Title of Invention

A PROSESS FOR THE PREPARATION OF HETEROBIFUNCTIONAL CROSSLINKING REAGENTS FOR THE IMMOBILIZATION OF BIOMOLECULES

Abstract

The present invention relates to a process for the preparation of heterobifunctional crosslinking reagents for the immobilization of biomolecules on the unmodified surface containing silanol functionalities, such as glass surfaces, without affecting the activity of the biomolecules. The reagents consist of two different chemically reactive groups; one reacts selectively with unmodified glass surface and the other one with nucleophilic groups present in the biomolecules to form a covalent linkage between the two. The use of these reagents makes the immobilization process a rapid and economical. S-CH2CH2CH2N(CH3)CH2CH2O-R I

Full Text

The present invention provides a process for the preparation of heterobifunctional crosslinking reagents of formula I for the immobilization of biomolecules. The reagents consist of two different chemically reactive groups, one reacts selectively with unmodified surfaces with silanol functionalities and the other one with nucleophilic groups present in the biomolecules (biomolecular ligand molecules). The basic purpose of these reagents is to form a covalent linkage between two different species. More precisely, the utility of this invention is to immobilize a variety of bioactive ligands on the unmodified glass surface. Hetero- and homobifunctional reagents have been finding a widespread use in the area of genomics and proteomics (Kreig et al., Proc. Natl. Acad. Sci. USA, 83 (1986) 8604; Hermanson, Bioconjugate Techniques, San Diego, Academic Press (1996) 284). These reagents are organic molecules which have two reactive functional groups either similar or identical (homobifunctional reagents) or different (heterobifunctional reagents). The functional groups react with nucleophilic groups (aminoalkyl- and mercaptoalkyl-) of the bioactive ligands. The reaction can occur either intramolecularly or intermolecularly. Simultaneously, bridges within a molecule or heteromolecular complexes can be formed. The general purpose of these reagents is to serve as molecular tools to attach biomolecules on the surface for various types of analytical and biological applications. The molecules, which can be attached through homo- and heterobifunctional reagents, are proteins, peptides, oligonucleotides, enzymes, polysaccharides, etc. These immobilized biomolecules can be used to analyze antigen-antibody interactions, structure of multi-enzyme complexes, the arrangement of proteins in membranes and many other biological applications. Microarray technology is one of the most recent and important applications of synthetic oligonucleotides (Podyminogin et al., Nucl. Acids Res., 29 (2001) 5090; Rehman et al., Nucl. Acids Res., 27 (1999) 649; Zhao et al., 29 (2001) 955; Timofeev et al., Nucl. Acids Res., 24 (1996) 3142). DNA microarray is a powerful tool, which allows the simultaneous detection of many different target molecules present in a sample. The technology deals with the covalent fixing of oligonucleotides (small fragments of DNA), cDNA, PCR fragments, etc on the solid or polymer surfaces. These DNA chips or biochips can be used for mutation detection, SNP analysis, disease diagnosis, monitoring of gene expression, etc. A number of methods have reported for the preparation of oligonucleotide arrays using homo- and heterobifunctional reagents. Immobilization can be accomplished in two ways, viz., (i) the reaction occurs between the reagent and suitably modified oligonucleotides followed by reaction with functional groups on the solid surface to obtain immobilized oligonucleotides, and (ii) the method involves reaction of the reagent with functional groups on the polymeric surface followed by reaction with suitably modified oligonucleotides to obtain immobilized oligonucleotides. In general, oligonucleotide arrays are solid surfaces consisting of hundreds or thousands of oligomers covalently attached at discrete locations, which are available for hybridization. Currently, two methods are being followed for the construction of oligonucleotide arrays. The first one involves direct synthesis of oligonucleotides at the pre-determined sites on the solid or polymeric surfaces using photolithographic technique (Fodor et al.. Science 251 (1991) 767). The second method is based on the immobilization of pre-synthesized oligonucleotides on the solid or polymeric surfaces using a suitable hetero- or homobifunctional reagent or a coupling reagent (Beier and Hoheisel, Nucl. Acids Res., 27 (1999) 1970). The method provides flexibility in the sense that it does not require expensive chemistry and sophisticated instrumentation. Modifications can easily be incorporated in the oligomers according to the functionalities present on the solid surface. This method is preferred for generating low to moderate density oligonucleotide arrays. Generally, linking units are added at 3'-end, internal positions or 5'-end. For hybridization assays, the position of attachment is irrelevant. Several surface materials have been tested so far, such as nylon, nitrocellulose, polypropylene, polystyrene, silicon, glass. Teflon, etc. Out of these, glass and polypropylene stand a good chance because these materials can easily be derivatized to generate functional groups on the surface, viz., aminoalkyl, carboxyl, aldehyde, mercaptoalkyl, etc. Moreover, glass slides are readily available, inexpensive and possess a relatively homogeneous chemical surface. Glass has an additional advantage in that the currently used laser scanners can also be used. A number of methods have been reported for the immobilization of oligonucleotides on the polymeric surfaces. In one approach, Beattie et al. (Mol. Biotech., 1995, 4, 213) have employed 3-glycidyloxypropyltriethoxysilane as a heterobifunctional reagent to immobilize aminoalkylated oligonucleotides. They generated electrophilic surfaces (epoxy groups) by reacting the unmodified glass slides with the reagent, 3-glycidyloxypropyltriethoxysilane, followed by spotting of the aminoalkylated oligonucleotides. Similarly, in another approach, mercaptoalkylated oligonucleotides were conjugated to 3-mercaptopropyltriethoxysilane via disulfide formation (Kumar et al.. Nucleic Acids Res., 28 (2000) e71). Subsequently, these silylated oligonucleotides were spotted on the virgin glass slide to obtain immobilized oligonucleotides. Further, they used these slides for hybridization experiments using complementary oligomers labeled with cy3 and cy5 fluorescent dyes. Some of the methods for immobilization of biomolecules have been reported using nitrenes and carbenes as reactive species generated upon UV irradiation. However, these species are extremely reactive and sometimes immediately react with water or organic compounds, organic solvents, etc. This process results in the loss of photoactivity. Recently, Koch et al. (Bioconjugate Chem., 11 (2000) 474) have demonstrated that anthraquinone linked oligonucleotides can be immobilized at any carbon-containing surfaces with active hydrogen atoms. They have synthesized a phosphoramidite reagent of the anthraquinone linker and finally coupled to oligomer chain after synthesis of a desired oligomer. Anthraquinone-oligonucleotide conjugates were then immobilized on polystyrene plates. The same conjugates can be immobilized on other polymeric surfaces, e.g. polycarbonates, polypropylene, polyethylene, etc. Photochemically immobilization of oligonucleotides appears to be superior to currently known chemical procedures. Since the above mentioned methods are applicable to immobilize ligands on polymeric or unmodified glass surfaces, it was then decided to develop heterobifunctional reagents of formula I, which could be used for immobilizing a variety of biomolecules, such as oligonucleotides, peptides, enzymes, proteins etc. on the surface of choice. The novelty of the present invention is in providing novel heterobifunctional reagents of formula I, where S stands for a chemical group, which can react with unmodified glass surfaces containing silanol functionalities and R stands for a chemically reactive group, which can react with the nucleophilic groups present in the bioactive ligands, such as oligonucleotides, peptides, enzymes, proteins, etc. Therefore, the proposed reagents can easily be used for the covalent fixing of the biomolecules on the commercially available unmodified glass surfaces containing silanol groups. Further, the use of these reagents makes the immobilization process a rapid and economical. (Formula Removed) The main objective of the present invention is, therefore, to provide novel heterobifuctional reagents suitable for attaching biomolecules on to the unmodified glass surfaces. Another objective of the present invention is to provide a rapid silanization method for the immobilization of a desired biomolecular ligand molecules on unmodified glass surfaces without any side product formation. Yet another objective is to provide a silanization method of fixing biomolecules on the glass surface without affecting the activity of the biomolecules. Accordingly, present invention provides a process for the preparation of heterobifunctional crosslinking reagents of formula I for the immobilization of biomolecules, which comprises: (i) reacting a silylating reagent such as herein described with N-methyl-2-aminoethanol in dry toluene at a temperature in the range of 35°C to 75°C to obtain N-(2-hydroxyethyl)-N(methyl)-trialkoxysilylpropy 1-3-amine; (ii) reacting N-(2-hydroxyethyl)-N(methyl)-trialkoxysilylpropyl-3-amine obtained in step (i) with a sulfonate ester in an organic solvent at ambient temperature for 2h followed by purification to obtain heterobifunctional reagent; (iii) reacting the heterobifunctional reagent obtained in step (ii) with the nucleophilic groups present in the biomolecules selected from enzymes, functional group bearing oligonucleotides, proteins, peptides, PNA, followed by concentration to obtain the residue; (iv) dissolving the residue obtained in step (iii) in an aqueous solution and spotting the solution on an untreated surface material and keeping the spotted surface material in a moist chamber at a temperature in the range of 50°C to 70°C for 15 to 30 min; (v) washing of the surface material incubated in step (iv) with buffer such as Phosphate buffer saline. In an embodiment to the present invention, the silylating reagent may be selected from 3- chloropropyltriethoxysilane, 3-bromopropyltrimethoxysilane, 3-iodopropyltriethoxysilane, 3- bromopropyltriethoxysilane, 3-chloropropyltrimethoxysilane, 3-iodopropyltrimethoxysilane, 3- chloropropyldimethylethoxysilane, 3-bromopropylmethyldiethoxysilane, 3- chloropropylmethyldimethoxysilane, 3-bromopropyldimethylmethoxysilane and the like. In yet another embodiment, the sulfonate esters may be selected from trifluoroethanesulfonyl, trifluoromethanesulfonyl, toluenesulfonyl, mesitylenesulfonyi and the like for the activation of hydroxyl group. In still another embodiment, the non-polar organic solvent may be selected from benzene, toluene, xylene and the like. In another embodiment, the surface material may be selected from glass, silicon wafers, silicon based optical fibres and the like. In yet another embodiment, the temperature required for the reaction may be in the range of 35° to 75°C. In another embodiment, the reaction time required for immobilization may be in the range of 10 min to 180min. In still another embodiment, the biomolecular ligand molecules may be selected from a group of enzymes, nucleophilic group bearing oligonucleotides, proteins, peptides, PNA and the like. Details of the invention The invention discloses a process for novel heterobifunctional crosslinking reagents of formula I suitable for attaching biomolecular ligand molecules or biomolecules on to the unmodified glass surfaces, which comprises : Preparation of N-(2-hvdroxvethvl)-N-(methvntriethoxvsilvlpropvl-3-amine : 3- Chloropropyltriethoxysilane (0.3 mol) and diisopropylethylamine (0.1 mol) was mixed together in dry toluene (70 ml) and heated in a oil bath to 70°C. In the hot solution, N-methyl-2-aminoethanol (0.1 mol) was added in one portion. Then the mixture was refluxed for 10h under N2 atmosphere and cooled to 4°C. After 16h, the solid, diisopropylethylamine hydrochloride, was removed by filtration. The filtrate was concentrated at reduced pressure and distilled under vacuum to obtain N-(2-hydroxyethyl)-N-(methyl)triethoxysilylpropyl-3-amine in 58% yield. Preparation of reagent [N-aryl or alkvlsulfonvloxvethvl)-N(methvl)triethoxvsilvlpropvl-3-amine1: N-(2-Hydroxyethyl)-N-(methyl)triethoxysilylpropyl-3-amine (5 mmol) was taken up in dry dichloromethane (20 ml) and triethylamine (5.5 mmol). After cooling the reaction mixture to 0-4°C, aryl- or alkylsulfonyl halide was added slowly. The reaction mixture was stirred at ambient temperature for 2h and then monitored on thin layer chromatography (TLC). The precipitated triethylamine hydrochloride was removed by filtration and solvent was removed under reduced pressure to obtain to obtain, [N-aryl or alkylsulfonyloxyethyl)-N(methyl)triethoxysilylpropyl-3-amine] in almost quantitative yield. Reaction with nucleophilic group bearing biomolecular ligand molecules : The nucleophilic groups bearing biomolecular ligand molecules were dissolved in an appropriate buffer, such as sodium acetate, potassium acetate, sodium phosphate, and mixed with the reagent, as obtained above. dissolved in a polar solvent, such as methanol, ethanol, propanol, etc. After 60 min, the reaction mixture was concentrated and redissolved in ddH2O. Immobilization of biomolecular ligand molecules : The resulting solution, as obtained in the above step, was spotted manually (0.5 µ1) on a glass slide. Then the plate was kept in a moist chamber at 50°C for 30 min followed by washing and the resulting spots of the immobilized ligand molecules were visualized by known methods. The following examples concerning the preparation of silyl group based silanization heterobifunctional reagents and their application for immobilizing biomolecules or biomolecular ligand molecules on the silica based surfaces are provided to illustrate the invention and should not be construed to limit the scope of the invention. Example 1 (a) Preparation of N-(2-hydroxyethyl)-N-(methyl)triethoxysilylpropyl-3-amine To a solution of anhydrous N-methyl-2-aminoethanol (0.3 mol) and diisopropylethylamine (0.1 mol) in hot toluene (70 ml) was added 3-chloropropyltriethoxysilane (0.1 mol) in one portion. The mixture was refluxed for l0h under inert atmosphere followed by cooling at 4°C for 16h. The amine-hydrochloride was removed by filtration and the residue washed with dry toluene. The combined filtrates were pooled together and concentrated under vacuum. The residual syrupy mixture was distilled under argon to obtain the title compound in 58% yield. (b) Preparation of N-(2-trifluoroethylsulfonyloxyethyl)-N-(methyl)triethoxysilylpropyI-3-amine To the distilled compound, N-(2-hydroxyethyl)-N-(methyl)triethoxysilylpropyl-3-amine, (5 mmol) in dichloromethane (20 ml) and triethylamine (5.5 mmol) was added trifluoroethanesulfonyl chloride (tresyl chloride) (6.0 mmol) dropwise at 0°C. The reaction was allowed to stir at room temperature for 2h. Then the amine hydrochloride was removed by filtration and the filtrate was concentrated in vacuum to syrupy mass. (c) Preparation of N-(2-hydroxyethyl)-N-(methyl)trimethoxysilylpropyl-3-amine To a solution of anhydrous N-methyl-2-aminoethanol (0.3 mol) and diisopropylethylamine (0.1 mol) in hot toluene (70 ml) was added 3-chloropropyltrimethoxysilane (0.1 mol) in one portion. The mixture was refluxed for 10h under inert atmosphere followed by cooling at 4°C for 16h. The amine-hydrochloride was removed by filtration and the residue washed with dry toluene. The combined filtrates were pooled together and concentrated under vacuum. The residual syrupy mixture was distilled under argon to obtain the title compound in 54% yield. (d) Preparation of N-(2-toluenesulfonyloxyethyl)-N-(methyl)trimethoxysilylpropyl-3-amine To the distilled compound, N-(2-hydroxyethyl)-N-(methyl)trimethoxysilylpropyl-3-amine, (5 mmol) in pyridine (20 ml) was added toluenesulfonyl chloride (tosyl chloride) (6.0 mmol) in one portion. The reaction was allowed to stir at room temperature for 4h. Then the solution was concentrated and redissolved in ethyl acetate and washed with 5% aq. sodium bicarbonate solution. The organic layer was separated and concentrated in vacuum to obtain the title compound in almost quantitative yield as a syrupy mass. Example 2 Covalent coupling of oligonucleotides onto glass slide : 5'-Aminopentyl-oligonucleotide [d(H2N—TTT TTT TTT TTT TTT TTT TT)] (2.0 O.D. at 260 nm) was dissolved in 100 mM sodium acetate buffer, pH 7.0 (50 µl). N-(2-Trifluoroethylsulfonyloxyethyl)-N-(methyl)triethoxysilylpropyl-3-amine (100 µl of 5% solution in ethanol) was added to the oligonucleotide solution and the reaction mixture was kept on a agitator for 2h. After stipulated time, the reaction mixture was concentrated and the residue obtained was redissolved in water and the suspension was centrifuged to remove the supernatant (10 µM). Oligonucleotide was spotted manually (0.5 µl) in triplicate on the glass slide and kept in a moist chamber at 60°C for 30 min. Then the slide was washed and the spots were hybridized with complementary oligomer d(Fluorescein—AAA AAA AAA AAA AAA AAA AA). After incubation at 35°C for 4h, the film was washed with 0.1M phosphate buffer containing sodium chloride (0.5M). Then the spots were visualized under laser scanner. Example 3 Covalent coupling of oligonucleotides onto glass slide : 5'-Mercaptohexyl-oligonucleotide [d(HS—TTT TTT TTT TTT TTT TTT TT)] (2.0 O.D. at 260 nm) was dissolved in 100 mM sodium acetate buffer (50 µl). N-(2-trifluoroethylsulfonyloxyethyl)-N-(methyl)triethoxysilylpropyl-3-amine (100 µl of 5% solution in ethanol) was added to the oligonucleotide solution and the reaction mixture was kept on a agitator for 2h. After stipulated time, the reaction mixture was concentrated and the residue obtained was redissolved in water and the suspension was centrifuged to remove the supernatant (10 µM). Oligonucleotide was spotted manually (0.5µl) in triplicate on the glass slide and kept in a moist chamber at 60°C for 15 min. Then the slide was washed and the spots were hybridized with complementary oligomer d(Fluorescein—AAA AAA AAA AAA AAA AAA AA). After incubation at 35°C for 4h, the film was washed with 0.1 M phosphate buffer containing sodium chloride (0.5M). Then the spots were visualized under laser scanner. Example 4 Covalent coupling of alkaline phosphatase onto silicon wafer : Alkaline phosphatase (1 mg) was dissolved in 50 mM sodium phosphate buffer (50 µl). N-(2-toluenesulfonyloxyethyl)-N-(methyl)trimethoxysilylpropyl-3-amine (100 |J.1 of 5% solution in ethanol) was added to the enzyme solution and the reaction mixture was kept on a agitator for 1.5h. After stipulated time, the reaction mixture was concentrated and the residue redissolved in water. The suspension was centrifuged and the supernatant collected. The conjugate solution was coated manually on a sheet of silicon wafer. The sheet was kept at room temperature for 3h and then it was washed. Subsequently, the sheet was treated with 10 mM solution of p-nitrophenylphosphate (pNPP). After incubation for 1h, the absorbance of the solution was measured at 410 nm. Example 5 Covalent coupling of PNA onto glass slide : A PNA oligomer, [H-TTT TTT TT-CONH2] (2.1 O.D. at 260 nm) was dissolved in 100 mM sodium acetate buffer (50 µl). N-(2-trifluoroethylsulfonyloxyethyl)-N-(methyl)triethoxysilylpropyl-3-amine (100 µl of 5% solution in ethanol) was added to the oligonucleotide solution and the reaction mixture was kept on a agitator for 2h. After stipulated time, the reaction mixture was concentrated and the residue obtained was redissolved in water and the suspension was centrifuged to remove the supernatant (10 µM). Oligonucleotide was spotted manually (0.5 µ1) in triplicate on the glass slide and kept in a moist chamber at 70°C for 15 min. Then the slide was washed and the spots were hybridized with complementary oligomer d(Fluorescein—AAA AAA AAA AAA AAA AAA AA). After incubation at 35°C for 4h, the film was washed with O.IM phosphate buffer containing sodium chloride (0.5M). Then the spots were visualized under laser scanner. Advantages of the invention 1. The method for the preparation is simple and rapid. 2. The reagent is sufficiently stable on storage at 4°C. 3. The reactions involving silylation are clean and do not result in any side product formation. 4. The reagents do not react with aqueous medium (e.g. water). 5. The reagents are highly specific for amino- and mercaptoalkylated ligands. 6. A variety of bioactive ligands can be immobilized without affecting their activity. 7. The reagents can be used in both the ways, i.e. silylation of the surface followed by reaction with the biomolecule and / or reaction of the reagents with biomolecules followed by silanization on the glass surface. We claim: 1. A process for the preparation of heterobifunctional crosslinking reagents of formula I described herein (Formula Removed) for the immobilization of biomolecules, which comprises: (i) reacting a silylating reagent such as herein described with N-methyl-2-aminoethanol in dry toluene at a temperature in the range of 35°C to 75°C to obtain N-(2-hydroxyethyl)-N(methyl)-trialkoxysilylpropyl-3-amine; (ii) reacting N-(2-hydroxyethyl)-N(methyl)-trialkoxysilylpropyl-3-amine obtained in step (i) with a sulfonate ester in an organic solvent at ambient temperature for 2h followed by purification to obtain heterobifunctional reagent; (iii) reacting the heterobifunctional reagent obtained in step (ii) with the nucleophilic groups present in the biomolecules selected from enzymes, functional group bearing oligonucleotides, proteins, peptides, PNA, followed by concentration to obtain the residue; (iv) dissolving the residue obtained in step (iii) in an aqueous solution and spotting the solution on an untreated surface material and keeping the spotted surface material in a moist chamber at a temperature in the range of 50°C to 70°C for 15 to 30 min; (v) washing of the surface material incubated in step (iv) with buffer such as Phosphate buffer saline. 2. A process as claimed in claim 1 wherein the silylating reagent is selected from 3- chloropropyltriethoxysilane, 3-bromopropyltrimethoxysilane, 3-iodopropyltriethoxysilane, 3- bromopropyltriethoxysilane, 3-chloropropyltrimethoxysilane, 3-iodopropyltrimethoxysilane, 3- chloropropyldimethylethoxysilane, 3-bromopropylmethyldiethoxysilane, 3- chloropropylmethyldimethoxysilane, 3-bromopropyldimethylmethoxysilane. 3. A process as claimed in claim 1, wherein the sulfonate ester for the activation of hydroxy! group is selected from trifluoroethanesulfonyl, trifluoromethanesulfonyl, toluenesulfonyl, mesitylenesulfonyl. 4. A process as claimed in claim 1, wherein the surface material is selected from glass, silicon wafers, silicon based optical fibres. 5. A process as claimed in claim 1, wherein the temperature for the reaction is preferably at 70°C. 6. A simple and rapid process for the preparation of heterobifunctional reagents for the immobilization of biomolecules substantially as described herein with reference to the examples.

Documents:

342-DEL-2003-Abstract-(22-10-2008).pdf

342-del-2003-abstract.pdf

342-DEL-2003-Claims-(22-10-2008).pdf

342-del-2003-claims.pdf

342-DEL-2003-Correspondence-Others-(22-10-2008).pdf

342-del-2003-correspondence-others.pdf

342-del-2003-correspondence-po.pdf

342-DEL-2003-Description (Complete)-(22-10-2008).pdf

342-del-2003-description (complete).pdf

342-DEL-2003-Form-1-(22-10-2008).pdf

342-del-2003-form-1.pdf

342-del-2003-form-18-(22-10-2008).pdf

342-del-2003-form-18.pdf

342-DEL-2003-Form-2-(22-10-2008).pdf

342-del-2003-form-2.pdf

342-del-2003-form-3.pdf