WO1996028438A1 - Derives non nucleosidiques de coumarine utilises comme agents de reticulation de polynucleotides - Google Patents

Derives non nucleosidiques de coumarine utilises comme agents de reticulation de polynucleotides Download PDF

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WO1996028438A1
WO1996028438A1 PCT/US1996/003134 US9603134W WO9628438A1 WO 1996028438 A1 WO1996028438 A1 WO 1996028438A1 US 9603134 W US9603134 W US 9603134W WO 9628438 A1 WO9628438 A1 WO 9628438A1
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compound
formula
group
carbon atoms
ring
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PCT/US1996/003134
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English (en)
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Michael L. Wood
Peter C. Cheng
Douglas Y. Thein
David Albagli
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Naxcor
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Priority claimed from US08/401,630 external-priority patent/US6005093A/en
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Priority to JP8527721A priority Critical patent/JPH11501927A/ja
Priority to EP96908709A priority patent/EP0827501A4/fr
Priority to AU51864/96A priority patent/AU719277B2/en
Publication of WO1996028438A1 publication Critical patent/WO1996028438A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
    • C07D407/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
    • C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
    • C07D311/16—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted in position 7
    • 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
    • C07F9/6552—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 six-membered ring
    • C07F9/65522—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 six-membered ring condensed with carbocyclic rings or carbocyclic 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
    • C07F9/6561—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

Definitions

  • This invention is related to photoactive nucleoside analogues that can be incorporated into synthetic oligonucleotides during automated DNA synthesis for use in crosslinking of complementary target nucleic acid sequences.
  • K. Yabusaki et al. compounds are based on furocoumarin (or psoralen) attached to existing polynucleotides (usually through adduct formation) and are satisfactory for many applications.
  • the crosslinking group/nucleoside adduct is difficult to synthesize, particularly in large quantities.
  • Saba et al. describe a photoactivatible nucleoside analogue comprising a coumarin moiety linked through its phenyl ring to the 1-position of a ribose or deoxyribose sugar moiety in the absence of an intervening base moiety.
  • the resulting nucleoside analogue is used as a photo-crosslinking group when inserted into a polynucleotide as a replacement for one or more of the complementary nucleoside bases present in a probe used in hybridization assays.
  • the current invention provides non-nucleosidic, stable, photoactive compounds that can be used as photo-crossliriking reagents in nucleic acid hybridization assays and therapeutic applications, as well as techniques and intermediates that can be used to prepare the final products.
  • the compounds comprise coumarinyl derivatives prepared by linking the phenyl ring of a coumarin molecule or derivative to a hydroxy or polyhydroxy hydrocarbon molecule, such as one of the terminal hydroxy groups of a glycerol molecule.
  • the (poly)hydroxy hydrocarbon moiety of the resulting compound is equivalent to the sugar of a nucleoside, while the coumarin moiety occupies the position of a base. Accordingly, the compounds can be inserted into growing polynucleotide chains using automated (or manual) techniques of polynucleotide synthesis.
  • the double bond between the 3 and 4 positions of the coumarin ring system is a photoactive group that covalently crosslinks to nucleosides in the complementary strand when an oligonucleotide containing this non-nucleoside analogue (the "probe") is used in a hybridization assay and/or therapeutic application.
  • the photoactive compound has the formula
  • the (poly)hydroxy hydrocarbon backbones give maximum flexibility and stability to the oligosaccharide structure in which they are located as well as good solubility in aqueous and organic media. DESCRIPTION OF SPECIFIC EMBODIMENTS
  • the present invention provides crosslinkable compounds that can be used as a photoactivatible non-nucleosidic crosslinker in oligonucleotide probes used in hybridization assays and/or therapeutic applications.
  • the compounds of the inventions are typically used as part of synthetic DNA or RNA oligonucleotides to determine the presence or absence of a specific DNA and RNA base sequence in a sample. More specifically, this invention provides coumarin derivatives attached to a stable, flexible, (poly)hydroxy hydrocarbon backbone unit that act as photoactive crosslinking compounds in hybridization assays.
  • Moiety here and elsewhere in this specification indicates a part of a molecule that performs the indicated function.
  • a given moiety is usually derived from another molecule by covalently linking together two or more molecules, with the identifiable remnants of the original molecules being referred to as "moieties. " For example, if a psoralen molecule is attached to a glycerin molecule with a divalent linker, such as a methylene group, the resulting single molecule is referred to as being formed of glycerin, methylene, and psoralen moieties. It is not necessary, however, that the three moieties actually arose from three separate molecules, as discussed below. Thus “derived from” can refer to theoretical, as well as actual, precursors.
  • crosslinking moiety will be derived from molecules having a fused benzopyrone structure, such as the following: (1) coumarin and its simple derivatives; (2) psoralen and its derivatives, such as 8-methoxypsoralen or
  • 5-methoxypsoralen (at least 40 other naturally occurring psoralens have been described in the literature and are useful in practicing the present invention);
  • the linking moiety will normally be formed from a precursor that contains from 1 to 100, preferably 1 to 25, more preferably 1 to 10, atoms with functional groups at two locations for attaching the other moieties to each other.
  • the total number of atoms in the shortest linking chain of atoms between the coumarin ring system and the backbone moiety (sugar substitute) is generally from 1 to 15, preferably 1 to 7, more preferably 1 to 3. Otherwise this part of the structure can vary widely, as this is essentially just a flexible linkage from the crosslinking moiety to the backbone moiety.
  • the linking moiety is most often a stable cyclic or acyclic moiety derived by reaction of a molecule bearing appropriate functional groups (usually at its termini) for linking the crosslinking molecule at one end and the backbone molecule at the other end.
  • appropriate functional groups usually at its termini
  • a precursor to the linking moiety need not be used (i.e., the backbone and crosslinking moieties can be connected by a covalent bond).
  • the linking moiety is considered to consist of atoms between the ring atom of the crosslinking moiety at the point of attachment and the last carbon atom that clearly forms part of the backbone structure in the moiety that replaces the sugar molecule, which is usually the carbon atom bearing a hydroxyl group (or reaction product of a hydroxyl group) that is closest to the crosslinking moiety.
  • the backbone moiety so called because it ultimately functions in place of the ribose or deoxyribose portion of the backbone of a polynucleotide, will generally have 1 to 3 (sometimes more) hydroxyl groups (or similar functional groups, as discussed below) attached to different sp 3 -hybridized carbon atoms.
  • the backbone moiety is generally uncharged so that it can function as a substitute for ribose or deoxyribose in the final modified nucleotide.
  • Backbone moieties include but are not limited to the following: (1) linear hydrocarbon moieties such as a three-carbon propane unit or a longer hydrocarbon chain with appropriate functional groups, usually selected from the group consisting of -OH, -NH_, -SH, -COOH, acid halides, and acid anhydrides, and (2) cyclic hydrocarbon moieties typically having a 5- to 7-membered carbon ring structure bearing one to three hydroxyl group or other functional groups as in (1) above.
  • the functional groups are shown in the preceding sentence in unreacted form and will be present as derivatives of the indicated functional groups in many embodiments.
  • the reactive functional groups mentioned above are generally present only in intermediates; however, after reacting with other functional groups, they become stable groups or form covalent bonds to other parts of the molecule.
  • one or more coupling moieties can be attached to the backbone moiety to facilitate formation of bonds to existing or growing polynucleotide chains.
  • the coupling moieties will typically comprise hydroxy coupling and/or protecting groups that are used in solution or solid-phase nucleic acid synthesis when the molecule in question is an intermediate being used in the preparation of a probe molecule.
  • Typical coupling groups include phosphoramidite, phosphate, H-phosphonate, phosphorothioate, methyl phosphonate, trityl, dimethoxytrityl, monomethoxytrityl, and pixyl groups.
  • Non-phosphorous coupling groups include carbamates, amides, and linear and cyclic hydrocarbon groups, typically connecting to the remainder to the molecule with heteroatom substituents, such as -COCH 3 , -CH 2 OH, -CF 3 , -NHCH 3 , and PO 2 CH 2 CH 3 .
  • heteroatom substituents such as -COCH 3 , -CH 2 OH, -CF 3 , -NHCH 3 , and PO 2 CH 2 CH 3 .
  • Preferred compounds of the invention have the formula:
  • B represents (1) a linear, branched, or cyclic hydrocarbon group containing from 2 to 15, preferably 3 to 10, more preferably 3 to 6, carbon atoms and, if cyclic, containing a 5- or 6-membered ring or (2) a heterocyclic aromatic ring system comprising a 5- or 6-membered ring, both of B(l) and B(2) being substituted with 1 , 2, or 3 groups of the formula OR,;
  • X represents (1) a linear, branched, or cyclic hydrocarbon group containing
  • n 0, 1 , 2, or 3;
  • each W independently represents a hydroxy, halogen, amino, amido, azido, nitro, thio, carboxy, carbonyl, perfluoromethyl, or cyano functional group; an unsubstituted hydrocarbyl group of 10 or fewer carbon atoms, preferably 6 or fewer, more preferably 3 or fewer; or such a hydrocarbyl group substituted with 1-3 of the functional groups or in which one carbon atom is replaced by an oxygen, sulfur, or nitrogen atom;
  • Y and Z independently represent H, F or lower alkyl (usually 5 of fewer carbons, preferably 3 or fewer);
  • each R independently represent H, F or a hydroxy-protecting or hydroxy-coupling group capable of protecting or coupling a hydroxy group during synthesis of a polynucleotide or one or two (preferably two) R, represent a nucleotide or a polynucleotide connected to the compound.
  • oxygen atom or other non-C atom (if present) of a functional group such as an ether or carboxylate
  • a functional group such as an ether or carboxylate
  • B moieties belong to a group of a first sub-formula
  • s 2 or 3;
  • R x , R y , and R z independently represent H or OR,
  • n, p, q, and r independently represent 0 or 1 ;
  • one hydrogen of the sub-formula is replaced by a covalent bond to the X group
  • the hydrogen atom of the sub-formula that is replaced by a covalent bond to the X group is usually a hydrogen of a hydroxyl group (i.e, at least one OR, would represent a hydroxyl group in such a precursor molecule).
  • a hydrogen of a hydroxyl group i.e, at least one OR, would represent a hydroxyl group in such a precursor molecule.
  • this preference is for convenience of synthesis only, as the resulting B-X linkage can readily be prepared from (poly)hydroxy hydrocarbon precursors, many of which are commercially available.
  • Other hydrogens can be replaced by the indicated covalent bond if desired.
  • the actual molecules used in synthesis are often still derived from a (poly)hydroxy compound in which one of the hydroxyl groups has been replaced by the functional group, often through a series of reactions.
  • a hydroxyl group can be replaced by a halogen atom or other leaving group, and the leaving group can participate in bond formation with an electron donating group in the precursor of
  • these compounds of the third sub-formula represent an acyclic, saturated, di- or tri-hydroxy hydrocarbon, especially glycerol and 1 ,2- or 1 ,3-dihydroxyalkanes of 3 to 5 carbons that are attached to the X group at their terminal position furthest from the indicated hydroxyl groups, such as 4,5-dihydroxypentyl, 3,5-dihydroxypentyl, 2,4-dihydroxy-2-methylbutyl, 3-hydroxy-2-(hydroxymethyl)propyl, and 2,3-dihydroxypropyl.
  • aromatic ring systems can be present in the B moiety. These include both hydrocarbon and hetererocyclic aromatic ring systems. Of these compounds in which B comprises a benzene or naphthalene ring system are preferred, especially 1 ,2-di(hydroxymethy)-substituted aromatics. The same substituents are preferred when
  • B comprises a heterocyclic ring system, such as a furan, pyran, pyrrole, pyrazole, imidazole, piperidine, pyridine, pyrazine, pyrimidine, pyrazidine, thiophene, acridine, indole, quinoline, isoquinoline, quinazoline, quinoxaline, xanthene or 1 ,2-benzopyran ring systems.
  • a heterocyclic ring system such as a furan, pyran, pyrrole, pyrazole, imidazole, piperidine, pyridine, pyrazine, pyrimidine, pyrazidine, thiophene, acridine, indole, quinoline, isoquinoline, quinazoline, quinoxaline, xanthene or 1 ,2-benzopyran ring systems.
  • B comprises a bridged hydrocarbon ring system, such as bicyclo [3.1.0] hexane or [2.2.1] heptane ring system.
  • B comprises a bridged hydrocarbon ring system
  • These molecules have configurations with reduced mobility so that various cis and trans substitution pattern can be easily prepared and maintained. See, for example, Ferguson, "Organic Molecular Structure,” Willard Grant, Boston, 1975, chapters 17-19, for a review of this chemistry and synthetic techniques.
  • compounds in which B comprises a spiro or dispiro hydrocarbon ring system are also within the scope of the invention.
  • the X linking group is not particularly restricted in structure, as it is not present in a part of the molecule that interacts either with the remainder of the backbone structure or with a complementary strand of a polynucleotide.
  • this part of the molecule such as the following, which can represent X, in either of the two possible orientations:
  • n 0 , 1 , or 2 .
  • X comprises a cyclic structure with a 5- or 6-membered carbon or heterocyclic ring (the latter containing one O, S, or N atom), such as cyclopentane, cyclohexene,
  • Y and Z generally have 5 or fewer carbons, preferably 3 or fewer, and are most preferably methyl if they are alkyl groups.
  • Compounds in which W, Y, and Z are all hydrogen are preferred, as are compounds in which W is a pyrone or furan ring fused to the phenyl ring of the formula.
  • These later compounds are preferably compounds in which all of the formula to the right of X in formula I represents coumarin, psoralen, cis- benzodipyrone, or trans-benzodipyrone or a derivative thereof within the formula.
  • the compounds of formula I in which a nucleotide or polynucleotide is connected to the compound are usually (but not always) connected via a
  • phosphorous-containing unking group Preferred phosphorous-containing linking groups, as well as other linking groups, are discussed elsewhere. Such compounds are preferred compounds of the invention, as they can be used directly in the assays and crosslinking processes that are the principal end use of this invention.
  • N ml Q m4 N m2 are integers (usually less than 200, preferably less than 100; one of ml and m2 is usually at least 14, preferably at least 17, most preferably at least 20); m3 is an integer from 1 to 10, preferably 1 to 5 (m3 is generally less than (m1 +m2)/10); each N independently represents a nucleotide of a desired polynucleotide sequence; Q represents the nucleotide- replacing molecule of the invention incorporated into the normal polynucleotide sequence; and m4 is 1-5, preferably 1-3.
  • Q can be present either in the interior of the polynucleotide or at one of its terminal positions. In an interior position, at least two R 1 groups must be present in order to allow the Q molecule to connect to ends of two separate strands; if Q is inserted at a terminal position, only one R 1 is required, although others may be present in both cases.
  • each N ml Q m4 N m2 can differ from each other in a polynucleotide sequence in which m3 is greater than 1 ; i.e. , multiple Q moieties can be present randomly along the length of a molecule, provided that the remaining parameters described above are complied with.
  • One group of preferred polynucleotides has a long sequence of
  • Q moieties can be either consecutive or can be interrupted with a few normal nucleotides.
  • Plural Q moieties in the middle of a probe also represents a preferred embodiment, with relatively long uninterrupted sequences to either side of the crosslinking Q units.
  • This uninterrupted sequence provides stability during the hybridization process so that proper recognition of the target will occur.
  • the factors that lead to stability and selectivity are the same in the present process as in any other hybridization process.
  • Uninterrupted sequences of complementary nucleotides followed by Q moieties are no different in this regard from uninterrupted sequences of target nucleotides followed by a non-complementary normal base.
  • the stability of polynucleotides containing the crosslinking moiety of the invention can readily be predicted from standard considerations of nucleic acid hybridization.
  • R1 groups are present in the B moiety and both represent a different hydroxyl-coupling or hydroxyl-protecting group, as such compounds are ready for use in the synthesis of a cross-linkable polynucleotide.
  • protecting and activating groups are also discussed elsewhere in this specification.
  • n 1 is 0 to 10 (preferably 0 to 5, more preferably 1 to 3);
  • n 2 is 0 to 5 (preferably 0 to 2, more preferably 0 or 1);
  • n 3 is 0 to 5 (preferably 0 to 2, more preferably 0 or 1);
  • each W is independently a small stable substituent containing up to 15 atoms (especially a lower hydrocarbyl group; a halogen, nitro, thio, cyano, carbonyl, carboxy, hydroxy, amino, amido, or polyfluoroalkyl group; or a hydrocarbyl substituent containing one or more hetero atoms (i.e., an atom other than carbon or hydrogen that forms a stable covalent bond with carbon at 25°C in water));
  • Y and Z independently represent H, F or a lower alkyl group
  • X is an organic group containing (a) 1 to 10 carbon atoms and (b) 0 to 10, preferably 0 to 2, hetero atom selected from the group consisting of O, S and N, and wherein X comprises a shortest linking chain of 1 to 10 atoms between the other atoms of the formula to which it is attached;
  • R 2 is H or OR,; and R 1 is H or a group capable of coupling with or protecting (the former preferably being located only on a terminal hydroxyl of the backbone moiety) a hydroxyl group during automated polynucleotide synthesis.
  • R 1 represents a nucleotide or polynucleotide linked to the compound by a
  • Preferred coupling groups include phosphorous containing groups such as phosphite, phospohramidite, phosphate, H-phosphonate, phosphorothioate, phosphorodithioate, and methyl phosphonate.
  • Non-phosphorous coupling groups include carbamates and amides.
  • Lower hydrocarbon groups include C 1 -C 6 alkenyl and alkenyl group as well as C 3 -C 6 cyclic groups, and preferably include C 1 -C 4 alkyl and alkenyl groups, especially methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, and tert-butyl.
  • Typical hydrocarbyl groups with hetero atom substituents include -COCH 3 , -CH 2 OH, -CF 3 , -NHCH 3 ,
  • Compounds of the invention are useful either as intermediates in the preparation of or as components of photoactivate polynucleotides used for example as probes in hybridization assays. Since the intention is that one or more of these molecules eventually form part of a polynucleotide, the backbone moiety that forms part of the molecules is derived either from glycerin or a different poly hydroxyl hydrocarbon molecule in most cases.
  • the glyceryl or other polyhydroxyl hydrocarbon molecule is incorporated at any position into the backbone of a nucleic acid typically by phosphodiester type linkage with the 3' and/or 5' hydroxyl groups of the adjacent nucleotides in the molecule, with the crosslinking moiety normally being attached to the backbone moiety prior to such incorporation.
  • the crosslinking moiety portion of the compound of the invention can be derived from coumarin itself or any number of substituted coumarins.
  • An organic functional group at the position in the crosslinking moiety precursor where glycerin or another backbone moiety will be attached is typically used to join the crosslinking moiety to the backbone moiety in the final product. Since final products can be often prepared by alternative synthetic routes, any given final product will likely have several possible precursors.
  • the linking moiety can arise from a separate molecule or be formed by reaction portions of the crosslinking moiety precursor and the backbone moiety precursor.
  • the coumarin (or other) ring system can be either unsubstituted or substituted.
  • Typical substitutents on the phenyl ring are small, stable substitutents normally found on aromatic rings in organic compounds.
  • Substitutents can be selected as desired to change the excitation wavelength of the coumarin.
  • Substitutents at the 3- and 4- positions are typically non-polar and are most often hydrocarbon substitutents, with methyl substitutents being most common. Although the location of coumarin substitutents can vary, substitutents are most often found at the 4-, 5-, 6-7, and 8-positions.
  • the coumarin moiety precursor prior to reaction with the backbone moiety precursor, will have the formula:
  • X 1 is a precursor of all or part of the X linking moiety. X, will react with an organic function group on the precursor of the linker moiety to form a covalent bond. Typical reactive functional groups include hydroxy, amine, halogen, thio, carbonyl, carboxy ester, carboxy amide, silyl and vinyl groups. These precursors can be synthesized by standard methods of organic synthesis from coumarin itself or from the many commercially available coumarin derivatives. In certain preferred embodiments the glycerol backbone moiety precursor has the formula:
  • R 1 , R 2 , and n 1 , and n 3 have the meaning previously defined and
  • X 2 is a precursor of all or part of the X linking group.
  • X 2 will react with an organic functional group on the coumarin moiety to form a covalent bond in the final linking X moiety.
  • X 2 typically will be selected from reactive functional groups and nucleophilic and electrophilic groups that are capable of undergoing nucleophilic or electrophilic substitution or addition.
  • Examples of specific functional groups include hydroxy, amino, halogen, thio, carbonyl, carboxy ester, carboxy amide, vinyl, and silicon derivatives.
  • This precursor can be synthesized by standard methods of organic synthesis from (poly)hydroxy hydrocarbons such as glycerin, commercial available 1 ,2- or 1 ,3- dihydroxy alkane derivatives, or such compounds with a protected hydroxyl group at the location of the indicated hydroxyl groups. See Misiura, K. , Durrant, I. , Evans, M.R. , and Gait, M.J., Nucleic Acids Res. (1990) 18, 4345-4354, which is herein incorporated by reference, for a discussion of attaching moieties having structures similar to those of the present backbone moieties to bases used in polynucleotide synthesis.
  • the reaction mixture was then diluted with 45 ml of ethyl acetate and 2.2 ml triethylamine, extracted with 10% aqueous sodium carbonate (2 ⁇ 30 ml), and with saturated sodium carbonate (2 ⁇ 30 ml), and with saturated sodium chloride (2 ⁇ 30 ml).
  • the organic phase was concentrated in vacuo.
  • the resulting product was purified by silica gel chromatography with a solvent system (methylene chloride/diethyl
  • an oligonucleotide was prepared via the /3-cyanoethylphosphoramidite method of D ⁇ A synthesis that was identical to a segment of human papilloma virus type 16, comprising nucleotides 397 to 417 of the E6 gene in which the 20th base (adenine) was replaced by 3-(7-coumarinylmethyl)glycerol.
  • oligonucleotides were prepared via the ⁇ -cyanoethylphosphoramidite method of DNA synthesis that were identical to segments of the genome of human papilloma virus type 16.
  • oligonucleotides were complementary to nucleotides 89-108 and 283-302 of the E6 gene, respectively (the sequence of which is herein incorporated by reference).
  • the 5' terminal nucleotide of the natural sequence (adenosine) was replaced by 3-(7-coumarinylmethyl) glycerol.
  • the 3' end terminated with a biotin moiety.
  • oligonucleotides had complementary sequences to either nucleotides 89-108 or 283-302 of the E6 gene; however, in these modified oligonucleotides
  • 3-(7-coumarinylmethyl) glycol was replaced by the nucleosidic coumarin derivative described in Saba et al. , U.S. Patent No. 5,082,934, by using the 3'-O-(N,N-diisopropyl phosphoramidite) 5'-O- (4,4'-dimethoxytrityl) derivative at the 5' position of the 2'-deoxyribonucleotide, herein referred to as the "Saba compound.”
  • the four oligonucleotides were cleaved from the solid support with 1 ml 30% NH 4 OH for 1.5 hours at room temperature.
  • the ammonia solution was then heated at 55°C for a further 1.5 hours.
  • the NH 4 OH was removed in vacuo.
  • the crude oligonucleotides were purified to homogeneity by high performance liquid chromatography.
  • This synthetic route requires less time to complete than the reaction sequence using 7-bromomethylcoumarin and provides a cost savings of about 50 percent compared to the 7-bromomethylcoumarin synthetic sequence.
  • the 7- hydroxycoumarin derivatives can be introduced into oligonucleotides and are more stable during deprotection of the oligonucleotides (exposure to concentrated ⁇ H 3 at room temperature) than compounds of U.S. Patent 5,082,934.
  • the 7-hydroxycoumarin derivatives exhibit a different absorption spectrum ( ⁇ maximum of 325 nm) compared to the 7-bromomethylcoumarin derivatives ( ⁇ maximum of 310 nm).
  • the 7-hydroxycoumarin derivatives are red shifted relative to the 7-bromomethylcoumarin derivatives, which reduces the effect of quenchers, such as nucleic acids.
  • the spectral shift also allows for more selective excitation of the 7-hydroxycoumarin derivatives.
  • the intermediate 7-glycidyl coumarin was prepared in a reaction flask equipped with a reflux condenser containing 16.2 g of 7-hydroxycoumarin, 15.8 g of epibromohydrin, 13.8 g of potassium carbonate and 270 ml of acetone
  • reaction solution The reaction solution was boiled and refluxed overnight, cooled, treated with 100 ml of 5 % NaOH aqueous solution, and extracted three times with 80 ml of methylene chloride. After evaporating the solvent a crude yellow solid was obtained. The crude solid (1.5g) was dissolved in a solution of
  • the coevaporated coumarinyl glycerol was added to 44 mg 4-dimethylaminopyridine, 330 ⁇ l triethylamine, 45 ml pyridine and 1.78 g of dimethoxytrityl chloride. The solution was stirred at room temperature for 3 hours. The reaction was stopped by adding 66 ml of deionized water. The reaction solution was then extracted three times with 35 ml of methylene chloride. The organic phase was dried over sodium sulfate. The crude product obtained by evaporating the solvent was purified by chromatography using a silica gel column and eluting with a solution of 70% hexane, 28% acetone and 2 % triethylamine. 2.6 g of purified product (1-O-(4,4' dimethoxytrityl)-3-O-(7-coumarinyl)glycerol) gave a single TLC spot with an R f of 0.43 using the same solvent system.
  • 1-O-4,4'-Dimethoxytrityl-3-O-(7-coumarinyl)glycerol (2.5g) was coevaporated with 12 ml of 75 % pyridine and 25 % methylene chloride two times. A solution of 5 ml methylene chloride and 5 ml pyridine was added to the dry viscous liquid (methylene chloride/coumarin solution). The methylene
  • oligonucleotides were prepared via the ⁇ -cyanoethylphosphoramidite method of D ⁇ A synthesis that were identical to segments of the cryptic plasmid of Chlamydia trachomatis.
  • oligonucleotides were complementary to nucleotides 876-900, 6857-6878, 7118- 7140, and 6725-6752 of the cryptic plasmid (the sequence of which is herein incorporated by reference), the first two oligonucleotides containing one crosslinking compound per oligonucleotide and the latter two oligonucleotides containing two crosslinking compounds per oligonucleotide.
  • the oligonucleotides were cleaved from the solid support and deprotected with 3 ml 30% NH 4 OH for 2 h at room temperature.
  • the NH 4 OH was removed in vacuo, and the crude oligonucleotide was purified to homogeneity by denaturing polyacrylamide gel electrophoresis.
  • the extent of crosslinking was determined by denaturing polyacrylamide gel electrophoresis followed by scintillation counting of the exicsed bands. The results are set forth in the following table:
  • Coumarin derivatives can be synthesized containing various side chains, including, (1) short side chains, such as glycerol, (2) long side chains, such as poly(ethylene glycols), (3) aromatic rings, and (4) aliphatic cyclic rings, such as ethylene-dioxy rings.
  • Such coumarin derivatives can be synthesized from the appropriate coumarin starting materials, such as, 7-methyl coumarin, 7-hydroxy coumarin, esculetin (6, 7-dihydroxy coumarin) or 7-glycidyl coumarin. Attached to each coumarin starting material is the desired side chain containing active functional groups.
  • This compound is not itself a compound within the general formulas described above, but is an intermediate that can be used to prepare such compounds via reaction of X and/or B unit precursors with the hydroxyl group that is activated by formation of a phosphoramidite in the last step of the reaction shown.
  • Esculetin (0.90g) was stirred with a solution of potassium carbonate (1.40g) and 200 ml of anhydrous acetone for 1 hour at room temperature.
  • Epibromohydrin (1.05g) was added to the solution. The yellow suspension solution was then refluxed overnight. Potassium hydroxide (0.70g) was then added and refluxed for one hour. The solution was then separated from the solids by centrifugation. The solution was then evaporated by a water aspirator. The resulting product was then dissolved in 50 ml of water. The aqueous solution was then extracted three times with 35 ml of methylene chloride. The organic solution was extracted twice with 50 ml of 2M sodium hydroxide. The resulting organic phase was dried over sodium sulfate. After evaporating the solvent, 200 mg of white solid was obtained. TLC in 50% acetone/hexane shows R f 0.42. The yield was 26% by weight.
  • N-bromosuccinimde (30.8g) was added to 140 ml of chloroform in a one liter flask and the suspension was refluxed overnight. The solution was diluted with 100 ml of chloroform. The resulting crude product was recrystallized from 750 ml of acetone. A white solid (21g) was obtained with a melting point of 172-176°C was obtained.
  • the dihydroxy coumarin derivative (230 mg) obtained as the product of Step CH was coevaporated with dry pyridine.
  • Dimethoxytritylchloride (320 mg), 60 ml of triethylamine and 10 mg of 4-dimethylaminopyridine were added to the coumarin derivative.
  • the solution was stirred at room temperature for 16 hours.
  • the solution was diluted with water and extracted with dichloromethane, then dried with sodium sulfate. After evaporating the solvent, the crude product was purified by a silica gel column with 40% (v/v) acetone/hexane as eluant.
  • chlorophosphoramidite and 2.4 eq of N,N-diisopropylethylamine are dissolved in 0.9 ml of dichloromethane in a glass container capped with a septum.
  • the coumarin precursor (1.0 eq) (such as the product of steps AH, BE or CIII) was dissolved 9.9 ml of pyridine and 0.9 ml of dichloromethane. While the chlorophosphoramidite solution is stirred, the coumarin solution is added. Stirring is continued for 2 hours. Ethylacetate is added, and the organic solution is washed with NaCl (aqueous) three times and dried with Na 2 SO 4 . After removing the solvent, the crude product is purified by column chromatography using

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Abstract

Cette invention concerne de nouveaux dérivés de coumarine comportant une fraction de coumarine liée à une fraction squelette non nucléosidique. Les molécules résultantes servent typiquement de groupements de réticulation photosensibles lorsqu'elles sont incorporées à des polynucléotides à la place d'une ou de plusieurs bases nucléotidiques complémentaires présentes à l'intérieur de sondes utilisées dans des procédures faisant appel à des réactions d'hybridation moléculaire d'acides nucléiques.
PCT/US1996/003134 1995-03-09 1996-03-08 Derives non nucleosidiques de coumarine utilises comme agents de reticulation de polynucleotides WO1996028438A1 (fr)

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JP8527721A JPH11501927A (ja) 1995-03-09 1996-03-08 ポリヌクレオチド架橋剤としての非ヌクレオシドクマリン誘導体
EP96908709A EP0827501A4 (fr) 1995-03-09 1996-03-08 Derives non nucleosidiques de coumarine utilises comme agents de reticulation de polynucleotides
AU51864/96A AU719277B2 (en) 1995-03-09 1996-03-08 Non-nucleosidic coumarin derivatives as polynucleotide-crosslinking agents

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US08/401,630 1995-03-09
US08/401,630 US6005093A (en) 1993-04-13 1995-03-09 Non-nucleosidic coumarin derivatives as polynucleotide-crosslinking agents

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Cited By (6)

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WO2000014281A2 (fr) * 1998-08-21 2000-03-16 Naxcor Dosages utilisant des acides nucleiques reticulables immobilises
WO2000027860A2 (fr) * 1998-11-10 2000-05-18 Naxcor Agents de reticulation de polynucleotides
US7868161B2 (en) 2005-07-29 2011-01-11 North Carolina State University Photocrosslinking probes and uses of the same
US8053777B2 (en) 2005-03-31 2011-11-08 General Electric Company Thin film transistors for imaging system and method of making the same
US10781175B2 (en) 2016-07-15 2020-09-22 Am Chemicals Llc Solid supports and phosphoramidite building blocks for oligonucleotide conjugates
US11034677B2 (en) 2015-01-20 2021-06-15 Biosearch Technologies, Inc. Coumarin-based compounds and related methods

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CN116535373A (zh) * 2023-05-06 2023-08-04 波顿香料股份有限公司 一种坚果香气化合物及其制备方法和坚果韵咖啡香型香基

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000014281A2 (fr) * 1998-08-21 2000-03-16 Naxcor Dosages utilisant des acides nucleiques reticulables immobilises
WO2000014281A3 (fr) * 1998-08-21 2000-08-24 Naxcor Dosages utilisant des acides nucleiques reticulables immobilises
US6696246B1 (en) 1998-08-21 2004-02-24 Naxcor, Inc. Assays using crosslinkable immobilized nucleic acids
WO2000027860A2 (fr) * 1998-11-10 2000-05-18 Naxcor Agents de reticulation de polynucleotides
WO2000027860A3 (fr) * 1998-11-10 2000-10-12 Naxcor Agents de reticulation de polynucleotides
US6303799B1 (en) 1998-11-10 2001-10-16 Naxcor Polynucleotide crosslinking agents
US8053777B2 (en) 2005-03-31 2011-11-08 General Electric Company Thin film transistors for imaging system and method of making the same
US7868161B2 (en) 2005-07-29 2011-01-11 North Carolina State University Photocrosslinking probes and uses of the same
US11034677B2 (en) 2015-01-20 2021-06-15 Biosearch Technologies, Inc. Coumarin-based compounds and related methods
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

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CA2214835A1 (fr) 1996-09-19
EP0827501A4 (fr) 1998-06-10
CN1181078A (zh) 1998-05-06
AU5186496A (en) 1996-10-02
EP0827501A1 (fr) 1998-03-11
JPH11501927A (ja) 1999-02-16
AU719277B2 (en) 2000-05-04

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