UA48150C2 - Amino acid or amino alcohol derivatives, oligonucleotide - Google Patents

Abstract

The present invention provides various novel oligonucleotide analogs having one or more properties that make the subject compounds superior to conventional oligonucleotides for use in procedures employing oligonucleotides. The compounds of the invention arc oligonucleotide analogs in which the furanose ring of a naturally occurring nucleic acid is replaced with an amino acid or a modified amino alcohol residue. Some embodiments of the novel compounds of the invention arc particularly useful for the antisense control of gene expression. The compounds of the invention may also be used as nucleic acid hybridization probes or as primers. Another aspect of the invention is to provide monomeric precursors of the oligonucleotide analogs of the invention. These monomeric precursors may be used to synthesize the subject polynucleotide analogs. Another aspect of the invention is to provide formulations of the subject polynucleotide analogs that arc designed for the treatment or prevention of disease conditions. Yet another aspect of the invention is to provide methods for treating or preventing diseases, particularly viral infections and cell growth disorders. The subject disease treatment methods comprise the step of administering an effective amount of the subject polynucleotide analogs for use as antisense inhibitors.

Description

Description of the invention

The present invention relates to analogs of polynucleotides that do not have a furanose ring. 2 Oligonucleotides, which specifically bind to complementary sequences of nucleotides (senseless strand) by hydrogen bonds to suppress gene expression, are colloquially called antisense oligonucleotides. Zty synthetic Oligonucleotide! bind to the target (matrix RNA), thereby suppressing the translation of the matrix RNA. Ztot anti-humorous principle (piItapp, E. ei a), Spet. Chemistry 5, 1990, 90, 543 - 584; and 5ieip, S.A. ей аЙ,, Sapseg Kevz., 1988, 48, 2659 - 2688) is used to control the expression of genes in nature. This antisense principle was used in the laboratory not only to suppress, but also to activate gene expression. In 1978, 7atespik and

Zierpepsop was the first to propose the use of synthetic oligonucleotides for therapeutic purposes (Bierpepsop M.Y.; apa latespik, R.S., Rgos. Mai. Asad. 5siI. OBA, 1978, 75, 280 apa 285). The specific suppression of antisense polynucleotides is based on the specific pairing of Watson-Crick 12 (uU/aivop-StiskK) between the heterocyclic bases of the antisense oligonucleotide and viral nucleic acid. The process of linking oligonucleotides to complementary nucleic acid is enhanced by hybridization. An oligomer having a sequence based on a complementary sequence based on an mRNA encoding a protein necessary for the progression of the disease is of particular interest. Specific hybridization with mRNA can disrupt the synthesis of proteins encoded by this mRNA.

In the past decade, the focus of attention of many research teams was the preparation of unmodified oligonucleotides, i.e. oligonucleotides having a DNA structure. Currently, the most effective method of preparing phosphodiester oligonucleotides is the synthesis using phosphoramidites according to Caruthers (MeVhide, II.). apa Sagciipegv, M.N., Teigapedop I eiv5., 1983, 24, 245), p. 29 originally proposed by Letsinger (and eivziIpdeg, K.I..; apa I spvioga, MU.V., u). Ateg. Spent 5os., 1976, 98, Ge) 3655) as a phosphite-trisphyra method. When promised, i.e. unmodified, oligonucleotide! are used as antisense oligonucleotides, problems arise!, associated with instability in relation to nucleases and insufficient degree of penetration through membranes". In order to make an anti-funny oligonucleotide! beaten in a state will suppress translation, it is necessary that they get inside the cell in an unchanged form. An oligonucleotide-antisense inhibitor should have the following properties: (i) (3) resistance to extracellular and intracellular enzymes; (ii) the ability to penetrate the cell membrane and (ii) the ability to hybridize with the target - DNA or RNA (Adagmai, K. And... hey hey, MysieYiss --

Asidz Kev., 1979, 6, 3009; AdauaI), 5. ei aiYi.,, Rgos. May Asad All together. BOTH, 1988, 85, 7079). Thus, it is useful to create analogues of polynucleotides, the properties of which, necessary for their use as antisense units, seeds or hybridization probes, are more pronounced. M

In the past, a modified polynucleotide was synthesized; modifications include methylphosphonates, phosphorylates, various amidates and sugar groups in nucleic acids. These substitutions in the skeleton provide some increase in stability, but bring a disadvantage: "the connecting phosphorus turns out to be in the position of the chiral center, which leads to the formation of 2"-70 diastereoisomers, where H is the number of modified diastereomer bonds in the oligomer. The presence of multiple diastereomers significantly weakens the ability of the modified oligonucleotide to hybridize with the target sequences. Some of these substituted ones also retain the ability to carry a negative charge, and the presence of charged groups weakens the ability of the compounds to penetrate the cell membrane. These modified bonds have a number of disadvantages, which are determined by the nature of each bond. synthesis of some analogues of oligonucleotides containing sugar modifications. The sugar modifications that were used to create structures that were considered more perfect, especially structures that are better absorbed by the cell, include o-DNA, homo-DNA, morpholine and thionucleoside and leptid nucleic acids (PNA). The general scheme of the synthesis of such analogues involves the involvement of 1 50 primary hydroxyl groups! of a nucleoside or a nucleotide bound to a polymeric carrier or c

With a nucleotide with a phosphorus atom and a certain sequence, in pentavalent or trivalent oxidation. Some of these joining procedures were named: phosphite trisphyre (phosphoramidite), phosphorous disphyre, and hydrophosphonate. The industry produces the necessary monomers for carrying out such procedures and the binding of monomers to polymeric carriers! with protected bases (о, А, С, 59 Т, О and other heterocycles) and protected by phosphorus atoms, which allows the monomer to be stored for a long time and

HF) prevents non-specific reactions during the compound reaction. Look! nucleic acids containing sugar modification, non-ionic skeletons or non-cyclic polyamides (PMA) possess, to one degree or another, one or more of the following properties useful for gene control: greater duplex stability (hybridization efficiency), greater 60 specificity in relation to targets, resistance to nucleases, good absorption in the cell and the ability to assist nucleic acids in important processes (for example,

H-ribonuclease activity, catalytic cleavage activity, the ability to stop hybridization, etc.). It is also suggested to use carbonate-dysphyria. However, these compounds are very unstable, and the carbonate-disphyric bond does not retain the tetrahedral form characteristic of phosphorus in phosphodisphyrics. Similarly, carbamate bonds, although they are not recognized as chiral, give triangular symmetry, and, as it turned out, poly-y4T with such bonds does not hybridize with poly-dA sufficiently strongly (Sociei,

MIND. ei ai., Teggapeadgop I ek5., 1987, 28, 745; ZYCgspak, E.R. ei ai., U. Ogu. Spet., 1987, 52, 4202).

Not so long ago, reports about noncyclic sugar analogs appeared in the literature (Atsdiziupv, K.A. ei ai.,

Mysieis Asiaz Kev., 1991, 19, 2587 - 2593). The inclusion of those non-cyclic nucleosides in an oligonucleotide! led to a decrease in melting temperatures, which depends on the number of linkers embedded in the oligonucleotide. These oligonucleotides turned out to be resistant to enzymes and were base-paired with a complementary sequence. Taking into account the disadvantages inherent in polynucleotides and known analogues of polynucleotides, it would be useful to create new analogues of polynucleotides, which 7/0 can be used in the course of antisense inhibition and other processes in which oligomers are used.

Attempts to modify both sugar and skeletal components give rise to a product that, as a medicine (and in other ways), has certain disadvantages. Therefore, it is necessary to significantly improve them before obtaining effective means of treatment and diagnostics /5 or analytical reagents. Thus, the need to create improved oligomeric analogues of oligonucleotides for use as medicines has long been ripe.

The present invention offers new oligonucleotides and their structural precursors, more resistant to digestion by nucleases, more stable in physiological conditions and capable of remaining neutral or carrying a positive charge, which facilitates penetration into the cell. In addition,

New oligonucleotides corresponding to the present invention have a stronger ability to hybridize with target nucleic acids.

Oligomers corresponding to the present invention are generally characterized by the fact that they contain a number of limiting linkers, or monomers, capable of binding with heterocyclic bases to a specific sequence of the target nucleic acid. Limiting linker), described in c g of this document, when included in the oligomer provide a bond strength that often exceeds the strength of a single hydrogen bond, contributing to the formation of a conformation competent for bonding. at

Nucleomonomers corresponding to the present invention are casually characterized as groups! or residues in which the furanose ring present in natural nucleotides is replaced by an amino acid or a modified amino alcohol residue. Examples of monomers and oligomers corresponding to the present invention are represented by formulas 1 - 41. Inclusion of those monomers described in this document in an oligonucleotide! makes it possible to synthesize compounds with improved properties, which include: (i) greater lipophilicity due to the elimination of the charge inherent in "- phosphodiester bonds" (Yoaid, 9U. (I) resistance to degradation by enzymes such as nucleases. Oligomers containing this monomer can be sufficiently stable for hybridization with target sequences and, in terms of a number of properties, are superior to unmodified nucleoside residues.

The essence of the invention

The present invention offers different, new analogues of oligonucleotides, possessing one or more properties that give them advantages over oligonucleotides, traditionally "Used in processes where oligonucleotides are used!". The compounds proposed by the present invention are analogues of oligonucleotides in which the furanose ring of a natural nucleic acid is replaced by an amino acid or a modified amino alcohol residue. Some of the new compounds proposed by the present invention are especially useful for the antisense control of gene expression. The compounds corresponding to the present invention can also be used as hybridization probes or as seeds. Another aspect of the present invention is monomeric precursors of oligonucleotide analogs corresponding to the present invention. These monomeric precursors can be used! for co-synthesis of analogs of polynucleotides corresponding to the present invention. - Another aspect of the present invention is the preparation of analogs of polynucleotides corresponding to the present invention, intended for the treatment or prevention of pathological conditions. Another aspect of the present invention is the treatment or prevention of diseases, in particular, viral infections and impaired cell growth. The methods of treatment of diseases corresponding to the present invention provide for the introduction of an effective amount of polynucleotide analogs corresponding to the present invention and their use as anti-humor inhibitors. 5B Brief explanations of the illustrative material

Fig. from 1 to 25 are schemes of chemical reactions, with the help of which it is possible to synthesize monomer and

F) oligonucleotides corresponding to the present invention. In particular, as Fig. 1 is the scheme of synthesis of thymine monomer phosphoramidite connected with I-serinol. Thymine and serinol of the connection -СН-СО-., in Fig. 2 is the scheme of the synthesis of thymine monomer phosphoramidite connected with I-serinol. Thymine and serinol are connected by the bond -СНо-СНо-.

Fig. C and 4 - schemes for the synthesis of substituted phosphoramidites of thymine monomer connected with

I-serinolum. Thymine and serinol are connected by the bond-СН-СО-.

Fig. 5 is a scheme for the synthesis of a T-T dimer, in which a nucleotide! connect! a bond 5 atoms long, in the center 65 of which is hydroxylamine, and thymine and serinol of the compound bond -СНо»-СО-.

Fig. b - this is the scheme of the synthesis of thymine monomer phosphoramidite, in which thymine is connected to c

M-ztilhydroxylamine by the bond СН-СО-.

Fig. 7 is a scheme for the synthesis of thymine monomer phosphoramidite connected with I-serinol, in which the MH group. I-serine is connected to a 2-hydroxyacetyl group, and the functional hydroxy group is blocked by the YUMT (4,4'-dimethoxytrityl) group. This construction unit is used to connect 2-5. This illustration also presents the synthesis of thymine monomer, in which the MH group of I1-serine is connected with a 2'-hydroxyzyl functional group.

Fig. 8 - the synthesis scheme of the T-T dimer, which has a hydroxamate skeleton with a 2-5 bond. In this dimer, one structural unit is formed by I-aspartic acid and thymine, and the other by I-serine and thymine. Two additional amide bonds are present in the 7/0 skeleton of this dimer.

Fig. 9 - the synthesis scheme of the T-T dimer, which has a hydroxamate skeleton with a 2-5 bond. In this dimer, one structural unit is formed by I-aspartic acid and thymine, and the other by I-serine and thymine. In the skeleton of this dimer, there is no amide bond between the monomers.

Fig. 10 - the synthesis scheme of thymine monomer phosphoramidite connected with I-serinol-b-alanine. 7/5 Thymine and serinol bind! through p-alanine.

Fig. 11 - the synthesis scheme of thymine monomer phosphoramidite connected with I-serinol-alkylamine.

Thymine and serinol binding through alkylamine.

Fig. 12 - the synthesis scheme of the T-T dimer, which has a hydroxamate skeleton with a 4-5 bond. The dimer is formed by two units of I -aspartic acid! and two units of thymine; thymine and aspartic acid are linked through acetyl.

Fig. 13 - the synthesis scheme of the T-T dimer, which has a hydroxamate skeleton with a 4-5 bond. The dimer is formed by two units of I -aspartic acid! and two units of thymine; thymine and aspartic acid are connected through ztyla.

Fig. 14 - scheme of the synthesis of thymine structural units connected with M-hydroxyamino acid. with

Fig. 15 - scheme of the synthesis of a thymine structural unit connected with I-aspartic acid.

Aspartic acid and thymine compounds! with the help of M-hydroxylamine. at

Fig. 16 - the synthesis scheme of the T-T dimer, which has a hydroxamate skeleton with a 4-5 bond. Here, the carboxylic acid group is connected to the thymine structural unit by an M-hydroxylamine bond.

Fig. 17 - the scheme of the synthesis of building units 150 (M-hydroxyamino acid, substituted with thymidine acetic acid (with acid) and its analogue 149. These monomeric building units are useful for the construction of nucleic acids with hydroxamate skeletons.

Fig. 18 - scheme of the synthesis of hydroxylamine substituted by thymidineacetic acid, containing the same amino acid structural units 157 and 158. These monomers are useful for the construction of nucleic acids with amide skeletons and hydroxylamine functional groups. co

Fig. 19 - the scheme of the synthesis of structural units 166 (thymidine, compounds with I-serinol), in which a hydroxylamine group is located between thymine and serinol. This building block is useful for building nucleic acids with 4'-5' linkages.

Fig. 20 - scheme of synthesis of thymidine monomer 174 connected with glutamic acid-glycine. This monomeric structural unit is useful for creating nucleic acids with amide skeletons and 2-5 bonds. shsh with Fig. 21 - the scheme of the synthesis of structural units 181 and 182 (thymidine, combined with glycinol-glycine), in which a hydroxylamine group is located between thymine and glycinol. These building blocks are useful for the preparation of nucleic acids with 2-5 bonds.

Fig. 22 - 24 - one hundred schemes! synthesis of structural units 191, 199 and 207 (thymidine, compound with ribosomal amino acid). These building blocks are useful for the preparation of oligonucleotides with ribosome skeletons.

Fig. 25 - scheme of solid-phase synthesis of oligonucleotide 211 with a ribosome backbone. because Fig. 26 - synthesis scheme of 1-O-(4,4-dimethoxytrityl)-2-amino(thyminylacetyl))|-I-propane-3-O-(M,M-diisopropyl)-p-cyanzthyl phosphoramidite.

Fig. 27 - synthesis scheme of the second 1-O-(4,4-dimethoxytrityl)-2-(amino(thyminylacetyl)|-O-propane-3-O-(M,M-diisopropyl)-VD 62-cyanoethylphosphoramidite.

Fig. 28 - synthesis scheme of 2-MK(B-(4, 4-dimethoxytrityl)-O-acetyl)amino|-3-thyminyl-y-propane-1-0O-(M,M-diisopropyl)-ZD-cyanylphosphoramidite. 22 Fig. 29 - synthesis scheme of M-(thyminylacetyl)-M-((2-isobutyryl)oxy|zyl|-O-benzylhydroxylamine.

Gee! Fig. 0) - synthesis scheme of (2K,45)-1-(tert-butyloxycarbonyl)-2-|(M z-benzoyl(thymin-1-yl)|methyl-4-phthalimide-pyrrolidine. ko Full description of preferred variants of the invention

A. Definitions and abbreviations When describing the present invention, we will use the following terms, which are defined below.

In this document, the term "antisense" therapy means the introduction or production of DNA or RNA oligomers or their analogs that specifically bind to the complementary sequence of the target nucleic acid. Binding can occur according to the well-known principle b5 complementarity of base pairs or due to the action of other mechanisms, for example, in the case of binding to double-stranded DNA - due to specific interactions in the major groove of the double helix. In general,

the term "antisense" refers to a whole range of technologies in this field based on the described principle and is suitable for any method of treatment that provides for specific binding to oligonucleotide sequences. Technologies of antisense control of genes are well known to specialists in the field of molecular biology; try it! antisense gene control can be found, for example, in US Patent 5,107,065, US Patent 5,166,195, US Patent 5,087,617, and StooK, Appia! By chemistry

RpagtasoYodu Tohisoiodu 1992 32; 329-376.

The terms "oligomer" and "oligonucleotide" are interchangeable and refer to both natural compounds such as DNA and RNA and their synthetic analogues, including the compounds proposed by the present invention. If not specified otherwise, the terms! "oligomer" and "oligonucleotide" refer to both DNA/RNA and their synthetic analogues. The term "oligomer" refers to compounds containing two or more nucleomonomers covalently linked to each other by a phosphodiester bond or a second substituent bond. Unless otherwise indicated, the term "oligomer" does not imply any length limitations. Thus, an oligomer can contain only two covalently linked nucleomonomers (dimer) or even longer. The oligomer can be competent in binding, i.e. base pairing with single-stranded and double-stranded nucleic acid sequences. Oligomer! (for example, dimer-hexameria) also apply! as synthons for longer oligomers, as described in this document.

Oligomer! may contain areas without basic and pseudonucleoside!.

Oligomers include oligonucleotides, oligonucleosides, polydeoxyribonucleotides (containing 2"-deoxy-O-ribose or its modified form), i.e. DNA, polyribonucleotides (containing U-ribose or its modified form), i.e. RNA and polynucleotides of any second type, namely M-glycoside!

C-glycosides of purines or pyrimidines are based. In this document, oligomers also mean compounds in which neighboring nucleomonomers are joined by hydroxamate bonds. Elements typical for oligomers, such as a furanose ring and/or a phosphodiester bond, can be replaced by any suitable functionally equivalent element. By the term "oligomer" is meant any structure that serves as a framework or a carrier for a base, which provides i) specific binding to the sequence of the target nucleic acid. Izvestne v presentely vremya oligomers! can be divided into four groups characterized as follows: (iii) oligomers! with phosphodiester or similar (phosphorothioate, methylphosphonate, etc.) bonds, ((i) ozo oligomers with substitutional bonds containing a non-phosphorus center of stereoisomerism (riboacetal, formacetal, carbamate, etc.), (ii) oligomer! with morpholine residues, carbocyclic residues or other furanose sugars, such as arabinose or hexose, instead of ribose or deoxyribose and (IM) oligomers containing a nucleomonomer connected by amide bonds, or noncyclic nucleomonomers, connected by any suitable substituent bond.

The term "nucleomonomer" in this document means a group containing (1) a base covalently linked to (2) a second group. Nucleomonomers include nucleoside), nucleotide! or bases, binding to an amino alcohol. Nucleomonomers can be combined into an oligomer) that specifically binds to a target, or a nucleic acid based on a complementary sequence.

The term "second group" here denotes a compound connected with a nucleomonomer and refers to the "amino/amino alcohol group; generally, it is serinol, aspartic acid, glutamic acid, glycine and compounds containing modified amino acid groups, for example, where one or more hydrogen atoms are replaced by other functional groups (see Formulas 24 - 41) or one of of carboxyl groups is converted into alcohol, amine, thysol, hydroxylamine, etc. The definition of nucleomonomer given here also includes a base connected to an amino acid or an amino alcohol and/or an analog of an amino alcohol having a free carboxyl/hydroxyl group, and/or these and 5" protected forms.

The term "nucleoside" in this document means an amino acid and amino alcohol derivative containing, as described below, a purine, pyrimidine or similar form, defined below, but not containing a connecting group, such as a phosphodiester analog or a modified 5p internucleoside bond. By "5"-nucleoside is meant a nucleoside that provides the linker with a connection point on carbon atom 5". The "5"-end of the linker connects to the 5'-position of the nucleoside. The "3"-end of the linker connects to the 3'-position of the next nucleoside. If there is a modified nucleoside in which there is no

From and/or 5'-carbon, specialist! they will understand what a symbol is! "3" and "5" indicate the polarity of the threads by analogy with p

DNA or RNA. 5B The term "nucleoside" in this document refers to a base covalently bound to an analogue of an amino alcohol/"amino acid. Between the base and the amino acid/(amino alcohol there is a linker. Under (F), the term "nucleoside" generally refers to ribonucleosides, deoxyribonucleosides and any other nucleosides, which are M-glycosides or C-glycosides is based.

In this document, the term "nucleotide" refers to a nucleoside containing a phosphate group or a 6o phosphate analog (a group in which phosphorus is oxidized to the same degree as in phosphate, for example, thiophosphate, amidate).

The term "base" in this document denotes a variety of nucleoside bases, including purine and pyrimidine heterocycles, as well as their heterocyclic analogues and tautomers. Purines include adenine, guanine and xanthine; an example of a purine analogue can be 8-oxo-M d-methyladenine 65 ml 7-deazaxanthin. Pyrimidines include uracil and cytosine and their analogs, such as 5-methylcytosine, 5-(1-propynyluracil), 5-(1-propynylcytosine), 5-methyluracil and 4,4-ztancytosine. Bases, attached to a suitable molecular framework, for example, a phosphodiester skeleton, are capable of pairing, which occurs in double-stranded DNA or other double-stranded nucleic acids of a similar structure. Bases can be capable of pairing in triple helices of nucleic acids.

The term "sugar modification" in this document refers to any amino acid or amino alcohol group other than 2'-deoxyribose!.

The term "amino acid/amino alcohol" herein refers to any naturally occurring amino acid or amino alcohol in the K or 5 isomeric form.

The term "nucleoside bond" as used herein refers to a bond that exists within a monomer. 70 The term "bond" in this document refers to a group used to connect a base to an amino acid/amino alcohol and their derivatives.

The term "internucleotide bond" in this document denotes a phosphodiester group (-О-Р(ОХ(0)-0-) or any other functionally equivalent group that covalently binds adjacent nucleomonomers!).

The term "substituent bond" in this document means any analogue of the native group or any suitable group that covalently binds adjacent nucleomonomers. Substituent bonds include analogs of the phosphodiester group, for example, such as phosphorothioate and methylphosphonate, and groups that do not contain phosphorus, for example, such as amide, hydroxamate, hydroxylamine. Substituent bonds include phosphorus-free bonds offered by the present invention (bonds 2.5, bonds 3.5, bonds 47.5).

The term "cross-linking group" in this document means a group in the oligomer that forms a covalent bond with the target nucleic acid. Cross-linking groups include groups that covalently bind the oligomer to the target nucleic acid or spontaneously (for example,

M7, M"-ztancytosine), or under the activating influence of light (for example, psoralen), etc.

The term "blocking groups!" in this document denotes a substituent group, different from H, covalently bonded to oligomers or nucleomonomers as protecting groups, connecting groups for synthesis, ОРО»z». or other traditional conjugates, such as a solid support, a label, an antibody, a monoclonal antibody or its fragment, etc. In this document, the expression "blocking group" denotes not only a protective group, as in the jargon, but also refers, for example, to "2 connecting groups, such as H-phosphonate or phosphoramidite.

The term "protecting group" in this document refers to any group capable of protecting the O-, 5- or M- atom to which it is attached from participating in a reaction or forming a bond. Protective "-groups for M- atoms, which are part of the base of the nucleomonomer, and the methods of their application are known to specialists. Examples of suitable protective groups can be the following, but not exclusively: diisobutylformamidine, benzoyl, silyl, etc. Suitable for the O- and 5- atoms are, for example, the protective group UMT, MMT, EMOS or complex Zphyr. In this document, "protecting group" means any group capable of protecting the O-, 5- or M- atom, to which it is attached, from entering into a reaction or forming a bond. Such protective groups! for atoms "

O-, 5- and M-, which are part of the nucleomonomers, are described in the literature, and the methods of their application are given to specialists. The number of protective groups also includes groups capable of preventing reactions and 0-s coupling on carboxyls, thiols, etc. and The term "linking group" in this document means any group suitable for forming a bond or a substitutional bond between nucleomonomers, for example, H-phosphonate and phosphoramidite.

The term "conjugate" or "conjugated group" in this document refers to any group, i.e., attached to the oligomer at its end or at another site. Conjugates include solids with bases, such as silica gel, porous glass, and polystyrene; labels, for example, fluorescent, chemiluminescent, radioactive atoms or molecules, enzyme groups and "reporter" groups; - means of transport of oligomers, such as polycations, whey proteins, glycoproteins, polymers, etc. sl 50 K conjugates also include O-cholesterol, polyethylene glycol (RES) (PZG), amino acids, intercalators, groups of polynucleotide derivatives, cross-linking groups, lipids, hydroxamates, alkylating "2 agents, etc.

The term "synthon" in this document denotes a structural unit in the composition of the oligonucleotide analog corresponding to the present invention.

The term "transfection" in this document refers to any method of effective delivery of oligomers into cells. o The term "object" in this document means a plant or an animal, including a mammal, in particular, a human.

The term "derivative" of oligomers and their monomer components has a general meaning for this area. For example, an oligonucleotide! can be covalently bonded to various groups, such as intercalators, compounds that interact specifically with the minor groove of the DNA double helix, and other arbitrary conjugates, for example, labels (radioactive, fluorescent, enzymatic, etc.)

From these additional groups, there may be (but not necessarily) a derivative with the participation of a modified skeletal bond; they can become part of the connection itself. For example, an intercalator, such as acridine, can be bound through K-CH 5-, attached to any available -OH or 5H, for example, at the terminal 5-positions of RNA or DNA, 2-positions of RNA or OH/ZH, by insertion into 5 positions of pyrimidines, for example, instead of 5-methyl in cytosine. That is, the derivative contains -СНХСНОСНоОН or -СнНХСнНХСН»ОЗН in position 5. Any substituent can be attached, including one connected by a common bond. Consequently, the indication of the OH group in the oligomer corresponding to formula (1) can be substituted! protect with phosphonate groups! standard protection groups, activation! to form additional bonds with other nucleotides or compounds! with conjugates. 5i-terminal OH is indifferently phosphorylated; 2-OH or OH substituents at the 3-end can also be phosphorylated. Hydroxyl! it is also possible to transfer to the standard protection group.

The term "analogue of phosphodiesterase" in this document means an analogue of a simple phosphodiesterase 7/0 bond, and it refers to other groups of bonds. Join other groups! include, but not exclusively, the variant when O-P(O)- is replaced by P(0)5, P(IO)MK", RIK, RIK, where K is H or alkyl (1-7C) and B is alkyl (1-7C). Analogues of phosphodiester in one oligomer are not necessarily necessary! beat identically; There is only one requirement for them: at least one of these bonds must be a modified internucleotide bond. "Analogous" forms! Purines and pyrimidines are forms well known to specialists, and many of them are used as chemotherapeutic agents. A non-exhaustive list of examples includes 4-acetylcytosine, 8-hydroxy-MO-methyladenine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thysuracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, Mr-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine,

3-methylcytosine, 5-methylcytosine, Mo-methyladenine, 7-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thysuracil, beta-O-mannosylguanosine, 5-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio -Me-isopentenyladenine, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, dceovipe, 2-thiouracil, 4-thysuracil, 5-methyluracil, M-uracil-5-oxyacetic acid methyl ester , uracil-5-oxyacetic acid, o pseudouracil, dceovzipe, 2-thiocytosine and 2,6-diaminopurine. A particularly preferred analogue is 5-methylcytosine (abbreviated as "Ste" in this document).

The term "isosteric" in this document refers to the spatial and orientational properties of an internucleoside bond and implies the fact that these properties are so similar (and) to the properties of a native phosphodiester bond that a modified oligonucleotide containing an isosteric bond can replace, replace, imitate a native oligonucleotide and/or hybridize with it. "-

The term "ribosoamide" in this document denotes an internucleotide bond existing between two nucleotide bases. Ribozoamide internucleotide linkage contains a combination of functional groups of ribose/(2'-deoxy) and amino acids. "AND

In this application, various abbreviations are used to denote functional groups and compounds.

Specialist! in the field of organic chemistry, the abbreviations will be easily understood. For example, "RI" means phenyl, "Me" means methyl, "(1-7С)3" means that this chain contains from 1 to 7 carbon atoms, etc. "

Description of the invention

The present invention offers new analogues of oligonucleotides containing modified amino acid/amino alcohol bonds between bases and skeletons (phosphodiester, phosphorothioate and others, listed in Table 1), also called modified nucleotide bonds. ,» Modifications or their functional equivalent!, consisting in the replacement of the sugar group located between the skeleton and the base, with an amino acid derivative, represented, for example, by formula 24.

This invention also offers! a new nucleomonomer and ways of incorporating them into an oligomer, i.e., containing nucleomonomers! With this present invention, we offer a variety of nucleomonomers! with a structure corresponding to formulas 1 - 23. - Basis

Fo Ar o y,

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Kk, b5 2

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B, U and c in A at 5

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Basis - -

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Fo Kk | and OA

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Oligomers corresponding to the present invention are polymers containing one or more of the monomeric compounds corresponding to the present invention, linked in such a way that a structural analog of DNA or RNA is obtained. Oligomer), corresponding to the present invention, contain two or more nucleomonomers and can contain almost any number of nucleomonomers, although it is more convenient to synthesize an oligomer! of 200 and less nucleomonomers. Compounds described by formulas 1 - 23 can be connected to each other through bonds 4-5, 3-5 and 2-5, as can be seen from formulas 24 - A1. b5

And And oh oh oh oh. Основание Основание Основание

O-6-0 Oghr-O0 O-v-O0

It is the Foundation, the Foundation, the Foundation, and no H (o) Y

I 24 24A 248

Nucleotid bonds in the compounds corresponding to the present invention are obtained from the amino acids /5 serine and glycine or their derivatives. Oligonucleotide), corresponding to the present invention, stable Ip mimo, stable! in relation to the effect of zndogenic nucleases and abilities! to hybridize with specified sequences of nucleotides. Examples of compounds corresponding to the present invention are represented by formulas 24 - 41. These compounds are conformationally more restrictive! in comparison with phosphodiester bonds of unmodified DNA or RNA. This conformational limitation may be one of the reasons for the enhancement of the ability of these substances to bind to specific complementary polynucleotide sequences; however, the application of the present invention is not based on the theory of increasing the ability to connect.

The second embodiment of the present invention proposes modification of oligonucleotides or their derivatives, where the furanose group of a natural nucleotide, for example, DNA or RNA, is replaced by an amino acid/amino alcohol and other groups, including amino acids with substitutions in certain positions (formulas 25 - 41). Internucleotide bonds between adjacent nucleomonomers are (3 bonds between positions 4" and 5 of adjacent nucleomonomers. In other words, a phosphodiester internucleotide bond or its functional equivalent connects the 5'-position of one nucleomonomer with the 4-position of the second, as in the compounds represented by the formulas 24 - 33: o

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Where each "K" is independent of the others - i.e. H, OH, 5H, CM, CH3, OSNU3, ЗСН3, ОМН», ОМН(СН»), РИ, -(СНо)х-Е; where "x" means 1 to 7 carbon atoms and "E" means MH, 5H, OH, COOH, OSH3, ZSNU, RI, MON,

MON(SNZz), ZMN»o, (O)MN», B(OKHO)MN», SNz, RY.

Where each "base" is independent of the others - it is a nucleoside base.

Where each "K/" is independent of the others - i.e. H, OH, ЗН, СМ, СН3, ОСН3, ЗСН3, ОМН»б, ОМН(СН»), РИ, -(СНо)х-Е; where "x" means 1 to 7 carbon atoms and "E" means MH, 5H, OH, COOH, OSH3, ZSNU, RY, MON,

MON(SNZz), ZMN»o, (O)MN», B(OKHO)MN», SNz, RY.

Where each "Ko" is independent of the others - i.e. H, OH, ZN, CM, CH3, OSH3, ЗСН3, ОМН»б, ОМН(СН»), РИ, -(СНо)х-Е; where "x" means 1 to 7 carbon atoms and "E" means MH", 5H, OH, COOH, OSH3, ZSNU, RY, MON, 20. МОонН(СНУ), 5МНо, Б(ОМН", Б( OKHOMN", SN, RA.

Where each "Ku" is independent of the others - i.e. H, OH, ЗН, СМ, СН3, ОСН3, ЗСН3, ОМН»б, ОМН(СН»), РИ, -(СНо)х-Е; where "x" means 1 to 7 carbon atoms and "E" means MH, 5H, OH, COOH, OSH3, ZSNU, RY, MON,

MON(SNZz), ZMN»o, (O)MN», B(OKHO)MN», SNz, RY.

Where each "K/" is independent of the others - i.e. H, OH, ЗН, СМ, СН3, ОСН3, ЗСН3, ОМН»б, ОМН(СН»), РИ, с -(СНо)х-Е; where "x" means 1 to 7 carbon atoms and "E" means MH, 5H, OH, COOH, OSH3, ZSNU, RI, MON, d)

MES(SZ), ZMN", (O)MN", B(OKHO)MN",; SNz, RA.

Where each "A" is independent of the others - zto (СНо)х, СО, С5, 5, З(0), З3(0)(0), МН, МОН, МСН»з, MK» and ze, where "х "- from 1 to 7 carbon atoms.

Where each "B" is independent of the others - i.e. (СНо)х, СО, С5, 5, З(О), З(О) (0), МН, МОН, МОН», MK» and ze, where o " - hundred 1 - 7 carbon atoms. yu

Where each "X" is independent of the others - i.e. (СНо)х, СО, С5, 0, 5, З(О), 5(О) (0), МН, МОН, МОН», and MK», where "х "- from 1 to 7 carbon atoms. --

Where each "7" is independent of the others - i.e. (СНо)х, СО, С5, 5, З(0), З(О) (0), МН, МОН, МОН», and MK»b, where "х" so - zto 1 - 7 carbon atoms.

Zo Kv - zto H, OH, OMe, CM, MH, MON, OMCH», OMN», ztyl, propyl, lower alkyl (1-73), Me, heteroalkyl « (1-7C), aryl(b-7C) , -"SNO)XE; where "x" means 1 - 7C and "E" independently of the others - means Н, ОН, ЗН, ОСН», СМ, ЗСН»

OMN", OMN(SNz), ZMN»o, B(O)MN", B(OHOOMN", SN"z, RY.

Where each "M" is independent of the others - that is, an analogue of phosphodiester, phosphorothioate, methylphosphonate, "phosphorodithioate), borophosphonate), selenophosphonate, phosphoramidate, acetamidate, oxyformamide, oxyacetamide, diisopropylsilyl, carbamate, dimethyl sulfide, dimethyl sulfoxide, dimethyl sulfone c) and /or an internucleoside bond with a length of 2-4 atoms from among carbon, nitrogen, oxygen, sulfur and selenium. "» An oligomer can contain from two to two hundred monomers, and even more. The preferred "modification of the internucleotide bond "U" is shown in Table 1

In addition, the compounds described by these formulas can be connected with one or more conjugate groups. The number of suitable conjugate groups includes O-cholesterol, polyethylene glycol, and amino acids, intercalators, splitting groups! (for example, imidazole), cross-linking O group! (for example, psoralen), lipid, peptide, alkylating agents, hydroxamate! and fluorescent labels.

A conjugate group can be independently replaced by one of K, Ku, Ko, Kz, Ku and Kb. - Another variant of the implementation of the present invention is an oligomeric structure, representation (layer 50 by formulas 34 - 36, and their derivatives: in, o g. -4 t Basis

I (in;

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In the compounds described by formulas 34 - 36, the bonds between adjacent nucleomonomers are 3-5 bonds.

Where each "K" is independent of the others - i.e. H, OH, 5H, CM, CH3, ОСН3, ЗСН3, ОМН», ОМН(СН»), РИ, -(СНо)х-Е; where "x" means 1 to 7 carbon atoms and "E" means MH, 5H, OH, COOH, OSH3, ZSNU, RY, MON,

МОН(СН»), МН», З(О)МН»о, З(ОХО)МН», СНз, РИ. 65 Where each "base" is independent of the others - it is a nucleoside base.

Where each "K/" is independent of the others - i.e. H, OH, ЗН, СМ, СН3, ОСН3, ЗСН3, ОМН»б, ОМН(СН»), РИ,

-(СНо)х-Е; where "x" means 1 to 7 carbon atoms and "E" means MH, 5H, OH, COOH, OSH3, ZSNU, RY, MON,

МОН(СН»), МН», З(О)МН»о, З(ОХО)МН», СНз, РИ.

Where each "Ko" is independent of the others - i.e. H, OH, 5H, CM, СН3, OSН3, ЗСНЗ, ОМН», ОМН(СН»), RA, -(СНо)х-Е; where "x" means 1 to 7 carbon atoms and "E" means MH, З5Н, ОН, СООН, ОСН»з, 5СНз, РИ, МОН,

МОН(СН»), МН», З(О)МН»о, З(ОХО)МН», СНз, РИ.

Where each "Ku" is independent of the others - i.e. H, OH, ЗН, СМ, СН3, ОСН3, ЗСН3, ОМН»б, ОМН(СН»), РИ, -(СНо)х-Е; where "x" means 1 to 7 carbon atoms and "E" means MH, 5H, OH, COOH, OSH3, ZSNU, RY, MON,

МОН(СН»), МН», З(О)МН»о, З(ОХО)МН», СНз, РИ. 70 Where each "K/" independently of the others is H, OH, ZN, CM, CHz, OSH»z, ZCHz, OMN", OMN(CH"), RY, "SNao)x-E; where "x" means 1 to 7 carbon atoms and "E" means MH», ZN, OH, COOH, OSN», CH», RI, MON, MON(CH»), ZMN»

Z(O)MN", (0) (O)MN", SNz, RA.

Where each "A" is independent of the others - i.e. (СНо)х, СО, С5, 5, З3(0), (000), МН, МОН, МОН», MK» and ze, where "zh - zto 1-7 .

Where each "B" is independent of the others - i.e. (СНо)х, СО, С5, 5, 300), З(О)(0), МН, МОН, МОН», MK» and ze, where "zh - zto 1 -7.

Where each "X" is independent of the others - i.e. (СНо)х, СО, С5, 0, 5, З(О), З(О) (0), МН, МОН, МОН», and MK», where "х "-from 1-7.

Where each "y" is independent of the others - i.e. (СНо)х, СО, С5, О, 5, З(О), З(О) (0), МН, МОН, МОН» and MK», where it is 7х- zto1-.

Where each "7" is independent of the others - i.e. (СНо)х, СО, С5, 5, З(О), З(О) (0), МН, МОН, MSН»з and MK, where "x" is -3т01 -7.

Kv - zto H, OH, OMe, CM, MH, MON, OMCH»z, OMN», ztyl, propyl, lower alkyl (1-73), Me, heteroalkyl (1-7C), aryl(b-7C), "SNO)XE; where "x" means 1-7C and "E" independently of the others - means Н, ОН, 5Н, ОСН», СМ, ЗСН», сх дв ОМНо, ОМН(СН3), ЗМН», З(О)МНо, Z(OKHO) MN", SN, RA.

Where each "M" is independent of the others - that is, an analog of phosphodiester, phosphorothioate, methylphosphonate, i) phosphorothioate, borophosphonate, selenophosphonate, phosphoramidate, and/or an internucleoside bond with a length of 2 to 4 atoms from among carbon, nitrogen, oxygen, sulfur and selena An oligomer can contain from two to two hundred monomers, or even more. Preferable modification of the internucleotide bond "M" about the results! in Table 1.

Another embodiment of the present invention is oligomers corresponding to formulas 37 and 41, or their variants. These oligomers contain a new internucleotide bond - bonds 2-5. Zty "- oligonucleotide! stable! By the way, they are more resistant to the effects of zndogenic nucleases and abilities! to hybridize with given oligonucleotide sequences. co

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Where each "K" is independent of the others - i.e. H, OH, 5H, CM, СН3, ОСН3, ЗСН3, ОШ», ОБИН(СН3), РИ, "СНо)хХ-Е; 09 where "X" means 1-7 carbon atoms and "E" means MH», ЗН, ОН, СООН, ОСсН», СН», 5РИ, МОН, МОН(СН»), МН», «I

Z(O)MN", (0) (O)MN", SNz, RA.

Where each "base" is independent of the others - it is a nucleoside base.

Where each "K/" is independent of the others - i.e. H, OH, ЗН, СМ, СНз, ОСНз, ЗСНУ, ОШ», ОМН(СНЗ), РИ, « -(СНо)х-Е; where "X" means 1 to 7 carbon atoms and "EE" means MH", ЗН, ОН, СООН, ОСН»5, 5СН3, РИ, МОН,

МОН(СН»), МН», Б(О)МН»б, Б(ОЧО)МН», СН3, RA. - c Where each "Ko" is independent of the others - i.e. H, OH, 5H, CM, СН3, OSН3, ЗСНЗ, ОМН», ОМН(СН»), RA, and -(СНо)х-Е; where "X" means 1 to 7 carbon atoms and "EE" means MH», ЗН, ОН, СООН, ОСН»5, 5СН3, РИ, МОН, "» МОН(СН»), МН», З(О )MN", (0) (O)MN", SN", RY.

Where each "Ku" is independent of the others - i.e. H, OH, ЗН, СМ, СН3, ОСН3, ЗСН3, ОМН»б, ОМН(СН»), РИ, -(СНо)Х-Е; where "X" means 1 to 7 carbon atoms and "EE" means МН», ЗН, ОН, СООН, ОСН3, 5СН3, РИ, МОН, Т» МОН(СН»), МН», З(О)МН "o, Z(OKHO)MN", SNz, RY. because Where each "K/" is independent of the others H, OH, ZN, CM, CH3, OSH»z, ZCH3, OMN», OMN(CH"), РЙ, "СНао)х-Е; where "X" means 1-7 carbon atoms and "E" means МН», ЗН, ОН, СОН, ОСсН», ЗОН», ЗРИ, МОН, МОН(СН»), ZМН», - 5(0О) MN", (0) (O)MN", SN»z, RY. sl 50 Where each "A" is independent of the others - that is (СНо)х, СО, С5, 5, З(0), З3(0)(0), МН, МОН, МСН»з, MK» and ze, where "x" is 1 to 7 carbon atoms. "2 Where each "B" is independent of the others - i.e. (СНо)х, СО, С5, 5, З(0), З(0(О), МН, МОН, МОН", ME» and ze, where "Х " - hundred 1 - 7 carbon atoms.

Where each "X" is independent of the others - i.e. (СНо)х, СО, С5, 0, 5, З(О), З(О) (0), МН, МОН, МОН», and MK», where "ХХ " - hundred 1 - 7 carbon atoms. o Where each "7" is independent of the others - i.e. (СНо)х, СО, С5, 5, З(О), З(О) (0), МН, МОН, МОН», and MK», where "x" - from 1 to 7 carbon atoms. име) Kv - zto H, OH, OMe, CM, MH, MON, OMCH», OMN», ztyl, propyl, lower alkyl (1-7C), Me, heteroalkyl (1-73), aryl (b6-7C) , "СНо)ХЭ; where "x" means 1 - 7C and "E" independently of the others - means Н, ОН, 5Н, ОСН», СМ, ЗСН», 6о ОМНН», ОМН(СНУз), МН», 5(О)МНо, Z(OKHO) MN", SNz, RY.

Where each "M" is independent of the others - that is, an analogue of phosphodisphyra, phosphorotysate, methylphosphonate, phosphorodate!, borophosphonate, selenophosphonate!, phosphoramidate! and/or an internucleoside bond with a length of 2-4 atoms from among carbon, nitrogen, oxygen, serb and selenium. An oligomer can contain from two to two hundred monomers, or even more. Preferred modified internucleotide linkage "M" 65 shown in Table 1.

Other embodiments of the present invention provide for the creation of an oligomer,

corresponding to the following formula (formula 42), and its monomeric components (formulas 85 - 90).

M v u zna i k i zu 42A i i i v

Kk in-x

In: 1 what and in them k-x td 428 s x-v i) yn vi "c) ziv -m oyu g, - vo s eu 426 chI . x-V at 72 Ky «

M v u - s hv ts o y?" х Ки

Kk u at t. 426 (ee) shk min o toi wa s w t n-e min v i v o h o 42E 60 b5 min

Ge) i

IN

Vk n-n t khz o i kh

Kk 70 i

Where where

X is absorbed from the group consisting of (СНо), where n is 1-3, СО(СНо)в, where n is 0-2, and (СНо)3О», where n is 1-2, and U is absorbed from the group, consisting of CH", CO, COOH, C5 and 50". "U is absorbed from the group consisting of CH", СО, СООН, С5 and 50". 7 is absorbed from the group consisting of О, 5, МН and СН".

K is selected from the group consisting of CHOOH, CHONO, CHONO, CHONO, and CHOOH.

B is a nucleoside base. the dream was

M in Kk cs 85 o u: v i 1 u v u d o. ; -x

H - 86 p. in x -V o

Her Mr

M so kv -x 88 1 Yehiv o ze u v nn n 89

F) at the end of the 1960s

Кк у 90 where X is absorbed from the group consisting of (СНо), where n - 1 - 3, СО(СНо)в, where n - 0-2 and (СНо)в50», where n - 1-2,

U is absorbed from the group consisting of СН», СО, СООН, С5 and 50». "U is absorbed from the group consisting of CH", СО, СООН, С5 and 50". 7 is absorbed from the group consisting of О, 5, МН and СН".

K is selected from the group consisting of CHOOH, CHONO, CHONO, CHONO, and CHOOH.

B is a nucleoside base.

The second aspect of the present invention is the means of treating diseases due to the presence of a nucleotide sequence, providing for the introduction of an object in need of such treatment, the above-mentioned modified oligonucleotides, capable of specifically binding to / about the sequence of nucleotides, in the amount necessary for inactivation of that sequence of nucleotides.

In the oligonucleotides corresponding to the present invention, at least one of the phosphodiester groups designated by "M" in formulas 24 - 41 is replaced by a modified internucleoside bond from among the descriptions in this document. It is desirable to replace the entire series of phosphodiester bonds 7/5 of the unmodified oligonucleotide with a modified internucleoside bond; if desired, various modified internucleotide linkages can be formed. In preferred embodiments of the present invention, these substitution bonds are not chiral, which increases the ability of the oligonucleotide to hybridize with a given target; however, among the useful compounds corresponding to the present invention, there are also those in which chiral bonds are present.

Preferable modification of internucleotide linkages, designation in formulas "U", shown in Table 1.

Table 1 -0- сч -5- -5(0)- (8) -5(0)(0)- -6е2е- -51- -с(0)- | "c) -с15,)- -МН- IF) -Mon- - -МСН3- -НЕ5- со -СН»- « - . and? sh" (ee) - 1 (42) ime) 60 b5

-0-сС85- -сн-0- -0-СНо-СНо- -сНн;-сСнН»-о- -СсСН-0О-СНо- -0-сно-о- -5-СНо- -сн.-8 - -8-снН.-СНи- -СН.-СНо-5- -снИ-8-СНо- -8-СНо-5- -0-снО-8- -8-снН-0- -510)-СН ;- -Сно-5(0)- -510)-СНо-СНа- -снНо-СНо-5(0)- -сН»-810)-СНа- -510)-сСН»-5(0)- - 0-сн5-5(0)- -510)-СНИ-О -5(0)(0)-СН;- -СН2-5(0) (0) - -5(0)(0)-СНо- СН»- -сн.-СНо-5(0)(0)- сч зв сно-5(0) (0) -СН5- -8(0) (0)-СсН»-5(0) (0) - (о) о-сно-5(03 (0)- -810) (0)-СН-0- -58-8- -8в(0)-50)- (ав) -510) (0)- 510)-(0;)- -ve-СВо- юю -Сно-5е- - -8е-СН»-СНо- -сн.-СсНИ-5е- (ee) -СН»-5е-СНо- з5 -Be -СНо-8Зе- - -0-сн.-Зе- -5е-сСн)-0- -8е(03-С8- -СНо-8е(0)- " du -5е(03-СНо-СНо- with - сН»-СНо-5е(0)- с -СНо-5е(0)-СНо- "» -Бе(0)-СН»-5е(0)- " -0-сСНо-5е(0)- -Бе (0)-СН»-0- -Be(0) (0)-СН- iz -СНо-5е(0) (03- -Бе(0)(0)3-СсНо-С8о- (о) -сно -СНо-бе (0) (0)- а -СНо-5е(0) (0)-СН85- -8е(0) (0)-СНо-5е(0) (0)- "сл 50 -е- 58e- "2

F) why 65

-Be(0)-be(0)- -Be(0) (0)-5e(0)-(0)- -0-СНо-5e(0) (0)- -ve(0) (03- СН»-0- -8-СН;-бБе- -Бе-СН-5- -5(0)-Сн.-5е(0)- -5е(0)-СН-8(0)- -84103 ( 0) -СНа-бе (0) (0) -

I -Be(0) (0) -СН2-5(0) (0)- -58-8- -510)-5(0)- -510) (0)-510) (0)- -5e- 8e- -be(0)-58e(0)- -be(0)0)-5e(0)(0)- -M(B5)-СНо- -sna.-M(B5)- -M(B5 ) -СН.-СНо- -СНО-СНО-М(БЕ5) - -СН.-М(Б5)-СНИ- -МК(85)-0- -0-М(Б5)- -М(Б5)- 0о-СН- -сн.-0о-М(Б5)- -сно-М(Б5)-0- in -0-М(Б5)-СТ- сч -0-СНа-М(Е5) - -М( Б5)-СН»-О- о -М(Б5)-5- -5-М(Б5)- -М(Б5)-5-СНе- о зо -сн-8-М(Б5)- -сн. -Щ(Б5) -5- oyu -8-М(Б5)-СНо- -5-СНО.-М(Б5)- -- -М(Б5)-СН-8- -М(Б5)-85( 0)- со -510)-К(В5)- " -Ш(В5)-5(03-СНо- -СН.-510)-М(Е5)- -СНО-М(В5)-510)- - 5(0)-М(Б5) -СНо- « -8(0)-сНо-М(Б5) - -М(Бе)-СНо-5(0)- - с -М(Б5)-550) ( 0)- and -5810)(0)-М(Б5)- ;2 -М(Б5)-5(0) (0)-СН5- -сСН.-510) (0) -М(Б5) - - СНо-М(В5)-5 (0) (0)- 15 -Б(0) (0)-М(В5)-СН- o -5(0) (0) -СНУ-М(В5) - o -M(E5)-СНо-5(0) (0) - -0-Ш(В5)-5- - -5-М(В5)-0- -0-m(В5)-510)- layer o "2 (F) ko 60 65

-5(0)-M(B5)-0- -0-M(E5)-5(0) (03- -5(0) (0)-M(B5)-0- and -0-8- 0- -0-8(0)-0- -0-8(0) (0)-0- -M(B5)-5-M(B5) - -M(B5)-5(0)-M (B5) - -M(B5)-5(0) (0) -M(B5)- -сН;-5-0- -сн2-5:190) -0- -сн»-5(0) ( 0)-0- -sn.-с(0)-0- -sn.-s(5)-0- -СНИ-М(Б5)-С2(0)-0- -СН.-М/(Б5 )-С(5)-0- -М(Бе)-С(0)-0-Стб- -Мм(Бв5)-С(5)-0-СН5- -0-с(0)-М(Б5 )-0- -0-с(8)-М(Б5)-0- -0-0(0)-М(БЕ5) -СНо- -0-с(535-М(Б5)-СНо- -0 -с(0)-СНо-М(Б5) - -0-с(5)-сНо-М(Б5)- -0-с(0)-сн3-0-М(Е5)- -0-с( 8)-сН-О-М(Б5)- сч -0-с(0)-М(Б5) -0-СНо- 29 -0-С(5)-М(Б5)-0-СН- о - 0-М(85)-С(0) -0-СНо- -0-М(85)-С(5) -0-СНо- -сСНо-0-С(0)-М (В) -0- -сни-0-Сс(5)-М(Б5)-0- о зо -сСНо-0-С (0) -М(Б5)-СНо- -СН-0-С (5) -М (Б5) -СНо- ИС о) -сни-0-С(0)-СНо-М(Б5) - -СнНи-о-с(5)-СНо-М(Б5) - -- -СНо-0-С(0 )-К(Б5)- со -СН»-0-С(5)-М(Б5)- -сн.-0-с(0)-М(Б5)-0- « -сН»-о-С (8)-M(85)-0- -sn"-o-M(B5)-C(0)-0- -sn".-o-M(B5)-C(5)-0- -sn »-М(В5)-с(0)-5- « -сн»-М(В5)-С(58)-5- -К(В5)-С(0)-8-СН»- o) с -К(В5)-с(5)-5-С85- . -8-С2(0)-М(Б5)-0- «» -0-с(53-М(Б5)-5- -8-0(0)-М(Б5)-СНо- -8-0 (8)-М(Б5)-СНо- 15 -8-с0(0)-СНо-М(Б8)- т -8-с(8)-СН»-М (Бе) - со -8-С( 0)-СН»-О-М (Бе) - -0-с(8)-СН»-5-М (Бе) - - -0-с2(03-М(Б5)-5-СНо- -8 -C(8)-K(B5)-0-C- 1 "2

F) ko bo b5

-5-М(Б5)-2(0)-0-СНИ- -0-М(Б5)-С(5)-5-СНо- -СН;-5-С2(0)-М(НК5)- 0- -сн.-0-с(5)-М(Б5)-5- -СН-5-С2(0) -М(Б5) -СНо- -СН;-5-С(5)-М ( Kv) -СНаи- -снНа-5-2(0)-СН.-М(Б5) - -сн;-8-02(5)-СНО-М(К5) - -СНИ-5-С2 (0) -М(Б5) - -сН;.-5-С(5)-М(Б5)- -сСн;-5-С2(0)-М(Б5)-0- -сн.-8-с(5 )-М(Б5)-0- -СН»-5-М(Б5)-С2(0)-0- -сН.-5-М(8Б5)-С(5) -0- -СН»- Оо-С10)-М(ВБ5)-5- -СНо-0-С(5)-М(Б5) -5- -сСН;-О-М(Б5)-С2(0)-5- -СсН- 0-М(Б5)-С(5)-5- -М(Б5)-М(Б5) - -М(Б5)-М(Б5)-СНа- -СНО.-М(К5) -М(К5 ) - -М-С МН») -М(К5)- -М(Б5) -М-С (МН) - -8410)-СНе-О- -0-сн.-5(0)- -8- СН(Б5)-0- Га -08-с8 (Е5)-5- -0-сно-сНеСн- i) -5Б-СсН.-СН-СН- -5-СНИ-С-с- -М(К5 )-СН2-М (ВК) - -М(В5)-2(0)-М(В5)- (ав) -М(В5)-С(5)-М(В5) - ю -М(ВК5) -C(0)-5- -M(B5)-C(5)-5- «- -M(B5)-C(5)-0- -M(85)-C2(0)-0- s -0-2(0)-M(B5)- -0-s(8)-M(B85)- З -5-0(0)-M(B5)- -8-06(5)-M( K5) -

Kv - zto H, OH, OMe, CM, MH, MON, OMCH»z, OMN», ztyl, propyl, lower alkyl (1-73), Me, heteroalkyl « 40. (1-73), aryl(b- 7C), "SNO)XE; where "x" means 1-7C and "E" independently of the others - means Н, ОН, 5Н, ОСН», СМ, СН», - с ОоМН», ОМН(СНУз), ZМН», З(О)МН »o, (OKHO)MN»o, SNz, RY. In addition, one or several conjugate groups can be attached to the bond to obtain an oligomeric conjugate. Suitable conjugate groups include O-cholesterol, polyethylene glycol, amino acid, intercalating, splitting groups! (for example, imidazole), cross-linking groups! (for example, psoralen), lipid, peptide, alkylating agent, hydroxamate! and fluorescent labels. Particularly preferred 4" - 5' bonds include phosphodiester, phosphorothioate, methylphosphonate, cocarboxamide, thiocarboxamide, hydroxamate, sulfonamide, hydroxylamine, and carbamate. The same modifications are preferred as bonds 2-5 and 3-5. - and Oligomers corresponding to the present invention are not necessarily oligomers with the same type of bonds, they may contain alternating or randomly distributed bonds, including bonds of type 2, 5. Since the oligomers corresponding to the present invention can be synthesized, each time adding to one nucleomonomer residue, each individual bond, substitutional bond, and each individual "base" type substituent can be selected independently of the others to obtain an oligonucleotide with a given sequence of nucleotides.

Oligomers corresponding to the present invention may contain any desired amount of substitutional bonds. These substitution bonds can be the same or different depending on the chosen "M", in addition, they can be different from the "M" provided by the present invention. име) Since the assembly of oligomers occurs sequentially, it is possible to use any alternation of types of bonds, substitutional bonds, basic and sugar modifications. 60 In preferred embodiments of the present invention, the substitutional bonds corresponding to the present invention alternate regularly. For example, one substitution bond is followed by two phosphodiester bonds, which are followed by one substitution bond corresponding to the present invention, etc. In addition, it is possible, for example, such an alternation, when the substitution bond is followed by an analogue of phosphodiester (for example, thioate, etc.), which is followed by a substitution bond corresponding to the present invention, which is followed by a phosphodiester analogue, etc. that. in the oligomer corresponding to the present invention, substitutional bonds of two types can alternate through one. In oligomers corresponding to the present invention, in which there are more than one type of bonds, it is possible to use any regular order of alternation of bonds of different types connecting subunits of the oligomer.

Modification of sugar can be performed in one or more residues of nucleomonomers of oligomers corresponding to the present invention, however, when including such modifications, it is desirable to use nucleotide bonds of 4-5, 3-5 and 2-5 between amino acid residues. In this case, a further abbreviation can be used to represent the sequence based on the oligonucleotide analog. For example, ordinary DNA or RNA is usually presented in the form of a sequence of only 70 bases, for example, ATO SOS TOA. As a rule, it is indicated in advance what the designation corresponds to: DNA or RNA. In this document, for the presentation of analogs of oligonucleotides with a given sequence, the corresponding system used is indicated.

Additional modification of nucleomonomers

Oligomers corresponding to the present invention, in addition to the substitution bonds corresponding to the present invention, may contain various modifications. Additional modifications include oligomers in which () one or several nucleomonomer residues are modified! in positions 2", 3, 4 and 5, (II) contains one or more covalent cross-linking groups, (II) contains other bonds not provided for by the present invention, (II) contains other analogues of the base, such as 8-oxo -MO-methyladenine and (M) conjugates are present, such as an intercalator or polylysine, which, respectively, increase the binding affinity with target nucleic acid sequences or enhance oligomer binding to the cell.

The ability of oligomers corresponding to the present invention to specifically bind to sequences in polynucleotides of single- and double-stranded targets is compatible with additional modifications. These additional modifications can give oligomers other useful properties, such as resistance to cleavage by nucleases (for example, in the domain of the oligomer corresponding to the present invention, containing phosphodiester bonds) or increased i) ability to penetrate through cell membranes! etc.

Oligomers corresponding to the present invention may contain one or more substituent bonds, such as sulfide or sulfone bonds (Veppeg, 5.A., International publication Mo M/O 89/12060), sulfamate bonds (International publication Mo UUO 91/ 15500), carbamate or other substitution bonds in oligomers with morpholine bonds (Zygspak, E.R.

Kez. 1989, 17, 6129 - 6141; Zuttepop, ., international publication Mo 216860) and related connections. h

Thus, among the examples of oligomers corresponding to the present invention are oligomers in which (1) there is at least one substitution bond and an amino acid connected to the adjacent monomer, (2) there is one or more substitution bonds not related to the present " And the invention, which is selected from the group including phosphorothioate, methylphosphonate and thionomethylphosphonate, and/or (3) has one or more phosphodiester bonds and/or (4) contains analogues of purines or pyrimidines that increase the affinity of binding to complementary "target sequences". The second examples include: (1) an oligomer with substitution bonds corresponding to the present invention at the 3 and/or 5' end and phosphorylated bonds in the second parts of the oligomer; (2) oligomers with substituted bonds corresponding to the present invention and standard purine or pyrimidine bases (for example, adenine, guanine, cytosine, thymine or uracil); (3) oligomers with substituted bonds corresponding to the present invention and one or several bases that increase the binding affinity or penetration ability of the oligomer (for example, 5-methylcytosine, 5(1-propynyl)uracil, "" 5-(1-propynyl)cytosine). Examples also include oligomers containing nucleomonomer residues, hydroxamate binding Synthesis of oligomers - Oligomers) corresponding to the present invention can be assembled only from the nucleomonomers corresponding to the present invention, or the ordinary nucleomonomer can also be used. which are currently recognized as industries trenches corresponding to the present invention can be fulfilled in accordance with the method providing for the synthesis of a nucleomonomer or oligomer synthon with a protective group, a base and a linking group capable of combining with a nucleomonomer or oligomer; connection of a nucleomonomer or synton oligomer with a nucleomonomer-acceptor or an oligomer-acceptor; o removal of the protective group; repeating the cycle the required number of times until the required oligomer is synthesized.

Oligomers corresponding to the present invention can have any length, including more than 40, 50, 100, 200 or 500 nucleomonomers. In general, the preferred oligomer contains 2-30 nucleomonomers. Lengths from 8 to 20 nucleomonomers and more can be useful for therapeutic and diagnostic applications, provided that these oligomers have the corresponding basic sequence. Short oligomers containing 2, 3, 4 or 5 nucleomonomers are included in the present invention and can be used as synthons. 65 Oligomer! with random sequences containing 6, 7 or 8 nucleomonomers can be used as primers in cloning or amplification technologies that use random sequence primers, provided that the oligomer contains 1 or 2 residues at the 3-end that can serve primers for polymerases or reverse transcriptases and do not interact with polymerases in any other way.

In addition to the bonds described for the first time in this document, the oligomers according to the present invention may contain simple phosphodiester bonds or, in addition to the substitutional bonds corresponding to the present invention, may contain other substitutional bonds, such as phosphoramidate bonds. The non-exhaustive list of these substitution bonds includes the variant where the group described by the formula -O-P(OX5)-O- ("phosphorothioate"), -O-P(O) (ME 277)-X2, -O-P(O ) (K"7)-0-, -O-P(5) 70. (87)-O- ("thionoalkylphosphonate"), -P. (0) (089)-X2, -0-C(0) -X? or -O-C(O) (MEV»17)-X2-, where B! is H (or a salt) or alkyl (1-12 carbon atoms, including methyl and ethyl), and K is that is alkyl (1-9 carbon atoms), and the connection of the compound with neighboring nucleomonomers Through -O- or -5-, the connection with the carbon of the nucleomonomer, and X2 - that is O or 5. Phosphorothioate and phosphodiester bonds are good! particularly preferred for use in the oligomers proposed by the present invention are phosphodisphosphorylated, phosphorylated, methylphosphonate and thionomethylphosphonate substituent bonds. only phosphorothioate or methylphosphonate substituent bonds.

Oligomers, offered by the present invention, and their fragments can be synthesized using methods known to specialists. The method of synthesis, accepted in the industry and described in this document, can be used for the synthesis of oligomers containing substitutional bonds corresponding to the present invention, as well as other bonds and substitutional bonds known in the industry, from the corresponding protection of nucleomonomers. The method of synthesis of oligomers with CM phosphorus-containing bonds can be found, for example, in Egoepieg, V., ei ai!., Mysiyevs Asidz Kez., 1986, 14, 5399 - o 5467; Mysieis Asidz Kez., 1988. 16, 4831 - 4839; Mysieovzides 5 MysieoNaevz, 1987, 6, 287 - 291; Egoepieg, V.,

Tegapedgop I Ave., 1986, 27, 5575 - 5578; Sagsshi(yehv, M.N. in OIPIDodeochupisieoiPdez Apiizepze IppIirShopv oi

Sepe Ehrgezviop, 1989. 9.5. Sopep, eapyog, SKS Rgezz, Vosa Kaiop, r/ - 24; Keese, S.V. ei a), Teigapedgop

Yev., 1985, 26, 2245 - 2248. Synthesis of oligomers with methylphosphonate bonds with the help of ("v") methylphosphonamidate chemistry is also described in Adgau!Yi, 5. ei aYi., Temapedhop Iev., 1987, 28, 3539 - 3542 ; yu

Kiet, K.E., ek am, International publication Mo UUO 92/07864).

Oligomers), containing the connections proposed by the present invention, can be conveniently synthesized by preparing dimeric or trimeric compounds in the liquid phase with subsequent conversion of the synthon to a derivative, which is included in the oligomer using solid-phase or liquid-phase procedures. A typical synthesis is the production of 3'-phosphonate or phosphoramidate, blocking at the 5-end with the help of UOMT or MMT, preparation by standard methods. (saii, M.). ed., OPdopisieoy de Zupipeviv; AND

Rgasiisa! Arrgoasi 1984, IC. Rgezv, Ozhkhgoga).

The number of synthons included in the scope of the present invention includes dimers, trimers, tetramers, "hexamers and longer oligomers") synthesized in the solid or liquid phase. Trimers and longer synthons can contain more than one type of bond. These synthons may contain any of the bases described above, and in positions 2, 3, 4, and 5 may contain groups such as OH, HOMTO, HMMTO, O-allyl, phosphate, phosphonate, or amidite, as described above. ribosome oligonucleotide! can be synthesized using the standard technology of solid-phase synthesis of peptides (Etos chemistry) (Fig. 26).

Blocking groups for the synthesis of compounds proposed by the present invention - 1. Connecting groups! o Suitable linking groups include, for example, H-phosphonate, methylphosphonamidite or phosphoramidite. Suitable phosphoramidites include D-cyanzyl phosphoramidite! (which are mutually exclusive). It is also possible to use methylphosphonamide, alkylphosphonamide. (including c 20 zylphosphonamidit! and propylphosphonamidit!). Examples of phosphoramidites are shown in fig. 1 - 21.

The number of suitable "connecting groups" in positions 2, 3, 4 or 5 for the synthesis of oligomers according to the principles of phosphoramidite trisphyr chemistry, which we will call here "amidite" chemistry, include

M,M-diisopropylamine-p-cyanoethoxyphosphine, M,M-diisopropylamine-methoxyphosphine,

M,M-disthylamine-cyanzthoxyphosphine and (M-morpholine)-methoxyphosphine (Mooge, M.R. ei aii, U Ogd Spet., 1985, 5920, 22 2019-2025; Ozpapek A.MU., ei a!ii, Teigayedhop Iez., 1987, 28, 3401 - 3404; Wiegdagde, K., ei ai, Mysi Asidv

GF) Kez., 1991, 19, 5843 - 5850; Bani, 0. 5yMig Kerogov, 1991, 11, 167 - 192). For the preparation of methylphosphonates, it is also possible to use related connecting groups, such as di M,M-diisopropylamine-methyl-phosphine or M,M-disthylamine-methyl-phosphine. Methylphosphonate oligomer is convenient to synthesize with the help of connecting groups, such as 60 M, M-diisopropylamine-methylphosphoramidite. The synthesis of nucleomonomeric amidites corresponding to the present invention can be performed by standard methods (for example, Sguagpom, 5.M., ei ai, Mysi

Asidz Kev., 1992, 20, 1879 - 1882; Miipauak, K., ei aiYi, Mysi Asidz Kez., 1992, 20, 1265 - 1269; 5iIpra, M.O., hers, Mysi Asiaz OKez., 1984, 12, 4539 - 4557 and other references, given here). 2. Protector groups! For protection of the cyclic nitrogen of the heterocycles of cytosine, adenine, or guanine, such protective groups as diisobutylformamide, benzoyl, isobutyryl, EMOS, dialkylformamide, dialkylacetamide, or other groups known to specialists can be used. Cytidine can be directly included in the oligomer! without a protective group on the cyclocyclic nitrogen in accordance with the described methods (Sguagpom, 5.M., ei ai, U Ateg Spemu. Bos., 1991, 113, 5876 - 5877; sguagpom, 5.M., ei ai, Misi Asiaz Kez ., 1992, 20, 1879 - 1882; Kypa, R.-R. e(a), Temigapeadgop I ek5., 1992, 33 , 5869 - 5872).

Suitable protecting groups include OMT (dimethoxytrityl), B2 (benzoyl), Vy (isobutyryl), phenoxyacetyl, MMT (monomethoxytrityl) or EMOS at the 5-terminus and/or H-phosphonate, methylphosphoramidite, methylphosphonamidite, rd-cyanosthylphosphoramidite, tvo (tert -butyldimethylsilyl) or TvVORB 70 (tert-butyldiphenylsilyl) at the 3'-end.

Protective groups B are preferred; (benzoyl), UOMT (dimethoxytrityl), MMT (monomethoxytrityl) or EMOS at the 5-end or in the 5-position and/or TV5, H-phosphonate, methylphosphoramidite, methylphosphonamidite, VD-cyanzthylphosphoramidite at the 3'-end. However, if necessary, the positions of the blocking groups can be changed (for example, attach phosphoramidite in position 5, and OMT in position 3). In general, 75 derivatives of the oligomers corresponding to the present invention, and such "bSlocating groups" can be obtained by methods known in this field, as can be seen from the corresponding formulas.

Conjugate!

The present invention also provides "conjugates" to oligomers corresponding to the present invention. Conjugate ok general oligomers of lime to specialists. For example, the oligomer corresponding to the present invention can be covalently connected with various groups, for example, with intercalators or compounds interacting specifically with the minor groove of the DNA double helix. Conjugation of oligomers with other groups is also possible, including labels (for example, radioactive, fluorescent, fermentable) or groups that facilitate connection with the cell due to the presence of cleavable linkers, etc. To suitable radioactive marks Ge! include ZR, 358, ZN, 191) and 142; suitable fluorescent labels include fluorescein, resorufin, rhodamine, VOSIRM (Moiesciag Rgobez) and Texas red; suitable enzyme labels include alkaline phosphatase and horseradish peroxidase. Other compounds can be used as covalently attached groups: biotin, antibodies or antibody fragments, asialoglycoprotein, transferrin, and HIV T protein. at

The derivative of these additional groups can be obtained with the help of any suitable groups. For example, intercalators such as acridine and psoralen can be linked to oligomers according to the present invention with the help of any available -OH or -ZH, for example, at the 5'-terminal position of the oligomer, "-2-position of RNA, or through OH , MH", COOH or 5H, contained in the 5-position of pyrimidines. Comfortable! with a derivative form containing, for example, СНСНСННОН or СНОСННСНОЗН in the 5-position of pyrimidines. Conjugates, including polylysine or lysine, prepared by the described methods, increase the binding affinity of an oligomer with a given sequence of nucleic acids! (I etaige, M. ei ai.,

Ros Mai! Asai 5si. BOTH, 1987, 84, 648 - 652; I eta ge, M. ei ai., Mysieovidez 8: Mysieop des, 1987, 6, 311 - 315).

You can add various substitutes, including those that connect! bonds or substitute bonds. -OH groups in oligomers can be replaced by phosphate groups, protected by standard protecting groups, combined with connecting groups to form additional bonds with o, with other nucleomonomers, or connected with a conjugated substituent. The -OH group at the 5-end can be phosphorylated; 2-OH or OH substitutes at the 3-end can also be phosphorylated. Hydroxyls can also be produced using standard protecting groups. The oligomer corresponding to the present invention can be covalently linked with groups that facilitate connection with the cell due to the presence of cleavable linkers in them. The number of suitable conjugates also includes a solid substrate for the synthesis of oligomers and facilitating the determination of the sequence of nucleotides in a nucleic acid. A non-exhaustive list of solid substrates includes silica gel, porous glass, polystyrene and magnetic glass beads. - Modifications of sugar with 50 Production can be obtained! at the expense of substituted sugars. Among the preferred derivatives of oligomers according to the present invention are 2-O-allyl and 3-allyl, which increase "2 penetration ability and resistance to the action of nucleases, but do not reduce the affinity of the oligomer to single- or double-stranded targets. In particular, in the oligonucleotide! different functional groups can be introduced with ribosome skeletons! (in positions 1", 2", 3, 4 and 5 of ribose) to improve the pharmacokinetic properties of the corresponding oligonucleotides.

Gee! Substitute communication

Oligomers offered by the present invention, in addition to the descriptions in this document of bonds 2 - where 5,3 - 5 and 4 - 5, may contain one or more "substituent bonds", known to specialists. These "substituent bonds" include phosphoroisoate, methylphosphonate, thionomethylphosphonate, 60 phosphorodate, alkylphosphonate, morpholine sulfamide, boranophosphate (-0О-Р (ОСН») (ВНз)-О-), siloxane (0-81 (Х7) ( ХУ) -0-); X7 is alkyl with 1-b carbon atoms or phenyl) and phosphoramidate (methoxyethylamine (-О-Р (ОСНСНОСН») (0)-0-), etc., which are synthesized as described in the public literature, including in the following works (Zosd, A., ei a 9). At. Spet. Zos., 1990, 112, 9000 - 9001; UMO 91/08213; MO 90/15065; UMO 91/15500; ZyTsgspak, E.R . eig aI MysieIis Asid Kev., 1989, 17, 6129 - 6141; US patent 5034506, because US patent 5142047; Nem/Ii, U. M. eyi a, Mysieozidev 8: Mysieoi dev, 1992, 11, 1661 - 1666; Ztsitepop, in her ai;

International publication Mo 216860). To substitute connections, which can be used! oligomers described in this document also include sulfonamide (0-5О 2-МН), sulfide (-СнНО-5-СН»), sulfonate (-0-5О»-СН»о), carbamate (0-С( 0)-МН-, -МН-С(0)-0-), dimethylhydrazine (-СНо-МСН3-), sulfamate (-0-5(0)(0)-М-, -М-5(О0) (0)-M-), 3-amine (-МН-СНо-), M-methylhydroxylamine (-СНо-МСН3-О-) and 25i-bonds (such as 2,5 carbamate (2-М(Н)- C(0)-0-5), 5,2 carbamate (2-0-C(0)-M(H)-5), 5,2 methylcarbamate (2-0-C (0)-M (CH3) -5) and 5,2 thioformacetal (2-0О-СН.О-5-5)00 In addition, amide bonds are acceptable as substitutional bonds, described by the authors of Vyspagai, o. /20702), and Sook, R.O. Hey! (International publication Mo M/O 92/20822), Oe Meztaekeg, A. 7/0 eGai., (International publication Mo MO 92/20823 and in PCT/O5Z92/04294.

Unless otherwise indicated, substitutional bonds, such as formacetal -O-СНо-О-, are attached to carbon 4, 3, or 2" of the nucleomonomer on the left, and to carbon 5 of the nucleomonomer on the right. The designations 4", 3,2, or 5' can be to change the image if a structure other than ribose, deoxyribose, or arabinose is attached to the nucleomonomer. Such structures include xylose, hexose, morpholine ring, 7/5 hydrocarbon ring (for example, cyclopentane), etc.

The use of carbamates, carbonates, sulfides, sulfoxides, sulfones,

M-methylhydroxylamine and dimethylhydrazine bonds in synthons or oligomers were described (Magzechg, 9-9. ei aiYi, 9U. Ateg. Spet. Bos., 1992, 114, 4006 - 4007; M/o 89/12060; MyvisKiI, V. ей а), U Ogd Spet., 1990, 55, 4231 - 4233; Keupoidv, K.S. ei aI., U Ogd Spet., 1992, 57, 2983 - 2985; Mepez, M.R., hey! ) Mea. Speth,., 1969, 12, 154 - 157; Mypoai!Y, M/.5., ei aii, 9 Ogd Spet., 1977, 42, 703 - 706; 5yNgspak, ER. ей аЙ 9U Oga

Spet., 1987, 52, 4202 - 4206; UMUapo, N., ей аЙ, Temygapedhop I eiv., 1991, 32, 7385 - 7388; International application Mo PCT OBO1/03680).

Substitute bonds are included in oligomers to solve a number of problems: further facilitating binding to the complementary sequence of the target nucleic acid and/or increasing the resistance of oligomers to nucleases.

Reasons i)

The number of bases that can be used as nucleotide bases in the compounds proposed by the present invention includes not only natural purine and pyrimidine bases, but also analogues of these heterocyclic bases and their tautomers. Such analogues include the alkylation of purine! or o z pyrimidines, acylation of purines or pyrimidines or other heterocycles!. These "purine-like" or "pyrimidine-like" compounds, or analogs of purines or pyrimidines, are well known to specialists, and some of them are used as pharmaceuticals. Their non-exhaustive "- list includes MM"-ztancytosine, 7-deazaxanthosine, 7-deazaguanosine, 8-oxo-MU-methyladenine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, so

B-carboxymethylaminomethyl-2-thysuracil, inosine, M'U-isopentenyladenine, 1-methyladenine, 2-methylguanine, "F 5-methylcytosine, Mb-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thysuracil, 2-tisuracil, 4-tisuracil, 5-(1-propynyl)-4-tisuracil, 5-(1-propynyl)-2-tisuracil, 5-(1-propynyl)-2-t-iocytosine, 2-thiocytosine and 2,6-diaminopurine. In addition to these analogues based on the oligomer proposed by the present invention, it is possible to include analogues of pyrimidines, which include b-azacytosine, b6-azatimidine and 5-trifluoromethyluracil, described in Sook, ts O.R., ei aii, in an international publication Mo UMO 92/02258, included in this document through LINKS. -» The inclusion of 4-thysuridine and 2-thiothymidine in the oligomer is described (Mikio, T.T., ei aiYi, Teigapeadgop Iev., 1992, 33., 2379 - 2382; Simio, R., ei aYi, Teiapeidop iev., 1992, 33: 65 - 68; MiIKMOhom, T.T., ei ai, THemapedtop I env., 1991, 32: 2505 - 2508; Khy, U.7., ei aI Teigapedop I eiv.,, 1991, 32: 2817 - 2820; Simio, and R., and others, Teiapedgop Ienvv., 1992, 33: 69 - 72; Soppou, V.A., and others, MysiI. Asiya Kev., 1989, 17, 4957 - o 4974) . Preferred bases include: adenine, guanine, thymine, uracil, cytosine, 5-methylcytosine, 5-(1-propynyl) uracil, 5-(1-propynyl)cytosine, 8-oxo-M5-methyladenine, 7-deaza- 7-methylguanine, - 7-deaza-7-methyladenine and 7-deazaxanthosine. p 50 Covalent binding group

Some oligomers corresponding to the present invention include a group capable of forming at least one covalent bond between the oligomer and the double-stranded nucleic acid.

Multiple covalent bonds can be formed due to the inclusion of a number of such cross-linking groups. In the preferred case, the residue of the base of the target thread 22 participates in the covalent bond, but other fragments of it, for example, saccharide or phosphodiesterol, may also participate.

F! The functional nature of the cross-linking group also determines its target in the double strand. Among the preferred cross-linking groups are acylating and alkylating agents, in particular, those that are located in relation to the part providing specificity in such a way that they do not interfere with the reaction with the target in the nucleic acid strand. 60 It is clear that a heterocycle is not necessarily a purine or a pyrimidine, and a pseudobase to which a functional group is attached is not necessarily a heterocycle at all. We will accept any means of joining the functional group, if its location is correct.

Polarity of oligomers.

In the most general form, the symbol 3'---5 denotes an oligomeric chain in which all bonds are formed between the hydroxyl of the 5' position of the amino acid residue of the nucleomonomer on the left and the hydroxyl of the 3' position (position 2"

for oligomers with bonds 2 - 5 and "4 for oligomers with bonds 4 - 5) of the amino acid residue of the nucleomonomer on the right (that is, the region of uniform polarity), and the hydroxyl of the 5-position of the amino acid residue of the far right nucleomonomer remains free for further binding. Similarly, the symbol 5---3 denotes an oligomer chain of the opposite orientation, where the bonds formed between the hydroxyl of position 3 of the amino acid residue of the left nucleomonomer and the hydroxyl of position 5' of the amino acid residue of the right nucleomonomer, and the hydroxyl of position 3 of the far right nucleomonomer remains free for further bonding. This is also true for oligomeric chains 5'----4.

Pharmaceutically acceptable salts 70 The present invention also provides various salts of all the compounds described in this document, including salts acceptable from a pharmaceutical point of view for administration to animals and humans.

Pharmaceutically acceptable salts and substances that form them are well known to specialists. In the preferred case, these are salts of oligomers proposed by the present invention, and ammonium and metals, for example, alkaline or alkaline earth metals: sodium, potassium, magnesium or calcium; easily 7/5 Crystallizable ammonium salts obtained by reaction with ammonia or organic amines, such as mono-, di- or tri-lower (alkyl, cycloalkyl or hydroxyalkyl)-amides, lower alkylenediamines or lower (hydroxyalkyl or arylalkyl)- aluminum ammonium bases, for example, methylamine, distylamine, tristhylamine, dicyclohexylamine, tristanolamine, ethylenediamine, tris-(hydroxymethyl)aminomethane or benzyltrimethylammonium hydroxide. Oligomers corresponding to the present invention can form salts with acids; preferably, salts of therapeutically acceptable inorganic and organic acids, such as stronger inorganic acids, for example, hydrophilic: hydrochloric, hydrobromic, sulfuric, phosphoric, -aliphatic or aromatic carboxylic or sulfonic acids!: formic, acetic, propionic, succinic, glycolic, lactic , apple, tartaric, gluconic, lemon, ascorbic, maleic, fumaric, hydroxymaleic, pyruvic, phenylacetic, c benzoic, 4-aminobenzoic, anthranilic, 4-hydroxybenzoic, salicylic, 4-aminosalicylic, methanesulfonic, ztansulfonic, hydroxyztansulfonic, benzenesulfonic, sulfanilic or i) cyclohexylsulfam, etc.

Therapeutic effect and application

Since the oligomer proposed by the present invention has significant cross-linking activity against single- and double-stranded target nucleic acids and forms stable double, triple and other structures with natural polynucleotides and their structural analogues, the oligomer proposed by the present invention can is not used in most processes in which ordinary oligomers are used. Yes, oligomers corresponding to the present invention can be used, for example, as a probe! hybridization of polynucleotides, primers for the polymerase chain reaction with 5 and similar cyclic amplification reactions, primers for sequencing, etc. Oligomers, offered by the present invention, can also be used for diagnosis and treatment of diseases. The therapeutic application of oligomers corresponding to the present invention provides for the specific suppression of gene expression (or suppression of translation of RNA sequences encoded by these genes) associated with the emergence and development of pathological conditions, with the help of antisense oligomers. Oligomer! corresponding to the present invention can be used in many ways! for the mediation of anti-funny suppression of a number of gene targets. Examples of genes or RNA encoded by those genes, which can be targets; Antisense suppression with the help of oligomers can serve as a gene/RNA encoding enzymes, hormones, serum proteins, transmembrane proteins, adhesion molecules! (GEA-1, (ZRIIBLYa, EGAM-1, 45. MASM-1, ISAM-1, E-zeysiop, etc.), receptor molecules, including cytokine receptors, cytokines (1I--1, their I/-2, 1-3, 1-4, 1/-6, etc.), oncogenes, growth factors and interleukins. These genes or RNA can be associated with any pathological conditions, caused by inflammation, cardiovascular diseases , with immune reactions, cancer, viral infections, bacterial infections, fungal infections, - parasitic invasions, etc.

Oligomers, a proposal of the present invention, adventures! for therapeutic use, both Ip o mimo and eh mimo. Indications for the use of ex mimo include the need to treat cells, such as bone marrow or peripheral blood, in conditions such as leukemia (chronic myeloid leukemia, acute lymphocytic leukemia) or viral infection. Genes or RNA encoded by these genes, which can serve as targets in cancer treatment, include oncogenes such as gas, K-gas, rcI-2, c-tub, reg, c-tus, c-abi, or in supersynthesized sequences, such as tata, oncostatin M, 1-6 (Kaposi's sarcoma), NEK-2, and translocations, such as reg-ari. Suitable targets are also viral gene sequences (F, or RNA, encoded by corresponding genes, such as polymerase or reverse transcriptase genes of herpes viruses, such as cytomegalovirus and herpes simplex virus 1 and 2; retroviruses, such as human T-cell leukemia virus -1, HIV-1, HIV-2; or other DNA or RNA viruses, such as HNV, because NRM, MAM, influenza virus, adenovirus, flavivirus, rhinovirus, etc.

Specific binding ooligomers can be used in combination with other therapeutic agents. The second areas of therapeutic application of oligomers proposed by the present invention include (1) modulation of inflammatory reactions due to modulation of the expression of genes, such as receptor 1-1, P-1ISAM or E-Zeiesiop genes, which play a certain role in 65-mediated inflammation (2 ) modulation of cell proliferation in conditions such as arterial occlusion (restenosis) after angioplasty due to modulation of the expression of (a) growth or cell division factors, such as non-cellular myosin, tus, OH, ROMA, ROSE or BOB or their receptors or (B) factors proliferation of cells, such as c-tur. In the treatment of psoriasis and other conditions, other proliferation factors or signaling factors, such as TOEx, II-6, dIME, protein kinase C, tyrosine kinase, can serve as targets! (such as p210 and p190). In addition, alleles of the main histocompatibility complex, TOR, or ESE receptor can serve as targets for autoimmune diseases.

The efficiency of delivery of the oligomers proposed by the present invention to cells can be determined by any suitable method, including by transfection with calcium phosphate, dimethyl sulfoxide, glycerol or dextran, by electroporation or by cationic, anionic 7/0 Miml of neutral lipid compounds or liposomes , as described in international publications MO UUO 90/14074, MO 91/16024, MO 91/17424 and US patent 4897355. Oligomer! it can be introduced into cells in the form of complexes with cationic lipids, such as SOTMA (which may or may not form liposomes); the input complex then interacts with the cell. A non-exhaustive list of acceptable cationic lipids includes M-(2,3-di(9-(7)-octadecenyloxyl))-prop-1-yl-M,M,M-trimethylammonium 7/5 (BOTMA) and its salts, 1 -O-oleyl-2-O-oleyl-3-dimethylaminopropyl-pD-hydroxyethylammonium and its salts and 2,2-bis(oleyloxy)-3-trimethylammonium)propane and its salts. It is also possible to increase the efficiency of the delivery of oligomers proposed by the present invention with the help of viruses such as the Sendai virus (Vagilai,

K., ViIoiesppo! Arri Biospet., 1989, 11, 133 - 135) or adenovirus (U/adpeg, E. eyi aYi, Rhos Mai! Asai 5siI. O5A, 1992, 89, 6099 - 6013); (II) polyamine or polycationic conjugates, such as polylysine, protamine or Ma,

Mi2-vps(ztyl)spermine (Uuadpeg, E. ei aiYi, Rhos Mai Asad zZsi. OA, 1991, 88, 4255 - 4259; Hepke, M., ei ai,

Rgos Mai Asai 5si. BOTH, 1990, 87, 3655 - 3659; Spapk, V.K., ei ai, Viospet Viorpuz Kez Sottyp., 1988, 157, 264 - 270; US Patent 5138045); (II) lipopolyamine complexes with such compounds as lipospermin (Vepg, 9.-R. ei a), Rgos Mai Asai si. BOTH, 1989, 86, 6982 - 6986; IoeSheg, 9.R., ey ai, 9. Meygospet., 1990, 54, 1812 - 1815); (Im) anionic, neutral, or pH-sensitive lipids, with the use of Compounds, which include anionic phospholipids, such as phosphatidylglycerol, cardiolipin, phosphatidic acid, or phosphatidylethanolamine (I ee, K.-O. e( ai, Viospet Viorpuz ASTA, 1992, 1103, 185 - i) 197; Spedaag, 0. ei aiY, Agspy Viospet Viorpuz, 1992, 294, 188 - 192; MovzpItiga, T., ei aYi, Viospet Ipi., 1990, 20, 697 - 706); (M) conjugation with such compounds as transferrin or biotin, or (mi) conjugation with proteins (including albumin or antibodies), glycoproteins or polymers (including "polyethylene glycol"), which improve the pharmacokinetic properties of oligomers, which I'm talking. In this document, transfection means any method acceptable for the delivery of oligomers into cells. Any substance, such as a lipid, or any agent, such as a virus, that can be used for transfection, in general! let's call it "an agent that improves penetrating ability". The delivery of oligomers to cells can be carried out by means of co-transfection with other nucleic acids, such as (i) an expressible DNA fragment encoding a protein/proteins or a protein fragment, or (its) translatable RNA encoding a protein/proteins or a fragment! a squirrel

Thus, the oligomer proposed by the present invention can be included in any suitable composition that increases the efficiency of their delivery to cells. Suitable pharmaceutical "compositions" also include those that are commonly used when the delivery of the compound to cells or tissues occurs due to external application of the composition. Compounds such as polyethylene glycol, propylene glycol, azone, "» nonoxyl-9, oleic acid, dimethyl sulfoxide, polyamine, or lipopolyamine can be used in preparations for external use, which also contain an oligomer.

Oligomers, offered by the present invention, are convenient to use as a reagent! for the analysis or preparation of compounds when it is necessary to suppress the expression of genes. Currently, there are very few reagents that effectively and specifically suppress the expression of a given gene due to some mechanism. Oligomers, which were reported to suppress the expression of a given gene, because they often gave non-specific effects and/or did not suppress the expression of a given gene to a sufficient extent (to less than 4095 from the non-inhibited level).

Thus, the oligomers described in this document represent a reagent that can be used to suppress the expression of a specific protein or proteins in a subject organism or in cells where the proteins are encoded by DNA sequences and translated from RNA sequences, in accordance with by methods providing: the introduction of the oligomer corresponding to the present invention into cells; creation of conditions under which the oligomer forms a double or triple structure c

DNA or RNA and thus suppresses the expression of a protein or proteins. Ability! and compounds proposed by the present invention are suitable for modulating gene expression in both prokaryotic and iF) zukaryotic cells, such as bacterial cells, fungal parasite cells, yeast and mammalian cells.

The use of substitution bonds proposed by the present invention makes it easy to construct an oligomer that is "competent" and "incompetent" in relation to ribonuclease H. Competent in relation to ribonuclease H oligomer! may contain one or more ribonuclease H-competent domains, consisting of mutually linked nucleomonomers competent in relation to ribonuclease H. Oligomers containing modifications such as 2-substitutions (2"-O-allyl, etc. d.) or an uncharged bond (methylphosphonate, phosphoramidate, etc.), generally incompetent! as a substrate for b5 ribonuclease H, which it recognizes and acts on. Competence in relation to ribonuclease H can enhance the antisense effect of the oligomer due to degradation of the target RNA into the structure of the RNA oligomer (Yuadie, 9U.M. ei ai, Mysi Asiyz Kev., 1990, 18, 4751 - 4757; MUaideg, 9.A. ei ai,

International publication Mo UUO 89/05358). The enzyme cleaves RNA in double RNA-DNA structures.

To preserve competence in relation to ribonuclease H, it is necessary that! the oligomer contained a domain competent in relation to ribonuclease H, consisting of three or more competent nucleomonomers connected to each other (Scapip, K.5., ei ai, Mysi Asiyaz Kev., 1989, 17, 7253 - 7262). In the structure of oligomers resistant to digestion by nucleases, there are terminal connections and modifications

Sakharov and/or basic, providing resistance to nucleases. Thus, it is possible to construct an oligomer containing modified nucleomonomeric residues at one of the ends 5 or 3 or at both ends and 7/0 Competent with respect to ribonuclease H domain in the middle. Examples of oligomers that have retained competence in relation to ribonuclease H are, as a rule, oligomer! with uniform polarity and 2 - 12 nucleomonomers at the 5'-end and at the 3'-end, giving them resistance to nucleases, and 3 - 26 nucleomonomers, forming competence in relation to ribonuclease

N domain and dispositions between incompetents in ribonuclease relations! H ends 3 and 5. Possible! /5 varieties of such oligomers containing (1) a shorter, more competent ribonuclease! H domain containing 1-2 ribonuclease-competent H bonds or substitutional bonds, (2) longer incompetent in ribonuclease relations H domain containing 1-20 or more substitutional bonds or nucleomonomers, (3) longer competent in ribonuclease relations! H domain containing 30-40 and more connections, (4) the only incompetent ribonuclease! H domain at the 3 end or at the 5 end.

Oligomers) containing up to 8 nucleomonomers can be used to suppress the expression of a given protein (proteins) due to their ability to form a double or triple structure! with specified sequences of nucleic acids. However, in the preferred case, there is a linear oligomer used to suppress the expression of a given protein due to the formation of double or triple structures, see Ddolzhn! contain from 10 to 20 nucleomonomeric residues.

Oligomers containing substitution bonds, offered by the present invention, can be given i) a ring form, as described in (International publication Mo MO 92/19732; KOOYI, ET U At Spet Zos., 1991, 113, 6265 - 6266; Rgakazy , 0. ei a), In At Spet 5os., 1992, 114, 3523 - 3527). Such an oligomer! suitable for binding to single-stranded or double-stranded target nucleic acids. The size of ringed oligomers can vary. You can prepare such an oligomer! with a length of 22 - 50 nucleomonomers. Ring oligomer! may contain three to six nucleomonomer residues separating the binding domain in the region of the ring closure! oligomer, as described in (Rgakavzi, ibid.). "-

It is possible to encircle oligomers with the help of enzymes (acting with a ligase on the terminal phosphate) or chemically - due to the linking of 5'- and 3-terminal sugars and/or bases. co

The oligomer can be used to modulate the expression of specific genes as an inhibitor of the interaction of proteins that bind to nucleic acids and are responsible for controlling transcription (Manpeg, /.., ei aiY, Zsiepse, 1989, 245, 725 - 730) or translation. Thus, the oligomer! acceptable! as sequence-specific agents that compete with nucleic acid-binding proteins (including ribosomes, RNA polymerases, DNA polymerases, translation initiators, factors that accelerate or suppress transcription, and protein-hormonal transcription factors and t.p.)00 Consequently, the corresponding oligomer construction! can be used to enhance the synthesis of a given protein at the expense of connection of the control area (or its vicinity), which is a factor! transcription is used to suppress expression, or due to suppression of expression of the repressor protein itself.

The oligomer proposed by the present invention and containing additional modifications that increase binding affinity can be designed so that they include secondary or tertiary structures, such as a pseudoknot! or pseudopoluzl (EskKeg, Yu..). Еий АЙЙ, 5th edition, 1992, p. 257, 958 - 961). Such structures can have a more stable secondary or tertiary structure than the corresponding unmodified oligomers. The increased stability of such structures is explained by the increased binding affinity between the complementary regions of one oligomer or the complementary regions of two or more oligomers forming this structure. Such a structure! can be used to imitate structures such as the human immunodeficiency virus TAK structure to prevent it from binding to the HIV Tai-protein (TAK-binding protein). A similar approach can be applied to other transcription or translation factors that recognize higher structures! nucleic acids, such as stems, loops, hairpins, knots, etc. In addition, the oligomers offered by the present invention can be used in many ways! for: (1) destruction or (2) binding of such structures in order to (1) prevent or (2) promote binding of proteins to nucleic acid structures. The oligomer proposed by the present invention can be used not only as antisense or triple-helix therapy, but also as a therapeutic and diagnostic agent, acting due to the direct displacement of one of the strands of double-stranded nucleic acids! Displace one of the strands of a natural double-stranded nucleic acid, such as chromosomal DNA or double viral DNA, RNA or an RNA/DNA hybrid can an oligomer with a high binding affinity, since its complementary sequence is not large enough to effectively displace a DNA or RNA strand from a double structure. The therapeutic effectiveness of oligomers acting due to O-loops is due to the high affinity of binding to the complementary sequence and provides modulation of normal biological function , associated with this nucleic acid. A non-exhaustive list of types of nucleic acids that can serve as targets include: (ii) gene sequences, including exons, introns, junctions of exons with nitrones, promoter/enhancer regions and 5- or 3-terminal untranslated regions, (ii) regions of nucleic acids on which the secondary structure functions (for example, the stem-loop element of HIV or tRNA), (ii) nucleic acids that perform structural or other functions, for example, telomeres, centromeres or sources of replication (viruses, bacteria, etc.) and (m) any other two-stranded section. It is clear that an oligomer can be synthesized! with discrete functional domains, such that one part of the oligomer binds to the target by forming O-planes, and the adjacent part binds to the target molecule, say, due to the formation of a triple 7/0 helix or binds to a protein as an aptamer. In addition, an oligomer forming O-loops can bind to each strand of a double-stranded structure, switching the strand with which it binds (i.e., one part of the oligomer binds to one strand, and the other part to a complementary strand). The controlling elements that dictate the nature of binding (triple helix or O-loop) are the sequence of the oligomer and the affinity built into it. The rules of recognition based on binding into a double 7/5 structure according to Watson-Crick differ from the rules of controlled binding into a triple structure according to Hogsten (Noodziweep). Therefore, the base sequence of the oligomer can be used to determine the type of binding rules that the oligomer will follow. In nature, O-loop structures are formed during processes mediated by enzymes (Naigtizv, I.O. ei ai!., U Vioi Spet., 1987, 262, 9285 - 9292), or binding to areas where DNA replication occurs, N.T. ei ai., Mysi Asiaz Kez., 1989, 17, 2o 8949 - 8966). O-loops formed when oligomers are linked are formed as a result of one- or two-step processes. Immediate displacement of the target thread leads to the formation of a U-loop in one step. However, the formation of O-loops can also occur during the formation of a triple helix, which facilitates the displacement of threads leading to the formation of O-loops.

For the design of forms with changes in characteristics, many designs can be used! ribozyme, c g stimulating substitutional bonds, proposed by the present invention. Descriptions of ribozymes cleaving single-stranded RNA or DNA (KorBbegisop, O.Yi., ei aYi., Maishge, 1990, 344, 467 - 468). There are (8) offers to use ribozim! for therapeutic purposes (Zagmeg, M. ei aI., Zsiepse, 1990, 247, 1222 - 1225;

International publication Mo UUO 91/04319). Secondary or tertiary structures, necessary for the functioning of ribozymes, can be created by constructing the corresponding oligomeric sequences. For example, a ribozyme) containing nuclease-resistant targeting domains, in which there are substitution bonds, proposed by the present invention, have a higher affinity, without losing the specificity of base pairing (with "- the target sequence. Due to the high affinity and/or stability of the substitution connections proposed by the present invention to the effect of nucleases/recognition domain in the ribozyme can be shorter (convenience of preparation), and this will lead to a more favorable metabolism of the substrate (advantage of ribozyme functioning).

Oligomers proposed by the present invention can be used as medicinal products! in various ways, suitable for the treatment of various constitutive due to the suppression of the expression of the corresponding target genes. Therefore, the oligomer can be prepared in the form of "preparations used in various ways, including preparations for systemic, external or local use. The composition of the preparations and the technology of preparation can be found in the latest edition

Ketipdiopye Rpaptaseshisa! Zsiepsez Megsk RybiizpIpu So., Eaviop, RA, Iaievi eShop. Active substance - ;" oligomer - usually combined with a carrier, such as a diluent or filler, for example, a dry filler, a binder, a moisturizer, a dispersant, a surface-active substance or a lubricant, depending on the method of administration and dosage forms. Typical dosage forms include tablets, powders, liquid forms, including suspensions, emulsions, and solutions; granule, capsule! and suppositories, as well as liquid preparations! for injections, including a liposomal drug! - The preferred method of systemic administration is injections, including intramuscular, intraperitoneal, intraperitoneal and subcutaneous. For injections, the oligomer proposed by the present invention is prepared in the form of liquid solutions, preferably in physiologically acceptable buffers, such as Kznks (Napkv) or Ringer's (Kipdeg) solution. In addition, the oligomer can be prepared as a solid, dissolved or weighed immediately before use. Lyophilization form is also possible. Preferable for systemic use are dose! ranging from about 0.01mg/kg to 5mg/kg once or twice a day. However, you can use different schemes! dosage depending on (ii) the degree of suppression of DNA or RNA-target activity by this oligomer, (II) severity or (Ф, degree of development of the pathological condition associated with this gene, or (I) pharmacokinetic properties of this oligomer.

With systemic use, it is possible to introduce the drug through the mucous membrane, skin or inside. When br is introduced through the skin and mucous membranes, substances that promote penetration are used in the preparations. Their behavior depends on the barrier through which they must penetrate. Ask the experts for lime substances. These include, for example, salts of bile acids and derivatives of fusidic acids! - for introduction through the mucous membrane. In addition, a detergent is used to increase penetration.

Administration through the mucous membrane can be carried out with the help of aerosols for the nose or, for example, 65 Suppositories. Oligomer for oral administration! prepared in the form of traditional medicinal forms for oral administration, for example, capsules, tablets and potions.

For external use, the oligomer offered by the present invention is prepared in the form of ointments, gels or creams by methods known to specialists. Preparations based on the oligomers proposed by the present invention, for introduction into the eyes, for example, with viral infections, are prepared in accordance with the technologies adopted in the industry.

The oligomers proposed by the present invention can be used not only as therapeutic agents, but also as diagnostic preparations, with the help of which the presence or absence of specific sequences of nucleic acids, with which the oligomers specifically bind, is determined.

The increased binding affinity inherent in the oligomers offered by the present invention, 7/0 is their advantage when using them as seeds and probes. Diagnostic tests are carried out by means of hybridization with the formation of a double or triple helix, which is then examined by traditional methods. For example, oligomers can be labeled with radioactive, fluorescent, or chromogenic labels and the presence of labels bound to a solid substrate can be determined. In addition, the presence of a double or triple helix can be determined with the help of antibodies that specifically recognize these forms. Means of analysis using such oligomers as lime probes! specialists

The use of oligomers with substitutional bonds, proposed by the present invention, as diagnostic drugs acting due to the formation of a triple helix is convenient, since triple helices are formed in mild conditions, and the analysis can be carried out without placing the analyzed 2000 samples in harsh conditions. Diagnostic tests based on detected RNA to detect sequences characteristic of bacteria, fungi, and protozoa often require isolation of RNA from samples or organisms grown in the laboratory, which is very complicated and time-consuming, since

RNA is extremely sensitive to ubiquitous nucleases.

Oligomernse probe! may contain additional modifications, such as modifications of Sakharov and/or c g Substituted bonds that make an oligomer! especially resistant to nucleases, and then they will be useful for tests carried out in the presence of cell or tissue extracts, where i) nuclease activity is normally present. Oligomers containing terminal modifications often retain the ability to bind to complementary sequences without losing specificity (Piphapp et al., SPpetisa! Chemiemuz5, 1990, 90, 543 - 584). As mentioned above, the probes proposed by the present invention may contain linkers that ensure specific binding to both strands of DNA (Egoepieg, V.S. et al., Viospetivighu, 1992, 31, 1603 - 1609; Note et al., In At Spet 5os., 1990, 112, 2435 - 2437). what)

The inclusion of analogues based on the present invention in the probe, which also contains covalent cross-linking groups, can increase sensitivity and reduce the background in diagnostic tests or detection tests. In addition, the use of cross-linking agents

35 is associated with a change in the analysis technique: (1) cross-linking increases the discrimination of the probe, (2) denaturing washing is carried out to reduce the background, (3) hybridization and cross-linking is carried out at a temperature close to the melting temperature of the hybrid to weaken the secondary structure of the target and to increase the specificity of the probe. Changing the conditions of hybridization is described in

Mysieis Asidz Kez., 1986, 14, 9943). "

Oligomers, offered by the present invention, are suitable for use in diagnostic tests performed according to methods in which either the oligomer or the nucleic acid to be detected is covalently bound to a solid substrate (US patent Mo 4775619). Ask;" oligomers are also suitable for use in diagnostic tests based on the polymerase chain reaction technique, which amplifies the target sequence, as described in European Patent Publication No. 0393744. Oligomers, proposed by the present invention, having a 3-end, which 5" can serve as a seed, are compatible! with polymerases used in polymerase chain reactions, such as Tad or Mepitm polymerases (Mem/Epdiapa Bioir5). Thus, the oligomers proposed by the present invention can be used as seeds in the protocols of polymerase chain reactions. The oligomer proposed by the present invention can be used! as seeds representing a discrete sequence, or as seeds with a random sequence. i-th Primers with a random sequence generally have a length of 6, 7 or 8 nucleomonomers. Such primers can be used in various nucleic acid amplification schemes (polymerase or ligase chain reactions, etc.) or in cloning procedures. Substituent bonds, offered by the present invention, generally do not weaken the ability of the oligomer to function as a seed. Oligomers), offered by the present invention, having 2'--modifications in areas different from the 3-terminal residue, and other modifications that give them incompetence in relation to ribonuclease H and F) or second-order insensitivity to the action of nucleases, are convenient to use as probes or primers for RNA or DNA sequences in cell extracts or other solutions containing nuclease.

Thus, oligomers can be used in nucleic acid amplification procedures in a sample by mixing an oligomer with a sample containing a given nucleic acid, followed by hybridization of the oligomer with a given nucleic acid and amplification of a given nucleic acid! by means of polymerase or ligase chain reactions or other suitable methods.

Derivatives of oligomers and chelating agents, such as ZDTA (EOTA), distylenetriaminepentaacetic acid (DTA) or analogs of 1,2-diaminocyclohexane acetic acid!, can be used in various diagnostic tests of Imo, as described in US patents MoMo 4772548, 4707440 and 4707352. In addition, the oligomers proposed by the present invention can be derivatized with the help of cross-linking agents, such as 5-(3-iodacetamidoprop-1-yl)2-deoxyuridine or 5-(3--4-bromobutyramide )prop-1-yl)-2-deoxyuridine, and use in various analytical kits and methods, as described in the international publication Mo MO

Z0/14353.

In addition to the above, the ability of oligomers to suppress gene expression can be tested in immunological systems by measuring expression levels in cells, and in recombinant systems by any suitable method (Sgaezztapp, Meiyi, Mysieis Asidz Kez., 1991, 19, 53 - 59).

The description of the invention has been given above, and in order to better explain it, we offer the following 7/0 examples. Try it! tribute! as an illustration of the present invention, and should not be construed as limiting the scope of the invention.

Try it!

Briefly about the synthesis of nucleomonomeric synthons and oligomers

The oligomers proposed by the present invention can be synthesized in the course of reactions known to specialists in the field of synthesis of derivative oligonucleotides. See, e.g., Riapdog, ) apa Mat, 5.U.,

Teigapedhop I Ave., 1990, 31, 597 - 600; Mayvop, K.). Spipa,

SK. egaI., U Ogd Spet., 1989, 54, 2767 - 2769.

As can be seen from Table 1, in which the various substitutional bonds, substitutional bonds offered by the present invention can be very diverse and contain one or more nitrogen, sulfur and/or oxygen atoms. The position of these atoms in the substitution bond can be 5-terminal, "middle" or 2-, 3'- or 4-terminal. This section is provided with diagrams of a number of characteristic synthesis reactions, indicating the "path" to various arrangements and combinations of nitrogen and oxygen atoms in substitution bonds.

Procedure! synthesis, illustrated in fig. 1 - 25, can be modified, as is known to specialists in the field of oligonucleotide chemistry. For example, base protection is not always indicated on synthesis schemes, but it may be needed, and then it can be implemented using reagents and technologies known to i) specialists. See e.g. RgoiesPme Sgotsrz Ip Ogdapis Zupipeziv (Teodoga MU. Sgeepe, dopp UMPeu apa 5opv, 1981). Similarly, in some cases, the use of protecting groups is reflected in the schemes, but it is not always necessary in the synthesis of oligomers proposed by the present invention. Example 1

The first five steps, shown in fig. 1, refer to the preparation of the amino alcohol serinol protected by isobutyryl. The sixth and subsequent batch! directions! for obtaining building units!- - where thymine is phosphoramidite replaced by serine.

In step 1 in fig. 1 amino group of the amino acid serine is protected by the reaction of compound 1 with di-tert-butyldicarbonate, giving compound 2. Other equivalent searchlight "g" groups can be used. In the next step, the p-hydroxyl group of compound 2 is blocked with dihydropyran and a fully protected amino acid 3 is obtained. Then the amino acid 3 is reacted with a diborane-dimethyl sulfide complex and alcohol 4 is obtained, which, reacting with isobutyryl chloride, gives compound 5. This reduction reaction can be carried out using isobutyl chloroformate and sodium borohydride (K. Katazatu, K.K. Oivep and T. Etegu Zupipeviv, 1982, 42). The reaction of compound 5 with trifluoroacetic acid, carried out for 30 minutes, followed by washing with Mansoit, also gives compound 6. "" Thyminacetic acid 7 was prepared as described in the literature (I. KozupKiIpa, MU. MUapd apa T.S. ShPapo ,

Tegapedgop I ev5., 1994, 35, 5173). The combination of substances / and b according to the principle of mixed anhydride gave 8. Dimethoxytritylation of compound 8 with the help of UOMTCI gave compound 9, which after hydrolysis with a base gave 10. Phosphitylation of 10 under standard conditions gave structural unit 11 - thymine, combined with serinol. Ztot synthon can be added to the growing oligomer by standard methods. Because any chemistry of DNA synthesis, for example, phosphoramidate or phosphonate, can be used to link monomers or dimers in a way similar to that described above.

Example 2 o As can be seen in Fig. 2, o thymine acetaldehyde 13 was obtained by treating thymine o bromoacetaldehyde-dimethylacetal with subsequent hydrolysis of 12 with the help of aqueous trifluoroacetic acid. Then aldehyde 13 and amine 6 were combined and the corresponding intermediate product was converted into phosphoramidite structural unit 17 by a method similar to the steps in fig. 1.

Example C

The starting material for the reaction shown in fig. C, is p-substituted amino acid 18. i) Substituted amino acid can be converted into phosphoramidite building unit 27, which corresponds to the procedure shown in fig. 1 and 2.

Example 4 60 According to fig. 4, the original amino alcohol 21 is oxidized with a mixture of CgO with/pyridine and aldehyde 28 is obtained. This aldehyde when reacted with an alkyl halide in the presence of a base gives compound 29. Amino alcohol 29 can be converted into structural unit 35 by a method similar to that shown in Fig. 1 and 2.

Example 5

The first four joints, shown in fig. 5, basically the same as the image in fig. 1, but instead of 65 serine, aspartic acid is used. Methyl zfir aspartic acid! 3b gave fully protected alcohol 40, which after selective deprotection with the help of acetic acid! gave 41.

Oxidation of 41 with a mixture of CgOz/pyridine gave the corresponding aldehyde 42. Reductive amination of aldehyde 42 with O-benzylhydroxylamine in the presence of sodium triacetoxyborohydride should give 43 (see

T. Koiaza apa M. u. Misheg, U Ogd Seiya., 1990, 55, 1711). Then alcohol 39 is converted to aldehyde 46, in OsHnOovNOM, under the same conditions as described above, but with an allyl protecting group for the hydroxyl functional group 39. The combination of aldehyde 46 with hydroxylamine 43 in the presence of sodium triacetoxyborohydride with subsequent removal of amino protecting groups should give bisamine 48.

Bisamine 48 can be converted to dimer 53 according to the procedure shown in Fig. 1.

Example 6 70 According to fig. b, the combination of alcohol 54 with O-benzylhydroxylamine 55 under Mitsunobu (Miivypovry) reaction conditions (see: O. Miivypovry, Zupipeziv, 1981, 1) gives compound 56. Intermediate compound 56 after hydrogenation and acetylation gives 57. Treatment of 57 with trifluoroacetic acid removes the protective group "TVOM5BI" and gives 58. The combination of 58 and 7 with subsequent dimethoxytritylation gives 60.

The finished structural unit 62 is obtained from 60 by basic hydrolysis followed by 75 phosphitylation.

Example 7

According to fig. 7, serinol 4 is converted into halide 59 and alkylated with thymine to obtain 63. Protecting groups are removed from compound 63, it is combined with oxyacetic acid protected by UOMT, phosphitylated and 66 is obtained.

Example 8

According to fig. 8, alcohol 64 is combined with M-hydroxylaminopropanoic acid 69 and 70 is obtained.

Alkylation of thymine with halide 7Z gives 74, which after removal of protecting groups, compound 76 and hydrolysis gives 78. Condensation of 78 with 70 followed by phosphitylation should give the hydroxamate dimer 80.

Example 9

According to fig. 9, M-hydroxylaminopropane aldehyde 81 is combined with alcohol 64. Dimer 88 is obtained from i) 83 and 86 in accordance with the procedure shown in fig. 8.

Example 10

According to fig. 10, alkylation (see T. Koiaza and M. y). MiPeg, U Ogu Spet.i 1990, 55, 4246) of methyl suso Zfira o-bromo-p-aminopropanoic acid 89 with thymine gives 90. Intermediate compound 90 after hydrolysis with sodium hydroxide gives acid 91, which is combined with b to obtain 92. Then the compound 92 is transformed into a phosphoramidite structural unit 95 in accordance with the solders shown in Fig. - where 1.

Example 11 so

According to fig. 11, thymine is alkylated with an alkylamine halide 96 (about prepared aminoalkyl halides /-«f see: K.K.OiYiIvep, K.Katazatu apa T. Etegu U. Ogd. Spet., 1984, 49, 3527 apa Iviat eii ai., 9. Med. Spet., 1994, 37, 293 - 304) and obtain 97. Treatment of compound 97 with trifluoroacetic acid followed by alkylation gives 100. Structural unit 103 is obtained from 100 by means of dimethoxytritylation, hydrolysis, and phosphitylation. "

Example 12 with Fig. 12 illustrates an alternative method of obtaining dimer 111 with a hydroxamate skeleton from M-hydroxylamine 43 and aldehyde 107, which, in turn, is obtained from aspartic acid. "" Example 13

As can be seen in fig. 13, dimer 115 is obtained from the intermediate compound 108 and 13 according to the procedure shown in fig. 2. "drive" Example 14

According to fig. 14, prepare M-hydroxylthymine (Kigp, S.O., ei aiYi., Teiapedgop I ev. 1992, 33, 25 - 28) so and, combining it with M-hydroxyphthalimide, obtain 117, which upon treatment with hydrazine in ethanol gives 118. - Treatment of compound 118 with glycerol epoxide 119, protected by OMT, gives 120. Then the intermediate 5p compound 120 is converted into phosphoramidite 121 according to the standard procedure. During the second and third process, compound 118 is combined with aminoaldehyde 122 under conditions of reductive amination and 123 is obtained. Protection of secondary functional amino groups with the help of EMOSSI with subsequent hydrolysis gives 125.

Example 15

According to fig. 15, 1,2-dihydroxypropanoic acid 126 is combined with M-hydroxylamine-thymine 118 and 127 is obtained, which is converted into phosphoramidite synthon 129 under standard conditions. Compound 118 and F) are also combined with adipic acid and converted into structural unit 133 nucleic acid. Example 16

According to fig. 16, first synthesize building unit 136 from 118 and 134, similarly as shown in fig. 1. Combining 139 and 118 gives 140. Treatment of 137 with the help of 118 gives 138, which after condensation with 140 gives dimer 141.

Example 17

According to fig. 17, aldehyde 142 is combined with glycine benzyl ester and 143 is obtained. Treatment of compound 143 with compound 7 gives 145, which, reacting with acetic acid, gives 148. 65 Alkylation of 148 under the conditions of the Mitsunobu (Mihyipovby) reaction with Bos-MH-O-acetylhydroxylamine gives 147, from which by means of hydrogenation it is possible to obtain building unit 150. A similar compound

143 with 13 and the above-mentioned reactions give synthon 149.

Example 18

As can be seen in fig. 18, reductive amination of aldehyde 142 with Bos-MH-O-benzylhydroxylamine gave 151. Hydrogenation of 151 with subsequent alkylation of 152 with glycolic acid 153 (V.S. Voheg apa o.

S. Vaiodn, Teigapedgop Iev., 1991, 32, 1039) gives 154. Due to the treatment of 154 with trifluoroacetic acid, the protecting group Bos is removed, then the connection is carried out and 155 is obtained. The hydroxyl protecting group of compound 155 can be removed with the help of acetic acid and 156 is obtained Compound 156 is converted into structural unit 157 using standard reactions. Similarly, the structural 7/0 unit 158 is obtained by combining 154 and 13 followed by the procedures necessary to obtain 157.

Example 19

According to fig. 19, alkylation of thymine-M-hydroxylamine 160 with alcohol 162 gives 163. Compound 163 can be converted into a phosphoramidite building block 166 by means of the procedures shown in FIG. 1.

Example 20

As can be seen in fig. 20, first in standard conditions from glutamic acid! synthesize intermediate compound 169. Alkylation of thymine with the help of 169 gives 170, which upon treatment with trifluoroacetic acid gives 171. Intermediate compound 171 can be combined with Bos-glycine and 2o obtain 173, which upon hydrolysis gives the monomeric synthon 174. 172 can be obtained in a similar way: using compound 118 with Bos-aminoacetic aldehyde with subsequent hydrolysis of benzyl sulfur.

Example 21

According to fig. 21, intermediate compound 177 is prepared under standard conditions

Bos-MH-O-benzylhydroxylamine and 175. Hydrogenation of 177 with subsequent connection with

M-hydroxythymine 116 gives 178. Removal of the TNR protecting group followed by i) dimethoxytritylation and phosphitylation gives synthon 181. Following the procedures described above, but using TNR-oxyacetic aldehyde instead of TNR-oxyacetic acid, 182 can be obtained.

Example 22 o z As can be seen in fig. 22, the building unit 191 can be obtained from the known starting material 183, following the reaction conditions given in the lower part of fig.22. i am

Example 23 "-

As can be seen in fig. 23, the synthesis of structural units 199 can be carried out using the starting material 183 and following the reaction conditions shown in the lower part of fig. 23. co

Example 2 4 "E

According to fig. 24, the starting material 200 is transformed into a building unit 207, following the reaction conditions shown in the lower part of fig. 24.

Example 25

Connections, uses and results in this example, show! in fig. 1. "

Series (1): thymine (37.8 g, 30 mmol) was dissolved in a solution of potassium hydroxide (64.5 g, 1150 mmol) in 200 ml of water. This solution was heated in a water bath at 40°C for 1 hour, and during this time a solution of bromoacetic acid (62.5 g, 450 mmol) in 100 ml of water was added to it. The reaction mixture was stirred at room temperature. temperature for another hour. Then it was left to cool at room temperature, and when the mixture cooled, the pH was brought to 5.5 with the help of concentrated HCl. Then the mixture was cooled in a refrigerator in

Duration of 2 hours. The formed precipitate (unreacted thymine) was removed by filtration. Then the pH of the mixture was brought to 2 with the help of concentrated HCl and the mixture was placed in the refrigerator for 2 hours.

A white precipitate was collected by filtration and dried in a vacuum oven at 40°C for 6 hours. The yield was 44 g (8896). ,66g, 700mmol) was suspended in 5% tetrahydrofuran/M,M-dimethylformamide (THER/OME) (100ml of each) at room temperature. Trisethylamine (11.13g, 110mmol) was added to this mixture while stirring, then di-tert- butyl dicarbonate (24.0 g, about 110 mmol) and continued stirring at room temperature for 30 minutes. Water (20 ml) was added and the mixture was stirred for 8 hours at room temperature. The mixture was evaporated to dryness. The residue was suspended in ethyl acetate (250 ml) and worked up with potassium hydrosulfate (0.25 M, 100 ml). The product was immediately extracted with ethyl acetate solution. The organic extract was washed with water (100 ml), brine (100 ml) and dried over anhydrous sodium sulfate. Evaporation of the organic solvent gave

F) oily residue in 26 bg (9095). M-Voc-I-serine M-Voc-I-Methyl sulfur (OTNR)(Z3): compound 2 (15 g, 68.49 mmol) was dissolved in dry СНоСи» (100 ml) and treated with 3,4-dihydro-2H-pyran (8.4 g, 100 mmol) and p-toluene with sulfonic acid in an amount sufficient for catalytic action (100 mg) at room temperature. The reaction mixture was stirred at room temperature for 12 hours, then evaporated to dryness. The residue was dissolved in ethyl acetate (200 ml), washed with a 595% solution of MnSO 3 (100 ml), water (5 ml) and saline (5 ml).

The organic extract was dried over anhydrous Ma»5O) and evaporated to dryness. The rest was quite clean and was used in the next step in this form. Output: 15 GHz (7290). 65 M-Boc-I-serine (OTNR) (4): serine methyl sulfur (OTNR) (10 g, 33 mmol) was dissolved in dry tetrahydrofuran (100 ml) and cooled to 0"C in an ice bath in an argon atmosphere. To this cold solution borane-methyl sulfide complex (2 M solution in tetrahydrofuran, 100 ml, 200 mmol) was added while stirring for 1 hour at 0"C. After adding borane, the reaction mixture was heated to room temperature and kept at a temperature of 40°C for several hours. The reaction mixture was cooled to 0°C, neutralized with the help of water and acetic acid to a pH of about 7, and extracted with zephyr (3 x 100 ml).

The yeast extract was washed with water (2 x 100 ml) and saline solution (100 ml), dried over anhydrous

Ma»5ZO,, was evaporated to dryness and a gray product was obtained in the form of an oil. Purification of the oil on an evaporative column with silica gel and hexane - acetone as a solvent gave 8 g (88905) of pure product.

M-Vos-I-serine (OTNR) O1IB(5): TEA (3.54g, 3.54g) was added to a solution of compound 4 (8g, 29.09mmol) in 7/0 SNOSII" (100ml) while stirring at 0"C. Zbmmol), then isobutyryl chloride (3.71 g, Zbmmol) in 30 minutes.

Then the reaction mixture was stirred at room temperature for 4 hours, then evaporated to dryness. The residue was dissolved in ethyl acetate (E(Ac) (200 ml), washed with 596 MnSOOz solution (5Oml), water (BOml) and brine (5Oml). The organic extract was dried over anhydrous Ma 2504, dried to dryness and a gray product was obtained in the form of an oil Purification of the oil on an evaporative column with silica gel and /5 Hexane - » acetone as a solvent gave 7.9 g (7995) of pure product.

I-serinol (OB) (b): compound 5 (10g, 28.98mmol) was dissolved in СНоСи» (100ml), stirred at room temperature with trifluoroacetic acid (50ml) for 1 hour, then evaporated to dryness.

The residue was dissolved in methanol (5 Oml) and evaporated again. The residue was dissolved in SiO2 (200 ml), washed with a saturated solution of MnSOO (2 x 100 ml), water (100 ml) and saline (50 ml). The organic extract was dried over anhydrous NaCl, evaporated to dryness and 4.5 g (96905) of the product was obtained as an oil.

M-(thyminylacetyl)-I-serinol (016) (8): thyminacetic acid 7 (7.3 g, 40 mmol) and M-methylmorpholine (4.4 ml, 4 mmol) were dissolved in 100 ml of M,M-dimethylformamide. The solution was cooled to -207"C in an argon atmosphere. Isobutyl chloroformate (5.2 ml, 40 mmol) was added to the cold solution with stirring in one go.

After 15 minutes, a solution of compound 6 (6.44 g, 400 mmol) in 3 mL of M,M-dimethylformamide was added (cooling chamber to the same temperature)). The reaction mixture was stirred at -20"C for 30 minutes, then warmed to room temperature! and stirring was continued for 1 hour. The reaction mixture was evaporated to dryness, and) and the residue was dissolved in СНоСи" (200 ml). The organic solution was washed with 595 Manso solution " (100 ml), water (100 ml) and saline solution (5 O ml). The organic extract was dried over anhydrous Ma»50O), evaporated to dryness and a gray product was obtained in the form of a stump. The gray product was purified on an evaporative column with ; - » with acetone as a solvent and obtained 12 g (92795) of pure product. 4,4-dimethoxytrityl-M-(thyminylacetyl)-I-serinol (OIVB)(9U): compound 8 (10 g, 30.58 mmol) was evaporated together with dry pyridine (3 x 50 ml) and dissolved in dry pyridine (100 Oml). To this solution was added TEA (3.54 g, ZbBmmol), then UMTC (11.83g, ZbBmmol) at room temperature in an argon atmosphere.

The reaction mixture was stirred for 12 hours, quenched with methanol (20 mL) and stirred for

30 minutes. The solution was evaporated to dryness and dissolved in SO2 (200 ml). The organic extract was washed with 590 g of Manso solution (100 ml), water (100 ml) and saline (5 ml). A layer of SNІІСІ" was brought over the anhydrous one

Ma»ZO), evaporated to dryness and obtained a gray product in the form of a stump. The gray product was purified on an evaporative column with silica gel and CH»oSi»-»acetone as a solvent, and 17 g (8890) of pure product were obtained. 1-0-(4,47..-dimesoxytrisyl)-2--amine (thyminylacetyl)| -I-propane-1,3-diol (10): compound 9 (10Hg, 15.89mmol) was dissolved in methanol (20ml). To this solution at 0°C was added 1 M Man solution (20 ml, pt» with 20 mmol). The reaction mixture was stirred for 1 hour, then quenched with acetic acid, bringing the pH to 7. The solution was extracted with EtOAc (2 x 100 ml). The organic extract was washed with 595% MansSoOz solution; (100ml), water (100ml) and saline solution (50Oml). The layer of EUAs was dried over anhydrous Ma»SO), evaporated to dryness and the solid product was obtained in the form of a stump. The crude product was purified on an evaporative column with bilicagel and SiOCl-acetone as a solvent, and 8.2 g (92795) of pure product were obtained. their 1-O-(4,4-dimethoxytrityl)-2-(amine (thyminylacetyl))|-I-propane-3'-O-(M,M-diisopropyl)-B-cyanosthylphosphoramidite (11): compound 10 (8.00 Hg,

Me 14.31 mmol) was evaporated together with dry pyridine (3 x 5 Oml) and dried over Mahon solid in vacuum overnight. The dry material was dissolved in dry SiO2 (100 ml) and cooled to 0°C in an argon atmosphere. Therefore

M,M-diisopropylethylamine (5.23g, 25mmol) was added to the cold solution in an argon atmosphere, then 2-cyanoethyl-M,M-diisopropylchlorophosphoramidite (4.72g, 20.00mmol). The reaction mixture was stirred for 1 hour at 0°C and for 1 hour at room temperature. The reaction mixture was diluted with CH2O2O (100 ml). The solution in CH2SO4 was washed with 595% Manso solution (100 ml), water (100 ml) and saline solution (5 Oml) - The SnSi2 layer was dried over anhydrous Ma2504, evaporated to dryness and a gray product was obtained in the form of a stump. (x95) of the pure product. The mass was dried over solid Mahon in a vacuum overnight. The mass was dissolved in CH2Si" (15 ml) and during the course of an hour in an argon atmosphere, a stirred solution of dry hexanes (2000 ml) was added dropwise to the co. After adding dissolved in CH A precipitate of 25% mass formed, which was stirred for another hour, then filtered, washed with dry hexanes (200 ml) and dried overnight over solid Mahon. Yield: 9.5 g (87905).

Example 26 (see Fig. 26)

M-(tert-butyloxycarbonyl)-O-benzyl-I! -serine (2): O-benzyl-I-serine 1 (10g, 51.28mmol) was suspended in tetrahydrofuran/H2O (8:2, 100ml) at room temperature. Tristhylamine (6.06 g, bOmmol) was added to this mixture while stirring, then di-tert-butyl dicarbonate (13.08g, bOmmol) and stirring was continued overnight at room temperature. The homogeneous solution was evaporated to dryness and the residue was dissolved in ethyl acetate (300 ml). The organic extract was washed with a 0.5 M potassium hydrosulfate solution (100 ml), water

(100ml) and saline solution (5Oml). The ethyl acetate extract was dried over anhydrous sodium sulfate, evaporated to dryness, and 14 g (9395) of an oily residue were obtained.

M-(tert-butyloxycarbonyl)-O-benzyl-I-serinol (3): M-(tert-butyloxycarbonyl)-O-benzyl-I-serine (2) (god, 20.34 mmol) was dissolved in dry tetrahydrofuran and cooled to -20"C in an argon atmosphere. TEA (2.32g, 233mmol) and isobutyl chloroformate (3.13Hg, 23mmol) were added to this cold solution with stirring. Stirring was continued for 30 minutes at -207"C in an argon atmosphere. The reaction mixture was immediately filtered under argon, and the precipitate was washed with dry tetrahydrofuran (5 Oml).

The combined filtrate was slowly added to a cold (07) solution of MavN; (7.4g, 200Omol) in 7/0 tetrahydrofuran/water (80:20, 200ml) for 10 minutes. After that, the reaction mixture was stirred for 2 hours at 0"C, then the pH was brought to 7 with the help of acetic acid. The solution was evaporated to dryness, partitioned between ethyl acetate/water (300:150 ml) and extracted with ethyl acetate. The organic extract was washed with saline solution (100 Oml ), dried over anhydrous sodium sulfate and evaporated to dryness. The raw product was purified on an evaporative chromatographic column with silica gel, with СНоСи»-»5 ЕИХАСС as a solvent. The pure product was combined, evaporated to dryness and 4.7 g (8295) of pure product were obtained in in the form of oil. "H NMR (SOSI 3): 5 1.41 (v, 9H, Bos), 3.60 - 3.70 (t, 4H), 3.82 (4, 2H), 4.53 (5 , 2H, OSNORI), 5.20 (bvv, 1H,

МН) and 7.30 - 7.40 (t, 5Н, РП).

M-(tert-butyloxycarbonyl)-O-benzyl-i-serinol-O-1p (4): Kk of the dried solution

M-(tert-butyloxycarbonyl)-O-benzyl-I-serinol C (4.3g, 14.3mmol) in dry pyridine (50ml) was added TEA (2.02g, 20mmol) at room temperature. Isobutyric anhydride (3.16 g, 2 mmol) was added to this solution while stirring, and stirring was continued overnight in an argon atmosphere. The reaction mixture was evaporated to dryness, partitioned between EtOAc (100 mL) and MnSO 3 (595 solution, 10 mL) and extracted

EUAs. The organic extract was washed with water (100 ml), saline (5 ml) and dried over anhydrous

Ma»zO). The dried solution was evaporated to dryness and a gray residue was obtained. The residue was purified by means of super-evaporative chromatography on silica gel with hexane-»EyudAs as a solvent. The purified fractions were combined, evaporated and an oily product in the amount of 4.5 g (8495) was obtained. "H NMR (SOCI5): 6 1.04 (a,

Yen, ISNU"), 1.39 (v, 9H, Vos), 2.46 (t, 1H, IBSN), 3.40 (t, 2H), 3.92 (t, 2H), 4.12 ( t, 1H), 4.46 (v, 2H, OSNORI), 6.84 (y, 1H, МН) and 7.24 - 7.40 (t, 5H, RI).

M-(thyminylacetyl)-O-benzyl-I-serinol-O-IB (6): M-(tert-butyloxycarbonyl)-O-benzyl-I-serinol-O-IB 4 (Z (4.3g, 12, 25 mmol) was stirred at room temperature in trifluoroacetic acid (200 ml) and CH2Cl2 (20 ml) for 30 minutes. The reaction mixture was evaporated to dryness, dissolved in dry CH2OH (1 ml) and evaporated again to dryness. The residue was dried over solid KOH under vacuum for 12 hours. The dried residue (/zh7) was used in this form, without research, in subsequent reactions.

Thymine acetic acid 5 (2.76 mg, 15 mmol) was dissolved in dry M,M-dimethylformamide (75 ml) and cooled to -20°C in argon. M-methylmorpholine (1.72 g, 17 mmol) was added to this cold solution with stirring. then isobutyl chloroformate (2.31 g, 17 mmol).

M-methylmorpholine (1.72 g, 17 mmol) and immediately added to a cold, stirred solution of thymine acetic acid. The reaction mixture was stirred at -20°C for 1 hour, warmed to room temperature, and stirring was continued overnight. The solution was evaporated to dryness, and the residue was dissolved in NaCl (250 mL) and water (100 mL) and extracted with NaCl - The organic extract was washed with 595 Manso solution (100ml), water (100ml) and saline solution (5Oml). The extract was dried, evaporated to dryness and a gray product was obtained. The gray product was purified by means of evaporative chromatography on silica gel with

СНоСИ» - » with acetone as a solvent. The necessary fractions were collected, evaporated and 4.8 g (94905) of pure product were obtained. The pure product was crystallized from CH2Cl2/hexane, melting point 122 - 12476. Ti NMR (SOCI5): 6 1.04 o (0, EN, TCHN»), 1.72 (v, ZN, SNU), 2.44 ( t, 1H, IBSN), 3.42 (t, 2H), 4.06 (t, 2H), 4.18 (t, 1H), 4.30 (v, 2H), 4.46 (v, 2H , OSNORNY), 7.24 - 7.40 (t, 6H, SeH and RI), 8.22 (d, IN, MH) and 11.22 (v, 1H, MH). - M-(thyminylacetoyl)-I -serinol-O-IvB (7): M-(thyminylacetyl)-O-benzyl-I| -serinol-O-Ir b (2.08 g, Bmmol) cl 20 was dissolved in ethanol (5 Oml). To this solution at room temperature was added PaY(OH)" (0.6 g) and cyclohexene (5 ml). The reaction mixture was heated to 70°C for 12 hours. The catalyst was filtered off and washed with methanol (20 ml). The filtrate was evaporated to dryness and a white solid was obtained. The white solid was dissolved in a minimal amount of MeOH and cooled to room temperature. The product recrystallized as a fine powder, melting point 196 - 19870. Yield: 1.48 g (91906). TN nNMR (Me, 3O-yv): 5 1.04 (a, 6H, IbCH»), 1.72 (v, 3H, CH» ), 2.42 (t, 1H, IBSN), 3.40 (t, 2H), 3.94 (t, 2H), 4.06 (t, (F) 1H), 4.28 (5, 2H ), 4.90 (i, 1H, OH), 7.20 (v, 1H, SeH), 8.12 (a, 1H, MH) and 11.22 (8, 1H, MH). r 4.4 -dimethoxytrityl-M-(thyminylacetyl)-I-serinol-O-II (8): M-(thyminylacetyl)-I-serinol-O-IB 7 (1.48g, 4.5mmol) was dissolved in dry pyridine (5Oml) under an argon atmosphere. TEA (0.51 g, BbBmmol) and M,M-dimethylamine-pyridine (0.10g) were added to this solution under stirring. After 10 minutes, 4,4-dimethoxytrityl chloride (1.69g, bmmol) was added and continued stirring at room temperature under argon atmosphere during the night. The reaction mixture was quenched with Meon (10 ml), stirred for 10 minutes and dried. The residue was dissolved in Eas (200ml), washed with 595% Manso's solution (100ml), water (100ml) and saline (50ml). The organic extract was dried over anhydrous Ma»5ZO) and evaporated to dryness. The pure product was purified by means of evaporative chromatography on a column with silica gel, p

SN»SI» - » EIOAs as a solvent. The pure fractions were combined, evaporated and 2.5 g (8896) of stump were obtained. "N

NMR (SOSI"): 5 1.04 (a, 6Н, ISNU"), 1.72 (8, ЗН, СН»), 2.40 (t, 1Н, IBSN), 3.38 (t, 2Н) . ), 7.20 - 7.40 (t, 12H, SeH and RI), 8.30 (d, 1H, MH) and 11.28 (c, 1H, MH). 1-0-(4,4-dimeooxyshrityl)-2-(amine (thyminylacetyl)|-I-prolan-1,3-diol (9): 4,4-dimethoxytrityl-M-(thyminylacetyl)-I -serinol- O-IV 8 (34g, 5.41mmol) was dissolved in Meon (30ml) and cooled to 0"C in an ice bath. To this cold solution, while stirring, 2M Mmaon (10ml, 20mmol) was added and stirring was continued for 30 minutes at 07C With the help of acetic acid, the pH of the mixture was brought to 7 and the mixture was evaporated to dryness. The residue was partitioned between water (5 Oml) and SiOCl (150 ml) and 7/0. washed with saline solution (5 Oml), dried and evaporated to dryness. The residue was purified by means of evaporative chromatography on a column with silica gel, with СНоSiO - » acetone as a solvent. Output:

Z, Oh (9996). "H NMR (SOCIz): 1.72 (v, ZN, SNU), 3.0 (t, 2H), 3.42 (t, 2H), 3.72 (v, 6H, 2.0СНa), 3 ,94 (t, 1H),, 4.32 (d, 2H), 4.68 (t, IN, OH), 6.84 (t, 4H, RI), 7.20 - 7.40 (t, 12H, SeH and RP), 8.06 (a, 1H, MH) and 11.28 (re, 1H, MH). 1-0-(44-dimeooxytrityl)-2-(amine (hymynylacesyl))-I- lrolan-3-O-(M,M-diisoprolyl)-pD-cyanosthylphosphoramidite (10): 1-O-(4,4-dimethoxytrityl)-2--damine(thyminylacetyl))|-I -propane-1,3 -diol 9 (3.1 g, 5.55 mmol) was dried over solids

Mahon in vacuum overnight, then dissolved in dry CHClCl (1000 ml). The solution was cooled to 0"C in an argon atmosphere. M,M-diisopropylethylamine (1.29 g, 1 mmol), then 2-cyanoethyl-M,M-diisopropylchlorophosphoramidite (1.96 g, 8.3 mmol) was added to this cold solution with stirring. The reaction mixture was stirred for 1 hour at 0"C and for 2 hours at room temperature. The reaction mixture was diluted with NaCl (100 ml) and the organic layer was washed with 596 NaCl solution (100 ml), water (100 ml) and brine (5 ml). The extract was dried in SiO2, evaporated to dryness, and an oily residue was obtained.

The residue was purified by means of evaporative chromatography on silica gel with СНоСиИ» -» ЕХОАс, containing «М 0.195 TEA, as a solvent. Pure fractions were combined, evaporated and a foam was obtained. The foam was dried for (5) nights over Masonry under vacuum. The dried foam was dissolved in dry SiO2 (20 ml) and added dropwise to stirred dry hexane (2000 ml) during an hour under an argon atmosphere. After that, the resulting precipitate was stirred for another hour, then filtered, washed with dry hexane (100 ml) and the solid was dried over Mahon solids in a vacuum for 4 hours. Output: 3.5g (83905). (av)

Example 27 (see fig. 27)

M-(tert-butyloxycarbonyl)-O-benzyl-SO-series (12): O-benzyl-O-serine 11 (5 g, 25.64 mmol) was suspended in tetrahydrofuran/water (8 : 2, 70 mL) at room temperature . 2: Trisethylamine (4.04 g, 4 mmol), then di-tert-butyl dicarbonate (6.54 g, 0 mmol) was added to this mixture while stirring, and stirring was continued overnight at room temperature. The homogeneous solution was evaporated to dryness and the residue was dissolved in ethyl acetate (150 ml). The organic extract was washed with a 0.5 M solution of potassium hydrosulfate (100 ml), water (100 ml) and saline solution (5 ml). The ethyl acetate extract was dried over anhydrous sodium sulfate, evaporated to dryness and 7.56 g (10095) of an oily residue was obtained.

M-(tert-butyloxycarbonyl)-O-benzyl-SO-serinol (13): M-(tert-butyloxycarbonyl)-O-benzyl-SO-serine (10) « (7.56 g, 25.63 mmol) was dissolved in dry tetrahydrofuran and cooled to -207C in an argon atmosphere. TEA (3.03 g, 0 mmol) and isobutyl chloroformate (4.08 g, 0 mmol) were added to this cold solution while stirring. Stirring was continued for 30 minutes at -20°C in an argon atmosphere. Then, the reaction mixture was filtered under an argon blanket, and the precipitate was washed with dry tetrahydrofuran (5 ml). and The combined filtrate was slowly added to a cold (07) solution of MavN; (7.4g, 200Omol) in tetrahydrofuran/water (80:20, 200ml) for 10 minutes. After that, the reaction mixture was stirred for 2 hours at 0"C, then with the help of acetic acid, the pH was brought to 7. The mixture was evaporated to dryness, partitioned between ethyl acetate/water (300 : 150 ml) and extracted with ethyl acetate. The organic extract was washed with brine. (100 ml), dried over anhydrous sodium sulfate and evaporated to dryness. The pure product was purified by means of evaporative chromatography on a column with silica gel, with СНоСи»-»5 EUAs as a solvent. The pure product was combined, evaporated to dryness and 6.68 g (9295) were obtained pure product in the form of 50 g of oil. "H NMR (SOCI3): 5 1.41 (b, 9H, Bos), 3.60 - 3.70 (t, 4H), 3.82 (d, 2H), 4 .53 (in, 2H, OSNORI), 5.20 (Be, 1H,

МН) and 7.30 - 7.40 (t, 5Н, РП). with M-(tert-butyloxycarbonyl)-O-benzyl-O-serinol-O-1r (14): Kk of the dried solution

M-(tert-butyloxycarbonyl)-O-benzyl-O-serinol 13 (6.6bg, 23.5mmol) in dry pyridine (50ml) was added TEA (3.03g, 00mmol) at room temperature. Isobutyric anhydride (4.74 g, ZOmmol) was added to this solution while stirring, and stirring was continued overnight in an argon atmosphere. Reactionary

HF) the mixture was evaporated to dryness, partitioned between EtOAc (200 ml) and MnSO 3 (595 solution, 10 Oml) and extracted

EUAs. The organic extract was washed with water (100ml), saline solution (50ml) and dried over anhydrous de Maazot. The dried solution was evaporated to dryness and a gray residue was obtained. The residue was purified by means of evaporative chromatography on silica gel with hexane - » EOAc as a solvent. Pure fraction 60 was combined, evaporated and an oily product was obtained in the amount of 8.0 g (9795). "H NMR (SOSIs): 5 1.04. (a,

EM, IvSN"), 1.39 (v, 9H, Bos), 2.46 (t, 1H, ISSN) 3.40 (t, 2H), 3.92 (t, 2H), 4.12 (t , 1H), 4.46 (v, 2H, OSNORI), 6.84 (y, 1H, МН) and 7.24 - 7.40 (t, 5H, RI).

M-(thyminylacetyl)-O-benzyl-O-serinol-O-Ir (15): M-(tert-butyloxycarbonyl)-O-benzyl-O-serinol-O-Ir 14 (5.0g, 14.25mmol) was stirred at room temperature in trifluoroacetic acid (20 ml) and СНоСі»бо (20 ml) for 30 minutes. The reaction mixture was evaporated to dryness, dissolved in dry SONH (1 Oml) and evaporated to dryness again. The residue was dissolved in CHClCl (150 ml), with the help of 595 ml of MansCoO solution, the pH was brought to 7 and the CHClCl solution was extracted. The organic layer was washed with water (5 Oml) and saline solution (5 Oml).

The extract was dried and evaporated to dryness. The resulting residue was dried in a vacuum over solid KOH for 12 hours. The dried residue was used in subsequent reactions without further investigation.

Thymine acetic acid 5 (2.57g, 14mmol) was dissolved in dry M,M-dimethylformamide (5Oml) and cooled to -20"C in an argon atmosphere. M-methylmorpholine (1.52g, 15mmol) was added to this cold solution while stirring. then isobutyl chloroformate (2.04 g, 15 mmol). After stirring for 15 minutes, a solution of the above-mentioned amine in dry

M,M-dimethylformamide (5 Oml). The reaction mixture was stirred at -20°C for 1 hour, warmed to room temperature, and stirring was continued overnight. The solution was evaporated to dryness, and the residue was dissolved in CH2SiO2 (250 ml) and water (100 ml) and extracted with SNCl2. The organic extract was washed with 595 with a solution of MansSoO»z (100 ml), water (10O0 ml) and saline solution (5 O ml). The extract was dried in CH 2 Si»i, /5 was evaporated to dryness and a crude product was obtained. as a solvent. The necessary fractions were collected, evaporated, and 2.8 g (5495) of the pure product were obtained by H NMR (SOCIz): , 2.44 (t, 1H, IBSN), 3.42 (t, 2H), 4.06 (t, 2H), 4.18 (t, 1H), 4.30 (v, 2H), 4, 46 (in, 2H, OSNORI), 7.24 - 7.40 (t, 6H, SeH and

RI), 8.22 (8, 1Н, МН) and 11.22 (8, 1Н, МН).

The substance indicated in the title was also prepared in accordance with the method described in connection with the preparation of the I-isomer. The following reagents were used: thymine acetic acid (2.2 g, 12 mmol), isobutyl chloroformate (1.77 g, 1 mmol), M-methylmorpholine (1.52 g, 15 mmol), trifluoroacetic acid salt (3.65 g, 10 mmol); M-methylmorpholine (1.5 g, 15 mmol) and dry M, M-dimethylformamide (10 Oml). Output: 3.5g (84905).

M-(thyminylacetyl)-O-serinol-O-Ir (16): M-(thyminylacetyl)-O-benzyl-O-serinol-O-Ir 15 (3.5g, 8.3µmol) was dissolved in ethanol (500ml ). To this solution at room temperature was added RaYAA(OH)" (1.00 g) and cyclohexene (1 Oml). The reaction mixture was heated to 70°C for 12 hours. The catalyst was filtered off and washed with methanol (20ml). The filtrate was evaporated to dryness and a white solid was obtained. Yield: 2.7g (98965). "IN NMR (Me»5O-iv): 5 1.04 (8, 6Н, ISNU»), 1.72 (8, ЗН, СН»), 2.42 (t, 1Н, IBSN), 3.40 (t, 2Н), 3.94 (t , 2H), 4.06 (t, 1H), 4.28 (8, 2H), 4.90 (Yi, 1H, OH), 7.20 (v, 1H, SeH), 8.12 ( 1H, MH) and 11.22 (in, 1H, MH). o 4,4-dimethoxysrytyl-M-(thyminylacetyl)-O-serinol-O-Ir (17): M-(thyminylacetyl)-YOO-serinol-O-Ir 16 (2.7 g, 8.2 mmol) was dissolved in dry pyridine (5 Oml) in an argon atmosphere. TEA (1.01 g, 7 mmol) was added to this solution while stirring, then 4,4-dimethoxytrityl chloride (3.38 g, 1 mmol) and stirring was continued overnight at room temperature in an argon atmosphere. The reaction mixture was quenched with Meon (1 Oml), then stirred for 10 minutes and evaporated to dryness. The remainder was dissolved in

Ace (250 ml), washed with 595 MansSoOz solution (100 ml), water (100 ml) and saline solution (500 ml). "

The organic extract was dried over anhydrous Ma»5O4 and evaporated to dryness. The pure product was purified by means of evaporative chromatography on a column with silica gel, with СНоСи»-»5 ЕИХАСС as a solvent.

The pure fractions were combined, evaporated and 5.0 g (9696) of stump were obtained. "H NMR (SOSI"): 5 1.04 (a, EM, ISNU), 1.72 « (85, ZN, SNU), 2.40 (t, 1H, IBSN), 3.38 (t, 2H ), 3.72 (v, 6H, 2.0CH»U), 4.12 (t, 2H), 4.20 (t, 1H), 4.32 (a, 2H), C 740 v,84(t ) (4-dimeoxytrityl)-2-4(amine (thyminylacetyl)|-O-propane-1,3-diol (18):

4,4-Dimethoxytrityl-M-(thyminylacetyl)-O-serinol-O-IB 17 (5.0 g, 7.95 mmol) was dissolved in Meon (3 mL) and cooled to 0 °C in an ice bath. 2M Mammon (10 ml, 20 mmol) was added to the stirred solution and stirring was continued for 30 minutes at 07°C. With the help of acetic acid, the pH of the mixture was brought to 7 and the mixture was dried. The residue was partitioned between water (500 ml) and SiO ) and extracted with СНоСі". The aqueous layer was also extracted with СНОСі" (5 Oml). The combined organic extract was washed with a saline solution (5 Oml), dried and evaporated to dryness. The residue was purified by further evaporative chromatography on a column with silica gel, with СНоSiO» - » acetone as a solvent. Yield: 4.0 g (9095). "H NMR (SOCI3): 1.72 (z, ZN, SNU), 3.0 (t, 2H), 3.42 (t, 2H), 3 ... RI), 7.20 - 7.40 (t, 12H, SeH and RI), 8.06 (y, 1H, MH) and 11.28 (bz, 1H, MH). o 1-0-(4,4-dimethoxytrityl)-2-(amine (thyminylacesyl)|-O-propane-3-0-(M,M-diisopropyl)- D-cyanzthyl phosphoramidite (19): 1-0-( 4,4-dimethoxytrityl)-2-amine (thyminylacetyl)|-O-propane-1,3-diol 18 (2.79 g, 5 mmol) was dried over Mahon solids in vacuum overnight, then dissolved in dry CHCl» (100ml). The solution was cooled to 0"C in an argon atmosphere. M,M-diisopropylethylamine (F) (1.29g, TOmol) was added to this cold solution while stirring, then 2-cyanoethyl-M,M-diisopropylchlorophosphoramidite (1, 96g, 8.mmol). The reaction mixture was stirred for 1 hour at 0"C and for 2 hours at room temperature. The reaction mixture was diluted with NaCl" (100 ml) and the organic layer was washed with a solution of 596 MnSO" (100 ml), water ( 100ml) and diluted with boline solution (50ml). The extract was dried in SiO2, evaporated to dryness and an oily residue was obtained.

The residue was purified by means of evaporative chromatography on silica gel with CHNOSi»-»5 EUAs containing 0.195 TEA as a solvent. The pure fractions were combined, evaporated and a foam was obtained. The foam was dried overnight over Masonry under vacuum. The dried foam was dissolved in dry SiO2 (20 ml) and added dropwise to stirred dry hexane (2000 ml) during an hour under an argon atmosphere. After that, the precipitate formed was stirred for another hour, then filtered, washed with dry hexane (100 ml), and the solid was dried over Mahon solids under vacuum for 4 hours. Entrance: Z, Zg (87905).

Example 2 8 (see fig. 28) 1-O-benzyl-2-tert-butyloxycarbonyl)amine|-3-(M3-benzoyl (thyminyl)-I-propanol (21): to a stirred solution of M3-benzoylthymine 20 (5 .75g, 25mmol) in dry tetrahydrofuran (200ml) in an argon atmosphere at room temperature, triphenylphosphine (10.48g, dOmmol) was added and

Mag-o-tert-butyloxycarbonyl-p-benzyloxy-I-serinol C (5.3g, 18.8bmmol). After 15 minutes, slowly (within

30 minutes) added distylazodicarboxylate (6.96 g, 40 mmol). The reaction mixture was covered with aluminum foil and stirred at room temperature in an argon atmosphere for 24 hours.

The solvent was evaporated to dryness, and the residue was dissolved in EtOAc (30 mL). The organic extract was washed with 595 70 solution of MansSoO»z (100 ml), water (100 ml) and saline solution (100 ml), then dried over anhydrous

Ma»ZO,). The dried extract in EtOAc was evaporated to dryness and an orange oil was obtained. The gray product was purified by evaporative chromatography on silica gel with hexane-»E(Ac) as a solvent. The fraction containing the desired product was combined and evaporated. A pale pink oil was obtained. Yield: 8.0 g (8695). "H NMR (SOCI3) : 1.41 (v, 9H, Bos), 1.72 (8, ЗН, СН3з), 3.56 (t, 2H), 4.20 (t, 2H), 4.32 (t, 1H), 4.52 (a, 75.2H, OSNORI), 5.20 (a, 1H, MH), 7.06 (c, 1H, SeH) and 7.20 - 7.60 (t, 1OH, RI). 2-tert-butyloxycarbonyl)amino!i-3-IM3-benzoyl (thyminyl)-1-propan-1-ol (22): 1-O-benzyl-2-tert-butyloxycarbonyl)amine-3-IMa-benzoyl ( thyminyl)|I-|-propanol 21 (4.93 g, 1 mmol) was dissolved in Meon (100 mL) and treated with Ra/s (10905, 1 g). The reaction mixture was hydrogenated at 344.7 kPa (hydrogen pressure) for 12 hours. The catalyst was filtered off, washed with MeonH (50 mL), and the filtrate was evaporated to dryness. The residue was crystallized from acetone/hexane to give 33.70 g (9295) of pure product, mp 156 - 15920. "H NMR (SOCI3): 1.42 (in, 9H, Bos), 1.94 (in, ЗН, СН), 3.64 (t, 4H), 3.84 (t, 1H), 4.14 (t, 1H), 5.22 (d, IN, MH), 7.18 (v, 1H, Sen), 7.48 (Her, 2H , RI), 7.62 (I, IN, RI) and 7.98 (9, 2H, RI). 1-O-isobutyryl-2-tert-butyloxycarbonyl)amine-3-(M3-benzoyl(thyminyl)-I-propanol (23): 2-tert-butyloxycarbonyl)amino-3-|M3-benzoyl(thyminyl)- I-propan-1-ol 22 (1.60 g, 3.97 mmol) was dissolved in DM dry pyridine (3 mL) and stirred at room temperature under an argon atmosphere. TEA (0.51 g, 5 mmol) and isobutyric anhydride (0.79 g, 1 mmol) were added to this solution while stirring. The reaction mixture was stirred at room temperature for 12 hours, then evaporated to dryness. The remainder was dissolved in

EtOAc (150 ml) and washed with 595% MansSO 2 solution (100 ml), water (100 ml) and saline solution (50 ml).

The organic extract was dried and evaporated to dryness. The residue was purified by means of an evaporator (2

Chromatographies on a column with silica gel, with СНоСио - » ЕИУАс as a solvent. The pure fractions were combined, evaporated and 1.6 bg (8595) of stump were obtained. The pure product was crystallized from acetone/hexane, melting point 165 - 167"С. "H NMR (SOCI"): 1.16 (a, EN, IBSsH»Z), 1.42 (8, 9Н, Bos), 1, 94 (v, ZN, CH"), 2.52 (t, 1H), 3.64 (t, 7 4H), 3.84 (t, 1H), 4.14 (t, 1H), 5.22 (9, 1H, MH), 7.18 (b, IN, SeH), 7.48 (b 2H, RH), 7.62 (B 1H, Ri) and 7.98 (4.2H, RY). so 1-O-isobutyryl-2-(p-hydroxyacetyl)amine-3-(M3-benzoyl (thyminyl)-I-propanol (24):

Zo 1-O-isobutyryl-2-tert-butyloxycarbonyl)amino|-3-IM 3z-benzoyl (thyminyl)-I-propanol 23 (1.6g, 3.38mmol) M was stirred in a mixture of trifluoroacetic acid (5ml) and СНоСи » (1 Oml) at room temperature for 30 minutes, then evaporated to dryness. The residue was dissolved in dry Meon (10 ml) and evaporated again. The resulting residue was dried overnight over solid Mahon in a vacuum. The dried material was used in this form for subsequent reactions. 1-Hydroxybenzotriazole (0.67 g, mmol) and /1-zyl-3-(3-dimethylaminopropyl) were added to a stirred solution of glycolic acid (0.53 g, 7 mmol) in dry M,M-dimethylformamide (50 ml) at 70 K. -carbodiyimide "with hydrochloride (EOS) (1.91g, 1bmmol) After 15-minute stirring at room temperature, TEA (1.01g, tTOmmol) and the above-mentioned trifluoroacetic acid salt were added! in M,M-dimethylformamide (2 Oml).

The reaction mixture was stirred for 12 hours, then evaporated to dryness. The rest was divided between them

SNiCl" (150ml) and water (100ml) and extracted the NiClSi". The organic extract was washed with a saline solution (BOml), dried and layered to dryness. The residue was purified by means of evaporative chromatography on o silica gel with СНоSiO»о - » acetone as a solvent. Fractions containing the desired product were collected, - evaporated and 1.35 g (92905) of stump were obtained. IN NMR (SOSI5): 1.16 (a, 6H, ISNU"), 1.94 (v, ZN, CH"), 2.52 (t, 1H), 3.20 (rev, 1H), 3, 80 - 4.30 (t, 6H), 4.56 (t, 1H), 7.14 (a, 2H, SeH and MH), 7.50 (i, 2H, RI), 7.64 (i, 1H, RI) and 7.94 sya 70 (d, 2nH, P). o 1-O-isobutyryl-2-((p-(4,4'-dimethoxytrityl)-O-acetyl) amine)| -3- |M3-benzoyl (thyminyl)-I-propanol (25): 1-O-isobutyryl-2-(K-hydroxyacetyl) /amino-3-(M3-benzoyl (thyminyl)-I-propanol 24 (1 .2g, 2.78mmol) was dissolved in dry pyridine (50ml) and stirred at room temperature in an argon atmosphere. TEA (0.35g, 3.5mmol) and 4,4-dimethoxytrityl chloride (1 , 18 Hg,

Z, Bmmol). The reaction mixture was stirred at room temperature for 12 hours, quenched at

F) aid Meon (TOml) and steamed to dryness. The residue was dissolved in EtOAc (150 ml) and washed with a 595 solution

GI Manso" (100ml), water (100ml) and saline solution (5Oml). The organic extract was dried over MA»zO and layered to dryness. The residue was purified by means of evaporative chromatography on silica gel with СНоСи»-»бо ЕУБдс as a solvent. The pure fractions were combined, evaporated and 1.7 g (83795) of the pure product was obtained. so on

NMR (SOSI»z): 1.16 (9, EN, ISNU»), 1.94 (in, ЗН, СН»), 2.52 (t, 1Н), 3.74 (in, 6Н, 2, 0СН»), 3.80 - 4.30 (t, 6Н), 4.56 (t, 1Н), 6.82 (9, 4Н, РИ), 7.14 (а, 2Н, SeН and МН) and 7.26 - 8.00 (t, 14H, RI). 2-I(8-(4,4-dimethoxytrityl)-O-acetyl)amino|-3-thyminyl-Y - propanol (26): 1-O-isobutyryl-2-|(B-(4,4'dimethoxytrityl )-O-acetyl)amino-3-|M z-benzoyl(thyminyl)-I-propanol 25 (1.55 g, 65 2.05 mmol) was dissolved in Meon (20 ml) and cooled to 0"C in an ice bath. K therefore, 2M Mahon (5ml, TOmmol) was added to the cold solution while stirring, and stirring was continued at 0"C for 30 minutes.

With the help of acetic acid! The pH of the mixture was adjusted to 7 and the mixture was evaporated to dryness. The residue was partitioned between water (ppm) and CHClCl (1500 ml) and extracted with CHClCl. The aqueous layer was also extracted with SiO 2 (5 Oml).

The combined organic extract was washed with saline solution (5 Oml), dried and evaporated to dryness.

The residue was purified by means of evaporative chromatography on a column with silica gel, with СНоСио - » acetone as a solvent. Output: 1.0g (9990). IN YAM (SOSI5): 1.94 (v, ZN, SNU"), 3.74 (v, 6H, 2.0СН»U), 3.80 - 4.30 (t, 6H), 4.56 ( t, 1H), 6.82 (a, 4H, RI), 7.14 (9, 2H, SeH and MH) and 7.26 - 8.00 (t, 14H, RI). 2-I(D-(4,4-dimethoxytrityl)-O-acetyl)amino|-3-thyminyl-I-propane-1-0-(M,M-diisopropyl)-B-cyanosthylphosphoramidite (27): 2- ((8-(4,4-Dimethoxytrityl)-O-acetyl)amino|-Z-thyminyl-I-propanol 26 (1.0OHg, 70 2.09mmol) was dried overnight in vacuum over Mahon solids, then dissolved in dry СНоСи" (50 ml).

The solution was cooled to 0"C in an argon atmosphere. To this cold solution, while stirring,

M,M-diisopropylethylamine (0.54g, 4.2mmol), then 2-cyanoethyl-M,M-diisopropylchlorophosphoramidite (0.7g,

C, mmol). The reaction mixture was stirred for 1 hour at 0"С and for 2 hours at room temperature. The reaction mixture was diluted with СНоСи" (100 ml) and the organic layer was washed with 595 solution 75 Manso)" (100 ml), water (100 ml) and brine ( 5 Oml). The extract was added to the SNHC, evaporated to dryness and an oily residue was obtained. The residue was purified by means of evaporative chromatography on silica gel with

SNІСІ» - » EUZAs, containing 0.195 TEA, as a solvent. The pure fractions were combined, evaporated and a foam was obtained. The foam was dried overnight over MOH solid in a vacuum. The dried foam was dissolved in dry SiOCl (100 ml) and added dropwise over 30 minutes to stirred dry hexane (800 ml) in an argon atmosphere. After that, the resulting precipitate was stirred for another 30 minutes, then filtered, washed with dry hexane (100 ml) and the resulting solid was dried in a vacuum over a Maoson furnace for 4 hours. Output: 1.3g (82905).

Example 29 (Fig. 29)

Maz-tert-butyloxycarbonyl-O-benzylhydroxylamine (28): O-benzyl-hydroxylamine hydrochloride (15.9g, "M

O0mmol) was suspended in a mixture of tetrahydrofuran (15Oml) and water! (5 Oml). TEA (15.15 g, 150 mmol), then di-tert-butyl dicarbonate (23.98 g, 110 mmol) was added to this solution while stirring (5). The reaction mixture was stirred at room temperature for 12 hours, then evaporated to dryness. The residue was partitioned between EAs (250ml) and water (200ml) and extracted with EAs. The extract in EUDAs was washed with potassium hydrosulfate (10 Oml) and saline solution (1O0 Oml), dried, evaporated to dryness, and 15 g (9195) of transparent (ab) oil were obtained. 1-chloro-2-(tetrahydropyranyl)oxy-ztane (29): 1-chloro-ztanol (8.0 mg, 100 mmol) was dissolved in dry CH2Cl2 (100 ml) and cooled to 0°C in an ice bath under an argon atmosphere. Dihydropyran (12.6 g, 1500 mmol) was added to this solution while stirring, then pyridinium-p-toluene-4-sulfonate (1.25 g, mmol) and stirring was continued overnight. The reaction mixture was evaporated to dryness and dissolved in EtOAc ( 200ml). The extract in EtOAc was washed with 595 MnSO3 solution (100ml), water (100ml) and saline solution "I (100ml). The organic extract was dried and evaporated to dryness. The raw material was purified by means of evaporative chromatography on silica gel with hexane - "СНоСи" as The pure fraction was collected, evaporated and 1 g (6795) of pure product was obtained.

M-tert-butyloxycarbonyl-M-((tetrahydropyranyl)oxy|zyl-O-benzylhydroxylamine (30). CC of the solution

M3-tert-butyloxycarbonyl-O-benzylhydroxylamine 28 (5.79g, 25.96bmmol) in dry M,M-dimethylformamide - with (5Oml) while stirring slowly, for 15 minutes, at 0"C in an argon atmosphere, added Man ( 60960, and 1.2 g, 00 mmol).The reaction mixture was stirred at 0°C for 30 minutes and at room temperature for 1 hour. 1-Chloro-2-(tetrahydropyranyl)oxystane 29 (4.95 g, 30 mmol) was added and the reaction mixture was heated to 80°C for 12 hours. The reaction mixture was cooled and evaporated to dryness. The residue was suspended in water (500 ml), The pH of the solution was brought to 7 and it was extracted with EtOAc (150 ml). The extract in EtOAc was washed with water and brine, dried and evaporated to dryness. The residue was purified by coevaporative chromatography on silica gel with hexane - "СНоСи" as a solvent. Required fractions combined, evaporated and obtained 6.0 g (6695) of an oily product. "H NMR (SOCI»z): 5 1.48 (5, 9M, Bos), 1.49 - - 1.84 (t, 6H, 3. CH"), 3.48 - 3.70 (t, 4H, 2.CH»5), 3.86 (t, 2H, CH"), 4.60 (i, IN, CH), 4.84 ( 5, 2Н, СНоРИ) and 7.32 - s 20 7.А2 (t, BN, RI).

M-tert-butyloxycarbonyl-M-|((2-hydroxy)zyl|-O-benzylhydroxylamine (31): a solution with M-tert-butyloxycarbonyl-M-(tetrahydropyranyl)oxy|zyl-O-benzylhydroxylamine 30 (3.51g , 1 mmol) in tetrahydrofuran:water"AcOnH (1:1:1, 100 ml) with stirring and heated to 70"C for 3 hours.

The reaction mixture was cooled to 0"C and the pH of the mixture was brought to 7 with the help of solid BiansCoOz. The reaction mixture was extracted into EAs (2 x 7Bml). The combined organic extract was washed with water (100ml) and

GF! saline solution (100 ml), dried and evaporated to dryness. The residue was purified by means of evaporative chromatography on silica gel with СНоСи» - » EUAs as a solvent. The clean fractions were combined, evaporated and 2.5 g (9490) of stump were obtained. 18. NMR (SOCI5) 85 1.48 (in, 9H, Bos), 3.60 (i, 2H, CH"), 3.74 (t, 2H, CH"), 4.84 (in, 2H, SNR) and 7.32 - 7.42 (t, 5H, RI). 60 M-tert-butyloxycarbonyl-M-((2-isobutyryl)oxy|zyl|-O-benzylhydroxylamine (32): Kc solution

M-tert-butyloxycarbonyl-M-((2-hydroxy)zyl|-O-benzylhydroxylamine 31 (4.2g, 16.bmmol) in dry pyridine (50ml) was added with stirring, TEA (2.02g, 20mmol), then isobutyric acid anhydride (3.16 g, 20 mmol) at room temperature under an argon atmosphere. The reaction mixture was stirred at room temperature for 12 hours, then evaporated to dryness. The residue was dissolved in EtOAc (200 mL), washed with 590 mL of Manso solution (100 mL), water and saline solution (50 Oml). The organic extract was dried and evaporated to dryness. The residue was purified by means of evaporative chromatography on silica gel with СНоСи" as a solvent. The pure fractions were combined, evaporated and 4.5 g (80965) of the pure compound were obtained. "H NMR (SOCIz): 5 1.04 (a, EM, ISSN"), 1.48 (5, 9H, Bos), 2.44 (t, 1H, IBSN), 3.60 (I, 2H, CH"), 3.74 (t, 2H, CH"), 4.84 (v, 2H, СНоОРИ) and 507.32 - 7.42 (t, 5Н, RI).

M-(thyminylacetyl)-M-I|(2-isobutyryl)oxy|zyl|-O-benzylhydroxylamine (33):

M-tert-butyloxycarbonyl-M-|(2-isobutyryl)oxy| Ztyl|1-O-benzylhydroxylamine 32 (5.0 g, 14.84 mmol) was dissolved in СНоСи» (1 Oml) and stirred in trifluoroacetic acid (12 ml) for 30 minutes.

The reaction mixture was evaporated to dryness and dissolved in dry methanol (1 mL). The mixture was again evaporated to dryness and dried overnight under vacuum over solid Mahon. The dried material in the same form, without research, was used in the next reaction.

Thymine acetic acid 5 (3.13g, 1/mmol) was dissolved in dry M,M-dimethylformamide (75ml) and cooled to -20"C in an argon atmosphere. M-methylmorpholine (2.02g, 20mmol) was added to this cold solution while stirring ), then isobutyl chloroformate (2.72 g, 20 mmol). After 15 minutes of stirring, a solution of 75 of the mentioned trifluoroacetic acid salt in dry M,M-dimethylformamide (5 Oml) was neutralized

M-methylmorpholine (2.02 g, 20 mmol) and immediately, in one step, added thymine acetic acid to the cold stirred solution. The reaction mixture was stirred at -20°C for 1 hour, warmed to room temperature, and stirring was continued overnight. The solution was evaporated to dryness, and the residue was dissolved in CH2Cl2 (250 ml) and water (100 ml) and extracted with CHCl2. The organic extract was washed 590 with MansSOOz solution (100ml), water (100ml) and brine (5Oml). The extract was dried in CH 25Si», evaporated to dryness and 4.0g (7095) of pure product was obtained. The pure product was crystallized from СНоSiO/hexane, melting point 185 - 1882. "H NMR (Me»5O-yv): 5 1.00 (a, EM, IBSNZ), 1.74 (8, ZN, CH), 2.44 (t, 1H, IBSN), 3, 92 (t, 2H), 4.18 (i, 2H), 4.68 (re, 2H), 4.98 (v, 2H), 7.34 (8, 1H, SeH), 7.40 - 7 .50 (t, 5H, RI) and 11.32 (rve, 1H, MN).

Example 30 (Fig. 30) with (2k, 4kK)-2-carbomethoxy-4-hydroxypyrrolidine (35): dry methanol (40ml) was placed in a round-bottom flask with a capacity of 250ml, equipped with a magnetic stirrer and a partial condenser of hot irrigation, and cooled in an ice bath in an argon atmosphere. Acetyl chloride (4.32 g, 55 mmol), then cis-4-hydroxy-O-proline 34 (5.00 g, 38.17 mmol) was added to this solution while stirring. The resulting solution was boiled for 7-8 hours, then cooled to room temperature. The solution was diluted with zephyr, and the resulting white solid was collected by suction, washed with zephyr and dried in a vacuum over a Mahon solid. Output: 6.9g (10090). IN NMR (SOCI3): 2.09 (2 aa, 1n), 2.34 (t, 1H), 3.49 - 3.73 (t, ЗН), 3.79 (8, ЗН, СН»), 4.34 (t, 2H). - (2k,4kK)-1-(tert-butyloxycarbonyl)-2-carbomeoxy-4-hydroxypyrrolidine (36): to a solution (2K, 4K) o -2-carbomethoxy-4-hydroxypyrrolidine 35 (6.9g, 38, 12mmol) in tetrahydrofuran/water (8:2, 1500ml) at 32 P. TEA (10.1g, 10mmol) was added with stirring, then di-tert-butyldicarbonate (10.9g, 10mmol) at room temperature. The reaction mixture was stirred at room temperature for two hours, then evaporated to dryness. The residue was dissolved in EtOAc (200ml) and washed with 0.5M potassium hydrosulfate (50ml), water (100ml) and brine (50ml). The organic extract was dried over NaCl, evaporated to dryness and 7.8 g (84956) of an oily product were obtained. From the oily product, after drying, 50 was obtained as a colorless solid, melting point 75 - 7770. IN NMR (SOCI5): 1.45 (v, 9H, Bos), 2.09 (2 aa, C s 1H), 2.34 ( t, 1H), 3.49 - 3.73 (t, ЗН), 3.79 (v, ЗН, СН»), 4.34 (t, 2Н). ; with" (2k,4kK)-1-(tert-butyloxycarbonyl)-2-hyroxymeotyl-4-hydroxypyrrolidine (37): (2,4k)-1-(tert-butyloxycarbonyl)-2-carbomethoxy-4-hydroxypyrrolidine 36 ( 7.0 g, 28.6 mmol) was dissolved in dry tetrahydrofuran (100 ml) and cooled in an ice-salt bath in an argon atmosphere. To this cold solution, lithium borohydride (1.88 g, 85.9 mmol) was added in small portions over 15 minutes. After the addition of lithium borohydride, the reaction mixture was stirred at 0"C for 1 hour, then at (oo) room temperature for 15 hours in an argon atmosphere. The mixture was cooled to "C and diluted with water (50ml), then the pH was adjusted using AcOH to 6. The reaction mixture was evaporated to dryness and dissolved in EtOAc (200 ml), washed with water (100 ml) and brine (10 ml). The extract was dried in EtOAc and evaporated to dryness. with 20 The residue was purified by means of evaporative chromatography on a column with silica gel, with СНоSiO»о -» ЕХАс as a solvent. The clear fractions were collected and evaporated to dryness. 5.00 g (8190) of transparent oil were obtained.

The oil was allowed to stand and a colorless solid was obtained, melting point 95 - 9770. "H NMR (SOCI"): 1.45 (5, 9H, Voe), 1.90 (aa, 1H), 2.34 (t, 1H ), 3.40 - 3.62 (t, ZN), 4.00 (t, 2H), 4.28 (B, 1H), 4.44 (t, 1H). (2k,4K)-1- (tert-butyloxycarbonyl)-2-(4,4-dimethoxytrityl)oxymethyl-4-hydroxypyrrolidine (38): 59 (2k,4K)-1-(tert-butyloxycarbonyl)-2-hydroxymethyl-4-hydroxypyrrolidine 37 (4, 4 g, 20.28 mmol) was dissolved in

HF) with dry pyridine (bOml) and stirred in an argon atmosphere. To this solution when mixed

TEA (2.53g, 25mmol) was added to HF, then 4,4"-dimethoxytrityl chloride (7.45g, 22mmol). The reaction mixture was stirred at room temperature for 12 hours, then quenched with MeonH (10ml). The solution was evaporated to dryness and was dissolved in EtOAc (200 ml). The EtOAc layer was washed with 595% NaCl solution (100 ml), water (100 ml) and brine. The organic extract was dried over anhydrous NaCl and evaporated to dryness.

The residue was purified by means of evaporative chromatography on silica gel with hexane - » E(Ac) as a solvent. The required fractions were combined, evaporated to dryness and 8.09 g (10095) of an orange stump were obtained. "H NMR (SOCI"): 1.45 (in, 9Н ) ,00 (t, 2H), 4.28 65 (p5, 1H), 4.44 (t, 1H), 6.82 (9, 4H, RI) and 7.26 - 8.00 (t, 9H, RP).

pyrrolidine (39): (2K,4K)-1-(tert-butyloxycarbonyl)-2-(4,4-dimethoxytrityl)oxymethyl-4-hydroxypyrrolidine 38 (8.09g, 20.27mmol) was dissolved in dry pyridine/CH2Cl (2 : 1, 200 ml) and cooled in an ice bath in an argon atmosphere. To this cold solution was added TEA (3.03 g, 30 mmol), then p-toluenesulfonyl chloride (5.77, 30 mmol). The reaction mixture was stirred at 0"C for 3 hours and at a temperature of

30"C for 8 hours. The reaction mixture was evaporated to dryness, partitioned between E(JuAc (200 ml) and 590 solution

Manso»z (100 ml) and extracted into EtOAc. The EUAs extract was washed with water (100 ml) and saline solution (100 ml), dried and evaporated to dryness. The gray product was purified by means of evaporative chromatography on silica gel with hexane - » E(UAs) as a solvent. The pure fractions were combined, evaporated and obtained 12.2 g 70 (89905) of orange oil. "IN YAM (SOCI5): 1.45 (in, 9Н , Bos), 1.90 (aa, 1H), 2.34 (t, 1H), 2.40 (5, ЗН, СН»), 3.40 - 3.62 (t, ЗН), 3.74 (in, EN, 2.0СН»), 4.00 (t, 2Н), 4.28 (rev, 1Н), 4.44 (t, 1Н), 6.82 (y, 4Н, RI) and 7, 26 - 8.00 (t, 1ZN, RI). (2K, 45) -1-(tert-butyloxycarbonyl)-2-(4,4'-dimethoxytrityl)oxymethyl-4-acyl-pyrrolidine (40): (2K . ) and diluted with water (bml). Sodium azide (0.65 g, 1 mmol) was added to this solution while stirring and heated to 80°C for 8 hours. The mixture was cooled and dried. The residue was partitioned between СНоСи» (200 ml) and water (100 ml) and extracted

SNOSI". The organic extract was washed with saline solution (50 ml), dried over NaCl, and evaporated to dryness.

The pure product was purified by means of evaporative chromatography on silica gel with hexane-» EtOAc as a solvent. The pure fractions were combined, evaporated and 3.8 g (92965) of transparent oil were obtained. "H NMR (SOSI"): 1.45 (v, 9H, Bos), 1.90 (da, 1), 2.34 (t, 1H), 3.40 - 3.62 (t, ЗН), 3.74 (v, 6H, 2.0CH»U), 4.00 (t, 2H), 4.28 (p5, 1H), 4.44 (t, 1H), 6.82 (9, 4H, RI ) and 7.26 - 7.80 (t, 9H, RI). (2k,45)-1-(tert-butyloxycarbonyl)-2-hydroxymethyl-4-amino-pyrrolidine (41): (2k,45)- 1-(tert-butyloxycarbonyl)-2-(4,4"-dimethoxytrityl)oxymethyl-4-azide-pyrrolidine 40 (2.72 g, mmol) in CM methanol (75 ml) was hydrogenated in the presence of 1095 palladium-coated coal (0.3 g) at room (5) temperature under a pressure of 5 atmospheres. After 12 hours, the catalyst was filtered off, washed with methanol (20 ml) and the solvent was removed under vacuum. Output: 1.0g (9395). "H NMR (SOCI3): 1.45 (b, 9H, Vos), 1.90 (av, 1H), 2.34 (t, 1H), 3.40 - 3.62 (t, ЗН), 4 .00 (t, 2H), 4.28 (rev, 1H) and 4.44 (t, 1H). (2k, 45)-1-(tert-butyloxycarbonyl)-2-hydroxymethyl-4-phthalimide-pyrrolidine ( 42): ("c") (2kK,45)-1-(tert-butyloxycarbonyl)-2-hydroxymethyl-4-amino-pyrrolidine 41 (1.00g, 4.63mmol) was dissolved in dry methanol (20ml) and M-zoxycarbonyl was treated with phthalimide (1.09 g, mmol) at room temperature. The reaction mixture was stirred for 2 hours and evaporated to dryness. The residue was purified by evaporative chromatography on silica gel with СНоСи»-» EUDAs as a solvent. The pure fractions were combined , evaporated and obtained 1.5 g (9495) of pure product in the form of a stump. "H NMR (SOCI5): 1.45 (v, 39 OH, Voe), 1.90 (aa, 1H), 2.34 (t, 1H), 3.40 - 3.62 (t, ЗН), 4.00 (t, 2H), 4.28 (rev, 1H), 4.44 (t, 1H) and 7.3 - - 7, 6 (t, 4H, RI). (2kK,45)-1-(tert-butyloxycarbonyl)-2- |M3-benzoyl (thymin-1-yl) |methyl-4-phthalimide-pyrrolidine (43): to a stirred solution of Ma-benzoylthymine 20 (1.15g , mmol) in dry tetrahydrofuran (7Oml) in an argon atmosphere at room temperature was added triphenylphosphine (2.62g, l1Omol) and 40. (28.45)-1-(tert-butyloxycarbonyl)-2-hydroxymethyl-4-phthalimide- pyrrolidine (1.4 g, 4.05 mmol). After 15 minutes, distilazodicarboxylate (1.74 g, 1 mmol) was added slowly over 10 minutes. The reaction mixture was covered with aluminum foil and stirred at room temperature in an argon atmosphere for 24 hours. The solvent was evaporated to dryness, and the residue was dissolved in EAs (150 ml). The organic extract was washed with a 595% solution of CaCl2O3 (100 ml), water (100 ml) and brine (100 ml), then dried over anhydrous Ma»5O). The dried extract in EOAc was evaporated to dryness and an orange oil was obtained. The resulting product was purified by means of evaporative chromatography on silica gel with hexane-EAc as a solvent. The fraction containing the desired product was combined and evaporated. A pale pink oil was obtained. with Output: 2.0g (8995). "H NMR (SOS33): 1.41 (b, 9H, Bos), 1.72 (b, 3H, CH), 1.90 (ad, 1H), 2.34 (t, 1H), 3.40 - 3.62 (t, ZN), 4.00 (t, 2H), 4.28 (B5, 1H), 4.44 (t, 1H), 7.06 (v, 1TN, SeH) and 7, 20 - 7.60 (t, 9H, RI). 4! 250 Example 31

Synthesis of oligonucleotides: Oligonucleotides containing a modified aminonucleotide skeleton were synthesized in an automatic DNA synthesizer (Arriei Viozuzietv, model 394) according to the principles of standard phosphoramidite chemistry. Beta-cyanidyl-phosphoramidite, reagents for synthesis and SRO polystyrene columns were purchased from Arriea Viozuvietv company (Rovieg SPU, SA). For the synthesis of 25 phosphorothioate oligonucleotides, the standard oxidizing flask was replaced with tetraethylthiuram

HF) with disulfide/acetonitrile, and for the post-salt thiatiation of phosphate bonds, the standard AVI phosphorotysate program was used. After splitting on a column made of porous glass, protective groups! was removed by treating oligonucleotides with concentrated ammonium hydroxide for 8 hours at 55°C. Purification of oligonucleotides (with OMT groups) was carried out by high-resolution liquid chromatography on a semi-preparative reversed-phase Ce (AVI) column with a linear gradient of 595 acetonitrile in 0 ,1M trisethylammonium acetate (buffer A) and acetonitrile (buffer B).

OMT protecting groups were cleaved by treatment with 8095 acetic acid, and the product was precipitated with ethanol. The purity of the compounds was checked using high-resolution liquid chromatography on an analytical column St (Vesktap). Aminonucleic monomer! were attached to the 3'-ends, as well as to the 5-ends and in the middle of the DNA sequences with 10095 attachment efficiency. A homopolymer containing 16 thymines modified with amino acids was also prepared without difficulty.

Example 32

Hybridization analysis: The ability of oligonucleotides modified with amino acids, proposed by the present invention, to hybridize with their complementary RNA and DNA sequences is determined by thermal analysis. The RNA complement was synthesized by the company (Zepzei! (Ia doiIa, SA) and purified by means of denaturing electrophoresis in a polyacrylamide gel with urea. K RNA-i

Natural antisense oligonucleotides or oligonucleotides containing 7/0 functional groups were added in stoichiometric concentrations to DNA complements for the formation of double hybrid structures! in certain places. The dependence of optical density (26bO0nm) and hyperchromism on temperature during the transition from a double structure to a random spiral was measured using an ultraviolet spectrophotometer Magiap Sagu IE OM-MiizIbie. Measurements were made in a buffer of 10 mM sodium phosphate, pH 7.4, 01 mM ZDTA and Mass, providing an ionic strength of either 0.1M or 1.0M. The analysis of the data was carried out by means of a graphical representation of 1/Hpl relative to Ip(CO, where Si is the total 7/5 concentration of oligonucleotides. This analysis allowed us to determine the thermodynamic parameters! Based on the data on the stability of the formed duplexes or heteroduplexes, the influence of the modified pyrimidine present in the oligonucleotides on the stability was evaluated spirals

Modifications that significantly change the stability of the hybrid correspond to a decrease or increase in free energy (delta C), and based on that, a decision is made about the antisense oligonucleotides used in them.

The hybridization analysis was carried out with oligonucleotides containing an aminonucleic skeleton! on 3-ends, as well as on 5-ends. Preliminary analysis showed that the modified oligonucleotide! form a duplex! with complementary RNA and DNA sequences, as well as an unmodified oligonucleotide. with

Example 33

Resistance to nucleases: The resistance of natural, phosphorylated and modified i) oligonucleotides proposed by the present invention to Kk serum nucleases was evaluated by incubating oligonucleotides in media containing fetal calf serum or adult human serum in various concentrations. The labeled oligonucleotide is kept in the media for different times, treated with protease K and analyzed by means of electrophoresis on 2095 polyacrylamide-urea denaturing gels followed by autoradiography or luminophore visualization. Quantitative assessment of autoradiograms is carried out using laser densitometry. Knowing the position of the modification and the length of the oligonucleotide, it is possible to determine the influence of one or another modification on degradation under the action of nucleases. For experiments with cytoplasmic nucleases, white water was used

From Cell line NO 60. The post-mitochondrial supernatant is prepared by means of differential "E" centrifugation; labeled oligonucleotides are kept in this supernatant for different times. Then the degradation of oligonucleotides is evaluated, as well as degradation under the action of serum nucleases. Quantitative evaluation of the results of autoradiography is carried out to compare unmodified ones, i.e. phosphorylated, modified oligonucleotides. "

Preliminary analysis of oligonucleotides modified with amino acids showed that they are resistant to the action of phosphodiesterases from snake venom.

Activation by means of a link;" All patents, patent applications and publications mentioned in this document are incorporated by reference.

Equivalent to 5" The above description is considered sufficient so that a specialist can put the invention into practice. Various modifications of the invention described above, obvious to specialists in the field of molecular biology, organic chemistry or related fields, are included in the scope of the following formulas! inventions with 50

Claims (8)

The formula of the invention 1. Production of amino acids or amino alcohol formulas; | in any of the groups |, YII Y x I ' o MA, with 1 where M denotes nitrogen, because Group X denotes CHKOOH, where K» denotes H, (lower alkyl)amine or (lower alkyl)imidazole; M denotes Vaze-(CHo), where Vaze denotes a non-halogenated variable nucleoside base, where n is 1 to 7; A denotes carbonyl; b5 U U U 7 denotes H or OK", where Kz denotes H, lower alkyl, (lower alkyl)iuamine or (lower alkyl) imidazole; M denotes SNADONE, where Ku denotes H, (lower alkyl) amine or (lower alkyl) imidazole; Group II A denotes carbonyl; X denotes Vase-(CH0)., where Vase denotes a non-halogenated variable nucleoside base, where n- 1 - C; M stands for -SNYON; M denotes -СНоОН; 70 7 denotes N. 2. Oligonucleotide, the composition of which includes! compounds according to claim 1 in such a way that the internucleotide bonds are between M and X in group II, M and Y in group II. Z. Oligonucleotide according to claim 2, where the internucleotide linkage contains phosphodiester. 4. Oligonucleotide according to claim 2, where the internucleotide linkage contains phosphorothioate. 5. Oligonucleotide according to claim 2, where the internucleotide linkage contains phosphoramidate. 6. Oligonucleotide according to claim 2, where the internucleotide linkage contains hydroxamate. 7. Oligonucleotide according to claim 2, in which a plurality of monomers can be distinguished, where at least one of the monomers contains a 2'-deoxyribose nucleoside, and additionally contains at least two different internucleotide bonds from the group consisting of phosphodiester, phosphorothioate, phosphoramidate and 2o hydroxamate . 8. Oligonucleotide according to claim 7, which has a 2'-deoxyribose nucleoside pentafuranosyl ring, in which the ring oxygen is replaced by one of the groups 5, СНо or MK, where KO is acetyl, lower alkyl, carbonyl, carbonyl (lower alkyl)amine or carbonyl (lower alkyl)imidazole. with points 6) "c) IF) "- (ee) " - and? sh» (ee) - 1 (42) ime) 60 b5 -BO0-