Process for the functionalization of a surface

Abstract

A functionalization is performed with a dissolved oligo- or polysaccharide derivative which contains at least one free functional group, especially an amino group, linked through a polar carbamate linkage and a spacer (X), according to the general formula I: ##STR00001##

Claims

1. A process for the functionalization of a surface, which comprises at least one of synthetic polymers, natural polymers, paper, glass, ceramic, silicon, metals, or metal oxides, and is contacted, for surface functionalization, with a solution containing at least one compound for forming a material composite with the surface material, wherein as said at least one compound for forming the material composite, a dissolved oligo- or polysaccharide derivative is employed which has at least one free functional group R.sup.1 linked through a polar carbamate linkage and a spacer (X), according to the general formula I: ##STR00006## with R.sup.1NH.sub.2, SH or OH; and R.sup.2 and (independently) R.sup.3H or ##STR00007## and ##STR00008##

2. The process according to claim 1, wherein a hydroxy group is provided as at least one functional group R.sup.1.

3. The process according to claim 1, wherein a thiol group is provided as at least one functional group R.sup.1.

4. The process according to claim 1, wherein an amino group is provided as at least one functional group R.sup.1.

5. The process according to claim 1, wherein a homo- or heteroglycan is used as said oligo- or polysaccharide.

6. The process according to claim 1, wherein glucan, cellulose or chitin, is used as said oligo- or polysaccharide.

7. The process according to claim 1, wherein an at least bifunctional amino-substituted oligo- or polysaccharide with a functional group of general formula II is provided:
C(O)NH(X)NH.sub.2,(II) wherein X represents any organic moiety which is optionally substituted, or a moiety X as disclosed under (I).

8. The process according to claim 1, wherein the oligo- or polysaccharide has a cellulose skeleton of general formula III ##STR00009## wherein the hydroxy groups of cellulose are at least in part substituted by OC(O)NH(X)NH.sub.2, in which X represents any organic moiety an alkyl and/or alkenyl moiety, which is optionally substituted or includes a moiety X as disclosed under (1).

9. The process according to claim 6, wherein a cellulose with an average degree of polymerization (DP), based on its molecular mass, of from 30 to 1500 is employed as said oligo- or polysaccharide.

10. The process according to claim 1, wherein the natural polymers are selected from the group consisting of polysaccharides and proteins.

11. The process according to claim 6, wherein the glucan is -1-4-glucan.

12. The process according to claim 7, wherein the organic moiety is selected from the group consisting of an aromatic moiety, a condensed aromatic moiety, a heterocyclic moiety, a heteroaromatic moiety, an alkyl moiety, and an alkenyl moiety.

13. The process according to claim 8, wherein the organic moiety is selected from the group consisting of an aromatic moiety, a condensed aromatic moiety, a heterocyclic moiety, a heteroaromatic moiety, an alkyl moiety, and an alkenyl moiety.

14. The process according to claim 9, wherein the average degree of polymerization is within a range of from 50 to 300.

Description

EXAMPLE 1

(1) Synthesis of Cellulose Phenyl Carbonates:

(2) Cellulose (Avicel, DP 220, 5 g, 30.8 mmol) was treated in 150 ml of dry DMAc with stirring at 120 C. for 2 hours; subsequently, 9 g of LiCl (212.3 mmol) was added at 90 C. The suspension was stirred until a clear solution is obtained (5-24 hours). The cellulose solution was cooled down to 0 C. in a 250 ml double wall reactor, and equivalent amounts of pyridine and carbonic acid phenyl ester chloride were added under an N.sub.2 atmosphere. After a reaction time of 4 hours at 0 C., the mixture was precipitated in 1.5 liters of ice-water, the precipitate was filtered off and washed twice with 1 liter of water and twice with 1 liter of ethanol. The product was dried under vacuum at 40 C. and subsequently reprecipitated from 120 ml of acetone.

(3) TABLE-US-00001 AGU/carbonic acid phenyl ester chloride molar ratio No. DS Yield (%) 1.0:1.0 1 0.84 92 1.0:1.5 2 1.17 94 1.0:3.0 3 1.49 99 1.0:5.0 4 1.75 98 1.0:10.0 5 1.98 94

(4) Spectroscopic Data from Sample 1:

(5) FT-IR (KBr): 3460 cm.sup.1 (OH), 3070 cm.sup.1 (CH.sub.arom), 2900 cm.sup.1 (CH), 1766 cm.sup.1 (CO), 1254 cm.sup.1 (COC)

(6) .sup.1H NMR (250 MHz, DMSO-d.sub.6): [ppm]=7.38-7.25 (H.sub.arom), 5.60-3.16 (H-1-H-6 and OH)

(7) .sup.13C NMR (250 MHz, DMSO-d.sub.6): [ppm]=153.4 (CO), 151.2 (C.sub.ipso), 130.0 (CH.sub.m), 126.6 (CH.sub.p), 121.8 (CH.sub.o), 102.7 (C-1), 79.1, 74.3, 73.0, 72.3 (C-4, C-5, C-3, C-2), 67.7 (C-6.sub.s), 60.8 (C-6).

EXAMPLE 2

(8) Aminolysis of Cellulose Phenyl Carbonates:

(9) a) With N-Tert-Butoxycarbonyldiamine:

(10) Cellulose phenyl carbonate (2 g) in 15 ml of DMF was mixed with a solution of the corresponding amine (see below) in 15 ml of DMF with vigorous stirring, and the reaction mixture was stirred at 60 C. for 24 hours. Subsequently, the product was precipitated in 400 ml of water, filtered off, washed twice with water, twice with sodium hydrogencarbonate solution, and then again twice with water. After drying under vacuum at 40 C., a white powder was obtained.

(11) b) With p-Aminobenzylamine:

(12) Cellulose phenyl carbonate (1 g) in 8 ml of DMF was mixed with a solution of p-aminobenzylamine (2-3 equivalents per carbonate group) in 8 ml of DMF with vigorous stirring, and the reaction mixture was stirred at 60 C. for 24 hours. Subsequently, the product was precipitated in 150 ml of 2-propanol, filtered off, and washed four times with 2-propanol. After drying under vacuum at 40 C., a white powder was obtained.

(13) TABLE-US-00002 DS.sub.carbonate Amine No. DS DS yield (%) 1.49 N-Boc-EDA 6 1.35 91 1.75 N-Boc-EDA 7 1.75 100 1.75 N-Boc-BDA 8 1.71 98 1.75 N-Boc-DA-10 9 1.69 97 1.98 N-Boc-EDA 10 1.77 89 1.75 p-APA 11 1.64 94

(14) N-Boc-EDA: N-tert-butoxycarbonyl-1,2-ethanediamine, N-Boc-BDA: N-tert-butoxycarbonyl-1,4-butanediamine, N-Boc-DA-10: N-tert-butoxycarbonyl-2,2-(ethylenedioxy)diethylamine, p-ABA: p-aminobenzylamine

(15) Spectroscopic Data from Sample 7:

(16) FTIR (KBr): 1701 cm.sup.1 (.sub.CO), .sup.13C NMR (100 MHz, DMSO-d.sub.6): [ppm]=156 (CO), 102.9 (C-1), 101.1 (C-1), 78.3 (CMe.sub.3), 82-72 (C-2, C-3, C-4, C-5), 63.4 (C-6), CH.sub.2 hidden by the solvent signal, 28.7 (CH.sub.3).

EXAMPLE 3

(17) Deprotection of the Boc Protecting Group:

(18) Cellulose carbamate (2 g) was dissolved in 40 ml of TFA and subsequently stirred at room temperature for 15 min. The product was isolated by precipitation in 400 ml of 2-propanol, and then washed four times with 100 ml each of the precipitant. After drying under vacuum at 40 C., the product was dissolved in 50 ml of water and treated over night with the ion-exchanger Amberlite IRA-410 (chloride form). Subsequently, the solution obtained was freeze-dried.

(19) Spectroscopic Data for Aminoethyl Cellulose Carbamates:

(20) FTIR (KBr): 1710 cm.sup.1 (.sub.CO), .sup.13C NMR (100 MHz, D.sub.2O): [ppm]=158 (CO), 102.9 (C-1), 101.8 (C-1), 81-71 (C-2, C-3, C-4, C-5), 63.0 (C-6), 39.6 (CH.sub.2), 38.1 (CH.sub.2).

EXAMPLE 4

(21) Coating of Gold Surfaces:

(22) Gold-coated silicon wafers were placed into piranha solution (sulfuric acid/hydrogen peroxide, 2:1) for 3 hours before the coating in order to clean the surface. Subsequently, the supports were thoroughly rinsed with distilled water. The wafers were coated by means of a spin coater (MicroTec Delta 10TT, Sss) at 200 rotations per minute with a 1% aqueous solution of aminocellulose ethyl carbamate, DS 1.43, and subsequently rinsed with distilled water. The contact angle (water) of the coated surface is 61.

EXAMPLE 5

(23) Coating of a Gold Single Crystal:

(24) An Au(111) substrate was treated with concentrated sulfuric acid for 48 hours, washed with distilled water and absolute ethanol, dried in a nitrogen flow, and subsequently heated with a butane gas burner for 5-10 min until it glowed. Then, the surface was measured with an atomic force microscope (Dualscope C-21, DME), and subsequently treated with a 0.1% solution of aminocellulose ethyl carbamate, DS 1.43. Subsequently, it was intensively washed with water. The roughness (RMS, root mean square) of the surface increased from 80 pm (Au(111) substrate) to 260 pm by the functionalization, which demonstrates the coating.

EXAMPLE 6

(25) Quantification of the Coating of Gold by Means of QCM:

(26) The adsorption of aminoethylcellulose carbamate on gold surfaces was demonstrated with a quartz crystal microbalance (Q-Sence). The QCM substrates were cleaned with piranha solution before the coating, rinsed with distilled water, and subsequently dried in a nitrogen flow. The crystals were flooded by a solution of the aminoethylcellulose carbamate (concentrations of 0.001-1%) and subsequently washed with water until the equilibrium frequency was reached. For concentrations of 0.1%, an adsorbed mass of 200 ng per cm.sup.2 was detected.

EXAMPLE 7

(27) Coating of Glass:

(28) Glass supports (beads 1.5 mm, KGM Fulda) were cleaned with piranha solution before the coating and subsequently rinsed with distilled water. The glasses were immersed in an aqueous solution of 0.1% aminocellulose ethyl carbamate, DS 1.43, for 15 min, and subsequently washed with distilled water.

(29) To determine the surface density of amino groups, they were derivatized with 4,4-dimethoxytrityl chloride, treated with methanolic perchloric acid, and the concentration of dimethoxytrityl cations was measured by UV/Vis spectroscopy (498 nm). For aminoethylcellulose carbamate layers on glass, a density of amino groups of 0.6-0.8 nmol per cm.sup.2 was found.

EXAMPLE 8

(30) Coating of Polyethylene:

(31) Polyethylene supports were treated in an oxygen plasma and thereafter placed in an aqueous solution of 0.1% aminocellulose ethyl carbamate, DS 1.43, for 15 min. Subsequently, the supports were washed with distilled water. The concentration of amino groups was 0.30 nmol per cm.sup.2.

EXAMPLE 9

(32) Immobilization of Anti-h CRP Antibodies (Clone 6404, Medix) on NH.sub.2-Functionalized Supports with the Homobifunctional Reagent Glutardialdehyde:

(33) 1. Activation of the NH.sub.2-Functionalized Surfaces by Glutardialdehyde:

(34) Ten NH.sub.2-functionalized PE microfilters (h=2.5 mm, =5 mm, type 180, Porex) were stirred with 2 ml of a 5% aqueous glutardialdehyde solution at room temperature for 15 minutes. After four washes with 3 ml each of water, the microfilters were available for immobilization.

(35) 2. Immobilization of Anti-h CRP Antibodies (Clone 6404, Medix) on the Glutaraldehyde-Activated Surfaces:

(36) To the PE microfilters treated as described under 1., 75 g of anti-h CRP antibodies (clone 6404, Medix) in 2 ml of bicarbonate buffer (0.1 M, pH 9.5) was added, and stirred at room temperature for four hours. To block the remaining activated surface, 5 l of ethanolamine was added to the immobilization solution after the immobilization, and stirring was continued for another two hours. After four washes with 3 ml each of water and subsequent drying in an air flow, CRP-affine surfaces were obtained. 6.0 g per microfilter was successfully immobilized, wherein 30% of the immobilized antibodies were active.

EXAMPLE 10

(37) Immobilization of Anti-h CRP Antibodies (Clone 6404, Medix) on COOH-Functionalized Supports by Means of EDC/s-NHS Chemistry:

(38) 1. Refunctionalization of the NH.sub.2-Functionalized Surfaces and Subsequent Activation of the COOH-Functionalized Surfaces:

(39) Ten NH.sub.2-functionalized PE microfilters were stirred in 2 ml of a solution of succinic anhydride (c=40 mg/ml) and 5 l of pyridine in N,N-dimethylformamide at room temperature for 16 hours. Carboxy groups were introduced on the PE microfilters by the reaction of the succinic anhydride with the amino groups of the surface. After repeated washes of the surfaces with 3 ml each of water, the carboxy groups could be activated by 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC) and sulfo-N-hydroxysuccinimide (s-NHS). Thus, the microfilters were stirred in 2 ml of a 2-(N-morpholino)ethanesulfonic acid buffered solution (0.1 M, pH 5.5) with EDC (2 mM) and s-NHS (5 mM), and stirred for 30 minutes.

(40) 2. Immobilization of Anti-h CRP Antibodies (Clone 6404, Medix) on the Activated COOH Surfaces:

(41) The immobilization of anti-h CRP antibodies (clone 6404, Medix) was effected after washing with water the microfilters treated as described under 1, followed by the addition of 75 g of anti-h CRP antibodies (clone 6404, Medix) in 2 ml of phosphate buffers (0.1 M, pH 7.4) and stirring at room temperature for two hours. The remaining activated carboxy groups were blocked by adding 5 l of ethanolamine to the immobilization solution and stirring for another 60 minutes. After four washes with 3 ml each of water and subsequent drying in an air flow, CRP-affine surfaces were obtained. 7.0 g per microfilter was successfully immobilized, wherein 30% of the immobilized antibodies were active.

EXAMPLE 11

(42) Immobilization of Anti-h CRP Antibodies (Clone 6404, Medix) on NH.sub.2-Functionalized Supports with the Reagent Ascorbic Acid:

(43) 1. Activation of the NH.sub.2-Functionalized Surfaces by Ascorbic Acid:

(44) Ten NH.sub.2-functionalized PE microfilters (h=2.5 mm, =5 mm, type 180, Porex) were stirred with 2 ml of a saturated solution of ascorbic acid in N,N-dimethylformamide at room temperature for 15 minutes. After four washes with 3 ml each of water, the microfilters were available for immobilization.

(45) 2. Immobilization of Anti-h CRP Antibodies (Clone 6404, Medix) on the Ascorbic Acid-Activated Surfaces:

(46) To the PE microfilters treated as described under 1., 75 g of anti-h CRP antibodies (clone 6404, Medix) in 2 ml of bicarbonate buffer (0.1 M, pH 9.5) was added, and stirred at room temperature for 16 hours. To block the remaining activated surface, 5 l of ethanolamine was added to the immobilization solution after the immobilization, and stirring was continued for another two hours. After four washes with 3 ml each of water and subsequent drying in an air flow, CRP-affine surfaces were obtained. 5.2 g per microfilter was successfully immobilized, wherein 35% of the immobilized antibodies were active.

EXAMPLE 12

(47) Immobilization of Anti-h CRP Antibodies (Clone 6404, Medix) on NH.sub.2-Functionalized Supports with the Reagent Benzoquinone:

(48) 1. Activation of the NH.sub.2-Functionalized Surfaces by Benzoquinone:

(49) Ten NH.sub.2-functionalized PE microfilters (h=2.5 mm, =5 mm, type 180, Porex) were stirred with 2 ml of a saturated solution of benzoquinone in N,N-dimethylformamide at room temperature for 15 minutes. After four washes with 3 ml each of DMF and then twice with water, the microfilters were available for immobilization.

(50) 2. Immobilization of Anti-h CRP Antibodies (Clone 6404, Medix) on the Benzoquinone-Activated Surfaces:

(51) To the PE microfilters treated as described under 1., 75 g of anti-h CRP antibodies (clone 6404, Medix) in 2 ml of bicarbonate buffer (0.1 M, pH 9.5) was added, and stirred at room temperature for 16 hours. To block the remaining activated surface, 5 l of ethanolamine was added to the immobilization solution after the immobilization, and stirring was continued for another two hours. After four washes with 3 ml each of water and subsequent drying in an air flow, CRP-affine surfaces were obtained. 6.0 g per microfilter was successfully immobilized, wherein 36% of the immobilized antibodies were active.

EXAMPLE 13

(52) Immobilization of anti-h CRP antibodies (clone 6404, Medix) on NH.sub.2-functionalized supports with the reagent 4,4-dihydroxybiphenyl diglycidyl ether:

(53) 1. Activation of the NH.sub.2-functionalized surfaces by 4,4-dihydroxybiphenyl diglycidyl ether:

(54) Ten NH.sub.2-functionalized PE microfilters (h=2.5 mm, =5 mm, type 180, Porex) were stirred with 2 ml of a 5% aqueous solution of 4,4-dihydroxybiphenyl diglycidyl ether at room temperature for 15 minutes. After four washes with 3 ml each of water, the microfilters were available for immobilization.

(55) 2. Immobilization of anti-h CRP antibodies (clone 6404, Medix) on the 4,4-dihydroxybiphenyl diglycidyl ether activated surfaces:

(56) To the PE microfilters treated as described under 1., 75 g of anti-h CRP antibodies (clone 6404, Medix) in 2 ml of bicarbonate buffer (0.1 M, pH 9.5) was added, and stirred at room temperature for 16 hours. To block the remaining activated surface, 5 l of ethanolamine was added to the immobilization solution after the immobilization, and stirring was continued for another two hours. After four washes with 3 ml each of water and subsequent drying in an air flow, CRP-affine surfaces were obtained. 5.5 g per microfilter was successfully immobilized, wherein 35% of the immobilized antibodies were active.

EXAMPLE 14

(57) Immobilization of Anti-h CRP Antibodies (Clone 6404, Medix) on COOH-Functionalized Supports by Adhesive Interactions:

(58) 1. Refunctionalization of the NH.sub.2-Functionalized Surfaces for Introducing Carboxy Groups on the Surface:

(59) Ten NH.sub.2-functionalized PE microfilters were stirred in 2 ml of a solution of succinic anhydride (c=40 mg/ml) and 5 l of pyridine in N,N-dimethylformamide at room temperature for 16 hours. Carboxy groups were introduced on the PE microfilters by the reaction of the succinic anhydride with the amino groups of the surface.

(60) 2. Immobilization of Anti-h CRP Antibodies (Clone 6404, Medix) on the COOH-Functionalized Surfaces:

(61) The immobilization of anti-h CRP antibodies (clone 6404, Medix) was effected after washing with water the microfilters treated as described under 1, followed by the addition of 75 g of anti-h CRP antibodies (clone 6404, Medix) in 2 ml of a 2-(N-morpholino)ethanesulfonic acid buffered solution (0.1 M, pH 7.4) and stirring at room temperature for two hours. The remaining activated carboxy groups were blocked by adding 10 l of a 10% aqueous bovine serum albumin (BSA) solution to the immobilization solution and stirring for another 60 minutes. After four washes with 3 ml each of water and subsequent drying in an air flow, CRP-affine surfaces were obtained. 5.9 g per microfilter was successfully immobilized, wherein 15% of the immobilized antibodies were active.

EXAMPLE 15

(62) Immobilization of Anti-h CRP Antibodies (Clone 6404, Medix) on SO.sub.3H-Functionalized Supports by Adhesive Interactions:

(63) 1. Refunctionalization of the NH.sub.2-Functionalized Surfaces for Introducing Sulfonic Acid Groups on the Surface:

(64) Ten NH.sub.2-functionalized PE microfilters were stirred in 2 ml of a saturated solution of 4,4-biphenyldisulfonic acid dichloride in dry diethyl ether at room temperature for 15 minutes. Sulfonic acid chloride groups were introduced on the PE microfilters by the reaction of the 4,4-biphenyldisulfonic acid dichloride with the amino groups of the surface. These amino groups were hydrolyzed by treatment with 2 ml of a 0.1 M hydrochloric acid over night to obtain sulfonic acid groups on the surface.

(65) 2. Immobilization of Anti-h CRP Antibodies (Clone 6404, Medix) on the SO.sub.3H-Functionalized Surfaces:

(66) The immobilization of anti-h CRP antibodies (clone 6404, Medix) was effected after washing with water the microfilters treated as described under 1, followed by the addition of 75 g of anti-h CRP antibodies (clone 6404, Medix) in 2 ml of a 2-(N-morpholino)ethanesulfonic acid buffered solution (0.1 M, pH 7.4) and stirring at room temperature for two hours. The remaining activated carboxy groups were blocked by adding 10 l of a 10% aqueous bovine serum albumin (BSA) solution to the immobilization solution and stirring for another 60 minutes. After four washes with 3 ml each of water and subsequent drying in an air flow, CRP-affine surfaces were obtained. 5.8 g per microfilter was successfully immobilized, wherein 15% of the immobilized antibodies were active.