JP2003535317A - Method for immobilizing oligonucleotide using cycloaddition bioconjugation method - Google Patents

Abstract

  (57) [Summary] The present invention discloses a novel method for immobilizing a molecule on a support. In particular, the present invention discloses a method of immobilizing a derivatized biomolecule, such as an oligonucleotide, using a cycloaddition reaction, such as a Diels-Alder reaction. Included in the present invention are novel immobilized biomolecules that can be produced according to the methods of the present invention.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention describes a novel method of immobilizing molecules on a support. In particular, the invention is
A method of immobilizing a biomolecule on a support using a cycloaddition reaction such as the Diels-Alder reaction is described.

BACKGROUND OF THE INVENTION The applications of surface-immobilized biomolecules are widespread and include nucleic acid sequencing, gene expression profiling, analysis of single nucleotide polymorphisms (SNPs), evaluation of hapten-antibody or ligand-target interactions. . An important subset of these technologies is Genome D
It involves immobilization of oligonucleotide probes, which utilize Watson-Crick hybridization in interaction with target nucleic acids such as NA, RNA, or cDNA produced by polymerase chain reaction (PCR) amplification of sample DNA. Recent techniques have arrayed oligonucleotide probes for such analysis into microarrays on glass slides, silicon chips or wafers, microtiter plates, or other supports containing polyacrylamide gel matrices. Often accompany.

There are a variety of methods for immobilizing biomolecules, including non-covalent (hydrophobic or ionic interactions) as well as covalent methods. Table 1 summarizes many of these methods. Methods involving covalent additions are generally considered preferable to (
In order to reduce non-specific ionic or hydrophobic binding (which enhances background signal) more stringent conditions can be applied to the immobilization system without concern for probe loss from the surface. . Commonly utilized covalent methods include surface condensation of amines with activated carbonyl groups, such as activated carboxylic acid esters, carbonates, or isocyanates or isothiocyanates. In addition, amine groups can condense with aldehydes under conditions of reductive amination, providing a secondary amine linkage between the surface and biomolecules. Additionally, amines can be fused with electron-deficient heterocycles via nucleophilic aromatic substitution as well as epoxide ring opening.

A cycloaddition reaction involves reacting atoms as the reaction proceeds along a reaction coordinate leading to a product (typically in a concerted manner, although various intermediates may be involved). Can be defined as the reaction (either intramolecular or intermolecular) of two (or more) moieties that form a circular array. The orbitals involved in this class of reactions are typically in the π system, but some σ orbitals may also be involved. The number of electrons involved in this type of reaction is of two types: 4n + 2 and 4n (where n = 0, 1, 2
3, 4, etc.). Typical examples of cycloaddition reactions include Diels-Alder cycloaddition reactions, 1,3-dipolar cycloadditions, and [2 + 2] cycloadditions.

The Diels-Alder reaction, the most studied cycloaddition to date, is
Cycloaddition reaction of a conjugated diene with an unsaturated molecule to form a cyclic compound with π electrons used to form new σ bonds. The Diels-Alder reaction is an example of a [4 + 2] cycloaddition reaction because it involves a system of 4π electrons (dienes) and a system of 2π (dienophile). This reaction can occur very quickly under mild conditions and for a wide variety of reactants. The Diels-Alder reaction has a wide range and is well known to those skilled in the art. A review of the Diels-Alder reaction is incorporated herein by reference, "Advanced Organic Chemistry" (March, J., ed.) 8
39-852 (1992) may be found in John Willie and Sons (NY).

It has been discovered that the rate of Diels-Alder cycloaddition reactions is enhanced by aqueous solvents (Rideout and Brestow (1980) J. Am. Chem. Soc. 102: 7816). Similar effects have also been observed in 1,3-dipolar cycloaddition reactions (Engberts (1995) Te
trahedron Lett. 36: 5389). This enhancement is probably due to the hydrophobicity of the diene and dienophile reactants (Breslow (1991) Acc. Chem. Res. 24: 159.
). This effect extends to the intramolecular Diels-Alder reaction (Blokzijl et al. (1
991) J. Am. Chem. Soc. 113: 4241). Not only the reaction rate is accelerated in water, but the endo / exo product ratio is also increased in some cases (Breslow and Maitra (1984) Tetrahedron Lett. 25). : 1239; Lub
ineau et al. (1990) J. Chem. Soc. Perkin Trans. I, 3011; Grieco et al.
1983) Tetrahedron Lett. 24: 1897). Lithium chloride (Breslow et al. (1983)
Tetrahedron Lett. 24: 1901), as well as monovalent phosphates (Pai and Smith (1995) J.
It has been observed that salts that enhance the hydrophobic effect in water, such as Org. Chem. 60: 3731), further accelerate the [4 + 2] cycloaddition.

In both cases, “Method for Solution Phase Sy
US patent application Ser. No. 09/051, entitled "Nthesis of Oligonucleotides)"
No. 449 (filed April 6, 1998); No. 08 / 843,820 (filed April 21, 1997) and No. 09 / 402,430 (filed October 7, 1997) deal- The alder cycloaddition reaction has been shown to be an ideal method for immobilizing oligonucleotides on a resin. The diene- or dienophile-derivatized resin is reacted with a dienophile- or diene-derivatized oligonucleotide, respectively, to yield a Diels-Alder cycloaddition product. In particular, the Diels-Alder reaction of a diene-derivatized oligonucleotide with a polymer resin derivatized with a maleimide group and phenyl-triazoline-dione (PTAD) is described. The resulting resin can be used as an affinity chromatography resin.

[Bioconjugation of Macromolecules]
US patent application Ser. No. 09 / 341,337 (filed Jul. 7, 1999) describes that cycloaddition reactions such as Diels-Alder reactions and 1,3-dipolar cycloaddition reactions are In general, it is shown to be an ideal alternative to existing methods of conjugating macromolecules to other molecular moieties. In particular, the Diels-Alder reaction is an ideal method for covalently bonding large water-soluble macromolecules to other compounds because the reaction rate can be accelerated in water and carried out at neutral pH. (Rideout and Brestow (19
80) J. Am. Chem. Soc. 102: 7816). In addition, the nature of this reaction allows post-synthesis modification of hydrophilic macromolecules without excess reagents or hydrolysis of reagents. When it comes to conjugation to oligonucleotides, this technique efficiently
Facilitated by the ability to synthesize'-O-diene-nucleotides, allowing conjugation sites to be diversified throughout the oligonucleotide and the option of having multiple conjugation sites.

The present invention describes a method of immobilizing molecules, particularly biomolecules, to a support using a cycloaddition bioconjugation method. Immobilization of biomolecules via cycloadditions, especially the Diels-Alder reaction, provides the following major advantages over the conventional methods (see Table 1): Shared between compounds to which the cycloaddition reaction is linked. And the establishment of a stable linkage; the reaction proceeds with a high degree of chemoselectivity; the functional groups of the biomolecule do not interfere with the cycloaddition reaction; It is orthogonal to the labeling protocol and is therefore capable of doubling the reaction in one reaction mixture; in contrast to the general techniques of organic synthesis as discussed above, Diels-Alder That the reaction can be carried out in the aqueous phase, that the Diels-Alder reaction is very accelerated in water and is very fast at room temperature or a little lower temperature; Progress in No by-products are formed during this reaction; no activators or additives are required to carry out this reaction, and the moieties involved in this reaction (dienes and dienophiles) are conjugated to biomolecules. Or it should be stable under the various reaction conditions used for immobilization.

It should be noted that various references are provided throughout this application. Each reference is specifically incorporated herein by reference in its entirety. SUMMARY OF THE INVENTION The present invention provides a novel, chemoselective for immobilizing molecules using cycloaddition reactions,
And describe a very efficient method. The method of the present invention offers advantages over existing immobilization methods that may suffer from cross-reactivity, low selectivity, mechanism ambiguity, and competitive hydrolysis of reactive groups.

In summary, the method of the invention comprises reacting a derivatized molecule with a derivatized support capable of reacting with said derivatized molecule via a cycloaddition reaction. In a preferred embodiment, the derivatized molecule is a biomolecule, preferably an oligonucleotide,
It can also be haptens, carbohydrates, oligosaccharides, peptides, and proteins (including antibodies). The support is preferably glass or controlled pore glass (CPG), but can also be polypropylene, polystyrene, polyacrylamide, or silicon. In a preferred embodiment, 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 the Diels-Alder reaction, ie, a diene or dienophile, and the support is derivatized with the appropriate pair of reactants, ie, dienophile or diene, respectively. .

The present invention includes reaction schemes for producing a wide variety of immobilized biomolecules using cycloaddition reactions as represented by the Diels-Alder cycloaddition reaction. The method of the invention can be extended to immobilize any molecule, especially biomolecules, on any support that can be properly derivatized.

The method of the present invention applies to all 4n and 4n + 2 (where n = 0, 1, 2, 3,
4, etc.) can be extended to cycloaddition. This includes, but is not limited to, Diels-Alder cycloadditions, 1,3-dipolar cycloadditions, ene cycloaddition reactions, and [2 + 2] additions such as ketone addition and photochemical [2 + 2] addition. Includes (4n-type) cycloaddition.

Also described herein is the addition of the present invention to a surface, preferably a microscope glass slide or CPG, by vapor deposition of a silane monolayer properly functionalized with the reaction elements of the cycloaddition reaction. It is a method that can be converted into a reaction element of the cyclization immobilization method.

Also included in the present invention are novel immobilization and derivatization molecules and derivatized supports produced by the methods of the invention. The method of the present invention is used for nucleic acid sequencing, gene expression profiling, single nucleotide polymorphism (SNP) analysis, hapten-antibody or ligand-target interaction evaluation, research, development and clinical diagnostic applications. It is applicable to the field of producing a molecular array.

DETAILED DESCRIPTION OF THE INVENTION The present invention includes a method of immobilizing a molecule to a support. In particular, the invention describes the use of cycloaddition reactions, in particular Diels-Alder cycloaddition reactions, to chemoselectively immobilize molecules on a support.

The method of the invention comprises reacting a derivatized molecule with a derivatized support capable of reacting with the derivatized molecule via a cycloaddition reaction. In a preferred embodiment, the derivatized molecule is a biomolecule, most preferably an oligonucleotide, and the support is preferably glass or controlled pore glass (CPG). In a preferred embodiment, the cycloaddition reaction is a Diels-Alder cycloaddition reaction. Thus, the biomolecule is derivatized with one component of the Diels-Alder reaction, a diene or dienophile, and the support is derivatized with the appropriate pair of reactants, a dienophile or diene, respectively.

The present invention includes reaction schemes for producing a wide variety of immobilized biomolecules using cycloaddition reactions as represented by the Diels-Alder cycloaddition reaction. The methods of the invention can be used to immobilize any molecule, especially biomolecules, on any support that can be properly derivatized.

Certain terms used to describe the invention are described herein as follows: “Oligonucleotide” means a polynucleotide formed from a plurality of linked nucleotide units. Nucleotide units each include nucleoside units linked together through a phosphate linking group. The term oligonucleotide also means a plurality of nucleotides linked via a linkage other than a phosphate linkage, such as a phosphorothioate linkage. Oligonucleotides can be naturally occurring or non-naturally occurring. In a preferred embodiment, the oligonucleotide of the invention has 1 to 1000 nucleotides.

For purposes of the present invention, “nucleobase” has the following definition. Nucleo bases are purine or pyrimidine bases. Nucleobases include all purines and pyrimidines known to those skilled in the art today, or chemical modifications thereof. Purine is attached to the ribose ring via the 9-position nitrogen of the purine ring, and pyrimidine is attached to the ribose ring via the 1-position nitrogen of the pyrimidine ring. Pyrimidine is the 5- of the pyrimidine ring.
Alternatively, it can be modified at the 6-position and the purines can be modified at the 2-, 6- or 8-positions of the purine ring. Certain modifications are incorporated herein by reference, "known and novel 2".
'Novel Method of Preparation for Modified Nucleophilic Substitution of Modified Pyrimidine
of Known and Novel 2 'Modified Pyrimidines Intramolecular Nucleophilic D
US patent application Ser. No. 08 / 264,029 (filed Jun. 22, 1994) entitled “Isplacement” and “Method for Palladium Catalyzed Carbon-Carbon Coupling and Products) "
U.S. Pat. No. 5,428,149. More specifically,
Nucleo bases include, but are not limited to, uracil, cytosine, N4-protected cytosine, 4-thiouracil, isocytosine, 5-methyluracil (thymine), 5-substituted uracil, adenine, N6-protected adenine, guanine, N2-. Included are protected guanine, 2,6-diaminopurine, halogenated purines, and heterocycles to mimic purine or pyrimidine rings, such as imidazole.

A “diene” is defined as a molecule that carries two conjugated double bonds. The diene
It can even be unconjugated if the geometry of the molecule is constrained to facilitate the cycloaddition reaction (Cookson (1964) J. Chem. Soc. 5416). The atoms forming these double bonds can be carbon or heteroatoms, or any combination thereof.

“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. Typically, the dienophile is a substituted alkene of the formula C = C-Z or Z'-C = C-Z, where Z and Z '.
Is, CHO, COR, COOH, COCl, CO aryl, CN, NO _{2, aryl, CH 2 OH, CH 2 Cl} , CH 2 NH 2, CH 2 CN, CH 2 COOH, the group consisting of halogen, or C = C Are electron withdrawing groups independently selected from In some cases, the group attached to the alkene unit includes, but is not limited to, a phenyl ring, a conjugated double bond, an alkyl group, an OMe group, or another X-alkyl moiety, where X is an electron donating group, It can be a donor group (these types of dienophiles undergo cycloaddition, commonly known as reverse electron demand cycloaddition). Examples of other dienophiles include compounds having the formula R ₂ C = X, where X is a heteroatom selected from the group consisting of oxygen, nitrogen, phosphorus, and sulfur. For example, a molecule bearing a primary amino group, such as an amino acid or a lysine-containing peptide, can be converted to an efficient dienophile by reaction with formaldehyde to yield its corresponding iminium salt as illustrated below. The latter undergoes Diels-Alder cycloaddition with diene-supported macromolecules under mild conditions in aqueous solvents.

[0023]

[Chemical 11] A "1,3-dipole" is defined as a compound containing a continuous series of three atoms, abc, where atom a contains a sextet electron in its outer shell, and c is
It contains an octet with at least one unshared electron pair in its shell. Molecules with six electrons in the outer shell of an atom are typically unstable, so the example of an abc atom is, in fact, one resonant hybrid of which at least one structure may be depicted. It is an exemplary structure. The 1,3-dipole can be divided into two main groups: 1) one of the exemplary forms has a double bond on the sextet atom (atom a) and the other exemplary form is on that atom. Systems with triple bonds:

[0024]

[Chemical 12] 2) Systems in which the dipole dilemma has a single bond on the hexat atom (atom a) and the other dimorphism has a double bond on the atom:

[0025]

[Chemical 13] See "Advanced Organic Chemistry" for an overview of this reaction type.
(March, J., ed.) 839-852 (1992) John Willie and Sons (NY)
) And “Frontier Orbitals and Organic Chem
ical Reactions) (I. Fleming) 148-161 (1976) John Willie & Sons, Inc. Typical examples of 1,3-dipoles include, but are not limited to
Nitrile ylide, nitrile imine, nitrile oxide, diazoalkane, azide, azomethine ylide, azomethine imine, nitrone, carbonyl ylide, carbonyl imine, and carbonyl oxide are included.

“1,3-Dipolarophile” is defined in the same manner as a dienophile (as described above) or a diene. Polymer is 1,3-
It may be attached to either (or both) the dipole or the 1,3-dipolarophile.

[0027]   A "1,3-dipolar cycloaddition reaction" can generally be represented as follows:

[0028]

[Chemical 14] An “ene reaction” can generally be represented as:

[0029]

[Chemical 15] The reaction partners in the ene reaction are "ene" and "enophile".
Is said. "Enofil" is defined in the same way as "Genofil" (
See above 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. Typically, "ene" the formula _{X-C = CH-CH 2} - is or X'-C = C-X- CXH- substituted alkenes, wherein X and X 'is an electron donating group. The macromolecule may be attached to either (or both) the ene component or the enophile component.

“Bioconjugate” means any macromolecule derivatized with another molecular entity, as defined herein. "Bioconjugation" or "conjugation" means derivatization of macromolecules with another molecular entity.

As used herein, “support” means controlled pore glass (CPG), glass slides, glass fibers, glass discs, glasses including without limitation glass, glass-containing metals and Silicon chips and wafers including without limitation composite materials; polystyrene (PS), polyethylene glycol (PEG), PS and PE
Polymers / resins including without limitation copolymers of G, copolymers of polyacrylamide and PEG, copolymers containing maleimide and maleic anhydride, polyvinyl alcohol, and non-immunogenic high molecular weight compounds; and , Polysaccharides such as cellulose,
By large biomolecules, including without limitation proteins, and nucleic acids. The support can, but need not be, a solid support.

As used herein, “immobilization” means attachment to a support via covalent bonds. Immobilization includes functional groups on the support or derivatized support. "Functionality", as used herein, refers to a func
cc.

As used herein, “derivatized” means a molecule and / or support functionalized with moieties capable of undergoing a cycloaddition reaction. Molecules or supports that bear moieties capable of undergoing cycloaddition reactions without functionalization also fall within this definition. Examples of moieties that can undergo a cycloaddition reaction include, but are not limited to, dienes,
Dienophile, 1,3-dipole, 1,3-dipolarophile, en, enophyll,
Alternatively, other moieties capable of undergoing a cycloaddition reaction are included.

The term “molecule” includes, but is not limited to, biomolecules, macromolecules, diagnostic detection molecules (DDM), and other small molecules, especially those used in combinatorial chemistry.

As used herein, “biomolecule” includes, but is not limited to, nucleic acids, oligonucleotides, proteins, peptides and amino acids, polysaccharides and saccharides, glycoproteins and glycopeptides (generally sugar conjugates. ) Alkaloids, lipids, hormones, drugs, prodrugs, antibodies, and metabolites.

The term “polymer,” as used herein, means the product of coupling of two macromolecules via cycloaddition. "Diagnostic detection molecules (DDM)" include, but are not limited to, fluorescent agents, chemiluminescent agents, radioisotopes and bioluminescent marker compounds; antibodies, biotin, and metal chelates.

As used herein, a “cycloaddition reaction” occurs between two reactants by the rearrangement of valence electrons through an activated complex, which is normally a cyclic transition state. Means any reaction. The orbitals involved in this class of reactions are typically in the π system, but some σ orbitals may also be involved. The number of electrons associated with this type of reaction is of two types: 4n + 2 and 4n (where n = 0, 1, 2, 3, 4, etc.). Typical examples of cycloaddition reactions include, but are not limited to, [1 + 2] cycloaddition, such as reaction between carbene and olefin, reaction between olefins, or [2 + 2] cycloaddition such as reaction between ketone and olefin. , [3 + 2] cycloaddition such as 1,3-dipolar cycloaddition, [2 + 4] cycloaddition such as Diels-Alder reaction and ene reaction,
Includes [4 + 6] cycloadditions, and cheleotropic reactions.
The types of reactants involved in the cycloaddition reaction include, but are not limited to, olefins including but not limited to alkenes, dienes with or without heteroatoms, alkynes with or without heteroatoms, anthracenes. Aromatic compounds such as 1,3-
Dipoles, carbene, and carbene precursors are included.

The “derivatized oligonucleotide” of the present invention is generally represented by the formula:

[0039]

[Chemical 16] [Wherein B is a nucleobase; A and A ′ are 2′-sugar substituents; W is an oligonucleotide having 1 to 1000 nucleobases, X, or H].
Independently selected from the group consisting of; and X is capable of undergoing a diene, dienophile, 1,3-dipole, 1,3-dipolarophile, ene, enophile, alkene, alkyne, or cycloaddition reaction , And when X is attached to B of the nucleobase, it can also be attached to a carbon atom, an exocyclic nitrogen, or an exocyclic oxygen].

In a preferred embodiment of the invention: A and A ′ are H, ² H, ³ H, Cl, F, OH, NHOR ¹ , NHOR ³ , NH
NHR ³ , NHR ³ , = NH, CHCN, CHCl ₂ , SH, SR ₃ , CFH ₂ , C
Independently selected from the group consisting of F ₂ H, CR ² ₂ Br, — (OCH ₂ CH ₂ ) _n OCH ₃ , OR ⁴ , and imidazole (“2′-modified pyrimidine molecule, incorporated herein by reference. Novel Method of Preparation of 2 '
Modified Pyrimidines Intramolecular Nucleophilic Displacement) ", U.S. patent application Ser. No. 08 / 264,029 (filed Jun. 22, 1994); R ¹ is selected from the group consisting of H and an alcohol protecting group. R ² is ═O, ═S, H, OH, CCl ₃ , CF ₃ , halide, optionally substituted C ₁ -C ₂₀ alkyl (including cyclic, straight chain, and branched chain), C ₂ -C ₂₀ alkenyl, C ₆ -C ₂₀ aryl, C1 to C20 acyl, C ₁ -C ₂₀ benzoyl, O
R ⁴ and an ester; R ³ is R ² , R ⁴ , CN, C (O) NH ₂ , C (S) NH ₂ , C (O) CF ₃ , S
Selected from the group consisting of O ₂ R ⁴ , amino acids, peptides, and mixtures thereof; R ⁴ is an optionally substituted hydrocarbon (C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, C ₂ -C) ₂₀ alkynyl), optionally substituted heterocycle, t-butyldimethylsilyl ether, triisopropylsilyl ether, nucleoside, carbohydrate, fluorescent label, and phosphate; most preferably A is , H, OH, NH ₂ , Cl, F, NHOR ³ , OR ⁴ , O
SiR ⁴ ₃ is selected from the group consisting of ( "2'-modified pyrimidines intramolecular nucleophilic substitution of a new production process (Novel Method of Preparation of 2 ' Modified Pyrimidines Intramole
US patent application Ser. No. 08/264, entitled "cular Nucleophilic Displacement)"
No. 029 (filed Jun. 22, 1994); and X includes, but is not limited to, alkyl or substituted alkyl groups bearing conjugated diene units, alkoxy or substituted alkoxy groups bearing conjugated diene units, CH ₂ CH ₂ C
H = CHCH ₂ CH ₂ O, maleimide-substituted alkoxy group, dienophile-substituted alkoxy group, alkylamino group or substituted alkylamino group bearing conjugated diene unit, maleimide-substituted alkylamino group or substituted alkylamino group, alkylamino group bearing dienophile moiety Alternatively, it includes a substituted alkylamino group, nitrile ylide, nitrile imine, nitrile oxide, diazoalkane, azide, azomethine ylide, azomethine imine, nitrone, carbonyl ylide, carbonyl imine, and carbonyl oxide.

All moieties of the alkyl groups listed above may have 1 to 50 carbons, preferably 1 to 30 carbons. As used herein, a "linking molecule" is a molecular entity that links two or more molecular entities through covalent interactions. More particularly, the linking molecule is a polyfunctional molecule that can be used to derivatize the molecule or support with dienes, dienophiles, or other moieties capable of undergoing cycloaddition reactions. The linking molecules of the invention are generally represented by the formula:

[0042]

[Chemical 17] [Wherein X is as defined above; n is an integer from 1 to 20; and L is the following general formula:

[0043]

[Chemical 18] Where: m, n, o are equal to 0, 1, 2, and Y is NH, O, NH (CO) O, NH (CS) O, NH (CO) NH, NH
(CO), S—S—S—, Si (OR) ₃ , and SiR ₂ where R is alkyl, aryl, substituted alkyl or substituted aryl, each having 1 to 50 carbon atoms. A linker that includes, but is not limited to, a compound of choice).

Other obvious substitutions for the above-listed substituents are also included within the scope of the invention, which is a generalized reaction format not limited to a particular one. Cycloaddition reactions, especially the Diels-Alder reaction, are uniquely suitable as general methods for immobilizing molecules, especially biomolecules, to a support. Cycloadditions of dienes and dienophiles are very chemoselective and only properly electron-oriented diene and dienophile pairs react. This reaction proceeds under mild conditions in a reasonable time frame. Biomolecules such as nucleic acids, oligonucleotides, proteins, peptides, carbohydrates, polysaccharides, glycoproteins, antibodies, and lipids,
Generally, it does not contain a moiety that can undergo such a cycloaddition reaction. Thus, the introduction of specific diene and dienophile reaction partners allows the immobilization of biomolecules to a support with unprecedented specificity.

The high selectivity of the reaction of the diene or dienophile with the corresponding dienophile or diene, respectively, eliminates the need to protect functional groups during the synthesis of biomolecules such as oligonucleotides or peptides. . This is because the limited selectivity of the protected chemistry often determines the yield of immobilization,
This is a very practical advantage over other functional groups used for immobilization in biomolecule synthesis. Furthermore, dienes and dienophiles are immune to side reactions typically encountered in known immobilization methods. Since they do not undergo hydrolysis or solvolysis, these reactions can proceed in aqueous media at near stoichiometric concentrations, thus saving valuable reagents. The low number of such side reactions makes it possible to immobilize biomolecules with unprecedented yield and purity. The Diels-Alder cycloaddition reaction is accelerated by aqueous solvents and is therefore uniquely suitable for derivatization or immobilization of hydrophilic biomolecules. Finally, this method is typically much less pH sensitive than most known alternatives.

In one aspect of the invention, 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. Therefore, the optimal reaction for immobilization of such groups proceeds readily in water and does not show side reactions with water, such as hydrolysis of the reactants. Based on these conditions for optimal and specific immobilization of oligonucleotides, the present disclosure provides Diels-Alder cycloaddition reactions for chemoselective and efficient immobilization of oligonucleotides to a support. Describe the use of. Thus, 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 would react with a support bearing either the dienophile or diene moiety, respectively. Can be done.

The diene or dienophile moiety is, for example, 5- (3,5-hexadiene)
The introduction of the 2'-deoxyuridine nucleoside can be incorporated into the oligonucleotide at any position on this chain (as is incorporated herein by reference, "
Novel production method of known and novel 2'-modified nucleosides by intramolecular nucleophilic substitution (Novel
Method of Preparation of Known and Novel 2 'Modified Nucleosides Intramo
US patent application Ser. No. 08/264 entitled "Lecular Nucleophilic Displacement)"
, 029 (filed June 22, 1994)). Alternatively, the diene or dienophile moiety can be introduced as a 2'-O- (3,5-hexadiene) -uridine nucleoside. The diene moiety is also 3,5-hexadiene-
It can be introduced into the oligonucleotide as a diene-supported non-nucleoside phosphoramidite, such as N, N-diisopropyl-2-cyanoethyl phosphoramidite. Reaction of a diene-modified oligonucleotide, such as the 5'-terminal 3,5-hexadiene phosphate oligonucleotide, with a dienophile-modified support, such as maleimide-derivatized glass, results in efficient immobilization of the oligonucleotide.

The method of the present invention can be extended without limitation to the immobilization of any molecule that can be derivatized with a diene, dienophile, or other reactive group capable of undergoing a cycloaddition reaction.
For example, the method can be extended to the immobilization of peptides and proteins with any support that can be derivatized. Peptides or proteins containing building blocks of amino acids derivatized with dienes or dienophiles, such as O-3,5-hexadiene-tyrosine or serine, or N-maleimidolysine, use the methods described herein. Then, it can be immobilized on any support without limitation. Natural molecules such as proteins can be derivatized with dienes or dienophiles bearing heterobifunctional cross-linking reagents, such as NHS ester of 3- (4-maleimidophenyl) -propionic acid (Pierce), This allows subsequent conjugation to the support bearing the corresponding diene or dienophile group.

Polyethylene glycol is often conjugated to biomolecules to give in v
Itro reduces its immunogenicity and increases its residence time. The method of the present invention is
A biomolecule, such as an oligonucleotide or a peptide, bearing a diene, dienophile, or other reactive group capable of undergoing a cycloaddition reaction, has one or several corresponding dienes, dienophiles, or cycloadditions. Allows immobilization with other polymers or resins, such as polyethylene glycol or polystyrene, bearing other groups capable of undergoing reaction.

Example 1 (Scheme 1) describes the synthesis of 5-hydroxymethylcyclohexa-1,3-diene (5). Example 2 (Scheme 2) shows NH from cyclohexadiene alcohol (5)
A method for producing the S reagent (10) will be described.

Example 3 (Scheme 3) describes the synthesis of a diene amidite linker (16) from an amino linker (12). The amino linker (12) is a primary (5'-
OH) and secondary hydroxyl groups (3'-OH) containing nucleosides were developed. This allows conjugation of the linker at either the 3'- or 5'-end of any oligonucleotide. Attachment of various moieties (eg, dienes or dyes) to the oligonucleotide is accomplished via the amino functionality of the linker. Therefore, compound (12) is considered a universal linker for conjugation of oligonucleotides.

Example 4 (Scheme 4) describes the synthesis of maleimide-trialkoxysilane (19) used to functionalize the glass surface. Example 5 is a diene-trialkoxysilane (for functionalizing the glass surface (
20) is described.

Example 6 includes three 5'functionalized oligonucleotides, compounds (23), (24
) And (26) are described. Compounds (23) and (24) (Scheme 6)
Is functionalized with a diene at the 5'end by reaction with a diene-linker amidite (16). Compound (26) (Scheme 7) is functionalized with maleimide at the 5'end by reaction with maleimidopropionic acid N-hydroxysuccinimide ester (Compound 18, Scheme 4).

Examples 7 and 8 describe a method of functionalizing two glass surfaces, especially glass slides and CPG, with both dienes and dienophiles. The glass surface is derivatized with a reactive moiety capable of undergoing a cycloaddition reaction by vapor deposition of a properly functionalized silane monolayer for the cycloaddition reaction. In particular, Example 7 describes the functionalization of two glass surfaces with maleimide dienophile (19) (Scheme 8
). Example 7 also describes a method for detecting maleimide functionalized surfaces. Maleimide-derivatized surfaces are detected by staining with a thiol-fluorescein reagent, which reacts with maleimide by Michael addition as illustrated in Scheme 9. The presence of surface-bound fluorescein was then detected by a molecular dynamics Typhoon Fluorescence Scanner using a green laser that excites surface-bound fluorescein, followed by 526
Detected by detection of emitted light using a nm filter.

Example 8 describes the functionalization of two glass surfaces with a diene (20) (
Scheme 10). Example 8 also describes a method for detecting functionalized surfaces. The diene derivatized surface is detected by the Diels-Alder reaction with fluorescein-maleimide as illustrated in Scheme 11.

Example 9 (Scheme 12) describes a 5'diene functionalized oligonucleotide (23).
And the Diels-Alder reaction with maleimide functionalized glass slides and microtiter plates. This reaction is based on the Diels-
Demonstrate Alder surface immobilization (Scheme 12). Detection of the surface-bound oligonucleotide was achieved by its hybridization to a fluorescein-labeled complementary sequence (27) and detection of fluorescein. The results of this example are shown in FIG.
Shown in.

Example 10 describes maleimide-CPG and diene derivatized oligonucleotides (24
) With the Diels-Alder reaction. In Example 9, conjugation of a diene oligonucleotide to a maleimide-derivatized support was detected by fluorescence of the labeled complementary sequence after hybridization. However, while this detection method gives qualitative results, it does not allow a quantitative determination of surface loading. In Example 10, different detection methods were selected for the determination of immobilized oligonucleotides, resulting in a quantitative determination of surface loading. For example, the diene (
DMT) -oligonucleotide (24) was used. After the Diels-Alder reaction of the diene (24) with the maleimide, the amount of surface-bound oligonucleotide (loading) was measured after cleavage of the DMT group, as shown in Scheme 12, after DMT group cleavage.
Calculated by photometric detection of T-cation.

Maleimide-derivatized CPG was incubated with increasing amounts of oligonucleotide (24) to show the relationship between oligonucleotide immobilization (as CPG loading) and the amount of diene-oligonucleotide in solution. Using this method, a typical range of oligonucleotide loading was calculated to be 0.8-1.7 micromol / g (Table 2, Figure 9). The results obtained from this experiment also show that not all maleimide moieties react with diene-oligonucleotides, and by achieving an exponential dependence instead of a linear curve fit, a higher amount of (24) was obtained. It was shown that the treatment can further increase the loading.

A kinetic relationship between loading and incubation time was also shown. An exponential curve was obtained as expected, but the plateau of this curve was not reached in the time frame applied in this experiment (Table 3, Figure 10). After about 100 minutes, saturation was achieved (due to consumption of all oligonucleotide (24) in solution). The most interesting result is that a loading of 0.4 micromol / g was obtained even after an incubation time of 5 minutes. Compared to the data from the first example, the concentration of (17) applied in this experiment is too low to saturate all the maleimide sites of the CPG support.

Example 11 (Scheme 14) describes the conjugation of 5′-maleimide derivatized oligonucleotides (26) to diene coated glass surfaces. Proof of conjugation was achieved by hybridization with labeled complementary sequence (27) and detection of fluorescence, as described above.

Example 12 (Scheme 15) describes the synthesis of diene-modified oligonucleotides (29) from NHS reagent (10), the synthesis of which is described in Example 2. Example 13 (Scheme 16) describes HPLC monitoring of Diels-Alder reactions utilizing cyclohexadiene derivatized oligonucleotides. To confirm the Diels-Alder reactivity of the diene conjugate (29),
Labeling was performed using commercially available maleimido dienophiles (30) and (31). The progress of this reaction was monitored by analytical anion exchange chromatography, with samples taken every 5 minutes. Treatment of (29) with N-ethylmaleimide (30) resulted in complete conversion to the adduct (32) within 5 minutes, whereas biotin maleimide (31) required 20 minutes.

Example 14 (Scheme 17) describes a diene-modified polyethylene glycol substrate (3
The synthesis of 4) is described. Example 14 (Scheme 18) also describes the synthesis of the anthracene derivative (36).

Example 15 (Scheme 19) is a dienophile CM5 BIAcore.
The method for producing the surface of the flow cell will be described. Example 16 (Scheme 20) describes a comparison of PEG-SH (Michael addition) versus PEG-diene (Diels-Alder reaction surface immobilization) for surface derivatization.

Example 17 (Scheme 21) provides Example 16 to provide compound (38).
3 describes the Diels-Alder reaction of a dienophile-derivatized CM5 BIAcore flow cell surface (37) with an anthracene derivative (36) (preparation described in Example 14) using the method described in.

Example 18 (Scheme 22) describes a dienophile-derivatized CM5 BIAcore flow cell surface (37) of cyclohexadiene modified oligonucleotides (29).
Immobilization to and subsequent hybridization with complementary oligonucleotide sequences is described.

Example 19 (Scheme 23) describes the synthesis of anthracene-silane reagent (42). Example 20 (Scheme 24) illustrates functionalization of glass slides with anthracene-silane reagent (42).

The following examples are provided to illustrate and illustrate the present invention and should not be taken as limiting the present invention.

[0068]

【Example】

Example 1 . Preparation of Cyclic Diene Alcohol (5) Scheme 1 shows the preparation of cyclohexadiene (5). Briefly, the diene-alcohol (5) synthesis was obtained from alcohol (1) in 4 steps by a scalable and very convenient method as outlined in Scheme 1. This diene
Roth et al. ((1993) Chem. Ber. 126: 2701-2715) have been synthesized by different routes.

[0069]

[Chemical 19] Synthesis of 4- (tert-butyldimethylsilyloxymethyl) -cyclohex-1- ene (2) . 1,2,3,6-Tetrahydrobenzyl alcohol (1) (1.12 g, 10 mmol) and imidazole (1.3) in DMF (10 mL).
To the stirred solution of 6 g, 20 mmol) was added TBDMSCl (1.81 g, 12 mmol). After 20 hours, the mixture was treated with brine (100 mL) and the product extracted with hexane (3 x 80 mL). Combine the organic layers with brine (100 m
L), dried over MgSO ₄ and evaporated in vacuo to give compound (2) (2.2
6 g, 100%) was obtained as a colorless liquid. _Rf 0.29 (hexane).

[0070]

[Chemical 20] trans-1,2-dibromo-3-(tert-butyldimethylsilyl Oki Shimechiru) - cyclohexanecarboxylic (3). Bromine (0.4 mL, 8 mmol) in CCl ₄ (1 mL) was added dropwise to a stirred solution of alkene (2) (1.81 g, 8 mmol) in CCl ₄ (15 mL). The resulting red mixture was diluted with CH ₂ Cl ₂ (50 mL), 10% Na ₂ S ₂ O ₃ solution (50 mL) and water (
It was washed with 50 mL). The organic phase was dried over Na ₂ SO ₄ and evaporated in vacuo to 1.
98 g (96%) of compound (3) was obtained as a yellow oil. R _f 0.20 (1
00: 1 hexane / EtOAc).

[0071]

[Chemical 21] Synthesis of 5-hydroxymethylcyclohexa-1,3-diene (5). THF (
Dibromide (3) (69.52, 0.18 mol) and Aliq in 500 mL)
uat 336 (1.46 g, 3.6 mmol, 0.02 eq) stirred 0
KOtBu (44.89 g, 0.4 mol, 2.2 eq) was added to the solution at 0 ° C. Immediate formation of a yellow precipitate occurred and after 5 minutes the cooling bath was removed. After 30 minutes, an aliquot of this mixture was taken and dried under a stream of argon. ¹ H-NMR analysis (
C ₆ D ₆ ) showed a trace of dibromide (3) remaining. After an additional 40 minutes, the mixture was evaporated in vacuo, diluted with hexane (300 mL),
Wash with saturated NH ₄ Cl solution (300 mL). After separation, the aqueous layer was mixed with hexane (20
0 mL) and the combined organic layers were washed with water (300 mL), dried over MgSO ₄ and evaporated in vacuo to give 65 g of crude diene (4). This material is Me
Dissolved in OH (600 mL) and treated with Dowex 50WX4-50 (65 g). After shaking the slurry at 200 rpm for 140 minutes, TLC showed no residue. The resin was filtered and washed with MeOH. The filtrate was evaporated in vacuo and the remaining residue was bulb to bulb distilled under reduced pressure to give diene (
5) (16.71 g, 84%) was obtained as a colorless liquid. Bp 47 ° C (2.2
Mbar) -50 <0> C (1.7 mbar); _Rf 0.49 (1: 1 hexane / EtOAc).

[0072]

[Chemical formula 22] Example 2 . Preparation of NHS Reagent (10) Scheme 2 shows the preparation of NHS Reagent (10) from diene (5).

[0073]

[Chemical formula 23] Synthesis of 13-N- (5-cyclohexa-1,3- dienemethoxycarbonyl ) -4,7,10-trioxa-1,13-tridecanediamine (8) . THF (
To a stirred solution of diene (5) (1.10 g, 10 mmol) in 20 mL) was added 1,1'-carbonyldiimidazole (CDI) (1.63 g, 10.05 mmol). After 40 minutes, a solution of 4,7,10-trioxa-1,13-tridecanediamine (7) (4.4 mL, 20 mmol) in THF (5 mL) was added. After 50 minutes, the reaction mixture was diluted with EtOAc (50 mL) and washed with brine (
50 mL) and water (2 x 50 mL). The organic phases were combined, dried over MgSO _4, and evaporated in vacuo. CH ₂ Cl ₂ / MeOH / Et ₃ N (15: 1: 0
． Purification via chromatography on the Biotage Flash 40 system eluting in 1) gave 2.62 g (74%) of the carbamate (8) as a colorless oil.

[0074]

[Chemical formula 24] 18-N-(5-cyclohex-1,3-diene-methoxycarbonyl) -18 - Synthesis of Amino-5-aza-4-keto-9,12,15-trioxa-18-Okutade Kang acid (9). Amine (8) in pyridine (20 mL) (1.43 g, 4
Succinic anhydride (0.44 g, 4.4 mmol) was added to a solution of (mmol) and N-methylimidazole (NMI) (0.64 mL, 8 mmol). The mixture was stirred for 2 hours, evaporated in vacuo and CH ₂ Cl ₂ / MeOH / Et ₃ N (10: 1).
: 0.1) and the residue was purified by silica gel chromatography on a Biotage Flash 40 system. Et ₃ N was removed and the product was washed with MeOH
It was dissolved in (20 mL) and treated with Dowex MWC-1 (2 g) for 1 hour. The solid was filtered and the filtrate evaporated in vacuo to give 1.50 g (82%) of acid (9) as a slightly yellow oil.

[0075]

[Chemical 25] 18-N-(5-cyclohex-1,3-diene-methoxycarbonyl) -18 - amino-5-aza-4-keto-9,12,15-trioxa-18-Okutade Kang acid · N-hydroxysuccinimide ester ( Synthesis of 10) . CH ₂ Cl ₂ (
Acid (9) (913 mg, 2 mmol), N-hydroxysuccinimide (230 mg, 2 mmol) and DMAP (12 mg, 0.1 mmol) in 10 mL).
To a stirred solution of dicyclohexylcarbodiimide (433 mg, 2
． 1 mmol) was added. After 5 minutes, the cooling bath was removed. After 2 hours, TLC showed incomplete conversion, so additional N-hydroxysuccinimide (46 mg, 0.
4 mmol) and dicyclohexylcarbodiimide (83 mg, 0.4 mmol) were added. After 22 hours, TLC analysis still showed incomplete conversion, so
Further N-hydroxysuccinimide (46 mg, 0.4 mmol) and dicyclohexylcarbodiimide (83 mg, 0.4 mmol) were added. After 3 hours
The mixture was filtered through Celite and the precipitate washed with CH ₂ Cl ₂ (10 mL). The filtrate was evaporated in vacuo, the residue was dissolved in EtOAc (5 mL), filtered again and evaporated in vacuo. Hexane / acetone gradient (1: 1 → 1: 0.8
Flash chromatographic purification using a Biotage Flash 40 system eluting at 1: 0.6) yielded 370 mg (33%) of NHS-ester (10) as a colorless oil. R _f 0.45 (10: 1: 0.2 C
HCl ₃ /MeOH/AcOH),0.32(1:2 hexane / acetone).

[0076]

[Chemical formula 26] Example 3 . Synthesis of Diene Phosphoramidite (16) The synthesis of diene-phosphoramidite (16) suitable for attachment to the 5'end of an oligonucleotide is outlined in Scheme 3. Briefly, starting from a commercially available enantiomerically pure alcohol (11), LiAlH ₄ reduction gave the generic linker (12). The crude amine was acylated with the diene-carbamate (6) generated in situ by reaction of the diene (5) with carbonyldiimidazole (CDI) as described in Example 1. Then, the crude product diol (
Acetylation of 13) allowed purification by flash-chromatography. After purification, the acetate was cleaved and the crude diol was selectively tritylated with 4,4'-dimethoxytrityl chloride (DMT) to give alcohol (15). The alcohol (15) is then added to 2-cyanoethyl-bis- (N, N-diisopropylamino) -phosphoramidite and 4,5-dicyanoimidazole (D
CI) to produce the amidite (16), which was then purified by flash-chromatography.

[0077]

[Chemical 27] Synthesis of (S) -5-amino-pentane-1,3-diol (12) . LiAl
H ₄ (26.5 g, 0.70 mol) was suspended in anhydrous THF (250 mL) and
Cooled to ° C. Alcohol (11) (20) dissolved in anhydrous THF (100 mL)
． 0 g, 0.13 mol) was added drop by drop. The mixture was warmed to room temperature and stirred overnight. After cooling to 0 ° C., the mixture was H ₂ O (12.6 mL, 0.70 mol), 10N NaOH (34.9 mL, 0.35 mol), and H ₂ O (37.
8 mL, 2.10 mol). The resulting solid was removed by filtration and the filtrate was concentrated in vacuo. The recovered material (12.7 g, 84%) was the desired amine-
It was consistent with the diol (12).

[0078] (S) -O- cyclohex-2,4 Jienirumechiru-N-(3,5-Jiase Tokishipenchiru) - Synthesis of carbamate (14). CDI to a solution of diene-alcohol (5) (2.0 g, 18.2 mmol) in DMF (17 mL) (3.
5 g, 21.6 mmol) was added to diene-carbamate (6) in si
generated with tu. After stirring for 2 hours, crude amine (12) (2.0 g, 16.8 mmol) was added and stirring was continued overnight. The mixture was concentrated in vacuo and the residue was CH ₂
Dissolved in Cl ₂ (55 mL). After addition of Et ₃ N (23.4 mL, 168 mmol), the mixture was cooled to 0 ° C. and treated with Ac ₂ O (15.9 mL, 168 mmol). The solution was warmed to room temperature and stirring was continued overnight. The reaction mixture was washed with saturated NaHCO ₃ solution (50 mL) and brine (50 mL), dried over MgSO ₄ and concentrated in vacuo. The crude product was then purified by flash chromatography on silica gel (Biotage Flash 40 system), eluting with EtOAc / CH ₂ Cl ₂ (1: 4 v / v) to give 1.9 g (33%) of carbamate ( 14) was obtained as a colorless oil:

[0079]

[Chemical 28] (S) -O- cyclohex-2,4 Jienirumechiru -N- [5- (4,4 '- dimethoxytrityl) -3-hydroxypentyl] - Synthesis of carbamate (15). Diacetate (14) (1.9 g, 5.6 mmol) was added to MeOH (30 m).
It was dissolved in L) and K ₂ CO ₃ (39 mg, 0.3 mmol) was added. After 2 hours, the mixture was concentrated in vacuo and the residue dried by azeotropic distillation with pyridine (2 x 20 mL). This crude diol was dissolved in pyridine (37 mL) and treated with 4,4'-dimethoxytrityl chloride (3.77 g, 11.1 mmol). The reaction was stirred overnight then concentrated to a residue. This reddish brown oil is EtO
Dissolve in Ac (50 mL), sat. NaHCO ₃ solution (50 mL) and brine (50
(mL). The organic layer was dried over MgSO _4, filtered, and concentrated in vacuo. The crude residue was purified by flash chromatography on silica gel (Biotage Flash 40 system), eluting with EtOAc / CH ₂ Cl ₂ (1: 9 v / v) to give 1.0 g (32%) of alcohol (15%). ) Was obtained as a slightly yellow oil:

[0080]

[Chemical 29] (S) -O- cyclohex-2,4 Jienirumechiru -N- {3 - [(2- sheet Anoetokishi) - diisopropylamino phosphite Fano] -5- (4,4'-dimethoxyethane Kishitorichiru)} - 3-hydroxypentyl ] -Synthesis of carbamate (16) .
A solution of alcohol (15) (1.00 g, 1.8 mmol) in anhydrous CH ₂ Cl ₂ (18 mL) was added to 2-cyanoethyl-bis- (N, N-diisopropylamino).
Treatment with phosphoramidite (0.60 g, 2.0 mmol) followed by 4,5-dicyanoimidazole (0.11 g, 0.9 mmol). After stirring for 1 hour, TLC analysis showed complete conversion of (15). This mixture was added to CH ₂ Cl ₂ (3
0 mL), washed with saturated NaHCO ₃ solution (30 mL) and brine (30 mL), dried over MgSO ₄ and concentrated in vacuo. This crude residue was treated with EtOA.
Purified by flash chromatography on silica gel (Biotage Flash 40 system), eluting with c / CH ₂ Cl ₂ (1: 9 v / v), 933
Obtained mg (68%) of the amidite (16) as a colorless foam:

[0081]

[Chemical 30] Example 4 . Synthesis of Maleimide-Silane Reagents Scheme 4 illustrates the synthesis of maleimido-silane reagents for functionalizing glass surfaces. Briefly in connection with Scheme 4, propylaminosilane (17)
Was reacted with a functionalized maleimide N-hydroxysuccinimide-ester (18) to give maleimide-silane (19) after aqueous work-up, which was used as the crude product of the derivatization of glass surfaces.

[0082]

[Chemical 31] 3- maleimido-N-(3-triethoxysilylpropyl -lH-propyl) - Puropion'a Synthesis of bromide (19). 3-Maleimidopropionic acid N-hydroxysuccinimide ester (18) (4.0 g, 15.03 mmol, 1.0 eq) was added to N, N-.
Dissolved in dimethylformamide (75 mL), added 3-aminopropyltriethoxysilane (17) (5.06 mL, 15.03 mmol, 1 eq) and allowed the reaction mixture to stir for 22 hours. An aliquot (0.15 mL) was taken in a syringe, concentrated in vacuo and the resulting oil was analyzed by ¹ H-NMR spectroscopy. This oil was consistent with the desired product. The reaction mixture was then concentrated in vacuo at 50-55 ° C. and the resulting residue was dissolved in CH ₂ Cl ₂ (75 mL), water (3 × 50 mL).
) And brine (50 mL), filtered through granular Na ₂ SO ₄ and concentrated under reduced pressure in vacuo to give 6.65 g (> 10 according to residual N, N-dimethylformamide).
0%) of crude maleimido-silane (19) was obtained as a light yellow oil. This crude product (19) was used without purification.

[0083]

[Chemical 32] Example 5 . Synthesis of Diene-Silane Reagents Scheme 5 illustrates the synthesis of diene-silane reagents for functionalizing glass surfaces. Briefly in relation to Scheme 5, the diene (5) was treated with carbonyldiimidazole as in the synthesis of the linker (13). The amine (17) was added to the imidazolate formed in situ. After stirring the product of this reaction mixture overnight, the diene-silane (20) was isolated and used as a crude product for glass surface functionalization.

[0084]

[Chemical 33] O- cyclohex-2,4 Jienirumechiru-N-(3- triethoxysilylpropyl silanylene Rupuropiru) - Synthesis of carbamate (20). Diene alcohol (5) (0.5
0 g, 4.54 mmol, 1.0 equivalent) was added to N, N-dimethylformamide (15
mL) and carbonyldiimidazole (0.77 g, 4.77 mmol,
1.05 eq) was added and the mixture was allowed to stir for 4 hours. The formation of the imidazolate intermediate (6) (Scheme 2) was confirmed by ¹ H-NMR spectroscopy of an aliquot (0.3 mL) of the reaction mixture concentrated in vacuo to an oil. This oil was consistent with the desired intermediate by ¹ H-NMR spectroscopy as evidenced by the chemical shift of the resonance of the methyl proton at 3.6 ppm to 4.3 ppm. Then, 3-aminopropyltriethoxysilane (17) (1.51 mL,
4.50 mmol, 1.0 eq) was added and the reaction mixture was stirred for 12 hours.
The formation of the desired product (20) was confirmed by ¹ H-NMR spectroscopy of an aliquot (0.3 mL) of the reaction mixture concentrated in vacuo to an oil. This oil was consistent with the desired product as evidenced by the chemical shift in resonance of the methylene protons at 4.3 ppm to 4.0 ppm. The reaction mixture was concentrated under vacuum vacuum overnight and the imidazole crystallized from the resulting brown oil. The resulting oil is separated from the crystals by pipette and transferred to a clean round bottom flask,
Obtained 1.5 g (93%) of the desired diene-silane (20) as a brown oil. This crude product (20) was used without purification.

[0085]

[Chemical 34] Example 6 . Synthesis of Labeled Oligonucleotides The synthesis of various labeled and unlabeled (control experiments) oligonucleotides is illustrated in Schemes 6 and 7 below. All syntheses were performed utilizing standard methods of solid phase oligonucleotide synthesis using a phosphoramidite building block and a CPG solid support. The 20-mer (22) (SEQ ID NO: 1) was chosen for illustration purposes. Referring to Scheme 6, a standard protocol was performed to synthesize a DNA oligonucleotide (22) on CPG, resulting in a CPG-bound oligonucleotide (21). An aliquot of (21) was deprotected and cleaved from the CPG support to give the crude control oligonucleotide (22). Extension of the 5'-end of (21) with a diene-linker amidite (16) resulted in diene-labeled oligonucleotide (2
3) was synthesized. After further treatment and detritylation / cleavage, the crude oligonucleotide was subjected to AX-HPLC to give 86% of pure (23).

In a second experiment, the diene-functionalized CPG linked oligonucleotide from step c was divided into two aliquots. The first aliquot was deprotected without detritylation to give the crude diene (DMT) -oligonucleotide (24) (Scheme 6) and the second aliquot was treated as above. The crude diene-oligonucleotide (23) was obtained without purification.

[0087]

[Chemical 35] Note: a: coupling; b: oxidation; c: capping; d: detritylation; e:
Deprotection and cleavage from CPG; f: Purification A maleimide functionalized oligonucleotide was synthesized as illustrated in Scheme 7 to perform the Diels-Alder cycloaddition in the opposite direction. Regarding Scheme 7, a canonical sequence was synthesized on a CPG support and extended with a protected amino-linker. After deprotection and cleavage from the CPG support, the 5'-aminohexyl oligonucleotide product (25) was acylated with maleimide NHS-ester (18). The crude maleimide-oligonucleotide (26) was obtained after filtration through Sephadex.

To allow fluorescent detection of immobilized oligonucleotides by hybridization with dye-labeled complementary sequences, a 20-mer 5′-fluorescein-CAC
ACACACACACACACACACA-3 '(27) (SEQ ID NO: 2) was synthesized on a CPG support utilizing standard protocols.

[0089]

[Chemical 36] Synthesis of 5'-TGTGTGTGTGTGTGTGTGTGTG-3 '(22) (SEQ ID NO: 1) . Oligonucleotide (22) was synthesized by standard phosphoramidite method on a 160 micromolar scale on an 8800 DNA / RNA synthesizer (Millipore). After the final detritylation and subsequent washing, an aliquot of CPG-conjugated oligonucleotide (21) was removed, dried and deprotected with 27% aqueous NH ₄ OH at 55 ° C. for 15 hours. Anion exchange HPLC analysis showed the crude oligonucleotide (22) to be 78% pure (at 260 nm).

Synthesis of 5'-diene-oligonucleotide (23) . The synthesis of compound (23) was carried out in a fluid bed conformation on a Millipore 8800 synthesizer. The CPG bound oligonucleotide (21) as described in (160 .mu.mol) of the amidite _{(16) (0.2M / CH 3} CN, 2.5 eq) and 4,5-dicyano-imidazole (1.0 M / CH ₃ CN, 16 eq) for 10 minutes,
Second amidite _{(16) (0.2M / CH 3} CN, 1.5 eq) was treated 10 min by adding together the 4,5-dicyano imidazole (1.0M / CH ₃ CN, 16 equiv). The support-bound oligonucleotides were oxidized with I ₂ in the presence of pyridine under standard conditions and detritylated by treatment with 10% dichloroacetic acid in CH ₂ Cl ₂ . Cleavage from the support and deprotection of the base was carried out with 27% NH ₄ OH.
It was carried out at 70 ° C. for 2 hours using an aqueous solution. The deprotected solution was then cooled to 4 ° C., then the CPG was filtered and washed with DI water to recover the crude oligonucleotide product, compound (23).

An aliquot (800 μL) of this crude oligonucleotide product was added to 10.6
Loaded on a DNAPac PA-100 4x250 mm anion exchange column at a concentration of mg / mL. 2-buffer system (where Buffer A is 10% CH ₃ CN
Containing 25 mM Trizma / 1 mM EDTA (Trizma-EDTA)
And buffer B is Trizma-containing 10% CH ₃ CN and 1M NaCl.
The product was eluted with a linear elution gradient utilizing EDTA). A temperature of 80 ° C. was maintained throughout the purification. The appropriate purified fractions were combined and desalted on a Nap25 column. After desalting, oligonucleotide (23) (designated as "23, pure") in deionized water at a concentration of 1.1 OD / mL (86% purity by anion exchange HPLC).
I chose MALDI-MS: M ⁺ 658.47 (calculated: 6585.1).

In the second synthesis of oligonucleotide (23), CPG-conjugated oligonucleotide (21) (25 μmol) was added to amidite (16) (0.2 M / CH ₃ CN).
, 15 equivalents) and 4,5-dicyanoimidazole (1.0 M / CH ₃ CN, 16
Equivalent) for 20 minutes. After oxidation and capping, the CPG binding oligonucleotide was fractionated into two aliquots. A first aliquot (0.25 g, 6 μmol) was subjected to cleavage from the support and deprotection of the base to give 17 mg (450 OD) of crude (60% pure by anion exchange HPLC) diene (DMT) -oligonucleotide. (24) was obtained after drying. Second aliquot (0.80g, 1
9 micromol) was detritylated prior to cleavage from the support and deprotection of the base to yield 89 mg (2400 OD) of crude oligonucleotide (23% crude by anion exchange HPLC) (23) (“23, crude ").

Synthesis of 5 ′-[N- (3-maleimidopropionyl) -6-aminohexyl] -TG TGTGTGTGTGTGTGTGTGG- 3 ′ DNA oligonucleotide (26 ) . 2-cyanoethyl -N, N'-diisopropyl-6- (4-methoxytrityl - amino) -. Hexyl phosphoramidite ^* (0.2M / CH ₃ CN, each 10 eq .Proligo Biochemie GmbH, Hamburg, Germany Product Number the M010282), 4,5-dicyano imidazole activator _{(1.0M / CH 3 CN, 16} 20 min in the presence of eq) was coupled to the CPG bound oligonucleotide (21) (32 micromolar). After standard oxidation and capping, a final monomethoxytrityl removal with 10% dichloroacetic acid in CH ₂ Cl ₂ was done. Cleavage of the oligonucleotide from the support in 27% aqueous NH ₄ OH and deprotection followed by drying gave 155 mg (4200
OD) of crude amino-oligonucleotide (25) (purity 55% by anion exchange HPLC) was obtained.

Amino-oligonucleotide (25) (2.6 μmol) was added to DMF (2 m
L). To this solution maleimide NHS-ester (18) (35 mg
, 130 μmol, 50 eq) was added and the reaction was stirred overnight at room temperature then placed in a high speed vacuum to remove the solvent. The dried pellet was then washed with H ₂ O (1
． 5 mL) and loaded onto four 1 mL G-25 spin columns to react the reaction maleimide-oligonucleotide product (26) to unreacted (18).
And by-products were removed.

Detection of maleimide by fluorescence . Deprotected 5-((2- and -3) -S- (acetylmercapto) succinoyl) maleimide-oligonucleotide (26) (15 nmol, about 500 OD)
Amino) fluorescein) (SAMSA fluorescein reagent). S
AMSA Fluorescein Reagent is a protected SAMSA Fluorescein Reagent (500
μM) with 0.1 M NaOH (50 μL), followed by a reaction at room temperature for 15 minutes, followed by 6N HCl buffered with 0.5 M Na ₂ HPO ₄ buffer (10 μL, pH = 7.0). Deprotection was achieved by neutralization with (0.7 μL). This completely deprotected SAMSA-fluorescein was converted to compound (26) (20 μm).
L, 15 nmol) and incubated at room temperature for 30 minutes. Unreacted SAMSA-fluorescein was removed by loading the reaction mixture onto a 1 mL G-25 spin column pre-equilibrated with phosphate buffered saline. The product fluorescein stained (26) (1, 2, and 3 μL) was loaded on a 15% polyacrylamide-Tris borate EDTA (TBE) gel and scanned on a Typhoon Molecular Dynamics scanner for detection. . After exciting the surface-bound fluorescein with a green laser, the emitted light was detected with a 526 nm filter (SP fluorescein filter). The photomultiplier tube was set to 600 V, the sensitivity was set to normal, and the focal plane was set to the surface. The result is shown in FIG. The presence of fluorescent signal indicates that (25) successfully reacted with reagent (18), creating the maleimide-oligonucleotide (26).

Synthesis of 5'-fluorescein-CACACACACACACACACACA-3'DNA oligonucleotide (27) (SEQ ID NO: 2) . Two separate 1 micromolar syntheses were prepared using the standard phosphoramidite protocol, Expe
It was performed on a site DNA / RNA synthesizer. 5'-fluorescein - phosphoramidite (0.1M / CH ₃ CN, 11.2 eq, Glen Research, VA,
Obtained from the USA, product number 105901, tetrazole (0.25M /
CH ₃ CN, 102 5 min in the presence of eq) was coupled to CPG- binding 20-mer. Anion exchange HPLC by deprotection with 27% aqueous NH ₄ OH.
This gave approximately 300 OD of crude product for purification. 20 mM NaClO ₄ (buffer A) containing 20 mM NaOAc and 10% by volume CH ₃ CN and 20 mM
Purification using a buffer system of 600 mM NaClO ₄ (buffer B) containing NaOAc and 10% by volume of CH ₃ CN yielded 75.7 ODs of oligonucleotide product (27) from Synthesis No. 1 (purity by anion exchange HPLC). 96.6%), and synthesis number 2 yielded an oligonucleotide product (27) of 79.3 OD (80% pure by anion exchange HPLC). Both oligonucleotides are Nap10
It was desalted on a column and finally evaporated on a high speed vacuum concentrator.

Example 7 Functionalization of the glass surface with maleimide Scheme 8 shows the maleimide-silane (1 of either glass slide or CPG).
9 illustrates the reaction with 9). The functionality of maleimide is described in maleimide-silane (19).
Is introduced onto the glass slide and CPG by condensation with the glass surface.

[0098]

[Chemical 37] The method used to detect the maleimide functionalization of the glass surface is to stain the glass surface with a thiol-containing fluorescein reagent, which reacts with a surface-bound maleimide via a Michael addition reaction, as illustrated in Scheme 9. Accompanied by. Thus, the presence of surface-bound fluorescein is detected by molecular dynamics typhoon Fluorescence Scanner using a green laser that excites surface-bound fluorescein, followed by detection of emitted light using a 526 nm filter. Can be detected.

[0099]

[Chemical 38] Pretreatment of micro slide glass . Method No. 1: Micro slide glass (Corning, No. 2947, description: flat plate, washed, 3 inch x 1 inch x 1 millimeter) was immersed in 2N NaOH for 2 hours at ambient temperature, washed with water and in boiling 2N HCl. It was soaked in water for 1 hour, washed with water and methanol, and then dried in a high vacuum oven at 100 ° C. for 2 hours under reduced pressure. Then this slide
Cooled in a vacuum dessicator until use. Method number 2: 2 micro slides
Immerse in N HCl at ambient temperature for 2 hours, then in boiling 2N HCl for 1 hour, wash with water and methanol, then in a high vacuum oven at 100 ° C. under reduced pressure.
It was dried at ° C for 2 hours. The slide was then cooled in a vacuum dessicator until use.

Maleimide functionalization of glass slides . Microslides (two pretreated according to method number 1 and two pretreated according to method number 2) were added to toluene (25
It was placed upright into a slide chamber containing a 1% (v / v) solution of the maleimido-silane reagent (19) in (mL). The amount of solution used was sufficient to soak only the bottom half of each slide (about 1.5 inches). After 16 hours, remove one of the slides (pretreated according to Method No. 1) from the chamber and wash with toluene,
Methanol, methanol / water (1: 1, v / v), water, methanol / water (1: 1)
, V / v), methanol, and ethyl acetate sequentially (both sides of the slide were pipetted with 3 × 2 mL of each solvent). The slide was then air dried.

Detection of surface-maleimide by fluorescence . A thiol-containing fluorescein reagent, 5-((2- and -3) -S- (acetylmercapto) succinoyl) amino) fluorescein) (SAMSA fluorescein, Molecular Probes)
Was used to assay maleimide functionalization of slides. S on one side of slide
AMSA Fluorescein Activation Solution (1 mL 0.1 M NaOH, 14 μL
6M HCl, 0.2mL 0.5M sodium phosphate buffer (pH = 7)
(Prepared from 10 mg in) and incubated for 30 minutes at ambient temperature. Then, the slide was immersed in 0.5 M sodium phosphate buffer, pH 7 for 30 minutes while shaking, blotted with a fine tissue paper and dried. The slide was then placed on the surface of a Molecular Dynamics Typhoon fluorescence scanner. After exciting the surface-bound fluorescein with a green laser, the emitted light was detected with a 526 nm filter (SP fluorescein filter). The photomultiplier tube was set to 600 V, the sensitivity was set to normal, and the focal plane was set to the surface.

This slide showed a strong reaction, mainly in the lower half of the slide, consistent with the functionality of maleimide (FIG. 2). A strong reaction with the midline of the slide and the clear line below it indicates successful maleimide functionalization of the slide. This slide also shows the maleimide on the edge of the slide, probably due to contact with the surface of the chamber, and slightly above the intermediate mark, where the slide may have come into contact with the solution while moving the slide in and out of the chamber. There was also a weak reaction consistent with functionality. After 19 hours, the remaining slides were washed as above in a solution of toluene containing the maleimido-silane reagent (19), then pyridine / THF (1: 9).
, V / v) for 5 minutes to cap free silanol groups. The slides were then washed with THF, methanol, and ethyl acetate (both sides of the slides were pipetted 3x2 of each solvent).
washed with mL), air dried and stored in a vacuum dessicator until use.

Pretreatment of native CPG-500 . Native CPG-500 was stirred in boiling 2N HCl for 2 hours, then collected in a glass frit funnel, washed with water and methanol and dried in a high vacuum oven under vacuum at 100 ° C. for 2 hours. The CPG was then cooled in a vacuum dessicator until used.

Maleimide functionalization of CPG . CPG (0.75 g, pretreated according to the method described above) was stirred for 51 h in a 1% by volume solution of the maleimido-silane reagent (19) in toluene (25 mL). The CPG was then collected in a glass frit funnel, toluene, methanol, methanol / water (1: 1, v / v), water, methanol / water (1: 1, v / v), methanol, and ethyl acetate ( 3x1 for each solvent
It wash | cleaned in order with 0 mL). Then, this powder was mixed with pyridine / THF (1: 9, v /
Treatment with a 5% by volume solution of chlorotrimethylsilane in v) for 2 minutes capped the free silanol groups. The CPG was then washed with THF, methanol, and ethyl acetate (3 x 10 mL of each solvent). The powder was air dried in a funnel under suction for 5 minutes, transferred to a beaker and placed in a vacuum dessicator for 44 hours. This light brown powder (0.6g) was then stored in a -20 ° C freezer until used.

Detection of surface maleimide by fluorescence . A thiol-containing fluorescein reagent,
5-((2- and -3) -S- (acetylmercapto) succinoyl) amino)
Fluorescein) (SAMSA Fluorescein) was used to assay the maleimide functionalization of derivatized CPGs. Place CPG (5 mg) in a centrifuge tube and use SAMSA.
An activating solution of fluorescein (0.5 mL) was added, the vial was shaken to mix the contents and allowed to stand for 30 minutes at ambient temperature. Native CPG-500 (5 mg) was also placed in the centrifuge tube and the activation solution of SAMSA fluorescein (0.5 mL of 0.1 M) was added.
Treated with NaOH, 7 μL of 6 M HCl, 0.1 mL of 5 M in 0.5 M sodium phosphate buffer (pH = 7)) and served as a control according to the same method. After 30 minutes, the mixture was centrifuged, the supernatant was pipetted off, and the CPG sample was suspended in water. The resulting mixture was centrifuged, the supernatant was again pipetted off and the CPG sample was resuspended in water. This procedure was repeated until the supernatant was clear and colorless (a total of 5 washes for each sample). Each powder was sprinkled on clear plastic wrap paper, which was folded to contain the powder. Both samples were then placed side by side on the surface of a Molecular Dynamics Typhoon fluorescence scanner. After exciting the surface-bound fluorescein with a green laser, the emitted light was detected with a 526 nm filter (SP fluorescein filter). The photomultiplier tube was set to 600 V, the sensitivity was set to normal, and the focal plane was set to the surface. CPG treated with maleimido-silane showed a strong response consistent with the functionality of maleimide, whereas the native CPG-500 control did not (Fig. 3). Native CPG
The strong reaction on CPG treated with maleimido-silane relative to the -500 control was:
It shows successful maleimide functionalization of CPG.

Example 8 Functionalization of a glass surface with a diene Scheme 10 shows a glass slide with a diene-silane (20) to provide a support-bound diene capable of undergoing Diels-Alder surface immobilization of dienophile and 6 illustrates the functionalization of CPG. As illustrated in Scheme 10, condensation of the diene-silane (20) with the glass surface introduces the functionality of the diene onto the glass slide and CPG.

[0107]

[Chemical Formula 39] The method used to detect the diene-functionalized glass surface is to stain the glass surface with a maleimide-containing fluorescein reagent that reacts with a surface-bound diene via a Diels-Alder addition reaction, as illustrated in Scheme 11. With that. Therefore, the presence of surface-bound fluorescein can be detected by molecular dynamics Typhoon fluorescence scanner using a green laser that excites surface-bound fluorescein, followed by detection of emitted light using a 526 nm filter.

[0108]

[Chemical 40] Functionalization of glass slides with diene-silane reagent (20) . A microslide glass (pretreated according to Method No. 1 described in Example 7) was placed upright into a slide chamber containing a 1% by volume solution of diene-silane reagent (20) in toluene (25 mL). placed. Use the amount of solution used in the bottom half of each slide (
Only about 1.5 inches) was enough to pickle. After 15 hours, as each slide was removed from the chamber, a "T" was carved in the right corner of its top. The slide was 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). Was washed with toluene (3 x 2 mL) with a pipette and the remaining washes were
This was done by soaking the slide in a Petri dish containing 10 mL of solvent).
. The slides were then air dried and treated with a 5% by volume 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 pipetted with 3 × 2 mL of each solvent), air dried, and stored in a vacuum dessicator until use.

Detection of surface dienes by fluorescence . Fluorescein-5-maleimide (Molecular Probes) was used to assay diene functionalization on one of the slides. Briefly, one side of the slide was completely coated 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 (4 x 10 mL), blotted with fine tissue and dried. The slide was then placed on the surface of a Molecular Dynamics Typhoon fluorescence scanner. After exciting the surface-bound fluorescein with a green laser, a 526 nm filter (
The emitted light was detected with a SP fluorescein filter). The photomultiplier tube was set to 600 V, the sensitivity was set to normal, and the focal plane was set to the surface. This slide showed a strong reaction in the lower half of the slide, consistent with the functionality of the diene (Figure 4). A strong reaction with the mid-mark of the slide and the underlying clear line indicate successful diene functionalization of the glass slide.

Diene functionalization of CPG . CPG (0.75 g, pretreated according to the method described in Example 7) was stirred for 52 hours in a 1% by volume solution of diene-silane reagent (20) in toluene (25 mL). The CPG was then collected in a glass frit funnel, toluene, methanol, methanol / water (1: 1, v / v), water, methanol / water (1: 1, v / v), methanol, and ethyl acetate ( 3x1 for each solvent
It wash | cleaned in order with 0 mL). Then, this powder was mixed with pyridine / THF (1: 9, v /
Treatment with a 5% by volume solution of chlorotrimethylsilane in v) for 2 minutes capped the free silanol groups. The CPG was then washed with THF, methanol, and ethyl acetate (3 x 10 mL of each solvent). The powder was air dried in a funnel under suction for 5 minutes, transferred to a beaker and placed in a vacuum dessicator for 44 hours. This white powder (0.6g) was then stored in a -20 ° C freezer until use.

Detection of surface dienes by fluorescence . Fluorescein-5-maleimide (Molecular Probes) was used to assay the diene functionalization of derivatized CPGs. C
PG (5 mg) was placed in 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 overnight at 6 ° C. The mixture was then centrifuged, the supernatant removed with a pipette and the CPG sample suspended in water. Centrifuge the resulting mixture,
The supernatant was pipetted off again and the CPG sample was resuspended in water. This method was repeated until the supernatant was clear and colorless (5 washes in total). This powder was sprinkled on clear plastic wrap paper, which was folded to contain the powder.
Native CPG-500 (5 mg) was also treated with fluorescein-5-maleimide to serve as a control. It was incubated in a 10 mM solution of fluorescein-5-maleimide in DMF (0.5 mL) for 2 hours at ambient temperature and then treated according to the above method of treating CPG with diene-silane. Then
Both samples were placed side by side on the surface of a Molecular Dynamics Typhoon fluorescence scanner. After exciting the surface-bound fluorescein with a green laser, the emitted light was detected with a 526 nm filter (SP fluorescein filter). Set the photomultiplier tube to 600V and set the sensitivity to normal,
The focal plane was set on the surface. CPGs treated with diene-silane showed a strong response consistent with the functionality of the diene (Figure 5A), whereas the native CPG-500 control showed no response (Figure 5B). Versus native CPG-500 control,
The strong reaction on CPG treated with diene-silane indicates that the diene functionalization of CPG was successful.

Example 9 Immobilization of Oligonucleotides via Diels-Alder Cycloaddition Scheme 12 illustrates the conjugation of diene oligonucleotides (23) to maleimide-functionalized glass slides and maleimide-coated microtiter plates. Proof of conjugation was achieved by hybridization with a labeled complementary sequence and detection of fluorescence.

[0113]

[Chemical 41] Conjugation of diene-oligonucleotide (23) to maleimide functionalized glass slides . Two maleimide coated slides (one pretreated according to Method No. 1 and one according to Method No. 2 as described in Example 7) were provided with three silicon rubber septa (one septa) (standard taper 14/20) was attached. The septum was secured to the slide using plastic ties. Two septa were fixed to the maleimide-functionalized lower half of each slide and one septum was fixed to the non-functionalized upper half of each slide. Septa were contoured into slides pretreated according to method number 2. 100 mM Na ₂ H
PO ₄ buffer (pH = 6.5) in the 5'-diene - oligonucleotides (23)
, Pure 4 pmol / μL solution (125 μL) was added to the septum located at the bottom of each slide to demonstrate surface immobilization of oligonucleotides on microslides (FIG. 6). A 4 picomole / μL solution (125 μL) of control oligonucleotide (22) (SEQ ID NO: 1) in 100 mM Na ₂ HPO ₄ buffer (pH = 6.5) was applied to the septum located in the center of each slide as a control. In addition, potential non-specific binding of the oligonucleotide to the functionalized portion of the slide was checked. 100 mM N
a ₅ HPO ₄ buffer (pH = 6.5) 5′-diene-oligonucleotide (2
3) Pure 4 pmol / μL solution (125 μL) was added to the septum placed on top of each slide as a control to check for potential non-specific binding of (23) to the non-functionalized portion of the slide. The slides were incubated at 37 ° C. for 1 hour, then the septum was removed and the slides were placed in Tween® 20 (TBST) containing TRIS-buffered saline (10 mM TRIS-Cl, pH 8, 150 mM NaCl, 0. Each was soaked 3 times in Petri dishes containing 1% Tween® 20).

Detection of oligonucleotide immobilization on glass slides . The slide was pulled from the buffer solution and placed in a Petri dish containing 0.1% sodium dodecyl sulfate (SDS).
) Containing 5X standard citrate saline (SSC) (750 mM NaCl, 75 m
M sodium citrate, pH = 7, 0.1% SDS) 4 pmol / μL solution of complementary 5′-fluorescein-oligonucleotide (27) (about 10 mL).
Immediately soak (to prevent slides from drying). This slide at 55 ℃ 3
Incubate for 0 minutes and then soak 3 times each in Petri dishes containing TBST. The slide was then placed on the surface of a Molecular Dynamics Typhoon fluorescence scanner. After exciting fluorescein with a green laser, emitted light was detected with a 526 nm filter (SP fluorescein filter). The photomultiplier tube was set to 800 V, the sensitivity was normal, and the focal plane was set on the surface. Each slide showed a strong reaction when the diene-oligonucleotide (23) was contacted with the maleimide functionalized portion of the glass slide (27) (Figure 7). The two controls on each slide showed no reaction, indicating no (22) nonspecific binding to the maleimide functionalized portion of the slide and no (23) nonspecific binding to the nonfunctionalized portion of the slide. Clarified The contour of the incised partition on the slide pre-treated according to method number 2 (slide "2") is visible in the scan.

Conjugation of diene-oligonucleotide ( 23) to maleimide coated microtiter plates . 200 μL of either 5′-diene-oligonucleotide (23), crude and pure, or control oligonucleotide (22) was added to 100 mM Na ₂ HPO ₄ buffer (pH = 5.5, 6 as indicated). .5, or 7.7) to a maleimide microtiter plate (Pierce, Cat # 15150ZZ). 100 mM Na ₂
Wells containing only HPO ₄ (pH = 6.5) were also included as a control for non-specific binding of labeled complementary oligonucleotide (27). Plates were incubated for 2 hours at 37 ° C. for conjugation via the Diels-Alder reaction. The well containing the immobilized oligonucleotide was aspirated, and TBST (10 mM
Tris-Cl, pH = 8.0, 150 mM NaCl, 0.1% Tween
20) and washed 3 times.

Detection of oligonucleotide immobilization on maleimide coated microtiter plates . An equimolar amount of labeled complementary oligonucleotide (27) was added to 5xSSC + 0.1.
% SDS (750 mM NaCl, 75 mM Na citrate, pH = 7.0)
Was added to the wells containing cells and hybridized at 55 ° C. for 30 minutes. After hybridization, all samples were washed with TBST (3 x 200 mL).
Place the plate and slide on the surface of a Molecular Dynamics Typhoon fluorescence scanner and excite the fluorescein with a green laser to 526 nm.
The emitted light was detected with a filter (SP fluorescein filter). The photomultiplier tube was set to 800 V, the sensitivity was set to normal, and the focal plane was set to the surface. As shown in FIG. 8, the immobilization of the oligonucleotide has a pH of 6.5.
Was the most efficient in. The control oligonucleotide (22) reaction set, like all buffer controls, showed no non-specific binding. Maleimide functionalized slides also demonstrate oligonucleotide immobilization as evidenced by the signal produced only from the area of the slide that reacted with the silane-maleimide reagent (19).

Example 10 Conjugation of 5'-diene-oligonucleotide (24) to maleimide-functionalized CPG Incubation of maleimide-CPG with oligonucleotide (24) forms a Diels-Alder adduct, as illustrated in Scheme 13. Immobilization was verified by photometric determination of DMT after cleavage with acid.

[0118]

[Chemical 42] Titration of maleimide-functionalized CPG with 5'-diene-oligonucleotide (24) . A series of increasing amounts of diene (DMT) -oligonucleotide (24) (Table 2
(See above) is added to Na ₂ HPO ₄ buffer (100 mM, pH = 6.5, 350 μL).
) Was added to maleimide-derivatized CPG (10 mg each) and weighed in a centrifuge tube. The mixture was incubated at 37 ° C. for 1 hour with shaking (to keep optimal mixing of CPG). Each of the mixtures was centrifuged and the supernatant was pipetted off. The CPG sample was suspended in water (1 mL), the resulting mixture was centrifuged, and the supernatant was again pipetted off. This washing operation was repeated 3 times for each sample and then dried in a high speed vacuum concentrator. The result of this titration is illustrated by the graph in FIG.

[0119] maleimide -CPG and diene (DMT) - the time dependence of the reaction of an oligonucleotide (24). Na ₂ HPO ₄ buffer (100 mM, previously equilibrated at 37 ° C.)
Maleimide-derivatized CPG (10 mg each) in pH = 6.5,100 μL)
Six samples were prepared by adding diene (DMT) -oligonucleotide (24) (37 nmol) to each of the six tubes containing the solution of. After various time intervals (see Table 3), the conjugation reaction was stopped by removal of the supernatant solution of (24) and washing of CPG as described above. The results are shown graphically in the graph of FIG.

Determination of loading . CH ₃ CN (0.75m
A 3% solution of p-toluenesulfonic acid in L) was added and the resulting mixture was shaken for 1 minute. For loading determination, the absorbance of the supernatant at 497 nm was determined photometrically using the following formula:

[0121]

[Chemical 43] Example 11 To diene coated glass surface 5'-maleimide - conjugation scheme 14 of oligonucleotide de (26), maleimide - shows the conjugation of the oligonucleotide (26) diene-functionalized glass surface. Proof of conjugation was achieved by hybridization with labeled complementary sequence (27) and detection of fluorescence, as described above.

[0122]

[Chemical 44] Conjugation of maleimide-oligonucleotide (26) to diene functionalized glass slides . One diene-coated slide was fitted with three silicon rubber septa (standard taper 14/20) orthogonal to the slide. The septum was compressed onto the slide between two pieces of 3x5x1 / 4 inch acrylic paper secured to both sides of the slide using thumb clamps. The top acrylic paper contains a 3/16 inch hole just above the center of the septum to allow needle access to the septum. Two septa were fixed to the diene functionalized lower half of the slide and one septum was fixed to the non-functionalized upper half of the slide. 100 mM Na ₂ HPO ₄ buffer (pH = 6.5)
Maleimide-oligonucleotide (26) in 4 pmol / μL solution (125
μL) was added to the septum located at the bottom of the slide to demonstrate surface immobilization of the oligonucleotide on the microslide. 100 mM Na ₂ HPO ₄ buffer (p
4 pmol / μL solution of control oligonucleotide (22) in H = 6.5) (1
25 μL) was added as a control to a septum located in the middle of the slide to check for potential non-specific binding of the oligonucleotide to the functionalized portion of the slide. 10
A 4 pmol / μL solution (125 μL) of the maleimide-oligonucleotide (26) in 0 mM Na ₂ HPO ₄ buffer (pH = 6.5) was added to the septum placed on top of each slide as a control, and The potential non-specific binding of the maleimide-oligonucleotide (26) to the functionalizing moiety was checked. Slide at 37 ℃
Incubate for 1 hour, then remove septum and place the slide on Tween.
(Registered trademark) 20 (TBST) -containing TRIS-buffered saline (10 mM T
RIS-Cl, pH = 8.0, 150 mM NaCl, 0.1% Tween (
Each was soaked 3 times in Petri dishes containing ™) 20).

Detection of oligonucleotide immobilization on glass slides . The slide was drawn from the buffer solution and placed in a Petri dish into a 4 pmol / μL solution (10 mL) of complementary 5'-fluorescein-oligonucleotide (27) in 5X SSC containing 0.1% SDS (from the slide to dryness. Immersed immediately (to prevent). The slide was incubated for 30 minutes at 55-60 ° C. The slides were then each dipped 3 times in a Petri dish containing TBST. The slides were analyzed using a Molecular Dynamics Typhoon fluorescence scanner and the results showed that the wash conditions were insufficient to remove non-specifically bound oligonucleotides from the plate (Figure 11, slide "1"). The slides were then washed 3 times in Petri dishes containing 1X PBS (phosphate buffered saline solution) with 0.1% SDS. Analysis of this slide again using a Molecular Dynamics Typhoon fluorescence scanner revealed that the maleimide-oligonucleotide (26) was only in contact with the diene functionalized portion of the glass slide (27) and then hybridized with (27). Showed a strong reaction (
FIG. 11, slide “2”). The two controls on the slide showed no reaction, the non-covalently bound oligonucleotide (22) was removed from the diene-functionalized part of the slide and the non-covalently bound maleimide-oligonucleotide (26) from the non-functionalized part of the slide. It was demonstrated that the previous wash conditions were sufficiently stringent to remove. The diene functionalized portion of the slide (bottom half) shows some fluorescence outside of the area that reacted with (26), leaving some non-specific binding of (27) to the diene functionalized portion of the slide. Was clearly stated.

Gongju Geshon of oligonucleotide (26) - [0124] maleimide to the diene-functionalized CPG. Diene-coated CPG (15 mg) was placed in a centrifuge tube. 100 mM N
a 4 pmol / μL solution of maleimide-oligonucleotide (26) in a ₂ HPO ₄ buffer (pH = 6.5) (125 μL) was added, the vial was shaken to mix the contents, then at 37 ° C. Incubated for 1 hour. The mixture was then centrifuged, the supernatant was removed by pipette and the CPG sample was washed with Twe.
en (registered trademark) 20-containing TRIS-buffered saline (TBST) (10 mM TRIS-Cl, pH = 8.0, 150 mM NaCl, 0.1% Tween).
n (registered trademark) 20). The resulting mixture was centrifuged, the supernatant removed again with a pipette and the CPG sample resuspended. This operation was repeated for all three washes. In addition, two control experiments were performed according to the method described above. One of the control experiments was to use a control oligonucleotide (22) instead of compound (26) to check for potential non-specific binding of this oligonucleotide to the diene-functionalized CPG. Another control experiment was to check for potential non-specific binding of maleimide-oligonucleotide (26) to non-functionalized CPG using non-functionalized CPG capped with chlorotrimethylsilane as a control. .

Detection of oligonucleotide immobilization on CPG . 5XS with 0.1% SDS
A 4 pmol / μL solution (125 μL) of complementary 5′-fluorescein-oligonucleotide (27) in SC was immediately added to each of the CPG samples obtained after reaction with maleimide-oligonucleotide (26) and washing. added. The sample was incubated at 55-60 ° C for 30 minutes. This sample was then treated with 1 × PBS (phosphate buffered saline solution) containing 0.1% SDS, as described above.
It was washed 3 times. After removing the last wash with a pipette, each sample was sprinkled onto clear plastic wrap and the plastic wrap folded to contain the powder. The three samples, each wrapped, were then placed side by side on the surface of a Molecular Dynamics Typhoon fluorescence scanner. The sample was analyzed using a Typhoon fluorescence scanner and the results showed that the wash conditions were insufficient to remove non-specifically bound (26) from the diene-functionalized CPG (Figure 12A). Then return the sample to the original centrifuge tube, 0.5X
Washed 3 times with SSC + 0.1% SDS (significant loss of each sample occurred during this transfer). The sample was then respread onto plastic wrap and analyzed using a Typhoon fluorescence scanner. The diene-functionalized CPG sample treated with maleimide-oligonucleotide (26) showed a strong reaction after hybridization with (27) (Fig. 12B, slide "2A"). The two controls showed little reaction, demonstrating that Diels-Alder surface immobilization of maleimide-oligonucleotides can be achieved on diene-functionalized CPGs.

Example 12 Preparation of Diene Modified Oligonucleotides Scheme 15 illustrates the preparation of diene modified oligonucleotides (29) from N-hydroxysuccinimide ester (10) whose synthesis is described in Example 2.

[0127]

[Chemical formula 45] A 5'-amine modified oligonucleotide (28) (ODN99225) was prepared utilizing standard solid phase automated synthesis on controlled pore glass (CPG) by the phosphoramidite method. After deprotection and cleavage from the CPG support, preparative anion exchange chromatography of a 200 mL Source 15Q column (quaternary ammonium functionalized monodisperse polystyrene beads) eluted with a gradient as described in Table 4. The crude amine-modified oligonucleotide was purified by chromatography.

Fractions containing product were combined and concentrated in vacuo. This material is C18 in water
The crude material was desalted by an HPLC method that included adsorbing the crude material to the column, washing with a 1M NaCl solution followed by water, and then eluting the material with EtOH. The amine oligonucleotide was coupled to the compound (10) utilizing about 4 equivalents of N-hydroxysuccinimide ester (10) in a mixture of 25 mM sodium borate buffer and acetonitrile (40%). Analytical reverse phase chromatography showed the final product to be 95.9% pure. The identity of this oligonucleotide conjugate (29) (observed MW = 6639.0; calculated MW = 6639) was confirmed by electrospray mass spectrometry.

Example 13 HPLC Monitoring of Diels -Alder Reaction Utilizing Cyclohexadiene Oligonucleotide To confirm the Diels-Alder reactivity of the diene conjugate (29), a commercially available maleimido dienophile (30) was prepared as illustrated in Scheme 16.
Labeling was performed using (Aldrich) and (31) (Molecular Bioscience). Briefly, 100 equivalents of compound (30) or (31) were added to a 1.5 mM solution of (29) in phosphate buffer (pH = 6.8). The progress of this reaction was monitored by analytical anion exchange chromatography, with samples taken every 5 minutes (as described below). N-ethylmaleimide (
Treatment of (29) with 30) resulted in complete conversion to the adduct (32) within 5 minutes, whereas biotin maleimide (31) required 20 minutes.

[0130]

[Chemical formula 46] The progress of the Diels-Alder reaction was monitored as follows: 2 μL of reaction solution was removed and 8 μL of 0.1 M NaO (to quench maleimide).
Treated with H, shaken vigorously for 30 seconds and diluted with 50 μL H ₂ O. This 50μ
50 μL of the L solution (about 16.7 μg of oligonucleotide) was injected onto a 5 micron C18 Jupiter column. This sample was treated with TEAA (pH 7.0; eluent A) and acetonitrile (eluent B) as outlined in Table 5.
) Buffer system.

Example 14 Preparation of Diene Modified Polyethylene Glycol Substrate Scheme 17 illustrates the synthesis of cyclohexadiene-PEG (34).

[0132]

[Chemical 47] The diene (10) was treated sequentially with carbonyldiimidazole and methoxypolyethylene glycol amine (MW = 5000). The resulting carbamate product was
It was purified by careful drop wise addition to cold ether and the precipitate collected by filtration. The resulting material was used for surface immobilization tests without further purification. Similarly, hydroxymethylanthracene (35) was converted to a similar PEG derivative (36) using the same method illustrated in Scheme 18.

[0133]

[Chemical 48] Example 15 Preparation of Maleimide Coated BIAcore Flow Cell Scheme 19 illustrates the preparation of a dienophile-derivatized CM5 BIAcore flow cell surface.

[0134]

[Chemical 49] By subjecting commercially available chips (coated with a matrix of carboxymethyl dextran) to a three-step derivatization method, as illustrated in Scheme 19,
A dienophile CM5 BIAcore flow cell surface was prepared. Briefly, the carboxy group as an N-hydroxysuccinimide ester (EDC / NH
After activation (via treatment with S), a diamine linker (ethylenediamine, which provides a reactive primary amine surface) was added and this was treated with a commercially available bifunctional crosslinking reagent. The cell surface of the resulting dienophile (37) was determined by the BIA for each experiment.
It was reproducibly manufactured in this manner, as evidenced by the measurement of the core sensorgram.

Allows real-time monitoring of the interaction of two or more molecules such as proteins or nucleic acids with small molecules such as signaling substances and pharmaceuticals (Pharmacia Biosensor AB, http: // www. Biacore 2000 instrument (Pharmacia Biosensor AB, Uppsala, Sweden. Lofas et al. (1991). Sens. Actuators
B 5: 79-84; Malmqvist (1993) Nature 361: 186-187) was used to obtain sensorgrams. This detection principle is based on the optical phenomenon of "surface plasmon resonance" (SPR), which detects changes in the refractive index of a solution that is close to the surface of the sensor chip (Brockman et al. (1999) J. Am. Chem. Soc. 121: 8044-8051 and references cited herein). This, in turn, is directly related to the concentration of surface solute. To perform a BIA analysis, one interactant is immobilized on the sensor chip forming the wall of one microflow cell. Then
A sample containing other interactants is injected over the surface in a controlled flow.
The change in surface concentration resulting from the interaction is detected as an SPR signal, which is expressed in resonance units (RU). Thus, a continuous display of RUs as a function of time, called the BIAcore sensorgram, provides an overall record of the progression of association and dissociation.

Protocol for Maleimide Derivatization (Khilko (1993) J. Bio. Chem. 268: 15425
-15434): Flow rate = 10 μL / min, PBS running buffer (pH 6.8). Injection sequence: 1.40 μL EDC-NHS mixture (from stock solution: NHS 11.5 m
g / mL, and EDC 75 mg / mL). 2. 100 μL of 1.0 M ethylenediamine.2HCl (pH 6.0). 3. 100 μL of 25 mM Sulfo EMCS (NHS / maleimide bifunctional reagent / running buffer, pH 6.8). The overlapping Biacore sensorgrams shown in Figure 13 show the consistent results of the method.

Example 16 Comparison of surface derivatization with PEG-SH (Michael addition) vs. PEG-diene (Diels- Alder surface immobilization)

[0138]

[Chemical 50] The surface of the dienophile flow cell described in Example 15 was treated with MeO-PEG-SH (M
W = 5000; pH = 6.8, phosphate buffer, flow rate: 10 μL / min) to confirm the functional activity of this surface, as evidenced by the Biacore sensorgram shown in FIG. .

Similarly, the addition of the PEG-diene substrate (34) prepared in Example 14 is shown in FIG.
The Biacore sensorgram shown in Figure 5 was provided, which confirmed the dienophile activity on the flow cell surface, suggesting that the relative rate of Diels-Alder surface immobilization was substantially slower than Michael addition utilizing PEG-thiol. (12 hours vs. 10 minutes).

Example 17 BIAcore Diels- Alder reaction with anthracene-PEG (36)

[0141]

[Chemical 51] Using the method described in Example 16, dienophile derivatized CM5 BIAco
Reaction of re flow cell surface (37) with anthracene derivative (36) (prepared as described in Example 14) is described in the literature as having improved aqueous Diels-Alder kinetics, compound (38). Brought. Using compound (36), enhanced surface immobilization was observed compared to compound (34). Figure 1
As seen in 6, comparable reactions were observed in almost half the time.

Example 18 Diels - second only to surface immobilization of hybridization cyclohexane diene modified oligonucleotides (29) of the BIAcore surface immobilized complementary sequence of Alder conjugation via diene modified oligonucleotide (29), with a complementary oligonucleotide sequence Hybridization is illustrated in Scheme 22. Briefly, the Diels-Alder reaction of dienophile (37) with diene (29) was performed using the method described in Example 16 to give the immobilized oligonucleotide, compound (39). The results of this sensorgram are shown in FIG.
The immobilized oligonucleotide (39) was then hybridized with its complementary sequence using standard means. The Biacore sensorgram shown in FIG.
The enhanced reaction was demonstrated when complementary oligonucleotide sequences were associated with immobilized oligonucleotides, thus supporting the above immobilization technique.

[0143]

[Chemical 52] Example 19 Synthesis of Anthracene-Silane Reagent (42) Scheme 23 illustrates the synthesis of anthracene silane reagent for functionalizing the glass surface. Briefly with respect to Scheme 23, hydroxymethylanthracene (35) was reacted with CDI to form the imidazolate (41). The imidazolate (41) was then reacted with propylaminosilane (17) to give the anthracene-silane reagent (42), which was then used for glass derivatization, as illustrated in Scheme 24 below.

[0144]

[Chemical 53] Synthesis of anthracene-silane reagent (42) . To a stirred solution of (35) (4.8 mmol, 1.0 g) in DMF (16 mL, 0.3 M), CDI (5.2
8 mmol, 0.856 g) was added. After 3 hours, CDI (1.06 mmol,
The reaction was driven to completion with the addition of 0.171 g). Imidazole (4
The complete formation of 1) was confirmed by ¹ H-NMR, which indicated that the alcohol methylene had shifted from 5.65 ppm to 6.45 ppm. This is characteristic of the expected shift. To this stirred solution was added aminopropyltriethoxysilane (17) (4.8 mmol, 1.06 g). The reaction was placed under an argon environment and stirred overnight. The reaction was complete by ¹ H-NMR, which showed that the imidazole methylene had shifted from 6.45 ppm to 6.15 ppm, which led to the expected carbamate product (42). Characteristic for conversion.

D ₂ O (200 μL) was added to the NMR sample in this CDCl ₃ . The tube was shaken vigorously for 1 minute, then a second ¹ H-NMR analysis was performed, and the compound (
42) remained untouched. Therefore, the reaction solution was concentrated under reduced pressure at 40 ° C. to give a dark orange oil. Add this oil to CH ₂ CH ₂ (
250 mL) and washed with H ₂ O (150 mL). This organic phase at 40 ° C
Concentrated under reduced pressure to give a dark orange oil. Upon standing, the oil turned into an orange crystalline solid.

[0146]

[Chemical 54] This crude material was used in the glass slide derivatization experiment illustrated in Example 20.

Example 20 . Anthracene - government functionalised scheme of the slide glass using a silane reagent (42) 24 (FIG. 19) is anthracene - illustrates the functionalization of a glass microscope slide using a silane reagent (42).

[0148]

[Chemical 55] Half of a clean microslide (without pretreatment, VWR) was washed with toluene (50
(mL) and CH ₂ CH ₂ (10 mL) in a suspension of reagent (42) (1.04 g) in 1 mL for 1 hour. The slide was then removed from the suspension and wiped dry with a piece of filter paper. Slide this slide into toluene, toluene / ethanol (
1: 1 v / v) and ethanol (100 mL each) in turn. To dry, slides were placed in an oven at 80 ° C overnight.

[0149]

[Table 1]

[0150]

[Table 2]

[0151]

[Table 3]

[0152]

[Table 4]

[0153]

[Table 5]

[0154]

[Table 6]

[Brief description of drawings]

FIG. 1 Fluorescein labeled compound (26) (see Example 6 for this synthesis).
As described in 1.) and a fluorescence scan of a polyacrylamide gel loaded with 1, 2, and 3 μL.

FIG. 2 shows maleimide functionalization in the lower half of the slide after treatment with maleimide-silane (19) and reaction with a thiol-containing fluorescein reagent (SAMSA reagent) as described in Example 7, It is a fluorescence scan of a slide glass.

FIG. 3 is a fluorescence scan of maleimide-derivatized CPG. In FIG. 3A, native CPG was first treated with maleimidosilane (19) before SAMSA.
Stained with reagent. In FIG. 3B, native CPG was treated with SAMSA-fluorescein and served as a control.

FIG. 4 This diene functionalized slide was treated with fluorescein-5-maleimide after treatment with a diene-silane reagent (20) as described in Example 8, diene functionalization in the lower half of the slide. It is a fluorescence scan of a slide glass showing the conversion.

FIG. 5 is a fluorescence scan of diene-functionalized CPG. In FIG. 5A, CPG was treated with diene-silane (20) and then stained with fluorescein-5-maleimide. In FIG. 5B, native CPG was treated with fluorescein-5-maleimide and served as a control.

FIG. 6 shows the placement of septa and the contents of the septa showing the Diels-Alder surface immobilization of oligonucleotides on maleimide-functionalized microslides as described in Example 9. It is a drawing of a slide.

FIG. 7 shows successful Diels-Alder surface immobilization of 5′-diene-oligonucleotides (23) on maleimide-functionalized microslide glasses,
6 illustrates a fluorescence scan of a glass slide. Slide "1" was pretreated with 2N NaOH followed by warm 2N HCl prior to maleimide functionalization. slide"
2 "was pretreated with 2N HCl only. The spot visible on each slide was where compound (23) was contacted with the maleimide functionalized portion of the slide prior to hybridization with the complementary 5'-fluorescein-oligonucleotide (27); contacted with control. The area of the slide showed no reaction.

FIG. 8: Complementary fluorescein labeling sequence, 5′-fluorescein- (CA
) ₁₀ (27) (SEQ ID NO: 2) after conjugation of diene-oligonucleotide (23) to maleimide-coated microtiter plates. Wells 1-3 are pure diene-oligonucleotides (
23) treated wells 4-6 with crude diene-oligonucleotide (23)
Processed in. Wells 1A-6A are controls for the corresponding oligonucleotides (22). Wells 7 and 7A are buffer controls. Wells 1, 1A, 4, 4A
Were incubated at pH = 5.5, wells 2, 2A, 5, 5A at pH = 6.5 and wells 3, 3A, 6, 6A at pH = 7.7.

FIG. 9 graphically illustrates the relationship between the loading of oligonucleotide-loaded CPG (10 mg) and the concentration of oligonucleotide (24) in solution.

FIG. 10 graphically depicts the relationship between loading of oligonucleotide-loaded CPG (10 mg) and incubation time of oligonucleotide (24).

FIG. 11 illustrates a fluorescence scan of a glass slide showing successful Diels-Alder surface immobilization of maleimide-oligonucleotide (26) on a diene-functionalized microslide glass. Slide "1" precedes the phosphate buffered saline (PBS) wash and demonstrates that this wash is required to remove non-covalently bound oligonucleotides from the glass surface. slide"
2 ”is after PBS washing. The single fluorescent reaction visible on this slide is where compound (26) was contacted with the diene functionalized portion of the slide prior to hybridization with the complementary 5'-fluorescein-oligonucleotide (27). . After the PBS wash, the areas of the slide that were in contact with the control showed no reaction.

FIG. 12: CPG showing Diels-Alder surface immobilization of an oligonucleotide (maleimide-oligonucleotide (26)) on a diene-functionalized CPG.
6 illustrates a fluorescence scan of a sample. FIG. 12A is prior to washing with a mixture of 5 × 0.3M sodium citrate and 3M sodium chloride (SSC), and FIG. 12B is after SSC washing. The fluorescent reaction visible in the sample labeled "2A" is where compound (26) was contacted with the diene-functionalized CPG prior to hybridization with the complementary 5'-fluorescein-oligonucleotide (27). After SSC washing, the control samples labeled "2B" and "2C" showed relatively little reaction.

FIG. 13 is an overlapping Biacore sensorgram illustrating the reproducibility of maleimide-coated BIAcore flow cell surface (37) formation described in Example 15.

FIG. 14: Biaco, a Michael addition product of a maleimide-coated BIAcore flow cell surface (37) and MeO-PEG-SH, as described in Example 16.
It is a re-sensorgram.

FIG. 15 is a Biacore sensorgram of the Diels-Alder reaction product of the maleimide-coated BIAcore flow cell surface (37) and PEG-diene substrate (34) described in Example 16.

FIG. 16 is a Biacore sensorgram of the Diels-Alder reaction product of the maleimide-coated BIAcore flow cell surface (37) and PEG-anthracene substrate (36) described in Example 17.

FIG. 17 is a Biacore sensorgram of the Diels-Alder reaction product of the maleimide-coated BIAcore flow cell surface (37) and cyclohexadiene-modified oligonucleotide (29) described in Example 18.

FIG. 18: Biacore sensor of Diels-Alder reaction product of maleimide-coated BIAcore flow cell surface (37) with cyclohexadiene modified oligonucleotide (29) when the immobilized sequence hybridizes with its complementary oligonucleotide sequence. It is gram.

FIG. 19 illustrates functionalization of microscope slides with anthracene-silane reagent (42).

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12M 1/00 C12M 1/00 A C12N 15/09 ZNA C12N 15/00 ZNAF (81) Designated country EP (AT , BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI) , CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, B , 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 (72) Inventor Sevesta, David Peas, USA 80501, Longmont, Katie Lane 1056 (72) Inventor Luke, Michael United States Colorado 80305, Boulder, Bellaire Drive 1235 (72) Inventor Latham-Timons, needle A. United States Colorado 80305, Boulder, Whitney Place 4375 (72) Inventor Pilon, John United States Colorado 80524, Fort Collins, East Elizabeth 526 (72) Inventor Husser, Gregory Em United States Colorado 80503 , Longmont, Gooseberry Drive 640, Number 903 F Term (reference) 4B024 AA11 AA19 CA01 CA11 HA19 4B029 AA07 AA23 BB15 BB17 CC03 CC08 FA15 4C057 LL10 LL17 LL18 LL23 BB29 AB20 BB46 AB12CBBAB 4C069 AC30 AD02 DB30 EB03 FB17 GB13

Claims (22)

[Claims] 1. A method of immobilizing a molecule on a support, comprising the step of reacting a derivatized molecule with a derivatized support capable of reacting with the derivatized molecule via a cycloaddition reaction. 2. The cycloaddition reaction is [1 + 2] -cycloaddition, [2 + 2]-
The method according to claim 1, wherein the method is selected from the group consisting of cycloaddition, [3 + 2] -cycloaddition, [2 + 4] -cycloaddition, [4 + 6] -cycloaddition, and chereotropic reaction. 3. The cycloaddition reaction is selected from the group consisting of 1,3-dipolar cycloaddition reaction, Diels-Alder reaction, ene reaction, and [2 + 2] photochemical cycloaddition reaction. The method described in. 4. The molecule is a biomolecule, a macromolecule, and a diagnostic detection molecule (DD).
The method of claim 1 selected from the group consisting of M). 5. The group of biomolecules consisting of nucleic acids, oligonucleotides, proteins, peptides and amino acids, polysaccharides and saccharides, glycoproteins and glycopeptides, alkaloids, lipids, hormones, drugs, prodrugs, antibodies and metabolites. The method of claim 4, selected from: 6. The method of claim 4, wherein the DDM is selected from the group consisting of fluorescent agents, chemiluminescent agents, radioisotopes, and bioluminescent marker compounds; antibodies, biotin, and metal chelates. 7. The method of claim 6, wherein the DDM is fluorescein. 8. The molecule has a diene or dienophile with or without heteroatoms, a 1,3-dipole or 1,3-dipolarophyl with or without heteroatoms, a heteroatom, or Selected from the group consisting of enes or enophiles with or without, C2-C50 alkenes with or without heteroatoms, C2-C50 alkynes with or without heteroatoms, aromatic compounds, carbene and carbene precursors. The method of claim 1, wherein the method is derivatized with a moiety. 9. The diene is: [In the formula: Z is Where: m, n, o are equal to 0, 1, 2, and Y is NH, O, NH (CO) O, NH (CS) O, NH (CO) NH, NH
(CO), S-S- S-, Si (OR) 3, and is selected from SiR _2, wherein, R represents an alkyl, aryl, substituted alkyl or substituted aryl)
Is a linker selected from the group consisting of] and the method according to claim 8. 10. The dienophile is represented by: [In the formula: Z is Where: m, n, o are equal to 0, 1, 2, and Y is NH, O, NH (CO) O, NH (CS) O, NH (CO) NH, NH
(CO), S-S- S-, Si (OR) 3, and is selected from SiR _2, wherein, R represents an alkyl, aryl, substituted alkyl or substituted aryl)
Is a linker selected from the group consisting of] and the method according to claim 8. 11. The derivatized molecule has the following formula: [Wherein B is a nucleobase; A and A ′ are 2′-sugar substituents; W is an oligonucleotide having 1 to 1000 nucleobases, X, or H].
Independently selected from the group consisting of; and X is capable of undergoing a diene, dienophile, 1,3-dipole, 1,3-dipolarophile, ene, enophile, alkene, alkyne, or cycloaddition reaction Another moiety, which in addition is attached to B of the nucleobase, it can also be attached to a carbon atom, an exocyclic nitrogen, or an exocyclic oxygen] is an oligonucleotide selected from the group of compounds The method of claim 1. 12. A and A ^{', H, 2 H, 3 H} , Cl, F, OH, NHOR 1, NHOR 3, NHNHR 3, NHR 3, = NH, CHCN, CHCl 2, SH,
_{SR 3, CFH 2, CF 2} H, CR 2 2 Br, - (OCH 2 CH 2) n OCH 3, OR 4,
Independently selected from the group consisting of and imidazole; R ¹ is selected from the group consisting of H and an alcohol protecting group; R ² is ═O, ═S, H, OH, CCl ₃ , CF ₃ , a halide , C ₁ -C ₂₀ optionally substituted alkyl (cyclic, linear, and branched), C ₂ -C ₂₀ alkenyl, C ₆ -C ₂₀ aryl, C1 to C20 acyl, C ₁ -C ₂₀ Benzoyl, O
R ⁴ and an ester; R ³ is R ² , R ⁴ , CN, C (O) NH ₂ , C (S) NH ₂ , C (O) CF ₃ , S
Selected from the group consisting of O ₂ R ⁴ , amino acids, peptides, and mixtures thereof; R ⁴ is an optionally substituted hydrocarbon (C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, C ₂ -C) ₂₀ alkynyl), optionally substituted heterocycle, t-butyldimethylsilyl ether, triisopropylsilyl ether, nucleoside, carbohydrate, fluorescent label, and phosphoric acid; and X is a conjugated diene unit. Alkyl or substituted alkyl group, alkoxy or substituted alkoxy group bearing a conjugated diene unit, CH ₂ CHCH = CHCH ₂ CH ₂ O, maleimide substituted alkoxy group, dienophile substituted alkoxy group, alkylamino group bearing a conjugated diene unit or substituted alkylamino group , Maleimide-substituted alkylamino groups or substituted alkyl groups Ruamino group, an alkylamino group or a substituted alkylamino group that carries a dienophile moiety, nitrile ylide, nitrile imine, nitrile oxide, diazoalkane, azide, azomethine ylide, azomethine imine,
12. The method of claim 11, selected from the group consisting of nitrone, carbonyl ylide, carbonyl imine, and carbonyl oxide. 13. The method of claim 1, wherein the support is selected from the group consisting of glass, polymers and resins, and large biomolecules. 14. The method of claim 13, wherein the glass is selected from the group consisting of controlled pore glass (CPG), glass slides, glass fibers, glass disks, and glass coating materials. 15. A copolymer containing polystyrene (PS), polyethylene glycol (PEG), a copolymer of PS and PEG, a copolymer of polyacrylamide and PEG, and a maleimide and maleic anhydride as the polymer and resin. 14. The method of claim 13, wherein the method is selected from the group consisting of :, polyvinyl alcohol, and an immunogenic high molecular weight compound. 16. The method of claim 13, wherein the large biomolecule is selected from the group consisting of polysaccharides, proteins and nucleic acids. 17. The method of claim 1, wherein the support is a solid support. 18. The support comprises diene or dienophile with or without heteroatoms, 1,3-dipole with or without heteroatoms, or 1,3-dipolarophyl, heteroatoms. Or selected from the group consisting of enes or enophiles with or without, C2-C50 alkenes with or without heteroatoms, C2-C50 alkynes with or without heteroatoms, aromatic compounds, carbene and carbene precursors. 2. The method of claim 1, wherein the method is derivatized with a moiety that is 19. The diene is: [In the formula: Z is Where: m, n, o are equal to 0, 1, 2, and Y is NH, O, NH (CO) O, NH (CS) O, NH (CO) NH, NH
(CO), S-S- S-, Si (OR) 3, and is selected from SiR _2, wherein, R represents an alkyl, aryl, substituted alkyl or substituted aryl)
It is a linker selected from the group consisting of] The method according to claim 18, selected from the group consisting of: 20. The dienophile is: [In the formula: Z is Where: m, n, o are equal to 0, 1, 2, and Y is NH, O, NH (CO) O, NH (CS) O, NH (CO) NH, NH
(CO), S-S- S-, Si (OR) 3, and is selected from SiR _2, wherein, R represents an alkyl, aryl, substituted alkyl or substituted aryl)
It is a linker selected from the group consisting of] The method according to claim 19, selected from the group consisting of: 21. An immobilized product formed by the method of claim 1. 22. A compound selected from the group consisting of: