Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
  • C07C271/06—Esters of carbamic acids
  • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
  • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
  • C07C271/12—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
  • C07C271/06—Esters of carbamic acids
  • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
  • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
  • C07C271/16—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by singly-bound oxygen atoms

Definitions

• the present invention relates to a substrate that is applicable to a biochip, having a molecular layer including an amine group with a low density on its surface, and more particularly to a compound used for formation of a molecular layer with a low density, and a method of preparing the same, and a substrate including a molecular layer having an amine group prepared from the compound, and a method of preparing the same.
• Silylation on a substrate surface has been applied to various processes such as fixation of bio-molecules like enzymes and an antibodies; inorganic catalyst fixation; modification of electrolyte; chromatography; and a building formation of various types of molecules having ionic polymers, optical nonlinear chromophoric groups,
• Whitesell et al. suggested that a single layer of aminotrithiol be piled on a gold surface so that polyalanine can have a helical structure.
• they suggested piling a double layer of polyphenyl-alanine on a gold surface so that the polyalanine can have a helical structure, since the polyphenyl-alanine has too small a space to form a helical structure (Science 261 , 73 (1993)).
• R includes phenyl; phenyl substituted with nitro, halogen or cyano group; naphthyl; or anthryl.
• a preparation method of the compound represented by Chemical Formula 1 in a preparation method of a carboxylic derivative represented by Chemical Formula 1 which comprises: a) cyanoethylation of tris(hydroxymethyl)aminomethane and acrylonitrile to prepare tris[(cyanoethoxy)methyl]aminomethane; b) refluxing of the tris[(cyanoethoxy)methyl]aminomethane added with concentrated hydrochloric acid in order to prepare tris[(carboxyethoxy) ethyl] methyl] aminomethane ; c) esterifcation of the tris[(carboxyethoxy)ethyl]methyl] aminomethane by addition of methanol in order to prepare tris[[(methoxycarbonyl)ethoxy]methyl]aminomethane; d) protecting of the tris[(methoxycarbonyl)ethoxy]methyl]aminomethane by addition of a compound represented by Chemical Formula 1 , which comprises: a
• R includes phenyl; phenyl substituted with nitro, halogen, or cyano group; naphthyl; or anthryl, [Chemical Formula 3]
• R includes phenyl; phenyl substituted with nitro, halogen, or cyano group; naphthyl; or anthryl; e) hydrolyzing the mixture after adding sodium hydroxide solution to the compound represented by Chemical Formula 3 in order to prepare a compound represented by Chemical Formula 4:
• R includes phenyl; phenyl substituted with nitro, halogen, o group; naphthyl; or anthryl, f) reacting the mixture after dissolving the compound represented by Chemical Formula 4 and the tris[(methoxycarbonyl)ethoxy]methyl]aminomethane in dimethylformamide (DMF) and adding dicyclohexylcarbodiimide (DCC) and hydroxybenzotriazole (HOBT) to the dissolved material in order to prepare a compound represented by Chemical Formula 5:
• R includes phenyl; phenyl substituted with nitro, halogen, or cyano group; naphthyl; or anthryl; g) hydrolyzing the mixture after adding sodium hydroxide solution to the compound represented by Chemical Formula 5 in order to prepare the compound represented by Chemical Formula 1.
• a substrate having a molecular layer prepared by reaction of amine groups on the surface of an aminosilylated substitute and a derivative compound having a carboxylic acid represented by Chemical Formula 1 on its surface may also be achieved.
• a preparation method of a substrate having a molecular layer with a controlled amine density and regular spacing on its surface which comprises: a) a step of preparing a substrate having an amino silane layer on its surface; and b) a step of reacting amine groups produced from the amino silane
• the end group of the derivative of the step b) includes
• both the carboxylic acid and the amine group, and more preferably, the derivative includes the compound represented by Chemical Formula 1 .
• Fig. 1 is a schematic diagram illustrating a preparation procedure for a substrate with a controlled amine density and regular spacing with use of a
• Fig. 2 is a schematic diagram in series with Fig. 1 illustrating a
• Fig. 3 is a graph showing a stability of the substrate of Example 2
• Fig. 4 is a graph showing a stability of the substrate of Example 2 relative to temperature
• Fig. 5 is a graph showing stability of the substrate of Example 3 relative to temperature
• Fig. 6 is a graph showing stability of the substrate of Example 3 relative to various pH levels
• Fig. 7 is an atomic force microscope (AFM) image analysis showing a CBZ group not-deprotected by neat trifluoroacetic acid;
• Fig. 8 is an AFM image analysis showing a CBZ group deprotected by neat trifluoroacetic acid
• Fig. 9 is UV-visible spectrum showing a molecular layer of a compound represented by Chemical Formula 1a, wherein “a” shows a CBZ group after deprotection, “b” shows formed 9-antraldehydroimine, and “c” is after hydrolysis;
• Fig. 10 is a fluorescence spectrum at concentrations of 20, 40, 60, 80, and 100 mM, wherein the insert represents a calibration curve showing the relation of fluorescence intensity versus 9-anthraldehyde.
• a substrate on which a molecular layer
• the molecular layer having amine groups with a certain spacing is formed by preparing a polymer derivative represented by Chemical Formula 1 with a regular molecular weight, and the polymer is a cone-shaped and hyperbranched molecule having one amine group and nine carboxylic groups.
• the molecular layer having amine groups with a low density and a certain spacing is formed by reacting the amine groups produced from an aminosilylated substrate with the polymer represented by Chemical Formula
• R includes phenyl; phenyl substituted with a nitro, halogen
• R includes phenyl of a
• b is the step of adding concentrated hydrochloric acid to the mixture and refluxing the solution for 3 hours
• c is the step of adding MeOH to the solution and stirring the resulting
• d is the step of adding the compound represented by Chemical Formula 2, NaHC0 3 , and H 2 0 to the stirred mixture and further reacting the mixture at 25 °C for 12 hours,
• e is the step of adding 1 N NaOH to the mixture of d at 25 ° C for 12
• a is the step of adding DCC, 1 -hydroxybenzotriazole, and DMF, and
• b is the step of adding 1 N NaOH to the mixture of a, and reacting the resulting mixture at 25 ° C for 12 hours.
• a representative compound among the compounds represented by Chemical Formula 1 is N-(benzyloxycarbonyl)-tris[((N'-(carbonyl)- tris((carboxyethoxy)methyl)methylamino)ethoxy)methyl]aminomethane (hereinafter referred to as N-CBZ-[1]amine-[9] acid) of Chemical Formula 1a, wherein the R is phenyl.
• N-CBZ-[1]amine-[9] acid is regarded as a
• the amine groups produced from the molecular layer formed on the substrate surface reacts with the compound represented by Chemical Formula 1 , such as N-CBZ-[1]amine-[9] acid, so that the amine density decreases by the desirable amount.
• the end group of the N-CBZ-[1]amine-[9] acid is protected by CBZ (carbobenzyloxy).
• the N-CBZ-[1]amine-[9] acid having a protected amine group is designed such that the amine group is not transformed by reactants during reaction on the substrate surface, so the unneeded reaction does not occur.
• the CBZ deprotects easily, and is capable of turning into the primary amine group.
• the method developed by Bruson is followed for preparation of tris[(cyanoethoxy)methyl]aminomethane through cyanoethylation of tris(hydroxyme.thyl)aminomethane and acrylonitrile.
• the tris(hydroxymethyl)aminomethane and potassium hydroxide should be dried sufficiently under a vacuum for use in the preparation, since the tris(hydroxymethyl)aminomethane and potassium hydroxide are hygroscopic.
• the amount of potassium hydroxide is critical.
• the amount of potassium hydroxide ranges from 5 to 20 wt% based on the amount of tris(hydroxymethyl)aminomethane, and is preferably 15 wt%. When the amount of potassium hydroxide is greater than 20 wt% the acrylonitrile may be excessively polymerized, but when the amount of potassium hydroxide is less than 5 wt% the cyanoethylation of tris(hydroxymethyl)aminomethane and acrylonitrile may not occur. After completion of the reaction, a specific nitrile peak appeared at 118.5 ppm of 13 C NMR, and the peak is identical to the spectrum of the compound prepared by Newkome.
• tris[(carboxyethoxy)methyl]aminomethane In order to obtain tris[(carboxyethoxy)methyl]aminomethane, tris[(cyanoethoxy)methyl]aminomethane is refluxed in hydrochloric acid for 3 hours, though the tris[(cyanoethoxy)methyl]aminomethane is separated through column chromatography, since it is soluble in organic solvents. The nitrile group is changed to carboxylic acid, and a large amount of NH 4 CI salt is obtained as a byproduct. After the NH 4 CI is filtered by dissolving it in
• protecting reagents are used in order to protect the
• the tris[(carboxyethoxy)methyl]aminomethane is an oily and acidic compound, and the mixture is esterificated when methanol is added to it. Tris[((methoxycarbonyl)ethoxy)methyl]aminomethane is prepared simply by the esterification of the mixture, and carboxylic acid, as an end group of the tris[((methoxycarbonyl)ethoxy)methyl]aminomethane, is protected.
• Newkome suggested a simple method in which hydrogen chloride was injected into tris[((methoxycarbonyl)ethoxy)methyl]aminomethane added to
• a repeating unit for preparation of the dendrimer is
• N-(benzyloxycarbonyl)- tris[(carboxyethoxy)methyl]aminomethane is easy to separate as an organic layer.
• the N-(benzyloxycarbonyl)- tris[(carboxyethoxy)methyl]aminomethane shows CBZ peaks at 128.7 ppm and 128.2 ppm, and a carbamate peak at 155.2 ppm.
• N-(benzyloxycarbonyl)-tris[((N'-(carbonyl)- tris(((methoxycarbonyl)ethoxy)methyl)methylamino)ethoxy)methyl]aminomet han-e is hydrolyzed in 1 N NaOH to obtain N-(benzyloxycarbonyl)-tris[((N'- (carbonyl)-tris-((carboxyethoxy)methyl)methylamino)ethoxy)methyl] aminomethane).
• a substrate surface is cleaned and dried.
• the . dried substrate is immersed in a solution of aminosilane compound and a solvent for a predetermined time in order to aminosilylate the substrate.
• the aminosilane compound may be a compound that does not produce an acidic byproduct, such as 3-aminopropyl-tri-ethoxysilane, 3-aminopropyl-di- ethoxymethyl silane, and 3-aminopropylethoxy-di-methyl silane, and the solvent includes toluene that dissolves the aminosilane compound.
• substrate includes silicon wafer, glass, silica and fused silica.
• the substrate is cleaned with the solvent
• a protecting group should be removed from the N-CBZ- [1]amine-[9] acid in order to reveal the amine group on the substrate surface.
• the substrate is subjected to dissolution in trifluoroacetic acid and is sonicated at room temperature so that the amine group is deprotected. Then, the substrate surface is cleaned with a copious amount of solvent such as methanol, and the trifluoroacetic acid and the separated protecting group, which are adsorbed physically on the surface, are removed from the surface.
• solvent such as methanol
• aminosilylated substrate surface is represented.
• the bond is stable at
• the surface of the substrate are all changed to primary amines
• the solid substrate may be applied to and take an important role in
• the DNA chip has a high stability and is easily prepared.
• Desired molecules may be fixed on a substrate surface by
• the procedure of the present invention, and the surface may be studied.
• the total weight of the yellow liquid was 1 .52 g, and the yield
• a cleaned silica substrate was dried under a 20 mTorr vacuum.
• silica substrate was added thereto in order to aminosilylate the substrate.
• the aminosilylated substrate was cleaned with toluene, and dried in an oven at 120 ° C for 30 minutes. The dried substrate was cooled to room
• the reacted substrate was dissolved in a sequence of methanol, a mixture of methanol and water in a volume ratio of 1 :1 , water, and methanol, and the dissolved substrate was sonicated for 3 minutes, and dried under vacuum.
• the silica substrate was dissolved in trifluoroacetic acid in order to remove the CBZ group from the silica substrate, and was sonicated at room
• the substrate was cleaned with a copious amount of methanol, and was further sonicated for 10
• the initial layer of aminosilane molecules was about 8 A in
• CBZ-[1]amine-[9] acid increased by approximately 10 A, to 18 to 19 A, and the surface density of the amine group decreased by 0.18 amines/ran 2 .
• Fig. 3 shows stability of the thin film in water according to various pH
• the thin film shows itself to be stable, but
• the thin film decreases substantially. Therefore, it is shown that the thin film is
• Fig. 4 shows
• the thickness of the thin film according to varying temperature.
• the thin film was soaked in solution for 30 minutes while varying the temperature
• thin film has thermal stability, and may be applied to bio-chips.
• the unit of thickness is A in Figs. 3 and 4.
• a substrate was treated in the same manner as in Example 2, except that a fused silica substrate was used instead of a silica substrate.
• the fused silica substrate has similar characteristics to those of the silica substrate in Example 2.
• toluene (20 mi) and a coupling agent (0.2 mi) were added through a septum.
• vials were placed in an oven, and heated at 1 10 ° C for 30 minutes.
• the plates were immersed in toluene, toluene-methanol (1 :1 in the volume ratio), and methanol in a sequential manner, and they were sonicated for 3 minutes at each washing step.
• Each washed plate was placed in a vial, and several of such vials were placed in a glass container with a large screw cap lined with an O-ring, and eventually the container was evacuated under pressure ranging from 30 to 40 Torr, for dryness.
• N- CBZ-[1]amine-[9] acid was dissolved in a mixed solvent of dimethylformamide (DMF) and deionized water in a volume ratio of 1 :1 to
• each substrate was sonicated for 3 minutes in deionized water, a mixture of deionized water-methanol in the volume ratio of 1 :1 , and methanol in a sequential manner. After the sonication, each substrate was placed in a vial, and the several of such vials were placed in a glass container with a large screw cap lined with an O-ring, and eventually the container was evacuated under pressure ranging from 30 to 40 Torr, for dryness.
• each plate was taken out of the flask, washed with methylene chloride, and placed in a glass vial. The vials were sonicated for
• the phenyl of CBZ protecting group did not increase the water contact angle upon self-assembly.
• fused silica substrates are transparent to UV-like rays, the
• the absorption band is broadened even if it is believed that the phenyl
• chromophores are separated from each other by a distance (ca. 20—30 A)
• Thickness of the aminosilylated substrate was 8( ⁇ 2) A, and that was increased to 18( ⁇ 2) A after being self-assembled with the N-CBZ-
• the pH of the solution was adjusted by adding an appropriate amount of 0.1 N NaOH and 0.1 N HCl, or a mixture thereof in a suitable amount.
• one or two plates were put in a vial of a particular pH. After leaving vials at room temperature for 3 hours, the plates were taken out of the solution, and washed with deionized water. The plates were sonicated for 3 minutes while they were immersed in deionized water and methanol in a volume ratio of 1 :1 , and methanol in a sequential manner. After the sonication, each piece of the substrates was placed in a vial, and several of such vials were placed in a glass container with a large screw cap lined with an O-ring, and eventually the container was evacuated under pressure ranging from 30 to
• the RNH 3 + group is deprotonated, respectively.
• the K a of acetic acid and RNH 3 + are 1 .8 x 10 "5 and ca. 10 "10,5 , respectively, the number of ions
• the thickness of the molecular layer was measured after washing the
• the plates were soaked in a test tube containing deionized water.
• the test tube was
• condition should be mild enough to keep the molecules intact on the surface.
• the thickness of the organic layer decreased typically by 2
• the substrate under investigation was placed in the solution at 50 ° C for 12
• nitrobenzenaldehyde is sufficient for the accurate detection of a density as
• absorptivity is 8.70 x 10 4 L/cm • mol qualifies. As the density reduces to
• substrate surface was impregnated with deionized water in order to
• each hyperbranched molecule occupies area in
• the amine density of the substrate surface is capable of being controlled and decreased, allowing the solid substrate having the controlled amine density to take an important role in development of DNA chips and biochips.
• a film according to the present invention is stable at various pH levels and high temperatures, and the stability results from a multiple bond among 9 carboxylic acids and the substrate surface. Compared to a single bond and a 3-point bond that are not stable, a 9-point bond is highly stable.
• the film is used for fixation of desired molecules on a substrate, and for a surface substrate in studies of surface characteristics.

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• 2000
  • 2000-09-05 KR KR10-2000-0052504A patent/KR100383080B1/ko not_active Expired - Fee Related
• 2001
  • 2001-09-05 EP EP01967840A patent/EP1317422A4/en not_active Withdrawn
  • 2001-09-05 WO PCT/KR2001/001501 patent/WO2002020469A1/en not_active Ceased
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