WO1994001446A2 - Support solide organique derive utilise pour effectuer la synthese d'acides nucleiques - Google Patents

Support solide organique derive utilise pour effectuer la synthese d'acides nucleiques Download PDF

Info

Publication number
WO1994001446A2
WO1994001446A2 PCT/US1993/006214 US9306214W WO9401446A2 WO 1994001446 A2 WO1994001446 A2 WO 1994001446A2 US 9306214 W US9306214 W US 9306214W WO 9401446 A2 WO9401446 A2 WO 9401446A2
Authority
WO
WIPO (PCT)
Prior art keywords
nucleoside
group
solid
support
protected
Prior art date
Application number
PCT/US1993/006214
Other languages
English (en)
Other versions
WO1994001446A3 (fr
Inventor
Parameswara M. Reddy
Maged A. Michael
Original Assignee
Beckman Instruments, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beckman Instruments, Inc. filed Critical Beckman Instruments, Inc.
Publication of WO1994001446A2 publication Critical patent/WO1994001446A2/fr
Publication of WO1994001446A3 publication Critical patent/WO1994001446A3/fr

Links

Classifications

    • 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

Definitions

  • This invention is directed to solid phase supports for oligodeoxyribonucleotide and oligoribonucleotide synthesis and methods for their use.
  • amplification reactions such as the polymerase chain reaction (PCR) and the ligase chain reaction (LCR), and primers for nucleic acid sequencing.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • DNA and RNA are both long polymeric molecules comprised of monomers known as nucleotides. Each nucleotide is composed of a nitrogenous organic base, a five-carbon sugar, and a phosphate residue. The chemical combination of the base and the sugar, which can exist separately, is known as a nucleoside.
  • the sugar is deoxyribose, while in RNA, it is ribose, which contains one more oxygen atom.
  • the bases are normally adenine (A), guanine (G), cytosine (C), and thymine (T), while in RNA, the bases are normally adenine, guanine, cytosine, and uracil (U).
  • A adenine
  • G guanine
  • C cytosine
  • T thymine
  • U uracil
  • polydeoxyribonucleotide for DNA and polyribonucleotide for RNA Shorter chains of DNA and RNA can also exist, either as a result of chemical synthesis or breakdown of a larger chain.
  • oligonucleotides oligodeoxyribonucleotides for DNA and oligoribonucleotides for RNA.
  • Natural DNA consists of two strands of
  • polydeoxyribonucleotides bound together The strands are held together by base pairing, so that an adenine always pairs with a thymine, and a cytosine always pairs with a guanine.
  • RNA generally occurs as a single stranded
  • polyribonucleotide it also can form double strands by the same base pairing rules, with U behaving in the same way as T in DNA.
  • the specificity of base pairing means that synthesis of DNA in the laboratory must be extremely accurate to be useful, as the insertion of even one wrong base would disrupt the structure and make the resulting product useless.
  • Oligodeoxyribonucleotide synthesis in the laboratory is typically carried out by one of two reaction sequences: the phosphotriester approach and the phosphite triester approach.
  • the general reaction sequences are described in "Oligonucleotide Synthesis: A Practical Approach,” M.J. Gait, ed., IRL Press, Oxford, 1984, incorporated herein by this reference. In either case, the reaction sequence starts with a 5'-protected nucleoside bound to a solid-phase support through the 3'-carbon of the sugar.
  • a 5'-OH moiety is available for reaction with the 3'-position of either a phosphotriester or a phosphite triester activated nucleoside (also referred to as "activated intermediates").
  • activated intermediates also referred to as "activated intermediates”.
  • the solid support plays a crucial role in the success of either reaction scheme. The solid support must not interfere with or contaminate the reaction and must make the nucleosides attached to it available for reaction with the activated intermediates. The choice of solid support is important in the maintenance of a high repetitive yield, which is an important factor in the efficient synthesis of
  • CPG inorganic material controlled pore glass
  • CPG has proven useful, it has some drawbacks. CPG does not permit high levels of nucleotide loading and can be responsible for spurious indications of coupling yields.
  • CPG is susceptible to degradation by reagents used in the deprotection step of synthesis, such as
  • CPG can support chain growth on its surface at sites other than those associated with the 5' -terminus of an attached nucleotide and thus give rise to extraneous protected products. Because of these drawbacks, in some applications it may be desirable to use an organic support. The use of an organic support would be desirable in order to provide a support compatible with a wide variety of commercially available nucleotide synthesis apparatus while providing efficient synthesis at lower cost than can be obtained with CPG. It would additionally be desirable for the user to be able to attach a variety of spacers of different yet defined length for optimal performance of the synthesis.
  • a solid-phase support according to the present invention, referred to as a "nucleotide synthesis intermediate,” comprises:
  • a particulate support comprising a porous polymer whose backbone comprises optionally substituted acrylate or methacrylate moieties;
  • a linker having a first and a second end, the first end being covalently attached to the particulate support and the second end being covalently attached to the
  • nucleoside the linker spacing the nucleoside at least 4 atoms away from the polymer.
  • the nucleoside is a protected deoxyribonucleoside.
  • the porous polymer is a methacrylatevinylidene copolymer.
  • the linker comprises at least one aliphatic diamine. Each aliphatic diamine
  • the aliphatic diamines have a total chain length of from about 3 to about 36 atoms, with the proviso that, if more than one diamine is present in the linker, the diamines are linked to each other by carbonyl groups.
  • the linker comprises at least one aliphatic diamine, it can be attached to the porous polymer by a
  • the nucleoside is then linked to the other end of the diamine, preferably by a succinyl ester group.
  • a linker comprising at least one aliphatic diamine can be linked attached to the porous polymer by the structure -CH 2 -CH(OCOCH 3 ) -CH 2 - .
  • the first amino group of the diamine moiety attached to the particulate support is substituted with an acetyl group.
  • the linker can comprise a polyethylene glycol moiety containing from about 3 to about 10 ethylene glycol monomers, preferably from about 4 to about 7 ethylene glycol monomers.
  • the linker can comprise a polyethylene glycol moiety containing from about 3 to about 10 ethylene glycol monomers, preferably from about 4 to about 7 ethylene glycol monomers.
  • polyethylene glycol moiety has a first end and a second end, with the first end covalently attached to the particulate support, and the second end terminating in an amine moiety.
  • the nucleoside is linked to the amine moiety at the second end of the polyethylene glycol linker by an amide linkage between the amine moiety and a carboxyl group in covalent linkage with the nucleoside.
  • Particular nucleotide synthesis intermediates according to the present invention include those shown below as Formulas I-VI.
  • (F) is a macroreticular methacrylate-vinylidene polymer
  • X is a nucleoside
  • Z is a 5' -protecting group.
  • a process of producing nucleotide synthesis intermediates according to the invention comprises generally:
  • a particulate support comprising a porous polymer whose backbone comprises optionally substituted acrylate or methacrylate moieties, with:
  • Nucleotide synthesis intermediates according to the present invention can be used for nucleotide synthesis in either the phosphite-triester or the phosphotriester synthesis method.
  • the phosphite-triester method for synthesis of DNA used with solid supports according to the present invention, comprises the steps of selecting a solid- phase intermediate containing a 5' -protected
  • deoxyribonucleoside removing the protecting group, coupling the free 5' -OH group with a phosphoramidite, oxidizing the phosphite triester linkage to a phosphotriester linkage, acetylating unreacted 5'-OH groups, and cleaving the
  • the phosphotriester method for synthesis of DNA used with solid supports according to the present invention, comprises the steps of selecting a solid-phase intermediate containing a 5'-protected deoxyribonucleoside, removing the protecting group, coupling a phosphorylated nucleotide to form a dinucleotide, and cleaving the
  • Nucleotide synthesis intermediates according to the present invention are capable of supporting efficient
  • nucleotides up to at least 1000 bases in length.
  • Figure 1 is a capillary gel electropherogram of a 24-base oligodeoxyribonucleotide synthesized using controlled pore glass;
  • Figure 2 is a capillary gel electropherogram of the same 24-base oligodeoxyribonucleotide synthesized using a solid support according to the present invention
  • Figure 3 is a capillary gel electropherogram of the same 24-base oligodeoxyribonucleotide synthesized using a different solid support according to the present invention
  • Figure 4 is a capillary gel electropherogram of a 35-base oligodeoxyribonucleotide synthesized using controlled pore glass
  • Figure 5 is a capillary gel electropherogram of the same 35-base oligodeoxyribonucleotide synthesized using a solid support according to the present invention
  • Figure 6 is a capillary gel electropherogram of the same 35-base oligodeoxyribonucleotide synthesized using a different solid support according to the present invention.
  • Figure 7 is a capillary gel electropherogram of the same 35-base oligodeoxyribonucleotide synthesized using another different support according to the present invention.
  • Figure 8 is a capillary gel electropherogram of the same 35-base oligodeoxyribonucleotide synthesized using another different support according to the present invention.
  • Figure 9 is a slab gel electropherogram of 24- and 35-base oligodeoxyribonucleotides synthesized with different solid supports according to the present invention, using different nucleotide synthesis apparatuses;
  • Figure 10 is a slab gel electropherogram of 35-base oligodeoxyribonucleotides synthesized with different solid supports according to the present invention, using different nucleotide synthesis apparatuses;
  • Figure 11 is a slab gel electropherogram of 35- and 51-base oligodeoxyribonucleotides synthesized with different solid supports according to the present invention, using different nucleotide synthesis apparatuses;
  • Figure 12 is a slab gel electropherogram of 51-
  • Figure 13 is a slab gel electropherogram of 24- and 35-base oligodeoxyribonucleotides synthesized with different solid supports according to the present invention, using different nucleotide synthesis apparatuses, showing different syntheses than depicted in Figure 11;
  • Figure 14 is a slab gel electropherogram of 35-base oligodeoxyribonucleotides synthesized with different solid supports according to the present invention, using different nucleotide synthesis apparatuses, showing different syntheses than depicted in Figure 10;
  • Figure 15 is a slab gel electropherogram of 51-base oligodeoxyribonucleotides synthesized with different solid supports according to the present invention, using different nucleotide synthesis apparatuses, showing different syntheses than depicted in Figures 10 and 14;
  • Figure 16 is a slab gel electropherogram of 51-base oligodeoxyribonucleotides synthesized with different solid supports according to the present invention, using different nucleotide synthesis apparatuses;
  • Figure 17 is a slab gel electropherogram of 101-base oligodeoxyribonucleotides synthesized with different solid supports according to the present invention.
  • Figure 18 is a slab gel electropherogram of 35-, 70-, and 101-base oligodeoxyribonucleotides synthesized with different solid supports according to the present invention
  • Figure 19 is a slab gel electropherogram of products from a polymerase chain reaction synthesis using a 17-base oligodeoxyribonucleotide produced with a solid support according to the present invention as a primer;
  • Figure 20 is a gel showing the result of a chain-terminating DNA sequencing reaction performed on the products from the polymerase chain reaction synthesis.
  • Polymer A polymer is a molecule comprised of a number of individual monomer units chemically linked together. The individual monomer units can be the same or different. A polymer whose individual monomer units are carbon-containing is an organic polymer, while a polymer whose individual monomer units do not contain carbon is an inorganic polymer.
  • Partially Hydrophilic A polymer is partially hydrophilic when at least about 25% of the exposed individual monomer units can interact with water.
  • a solvent is polar when it can mix with water and has molecules which are polarized to produce partial positive and negative electrical charges at opposite ends of the molecule.
  • a solvent is aprotic when it has no proton that can be removed by the action of a base in an aqueous solution.
  • substituent groups and the molecule with the substituent groups are included.
  • the optional substituents are of a type that do not alter the overall chemical reactivity of the polymer or other molecule.
  • “Aliphatic” An aliphatic molecule is an organic molecule whose backbone contains carbon and hydrogen with no carbon-carbon double bonds or triple bonds.
  • “Diamine” An organic molecule containing two amine (-NH 2 ) groups, typically at opposite ends of the chain.
  • diamine includes derivatives of aliphatic diamines optionally containing up to 2 heteroatoms. In such derivatives, the heteroatoms replace carbon atoms.
  • Porous A polymer is porous when it contains pores that can be penetrated by a solvent into which the polymer is placed.
  • “Monoderivatized” A molecule is monoderivatized when it contains one substituent of a particular type, as distinguished from two or more substituents.
  • acylatin ⁇ Reacting an alcohol or amino group with a reagent containing an acyl (RCO-) group.
  • RCO- acyl
  • Capping A process for ensuring that hydroxy (-OH) residues remaining on a polymer are blocked, typically by forming an ester linkage.
  • Space or “Linker”: These terms, used interchangeably, both refer to a portion of a composite structure that spatially separates a reactive molecule from a large polymer so that reagents can react readily with the reactive molecule without interference from the polymer.
  • Activated Nucleoside A nucleoside one of whose 3'- or 5'- positions is substituted with a group that can react to form a bond with another nucleoside is known as
  • an activated nucleoside incorporates an acid group such as an ester function.
  • Exocyclic Amino Protecting Group Certain of the bases of the nucleotides, in particular adenine, guanine, and cytosine, have amino groups whose reactivity could interfere with oligodeoxyribonucleotide synthesis . These amino groups must be blocked or protected, typically by acylation. The group used for protection is referred to as an exocyclic amino protecting group.
  • Nucleotide Synthesis Intermediate A substituted polymer according to the present invention incorporating an activated nucleotide and suitable for oligonucleotide
  • nucleotide synthesis is designated a nucleotide synthesis intermediate.
  • the polymers useful for synthesis according to the present invention are porous organic polymers, at least partially hydrophilic and having hydroxy residues, whose backbone comprises optionally substituted acrylate or
  • the backbone comprises straight-chain acrylate or methacrylate moieties.
  • optional substitutions can include any groups that do not significantly react with any of the other reagents employed for coupling or subsequent DNA synthesis, such as 1,1'-carbonyldiimidazole or oxidizing agents.
  • groups include, but are not limited to, aryl groups, alkoxy groups, and heterocyclic groups.
  • the polymers are substantially free of pyranose or furanose moieties or their derivatives.
  • a substantially hydrophobic monomer selected from the group consisting of ethylene dimethacrylate, ethylene diacrylate, methylenebisacrylamide, diethylene glycol methacrylamide, poly (ethyleneglycol) methacrylamide,
  • Preferred organic polymers are the macroreticulated methacrylate-vinylidene copolymers. As described below, in a particularly preferred embodiment, the polymers are either extended with
  • alkanediamine spacers or derivatized with polyethylene glycol to provide a framework for attachment of the protected
  • deoxyribonucleoside is also possible to directly attach the protected deoxyribonucleoside to the hydroxy groups of the polymer.
  • nucleotide synthesis intermediate produced by this process comprises:
  • a particulate support comprising a porous polymer whose backbone comprises optionally substituted acrylate or methacrylate moieties;
  • a linker having a first and a second end, the first end being covalently attached to the particulate support and the second end being covalently attached to the
  • deoxyribonucleoside at least 4 atoms away from the polymer.
  • the linker is used to separate the first nucleoside from the support, so that the nucleoside is reactive to form a bond with a second nucleoside, thus beginning the process of oligonucleotide synthesis.
  • organic polymers defined and disclosed herein can be used in a process for producing a particulate porous organic solid-phase support derivatized with, e.g., a
  • this process involves: (1) attaching one end of the spacer to the polymer; (2) attaching the nucleoside to the other end of the spacer so that the nucleoside is separated from the polymer by the spacer; and (3) blocking any reactive groups on the polymer that might interfere with subsequent nucleotide synthesis reactions.
  • One version of the process results in a support incorporating an alkanediamine spacer.
  • this process comprises the steps of:
  • a porous solid-phase particulate support comprising a porous polymer, at least partially hydrophilic and having hydroxy residues, whose backbone comprises
  • the porous polymer being extended with a linear polymer having first and second ends, the linear polymer being covalently linked through the first end to the porous polymer, the linear polymer comprising at least one aliphatic diamine, the linear polymer having an amino group at its second end, with:
  • nucleoside (b) a nucleoside, such that the nucleoside is covalently linked to the amino group at the second end of the linear polymer;
  • Each aliphatic diamine can optionally contain up to 2 heteroatoms replacing carbon atoms.
  • the heteroatoms are selected from the group consisting of oxygen and nitrogen.
  • the heteroatoms are bonded solely to carbon and are separated from the terminal amino group of the diamine by at least one carbon atom.
  • the aliphatic diamines have a total chain length of from about 3 to about 36 carbon atoms. If more than one diamine is present in the linker, the diamines are linked to each other by carbonyl groups.
  • nucleotide synthesis intermediate produced by this process comprises:
  • a porous solid-phase particulate support comprising a porous organic polymer, at least partially hydrophilic and having hydroxy residues, whose backbone comprises straight-chain optionally substituted acrylate or methacrylate moieties;
  • linear polymer having first and second ends, the linear polymer being covalently linked through the first end to the particulate support, the linear polymer comprising at least one aliphatic diamine as described above, the linear polymer having an amino group at its second end available for reaction with an activated protected deoxyribonucleoside, the linkage between the particulate support and the first end of the linear polymer comprising a carbamate moiety;
  • This step involves the condensation or coupling of at least one alkanediamine moiety to the polymer to produce a polymer extended with a spacer or linker.
  • the spacer or linker is subsequently coupled to a nucleoside, resulting in the nucleotide synthesis intermediate.
  • Preferred polymers for assembly of the solid-phase support are the macroreticulated methacrylate-vinylidene copolymers TOYOPEARLTM (available from Tosohaas) and as
  • FRACTOGELTM available from Merck.
  • Particularly preferred polymers are such methacrylate-vinylidene copolymers having particles of from about 5 microns to about 200 microns in mean diameter, preferably from about 30 microns to about 70 microns; most preferred are particles having a mean diameter of about 45 microns. These values are mean values, and individual particles vary somewhat in size.
  • These preferred polymers can have a range of porosities; most preferred are polymers having pores of a size that excludes proteins of molecular weight greater than 5 ⁇ 10 6 . Also particularly preferred are polymers having pores of a size that excludes proteins of a molecular weight greater than about 5 ⁇ 10 7 .
  • diamine includes derivatives of aliphatic diamines optionally containing up to 2 heteroatoms.
  • the heteroatoms replace carbon atoms.
  • the heteroatoms are selected from the group consisting of oxygen and nitrogen.
  • the heteroatoms are bonded solely to carbon and are separated from the terminal amino group of the diamine by at least one carbon atom.
  • the diamines can be either straight-chain or
  • the diamines are straight-chain.
  • the diamine linker can have a total chain length, counting only methylene (-CH 2 -) groups and heteroatoms
  • substituting for carbon and not amino or carbonyl groups of from 3 to 36 atoms, more preferably 6 to 18 carbon atoms.
  • Each diamine moiety preferably has a chain length of from 3 to 12 atoms. If there is more than one diamine moiety, the diamine moieties can have the same or different chain lengths. If branched-chain diamines are included, the chain length intended is the longest continuous chain present.
  • Particularly preferred diamine arrangements are the following:
  • the coupling reaction is preferably performed in a polar, aprotic solvent such as acetonitrile.
  • the polymer to be derivatized is suspended in the solvent at a concentration of from about 0.05-0.2 g/ml of solvent.
  • the polymer is suspended at about 0.1 g/ml of solvent.
  • the coupling reaction is performed using a coupling agent.
  • the coupling agent is 1,1'-carbonyldiimidazole, and is used at a concentration of about 1.0 mole/l.
  • Other coupling agents, such as carbodiimides, are also well known in the art.
  • the coupling reaction results in the coupling of some of the hydroxy residues of the polymer to diamines.
  • the hydroxy residues not coupled to diamines are blocked, as described below, to prevent interference with the remaining reactions.
  • the first stage in a preferred coupling reaction is the reaction of 1,1'-carbonyldiimidazole, at a concentration of about 1.0 mole/l, with the polymer suspended in acetonitrile at about 0.1 g/ml.
  • This reaction occurs at a temperature of about 15°-30°C, preferably at room temperature.
  • the reaction is allowed to proceed from about 2-6 hours, more preferably for about 4 hours. After this reaction is substantially complete, the polymer can be used for
  • the first alkanediamine to be coupled to the polymer is then added to the reaction mixture to a concentration of about 0.5-2 mole/l, preferably at about 1.0 mole/l.
  • the reaction is allowed to occur overnight.
  • the remaining hydroxy-containing sites on the polymer are blocked by the addition of a quantity of an aliphatic monoamine sufficient to react with all remaining activated hydroxyl groups.
  • the aliphatic monoamine preferably, the aliphatic
  • the monoamine is selected from the group consisting of ethylamine, n-propylamine, n-butylamine, and piperidine. Most preferably, the aliphatic monoamine is n-propylamine. Most preferably, the blocking reaction is carried out prior to the coupling of additional alkanediamine residues, if any.
  • the blocked, monoderivatized polymer is suspended in a halogen-containing solvent such as dichloromethane.
  • the coupling reagent p-nitrophenylchloroformate is then added to a concentration of about 0.5-2 mole/l, preferably about 1 mole/l.
  • a methylated pyridine derivative serving as a base, is added to a concentration of about 0.5 to about 3.0 moles/l, preferably about 1.5 moles/l.
  • the methylated pyridine derivative is collidine.
  • the reaction of the monoderivatized polymer is allowed to occur overnight at a temperature between about 15°C and 30°C, preferably room temperature.
  • a second alkanediamine residue can be added by means of reaction with carbonyldiimidazole.
  • the monoderivatized solid support is suspended in dry
  • the support is then filtered and washed with
  • the second alkanediamine to be coupled is added at a concentration of about 0.5 mole/l to about 1.5 mole/l, preferably about 1.0 mole/l, and the mixture shaken overnight.
  • the diderivatized support is washed extensively. A preferable washing routine is 10 times with acetonitrile, 20 times with distilled water,3 times with acetonitrile, 3 times with dichloromethane, and twice with ether. The support is then dried under vacuum to remove the remaining ether.
  • the nucleoside coupled is preferably an activated aryl ester of a protected deoxyribonucleoside.
  • the carboxyl function is being sufficiently activated to create a linkage between the deoxyribonucleoside and the solid support.
  • the process of the present invention can be used to produce nucleotide synthesis intermediates containing either a deoxyribonucleoside, for DNA synthesis, or a ribonucleoside, for RNA synthesis.
  • the nucleoside can be protected at either its 5'-end or its 3'-end.
  • the nucleoside is protected at the 5'-end, for synthesis in the 3'- to 5'- direction.
  • the nucleoside can also be protected at the 3' -end, for synthesis in the 5'- to 3'-direction.
  • protecting group thereon Protection of either the 5'-hydroxyl group or 3'-hydroxyl group of all of the four common deoxyribonucleosides and the four common ribonucleosides is also well understood in the art.
  • the exocyclic amino groups are protected by acylation.
  • the benzoyl group is used to protect both adenine and cytosine, while the isobutyryl group is used to protect guanine.
  • Other alternative protecting groups are known, such as phthaloyl or di-N-butylaminomethylene for the amino group of adenine.
  • dimethoxytrityl (dimethoxytrityl).
  • the dimethoxytrityl protecting group is generally preferred.
  • the protected deoxyribonucleoside used in the coupling reaction is protected at the 5' -hydroxyl group.
  • the coupling of the activated aryl ester of the protected deoxyribonucleoside with the support typically occurs in two stages: (i) formation of the activated aryl ester from a carboxyl-containing derivative of the protected nucleotide; and (ii) reaction of the activated aryl ester with the amino-terminated derivatized support.
  • Other coupling reactions are possible that result in the coupling of the protected deoxyribonucleoside to the amino terminus of the support. See, for example, M.J. Gait, "Oligonucleotide
  • the activated aryl ester is preferably an ester of a succinyl or oxalyl derivative of the protected nucleoside with a phenol substituted with at least one electron-withdrawing substituent. Such a substituent activates the derivative for reaction with an electron-rich group like a hydroxyl group.
  • the activated aryl ester is a succinyl derivative and the substituted phenol is p-nitrophenyl.
  • the compound coupled is most preferably a p-nitrophenyl ester of a succinylated protected nucleoside.
  • concentration of the p-nitrophenyl ester can be varied to give different levels of loading the support, as shown below in Table I.
  • the support on which the deoxyribonucleoside has been loaded is then washed, as with N,N-dimethylformamide (five times), methanol (five times), and ether (twice), and then dried under vacuum.
  • Table I shows that the quantity of nucleoside loaded per gram of support varies with the time of reaction and the concentration of activated protected nucleoside used. The longer the time or the greater the concentration, the greater is the quantity of nucleoside loading that results.
  • the final step of the reaction is the capping of all remaining unreacted amino groups on the
  • nucleoside-loaded solid support Preferably, the capping is accomplished by acylation. Most preferably, the capping is performed by acetylation.
  • the support is resuspended in dry pyridine (1 g support per 10 ml pyridine). N,N-dimethylaminopyridine is added to 0.2 mole/l, followed by acetic anhydride to 2.5 mole/l. The reaction mixture is shaken overnight at a temperature of 15°-30°C, preferably room temperature. The solid support is filtered and washed
  • the derivatized, capped solid support is then dried under vacuum.
  • the derivatized support is stable and can be stored.
  • X represents a nucleoside
  • Z represents a 5'-protecting group for the nucleoside.
  • the diagonal lines by convention, represent methylene (-CH 2 -) groups.
  • the support can be prepared with any DNA or RNA base attached to the support: adenine, thymine, guanine, or cytosine for DNA, and uracil instead of thymine in the case of RNA.
  • the quantity of protected nucleoside derivatized per gram of solid support ranges from about 5 ⁇ mole/g to about 200 ⁇ mole/g. Typically, the quantity is about 30 ⁇ mole/g.
  • the level of loading of nucleoside can be varied by varying the time of reaction of the p-nitrophenyl ester with the support and varying the concentration of p-nitrophenyl ester used for reaction.
  • the fully derivatized support is thoroughly washed and filtered, then dried. Several different washing regimes are possible.
  • the support can be washed five times with dichloromethane, ten times with acetone, five times with acetonitrile, and five times again with dichloromethane.
  • the last dichloromethane filtrate is then tested qualitatively for amino groups using the ninhydrin test, in which a positive result (i.e., the presence of amino groups) yields a blue color and a negative result yields no color.
  • the ninhydrin test should be negative.
  • the support is then washed twice with ether and dried under vacuum, preferably about five hours.
  • the solid support can be washed with dichloromethane and then 95% ethanol.
  • the filtrates can be collected and assayed by absorbance at 402 nanometers (A 402 ) for determining p-nitrophenol, which is a measure of the quantity of amino groups attached to the support.
  • a 402 nanometers
  • Other washing schemes are possible, as long as they substantially remove unreacted reagents.
  • this process preferably comprises the steps of:
  • a porous solid-phase support comprising a methacrylate-vinylidene copolymer extended with polyethylene glycol, the polyethylene glycol having from about 2 to about 20 ethylene glycol moieties and containing a terminal amino group, with:
  • the nucleoside is a protected deoxyribonucleoside.
  • nucleoside being linked to the terminal amino group of the polyethylene glycol-derivatized polymer. Accordingly, the nucleotide synthesis intermediate produced thereby comprises:
  • a porous solid-phase particulate support comprising a methacrylate-vinylidene copolymer
  • a polyethylene glycol linker having from about 2 to about 20 ethylene glycol moieties and having a first end and a second end, the first end covalently attached to the particulate support, the second end terminating in an amine moiety;
  • nucleoside linked to the amine moiety at the second end of the polyethylene glycol linker by an amide linkage between the amine moiety and a carboxyl group in covalent linkage with the nucleoside.
  • the length of the polyethylene glycol linker is preferably from about 3 to about 10 monomers, most preferably from about 4 to about 7 monomers.
  • the particles of the porous particulate support are from about 10 microns to about 200 microns in mean diameter; preferably from about 30 microns to about 70 microns; most preferably, they are about 60 microns in mean diameter.
  • the activated aryl ester is a p-nitrophenyl ester
  • deoxyribonucleoside is succinylated, the formation of the p-nitrophenyl ester of the succinylated protected
  • deoxyribonucleoside is described in Section 11(A) (2), supra.
  • the p-nitrophenyl ester is reacted with the amino-derivatized support by the following procedure: The support is suspended in dry N,N-dimethylformamide. The p-nitrophenyl ester is added as a supernatant from the coupling procedure, and triethylamine is added (1 ml per 2 ml
  • the reaction mixture is shaken at a temperature between about 15°C and about 30°C for about 15 minutes to about one hour. Preferably, the reaction is performed at room temperature for about 30 minutes.
  • the acetylation reaction is performed as detailed in Section 11(A) (2), supra. in dry pyridine, with the addition of N,N-dimethylaminopyridine and acetic anhydride.
  • the acetylated support is washed successively with N,N-dimethylformamide, acetonitrile, and dichloromethane, and dried under vacuum.
  • the derivatized solid support produced by this method has the schematic structure shown below as Formula V.
  • Another version of the process first reacts the support with epichlorohydrin (1-chloro-2,3 epoxypropane), and then reacts the epoxy function of the epichlorohydrin with an alkanediamine.
  • this version of the process comprises the steps of: (1) condensing a porous solid-phase particulate support comprising a porous polymer as described above, with epichlorohydrin;
  • modified solid phase support containing at least one diamine linked to the solid support by a spacer with the structure -O-CH 2 -CHOH-CH 2 -, the modified solid phase support having a terminal amino group available for reaction with a nucleoside;
  • the nucleoside is a protected deoxyribonucleoside
  • the nucleotide synthesis intermediate thus produced comprises:
  • a porous solid-phase particulate support comprising a porous organic polymer, at least partially hydrophilic and having hydroxy residues, whose backbone comprises straight-chain optionally substituted acrylate or methacrylate moieties;
  • aliphatic diamines having a total chain length of from about 6 to about 36 carbon atoms, the linkage between the particulate support and the first end of the linear polymer comprising a spacer with the structure -CH 2 -CH(OCOCH 3 )-CC 2 - , with the first amino group of the diamine moiety attached to the particulate support being substituted with an acetyl group;
  • the linear polymer that links the particulate support and the nucleoside has an amino group at its second end available for reaction with the nucleoside. If more than one diamine is present in the linear polymer, the diamines are linked to each other by carbonyl groups.
  • the single diamine has a chain length of twelve carbon atoms.
  • Preferred polymers in this version are the same as those described above (Section 11(A) (I) (a), supra).
  • This polymer has a mean particle diameter of about 45 microns.
  • This polymer has pores of a size that exclude proteins of molecular weight greater than about 5 ⁇ 10 6 .
  • the hydroxy-terminated polymer is slightly moistened with a small amount of water and suspended in a 1.5 N-2.5 N solution of alkali metal hydroxide.
  • the hydroxy-terminated polymer is slightly moistened with a small amount of water and suspended in a 1.5 N-2.5 N solution of alkali metal hydroxide.
  • alkali metal hydroxide concentration of alkali metal hydroxide is about 2 N and the alkali metal hydroxide is sodium hydroxide.
  • Epichlorohydrin is added to a final concentration of from about 2.4 mole/l to about 2.7 mole/l; preferably, the concentration of
  • epichlorohydrin is about 2.56 mole/l.
  • the mixture is shaken for about 2-4 hours, preferably about 3 hours.
  • the epichlorohydrin-derivatized polymer is subsequently condensed with an alkanediamine moiety.
  • the epichlorohydrin-derivatized polymer is resuspended in N,N-dimethylformamide, and the alkanediamine is added to a concentration of about 0.5 mole/l.
  • the mixture is shaken overnight at a temperature from about 15°-30°C, preferably room temperature. If desired, additional alkanediamine moieties can be added as described above.
  • Ml unreacted epichlorohydrin moieties remaining on the solid support are then blocked by reaction with a short-chain aliphatic primary amine.
  • the primary amine is n-propylamine.
  • the blocking step is carried out as follows: The short-chain aliphatic primary amine is added to a concentration of about 1 mole/l and the reaction mixture is shaken for an additional period of about two hours.
  • the solid support is filtered and washed successively with, e.g., dimethylformamide (five times), acetone (five times), acetonitrile (five times), dichloromethane (five times) and ether (two times).
  • the support is then dried under vacuum.
  • the coupling step is carried out as described above in Section A.
  • the quantity of nucleoside loading can be varied by varying the concentration of activated nucleoside and the time of reaction, as shown in Table II.
  • the final step of the process is a capping step, in which all remaining unreacted amino groups and hydroxyl groups are capped by acetylation. This is carried out as described above in Section 11(A) (2), supra. After acetylation, the support is filtered and washed successively with N,N- dimethylformamide, acetonitrile, dichloroethane (five times each), then twice with ether, and dried under vacuum for about five hours. The resulting derivatized support has the structure shown below in Formula VI. In Formula VI, Ac represents acetyl (-COCH 3 ).
  • the solid-phase supports of the present invention are particularly suitable for use with either the phosphite- triester or the phosphotriester method of oligonucleotide synthesis.
  • the phosphite-triester method comprises the steps of:
  • nucleotide synthesis intermediate prepared as described above, the nucleotide synthesis intermediate containing a 5' -protected deoxyribonucleoside; (2) removing the protecting group of the 5'-protected deoxyribonucleoside attached to the solid support by treating the intermediate with acid to yield a free 5'-OH group;
  • the phosphotriester method comprises the steps of:
  • nucleotide synthesis intermediate prepared as described above, the nucleotide synthesis intermediate containing a 5'-protected deoxyribonucleoside;
  • the coupling agent is preferably 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole and the catalyst 1-methylimidazole.
  • Solid supports according to the present invention can be used for synthesis in automated equipment, including, but not limited to, the Gene AssemblerTM (Pharmacia, Uppsala, Sweden), the BiosearchTM 8750 (Milligen Biosearch, San Rafael, California), and the ABI PCR MateTM (ABI, Foster City
  • the spacer, 1,12-diaminododecane (20 g, to make a solution of 1 mole/l) was added and the reaction mixture was shaken at room temperature overnight (i.e., for 16-20 hours).
  • the blocking agent n-propylamine (8.2 ml, to make a solution of 1 mole/l) was added to the above mixture. The mixture was then shaken for an additional hour.
  • the support was filtered and washed successively five times with 100 ml each time of dichloromethane, ten times with 100 ml each time of acetone, five times with 100 ml each time of acetonitrile, and five times with 100 ml each time of dichloromethane. After the last of the dichloromethane washes, the filtrate was tested qualitatively by reaction with ninhydrin.
  • the support was then washed twice with 100 ml each time of ether. The support was dried under vacuum for about five hours.
  • Reagent 1 was 0.01 mole/l aqueous KCN (2 ml) diluted to 100 ml with pyridine.
  • Reagent 2 was a 5% ninhydrin solution in n-butanol.
  • Reagent 3 was 80% phenol in n-butanol.
  • An aliquot (about 3-5 mg) of the solid support was placed in a test tube and equal amounts (about 5 drops each) of Reagents 1, 2, and 3 were successively added.
  • the test tube was placed in a preheated block at 100°C for 5 minutes. The beads gave a dark blue color, which indicated a positive amine reaction.
  • the amino groups on the solid support were quantitated by a quantitative picric acid test.
  • the reagents used were Reagent A, 5% (v/v) diisopropylethylamine in dichloromethane; and Reagent B, 0.1 mole/l picric acid in dichloromethane.
  • An accurately weighed amount of the solid support (5-10 mg) was placed in a small column fitted with a TeflonTM stopcock. The solid support was washed twice with 2 ml each of dichloromethane and then treated twice with 2 ml of Reagent A for one minute each. The support was then washed five times with 1 ml each of dichloromethane for one minute each time.
  • the solid support was then treated twice with Reagent B, 2 ml each time, for one minute each time.
  • the support was washed five times with dichloromethane, 1 ml each time, for one minute each time.
  • the picrate was eluted twice with Reagent A (2 ml each time, 1 minute each).
  • the picrate was diluted with 95% ethanol to give an absorbance measurable at 358 nm in the spectrophotometer.
  • the amino group content on the solid support was calculated using the following formula:
  • a 358 is the measured absorbance at 358 nm
  • V is the volume of the solution in milliliters
  • E ⁇ 10 -3 is 14.5
  • W is the weight of the sample used in the test in milligrams.
  • the amino group content on the solid support was typically 300-400 ⁇ mole/g.
  • the next step in the preparation of the derivatized solid support was loading the amino-containing solid support with 5'-dimethoxytrityl-thymidine. Different concentrations of the 5'-dimethoxytrityl-thymidine succinates were prepared according to the required nucleoside loading. The particular procedure set forth here was used for preparing a 0.05 mole/l solution to load 5 g of the solid support.
  • the succinylated 5'-dimethoxytrityl-thymidine (2.5 mmole) was dissolved in 22 ml of dry dioxane containing 1 ml of dry pyridine and 350 mg of p-nitrophenol (25 nmole).
  • the condensing agent (1,3-dicyclohexylcarbodiimide; 1.29 g, 6.25 mmole) was then added. After a few minutes dicyclohexylurea began to precipitate.
  • the reaction mixture was shaken at room temperature for three hours.
  • the dicyclohexylurea was then removed by filtration and the supernatant was added to the amino-derivatized solid support (5 g) suspended in dry N,N-dimethylformamide (22 ml).
  • Triethylamine (5 ml) was added to the above mixture and the mixture was shaken briefly by hand. A bright yellow color developed, due to the release of p-nitrophenol. The reaction was shaken for a period of time sufficient to give the required nucleoside loading. (See Table I above). The supernatant was removed and used to derivatize another aliquot (2-3 g) of amino-derivatized solid support because it still contained activated deoxyribonucleoside.
  • the deoxyribonucleoside-derivatized solid support was washed extensively: 5 times with 100 ml each of N,N-dimethylformamide, 5 times with 100 ml each of methanol, and twice with 100 ml each of ether. The solid support was then dried under vacuum. The remaining amino groups on the
  • deoxyribonucleoside-loaded solid support were then capped by acetylation.
  • the solid support (5 g) from the previous step was suspended in 50 ml of dry pyridine.
  • N,N-dimethylaminopyridine (1.25 g, to make an 0.2 mole/l solution) was added followed by the addition of acetic anhydride (12.5 ml, to make a solution of 2.5 mole/l).
  • the mixture was shaken at room temperature overnight.
  • the solid support was filtered and washed successively with 5 ⁇ 50 ml each of N,N-dimethylformamide, acetonitrile, and dichloromethane, and once with ether. The support was then dried under vacuum for about 5 hours. The amine-capped solid support was tested by the ninhydrin test for complete capping. The test showed that there were no free (i.e., uncapped) amino groups remaining on the solid support.
  • a solid support derivatized with a diaminododecane spacer coupled to deoxycytidine was prepared as in Example 1, except that the protected deoxyribonucleoside used was
  • a solid support derivatized with a diaminododecane spacer coupled to deoxyguanosine was prepared as in Example 1, except that isobutyryl-dimethoxytrityl-deoxyguanosine
  • Example 1 To Deoxyadenosme A solid support derivatized with a diaminododecane spacer coupled to deoxyadenosme was prepared as in Example 1, except that benzoyl-dimethoxytrityl-deoxyadenosme succinate was substituted for dimethoxytrityl-thymidine succinate, as used in Example 1.
  • the nucleoside loading on the solid supports for Examples 1-4 was determined using two methods. The first method was quantitating the released dimethoxytrityl color. The second was by quantitating the released nucleoside. The released nucleoside was quantitated by determining its
  • the nucleoside loading was estimated from the DMT assay using the following formula:
  • a 500 is the measured absorbance at 500 nanometers
  • V is the volume in milliliters of dichloroacetic acid solution used
  • 80 is the molar absorbance times 1 ⁇ 10 -3 of DMT at 500 nm
  • W is the weight in mg of the solid support.
  • the solid support from step two of the first method was washed with 5 ml of dichloromethane and 5 ml of acetonitrile, and then dried.
  • the solid support was treated with 1 ml of ammonia at room temperature for one hour to bring about hydrolysis of the 3'-succinate bond.
  • the ammonia solution was transferred to a screw-cap vial, capped tightly, and sealed well with parafilm.
  • the vial containing the nucleoside was placed in a water bath at 65°C for three hours. The vial was cooled before opening, left open at room temperature for at least 30 minutes, and then evaporated to dryness using a vacuum concentrator. The residue was dissolved in 1 ml of double distilled water.
  • the ultraviolet absorbance was read at the following wavelengths: 264 nm for thymidine; 260 nm for deoxyadenosine; 269 nm for deoxycytidine; and 255 nm for deoxyguanosine.
  • Nucleoside loading was determined according to the following formula:
  • E is the extinction coefficient for the particular deoxyribonucleoside in ⁇ mole/ml; 15.26 for deoxyadenosine;
  • W is the weight of the solid support in mg.
  • FRACTOGELTMTM-65M AF-amino-650M, Tosohaas, Philadelphia, Pennsylvania
  • This macroreticular methacrylate-vinylidene copolymer has amino-terminated polyethylene glycol residues attached thereto.
  • the solid support was filtered and successively washed with N,N-dimethylformamide (3 times with 10 ml each), methanol (twice with 10 ml each), and ether (twice with 10 ml each), and dried under vacuum for about five hours.
  • the solid support from the previous step (1 g) was suspended in 10 ml of dry pyridine.
  • N,N-dimethylaminopyridine 250 mg, to make a solution of 0.2 mole/l
  • acetic anhydride to make a 2.5 mole/l solution.
  • the reaction mixture was shaken at room temperature overnight.
  • the solid support was filtered and washed successively with N,N-dimethylformamide,
  • the solid support was tested for complete capping by the ninhydrin test. A negative result was obtained, (i.e., no blue color), indicating that there were no free amino groups remaining.
  • the nucleoside loading on the solid support was estimated by quantitating the released DMT color as described in Example 1. The nucleoside loading was determined to be 28 ⁇ mole/g.
  • Example 5 A procedure similar to that disclosed in Example 5 was used to couple two diaminohexane residues to an hydroxyl-terminated macroreticular methacrylate-vinylidene copolymer.
  • FRACTOGELTMTM-65F (10 g dry weight) was suspended in 100 ml of dry acetonitrile.
  • the solid support was filtered on a sintered glass funnel and washed four times with 100 ml each time of dry acetonitrile. The washed solid support was then resuspended in 100 ml of dry acetonitrile.
  • the first diaminohexane spacer was coupled by adding 11.6 g of 1,6-diaminohexane to make a 1 mole/l solution.
  • the solid support was then filtered and washed successively ten times with 100 ml each of acetonitrile, twenty times with 100 ml each of distilled water, three times with 100 ml each of acetonitrile, three times with 100 ml each of dichloromethane, and twice with 100 ml each with ether.
  • the solid support was then dried under a vacuum for about five hours.
  • the picric acid assay showed that the amino group loading was about 350-400 ⁇ mole/g.
  • the next step was the loading of the amino-terminated solid support with thymidine.
  • Triethylamine (5 ml) was added to the above reaction mixture, which was then shaken briefly by hand. A bright yellow color developed due to the release of p-nitrophenol. The reaction mixture was then shaken at room temperature for thirty
  • the next step was the capping of the remaining amino groups on the nucleoside-loaded solid support by acetylation.
  • the solid support from the previous step (5 g) was suspended in 50 ml of dry pyridine.
  • N,N-dimethylaminopyridine (1.25 g, to make a 0.2 mole/l solution) was added followed by 12.5 ml of acetic anhydride (to make a 2.5 mole/l solution).
  • the reaction mixture was shaken overnight at room temperature.
  • the solid support was filtered and washed successively five times with 50 ml each time of N,N-dimethylformamide,
  • the solid support FRACTOGELTM-75F (exclusion limit 5 ⁇ 10 7 molecular weight for proteins; particle size 30-60 microns) (1 g dry weight) was suspended in 20 ml of dry acetonitrile.
  • the coupling agent 1,1'-carbonyldiimidazole (3.24 g, to make a solution of 1 mole/l) was added and the mixture was shaken at room temperature for four hours.
  • the solid support was filtered on a sintered glass funnel and washed five times with dry acetonitrile (10 ml each time).
  • the solid support was then resuspended in 20 ml of dry acetonitrile.
  • the first diamine moiety, 1,6-diaminohexane (2.32 g, to make a solution of 1 mole/l) was added and the mixture shaken at room temperature overnight.
  • the blocking agent n-propylamine (1.64 ml) was added to the above mixture.
  • the mixture was then shaken for an additional hour.
  • the solid support was filtered and washed successively ten times with 10 ml of acetonitrile each time, twenty times with 10 ml of distilled water each time, three times with 10 ml of
  • the solid support was further washed five times with methanol (10 ml each time), five times with acetonitrile (10 ml each time), five times with dichloromethane (10 ml each time), and twice with 10 ml each time of ether.
  • the support was dried under vacuum for about five hours.
  • the p-nitrophenol-diaminohexane salt solution obtained from the above reaction was appropriately diluted and read for absorbance at 402 nm.
  • the amount of p-nitrophenol released in the reaction which corresponds to the amount of amino groups attached, was calculated from the formula:
  • a 402 is the absorbance at 402 nm
  • V is the final volume in milliliters of the solution used to measure the absorbance
  • 21 is the extinction coefficient ⁇ 10 -3 of the p-nitrophenol amine complex at 402 nm
  • W is the weight of the solid support in grams.
  • the assay indicated that the amount of p-nitrophenol released was 200 to 240 ⁇ mole/g.
  • the amino group loading was 190 to 240 ⁇ mole/g.
  • the next step was the loading of the amino-terminated solid support with thymidine.
  • Succinylated 5'-dimethoxytrityl-thymidine (1.29 g, 2 mmole) was dissolved in 4 ml of dry dioxane containing 0.4 ml of dry pyridine and 280 mg of p-nitrophenol (2 mmole).
  • the coupling agent 1,3-dicyclohexylcarbodiimide (1.30 g, 5 mmole) was then added.
  • the dicyclohexylurea was removed by filtration and the supernatant was added to the amino-derivatized solid support (1 g) suspended in 4 ml of dry N,N-dimethylformamide.
  • Triethylamine (2 ml) was added to the above reaction mixture.
  • the reaction mixture was then shaken at room temperature overnight .
  • the solid support was then filtered and successively washed 3 times with 10 ml each time of N,N-dimethylformamide, twice with 10 ml each time of methanol, and twice with 10 ml each time of ether.
  • the solid support was then dried under vacuum for about 5 hours.
  • the final step was the capping of the remaining amino groups on the nucleoside-loaded solid support by
  • the solid support (1 g) from the previous step was suspended in 10 ml of dry pyridine. N,N-dimethylaminopyridine (250 mg, to make a solution of 0.2 mole/l) was added, followed by the addition of 2.5 ml acetic anhydride (to make a solution of 2.5 mole/l). The reaction mixture was shaken at room temperature overnight. The solid support was then filtered and washed successively 5 times with 10 ml each time with the following reagents: N,N-dimethylformamide, acetonitrile, and dichloromethane. The solid support was then washed twice with 10 ml each time of ether and then dried under vacuum for about 5 hours. The nucleoside loading on the solid support was 28 to 30 ⁇ mole/g, estimated by quantitating the released DMT color.
  • the solid support was filtered and washed with distilled water in a sintered glass funnel until the washes were neutral to pH paper.
  • the solid support (slightly wet) was resuspended in 100 ml of N,N-dimethylformamide.
  • the diamine spacer, powdered 1,12-diaminododecane (10 g, to make a solution of 0.5 mole/l) was added and the mixture was shaken at room temperature overnight (i.e., about 16 to 20 hours).
  • the blocking agent n-propylamine (8.2 ml, to make a solution of 1 mole/l) was added to the above mixture. The mixture was then shaken for an additional 2 hours.
  • the solid support was then filtered and washed successively 5 times with 100 ml each time of N,N-dimethylformamide, 5 times with 100 ml each time of acetone, 5 times with 100 ml each time of acetonitrile, and 5 times with 100 ml each time of dichloromethane. The last washing with dichloromethane gave a negative ninhydrin test. Finally, the solid support was washed twice with 100 ml each time of ether and dried under vacuum for about 5 hours. The picrate acid assay showed that the amino group loading was about 700 to 800 ⁇ mole/g.
  • the next step was the loading of the amino- terminated solid support with thymidine.
  • concentrations of the activated thymidine succinate could be used, according to the required nucleoside loading.
  • a 0.05 mole/l reagent was prepared to load 1 g of the solid support.
  • succinylated 5'-dimethoxytritylthymidine (323 mg, 0.5 mmole) was dissolved in 4 ml of dry dioxane containing 0.2 ml dry pyridine and p-nitrophenol (70 mg, 0.5 mmole).
  • the coupling agent 1,3-dicyclohexylcarbodiimide (258 mg, 1.25 mmole) was added and the reaction mixture was shaken at room temperature for 3 hours.
  • the dicyclohexylurea was removed by filtration and the supernatant was added to the amino-derivatized solid support (1 g) suspended in 4 ml dry N,N-dimethylformamide.
  • N,N- triethylamine (2 ml) was added to the reaction mixture.
  • the reaction mixture was shaken for an appropriate time to obtain the required loading of nucleoside.
  • the loaded solid support was then filtered and successively washed 3 times with 10 ml each time of N,N-dimethylformamide, twice with 10 ml each time of methanol, and twice with 10 ml each time of ether. It was then dried under vacuum for about 5 hours.
  • the nucleoside loading on the solid support was estimated by quantitating the released DMT color.
  • the last step was the capping of the remaining amino groups on the nucleoside-loaded solid support by acetylation.
  • the solid support (1 g) from the previous step was suspended in 10 ml dry pyridine. N,N-dimethylaminopyridine (250 mg, to make a solution of 0.2 mole/l) was added followed by acetic anhydride (2.5 ml, to make a solution of 2.5 mole/l). The reaction mixture was shaken at room temperature overnight. The solid support was filtered and washed successively with 5 ⁇ 10 ml each time of N,N-dimethylformamide, acetonitrile, and dichloromethane, then twice with 10 ml each time of ether. The solid support was dried under vacuum for about 5 hours.
  • the amino-capped solid support was tested by the ninhydrin test for complete capping.
  • the ninhydrin test gave a negative result, which indicated complete capping of the solid support.
  • Table IV shows that the solid supports of Examples 1 and 5 are at least as efficient as CPG for synthesis of oligonucleotides ranging in length from 17 bases to 101 bases ("NT" means "not tested”). In particular, the solid supports of Examples 1 and 5 proved more efficient than CPG for
  • Table V shows that solid supports according to the present invention can be used to synthesize
  • deoxyribonucleotides efficiently with any of the four common DNA nucleotides attached to the solid support.
  • Example 1 The solid support of Example 1 was similarly used for synthesis with the Pharmacia Gene Assembler.
  • the trityl coupling efficiency is shown in Table IV above.
  • oligodeoxyribonucleotide as is controlled pore glass.
  • a 24-nucleotide DNA sequence 5'-ATTGAGAAAGCGCCACGCTTCCCT-3' (SEQ ID NO: 2), was synthesized using derivatized solid supports as described above and in vitro DNA synthesis apparatus, also as described above.
  • the 24-mer described above was synthesized using controlled pore glass of 500 ⁇ porosity, as a control, on the Pharmacia Gene Assembler as described above.
  • the trityl coupling efficiency for this synthesis is shown in Table IV.
  • the DNA synthesized was analyzed by capillary gel electrophoresis.
  • the capillary gel column used was from
  • Tris-HCI Tris-hydroxymethylaminomethane-HCl
  • the same oligonucleotide was synthesized using the solid support of Example 1 on the Pharmacia Gene Assembler DNA synthesis apparatus.
  • the trityl coupling efficiency is shown in Table IV, and the capillary gel electropherogram is shown in Figure 2.
  • the electropherogram showed a single sharp spike. This indicates that the synthesized nucleotide was substantially homogeneous and contained fewer contaminants of differing molecular weight than the nucleotide synthesized with controlled pore glass.
  • Stepwise coupling efficiency e [InY/(n-1)] where Y is (absorbance of color released after n-1 couplings) / (absorbance of color released after the first coupling) and n is the number of nucleotides.
  • the trityl coupling efficiency is shown in Table VI below ("NT" means "not tested”).
  • oligodeoxyribonucleotide of 35 residues 5'-GATGCCAGTTCGGTCATACACGTAGTACTACGACT-3' (SEQ ID NO: 3), was synthesized using the solid supports described above on various DNA synthesis apparatuses.
  • Example 1 The solid support of Example 1 was used for synthesis with the Pharmacia Gene Assembler. The trityl coupling efficiency is given in Tables IV and V. A capillary gel electropherogram of the synthesized DNA is shown in Figure 5. (c) Synthesis Using Solid Support of Example 2 with Pharmacia Gene Assembler
  • Example 2 The solid support of Example 2 was similarly used for synthesis with the Pharmacia Gene Assembler to synthesize the 35-nucleotide sequence described above. Because the nucleoside attached to the solid support is deoxycytidine for Example 2, the sequence actually synthesized had a C at its 3'-terminal end instead of a T. The trityl coupling
  • Example 4 The solid support of Example 4 was used to synthesize the same 35-residue oligodeoxyribonucleotide.
  • Example 4 Because the solid support of Example 4 has an deoxyadenosine residue attached to the solid support, the synthesized DNA had an A at its 3' -terminus instead of a T.
  • the trityl coupling efficiency for this synthesis is shown in Table V.
  • Example 5 The solid support of Example 5 was used for synthesis with the Pharmacia Gene Assembler for synthesis of the 35-residue oligodeoxyribonucleotide whose sequence is given above. (For this and all other portions of Example 11, the solid support has a thymidine residue attached, so the sequence synthesized is identical to the one set out initially at the beginning of the Example.) The trityl coupling efficiency for this synthesis is shown in Tables IV and V. A capillary gel electropherogram of the synthesized DNA is shown in Figure 6.
  • Example 6 The solid support of Example 6 was used for the synthesis of the 35-residue oligodeoxyribonucleotide given above with the Pharmacia Gene Assembler. A capillary gel electropherogram of the synthesized DNA is shown in Figure 7.
  • Example 8 The solid support of Example 8 was similarly used with the Pharmacia Gene Assembler for synthesis of the 35-residue oligodeoxyribonucleotide.
  • a capillary gel A capillary gel
  • Example 1 The solid support of Example 1 was used for synthesis with the Biosearch 8750 DNA synthesis apparatus to synthesize the 35-residue oligodeoxyribonucleotide described above.
  • the trityl coupling efficiency is shown in Table V.
  • Example 5 The solid support of Example 5 was used for synthesis of the same 35-residue oligodeoxyribonucleotide on the Biosearch 8750 synthesis apparatus. The trityl coupling efficiency for this synthesis is shown in Table V.
  • Example 6 The solid support of Example 6 was used for synthesis of the same 35-residue oligodeoxyribonucleotide on the Biosearch 8750 DNA synthesis apparatus.
  • the trityl coupling efficiency is shown in Table V.
  • Example 7 Use of the Solid Support of Example 7 for Synthesis with the Biosearch Synthesis Apparatus
  • the solid support of Example 7 was used for synthesis of the same 35-residue oligodeoxyribonucleotide with the Biosearch 8750 DNA synthesis apparatus.
  • the trityl coupling efficiency is given in Table V.
  • Example 8 The solid support of Example 8 was used for synthesis of the same 35-residue oligodeoxyribonucleotide on the Biosearch 8750 DNA synthesis apparatus.
  • the trityl coupling efficiency is given in Table V.
  • controlled pore glass 500 A porosity was used for synthesis of the 35-residue
  • Example 1 The solid support of Example 1 was used for synthesis of the same 35-residue oligodeoxyribonucleotide on the ABI PCR Mate DNA synthesis apparatus.
  • Example 12 The solid support of Example 5 was used for synthesis of the same 35-residue oligodeoxyribonucleotide with the ABI PCR Mate DNA synthesis apparatus.
  • Example 12 The solid support of Example 5 was used for synthesis of the same 35-residue oligodeoxyribonucleotide with the ABI PCR Mate DNA synthesis apparatus.
  • a 51-residue oligodeoxyribonucleotide 5'- TCCATGGC.AACTGTCAAGGCACTGGCTCGTAGCCTACTGGCTTGACCGTAT-3' (SEQ ID NO: 4), was synthesized using the solid supports described above in various DNA synthesis apparatus.
  • Example 1 The solid support of Example 1, above, was used for synthesis of the 51-residue oligodeoxyribonucleotide described above with the Pharmacia Gene Assembler DNA synthesis
  • Example 5 The solid support of Example 5 was used for synthesis of the 51-residue oligodeoxyribonucleotide whose sequence is given above with the Pharmacia Gene Assembler DNA synthesis apparatus. The trityl coupling efficiency for the synthesis is given in Table IV.
  • Example 8 The solid support of Example 8, above, was used for synthesis of the same 51-residue oligodeoxyribonucleotide with the Pharmacia Gene Assembler DNA synthesis apparatus.
  • controlled pore glass 500 A porosity was used for synthesis of the 51-residue
  • Example 1 The solid support of Example 1, above, was used for synthesis of tne 51-residue oligodeoxyribonucleotide whose sequence is given above with the Biosearch 8750 DNA synthesis apparatus.
  • the trityl coupling efficiency is shown in Table V.
  • Example 8 The solid support of Example 8, above, was used for synthesis of the 51-residue oligodeoxyribonucleotide whose sequence is given above on the Biosearch 8750 DNA synthesis apparatus.
  • the trityl coupling efficiency for this synthesis is given in Table V.
  • Example 8 The solid support of Example 8, above, was used for synthesis of the 51-residue oligodeoxyribonucleotide whose sequence is given above on the Biosearch 8750 DNA synthesis apparatus.
  • the trityl coupling efficiency for this synthesis is given in Table V.
  • a 70- esidue oligodeoxyribonucleotide with the sequence of
  • TAACGTAGGAT-3' (SEQ ID NO: 5), was synthesized using the solid supports described above with various DNA synthesis apparatus.
  • oligodeoxyribonucleotide whose sequence is given above was synthesized using controlled pore glass (1000 ⁇ porosity), on the Pharmacia Gene Assembler DNA synthesis apparatus.
  • the trityl coupling efficiency for this synthesis is given in Table IV.
  • oligodeoxyribonucleotide whose sequence is given above was synthesized using controlled pore glass (1000 ⁇ porosity) with the Biosearch 8750 DNA synthesis apparatus.
  • the trityl coupling efficiency for the synthesis is given in Table V.
  • Biosearch Synthesis Apparatus The solid support of Example 7, above, was used for synthesis of the 70-residue oligodeoxyribonucleotide whose sequence is given above with the Biosearch 8750 DNA synthesis apparatus.
  • the trityl coupling efficiency is shown in Table V.
  • a oligodeoxyribonucleotide of 101 residues with the sequence of 5'- AACGTCGGTAACGTACACGGTAGCTACGGACACCGTGGCAATACGACAGGTAACCTGTGGAA CGTACACGGAAGAGACTAGGGATGGGAGTACGGATGGGT-3' (SEQ ID NO: 6), was synthesized using various solid supports as described above and various DNA synthesis apparatuses.
  • Control With Pharmacia Synthesis Apparatus
  • the 101-residue oligodeoxyribonucleotide whose sequence is given above was synthesized using the solid support of Example 1 with the Pharmacia Gene Assembler DNA synthesis apparatus.
  • the trityl coupling efficiency for this synthesis is given in Table IV.
  • the 101-residue oligodeoxyribonucleotide whose sequence is given above was synthesized using the solid support of Example 5 with the Pharmacia Gene Assembler DNA synthesis apparatus.
  • the trityl coupling efficiency for the synthesis is given in Table IV.
  • the 101-residue oligodeoxyribonucleotide whose sequence is given above was synthesized using the solid support of Example 6 with the Pharmacia Gene Assembler DNA synthesis apparatus. (e) Use of Solid Support of Example 8 With Pharmacia
  • the 101-residue oligodeoxyribonucleotide whose sequence is given above was synthesized using the solid support of Example 8 with the Pharmacia Gene Assembler DNA synthesis apparatus.
  • oligodeoxyribonucleotide whose sequence is given above was synthesized using controlled pore glass (1000 A porosity) with the Biosearch 8750 DNA synthesis apparatus.
  • the trityl coupling efficiency is shown in Table V.
  • the 101- residue oligodeoxyribonucleotide whose sequence is given above was synthesized using the solid support of Example 7, above, with the Biosearch 8750 DNA synthesis apparatus.
  • the trityl coupling efficiency for the synthesis is shown in Table V.
  • Polyacrylamide slab gel electrophoresis of the synthesized oligodeoxyribonucleotides was performed according to the following protocol.
  • a 22 cm ⁇ 16.5 cm denaturing gel was prepared by adding 107.3 ml of water to a 100 g bottle of premixed acrylamide/methylenebisacrylamide (29:1) to make a 50% stock solution. The following were mixed: (1) 20 ml of the 50% acrylamide stock solution; (2) 22.5 g urea; and (3) 5 ml of 10 x TBE (0.9 mole/l Tris-borate, pH 8.0, 0.02 mole/l EDTA). The volume was brought to 50 ml with water, and the solution was heated and stirred to dissolve the urea.
  • oligodeoxyribonucleotide 20 mg was added to initiate polymerization.
  • the gel was poured into the plates, and allowed to polymerize for 1 hour.
  • the gel was then prerun with 1 ⁇ TBE at 20 mA for 1 hour.
  • the appropriate quantity of oligodeoxyribonucleotide (0.2-1 A 260 unit) was mixed in 10 ⁇ l of water with 10 ⁇ l of 10 mole/l urea.
  • the resulting 20- ⁇ l mixture was loaded onto the gel and run at 28 mA for 2-4 hours, depending on the length of the oligodeoxyribonucleotide.
  • the oligodeoxyribonucleotide bands were visualized by UV shadowing on a thin layer
  • Plate I Figure 9: samples from Examples 10(a), 10(b), 10(c), 11(a), 11(b), 11(f), 11 (o), 11 (p), 11 (p) (2nd batch), and 11 (q)).
  • Plate II Figure 10: samples from Examples 11(a), 1Kb), 11(c), 11(d), and 11(e)).
  • Plate VII ( Figure 15: samples from Examples 11(e), 11(h), 11 (i), 11(1), 11 (n), 11 (i), and 11(n)).
  • Plate VIII ( Figure 16: samples from Examples 12(a), 12(e), 12(b), and 12(d)).
  • Plate X was done with ethidium bromide staining; the others were done with fluorescent visualization.
  • Reverse phase high pressure chromatography separates the desired oligodeoxyribonucleotide, which is expected to carry a lipophilic dimethoxytrityl (DMT group) from failed partial sequences that are not expected to carry the DMT group.
  • DMT group lipophilic dimethoxytrityl
  • the HPLC column used was C 18 Ultrasphere (Beckman Instruments, Fullerton, California), 5 ⁇ particles, 4.6 mm ⁇ 25 cm. Buffers used were 0.1 mole/l ammonium
  • the elution program at a flow rate of 1 ml/min, was a gradient to 15% Buffer B from zero time to 20 minutes, a gradient to 25% Buffer B from 20 minutes to 25 minutes, a gradient to 50% Buffer B from 25 minutes to 27 minutes, 50% Buffer B from 27 minutes to 30 minutes, and 0% Buffer B from 30 to 35 minutes.
  • the two enzymes used were phosphodiesterase I reconstituted with 5 ml of 40 mmole/l Tris, 10 mmole/l MgCl 2 , pH 7.5) and alkaline phosphatase from Escherichia coli. at 200 units/1.8 ml.
  • the assay used 0.01 units of phosphodiesterase I and 0.2 units of alkaline phosphatase per assay, which was 25 ⁇ l of phosphodiesterase and 2 ⁇ l of phosphatase per assay.
  • PCR polymerase chain reaction
  • PCR was performed according to the published instructions for the Perkin-Elmer-Cetus amplification reagent kit (Perkin-Elmer-Cetus, Norwalk, Connecticut). Initial melting was at 95°C for 7 minutes, and 25 cycles were then run on the DNA thermal cycler with the following cycle profile: segment 1, 94°C for 1 second; segment 2, 94°C for 1 minute; segment 3, 37°C for 1 second; segment 4, 37°C for 2 minutes; segment 5, 72°C for 1 second; and segment 6, 72°C for 3 minutes.
  • the 957 base pair PCR products were electrophoresed on a 1% agarose gel in TAE (0.04 mole/l Tris-acetate, 0.001 mole/l EDTA), and stained with ethidium
  • lane 1 is the product from the CPG-synthesized primers
  • lanes 2 and 3 are the products from the primers synthesized by the solid support of Example 1
  • lane 4 is a gel marker consisting of
  • bacteriophage ⁇ DNA digested with the restriction endonuclease Hind III, showing 2322 and 2027 base pair markers
  • lane 5 is a gel marker consisting of plasma pBR 322 DNA digested with the restriction endonuclease Hinf I, showing 1632 and 506 base pair markers.
  • DNA sequencing was performed using the same purified 18-mer primer synthesized in Example 18.
  • the template was M13mpl8 single-stranded DNA (New England Biolabs).
  • the published protocol using ⁇ - [ 35 S] -dATP was followed for chain-termination sequencing using the SequenaseTM enzyme (United States Biochemical, Cleveland, Ohio).
  • Example 20 synthesized using the primers originally synthesized with controlled pore glass and the solid support of Example 1 were compared by gel electrophoresis ( Figure 20). The results were substantially identical.
  • Example 20
  • Example 18 A 22-mer as synthesized in Example 18 for the polymerase chain reaction on controlled pore glass and the solid support of Example 1 were purified by Sep Pak (Waters Chromatography Division of Millipore, Milford, Mass.) C 18 cartridge before the kinase reaction by diluting the
  • oligonucleotide 1:2 into 0.5 mole/l ammonium acetate loading it onto the cartridge, washing the cartridge with water, diluting the oligonucleotide with 60% methanol in water, and evaporating the methanol :water solvent.
  • the oligonucleotides were then resuspended in water to a concentration of 1 ⁇ g/ ⁇ l.
  • oligonucleotide was mixed with 10 ⁇ l of 10 ⁇ kinase buffer (0.5 mole/l Tris-HCI, pH 7.6, 0.1 mmole/l MgCl 2 , 50 mmole/l dithiothreitol, and 1 mmole/l EDTA), 10 ⁇ l of ATP (10 mmole/l), and 30 units T4 polynucleotide kinase (Boehringer Mannheim, Mannheim, Germany) and the volume was brought to 100 ⁇ l with water. The reaction mixtures were incubated at 37°C for 45 minutes, then heated at 75°C for 5 to 10 minutes to inactivate the enzymes.
  • 10 ⁇ kinase buffer 0.5 mole/l Tris-HCI, pH 7.6, 0.1 mmole/l MgCl 2 , 50 mmole/l dithiothreitol, and 1 mmole/l EDTA
  • the reaction mixtures were analyzed by capillary gel electrophoresis as described above with injection for 10 seconds at 10 kV and separate at 11 kV for 45 minutes at 20°C.
  • the phosphorylated oligonucleotides were differentiated from non-phosphorylated oligonucleotides by spiking the kinase reaction mix 1:2 with 50 ⁇ g oligonucleotides and analyzed on capillary gel electrophoresis in the same manner. Both the oligonucleotides synthesized on the solid support of Example 1 and the controlled pore glass were phosphorylated at the 5'- ends quantitatively.
  • transferase reactions were performed. The following were mixed: 2.5 A 260 of the oligonucleotide in 150 ⁇ l water, 5 mg TTP (Sigma Chemical Company, St. Louis, Missouri), 5 ⁇ l terminal deoxynucleotidyl transferase (15 units/ ⁇ l) (Bethesda Research Labs, Bethesda, Maryland), and 50 ⁇ l tailing buffer (Bethesda Research Labs). The reactions were incubated overnight at 37°C.
  • the reacted nucleotides were purified by Sep Pak C 18 cartridge by diluting the reaction mixture 1:2 in 0.5 mmole/l ammonium acetate, loading on the cartridge, washing the cartridge with water, and eluting the oligonucleotides with 60% methanol in water.
  • the eluted oligonucleotides were analyzed by capillary get electrophoresis in the same manner as described above. Both the oligonucleotides synthesized on the controlled pore glass and on the solid support of Example 1 were substantially quantitatively extended at the 3' -end by the terminal transferase.
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 7: CGCCAGGGTT TTCCCAGT 18
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

Abstract

Intermédiaire de synthèse nucléotidique en phase solide utile pour effectuer la synthèse chimique d'oligodésoxyribonucléotides et d'oligonucléotides comprenant: (1) un support particulaire constitué d'un polymère poreux dont le squelette comprend des fractions acrylate ou méthacrylate facultativement substituées; (2) un nucléoside; et (3) un segment de liaison comportant des première et deuxième extrémités qui sont respectivement liées de manière covalente au support particulaire et au nucléoside, le segment de liaison éloignant le nucléoside à au moins trois atomes du polymère. Ledit segment de liaison peut comporter au moins une diamine aliphatique facultativement substituée ou bien il peut comprendre une fraction de polyéthylène glycol. Le polymère poreux est de préférence un polymère méthacrylate-vinylidène. On peut utiliser le support en phase solide produit de la sorte pour effectuer la synthèse d'oligodésoxyribonucléotide à l'aide des processus phosphite-triester ou phosphotriester.
PCT/US1993/006214 1992-07-09 1993-06-29 Support solide organique derive utilise pour effectuer la synthese d'acides nucleiques WO1994001446A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US91022392A 1992-07-09 1992-07-09
US07/910,223 1992-07-09

Publications (2)

Publication Number Publication Date
WO1994001446A2 true WO1994001446A2 (fr) 1994-01-20
WO1994001446A3 WO1994001446A3 (fr) 1994-03-03

Family

ID=25428489

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/006214 WO1994001446A2 (fr) 1992-07-09 1993-06-29 Support solide organique derive utilise pour effectuer la synthese d'acides nucleiques

Country Status (1)

Country Link
WO (1) WO1994001446A2 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995011912A2 (fr) * 1993-10-29 1995-05-04 Beckman Instruments, Inc. Synthese de biopolymeres utilisant des polymeres organiques actives en surface
US5723599A (en) * 1990-10-26 1998-03-03 Genta Incorporated Organic polymer reagents for solid phase synthesis of oligonucleotides
EP0920440A2 (fr) * 1996-02-09 1999-06-09 Cornell Research Foundation, Inc. Detection de differences entre sequences d'acide nucleique faisant appel a la reaction de detection de ligase et a des reseaux adressables
US6238927B1 (en) 1998-10-05 2001-05-29 Mosaic Technologies, Incorporated Reverse displacement assay for detection of nucleic acid sequences
WO2002046205A3 (fr) * 2000-12-05 2002-08-15 Avecia Ltd Procede de preparation d'oligonucleotides phosphorothionate
US6768005B2 (en) 2000-12-20 2004-07-27 Avecia Limited Process
US6852487B1 (en) 1996-02-09 2005-02-08 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
US7097980B2 (en) 1996-05-29 2006-08-29 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
US7205399B1 (en) 2001-07-06 2007-04-17 Sirna Therapeutics, Inc. Methods and reagents for oligonucleotide synthesis
US7279261B2 (en) 2005-01-13 2007-10-09 Xerox Corporation Emulsion aggregation toner compositions
US7455965B2 (en) 2000-04-14 2008-11-25 Cornell Research Foundation, Inc. Method of designing addressable array for detection of nucleic acid sequence differences using ligase detection reaction
US7777023B2 (en) 1998-10-23 2010-08-17 Sirna Therapeutics, Inc. Method for the chemical synthesis of oligonucleotides

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1982003079A1 (fr) * 1981-03-10 1982-09-16 Inc Bioresearch Synthese rapide en phase solide d'oligonucleotides par l'activation d'oxychlorure de phosphore
WO1985001051A1 (fr) * 1983-09-02 1985-03-14 Molecular Biosystems, Inc. Systeme de support polymere d'oligonucleotides
US4659774A (en) * 1985-11-01 1987-04-21 American Hoechst Corporation Support for solid-phase oligonucleotide synthesis
WO1992007882A1 (fr) * 1990-10-26 1992-05-14 Genta Incorporated Reactifs polymeres organiques non aromatiques pour la synthese en phase solide d'oligomeres
WO1992007864A1 (fr) * 1990-10-26 1992-05-14 Genta Incorporated Procede ameliore de synthese d'oligomeres

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1982003079A1 (fr) * 1981-03-10 1982-09-16 Inc Bioresearch Synthese rapide en phase solide d'oligonucleotides par l'activation d'oxychlorure de phosphore
WO1985001051A1 (fr) * 1983-09-02 1985-03-14 Molecular Biosystems, Inc. Systeme de support polymere d'oligonucleotides
US4659774A (en) * 1985-11-01 1987-04-21 American Hoechst Corporation Support for solid-phase oligonucleotide synthesis
WO1992007882A1 (fr) * 1990-10-26 1992-05-14 Genta Incorporated Reactifs polymeres organiques non aromatiques pour la synthese en phase solide d'oligomeres
WO1992007864A1 (fr) * 1990-10-26 1992-05-14 Genta Incorporated Procede ameliore de synthese d'oligomeres

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TETRAHEDRON vol. 45, no. 9 , 1989 , OXFORD, GB pages 2777 - 2792 KATZHENDLER J. ET AL 'THE EFFECT OF SPACER, LINKAGE AND SOLID SUPPORT ON THE SYNTHESIS OF OLIGONUCLEOTIDES' cited in the application *

Cited By (43)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5723599A (en) * 1990-10-26 1998-03-03 Genta Incorporated Organic polymer reagents for solid phase synthesis of oligonucleotides
WO1995011912A2 (fr) * 1993-10-29 1995-05-04 Beckman Instruments, Inc. Synthese de biopolymeres utilisant des polymeres organiques actives en surface
WO1995011912A3 (fr) * 1993-10-29 1995-06-08 Beckman Instruments Inc Synthese de biopolymeres utilisant des polymeres organiques actives en surface
EP2332957A1 (fr) * 1996-02-09 2011-06-15 Cornell Research Foundation, Inc. Détection de différences entre séquences d'acide nucléique faisant appel à la réaction de détection par ligase et à des réseaux adressables
EP2368897A1 (fr) * 1996-02-09 2011-09-28 Cornell Research Foundation, Inc. Detection de différences entre séquences d'acide nucleique faisant appel à la réaction de détection par ligation et d'hybridation sur des réseaux adressables d'oligonucléotides
US9234241B2 (en) 1996-02-09 2016-01-12 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
US9206477B2 (en) 1996-02-09 2015-12-08 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
US8624016B2 (en) 1996-02-09 2014-01-07 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
EP0920440A4 (fr) * 1996-02-09 2004-08-25 Cornell Res Foundation Inc Detection de differences entre sequences d'acide nucleique faisant appel a la reaction de detection de ligase et a des reseaux adressables
EP1958955A1 (fr) * 1996-02-09 2008-08-20 Cornell Research Foundation, Inc. Réseaux d'oligonucleotides adressables spatialement et méthode pour leur fabrication.
US7083917B2 (en) 1996-02-09 2006-08-01 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
EP2574617A1 (fr) * 1996-02-09 2013-04-03 Cornell Research Foundation, Inc. Détection de différences entre séquences d'acide nucléique faisant appel à la réaction de détection de ligase et à des réseaux adressables
EP2573101A1 (fr) * 1996-02-09 2013-03-27 Cornell Research Foundation, Inc. Détection de différences entre séquences d'acide nucléique faisant appel à la réaction de détection de ligase et à des réseaux adressables
US8288521B2 (en) 1996-02-09 2012-10-16 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
EP0920440A2 (fr) * 1996-02-09 1999-06-09 Cornell Research Foundation, Inc. Detection de differences entre sequences d'acide nucleique faisant appel a la reaction de detection de ligase et a des reseaux adressables
EP2332958A1 (fr) * 1996-02-09 2011-06-15 Cornell Research Foundation, Inc. Détection de différences entre séquences d'acide nucléique faisant appel à la réaction de détection par ligase et à des réseaux adressables
US7892747B2 (en) 1996-02-09 2011-02-22 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
US7893233B2 (en) 1996-02-09 2011-02-22 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
US7892746B2 (en) 1996-02-09 2011-02-22 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
US7888009B2 (en) 1996-02-09 2011-02-15 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
US8703928B2 (en) 1996-02-09 2014-04-22 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
US6852487B1 (en) 1996-02-09 2005-02-08 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
US7364858B2 (en) 1996-05-29 2008-04-29 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
US7166434B2 (en) 1996-05-29 2007-01-23 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
US7332285B2 (en) 1996-05-29 2008-02-19 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
US7097980B2 (en) 1996-05-29 2006-08-29 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
US7429453B2 (en) 1996-05-29 2008-09-30 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
US7312039B2 (en) 1996-05-29 2007-12-25 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
US7320865B2 (en) 1996-05-29 2008-01-22 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
US6238927B1 (en) 1998-10-05 2001-05-29 Mosaic Technologies, Incorporated Reverse displacement assay for detection of nucleic acid sequences
US7777023B2 (en) 1998-10-23 2010-08-17 Sirna Therapeutics, Inc. Method for the chemical synthesis of oligonucleotides
US9340834B2 (en) 2000-04-14 2016-05-17 Cornell Research Foundation, Inc. Method of designing addressable array suitable for detection of nucleic acid sequence differences using ligase detection reaction
US10131938B2 (en) 2000-04-14 2018-11-20 Cornell Research Foundation, Inc. Method of designing addressable array suitable for detection of nucleic acid sequence differences using ligase detection reaction
US9725759B2 (en) 2000-04-14 2017-08-08 Cornell Research Foundation, Inc. Method of designing addressable array suitable for detection of nucleic acid sequence differences using ligase detection reaction
US7455965B2 (en) 2000-04-14 2008-11-25 Cornell Research Foundation, Inc. Method of designing addressable array for detection of nucleic acid sequence differences using ligase detection reaction
US8492085B2 (en) 2000-04-14 2013-07-23 Cornell Research Foundation, Inc. Method of designing addressable array suitable for detection of nucleic acid sequence differences using ligase detection reaction
JP2004517089A (ja) * 2000-12-05 2004-06-10 アベシア・リミテッド ホスホロチオエートオリゴヌクレオチドの調製方法
CN1302005C (zh) * 2000-12-05 2007-02-28 艾夫西亚有限公司 硫代磷酸寡核苷酸的制备方法
WO2002046205A3 (fr) * 2000-12-05 2002-08-15 Avecia Ltd Procede de preparation d'oligonucleotides phosphorothionate
US7227017B2 (en) 2000-12-05 2007-06-05 Avecia Limited Process for the preparation of phosphorothioate oligonucleotides
US6768005B2 (en) 2000-12-20 2004-07-27 Avecia Limited Process
US7205399B1 (en) 2001-07-06 2007-04-17 Sirna Therapeutics, Inc. Methods and reagents for oligonucleotide synthesis
US7279261B2 (en) 2005-01-13 2007-10-09 Xerox Corporation Emulsion aggregation toner compositions

Also Published As

Publication number Publication date
WO1994001446A3 (fr) 1994-03-03

Similar Documents

Publication Publication Date Title
US5348868A (en) Methods and reagents for cleaving and deprotecting oligonucleotides
EP1379698B1 (fr) Polymeres de nucleobase a mobilite modifiee et procedes d'utilisation des memes
EP0971943B1 (fr) Complexe polymerisable contenant de l'acide nucleique
US6590093B1 (en) Orthoester protecting groups
EP0786468B1 (fr) Réactifs sur supports solides pour la synsthèse directe de 3'-polynucléotides marqués
CA2087818C (fr) Analogues d'oligonucleotides; preparation et utilisation
US4948882A (en) Single-stranded labelled oligonucleotides, reactive monomers and methods of synthesis
JP2533100B2 (ja) ポリヌクレオドの誘導方法
EP1585755B1 (fr) Procedes et compositions pour la synthese en tandem d'au moins deux oligonuleotides sur le meme support solide
EP1247815A2 (fr) Oligonucléotides modifiés et leurs utilisations
JPH06500107A (ja) オリゴ（α―アラビノフラノシル・ヌクレオチド）およびそれらのα―アラビノフラノシル前駆体
WO1996015270A1 (fr) Sonde en fluorescence a auto-extinction et methode de fonctionnement
WO1996006104A1 (fr) Reactifs polynucleotidiques contenant des fractions non nucleotidiques, et procedes de synthese et d'utilisation associes
WO1994001446A2 (fr) Support solide organique derive utilise pour effectuer la synthese d'acides nucleiques
WO1992009615A1 (fr) Procede et systeme permettant de realiser la synthese d'oligonucleotides
CA2087817C (fr) Analogues, derives de substitution non nucleotidique en 3' d'oligonucleotides; preparation et utilisation
CN101547928A (zh) 合成和纯化寡核苷酸的化合物和方法
EP1244812A1 (fr) Compose ramifie s'utilisant dans des reactions de detection et d'analyse d'acide nucleique
CA2190145A1 (fr) Adn et toxines complementaires
Reddy et al. New and efficient solid support for the synthesis of nucleic acids
WO1999011820A1 (fr) Compositions et procedes d'identification et de quantification d'oligonucleotides a sequence de deletion dans des preparations d'oligonucleotides de synthese
KR20040015107A (ko) 고체 지지체 상에 올리뉴클레오티드의 고정화
RU2124522C1 (ru) Ковалентно-связанные комплексы олигонуклеотид-нуклеиновая кислота, способ их получения
Kumar Nucleic Acids
AU695561C (en) Self-quenching fluorescence probe and method

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): JP

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

AK Designated states

Kind code of ref document: A3

Designated state(s): JP

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase