Classifications

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  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • A61K49/0013—Luminescence
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  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
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  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
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  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/531—Production of immunochemical test materials
  • G01N33/532—Production of labelled immunochemicals
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  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
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  • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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  • Y10S977/724—Devices having flexible or movable element
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Definitions

• the present invention relates to a pentopyranosyl nucleoside of the formula (I) or of the formula (II)
• Pyranosyl nucleic acids are generally structure types isomeric to natural RNA, in which the pentose units are in the pyranose form and are repetitively linked by phosphodiester groups between positions C-2 'and C-4' (Fig. 1) .
• “Nucleobase” here means the canonical nucleobases A, T, U, C, G, but also the pairs isoguanine / isocytosine and 2,6-diaminopurine / xanthine and, within the meaning of the present invention, also other purines and pyrimidines.
• p-NAs namely the p-RNAs derived from the ribose, were first described by Eschenmoser et al.
• a suitable protected nucleobase was reacted with the anomer mixture of tetrabenzoyl-ribopyranose by the action of B ⁇ s (trimethylsilyl) acetam ⁇ d and a Lewis acid such as trimethylsilyl-trifluoromethanesulfonate (analogue H Vorbruggen, K Krolikiewicz, B Bennua, Chem Ber 1981, 1 14, 1234)
• bases NaOH in THF methanol / water in the case of Pu ⁇ ne, saturated ammonia in MeOH in the case of pyrimidines
• the acyl protective groups were split off from the sugar, and the product under acidic catalysis with p-anisaldehyde dimethyl acetal in 3 ', 4 '-Position protected
• the mixture of diastereomers was acylated in the 2'-position, the 3', 4'-methoxybenzylidene-protected 2'-benzoate was deace
• 5- (4-nitrophenyl) -IH-tetrazole is used as a coupling reagent in the automated p-RNA synthesis.
• concentration of this reagent in the solution of tetrazole in acetonitrile is so high that the 5- (4-nitrophenyl) lH-tetrazole regularly crystallizes out in the thin tubes of the synthesizer and the synthesis thus comes to an early end. It was also observed that the oligomers were contaminated with 5- (4-nitrophenyl) -IH-tetrazole.
• Thymidine content not always in the oligomer.
• a biomolecule e.g. B. DNA or RNA
• a DNA molecule to be analyzed in its sequence is immobilized on the one hand on a solid support via such a non-covalent DNA linker, and on the other hand bound to a signal-enhancing branchedDNA molecule (bDNA)
• bDNA signal-enhancing branchedDNA molecule
• a major disadvantage of the systems described last is that they have so far been inferior in sensitivity to the methods for nucleic acid diagnosis by polymerase chain reaction (PCR) (K. Mullis, Methods Enzymol. 1987, 155, 335).
• the object of the present invention was therefore to provide new biomolecules and a method for their production, in which the disadvantages described above can be avoided.
• p-NAs as an orthogonal pairing system, which does not interfere with the DNA or RNA pairing process, solves this problem in an advantageous manner, as a result of which the sensitivity of the analytical methods described can be significantly increased.
• the present invention therefore relates to the use of pentopyranosyl nucleosides or pentopyranosyl nucleic acids, preferably in the form of a conjugate containing a pentopyranosyl nucleotide or a pentopyranosyl nucleic acid and a biomolecule for producing an electronic component, in particular in the form of a diagnostic agent.
• conjugates are covalently bound hybrids of p-NA's and other biomolecules, preferably a peptide, protein or a nucleic acid, for example an antibody or a functional part thereof or a DNA and / or RNA functional part of naturally occurring Antibodies are, 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 Sei USA, 85, 5879) or Fab fragments (Better et al (1988) Science 240, 1041)
• Biomolecule in the sense of the present invention means a naturally occurring substance or a substance derived from a naturally occurring substance
• these are p-RNA / DNA or p-RNA / RNA conjugates
• Conjugates are preferably used when the functions
• p-NA's and especially the p-RNA's form stable duplexes with one another and generally do not pair with the DNA 's and RNA's occurring in their natural form. This property makes p-NA's preferred pairing systems
• Such pairing systems are supramolecular systems of non-covalent interaction, which are characterized by selectivity, stability and reversibility, and whose properties are preferably influenced thermodynamically, ie by temperature, pH and concentration.
• Such pairing systems can also be used, for example, as a “molecular adhesive” due to their selective properties 'can be used to combine different metal clusters into cluster assemblies with potentially new properties [see, for example, BRL Letsinger, et al, Nature 1996, 382, 607-9, PG Schultz et al, Nature 1996, 382, 609-11]
• p-NAs are also suitable for use in the field of nanotechnology, for example for the production of new materials, diagnostics and therapeutics as well as microelectronic, photonic or optoelectronic components and for the controlled assembly of molecular species into supramolecular units, such as for the (combinatorial) construction of protein assemblies [see, for example, Lombardi, JW Bryson, WF DeGrado, Biomolek
• p-RNA oligomers with amino terminal linkers and, for example, DNA oligomers with, for example, thiol linkers are synthesized in separate processes.
• the i-acetylation of the p-RNA oligomer is then preferably carried out and the coupling of the two units Protocols known from the literature (T. Zhu et al., Bioconjug. Chem 1994, 5, 312). Convergent methods have proven to be particularly preferred on account of their flexibility
• conjugate in the sense of the present invention also means so-called arrays.
• arrays are arrays of immobilized recognition species that play an important role in the simultaneous determination of analytes, especially in analysis and diagnostics. Examples are peptide arrays (Fodor et al, Nature 1993, 364, 555) and nucleic acid arrays (Southern et al Genomics 1992, 13, 1008, Heller, US Pat. No. 5,632,957) Greater flexibility of these arrays can be achieved by binding the recognition species to coding oligonucleotides and the associated complementary strands at specific positions on a fixed support by applying the coded ones Recognition species on the "anti-coded" fixed supports and setting of hybridization conditions, the recognition species are non-covalently bound at the desired positions.
• carrier is understood to mean material, in particular chip material, which is in solid or gel-like form.
• suitable carrier materials are ceramics, metal, in particular noble metal, glazers, plastics, small-scale materials or thin layers of the carrier, especially the materials mentioned, or (b ⁇ o) molecular filaments such as cellulose, framework proteins
• the present invention therefore also relates to the use of pentopyranosyl nucleic acids, preferably ribopyranosyl nucleic acids for coding recognition species, preferably natural DNA or RNA strands or proteins, in particular antibodies or functional parts of antibodies.
• pentopyranosyl nucleic acids preferably ribopyranosyl nucleic acids for coding recognition species, preferably natural DNA or RNA strands or proteins, in particular antibodies or functional parts of antibodies.
• the analyte becomes If, for example, a biological sample such as serum or the like is applied, then the species to be detected are bound in a specific pattern on the array, which is then indirect (for example by fluorescent labeling of the recognition species) or directly (for example by measuring the impedance at the connecting point) If the codons are registered) then the hybridization is canceled by a suitable condition (temperature, salts, solvents, electrophoretic processes) so that only the carrier remains with the codons. This is then loaded again with other recognition species and becomes, for example, the same analyte used for the determination of another pattern
• a biological sample such as serum or the like
• the pentopyranosyl nucleoside is a compound of the formula (I)
• R, ⁇ is H, OH, shark with shark is Br or Cl or a residue selected from
• R 12 , R 13 , R 14 and R 1 are, independently of one another, identical or different, in each case H, OR 7 , where R 7 has the meaning given above, or C n H 2n + ⁇ , or C “H 2n - ⁇ , where n has the meaning given above, mean and
• R 1 is H, OH, shark with shark is Br or Cl, or a radical selected from
• the pentopyranosyl nucleoside is generally a ribo-, arabino-, lyxo- and / or xylopyranosyl-nucleoside, preferably a ribopyranosyl-nucleoside, whereby the pentopyranosyl part can be D-configured, but also L-configured
• the pentopyranosyl nucleoside according to the invention is usually a pentopyranosyl-purine, -2,6-diaminopurine, -6-purinthiol, -pyridine, -pyrimidine, - adenosine, -guanosine, -isoguanosine.
• -6-thioguanosine -xanthine, -hypoxanthine, -thymidine, - cytosine, -isocytosine, -indole, -tryptamine, -N-phthaloyltryptamine, -uracil, -caffeine, - theobromine, -theophylline, -benzotriazole or -acridine, in particular a pentopyranosyl purine, pyrimidine, adenosine, guanosine, thymidine, cytosine, tryptamine, N-phthalotryptamine or uracil
• the compounds also include pentopyranosyl nucleosides which can be used as linkers, i.e. as compounds with functional groups that are covalently attached to biomolecules, such as, for example, naturally occurring or modified nucleic acids, such as DNA, RNA, but also p-NAs, preferably pRNAs. can bind This is surprising since no linkers are known for p-NAs
• Uracil-based linkers are preferred according to the present invention, for example, in which the 5-position of the uracil has preferably been modified, for example N-phthaloylaminoethyluracil, but also indole-based linkers, preferably tryptamine derivatives, such as, for example, B N-phthaloyltryptamine
• the present invention also provides more manageable pentopyranosyl-N, N-diacyl nucleosides, preferably purines, in particular adenosine, guanosine or 6-thioguanosine, the nucleobase of which can be completely deprotected in a simple manner. Therefore, the invention also includes Pentopyranosyl nucleosides in which R 2 , R 3 , R 4 , R 2 ' , R 3' and / or R 4 'is a radical of the formula -N [C (O) R 9 ] 2, in particular N 6 , N 6 -dibenzoyl-9- ( ⁇ -D-ribopyranosyl) adenosine.
• the present invention provides pentopyranosyl nucleosides which carry a protective group, preferably a base- or metal-catalyzed protective group, especially an acyl group, particularly preferably a benzoyl group, exclusively on the 3'-oxygen atom of the pentopyranoside part.
• a protective group preferably a base- or metal-catalyzed protective group, especially an acyl group, particularly preferably a benzoyl group, exclusively on the 3'-oxygen atom of the pentopyranoside part.
• These connections serve e.g. B. as starting materials for the direct introduction of a further protective group, preferably an acid- or base-labile protective group, in particular a trityl group, particularly preferably a dimethoxytrityl group, to the 4'-oxygen atom of the pentopyranoside part without additional steps which reduce the yield, such as, for. B. additional cleaning steps.
• the present invention provides pentopyranosyl nucleosides which carry a protective group, preferably an acid- or base-labile protective group, in particular a trityl group, particularly preferably a dimethoxytrityl group, exclusively on the 4'-oxygen atom of the pentopyranoside part.
• a protective group preferably an acid- or base-labile protective group, in particular a trityl group, particularly preferably a dimethoxytrityl group, exclusively on the 4'-oxygen atom of the pentopyranoside part.
• These connections also serve e.g. B. as starting materials for the direct introduction of a further protective group, preferably a base- or metal-catalyzed removable protective group, in particular an acyl group, particularly preferably a benzoyl group, for. B. on the 2'-oxygen atom of the pentopyranoside part, without additional, the yield-reducing steps such. B. additional cleaning steps.
• the pentopyranoside nucleosides according to the invention can be reacted in a so-called one-pot reaction, which increases the yields and is therefore particularly advantageous.
• A) [2 ', 4'-di-O-benzoyl) -ß-ribopyranosyl] nucleosides in particular a [2', 4'-di-O-benzoyl) -ß-ribopyranosyl] -adenine, - guanine, - cytosine, thymidine, uracil, xanthine or hypoxanthine, and an N-benzoyl-2 ', 4' -di-O-benzoyl-ribopyranosyl nucleoside, in particular an -adenine, -guanine or -cytosine, and an N -Isobutyroyl-2 ', 4'-di-O- benzoyl-ribopyranosyl-nucleoside, especially an -adenine, -guanine or -cytosine, as well as an O 6 - (2-cyanoethyl) -
• ⁇ -ribopyranosyl nucleosides in particular a ⁇ -ribopyranosyl adenine, guanine, cytosine, thymidine or uracil, xanthine or hypoxanthine, and an N-benzoyl, N-isobutyroyl, O 6 - (2 -Cyanoethyl) - or O 6 - (2- (4-nitrophenyl) ethyl) -N 2 -isobutylroyl-ß-ribopyranosyl nucleoside.
• 4'-DMT-pentopyranosyl nucleosides preferably a 4'-DMT-ribopyranosyl nucleoside, in particular a 4'-DMT-ribopyranosyl adenine, guanine, cytosine, thymidine, uracil, xanthine or hypoxanthine , and an N-benzoyl-4'-DMT-ribopyranosyl nucleoside, in particular an N-benzoyl-4'-DMT-ribopyranosyl adenine, guanine or - cytosine, and an N-isobutyroyl-4'-DMT-ribopyranosyl- Nucleoside, in particular an N-isobutyroyl-4'-DMT-ribopyranosyl-adenine, guanine or -cytosine and an O 6 - (2-cyanoethyl) -N 2 -iso
• Suitable precursors for oligonucleotide synthesis are, for example, 4'-DMT-pentopyranosyl-nucleosides-2'-phosphitamide / -H-phosphonate, preferably a 4'DMT-ribopyranosyl-nucleoside-2'-phosphitamide / -H-phosphonate, in particular a 4 '-DMT- ribopyranosyl-adenine, -guanine, cytosine, -thymidine, -xanthine, -hypoxanthine-, or - uracil-2'-phosphitamide / -H-phosphonate and an N-benzoyl-4'- DMT-ribopyranosyl-adenine, -guanine or -cytosine-2'-phosphitamide / -H-phosphonate and an N-isobutylroyl-4'-DMT-ribo
• the pentopyranosyl nucleosides can be produced particularly advantageously by starting from the unprotected one
• This method is not limited to the nucleobases described in the cited literature, but can surprisingly be carried out successfully with a large number of natural and synthetic nucleobases.
• it is particularly surprising that the process according to the invention can be carried out in large yields and with an average time saving of 60% compared to the process known from the literature, which is particularly advantageous for industrial use.
• the cleaning steps required in the method described in the literature, e.g. B. intermediate chromatographic purifications, not necessary and the reactions can sometimes be carried out as a so-called one-pot reaction, which significantly increases the space / time yields.
• the protective group in the case of a 2 'protected position, is rearranged from the 2' position to the 3 'position, which is generally carried out in the presence of a base, in particular in the presence of N-ethyldiisopropylamine and / or triethylamine .
• this reaction can be carried out particularly advantageously in the same reaction container as a one-pot reaction.
• the pyranosyl nucleoside is protected by an acid-labile, base-labile or metal-catalyzed protective group S c ⁇ , S e2 , S c v or Sc2-, the protective groups S c ⁇ and S c r being preferably protected by the protective groups S c2 or S C 2 'are different.
• the protective groups mentioned are an acyl group, preferably an acetyl, benzoyl, nitrobenzoyl and / or methoxybenzoyl group, trityl groups, preferably a 4, 4'-dimethoxytrityl (DMT) group or a ⁇ -eliminable group, preferably a group of the formula - OCH 2 CH 2 R where R is a cyano or p-nitrophenyl radical or a fluorenylmethyloxycarbonyl (Fmoc) group.
• acyl group preferably an acetyl, benzoyl, nitrobenzoyl and / or methoxybenzoyl group
• trityl groups preferably a 4, 4'-dimethoxytrityl (DMT) group or a ⁇ -eliminable group, preferably a group of the formula - OCH 2 CH 2 R where R is a cyano or p-nitrophenyl radical or a flu
• the 2 'or 3' position is protected by a base-labile or metal-catalyzed protective group, preferably by an acyl group, in particular by an acetyl, benzoyl, nitrobenzoyl and / or methoxybenzoyl group, and / or that 4 'position is protected by an acid- or base-labile protecting group, preferably by a trityl and / or Fmoc group, in particular by a DMT group.
• a base-labile or metal-catalyzed protective group preferably by an acyl group, in particular by an acetyl, benzoyl, nitrobenzoyl and / or methoxybenzoyl group, and / or that 4 'position is protected by an acid- or base-labile protecting group, preferably by a trityl and / or Fmoc group, in particular by a DMT group.
• this process does not require any acetal protecting groups, such as acetals or ketals, which avoids additional chromatographic intermediate purifications and consequently allows the reactions to be carried out as one-pot reactions with surprisingly high space / time yields.
• the protective groups mentioned are preferably introduced at low temperatures, since surprisingly they can thereby be introduced selectively.
• a benzoyl group is introduced by reaction with benzoyl chloride in pyridine or in a pyridine / methylene chloride mixture at low temperatures.
• the introduction of a DMT group can, for example, by reaction with DMTC1 in the presence of a base, e.g. B. of N-ethyldiisopropylamine (Hünig base), and z. B. of pyridine, methylene chloride or a pyridi / methylene chloride mixture at room temperature.
• a base e.g. B. of N-ethyldiisopropylamine (Hünig base)
• the reaction products are purified by chromatography. Purification after the tetylation is not necessary according to the process of the invention, which is particularly advantageous
• the end product can be further purified by crystallization
• a protected nucleobase is first reacted with a protected ribopyranose, then
• step (b) the protective groups are removed from the ribopyranosyl part of the product from step (a), and then
• step (c) the product from step (b) is reacted according to the method described in more detail above
• pentopyranoses such as, for example, tetrabenzoyl-pentopyranoses, preferably ⁇ -tetrabenzoyl- ⁇ bopyranoses (R Jeanloz, J Am Chem Soc 1948, 70, 4052)
• a linker of the formula (II) in which R 4 is (C “H 2 n) NR 10 R n and R 10 R ⁇ is linked via a radical of the formula (III) with the meaning already described following method advantageously manufactured
• reaction product from (b) is reacted with a corresponding phthahmide, for example N-ethoxycarbonylphthahmid, and (d) the reaction product from (c) is reacted with an appropriate protected pyranose, for example ribosetetrabenzoate, and finally
• indole derivatives as linkers have the advantage of fluorescence capability and are therefore particularly preferred for nanotechnology applications, which may involve the detection of the smallest amounts of substance.
• indole-1-ribosides were described by NN Suvorov et al., Biol Aktivn Soedin., Akad Nauk SSSR 1965, 60 and Tetrahedron 1967, 23, 4653 have already been described.
• 3-substituted derivatives there is no analogous process for producing 3-substituted derivatives. In general, they are prepared by forming an amine of the unprotected sugar component and an indoline. which is then converted into the indole-1-riboside by oxidation.
• indole-1-glucoside and -1-arabinoside (YV Dobriynin et al, Khim -Farm Zh 1978, 12, 33), their 3 -substituted derivatives, have mostly been described were produced via a Dahlsmeier reaction.
• this method of introducing aminoethyl units into the 3-position of the indole is too expensive for industrial use
• this method can be used not only for ribopyranoses, but also for ribofuranoses and 2'-deoxyribofuranoses or 2'-deoxyribopyranoses, which is particularly advantageous.
• the sugar is preferred as the nucleosidation partner Tryptamine, especially N-Acylde ⁇ vate des Tryptamin, ⁇ or all N-Phthaloyltryptamm used
• the 4'-protected, preferably the 3 ', 4'-protected, pentopyranosyl nucleosides are phosphitylated in a further step or bound to a solid phase
• the phosphity is carried out, for example, by phosphoric acid monoallyl ester dnsopropyl amide chloride in the presence of a base, for example N-ethyl dnsopropylamine or by phosphor toluene and imidazole or tetrazole and subsequent hydrolysis with addition of base.
• a base for example N-ethyl dnsopropylamine
• phosphor toluene and imidazole or tetrazole and subsequent hydrolysis with addition of base.
• the product is a phosphoramidite
• a protected pentopvranosyl nucleoside according to the invention can be bound to a solid phase, for example "long-chain-alkylamino-controlled pore glass" (CPG, Sigma Chemie, Kunststoff), for example as described in Eschenmoser et al (1993)
• the compounds obtained are used, for example, for the production of pentopyranosyl nucleic acids, preferably
• step (b) in a second step, the 3'-, 4'- protected pentopyranosyl nucleoside bound to a solid phase according to step (a) is extended by a phosphity-protected 3'-, 4 '-protected pentopyranosyl nucleoside and then, for example, by an aqueous iodine solution is oxidized, and
• Step (b) is repeated with the same or different phosphityherten 3'-, 4'- protected pentopyranosyl nucleosides until the desired pentopyranosyl nucleic acid is present
• Acid activators such as pyridinium hydrochloride are particularly suitable as coupling reagents when phosphoramidites are used, especially benzimidazolium triflate, preferably after recrystallization in acetonitrile and after loosening in acetonitrile, since, in contrast to 5- (4-nitrophenyl) 1H-tetrazole, the coupling reagent is not clogged as a coupling reagent. Pipes and contamination of the product takes place Aryl sulfonyl chlorides, diphenyl chlorophosphate, pivaloyl chloride or adamantoyl chloride are particularly suitable as coupling reagents when using H-phosphonates
• a salt such as sodium chloride
• ogonucleotides in particular p-NA's, preferably p-RNA's, pyrimidine bases, especially uracil and thymine, which prevent this Oligonucleotide has been destroyed
• Allyloxy groups can preferably be split off by palladium [Pd (0)] complexes, for example before hydrazinolysis
• pentofuranosyl nucleosides for example adenosine, guanosine, cytidine, thymidine and / or uracil, which occur in their natural form, can also be incorporated in step (a) and / or step (b), which, for example, also includes a mixed p-NA-DNA or p-NA-RNA
• an allyloxy linker of the formula can be used in a further step
• S c4 and S c independently of one another, the same or different, each have a protective group selected in particular from Fmoc and / or DMT,
• a particularly preferred allyloxy linker is (2- (S) - ⁇ -Fmoc-O -DMT-O - allyloxydiisopropylaminophosph ⁇ nyl-6-amino-1, 2-hexanediol)
• amino-terminal linkers can be built up in a few reaction steps, which are both an activatable phosphorus compound and a acid-labile protecting group, such as DMT, and can therefore be easily used in automatable oligonucleotide synthesis (see, for example, Nelson et al., Nucleic Acid Res. 1989, 17, 7179; LJ Arnold et al., WO 8902439).
• the repertoire was expanded in the present invention by a lysine-based linker, in which an allyloxy group was introduced instead of the usual cyanoethyl group on the phosphorus atom, and which can therefore be used advantageously in the Noyori oligonucleotide method ( R. Noyori, J. Am. Chem. Soc. 1990, 112, 1691-6).
• Another object of the present invention also relates to an electronic component, in particular in the form of a diagnostic agent, containing an above-described pentopyranosyl nucleoside or a pentopyranosyl nucleic acid in the form of a conjugate, and a method for producing a conjugate in which a pentopyranosyl nucleoside or a pentopyranosyl nucleic acid is associated with a biomolecule, as already described in more detail above.
• RNA 1 shows a section of the structure of RNA in its naturally occurring form (left) and in the form of a p-NA (right).
• the G-triol A (393 mg, 1.0 mmol) was dissolved in 4 ml of dry dichloromethane. Trimethylorthoester (0.52 ml, 3.0 mmol) and camphorsulfonic acid (58 mg, 0.25 mmol) were added and the mixture was stirred at room temperature for 15 h. The mixture was then cooled to 0 ° C. and 2 ml of a mixture of acetonitrile, water and trifluoroacetic acid (50: 5: 1) precooled to 0 ° C. were added. The mixture was stirred for 10 min and the solvent was removed in vacuo.
• the diol B (101 mg, 0 2 mmol) was suspended in 3 2 ml of dry dichloromethane. 171 ⁇ l (1 0 mmol) of N-ethyldiisopropylamine, 320 ⁇ l (3 96 mmol) of pyridine and 102 mg (0 3 mmol) of DMTC1 were added and stirred at room temperature After 24 h, a further 102 mg (0 3 mmol) of DMTC1 were added and the mixture was stirred again for 24 h. Then the mixture was diluted with 30 ml of dichloromethane.
• hydroxyethyluracil 28 succeeds on a large scale by known method (JD Fissekis, A Myles, GB Brown, J Org Chem 1964, 29, 2670)
• g-butyrolactone 25 was formylated with methyl formate, the sodium salt 26 was converted to the urea derivative 27 and this to Hydroxyethyluracil 28 cyclized (Scheme 4)
• Hydroxyethyluracil 28 was mesylated with methanesulfonyl chloride in pyridine to 29 (J.D. Fissekis, F Sweet, J Org. Chem 1973, 38, 264)
• reaction mixture was then heated to 50 ° C. After stirring for 5 hours at 50 ° C. (TLC control) cooled to rt, on an ice-cold mixture of 250 ml of AcOEt and 190 ml of saturated NaHC0 3 solution and thoroughly stirred for 10 min. The mixture was washed again with 100 ml of NaHCO 3 solution and the aqueous phases were extracted again with 100 ml of AcOEt dry with MgSO 4 t and the solvent IV i RV removed.
• TLC control cooled to rt, on an ice-cold mixture of 250 ml of AcOEt and 190 ml of saturated NaHC0 3 solution and thoroughly stirred for 10 min.
• the mixture was washed again with 100 ml of NaHCO 3 solution and the aqueous phases were extracted again with 100 ml of AcOEt dry with MgSO 4 t and the solvent IV i RV removed.
• N-phthaloyltryptamine is obtained from phthalic anhydride and tryptamine as described (Kuehne et al J. Org. Chem. 43, 13, 1978, 2733-2735). This is reduced to indoline with borane-THF (analogously to A. Giannis, et al., Angew. Chem. 1989, 101, 220).
• the 3-substituted indoline is first converted to the nucleoside triol with ribose and then to the triacetate with acetic anhydride.
• Oxidized with 2,3-dichloro-5,6-dicyanoparachinone and the acetates are cleaved with sodium methylate, benzoylated in the 2'-position, DM-tritylated selectively in the 4'-position, and carries out the migration reaction to the 3 '- benzoate.
• the phosphoramidite is formed as usual. This can be used for automated oligonucleotide synthesis without changing the synthesis protocols.
• 6-Am ⁇ no-2 (S) -hydroxyhexanoic acid (1) was prepared in a manner known from the literature by diazotization and subsequent hydrolysis from L-lysine (K -I Aketa, Chem Pharm Bull. 1976, 24, 621)
• indollinker phosphoramidite and 244 mg A phosphoramidite are weighed into synthesizer bottles and left for 3 h in the desiccator over KOH together with the pill filled with 28.1 mg CPG carrier, loaded with A building block, at the HV.
• the phosphoramidites are in 1 ml (Indollinker) or 2.5 ml (A-phosphoramidite) acetonitrile dissolved and a few balls added from the molecular sieve and left closed in the desiccator over KOH
• the carrier is slurried with aqueous 0.1 molar sodium diethyldithiocarbamate solution and left at RT for 45 min. You suck off, wash with water, acetone,
• Triethylammonium bicarbonate buffer (TEAB buffer) diluted to 7 ml About one
• Triethylammonium acetate buffer Triethylammonium acetate buffer
• Usual desalination Waters Sep-Pak Cartridge
• a p-RNA oligomer of sequence A 8 is produced on the Eppendorf Ecosyn D 300+ and then the following reagents are exchanged 6% dichloroacetic acid for 2% trichloroacetic acid, iodine in Collidine against iodine in pyridine, benzimidazolium triflate solution against tetrazole solution
• a DNA oligomer of the sequence GATTC is further synthesized according to known methods (MJ Gait, Oligonucleotide Synthesis, IRL Press, Oxford, UK 1984). Deallylation, hydrazinolysis, HPL chromatography and Desalination is carried out as described for the p-RNA oligomer (see above) and provides the desired conjugate.
• Example 2 convergent procedure As described in Example 2, a p-RNA oligomer with the sequence 4'-indollinker-A 8 -2 'is prepared, purified, and iodoacetylated.
• a DNA oligomer of the sequence GATTC thiol linker is synthesized and purified by known methods (MJ Gait, Oligonucleotide Synthesis, IRL Press, Oxford, UK 1984) (3'-thiol linker from Glen Research: No. 20-2933) .
• the two fragments T. Zhu et al., Bioconjug. Chem. 1994, 5, 312
• the conjugate is formed, which is then purified by HPLC.
• Zuert was analogous to that described in Example 6, a p-RNA oligomer of the sequence TAGGCAAT, which at the 4 'end using the 5'-amino modifier 5 from Eurogentec (2- (2- (4-monomethoxytrityl) aminoethoxy) ethyl- (2nd -cyanoethyl) - (N, N-diisopropyl) phosphorus amidite) is provided with an amino group, synthesized and worked up.
• the oligonucleotide (17.4 OD, 0.175 ⁇ mol) was taken up in 0.5 ml of basic buffer, 1.14 mg (2.5 ⁇ mol) of biotin-N-hydroxysuccinimide ester was dissolved in 114 ⁇ l of DMF (abs.) And left to stand at RT for 1 hour .
• the resulting conjugate was purified by preparative HPLC and the pure product was desalted with a Sepak.
• a synthesis cycle consists of the following steps (a) Detritylation: 5 minutes with 6% DCA (dichloroacetic acid) in CH 2 C1 2 (79 ml),
• the last DMT (dimethoxyt ⁇ tyl) or MMT (monomethyoxytrityl) protective group of biotin or cyanine monomers was not removed.
• the last coupling with the modified phorphoramidites is detected after synthesis with 1% of the Resin by trityl cation absorption in UV (503 nm)
• the allyl ether protecting groups were split off with a solution of tetrakis (triphenylphosph ⁇ n) palladium (272mg), triphenylphosphine (272 mg) and diethylammonium hydrogen carbonate in CH 2 C1 2 (15ml) after 5 hours at RT.
• the glass supports are then washed with CH 2 CL 2 (30ml) , Acetone (30 ml) and water (30 ml) washed
• the resin was rinsed with an aqueous 0.1 M sodium diethyldithiocarbamate hydrate solution. The above-mentioned washing operation was carried out again in a reverse sequence.
• the resin was then dried under high vacuum for 10 minutes.
• the cleavage step from the glass support with simultaneous debenzoylation was carried out in 24% hydrazine hydrate solution (6 ml) at 4 ° C.
• the “Trityl ON” oligonucleotide was activated using an (acetonitrile, 20 ml) water Sep-Pak Cartridge Free of Hydrazine
• the hydrazine was washed with TEAB 0.1M (30ml) and the oligonucleotide was then washed with acetonitrile / TEAB 0.1M (10ml) eluted.
• the oligos were freeze-dried for storage.
• Buffer A 0.1 molar triethylammonium acetate buffer in water
• Buffer B 0.1 molar triethylammonium acetate buffer in water: acetonitrile 1: 4
• Retention time of the products in this case 23.1 minutes
• the mixture was diluted to four times the volume with water.
• a Waters Sep-Pak cartridge RP-18 (from 15 OD 2 g filling) was activated with 2 x 10 ml acetonitrile and 2 x 10 ml water, the oligo was applied, let it sink in, the reaction vessel was washed with 2 x 10 ml Water, rinse with 3 x 10 ml water to remove salt and reagent, and elute first with 5 x 1 ml 50: 1 water: acetonitrile and then 1: 1. The product eluted in the 1: 1 fractions in very good purity. The fractions were concentrated in the cold and in the dark, combined, and concentrated again.
• Buffer system Borax / HCl buffer from Riedel-de Haen, pH 8.0, was mixed in a ratio of 1: 1 with a 10 millimolar solution of EDTA disodium salt in water and adjusted to pH 6.3 with HC1. This gave a solution containing 5 mM Na 2 EDTA.
• Buffer A 0.1 molar triethylammonium acetate buffer in water
• Buffer B 0.1 molar triethylammonium acetate buffer in water: acetonitrile 1: 4 gradient: starting from 10% B to 50% B in 40 minutes
• Buffer system Borax / HCl buffer from Riedel-de Haen, pH 8.0 was mixed in a 1 1 ratio with a 10 millimolar solution of EDTA disodium salt in water and adjusted to pH 6.6 with HCl This gave a solution containing 5 mM Na 2 EDTA.
• the standard conditions of the analytical HPLC are - buffer A 0.1 molar triethylammonium acetate buffer in water buffer B 0.1 molar triethylammonium acetate buffer in water acetonitrile 14 gradient from 10% B starting at 50% B in 40 minutes

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