PL219355B1 - Modified deoxyribozyme 10-23 with hydrolytic stability and increased catalytic activity and process for the preparation thereof - Google Patents

Description

Description of the invention

The subject of the invention is a modified deoxyribozyme 10-23 (DNAzyme 10-23) of the general formula 1, containing at least one internucleotide phosphorothioate bond located in selected P positions of the catalytic domain P1 to P15, especially in position P1, P7, P8, P14 and / or P15, preferably with a defined absolute configuration on chiral phosphorus atoms and / or at least one modified N nucleoside residue represented by general formula 2, wherein B is a 5-modified uracil residue or a 6-modified adenine residue represented by the formula 2 'or 2, where R2 is an alkylamine or amino acid residue, preferably represented by the formula 2a, 2b, 2c and / or 2d, and / or at least one modified nucleoside residue at the N position represented by the general formula 3, wherein B1 is a thymine residue, cytosine, adenine or guanine, and R1 at the 2'-deoxyribose position is -F or an O-alkyl group, most preferably an OMe group at selected positions h N of the deoxyribozyme, especially in the N2, N7, N8, N11, N14 and / or N15 positions of the catalytic domain, and in the terminal positions of the substrate recognition arms (at the 3 'and 5' ends of the oligonucleotide), with stabilized resistance to hydrolysis by cell nucleases and increased catalytic activity.

Unmodified deoxyribozyme 10-23 (DNAzyme 10-23) is represented by formula 1, in which the nucleobases of all N nucleosides from 1 to 15 of the catalytic domain are identical to the units contained in the original in vitro selected DNAase 10-23 (according to SW Santoro, GF Joyce , 1997, Proc. Natl. Acad. Sci. USA, 94, 4262), and have the following sequence:

5'-G1G2C3T4A5G6C7T8A9C10A11A12C13G14A15-3 'where this deoxyribozyme is designed for the selected mRNA sequence of the BACE1 protein, which means that the enzyme recognition arms are complementary according to the Watson-Crick pairing principles to the selected mRNA sequence of the BACE1 protein.

The modified deoxyribozyme according to the invention has favorable biological properties, showing increased catalytic activity and noticeably higher stability to nuclease hydrolysis in the environment of a cell extract from HeLa cells.

State of the art

Nucleic acid fragments that under appropriate conditions show the ability to catalyze chemical reactions without protein factors are classified as catalytic nucleic acids, or otherwise, to nucleic acids with catalytic properties. If the catalysis involves the sequentially specific hydrolysis of a phosphodiester bond in RNA, catalytic nucleic acids can be next generation drugs. The mechanism of action of this group of drugs is based on modulating the process of viral gene expression and inhibiting the biosynthesis of pathogenic proteins.

Several possible strategies for the use of synthetic oligonucleotides for therapeutic purposes have been developed. According to the antisense strategy, complementary (according to the Watson-Crick rules) oligonucleotides interact with single-stranded mRNA or pre-mRNA to form heteroduplexes recognized by protein degrading enzymes (E. Uhlmann, A. Peyman, 1990, Chem. Rev. 90, 544). The sequence antisense-type recognition strategy is also used in the degradation of mRNA using oligonucleotide enzymes - identified natural ribozymes (OC Uhlenbeck, 1987, Nature 328, 596) and deoxyribozymes selected in vitro (SW Santoro, GF Joyce, 1997, Proc. Natl. Acad. Sci. USA 94, 4262; T. Kuwabara, M. Warashina, T. Tanabe, K. Tani, S. Asano, K. Taira, 1997, Nucleic Acids Res. 25, 3074). Another approach is the so-called antigen therapy in which synthetic oligonucleotides recognize double-stranded DNA sequences according to Hoogsteen's rules and influence the process of gene transcription (C. Giovannangeli, C. Helene 1997, Antisense Nucleic Acids Drug Dev., 7, 413). Another strategy for inhibiting protein activity is the use of the so-called protein aptamers - oligonucleotides selected in the in vitro selection process and forming stable complexes with proteins (C. Tuerk, L. Gold, 1990, Science, 249, 505). The recently discovered effect of RNA interference (A. Fire, S. Xu, MK Montgomery, SA Kostas, SE, Driver, CC Mello 1998, Nature 391, 806) allows to silence the expression of unwanted genes by using short interfering RNAs - siRNAs (S. Elbashir , T. TuschI 2001, Nature 411, 494). Double-stranded RNAs of 21-23 nt in length are promising tools of particularly important therapeutic importance (M. Miyagishi, K. Taira 2002, Nat. Biotech. 19, 497; H. Xia, Q. Mao, HL Paulson, BL Davidson 2002, Nat. Biotech. 20, 1006).

The search for new therapeutics is focused on the research of oligonucleotide analogues showing increased potential for modulation of gene expression, increased affinity for target molecules, or characterized by increased resistance to the action of nucleolytic enzymes involved in in vivo processes.

Synthetic oligonucleotides constituting potential therapeutics are oligonucleotide analogs containing variously modified elements of the structure of natural nucleic acids. The group of oligonucleotide analogues containing the modified phosphate backbone are oligonucleotide phosphorothiates (WJ Stec, G. Zon, W. Egan, B. Stec 1984, J. Am. Chem. Soc., 106, 6077), methylphosphonates (PS Miller, ND Annan, SM McParland 1980, J. Biol. Chem., 255, 9659; PS Miller, ND Annan, SM McParland, KB Pulford 1982, Biochemistry, 21, 2507), phosphoramidates (SM Gryaznov, T. Skorski, C. Cucco, M. Nieborowska -Skorska, CY Chiu, DH Lloyd, J. Chen, M. Koziołkiewicz, B. Calabretta, 1996, Nucleic Acids Res., 24, 1508), borate phosphates (BR Shaw 2007, Nucleic Acids Symp. Series (Oxf). 51, 117), and dithiophosphates (Y. Xianbin 2008, Nucleic Acids Symp. Series, (Oxf.) 52 (1), 341).

Oligonucleotide phosphorothioates were the first to be introduced into therapy. One of them is Fomivirsen, an antisense oligonucleotide targeting the cytomegalovirus gene. This medicine is injected into the eye in patients with AIDS syndrome. Another drug with an oligonucleotide phosphorothioate structure approved for sale in both the US and European markets is Macugen, an aptamer for the VEGF protein whose deactivation is important in the treatment of age-related macular degeneration (AMD).

Deoxyribozymes are single-stranded oligodeoxyribonucleotides that exhibit catalytic activity on RNA molecules. It is analogous to the activity of protein nucleases that catalyze the hydrolysis of the phosphodiester bond in RNA and to the activity of natural ribozymes that catalyze the hydrolysis of the phosphodiester bond in RNA. Deoxyribozymes bind in a sequentially-dependent manner to the complementary strand of RNA and catalyze the hydrolysis of the internucleotide bond in the presence of divalent ions, most often magnesium (II). This property determines the use of deoxyribozymes as molecular tools for manipulating RNA and as compounds with a therapeutic effect (C. Hobartner, SK Silverman 2007, Biopolymers, 87, 279; SK Silverman 2005, Nucleic Acids Res., 33, 6151; SK Silverman, 2004, Org. Biomol. Chem., 2, 2701; RR Breaker, GM Emilsson, D. Lazarev, S. Nakamura, IJ Puskarz, A. Roth, N. Sudarsan 2003, RNA, 9, 949; Y. Isaka 2007, Curr. Opin. Mol. Ther., 9, 132; R. Bhindi, RG Fahmy, HC Lowe, CN Chesterman, CR Dass, MJ. Cairns, EG Saravolac, LQ Sun, LM Khachigian 2007, Am. J. Pathol., 171, 1079).

The best characterized is deoxyribozyme 10-23, selected by Joyce (RR Breaker, GF Joyce 1994, Chem. Biol., 1, 223; SW Santoro, GF Joyce 1997, Proc. Natl. Acad. Sci. USA 94, 4262) by in vitro selection reaction (C. Tuerk, L. Gold, Science, 1990, 249, 505-510; AD Ellington, JW Szostak 1990, Nature, 346, 818; DS Wilson, JW Szostak, 1999, Annu. Rev. Biochem ., 68, 611). The deoxyribozyme 10-23 contains a 15 nucleotide (nt) conserved (invariant) catalytic domain and substrate recognition arms of 6 to 12 nt in length and a variable sequence designed to be complementary to the target RNA molecule. Hydrolysis of the phosphodiester bond takes place between an unpaired purine nucleoside involved in the Watson-Crick pair and a pyrimidine nucleoside according to the transesterification reaction taking place in the presence of a divalent metal ion. The reaction products are two RNA fragments: the 5'-terminal with a 3'-end nucleotide with a 2 ', 3'-cyclic phosphate group and the 3'-terminal with a free 5'-OH group (SW Santoro, GF Joyce 1998, Biochemistry, 37, 13330; GF Joyce, 2001, Methods Enzymol. 341, 503). The mechanism of action of deoxyribozyme is not fully understood, and the spatial structure of this catalyst is also unknown. Attempts to obtain such a structure so far have resulted in obtaining a crystal of a complex of two deoxyribozyme 10-23 molecules and two non-hydrolyzable RNA substrate molecules, in which the components did not form a functionally active catalytic domain (J. Nowakowski, PJ Shim, GF Joyce, CD Stout, 1999, Acta Crystallogr., Sect. D: Biol. Crystallogr. D55, 1885; J. Nowakowski, PJ Shim, GC Prasad, CD Stout, GF Joyce 1999, Nat. Struct. Biol., 6, 151). Numerous studies have been carried out to determine the dependence of the activity of deoxyribozyme 10-23 on its structure, and it has been shown that the nucleosides at positions 1-6 and 13-14 are essential for enzymatic activity, and the nucleoside at position 8 (thymidine) can be deleted from the catalytic domain or substituted by another nucleoside, without loss of enzyme catalytic activity (SW Santoro, GF Joyce 1997, Proc. Natl. Acad. Sci. USA, 94, 4262; SW Santoro, GF Joyce Biochemistry 1998, 37, 13330; GF Joyce 2001, Methods Enzymol. 341, 503; Z. Zaborowska, VA Erdmann, J. Kurreck 2002, J. Biol. Chem., 277, 40617; Z. Zaborowska,

PL 219 355 B1

S. Schubert, J. Kurreck, V.A. Erdmann 2005, FEBS Lett. 579, 554). Substitution of the 8-position with the thymidine residue containing the photolabile 6-nitropiperonyloxymethyl substituent (NPOM) on the nitrogen atom in the 3-position (N3) of thymine made it possible to obtain an enzyme with activity independent of UV irradiation, contrary to the situation when the modified unit was introduced in position 4 of the domain catalytic enzyme (H. Lusic, DD Young, MO Lively, A. Deiters 2007, Organic Lett., 9, 1903). In order to increase the stability in the presence of cellular nucleases, deoxyribozyme 10-23 was modified both within the catalytic domain and in the substrate recognition arm. The insertion of 6 nucleoside residues containing the 2'-OMe group into positions 2, 7, 8, 11, 14 and 15 of the catalytic domain increased the nucleolytic stability of the enzyme with only a slight reduction in its catalytic activity (S. Schubert, D. GOl, HP. Grunert , H. Zeichhardt, VA Erdmann, J. Kurreck 2003, Nucleic Acids Res. 31, 5882). 2'-OMe and LNA (locked nucleic acids) type modified in the sugar part and modified bonds such as: phosphorothioate or 3'-3 'reverse are used to increase the enzyme resistance to 3'- and 5'-exonuclease (S Schubert, D. GOl, HP Grunert, H. Zeichhardt, VA Erdmann, J. Kurreck 2003, Nucleic Acids Res. 31, 5882; B. Vester, LB Lundberg, MD Sorensen, BR Babu, S. Douthwaite, J. Wengel 2002, J. Am. Chem. Soc., 124, 13682; B. Vester, LH Hansen, LB Lundberg, BR Babu,

M.D. Sorensen, J. Wengel, S. Douthwaite 2006, BMC Mol. Biol., 7, 19; C.R. Dass, E.G. Saravolac, Y. U and L-Q Sun 2002, Antisense Nucleic Acid Drug Dev. 12, 289).

So far, the use of deoxyribozymes 10-23 modified within internucleotide bonds with a phosphorothioate group in order to determine the architecture of the catalytic domain and the method of metal ion chelation has been described (B. Nawrot, K. Widera, M. Wójcik, B. Rebowska, G. Nowak, WJ Stec 2007, FEBS J., 274, 1062). These studies showed that the presence of a single phosphorothioate bond (in the form of a mixture of P-epimers) in some positions of the catalytic domain (especially P1 and P8) increases the activity of the enzyme both in the presence of magnesium (II) and manganese (II) ions. On the other hand, no differentiation in catalytic activity was demonstrated between individual diastereomeric phosphorothioate enzymes containing phosphorothioate bonds in P1 and P8 positions, and enzymes containing more than one (apart from the P1 / 8 enzyme) phosphorothioate linkage in the catalytic domain were not tested.

The subject of the invention are deoxyribozymes modified within the catalytic domain with amino acid residues, showing enhanced catalytic properties as compared to unmodified deoxyribozyme 10-23. Compounds of this type of general formula I, where the units in selected positions of the catalytic domain are nucleosides of the general formula according to the invention, have not been described in the literature so far, although a number of other deoxyribozymes are known with amino acid residues in their catalytic domain, usually basic (M Hollenstein, Ch. J. Hipolito, CH Lam, DM Perrin 2009, Nucleic Acids Res., 37, 1; R. Ting, RM Thomas, L. Lermer, DM Perrin 2004, Nucleic Acids Res., 32, 6660; DM Perrin, T. Garestier, C. Helene 2001, J. Am. Chem. Soc., 123, 1556; M. Hollenstein, C. Hipolito, C. Lam, DM Perrin 2009, Chembiochem, 10, 1988; A. Sidorov, JA Grasby, DM Williams 2004, Nucleic Acids Res., 32, 1591). Some of the new deoxyribozymes show activity without a metal ion, but the change in activity is small compared to unmodified deoxyribozyme 10-23. Thus, deoxyribozyme 925-11 containing 31 nt, including 18 forming the catalytic domain, including four 8-histaminyl-2'-deoxyadenosine residues and six 5-allylamino-2'-deoxyuridine residues, shows high activity under conditions of single and multiple rotation without presence of divalent metal ions (L. Lermer, Y. Roupioz, R. Ting, DM Perrin 2002, J. Am. Chem. Soc. 124, 9960), while deoxyribozyme 9-33 obtained by in vitro selection using three units modified: 8-histaminyl-2'-deoxyadenosine, 5-allylamino-2'-deoxycytidine and 5-guanidino-allyl-2'-deoxyuridine hydrolyzes the bond adjacent to the cytidine unit in the RNA substrate without the participation of divalent ions with the catalysis constant k = 0, 05 min ^-1 (M. Hollenstein, C. Hipolito, C. Lam, Perrin, D., 2008, Nucleic Acids Res. Symp. Ser. 52, 73). The highest catalytic activity without the participation of metal ions in the hydrolysis of the phosphodiester bond in DNA is achieved by deoxyribozyme 9-86 containing three units: 5-guanidino-allyl-2'-deoxyuridine, 8-histaminyl-2'-deoxyadenosine and 5-allylamino-2'-deoxyuridine (M. Hollenstein, C. Hipolito, C. Lam, DM Perrin 2009, Nucleic Acids Res. 37, 1). This enzyme is deactivated by the presence of metal ions at a concentration above 0.5 mM. Similarly modified deoxyribozyme 10-66 selected against the RNA substrate is active without the participation of divalent metal ions, but shows high activity in the presence of millimolar concentrations of monovalent metal ions and is 1000 times more active than

Unmodified enzyme (M. Hollenstein, C. Hipolito, C. Lam, D. M. Perrin 2009, ChemBioChem, 10, 1988).

The so far known and described deoxyribozymes containing modifications with amino acid residues, and showing activity without the participation of divalent metal ions, were obtained only by in vitro selection, and not by chemical modification of deoxyribozyme 10-23.

The essence of the invention

Modified deoxyribozyme 10-23 of general formula 1, comprising at least one internucleotide phosphorothioate linkage of formula 4a and / or 4b, located at selected positions in the catalytic domain P1 to P15, especially at position P1, P7, P8, P14 and / or P15, preferably with a defined absolute configuration on chiral phosphorus atoms, and / or at least one nucleoside residue of general formula 2, wherein B is a 5-modified uracil residue or a 6-modified adenine residue represented by 2 'or 2' ', where R2 is an alkylamine or amino acid residue, preferably a histamine or glycine residue, and / or at least one nucleoside residue of formula 3, wherein B1 is a thymine, uracil, cytosine, adenine and / or guanine residue and R1 is F or an alkoxy group -OR, where R is a group of the formula -CnH2n + 1, and n is a natural number from 1 to 5, preferably -OR is a methoxy group with said nucleoside residues in the position of one selected from the group consisting of N2, N7, N8, N11, N14 and N15.

The modified deoxyribozyme 10-23 of general formula 1 according to the invention comprises at least one internucleotide phosphorothioate bond of formula 4a and / or 4b, located at selected positions in the catalytic domain P1 to P15, especially in position P1, P7, P8, P14 and / or P15, preferably with a defined absolute configuration on chiral phosphorus atoms, and / or at least one nucleoside residue of formula 2a, 2b, 2c and / or 2d, and / or at least one nucleoside residue of formula 3a, 3b, 3c and / or 3d, in which R1 is a fluorine atom or an alkoxy group of the formula -OR, where R is a group of the formula -CnH2n + 1, and n is a natural number from 1 to 5, preferably -OR is a methoxy group, said nucleoside residues are on a position selected from the group consisting of N2, N7, N8, N11, N14 and N15 of a deoxyribozyme of general formula 1.

Deoxyribozymes with modifications being the subject of the present invention have a stabilized resistance to hydrolysis by cell nucleases, in which selected deoxyribozyme units with the structure of the catalytic domain of the known and previously selected deoxyribozyme 10-23 are obtained by chemical synthesis consisting in converting at least one nucleoside residue into of a specific position of the catalytic domain to another amino acid residue and / or at least one internucleotide linkage within the nucleobase to a stereophosphate linkage in a stereophosphate configuration or as mixtures of β-epimers.

The deoxyribozyme of the general formula I according to the invention is an oligonucleotide of 27 to 39 nt in length and a conserved 15-nt catalytic domain described in the literature as domain 10-23, with the proviso that at selected positions in the catalytic domain there are nucleobases of all nucleosides of the domain identical to the units contained in the originally selected deoxyribose 10-23 (Santoro and Joyce, 1997), except for nucleosides at selected positions containing the unit of general formula 2, preferably formula 2a, 2b, 2c or 2d, where B is a residue uracil modified at position 5 or adenine residue modified at position 6 of the nucleobase ring with R2 being a basic amino acid residue or an alkylamine residue, most preferably a residue selected from the group consisting of histidine or glycine.

Deoxyribzymes of formula 1 and units of formula 2, located at a selected position of the catalytic domain, most preferably at the 8-position, when B is a uracil residue having a histamine or alkyl-glycylamide substituent at the C-5 position of the nucleobase ring, exhibit favorable properties of gene expression inhibitors, that is, they are several times more active than the starting deoxyribozyme (unmodified), for reactions carried out in a single rotation with a 100-fold excess of deoxyribozyme in relation to the RNA substrate or its chimeric analog, containing only RNA dimer in the hydrolysis site, and the remaining fragments are built in residues 2'-deoxyribonucleotide, and in the presence of divalent metal ions, most preferably magnesium (II) ion used at millimolar concentrations.

Deoxyribozymes of formula 1 and units of formula 2 located in a selected position of the catalytic domain, most preferably in the 8-position, when B is a uracil residue having a histamine or alkylglycylamide substituent in the C-5 position of the nucleobase ring, show catalytic activity under single rotation conditions, i.e. 100-fold excess of the enzyme on the substrate and without the participation of divalent metal ions. According to the invention, it is most preferably in buffer

TRIS and PIPES pH greater than 7, used up to molar concentrations, most preferably 20 to 50 mM concentrations, for reactions ranging from 25 to 43 ° C, most preferably 37 ° C.

Deoxyribzymes of general formula I, in which there is a unit of general formula II, where the side chain is derived from an amino acid residue, most preferably according to the invention, such as histidine, glycine or other basic amino acids or alkylamines in the absence of divalent metal ions are only active in relation to certain substrates. Our research shows that these enzymes are specific only for the substrate having 5'-GpU-3 'dimer in the hydrolysis site, so hydrolysis takes place between the 5'-flanking guanosine unit and the 3'-flanking uridine unit. The substrate specificity of the deoxyribozymes 1 according to the invention is broadened relative to the substrate specificity of the unmodified deoxyribzyme.

A method for the preparation of deoxyribozymes with stabilized resistance to hydrolysis and increased catalytic activity, represented by the formula 1, containing in selected P positions of the catalytic domain, at least one internucleotide phosphorothioate bond of formula 4a or 4b, and / or in selected N positions, at least one nucleoside residue of general formula II, in which B is a uracil residue optionally modified at position 5 or an adenine residue modified at position 6, represented by formula 2 'or 2, where R2 is an alkylamine or amino acid residue, preferably a histamine or glycine residue, and / or at least one nucleoside residue of formula 3, wherein B1 is a residue of thymine, uracil, cytosine, adenine and / or guanine, and R 1 is F or an alkoxy group -OR, wherein R is a group of the formula -C _n H _2n + n represents a natural number from 1 to 5, preferably -OR represents a methoxy group, according to the invention, consists in the phosphorothioate bond, preferably stereo defined is introduced into an oligonucleotide by the use of a procedure that allows the synthesis of a stereodefined dinucleoside phosphorothioate, using stereocontrolled opening of the chiral 3'-nucleoside oxathiophospholate with the 5'-hydroxyl group of the second nucleoside component, followed by removal of the base-labile protecting group from the 3 'function -hydroxy dimer and protection of the phosphorothioate function with an alkylaryl group, activation of the 3'-hydroxyl group of the dinucleoside phosphorothioate is carried out with a phosphoramidite residue to obtain a monomer which is then used, in addition to phosphoramidites, canonical units and / or 2 and / or 3 modi fi ed units, for the synthesis of an oligonucleotide with the deoxyribozyme sequence 10-23 by using the standard phosphoramidite procedure, with extended condensation time and an increased excess of modified phosphoramidites compared to monomers of canonical units and the use of an appropriate procedure for removing the oligonucleotide from the fixed bed and deprotecting, including deprotecting from the phosphorothioate function, using a thiophenol-containing solvent system.

A method for the preparation of deoxyribozymes with stabilized resistance to hydrolysis and increased catalytic activity, represented by the formula 1, containing in selected P positions of the catalytic domain, at least one internucleotide phosphorothioate bond of formula 4a or 4b, and / or in selected N positions, at least one nucleoside residue of general formula II in which B is a 5-modified uracil residue or a 6-modified adenine residue represented by the formula 2 'or 2, wherein R2 is an alkylamine or amino acid residue, preferably a histamine or glycine residue, according to the invention, that the appropriately modified nucleoside units of the general formula 2 are prepared by the activation of the test function of adenine and condensation with an active reagent bearing in its structure an amino acid or alkylamine residue, or by activation of the 5-position of the thymine ring and subsequent attachment of a residue bearing an amino acid moiety w or an alkylamino group, and then incorporation of the unit modified in this way into the oligonucleotide chain using modified N units activated with a phosphoramidite function and units of the general formula 25 bearing the stereo-defined phosphorothioate functions of formula 4a or 4b.

For the preparation of deoxyribozymes of formula 1 containing units of formula 2 in a selected position of the catalytic domain, where B is a nucleobase residue modified with an amino acid residue group or other alkylamino group, the synthesis procedure according to the method described in the literature and based on the synthesis of oligodeoxyribonucleotides on the LCA CPG polymer support is used. using standard nucleotide monomers in the form of 3'-O- (2-cyanoethyl-N, N-diisopropylamido) phosphites 5'-DMTr protected nucleosides and 3'-O- (2-cyanoethyl-N, N-diisopropylamido) phosphite modified unit 2, obtained according to scheme I by reaction

The activation of the test function by means of an aryloxycarbonyl group, most preferably phenoxycarbonyl, when the nucleoside is 2'-deoxyadenosine, followed by replacement of the substituent with an amino acid residue according to the previously described procedure (RW Adamiak, E. Biała, K. Grześkowiak, R. Kierzek, A. Kraszewski, WT Markiewicz, J. Okupniak, J. Stawiński, M. Wiewiórowski 1978, Nucleic Acids Res. 5, 1889), or according to scheme II in the oxidation of the 5-methyl group when the rest of the nucleoside is thymidine, followed by the reaction reductive amination of 5-formyl-thymidine with primary amine reagents containing an amino acid substituent according to a previously developed procedure (E. Sochacka, D. Smuga 2007, Tetrahedron Lett., 48, 1363; E. Sochacka, D. Smuga, 2008, Nucleosides Nucleotides Nucleic Acids, 27, 1045). The obtained units 10 and 18, protected on the 5'-hydroxyl function with the 4,4'-dimethoxytrityl residue (DMTr), are subjected to the phosphitylation reaction with chloro-2-cyanoethyl-N, N-diisopropylphosphoramidite and in this form (as compounds 11 and 19) used for the synthesis of oligonucleotides with the deoxyribozyme sequence 10-23, incorporated in the strictly designed position of the catalytic domain. The synthesis of monomers of formula 2 is shown in Scheme I and Scheme II, and exemplary synthetic procedures are given in the Examples of the invention. The synthesis of oligonucleotides containing 2 units according to the invention is carried out as described in the publicly available literature (MH Caruthers, 1985, Science, 230, 281), selecting, according to the invention, the LCA CPG (succinyl, oxalyl or sarcosinyl) resin linker, respectively, the condensation time of the monomers (from 60 to 600 s) and appropriate conditions for the removal of protecting groups (for example 28% aqueous ammonia solution, time from 2 to 16 hours, temperature 25-55 ° C) and oligomer removal from the LCA CPG bed (for example 28% aqueous ammonia solution, time 30-120 minutes), as well as a two-step method of purifying the oligomer according to the method of Zona and Stec by reversed-phase high performance liquid chromatography (G. Zon, WJ Stec Phosphorothioate oligonucleotides. In: Oligonucleotides and Analogues; A practical Approach. Eckstein, F. ( editor. Oxford; IRL Press, 1991, pp. 87-108)).

The molecular weights of the oligonucleotides are confirmed by MALDI-TOF mass spectrometry and the purity is> 95% by polyacrylamide gel electrophoresis with the addition of 7M urea.

Deoxyribozymes of formula 1 containing units of formula 2 at a selected position of the catalytic domain, where B is a nucleobase residue modified with an amino acid residue group or other alkylamino group according to formula 2, are hybridized with a complementary oligonucleotide with a sequence homologous to the beta-secretase mRNA (BACE1 ), a protein involved in the pathogenesis of Alzheimer's disease (B. Nawrot, S. Antoszczyk, M. Maszewska, T. Kuwabara, M. Warashina, K. Taira, WJ Stec, 2003, Eur. J. Biochem. 270, 3962; B. Nawrot, 2004, Acta Biochim. Pol. 51 (2), 431; M. Sierant, K. Kubiak, J. Kazmierczak-Baranska, M. Warashina, T. Kuwabara, B. Nawrot, 2009, International Journal of Alzheimer's Disease Article ID 257403) and tested for their physico-chemical and biological properties.

The deoxyribozymes of general formula I can be designed as inhibitors of gene expression of selected molecular targets, such as mRNA of human proteins responsible for the pathogenesis of diseases, or as inhibitors of viral replication targeting viral RNA. Deoxyribozymes can also be designed as RNA nucleases for arbitrary sequences. In such cases, the sequence of the substrate recognition domains changes, while the catalytic part, i.e. the sequence of the domain 10-23 with modifications according to the invention, remains unchanged.

The invention also relates to deoxyribozymes of the general formula I, which according to the claim at selected positions in the catalytic domain contain the nucleic bases of all nucleosides of the catalytic domain identical to the units contained in the originally selected deoxyribzyme 10-23 (SW Santoro, GF Joyce 1997, Proc. Natl. Acad. Sci. USA 94, 4262), or all except the case where at selected positions of the invention there is a unit of general formula 3 in which the 2'-deoxyribose residue is replaced with a 2'-substituted ribose residue, according to the invention with a R1 substituent such as: F or O-alkyl, most preferably with an OMe group, which according to the invention have modifications of the internucleotide bonds at selected positions in the catalytic domain consisting in replacing one or more phosphate bonds with phosphorothioate bonds, i.e. one in which one of the non-bridging oxygen atoms is replaced with a sulfur atom, and the configuration of the chiral center is defined as R _p or S _p of formula 4 or deoxyribozyme is a mixture of P-epimers Rp + Sp (mix) at the selected chiral phosphorus atom, most preferably deoxyribozyme 1 contains a single phosphorothioate bond with _Rp configuration at P1 or two phosphorothioate bonds at P1 configuration R _p and in position P8 with configuration S _p , while all jed8

The nucleoside units of the catalytic domain are identical to the unmodified enzyme 10-23, or the deoxyribozymes of general formula I, which have one or more mix-configuration phosphorothioate bonds, most preferably two phosphorothioate bonds in the P1 and P8 positions or five phosphorothioate bonds in the positions P1, P7, P8, P14 and P15 and all nucleoside units of the catalytic domain identical to the unmodified enzyme 10-23, or some catalytic domain units replaced by units of general formula 3 in which the residue R1 2'-deoxyribose is replaced with the residue 2'- substituted ribose according to the invention the 2'-R1 substituents such as F or -OR where R is an alkyl C _n H _2n + ₁ and n = 1 to 5, more preferably the group OMe at positions N2, N7, N8, N11, N14 and N15.

Compounds of general formula I, which contain phosphorothioate bonds in their structure, have favorable biophysical and biological properties, including increased resistance to hydrolysis by cell nucleases and different hydrolytic activity in relation to complementary RNA fragments. In the most advantageous situation, deoxyribozyme 1, in which there is one phosphorothioate bond with the Rp configuration (IC50 = 0.008 pM), is more than 60 times more active as a catalyst for the hydrolysis of the phosphodiester bond in a complementary RNA substrate than its P-epimer, deoxyribozyme 1, in which has one phosphorothioate bond with S _p configuration (IC ₅₀ = 0.530 pM) and deoxyribozyme 1 containing two phosphorothioate bonds with Rp configuration in P1 position and Sp configuration in P8 position showing activity several times higher than other P-diastereomers and unmodified deoxyribozyme 10- 23.

Compounds of general formula I which have one or more phosphorothioate bonds in the mix configuration, most preferably two phosphorothioate bonds in positions P1 and P8 or five phosphorothioate bonds in positions P1, P7, P8, P14 and P15 and all nucleoside units of the catalytic domain identical to the unmodified enzyme 10-23, or some units replaced with units of general formula 3, in which the 2'-deoxyribose residue is replaced with a 2'-substituted ribose residue with a R1 group, most preferably a 2'-OMe group at positions N2, N7, N8, N11 , N14 and N15 of the catalytic domain and at the 3'- and 5'-end of the oligonucleotide have favorable biological properties, showing a noticeably higher stability to nuclease hydrolysis in the environment of a cell extract from HeLa cells (e.g., the half-life of deoxyribozyme 1 containing five phosphorothioate bonds at positions P1, P7, P8, P14 and P15 and units of general formula 3 in which the 2'-deoxyribose residue is replaced with the 2 'residue -substituted ribose with the R1 group, most preferably the 2'-OMe group at positions N2, N7, N8, N11, N14 and N15 is 4.6 h compared to 2.5 h for the unmodified enzyme) and increased in vitro efficiency in hydrolysis phosphodiester binding in a complementary radiolabeled RNA substrate with a sequence homologous to the mRNA of the BACE1 protein (B. Nawrot, K. Widera, M. Wojcik, B. Rebowska, G. Nowak, WJ. Stec, 2007, FEBS J., 274, 1062) (2 to 3 times higher efficiency defined as the ratio of stability to catalytic activity compared to the unmodified enzyme) and in the cell system (50% inhibition of BACE1 gene expression compared to only 20% activity of the unmodified enzyme used at a concentration of 50 nM for the experiment carried out in HeLa cells with overexpression of the BACE / GFP fusion protein gene, proceeding from the p-BACE / GFP plasmid (K. Sipa, E. Sochacka, J. Kazmierczak-Baranska, M. Maszewska, M. Janicka, G. Nowak, B. Nawrot, 2007, RNA 13 (8): 1301).

A method for the preparation of deoxyribozymes of formula I, stable to hydrolysis by cell nucleases, containing at least one internucleotide phosphorothioate bond of formula 4a or 4b, preferably with a defined absolute configuration on chiral phosphorus atoms, located in selected P positions of the catalytic domain, especially in positions P1, P7, P8, P14 and / or P15, and optionally at least one modified N nucleoside residue represented by general formula 3, wherein B1 is uracil, cytosine, adenine and / or guanine residue and R1 is fluoro or alkoxy, preferably methoxy, especially at the positions N2, N7, N8, N11, N14 and / or N15 of the catalytic domain, or at least one modified nucleoside residue N represented by formula 2, wherein B is an optionally substituted uracil or adenine residue, preferably represented by formula 2a , 2b, 2c and / or 2d, according to the invention, consists in introducing a stereo-defined phosphorothioate bond into the oligo nucleotide by using the procedure shown in Scheme III allowing the synthesis of the stereodefined dinucleoside-thiophosphate 22, preferably formed from 5'-O-DMTr and N-protected 2'-deoxyribonucleoside-3'-O- (2'-thio-spiro-pentamethylene-1 , 3,2-oxathiaphospholate) of formula 20, wherein B3 is a, b, c or d group and PG is an acyl protecting group routinely used in the synthesis of oligonucleotides by

PL 219 355 B1 phosphoramidite, most preferably benzoyl, isobutyryl, phenoxyacetyl (P. Guga, and WJ Stec, 2003. Current Protocols in Nucleic Acid Chemistry; editors: SL Beaucage, DE Bergstrom, GD Glick, RA Jones, pp. 4.17.1- 4.17.28 John Wiley & Sons, Hoboken: B. Nawrot, B. Rębowska, K. Cieślińska, WJ Stec. 2005, Tetrahedron Lett. 46, 6641; B. Nawrot, B. Rębowska, 2009; Current Protocols in Nucleic Acid Chemistry Chapter 4: Unit 4.34),) by opening the oxathiophospholate ring with a 3'-protected aryl residue, preferably an isopropoxyacetyl residue, a nucleoside of formula 21, in which B4 is a group of formula a, b, c or d, and PG is acyl protecting group with blocked test residues (for dA, dC and dG) in anhydrous acetonitrile in the presence of DBU (Scheme III). The resulting dimer of formula 22 is treated with a weak alkaline medium, preferably with an aqueous solution of ammonia (28%) in dioxane (1: 1) for 25-30 min to remove the isopropoxyacetyl protecting group from the 3'-hydroxyl function, followed by the compound with Formula 23 is reacted with an aralkyl halide, preferably 2-nitrobenzyl bromide, to effect an S-alkylation reaction. After activation of the 3'-hydroxyl group in the compound of formula 24 with 2-cyanoethyl bis- (N, N-diisopropyl) phosphoramidite in the presence of S-ethylthiotetrazole in anhydrous acetonitrile, the synthesized monomer of formula 25 is then used to synthesize the oligonucleotide of sequence deoxyribozyme 10-23 with a phosphorothioate linkage of formula 4a or 4b, by the use of a procedure typical for the synthesis of oligonucleotides by the phosphoramidite method (MH Caruthers, 1985, Science, 230, 281), but the phosphoramidite of formula 25 is fed in excess of relative to the monomers of the canonical units, the condensation reactions are carried out for an extended period of up to 600 s, and the synthesis of the oligonucleotide is performed leaving the 5'-terminal DMTr group.

The deblocking procedure must be safe to maintain the phosphorothioate functions in the oligonucleotide. Therefore, in the first step, the oligonucleotide is treated with a 1 M solution of piperidine in acetonitrile, during which the 2-cyanoethyl groups are removed from the phosphate residues and the evolving acrylonitrile is removed from the reaction mixture. According to this procedure, there is no post-reaction, i.e. Michael addition of acrylonitrile to the phosphorothioate group (S., Alefelder, B.K., Patel, and F. Eckstein, 1998. Nucleic Acid Res. 26: 4983-4988). In the next step, the oligonucleotide is treated for 5-7 min with a solution of dioxane, triethylamine and thiophenol (1/2/2, v / v / v) to remove the protecting group from the PS function, and then with 20% ethanolic ammonia solution to remove the groups. base-labile. After purification of the oligonucleotide by reverse-phase HPLC, the oligomer is treated with 50% acetic acid solution and, after concentration of the solution under reduced pressure, again subjected to RP-HPLC purification. The molecular weight of the oligonucleotides was confirmed by the MALDI-TOF method, and the purity by electrophoresis on a 20% polyacrylamide gel with the addition of 7M urea. The absolute configuration was confirmed enzymatically using snake venom phosphodiesterase (PDE I) and P1 3'-endonuclease (nP1) to hydrolyze the oligonucleotides in a known manner (P.MJ., Burgers, F., Eckstein, and D.HJ. Hunneman, 1979. J. Biol. Chem. 254: 7476-7478; BV Potter, and F. Eckstein, 1984. J. Biol. Chem. 259: 14243-14248; BV, Potter, BA, Connolly, and F. Eckstein, 1983, Biochemistry 22: 1369-1377). The hydrolysis products were analyzed by HPLC and compared with commercial standards.

A method for the preparation of deoxyribozymes of formula I, with increased catalytic activity, containing at least one internucleotide phosphorothioate bond of formula 4a or 4b, preferably with a defined absolute configuration on chiral phosphorus atoms, located in selected P positions of the catalytic domain, especially P1, P7 , P8, P14 and / or P15, and optionally at least one modified nucleoside residue N represented by formula 2, in particular by formula 2a, 2b, 2c and / or 2d, according to the invention, consists in preparing a suitably modified nucleoside unit by reaction activation of the test function of adenine and condensation with an active reagent bearing an amino acid or alkylamine residue in its structure, or by activating the 5-position of the thymine ring and subsequent attachment of a residue bearing an amino acid or alkylamino group, and then incorporation of such modified units into the oligonucleotide chain with the use ofphosphoramidite function activated N units and units of the general formula bearing the stereotyped phosphorothioate function of formula 4a or 4b.

For the production of compounds of general formula 1, the known and described method of synthesizing oligodeoxyribonucleotides on the LCA CPG polymer support and standard monomers in the form of 3'-O- (2-cyanoethyl-N, N-diisopropylamido) phosphites 5'-DMTr protected nucleosides and, in the case of the synthesis of stereo-defined analogs of phosphorothioate oligonucleotides is used

PL 219 355 B1 additionally prepared according to the known procedure of appropriately protected 3'-O- (2-cyanoethyl-N, N-diisopropylamido) dinucleoside-thiophosphate phosphite (B. Nawrot, B. Rębowska, K. Cieślińska, WJ Stec. 2005, Tetrahedron Lett. 46, 6641; B. Nawrot, B. Rębowska, 2009; Current Protocols in Nucleic Acid Chemistry Chapter 4: Unit 4.34), and in the synthesis of phosphorothioate oligonucleotides in the mix configuration instead of the oxidation of the phosphite function in a given internucleotide bond carried out routinely in the presence of The I2 / H2O / Py is sulfatized, in a known manner, with commercially available sulfatizing reagents (e.g. Beaucage's reagent). Removal of the oligonucleotides from the fixed bed and removal of all protecting groups are carried out in a known manner.

Embodiments of the invention are presented below.

P r z k ł a d I

Synthesis of 3 ', 5'-di-O-acetyl-N ⁶ -phenoxycarbonyl-2'-deoxyadenosine of formula 7 by the method using 1-phenoxycarbonylotetrazole (scheme I)

3 ', 5'-di-O-Acetyl-2'-deoxyadenosine (Formula 6) obtained according to the general recipe (W. Andersen, DH Hayes, AM Michelson, AR Todd 1954, J. Chem. Soc. (Resumed), 1882) (1.16 g, 3.5 mmol) was dissolved in anhydrous dioxane (35 mL) and reacted with 1-phenoxycarbonylotetrazole (2.0 g, 10.5 mmol) at 35 ° C. After 24 h, when TLC control indicated complete consumption of the starting material, the solvent was evaporated under reduced pressure and the crude product was purified by column chromatography over silica gel (eluent: 0-1% MeOH in CHCl ₃ v / v). The nucleoside of formula 7 was obtained as a white foam in 2nd Ed. 75% (1.20 g).

P r z x l a d II

Synthesis of 3 ', 5'-di-O-acetyl-N ⁶ -phenoxycarbonyl-2'-deoxyadenosine of formula 7 by method ₁ using 1-phenoxycarbonyl-N ^1- methylimidazolium chloride

3 ', 5'-di-O-Acetyl-2'-deoxyadenosine (6) (1.68 g, 5.0 mmol) was dissolved in anhydrous ₁

CH ₂ Cl ₂ (50 mL) was then added 1-phenoxycarbonyl-methylimidazolium chloride (3.6 g, 15.0 mmol). The suspension was stirred for 2 hours. at room temperature under argon atmosphere. After this time, the precipitate had partially dissolved and TLC monitoring of the reaction progress indicated conversion of the starting material. The precipitate was filtered off and washed with anhydrous CH ₂ Cl ₂ (2x5 mL). The filtrate was evaporated under reduced pressure and the crude product was purified by column chromatography on silica gel using ethyl acetate as an eluent. The nucleoside of formula 7 (1.71 g, 75% Ed) was obtained.

TLC Rf: 0.63 (CHCl3 / MeOH 90:10 v / v), 0.42 (AcOEt);

¹ H NMR (250 MHz, CDCl ₃ ) δ 2.07 (s, 3H, CH3COO), 2.14 (s, 3H, CH3COO), 2.67 (ddd, 1H, JH2 ', H3' = 2.5 Hz, JH2 ', H1' = 6.0 Hz, Jgem = 14.2 Hz, H2 '), 2.98 (ddd, 1H, JH2', H3 '= 6.5 Hz, JH2', H1 '= 7.7 Hz, Jgem = 14.2 Hz, H2''), 4.27 - 4.49 (m, 3H, H4 ', H5', H5 ''), 5.44 (m, 1H, H3 '), 6.49 (dd, 1H, JH1', H2 '= 6.1 Hz, JH1', H2 '' = 7.7 Hz , H1 '), 6.80-7.47 (m, 5H, PhO), 8.26 (s, 1H, H2), 8.81 (s, 1H, H8), 9.34 (bs, 1H, CONH);

P r x l a d III

Synthesis of 3 ', 5'-di-O-acetyl-N ⁶ - [(1S, 2R) -1 - {{2- (trimethylsilyl) ethoxy] carbonyl} -2 {[(tert-butyl) dimethylsilyl] oxy} propylaminocarbonyl ] -2'-deoxyadenosine of formula 8a

3 ', 5'-Di-O-acetyl-N ⁶ -phenoxycarbonyl-2'-deoxyadenosine (of formula 7) (650mg, 1.4mmol), after drying by evaporation twice with anhydrous pyridine (2x10mL), dissolved in the same solvent (10 mL) and then treated with O-tert-butyldimethylsilyl-L-threonine trimethylsilylethyl ester trifluoroacetate (1.25 g, 2.8 mmol). After 72 hours After stirring at 35 ° C, when the control of the reaction progress by TLC indicated complete conversion of the substrate, the solution was evaporated under reduced pressure, and then three times with anhydrous toluene (3x5 mL). The crude product was purified by column chromatography over silica gel (eluent: 0-2% MeOH in CHCl ₃ v / v). Nucleoside 8a was obtained as white foam (689 mg, 69% yield).

TLC Rf: 0.09 (CHCl3 / MeOH 98: 2 v / v), 0.40 (AcOEt / MeOH 98: 2 v / v);

m / z (MS, FAB) 695.4 [M + H] ⁺ , 693.4 [MH] ^- ; (694.3 calcd for C30H50Si2N6O9) m / z (HRMS, FAB) 695.3233 ([M + H] + C ^^ and ^ Og calcd 695.3256);

¹ H NMR (250 MHz, CDCl3) δ 0.03 (s, 9H, (CH3) 3Si), 0.04 (s, 3H, CH3Si), 0.09 (s, 3H, CH3Si),

0.94 (s, 9H, (CH3) ₃ CSi), 0.96 - 1.06 (m, 2H, SiCH ₂ CH ₂ ), 1.27 (d, 3H, J _C h _y , ch _p = 6.3 Hz, CH ₃ - _y ), 2.09 (s, 3H, CH3COO), 2.15 (s, 3H, CH3COO), 2.65 (ddd, 1H, JH2 ', H3' = 2.6 Hz, JH2 ', H1' = 6.0 Hz, Jgem = 14.2 Hz, H2 ') , 3.02 (ddd, 1H, JH2 '', H3 '= 6.3 Hz, JH2'',H1' = 7.9 Hz, Jgem = 14.0 Hz, H2 ''), 4.10 - 4.64 (m, 7H, CH ₂ CH ₂ O , H4 ', H5', H5 ', CH-a.CH-β), 5.45 (m, 1H, H3'), 6.45 (dd, 1H, Jhi'H2 '= 6.1 Hz, Jhi'H2' = 7.7 Hz, H1 '), 8.17 (s, 1H, H 2), 8.22 (bs, 1H, NHCO), 8.54 (s, 1H, H8), 10.04 (d, 1H, 1H, H 2 H 2);

PL 219 355 B1 ¹³ C NMR (63 MHz, CDCis) δ -5.19 (CH ₃ Si), -4.10 (CH ₃ Si), -1.44 ((CH ₃ ) ₃ Si), 17.45 ((CH ₃ ) ₃ CSi) ,

17.99 (SiCH ₂ CH ₂ ), 20.86 (CH3COO), 21.04 (CH3COO), 21.28 (Ογ), 25.72 <(CH3) 3CSi), 37.40 (C2 '),

59.70 (Ca), 63.77 (CH ₂ CH ₂ O), 63.86 (C5 '), 68.98 (Cp), 74.69 (C3'), 82.83 (C4 '), 84.92 (Cl'), 121.17 (C5), 141.84 ( C8), 150.77 (C4), 151.23 (NHCONH), 151.39 (C2), 154.76 (C6), 170.37 (CH3COO),

170.57 (CH3COO), 171.20 (COOCH2);

P r x l a d IV

Synthesis of N ⁶ - [(1S, 2R) -1 - {{2- (trimethylsilyl) ethoxy] carbonyl} -2 {[(tert-butyl) dimethylsilyl] oxy} propylaminocarbonyl] -2'-deoxyadenosine 9a

Blocked nucleoside 8a (556 mg, 0.8 mmol) was treated with NH ₃ saturated methanol (~ 10M, 17 mL) for 24 h. at room temperature. Then the solvent was evaporated under reduced pressure and the obtained crude product was purified by column chromatography over silica gel (eluent: 0-10% MeOH in CHCl ₃ v / v). Deacetylated nucleoside 9a was obtained as a white foam (366 mg, 75% yield).

TLC R _f : 0.34 (CHCl 2 / MeOH 90:10 v / v), 0.36 (AcOEt / MeOH 90:10 v / v);

MS Cl m / z: 611.3 [M + H] + (calc. 611.30 for C 19 Y 4 Oy Sh);

¹ H NMR (250 MHz, CDCl3) δ 0.00 (s, 9H, (CH3) 3Si), 0.01 (s, 3H, CH3Si), 0.07 (s, 3H, CH3Si), 0.90 (s, 9H, (C ^^ CSi), 0.92-1.02 (m, 2H, SiCH ₂ CH ₂ ), 1.25 (d, 3H, Jch ^ ch _p = 6.2 Hz, CHs-γ), 2.37 (m, 1H, H2 '), 3.02 (m, 1H, H2 "), 3.80-4.56 (m, 7H, CH ₂ CH ₂ O, H4 ', H5', H5", CH-a, CH-p), 4.79 (m, 1H, H3 '), 6.44 (dd, 1H, JH1 ', H2' = 5.6 Hz, JH1, H2 '= 9.0 Hz, H1'), 8.17 (s, 1H, H2), 8.47 (s, 1H, H8), 8.64 (bs, 1H , NHCO), 10.00 (d, 1h, JNHa, CHa = 9.1 Hz, NH-α);

P r z k ł a d V

Synthesis of 3 ', 5'-di-O-acetyl-N ⁶ - {2- [N ¹ - (4-methoxytrityl) imidazol-4-yl] ethylaminocarbonyl} -2'-deoxyadenosine of formula 8b

3 ', 5'-Di-O-acetyl-W ⁶ -phenoxycarbonyl-2'-deoxyadenosine (7) (271 mg, 0.60 mmol) was dried by evaporation twice with anhydrous pyridine (2x5 mL) and dissolved in the same solvent (4 mL). N ^m - (4-methoxytrityl) histamine (596 mg, 1.20 mmol, 2 eq.), Also previously dried by evaporation twice with anhydrous pyridine (2x5 mL), was then added. After 24 hours After stirring at 35 ° C, control of the reaction progress by TLC indicated the substrate had reacted. The reaction mixture was evaporated under reduced pressure then three times with toluene (3 x 5 mL) to remove traces of pyridine. The crude product was purified by column chromatography over silica gel (eluent: 0-5% MeOH in CHCl ₃ v / v). Nucleoside 8b was obtained as a cream foam (352 mg, 78% Ed).

TLC Rf: 0.44 (CHCl3 / MeOH 90:10 v / v), 0.28 (AcOEt / MeOH 90:10 v / v); m / z (MS, FAB) 745.3 [M + H] ⁺ , 743.1 [MH] ^- (C40H40N8O7 calcd 744.3)

HRMS FAB m / z: 745.3078 ([M + H] ⁺ C40H41N8O7 calc. 745.3098);

¹ H NMR (250 MHz, CDCl3) δ 2.08 (s, 3H, CH3COO), 2.14 (s, 3H, CH3COO), 2.63 (ddd, 1H, JH2 ', H3' = 2.5 Hz, JH2 ', H1' = 6.0 Hz, Jgem = 14.2 Hz, H2 '), 2.90 (t, 2H, J = 6.7 Hz, CH2-p), 3.02 (ddd, 1H, Jh2''H3' = 6.3 Hz, Jh2 ', h1' = 8.0 Hz, J ₉ em = 14.2 Hz, H2 ''), 3.66 - 3.76 (m, 2H, CH ₂ -a), 3.77 (s, 3H, OCH3), 4.31 - 4.47 (m, 3H, H4 ', H5' , H5 ''), 5.46 (dt, 1H, JH3 ', H2' = JH3 ', H4' = 2.3 Hz, JH3 ', H2''= 6.1 Hz, H3'), 6.46 (dd, 1H, JH1 ', H2 '= 5.9 Hz, JH1', H2 '' = 8.0 Hz, H1 '), 6.66 (d, 1H, ⁴ JH5, H2 = 1.4 Hz, Im-CH-5), 6.73 - 7.35 (m, 14H, MMTr ), 7.39 (d, 1 H, ⁴ JH2, H5 = 1.4 Hz, Im-CH-2), 8.27 (s, 1H, H2), 8.31 (bs, 1H, NHCO), 8.34 (s, 1H, H8), 9.56 (t, 1H, JnH <&gt; Ch &lt; x = 5.6Hz, NH-α);

¹³ C NMR (63 MHz, CDCl3) δ 20.97 (CH3COO), 21.13 (CH3COO), 28.89 (Cp), 37.57 (C2 '), 40.17 (Ca), 55.44 (OCH3), 63.91 (C5'), 74.71 (C3 '), 75.00 (MMTr-CPh3), 82.90 (C4'), 84.92 (Cl '), 113.38 (MMTr-o'), 118.92 (Im-CH-5), 121.02 (C5), 128.13 (MMTR-o, p), 129.82 (MMTr-m), 131.29 (MMTr-m '), 134.69 (Im-CH-4), 138.64 (Im-CH-2), 138.75 (MMTr-i'), 141.22 (C8), 142.93 (MMTr-i), 150.13 (C4), 150.64 (NHCONH), 151.43 (C2), 154.01 (C6), 159.23 (MMTr-p '), 170.47 (CH3COO), 170.62 (CH3COO);

P r x l a d VI

Synthesis of N ⁶ - {2- [N ¹ - (4-methoxytrityl) imidazol-4-yl] ethylaminocarbonyl} -2'-deoxyadenosine of formula 9b

The acetylated nucleoside 8b (335 mg, 0.45 mmol) was dissolved in NH ₃ saturated MeOH (~ 10M, 9.5 mL). After 24 hours at room temperature, control of the reaction progress (TLC) showed the substrate had reacted. After evaporating the solvent under reduced pressure, the crude product was purified by column chromatography over silica gel (eluent: 0-15% MeOH in CHCl ₃ v / v). Nucleoside 9b was obtained as a cream foam (230 mg, 78% Ed).

PL 219 355 B1

TLC Rf: 0.45 (CHCl3 / MeOH 85:15 v / v), 0.25 (AcOEt / MeOH 80:20 v / v);

MS FAB m / z: 663.5 [M + 3H] ⁺ (calc. 663.30 for C36H39N8O5);

¹ H NMR (250 MHz, CDCl ₃ ) δ 2.42 (ddd, 1H, Jh2 ', h3' = 1.9 Hz, Jh2 ', hi' = 5.7 Hz, Jgem = 13.5 Hz,

H2 '), 2.91 (ddd, 1H, JH2' ', H3' = 5.2 Hz, JH2 '', H1 '= 7.7 Hz, Jgem = 13.5 Hz, H2' '), 3.05 (t, 2H, J = 6.5 Hz ,

CH2-P), 3.77 (s, 3H, OCH 3), 3.78 - 4.03 (m, 4H, CH ₂ s, H5 '. H5'). 4.19 (m, 1H. H4 '), 4.84 (m. 1H. H3'),

6.42 (dd, 1H, JH1 ', H2' = 5.9 Hz, JH1 ', H2''= 7.7 Hz, H1'), 6.75 (d, 1H, ⁴ JH5, H2 = 0.7 Hz, Im-CH-5), 6.76 - 7.38 (m, 15H, MMTr, Im-CH-2), 8.32 (s, 1H, H2), 8.40 (s, 1H, H8), 8.55 (bs, 1H, NHCO), 9.58 (t, 1H, dNHa, CHa = 5.5 Hz, NH- a);

Example VII ₁

Synthesis of 3 ', 5'-di-O-acetyl-5- {2- [N ¹ - (4-metoksytrytylo) imidazole-4-yl] aminomethyl} -2'-deoxyuridine of formula 15 ^a (Scheme II)

3 ', 5'-Di-O-acetyl-5-formyl-2'-deoxyuridine (14) (340 mg, 1.0 mmol) was dried by evaporation with anhydrous CH ₂ Cl ₂ (2x10 mL), then the nucleoside was dissolved in the same solvent (7 mL). ^{W m} -4-methoxytrityl) histamine (596 mg, 1.2 mmol), Et ₃ N (167 μί, 1.2 mmol) and NaBH (OAc) ₃ (254 mg, 1.2 mmol) were added sequentially. After 2 hours After stirring at room temperature, when the TLC control performed showed the presence of a small amount of the starting nucleoside, another portion of NaBH (OAc) ₃ (106 mg, 0.50 mmol) was added. After another 2 hours, when the substrate was completely consumed (TLC), CH ₂ Cl ₂ (20 mL) was added to the reaction mixture and washed with a 5% aqueous NaHCO ₃ solution (10 mL). The aqueous layer was then extracted 3 × CH ₂ Cl ₂ (20 mL) and the combined organic fractions were dried with MgSO ₄ . After the drying agent was filtered off and the solvent was evaporated, two yellow foam products were isolated from the crude mixture by silica gel column chromatography (eluent: 0-25% MeOH in CHCl ₃ ): Formula 15a (390 mg, 55% Ed.) and formula 16a as a mixture of Z and E isomers (290 mg, 41% Ed)

Data for 15a

TLC Rf: 0.32 (CHCl3 / MeOH 90:10 v / v), 0.11 (AcOEt / MeOH 90:10 v / v);

HRMS FAB m / z. 708.3052 ([M + H] + C3 _{8 H 4 2} N ₅ O 8 calcd. 708.3033);

UV (CHCl ₃ ): X _max = 266.0 nm (ε = 7 700 M ^-1 cm ^-1 ), X _min = 251.5 nm;

¹ H NMR (250 MHz, CDCl) δ 2.10 (s, 3H, CH3COO), 2.11 (s, 3H, CH3COO), 2.30 (ddd, 1H, JH2 'hs' = 6.4 Hz, JH2', hi · = 8.4 Hz, Jgem = 14.5 Hz, H2 '), 2.43 (ddd, 1H, Jh2 ", h3' = 1.8 Hz, Jh2", hi · = 5.7 Hz, Jgem = 14.3 Hz, H2 "), 2.80 (t, 2H , J = 7.0 Hz, CH ₂ -3), 2.99 (t, 2H, J = 7.0 Hz, CH ₂ -a), 3.57 (d, 1H, Jgem = 13.7 Hz, one of CH ₂ -7), 3.66 ( d, 1H, Jgem = 13.8 Hz, one of CH ₂ -7), 3.81 (s, 3H, OCH3), 4.21 (m, 1H, H4 '), 4.28 (dd, 1H, Jh ₅ -, h4' = 3.5 Hz, Jgem = 12.0 Hz, H5 ''), 4.39 (dd, 1H, Jh5 ', h4' = 4.7 Hz, Jgem = 12.0 Hz, H5 '), 5.22 (dt, 1H, Jh3', h2 '' = Jh3 ', h4' = 2.0 Hz, Jh3 ', h2' = 6.3 Hz, H3 '), 5.77 Hz, Jhi', h2 '= 8.6 Hz, HI'), 6.59 (d, 1H, ⁴ Jh5, h2 = 1.2 Hz , Im-CH-5), 6.77-7.37 (m, 14H, MMTr), 7.38 (d, 1H, ⁴ Jh2, h5 = 1.4Hz, Im-CH-2), 7.66 (s, 1H, H6);

¹³ C NMR (63 MHz, CDCis), δ 21.08 (2xCH3COO), 27.56 (Οβ). 37.34 (C2 '), 45.52 (C7), 48.85 (Ca), 55.47 (OCH3), 64.03 (C5'), 74.58 (C3 '), 7505 (MMTr-CPh3), 82.52 (C4'), 85.30 (Cl ' ), 111.73 (C5), 113.45 (MMTr-o '), 118.74 (Im-CH-5), 128.19 (MMTr-o, p), 129.82 (MMTr-m), 131.30 (MMTr-m'), 134.64 ( Im-CH-4), 138.13 (MMTr-I '), 138.64 (C6), 138.71 (Im-CH-2), 142.88 (MMTr-I), 150.43 (C2), 159.25 (MMTr-p'), 163.47 (C4), 170.51 (CH3COO), 170.65 (CH3COO);

Data for the mixture of isomers 16a (Z and E)

TLC Rf: 0.48, 0.63 (CHCl2 / MeOH 90:10 v / v), 0.23, 0.41 (AcOEt / MeOH 90:10 v / v);

HRMS FAB m / z: 708.3048 ([M + H] + C39H42N5O8 calc. 708.3033);

UV (CHCl ₃ ): X _max = 305.0 nm (ε = 12,600 M ^-1 cm ^-1 ): X _min = 258.5 nm;

¹ H NMR (250 MHz, CDCh) δ 2.08 (s, 3H, CH3COO from Z), 2.09 (s, 3H, CH3COO from Z), 2.03 2.19 (m, 10H, 2xCH3COO from E, H2 '. H2 "from Z and E), 2.77 (t, 2H, J = 6.7 Hz, CH ^ -β from Z), 2.79 - 2.88 (m, 2H, CHz-β from E), 3.48 - 3.60 (m, 4H, CH2-a from Z and E), 3.81 (s, 3H, OCH3 from Z), 3.82 (s, 3H, OCH3 from E), 3.68 - 3.98 (m, 4H, CH2-6 from Z and E), 3.99 - 4.39 (m, 6H, H4 '. H5'. H5 "from Z and E), 5.06 - 5.19 (m, 3H, H3 'from Z and E, NH-a from E), 6.30 (dd, 1H, Jhi', h2 '' = 5.9 Hz, Jhi ', h2' = 8.9 Hz, HI 'from Z), 6.41 (dd, 1H, Jhi', h2 '' = 5.9 Hz, Jhi ', h2' = 8.8 Hz, HI 'from E), 6.60 (d, 1H, ⁴ Jh5, h2 = 1.0 Hz, Im-CH-5 from Z), 6.61 (d, 1H, ⁴ JH5, H2 = 1.0 Hz, Im-CH-5 from E), 6.70 (d, 1H, JH7, NHa = 13.1 Hz, H7 from Z), 6.79 - 7.41 (m, 31H, MMTr, NH-3 from Z and E, Im-CH-2 from E), 7.43 (d, 1H, ⁴ Jh2, h5 = 1.3 Hz, Im-CH-2 from Z), 7.46 (m, 1H, H7 from E),

8.43 (m, 1H, NH- a from Z);

¹³ C NMR (63 MHz, CDCh) δ 21.00 (CH3COO from E and Z), 21.06 (CH3COO from E and Z), 28.90 ^ β from E), 30.09 ^ β from Z), 32.69 (C2 'from E), 33.30 (C2 'from Z), 36.84 (C6 from E), 40.32 (C6 from Z), 48.75 (Ca from Z), 49.34 (Ca from Z), 49.34 (Ca from E), 55.42 (OCH3 from Z and E ), 63.94 (C5 'from E), 64.16 (C5' from Z),

PL 219 355 B1

74.34 (C3 'from E), 74.46 (C3' from Z), 75.10 (MMTr-CPh ₃ from Z), 75.22 (MMTr-CPh ₃ from E), 80.44 (C4 'from Z), 80.66 (C4' from E ), 84.20 (C1 'from Z), 84.59 (C1' from E), 85.00 (C5 from Z), 87.43 (C5 from Z), 113.44 (MMTr-o 'from Z and E), 118.91 (Im-CH- 5 from E), 119.60 (Im-CH-5 from Z), 128.17 (MMTr-o from Z and E, MMTr-p from Z), 128.44 (MMTr-p from E), 129.74 (MMTr-m from Z and E), 131.23 131.30 (MMTr-m 'from Z and E), 134.31 (Im-CH-4 from E), 134.43 (Im-CH-4 from Z), 137.18 (MMTr-i' from Z), 137.75 ( MMTr-i 'from E), 138.57 (Im-CH-2 from E), 138.83 (Im-CH-2 from Z), 142.61 (MMTr-i from E), 142.70 (MMTr-i from Z), 146.72 ( C7 from E), 150.15 (C7 from Z), 153.09 (C2 from E), 153.81 (C2 from Z), 159.25 (MMTr-p 'from Z and E), 164.91 (C4 from E), 166.71 (C4 from Z) ), 170.63 (CH3COO from Z and E), 171.09 (CH3COO from Z and E);

Example VIII ₁

Synthesis of 5- {2- [N ¹ - (4-methoxytrityl) imidazol-4-yl] ethylaminomethyl} -2'-deoxyuridine of formula 17a

Acetylated nucleoside 15a (354 mg, 0.50 mmol) was treated with a solution of Et ₃ N in MeOH (1: 9 v / v, 4 ml) at room temperature for 24 h. After this time, control of the reaction progress (TLC) showed complete conversion of the starting material. Toluene (4 mL) was added to the reaction mixture and the solvents were evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: 0-15% MeOH in CHCl ₃ ) to give 17a as a yellow foam (224 mg, 72% Ed).

TLC Rf: 0.24 (CHCl3 / MeOH 85:15 v / v), 0.05 (AcOEt / MeOH 80:20 v / v);

MS FAB m / z: 624.3 [M + H] ⁺ (calc. 624.27 for C35H38N5O6);

¹ H NMR (250 MHz, CDCfe) δ 2.24 (m, 1H, H2 '), 2.43 (ddd, 1H, JH2', H3 '= 1.4 Hz, JH2', h1 = 5.5 Hz, J _g em = 14.0 Hz, H2 "), 2.75 (t, 2H, J = 6.7 Hz, CH ₂ -3), 2.93 (t, 2H, J = 6.8 Hz, CH ₂ -a), 3.52 '(d, 1H, Jgem = 14.1 Hz , one of CH2-7), 3.60 (d, 1H, Jgem = 14.1 Hz, one of CH2-7), 3.81 (s, 3H, OCH3), 4.19 - 4.32 (m, 2H, H4 ', H5'') , 4.37 (dd, 1H, JH5 ', H4' = 4.4 Hz, Jgem = 12.0 Hz, H5 '), 5.21 (dt, 1H, JH3', H2 '' = JH3 ', H4' = 2.0 Hz, JH3 ', H2 '= 6.5 Hz, H3'), 6.30 (dd, 1H, JH1 ', H2' = 5.6 Hz, JH1 ', H2' = 8.7 Hz, H1 '), 6.58 (d, 1H, ⁴ JH5, H2 = 1.2 Hz, Im-CH-5), 6.77-7.34 (m, 14H, MMTr), 7.35 (d, 1H, ⁴ JH5, H2 = 1.2 Hz, Im-CH-2), 7.56 (s, 1H, H6);

P r x l a d IX

Synthesis of 3 ', 5'-di-O-acetyl-5-methoxycarbonylaminomethyl-2'-deoxyuridine 15b by direct reductive amination of 3', 5'-di-O-acetyl-5-formyl-2'-deoxyuridine with ester hydrochloride glycine methyl

3 ', 5'-Di-O-acetyl-5-formyl-2'-deoxyuridine (of formula 14) (340 mg, 1.0 mmol) was dried by evaporation with anhydrous CH2Cl2 (2x10 mL), then the nucleoside was dissolved in the same solvent (7 mL). Glycine methyl ester hydrochloride (180 mg, 1.5 mmol), Et ₃ N (210 gL, 1.5 mmol) and NaBH (OAc) ₃ (254 mg, 1.2 mmol) were added sequentially. After 2 hours After stirring at room temperature, when TLC control (CHCl3 / MeOH 90:10 v / v) showed the presence of a small amount of starting nucleoside, another portion of NaBH (OAc) ₃ (106 mg, 0.50 mmol) was added. After another 2 hours It said complete consumption of starting material (TLC) to terminate the reaction and the reaction mixture was added 5% aqueous solution of NaHCO3 (10 mL) and the aqueous layer was extracted with 3 x CH ₂ Cl ₂ (20 mL). The combined organic fractions were dried with MgSO ₄ . After the drying agent was filtered off and the solvent was evaporated, two products were isolated from the crude mixture by column chromatography on silica gel (eluent; 0-5% MeOH in CHCl ₃ ): 15b (339 mg, 82% Ed) and 16b as mixture of E and Z isomers (33 mg, 8% Ed).

Data for 15b

TLC R _f : 0.45 (CHCl 2 / MeOH 90:10 v / v), 0.22 (AcOEt / MeOH 95: 5 v / v); m / z: (MS, FAB) 414.2 [M + H] ⁺ , 412.2 [MH] (calc. for C17H23N3O9 413.1) m / z: (HRMS, FAB) 414.1513 (calc. [M + H] ⁺ C17H24N3O7 414.1507);

¹ H NMR (250 MHz, CDCl3) δ 2.12 (s, 3H, CH3COO), 2.14 (s, 3H, CH3COO), 2.20 (m, 1H, H2 '), 2.47 (ddd, 1H, J ..3 .. = 1.6 Hz, Jh-, h1 = 5.5 Hz, Jgem = 14.2 Hz, H2 "), 3.42 (s, 2H, CH ₂ -a), 3.56 (s, 2H, CH2-7), 3.72 (s, 3H, OCH3), 4.22 - 4.35 (m, 2H, H4 ', H5''), 4.40 (dd, 1H, JH5', H4 '= 4.4 Hz, Jgem = 12.1 Hz, H5'), 5.23 (dt, 1H , JH3 ', H2''=JH3', H4 '= 1.9 Hz, JH3', H2 '= 6.6 Hz, H3'), 6.34 (dd, 1H, JH1 ', H2''= 5.5 Hz, JH1', H2 '= 8.8 Hz, H1'), 7.53 (s, 1H, H6);

¹³ C NMR (63 MHz, CDCl3), δ 20.60 (CH3COO), 20.72 (CH3COO), 37.12 (C2 '), 45.27 (C7), 49.67 (Ca), 51.66 (OCH3), 63.80 (C5'), 74.29 ( C3 '), 82.09 (C4'), 84.88 (C1 '), 112.77 (C5), 136.52 (C6), 150.41 (C2), 163.43 (C4), 170.25 (CH3COO), 172.27 (COOCH3);

Data for 16b

PL 219 355 B1

TLC Rf: 0.50, 0.60 (CHCl3 / MeOH 90:10 v / v), 0.32, 0.50 (AcOEt / MeOH 95: 5 v / v); m / z (MS FAB) 414.3 [M + H] ^+, 412.2 [M-H] ^- (calcd for C17H23N3O9 413.14) ¹ H NMR (250 MHz, CDCfe) δ 2.07 - 2.22 (m, 16H, 2 xCH ₃ COO, H2 ', H2''from Z and E), 3.63 - 3.88 (m, 2H, one of CH2-6 from Z and E), 3.77 (s, 3H, OCH3 from E), 3.78 (s, 3H, OCH3 from Z), 3.90 - 4.40 (m, 11H, one from CH ₂ -6, CH ₂ -a, H4 'from Z and E, H5', H5 'from Z, H5' from E), 4.47 (dd, 1H, Jh5w = 7.0 Hz, Jgem = 11.1 Hz, H5 'from E), 5.06 - 5.19 (m, 2H, H3' from Z and E), 5.61 (m, 1H, NH from E), 6.31 (dd , 1H, Jhi ', h2 ”= 6.1 Hz, Jhi', h2 · = 8.6 Hz, H1 'from Z), 6.40 (m, 1H, H1' from E), 6.65 (d, 1H, Jh7, nho, = 12.8 Hz, H7 from Z), 7.38 (dt, 1H, ⁴ Jh7, h6 = 1.4 Hz, J .. = 14.4 Hz, H7 from E), 7.45 (s, 1H, NH-3 from Z), 7.47 (s , 1H, NH-3 from E), 8.48 (M, 1H, NH- a from Z);

¹³ C NMR (63 MHz, CDCl3) δ 21.04 (CH3COO from Z and E), 21.10 (CH3COO from Z and E), 33.40 (C2 'from Z and E), 36.35 (C6 from E), 40.25 (C6 from Z ), 49.40 (Ca from Z), 49.50 (Ca from E), 52.52 (OCH3 from E), 52.68 (OCH3 from Z), 64.12 (C5 'from E), 64.17 (C5' from Z), 74.37 (C3 ' from Z), 74.42 (C3 'from E), 80.50 (C4' from Z), 81.18 (C4 'from E), 84.26 (C1' from Z), 84.54 (C1 'from E), 88.19 (C5 from Z) , 90.52 (C5 from E), 145.68 (C7 from E), 149.18 (C7 from Z), 152.60 (C2 from E), 153.37 (C2 from Z), 164.88 (C4 from E), 166.93 (C4 from Z), 170.70 (CH3COO from Z and E), 170.77 (CH3COO from Z and E), 171.81 (COOCH3 from Z and E);

P r z k ł a d X

Synthesis of 5-methoxycarbonylaminomethyl-2'-deoxyuridine of formula 17b

Nucleoside 11b (320 mg, 0.77 mmol) was treated with a solution of Et ₃ N in MeOH (1: 9 v / v, 6 ml) at room temperature for 24 h. After this time, control of the reaction progress (TLC) showed complete consumption of the starting material. Toluene (6 mL) was added to the reaction mixture and the solvents were evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (eluent: 0-20% MeOH in CHCl ₃ ) to give 17b as a white foam (167 mg, 66% Ed).

TLC Rf: 0.21 (CHCl3 / MeOH 85:15 v / v), 0.13 (AcOEt / MeOH 85:15 v / v);

MS FAB m / z: 330.2 [M + H] ⁺ (calc. 330.12 for C13H20N3O7);

¹ H NMR (250 MHz, D2O) δ 2.28 - 2.48 (m, 2H, H2 ', H2''), 3.50 (s, 2H, CH2-a), 3.57 (s, 2H, CH2-7), 3.73 ( s, 3H, OCH3), 3.76 (m, 1H, H5 ''), 3.85 (dd, 1H, JH5 ', H4' = 3.2 Hz, Jgem = 12.5 Hz, H5 '), 4.04 (m, 1H, H4' ), 4.47 (m, 1H, H3 '), 6.27 (t, 1H, JH1', H2 '' = 6.6 Hz, H1 '), 7.86 (s, 1H, H6);

P r z x l a d XI

General Nucleoside 5'-O-dimethoxytritylation Procedure

The starting nucleoside (0.35 mmol) was evaporated twice with anhydrous pyridine (2x4 mL) to remove traces of water, then dissolved in the same solvent (4 mL) and 4,4'-dimethoxytrityl chloride (124 mg, 0.39 mmol) was added ). The reaction mixture was stirred for 16 h. at room temperature. After this time, when TLC control indicated the presence of a small amount of starting nucleoside, another portion of 4,4'-dimethoxytrityl chloride (11 mg, 0.035 mmol) was added. After another 4 hours, when the substrate was completely consumed (TLC), water (7 mL) and CHCl ₃ (15 mL) were added to the reaction mixture, and then the aqueous fraction was extracted three times with CHCl ₃ (3 × 15 mL) and the combined organic fractions were dried. with MgSO ₄ . After filtering off the drying agent and evaporating the solvent, the crude product was evaporated three times with anhydrous toluene (3x10 mL) and then purified by silica gel column chromatography (eluent: 0-10% MeOH in CHCl ₃ ) to give the corresponding 5'-O-dimethoxitrile light yellow foam derivatives.

5'-O- (4,4'-dimethoxytrityl) -N6 - [(1S, 2R) -1 - {{2-trimethylsilyl) ethoxy] carbonyl} -2 - {[(tert-butyl) dimethylsilyl} propylaminocarbonyl] - 2'-deoxyadenosine 10a

225 mg, 71% yield; TLC Rf: 0.64 (CHCl3 / MeOH 90:10 v / v), 0.54 (AcOEt / MeOH 95: 5 v / v);

¹ H NMR (250 MHz, CDCl3) δ 0.03 (s, 9H, CH3) 3Si), 0.05 (s, 3H, CH3Si), 0.10 (s, 3H, CH3Si), 0.95 (s, 9H, (CH3) 3CSi) , 1.01 (t, 2H, J = 8.5 Hz, SiCH ₂ CH ₂ ), 1.28 (d, 3H, J _C h _y , ch _p = 6.2, CHs-γ), 2.57 (m, 1H, H2 '), 2.85 (m, 1H, H2 ''), 3.35 (dd, 1H, JH5 '', H4 '= 3.3Hz, Jgem = 10.4Hz, H5''), 3.42 (dd, 1H, JH5', H4 '=

5.3 Hz, Jgem = 10.3 Hz, H5 '), 3.79 (s, 6H, 2xOCH3), 4.12 - 4.62 (m, 5H, CH ₂ CH ₂ O, CH-β, CH-a, H4'), 4.69 (m , 1H, H3 '), 6.46 (t, 1H, JH1', H2 '= JH1', H2 '' = 6.5 Hz, H1 '), 6.79-7.41 (m, 13H, DMTr), 7.96 (bs, 1H, NHCO), 8.07 (s, 1H, H 2), 8.48 (s, 1H, H8), 10.03 (d, 1H, JNHa, CHa = 9.4Hz, NH- a);

5'-O- (4,4'-dimethoxytrityl) -N ⁶ - {2- [N1- (4-methoxytrityl) imidazol-4-yl] ethylaminocarbonyl} -2'-deoxyadenosine of formula 10b

212 mg, 63% yield; TLC Rf: 0.45 (CHCl3 / MeOH 90:10 v / v), 0.41 (AcOEt / MeOH 85:15 v / v);

¹ H NMR (250 MHz, CDCfe) δ 2.56 (m, 1H, H2 '), 2.91 (m, 1H, H2''), 3.05 (t, 2H, J = 6.5 Hz, CH ₂ -3), 3.40 - 3.43 (m, 2H, CH2-a), 3.76 (s, 9H, 3xOCH3), 3.64 - 3.84 (m, 2H, H5 ', H5''), 4.13 (m, 1H, H4'),

PL 219 355 B1

4.72 (m, 1H, H3 '), 6.47 (m, 1H, H1'), 6.75 (s, 1H, Im-CH-5), 6.74 - 7.40 (m, 28H, DMTr, MMTr, Im-CH-2 ),

7.91 (bs, 1H, NHCO), 8.10 (s, 1H, H 2), 8.28 (s, 1H, H8), 9.50 (t, 1H, 1H, NH 4a = 5.7Hz, NH?);

₁

5'-O- (4,4'-dimethoxytrityl) -5- {2- [N ¹ - (4-methoxytrityl) imidazol-4-yl] ethylaminomethyl} -2'-deoxyuridine of formula 18a

197 mg, 61% yield; TLC Rf: 0.53 (cHCl3 / MeOH 85:10 v / v), 0.32 (AcOEt / MeOH 80:20 v / v);

¹ H NMR (250 MHz, CDCis) δ 2.19 - 2.42 (m, 2H, H2 ', H2 "), 2.48 - 2.67 (m, 4H, C6 -β, CH2-a), 3.05 (d, 1H, Jgem = 14.0 Hz, one of cH2-7), 3.11 (d, 1H, Jgem = 14.0 Hz, one of cH2-7), 3.35 (dd, 1H, JH5 '', H4 '= 3.8 Hz, Jgem = 10.6 Hz) , 3.42 (dd, 1H, JH5 ', H4' = 3.8 Hz, Jgem = 10.7 Hz), 3.74 (s, 6H, 2xOCH3), 3.80 (s, 3H, OCH3), 3.99 (q, 1H, JH4 ', H5 '= JH4', H5 '' = 3.5 Hz, H4 '), 4.50 (m, 1H, H3'), 6.31 (t, 1H, JH1 ', H2''=JH1', H2 '= 6.5 Hz, H1' ), 6.52 (d, 1H, ⁴ JH5, H2 = 1.0 Hz, Im-CH-5), 6.75-7.43 (m, 28H, DMTr, MMTr, ImCH-2), 7.57 (s, 1H, H6);

5'-O- (4,4'-dimethoxytrityl) -5-methoxycarbonylaminomethyl-2'-deoxyuridine of the formula

18b

150 mg, 68% yield; TLC Rf: 0.32 (CHCl3 / MeOH 90:10 v / v), 0.22 (AcOEt / MeOH 95: 5 v / v);

¹ H NMR (250 MHz, CDCl3) δ 2.29 (m, 1H, H2 '), 2.40 (ddd, 1H, JH2', H3 '= 4.1 Hz, JH2', H1 '= 6.4 Hz, Jgem = 14.4 Hz , H2 ''), 2.93 - 3.15 (m, 4H, CH2-a, CH2-7), 3.37 (dd, 1H, JH5 ', H4''= 3.4 Hz, Jgem = 10.5 Hz, H5''), 3.47 (dd, 1H, JH5 ', H4' = 3.6 Hz, Jgem = 10.6 Hz, H5 '), 3.65 (s, 3H, OCH3), 3.79 (s, 6H, 2xOCH3), 4.02 (m, 1H, H4') , 4.55 (m, 1H, H3 '), 6.36 (t, 1H, JH1', H2 '' = JH1 ', H2' = 6.7 Hz, H1 '), 6.79 - 7.44 (m, 13H, DMTr), 7.66 ( s, 1H, H6);

P r x l a d XII

General procedure for 3'-O-phosphitylation of 5'-blocked modified nucleosides

The starting 5'-O-dimethoxytritylated nucleoside (0.20 mmol) was dried under reduced pressure for 12 hours and then dissolved with anhydrous CH2Cl2 (2 mL). The solution was placed under argon and anhydrous diisopropylethylamine (140 gL, 0.80 mmol) was added followed by dropwise addition of 2-cyanoethoxy-H, H-diisopropylaminochlorophosphine (67 gL, 0.3 mmol). After 1 hour at room temperature, the substrate was completely consumed (TLC) and a small amount of MeOH (150 gL) was added. After 2 min. CH2Cl2 (15 mL) was added and the reaction mixture was washed with a 5% NaHCO3 solution (5 mL). The organic fraction was dried with MgSO4, the drying agent was filtered off and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel flash column chromatography using appropriate elution systems (listed below). Appropriate fractions containing pure product were combined and evaporated to dryness to give the corresponding 3'-O-phosphoramidite as a white foam.

3'-O- (2-cyanoethyl-N, N-diisopropylaminophosphinoxy) -5'-O- (4,4'-dimethoxytrityl) -N ⁶ - [(1S, 2R) -1 - {[2- (trimethylsilyl) ethoxy] carbonyl} -2 - {[(tert-butyl) dimethylsilyl] oxy} propylaminocarbonyl] -2'-deoxyadenosine of formula 11a

Elution mixture CH2Cl2: ethyl acetate: triethylamine (10: 10: 1);

127 mg, 57% yield;

TLC Rf: 0.32 (CH2Cl2 / AcOEt / Et3N 10: 10: 1 v / v);

HRMS FAB m / z: 113.5402 ([M + H] ⁺ C56H82Si2PN8O10 calc. 113.5430);

³¹ P NMR (101 MHz, C6D6) δ 148.9, 149.2;

3'-O- (2-cyanoethyl-N, N-diisopropylaminophosphinoxy) -5'-O- (4,4'-dimethoxytrityl) -N ⁶ - {2- [N ¹ - (4-methoxytrityl) imidazole-4- yl] ethylaminocarbonyl} -2'-deoxyadenosine of formula 11b

Elution mixture: Petroleum ether: acetone 3: 1, 2: 1, 1: 1, 2: 1 v / v (100 mL each);

137 mg, 59% yield;

TLC Rf: 0.15 (CH2Cl2 / AcOEt / Et3N 10: 10: 1 v / v);

HRMS FAB m / z: 1163.5235 ([M + H] ⁺ C66H72PN10O8 calcd. 1163.5272);

³¹ P NMR (101 MHz, C6D6) δ 149.2, 149.3;

₁

3'-O- (2-cyanoethyl-N, N-diisopropylaminophosphinoxy) -5'-O- (4,4'-dimethoxytrityl) -5- {2- [N ¹ - (4-methoxytrityl) imidazol-4-yl ] ethylaminomethyl} -2'-deoxyuridine of formula 19a

Before chromatographic purification, the crude product was precipitated from petroleum ether at -78 ° C. Elution system: CH2Cl2 / MeOH / Et3N 93: 5: 2 v / v; 126 mg, 56% yield;

TLC Rf: 0.09 (benzene / CH2Cl2 / Et3N 7: 2: 1 v / v);

HRMS FAB m / z: 1126.5172 ([M + H] ⁺ C65H73PN7O9 calc. 1126.5207);

³¹ P NMR (101 MHz, C6D6) δ 148.0, 148.8;

3'-O- (2-cyanoethyl-N, N-diisopropylaminophosphinoxy) -5'-O- (4,4'-dimethoxytrityl) -5-methoxycarbonylaminomethyl-2'-deoxyuridine of formula 19b

PL 219 355 B1

Elution mixture: Petroleum ether: acetone 3: 1, 2: 1, 1: 1, 2: 1 v / v (100 mL each);

mg, 56% yield;

TLC Rf: 0.29 (benzene / CH2Cl2 / Et3N 7: 2: 1 v / v);

HRMS FAB m / z: 832.3655 ([M + H] ⁺ C43H55PN5O10 calc. 832.3687);

³¹ P NMR (101 MHz, C6D6) δ 149.1, 149.2;

P r x l a d XIII

Synthesis of oligonucleotides with deoxyribozyme 1 sequence modified with type 2 unit

Oligonucleotides were synthesized at 1 micromolar scale on an Applied Biosystems 394 instrument according to the manufacturer's standard procedure. For the synthesis, 0.10 M solutions of the phosphoramidites of canonical nucleosides (Glen Research, Sterling, VA) and 0.15 M solutions of phosphoramidites of protected units 2 (compounds 11a, 11b, 19a or 19b according to schemes I and II) in anhydrous acetonitrile were used. After synthesis, protecting groups from unmodified oligonucleotides were removed with a 3: 1 (v / v) mixture of aqueous ammonia and ethanol during 4 hours. at 55 ° C, while with oligonucleotides modified overnight at room temperature. All oligonucleotides containing DMTr groups at the 5'-end were purified by reverse phase high performance liquid chromatography (RP-HPLC) with a 0-40% acetonitrile gradient in 0.10M triethylammonium bicarbonate at pH 7.0. The purified oligonquettes were treated with 50% aqueous acetic acid for 0.5 h. at room temperature in order to remove DMTr groups, and then, after evaporation of the reagents, it is subjected to further purification with PRHPLC. The molecular weight of the oligonucleotides was confirmed by MALDI-TOF MS and the purity by denaturing polyacrylamide gel electrophoresis (20%, 7M urea). Characteristics of exemplary oligonucleotides with deoxyribozyme 1 sequence modified with type 2 units, obtained according to the above-mentioned procedure, are presented in Table 1.

P r x l a d XIV

Synthesis of oligonucleotides with deoxyribozyme 1 sequence containing mix phosphorothioate bonds and possibly containing type 3 units

Oligonkieotides were synthesized at 1 micromolar scale on an Applied Biosystems 394 apparatus according to the manufacturer's procedure. For the synthesis, 0.10 M solutions of the phosphoramidite canonical nucleosides and 2'-OMe nucleosides (units 2) (Glen Research, Sterling, VA) in anhydrous acetonitrile were used. During the synthesis at the site of the planned phosphorothioate bond in the mix configuration, oxidation of the phosphite function was carried out with commercially available sulfating reagents (e.g. Beaucage's reagent) in a known manner. The remaining phosphite functions were routinely oxidized in the presence of I2 / H2O / Py. Removal of the oligonucleotides from the fixed bed and removal of all protecting groups were carried out in a known manner. The molecular weight of the oligonucleotides was confirmed by MALDI-TOF MS and the purity by denaturing polyacrylamide gel electrophoresis (20%, 7M urea).

Characteristics of exemplary oligonucleotides with deoxyribozyme 1 sequence modified with units 2 and / or containing phosphorothioate linkages in the mix configuration are presented in Table 2.

P r x l a d XV

Synthesis of 2'-deoxyribonucleoside-3'-O- (2'-thio-spiro-4,4-pentamethylene-1,3,2-oxathiaphospholane) of formula 20

The synthesis is carried out according to a known procedure (WJ, Stec B., Karwowski, P., Guga, M., Koziołkiewicz M., Boczkowska, MW, Wieczorek, J., Błaszczyk, M., Sochacki. 1998, J. Am. Chem. Soc. 120, 7156). 1 mmol of a 5'-DMTr-derivative of an N-protected nucleoside was dried on a vacuum line for 12 h. After removing the traces of water, argon was pushed into the nucleoside flask, and then the substrate was dissolved in 3 mL of anhydrous CH ₂ Cl ₂ , followed by the addition of 1.1 mmol of diisopropylethylamine. Then 1.1 mmol of 2-chloro-4,4-spiro-pentamethylene-1,3,2-oxathiaphospholane were added dropwise with stirring. The phosphitylation reaction lasted 4 hours. After this time, 2 mmol of elemental sulfur were added. The reaction was continued for the next 8 hours. After completion of the phosphitylation reaction, excess sulfating reagent was filtered off and the solvent was evaporated. The product was purified on silica gel 60, eluting with CHCl ₃ distilled with pyridine.

Data for 20c

5'-DMTr-dA-N-protected-3'-O- (2'-thio-spiro-4,4-pentamethylene-1,3,2-oxathiaphospholane) ³¹ P NMR 104.7 ppm and 105.1 ppm at CD3CN

PL 219 355 B1

Data for 20b

5'-DMTr-dC-N-protected-3'-O- (2'-thio-spiro-4,4-pentamethylene-1,3,2-oxathiaphospholane) ³¹ P NMR 105.3 ppm and 105.6 ppm at CD3CN.

The purified and characterized diastereomeric mixtures of compounds were separated into diastereoisomers by column chromatography on silica gel 60H. The highest partition efficiencies were obtained when the eluent was used:

for derivative 20c ethyl acetate / butyl acetate = 2/1 where

Rf for the isomer Rp = 0.54; ³¹ P NMR 104.7 ppm

Rf for the isomer Sp = 0.46; ³¹ P NMR 105.1 ppm for derivative 20b ethyl acetate / butyl acetate = 2/1 where Rf for the isomer Rp = 0.6; ³¹ P NMR 105.3 ppm Rf for the isomer Sp = 0.4; ³¹ P NMR 105.6 ppm

P r x l a d XVI

Synthesis of the 3'-isopropoxyacetyl derivative of 2'-deoxyribonucleosides (compounds of formula 21) mmol of the 5'-DMTr derivative of the N-protected nucleoside dC, dA, dG or thymidine are dissolved in 25 mL of anhydrous pyridine and 1.3 mmol of anhydride are added in two portions isopropoxyacetyl (SinoChemexper). After 2 hours After stirring, the solvent was removed under reduced pressure and the residue was chromatographed on silica gel, eluting the column with chloroform. The obtained product is treated with a solution of para-toluenesulfonic acid (3 mmol) in methylene chloride (30 mL) for 30 min. at room temperature, and then added 10 mL of THF containing 1% water. After concentration under reduced pressure, the residue is purified by silica gel column chromatography with a gradient to 10% methanol in chloroform. The obtained product 21 is subjected to chromatographic and spectral analysis.

Data for 21a: 65% yield, R _f = 0.4 (9: 1, v / v, CHCl ₃ / MeOH), FAB MS: m / z 343 (MW 342.2)

Data for 21d: 65% yield, R _f = 0.5 (9: 1, v / v, CHCl ₃ / MeOH), FAB MS: m / z 438 (MW 437.7)

E xample XVII

Synthesis of dinucleoside-phosphorothioate with the stereoside or Sp configuration on a chiral phosphorus atom (compound of formula 22) (Scheme III) mmol 5'-DMTr of a diastereomerically pure nucleoside 3'-oxathiaphospholate derivative (N-protected dC, dA, dG or thymidine) 20 and 1.5 mmol of compound 21 was dissolved in 3 mL of anhydrous acetonitrile under argon and 50 μΜ (0.33 mmol) of DBU was added. The reaction mixture was stirred at room temperature for 2 h. then the solvent was evaporated, and the residue was subjected to silica gel column chromatography. The product was eluted with CHCl3 / MeOH / pyridine (94: 5: 1, v / v / v) to give a white foam after evaporation.

Data for compound 22 B3 = b, B4 = a, R _p isomer: 72% yield, ( ³¹ P NMR 54.19 ppm in CD ₃ CN, MALDITOF MS m / z = 1052.4; molecular weight calculated 1052.4)

Data for compound 22 B3 = b, B4 = a, S _p- isomer: 41% yield ( ³¹ P NMR 55.64 ppm in CD ₃ CN, MALDI TOF MS m / z = 1052.4; molecular weight calculated 1052.4)

Data for compound 22 B3 = c, B4 = d, R _p isomer: 68% yield, ( ³¹ P NMR 55.71 ppm in CD ₃ CN, MALDI TOF m / z = 1171.4; Molecular weight calculated 1172)

Data for compound 22 B3 = c, B4 = d, S _p isomer: yield 49% ( ³¹ P NMR 55.35 ppm in CD ₃ CN, MALDI TOF m / z = 1171.7; Molecular weight calculated 1172)

E xample XVIII

Removal of the 3'-isopropoxyacetyl protecting group from compound 22

Derivative 22 (0.5 mmol) is dissolved in dioxane (1 mL) and then 28% aqueous ammonia solution in the amount of 1 mL is added. The resulting solution is stirred for 25-30 min. At this time, the mixture was concentrated under reduced pressure and column chromatographed on silica gel, eluting pure product of formula 23 with CHCl ₃ / MeOH / pyridine (94: 5: 1, v / v / v). The product 23 after evaporation is a white foam.

Data for compound 23 B3 = b, B4 = a, R _p isomer: 82% yield, (MALDI TOF MS m / z = 952.4; MW calculated 952)

Data for compound 23 B3 = b, B4 = a, S-isomer _p : yield 80%, (MALDI TOF MS m / z = 952.4; MW calculated 952)

PL 219 355 B1

Data for compound 23 B3 = c, B4 = d, R _p isomer: 78% yield, (MALDI TOF MS m / z = 1071.4;

Molecular weight calculated 1072)

Data for compound 23 B3 = c, B4 = d, S-isomer _p : 77% yield, (MALDI TOF MS m / z = 1071.4;

Molecular weight calculated 1072)

E xample XIX

Blocking of the phosphorothioate function with 2-nitrobenzyl bromide

The substrate 23 (1 mmol) and 2-nitrobenzyl bromide (2 mmol) were dried on a vacuum pump for 12 h. After the trace of moisture had been removed, dry argon was purged into the flask, followed by the addition of 1 mL of anhydrous CH ₃ CN and Et 3 N (2 mmol). The flask was protected against light with aluminum foil. The reaction was stirred for 8 h. in room temperature. The reaction product was purified by column chromatography on silica gel using CHCl ₃ as an eluent.

Data for compound 24 B3 = b, B4 = a

Yield> 98% for the Rp and Sp isomers

Rp isomer ³¹ P NMR 27.67 ppm, CD ₃ CN

Sp ³¹ P NMR isomer 26.72 ppm in CD ₃ CN

Data for compound 24 B3 = c, B4 = d

Yield> 97% for the Rp and Sp isomers

Rp isomer ³¹ P NMR 28.18 ppm, CD ₃ CN

Sp ³¹ P NMR isomer 28.47 ppm in CD ₃ CN

P r z x l a d XX

Phosphitylation of dinucleoside-phosphorothioates of formula 24 under "in situ" conditions

The substrate 24 (20 µmol) was dried on a vacuum line for 12 h. and dissolved in 40 μL of anhydrous CH ₃ CN. 42 μL of a 0.5 M solution of S-ethylthiotetrazole in CH ₃ CN and 6.3 μL of 2-cyanoethyl-bis- (N, N-diisopropyl) phosphoramidite were added dropwise to the flask. The reaction was carried out for 35 min. until the white crystals of the tetrazolium salt precipitate. After this time, 90 μL of a 0.5 M S-ethylthiotetrazole solution in CH ₃ CN was added and used for the next stage of oligonucleotide synthesis.

P r x l a d XXI

Synthesis of oligonucleotides with deoxyribozyme 1 sequence containing stereodefined phosphorothioate bonds with Rp or Sp configuration

Oligonkieotides were synthesized in the 0.5 μmol scale according to a known procedure (B. Nawrot, B. Rębowska, K. Cieślińska, WJ Stec. 2005, Tetrahedron Lett. 46, 6641; B. Nawrot, B. Rębowska, 2009; Current Protocols in Nucleic Acid Chemistry Chapter 4: Unit 4.34) on an Applied Biosystems 394 apparatus. For the synthesis, 0.10 M solutions of canonical nucleoside phosphoramidites (Glen Research, Sterling, VA) and 0.15 M solutions of dinucleoside phosphoramidites in anhydrous acetonitrile were used.

The obtained chimeric oligonucleotides suspended on the LCA CPG resin were subjected to the subsequent deprotection steps according to the following scheme: 2 mL of piperidine solution in CH ₃ CN, 2 mL of CH ₃ CN, 2 mL of the dioxane / thiophenol / triethylamine mixture in the ratio 2/1/2 were passed through the reaction column. (v / v / v) and finally 2 mL of dioxane. In this way, protecting groups from phosphate and phosphorothioate functions were removed. Remaining protecting groups were removed in a known manner and the oligonucleotides were purified in two steps by reverse phase high performance liquid chromatography (RP HPLC) in a 0-40% acetonitrile gradient in 0.10 M triethylammonium bicarbonate at pH 7.0. The purified oligonquotes were treated with 50% aqueous acetic acid for 0.5 h at room temperature to remove DMTr groups, and then, after evaporation of the reagents, subjected to further purification by RP HPLC. The molecular weight of the oligonucleotides was confirmed by MALDI TOF MS and the purity by denaturing polyacrylamide gel electrophoresis (20%, 7M urea). Characteristics of exemplary oligonucleotides with deoxyribozyme 1 sequence containing phosphorothioate linkages with Rp or Sp configuration are provided in Table 3.

E xample XXII

Phosphorylation of the 5'-hydroxyl group of a substrate oligonucleotide with [γ- P] ATP and polynucleotide kinase

A 20 μL mixture containing 2 μL of the concentrated buffer for PNK T4, 0.1 OD (30 μM) of the substrate oligonkieotide with the following sequence: 5'-d (ACAGATGA) G U d (CAACCCT) -3 ', 15 uCi

[γ- P] ATP (2 (uL) and 5 units of polynucleotide kinase from T4 phage were incubated for 0.5 h

It was then thermally denatured at 95 ° C for 5 minutes. The samples were stored at -20 ° C for a maximum of 7 days.

E xample XXIII

Hydrolysis of a substrate oligonucleotide by deoxyribozymes of the general formula I with or without divalent metal ions

Substrate hydrolysis reactions are performed under single turnover conditions using a 100-fold molar excess of enzyme to substrate. Radioactively labeled substrate oligonucleotide (0.1 gM) is incubated with deoxyribozyme 1 in PBS buffer at pH 7.0 or in TRIS or PIPES buffer (most preferably 20 or 50 mM) containing 100 mM NaCl and magnesium ions at a concentration of 10, 3.0 or 1.0 mM or without magnesium ions but with 4 mM EDTA for 24 h and 37 ° C. The reaction in the presence of metal ions is stopped by adding EDTA (33 mM) and cooling the reaction mixture. The sample (10 gL), after mixing with 8 gL of the loading dye: 0.05% cyanoxylene and 0.05% bromophenol blue in formamide, is applied to a denaturing 20% polyacrylamide gel with 7M urea and electrophoresis is carried out and then evaluated with a Phosphorlmager. is the percentage of substrate hydrolysis.

Fig. 1 shows an exemplary plot of substrate hydrolysis in the presence of deoxyribozymes 1 modified with units of formula 2 for the reaction carried out in a PBS solution with the addition of 10 and 3 mM magnesium ions.

Example XXIV

Analysis of the kinetics of the substrate oligonucleotide hydrolysis reaction using deoxyribozymes of the general formula 1

The substrate hydrolysis reaction catalyzed by the deoxyribozyme of general formula 1 was carried out under single turnover conditions with a 10-fold or 100-fold excess of the enzyme over the substrate. Radioactively labeled substrate (0.1 gM) was incubated with deoxyribozyme 1 (1 gM) in 20 mM Tris-HCl or PIPES (pH 7.5 or 8.0) containing 100 mM NaCl, 1 mM MgCl2 (or without divalent metal ions and then with the addition of 4 mM EDTA) at 37 ° C. At each time interval, 10 gL of the reaction mixture were taken, and 1.2 gL of 50 mM EDTA was added to this sample. All samples were kept in an ice bath. After dissolving the dyes in the solution (loading dye: 0.05% cyanoxylene and 0.05% bromophenol blue in formamide), the samples were applied to a denaturing 20% polyacrylamide gel with 7M urea and electrophoresed. The quantitative analysis of the hydrolysis products was performed with a Phosphorlmager reader using the ImageQuant program. The kobs value was calculated in Graph Pad Prism and determined from the formula:

Y = [EP] (1 - exp (-kobs x t) where:

Y - the percentage of the obtained product at time t

EP - end point at which 80% -90% hydrolysis product was obtained

Exemplary kobs values determined for the hydrolysis of chimeric RNA / DNA substrates catalyzed with deoxyribozymes of general formula 1 modified with X, Y units, or with phosphorothioate (PS) groups are given in Table 4.

Deoxyribozyme 1 characterized in items 1-11 refers to the following substrate target sequence;

5'-d (ACAGATGA) G U d (CAACCCT) -3 '

Deoxyribozyme 1 characterized in items 12-17 and 24-32 refers to the following substrate target sequence;

5'-d (CTGTACACAG) G C d (AGTCTCTGG) -3 '

Deoxyribozyme 1 characterized in items 18-23 refers to the following substrate target sequence;

5'-d (TCTTTTCTTA) G U d (TTCAGAAGT) -3 '

P r z o m a d XXV

Determination of deoxyribozyme 1 stability in HeLa cell extract

Deoxyribozymes are radiolabeled at the 5'-end with T4 kinase in the presence

[γ-P] ATP i is administered to the cell extract containing 2.5, 5 and 10 gg of protein, respectively, in the presence of the phosphatase inhibitor Phos Stop in PBS. The reactions are carried out at intervals of: 0, 15, 30, 60, 120, 240, 360 and 1440 min. at 37 ° C. Samples taken at individual time intervals containing 10 gL of the reaction mixture are dissolved in the dye solution (loading dye:

PL 219 355 B1

0.05% cyanoxylene and 0.05% bromophenol blue in formamide) and applied to a denaturing 20% polyacrylamide gel with 7M urea and performed electrophoresis. Quantitative analysis of products is performed using the PhosphorImager reader using the ImageQuant program.

Electrophoretic analysis of the exemplary oligonucleotides showed that the greatest differences in stability between the unmodified deoxyribozyme and the one with the most PS modification and the 2'-OMe in the catalytic domain are in relation to the extract containing 10 pg of protein (Fig. 2). Such optimized reaction conditions are used to test the stability of deoxyribozymes of formula 1. Among the series of DNAzymes Dz2 (numbers from 12 to 17 in Table 4) and Dz4 (numbers from 18 to 23) the deoxyribzymes Dz2 (2) (in Table 4, no. 14) and Dz4 (2) (in Table 4 No. 20). These are enzymes with five PSmix modifications in the catalytic domain. The additional introduction of the six 2-OMe modifications to the catalytic domain of these deoxyribozymes showed their highest stability in the cell extract (No. 17 and 23 in Table 4, respectively).

Example XXVI

Activity of deoxyribozymes 1 modified as in Table 2 in HeLa cells overexpressing the BACE1 / GFP fusion protein gene

HeLa cells and two plasmids encoding fluorescent proteins were used in the experiments. The plasmid encoding the RFP reference protein was pDsRed2-N1, while the plasmid encoding the BACE-GFP fusion protein was pBACE-GFP.

hours prior to transfection, HeLa cells were seeded (15,000 / well) in clear bottom, black walled 96 well plates (Perkin Elmer). HeLa cells were grown in RPMI 1640 medium (Gibco) with 10% addition of fetal bovine serum, FBS and antibiotics in concentrations: penicillin (Polfa Tarchomin) 100 U / L and streptomycin 100 pg / mL. One hour prior to transfection, the medium was changed to medium without antibiotics (60 pL / well).

Cells were transfected simultaneously with plasmids and deoxyribozymes using lipofectamine (Invitrogen) in the proportion of 1 µL of transfection reagent per 1 µg of nucleic acids. Transfection mixtures were prepared in reduced OPTT-MEM medium (Gibco). Separately (20 pL OPTI-MEM / well) appropriate dilutions of the transfection reagent and plasmid DNA (dDsRed2-N1, 15 ng / well and p-BACE-GFP, 70 ng / well) and DNAzymes (in an amount appropriate to achieve concentrations of 10 -50 nM). After 5 min. of incubation, the solutions were combined and allowed to incubate further for 20 min. in room temperature. 40 µL / well of the resulting transfection mixtures were added to 60 µL of medium. Transfection was carried out for 6 hours at 37 ° C, 5% CO ₂ .

After the transfection time had elapsed, the medium above the cells was replaced with antibiotic-containing culture medium (100 µL) and the cells were further incubated for 36 h (post-incubation time). The cells were then washed 3 times with PBS phosphate buffer (100 µL). After the final rinse, 100 µL of the solution was left over the cells and 50 µL of NP-40 lysis buffer (150 mM NaCl, 1% IGEPAL, 50 mM Tris-HCl (pH 7), 1 mM PMSF) was added at 37 ° C overnight. in the dark. Lysates were used to read fluorescence directly from the plates. Protein fluorescence was measured in a Synergy ™ HT plate reader (BioTek) operated by the KC4 program. Readings were carried out in the ranges of excitation and emission wavelengths characteristic for green fluorescence (GFP and BACE-GFP) λ _ΕΧ = 485/20 nm and X _EM = 528/20 nm and for red fluorescence (RFP) λ _ΕΧ = 530/25 nm and ź ~ _EM = 590/30 nm. DNAzyme activity was calculated on the basis of the ratio of GFP fluorescence (BACE-GFP) values to the RFP fluorescence level. Each experiment was performed in 8 parallel replications. Cells transfected with plasmids and DNA of the same length as deoxyribozymes constituted a positive control. The DNA used for the positive control was not complementary to the BACE target sequence.

Figure 3 shows an exemplary result for the intracellular activity of deoxyribozymes 1 numbered 12-17 (2.2 (1), 2 (2), 2 (3), 2 (4) and 2 (5), respectively) and 18-23 ( 4.4 (1), 4 (2), 4 (3), 4 (4) and 4 (5) respectively of Table 4.

Claims (10)

Patent claims 1. Modified deoxyribozyme 10-23 of general formula 1 with stabilized resistance to hydrolysis and increased catalytic activity, containing at least one internucleotide phosphorothioate bond of formula 4a and / or 4b, located in selected positions P1 to P15, catalytic domain, especially at position P1, P7, P8, P14 and / or P15, preferably with a defined absolute configuration on chiral phosphorus atoms, and / or at least one nucleoside residue of general In formula 2, wherein B is a 5-modified uracil residue or a 6-modified adenine residue represented by the formula 2 'or 2, wherein R2 is an alkylamine or amino acid residue, preferably a histamine or glycine residue, and / or at least one nucleoside residue of formula 3, wherein B1 is a residue of thymine, uracil, cytosine, adenine and / or guanine, and R 1 is F or an alkoxy group -OR, wherein R is a group of the formula -C _n H _2n + _1, and n is a natural number from 1 to 5, preferably -OR is methoxy, said nucleoside residues being at selected N positions. 2. A deoxyribozyme according to claim 1 The method of claim 1, characterized in that it contains at least one internucleotide phosphorothioate linkage of formula 4a and / or 4b, located in selected P positions of the catalytic domain, especially in position P1, P7, P8, P14 and / or P15, and / or at least one nucleoside residue of formula 2, wherein B and R1 are as defined above, located at a position selected from the group consisting of N2, N7, N8, N11, N14 and / or N15. 3. The deoxyribozyme according to claim 1 The method according to claim 2, characterized in that it contains at least one internucleotide phosphorothioate linkage of formula 4a and / or 4b, located at selected positions in the catalytic domain P, and / or at least one nucleoside residue of formula 2a, 2b, 2c and / or 2d located in in position N. 4. The deoxyribozyme according to claim 1 The method according to claim 1, characterized in that in said P positions of the catalytic domain it contains at least one internucleotide phosphorothioate bond of formula 4a and / or 4b located in selected P positions of the catalytic domain, especially in position P1, P7, P8, P14 and / or P15 , and optionally at least one modified N nucleoside residue represented by formula 3, wherein B and R1 are as defined above. 5. The deoxyribozyme according to claim 1 4. The method according to claim 4, characterized in that it comprises at least one internucleotide phosphorothioate bond of formula 4a and / or 4b, located in selected P positions of the catalytic domain, P1 to P15, especially in position P1, P7, P8, P14 and / or P15, and / or at least one nucleoside residue at the N position of formula 3a, 3b, 3c and / or 3d in which R1 is alkoxy, especially methoxy, said nucleoside residues being at a position selected from the group consisting of N2, N7, N8 , N11, N14 and / or N15. 6. A deoxyribozyme according to claim 1 The method of claim 1, wherein at least one modified nucleoside residue N is represented by the formula 2c or 2d at position N8. 7. The deoxyribozyme according to claim 1 The method of claim 1, characterized in that it contains a single phosphorothioate bond with Rp configuration at position P1, and all nucleoside units of the catalytic domain are identical to the unmodified enzyme 10-23. 8. The deoxyribozyme according to claim 1 A method according to claim 1, characterized in that it contains two phosphorothioate bonds, in position P1 of Rp configuration and in P8 position of Sp configuration, and all nucleoside units N of the catalytic domain are identical to unmodified enzyme 10-23. 9. A method of producing deoxyribozymes with stabilized resistance to hydrolysis and increased catalytic activity, represented by formula 1, containing at selected P positions of the catalytic domains from P1 to P15, especially in position P1, P7, P8, P14 and / or P15, at least one internucleotide a phosphorothioate bond of formula 4a or 4b, and / or at selected N positions, at least one nucleoside residue of general formula 2, in which B is a 5-modified uracil residue or a 6-modified adenine residue represented by 2 'or 2 , where R2 is an alkylamine or amino acid residue, preferably a histamine or glycine residue, and / or at least one nucleoside residue of formula 3, wherein B1 is a thymine, uracil, cytosine, adenine and / or guanine residue and R1 is F or an alkoxy group -OR, wherein R is a group of the formula -C _n H _2n + ₁ and n is an integer from 1 to 5, preferably -OR represents methoxy, characterized in that the binding e phosphorothioate, preferably stereodefined, is introduced into an oligonucleotide by the use of a procedure allowing the synthesis of a stereodefined dinucleoside thiophosphate, using stereocontrolled opening of the chiral 3'-nucleoside oxathiophosphate ring with the 5'-hydroxyl group of the second nucleoside-protective group, followed by removal of from the 3'-hydroxyl function of the dimer and the protection of the phosphorothioate function with an alkylaryl group, activation of the 3'-hydroxyl group of dinucleoside phosphorothioate is carried out with a phosphoramidite residue to obtain a monomer which is then used, in addition to phosphoramidites, canonical units and / or 2 and / or modified units or 3, for the synthesis of an oligonucleotide with the deoxyribozyme sequence 10-23 by using the standard phosphoramidite procedure, with extended condensation time and an increased excess of phosphoramidite modified units in po22 By equation to the monomers of the canonical units and the use of an appropriate procedure for cleavage of the oligonucleotides from the fixed bed and deprotection, including deprotection of the phosphorothioate function, which is carried out with a thiophenol-containing solvent system. A method for the preparation of deoxyribozymes with stabilized resistance to hydrolysis and increased catalytic activity, represented by the formula 1, containing at selected P positions of the catalytic domains P1 to P15, especially in position P1, P7, P8, P14 and / or P15, at least one internucleotide linkage phosphorothioates of formula 4a or 4b, and / or at selected N positions, at least one nucleoside residue of general formula 2, wherein B is a 5-modified uracil residue or a 6-modified adenine residue represented by 2 'or 2, where R2 represents an alkylamine or amino acid residue, preferably a histamine or glycine residue, characterized in that the appropriately modified nucleoside units of the general formula 2 are prepared by activation of the test function of adenine and condensation with an active reagent bearing an amino acid or alkylamine residue in its structure, or by activating the 5-position of the thymine ring and subsequent attachment removing a residue bearing an amino acid moiety or an alkylamino group, and then incorporation of such a modified unit into the oligonucleotide chain using modified N units activated with a phosphoramidite function and units of the general formula 25 bearing the stereophosphate thiophosphate functions of formula 4a or 4b.