WO1992002532A1 - Reactifs de liaison ameliores a base de non nucleotide pour oligomeres - Google Patents

Reactifs de liaison ameliores a base de non nucleotide pour oligomeres Download PDF

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Publication number
WO1992002532A1
WO1992002532A1 PCT/US1991/008769 US9108769W WO9202532A1 WO 1992002532 A1 WO1992002532 A1 WO 1992002532A1 US 9108769 W US9108769 W US 9108769W WO 9202532 A1 WO9202532 A1 WO 9202532A1
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Prior art keywords
nucleotide
reagent according
skeleton
monomeric unit
oligomer
Prior art date
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PCT/US1991/008769
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English (en)
Inventor
Mark A. Reynolds
Morteza M. Vaghefi
Lyle J. Arnold, Jr.
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Genta Incorporated
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Publication date
Application filed by Genta Incorporated filed Critical Genta Incorporated
Priority to JP92502337A priority Critical patent/JPH05507419A/ja
Priority to AU90710/91A priority patent/AU664542B2/en
Priority to KR1019930700371A priority patent/KR930701468A/ko
Publication of WO1992002532A1 publication Critical patent/WO1992002532A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
    • 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
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/22—Amides of acids of phosphorus
    • C07F9/26—Amides of acids of phosphorus containing P-halide groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/02—Ortho- or ortho- and peri-condensed systems
    • C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
    • C07C2603/06—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members
    • C07C2603/10—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings
    • C07C2603/12—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings only one five-membered ring
    • C07C2603/18—Fluorenes; Hydrogenated fluorenes

Definitions

  • Nucleotide Probes PCT WO 8902439 assigned to Gen-Probe, Inc.
  • those non-nucleotide reagents were of mixed chirality.
  • Those reagents are coupled into the phosphodiester backbone of an oligonucleotide by chemical synthesis as disclosed therein.
  • One aspect of the present invention provides nonnucleotide reagents which are suitable for preparing
  • nucleotide/non-nucleotide polymer comprises at least one nucleotide monomeric unit.
  • the ligand moiety may comprise a linker-arm group which may participate in conjugation reactions upon its activation or deprotection, a detectable chemical moiety or label or a side arm to which a detectable chemical moiety or label has been attached prior to initiating synthesis of the polymer.
  • Suitable chemical moieties include detectable labels, chelators, catalysts, nucleolytic moieties, drug carriers, hormone receptors,
  • Suitable chemical moieties include psoralen and analogs thereof, acridines and analogs thereof, porphyrins and porphyrin analogs, cyclic chelators and the like.
  • deoxyribooligonucleotide is an oligonucleotide consisting of deoxyribonucleotide monomers.
  • nucleotide multimer probe is a nucleotide multimer having a nucleotide sequence complementary with a target
  • the probe usually is selected to be perfectly complementary to the corresponding base in the target sequence. However, in some cases it may be adequate or even desirable that one or more nucleotides in the probe not be complementary to the corresponding base in the target sequence.
  • non-nucleotide monomeric unit refers to a monomeric unit which does not significantly participate in hybridization of a polymer. Such monomeric units must not, for example,
  • nucleotide/non-nucleotide polymer refers to a polymer comprised of nucleotide and non-nucleotide monomeric units.
  • oligonucleotide analogs such as phosphotriesters, and other oligonucleotide analogs and modified oligonucleotides, and also includes nucleotide/non-nucleotide polymers.
  • the term also includes nucleotide/non-nucleotide polymers wherein one or more of the phosphorous group linkages between monomeric units has been replaced by a non-phosphorous group linkage such as a formacetal linkage or a carbamate linkage.
  • alkyl- or aryl-phosphonate oligomer refers to nucleotide oligomers (or nucleotide/non-nucleotide polymers) having internucleoside (or intermonomer) phosphorus group linkages wherein at least one alkyl- or aryl- phosphonate linkage replaces a phosphodiester linkage.
  • methylphosphonate oligomer refers to nucleotide oligomers (or nucleotide/non-nucleotide polymer) having internucleoside (or intermonomer) phosphorus group linkages wherein at least one methylphosphonate internucleoside linkage replaces a phosphate diester
  • nucleoside includes a nucleosidyl unit and is used interchangeably therewith
  • Figures 1A, 1B and 1C depict the formulas of non-nucleotide reagents of the present invention having Fmoc-procected linker arms.
  • Figure 2 depicts a synthetic scheme for preparing non-nucleotide reagents of the present invention having C2, C4 and C6 linker arms.
  • Figure 3 depicts a synthetic scheme for preparing non-nucleotide reagents of the present invention having C8 , C10 and C12 linker arms.
  • Figure 4 depicts a synthetic scheme for a psoralen reagent which may form a psoralen conjugate with the- linker arm of one of the non-nucleotide reagents of the present invention.
  • chemistries already known in the art include intercalators, alkylators, photoactivated reactive moieties such as psoralens, chelating agents, etc. We believe that by applying such chemistries, we will be able to increase the potency of methylphosphonate oligomers as
  • the non-nucleotide based linking reagents are prepared in a chirally pure form. Moreover, these non-nucleotide reagents remain chirally pure when incorporated in oligomers (non-nucleotide/nucleotide polymers). This advantage may be critical when it is desired or desirable to direct a label to a particular location when the oligomer is hybridized to a corresponding carget nucleic acid.
  • the hydrocarbon skeleton of these reagents comprises a reduction product of threonine. Since the four enantiomers of threonine are commercially available, non-nucleotide reagents having a chirally pure skeletons derived from any one of the four
  • stereoisomers of threonine may be prepared.
  • threonine as the starting material to supply the chirally pure skeleton for some of these reagents has additional advantages.
  • non-nucleotide reagents in such a way that an alkyl- or aryl- phosphonate diester linkage results when these non-nucleotide reagents are inserted into the phosphorus backbone of the oligomer.
  • Such reagents couple to nucleotides or other non-nucleotide reagents in high yield.
  • these non-nucleotide reagents can be used for the attachment of psoralen analogs to the oligomers. These psoralen analogs are attached to a non-nucleotide reagent using a novel chemistry (See Example 13).
  • the present invention provides a non-nucleotide reagent, with a non-nucleotide monomeric unit which can be coupled synthetically with specific nucleotide monomeric units from nucleotide reagents, to produce a defined sequence polymer which is comprised of nucleotide and non-nucleotide monomeric units.
  • Said non-nucleotide reagent also possesses a ligand which may comprise a linker-arm moiety which may
  • the techniques for linking moieties to the linker arm may be similar to the techniques for linking labels to groups on proteins. However, modifications of such techniques may be required.
  • useful chemistries include the reaction of alkylamines with active esters, active imines, arylhalides, or isothiocyanates, and the reaction of tbiols with maleimides, haloacetyls, etc. (for further potential techniques see G.M. Means and R.E.
  • Suitable protecting groups which can be used to protect the linker arm functional group during formation of a polymer are also similar to those used in protein chemistry (see for example, “The Peptides: Analysis and Synthesis, Biology,” Vol. 3, ed. E. Gross and J. Meienhofer, Academic Press, 1971). Due to the chemical nature of the non-nucleotide reagent, it may be placed at any desired position within the nucleotide monomer sequence. This makes it possible to design a wide variety of properties into polymers which contain nucleotide monomers. These include: (1) attachment of specific chemical moieties at any desired location within the polymer, such moieties can include (but are not limited to) detectable labels, intercalating agents,
  • nucleolytic agents drugs, hormones, proteins, peptides, haptens, radical generators, nucleolytic agents, proteolytic agents, catalysts, receptor binding substances, and other binding substances of biological interest, and agents which modify DNA transport across a biological barrier (such as a membrane), and substances which alter solubility of a nucleotide multimer.
  • a biological barrier such as a membrane
  • nucleotide affinity supports it is possible to position such labels and intercalating agents adjacent to any desired nucleotide; (2) the ability to immobilize the defined sequence to a solid support employing its linker-arm for conjunction to a chemical moiety of said support in order to construct, for example, nucleotide affinity supports; (3) the ability to attach multiple chemical moieties to the polymer through linker-arms by incorporating multiple non-nucleotide monomeric units into the polymers; (4) the ability to construct polymers which differ from naturally occurring polynucleotides in that they have altered activities with proteins and enzymes which act on polynucleotides.
  • non-nucleotide monomeric unit on the 3 ' terminus of an otherwise pure polynucleotide imparts resistance to degradation by snake venom phosphodiesterase.
  • non-nucleotide monomeric units may create specific cleavage sites for other nucleases; (5) the ability to construct hybridization probes by interspersing hybridizable nucleotide and non-nucleotide monomeric units.
  • a mixed block synthesis of nucleotide and non-nucleotide monomers can be produced, whereby a defined sequence of
  • nucleotide monomers are synthesized followed by a stretch of the one or more non-nucleotide monomeric units followed by second block of defined sequence nucleotide monomers; (6) the ability to construct synthetic probes which simultaneously detect target nucleotide multimers which differ by one or more base pairs.
  • labelled oligomers are constructed with a defined sequence comprised of nucleotide and non-nucleotide monomers.
  • the non-nucleotide monomeric units are used to connect two or more defined sequence nucleotide
  • non-nucleotide monomeric units are chemically labelled for use in cross-linking reactions.
  • the non-nucleotide reagent is constructed in a manner to permit it to be added in a step-wise fashion to produce a mixed nucleotide/non-nucleotide polymer employing one of the current DNA synthesis methods.
  • Such nucleotide and non-nucleotide reagents normally add in a step-wise fashion to attach their corresponding monomeric units to a growing oligomer chain which is covalently immobilized to a solid support.
  • the first nucleotide is attached to the support through a cleavable ester linkage prior to the initiation of synthesis. Step-wise extension of the oligomer chain is normally carried out in the 3' to 5 ' direction.
  • the present invention provides a non-nucleotide reagent for preparing polymers which contain a mixture of nucleotide and non-nucleotide monomeric units.
  • Said non-nucleotide monomers additionally may contain one or more
  • linker-arms or one or more linker-arms conjugated to a desired chemical moiety such as a label, a cross-linking agent or an intercalating agent.
  • Such a non-nucleotide monomer additionally possesses two coupling groups so as to permit its step-wise inclusion into a polymer of nucleotide and non-nucleotide monomeric units.
  • a first one of said coupling groups has the property that it can couple efficiently to the terminus of a growing chain of
  • the second of said coupling groups is capable of further extending, in a step-wise fashion, the growing chain of mixed nucleotide and non-nucleotide monomers. This requires that the second coupling group be inactivated while the first coupling group is coupling, so as not to substantially couple at that time , but can thereafter be activated so as to then couple the non-nucleotide monomeric unit.
  • the "inactivation” is preferably accomplished with a blocking group on the second coupling group, which can be removed to "activate" the second coupling group.
  • inactivation and activation might be accomplished simply by changing reaction conditions (e.g., pH, temperature, altering the concentration of some other component in the reaction system) with second coupling groups of a suitable chemical structure, which also lend themselves to inactivation and activation by such techniques.
  • Said coupling groups permit the adjacent attachment of either nucleotide or non-nucleotide monomeric units.
  • said coupling groups operate through coupling and deblocking and deprotection steps which are compatible with one of the standard DNA synthesis methods.
  • the non-nucleotide monomer preferably has a skeleton, to the ends of which the coupling groups are linked.
  • the skeleton is preferably an acyclic one to twenty atom chain, and preferably an acylic hydrocarbon chain of from one to twenty carbon atoms. PREFERRED NON-NUCLEOTIDE REAGENTS
  • Preferred non-nucleotide reagents comprise non- nucleotide monomeric units in which the skeleton has a backbone of up to about 10 carbon atoms in which said backbone comprises at least one asymmetric carbon which remains chirally pure upon being coupled into a nucleotide/non-nucleotide polymer.
  • Skeletons having backbones of about three carbons are preferred, in part, because such backbones resemble the three-carbon spacing of deoxyribose groups.
  • One preferred aspect of the present invention is directed to chirally pure non-nucleotide reagents which when incorporated in an oligomer comprise a chirally pure non- nucleotide monomeric unit of the formula:
  • SKEL comprises a chirally pure non-nucleotide skeleton of from about 1 to about 20 carbon atoms, wherein -NHL, Y and Z are covalently linked to a carbon atom of SKEL, L is a ligand, Y is - CH 2 -, -O-, -S- or -NH- and Z is -O-, -S- or -NH-.
  • SKEL further comprises a backbone of about 1 to about 10 carbon atoms separating Y and Z . Examples of non-nucleotide monomeric units incorporating these preferred SKEL groups include:
  • X s groups are independently selected from hydrogen or alkyl and may be the same or different, and q and r are
  • these preferred non-nucleotide reagents may be represented by the general formula:
  • X is halogen or substituted amino
  • X 2 is halogen, amino, or substituted amino, or O-
  • R 5 is alkyl, optionally substituted alkoxy or optionally substituted aryloxy
  • R 6 is alkyl, optionally substituted alkoxy or optionally substituted aryloxy, or if X 2 is O-, optionally hydrogen
  • U is oxygen, sulfur or imino
  • W is alkyl, aryl, alkoxy, aryloxy, alkylthio, arylthio, S-, O-, amino or substituted amino
  • V is alkoxy, alkylthio, amino or substituted amino.
  • non-nucleotide reagents which are capable of forming alkyl- or aryl-phosphonate, and in particular methylphosphonate, diester linkages between monomeric units, especially preferred non-nucleotide reagents include those wherein the first coupling group, -YCp 1 , is selected from
  • X 1 is chloro or secondary amino and R 5 is alkyl; X 2 is substituted amino, halogen or O- and R 6 is alkyl.
  • the ligand moiety, L is preferably selected from a functional moiety or from a protected linking arm which can be deprotected under non adverse conditions so as to capable of then linking with a functional moiety (under non-adverse
  • L comprises a protecting group, Pr, or protected linker arm which can be deprotected under non-adverse conditions so as to be capable of then linking with a functional moiety, including a cross linking agent such as psoralen, or a drug carrier molecule.
  • Preferred linker arms include those having one of the following formulas:
  • n and m are independently integers between 1 and 15, preferably between 1 and 5, and Pr is a protecting group
  • One group of particularly preferred non-nucleotide reagents has a skeleton derived from the amino acid threonine. These preferred reagents comprise a 3-carbon backbone having two asymmetric carbons, each of which remains chirally pure when incorporated in a nucleotide/non-nucleotide polymer.
  • these reagents having threonine-derived backbones advantageously have a primary hydroxyl and a secondary hydroxyl, which due to their differing reactivities allow selectivity and high yields in the subsequent protection, deprotection, blocking, deblocking and derivatization steps.
  • the first coupling group is associated with the secondary hydroxyl group and the second coupling group is associated with the primary hydroxyl.
  • the threonine-based non-nucleotide is threonine-based non-nucleotide
  • * C denotes an asymmetric carbon which is chirally pure, and wherein one of R 1 and R 2 is hydrogen and the other is -NH-L where L is a ligand moiety as hereinafter defined; one of R 3 and R 4 is hydrogen and the other is lower alkyl of about 1 to about 10 carbon atoms, -Y-Cp 1 is a first coupling group, and -ZCp 2 is a blocked second coupling group, wherein:
  • X is halogen or substituted amino
  • X 2 is halogen, amino, or substituted amino, or O-
  • R 5 is alkyl, optionally substituted alkoxy or optionally substituted aryloxy
  • R 6 is alkyl, optionally substituted alkoxy or optionally substituted aryloxy, or if X 2 is O-, optionally hydrogen
  • U is oxygen or sulfur
  • W is alkyl, aryl, alkoxy, alkylthio, aryloxy, arylthio, O-, S-, amino or substituted amino
  • V is alkoxy, alkylthio, amino or substituted amino
  • the ligand moiety, L is prererably selected from a functional moiety or from a protected linking arm which can be deprotected under non-adverse conditions so as to be capable of then linking with a functional moiety (under non-adverse conditions). Since non-nucleotide reagents which are capable of forming alkyl- or aryl-phosphonate, and in particular
  • methylphosphonate, diester linkages between monomeric units are preferred especially preferred non-nucleotide reagents include those wherein the first coupling group, -YCp 1 , is selected from
  • X is chloro or secondary amino and R 5 is alkyl; X 2 is substituted amino, halogen or O- and R 6 is alkyl.
  • L comprises a protecting group, Pr, or a protected linker arm which can be deprotected under non-adverse conditions so as to be capable of then linking with a functional moiety, including a cross linking agent such as psoralen, or a drug carrier molecule.
  • Preferred linker arms include those having one of the following formulas:
  • n and m are independently integers between 1 and 15, preferably between 1 and 5, and Pr is a protecting group
  • Suitable protecting groups, Pr include 9-fluorenylmethoxycarbonyl ("Fmoc”), trifluoroacetyl, phenoxyacetyl and the like. See, e.g., Chapter 7 of Greene, Theodore W. , "Protective Groups in Organic Synthesis", John Wiley & Son, New York, 1981. These linker arms may be conveniently prepared according to the reaction schemes outlined in Figures 2 and 3 and described the Examples herein.
  • Fmoc 9-fluorenylmethoxycarbonyl
  • Non-nucleotide reagents are useful in preparing oligomers haying ligand moieties conjugated to the oligomer without adversely affecting the nor-al base pairing associated with hybridization to a target nucleic acid.
  • These ligand moieties may comprise functior al groups or protected linker arms which may later (after synthesis of the oligonucleotide) be deprotected and react with a labelling reagent to give a linker arm - labelling reagent complex.
  • Functional groups of particular utility may include detectable labels, agents which react with a target nucleic acid such as cross-linking agents, agents which cleave the target nucleic acid, agents which increase the uptake of oligomers into cells or skin and agents which slow the excretion of oligomers from the body.
  • a target nucleic acid such as cross-linking agents
  • agents which cleave the target nucleic acid agents which increase the uptake of oligomers into cells or skin and agents which slow the excretion of oligomers from the body.
  • non-nucleotide reagents are incoproated into oligomers having alkyl- or aryl- phosphonate linkages between monomeric units, it may be
  • nucleotide monomeric units having modified ribosyl moieties advantageous to incorporate nucleotide monomeric units having modified ribosyl moieties.
  • the incorporation of nucleotide units having 2'-o-alkyl-, in particular 2'-O-methyl, ribosyl moieties into alkyl- or aryl-phosphonate oligomers advantageously may improve hybridization of the oligomer to its complimentary target sequence.
  • the reaction mixture was cooled to 5-10o C and quenched with dropwise addition of 0.25 M NaOH (100 ml).
  • the mixture was evaporated to remove over 90% of THF and the residue was diluted with 100 ml of dimethylformamide which facilitates the filtration.
  • the mixture was then filtered through a Whatman #1 paper using aspirator vacuum. The filtrate was evaporated to dryness and the residue was purified on a flash Silica Gel column. The column was packed with dichloromethane and the product was eluted with 50% methanol in dichloromethane.
  • FMOC-GLYCYLAMIDO-CAPROIC ACID C8 .
  • the desired non-nucleotide reagent (6 mmol) , which was made according to Examples 3 and 5 above, was dried by co- evaporation with dry pyridine and dissolved in 15 ml of dry pyridine. A solution of 2.2 g of dimethoxytrityl chloride in 20 ml of CH 2 Cl 2 /pyridine (1:1) was added dropwise with stirring.
  • reaction continued at room temperature for 45 min. The progress of the reaction was monitored by TLC. After the completion of the reaction it was quenched by the addition of 2 ml methanol which was stirred for 10 min. The solvents were removed under reduced pressure and the residue was dissolved in 50 ml of dichloromethane and extracted with saturated sodium hydrogen carbonate (2 x 50 ml) followed by water (30 ml). The organic phase was dried (MgSOJ and filtered. After the
  • a 4 mmol portion of a dimethoxytrityl (DMT) -blocked non-nucleotide reagent having a C6 linker arm was dried by co-evaporation with dry pyridine. The residue was dissolved in 20 ml of anhydrous dichloromethane. Under a closed argon
  • a phosphate diester oligodeoxyribonucleotide was synthesized which incorporated a C8 methoxyphosphoramidite non-nucIeotide reagent in the following sequence:
  • the C8 methoxyphosphoramidite non-nucleotide reagent (1-O-dimethcxytrityl-2-N[N'-(N"-fluorenyl-methoxycarbonyl-6-aminohexanoyl)-2-aminoacetyl]-3-O-[N,N-diisopropylmethoxy-phosphinyl]-2-amino-1,2-dihydroxybutane) was dissolved in dry acetonitrile at a concentration of 100 mM and coupled into the oligonucleotide sequence using a Biosearch Model 8750 DNA synthesizer by standard phosphoramidite chemistry (M.H.
  • Methylphosphonate oligomers which incorporated non-nucleotide reagents of the present invention were synthesized using methylphosphonamidite monomers and non-nucleotide
  • Non-nucleotide linker reagents were dissolved in acetonitrile at a concentration of 120 mM.
  • DEBLOCK reagent 2.5% dichloroacetic acid in dichloromethane.
  • OXIDIZER reagent 25 g/L iodine in 2.5% water, 25% 2,6-lutidine, 72.5% tetrahydrofuran.
  • CAP A 10% acetic anhydride in acetonitrile.
  • CAP B 0.625% N,N-dimethylaminopyridine in pyridine. The 5'-dimethoxytrityl protecting group was left on at the end of the synthesis to facilitate purification of the oligomers, as
  • incorporating methylphosphonate oligomers were removed from the solid support by mixing with concentrated ammonium hydroxide for two hours at room temperature.
  • the solution was drained from the support using an Econo-ColumnTM (Bio-Rad, Richmond, CA) and the support was ,washed five times with 1:1 acetonitrile/water.
  • the eluted oligomer was then evaporated to dryness under vacuum at room temperature.
  • the protecting groups were removed from the bases with a solution of ethylenediamine/ethanol/acetonitrile/water (50:23.5:23.5:2.5) for 6 hours at room temperature. The resulting solutions were then evaporated to dryness under vacuum.
  • the phosphate diester oligonucleotide of Example 9 (19 nmoles) was suspended in 115 ⁇ l of 0.15 M HEPES buffer (pH 8.0).
  • NHS-LC-biotin (Pierce Chemical Co., Rockford, IL) was added (10 ⁇ l of a 100 mM solution in dimethylsulfoxide). The solution was heated at 37oC for 30 minutes. Additional NHS-LC-biotin solution was added (10 ⁇ l) and the reaction was continued for 30 minutes at 37oC.
  • the product was then dissolved in water (100 ⁇ l) and purified by reverse-phase HPLC chromatography according to the following method.
  • the HPLC apparatus consisted of a Beckman System Gold Model 126 Solvent Module and Model 167 Detector interfaced to an IBM compatible computer.
  • a PLRP-S column was used (Polymer Laboratories, 8 ⁇ , 300 A pore size, 4.6 mm i.d. x 250 mm long).
  • Buffer B 50% acetonitrile in 50 mM triethylammonium acetate (pH 7) .
  • a linear gradient of 20-60% B was run over 30 minutes at a flow rater of 1.5 ml/minute. Under these conditions, the biotinylated product oligonucleotide eluted at 10.3 minutes.
  • the 32P-labelled biotinylated oligonucleotide (10,000 cpm) was dissolved in 0.5 ml of 50 mM sodium phosphate (pH 6.8), 0.5 M sodium chloride, 2 mM EDTA in a 1.5 ml microcentrifuge tube. Controls were also prepared containing the same components plus l mg/ml biotin (Calbiochem Corp., San Diego, CA) . Next, 50 -1 of steptavidin-agarose (Pierce Cher sal Co., Rockfo: IL) was added to each tube and the contents wt.re mixed on a vcrtexer for 15 minutes.
  • LINKER NON-NUCLEOTIDE MONOMER WITH PSORALEN A C4 linker-modified methylphosphonate oligomer was prepared having the following sequence:
  • HPLC purification of the solution of crude psoralenoligomer conjugate described above was conducted as follows: A Beckman System Gold analytical HPLC system was used with a
  • Buffers used for elution were: Buffer A - 50 mM triethylammonium acetate (pH 7) ; Buffer B - 50% acetonitrile in 50 mM triethylammonium acetate (pH 7).
  • the sample was loaded onto the column in five 100 ⁇ l portions at two minute intervals with a 500 ⁇ l sample loop while the column was flowing at 1.5 ml/min with 10% Buffer B. Next, a linear gradient from 10 - 70% Buffer B was run over 30 minutes. Fractions were collected at 0.5 minute intervals.
  • the psoralen-modified methylphosphonate oligomer prepared according to the procedure of Example 13, was labeled at its 5'-end with 32P as described above (See Example 12).
  • the resulting autoradiograph revealed upper bands for samples containing diester target oligonucleotide which migrated slower in the gel than the bands corresponding to psoralen-modified methylphosphonate oligomer alone.
  • the bands were then excised from the wet gel with a scalpel and counted in a scintillation counter. Based on this method, it was determined that cross-linking of psoralen-modified methylphosphonate oligomer to its complementary diester target was greater than 95%.

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Abstract

L'invention décrit de nouveaux réactifs non nucléotides destinés à s'intégrer à des oligomères, qui possèdent éventuellement un squelette chiralement pur, ainsi que des réactifs non nucléotides qui peuvent s'intégrer à un polymère non nucléotide/nucléotide. Ces réactifs possèdent éventuellement un squelette chiralement pur et peuvent comporter un bras de liaison auquel peut se conjuguer un marqueur détectable ou un agent de réticulation.
PCT/US1991/008769 1990-08-09 1991-08-09 Reactifs de liaison ameliores a base de non nucleotide pour oligomeres WO1992002532A1 (fr)

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JP92502337A JPH05507419A (ja) 1990-08-09 1991-08-09 オリゴマーのための改善された非ヌクレオチド系リンカー試薬
AU90710/91A AU664542B2 (en) 1990-08-09 1991-08-09 Improved non-nucleotide-based linker reagents for oligomers
KR1019930700371A KR930701468A (ko) 1990-08-09 1991-08-09 비-누클레오티드를 기초로 한 올리고머용 링커 시약

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US565,307 1990-08-09

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AU (1) AU664542B2 (fr)
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IE (1) IE912805A1 (fr)
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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0542887A1 (fr) * 1990-08-09 1993-05-26 Genta Incorporated Oligonucleotides de methylphosphonate conjugues avec du psoralene utilises en tant qu'agents therapeutiques contre la leucemie myelogene chronique
WO1994012518A2 (fr) * 1992-11-25 1994-06-09 Gabor Igloi Reactif pour le couplage de diverses substances a des acides nucleiques et procede pour sa preparation
WO1994027615A1 (fr) * 1993-05-26 1994-12-08 Genta Incorporated Conjugaisons oligomeres et leur utilisation
WO1995013833A1 (fr) * 1993-11-16 1995-05-26 Genta Incorporated Oligomeres synthetiques comprenant des liaisons phosphonate internucleosidyle de chiralite indefinie melangees avec des liaisons non phosphonate internucleosidyle
WO1995014030A1 (fr) * 1993-11-16 1995-05-26 Genta Incorporated Oligomeres synthetiques ayant des liaisons internucleosidyle phosphonate chiralement pures melangees avec des liaisons internucleosidyle non phosphonate
WO1995018820A1 (fr) * 1994-01-11 1995-07-13 Isis Pharmaceuticals, Inc. Diols monomeres et oligomeres a liaison phosphate formes a partir de ces derniers
WO1996015105A1 (fr) * 1994-11-15 1996-05-23 Italfarmaco S.P.A. Derives fluorenyl-hydroxamiques possedant une activite immunodepressive et anti-inflammatoire
US5837856A (en) * 1993-11-16 1998-11-17 Genta, Incorporated Chirally enriched synthetic phosphonate oligomers
US5854410A (en) * 1994-03-31 1998-12-29 Genta Incorporated Oligonucleoside cleavage compounds and therapies
US5886177A (en) * 1994-01-11 1999-03-23 Isis Pharmaceuticals, Inc. Phosphate linked oligomers
US5955597A (en) * 1993-11-16 1999-09-21 Genta, Incorporated Chirally enriched synthetic phosphate oligomers
EP0959077A1 (fr) * 1998-05-06 1999-11-24 Tosoh Corporation Sonde optiquement active d'ADN avec une liaison diester phosphonique
US6008398A (en) * 1995-01-18 1999-12-28 Genzyme Corporation Non-nucleotide phosphorus ester oligomers
US6184389B1 (en) 1994-01-11 2001-02-06 Isis Pharmaceuticals, Inc. Combinatorial libraries having aminodiol monomer subunits
US6828427B1 (en) 1994-01-11 2004-12-07 Isis Pharmaceuticals, Inc. Oligomeric aminodiol-containing compounds, libraries thereof, and process of preparing the same
WO2011109398A2 (fr) 2010-03-02 2011-09-09 President And Fellows Of Harvard College Procédés et compositions pour le traitement du syndrome d'angelman et des troubles du spectre autistique
WO2012174446A1 (fr) 2011-06-17 2012-12-20 President And Fellows Of Harvard College Frizzled 2 en tant que cible pour des anticorps thérapeutiques dans le traitement du cancer
WO2013003112A1 (fr) 2011-06-27 2013-01-03 The Jackson Laboratory Procédés et compositions pour le traitement du cancer et d'une maladie auto-immune
US10450334B2 (en) 2013-03-28 2019-10-22 Japan Science And Technology Agency Photoresponsive nucleotide analogue having photocrosslinking ability
US11078211B2 (en) 2017-07-26 2021-08-03 Japan Advanced Institute Of Science And Technology Photoresponsive nucleotide analog capable of photocrosslinking in visible light region
US11214590B2 (en) 2017-07-26 2022-01-04 Japan Advanced Institute Of Science And Technology Photoresponsive nucleotide analog capable of photocrosslinking in visible light region

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US20140178877A1 (en) * 2012-12-20 2014-06-26 Roche Molecular Systems, Inc. Labeled Oligonucleotide Probes Used for Nucleic Acid Sequence Analysis

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WO1986000074A1 (fr) * 1984-06-15 1986-01-03 Institut National De La Sante Et De La Recherche M Acides nucleiques marques chimiquement, leur utilisation et necessaire pour sa mise en oeuvre
US4757141A (en) * 1985-08-26 1988-07-12 Applied Biosystems, Incorporated Amino-derivatized phosphite and phosphate linking agents, phosphoramidite precursors, and useful conjugates thereof

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FR2642074B1 (fr) * 1989-01-20 1994-04-29 Oris Ind Derives de molecules polyhydroxylees permettant l'introduction d'au moins une ramification dans un oligonucleotide

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WO1986000074A1 (fr) * 1984-06-15 1986-01-03 Institut National De La Sante Et De La Recherche M Acides nucleiques marques chimiquement, leur utilisation et necessaire pour sa mise en oeuvre
US4757141A (en) * 1985-08-26 1988-07-12 Applied Biosystems, Incorporated Amino-derivatized phosphite and phosphate linking agents, phosphoramidite precursors, and useful conjugates thereof

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Cited By (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0542887A4 (en) * 1990-08-09 1993-09-08 Genta Incorporated Psoralen conjugated methylphosphonate oligonucleotides as therapeutic agents for chronic myelogenous leukemia
EP0542887A1 (fr) * 1990-08-09 1993-05-26 Genta Incorporated Oligonucleotides de methylphosphonate conjugues avec du psoralene utilises en tant qu'agents therapeutiques contre la leucemie myelogene chronique
WO1994012518A2 (fr) * 1992-11-25 1994-06-09 Gabor Igloi Reactif pour le couplage de diverses substances a des acides nucleiques et procede pour sa preparation
WO1994012518A3 (fr) * 1992-11-25 1994-07-21 Gabor Igloi Reactif pour le couplage de diverses substances a des acides nucleiques et procede pour sa preparation
WO1994027615A1 (fr) * 1993-05-26 1994-12-08 Genta Incorporated Conjugaisons oligomeres et leur utilisation
US5955597A (en) * 1993-11-16 1999-09-21 Genta, Incorporated Chirally enriched synthetic phosphate oligomers
WO1995013833A1 (fr) * 1993-11-16 1995-05-26 Genta Incorporated Oligomeres synthetiques comprenant des liaisons phosphonate internucleosidyle de chiralite indefinie melangees avec des liaisons non phosphonate internucleosidyle
WO1995014030A1 (fr) * 1993-11-16 1995-05-26 Genta Incorporated Oligomeres synthetiques ayant des liaisons internucleosidyle phosphonate chiralement pures melangees avec des liaisons internucleosidyle non phosphonate
US6028188A (en) * 1993-11-16 2000-02-22 Genta Incorporated Synthetic oligomers having chirally pure phosphonate internucleosidyl linkages mixed with non-phosphonate internucleosidyl linkages
US5792615A (en) * 1993-11-16 1998-08-11 Genta, Incorporated Synthetic oligomers having chirally pure phoshonate internucleosidyl linkages mixed with non-phosphonate internucleosidyl linkages
US5837856A (en) * 1993-11-16 1998-11-17 Genta, Incorporated Chirally enriched synthetic phosphonate oligomers
US5986083A (en) * 1993-11-16 1999-11-16 Genta, Inc. Synthetic oligomers having phosphonate internucleosidyl linkages of undefined chirality mixed with non-phosphonate internucleosidyl linkages
WO1995018820A1 (fr) * 1994-01-11 1995-07-13 Isis Pharmaceuticals, Inc. Diols monomeres et oligomeres a liaison phosphate formes a partir de ces derniers
US6448373B1 (en) 1994-01-11 2002-09-10 Isis Pharmaceuticals, Inc. Phosphate linked oligomers formed of monomeric diols and processes for preparing same
US6828427B1 (en) 1994-01-11 2004-12-07 Isis Pharmaceuticals, Inc. Oligomeric aminodiol-containing compounds, libraries thereof, and process of preparing the same
US6184389B1 (en) 1994-01-11 2001-02-06 Isis Pharmaceuticals, Inc. Combinatorial libraries having aminodiol monomer subunits
US5886177A (en) * 1994-01-11 1999-03-23 Isis Pharmaceuticals, Inc. Phosphate linked oligomers
US5854410A (en) * 1994-03-31 1998-12-29 Genta Incorporated Oligonucleoside cleavage compounds and therapies
WO1996015105A1 (fr) * 1994-11-15 1996-05-23 Italfarmaco S.P.A. Derives fluorenyl-hydroxamiques possedant une activite immunodepressive et anti-inflammatoire
US6008398A (en) * 1995-01-18 1999-12-28 Genzyme Corporation Non-nucleotide phosphorus ester oligomers
EP1340766A1 (fr) * 1998-05-06 2003-09-03 Tosoh Corporation Sonde optiquement active d'ADN avec une liaison diester phosphonique
US6211354B1 (en) 1998-05-06 2001-04-03 Tosch Corporation Optically active DNA probe having phosphonic diester linkage
EP0959077A1 (fr) * 1998-05-06 1999-11-24 Tosoh Corporation Sonde optiquement active d'ADN avec une liaison diester phosphonique
WO2011109398A2 (fr) 2010-03-02 2011-09-09 President And Fellows Of Harvard College Procédés et compositions pour le traitement du syndrome d'angelman et des troubles du spectre autistique
WO2012174446A1 (fr) 2011-06-17 2012-12-20 President And Fellows Of Harvard College Frizzled 2 en tant que cible pour des anticorps thérapeutiques dans le traitement du cancer
EP3281640A1 (fr) 2011-06-17 2018-02-14 President and Fellows of Harvard College Frizzled 2 en tant que cible pour des anticorps thérapeutiques dans le traitement du cancer
WO2013003112A1 (fr) 2011-06-27 2013-01-03 The Jackson Laboratory Procédés et compositions pour le traitement du cancer et d'une maladie auto-immune
US10450334B2 (en) 2013-03-28 2019-10-22 Japan Science And Technology Agency Photoresponsive nucleotide analogue having photocrosslinking ability
US11078211B2 (en) 2017-07-26 2021-08-03 Japan Advanced Institute Of Science And Technology Photoresponsive nucleotide analog capable of photocrosslinking in visible light region
US11214590B2 (en) 2017-07-26 2022-01-04 Japan Advanced Institute Of Science And Technology Photoresponsive nucleotide analog capable of photocrosslinking in visible light region

Also Published As

Publication number Publication date
IL99068A (en) 1998-01-04
CA2089087A1 (fr) 1992-02-10
JPH05507419A (ja) 1993-10-28
EP0542928A1 (fr) 1993-05-26
AU9071091A (en) 1992-03-02
IL99068A0 (en) 1992-07-15
NZ239250A (en) 1993-12-23
AU664542B2 (en) 1995-11-23
EP0542928A4 (en) 1994-07-27
IE912805A1 (en) 1992-02-12
KR930701468A (ko) 1993-06-11

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