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    • 专利摘要:
    The present invention relates to a process for the preparation of 3'4'-cyclic acetals of pentopyranosyl nucleosides comprising reacting the pentopyranosyl nucleosides with aldehydes, ketones, acetals or ketals under reduced pressure.
    公开号:KR20010074475A
    申请号:KR1020007011147
    申请日:1999-04-07
    公开日:2001-08-04
    发明作者:에쉔모저알베르트；피취슈테판；벤데보른세바슈티안
    申请人:렐케；아벤티스 레제아르히 운트 테히놀로기스 게엠베하 운트 콤파니 카게；
    IPC主号:
    专利说明:

    Method for the production of pentopyranosyl nucleosides
    [1] The present invention relates to a process for the preparation of 3 ', 4'-cyclic acetals of pentopyranosyl nucleosides comprising reacting pentopyranosyl nucleosides with aldehydes, ketones, acetals or ketals under reduced pressure.
    [2] Pyranosylnucleic acid (p-NA) is generally an isomer to natural RNA in which the pentose units are in pyranose form and are repeatedly linked by phosphodiester groups between the C-2 'and C-4' positions. Represents a structural type that is in form (FIG. 1). In this context, “nucleobase” refers herein to standard nucleobases A, T, U, C, G, but within the scope of the present invention isoguanine / isocytosine and 2,6-diaminopurine It is understood to mean not only / xanthine pairs but also other purines and pyrimidines. p-NA, ie, p-RNA derived from ribose, was first described by Eschenmoser et al., S. Pitsch et al., Helv. Chim. Acta 76 , 2161 (1993); S. Pitsch et al., Helv. Chim. Acta 78 , 1621 (1995); Angew. Chem. 108 , 1619-1623 (1996). They form only so-called Watson-Crick paired, ie, purine-pyrimidine and purine-purine paired antiparallelically reversible "melting" quasi-linear stable duplexes. Homochiral p-RNA strands of the opposite concept of chirality also form pairs at an adjustable level and are strictly non-helical in the duplex formed as described above. This specificity is useful for the preparation of supramolecular units, with the relatively low flexibility of these ribopyranose phosphate backbones and the strong slope of the base plane relative to the strand axis and the internal chain bases attached to the resulting double helix. And a 2 ', 4'-cis-disubstituted ribopyranose ring may eventually be involved in the preparation of the main chain. Because of these significantly better pairing properties, p-NA pairing systems are more preferred compared to DNA and RNA for use in the preparation of supramolecular units. They form orthogonal pairing systems for native nucleic acids, ie they do not pair with native forms of DNA and RNA, which is particularly important in the field of diagnostics.
    [3] EssenMoss et al. First produced p-RNAs as shown in FIG. 2 and as illustrated in S. Pitsch et al. (1993), supra].
    [4] In this context, appropriately protected nucleobases are anomers of tetrabenzoylibopyranose by the action of bis (trimethylsilyl) acetamide and Lewis acids such as trimethylsilyl trifluoromethanesulfonate. React with the mixture. See, eg, Vorbruggen, H. et al., Chem. Ber. 114 , 1234 (1981). Under the action of a base (NaOH in THF / methanol / water for purine; saturated ammonia in MeOH for pyrimidine), the acyl protecting group is removed from the sugar and this product is acidified by p-anisaldehyde dimethyl acetal. Protected in the 3 ', 4'-position under catalysis. This diastereomeric mixture is acylated at the 2'-position, and the 3 ', 4'-methoxybenzylidene-protected 2'-benzoate is deacidified, for example, by trifluoroacetic acid in methanol. Acetalization followed by reaction with dimethoxytrityl chloride. The 2 '→ 3' shift of the benzoate is initiated by treatment with p-nitrophenol / 4-dimethylaminopyridine / triethylamine / pyridine / n-propanol. Almost all reactants are worked up by column chromatography. Subsequently, the main unit synthesized in this way, namely the 4'-DMT-3'-benzoyl-1'-nucleobase derivative of ribopyranose, is partially phosphtylated and then bound to the solid phase via a linker. Let's do it.
    [5] In subsequent automated oligonucleotide synthesis, the carrier-linked component at the 4'-position is repeatedly deprotected under acidic conditions and the phosphoramidite is coupled under the action of a coupling agent, for example a tetrazole derivative. The acetylated 4'-oxygen atom, which is still free, is then oxidized to the extent that an oligomeric product is obtained. The remaining protecting group is then removed and the product is purified and then desalted by HPLC.
    [6] However, the above-described method such as Equation Moser cannot be reproduced in the yields presented, and thus it is almost impossible to apply on an industrial scale.
    [7] Accordingly, it is an object of the present invention to make available methods for producing pentopyranosyl nucleosides on an industrial scale.
    [8] Surprisingly, according to the invention, the 3 ', 4'-cyclic acetal of pentopyranosyl nucleoside, which is an intermediate in the epoch moiety synthesis, is reacted only when the pentopyranosyl nucleoside is reacted with aldehyde or ketone or acetal or ketal under reduced pressure. It has been found to be produced in significant yield.
    [9] Accordingly, an object of the present invention is a process for the preparation of 3 ', 4'-cyclic acetals of pentopyranosyl nucleosides comprising reacting pentopyranosyl nucleosides with aldehydes, ketones, acetals or ketals under reduced pressure. .
    [10] Decompression is understood according to the invention, in particular, to mean a pressure of less than about 500 mbar, preferably less than about 100 mbar, in particular less than about 50 mbar, more particularly about 30 mbar.
    [11] Aldehyde is, for example, formaldehyde, acetaldehyde, benzaldehyde or 4-methoxybenzaldehyde, and acetal is formaldehyde dimethyl acetal, acetaldehyde dimethyl acetal, benzaldehyde dimethyl acetal or 4-methoxybenzaldehyde and acetyl acetone, dimethyl acetone , Cyclopentanone or cyclohexanone, the ketal is acetone dimethyl ketal, cyclopentanone dimethyl ketal, cyclohexanone dimethyl ketal or in the form of 2-methoxypropene.
    [12] In a particular embodiment, the pentopyranosyl nucleosides are purified prior to the reaction, for example on SiO 2 , preferably on SO 2 in the form of silica gel. For example, purification on silica gel chromatography columns is suitable for this purpose. For elution of pentopyranosyl nucleosides, a gradient of, for example, about 1-20% or about 5-15% methanol in dichloromethane is suitable. It is especially advantageous to neutralize the pentopyranosyl nucleoside prior to purification, for example with a 1% concentration of hydrochloric acid solution or solid ammonium chloride, and optionally remove the solvent.
    [13] Suitable pentopyranosyl nucleosides are generally ribo-, arabino-, lyxo- or xylo-pyranosylnucleosides. Examples of suitable pentopyranosyl nucleosides include pentopyranosylpurine, -2,6-diaminopurine, -6-furinthiol, -pyridine, -pyrimidine, -adenosine, -guanosine, -isoguanosine,- 6-thioguanosine, -xanthine, -hypoxanthine, -thymidine, -cytosine, -isocytosine, -indole, -tryptamine, -N-phthaloyl tryptamine, -uracil, -caffeine, -theobromine, -Theophylline, -benzotriazole or -acridine.
    [14] Pentopyranosyl nucleosides may be compounds of Formula I or Formula II:
    [15]
    [16]
    [17] In Chemical Formulas I and II,
    [18] R 1 is H, OH or Hal, where Hal is Br or Cl,
    [19] R 2 , R 3 and R 4 are independently the same or different from each other, and H, Hal (where Hal is Br or Cl), NR 5 R 6 , OR 7 , SR 8 , wherein R 5 , R 6 , R 7 and R 8 are independently the same or different from each other, and H, C n H 2n + 1 or C n H 2n-1 , wherein n is 1 to 12, preferably 1 to 8, in particular 1 to Is an integer of 4), -C (O) R 9 , wherein R 9 is a straight or branched, substituted or unsubstituted alkyl or aryl radical, preferably a phenyl radical], = O, C n H 2n +1 where n is as defined above or (C n H 2n ) NR 10 R 11 wherein R 10 and R 11 are H or C n H 2n + 1 or R 10 R 11 is Radical):
    [20] X, Y and Z are independently the same or different from each other, and each = N-, = C (R 16 )-or -N (R 17 )-, wherein R 16 and R 17 are independently the same or different Are each H or C n H 2n + 1 or (C n H 2n ) NR 10 R 11 as defined above),
    [21] R 1 ′ is H, OH or Hal, where Hal is Br or Cl,
    [22] R 2 ' , R 3' and R 4 ' are the same or different independently from each other and are each H, Hal (where Hal is Br or Cl), = 0, C n H 2n + 1 or OC n H 2n- 1 or (C n H 2n ) NR 10 ' R 11' , wherein R 10 ' and R 11' are independently of each other R 10 or R 11 as defined above,
    [23] X 'is = N-, = C (R 16' )-or -N (R 17 ' )-, where R 16' and R 17 ' are independently of each other R 16 or R 17 as defined above to be.
    [24]
    [25] In Chemical Formula III,
    [26] R 12 , R 13 , R 14 and R 15 are independently the same or different from each other and are each H, OR 7 (wherein R 7 is as defined above) or C n H 2n + 1 or C n H 2n-1 (Where n is as defined above).
    [27] The process according to the invention is generally carried out at a temperature of about 40 to 70 ° C, preferably about 50 to 60 ° C, in particular about 50 to 55 ° C. Moreover, the reaction is generally carried out under acidic catalysis, for example p-toluenesulfonic acid, methanesulfonic acid, tetrafluoroboric acid, sulfuric acid, acidic ion exchangers [e.g. acidic Amberlite R (Rom and Haas) Rohm & Haas))] and / or Lewis acids (eg zinc chloride, trimethylsilyl triflate or pyridinium paratoluene sulfonate). The reaction time is usually about 1 to 1.5 hours, preferably about 1.5 hours.
    [28] In a further aspect of the process according to the invention, in a further step the 3 ', 4'-cyclic acetal of the pentopyranosyl nucleoside obtained according to the invention can be protected at the 2' position. The 2 'position is preferably a protecting group, preferably base-labile or which can be removed by metal catalysis, according to methods known to those skilled in the art, for example using benzoyl chloride in a dimethylaminopyridine / pyridine solution at room temperature. Preferably protected by acyl groups, in particular acetyl, benzoyl, nitrobenzoyl and / or methoxybenzoyl groups.
    [29] In a further aspect of the method according to the invention, the 3 ', 4'-cyclic acetal of the pentopyranosyl nucleoside protected at the 2' position can be deketalized. In general, deketalization is carried out in the presence of an acid, preferably a strong acid such as trifluoroacetic acid. The workup of the reaction product obtained is preferably carried out in the presence of anhydrous basic conditions, for example solid hydrogen carbonate, carbonates and / or basic ion exchangers such as basic amberlites (Roam and Haas). The workup reaction product can then be purified, for example, on SiO 2 , in particular on SiO 2 in the form of silica gel.
    [30] In a further aspect of the method according to the invention, in a further step the 4 'position can also be protected. Suitable protecting groups are generally acid- or base-labile protecting groups, preferably trityl groups, in particular DMT groups, and / or β-removable groups, in particular Fmoc groups. Introduction of the protecting group is generally carried out by known methods, for example by dimethoxytrityl chloride in the presence of N-ethyldiisopropylamine [Hunig's base].
    [31] In a further aspect of the method according to the invention, in a further step the protective group can be rearranged from the 2 'position to the 3' position. In general, rearrangements are generally known in the presence of a base, in particular N-ethyldiisopropylamine and / or triethylamine, for example N-ethyldiisopropylamine, isopropanol, p-nitrophenol in pyridine And elevated temperature (eg, about 60 ° C.) in the presence of a mixture of dimethylaminopyridine. The product obtained can then be purified by chromatography and / or crystallization on SiO 2 , in particular SiO 2 in the form of silica gel.
    [32] The starting compounds of the described process according to the invention, ie pentopyranosyl nucleosides, are for example first reacted with a protected ribopyranose followed by removal of the protecting group from the ribopyranosyl residue. It can manufacture. This method can be performed, for example, as described in Pitsch et al. (1993), supra, or Pitsch et al. (1995), supra. In order to avoid further time- and material-consuming chromatography, only anomerically pure protected pentopyranose, such as tetrabenzoylpenttopyranose, preferably β-tetrabenzoyl ribopyranose, see R. Jeanloz , J. Am. Chem. Soc. 1948, 70, 4052 is advantageous.
    [33] Thus, another object of the present invention is also
    [34] (a) reacting a protected nucleobase with a protected pentopyranose,
    [35] (b) removing the protecting group from the ribopyranosyl residue that is the product of step (a),
    [36] (c) a process for preparing ribopyranosyl nucleosides, comprising reacting the product of step (b) according to the process of the invention described in more detail above.
    [37] For the preparation of pentopyranosylnucleic acid, the obtained pentopyranosyl nucleosides are phosphitylated for oligomerization or bound to the solid phase for solid phase synthesis in a further step. Phosphotylation can be carried out, for example, by allyl N-diisopropylchlorophosphoramidite in the presence of a base such as N-ethyldiisopropylamine. The binding of the protected pentopyranosyl nucleosides according to the invention to the solid phase [eg long chain alkylamino controlled pore glass [CPG, manufactured by Sigma Chemie, Munich] is described, for example, in literature. [Pitsch et al. (1993), supra.
    [38] Thus, another subject of the present invention
    [39] (a) a first step of preparing pentopyranosyl nucleosides according to the method according to the invention described above,
    [40] (b) a second step of binding the pentopyranosyl nucleoside prepared according to step (a) to a solid phase,
    [41] (c) an additional step of stretching the pentopyranosyl nucleoside bound to the solid phase according to step (b) with a phosphitylated 3 ', 4'-protected pentopyranosyl nucleoside and
    [42] (d) repeating step (c) with the same or different phosphitylated 3 ', 4'-protected pentopyranosyl nucleosides until the desired pentopyranosyl nucleoside is obtained, A method for producing pentopyranosylnucleic acid.
    [43] In certain embodiments, pentofuranosylnucleosides, such as adenosine, guanosine, cytidine, thymidine, which are customary for nucleic acid synthesis generally known in steps (b) and / or (c), and And / or uracil [Uhlmann, E. & Peyman, A. (1990). Chemical Reviews, 90, 543-584 No. 4] can also be introduced in such a way that mixed nucleic acids consisting of pentopyranosyl nucleosides and pentofuranosylnucleosides with novel properties are formed.
    [44] The coupling agent used for the extension according to step (c) is generally an acidic activator, preferably 5- (4-nitrophenyl) -1H-tetrazole, in particular benzimidazolium triflate, as a coupling agent 5 In contrast to-(4-nitrophenyl) -1H-tetrazole, benzimidazolium triflate does not cause blocking of the coupling reagent line and contamination of the product.
    [45] It is also advantageous to protect nucleobases, in particular pyrimidine bases, in particular uracil and thymine, from ring-opening reactions which can add oligonucleotides to salts such as sodium chloride in the hydrazine degradation reaction to remove the protecting groups. The allyloxy group can preferably be removed prior to the hydrazine decomposition reaction, for example by a palladium [Pd (O)] complex.
    [46] In addition, nucleic acids formed from the solid phase are generally removed by hydrazine degradation reactions.
    [47] In a further aspect according to the invention, the protecting group and pentopyranosylnucleic acid formed in further step (e) are removed from the solid phase.
    [48] In general, pentopyranosylnucleic acid prepared according to the invention is purified on chromatography, for example on alkylsilylated silica gel, preferably on RP-C 18 silica gel.
    [49] 3 provides an exemplary general schematic of a method according to the invention comprising further aspects.
    [50] Pentopyranosylnucleic acids prepared according to the invention are suitable, for example, for the preparation of pairing systems or conjugates.
    [51] Such pairing systems are supramolecular systems of non-covalent interactions distinguished by selectivity, stability and reversibility, the properties of which are preferably thermodynamically ie affected by temperature, pH and concentration. In view of the optional properties of the fairing system, such a system is also used as a "molecular coalescing agent" for, for example, bridging different metal clusters together to provide cluster combinations with potentially novel properties. May be used. See, for example, BRL Letsinger, et al., Nature 1996, 382, 607-9; P.G. Schultz et al., Nature 1996, 382, 609-11. As a result, p-NA is also suitable for use in the field of nanotechnology, for example, to manufacture novel materials, diagnostics and therapeutics, and also to manufacture components for microelectronics, photonics or optoelectronics. Suitable for use in deriving together molecular species to provide supramolecular units such as, for example, (combinational) synthesis of protein assemblies in BA Lombardi, JW. Bryson, W. F. DeGrado, Biomolekuls (Pept. Sci.) 1997, 40, 495-504, because p-NAs form a potent and thermodynamically adjustable pairing system. Thus, due to the possibility of providing functional units, preferably biological units such as proteins or DNA / RNA fractions, having a p-NA code that does not interfere with natural nucleic acids, in particular in the field of diagnostics and drug discovery It may also be applied in addition to WO 93/20242.
    [52] In addition, if a biomolecule, for example DNA or RNA, contains both fractions that can bind to each other by the formation of a hydrogen bridge due to the complementary sequence of the nucleobase, another biomolecule, for example DNA or RNA, It can be used to non-covalently bind (link) to DNA or RNA. Biomolecules of this type are used, for example, in assay systems for signal amplification, wherein a DNA molecule having the sequence to be analyzed is immobilized on a solid support on the one hand through the non-covalent DNA linker on the one hand and on the other hand To a signal-amplified branched DNA molecule (bDNA). FIG. 3; S. Urdea, Biol / Technol. 1994, 12, 926 or US Pat. No. 5,624,802]. The main disadvantage of the last mentioned systems is that these systems are still inferior to nucleic acid diagnostic methods by polymerase chain reaction (PCR) in terms of sensitivity. K. Mullis, Methods Enzymol. 1987, 155, 335]. This is especially true because the non-covalent binding of the solid support to the DNA molecule to be analyzed results in a mixture of "sequence recognition" and "non-covalent bond" functions, such as the non-covalent bond of the DNA molecule to be analyzed. This is due to the fact that it does not always occur specifically. The use of p-NA as an orthogonal pairing system that does not interfere with DNA or RNA pairing methods solves this problem in an advantageous way, since the sensitivity of the analytical methods described above can be significantly increased.
    [53] Within the scope of the present invention the conjugate is a p-NA and other biomolecules, preferably peptides, proteins or nucleic acids, such as antibodies or functional residues thereof, or covalently linked hybrids of DNA and / or RNA in their native form. to be. Functional residues of an antibody include, for example, Fv fragments (Skerra & Pluckthun (1988) Science 240, 1038), single chain Fv fragments [scFv; Bird et al. (1988), Science 242, 423; Huston et al. (1988) Proc. Natl. Acad. Sci. USA, 85, 5879] or Fab fragments [Better et al. (1988) Science 240, 1041. In general, p-RNA / DNA or p-RNA / RNA conjugates are preferred.
    [54] The conjugate is preferably used only when the "sequence recognition" function and the "non-covalent bond" function have to be performed in one molecule, since the conjugate contains two pairing systems orthogonal to each other.
    [55] Conjugates within the scope of the present invention are also recognized to mean so-called arrays. An array is an array of immobilized recognized species that plays an important role, in particular, for simultaneous measurement of analytes during analysis and diagnosis. Examples thereof include peptide arrays (Fodor et al., Nature 1993, 364, 555) and nucleic acid arrays (Southern et al., Genomics 1992, 13, 1008; Heller, US Patent No. 5,632,957. Higher flexibility of these arrays can be achieved by combining the recognition species encoding the oligonucleotides with the complementary strands bound at specific locations on the solid support. By applying the recognition species encoded as above to a "anti-encrypted" solid support and setting the hybridization conditions, the recognition species are non-covalently bound to the desired position. As a result, various types of recognition species, such as DNA fractions, antibodies, and the like can be arranged simultaneously on a solid support using hybridization conditions. However, as a prerequisite for this, codons and anticodons are needed to maintain coding fractions as short as possible, which are very powerful, selective and do not interfere with natural nucleic acids. p-NA, preferably p-RNA, is particularly advantageously suitable for this purpose.
    [56] Both sequential and convergent methods are suitable for the preparation of the conjugates, which is particularly preferred because of its flexibility.
    [57] In the sequential method, p-RNA oligomers are automatically synthesized directly on the same synthesizer, after adjusting the reagent and coupling protocol, for example further synthesis of DNA oligonucleotides. This method may also be performed in the reverse order.
    [58] In a convergent method, for example, a p-RNA oligomer having an amino-terminal linker and a DNA oligomer having a thiol linker, for example, are synthesized by separate methods. Then, see T. Zhu et al., Bioconjug. Chem. 1994, 5, 312, it is preferred to carry out the coupling of the two units and the iodoacetylation of the p-RNA oligomers according to a protocol known from.
    [59] Particularly preferred amino-terminal linkers are allyloxy linkers of formula IV:
    [60] S c1 NH (C n H 2n ) CH (OPS c2 S c3 ) C n H 2n S c4
    [61] In Formula IV,
    [62] S c1 and S c4 , independently of one another, are the same or different and each is a protecting group selected from a protecting group, in particular Fmoc and / or DMT,
    [63] S c2 and S c3 are the same or different independently of each other, and are each allyloxy and / or diisopropylamino group,
    [64] n is an integer of 1 to 12, preferably 1 to 8, in particular 1 to 4.
    [65] Particularly preferred allyloxy linkers are 2- (S) -N-Fmoc-O 1 -DMT-O 2 -allyloxydiisopropylaminophosphinyl-6-amino-1,2-hexanediol.
    [66] 2- (S) -N-Fmoc-O 1 -DMT-O 2 -allyloxydiisopropylaminophosphinyl-6-amino-1,2-hexanediol is, for example, 6-amino-2 (S) It can be prepared from hydroxyhexanoic acid. 6-Amino-2 (S) -hydroxyhexanoic acid is described by K.-I. Aketa, Chem. Pharm. Bull. 1976, 24, 621 can be prepared from L-lysine by diazotization in a manner known in the art and then by hydrolysis. This was then reacted with FmocCl to afford 2- (S) -N-Fmoc-6-amino-1,2-hexanediol, which was then DM-tritylated according to WO 89/02439 to give 2- ( S) -N-Fmoc-O 1 -DMT-6-amino-1,2-hexanediol can be obtained. It is reacted, for example, in the presence of ethyldiisopropylamine and chloro-N, N-diisopropylaminoallyloxyphosphine, so that 2- (S) -N-Fmoc-O 1 -DMT-O 2 -allyl Obtain oxydiisopropylaminophosphinyl-6-amino-1,2-hexanediol.
    [67] In some reaction steps, for example, starting from lysine, an amino-terminal linker carrying both an activatable phosphorus compound and an acid-labile protecting group such as DMT can be synthesized, which results in automated oligonucleotide synthesis. Can be readily used. See BPS Nelson et al., Nucleic Acid Res. 17 , 7179 (1989); LJ Arnold et al., WO 89/02439. Lysine-based linkers in which allyloxy groups have been introduced onto the personnel instead of other conventional cyanoethyl groups can be advantageously used in the Noyori oligonucleotide method. See R. Noyori, J. Am. Chem. Soc. 112 , 1691-6 (1990).
    [68] In a further embodiment of the process according to the invention for producing pentopyranosnonucleic acid, in a further step an allyloxy linker of formula IV is introduced:
    [69] Formula IV
    [70] S c1 NH (C n H 2n ) CH (OPS c2 S c3 ) C n H 2n S c4
    [71] In Formula IV,
    [72] S c1 and S c4 , independently of one another, are the same or different and each is a protecting group selected from a protecting group, in particular Fmoc and / or DMT,
    [73] S c2 and S c3 are the same or different independently of each other, and are each allyloxy and / or diisopropylamino group,
    [74] n is an integer of 1 to 12, preferably 1 to 8, in particular 1 to 4.
    [75] Indole derivatives as linkers (compounds of formula (I) combined with compounds of formula (III)) also have the advantage of the ability to fluoresce and are therefore particularly preferred for nanotechnology applications in which trace amounts of substances must be detected. For example, already described in NN Suvorov et al., Biol. Aktivn. Soedin., Akad. Indole-1-ribosides as described in Nauk SSSR, 60 (1965) and Tetrahedron 23 , 4653 (1967) are suitable. In general, 3-substituted derivatives are prepared through the amine formation of the unprotected sugar component and indolin, and then converted to indole-1-riboside by oxidation. For example, indole-1-glucoside and -1-arabinosides are described in YV Dobriynin et al. Khim.-Farm Zh. 12 , 33 (1978), the 3-substituted derivatives thereof can be prepared conventionally via the Vielsmeier's reaction.
    [76] To prepare indole linkers, for example, the starting materials used are phthalic anhydride and trytamine, which react to form N-phthaloyltrytamine. See Kuehne et al., J. Org. Chem. 43 , 13, 1978, 2733-2735 (1987). This is reduced to indole, for example using borane-THF. See A. Giannis et al., Angew. Chem. 101 ; 220 (1989). Subsequently, the 3-substituted indolin is first reacted with ribose to give a nucleoside triol, followed by acetic anhydride to give triacetate. This is then oxidized using, for example, 2,3-dichloro-5,6-dicyanoparaquinone, the acetate is cleaved, for example with sodium methoxide, and then optionally benzoylated at the 2'-position. And optionally DM-tritylated at the 4'-position, followed by a transfer reaction to give 3'-benzoate. Phosphoramidites are formed according to known methods. It can be used for automated oligonucleotide synthesis without altering the synthesis protocol.
    [77] Further linkers suitable for the process according to the invention (see eg compounds of formula II in combination with compounds of formula III) are uracil-based linkers in which the 5 'position of uracil is modified. Suitable examples are N-phthaloylaminoethyluracil, which can be obtained from hydroxyethyluracil.
    [78] Hydroxyethyluracil is known from known methods [JD Fissekis, A. Myles, GB Brown, J. Org. Chem. 29 , 2670 (1964). Then, for example, the obtained hydroxyethyluracil is mesylated with methanesulfonyl chloride in pyridine. See JD Fissekis, F. Sweet, J. Org. Chem. 38 , 264 (1973). Generally, the reaction product is reacted with sodium azide in DMF to give an azide, which is reduced with triphenylphosphine in pyridine to give aminoethyluracil. The amino functionality is finally protected using, for example, N-ethoxycarbonylphthalimide. Ribose tetrabenzoate is nucleosided with N-phthaloylaminoethyluracil to give, for example, ribose tribenzoate linker in good yield. Subsequently, NaOMe in MeOH can be used to remove the benzoate protecting group to give a linker triol, which can be reacted with benzoyl chloride at -78 ° C in pyridine / dichloromethane 1:10 in the presence of DMAP. In addition to the desired 2'-benzoate (64%), a 2 ', 4'-dibenzoylation product (22%) is obtained, which can be collected and then converted back to triol. The 2'-benzoate is tritylated at the 4'-position with dimethoxytrityl chloride in excess of 90%, for example in the presence of Hunig base in dichloromethane. Rearrangement of 4'-DMT-2'-benzoate to 4'-DMT-3'-benzoate, for example, of DMAP, p-nitrophenol and Hunig base in n-propanol / pyridine 5: 2 Perform in presence. After chromatography, 4'-DMT-3'-benzoate is obtained, which is finally reacted with ClP (OAll) N (iPr) 2 , for example in the presence of Hunig's base to form phosphoramidite. To obtain. It can be used for automated oligonucleotide synthesis without changing the synthesis protocol.
    [79] The following figures and examples are intended to describe the invention in more detail without restricting the invention.
    [80] Figure 1 shows a cross section of a native form of RNA (left) and a p-NA form of RNA (right).
    [81] 2 is a schematic representation of the synthesis of p-ribo (A, U) -oligonucleotides according to Pitsch et al. (1993).
    [82] 3 diagrammatically shows a method according to the invention comprising further aspects.
    [83] Definition of Acronyms
    [84] BSA is bis (trimethylsilyl) acetamide,
    [85] TMS-OTf is trimethylsilyl trifluoromethylsulfonate,
    [86] AllOH is allyl alcohol,
    [87] DMF is dimethylformamide,
    [88] Bz is benzoyl,
    [89] ibu is isobutyryl,
    [90] THF is tetrahydrofuran,
    [91] TsOH is toluenesulfonic acid,
    [92] DMT is dimethoxytrityl,
    [93] DMAP is dimethylaminopyridine,
    [94] CPG is controlled pore glass.
    [95] Example
    [96] Sequence G 3CG 3Synthesis of oligomers of C
    [97] Produce:
    [98] The amount of phosphoramidite required for the synthesis of the planned sequence (320 μl / coupling step) and the amount of tetrazole mixture required (650 μl / 0.35M tetrazol / 0.15M p-nitrophenyltetrazole solution) ) Are weighed into synthesizer vials and dried in KOH flakes (dryer) under high vacuum for at least 14 hours. The mixture is then dissolved in the required volume of CH 3 CN and mixed with about 5 beads of 4 μs of activated molecular sieve. The vial is sealed with a septum and further stored at room temperature for at least 14 hours.
    [99] synthesis:
    [100] Sequence synthesis on a Pharmacia gene assembler is carried out in principle identically to the synthesis of DNA oligonucleotides according to the standard conditions of the automated equipment manufacturer PHARMACIA, D-Freiburg. In synthesis, the final trityl group remains on the oligonucleotide (trityl residues). The following changes are introduced for standard conditions:
    [101] 1. Detritylation time is extended to 7 minutes.
    [102] 2. A 6% concentration (instead of 3% concentration) solution of dichloroacetic acid in dichloroethane is used.
    [103] 3. The coupling time is extended to 30 minutes.
    [104] For the synthesis, the starting material used was 400 mg (= 10 μmol) of support loaded with p-ribo-C component.
    [105] Deprotection:
    [106] After synthesis, the support in the cartridge was dried under vacuum (about 30 minutes), 60 mg (66 μmol) of tetrakis (triphenylphosphine) palladium (O) in 4.5 ml of CH 2 Cl 2 , 60 mg (225 μmol) of triphenylphosphine and Treated with a solution of 60 mg (170 μmol) of diethylammonium hydrogencarbonate. The mixture is shaken for 5 hours at room temperature (RT), then the support is filtered and washed successively with 20 ml of CH 2 Cl 2 and 25 ml of acetone. It is dissolved in 4.5 ml of 0.1 M sodium N, N-diethyldithiocarbamate solution and left to stand at room temperature for 30 minutes, then filtered again, followed by continuous washing with 10 ml of H 2 O, 15 ml of acetone and 10 ml of EtOH. .
    [107] The support is then dissolved in 3.6 ml of H 2 O / 0.9 ml of hydrazine hydrate and circulated at 4 ° (cold temperature) by a motor for 30 minutes. Then chromatograph on 1 × 4 cm RP-C 18 silica gel. To this end, the column material is suspended in CH 3 CN and packed in a column (1 × 5 cm). This is adjusted with 50 ml of CH 3 CN, 50 ml of 2% NEt 3 in CH 3 CN, followed by 50 ml of 0.1 M TEAB buffer. The sample is then applied to 0.1 M TEAB buffer and eluted with 50 ml of 0.1 M TEAB buffer, 30 ml of H 2 O, followed by H 2 O → H 2 O / CH 3 CN (1: 1). The product is detected by UV photometry. The product-containing fractions were evaporated, dissolved in 15 ml of H 2 O / HCOOH (1: 4), left at room temperature for 15 minutes, evaporated to 5 ml of H 2 O, then evaporated again and Sepak ( Chromatography on Waters). To do this, the cartridge is washed with 15 ml of CH 3 CN and then 15 ml of 0.1 M TEAB buffer. The oligonucleotide containing solution is applied to 0.1 M TEAB buffer, then eluted with 10 ml of 0.01 M TEAB buffer, followed by a H 2 O / CH 3 CN gradient (0 → 50%). 1.5 ml fractions are collected and analyzed in UV.
    [108] The product containing fractions are evaporated and purified by HPLC chromatography. The combined product-containing fractions are desalted on Sepak as described above. The product is stored frozen as an aqueous solution (10 ml). Yield by UV spectroscopy was 25% (= 250 o.D.).
    [109] Specialization:
    [110] HPLC: retention time on Aquapore RP-300 15.3 min, 7 μm, 220 × 4.6 mm, flow 1 ml / min; Buffer A: H 2 O in 0.1M NEt 3 /0.1M AcOH (pH 7.0 ), Buffer B: H 2 O / CH 3 CN (1: 4) in 0.1M NEt 3 /0.1M AcOH (pH 7.0 ); Gradient: 100% A → 70% A / 30% B. Detection 260 nm.
    [111] UV: λ max : 257 nm.
    [112] MALDI-TOF-MS: [M-1] calculated = 2617; [M-1] observation = 2617.
    权利要求:
    Claims (31)
    [1" claim-type="Currently amended] A process for preparing 3 ', 4'-cyclic acetal of pentopyranosyl nucleosides comprising reacting pentopyranosyl nucleosides with aldehydes, ketones, acetals or ketals under reduced pressure.
    [2" claim-type="Currently amended] The process according to claim 1, wherein the reaction is carried out at less than about 500 mbar, preferably less than about 100 mbar, in particular less than about 50 mbar, more particularly at about 30 mbar.
    [3" claim-type="Currently amended] The process of claim 1 or 2, wherein the aldehyde is selected from formaldehyde, acetaldehyde, benzaldehyde and 4-methoxybenzaldehyde, and acetal is formaldehyde dimethyl acetal, acetaldehyde dimethyl acetal, benzaldehyde dimethyl acetal and 4-methoxybenzaldehyde Selected from dimethyl acetal, ketone is selected from acetone, cyclopentanone and cyclohexanone, ketal is selected from acetone dimethyl ketal, cyclopentanone dimethyl ketal and cyclohexanone dimethyl ketal or in the form of 2-methoxypropene Way.
    [4" claim-type="Currently amended] The method of claim 1, wherein the pentopyranosyl nucleoside is purified prior to reaction.
    [5" claim-type="Currently amended] The process according to claim 4, wherein the pentopyranosyl nucleoside is purified on SiO 2 , preferably in neutralized form.
    [6" claim-type="Currently amended] The method according to any one of claims 1 to 5, wherein the pentopyranosyl nucleosides used are ribo-, arabino-, lyxo- or xylopyranosylnucleosides.
    [7" claim-type="Currently amended] The pentopyranosyl nucleoside to be used is pentopyranosylpurine, -2,6-diaminopurine, -6-purinethiol, -pyridine, -pyrimidine, -Adenosine, -Guanosine, -Isoguanosine, -6-Tioguanosine, -Xanthine, -Hypoxanthine, -Thymidine, -Cytocin, -Isocytosine, -Indole, -Tryptamine, -N-phthalo Yltriptamine, -uracil, -caffeine, -theobromine, -theophylline, -benzotriazole or -acridine.
    [8" claim-type="Currently amended] 8. The method of any one of claims 1 to 7, wherein the pentopyranosyl nucleoside is a compound of formula (I) or formula (II).
    Formula I

    Formula II

    In Chemical Formulas I and II,
    R 1 is H, OH or Hal, where Hal is Br or Cl,
    R 2 , R 3 and R 4 are independently the same or different from each other, and H, Hal (where Hal is Br or Cl), NR 5 R 6 , OR 7 , SR 8 , wherein R 5 , R 6 , R 7 and R 8 are independently the same or different from each other, and H, C n H 2n + 1 or C n H 2n-1 , wherein n is 1 to 12, preferably 1 to 8, in particular 1 to Is an integer of 4), -C (O) R 9 , wherein R 9 is a straight or branched, substituted or unsubstituted alkyl or aryl radical, preferably a phenyl radical], = O, C n H 2n +1 where n is as defined above or (C n H 2n ) NR 10 R 11 wherein R 10 and R 11 are H or C n H 2n + 1 or R 10 R 11 is Radical):
    X, Y and Z are independently the same or different from each other, and each = N-, = C (R 16 )-or -N (R 17 )-, wherein R 16 and R 17 are independently the same or different Are each H or C n H 2n + 1 or (C n H 2n ) NR 10 R 11 as defined above),
    R 1 ′ is H, OH or Hal, where Hal is Br or Cl,
    R 2 ' , R 3' and R 4 ' are the same or different independently from each other and are each H, Hal (where Hal is Br or Cl), = 0, C n H 2n + 1 or OC n H 2n- 1 or (C n H 2n ) NR 10 ' R 11' wherein R 10 ' and R 11' are independently of each other R 10 or R 11 as defined above,
    X 'is = N-, = C (R 16' )-or -N (R 17 ' )-, where R 16' and R 17 ' are independently of each other R 16 or R 17 as defined above to be.
    Formula III

    In Chemical Formula III,
    R 12 , R 13 , R 14 and R 15 are independently the same or different from each other and are each H, OR 7 (wherein R 7 is as defined above) or C n H 2n + 1 or C n H 2n-1 (Where n is as defined above).
    [9" claim-type="Currently amended] 9. The process according to claim 1, wherein the reaction is carried out at a temperature of about 40 to 70 ° C., preferably about 50 to 60 ° C., in particular about 50 to 55 ° C. 10.
    [10" claim-type="Currently amended] The process according to claim 1, wherein the reaction is carried out in the presence of an acid.
    [11" claim-type="Currently amended] The method of claim 10, wherein the acid is selected from p-toluenesulfonic acid, methanesulfonic acid, tetrafluoroboric acid, sulfuric acid, acidic ion exchangers and / or Lewis acids.
    [12" claim-type="Currently amended] The method according to any one of claims 1 to 11, wherein the 3 ', 4'-cyclic acetal of the pentopyranosyl nucleoside obtained is protected at the 2' position in a further step.
    [13" claim-type="Currently amended] 13. The position according to claim 12, wherein the 2 'position is protected by a protecting group, preferably an acyl group, in particular an acetyl, benzoyl, nitrobenzoyl and / or methoxybenzoyl group, which may be base-labile or removed by metal catalysis. Way.
    [14" claim-type="Currently amended] A process for preparing pentopyranosyl nucleosides, comprising deketalizing 3 ', 4'-cyclic acetals of a protected pentopyranosyl nucleoside at the 2' position as described in claim 12 or 13. .
    [15" claim-type="Currently amended] 15. The process according to claim 14, wherein the deketalization is carried out in the presence of an acid, preferably a strong acid.
    [16" claim-type="Currently amended] The process according to claim 14 or 15, wherein the reaction product obtained is worked up under anhydrous basic conditions.
    [17" claim-type="Currently amended] 17. The process of claim 16, wherein the anhydrous basic workup is performed in the presence of solid hydrogen carbonate, carbonate and / or basic ion exchanger.
    [18" claim-type="Currently amended] 18. The method of any one of claims 14-17, wherein the 4 'position is protected in a further step.
    [19" claim-type="Currently amended] 19. The method according to claim 18, wherein the 4 'position is protected by an acid- or base-labile protecting group, preferably a trityl group, in particular a DMT group, and / or a β-removable group, in particular a Fmoc group.
    [20" claim-type="Currently amended] 20. The method of claim 18 or 19, wherein rearrangement of the protecting group from the 2 'position to the 3' position is performed in a further step.
    [21" claim-type="Currently amended] The process according to claim 20, wherein the rearrangement is carried out in the presence of a base, in particular N-ethyldiisopropylamine and / or triethylamine.
    [22" claim-type="Currently amended] (a) reacting a protected nucleobase with a protected pentopyranose,
    (b) removing the protecting group from the pentopyranosyl residue of the product of step (a),
    (c) reacting the product of step (b) according to the method according to any one of claims 1 to 21, a process for preparing pentopyranosyl nucleosides.
    [23" claim-type="Currently amended] 23. The process according to any one of claims 20 to 22, wherein the pentopyranosyl nucleoside obtained is phosphtylated or bound to a solid phase in a further step.
    [24" claim-type="Currently amended] (a) a first step of preparing a pentopyranosyl nucleoside according to any one of claims 20 to 22,
    (b) a second step of binding the pentopyranosyl nucleoside prepared according to step (a) to a solid phase,
    (c) expanding the 3 ', 4'-protected pentopyranosyl nucleoside bound to the solid phase according to step (b) by phosphitylated 3', 4'-protected pentopyranosyl nucleoside Additional steps and
    (d) repeating step (c), a method for producing pentopyranosylnucleic acid.
    [25" claim-type="Currently amended] The method of claim 24, wherein at least one pentofuranosyl nucleoside is introduced further into step (b) and / or step (c).
    [26" claim-type="Currently amended] Process according to claim 24 or 25, wherein the coupling agent used for expansion according to step (c) is an acidic activator, preferably p-nitrophenyltetrazole, in particular benzimidazolium triflate.
    [27" claim-type="Currently amended] 27. The process according to any one of claims 24 to 26, wherein the protective groups and oligomers formed are removed from the solid phase in a further step (e).
    [28" claim-type="Currently amended] The method of claim 27, wherein the removal reaction is performed by a hydrazine decomposition reaction.
    [29" claim-type="Currently amended] The method according to claim 27 or 28, wherein the oligomer obtained is purified by chromatography.
    [30" claim-type="Currently amended] 30. The process according to claim 29, wherein the chromatography purification is carried out on alkylsilylated silica gel, preferably RP-C 18 silica gel.
    [31" claim-type="Currently amended] 31. The process of any of claims 24-30, wherein the allyloxylinker of formula IV is introduced in a further step.
    Formula IV
    S c4 NH (C n H 2n ) CH (OPS c5 S c6 ) C n H 2n S c7
    In Formula IV,
    S c4 and S c7 are the same or different independently from each other, and are each a protecting group selected from a protecting group, in particular Fmoc and / or DMT,
    S c5 and S c6 , independently of one another, are the same or different and each is an allyloxy and / or diisopropylamino group,
    n is an integer of 1 to 12, preferably 1 to 8, in particular 1 to 4.
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    同族专利:
    公开号 | 公开日
    JP2002511479A|2002-04-16|
    CA2327436A1|1999-10-21|
    WO1999052923A3|2000-01-13|
    AT259824T|2004-03-15|
    EP1070079B1|2004-02-18|
    AU755533B2|2002-12-12|
    US6545134B1|2003-04-08|
    AU3706499A|1999-11-01|
    DE19815901A1|1999-10-14|
    EP1070079A2|2001-01-24|
    BR9909909A|2000-12-26|
    WO1999052923A2|1999-10-21|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1998-04-08|Priority to DE19815901A
    1998-04-08|Priority to DE19815901.3
    1999-04-07|Application filed by 렐케, 아벤티스 레제아르히 운트 테히놀로기스 게엠베하 운트 콤파니 카게
    1999-04-07|Priority to PCT/EP1999/002356
    2001-08-04|Publication of KR20010074475A
    优先权:
    申请号 | 申请日 | 专利标题
    DE19815901A|DE19815901A1|1998-04-08|1998-04-08|Process for the preparation of pentopyranosyl nucleosides|
    DE19815901.3|1998-04-08|
    PCT/EP1999/002356|WO1999052923A2|1998-04-08|1999-04-07|Method for the production of pentopyranosyl nucleosides|
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