RU2243231C2 - Bicyclic analogues of nucleosides, nucleotides and oligonucleotides - Google Patents

Abstract

FIELD: organic chemistry, biochemistry, nucleic acids.

SUBSTANCE: invention elates to LNA-modified oligonucleotide comprising at least one nucleoside analogue (LNA) of the general formula (I)

wherein X means -O-; B means a nitrogen (nucleotide) base; P is a point for joining inter-nucleoside "bridge" or 5'-terminal group that is taken among hydroxyl, monophosphate, diphosphate and triphosphate; R³ or R^3* is inter-nucleoside "bridge" by 3'-terminal group; R²* and R^4* means biradical taken among -(CR*R*)_r-O-(CR*R*)_s-, -(CR*R*)_r-S-(CR*R*)_s-, -(CR*R*)_r-N(R*)-(CR*R*)_s-. Each among R¹*, R², R³*, R³, R⁵* and R⁵ not taking part in formation of biradical or inter-nucleoside "bridge" means hydrogen, halogen atom, hydroxy-, mercapto-, amino-, azido-group; or R² and R³ mean biradical -(CR*R*)_r-O-(CR*R*)_s- wherein R²* is taken among hydrogen atom, hydroxy-group and optionally substituted (C₁-C₆)-alkoxy-group; R¹*, R⁴*, R⁵ and R⁵* mean hydrogen atom wherein each among r and s = 0-4 under condition that sum r + s = 1-4 and each R* represents hydrogen atom or (C₁-C₆)-alkyl; or its basic salt or acid-additive salt. Also, invention proposes the corresponding nucleoside analogue (LNA) and the solid backing material with immobilized LNA. LNA-modified oligonucleotides elicit the enhanced affinity and specificity level with respect to complementary DNA and RNA-oligomers.

EFFECT: valuable properties of analogues.

62 cl, 44 dwg, 14 tbl, 161 ex

Description

Field of Invention

The invention relates to bicyclic and tricyclic nucleoside analogs and the synthesis of such nucleoside analogues that are applicable for the production of synthetic oligonucleotides capable of forming context-specific duplexes and triplexes with single-stranded and double-stranded nucleic acids. Such complexes exhibit higher thermal stability compared to the corresponding complexes formed by conventional nucleic acids. The present invention also relates to bicyclic and tricyclic nucleoside analogs and the synthesis of such nucleosides that could be used as drugs and which could be integrated into oligonucleotides using matrix dependent polymerase nucleic acid polymerases.

BACKGROUND OF THE INVENTION

Synthetic oligonucleotides are compounds widely used in a wide variety of fields, such as molecular biology and DNA-based diagnostics and therapy.

Medicines

In drugs, for example, oligonucleotides have been successfully used to block in vivo translation of specific mRNAs, thereby preventing the synthesis of proteins that are undesirable or harmful to the cell or organism. This oligonucleotide-mediated translation blocking approach is known as the “antisense method”. From the point of view of the kinetics of the process, a hybridizing oligonucleotide is believed to exert its effect either by inducing a physical barrier to translation or by activating cellular enzymes that specifically destroy the mRNA component of a duplex (such as RNase-H).

Recently, oligoribonucleotides and oligodeoxyribonucleotides and their analogues, which combine RNase catalytic activity with the ability to contextually specific interaction with a complementary target RNA, (ribozymes) have attracted much attention as antisense probes. Accordingly, for ribozymes, efficacy was demonstrated both against viral targets and against oncogenes when modeling on cell cultures.

To completely suppress the synthesis of a specific protein using the “antisense method”, it is necessary to block / destroy all mRNAs that encode such a protein, and in many cases the amount of such mRNA is very large. Typically, mRNAs that encode a particular protein are transcribed from one or more genes. Therefore, by labeling such a gene (the “antigenic method”), and not the mRNA product formed from it, it will be possible to either block the production of the protein encoded by it with higher efficiency, or to achieve a significant reduction in the number of oligonucleotides necessary to achieve the desired effect. To block transcription, the oligonucleotide must be able to hybridize with double-stranded DNA, showing specificity in relation to the context of the nucleotide sequence. In 1953, Watson and Crick showed that deoxyribonucleic acid (DNA) consists of two chains (Watson & Crick, 1953, Nature, 171, 737), which are held together in a spiral conformation due to hydrogen bonds formed between opposite ones other complementary bases of two such chains. The four bases usually found in DNA are guanine (G), adenine (A), thymine (T) and cytosine (C): the base G is complementary to C, and the base A is complementary to T. In RNA, the thymine base is replaced uracil (U) base, which, like T, is complementary to base A. The chemical groups of the bases that participate in the formation of the standard duplex represent the “surface” of the Watson-Crick model. In 1959, Hoogsten showed that purine bases (G and A), in addition to the participation of their surface in the Watson-Crick model, are also characterized by a “Hoogsten surface”, which can be recognized from the outside of the duplex, and used them for bonding pyrimidine oligonucleotides by hydrogen bonds, thereby forming a triple helix. Although the development of the “antigenic approach” is in principle possible from the point of view of practical application in the formation of three-chain oligomers, it is currently limited by a number of factors, including the need for homopurine motifs in the sequence of the target gene and the need to create non-physiological high ionic strength and low values pH in order to stabilize the three-chain complex.

The use of oligonucleotides known as “aptamers” is also being actively investigated. This promising therapeutic class of a new class of oligonucleotides is selected in vitro for specific binding on a specific target with a high level of affinity for it, such as, for example, ligand receptors. Their binding characteristics are apparently a reflection of the ability of oligonucleotides to form three-dimensional structures supported by intramolecular nucleotide interaction.

Nucleosides and nucleoside analogues have also been shown to be effective in chemotherapy of a variety of viral infections and malignant tumors.

Also, various types of double-stranded RNAs have been shown to effectively inhibit the growth of certain types of malignant tumors.

Diagnostics

In molecular biology, oligonucleotides are usually used for various purposes, such as, for example, (1) the use of target nucleic acids as hybridization probes, (2) as affinity probes in the purification of nucleic acids targets, (3) as seeds for sequencing reactions and amplification processes of targets, such as polymerase chain reaction (PCR), (4) for cloning and mutation of nucleic acids, and (5) as building blocks for assembling a macromolecule GOVERNMENTAL structures.

In diagnostics, many of the above methods are used, based on the use of oligonucleotides, in particular, those that lend themselves to simple automation and provide reproducible results at high sensitivity. The subject of this analysis is the use of oligonucleotide-based methods, for example, for (1) testing people, animals and food for the presence of pathogenic microorganisms, (2) testing a genetic predisposition to a disease, (3) identifying hereditary and acquired genetic pathologies, (4 ) searching for a connection between biological objects and suspects in investigative practice; and (5) checking the presence of microorganisms involved in the production of food and drinks.

Key considerations

In order for them to be used to solve a wide range of tasks outlined above, oligonucleotides must satisfy a significant number of different conditions. In “antisense therapy”, for example, the oligonucleotide used must be able to penetrate into the cell through its membrane, have significant resistance to the effects of extra- and intracellular enzymes, and preferably have the ability to recruit endogenous enzymes such as RNase-H. Other properties, such as, for example, the ability of oligonucleotides to act as effective substrates for a wide range of different enzymes associated with natural nucleic acids, such as, for example, polymerases, kinases, are important in the diagnosis and in the sections of molecular biology based on the use of DNA. ligases and phosphatases. However, the fundamental property of oligonucleotides, which is necessary in all their applications, is the ability to recognize and hybridize contextually specific sequences with complementary single-stranded nucleic acids involving either hydrogen bonds in accordance with the Watson-Crick model (AT and CG), or other hydrogen variants connections such as the Hoogsten variant. There are two important terms, “affinity” and “specificity,” which are commonly used to characterize the hybridizability parameters of a particular oligonucleotide. Affinity is understood as a measure of the binding strength of an oligonucleotide with a target sequence complementary to it (expressed in the magnitude of the thermal stability of the duplex - T _m ). Each base pair in the duplex contributes to the value of thermal stability and thus affinity increases with increasing size (number of bases) in the oligonucleotide. Specificity is a measure of the ability of a given oligonucleotide to distinguish between a fully complementary and an incorrect target sequence. In other words, specificity is a measure of the loss of affinity due to the presence of incorrect bases in the composition of the target. With a constant size of the oligonucleotide, specificity increases in parallel with an increase in the number of discrepancies between the oligonucleotide and its targets (i.e., with an increase in the error rate). On the other hand, specificity decreases when the size of a given oligonucleotide increases with a constant number of mismatches (i.e., the error rate decreases). Formulated differently, an increase in the affinity of an oligonucleotide occurs “due to” specificity, and vice versa.

This property of oligonucleotides creates a number of problems in their practical application. In the case of very long diagnostic procedures, for example, it is necessary that the oligonucleotide is characterized both by high affinity in terms of ensuring adequate sensitivity throughout the test and by a high level of specificity in order to prevent false-positive results. The oligonucleotide used as an antisense probe should also be characterized by high affinity for the corresponding mRNA target in order to effectively suppress its translation, and a high level of specificity in order to prevent unintentional blocking of the expression of other proteins. In enzymatic processes, such as, for example, amplification by PCR, the affinity of the oligonucleotide seed must be high enough so that the duplex “seed-target” is stable in the temperature regime in which the enzyme is active, and the specificity must be sufficiently high in order to be sure that the target sequence is amplified.

Taking into account the limitations typical for the use of native oligonucleotides, new approaches to increasing the levels of specificity and affinity should be developed in connection with the use of DNA-based methods in therapy, diagnostics, and molecular biology in general.

Conformationally Restricted Nucleosides

It is known that oligonucleotides undergo a conformational transformation during hybridization with the target sequence from a relatively free helical structure characteristic of a single-chain state to an ordered structure typical of a double-stranded state.

A number of conformationally restricted oligonucleotides, including bicyclic and tricyclic nucleoside analogues (FIGS. 1A and 1B, on which B = nucleotide base) were synthesized, inserted into the oligonucleotide and oligonucleotide analogues, and tested for their hybridizability and other parameters.

Bicyclo [3.3.0] nucleosides (bsDNA) with additional C-3 ', C-5'-two-carbon “bridges” (A and B) were synthesized for all five bases (G, A, T, C and U), in while (C) was synthesized only with bases T and A (M.Tarkoy, M. Bolli, B. Schweizer & C. Leumann, 1993, Helv. Chim. Acta, 76, 481; M.Tarkoy & C. Leumann , 1993, Angew. Chem. Int. Ed. Engl., 32, 1432; M. Egli, P. Lubini, M. Dobler & C. Leumann, 1993, J. Amer. Chem. Soc., 115, 5855; M .Tarkoy, M. Bolli & C. Leumann, 1994, Helv. Chim. Acta, 77, 716; M. Bolli & C. Leumann, 1995, Angew. Chem. Int. Ed. Engl., 34, 694; M. Bolli, P. Lubini & C. Leumann, 1995, Helv. Chim. Acta, 78, 2077; JCLitten, C. Apple & C. Leumann, 1995, Bioorg. Med. Chem. Lett., 5, 1231; JCLitten & C. Leumann, 1996, Helv. Chim. Acta, 79, 129; M. Bolli, JCLitten, R. Schultz & C. Leumann, 1996, Chem. Biol., 3, 1 97; M. Bolli, H. U. Trafelet & C. Leumann, 1996, Nucl. Acids Res., 24, 4660). DNA oligonucleotides, including several of these analogues or completely consisting of them, in most cases are able to form duplexes according to the Watson-Crick model with complementary DNA and RNA oligonucleotides. The thermal stability of the resulting duplexes, however, is either significantly inferior to (C), or somewhat lower than (A), or comparable (B) to that of natural DNA and RNA variants. All bcDNA oligomers are characterized by a clearly higher sensitivity to the ionic strength of the hybridization medium compared to native variants. α-bc-DNA (B) is characterized by greater stability with respect to the 3’-exonuclease activity of snake venom phosphodiesterase compared to β-bcDNA, which is only slightly superior in stability to unmodified oligonucleotides.

Bicarbocyclo [3.1.0] nucleosides with an additional C-1 ', C-6'- or C-6', C-4'-carbon "bridge" in the cyclopentane ring (respectively, D and E) were synthesized for all five bases (T, A, G, C and U). However, only T analogs were included in the oligomers. The introduction of 1-10 D monomers into the mixed polypyrimidine-DNA oligonucleotide caused a significant decrease in the affinity for both DNA and RNA oligonucleotides compared to the unmodified control oligonucleotide. This decrease was more pronounced with respect to single-stranded DNA compared to single-stranded RNA. The introduction of one monomer E into two different polypyrimidine-DNA oligonucleotides led to a slight increase in the melting temperature (by 0.8 ° C and 2.1 ° C) for duplexes in relation to ssRNA compared with unmodified control duplexes. When a 15-dimensional oligonucleotide having exclusively phosphotioate bonds between nucleosides was included, 10 T analogues were included, the value of T _m with respect to the complementary RNA oligonucleotide increased by approximately 1.3 ° C for each modification compared to the same unmodified phosphotioate sequence. In contrast to the control sequence, the oligonucleotide comprising the bicyclic nucleoside E did not provide cleavage mediated by RNase-H. The hybridization parameters of oligonucleotides, including analogues of G, A, C, and U according to Formula E, were not previously reported. In addition, the chemistry of these analogs alone did not in itself cause further studies of fully modified oligonucleotides (K.-H. Altmann, R. Kesselring, E. Francotte & G. Rihs, 1994, Tetrahedron Lett., 35, 2331; K .-H. Altmann, R. Imwinkelried, R. Kesselring & G. Rihs, 1994, Tetrahedron Lett., 35, 7625; VEMarquez, MASiddiqui, A. Ezzitouni, P. Russ, J. Wang, RWWagner & MD Matteucci, 1996, J. Med. Chem., 39, 3739; A. Ezzitouni & VEMarquez, 1997, J. Chem. Soc., Perkin Trans., 1, 1073).

Bicyclo [3.3.0] a nucleoside comprising an additional C-2 ', C-3'-dioxalane ring was synthesized as a dimer with an unmodified nucleoside, in which the additional ring is part of the internucleoside “bridge” replacing the usual phosphodiester bond (F) . This analogue was synthesized only in thymine-thymine or thymine-5-methylcytosine dimer variants. The 15-dimensional polypyrimidine sequence, including seven such dimeric “blocks” and characterized by alternating phosphodiester and riboacetal “bridges”, showed a significantly reduced value of T _m with respect to complementary single-stranded RNA compared to a control sequence characterized by exclusively native phosphodiester bonds between nucleosides ( , S. Swaminathan, JF Millagan, S. Wad-wani, BS Froehler & M. Matteucci, 1993, J. Amer. Chem. Soc., 115, 9816).

Two dimers (G and H) with additional C-2 ', C-3'-dioxane rings forming a bicyclo [4.3.0] system as part of the internucleoside “bridges” of the acetal type, were synthesized as dithymine dimers and included in the middle part 12-dimensional polypyrimidine oligonucleotides. Oligonucleotides comprising either G or H formed substantially less stable duplexes with single-stranded RNA and DNA complementary to them compared to the unmodified control oligonucleotide (J. Wang & M. D. Matteucci, 1997, Bioorg. Med. Chem. Lett., 7 , 229).

Dimers including bicyclo [3.1.0] nucleosides with a C-2 ', C-3'-carbon "bridge", which is part of the internucleoside "bridges" of the amide and sulfonamide types (I and J), were synthesized and incorporated into the oligonucleotides. Oligonucleotides comprising one or more of these analogs showed a significant decrease in T _m compared to the unmodified native oligonucleotide (CGYannopoulos, WQZhou, P.Nower, D. Peoch, YSSanghvi & G. Just, 1997, Synlett., 378).

A trimer having formational bonds between nucleosides and including in the middle of a bicyclo [3.3.0] glucose derivative nucleoside analog (K) was synthesized and attached to the 3'-end of the oligonucleotide. The value of T _m in relation to complementary single-stranded RNA decreased by 4 ° C compared with the control sequence and 1.5 ° C compared with the sequence including two 2 ', 5'-formacetal bonds in the 3'-end (CGYannopoulos, WQZhou , P. Nower, D. Peoch, YSSanghvi & G. Just, 1997, Synlett., 378).

More recently, it has been reported that oligomers including tricyclic nucleoside (L) analogues provide higher duplex stability compared to native DNA (R. Steffens & C. Leumann [Poster SB-B4], 1997, Chimia, 51, 436 )

Three bicyclic ([4.3.0] and [3.3.0]) nucleosides with additional C- 2 ', C-3' attached six- (M and N) or pentatomic (O) rings were synthesized as thymine analogues . The bicyclic nucleosides M and N were included, one or two, each in the 14-dimensional oligo-T sequences. The value of T _m in relation to single-stranded RNA and DNA decreased by 6-10 ° C per modification compared to unmodified control sequences. Fully modified oligonucleotides of analogue O were characterized by an increase in T _{m of} about 1.0 ° C per modification with respect to the RNA oligonucleotide compared to the control DNA oligonucleotide. Also, the fully modified sequence was significantly more resistant to hydrolysis by snake venom phosphodiesterase compared to the unmodified control sequence. Partially modified oligonucleotides, which contain up to four analogs of type O, inclusive, however, were less thermostable compared to the corresponding unmodified oligonucleotides. All oligonucleotides, including analog O (both fully and partially modified), were characterized by significantly reduced thermal stability with respect to complementary DNA oligonucleotides in comparison with unmodified oligonucleotides (P. Nielsen, HMPfundheller & J. Wengel, 1997, Chem. Commun., 826 ; P. Nielsen, HMPfundheller & J. Wengel, 1996, XII Intern. Roundtable: "Nucleosides, Nucleotides and Their Biological Applications", La Jolla, CA, Sept. 15-19, [Poster PPI-43]).

An attempt to obtain bicyclic analogues of uridine (Q) nucleoside, which was intended to include an additional C-2 ', C-4'-five-membered ring starting with 4'-C-hydroxymethyl nucleoside (P), was unsuccessful (KDNielsen, 1995, Specialerapport, Odense Univ., Denmark).

So far, work with conformationally restricted nucleosides used to construct synthetic oligonucleotides to give them improved hybridization characteristics has been little success. In most cases, oligonucleotides including such analogues form less stable duplexes with complementary nucleic acids compared to unmodified oligonucleotides. In other cases, when there is a slight improvement in the stability of the duplex, this applies only to either the DNA or target RNA, or it refers to fully, but not partially modified oligonucleotides, and vice versa. Further analysis of most of the described analogues is complicated by the lack of data on nucleoside analogues G, A and C and the lack of data characterizing the specificity and type of hybridization. In many cases, the synthesis of the described nucleoside analogs is very complicated, while in other cases, the synthesis of fully modified oligonucleotides is incompatible with commonly used standard phosphoramidite synthesis methods.

SUMMARY OF THE INVENTION

From the point of view of overcoming the limitations inherent in the known nucleoside analogues, the applicants present new nucleoside analogues (LNA) and oligonucleotides, which include LNA nucleosides. New nucleoside analogs of LNA are presented for all common bases, thereby providing a complete set of nucleoside analogues required for incorporation into oligonucleotides. As will be seen from the following, the nucleoside analogues of LNA and LNA-modified oligonucleotides open up the possibility for a wide range of options for improving oligonucleotides used in diagnosis and therapy. Moreover, the nucleoside analogs of LNA and the oligonucleotides modified by them also represent completely new perspectives in the diagnosis and therapy based on the use of nucleosides and oligonucleotides.

Thus, the present invention relates to oligomers comprising at least one nucleoside analogue

(hereinafter referred to as "LNA") of the basic formula I:

where X is selected from —O—, —S—, —N (R ^{N *} ) -, —C (R ⁶ R ^{6 *} ) -, —OC (R ⁷ R ^{7 *} ) -, —C (R ⁶ R ^{6 *} ) -O-, -SC (R ⁷ R ^{7 *} ) -, -C (R ⁶ R ^{6 *} ) -S-, -N (R ^{N *} ) -C (R ⁷ R ^{7 *} ) -, -C (R ⁶ R ^{6 *} ) —N (R ^{N *} ) - and —C (R ⁶ R ^{6 *} ) —C (R ⁷ R ^{7 *} ) -;

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

P denotes the position of the radical for the formation of the internucleoside “bridge” with the next monomer or 5'-terminal group, while such a bond or 5'-terminal group optionally includes a substituent R ⁵ ;

one of the substituents R ² , R ² *, R ³ and R ³ * is a P * group that forms an internucleoside “bridge” with the preceding monomer or a 3'-terminal group;

one or two pairs of non-heminal substituents (i.e., “non-double” at one carbon atom) are selected from the available substituents R ¹ *, R ⁴ *, R ⁵ , R ⁵ *, R ⁶ , R ⁶ *, R ⁷ , R ⁷ *, R ^N *, and each of the substituents R ² , R ² *, R ³ and R ³ *, not involved in P *, forms a biradical of 1-8 groups / atoms selected from –C (R ^a R ^b ) -, -C (R ^a ) = C (R ^a ) -, -C (R ^a ) = N-, -О-, -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 ^a and R ^{b are} independently selected from hydrogen, optionally substituted with C _1-12 -alkyl, optionally substituted C _2-12 -alkenyl, optionally substituted C _2-12 -alkynyl, neobyaz Tel'nykh substituted with hydroxyl, C _1-12 alkoxy, C _2-12 -alkeniloksigruppy, carboxy, C _1-12 -alkoxycarbonyl, C _1-12 -alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino groups, mono- and di- (C _1-6 -amino) amino groups, carbamoyl, mono- and di- (C _1-6 -alkyl) -aminocarbonyl, amino-C _1-6 -alkyl-aminocarbonyl, mono- and di- (C _1-6 alkyl) amino C _1-6 alkylaminocarbonyl, C _1-6 alkylcarbonylamino groups, urea, C _1-6 alkanoyloxy groups, sulfonic th group, C _1-6 alkylsulfonyloxy groups, nitro groups, azido groups, sulfanyl, C _1-6 alkylthio groups, halogen, DNA intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands, where aryl and heteroaryl can be optionally substituted, and where two geminal substituents R ^a and R ^b together can form optionally substituted methylene (= CH ₂ ), and while two geminal or non-heminal substituents selected from R ^a , R ^b and any of the substituents R ^{1 *} , R ² , R ^{2 *} , R ³ , R ^{3 *} , R ^{4 *} , R ⁵ , R ^{5 *} , R ⁶ and R ^{6 *} , R ⁷ and R ^{7 *} , which are present and are not involved in P, P * or the diradical (diradicals), together can form a linked diradical selected from the same type of diradicals that were . defined above;

said pair (s) of non-heminal substituents thereby form a mono- or bicyclic moiety together with (i) atoms to which the non-heminal substituents are attached, and (ii) with any intermediate atoms; and

each of the substituents R ^{1 *} , R ² , R ^{2 *} , R ³ , R ^{3 *} , R ^{4 *} , R ⁵ , R ^{5 *} , R ⁶ and R ^{6 *} , R ⁷ and R ^{7 *} , which are present and not involved in P, P * or diradical (diradicals), independently selected from hydrogen, optionally substituted C _1-12 alkyl, optionally substituted C _2-12 alkenyl, optionally substituted C _2-12 alkynyl, optionally substituted hydroxyl, C _{1 -12} alkoxy, C _2-12 -alkeniloksigruppy, carboxy, C _1-12 -alkoxycarbonyl, C _1-12 -alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, het roariloksigruppy, heteroarylcarbonyl, amino, mono- and di- (C _1-6 -amino) 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 -alkilkarbonilaminogruppy, carbamido, C _1-6 -alkanoiloksigruppy, sulfone group, C _1-6 -alkilsulfoniloksigruppy, nitro, azido , sulfanyl, C _1-6 alkylthio, halogen, DNA-intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands where aryl and heteroaryl can be optionally substituted and where two geminal substituents together can define an oxo group, a thioxo group, an imino group or an optionally substituted methylene, or together they can form a spiro radical, including a 1-5-carbon alkylene chain, which is optionally interrupted and (or) terminated by one or several heteroatoms / groups selected from —O—, —S— and - (NR ^N ) -, where R ^{N is} selected from H and C _1-4 alkyl and where two adjacent (but geminal) substituents can form an additional double connection ; and R ^{N *} , when present and not included in the diradical, is selected from H and C _1-4 alkyl;

and their basic salts and acid addition salts, provided that

(i) R ² and R ³ do not simultaneously mean a biradical selected from —O — CH ₂ —CH ₂ - and —O — CH ₂ —CH ₂ —CH ₂ -, where LNA is a bicyclic nucleoside analogue;

(ii) R ³ and R ⁴ do not simultaneously mean a diradical selected from —CH ₂ —CH ₂ - and —O — CH ₂ -, where LNA is a bicyclic nucleoside analogue;

(iii) R ³ , R ⁵ and R ^{5 *} do not simultaneously mean the triradical —CH ₂ —CH (-) - CH ₂ -, where LNA is a tricyclic nucleoside analogue;

(iv) R ^{1 *} and R ^{6 *} at the same time do not mean the biradical –CH ₂ -, where LNA is a bicyclic nucleoside analogue; and

(v) R ^{4 *} and R ^{6 *} at the same time do not mean the biradical –CH ₂ -, where LNA is a bicyclic nucleoside analogue.

The present invention further provides nucleoside analogues (hereinafter LNA) of the general formula II:

where Deputy B is selected from nucleotide bases, DNA intercalating groups, 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 *} ) -;

one of the substituents R ² , R ² *, R ³ and R ³ * is a group Q ^* ;

each of Q and Q ^{* is} independently selected from hydrogen, azido group, halogen, cyano group, nitro group, hydroxyl, Prot-O-, Act-O-, mercapto group, Prot-S-, Act-S-, C _1-6 alkylthio group, amino groups, Prot-N (RH) -, Act-N (RH) -, mono- or di- (C _1-6 alkyl) amino groups, optionally substituted C _1-6 alkoxy groups, optionally substituted C _1-6 alkyl optionally substituted C _2-6 -alkenyl, optionally substituted C _2-6 -alkeniloksigruppy optionally substituted C _2-6 -alkynyl, optionally substituted C _2-6 -alkiniloksigruppy, monophosphate, diphosphate, triphosphate, DNA intercalators boiling groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands, carboxy, sulfonic groups, 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, and “Act” is the activation group for —OH, —SH and —NH (R ^H ), respectively, a R ^{H is} selected from H and C _1-6 alkyl;

(i) R ^{2 *} and R ^{4 *} together denote a biradical selected from —O—, - (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-, -SO (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 *) -;

(ii) R ² and R ³ simultaneously mean a biradical selected from —O—, - (CR * R *) _{r + s} -, - (CR * R *) _r —O- (CR * R *) _s -, (CR * R *) _r -S- (CR * R *) _s - and - (CR * R *) _r -N (R *) - (CR * R *) _s -;

(iii) R ^{2 *} and R ³ together form a biradical selected from —O—, - (CR * R *) _{r + s} -, - (CR * R *) _r —O- (CR * R *) _s - , - (CR * R *) _r -S- (CR * R *) _s - and - (CR * R *) _r -N (R *) - (CR * R *) _s -;

(iv) R ³ and R ⁴ * simultaneously mean a biradical selected from - (CR * R *) _r -O- (CR * R *) _s -, - (CR * R *) _r -S- (CR * R *) _s - and - (CR * R *) _r -N (R *) - (CR * R *) _s -;

(v) R ³ and R ⁵ simultaneously mean a biradical selected from - (CR * R *) _r -O- (CR * R *) _s -, - (CR * R *) _r -S- (CR * R * ) _s - and - (CR * R *) _r -N (R *) - (CR * R *) _s -; or

(vi) R ^{1 *} and R ^{4 *} simultaneously mean a biradical selected from - (CR * R *) _r -O- (CR * R *) _s -, - (CR * R *) _r -S- (CR * R *) _s - and - (CR * R *) _r -N (R *) - (CR * R *) _s -;

(vii) R ¹ * and R ² * simultaneously mean a biradical selected from (CR * R *) _r -O- (CR * R *) _s -, (CR * R *) _r -S- (CR * R * ) _s - and - (CR * R *) _r -N (R *) - (CR * R *) _s -;

while each R * is independently selected from hydrogen, halogen, azido group, cyano group, nitro group, hydroxyl, mercapto group, amino group, mono- or di- (C _1-6 alkyl) amino group, optionally substituted with C _1-6 -alkoxy groups, optionally substituted C _1-6 alkyl, DNA intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands, and (or) two adjacent (non-heminal) R * can together form a double bond, and in each case r and s are 0-3 provided that with MMA r + s is 1-4;

each of the substituents R ¹ *, R ² , R ² *, R ³ , R ⁴ *, R ⁵ and R ⁵ *, which are not involved in Q, Q * or a diradical, is independently selected from hydrogen, optionally substituted with C _1-12 -alkyl, optionally substituted C _2-12 alkenyl, optionally substituted C _2-12 alkynyl, optionally substituted hydroxyl, C _1-12 alkoxy, C _2-12 alkenyloxy, carboxy, C _1-12 alkoxycarbonyl, C _{1 -12-} alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy groups, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy groups, heteroarylcarbonyl, amine groups, mono- and di- (C _1-6 amino) amino groups, 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 -alkylcarbonylamino, carbamido, C _1-6 -alkanoyloxy, sulfonic, C _1-6 -alkylsulfonyloxy, nitro, azido, sulfanyl, C _{1 -6-} alkylthio groups, halogen, DNA intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands, where aryl and heteroaryl may be optional fully substituted and where two geminal substituents together can define an oxo group, thioxo group, imino group or optionally substituted methylene or together they can form a spiradio radical including a 1-5-carbon alkylene chain, which is optionally interrupted and (or) terminated by one or more heteroatoms / groups selected from —O—, —S— and - (NR ^N ) -, where R ^{N is} selected from hydrogen and C _1-4 alkyl and where two adjacent (but heminal) substituents can form an additional double bond; and R ^{N *} , when present and not included in the diradical, is selected from hydrogen and C _1-4 alkyl; and their basic salts and acid addition salts under the first condition that

(i) R ² and R ³ together do not form a biradical selected from —O — CH ₂ —CH ₂ - and —O — CH ₂ —CH ₂ —CH ₂ -, where LNA is a bicyclic nucleoside analogue;

(ii) R ³ and R ⁵ together do not form a biradical selected from —CH ₂ —CH ₂ -, —O — CH ₂ -, and —O — Si ( ⁱ Pr) ₂ —O — Si ( ⁱ Pr) ₂ —O -;

and under the second condition that any chemical group (including any nucleotide bases) that is reactive under the conditions that occur during the synthesis of oligonucleotides does not have to be protected in its functional component.

The present invention also provides the use of nucleoside analogues (LNAs) in the construction of oligomers and the use of such oligomers, as well as nucleoside analogues (LNAs) in diagnostics, molecular biological studies and therapy.

Brief Description of the Drawings

1A and 1B show known conformationally restricted nucleotides.

Figure 2 shows the nucleotide and nucleoside analogs of the present invention.

Figure 3 shows a method for using LNA-modified oligonucleotides in sequence-specific selection of PCR amplicons.

Figures 4A and 4B show that LNA-modified oligonucleotides are able to recognize their related PCR amplicon due to chain invasion.

Figure 5 shows that LNA-modified oligonucleotides immobilized on a solid surface actively function in sequence-specific selection of a PCR amplicon.

Figure 6 shows that LNA-modified oligonucleotides can act as substrates for T4 polynucleotide kinase.

Figure 7 shows that LNA-modified oligonucleotides can function as seeds for nucleic acid polymerases.

Figure 8 shows that LNA-modified oligonucleotides can function as seeds in target amplification processes.

Figure 9 shows that LNA-modified oligonucleotides bearing 5′-anthraquinone can be covalently immobilized onto a solid support by irradiation and that the immobilized oligomer is effective in selecting a complementary DNA oligomer.

Figure 10 shows that LNA-thymidine-5’-triphosphate (LNA-TTP) can act as a substrate for terminal deoxynucleotidyl transferase (TdT).

The figure 11 illustrates the hybridization and detection of motif using various LNA-modified Cγ 3-labeled octonucleotides.

Figures 12 and 13 illustrate hybridization and detection of terminal errors in the motif using various LNA-modified Cγ 3-labeled octonucleotides.

Figure 14 illustrates LNA blocking of [D-Ala ₂ ] -deltorphin-induced analgesia in “conscious” rats in a warm water irritation test.

Figures 15A, 15B, and 15C illustrate hybridization and detection of terminal errors in a motif using AT and all LNA-modified Cγ 3-labeled octonucleotides.

Figures 16 and 17 show that LNA can be introduced into living MCF-7 cells of a human breast tumor.

Figures 18 and 19 show a method of using labeled [α- ³³ P] -ddNTP and ThermoSequenase ™ DNA polymerase reagents in sequencing DNA matrices including LNA type T monomers.

Figures 20 and 21 show that exonuclease-free DNA polymerase I (Clenov fragment) can incorporate adenosine, cytosine, guanosine, and uridine-5′-LNA-type triphosphates into a DNA strand.

The figure 22 shows the ability of terminal deoxy nucleotidyl transferase (TdT) to attach at the end position of LNA-modified oligonucleotides.

Figures 23A and 23B show that fully mixed LNA monomers can be used to significantly increase the involvement of immobilized biotin-labeled DNA oligomers in sequence-specific selection of PCR amplicons.

Figures 24-41 show possible routes for the synthesis of LNA monomers of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “LNA” (Locked Nucleoside Analogues) refers to the bi- and tricyclic nucleoside analogues of the present invention, either included in the oligomer of the present invention (according to basic formula I), or existing as a discrete chemical units (according to the basic formula II). The term “monomeric LNA” is particularly applicable to the latter case.

Oligomers and nucleoside analogues

As noted above, the present invention, among other things, provides new oligomers (oligonucleotides) comprising one or more bi-, tri- or polycyclic nucleoside analogs (hereinafter referred to as “LNA”). It has been found that the introduction of such LNAs in place of and in addition to, for example, known nucleosides, gives the resulting oligonucleotide interesting and promising properties. Bi- and tricyclic, and especially bicyclic, LNAs are apparently particularly interesting in the scope of the present invention.

Each of the probable LNAs included in the oligomer (oligonucleotide) corresponds to formula I:

where X is selected from —O— (furanose motif), —S—, —N (R ^{N *} ) -, —C (R ⁶ R ⁶ *) -, —OC (R ⁷ R ⁷ *) -, —C ( R ⁶ R ^{6 *} ) -O-, -SC (R ⁷ R ^{7 *} ) -, -C (R ⁶ R ^{6 *} ) -S-, -N (R ^{N *} ) -C (R ⁷ R ^{7 *} ) -, -C (R ⁶ R ^{6 *} ) -N (R ^{N *} ) - and -С (R ⁶ R ^{6 *} ) -С (R ⁷ R ^{7 *} ) -, where R ⁶ , R ⁶ *, R ⁷ , R ⁷ * and R ^N * are as defined below.

Thus, the LNAs introduced into the oligomer composition can include either a five or six ring atom as an essential part of a bi-, tri- or polycyclic structure. We can assume that the 5-atomic rings (X is represented by -O-, -S-, -N (R ^N *) -, -C (R ⁶ R ⁶ *) -) are especially interesting due to the fact that they are able to occupy essentially the same conformations (however, closed by the introduction of one or more biradicals - see below) as the native furanose ring in naturally occurring nucleosides. Among the likely 5-atomic rings, the most interesting cases are those in which X is represented by —O—, —S— and —N (R ^N *) -, and the variant in which X is represented by —O— is considered to be particularly interesting.

Substituent B may represent a group which, when the oligomer is complexed with DNA or RNA, is capable of interacting (for example, by forming a hydrogen bond or a covalent bond, or by electronic interaction) with DNA or RNA, especially DNA or RNA nucleotides. Alternatively, substituent B may represent a group that serves as a label or reporter, or substituent B may represent a group (eg, hydrogen) that is expected to interact weakly or not at all with DNA or RNA. Thus, substituent B is preferably selected from hydrogen, hydroxyl, optionally substituted C _1-4 alkoxy, optionally substituted C _1-4 alkyl, optionally substituted C _1-4 acyloxy, nucleotide bases, DNA intercalating groups, photochemically active groups , thermochemically active groups, chelating groups, reporter groups and ligands.

In the context of the present invention, the term “nucleotide base” includes native bases, as well as non-natural bases. It will be understood by those skilled in the art that various bases, which were previously designated as “unnatural,” can subsequently be found in nature. Thus, the concept of “nucleotide base” includes not only known purine and pyrimidine heterocycles, but also heterocyclic analogues and their tautomers. Illustrative examples of the bases are adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurin, 8-oxo-N ^6- methyladenine, 7-deazaxanthin, 7-deazaguanine, N ⁴ , N ^4- ethancitosine, N ⁶ , N ⁶ -ethane-2,6-diaminopurin, pseudoisocytosine, 2-hydroxy-5-methyl-4-thiazolopyridine, isocytosine, isoguanine, inosine and the “unnatural” bases described by Benner et al. (Benner et al .: US patent No. 5432272). It is understood that the term “nucleotide base” encompasses each individually and collectively these examples, as well as their analogues and tautomers. Particularly interesting bases are adenine, guanine, thymine, cytosine and uracil, which are considered as natural bases in connection with their therapeutic and diagnostic use in medicine.

As used in this text, the term “DNA intercalating group” means a group that can intercalate a helix, duplex or triplex of DNA or RNA. Examples of the functional constituents of DNA intercalating groups are acridines, anthracene, quinones, such as anthraquinone, indole, quinoline, isoquinoline, dihydroquinones, anthracyclines, tetracyclines, methylene blue, anthracyclinone, psoralen, coumarins, ethidium halides, metal, dynamin, , 10-phenanthroline-copper, tris (4,7-diphenyl-1,10-phenanthroline) ruthenium-cobalt-endinines, 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 this invention, the term “photochemically active groups” includes compounds that are capable of undergoing chemical transformations when irradiated with visible light. Illustrative examples of such functional groups are quinones, especially 6-methyl-1,4-naphthoquinone, anthraquinone, naphthoquinone and 1,4-dimethylanthraquinone, diazirines, aromatic azides, benzophenones, psoralen, diazo compounds and diazirin compounds.

In the context of this invention, the term “thermochemically reactive groups” defines a functional group that is capable of undergoing thermochemically induced covalent bonding with other groups. Illustrative examples of the functional constituents of thermochemically reactive groups are carboxylic acids, carboxylic acid esters such as activated esters, carboxylic acid halides such as fluorides, chloro, bromo and iodohydrides of acids, azides of carboxylic acids, hydrazides-carboxylic acids, sulfonic acids, sulfonic acid esters, sulfonic acid halides, semicarbazides, thiosemicarbazides, aldehydes, ketones, primary alcohols, secondary alcohols, tertiary alcohols, phenols, alkyl halogen rows, disulfides, thiols, primary amines, secondary amines, tertiary amines, hydrazines, epoxides, maleimides, and boronic acid derivatives.

In the context of this invention, the term “chelating group” means a molecule that includes more than one binding site and often binds to another molecule, atom or ion at more than one binding site at the same time. Examples of the functional constituents of the chelating groups are iminodiacetic acid, nitrilotriacetic acid, ethylenediaminetetraacetic acid (EDTA), aminophosphonic acid, and the like.

In the context of the present invention, the term “reporter group” means a group that can be detected on its own or during a detection analysis. Examples of functional reporter groups are biotin, digoxygenin, fluorescence groups (groups that are capable of absorbing electromagnetic radiation, such as visible light or x-rays, of various wavelengths, followed by remission of the absorbed energy in the form of radiation with a longer wavelength: illustrative examples are DANSYL [( 5-dimethylamino) -1-naphthalenesulfonyl], DOXYL [N-oxyl-4,4-dimethyloxazolidine], PROXYL [N-oxyl-2,2,5,5-tetramethylpyrrolidinyl], TEMPO [M-oxyl-2,2, 6,6-tetramethylpiperidine], dinitrophenol, acridines, coumarin s, Cγ 3 and Cγ 5 (trademarks of Biological Detection Systems Inc.), erythrosine, coumaric acid, umbelliferone, Texas red, rhodamine, tetramethylrodamine, Rox reagent, 7-nitrobenzo-2-oxa-1-diazole (NBD) , pyrene, fluorescein, europium, ruthenium, samarium and other rare earth metals), radioisotope tags, chemoluminescent tags (tags that are detected by the emission of visible light during a chemical reaction), spin tags (free radical, for example, substituted organic nitroxide, or other paramagnetic probes, e.g., Cu ^2+, Mg ^2+, connectedness f with a biological molecule that can be detected using electron spin resonance spectrometry), enzymes (such as peroxidases, alkaline phosphatases, β-galactosidases, and glycosoxidases), antigens, antibodies, haptens (groups that can combine with the antibody, but which cannot determine the immune response on their own, such as peptides and steroid hormones), carrier systems designed to penetrate cell membranes, such as residues of fatty acids, steroid components (choles Erin), vitamin A, vitamin D, vitamin E, folic acid, ligands specific receptors, groups for mediating endocytose, epidermal growth factor (EGF), bradykinin, and platelet derived growth factor (PDGF). Particularly interesting examples are biotin, fluorescein, Texas red, rhodamine, dinitrophenol, digoxygenin, ruthenium, europium, Cγ 5, Cγ 3, and the like.

In the context of this invention, the term “ligand” means something that can bind. Ligands can be functional groups, such as aromatic groups (such as benzene, pyridine, naphthalene, anthracene and phenanthracene), heteroaromatic groups (such as thiophene, furan, tetrahydrofuran, pyridine, dioxane and pyrimidine), carboxylic acids, complex 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 alcohols mouths, tertiary alcohols, phenols, alkyl halides, thiols, disulfides, primary amines, secondary amines, tertiary amines, hydrazines, epoxides, maleic acid imides, C _1-20 alkyl groups optionally interrupted or terminated by one or more heteroatoms such as atoms oxygen nitrogen atoms and / or sulfur atoms optionally containing aromatic or mono / polyunsaturated hydrocarbons, polyoxyethylene such as polyethylene glycol, oligo / polyamides such as poly-β-alanine, polyglycine, polylysine, peptides, oligo / polysaccharide , Oligo / polyphosphates, toxins, antibiotics, cell poisons, and steroids, as well as "affinity ligands", ie functional groups or biological molecules characterized by specific affinity for the sites of specific proteins, antibodies, poly- and oligosaccharides and other biological molecules.

It should be clear to those skilled in the art that the above specific examples of DNA intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands correspond to the “active-functional” part of these considered groups. It should also be understood by a person skilled in the art that DNA intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands are usually presented in the form of M-K-, where M is “active is the “functional” part of the group under consideration, and K is the spacer through which the “active-functional” part is attached to the 5- or 6-atom ring. Thus, it should be understood that group B, in the case when B is selected from DNA intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands, has the form M-K-, in which M is The "active-functional" part of the DNA intercalating group, the photochemically active group, the thermochemically active group, the chelating group, the reporter group, and the ligand, respectively, and K is an optional spacer consisting of 1-50 atoms, preferably 1-30 atoms, in private whith 1-15 atoms, located between the 5- or 6-atom ring and "active-functional" part.

, а в некоторых случаях предпочтительно составляет менее 100

. Таким образом, спейсер включает цепочку атомов углерода, необязательно прерываемую или терминируемую одним или несколькими гетероатомами, такими как атомы кислорода, атомы азота и(или) атомы серы. Таким образом спейсер К может включать одну или несколько функциональных групп амидов, сложных эфиров, аминогрупп, простых эфиров и(или) тиоэфиров, а также необязательно ароматические или моно/полиненасыщенные углеводороды, полиоксиэтилен, такой как полиэтиленгликоль, олиго/полиамиды, такие как поли-β -аланин, полиглицин, полилизин, пептиды в целом, олигосахариды, олиго/полифосфаты. Более того, спейсер может включать их комбинированные сочетания. Длина спейсера может варьироваться, принимая во внимание желательное или являющееся необходимым расположение и пространственную ориентацию “активно-функциональной” части рассматриваемой группы по отношению к 5- или 6-атомному кольцу. В конкретных представляющих интерес вариантах спейсер включает химически отщепляемую группу. Примеры таких химически отщепляемых групп включают дисульфидные группы, отщепляемые в восстановительных условиях, пептидные фрагменты, отщепляемые с участием пептидаз, и т.п.In the context of the present invention, the term “spacer” means a thermochemically and photochemically inactive, creating a “gap” group, which is used to connect two or more different components described above. Spacers are selected based on a number of parameters, including their hydrophobicity, hydrophilicity, molecular flexibility and size (see, for example, Hermanson et al., 1992, Immobilized Affinity Ligand Techniques, Acad. Press, San Diego, CA, pp. 137 -ff). In general, the spacer length is less than or approximately equal to 400

and in some cases is preferably less than 100

. Thus, the spacer includes a chain of carbon atoms, optionally interrupted or terminated by one or more heteroatoms, such as oxygen atoms, nitrogen atoms and (or) sulfur atoms. Thus, spacer K may include one or more functional groups of amides, esters, amino groups, ethers and / or thioethers, as well as optionally aromatic or mono / polyunsaturated hydrocarbons, polyoxyethylene such as polyethylene glycol, oligo / polyamides, such as poly β-alanine, polyglycine, polylysine, peptides in general, oligosaccharides, oligo / polyphosphates. Moreover, the spacer may include combinations thereof. The length of the spacer can vary, taking into account the desired or necessary location and spatial orientation of the “active-functional” part of the group under consideration with respect to the 5- or 6-atom ring. In particular embodiments of interest, the spacer includes a chemically cleavable group. Examples of such chemically cleavable groups include disulfide groups cleaved under reducing conditions, peptide fragments cleaved with the participation of peptidases, and the like.

In one embodiment of the present invention, K represents a single bond in such a way that the “active-functional” part of the group in question is directly attached in a 5- or 6-atom ring.

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

In the oligomers of the present invention (Formula I), P denotes the position of the radical for the internucleoside “bridge” followed by a monomer or a 5'-terminal group. The first of these possibilities is realized when the LNA under consideration is not a 5'-terminal monomer, while the second possibility corresponds to the fact that the LNA under consideration is such a monomer. It should be understood (which also follows clearly from the definition of the internucleoside “bridge” and the 5'-terminal group given below) that such an internucleoside “bridge” and the 5'-terminal group may include the substituent R ⁵ (or equally applicable and substituent R ⁵ *), resulting in a double bond with the R.

On the other hand, the internucleoside “bridge” with the preceding monomer or the 3'-terminal group (P *) can be formed from the positions defined by the substituents R ² , R ² *, R ³ and R ³ *, preferably from the positions defined by one of the substituents R ³ and R ³ *. Similarly, the first possibility is realized when the LNA under consideration is not a “3'-terminal monomer," while the last of these possibilities corresponds to the fact that the LNA under consideration is such a monomer. (The term “3'-terminal” denotes the position corresponding to the carbon atom 3 'in the ribose component of the nucleoside).

In the context of this invention, the term “monomer” refers to naturally occurring nucleosides that are not found under natural conditions nucleosides, RNA, etc., as well as LNA. Thus, the term “downstream monomer” refers to the adjacent monomer in the 5 ’direction, and the term“ upstream monomer ”refers to the adjacent monomer in the 3’ direction. Such subsequent and preceding monomers, located so in relation to the LNA monomer, can be natural or unnatural nucleosides and even other LNAs.

Therefore, in the context of the present invention (which can be understood from the above), the term “oligomer” means an oligonucleotide modified by the inclusion of one or more LNAs.

A key part of the present invention is related to the presence of one or more rings connected by a 5- or 6-atomic ring, as illustrated by the basic formula I. Thus, one or two pairs of non-heminal substituents selected from R ¹ *, R ⁴ *, R ⁵ , R ⁵ *, R ⁶ , R ⁶ *, R ⁷ , R ⁷ *, R ^H * and the substituents R ² , R ² *, R ³ and R ³ *, which do not form P *, in each case form a biradical consisting of from 1-8 groups / atoms, preferably from 1-4 groups / atoms, independently 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. (The term “present” indicates that the existence of certain substituents, for example, R ⁶ , R ⁶ *, R ⁷ , R ⁷ *, R ^N *, depends on whether X includes such substituents).

In the groups forming the biradical (biradicals), Z is selected from —O—, —S— and –N (R ^a ) -, and each of R ^a and R ^{b is} independently selected from hydrogen, optionally substituted with C _1-12 -alkyl, optionally substituted C _2-12 alkenyl, optionally substituted C _2-12 alkynyl, optionally substituted hydroxyl, C _1-12 alkoxy, C _2-12 alkenyloxy, carboxy, C _1-12 alkoxycarbonyl, C _{1 -12} alkyl-carbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, heteroaryl Arbon, amino, mono- and di- (C _1-6 -amino) 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 alkylcarbonylamino groups, carbamido, carbamido, C _1-6 alkanoyloxy groups, sulfonic groups, C _1-6 alkylsulfonyloxy groups, nitro groups, azido groups, sulfanyl, C _1-6 alkylthio group, halogen, DNA intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands (where the last group The substrates may include a spacer as defined for substituent B), wherein aryl and heteroaryl may be optionally substituted and where two geminal substituents R ^a and R ^b together may be optionally substituted methylene (= CH ₂ optionally substituted once or twice with substituents according to the definition as arbitrary substituents for aryl), and despite the fact that two geminal or non-heminal substituents selected from R ^a , R ^b and any of the substituents R ¹ *, R ² , R ² *, R ³ , R ³ * , R ⁴ *, R ⁵ , R ⁵ *, R ⁶ and R ⁶ *, R ⁷ and R ⁷ *, which are present exist and are not involved in P, P * or a biradical (biradicals), together they can form a linked biradical selected from the same type of biradicals as defined above. It should be understood that each of the pairs of non-heminal substituents thereby forms a mono- or bicyclic moiety with the participation of (1) atoms to which the non-heminal substituents are linked, and (2) with any intermediate atoms.

We can assume that biradicals that are bonded to ring atoms in 5- or 6-atomic rings are preferred in the sense that the inclusion of R ⁵ and R ⁵ * substituents may cause undesirable steric interactions with the internucleoside bridge. Therefore, it is preferable that one or two pairs of non-heminal substituents that form one or two biradicals, respectively, are selected from the substituents R ¹ *, R ⁴ *, R ⁵ , R ⁵ *, R ⁶ , R ⁶ *, R ⁷ , R ⁷ *, R ^N *, and the substituents R ² , R ² *, R ³ and R ³ * did not form P *.

It is preferable that the LNAs included in the oligomers contain only one diradical formed by a pair of non-heminal substituents. In particular, it is preferred that R ³ * forms P * and that such a biradical forms between R ² * and R ⁴ * or between R ² and R ³ .

As already mentioned, it should be clear (especially with due attention to the known bi- and tricyclic nucleoside analogues, see the "Background of the invention" section) that the present invention does not apply to oligomers comprising the following bi- or tricyclic nucleoside analogues:

(i) R ² and R ³ simultaneously mean a diradical selected from —O — CH ₂ —CH ₂ - and —O — CH ₂ —CH ₂ —CH ₂ -, where LNA is a bicyclic nucleoside analogue;

(ii) R ³ and R ⁴ simultaneously mean a diradical selected from —CH ₂ —CH ₂ - and —O — CH ₂ -, where LNA is a bicyclic nucleoside analogue;

(iii) R ³ , R ⁵ and R ⁵ * simultaneously mean the triradical –CH ₂ —CH (-) - CH ₂ -, where LNA is a tricyclic nucleoside analogue;

(iv) R ¹ * and R ⁶ * simultaneously mean diradical -CH ₂ -, where LNA is a bicyclic nucleoside analogue; and

(v) R ⁴ * and R ⁶ * simultaneously mean diradical -CH ₂ -, where LNA is a bicyclic nucleoside analogue;

with the exception that such bi- or tricyclic nucleoside analogs are combined with one or more new LNAs as defined by the present invention.

In the context of the present invention, i.e. in this detailed description and in the claims, the orientation of the biradicals is such that the side on the left hand is occupied by the substituent with the lowest number, and the side on the right hand is occupied by the substituent with the highest number: therefore, when R ³ and R ⁵ simultaneously mean the “-O biradical -CH ₂ - ”, it is understood that the oxygen atom corresponds to the substituent R ³ , i.e. an oxygen atom is attached at the position of the substituent R ³ , and the methylene group is a substituent R ⁵ .

As regards the numerous interesting structural capabilities of the biradical (biradicals) in the LNA oligomers of the present invention, it can be considered that the biradical (biradicals) formed by a pair of non-heminal substituents is preferably selected from (CR * R *) _r - (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 *) -,> C = O, -C (= O) -N (R *) - and -N (R *) - С (= O) -, where each R * is independently selected from H, halogen, azido group, cyano group, nitro group, hydroxyl, mercapto group, amino group s, mono- or di (C _1-6 -alkyl) amino, optionally substituted C _1-6 alkoxy, optionally substituted C _1-6 -alkyl, DNA-intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands, and (or) two neighboring (non-heminal) R * can form a double bond; and each of r and s is 0-4 provided that the sum of r + s is 1-5. Particularly interesting options are those 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-, despite the fact that each of r and s is 0-3, provided that the sum of r + s is 1-4.

Regarding the position of the biradical in the LNA, it can be considered (based on preliminary data - see examples) that the following options are of the greatest interest, such as: R ² * and R ⁴ * simultaneously mean a biradical selected from -Y-, - (CR * R *) _{r + s + 1} -, - (CR * R *) _r -Y- (CR * R *) _s - and –Y- (CR * R *) _{r + s} -Y-; R ² and R ³ simultaneously mean a biradical selected from -Y-, - (CR * R *) _{r + s} -, - (CR * R *) _r -Y- (CR * R *) _s - and –Y- (CR * R *) _{r + s} -Y-; R ² * and R ³ simultaneously mean a biradical selected from -Y-, (CR * R *) _{r + s} -, - (CR * R *) _r -Y- (CR * R *) _s - and –Y- (CR * R *) _{r + s} -Y-; R ³ and R ⁴ * simultaneously mean a biradical selected from -Y-, - (CR * R *) _{r + s} -, - (CR * R *) _r -Y- (CR * R *) _s - and –Y - (CR * R *) _{r + s} -Y-; R ³ and R ⁵ simultaneously mean a biradical selected from -Y'-, - (CR * R *) _{r + s + 1} -, - (CR * R *) _r -Y- (CR * R *) _s - and –Y- (CR * R *) _{r + s} -Y-; R ¹ * and R ⁴ * simultaneously mean a biradical selected from -Y'-, - (CR * R *) _{r + s + 1} -, - (CR * R *) _r -Y- (CR * R *) _s - and –Y- (CR * R *) _{r + s} —NR * -; R ¹ * and R ² * simultaneously mean a biradical selected from -Y-, - (CR * R *) _{r + s} -, - (CR * R *) _r -Y- (CR * R *) _s - and - Y- (CR * R *) _{r + s} -Y-; despite the fact that each r and s is 0-3, provided that the sum r + s is 1-4, Y is as defined above, and Y 'is selected from -NR * -C (= O) - and -C (= O) -NR * -.

Of particular interest, oligomers are those for which one of the following criteria is met for at least one LNA included in the oligomer: R ² * and R ⁴ * simultaneously mean a diradical 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 *), R ² and R ³ simultaneously mean a biradical selected from -O-, (CR * R *) _{r + s} -, - (CR * R *) _r -O- (CR * R *) _s -, - (CR * R *) _r -S- (CR * R *) _s - and (CR * R *) _r -N (R *) - (CR * R *) _s -; R ² * and R ³ simultaneously mean a biradical selected from -O-, - (CR * R *) _{r + s} -, - (CR * R *) _r -O- (CR * R *) _s -, (CR * R *) _r -S- (CR * R *) _s - and - (CR * R *) _r -N (R *) - (CR * R *) _s -; R ³ and R ⁴ * simultaneously mean a biradical selected from (CR * R *) _r -O- (CR * R *) _s -, - (CR * R *) _r -S- (CR * R *) _s - and - (CR * R *) _r -N (R *) - (CR * R *) _s -; R ³ and R ⁵ simultaneously form a biradical selected from (CR * R *) _r -O- (CR * R *) _s -, (CR * R *) _r -S- (CR * R *) _s - and - (CR * R *) _r -N (R *) - (CR * R *) _s -; R ¹ * and R ⁴ * simultaneously mean a biradical selected from - (CR * R *) _r -O- (CR * R *) _s -, - (CR * R *) _r -S- (CR * R *) _s - and (CR * R *) _r -N (R *) - (CR * R *) _s -; or R ¹ * and R ² * simultaneously mean a biradical selected from - (CR * R *) _r -O- (CR * R *) _s -, - (CR * R *) _r -S- (CR * R * ) _s - and - (CR * R *) _r -N (R *) - (CR * R *) _s -, despite the fact that each r and s is 0-3 provided that the sum r + s is 1 -4, and while R ^H is hydrogen or C _1-4 alkyl.

It is further preferred that one of R * is selected from hydrogen, hydroxyl, optionally substituted C _1-6 alkoxy, optionally substituted C _1-6 alkyl, DNA intercalating groups, photochemical active groups, thermochemically active groups, chelating groups, reporter groups and ligands, and all other R * were hydrogen.

In one preferred embodiment, one R * group in the biradical of at least one of the LNAs is selected from DNA intercalating groups, photochemical active groups, thermochemically active groups, chelating groups, reporter groups, and ligands (where the latter groups may include a spacer in accordance with specific to substituent B).

As for the substituents R ¹ *, R ² , R ² *, R ³ , R ³ *, R ⁴ *, R ⁵ , R ⁵ *, R ⁶ and R ⁶ *, R ⁷ and R ⁷ *, which are present and not involved in the formation of P, P * or a diradical (diradicals), they are independently selected from hydrogen, optionally substituted C _1-12 alkyl, optionally substituted C _2-12 alkenyl, optionally substituted C _2-12 alkynyl, hydroxyl, C _1-12 alkoxy groups, C _2-12 alkenyloxy groups, carboxy groups, C _1-12 alkoxycarbonyl, C _1-12 alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy group, arylcarbonyl, heteroaryl, heteroaryloxy carbonyl, heteroaryloxy groups, heteroarylcarbonyl, amino groups, mono and di (C _1-6 amino) amino groups, 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 alkylcarbonylamino groups, carbamido, C _1-6 alkanoyloxy groups, sulfonic groups, C _1-6 alkylsulfonyloxy groups, nitro groups, azido groups, sulfanyl, C _1-6 alkylthio groups, halogen, DNA intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter g Rupp and ligands (where the latter groups may include a spacer as defined for substituent B), where aryl and heteroaryl may optionally be substituted, and where two geminal substituents together may form an oxo group, thioxo group, imino group or optionally substituted methylene, or together they can form a spiro radical. including a 1-5-carbon alkylene chain, which is optionally interrupted and (or) terminated by one or more heteroatoms / groups selected from —O—, —S— and - (NR ^N ) -, where R ^{N is} selected and h of hydrogen and C _1-4 alkyl and where two adjacent (but geminal) substituents can form an additional double bond; and R ^N *, when present and not included in the diradical, is selected from hydrogen and C _1-4 alkyl.

Preferably, each of the substituents R ¹ *, R ² , R ² *, R ³ , R ³ *, R ⁴ *, R ⁵ , R ⁵ *, R ⁶ and R ⁶ *, R ⁷ and R ⁷ * in the composition of the LNA, which are present and not involved in the formation of P, P * or diradical (diradicals) are independently selected from hydrogen, optionally substituted C _1-6 alkyl, optionally substituted C _2-6 alkenyl, hydroxyl, C _1-6 alkoxy, C _2-6- alkenyloxy groups, carboxyl, C _1-6 -alkoxycarbonyl, C _1-6 -alkylcarbonyl, formyl, amino groups, mono- and di (C _1-6 -alkyl) amino groups, carbamoyl, mono- and di (C _{1- 6} -alkyl) aminocarbonyl, C _1-6 -alkilkarbonilaminogruppy, carbo ogruppy, azido, C _1-6 -alkanoiloksigruppy, a sulfonic group, sulfanyl, C _1-6 alkylthio, DNA-intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, and halogen, where two geminal substituents simultaneously form an oxo group and where R ^N *, in the case of presence and non-participation in the formation of a biradical, is selected from H and C _1-4 alkyl.

In a preferred embodiment of the present invention, X is selected from —O—, —S— and - (NR ^N ) -, in particular it is —O—, and each of the substituents R ¹ *, R ² , R ² *, R ³ , R ³ *, R ⁴ *, R ⁵ , R ⁵ *, R ⁶ , R ⁶ *, R ⁷ and R ⁷ * in the LNA, which are present and are not involved in P, P * or biradical (biradicals), are represented by hydrogen .

In an even more preferred embodiment of the present invention, R ² * and R ⁴ * from the LNA included in the oligomer, simultaneously mean biradical. Preferably X is O, R ^{2 is} selected from hydrogen, hydroxyl and an optionally substituted C _1-6 alkoxy group, and R ¹ *, R ³ , R ⁵ and R ⁵ * are hydrogen, and more specifically, such a diradical is 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 . In general, with due attention to the results, it is preferable that the biradical formed by the substituents R ² * and R ⁴ * form a two-carbon “bridge”, i.e. so that this diradical forms a 5-atom furanose ring (X is O).

In another embodiment of the present invention, R ² and R ³ in the LNA included in the oligomer, simultaneously mean biradical. Preferably X is O, R ² * is selected from halogen, hydroxyl and an optionally substituted C _1-6 alkoxy group, R ¹ *, R ⁴ *, R ⁵ and R ⁵ * are hydrogen, and more specifically, such a diradical is selected from - (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 ) _1-4 -, in particular from —O — CH ₂ -, —S — CH ₂ - and –NR ^H —CH ₂ -. In the latter case, amino- and thio-variants are of particular interest.

In another embodiment of the present invention, R ² * and R ³ in the LNA included in the oligomer, simultaneously mean biradical. Preferably X is O, R ^{2 is} selected from halogen, hydroxyl and an optionally substituted C _1-6 alkoxy group, R ¹ *, R ⁴ *, R ⁵ and R ⁵ * are hydrogen, and more specifically, such a diradical is selected from (CH ₂ ) _0-1 -O- (CH ₂ ) _1-3 - and - (CH ₂ ) _2-4 -.

In yet another embodiment of the present invention, R ³ and R ⁴ * in the LNA included in the oligomer, simultaneously mean biradical. Preferably X is O, R ² * is selected from halogen, hydroxyl and an optionally substituted C _1-6 alkoxy group, R ¹ *, R ² , R ⁵ and R ⁵ * are hydrogen, and more specifically, such a diradical is selected from (CH ₂ ) _0-2 -O- (CH ₂ ) _0-2 -.

In a further embodiment of the present invention, R ³ and R ⁵ * in the LNA included in the oligomer simultaneously mean a diradical. Preferably X is O, R ² * is selected from halogen, hydroxyl and an optionally substituted C _1-6 alkoxy group, R ¹ *, R ² , R ⁴ and R ⁵ are hydrogen, and more specifically, such a diradical is selected from —O— (CHR *) _2-3 - and - (CHR *) _1-3 -O- (CHR *) _0-3 -.

In a further embodiment of the present invention, R ¹ * and R ⁴ * in the LNA included in the oligomer, simultaneously mean biradical. Preferably X is O, R ² * is selected from halogen, hydroxyl and an optionally substituted C _1-6 alkoxy group, R ² , R ³ , R ⁵ and R ⁵ * are hydrogen, and more specifically, such a diradical is selected from - (CH ₂ ) _0-2 -O- (CH ₂ ) _0-2 -.

In these embodiments, it is further preferred that at least one LNA included in the oligomer comprise a base (substituent B) selected from adenine and guanine. In particular, it is preferable that the oligomer has an LNA included therein, including at least one base selected from thymine, uracil and cytosine, and at least one base selected from adenine and guanine. For LNA monomers, it is particularly preferred that such a base is selected from adenine and guanine.

For these options of interest, it is also preferred that the LNA is in accordance with the basic formula Ia (see below).

In one branch of these variants of interest, all oligonucleotide monomers are LNA monomers.

As should be obvious from formula I (LNA in the oligomer) (and basic formula II for monomeric LNA - see below) and its definitions, one or more asymmetric carbon atoms may be present in oligomers (and LNA monomers), which depends on the nature of the substituents and probable biradicals, see below. The oligomers constructed in accordance with the method of the present invention, as well as the oligomers themselves, must include all stereoisomers due to the presence of any of and all isomers of the individual monomeric fragments, as well as mixtures thereof, including mixtures of racemates. As for 5- or 6-atom rings, it can be considered that some stereochemical configurations will be of particular interest, for example, the following:

in which wavy lines indicate the possibility of the existence of both diastereomers arising from the interchange of the two considered substituents.

Of particular interest in connection with stereoisomerism is the case in which the LNA is characterized by the following formula Ia:

Also a separate, interesting aspect of the present invention is a variant of formula Ia, in which B is in the α-configuration.

In these cases, as well as in general, R ³ * preferably forms P *.

Oligomers in accordance with the present invention typically consist of 1-10000 LNA basic formula I (or more detailed formula Ia) and 0-10000 nucleosides selected from native nucleosides and nucleoside analogues. The sum of the number of nucleosides and the amount of LNA is at least 2, preferably at least 3, in particular at least 5, particularly preferably at least 7, and is in the range of 2-15000, more preferably in the range of 2-100, also 3 -10, in particular in the range of 2-50, as well as 3-50 or 5-50, or 7-50.

Preferably, at least one LNA includes a nucleotide base as a B substituent.

In the context of the present invention, the term “nucleoside” means a heterocyclic base glycoside. The term “nucleoside” is widely used to refer to nucleosides, native nucleosides, and also other nucleoside analogues that are not found in natural conditions. Illustrative examples of nucleosides are ribonucleosides, including a ribose residue, as well as deoxyribonucleosides, including a deoxyribose residue. As for the base in such nucleosides, it should be understood that it can be any of the naturally occurring bases, for example, adenine, guanine, cytosine, thymine and uracil, as well as any modified versions thereof or any other non-native bases.

When considering the definitions and known nucleosides (native and not found in vivo) and nucleoside analogues (including known bi- and tricyclic analogues), it is clear that the oligomer can include one or more LNAs (which can be identical or differentiated from the point from the point of view of the choice of substituents, and from the point of view of the choice of a biradical), as well as one or more nucleosides and (or) nucleoside analogues. In the context of the present invention, the term “oligonucleotide” refers to a sequential chain of nucleosides connected via internucleoside “bridges”, however, it should be understood that the nucleotide base in one or more units (monomers) of the oligomer (oligonucleotide) can be modified using substituent B in as defined above.

Oligomers can be linear, branched or cyclic. In the case of a branched oligomer, branch points can be located in the nucleoside, in the internucleoside “bridge” or, as in one of the peculiar variants, in the LNA. We can assume that in the latter case, the substituents R ² , R ² *, R ³ and R ³ * can form two groups P *, each of which forms an internucleoside “bridge” with the previous monomer: in particular, one of R ² and R ² * form one P *, and one of R ³ and R ³ * forms the next P *.

As noted above, the LNAs in the oligomer bind to other monomers via the internucleoside “bridge”. In the context of the present invention, the term “internucleoside“ bridge ”” 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 ^{n is} selected from H and C _1-4 alkyl, and R ”is selected from C _1-6 alkyl and phenyl. Illustrative examples of such internucleoside “bridges” are —CH ₂ —CH ₂ —CH ₂ -, —CH ₂ —CO — CH ₂ -, —CH ₂ —CHON — CH ₂ -, —O — CH ₂ —O—, —O —CH ₂ —CH ₂ -, —O — CH ₂ —CH = (including R ⁵ when 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 -ON = (including R ⁵ when used as a link to the 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-N R ^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 when 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-, -OP (O, S) -O-, -OP (S) ₂ -O-, -SP (O) ₂ -O-, -SP (O, S) -O -, -SP (S) ₂ -O-, -OP (O) ₂ -S-, -OP (O, S) -S-, -OP (S) ₂ -S-, -SP (O) ₂ - S-, -SP (O, S) -S-, -SP (S) ₂ -S-, -O-PO (R``) - O-, -O-PO (OCH ₃ ) -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 -, -OP (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 them —CH ₂ —CO — NR ^H -, —CH ₂ —NR ^H —O—, —S — CH ₂ —O—, —OP (O) ₂ —O—, —OP (O, S) —O -, -OP (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 H and C _1-4 alkyl and R ″ is selected from C _1-6 alkyl and phenyl are most preferred. Further illustrative examples are provided by Messmaker et al. (Mesmaeker et al., 1995, Cur. Opinion Struct. Biol., 5, 343-355). The side of the internucleoside “bridge”, located on the left hand, is connected to the 5 or 6-atom ring as a substituent P *, while its side, located on the right hand, is connected to the 5'-site of the previous monomer.

It is also clear from the foregoing that the P group can also constitute a 5'-terminal group when the LNA in question is a 5'-terminal monomer. Examples of such 5'-terminal groups are hydrogen, hydroxyl, optionally substituted C _1-6 alkyl, optionally substituted C _1-6 alkoxy group, optionally substituted C _1-6 alkylcarbonyloxy group, optionally substituted aryloxy group, monophosphate, diphosphate, triphosphate and group -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 intercalating groups , photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands (where the latter groups may include the spacer in accordance with the previously defined for substituent B).

In the present description and claims, the terms “monophosphate”, “diphosphate” and “triphosphate” mean groups having the formula —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 particular embodiment of interest, the P group forms 5'-terminal groups selected from monophosphate, diphosphate and triphosphate. Especially attractive is the case when the substrate is the variant with triphosphate.

Similarly, the P * group can form a 3'-terminal group when the LNA in question is a 3'-terminal monomer. Examples of such 3'-terminal groups are hydrogen, hydroxyl, an optionally substituted C _1-6 alkoxy group, an optionally substituted C _1-6 alkylcarbonyloxy group, an optionally substituted aryloxy group and a group-W-A ', where W is selected from -O-, - S- and -N (R ^H ) -, where R ^{H is} selected from H and C _1-6 -alkyl, and where A 'is selected from DNA intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands (where the latter groups may include a spacer as previously defined for substituent B).

In a preferred embodiment of the present invention, the oligomer is characterized by the following formula V:

where q is 1-50;

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

each of m (1), ....., m (q) independently of each other is equal to 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 native nucleosides and nucleoside analogues;

each LNA is independently a nucleoside analogue;

each L denotes an internucleoside “bridge” between two groups selected from Nu and LNA, or L together with G * forms a 3’-terminal group; and each LNA-L independently denotes a nucleoside analog of basic formula I as defined above or, more preferably, basic formula Ia as defined above.

In this embodiment, as well as in general in the present invention, there is a promising possibility of combining LNA with various (i.e., purine and pyrimidine) bases: in particular, and bases selected from thymine, cytosine and uracil, and with bases selected from adenine and guanine.

In another embodiment of the present invention, the oligomer further comprises a mono- or oligomeric PNA segment of the formula:

while B corresponds to the previous definition for formula I, AASC is hydrogen or an amino acid side chain, t is 1-5 and w is 1-50.

In the context of the present invention, the term “amino acid side chain” means a group attached to the α-atom of α-amino acids, i.e. corresponding to the considered α-amino acid without a glycine moiety, preferably a natural or readily available α-amino acid. Illustrative examples are hydrogen (glycine itself), deuterium (deuterated glycine), methyl (alanine), cyanomethyl (β-cyanoalanine), ethyl, 1-propyl (norvaline), 2-propyl (valine), 2-methyl-1 propyl (leucine), 2-hydroxy-2-methyl-1-propyl (β-hydroxyleucine), 1-butyl (norleucine), 2-butyl (isoleucine), methylthioethyl (methionine), benzyl (phenylalanine), n-aminobenzyl (n-aminophenylalanine), n-iodobenzyl (n-iodophenylalanine), n-fluorobenzyl (n-fluorophenylalanine), n-bromobenzyl (n-bromophenylalanine), n-chlorobenzyl (n-chlorophenylalanine), n-nitrobenzyl (n-nitrophenyl) 3-pyridylmethyl (β - [3-pyridyl] -alanine), 3,5-diiod-4-hydroxybenzyl (3,5-diiodotyrosine), 3,5-dibromo-4-hydroxybenzyl (3,5-dibromothyrosine), 3,5- dichloro-4-hydroxybenzyl (3,5-dichlorothyrosine), 3,5-difluoro-4-hydroxybenzyl (3,5-difluorothyrosine), 4-methoxybenzyl (O-methyl tyrosine), 2-naphthylmethyl (β - [2-naphthyl] -alanine), 1-naphthylmethyl (β - [1-naphthyl] -alanine), 3-indolylmethyl (tryptophan), hydroxymethyl (serine), 1-hydroxyethyl (threonine), mercaptomethyl (cysteine), 2-mercapto-2-propyl (penicillamine), 4-hydroxybenzyl (tyrosine), aminocarbonylmethyl (asparagine), 2-aminocarbonylethyl (glutamine), carboxylmethyl (asparagus) new acid), 2-carboxyethyl (glutamic acid), aminomethyl (α, β-diaminopropionic acid), 2-aminoethyl (α, γ-diaminobutyric acid), 3-aminopropyl (ornithine), 4-amino-1-butyl (lysine ), 3-guanidino-1-propyl (arginine) and 4-imidazolylmethyl (histidine).

The mono- and oligomeric PNA segment can be included in the oligomer as described in EP 0672677 A2.

The oligomers of the present invention are also intended to include chimeric oligomers. The term “chimeric oligomers” means two or more oligomers, including monomers of various origins, connected directly to each other or via a spacer. Illustrative examples of such oligomers that can be recombined are peptides, RNA oligomers containing LNA oligomers and oligonucleotide oligomers.

In addition to the oligomers described above, the present invention also provides monomeric LNAs useful, for example, for the synthesis of oligomers, as substrates for, for example, nucleic acid polymerases, polynucleotide kinases, terminal transferases and as therapeutic agents: see below. Monomeric LNAs in their general structure (especially regarding possible diradicals) correspond to those LNAs that are defined as components of oligomers, however, as for the P and P * groups, monomeric LNAs are slightly different, which is explained below. In addition, monomeric LNAs can be protected in terms of their functional components, especially when monomeric LNAs are to be included in oligomers using chemical synthesis methods.

A subgroup of monomeric LNAs of interest includes bicyclic nucleoside analogues (LNAs) of the general formula II:

where Deputy B is selected from bases, DNA intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands; X is selected from —O—, —S—, —N (R ^H *) - and —C (R ⁶ R ⁶ *) -, preferably from —O—, —S— and —N (R ^H *) -; one of the substituents R ² , R ² *, R ³ and R ³ * is a group Q *;

each of Q and Q * is independently selected from hydrogen, azido group, halogen, cyano group, nitro group, hydroxyl, Prot-O-, Act-O-, mercapto group, Prot-S-, Act-S-, C _1-6 alkylthio group, amino groups, Prot-N (R ^H ) -, Act-N (R ^H ) -, mono- or di (C _1-6 alkyl) amino groups, optionally substituted C _1-6 alkoxy groups, optionally substituted C _1-6 - alkyl, optionally substituted C _2-6 -alkenyl, optionally substituted C _2-6 -alkeniloksigruppy optionally substituted C _2-6 -alkynyl, optionally substituted C _2-6 -alkiniloksigruppy, monophosphate, diphosphate, triphosphate, DNA-intercalation uyuschih groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands, carboxy, sulfonic groups, 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, and “Act” is the activation group for —OH, —SH and —NH (R ^H ), respectively, a R ^{H is} selected from H and C _1-6 alkyl;

R ² * and R ⁴ * simultaneously mean a biradical selected from -O-, -S-, -N (R *) -, - (CR * R *) _{r + s + 1} -, - (CR * R *) _r -O- (CR * R *) _s -, (CR * R *) _r -N- (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 -N- (CR * R *) _{r + s} -N (R *) -; R ² and R ³ simultaneously mean a biradical selected from -O-, (CR * R *) _{r + s} , - (CR * R *) _r -O- (CR * R *) _s -, - (CR * R *) _r -S- (CR * R *) _s - and - (CR * R *) _r -N (R *) - (CR * R *) _s -; R ² * and R ³ simultaneously mean a biradical selected from —O—, - (CR * R *) _{r + s} -, - (CR * R *) _r —O- (CR * R *) _s -; (CR * R *) _r -S- (CR * R *) _s - and (CR * R *) _r -N (R *) - (CR * R *) _s -; R ³ and R ⁴ * simultaneously mean a biradical selected from (CR * R *) _r -O- (CR * R *) _s -, - (CR * R *) _r -S- (CR * R *) _s - and - (CR * R *) _r -N (R *) - (CR * R *) _s -; R ³ and R ⁵ simultaneously mean a biradical selected from - (CR * R *) _r -O- (CR * R *) _s -, - (CR * R *) _r -S- (CR * R *) _s - and - (CR * R *) _r -N (R *) - (CR * R *) _s -; R ¹ * and R ⁴ * simultaneously mean a biradical selected from - (CR * R *) _r -O- (CR * R *) _s -, - (CR * R *) _r -S- (CR * R *) _s - and (CR * R *) _r -N (R *) - (CR * R *) _s -; R ¹ * and R ² * simultaneously mean a biradical selected from - (CR * R *) _r -O- (CR * R *) _s -, - (CR * R *) _r -S- (CR * R *) _s - and (CR * R *) _r -N (R *) - (CR * R *) _s -; where R * corresponds to the above for the oligomers and each of the substituents R ¹ *, R ² , R ² *, R ³ , R ⁴ *, R ⁵ and R ⁵ *, which are not involved in the formation of Q, Q * or biradical, correspond to what has been determined for oligomers.

It should also be understood, given due attention to known bicyclic nucleoside analogues, that R ² and R ³ together do not form a biradical selected from —O — CH ₂ —CH ₂ - and —O — CH ₂ —CH ₂ —CH ₂ - ; and R ³ and R ⁵ together do not form a diradical selected from —CH ₂ —CH ₂ -, —O — CH ₂ - and —O — Si ( ⁱ Pr) ₂ —O — Si ( ⁱ Pr) ₂ —O—.

Monomeric LNAs also include their basic salts and acid addition salts.

Moreover, it should be understood that any chemical group (including any base) that is reactive under those conditions that occur during the chemical synthesis of oligonucleotides, optionally can be protected in accordance with the well-known in the art. This means that groups such as hydroxyl amino group, sulfonic group, carboxyl group and mercapto group, as well as nucleotide bases in the composition of monomeric LNA may not necessarily be protected in their functional components. The attachment of the protective group (as well as the release from it) is carried out using methods known to specialists in this field of technology (see, for example, TWGreene & PGMWuts, 1991, "Protective Groups in Organic Synthesis", 2 ^nd ed., J. Wiley, NY and MJGait, 1984, "Oligonucleotide Synthesis", IRL Press).

Illustrative examples of protecting groups for a hydroxyl group are optionally substituted trityl, such as 4,4'-dimethoxytrityl (DMT), 4-monomethoxytrityl (MMT), and trityl, optionally substituted with 9- (9-phenyl) xanthenyl (pixel), optionally substituted ethoxycarbonyloxy group, p-phenylazophenyloxycarbonyloxy group, tetrahydropyronyl (THP), 9-fluorenylmethoxycarbonyl (Fmoc), methoxytetrahydropyranyl (MTHP), silyloxy group, such as trimethylsilylpropyltrimethylsilyl (TMS) il, benzyloxycarbonyl or substituted benzyloxycarbonyl esters such as 2-bromobenzyloxycarbonyl, tert-butyl esters, alkyl esters such as methyl ether, acetals (including two hydroxyl groups), an acyloxy group such as acetyl or halosubstituted acetyls, for example chloroacetyl or , isobutyryl, pivaloyl, benzoyl and substituted benzoyls, methoxymethyl (MOM), benzyl esters or substituted benzyl esters such as 2,6-dichlorobenzyl (2,6-Cl ₂ Bzl). Alternatively, the hydroxyl group may be protected by attachment to a solid support, optionally via a linker.

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

Illustrative examples of protecting groups for the carboxyl group are allyl ethers, methyl ethers, ethyl ethers, 2-cyanoethyl ethers, trimethylsilylethyl ethers, benzyl ethers (Obzl), 2-adamantyl ethers (O-2-Ada), cyclohexyl ethers (OsNex) , 3-oxazolines, oxazole, 1,3-oxazolidines, amides or hydrazides.

Illustrative examples of protecting groups for mercapto groups are trityl (Trt), acetamidomethyl (AFM), trimethylacetamidomethyl (Tcm), 2,4,6-trimethoxybenzyl (Tmob), tert-butylsulfenyl (StBu), 9-fluorenylmethyl (Fm), 3-nitro -2-pyridinesulfenyl (Npys) and 4-methylbenzyl (Meb).

Further, it may be necessary or desirable to protect any of the bases included in the monomeric LNA, especially when such a monomeric LNA is to be included in the oligomer of the present invention. In the context of the present invention, the term “protected bases” means that the base in question carries a protecting group selected from groups that are well known to those skilled in the art (see, for example, "Protocols for Oligonucleotides and Analogs", vol. 20, ed. S. Agrawal, Humana Press, Totowa, NJ, 1993; SLBeaucage & RPIyer, 1993, Tetrahedron, 49, 6123; SLBeaucage & RPIyer, 1992, Tetrahedron, 48, 2223; and E. Uhlmann & A. Payman, Chem. Rev. 90, 543). Illustrative examples are benzoyl, isobutyryl, gret-butyl, tert-butyloxycarbonyl, 4-chlorobenzyloxycarbonyl, 9-fluorenylmethyl, 9-fluorenylmethyloxycarbonyl, 4-methoxybenzoyl, 4-methoxytriphenylmethyl, optionally substituted triazole, p-toluenesulfonyl, n-optionally substituted triazolephenol, optionally substituted amidines, optionally substituted trityl, phenoxyacetyl, optionally substituted acyl, pixel, tetrahydropyranyl, optionally substituted silyl ethers and 4-methoxybenzyloxycarbonyl. Other suitable examples are presented in Chapter 1 of the monograph "Protocols for oligonucleotide conjugates", "Methods in Molecular Biology", vol. 26, ed. S. Agrawal, Humana Press, Totowa, NJ, 1993; S. L. Beaucage & R. P. Iyer, 1992, Tetrahedron, 48, 2223.

In a preferred embodiment, the monomer LNA group B is preferably selected from nucleotide bases and protected bases.

In the monomeric LNA embodiment of the present invention, one of the groups Q and Q *, preferably Q *, form a group selected from Act-O-, Act-S-, Act-N (R ^H ) -, Act-O-CH ₂ - , Act-O-CH ₂ -, Act-N (R ^H ) -CH ₂ -, and the other Q and Q *, preferably Q *, form a group selected from hydrogen, azido group, halogen, cyano group, nitro group, hydroxyl, Prot -O-, mercapto groups, Prot-S-, C _1-6 alkylthio groups, amino groups, Prot-N (R ^H ) -, mono- or di (C _1-6 -alkyl) amino groups, optionally substituted with C _1-6 - alkoxy, optionally substituted C _1-6 -alkyl, optionally substituted C _2-6 -alkenyl, optionally replaced ennoy -alkeniloksigruppy C _2-6, optionally substituted C _2-6 -alkynyl, optionally substituted C _2-6 -alkiniloksigruppy, monophosphate, diphosphate, triphosphate, DNA-intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups , ligands, carboxyl, sulfonic group, 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 case, the group “Prot” denotes a protecting group for —OH, —SH and —NH (R ^H ), respectively. Such protecting groups are selected from the same groups as defined above for hydroxyl protecting groups, mercapto protecting groups and amino protecting groups, respectively, taking into account the need to ensure stability and reversibility of the protecting groups. However, it is preferable that any protecting group for hydroxyl be selected optionally substituted trityl, such as dimethoxytrityl (DMT), monomethoxytrityl (MMT) and trityl, and 9- (9-phenyl) xanthenyl (pixel), optionally substituted tetrahydropyranyl (THP) (other hydroxyl protecting groups suitable for carrying out phosphoramidite synthesis of oligonucleotides are described in 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, ed. S. Agrawal, Humana Press, Totowa, NJ, 1993) or was protected an acetal; so that any protecting group for —SH is selected from trityl such as dimethoxytrityl (DMT), monomethoxytrityl (MMT) and trityl, and 9- (9-phenyl) xanthenyl (pixel), optionally substituted tetrahydropyranyl (THP) (other protecting groups for the mercapto group suitable for carrying out phosphoramidite synthesis of oligonucleotides are described in Agrawal - cit. above); and that any protecting group for the –NH (R ^H ) group is selected from trityl such as dimethoxytrityl (DMT), monomethoxytrityl (MMT) and trityl, and 9- (9-phenyl) xanthenyl (pixyl), optionally substituted tetrahydropyranyl (THP) (other amino protecting groups suitable for the phosphoramidite synthesis of oligonucleotides are described in Agrawal cit. above).

In the embodiment described above, as well as for all variants of the monomeric LNA described herein, the “Act” group refers to the activation group for —OH, —SH, and –N (R ^H ), respectively. Such activation groups are selected, for example, from optionally substituted O-phosphoramidite, optionally substituted O-phosphorus diester, optionally substituted O-phosphorus diester, optionally substituted P-phosphonate and optionally substituted O-phosphonate.

In the context of the present invention, the term “phosphoramidite” means a group of the formula —P (OR ^x ) —N (R ^v ) ₂ , where R ^x is an optionally substituted alkyl group, for example methyl, 2-cyanoethyl or benzyl, and each R ^v group is optional substituted alkyl groups, for example ethyl or isopropyl, or the –N (R ^v ) ₂ group forms the morpholino group —N (CH ₂ CH ₂ ) ₂ O. Rx is preferably 2-cyanoethyl and the two R ^v groups are preferably identical and form isopropyl . Thus, the most suitable phosphoramidite is N, N-diisopropyl-O- (2-cyanoethyl) phosphoramidite.

It should be understood that the protective groups used in the present invention for individual LNA monomers or several monomeric LNAs can be selected in such a way that when the LNAs are added to the oligomer of the present invention, it is possible either to simultaneously release the protective group or sequential deprotection of protected functional groups. The latter situation opens up the possibility of introducing selectively at a specific site one or more “active / functional” groups, such as DNA intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands, when such groups can be attached via a spacer as described above.

In a preferred embodiment, Q is selected from hydrogen, azido group, halogen, cyano group, nitro group, hydroxyl, Prot-O-, mercapto group, Prot-S-, C _1-6 alkylthio group, amino group, Prot-N (R ^H ) -, mono or di (C _1-6 alkyl) amino group, optionally substituted C _1-6 alkoxy group, optionally substituted C _1-6 alkyl, optionally substituted C _2-6 alkenyl, optionally substituted C _2-6 alkenyloxy group, optionally substituted C _2-6 alkynyl, optionally substituted C _2-6 alkynyloxy groups, monophosphate, diphosphate, triphosphate, DNA intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands, carboxyl, sulfonic group, hydroxymethyl, Prot-O-CH ₂ -, aminomethyl, Prot-N (R ^H ) -CH ₂ -, carboxymethyl, sulfonomethyl, where “Prot” is a protecting group for —OH, —SH and —NH (R ^H ), respectively, a R ^{H is} selected from H and C _1-6 alkyl; and Q * are selected from hydrogen, azido group, halogen, cyano group, nitro group, hydroxyl, Act-O-, mercapto group, Act-S-, C _1-6 alkylthio group, amino group, 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 -alkeniloksigruppy optionally substituted C _2-6 alkynyl, optionally substituted C _2-6 alkynyloxy group, monophosphate, diphosphate, triphosphate, DNA intercalating groups, photochemically active groups pp, thermochemically active groups, chelating groups, reporter groups, ligands, carboxyl, sulfonic group, hydroxymethyl, Prot-O-CH ₂ -, aminomethyl, Prot-N (R ^H ) -CH ₂ -, carboxymethyl, sulfonomethyl, where “Act ”Is an activation group for —OH, —SH and —NH (R ^H ), respectively, and R ^{H is} selected from hydrogen and C _1-6 alkyl.

Monomeric LNAs of basic formula II may, despite the fact that they are introduced into the oligomers, represent various stereoisomers. Therefore, the stereochemical variants described above for LNAs introduced into oligomers can be considered equally applicable to the variant with monomeric LNAs (however, it should be noted that P must be replaced by Q in this case).

In a preferred embodiment of the present invention, monomeric LNA is characterized by basic formula IIa:

where the substituents correspond to the above definitions.

Moreover, in connection with the definitions of substituents, biradicals, R * and the like, the same preferred embodiments described above for the oligomer of the present invention are also applicable to the case of monomeric LNAs.

In a particular preferred embodiment, the monomeric LNAs of the present invention B correspond to a base, and preferably the base is selected from thymine, cytosine, uracil, adenine and guanine (in particular from adenine and guanine), X is —O—, R ² * and R ⁴ * at the same time mean 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-, R ³ * is Q *, which forms Act-OH, and each of R ¹ *, R ² , R ³ , R ⁵ and R ⁵ * is hydrogen . In this embodiment, R ^N may also be selected from DNA intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands.

In yet another embodiment of particular interest, the monomeric LNAs of the present invention are represented by a base, preferably a base selected from thymine, cytosine, uracil, adenine and guanine (in particular adenine and guanine), X is —O—, R ² *, and R ⁴ * simultaneously means 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 H and C _1-4 alkyl; Q is selected from hydroxyl, mercapto group, C _1-6 alkylthio group, amino group, mono- or di (C _1-6 alkyl) amine ogruppy optionally substituted C _1-6 alkoxy, optionally substituted C _2-6 -alkeniloksigruppy optionally substituted C _2-6 -alkiniloksigruppy, monophosphate, diphosphate, and triphosphate, R ³ * - is Q *, is selected from hydrogen, azido, halogen , cyano groups, nitro groups, hydroxyl, mercapto groups, C _1-6 alkylthio groups, amino groups, mono or di (C _1-6 alkyl) amino groups, optionally substituted C _1-6 alkoxy groups, optionally substituted C _1-6 alkyl, optionally substituted C _2-6 -alkenyl, optionally substituted C _2-6 -alkeniloksigrupp Optionally substituted C _2-6 -alkynyl and optionally substituted C _2-6 -alkiniloksigruppy, 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 each of R ¹ *, R ² , R ⁵ and R ⁵ * are hydrogen. Also in this case, R ^N may be selected from DNA intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands.

In yet another particular embodiment of the monomeric LNA of the present invention, B is represented by base, X is —O—, R ² and R ^{3 are} both a diradical selected from (CH ₂ ) _0-1 —O — CH = CH—, (CH ₂ ) _0-1 -S-CH = CH- and (CH ₂ ) _0-1 -N (R ^N ) -CH = CH-, where R ^{N is} selected from H and C _1-4 -alkyl, Q is selected from hydroxyl , mercapto groups, C _1-6 alkylthio groups, amino groups, mono- or di (C _1-6 alkyl) amino groups, optionally substituted C _1-6 alkoxy groups, optionally substituted C _2-6 alkenyloxy groups, optionally substituted C _2-6 -alkynyloxy groups, monophosphate, diphos ata and triphosphate, R ³ * - is Q *, is selected from hydrogen, azido, halogen, cyano, nitro, hydroxyl, 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-} alkynyloxy groups, and each of R ¹ *, R ² *, R ⁵ and R ⁵ * are hydrogen.

In one aspect of the present invention, various LNA derivatives for solid or liquid incorporation into an oligomer are provided. Cited as illustrative examples of monomers suitable for the introduction of (1S, 3R, 4R, 7S) -7-hydroxy-1-hydroxymethyl-3- (thymin-1-yl) -2,5-dioxabicyclo [2.2.1] heptane, (1S, 3R, 4R, 7S) -7-hydroxy-1-hydroxymethyl-3- (cytosin-1-yl) 2,5-dioxabicyclo [2.2.1] heptane, (1S, 3R, 4R, 7S) -7 -hydroxy-1-hydroxymethyl-3- (uracil-1-yl) 2,5-dioxabicyclo [2.2.1] heptane, (2S, 3R, 4R, 7S) -7-hydroxy-1-hydroxymethyl-3- ( guanine-1-yl) 2,5-dioxabicyclo [2.2.1] heptane, [1S, 3R, 4R, 7S) -7-hydroxy-1-hydroxymethyl-3- (adenine-1-yl) 2,5-dioxabicyclo [2.2.1] heptane using the phosphoramidite method, the phosphotriether method and hydrogen phosphon methods, respectively, are (1R, 3R, 4R, 7S) -7- (2-cyanoethoxy (diisopropylamino) -phosphinoxy) -1- (4,4'-dimethoxy-nitroxymethyl) -3- (thymin-1-yl) - 2,5-dioxabicyclo [2.2.1] heptane, (1R, 3R, 4R, 7S) -7- (4,4'-dimethoxytrityloxymethyl) -3- (thymin-1-yl) 2,5-dioxabicyclo- [2.2 .1] heptane-7-O- (2-chlorophenylphosphate) and [1R, 3R, 4R, 7S) -7- (4,4'-dimethoxytrityloxymethyl) -3- (thymin-1-yl) 2,5-dioxabicyclo [2.2.1] heptan-7-O- (hydrogen phosphonate), as well as their analogues 3- (cytosin-1-yl), 3- (uracil-1-yl), 3- (adenine-1-yl) and 3 - (guanine-1-yl), respectively. In addition, analogs in which methyleneoxy diradical monomers are replaced by methylenedio, methyleneamino or 1,2-ethylene radicals are also considered as interesting variants of the present invention. Methylenedio- and methyleneamine analogues are also considered equally applicable as methylene oxides: respectively, specific reagents corresponding to the introduction of (1S, 3R, 4R, 7S) -7-hydroxy-1-hydroxymethyl-3- (thymin-1-yl) -2,5-dioxabicyclo [2.2.1] heptane, (1S, 3R, 4R, 7S) -7-hydroxy-1-hydroxymethyl-3- (cytosin-1-yl) 2,5-dioxabicyclo [2.2.1] -heptane, (1S, 3R, 4R, 7S) -7-hydroxy-1-hydroxymethyl-3- (uracil-1-yl) 2,5-dioxabicyclo [2.2.1] heptane, (1S, 3R, 4R, 7S ) -7-hydroxy-1-hydroxymethyl-3- (guanin-1-yl) 2,5-dioxabicyclo [2.2.1] heptane, (1S, 3R, 4R, 7S) -7-hydroxy-1-hydroxymethyl-3 - (adenin-1-yl) 2,5-dioxabicyclo [2.2.1] g Heptane should also be considered as specific, of interest to the reactive monomers of the present invention. For the methyleneamine analog, it should be noted that the secondary amine may include 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 .

In a particular embodiment of the present invention, an oligomer comprising at least one LNA of basic formula Ia is provided:

where X is represented by —O—; B is selected from bases, DNA intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands; P represents the position of the radical for the internucleoside “bridge” followed by a monomer or 5'-terminal group, with each internucleoside “bridge” or 5'-terminal group optionally including a substituent R ⁵ ; R ³ * is represented by the group P *, which forms the internucleoside “bridge” with the preceding monomer or 3'-terminal group; R ² * and R ⁴ * simultaneously form 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 *) _s -S- and -S- (CR * R *) _{r + s} -N (R *) -; where each R * is independently selected from hydrogen, halogen, azido group, cyano group, nitro group, hydroxyl, mercapto group, amino group, mono- or di (C _1-6 alkyl) amino group, optionally substituted C _1-6 alkoxy groups, optionally substituted C _1-6 alkyl, DNA intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands, and (or) two adjacent (non-heminal) R * can together form a double bond, and each of r and s is 0-3 provided that the sum r + s is 1-4; each of the substituents R ¹ *, R ² , R ³ , R ⁵ and R ⁵ * is independently selected from hydrogen, optionally substituted C _1-6 alkyl, optionally substituted C _2-6 alkenyl, hydroxyl, C _1-6 alkoxy , C _2-6 alkenylalkoxy, carboxyl, 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 _2-6 -alkilkarbonilaminogruppy, carbamido, azido, C _1-6 -alkanoiloksigruppy, a sulfonic group, sulfanyl, C _1-6 alkylthio, a DNA intercalating group, a photochemically tive groups, thermochemically active groups, chelating groups, reporter groups, and ligands, and halogen, where two geminal substituents together may form an oxo; as well as their salts (molecular compounds with acids and bases). In particular, R * is selected from hydrogen, hydroxyl, an optionally substituted C _1-6 alkoxy group, an optionally substituted C _1-6 alkyl group, DNA intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands, and any of the other R * substituents are hydrogen. Specifically, the biradical is 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 another specific embodiment of interest, the present invention provides an LNA of basic formula IIa:

where X is -O-; B is selected from bases, DNA intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands; R ³ * is represented by the group Q *; each of Q and Q * is independently selected from hydrogen, azido group, halogen, cyano group, nitro group, hydroxyl, Prot-O-, Act-O-, mercapto group, Prot-S-, Act-S-, C _1-6 alkylthio group, amino groups, Prot-N (R ^H ) -, Act-NtR ^H ) -, a mono or di (C _1-6 alkyl) amino group, an optionally substituted C _1-6 alkoxy group, an optionally substituted C _1-6 alkyl, optionally substituted C _2-6 -alkenyl, optionally substituted C _2-6 -alkeniloksigruppy optionally substituted C _2-6 -alkynyl, optionally substituted C _2-6 -alkiniloksigruppy, monophosphate, diphosphate, triphosphate, DNA intercalators constituent groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands, carboxy, sulfonic groups, 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, and “Act” is the activation group for —OH, —SH and —NH (R ^H ), respectively, a R ^{H is} selected from hydrogen and C _1-6 alkyl; R ² * and R ⁴ * simultaneously mean 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 each R * is independently selected from hydrogen, halogen, azido group, cyano group, nitro group, hydroxyl, mercapto group, amino group, mono- or di (C _1-6 alkyl) amino group, optionally substituted C _1-6 alkoxy groups, optionally substituted C _1-6 alkyl, DNA intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands, and (or) two adjacent (non-heminal) R * can together form a double bond, and each of r and s is 0-3 provided that the sum r + s is 1-4; each of the substituents R ¹ *, R ² , R ³ , R ⁵ and R ⁵ * is independently selected from hydrogen, optionally substituted C _1-6 alkyl, optionally substituted C _2-6 alkenyl, hydroxyl, C _1-6 alkoxy , C _2-6 alkenylalkoxy, carboxyl, 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 -alkilkarbonilaminogruppy, carbamido, azido, C _1-6 -alkanoiloksigruppy, a sulfonic group, sulfanyl, C _1-6 alkylthio, a DNA intercalating group, a photochemically tive groups, thermochemically active groups, chelating groups, reporter groups, and ligands, and halogen, where two geminal substituents together may form an oxo; as well as their salts (molecular compounds with acids and bases); provided that any chemical group (including any of the bases) that is reactive under the conditions under which the synthesis of the oligonucleotide can be optionally protected by its functional components. Preferably, one of R * is selected from hydrogen, hydroxyl, an optionally substituted C _1-6 alkoxy group, an optionally substituted C _1-6 alkyl, DNA intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands, and any of the other substituents R * is hydrogen. Specifically, the biradical is 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 general, the present invention provides oligomers characterized by surprisingly high hybridization parameters in terms of affinity and specificity. Therefore, the present invention provides an oligomer comprising at least one nucleoside analog that gives the oligomer T _m with respect to a complementary DNA oligonucleotide that is at least 2.5 ° C higher, preferably at least 3.5 ° C. higher, specifically at least 4.0 ° C higher, particularly preferably at least 5.0 ° C higher than the corresponding modified control oligonucleotide, which does not include any nucleoside analogues. In particular, the T _{m of the} oligomer is at least 2.5 × N ° C higher, preferably at least 3.5 × N ° C higher, specifically at least 4.0 × N ° C higher, particularly preferably at least 5.0 × N ° C higher, where N is the number of nucleoside analogues in the oligomer.

In the case of hybridization with complementary RNA oligonucleotides, at least one nucleoside analog gives the oligomer T _m with respect to the complementary DNA oligonucleotide, which is at least 4.0 ° C higher, preferably at least 5.0 ° C higher, specifically at least 6.0 ° C higher, particularly preferably at least 7.0 ° C higher than the corresponding modified control oligonucleotide that does not include any nucleoside analogues. In particular, the T _{m of the} oligomer is at least 4.0 × N ° C higher, preferably at least 5.0 × N ° C higher, specifically at least 6.0 × N ° C higher, particularly preferably at least 7.0 × N ° C higher, where N is the number of nucleoside analogues in the oligomer.

The term “corresponding unmodified control oligonucleotide” is intended to mean an oligonucleotide entirely consisting of natural nucleotides that represent the same bases in exactly the same order (and in the same orientation).

The value of the melting temperature (dissociation of the duplex) T _m is determined in one of the conditions (for example, as described in example 129):

1) 10 mm Na ₂ HPO ₄ - pH 7.0, 100 mm NaCl, 0.1 mm EDTA;

2) 10 mm Na ₂ HPO ₄ - pH 7.0, 0.1 mm EDTA;

3) 3 M tetramethylammonium chloride (TMAC), 10 mM Na ₂ HPO ₄ - pH 7.0, 0.1 mM EDTA;

preferably under conditions (1), at equimolar amounts (usually 1, μM) of the oligomer and the complementary DNA oligonucleotide.

Preferably, the oligomer is as defined above, i.e. in it, at least one nucleoside analog is characterized by the formula I, and B is represented by a nucleotide base. Particular cases of interest are those where at least one nucleoside analogue includes a base selected from adenine and guanine.

Further, with regard to specificity and affinity levels, the oligomer when hybridizing with a partially complementary DNA oligonucleotide or with a partially complementary RNA oligonucleotide having one or more inconsistencies with the named oligomer should exhibit a decrease in T _m due to such inconsistencies, which will turn out to be the same or more significant than what would be observed for the corresponding unmodified control oligonucleotide, which does not include any nucleoside analogues. Also, such an oligomer should essentially have the same level of sensitivity of the value of T _m to the ionic strength of the hybridization buffer as the corresponding unmodified control oligonucleotide.

The oligomers of the present invention are usually modified by at least 30%, more preferably modified by at least 50%, in particular, modified by 70%, and in the most preferred embodiments, modified by 100%.

The oligomers of the present invention are characterized by significantly higher stability with respect to the action of 3'-exonucleases compared with the corresponding unmodified control oligonucleotide. This important property can be tested as described in Example 136.

Definitions

In the context of the present invention, the term “C _1-12 -alkyl” means a linear, cyclic or branched hydrocarbon group containing 1-12 carbon atoms, such as methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, tert-butyl, isobutyl, cyclobutyl pentyl, cyclopentyl, hexyl, cyclohexyl and dodecyl. Similarly, the term “C _1-6 -alkyl” denotes a linear, cyclic or branched hydrocarbon group containing 1-6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, pentyl, cyclopentyl, hexyl, cyclohexyl, and the term “C _1-4- alkyl ”means a linear, cyclic or branched hydrocarbon group containing 1-4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl.

Preferred examples of “C _1-6 alkyls” are methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, isobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, in particular methyl, ethyl, propyl, isopropyl, tert-butyl, isobutyl and cyclohexyl. Preferred examples of “C _1-4 alkyls” are methyl, ethyl, propyl, isopropyl, butyl, tert-butyl and isobutyl.

Similarly, the term “C _2-12 alkenyl” means a linear, cyclic or branched hydrocarbon group having 2-12 carbon atoms and having one unsaturated bond. Examples of alkenyl groups are vinyl, allyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, dodecaenyl. Similarly, the term “C _2-6 alkenyl” is intended to mean a linear, cyclic or branched hydrocarbon group containing 2-6 carbon atoms and having one unsaturated bond. Preferred examples of alkenyls are vinyl, allyl, butenyl, and especially allyl.

Similarly, the term “C _2-12 alkynyl” means a linear, cyclic or branched hydrocarbon group comprising 2-12 carbon atoms and having a triple bond. Examples thereof are ethynyl, propynyl, butynyl, octynyl and dodecanyl.

In the context of the present invention, i.e. in connection with the terms “alkyl”, “alkenyl” and “alkynyl”, the term “optionally substituted” means that the group in question can be substituted once or more, preferably 1-3 times, by groups selected from hydroxyl (which, in when bonded to an unsaturated carbon atom, it may have a tautomeric ketone form), C _1-6 alkoxy groups (e.g. C _1-6 alkyloxy), C _2-6 alkenyloxy groups, carboxyl, oxo groups (forming a ketone or aldehyde moiety), C _1-6 alkoxycarbonyl, the C _1-6 alkylcarbonyl, formyl, aryl, ariloksikar Onila, 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-} alkylaminocarbonyl, mono- and di (C _1-6 -alkyl) amino-C _1-6 -alkylaminocarbonyl, C _1-6 -alkylcarbonylamino groups, cyano groups, guanidino groups, urea groups, C _1-6 alkanoyloxy groups, sulfonic groups, C _1-6 alkylsulfonyloxy groups, nitro groups, sulfanyl, C _1-6 alkylthio groups, halogen, where any aryl and heteroaryl can be be substituted as described below for “optionally substituted aryl and heteroaryl”.

Preferably, the substituents are selected from hydroxyl, C _1-6 alkoxy, carboxyl, C _1-6 alkoxycarbonyl, C _1-6 alkylcarbonyl, formyl, aryl, aryloxycarbonyl, arylcarbonyl, heteroaryl, amino, mono and di (C _1-6 -alkyl) amino groups, 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 alkylcarbonylamino groups, cyano groups, urea groups, halogen, where aryl and heteroaryl can be substituted 1-5 times, preferably 1-3 times, C _1-4 alkyl, C _1-4 alkoxy, n itro group, cyano group, amino group or halogen. Most preferred examples are hydroxyl, C _1-6 -alkoxy, carboxyl, aryl, heteroaryl, amino, mono- and di (C _1-6 alkyl) amino, and halogen, where aryl and heteroaryl may be substituted 1-3 times ₁ C _-4- alkyl, C _1-4 -alkoxy, nitro, cyano, amino or halogen.

In the context of the present invention, the term “aryl” means a fully or partially aromatic carbocyclic ring or ring system such as phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, anthracyl, phenanthracil, pyrenyl, benzopyrenyl, fluorenyl and xanthenyl, among which is preferred an example is phenyl.

The term “heteroaryl” means a fully or partially aromatic carbocyclic ring or ring system in which one or more carbon atoms are replaced with heteroatoms, for example, nitrogen atoms (= N- or —NH), sulfur and (or) oxygen. Examples of such heteroaryl groups are oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, piperidinyl, coumaryl, furyl, quinolyl, benzothiazolazolazolazolazolazolazolazolazolazolazolazolazolazolazolazolazolazolazolazolazolazolazolazolazolazolitholazolazolazolazolazolithol , acridinyl, carbazolyl, dibenzazepinyl, indolyl, benzopyrazolyl, fenoxarzonil.

In the context of the present invention, i.e. in connection with the terms “aryl” and “heteroaryl”, the term “optionally substituted” means that such a group in question is substituted once or more, preferably 1-5 times, in particular 1-3 times, by a group (s) selected from hydroxyl (which, if present in the enol system, can have a tautomeric ketone form), C _1-6 alkyl, C _1-6 alkoxy groups, oxo groups (which can be represented by a tautomeric enol form), carboxyl, C _{1 -6-} alkoxycarbonyl, C _1-6 -alkylcarbonyl, formyl, aryl, aryloxy groups, ari oxycarbonyl, arylcarbonyl, heteroaryl, 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 alkylcarbonylamino group, cyano group, guanidino group, urea group, C _1-6 alkanoyloxy group, sulfonic group, C _1-6 alkylsulfonyloxy group, nitro groups, sulfanyl, dihalo-C _1-4 -alkyl, trihalo-C _1-4 -alkyl, halogen, where aryl and heteroaryl substituents can be substituted 1-3 times by C _1-4 -alkyl, C _1-4 -a alkoxy group, nitro group, cyano group, amino group or halogen. Preferred examples are hydroxyl, C _1-6 alkyl, C _1-6 alkoxy, carboxyl, C _1-6 alkoxycarbonyl, C _1-6 alkylcarbonyl, aryl, amino, mono and di (C _1-6 alkyl ) an amino group and a halogen, where aryl may be substituted 1-3 times by C _1-4 alkyl, C _1-4 alkoxy, nitro, cyano, amino or halogen.

The term “halogen” includes fluoro, chloro, bromo and iodo.

It should be understood that oligomers (which include LNAs) and LNAs themselves include their possible salts, among which pharmaceutically acceptable salts are most preferred. Salts include basic and acid addition salts. Examples of acid salts are hydrochlorides, sodium salts, calcium salts, potassium salts, and the like. Examples of basic salts are salts in which the ion to be attached is selected from alkali metal ions such as sodium and potassium, alkaline earth metals such as calcium, and ammonium ions ( ⁺ N (R ^g ) ₃ R ^h , where each of R ^g and R ^h independently denotes optionally substituted C _1-6 alkyl, optionally substituted C _2-6 alkenyl, optionally substituted aryl or optionally substituted heteroaryl). Pharmaceutically acceptable salts are those salts which are described in Remington's Pharmaceutical Sciecnes, 17 ^th edition, ed. ARGennaro, Mack Publ. Co., Easton, PA, 1985 and in later editions, as well as in the Encyclopedia of Pharmaucetical Technology encyclopedia. Thus, the term “their salts (molecular compounds with acids and bases)” in this text is intended to denote such salts. Moreover, oligomers and LNAs, as well as any intermediates or starting materials for them, can also be in the form of hydrates.

Preparation of Monomers

In a preferred embodiment, nucleosides including an additional 2’-O, 4’-C-linked ring were synthesized using the following procedure.

The synthesis of certain 4'-C-hydroxymethyl nucleosides has been previously described (RDYoussefyeh, JPHerheyden & JGMoffatt, 1979, J. Org Chem., 44, 1301; GHJones, M. Taniguchi, D.Tegg & JGMoffatt, 1979, J. Org. Chem. , 44, 1309; CO-Yang, HYWu, EBFraser-Smith & KAMWalker, 1992, Tetrahedron Lett., 33, 37; H.Thrane, J. Fensholdt, M. Regner & J. Wengel, 1995, Tetrahedron, 51, 10389 ; KDNielsen, F. Kirpekar, P. Roepstorff & J. Wengel, 1995, Bioorg. Med. Chem., 3, 1493). As an example of the synthesis of 2'-O, 4'-C-linked bicyclic nucleosides, we have chosen a strategy starting with a 4'-C-hydroxymethylfuranose derivative (31). Benzylation, acetylation and acetolysis, followed by additional acetylation, resulted in furanose (33), which is a key intermediate in the nucleoside combination. In a stereoselective reaction using silylated thymine, compound 34 was obtained, which was deacetylated to give nucleosidiol (35). Base tosylation followed by ring closure resulted in a 2'-O, 4'-C-linked bicyclic nucleoside derivative (36). As a result of debenzylation, a bicyclic nucleoside analogue 37 freed from protection was obtained, which was transformed into the 5'-O-4,4'-analog 38 protected by dimethoxytrityl and then to the phosphoramidite derivative 39 necessary for the synthesis of the oligonucleotide. A similar procedure was used to synthesize the corresponding uracil, adenine, cytosine and guanine nucleosides, which is discussed in the examples section. This combination method is only one of a number of possible procedures, which should be obvious to a person skilled in the art. It is also possible to apply a strategy starting with a transformed nucleoside. Thus, for example, the conversion of the uridine derivative 62 to the derivative 44 was successfully accomplished by tosylation, deisopropylidination and base-induced ring closure. In another example, the conversion of nucleoside 67 into nucleoside 61B is carried out in accordance with Fig. 34. The conversion of the bicyclic thymine nucleoside 37 to the corresponding 5-methylcytosine nucleoside 65 was carried out by a series of known reactions using triazole and POCl ₃ followed by benzylation and treatment with ammonia. A similar procedure is applicable for the synthesis of 57C and 44. As an example of other possible synthesis strategies, a combination of pre-cyclized furanose derivatives, which already include an additional ring, with nucleotide derivatives can be given. This strategy, in addition, allows to obtain the corresponding α-nucleoside analogues. During the coupling reaction with the protected 4-C, 2-O-methylene-D-ribofuranose methylenefuranoside, an open ring product was mainly obtained. However, the combination of 1-O-acetylfuranose (207) or thiophenylfuranose (212) leads to the successful production of LNA nucleosides, and α-anomers are one of the obtained products. The incorporation of such (x-LNA nucleosides will be possible using standard oligomerization techniques (also applicable to LNAs including Z) to produce α-LNA oligomers. In addition, a synthesis strategy is possible by nucleoside coupling using 4'- C-hydroxymethylfuranose, which is already activated by ring closure (for example, due to the presence of a mesyl or tosyl group in the composition of the 4'-C-hydroxymethyl group), for example, the conversion of furanose (78) to nucleoside 79 s a further deprotection and ring closure to 36. Another suitable strategy is the chemical or catalytic transglycosylation or anomerization of suitable furanose derivatives or nucleosides.These and other strategies provide for the synthesis of bicyclic nucleosides, including other nucleotides or their analogues, by or in combination with these nucleotides or analogs, or starting with preformed nucleoside derivatives of the compounds.

The described examples are intended to be illustrative for the procedures and examples of the present invention. To verify the structure of the synthesized compounds, one- and two-dimensional NMR were used, in particular, in the NOE experiment.

An additional embodiment of the present invention relates to the representation of bicyclic nucleosides, including additional rings of various sizes and different chemical structures. For specialists in the field of organic synthesis, based on the above methodological description, it should be clear that the cyclization of other nucleosides is possible using similar procedures, including nucleosides having different C-branches. It is not difficult for a person skilled in the art to find in the scientific literature guidelines for the preparation of intermediates such as substituted nucleoside analogues: see, for example, international application WO 96/14329. As for rings of different chemical composition, it is clear that the use of similar procedures or procedures generally well developed in the field of organic chemistry, the synthesis of, for example, thio analogs, cited as an example of oxo analogs, is possible to the same extent as the synthesis of the corresponding amino analogs (for example using nucleophilic substitution reactions or reductive alkylation reactions).

The examples section describes the synthesis of amino-LNA analogues (73-74F). Conversion of 74 and 74D in a standard manner arranged blocks for oligomerization is possible using 5’-O-DMT protection and 3’-O-phosphitylation followed by standard procedures. In the case of amino-LNA analogues, protection of the 2’-amino functional group is necessary for reasons of control over linear oligomerization. Such protection can be carried out using standard amino protecting groups such as Fmoc, trifluoroacetyl or BOC. Alternatively, an N-alkyl group (e.g., benzyl, methyl, ethyl, propyl or functionalized alkyl) may be retained during nucleoside transformations and oligomerization. Figures 35 and 36 show strategies based on the use of N-trifluoroacetyl and N-methyl derivatives. As can be seen from Fig. 37, the conversion of nucleoside 75 into a 2'-thio-LNA-nucleoside analogue 76D was successfully carried out, as well as the subsequent synthesis of its phosphoramidite derivative 76F. Compound 76F is characterized by the structure necessary for the automatic synthesis of 2’-thio-LNA oligonucleotides. Sinton N-trifluoroacetyl-2’-amino-LNA (74A) is characterized by the structure necessary for the automatic synthesis of 2’-amino-LNA-oligonucleotides.

The synthesis of the corresponding cytosine, guanine and adenine derivatives of 2'-thio- and 2'-amino-LNA nucleosides can be carried out using strategies similar to those shown in Figs. 35, 36 and 37. On the other hand, stereochemistry around the atom C-2 'can be inverted prior to cyclization, either using a furanose synthon having a standard configuration (e.g., arabinoconfiguration), or by inverting the configuration around a C-2' carbon atom, starting with a nucleoside / furanose having a riboconfiguration. Subsequent activation of 2'-β-OH, for example, by tosylation, and double nucleophilic substitution, as in the above example with uracil / thymine, can lead to the desired 2'-thio-LNA or 2'-amino-LNA nucleosides . The protected cytosine, guanine, and adenine analogs thus obtained can be prepared for oligomerization using standard reactions (DMT protection and phosphitylation) as described above for other examples.

Obtaining oligomers

Linear, branched (M. Groetli & BSSproat, 1995, J. Chem. Soc., Chem. Commun., 495; RHE Hudson & MJ Damha, 1993, J. Amer. Chem. Soc., 115, 2119; M. Von Buren, GV Petersen, K. Rasmussen, G. Brandenburg, J. Wengel & F. Kirpekar, 1995, Tetrahedron, 51, 8491) and cyclic (G. Prakash & ETKool, 1992, J. Amer. Chem. Soc. 114, 3523) the oligo and polynucleotides of the present invention can be prepared using polymerization methods well known in the field of organic chemistry for nucleic acid chemistry. For this purpose, phosphoramidate chemistry was used (SLBeaucage & RPIyer, 1993, Tetrahedron, 49, 6123; SLBeaucage & RPIyer, 1992, Tetrahedron, 48, 2223), but methods were also used, for example, hydrogen-phosphonate chemistry, phosphotriether chemistry or catalytic synthesis. In general, standard coupling conditions and the phosphoramidite method were used, although for some monomers of the present invention, longer coupling reaction periods and / or repetition of the coupling reaction with fresh reagents were used and / or more concentrated coupling reaction reagents were used. As another possibility, activators more active than 1H-tetrazole can also be used, allowing to increase the rate of the coupling reaction. All phosphoramidite compounds 39, 46, 53, 57D, 61D and 66 provide a combination with a satisfactory yield of> 95%. An LNA oligomer based only on phosphotioate (Table 7) was synthesized using standard procedures. Thus, the phosphothioate LNA oligomer was efficiently synthesized (combination reaction yield ≥ 98%) by eliminating normal, for example, using iodine / pyridine / hydrogen peroxide, the oxidation used in the synthesis of phosphodiester oligomers, with oxidation by Buccage reagent (available commercially basis). The 2’-amino-LNA and 2’-methylamino-LNA oligonucleotides (Table 9) were efficiently synthesized (coupling reaction yield ≥ 98%) using amidites 74A and 74F. 2’-thio-LNA oligonucleotides (Table 8) were efficiently synthesized using amidite 76F using a standard phosphoramidite procedure as described for LNA oligonucleotides. After synthesis of the desired sequence, it was processed under standard conditions (cleavage from a solid support and removal of the protective groups using 30% ammonia at 55 ° C for 5 hours). Purification of LNA oligonucleotides was carried out using reverse phase cartridges for purification and (or) reverse phase HPLC and / or by precipitation with ethanol or butanol. Capillary gel electrophoresis, reverse phase HPLC, and MALDI mass spectrometry were used to verify the purity of the synthesized oligonucleotide analogs and to verify that the desired number of bicyclic nucleoside analogs of the present invention were included.

An additional aspect of the present invention relates to the presentation of methods for producing oligonucleotide analogues, including LNAs, which contain unnatural internucleoside “bridges”. For example, the synthesis of the corresponding phosphothioate or phosphoramidate analogues is possible using strategies well known in the field of oligonucleotide chemistry ("Protocols for Oligonucleotides and Analogs", vol. 20, ed. S. Agrawal, Humana Press, Totowa, NJ, 1993; SLBeaucage & RPIyer, 1993, Tetrahedron, 49, 6123; SLBeaucage & RPIyer, 1992, Tetrahedron, 48, 2223; E. Uhlmann & A. Payman, Chem. Rev., 90, 543).

Thus, the present invention also provides the use of the LNAs provided herein for the preparation of LNA-modified oligonucleotides. It should be understood that LNA-modified oligonucleotides may include normal nucleosides (i.e., those found under natural conditions, such as ribonucleosides and / or deoxyribonucleosides), as well as modified nucleosides different from those described by general formula II. In a specific embodiment, the incorporation of LNA modulates the ability of the oligonucleotide to serve as a substrate for enzymes active against nucleic acids.

Moreover, solid support materials onto which an optionally 5 О OH nucleotide protected and optionally 5 О OH LNA are immobilized are particularly interesting as a material for the synthesis of LNA-modified oligonucleotides in which the LNA monomer is incorporated at the 3 ’end. In this example, the solid support material is preferably CPG, for example, readily (commercially) available CPG material on which a 3'-functionalized, optionally protected nucleotide and optionally 5'-OH protected LNA binds using conditions specified by the manufacturer of the particular material. For example, BioGenex Universal CPG Support (BioGenex, USA) can be used. A protecting group for 5’-OH. may be, for example, a DMT group. A functional 3’-group should be selected taking into account the conditions proposed for the CPG material in question.

Application

The present invention describes an unexpected finding that various new derivatives of bicyclic nucleoside monomers (LNAs), when incorporated into oligonucleotides, greatly increase the affinity of such modified oligonucleotides for both single-stranded DNA and single-stranded RNAs compared to unmodified oligonucleotides . Further, an unexpected finding is presented that both fully and partially LNA-modified oligonucleotides exhibit sharply enhanced hybridization properties with respect to their complementary nucleic sequences. Depending on the specific application, the use of these LNAs provides a promising opportunity to either significantly increase the affinity level of a standard oligonucleotide without corresponding loss in specificity (at a constant oligonucleotide size), or significantly increase the specificity level without corresponding losses in affinity (decrease in oligonucleotide size). Also, the present invention provides an unexpected finding that LNA-modified oligonucleotides, in addition to a significant improvement in hybridizability parameters, exhibit a number of valuable physicochemical properties characteristic of normal DNA and RNA oligonucleotides. Examples given here include excellent solubility, the response of LNA-modified oligonucleotides to salts such as sodium chloride and trimethylammonium chloride, resembling those of unmodified oligonucleotides, the ability of LNA-modified oligonucleotides to act as seeds for various polymerases, and the ability of LNA-modified oligonucleotides to serve as seeds amplification of targets using thermostable DNA polymerases, the ability of LNA-modified oligonucleotides to perform p the role of the substrate for T4 polynucleotide kinase, the ability of biotinylated LNAs to select sequence-specific PCR amplicons on a streptavidin-coated solid substrate, the ability of immobilized LNA-modified oligonucleotides to sequence-specific selection of amplicons, and, very importantly, the ability of LNA-modified oligon specific sequence recognition of double-stranded DNA by the mechanism of chain invasion. Therefore, it will be understood by those skilled in the art that the new nucleoside analogs are characterized by their exceptional applicability as a means to improve the parameters and characteristics of techniques based on the use of oligonucleotides and used in therapy, diagnostics, and molecular biology.

An object of the present invention is to provide monomeric LNAs in accordance with the present invention, which can be incorporated into oligonucleotides using techniques and equipment well known to those skilled in the art of oligonucleotide synthesis.

Another object of the present invention is the presentation of fully or partially LNA-modified oligonucleotides (oligomers), which are able to hybridize in a specific nucleotide sequence type with complementary oligonucleotides to form either duplexes or triplexes, showing a significantly higher level of affinity compared to the corresponding complexes formed by unmodified oligonucleotides.

Another object of the present invention is the use of LNA to increase the level of specificity of normal oligonucleotides without a corresponding loss in the level of affinity. This can be achieved by reducing the length (and thereby affinity) of the normal oligonucleotide to the extent that the level of affinity achieved by the incorporation of LNA is achieved.

Another object of the present invention is the provision of fully or partially modified oligonucleotides, including both LNA and normal nucleosides, as well as other nucleoside analogues.

The next object of the present invention is the application of the high affinity property of LNA to create modified oligonucleotides with exceptional affinity properties that would be able to bind to the corresponding target sequences “within” the double-stranded DNA molecule by the “chain-replacing” mechanism.

The next object of the present invention is the presentation of various classes of LNA, which, if included in the oligonucleotides, will vary in their affinity for the corresponding complementary nucleosides. In accordance with the present invention, this can be achieved either by replacing the normal nucleotides G, A, T, C, and U with their derivatives, characterized, for example, by an altered ability to form hydrogen bonds, or by using LNAs that differ in their basic structure. The availability of such different LNAs allows the fine tuning of affinity levels of modified oligonucleotides.

Another object of the present invention is the presentation of LNA-modified oligonucleotides, which are characterized by a higher resistance to the action of nucleases compared with the corresponding unmodified oligonucleotides.

Another object of the present invention is the provision of LNA-modified oligonucleotides that are able to recruit RNase-H.

An additional object of the present invention is the presentation of LNA, which may be drugs. Numerous examples of therapeutically useful nucleoside analogs are known, and similar derivatives of the nucleoside analogs provided herein can be synthesized using methods known from the scientific literature (E. De Clercq, 1995, J. Med. Chem., 38, 2491; P. Herdewijn & E .De Clercq, 1996, ″ Classical antiviral agents and design of new antiviral agents ". In ″ A Textbook of Drug Design and Development", eds. P. Krogsgaard-Larsen, T. Liljefors & U. Madsen, Harwood Acad. Publ. , Amsterdam, p. 425; IKLarsen, 1996, ″ Anticancer agents ". In" A Textbook of Drug Design and Development ", eds. P. Krogsgaard-Larsen, T. Liljefors & U. Madsen, Harwood Acad. Publ., Amsterdam, p. 460).

Double-stranded RNA has been shown to exhibit antiviral activity and tumor suppressor activity (Sharp et al., 1995, Eur. J. Biochem., 230, 97-103; Lengyel-P et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 5893-5895; and Laurent-Crawford et al., 1992, AIDS Res. Human Retroviruses, 8, 285-290). Double-stranded LNAs are likely to mimic the effect of therapeutically active double-stranded RNAs: accordingly, such double-stranded LNAs have potential as drugs.

As used in this text, the term “natural nucleic acid” refers to nucleic acids in the broad sense of the word, for example, nucleic acids of intact cells of any origin or nucleic acids isolated from such sources using chemical or physical methods, or nucleic acids derived from such primary sources through amplification reactions Native nucleic acids can be single-stranded, double-stranded or partially double-stranded and can be relatively “pure” forms, or a mixture of different nucleic acids. They can also be components of an untreated biological sample containing 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 methods.

The present invention also provides the use of LNA-modified oligonucleotides in nucleic acid-based therapeutics, diagnostics, and molecular biology. LNA-modified oligonucleotides can be used in the detection, identification, selection, characterization, quantification and fragmentation of natural or synthetic nucleic acids, as well as in vivo and in vitro translation and transcription blocking agents. In many cases, it may be of interest to attach various molecules to LNA-modified oligonucleotides. Such molecules can be attached either to the end of the oligonucleotide, or they can be attached at one or more internal positions. Alternatively, they can be attached to this oligonucleotide indirectly via spacers attached at the 5 ′ or 3 ′ end. Representative groups of such molecules are DNA intercalating groups, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands. Typically, all methods for labeling unmodified DNA and RNA oligonucleotides using such molecules can also be used to label LNA-modified oligonucleotides. All methods used to detect labeled oligonucleotides are, in principle, also applicable to appropriately labeled LNA-modified oligonucleotides.

Therapy

The term “chain substitution” means a process in which an oligonucleotide binds to its complementary target sequence of double-stranded DNA or RNA in such a way as to replace another chain on said target chain.

In one aspect of the present invention, LNA-modified oligonucleotides capable of “chain displacement” are used in the development of new drugs used in a series of “antigen method” techniques. In contrast to oligonucleotides capable of forming three-stranded helices, such oligonucleotides “substituting a chain” are capable of interacting with any sequence in a double-stranded DNA chain at physiological values of ionic strength and pH.

Oligonucleotides “for a substitute chain” can also be highly efficiently used in “antisense techniques” - i.e. in cases where the target RNA sequence is unavailable due to the presence of intramolecular hydrogen bonds. Such intramolecular structures can occur in mRNA and can cause significant problems when trying to “stop” the translation of such mRNA by “antisense methods”.

Other classes of cellular RNA, such as, for example, tRNA, rRNA, mRNA and mcRNA, carry intramolecular structures that are necessary for their functioning. These classes of complex RNAs do not encode proteins, but play a significant role (in the form of nucleoprotein formations) involved in providing a number of cellular functions, such as mRNA splicing, polyadenylation, translation, “RNA editing”, maintaining the stability of chromosome ends, etc. Due to their complex spatial structure, which does not allow their hybridization properties to partially or even completely show oligonucleotides, these RNA classes still have not attracted the attention of developers of “antisense methods”.

The use of high affinity LNA monomers should ensure the construction of antisense probes characterized by significant thermal stability, effective hybridization with such RNA targets. Therefore, in a preferred embodiment, LNAs are used to provide a significant level of affinity for the oligonucleotide, which ensures its hybridizability with RNAs of these classes: in this way, modulation of the qualitative and (or) quantitative parameters of the particles that comprise these RNAs can be achieved.

In some cases, it may be important to reduce the level of expression of a certain gene, while in other cases, activation may be appropriate. As shown by Mellegard et al. (N.E. Moellegaard, O.Buchardt, M.Egholm, P.E. Nielsen, 1994, Proc. Natl. Acad. Sci. USA, 91, 3892), oligomers capable of causing “chain substitution” can function as RNA transcriptional activators. In one aspect of the present invention, LNAs capable of causing “chain displacement” are useful for activating genes of therapeutic interest.

For chemotherapy of various viral infections and malignant tumors, the effectiveness of nucleosides and nucleoside analogues has been confirmed. LNA nucleosides can potentially be used as such nucleoside-based drugs.

Different types of double-stranded RNAs inhibit the growth of certain types of malignant tumors. Duplexes containing one or more LNA oligonucleotides are potentially useful as such double-stranded drugs.

The present invention also provides a pharmaceutical composition comprising a pharmaceutically active LNA-modified oligonucleotide or a pharmaceutically active 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, intraocular or intrapulmonary administration, preferably in a form suitable for oral administration, and such compositions can be prepared using techniques well known to those skilled in the art, for example, in accordance with the general descriptions in "Remington's Pharmaceutical Sciecnes", 17 ^th ed., ed. ARGennaro, Mark Publ. Co., Easton, PA, 1985 and later, as well as monographs of the Drugs and Pharmaceutical Sciences series by M.Dekker.

Diagnostics

Several diagnostic and molecular biological procedures have been developed in such a way as to utilize sets of different oligonucleotides in order to simultaneously analyze the target nucleic acid for the presence of an excess of possible mutations. Typically, oligonucleotide sets are immobilized in a predetermined order on a solid substrate so that the presence of a particular mutation in the target nucleic acid can be determined by its location on the solid substrate after its hybridization. One of the important prerequisites for the successful application of this set of different oligonucleotides in nucleic acid analysis is that all of them are specific for a specific target sequence under the same hybridization conditions used in a particular case. Since the affinity and specificity of standard oligonucleotides for the corresponding complementary target sequences is largely dependent on their nucleotide sequence and overall length, these criteria have so far been difficult to fully satisfy.

In a preferred embodiment, therefore, LNAs are used as means for increasing the affinity and / or specificity of the probes, and also as means for leveling the affinity levels of different oligonucleotides for their respective complementary sequences. It appears in the present invention, such a modulation of the level of affinity can be carried out, for example, by replacing selected nucleosides in the oligonucleotide with an LNA bearing the same base. As will be shown hereinafter, different classes of LNA show different levels of affinity for their respective complementary nucleosides. For example, an LNA with 2-3 “bridges” connected to the base T exhibits less affinity for adenosine compared to the corresponding LNA having 2-4 “bridges”. Therefore, the use of different classes of LNA provides an additional opportunity to fine-tune the affinity levels of the oligonucleotide.

In another preferred embodiment, high levels of affinity and specificity of LNA-modified oligonucleotides are used in sequence-specific selection and purification of natural or synthetic nucleic acids. In one aspect, natural and synthetic nucleic acids are contacted with an LNA-modified oligonucleotide immobilized on a solid surface. In this case, hybridization and selection occur simultaneously. The selected nucleic acids may, for example, be detected, characterized, quantified or amplified directly on the surface using various methods well known in the art, or they may be removed from this surface before such characterization is carried out or amplification by placing immobilized modified oligonucleotide and the nucleic acid selected with its help in dehydrating conditions, for example, by heating or using buffers characterized by low ionic strength.

The solid support can be selected from a wide range of polymeric materials, such as, for example, CPG (glass with adjustable pores), polypropylene, polystyrene, polycarbonate or polyethylene, and it can have a different shape, such as, for example, a test tube, microtiter plate, stick , ball, filter, etc. The LNA-modified oligonucleotide can be immobilized on a solid support using its 5'- or 3'-end (or at the terminal components of the linker attached to the 5'- or 3'-end of the oligonucleotide) using various chemical or photochemical methods commonly used for immobilizing oligonucleotides, or using a non-covalent combination, for example, by binding biotinylated LNA-modified oligonucleotides on various solid substrates on immobilized streptavidin. One of the preferred methods for immobilizing LNA-modified oligonucleotides on various solid substrates is the photochemical method, which uses a photochemically active anthraquinone covalently attached to the 5'- or 3'-end of the modified oligonucleotide (optionally via linkers) as described in the international application WO 96/31557. Thus, the present invention also provides a surface bearing an LNA-modified oligonucleotide.

In another aspect of the present invention, an LNA-modified oligonucleotide carries a ligand covalently attached to its 5'- or 3'-end. In this case, an LNA-modified oligonucleotide. contact with native or synthetic nucleic acids in solution, after which the formed hybrid molecules are selected on a solid support that carries molecules that can specifically bind a particular ligand.

In yet another aspect, LNA-modified oligonucleotides capable of providing “chain substitution” are used to select native and synthetic nucleic acids without first denaturing them. Such modified oligonucleotides, in particular, are applicable in those cases where access to the target sequence using conventional oligonucleotides is difficult or even impossible due to the rapid formation of stable intramolecular structures. Examples of nucleic acids including such structures are rRNA, tRNA, snRNA and mcRNA.

In another preferred embodiment, LNA-modified oligonucleotides designed to provide a high level of specificity are used as seeds for nucleic acid sequencing and as seeds for any of a number of well-known amplification reactions, such as PCR. As shown in this application, the design of LNA-modified oligonucleotides determines whether they will provide an exponential or linear type of amplification of the target. The amplification reaction products can be analyzed using various methods applicable to the analysis of amplification products obtained on conventional DNA seeds. In the specific case, when seeds based on LNA-modified oligonucleotides are designed to provide linear amplification, the resulting amplicons will have single-stranded ends that can be labeled with complementary probes without denaturation. Such ends can be used, for example, to select amplicons using other complementary LNA-modified oligonucleotides attached to a solid surface.

In another aspect, LNA-modified oligomers capable of providing “chain substitution” are used as seeds in either exponential or linear amplification reactions. The use of such oligomers is expected to result in an increase in the overall output of amplicons due to effective competition in the hybridization of amplicons in the late stages of the amplification reaction. It has been proposed (Demers et al., 1995, Nucl. Acids Res., 23, 3050-3055) the use of high affinity unfinished oligomers in order to increase the overall yield in the PCR reaction. We can assume that oligomers will cause such an effect due to the prevention of amplicon rehybridization in the late stages of the PCR reaction. It can be expected that LNA-modified oligomers blocked at their 3’-end will provide a similar advantage. Blocking at the 3'-end can be achieved in a variety of ways, such as, for example, by replacing a 3'-hydroxyl group with hydrogen or phosphate. Such 3’-terminally blocked LNA-modified oligomers can also be used to selectively amplify closely related nucleotide sequences in a manner similar to that described previously (Yu et al., 1997, Biotechniques, 23, 714-716).

In recent years, new classes of probes have been introduced that can be used, for example, to identify amplicons obtained in target amplification reactions in real time. One of the classes of such probes was called "Molecular Beacons". These probes are synthesized as partially auto-complementary oligonucleotides that include a fluorophore at one end and a quencher molecule (“fluorescence quencher”) at the other end. Being in solution in a free form, such a probe is packed into a “hairpin” structure (due to the interaction of its auto-complementary regions): in this case, the quencher is close enough to the fluorophore to extinguish the fluorescent signal. Under conditions of hybridization with the corresponding target nucleic acid, “opening of the hairpin” occurs, resulting in a “separation” of the fluorophore and kvencher and the manifestation of the fluorescent signal.

Another class of probes was called “Taqman probes”. These probes also include a fluorophore and a quencher molecule. However, unlike the Molecular Beacons probes, the ability of the quencher to suppress the fluorescent signal of the fluorophore is also supported after the hybridization of the probe with its target sequence. Instead, the fluorescent signal is generated after hybridization by physically removing either the quencher or the fluorophore from the probe as a result of the 5’-exonuclease activity of the polymerase, which initiates the synthesis from the seed located on the 5 ′ side of the Taqman probe binding site.

A high level of affinity for the target site is an important property of both of these types of probes: therefore, such probes tend to accumulate too much (usually from 30 to 40 nucleotides). As a result, serious problems arise in obtaining highly informative probes. In a preferred embodiment of the present invention, therefore, LNA is used to improve the parameters and corresponding application characteristics of Taqman probes and Molecular Beacons probes by reducing their size while maintaining the original level of affinity.

In a further aspect of the present invention, LNAs are used to construct novel affinity probe pairs (or fully, partially modified oligonucleotides). Affinity levels can be easily selected over a wide range, and a very large number of affinity pairs can be determined and synthesized. One of the components of the affinity pair can be attached to the molecule of interest (such as proteins, amplicons, enzymes, polysaccharides, antibodies, haptens, peptides, RNA, etc.) using standard methods, while the other component of the affinity pair can be attached, for example, to a solid support such as spheres, membranes, microtiter plates, sticks, tubes, etc. The solid support can be selected from a wide range of polymeric materials, such as, for example, polypropylene, polystyrene, polycarbonate or polyethylene. Affinity pairs can be used in the selective isolation, purification, selection and detection of a variety of target molecules, indicated above.

The principle of selecting an LNA-labeled molecule by interacting with another complementary LNA oligonucleotide (either fully or partially modified) can be applied to create an unlimited number of new affinity probe pairs.

In another preferred embodiment, high levels of affinity and specificity for LNA-modified oligonucleotides are used in the construction of probes useful in in situ hybridization. For example, LNA can be used to reduce the size of traditional DNA probes while maintaining the required level of affinity, which will thereby improve the kinetic parameters of this probe and its ability to penetrate into the sample. The ability of LNA-modified oligonucleotides to "chain invasion" in double-stranded nucleic acid molecules also provides significant advantages in situ hybridization due to the fact that they provide hybridization without prior denaturation of the DNA / RNA targets.

In another preferred embodiment, LNA-modified oligonucleotides intended for use in “antisense therapy” are designed to have both a high level of affinity and the ability to initiate RNase-H. This can be achieved, for example, by the presence of LNA segments flanking an unmodified, centrally located DNA segment.

The present invention also provides a kit for isolating, purifying, amplifying, detecting, identifying, quantifying or selecting natural or synthetic nucleic acids, the kit comprising a reaction device and one or more LNA-modified oligonucleotides (oligomers) as described herein . Preferably, the LNA-modified oligonucleotides are immobilized on the surface of said reaction device.

The present invention also provides a kit intended for isolation, purification, amplification, detection, identification, quantification or selection of natural or synthetic nucleic acids, despite the fact that this kit includes a reaction device and one or more. LNA as described here. Preferably, the LNAs are immobilized onto the surface of said reaction device (for example, using the immobilization methods described above).

For the kits of the present invention, the reaction device is preferably a solid support material, for example, selected from borosilicate glass, sodium-calcium glass, polystyrene, polycarbonate, polypropylene, polyethylene, polyethylene glycol terephthalate, polyvinyl acetate, polyvinyl pyrrolidinone, polymethyl methacrylate and polyvinyl chloride, and preferably. The reaction device may take the form of a test tube, ampoule, glass slide, plate, film, ball, granule, disk, plate, ring, rod, mesh, filter, tray, microtiter plate, stick or multi-vane stick.

Kits are usually accompanied by written instructions that describe the optimal conditions for using this kit.

The diagnostic and therapeutic aspects of the present invention described above are further illustrated by the following examples.

experimental part

Basic information

All reagents were obtained from commercial sources and were used without further purification. After the organic phases were dried over Na ₂ SO ₄ , filtration was performed. Silica gel used in chromatography (pores 0.040-0.63 mm) was purchased from Merck. NMR spectra were recorded at 300 MHz or 250 MHz for ¹ H-NMR, or at 62.9 MHz for ¹³ C-NMR, or at 202.33 MHz for ³¹ P-NMR. Values of δ are indicated in ppm. in relation to tetramethylsilane, which is an internal standard ( ¹ H-NMR and ¹³ C-NMR) and in relation to 85% H ₃ PO ₄ taken as an external standard ( ³¹ P-NMR). Designations of the NMR peaks are given in accordance with the standard nomenclature of nucleosides. EI mass spectra, FAB mass spectra, and PD (Plasma Desorption) mass spectra were determined to obtain information on the molecular weights of the synthesized compounds. Oligonucleotide analogues were synthesized using phosphoramidite technology. 5'-O-DMT-including and 5'-O-DMT-released oligonucleotide analogues were purified using replaceable reverse phase chromatography cartridges and, if necessary, reverse phase HPLC. The method of laser desorption mass spectrometry on a matrix was used to verify the molecular weight and composition of monomers in representative samples of oligonucleotides. The method of capillary gel electrophoresis was used to verify the purity of representative samples of oligonucleotides.

The specific descriptions below are accompanied by figures 2-41 and tables 1-10. Unless otherwise specified in the following examples, “LNA” refers to the 2’-4 ’“ bridge ”variant illustrated by the formula Z in FIG. 2.

Obtaining LNA Monomers

Example 1

3-C-allyl-1,2-O-isopropylidene-α-D-ribofuranose (0A)

Method 1. Solution of 5-O-tert-butyldimethylsilyl-1,2-O-isopropylidene-α-D-ribofuran-3-ulose (Y. Yoshimura, T.Sano, A. Matsuda & T. Ueda, 1988, Chem. Pharm. Bull., 36, 162) (17.8 g, 58.9 mmol) in anhydrous THF (980 cm ³ ) was stirred at 0 ° C. and 1 M magnesium allyl bromide in anhydrous ether (130 cm ³ , 130 was added dropwise). mmol). After stirring for 2 hours, a saturated aqueous solution of ammonium chloride (800 cm ³ ) was added and the resulting mixture was extracted with dichloromethane (3 × 400 cm ³ ). The organic phase was washed with brine (3 × 450 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure, and the residue was dissolved in anhydrous THF (700 cm ³ ). A 1.1 M solution of tetrabutylammonium fluoride in THF (54.4 cm ³ , 59.8 mmol) was added, the resulting mixture was stirred at room temperature for 1 hour and evaporated to dryness. The residue was dissolved in dichloromethane (1700 cm ³ ) and washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 500 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (98: 2 volume ratio) as eluent to obtain furanose (0A) as a white solid (9.42 g, 69%). Method 2. Furanose (0A) was similarly synthesized from 5-O-tert-butyl diphenylsilyl-1,2-O-isopropylidene-α-D-ribofuran-3-uloses (T.F. Tam and B. Fraser-Reid, 1980, J Chem. Soc., Chem. Commun., 556) (9.5 g, 22.2 mmol) using: anhydrous THF (425 cm ³ ), 1 M solution of magnesium allyl bromide in anhydrous ether (130 cm ³ , 130 mmol ), a saturated aqueous solution of ammonium chloride (490 cm ³ ), ether for extraction (350 + 2 × 160 cm ³ ), a 1.1 M solution of tetrabutylammonium fluoride in THF (22.3 cm ³ , 24.6 mmol), anhydrous THF (400 cm ³ ), dichloromethane (1400 cm ³ ), saturated aqueous sodium hydrogen carbonate (3 × 500 cm ³ ), brine (500 cm ³ ) and Na ₂ SO ₄ . δ _H ((CD ₃ ) ₂ SO) 5.84 (1H, m, 2'-H), 5.65 (1H, d, J 3.8, 1-H), 5.12 (1H, d, J 6.1, 3'-H _a ), 5.06 (1H, br, 3'-H _b ), 4.76 (1H, s, 3-OH), 4.64, (1H, t, J 5.4, 5-OH), 4.16 (1H, d, J 3.8, 2-H), 3.84 (1H, dd, J 2.2, 8.1, 4-H), 3 56 (1H, ddd, J 2.3, 5.6, 11.8, 5-H _a ), 3.42 (1H, m, 5-H _b ), 2.16 (1H, dd, J 6 , 1, 14.3, 1'-H _a ), 1.98 (1H, dd, J 8.2, 14.3, 1'-H _b ), 1.46 (3H, s, CH ₃ ), 1.25 (3H, s, CH ₃ ). δ _C (CDCl ₃ ) 133.5 (C-2 '), 117.9 (C-3 ⁷ ), 110.8 (C (CH ₃ ) ₂ ), 102.9 (C-1), 82.6 , 81.0, 77.7, (C-2, C-3, C-4), 59.4, (C-5), 36.4 (C-1 '), 26.4, 26.3 (CH ₃ ). Received: C-57.4, H-8.0. For C ₁₁ H ₁₈ O _{5 it is} calculated: C - 57.4, H - 7.9%.

Example 2

3-C-allyl-3,5-di-O-benzyl-1,2-O-isopropylidene-α-D-ribofuranose (0B)

A 60% suspension of sodium hydride (4.9 g, 123 mmol) in anhydrous DMF (100 cm ³ ) was stirred at 0 ° C and a solution of furanose 0A (9.42 g, 40.9 mmol) was added dropwise over 45 minutes. ) in anhydrous DMF (65 cm ³ ). The solution was stirred for 1 hour at 50 ° C. and cooled to 0 ° C. A mixture of benzyl bromide (14.5 cm ³ , 121 mmol) and anhydrous DMF (14.5 cm ³ ) was added dropwise, and the resulting mixture was stirred at room temperature in within 18 hours. The reaction mixture was evaporated to dryness and a solution of the residue in dichloromethane (700 cm ³ ) was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 450 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using petroleum ether / ethyl acetate (v / v, 9: 1) as an eluent to give 0B as an oily substance (14.5 g, 86%). δ _H (CDCl ₃ ) 7.39-7.21 (10H, m, Bn), 5.92 (1H, m, 2'-H), 5.71 (1H, d, J 3.8, 1- H), 5.17-5.09 (2H, m, 3'-H _a , 3'-H _b ), 4.67 (2H, m, Bn), 4.60 (1H, d, J 12, 2, Bn), 4.52 (1H, d, J 12.1, Bn), 4.43 (1H, m, 4-H), 4.42 (1H, d, J 3.8, 2-H ), 3.73 (1H, dd, J 3.2, 10.8, 5-H _a ), 3.66 (1H, dd, J 7.4, 10.8, 5-H _b ), 2, 50 (1H, dd, J 7.7, 14.9, 1'-H _a ), 2.39 (1H, dd, J 6.5, 14.9, 1'-H _b ), 1.60 ( 3H, s, CH ₃ ), 1.34 (3H, s, CH ₃ ). δ _C (CDCl ₃ ) 138.7, 138.1 (Bn), 132.6 (C-2 '), 128.3, 128.2, 127.7, 127.5, 127.4, 127.4 (Bn), 118.5 (C-3 ^' ), 112.6 (C (CH ₃ ) ₂ ), 104.1 (C-1), 86.5, 82.1, 80.4 (C-2 , C-3, C-4), 73.4, 68.6 (Bn), 67.0 (C-5), 35.8 (C-1 '), 26.8, 26.6 (CH ₃ ) FAB-MS m / z 433 [M + Na] ⁺ . Received: C, 73.4; H, 7.4. For C ₂₅ H ₃₀ O ₅ calculated: C - 73.2; H - 7.4%.

Example 3

3-C-allyl-1,2-di-O-acetyl-3,5-di-O-benzyl-D-ribofuranose (0C)

A solution of furanose 0B (12.42 g, 30.3 mmol) in 80% aqueous acetic acid (150 cm ³ ) was stirred at 90 ° C for 3 hours. The solvent was removed under reduced pressure and the residue was evaporated together with ethanol (3 × 75 cm ³ ), toluene (3 × 75 cm ³ ) and anhydrous pyridine (2 × 75 cm ³ ) and re-dissolved in anhydrous pyridine (60 cm ³ ). Acetic anhydride (46 cm ³ ) was added and the resulting solution was stirred at room temperature for 48 hours. A mixture of ice and water (300 cm ³ ) was added and the resulting mixture was extracted with dichloromethane (2 × 300 cm ³ ). The combined organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 200 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was evaporated and the residue was chromatographed on a silica gel column using petroleum ether / ethyl acetate (v / v, 4: 1) as an eluent to obtain the 0C anomeric mixture (β: α ≈ 2: 1) as an oily substance (13.3 g, 97%). δ _C (CDCl ₃ ) 169.7, 169.6 (C = O), 138.7, 138.4, 137.7, 137.6 (Bn), 132.4, 132.2 (C-2 ' ), 128.4 128.4, 128.2, 128.2, 127.8, 127.7, 127.7, 127.6, 127.3, 127.3, 126.9, 126.8 (Bn ), 118.5 (С-3 '), 99.4, 93.5 (С-1), 84.8, 83.7, 83.2, 82.0, 79.1, 75.5 (С -2, C-3, C-4), 73.7, 73.5, 69.3, 68.7 (Bn), 66.1 (C-5), 35.5, 34.9 (C- 1), 21.1, 21.0, 20.7, 20.6 (CH ₃ ). Received: C - 68.7; H - 6.7. For C ₂₆ H ₃₀ O ₇ calculated: C - 68.8; H - 6.6%.

Example 4

1- (2-O-acetyl-3-C-allyl-3,5-di-O-benzyl- (β-D-ribofuranosyl) thymine (1)

To a stirred solution of an anomeric mixture 0С (β: α ≈ 2: 1, 11.8 g, 26.0 mol) (P. Nielsen, HMPfundheller & J. Wengel, 1997, Chem. Commun., 825; P. Nielsen, HMPfundheller , CEOlsen & J. Wengel, 1997, J. Chem. Soc., Perkin Trans. 1, in press) and thymine (6.55 g, 52.0 mmol) in anhydrous acetonitrile (250 cm ³ ) were added N, O- bis (trimethylsilyl) acetamide (44.9 cm ³ , 182 mmol). The reaction mixture was stirred for 1 hour under reflux and cooled to 0 ° C. Trimethylsilyl triflate (8.00 cm ³ , 44.0 mmol) was added dropwise and the resulting solution was stirred at room temperature for 12 hours. A saturated aqueous solution of sodium hydrogencarbonate (270 cm ³ ) cooled on ice was added and the mixture was extracted with dichloromethane (3 × 125 cm ³ ). The organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (2 × 125 cm ³ ) and brine (2 × 125 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed and the residue was chromatographed on a silica gel column using dichloromethane / methanol (v / v, 98: 2) as eluent to give nucleoside 1 as a white solid (11.6 g, 86%). δ _H (CDCl ₃ ) 8.64 (1H, br s, NH), 7.75 (1H, d, J 1.1, 6-H), 7.41-7.25 (10H, m, Bn), 6.43 (1H, d, J 8.2, 1'-H), 5.88 (1H, m, 2 '' - H), 5.66 (1H, d, J 8.2, 2'- H), 5.12 (1H, s, 3 '' - H _a ), 5.07 (1H, dd, J 1.5, 8.5, 3 '' - H _b ), 4.85 (1H, d, J 11.2, Bn), 4.64 (2H, s, Bn), 4.63 (1H, d, J 11.2, Bn), 4.33 (1H, br, 4'-H) 3.81 (1H, dd, J 2.7, 11.1, 5'-H _a ), 3.65 (1H, m, 5'-H _b ), 2.81-2.65 (2H, m, 1 '' - H _a , 1 '' - H _b ), 2.08 (3H, s, COSH ₃ ), 1.52 (3H, d, J 0.8, CH ₃ ). δ _C (CDCl ₃ ) 170.1 (C = 0), 163.6 (C-4), 150.9 (C-2), 138.1, 136.6 (Bn), 136.0 (C- 6), 131.6 (C-2``), 128.8, 128.4, 128.3, 127.6, 127.5, 127.1 (Bn), 118.5 (C-3 '' ), 111.1 (C-5), 84.2, 83.4, 83.1, 77.4 (C-1 ', C-2', C-3 ', C-4'), 73, 6, 69.2 (Bn), 65.6 (C-5 ', 33.7 (C-1''), 20.8 (COSH ₃ ), 11.9 (CH ₃ ). Received: C - 66 , 8; H - 6.3; N - 5.1. For C ₂₉ H ₃₂ N ₂ O ₇ calculated: C - 66.9; H - 6.2; N - 5.4%.

Example 5

1- (3-C-allyl-3,5-di-O-benzyl-β-D-ribofuranosyl) thymine (2)

To a stirred solution of nucleoside 1 (11.6 g, 22.3 mmol) in methanol (110 cm ³ ) was added sodium methoxide (3.03 g, 55.5 mmol). The reaction mixture was stirred at room temperature for 16 hours and neutralized with dilute hydrochloric acid. The solvent was partially evaporated and the residue was dissolved in dichloromethane (2 × 400 cm ³ ). The organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (2 × 125 cm ³ ) and brine (2 × 125 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure to give compound 2 as a white solid (10.1 g, 95%). δ _H (COCl ₃ ) 8.77 (1H, br s, NH), 7.58 (1H, d, J 1.2, 6-H), 7.41-7.25 (10H, m, Bn), 6.14 (1H, m, 2``-H), 6.12 (1H, d, J 7.8, 1'-H), 5.23 (1H, m, 3 '' - H <sub>a</sub> ), 5.17 (1H, br, 3``-H _b ), 4.68 (1H, d, J 10.8, Bn), 4.59 (2H, s, Bn), 4.55 (1H, d , J 10.9, Bn), 4.39 (1H, brs, 4'-H), 4.26 (1H, dd, J 7.8, 10.7, 2'-H), 3.84 ( 1H, dd, J 3.1, 11.0, 5'-H _a ), 3.58 (1H, dd, J 1.4, 11.0, 5'-H _b ), 3.04 (1H, d, J 10.8, 2'-OH), 2.82-2.78 (2H, m, 1 '' - H _a , 1 '' - H _b ), 1.51 (3H, d, J 1 , 0, CH ₃ ). δ _C (CDCl ₃ ) 163.5 (C-4), 151.1 (C-2), 137.3, 136.7 (Bn), 136.0 (C-6), 132.1 (C- 2``), 128.8, 128.5, 128.3, 127.9, 127.6 (Bn), 118.4 (C-3 ''), 111.1 (C-5), 87, 4, 82.6, 81.1, 79.3 (C-1 ', C-2', C-3 ', C-4'), 73.7, 69.8 (Bn), 64.7 ( C-5 '), 35.1 (C-1''), 11.9 (CH ₃ ). Received: C, 67.8; H, 6.1; N is 5.5. For C ₂₇ H ₃₀ H ₂ O ₆ calculated: C - 67.8; H 6.3; N - 5.9%.

Example 6

1- (3-C-allyl-3,5-di-O-benzyl-2-O-methanesulfonyl-β-D-ribofuranosyl) thymine (3)

To a stirred solution of nucleoside 2 (3.50 g, 7.31 mmol) in anhydrous pyridine (23 cm ³ ) was added methanesulfonyl chloride (1.69 cm ³ , 21.89 mmol) at 0 ° C. The reaction mixture was stirred at room temperature in for 1 hour, water (100 cm ³ ) was added and extraction was carried out with dichloromethane (3 × 150 cm ³ ). The organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 200 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (v / v 99: 1) as an eluent to give compound 3 as a white solid (3.64 g, 89%). δ _n (CDCl ₃ ) 8.95 (1H, brs, NH), 7.71 (1H, d, J 1.1, 6-H), 7.39-7.25 (10H, m, Bn), 6.52 (1H, d, J 8.0, 1'-H), 5.90 (1H, m, 2 '' - H), 5.34 (1H, d, J 7.9, 2'- H), 5.20-5.09 (2H, m, 3 '' - H _a , 3 '' - H _b ), 4.91 (1H, d, J 11.2, Bn), 4.68 ( 1H, d, J 11.3, Bn), 4.64 (2H, s, Bn), 4.33 (1H, br, 4'-H), 3.81 (1H, dd, J 2.5, 11.1, 5'-H _a ), 3.73 (1H, dd, J 1.1, 11.1, 5'-H _b ), 3.08 (1H, dd, J 5.5, 5, 7, 1 '' - H _a ), 2.99 (3H, s, CH ₃ ), 2.68 (1H, m, 1 '' - H _b ), 1.51 (3H, d, J 0.8 , CH ₃ ). δ _C (CDCl ₃ ) 163.4 (C-4), 150.8 (C-2), 137.9, 136.3 (Bn), 135.5 (C-6), 131.0 (C- 2``), 128.8, 128.3, 127.5, 127.2 (Bn), 119.3 (C-3 ''), 111.6 (C-5), 84.1, 83, 6, 82.4, 82.2 (C-1 ', C-2', C-3 ', C-4'), 73.7, 68.9 (Bn), 66.2 (C-5 ' ), 38.7 (CH ₃ ), 33.0 (C-1 ″), 11.9 (CH ₃ ). Received: C - 60.5; H - 5.8; N - 4.9. For C ₂₈ H ₃₂ N ₂ O ₈ S calculated: C - 60.4; H - 5.8; N - 5.0%.

Example 7

1- (3-C-allyl-3,5-di-O-benzyl-β-D-arabinofuranosyl) thymine (4)

A solution of nucleoside 3 (3.59 g, 6.45 mmol) in ethanol (72 cm ³ ), water (72 cm ³ ) and 1 M aqueous sodium hydroxide (20.6 cm ³ ) was stirred under reflux for 18 hours. After neutralization with dilute hydrochloric acid, the solvent was removed under reduced pressure and the residue was dissolved in dichloromethane (3 × 150 cm ³ ). The organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 200 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (v / v 99: 1) as an eluent to give compound 4 as a white solid (2.32 g, 74%). δ _n (CDCl ₃ ) 7.60 (1H, d, J 1.2, 6-H), 7.50-7.23 (10H, m, Bn), 6.22 (1H, d, J 2, 9, 1'-H), 5.80 (1H, m, 2 '' - H), 5.15-5.08 (2H, m, 3 '' - H _a , 3 '' - H _b ), 4.86-4.33 (6H, m, 2 × Bn, 2'-H, 4'-H), 3.82-3.71 (2H, m, 5'-H _a , 5'-H _b ), 2.72 (1H, m, 1``-H <sub>a</sub> ), 2.52 (1H, dd, J 7.6, 16.1, 1 '' - H <sub>b</sub> ), 1.70 (3H, d J 0.9, CH <sub>3</sub> ). δ <sub>s</sub> (CDCl <sub>3</sub> ) 165.1 (C-4), 150.4 (C-2), 138.4, 136.8 (Bn), 137.7 (C-6), 132.3 (C- 2``), 128.77, 128.4, 128.3, 128.0, 127.9, 127.6 (Bn), 118.5, (C-3 ''), 107.8 (C- 5), 88.0, 87.8, 83.7 (C-1 ', C-3', C-4 '), 73.7, 72.9, 69.4 (Bn, C-2') 64.7 (C-5 '), 31.1 (C-1''), 12.4 (CH ₃ ). Received: C, 67.5; H 6.3; N is 5.3. For C ₂₇ H ₃₀ N ₂ O ₆ · 0.25 H ₂ O, C - 67.1 was calculated; H, 6.4; N - 5.8%.

Example 8

1- (3,5-di-O-benzyl-3-C- (2-hydroxyethyl) -β-D-arabinofuranosyl) thymine (5)

To a stirred solution of nucleoside 4 (2.26 g, 4.68 mmol) in THF (12 cm ³ ) and water (12 cm ³ ) was added sodium periodate (3.04 g, 14.2 mmol) and a 2.5% solution osmium tetroxide in tert-butanol (w / v, 0.603 cm ³ , 40 μmol). The solution was stirred at room temperature for 45 minutes. Water (25 cm ³ ) was added and the solution was extracted with dichloromethane (2 × 50 cm ³ ). The organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 30 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure and the residue was redissolved in THF (12 cm ³ ) and water (12 cm ³ ). The resulting mixture was stirred at room temperature and sodium borohydride (182 mg, 4.71 mmol) was added. After stirring for 1.5 hours, water (25 cm ³ ) was added and the solution was extracted with dichloromethane (2 × 50 cm ³ ). The organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 30 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (v / v 98: 2) as an eluent to give compound 5 as a white solid (1.13 g, 49%). δ _n (CDCl ₃ ) 9.29 (1H, brs, NH), 7.47 (1H, d, J 1.1, 6-H), 7.38-7.25 (10H, m, Bn), 6.22 (1H, d, J 3.4, 1'-H), 4.62 (2H, s, Bn), 4.60 (1H, m, 4'-H), 4.46 (2H, s, Bn), 4.35 (1H, dd, J 3.4, 7.5, 2'-H), 3.83-3.73 (4H, m, 2 × 5'-H, 2 × 2 '' -H), 2.67 (1H, br, OH), 2.07-2.01 (2H, m, 2 × 1 '' - H), 1.77 (3H, d, J 0.5 , CH ₃ ). δ _s (CDL ₃ ) 164.3 (C-4), 150.3 (C-2), 137.6, 137.4 (Bn, C-6), 136.7 (Bn), 128.6, 128.4, 128.2, 127.8, 127.6, 127.3, 127.1 (Bn), 108.4 (C-5), 88.0, 87.7, 81.6, 74, 7 (C-1 ', C-2', C-3 ', C-4'), 73.7, 69.6 (Bn), 64.6 (C-5 '), 57.7 (C- 2``), 28.6 (C-1 ''), 12.4 (CH ₃ ). FAB-MS m / z 483 [M + H] ⁺ , 505 [M + Na] ⁺ . Received: C, 63.6; H, 6.2; N is 5.4. For C ₂₆ H ₃₀ N ₂ O ₇ · 0.5H ₂ O, C — 63.5; H, 6.4; N - 5.7%.

Example 9

(1S, 5R, 6R, 8R) -5-hydroxy-6- (hydroxymethyl) -8- (thymin-1-yl) -2,7-dioxabicyclo [3.3.0] octane (6)

A solution of nucleoside 5 (1.08 g, 2.20 mmol) in anhydrous pyridine (5.0 cm ³ ) was stirred at 0 ° C and a solution of p-toluenesulfonyl chloride (462 mg, 2.47 mmol) in anhydrous pyridine ( 2.0 cm ³ ). After stirring at room temperature for 20 hours and adding a mixture of water and ice (70 cm ³ ), extraction was carried out with dichloromethane (2 × 75 cm ³ ). The organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 50 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (v / v, 99: 1) as an eluent to give an intermediate, which, after evaporation, was dissolved in anhydrous DMF (4.0 cm ³ ). This solution was added dropwise to a stirred suspension of 60% sodium hydride (203 mg, 4.94 mmol) in anhydrous DMF (4.0 cm ³ ) at 0 ° C. The resulting mixture was stirred for 18 hours and water was added (20 cm ³ ). After neutralization with hydrochloric acid, dichloromethane (75 cm ³ ) was added. The organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 50 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (v / v 98: 2) as an eluent to give a white solid (858 mg). A solution of this white solid in ethanol (10.0 cm ³ ) was stirred at room temperature and 20% palladium hydroxide on carbon (400 mg) was added. The resulting mixture was degassed with argon and placed in a hydrogen atmosphere. After stirring for 2 hours, the mixture was directly chromatographically purified on silica gel using dichloromethane / methanol (v / v, 97: 3) as eluent to give 6 as a white solid (444 mg, 82%). δ _n ((CD ₃ ) 2SO) 11.3 (1H, brs, NH), 7.36 (1H, d, J 1.1, 6-H), 5.80 (1H, d, J 4.3 , 1'-H), 5.61 (1H, s, OH), 4.86 (1H, m, 5'-H _a ), 3.89 (1H, d, J 4.2, 2'-H ), 3.85 (1H, m, 2``-H <sub>a</sub> ), 3.83-3.64 (3H, m, 4'-H, 5'-H <sub>b</sub> , 2 '' - H <sub>b</sub> ), 2 14 (1H, m, 1 '' - H <sub>a</sub> ), 1.81 (1H, m, 1 '' - H <sub>b</sub> ), 1.78 (3H, d, J 1.0, CH <sub>3</sub> ). δ <sub>c</sub> (CD <sub>3</sub> OD) 166.7 (C-4), 152.2 (C-2), 139.7 (C-6), 110.1 (C-5), 89.4, 89.1 85.5 85.2 (C-1 ', C-2', C-3 ', C-4'), 71.4 (C-2 ''), 61.6 (C-5 ') 37.0 (C-1``), 12.7 (CH ₃ ). Received: C - 47.4; H - 5.7; N - 9.0. For C ₁₂ H ₁₆ N ₂ O ₆ · H ₂ O calculated: C - 47.7; H - 6.0; N - 9.3%.

Example 10

(1S, 5R, 6R, 8R) -6- (4,4’-dimethoxytrityloxymethyl) -5-hydroxy-8- (thymin-1-yl) -2,7-dioxabicyclo [3.3.0] nonane (7)

A solution of nucleoside 6 (310 mg, 1.09 mmol) in anhydrous pyridine (2.5 cm ³ ) was stirred at room temperature and 4.4'-dimethoxytrityl chloride (593 mg, 1.83 mmol) was added. After stirring for 3 additional hours, 4,4'-dimethoxytrityl chloride (100 mg, 0.310 mmol) was added. After stirring for another 2 hours, methanol (0.5 cm ³ ) was added and the resulting mixture was evaporated. The residue was dissolved in dichloromethane (5 cm ³ ) and washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 5 cm ³ ). The organic phase was dried over Na ₂ SO ₄ and evaporated under reduced pressure. The residue was chromatographed on a silica gel column using dichloromethane / methanol (v / v, 99: 1) as an eluent to give compound 7 as a white solid (618 mg, 97%). δ _n (CDCl ₃ ) 9.04 (1H, brs, NH), 7.47-7.16 (10H, m, 6-H, DMT), 6.86-6.82 (4H, m, DMT) 6.06 (1H, d, J 4.1, 1'-H), 4.35 (1H, d, J 4.1, 2'-H), 4.03 (1H, m, 4'- H), 3.89 (1H, m, 2 '' - H _a ), 3.79 (6H, s, 2 × OCH ₃ ), 3.61 (1H, m, 5'-H _a ), 3, 32-3.26 (2H, m, 5'-H _b , 2 '' - H _b ), 1.94-1.69 (2H, m, 1 '' - H _a , 1 '' - H _b ) 1.89 (3H, s, CH ₃ ). δ _s (CDL ₃ ) 163.4 (C-4), 158.6 (DMT), 150.1 (C-2), 144.3 (DMT), 137.2 (C-6), 135.6 , 135.3, 129.9, 129.9, 128.9, 128.1, 127.9, 126.9, 125.2, 113.2 (DMT), 109.3 (C-5), 88 7, 87.3, 86.9, 83.5, 81.0 (DMT, C-1 ', C-2', C-3 ', C-4'), 69.7 (C-2 ''), 62.1 (C-5'), 55.1 (OCH ₃ ), 36.5 (C-1 ''), 12.5 (CH ₃ ).

Example 11

(1S, 5R, 6R, 8R) -5- (2-cyanoethoxy (diisopropylamino) phosphinoxy) -6- (4,4'-dimethoxytrityloxymethyl) -8- (thymin-1-yl) -2,7-dioxabicyclo [3.3 .0] nonane (8)

A solution of nucleoside 7 (436 mg, 0.743 mmol) in anhydrous dichloromethane (2.2 cm ³ ) and diisopropylethylamine (0.62 cm ³ ) was stirred at room temperature and 2-cyanoethyl-N, N-diisopropylphosphoamidochloridite (0.33 cm ³ ) was added. 1.46 mmol). After stirring for 1.5 hours, methanol (0.4 cm ³ ) and ethyl acetate (5 cm ³ ) were added, and the resulting mixture was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 5 cm ³ ) and saturated brine (3 × 5 cm) ³ ). The organic phase was dried over Na ₂ SO ₄ and evaporated under reduced pressure. The residue was chromatographed on a silica gel column using dichloromethane / methanol (v / v 97: 3) as an eluent and the solvents were evaporated to give an oily substance, which was dissolved in toluene (1 cm ³ ) and precipitated with hexane at -30 ° С to obtain Compound 8 as a white solid (517 mg, 88%). δ _p (CDCl ₃ ) 142.0, 141.9.

Example 12

1- (3,5-di-O-benzyl-3-C (2-hydroxyethyl) -β-D-ribofuranosyl) thymine (9)

Sodium periodate (1.34 g, 6.27 mmol) and 2 were added to a stirred solution of nucleoside 2 (1.00 g, 2.09 mmol) in THF (5.4 cm ³ ) and water (5.4 cm ³ ) 5% solution of osmium tetrooxide in greg-butanol (w / v, 0.265 cm ³ , 19 μmol). The solution was stirred at room temperature for 45 minutes. Water (25 cm ³ ) was added and the solution was extracted with dichloromethane (2 × 50 cm ³ ). The organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 30 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure and the residue was redissolved in THF (5.4 cm ³ ) and water (5.4 cm ³ ). The mixture was stirred at room temperature and sodium borohydride (79 mg, 2.08 mmol) was added. After stirring for 1.5 hours, water (25 cm ³ ) was added and the resulting solution was extracted with dichloromethane (2 × 50 cm ³ ). The organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 30 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (v / v, 98: 2) as an eluent to give compound 9 as a white solid (488 mg, 48%). δ _n (CDCl ₃ ) 9.14 (1H, br s, NH), 7.60 (1H, d, J 1.1, 6-H), 7.40-7.22 (10H, m, Bn), 6.25 (1H, d, J 7.7, 1'-H), 4.59 (1H, d, J 7.1, Bn), 4.49 (1H, d, J 7.1 Bn), 4.39-3.30 (m, 8H, 4'-H, 2'-H, Bn, 5'-H _a , 5'-H _b , 2 '' - H _a , 2 '' - H _b ) 2.23-2.00 (2H, m, 1 '' - H _a , 1 '' - H _b ), 1.49 (3H, d, J 0.7, CH ₃ ). δ _s (CDCl ₃ ) 163.5 (C-4), 151.2 (C-2), 137.1, 136.5 (Bn), 135.7 (C-6), 128.7, 128, 5, 128.2, 127.8, 127.6, 127.2 (Bn), 111.3 (C-5), 87.0, 82.7, 81.1, 78.3 (C-1 ' , C-2 ', C-3', C-4 '), 73.7, 69.6 (Bn), 64.4 (C-5'), 57.0 (C-2 ''), 32 4 (C-1``), 11.8 (CH ₃ ). Received: C, 63.9; H 6.3; N is 5.4. For C ₂₆ H ₃₀ N ₂ O ₇ · 0.25H ₂ O, calculated: C — 64.1; H 6.3; N - 5.75%.

Example 13

1- [3-C- (2-O-tert-butyldimethylsilyloxyethyl) -3,5-di-O-benzyl-β-D-ribofuranosyl] thymine (10)

A mixture of nucleoside 9 (1.80 g, 3.4 mmol) and tert-butyldimethylsilyl chloride (0.585 g, 3.9 mmol) was dissolved in anhydrous pyridine (20 cm ³ ). After standing for 2 hours at room temperature, the reaction mixture was evaporated to dryness, evaporated twice with toluene (2 × 10 cm ³ ) and redissolved in dichloromethane (150 cm ³ ). The solution was washed with a saturated aqueous solution of sodium hydrogencarbonate (2 × 50 cm ³ ) and evaporated to give a foam. The resulting material was purified by preparative HPLC in silica gel using an elution gradient (0-3% methanol in dichloromethane: o / o) to give nucleoside 10 as a white solid (1.86 g, 92%). δ _n (CDCl ₃ ) 7.61 (1H, d, J 1.1, 6-H), 7.35-7.20 (10H, m, Bn), 6.27 (1H, d, J 7, 9, 1'-H), 4.65-4.40 (4H, m, Bn, 2'-H), 4.37 (1H, s, Bn), 4.28 (1H, t, J 7, 9, 4'-H), 4.35-3.55 (4H, m, 2 '' - H _a , 2 '' - H _b , 5'-H _a , 5'-H _b ), 2.30 -2.05 (2H, m, 1 '' - H _a , 1 '' - H _b ), 1.46 (3H, s, 5-CH ₃ ), 0.90 (9H, m, CH ₃ -C -Si), 0.08 (6H, m, CH ₃ -Si). δ _s (CDCl ₃ ) 163.6 (C-6), 151.0 (C-2), 137.5, 136.6, 135.8 (C-5, Bn), 128.3, 128.1 , 127.8, 127.2, 127.1, 126.8, 126.7 (Bn), 110.7 (C-4), 86.8, 82.5, 81.6, 78.3 (C -1 ', C-2', C-3 ', C-4'), 73.3, 69.8 (Bn), 64.46 (C-5 '), 58.2 (C-2'' ), 32.9 (C-1``), 25.6, 25.4, 17.9, -3.9, -5.7 (TBDMS), 11.6 (CH ₃ ). FAB ⁺ -MS: m / z 597.19 [M + H] ⁺ , 619.18 [M + Na] ⁺ . Received: C, 64.2; H - 7.4; N is 4.2. For C ₃₂ H ₄₄ O ₇ N ₂ Si calculated: C - 64.4; H - 7.4; N - 4.7%.

Example 14

1- [3-C- (2-O-tert-butyldimethylsilyloxyethyl) -3,5-di-O-benzyl-β-D-erythropentofuran-2-ulosyl] thymine (11)

A mixture of nucleoside 10 (2.14 g, 3.59 mmol), pyridinium dichromate (1.48 g, 3.95 mmol) and activated 3A molecular sieve powder (4 g) was suspended in anhydrous dichloromethane (80 cm ³ ). After cooling the mixture to −10 ° C. with vigorous stirring, acetic anhydride (10 cm ³ , 98 mmol) was added dropwise. The suspension was allowed to warm to room temperature and stirring was continued for 1.5 hours until the reaction was stopped by the addition of triethylamine (20 cm ³ ). The resulting mixture was diluted with dichloromethane to 300 cm ³ and washed with water (2 × 200 cm ³ ). The organic phase was evaporated and the obtained residue was chromatographically purified in silica gel using a gradient of 1.0%, 1.2%, 1.3%, 1.4% and 1.5% methanol in dichloromethane (v / v, total volume 250 cm ³ at each step of the gradient) to obtain nucleoside 11 (1.89 g, 84.4%) as a white solid. δ _n (CDCl ₃ ) 7.35-7.20 (11H, m, Bn, 6-H), 6.40 (1H, s, 1'-H), 4.57 (1H, s, Bn), 4.52 (1H, s, Bn), 4.46 (1H, d, J 11.0, Bn), 4.29 (1H, d, J 11.0, Bn), 4.07 (1H, dd , J 0.5, 2.2, 4'-H), 3.95-3.70 (4H, m, 2 '' - H _a , 2 '' - H _b , 5'-H _a , 5 ' -H _b ), 2.05 (1H, m, 1 '' - H _a ), 2.42 (1H, m, 1 '' - H _b ), 1.42 (3H, d, J 1.1, 5-CH ₃ ), 0.86 (9H, s, CH ₃ -C-Si), 0.01 (6H, s, CH ₃ -Si). δ _s (CDCl ₃ ) 202.6 (C-2 '), 163.7 (C-4), 151.2 (C-2), 137.7, 136.6, 136.5 (Bn, C- 6), 128.7, 128.5, 128.2, 128.1, 127.7, 126.4, 126.3 (Bn), 110.9 (C-5), 84.5, 81.3 80.2 (C-1 ', C-3', C-4 '), 73.6, 70.4 (Bn), 66.0 (C-5'), 57.6 (C-2 ''), 27.3 (C-1``), 25.9, 25.7, 18.2, -5.8, -5.9 (TBDMS), 11.7 (CH ₃ ). FAB-MS m / z 595.14 [M + H] ⁺ . Received: C, 64.1; H - 6.9; N is 4.5. For C ₃₂ H ₄₂ N ₂ Si calculated: C - 64.6; H - 7.1; N - 4.7%.

Example 15

(1S, 5R, 6R, 8R) -1-hydroxy-5-benzyloxy-6-benzyloxymethyl-8- (thymin-1-yl) -2,7-dioxabicyclo [3.3.0] octane (12)

Compound 11 (1.80 g, 30.3 mmol) was dissolved in 0.5% HCl in methanol (w / v, 20 cm ³ ) and the resulting mixture was stirred for 30 minutes at room temperature. After evaporation to dryness, the residue was dissolved in dichloromethane (100 cm ³ ) and washed with a saturated aqueous solution of sodium hydrogencarbonate (2 × 40 cm ³ ). The organic phase was evaporated and the residue was chromatographed on silica gel using 2% methanol in dichloromethane (v / v) to give nucleoside 12 (1.35 g, 93.5%) as a white solid. δ _n (CDCl ₃ ) 7.37-7.27 (11H, m, Bn, 6-H), 5.87 (1H, s, 1'-H), 4.71 (2H, s, Bn), 4.64 (1H, d, J 12.0, Bn), 4.56 (1H, d, J 12.0, Bn), 4.36 (1H, t, J 5.7, 4'-H) 4.16 (1H, m, 2 '' - H _a ), 3.96 (1H, m, 2 '' - H _b ), 3.74 (2H, m, 5'-H _a , 5'- H _b ), 2.35-2.15 (2H, m, 1``-H _a , 1 '' - H _b ), 1.88 (3H, s, CH ₃ ). δ _s (CDCl ₃ ) 163.7 (C-4), 151.4 (C-2), 137.8, 137.3, 136.7 (Bn, C-6), 128.5, 128.4 , 128.0, 127.8, 127.5 (Bn), 109.9 (C-5), 108.6 (C-2 '), 88.8, 87.1, 80.9 (C-1 ', C-3', C-4 '), 73.6, 68.5, 68.1, 67.9 (C-5', C-2 '', Bn), 30.9 (C-1 ''), 12.6 (CH ₃ ). FAB-MS: m / z 481.03 [M + H] ⁺ , 503.02 [M + Na] ⁺ . Received: C, 64.6; H - 5.8; N - 5.7. For C ₂₆ H ₂₈ O ₇ N ₂ calculated: C - 65.0; H - 5.9; N - 5.8%.

Example 16

(1S, 5R, 6R, 8R) -1,5-dihydroxy-6-hydroxymethyl-8- (thymin-1-yl) -2,7-dioxabicyclo [3.3.0] octane (13)

Compound 13 was sequentially obtained from compound 12 by catalyzing the protective benzyl group in the same manner as described for compound 6. Purification 13 was carried out by chromatography on a silica gel column in a methanol gradient (6 to 14%) in dichloromethane. Analytical amounts of compound 13 (up to 15 mg) were further purified by reverse phase HPLC on a column (10 × 250 mm) loaded with Nucleosil C18 (10 μm). The flow rate is 8 cm ³ / min. Eluent - 0-10% acetonitrile. Time is 60 minutes. The yield was 82%. δ _n (CD ₃ OD) 7.44 (1H d, J 1.2, 6-H), 5.83 (1H, s, 1'-H), 4.10-3.80 (5H, m, 5'-H _a , 5'-H _b , 2 '' - H _a , 2 '' - H _b , 4'-H), 2.39-2.25 (1H, m, 1 '' - H _a ), 2.00-1.90 (1H, m, 1 '' - H _b ), 1.87 (3H, d, J 1.2, CH ₃ ). δ _s (CD ₃ OD) 166.3 (C-4), 152.7 (C-2), 139.8 (C-6), 110.0, 109.6 (C-2 ', C-5 ), 87.8, 85.8, 84.6 (C-1 ', C-3', C-4 '), 68.8, 61.6 (C-5', C-2 ''), 35.6 (C-1``), 12.4 (CH ₃ ). FAB-MS: m / z 301.03 [M + H] ⁺ . Received: C, 46.6; H - 5.7; N is 8.5. For C ₁₂ H ₁₆ O ₇ N ₂ calculated: C - 48.0; H - 5.4; N - 9.3%.

Example 17

(1S, 5R, 6R, 8R) -5-benzyloxy-6-benzyloxymethyl-1-methoxy-8- (3-N-methylthymin-1-yl) -2,7-dioxabicyclo [3.3.0] octane (14) , (1S, 5R, 6R, 8R) -5-benzyloxy-6-benzyloxymethyl-1-hydroxy-8- (3-N-methylthymin-1-yl) -2,7-dioxabicyclo [3.3.0] octane (15 ) and (1S, 5R, 6R, 8R) -5-benzyloxy-6-benzyloxymethyl-1-methoxy-8- (thymin-1-yl) -2,7-dioxabicyclo [3.3.0] octane (16)

A mixture of compound 12 (1.04 g, 2.16 mmol) and sodium hydride (171 mg of a 60% suspension in mineral oil, 4.30 mmol) was dissolved in anhydrous dichloromethane (4 cm ³ ) for 10 minutes with stirring. Methyl iodide (1 cm ³ , 16 mmol) was added and the resulting reaction mixture was incubated at 36 ° C for 23 hours. After evaporation, the residue was chromatographed on a silica gel column using a gradient of 0.4-2.4% methanol in dichloromethane (v / v) as eluent to give products 14, 15 and 16, as well as starting compound 12 (212 mg, 20 ,5%). Compound 14 (47 mg, 4.3%). δ _n (CDCl ₃ ) 7.25-7.37 (11H, m, Bn, 6-H), 6.15 (1H, s, 1'-H), 4.74 (1H, d, J 11, 5, Bn), 4.67 (1H, d, J 11.3, Bn), 4.62 (1H, d, J 12.1, Bn), 4.55 (1H, d, J 11.9, Bn), 4.34 (1H, t, J 5.6, 4'-H), 3.99 (1H, m, 2 '' - H _a ), 4.22 (1H, t, 2 '' - H _b ), 3.72 (2H, m, 5'-H _a , 5'-H _a ), 3.41 (3H, s, CH ₃ -O), 3.35 (3H, s, CH ₃ - N ³ ), 2.27 (1H, m, 1 '' - H _a ), 2.41 (1H, m, 1 '' - H _b ), 1.93 (3H, s, 5-CH ₃ ). δ _s (CDCl ₃ ) 163.3 (C-4), 151.0 (C-2), 138.2, 137.3, 135.7 (Bn, C-6), 128.3, 128.2 , 127.8, 127.6, 127.4, 126.9 (Bn), 111.8 (C-5), 108.5 (C-2 '), 89.1, 84.8, 79.5 (C-1 ', C-3', C-4 '), 73.5, 68.4, 68.2, 67.3 (Bn, 5 C-5', C-2``), 50, 8 (CH <sub>3</sub> -O), 32.6 (C-1``), 27.9 (CH ₃ -N), 13.2 (CH ₃ ). FAB-MS: m / z 508.88 [M + H] ⁺ . Received: C, 65.7; H - 6.9; N - 4.8. For C ₂₈ H ₃₂ O ₇ N ₂ calculated: C - 66.1; H 6.3; N - 5.5%. Compound 15 (97 mg, 9.1%). δ _n (CDCl ₃ ) 7.37-7.28 (11H, m, Bn, 6-H), 5.86 (1H, s, 1'-H), 4.72 (2H, s, Bn), 4.64 (1H, d, J 11.9, Bn), 4.58 (1H, d, J 11.9, Bn), 4.37 (1H, t, J 5.6, 4'-H) 4.13 (1H, m, 2``-H <sub>a</sub> ), 3.93 (1H, m, 2 '' -H <sub>b</sub> ), 3.75 (2H, m, 5'-H <sub>a</sub> , 5'- H <sub>b</sub> ), 3.34 (1H, s, CH <sub>3</sub> -N), 2.32-2.16 (2H, m, 1 '' - H <sub>a</sub> , 1 '' - H <sub>b</sub> ), 1.93 (3H , s, CH <sub>3</sub> ). δ <sub>s</sub> (CDL <sub>3</sub> ) 163.2 (C-4), 151.9 (C-2), 137.5, 137.1, 134.0 (Bn, C-6), 128.4, 128.3 , 128.1, 127.9 127.7, 127.6, 127.3 (Bn), 108.8, 108.5 (C-2 ', C-5), 88.7 (C-1') , 88.0, 81.0 (C-3 ', C-4'), 73.5, 68.3, 67.9, 67.7 (Bn, C-5 ', C-2''), 30.6 (C-1``), 27.8 (CH ₃ -N), 13.2 (CH ₃ ). FAB-MS m / z 495.28 [M + H] ⁺ , 517.24 [M + Na] ⁺ . Compound 16 (665 mg, 62.3%). δ _n (CDCl ₃ ) 7.35-7.25 (11H, m, Bn, 6-H), 6.06 (1H, s, 1'-H), 4.73 (1H, d, J 11, 5, Bn), 4.6, (1H, d, J 11.3, Bn), 4.61 (1H, d, J 11.9, Bn), 4.55 (1H, d, J 12.0 , Bn), 4.34 (1H, t, J 5.6, 4'-H), 4.20 (1H, m, 2``-H <sub>a</sub> ), 3.98 (1H, m, 2 '' -H <sub>b</sub> ), 3.72 (2H, m, 5'-H <sub>a</sub> , 5'-H <sub>b</sub> ), 3.40 (3H, s, CH <sub>3</sub> -O), 2.45-2.35 (1H, m 1 '' - H <sub>a</sub> ), 2.30-2.20 (1H, m, 1 '' - H <sub>b</sub> ), 1.90 (3H, d, J 1.1, CH <sub>3</sub> ). δ <sub>s</sub> (CDL <sub>3</sub> ) 163.2 (C-4), 150.1 (C-2), 138.2, 137.9, 137.3 (Bn, C-6), 128.4, 128.2 , 127.8, 127.6 127.4, 127.1 (Bn), 110.8 (C-5), 109.3 (C-2 '), 89.2, 84.2, 79.6 ( C-1 ', C-3', C-4 '), 73.6, 68.5, 68.3, 67.4 (Bn, C-5', C-2``), 50.8 ( CH ₃ -O), 32.6 (C-1 ″), 12.5 (CH ₃ ). FAB-MS m / z 495.22 [M + H] ⁺ , 517.23 [M + Na] ⁺ . Received: C, 66.2; H - 7.2; N is 4.4. For C ₂₇ H ₃₀ O ₇ N ₂ calculated: C - 65.6; H, 6.1; N - 5.7%.

Example 18

[1S, 5R, 6R, 8R) -5-hydroxy-6-hydroxymethyl-1-methoxy-8- (thymin-1-yl) -2,7-dioxabicyclo [3.3.0] octane (17)

To a solution of nucleoside 16 (1.20 g, 2.43 mmol) in methanol (10 cm ³ ) was added 20% palladium hydroxide on carbon (250 mg) and the resulting mixture was carefully degassed under reduced pressure. A hydrogen atmosphere was used and stirred for 12 hours. After that, the catalyst was removed by filtering the reaction mixture through a glass column (1 × 8 cm) loaded with silica gel in methanol. This column was further washed with methanol (20 cm ³ ). All fractions were collected, evaporated to dryness and evaporated together with petroleum ether to give a glassy solid. This residue was chromatographically purified on silica gel using a gradient of 5-10% methanol in dichloromethane (v / v) for elution. These fractions, including the desired product, were collected, combined and evaporated to dryness. The residue was dissolved in anhydrous methanol (5 cm ³ ) and anhydrous benzene (100 cm ³ ) was added. Lyophilization yielded nucleoside 17 (0.61 g, 79%) as a white solid. δ _n (CD ₃ OD) 7.45 (1H, s, 6-H), 5.93 (1H, s, 1'-H), 4.15-3.81 (5H, m, 5'-H _a , 5'-H _b , 2 '' - H _a , 2 '' - H _b , 4'-H), 3.43 (3H, s, CH ₃ -O), 2.47-2.40 ( 1H, m, 1 '' - H), 2.03-1.93 (1H, m, 1 '' - H _b ), 1.92 (3H, s, CH ₃ ). δ _C (CD ₃ OD) 164.1 (C-4), 150.1 (C-2), 138.3 (C-6), 109.6 (C-5), 108.3 (C-2 '), 84.4, 84.1, 82.4 (C-1', C-3 ', C-4'), 68.0, 59.5 (C-5 ', C-2'') 49.6 (CH ₃ -O), 34.0 (C-1``), 10.5 (CH ₃ ). FAB-MS m / z 315.13 [M + H] ⁺ , 337.09 [M + Na] ⁺ . Received: C - 49.9; H - 5.7; N - 8.2. For C ₁₃ H ₁₈ O ₇ N ₂ calculated: C - 49.7; H - 5.8; N - 8.9%.

Example 19

(1S, 5R, 6R, 8R) -6- (4,4'-dimethoxytrityloxymethyl) -5-hydroxy-1-methoxy-8- (thymin-1-yl) -2,7-dioxabicyclo [3.3.0] octane (18)

A mixture of compound 17 (0.95 g, 3.03 mmol) and 4,4'-dimethoxytrityl chloride (1.54 g, 4.77 mmol) was dissolved in anhydrous pyridine (20 cm ³ ) and stirred for 4 hours at room temperature . The reaction mixture was evaporated to give an oily residue, which was evaporated together with toluene (2 × 20 cm ³ ). Dichlorometes (50 cm ³ ) and a saturated aqueous solution of sodium hydrogencarbonate (50 cm ³ ) were added, the organic phase was isolated and evaporated, and the obtained residue was purified by HPLC in silica gel (the residue was dissolved in a minimum amount of dichloromethane containing 0.5% triethylamine (o / o), which was applied to a column balanced with the same solvent). The column was washed with ethyl acetate / petroleum ether / triethylamine (15: 84.5: 0.5, v / v / v, total volume 1000 cm ³ ) and the resulting product was eluted in a methanol gradient (0-2%) in dichloromethane containing 0, 5% triethylamine (v / v) to give compound 18 (1.71 g, 92.8%) as a white solid. δ _n (CDCl ₃ ) 7.51-7.17 (10H, m, DMT, 6-H), 6.79-6.85 (4H, m, DMT), 6.04 (1H, s, 1 ' -H), 4.12-3.98 (3H, m, 5'-H _a , 5'-H _b , 4'-H), 3.77 (6H, s, CH ₃ -DMT), 3, 49 (3H, s, CH ₃ -O), 3.45-3.32 (2H, m, 2 '' - H _a , 2 '' - H _b ), 2.11-2.01 (1H, m , 1 '' - H _a ), 1.94-1.87 (1H, m, 1 '' - H _b ), 1.93 (3H, s, CH ₃ ). δ _s (CDCl ₃ ) 164.2 (C-4), 158.6, 144.7, 135.7, 130.1, 128.2, 127.9, 126.8, 113.2 (DMT), 150.7 (C-2), 137.7 (C-6), 109.8, 109.7 (C-5, C-2 '), 86.5, 85.3, 85.0, 81, 4 (DMT, C-1 ', C-3', C-4 '), 69.2, 62.4 (C-5', C-2 ''), 55.2 (CH ₃ -DMT), 51.7 (CH ₃ -O), 35.5 (C-1``), 12.7 (CH ₃ ). FAB-MS m / z 617.26 [M + H] ⁺ , 639.23 [M + Na] ⁺ . Received: C, 66.4; P is 6.1; N is 4.2. For C ₃₄ P ₃₆ O ₉ N ₂ calculated: C - 66.2; P - 5.9; N - 4.5%.

Example 20

(1S, 5R, 6R, 8R) -5- (2-

cyanoethoxy (diisopropylamimo) phosphinoxy) -6- (4,4’-dimethoxytritylmethyl) -1-methoxy-8- (thymin-1-yl) -2,7-dioxabicyclo [3.3.0] octane (19)

Compound 18 (1.2 g, 1.95 mmol) was dissolved in anhydrous dichloromethane (10 cm ³ ). N, N-diisopropylethylamine (1.35 cm ³ , 7.8 mmol) and 2-cyanoethyl-N, N-diisopropylphosphoramidochloridite (0.92 g, 3.9 mmol) were added under stirring at room temperature. After 72 hours, the resulting mixture was diluted to 100 cm ³ in dichloromethane and washed in a saturated aqueous solution of hydrogen carbonate (50 cm ³ ). The organic phase was evaporated and applied to a silica gel HPLC column using a gradient of eluent B (petroleum ether / dichloromethane / ethyl acetate / pyridine - 45:45:10; 0.5 - v / v) in eluent A (petroleum ether / dichloromethane / - pyridine - 50: 50: 0.5 - o / o). Fractions containing the desired product were concentrated, evaporated together with toluene (10 cm ³ ) and dried under reduced pressure. The residue was dissolved in anhydrous benzene (20 cm ³ ) and precipitated by adding this solution to anhydrous petroleum ether (400 cm ³ ) with stirring. The resulting white solid was isolated by filtration and dried to give compound 19 (0.96 g, 60.3%). δ _p (CDCl ₃ ) 142.64, 142.52. FAB-MS m / z 817.26 [M + H] ⁺ , 839.24 [M + H] ⁺ . Received: C - 62.8; P - 6.4; N, 6.9. For C ₄₃ P ₅₃ O ₁₀ N ₄ P it is calculated: C - 63.2; H - 6.5; N - 6.9%.

Example 21

1,2-O-isopropylidene-3-C-vinyl-α-D-ribofuranose (20)

A solution of 5-O-tert-butyldimethylsilyl-1,2-O-isopropylidene-α-D-erythropent-3-olofuranose (Y. Yoshimura, T.Sano, A. Matsuda, T. Ueda, 1988, Chem. Pharm. Bull ., 36, 162) (6.05 g, 0.020 mol) in anhydrous THF (250 cm ³ ) was stirred at 0 ° C and a 1 M solution of magnesium vinyl bromide in ether (44 cm ³ , 44 mmol) was added dropwise. The resulting reaction mixture was stirred at room temperature for 2 hours, after which a saturated aqueous solution of ammonium chloride (200 cm ³ ) was added and extraction was carried out with dichloromethane (3 × 300 cm ³ ). The combined extracts were washed with brine (3 × 250 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed and the residue was redissolved in anhydrous THF (225 cm ³ ). To this mixture was added a 1 M solution of tetrabutylammonium fluoride in THF (22 cm ³ , 22 mmol), stirred at room temperature for 20 minutes, after which the mixture was evaporated under reduced pressure. The residue was dissolved in dichloromethane (500 cm ³ ) and washed with a saturated solution of sodium hydrogencarbonate (2 × 200 cm ³ ). The aqueous phase was extracted using continuous 12-hour extraction and the combined extracts were dried over Na ₂ SO ₄ and evaporated. The residue was chromatographed on silica gel using dichloromethane / methanol (99: 1, v / v) as an eluent to give furanose 20 as a white solid (3.24 g, 75%). δ _n (CDCl ₃ ) 5.84 (1H, d, J 3.7, 1-H), 5.74 (1H, dd, J 11.0, 17.2, 1'-H), 5.52 (1H, dd, J 1.6, 17.1, 2'-H _a ), 5.29 (1H, dd, J 1.3, 11.0, 2'-H _b ), 4.21 (1H d, J 3.7, 2-H), 3.98 (1H, t, J 5.7, 4-H), 3.68-3.64 (2H, m, 5-H _a , 5- H _b ), 2.88 (1H, s, 3-OH), 1.99 (1H, t, J 6.3, 5-OH), 1.60 (3H, s, CH ₃ ), 1.35 (3H, s, CH ₃ ). δ _s (CDCl ₃ ) 133.6 (C-1 '), 116.2 (C-2'), 113.0 (C (CH ₃ ) ₂ ), 103.8 (C-1), 83.4 82.4 (C-4, C-2), 79.6 (C-3), 61.3 (C-5), 26.5, 26.4 (CH ₃ ).

Example 22

3,5-di-O-benzyl-1,2-O-isopropylidene-3-C-vinyl-α-D-ribofuranose (21)

A 60% suspension of sodium hydride (w / v, 1.78 g, 44.5 mmol) in anhydrous DMF (50 cm ³ ) was stirred at 0 ° C and a solution of furanose 20 (3.20 g, 14.8 mmol) in anhydrous DMF (35 cm ³ ) was added dropwise over 30 minutes. The mixture was stirred at 50 ° C. for 1 hour and then cooled to 0 ° C. A solution of benzyl bromide (5.3 ml, 44.5 mol) in anhydrous DMF (5.3 cm ³ ) was added dropwise and the resulting mixture was stirred at room temperature. temperature for 20 hours. The reaction mixture was evaporated and redissolved in dichloromethane (300 cm ³ ), washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 200 cm ³ ) and dried over Na ₂ SO ₄ . The solvents were removed under reduced pressure and the residue was subjected to chromatographic purification with petroleum ether / ethyl acetate (9: 1 v / v) as an eluent to obtain furanose 21 as a white solid (5.36 g, 91%). δ _n (CDCl ₃ ) 7.40-7.26 (10H, m, Bn), 5.90 (1H, d, J 3.6, 1-H), 5.72 (1H, dd, J 11, 1, 17.9, 1'-H), 5.41 (1H, dd, J 0.7, 11.1, 2'-H _a ), 5.30 (1H, dd, J 0.5, 17 8, 2'-H _b ), 4.70-4.45 (6H, m, Bn, 2-H, 4-H), 3.69 (1H, dd, J 2.6, 10.8, 5-H _a ), 3.50 (1H, dd, J 7.9, 10.9, 5-H _b ), 1.64 (3H, s, CH ₃ ), 1.40 (3H, s, CH ₃ ). δ _s (CDCl ₃ ) 138.6, 138.3 (Bn), 134.5 (C-1 '), 128.3-127.4 (Bn), 118.2 (C-2'), 112, 9 (C (CH ₃ ) ₂ ), 104.7 (C-1), 84.7, 81.1, 81.0 (C-2, C-3, C-4), 73.3 (C- 5), 69.4, 67.0 (Bn), 26.8, 26.6 (CH ₃ ).

Example 23

3,5-di-O-acetyl-3,5-di-O-benzyl-3-C-vinyl-α, β-D-ribofuranose (22)

A solution of furanose 21 (4.40 g, 11.1 mmol) in 80% aqueous acetic acid (50 cm ³ ) was stirred at 90 ° C for 8 hours. The solvents were removed and the resulting residue was evaporated together with 99% ethanol (3 × 25 cm ³ ), toluene (3 × 25 cm ³ ) and anhydrous pyridine (2 × 25 cm ³ ) and re-dissolved in anhydrous pyridine (20 cm ³ ) . Acetic anhydride (17 cm ³ ) was added and the resulting solution was stirred at room temperature for 48 hours. The reaction was stopped with ice-cold water (10 cm ³ ) and extracted with dichloromethane (2 × 100 cm ³ ). The combined extracts were washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 100 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was evaporated and the residue was chromatographed on silica gel using petroleum ether / ethyl acetate (4: 1, v / v) as an eluent to obtain furanose 22 as an oily substance (4.27 g, 87%, α: β ≈ 1: 1). δ _s (CDCl ₃ ) 169.9, 169.8 (C = O), 139.0, 138.6, 138.0, 137.8 (Bn), 133.3, 132.4 (C-1 ' ), 128.4-126.8 (Bn), 119.6, 119.5 (C-2 '), 99.5, 94.0 (C-1), 85.4, 85.0, 84, 3, 83.6, 77.7, 73.6, 73.5, 73.3, 70.0, 69.2, 67.5, 67.2 (C-2, C-3, C-4, C-5, Bn), 21.0, 20.9, 20.6, 20.4 (CH ₃ ).

Example 24

1- (2-O-acetyl-3,5-di-O-benzyl-3-C-vinyl-β-D-ribofuranosyl) thymine (23)

To a stirred solution of compound 22 (4.24 g, 9.6 mmol) and thymine (2.43 g, 19.3 mmol) in anhydrous acetonitrile (100 cm ³ ) was added N, O-bis (trimethylsilyl) acetamide (11, 9 cm ³ , 48.1 mmol). The resulting reaction mixture was stirred under reflux for 30 minutes. After cooling to 0 ° C., trimethyl silyl triflate (3.2 cm ³ , 16.4 mmol) was added dropwise and the resulting solution was stirred at room temperature for 24 hours. The reaction was stopped with a cold saturated aqueous solution of sodium hydrogencarbonate (10 cm ³ ) and the resulting mixture was extracted with dichloromethane (3 × 50 cm ³ ). The combined extracts were washed with saturated aqueous solution of sodium hydrogencarbonate (2 × 50 cm ³ ) and brine (2 × 50 cm ³ ) and dried over Na ₂ SO ₄ . The extract was evaporated under reduced pressure and the residue was chromatographed on silica gel using dichloromethane / methanol (99: 1, v / v) as an eluent to give nucleoside 23 as a white foam (4.03 g, 83%). δ _n (CDCl ₃ ) 8.78 (1H, brs, NH), 7.75 (1H, s, 6-H), 7.38-7.26 (10H, m, Bn), 6.49 (1H d, J 8.1, 1'-H), 5.99-5.88 (2H, m, 2'-H and 1``-H), 5.54-5.48 (2H, m, 2 '' - H <sub>a</sub> , 2 '' - H <sub>b</sub> ), 4.91-4.50 (4H, m, Bn), 4.34 (1H, s, 4'-H), 3.80 (1H, m, 5'-H <sub>a</sub> ), 3.54 (1H, m, 5'-H <sub>b</sub> ), 2.11 (3H, s, COCH <sub>3</sub> ), 1.48 (3H, s, CH <sub>3</sub> ). δ <sub>s</sub> (CDCl <sub>3</sub> ) 170.1 (C = O), 163.8 (C-4), 151.0 (C-2), 138.9, 136.9 (Bn), 136.1 (C- 6), 132.0 (C-1``), 128.7, 128.5, 128.2, 127.8, 127.7, 127.5, 127.5, 127.1 (Bn), 120 7 (C-2 ''), 111.3 (C-5), 85.4 (C-1 '), 85.2 (C-3'), 84.3 (C-4 '), 76 0 (C-2 '), 73.7 (C-5'), 69.3, 67.6 (Bn), 20.6 (COSH ₃ ), 11.7 (CH ₃ ). Received: C, 66.3; H - 6.0; N is 5.1. For C ₂₈ H ₃₀ N ₂ O ₇ calculated: C - 66.4; H - 6.0; N - 5.5%.

Example 25

1- (3,5-di-O-benzyl-3-C-vinyl-β-D-ribofuranosyl) thymine (24)

To a stirred solution of nucleoside 23 (3.90 g, 1.1 mmol) in anhydrous methanol (40 cm ³ ) was added sodium methoxide (0.83 g, 15.4 mmol). The mixture was stirred at room temperature for 42 hours and then neutralized with dilute aqueous hydrochloric acid. The resulting mixture was extracted with dichloromethane (2 × 150 cm ³ ) and the combined extracts were washed with saturated aqueous sodium hydrogen carbonate (3 × 100 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure to give nucleoside 24 as a white foam (3.48 g, 97%). δ _n (CDCl ₃ ) 8.89 (1H, br s, NH), 7.60 (1H, d, J 0.9, 6-H), 7.36-7.26 (10H, m, Bn), 6.23 (1H, d, J 7.8, 1'-H), 5.98 (1H, dd, J 11.2, 17.7, 1 '' - H), 5.66 (1H, d J 17.7, 2 '' - H _a ), 5.55 (1H, d, J 11.5, 2 '' - H _b ), 4.75-4.37 (6H, m, 2'- H, 4'-H, Bn), 3.84 (1H, dd, J 2.7, 10.8, 5'-H _a ), 3.58 (1H, d, J 11.2, 5'- H _b ), 3.23 (1H, d, J 10.6, 2'-OH), 1.50 (3H, s, CH ₃ ). δ _s (CDCl ₃ ) 163.7 (C-4), 151.3 (C-2), 138.0, 136.9 (Bn), 136.0 (C-6), 131.2 (C- 1``), 128.8, 128.6, 128.3, 127.8, 127.7, 127.3 (Bn), 120.7 (C-2 ''), 111.3 (C-5 ), 87.3 (C-1 '), 84.6 (C-3'), 81.4 (C-4 '), 78.0 (C-2'), 73.7 (C-5 ' ), 70.0, 66.4 (Bn), 11.8 (CH ₃ ). Received: C, 66.8; H, 6.2; N - 5.9. For C ₂₆ H ₂₈ H ₂ O ₆ calculated: C - 67.2; H, 6.1; N - 6.0%.

Example 26

1- (3,5-di-O-benzyl-2-O-methanesulfonyl-3-C-vinyl-β-D-ribofuranosyl) thymine (25)

Nucleoside 24 (2.57 g, 5.53 mmol) was dissolved in anhydrous pyridine (18 cm ³ ) and cooled to 0 ° C. Methanesulfonyl chloride (1.28 cm ³ , 16.6 mmol) was added dropwise and the resulting mixture was stirred at room temperature for 30 minutes. The reaction was stopped with water (5 cm ³ ) and the resulting mixture was extracted with dichloromethane (3 × 80 cm ³ ). The combined extracts were washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 120 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was evaporated under reduced pressure and the residue was chromatographed on silica gel using dichloromethane / methanol (99: 1, v / v) as an eluent to give nucleoside 25 as a yellow foam (2.53 g, 84%). δ _n (CDCl ₃ ) 8.92 (1H, brs, NH), 7.71 (1H, d, J 1.4, 6-H), 7.41-7.28 (10H, m, Bn), 6.57 (1H, d, J 7.8, 1'-H), 5.99-5.61 (4H, m, 2'-H, 1 '' - H and 2 '' - H _a , 2 '' -H _b ), 4.86-4.50 (4H, m, Bn), 4.37 (1H, dd, J 1.5, 2.4, 4'-H), 8.82 (1H dd, J 2.6, 11.0, 5'-H _a ), 3.55 (1H, dd, J 1.2, 11.0, 5'-H _b ), 3.02 (3H, s , CH ₃ ), 1.47 (3H, d, J 1.1, CH ₃ ). δ _s (CDCl ₃ ) 163.7 (C-4), 151.5 (C-2), 138.7, 136.7 (Bn), 135.7 (C-6), 130.9 (C- 1``), 128.8, 128.5, 128.4, 127.6, 127.0 (Bn), 121.8 (C-2 ''), 111.9 (C-5), 85, 1 (C-1 '), 84.5 (C-3'), 84.0 (C-4 '), 80.7 (C-2'), 73.7 (C-5 '), 69, 2, 67.7 (Bn), 38.9 (CH ₃ ), 11.8 (CH ₃ ).

Example 27

1- (3,5-di-O-benzyl-3-C-vinyl-β-D-arabinofuranosyl) thymine (26)

A solution of nucleoside 25 (2.53 g, 4.66 mmol) in a mixture of ethanol (50 cm ³ ), water (50 cm ³ ) and 1 M aqueous sodium hydroxide solution (15 cm ³ ) was stirred under reflux for 16 hours. The mixture was neutralized with dilute aqueous hydrochloric acid, the solvent was evaporated under reduced pressure, and the resulting residue was extracted with dichloromethane (3 × 120 cm ³ ). The combined extracts were washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 150 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was evaporated under reduced pressure, and the residue was chromatographed on a silica gel column using dichloromethane / methanol (99: 1) as an eluent to give compound 26 as a white foam (1.61 g, 74%). δ _n (CDCl ₃ ) 9.89 (1H, brs, NH), 7.50 (1H, d, J 1.1, 6-H), 7.41-7.26 (Bn), 6.28 ( 1H, d, J 2.8, 1'-H), 6.05 (1H, dd, J 11.1, 17.9, 1 '' - H), 5.58-5.50 (2H, m , 2 '' - H _a , 2 '' - H _b ), 4.98 (1H, d, J 9.0, 2'-OH), 4.64-4.31 (6H, m, 2'- H, 4'-H, Bn), 3.73 (2H, m, 5'-H _a , 5'-H _b ), 1.73 (1H, d, J 0.6, CH ₃ ). δ _s (CDCl ₃ ) 165.1 (C-4), 150.5 (C-2), 138.4, 138.0, 136.7 (C-6, Bn), 130.4 (C-1 ``), 128.8, 128.6, 128.5, 128.1, 128.0, 127.8 (Bn), 120.6 (C-2 ''), 108.1 (C-5) , 88.6 (C-1 '), 87.9 (C-3'), 87.2 (C-4 '), 73.7 (C-2'), 71.8 (C-5 ') 69.7, 66.3 (Bn), 12.3 (CH ₃ ). Received: C, 66.8; H, 6.2; N - 5.9. For C ₂₆ H ₂₈ N ₂ O ₆ calculated: C - 67.2; H, 6.1; N - 6.0.

Example 28

1- (3,5-di-O-benzyl-3-C-hydroxymethyl-β-D-arabinofuranosyl) thymine (27)

To a solution of nucleoside 26 (2.00 g, 4.31 mmol) in a mixture of THF (15 cm ³ ) and water (15 cm ³ ) was added sodium periodate (2.76 g, 12.9 mmol) and 2.5% solution of osmium tetraoxide in tert-butanol (w / v, 0.54 cm ³ , 43 μmol). The reaction mixture was stirred at room temperature for 18 hours, the reaction was stopped with water (50 cm ³ ) and the resulting mixture was extracted with dichloromethane (2 × 100 cm ³ ). The combined extracts were washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 75 cm ³ ), dried over Na ₂ SO ₄ and evaporated under reduced pressure. The residue was redissolved in a mixture of THF (15 cm ³ ) and water (15 cm ³ ) and sodium borohydride (488 mg, 12.9 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour, water (50 cm ³ ) was added, and the resulting mixture was extracted with dichloromethane (2 × 100 cm ³ ). The combined extracts were washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 75 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed and the residue was chromatographed on a silica gel column using dichloromethane / methanol (98: 2) as eluent to give nucleoside 27 as a white foam (732 mg, 36%). δ _n (CDCl ₃ ) 11.09 (1H, brs, NH), 7.41 (1H, d, J 1.0, 6-H), 7.38-7.26 (Bn), 6.16 ( 1H, d, J 2.6, 1'-H), 5.12 (1H, d, J 5.4, 2'-OH), 4.66-4.29 (6H, m, 2'-H 4'-H, Bn), 4.02-3.96 (2H, m, 1 '' - H _a , 1 '' - H _b ), 3.90 (1H, dd, J 7.2, 9 7, 5'-H _a ), 3.79 (1H, dd, J 5.6, 9.7, 5'-H _b ), 2.49 (1H, t, J 6.4, 1 '' -OH) 1.68 (3H, d, J 0.6, CH ₃ ). δ _s (CDL ₃ ) 166.1 (C-4), 150.6 (C-2), 139.0, 137.9, 137.0 (C-6, Bn), 128.7, 128.6 , 128.4, 128.3, 128.0 (Bn), 107.5 (C-5), 88.2 (C-1 '), 88.1 (C-3'), 84.2 (C -4 '), 73.7 (C-5'), 72.1 (C-2 '), 69.3, 65.4 (Bn), 58.6 (C-1''), 12.3 (CH ₃ ).

Example 29

(1R, 2R, 4R, 5S) -1-benzyloxy-2-benzyloxymethyl-4- (thymin-1-yl) -3,6-dioxabicyclo [3.2.0] heptane (28)

A solution of compound 27 (2.26 g, 4.83 mmol) in anhydrous pyridine (20 cm ³ ) was stirred at -40 ° C and a solution of methanesulfonyl chloride (0.482 cm ³ , 4.83 mmol) in anhydrous pyridine (10 cm ³ ) was added. . The reaction mixture was stirred at room temperature for 17 hours, water (50 cm ³ ) was added and the resulting mixture was extracted with dichloromethane (2 × 100 cm ³ ). The combined extracts were washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 100 cm ³ ), dried over Na ₂ SO ₄ and evaporated under reduced pressure. The residue was chromatographed on a silica gel column using dichloromethane / methanol (99: 1 v / v) as an eluent to give an intermediate which, after evaporation of the solvents, was dissolved in anhydrous DMF (15 cm ³ ). The resulting solution was added dropwise to a suspension of 60% sodium hydride (461 mg, 11.5 mmol) in anhydrous DMF (15 cm ³ ) at 0 ° C. The reaction mixture was stirred at room temperature for 30 minutes, then the reaction was stopped with water (60 cm ³ ). After neutralization with dilute aqueous hydrochloric acid, the mixture was dissolved in dichloromethane (150 cm ³ ), washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 100 cm ³ ) and dried over Na ₂ SO ₄ . The solvents were evaporated and the residue was chromatographed on a silica gel column using dichloromethane / methanol (99: 1) as eluent to give nucleoside 28 as a white foam (2.00 g, 93%). δ _n (CDCl ₃ ) 9.13 (1H, brs, NH), 7.55 (1H, d, J 1.4, 6-H), 7.40-7.26 (Bn), 5.99 (1H, d, J 2.5, 1'-H), 5.30 (1H, d, J 2.7, 2'-H), 4.88-4.57 (6H, m, 1 '' -H _a , 1 '' - H _b , Bn), 4.22-4.19 (1H, m, 4'-H), 3.92 (1H, dd, J 6.2, 10.8, 5 '-H _a ), 3.82 (1H, dd, J 3.7, 10.8, 5'-H _b ), 1.91 (3H, d, J 1.3, CH ₃ ). δ _c (CDCl ₃ ) 163.8 (C-4), 150.3 (C-2), 137.6 (C-6), 137.5, 137.0 (Bn), 128.7, 128, 6, 128.2, 128.0, 127.8, 127.3 (Bn), 109.8 (C-5), 85.7 (C-3 '), 84.1 (C-1'), 83.5 (C-4 '), 79.7 (C-1''), 73.9 (C-2'), 73.6 (C-5 '), 68.6, 10 67.8 ( Bn), 12.4 (CH ₃ ). FAB m / z 451 [M + H] ⁴ , 473 [M + Na] ⁺ . Received: C, 66.3; H - 5.9; N, 6.1. For C ₂₅ H ₂₆ H ₂ O _{6 it is} calculated: C - 66.7; H - 5.8; N - 6.2%.

Example 30

[1R, 2R, 4R, 5S) -1-hydroxy-2-hydroxymethyl-4- (thymin-1-yl) -3,6-dioxabicyclo [3.2.0] heptane (29)

To a stirred solution of nucleoside 28 (180 mg, 0.40 mmol) in ethanol (3 cm ³ ) was added 10% palladium hydroxide on carbon (90 mg). The mixture was degassed several times with argon and placed in a hydrogen atmosphere. The reaction mixture was stirred at room temperature for 6 hours, then filtered through celite. The filtrate was evaporated under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (96: 4) as an eluent to give nucleoside 29 as a white solid (92 mg, 86%). δ _n (CD ₃ OD) 7.79 (1H, d, J 1.2, 6-H), 5.91 (1H, d, J 2.5, 1'-H), 4.96 (1H, d, J 2.5, 2'-H), 4.92 (1H, d, J 7.4, 1 '' - H _a ), 4.58 (1H, dd, J 0.9, 7.4 , 1 '' - H _b ), 3.98 (1H, dd, J 7.3, 12.8, 5'-H _a ), 3.87-3.82 (2H, m, 4'-H, 5'-H _b ), 3.34 (2H, s, 3'-OH, 5'-OH), 1.87 (3H, d, J 1.3, CH ₃ ). δ _s (CD ₃ OD) 166.5 (C-4), 152.1 (C-2), 140.1 (C-6), 110.1 (C-5), 91.2 (C-2 '), 85.1 (C-1'), 84.0 (C-4 '), 79.6 (C-3'), 78.6 (C-1 ''), 61.1 (C- 5 '), 12.3 (CH ₃ ).

Example 31

(1R, 2R, 4R, 5S) -1- (2-cyanoethoxy (diisopropylamino) phosphinoxy) -2- (4,4'-dimethoxytrityloxymethyl) -4- (thymin-1-yl) -3,6-dioxabicyclo [3.2 .0] heptane (30)

To a solution of diol 29 (250 mg, 0.925 mmol) in anhydrous pyridine (4 cm ³ ) was added 4,4'-dimethoxytrityl chloride (376 mg, 1.11 mmol) and the resulting mixture was stirred at room temperature for 18 hours. The reaction was stopped with methanol (1.5 cm ³ ) and the mixture was evaporated under reduced pressure. A solution of the residue in dichloromethane (30 cm ³ ) was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 20 cm ³ ), dried over Na ₂ SO ₄ and evaporated. The residue was subjected to silica gel column chromatography using dichloromethane / methanol (98: 2, v / v) as an eluent to obtain an intermediate compound, which was dissolved in anhydrous dichloromethane (7.0 cm ³ ). First, N, N-diisopropylethylamine (0.64 cm ³ , 3.70 mmol) was added and then 2-cyanoethyl-N, N-diisopropylphosphoramidochloridite (0.41 cm ³ , 1.85 mmol) was added and the resulting mixture was stirred at room temperature in within 25 hours. The reaction was stopped with methanol (3 cm ³ ) and the resulting mixture was dissolved in ethyl acetate (70 cm ³ ), washed with saturated aqueous sodium hydrogen carbonate (3 × 50 cm ³ ) and brine (3 × 50 cm ³ ), dried over Na ₂ SO ₄ and evaporated under reduced pressure. The residue was chromatographed on a silica gel column using petroleum ether / dichloromethane / ethyl acetate / triethylamine (100: 45: 45: 10 - v / v) as an eluent. The resulting residue was dissolved in toluene (2 cm ³ ) and precipitated with stirring from petroleum ether at -50 ° C. After evaporation of the solvents, the residue was evaporated with anhydrous acetonitrile (4 × 5 cm ³ ) to give compound 30 as a white foam (436 mg, 61%). ³¹ P-NMR (CDCl ₃ ) 146.6.

Example 32

3,5-di-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose (31)

To a solution of 3-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose (RDYoussefyeh, JPHerheyden & JGMoffatt, 1979, J. Org. Chem., 44, 1301) (20, 1 g, 0.064 mol) in anhydrous DMF (10 cm ³ ) at -5 ° C was added a suspension of sodium hydride (60% in mineral oil: w / v) in four portions over 1.5 hours, the total amount of 2.85 g, 0.075 mol. Benzyl bromide (8.9 cm ³ , 0.075 mol) was added dropwise and stirring was continued at room temperature for 3 hours, after which a mixture of ice and water (50 cm ³ ) was added. The resulting mixture was extracted with EtOAc (4 × 100 cm ³ ) and the combined organic phase was dried over Na ₂ SO ₄ . After evaporation, the residue was chromatographed on a silica gel column eluting with 5% EtOAc in petroleum ether (v / v) to give compound 31 (18.5 g, 71%). δ _s (CDCl ₃ ) 138.0, 137.4, 128.5, 128.3, 128.0, 128.0, 127.8, 127.6 (Bn), 113.5 (C (CH ₃ ) ₂ ), 104.4 (C-1), 86.5 (C-4), 78.8, 78.6 (Bn), 73.6, 72.6, 71.6 (C-2, C- 3, C-5), 63.2 (C-1 '), 26.7, 26.1 (CH ₃ ).

Example 33

4-C- (acetoxymethyl) -3,5-di-O-benzyl-1,2-O-isopropylidene-α-D-ribofuranose (32)

To a solution of furanose 31 (913 mg, 2.28 mmol) in anhydrous pyridine (4.5 cm ³ ) was added dropwise acetic anhydride (1.08 cm ³ , 1.4 mmol) and the resulting reaction mixture was stirred at room temperature for 3 hours. The reaction was stopped by adding a mixture of ice and cold water (50 cm ³ ) and extraction was carried out with dichloromethane (3 × 50 cm ³ ). The combined organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (2 × 50 cm ³ ), dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was chromatographed on a silica gel column using dichloromethane as eluent to give compound 32 as a clear oily substance (911 mg, 90%). δ _n (CDCl ₃ ) 7.34-7.25 (10H, m, Bn), 5.77 (1H, d, J 3.6, 1-H), 4.78-4.27 (8H, m , Bn, H-5 _a, H-5 _b, H-3, H-2), 3.58 (1H, d, J 10,3, H-1 _'a), 3,48 (1H, d, J 10.5, H-1 ' _b ), 2.04 (3H, s, COCH ₃ ), 1.64 (3H, s, CH ₃ ), 1.34 (3H, s, CH ₃ ). δ _s (CDL ₃ ) 171.1 (C = O), 138.2, 137.9, 128.6, 128.1, 128.0, 128.0, 127.8 (Bn), 114.0 ( C (CH ₃ ) ₂ ), 104.5 (C-1), 85.4 (C-4), 79.3, 78.6 (C-2, C-3), 73.7, 72.7 71.2 (Bn, C-5), 64.9 (C-1 '), 26.7, 26.3 (C (CH ₃ ) ₂ ), 21.0 (COCH ₃ ). Received: C, 67.0; H - 6.5. For C ₂₅ H ₃₀ O ₇ · 0.25 H ₂ O it was calculated: C - 67.2; H - 6.9%.

Example 34

4-C- (acetoxymethyl) -1,2-di-O-acetyl-3,5-di-O-benzyl-D-ribofuranose (33)

A solution of furanose 32 (830 mg, 1.88 mmol) in 80% acetic acid (10 cm ³ ) was stirred at 90 ° C for 4 hours. The solvent was removed under reduced pressure and the residue was evaporated together with ethanol (3 × 5 cm ³ ), toluene (3 × 5 cm ³ ) and anhydrous pyridine (3 × 5 cm ³ ) and redissolved in anhydrous pyridine (3.7 cm ³ ) . Acetic anhydride (2.85 cm ³ ) was added and the resulting solution was stirred for 72 hours at room temperature. The solution was poured into a mixture of ice and water (20 cm ³ ) and the resulting mixture was extracted with dichloromethane (2 × 20 cm ³ ). The combined organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (2 × 20 cm ³ ), dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was chromatographed on a silica gel column using dichloromethane as eluent to give compound 33 (β: α ≈ 1: 3) as a clear oily substance (789 mg, 86%). δ _s (CDCl ₃ ) 171.0, 170.3, 170.0, 169.3 (C = O), 138.1, 137.6, 136.3, 128.9, 128.6, 128.2 , 128.0, 128.0, 127.9, 127.7, 124.0 (Bn), 97.8, 97.8 (C-1), 87.0, 85.0, 78.9, 74 5, 74.4, 73.8, 73.6, 72.0, 71.8, 71.0, 70.9, 64.6, 64.4 (C-2, C-3, C-4 , Bn, C-5, C-1 '), 21.0, 20.8, 20.6 (COCH ₃ ). Received: C, 64.2; H - 6.3. For C ₂₆ H ₃₀ O ₉ calculated: C - 64.2; H - 6.2%.

Example 35

1- (4-C- (acetoxymethyl) -2-O-acetyl-3,5-di-O-benzyl-β-D-ribofuranosyl) thymine (34)

To a stirred solution of an anomeric mixture of 33 (736 mg, 1.51 mmol) and thymine (381 mg, 3.03 mmol) in anhydrous acetonitrile (14.5 cm ³ ) was added N, O-bis- (trimethylsilyl) acetamide (2, 61 cm ³ , 10.6 mmol). The reaction mixture was stirred under reflux for 1 hour, then cooled to 0 ° C. Trimethylsilyl triflate (0.47 cm ³ , 2.56 mmol) was added dropwise with stirring, and the resulting solution was stirred at 65 ° C. for 2 hours . The reaction was stopped with a cold saturated aqueous solution of sodium hydrogencarbonate (15 cm ³ ) and extraction was carried out with dichloromethane (3 × 10 cm ³ ). The combined organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (2 × 20 cm ³ ) and brine (2 × 20 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (98: 2, v / v) as an eluent to give nucleoside 34 as a white solid (639 mg, 76%). δ _n (CDCl ₃ ) 8.98 (1H, brs, NH), 7.39-7.26 (11H, m, Bn, 6-H), 6.22 (1H, d, J 5.3, 1 '-H), 5.42 (1H, t, J 5.4, 2'-H), 4.63-4.43 (5H, m, 3'-H, Bn), 4.41 (1H, d, J 12.2, 5'-H _a ), 4.17 (1H, d, J 12.1, 5'-H _b ), 3.76 (1H, d, J 10.2, 1 '' -H _a ), 3.51 (1H, d, J 10.4, 1 '' - H _b ), 2.09 (3H, s, COSH ₃ ), 2.03 (3H, s, COSH ₃ ), 1.53 (3H, d, J 0.9, CH ₃ ). δ _s (CDCl ₃ ) 170.8, 170.4 (C = O), 163.9 (C-4), 150.6 (C-2), 137.4 (C-6) 137.4, 136 , 1, 128.9, 128.8, 128.4, 128.2, 127.9 (Bn), 111.7 (C-5), 87.2, 87.2, 86.1 (C-1 ', C-3', C-4 '), 77.6 (C-2'), 74.8, 73.9, 71.1, 63.8 (Bn, C-1 '', C-5 '), 20.9, 20.8 (COCH ₃ ), 12.0 (CH ₃ ). FAB-MS m / z 553 [M + H] ⁺ . Received: C - 62.7; H - 5.9; N - 4.7. For C ₂₉ H ₃₂ N ₂ O _{9 it was} calculated: C - 63.0; H - 5.8; N - 5.1%.

Example 36

1- (3,5-di-O-benzyl-4-C- (hydroxymethyl) -β-D-ribofuranosyl) thymine (35)

To a stirred solution of nucleoside 34 (553 mg, 1.05 mmol) in methanol (5.5 cm ³ ) was added sodium methoxide (287 mg, 5.25 mmol). The reaction mixture was stirred at room temperature for 10 minutes, then neutralized with dilute hydrochloric acid. The solvent was partially evaporated and extraction was carried out with dichloromethane (3 × 20 cm ³ ). The combined organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 20 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure to give compound 35 as a white solid (476 mg, 97%). δ _H (CDCl ₃ ) 7.47 (1H, d, J 1.0 6-H), 7.36-7.22 (10H, m, Bn), 6.07 (1H, d, J 3.8 , 1'-H), 4.87 (1H, d, J 11.7, Bn), 4.55 (1H, d, J 11.7, Bn), 4.50-4.32 (4H, m , Bn, 2'-H, 3'-H), 3.84-53.53 (4H, m, 5'-H _a , 5'-H _b , 1 '' - H _a , 1 '' - H _b ) 1.50 (3H, d, J 1.1, CH ₃ ). δ _C (CDCl ₃ ) 164.3 (C-4), 151.3 (C-2), 137.6 (C-6) 136.4, 136.3, 128.8, 128.6, 128, 4, 128.3, 127.9 (Bn), 111.1 (C-5), 91.1, 91.0, 88.1 (C-1 ', C-3', C-4 '), 77.4 (C-2 '), 74.8, 73.8, 71.4, 63.2 (Bn, C-5', C-1 ''), 12.0 (CH ₃ ). FAB-MS m / z 491 [M + Na] ⁺ . Received: C, 63.4; H - 6.0; N is 5.5. For C ₂₅ H ₂₈ N ₂ O ₇ · 0.25 H ₂ O, it was calculated: C - 63.5; H, 6.1; N - 5.9%.

Example 37

Intermediate 35A

A solution of nucleoside 35 (225 mg, 0.48 mmol) in anhydrous pyridine (1.3 cm ³ ) was stirred at 0 ° C and p-toluenesulfonyl chloride (118 mg, 0.62 mmol) was added in small portions. The solution was stirred at room temperature for 16 hours and p-toluenesulfonyl chloride (36 mg, 0.19 mmol) was added again. After stirring for another 4 hours and adding a mixture of ice and water (15 cm ³ ), extraction was carried out with dichloromethane (2 × 15 cm ³ ). The combined organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 15 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (99: 1, v / v) as an eluent to give intermediate 35A (140 mg), which was used for further purification, which was carried out in the next step .

Example 38

(1S, 3R, 4R, 7S) -7-benzyloxy-1-benzyloxymethyl-3- (thymin-1-yl) -2,5-dioxabicyclo [2.2.1] heptane (36)

Intermediate 35A (1.59 mg) was dissolved in anhydrous DMF (0.8 cm ³ ). The solution was added dropwise to a stirred suspension of 60% sodium hydride in mineral oil (w / v, 32 mg, 0.80 mmol) in anhydrous DMF (0.8 cm ³ ) at 0 ° C. The resulting mixture was stirred at room temperature for 72 hours and then concentrated under reduced pressure. The residue was dissolved in dichloromethane (10 cm ³ ), washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 15 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (99: 1, v / v) as an eluent to give bicyclic nucleoside 36 as a white solid (65.7 mg, 57%) . δ _H (CDCl ₃ ) 9.24 (1H, br s, NH), 7.49 (1H, s, 6-H), 7.37-7.26 (10H, m, Bn), 5.65 (1H s, 1'-H), 4.70-4.71 (5H, m, Bn, 2'-H), 4.02-3.79 (5H, m, 3'-H, 5'-H _a , 5'-H _b , 1 '' - H _a , 1 '' - H _b ), 1.63 (3H, s, CH ₃ ). δ _C (CDCl ₃ ) 164.3 (C-4), 150.1 (C-2), 137.7, 137.1 (Bn), 135.0 (C-6), 128.8, 128, 7, 128.4, 128.0, 127.9 (Bn), 110.4 (C-5), 87.5, 87.3 (C-1 ', C-3'), 76.7, 75 8, 73.9, 72.3, 72.1 (Bn, C-5 ', C-2', C-4 '), 64.5 (C-1''), 12.3 (CH ₃ ) FAB-MS m / z 451 [M + H] ⁺ .

Example 39

(1S, 3R, 4R, 7S) -7-hydroxy-1-hydroxymethyl-3- (thymin-1-yl) -2,5-dioxabicyclo [2.2.1] heptane (37)

A solution of nucleoside 36 (97 mg, 0.215 mmol) in ethanol (1.5 cm ³ ) was stirred at room temperature and 20% palladium hydroxide on carbon (50 mg) was added. The mixture was degassed several times using argon and placed in a hydrogen atmosphere in a cylinder. After stirring for 4 hours, the mixture was chromatographed on a silica gel column using dichloromethane / methanol (97: 3, v / v) as an eluent to give bicyclic nucleoside 37 as a white solid (57 mg, 98%). δ _H ((CD ₃ ) ₂ SO) 11.33 (1H, br s, NH), 7.62 (1H, d, J 1.1 Hz, 6-H), 5.65 (1H, d, J 4 4 Hz, 3'-OH), 5.41 (1H, s, 1'-H), 5.19 (1H, t, J 5.6 Hz, 5'-OH), 4.11 (1H, s, 2'-H), 3.91 (1H, d, J 4.2 Hz, 3'-H) -, 3.82 (1H, d, J 7.7 Hz, 1 '' - H ,, _a ), 3.73 (1H, s, H'-5 _a ), 3.76 (1H, g, 5'-H _b ), 3.63 (1H, d, J, 7.7 Hz, 1'- H _b ), 1.78 (3H, d, J 0.7 Hz, CH ₃ ). δ _C (CDCl ₃ ) 166.7 (C-4), 152.1 (C-2), 137.0 (C-6), 110.9 (C-5), 90.5, 88.4 ( C-1 ', C-4'), 80.9, 72.5, 70.4 (C-2 ', C-3', C-5 '), 57.7 (C-1''), 12.6 (CH ₃ ). EI-MS m / z 270 [M] ⁺ .

Example 40

(1R, 3R, 4R, 7S) -1- (4,4’-dimethoxytrityloxymethyl) -7-hydroxy-3-thymin-1-yl) -2,5-dioxabicyclo [2.2.1] heptane (38)

To a solution of nucleoside 37 (1.2 g, 4.44 mmol) in anhydrous pyridine (5 cm ³ ) was added 4,4'-dimethoxytrityl chloride (2.37 g, 7.0 mmol) at 0 ° C. The solution was stirred at room temperature for 2 hours, after which the reaction was stopped with a mixture of ice and water (10 cm ³ ) and extraction was carried out with dichloromethane. (3 × 15 cm ³ ). The combined organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 10 cm ³ ) and brine (2 × 10 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (98: 2, v / v) as an eluent to give nucleoside 38 as a white solid (2.35 g, 93%). δ _H (CDCl ₃ ) 9.89 (1H, brs, NH), 7.64 (1H, s, 6-H), 7.47-7.13 (9H, m, DMT), 6.96-6 80 (4H, m, DMT), 5.56 (1H, s, 1'-H), 4.53 (1H, br, 2'-H), 4.31 (1H, m, 3'-H ), 4.04-3.75 (9H, m, 1 '' - H _a , 1 '' - H _b , 3'-OH, OCH ₃ ), 3.50 (2H, br, 5'-H _a 5'-H _b ), 1.65 (3H, s, CH ₃ ). δ _C (CDCl ₃ ) 164.47 (C-4), 158.66 (DMT), 150.13 (C-2), 144.56, 135.46, 135.35, 134.78, 130.10 , 129.14, 128.03, 127.79, 127.05 (C-6, DMT), 113.32, 113.14 (DMT), 110.36 (C-5), 89.17, 88, 16, 87.05 (C-1 ', C-4', DMT), 79.36, 71.81, 70.25, 58.38 (C-2 ', C-3', C-5 ', C-1 ''), 55.22 (OCH ₃ ), 12.57 (CH ₃ ). FAB-MS m / z 595 [M + Na] ⁺ , 573 [M + H] ⁺ .

Example 41

(1R, 3R, 4R, 7S) -7- (2-cyanoethoxy (diisopropylamino) phosphinoxy) -1- (4,4'-dimethoxytrityloxymethyl) -3- (thymin-1-yl) -2,5-dioxabicyclo [2.2 .1] heptane (39)

To a solution of nucleoside 38 (2.21 g, 3.86 mmol) in anhydrous dichloromethane (6 cm ³ ), N, N-diisopropylethylamine (4 cm ³ ) and 2-cyanoethyl-N, N-diisopropylphosphoramidochloridite (1 cm) were added at room temperature ³ , 4.48 mmol) and stirring was continued for 1 hour. MeOH (2 cm ³ ) was added and the resulting mixture was diluted with ethyl acetate (10 cm ³ ) and washed successively with a saturated aqueous solution of sodium hydrogencarbonate (3 × 5 cm ³ ) and brine (2 × 5 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was evaporated under reduced pressure and the residue was chromatographed on alkaline alumina columns using dichloromethane / methanol (99: 1, v / v) as an eluent to give compound 39 as a white foam. This residue was dissolved in dichloromethane (2 cm ³ ) and the resulting product was precipitated from petroleum ether (100 cm ³ , cooled to -30 ° C) with vigorous stirring. The precipitate was collected by filtration and dried to give nucleoside 39 as a white solid (2.1 g, 70%). δ _P (CDCl ₃ ) 149.06, 148.74. FAB-MS m / z 795 [M + Na] ⁺ , 773 [M + H] ^+.

Example 42

1- (2-O-acetyl-4-C-acetoxymethyl-3,5-di-O-benzyl-β-D-ribofuranosyl) uracil (40)

To a stirred solution of an anomeric mixture of 33 (3.0 g, 6.17 mmol) and uracil (1.04 g, 9.26 mmol) in anhydrous acetonitrile (65 cm ³ ) was added N, O-bis (trimethylsilyl) acetamide (9 , 16 cm ³ , 37.0 mmol). The reaction mixture was stirred for 1 hour at room temperature and cooled to 0 ° C. Trimethylsilyl triflate (1.8 cm ³ , 10.0 mmol) was added dropwise and the resulting solution was stirred at 60 ° C. for 2 hours. The reaction was stopped by the addition of a saturated aqueous solution of sodium hydrogencarbonate (10 cm ³ ) at 0 ° C and extraction was carried out with dichloromethane (3 × 20 cm ³ ). The combined organic phase was washed with brine (2 × 20 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (99: 1, v / v) as an eluent to give nucleoside 40 as a white solid (2.5 g, 7S%). δ _H (CDCl ₃ ) 9.57 (1H, br s, NH), 7.63 (1H, d, J 8.2, 6-H), 7.40-7.24 (10H, m, Bn), 6.18 (1H, d, J 4.5, 1'-H), 5.39-5.32 (2H, m, 2'-H, 5-H), 4.61 (1H, d, J 11.6, Bn), 4.49-4.40 (5H, m, 3'-H, Bn, 1 '' - H _a ), 4.37 (1H, d, J 12.3, 1 '' -H _b ), 3.76 (1H, d, J 10.1, 5'-H _a ), 3.49 (1H, d, J 10.1, 5'-H _b ), 2.09 (3H , s, COCH ₃ ), 2.04 (3H, s, COCH ₃ ). δ _C (CDCl ₃ ) 170.47, 169.94 (C = 0), 163.32 (C-4), 150.30 (C-2), 140.24 (C-6), 137.15, 136.95, 128.65, 128.52, 128.32, 128.19, 128.02, 127.77 (Bn), 102.57 (C-5), 87.41, 86.14 (C- 1 ', C-4'), 77.09, 74.84, 74.51, 73.75, 70.60, 63.73 (C-2 ', C-3', C-5 ', C- 1 '', Bn), 20.79, 20.68 (COSH ₃ ). FAB-MS m / z 539 [M] ⁺ .

Example 43

1- (3,5-di-O-benzyl-4-C-hydroxymethyl-β-D-ribofuranosyl) uracil (41)

To a stirred solution of nucleoside 40 (2.0 g, 3.7 mmol) in methanol (25 cm ³ ) was added sodium methoxide (0.864 g, 95%, 16.0 mmol). The reaction mixture was stirred at room temperature for 10 minutes and neutralized with 20% aqueous hydrochloric acid. The solvent was partially evaporated and the residue was extracted with ethyl acetate (3 × 50 cm ³ ). The combined organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate ( ³ × 20 cm ³ ) and dried over Na ₀ SO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (98.5: 1.5, v / v) as an eluent to give nucleoside 41 as a white solid (1.58 g, 95 %). δ _H (CDCl ₃ ) 9.95 (1H, br s, NH), 7.69 (d, J 8.1, 6-H), 7.35-7.17 (10H, m, Bn), 6, 02 (1H, d, J 2,3, 1'-H), 5.26 (1H, d, J 8.1, 5-H), 4.80 (1H, d, J 11.7, Bn) 4.47 (1H, d, J 11.7, Bn), 4.45-4.24 (4H, m, Bn, 2'-H, 3'-H), 3.81 (1H, d, J 11.9, 1 '' - H _a ), 3.69 (2H, br, 2'-OH, 1 '' - OH), 3.67 (2H, m, 5'-H _a , 1 '' -H _b ), 3.48 (1H, d, J 10.3, 5'-H _b ). δ _C (CDCl ₃ ) 163.78 (C-4), 150.94 (C-2), 140.61 (C-6), 137.33, 137.22, 128.59, 128.18, 128 01 (Bn), 102.16 (C-5), 91.46, 88.36 (C-1 ', C-4'), 76.73, 74.66, 73.71, 73.29, 70.81, 62.81 (C-2 ', C-3', C-5 ', C-1'', Bn). FAB-MS m / z 455 [M + H] ⁺ .

Example 44

The intermediate connection 42

A solution of nucleoside 41 (1.38 g, 3.0 mmol) in anhydrous pyridine (2 cm ³ ) and anhydrous dichloromethane (6 cm ³ ) was stirred at -10 ° C and p-toluenesulfonyl chloride (0.648 g, 3.4) was added in small portions. mmol) for 1 hour. The resulting solution was stirred at -10 ° C for 3 hours. The reaction was stopped by adding a mixture of ice and water (10 cm ³ ) and the mixture was extracted with dichloromethane (3 × 50 cm ³ ). The combined organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 20 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (99: 1, v / v) as an eluent to give intermediate 42 (0.9 g), which was used without further purification in the next step .

Example 45

(1S, 3R, 4R, 7S) -7-benzyloxy-1-benzyloxymethyl-3- (uracil-1-yl) -2,5-dioxabicyclo [2.2.1] heptane (43)

Compound 42 (0.7 g) was dissolved in anhydrous DMF (3 cm ³ ), a 60% suspension of sodium hydride (w / v, 0.096 g, 24 mmol) was added in four portions over 10 minutes at 0 ° C and the reaction mixture stirred at room temperature for 12 hours. The reaction was stopped with methanol (10 cm ³ ) and the solvents were removed under reduced pressure. The residue was dissolved in dichloromethane (20 cm ³ ), washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 6 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / ethanol (99: 1, v / v) as an eluent to give nucleoside 43 (0.30 g, 60%). δ _H (CDCl ₃ ) 9.21 (1H, br s, NH), 7.70 (1H, d, J 8.2, 6-H), 7.37-7.24 (10H, m, Bn), 5.65 (1H, s, 1'-H), 5.52 (1H, d, J 8.2, 5-H), 4.68-4.45 (5H, m, 2'-H, Bn ), 4.02-3.55 (5H, m, 3'-H, 5'-H _a , 1 '' - H _a , 5'-H _b , 1 '' - H _b ). δ _C (CDCl ₃ ) 163.33 (C-4), 149.73 (C-2), 139.18 (C-6), 137.46, 136.81, 128.58, 128.54, 128 21, 128.10, 127.79, 127.53 (Bn), 101.66 (C-5), 87.49, 87.33 (C-1 ', C-4'), 76.53, 75.71, 73.77, 72.33, 72.00, 64.35 (C-2 ', C-3', C-5 ', C-1'', Bn). FAB-MS m / z 459 [M + Ma] ⁺ .

Example 46

(1S, 3R, 4R, 7S) -7-hydroxy-1-hydroxymethyl-3- (uracil-1-yl) -2,5-dioxabicyclo [2.2.1] heptane (44)

To a solution of compound 43 (0.35 g, 0.8 mmol) in absolute ethanol (2 cm ³ ) was added 20% palladium hydroxide on carbon (0.37 g) and the resulting mixture was degassed several times with hydrogen and stirred in a hydrogen atmosphere for 4 hours. The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (9: 1, v / v) as an eluent to give nucleoside 44 as a white solid (0.16 g, 78%). δ _H (CD ₃ OD) 7.88 (1H, d, J 8.1, 6-H), 5.69 (1H, d, J 8.1, 5-H), 5.55 (1H, s , 1'-H), 4.28 (1H, s, 2'-H), 4.04 (1H, s, 3'-H), 3.96 (1H, d, J 7.9, 1 ''-H _a ), 3.91 (2H, s, 5'-H), 3.76 (1H, d, J 7.9, 1''- H _b ). δ _C (CD ₃ OD) 172.95 (C-4), 151.82 (C-2), 141.17 (C-6), 101.97 (C-5), 90.52, 88.50 (C-1 ', C-4'), 80.88, 72.51, 70.50, 57.77 (C-2 ', C-3', C-5 ', C-1''). FAB-MS m / z 257 [M + H] ⁺ .

Example 47

[1R, 3R, 4R, 7S) -1- (4,4’-dimethoxytrityloxymethyl) -7-hydroxy-3- (uracil-1-yl) -2,5-dioxabicyclo [2.2.1] heptane (45)

To a solution of compound 44 (0.08 g, 0.31 mmol) in anhydrous pyridine (0.5 cm ³ ) was added 4,4'-dimethoxytrityl chloride (0.203 g, 0.6 mmol) at 0 ° C and the resulting mixture was stirred at room temperature for 2 hours. The reaction was stopped with a mixture of water and ice (10 cm ³ ) and extraction was carried out with dichloromethane (3 × 4 cm ³ ). The combined organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 3 cm ³ ) and brine (2 × 3 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (98: 2, v / v) as an eluent to give nucleoside 45 as a white solid (0.12 g, 69%). δ _H (CDCl ₃ ) 9.25 (1H, br s, NH), 7.93 (1H, d, J 7.2, 6-H), 7.50-7.15 (9H, m, DMT), 6.88-6.78 (4H, m, DMT), 5.63 (1H, s, 1'-H), 5.59 (1H, d, J 8.0, 5-H), 4.48 (1H, s, 2'-H), 4.26 (1H, s, 3'-H), 3.88 (1H, d, J 8.1, 1 '' - H _a ), 3.85- 3.55 (7H, m, 1 '' - H _b , OCH ₃ ), 3.58-3.40 (2H, m, 5'-H _a , 5'-H _b ). δ _C (CDCl ₃ ) 164.10 (C-4), 158.60 (DMT), 150.45 (C-2), 147.53 (DMT), 144.51 (C-6), 139.72 , 135.49, 135.37, 130.20, 129.28, 128.09, 127.85, 127.07 (DMT), 113.39, 113.17 (DMT), 101.79 (C-5 ), 88.20, 87.10, 86.87 (C-1 ', C-4', DMT), 79.25, 71.79, 69.70, 58.13 (C-2 ', C- 3 ', C-5', C-1 ''), 55.33 (OCH ₃ ). FAB-MS m / z 559 [M + H] ⁺ .

Example 48

(1R, 3R, 4R, 7S) -4- (2-cyanoethoxy (diisopropylamino) phosphinoxy) -1- (4,4'-dimethoxytrityloxymethyl) -3- (uracil-1-yl) -2,5-dioxabicyclo [2.2 .1] heptane (46)

To a solution of compound 45 (0.07 g, 0.125 mmol) in anhydrous dichloromethane (2 cm ³ ), N, N-diisopropylethylamine (0.1 cm ³ ) and 2-cyanoethyl-N, N-diisopropylphosphoramidochloridite (0, 07 cm ³ , 0.32 mmol). After stirring for 1 hour, the reaction was stopped by the addition of MeOH (2 cm ³ ) and the resulting mixture was diluted with ethyl acetate (5 cm ³ ) and washed successively with a saturated aqueous solution of sodium hydrogencarbonate (3 × 2 cm ³ ) and brine (3 × 2 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was evaporated under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (99: 1, v / v) as an eluent to give a white foam. This foam was dissolved in dichloromethane (2 cm ³ ) and the resulting product was precipitated from petroleum ether (10 cm ³ , cooled to -30 ° C) with vigorous stirring. The precipitate was collected by filtration and dried to give compound 46 as a white solid (0.055 g, 58%). δ _P (CDCl ₃ ) 149.18, 149.02.

Example 49

9- (2-O-acetyl-4-C-acetoxymethyl-3,5-di-O-benzyl-β-D-ribofuranosyl) -2-N-isobutyryl guanine (47)

To a stirred suspension of an anomeric mixture of 33 (1.28 g, 5.6 mmol) and 2-N-isobutyryl guanine (1.8 g, 3.7 mmol) in anhydrous dichloroethane (600 cm ³ ) was added N, N-bis (trimethylsilyl ) acetamide (4 cm ³ , 16.2 mmol). The reaction mixture was stirred under reflux for 1 hour. Trimethylsilyl triflate (1.5 ml, 8.28 mmol) was added dropwise, and the resulting solution was stirred under reflux for 2 hours while stirring. The reaction mixture was allowed to cool to room temperature over 1.5 hours. After dilution with dichloromethane to 250 cm ^{3, the} mixture was washed with a saturated aqueous solution of sodium hydrogencarbonate (200 cm ³ ) and water (250 cm ³ ). The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using 1.25% (200 cm ³ ) and 1.5% (750 cm ³ ) methanol in dichloromethane (v / v) as eluents to give 2.10 g (87%) of a white solid, which, based on ¹ H-NMR, consists of three isomers (their ratio is 12.5: 2.5: 1). The main product formed under the described conditions is obviously Compound 47 (P. Garner, S. Ramakanth, 1988, J. Org. Chem., 53, 1294; H. Vorbruggen, K. Krolikiewicz, B. Bennua, 1981, Chem Ber. 114, 1234). Individual isomers were not isolated, and the resulting mixture was used in the following steps. For compound 47: δ _H (CDCl ₃ ) 12.25 (ss, NHCO), 9.25 (ss, NH), 7.91 (s, 8-H), 7.39-7.26 (m, Bn ), 6.07 (d, J 4.6, 1'-H), 5.80 (dd, J 5.8, 4.7, 2'-H), 4.72 (d, J 5.9 , 3'-H), 4.59-4.43 (m, Bn, 1 '' - H _a ), 4.16 (d, J 12.1, 1 '' - H _b ), 3.70 ( d, J 10.1, 5'-H _a , 3.58 (d, J 10.1, 5'-H _b ), 2.65 (m, CHCO), 2.05 (s, COSH ₃ ), 2.01 (s, COSH ₃ ), 1.22 (d, J 6.7, CH ₃ CH), 1.20 (d, J 7.0, CH ₃ CH). Δ _C (CDCl ₃ ) 178, 3 (SOSN), 170.6, 179.8 (SOSN ₃ ), 155.8, 148.2, 147.6 (guanine), 137.6, 137.2 (guanine, Bn), 128.5, 128 4, 128.2, 128.1, 128.0, 127.8, 127.7 (Bn), 121.2 (guanine), 86.2, 86.0 (C-1 ', C-4' ), 77.8 (С-3 '), 74.9, 74.5, 73.7, 70.4 (Bn, C-2', C-5 '), 63.5 (С-1') , 36.3 (ССОН), 20.8, 20.6 (ССОН ₃ ), 19.0 (СН ₃ СН) For a mixture of isomers: FAB-MS m / z 648 [M + H] ⁺ , 670 [M + Na] ⁺ . Calculated: C - 60.8; H - 6.0; N - 10.4. For C ₃₃ H ₃₆ N ₅ O ₉ calculated: C - 61.3; H - 5.6; N - 10.8%.

Example 50

9- (3,5-di-O-benzyl-4-C-hydroxymethyl-β-D-ribofuranosyl) -2-N-isobutyryl guanine (48)

A solution of the mixture obtained in example 49 containing compound 47 (2.10 g, 3.25 mmol) in THF / pyridine / methanol (2: 3: 4, v / v) (40 cm ³ ) was cooled to -10 ° C and sodium methoxide (320 mg, 5.93 mmol) was added to the stirred solution. The resulting reaction mixture was stirred at 10 ° C for 30 minutes and neutralized by adding 2 cm ^{3 of} acetic acid. The solvent was evaporated under reduced pressure, and the residue was extracted twice with dichloromethane / water (2 × 100 cm ³ ). The organic fractions were combined and evaporated under reduced pressure. After evaporation with toluene, the residue was chromatographed on a silica gel column in a gradient (2-7%) of methanol in dichloromethane (v / v) to give a white solid (1.62 g). According to ¹ H-NMR, it contains three isomers (in a ratio of 13.5: 1.5: 1). For the main product 48: δ _H (CD ₃ OD) 8.07 (s, 8-H) 7.36-7.20 (m, Bn), 6.05 (d, J 3.9, 1'-H ), 4.81 (d, J 11.5, Bn), 4.75 (m, 2'-H), 4.56 (d, J 11.5, Bn), 4.51-4.43 ( m, Bn, 3'-H), 3.83 (d, J 11.7, 1 '' - H _a ), 3.65 (d, J 11.7, 1 '' - H _b ), 3, 64 (d, J 10.6, 5'-H _a ), 3.57 (d, J 10.3, 5'-H _b ), 2.69 (m, CHCO), 1.20 (6H, d J 6.8; CH ₃ CH). δ _c (CD ₃ OD) 181.6 (SOS), 157.3, 150.2, 149.5 (guanine), 139.4, 139.3, 139.0 (guanine, Bn), 129.5, 129.4, 129.3, 129.2, 129.1, 129.0, 128.9, 128.8 (Bn), 121.2 (guanine), 90.7, 89.6 (C-1 ' , C-4 '), 79.2 (C-3'), 75.8, 74.5, 74.3, 72.2 (Bn, C-2 ', C-5'), 63.1 ( C-1 ''), 36.9 (COSH), 19.4 (CH ₃ CH), 19.3 (CH ₃ CH). For a mixture of isomers: FAB-MS m / z 564 [M + H] ⁺ .

Example 51

The intermediate connection 49

A solution of the mixture obtained in Example 50 containing compound 48 (1.6 g) in anhydrous pyridine (6 cm ³ ) was stirred at -20 ° C and p-toluenesulfonyl chloride (0.81 g, 4.27 mmol) was added. The solution was stirred at -20 ° C for 1 hour and at -25 ° C for 2 hours. Then the mixture was diluted with dichloromethane to 100 cm ³ and immediately washed with water (2 × 100 cm ³ ). The organic phase was separated and evaporated under reduced pressure. The residue was subjected to chromatographic purification using dichloromethane / methanol as eluent (1-2%, v / v) to give intermediate 49 (980 mg). After eluting Compound 49 from the column, an initial mixture containing Compound 48 (510 mg) was eluted with 8% methanol in dichloromethane (v / v). The resulting material was concentrated, dried under reduced pressure, and worked up in the same manner as described above to give an additional 252 mg of the same intermediate. The intermediate compound (1.23 g) was purified by HPLC in silica gel (PrepPak cartridge loaded with beads, 15-20 μm, 125A, flow rate 60 cm ³ / min, eluent 0-4% methanol in dichloromethane, v / v, elution duration 120 minutes). Fractions containing intermediate 49 were combined and concentrated to give a white solid (1.04 g). According to ¹ H-NMR, it contains two main products, namely 1 ″ —O- and 2 ″ —O-monosilylated derivatives in a ratio of about 2: 1. FAB-MS m / z 718 [M + H] ⁺ . Calculated: C - 60.4; P - 5.8; N - 9.3. For C ₃₆ H ₃₉ N ₅ O ₉ S calculated: C - 60.2; H - 5.5; N - 9.8%.

Example 52

(1S, 3R, 4R, 7S) -7-benzyloxy-1-benzyloxymethyl-3- (2-N-isobutyrylguanin-9-yl) -2,5-dioxabicyclo [2.2.1] heptane (50)

To a solution of intermediate 49 (940 mg) in anhydrous THF (20 cm ³ ) was added a 60% suspension of sodium hydride (w / v, 130 mg, 3.25 mmol) and the resulting mixture was stirred at room temperature for 1 hour. Acetic acid (0.25 ml) was added and the mixture was concentrated under reduced pressure. The residue was dissolved in dichloromethane (100 cm ³ ) and washed with water (2 × 100 cm ³ ). The organic phase was separated and evaporated under reduced pressure. The residue was purified by silica gel column chromatography using methanol / dichloromethane (1-1.5%, v / v) as an eluent to give nucleoside 50 as a white solid (451 mg, 57%). δ _H (CDCl ₃ ) 12.25 (1H, brs, NHCO), 10.12 (1H, br, NH), 7.84 (1H, s, 8-H), 7.31-7.15 (10H m, Bn), 5.72 (1H, s, 1'-H), 4.60-4.46 (5H, m, Bn, 2'-H), 4.14 (1H, s, 3 ' -H), 4.02 (1H, d, J 7.9, 1'-H _a ), 3.85 (1H, d, J 7.9, 1'-H _b ), 3.78 (2H, s, 5'-H), 2.81 (1H, m, CHCO), 1.24 (3H, d, J 6.8, CH ₃ CH), 1.22 (3H, d, J 6.4, CH ₃ CH). δ _C (CDCl ₃ ) 179.5 (COSH), 155.6, 148.1, 147.3 (guanine), 137.3, 136.9, 136.0 (guanine, Bn), 128.4, 128 , 3, 127.9, 127.8, 127.5, 127.4 (Bn), 121.2 (guanine), 87.1, 86.2 (C-1 ', C-4'), 77, 0 (С-3 '), 73.6, 72.5, 72.1 (Вn, С-2', С-5 '), 64.9 (C-1``), 36.1 (ССОН) 19.0 (CH ₃ CH); 18.9 (CH ₃ CH). FAB-MS m / z 546 [M + H] ⁺ . Received: C, 63.3; H - 5.9; N - 12.5. For C ₂₉ H ₃₀ N ₅ O _{6 it is} calculated: C - 64.0; H - 5.6; N - 12.9%.

An alternative way to obtain compounds 50. G1AQ.

To a suspension of compound 78 (1.5 g, 2.51 mmol) and N-2-isobutyryl guanine (0.93 g, 4.06 mmol) in dry DCM (50 ml) was added BSA (N, O-bistrimethylsilylacetamide: 3, 33 ml, 13.5 mmol) and the resulting mixture was heated under reflux for 2 hours. Trimethylsilyl triflate (1.25 ml, 6.9 mmol) was added to this mixture, and the resulting mixture was further heated under reflux for another 2 hours. The mixture was allowed to cool to room temperature, diluted with DCM to 200 ml and washed with saturated aqueous sodium hydrogen carbonate NaHCO ₃ and water. After chromatographic purification on a silica gel column (1-2.5% methanol in dichloromethane), 1.05 g (55%) of the desired G1AQ isomer was obtained, and another 380 mg of the higher mobility isomers were converted to G1AQ by repeating the above procedure. Ammonium hydroxide (12 ml of a 25% aqueous solution) was added to a G1AQ solution (1.05 g in 12 ml of methanol) and the resulting mixture was stirred at room temperature for 1 hour. After concentration, the product was purified by chromatography on a silica gel column (1-3% methanol in dichloromethane) to give 700 mg of compound G3 as a white solid. 70 mg of G3 in anhydrous THF (15 ml) was treated with sodium hydride (225 mg of a 60% suspension in mineral oil). After half an hour, the reaction was stopped by adding 1.25 ml of acetic acid and the resulting mixture was concentrated under reduced pressure. The residue was dissolved in dichloromethane, washed in NaHCO ₃ and water and chromatographed on silica gel in a gradient of 0.5-3% methanol in dichloromethane. The yield of compound 50 was 400 mg (75%).

Example 53

(1S, 3R, 4R, 7S) -7-hydroxy-1-hydroxymethyl-3- (2-N-isobutyrylguanin-9-yl) -2,5-dioxabicyclo [2.2.1] heptane (51)

A mixture of nucleoside 50 (717 mg, 1.31 mol) and 10% palladium on carbon (500 mg) was suspended in methanol (8 cm ³ ) at room temperature. The resulting mixture was degassed several times under reduced pressure and placed in a hydrogen atmosphere. After stirring for 24 hours, the mixture was chromatographed using methanol / dichloromethane (8-20%, v / v) as eluent to give nucleoside 51 as a glassy substance (440 mg, 92%). δ _H (CD ₃ OD) 8.12 (1H, br, 8-H), 5.86 (1H, s, 1'-H), 4.50 (1H, s, 2'-H), 4, 30 (1H, s, 3'-H), 4.05 (1H, d, J 8.0, 1 '' - H _a ), 3.95 (2H, s, 5'-H), 3.87 (1H, d, J 7.9, 1``-H <sub>b</sub> ), 2.74 (1H, m, CHCO), 1.23 (6H, d, J 6.9, CH <sub>3</sub> CH). δ <sub>C</sub> (CD <sub>3</sub> OD, hydrocarbon signals) 90.2, 87.6 (C-1 ', C-4'), 81.1 (C-3 '), 72.9, 71.3 (C -2 ', C-5'), 58.2 (C-1``), 37.1 (COSH), 19.5 (CH ₃ CH). FAB-MS m / z 366 [M + H] ⁺ .

Example 54

(1R, 3R, 4R, 7S) -1- (4,4'-dimethoxytrityloxymethyl) -7-hydroxy-3- (2-N-isobutyrylguanine-9-yl) -2,5-dioxabicyclo [2.2.1] heptane (52)

A mixture of compound 51 (440 mg, 1.21 mmol) and 4.4'-dimethoxytrityl chloride (573 mg, 1.69 mmol) was dissolved in anhydrous pyridine (7 cm ³ ) and stirred at room temperature for 4 hours. The mixture was evaporated under reduced pressure to give an oil. Extraction was carried out in a dichloromethane / water system (1: 1, v / v, 40 cm ³ ). The organic phase was separated and concentrated to give a solution of a minimum amount of dichloromethane containing 0.5% pyridine (v / v), which was loaded onto a silica gel chromatography column equilibrated with the same solvent. The product was eluted in a gradient of methanol (0.6-2%, v / v) in dichloromethane containing 0.5% pyridine (v / v) to give compound 52 as a white solid (695 mg, 86%). δ _H (CDCl ₃ ) 12.17 (1H, brs, NHCO), 10.09 (1H, br, NH), 7.87 (1H, s, 8-H), 7.42-6.72 (13H) , m, DMT), 5.69 (1H, s, 1'-H), 4.59 (1H, s, 2'-H), 4.50 (1H, s, 3'-H), 3, 98 (1H, d, J 8.1, 1``-H _a ), 3.69-3.39 (9H, m, DMT, 5'-H, 1 '' - H _b ), 2.72 ( 1H, m, CHCO), 1.17 (6H, d, J 6.8, CH ₃ CH). δ _C (CDCl ₃ ) 179.8 (COSH), 158.8, 144.5, 135.6, 135.5, 130.1, 128.1, 127.7, 126.9, 113.2 (DMT ), 155.8, 147.9, 147.5, 137.0, 120.8 (guanine), 87.6, 86.4, 86.1 (C-1 ', C-4', DMT), 79.7 (C-3 '), 72.6, 71.4 (C-2', C-5 '), 59.8 (C-1''), 55.2 (DMT), 36.1 (COSH), 19.1, 18.8 (CH ₃ CH). FAB-MS m / z 668 [M + H] ⁺ .

Example 55

(1R, 3R, 4R, 7S) -7- (2-cyanoethoxy (diisopropylamino) phosphinoxy) -1- (4,4'-dimethoxytrityloxymethyl) -3- (2-N-isopropionylguanine-9-yl) -2.5 dioxabicyclo [2.2.1] heptane (53)

Compound 52 (670 mg, 1.0 mol) was dissolved at room temperature in anhydrous dichloromethane (5 cm ³ ) containing N, N-diisopropylethylamine (0.38 cm ³ , 4 mmol). 2-Cyanoethyl-N, N-diisopropylphosphoramidochloridite (0.36 cm ³ , 2.0 mmol) was added dropwise with stirring. After 5 hours, methanol (2 cm ³ ) was added and the resulting mixture was diluted with dichloromethane to 100 cm ³ and washed with a saturated aqueous solution of sodium hydrogencarbonate (50 cm ³ ). The organic phase was separated and the solvent was evaporated under reduced pressure. The residue was dissolved in a minimal amount of a mixture of dichloromethane and petroleum ether (1: 1, v / v) containing 0.5% pyridine (v / v), and a silica gel column equilibrated with the same solvent was loaded. The column was washed with dichloromethane / petroleum ether / pyridine (75: 25: 0.5, v / v, 250 cm ³ ) and the product was eluted using a gradient of methanol (0-1%, v / v) in dichloromethane containing 0.5% pyridine (o / o). Fractions containing the main product were evaporated and then evaporated with toluene. The residue was dissolved in anhydrous dichloromethane (5 cm ³ ) and precipitated with petroleum ether (10 cm ³ ) to give compound 53 as a white solid (558 mg, 64%) after filtration and drying. δ _P (CDCl ₃ ) 148.17, 146.07. FAB-MS m / z 868 [M + 1] ⁺ .

Example 56

1- (2-O-acetyl-4-C-acetoxymethyl-3,5-di-O-benzyl-β-D-ribofuranosyl) -4-N-benzoylcytosine (54)

To a stirred solution of an anomeric mixture of 33 (4.0 g, 8.22 mmol) and 4-N-benzoylcytosine (2.79 g, 13.0 mmol) was added N, O-bis (trimethylsilyl) acetamide (8.16 cm ³ , 33.0 mmol). The reaction mixture was stirred at room temperature for 1 hour and cooled to 0 ° C. Trimethylsilyl triflate (3.0 cm ³ , 16.2 mmol) was added dropwise and the resulting mixture was stirred at 60 ° C. for 2 hours. A saturated aqueous solution of sodium hydrogencarbonate (3 × 20 cm ³ ) and brine (2 × 20 cm ³ ) were successively added and the separated organic phase was dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (99: 1, v / v) as an eluent to give compound 54 as a white solid (3.9 g, 74%). δ _H (CDCl ₃ ) 8.28 (1H, d, J 7.5, 6-H), 7.94-7.90 (2H, m, Bz), 7.65-7.25 (13H, m , Bn, Bz), 7.16 (1H, d, J 7.1, 5-H), 6.22 (1H, d, J 2.8, 1'-H), 5.51 (1H, dd , J 2.8, 5.8, 2'-H), 4.62 (1H, d, J 11.6, Bn), 4.51 (1H, d, J 12.3, 1 '' - H _a ) 4.49-4.34 (4H, m, 3'-H, Bn), 4.21 (1H, d, J 12.3, 1 '' - H _b ), 3.85 (1H, d, J 10.3, 5'-H _a ), 3.47 (1H, d, J 10.3, 5'-H _b ), 2.13 (3H, s, COSH ₃ ), 2.06 ( 3H, s, COCH ₃ ). δ _C (CDCl ₃ ) 170.52, 169.61 (C = O), 166.83, 162.27 (C-4, C = O), 154.26 (C-2), 145.26 (C -6), 137.25, 136.93, 133.18, 129.0, 128.75, 128.51, 128.45, 128.18, 128.10, 127.89, 127.71 (Bn, Bz), 96.58 (C-5), 89.42, 86.52 (C-1 ', C-4'), 76.21, 75.10, 74.17, 73.70, 69.70 63.97 (C-2 ', C-3', Bn, C-5 ', C-1''), 20.82 (COSH ₃ ). FAB-MS m / z 664 [M + Na] ⁺ , 642 [M + H] ⁺ . Received: C, 65.0; H - 5.7; N is 6.5. For C ₃₅ H ₃₅ N ₃ O ₉ calculated: C - 65.5; H - 5.5; N - 6.5%.

Example 57

1- (3-, 5-di-O-benzyl-4-C-hydroxymethyl-β-D-ribofuranosyl) -4-N-benzoylcytosine (55)

To a stirred solution of nucleoside 54 (3.4 g, 5.3 mmol) in methanol (20 cm ³ ) was added sodium methoxide (0.663 g, 11.66 mmol). The reaction mixture was stirred at room temperature for 10 minutes and then neutralized with 20% aqueous hydrochloric acid. The solvent was partially evaporated and the residue was extracted with dichloromethane (3 × 50 cm ³ ).

The combined organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 20 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (98.5: 1.5, v / v) as an eluent to give compound 55 as a white solid (1.6 g, 54%). δ _H (CDCl ₃ ) 9.95 (1H, brs, NH), 8.33 (1H, d, J 7.4, 6-H), 7.98 (2H, m, Bz), 7.60- 7.12 (14H, m, Bn, Bz, 5-H), 6.17 (1H, d, J 1.6, 1'-H), 4.78 (1H, d, J 11.8, Bn ), 4.48-4.27 (5H, m, Bn, 2'-H, 3'-H), 3.85 (1H, d, J 11.8, 5'-H _a ), 3.66 -3.61 (2H, m, 5'-H _b , 1 '' - H _a ), 3.47 (1H, d, J 10.4, 1 '' - H _b ). δ _C (CDCl ₃ ) 167.5, 162.31 (C-4, C = O), 155.36 (C-2), 145.34 (C-6), 137.49, 137.08, 133 09, 133.01, 128.94, 128.67, 128.48, 128.30, 128.01, 127.90, 127.80 (Bn, Bz), 96.53 (C-5), 93 97, 89.35 (C-1 ', C-4'), 76.06, 75.28, 73.70, 72.76, 70.26, 62.44 (C-2 ', C-3 ', Bn, C-5', C-1 ''). FAB-MS m / z 558 [M + H] ⁺ .

Example 58

The intermediate connection 56

A solution of nucleoside 55 (2.2 g, 3.94 mmol) in anhydrous tetrahydrofuran (60 cm ³ ) was stirred at -20 ° C and a suspension of 60% sodium hydride in mineral oil (w / v, 0.252 g, 6.30 mmol) was added in seven portions over 45 minutes. The solution was stirred at −20 ° C. for 15 minutes, and then p-toluenesulfonyl chloride (0.901 g, 4.73 mmol) was added in small portions. The resulting solution was stirred for 4 hours at −20 ° C. Sodium hydride (0.252 g, 6.30 mmol) and p-toluenesulfonyl chloride (0.751 g, 3.93 mmol) were additionally added. The reaction mixture was kept at -20 ° C for 48 hours. The reaction was stopped by adding a mixture of ice and water (50 cm ³ ), after which extraction was carried out with dichloromethane (3 × 60 cm ³ ). The combined organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 20 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was evaporated under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (99: 1, v / v) as an eluent to give intermediate 56 (1.80 g).

Example 59

(1S, 3R, 4R, 7S) -7-benzyloxy-1-benzyloxymethyl-3- (4-N-benzoylcytosin-1-yl) -2,5-dioxabicyclo [2.2.1] heptane (57)

Intermediate 56 (1.80 g) was dissolved in anhydrous DMF (30.0 cm ³ ) and a 60% suspension of sodium hydride in mineral oil (w / v, 0.16 mg, 3.9 mmol) was added in five portions for 30 minutes at 0 ° C. The reaction mixture was stirred at room temperature for 36 minutes. The reaction was stopped by adding a mixture of ice and water (70 cm ³ ) and the resulting mixture was extracted with dichloromethane (3 × 50 cm ³ ). The combined organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 30 cm ³ ) and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (99.5: 0.5, v / v) as an eluent to give compound 57 as a white solid (1.08 g, 79%). δ _n (CDCl ₃ ) 8.95 (1H, brs, NH), 8.20 (1H, d, J 7.5, 6-H), 7.95-7.92 (2H, m, Bz), 7.66-7.22 (14H, m, Bn, Bz, 5-H), 5.78 (1H, s, 1'-H), 4.70-4.65 (3H, m, 2'- H, Bn), 4.60 (1H, d, J 11.6, Bn), 4.47 (1H, d, J 11.6, Bn), 4.05-3.78 (5H, m, 3 '-H, 5'-H _a , 1''- H _a , 5'-H _b , 1''- H _b ). δ _C (CDCl ₃ ) 167.0, 162.36 (C-4, C = O), 154.5 (C-2), 144.58 (C-6), 137.46, 136.93, 133 , 35, 132.93, 129.11, 128.67, 128.50, 128.16, 128.11, 127.68, 127.60 (Bn), 96.35 (C-5), 88.38 87.67 (C-1 ', C-4'), 76.14, 75.70, 73.79, 72.27, 72.09, 64.34 (Bn, C-5 ', C-1 '', C-2 ', C-3'). FAB-MS m / z 540 [M + H] ⁺ . Received: C, 68.0; H, 5.5; N, 7.5. For C ₃₁ H ₂₉ N ₃ O ₆ calculated: C - 69.0; H - 5.4; N - 7.8%.

Example 60

(1S, 3R, 4R, 7S) -7-hydroxy-1-hydroxymethyl-3- (cytosin-1-yl) -2.5-2.5-dioxabicyclo [2.2.1] heptane (57A)

To a solution of nucleoside 57 (0.3 g, 0.55 mmol) in anhydrous methanol (22 cm ³ ) was added 1,4-cyclohexadiene (5.0 cm ³ ) and 10% palladium on carbon (0.314 g). The resulting mixture was stirred under reflux for 18 hours while stirring. An additional 10% palladium on carbon (0.380 g) and 1,4-cyclohexadiene (5.5 cm ³ ) were added and the mixture was heated under reflux for 54 hours. The reaction mixture was filtered through a pad of silica gel, which was then washed with methanol (1500 cm ³ ). The combined filtrate was evaporated under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (92.5: 7.5, v / v) as an eluent to give compound 57A as a white solid (0.051 g, 36%). δ _H ((CD ₃ ) ₂ SO) 7.73 (1H, d, J 7.7, 6-H), 7.12-7.20 (2H, br s, NH ₂ ), 5.74 (1H, d, J 7.7, 5-H), 5.61 (1H, br, 3'-OH), 5.39 (1H, s, 1'-H), 5.12 (1H, m, 5 ' -OH), 4.08 (1H, s, 2'-H), 3.80 (1H, d, J 7.7, 1 '' - H _a ), 3.81 (1H, s, 3'- H), 3.74 (2H, m, 5'-H _a , 5'-H _b ), 3.63 (1H, d, J 7.7, 1 '' - H _b ). δ _C ((CO ₃ ) ₂ SO) 165.66 (C-4), 154.58 (C-2), 139.68 (C-6), 93.19 (C-5), 88.42, 86.73 (C-1 ', C-4'), 78.87, 70.85, 68.32, 56.04 (C-2 ', C-1'',C-3', C-5 '). FAB-MS m / z 256 [M + H] ⁺ .

Example 61

Intermediate 57B

To nucleoside 57A (0.030 g, 0.11 mmol) suspended in anhydrous pyridine (2.0 cm ³ ), trimethylsilyl chloride (0.14 cm ³ , 1.17 mmol) was added and stirred at room temperature for 1 hour. Benzoyl chloride (0.07 cm ³ , 0.58 mmol) was added at 0 ° C and the resulting mixture was stirred at room temperature for 2 hours. After cooling the reaction mixture to 0 ° C., water (3.0 cm ³ ) was added. After stirring for 5 minutes, aqueous ammonia solution (1.5 cm ³ , 32%, w / v) was added and stirring was continued at room temperature for 30 minutes. The resulting mixture was evaporated under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (97.5: 2.5, v / v) as an eluent to give intermediate 57B as a white solid (0.062 g) .

Example 62

(1R, 3R, 4R, 73) -1- (4,4'-dimethoxytrityloxymethyl) -7-hydroxy-3- (4-N-benzoylcytosin-1-yl) -2,5-dioxabicyclo [2.2.1] - heptane (57C)

To a solution of intermediate 57B (0.042 g, 0.11 mmol) in anhydrous pyridine (1.5 cm ³ ) was added 4,4'-dimethoxytrityl chloride (0.06 g, 0.17 mmol). The reaction mixture was stirred at room temperature for 3.5 hours, cooled to 0 ° C. and a saturated aqueous solution of sodium hydrogencarbonate (20 cm ³ ) was added. The extraction was carried out with dichloromethane (3 × 10 cm ³ ). The combined organic phase was dried over Na ₂ SO ₄ and evaporated to dryness under reduced pressure. The residue was chromatographed on a silica gel column using dichloromethane / methanol / pyridine (98.0: 1.5: 0.5, v / v) as an eluent to give 57C nucleoside as a white solid (0.031 g, ≈ 63 % from 57A). δ _H (C ₅ D ₅ N) 12.32 (1H, br, NHCO), 8.75-7.06 (20H, m, DMT, Bz, H-5, H-6), 6.24 (1H , s, 1'-H), 5.11 (1-H, s, 2'-H), 4.90 (1H, s, 3'-H), 4.38 (1H, d, J 7, 6, 1 '' - H _a ), 4.10 (1H, d, J 7.6, 1 '' - H _b ), 4.02 (1H, d, J 10.6, 5'-H _a ) 3.87 (1H, d, J 10.6, 5'-H _b ), 3.77, 3.76 (2 × 3H, 2 × s; 2 × OCH ₃ ). δ _C (C ₅ D ₅ N) 169.00 (NHCO), 164.24 (C-2), 159.39 (DMT), 150.5, 145.62 (DMT), 144.31, 132.89 , 130.82, 130.72, 129.09, 128.89, 128.60, 113.96 (DMT), 96.96, 89.01, 87.18, 79.91, 72.56, 70, 25 (C-5, C-1 ', C-4', C-2 ', C-1'',C-3'), 59.51 (C-5 '), 55.33 (OCH ₃ ) . FAB-MS m / z 662 [M + H] ⁺ .

Example 63

(1R, 3R, 4R, 7S) -7- (2-cyanoethoxy (diisopropylamino) phosphinoxy) -1- (4,4'-dimethoxytrityloxymethyl) -3- (4-N-benzoylcytosin-1-yl) -2.5 dioxabicyclo [2.2.1] heptane (57D)

To a solution of 57C nucleoside (0.025 g, 0.03 mmol) in anhydrous dichloromethane (1.5 cm ³ ) was added N, N-diisopropylethylamine (0.03 cm ³ , 0.17 mmol), and then 2- cyanoethyl-N, N-diisopropylphosphoramidochloridite (0.02 cm ³ , 0.09 mmol). After stirring at room temperature for 5 hours, the reaction mixture was cooled to 0 ° C, dichloromethane / pyridine (10.0 cm ³ , 99.5: 0.5, v / v) was added and washing was carried out using a saturated aqueous solution of carbonic acid sodium (3 × 8 cm ³ ). The organic phase was separated, dried over Na ₂ SO ₄ and evaporated to dryness under reduced pressure. The residue was subjected to chromatographic purification on a silica gel column using dichloromethane / methanol / pyridine (99.0: 0.5: 0.5, v / v) as an eluent to give amidite 57D as a light yellow oily substance (0.038 g). δ _P (CDCl ₃ ) 147.93.

Example 64

9- (2-O-acetyl-4-C-acetyloxymethyl-3,5-di-O-benzyl-β-D-ribofuranosyl) -6-N-benzoyladenine (58)

To a stirred suspension of an anomeric mixture of 33 (5.0 g, 10.3 mmol) and 6-N-benzoyladenine (3.76 g, 15.7 mmol) in anhydrous dichloromethane (200 cm ³ ) was added N, O-bis (trimethylsilyl ) acetamide (15.54 cm ³ , 61.8 mmol). The reaction mixture was stirred under reflux for 1 hour and then cooled to room temperature. Trimethylsilyl triflate (7.0 cm ³ , 38.7 mmol) was added dropwise and the resulting mixture was heated under reflux for 20 hours. The reaction mixture was then allowed to cool to room temperature and the volume of the mixture was reduced by 75% under reduced pressure. Dichloromethane (250 cm ³ ) was added and the resulting solution was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 50 cm ³ ) and water (50 cm ³ ). The organic phase was dried over Na ₂ SO ₄ and evaporated under reduced pressure. The residue was chromatographed on a silica gel column using dichloromethane / methanol (99.5: 0.5, v / v) as an eluent to give nucleoside 58 as a white solid (3.65 g, 52%). δ _H (CDCl ₃ ) 9.25 (1H, brs, NH), 8.71 (1H, s, 8-H), 8.24 (1H, s, 2-H), 8.0 (2H, d , J 7.5, Bz), 7.60-7.23 (13H, m, Bn, Bz), 6.35 (1H, d, J 4.6, 1'-H), 5.99 (1H dd, J 4.9, 5.3, 2'-H), 4.78 (1H, d, J 5.6, 3'-H), 4.64-4.42 (5H, m, Bn , 1 '' - H _a ), 4.25 (1H, d, J 12.1, 1 '' - H _b ), 3.72 (1H, d, J 10.1, 5'-H _a ), 3.56 (1H, d, J 10.1, 5'-H _b ), 2.07 (3H, s, COCH ₃ ), 2.02 (3H, s, COCH ₃ ). δ _C (CDCl ₃ ) 170.42, 169.72 (COCH ₃ ), 164.60 (NHCO), 152.51 (C-6), 151.45 (C-2), 149.46 (C-4 ), 141.88 (C-8), 137.04, 137.00, 133.50, 132.60, 128.86, 128.66, 128.53, 128.41, 128.38, 128.18 , 128.06, 127.91, 127.88, 127.79, 127.63, 123.26 (Bz, Bn, C-5), 86.38 (C-1 '), 86.25 (C- 4 '), 77.74, 74.74, 74.44, 73.48 (C-2', C-3 ', 2 × Bn), 70.11 (C-1''), 63.42 ( C-5 '), 20.70, 20.54 (COSH ₃ ). FAB-MS m / z 666 [M + H] ⁺ .

Example 65

9- (3,5-di-O-benzyl-4-C-hydroxymethyl-β-D-ribofuranosyl) -6-N-benzoyladenine (59)

To a stirred solution of nucleoside 58 (4.18 g, 6.28 mmol) in methanol (50 cm ³ ) was added sodium methoxide (0.75 g, 13.8 mmol) at 0 ° C. The reaction mixture was stirred for 2 hours and ice was added. The mixture was neutralized using a 20% aqueous hydrochloric acid solution. Extraction was carried out with dichloromethane (3 × 75 cm ³ ), the organic phase was separated, dried over Na ₂ SO ₄ and evaporated under reduced pressure. The residue was chromatographed on a silica gel column using dichloromethane / methanol (98.5: 1.5 v / v) as eluent to give nucleoside 59 as a white solid (2.68 g, 73%). δ _H (CDCl ₃ ) 9.42 (1H, brs, NH), 8.58 (1H, s, H-8), 8.16 (1H, s, 2-H), 7.96 (2H, d , J 7.2, Bz), 7.52-7.08 (13H, m, Bn, Bz), 6.18 (1H, d, J 2.5, 1'-H), 4.85-4 38 (4H, m, Bn, 2'-H, 3'-H), 4.33 (2H, s, Bn) 3.90 (1H, d, J 11.9, 1 '' - H _a ) 3.71 (1H, d, J 1.8, 1 '' - H _b ); 3.50-3.39 (2H, m, 5-H). δ _C (CDCl ₃ ) 164.98 (NHCO), 152.19 (C-6), 151.00 (C-2), 149.34 (C-4), 142.28 (C-8), 137 32, 137.25, 133.46, 132.70, 128.69, 128.49, 128.40, 128.11, 128.03, 127.94, 127.83, 127.62, (Bz, Bn), 122.92 (C-5), 90.94, 88.75 (C-1 ', C-4'), 77.65, 74.08, 73.44, 73.20, 71.12 62.39 (C-1 ’, C-5 ′, C-2 ′, C-3 ′, 2 × Bn). FAB-MS m / z 582 [M + H] ⁺ . Received: C, 65.6; H - 5.5; N, 11.1; For C ₃₂ H ₃₁ N ₅ O _{6, the} calculated: C, 66.1; H - 5.4; N - 12.0%.

Example 66

The intermediate connection 60

A solution of nucleoside 59 (2.43 g, 4.18 mmol) in anhydrous tetrahydrofuran (25 cm ³ ) was stirred at -20 ° C and a 60% suspension of sodium hydride in mineral oil (w / v, 0.28 g, 7 , 0 mmol) was added in four portions over 30 minutes. After stirring for 1 hour, p-toluenesulfonyl chloride (1.34 g, 7.0 mol) was added in small portions. The resulting mixture was stirred at -10 ° C for 15 hours. Chilled ice water (50 cm ³ ) was added and extraction was performed with dichloromethane (3 × 50 cm ³ ). The combined organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (2 × 25 cm ³ ), dried over Na ₂ SO ₄ and evaporated under reduced pressure. The residue was subjected to silica gel column chromatography using dichloromethane / methanol (99: 1, v / v) as an eluent, to give intermediate 60 (1.95 g).

Example 67

(1S, 3R, 4R, 7S) -7-benzyloxy-1-benzyloxymethyl-3- (6-N-benzoyladenin-9-yl) -2,5-dioxabicyclo [2.2.1] heptane (61)

Intermediate 60 (1.90 g) was dissolved in anhydrous DMF (20 cm ³ ) and a 60% suspension of sodium hydride in mineral oil (w / w, 0.16 g, 3.87 mmol) was added in small portions at 0 ° C. The resulting mixture was stirred at room temperature for 10 hours and then concentrated under reduced pressure. The residue was dissolved in dichloromethane (75 cm ³ ), washed with a saturated aqueous solution of sodium hydrogencarbonate (2 × 25 cm ³ ), dried over Na ₂ SO ₄ and evaporated under reduced pressure. The residue was chromatographed on a silica gel column using dichloromethane / methanol (99: 1, v / v) as an eluent to give nucleoside 61 as a white solid (1.0 g, ≈ 44% of 59). δ _H (CDCl ₃ ) 8.71 (H, s, 8-H), 8.23 (1H, s, 2-H), 8.02 (2H, m, J 7.0, Bz), 7, 99-7.19 (13H, m, Bn, Bz), 6.08 (1H, s, 1'-H), 4.78 (1H, s, 2'-H), 4.61-4.50 (4H, m, 2 × Bn), 4.24 (1H, s, 3'-H), 4.12 (1H, d, J 7.8, 1 '' - H _a ), 4.00 (1H d, J 7.9, 1 '' - H _b ), 3.85-3.78 (2H, m, 5'-H _a , 1 '' - H _b ). δ _C (CDCl ₃ ) 164.61 (NHCO), 152.32 (C-6), 150.61 (C-2), 149.35 (C-4), 140.67 (C-8), 137 24, 136.76, 133.33, 132.66, 128.68, 128.39, 128.29, 127.94, 127.77, 127.51 (Bn, Bz), 123.43 (C- 5), 87.14, 86.52 (C-1 ', C-4'), 77.21, 76.77, 73.56, 72.57, 72.27, 64.65 (C-2 ' , C-3 ', C-1'', 2 × Bn, C-5'). FAB-MS m / z 564 [M + H] ⁺ . Received: C, 66.2; H - 5.5; N - 11.4. For C ₃₂ H ₂₉ N ₅ O ₅ calculated: C - 66.2; H - 5.2; N - 12.4%.

Example 68

(1S, 3R, 4R, 7S) -7-hydroxy-1-hydroxymethyl-3- (adenin-9-yl) -2,5-dioxabicyclo [2.2.1] heptane (61A)

To a stirred solution of nucleoside 61 (0.80 g, 1.42 mmol) in anhydrous dichloromethane (30 cm ³ ) at -78 ° C was added dropwise BCl ₃ (1 M solution in hexane, 11.36 cm ³ , 11.36 mmol) for 30 minutes. The resulting mixture was stirred at -78 ° C for 4 hours, BCl ₃ (1 M solution in hexane, 16.0 cm ³ , 16.0 mmol) was added dropwise again and the mixture was stirred at -78 ° C for 3 hours. Then the temperature of the reaction mixture was slowly increased to room temperature and stirring was continued for 30 minutes. Methanol (25.0 cm ³ ) was added at -78 ° C and the mixture was stirred at room temperature for 12 hours. The resulting mixture was evaporated under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol (92: 8, v / v) as an eluent to give 61A nucleoside as a white solid (0.332 g, 84%). δ _H ((CD ₃ ) ₂ SO) 8.22 (1H, s, 8-H), 8.15 (1H, s, 2-H), 7.33 (2H, s, NH ₂ ), 5, 89 (1H, s, 1'-H), 5.83 (1H, d, J 4.2, 3'-OH), 5.14 (1H, t, J 5.9, 5'-OH), 4.14 (1H, s, 2'-H), 4, -25 (1H, D, J 4.2, 3'-H), 3.92 (1H, d, J 7.8, 1 '' -H _a ), 3.81-3.41 (3H, m, 5'-H _a , 5'-H _b , 1 '' - H _b ). δ _C ((CO ₃ ) ₂ SO) 155.90 (C-6), 152.64 (C-2), 148.35 (C-4), 137.72 (C-8), 118.94 ( C-5), 88.48, 85.17 (C-1 ', C-4'), 79.09, 71.34, 69.83, 56.51 (C-2 ', C-3', C-1 '', C-5 '). FAB-MS m / z 280 [M + H] ⁺ .

Example 69

(1S, 3R, 4R, 7S) -7-hydroxy-1-hydroxymethyl-3- (6-N-benzoyladenin-9-yl) -2,5-dioxabicyclo [2.2.1] heptane (61B)

To a stirred solution of 61A nucleoside (0.32 g, 1.15 mmol) in anhydrous pyridine (1 cm ³ ) was added trimethylsilyl chloride (0.73 cm ³ , 5.73 mmol) and the resulting mixture was stirred at room temperature for 20 minutes. Benzoyl chloride (0.67 cm ³ , 5.73 mmol) was added at 0 ° C and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was cooled to 0 ° C. and ice-cold water (15.0 cm ³ ) was added. After stirring for 5 minutes, a 32% (w / v) aqueous ammonia solution (1.5 cm ³ ) was added and the mixture was stirred for 30 minutes. The resulting mixture was evaporated to dryness and the residue was dissolved in water (25 cm ³ ). After the mixture was evaporated under reduced pressure, the residue was chromatographed on a silica gel column using dichloromethane / methanol (97: 3, v / v) as an eluent to give 61B nucleoside as a white solid (0.55 g). FAB-MS m / z 384 [M + H] ⁺ .

Example 70

(1R, 3R, 4R, 7S) -7-hydroxy-1- (4,4'-dimethoxytrityloxymethyl) -3- (6-N-benzoyladenin-9-yl) -2,5-dioxabicyclo [2.2.1] - heptane (61C)

To a stirred solution of compound 61B (0.50 g) in anhydrous pyridine (20 cm ³ ) was added 4,4'-dimethoxytrityl chloride (0.71 g, 2.09 mmol) and 4-N, N-dimethylaminopyridine (DMAP) (0 , 1 g). After stirring at room temperature for 2 hours and for 1 hour at 50 ° C., the reaction mixture was cooled to 0 ° C. and a saturated aqueous solution of sodium hydrogencarbonate (100 cm ³ ) was added. After extraction with dichloromethane (3 × 50 cm ³ ), the combined organic phase was dried over N ₂ SO ₄ and evaporated under reduced pressure. The residue was chromatographed on a silica gel column using dichloromethane / methanol / pyridine (98.0: 1.5: 0.5) as an eluent to give 61C nucleoside as a white solid (0.36 g, ≈ 50% of 61A). δ _H (C ₅ D ₅ N) 12.52 (NHCO), 9.10 (2H, d, J 7.7, Bz), 8.88 (1H, s, 8-H), 8.50-7 11 (17H, m, DMT, Bz, 2-H), 6.65 (1H, s, H-1 '), 5.25 (2H, s, H-2', H-3 '), 4 71 (1H, d, J 7.8, 1``-H _a ), 4.56 (1H, d, J 7.8, 1 '' - H _b ), 4.20 (1H, d, J 10.8, 5'-H _a ), 4.07 (1H, d, J 10.8, 5'-H _b ), 3.82, 3.81 (2 × 3H, 2 × s, 2 × OCH ₃ ). δ _C (C ₅ D ₅ N) 167.56 (NHCO), 159.24 (C-6), 152.50, 152.08, 151.81, 145.84, 141.45, 136.52, 136 , 28, 132.55, 130.76, 130.70, 129.32, 128.85, 128.76, 128.46, 127.38, 126.33 (DMT, Bz, C-2, C-4 , C-8), 113.84 (C-5), 88.64, 87.20, 86.85, 80.52, 73.13, 72.16, 60.86 (C-1 ', C- 4 ', DMT, C-2', C-3 ';C-1'',C-5'), 55.24 (OCH ₃ ). FAB-MS m / z 686 [M + H] ⁺ . Received: C, 68.3; H - 5.0; N - 9.7. For C ₃₉ H ₃₅ N ₅ O ₇ calculated: C - 68.3; H - 5.1; N - 10.2%.

Example 71

(1R, 3R, 4R, 7S) -7- (2-cyanoethoxy (diisopropylamino) phosphinoxy-1- (4,4'-dimethoxytrityloxymethyl) -3- (6-N-benzoyladenin-9-yl) -2,5- dioxabicyclo [2.2.1] heptane (61D)

To a solution of compound 61C (190 mg, 0.277 mmol) in anhydrous dicloromethane (1.5 cm ³ ) was added N, N-diisopropylethylamine (0.16 cm ³ , 0.94 mmol) and 2-cyanoethyl-N, N-diisopropylphosphoramidochloridite ( 0.1 cm ³ , 0.42 mmol) at 0 ° C. The mixture was allowed to warm to room temperature and was stirred for 5 hours. The solution was diluted with dichloromethane (50 cm ³ ), washed with a saturated aqueous solution of sodium hydrogencarbonate (2 × 30 cm ³ ) and evaporated under reduced pressure. Products were isolated by HPLC in silica gel: PrepPak cartridge, 25 × 100 mm, Prep Nova-Pak® HR Silica, 6 μm loaded, solution B gradient in solution A (from 0% to 15% for 25 minutes and from 15% to 100 % for another 25 minutes; solution A - petroleum ether / dichloromethane / pyridine - 60: 39.6: 0.4, o / o / o; solution B ethyl acetate). Fractions containing two main products (retention time 30-40 minutes) were combined, concentrated under reduced pressure, evaporated together with anhydrous toluene (2 × 40 cm ³ ) and dried in vacuum overnight. The residue was dissolved in anhydrous dichloromethane (4 cm ³ ) and precipitated by adding this solution to anhydrous petroleum ether (80 cm ³ ) with vigorous stirring. The precipitate was collected by filtration, washed with petroleum ether (2 × 20 cm ³ ) and dried under reduced pressure to obtain compound 61D (178 mg, 73%) as a white solid. δ _p (CD ₃ CN) 148.42, 147.93.

Example 72

1- (2,3-O-isopropylidene-4-C- (4-toluenesulfonyloxymethyl) -β-D-ribofuranosyl) uridine (62)

To a stirred solution of 1- (2,3-O-isopropylidene-4'-C-hydroxymethyl-β-O-ribofuranosyl) uridine (2.0 g, 6.4 mmol) (R. Youssefyeh, D.Tegg, JPH Verheyden, GHJones & JGMoffat, 1977, Tetrahedron Lett., 5, 435; GHJones, M. Taniguchi, D.Tegg & JGMoffat, 1979, J. Org. Chem., 44, 1309) in p-pyridine (28 cm ³ ) was added p- toluenesulfonyl chloride (1.46 g, 7.3 mmol) dissolved in anhydrous pyridine (10 cm ³ ) at −30 ° C. After 30 minutes, the reaction mixture was allowed to warm to room temperature and stirring was continued at room temperature for 12 hours. The reaction was stopped with methanol (4 cm ³ ), silica gel (2 g) was added and the solvent was removed under reduced pressure. The residue was chromatographed on a silica gel column using a gradient of 0-3% methanol in dichloromethane (v / v) as eluent to give nucleoside 62 as a pale red solid (1.8 g, 60%). δ _H (CDCl ₃ ) 9.80 (1H, brs, NH), 7.80 (2H, d, J 8.3, Ts), 7.46 (1H, d, J 8.1, 6-H) 7.35 (2H, d, J 8.01, Ts), 5.72 (1H, d, J 8.0, 5-H), 5.54 (1H, d, J 3.5, 1 ' -H), 5.08 (1H, dd, J 3.5, 6.4, 2'-H), 4.94 (1H, d, J 6.4, 3'-H), 4.18 ( 2H, s, 1 ”-H), 3.82-3.70 (2H, m, 5'-H), 2.45 (3H, s, Ts), 1.46, 1.29 (6H, s , CH ₃ ). δ _C (CDCl ₃ ) 163.6 (C-4), 150.4 (C-2), 145.2 (C-6), 142.9, 132.5, 129.9, 128.0 (Ts ), 114.7 (CCA), 102.6 (C-5), 94.9, 87.6, 83.9, 81.5 (C-4 ', C-1', C-3 ', C -2 '), 68.7, 63.5 (C-1``, C-5'), 26.4, 24.7 (CH ₃ ), 21.7 (Ts). FAB-MS m / z 469 [M + H] ⁺ .

Example 73

1- (4-C- (p-toluenesulfonyloxymethyl-β-D-ribofuranosyl) -uridine (63)

Nucleoside 62 (1.33 g, 2.83 mmol) was dissolved in 80% acetic acid (25 cm ³ ) and stirred at 80 ° C for 3 hours, after which the solvent was removed under reduced pressure. The residue was evaporated together with ethanol (10 cm ³ ) and purified by silica gel column chromatography using a gradient of 0-2% methanol in dichloromethane (v / v) as an eluent to give nucleoside 63 as a white solid (391 mg, 33 %). δ _H (CD ₃ OD) 7.81 (1H, d, J 8.1, 6-H), 7.77 (1H, d, J 8.4, Ts), 7.40 (2H, d, J 8.6, Ts), 5.74 (1H, d, J 6.6, 1'-H), 5.69 (1H, d, J 8.1, 5-H), 4.17-4, 33 (2H, m, 2'-H, 3'-H), 3.67-3.62 (2H, m, 1 '' - H), 3.26-3.20 (2H, m, 5 ' -H), 2.43 (3H, s, Ts). δ _C (CD ₃ OD) 166.0 (C-4), 153.0 (C-2), 146.5 (C-6), 142.5, 130.9, 129.15 (Ts), 103 , 1 (C-5), 89.0, 87.2 (C-1 ', C-4'), 75.1, 72.7, 71.3, 63.8 (C-1 '', C -3 ', C-2', C-5 '), 21.6 (Ts).

Example 74

(1S, 3R, 4R, 7S) -7-hydroxy-1-hydroxymethyl-3- (uracil-1-yl) -2,5-dioxabicyclo [2.2.1] heptane (44)

To a stirred solution of nucleoside 63 (64 mg, 0.14 mmol) in anhydrous DMF (2 cm ³ ) was slowly added a hybrid of sodium (8.4 mg, 21 mol, 60% suspension in mineral oil, w / v) in anhydrous DMF (2 cm ³ ) at 0 ° C. The reaction mixture was then heated to 120 ° C. for 6 hours. After stopping the reaction with water (2 cm ³ ), the solvents were removed under reduced pressure, and the residue was subjected to chromatographic purification on a silica gel column using a gradient of 5-7% methanol in dichloromethane (v / v) to obtain nucleoside 44 as a white solid (9 mg, 29%). Nuclear magnetic resonance data are consistent with those previously given for compound 44.

Example 75

(1S, 3R, 4R, 7S) -7-acetoxy-1-acetoxymethyl-3- (thymin-1-yl) -2,5-dioxabicyclo [2.2.1] heptane (64)

To a stirred solution of nucleoside 37 (209.8 mg, 0.78 mmol) in anhydrous pyridine (2.5 cm ³ ) was added acetic anhydride (0.3 cm ³ , 3.23 mmol) and a catalytic amount of DMAP (5 mg). After stirring for 2 hours, ethanol (4 cm ³ ) was added and the resulting mixture was evaporated under reduced pressure. The residue was redissolved in dichloromethane and washed with a saturated aqueous solution of sodium hydrogencarbonate (7 cm ³ ). The organic phase was dried over Na ₂ SO ₄ and evaporated under reduced pressure. The residue was chromatographed on a silica gel column using dichloromethane / methanol (93: 3, v / v) as eluent to give compound 64 as a white solid (249 mg, 90%). δ _C (CDCl ₃ ) 169.59, 163.20, 149.50, 133.55, 110.64, 87.05, 85.38, 77.84, 71.70, 71.02, 58.60, 20.62, 20.53, 12.78. FAB-MS m / z 355 [M + H] ⁺ .

Example 76

(1S, 3R, 4R, 7S) -1-hydroxymethyl-3- (5-methyl-4-N-benzoylcytosin-1-yl) -7-hydroxy-2,5-dioxabicyclo [2.2.1] heptane (65)

To a solution of nucleoside 64 (232.7 mg, 0.66 mmol) in anhydrous acetonitrile (3 cm ³ ) was added a solution of 1,2,4-triazole (420 mg, 6.1 mmol,) and POCl ₃ (0.12 cm ³ , 1.3 mmol) in anhydrous acetonitrile (5 cm ³ ). The reaction mixture was cooled to 0 ° C. and anhydrous triethylamine (0.83 cm ³ ) was added, after which the mixture was kept at room temperature for 90 minutes. Triethylamine (0.54 cm ³ ) and water (0.14 cm ³ ) were added and the resulting reaction mixture was stirred for 10 minutes and evaporated under reduced pressure. The residue was dissolved in EtOAc and washed with a saturated aqueous solution of sodium hydrogencarbonate (2 × 9 cm ³ ) and water (9 cm ³ ). The aqueous phase was extracted with dichloromethane (3 × 20 cm ³ ). The combined organic phase was evaporated under reduced pressure and the resulting residue was redissolved in dioxane (4 cm ³ ), after which a 32% aqueous ammonia solution (0.7 cm ³ ) was added. After stirring for 3 hours, the reaction mixture was evaporated under reduced pressure and evaporated together with anhydrous pyridine. The residue was dissolved in anhydrous pyridine (3.6 cm ³ ) and benzoyl chloride (0.42 cm ³ , 3.6 mmol) was added. After stirring for 2 hours, the reaction was stopped by adding water (1 cm ³ ) and the reaction mixture was evaporated under reduced pressure. The residue was then redissolved in EtOAc and washed with water (3 × 7 cm ³ ). The organic phase was evaporated to dryness under reduced pressure and the residue was dissolved in pyridine / methanol / water (13: 6: 1, o / o / o 14 cm ³ ) at 0 ° C and a 2 M solution of sodium hydroxide in pyridine / methanol / water was added ( 13: 6: 1, o / o / o 7 cm ³ ). After stirring for 20 minutes, the reaction mixture was evaporated under reduced pressure. The residue was chromatographed on a silica gel column using dichloromethane / methanol (95: 5, v / v) as eluent to give nucleoside 65 as a yellow foam (94.6 mg, 38%). δ _H (CD ₃ OD) 8.21 (1H, br), 8.02 (1H, m), 7.84-7.9 (1H, m), 7.41-7.58 (4H, m) 5.61 (1H, s), 4.36 (1H, s), 4.10 (1H, s), 3.98 (1H, d, J 8.0), 3.94 (2H, s) 3.78 (1H, d, J 7.9); 2.11 (3H, d, J 1.0). δ _C (CD ₃ OD, selective signals) 133.66, 132.90, 130.63, 129.50, 129.28, 128.65, 90.71, 88.86, 80.57, 72.47, 70.22, 57.53, 14.01. FAB-MS m / z 374 [M + H] ⁺ .

Example 77

(1R, 3R, 4R, 7S) -1- (4,4'-dimethoxytrityloxymethyl) -3- (5-methyl-4-N-benzoylcytosin-1-yl) -7-O- (2-cyanoethoxy (diisopropylamino) phosphinoxy) 2,5-dioxabicyclo [2.2.1] heptane (66)

To a stirred solution of nucleoside 65 (82 mg, 0.22 mmol) in anhydrous pyridine (1.5 cm ³ ) was added 4,4'-dimethoxytrityl chloride (80 mg, 0.24 mmol) and stirring was continued at room temperature for 12 hours . Additionally, 4,4'-dimethoxytrityl chloride (80 mg, 0.24 mmol) was added and stirring was continued for another 12 hours. Methanol (0.5 cm ³ ) was added and the reaction mixture was evaporated under reduced pressure. The residue was chromatographed on a silica gel column using dichloromethane / methanol / pyridine (98.5: 1.0: 0.5, v / v / v. Intermediate obtained (FAB-MS m / z 676) (50.2 mg ) was evaporated with anhydrous acetonitrile and dissolved in anhydrous dichloromethane (0.62 cm ³ ) N, N-diisopropylethylamine (0.1 cm ³ ) was added followed by 2-cyanoethyl-N, N-diisopropylphosphoramidochloridite (0.3 cm ³ , 0.11 mmol). After stirring at room temperature, water was added during 3 hours (1 cm ³⁾ and the resulting mixture was diluted ethyl acetate (10 cm ^3), washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 6 cm ³⁾ and brine (3 × 6 cm ^3). The organic phase was dried over Na ₂ SO ₄ and evaporated under reduced pressure. The residue was purified by HPLC silica gel As a result of the precipitation described above in the preparation of compound 53, compound 66 was obtained as a white solid (29.5 mg, 0.03 mmol, 14%). δ _P (CH ₃ CN) 148.46, 147 , 49.

Example 78

9- (4-hydroxymethyl) -2,3-O-isopropylidene-β-D-ribofuranosyl) -6-N-benzoyladenine (67)

A mixture of oxalyl chloride (0.93 ml, 10.75 mmol) and dichloromethane (25 ml) was cooled to -70 ° C. Dimethyl sulfoxide (DMSO, 1.7 ml, 22 mmol) was added dropwise with vigorous stirring. The resulting mixture was stirred for 10 minutes at -70 ° C and a solution of 9- (2,3-O-isopropylidene-β-D-ribofuranosyl) -6-N-benzoyladenine (3.4 g, 8, 27 mmol) in dimethyl sulfoxide / dichloromethane (1: 9, v / v, 20 ml). The mixture was stirred at -60 ° C for 30 minutes. Triethylamine (7 ml, 50.3 mmol) was added and the resulting mixture was allowed to warm to room temperature. The solution was diluted with dichloromethane (50 ml) and washed with water (3 × 100 ml). The aqueous fraction was further washed with dichloromethane (100 ml). The organic phase was concentrated to an oily mass, evaporated with dioxane (50 ml) and redissolved in dioxane (30 ml). A 37% aqueous formaldehyde solution (2.95 ml, 33.4 mmol) and a 2 M aqueous sodium hydroxide solution (8.26 ml) were added, then the mixture was stirred at room temperature for 10 minutes and cooled to 0 ° C. Sodium borohydride (640 mg, 16.9 mmol) was added and the reaction mixture was allowed to warm to room temperature for 15 minutes. The reaction was stopped by the addition of acetic acid (5 ml) and dichloromethane and a saturated aqueous solution of sodium hydrogencarbonate (100 ml each) were added to the mixture. The organic phase was washed with water (100 ml), concentrated in vacuo, and the obtained product was isolated by chromatography on a silica gel column (2.5 × 25 cm) using a gradient of 2-3.2% methanol in dichloromethane (v / v) as eluent. The yield of compound 67 as a white solid was 1.85 g (50.7%). δ _H (CDCl ₃ ) 8.72 (1H, s), 8.14 (1H, s), 8.03-8.00 (2H, m), 7.60-7.57 (1H, m), 7.56-7.46 (2H, m), 6.00 (1H, d, J 4.9), 5.35 (1H, dd, J'5.8, J''5.0), 5 13 (1H, d, J 5.8), 3.87-3.78 (4H, m), 1.65 (3H, s), 1.38 (3H, s). δ _C (CDCl ₃ ) 164.8, 152.2, 150.4, 150.2, 142.6, 133.3, 132.9, 128.8, 128.0 124.1, 114.7, 93 3, 90.2, 83.8, 82.5, 65.3, 62.9, 27.3, 25.1. FAB-MS m / z 442 [M + H] ⁺ , 464 [M + Na] ⁺ .

Alternative Synthesis of Compound 67

To a solution of 2 ’, 3’-O-isopropylidenadenosine (15 g) in anhydrous pyridine (250 ml) was added trimethylsilyl chloride (15.5 ml). The reaction mixture was stirred at room temperature for 20 minutes and cooled to 0 ° C. Benzoyl chloride (17 ml) was added dropwise and the resulting mixture was kept at room temperature for 2-3 hours. Water (50 ml) and a 25% aqueous solution of ammonium hydroxide (100 ml) were added and stirring was continued for 3 hours. The mixture was then concentrated under reduced pressure, evaporated with toluene (2 × 200 ml) and dichloromethane and a saturated aqueous solution of sodium hydrogencarbonate was added. The organic phase was evaporated to dryness to give a yellow solid. Recrystallization from ethanol gave the intermediate compound as a white solid (12.5 g, ≈ 80%). Oxalyl chloride (4.68 ml) in dry dichloromethane (120 ml) was cooled to -70 ° C. DMSO (8.5 ml) was added with vigorous stirring. Later (after 10 minutes), a solution of the intermediate compound (17 g) in 10% DMSO / dichloromethane (100 ml) was added dropwise over 20 minutes, the synthesis of which was described above. The mixture was allowed to rise to -50 ° C for a period of 30 minutes after the reaction was stopped by the addition of triethylamine (35 ml). Dichloromethane (200 ml) was added to the reaction mixture, which was washed with water (3 × 200 ml). The intermediate was concentrated in vacuo, evaporated with dioxane and redissolved in dioxane (120 ml). Formaldehyde (37%) and a 2 M aqueous solution of sodium hydroxide (40 ml) were added and the reaction mixture was stirred for 1 hour. The mixture was neutralized with acetic acid (6 ml) and dichloromethane (400 ml) and a saturated aqueous solution of sodium hydrogencarbonate (400 ml) were added. The organic phase was concentrated. Compound 67 was purified by chromatography on silica gel in a gradient of 1.5-5.0% methanol in dichloromethane. The yield of compound 67 was 8.5 (46%). The data were consistent with what was previously defined in this example.

Example 79

9- (2,3-O-isopropylidene-4- (p-toluenesulfonyloxymethyl) -β-D-ribofuranosyl) -6-N-benzoyladenine (68) and 9- (4-hydroxymethyl-2,3-O-isopropylidene 5-O- (p-toluenesulfonyl) -β-D-ribofuranosyl) -6-N-benzoyladenine

A mixture of compound 67 (1.95 g, 4.42 mmol) and p-toluenesulfonyl chloride (1.26 g, 6.63 mmol) was dissolved in 10 ml of anhydrous pyridine at 0 ° C. The reaction mixture was stirred for 4 hours and then diluted dichloromethane (100 ml), washed with water (2 × 100 ml) and concentrated under reduced pressure. The mixture was purified on a chromatographic silica gel column using a gradient (1-4%) of methanol in dichloromethane, which made it possible to isolate the starting material 67 (360 mg, 18.5%) and two structural isomers, namely 68 (less polar isomer; 971 mg , 36.7%) and 9- (4-hydroxymethyl-2,3-O-isopropylidene-5-O- (4'-toluenesulfonyl) -β-D-ribofuranosyl-N ^6- benzoyladenine (more polar isomer; 352 mg , 13.1) as a white solid. For 68: δ _H (CDCl ₃ ) 8.69 (1H, s), 8.11 (1H, s), 8.00 (2H, m), 7, 79 (2H, m), 7.58-7.55 (1H, m), 7.50-7.46 (2H, m), 7.34-7.32 (2H, m), 5.88 ( 1H, d, J 4.9), 5.35 (1H, dd, J'5.8, J''5.0), 5.13 (1H, d, J 5.8), 3.87- 3,7 8 (4H, m), 1.65 (3H, s), 1.38 (3H, s). Δ _C (CDCl ₃ ) 164.7, 152.0, 150.2, 150.1, 144.9 , 142.5, 133.2, 132.7, 132.3, 129.6, 128.6, 127.9, 127.8, 123.9, 114.6, 93.1, 87.9, 83 4, 81.6, 68.3, 64.4, 27.1, 25.0, 21.5. FAB-MS: m / z 596 [M + H] ⁺ .

Example 80

9- (4- (p-toluenesulfonyloxymethyl) -β-D-ribofuranosyl) -6-N-benzoyladenine (69)

A solution of compound 68 (940 mg, 1.58 mmol) in 10 ml of 90% aqueous trifluoroacetic acid was kept at room temperature for 30 minutes and concentrated in vacuo to an oily mass. After evaporation together with methanol (2 × 20 ml) and toluene (20 ml), the mixture was purified on a chromatographic silica gel column (2 × 25 cm) in a gradient of methanol (2-5%) in dichloromethane to give the compound. 69 (825 mg, 94%) as a white solid. δ _H (methanol-d ₄ ) 8.67 (1H, s), 8.53 (1H, s), 8.05 (2H, d, J 7.7), 7.75 (2H, d, J 8 , 2), 7.63 (1H, m), 7.53 (2H, m), 7.32 (2H, d, J 8.0), 5.94 (1H, d, J 7.1), 4.92 (1H, dd, J'7.1, J''5.3), 4.41 (1H, d, J 5.1), 4.38 (1H, d, J 10.2), 4.28 (1H, d, J 10.2), 3.80 (1H, d, J 12.0), 3.68 (1H, d, J 12.0), 2.35 (3H, s) . δ _C (methanol-d ₄ ) 168.2, 152.9, 150.8, 151.2, 146.4, 144.9, 134.7, 134.1, 134.0, 130.8, 129, 7, 129.4, 129.1, 125.1, 90.0, 88.4, 75.3, 73.1, 71.1, 64.2, 21.6. FAB-MS: m / z 556 [M + H] ⁺ .

Example 81

9- (4-p-toluenesulfonyloxymethyl) -3,5-O- (tetraisopropyl disiloxane-1,3-diyl) -β-D-ribofuranosyl) -6-N-benzoyladenine (70)

To a solution of compound 69 (780 mg, 1.40 mmol) in anhydrous pyridine (7 ml) was added 1,3-dichloro-1,1,3,3-tetraisopropyl disiloxane (500 ml, 1.57 mmol) at 0 ° C. After stirring for 2 hours at 0 ° C., additional 1,3-dichloro-1,1,3,3-tetraisopropyl disiloxane (50 ml, 0.16 mmol) was added. The reaction mixture was allowed to warm to room temperature, diluted with dichloromethane (100 ml) and washed with water (2 × 100 ml). The organic phase was concentrated and the residue was purified by preparative HPLC (PrepPak cartridge, Porasil, 15-20 μm, 125 A; eluent - 0-3% methanol in dichloromethane (v / v) for 120 minutes; flow rate 60 ml / min). Concentration in vacuo gave 870 mg (78%) of compound 70 as a white solid. δ _H (CDCl ₃ ) 8.65 (1H, s), 8.03 (2H, m), 8.00 (1H, s), 7.83 (2H, d, J 8.4), 7.58 (1H, m), 7.49 (2H, m), 7.34 (2H, d, J 8.4), 5.87 (1H, s), 5.70 (1H, d, J 6.2 ), 4.68 (1H, d, J 6.2), 4.59 (1H, d, J 10.8), 4.31 (1H, d, J 11.0), 3.91 (2H, s), 2.45 (3H, s), 1.03-0.84 (28H, m). δ _C (CDCl ₃ ) 164.9, 152.2, 150.5, 150.0, 144.7, 142.9, 133.5, 132.9, 132.8, 129.7, 128.8, 128.1, 128.0, 123.6, 90.3, 85.3, 76.0, 75.0, 68.7, 65.2, 21.6, 17.5, 17.4, 17, 2, 17.1, 17.0, 16.9, 13.1, 13.0, 12.5, 12.4. FAB-MS: m / z 798 [M + H] ⁺ .

Example 82

9- (2-O, 4-C-methylene-3,5-O- (tetraisopropyl disiloxa-1,3-diyl) -β-D-ribofuranosyl) -6-N-benzoyladenine (71)

A solution of compound 70 (540 mg, 0.68 mmol) in anhydrous THF (20 ml) was cooled to 0 ° C and sodium hydride (130 mg of a 60% suspension in mineral oil, 3.25 mmol) was added with stirring. The reaction mixture was stirred for 30 minutes and then neutralized by the addition of 750 μl of acetic acid. Dichloromethane (50 ml) was added, the reaction mixture was washed with a saturated aqueous solution of sodium hydrogencarbonate (2 × 50 ml) and concentrated under reduced pressure. The residue was loaded onto a silica gel column (2.5 × 25 cm) and the product was eluted using a gradient of methanol (0.5-1.2%) in dichloromethane as an eluent to give compound 71 (356 mg, 84%) as a foam white color. δ _H (CDCl ₃ ) 8.77 (1H, s), 8.28 (1H, s), 8.03 (2H, m), 7.59 (1H, m), 7.50 (2H, m) 6.08 (1H, s), 4.86 (1H, s), 4.56 (1H, s), 4.14 (1H, d, J 13.2), 4.06 (1H, d, J 7.7), 3.97 (1H, d, J 13.2), 3.89 (1H, d, J 7.7), 1.12-0.95 (28H, m). δ _C (CDCl ₃ ) 164.8, 152.6, 150.5, 149.6, 140.7, 133.6, 132.7, 128.7, 127.9, 123.1, 89.4, 86.5, 78.9, 71.7, 71.2, 56.7, 17.3, 17.1, 17.0, 16.9, 16.8, 13.3, 13.1, 12, 5, 12.2. FAB-MS: m / z 626 [M + H] ⁺ .

Example 83

7-hydroxy-1-hydroxymethyl-3- (6-N-benzoyladenin-9-yl) -2,5-dioxabicyclo [2.2.1] heptane (61B)

Triethylamine trishydrofluoride (300 ml, 1.84 mmol) was added to a solution of compound 71 (420 mg, 0.067 mmol) in anhydrous THF (7 ml). The reaction mixture was kept at room temperature for 1 hour and concentrated in an oily substance, which was purified on a chromatographic silica gel column (2 × 25 cm) using a gradient of 4-10% methanol in dichloromethane (o / o) for elution. The yield of compound 61B as a white solid was 232 mg (92%). Nuclear magnetic resonance data were identical to those obtained previously for 61V.

Example 84

1- (3,5-di-O-benzyl-4-C- (p-toluenesulfonyloxymethyl) -2-O-p-toluenesulfonyl-β-D-ribofuranosyl) thymine (72)

Solution 1- (3,5-di-O-benzyl-4-C- (hydroxymethyl) -β-D-ribofuranosyl) thymine 35 (1.48 g, 3.16 mmol), DMAP (1.344 g, 0.011 mol ) and p-toluenesulfonyl chloride (1.45 g, 7.6 mmol) in dichloromethane (20 ml) was stirred at room temperature for 3 hours. The reaction mixture was diluted with dichloromethane (30 ml) and washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 20 ml) and sodium chloride (2 × 25 ml). The organic phase was dried over Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was chromatographed on a silica gel column using methanol / dichloromethane (1:99, v / v) as eluent to give 72 nucleoside (1.95 g, 80%) as a white solid. FAB-MS m / e 776. δ _C (CDCl ₃ ) 162.9, 149.8 (C-2, C-4), 145.8, 145.2 (2 × Ts), 136.9, 136, 8 (2 × Bn), 134.3 (C-6), 132.1, 132.0, 130.0, 129.9, 129.0 128.9, 128.4, 128.3, 128.2 , 128.0, 127.7 (2 × Ts, 2 × Bn), 111.2 (C-5), 85.3, 84.0 (C-1 ', C-4'), 78.9, 78.3, 75.2, 74.3, 72.7 69.1 (C-2 ', C-3', C-5 ', C-1'', 2 × Bn), 21.7 (2 × CH ₃ ), 11.9 (CH ₃ ); Analytical calculation for C ₃₉ H ₄₀ N ₂ S ₂ O ₁₁ : C - 60.30; H - 5.19; N - 3.61. Received: C, 59.95; H, 5.11; N, 3.81.

Example 85

1- (2-benzylamino-2-deoxy-3,5-di-O-benzyl-2-N, 4-C-methylene-β-D-ribofuranosyl) thymine (73)

A solution of compound 72 (8.6 g, 11.1 mol) in benzylamine (10 ml) was stirred at 130 ° C. for 36 hours. The reaction mixture was directly chromatographed on silica gel using methanol / dichloromethane (1:99, v / v) as eluent to give nucleoside 73 (1.79 g, 30%) as a white solid. FAB-MS m / e 540. δ _C (CDCl ₃ ) 163.9, 149.8 (C-2, C-4), 139.2, 137.6, 137.3 (3 × Bn), 135, 6 (C-6), 128.5, 128.4, 128.3, 128.2, 128.0, 127.7, 127.0 (3 × Bn), 109.6 (C-5), 88 2, 86.3 (C-1 ', C-4'), 76.7, 73.8, 72.0, 66.0, 63.8, 57.9, 57.8 (C-2 ' , C-3 ′, C-5 ′, C-1 ″, 3 × Bn), 12.2 (CH ₃ ). Analytical calculation for C ₃₂ H ₃₃ N ₃ O ₅ · 0.5H ₂ O: C - 70.06; H, 6.25; N - 7.66. Received: C - 70.00; H - 6.06; N - 7.50;

Example 86

1- (2-amino-2-deoxy-2-N, 4-C-methylene-β-D-ribofuranosyl) thymine (74)

To a solution of nucleoside 73 (1.62 g, 0.003 mol) in ethanol (1.50 ml) was added 20% palladium hydroxide on carbon (3 g) and the resulting suspension was stirred for 5 days in a hydrogen atmosphere. The catalyst was filtered through silica gel and washed with methanol (20 ml). The combined filtrate was concentrated under reduced pressure to give a white solid, which was filtered and washed with methanol / dichloromethane (1: 4, v / v) to give a monobenzylated intermediate (0.82 g, 76%). FAB-MS m / e 360 [M + H] ⁺ . ¹³ C-NMR (DMSO-d ₆ , 250 MHz): 163.7, 149.8 (C-2, C-4), 138.2 (Bn), 134.9 (C-6), 128.2 , 127.5, 127.4 (Bn), 107.8 (C-5), 87.8, 87.6 (C-1 ', 0-4'), 72.7, 68.9, 65, 9, 61.7, 49.4 (C-2 ', C-3', C-5 ', C-1'', Bn), 11.9 (CH ₃ ). Analytical calculation for C ₁₈ H ₂₁ N ₃ O ₆ : C - 60.16; H - 5.89; N - 11.69. Received: C, 59.86; H - 5.61; N - 11.56. A mixture of these intermediates (0.1 g, 0.29 mmol), ammonium format (0.085 g, 1.35 mmol), 10% palladium on carbon (130 mg) in anhydrous methanol (7 ml) was heated under reflux for 2 hours. The catalyst was filtered through silica gel and washed with methanol (15 ml); the combined filtrate was concentrated to dryness under reduced pressure. The residue was chromatographed using methanol / dichloromethane (1: 9, v / v) to elute to give the title compound 74 (0.053 g, 71%) as a white solid. FAB-MS m / e 270.

δ _H (DMSO-d ₆ ) 11.29 (br s, 1H, NH), 7.73 (d, 1H, J 1.1, 6-H), 5.31 (s, 1H, 1'-H) 5.29 (br s, 1H, 3'-OH), 5.13 (m, 1H, 5'-OH), 3.81 (s, 1H, 3'-H), 3.69 (m, 2H 5'-H), 3.23 (s, 1H, 2'-H), 2.88 (d, 1H, J 9.8, 1``-H _a ), 2.55 (d, 1H, J 9.8, 1 '' - H _b ), 1.77 (d, 3H, J 0.8, CH ₃ ). δ _C (DMSO-d ₆ ) 164.0, 150.1 (C-2, C-4), 135.6 (C-6), 107.8 (C-5), 89.5, 87.9 (C-1 ', C-4'), 68.7, 61.9, 57.1, 49.4, (C-2 ', C-3', C-5 ', C-1'') . Analytical calculation for C ₁₁ H ₁₅ N ₃ O ₅ · 0.5H ₂ O: C - 47.48; H - 5.80; N - 15.10. Received: C, 47.54; H - 5.30; N - 14.79.

Alternative Conversion Method 73 to 74

To a solution of compound 73 (0.045 g, 0.0834 mmol) in methanol (6 ml) was added 10% palladium on carbon (0.118 g) and an ammonium format (0.145 g, 0.0023 mol) was also added in three portions over 3 hours. The suspension was heated under reflux for 4.5 hours. The catalyst was filtered through silica gel and washed with methanol (4 × 3 ml). The combined filtrate was concentrated and the residue was chromatographed on a silica gel column using methanol / dichloromethane (1: 9, v / v) to elute to give nucleoside 74 (0.015 g, 67%). The spectral analysis data were consistent with the data previously given in the example describing compound 74.

Example 87

1- (2-amino-2-deoxy-2-N, 4-C-methylene-2-N-trifluoroacetyl-β-D-ribofuranosyl) thymine (74-СОСОF ₃ )

To a suspension of nucleoside 74 (0.050 g, 0.186 mmol) in methanol (2 ml) was added DMAP (0.013 mg, 0.106 mmol) and ethyl trifluoroacetate (0.029 ml, 0.242 mmol) and the resulting mixture was stirred at room temperature for 2.5 hours. The solvent was removed under reduced pressure and the residue was chromatographed on a silica gel column using methanol / dichloromethane (2.5: 97/5, v / v) to elute to give the title nucleoside 74-COFF ₃ as a white solid after evaporation of the solvents under reduced pressure (0.055 g, 81%). FAB-MS m / z 366 [M + H] ⁺ . ¹³ C-NMR (CD ₃ OD, 62.9 MHz) δ 166.5, 157.7 (q, ² J _{C, F} 37.5 Hz, COCF ₃ ), 157.6 (q, ¹ J _{C, F} 37.2 Hz, СОСF ₃ ), 151.8, 136.8, 136.8, 117.6 (d, ¹ J _{C, F} 287.5 Hz, СF ₃ ), 117.5 (d, ² J _{C , F} 286.5 Hz, CF ₃ ), 110.8, 110.8, 90.7, 89.3, 87.7, 87.3, 70.1, 68.6, 66.2, 66.2 , 64.5, 57.9, 53.3, 12.7. Analytical calculation for C ₁₃ H ₁₄ N ₃ O ₆ F ₃ : C - 42.8; H - 3.9; N - 11.5. Received: C, 42.5; H - 4.0; N - 11.2.

Example 88

1- (2-amino-2-deoxy-5-O-4,4’-dimethoxytrityl-2-N, 4-C-methylene-2-N-trifluoroacetyl-β-D-ribofuranosyl) thymine (74-DMT)

To a solution of 74-COCF ₃ nucleoside (0.030 g, 0.082 mmol) in anhydrous pyridine (0.6 ml) at 0 ° C, 4.4'-dimethoxymethyl chloride (0.054 g, 0.159 mmol) was added dropwise, dissolved in anhydrous pyridine / dichloromethane (0.6 ml, 1: 1, v / v) and the resulting mixture was stirred for 10 hours at room temperature. A mixture of ice and water (5 ml) was added, and the resulting mixture was extracted with dichloromethane (3 × 5 ml). The combined organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 2 ml), dried over Na ₂ SO ₄ and filtered. The filtrate was evaporated to dryness under reduced pressure, and the residue was chromatographed on silica gel using methanol / dichloromethane / pyridine (1.5: 98.0: 0.5, v / v) as eluent to give 74-DMT nucleoside as a solid white after evaporation of the solvents under reduced pressure (0.051 g, 93%). FAB-MS m / z 667 [M] ⁺ , 668 [M + H] ⁺ . FAB-HRMS, calculation for C ₃₄ H ₃₂ N ₃ O ₈ F $\genfrac{}{}{0ex}{}{\text{+}}{\text{3}}$ : 667.2142. Received: 667.2146. ¹³ C-NMR (C ₅ D ₅ N, 100.6 MHz) δ 165.1, 165.0, 159.5, 159.5, 151.4, 145.7, 136.3, 136.1, 134 , 8, 134.6, 130.9, 130.9, 130.9, 128.9, 128.9, 128.7, 128.7, 128.4, 127.7, 123.2, 114.1 , 114.1, 114.0, 110.4, 89.4, 87.9, 87.5, 87.4, 87.2, 70.8, 69.0, 66.0, 64.4, 60 5, 60.2, 55.5, 53.6, 53.4, 49.9, 13.2, 13.1.

Example 89

1- (2-amino-3-O- (2-cyanoethoxy (diisopropylamino) phosphino-2-deoxy) -5-O-4,4'-dimethoxytrityl-2-N, 4-C-methylene-2-N- trifluoroacetyl-β-D-ribofuranosyl) thymine (74A)

To a solution of 74-DMT nucleoside (0.121 g, 0.181 mmol) in anhydrous dichloromethane (2 ml) was added N, N-diisopropylethylamine (0.093 ml, 0.54 mmol) and 2-cyanoethyl-N, N-diisopropylphosphoramidochloridite (0.057 ml, 0 , 26 mmol) at 0 ° C and the resulting mixture was stirred at room temperature for 10 hours. The mixture was diluted with dichloromethane (20 ml), extracted with a saturated aqueous solution of sodium hydrogencarbonate (3 × 10 ml), dried over N ₂ SO ₄ and filtered. The filtrate was evaporated to dryness under reduced pressure and the residue was chromatographed on silica gel using methanol / dichloromethane / pyridine (1.5: 98.0: 0.5, o / o / o) as eluent to give a crude product (0.107 g) after evaporation of the solvents under reduced pressure. The residue was dissolved in anhydrous dichloromethane (1 ml) and was added dropwise with vigorous stirring in petroleum ether (60-80 ° C, 30 ml) at -30 ° C, nucleotide 74A precipitated to obtain a white solid after filtration (0.090 g, 57%). FAB-MS m / z 868 [M + H] ⁺ , 890 [M + Na] ⁺ . ³¹ P-NMR (CD ₃ CN, 121.5 MHz) δ 150.4, 150.2, 148.8, 149.1.

Example 90

1- (2-amino-2-N, 4-C-methylene-3,5-O- (tetraisopropyl disiloxane-1,3-diyl) -β-D-ribofuranosyl) thymine (74B)

To a solution of nucleoside 74 (0.20 g, 0.74 mmol) in anhydrous pyridine (3 ml) at -15 ° C was added dropwise (over 3 hours) 1,3-dichloro-1,1,3,3- tetraisopropyl disiloxane (0.305 ml, 0.0011 mol) and the resulting mixture was stirred at room temperature for 10 hours. MeOH (3 ml) was added and the mixture was evaporated to dryness under reduced pressure. The residue was chromatographed on silica gel using methanol / dichloromethane (1:99, v / v) as eluent to give 74B nucleoside as a white solid after evaporation of the solvents under reduced pressure (0.370 mg, 97%). FAB-MS m / z 512 [M + H] ⁺ . ¹³ H-NMR ((CD ₃ ) ₂ SO, 400 MHz) δ 11.37 (br, 1H), 7.48 (s, 1H), 5.32 (s, 1H), 4.06 (d, 1H) , J 13.5 Hz), 4.00 (s, 1H), 3.84 (d, 1H, J 13.5 Hz), 3.41 (s, 1H), 2.92 (d, 1H, J 10.2 Hz), 2.64 (d, 1H, J 10.2 Hz), 1.74 (s, 3H), 1.10-0.92 (m, 28H). ¹³ C-NMR ((CD) ₃ SO ₂ , 62.9 MHz) 6 163.8, 149.8, 134.1, 107.9, 89.5, 87.9, 70.1, 61.1, 57.9, 49.3, 17.2, 17.2, 17.0, 16.9, 16.8, 16.7, 12.6, 12.2, 11.7. Analytical calculation for С ₂₃ Н ₄₁ N ₃ О ₆ Si ₂ : С - 54.0; H - 8.1; N - 8.2. Received: C, 54.0; H - 8.3; N - 7.8.

Example 91

1- (2-deoxy-2-methylamino-2-N, 4-C-methylene-3,5-O- (tetra-isopropyldisiloxane-1,3-diyl) -β-D-ribofuranosyl) thymine (74C)

To a solution of nucleoside 74B (0.33 g, 0.64 mmol) in anhydrous THF / dichloromethane (4: 1, v / v) at -10 ° C, 1.8-diazabicyclo was added dropwise (over 30 minutes) [5.4 .0] undecene-7 (DBU, 0.125 ml, 0.836 mmol) and methyl iodide (0.05 ml, 0.79 mmol) and the resulting mixture was stirred for 48 hours at 10 ° C. Also dropwise (within 15 minutes) DBU (0.05 ml, 0.33 mmol) and methyl iodide (0.02 ml, 0.32 mmol) were added to the reaction mixture and stirred for 24 hours at 10 ° C. The mixture was evaporated to dryness under reduced pressure and the residue was subjected chromatographic purification in silica gel using Hovhan eluent methanol / dichloromethane (1:99, v / v) to give nucleoside 74C as a white solid after evaporation of solvents (0.25 g, 74%). FAB-MS m / z 526 [M + H] ⁺ . ¹ H-NMR (CDCl ₃ , 400 MHz) δ 8.19 (br, 1H), 7.65 (d, 1H, J 1.3 Hz), 5.59 (s, 1H), 4.11 (s , 1H), 4.05 (d, 1H, J 13.2 Hz), 3.87 (d, 1H, J 13.2 Hz), 3.44 (s, 1H), 2.98 (d, 1H , J 9.5 Hz), 2.71 (d, 1H, J 9.5 Hz), 2.72 (s, 3H), 1.91 (d, 1H, J 1.1 Hz), 1.12 -0.96 (m, 28H), ¹³ C-NMR (CDCl ₃ , 62.9 MHz) δ 163.7, 149.6, 135.2, 109.7, 90.9, 85.7, 71, 4, 67.3, 58.6, 58.2, 41.2, 17.5, 17.4, 17.3, 17.2, 17.1, 16.9, 13.3, 13.1, 13.0, 12.6, 12.1. Analytical calculation for C ₂₄ H ₄₄ N ₃ O ₆ Si ₂ · 0.25H ₂ O: C - 54.4; H - 8.3; N - 7.9. Received: C, 54.4; H - 8.1; N - 7.7.

Example 92

1- (2-deoxy-2-methylamino-2-N, 4-C-methylene-β-D-ribofuranosyl) thymine (74D)

To a solution of nucleoside 74C (0.40 g, 0.76 mmol) in THF at room temperature was added a solution of tetrabutylammonium fluoride in THF (1.0 M, 2.2 ml, 2.2 mmol) and the resulting reaction mixture was stirred for 20 minutes, after which a mixture of pyridine, water and methanol (6 ml, 3: 1: 1, v / v / v) was added. The reaction mixture was added to Dowex 50 × 200 resin (2.2 g, H ⁺ (pyridinium) form, 100-200 mesh) suspended in pyridine / water / methanol (6 ml, 3: 1: 1, v / v) and the resulting mixture was stirred for 20 minutes. After filtration, the residue was washed in pyridine / water / methanol (3 × 3 ml, 3: 1: 1, v / v) and the combined filtrate was evaporated to dryness under reduced pressure to give an oily residue after coevaporation with methanol (2 × 5 ml). As a result of chromatographic purification on a silica gel column using methanol / dichloromethane (1:49, v / v) as the eluent, nucleoside 74D was obtained as a white solid after evaporation of the solvents under reduced pressure (0.17 g, 79%). FAB-MS m / z 284 [M + H] ⁺ . FAB-HRMS, calculation for C ₁₂ H ₁₈ N ₃ O $\genfrac{}{}{0ex}{}{\text{+}}{\text{5}}$ : 284.12465. Received: 284.12402. ¹ H-NMR ((CD ₃ ) ₂ SO, 400 MHz) δ 11.3 (br, 1H, NH), 7.70 (d, 1H, J 1.1 Hz, 6-H), 5.50 ( s, 1H, 1'-H), 5.26 (d, 1H, J 4.9 Hz, 3'-OH), 5.12 (t, 1H, J 5.7 Hz, 5'-OH), 3.87 (d, 1H, J 4.8 Hz, 3'-H), 3.67 (d, 2H, J 5.5 Hz, 5'-H), 3.12 (s, 1H, 2 ' -H), 2.87 (d, 1H, J 9.3 Hz, 5 '' - H _a ), 2.56 (s, 3H, NCH ₃ ), 2.52-2.49 (1H, m, 5 '' - H _b ), 1.77 (s, 3H, CH ₃ ). ¹ H-NMR (CD ₃ OD, 400 MHz) δ 7.80 (d, 1H, J 1.3 Hz, 6-H), 5.71 (s, 1H, 1'-H), 4.07 ( s, 1H, 3'-H), 3.83 (s, 2H, 5'-H), 3.36 (s, 1H, 2'-H), 3.08 (d, 1H, J 9.9 Hz, 5 '' - H _a ), 2.68 (s, 3H, NCH ₃ ), 2.57 (d, 1H, J 9.9 Hz, 5 '' - H _b ), 1.88 (d, 3H, J 1.1 Hz, CH ₃ ). ¹³ C-NMR (CD ₃ OD, 62.9 MHz) δ 166.6, 151.9, 137.4, 110.4, 91.3, 85.2, 71.4, 69.1, 59.4 58.7, 40.2, 12.2.

Example 93

1- (2-deoxy-5-O-4,4’-dimethoxytrityl-2-methylamino-2-N, 4-C-methylene-β-D-ribofuranosyl) thymine (74E)

To a solution of nucleoside 74D (0.135 g, 0.477 mmol) in anhydrous pyridine (1.5 ml) at 0 ° C., a solution of 4,4'-dimethoxytrityl chloride (0.238 g, 0.702 mmol) in anhydrous pyridine was added dropwise. / dichloromethane (1.0 ml, 1: 1, v / v) and the resulting reaction mixture was stirred for 10 hours at room temperature. A mixture of water and ice (5 ml) was added and the resulting mixture was extracted with dichloromethane (3 × 10 ml). The combined organic phase was washed with saturated aqueous sodium bicarbonate (3 × 5 ml), dried over N ₂ SO ₄ and filtered. The filtrate was evaporated to dryness and the residue was chromatographed on a silica gel column using methanol / dichloromethane / pyridine (1: 98: 1, v / v / v) as eluent to give 74E nucleoside as a white solid after evaporation of the solvents under reduced pressure (0.20 g, 72%). FAB-MS m / z 586 [M + H] ⁺ . ¹ H-NMR (C ₅ D ₅ N, 400 MHz) δ 13.2 (br, 1H), 7.98 (d, 1H, J 1.3 Hz), 7.98-7.00 (m, 13H ), 6.12 (s, 1H), 4.78 (d, 1H, J 3.7 Hz), 3.88-3.79 (m, 4H), 3.71 (s, 3H), 3, 71 (s, 3H), 3.29 (d, 1H, J 9.3 Hz), 2.84 (d, 1H, J 9.3 Hz), 2.81 (s, 3H), 1.85 ( d, 3H, J 0.9 Hz). ¹³ C-NMR (C ₅ D ₅ N, 62.9 MHz) δ 165.1, 159.2, 151.4, 145.9, 136.5, 136.4, 130.8, 130.7, 128 , 7, 128.4, 127.4, 113.8, 109.6, 89.8, 86.8, 85.1, 72.0, 68.7, 60.9, 59.4, 55.2 , 40.1, 13.1. Analytical calculation for C ₃₃ H ₃₅ N ₃ O ₇ · 0.25H ₂ O: C, 67.2; H, 6.1; N is 7.1. Received: C, 67.2; H, 6.2; N, 6.9.

Example 94

1- (3-O- (2-cyanoethoxy (diisopropylamino) phosphino) -5-O-4,4'-dimethoxytrityl-2-methylamino-2-N, 4-C-methylene-2-deoxy-β-D- ribofuranosyl) thymine (74F)

To a solution of nucleoside 74E (0.130 g, 0.222 mmol) in anhydrous dichloromethane (2 ml) at 0 ° C was added N, N-diisopropylethylamine (0.088 ml, 0.514 mmol) and 2-cyanoethyl-N, N-diisopropylphosphoramidochloridite (0.065 ml, 0.291 mmol) and the resulting mixture was stirred for 10 hours at room temperature. Dichloromethane (30 ml) was added and the reaction mixture was extracted with a saturated aqueous solution of sodium hydrogencarbonate (3 × 10 ml), dried over Na ₂ SO ₄ and filtered. The filtrate was evaporated to dryness and the residue was chromatographed on a silica gel column using methanol / dichloromethane / pyridine (0.5: 98.5: 1.0, o / o / o) as eluent to give a crude product (0.120 g) after evaporation solvents under reduced pressure. The residue was dissolved in anhydrous dichloromethane (1 ml) and, with vigorous stirring, dropwise adding petroleum ether (60-80 ° C, 30 ml) at -30 ° C, nucleotide 74F was precipitated to give a white solid after filtration (0.090 g, 52%). ³¹ P-NMR (CD ₃ CN, 121.5 MHz) δ 147.7.

Example 95

1- (3,5-di-O-benzyl-4-C- (p-toluenesulfonyloxymethyl) -2-O-p-toluenesulfonyl-β-D-ribofuranosyl) uracil (75)

To a stirred solution of 1- (3,5-di-O-benzyl-4-C-hydroxymethyl-β-D-ribofuranosyl) uracil 41 (3.55 g, 7.81 mmol) in dichloromethane (50 cm ³ ) was added DMAP (3.82 g) and p-toluenesulfonyl chloride (4.47 g, 23.5 mmol) at room temperature. The mixture was stirred for 2 hours and dichloromethane (100 cm ³ ) was added. The reaction mixture was washed in a saturated aqueous solution of sodium hydrogencarbonate (2 × 75 cm ³ ) and dried over Na ₂ SO ₄ . The organic phase was evaporated under reduced pressure and chromatographed on a silica gel column using dichloromethane / methanol (99.5: 0.5, v / v) as eluent to give nucleoside 75 (4.65 g, 78%) as a solid white matter. δ _H (CDCl ₃ ) 8.49 (1H, brs, NH), 7.67 (1H, d, J 8.3, 6-H), 7.51-7.03 (18H, m, Bn, Ts ), 6.0 (1H, d, J 7.6, 1'-H), 5.05 (1H, m, 2'-H), 4.91 (2H, m, 5-H, Bn), 4.56 (2H, m, Bn), 4.42 (1H, d, J 10.4, Bn), 4.31 (1H, d, J 4.9, 3'-H), 4.05 ( 2H, m, 1 '' - H), 3.75-3.64 (2H, m, 5 '' - H), 2.41 (3H, s, CH ₃ ), 2.34 (3H, s, CH ₃ ). δ _C (CDCl ₃ ) 162.2 (C-4), 149.5 (C-2), 146.0, 145.3 (Ts), 139.0 (C-6), 136.7, 131, 9, 130.0, 129.9, 128.9, 128.7, 128.5, 128.4, 128.3, 128.2, 128.0, 127.6 (Bn, Ts) 102.7 ( C-5), 85.5 (1'-C), 84.4 (4'-C), 79.2, 78.3, 75.1, 74.3, 72.4, 69.1 (Bn , 3'-C, 2'-C, 5'-C, 1 '' - C), 21.7, 21.6 (Ts). FAB-MS m / z 763. Received: C, 61.2; H - 4.4; N is 3.3. For C ₃₈ H ₃₈ N ₂ O ₁₁ S ₂ calculated: C - 59.8; H - 5.0; N is 3.6.

Example 96

1- (2-deoxy-3,5-di-O-benzyl-2S, 4-C-methylene-2-mercapto-β-D-ribofuranosyl) thymine (76)

To a stirred solution of nucleoside 75 (3.70 g, 4.86 mmol) in DMF (40 cm ³ ) was added potassium thioacetate (0.83 g, 7.28 mmol). The resulting mixture was stirred and heated to 110 ° C for 80 hours. After evaporation under reduced pressure, water (100 cm ³ ) was added. Extraction was carried out with dichloromethane (4 × 50 cm ³ ) and the combined organic phase was dried over Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was chromatographed on silica gel using dichloromethane / methanol (99.6: 0.4, v / v) as eluent to give nucleoside 76 (1.65 g, 75%) as a white solid. δ _H (CDCl ₃ ) 9.08 (1H, brs, NH), 7.98 (1H, d, J 8.1, 6-H), 7.39-7.20 (10H, m, Bn), 5.85 (1H, s, 1'-H), 5.26 (1H, d, J 8.1, 5-H), 4.61 (1H, d, J 11.4, 5'-H) 4.56 (2H, s, Bn), 4.45 (1H, d, J 11.4, Bn), 4.14 (1H, d, J 1.7, 3'-H), 3.82 (2H, m, Bn), 3.72 (1H, d, J 1.9, 2'-H), 3.02 (1H, d, J 9.9, 1 '' - H _a ), 2, 78 (1H, d, J 9.9, 1 '' - H _b ). δ _C (CDCl ₃ ) 163.4 (C-4), 150.0 (C-2), 139.9 (C-6), 137.2, 136.8, 128.6, 128.5, 128 2, 127.9, 127.7 (Bn), 100.8 (C-5), 90.8, 88.8 (C-1 ', C-4'), 76.5, 73.8, 72.0, 70.0 (2 × Bn, C-3 ', C-5'), 49.52 (C-2 '), 35.63 (C-1''). FAB-MS m / z 453. Received: C, 63.4; H - 5.1; N - 5.9. For C ₂₄ H ₂₄ N ₂ O ₂ S calculated: C - 63.7; H - 5.3; N, 6.1.

Example 97

1- (2-O-p-toluenesulfonyl-4-C- (p-toluenesulfonyloxymethyl) -β-D-ribofuranosyl) uracil (76A)

To a solution of compound 75 (0.80 g, 1.0 mmol) in absolute ethanol (2 cm ³ ) was added 20% palladium hydroxide on carbon (0.80 g) and the resulting mixture was degassed several times with hydrogen and stirring was continued in a hydrogen atmosphere in within 48 hours. The catalyst was filtered off and the resulting filtrate was evaporated under reduced pressure. The residue was chromatographed on a silica gel column using dichloromethane / methanol (99: 1, v / v) as eluent to give nucleoside 76A (0.30 g, 49%) as a white solid. δ _H (CD ₃ OD) 7.67 (4H, m), 7.45 (1H, d, J 8.2 Hz), 7.34 (4H, m), 5.86 (1H, d, J 8 , 0 Hz), 5.40 (1H, d, J 8.1 Hz), 4.95 (1H, m), 4.35 (1H, d, J 5.0 Hz), 4.17 (2H, m), 3.61 (2H, s), 2.40 (6H, s). δ _C (CD ₃ OD) 165.4, 151.6, 147.5, 146.6, 141.3, 134.0, 133.8, 131.4, 130.9, 129.2, 128.9 , 103.7, 88.0, 85.4, 80.7, 72.4, 71.0, 64.3, 21.7, 21.6. FAB-MS m / z 583 [M + H] ⁺ .

Example 98

1- (3,5-O- (tetraisopropyl disiloxa-1,3-diyl) -2-O-p-toluenesulfonyl-4-C- (p-toluenesulfonyloxymethyl) -β-D-ribofuranosyl) uracil (76B)

To a stirred solution of nucleoside 76A (0.27 g, 0.46 mmol) in anhydrous pyridine (4 cm ³ ) was added 1,3-dichloro-1,1,3,3-tetraisopropyl disiloxane (0.22 cm ³ , 0.70 mmol). After 48 hours of stirring, the reaction mixture was cooled to 0 ° C. and a saturated aqueous solution of sodium hydrogencarbonate (15 cm ³ ) was added. The mixture was extracted with dichloromethane (3 × 10 cm ³ ) and the combined organic phase was dried over N ₂ SO ₄ and filtered. The solvents were evaporated under reduced pressure and the residue was chromatographed on silica gel using dichloromethane / methanol (99.5: 0.5, v / v) as eluent to give 76B nucleoside (0.37 g, 97%) as a solid white color. δ _H (CDCl ₃ ) 8.70 (1H, br), 7.80 (4H, m), 7.36 (4H, m), 6.98 (1H, d, J 8.1 Hz), 5, 64 (1H, d, J 8.0 Hz), 5.18 (2H, m), 4.98 (1H, d, J 7.0 Hz), 4.39-4.32 (2H, m), 3.92-3.76 (2H, s), 2.45 (6H, s), 1.27-0.66 (28H, m). δ _C (CDCl ₃ ) 162.9, 149.3, 145.6, 144.8, 143.9, 132.9, 130.1, 129.9, 128.2, 128.1, 102.2, 94.6, 84.7, 80.4, 72.8, 67.8, 64.6, 21.7, 17.3, 17.2, 17.1, 16.9, 16.8, 13, 1, 12.8, 12.3. FAB-MS m / z 825 [M + H] ⁺ .

Example 99

1- (2-deoxy-2-mercapto-2-S, 4-C-methylene-3,5-O- (tetraisopropyl disiloxa-1,3-diyl) -β-D-ribofuranosyl) uracil (76C)

To a stirred solution of nucleoside 76B (0.26 g, 0.32 mmol) in DMF (5 cm ³ ) was added potassium thioacetate (0.054 g, 0.47 mmol). The resulting reaction mixture was stirred at 110 ° C for 20 hours. After the reaction mixture was evaporated under reduced pressure, water (20 cm ³ ) was added. Extraction was carried out with dichloromethane (3 × 10 cm ³ ) and the combined organic phase was dried over Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was chromatographed on silica gel using dichloromethane / methanol (99.25: 0.75, v / v) as eluent to give 76C nucleoside (0.125 g, 77%) as a white solid. δ _H (CDCl ₃ ) 8.55 (1H, bs), 8.02 (1H, d, J 8.1 Hz), 5.82 (1H, s, 1'-H), 5.65 (1H, d, J 8.1 Hz), 4.37 (1H, d, J 2.1 Hz), 4.10 (1H, d, J 13.2 Hz), 3.90 (1H, d, J 13, 1 Hz), 3.53 (1H, d, J 2.1 Hz), 2.92 (1H, d, J 10.1 Hz), 2.74 (1H, d, J 10.0 Hz), 1 30-0.80 (28H, m). δ _C (CDCl ₃ ) 163.2, 149.8, 139.6, 100.9, 91.4, 90.7, 71.5, 59.8, 51.5, 34.4, 17.5, 17.3, 17.1, 16.9, 15.5, 13.6, 13.3, 13.1, 12.9, 12.3. FAB-MS m / z 515 [M + H] ⁺ .

Example 100

1- (2-deoxy-2-mercapto-2-S, 4-C-methylene-β-D-ribofuranosyl) uracil (76D)

To a stirred solution of 76C nucleoside (25 mg, 0.049 mmol) in THF (1.0 cm ³ ) was added a solution of tetrabutylammonium fluoride (0.20 cm ³ in a 1 M solution of THF, 0.20 mmol) at 0 ° C. After stirring the reaction the mixture at 0 ° C over 1 hour was added water (5 cm ³ ) and the mixture was evaporated. The residue was chromatographed on silica gel using dichloromethane / methanol (97: 3, v / v) as eluent to give 76D nucleoside (9.0 mg, 69%) as a white solid. δ _H (CD ₃ OD) 8.19 (1H, d, J 8.1 Hz, 6-H), 5.77 (1H, s, 1'-H), 5.65 (1H, d, J 8 , 1 Hz, 5-H), 4.31 (1H, d, J 2.1 Hz, 3'-H), 3.86 (2H, s, 5'-H), 3.53 (1H, d , J 2.2 Hz, 2'-H), 2.93 (1H, d, J 10.3 Hz, 1 '' - N _a ), 2.73 (1H, d, J 10.3 Hz, 1 '' -H _b ). δ _C (CD ₃ OD) 166.5, 152.0, 141.7, 101.2, 92.1, 92.0, 71.4, 59.9, 53.6, 35.4. FAB-MS m / z 273 [M + H] ⁺ .

Example 101

1- (2-deoxy-5-O- (4,4′-dimethoxytrityl) -2-mercapto-2-S, 4-C-methylene-β-D-ribofuranosyl) uracil (76E)

To a solution of compound 76D (0.2 g, 0.37 mmol) in anhydrous pyridine (5 cm ³ ) was added 4,4'-dimethoxytrityl chloride (0.186 g, 0.55 mmol) at room temperature. The resulting solution was stirred for 5 hours, after which the reaction mixture was cooled to 0 ° C. A saturated aqueous solution of sodium hydrogencarbonate (30 cm ³ ) was added and the resulting mixture was extracted with dichloromethane (3 × 50 cm ³ ). The combined organic phase was separated and dried over Na ₂ SO ₄ . The solvents were removed under reduced pressure and the residue was chromatographed on a silica gel column using dichloromethane / methanol / pyridine (98.5: 1.0: 0.5, o / o / o) as eluent to give nucleoside 76E as a solid brownish-white color (0.175 g, 83%). δ _C (CDCl ₃ ) 164.5, 159.4, 151.6, 145.7, 139.9, 136.4, 136.0, 135.6, 130.9, 130.8, 128.8, 128.5, 128.4, 127.5, 127.4, 122.7, 113.9, 101.5, 91.7, 90.2, 87.6, 71.8, 61.9, 55, 3, 53.7, 36.2, 30.6. FAB-MS m / z 574 [M] ⁺ , 575 [M + H] ⁺ . Received: C, 65.2; H - 5.4; N is 5.0. For C ₃₁ H ₃₀ N ₂ O ₇ S calculated: C - 64.8; H - 5.3; N - 4.9%.

Example 102

1- (3-O- (2-cyanoethoxy (diisopropylamino) phosphino) - (2-deoxy-5-O- (4,4'-dimethoxytrityl) -2-mercapto-2-S, 4-C-methylene-β -D-ribofuranosyl) uracil (76F)

To a solution of compound 76E (0.160 g, 0.28 mmol) in anhydrous dichloromethane (2 cm ³ ) at 0 ° C was added N, N-diisopropylethylamine (0.27 cm ³ ) and 2-cyanoethyl-N, N-diisopropylphosphoramidochloridite (97 mg, 0.42 mmol). Stirring was continued at room temperature for 5 hours. The reaction mixture was cooled to 0 ° C. and a saturated aqueous solution of sodium hydrogencarbonate (30 cm ³ ) was added. Extraction was carried out with dichloromethane (3 × 20 cm ³ ) and the combined organic phase was dried over Na ₂ SO ₄ and evaporated to dryness under reduced pressure. The residue was chromatographed on a silica gel column using dichloromethane / methanol / pyridine (99: 0.5: 0.5, v / v) as an eluent to give a white foam. This residue was dissolved in dichloromethane (2 cm ³ ) and the resulting product was precipitated from light fractions of oil distillation (100 cm ³ , cooled to -40 ° C) with vigorous stirring. The precipitate was collected by filtration and finally dried to obtain 76F nucleoside as a white solid (95 mg, 44%). δ _P (CDCl ₃ ) 148.9, 149.0.

Example 103

3,5-di-O-benzyl-1,2-O-isopropylidene-4-C- (p-toluenesulfonyloxymethyl) -β-D-ribofuranose (77)

A solution of 3,5-di-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose 31 (15.38 g, 38.4 mmol), anhydrous pyridine (20 cm ³ ) and anhydrous dichloromethane (80 ml) was stirred at -5 ° C. p-Toluenesulfonyl chloride (8.75 g, 46.0 mmol) dissolved in anhydrous dichloromethane (8 cm ³ ) was added over 15 minutes. The resulting solution was stirred at room temperature for 17 hours. The reaction was stopped with ice-cold water (200 cm ³ ). Extraction was performed with dichloromethane (5 × 150 cm ³ ) and the combined organic phase was washed with saturated aqueous sodium hydrogen carbonate (3 × 100 cm ³ ) and brine (3 × 100 cm ³ ), dried over Na ₂ SO ₄ , filtered and evaporated at reduced pressure. The residue was chromatographed on a silica gel column using dichloromethane / methanol (98.5: 1.5, v / v) as eluent to give compound 77 as a clear oily substance (17.4 g, 82%). δ _H (CDCl ₃ ) 7.79-7.19 (14H, m, Bn), 5.66 (1H, d, J 3.6, 1-H), 4.69-4.20 (8H, m , Bn, 5-H _a , 5-H _b , 3-H, 2-H), 3.53 (1H, d, J 10.3, 1'-H _a ), 3.46 (1H, d, J 10.3, 1'-H _b ), 2.40 (3H, s, CH ₃ ), 1.29 (3H, s, CH ₃ ), 1.26 (3H, s, CH ₃ ). δ _C (CDCl ₃ ) 144.6, 137.9, 137.3, 133.0, 129.8, 128.4, 128.3, 128.1, 128.0, 127.9, 127.7, 127.6 (aromatic), 113.6 (C (CH ₃ ) ₂ ), 104.2, (C-1), 84.7 (C-4), 79.0, 78.7, 73.7, 72.7, 70.7, 70.2 (Bn, C-2, C-3, C-5, C-1 '), 26.3, 26.0 (C (CH ₃ ) ₂ ), 21, 6 (CH ₃ ). FAB-MS m / z 555 [M + H] ⁺ . Received: C, 64.8; H, 6.2. For C ₃₀ H ₃₄ O ₈ S calculated: C - 64.9; H - 6.1%.

Example 104

1,2-di-O-acetyl-3,5-di-O-benzyl-4-C- (p-toluenesulfonyloxymethyl) -α, β-D-ribofuranose (78)

A solution of furanose 77 (17.4 g, 31.4 mmol) in 80% acetic acid (250 cm ³ ) was stirred at 60 ° C for 20 hours. The solvent was removed in vacuo and the residue was evaporated with toluene (3 × 20 cm ³ ). The residue was redissolved in anhydrous pyridine (100 cm ³ ). Acetic anhydride was added and the resulting solution was stirred at room temperature for 15 hours. The reaction was stopped by adding a mixture of ice and water (200 cm ³ ) and the reaction mixture was extracted with dichloromethane (4 × 150 cm ³ ). The combined organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (2 × 125 cm ³ ) and brine (3 × 150 cm ³ ), dried over Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was chromatographed on a silica gel column using dichloromethane / methanol (98.5: 1.5, v / v) as eluent to give compound 78 (ratio α: β ≈ 1: 1 as a clear oily substance (13.5 g, 72%). δ _C (CDCl ₃ ) 169.8, 169.6, 69.4, 168.8 (C = O), 144.7, 137.7, 137.5, 132.8, 129 7, 129.6, 128.5, 128.4, 128.3, 128.2, 128.0, 127.8, 127.7, 127.6 (Bn), 97.4, 94.2 ( C-1), 86.4, 84.2 (C-4), 78.9, 77.5, 74.5, 74.1, 73.7, 73.5, 71.8, 70.6, 70.5, 69.6, 69.5 (Bn, C-2, C-3, C-1 '), 21.6, 21.0, 20.8, 20.6, 20.4 (COS ₃ , C (CH ₃ ) ₂ ). FAB-MS m / z 599 [M + H] ⁺ .

Alternative Procedure for Compound 78

3-O-benzyl-1,2: 5,6-di-O-isopropylidene-α-O-glucofuranose (30B)

To a solution of 1,2: 5,6-di-O-isopropylidene-α-D-allofuranose (30A) (obtained from Pfanstiehl Lab. Inc.) (40 g) in dimethylformamide at 0 ° C. was added in small portions of sodium hydride. The reaction mixture was stirred for 1 hour and benzyl bromide was added dropwise over 1 hour. The resulting reaction mixture was stirred at room temperature for 16 hours. To stop the reaction, methanol was added and dimethylformamide was removed under pressure. The resulting syrup was extracted with ethyl acetate and washed with brine. After evaporation of ethyl acetate, a semi-solid (93%) was obtained. Homogenized using TLC.

3-O-benzyl-1,2-O-isopropylidene-α-D-glucofuranose (30C)

Partial hydrolysis of compound 30B (50 g) was carried out in 75% acetic acid for 20 hours. Concentration to a smaller volume and extraction with ethyl acetate gave compound 30C (40 g, 90%). Homogenized using TLC.

3-O-benzyl-1,2-O-isopropylidene-α-D-ribopentodialdofuranose (30D)

A solution of compound 30C (40 g) in water / methanol (1: 1) was slowly added with stirring to a solution of sodium periodate in water at 0 ° C. The reaction mixture was stirred for 2 hours, ethylene glycol was added and the mixture was extracted with ethyl acetate. The dried extract was evaporated to give compound 30D (32 g, 89%). Homogenized using TLC. At this stage, the addition of methanol is important, which ensures the completion of the reaction.

3-O-benzyl-4- (hydroxymethyl) -1,2-O-isopropylidene-α-D-erythropentofuranose (30E)

An aqueous 37% formaldehyde solution and a 1N sodium hydroxide solution were added at 0 ° C to a stirred solution of compound 30D (32 g) in water and tetrahydrofuran (1: 1), the reaction was continued for 16 hours, extracted with ethyl acetate and washed with brine. After evaporation, the organic phase formed a syrup, which crystallized from a mixture of ether and petroleum ether as a white solid (23 g), while the filtrate was an oily substance that crystallized into a low melting solid (10 g), with a total yield of the compound 30E 92%: 23 g (a white solid was purified up to 99% by thin layer chromatography), 10 g of a low melting solid (includes impurities more mobile in TLC, approximate purity 75%). At this stage, the addition of tetrahydrofuran is very important from the point of view of completing the reaction.

3,5-di-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose (31)

As a result of benzylation of compound 30E (20 g) using sodium hydride (60%) and benzyl bromide at -10 ° C, a mixture of two isomers was obtained. Using column chromatography, the basic isomer of compound 31 (14 g, 54%) was obtained. Homogenized using TLC.

3,5-di-O-benzyl-1,2-O-isopropylidene-4-C-tosyl-α-D-ribofuranose (77)

A solution of compound 31 (12.5 g) in pyridine at 0 ° C was treated with p-toluenesulfonyl chloride and the reaction mixture was kept at room temperature for 14-16 hours. As a result of pyridine removal, extraction with methylene chloride and saturated sodium bicarbonate solution, 77 (14 g, 80%) were obtained. Homogenized using TLC.

1,2-di-O-acetyl-3,5-O-benzyl-4-C-tosyl-D-ribofuranose (78)

Hydrolysis of compound 77 (14 g) with 75% acetic acid was carried out at 65 ° C for 18 hours. The solvent was removed under pressure and the residue was treated with ethanol (3 × 100 ml), toluene (3 × 50 ml) and anhydrous pyridine (2 × 50 ml). (This compound 78 was crystallized from petroleum ether as a white solid). The residue was taken up in dry pyridine and treated with acetic anhydride at room temperature for 8 hours. As a result of extraction with ethyl acetate and saturated sodium bicarbonate, followed by washing in brine, compound 78 was obtained as a mixture of α and β anomers (12 g, 83%). A direct comparison of representative samples of compound 78 (TLC, HPLC, NMR) confirmed its identity and purity.

Example 105

1- (2-O-acetyl-35-di-O-benzyl-4-C- (p-toluenesulfonyloxymethyl) -β-D-ribofuranosyl) thymine (79)

To a stirred solution of anomeric mixture of 78 (12.8 g, 21.4 mmol) and thymine (5.38 g, 42.7 mmol) in anhydrous acetonitrile (182 cm ³ ) was added N, O-bis (trimethylsilyl) acetamide (31 68 ml, 128.23 mmol). The resulting reaction mixture was stirred for 1 hour at room temperature, and then stirring was continued at 60 ° C. for 1.5 hours. After cooling to 0 ° C, trimethylsilyl triflate (6.57 ml, 30.33 mmol) was added dropwise and the reaction mixture was stirred at 60 ° C for 10 hours. The reaction mixture was neutralized using an ice-cold saturated aqueous solution of sodium hydrogencarbonate (90 ml). The reaction mixture was filtered and the resulting filtrate was concentrated under reduced pressure to half the original volume. Extraction was carried out with dichloromethane (4 × 200 cm ³ ). The combined organic phase was washed with saturated aqueous sodium hydrogen carbonate (3 × 150 cm ³ ) and brine (3 × 150 ml), dried over Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was chromatographed on a silica gel column using dichloromethane / methanol (99: 1 to 98: 2, v / v) as eluent to give nucleoside 79 as a white solid (13.1 g, 92%). δ _H (CDCl ₃ ) 9.04 (s, 1H, NH), 7.73-7.19 (15H, m, 6-H, aromatic), 5.94 (1H, d, J 5.5, 1 '-H), 5.37 (1H, d, J 5.6, 2'-H), 4.57-4.40 (5H, m, 3'-H, 5'-H _a , 5'- H _b , Bn), 4.14 (2H, s, Bn), 3.75 (1H, d, J 10.2, 1 '' - H _a ), 3.57 (1H, d, J 10.2 , 1 '' - H _b ), 2.41 (3H, s, CH ₃ C ₆ H ₅ ), 2.02 (3H, s, COCH ₃ ), 1.54 (3H, s, CH ₃ ). δ _C (CDCl ₃ ) 169.8 (C = O), 163.5 (C-4), 150.2 (C-2), 145.0, 136.8, 135.6, 132.1, 129 7, 128.5, 128.0, 127.9, 127.8, 127.5 (aromatic), 113.5 (C-5), 86.8, 85.3, 77.6, 74.6 , 74.3, 73.6, 70.8, 68.8 (Bn, C-1 ', C-3', C-2 ', C4'), 21.3 (CH ₃ ), 20.5 ( POSS ₃ ), 11.8 (CH ₃ ). FAB-MS m / z 665 [M + H] ⁺ . Received: C, 61.2; H 5.3; N, 4.1; S is 4.7. For C ₃₄ H ₃₆ O ₁₀ N _S calculated: C - 61.4; H - 5.4; N, 4.2; S - 4.8.

Example 106

1- (3,5-di-O-benzyl-4-C- (p-toluenesulfonyloxymethyl) -β-D-ribofuranosyl) thymine (80)

Nucleoside 79 (13.1 g, 19.7 mmol) was dissolved in a solution of ammonia in methanol (200 cm ³ , prepared by diluting a saturated solution of ammonia in methanol with an equal volume of methanol) and stirred at room temperature for 4 hours. After that, the reaction mixture was evaporated and the residue was dissolved in dichloromethane (400 cm ³ ). The organic phase was washed with brine (3 × 150 cm ³ ), dried over Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was chromatographed on a silica gel column using dichloromethane / methanol (99.5: 0.5, v / v) as eluent to give nucleoside 80 as a white solid (10.7 g, 87%). δ _H (CDCl ₃ ) 9.66 (s, 1H, NH), 7.71-7.21 (15H, m, 6-H, aromatic), 5.72 (1H, d, J 5.1, 1 '-H), 4.75, 4.55 (2H, each d, J 11.5, Bn), 4.51 (2H, s, Bn), 4.37 (1H, t, J 5.4, 2'-H), 4.30-4.12 (3H, m, 3'-H, Bn), 3.76 (1H, d, J 10.2, 1 '' - H _a ), 3.59 (1H, d, J 10.2, 1 '' - H _b ), 2.39 (3H, s, CH ₃ C ₆ H ₅ ), 1.48 (3H, s, CH ₃ ). δ _C (CDCl ₃ ) 163.8 (C-4), 150.9 (C-2), 145.0, 137.0, 136.9, 135.9, 132.3, 129.8, 128, 7, 128.6, 128.2, 128.1, 128.0, 127.6 (aromatic), 111.0 (C-5), 89.6, 85.3, 78.4, 74.5, 73.8, 71.1, 69.7 (Bn, C-1 ', C-3', C-2 ', C-4', C-1 ''), 21.6 (CH ₃ ), 12 0 (CH ₃ ). FAB-MS m / z 623 [M + H] ⁺ . Received: C, 61.5; H - 5.2; N - 4.4; S is 5.2. For C ₃₂ H ₃₄ O ₉ N ₂ S calculated: C - 61.7; H - 5.4; N - 4.5; S is 5.1.

Example 107

(1S, 3R, 4R, 7S) -7-benzyloxy-1-benzoyloxymethyl-3- (thymin-1-yl) -2,5-dioxabicyclo [2.2.1] heptane (36)

To a stirred solution of nucleoside 80 (10.65 g, 17.1 mmol) in anhydrous DMF (150 cm ³ ), a 60% suspension of sodium hydride in mineral oil (0.9 g, 22.2 mmol) was added in small portions at 0 ° C. The reaction mixture was stirred at 0 ° C for 15 hours, after which an additional 60% sodium hydride (0.205 g, 5.12 mmol) was added and the reaction mixture was stirred for another 22 hours at 0 ° C. Methanol was added (20 cm ³ ) and the reaction mixture was then concentrated under reduced pressure to half the original volume. Chilled ice water (300 cm ³ ) was added and extraction was carried out with dichloromethane (5 × 150 cm ³ ). The combined organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (3 × 40 cm ³ ) with brine (3 × 40 cm ³ ), dried over Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was chromatographed on a silica gel column using dichloromethane / methanol (99.5: 0.5, v / v) as eluent to give nucleoside 36 as a white solid (7.1 g, 92%). The data of spectral analysis correspond to the data previously obtained for compound 36. Received: C - 66.2; H - 5.8; N, 6.1. For C ₂₅ H ₂₆ N ₂ O ₆ calculated: C - 66.6; H - 5.8; N, 6.2.

Example 108

3,5-di-O-benzyl-1,2-O-isopropylidene-4-C-methanesulfonyloxymethyl-α-D-ribofuranose (200)

To a stirred solution of furanose 31 (2.16 g, 5.39 mmol) in anhydrous pyridine (3 ml) at 0 ° C. methanesulfonyl chloride (0.61 ml, 16.0 mmol) was added dropwise. The reaction mixture was stirred for 20 minutes at room temperature, the reaction was stopped with a mixture of ice and water (300 ml) and was extracted with dichloromethane (2 × 300 ml). The combined extracts were washed with a saturated aqueous solution of sodium hydrogencarbonate (300 ml) and dried over MgSO ₄ . The solvent was removed by distillation under reduced pressure, and the residue was chromatographed on silica gel using dichloromethane as an eluent to give compound 200 as an oily substance (2.55 g, 99%). ¹ H-NMR (CDCl ₃ ): δ 7.37-7.24 (10H, m, Bn), 5.78 (1H, d, J 3.8 Hz, H-1), 4.85 (1H, d, J 11.7 Hz, Bn), 4.73 (1 H, d, J 11.9 Hz, Bn), 4.64 (1 H, dd, J 4.0, 5.3 Hz, H-2) 4.54 (1H, d, J 11.9 Hz, H-5 '), 4.52 (1H, d, J 11.9 Hz, Bn), 4.46 (1H, d, J 11.9 Hz, H-5 '), 4.41 (1H, d, J 11.8 Hz, Bn), 3.60 (1H, d, J 10.4 Hz, H-5), 3.50 (1H, d, J 10.5 Hz, H-5), 3.06 (3H, s, SO ₂ CH ₃ ), 1.68 (3H, s, CH ₃ ), 1.34 (3H, s, CH ₃ ) . ¹³ C-NMR (CDCl ₃ ): δ 137.79, 137.31, 128.54, 128.48, 128.16, 128.01, 127.87, 127.79 (Bn), 113.66 (C (CH ₃ ) ₂ ), 104.46 (C-1), 84.88 (C-4), 78.48, 78.41 (C-2, C-3), 73.65, 72.63, 70.78, 70.16 (Bn, C-5, C-5 '), 37.84 (SO ₂ CH ₃ ), 26.20 (CH ₃ ), 25.69 (CH ₃ ). MS FAB: 501 (M + Na, 100%). Received: C, 60.37; H, 6.29; S - 6.53. For C ₂₄ H ₃₀ O ₈ S, it was calculated: C - 60.24; H, 6.32; S - 6.70%.

Example 109

Methyl-3,5-di-O-benzyl-4-C-methanesulfonyloxymethyl-α-D-ribofuranose (201)

A solution of furanose 200 (1.133 g, 2.37 mmol) in methanol hydrochloric acid (20%, w / v, 31.7 ml) and water (4.4 ml) were stirred at room temperature for 2 hours. After neutralization with sodium bicarbonate (saturated solution), the resulting mixture was extracted with dichloromethane (2 × 150 ml). The combined extracts were washed with water (150 ml) and dried over MgSO ₄ . The solvent was removed by distillation under reduced pressure, and the residue was chromatographed on silica gel using dichloromethane / methanol (99: 1) as eluent to give compound 201 (in the mixture of anomers: α: β ≈ 1: 2) as a clear oily substance (1,018 g, 95%). ¹ H-NMR (CDCl ₃ ): δ 7.39-7.22 (m, Bn), 4.86 (bs, Bn), 4.69-3.99 (m, Bn, H-5 ', H -1, H-2, H-3), 3.68 (d, J 8.9 Hz, H-5 β), 3.51 (d, J 9.8 Hz, H-5 α), 3, 46 (s, OCH ₃ α), 3.34 (d, J 9.1 Hz, H-5 β), 3.32 (d, J 9.7 Hz, H-5 α), 3.28 (s , OCH ₃ β), 2.97 (3H, s, SO ₂ CH ₃ β), 2.93 (3H, s, SO ₂ CH ₃ α). ¹³ C-NMR (CDCl ₃ ): δ 137.74, 136.98, 128.70, 128.64, 128.58, 128.56, 128.37, 128.21, 128.15, 128.09, 127.98, 127.86, 127.83 (Bn), 107.54 (C-1 β), 103.39 (C-1 α), 84.65, 83.18, 81.90, 78.87 (C-4, C-3), 75.04, 74.07, 73.73, 73.70, 73.38, 72.56, 72.11, 70.85, 70.55, 70.20 ( C-2, Bn, C-5, C-5 '), 55.90 (OCH ₃ α), 54.96 (OCH ₃ β), 37.18 (SO ₂ CH ₃ β), 37.07 (SO ₂ CH ₃ α). MS FAB: 475 (M + Na, 25%). Received: C, 58.40; H, 6.33. For C ₂₄ H ₃₀ O ₈ S calculated: C - 58.39; H - 6.24%.

Example 110

(3R) - and (3S) - (1S, 4R, 7S) -7-benzyloxy-1-benzyloxymethyl-3-methoxy-2,5-dioxabicyclo [2.2.1] heptane (202 and 203)

A solution of compound 201 (3.32 g, 7.34 mmol) in anhydrous DMF (25 ml) was stirred at 0 ° C and a 60% suspension of sodium hydride in mineral oil (700 mg, 16.9 mmol) was added. The resulting reaction mixture was stirred at room temperature for 90 minutes, the reaction was stopped with water (300 ml) and extracted with diethyl ether (2 × 300 ml). The combined extracts were washed with water (200 ml), dried over MgSO ₄ . The solvent was removed by distillation under reduced pressure, and the residue was chromatographed on silica gel using dichloromethane as an eluent to give compounds 202 and 203 as clear oily substances (1.571 g, 60% and 0.111 g, 30%, respectively. (1S, 3R , 4R, 7S) -7-benzyloxy-1-benzyloxymethyl-3-methoxy-2,5-dioxabicyclo [2.2.1] heptane (202). ¹ H-NMR (CDCl ₃ ): δ 7.36-7.26 (10H, m, Bn), 4.81 (1H, s, H-1), 4.65 (1H, d, J 11.9 Hz, Bn), 4.61 (2H, s, Bn), 4 56 (1H, d, J 11.9 Hz, Bn), 4.11 (1H, s, H-2), 4.09 (1H, s, H-3), 4.01 (1H, d, J 7.5 Hz, H-5 '), 3.80-3.77 (3H, m, H-5', H-5), 3.39 (3H, s, OCH ₃ ). ¹³ C-NMR (CDCl ₃ ): δ 138.05, 137.36, 128.47, 128.44, 127.88, 127.73, 127.63 (Bn), 104.97 (C-1), 85.13 (C-4), 79.16 (C-3), 77.18 ( C-2), 73.64 (Bn), 72.26, 72.10 (Bn, C-5 '), 66.50 (C-5), 55.34 (OCH ₃ ). MS FAB: 379 ( M + Na, 28%). Received: C - 70.55; H - 6.97. For C ₂₁ H ₂₄ O ₅ calculated: C - 70.77; H - 6.79%. (1S, 3S, 4R , 7S) -7-benzyloxy-1-benzyloxymethyl-3-methoxy-2,5-dioxabicyclo [2.2.1] heptane (203). ¹ H-NMR (CDCl ₃ ): δ 7.36-7.26 (10H, m, Bn), 5.00 (1H, s, H-1), 4.67-4.54 (4H, m, Bn), 4.18 (1H, s, H-2), 3.99 (1H, s, H-3), 3.99-3.90 (2H, m, H-5 '), 3.75 -3.68 (2H, m, H-5); 3.49 (3H, s, OCH ₃ ). ¹³ C-NMR (CDCl ₃ ): δ 137.83, 137.53, 128.51, 128.48, 127.96, 127.82, 127.71, 127.62 (Bn), 104.05 (C -1), 88.44 (C-4), 79.54 (C-3), 77.16 (C-2), 73.68 (Bn), 72.61 (C-5 '), 72, 24 (Bn), 65.73 (C-5), 56.20 (OCH ₃ ). MS FAB: 379 (M + Na, 100%).

Example 111

(1R, 2S, 3S) -2-benzyloxy-3-benzyloxymethyl-1- (methoxy (thymin-1-yl) methyl) -3-trimethylsilyloxytetrahydrofuran (204)

A solution of compound 202 (216 mg, 0.606 mmol) and thymine (153 mg, 1.22 mmol) in anhydrous acetonitrile (9.3 ml) was added (N, O-bis (trimethylsilyl) acetamide (BSA, 0.90 ml, 3 , 6 mmol) and stirred under reflux for 15 minutes.The solution was cooled to 0 ° C and trimethylsilyl triflate (0.153 ml, 0.777 mmol) was added dropwise. After stirring at room temperature for 18 hours and at 60 ° C, for 24 hours, the reaction was stopped with a saturated aqueous solution of sodium hydrogencarbonate (20 ml) and extraction was carried out with dichloromethane (2 × 50 ml). The extracts were washed with a saturated aqueous solution of sodium hydrogencarbonate (50 ml) and dried over MgSO _{4. The} solvent was removed under reduced pressure and the residue was chromatographed on silica gel using dichloromethane / methanol (98: 2) as an eluent to give compound 204 (mixture diastereomers ≈ 1.7: 1) as a solid (196 mg, 67%). ¹ H-NMR (CDCl ₃ ): δ 7.36-7.14 (m, Bn, H-6), 5.77 (1H, d, J 7.9 Hz, H-1 '), 5.57 (1H, d, J 5.8 Hz, H-1'), 4.68-4.43 (m, Bn, H -2 '), 4.12-3.68 (m, H-5', H-5 ', H-3'), 3.32 (s, OCH ₃ ), 3.24 (s, OCH ₃ ) , 1.93 (d, J 0.9 Hz, CH ₃ ), 1.86 (d, J 1.1 Hz, CH ₃ ), 0.14 (s, Si (CH ₃ ) ₃ ), 0.12 (s, Si (CH ₃ ) ₃ ). ¹³ C-NMR (CDCl ₃ ): δ 163.68, 163.55 (C-4), 151.58, 151.07 (C-2), 137.84, 137.74, 137.32 (Bn) , 135.93, 135.10 (C-6), 128.57, 128.42, 128.41, 128.10, 127.95, 127.85, 127.77, 127.74 (Bn), 111 38, 111.01 (C-5), 86.89, 85.61, 85.40, 84.72, 83.40, 83.31, 82.10 (C-1 ', C-2', C-3 ', C-4'), 75.20, 73.98, 73.62, 73.59, 72.55, 72.13, 71.04, 70.74 (Bn, C-5 ', C-5 ''), 56.82, 56.54 (OCH ₃ ), 12.47, 12.38 (CH ₃ ), 1.72, 1.69 (Si (CH ₃ ) ₃ ). MS FAB: 555 (M + H, 65%), 577 (M + Na, 70%). Received: C, 62.76; H - 6.88; N - 4.94. For C ₂₉ H ₃₈ N ₂ O ₇ Si calculated: C — 62.79; H, 6.90; N - 5.05%.

Example 112

(1R, 2S, 3S) -2-benzyloxy-3-benzyloxymethyl-1- (methoxy (6-N-benzoyladeninin-9-yl) methyl) -3-trimethylsilyloxytetrahydrofuran (205)

A solution of compound 202 (240 mg, 0.673 mmol) and 6-N-benzoyladenine (301 mg, 1.26 mmol) in anhydrous acetonitrile (8.2 ml) was added BSA (0.67 ml, 2.7 mmol) and stirred at room temperature for 1 hours. The solution was cooled to 0 ° C. and trimethylsilyl triflate (0.25 ml, 1.33 mmol) was added dropwise. After stirring at 65 ° C for 18 hours, the reaction was stopped by the addition of a saturated aqueous solution of sodium hydrogencarbonate (50 ml), extracted with dichloromethane (2 × 50 ml). The combined extracts were dried over MgSO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on silica gel using dichloromethane / methanol (98: 2) as eluent to give compound 205 (mixture of diastereomers ≈ 1.8: 1) as a solid (185 mg, 41%) . ¹ H-NMR (CDCl ₃ ): δ 8.78 (s, H-8), 8.21 (s, H-2), 8.17 (s, H-2), 8.03-8.00 (m, Bz), 7.61-7.49 (m, Bz), 7.36-7.23 (m, Bn), 7.07-7.04 (m, Bz), 5.85 (1H d, J 7.9 Hz, H-1 '), 5.76 (1 H, d, J 6.0 Hz, H-1'), 4.74-4.40 (m, Bn, H-2 '), 4.22-3.62 (m, H-5', H-5 ', H-3'), 3.33 (s, OCH ₃ ), 3.24 (s, OCH ₃ ), 0 15 (s, Si (CH ₃ ) ₃ ), 0.14 (s, Si (CH ₃ ) ₃ ). ¹³ C-NMR (CDCl ₃ ): δ 164.68 (HNC = O), 153.17, 152.99 (C-6), 149.47 (C-2), 141.82, 141.66 (C -8), 137.74, 137.71, 137.65 (Bn), 133.87, 132.87, 132.78 (Bz), 128.97, 128.93, 128.45, 128.42, 128.38, 128.14, 127.97, 127.88, 127.82, 127.78 (Bn, Bz), 123.66, 122.85 (C-5), 86.41, 86.23, 85.70, 85.24, 84.78, 83.73, 83.58, 82.79 (C-1 ', C-2', C-3 ', 0-4'), 75.32, 74 55, 73.61, 72.18, 71.98, 70.85, 70.59 (Bn, C-5 ', C-5``), 57.23, 57.04 (OCH ₃ ), 1 78 (Si (CH ₃ ) ₃ ). MS FAB: 668 (M + H, 50%), 690 (M + Na, 100%). Received: C, 64.07; H 6.01; N, 9.94. For C ₂₉ H ₃₈ N ₂ O ₇ Si · 0.5H ₂ O, it was calculated: C - 63.88; H, 6.25; N - 10.34%.

Example 113

(1R, 2R, 3R) -2-benzyloxy-3-benzyloxymethyl-3-hydroxytetrahydrofurfural (206)

A solution of compound 202/203 (252 mg, 0.707 mmol) in 80% acetic acid (3.8 ml) was stirred at 90 ° C. for 2 hours, after which the solvent was removed by distillation under reduced pressure. The residue was evaporated with toluene (3 × 10 ml) to give compound 206 as an oily substance (242 mg, 100%). ¹ H-NMR (CDCl ₃ ): δ 9.66 (1H, d, J 0.8 Hz, H-1), 7.36-7.25 (10H, m, Bn), 4.68 (1H, d, J 11.9 Hz, Bn), 4.60-4.39 (5Н, m, Bn, Н-2, Н-3), 3.98-3.92 (2Н, m, Н-5) 3.85 (1H, d, J 9.3 Hz, H-5 '); 3.52 (1H, d, J 9.2 Hz, H-5'). ¹³ C-NMR (CDCl ₃ ): δ 203.64 (C-1), 137.39, 137.19, 128.61, 128.54, 128.29, 128.12, 127.87, 127.83 (Bn), 87.17, 87.05 (C-4, C-2), 80.98 (C-3), 75.00, 73.70, 71.86 (Bn, C-5 '), 67.84 (C-5). MS FAB: 707 (2 × M + Na, 100%).

Example 114

(2S, 3S, 4R, 7S) -3-acetoxy-7-benzyloxy-1-benzyloxymethyl-2,5-dioxabicyclo [2.2.1] heptane (207)

To a stirred solution of compound 206 (230 mg, 0.672 mmol) in anhydrous pyridine (2.0 ml) was added acetic anhydride (0.18 ml, 1.91 mmol). The resulting reaction mixture was stirred at room temperature for 23 hours, water (0.13 ml) was added, and the solvent was removed by distillation under reduced pressure. The residue was evaporated with toluene (3 × 10 ml) and chromatographed on silica gel using dichloromethane / methanol (99: 1) as eluent to give compound 207 as a clear oily substance (56.7 mg, 23%). ¹ H-NMR (CDCl ₃ ): δ 7.38-7.26 (10H, m, Bn), 6.00 (1H, s, H-1), 4.68 (1H, d, J 12.0 Hz, Bn), 4.62 (1H, d, J 12.2 Hz, Bn), 4.60 (1H, d, J 12.4 Hz, Bn), 4.56 (1H, d, J 12, 2 Hz, Bn), 4.17 (1H, s, H-2), 4.14 (1H, s, H-3), 4.01 (1H, d, J 7.7 Hz, H-5 ' ), 3.81-3.78 (3H, m, H-5 ', H-5), 20.06 (3H, s, COCH ₃ ). ¹³ C-NMR (CDCl ₃ ) δ 169.18 (C = O), 137.92, 137.48, 128.52, 128.45, 128.03, 127.77, 127.73, 127.68 ( Bn), 95.95 (C-1), 86.49 (C-4), 78.27, 76.58 (C-3, C-2), 73.65 (Bn), 72.26, 71 96 (Bn, C-5 '), 65.49 (C-5), 20.98 (COSH ₃ ). MS FAB: 407 (M + Na, 55%). Received: C, 68.80; H, 6.11. For C ₂₂ H ₂₄ O ₆ , calculated: C - 68.74; H - 6.29%.

Example 115

(1S, 3S, 4R, 7S) -3- (6-N-benzoyladenin-9-yl) -7-benzyloxy-1-benzyloxymethyl-2,5-dioxabicyclo [2.2.1] heptane (208)

To a solution of furanose 207 (167 mg, 0.434 mmol) and 6-N-benzoyladenine (194 mg, 0.813 mmol) in anhydrous acetonitrile (5.3 ml) was added BSA (0.43 ml, 1.76 mmol) and stirred at room temperature for 1 hour. The resulting solution was cooled to 0 ° C. and trimethylsilyl triflate (0.16 ml, 0.86 mmol) was added dropwise. After stirring at 65 ° C for 2 hours, the reaction was stopped with a saturated aqueous solution of sodium hydrogencarbonate (40 ml) and the reaction mixture was extracted with dichloromethane (2 × 50 ml). The combined extracts were dried over MgSO ₄ . The solvent was removed under reduced pressure and the residue was chromatographed on silica gel using dichloromethane / methanol (98: 2) as eluent to give compound 208 as a solid (111 mg, 45%). ¹ H-NMR (CDCl ₃ ): δ 8.82 (1H, s, H-8), 8.14 (1H, s, H-2), 7.59-7.26 (15H, m, Bz, Bn), 6.74 (1H, s, H-1 '), 4.92 (1H, s, H-2'), 4.74-4.39 (4H, m, Bn), 4.42 ( 1H, s, H-3 '), 4.19-4.10 (2H, m, H-5'), 3.92 (1H, d, J 11.8 Hz, H-5 '), 3, 88 (1H, d, J 11.5 Hz, H-5 '). MS FAB: 564 (M + H, 100%).

Example 116

Methyl-2-O-acetyl-3,5-di-O-benzyl-4-C-methanesulfonyloxymethyl-D-ribofuranoside (209)

Acetic anhydride (0.43 ml, 4.56 mmol) was added dropwise to a stirred solution of compound 201 (687 mg, 1.52 mmol) in anhydrous pyridine (4 ml) at 0 ° C. The reaction mixture was stirred at room temperature for 2 days, the reaction was stopped with a saturated aqueous solution of sodium hydrogencarbonate (75 ml) and extracted with dichloromethane (150 + 75 ml). The combined extracts were dried over MgSO ₄ , the solvent was removed by distillation under reduced pressure, and the residue was chromatographed on silica gel using dichloromethane as an eluent to give compound 209 as a clear oily substance (β: α ≈ 3: 1, 750 mg, 100% ) MS FAB: 463 (M-OCH ₃ , 100%), 517 (M + Na, 28%). Received: C, 58.53; H, 6.16. For C ₂₄ H ₃₀ O ₉ S, C - 58.29; H - 6.11%. Methyl-2-O-acetyl-3,5-di-O-benzyl-4-C-methanesulfonyloxymethyl-β-D-ribofuranoside (209β). ¹ H-NMR (CDCl ₃ ): δ 7.36-7.18 (10H, m, Bn), 5.27 (1H, d, J 4.9 Hz, H-2), 4.88 (1H, s, H-1), 4.55-4.44 (6H, m, H-5 ', Bn), 4.35 (1H, d, J 5.0 Hz, H-3), 3.73 ( 1H, d, J 9.2 Hz, H-5), 3.38 (1H, d, J 9.3 Hz, H-5), 3.30 (3H, s, OCH ₃ ), 2.95 ( 3H, s, SO ₂ CH ₃ ), 2.11 (3H, s, OCCH ₃ ). ¹³ C-NMR (CDCl ₃ ) δ 169.91 (C = O), 137.83, 137.28, 128.49, 128.44, 127.99, 127.87, 127.77 (Bn), 105 40 (C-1), 82.65, 81.05, 74.55, 73.62, 73.56, 71.86, 70.22 (C-2, C-3, C-4, C- 5, C-5 ', Bn), 55.03 (OCH ₃ ), 37.14 (SO ₂ CH ₃ ), 20.73 (OCHN ₃ ). Methyl-2-O-acetyl-3,5-di-O-benzyl-4-C-methanesulfonyloxymethyl-α-D-ribofuranoside (209α). ¹ H-NMR (CDCl ₃ ): δ 7.36-7.18 (10H, m, Bn), 5.09 (1H, d, J 4.5 Hz, H-1), 4.95 (1H, dd, J 4.5, 6.8 Hz, H-2), 4.65-4.44 (6H, m, H-5 ', Bn), 4.27 (1H, d, J 6.6 Hz , H-3), 3.49 (1H, d, J 9.9 Hz, H-5), 3.46 (3H, s, OCH ₃ ), 3.36 (1H, d, J 9.9 Hz , H-5), 2.92 (3H, s, SO ₂ CH ₃ ), 2.14 (3H, s, OCCH ₃ ). ¹³ C-NMR (CDCl ₃ ) δ 170.41 (C = O), 137.59, 137.28, 128.56, 128.51, 128.49, 128.44, 127.98, 127.88 ( Bn), 102.35 (C-1), 84.25, 77.53, 74.66, 73.67, 72.12, 70.39, 70.28 (C-2, C-3, C- 4, C-5, C-5 ', Bn), 56.07 (OCH ₃ ), 36.94 (SO ₂ CH ₃ ), 20.63 (OCHN ₃ ).

Example 117

Phenyl-2-O-acetyl-3,5-di-O-benzyl-4-C-methanesulfonyloxymethyl-1-thio-β-D-ribofuranoside (210)

Method a. To a stirred solution of compound 209 (738 mg, 1.49 mmol) in anhydrous dichloromethane (6.4 ml) was added phenylthiotrimethylsilane (2.42 ml, 12.8 mmol) and cooled to 0 ° C. Trimethylsilyl triflate (0, 67 ml, 3.67 mmol) and the resulting solution was stirred at room temperature for 4 hours. The reaction was stopped by adding a saturated aqueous solution of sodium hydrogencarbonate (100 ml) and was extracted with dichloromethane (2 × 200 ml). The combined extracts were dried over MgSO4 and the solvent was removed by distillation under reduced pressure. The residue was chromatographed on silica gel using dichloromethane as an eluent to give compound 210 as a clear oily substance (564 mg, 86%) and unreacted starting material (191 mg, 26%). Method b. To a stirred solution of compound 211 (86 mg, 0.165 mmol) in anhydrous dichloromethane (0.49 ml) was added phenylthiotrimethylsilane (0.16 ml, 0.825 mmol) and cooled to 0 ° C. Trimethylsilyl triflate (0.037 ml, 0.206 mol) was added and the resulting the solution was stirred at room temperature for 2 hours. The reaction was stopped by the addition of a saturated aqueous solution of sodium hydrogencarbonate (15 ml) and the resulting mixture was extracted with dichloromethane (2 × 25 ml). The combined extracts were dried over MgSO ₄ and the solvent was removed by distillation under reduced pressure. The residue was chromatographed on silica gel using dichloromethane as an eluent to give compound 210 as a clear oily substance (75 mg, 79%). ¹ H-NMR (CDCl ₃ ): δ 7.47-7.19 (15H, m, Bn, SPh), 5.48 (1H, d, J 3.6 Hz, H-2), 5.34 ( 1H, dd, J 3.7, 5.2 Hz, H-1), 4.54-4.36 (7H, m, H-3, H-5 ', Bn), 3.66 (1H, d , J 9.7 Hz, H-5), 3.48 (1H, d, J 9.5 Hz, H-5), 2.89 (3H, s, SO ₂ CH ₃ ), 2.09 (3H , s, OSSN ₃ ). ¹³ C-NMR (CDCl ₃ ): δ 169.93 (C = O), 137.69, 137.08, 132.65, 132.45, 129.15, 128.53, 128.52, 128.18 , 128.14, 128.08, 127.91, 127.85 (Bn, SPh), 87.99, 84.35, 80.34, 75.33, 74.20, 73.67, 70.83, 69.34 (C-1, C-2, C-3, C-4, C-5, C-5 ', Bn), 37.27 (SO ₂ CH ₃ ), 20.68 (OCCH ₃ ). MS FAB: 463 (M-SPh, 100%), 595 (M + Na, 24%). Received: C, 61.17; H - 5.55. For C ₂₉ H ₃₂ O ₈ S ₂ calculated: C - 60.82; H - 5.63%.

Example 118

1,2-di-O-acetyl-3,5-di-O-benzyl-4-C-methanesulfonyloxymethyl-D-ribofuranose (211)

A solution of compound 201 (150 mg, 0.313 mmol) in 80% acetic acid (1.5 ml) was stirred at 90 ° C for 3 hours. The solvent was removed by distillation under reduced pressure, and the residue was evaporated together with ethanol (3 × 5 ml), toluene (3 × 5 ml) and pyridine (3 × 5 ml). The residue was redissolved in anhydrous pyridine (0.62 ml), acetic anhydride (0.47 ml) was added and the resulting solution was stirred at room temperature for 16 hours. The reaction was stopped with water (50 ml) and the resulting reaction mixture was extracted with dichloromethane (2 × 50 ml). The combined extracts were washed with a saturated aqueous solution of sodium hydrogencarbonate (50 ml) and dried over MgSO ₄ . The solvent was evaporated and the residue was chromatographed on a silica gel column using dichloromethane as an eluent to give compound 211 as an oily substance (99 mg, 60%). ¹ H-NMR (CDCl ₃ ): δ 7.39-7.21 (m, Bn), 6.38 (d, J 4.6 Hz, H-1 β), 6.15 (s, H-1 α), 5.35 (d, J 4.9 Hz, H-2 α), 5.17 (dd, J 6.3, 4.9 Hz, H-2 β), 4.69-4.23 (m, H-3, Bn), 3.64 (d, J 9.7 Hz, H-5 α), 3.52 (d, J 10.1 Hz, H-2 β), 3.45 ( d, J 9.7 Hz, H-5 α), 3.39 (d, J 9.9 Hz, H-2 β), 2.99 (s, SO ₂ CH ₃ α), 2.96 (s , SO ₂ CH ₃ β), 2.14, 2.13, 2.06, 1.90 (4 × s, COCH ₃ ). ¹³ C-NMR (CDCl ₃ ): δ 169.68, 169.00 (C = O), 137.68, 137.05, 128.60, 128.55, 128.50, 128.21, 128.12 , 128.04, 127.94, 127.82, 127.79 (Bn), 99.35 (C-1 α), 94.24 (C-1 β), 86.36 (C-4 β), 84.28 (C-4α), 79.15, 77.47, 74.58, 74.06, 73.73, 73.56, 71.67, 70.57, 70.19, 69.84 ( Bn, C-2, C-3, C-5, C-5 '), 37.61 (SO ₂ CH ₃ β), 37.48 (SO ₂ CH ₃ α), 21.07, 20.74, 20.63, 20.39 (SOSN ₃ ). MS FAB: 545 (M + Na, 13%). Received: C, 57.70; H - 5.56. For C ₂₅ H ₃₀ O ₁₀ S calculated: C - 57.46; H - 5.79%.

Example 119

(3R) - and (3S) - (1S, 4R, 7S) -7-benzyloxy-1-benzyloxymethyl-3-phenylthio-2,5-dioxabicyclo [2.2.1] heptane (212)

A solution of compound 210 (553 mg, 0.966 mmol) in methanol saturated with ammonia (35 ml) was stirred at room temperature for 2 hours, after which the solvent was removed by distillation under reduced pressure. The residue was redissolved in anhydrous DMF (3.5 ml) and the solution was stirred at 0 ° C. A 60% suspension of sodium hydride (118 mg, 2.88 mmol) was added and the resulting reaction mixture was stirred at room temperature for 12 hours. The reaction was stopped by the addition of a saturated aqueous solution of sodium hydrogencarbonate (100 ml) and the resulting mixture was extracted with dichloromethane (2 × 100 ml). The combined extracts were dried over MgSO ₄ and the solvent was removed by distillation under reduced pressure. The residue was chromatographed on silica gel using dichloromethane as an eluent to give compound 212 as a clear oily substance (404 mg, 96%). MS FAB: 435 (M + H, 35%), 457 (M + Na, 16%). Received: C, 71.76; H, 6.18. For C ₂₆ H ₂₆ O ₄ S calculated: C - 71.86; H - 6.03%. (1S, 3R, 4R, 7S) -7-benzyloxy-1-benzyloxymethyl-3-phenylthio-2,5-dioxabicyclo [2.2.1] heptane (212β). ¹ H-NMR (CDCl ₃ ): δ 7.46-7.26 (15H, m, Bn, SPh), 5.35 (1H, s, H-1), 4.68-4.56 (4H, m, Bn), 4.31 (1H, s, H-2), 4.10 (1H, s, H-3), 4.09 (1H, d, J 7.3 Hz, H-5 ') 3.93 (1H, d, J = 7.8 Hz, H-5 '), 3.79 (2H, m, H-5). ¹³ C-NMR (CDCl ₃ ): δ 138.03, 137.45, 133.42, 132.36, 129.19, 128.55, 128.46, 128.05, 127.84, 127.83, 127.76 (Bn, SPh), 89.96 (C-1), 87.18 (C-4), 79.71 (C-2), 79.40 (C-3), 73.64 (Bn ), 73.23 (C-5 '), 72.30 (Bn), 66.31 (C-5). (1S, 3S, 4R, 7S) -7-benzyloxy-1-benzyloxymethyl-3-phenylthio-2,5-dioxabicyclo [2.2.1] heptane (212α). ¹ H-NMR (CDCl ₃ ): δ 7.52-7.19 (15H, m, Bn, SPh), 5.52 (1H, s, H-1), 4.70-4.50 (4H, m, Bn), 4.41 (1H, s, H-2), 4.18 (1H, d, J 7.8 Hz, H-5 '), 4.08 (1H, d, J 8.4 Hz, H-5 '), 4.07 (1H, s, H-3), 3.78 (1H, d, J 11.3 Hz, H-5), 3.72 (1H, d, J 11 5 Hz, H-5). ¹³ C-NMR (CDCl ₃ ): δ 137.89, 137.46, 135.29, 130.93, 129.13, 128.99, 128.57, 128.48, 127.81, 127.76, 127.58, 126.95 (Bn, SPh), 91.87 (C-1), 88.59 (C-4), 80.07, 79.14 (C-2, C-3), 73, 65, 73.40, 72.04 (Bn, C-5 '), 65.62 (C-5).

Example 120

(3R) - and (3S) - (1S, 4R, 7S) -7-benzyloxy-1-benzyloxymethyl-3- (thymin-1-yl) 2,5-dioxabicyclo [2.2.1] heptane (36 + 213)

Thymine (175 mg, 1.38 mmol) was stirred in hexamethyldisilazane (6.8 ml) under reflux and ammonium sulfate (5 mg) was added. After stirring for 16 hours, the clear solution was cooled to 40 ° C. and the solvent was removed by distillation under reduced pressure. To the residue was added a solution of compound 212 (201 mg, 0.463 mmol) in anhydrous dichloromethane (4.6 ml) and a molecular sieve with a pore diameter of 4A (180 mg). After stirring at room temperature for 10 minutes, NBS (107 mg, 0.602 mmol) was added and the resulting reaction mixture was stirred for another 30 minutes. The reaction was stopped with a saturated aqueous sodium thiosulfate solution (25 ml) and the extract was dried over MgSO ₄ and evaporated, and the residue was chromatographed on silica gel using dichloromethane / methanol (97: 3) as eluent to obtain 36 + 213 as a mixture of anomers (β: α ≈ 1: 2) (127 mg, 61%). ¹ H-NMR (CDCl ₃ ): δ 7.49 (d, J 0.9 Hz, H-6 β), 7.46 (d, J 1.0 Hz, H-6 α), 7.39- 7.25 (m, Bn), 5.94 (s, H-1 'α), 5.64 (s, H-1' β), 4.71-4.50 (m, Bn, H-2 '), 4.23 (s, H-3' α), 4.16 (d, J 8.6 Hz, H-5 '' α), 4.09-3.78 (m, H-5 ' , H-5 '', H-3 'β), 1.94 (d, J 0.9 Hz, CH ₃ α), 1.62 (d, J 1.2 Hz, CH ₃ β). MS FAB: 551 (M + H, 96%).

Example 121

(3R) - and (3S) - (1S, 4R, 7S) -7-hydroxy-1-hydroxymethyl-3- (thymin-1-yl) 2,5-dioxabicyclo [2.2.1] heptane (37 + 214)

A solution of compounds 36 + 213 (175 mg, 0.39 mmol) in ethanol (2.7 ml) was stirred at room temperature and 20% palladium hydroxide on carbon (50 mg) was added. The resulting mixture was degassed several times with argon and placed in a hydrogen atmosphere. After stirring for 18 hours, the reaction mixture was chromatographed on a silica gel column using dichloromethane / methanol (95: 5) as an eluent to give a mixture of compounds 37 and 214 (1: 1.2) (26 mg, 25%). ¹ H-NMR (CD ₃ OD): δ 7.78 (d, J 1.3 Hz, H-6 α), 7.73 (d, J 1.2 Hz, H-6 β), 5.88 (s, H-1 'α), 5.53 (s, H-1' β), 4.38 (s, H-2 'α), 4.34 (s, H-3' α), 4 26 (s, R-2 'β), 4.08-3.69 (m, H-5', H-5 '', H-3 'β), 1.92 (d, J 1.2 Hz, CH ₃ α), 1.88 (d, J 1.1 Hz, CH ₃ β). ¹³ C-NMR (CD ₃ OD): δ 138.00 (C-6 α), 136.96 (C-6 β), 110.80 (C-5 β), 110.08 (C-5 α) , 92.49, 89.01 (C-4 ', C-1' α), 90.46, 88.37 (C-4 ', C-1'β), 80.89, 74.27, 73 34 (C-2 ', C-3', C-5 'α), 80.59, 72.47, 70.39 (C-2', C-3 ', C-5' β), 59 29 (C-5 '' α), 57.61 (C-5 '' β), 12.52 (CH ₃ α), 12.39 (CH ₃ β). MS EI: 270 (M +, 100%).

Obtaining LNA-phosphoramidites

Example 122

4-N-benzoyl-LNA-C [(1R, 3R, 4R, 7S) -3- (4-N-benzoylcytosin-1-yl) - (hydroxymethyl) -7-hydroxy-2,5-dioxabicyclo {2.2. 1} -heptane]

LNA-C (formula Z) was placed in absolute ethanol and heated under reflux. Benzoic anhydride (2 equivalents) was added to the heated solution and the reaction mixture was subjected to HPLC (eluent: 20% acetonitrile in 0.1 M TEAA, pH 7.0, flow rate 1 ml / min, Novapak C-18 analytical column). Anhydride was additionally added at intervals of 0.5–2 hours until the increase in the amount of the obtained product ceased during HPLC. The reaction mixture was concentrated on a rotary evaporator. The residue was washed again with ether, filtered and dried to give a white solid. The yield was 45%.

The main method of dimethoxytritylation of base-protected LNA nucleosides (LNA-C ^Bz , LNA-T, LNA-G ^iBu , LNA-A ^Bz ).

Base-protected LNA nucleosides were evaporated together with pyridine (2 ×) and mixed with dimethoxytrityl chloride (1.5 equivalents) in pyridine (≈ 10 ml / g nucleoside). The reaction was continued by HPLC (50% acetonitrile in 0.1 M TEAA, pH 7.0 for 5 minutes, 50-100% acetonitrile for 10 minutes and 100% acetonitrile for 5 minutes; flow rate 1 ml / min; column Novapak C-18). When more than 95% of the starting material turned out to be reacted, the reaction mixture was placed on ice. The reaction was stopped by the addition of cold saturated NaHCO ₃ (≈ 15 ml × volume of pyridine). The mixture was extracted with dichloromethane (3 x half volume of sodium bicarbonate). The organic extracts were combined, dried over Na ₂ SO ₄ , filtered and concentrated on a rotary evaporator.

The residue was dried in vacuo and chromatographed on silica gel using 0.5% pyridine and 0-2% methanol in dichloromethane as eluents. Fractions containing purified products were combined and concentrated on a rotary evaporator. The residue was evaporated with anhydrous acetonitrile (3x) and dried in vacuo.

The main method of phosphitylation of protected LNA nucleosides

Dimethoxytrityl-LNA nucleosides protected at their base were evaporated together with anhydrous dichloromethane (2x) and placed in anhydrous dichloromethane (10 ml per 1 g of nucleosides in variants A, G and T and ≈ 30 ml / g for variant C). To this mixture was first added bis (diisopropylamino) (2-cyanoethyl) phosphite (1.05-1.10 equivalents), and then tetrazole (0.95 equivalents). The mixture was stirred at room temperature and the reaction continued in HPLC (70% acetonitrile in 0.1 M TEAA, pH 7.0 for 2 minutes, 70-100% acetonitrile for 8 minutes and 100% acetonitrile for 5 minutes; flow rate 1 ml / min; Novapak C-18 column). When more than 90% of amidite formed in the reaction and its amount ceased to increase, the reaction mixture was placed on ice. It was diluted with dichloromethane (approximately 15-20 times the volume relative to the original volume) and washed with cold saturated sodium bicarbonate solution (2x) and then with cold brine (1x). The organic phase was dried over Na ₂ SO _{4 was} filtered and concentrated on a rotary evaporator. The residue was evaporated with anhydrous acetonitrile (3x) and dried in vacuo overnight. HPLC purity ranged from 93-98%.

Obtaining LNA-nucleoside-5’-triphosphates

Example 123

Synthesis of LNA-Nucleoside-5’-Triphosphates (Tetrahedron Lett., 1988, 29, 4525)

In a 13 × 100 mm polypropylene tube, nucleosides 37, 44, 51, 4-N-benzoylated 57A or 61B (93.8 μmol) were suspended in 1 ml of pyridine (dried with CaH ₂ ). The solution was evaporated to dryness in a vacuum centrifuge in high vacuum. The residue was resuspended twice in acetonitrile (dried with CaH ₂ ) and evaporated to dryness. The nucleoside was suspended in 313 μl of trimethyl phosphate (dried using a 4A molecular sieve), to which 30.1 mg of Proton Sponge ™ (1.5 equivalents) was added. The mixture was compacted, centrifuged, and cooled to 0 ° C. POCl ₃ (9.8 μl, 1.1 equivalent) was added by centrifugation. The reaction was carried out at 0 ° degrees for 2.5 hours. At this time, 469 μmol sodium pyrophosphate (5 equivalents) was dissolved in 5 ml of water and passed through 5 ml of Dow 50 H ⁺ ion exchange resin. When the effluent became acidic, it was taken in 220 μl tributylamine and evaporated to form a syrup. TBA pyrophosphate was evaporated three times with dry acetonitrile. Finally, dry pyrophosphate was dissolved in 1.3 ml of DMF (4A-sieve). After 2.5 hours, during which the reaction took place, TBA pyrophosphate and 130 μl tributylamine were added to the nucleoside solution under vigorous rotation. After 1 minute, the reaction was stopped by adding 3 ml of 0.1 M ammonium triethyl acetate (pH 7.5). According to Mono-Q chromatography, the presence of 49% nucleoside 5'-triphosphates was shown. The reaction mixture was diluted to 100 ml with water and adsorbed onto a Q-sepharose ion exchange column, washed with water and eluted using a linear gradient of 0-700 mM NaCl in 5 mM sodium phosphate (pH 7.5). Fractions containing triphosphates were tested by ion exchange chromatography with Mono-Q columns. Fractions containing triphosphates were combined and concentrated to saturation with NaCl. The product was desalted using a C ₁₈ cartridge. Quantitative analysis of triphosphates was carried out using UV spectroscopy and formed in the form of a final solution with a concentration of 10 mm. The yields amounted to 17-44%. In the described method, LNA nucleosides of bases U, T, A, G, and C were obtained.

Obtaining LNA-modified oligonucleotides

Example 124

Synthesis of Oligonucleotides Including LNA of Formulas V, X, Y and Z ^T , Z ^U , Z ^G , Z ^C , Z ^A , Z ^MeC

Bicyclic nucleoside-3'-O-phosphoramidite analogs 8, 19, 30, 39, 46, 53, 57D, 61D and 66, as well as commercially available 3'-O-phosphoramidites, were used to synthesize the exemplary LNA- variants oligonucleotides of the present invention (in a quantitative scale of 0.2-5 μmol), which would contain one or more LNA variants V, X, Y and Z ^T , Z ^U , Z ^G , Z ^C , Z ^A , Z ^MeC . The purity and composition of the synthesized LNA oligonucleotides was checked using the method of capillary gel electrophoresis and (or) HPLC, and (or) MALDI-MS. In general, satisfactory parameters of coupling reactions were confirmed for all monomers. The highest level of coupling efficiency (approximately 95-100%) was observed for nucleosides 39, 46, 53, 57D, 61D and 66 (corresponding to LNA monomers of formula Z), for which the most satisfactory results were obtained when the synthesis of fully LNA was carried out -modified oligonucleotides or when LNAs were incorporated into DNA or RNA oligonucleotides not modified by any other means, or LNAs were introduced into an oligonucleotide entirely consisting of phosphothioate monomers. LNA oligonucleotides were dissolved in pure water and the concentration was determined in units of OD ₂₆₀ . Solubility in all cases was perfect. For the synthesis of standard DNA / RNA oligonucleotides and partially modified LNA oligomers, standard CPG or polystyrene substrates were used. For the synthesis of fully modified LNA oligomers (for example, such as 5'-d (GTGATATGC) -3 '), a special solid substrate BioGenex Universal CPG Support (BioGenex, USA) was used or LNA-derived substrates were used.

Example 125

Synthesis of Phosphothioate-LNA Oligonucleotides

Fully phosphothioate-LNA oligonucleotides (Table 7) were synthesized using an automatic DNA synthesizer using approximately the same conditions as previously described (Example 124). Bucage reagent was used as the sulfonating agent. The reaction yields of a phased combination of monomers exceeded 98%. After completion of the synthesis, the release from the protective groups and the separation from the solid support were effectively carried out using concentrated ammonia solution (55 ° C, 14 hours).

Example 126

Synthesis of 2’-thio-LNA oligonucleotides

1'-thio-LNA oligonucleotides (including the monomer U ^S “thio-variant” of the formula Z shown in FIG. 2; FIG. 37, table 8) were synthesized using an automatic DNA synthesizer based on standard protocols (Example 124). The yield of the stepwise coupling for amidite 76F was approximately 85% (the duration of the coupling reaction was 12 minutes; it is expected that an increase in the purity of amidite 76F should result in an increase in the yield of the coupling reaction). Upon completion of the synthesis, the release from the protective groups and the separation from the solid support were effectively carried out using concentrated ammonia solution (55 ° C, 8 hours).

Example 127

Synthesis of 2’-amino-LNA oligonucleotides

Using procedures similar to those described in Example 126, 2'-amino-LNA oligonucleotides (including T ^NH monomer and T ^NMe monomer “amino variants” of formula Z shown in FIG. 2; FIGS. 35 and 36 ) were efficiently synthesized on an automatic DNA synthesizer using amidites 74A and 74F (phased combination yields ≥ 98%).

Example 128

Labeling LNA Oligomers with Fluorescein

LNA oligomers (of the formula Z shown in FIG. 2) AL16 (5'-d (TGTG-TGAAATTGTTAT) -3 '; bold selection refers to LNA-modified oligonucleotides) and AL17 (5'-d (ATAAAGTGTAAAG) -3'; bold isolation indicates LNA-modified oligonucleotides) were successfully labeled with fluorescein using the FluoroAmp T4 Kinase Green Oligonucleotide Labeling System reagent kit used in accordance with the manufacturer's recommendations (Promega). Briefly, 16 nmol of an LNA oligomer (either AL16 or AL17) was labeled with 5′-thiophosphate in 50 μl of reaction buffer containing T4 kinase and γ-A-ATP. The reaction was incubated at 37 ° C for 2 hours. Thiophosphorylated LNA oligomers were precipitated by adding 5 μl of precipitate for oligonucleotides (Promega) and 165 μl of ice-cold (up to -20 ° C) 95% ethanol. After centrifugation, the centrifuges were washed with 500 μl of ice-cold (up to -20 ° C) 70% ethanol and dissolved in 25 μl of PBSE buffer. A freshly prepared solution of 5-maleimidofluorescein (50 μg in 5 μl DMSO) was added to thiophosphorylated LNA oligomers and the reaction mixture was incubated at 68 ° C for 30 minutes. To each LNA oligomer was added 5-maleimidofluorescein (50 μg in 5 μl DMSO) and the reaction mixture was incubated for another 60 minutes. After incubation, 10 μl of precipitate for oligonucleotides were added to each reaction mixture, followed by 180 μl of ice-cold (up to -20 ° С) 70% ethanol and 100 μl of N, N-dimethylformamide. Fluorescein-labeled LNA oligomers were purified by reverse phase HPLC with the following parameters: Delta-Pak C-18 column, 300 A pores, 0.4 × 30 cm; eluent 0-50% acetonitrile in 0.04 M triethylammonium buffer (pH 7.0); flow rate 1.5 ml / min. Fractions containing LNA oligomers were combined and evaporated under reduced pressure (using a vacuum centrifuge equipped with an oil pump) for 12 hours.

Hybridization Data

Example 129

Thermostability of oligonucleotides, including monomers of the formulas V, X, Y and Z ^T , Z ^U , Z ^G , Z ^C , Z ^A , Z ^MeC

The thermal stability of LNA-modified oligonucleotides was determined spectrophotometrically using a spectrophotometer equipped with a Peltier thermoregulated element. 1 ml hybridization mixtures were prepared using one of three buffers (10 mM Na ₂ HPO ₄ , pH 7.0, 100 mM NaCl, 0.1 mM EDTA / 10 mM Na ₂ HPO ₄ , pH 7.0, 0.1 mM EDTA / 3 M tetramethylammonium chloride, 10 mM Na ₂ HPO ₄ , pH 7.0, 0.1 mM EDTA) and equimolar (1 μM or 1.5 μM) amounts of various LNA-modified oligonucleotides and complementary to them or including DNA mismatches RNA oligonucleotides. Identical hybridization mixtures using unmodified oligonucleotides were prepared as controls. The values of T _{m were} determined as the first derivative of the dissociation curve. In the table. 1-4 summarized the results (LNA options are shown in bold). Figure 2 shows the use of monomeric LNA. The nomenclature V, X, Y, and Z ^T , Z ^U , Z ^G , Z ^C , Z ^A , Z ^MeC corresponds to the structures V, X, Y, and Z shown in FIG. The tables indicate the bases that make up the LNA monomers. In addition, for the thio and amino variants of the LNA structures, the formula Z in the last two tables, according to the nomenclature used, denotes Z ^TS and Z ^TNH , respectively.

LNA-incorporating Z-structures were tested almost completely (see table. 1). When three Z ^T residues were introduced into the mixed sequence oligonucleotide, the T _m value determined in the buffer containing NaCl for both complementary DNA oligonucleotides (10) and for complementary RNA oligonucleotides (16) was significantly higher ( rough estimates for RNA and DNA are 7 ° C and 5 ° C, respectively, per 1 modification) compared to the value of T _{m of the} corresponding duplexes formed with unmodified oligonucleotides (1 and 8). Similar results were obtained in the analysis of LNA-containing Z ^T residues, as well as one of the residues Z ^G (21 and 24B) or Z ^U (25), Z ^C (69), Z ^MeC (65) and Z ^A (58) . When errors were introduced into the composition of RNA or DNA oligonucleotide targets, the value of T _m for LNA-modified oligonucleotides in all cases decreased significantly (11-15A and 17; 18-20 and 22-24A; 26-31; 57 and 59- 60; 63-64 and 66, and 67): this clearly indicates that LNA-modified oligonucleotides hybridize with their target sequences, obeying the rules of the Watson-Crick model based on the formation of hydrogen bonds. In all cases, the decrease in T _m for LNA-modified oligonucleotides in response to errors in the target was the same or even more pronounced compared to that determined for the corresponding unmodified oligonucleotides (2-7 and 9; 33-38): this confirms that LNA-modified oligonucleotides are at least as specific as their unmodified analogues. A decrease in the ionic strength of the hybridization solution (from 10 mM Na ₂ HPO ₄ , pH 7.0, 100 mM NaCl, 0.1 mM EDTA to 10 mM Na ₂ HPO ₄ , pH 7.0, 0.1 mM EDTA) results in a decrease T _m for LNA-modified oligonucleotides in relation to their complementary DNA oligonucleotides (40, 41) or RNA oligonucleotides (49A, 41A). A similar effect is observed in the analysis of unmodified oligonucleotides and their complementary DNA oligonucleotides (39) or RNA oligonucleotides (39A).

Adding 3 M tetramethylammonium chloride (TMAC) to the hybridization buffer leads to a significant increase in T _m LNA-modified oligonucleotides relative to their complementary DNA oligonucleotides (10, 21, 25). Moreover, the presence of TMAC leads to smoothing out the differences in the values of T _m determined for different oligonucleotides that were detected using NaCl-containing buffer (the range of the lowest and highest T _m 44 ° C and 49 ° C, respectively, for NaCl buffer against, respectively 56 ° C and 57 ° C in TMAC buffer). The introduction of errors leads to a significant decrease in T _m LNA-modified oligonucleotides in relation to their DNA targets (11-13, 18-20 and 26-28). A similar picture is observed in the analysis of unmodified control oligonucleotides (1-4 and 32-35).

The data obtained in the variant with low-salt buffers show that LNA-modified oligonucleotides exhibit sensitivity to the ionic strength of the hybridization buffer, similar to that characteristic of normal oligonucleotides. Based on the data obtained with the TMAC buffer, it was shown that TMAC has the effect of alignment of T _m for LNA-modified oligonucleotides, similar to the same effect found in the analysis of unmodified LNA oligonucleotides. LNA-modified oligonucleotides in both of these hybridization buffers retain their highly specific specificity.

Fully LNA-modified oligonucleotides, including all four monomers (71 and 75), almost completely modified LNA-oligonucleotides (modified in all positions, except for the 3'-terminal DNA nucleoside), including both Z ^G and Z ^T (41 and 41A), and partially modified oligonucleotides, including the central block Z ^T and Z ^G (40 and 40A), also exhibit a significantly increased level of affinity compared to the unmodified control oligonucleotide (39 and 39A; 1 and 8). This indicates that LNAs of formula Z are promising from the point of view of designing both fully and partially modified oligonucleotides. Applicants note that an almost completely modified oligomer (41 and 41A) exhibits an unprecedented high level of affinity for both RNA- (> 93 ° C) and DNA oligonucleotides (83 ° C). A similar exceptionally high affinity (for both types of oligonucleotides) was revealed in the analysis of an almost completely modified LNA oligomer, including exclusively the Z ^T variant (Tables 1: 52 and 53), and a fully modified LNA oligomer (71 and 75). The affinity of the partially modified poly-T-oligonucleotide depended on the positions occupied by the Z ^T introduced and on their number (44-51). Although the T _m values for target RNAs (45, 47, 49, and 51) were in all cases higher in comparison with the corresponding unmodified oligonucleotides (43), the same variants exhibit lower T _m for the target DNA (46). Since an oligonucleotide with a mixed sequence, including three Z ^T residues, showed a significantly increased level of affinity for its DNA (10) and RNA targets (16) compared to unmodified control oligonucleotides (1 and 8), this confirms that others, in contrast to the Watson-Crick model characteristic, motifs (such as, for example, the Hogsten binding motif) are “open” to poly-T-oligonucleotides, and also that such binding motifs are at least somewhat susceptible to specific st the structure of the modified oligonucleotide. In all cases, the introduction of individual erroneous (inappropriate) bases into the complex formed by completely modified Z ^T residues with a poly-T-oligonucleotide and target DNA (54-56) caused a significant decrease in Т _m .

Oligonucleotides, including LNA formulas of either V (Table 2), X (Table 3), or Y (Table 4), were analyzed in the context of the analysis of fully and partially modified poly-T sequences. Fully modified oligonucleotides of the formula V and Y show an increase in T _m (although less pronounced compared to Z ^T -modified oligonucleotides) with respect to both RNA targets (Tables 2, 14, and Tables 4, 14), and to DNA targets (Tables 2, 13, and Tables 4, 13) compared to unmodified oligonucleotides (Tables 1, 42, and 43). Partially modified oligonucleotides comprising monomers of formulas V and Y showed similar properties with respect to partially modified oligonucleotides including Z ^T : this is apparently due to the homopolymer nature of the sequence as defined above. Oligonucleotides including X ^T in all cases exhibit a significantly reduced T _m compared to control DNA oligonucleotides.

Example 130

The formation of stable hybrids with a fully modified LNA oligonucleotide with complementary DNA in both parallel and antiparallel orientations

A fully modified LNA oligonucleotide was hybridized with its complementary DNA in both orientations - parallel and antiparallel. The composition of the hybridization solutions (1 ml) included 10 mm Na ₂ HPO ₄ (pH 7.0), 100 mm NaCl and 0.1 mm EDTA and 1 μm of each of the two oligonucleotides. As shown in the table. 1, both antiparallel (71) and parallel binding orientations (77) led to the formation of stable duplexes. Of these two options, the option with antiparallel orientation led to the greatest stability. However, even a duplex with a parallel orientation is significantly more stable than the corresponding antiparallel duplex with unmodified DNA oligonucleotides (Table 1, 1).

Example 131

The possibility of using LNA monomers to increase the affinity of RNA oligomers for complementary nucleic acids

The thermal stability of the complexes formed by a 9-dimensional RNA oligonucleotide, including three LNA-T monomers (Z ⁷ ), and complementary DNA or RNA oligonucleotides, was determined spectrophotometrically. The composition of the hybridization solutions (1 ml) included 10 mm Na ₂ HPO ₄ (pH 7.0), 100 mm NaCl and 0.1 mm EDTA and 1 μm of each of the two oligonucleotides. As a control, identical hybridization mixtures using unmodified RNA oligonucleotides were formed. As can be seen from the table. 5, the LNA-modified RNA oligonucleotide hybridizes with both the complementary DNA oligonucleotide (1) and the complementary RNA oligonucleotide (3). As was previously established for LNA-modified DNA oligonucleotides, the affinity for binding of LNA-modified RNA oligonucleotides is most pronounced for RNA complement (3). In both cases, the affinity of the LNA-modified RNA oligonucleotide is significantly higher compared to the unmodified control (2 and 4). In the table. 5 also shows that in the case of LNA-modified RNA oligonucleotides, specificity for both DNA and RNA targets is maintained.

Example 132

Base pairing LNA-LNA

RNA or DNA oligonucleotides comprising three Z ^T -LNA monomers, or oligonucleotides fully modified by LNA-Z monomers, were hybridized with complementary unmodified DNA oligonucleotides or DNA oligonucleotides comprising three LNA-Z ^A monomers: T _m the resulting hybrids were determined spectrophotometrically. The composition of the hybridization solutions (1 ml) consisted of 10 mm Na ₂ HPO ₄ (pH 7.0), 100 mm NaCl and 0.1 mm EDTA and 1 μm of each of the two oligonucleotides. As can be seen from the table. 6, all LNA-modified oligonucleotides hybridized with complementary unmodified DNA oligonucleotides (2 and 3), as well as with complementary LNA-modified oligonucleotides (4, 5 and 6). As was established earlier, the presence of LNA monomers in one of the hybrid chains (2 and 3) causes a significant increase in T _m compared to the unmodified control molecular hybrid (1). The presence of LNA-LNA base pairs in a hybrid duplex causes an even greater increase in T _m (4 and 5). Moreover, a hybrid characterized by a high level of stability can be formed between a fully modified LNA oligonucleotide and an oligonucleotide partially modified with LNA-Z ^A residues (6). This is the first example of pairing in pairs of LNA-LNA in a hybrid molecule.

Example 133

Manifestation of a fully LNA-phosphomonothioate oligonucleotide with a relatively lower decrease in thermal stability with respect to complementary DNAs and RNAs compared with a fully DNA-phosphotioate DNA oligonucleotide

The thermal stability of a fully DNA phosphomonothioate oligonucleotide comprising three LNA-Z ^T monomers (LNA oligonucleotide) and the corresponding fully DNA phosphomonothioate control oligonucleotide with respect to complementary DNA and RNA were evaluated under the same conditions as described in example 132, however EDTA was excluded (Table 7). It was found that a fully LNA-phosphomonothioate oligonucleotide, including the three LNA-Z ^T monomers, shows only slightly reduced thermal stability (Tables 7, 3 and 4) when compared with the corresponding control LNA-oligonucleotide (Tables 1, 10 and 16 ) The corresponding fully DNA phosphomonothioate oligonucleotide (Tables 7, 1 and 2) showed a significant decrease in thermal stability when compared with the corresponding control DNA oligonucleotide (Tables 1, 1 and 8). This may have the prospective use of fully or partially LNA-phosphomonothioate oligonucleotides in “antisense therapy” and other therapeutic approaches. Thus, the compatibility of LNA monomers and unmodified monomers in the phosphomonothioate oligonucleotide was confirmed. It can be expected that such constructs will exhibit both RNase-H activity and nuclease resistance in addition to improving hybridization parameters due to the presence of LNA.

Example 134

Manifestation of 2’-thio-LNA nucleic acid recognition properties comparable to those of the LNA itself (Z-monomer)

Hybridization conditions were consistent with those described in example 132, with the exception of EDTA. The results obtained for 2'-thio-LNA (Table 8) clearly show the presence of a positive effect on the thermal stability of duplexes with respect to both DNA and RNA, as a result of the introduction of the 2'-thio-LNA monomer U ^S (monomers corresponding to the formula Z shown in FIG. 2, despite the fact that the “bridge” of the methyleneoxy group is replaced by the “bridge” of the methylenetio group). This effect (Δ Т _m ≈ + 5 ° С per one modification, in relation to the target DNA; Δ Т _m ≈ + 8 ° С per one modification, in relation to the target RNA) is comparable to that which was detected for the original LNA. This picture is somewhat confused with the simultaneous introduction of two modifications (2'-thio group and uracil instead of thymine). However, taking into account the fact that earlier the applicants revealed the identity of dissociation temperatures of the LNA monomers of thymine and uracil, and taking into account the fact that the presence of 2'-deoxyuridine in the control molecules instead of thymidine should lead to the manifestation of lower T _m values, such a comparison is acceptable.

Example 135

The manifestation of 2'-amino-LNA (monomer 2 ^TNH ) and 2'-methylamino-LNA (monomer Z ^TNMe ) recognition properties of nucleic acids, comparable to those characteristic of the original LNA (monomer Z)

Hybridization conditions were consistent with those described in example 132, with the exception of EDTA. The data on duplex dissociation parameters for variants with 2'-amino-LNA (Table 9) clearly show the presence of a positive effect on the thermal stability of duplexes involving both DNA and RNA due to the addition of 2'-amino-LNA monomers T ^NH or T ^NMe (monomers corresponding to the formula Z shown in FIG. 2, while the “bridge” of the methyleneoxy group is replaced by the “bridge” of the methyleneamino group or the “bridge” of the methylene (N-methyl) amino group, respectively). This effect (Δ Т _m ≈ + 3 ° С per one modification, in relation to the target DNA; Δ Т _m ≈ from + 6 ° С to 8 ° С per one modification, in relation to the target RNA) is comparable to the one that was identified for the original LNA. It is also noteworthy that increased thermal stability is also observed in the variant with an oligomer comprising a mixture of 2'-alkylamino-LNA monomers and non-alkylated 2'-amino-LNA monomers.

LNA and LNA-modified oligonucleotides as substrates for enzymes.

Example 136

3'-exonucleolytic stability of 5'-V oligomers $\genfrac{}{}{0ex}{}{\text{T}}{\text{thirteen}}$ T and 5'-Z $\genfrac{}{}{0ex}{}{\text{T}}{\text{thirteen}}$ T

A solution of oligonucleotides (0.2 OD) in 2 ml of buffer (0.1 M Tris-HCl, pH 8.6, 0.1 M NaCl, 14 mm MgCl ₂ ) was treated at 1.2 ° C with 1.2 units SVPDE (snake venom phosphodiesterase). During processing, an increase in absorption at 260 nm was noted. While unmodified polythymidine-14 was completely destroyed in 10 minutes of the enzyme, 5'-V variants $\genfrac{}{}{0ex}{}{\text{T}}{\text{thirteen}}$ T and 5'-Z $\genfrac{}{}{0ex}{}{\text{T}}{\text{thirteen}}$ T remained intact for 60 minutes.

Example 137

LNA-modified oligomers as substrates for T4 polynucleotide kinase.

20 picomoles of each seed (FP2: 5'-GGTGGTTTGTTTG-3 ': DNA probe), (AL2: 5'-GGTGGTTTGTTTG-3': LNA nucleosides are shown in bold) and (AL3: 5'-GGTGGTTTGTT : LNA nucleosides in bold) were mixed with T4 polynucleotide kinase (5 units: New England Biolabs) and 6 μl of γ - ^32P - ^ATP (300 Ci / mmol, Amershaiti) in a buffer containing 70 mM Tris-Hcl (pH 7, 6), 10 mM MgCl ₂ , 5 mM dithiothreutol (final volume 20 μl). Samples were incubated at 37 ° C for 40 minutes and then heated to 65 ° C for 5 minutes. To each reaction mixture were added 2 μl of tRNA (1 μg / μl), 29 μl of 3 M ammonium acetate and 100 μl of ethanol. The reaction mixtures were incubated at -20 ° C for 30 minutes and the labeled oligonucleotides were precipitated by centrifugation at 15000g for 30 minutes. The pellet was resuspended in 20 μl of water. Samples (1 μl) were mixed with loading buffer (formamide [pH 8.0], 0.1% xylencyanol FF, 0.1% bromophenol blue and 10 mm EDTA) and subjected to denaturing polyacrylamide gel electrophoresis (16% acrylamide / bisacrylamide solution , 7 M urea, 1xTBE and 0.1 mM EDTA) in TBE buffer (90 mM Tris-Hcl [pH 8.3], 90 mM boric acid and 2.5 mM disodium EDTA). The gel was dried in a gel dryer (BioRad, Model 583) and subjected to radiographic analysis using an X-ray film (CL-XPosure film, Pierce 34075) for 20 minutes. The results are shown in Fig.6 (FP2 - tracks 1 and 2; AL2 - tracks 3 and 4; AL3 - tracks 5 and 6). Based on the experimental data, three conclusions can be drawn. Firstly, it can be concluded that partially and fully modified LNA oligomers are effective mimics of native nucleic acids in terms of their ability to serve as a substrate for enzymes specific for nucleic acids, such as polynucleotide kinase. Secondly, it can be concluded that LNA-modified oligomers can be efficiently precipitated in techniques commonly used to precipitate common nucleic acids. Indeed, relative signal intensity levels for unmodified (lanes 1, 2), partially modified (lanes 3, 4), and fully modified oligomers (lanes 5, 6) in X-ray diffraction patterns confirm that the larger the LNA nucleosides are part of standard DNA oligonucleotide, the more effective it can be precipitated in the salt-alcohol procedure. Thirdly, the similar positions of the signals in the X-ray diffraction patterns corresponding to unmodified, partially and completely modified oligonucleotides shows that the incorporation of LNA nucleosides into DNA oligomers does not lead to a change in its electrophoretic mobility in the polyacrylamide gel.

Example 138

3’-terminal labeling of LNA-containing oligonucleotides using terminal deoxynucleotidyl transferase

Oligonucleotides comprising LNA monomers were 3’-labeled using the terminal deoxynucleotidyl transferase enzyme. The sequences and degree of modifications using LNA were as follows (LNA monomers are indicated in bold):

An oligonucleotide (50 pcmol) was incubated with 250 μCi α - ³² R-ddATP (3000 Ci / mmol) and 100 units. terminal deoxynucleotidyl transferase in 250 μl of 100 mm cacodilate buffer, pH 7.2, 2 mm CoCl ₂ and 0.2 mm 2-mercaptoethanol at 37 ° C for 2 hours. The reaction was stopped by adding formamide-containing loading buffer and heating to 100 ° C for 5 minutes before being placed on ice. Samples (0.2 pmol) were passed through a 19% acrylamide gel containing 7 M urea: the level of radioactivity incorporation into oligonucleotide bands was quantitatively determined using a phosphorus-sensitive sensor (Molecular Dynamics). The results showed the presence of radioactivity inclusion in all tested cases, including the oligonucleotide, characterized by the highest amount of LNA: control - 94.9%, (1) - 39.7%, (2) - 83.7%, (3) - 31, 7% It is concluded that LNA-modified oligonucleotides are a substrate for the terminal deoxynucleotidyl transferase enzyme.

Example 139

The ability of terminal deoxynucleotidyl transferase (TdT) to provide terminal attachments in modified oligonucleotides depending on the design of the oligomer.

The following 15 nucleotide seeds and a mixture of 8-32-dimensional oligonucleotide markers were labeled at the 5'-end of [γ - ³³ P] -ATP and T4 polynucleotide kinase (where LNA monomers are shown in bold):

The reaction mixtures were boiled for 5 minutes after labeling to exclude residual polynucleotide kinase activity. 4 pcmol of each marked seed, 25 units. terminal deoxynucleotidyl transferase and 16 μM dATP were incubated in 25 μl of 100 mM cacodylate buffer (pH 7.2), 2 mM CoCl ₂ and 0.2 mM 2-mercaptoethanol for 90 minutes at 37 ° C. Reactions were stopped by the addition of formamide-containing stop solution and reaction products were passed through a 19% polyacrylamide gel containing 7 M urea and labeled markers. An autoradiographic analysis of the dried gel was carried out using a Biomax film. As shown in Fig. 22, for variants P1 (lane 2), P2 (lane 4) and PZ1 (lane 3), in all cases, a tail extension estimated at more than 70 nucleotides is detected, taking into account the parameters of 8-32-nucleotide marker fragments (lanes 1 and 6). Under the same conditions, the terminal completion reaction of the PZ2 variant does not occur. The conclusion is made that the TdT enzyme will be tolerant to the presence of LNA monomers within the oligonucleotide, but not at its 3'-end.

Example 140

LNA-thymidine-5’-triphosphate (LNA-TTP) as a substrate for terminal deoxynucleotidyl transferase (TdT)

In order to analyze the ability of the triphosphate in the composition of LNA-TTP (Example 123) to be recognized by the terminal deoxynucleotidyl transferase as a substrate, an “terminal extension” of the oligonucleotide was carried out. A 15-nucleotide seed (its sequence: 5'-TGC ATG TGC TGG AGA-3 ') and a mixture of 8-32-dimensional oligonucleotides were labeled at the 5'-ends using [γ - ³³ P] -ATP and T4 polynucleotide kinase. The reaction mixtures were boiled for 5 minutes after labeling to exclude residual polynucleotide kinase activity. 4 pmol of labeled seed, 25 units terminal deoxynucleotidyl transferase and 32, 64 or 128 μM dTTP or LNA-TTP were incubated in 25 μl of 100 mM cacodylate buffer (pH 7.2), 2 mM CoCl ₂ and 0.2 mM 2-mercaptoethanol for 90 minutes at 37 ° C. Reactions were stopped by the addition of formamide-containing stop solution and reaction products were passed through a 19% polyacrylamide gel containing 7 M urea and labeled markers. An autoradiographic analysis of the dried gel was carried out using a Biomax film. As shown in FIG. 10, in all cases, in reactions with 32 μM dTTP (lane B), 64 μM dTTP (lane C) or 128 μM dTTP (lane D), the “tail extension” of the oligonucleotide occurred, as indicated by comparison with 8- 32-dimensional oligonucleotide markers (extreme left and right tracks), the size of which is estimated at more than 100 nucleotides. Reactions with LNA-TTP (32 μM dTTP (lane E), 64 μM dTTP (lane F) or 128 μM dTTP (lane G) in all cases led to an extension of the seeds by one base, and in about half the cases there was another nucleotide . These results are largely similar to those obtained for ribonucleotides and TdT. It is concluded that LNA-derived triphosphates can be recognized and incorporated into a DNA oligonucleotide with the participation of the TdT enzyme. This observation, according to which LNA-TTP can bind to polymerase, confirms the prospects of the successful use of derivatives of LNA monomers as nucleoside-containing drugs.

Example 141

Possibility of incorporation of LNA-adenosine, cytosine, guanosini and uridine 5'-triphosphates (LNA-ATP, LNA-CTF, LNA-GTP, LNA-UTP) into the DNA strand under the control of a Klenov fragment without exonuclease activity DNA polymerase I.

The seed completion test was used to evaluate LNA-NTP (see Example 123) and ribonucleotides as potential substrates for the free Maple fragment of DNA polymerase I. In this test, the 5'-terminal ³³ P-labeling of a 15-nucleotide seed was used, hybridizable to one of four different 24-nucleotide matrices. The seed and matrix sequences are as follows (LNA monomers are shown in bold):

One pmol of ³³ P-labeled seeds was hybridized with 2 pmol of the matrix in × 2 Klenov buffer. To this mixture was added either 4 μM dNTPα S, or 500 μM LNA-HTP, or a mixture of 4 μM dNTPα S and 500 μM LNA-HTP. 2 units were added to each reaction. Klenov fragment of DNA polymerase. To each of the reaction mixtures was added 2 ppm. inorganic pyrophosphatase. Seed, matrix and enzyme were also combined in the control variant. All reactions were carried out with a total volume of 20 μl. The reaction mixtures were incubated at 37 ° C for 3 minutes. Then the reaction was stopped by adding 10 μl of stop solution of formamide-EDTA. The reaction products were separated in a 19% polyacrylamide gel with the addition of 7 M urea and the obtained fragments were diagnosed by size, comparing with the “ladder” ³³ P-labeled 8-32 oligonucleotide standards and using a Kodak Biomax X-ray film for shooting.

On Fig shows the experience with LNA-UTP for matrix 1. Shown tracks (1-12) correspond to the following reactions: the introduction of LNA-UTP with the participation of the Klenov fragment. Lane 1 - seed, matrix and enzyme. Lane 2 is the same plus dTTPα S. Lane 3 is plus LNA-UTP. Lane 4 - plus dTTPα S and dGTFα S. Lane 5 - plus LNA-UTP and dGTPα S. Lane 6 - plus dATPα S, dGTPα S and dTTPα S. Lane 7 - plus LNA-UTP, dTTPα S, dGTPα S and dTTPα S and dTTPα S Lane 8 - plus dGTPα S. Lane 9 - plus dTTPα S, dGTPα S and dTTPα S. Lane 10 - plus LNA-UTP, dATPα S, dTTPα S and dGTPα S. Lane 11 - plus dATPα S, dTTPα S and dGTPα S and dGTPα S Lane 12 — all 4 dNTPhs S. The extreme lanes correspond to 8-32-dimensional oligonucleotide markers used to determine the length of the tested products.

As can be seen from FIG. 20, LNA-UTP is specifically incorporated as nucleotide “T”. Further completion at the 3’-end, on which the LNA-UTP is located, is significantly slowed down.

The figure 21 shows the results of experiments with LNA-ATP, LNA-CTF and LNA-GTP using matrices 2-4. Tracks (1-21) correspond to the following reactions. Lanes 1, 7, 13, and 17 — seed, matrix, and enzyme. Lane 2 is the same plus dGTPα S. Lane 3 is plus dATPα S and dGTPα S. Lane 4 is plus LNA-GTP. Lane 5 - plus dGTPα S and LNA-ATP. Lane 6 - plus LNA-ATP and LNA-GTP. Lane 8 - plus dATPα S. Lane 9 - plus dATPα S and dTTPα S. Lane 10 - plus LNA-ATP. Lane 11 - plus dTTPα S and LNA-ATP. Lane 12 plus DATPα S and LNA-CTF. Lane 14 plus dTTPα S. Lane 15 plus dGTPα S and dTTPα S. Lane 16 plus dTTPα S and LNA-GTP. Lane 18 - plus dTTPα S. Lane 19 - plus dTTPα S and dTTPα S. Lane 20 - plus LNA-CTF. Lane 21 — dTTPα S and LNA-CTF. The extreme lanes correspond to 8-32-dimensional oligonucleotide markers used to determine the length of the tested products.

Experiments using matrix 2 (tracks 1-6) show that LNA-GTP are able to form the product “+1” with effective lengthening of the seed (4th track). The addition of dGTPα S and LNA-ATP in most cases leads to the formation of the product “-1-2” (5th track). This is due to the inclusion of dGTPα S with the formation of the product “+1” and subsequent completion due to LNA-ATP. There is an indication of the presence of a small amount of “+3” product, which is due to the subsequent inclusion of LNA-ATP. Matrix 3 experiments (tracks 7-12) show that LNA-ATP is effectively incorporated to form the “+1” product (track 10). The completion of this product involving dTCPα S is slowed down (track 11). Adding dATPα S and LNA-CTF leads to the formation of “+2” and “+3” products (12th track). The absence of any significant amount of the “+1” product indicates that the addition of the first LNA-CTF monomer is effective, while the addition of the second LNA-CTF residue is delayed. The results of experiments with matrices 1 (tracks 13-16) and 4 (tracks 17-21) indicate the presence of trends similar to those of other matrices. LNA-CTF is effectively incorporated with the formation of the product “+1” when using the matrix (20th track). The completion of this product with the participation of dTTPα S is again delayed (track 21). The addition of LNA-GTP and dTTPα S in the reaction with matrix 1 determines the formation of the “+2” product (16th track). Again, these data indicate that the addition of a single LNA triphosphate is very effective, but the addition of subsequent LNA triphosphate is slow.

Example 142

The possibility of using LNA monomers to increase the resistance of the oligonucleotide to exonuclease III cleavage.

In order to test the resistance parameters of LNA-containing oligonucleotides to cleavage by exonuclease III, the following reaction was carried out. The following 15 nucleotide primers and 8-32-dimensional oligonucleotide standards were subjected to 5'-end labeling using [γ - ³³ P] -ATP and T4 polynucleotide kinase (where LNA monomers are shown in bold):

The reaction mixtures were boiled for 5 minutes after labeling to exclude residual polynucleotide kinase activity. 8 pmol of each seed was hybridized with 25 pmol of the matrix (its sequence: 3’-ACG TAC ACG ACC TST ACC TTG STA-5 ’) in 2 Klenov buffer. 10 units were added to each reaction. exonuclease III. Control variants were also set up, in which 1 μl of water was added instead of exonuclease. The reaction mixtures were incubated at 37 ° C for 5 minutes. Reactions were stopped by the addition of 10 μl of formamide-EDTA stop solution. The reactions were heated to 95 ° C for 3 minutes, followed by loading into a 19% polyacrylamide gel containing 7 M urea. The gel was fixed with a mixture of 10% acetic acid and 10% methanol, followed by transfer to ZMM paper and drying. The dried gel was analyzed on a phosphorus sensor for 3 hours. This scanner was powered by the Molecular Dynamics Storm 860 equipped with ImageQuant. A gel scan showed that, in the absence of exonuclease, the signal intensity for full-size P2 and PZ2 seeds was, respectively, 99% and 96%. In the presence of the enzyme after a 5-minute incubation, only 20% of the signal of the full-size P2 seed was preserved. However, for the full-size PZ2 seed, at the same processing mode, 62% of the signal intensity was preserved. This indicates that the only LNA monomer attached at the 3’ end of the oligonucleotide gives it increased resistance to exonuclease III.

Application in PCR.

Example 143

LNA monomers can be used to significantly increase the efficiency of using biotinylated DNA oligonucleotides for sequence-specific selection (“capture”) of PCR amplifiers in the MTP format. Two digoxigenin-labeled amplicons based on the sequence of plasmid pUC19 were obtained by PCR as follows.

PCR reaction mixture for 1st amplicon:

1 μl pUC19 (1ng / μl),

1 μl of back seed (5’-AACAGCTATGACCATG-3 ’) (20 μM),

1 μl of direct seed (5'-GTAAAACGACGGCCAGT-3 ⁷ ) (20 μm),

10 μl dNTP-mix (2 mM dATP, 2 mM dTSP, 2 mM dGTP and 6 mM DUTP),

1.5 μl of digoxigenin-11-dUTP (1 mm),

10 μl of 10x Taq buffer (Boehringer Mannheim, includes MgCl ₂ ),

1 μl Taq polymerase (Boehringer Mannheim) (5 units / μl),

distilled water 100 μl.

PCR reaction mixture for 2nd amplicon:

1 μl pUC19 (1ng / μl),

0.4 μl of seed 3 (5’-GATAGGTGCCTCACTGAT-3 ’) (50 μM),

0.4 μl of seed 4 (5’-GTCGTTCGCTCCAAGCTG-3 ’) (50 μM),

10 μl dNTF-mix (2 mM dATP, 2 mM dTTP, 2 mM dGTP and 6 mM dUTP),

1.5 μl of digoxigenin-11-dUTP (1 mm),

10 μl of 10x Taq buffer (Boehringer Mannheim, includes MgCl ₂ ),

1 μl Taq polymerase (Boehringer Mannheim) (5 units / μl),

distilled water 100 μl.

PCR parameters (Perkin-Elmer 9600 model is used): 94 ° С - 5 minutes; addition of polymerase; 94 ° C - 1 minute, 45 ° C - 1 minute, 70 ° C - 2 minutes (29 cycles); 72 ° C - 10 minutes.

10 μl of each PCR was analyzed on a standard agarose gel in which the expected fragments of 100 and 500 nucleotides in length were detected.

10 μl of digoxigenin-labeled amplicon 1 or amplicon 2 was mixed with 5 pmol of a 5'-biotinylated selection probe in 1xSSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0) with a total volume of 450 μl. The following screening probes were used: B-DNA1 (Biotin-ATGC-CTGCAGGT-CGAC-3 '; DNA probe specific for 1st amplification), B-DNA2 (Biotin-GGTGGTTTGTTTG-3'; DNA probe, specific in relation to the 2nd amplicon), B-LNA2 (biotin-GGTGGT-TTGTTTG-3 '; LNA nucleosides are shown in bold; LHA probe specific for the 2nd amplicon). The reactions were heated to 95 ° C for 5 minutes to denature the amplicons and allowed to cool at 25 ° C for 15 minutes, providing hybridization between the probe and the target amplicon sequence. After hybridization, 190 μl of each reaction mixture was transferred onto a streptavidin-coated plate (Pierce, Catalog No. 15124) and incubated for 1 hour at 37 ° C. After washing the plate with phosphate-buffered saline (FSBT: 0.15 M Na ⁺ , pH 7 , 2, 0.05% Tween-20, 3 × 300 μl) was added 200 μl of antidigoxygenin antibodies labeled with peroxidase (Boehringer Mannheim, 1: 1000 dilution in phosphate-buffered saline). The plates were incubated for 30 minutes at 37 ° C and washed (FSBT, 3 × 300 μl). Wells were tested for peroxidase activity by adding 100 μl of a substrate solution (0.1 M citrate phosphate buffer, pH 5.0, 0.66 mg / ml ortho-phenylenediamine dihydrochloride, 0.012% hydrogen peroxide). The reaction was stopped after 8 minutes by adding 10 μl of sulfuric acid (0.5 M) and the absorbance parameters were read at 492 nm using a microplate reader. As shown in FIG. 3, unmodified biotin-DNA-selection probes (B-DNA1 and B-DNA2) in both cases behave as expected, i.e. each of them selects only “its” PCR amplicon. Compared to the B-DNA1 probe, the B-DNA2 probe is characterized by greater inefficiency in the selection of “own” amplificon. However, the selection ability of the B-DNA2 probe can be dramatically increased by replacing 12 of 13 deoxyribonucleosides with the corresponding LNA nucleosides. As shown in FIG. 3, the use of a B-LNA2 probe instead of a B-DNA2 probe causes a more than 10-fold increase in sensitivity in the test. At the same time, the B-LNA2 probe retains the ability inherent in the unmodified B-DNA2 probe to efficiently distinguish between related and unrelated amplicons, thereby ensuring the specificity of LNA oligomers. It is concluded that (1) biotin covalently attached to the LNA-modified oligomer retains the ability to bind to streptavidin, (2) LNA-modified oligomers are effective in the MTP-mediated amplicon assay, and (3) LNA provides a sharp increase in efficiency the use of standard DNA oligonucleotides for affinity selection of PCR amplicons.

Example 144

The ability of LNA-substituted oligomers to select their corresponding PCR amplicons according to the “chain invasion” mechanism.

Two identical compositions of 10 μl of reactions with amplicon 1 or 2 (prepared in the same way as described in Example 143) were mixed with 1, 5, or 25 pmol of the B-LNA2 selection probe (Biotin-GGTGGTTTGTTTG-3 '; LNA nucleosides are shown in bold ; LHA probe specific for the 2nd amplicon) in 1xSSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0) with a total volume of 450 μl. One set of reactions was heated to 95 ° C for 5 minutes to denature the amplicons and allowed to cool at 25 ° C for 15 minutes, ensuring hybridization between the probe and the target amplicon sequence. Another set of reactions was left without a denaturation step. 190 μl from each reaction was transferred onto a streptavidin-coated plate (Pierce, Catalog No. 15124) and incubated for 1 hour at 37 ° C. After washing the plate with phosphate-buffered saline (FSBT: 0.15 M Na ⁺ , pH 7, 2, 0.05% Tween-20, 3 × 300 μl) 200 μl of peroxidase-labeled antidigoxygenin antibodies (Boehringer Manneim 1: 1000 dilution in phosphate-buffered buffer) was added. The plates were incubated for 30 minutes at 37 ° C and washed (FSBT, 3 × 300 μl). Wells were tested for peroxidase activity by adding 100 μl of a substrate solution (0.1 M citrate phosphate buffer, pH 5.0, 0.66 mg / ml ortho-phenylenediamine dihydrochloride, 0.012% hydrogen peroxide). The reaction was stopped after 10 minutes by adding 10 μl of sulfuric acid (0.5 M) and the absorbance parameters were read at 492 nm using a microplate reader. When the amplicons were denatured before hybridization with a selection probe (Fig. 4A), the same efficiency and sequence specificity were determined as in the experiment described in Example 143. An increase in the concentration of the B-LNA2 probe from 1 to 5 pmol leads to an increase in the selection efficiency. A further increase to 25 pcmol of the probe leads to a decrease in signal intensity. This observation corresponds to the saturation of potential binding sites of biotin with streptavidin-MTP. In an embodiment where amplicons were not denatured on the eve of hybridization with a selection probe (Fig. 4B), efficient and sequence-specific selection of amplicon was also noted. Indeed, the data obtained show that selection of amplicons without prior denaturation is as effective and specific as selection after denaturation. This clearly confirms that the Bio-LNA2 probe is able to bind to its target sequence via a “chain invasion” mechanism. It seems that the first example of a sequence-specific recognition of double-stranded DNA in physiological conditions according to salt concentration using a mixed purine-pyrimidine probe is presented. In addition to the potential significant simplification of a number of basic research and diagnostic procedures, this unexpected property of LNA-modified oligonucleotides can be promising from the point of view of fundamentally improving methods for creating new highly effective drugs used in the technology of “antisense therapy”, in particular, the so-called "Antigenic approach."

Example 145

Effective sequence-specific selection of a PCR amplicon by an LNA-substituted oligomer immobilized on a solid support.

Wells of a streptavidin-coated microtiter plate (Boehringer Mannheim) were incubated for 1 hour with either 5 pmol of the B-DNA2 probe (Biotin-GGTGGTTTGTTTG-3 '; DNA probe specific for the 2nd amplicon) or B-LNA2 probe (biotin -GGTGGTTTGTTTG-3 '; LNA nucleosides are shown in bold; LHA probe specific for the 2nd amplicon) with a total volume of 100 μl in 1xSSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0). Altogether, four wells were incubated with a B-DNA2 probe, four wells with a B-LNA2 probe, and four wells were incubated with a “clean” buffer. After incubation, the wells were washed three times in 1xSSC. Digikigenin-labeled amplicon-1 (60 μl) or amplificon-2 (60 μl) was mixed with 540 μl of 1xSSC, denatured by heating to 95 ° C for 5 minutes and transferred (100 μl) to the wells of a microtiter plate. Two wells containing either B-DNA2 or B-LNA2 or not containing a selection probe “received” amplicon-1 and two wells containing either B-DNA2 or B-LNA2 or not containing a selection probe, “ received "amplicon-2. After 1 hour at 37 ° C, the plate was washed three times with phosphate-buffered saline (FSBT: 0.15 M Na ⁺ , pH 7.2, 0.05% Tween-20, 3 × 300 μl) and 200 μl of antidigoxygenin antibodies were added, labeled with peroxidase (Boehringer Mannheim, 1: 1000 dilution in phosphate-buffered saline). The plates were incubated for 30 minutes at 37 ° C and washed (FSBT, 3 × 300 μl). Wells were tested for peroxidase activity by adding 100 μl of a substrate solution (0.1 M citrate phosphate buffer, pH 5.0, 0.66 mg / ml ortho-phenylenediamine dihydrochloride, 0.012% hydrogen peroxide). The reaction was stopped after 6 minutes by adding 10 μl of sulfuric acid (0.5 M) and the absorbance parameters were read at 492 nm using a microplate sensor. As shown in FIG. 5, an LNA-modified selection probe (B-LNA2) selects a specific amplicon (amplicon-2) with a very high degree of efficiency and significantly better (with a sensitivity greater than about 5 times) than the corresponding unmodified DNA selection probe (B-DNA2). No signal was detected in the variant when the B-LNA2 probe was incubated with a “foreign” amplicon (amplicon-1), which confirms the very precise specificity of the B-LNA2 probe. The conclusion is made that LNA-amplified oligonucleotides are effective in sequence-specific selection of PCR amplicons when they are immobilized on a solid surface. It is also concluded that the use of LNA-amplified oligonucleotides instead of standard DNA oligonucleotides will increase the received signals above the background noise level. Thus, LNAs provide a way to significantly improve the applicability of modern DNA-based tests that use immobilized selection probes, for example, in the format of “immobilized kits” in which multiple probes are used to simultaneously detect the presence of several different nucleotide sequences in a sample.

Example 146

The possibility of using fully mixed LNA monomers to significantly increase the efficiency of using immobilized biotinylated DNA oligonucleotides for sequence-specific selection of PCR amplicons in MTP format.

Three digoxigenin-labeled amplicons from the N-ras oncogene sequence (see: Nucl. Acids Res., 1985, 13 [14], 52-55) were generated using PCR amplification.

PCR Seeding:

Direct seed 5’-CCAGCTCTCAGTAGTTTAGTACA-3 ’, nucleotides 701-723 in accordance with the published sequence.

Reverse seed 910-bp: 5’-GTAGAGCTTTCTGGTATGACACA-3 ’, nucleotides 1612-1590 (reverse sequence according to published sequence).

Reverse seed 600-bp: 5’-TAAGTCACAGACGTATCTCAGAC-3 ’nucleotides 1331-1308 (reverse sequence according to published sequence).

200-bp seed: 5’-CTCTGTTTCAGACATGAACTGCT-3 ’nucleotides 909-886 (reverse sequence according to published sequence).

PCR reaction mixture for N-ras amplicons. 2.3 μl of human placenta genomic DNA (440 ng / μl), 50 μl 10 × PCR buffer (without MgCl ₂ , Perkin-Elmer), 30 μl 25 mM MgCl ₂ , 50 μl dNTP-mix nucleotide mixture (2 mM each) dATP, dGTP, dTTP and 1.8 mM dTTP), 10 μl 1 mM digoxigenin-11-dUTP, 10 μl 25 μM direct seed, 10 μl 25 μM back seed, 5 μl 3 units / μl AmpliTaq Gold reagent (Perkin Elmer ) and water up to 500 μl. PCR conditions. The above reaction mixture was prepared for each of the N-ras PCR products. The only difference was the inclusion of different 910-bp, 600-bp and 200-bp back seeds. PCR mixtures were aliquoted in ten PCR tubes and placed in a Perkin-Elmer 9600 model amplifier, observing the following amplification conditions: 95 ° C for 3 minutes; 95 ° С - 1 minute, 55 ° С - 2 minutes, 72 ° С - 3 minutes (30 cycles); 72 ° C - 10 minutes; 4 ° C - exposure. 10 μl of each PCR was analyzed on a standard agarose gel and the presence of fragments of the expected size — 910, 60, and 200 pairs of nucleotides — was observed. Testing conditions. Wells of a streptavidin-coated microtiter plate (Boehringer Mannheim; binding capacity of 20 pmol biotin per well) were incubated for 1 hour in 5xSSCT (0.75 M NaCl, 75 mm citrate, pH 7.0, 0.1% Tween-20) at 37 ° C with 1 pmol of the probe DNA-Nras-Cap-A (biotin-5'-ТТССАСАГСАСАА-3 '), LNA / DNA-Nras-Сар-A (biotin-5'-ТТССАСАГСАСАА-3'), LNA-Nras -Cap-A (biotin-5'-TTCCACAGCACAA-3 '), DNA-Nras-Cap-B (biotin-5'-AGAGCGATAAAS-3'), LNA / DNA-Nras-Cap-B (biotin-5'- AGAGCCGATAACA-3 ') or LNA-Nras-Cap-B (Biotin-5'AGAGCCGATAACA-3'); LNA nucleosides are highlighted in bold. Nras-Cap-A selection probes provide the selection of Nras-910, Nras-600 and Nras-200 amplicons. Nras-Cap-B sampling probes allow selection of the Nras-910 and Nras-600 amplicons. After incubation of the various selection probes, the wells were washed with 5xSSCT buffer and 5 μl of native or denatured (95 ° C for 5 minutes and 10 minutes on ice) digoxigenin-labeled amplicons (Nras-910, Nras-600 or Nras-200) in 95 μl of 1xSSCT (0.15 M NaCl, 15 mM citrate, pH 7.0, 0.1% Tween-20) was added per well and incubated for 1 hour at 37 ° C. The wells were washed three times with phosphate-buffered saline ( 1x PBS: 0.15 M Na ⁺ , pH 7.2, 0.05% Tween-20) and incubated for half an hour at 37 ° C with 200 μl of peroxidase-labeled anti-digoxigenin antibodies (Boehringer Mannheim, dilution ) 1: 1000 in 1xFSBT). Finally, the wells were washed three times in 1x FSBT and tested for peroxidase activity by adding 100 μl of a substrate solution (0.1 M citrate-phosphate buffer, pH 5.0, 0.66 mg / ml ortho-phenylenediamine dihydrochloride, 0.012% hydrogen peroxide). The reaction was stopped after 9 minutes by adding 100 μl of 0.5 M sulfuric acid and diluted four times in H ₂ SO ₄ , followed by reading the absorption parameters at 492 nm using a microplate reader. As shown in figa, selection probes containing 12 LNA nucleosides (LNA-Nras-Cap-A and LNA-Nras-Сар-В), highly specific amplificons are selected without preliminary denaturation (native amplicons) . Selection probes containing 4 LNA nucleosides each (LNA / -DNA-Nras-Cap-A and LNA / DNA-Nras-Cap-B) select the same amplifiers with lower efficiency, and “pure” selection DNAs probes (DNA-Nras-Cap-A and DNA-Nras-Sar-B) do not select amplicons specific for them at all. The control amplicon (Nras-200) is not sampled by the LNA-Nras-Cap-B and LNA / DNA-Nras-Cap-B probes: this confirms the highly accurate specificity of the LNA-including selection probes. On figv shows the data of the same experiment conducted on the material of denatured amplicons. In fact, the same picture differs from the previous one only in that the efficiency of selection of amplicons as a whole increases. It is concluded that LNA-modified oligonucleotides provide a method for constructing selection probes that will efficiently select amplicons without prior denaturation, i.e. carry out the selection according to the chain displacement mechanism. Such their ability provides a high level of specificity in modern amplicon detection formats based on DNA sequences.

Example 147

LNA-modified oligonucleotides as seeds for nucleic acid polymerases

The ability of an LNA-modified oligonucleotide (5'-GGTGGTTTGTTTG-3 ': LNA nucleosides in bold) to seed for matrix-dependent catalytic elongation (extension) was studied using three different types of polymerases. This is the reverse transcriptase of the M-MuLV virus (Boehringer Mannheim), which can be used with both the RNA matrix and the DNA matrix, the Klenov fragment (Escherichia coli DNA polymerase), which is a type of standard DNA polymerase, and thermostable polymerase BM-Taq (Boehringer Mannheim). As a control, completion reactions were performed on an identical unmodified DNA seed (5'-GGTGGTTTGTTTG-3 '). LNA and DNA seeds were labeled using γ- ^32P - ^ATP as described above in Example 137. A 50-mer DNA oligonucleotide - 5'-AAAAATCGACGCTCAAGTC-AGAAAAGCATCTCACAAACAAACAAACCACC-3 'was used as a template. The reaction with M-MuLV polymerase (Boehringer Mannheim) included 2 μl of labeled LNA seed or DNA seed (10 μM), 2 μl of DNA template (10 μM), 2 μl of 2 mM dNTP, 2 μl of 10x buffer (500 mm Tris-HCl, 300 mM KCl, 60 mM MgCl ₂ , 100 mM dithiothreitol, pH 8.3 (37 ° C)), 1 μl of enzyme (20 units / μl) and water up to 20 μl. The reaction mixtures were incubated at 37 ° C for 60 minutes. The reaction with Klenov polymerase included 2 μl of labeled LNA seed or DNA seed (10 μM), 2 μl of DNA template (10 μM), 2 μl of 2 mM dNTP, 2 μl of 10x buffer (100 mM Tris-HCl, 50 mM MgCl ₂ , 75 mM dithiothreitol, pH 7.5), 1 μl of the enzyme (10 units / μl) and water up to 20 μl. The reaction mixtures were incubated at 37 ° C for 60 minutes. Reactions with BM-Taq polymerase (Boehringer Mannheim) included 2 μl of labeled LNA seed or DNA seed (10 μM), 2 μl of DNA template (10 μM), 2 μl of 2 mM dNTP, 2 μl of 10x buffer (100 mM Tris -HCl, 50 mM KCl, 15 mM MgCl ₂ , pH 8.3), 1 μl of the enzyme (5 units / μl) and water up to 20 μl. The reaction mixtures were incubated at an initial temperature of 37 ° C, which was increased at a rate of 1 ° C per minute to 60 ° C, at which the mixture was kept for 30 minutes. At the end of the incubation, the reactions were stopped by adding 10 μl of loading buffer (0.25% bromophenol blue - w / v, 0.25% xylencyanol - w / o, 80% formamide - o / o). Samples were heated to 95 ° C for 1 minute, placed on ice, and 2 μl was loaded into an 8% polyacrylamide gel, which was subjected to electrophoresis on Life Technologies Inc. equipment BRL, model 52. After electrophoresis, the gel was dried on a plate and subjected to autoradiographic analysis (X-ray film Kodak X-Omat AR). As shown in FIG. 7, distinct similar products are detected with both types of seeds (LNA and DNA) using a Klenov fragment (lane 3) or BM-Taq polymerase (lane 5). When using M-MuLV reverse transcriptase (lane 2), the completion product can only be detected in the LNA primer variant. Labeled LNA and DNA primers that have not undergone catalytic completion are shown in lanes 1, 4, and 6. It is concluded that the incorporation of LNA nucleosides into standard DNA oligonucleotides does not prevent polymerase recognition of nucleic acids formed with the participation of duplexes “Oligonucleotide / matrix”. It also appears that LNA-modified oligonucleotides can be as effective as seeds as unmodified DNA oligonucleotides.

Example 148

LNA-modified oligonucleotides as seeds for matrix amplification processes

The ability of LNA-modified oligonucleotides to serve as seeds for PCR amplification was analyzed using three oligomers that differ only in the number of LNA nucleosides introduced into their composition, and they included: 4 LNA nucleosides (seed AL2 - 5'-GGTGGTTTGTTTG-3 '' : LNA nucleosides are shown in bold), 1 LNA nucleoside (seed AL10 - 5'-GGTGGTTTGTTTG-3 ': LNA nucleosides are shown in bold) and with no LNA nucleosides (seed FP2 - 5'-GGTGGTTTTTT) . PCR reactions (100 μl) either did not have a matrix (control), or they included 0.01 ng, 0.1 ng or 1 ng of a matrix (plasmid pUC19), 0.2 μM back seed (5'-GTGGTTCG-CTCCAAGCTG-3 '), 0.2 μM direct seed AL2, AL10 or FP2, 200 μM dATP, dGTP, dTSP and dTTP, 10 mM Tris-Hcl (pH 8.3), 1.5 mM MgCl ₂ , 50 mM KCl and 2 , 5 units polymerase BM-Taq. A total of 50 cycles were carried out: 94 ° C - 1 minute, 45 ° C - 1 minute, 72 ° C - 1.5 minutes (after the first 30 cycles, 2.5 units of Taq polymerase were added); used a Techne Genius thermocycler. At the end of the last cycle, the reactions were incubated at 72 ° C for 3 minutes and left at 4 ° C overnight. To 30 μl of each reaction mixture, 6 μl of loading buffer (0.25%, w / v bromphenol blue and 40%, w / o glycerol) was added and samples (together with Amplisize ™ size marker) were loaded into a 2% agarose gel and subjected electrophoresis for 45 minutes at a voltage of 150 V. Finally, the gel was stained with ethidium bromide and photographed. As shown in FIG. 8, in a PCR using unmodified direct primer FP2 and unmodified reverse primer, detectable amplicons of the correct size were formed for all amounts of the matrix tested (lane 9 - 0.01 ng of the template; lane 10 - 0.1 ng; and lane 11 - 1 ng). In the control (without matrix) reaction, the signal was completely absent (lane 12). In the variant with replacing the direct primer FP2 with a primer comprising in the middle 1 LNA nucleoside (AL10), amplicons are also detected with all tested amounts of the matrix (lane 5 - 0.01 ng of the matrix; lane 6 - 0.1 ng; and lane 7 - 1 ng). This clearly indicates that the AL10 seed supports exponential amplification, i.e. The AL10 seed can be either completed or used as a matrix on its own. Again, in the control reaction without a matrix (lane 8), amplificons were not formed. In the variant with replacing the direct seed of FP2 with a seed containing in the middle of 4 LNA nucleosides (AL2), amplicons of the correct size were not detected in any of the reactions (lane 1 - 0.01 ng of the template; lane 2 - 0.1 ng; lane 3 - 1 ng; lane 4 - no matrix). However, at the highest matrix concentration (1 ng), a band of a high molecular weight fragment is detected in the gel (lane 3). Apparently, this is an artifact of the influence of the seed RP1, as indicated by the control reaction in which each of the seeds AL2 (lane A), AL10 (lane B), FP2 (lane C) and RP1 (lane D) were tested for their ability form an amplicon with the highest amount of matrix (1 ng). Since the ability to serve as a seed was shown for AL2 (Example 147), the absence of detectable amplicons clearly confirms that it cannot work as a matrix, i.e. the presence of a block of 4 neighboring LNA nucleosides prevents the progression of the polymerase and thereby “translates” the reaction into linear amplification (the product of which cannot be detected by the applied experimental techniques). It is concluded that LNA-modified oligonucleotides can be used as seeds in PCR amplification. It also appears that the degree of amplification (graded from fully exponential to linear amplification) can be controlled by the design parameters of the LNA-modified oligonucleotide. Applicants note that the possibility of blocking the progress of polymerase by introducing LNA nucleosides into the seed composition will provide the formation of amplificons bearing single-stranded (“protruding”) ends. Such ends can be effectively used in hybridization without amplicon denaturation, and this property can find very wide application.

Example 149

The possibility of covalent immobilization of the LNA-modified oligomer, including 5'-anthraquinone, on a solid substrate using irradiation and the effectiveness of the immobilized oligomer in the selection of a complementary DNA oligonucleotide 25 pmol / μl or 12.5 pmol / μl anthraquinone-DNA oligomer (5'- AQ-CAG CAG TCG ACA GAG-3 ') or anthraquinone-LNA-modified DNA oligomer (5'-AQ-CAG CAG TCG ACA GAG-3': LNA oligomer underlined) was digested (1 μl / spot) at 0, 2 M LiCl on a polycarbonate plate (Nunc). The oligomers were irradiated for 15 minutes with soft ultraviolet light. After irradiation, the plate was washed three times with Milli-Q water and dried in air. 25 ml 0.5 pmol / μl of the complementary biotinylated oligomer (5'-biotin-CTC TGT CGA CTG CTG-3 ') was hybridized with immobilized oligomers in 5xSSCT (75 mM citrate, 0.75 M NaCl, pH 7.7, 0, 1% Tween-20) at 50 ° C for 2 hours. After washing four times in 1xSSCT and washing once in phosphate-buffered saline (FSBT: 0.15 M Na ⁺ , pH 7.2, 0.05% Tween-20), 25 ml of FSBT containing 0.06 μg / ml was applied to the plate combined with streptavidin horseradish peroxidase and 1 μg / ml streptavidin. The plate was incubated for 30 minutes and washed 4 times in 25 ml of FSBT. The plate was then developed with a chemoluminescent substrate (SuperSignal; Pierce) in accordance with the manufacturer's recommendations and an X-ray film was used (CL-XPosure, Pierce 34075). As shown in FIG. 9, both the AQ-DNA oligomer and the AQ-LNA-modified DNA oligomer give a distinct signal. It is concluded that LNA-modified DNA oligomers connected to anthraquinone can be efficiently attached to a solid surface by irradiation, while such oligomers attached in this way are able to hybridize with their complementary DNA oligonucleotide targets.

Example 150

Hybridization and typesetting using various LNA-modified Cγ 3-labeled octomers.

Cooking records. Glass plates were aminosilanated using a 10% solution of aminopropyltriethoxysilane in acetone, followed by washing with acetone. The following oligonucleotides were dug onto the plates:

For each oligonucleotide, 10 repeating spots of approximately 1 nl (each spot) from each pipette onto each of 12 plates were applied.

Plates and hybridization conditions:

plates 1, 2 and 3 were hybridized with aZ1 probe @ 300 fcmol / μl, 30 fcmol / μl, 3 fcmol / μl;

plates 4, 5 and 6 were hybridized with aZ2 probe @ 300 fcmol / μl, 30 fcmol / μl, 3 fcmol / μl;

plates 7, 8 and 9 were hybridized with aZ3 probe @ 300 fcmol / μl, 30 fcmol / μl, 3 fcmol / μl;

plates 10, 11 and 12 were hybridized with a sequence of 16 @ 300 fcmol / μl, 30 fcmol / μl, 3 fcmol / μl.

A probe diluted in 30 μl of hybridization buffer (5 × SSC, 7% sodium laurylsarcosine) was instilled along the length of each plate, covered with a coverslip and placed in a plastic container on the surface of the plastic insert, which is located on a paper towel moistened with water. The containers were sealed with aluminum foil to prevent light from entering and incubated at + 4 ° C overnight.

Flushing the plates. The coverslips were removed and the plates were inserted into the holder (6 plates per holder), which was placed in glass cups wrapped in foil:

After washing, the plates were dried with a stream of air and scanned. The fluorescence level was determined on a slide scanner and the obtained data were analyzed using the ImageQuant program (Molecular Dynamics). As shown in FIG. 11, the lack of binding of the Cγ 3 probes is observed with respect to the oligonucleotide 3, including the error, when using both the unmodified probe (plates 10-12) and the LNA-modified probe aZ1 (plates 1-3), and a single LNA-modified probe aZ2 (plates 4-6), and a three-time modified probe aZ3 (plates 7-9) (i.e., the detected signal with respect to the erroneous oligonucleotide is comparable to the background signal). The intensity of these signals clearly correlates with the amount of LNA residues present in the probes, and with the concentration of these probes used. Each LNA-T residue increases the signal intensity by about a factor of two compared to the signal of a normal DNA oligonucleotide probe: i.e. the probes aZ1 and aZ2 give a double signal of sequence 16, and the probe aZ3 gives an 8 times stronger signal of sequence 16. The distinction between correct and erroneous targets is good when using LNA-T substitutions, and an increase in signal intensity provides better recognition of erroneous targets.

Example 151

Hybridization and detection of terminal errors in a typesetting test using LNA-modified Cγ 3-labeled octomers.

Cooking records. Glass plates were aminosilanated using a 10% solution of aminopropyltriethoxysilane in acetone, followed by washing with acetone. The following oligonucleotides were dug into the plates in an amount of 1 pmol / μl:

For each oligonucleotide, 10 repeating spots of approximately 1 nl volume (each spot) were applied from each of 6 pipettes to each of 12 plates.

The prefix 35Z was also included in the designation of probes including LNA monomers, which is also included in the sequence identification number.

Individual LNA monomers are identified by italics / bold and are located at the 3 ′ and 5 ′ ends of the LNA monomers.

Plates and hybridization conditions. Each probe was hybridized on a separate plate and the concentration of all probes was 1 fcmol / μl. Each probe was diluted in a hybridization buffer (5 × SSC, 7% sodium laurylsarcosine), 30 μl of which was dug along the length of the plate, which was then covered with coverslip and placed in a plastic container on the surface of the plastic insert, which is located on a paper towel moistened with water. The containers were sealed with aluminum foil to prevent light from entering and incubated at + 4 ° C overnight.

Flushing the plates.

Cover glasses were removed and the plates were placed in a holder (8 plates per holder, which were placed in glass cups wrapped in foil. All plates were washed in 5 × SSC twice for 5 minutes at a temperature of + 4 ° C. After washing, the plates were dried in an air stream and scanned. The level of fluorescence was analyzed in Image Quant Molecular Dynamics).

Conclusion As shown in FIGS. 12 and 13, probes including LNA nucleosides at their 5 ′ and 3 ’ends, in most cases, exhibit significantly better parameters in distinguishing between correct and erroneous target sequences compared to their corresponding unmodified oligonucleotides.

For DNA oligonucleotides, C = T errors are most difficult to distinguish, for example, when a probe characterized by sequence 1 hybridizes to a target sequence 132 and when a probe characterized by sequence 5 hybridizes to a target sequence 134. Other errors are detected, such as errors T = T and G = T, although the signals in these cases turn out to be weaker: for example, in cases where the probes characterized by sequences 5 and 6 hybridize with the target sequence 135. LNA oligomers lead to substantial ennomu reduce the intensity of these error signals to the C = T and T = T to levels comparable to the levels typical for other types of errors. The relative signal intensities for probes having sequences 1, 2, and 3 turn out to be similar for DNA and LNA oligomers. However, for probe sequences 4, 5, and 6, LNA oligomers exhibit significantly higher signal intensities when they hybridize with their “correct” target sequences, 9, 133, and 134, respectively.

Example 152

Hybridization and detection of terminal errors in a typesetting test using AT- and fully LNA-modified Cγ 3-labeled octomers

Cooking records.

Glass plates were aminosilanated using a 10% solution of aminopropyltriethoxysilane in acetone, followed by washing with acetone. The following oligonucleotides were dug into the plates in an amount of 1 pmol / μl:

For each oligonucleotide, 10 repeating stains of approximately 1 nl volume (each stain) were applied from each of 6 pipettes to each of 36 plates.

In the designations of probes, including LNA monomers, the ATZ or A11Z- prefixes are used, which are part of the sequence number. Individual LNA monomers are indicated in italics in the descriptions of LNA oligomers.

Plates and hybridization conditions. Each probe was hybridized on a separate plate and the concentration of all probes was 1 fcmol / μl. Each probe was diluted in a hybridization buffer (5 × SSC, 7% sodium laurylsarcosine), 30 μl of which was dug along the length of the plate, which was then covered with coverslip and placed in a plastic container on the surface of the plastic insert, which is located on a paper towel moistened with water. The containers were sealed with aluminum foil to prevent light from entering and incubated at room temperature overnight.

Flushing the plates. The coverslips were removed and the plates were placed in a holder (9 plates per holder, which were placed in glass cups wrapped in foil. All plates were washed in 5 × SSC twice for 5 minutes at room temperature. After washing, the plates were dried in an air stream and scanned. The fluorescence level was analyzed using a slide scanner and the obtained data were analyzed using ImageQuant (Molecular Dynamics).

Conclusion As shown in figa, 15B and 15C, the average level of intensity of DNA hybridization at room temperature was approximately 10% of the level of intensity achieved using oligonucleotides LNA-modified at AT or all nucleosides. No signals were detected on the plates that hybridized with DNA probes 5 and 6. Therefore, such conditions are not optimal for DNA probes. However, recognition of correct and erroneous sequences when used in substitutions of LNA nucleosides at bases A and T was highly effective. The stringency of hybridization conditions for fully LNA-modified oligonucleotides may not be as high as to distinguish between correct and erroneous sequences, which is required for equally good discrimination using AT-LNA oligonucleotides. Oligonucleotides, including LNA modifications, work very well, and the most difficult to identify are the following errors:

probe 1 with respect to the target sequence 135 = CT error;

probe 2 regarding target sequence 131 = GT error;

probe 3 in relation to the target sequence 15 = AA error;

probe 4 in relation to the target sequence 131 = CT error;

probe 5 with respect to the target sequence 135 = TT error;

probe 6 in relation to the target sequence 135 = GT error;

probe 6 with respect to the target sequence 133 = GA error.

When using AT-LNA oligonucleotides, good discrimination results were obtained when the intensity of the erroneous signals was usually 50% of the intensity of the correct signals. For such errors, fully LNA-modified oligomers exhibit signal intensity at errors of 50-70% of the level of intensity of the correct signals. In general, modification using LNA allows the use of higher temperatures for hybridization and washing, while end errors will be determined. These results are at least as good as those obtained with DNA probes hybridizing at 4 ° C (see Example 151).

Example 153

The use of [α - ³³ P] ddNTPs and the ThermoSequenase ™ DNA polymerase system for sequencing DNA matrices including LNA-T monomers.

Radiolabeled end-sequencing reactions were generated to determine the ability of the LNA-T monomer to serve as a template for DNA polymerase. A 15-dimensional seed (sequence: 5’-TGC ATG TGC TGG AGA-3 ’) was used to prime the following short oligonucleotide sequences (LNA monomer in bold):

The following reaction mixtures were formed:

Mix for matrix 1:

× 16 ThermoSequenase buffer 2 μl

1 pmol / μl matrix 1 6 μl

water 4 μl

ThermoSequenase DNA polymerase (4 units / μl) 2 μl

Total volume 20 μl

TZ1 matrix mixture: × 16 ThermoSequenase buffer 2 μl

seed, 2 pmol / μl 6 μl

TZ1 matrix, 1 pmol / μl 6 μl

water 4 μl

ThermoSequenase DNA polymerase (4 units / μl) 2 μl

Total volume 20 μl

To each Eppendorf tube, 2 μl of a mixture of nucleotides (7.5 μM of each of the dNTPs) was added. 0.5 μl of [α - ³³ P] -ddATP were added to tubes 1 and 5. 0.5 μl of [α - ³³ P] -ddCTP were added to tubes 2 and 6. 0.5 μl of [α - ³³ P] -ddGTP were added to tubes 3 and 7. 0.5 μl of [α - ³³ P] -ddTTP were added to tubes 4 and 8. 4.5 μl of the mixture for matrix 1 was added to each of tubes 1-4. 4.5 μl of the mixture for matrix TZ1 was added to each of tubes 1-4. All reactions were incubated at 60 ° C for 3 minutes. Reactions were stopped by adding 4 μl of formamide / EDTA stop solution. The reaction mixtures were heated to 95 ° C for 3 minutes, followed by loading into a 19% polyacrylamide gel containing 7 M urea. The gels were fixed in 10% acetic acid and 10% methanol, followed by transfer to ZMM paper and dried. Dried gels were exposed using a Kodak Biomax X-ray film.

The results are presented in Fig. 18 (lanes 1-4) and Fig. 19 (lanes 5-8). These tracks correspond to the following reactions: (Figure 18): Track 1 - ddATP; lane 2 - ddCTP; lane 3 - ddGTP; lane 4 - ddTTP; lane 5 — oligonucleotide markers consisting of 8-32 bases; (figure 19): lane A - oligonucleotide markers consisting of 8-32 bases; lane 5 - ddATP; lane 6 - ddCTP; lane 7 - ddCTP; lane 8 - ddTTP.

As can be seen from FIGS. 18 and 19, the complete sequences of both matrices can be easily read and scanned. The desired sequence is the 5’-TGG AAC GTA-3 ’sequence, which corresponds to the sequence of the 3’-ACC TTG STA-5’ matrix. This indicates that a single LNA-T monomer can serve as a template for DNA polymerases. The LNA-T monomer is specifically defined as “T" with regard to ddATP incorporation.

Use in therapy

Example 154

Possibility of introducing LNA-modified oligomers into cells. Experiments with radioactively labeled LNA oligomers 10 pmol oligodeoxyribonucleotide (ODN) (ODN 10: 5'-TTA ACG TAG GTG CTG GAC TTG TCG CTG TTG TAC TT-3 ': 35-nucleotide, complementary to the human cathepsin-D gene sequence) and 10 pcmol of two LNA oligonucleotides - AL16 (5'-d [TGT GTG AAA TTG TTA T] -3 '; LNA nucleosides in bold) and AL17 (5'-d [ATA AAG TGT AAA G] -3 '; LNA nucleosides in bold) were mixed in T4 polynucleotide kinase (10 units; BRL, catalog number 510-8004SA), 5 μl γ - ³² P-ATP (5000 Ci / mmol, 10 μCi / μl, Amersham ) in kinase buffer (50 mM Tris-HCl, pH 7.6, 10 mM MgCl _2, 5 mM dithiothreitol, 0.1 mM EDTA). Samples were incubated for 45 minutes at 37 ° C and then heated to 68 ° C for 10 minutes and then transferred to + 0 ° C. Unincorporated nucleotides were removed by passing through Chroma Spin TE-10 columns (Clontech, Catalog No. K1320 -1). The yields were 5 × 10 ⁵ pulse / min per 1 μl, 2 × 10 ⁵ pulse / min per 1 μl and 0.8 × 10 ⁵ pulse / min per 1 μl for, respectively, ODN No. 1, AL16 and AL17. Human breast breast MCF-7 cells, originally obtained from the Human Cell Culture Bank (Mason Res. Inst., Rockville, USA), were cultured in DME / F12 (1: 1) culture medium, to which 1% heat inactivated was added. fetal calf serum (Gibco BRL), 6 ng / ml bovine insulin (Novo) and 2.5 mM glutamax (Life Technologies) in 25 cm ² cell culture flasks (Nunclon, NUNC) and incubated in a wet incubator at 37 ° C in an atmosphere of 5% CO ₂ , 20% O ₂ , 75% N ₂ . MCF-7 cells at the time of the experiment were at the stage of 40% fusion. A small amount (less than 0.1 pcmol) of kinase-treated oligonucleotides was mixed with 1.5 μg of pEGFP-NI plasmid (Clontech, catalog No. 60851) and mixed with 100 μl of diluted transfection agent FuGENE6 (Boehringer Mannheim, catalog No. 1-814443) : dilution - 5 μl of FuGENE6 in 95 μl of serum-free DME / F12 culture medium. The FuGENE6 / DNA / oligomer mixture was directly added to the culture medium (5 ml) with growing MCF-7 cells and incubated with the cells for 18 hours, exactly following the manufacturer's recommendations. Three groups of experiments were performed: (1) ODN No. 10 + pEGFP-NI; (2) AL16 + pEGFP-NI; (3) AL17 + pEGFP-NI. Cell uptake of DNA / LNA material was analyzed by removing the culture medium containing the FuGENE6 / AHK / onHroMep mixture (an aliquot was transferred to a container for scintillation counting). The cells were rinsed once with phosphate-buffered saline (PBS), fresh culture medium was added, and the cells were analyzed using a fluorescence microscope. Approximately 30% of the transfected cells contained green fluorescent material: this indicates that approximately 30% of these cells absorbed the pEGFP-NI plasmid and expressed the reporter green fluorescent protein encoded by this plasmid. After fluorescence microscopy, the merged MCF-7 cells were removed from the culture flasks. Briefly, the culture medium was removed, then the cells were washed with a solution of 0.25% trypsin (Gibso BRL), 1 mM EDTA in PBS (without Mg ²⁺ and Ca ^{2+ ions} ), 1 ml of trypsin / EDTA was added and the cells were incubated for 10 minutes at 37 ° C. During the incubation, the cells “softened” and could be easily resuspended and transferred to a scintillation counter container. Then the cells were completely suspended by adding 10 ml of Optifluor scintillation mixture (Packard, catalog number 6013199), and the material was counted on a Wallac-1409 scintillation counter. The following results were obtained: (1) ODN No. 10 + pEGFP-NI: approximately 1.4% of the added radioactivity was associated with cellular material; (2) AL16 + pEGFP-NI: approximately 0.8% of incremental radioactivity was associated with cellular material; (3) AL17 + pEGFP-NI: approximately 0.4% of the additional radioactivity was associated with cellular material. It is concluded that 0.4-0.8% of the added LNA oligomers were absorbed by the cells.

Example 155

Effective LNA delivery to living cells of a human breast tumor MCF-7.

In order to increase the efficiency of LNA uptake by human MCF-7 cells, various transfection agents were tested and different concentrations of 5’F1TC-labeled LNA and DNA were used. The oligonucleotides described in the table were investigated.

Probes AL16 and AL17 were catalytically labeled with FITC as described in Example 128. Oligonucleotides EQ3009-01 and EQ3008-01 were labeled with FITC using standard solid state chemical reactions. Three transfection agents were tested - FuGENE6 (Boehringer Mannheim, catalog number 1-814443), SuperFect (Quiagen, catalog number 301305) and Lipofectin (Gibco BRL, catalog number 18292-011). Human breast breast MCF-7 cells were cultured as previously described (Example 154). Three days before the experiments, the cells were sown at a density of approximately 8 thousand cells per 1 cm ² . Depending on the type of experiment, MCF-7 cells were seeded in standard T25 flasks (Nunc, Life Technologies, catalog number 163371A), 24-well plates (Nunc, Life Technologies, catalog number 143982A) or slide bottles (Nunc, Life Technologies , Catalog number 170920A). The experiments were carried out at the stage of 30-40% cell fusion. Absorption by LNA and DNA cells was analyzed under serum-free conditions, i.e. normal serum-containing DME / F12 culture medium was removed and replaced with serum-free DME / F12 medium before the transfection mixture was added to the cells. Under these conditions, SuperFect showed toxicity to MCF-7 cells. Transfection mixtures containing SuperFect and plasmid DNA (pEGFP-N1, Clontech, Catalog No. 6085-1), a DNA oligomer or LNA oligomer, were equally toxic to MCF-7 cells. In contrast to the SuperFect agent, FuGene6 and Lipofectin agents worked efficiently against plasmid DNA (pEGFP-N1). However, only lipofectin was able to provide efficient delivery of oligonucleotides to living MCF-7 cells. Briefly, efficient delivery of FITC-labeled LNAs and DNA to MCF-7 cells was observed when cells were cultured in DME / F12 culture medium containing 1% fetal calf serum to about 40% fusion. The Lipofectin agent was then diluted 40 times in serum-free DME / F12 culture medium and combined with an oligomer to a concentration of 750 nM of this oligomer. The oligonucleotide lipophectin complex was allowed to form for 15 minutes at room temperature and then diluted with serum-free culture medium to a final oligomer concentration of 250 nM and 0.8 μg / ml lipofectin. Then the culture medium was removed from the cells and replaced with a culture medium containing an oligonucleotide lipophectin complex. Cells were incubated at 37 ° C for 6 hours, rinsed once in serum-free DME / F12 medium and incubated for another 18 hours in serum DME / F12 medium (1% FCS) at 37 ° C. The experimental results were evaluated either directly on live cells in culture flasks or on 24-well plates, or on cells cultured in slide bottles and fixed in 4% ice-cold PFA. In all cases, a Leica DMRB model fluorescence microscope equipped with a high-resolution CCD camera was used. The results obtained on living cells are shown in Fig. 16, and the results obtained on fixed cells cultured in slide vials are shown in Fig. 17. In all cases, the cells shown in FIGS. 16 and 17 were transfected with the FITC-labeled LNA molecule AL16. By counting the total number of cells and green fluorescent cells, it was found in several visual fields that the FITC-labeled LNA molecules AL16 were transfected in approximately 35% of MCF-7 cells. Importantly, the preferred localization of LNA in cell nuclei has been shown (FIG. 17). This is important because the nuclear uptake of fluorescent oligonucleotides correlates with their potential antisense activity (Stein C.A. et al., 1997, “Making sense of antisense: A debate", b ″ HMS Beagle: a BioMedNet Publ. "- hmsbeagle. com / 06 / cutege / overwiev.htm (Internet address)). An increase in the number of oligomer and lipofectin to a final concentration of 1250 nM oligomer and 4 μg / ml lipofectin leads only to a slight increase in the proportion of green fluorescent cells. An increase in this concentration further becomes toxic to the cells. Similar results were obtained by analyzing other LNA and FITC-labeled DNA oligonucleotides (see table above). It is concluded that: (1) LNA can be efficiently delivered to live breast tumor MCF-7 cells using lipofectin-mediated transfection; (2) a constantly high proportion of cells (30% or more) is transfected using a final concentration of 250 nM LNA and 0.8 μg lipofectin per 1 ml of serum-free culture medium. An increase in the concentration of LNA and lipofectin up to a 5-fold increase in transfection efficiency does not increase significantly; (3) the procedure provides transfection of LNA into the nuclei of cells, which, in accordance with the literature, indicates the promise of transfection of LNA in order to achieve antisense effects in cells.

Example 156

Possibility of transfection of LNA-modified oligonucleotides into cells. Experiment with LNA oligomers labeled with fluorescein

Two AL16 LNA oligomers (5'-TGT GTG AAA TTG TTA T-3 '; LNA nucleosides are shown in bold) and AL17 (5'-ATA AAG TGT AAA G-3': LNA nucleosides are shown in bold) are marked with fluorescein as described in Example 128. MCF-7 cells of a human breast tumor were cultured as described in Example 154. Three experimental options were delivered: (1) approximately 1.5 μg of FITC-labeled AL16; (2) approximately 1.5 μg of FITC-labeled AL17; and (3) approximately 0.75 μg of FITC-labeled AL16 and 0.75 μg of the plasmid pRSV-β Gal (plasmid expressing the bacterial lacZ gene that encodes the β-galactosidase enzyme: Tulchinsky et al., 1992, Proc. Natl. Acad . Sci. USA, 89, 9146-9150). Two LNA oligomers and a mixture of LNA plasmids were mixed with FuGENE6 and added to MCF-7 cells as described in Example 154. After incubation for 18 hours, the cell uptake of the LNA oligomers was evaluated using a fluorescence microscope for cell cultures. In the part of the treated cells, the presence of a green fluorescent signal was observed (see FIG. 16): this indicates that the cells absorbed fluorescein-labeled LNA. In this regard, labeled with fluorescein AL16 had preferred parameters with respect to labeled with fluorescein AL17. After fluorescence microscopy, the culture medium was removed from cells treated with both labeled AL16 fluorescein and plasmid pRSV-β Gal. The cells were washed once with PBS, fixed with 2% (o / o) formaldehyde, 0.2% (o / o) glutaraldehyde at 4 ° C for 5 minutes, and the cells containing β-galactosidase were stained blue when exposed to X- reagent gal (5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside), which a colorless object turns blue in the presence of β -galactosidase activity. Staining with X-gal reagent showed that the plasmid pRSV-β Gal is efficiently transfected into cells. It is concluded that fluorescein-labeled LNA oligomers were absorbed by cells.

Example 157

Relative stability of LNA-modified oligonucleotides in cell culture conditions.

After fluorescence microscopy described in Example 156, cells transfected with only labeled AL16-LNA fluorescein were allowed to incubate for 3 additional days. During this period of time, the number of green fluorescent cells did not change. It is concluded that fluorescein-labeled LNA oligomers are characterized by good stability parameters under conditions existing in cell culture.

Example 158

Blocking by antisense “closed nucleic acids” (LNAs) of anesthetized [D-Ala ₂ ] -deltorphin anesthesia in “conscious” rats in the test for “warm-water tail click”.

Male Sprague-Dawley rats (300 g body weight) were implanted with a polyethylene catheter intrashell, followed by a recovery period of 5 days, followed by injection (including control). Antisense LNA constructs (12.5 and 2.5 μg per injection) were administered in a volume of 5 μl twice a day (at 8 a.m. and 5 p.m.) for 3 days. No nonspecific manifestations or toxicity were detected, as indicated by observations of locomotor activity and animal weighing. The day after the last injection, the rats were injected with [D-Ala ₂ ] -deltorfin (60 μg, intracranial) and tested for a “warm click” (the reaction of the tail of an animal entering water heated to 52 ° C), which allows to evaluate the anesthesia mediated δ-opioid receptors. Fig. 14 shows data that are values averaged over 6-8 animals within each of the experimental groups (data are converted to the percentage of the most likely response,% MPE). Statistical analysis was carried out according to Kruskall-Wallace in the ANOVA program, followed by a comparison of experimental options with control. As shown in FIG. 14, deltorfin provides a powerful analgesic effect in animals that were injected with saline. This effect was suppressed at a statistically significant level by both groups of antisense LNA constructs (12.5 and 2.5 μg) compared with the control, which was injected with simulated saline.

LNA-solid substrates

Example 159

The basic method for producing DMT-LNA nucleoside succinates.

Base-protected DMT-LNA nucleosides and succinic anhydride (1.5 equivalents) were placed in anhydrous ethylene dichloride (≈ 10 ml / g nucleoside). Triethylamine (2 equivalents) was added to this mixture, and the resulting mixture was stirred at room temperature. After that, the reaction mixture was subjected to HPLC (under the same conditions as in the tritylation reaction). After completion of the reaction (> 95%), the reaction mixture was concentrated, evaporated with ethylene dichloride and acetonitrile and dried in vacuo to remove triethylamine. The residue was dissolved in ethylene dichloride or ethyl acetate (≈ 100 ml / g of the starting nucleoside), washed with cold 10% citric acid (3 × 80 ml / g) and cold water (3 × 80 ml / g). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in the presence of 1-2 equivalents of triethylamine or without it. The remaining solid was evaporated with anhydrous acetonitrile (2-3x) and dried in vacuo to give the product as a white solid. The basic method for producing LNA nucleoside substrates. Base-protected DMT-LNA nucleoside succinate (as free acid or its triethylammonium salt, 65 μmol per 1 g of substrate), amino derivative (Primer Support ™ 30HL, 160 μmol of amino groups per g of substrate), DMAP (3 mg / g) and 1- (3- [dimethylamino] propyl) -3-ethylcarbodiimide hydrochloride (80 mg per g of support) was placed in anhydrous pyridine (6 ml / g of support). Triethylamine (16 μl / g support) was added to this mixture, and the resulting mixture was kept in a shaker at 150 rpm overnight. The substrate was filtered, washed with methanol (3 × 10 ml / g of substrate) and dichloromethane (3 × 10 ml / g of substrate). After air drying, the substrate was dried in vacuo for half an hour. To this material was added 6% DMAP in anhydrous acetonitrile (mixture-A, ≈ 3 ml / g of substrate) and a mixture of 20% acetic anhydride / 30% 2,4,6-collidine / 50% acetonitrile (mixture-B, ≈ 3 ml / g of substrate). The mixture was placed in a shaker for 5 hours. The substrate was filtered off, washed with anhydrous dichloromethane (2 × 10 ml / g of substrate) and dried as before. It was resuspended in mixed mixture-A and mixture-B (total volume 6 ml per 1 g of substrate) and kept in a shaker overnight. The substrate was filtered, washed with methanol (6 × 10 ml / g of substrate), dichloromethane (3 × 10 ml / g of substrate) and dried in air. Then it was dried in vacuum for 5-6 hours. Quantitative loading parameters were determined in the dimethoxytrityl test: they were determined at a level of approximately 40 μmol / g.

Example 160

Synthesis of the first cDNA strand using poly-dT seed, including LNA-T monomers.

The reactions were formed in order to determine the ability of polydeoxyribotimidine seeds, including LNA-T monomers, to seed the synthesis of the first cDNA chain. The following poly-dT seeds were tested (LNA monomers are shown in bold):

As a control, we used poly-dT seed, which is part of the cDNA labeling reagent kit - RPK0140 kit Сγ Dye cDNA label-ling kit (Amersham Pharmacia Biotech).

Reactions were generated for each of the above seeds as follows:

Arabidopsis rhizome mRNA, 0.5 μg / μl 1 μl

poly-dT seed, 8 pmol / μl 2 μl

× 5 reverse transcriptase buffer AMV 4 μl

water 1 μl

total volume 8 μl

This mixture was then heated to 75 ° C for 3 minutes and then allowed to cool to room temperature for at least 10 minutes.

Then, the following was added to each reaction mixture:

sodium pyrophosphate, 80 mm 1 μl

human placenta ribonuclease inhibitor, 20 units / μl 1 μl

dNTP solution, 0.5 mM 7 μl

[α - ³³ P] -dATP, 10 mCi / ml, 3000 Ci / mmol 2 μl

AMV reverse transcriptase, 20 units / μl 1 μl

Total volume 20 μl

The reactions were incubated at 42 ° C for 2 hours. Then they were heated to 95 ° C for 3 minutes, followed by loading into a 6% polyacrylamide gel containing 7 M urea. The gels were fixed in 10% acetic acid + 10% methanol, followed by 3MM transfer to paper and drying. The dried gels were exposed on a Kodak Biomax X-ray film overnight.

The fingerprints clearly showed that the LNA-incorporating oligonucleotide primers RTZ1-4 were ways to efficiently seed cDNA synthesis. The number and intensity of cDNA products formed in the reactions were the same as those obtained using attached control dT oligonucleotides. A small amount of cDNA was also formed from the RTZ 5 seed, however, the yield was significantly lower compared to the yield from the control oligonucleotide seed.

Example 161

Effective functioning of LNA-modified oligonucleotides covalently attached to sepharose beads with sequence-specific selection of RNA molecules.

Three oligonucleotides were synthesized by chemical methods (Amy Mueller), which were used to evaluate binding to poly-p (A):

- NH ₂ - (T8) -T - control,

- NH ₂ - (T15) -T - control,

- NH ₂ (LNA-T8) -T - test

200 nmol of each oligonucleotide was combined with 50 mg of preparative Sepharose-4B cyanobromide activated (Pharmacia) according to manufacturers instructions. To block unreacted binding sites on sepharose, 100 nM Tris, pH 8.0, was used.

Resins with bound oligonucleotides were divided into two portions (≈ 25 mg of resin in each) to analyze the binding of polyriboadenosine in duplicate. Poly- (rA) Pharmacia test reagents, catalog No. 27-4110-01 were used for binding: dissolved at an absorption density of 28.2 units / ml (A ₂₆₀ ) in the binding buffer. Five A ₂₆₀ units were linked to duplicate portions of 25 mg for each resin variant with oligonucleotides bound on it per SOP QC 5543. Unbound “missed” poly- (riboA) was quantified by absorption at A ₂₆₀ and used to quantify bound reagent. The nature of the bound poly- (riboA) was determined by washing in a low-salt buffer and several elutions. As shown in the table. 10, the beads coated with both LNA and DNA were effective in connection with the selection (self-binding) of polyriboadenosine target molecules. However, LNA coated beads bound the poly (riboA) target more tightly than DNA coated beads: this is indicated by differences in elution parameters of different bead variants. It is concluded that (2) the LNA nonatimidine oligonucleotide is effective in selecting RNA molecules including a motif consisting of adenine residues, and (2) the selected (bound) RNA molecules bind more strongly to the LNA-T9 oligomer compared to strength binding to control 9- and 16-thymine DNA oligonucleotides.

Claims (64)

1. LNA-modified oligonucleotide comprising at least one nucleoside analogue (LNA) of the General formula I

where X is —O—; B represents a nucleotide base; P denotes the position of the radical for the formation of the internucleoside “bridge” with the following monomer or 5’-terminal group, where the specified 5’-terminal group is selected from hydroxyl, monophosphate, diphosphate and triphosphate; R ³ or R ³ * represent a group P *, which denotes the internucleoside bridge with the previous monomer or a 3'-terminal group; and non-heminal substituents R ² * and R ⁴ * denote a biradical selected from - (CR * R *) _r -O- (CR * R *) _s -, - (CR * R *) _r -S- (CR * R * ) _s -, - (CR * R *) _r -N (R *) - (CR * R *) _s -, with each of the substituents R ¹ *, R ² , R ³ *, R ³ , R ⁵ * and R ⁵ not involved in the formation of the biradical or internucleoside “bridge” is hydrogen, halogen, hydroxy, mercapto, amino, azido, or non-heminal substituents R ² and R ³ are the diradical - (CR * R *) _r -O- (CR * R *) _S -, while R ² * is selected from hydrogen, hydroxy and optionally substituted C _1-6 alkoxy groups, and R ¹ *, R ⁴ *, R ⁵ and R ⁵ * are hydrogen; where each of r and s is 0 to 4, provided that the sum of r + s is 1 to 4 and each R * is hydrogen or C _1-6 alkyl; or its basic salt or acid addition salt; moreover, the oligonucleotide may include a photochemically active group, a thermochemically active group, a chelating group, a reporter group or a ligand, which cause its direct or indirect detection or immobilization on a solid support. 2. The oligonucleotide according to claim 1, comprising 1-100 LNAs of the general formula I and 0-100 natural nucleosides, provided that the sum of the amounts of nucleosides and LNA is in the range of 2-150. 3. The oligonucleotide according to any one of paragraphs. 1 and 2, while one of the substituents R ³ and R ³ * is P *. 4. The oligonucleotide according to any one of paragraphs. 1-3, despite the fact that LNA is characterized by the following formula Ia:

despite the fact that P, P *, B, X, R ¹ *, R ² , R ² *, R ³ , R ⁴ *, R ⁵ and R ⁵ * are defined in claim 1. 5. The oligonucleotide of claim 4, wherein R ³ * is P *. 6. The oligonucleotide according to claim 5, wherein R ² * and R ⁴ * together represent a diradical. 7. The oligonucleotide according to claim 6, while the biradical is 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 *) - (CH ₂ ) _1-3 -. 8. The oligonucleotide according to claim 7, although the biradical is selected from —O — CH ₂ -, —S — CH ₂ -, and —N (R *) —CH ₂ -. 9. The oligonucleotide according to claim 8, wherein such an oligonucleotide comprises at least one LNA, while B is selected from adenine and guanine, and at least one LNA, while B is selected from thymine, cytosine, 5-methylcytosine and uracil. 10. The oligonucleotide according to claim 5, wherein R ² and R ³ together represent a diradical. 11. The oligonucleotide according to claim 10, despite the fact that the biradical is - (CH ₂ ) _0-1 -O- (CH ₂ ) _1-3 -. 12. The oligonucleotide according to any one of paragraphs. 1-11, despite the fact that any internucleoside “bridge” of the LNA is selected from —O — P (O) ₂ —O—, —OP (O, S) —O—. 13. The oligonucleotide according to any one of paragraphs. 1-11, despite the fact that each of the substituents R ¹ *, R ² , R ³ , R ³ *, R ⁵ and R ⁵ * in the LNA, which are present and are not involved in P, P * or biradical, is hydrogen , hydroxy, mercapto, amino or azido. 14. The oligonucleotide according to any one of paragraphs. 1-13, while P represents a 5’-terminal group selected from hydroxyl, monophosphate, diphosphate, triphosphate. 15. The oligonucleotide according to any one of paragraphs. 1-14, while P * represents a 3’-terminal group, which is hydroxyl. 16. The oligonucleotide according to any one of paragraphs. 1-15 having the formula V G- [Nu-L] _{n (0)} - {[LNA-L] _{m (q)} - [Nu-L] _{n (q)} } _q -G * V where q is 1-50; each of n (0), ..., n (q) is independently 0-100; each of m (l), ..., m (q) is independently 1-100; provided that the sum of n (0), ..., n (q) and m (1), ..., m (q) is 2-150; G represents a 5’-terminal group; each Nu is independently a natural nucleoside; each LNA is independently a nucleoside analogue; each L independently represents an internucleoside “bridge” between two groups selected from Nu and LNA, or L together with G * forms a 3’-terminal group; and each LNA-L independently denotes a nucleoside analog of the general formula I

despite the fact that the substituents B, P, P *, R ¹ *, R ² , R ² *, R ³ , R ⁵ and R ⁵ * and X are defined in paragraphs. 1-15. 17. The oligonucleotide according to any one of paragraphs. 1-16, which is characterized by increased specificity for complementary single-stranded RNA and single-stranded DNA compared with natural oligonucleotides. 18. The oligonucleotide according to any one of paragraphs. 1-16, which is characterized by increased affinity for complementary single-stranded RNA and single-stranded DNA compared to a natural oligonucleotide. 19. The oligonucleotide according to any one of paragraphs. 1-16, which is able to bind to the target sequence, which is part of a double-stranded DNA or double-stranded RNA molecule by the mechanism of "chain replacement" or due to the formation of triplex. 20. The oligonucleotide according to any one of paragraphs. 1-16, which is more resistant to the action of nucleases in comparison with the natural oligonucleotide. 21. The oligonucleotide according to claim 1, comprising at least one nucleoside analogue, characterized by formula I, which affects the T _m oligonucleotide with a complementary DNA oligonucleotide, which is at least 2.5 ° C higher than the corresponding unmodified a control oligonucleotide that does not include any nucleoside analogues. 22. The oligonucleotide according to claim 21, despite the fact that T _m is at least 2.5 × N ^o C higher, where N is equal to the number of nucleoside analogues. 23. The oligonucleotide according to claim 1, comprising at least one nucleoside analog characterized by formula I, which affects a T _m oligonucleotide with a complementary RNA oligonucleotide that is at least 4.0 ° C higher than the corresponding unmodified control oligonucleotide, not including any nucleoside analogues. 24. The oligonucleotide of claim 23, wherein T _m is at least 4.0 × N ^o C higher, where N is the number of nucleoside analogues. 25. The oligonucleotide according to claim 21, wherein said oligonucleotide, being hybridized with a partially complementary DNA oligonucleotide, characterized by one or more mismatches with said oligonucleotide, exhibits a decrease in T _m due to said mismatches, which is equal to or greater than such a decrease that may be detected for the corresponding unmodified control oligonucleotide that does not include any nucleoside analogues. 26. The oligonucleotide according to claim 23, wherein said oligonucleotide, being hybridized with a partially complementary RNA oligonucleotide, characterized by one or more mismatches with said oligonucleotide, exhibits a decrease in T _m due to said mismatches, which is equal to or greater than such a decrease that can be detected for the corresponding unmodified control oligonucleotide that does not include any nucleoside analogues. 27. The oligonucleotide according to claim 21 or 23, which is characterized by essentially the same sensitivity of the T _m value to the ionic strength of the hybridization buffer as the corresponding unmodified control oligonucleotide. 28. The oligonucleotide according to claim 21 or 23, which is modified by at least 30%. 29. The oligonucleotide according to claim 21 or 23, which is characterized by significantly higher 3'-exonucleolytic stability compared with the corresponding unmodified control oligonucleotide. 30. The oligonucleotide according to any one of paragraphs. 1-29 for use in anti-sense therapeutic methods. 31. The oligonucleotide according to claim 30, wherein the LNA-modified oligonucleotide includes at least one internucleoside “bridge” that is not a phosphodiester linkage. 32. The oligonucleotide according to any one of paragraphs. 1-29 for the isolation, purification, amplification, detection, identification, quantification or selection of natural or synthetic nucleic acids. 33. The oligonucleotide according to claim 32, wherein the oligonucleotide comprises a photochemically active group, a thermochemically active group, a chelating group, a reporter group or a ligand, which cause direct or indirect detection of this oligonucleotide or provide immobilization of the oligonucleotide on a solid support. 34. The oligonucleotide according to claim 33, wherein the photochemically active group, the thermochemically active group, the chelating group, the reporter group, or the ligand include a spacer (K), wherein said spacer includes a chemically cleavable group. 35. The oligonucleotide according to claim 32 for the purpose of selection and detection of natural or synthetic double-stranded or single-stranded nucleic acids, such as RNA or DNA. 36. The oligonucleotide according to claim 32, for purification of natural double-stranded or single-stranded nucleic acids, such as RNA or DNA. 37. The oligonucleotide according to claim 32 for use as a seed in a nucleic acid amplification reaction. 38. Nucleoside analogue (LNA) of formula II

where X is —O—; B is a nucleotide base which is optionally protected in terms of its functional components; R ³ or R ³ * represents a group Q *, which is selected from hydroxy, Prot-O-, Act-O; Q represents hydroxyl, monophosphate, diphosphate, triphosphate, Prot-O- or Act-O, where Prot represents a protecting group for —OH; Act denotes an activation group for —OH; non-heminal substituents R ² * and R ⁴ * denote a biradical selected from - (CR * R *) _r -O- (CR * R *) _s -, - (CR * R *) _r -S- (CR * R * ) _s -, - (CR * R *) _r -N (R *) - (CR * R *) _s -, with each of the substituents R ¹ *, R ² , R ³ *, R ³ , R ⁵ * and R ⁵ not involved in the formation of the biradical, Q or Q *, is hydrogen, hydroxy, mercapto, amino, azido; or non-heminal substituents R ² and R ³ are diradical - (CR * R *) _r -O- (CR * R *) _S -, while R ² * is selected from hydrogen, hydroxyl and optionally substituted C _1-6 alkoxy groups, and R ¹ *, R ⁴ *, R ⁵ and R ⁵ * are hydrogen; Where each of r and s is 0 to 4, provided that the sum of r + s is 1 to 4 and each R * is hydrogen or C _1-6 alkyl; or its basic salt or acid addition salt. 39. The nucleoside analogue of claim 38, wherein the Act activation group is selected from the group consisting of optionally substituted O-phosphoramidite, optionally substituted O-phosphoric ester, optionally substituted O-phosphorodiester, optionally substituted H-phosphonate, and optionally substituted O-phosphonate. 40. The nucleoside analogue of claim 39, wherein the Act activation group is an optionally substituted O-phosphoramidite of the formula —OP (OR ^X ) —N (R ^Y ) 2, where R ^X is an optionally substituted alkyl group and each of R ^Y is optionally substituted alkyl groups, or the group —N (R ^Y ) ₂ forms a morpholino group (—N (CH ₂ CH ₂ ) ₂ O). 41. The nucleoside analogue of claim 40, wherein R ^X is 2-cyanoethyl and two R ^{Y are the} same and are isopropyl, i.e. a group of the formula —OP (OR ^X ) —N (R ^Y ) ₂ is N, N-diisopropyl-O- (2-cyanoethyl) phosphoramidite. 42. The nucleoside analogue of claim 40, selected from (lR, 2R, 4R, 5S) -l- (2-cyanoethoxy (diisopropylamino) phosphoxy) -2- (4,4'-dimethoxytrityloxymethyl) -4- (thymin-1-yl) -3,6-dioxabicyclo [3.2 .0] heptane, (lR, 3R, 4R, 7S) -7- (2-cyanoethoxy (diisopropylamino) phosphoxy) -l- (4,4'-dimethoxytrityloxymethyl) -3- (thymin-1-yl) -2,5-dioxabicyclo [2.2 .1] heptane, (lR, 3R, 4R, 7S) -7- (2-cyanoethoxy (diisopropylamino) phosphoxy) -l- (4,4'-dimethoxytrityloxymethyl) -3- (2-N-isopropionylguanine-9-yl) -2.5 -dioxabicyclo [2.2.1] heptane, (1R, 3R, 4R, 7S) -7- (2-cyanoethoxy (diisopropylamino) phosphinoxy) -1- (4,4'-dimethoxytrityloxymethyl) -3- (4-N-benzoylcytosin-1-yl) -2.5 -dioxabicyclo [2.2.1] heptane, (1R, 3R, 4R, 7S) -7- (2-cyanoethoxy (diisopropylamino) phosphinoxy) -1- (4,4'-dimethoxytrityloxymethyl) -3- (6-N-benzoyladenin-9-yl) -2.5 -dioxabicyclo [2.2.1] heptane, (1R, 3R, 4R, 7S) -7- (2,2-dimethoxytrityloxymethyl) -3- (5-methyl-4-N-benzoylcytosin-1-yl) -2,5-dioxabicyclo [2.2.1] heptane, 1- (2-amino-3-O- (2-cyanoethoxy (diisopropylamino) phosphino-2-deoxy) -5-O-4,4'-dimethoxytrityl-2-N, 4-C-methylene-2-N- trifluoroacetyl-β-D-ribofuranosyl) thymine, 1- (3-O- (2-cyanoethoxy (diisopropylamino) phosphino) -5-O-4,4'-dimethoxytrityl-2-methylamino-2-N, 4-C-methylene-2-deoxy-β-D- ribofuranosyl) thymine, 1- (3-O- (2-cyanoethoxy (diisopropylamino) phosphino) -2-deoxy-5-O- (4,4'-dimethoxytrityl) -2-mercapto-2-S, 4-C-methylene-β- D-ribofuranosyl) uracil. 43. The nucleoside analogue of claim 38 of general formula IIa

where the substituents Q, B, R ¹ *, R ² , R ² *, R ³ , R ³ *, R ⁴ *, R ⁵ and R ⁵ * are defined in § 38. 44. The nucleoside analogue of claim 43, wherein R ² * and R ⁴ * together form a diradical. 45. The nucleoside analogue of claim 44, wherein the biradical is selected from —O — CH ₂ -, —S — CH ₂ -, and —N (R *) —CH ₂ -. 46. The nucleoside analogue according to any one of paragraphs. 44-45, while B is selected from nucleotide bases. 47. The nucleoside analogue of claim 44, wherein the biradical is —O — CH ₂ -. 48. The nucleoside analogue of claim 43, wherein R ² and R ³ together form a diradical. 49. The nucleoside analogue of claim 48, wherein R ¹ *, R ² *, R ⁴ *, R ⁵ and R ⁵ * are hydrogen. 50. The nucleoside analogue of claim 49, wherein the biradical is the group - (CH ₂ ) _0-1 —O— (CH ₂ ) _1-3 -. 51. The nucleoside analogue of claim 38, which is selected from (1R, 3R, 4R, 7S) -7- (2-cyanoethoxy (diisopropylamino) phosphinoxy) -1- (4,4'-dimethoxytrityloxymethyl) -3- (thymin-1-yl) -2,5-dioxa-bicyclo [2.2.1] heptane, (lR, 3R, 4R, 7S) -7-hydroxy-l- (4,4'-dimethoxy-nitroxymethyl) -3- (thymin-l-yl) -2,5-dioxabicyclo [2.2.1] heptane-7- 0- (2-chlorophenyl phosphate) and (lR, 3R, 4R, 7S) -7-hydroxy-l- (4,4'-dimethoxy-nitroxymethyl) -3- (thymin-l-yl) -2,5-dioxabicyclo [2.2.1] heptane-7- 0- (H-phosphonate) and the corresponding -3- (cytosin-1-yl), -3- (uracil-1-yl), -3- (adenin-1-yl) and -3- (guanine-3-yl) analogues . 52. The nucleoside analogue according to any one of paragraphs. 38-51 to obtain an LNA-modified oligonucleotide corresponding to any one of paragraphs. 1-29. 53. The nucleoside analogue of claim 52, wherein the addition of LNA modulates the ability of such an oligonucleotide to serve as a substrate for enzymes active against nucleic acids. 54. The nucleoside analogue according to any one of paragraphs. 38-51 as a substrate for enzymes active against nucleic acids. 55. The nucleoside analogue of claim 54, wherein the substituent Q in Formula I in claim 38 is triphosphate. 56. The nucleoside analogue of claim 54, wherein LNA is used as a substrate for DNA and RNA polymerases. 57. The nucleoside analogue according to any one of paragraphs. 38-51 for constructing a solid support to which LNA-modified oligonucleotides having different sequences are attached. 58. The nucleoside analogue of claim 57, wherein the LNA-modified oligonucleotides are attached at their specified location. 59. The nucleoside analogue of claim 57, wherein the LNAs are used to equalize the T _m values of the corresponding unmodified control oligonucleotides. 60. The nucleoside analogue of claim 57, wherein the LNA-modified oligonucleotides are characterized by increased affinity for complementary single-stranded DNA or single-stranded RNA compared to a natural oligonucleotide. 61. The nucleoside analogue of claim 57, wherein the LNA-modified oligonucleotides are characterized by increased specificity for complementary single-stranded DNA or single-stranded RNA compared to a natural oligonucleotide. 62. The material of the solid support with it immobilized on it is not necessarily protected at its base and is not necessarily protected according to 5’-OH LNA according to claim 38. Priority on points and signs: 09/12/1997 according to claims 1, 38, 62; 12/19/1997, 01/16/1998, 03/03/1998, 04/29/1998, 06/05/1998, 08/08/1998, 07/28/1998 - refinement of signs according to paragraphs. 1-62 of the claims.

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