that is also able to synthesize DNA using RNA as a template.

The enzymatic effect of telomerase depends on the correct position of the free 3'-end of the telomere on the RNA molecule to extend the telomere. Molecules that are capable of specifically interacting with either the farthest end of the telomere, or possibly the RNA component of telomerase, will inhibit the enzyme. α-L-ribo-ZNA can be designed to meet such criteria. This approach may be of interest, for example, in the treatment of cancer, since, with the exception of stem cells, telomerase activity is not detected in normal somatic cells, which sharply distinguishes them from cancer cells, most of which contain easily detectable telomerase activity. Cancer cells are immortalized, i.e. they do not age, but continue to proliferate and form a tumor mass until the death of the body. Currently available data suggest that telomerase activity is required for the immortalization of cancer cells. Interestingly, cancer cell telomeres are significantly shorter than stem cell telomeres, and this fact indicates that cancer cells must reach the “telomere length barrier” earlier than stem cells, and it can be assumed that a drug that specifically inhibits telomerase activity , will be useful as an anti-cancer agent.

In light of the above, an important aspect is the use of the exceptional properties of α-L-ribo-ZNA oligomers to construct short α-L-ribo-ZNA oligomers directed against specific parts of the RNA component of telomerase, in order to inhibit the telomerase activity of human cancer cells.

Another embodiment of the present invention relates to the use of L-ribo-ZNA oligonucleotides, especially α-L-ribo-ZNA oligonucleotides, as aptamers. This new promising class of therapeutic oligonucleotides has been selected based on in vitro studies as specifically binding to this target with high affinity, such as, for example, ligand receptors. The binding characteristics of these molecules most likely reflect the ability of oligonucleotides to form three-dimensional structures held together by intramolecular pairing of nucleotide bases. It is likely that aptamers containing α-L-ribo-ZNA oligonucleotides can exhibit beneficial characteristics, which, in turn, can be used for therapeutic purposes.

Buchardt, О.; Egholm, М.; Nielsen, P.E. Ргос. Natl. Acad. Scl. U.S.A. 1994, 91, 3892), олигомеры, способные к «замещению цепи», могут функционировать как активаторы транскрипции РНК. В одном аспекте настоящего изобретения ЗНК, способные к «замещению цепи», используют для активации генов, интересных с точки зрения возможного лечения.In some cases, it may be beneficial to downregulate gene expression, while in other cases it may be beneficial to activate it. As shown by Mollegaard et al. (

Buchardt, O .; Egholm, M .; Nielsen, PE Proc. Natl. Acad. Scl. USA 1994, 91, 3892), oligomers capable of "chain substitution" can function as activators of RNA transcription. In one aspect of the present invention, LNAs capable of “chain replacement” are used to activate genes that are interesting in terms of possible treatment.

Nucleosides and nucleoside analogues have been shown to be effective in chemotherapy of various viral infections and cancer. L-ribo-ZNA nucleosides should be considered as potentially useful as such drugs of nucleoside nature.

There are reports that in some cases double-stranded RNA (ds-RNA) has a specific pharmaceutical activity. Duplexes, including fully L-ribo-LNA modified nucleoside (s), can be used as such double-stranded drugs and, in addition, it is likely that double-stranded α-L-ribo-LNA oligonucleotides will make an important contribution to the range of functions biologically active molecules such as double-stranded RNA.

Based on the foregoing, the therapeutic potential of double-stranded ZNA (ds-ZNA) may include the treatment of cancer or viral infections, which will be discussed below.

There is evidence that various types of DC-NK, either alone or in synergistic combination with gamma interferon, inhibit the growth of several types of cancer cells (Borecky et al. Tech. Rep. Blol. Med., 1981, 41 575; Sharp et al. Eur. J. Biochem., 1995, 230 (1), 97). DC-RNAs inhibit the growth of cancer cells in culture, as well as in tumors of experimental animals. Apparently, at least two enzymes activated by double-stranded RNA are involved in the tumor-suppressing activity of ds RNA: activated double-stranded RNA protein kinase (RCC) and ribonuclease L (Lengyel-P, Proc. Natl. Acad. Scl USA, 1993, 90 (13 ), 5893). While RCC is directly activated by dsRNA, RNAse L is activated by dsRNA via (2'-5 ') oligoadenylate synthetase, which remains latent until activated by dsRNA. Dc RNA also induces the activity of a natural killer cell (PC), and this activity also seems to contribute to the antitumor effect of dc RNA.

Despite the fact that natural ds-RNA is typically associated with a viral infection, it has been shown that ds-RNA also has antiviral activity. DC-RNA exhibits its antiviral activity against human immunodeficiency virus HIV-1 and HIV-2 (Haines et al. J. Cell. Biochem., 1991, 46 (1), 9). Ds-RNA and also ds-ZNA can, in this regard, be possible candidates for their use as medicines in the treatment of AIDS.

DC-RNA must prove its effectiveness in practice. However, mammalian cells contain many specific ds-RNA nucleases and, possibly, because of this, the indicated ds-RNA activity is rapidly eliminated in the patient’s body. ZNA is in many ways similar to RNA, and it is characterized by most of the chemical characteristics of RNA (Koshkin et al., Tetrahedron, 1998, 54, 3607), and in addition, ZNA forms stable duplexes, and the structural differences between RNA and ZNA are weakly distinguishable. Thus, it is likely that adequate double-stranded ZNAs can mimic the effects of some ds-RNAs and, accordingly, activate RCC and / or (2'-5 ') oligoadenylate synthetase, and since ZNA itself has been shown to have exonucleolytic activity (Singh et al ., Chem. Commun., 1998,455), it is possible that ds-ZNA molecules may exhibit improved therapeutic efficacy compared to ds-RNA.

The present invention also relates to a pharmaceutical composition comprising a pharmaceutically active L-ribo-LNA modified oligonucleotide or a pharmaceutically active L-ribo-LNA monomer, as defined above, in combination with a pharmaceutically acceptable carrier.

Such compositions may be in a form adapted for oral, parenteral (intravenous, intraperitoneal), intramuscular, rectal, intranasal, cutaneous, vaginal, buccal, ophthalmic or intrapulmonary administration, preferably in a form adapted for oral administration, and such compositions can be prepared by a method well known to those skilled in the art, in particular from those described in the literature ("Remington's Pharmaceutical Sciences", 17. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, U .S.A., 1985 and later, and in monographs of the Drugs and the Pharmaceutical Sciences series, Marcel Dekker).

Diagnostics

Several diagnostic and molecular biological procedures have been developed that use a set of different oligonucleotides to simultaneously analyze the target nucleic acid in order to assess the presence of possible mutations. Typically, sets of oligonucleotides are immobilized in a predetermined manner on a solid support, so that the presence of a particular mutation in the target nucleic acid can be detected by the position on the solid support on which the hybridization takes place. One of the important conditions for the successful use of a set of different oligonucleotides in nucleic acid analysis is that all of them must be specific for a particular target sequence under specific hybridization conditions. Since the affinity and specificity of standard oligonucleotides for their complementary target sequences substantially depends on their sequence and size, these criteria are difficult to fulfill.

In addition, many techniques have been developed so that the different types of RNA that the cells may contain can be characterized. Nucleic acid hybridization is a general approach for characterizing them, and examples of such hybridization methods include in situ hybridization, dot blot hybridization, dot blot hybridization, Northern hybridization, and reverse transcriptase polymerase chain reaction (PCR). Often, these methods are carried out on samples containing both DNA and RNA, which often creates problems in the determination, which are easy to avoid if you have probes in which an adequate separation of DNA and RNA has been carried out. In particular, this problem is characteristic of in situ hybridization using various tissue samples. Due to the pronounced hybridization preference for RNA α-L-ribo-LNA oligonucleotides for RNA, α-L-ribo-LNA oligonucleotides can be constructed that specifically hybridize with RNA in the sample, thereby eliminating the possibility of obtaining erroneous results in the case of hybridization with irrelevant DNA having the same nucleotide sequence.

In a preferred embodiment of the invention, L-ribo-ZNAs are used as a means of increasing the affinity and / or specificity of the probes, and also as a means of aligning the affinity of various oligonucleotides to their complementary sequences. As disclosed herein, such affinity modulation can be accomplished, for example, by replacing selected nucleotides in an L-ribo-ZNA oligonucleotide bearing a similar nucleotide base. In particular, this relates to α-L-ribo-ZNA ligonucleotides.

In another preferred embodiment of the present invention, the high affinity and specificity of L-ribo-LNA modified oligonucleotides is used to capture sequence-specific natural or synthetic nucleic acids and purify them. In one aspect, natural and synthetic nucleic acids are contacted with L-ribo-LNA modified oligonucleotide immobilized on a solid support. In this case, hybridization and capture occur simultaneously. In this case, the captured nucleic acid can, for example, be detected, characterized, quantified or amplified directly on the surface using a variety of methods known in the art, or it can be released from the surface prior to carrying out such characterization or amplification by placing an immobilized modified oligonucleotide and captured nucleic acid under conditions not conducive to hybridization, such as, for example, heating or using low io buffers Noah force.

The solid substrate can be selected from a wide range of polymeric materials, such as, for example, CPG (glass with controlled porosity), polypropylene, polystyrene, polycarbonate or polyethylene, each of these materials may have a different shape, such as, for example, a tube, microtiter plate, rod, ball, filter, etc. L-ribo-ZNA modified oligonucleotide can be immobilized on a solid support through its 5'- or its 3'-end (or through the end of linkers attached to the 5'- or 3'- end) using a lot of chemical scopic or photochemical methods usually employed in the immobilization of oligonucleotides or by non-covalent compound such as, for example, in the case of the binding of biotinylated L-ribo-ZNK modified oligonucleotide to immobilized streptavidin. One preferred method for immobilizing L-ribo-LNA modified oligonucleotides on various solid supports is a photochemical method using a photochemically active anthraquinone covalently attached to the 5'- or 3'-end of the modified oligonucleotide (optionally via linkers), as described in WO 96 / 31557. Thus, the present invention also relates to a surface bearing an LNA modified oligonucleotide.

In another aspect, the L-ribo-ZNA-modified oligonucleotide carries a ligand covalently attached to either the 5'- or 3'-end. In this case, the L-ribo-ZNA-modified nucleotide is contacted with natural or synthetic nucleic acids in solution, while the formed hybrids are captured on a solid carrier carrying molecules that can specifically bind the ligand.

In yet another aspect, L-ribo-ZNA-modified oligonucleotides capable of "chain substitution" are used to capture natural and synthetic nucleic acids without prior denaturation. Such modified oligonucleotides are particularly suitable for use in cases where it is difficult or impossible to reach the target sequence using normal oligonucleotides in connection with the rapid formation of stable intramolecular structures. Examples of nucleic acids including such structures / are rRNA, tRNA, snRNA and mcRNA.

In another preferred embodiment, L-ribo-ZNA-modified oligonucleotides designed to achieve high specificity are used as primers for nucleic acid sequencing and as primers for any of a number of known amplification reactions, such as PCR. As shown in the present description, the construction of L-ribo-ZNA-modified oligonucleotides allows you to determine whether they will support the target amplification in exponential or linear mode. The amplification reaction products can be further analyzed using a variety of methods applicable to the analysis of amplification products obtained with normal DNA primers. In the specific case, when the L-ribo-ZNA-modified oligonucleotide primers are designed to support linear amplification, the resulting amplicons will contain single-stranded ends that can be specific for complementary probes without denaturation. Such ends can, for example, be used to capture amplicons with other complementary L-ribo-ZNA-modified oligonucleotides attached to a solid surface.

In another aspect, L-ribo-ZNA-modified oligonucleotides capable of "chain replacement" are used as primers for a linear or exponential amplification reaction. The use of such oligonucleotides is expected to increase the overall output of amplicons due to effective competition with amplicon in repeated hybridization at later stages of the amplification reaction. Demers et al. (Demers et al., Nucl. Acid Res. 1995, 23, 3050-3055) discloses the use of high affinity, non-extendable oligomers as a means of increasing the overall yield of PCR. It is believed that oligomers exhibit these effects due to an obstacle to the re-hybridization of amplicon in the late stages of the PCR reaction. It is expected that L-ribo-ZNA-modified oligonucleotides blocked at its 3'-end will have the same advantage. Blocking the 3'-end can be achieved in various ways, such as, for example, exchanging the 3'-hydroxyl group for hydrogen or phosphate. Such 3'-blocked L-ribo-ZNA-modified oligonucleotides can also be used to selectively amplify very close nucleic acid sequences in a manner similar to that described by Yu et al. (Yu et al., Biotechnigues, 1997, 23, 714-716).

In recent years, new classes of probes have been invented that can be used, for example, to detect in real time the amplicons generated in target amplification reactions. One such class of probes has been called "molecular buoys" ("Molecular Beacons"). These probes are synthesized as partially self-complementary oligonucleotides, including a fluorophore at one end and a quencher molecule at the other end. In the free state in the solution, the probe is folded into a hairpin structure (under the control of self-complementary sites), in which the absorber is located in a position sufficiently close to the fluorophore to quench its fluorescent signal. Upon hybridization with its target nucleic acid, the hairpin opens, thereby separating the fluorophore and quencher and terminating the fluorescent signal.

Another class of probes was called "Taqman probes". These probes also include a fluorophore and a quencher molecule. However, unlike molecular buoys, the ability of absorbers to damp the fluorescent signal from the fluorophore is maintained after the probe hybridizes with its target sequence. Instead, the fluorescence signal is generated after hybridization by physically separating the quencher or fluorophore and probe using the 5'-exonuclease activity of the polymerase, which initiates the synthesis of a primer located in the 5'-direction to the Taqman probe binding site.

High affinity for the target site is an important feature of both types of probes, and therefore, such probes tend to be relatively large (typically from 30 to 40 measures). As a result of this, significant problems arise in the production of high-quality probes. In this regard, in a preferred embodiment of the invention, ZNKs are used to improve the formation and subsequent functioning of such Taqman probes and molecular buoys by reducing their size while maintaining the required affinity.

In yet another aspect, L-ribo-LNAs are used to construct novel affinity pairs (fully or partially modified oligonucleotides). Affinity constants can be easily adjusted over a wide range, and a large number of affinity pairs can be developed and synthesized. In this case, one part of affinity pairs can be attached to the molecule in question (for example, proteins, amplicons, enzymes, polysaccharides, antibodies, haptens, peptides, PNAs, etc.) using standard methods, while the other part of affinity pairs can be attached, for example, to a solid substrate, such as balls, membranes, microtiter plates, rods, tubes, etc. The solid substrate can be selected from a wide range of polymeric materials, such as, for example, polypropylene, polystyrene, polycarbonate or polyethylene en. Affinity pairs can be used in the selective isolation, purification, capture and detection of many different target molecules mentioned above.

The principle of capture of an L-ribo-LNA-labeled molecule through interaction with another complementary L-ribo-LNA oligonucleotide (either fully or partially modified) can be used to create an infinite number of new affinity pairs.

In another preferred embodiment of the invention, the high affinity and specificity of L-ribo-ZNA-modified oligonucleotides is used in the construction of probes used for in situ hybridization. So, for example, L-ribo-ZNA can be used to reduce the size of traditional DNA probes while maintaining the desired affinity by increasing the kinetics of the probe and its ability to penetrate into the sample.

Cleaning

In yet another embodiment, the present invention relates to the use of L-ribo-LNA oligonucleotides, in particular α-L-ribo-LNA oligonucleotides, in purification procedures specific for RNA. Methods conventionally used to isolate nucleic acids from prokaryotic cells, eukaryotic cells, or a complex of biological samples include the use of organic solvents such as phenol and chloroform. In this case, nucleic acid isolation typically starts with enzymatic cleavage of the sample with proteases, followed by lysis of the cell with ionic detergents, and then extraction with phenol and / or phenol / chloroform is carried out. The organic and aqueous phases are further separated, and the nucleic acids that have entered the aqueous phase are precipitated with alcohol. However, phenol or a phenol / chloroform mixture has a destructive effect on human skin and is a hazardous substance that must be handled very carefully and appropriately. In addition, standard extraction methods using phenol / chloroform isolation methods produce a mixture of RNA and DNA. In this regard, when carrying out the isolation of nucleic acids, it would be advantageous to use the ability of α-L-ribo-ZNA to distinguish between RNA and DNA, which will allow obtaining samples of pure RNA.

Sets

The present invention also relates to a kit for the isolation, purification, amplification, detection, identification, quantification or capture of natural or synthetic nucleic acids, said kit comprising a reaction substrate and one or more L-ribo-ZNA modified oligonucleotides (oligomer) as defined in the present description. L-ribo-ZNA modified oligonucleotides are preferably immobilized on said reaction substrate.

The present invention also relates to a kit for the isolation, purification, amplification, detection, identification, quantification or capture of natural or synthetic nucleic acids, said kit comprising a reaction substrate and one or more L-ribo-ZNAs as defined herein. L-ribo-ZNAs are preferably immobilized on said reaction substrates (for example, using the immobilization methods described above).

In the kits of the present invention, the reaction support is preferably a solid support material, for example selected from borosilicate glass, soda-lime glass, polystyrene, polycarbonate, polypropylene, polyethylene, polyethylene glycol terephthalate, polyvinyl acetate, polyvinyl pyrrolidinone, polymethyl methacrylate, and polyvinyl chloride . The reaction substrate may take the form of a tube for a sample, vial, plate, sheet, film, ball, small ball, disk, plate, ring, rod, network, filter, tray, microtiter plate, stick or stick with many blades.

Kits are typically accompanied by an insert with instructions that indicate the optimal conditions for using the kit.

EXPERIMENTAL PART

General directions

The reactions are carried out in a nitrogen atmosphere when anhydrous solvents are used. Column chromatography was performed on glass columns with silica gel 60 (0.040-0.063 mm). After column chromatography, the fractions containing the product were combined, evaporated to dryness under reduced pressure and dried in vacuo to give the product. After drying the organic phases with Na ₂ SO ₄ , filtering is carried out. Use petroleum ether in the distillation range of 60-80 ° C. Chemical shifts δ are expressed as parts per million (ppm) relative to tetramethylsilane used as the internal standard ( ¹ H and ¹³ C NMR) and relative to 85 ° H ₃ PO ₄ ( ³¹ P NMR). Microanalyses are performed at the Microanalytical Laboratory, Department of Chemistry, University of Copenhagen, in the microanalysis laboratory of the chemistry department of the University of Copenhagen.

The following specific descriptions are accompanied by figures 1-4 and tables 1-3.

Obtaining L-ribo-ZNA monomers

Example 1

5-O-benzoyl-4-C-benzoyloxymethyl-3-O-benzyl-1,2-O-isopropylidene-α-D-glucofuranose (2)

To an ice-cooled solution of 3-O-benzyl-4-C-hydroxymethyl-1,2-isopropylidene-α-D-glucofuranose (1) (5.00 g; 0.016 mol) in anhydrous pyridine (60 cm ³ ) was added benzoyl chloride (4.1 cm ³ ; 0.035 mol). After stirring at room temperature for 4 hours, the reaction mixture was cooled to 0 ° C, H ₂ O (50 cm ³ ) was added and the mixture was extracted with dichloromethane (100 cm ³ × 3). The combined organic phase was washed with saturated aqueous solutions of sodium bicarbonate (30 cm ³ × 3) and salt (20 cm ³ × 3), dried (Na ₂ SO ₄ ) and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography, using, first, petroleum ether / dichloromethane (1: 1, vol / vol) and then dichloromethane / methanol (99: 1, vol / vol) to obtain, after evaporation of the solvents under reduced pressure furanose 2 (7.50 g; 90 °) as a yellowish oil.

δ _H (CDCl ₃ ) 8.02-7.23 (15H, m), 6.08 (1H, d, J 4.2), 4.81-4.50 (7H, m), 4.22 ( 1H, d, J 1.0), 1.59 (3H, s), 1.37 (3H, s).

δ _c (CDCl ₃ ) 166.1, 165.8, 136.7, 133.1, 133.0, 129.9, 129.7, 129.6, 129.5, 128.5, 128.4, 128.3, 128.0, 127.9, 113.3, 105.4, 86.4, 85.1, 83.8, 72.3, 64.3, 63.8, 27.0, 26, 4. FAB-MS m / z 521 [M + H] ⁺ . Found (%) C, 69.1; H, 5.9; C ₃₀ H ₂₂ O ₈ , calculation C, 69.2; H, 6.2.

Example 2

5-O-benzoyl-4-C-benzoyloxymethyl-3-O-benzyl-1,2-di-O-acetyl-P-glucofuranose (3)

A solution of furanose 2 (7.40 g; 0.014 mol) in 80% acetic acid (60 cm ³ ) was stirred for 9 hours at 90 ° C. The mixture was evaporated to dryness under reduced pressure, and the residue was evaporated together with toluene (10 cm ³ × 3) and dissolved in anhydrous pyridine (80 cm ³ ). Acetic anhydride (5.5 cm ³ ) was added and the solution was stirred for 46 hours at room temperature. The mixture was evaporated to dryness under reduced pressure, and the residue was evaporated together with toluene (10 cm ³ × 3) and dissolved in dichloromethane (150 cm ³ ). The solution was washed with saturated aqueous solutions of sodium bicarbonate (30 cm ³ × 3) and salt (30 cm ³ × 3), dried (Na ₂ SO ₄ ) and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using, first, petroleum ether / dichloromethane (1: 1; vol / vol) and then dichloromethane / bristhanol (99: 1; vol / vol) to obtain, after evaporation of the solvents under reduced pressure anomer mixture 3 (α: β = 3: 1; 7.33 g; 92%) as a clear oil. The specified oil is used in the next step without further purification.

δ _s (CDCl ₃ ) 169.4, 169.0, 165.8, 165.6, 137.0, 133.2, 133.1, 133.0, 129.6, 129.5, 129.2, 128.3, 127.8, 127.7, 127.4, 99.4, 92.3, 87.0, 83.2, 82.2, 80.7, 77.4, 76.9, 76, 3, 73.2, 72.4, 20.9, 20.8, 20.6, 20.3. FAB-MS m / z 562 [M] ⁺ .

Example 3

1- (2-O-acetyl-5-O-benzoyl-4-C-benzoyloxymethyl-3-O-benzyl-β-D-xylofuranosyl) thymine (4)

To a stirred suspension of an anomeric mixture of 3 (7.26 g; 0.013 mol) and thymine (3.25 g; 0.028 mol) in anhydrous acetonitrile (80 cm ³ ) add N, O-bis (trimethylsilyl) acetamide (19.1 cm ³ ; 0.077 mol). The reaction mixture was stirred at 60 ° C for 1 hour and then cooled to 0 ° C. Trimethylsilyl triflate (4.1 cm ³ ; 0.023 mol) was added dropwise over 10 minutes and the mixture was subsequently heated for 22 hours at reflux temperature. After cooling to room temperature, saturated aqueous sodium hydrogen carbonate solution (30 cm ³ ) was added and extraction was carried out with dichloromethane (100 cm ³ × 3). The combined organic phase is washed with saturated aqueous solutions of sodium bicarbonate (30 cm ³ × 3) and salt (50 cm ³ × 3), dried (Na ₂ SO ₄ ) and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a dichloromethane / methanol mixture (0.5-2.0% methanol, vol / vol) as eluent to obtain, after evaporation of the solvents under reduced pressure, nucleoside 4 (6.88 g; 85%) in the form of a white solid material.

δ _H (CDCl ₃ ) 8.97 (1H, br s), 8.04-7.23 (16H, m), 6.37 (1H, d, J 3.6), 5.42 (1H, t, J 3.1), 4.89-4.56 (6H, m), 4.22 (1H, d, J 2.6), 2.13 (3H, s), 1.74 (1H, d, J 0.8).

δ _c (CDCl ₃ ) 169.9, 166.0, 165.7, 163.4, 150.4, 136.2, 135.2, 133.5, 133.4, 129.8, 129.7, 129.6, 129.5, 129.0, 128.6, 128.4, 128.2, 112.0, 87.4, 86.0, 81.3, 80.3, 72.6, 63, 1, 62.9, 20.8, 12.3. FAB-MS m / z 629 [M + H] ⁺ . Found (%) C, 64.4; H, 4.9; N, 4.4; C ₃₄ H ₃₂ N ₂ O ₁₀ , 25H ₂ O, Calcd. C, 64.5; H, 5.1; N, 4.4.

Example 4

1- (3-O-benzyl-4-C-hydroxymethyl-β-D-xylofuranosyl) thymine (5)

To a stirred solution of nucleoside 4 (9.00 g; 0.014 mol) in methanol (130 cm ³ ) was added sodium methoxide (3.87 g; 0.0716 mol). The reaction mixture was stirred at room temperature for 4 hours and then neutralized with dilute hydrochloric acid. The mixture was evaporated to dryness under reduced pressure, followed by coevaporation with toluene (15 cm ³ × 3). The residue was purified by silica gel column chromatography using a dichloromethane / methanol mixture (4-15% methanol, vol / vol) as eluent to obtain, after evaporation of the solvents under reduced pressure, nucleoside triol 5 (4.82 g; 89%) as white solid material.

δ _H (CD ₃ OD) 7.89 (1H, d, J 1.2), 7.40-7.24 (5H, m), 5.97 (1H, d, J 6.2), 4, 83-4.65 (2H, m), 4.53 (1H, t, J 6.2), 4.21 (1H, d, J 6.2), 3.84 (1H, d, J 12, 0), 3.63 (1H, d, J 12.0), 3.59 (2H, d, J 2.6), 1.82 (1H, d, J 1.1). δ _s (CD ₃ OD) 164.4, 150.9, 137.5, 136.6, 127.5, 127.0, 126.9, 109.8, 86.7, 86.4, 82.8 78.0, 72.1, 62.3, 61.1, 10.5 (CH,). FAB-MS m / z 379 [M + H] ⁺ . Found (%) C, 56.2; H, 6.0; N, 7.0; C ₁₈ H ₂₂ N ₂ O ₇ , 0.25H ₂ O Calculation C, 56.5; H, 5.9; N, 7.3.

Example 5

1- (3-O-benzyl-4-C- (4,4'-dimethoxytrityloxymethyl) - (3-D-xylofuranosyl) thymine (6)

To a solution of 1- (3-O-benzyl-4-C-hydroxymethyl-β-D-xylofuranosyl) thymine 5 nucleoside (5.38 g; 14.2 mmol) in anhydrous tetrahydrofuran (400 cm ³ ), AgNO ₃ (2 66 g, 15.7 mmol) and then anhydrous pyridine (5.7 cm ³ ) and 4.4'-dimethoxytrityl chloride (5.30 g, 15.6 mmol). The mixture was stirred in the dark under nitrogen for 18 hours at room temperature. The reaction is then quenched by the addition of a saturated aqueous sodium hydrogen carbonate solution (10 cm ³ ) and the resulting mixture is extracted with dichloromethane. The combined organic phase was evaporated to dryness under reduced pressure, and the residue was evaporated together with toluene and then purified by silica gel column chromatography using dichloromethane / methanol / pyridine as eluent (0.5% methanol, 0.5% pyridine, v / v) to obtain, after evaporation of the solvents, nucleoside 6 (3.13 g; 31%) as a white foam.

δ _s ((CD) ₂ SO) 164.1 (C-4), 158.4, 145.1, 138.5, 137.0, 135.9, 135.7, 130.1, 130.1, 129.2, 128.5, 128.5, 128.2, 128.1, 127.7, 127.6, 127.0, 125.7, 113.5 (DMT, benzyl, C-6), 151 4 (C-2), 110.1 (C-5), 85.8, 85.2, 84.6, 83.5 (C-1 ', C-3', C-4 ', DMT) , 76.8 (C-2 '), 72.3 (CH ₂ Ph), 65.2 (C-5''), 62.1 (C-5'), 55.4 (2 × CH ₃ O ), 12, 6 (5-CH ₃ ).

Example 6

1- (3-O-benzyl-4-C- (4,4'-dimethoxytrityloxymethyl) -2,5-di-O- (p-toluenesulfonyl) -β-D-xylofuranosyl) thymine (7)

To a solution of nucleoside 6 (2.79 g; 3.9 mmol) in anhydrous pyridine (50 cm ³ ) was added a catalytic amount of 4- (N, N-dimethylamino) pyridine and p-toluenesulfonyl chloride (6.50 g; 34 mmol). The reaction mixture was stirred for 24 hours at room temperature in a nitrogen atmosphere. The reaction was quenched by the addition of a saturated aqueous sodium hydrogen carbonate solution (100 cm ³ ) and the resulting mixture was extracted with dichloromethane. The combined organic phase is washed with saturated aqueous solutions of sodium bicarbonate (3 × 75 cm ³ ) and sodium chloride (2 × 75 cm ³ ). Next, the separated organic phase is evaporated (Na ₂ SO ₄ ) and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane / methanol / pyridine (0.5% methanol; 0.5% pyridine; vol / vol) as eluent to obtain nucleoside 7 (2.40 g; 62%) after evaporation of the solvents ) in the form of a yellow foam.

δ _c ((CD ₃ ) ₂ SO) 163.2 (C-4), 158.2, 145.9, 145.1, 144.3, 136.8, 135.0, 134.9, 134.8 , 131.8, 131.6, 130.2, 130.0, 129.7, 128.2, 127.9, 127.8, 127.6, 127.5, 127.5, 127.4, 126 8, 113.3 (DMT, C-6, 2 × Ts, benzyl), 150.2 (C-2), 110.8 (C-5), 95.0, 86.2 (DMT, C- 4 '), 82.2, 81.9, (C-1', C-2 '), 81.2 (C-3'), 72.9 (CH ₂ Ph), 79 (C-5 '' ), 64 (C-5 '), 55.1 (2 × CH ₃ O), 21.2, 21.2 (2 × CH ₃ ), 12.0 (5-CH ₃ ).

Example 7

(1R, 3R, 4S, 7R) -7-benzyloxy-1- (4,4'-dimethoxytrityloxymethyl) -3- (thymin-1-yl) -2,5-dioxabicyclo [2.2.1] heptane (8)

An aqueous solution of NaOH (2M, 8 cm ³ ) was added to a solution of nucleoide 7 (3.87 g, 3.92 mmol) in a mixture of ethanol and H ₂ O (1: 1, v / v). The mixture was heated at reflux for 24 hours and, after cooling, it was extracted with dichloromethane. The combined organic phase was washed with saturated aqueous sodium hydrogen carbonate (2 × 75 cm ³ ) and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography using a dichloromethane / methanol / pyridine mixture (0.5% methanol; 0.5% pyridine, vol / vol) as eluent to obtain nucleoside 8 (2.10 g, 81%) after evaporation of the solvents ) in the form of a white foam.

δ _c ((CD ₃ ) ₂ SO) 163.8 (C-4), 158.2, 158.1, 144.7, 137.7, 135.9, 135.2, 135.1, 129.8 , 129.7, 128.3, 127.9, 127.7, 127.7, 127.4, 126.7, 113.35 (DMT, benzyl, C-6), 150.3 (C-2) , 108.1 (C-5), 88.4, 85.5 (C-4 ', DMT), 86.4 (C-1'), 79.5 (C-2 '), 76.3 ( C-3 '), '72, 6 (C-5'), 71.2 (CH ₃ Ph), 58.9 (C-5 ''), 55.1 (2 × CH ₃ O), 12, 4 (5-CH ₃ ).

Example 8

(1R, 3R, 4S, 7R) -1- (4,4'-dimethoxytrityloxymethyl) -7-hydroxy-3- (thymin-1-yl) -2,5-dioxabicyclo [2.2.1] heptane (9)

Ammonium formate (0.33 g, 5.29 mmol) was added to a solution of nucleoside 8 (1.09 g, 1.65 mmol) in methanol (30 cm ³ ). Then, a catalytic amount of Pd / C suspended in methanol (10 cm ³ ) was added to the mixture, and the mixture was heated for 2 hours at reflux temperature. After cooling to room temperature, the mixture was evaporated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography using a dichloromethane / methanol / pyridine mixture (2% methanol, 0.5% pyridine, v / v) as an eluent, to give solvents after evaporation nucleoside 9 (0.76 g, 80%) as a white solid.

δ _c ((CD ₃ ) ₂ SO) 163.9 (C-4), 158.2, 144.8, 135.8, 135.4, 135.3, 129.8, 127.9, 127.7 , 126.8, 113.3 (DMT, C-6), 150.4 (C-2), 108.0 (C-5), 89.2, 85.4 (C-4 ', DMT), 86.4 (C-1 '), 78.9 (C-2'), 72.9 (C-3 '), 72.3 (C-5'), 59.9 (C-5 '') 55.1 (2 × CH ₃ O); 12.5 (5-CH ₃ ).

Example 9

(1R, 3R, 4S, 7R) -7- (2-cyanoethoxy (diisopropylamino) phosphinoxy) -1- (4,4'-dimethoxytrityloxymethyl) -3- (thymin-1-yl) -2,5-dioxabicyclo [2.2 .1] heptane (10)

To a solution of nucleoside 9 (420 g, 0.73 mmol) in anhydrous dichloromethane (4 cm ³ ) was added N, N-diisopropylethylamine (0.4 cm ³ ) and 2-cyanoethyl N, N-diisopropylphosphoramidochloridite (0.4 cm ³ ) . The mixture was stirred in the dark under nitrogen for 18 hours at room temperature. The reaction was then quenched by the addition of methanol and the mixture was diluted with ethyl acetate (10 cm ³ ), washed with saturated aqueous solutions of sodium hydrogen carbonate (3 × 10 cm ³ ) and sodium chloride (2 × 10 cm ³ ) and evaporated to dryness under reduced pressure. The residue was evaporated together with anhydrous acetonitrile and purified by silica gel column chromatography using petroleum ether / ethyl acetate / pyridine (30-40% ethyl acetate, 0.2% pyridine, vol / vol) to obtain an oil. The specified oil is dissolved in dichloromethane (1 cm ³ ) and the product precipitated from petroleum ether (20 cm ³ ) at -40 ° C with vigorous stirring. The precipitate was collected by filtration and evaporated together with anhydrous acetonitrile to give nucleoside 10 (117 mg, 21%) as a white foam. δ _p (CH ₃ CN) 149.9, 149.3.

Obtaining ZNA oligonucleogides

Example 10

Synth-unmodified oligonucleotides and oligonucleotides, including L-ribo-ZNA, obtained from phosphoramidite 10 (formula X).

L-ribo-ZNA and control oligonucleotides are obtained on a Biosearch 8750 DNA synthesizer. Amidite 10 is attached by “manual binding” (amidite and activator are mixed with acetonitrile in a syringe and then passed through the reactor column at a rate of about two times per minute for the whole time binding; use solid CPG substrates). The synthesis of L-ribo-ZNA is carried out using pyridine hydrochloride as an activator (10-30 minute binding: the yield of amidite 10 with stepwise binding is 96-99%). Unmodified 2-deoxynucleoside 2-cyanoethyl N, N-diisopropylphosphoramidites are coupled using a standard DNA program for the synthesizer, except that the binding immediately after the X monomer is carried out in accordance with the RNA program for the synthesizer. After completion of the sequence formation and removal of the protective groups using concentrated ammonia in methanol (32%, v / v, room temperature, 12 hours) from 5'-O-DMT-ON oligonucleotides, followed by purification with phase reversal (using commercially available interchangeable cartridges (Cruachem) and with the inclusion in the procedure of detritylation) receive finished oligomeric products. In the case of all unmodified oligonucleotides and L-ribo-ZNA, including only one X monomer, the 5'-O-DMT group is removed on the synthesizer immediately after completion of the sequence formation. Subsequent treatment with concentrated ammonia in methanol (32%, v / v, 12 hours, 55 ° C.) and ethanol precipitation afforded oligomeric products. To analyze the purity of the synthesized L-ribo-ZNA, capillary gel electrophoresis is used.

Hybridization Data

Example 11

Thermostability of oligonucleotides including X monomer

The thermal stability of L-ribo-ZNA modified oligonucleotides is determined spectrophotometrically using a spectrophotometer equipped with a Ptier element (Ptier). 1 ml hybridization mixtures are prepared using a medium containing saline buffer solution (10 mM Na ₂ HPO ₄ , pH 7.0, 100 mM NaCl, 0.1 mM EDTA) and equimolar (1 μM or 1.5 μM) amounts of various L-ribo-LNA modified oligonucleotides and their complementary DNA or RNA oligonucleotides. As a control, identical hybridization mixtures containing unmodified oligonucleotides are obtained. The absorbance is recorded at 260 nm with a linear increase in temperature in the range from 10 to 90 ° C (1 ° C / min.). The values of the melting temperature (values T _PL ) are obtained as the maximum value (+/- 1 ° C) of the first derivative of the melting curves. Tables 1-3 summarize the results obtained (L-ribo-ZNAs are shown in bold). Figure 2 illustrates the use of monomeric L-ribo-ZNA.

Table 1 shows that the inclusion of one or more consecutive α-L-ribo-ZNA monomers X in the oligonucleotide sequence (A) or (B) does not change the binding affinity of α-L-ribo-ZNA for complementary DNA, whereas its binding affinity for complementary RNA increases significantly.

Table 2 shows the results of studying the binding of homotymine diastereoisomeric ZNAs to RNA (rA ₁₄ ), to RNA with one erroneous base pairing (5'-r (A ₆ CA ₇ )), to enantiomeric RNA (ent-rA ₁₄ ) and to enantiomeric RNA with one erroneous pairing (ent-5'-r (A ₆ CA ₇ )).

Table 3 shows the results of studying the binding of a mixed 9-dimensional sequence of DNA, ZNA and a-L-ribo-ZNA.

Alternative method

Example 12

1- (3-O-benzyl-2,5-di-O-methanesulfonyl-4-C- (methanesulfonyloxymethyl) -β-D-xylofuranosyl) thymine (11)

Anhydrous pyridine (5 cm ³ ) and then methanesulfonyl chloride (1.2 ml, 15.5 mmol) are added to a solution of nucleoside 5 (1100 mg; 2.91 mmol) in anhydrous tetrahydrofuran (20 cm ³ ). The mixture was stirred under nitrogen for 18 hours at room temperature. The reaction mixture was evaporated to dryness under reduced pressure and dissolved in ethyl acetate. The organic phase is washed with saturated aqueous sodium hydrogen carbonate (3 × 10 cm ³ ) and dried (Na ₂ SO ₄ ). The organic phase is evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane / methanol (2% methanol, v / v) as eluent to give nucleoside 11 (908 mg, 51%).

δ _c (CDCl ₃ ) 163.3, 150.6, 135.6, 134.6, 128.7, 128.3, 112.2, 87.9, 85.0, 83.1, 80.9, 77.2, 76.9, 76.6, 73.3, 66.6, 66.2, 38.6, 37.6, 37.6, 12.2.

Example 13

(1R, 3R, 4S, 7R) -1- (hydroxymethyl) -7-benzyloxy-3- (thymin-1-yl) -2,5-dioxabicyclo [2.2.1] heptane (12)

To a solution of nucleoside 11 (329 mg, 0.54 mmol) in ethanol / water (10 cm ³ , 1: 1, v / v) was added 6 M NaOH (aq) (0.9 ml, 5.4 mmol). The mixture was refluxed at 80 ° C. for 43 hours and then evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane / methanol (2.4% methanol, v / v) as eluent to give nucleoside 12 (8.5 mg, 44%).

δ _c ((CD ₃ ) ₂ SO) 163.8, 150.3, 138.0, 135.8, 128.3, 127.7, 127.5, 108.0, 90.2, 86.5, 86.4, 79.3, 76.5, 72.5, 71.2, 57.2, 40.2, 40.0, 39.8, 39.6, 39.4, 39.2, 39, 0, 12.3.

Example 14

Synthesis of nucleoside 8 from nucleoside 12

The standard method of DMT protection of the primary hydroxy group in nucleoside 12 (for example, using the same procedure as in the preparation of nucleoside 6 for DMT protection of the primary hydroxy group in nucleoside 5) yields nucleoside 8, which can be used in the synthesis of the α- phosphoramidite derivative L-ribo-ZNA nucleoside 10 (see FIG. 2 and related examples).

Example 15

9- (2-O-acetyl-5-O-benzoyl-4-C- (benzoyloxymethyl) -3-O-benzyl-α-L-ribofuranosyl) -6-N-benzoyladenine (14)

Sugar 3 (2.05 g) was dissolved in anhydrous acetonitrile (30 ml). N-6-benzoyladenine (1.86 g) was added and then SnCl ₄ (1.3 ml) was added and the resulting mixture was stirred at room temperature for 3.7 hours, after which a saturated aqueous NaHCO ₃ solution was added until neutralization. It is filtered through celite and the filtrate is washed successively with saturated aqueous NaHCO ₃ (3 × 150 ml) and H ₂ O (2 × 150 ml), dried (Na ₂ SO ₄ ) and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (40-60% NaOAc in petroleum ether) to give a fully protected nucleoside intermediate (1.40 g, 52% yield). The indicated intermediate (1.29 g) was dissolved in methanol (35 ml) and a saturated solution of NH ₃ in methanol (35 ml) was added. After stirring at 0 ° C for 2.3 hours, the mixture was evaporated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (1% methanol in dichloromethane) to obtain an intermediate compound, which was dissolved in anhydrous dichloromethane (40 ml). After cooling to −50 ° C., anhydrous pyridine (3 ml) was added together with trifluoromethanesulfonic acid anhydride (0.65 ml). After stirring for 50 minutes, more trifluoromethanesulfonic acid anhydride (0.65 ml) was added and stirring was continued at -10 ° C for 1 hour. Then dichloromethane (100 ml) was added and washing was carried out using a saturated aqueous solution of NaHCO ₃ (3 × 100 ml). The separated organic phase was dried (Na ₂ SO ₄ ) and evaporated to dryness under reduced pressure to give an intermediate. The specified intermediate was dissolved in toluene (20 ml), KOAc (0.85 g) and 18-crown-6 (0.92 g) were added and the resulting mixture was stirred at 80 ° C for 7 hours, after which it was evaporated to dryness under reduced pressure to obtain a residue, which is purified by silica gel column chromatography (0-1.5% methanol in dichloromethane) to obtain nucleoside 14 (1.1 g, 84% in three stages).

δ _c (CDCl ₃ ) 168.8, 165.8, 142.7, 136.0, 133.5, 133.3, 132.7, 129.6, 129.6, 128.8, 128.6, 128.5, 128.4, 128.4, 128.1, 127.8, 83.8, 82.2, 78.4, 74.3, 70.8, 64.7, 63.4, 20, 5. MS (m / z) 742.0 [M + H] ⁺ .

Example 16

(1R, 3R, 4S, 7R) -7-benzyloxy-1-hydroxymethyl-3- (N-6-benzoyladenin-9-yl) -2,5-dioxabicyclo [2.2.1] heptane (15)

Nucleoside 14 (3.05 g) was dissolved in a saturated solution of NH ₃ in methanol (200 ml) and stirred at room temperature for 4 days, after which a 33% aqueous solution of NH ₃ (60 ml) was added and stirring was continued for another 4 hours. The mixture was evaporated to dryness under reduced pressure to give an intermediate which was dissolved in anhydrous pyridine (100 ml). TMSC1 (7.8 ml) was added and stirring was continued at room temperature for 5 hours. After cooling to 0 ° C., benzoyl chloride (2.4 ml) was added and stirring was continued at room temperature for 16 hours. H ₂ O (50 ml) was added and then after 5 minutes a 25% saturated aqueous solution of NH ₃ (25 ml). After stirring for 20 minutes at room temperature, the mixture was evaporated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (2-5% methanol in dichloromethane) to obtain an intermediate product (1.76 g, 87% in two stages). The indicated intermediate (326 mg) was dissolved in anhydrous pyridine (50 ml) and mesyl chloride (0.11 ml) was added at 0 ° C. with stirring. After stirring for 2 hours, H ₂ O (5 ml) was added and the volume of the mixture was reduced by about 50% by evaporation under reduced pressure. Dichloromethane (100 ml) was added and washing was carried out with saturated aqueous NaHCO ₃ solution (3 × 20 ml). The organic phase is dried (Na ₂ SO ₄ ) and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (2-4% methanol in dichloromethane) to give an intermediate (284 mg). The indicated intermediate compound (354 mg) was dissolved in a mixture of dioxane (15 ml), H ₂ O (15 ml) and 2M NaOH (5.5 ml). After stirring for 72 hours under reflux, a 7% (w / w) solution of HCl in dioxane was added until neutralization. The washing is carried out with a saturated aqueous solution of NaHCO ₃ (2 × 100 ml), and the organic phase is dried (Na ₂ SO ₄ ) and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0-4% methanol in dichloromethane) to give 15 bicyclic nucleoside (24 mg).

δ _c ((CD ₃ ) ₂ SO) 156.0, 152.6, 149.4, 138.8, 138.0, 128.3, 127.7, 127.5, 118.3, 89.7, 83.9, 79.7, 77.0, 73.0, 71.2, 57.2. δ _n ((CD ₃ ) ₂ SO) 8.38 (1H, s), 8.14 (1H, s), 7.40-7.30 (7H, m), 6.37 (1H, s), 5.06 (1H, t, J 5.8 Hz), 4.73-5.66 (3H, m), 4.46 (1H, s), 4.15 (1H, d, J 8.4 Hz) ), 4.04 (1H, d, J 8.2 Hz), 3.75 (2H, d, J 5.7 Hz).

Example 17

(1S, 3R, 4S, 7R) -7- (2-cyanoethoxy (diisopropylamino) phosphinoxy) -1- (4,4'-dimethoxytrityloxymethyl-3- (6-N-benzoyladenin-9-yl) -2,5- dioxabicyclo [2.2.1] heptane (16)

The DMT protection of nucleoside 15 followed by debenzylation and 3'-O-phosphitylation is expected to produce a phosphoramidite derivative 16. Another possible way to obtain 16 from nucleoside 15 involves debenzylation 15 followed by selective DMT protection of the primary hydroxyl group and then 3 ' -O-phosphitylation. The reactions indicated in the example are carried out in accordance with standard procedures described in the literature (see, for example, Koshkin, AA, Singh, SK, Nielsen, P., Rajwanshi, VK, Kumar, R., Meldgaard, M., Olsen, CE, Wengel, J. Tetrahedron 1998, 54, 3607).

Claims (15)

1. The oligomer comprising at least one nucleoside analogue of L-ribo-ZNA of General formula Ia

where X is —O—; B is a nucleotide base; P denotes the position of the radical in the internucleoside bond with the subsequent monomer or 5'-terminal hydroxy group; P * denotes an internucleoside bond with the preceding monomer or a 3'-terminal hydroxy group; R ^{2 *} and R ^{4 *} together represent the diradical - (CH ₂ ) _0-1 -O- (CH ₂ ) _1-3 -, - (CH ₂ ) _0-1 -S- (CH ₂ ) _1-3 - or - (CH ₂ ) _0-1 -NR- (CH ₂ ) _1-3 -, where R is hydrogen, alkyl or acyl; R ^{1 *} , R ² , R ^{3 *} , R ⁵ , R ^{5 *} are hydrogen; or its basic or acid addition salt. 2. The oligomer according to claim 1, comprising 1-10000 L-ribo-ZNA of the general formula Ia and 0-10000 nucleosides selected from natural nucleosides and nucleoside analogues, provided that the sum of the number of nucleosides and the number of L-ribo-ZNA is at least 2 in an interval such as 2-10000. 3. The oligomer according to claim 2, characterized in that the oligonucleotide comprises at least 7 consecutive L-ribo-ZNA monomers. 4. The oligomer according to claim 2, characterized in that all nucleoside monomers in the oligomer are L-ribo-ZNA. 5. The oligomer according to any one of claims 1 to 4, characterized in that the biradical is selected from —O — CH ₂ -, —S — CH ₂ -, and —NR — CH ₂ -. 6. The oligomer according to any one of claims 1 to 5, characterized in that any internucleoside bond is —O — P (O ₂ ) —O— or —OP (O, S) —O—. 7. The oligomer according to any one of claims 1 to 6, having the following formula III:

where q is 1-50; each of n (0), ..., n (q) is independently 0-10000; each of m (1), ..., m (q) is independently 1-10000; provided that the sum of n (0), ..., n (q) and m (1), ..., m (q) is 2-10000; G represents a 5'-terminal hydroxy group; each Nu independently represents a nucleoside selected from natural nucleosides and nucleoside analogues; each L-ribo-ZNA independently denotes a nucleoside analogue; each L independently represents an internucleoside bond between two groups selected from Nu and L-ribo-ZNA, or L together with G * represents a 3'-terminal hydroxy group; and each L-ribo-3HK-L independently denotes a nucleoside analogue of the general formula Ia. 8. The nucleoside analogue of L-ribo-ZNA of General formula II

where X is —O—; B is a nucleotide base whose functional groups are optionally protected; Q and Q * are independently selected from hydroxy, Prot-O- and Act-O-; R ^{3 *} represents a group which is hydroxyl, Prot-O- or Act-O-; where "Prot" denotes a protecting group for —OH, “Act” denotes an activating group for —OH; R ^{2 *} and R ^{4 *} together represent the diradical - (CH ₂ ) _0-1 -O- (CH ₂ ) _1-3 -, - (CH ₂ ) _0-1 -S- (CH ₂ ) _1-3 - or - (CH ₂ ) _0-1 -NR- (CH ₂ ) _1-3 -, where R is hydrogen, alkyl or acyl; R ^{1 *} , R ² , R ^{3 *} , R ⁵ , R ^{5 *} are hydrogen, or its basic or acid addition salt. 9. The nucleoside analogue of claim 8, wherein the biradical is selected from —O — CH ₂ -, —S — CH ₂ -, and —NR — CH ₂ -. 10. L-ribo-ZNA according to claim 8 or claim 9 to obtain the L-ribo-ZNA-modified oligomer according to any one of claims 1 to 7. 11. L-ribo-ZNA according to claim 10, characterized in that the inclusion of L-ribo-ZNA modulates the ability of the oligomer to act as a substrate for active enzymes specific for nucleic acids. 12. The L-ribo-ZNA-modified oligomer according to any one of claims 1 to 7 as an antisense, antigenic or gene activating agent. 13. The L-ribo-ZNA-modified oligomer according to any one of claims 1 to 7, characterized in that said oligomer mobilizes RNazuH. 14. The α-L-ribo-ZNA-modified oligomer according to any one of claims 1 to 7 for specific interaction with RNA selected from tRNA, rRNA, snRNA and mcRNA. 15. The L-ribo-ZNA-modified oligomer according to any one of claims 1 to 7 for hybridization of non-coding protein cellular RNAs selected from tRNA, rRNA, snRNA and mcRNA, in vivo or in vitro.

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