WO2001084234A1 - Procede d'immobilisation d'oligonucleotides au moyen du procede de bioconjugaison de cycloaddition - Google Patents

Procede d'immobilisation d'oligonucleotides au moyen du procede de bioconjugaison de cycloaddition Download PDF

Info

Publication number
WO2001084234A1
WO2001084234A1 PCT/US2001/013956 US0113956W WO0184234A1 WO 2001084234 A1 WO2001084234 A1 WO 2001084234A1 US 0113956 W US0113956 W US 0113956W WO 0184234 A1 WO0184234 A1 WO 0184234A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
diene
oligonucleotide
maleimide
reaction
Prior art date
Application number
PCT/US2001/013956
Other languages
English (en)
Inventor
Wolfgang Pieken
Andreas Wolter
David P. Sebesta
Michael Leuck
Hallie A. Latham-Timmons
John Pilon
Gregory M. Husar
Original Assignee
Proligo Llc
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 Proligo Llc filed Critical Proligo Llc
Priority to JP2001580595A priority Critical patent/JP2003535317A/ja
Priority to AU2001259285A priority patent/AU2001259285A1/en
Priority to EP01932784A priority patent/EP1287404A4/fr
Publication of WO2001084234A1 publication Critical patent/WO2001084234A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C215/00—Compounds containing amino and hydroxy groups bound to the same carbon skeleton
    • C07C215/02—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C215/04—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated
    • C07C215/06—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and acyclic
    • C07C215/10—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and acyclic with one amino group and at least two hydroxy groups bound to the carbon skeleton
    • 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
    • C07C271/16—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 singly-bound oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic System
    • C07F7/02—Silicon compounds
    • C07F7/08—Compounds having one or more C—Si linkages
    • C07F7/18—Compounds having one or more C—Si linkages as well as one or more C—O—Si linkages
    • C07F7/1804—Compounds having Si-O-C linkages
    • 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 System
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/22—Amides of acids of phosphorus
    • C07F9/24—Esteramides
    • C07F9/2404—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/2408—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic of hydroxyalkyl compounds
    • 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
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
    • C40B50/18—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support using a particular method of attachment to the solid support
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11—Compounds covalently bound to a solid support
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated

Definitions

  • This invention describes a novel method for immobilizing molecules on a support. Particularly, this invention describes a method for immobilizing biomolecules on a support using cycloaddition reactions, such as the Diels-Alder reaction.
  • oligonucleotide probes that employ Watson-Crick hybridization in the interaction with target nucleic acids such as genomic DNA, RNA or cDNA prepared via Polymerase Chain Reaction (PCR) amplification of sample DNA.
  • PCR Polymerase Chain Reaction
  • Current technologies often involve formatting oligonucleotide probes for such analyses into microarrays on glass slides, silicon chips or wafers, micro titer plates or other supports including polyacrylamide gel matrices.
  • Cycloaddition reactions can be defined as any reaction between two (or more) moieties (either intra or intermolecular) where the orbitals of the reacting atoms form a cyclic array as the reaction progresses (typically in a concerted fashion although intermediates may be involved) along the reaction coordinate leading to a product.
  • the orbitals involved in this class of reactions are typically ⁇ systems although certain ⁇ orbitals can also be involved.
  • Typical examples of cycloaddition reactions include Diels-Alder cycloaddition reactions, 1,3-dipolar cycloadditions and [2+2] cyclo additions.
  • the Diels-Alder reaction by far the most studied cycloaddition, is the cycloaddition reaction between a conjugated diene and an unsaturated molecule to form a cyclic compound with the ⁇ -electrons being used to form the new ⁇ -bonds.
  • the Diels- Alder reaction is an example of [4+2] cycloaddition reaction, as it involves a system of 4 ⁇ - electrons (the diene) and a system of 2 ⁇ -(the dienophile).
  • the reaction can be made to occur very rapidly, under mild conditions, and for a wide variety of reactants.
  • the Diels- Alder reaction is broad in scope and is well known to those knowledgeable in the art.
  • a review of the Diels-Alder reaction can be found in "Advanced Organic Chemistry” (March, J., ed.) 839-852 (1992) John Wiley & Sons, NY, which is incorporated herein by reference. It has been discovered that the rate of Diels-Alder cycloaddition reactions is enhanced in aqueous solvents. (Rideout and Breslow (1980) J. Am. Chem. Soc. 102:7816).
  • Bioconjugation of Macromolecules illustrates that cycloaddition reactions in general, such as the Diels-Alder reaction and 1,3-dipolar cycloaddition reactions, are an ideal replacement for current methods of conjugating macromolecules with other molecular moieties.
  • the Diels-Alder reaction in particular, is an ideal method for covalently linking large water soluble macromolecules with other compounds as the reaction rate is accelerated in water and can be run at neutral pH. (Rideout and Breslow (1980) J. Am. Chem. Soc. 102:7816). Additionally, the nature of the reaction allows post-synthetic modification of the hydrophilic macromolecule without excess reagent or hydrolysis of the reagent.
  • conjugation to oligonucleotides With respect to conjugation to oligonucleotides, this technology has been aided by the ability to efficiently synthesize 2 -O-diene-nucleosides, which allows the conjugation site to be varied throughout the oligonucleotide or the option of having multiple conjugation sites.
  • the present invention describes a method for immobilizing molecules, particularly biomolecules, to a support using the cycloaddition bioconjugation method.
  • Immobilization of biomolecules via cycloaddition, particularly Diels-Alder reactions, offers the following major advantages over conventional methods (cf.
  • cycloaddition reactions establish a covalent and stable linkage between the linked compounds; the reaction proceeds with high chemoselectivity; functional groups of biomolecules do not interfere with the cycloaddition reaction; the cycloaddition reaction is orthogonal to other immobilization/labeling protocols, thus two-fold reactions are possible in one reaction mixture; in contrast to general techniques in organic synthesis, as discussed above, Diels-Alder reactions, can be carried out in aqueous phase, the Diels-Alder reaction is tremendously accelerated in water and is very fast at room temperature or slightly below; the cycloaddition reaction proceeds under neutral conditions in a one-step procedure; no by-products are formed during the reaction; no activators or additives are necessary to run the reaction and the moieties involved in the reaction (dienes and dienophiles) are stable under various reaction conditions employed for conjugation or immobilization of biomolecules
  • the present invention describes a novel, chemoselective and highly efficient method for immobilizing molecules using cycloaddition reactions.
  • the method of the invention offers advantages over existing immobilization methods that can suffer from cross-reactivity, low selectivity, mechanistic ambiguity and competitive hydrolysis of reactive groups.
  • the method of the instant invention comprises the step of reacting a derivatized molecule with a derivatized support capable of reacting with said derivatized molecule via a cycloaddition reaction.
  • the derivatized molecule is a biomolecule, preferably an oligonucleotide, but could also be a hapten, carbohydrate, oligosacchari.de, peptide and protein (including an antibody).
  • the support is preferably glass or controlled pore glass (CPG), but could also be polypropylene, polystyrene, polyacrylamide or silicon.
  • the cycloaddition reaction is a Diels-Alder cycloaddition reaction between the support and the biomolecule. Therefore, the biomolecule is preferably derivatized with one component of a Diels-Alder reaction, i.e., a diene or a dienophile, and the support is derivatized with the appropriate counterpart reactant, i.e., a dienophile or a diene, respectively.
  • This invention includes a reaction scheme for producing a wide variety of immobilized biomolecules using cycloaddition reactions as typified by the Diels-Alder cycloaddition reaction.
  • the method of this invention can be extended to the immobilization of any molecule, particularly biomolecules on any support that can be appropriately derivatized.
  • the method of the invention is applicable to the fields of biomolecule array fabrication for research, development and clinical diagnostic applications relating to nucleic acid sequencing, gene expression profiling, analysis of single nucleotide polymorphisms (SNPs) and evaluation of hapten-antibody or ligand-target interactions.
  • SNPs single nucleotide polymorphisms
  • Figure 1 is a fluorescence scan of polyacrylamide gel loaded with 1, 2 and 3 ⁇ L of fluorescein labeled compound (26), the synthesis of which is described in Example 6.
  • Figure 2 is a fluorescence scan of a glass slide showing maleimide functionalization on the lower half of the slide, that was treated with maleimide-silane (19), followed by reaction with a thiol containing-fluorescein reagent (SAMSA-reagent), as described in Example 7.
  • SAMSA-reagent a thiol containing-fluorescein reagent
  • Figure 3 is a fluorescence scan of maleimide derivatized CPG.
  • a native CPG was first treated with maleimide silane (19), followed by staining with SAMSA- reagent, hi Figure 3B native CPG was treated with SAMSA-fluorescein to serve as a control.
  • Figure 4 is a fluorescence scan of a glass slide showing diene functionalization on the lower half of the slide that was treated with diene-silane reagent (20), followed by reaction of the diene functionalized slide with fluorescein-5-maleimide, as described in Example 8.
  • Figure 5 is a fluorescence scan of diene functionalized CPG.
  • a CPG was treated with diene-silane (20), followed by staining with fiuorescein-5-maleimide.
  • Figure 5B native CPG was treated with fluorescein-5-maleimide to serve as a control.
  • Figure 6 shows a diagram of a slide showing placement of septa and contents of septa for demonstration of Diels-Alder surface immobilization of an oligonucleotide on maleimide functionalized glass micro slides as described in Example 9.
  • Figure 7 illustrates fluorescence scans of glass slides showing successful Diels-
  • Figure 8 illustrates the conjugation of diene-oligonucleotide (23) to maleimide- coated micro titer plates after hybridization with complementary fluorescein labeled sequence 5 , -fluorescein-(CA) 10 (27) (SEQ JO NO:2).
  • Wells 1-3 were treated with diene- oligonucleotide (23), pure, and wells 4-6 were treated with diene-oligonucleotide (23), crude.
  • Wells 1 A-6A are the corresponding oligonucleotide (22) controls.
  • Wells 7 and 7A are the buffer controls.
  • Figure 9 illustrates graphically the relation between the loading of CPG (10 mg) with oligonucleotide (24) and the concentration of oligonucleotide (24) in solution.
  • Figure 10 illustrates graphically the relation between the loading of CPG (10 mg) with oligonucleotide and the incubation time with oligonucleotide (24).
  • Figure 11 illustrates fluorescence scans of glass slides showing successful Diels- Alder surface immobilization of maleimide-ohgonucleotide (26) on diene functionalized glass micro slides.
  • Slide “1” is prior to the wash with phosphate buffered saline (PBS) illustrating the necessity of the wash to remove non-covalently bound oligonucleotide from the glass surface.
  • Slide “2” is after the PBS wash.
  • the single fluorescent response visible on the slide is where compound (26) came into contact with the diene- functionalized portion of slide prior to hybridization with complementary-5'-fluorescein- oligonucleotide (27). After the PBS wash, areas of the slide that came into contact with controls showed no response.
  • Figure 12 illustrates fluorescence scans of CPG samples showing Diels-Alder surface immobilization of an oligonucleotide (maleimide-ohgonucleotide (26)) on diene- functionalized CPG.
  • Figure 12A is prior to the wash with a mixture of 5x0.3 M sodium citrate and 3 M sodium chloride (SSC) and Figure 12B is after the SSC wash.
  • SSC sodium citrate
  • Figure 12B is after the SSC wash.
  • the fluorescent response visible on the sample labeled "2A” is where compound (26) came into contact with diene-functionalized CPG prior to hybridization with complementary 5'- fluorescein-oligonucleotide (27).
  • the control samples labeled "2B” and "2C” showed relatively little response.
  • Figure 13 is an overlaid Biacore sensorgram illustrating the reproducibility of the formation of the maleimide-coated BIAcore flow cell surface (37) described in Example 15.
  • Figure 14 is a Biacore sensorgram of the product of the Michael-addition between maleimide-coated BIAcore flow cell surface (37) and MeO-PEG-SH, which is described in Example 16.
  • Figure 15 is a Biacore sensorgram of the product of the Diels-Alder reaction between maleimide-coated BIAcore flow cell surface (37) and PEG-diene substrate (34), described in Example 16.
  • Figure 16 is a Biacore sensorgram of the product of the Diels-Alder reaction between maleimide-coated BIAcore flow cell surface (37) and PEG-anthracene substrate (36), described in Example 17.
  • Figure 17 is a Biacore sensorgram of the product of the Diels-Alder reaction between maleimide-coated BIAcore flow cell surface (37) and cyclohexadiene modified oligonucleotide (29), described in Example 18.
  • Figure 18 is a Biacore sensorgram of the product of the Diels-Alder reaction between maleimide-coated BIAcore flow cell surface (37) and cyclohexadiene modified oligonucleotide (29), upon hybridization of the immobilized sequence with its complementary oligonucleotide sequence.
  • Figure 19 illustrates the functionalization of glass microscope slides with anthracene-silane reagent (42).
  • the present invention includes a method for immobilizing molecules on a support.
  • the present invention describes the use of cycloaddition reactions, in particular the Diels-Alder cycloaddition reaction for the chemoselective immobilization of molecules on a support.
  • the method of the instant invention comprises the step of reacting a derivatized molecule with a derivatized support capable of reacting with said derivatized molecule via a cycloaddition reaction.
  • the derivatized molecule is a biomolecule, most preferably an oligonucleotide and the support is preferably glass or controlled pore glass (CPG).
  • the cycloaddition reaction is a Diels-Alder cycloaddition reaction. Therefore, the biomolecule is derivatized with one component of a Diels-Alder reaction, i.e., a diene or a dienophile, and the support is derivatized with the appropriate counterpart reactant, i.e., a dienophile or a diene, respectively.
  • This invention includes a reaction scheme for producing a wide variety of immobilized biomolecules using cycloaddition reactions as typified by the Diels-Alder cycloaddition reaction.
  • the method of this invention can be used to immobilize any molecule, particularly biomolecules, on any support that can be appropriately derivatized.
  • Certain terms used to describe the invention are described herein as follows: "Oligonucleotide” refers to a polynucleotide formed from a plurality of linked nucleotide units. The nucleotide units each include a nucleoside unit linked together via a phosphate linking group.
  • oligonucleotide also refers to a plurality of nucleotides that are linked together via linkages other than phosphate linkages such as phosphorothioate linkages.
  • the oligonucleotide may be naturally occurring or non- naturally occurring.
  • the oligonucleotides of this invention have between 1-1,000 nucleotides.
  • nucleobase will have the following definition.
  • a nucleobase is a purine or a pyrimidine base.
  • Nucleobase includes all purines and pyrimidines currently known to those skilled in the art or any chemical modifications thereof.
  • the purines are attached to the ribose ring through the nitrogen in the 9 position of the purine ring and the pyrimidines are attached to the ribose ring through the nitrogen in the 1 position of the pyrimidine ring.
  • the pyrimidine can be modified at the 5- or 6- position of the pyrimidine ring and the purine can be modified at positions 2-, 6- or 8- of the purine ring.
  • nucleobase includes, but is not limited to, uracil, cytosine, N4-protected cytosine, 4- thiouracil, isocytosine, 5-methyluracil (thymine), 5-substiruted uracils, adenine, N6- protected adenine, guanine, N2-protected guanine, 2,6-diaminopurine, halogenated purines as well as heterocycles meant to mimic the purine or pyrimidine ring, such as imidazole.
  • a “diene” is defined as a molecule bearing two conjugated double bonds. The diene may even be non-conjugated, if the geometry of the molecule is constrained so as to facilitate a cycloaddition reaction (Cookson (1964) J. Chem. Soc. 5416). The atoms forming these double bonds can be carbon or a heteroatom or any combination thereof.
  • a "dienophile” is defined as a molecule bearing an alkene group, or a double bond between a carbon and a heteroatom, or a double bond between two heteroatoms.
  • the dienophile can be any group, including but not limited to, a substituted or unsubstituted alkene, or a substituted or unsubstituted alkyne.
  • the groups attached to the alkene unit can be electron donating groups, including but not limited to phenyl rings, conjugated double bonds, alkyl groups, OMe groups or other X-alkyl moieties wherein X is an electron donating group (these type of dienophiles undergo cycloadditions that are known generally as reverse electron demand cycloadditions).
  • molecules bearing a primary amino group such as amino acids or a lysine containing peptide
  • the latter undergo Diels-Alder cycloaddition with macromolecules bearing a diene group under mild conditions in aqueous solvents.
  • a "1,3-dipole” is defined as a compound that contains a consecutive series of three atoms, a-b-c, where atom a contains a sextet of electrons in its outer shell and atom c contains an octet with at least one unshared pair of electrons in its outer shell. Because molecules that have six electrons in the outer shell of an atom are typically unstable, the a- b-c atom example is actually one canonical structure of a resonance hybrid, where at least one structure can be drawn. 1,3-dipoles can be divided into two main groups:
  • 1,3-dipoles include, but are not limited to nitrile ylids, nitrile imines, nitrile oxides, diazoalkanes, azides, azomethine ylids, azomethine imines, nitrones, carbonyl ylids, carbonyl imines and carbonyl oxides.
  • a "1,3-dipolarophile” is defined in the same manner as a “dienophile” or “diene” (as described above).
  • the macromolecule can be attached to either (or both) the 1,3- dipole or the 1,3-dipolarophile.
  • a "1,3-dipolar cycloaddition reaction” can be generally represented as follows:
  • An “Ene reaction” can be generally represented as follows:
  • an "ene” is defined in the same manner as a “dienophile” (see the above description for dienophile).
  • An “ene” can be any unsaturated group, including but not limited to, a substituted or unsubstituted alkene, or a substituted or unsubstituted alkyne.
  • the macromolecule can be attached to either (or both) the ene component or the enophile component.
  • Bioconjugate refers to any macromolecule which has been derivatized with another molecular entity.
  • Bioconjugation or “Conjugation” refers to the derivatization of a macromolecule with another molecular entity.
  • a “support” refers to glass, including but not limited to controlled pore glass (CPG), glass slides, glass fibers, glass disks, materials coated with glass, silicon chips and wafers including, but not limited to metals and composites containing glass; polymers/resins, including but not limited to polystyrene (PS), polyethylene glycol (PEG), copolymers of PS and PEG, copolymers of polyacrylamide and PEG, copolymers containing maleimide or maleic anhydride, polyvinyl alcohol and non-immuno genie high molecular weight compounds; and large biomolecules, including but not limited to polysaccharides, such as cellulose, proteins and nucleic acids.
  • CPG controlled pore glass
  • PEG polyethylene glycol
  • copolymers of PS and PEG copolymers of polyacrylamide and PEG
  • copolymers containing maleimide or maleic anhydride polyvinyl alcohol and non-immuno genie high molecular weight compounds
  • the support can be, but is not necessarily, a solid support.
  • immobilization refers to the attachment, via covalent bond, to a support. Immobilization includes a functionality on the support or a derivatized support.
  • functionality refers to functional groups, including but not limited to alcohols, carboxylates, amines, sulfonic acids and halides, that allow the attachment of one component of the cycloaddition system (e.g. a diene or a dienophile).
  • cycloaddition system e.g. a diene or a dienophile
  • derivatized refers to molecules and/or supports that have been functionalized with a moiety capable of undergoing a cycloaddition reaction.
  • a molecule or support that bears a moiety capable of undergoing a cycloaddition reaction without functionalization also falls within this definition.
  • moieties capable of undergoing a cycloaddition reaction include but are not limited to a diene, dienophile, 1,3- dipole, 1,3-dipolarophile, ene, enophile or other moiety capable of undergoing a cycloaddition reaction.
  • molecule includes, but is not limited to biomolecules, macromolecules, diagnostic detector molecules (DDM's) and other small molecules, particularly small molecules for use in combinatorial chemistry.
  • biomolecules include, but are not limited to nucleic acids, oligonucleotides, proteins, peptides and amino acids, polysaccharides and saccharides, glycoproteins and glycopeptides (in general, glycoconjugates) alkaloids, lipids, hormones, drugs, prodrags, antibodies and metabolites.
  • macromolecules refers to the product of the coupling of two macromolecules via cycloaddition.
  • DDM Diagnostic detector molecules
  • DDM's include, but are not limited to fluorescent, chemiluminescent, radioisotope and bioluminescent marker compounds; antibodies, biotin and metal chelates.
  • cycloaddition reaction refers to any reaction that occurs between two reactants by a reorganization of valence electrons through an activated complex, which is usually a cyclic transition state.
  • the orbitals involved in this class of reactions are typically ⁇ -systems although certain ⁇ -orbitals can also be involved.
  • cycloaddition reactions include, but are not limited to [1+2] -cycloaddition, such as reaction between carbenes and olefins, [2+2] -cycloaddition, such as reaction between olefins or reaction between ketenes and olefins, [3+2]- cycloaddition, such as 1,3-dipolar cycloaddition, [2+4] -cycloaddition, such as the Diels- Alder reaction and ene reaction, [4+6] -cycloaddition, and cheleotropic reactions.
  • [1+2] -cycloaddition such as reaction between carbenes and olefins
  • [2+2] -cycloaddition such as reaction between olefins or reaction between ketenes and olefins
  • [3+2]- cycloaddition such as 1,3-dipolar cycloaddition
  • Types of reactants involved in cycloaddition reactions include, but are not limited to, olefins, including but not limited to alkenes, dienes etc with or without heteroatoms, alkynes, with and without heteroatoms, aromatic compounds, such as anthracene, 1,3-dipoles, carbenes and carbene-precursors.
  • the "derivatized oligonucleotides” of this invention are generally represented by the following formulas:
  • B is a nucleobase
  • a and A' are 2'-sugar substituents;
  • W is independently selected from the group consisting of an oligonucleotide having between 1-1000 nucleobases, X or H;
  • X is a diene, dienophile, 1,3-dipole, 1,3 dipolarophile, ene, enophile, alkene, alkyne or other moiety capable of undergoing a cycloaddition reaction, additionally when X is attached to nucleobase B it can be attached to a carbon atom, an exocyclic nitrogen or an exocyclic oxygen.
  • R 1 is selected from the group consisting of H and an alcohol protecting group
  • R 3 is selected from the group consisting of R 2 , R 4 , CN, C(O)NH 2 , C(S)NH 2 , C(O)CF 3 , SO 2 R 4 , amino acid, peptide and mixtures thereof;
  • R 4 is selected from the group consisting of an optionally substituted hydrocarbon
  • A is selected from the group consisting of H, OH, NH 2 , Cl, F, NHOR 3 , OR 4 , OSiR 4 3 .
  • alkyl groups on all the above listed moieties can have between 1-50 carbons, preferably 1-30 carbons.
  • linking molecule is a molecular entity that connects two or more molecular entities through covalent interactions. More specifically a linking molecule is a multifunctional molecule that can be used to derivatize a molecule or support with a diene, dienophile or other moiety capable of undergoing a cycloaddition reaction.
  • the linking molecules of this invention are generally represented by the following formulas:
  • X is as defined above; n is an integer from 1-20; and
  • L is a linker which includes, but is not limited to, compounds of the following general formula:
  • Y is selected from NH, O, NH(CO)O, NH(CS)O, NH(CO)NH, NH(CO), S-S-S-, Si(OR) 3 and SiR 2 wherein
  • R is selected from alkyl, aryl, substituted alkyl or substituted aryl, each having between 1-50 carbon atoms.
  • substituents described above are also included within the scope of this invention, which is not limited to the specific, but rather the generalized formula of reaction.
  • Cycloaddition reactions are uniquely suited as a general method for the immobilization of molecules, particularly biomolecules to a support.
  • the cycloaddition of a diene and a dienophile is highly chemoselective and only a suitably electronically configured diene and dienophile pair will react.
  • the reaction proceeds under mild conditions in a reasonable time-frame.
  • Biomolecules such as nucleic acids, oligonucleotides, proteins, peptides, carbohydrates, polysaccharides, glycoprotems, antibodies and lipids generally do not contain moieties that can undergo such a cycloaddition reaction.
  • the biomolecule is an oligonucleotide.
  • the solvent of choice for the derivatization of oligonucleotides is water, due to the highly anionic nature of these molecules.
  • an optimal reaction for the immobilization of such groups proceeds readily in water, and displays no side reactions with water, such as hydrolysis of any of the reactants.
  • this disclosure describes the use of Diels-Alder cycloadditions for the chemoselective and efficient immobilization oligonucleotides on a support.
  • an oligonucleotide bearing either a diene modified nucleoside or non- nucleoside phosphate diester group, or a dienophile modified nucleoside or non-nucleoside phosphate diester group can be reacted with a support bearing either a dienophile or a diene moiety, respectively.
  • the diene or dienophile moiety can be incorporated into the oligonucleotide at any position in the chain, for instance, by introduction of a 5-(3,5-hexadiene)-2'-deoxyuridine nucleoside (See U.S. Patent Application Serial No.
  • the diene or dienophile moiety can be introduced as a 2'-O- (3,5-hexadiene)-uridine nucleoside.
  • a diene moiety can also be introduced to the oligonucleotide as a diene-bearing non-nucleoside phosphoramidite, such as 3,5- hexadiene-N,N-diisopropyl-2-cyanoethyl phosphoramidite.
  • the diene modified oligonucleotide such as a 5'-terminal 3,5-hexadienephosphate oligonucleotide
  • the dienophile modified support such as maleimide derivatized glass
  • the method of this invention can be extended to the immobilization of any molecule that can be derivatized with a diene, dienophile or other reactive group capable of undergoing a cycloaddition reaction without limitation.
  • the method can be extended to the immobilization of peptides and proteins with any support capable or being derivatized.
  • a peptide or protein that contains an amino acid building block which has been derivatized with the diene or dienophile, such as O-3.5-hexadiene-tyrosine or serine, or N-maleimidolysine, can be immobilized on any support using the method described herein, without limitation.
  • Natural molecules, such as proteins, can be derivatized with a diene or dienophile bearing a heterobifunctional crosslinking reagent, such as the NHS ester of 3-(4-maleimidophenyl)-propionic acid (Pierce), which allows subsequent conjugation to a support bearing a corresponding diene or dienophile group.
  • Polyethylene glycol is often conjugated to biomolecules to reduce their immunogenicity and to increase their residence time in vitro.
  • the method of this invention allows immobilization of biomolecules, such as oligonucleotides or peptides, bearing a diene, dienophile or other reactive group capable of undergoing a cycloaddition reaction with another polymer or resin, such as polyethyleneglycol or polystyrene bearing one or several corresponding diene, dienophile or other groups capable of undergoing cycloaddition reactions.
  • Example 1 describes the synthesis of 5-hydroxymethylcyclohexa-l,3- diene (5).
  • Example 2 describes the preparation of NHS reagent (10) from cyclohexadiene alcohol (5).
  • Example 3 describes the synthesis of the diene amidite linker (16) from amino-linker (12).
  • Amino-linker (12) was developed in mimesis of a nucleoside containing a primary (as 5'-OH) and a secondary hydroxyl group (as 3'-OH). This allows the conjugation of the linker on either the 3'- or 5'-end of any oligonucleotide.
  • oligonucleotide e.g., dienes or dyes
  • linker the linker's amino functionality. Therefore, compound (12) is recognized as a universal linker for conjugation of oligonucleotides.
  • Example 4 describes the synthesis of maleimide-trialkoxy silane (19), which is used to functionalize glass surfaces.
  • Example 5 describes the synthesis of diene-trialkoxy silane (20) for the functionalization of glass surfaces.
  • Example 6 describes the synthesis of three 5' functionalized oligonucleotides, compounds (23), (24) and (26).
  • Compounds (23) and (23) (Scheme 6) are functionalized at the 5' end with a diene by reaction with diene-linker amidite (16).
  • Compound (26) (Scheme 7) is functionalized at the 5' end with a maleimide by reaction with maleimidopropionic acid N-hydroxysuccimide ester (compound 18, Scheme 4).
  • Examples 7 and 8 describe a method for the functionalization of two glass surfaces, specifically glass slides and CPG, with both a diene and a dienophile.
  • Example 7 describes the functionalization of the two glass surfaces with maleimide dienophile (19) (Scheme 8).
  • Example 7 also describes methods for the detection of the maleimide functionalized surfaces.
  • the maleimide derivatized surfaces are detected by staining with a thiol-fluorescein reagent, which reacts with the maleimide via a Michael-addition as illustrated in Scheme 9.
  • the presence of fluorescem bound to the surfaces is then detected with a Molecular Dynamics' Tyhpoon Fluorescence Scanner using a green laser to excite the surface-bound fluorescein followed by detection of emission using a 526 nm filter.
  • Example 8 describes the functionalization of the two glass surfaces with diene (20) (Scheme 10).
  • Example 8 also describes methods for the detection of the functionalized surfaces.
  • the diene derivatized surfaces are detected by Diels-Alder reaction with a fluorescein maleimide as illustrated in Scheme 11.
  • Example 9 describes the Diels-Alder reaction between the 5' diene functionalized oligonucleotide (23) and maleimide functionalized glass slides and microtiter plates. This reaction demonstrates the Diels-Alder surface immobilization of an oligonucleotide (Scheme 12). Detection of the surface-bound oligonucleotide was achieved by its hybridization to a fluorescein-labeled complementary sequence (27) and detection of fluorescence. The results of this Example are set forth in Figure 8.
  • Example 10 describes the Diels-Alder reaction between maleimide-CPG and diene derivatized oligonucleotide (24).
  • Example 9 the conjugation of the diene- oligonucleotide to the maleimide derivatized support was detected by the fluorescence of a labeled complementary sequence after hybridization. This method of detection, however, gives a qualitative result, but does not allow the quantitative determination of surface loading.
  • a different detection method was selected for determination of immobilized oligonucleotide in order to obtain a quantitative determination of surface loading.
  • diene(DMT)-oligonucleotide (24) was used.
  • the surface bound amount of oligonucleotide (loading) is calculated after the cleavage of the DMT-group by photometric detection of the DMT-cation as illustrated in Scheme 12.
  • Maleimide-derivatized CPG was incubated with increasing amounts of oligonucleotide (24) in order to demonstrate the relation between oligonucleotide immobilization (as CPG loading) and the amount of diene-oligonucleotide in solution.
  • the typical range of oligonucleotide loading was calculated to be 0.8- 1.7 ⁇ mol/g (Table 2, Figure 9).
  • the results obtained from this experiment also showed that not all maleimide-sites were reacted with diene-oligonucleotide and that the loading can be further increased be treatment with larger amounts of (24) by achieving an exponential dependence instead of a linear curve fit. The kinetic relationship between loading and incubation time was also demonstrated.
  • Example 11 describes the conjugation of 5'-maleimide derivatized oligonucleotide (26) to diene coated glass surfaces. The demonstration of conjugation was achieved by hybridization with the labeled complementary sequence (27) and the detection of fluorescence as described above.
  • Example 12 describes the synthesis of diene modified oligonucleotide (29) from NHS reagent (10), the synthesis of which is described in Example 2.
  • Example 13 describes the HPLC monitoring of a Diels-Alder reaction employing a cyclohexadiene derivatized oligonucleotide.
  • Diels-Alder reactivity of diene conjugate (29) labeling with commercially available maleimide dienophiles (30) and (31) were carried out. The progress of the reaction was monitored by analytical anion exchange chromatography with samples taken every 5 minutes. Treatment of (29) with N-ethyl maleimide (30) resulted in complete conversion to adduct (32) within 5 minutes, while biotin maleimide 31 required 20 minutes.
  • Example 14 (Scheme 17) describes the synthesis of diene modified polyethylene glycol substrates (34).
  • Example 14 (Scheme 18) also describes the synthesis of anthracene derivative (36).
  • Example 15 (Scheme 19) describes the preparation of a dienophile CM5 BIAcore flow cell surface.
  • Example 16 describes a comparison of surface derivatization via
  • Example 17 describes the Diels-Alder reaction of dienophile derivatized CM5 BIAcore flow cell surface (37) with anthracene derivative (36) (preparation described in Example 14), using the method described in Example 16 to provide compound (38).
  • Example 18 describes the surface immobilization of the cyclohexadiene modified oligonucleotide (29) to the dienophile derivatized CM5 BIAcore flow cell surface (37), followed by hybridization with the complementary oligonucleotide sequence.
  • Example 19 describes the synthesis of anthracene-silane reagent (42).
  • Example 20 illustrates the functionalization of glass slides with anthracene-silane reagent (42).
  • Scheme 1 illustrates the preparation of cyclohexadiene (5). Briefly, the synthesis of diene-alcohol (5) was obtained from alcohol (1) in four steps by a scaleable and very convenient procedure as outlined in Scheme 1. This diene has been synthesized via a different route by Roth et al. ((1993) Chem. Ber. 126:2701-2715).
  • Scheme 2 illustrates the preparation of NHS reagent (10) from diene (5).
  • Scheme 4 illustrates the synthesis of a maleimide-silane reagent for the functionalization of glass surfaces.
  • propylaminosilane (17) was reacted with the functionalized maleimide N- hydroxysuccinimide-ester (18), to provide after aqueous work-up maleimide-silane (19), which was used as a crude product for derivatization of glass surfaces.
  • Scheme 5 illustrates the synthesis of a diene-silane reagent for the functionalization of glass surfaces.
  • diene (5) was treated with carbonyldiimidazole.
  • amine (17) was added to the imidazolate formed in situ.
  • diene- silane (20) was separated and used as a crude product for glass surface functionalizations.
  • the reaction mixture was concentrated at reduced pressure, in vacuo overnight and imidazole crystallized out of the resulting brown oil.
  • the oil obtained was separated from the crystals by pipette and transferred to a clean round-bottom flask to provide 1.5 g (93%) of the desired diene-silane (20) as a brown oil. Crude (20) was used without further purification.
  • Example 6 Synthesis of Labeled Oligonucleotides
  • the synthesis of various labeled and unlabeled (for control experiments) oligonucleotides is illustrated in Schemes 6 and 7 below. All syntheses were carried out employing standard procedures for solid phase oligonucleotide synthesis using phosphoramidite building blocks and a CPG solid support. The 20 mer (22) (SEQ ID NO:l) was chosen for purposes of illustration. With reference to Scheme 6, DNA oligonucleotide (22) was synthesized on CPG performing the standard protocol to give the CPG bound oligonucleotide (21).
  • the diene-functionalized CPG bound oligonucleotide obtained from step c was divided into two portions. One portion was cleaved and deprotected without detritylation to give crude diene(DMT)-oligonucleotide (24) (Scheme 6) and the second portion was processed as described above, but without purification to give crude diene-oligonucleotide (23).
  • a maleimide functionalized oligonucleotide was synthesized as illustrated in Scheme 7.
  • the standard sequence was synthesized on a CPG support and extended with a protected amino-linker.
  • the 5'-aminohexyloligonucleotide product (25) was acylated with the maleimide NHS-ester (18).
  • the crude maleimide- ohgonucleotide (26) was obtained.
  • the support bound oligonucleotide was oxidized with I 2 in the presence of pyridine under standard conditions and detritylated by treatment with 10% dichloroacetic acid in CH C1 2 . Cleavage from the support and base deprotection was performed with 27% aqueous NH 4 OH for 2 hours at 70 °C. The deprotection solutions were then cooled to 4 °C before the CPG was filtered and washed with DI water to recover the crude oligonucleotide product, compound (23).
  • oligonucleotide (23) (designated as "23, pure") was provided in deionized- water at a concentration of 1.1 OD/mL (86% purity by anion exchange HPLC).
  • MALDI-MS M + 6585.47 (6585.1 calcd).
  • CPG bound oligonucleotide (21) 25 ⁇ mol was treated with amidite (16) (0.2 M in CH 3 CN, 15 equiv.) and 4,5- dicyanoimidazole (1.0 M in CH 3 CN, 16 equiv.) for 20 minutes. After oxidation and capping the CPG bound oligonucleotide was partitioned into two aliquots.
  • the first aliquot (0.25 g, 6 ⁇ mol) was subjected to cleavage from the support and base deprotection to give 17 mg, (450 OD) of crude (purity 60% by anion exchange HPLC) diene (DMT)- oligonucleotide (24) after drying.
  • the second aliquot (0.80 g, 19 ⁇ mol) was detritylated prior to cleavage from the support and base deprotection to give 89 mg (2400 OD) of crude (61% purity by anion exchange HPLC purity) oligonucleotide (23) (designated as "23, crude”).
  • Prod.# M010282 was coupled to CPG bound oligonucleotide (21) (32 ⁇ mole) in the presence of 4,5-dicyanomidazole activator (1.0 M in CH 3 CN, 16 equiv.) for 20 minutes. Standard oxidation and capping preceded the final monomethoxytrityl removal with 10% dichloro acetic acid in CH 2 C1 . Cleavage and deprotection of the oligonucleotide from the support in 27% aqueous NH OH and consequential drying provided 155 mg (4200 OD) of crude amino-oligonucleotide (25) (55% purity by anion exchangeHPLC).
  • Amino-oligonucleotide (25) (2.6 ⁇ mol) was resuspended in DMF (2 mL).
  • maleimide NHS-ester (18) (35 mg, 130 ⁇ mol, 50 equiv.) was added and the reaction was stirred at room temperature overnight then placed in speed vacuum to remove the solvent.
  • the dried pellet was then resuspended in H 2 O (1.5 mL) and loaded onto four 1 mL G-25 spin columns to remove un-reacted (18) and side products from the product of the reaction maleimide-ohgonucleotide (26). Detection of maleimide by fluorescence.
  • the photomultiplier tube settings were 600 V, sensitivity set to normal and the focal plane was set at the surface.
  • the results are set forth in Figure 1.
  • the presence of a fluorescent signal demonstrates that (25) was successfully reacted with reagent (18) to create the maleimide-ohgonucleotide (26).
  • Scheme 8 illustrates the reaction of either glass slides or CPG with the maleimide- silane 19.
  • the maleimide functionality is introduced onto the glass slides and CPG by condensation of maleimide-silane (19) with the glass surfaces.
  • the method used to detect maleimide functionalization of the glass surfaces involves staining the glass surface with a thiol-containing fluorescein reagent, which reacts with the surface-bound maleimide via a Michael-addition reaction, as illustrated in Scheme 9.
  • the presence of fluorescein bound to the surfaces can then be detected with a Molecular Dynamics' Tyhpoon fluorescence Scanner using a green laser to excite the surface-bound fluorescein followed by detection of emission using a 526nm filter.
  • Procedure #1 Glass micro slides (Corning no. 2947, description: plain, pre-cleaned, 3 inch x 1 inch x 1 millimeter) were soaked in 2 N NaOH for 2 hours at ambient temperature, washed with water, soaked in boiling 2 N HCl for 1 hour, washed with water and methanol, and then dried under reduced pressure in a high vacuum oven at 100° C for 2 hours. The slides were then allowed to cool in a vacuum desiccator until use.
  • Procedure #2 Micro slides were soaked in 2 N HCl at ambient temperature for 2 hours then in boiling 2 N HCl for 1 hour, washed with water and methanol, and then dried under reduced pressure in a high vacuum oven at 100 °C for 2 hours. The slides were then allowed to cool in a vacuum desiccator until use.
  • one of the slides was removed from the chamber and washed sequentially with toluene, methanol, methanol/water (1:1, v/v), water, methanol/water (1:1, v/v), methanol and ethyl acetate (both sides of the slide were washed with 3x2 mL of each solvent by pipette). The slide was then allowed to air dry.
  • SAMSA fluorescein 5-((2-(and-3)-S-(acetyl- mercapto)succinoyl)amino)fluorescein
  • SAMSA fluorescein 5-((2-(and-3)-S-(acetyl- mercapto)succinoyl)amino)fluorescein
  • the slide was then soaked in 0.5 M sodium phosphate buffer, pH 1, for 30 minutes with agitation and blotted dry with a fine paper tissue.
  • the slide was then placed on the surface of a Molecular Dynamics' Typhoon fluorescence scanner. Surface bound fluorescein was excited with a green laser followed by emission detection with a 526 nm filter (SP fluorescein filter).
  • the photomultipher tube settings were 600 V, sensitivity set to normal and the focal plane was set at the surface.
  • the slide showed a strong response consistent with maleimide functionality primarily on the lower half of the slide ( Figure 2).
  • the distinct line and intense response at and below the halfway mark on the slide show that the maleimide functionalization of the glass slide was successful.
  • the slide also showed a weak response consistent with maleimide functionality on the edges of the slide, possibly due to the contact with the sides of the chamber, and slightly above the halfway mark where the slide may have come in contact with the solution during addition or removal of the slide from the chamber.
  • Native CPG-500 was stirred in boiling 2 N HCl for 2 hours, then collected on a glass-fritted funnel, washed with water and methanol, and dried under reduced pressure in a high vacuum oven at 100 °C for 2 hours. The CPG was then allowed to cool in a vacuum desiccator until use.
  • CPG Maleimide functionalization of CPG.
  • CPG (0.75 g, pre-treated according to the procedure described above) was stirred in a 1 vol-% solution of the maleimide-silane reagent (19) in toluene (25 mL) for 51 hours.
  • the CPG was then collected on a glass-fritted funnel and washed sequentially with toluene, methanol, methanol/water (1:1, v/v), water, methanol/water (1:1, v/v), methanol and ethyl acetate (3x10 mL of each solvent).
  • the powder was then treated with a 5 vol-% solution of chlorotrimethylsilane in pyridine/THF (1 :9, v/v) for 2 minutes to cap free silanol groups
  • the CPG was then washed with THF, methanol and ethyl acetate (3x10 mL of each solvent).
  • the powder was allowed to air dry in the funnel under suction for 5 minutes, transferred to a beaker and placed in a vacuum desiccator for 44 hours.
  • the light tan powder (0.6 g) was then stored in a -20 °C freezer until use.
  • the derivatized CPG was assayed for maleimide functionalization using the thiol-containing fluorescein reagent, 5-((2-(and-3)- S-(acetylmercapto)succinoyl)amino)fluorescein (SAMSA fluorescein).
  • SAMSA fluorescein 5-((2-(and-3)- S-(acetylmercapto)succinoyl)amino)fluorescein
  • Scheme 10 illustrates the functionalization of glass slides and CPG with diene- silane (20) to provide support-bound dienes capable of undergoing Diels-Alder surface immobilization of dienophiles.
  • the diene functionality is introduced onto the glass slides and CPG by condensation of diene-silane (20) with the glass surfaces as shown in Scheme 10.
  • the method used to detect diene functionalized glass surfaces involves staining the glass surfaces with a maleimide-containing fluorescein reagent, which reacts with the surface-bound diene via a Diels-Alder addition reaction as illustrated in Scheme 11.
  • the presence of fluorescein bound to the surface can then be detected with a Molecular Dynamics' Tyhpoon fluorescence scanner using a green laser to excite the surface-bound fluorescein followed by detection of emission using a 526 nm filter.
  • the slides were washed sequentially with toluene, methanol, methanol/water (1:1, v/v), water, methanol/water (1:1, v/v), methanol and ethyl acetate (both sides of each slide were washed with toluene (3x2 mL) by pipette and the remaining washes were done by soaking the slides in a petri dish containing 10 mL of the solvent).
  • the slides were then allowed to air dry and were treated with a 5 vol-% solution of chlorotrimethylsilane in pyridine/THF (1 :9, v/v) for 2 minutes to cap free silanol groups
  • the slides were then washed with THF, methanol and ethyl acetate (both sides of the slide were washed with 3x2 mL of each solvent by pipette), allowed to air dry and were stored in the vacuum desiccator until use.
  • Detection of surface diene by fluorescence One of the slides was assayed for diene functionalization using fluorescein-5-maleimide (Molecular Probes). Briefly, one side of the slide was completely covered with a 10 mM solution of fluorescein-5-maleimide in N,N-dimethylformamide and incubated at 6 °C overnight. The slide was then washed with water (4x10 mL) and blotted dry with a fine paper tissue. The slide was then placed on the surface of a Molecular Dynamics' Typhoon fluorescence scanner. Surface bound fluorescein was excited with a green laser followed by emission detection with a 526 nm filter (SP fluorescein filter).
  • SP fluorescein filter 526 nm filter
  • the photomultipher tube settings were 600 V, sensitivity set to normal and the focal plane was set at the surface.
  • the slide showed a strong response consistent with diene functionality on the lower half of the slide ( Figure 4).
  • the distinct line and intense response at and below the halfway mark on the slide show that the diene functionalization of the glass slide was successful.
  • CPG Diene functionalization of CPG.
  • CPG (0.75 g, pre-treated according to the procedure described in Example 7) was stirred in a 1 vol-% solution of the diene-silane reagent (20) in toluene (25 mL) for 52 hours.
  • the CPG was then collected on a glass-fritted funnel and washed sequentially with toluene, methanol, methanol/water (1:1, v/v), water, methanol/water (1:1, v/v), methanol and ethyl acetate (3x10 mL of each solvent).
  • the powder was then treated with a 5 vol-% solution of chlorotrimethylsilane in pyridine/THF (1 :9, v/v) for 2 minutes to cap free silanol groups
  • the CPG was then washed with THF, methanol and ethyl acetate (3x10 mL of each solvent).
  • the powder was allowed to air dry in the funnel under suction for 5 minutes, transferred to a beaker and placed in a vacuum desiccator for 44 hours.
  • the white powder (0.6 g) was then stored in a -20 °C freezer until use.
  • the derivatized CPG was assayed for diene functionalization using fluorescein-5-maleimide (Molecular Probes).
  • the CPG (5 mg) was placed into a centrifuge tube, a 10 mM solution of fluorescein-5 -maleimide in DMF (0.5 mL) was added, the vial was shaken to mix the contents and incubated at 6 °C overnight. The mixture was then centrifuged, the supernatant was removed by pipette, and the CPG sample was suspended in water. The resulting mixture was centrifuged, the supernatant again removed by pipette, and the CPG sample resuspended in water.
  • Scheme 12 illustrates the conjugation of diene oligonucleotide (23) to maleimide functionalized glass slides and maleimide coated microtiter plates. The demonstration of conjugation was achieved by hybridization with a labeled complementary sequence and the detection of fluorescence.
  • SSC 5X standard saline citrate
  • SDS sodium dodecyl sulfate
  • the slides were then placed on the surface of a Molecular Dynamics' Typhoon fluorescence scanner.
  • the fluorescein was excited with a green laser followed by emission detection with a 526 nm filter (SP fluorescein filter).
  • the photomultipher tube settings were 800 V, sensitivity was normal, and the focal plane was set at the surface.
  • the slides each showed a strong response where diene-oligonucleotide (23) came into contact with the maleimide-functionalized portion of the glass slide (27) ( Figure 7).
  • the two controls on each slide showed no response indicating the lack of non-specific binding of (22) to the maleimide-functionalized portion of the slide and the lack of non-specific binding of (23) to the non-functionalized portion of the slide.
  • the outlines of the septa etched on the slide pre-treated according to procedure #2 are faintly visible in the scan.
  • diene-oligonucleotide (23) to maleimide coated microtiter plates.
  • Scheme 14 shows the conjugation of maleimide-ohgonucleotide (26) with diene functionalized glass surfaces. The demonstration of conjugation was achieved by hybridization with the labeled complementary sequence (27) and the detection of fluorescence as described above.
  • oligonucleotide immobilization on glass slides The slide, pulled from the buffer solution, was immediately (to prevent the slide from drying out) immersed in a 4 pmol/ ⁇ L solution of complementary 5'-fluorescein-oligonucleotide (27) in 5X SSC containing 0.1 %> SDS (10 mL) in a petri dish. The slide was incubated for 30 minutes at 55-60 °C. The slide was then soaked 3 times each in a petri dish containing TBST. The slide was analyzed using the Molecular Dynamics Typhoon fluorescence scanner, but the results indicated that the washing conditions were insufficient to remove non-specifically bound oligonucleotide from the plate ( Figure 11, slide "1").
  • the slide was then washed 3 times in a petri dish containing IX PBS (phosphate-buffered saline solution) with 0.1% SDS.
  • the slide was again analyzed using the Molecular Dynamics Typhoon fluorescence scanner and showed a strong response only where maleimide-ohgonucleotide (26) came into contact with the diene-functionalized portion of the glass slide and then hybridized with (27) ( Figure 11, slide "2").
  • the two controls on the slide showed no response indicating that the washing conditions were sufficiently stringent to remove non- covalently bound oligonucleotide (22) from the diene-functionalized portion of the slide and non-covalently bound maleimide-ohgonucleotide (26) from the non-functionalized portion of the slide.
  • the diene-functionalized portion of the slide shows some fluorescence outside of the area that was reacted with (26) indicating there is some residual non-specific binding of (27) to the diene-functionalized portion of the slide.
  • the resulting mixture was centrifuged, the supernatant again removed by pipette, and the CPG sample resuspended in TBST. This procedure was repeated for a total of 3 washes.
  • two control experiments were run following the above procedure.
  • control oligonucleotide (22) in place of compound (26) to check for potential non-specific binding of the oligonucleotide to diene-functionalized CPG.
  • control experiment involved using non-functionalized CPG capped with chlorotrimethylsilane as a control to check for potential non-specific binding of the maleimide-ohgonucleotide (26) to non-functionalized CPG.
  • oligonucleotide immobilization on CPG A 4 pmol/ ⁇ L solution of complementary 5'-fluorescein-oligonucleotide (27) in 5X SSC containing 0.1% SDS (125 ⁇ L) was immediately added to each of the CPG samples obtained after reaction with maleimide-ohgonucleotide (26) and washings. The samples were incubated for 30 minutes at 55-60 °C. The samples were then washed 3 times as described above with IX PBS (phosphate-buffered saline solution) with 0.1% SDS. After the final wash was removed by pipette, each sample was dispersed onto a clean sheet of plastic wrap and the plastic wrap was folded to contain the powder.
  • IX PBS phosphate-buffered saline solution
  • the samples were analyzed using the Typhoon fluorescence scanner, but the results indicated that the washing conditions were insufficient to remove non-specifically bound (26) from the diene-functionalized CPG ( Figure 12A).
  • the samples were then transferred back into their original centrifuge tubes and washed 3 times with 0.5X SSC + 0.1%) SDS (significant loss of each sample occurred during transfers).
  • the samples were then again dispersed onto sheets of plastic warp and analyzed using the Typhoon fluorescence scanner.
  • 5'-Amine modified oligodeoxynucleotide (28) (ODN99225) was prepared employing standard solid phase automated synthesis on controlled pore glass (CPG) via the phosphoramidite method . After deprotection and cleavage from the CPG support, the crude amine-modified oligonucleotide was purified by preparative anion exchange chromatography on a 200 mL Source 15Q column (quaternary ammonium functionalized, monodisperse polystyrene beads) eluting with the gradient set forth in Table 4.
  • Example 14 Preparation of diene modified polyethylene glycol substrates.
  • Scheme 17 illustrates the synthesis of a cyclohexadiene-PEG (34) .
  • Scheme 19 illustrates the preparation of a dienophile derivatized CM5 BIAcore flow cell surface.
  • the dienophile CM5 BIAcore flow cell surface was prepared by subjecting the commercially available chip (coated with a matrix of carboxymethyl dextran) to a 3 step derivatization procedure, as illustrated in Scheme 19. Briefly, the carboxy groups were activated as the N-hydroxysuccinimide esters (via treatment with EDC/NHS), followed by the addition of diamine linker (ethylene diamine, to provide a reactive primary amine surface), which was treated with a commercially available bifunctional crosslinking reagent. The resulting dienophile cell surface (37) was reproducibly prepared in this manner as evidenced by the measurement of the Biacore sensorgram with each experiment.
  • the sensorgrams were obtained using a BIAcore 2000 instrument (Pharmacia Biosensor AB, Uppsala, Sweden. Lofas et al. (1991) Sens. Actuators B 5:79-84;
  • EDC-NHS mixture from stock solutions: NHS at 11.5 mg/mL and EDC at 75 mg/mL.
  • Scheme 23 illustrates the synthesis of an anthracene silane reagent for the functionalization of glass surfaces. Briefly, with reference to Scheme 23 hydroxymethylanthracene (35) was reacted with CDI to form imidazolate (41). Imidazolate (41) was then reacted with propylamino silane (17), to provide anthracene- silane reagent (42), which was then used for glass derivatization as illustrated in Scheme 24 below.
  • Scheme 24 ( Figure 19) illustrates the functionalization of glass microscope slides with anthracene-silane reagent (42).
  • One half of a clean microslide (not pre-treated, VWR) was dipped into a suspension of reagent 42 (1.04 g) in toluene (50 mL) and CH C1 (10 mL) for 1 hour. The slide was then removed from the suspension and blotted dry on a piece of filter paper. The slide was sonicated sequentially in toluene, toluene/ethanol (1:1, v/v) and ethanol (100 mL, each). For drying the slide was placed in an oven at 80 °C overnight.
  • Buffer B 2M NaBr solution with 10% EtOH.

Abstract

La présente invention concerne un nouveau procédé d'immobilisation de molécules sur un support. Plus précisément, cette invention concerne un procédé d'immobilisation de biomolécules dérivatisées, telles que des oligonucléotides, au moyen de réactions de cycloaddition, telles que la réaction de Diels-Alder. Cette invention concerne également les nouvelles molécules immobilisées pouvant être préparées conformément au procédé selon la présente invention.
PCT/US2001/013956 2000-05-01 2001-05-01 Procede d'immobilisation d'oligonucleotides au moyen du procede de bioconjugaison de cycloaddition WO2001084234A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP2001580595A JP2003535317A (ja) 2000-05-01 2001-05-01 付加環化バイオコンジュゲーション法を利用してオリゴヌクレオチドを固定化する方法
AU2001259285A AU2001259285A1 (en) 2000-05-01 2001-05-01 Method for immobilizing oligonucleotides employing the cycloaddition bioconjugation method
EP01932784A EP1287404A4 (fr) 2000-05-01 2001-05-01 Procede d'immobilisation d'oligonucleotides au moyen du procede de bioconjugaison de cycloaddition

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US20156100P 2000-05-01 2000-05-01
US60/201,561 2000-05-01
US26502001P 2001-01-30 2001-01-30
US60/265,020 2001-01-30

Publications (1)

Publication Number Publication Date
WO2001084234A1 true WO2001084234A1 (fr) 2001-11-08

Family

ID=26896865

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/013956 WO2001084234A1 (fr) 2000-05-01 2001-05-01 Procede d'immobilisation d'oligonucleotides au moyen du procede de bioconjugaison de cycloaddition

Country Status (4)

Country Link
EP (1) EP1287404A4 (fr)
JP (1) JP2003535317A (fr)
AU (1) AU2001259285A1 (fr)
WO (1) WO2001084234A1 (fr)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002012566A2 (fr) * 2000-08-09 2002-02-14 Motorola, Inc. Utilisation et evaluation d'une photocycloaddition [2+2] dans une immobilisation d'oligonucleotides sur une matrice hydrogel tridimensionnelle
US6664061B2 (en) 1999-06-25 2003-12-16 Amersham Biosciences Ab Use and evaluation of a [2+2] photoaddition in immobilization of oligonucleotides on a three-dimensional hydrogel matrix
WO2004058794A1 (fr) * 2002-12-31 2004-07-15 Proligo Llc Procedes et compositions pour la synthese en tandem d'au moins deux oligonuleotides sur le meme support solide
WO2005056636A2 (fr) * 2003-12-03 2005-06-23 Nektar Therapeutics Al, Corporation Procede de preparation de polymeres fonctionnalises par maleimide
WO2005062049A2 (fr) * 2003-12-22 2005-07-07 Interuniversitair Microelektronica Centrum (Imec) Utilisation de structures micro-electronique pour le depot de molecules sur des surfaces selon un trace
US7098326B2 (en) 2002-01-23 2006-08-29 Sigma-Aldrich Co. Methods for the integrated synthesis and purification of oligonucleotides
WO2008037036A2 (fr) * 2006-09-29 2008-04-03 Katholieke Universiteit Leuven Réseaux d'oligonucléotides
US7427678B2 (en) 1998-01-08 2008-09-23 Sigma-Aldrich Co. Method for immobilizing oligonucleotides employing the cycloaddition bioconjugation method
US7872082B2 (en) 2005-07-19 2011-01-18 Nektar Therapeutics Method for preparing polymer maleimides
EP2368875A1 (fr) * 2008-12-02 2011-09-28 Wako Pure Chemical Industries, Ltd. Photogénération de base
US9994687B2 (en) 2013-07-01 2018-06-12 Illumina, Inc. Catalyst-free surface functionalization and polymer grafting
CN108911941A (zh) * 2018-05-25 2018-11-30 凯莱英生命科学技术(天津)有限公司 1，3-环己二烯连续化合成方法
US10781175B2 (en) 2016-07-15 2020-09-22 Am Chemicals Llc Solid supports and phosphoramidite building blocks for oligonucleotide conjugates

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2005085857A1 (ja) * 2004-03-05 2008-01-24 日清紡績株式会社 固定化生体分子及び生体分子と相互作用し得る物質の検出法
JP4581571B2 (ja) * 2004-09-06 2010-11-17 富士ゼロックス株式会社 粒子のマレイミジル基量を測定する定量方法
WO2013176845A1 (fr) * 2012-05-21 2013-11-28 Agilent Technologies, Inc. Réaction de rétro-diels-alder comme lieur clivable dans des applications adn/arn
JP5986872B2 (ja) * 2012-10-05 2016-09-06 公益財団法人微生物化学研究会 化合物、該化合物の製造方法、アセテート誘導体の製造方法、及びアトルバスタチンの製造方法、並びに不斉配位子の回収方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5580697A (en) * 1993-01-21 1996-12-03 State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of The University Of Oregon Chemical functionalization of surfaces
WO1998030575A1 (fr) * 1997-01-08 1998-07-16 Proligo Llc Bioconjugaison de macromolecules

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5874532A (en) * 1997-01-08 1999-02-23 Nexstar Pharmaceuticals, Inc. Method for solution phase synthesis of oligonucleotides and peptides
AU7152098A (en) * 1997-04-21 1998-11-13 Nexstar Pharmaceuticals, Inc. Method for solution phase synthesis of oligonucleotides
US6171797B1 (en) * 1999-10-20 2001-01-09 Agilent Technologies Inc. Methods of making polymeric arrays

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5580697A (en) * 1993-01-21 1996-12-03 State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of The University Of Oregon Chemical functionalization of surfaces
WO1998030575A1 (fr) * 1997-01-08 1998-07-16 Proligo Llc Bioconjugaison de macromolecules

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KOCH ET AL.: "Photochemical immobilization of anthraquinone conjugated oligonucleotides and PCR amplicons on solid surfaces", BIOCONJUGATE CHEM., vol. 11, 2000, pages 474 - 483, XP002944640 *
KOCH ET AL.: "Polystyrene or polycarbonate plates/anthraquinone oligos or cDNA with irradiation", EXIQON, 1999, pages 1 - 3, XP002944639 *
See also references of EP1287404A4 *

Cited By (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7427678B2 (en) 1998-01-08 2008-09-23 Sigma-Aldrich Co. Method for immobilizing oligonucleotides employing the cycloaddition bioconjugation method
US6664061B2 (en) 1999-06-25 2003-12-16 Amersham Biosciences Ab Use and evaluation of a [2+2] photoaddition in immobilization of oligonucleotides on a three-dimensional hydrogel matrix
WO2002012566A3 (fr) * 2000-08-09 2003-01-09 Motorola Inc Utilisation et evaluation d'une photocycloaddition [2+2] dans une immobilisation d'oligonucleotides sur une matrice hydrogel tridimensionnelle
WO2002012566A2 (fr) * 2000-08-09 2002-02-14 Motorola, Inc. Utilisation et evaluation d'une photocycloaddition [2+2] dans une immobilisation d'oligonucleotides sur une matrice hydrogel tridimensionnelle
US7098326B2 (en) 2002-01-23 2006-08-29 Sigma-Aldrich Co. Methods for the integrated synthesis and purification of oligonucleotides
WO2004058794A1 (fr) * 2002-12-31 2004-07-15 Proligo Llc Procedes et compositions pour la synthese en tandem d'au moins deux oligonuleotides sur le meme support solide
US7615629B2 (en) 2002-12-31 2009-11-10 Sigma-Aldrich Co. Methods and compositions for the tandem synthesis of two or more oligonucleotides on the same solid support
US8039579B2 (en) 2003-12-03 2011-10-18 Nektar Therapeutics Intermediates useful in the preparation of maleimide functionalized polymers
US8258324B2 (en) 2003-12-03 2012-09-04 Nektar Therapeutics Intermediates useful in the preparation of maleimide functionalized polymers
US8895759B2 (en) 2003-12-03 2014-11-25 Nektar Therapeutics Intermediates useful in the preparation of maleimide functionalized polymers
WO2005056636A3 (fr) * 2003-12-03 2005-10-06 Nektar Therapeutics Al Corp Procede de preparation de polymeres fonctionnalises par maleimide
US7790835B2 (en) 2003-12-03 2010-09-07 Nektar Therapeutics Method of preparing maleimide functionalized polymers
US8653286B2 (en) 2003-12-03 2014-02-18 Nektar Therapeutics Intermediates useful in the preparation of maleimide functionalized polymers
WO2005056636A2 (fr) * 2003-12-03 2005-06-23 Nektar Therapeutics Al, Corporation Procede de preparation de polymeres fonctionnalises par maleimide
US8758688B2 (en) 2003-12-22 2014-06-24 Imec Microelectronic structures for patterned deposition of molecules onto surfaces
WO2005062049A2 (fr) * 2003-12-22 2005-07-07 Interuniversitair Microelektronica Centrum (Imec) Utilisation de structures micro-electronique pour le depot de molecules sur des surfaces selon un trace
WO2005062049A3 (fr) * 2003-12-22 2005-12-15 Imec Inter Uni Micro Electr Utilisation de structures micro-electronique pour le depot de molecules sur des surfaces selon un trace
US7872082B2 (en) 2005-07-19 2011-01-18 Nektar Therapeutics Method for preparing polymer maleimides
US8227558B2 (en) 2005-07-19 2012-07-24 Nektar Therapeutics Method for preparing polymer maleimides
US8058385B2 (en) 2005-07-19 2011-11-15 Nektar Therapeutics Method for preparing conjugates using polymer maleimides
WO2008037036A3 (fr) * 2006-09-29 2008-11-06 Univ Leuven Kath Réseaux d'oligonucléotides
WO2008037036A2 (fr) * 2006-09-29 2008-04-03 Katholieke Universiteit Leuven Réseaux d'oligonucléotides
US8957212B2 (en) 2008-12-02 2015-02-17 Wako Pure Chemical Industries, Ltd. Photobase generator
EP2368875A1 (fr) * 2008-12-02 2011-09-28 Wako Pure Chemical Industries, Ltd. Photogénération de base
EP2368875A4 (fr) * 2008-12-02 2012-08-08 Wako Pure Chem Ind Ltd Photogénération de base
TWI474997B (zh) * 2008-12-02 2015-03-01 Wako Pure Chem Ind Ltd 光鹼產生劑
US9994687B2 (en) 2013-07-01 2018-06-12 Illumina, Inc. Catalyst-free surface functionalization and polymer grafting
US10975210B2 (en) 2013-07-01 2021-04-13 Illumina, Inc. Catalyst-free surface functionalization and polymer grafting
US11618808B2 (en) 2013-07-01 2023-04-04 Illumina, Inc. Catalyst-free surface functionalization and polymer grafting
US10781175B2 (en) 2016-07-15 2020-09-22 Am Chemicals Llc Solid supports and phosphoramidite building blocks for oligonucleotide conjugates
US11447451B2 (en) 2016-07-15 2022-09-20 Am Chemicals Llc Solid supports and phosphoramidite building blocks for oligonucleotide conjugates
CN108911941A (zh) * 2018-05-25 2018-11-30 凯莱英生命科学技术(天津)有限公司 1，3-环己二烯连续化合成方法

Also Published As

Publication number Publication date
EP1287404A4 (fr) 2003-10-15
JP2003535317A (ja) 2003-11-25
AU2001259285A1 (en) 2001-11-12
EP1287404A1 (fr) 2003-03-05

Similar Documents

Publication Publication Date Title
US7427678B2 (en) Method for immobilizing oligonucleotides employing the cycloaddition bioconjugation method
WO2001084234A1 (fr) Procede d'immobilisation d'oligonucleotides au moyen du procede de bioconjugaison de cycloaddition
Hill et al. Diels− Alder bioconjugation of diene-modified oligonucleotides
Scaringe RNA oligonucleotide synthesis via 5′-silyl-2′-orthoester chemistry
CA2627216C (fr) Reactif de marquage de polynucleotide
JP2818650B2 (ja) トリスヒドロキシメチルエタン化合物
JPS58180500A (ja) Ｄｎａの化学的合成方法
JP3161730B2 (ja) オリゴヌクレオチド合成の方法および手段
US5410068A (en) Succinimidyl trityl compounds and a process for preparing same
EP0451213A1 (fr) Derives de molecules polyhydroxylees permettant l'introduction d'au moins une ramification dans un oligonucleotide.
JP2008214343A (ja) 光分解性化合物と前記化合物がカップリングしたオリゴマープローブアレイ用基板、オリゴマープローブアレイおよびその製造方法
EP0877751B1 (fr) Bras de liaison pour la synthese d'oligonucleotides sur un support solide et procede pour le former
Matulic-Adamic et al. Synthesis and Structure of 1-Deoxy-1-phenyl-bD-ribofuranose and Its Incorporation into Oligonucleotides
Fang et al. Fluoride‐cleavable biotinylation phosphoramidite for 5′‐end‐labeling and affinity purification of synthetic oligonucleotides
Wada et al. A new boranophosphorylation reaction for the synthesis of deoxyribonucleoside boranophosphates
Korshun et al. New pyrene derivatives for fluorescent labeling of oligonucleotides
Chaix et al. Oligonucleotide synthesis on maleic anhydride copolymers covalently bound to silica spherical support and characterization of the obtained conjugates
Sojka et al. Evaluating the quality of oligonucleotides that are immobilized on glass supports for biosensor development
Vigovskaya et al. Photo-induced chemistry for the design of oligonucleotide conjugates and surfaces
WO2005103247A1 (fr) Sonde oligonucléotidique
Lyttle et al. New reagents and methods for the synthesis of internal and 3 ‘-labeled DNA
US20030195351A1 (en) Methods for the integrated synthesis and purification of oligonucleotides
US8026348B2 (en) Nucleoside derivative, modified oligonucleotide, method for their synthesis and applications thereof
US20110201080A1 (en) Biomolecules having multiple attachment moieties for binding to a substrate surface
Yamamoto et al. Bicyclo [2.2. 2] octane-2, 3-diol as an universal linker for the solid-phase synthesis of oligonucleotides

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

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

Ref document number: 2001932784

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2001932784

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 2001932784

Country of ref document: EP