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WO2008141799A1 - Oligophosphoramidates - Google Patents

Oligophosphoramidates Download PDF

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Publication number
WO2008141799A1
WO2008141799A1 PCT/EP2008/004022 EP2008004022W WO2008141799A1 WO 2008141799 A1 WO2008141799 A1 WO 2008141799A1 EP 2008004022 W EP2008004022 W EP 2008004022W WO 2008141799 A1 WO2008141799 A1 WO 2008141799A1
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group
atom
moiety
branched
aryl
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PCT/EP2008/004022
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WO2008141799A8 (fr
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Dieter Heindl
Christoph Seidel
Wilma Thuer
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Roche Diagnostics Gmbh
F. Hoffmann-La Roche Ag
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Publication of WO2008141799A8 publication Critical patent/WO2008141799A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G79/00—Macromolecular compounds obtained by reactions forming a linkage containing atoms other than silicon, sulfur, nitrogen, oxygen, and carbon with or without the latter elements in the main chain of the macromolecule
    • C08G79/02—Macromolecular compounds obtained by reactions forming a linkage containing atoms other than silicon, sulfur, nitrogen, oxygen, and carbon with or without the latter elements in the main chain of the macromolecule a linkage containing phosphorus
    • C08G79/04—Phosphorus linked to oxygen or to oxygen and carbon
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/535—Organo-phosphoranes
    • C07F9/5355—Phosphoranes containing the structure P=N-
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
    • C07F9/576—Six-membered rings
    • C07F9/58—Pyridine rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
    • C07F9/576—Six-membered rings
    • C07F9/62—Isoquinoline or hydrogenated isoquinoline ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
    • C07F9/6503—Five-membered rings
    • C07F9/6506—Five-membered rings having the nitrogen atoms in positions 1 and 3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
    • C07F9/65515—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a five-membered ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having sulfur atoms, with or without selenium or tellurium atoms, as the only ring hetero atoms
    • C07F9/655363—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having sulfur atoms, with or without selenium or tellurium atoms, as the only ring hetero atoms the sulfur atom being part of a six-membered ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65583—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system each of the hetero rings containing nitrogen as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • 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

Definitions

  • the present invention is directed to oligomeric compounds consisting of monomeric units having a spacer segment covalently bound to a phosphoramidate moiety. Substituents are attached independently at both the spacer segment, and at the phosphoramidate moiety.
  • the oligomers consist of either a random or a predefined sequence of units. A plurality of individual sequences can be created by varying the substituents of the incorporated monomers. The substituents differ from each another due to the choice of the preparator and the purpose of the prepared oligomers. The individual substituents can be selected from a broad range of chemical functionalities.
  • EP 0 751 948 and Fathi, R., et al., J. Org. Chem. 61 (1996) 5600-5609 provides compounds and processes which are essentially based on the long known standard phosphoramidate modification of oligonucleotides (Vorob'ev, O., E., et al., Doklady Akademii Nauk SSSR 166 (1966) 95-98.).
  • Synthesis is based on the strategy of converting an H-Phosphonate with CC14 in the presence of an nucleophilic amine to the corresponding phosphoramidate (Froehler, B., C, Tetrahedron Letters 27 (1986) 5575-5578).
  • Acceptor-substituted amines could not be used since they are not nucleophilc and will not react with the dichlorophosphonate intermediate.
  • the monomeric units themselves are build of two parts, a phosphate containing part and a spacer part.
  • the monomeric units themselves contain two groups Rl and R2 where Rl is connected to the phosphate containing part and R2 is connected to the spacer part of the monomeric unit.
  • n is an integer denoting the number of monomers, and n is equal to or higher than 1, in which Ace is an electron acceptor with a Hammett constant ⁇ p which exceeds the value of preferably 0.30, more preferred 0.45, even more preferred 0.60, in which X is a moiety selected from the group consisting of 2-16 atoms branched alkyl, alkenyl, alkinyl, aryl, heteroaryl, cycloalkyl or cyclo- heteroalkyl structure, whereby the atom or atoms of X which are connected to phosphoramidate are sp 3 C atoms, in which Rl is selected independently from R2, and Rl is directly or via a tether Tl attached to Ace, in which R2 is selected independently from Rl, and R2 is directly or via a tether T2 to a C atom or (if present) N atom of X, whereby Rl and R2 are moieties selected from the group consisting of
  • - a hydrogen atom, a core moiety selected from the group consisting of ⁇ a linear or branched C1-C6 alkyl group,
  • each heteroatom is independently selected from the group consisting of N, O, and S,
  • tethers Tl and T2 are independent from each other and a tether consists of a linear, branched or cyclic organic moiety comprising 1 - 30 C- atoms and between 0 and 5 heteroatoms selected from N, O, and S, or a subunit selected from an amide moiety and a urea moiety.
  • Another embodiment of the invention is a process for producing a compound according to the invention, compring the steps
  • each heteroatom is independently selected from the group consisting of N, O, and S,
  • tethers Tl and T2 are independent from each other and a tether consists of a linear, branched or cyclic organic moiety comprising 1 - 30 C- atoms and between 0 and 5 heteroatoms selected from N, O, and S, or a subunit selected from an amide moiety and a urea moiety, whereby reactive groups of Rl and R2 selected from the group consisting of carboxyl, formyl, hydroxyl, amino, amido, sulfhydryl, phosphatyl, imidazolyl, indolyl and guanidyl groups are protected by protective groups.
  • the protective group PGl is selected from trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, trialkylsilyl, allyl, 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX), nitroveratryl and NPPOC, whereby the protective group PG2 is selected from betacyanoethyl, methyl, betanitroethyl and allyl, and R3 and R4 are independent from each other and are selected from Cl - C6 alkyl, whereby R3 and R4 can be linked to each other by methylen or an O-atom to form a ring structure,
  • step (c) reacting the phosphotriester of step (b) with Rl-Tl-Acc-azide (Formula III), whereby Ace is an electron acceptor with a Hammett constant ⁇ p which exceeds the value of preferably 0.30, more preferred 0.45, even more preferred 0.60
  • step (e) cleaving of the temporary protective group PG 1 , (f) reacting the new deliberated hydroxyl group from step (e) with a second monomer according to Formula II,
  • oligophosphoramidates obtainable by the process according to the invention.
  • Exemplary oligophosphoramidates are the oligophosphoramidates 1 to 11 as shown in Figures 6 to 16.
  • the central idea of the present invention was in this connection to start with a compound comprising a trivalent phosphorus atom and to react the trivalent phosphorous atom with a reagent in such a manner that a stable phosphate mimetic is formed as shown in Scheme 1 below.
  • the phosphoramidites according to Formula II with a phosphorus atom containing at least one hydroxyl residue which is provided with a protective group are reacted for this purpose with a free hydroxyl group:
  • the hydroxyl group is linked to a solid support via a cleavable or non cleavable linker.
  • protective groups PG2 and further protecting groups which are attached if neccessary to Rl and R2 are cleaved off.
  • Phosphoramidites comprising the substituted spacer unit, to which a protected hydroxyl group (e.g. dimethoxytrityl protected) is attached are useful starting materials to introduce a monomeric unit during solid phase synthesis of a oligophosphoamidate.
  • Phosphoramidites are activated by a weak acid, e.g. tetrazol or dicyanoimidazol, and than reacted with a hydroxylgroup of a monomeric unit which is already attached either via a cleavable or non cleavable linker to a solid support.
  • Oligophosphoamidate synthesis can then be subsequently continued by releasing the protective group (e.g. dimethoxytrityl) from the newly attached monomeric unit and reacting with a further phosphoramidite.
  • the protective group e.g. dimethoxytrityl
  • the oligomer is cleaved from the solid support, e.g. by ammonia.
  • all other protecting groups are removed, too.
  • Stable polyphosphoramidates are obtained as end product which are modified in almost any manner on one or more phosphoramidate residues and on the spacer units linking the phosphoamidate moieties.
  • the protective groups are removed as described above but the oligophosphoamidate remains attached to the solid support. Notably, this is useful for preparation of arrays of oligophosphoamidates
  • protective group denotes molecular assemblies which are connected to one or more functional groups of a molecule such that, as part of a multistep synthesis reaction, only one particular, non-protected functional group can react with the desired reaction partner.
  • the skilled person differentiates between temporary and permanent protective groups. The first protects the side chains of the growing oligomer until the synthesis is finalized; the second protects the growing chain end of the oligomer and is removed before each prolongation step. It is reintroduced with the incorporation of the next monomer.
  • the use of one or more protective groups assures that oligomer synthesis proceeds in the desired way.
  • Examples of frequently used protective groups to protect hydroxyl groups are trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, trialkylsilyl, allyl, 9- phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX), beta- cyano-ethyl and others which are known to the skilled person.
  • Protective groups for protecting amino groups are trifluoroacetyl, BOC, benzyloxycarbonyl, Fmoc and others. Other possible protective groups are summarized in standard text books (Greene, T., W., Protective groups in organic synthesis, John Wiley&Sons, Inc.
  • spacer denotes the linkage between two phosphoamidate moieties.
  • a spacer usually contains a characterizing substituent.
  • the spacer is a trifunctional moiety where two of the arms are connected to the O-atoms of the adjacent phosphoamidates when an oligomer is formed.
  • the third arm of the spacer contains a substituent from the group defined below. It may be a branched alkyl, heteroalkyl (an alkyl residue which additionally comprises one or more N atoms), alkenyl, alkinyl, aryl, heteroaryl, cycloalkyl or cyclo-heteroalkyl structure with three connectivities. Between the substituent and this branched structure a tether is optionally incorporated.
  • one O-atom and/or the spacer is linked to a hydrogen, to a solid phase (optionally via a linker), to a detectable moiety or to phosphate or phosphoamidate.
  • Phosphoramidites are molecules containing a trivalent phosphorus atom which can be coupled to a hydroxyl group. Examples are beta-cyanoethyl-bis- diisopropylamino-phosphoramidite very well known from standard oligonucleotide synthesis. Especially useful are intermediates and phosphoramidites described in EP 1 186 613 (aminopropane spacer), Kawakami, J., et al, Chemistry
  • the present invention concerns in particular embodiments in which the Hammett constant ⁇ p exceeds a certain value of 0.30, preferably 0.45 and particularly preferably 0.60 (Hansch, C, et al., Chem. Rev. 91 (1991) 165-195).
  • the electron acceptor must additionally be compatible with all chemical reactions in oligophorphoramidate synthesis i.e.
  • acceptors can also be bound to the nitrogen atom in a vinylogous or phenylogous manner.
  • nitro- and cyano-acceptors can be bound to the nitrogen atom in a vinylogous or phenylogous manner.
  • substituted means that the structure that is referred to as being substituted contains another residue at any position provided this position is not defined in more detail.
  • optionally substituted denotes that the structure referred to in this manner comprises embodiments with and without an additional residue.
  • N + -heterocycle encompasses N-heterocycles which are alkylated on an sp 2 nitrogen such that the overall charge of the heterocycle is positive. Examples of this are pyridinium, pyrimidinium and quinolinium. Such hetrocycles are known in the art to be electron deficient.
  • Bracketed portion is herein referred to as a monomeric unit.
  • a monomeric unit is comprised of a spacer segment with a phosphoramidate attached thereto.
  • Compounds of the present invention are made up of at least 2 of these monomeric units. Included in a monomeric unit is a phosphoramidate moiety that, in turn, is capable of bearing functional groups thereon.
  • the phosphoramidate moiety is covalently bonded to a spacer segment which may also be capable of including a variety of functional groups covalently bonded thereto.
  • Functional groups are covalently bonded directly to the backbone segment and the phosphoramidate, or via an optional tether group.
  • the spacer segment and phosphoramidate moiety serve as sites for connecting certain other groups that impart "functional" properties to the oligomeric compounds of the invention. By varying these functional groups - diversity is incorporated into the compounds of the invention.
  • tether denotes a carbon chain having a length of 1 - 30 C- atoms or can also be a bisconnectable cyclic structure.
  • a tether can also contain one or more internal heteroatom like nitrogen, oxygen, and/or sulphur and may thus comprise an amide or urea moeties. Tethers can also be branched, e.g. be dendritic.
  • a tether interconnects a spacer or a phosphoramidite moiety with, e.g. a substituent, a functional group or a detectable unit which may optionally be protected by one or more protective groups.
  • internal heteroatoms with the exception of a disulfide bond must be separated from each other by a minimum of two carbon atoms.
  • the groups Rl and R2 can be "reactive” or "non-reactive.” By reactive, it is meant that they will interact with a target molecule in some manner (that need not but can be predefined). By nonreactive, it is meant that they are not designed to primarily interact with a target molecule, and in fact while they may interact with the target molecule, the primary purpose of the non-reactive moieties are to impart other properties to the molecule such as effecting uptake, distribution, metabolism or identification.
  • Tethers are bivalent or polyvalent groups Such tethers can be used to position Rl and R2 in space with respect to the linear backbone or the phosphoramidate moiety of the oligomeric compound synthesized or to link Rl and/or R2 to the spacer or phosphoramidate moiety that themselves are not bindable to the parts of the monomeric unit.
  • Aryl groups according to the invention include but are not limited to substituted and unsubstituted aromatic hydrocarbyl groups such as phenyl and naphthyl groups.
  • Aralkyl groups include but are not limited to groups having both aryl and alkyl functionality, such as benzyl and xylyl groups.
  • a number of functional groups can be introduced into compounds of the invention containing protective groups.
  • Solid supports useful for synthesis of compounds according to the invention include controlled pore glass (CPG), oxalyl-controlled pore glass (see, e.g., Alul, R. H. et al., Nucleic Acids Research 19 (1991) 1527-1532), TentaGel Support - an aminopolyethyleneglycol derivatized support (see, e.g., Wright, P., et al.,
  • Another aspect of the invention is the use of a compound according to the invention for interacting with a target molecule.
  • another aspect of the invention is the use of a compound obtainable by a process according to the invention for interacting with a target molecule. According to the invention this interaction occurs in a solution, preferably in an aqueous solution.
  • the compounds of the invention can easily be synthesized in a great variety.
  • compounds can be synthesized which are capable of interacting with a target molecule.
  • a combinatorial library of separately synthesized compounds according to the invention is contacted with a target molecule and interaction is assayed, thereby detecting interaction of one or more compounds and the target molecule.
  • a compound capable of interacting with the target molecule may undergo further refinement, preferably by exchanging one or more substituents such as Rl and R2 or by introducing modifications in Tl and T2, according to the invention.
  • refinement can be used to fine-tune the interaction between the compound according to the invention and the target molecule.
  • the compound of the invention may physically interact with the target molecule.
  • the interaction between the target molecule and the compound of the invention is specific.
  • the interaction of a compound according to the invention with an enzyme as target molecule can be strong enough that the interaction can modify the enzymatic activity.
  • Modification of an enzymatic activity can result from (i) interaction of the compound with the substrate that is targeted by the enzymatic activity, (ii) interaction of the compound with a co-factor of the enzyme, provided that such a co-factor is present in the enzymatic reaction, and (iii) interaction of the compound with the enzyme itself. Modification can occur characterized in that the enzymatic activity is enhanced or inhibited.
  • such modification is the effect of a specific interaction between the compound of the invention and the target.
  • the invention encompasses a method to modify the activity of an enzyme by contacting a mixture comprising an enzyme, a substrate and optionally a co-factor with a compound according to the invention under conditions permitting activity of the enzyme.
  • the activity of the enzyme is enhanced compared to the mixture without the compound according to the invention.
  • the activity of the enzyme is reduced compared to the mixture without the compound according to the invention.
  • compositions comprising (i) a compound according to the invention or a compound obtainable by a process according to the invention, (ii) a polypeptide with enzymatic activity, (iii) a substrate capable of being converted by the enzymatic activity.
  • the composition additionally comprises (iv) a co-factor.
  • one single compound according to the invention is present in the composition.
  • the composition comprises one or more compounds according to the invention.
  • the preferred concentration of the compound (or compounds) is in the range between 100 ⁇ M and 100 pM. Particularly preferred ranges include between 100 ⁇ M and 100 nM and between 100 nM and 100 pM.
  • the oligophosphoramidate is synthesized in 1 ⁇ mol scale on ABI 394 synthesizer using the phophoramidite Uni-Link-Aminomodifier (BD Biosciences 5190-2) and PAl .
  • As solid support is used a Phospholink-CPG (Roche Id 12239809). All chemicals for the standard protocol are from Proligo or ABI. The synthesis follows the standard protocol except for the oxidation.
  • First oxidation occurs with 0.1 M 2- Azido-N-ethyl-pyridinium-tetrafluoroborate (RareChemicals) in acetonitrile 2 times for 30 min.
  • second oxidation with 0.1 M 4-Acetamidobenzenesulfonyl azide (Fluka) in acetonitrile 2 times for 30 min.
  • the product is cleaved form the solid support and deprotected with 30 % aqueous NH 3 (4h 55°C).
  • the Oligophosphoramidates (12-20) were synthesized in 1 mmol scale on an ABI 394 synthesizer using purchasable (GlenResearch), published (EP 1538221) or above described phosphoramidites. As solid support were used 3 V -Spacer-C3-CPG (GlenResearch) or Phospholink-CPG (GlenResearch) or 3 N -TFA-Aminomodifier- C7-CPG (Chem Genes). All chemicals for the standard protocol were from Proligo or ABI. The synthesis follows the standard protocol with a for 10 min. prolonged phosphoramidite coupling step.
  • the oxidation of the trivalent phosphorus occurs with purchasable (Tolensulfonyl azide (VeZerf Laborsynthesen GmbH), 2-Azido-l- ethylpyridinium tetrafluoro borate, 4-Azido-l,2,6-trimethylpyridinium tetrafluoro borate (Rare Chemicals), 2-azido-5-nitro-pyrimidine (Toslab BB), 4- Acetamidobenzenesulfonyl azide (Fluka), KA3205 (Aurora)) or above described Azidocompounds (0.05 M in Acetonitril) 2 times for 30 min. instead of the standard procedure. The products were cleaved form the solid support and deprotected with 30 % NH 3 (2-4 h at r.t.).
  • the experiment is performed using the enzyme beta-galactosidase in an ELISA immunoassay (Roche Cat. No. 11539 426 001) with ABTS as substrate. Each assay is performed according to the pack insert. Beta-galactosidase (available from Roche Applied Science, Roche Diagnostics GmbH, Mannheim, Germany) is used as standard. A calibration curve for one or more assays as described in the instruction leaflet is established by measuring the absorbance at 405 nm during the beta- galactosidase assay.
  • each experiment is performed (i) in the presence and (ii) in the absence of the oligophosphoramidate or the mixture of two or more oligophosphoramidates as the activity modifying agent(s).
  • the oligophosphoramidate or the mixture of two or more oligophosphoramidates (preferably between 2 and 10 different oligophosphoramidate compounds) is added in an amount to have a final concentration in the range between 100 ⁇ M and 100 pM (in the case of a mixture the concentration of the oligophosphoramidates combined is in the range between 100 ⁇ M and 100 pM).
  • Changes in absorbance at 405 nm are monitored in comparison to the calibration curve.
  • the experiment is performed using the enzyme alkaline phosphatase in an ELISA immunoassay with AttoPhos as substrate (Roche Cat. No. 11681982001). Each assay is performed according to the pack insert. Alkaline phosphatase (available from Roche Applied Science, Roche Diagnostics GmbH, Mannheim, Germany) is used as standard. A calibration curve for one or more assays as described in the instruction leaflet is established by measuring the emission at 550 nm during the alkaline phosphatase assay.
  • each experiment is performed (i) in the presence and (ii) in the absence of the oligophosphoramidate or the mixture of two or more oligophosphoramidates as the activity modifying agent(s).
  • the oligophosphoramidate or the mixture of two or more oligophosphoramidates is added in an amount to have a final concentration in the range between 100 ⁇ M and 100 pM (in the case of a mixture the concentration of the oligophosphoramidates combined is in the range between 100 ⁇ M and 100 pM). Changes in emission at 550 nm are monitored in comparison to the calibration curve.
  • the experiment is performed with a protease (selected from pronase and trypsin) assay using resorufin-labeled Casein as substrate (Roche Cat. No. 11080733 001). Each assay is performed according to the pack insert. Pronase or trypsin (available from Roche Applied Science, Roche Diagnostics GmbH, Mannheim, Germany) are used as standards. A calibration curve for one or more assays as described in the instruction leaflet is established by measuring the emission at 584 nm during the protease assay.
  • each experiment is performed (i) in the presence and (ii) in the absence of the oligophosphoramidate or the mixture of two or more oligophosphoramidates as the activity modifying agent(s).
  • the oligophosphoramidate or the mixture of two or more oligophosphoramidates is added in an amount to have a final concentration in the range between 100 ⁇ M and 100 pM (in the case of a mixture the concentration of the oligophosphoramidates combined is in the range between 100 ⁇ M and 100 pM). Trypsin and pronase are assessed separately. Changes in emission at 584 nm are monitored in comparison to the respective calibration curve.
  • PCR reactions in the presence or absence of 1 ⁇ M -100 ⁇ M of one or more oligophosphoramidates is performed in 50 ⁇ l reactions containing 25 ng, 10 ng, 5 ng, 1 ng and 0 ng of human genomic DNA, 30 mM Tris-HCl, pH 8.6, 1.5 mM MgCl 2 , 50 mM KCl, 0.2 mM dNTP's each, 0.4 ⁇ M primers (SEQ ID NO: 1 ATT AGA GAA CCA TGT TAA CAC TAC CG and SEQ ID NO: 2 GAG GTG AAT GAC CAC TGT TTA TTT TC ) and 2.5 units Taq DNA polymerase.
  • the following cycle conditions are used: Initial denaturation for 4 min at 94°C and 35 cycles with 20 seconds denaturation at 94°C,

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Abstract

L'atome de phosphore trivalent d'un composé est mis à réagir avec un réactif de telle sorte qu'un mimétique de phosphate stable est formé. Des phosphoramidites avec un atome de phosphore contenant au moins un reste hydroxyle qui comporte un groupe protecteur sont mis à réagir dans ce but avec un groupe hydroxyle libre. Dans le premier cycle de synthèse, le groupe hydroxyle est lié à un support solide par l'intermédiaire d'un liant clivable ou non clivable. Dans d'autres cycles de synthèse, le groupe hydroxyle est créé par clivage du groupe protecteur à partir de l'oligomère en croissance. Ceci conduit à la formation d'un triester d'acide phosphoreux qui est mis à réagir avec des azotures. On obtient des composés de formule (I).
PCT/EP2008/004022 2007-05-24 2008-05-20 Oligophosphoramidates WO2008141799A1 (fr)

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WO2014088923A1 (fr) * 2012-12-06 2014-06-12 Merck Sharp & Dohme Corp. Compositions et méthodes de dérivation de kinases nucléosidiques
JP2016507484A (ja) * 2012-12-06 2016-03-10 メルク・シャープ・アンド・ドーム・コーポレーションＭｅｒｃｋ Ｓｈａｒｐ ＆ Ｄｏｈｍｅ Ｃｏｒｐ． ジスルフィドマスキングプロドラッグ組成物および方法
WO2018156056A1 (fr) * 2017-02-21 2018-08-30 Дмитрий Александрович СТЕЦЕНКО Oligonucléotides modifiés activant l'arnase n
US10781175B2 (en) 2016-07-15 2020-09-22 Am Chemicals Llc Solid supports and phosphoramidite building blocks for oligonucleotide conjugates
JPWO2019039403A1 (ja) * 2017-08-22 2020-11-26 国立大学法人東海国立大学機構 修飾ポリヌクレオチド
US11208430B2 (en) 2014-08-22 2021-12-28 Noogen Llc Modified oligonucleotides and methods for their synthesis

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EP2370450A1 (fr) * 2008-11-27 2011-10-05 Roche Diagnostics GmbH Synthèse dirigée de stéréoisomères d'oligophosphoramidates
CN104955818B (zh) * 2012-12-06 2017-08-04 默沙东公司 核苷激酶旁路组合物和方法
US20150315221A1 (en) * 2012-12-06 2015-11-05 Merck Sharp & Dohme Corp. Nucleoside kinase bypass compositions and methods
JP2016507484A (ja) * 2012-12-06 2016-03-10 メルク・シャープ・アンド・ドーム・コーポレーションＭｅｒｃｋ Ｓｈａｒｐ ＆ Ｄｏｈｍｅ Ｃｏｒｐ． ジスルフィドマスキングプロドラッグ組成物および方法
JP2016509575A (ja) * 2012-12-06 2016-03-31 メルク・シャープ・アンド・ドーム・コーポレーションＭｅｒｃｋ Ｓｈａｒｐ ＆ Ｄｏｈｍｅ Ｃｏｒｐ． ヌクレオシドキナーゼバイパス組成物および方法
EP2928876A4 (fr) * 2012-12-06 2016-08-17 Merck Sharp & Dohme Compositions et méthodes de dérivation de kinases nucléosidiques
US9624249B2 (en) 2012-12-06 2017-04-18 Merck Sharp & Dohme Corp. Nucleoside kinase bypass compositions and methods
WO2014088923A1 (fr) * 2012-12-06 2014-06-12 Merck Sharp & Dohme Corp. Compositions et méthodes de dérivation de kinases nucléosidiques
CN104955818A (zh) * 2012-12-06 2015-09-30 默沙东公司 核苷激酶旁路组合物和方法
AU2013356383B2 (en) * 2012-12-06 2017-08-31 Merck Sharp & Dohme Corp. Disulfide masked prodrug compositions and methods
AU2013356386B2 (en) * 2012-12-06 2017-09-07 Merck Sharp & Dohme Corp. Nucleoside kinase bypass compositions and methods
US11208430B2 (en) 2014-08-22 2021-12-28 Noogen Llc Modified oligonucleotides and methods for their synthesis
US10781175B2 (en) 2016-07-15 2020-09-22 Am Chemicals Llc Solid supports and phosphoramidite building blocks for oligonucleotide conjugates
US11447451B2 (en) 2016-07-15 2022-09-20 Am Chemicals Llc Solid supports and phosphoramidite building blocks for oligonucleotide conjugates
WO2018156056A1 (fr) * 2017-02-21 2018-08-30 Дмитрий Александрович СТЕЦЕНКО Oligonucléotides modifiés activant l'arnase n
RU2740501C2 (ru) * 2017-02-21 2021-01-14 Федеральное государственное бюджетное учреждение науки Институт химической биологии и фундаментальной медицины Сибирского отделения Российской академии наук (ИХБФМ СО РАН) МОДИФИЦИРОВАННЫЕ ОЛИГОНУКЛЕОТИДЫ, АКТИВИРУЮЩИЕ РНКазу Н
US11236334B2 (en) * 2017-08-22 2022-02-01 National University Corporation Nagoya University Modified polynucleotide
JPWO2019039403A1 (ja) * 2017-08-22 2020-11-26 国立大学法人東海国立大学機構 修飾ポリヌクレオチド
JP7282379B2 (ja) 2017-08-22 2023-05-29 国立大学法人東海国立大学機構 修飾ポリヌクレオチド

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