WO1999015540A2 - Verfahren zur herstellung eines pentopyranosyl-nucleosids - Google Patents

Verfahren zur herstellung eines pentopyranosyl-nucleosids Download PDF

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Publication number
WO1999015540A2
WO1999015540A2 PCT/EP1998/005998 EP9805998W WO9915540A2 WO 1999015540 A2 WO1999015540 A2 WO 1999015540A2 EP 9805998 W EP9805998 W EP 9805998W WO 9915540 A2 WO9915540 A2 WO 9915540A2
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WIPO (PCT)
Prior art keywords
ribopyranosyl
nucleoside
group
benzoyl
dmt
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PCT/EP1998/005998
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German (de)
English (en)
French (fr)
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WO1999015540A3 (de
Inventor
Christian Miculka
Norbert Windhab
Tilmann Brandstetter
Gerhard Burdinski
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Aventis Research & Technologies Gmbh & Co Kg
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Application filed by Aventis Research & Technologies Gmbh & Co Kg filed Critical Aventis Research & Technologies Gmbh & Co Kg
Priority to JP2000512845A priority Critical patent/JP4601817B2/ja
Priority to US09/509,010 priority patent/US6613894B1/en
Priority to CA002302414A priority patent/CA2302414A1/en
Priority to AT98950062T priority patent/ATE233272T1/de
Priority to EP98950062A priority patent/EP1019423B1/de
Priority to BR9812501-0A priority patent/BR9812501A/pt
Priority to AU96271/98A priority patent/AU751058B2/en
Priority to KR1020007002982A priority patent/KR20010024202A/ko
Priority to DE59807330T priority patent/DE59807330D1/de
Publication of WO1999015540A2 publication Critical patent/WO1999015540A2/de
Publication of WO1999015540A3 publication Critical patent/WO1999015540A3/de

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • 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
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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    • Y10S977/70—Nanostructure
    • Y10S977/724—Devices having flexible or movable element
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    • Y10S977/728—Nucleic acids, e.g. DNA or RNA
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    • Y10S977/788—Of specified organic or carbon-based composition
    • Y10S977/797—Lipid particle
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    • Y10S977/799—Containing biological material
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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.
  • This specificity which is valuable for the construction of supramolecular units, is related to the relatively low flexibility of the ribopyranosephosphate backbone as well as the strong inclination of the base plane to the strand axis and the resulting tendency towards intercatenary base stacking in the resulting duplex and can ultimately be attributed to the participation of a 2 ⁇ 4 '-cis-disubstituted ribopyrano rings on the structure of the backbone.
  • These significantly better pairing properties make p-NAs preferred pairing systems over DNA and RNA for the application of the construction of supramolecular units. They form a pairing system orthogonal to natural nucleic acids, ie they do not pair with naturally occurring DNA's and RNA's, which is particularly important in the diagnostic field.
  • a suitable protected nucleobase was mixed with the anomer mixture of tetrabenzoyl ribopyranose by the action of bis (trimethylsilyl) acetamide and a Lewis acid such as. B. trimethylsilyl trifluoromethanesulfonate reacted (analog H. Vorbrüggen, K. Krolikiewicz, B. Bennua, Chem. Ber. 1981, 1 14, 1234.).
  • the carrier-bound component was repeatedly deprotected in the 4 'position, a phosphoramidite under the action of a coupling reagent, e.g. B. a tetrazole derivative, coupled, still free 4'-oxygen atoms acetylated and the phosphorus atom oxidized so as to obtain the oligomeric product.
  • a coupling reagent e.g. B. a tetrazole derivative
  • 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.
  • the object of the present invention was therefore to provide a new process for the preparation of pentopyranosyl nucleosides, which is intended to enable the production of known and new pentopyranosyl nucleosides on a larger scale than by known processes and to avoid the disadvantages described above.
  • An object of the present invention is therefore a process for the preparation of a pentopyranosyl nucleoside, in which starting from the unprotected pentopyranoside
  • R, R, R and R independently of one another, identical or different, in each case H, C n H 2n + ⁇ , or
  • R 9 is a linear or branched, optionally substituted alkyl, aryl, preferably phenyl radical,
  • Protecting group selected from an acyl, trityl or allyloxycarbonyl group, preferably a benzoyl or 4, 4 , -dimethoxytrityl (DMT) group,
  • R 1 is H, OH, shark with shark is Br or Cl or a residue selected from
  • the pentopyranosyl nucleoside according to the invention 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 especially a pentopyranosyl-purine, - pyrimidine. adenosine, guanosine, thymidine. -cytosine, tryptamine. -N-phthalotryptamine or - uracil.
  • Pentopyranosyl nucleosides also fall under the compounds. which can be used as linkers, ie as compounds with functional groups that are covalently attached to biomolecules, such as. B. occurring or modified nucleic acids in their natural form. like DNA, RNA but also p-NA's. preferably can bind pRNA's. This is surprising since no linkers are known for p-NA's.
  • this includes pentopyranosyl nucleosides.
  • R 2 . R 3 , R 4 , R 2 . R 3 and / or R 4 is a 2-phthalimidoethyl or allyloxy radical.
  • Preferred according to the present invention are, for example, uracil-based linkers, in which the 5-position of the uracil has preferably been modified, e.g. B. N-phthaloylaminoethyl uracil, but also indole-based linkers, preferably tryptamine derivatives, such as. 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.
  • the invention therefore 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, in particular an acyl group, particularly preferably a benzoyl group, only on the 3 'oxygen atom of the pentopyranoside part.
  • a protective group preferably a base- or metal-catalyzed protective group, in particular an acyl group, particularly preferably a benzoyl group
  • 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.
  • guanine or cytosine and an N-isobutyroyl-2 ', 4'-di-O-benzoyl-ribopyranosyl nucleoside, in particular an aadenine, guanine or cytosine, and an O 6 - (2-cyanoethyl) N 2 -isobutyroyl-2 ', 4'-di-O-benzoyl-ribopyranosyl nucleoside, in particular a guanine and an O 6 (2- (4-nitrophenyl) ethyl) -N 2 -isobutyroyl-2 ', 4'-di-O-benzoyl-ribopyranosyl nucleoside, in particular a guanine.
  • ⁇ -ribopyranosyl nucleosides in particular a ⁇ -ribopyranosyl adenine, guanine, cytosine, thymidine or uracil, xanthine or hypoxanthine, and an N-benzoyl, N
  • 4'-DMT-pentopyranosyl nucleosides preferably a 4'-DMT-ribopyranosyl nucleoside, in particular a 4'-DMT-ribopyranosyl adenine, guanine.
  • a 4'-DMT-ribopyranosyl nucleoside in particular a 4'-DMT-ribopyranosyl adenine, guanine.
  • N-isobutyroyl-4 ' -DMT-ribopyranosyl-nucleoside in particular an N-isobutyroyl-4'-DMT-ribopyranosyl-adenine, guanine or -cytosine as well as an O 6 - (2-cyanoethyl ) -N-isobutyroyl-4'-DMT-ribopyranosyl-nucleoside, in particular an O 6 - (2-cyanoethyl) -N 2 -isobutyroyl-4'-DMT-ribopyranosyl-guanine, and an O 6 - (2 - (- 4-nitrophenyl) ethyl) -N 2 -isobutyroyl-4'-DMT-ribopyranosyl-nucleoside, especially an O 6 - (2 - (- 4-nitrophenyl) ethyl) -N 2 -isobutyroyl-4'
  • 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 ribopyranosyl
  • the method according to the invention 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 c2 , S c p or S C 2 ', the protective groups S c ⁇ and S c r preferably being protected by the protective groups S C 2 and S c2 - 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 by one ⁇ -eliminable group, preferably a group of the formula OCH 2 CH 2 R 18 where R 18 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 by one ⁇ -eliminable group, preferably a group of the formula OCH 2 CH 2 R 18 where R 18 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 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
  • an acid- or base-labile protecting group preferably by a trityl and / or Fmoc group, in particular by a DMT group.
  • the process according to the invention manages without acetal protective 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. pyridine, methylene chloride or a pyridine / methylene chloride mixture at room temperature.
  • reaction products are purified by chromatography after the acylation and / or after the possible rearrangement from the 2 'to the 3' position. Cleaning after tritylation is not necessary in accordance with the method according to the invention, which is particularly advantageous.
  • the end product can be further purified by crystallization.
  • Another object of the present invention is a method for producing a ribopyranosyl nucleoside, in which (a) a protected nucleobase is reacted with a protected ribopyranose,
  • step (b) the protective groups are removed from the ribopyranosyl part of the product from step (a), and
  • step (c) the product from step (b) is reacted according to the method described in more detail above.
  • pentopyranoses such as, for. B. tetrabenzoyl pentopyranoses, preferably ⁇ -tetrabenzoyl ribopyranoses (R. Jeanloz, J. Am. Chem. Soc. 1948, 70, 4052).
  • a linker of the formula (II) in which R 4 is (C n H 2n ) NR 10 R ⁇ r and R 10 ' R " ' is linked via a radical of the formula (III) to the meaning already described advantageously produced by the following process:
  • indole derivatives as linkers have the advantage of fluorescence capability and are therefore particularly preferred for nanotechnology applications, which may involve the detection of very small amounts of substances.
  • indole-1-ribosides were found in NN Suvorov et al., Biol. Aktivn. Soedin., Akad. Nauk SSSR 1965, 60 and Tetrahedron 1967, 23, 4653 already described.
  • there is no analogous process for producing 3-substituted derivatives In general, their preparation takes place via the formation of an amine unprotected sugar component and an indoline, which is then converted into the indole-1-riboside by oxidation. Have been described for. B.
  • this method can be used not only for ribopyranoses, but also for ribofuranoses and 2'-deoxyribofuranoses or 2'-deoxy ribopyranoses. which is particularly beneficial.
  • Tryptamine in particular N-acyl derivatives of tryptamine, especially N-phthaloyltryptamine, is preferably used as the nucleosidation partner of the sugars.
  • the 4'-protected, preferably the 3 ', 4'-protected pentopyranosyl nucleosides are phosphitylated in a further step or bound to a solid phase.
  • the phosphitylation is carried out, for example, by phosphorous acid monoallyl ester diisopropyl amide chloride in the presence of a base, for. B. N-ethyldiisopropylamine or by phosphorus trichloride and imidazole or tetrazole and subsequent hydrolysis with addition of base.
  • a base for. B. N-ethyldiisopropylamine or by phosphorus trichloride and imidazole or tetrazole and subsequent hydrolysis with addition of base.
  • the product is a phosphoramidite and in the second case an H-phosphonate.
  • Binding of a protected pentopyranosyl nucleoside according to the invention to a solid phase e.g. B. "long-chain-alkylamino-controlled pore glass" (CPG, Sigma Chemie, Kunststoff) can be carried out, for example, as described in Eschenmoser et al. (1993
  • the compounds obtained serve e.g. B. for the production of pentopyranosyl nucleic acids.
  • Another object of the present invention is therefore a method for producing a pentopyranosyl nucleic acid, with the following steps: (a) in a first step, a protected pentopyranosyl nucleoside is bound to a solid phase, as already described above, and
  • 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 phosphitylated 3 ' -, 4 ' -protected pentopyranosyl nucleoside and then z.
  • B. is oxidized by an aqueous iodine solution, and
  • step (c) Repeat step (b) with the same or different phosphitylated 3'-, 4 " -protected pentopyranosyl nucleosides until the desired pentopyranosyl nucleic acid is present.
  • Acidic activators such as pyridinium hydrochloride are particularly suitable as coupling reagents when using phosphoramidites, especially benzimidazolium triflate, preferably after recrystallization in acetonitrile and after dissolving in acetonitrile, since unlike 5- (4-nitrophenyl) -1 H-tetrazole as coupling reagent there is no blockage of the coupling reagent - Lines and contamination of the product occur.
  • Arylsulfonyl chlorides, diphenyl chlorophosphate, pivaloyl chloride or adamantoyl chloride are particularly suitable as coupling reagents when using H-phosphonates.
  • oligonucleotides in particular p-NAs, preferably p-RNAs, preferably p-RNAs, especially uracil and thymine, which would destroy the oligonucleotide .
  • Allyloxy groups can preferably be formed by palladium [Pd (0)] complexes e.g. B. be split off before hydrazinolysis.
  • pentofuranosyl nucleosides for. B. the naturally occurring adenosine, guanosine, cytidine, thymidine and / or uracil, which z. B. leads to a mixed p-NA-DNA or p-NA-RNA.
  • an allyloxy linker of the formula can be used in a further step
  • S c and S c7 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) -N-Fmoc- ⁇ '-DMT-O 2 - allyloxydiisopropylaminophosphinyl-6-amino-1, 2-hexanediol).
  • Lysine can thus be built up in a few reaction steps amino-terminal linkers which carry both an activatable phosphorus compound and an acid-labile protective group, such as DMT, and can therefore be easily used in the automatable oligonucleotide synthesis (see, for example, Nelson 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 on the phosphorus atom instead of the usual cyanoethyl group, and which can therefore be used advantageously in the Noyori oligonucleotide method ( R. Noyori, J. Am. Chem. Soc. 1990, 1 12, 1691-6).
  • p-NA's and especially the p-RNA's form stable duplexes with each other and generally do not pair with the DNA's and RNA's occurring in their natural form.
  • This property makes p-NA 's the preferred pairing system.
  • Such pairing systems are supramolecular systems of non-covalent interaction that are characterized by selectivity. Characterize stability and reversibility, and their properties preferably thermodynamic. ie influenced by temperature, pH and concentration.
  • Such pairing systems can e.g. B. due to their selective properties, they can also be used as a “molecular adhesive for the merging of different metal clusters into clusters with potentially new properties [see e.g.
  • the p-NA 's 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 merging of molecular species into supramolecular units, such as B. for the (combinatorial) structure of protein assemblies [see, for. BA Lombardi, JW Bryson. WF DeGrado. Biomolecule (Pept. Sci.) 1997, 40, 495-504].
  • p-NA's form pairing systems that are strong and thermodynamically controllable. Another application therefore arises especially in the diagnostic and drug discovery area due to the possibility of being functional. preferably biological units such as proteins or DNA / RNA sections to be provided with a p-NA code which does not interfere with the natural nucleic acids (see, for example, WO93 / 20242).
  • a biomolecule e.g. B. DNA or RNA. can for non-covalent connection (linkage) with another biomolecule, e.g. B. DNA or RNA, are used if both biomolecules contain sections that can bind to each other due to complementary sequences of nucleobases by forming hydrogen bonds.
  • Such biomolecules find z. B. in analytical systems for signal amplification use, where 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) (see, e.g., BS Urdea, Bio / Technol. 1994, 12, 926, or U.S.
  • bDNA signal-enhancing branchedDNA molecule
  • Patent No. 5,624,802 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). This is partly due to the fact that the non-covalent binding of the solid support to the DNA molecule to be analyzed, as well as the non-covalent binding of the DNA molecule to be analyzed, is not always takes place specifically, which results in a mixing of the functions .. sequence recognition "and" non-covalent binding ".
  • the use of 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 pentopyranosyl nucleosides or pentopyranosyl nucleic acids prepared by the process according to the invention are therefore suitable for the production of a medicament, such as. B. a therapeutic, diagnostic and / or electronic component, for example in the form of a conjugate, d. H. in combination with a biomolecule.
  • 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 occurring in its natural form.
  • Functional parts of antibodies are, for example, Fv fragments (Skerra & Plückthun (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).
  • biomolecule means a naturally occurring substance or a substance derived from a naturally occurring substance.
  • these are p-RNA / DNA or p-RN A / RN A conjugates.
  • Conjugates are preferably used when the functions "sequence recognition” and “non-covalent binding” have to be realized in one molecule, since the conjugates according to the invention contain two mutually orthogonal pairing systems.
  • 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, U.S. Patent No. 5,632,957).
  • a higher flexibility of these arrays can be achieved by binding the recognition species to coding oligonucleotides and the associated, complementary strands at certain positions on a solid support.
  • the recognition species are non-covalently bound at the desired positions.
  • different types of recognition species such as, for example, DNA segments, antibodies, can only be bound by
  • the application of hybridization conditions can be arranged simultaneously on a solid support (see FIG. 3), but this requires extremely strong, selective - in order to keep the coding sections as short as possible - and codons and anticodons which do not interfere with natural nucleic acid, p-NAs p-RNAs are particularly suitable for this in a particularly advantageous manner.
  • the term “carrier” means material, in particular chip material, which is in solid or gel-like form.
  • suitable carrier materials are ceramic, metal, in particular noble metal, glasses, plastics, crystalline materials or thin layers of Carrier, in particular the materials mentioned, or (bio) 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. These can then be hybridized according to FIG. 4 with the associated codons on a solid support.
  • Another object of the present invention therefore relates in particular to a diagnostic agent containing a pentopyranosyl nucleoside described above or a conjugate according to the invention, as already described in more detail above.
  • RNA shows a section of the structure of RNA in its naturally occurring form (left) and in the form of a p-NA (right).
  • 2 schematically shows the synthesis of a p-ribo (A, U) oligonucleotide according to Eschenmoser et al. (1993).
  • FIG. 3 schematically shows an arrangement of immobilized recognition structures (arrays) on a solid support.
  • the reaction mixture was mixed with 2.46 g (20.5 mmol; 0.1 eq.) 4-dimethylaminopyridine (DMAP), cooled to -6 ° C and between -6 and -1 ° C within 15 min 27.9 ml (0.24 mol; 1.2 eq.) of benzoyl chloride (BzCl) in 30 ml of pyridine were added dropwise and the mixture was subsequently stirred for 10 min. To complete the reaction, 2.8 ml (24 mmol; 0.12 eq.) Of BzCl were then added every 25 minutes with cooling and finally stirred for 20 min.
  • DMAP 4-dimethylaminopyridine
  • BzCl benzoyl chloride
  • N 4 -benzoyl-l- ( ⁇ -D-ribopyranosyl) cytosine 1 were dried in 830 ml of dimethylformamide (DMF) and 1.5 l of pyridine (both solutions and dried over molecular sieve 3 ⁇ ) dissolved while heating to 124 ° C.
  • DMF dimethylformamide
  • pyridine both solutions and dried over molecular sieve 3 ⁇
  • N, N -dibenzoyl-9- ( ⁇ -D-ribopyranosyl) adenine 3 Under a ⁇ atmosphere, 16.8 g (62.9 mmol) 2 were suspended in 500 ml of anhydrous pyridine and cooled to -4 to -10 ° C. 40 ml (199 mmol; 5 eq.) Of trimethylchlorosilane were added dropwise within 20 min and the mixture was stirred for 2.5 h while cooling. At -10 to -15 ° C, 36.5 ml (199 mmol; 5 eq.) Of benzoyl chloride, dissolved in 73 ml of pyridine, were added within 25 min, 10 min with cooling and 2 h at RT.
  • 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.
  • Diol B (101 mg, 0.2 mmol) was suspended in 3.2 ml dry dichloromethane. 171 ⁇ l (1.0 mmol) of N-ethyldiisopropylamine, 320 ⁇ l (3.96 mmol) of pyridine and 102 mg (0.3 mmol) of DMTCl were added, and the mixture was stirred at room temperature. After 24 h, a further 102 mg (0.3 mmol) of DMTCl were added and the mixture was stirred for another 24 h. The mixture was then diluted with 30 ml of dichloromethane.
  • Hydroxyethyluracil 28 can be prepared on a large scale by known methods (JD Fissekis, A. Myles, GB Brown, J. Org. Chem. 1964, 29, 2670).
  • g-Butyrolactone 25 was formylated with methyl formate, the sodium salt 26 converted to the urea derivative 27 and this cyclized to the hydroxyethyl uracil 28 (Scheme 4).
  • Hydroxyethyluracil 28 was mesylated with methanesulfonic acid chloride in pyridine to 29 (J.D. Fissekis, F. Sweet, J. Org. Chem. 1973, 38, 264).
  • 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. It is oxidized with 2,3-dichloro-5,6-dicyanoparachinone and the acetates are cleaved with sodium methylate, benzoylated selectively in the 2'-position, DM-tritylated selectively in the 4'-position, and the migration reaction to the 3'-benzoate is carried out.
  • the phosphoramidite is formed as usual. This can be used for automated oligonucleotide synthesis without changing the synthesis protocols.
  • 6-Amino-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 of A-phosphoramidite are weighed into the synthesis glass and left for 3 hours in a desiccator over KOH together with the column filled with 28.1 mg CPG carrier, loaded with A building block, at the HV.
  • the phosphoramidites are dissolved in 1 ml (Indollinker) or 2.5 ml (A-phosphoramidite) acetonitrile and a few beads are added from the molecular sieve and left closed in the desiccator via KOH.
  • the carrier is slurried with aqueous 0.1 molar sodium diethyldithiocarbamate solution and at RT for 45 min. leave. It is suctioned off, washed with water, acetone, ethanol and dichloromethane.
  • the carrier is suspended in 1.5 ml of 24% hydrazine hydrate solution, shaken at 4 ° C. for 24-36 h and with 0.1 molar Triethylammonium hydrogen carbonate buffer (TEAB buffer) diluted to 7 ml. Hydrazine-free washing was carried out over a Waters Sep-Pak cartridge. 5 ml of an 80% formic acid solution are added, and the mixture is evaporated to dryness after 30 min.
  • TEAB buffer Triethylammonium hydrogen carbonate buffer
  • a p-RNA oligomer of sequence A 8 is produced on the Eppendorf Ecosyn D 300+ and then the following reagents are replaced: 6% dichloroacetic acid for 2% trichloroacetic acid, iodine in collidine against iodine in pyridine, benzimidazolium triflate solution against tetrazo solution.
  • a DNA oligomer of the sequence GATTC is further synthesized according to known methods (MJ Gait. Oligonucleotide Synthesis, IRL Press, Oxford, UK 1984). The deallylation, hydrazinolysis, HPL chromatography and desalting are carried out as described for the p-RNA oligomer (see above) and provide the desired conjugate.
  • 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 conjugate is formed when the two fragments (T. Zhu et al., Bioconjug. Chem. 1994, 5, 312) are left to stand in buffered solution. which is then purified using 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- (2-cyanoethyl) - (N, N-diisopropyl) -phosphoramidite) with one Amino group is provided, synthesized and worked up.
  • the oligonucleotide (17.4 OD, 0.175 ⁇ mol) was taken up in 0.5 ml basic buffer, 1.14 mg (2.5 ⁇ mol) biotin-N-hydroxysuccinimide ester was dissolved in 1 14 ⁇ l DMF (abs.) And lh at RT ditched. The resulting conjugate was purified by preparative HPLC and the pure product was desalted with a Sepak.
  • the last DMT (dimethoxytrityl) or MMT (monomethoxytrityl) protective group was not split off from biotin or cyanine monomers.
  • the last coupling with the modified phosphoramidites is detected after synthesis with 1% of the resin by means of trityl cation absorption in UV (503 nm).
  • the allyl ether protecting groups were split off with a solution of tetrakis (triphenylphosphine) palladium (272mg). Triphenylphosphine (272 mg) and diethylammonium hydrogen carbonate in CH 2 CI 2 (15 ml) after 5 hours at RT. The glass slides are then washed with CH 2 CL 2 (30ml), acetone (30ml) and water (30ml). To remove residual palladium complex, 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 freed of the hydrazine using an activated (acetonitrile, 20 ml) Waters Sep-Pak cartridge.
  • the hydrazine was washed with TEAB 0.1 M (30 ml)
  • the oligonucleotide was then eluted with acetonitrile / TEAB 0.1M (10ml) HPLC (to separate termination sequences) and performed the DMT deprotection (30 ml 80% aqueous formic acid).
  • Final desalination via Sep-Pak Kartuche, with TEAB buffer 0, IM / acetonitrile: 1/1) provided the pure 'cyanine or biotin-labeled oligomers.
  • the oligos were freeze-dried for storage.
  • the p-RNA was dissolved in a 0.1 molar sodium hydrogen carbonate solution (pH 8.4) (1 ml per 350 nmol) and a solution of N- (iodoacetyloxy) succinimide in DMSO (40 ⁇ l per mg) was added. The mixture was darkened with aluminum foil and left at room temperature for 30-90 minutes.
  • 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, wash with 3 x 10 ml of water to remove salt and reagent, and elute first with 5 x 1 ml of 50: 1 water: acetonitrile and then with 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 B 0.1 molar triethylammonium acetate buffer in water: acetonitrile 1: 4 gradient: starting from 10% B to 50% B in 40 minutes
  • the fractions were concentrated in the cold and in the dark, combined, and concentrated again. They were taken up in water and desalted.
  • 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, wash with 3 x 10 ml of water to remove the salt, and elute with water: acetonitrile 1: 1.
  • the product fractions were concentrated, combined and concentrated again. The yields were determined by means of UV absorption spectrometry at 260 nm. They achieved 70-95% of theory.
  • Buffer system Borax / HCl buffer from Riedel-de Haen, pH 8.0, was mixed 1: 1 with a 10 millimolar solution of EDTA disodium salt in water and adjusted to pH 6.6 with HC1. This gave a solution containing 5 mM Na 2 EDTA.
  • the mixture was diluted to four times the volume with water.
  • a Waters Sep-Pak cartridge RP-18 (from 15 OD 2g filling) was activated with 3 x 10 ml acetoniril and 3 x 10 ml water, the oligo was applied. let it sink in, washed the reaction vessel with 2 x 10 ml of water, washed the cartridge with 3 x 10 ml of water to remove salt and excess peptide, and eluted with 1: 1 water: acetonitrile until no spectroscopy was observed by UV spectroscopy eluted more. The frankings were concentrated in the cold and in the dark, combined and then concentrated again.
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WO1999052923A2 (de) * 1998-04-08 1999-10-21 Aventis Research & Technologies Gmbh & Co. Kg Verfahren zur herstellung von pentopyranosyl-nucleosiden
US6613894B1 (en) * 1997-09-22 2003-09-02 Nanogen Recognomics Gmbh Method for producing a pyranosyl nucleic acid conjugate
US6893822B2 (en) 2001-07-19 2005-05-17 Nanogen Recognomics Gmbh Enzymatic modification of a nucleic acid-synthetic binding unit conjugate
US7700761B2 (en) 1998-08-18 2010-04-20 Nanogen Recognomics Gmbh 3-deoxypentopyranosyl nucleic acid, its production and its use

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DE19741739B4 (de) * 1997-09-22 2006-04-27 Nanogen Recognomics Gmbh Supramolekulares Paarungssystem, dessen Herstellung und Verwendung
US20010049111A1 (en) * 1999-08-13 2001-12-06 Norbert Windhab Methods, procedures, and formats for using microelectronic array devices to perform multiplex immunoassay analyses
DE10010118A1 (de) * 2000-03-03 2001-09-20 Henkel Kgaa Beschichtungssystem auf der Basis von Pyranosyl-Nukleinsäuren
DE10111681A1 (de) * 2001-03-09 2002-10-02 Nanogen Recognomics Gmbh Verbessertes Verfahren zur Herstellung von Pentopyranosyl-Nucleosiden
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