JP2010151828A - Substrate, manufacturing method, diagnostic system, and detecting method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a substrate where the size of a molecule binding to a ligand is controlled, and preferably conical compound is bound. <P>SOLUTION: An application of a substrate is disclosed, which includes a molecular film of dendrimer macromolecule in which the size of a uniform spacer is controlled, which includes a polymer including a branched portion and a linear portion, wherein abundant terminals of the branched potion bind to the substrate and the terminal of the linear portion is functionalized. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to highly branched macromolecules. The present invention relates to a functionalized substrate in which the macromolecule is immobilized on the surface. The present invention also provides a dendrimer with a controlled molecular size in which a functional substrate is bound to one side of a dendron and a target-specific ligand is bound to the other side. size-controlled dendrimer) and dendrons. The present invention also relates to the field of combinatorial chemistry using a functionalized substrate to which a highly branched macromolecule to which a probe biomolecule is bound, a specific protein detection method, The present invention relates to a method for detecting specific nucleic acid hybridization or nucleic acid / peptide hybridization.

  In order to enable large-scale analysis of DNA order, genetic variation, and gene expression, DNA microarrays were first reported [Fodor et al. , Nature 364, 555-556 (1993); Saiki et al. , Proc. Natl. Acad. Sci. USA 86, 6230-6234 (1986)] has been of great interest. It is said that due to the aforementioned standardization and application to human genetic analysis, improvements are required in terms of essential accuracy, reproducibility, and spot homogeneity [Hackett et al. , Nature Biotechnology 21, 742-743 (2003)]. Such aspects are mainly due to the nature of the surface and molecular interlayer structure that is different from the ideal shape. Similarly, the mass target detection system field includes immobilized bioactive molecules and biological analysis utilizing biomolecules.

  A DNA microarray fabricated on a nano-sized surface according to the present specification can discriminate pairs mistakenly combined with a single base to the same extent as DNA in solution. Such access is ideal DNA with minimal steric hindrance, giving each probe DNA strand enough space to interact with the target DNA. Providing a microarray. The significantly increased discrimination efficiency can provide a highly reliable human genetic analysis. Furthermore, such an approach is so general that it can be applied to various biological analyzes using immobilized bioactive molecules and biomolecules.

  Affinity purification is a well-known technique for the separation and identification of ligand binding proteins [Cuatrecasas et al. , Proc. Natl. Acad. Sci. U. S. A. 1968, 61, 636-643]. The specific interaction of the ligand covalently bound to the insoluble substrate and the complementary target protein provides the specificity required to separate biomolecules from complex mixtures. However, the various uses of the technique are limited due to the constrained choice and instability of traditional substrates. The remarkable non-specific binding of proteins to many solid supports has always been a problem in establishing new substrates (Cuatrecasas, PJ. Biol. Chem. 1970, 245, 3059-3065). ). Accordingly, it has been required to find new substrates that are comparable to traditional substrates in terms of stability due to environmental changes and specificity that is precisely defined and exhibits convenient ligand binding.

  It has been shown that AMPCPG (aminopropyl-controlled pore glass), which has been traditionally used for solid peptide synthesis, has many favorable points. However, the surface of the CPG (controlled pore glass) was polar and maintained a partial negative charge even when coated (Hudson, DJ Comb. Chem. 1999, 1, 403-457). . In conjunction with the remarkable non-specific binding of proteins, morphology plays the most important role. Thus, applications for affinity chromatography and solid peptide synthesis have been limited. When these shortcomings are overcome, various uses of the material should be expected.

  Ligand accessibility is an important factor in determining binding capacity. The introduction of spacer molecules and increasing the concentration of ligand was the traditional method used to better expose the ligand to the surface (Rusin, et al., Biosensors & Bioelectronics 1992, 7, Suen et al., Ind. Eng.Chem.Res.2000, 39, 478-487; Penzol et al., Biotechnol and Bioeng.1998, 60, 518-523; Spinke et al., J. Chem. Phys. 1993, 99, 7012-7019). Although the method is effective to a certain extent, inappropriate spacing between ligands and disordered distribution of capture molecules on the surface are still unsolved problems (Hearn et al., J Chromatogr. A. 1990, 512, 23-39; Murza et al., J. Chromatogr.B.2000,740, 211-218; Xiao et al., Langmuir 2002, 18, 7728-7739). Recently, two methods for improving such problems have been provided. One method uses a giant substance such as a protein as a substance for fixing the position. The protein is bound to the substrate, and the substance for fixing the position is removed and washed. In this manner, a specific distance between the connected substances remaining on the substrate is ensured. Nevertheless, the selection of the substance for position fixing and the route for removing it must be finely optimized for all other situations (Hahn et al., Anal. Chem. 2003). 75, 543-548). Another approach is to provide a self-assembled monolayer that is precisely aligned, and a cone-shaped dendron that uses an activated functional group at the top of the dendron. (Xiao et al., Langmuir 2002, 18, 7728-7739; Whitesell et al., Langmuir 2003, 19, 2357-2365).

The present invention presents the properties of surface materials by modification of AMPCPG using dendron, additional binding of GSH at the top of the dendron, and binding of GST protein. A dendron having 3 to 9 carboxylic acids at the ends and one amine group at the top was introduced into the matrix. The carboxylic acid is covalently bound to the solid surface. With the understanding and widespread use of the GST (glutathione S-transfer) gene fusion system, glutathione was selected as a ligand for dendron-treated substrates. The degree of ligand binding of the substrate was measured using GST and two types of fusion protein ligands (GST-PX ^P47 , GST-Munc-18) (Smith et al., Gene 1988, 67, 31-40; Sebastian). et al., Chromatogr.B. 2003,786,343-355; Wu et al., Chromatogr.B.2003,786,177-185; DeCarlos et al., J.Chromatogr.B.2003,786,7- 15).

Fodor et al. , Nature 364, 555-556 (1993); Saiki et al. , Proc. Natl. Acad. Sci. USA 86, 6230-6234 (1986) Hackett et al. , Nature Biotechnology 21, 742-743 (2003). Cuatrecasas et al. , Proc. Natl. Acad. Sci. U. S. A. 1968, 61, 636-643 Cuatrecasas, P.A. J. et al. Biol. Chem. 1970, 245, 3059-3065 Hudson, D.M. J. et al. Comb. Chem. 1999, 1,403-457 Rusin, et al. , Biosensors & Bioelectronics 1992, 7, 367-373 Suen et al. , Ind. Eng. Chem. Res. 2000, 39, 478-487 Penzol et al. , Biotechnol and Bioeng. 1998, 60, 518-523 Spinke et al. , J .; Chem. Phys. 1993, 99, 7012-7019 Hearn et al. , J .; Chromatogr. A. 1990, 512, 23-39 Murza et al. , J .; Chromatogr. B. 2000, 740, 211-218 Xiao et al. , Langmuir 2002, 18, 7728-7739. Hahn et al. , Anal. Chem. 2003, 75, 543-548 Xiao et al. , Langmuir 2002, 18, 7728-7739. Whitesell et al. , Langmuir 2003, 19, 2357-2365. Smith et al. Gene 1988, 67, 31-40. Sebastian et al. , Chromatogr. B. 2003,786,343-355 Wu et al. , Chromatogr. B. 2003,786,177-185 DeCarlos et al. , J .; Chromatogr. B. 2003,786,7-15

  The present invention provides a substrate in which the size of the molecule bound to the ligand is controlled, preferably to which a conical compound is bound.

In the present invention, the multiple ends of the branched sites are covalently bonded to the substrate, and the ends of the linear sites are branched with functional groups capable of binding to organic moities. The present invention relates to a substrate including a molecular layer of a conical macromolecule whose molecular size is regularly distributed and includes a dendron including a region and a linear region.
The linear part includes a spacer domain (L) and a functional group (W), and the L is NH— (CH ₂ ) ₃ C (O), NH— (CH ₂ CH ₂ O) ₂ CH. _{2 C (O), NH- (} cyclohexyl) (CO), NH- (CH 2) 6 NHC (O), NH- (CH 2) 7 C (O), NH- (CH 2) 6 C (O) , NH- (CH ₂ ) ₆ NH (CO), NH- (cyclopropyl) (CO), NH— (CH ₂ ) ₆ NHC (O) CH ₂ , NH— (CH ₂ ) ₅ , NH- (cyclopropyl) ( CO) and NH— (CH ₂ ) ₆ C (O), and W is selected from the group consisting of NH, CH ₂ , and O.

The macromolecules are distributed at regular intervals on the substrate.
Specifically, the macromolecules are distributed so that the spacing between the functional groups at the linear sites is a regular spacing of about 0.1 nm to about 100 nm.
More specifically, the macromolecules are distributed at regular intervals of about 10 nm.

In the substrate, the end of the branched site is selected from the group consisting of OH, OMe, NH ₂ and OBzl.

  In addition, a protecting group is bound to the end of the linear site. The protecting group is selected from the group consisting of anthracenemethyl (A), Boc, Fmoc, and Ns.

In the substrate, the dendron is a polymer represented by the following formula.
However, in the above formula,
L _{is, NH- (CH 2) 3 C} (O), NH- (CH 2 CH 2 O) 2 CH 2 C (O), NH- (cyclohexyl) (CO), NH- (CH 2) 6 NHC ( _{O), NH- (CH 2)} 7 C (O), NH- (CH 2) 6 C (O), NH- (CH 2) 6 NH (CO), NH- (cyclopropyll) (CO), NH- Selected from the group consisting of (CH ₂ ) ₆ NHC (O) CH ₂ , NH— (CH ₂ ) ₅ , NH— (cyclopropyl) (CO), and NH— (CH ₂ ) ₆ C (O). Yes,
W is selected from the group consisting of NH, CH ₂ , and O;
X is selected from the group consisting of anthracenemethyl (A), Boc, Fmoc, and Ns;
_{R 1~3, R 11~13, R 21~23} , R 31~33, R 111~113, R 121~123, R 131~133, R 211~213, R 221~223, R 231~233, _R 311~313, R 321~323, R 331~333 _{is, CH 2 O (CH 2)} 2 C (O), (CH 2) 2 - (cyclohexyl) -C (O), CH 2 -C = C _{-CH 2 C (O), (} CH 2) 7, (CH 2) 2 C (O), CH 2 OCH (CH 3) CH 2 C (O), (CH 2) 2, CH 2 OCH 2 CH ( Selected from the group consisting of CH ₃ ) C (O), and CH ₂ OCH (CH ₃ ) CH ₂ C (O),
T is selected from the group consisting of OH, OMe, NH ₂ and OBzl.

  In yet another specific example of the present invention, the substrate can bind a target-specific ligand to the end of the linear site. In more detail, the target-specific ligand may be a compound, DNA, RNA, PNA, aptamer, peptide, polypeptide, carbohydrate, antibody, antigen, biomimetics, nucleotide analog, or these A mixture of The distance between target-specific ligands bound to the linear sites of the macromolecule is about 0.1 nm to about 100 nm.

  In another specific example of the present invention, the substrate may be a semiconductor, a synthetic organic metal, a synthetic semiconductor, a metal, an alloy, a plastic ( plastic, silicon, silicate, glass, or ceramic. In detail, the substrate may be a slide, a particle, a bead, a microwell, or a porous material, but is not limited thereto. The porous material is a membrane, gelatin, or hydrated gel. More specifically, the bead is a controlled pore bead.

  The present invention also relates to a method for producing a molecular layer of a conical macromolecule having a controlled size of regularly distributed molecules, including a polymer containing branched sites and linear sites. A plurality of ends of the branched sites are covalently bonded to the substrate, and ends of the linear sites have a functional group capable of binding to organic moiety, and the method includes: (Ii) functionalizing the substrate such that it can react with the end of the macromolecule; and (ii) contacting the macromolecule with the substrate to provide a gap between the end of the macromolecule and the substrate. Forming a bond of:

  In the manufacturing method, the substrate includes a semiconductor, a synthetic organic metal, a synthetic semiconductor, a metal, an alloy, a plastic, a silicon, a silicate, a glass ( glass) or ceramic, but is not limited thereto. The substrate is a slide, a bead, a microwell, or a porous material. The porous material is a hydrated gel, gelatin, or membrane. The beads are beads having controlled pores.

  In the production method as described above, the target-specific ligand can be bound to the end of the linear site, and the method comprises (i) from the end of the linear site of the macromolecule on the substrate. Removing a protecting group; and (ii) a linker molecule selected from the group consisting of a target-specific ligand or a homotypic homofunctional linker or a heterobifunctional linker linked to said ligand Contacting the ends of the linear sites of the macromolecule on the substrate such that the ligand or linker molecule and the ends form a bond.

  In the production method, the macromolecule on the substrate can minimize the obstacle in the binding between the end of the linear site and the target-specific ligand. Further, in the production method, the macromolecule on the substrate can minimize obstacles in detecting a specific target of a target-specific ligand. In the production method, the target-specific ligands are distributed at regular intervals. In the production method, the target-specific ligand is located on the substrate at a low density. In the production method, the target-specific ligand is a compound, DNA, RNA, PNA, aptamer, peptide, polypeptide, enzyme, carbohydrate, polysaccharide, antibody, antigen, biomimetic, nucleotide analog, or a mixture thereof. is there.

  In another specific example of the present invention, the present invention relates to a diagnostic system for detecting a gene mutation containing the substrate, wherein the substrate has a linear site terminal at a target-specific oligonucleotide. It is fixed. Such oligonucleotides are cancer-related genes. In particular, the cancer-related gene is p53.

  In yet another specific example of the present invention, the present invention includes the step of contacting the substrate with a sample containing the gene to be analyzed, wherein the substrate has a linear site at the end of the target-specific oligonucleotide. It is related with the detection method of the presence or absence of the variation | mutation of the gene currently fixed to. In this method, the gene is a cancer-related gene. The gene is p53.

  Such objects of the invention will be more clearly understood from the following detailed description of the invention, the accompanying drawings, and the appended claims.

  According to the present invention, it is possible to provide a substrate in which the size of a molecule bound to a ligand is controlled, and preferably a conical compound is bound.

Structure I showing branched / linear polymers or macromolecules with controlled molecular size. FIG. 3 is a schematic diagram of a reaction for producing a dendron, and X is a protecting group. It is the schematic for surface modification and hybridization using a dendron. It shows the molecular structure of the dendron used in the present invention. It shows the order of probe DNA and the order of target DNA muscles. Probe oligonucleotides are probes 1: 5′-NH ₂ -C ₆ -CAT TCC GNG TGT CCA-3 ′ (sequence number 1) and probe 2: 5′-NH ₂ -C _6- (T) ₃₀ -CAT TCC GNG TGT CCA-3 '(order number 2) is included. Target nucleotides are target 1: 5′-Cy3-TGG ACA CTC GGA ATG-3 ′ (sequence number 3) and target 2: 5′-Cy3-CCT ACG AAA TCT ACT GGA ACG AAA TCT ACT TGG ACA CTC GGA ATG- 3 '(order number 4). (A) shows the ultraviolet spectrum after each reaction stage. EG / GPDS and dendron show the spectra obtained before and after introducing the dendron on the ethylene glycol modified substrate, and deblock shows the spectrum after the deprotection step. is there. (B) shows a stability experiment, and the spectrum obtained after stirring with DMF for 1 day at room temperature is indicated by 'washing'. 1 shows a tapping mode atomic force microscope (TM-AFM) image of a dendron-modified surface. A Nanoscope IIIa AFM (Digital Instruments) equipped with an 'E' type scanner was used. The scanned area is 1.0 × 1.0 μm ² . The fluorescence image after hybridization is shown. (A) and (b) show images obtained after hybridization between (a) probe 1 and target 1 or (b) probe 1 and target 2 on a dendron modified surface. (C) and (d) show images recorded after hybridization between (c) target 1 and probe 1 or (d) target 1 and probe 2 on an APDES modified surface. It shows the intensity difference between a pair of oligonucleotide pairs or a pair of oligonucleotide pairs mistaken internally. The upper images (a) to (c) show 4 × 4 array fluorescent images, and the lower images (d) to (f) are images showing one spot extracted from 16 spots. (A) and (b) relate to a dendron modified microarray having a DSC linker, (b) and (e) relate to an APDES modified microarray having a PDITC linker, (c) and (F) relates to an APDES modified microarray with a DSC linker. Fluorescence images for dendron modified microarray with DSC linker and APDES modified microarray with PDITC linker showed a coefficient of variation of less than 10% (CV) and uniform fluorescence signal in one spot . On the other hand, the fluorescence image for APDES modified microarray with DSC linker showed much smaller spot size, coefficient of variation (CV) greater than 20% and non-uniform fluorescence signal in one spot. The size of each pixel is 10 × 10 μm ² . [9] shows a fluorescent image obtained after hybridization of a target DNA sample and a p53-specific oligonucleotide probe for detecting single mutation of p53 using acid dendron. [27] shows a fluorescent image obtained after hybridization of a target DNA sample and a p53-specific oligonucleotide probe for detecting single mutation of p53 using acid dendron. FIG. 5 shows simultaneous detection of seven hot spots of p53 gene. FIG. FIG. 5 shows simultaneous detection of seven hot spots of p53 gene. FIG. Sample E1 (Fmoc- [3] -acid) and Sample E3 (Fmoc- [9] -acid) were prepared using dendron on an AMPCPG matrix and the Fmoc group was deprotected with 20% piperidine dissolved in DMF. FIG. 3 is a schematic view showing mixing of glutathione. FIG. 4 shows the binding of purified GST and GST lysate utilizing three forms of beads. M means a marker. As a comparative example, GST lysate was flowed directly (lane 1). As a control group, binding to purified GST matrix (A, E1, and E3) was tested (lanes 2, 3, and 4). Finally, cell lysate binding was tested to investigate the efficiency of the matrices (A, E1, and E3) (lanes 5, 6, and 7). Protected first generation functionalized dendron (E1, Fmoc- [3] -acid) and protected second generation functionalized dendron (E3, Fmoc- [9] -acid) is there. The binding of GST obtained from cell lysates to two control beads (CL and CS) is recorded in comparison with E1 and E3. M means a marker, lane 1 means CL, lane 2 means CS, lane 3 means E1, and lane 4 means E3. The results of measuring the binding force change using three fusion GST proteins, GST (28 kDa), GST-PXp ⁴⁷ (41 kDa), and GST-Mucnc18 (98 kDa) are shown. The relative binding force of the three matrices was measured with a Densitometer. The binding strength of all matrices is 100% with respect to GST.

  In this specification, 'a' and 'an' are used to mean all singular and plural.

  As used herein, “aptamer” refers to a single muscle that can specifically recognize a non-oligotide molecule or a group of molecules selected by a mechanism other than Watson-Crick base pair formation or triple muscle formation. , Means partially single muscle, partially double muscle, or double muscle nucleotide sequence, advantageously replicable nucleotide sequence, and the like.

  As used herein, 'bifunctional or bifunctional', 'trifunctional or trifunctional', and 'multifunctional or multifunctional' are synthetic polymers or multifunctional. When used to indicate a valent homopolymer or heteropolymeric hybrid structure, it means divalent, trivalent, or multivalent, or two, three, or more specific recognition elements are defined Or containing binding sheets.

  As used herein, 'biomimetic' means a molecule, group, multimolecular structure, or method that mimics a biological molecule, molecular group, or structure.

  In the present specification, the term “dendritic molecule” means a molecule showing a regular tenderic molecule formed by continuous addition or generation addition of a layer branched from the center or toward the center side.

As used herein, a 'dendritic polymer'
By a polymer is meant a polymer exhibiting regular tendoric molecules formed by continuous or generational addition of layers branched from the center or toward the center. Dendritic polymers include 'dendrimers' characterized by a center, at least one or more inner branch layers, and a surface branch layer [see, e. g. Petar et al. Pages 641-645 In Chem. in Britain, (August 1994)]. “Dendron” means a type of dendrimer having a molecule emitted from a focal point that can be linked to the center to form a dendrimer, either directly or through a linking moiety. Many dendrimers contain two or more dendrons linked to a common center. However, the dendrimer can be used broadly in the sense of including one dendron.

Dendritic polymers include, but are not limited to, symmetric or asymmetric branched dendrimers, caskates, and arbor. As a preferred embodiment, the branched branches may have the same length. For example, the branching can occur without restriction of the terminal NH ₂ group hydrogen atom located on the normal subsequent branching. However, asymmetric dendrimers can also be used. For example, dendrimers of lysine origin are asymmetric. That is, the branched branches of the dendrimer have different lengths. One branch can occur at the epsilon nitrogen of the lysine molecule and another branch can occur at the alpha nitrogen adjacent to the reactive carboxy group that attaches the molecule to the previously formed branch. it can.

  Also, highly branched polymers, such as highly branched polyols, even if not formed by the addition of a regular continuous branch layer, are branched dendrimers. It can be equivalent to a dendritic polymer that exhibits a branched mode with regularity approaching the mode.

  As used herein, the term 'hyper branched' or 'branched' used to describe macromolecules or dendron structures is defined by covalent or ionic bonds. By many polymers having multiple ends that can be bonded to the substrate is meant. In one specific example, the branched or highly branched macromolecule is pre-made and bound to a substrate. Thus, the macromolecules of the present invention exclude polymer cross-linking methods such as those described in US Pat. No. 5,624,711 (Sundberg et al.).

  As used herein, the term 'immobilized' means insolubilized, insoluble, partially insoluble, colloidal, particulate, dispersed, suspended and / or dehydrated material, Alternatively, it means a molecule that binds to or includes a solid support, or that is operably associated with, including, or bound to a solid.

  As used herein, a “library” is a mixture, molecule, material, surface, structural form, surface feature, or a variety of chemical entities that are selective and non-limiting, monomers, polymers, Means a random or non-random mixture, assembly, or classification of structures, precursors, products, variants, derivatives, substances, forms, patterns, features.

  As used herein, 'ligand' refers to a selected molecule that can specifically bind to other molecules through the attraction of an affinity substrate that includes complementary base pair binding. The ligand may be a nucleic acid, various synthetic compounds, a receptor agonist, a partial agent, a mixed agent, an antagonist, a response-inducing molecule, or a response promoting molecule. -Stimulus molecule, drugs, hormones, pheromones, neurotransmitters, autocoids, growth factors, cytokines, prosthetic groups, coenzymes, coenzymes Factors, substrates, precursors, vitamins, toxins, regulators regulatory factor, antigen, hapten, carbohydrate, molecular mimic, structural molecule, effector molecule, selectable molecule, biomolecule, injectable molecule library selected non-oligonucleotide molecules capable of performing selected target specific binding as well as digoxigenin, crossreactants, analogs, competitors, and derivatives of these molecules And conjugates formed by the binding of such substances to secondary molecules Including, but not limited to

  As used herein, 'linker molecule' and 'linker' refer to a branched portion of a macromolecule with a controlled molecular size, such as a branched / linear polymer, as a protecting group or ligand. Means a molecule that binds to For example, linkers include, but are not limited to, selected molecules, spacer molecules, and the like that can attach a ligand to a dendron.

As used herein, “low density” means that the number of probes is about 0.01 to about 0.5 / nm ² , preferably about 0.05 to about 0.2 / nm ² , More preferably it means from about 0.075 to about 0.15 / nm ² , most preferably about 0.1 / nm ² .

  As used herein, “molecular mimics” and “mimetics” refer to the structure or function of other molecules or groups of molecules such as, for example, natural, biological, or sorted molecules. A natural or synthetic nucleotide or non-nucleotide molecule or group of molecules designed, selected, fabricated, modified or engineered to have an equivalent or similar structure or function. A molecular replica is a molecule that can act as an alternative, alternative, functional enhancer, functional improver, structural analog, or functional analog to a natural, synthetic, selected, or biological molecule. Or a multimolecular structure.

  As used herein, a “nucleotide analog” is used in place of naturally occurring bases in nucleic acid synthesis and processing, preferably not only in chemical synthesis and processing, but also in enzymatic synthesis and processing. Molecules that can be formed, particularly modified nucleotides capable of forming base pairs, and any synthesized bases that do not contain adenine, guanine, cytosine, thymidine, uracil, or minority bases. Such terms include modified purines and pyrimidines, minor bases, convertible nucleosides, structural analogs of purines and pyrimidines, labeled, derivatized, and modified nucleosides and nucleotides, conjugated. Including, but not limited to, nucleosides and nucleotides, sequence modifiers, terminal modifiers, spacer modifiers, and backbone modified nucleotides, and ribose modified nucleotides, phosphoramidates, phosphoro Thionate, Phosphonamidate, Methyl Phosphenate, Methyl Phosphoamidate, Methyl Host Amidate, 5'-β-Cyanoethyl Phosphoamidate, Methylene Phosphonate, Phosphodiothioate, Peptide, Nucleic Acid, Non-Optoselectivity (Achiral) and Including, but not limited to, neutral nucleotide linkages, and non-nucleotide linkages such as polyethylene glycols, aromatic polyamides and fats.

  As used herein, the terms 'polymer or polymer' or 'branched / linear polymer or polymer' have a structure branched at one end of the molecule and the other end. Means a molecule that has a linear portion, where the branched region binds to the substrate and the linear region binds to a ligand, probe, or protecting group.

  In the present specification, “polypeptide”, “peptide”, and “protein” mean a polymer composed of amino acid residues, and are used together. This term applies not only to polymers composed of naturally occurring amino acids, but also to the case where one or more residues are arbitrarily synthesized analogs corresponding to naturally occurring amino acids. The The term is also used to include a variety of variants to typical peptide bonds that link the amino acids that form the polypeptide.

  As used herein, 'protecting group' refers to a group attached to a reactive group (eg, a hydroxyl group or an amine group) of a molecule. The protecting group means a group selected to prevent reaction of a specific radical by one or more chemical reactions. In general, a particular protecting group refers to one that is selected for subsequent removal to recover a particular reactive group without altering other reactive groups present in the molecule. The choice of protecting group is made taking into account the function of the particular radical to be protected and the compound on which it is presented. The technology relating to the selection of the protecting group is, for example, Protective Groups in Organic Synthesis [2nd edition, such as Greene, John Wiley & Sons, Inc.]. Somerset, N .; J. et al. (1991)].

  As used herein, 'protected amine' means an amine that has reacted with an amino protecting group. The amino protecting group prevents the amide reaction from occurring while the branched end is attached to the solid support when the linear end functional group is an amino group. The amino protecting group is removed in the next step while maintaining the amino group in the molecule without change of other reactive groups. For example, the exocyclic amine can be reacted with dimethylformamide diethyl acetal to perform a dimethylaminomethyleneamino reaction. Amino protecting groups generally include carbamates, benzyl radicals, imidates, and others well known to those skilled in the relevant arts. Preferred amino protecting groups include, but are not limited to, p-nitrophenylethoxycarbonyl and dimethylaminomethyleneamino.

  In this specification, the term 'regular interval' means that the space between the ends of macromolecules of size-controlled dendrimers whose size of the molecule is controlled is regular. It means that the distance between them is about 1 nm to about 100 nm, which is the space for interaction between the target specific ligand and the target to occur without any practical hindrance. Therefore, the macromolecular layer on the substrate must not be too dense so that specific molecular interactions can occur.

  In the present specification, the term “solid support” means a structure composed of an immobilized substrate. Said immobilized substrate is an insoluble material, solid, surface, substrate, layer, coating, woven or non-woven, matrix, crystal, membrane, insoluble polymer, plastic, glass, biological or Includes, but is not limited to, biocompatible, bioerodible, or biodegradable polymers, or substrates, microparticles, or nanoparticulates. Solid supports include, for example, monolayers, bilayers, commercial membranes, resins, matrices, fibers, separation media, chromatographic supports. support), polymers, plastics, glass, mica, gold, beads, microspear, nanospear, silicon, gallium arsenide, organic and inorganic metals, semiconductors, insulators, microstructures and nanostructures, but not limited to . Microstructures and nanostructures are ultra-small and nanometer-scale, supramolecular sized probes, tips, bars, wedges, caps, rods, sleeves, lines ( wire), filaments, and tubes, but are not limited thereto.

  As used herein, a “spacer molecule” refers to one or more nucleotide or non-nucleotide molecules, functional groups selected or devised so that two nucleotides or nucleotides and non-nucleotides can bind to each other. , Spacer arms, preferably those that can change or adjust the distance between nucleotides or between nucleotides and non-nucleotides.

  As used herein, “specific binding” refers to a measurable and reproducible degree of reproducibility between a ligand and its specific binding partner or between a defined sequence fragment and a selected molecule or selected nucleic acid sequence. It means attraction. The degree of attraction need not be maximal. Weak, medium, or strong attractive forces can be applied in various ways. The specific binding that occurs in such interactions is well known to those skilled in the art. With respect to defined synthetic sequence fragments, it is a synthetic aptamer, synthetic heteropolymer, nucleotide ligand, nucleotide receptor, shape recognition element, and specifically bindable surface. Said 'specific binding' includes those that specifically recognize structural shapes and surface features. Specific binding is a third that shares chemical identity (ie, one or more identical chemical groups) or molecular recognition ability (ie, molecular binding specificity) with a specific binding partner. Clearly means a specific, saturable, non-covalent interaction between two molecules (ie, specific binding partners) that is competitively inhibited by a molecule (ie, a competitor). The competitor is, for example, a cross reactant, an antibody or antigen analog thereof, a ligand or receptor thereof, or an aptamer or target thereof. For example, specific binding between antigen antibodies is competitively inhibited by cross-reacting antibodies or antigens. The “specific binding” is a subset of specific recognition including all of specific binding and structural shape recognition, and can be used for convenience in an abbreviation or close sense.

  As used herein, “substrate” refers to a non-biological, synthetic, non-living, flat when used on a substance, structure, surface or material. Composition comprising a spherical or flat surface, multiple recognition sheets transcending various molecular species including surfaces, structures, or materials, specific binding, hybridizing or catalytic recognition sheets, or multiple Including various recognition sheets. The substrate is, for example, a semiconductor, a synthetic (organic) metal, a synthetic semiconductor, an insulating agent, a dopant; a metal, an alloy, an element, a compound, a mineral; a composition, a cutting, an etching, a lithography, a printing, a mechanization, or a fine Manufactured slides, devices, structures and surfaces; industrial polymers, plastics, membranes, silicon, silicates, glass, metals and ceramics; wood, paper, cardboard, cotton, wool, clothing, textiles or nonwovens, materials ( material), fabrics; including but not limited to nanostructures or microstructures that utilize branched / linear polymers and are not modified by immobilization of probe molecules.

  As used herein, 'target-probe binding' refers to two or more molecules in which at least one selected molecule is bound to another one in a specific manner. is there. In general, the first selected molecule is indirectly with the second selected molecule, i.e., for example, by intervention of a spacer arm, spacer group, molecule, bridge, carrier, or specific recognition partner. Or directly, for example, without the intervention of spacer arms, groups, molecules, bridges, carriers, or specific recognition partners. Other non-covalent binding refers to the binding of nucleotides and non-nucleotide molecules such as ionic binding, hydrophobic interaction, ligand nucleotide binding, chelating agent / metal ion pairing, or specific binding pairs such as avidin / Biotin, straptabidine / biotin, anti-fluorescein / fluorescein, anti-2,4-dinitrophenol (anti-2,4-dinitrophenol) / dinitrophenol (DNP), anti-peroxidase / peroxidase , Anti-digoxigenin / digoxigenin, or a more general receptor / ligand binding. For example, alkaline phosphatase, phosphorase peroxydase, β-calactosidase, urease, luciferase, rhodamine, fluorescein, or fluorescein Luminol, isoluminol, acridinium ester, or a molecule selected by a reporter molecule such as a fluorescent microspear used for labeling purposes or a selected nucleic acid sequence, avidin / biotin, streptavidin / biotin (Streptavidin / biotin), anti-fluosei Or fluorescein, anti-peroxydase / peroxysoyde, anti-2,4-dinitrophenol (anti-2,4-dinitrophenol) / dinitrophenol (DNP), anti-digoxigenin / digoxigenin, or It is also possible to bind to a selected molecule or selected nucleic acid sequence through a specific binding pair by receptor / ligand binding (ie, directly and covalently).

[Formulation of macromolecular polymer]
FIG. 1 is a view for explaining the structural formula I of the polymer of the present invention. A variety of R, T, W, L, and X group variants are shown in FIG. The macromolecular polymers of the present invention include branched, highly branched, or symmetric or asymmetric polymers. The branched ends of the polymer can be bonded to the substrate, preferably through multiple ends. The linear end of the polymer can have a functional group capable of binding to a protecting group or a target specific ligand. The distance between probes between multiple polymers on the substrate is from about 0.1 nm to about 100 nm, preferably from about 1 nm to about 100 nm, more preferably from about 2 nm to about 70 nm, more preferably from about 2 nm to About 60 nm, most preferably about 2 nm to about 50 nm.

<R working group>
According to Structural Formula I described in FIG. 1, the polymer generally includes a branched site, and multiple ends are functionalized and bonded to the substrate. In sites which are branched the branching, first generation action substituent _R x branched _{(R 1, R 2, R} 3) , the second-generation functional group branched by functional group W _R xx _(R 11, _{R 12} , R ₁₃ , R ₂₁ , R ₂₂ , R ₂₃ , R ₃₁ , R ₃₂ , R ₃₃ ). The branched second generation functional groups are branched by a functional group W to generate a third generation functional group R _xxx (R ₁₁₁ , R ₁₁₂ , R ₁₁₃ , R ₁₂₁ , R ₁₂₂ , R ₁₂₃ , R ₁₃₁ , R ₁₃₂ , R ₁₃₃ , R ₂₁₁ , R ₂₁₂ , R ₂₁₃ , R ₂₂₁ , R ₂₂₂ , R ₂₂₃ , R ₂₃₁ , R ₂₃₂ , R ₂₃₃ , R ₃₁₁ , R ₃₁₂ , R ₃₁₃ , R ₃₂₁ , R ₃₂₂ , R ₃₂₃ , R ₃₃₁ , R ₃₃₂ , R ₃₃₃ ). The additional fourth generation working group is then linked to the branched third generation working group in the same manner. The terminal R group is functionalized so that it can bind to the substrate.

All generations of R groups are the same or different. Usually, the R group is a linear or branched organic moiety such as, but not limited to, repeating units, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, ether, polyether, ester, aminoalkyl, and the like. However, it is not necessary for all R groups to be the same repeating unit, or for every atom position of the R group to be a repeating unit. For example, in the first generation branches R _x , R ₁ , R ₂ , R ₃ , all R groups at the branch level may be the same repeating unit. Alternatively, R ₁ can be a repeating unit, and R ₂ and R ₃ can be H or any other chemical entity. Alternatively, R ₂ can be a repeating unit and R ₁ and R ₃ can be H or all other chemical groups. Analogously, in the case of second and third generation branches, the R group can be a repeating unit, H, or any other chemical group.

Therefore, polymers having various shapes can be manufactured in this manner. For example, if R ₁ , R ₁₁ , R ₁₁₁ , R ₁₁₂ , and R ₁₁₃ are the same repeating unit and all other R groups are H ′ or many other neutral small molecules or atoms A very long and thin polymer is formed having branches with three functional termini for R ₁₁₁ , R ₁₁₂ , and R ₁₁₃ . A wide variety of other chemical sequences are possible. Thus, from about 3 to about 81 termini having functional groups capable of binding to the substrate can be obtained. Preferred terminal numbers are from about 3 to about 75, from about 3 to about 70, from about 3 to about 65, from about 3 to about 60, from about 3 to about 55, from about 3 to About 50, about 3 to about 45, about 3 to about 40, about 3 to about 35, about 3 to about 30, about 3 to about 27, about 3 to about 25 , About 3 to about 21, about 3 to about 18, about 3 to about 15, about 3 to about 12, about 3 to about 9, or about 3 to about 6 It is.

<T terminal group>
The terminal group T is a functional group that is sufficiently reactive to cause an addition or substitution reaction to occur. Examples of such functional groups include amino, hydroxyl, mercapto, carboxyl, alkenyl, allyl, vinyl, imide, halo, urea, ocsilanyl, aziridinyl, oxazole linyl, imidazole linyl, sulfonate, phosphonate, isocyanate, isothiocyanate, Including, but not limited to, silanyl, halogenyl and the like.

<W working group>
In Structural Formula I of FIG. 1, W is any functional group that can link the polymer to another (or any other divalent organic moiety), ether, ester, amide, ketone, urea, Urethane, imide, carbonate, carboxylic anhydride, carbodiimide, imine, azo group, amidine, thiocarbonyl, organic sulfide, disulfide, polysulfide, organic sulfoxide, sulfite, organic sulfone, sulfonamide, sulfonate, organic sulfate, amine , Organic phosphoros groups, alkylenes, alkylene oxides, alkylene amines and the like.

<L spacer or linker>
In FIG. 1, the linear portion of the polymer includes spacer domains that include linker sites optionally interspersed with functional groups. The linker moiety is composed of various polymers. The length of the linker is determined by a variety of factors, such as the number of branched functional groups attached to the substrate, the length of the bond with the substrate, the form of the R group used. In particular, the form of repeating units used, the form of target-specific ligands or protecting groups that bind to the ends of the linear sites of the polymer, etc. Thus, it can be seen that the linker is not limited to a particular type of polymer or a particular length. However, as a general rule, the linker length is from about 0.5 nm to about 20 nm, preferably from about 0.5 nm to about 10 nm, and most preferably from about 0.5 nm to about 5 nm.

  The chemical structure of the linker may be substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, ether, polyether, ester, aminoalkyl, polyalkenyl glycol, etc. It can include, but is not limited to, linear or branched organic moieties. The linker may additionally contain a functional group as described above, and is not limited to any specific structure as described above.

  A linker functionalized at the end contains a protecting group. Accordingly, in one aspect, the present invention relates to a substrate with a number of branched / linear polymers that includes a linear end with a protecting group. Such substrates can react chemically to remove protecting groups that are replaced by target-specific ligands. Thus, in a functional application of the system of the present invention, a substrate coupled with a branched / linear polymer linked to a target specific ligand at one end is provided.

<X protecting group>
The choice of protecting group depends on a number of factors such as the preferred acid or base reactivity. Thus, the present invention provides the ability to prevent the functional group from reacting with other chemical functional groups, and to any particular protecting group, so long as it is removed under the desired specialized conditions. It is not limited. The protecting group is preferably easily removed. Examples of such protecting groups used in the present invention include, but are not limited to:

  Amino acid protecting group: methyl, formyl, ethyl, acetyl, t-butyl, anisyl, benzyl, trifluoroacetyl, N-hydroxysuccinimide, t-butyloxycarbonyl, benzoyl, 4-methylbenzyl, thioanidyl, thiocresyl, benzyloxymethyl, 4-nitrophenyl, benzyloxycarbonyl, 2-nitrobenzoyl, 2-nitrophenylsorbyl, 4-toluenesulfonyl, pentafluorophenyl, diphenylmethyl (Dpm), 2-chlorobenzyloxycarbonyl, 2,4,5-tri Chlorophenyl, 2-bromobenzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, triphenylmethyl, 2,2,5,7,8-pentamethyl-chroman-6-sulfonyl, phthaloyl, 3-ni A Rofutaoiru, 4,5-dichloro-phthaloyl, tetrabromobisphenol follower Taro yl, tetrachloro phthaloyl.

  Alcohol protecting groups: p-anisyloxymethyl (p-AOM), benzyloxymethyl (BOM), t-butoxymethyl, 2-chlorotetrahydrofuran (THF), guaiacolmethyl (GUM), (1R) -ment Methoxymethyl (MM), p-methoxybenzyloxymethyl (PMBM), methoxyethoxymethyl (MEM), methoxymethyl (MOM), o-nitrobenzyloxymethyl, (phenyldimethylsilyl) methoxymethyl (SMOM), 2 -(Trimethylsilyl) ethoxymethyl (SEM).

  DNA, RNA protection reagent: 2'-OMe-Ac-C-CE phosphoramidite, 2'-OMe-Ac-RNA CPG, 2'-OMe-I-CE phosphoramidite, 2'-OMe-5-Me-C-CE Phosphoramidite, Ac-C-CE phosphoramidite, Ac-C-RNA 500, dmf-dG-CE phosphoramidite, dmf-dG-CPG 500, 2-amino-dA-CE phosphoramidite (MP Reddy, NB Hanna) , And F. Farooqui, tetrahedron Lett., 1994, 35, 4311-4314; BP Monia, et al., J. Biol. Chem., 1993, 268, 14514-14522).

  Common protecting reagents in organic synthesis: (dimethyl-t-butylsilyloxy) methyl chloride (SOMCl), ethoxyethyl chloride (EECl), -chloroethers, o-nitrobenzyloxymethyl chloride, b, b, b- Trichloroethoxymethyl chloride (TCEMCl), (−)-menthyl ester, (P) -benzyl ester, 1,1,1,3,3,3-hexafluoro-2-phenyl-2-propyl ether, 1 , 1,3,3-tetramethyl-1,3,2-tisilazan, 1,2,4-dithiazolelysine-3,5-dione, 1,2-dibromide, 1,2-dichloride, 1,2 -Diol mono-4-methoxybenzyl ether, 1,2-diol mono-t-butyl ether, 1,2-dioe Monoacetate ester, 1,2-diol monoallyl ether, 1,2-diol monobenzoate ester, 1,2-diol monobenzyl ether, 1,2-diol monotosylate, 1,3-benzodithiolane, 1,3 -Benzodithiolan-2-yl ether, 1,3-diol mono-4-methoxybenzyl ether, 1,3-diol monobenzoate ester, 1,3-diol monobenzyl ether, 1,3-dioxane, 1- (2 -(Trimethoxysilyl) ethoxy) ethyl ether, 1-adamantyl ester, 1-benzoyl-1-propen-2-ylamine, 1-ethoxyethyl ether, 1-methoxyethylidene acetal, 1-methyl-1-methoxyethyl ether, 1-phenyl-3,5-di-t-butyl Lohexadiene-4-oneylamine, 1-phenylethyl ester, 2,2,2-trichloroethoxymethyl ether, 2,2,2-trichloroethyl carbonate, 2,2,2-trichloroethyl ester, 2,2,2- Trichloroethyl phosphate, 2,2,5,7,8-pentamethylchroman-6-sulfonamide, 2,2-dimethyl-4-pentenoate ester, 2,3,6-trimethyl-4-methoxybenzenesulfonamide 2,4,6-trimethylbenzenesulfonamide, 2,4-DNP hydrazone, 2,5-dichlorophenyl phosphate, 2,5-dimethylpyrrole, 2- (2-methoxyethoxy) ethyl ester, 2- (4-nitro Phenyl) ethyl ether, 2- (4-nitrophenyl) ethyl phosphate 2- (4-toluenesulfonyl) ethyl ester, 2- (dibromomethyl) benzoate ester, 2- (trimethylsilyl) ethyl carbonate, 2- (trimethylsilyl) ethyl ester, 2- (trimethylsilyl) ethyl ether, 2-benzenesulfonyl Ethyl thioether, 2-bromoethyl ester, 2-chloroethyl ester, 2-chlorophenyl phosphate, 2-cyanoethyl phosphate, 2-methoxyethyl ester, 2-nitrobenzenesulfenamide, 2-nitrobenzenesulfonamide, 2-oxazoline, 2- Phenylethyl ester, 2-pyridyl disulfide, 2-tetrahydropyranylamine, 4-chlorobenzoate ester, 4-chlorobutyl ester, 4-methoxybenzamide, 4- Toxibenzoate ester, 4-methoxybenzylamine, 4-methoxybenzyl ester, 4-methoxybenzyloxymethyl ether, 4-nitrobenzamide, 4-nitrobenzoate ester, 4-nitrobenzyl ester, 4-nitrobenzyl ether, 4-nitro Benzyl phosphate, 4-nitrophenyl ester, 4-nitrophenyl hydrazone, 4-toluenesulfonamide, 4-toluenesulfonate, 9-fluorenylmethyl carbonate, 9-fluorenylmethyl ester, allyl carbonate, allyl ester , Benzenesulfonamide, benzenesulfonate, benzyl carbonate, benzyl ester, BOM ether, DMTr ether, MEM ether, methanesulfonamide, methanesulfonic acid , Ethyl carbonate, MMTr ether, MOM carbonate, MOM ester, MOM ether, MTHP ether, MTM ester, MTM ether, N-4-methoxybenzylamide, N-4-tolylamide, N-benzenesulfonylamide, N-benzylimine, n-butyl ester, n-butyl ether, O-4-methoxybenzyl covermate, O-9-fluorenylmethyl covermate, phenylthioether, phenylthiol ester piperidine amide, PMB ether, SEM ester, SEM ether, succinate Ester, t-butyl carbonate, t-butyl ester, t-butyl ether, t-butyl phosphate, t-butyl thioether, t-butyl thiol ester, TBDMS ester, TBDM S ether, TBDPS ether, TES ether, THF ether, THP ether, TIPDS diether, TIPS ether, TMS ester, TMS ether, TMS thioether, tosylhydrazone, TPS ether, trifluoroacetamide.

  A list of commercially available protecting groups can be found in the Sigma-Aldrich (2003) catalog, the contents of which are related to the protecting group substrate and are hereby incorporated by reference in their entirety. .

  In general, in one aspect of the present invention, the protecting group used in the present invention is one that is used to add one or more consecutive amino acids or appropriately protected amino acids to a growing peptide chain, etc. is there. Usually, the amino group or carboxyl group of the first amino acid is protected by a suitable protecting group.

  In a particularly preferred way, the amino-functional group is protected by an acid-sensitive or base-sensitive functional group. Such protecting groups must be suitable for linkage formation conditions and have properties that are easily removed without disruption of the growing branched / linear polymer. Such suitable protecting groups include 9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), non-penylisopropyl-oxycarbonyl, t-amyloxy Examples include, but are not limited to, carbonyl, isobornyloxycarbonyl, (α, α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl and the like.

  Examples of particularly preferred protecting groups are also 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), p-toluenesulfonyl, 4-methoxybenzenesulfonyl, adamantyloxycarbonyl, benzyl, o- Bromobenzyloxycarbonyl, 2,6-dichlorobenzyl, isoprofile, t-butyl (t-Bu), cyclohexyl, cyclophenyl and acetyl (Ac), 1-butyl, benzyl and tetrahydropyranyl, benzyl, p-toluenesulfonyl , And 2,4-dinitrophenyl.

  In an additional method, the branch ends of a linear / branched polymer are attached to a suitable solid support. Suitable solid supports that can be used for the synthesis are substances that are not only insoluble in the medium used, but also inert to the conditions and reagents of the sequential condensation-deprotection reaction.

  Removal of protecting groups such as Fmoc from the linear ends of branched / linear polymers can be accomplished by treatment with a secondary amine, preferably piperidine. The protected moiety can be introduced in about a 3-fold excess of moles, and it may be preferred to perform the coupling in DMF. Coupling agents were O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyluronium hexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxy-benzotriazole (HOBT, 1 equivalent). However, it is not limited to this.

  The polymer can be deprotected in a continuous operation or a single operation. Polypeptide removal and deprotection can be performed in a single operation by treating the substrate binding polypeptide with a cleavage reagent, which includes, for example, thiazole, water, Ethanedithiol, trifluoroacetic acid, and the like can be used.

Table 1 below lists various types of exemplary compounds. However, it is understood that mutations in X, L, W, R, and T are also included in the present invention.

[Target-Specific Ligand or Probe]
A target-specific ligand, also called a probe, is a moiety that binds to the linear end of the branched / linear polymer, and includes a variety of chemical substances, biochemical substances, and living compounds. Contains compounds. In this context, the ligand is a nucleic acid, oligonucleotide, RNA, DNA, PNA, or aptamer. The oligonucleotide is a natural nucleic acid or an analog thereof. Therefore, the ligand is a polypeptide consisting of natural amino acids or synthetic amino acids. The ligand is a combination of nucleic acids, amino acids, carbohydrates, or any other chemical that can bind to a linear portion of a branched / linear polymer. In particular, the ligand is a chemical based on a triazine skeleton or the like, which can be used as a component of a combinatorial chemical library, particularly a triazine labeled library.

[Substrate]
Substrate means any solid surface to which the branched / linear polymer can be bound by covalent or ionic bonds. The substrate is functionalized such that a bond occurs between the branched ends of the branched / linear polymer. The surface of the substrate may have various surfaces according to the needs of a person skilled in the art to which the present invention belongs. If a microarray or biochip format is required, typically an oxidized silicon wafer, fused silica, or glass can be used as the substrate. The substrate is preferably a glass slide. Other examples of substrates include, but are not limited to, membrane filters such as nitrocellulose or nylon. The substrate can be hydrophilic or polar and is negatively charged or positively charged before or after coating.

[Microarray]
To improve the performance of DNA microarrays, probe design, reaction conditions between spotting, hybridization and washing conditions, suppression of non-specific binding, distance between biomolecules and surfaces, and immobilized biomolecules Factors such as the space between them must be considered. Since most of these factors are related to the surface properties of the microarray, surface optimization has become a major objective in microarray research. Whitesell and Chang have shown that the alpha helix formation of immobilized oligopeptides is promoted on a space-controlled gold surface [Whitesell et al. , Science 261, 73-76 (1993)].

  The present invention provides a DNA microarray having a single nucleotide polymorphism (SNP) discrimination efficiency close to a lysis value (1: 0.01) by a conical dendron and having reduced non-specific binding of DNA. The

  FIG. 2 is a schematic diagram showing the synthesis of dendron. A variety of starting materials, intermediate compounds, and dendron compounds are shown, where 'X' in the drawing is all protecting groups such as anthracenemethyl (A), Boc, Fmoc, Ns, etc. FIG. 3a shows a modification of the glass surface with dendron (FIG. 3b) and a single base mistakenly combined while selectively hybridizing a target nucleotide labeled with a fluorophore with an appropriate oligonucleotide probe. We can show that these pairs can be effectively distinguished with dendron-modified surfaces.

  Second generation branched dendrons having surface reactive functional groups at the ends of the branches can be used, which are self-assembleable and provide adequate space between them. Studies to date have successfully produced a well-behaved monolayer by multiple ionic attraction between the cation on the glass substrate and the carboxylate, the anion at the end of the dendron. It has been shown that the spacing between ligands can be ensured to be 24 cm or more [Hong et al. , Langmuir 19, 2357-2365 (2003)]. In order to facilitate deprotection and enhance the reactivity of the deprotected end amine, we modified the structure as shown in FIG. 3b. The inventor has also observed that the formation of a covalent bond between the carboxylic acid group of the dendron and the hydroxy group on the surface is effective for the degree of ionic bond and promotes terminal stability. In addition, an oligoether interlayer was effective in suppressing non-specific oligonucleotide binding.

  Hydroxylated surfaces were made using previously reported methods [Maskis et al. , Nucleic Acids Res. 20, 1679-1684 (1992)]. A substrate containing oxidized silicon wafer, fused silica, and glass slide was modified with (3-glycidoxypropyl) methyldiethoxylane (GPDES) and ethylene glycol (EG). The use of 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride (EDC) or 1,3-dicyclohexylcarbodiimide (DCC) in the presence of 4-dimethylaminopyridine (DMAP). Dendron was introduced into the substrate by a binding reaction between the carboxylic acid group and the hydroxy group of the substrate [Boden et al. , J .; Org. Chem. 50, 2394-2395 (1985); Dhaon et al. , J .; Org. Chem. 47, 1962-1965 (1982)]. The increase in thickness after introduction of the dendron is 11 ± 2 mm, which is comparable to the existing value observed for ionic binding [Hong et al. , Langmuir 19, 2357-2365 (2003)]. After immobilization, the absorption peak generated in the dendron anthracene moiety was observed to be 257 nm. The molecular layer is stable and shows no change in thickness and adsorption properties when stirred with dimethylformamide for 1 day (FIG. 4). Local images obtained by a tapping mode atomic force microscope (TM-AFM) also showed that the resulting layer was very smooth and homogeneous without aggregation or pores (FIG. 5).

  In order to prepare a DNA microarray, the immobilized dendron was activated to generate primary amine groups through a deprotection process. After deprotection with 1.0 M trifluoroacetic acid (TFA) [Kornblum et al. , J .; Org. Chem. 42, 399-400 (1977)], the absorption peak at 257 nm disappeared without any other detrimental changes in surface properties (FIG. 4a). Such observations indicated that the protecting groups were successfully removed without chemical damage to the layer, and that the thickness was slightly reduced upon removal of the protecting groups.

  Already established methods [Beier et al. , Nucleic Acids Res. 27, 1970-1977 (1999)] after modification using di (N-succinimidyl) carbonate (DSC) and then using a Microsys 5100 Microarrayer (Cartesian Technologies, Inc.) to a class 10,000 clean room. Probe oligonucleotides were activated by spotting a 50 mM sodium non-carbonate buffer (10% dimethyl sulfoxide (DMSO), pH 8.5) solution of an appropriately amine-tethered oligonucleotide (20 μM). Immobilized on the glass slide surface. Usually, a substrate having a reactive amine group surface has a thiolated oligonucleotide and succinimidyl 4-maleimidobutyrate (SMB), or sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane- An atypical heterofunctional linker such as 1-carboxylate (SSMCC) is used [Oh et al. Langmuir 18, 1764-1769 (2002); Frutos et al. , Langmuir 16, 2192-2197 (2000)]. In contrast, the dendron-modified surface ensures a certain distance between the amine functional groups, so there is no problem using a homotypic, bifunctional linker such as DSC. . As a result, amine-linked oligonucleotides can be used for spotting. Aside from cost efficiency, the use of thiol-linked oligonucleotides, which are useful under certain conditions, but are readily oxidized, can be avoided.

The DNA microarray is fabricated to evaluate the discrimination efficiency between a complementary pair (A: T) and a pair (T: T, G: T, C: T) in which three internal single bases are mistakenly combined. can do. After spotting the probe oligonucleotides in 4/4 format, the microarray was incubated for 12 hours in a humidity chamber (80% humidity) to provide sufficient reaction time for the amine-conjugated DNA. Thereafter, the slides were buffered at 37 ° C. for 3 hours (2 × SSPE buffer (pH 7.4), 7.0).
non-specifically bound oligonucleotides were removed by stirring for 5 minutes with boiling water and with 5 mM sodium dodecyl sulfate). Finally, the DNA functionalized microarray was dried under a stream of nitrogen for the next step. Other types of probes were floated on one plate for proper comparison.

To hybridize, 15 base oligonucleotides (target 1) or 45 base oligonucleotides (target 2) were used (FIG. 3c). Hybridization was performed using GeneTAC ^™ HybStation (Genomic Solution, Inc.) for 1 hour at 50 ° C. with the washing buffer solution containing the target oligonucleotide (1.0 nM) labeled with Cy3 fluorescent dye. To remove excess target oligonucleotide, the microarray was washed 4 times with buffer solution at 37 ° C. and dried with nitrogen. The fluorescence signal on each spot was measured using ScanArray Lite (GSI Lumonics) and analyzed using Imagene 4.0 (Biodiscovery).

In the case of a 15 base target oligonucleotide, the image obtained showed a significant difference in intensity between the correctly combined pair and the incorrectly combined pair (FIG. 6A). The standardized fluorescence signal ratios (or ratios of single base internal mistakes to fully combined pairs, ie MM / PM) are 0.005, 0.008, and 0.006. (T: T, G: T, and C: wrong combinations inside T) (FIG. 6A and Table 2). The observed selectivity was significantly improved over existing conventional methods, and a very large increase in selectivity (20- to 82-fold) was recorded when compared to typical on-surface microarrays (Table 2). Already, the inventor has also demonstrated that the selectivity ratio of microarrays fabricated on various amine surfaces including mixed self-assembled monolayers (ie mixed SAMs) is 1: 0.19 to 0.57. Observed [Oh et al. , Langmuir 18, 1764-1769 (2002)]. In addition, other researchers have reported that the surface can be modified to develop better detection methods and improve the performance of DNA microarrays, but as far as fluorescence detection methods are concerned, None of these could show such an improved ratio [Zhao et al. , J .; Am. Chem. Soc. 125, 12531-12540 (2003); Chakrabarti et al. , J .; Am. Chem. Soc. 125, 12531-12540 (2003); Benters et al. , Nucleic Acids Res. 30, e10 (2002); Guschin et al. , Analytical Biochemistry 250, 203-211 (1997); Taton et al. , Science 289, 1757-1760 (2000); Wang et al. , Nucleic Acids Res. 30, e61 (2002)]. For example, in a ternary hybridization / detection system (capturer / target / probe), a successful discrimination ratio of 1: 0.07 has been reported (Zhao et al., J. Am. Chem. Soc. 125). 12531-12540 (2003)] Even when peptide nucleic acids (PNAs) capable of increasing the selectivity are used, the selectivity on the gold thin film and the gold nanoparticle is 1: 0.14 and 1: 1, respectively. It was 0.07 [Chakrabarti et al., J. Am. Chem. Soc. 125, 12531-12540 (2003)].

  To achieve a more practical system, a 45 base target oligonucleotide was used. The ratios of MM / PM to base pairs mistakenly combined inside T: T, G: T, and C: T were 0.006, 0.009, and 0.009 (FIG. 6B and Table 2). ). Such a result indicates that when the length of the target oligonucleotide is long, it exhibits a clear selectivity. The efficiency of the DNA microarray appears to be due to the peculiarities of the dendron modified surface, ie mesospacing between immobilized DNA-strands.

  For comparison, a DNA microarray was fabricated on a substrate modified to (3-aminopropyl) diethoxymethylsilane (APDES) (Oh et al., Langmuir 18, 1764-1769 (2002)). This is a common substrate used for DNA or protein microarrays. The selectivity was tested as in the dendron modified DNA microarray except that a 1,4-phenylene diisothiocyanate (PDITC) linker was used. Amine-treated oligonucleotides are described in Guo et al. Nucleic acids Res. 22, 2121-2125 (1994). For T: T, G: T, and C: T, the measured MM / PM ratios were 0.41, 0.38, and 0.26 (FIG. 6C and Table 2). Using a DSC linker on an APDES modified substrate showed high coefficient of variation (CV) values (> 20%), indicating the degree of variation per spot and non-uniform fluorescence intensity within each spot. Is. On the other hand, like the dendron modified surface with DSC linker, PDITC linker showed better variation coefficient value (CV) (<15%) and uniform fluorescence intensity within a single spot (FIG. 7).

For additional comparison, a probe 2 oligonucleotide with an additional (T) ₃₀ spacer at the 5 'end of the oligomer was used for SNP discrimination experiments. In this case, the probe with additional spacers was immobilized on the APDES modified surface. The measured MM / PM ratios for the T: T, G: T, and C: T cases were 0.17, 0.18, and 0.12 (FIG. 6D and Table 2). As compared to a probe DNA having a C ₆ spacer, although the selectivity was greatly improved, it was still lower than dendron modified DNA microarray.

There are various problems in surface hybridization, making adjustment and prediction difficult when performing accurate microarray screening. In addition to Gibbs free energy for double muscle formation and melting temperature (Tm, melting temperature), non-specific binding, replacement effect, electrostatic effect, and condition change must be considered in the washing stage. Don't be. Between internally mispaired base pairs (of 15-mers, T: T, G: T, and C: T are defects miscombined internally) and fully paired base pairs The difference in Gibbs free energy is 2.67, 1.75, and 3.05 kcal / mol at 50 ° C. Gibbs free energy was calculated with H _Y T _HER ^™ software (http://ozone2.chem.wayne.edu). Thus, the theoretical fluorescence ratio (MM / PM) was 0.016, 0.065, and 0.009, respectively. Moreover, according to the research result on the solution using the molecular signal, the SNP discrimination ratio was as low as 1: 0.01 (Tatton et al., Science 289, 1757-1760 (2000)). These data illustrate that the dendron modified DNA microarray of the present invention is ideal for reaching or exceeding the thermodynamic limit. In particular, in the case of G: T, the discrimination efficiency in the microarray method is superior to the numerical value calculated on the solution. Although the main factors for increasing the selectivity were not investigated, strict conditions are expected to play an important role in the washing stage.

[P53 SNP detection]
In the biological world, p53 tumor suppressor genes play an important role in cell regulation, gene transcription, genome stability, DNA repair, and cell killing (Velculescu et al, 1996, Clin. Chem., 42: 858-868). , Harris et al, 1996, 88: 1442-1455, Sitransky et al, Annu, Rev. Med., 1996, 47: 285-301). Tumors are induced when p53 loses its original function, and mutations in p53 are the most common genetic mutations in human cancers such as rectal and lung cancer (Greenblatt, 1994, 54: 4855-4878).

  [9]-A DNA microarray on an acid dendron modified substrate is used to detect a single mutation of the p53 tumor suppressor gene from a cancer cell line. A target DNA sample (200-400 bases) containing 175 codons was produced by a Rendon primer method on a genomic DNA template and hybridized to a dendron-modified surface to which a probe oligonucleotide composed of 18 bases was immobilized. . The ratio of MM / PM to the wrong combination of A: C, T: C, and C: C was 0.028, 0.031, and 0.007 (FIG. 8a). Such a result shows that there is a clear selectivity for the actual target DNA.

  [27] -A DNA microarray on an acid dendron modified substrate is produced by the same method as the above [9] -acid dendron and applied to detection of a single base mutation at 175 codons in the p53 tumor suppressor gene did. The ratios of MM / PM to bases incorrectly combined in A: C, T: C, and C: C were 0.066, 0.01, and 0.005 (FIG. 8b). These results indicate that the DNA microarray on the [27] -acid dendron modified substrate also has a clear selectivity for single base mutations in the actual target DNA.

<Detection of 7 hot spot mutations in p53 gene using dendron modified surface>
A dendron modified substrate was used to detect single base mutations in the p53 tumor suppressor gene in cancer cell lines. A target DNA sample (200-400 base pairs) containing seven hot spot codons (175, 215, 216, 239, 248, 273, and 282) is amplified from DNA extracted from tumor cells by the Rendon primer method. Hybridized with capture probes (oligonucleotides 15-25mer) corresponding to the seven immobilized hot spots (Figures 9a and 9b). As a result, very good SNP discrimination efficiency was obtained.

  The present inventor can successfully produce a DNA microarray with the highest reproducibility through spatial modulation between probe DNAs, and the efficiency of SNP discrimination can be improved much more than the numerical value in solution. It was. The measured discrimination efficiency made the method according to the invention highly reliable for genetic diagnosis. It is expected that the method of the present invention can be applied to a variety of biological analyzes that utilize immobilized biomolecules.

[Glass beads with controlled pores]
Chromatographic carriers that are widely used in affinity chromatography are natural polymers such as dextrin and agarose. Sepharose 6B, 4B, and 2B are chromatographic materials composed of cross-linked agarose with a very low non-specific binding rate. Despite the widespread use of the materials, common beaded agarose gels have several disadvantages. For example, due to the soft nature of the material, the flow rate (flow rate) is low, it shrinks significantly and is irreversible, so it cannot be frozen or dried and is vulnerable to some organic solvents (Cuatrecasas, PJ Biol. Chem. 1970, 245, 3059-3065; Kim et al., Biochemistry 2002, 41, 3414-3421). In comparison, glass with controlled pores (CPG) exhibits a variety of special properties as a carrier: 1) mechanical stability, 2) a fixed three-dimensional structure, that is, it expands with environmental changes 3) Chemical stability under pH 1 to pH 14 conditions, 4) Stability to a wide range of nucleophilic and electrophilic reagents, 5) Thermal stability, 6) Excellent flowability (or elution) 7) Low adsorptivity to the container surface. In addition, reagents and by-products can be rapidly removed through washing after the transformation step. All these properties provide potential utility in gel permeation chromatography, solid state synthesis, affinity separation, and various other fields.

Pore size: The effective porosity of the CPG for the adsorbed material was measured using the guest accessibility to the host surface. As an initial approach, the CPG accessibility to the guest is dependent on geometric factors that are correlated to the relative size of the host relative to the size of the guest. The size of the molecules larger than the pores that induce the internal surface, adsorption, and interaction of the guest is possible only if it has an external surface that is much smaller than the internal surface area of the pore material under investigation (Poscharko et al., J Am. Chem. Soc. 2003, 125, 13415-13426; Ottaviani et al., J. Phys Chem. B. 2003, 107, 2046-2053). Considering these facts, the adsorption force and degree of adsorption of the guest on CPG may depend on the following factors: CPG pore size, overall host surface area, and host The chemical composition of the accessible surface. In the present invention, three types of GST fusion proteins (GST (28 kDa), GST-PX ^P47 (41 kDa), and GST-Munc18 fragment (98 kDa)) were used. The molecular dimension GST-Munc1 must be similar to the fusion GST 100 kDa, GST-DREF (140 × 40 × 93 cm) (Hirose et al., J. Biol. Chem. 1996, 271, 3930-3937; Zhan et al., Gene 2001, 218, 1-9). In order to reach a balance between porosity and surface area, the porosity of the carrier must be optimized to suit each specific protein. Since it is a well-known fact that CPG having pores of about 50 nm contains a complex of molecular subunits generally present in proteins, this study was conducted using 50 nm CPG. At the same time, as long as the protein was used, CPG with larger pores (300 nm) confirmed the efficiency of 50 nm CPG. (Collins et al., Anal. Biochem. 1973, 54, 47-53; Haller, W. J. Chromatogr. 1973, 85, 129-131).

Glutathione CPG variants (samples E1, E3, A, CS, and CL): The most important concern in the affinity matrix is the degree of non-specific binding (or NSB). This is a very unique problem in affinity separation and solid state synthesis. In general, the most important factor for suppressing non-specific binding is to eliminate hydrophilic groups in the matrix to increase hydrophilicity (Sigal et al, J. Am. Chem. Soc. 1998, 120, Chapman et al., Langmuir 2000, 16, 6927-6936; Chapman et al., J. Am. Chem. Soc. 2000, 122, 8303-8304; Ostuni et al., Langmuir 2001, 17, 6336-6343; Chapman et al., Langmuir 2001, 17, 1225-1233; Ostuni et al., Langmuir 200; , 17,5605-5620). Even when transformed to an aminoalkyl group, the CPG surface is polar and partially negatively charged (Hudson, DJ Comb. Chem. 1999, 1, 403-457). It has been reported that the use of diepoxide as a spacer can minimize matrix hydrophilicity and non-specific binding (Suenetal., Ind. Eng. Chem. Res. 2000, 39, 478-487; Sundberg). et al., J. Chromatogr. B. 1974, 90, 87-98; Shimizu et al., Nature Biotechnology 2000, 18, 877-881). Therefore, samples E1 and E3 were prepared by modifying the surface to 1,4-butanediol diglycidyl ether (or BUDGE). An important property of using BUDGE is that it produces a very stable ether bond against hydrolysis, has improved flexibility due to a long spacer arm, and suppresses non-specific binding to some extent. That's what it means. The last advantage is explained by a structural motif similar to polyethylene glycol. Diepoxides can be used to link molecules and surfaces with nucleophilic groups (eg amines and thiols). In the ring-opening process, not only β-hydroxy groups but also bonds between stable carbon heteroatoms are formed. By using a linker before and after the dendron modification step, the flexibility of the modified GSH can be ensured. The transformation stage is summarized and shown schematically in FIG. Common reagents called EDC and NHS were used to bind the dendron to the matrix surface. After changing the substrate with dendron, it was introduced into acetic anhydride to capping all remaining amine groups. Finally, the matrix was treated with 20% piperidine for 30 minutes to deblock additional variants of the dendron Fmoc group. After another extension reaction with BUDGE, GSH was immobilized using a bond between thiol and epoxide.
Sample A was produced as a control group. A BUDGE, 1,3-diaminopropane, and BUDGE-provided surface material was continuously transformed in a manner substantially similar to E1 and E3, except that there was no dendron. GSH was immobilized using a ring-opening reaction between epoxide and thiol. Other heterogeneous control beads (samples CL and CS) were prepared using a heterobifunctional linker called GMBS and conjugated with GSH and AMCPG or LCAA-CPG. AMPCPG has a short arm composed of C3 hydrocarbons on the surface, and LCAA-CPG has a long arm composed of C15 aliphatic chains. After amide formation with GMBS, the beads were treated with GSH. By adding a thiol group to the maleimide group, a covalent bond was formed between the carbon and sulfur atoms. Forming covalent bonds in the two-step process yielded glass beads with controlled pores, CS and CL, to which GSH was bound.

Ligand density measurement: Since it is difficult to directly measure the amount of immobilized glutathione, use an indirect method to determine the density of the ligand by measuring the amount of dibenzofulvene released in the deprotection step did. The 9-fluoromethoxycarbonyl (Fmoc) protecting group attached to the apex of the dendron is acid stable or easily cleaved by a variety of bases. In this example, 20% piperidine of DMF was used to deprotect the Fmoc group. Piperidine produces dibenzofulvene and an adduct, which absorbs the adduct at 301 nm (， ye et al., J. Phys. Chem. B. 2003, 107, 3496-3499). On the other hand, when the absorbance of the solution obtained in the deprotection step with 20% piperidine appears at 301 nm, this means that deprotection has occurred as intended.
The density of the ligand obtained by such a method was 8.3 μmol / g for E1 and 5.6 μmol / g for E3. The density is reduced by a factor of 11.1 when modified with Fmoc (3) acid, and the value is additionally reduced by a factor of 1.5 when using larger dendrons. Thus, in a specific example of the present invention, a smaller dendron is more effective at obtaining a higher density than a larger dendron.

Analysis of GST binding: Purified GST and cell lysates were used to analyze the binding properties of samples A, E1, and E3 (lanes 2, 3, and 4 in FIG. 11). Lane 1 shows that the lysate was successfully produced. It is clear that the three matrices bind effectively to purified GST. When cell lysates were introduced into the beads (lanes 5, 6, and 7), considerable differences were observed between A and E1 or E3 samples. In the case of sample A, severe non-specific binding was observed despite the introduction of the BUDGE linker. Furthermore, non-specific protein binding was effectively suppressed when dendron was introduced into the matrix. It is noteworthy that non-specific binding to the solid support was effectively suppressed by the self-assembly of one of the first and second generation dendrons. The extended spacer between dendron and GSH maintained the activity of the bound tripeptide.
In FIG. 12, in one example of the present invention, the ether and amide groups form the main skeleton of the structure, and the amide bond is again generated by dendron fixation. The wide range of dendrons is also an important factor for success.
The density of E1 ligand is 1.48 times higher than E3. In other words, E1 recorded a ligand concentration of 148% (Table 3). To test the binding efficiency of the two types of beads, the sample mass was adjusted to have the same number of GSH for each sample. According to the density meter, the utilization of the ligands in the two cases was very close (29%, 31%). The wider spatialization of E3 failed to improve the binding efficiency of GST, probably because the experimental protein was larger than the spatialization of E1 and E3.

  Control experiment: GSH density was 14.5 μmol / g for CS and 11.9 μmol / g for CL. In order to compare the efficiency of beads in terms of specific binding of GST, proteins captured with CS (5.7 mg) and CL (7.0 mg) beads were labeled E1 (10.0 mg) and E3 (14. Analysis with samples from 8 mg) beads. The utilization was adjusted to approximately the same number of GSH. It is clearly shown by chromatography that CS and CL beads not only exhibit low binding capacity but also unfavorable selectivity (FIG. 13). Such a result highlights the importance of dendrons to ensure effective suppression of non-specific binding as well as improved GST accessibility to immobilized GST.

Dependence on molecular weight: In order to provide a surface with a regulated space between ligands immobilized by dendron modification, the binding ability to proteins having various molecular weights is combined. In particular, it is known that a space of 24 cm or more can be secured when using a second-generation dendron (Cardona et al., J. Am. Chem. Soc. 1998, 120, 4023-4024). As a specific experiment, GST protein (28 kDa), GST-PX ^p47 (41 kDa), and a GST-munc-18 fragment (98 kDa) obtained from a wild lysate were produced. As shown in FIG. 14, the binding ability of the beads (E1, E3, and Sepharose 4B) decreases rapidly as the protein molecular weight increases. The degree of reduction is the same in all three cases. The interesting thing is that the degree of reduction is the same in three different cases. When the binding ability for E1 was adjusted to 100% for GST, GST-PX ^p47 had a relative binding ability of 92% and 22% for GST-munc18. For E3 beads, GST was 85% and GST-munc18 was 23%. This indicates that the dependence on the molecular weight of the protein was also observed with glutathione sepharose 4B. The binding ability of GST-PX ^p47 to glutathione sepharose 4B was 104%, and GST-munc18 was 17%. The only difference is that GST and GST-PX ^p47 show a certain binding capacity against commercially available matrices. The difference may reflect non-uniform spacing of Sepharose 4B. From these materials, there are various spacings between the GSHs, so that the matrix can bind to the fused GST as much as natural GST. For a much larger protein, GST-munc18, the spacing should be smaller. In this respect, the constant decrease in the binding capacity of the dendron-treated beads confirms the constant spacing of GSH on the surface.
In summary, the dendron modified matrix exhibits a sensitivity as high as that of a commercially available matrix (eg Sepharose 4B), and exhibits a molecular weight dependence of the same degree as that of a commercially available product. Introducing dendrons into the AMPCPG matrix not only effectively reduces non-specific binding, but also maintains the binding power of GSH. Observing that the binding force decreases steadily in proportion to the increase in protein molecular weight, this phenomenon is thought to be consistent with the regular spacing between immobilized GSHs. Not only is it easy to adjust the spacing, it has a variety of advantages such as mechanical stability, wide compatibility with various chemical environments, and ease of various anticipated applications.

  The scope of protection of the present invention is not limited to the specific examples described in the present specification, and various modifications other than the specific examples described in the present specification are described in the detailed description of the present invention and attached. It will be clear to the expert in this technical field from the drawings. Such modifications also fall within the protection scope of the claims of the present invention. The following examples are intended to illustrate the invention and are not intended to be limiting.

  In the following examples, the numbering scheme of compounds was named as Compound 1, Compound 2, I, II, III, IV, V, and the like. However, the compound numbering system is intended to be used consistently, limited in the specific examples cited. For example, Compound 1 cited again in Example 2 is not necessarily the same as Compound 1 in Example 3.

Example 1: Size-Controlled Macromolecule (Size-Controlled Macr
microarray manufacturing method using
In Example 1, as shown in FIG. 2, the designations I, II, III, IV, and V refer to various compounds and intermediate compounds shown in FIG.

<Example 1.1: Material>
Silane coupling agents (3-glycitoxypropyl) methyldiethoxylane (GPDES) and (3-aminopropyl) diethoxymethylsilane (APDES) were purchased from Gelest, Inc. and others The compounds were purchased in reagent grade from Sigma-Aldrich. The reaction solvent for silylation is an anhydride purchased from Aldrich (Sure / Seal bottles). All wash solvents for the substrate were purchased in HPLC grade from Mallinckrod Laboratories Chemicals. UV grade fumed silica plates (30 mm × 10 mm × 1.5 mm) were purchased from CVI Laser Corporation. Polished primary Si wafers (100) (dopant, phosphorous; resiliency, 1.5-2.1 Ω.cm) were purchased from MEMC Electronic Materials, Inc. Glass slides (2.5 × 7.5 cm) were purchased from Corning Co. All oligonucleotides were purchased from Metabion. Ultrapure water (18 MΩ.cm) was obtained from a Milli-Q purification system (Millipore).

<Example 1.2: Apparatus>
Film thickness was measured with an ellipsometer spectrometer (JA Woollam Co. Model M-44). The UV-vis spectrum was measured using a Hewlett-Packard spectrophotometer (Hewlett-Packard diodearray 8453 spectrophotometer). Tapping mode AFM experiments (Tapping mode AFM experiments) were performed using a Nanoscope IIIa AFM (Digital Instruments) instrument with an 'E' type scanner.

<Example 1.3: Cleaning of substrate>
Substrates such as silicon wafers, fumed silica and glass slides are immersed in a piranha solution (piranha solution, concentration H ₂ SO ₄ : 30% H ₂ O ₂ = 7: 3 (v / v)) The reaction vessel containing the substrate was sonicated for 1 hour (Note: The piranha solution may oxidize explosive organic materials, thus avoiding contact with oxidizing materials). After sonication, the plate was washed and rinsed with an excess of deionized water. The washed substrate was dried in a 30-40 mTorr vacuum chamber for the next step.

Example 1.4: Production of hydroxylated substrate
The washed substrate was immersed in 160 ml of toluene solution in which 1.0 ml of 3-glycidoxypropylmethyldiethoxysilane (GPDES) was dissolved for 10 hours. After performing a self-assembly reaction, the substrate was briefly washed with toluene and then placed in an oven and heated at 110 ° C. for 30 minutes. The plate was sonicated with toluene, toluene-methanol (1: 1 (v / v)), and methanol sequentially for 3 minutes at each wash step. The washed plate was dried in a vacuum chamber (30-40 mTorr). The GPDES modified substrate was immersed in a pure ethylene glycol (EG) solution with 2-3 drops of 95% sulfuric acid at 80-100 ° C. for 8 hours. After cooling, the substrate was sonicated with ethanol and methanol sequentially for 3 minutes each. The cleaned substrate was dried in a vacuum chamber (30-40 mTorr).

<Example 1.5: Production of dendron modified substrate>
The hydroxylated substrate is dendron (1.2 mM) and 1-[-3- (dimethylamino) propyl]-which is a coupling agent in the presence of 4-dimethylaminopyridine (DMAP) (0.82 mM). It was immersed in a methylene chloride solution in which 3-ethylcarbodiimide hydrochloride (EDC) or 1,3-dicyclylcyclohexylcarbodiimide (DCC) (11 mM) was dissolved. After 3 days at room temperature, the plate was sonicated sequentially with methanol, water, and methanol for 3 minutes each. For the next step, the washed plate was dried in a vacuum chamber (30-40 mTorr).

<Example 1.6: Production of NHS modified substrate>
The dendron modified substrate was immersed in a methylene chloride solution containing 1.0 M trifluoroacetic acid (TFA). After 3 hours, it was immersed in a methylene chloride solution containing 20% (v / v) diisopropylethylamine (DIPEA) for 10 minutes. The plates were sonicated for 3 minutes each with methylene chloride and methanol. Thereafter, it was dried in a vacuum chamber, and the unprotected substrate was reacted with an acetonitrile solution containing di (N-succinimidyl) carbonate (DSC) (25 mM) and DIPEA (1.0 mM). After reacting for 4 hours under a nitrogen atmosphere, the plate was placed in a dimethylformamide solution for 30 minutes and washed briefly with methanol. The washed plate was dried in a vacuum chamber (30-40 mTorr) for the next step.

<Example 1.7: Sequence oligonucleotide on NHS modified substrate>
Probe oligonucleotides dissolved in 50 mM NaHCO ₃ buffer solution (pH 8.5) were spotted in 4 by 4 format on NHS modified substrate. The microarray was reacted for 12 hours in a humidity chamber (80% humidity) to ensure sufficient reaction time for the aminated-thetherized DNA. Slides were agitated in a hybridization buffer solution (2 × SSPE buffer solution containing 7.0 mM sodium dodecyl sulfate (pH 7.4)) at 37 ° C. for 1 hour and then stirred in boiling water for 5 minutes. Then, non-specifically bound oligonucleotides were removed. Finally, the DNA-functionalized microarray was dried under a nitrogen atmosphere for the next step. For complete comparison, various types of probes were spotted on the single plate.

<Example 1.8: Hybridization>
Hybridization was performed at 50 ° C. for 1 hour with GeneTAC ™ HybStation (Genomic Solutions, Inc.) in a hybridization buffer solution containing target oligonucleotide (1.0 nM) labeling using Cy3 fluorescent dye. The microarray was rinsed using a hybridized buffer solution to remove the target oligonucleotide and then dried using nitrogen. The fluorescence signal on each spot was measured using ScanArray Lite (GSI Lumonics), and the result was analyzed using Imagene 4.0 (Biodiscovery).

<Example 1.9: Synthesis of dendron>
«Example 1.9.1: Production of 9-anthrylmethyl N- (3-carboxylpropyl) covermate (I) (Compound I)»
4-Aminobutyricane (0.50 g, 4.8 mmol, 1.0 equiv) and triethylamine (TEA) (1.0 ml, 7.3 mmol, 1.5 equiv) were dissolved in N, N-dimethylformamide (DMF). And stirred at 50 ° C. 9-Anthrylmethyl p-nitrophenyl carbonate (1.81 g, 4.8 mmol, 1.0 equiv) was gradually added to the solution with stirring. After stirring for 2 hours at 50 ° C., the solution was evaporated to dryness and basified with 0.50 N sodium hydroxide solution (NaOH). The aqueous solution was washed with ethyl acetate (EA), stirred in an ice bath, and acidified with dilute hydrochloric acid (HCI). The product was extracted with EA, the organic solution was dried over anhydrous MgSO ₄ , filtered and the solvent was evaporated. The total amount of final yellow powder was 1.06 g, and the yield was 65%.
¹ H NMR (CDCl ₃ )
δ 11.00-9.00 (br, CH ₂ COOH, 1 H), 8.41 (s, C ₁₄ H ₉ CH ₂ , 1 H), 8.31 (d, C ₁₄ H ₉ CH ₂ , 2H), 7.97 (d, C ₁₄ H ₉ CH ₂ , 2H), 7.51 (t, C ₁₄ H ₉ CH ₂ , 2H), 7.46 (t, C ₁₄ H ₉ CH ₂ , 2H), 6.08 (s, C ₁₄ H ₉ CH ₂ O, 2H) , 5.01 (t, OCONHCH ₂ , 1 H), 3.23 (q, NHCH ₂ CH ₂ , 2H), 2.34 (t, CH ₂ CH ₂ COOH, 2H), 1.77 (m, CH ₂ CH ₂ CH ₂ , 2H) .
¹³ C NMR (CDCl ₃ )
δ 178.5 (CH ₂ COOH), 157.9 (OCONH), 132.1 (C ₁₄ H ₉ CH ₂ ), 131.7 (C ₁₄ H ₉ CH ₂ ), 129.7 (C ₁₄ H ₉ CH ₂ ), 129.7 (C ₁₄ H ₉ CH ₂ ), 127.3 (C ₁₄ H ₉ CH ₂ ), 126.8 (C ₁₄ H ₉ CH ₂ ), 125.8 (C ₁₄ H ₉ CH ₂ ), 124.6 (C ₁₄ H ₉ CH ₂ ), 60.2 (C ₁₄ H ₉ CH ₂ ), 41.0 (NHCH ₂ CH ₂ ), 31.7 (CH ₂ CH ₂ COOH), 25.6 (CH ₂ CH ₂ CH ₂ ).

Example 1.9.2: Preparation of 9-anthrylmethyl N-{[(tris {[2- (methoxycarbonyl) ethoxy] methyl} methyl) amino] carbonyl} propyl carbonate (II) (compound II) >>
9-anthrylmethyl N- (3-carboxylpropyl) covermate (0.65 g, 1.93 mmol, 1.5 equiv), 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride (EDC ) (0.37 g, 1.93 mmol, 1.5 equiv) and 1-hydroxybenzotriazole hydrate (HOBT) (0.261 g, 1.93 mmol, 1.5 equiv) were dissolved in acetonitrile and stirred at room temperature. . Tris {[(methoxycarbonyl) ethoxy] methyl} aminomethane (0.49 g, 1.29 mmol, 1.0 equiv) dissolved in acetonitrile was added to the solution with stirring. After stirring for 12 hours at room temperature, acetonitrile was evaporated. The crude product was dissolved in EA and washed with 1.0 N HCl and saturated sodium bicarbonate solution. After drying over anhydrous MgSO ₄ and filtration, the solvent was evaporated, the crude product was loaded onto a column packed with silica gel, and column chromatography (developing solvent: ethyl acetate: hexane = 5: 1 (v / v )) To give a viscous yellow liquid. The total amount of yellow liquid was 0.67 g (74% yield).
¹ H NMR (CDCl ₃ )
δ 8.43 (s, C ₁₄ H ₉ CH ₂ , 1 H), 8.36 (d, C ₁₄ H ₉ CH ₂ , 2H), 7.99 (d, C ₁₄ H ₉ CH ₂ , 2H), 7.53 (t, C ₁₄ H ₉ CH ₂ , 2H), 7.47 (t, C ₁₄ H ₉ CH ₂ , 2H), 6.15 (s, CONHC, 1H), 6.08 (s, C ₁₄ H ₉ CH ₂ O, 2H), 5.44 (t, OCONHCH ₂ , 1 H), 3.63-3.55 (m, CH ₂ OCH ₂ CH ₂ COOCH ₃ , 21 H), 3.27 (q, NHCH ₂ CH ₂ , 2H), 2.46 (t, CH ₂ CH ₂ COOCH ₃ , 6H ), 2.46 (t, CH ₂ CH ₂ CONH, 2H), 1.81 (m, CH ₂ CH ₂ CH ₂ , 2H).
¹³ C NMR (CDCl ₃ )
δ173.2 (CH ₂ CONH), 172.7 (CH ₂ COOCH ₃ ), 157.4 (OCONH), 132.9 (C ₁₄ H ₉ CH ₂ ),
131.5 (C ₁₄ H ₉ CH ₂ ), 129.5 (C ₁₄ H ₉ CH ₂ ), 129.4 (C ₁₄ H ₉ CH ₂ }, 127.5 (C ₁₄ H ₉ CH ₂ ), 127.0 (C ₁₄ H ₉ CH ₂ ), 125.6 (C ₁₄ H ₉ CH ₂ ), 124.7 (C ₁₄ H ₉ CH ₂ ), 69.6 (NHCCH ₂ O), 67.2 (C ₁₄ H ₉ CH ₂ ), 60.1 (OCH ₂ CH ₂ ), 59.4 (NHCCH ₂ ) , 52.1 (OCH ₃ ), 40.8 (NHCH ₂ CH ₂ ), 35.1 (OCH ₂ CH ₂ ), 34.7 (CH ₂ CH ₂ CONH), 26.3 (CH ₂ CH ₂ CH ₂ ).
^{_{.. Anal Calcd for C 36 H}} 46 N 2 O 12 0.5 H 2 O: C 61.18, H 6.65, N 4.03; Found: C61.09, H 6.69, N 3.96.

Example 1.9.3: Preparation of 9-Antromethyl N-[({Tris [(2-carboxyethoxy) methyl] methyl} amino) carbonyl] propyl covermate (III) (Compound III) >>
9-anthrylmethyl N-{[(tris {[2- (methoxycarbonyl) ethoxy] methyl} methyl) amino] carbonyl} propyl carbonate (0.67 g, 0.93 mmol) in acetone (30 ml) and 0.20 NNaOH (30 ml, 6 mmol).
After stirring at room temperature for 1 day, the acetone was evaporated. The resulting aqueous solution was washed with EA and acidified with dilute hydrochloric acid while stirring in an ice bath. The product was extracted using EA, the organic solution was dried over anhydrous MgSO ₄ , filtered and the solvent was evaporated. The product was solidified with acetone and ether at −20 ° C. to give a yellow powder. The final amount of light yellow powder obtained was 0.54 g (88% yield).
¹ H NMR (CDCl ₃ )
δ 11.00-9.00 (br, CH ₂ COOH, 3H}, 8.61 (s, C ₁₄ H ₉ CH ₂ , 1H}, 8.47 (d, C ₁₄ H ₉ CH ₂ , 2H), 8.11 (d, C ₁₄ H ₉ CH ₂ , 2H), 7.60 (t, C ₁₄ H ₉ CH ₂ , 2H}, 7.52 (t, C ₁₄ H ₉ CH ₂ , 2H), 6.63 (s, CONHC, 1 H), 6.36 (t, OCONHCH ₂ , 1 H), 6.12 (s, C ₁₄ H ₉ CH ₂ O, 2H). 3.40-363 (m, CH ₂ OCH ₂ CH ₂ COOH, 12H), 3.20 (q, NHCH ₂ CH ₂ , 2H), 2.52 (t, CH ₂ CH ₂ COOH, 6H), 2.17 (t, CH ₂ CH ₂ CONH, 2H), 1.75 (m, CH ₂ CH ₂ CH ₂ , 2H).
¹³ C NMR (CDCl ₃ )
δ 172.2 (CH ₂ COOH), 172.0 (CH ₂ CONH), 156.7 (OCONH), 131.2 (C ₁₄ H ₉ CH ₂ ), 130.7 (C ₁₄ H ₉ CH ₂ ), 128.6 (C ₁₄ H ₉ CH ₂ ), 128.4 (C ₁₄ H ₉ CH ₂ ), 127.3 (C ₁₄ H ₉ CH ₂ ), 126.2 (C ₁₄ H ₉ CH ₂ ), 124.8 (C ₁₄ H ₉ CH ₂ ), 124.0 (C ₁₄ H ₉ CH ₂ ), 68.6 (NHCCH ₂ O), 66.5 (C ₁₄ H ₉ CH ₂ ), 59.5 (OCH ₂ CH ₂ ), 58.0 (NHCCH ₂ ), 40.0 (NHCH ₂ CH ₂ ), 34.0 (OCH ₂ CH ₂ ), 33.5 (CH ₂ CH ₂ CONH), 25.8 (CH ₂ CH ₂ CH ₂ ).
^{_{.. Anal Calcd for C 33 H}} 40 N 2 O 12 1.5 H 2 O: C 57.97, H 6.34, N 4.10; Found: C 57.89, H 6.21, N 4.09.

Example 1.9.4: 9-anthrylmethyl N-[({Tris [(2-{[(Tris {[2- (methoxycarbonyl) ethoxy] methyl} (methyl) amino) carbonyl] ethoxy} methyl ) Preparation of methyl] amino} carbonyl) propyl covermate (IV) (compound IV) >>
9-anthrylmethyl N-[({tris [(2-carboxyethoxy) methyl] methyl} amino) carbonyl] propyl covermate (0.54 g, 0.82 mmol, 1.0 equiv), EDC (0.55 g, 2 .87 mmol, 3.5 equiv) and HOBT (0.39 g, 2.89 mmol, 3.5 equiv) were dissolved in acetonitrile and stirred at room temperature. Thereafter, tris {[(methoxycarbonyl) ethoxy] methyl} aminomethane (0.96 g, 2.53 mmol, 3.1 equiv) dissolved in acetonitrile was added to the solution with stirring. After stirring for 36 hours at room temperature, the acetonitrile was evaporated. The crude product was dissolved in EA, washed with 1.0 N HCL and saturated with sodium bicarbonate solution. After drying over anhydrous MgSO4, filtration and evaporation, the crude product was loaded onto a column packed with silica gel. As a result of purification using a column (developing solvent: ethyl acetate: methanol = 20: 1 (v / v)), a viscous yellow liquid was obtained. The total weight of the yellow liquid was 1.26 g (88% yield).
¹ H NMR (CDCl ₃ )
δ 8.47 (s, C ₁₄ H ₉ CH ₂ , 1 H), 8.39 (d, C ₁₄ H ₉ CH ₂ , 2H), 8.02 (d, C ₁₄ H ₉ CH ₂ , 2H), 7.53 (t, C ₁₄ H ₉ CH ₂ , 2H), 7.47 (t, C ₁₄ H ₉ CH ₂ , 2H), 6.60 (s, CH ₂ CH ₂ CH ₂ CONHC, 1 H), 6.13 (s, OCH ₂ CH ₂ CONHC, 3H) , 6.11 (s, C ₁₄ H ₉ CH ₂ O, 2H), 5.79 (t, OCONHCH ₂ , 1 H), 3.65-3.60 (m, CH ₂ OCH ₂ CH ₂ CONH, CH ₂ OCH ₂ CH ₂ COOCH ₃ , 75H), 3.29 (q, NHCH ₂ CH ₂ , 2H), 2.50 (t, CH ₂ CH ₂ COOCH ₃ , 18H), 2.36 (t, OCH ₂ CH ₂ CONH, 6H), 2.27 (t, CH ₂ CH ₂ (CH ₂ CONH, 2H), 1.85 (m, CH ₂ CH ₂ CH ₂ , 2H).
¹³ C NMR (CDCl ₃ )
δ 173.3 (OCH ₂ CH ₂ CONH), 172.5 (CH ₂ CH ₂ CH ₂ CONH), 171.6 (CH ₂ COOCH ₃ ), 157.2 (OCONH), 131.8 (C ₁₄ H ₉ CH ₂ ), 131.5 (C ₁₄ H _{9 CH 2), 129.4 (C 14} H 9 CH 2), 129.3 (C 14 H 9 CH 2), 127 .6 (C 14 H 9 CH 2), 127.0 (C 14 H 9 CH 2), 125.6 (C 14 H ₉ CH ₂ ), 124.7 (C ₁₄ H ₉ CH ₂ ), 69.5 (NHCCH ₂ OCH ₂ CH ₂ COOCH ₃ ), 67.9 (NHCCH ₂ OCH ₂ CH ₂ CONH), 67.2 (C ₁₄ H ₉ CH ₂ ), 60.3 (OCH ₂ CH ₂ CONH), 60.2 (OCH ₂ CH ₂ COOCH ₃ ), 59.2 (NHCCH ₂ OCH ₂ CH ₂ COOCH ₃ , NHCCH ₂ OCH ₂ CH ₂ CONH), 52.1 (OCH ₃ ), 41.0 (NHCH ₂ CH ₂ ), 37.6 (OCH ₂ CH ₂ CONH), 35.1 (OCH ₂ CH ₂ COOCH ₃ ), 34.7 (CH ₂ CH ₂ CH ₂ CONH), 26.3 (CH ₂ CH ₂ CH ₂ ).
^{_{.. Anal Calcd for C 81 H}} 121 N 5 O 36 H 2 O: C 55.31, H 7.05, N 3.98; Found: C 55.05, H 7.08, N 4.04.
MALDI- TOF-MS: 1763.2 (MNa +), 1779.2 (MK +).

Example 1.9.5: 9-anthrylmethyl N-({[tris ({2-[({tris [(2-carboxyethoxy) methyl] methyl} amino) carbonyl] ethoxy} methyl) methyl] amino } Production of Carbonyl) propyl Covermate (V) (Compound V) >>
9-anthrylmethyl N-[({tris [(2-{[(tris {[2- (methoxycarbonyl) ethoxy] methyl} methyl) amino] carbonyl} ethoxy) methyl] methyl} amino) carbonyl] propyl cover Mate (0.60 g, 0.34 mmol) was dissolved in acetone (30 ml) and 0.20 N NaOH (30 ml). After stirring at room temperature for 1 day, acetone was evaporated. The resulting aqueous solution was washed with EA and acidified with dilute hydrochloric acid while stirring in an ice bath. The product was extracted using EA, the organic solution was dried over anhydrous MgSO ₄ and filtered, and then the solvent was evaporated. The final amount of the obtained yellow powder was 0.37 g (68% yield).
¹ H NMR (DMSO)
δ 13.00-11.00 (br, CH ₂ COOH, 9H), 8.66 (s, C ₁₄ H ₉ CH2, 1 H), 8.42 (d, C ₁₄ H ₉ CH ₂ , 2H), 8.13 (d, C ₁₄ H ₉ CH2, 2H), 7.62 (t, C ₁₄ H ₉ CH ₂ , 2H), 7.54 (t, C ₁₄ H ₉ CH ₂ , 2H), 7.12 (t, OCONHCH ₂ , 1H), 7.10 (s, OCH ₂ CH ₂ CONHC, 3H), 7.06 (s, CH ₂ CH ₂ CH ₂ CONHC, 1 H), 6.06 (s, C ₁₄ H ₉ CH ₂ O, 2H), 3.57-3.55 (m, CH ₂ OCH ₂ CH ₂ CONH , CH ₂ OCH ₂ CH ₂ COOH, 48H), 3.02 (q, NHCH ₂ CH ₂ , 2H), 2.42 (t, CH ₂ CH ₂ COOH, 18H), 2.32 (t, OCH ₂ CH ₂ CONH, 6H), 2.11 (t, CH ₂ CH ₂ CH ₂ CONH, 2H), 1.60 (m, CH ₂ CH ₂ CH ₂ , 2H). ¹³ C NMR (DMSO)
δ 172.8 (CH ₂ COOH), 172.2 (CH ₂ CH ₂ CH ₂ CONH), 170.5 (OCH ₂ CH ₂ CONH), 156.5 (OCONH), 131.0 (C ₁₄ H ₉ CH ₂ ), 130.6 (C ₁₄ H ₉ CH ₂ ), 129.0 (C ₁₄ H ₉ CH ₂ ), 128.7 (C ₁₄ H ₉ CH ₂ ), 127.6 (C ₁₄ H ₉ CH ₂ ), 126.7 (C ₁₄ H ₉ CH ₂ ), 125.4 (C ₁₄ H ₉ CH ₂ ), 124.3 (C ₁₄ H ₉ CH ₂ ), 68.3 (NHCCH ₂ OCH ₂ CH ₂ COOH), 67.4 (NHCCH ₂ OCH ₂ CH ₂ CONH), 66.8 (C ₁₄ H ₉ CH ₂ ), 59.8 (OCH ₂ CH ₂ COOH), 59.6 (OCH ₂ CH ₂ CONH), 57.9 (NHCCH ₂ OCH ₂ CH ₂ CONH), 55.9 (NHCCH ₂ OCH ₂ CH ₂ COOH), 36.4 (NHCH ₂ CH ₂ ), 34.6 (OCH ₂ CH ₂ COOH ), 30.8 (OCH ₂ CH ₂ CONH), 29.7 (CH ₂ CH ₂ CH ₂ CONH), 25.9 (CH ₂ CH ₂ CH ₂ ).

[Example 2: Method for producing dendron macromolecule as an alternative starting material (Fmoc-Spacer- [9] -acid)]
In Example 2, compounds with various names were named compounds 1, 2 and so on. First, the spacer 6-azidohexylamine (1) was obtained from 1,6-dibromohexane according to the literature method described below (Lee, JW; Jun, SI; Kim, K. Tetrahedron Lett. , 2001, 42, 2709).

The spacer was attached with repeating units through an unsymmetrical urea formation and a fabricated N ₃ -spacer- [3] ester (3) as shown below. The repeating unit was synthesized by condensation of Tris using tert-butyl acrylate (Which had been reported in Cardona, CM; Gawley, REJ Org. Chem. 2002, 67, 141).

The triester, through the combined hydrolysis and triester (2) a peptide coupling conditions, N 3 _- spacer - was transformed into [3] acid (4). After reduction of the azide to the amine group and protection of the amine with Fmoc group, hydrolysis of the nona ester provided the Fmoc-spacer- [9] acid (5). The structure of Fmoc-spacer- [9] acid (5) is shown below.

<N- (6-azidohexyl) -N-tris {[2- (tert-butoxycarbonyl) ethoxy] methyl} -methylurea (3)>
Triphosgene (1.3 g, 4.3 mmol) was dissolved in anhydrous CH ₂ Cl ₂ (20 mL). Anhydrous _CH 2 _Cl 2 (35mL), dissolved in 6-azido hexylamine (1) (1.6g, 12mmol) and N, the mixture syringe pump N- diisopropylethylamine (DIEA, 2.4mL, 13.8mmol) Was added dropwise to the stirred triphosphine solution with a period of 7 hours or more. After further stirring for 2 hours, a solution of the compound (2) (6.4 g, 13 mmol) and DIEA (2.7 mL, 15.2 mmol) dissolved in anhydrous CH ₂ Cl ₂ (20 mL) was added. The reaction mixture was stirred at room temperature for 4 hours under a nitrogen atmosphere and washed using 0.5 M HCl and salt. The organic layer was dried over anhydrous MgSO ₄ and the solvent was removed by evaporation. Purification using column chromatography (silica, 1: 1 EtOAc / hexane) gave a colorless oil (3.0 g, 40%).
¹ H NMR (CDCl ₃ , 300 MHz): δ 1.45 (s, (CH ₃ ) ₃ C, 27H); 1.36-1.58 (m, CH ₂ CH ₂ CH ₂ CH ₂ , 8H); 2.46 (t, CH ₂ CH ₂ O, J = 6.4 Hz, 6H), 3.13 (m, CONHCH ₂ , 2H), 3.26 (t, N ₃ CH ₂ , J = 6.9 Hz, 2H), 3.64-3.76 (m, CCH ₂ O and CH ₂ CH ₂ O, 12H); 5.00 (t, CH ₂ NHCO, J = 6.7 Hz, 1H), 5.29 (s, CONHC, 1H).
¹³ C NMR (CDCl ₃ , 75 MHz): δ 26.52, 26.54, 28.81, 30.26 (CH ₂ CH ₂ CH ₂ CH ₂ ); 28.14 ((CH ₃ ) ₃ C); 36.20 (CH ₂ CH ₂ O); 39.86 (CONHCH ₂ ); 51.40 (N ₃ CH ₂ ); 58.81 (CCH ₂ O); 67.16 (CH ₂ CH ₂ O); 69.23 (CCH ₂ O); 80.58 ((CH ₃ ) ₃ C); 157.96 (NHCONH) ; 171.26 (COOt-Bu).
FAB-MS: 674.26 (M ⁺ ).

<N- (6-azidohexyl) -N-tris {[2-carboxyethoxy] methyl} methylurea (4)>
N ₃ -spacer- [3] ester (3) (0.36 g, 0.56 mmol) was stirred with 6.6 mL of 96% formic acid for 24 hours. Formic acid was removed by reduced pressure at 50 ° C. to produce a colorless oil in quantitative yield.
¹ H NMR (CD ₃ COCD ₃ , 300 MHz): δ 1.34-1.60 (m, CH ₂ CH ₂ CH ₂ CH ₂ , 8H); 2.53 (t, CH ₂ CH ₂ O, J = 6.4 Hz, 6H), 3.07 (t, CONHCH ₂ , J = 6.9 Hz, 2H), 3.32 (t, N ₃ CH ₂ , J = 6.9 Hz, 2H), 3.67-3.73 (m, CCH ₂ O and CH ₂ CH ₂ O, 12H) .
¹³ C NMR (CD ₃ COCD ₃ , 75 MHz): δ 27.21, 29.54, 31.02 (CH ₂ CH ₂ CH ₂ CH ₂ ); 35.42 (CH ₂ CH ₂ O); 40.27 (CONHCH ₂ ); 52.00 (N ₃ CH ₂ ); 59.74 (CCH ₂ O); 67.85 (CH ₂ CH ₂ O); 70.96 (CCH ₂ O); 158.96 (NHCONH); 173.42 (COOH).
FAB-MS: 506.19 (MH ⁺ ).

<N- (6-azidohexyl) -N'-tris [(2-{[(tris {[2- (tert-butoxycarbonyl) ethoxy] -methyl} methyl) amino] carbonyl} ethoxy) methyl] methylurea (4. 1)>
HOBt (0.20 g, 1.5 mmol), DIEA (0.30 mL, 1.8 mmol), and EDC (0.33 g, 1.8 mmol) dissolved in 5.0 mL of dry acetonitrile (4) (0. 25 g, 0.50 mmol). Thereafter, the amine was dissolved in dry acetonitrile 2.5mL (2) (1.14g, 2.3mmol ) was added and the reaction mixture was stirred under 48 hours _{N 2.} After removing the solvent under reduced pressure, the residue was dissolved in MC and washed with 0.5 M HCl and salt. After the organic layer was dried over MgSO ₄ , the solvent was removed in vacuo and purified by column chromatography (SiO ₂ , 2: 1 EtOAc / hexane) to give a colorless oil (0.67 g, 70%). .
¹ H NMR (CDCl ₃ , 300 MHz): δ1.45 (s, (CH ₃ ) ₃ C, 81H); 1.36-1.58 (m, CH ₂ CH ₂ CH ₂ CH ₂ , 8H); 2.40-2.47 (m , CH ₂ CH ₂ O gen. 1 & 2, 24H), 3.13 (m, CONHCH ₂ , 2H), 3.26 (t, N ₃ CH ₂ , 6.9 Hz, 2H), 3.62-3.69 (m, CCH ₂ O gen 1 & 2, CH ₂ CH ₂ O gen. 1 & 2, 48H); 5.36 (t, CH ₂ NHCO, J = 6.7 Hz, 1H), 5.68 (br, CONHC, 1H), 6.28 (br, amide NH , 3H).
¹³ C NMR (CDCl ₃ , 75 MHz): δ 26.59, 26.69, 28.91, 30.54 (CH ₂ CH ₂ CH ₂ CH ₂ ); 28.22 ((CH ₃ ) ₃ C); 36.20 (CH ₂ CH ₂ O gen. 2 ); 37.43 (CH ₂ CH ₂ O gen. 1); 39.81 (CONHCH ₂ ); 51.47 (N ₃ CH ₂ ); 58.93 (CCH ₂ O gen. 1); 59.89 (CCH ₂ O gen. 2); 67.15 ( CH ₂ CH ₂ O gen. 2); 67.68 (CH ₂ CH ₂ O gen. 1); 69.23 (CCH ₂ O gen. 2); 70.12 (CCH ₂ O gen. 1); 80.57 ((CH ₃ ) ₃ C ); 158.25 (NHCONH); 171.01 (COOt-Bu) 171.41 (CONH amides).
MALDI-MS: 1989.8 (MNa ⁺ ), 2005.8 (MK ⁺ ).

<N- (6-aminohexyl) -N'-tris [(2-{[(tris {[2- (tert-butoxycarbonyl) ethoxy] -methyl} methyl) amino] carbonyl} ethoxy) methyl] methylurea (4 .2)>
Nona-tert-butyl ester (4.1) (0.37 g, 0.20 mmol) dissolved in ethanol (20.0 mL) with 10% Pd / C (37.0 mg) at room temperature under H ₂ for 12 hours. Stir. After confirming the completion of the reaction by TLC, the mixture was filtered through a 0.2 mm Millipore filter. After rinsing the filter paper with CH ₂ Cl ₂ , the mixed solvent was removed in vacuo to give a colorless oil.

<N- {6- (9-fluorenylmethoxycarbonyl) aminohexyl} -N'-tris [(2-{[(tris {[2- (tert-butoxycarbonyl) ethoxy] methyl} methyl) amino] carbonyl } Ethoxy) methyl] methylurea (4.3)>
Amine (4.2) (0.33 g, 0.17 mmol) and DIEA (33 mL, 0.19 mmol) were dissolved in 5.0 mL of CH ₂ Cl ₂ and stirred for 30 minutes under a nitrogen atmosphere. 9-Fluorenylmethyl chloroformate (48 mg, 0.19 mmol) dissolved in 2.0 mL of CH ₂ Cl ₂ was added and the reaction mixture was stirred at room temperature for 3 hours. The solvent was removed under reduced pressure and washed with 0.5 M HCl and salt (0.18 g, 64%). The residue was purified by column chromatography (silica, EtOAc) to give a colorless oil.
¹ H NMR (CDCl ₃ , 300 MHz): δ 1.45 (s, (CH ₃ ) ₃ C, 81H); 1.23-1.58 (m, CH ₂ CH ₂ CH ₂ CH ₂ , 8H); 2.37-2.47 (m, CH ₂ CH ₂ O gen. 1 & 2, 24H); 3.10-3.22 (m, CONHCH ₂ , 4H); 3.62- 3.70 (m, CCH ₂ O gen. 1 & 2, CH ₂ CH ₂ O gen. 1 & 4.22 (t, CH (fluorenyl) -CH ₂ , J = 7.1 Hz, 1H); 4.36 (d, fluorenyl-CH ₂ , J = 7.1 Hz, 2H); 5.27-5.35 (m, CH ₂ NHCO, 2H); 5.67 (br, CONHC, 1H); 6.25 (br, amide, 3H); 7.28 -7.77 (fluorenyl, 8H).
¹³ C NMR (CDCl ₃ , 75 MHz): δ 26.85, 27.02, 30.27, 30.88 (CH ₂ CH ₂ CH ₂ CH ₂ ); 28.49 ((CH ₃ ) ₃ C); 36.48 (CH ₂ CH ₂ O gen. 2 ); 37.73 (CH ₂ CH ₂ O gen. 1); 40.03, 41.34 (CONHCH ₂ ); 47.68 (CH (fluorenyl) -CH ₂ ); 59.22 (CCH ₂ O gen. 1); 60.16 (CCH ₂ O gen. 2); 66.87 (fluorenyl-CH ₂ ); 67.43 (CH ₂ CH ₂ O gen. 2); 67.98 (CH ₂ CH ₂ O gen. 1); 69.52 (CCH ₂ O gen. 2); 70.42 (CCH ₂ O gen.1); 80.84 ((CH ₃ ) ₃ C); 120.28, 125.52, 127.38, 127.98, 141.65, 144.48 (fluorenyl); 156.88 (OCONH); 158.52 (NHCONH); 171.27 (COOt-Bu) 171.65 (amide CONH ).
MALDI-MS: 2186.8 (MNa ⁺ ), 2002.8 (MK ⁺ ).

<N- {6- (9-fluorenylmethoxycarbonyl) aminohexyl} -N'-tris [(2-{[(tris {[2-carboxyethoxy] methyl} methyl) amino] carbonyl} ethoxy) methyl] -Methylurea (5)>
Nona-tert-butyl ester with protecting group (4.3) (0.12 g, 72 mmol) was stirred with 10 mL of 96% formic acid for 18 hours. The formic acid was removed at 50 ° C. under reduced pressure to produce a colorless oil in quantitative yield.
¹ H NMR (CD ₃ COCD ₃ , 300 MHz): δ 1.23-1.51 (m, CH ₂ CH ₂ CH ₂ CH ₂ , 8H); 2.44-2.58 (m, CH ₂ CH ₂ O gen. 1 & 2, 24H ); 3.15-3.18 (m, CONHCH ₂ , 4H); 3.61-3.75 (m, CCH ₂ O gen. 1 & 2, CH ₂ CH ₂ O gen. 1 & 2, 48H); 4.23 (t, CH (fluorenyl) -CH ₂ , J = 7.0 Hz, 1H); 4.35 (d, fluorenyl-CH ₂ , J = 7.0 Hz, 2H); 5.85, 6.09 (br, CH ₂ NHCO, 2H); 6.57 (br, CONHC, 1H); 6.88 (br , amide NH, 3H); 7.31-7.88 (fluorenyl, 8H).
¹³ C NMR (CD ₃ COCD ₃ , 75 MHz): δ 27.21, 27.33, 30.69, 30.98 (CH ₂ CH ₂ CH ₂ CH ₂ ); 35.31 (CH ₂ CH ₂ O gen. 2); 37.83 (CH ₂ CH ₂ O gen. 1); 40.56, 41.54 (CONHCH ₂ ); 48.10 (CH (fluorenyl) -CH ₂ ); 59.93 (CCH ₂ O gen. 1); 61.10 (CCH ₂ O gen. 2); 66.86 (fluorenyl-CH ₂ ); 67.81 (CH ₂ CH ₂ O gen. 2); 68.37 (CH ₂ CH ₂ O gen. 1); 69.80 (CCH ₂ O gen. 2); 70.83 (CCH ₂ O gen. 1); 120.84, 126.13 , 127.98, 128.56, 142.10, 145.16 (fluorenyl); 157.50 (OCONH); 159.82 (NHCONH); 173.20 (amide CONH); 173.93 (COOH).

[Example 3: Additional dendron compound]
Specific protecting groups are shown below, but it should be well known that the compounds are not limited. Also, as long as the various chains and spacers are depicted showing the exact molecular structure, modification for controlled density, preferably low density arrays on the substrate surface is an accepted chemical modification method. (Chemical modification methods). As a reference to the short-hand description of compounds, the majority of the left side of the compounds shown below indicate protecting groups; the numbers in brackets indicate the number of branched termini; Most show chemical chemistry on the end of the chain of the chemical entity.
In the examples, “A- [27] -acid” means an anthrylmethyl protecting group; the terminal 27 and an acid group at the terminal.

[Example 3.1: Manufacturing method]
1. A- [3] -OEt (Compound 3)
Compound 1 was reacted at 80 ° C. with NaC (CO ₂ Et) ₃ (Compound 2).

2. A- [3] -OMe (Compound 5)
A- [3] -OEt (compound 3) was reacted with LiAlH ₄ or LiBH ₄ dissolved in ether, reacted with chloroacetic acid in the presence of t-BuOK / t-BUOH and esterified with MeOH.

3. A- [3] -OTs (Compound 7)
Triol compound 6 tosylated to compound 7 is obtained by reaction of A- [3] -OMe (compound 5) and LiAlH ₄ with ether.

4). A- [9] -OEt (Compound 8)
The nonaester (compound 8) as the target compound is produced by treating A- [3] -OTs (compound 7) with NaC (CO ₂ Et) ₃ dissolved in C ₆ H ₆ -DMF.

5). A- [27] -OH (Compound 9)
A- [9] -OEt (Compound 8) was treated with tris (hydroxymethyl) aminomethane and K ₂ CO ₃ dissolved in DMSO at 70 ° C.

[Example 3.2]
1. Boc- [2] -OMe (Compound 3)
Compound 1 and methyl acrylate (compound 2) were reacted in a methanol solvent at a temperature of 50 ° C. or lower. Excess reagents and solvents were removed under high vacuum at 55 ° C or lower.

2. Boc- [4] -NH2 (Compound 5)
Boc- [2] -OMe (compound 3) and a very excessive amount of ethylenediamine (EDA) (compound 4) were reacted in a methanol solvent at a temperature of 50 ° C. or lower. Excess reagents and solvents were removed under high vacuum at 55 ° C or lower.

3. Boc- [8] -OMe (Compound 6)
Boc- [4] -NH ₂ (Compound 5) and methyl acrylate (Compound 2) were reacted in a methanol solvent at a temperature of 50 ° C. or lower. Excess reagents and solvents were removed under high vacuum at 55 ° C or lower.

[Example 3.3]
1. Boc- [2] -OH (compound 3)
Compound 1, 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole hydrate (HOBT) were dissolved in acetonitrile and stirred at room temperature. L-glutamic acid-diethyl ethyl ester (H ₂ NCH (CO ₂ Et) CH ₂ CH ₂ CO ₂ Et) dissolved in acetonitrile was added to the solution with stirring. After stirring for 12 hours at room temperature, acetonitrile was evaporated. The crude product was dissolved in EA and washed with 1.0 N HCl and saturated sodium bicarbonate solution. After drying over anhydrous MgSO ₄ , filtration and evaporation of the solvent gave the crude product in a column filled with silica gel. Purification by column chromatography (developing solvent: ethyl acetate: hexane) gave a viscous yellow liquid.
Compound 2 was hydrolyzed with NaOH. After stirring for 1 day at room temperature, the organic solution was evaporated. The aqueous solution was washed with EA, stirred in an ice bath and acidified with HCl. After extracting the product using EA, the organic solution was dried over anhydrous MgSO ₄ , filtered and the solvent evaporated.

2. Boc- [4] -OH (Compound 3)
Compound 3, 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride (EDC), and 1-hydroxybenzotriazole hydrate (HOBT) were dissolved in acetonitrile and stirred at room temperature. L-glutamic acid-diethyl ester (H ₂ NCH (CO ₂ Et) CH ₂ CH ₂ CO ₂ Et) dissolved in acetonitrile was added to the solution with stirring. After stirring for 12 hours at room temperature, acetonitrile was evaporated. The crude product was dissolved in EA and washed with 1.0 N HCl and saturated sodium bicarbonate solution. After drying over anhydrous MgSO ₄ , filtration and evaporation of the solvent gave the crude product in a column filled with silica gel. Purification by column chromatography (developing solvent: ethyl acetate: hexane) gave a viscous yellow liquid.
Compound 4 was hydrolyzed with NaOH. After stirring for 1 day at room temperature, the organic solution was evaporated. The aqueous solution was washed with EA, stirred in an ice bath and acidified with HCl. After extracting the product using EA, the organic solution was dried over anhydrous MgSO ₄ , filtered and the solvent evaporated.

3. Boc- [8] -OH (Compound 3)
Compound 5, 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride (EDC), and 1-hydroxybenzotriazole hydrate (HOBT) were dissolved in acetonitrile and stirred at room temperature. L-glutamic acid-diethyl ester (H ₂ NCH (CO ₂ Et) CH ₂ CH ₂ CO ₂ Et) dissolved in acetonitrile was added to the solution with stirring. After stirring for 12 hours at room temperature, acetonitrile was evaporated. The crude product was dissolved in EA and washed with 1.0 N HCl and saturated sodium bicarbonate solution. After drying over anhydrous MgSO ₄ , filtration and evaporation of the solvent gave the crude product in a column filled with silica gel. Purification by column chromatography (developing solvent: ethyl acetate: hexane) gave a viscous yellow liquid.
Compound 6 was hydrolyzed with NaOH. After stirring for 1 day at room temperature, the organic solution was evaporated. The aqueous solution was washed with EA, stirred in an ice bath and acidified with HCl. After extracting the product using EA, the organic solution was dried over anhydrous MgSO ₄ , filtered and the solvent evaporated.

[Example 3.4]
1. Boc- [2] -CN (compound 3)
Compound 1 was dissolved in acetonitrile at room temperature. Crystalline acetic acid was added and the solution was heated at reflux for 24 hours. Excess acetonitrile was removed by distillation under vacuum, the residue was extracted with chloroform, and then a concentrated ammonia solution was added. After separating the organic state, it was washed with water and dried over sodium sulfate.

2. Boc- [2] -NH ₂ (Compound 4)
Boc- [2] -CN (compound 3) was dissolved in methanol and cobalt (II) chloride hexahydrate was added. Sodium borohydride was added in portions. The resulting mixture was stirred for 2 hours at room temperature and then carefully acidified with concentrated hydrochloric acid. The solvent was removed under vacuum and concentrated. After separating the organic state, it was washed with water and dried over sodium sulfate.

3. Boc- [4] -CN (compound 5)
Boc- [2] -NH ₂ (compound 4) was dissolved in acetonitrile at room temperature. Crystalline acetic acid was added and the solution was heated at reflux for 24 hours. Excess acetonitrile was removed by distillation under vacuum, the residue was extracted with chloroform, and then a concentrated ammonia solution was added. After separating the organic state, it was washed with water and dried over sodium sulfate.

4). Boc- [4] -NH ₂ (Compound 6)
Boc- [4] -CN (compound 5) was dissolved in methanol and cobalt (II) chloride hexahydrate was added. Sodium borohydride was added in portions. The resulting mixture was stirred for 2 hours at room temperature and then carefully acidified with concentrated hydrochloric acid. The solvent was removed under vacuum and concentrated. After separating the organic state, it was washed with water and dried over sodium sulfate.

5). Boc- [8] -CN (Compound 7)
Boc- [4] -NH ₂ (Compound 6) was dissolved in acetonitrile at room temperature. Crystalline acetic acid was added and the solution was heated at reflux for 24 hours. Excess acetonitrile was removed by distillation under vacuum, the residue was extracted with chloroform, and then a concentrated ammonia solution was added. After separating the organic state, it was washed with water and dried over sodium sulfate.

6). Boc- [8] -NH ₂ (Compound 8)
Boc- [8] -CN (compound 7) was dissolved in methanol and cobalt (II) chloride hexahydrate was added. Sodium borohydride was added in portions. The resulting mixture was stirred for 2 hours at room temperature and then carefully acidified with concentrated hydrochloric acid. The solvent was removed under vacuum and concentrated. After separating the organic state, it was washed with water and dried over sodium sulfate.

7). Boc- [16] -CN (Compound 9)
Boc- [8] -NH ₂ (Compound 8) was dissolved in acetonitrile at room temperature. Crystalline acetic acid was added and the solution was heated at reflux for 24 hours. Excess acetonitrile was removed by distillation under vacuum, the residue was extracted with chloroform, and then a concentrated ammonia solution was added. After separating the organic state, it was washed with water and dried over sodium sulfate.

8). Boc- [16] -NH ₂ (Compound 10)
Boc- [16] -CN (compound 9) was dissolved in methanol and cobalt (II) chloride hexahydrate was added. Sodium borohydride was added in portions. The resulting mixture was stirred for 2 hours at room temperature and then carefully acidified with concentrated hydrochloric acid. The solvent was removed under vacuum and concentrated. After separating the organic state, it was washed with water and dried over sodium sulfate.

[Example 3.5]
1. A- [3] -Alkene (Compound 3)
A- [1] -SiCl ₃ (Compound 1) was refluxed for 4 hours using an excess amount of 10% allylmagnesium bromide dissolved in diethyl ether, then cooled at 0 ° C. and 10% NH ₄ Cl. Hydrolyzed with aqueous solution. The organic layer was washed with water, dried using MgSO ₄ and concentrated.

2. A- [3] -SiCl ₃ (Compound 4)
A- [3] -alkene (compound 3), HSiCl ₃ , and conventional platinum-based hydrosilylation catalysts such as H ₂ PtCl ₆ (Speier's catalyst) dissolved in propan-2-ol, or platinum divinyl The mixture of siloxane complex (Karstedt's catalyst) was stirred at ambient temperature for 24 hours. When the reaction was over, excess HSiCl ₃ was removed in vacuo.

3. A- [9] -Alkene (Compound 5)
A- [3] -SiCl ₃ (Compound 4) was refluxed for 4 hours using an excessive amount of 10% allylmagnesium bromide dissolved in diethyl ether, and then cooled at 0 ° C. Followed by hydrolysis with 10% _NH 4 Cl solution. The organic layer was washed with water, dried using MgSO ₄ and concentrated.

4). A- [9] -SiCl ₃ (Compound 6)
A- [9] -alkene (compound 5), HSiCl ₃ , and conventional platinum-based hydrosilylation catalysts such as H ₂ PtC ₁₆ (Speier's catalyst) or platinum divinylsiloxane complex dissolved in propan-2-ol The mixture of compounds (Karstedt's catalyst) was stirred at ambient temperature for 24 hours. When the reaction was over, excess HSiCl ₃ was removed using a vacuum pump.

[Example 3.6]
1. [1] -Acid- [3] -triol (Compound 3)
In step (a), the triol 1 was cyanoethylated to prepare the nitrile compound 2. Acrylonitrile, nBu ₃ SnH, and azobisisobutyronitrile were added to PhCH ₃ containing Compound 1 at 110 ° C. In step (b), the nitrile compound 2 was hydrolyzed under reaction conditions such as KOH, EtOH / H ₂ O, H ₂ O ₂ , and Δ to prepare compound 3 and carboxylic acid.

2. A- [3] -triol (compound 5)
In step (c), [1] -acid- [3] -triol is converted to 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole hydrate (HOBT). Was coupled to compound 4 through an amide coupling reaction.

3. A- [3] -Tribromide (Compound 6)
In step (d), HBr / H 2 _S O ₄ was brominated at 100 ° C., and the alcohol was used to synthesize tribromide.

4). [1] -CN- [3] -OBzl (Compound 8)
In step (e), triol (1) was treated with benzyl chloride to produce a triether using Me ₂ SO and KOH. In step (f), the triester (compound 8) was cyanoethylated to produce a nitrile compound (compound 9). Acrylonitrile, nBu ₃ SnH, and azobisisobutyronitrile were added to PhCH ₃ containing compound 8 at 110 ° C.

5). [1] -OH- [3] -OBzl (Compound 11)
In step (g), nitrile compound 9 (compound 9) was hydrolyzed with carboxylic acid in an environment such as KOH, EtOH / H ₂ O, H ₂ O ₂ , Δ to produce compound 10. In step (h), compound 10 and carboxylic acid were reacted with a BH ₃ THF solution exceeding a concentration of 1.0 M in order to convert the carboxylic acid into an alcohol.

6). [1] -Alkyne- [3] -OBzl (Compound 13)
In step (i), the alcohol was converted to chloride (CH ₂ Cl ₂ ) under an excessive amount of SOCl ₂ and pyridine catalyst. In step (j), the chloride was reacted with lithium acetylide ethylenediamine complex at 40 ° C. under dimethyl sulfoxide.

7). A- [3] -Alkyne- [9] -OBzl (Compound 14)
In step (k), the compound 13 having a block forming 4 equivalents of an alkyne terminus, hexamethylphosphoric triamide (HMPA), lithium diisopropylamide (LDA), and tetramethylethyleneamine (TMED) are used at 0 ° C. to 40 ° C. For 1.5 hours to alkylate the A- [3] -OBz (Compound 6).

[Example 3.7]
1. A- [9] -OH (Compound 15)
A- [3] -alkyne- [9] -OBzl (compound 14) was prepared with 60% Pd-C / H under EtOH and 10% THF solution to produce A- [9] -OH (compound 15). The reaction was carried out at 4 ° C. for 4 days, followed by reduction and deprotection.

2. A- [27] -COOH (Compound 17)
The alcohol was slowly converted to nonbromide using SOBr ₂ under chloride (CH ₂ Cl ₂ ) at 40 ° C. for 12 hours. After the above process, to produce 49% A- [9] -alkyne- [27] -OBzl (compound 16), the 12-valent [1] -alkyne- [3] -OBzl (compound 13) Non-bromide compounds were alkylated. A- [9] -Alkyne- [27] -OBzl (Compound 16) reacted with Pd-C / H for 4 hours at 60 ° C. in EtOH and THF solution containing 10% Pd—C / H. Reduction and deprotection through one step yielded 89% A- [27] -OH. A- [27] _NH 4 OH or -OH _{(CH 3)} is oxidized _with 4 under NOH RuO4, of 85% A- [27] was produced -COOH (Compound 17).

[Example 3.8]
1) [G1]-(OMe) ₂ (Compound 3)
Compound 1 (1.05 mol equiv.), 3,5-dimethoxybenzyl bromide (1.00 mol equiv. Compound 2), potassium carbonate (1.1 mol equiv.), And 18-c-6 (0.2 mol equiv.) The mixture was heated under dehydrated acetone for 48 hours until refluxed under nitrogen. The mixture was cooled and then distilled to dryness, and the residue was partitioned between chloride (CH ₂ Cl ₂ ) and water. The aqueous layer was extracted with chloride (CH ₂ Cl ₂ ) (3 ×), and the combined organic layer was dried and evaporated to dryness. To obtain compound 3, it was purified using flash chromatography (flash Chromatography) to _{EtOAc-CH 2} Cl 2 as eluent (eluent) to the crude resultant structure.

2) [G1]-(OH) ₂ (Compound 4)
The methyl ether group of compound ₃ was eliminated with BBr ₃ under EtOAc solution for 1 hour. The resulting crude product was purified using flash chromatography using MeOH-EtOAc as an eluent to give compound 4.

3) [G2]-(OMe) ₄ (Compound 5)
[G1]-(OH) ₂ (1.00 mol equiv. Compound 4), 3,5-dimethoxybenzyl bromide (2.00 mol equiv. Compound 2), potassium carbonate (2.1 mol equiv.) And 18-c-6 (0.2 mol equiv.) Mixture was heated under dehydrated acetone for 48 hours under nitrogen until refluxed. The mixture was cooled and then distilled to dryness, and the residue was partitioned between chloride (CH ₂ Cl ₂ ) and water. The aqueous layer was extracted with chloride (CH ₂ Cl ₂ ) (3 ×), and the combined organic layer was dried and evaporated to dryness. In order to obtain Compound 5, the crude product was purified using flash chromatography using EtOAc-CH ₂ Cl ₂ as an eluent.

4) [G2]-(OH) ₄ (Compound 6)
The methyl ether group of compound 5 was eliminated with BBr ₃ under EtOAc solution for 1 hour. The crude product was purified using flash chromatography with MeOH-EtOAc as an eluent to give compound 4.

5) [G3]-(OMe) ₈ (Compound 7)
[G2]-(OH) ₄ (1.00 mol equiv. Compound 6), 3,5-dimethoxybenzobromide (4.00 mol equiv. Compound 2), potassium carbonate (4.1 mol equiv.) And 18-c-6 (0.2 mol equiv.) Mixture was heated under dehydrated acetone for 48 hours under nitrogen until refluxed. The mixture was cooled and then distilled to dryness, and the residue was partitioned between chloride (CH ₂ Cl ₂ ) and water. The aqueous layer was extracted with chloride (CH ₂ Cl ₂ ) (3 ×), and the combined organic layer was dried and evaporated to dryness. In order to obtain the compound 7, the crude result was purified using flash chromatography using EtOAc-CH ₂ Cl ₂ as an eluent.

6) [G3] - (OH ) 8 ( Compound 8)
The methyl ether group of compound 7 was eliminated with BBr _{3 in} EtOAc solution for 1 hour. To obtain compound 8, flash chromatography was performed on the crude product using MeOH-EtOAc as an eluent.

[Example 4: Assembly of dendron on substrate]
Instead of APDES, TMAC (N-trimethoxysilylpropyl-N, N, N-trimethylammonium chloride) was self-assembled on glass oxide. It was not necessary to cap the amine residues of the dendrimer layer on the TMAC.

<Aminosilylation with TMAC>
A clean substrate (slide glass) was immersed in a mixed solution of TMAC (2 mL) and acetone (100 mL) for 5 hours. After self-assembly, the substrate was removed from the flask and washed with acetone. The substrate was placed in an oven and heated at 110 ° C. for 40 minutes. After soaking in acetone, the substrate was sonicated for 3 minutes. The cleaned substrate is placed in a Teflon container, placed in a glass container having a large screw cap connected to an O-hook, and the glass container is vacuum processed to allow the TMAC to be self-assembled. Straight dried.
The chemical structure of TMAC is shown below.

  The Fmoc-spacer- [9] acid was self-assembled under the same conditions as the CBz- [9] acid except that the amine residue was capped with acetic anhydride.

<Fmoc-Spacer- [9] Self-assembly of Acid (Compound 5)>
A certain amount of Fmoc-spacer- [9] acid (compound 5) was dissolved in a mixed solvent (DMF: deionized water = 1: 1 (v / v)) to prepare a 20 mL solution. The solution was put in a Teflon (registered trademark) container, and the aminosilylated slide glass piece prepared above was immersed in the solution. Each piece of substrate was removed from the solution after 1 day as the flask was placed at ambient temperature and allowed to self-assemble. The plate was washed with deionized water immediately after removing the strip. Each substrate was sonicated sequentially for 3 minutes with deionized water, deionized water-methanol mixed solvent (1: 1 (v / v)), and methanol. After sonication, the substrate is dipped in a Teflon (registered trademark) container, and again placed in a glass container having a large screw kit with an O-hook connected thereto, and the glass container is evacuated (30- 40 mTorr) The substrate was dried.

<Fmoc-Spacer-Fmoc Deprotection of [9] Acid (Compound 5)>
A Teflon container with 5% piperidine in DMF was facilitated. The self-assembled substrate was immersed in the Teflon container and stirred for 20 minutes. Each substrate was sonicated sequentially with acetone and methanol for 3 minutes and dried in a vacuum chamber (30-40 mTorr).

[Example 5: dendron (9-acid and 27-acid)] p53 microarray on modified surface]
Seven codons that were found to have a very high frequency of missense mutation hotspots, 175, 215, 216, 239, 248, 273, and 282, were selected and used in the experiments. Of the seven codons, codons 175, 248, 273, and 282 are obtained from the international P53 mutation database (IARC, http: //: www-p53.iarc.fr/p53DataBase.htm) and the remaining three codons, 215, 216, and 239 were obtained from the Korean p53 mutation hotspot database. Capture probe sequences for the six codons (DNA immobilized on the dendron-modified surface) were produced by software, their length varied from 15-23mer, and Tm was adjusted to about 55 ° C.

<Example 5.1: Detection of seven hot spot mutations in p53 gene using dendron modified surface>
A dendron modified substrate was used to detect a single mutation in the p53 gene of a cancer cell line. A target DNA sample (100-200 mer) containing 7 hot spot codons (175, 215, 216, 239, 248, 273, and 282) was amplified from DNA extracted from cancer cells using a random priming method. (See Example 5.8) and hybridized with a capture probe (oligonucleotide of 15-25mer) corresponding to 7 immobilized hot spot codons. The fluorescence intensity of each hybridized spot was measured with a confocal laser scanner and the SNP discrimination ability was calculated. According to this example, the quality of a DNA microarray having a dendron modified surface indicates that it can actually detect a single mutation present in the target sample.

<Example 5.2: Effect of length of probe oligonucleotide having T30 on hybridization efficiency and SNP discrimination>
The effect of capture probe length on SNP discrimination was tested using various length capture probes with T30. After fixing the capture oligonucleotide containing T30 and corresponding to codons 175 and 239 to the 5 ′ end and the primary amino group at the end of the dendron modified surface, the p53 target DNA was hybridized and fluorescent The strength was measured. This example showed the dependence of SNP discrimination ability and signal strength on capture probe length.

<Example 5.3: Capture probe concentration vs intensity (intensity); and capture probe vs SNP distinction>
The dependence of signal intensity and SNP discrimination ability on capture probe concentration was investigated. The capture probe on the dendron-modified surface was mixed with target DNA at various concentrations, and the fluorescence intensity and SNP discrimination ability were measured. The optimal concentration of capture probe for p53 was determined.

<Example 5.4: Probe concentration vs intensity; and target probe concentration vs SNP distinction>
The dependence of signal intensity and SNP discrimination ability on target probe concentration was investigated. The target DNA was hybridized at various concentrations, and the fluorescence intensity and SNP discrimination ability were measured. This example provided a diverse range of DNA microarrays with dendron modified surfaces.

<Example 5.5: Detection of mutation in mixed target sample>
Target samples with point mutations (5 or 10%) can be detected, where the mutated target sequence is present in a small part compared to the normal sequence. Target samples containing two types of target DNA were produced and hybridized at different molar concentrations to detect single point mutations at specific codons in a normal ordered mixture as well as mutated target DNA. This example has very important clinical significance in diagnosing early stage cancer.

<Example 5.6: Mutation detection of 10 unknown rectal cancer cell lines>
A system designed to detect unknown mutations in cancer cell lines.

<< Example 5.6.1: Cell culture and genomic DNA extraction >>
Rectal cancer cell lines SNU-C1, SNU-C5, COLO 201, COLO 205, DLD-1, LS 513, HCT-15, LS 174T, HCT 116, and SW 480 were KCLB (Korea Cell Line Bank, Seoul, Korea) Purchased from. Cells were inoculated into RPMI 1640 supplemented with 10% Utea serum (FBS), 100 μg / ml streptomycin, and 100 U penicillin (GibcoBRL, Carlsbad, CA) and cultured at 37 ° C. with 5% CO ₂ . did. Rectal cancer cells (2 × 10 ⁶ cells) were obtained according to the user manual of Invisorb® spin cell mini kit (Invitek, Berlin, Germany) and used for genomic DNA analysis. P53 target DNA was produced from these genomic DNAs (see Example 5.8.2), and DNA microarray experiments were performed in the same manner as described above.

<Example 5.7: Effect of target probe length on hybridization efficiency and SNP discrimination>
Target DNAs having various lengths were produced by various methods such as random primer method, PCR, and Dnase degradation, and the influence of target probe length on hybridization efficiency and SNP discrimination was investigated.

<Example 5.8: Experimental method>
<< Example 5.8.1: Genomic DNA Sample >>
Genomic DNA of SNU cell lines (SNU-61, 216, 475, 563, 601, 668, 761, and 1040) was obtained from Park Jegap of Seoul National University School of Medicine. The SNU cell line is a human tumor cell line collected from a Korean patient. The characteristics of these cell lines were identical to those previously described and were used in various studies (Bae IS et al., 2000, Park JG et al., 1997, Kang MS et al., 1996, Yuan Y et al., 1997, 378-87).

<< Example 5.8.2: Subcloning and sequence analysis >>
For each cell line, the p53 gene, in particular the p53 gene present between exon 5 and exon 8, was amplified using two pairs of synthetic oligonucleotide primers: exon 5Fwd I, 5′-CTG ACT TTC AAC TCT GTC TCC T-3 ′ (SEQ ID NO: 5); Exon 5Fwd II, 5′-TAC TCC CCT GCC CTCAAC AA-3 ′ (SEQ ID NO: 6); Exon 8 Rev I, 5′-TGC ACC CTT GGT CTC CTC CAC-3 ′ (SEQ ID NO: 7); Exon 8 Rev II, 5′-CTC GCT TAG TGC TCC CGG G-3 ′ (SEQ ID NO: 8) (Kang MS et al., 1996). Under the following conditions, 250 μM dNTP mix, 2.5 U Taq polymerase (NEB), 10 pmole in 1 × ThermoPol buffer (supplemented Taq polymerase) so that the reaction volume of the entire Multiblock System (Hybaid, UK) becomes 20 μl. Each genomic DNA was amplified using the first primer pair (exon 5Fwd I and exon 8 Rev I corresponding to 4 and 8), 250 μM dNTP mix, 2.5 U Taq polymerase (NEB): polymerase initial activation 20 cycles, 95 ° C. for 30 seconds; 58 ° C. for 30 seconds; 72 ° C. for 90 seconds; 72 ° C. for 5 minutes for a final extension. The first PCR product was diluted and used as a template for the second PCR. Except that the PCR amplification product of genomic DNA was diluted 20-fold and the PCR primer was used with 10 pmol of the second primer pair (exon 5 Fwd II and exon 8 Rev II corresponding to exon 5 and 8). A second nested PCR was performed under the same conditions as in the previous step. The amplification period was increased to 25 periods. The final nested PCR product was purified by gel extraction. The PCR product obtained from genomic DNA was ligated to the pGEM T-easy vector (Promega) and transformed into DH5a cells. The subcloned plasmid was isolated with QIAGEN Plasmid Min kit (QIAGEN Inc., Valencia, Calif.) And used for rank analysis. pUC / M13 forward and reverse sequence analysis was performed using the following primer pairs: M13 FWD 5'-GTT TTC CCA GTC ACG ACG TTG-3 '(SEQ ID NO: 9) and M13 REV 5'- TGA GCG GAT AAC AAT TTC ACA CAG-3 ′ (SEQ ID NO: 10).

Example 5.8.3: Production of target probe
50 ng template DNA, 50 U Klenow enzyme (NEB), 1 × EcoPol buffer (Klenow enzyme supplement), 6 μg random octamer (synthesized by Bionics) Then, the whole volume was made 20 μl with Multiblock System (Hybaid, UK) containing low concentration dT dNTP mix (100 μM dA, G, CTP / 50 μM dTTP) and 50 Cyanine3-dUTP (NEN), and this was performed at 37 ° C. for 2 hours. Non-merged nucleotides were isolated using the QIAGENMinElute PCR purification kit (QIAGEN Inc., Valencia, Calif.). After quantitative and qualitative analysis (specific activity, number of nucleotises per bound fluorescent dye) using a UV / Vis spectrophotometer, the analyzed product was used for hybridization.

<< Example 5.8.4: Cell culture and genomic DNA extraction >>
Rectal cancer cell lines SNU-C1, SNU-C5, COLO 201, COLO 205, DLD-1, LS 513, HCT-15, LS 174T, HCT 116, and SW480 were purchased from KCLB. RPMI 1640 supplemented with Utea serum (FBS), 100 μg / ml streptomycin, and 100 U penicillin (GibcoBRL, Carlsbad, Calif.) Was inoculated and reacted at 37 ° C. with 5% CO ₂ . The rectal cancer cell line (2 × 10 ⁶ cells) was harvested by the method according to the user manual using Invisorb (trademark) spin cell mini kit (Invitek, Berlin, Germany) and used for genomic DNA extraction.

[Example 6: Protein probe immobilized on dendron]
<Example 6.1: Immobilization of NHS biotin on dendrimer-modified slide glass>
A spotting solution was prepared containing succinimidyl D-biotin (1.0 mg) in 1 mL sodium bicarbonate buffer 50 mM and DMSO (40% v / v). NHS biotin was immobilized on dendrimer-modified glass slides in a Class 10,000 clean room using a Microsys 5100 microarrayer (Cartesian Technologies, Inc, USA). After immobilization and reaction for 1 hour in a humid chamber (75% humidity), the biotin microarray was treated with DMF (50 ° C.) THF and MBST (50 mM MES, 100 mM NaCl, 0.1% Tween-20, pH 6.0). ) In order for 2 hours. The slide was rinsed with distilled water, dried and used immediately or stored at room temperature for several days before use.

<Example 6.2: Detection of protein / ligand interaction>
This example is described by Hergenroter, P .; J. et al. Depew, K .; M.M. Schreiber, S .; L. J. et al. Am. Chem. Soc. 2000, 122, 7849. Blocked with 3% Utea serum supplemented MBST for 1 hour before adding Cy3-labeled streptavitin solution. After a slight rinse, the slides were exposed to a Cy3 labeled streptavitin solution for 30 minutes at room temperature. The solution was prepared to 1 mg / mL by diluting an appropriate protein stock solution with MBST supplemented with 1% BSA. After the reaction, the slide was washed once with MBST, and gently stirred for 12 minutes while changing MBST four times. Slides were dried and scanned using a confocal scanner Scan Array® Lite (GSI Lumonics). Image and fluorescence intensity analysis was performed using the microarray quantitative analysis software ImaGene (BioDiscovery, Inc.).

Example 7: Glass beads with controlled pores, including macromolecules with controlled pores
Pore-controlled glass beads with attached aminopropyl groups (AMPCPG; 80-120 mesh; average pore diameter, 50 nm or 300 nm) and glass beads with controlled pores modified with long chain aminoalkyl groups (LCAA-CPG; 80-120 mesh; mean pore diameter, 50 nm) was purchased from CPG, Inc. 1,4-butanediol diglycidyl ether, 1,3-diaminopropane, reduced glutathione (GSH), N- (3-methylaminopropyl) -N′-ethylcarbimide (EDC), N-hydroxysuccinimide (NHS) N- (9-fluoromethoxycarbonyloxy) chloride (Fmoc-Cl), piperidine, 4-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS), phosphate buffered saline (PBS) were purchased from Sigma-Aldrich. Other compounds were purchased in analytical reagent grade and used without further purification. Distilled water was treated with a Barnstead E-pure 3-Module system to produce deionized water (18 MΩ.cm). UV-vis spectra were recorded on a Hewlett-Packard diode-array 8453 spectrophotometer.

<Example 7.1: Immobilization of glutathione on dendron-modified CPG (samples E1 and E3)>
(I) Modification with Fmoc- [3] -acid: AMPCPG (dry weight 0.70 g) was washed with acetone on a glass filter. After vacuum drying, a mixed solution of 1,4-butanesyldiglycidyl ether (1.0 mL) and carbonate buffer (2.0 mL, pH = 11) was added to AMPCPG (surface capacity: 91.8 μmol / g, surface area). : 47.9 m ² / g). After thoroughly shaking for 24 hours at room temperature, the resulting beads were filtered and washed sequentially with deionized water and acetone. To the vial containing the sample, a mixture of 1,3-diaminopropane (1.0 mL) and carbonate buffer (pH = 11) was added and mixed at room temperature for 24 hours. After thorough washing, the reaction of the epoxy groups remaining on the surface is blocked with a mixture of 2-molcaptoethanol (1.0 mL) and aqueous sodium bicarbonate solution (2.0 mL, pH = 8.5). I let you. Dimethylformamide (30% DMF (v / v)), N- (3-methylaminopropyl) -N′-ethylcarboimide (15 mg) in which Fmoc- [3] -acid (14 mg, 21.3 μmol) was dissolved , 77 μmol), and N-hydroxysuccinimide (9.0 mg, 77 μmol) were added to the vial containing the beads. The mixture was shaken for 11 hours at room temperature and then thoroughly washed with deionized water and acetone sequentially.
(Ii) Blocking step: Acetic anhydride (1.0 mL) was used in anhydrous methylene chloride (2.0 mL) to block amine groups remaining overnight at room temperature.
(Iii) Deprotection step: Wash the beads sequentially with methylene chloride and acetone, add 20% piperidine in DMF (3.0 mL) to the vial containing the beads, and shake the vial for 30 minutes to mix. It was.
(Iv) Ligand immobilization step: Add a mixture of 1,4-butanesyldiglycidyl ether (1.0 mL) and carbonate buffer (2.0 mL, pH = 11) to the vial and mix at ambient temperature for 24 hours. It was. After sequential washing with deionized water and acetone, sodium bicarbonate solution (3.0 mL, pH 8.5) and reduced glutathione (GSH, 5.4 mg, 17.6 μmol) are added to the vial containing the beads, Shake for 24 hours at room temperature and mix. After washing the beads, a mixture of 2-molcaptoethanol (1.0 mL) and aqueous sodium bicarbonate solution (2.0 mL, pH = 8.5) was added to the vial containing the beads. Finally, the beads were separated and washed, dried in vacuum and stored at 4 ° C. under a nitrogen atmosphere. The same steps were performed to prepare sample E3 except that Fmoc- [9] -acid was used instead of Fmoc- [3] -acid.

Example 7.2: Production of different GSH binding matrices for control experiments (samples CS, CL, and A):
(I) Sample CS and CL: GSH was directly immobilized on AMPCPG and LCAA-CPG using GMBS linker. The beads (0.10 g) were washed with acetone using a glass filter. After drying in vacuum, sodium bicarbonate buffer (1.0 mL, 3: 7 (v / v) in which DMF and 4-maleimidobutyric acid, N-hydroxysuccinimide ester (GMBS, 3.0 mg, 11 μmol) were dissolved. ), PH = 8.5) was added to the vial containing the beads. After mixing by shaking at room temperature for 4 hours, the obtained beads were separated by filtration, washing with deionized water and acetone. Finally, the amine groups remaining in the matrix were reacted with acetic anhydride (1.0 mL) in anhydrous methylene chloride (2.0 mL). After washing, GSH (3.4 mg, 11 μmol) in PBS buffer (1.0 mL) was added to the vial containing the beads and mixed by shaking for 12 hours at room temperature. The maleimide group remaining in 2-molcaptoethanol (1.0 mL) was blocked, the beads were separated, washed and vacuum dried.
(Ii) Sample A: AMPCPG was modified with 1,4-butanedyl diglycidyl ether and 1,3-diaminopropane by the same modification method as E1 and E3. After capping with 2-molcaptoethanol, an epoxy group was produced with 1,4-butanesyl diglycidyl ether. Finally, the clithion was fixed, and the ring of the epoxy group remaining on the beads was opened with 2-molcaptoethanol.

<Example 7.3: Measurement of amine density on modified beads>
Beads undergoing E1 or E3 modification or control experimental beads (10 mg) were placed in an e-tube. At the same time, 9-fluoromethyl chloroformate (Fmoc-Cl, 1.75 mg) and Na ₂ CO ₃ (1.45 mg) were placed in a separate glass vial, and a mixed solvent of 1,4-dioxane and water (2: 1 (v / v)) was added to dissolve the reagent. One fifth of the solution was taken and added to the e-tube containing the beads. The tube was placed in a vial and the vial was shaken at room temperature for 12 hours to mix. The beads were separated using a glass filter, and the porous material was washed sequentially with deionized water and acetone. After drying in vacuo, 20% piperidine in DMF (0.50 mL) was placed in the e-tube containing the beads to react with piperidine for 30 minutes. The remaining solution was carefully transferred from the tube to a new e-tube and the beads were washed twice with 20% piperidine in DMF (0.25 mL). The entire solution was added to the previous e-tube. The solution was mixed again with methanol to adjust the final absorbance. Absorbance was measured at 310 nm using a UV / Vis spectrometer and the relevant solvent was used to correct for background errors. In order to improve reliability, measurements were performed on five different samples.
For measurement, 20% piperidine in a series of DMF and N-Fmoc-ethanolamine (or 9-fluoromethyl N- (2-hydroxyethyl) covermate) (30 μM-70 μM) were prepared. After reacting for 30 minutes, the absorbance was measured using a solution containing dibenzofulvene, and the extinction coefficient was calculated.

<Example 7.4: Production of GST fusion protein lysate>
GST-fusion proteins were prepared as described in the known literature (Kim, JH; Lee, S .; Kim, JH; Lee, TG; Hirata, M .; Suh, Ry, SH, Biochemistry 2002, 41, 3414-3421, the entirety of which is incorporated herein by reference). For large scale culture, a single colony containing the recombined pGEX plasmid was cultured in 200 ml 2X YTA medium. After culturing to the logarithmic growth phase, gene expression was induced with IPTG for 6 hours. The cells were then collected by centrifugation and washed with 1X PBS. Thereafter, E. coli was added with 10 mL hypotonic buffer (20 mM Tris, 150 mM NaCl, 1.0 mM MgCl ₂ , 1.0 mM EGTA, pH 7.4) containing 0.50 mM PMSF. E. coli was sonicated. The insoluble material was removed to obtain a protein.

<Example 7.5: Binding analysis>
(I) Effect of chain length: 0.8 mL of reaction buffer with the prepared beads CL (5.72 mg), CS (6.97 mg), E1 (10.0 mg), and E3 (14.8 mg) liquid _{(20mM Tris, 150mM NaCl, 1.0mM} MgCl 2, 1.0mM EGTA, 1% TX-100,0.10mM PMSF, pH7.4,0.50mM PMSF) mixed solution and separately containing the GST lysate in And was washed 3 times with 10 bed volumes of reaction buffer and 100 μL SDS sample buffer was added. After the tube was placed at 95 ° C. for 5 minutes, a 20 μL sample was taken and used for SDS-PAGE analysis and the resulting gel was stained with CBBG-250 staining solution.
(Ii) Selectivity of dendron-treated matrix: In addition to 10 mg of samples A, E1, and E3, 100 μg of purified GST or GST fusion protein lysate was used in the experiment. The other steps are the same as the above steps.

<Example 7.6: GST fusion protein elution from glutathione sepharose-4B, E1, and E3>
Glutathione Sepharose-4B, E1, and E3 were performed as described in the binding analysis item. The amount of protein bound to the beads was quantified with Image gauge V3.12 (FUJI PHOTO FILM CO., LTD.). The same steps were performed on the p47 ^phox PX domain and the Munc-18 fragment lysate (FIG. 13).

  All documents referred to in this specification are incorporated herein in their entirety. Those having ordinary skill in the art will be able to recognize and arrive at numerous inventions that are equivalent to the specific examples of the present invention simply through routine experimentation. Such equivalent invention is included in the protection scope of the claims.

Claims (33)

A substrate including a molecular layer in which conical macromolecules with controlled molecular size are regularly distributed,
The macromolecule comprises a dendron comprising a branched site and a linear site,
A plurality of ends of the branched site are covalently bonded to the substrate, and a terminal of the linear site has a functional group capable of binding to organic moiety;
The linear portion includes a spacer domain (L) and a functional group (W),
Wherein L _{is, NH- (CH 2) 3 C} (O), NH- (CH 2 CH 2 O) 2 CH 2 C (O), NH- (cyclohexyl) (CO), NH- (CH 2) 6 NHC (O), NH— (CH ₂ ) ₇ C (O), NH— (CH ₂ ) ₆ C (O), NH— (CH ₂ ) ₆ NH (CO), NH— (cyclopropryl) (CO), NH Selected from the group consisting of — (CH ₂ ) ₆ NHC (O) CH ₂ , NH— (CH ₂ ) ₅ , NH— (cyclopropyl) (CO), and NH— (CH ₂ ) ₆ C (O) And
The substrate, wherein W is selected from the group consisting of NH, CH ₂ and O.   The substrate according to claim 1, wherein the macromolecules are distributed at regular intervals.   The macromolecules are distributed such that the spacing between functional groups of the functionalized linear sites of the macromolecule is a regular spacing of about 0.1 nm to about 100 nm. 2. The substrate according to 2.   4. The substrate of claim 3, wherein the macromolecules are distributed at regular intervals of about 10 nm. The substrate according to claim 1, wherein the end of the branched portion is selected from the group consisting of OH, OMe, NH ₂ and OBzl.   The substrate according to claim 1, wherein a protecting group is bonded to an end of the linear part.   The substrate according to claim 6, wherein the protecting group is selected from the group consisting of anthracenemethyl (A), Boc, Fmoc, and Ns. The substrate according to claim 1, wherein the dendron is a polymer represented by the following formula.
However, in the above formula,
L _{is, NH- (CH 2) 3 C} (O), NH- (CH 2 CH 2 O) 2 CH 2 C (O), NH- (cyclohexyl) (CO), NH- (CH 2) 6 NHC ( _{O), NH- (CH 2)} 7 C (O), NH- (CH 2) 6 C (O), NH- (CH 2) 6 NH (CO), NH- (cyclopropyll) (CO), NH- Selected from the group consisting of (CH ₂ ) ₆ NHC (O) CH ₂ , NH— (CH ₂ ) ₅ , NH— (cyclopropyl) (CO), and NH— (CH ₂ ) ₆ C (O). Yes,
W is selected from the group consisting of NH, CH ₂ , and O;
X is selected from the group consisting of anthracenemethyl (A), Boc, Fmoc, and Ns;
_{R 1~3, R 11~13, R 21~23} , R 31~33, R 111~113, R 121~123, R 131~133, R 211~213, R 221~223, R 231~233, _R 311~313, R 321~323, R 331~333 _{is, CH 2 O (CH 2)} 2 C (O), (CH 2) 2 - (cyclohexyl) -C (O), CH 2 -C = C _{-CH 2 C (O), (} CH 2) 7, (CH 2) 2 C (O), CH 2 OCH (CH 3) CH 2 C (O), (CH 2) 2, CH 2 OCH 2 CH ( Selected from the group consisting of CH ₃ ) C (O), and CH ₂ OCH (CH ₃ ) CH ₂ C (O),
T is selected from the group consisting of OH, OMe, NH ₂ and OBzl.   The substrate according to claim 1, wherein a target-specific ligand is bound to an end of the linear site.   The target-specific ligand is selected from the group consisting of compounds, DNA, RNA, PNA, aptamers, peptides, polypeptides, carbohydrates, antibodies, antigens, biomimetics, nucleotide analogs, and mixtures thereof. 10. A substrate according to claim 9, characterized in that   The substrate of claim 9, wherein the distance between target-specific ligands bound to linear sites of the macromolecule is from about 0.1 to about 100 nm.   2. The substrate of claim 1, wherein the substrate is made of a material selected from the group consisting of a semiconductor, a synthetic organic metal, a synthetic semiconductor, a metal, an alloy, plastic, silicon, silicate, glass, and ceramic. Substrate.   The substrate of claim 12, wherein the substrate is selected from the group consisting of slides, particles, beads, microwells, and porous materials.   The substrate according to claim 13, wherein the porous material is selected from the group consisting of membrane, gelatin, and hydrated gel.   The substrate according to claim 13, wherein the beads are beads having controlled pores. It is a manufacturing method of the substrate according to claim 1, Comprising:
(I) functionalizing the substrate such that it can react with the end of the macromolecule; and (ii) contacting the macromolecule with the substrate so that the end of the macromolecule and the substrate are A method for producing a bond.   The substrate of claim 16, wherein the substrate is made of a material selected from the group consisting of a semiconductor, a synthetic organic metal, a synthetic semiconductor, a metal, an alloy, plastic, silicon, silicate, glass, and ceramic. Manufacturing method.   The manufacturing method according to claim 16, wherein the substrate is selected from the group consisting of a slide, a bead, a microwell material, and a porous material.   The method according to claim 18, wherein the porous material is selected from the group consisting of hydrated gel, gelatin, and membrane.   The manufacturing method according to claim 18, wherein the beads are beads having controlled pores. (I) removing a protecting group from the end of the linear site of the macromolecule on the substrate; and (ii) a target-specific ligand, or a homotypic amphoteric or linked to the target-specific ligand, or Contacting an atypical amphoteric linker molecule with the end of a linear site of a macromolecule on the substrate, such that the ligand or linker molecule and the end form a bond; The production method according to claim 16, wherein the target-specific ligand is immobilized at the end of the linear site.   The method of claim 21, wherein the macromolecule on the substrate minimizes obstacles in binding of the end of the target-specific ligand and a linear site.   The method of claim 21, wherein the macromolecule on the substrate minimizes obstacles in detecting a specific target of the target-specific ligand.   The method according to claim 21, wherein the target-specific ligands are distributed at regular intervals.   The method of claim 21, wherein the substrate comprises a low density target-specific ligand. 26. The method of claim 25, wherein the density of the low density target-specific ligand is in the range of about 0.01 probe / nm ^{2 to} about 0.5 probe / nm ² .   The target-specific ligand is selected from the group consisting of a compound, DNA, RNA, PNA, aptamer, peptide, polypeptide, enzyme, carbohydrate, polysaccharide, antibody, antigen, biomimetic, nucleotide analog, or a mixture thereof The manufacturing method according to claim 21, wherein the manufacturing method is one.   The diagnostic system for the detection of the variation | mutation in a gene characterized by including the substrate of Claim 1 by which the terminal of a linear site | part is fixed to the target-specific oligonucleotide.   The diagnostic system according to claim 28, wherein the substrate includes an oligonucleotide specific for a cancer-related gene.   30. The diagnostic system according to claim 29, wherein the cancer-related gene is p53.   The presence of a mutation in the gene comprising the step of contacting the substrate according to claim 1 with a sample containing the gene to be analyzed, wherein the end of the linear site is fixed to the target-specific oligonucleotide. Detection method.   32. The detection method according to claim 31, wherein the gene is a cancer-related gene.   The detection method according to claim 32, wherein the gene is p53.