Silane compounds and use of same for functionalizing solid supports and immobilizing biological molecules on these supports

Abstract

The invention relates to novel silane compounds corresponding to the formula (I) below:
A-E-X (I)
in which: X represents a silyl group capable of creating a covalent bond after reaction with the hydroxyl or hydride functional groups of a support; E represents an organic spacer group; A represents a group chosen from the groups of formulae below: ##STR00001##
in which: Z.sub.1 to Z.sub.5 independently represent a hydrogen atom or a halogen atom; Z.sub.6 and Z.sub.7 represent a group for protecting the phosphonic acid functional group, a hydrogen atom or a monovalent cation; Z.sub.8 to Z.sub.12 independently represent a group for protecting the carboxylic acid functional group, a hydrogen atom or a monovalent cation; and Z.sub.13 represents an imidazole, N-hydroxysuccinimide, nitrophenyl, pentafluorophenyl or acid anhydride group. Use of these silane compounds for functionalizing solid supports and for immobilizing biological molecules on these supports.

Claims

1. A compound of formula (II) below: ##STR00039##

Description

SUMMARY OF PARTICULAR EMBODIMENTS

Example 1

(1) This example illustrates the preparation of a silane compound conforming to the invention: 11-pentafluorophenyl ether undecyltrimethoxysilane according to the following reaction scheme:

(2) ##STR00014##

(3) The pentafluorophenyl ether functional group is obtained by reaction of 11-bromoundecene with pentafluorophenol in the presence of potassium carbonate. Next, the incorporation of the silyl group was carried out by a hydrosilylation reaction in the presence of a Karstedt catalyst.

(4) This example also illustrates the silanization of a silicon support by the silane compound (II).

a) Step 1: Synthesis of 11-pentafluorophenyl ether undec-1-ene

(5) ##STR00015##

(6) Added to a solution of pentafluorophenol (3.77 g; 21 mmol; 1 eq.) dissolved in 75 ml of DMF, were 11-bromoundecene (95%) (5.02 g; 4.7 ml; 21 mmol) and potassium carbonate (2.83 g; 21 mmol; 1 eq.). The reaction was carried out under reflux for 3 hours. After evaporating the DMF and taking up in dichloromethane, the reaction mixture was successively washed with distilled water (two times) and with a saturated sodium chloride solution, dried over anhydrous magnesium sulphate then concentrated to give a colourless liquid.

(7) The characteristics of the product obtained were the following:

(8) Mass obtained: 5.79 g

(9) Yield: 84%

(10) .sup.1H NMR (200 MHz; CDCl.sub.3): 1.35 (12H; m; H.sup.914); 1.79 (2H; m; H.sup.8); 2.08 (2H; m; H.sup.15); 4.19 (2H; t; H.sup.7; .sup.3J.sub.HH=6.5 Hz); 5 (2H; m; H.sup.17); 5.86 (1H; m; H.sup.16)

(11) .sup.13C NMR (200 MHz; CDCl.sub.3): 25.16; 29.23; 29.28; 29.42; 29.49; 29.63; 33.72; 34.18; 76.16 (t; C.sup.7; .sup.3J.sub.CC=3.4 Hz); 114.55 (C.sup.17); 134.46 (C.sup.4); 138.22 (2c; C.sup.3+5; .sup.3J.sub.CF=259 Hz); 139.51 (C.sup.16); 139.75 (C.sup.1); 142.38 (2c; C.sup.2+6; .sup.3J.sub.CF=243 Hz).

b) Step 2: Synthesis of 11-pentafluorophenyl ether undecyltrimethoxysilane (II)

(12) ##STR00016##

(13) The pentafluorophenyl ether undec-1-ene (5.79 g; 17 mmol) was mixed with trimethoxysilane (90%) (3 g; 3.1 ml; 22 mmol; 1.3 eq.). The Karstedt catalyst (0.04 g; 0.04 mmol; 0.0025 eq.) was added very slowly. The reaction took place at ambient temperature over 12 hours. The crude reaction product was purified by distillation to give a colourless liquid.

(14) The characteristics of the product obtained were the following:

(15) Mass obtained: 5.69 g

(16) Boiling point: 130-135 C. at 0.5 mbar

(17) Yield: 72%

(18) .sup.1H NMR (200 MHz; CDCl.sub.3): 0.64 (2H; m; H.sup.17); 1.27 (16H; m; H.sup.916); 1.75 (2H; m; H.sup.8); 3.56 (9H; s; H.sup.18); 4.13 (2H; t; H.sup.7; .sup.3J.sub.HH=6.5 Hz)

(19) .sup.13C NMR (200 MHz; CDCl.sub.3): 9.45 (C.sup.17); 22.94; 25.85; 29.57; 29.6; 29.83 (2C); 29.89; 30.17; 33.48; 50.67 (3C; C.sup.18); 76.16 (t; C.sup.7; .sup.3J.sub.CC=3.4 Hz); 134.46 (C.sup.4); 138.22 (2C; C.sup.3+5; .sup.3J.sub.CF=259 Hz); 139.51 (C.sup.16); 142.38 (2C; C.sup.2+6; .sup.3J.sub.CF=243 Hz)

(20) Si NMR (200 MHz; CDCl.sub.3): 41.29 (s)

c) Silanization of a Silicon Support by Compound (II)

(21) First, the silicon support, covered with a 5000 thick layer of thermal oxide, is subjected to a hydroxylation by bringing into contact with a 3.5M sodium hydroxide solution over two hours.

(22) A solution comprising the silane compound prepared above at a concentration of 10.sup.2M in anhydrous trichloroethylene was used, and the silanization reactions were carried out at a controlled temperature of 2 C. over 24 hours.

(23) The grafted support had the following configuration:

(24) ##STR00017##

Example 2

(25) This example illustrates the preparation of a silane compound conforming to the invention: 11-(diethylphosphonate)undecyltrimethoxysilane (III) according to the following reaction scheme:

(26) ##STR00018##

(27) The phosphonic acid functional group was protected in the diethylphosphonate form after reaction of 11-bromoundecene with triethylphosphate at high temperature.

(28) Next, the incorporation of the silyl group was carried out by a hydrosilylation reaction in the presence of a Karstedt catalyst.

(29) This example also illustrates the silanization of a silicon support by the silane compound (III).

a) Step 1: Synthesis of 11-(diethylphosphonate)undec-1-ene

(30) ##STR00019##

(31) 11-Bromoundecene (95%) (12.64 g; 11.8 ml; 52 mmol) was mixed with triethylphosphate (98%) (17.24 g; 17.8 ml; 102 mmol; 2 eq.). The solution was heated at 170 C. for 24 hours, then the crude reaction product was purified by distillation to give a colourless liquid.

(32) The characteristics of the product obtained were the following:

(33) Mass obtained: 13.55 g

(34) Boiling point: 115-120 C. at 0.5 mbar

(35) Yield: 91%

(36) .sup.1H NMR (200 MHz; CDCl.sub.3): 1.29 (12H; m; H.sup.712); 1.32 (6H; t; H.sup.1+3; .sup.3J.sub.HH=7.1 Hz); 1.51-1.94 (4H; m; H.sup.5+6); 2.04 (2H; m; H.sup.13); 4.09 (4H; m; H.sup.2+4); 4.97 (2H; m; H.sup.15); 5.81 (1H; m; H.sup.14)

(37) .sup.13C NMR (200 MHz; CDCl.sub.3): 16.75 (2C; d; C.sup.1+3; .sup.3J.sub.CP=6.2 Hz); 21.25; 22.69 (d; C.sup.7; .sup.3J.sub.CP=5.4 Hz); 24.58-27.37 (d; C.sup.5; .sup.1J.sub.CP=140.3 Hz); 29.20; 29.37; 29.60; 29.69; 30.88 (d; C.sup.6; .sup.2J.sub.CP=16.9 Hz); 34.09; 61.60 (2C; d; C.sup.2+4; .sup.2J.sub.CP=5.9 Hz); 114.43 (C.sup.15); 139.35 (C.sup.14).

b) Step 2: Synthesis of 11-(diethylphosphate)undecyltrimethoxysilane (III)

(38) ##STR00020##

(39) 11-(Diethylphosphonate)undec-1-ene (4.99 g; 17 mmol) was mixed with trimethoxysilane (90%) (3.3 g; 3.3 ml; 23 mmol; 1.3 eq.). The Karstedt catalyst (0.04 g; 0.04 mmol; 0.0025 eq.) was added very slowly. The reaction took place at ambient temperature over 16 hours. The crude reaction product was purified by distillation to give a colourless liquid.

(40) The characteristics of the product obtained were the following:

(41) Mass obtained: 4.22 g

(42) Boiling point: 165-170 C. at 0.5 mbar

(43) Yield: 60%

(44) .sup.1H NMR (200 MHz; CDCl.sub.3): 0.65 (2H; m; H.sup.15); 1.26 (16H; m; H.sup.714); 1.32 (6H; t; H.sup.1+3; .sup.3J.sub.HH=7 Hz); 1.53-1.81 (4H; m; H.sup.5+6); 3.56 (9H; s; H.sup.16); 4.09 (4H; m; H.sup.2+4)

(45) .sup.13C NMR (200 MHz; CDCl.sub.3): 9.52 (C.sup.15); 16.87 (2C; d; C.sup.1+3; .sup.3J.sub.CP=6.1 Hz); 22.74; 22.91 (d; C.sup.7; .sup.3J.sub.CP=6.1 Hz); 24.69-27.47 (d; C.sup.5; .sup.1J.sub.CP=140.3 Hz); 29.48; 29.63; 29.76; 29.86; 29.97; 31.01 (d; C.sup.6; .sup.2J.sub.CP=17 Hz); 33.53; 50.87 (3C; C.sup.16); 61.74 (2C; d; C.sup.2+4; .sup.2J.sub.CP=6.2 Hz).

c) Silanization of a Silicon Support by the Compound (III)

(46) First, the silicon support, covered with a 5000 thick layer of thermal oxide, is subjected to a hydroxylation by bringing into contact with a 3.5M sodium hydroxide solution over two hours.

(47) A solution comprising the silane compound prepared above at a concentration of 10.sup.2M in anhydrous trichloroethylene was used, and the silanization reactions were carried out at a controlled temperature of 2 C. over 24 hours.

(48) The grafted support had the following configuration:

(49) ##STR00021##

(50) The grafted support was then brought into contact with a solution of iodotrimethylsilane in order to release the phosphonic acid functional group, which will in turn react with an aqueous sodium hydroxide solution to give the desired phosphonate functional group according to the following reaction scheme:

(51) ##STR00022##

(52) The support thus charged may be used to adsorb, in a specific manner, charged proteins (such as protein markers) via ionic interaction.

Example 3

(53) This example illustrates the preparation of a silane compound conforming to the invention: trimethoxysilanylundecyl-10-iminodiacetic acid methyl ester (IV) according to the following reaction scheme:

(54) ##STR00023##

(55) The amine functional group is incorporated via a Williamson type reaction between the dimethyliminodiacetate and the 11-bromoundecene in the presence of potassium carbonate.

(56) Next, the incorporation of the silyl group was carried out by a hydrosilylation reaction in the presence of a Karstedt catalyst.

(57) This example also illustrates the silanization of a silicon support by the silane compound (IV).

a) Step 1: Synthesis of undec-1-eneiminodiacetic acid methyl ester

(58) ##STR00024##

(59) Added to a solution of dimethyliminodiacetate (hydrochloride) (8.53 g; 43 mmol; 1 eq.) dissolved in 250 ml of DMF, were triethylamine (4.35 g; 6 ml; 43 mmol; 1 eq.), 11-bromoundecene (95%) (10.53 g; 9.9 ml; 43 mmol) and potassium carbonate (5.95 g; 43 mmol; 1 eq.). The reaction was heated at 80 C. over 36 hours. After evaporating the DMF and taking up in ethyl acetate, the reaction mixture was washed successively with distilled water (two times) and with a saturated sodium chloride solution and dried over anhydrous magnesium sulphate. The residue was purified by chromatography over silica gel (cyclohexane/ethyl acetate (75/25)) to give a colourless liquid.

(60) The characteristics of the product obtained were the following:

(61) Mass obtained: 6 g

(62) Yield: 84%

(63) .sup.1H NMR (200 MHz; CDCl.sub.3): 1.28 (12H; m; H.sup.612); 1.59 (2H; m; H.sup.5); 2.03 (2H; m; H.sup.12); 3.74 (6H; s; H1); 4.09 (4H; t; H.sup.3+4; .sup.3J.sub.HH=6.8 Hz); 4.16 (2H; s; H.sup.3); 4.97 (2H; m; H.sup.14); 5.81 (1H; m; H.sup.16)

(64) .sup.13C NMR (200 MHz; CDCl.sub.3): 26.14; 29.20; 29.29; 29.48; 29.59; 29.77; 29.84; 34.18; 49.51 (C.sup.3); 52.54 (C.sup.1); 66.89 (C.sup.4); 114.52 (C.sup.14); 139.57 (C.sup.13); 170.40 (C.sup.2).

b) Step 2: Synthesis of trimethoxysilanylundecyl-10-iminodiacetic acid methyl ester

(65) ##STR00025##

(66) Undec-1-eneiminodiacetic acid methyl ester (6 g; 19 mmol) was mixed with trimethoxysilane (90%) (3.57 g; 3.7 ml; 26 mmol; 1.4 eq.). The Karstedt catalyst (0.05 g; 0.05 mmol; 0.0025 eq.) was added very slowly. The reaction took place at ambient temperature over 16 hours. The crude reaction product was purified by distillation to give a colourless liquid.

(67) The characteristics of the product obtained were the following:

(68) Mass obtained: 3.83 g

(69) Boiling point: 185-190 C. at 0.5 mbar

(70) Yield: 46%

(71) .sup.1H NMR (200 MHz; CDCl.sub.3): 0.65 (2H; m; H.sup.14); 1.26 (16H; m; H.sup.613); 1.60 (2H; m; H.sup.5); 3.57 (9H; s; H.sup.15); 3.74 (6H; s; H.sup.1); 4.09 (4H; t; H.sup.3+4; .sup.3J.sub.HH=6.8 Hz); 4.16 (2H; s; H.sup.3)

(72) .sup.13C NMR (200 MHz; CDCl.sub.3): 9.42 (C.sup.14); 22.95; 26.12; 29.18; 29.54; 29.61; 29.88 (2C).sup.2; 29.92; 33.49; 49.49 (C.sup.3); 50.83 (3C; C.sup.15); 52.50 (C.sup.1); 66.86 (C.sup.4); 170.29 (C.sup.2);

(73) Si NMR (200 MHz; CDCl.sub.3): 41.27 (s)

c) Silanization of a Silicon Support by the Compound (IV)

(74) First, the silicon support, covered with a 5000 thick layer of thermal oxide, is subjected to a hydroxylation by bringing into contact with a 3.5M sodium hydroxide solution over two hours.

(75) A solution comprising the silane compound prepared above at a concentration of 10.sup.2M in anhydrous trichloroethylene was used, and the silanization reactions were carried out at a controlled temperature of 2 C. over 24 hours.

(76) The grafted support had the following configuration:

(77) ##STR00026##

(78) The iminodiacetic acid functional group was then released by reaction of the modified support with 12N HCl, then the thus treated support was reacted with an aqueous copper sulphate solution to enable the copper complexation according to the following reaction scheme:

(79) ##STR00027##

(80) Such a complex may advantageously be used to ensure the retention of a protein comprising a sequence known as Histidine-Tag. Histidine-Tag is a sequence corresponding to a consecutive linking of 5 to 6 histidines placed in the terminal C or N position of a protein and comprising an imidazole functional group in its side chain. This functional group is capable of chelating with the free coordination site of a metallic ion that is itself chelated to an iminodiacetic acid or nitrilodiacetic acid group (in this case the metallic ion is Cu.sup.2+). Metallic ions that can also be envisaged may be Ni.sup.2+, Zn.sup.2+ or Co.sup.2+.

(81) The grafted silane compounds described above may therefore be used for the separation and purification of proteins bearing the Histidine-Tag sequence.

Example 4

(82) This example illustrates the preparation of a silane compound conforming to the invention: 4-nitrophenylundecyltrimethoxysilane ester (V) according to the following reaction scheme:

(83) ##STR00028##

(84) The ester functional group is synthesized by reaction between undecenoyl chloride and 4-nitrophenol.

(85) Next, the incorporation of the silyl group is carried out by a hydrosilylation reaction in the presence of a Karstedt catalyst.

a) Step 1: Synthesis of 4-nitrophenylundec-1-ene ester

(86) ##STR00029##

(87) Added to a solution of 4-nitrophenol (3.25 g; 23 mmol; 1 eq.) dissolved in 50 ml of anhydrous ether, was pyridine (1.85 g; 1.9 ml; 23 mmol; 1 eq.). The solution was heated under reflux of ether and undecenoyl chloride (97%) (4.9 g; 5.2 ml; 23 mmol; 1 eq.) was added very slowly (over a period of around 1 hour). The reaction was continued under reflux of ether for 1 hour. The reaction mixture was filtered, then the filtrate was concentrated to give a yellowish liquid.

(88) The characteristics of the product obtained were the following:

(89) Mass obtained: 7.1 g

(90) Yield: 100%

(91) .sup.1H NMR (200 MHz; CDCl.sub.3): 1.33 (10H; m; H.sup.1014); 1.74 (2H; m; H.sup.9); 2.04 (2H; m; H.sup.15); 2.60 (2H; t; H.sup.8); .sup.3J.sub.HH=7.5 Hz); 4.98 (2H; m; H.sup.17); 5.80 (1H; m; H.sup.16); 7.27 (2H; d; H.sup.3+5; .sup.3J.sub.HH=9.2 Hz); 8.27 (2H; d; H.sup.2+6; .sup.3J.sub.HH=9.2 Hz)

(92) .sup.13C NMR (200 MHz; CDCl.sub.3): 25.13; 29.28; 29.43 (2C); 29.56; 29.66; 34.17; 34.73; 114.61 (C.sup.17); 122.84 (2C; C.sup.3+5) 125.59 (2C; C.sup.2+6); 139.52 (C.sup.16); 145.66 (C.sup.1) 155.96 (C.sup.4); 171.73 (C.sup.7).

b) Step 2: Synthesis of 4-nitrophenylundecyltrimethoxysilane ester (V)

(93) ##STR00030##

(94) 4-Nitrophenylundec-1-ene ester (7.54 g; 25 mmol) was mixed with trimethoxysilane (90%) (4.25 g; 4.5 ml; 31 mmol; 1.3 eq.). The Karstedt catalyst (0.06 g; 0.06 mmol; 0.0025 eq.) was added very slowly and the whole mixture was heated at 140 C. under argon over 24 hours. The crude reaction product was purified by distillation to give a yellowish liquid.

(95) The characteristics of the product obtained were the following:

(96) Mass obtained: 5.02 g

(97) Boiling point: 170-175 C. at 0.5 mbar

(98) Yield: 48%

(99) .sup.1H NMR (200 MHz; CDCl.sub.3): 0.65 (2H; m; H.sup.17); 1.33 (10H; m; H.sup.1016); 1.74 (2H; m; H.sup.9); 2.60 (2H; t; H.sup.8; .sup.3J.sub.HH=7.5 Hz); 3.57 (9H; s; H.sup.18); 7.28 (2H; d; H.sup.3+5; .sup.3J.sub.HH=9.2 Hz); 8.25 (2H; d; H.sup.2+6; .sup.3J.sub.HH=9.2 Hz)

(100) .sup.13C NMR (200 MHz; CDCl.sub.3) 9.48 (C.sup.17); 22.95; 25.07; 29.39; 29.57 (2C); 29.78 (2C); 33.46; 34.59; 50.78 (3C; C.sup.18); 122.82 (2C; C.sup.3+5); 125.47 (2C; C.sup.2+6); 145.53 (C.sup.1) 155.91 (C.sup.4) 171.62 (C.sup.7)

(101) Si NMR (200 MHz; CDCl.sub.3): 41.22 (s)

c) Silanization of a Silicon Support by the Compound (V)

(102) First, the silicon support, covered with a 5000 thick layer of thermal oxide, is subjected to a hydroxylation by bringing into contact with a 3.5M sodium hydroxide solution over two hours.

(103) A solution comprising the silane compound prepared above at a concentration of 10.sup.2M in anhydrous trichloroethylene was used, and the silanization reactions were carried out at a controlled temperature of 2 C. over 24 hours.

d) Immobilization of an Oligonucleotide onto the Grafted Support

Hybridization and Analysis

(104) Deposits of a solution of oligonucleotides having the following sequence: 5 TTT TTGATA AAC CCC 3
modified at 5 by an amine functional group and deposits of a solution of oligonucleotides having the following sequence:

(105) TABLE-US-00001 5TTT TTGATA AAC CCC 3
that were unmodified were carried out on the support obtained in accordance with what was described in paragraph c), these deposits being carried out manually in an amount of 0.2 l. The oligonucleotide concentration of the solutions used was 10 M in a 0.1M NaCl buffer.

(106) After an incubation time of 16 hours in a humid chamber, the supports were hybridized with a solution of complementary targets having the following sequence:

(107) TABLE-US-00002 5CAT AGA GTG GGT TTA TCC A 3
having a concentration of 0.05 M, labelled with a fluorescent Cy3 group.

(108) The fluorescence signals were measured on a scanner sold under the name GenePix by Axon.

(109) The results show that the supports grafted in accordance with the invention make it possible to achieve the immobilization of oligonucleotides comprising an amine functional group and of unmodified oligonucleotides, that is to say those bearing a free OH functional group.

Example 5

(110) This example illustrates the preparation of a silane compound conforming to the invention: pentafluorophenylundecyltrimethoxysilane ester (VI) according to the following reaction scheme:

(111) ##STR00031##

(112) The ester functional group was synthesized by reaction between undecenoyl chloride and pentafluorophenol.

(113) Next, the incorporation of the silyl group was carried out by a hydrosilylation reaction in the presence of a Karstedt catalyst.

a) Step 1: Synthesis of pentafluorophenylundec-1-ene ester

(114) ##STR00032##

(115) Added to a solution of pentafluorophenol (6.51 g; 35 mmol, 1 eq.) dissolved in 60 ml of anhydrous ether was pyridine (2.8 g; 2.9 ml; 35 mmol; 1 eq.). The solution was heated under reflux of ether and undecenoyl chloride (97%) (7.4 g; 7.8 ml; 35 mmol; 1 eq.) was added very slowly (over a period of around 1 hour). The reaction was continued under reflux of ether for 1 hour. The reaction mixture was filtered, then the filtrate was concentrated to give a colourless liquid.

(116) The characteristics of the product obtained were the following:

(117) Mass obtained: 12.36 g

(118) Yield: 100%

(119) .sup.1H NMR (200 MHz; CDCl.sub.3): 1.37 (10H; m; H.sup.1014); 1.80 (2H; m; H.sup.9); 2.09 (2H; m; H.sup.15); 2.70 (2H; t; H.sup.8; .sup.3J.sub.HH=7.4 Hz) 5.01 (2H; m; H.sup.17); 5.85 (1H; m; H.sup.16)

(120) .sup.13C NMR (200 MHz; CDCl.sub.3): 25.16; 29.23; 29.28; 29.42; 29.49; 29.63; 33.72; 34.18; 114.55 (C.sup.17); 135.78-140.77 (2C; m; C.sup.3+5; .sup.1J.sub.C+F=251 Hz); 137.29-142.31 (2C; m; C.sup.2+6; .sup.1J.sub.CF=253 Hz); 138.99 (C.sup.4); 139.51 (C.sup.16); 144.07 (C.sup.1); 169.97 (C.sup.7)

(121) F NMR (400 MHz; CDCl.sub.3): 153.38 (2F; d; F.sup.3+4; .sup.3J.sub.FF=17.1 Hz); 158.82 (t; F.sup.1; .sup.3J.sub.FF=21.6 Hz; 163.02 (2F; t; F.sup.2+5; .sup.3J.sub.FF=16.8 Hz)

b) Step 2: Synthesis of the pentafluorophenylundecyltrimethoxysilane ester (VI)

(122) ##STR00033##

(123) The pentafluorophenylundec-1-ene ester (10.86 g; 31 mmol) was mixed with trimethoxysilane (90%) (5.65 g; 5.9 ml; 42 mmol; 1.4 eq.). The Karstedt catalyst (0.07 g; 0.08 mmol; 0.0025 eq.) was added very slowly. The reaction took place at ambient temperature over 16 hours. The crude reaction product was purified by distillation to give a colourless liquid.

(124) The characteristics of the product obtained were the following:

(125) Mass obtained: 10.69 g

(126) Boiling point: 135-140 C. at 0.5 mbar

(127) Yield: 73%

(128) .sup.1H NMR (200 MHz; CDCl.sub.3): 0.68 (2H; m; H.sup.17); 1.32 (14H; m; H.sup.1016); 1.80 (2H; m; H.sup.9); 2.69 (2H; t; H.sup.8; .sup.3J.sub.HH=7.4 Hz); 3.60 (9H; m; H.sup.18)

(129) .sup.13C NMR (200 MHz; CDCl.sub.3): 9.52 (C.sup.17); 22.99; 25.16; 29.25; 29.51; 29.60; 29.78 (2C); 33.51; 33.75; 50.84 (3C; C.sup.18); 135.73-140.76 (2C; m; C.sup.3+5; .sup.1J.sub.CF=253 Hz); 137.27-142.25 (2C; m; C.sup.2+6; .sup.1J.sub.CF=251 Hz); 138.98 (C.sup.4); 144.06 (C.sup.1); 169.95 (C.sup.7)

(130) F NMR (400 MHz; CDCl.sub.3): 153.31 (2F; d; F.sup.3+4; .sup.3J.sub.FF=17.4 Hz=; 158.76 (t; F.sup.1; .sup.3J.sub.FF=21.7 Hz); 162.98 (2F; t; F.sup.2+5; .sup.3J.sub.FF=16.4 Hz)

(131) Si NMR (200 MHz; CDCl.sub.3): 41.22 (s)

c) Silanization of a Silicon Support by the Compound (VI)

(132) First, the silicon support, covered with a 5000 thick layer of thermal oxide, is subjected to a hydroxylation by bringing into contact with a 3.5M sodium hydroxide solution over two hours.

(133) A solution comprising the silane compound prepared above at a concentration of 10.sup.2M in anhydrous trichloroethylene was used, and the silanization reactions were carried out at a controlled temperature of 2 C. over 24 hours.

d) Immobilization of an Oligonucleotide onto the Grafted Support

Hybridization and Analysis

(134) Deposits of a solution of oligonucleotides having the following sequence:

(135) TABLE-US-00003 5TTT TTGATA AAC CCC 3
modified at 5 by an amine functional group and deposits of a solution of oligonucleotides having the following sequence: 5 TTT TTGATA AAC CCC 3
that were unmodified were carried out on the support obtained in accordance with what was described in paragraph c), these deposits being carried out either manually in an amount of 0.2 l, or using a piezoelectric ejection robot sold under the name BCA1 by Perkin Elmer, in an amount of 300 l. The oligonucleotide concentration of the solutions used was 10 M in a 0.1M NaCl buffer or a 0.3M Na.sub.2HPO.sub.4 buffer+6% glycerol+4% butanol.

(136) After an incubation time of 16 hours in a humid chamber or in a dry chamber, the supports were hybridized with a solution of complementary targets having the following sequence:

(137) TABLE-US-00004 5CAT AGA GTG GGT TTA TCC A 3
having a concentration of 0.05 M, labelled with a fluorescent Cy3 group.

(138) The fluorescence signals were measured on a scanner sold under the name GenePix by Axon.

(139) The results show that the supports grafted in accordance with the invention make it possible to achieve the immobilization of oligonucleotides comprising an amine functional group and of unmodified oligonucleotides, that is to say those bearing a free OH functional group.

e) Immobilization of Streptavidine Cy3

(140) On a support obtained in accordance with what was described in paragraph c), deposits of a streptavidine solution were carried out manually in an amount of 0.2 l. The protein concentration of the solutions used was 0.001 mg/ml in a 0.01M PBS buffer (phosphate buffered saline).

(141) After an incubation time of 16 hours in a humid chamber, the supports were analysed with a scanner sold under the name GenePix by Axon.

(142) The results show that the modified supports make it possible to achieve the immobilization of this protein.

Example 6

(143) This example illustrates the preparation of a silane compound conforming to the invention: 1-trimethoxysilanyl-10-amidoundecyl-11-iminodiacetic acid methyl ester of formula (VII) according to the following reaction scheme:

(144) ##STR00034##

(145) The amide functional group is synthesized by reaction between undecenoyl chloride and dimethyliminodiacetate. The incorporation of the silyl group is carried out by a hydrosilylation reaction.

a) Step 1: Synthesis of 10-amidoundec-1-eneiminodiacetic acid methyl ester

(146) ##STR00035##

(147) Added to a solution of dimethyliminodiacetate (hydrochloride) (3.08 g; 16 mmol) dissolved in 50 ml of anhydrous ether was pyridine (2.46 g; 2.59 ml; 35 mmol; 2 eq.). The solution was heated under reflux of ether and the undecenoyl chloride (97%) (3.48 g, 3.7 ml, 17 mmol, 1 eq.) was added very slowly (approximately over a period of one hour). The reaction was continued under reflux of ether for 1 hour. The reaction mixture was filtered, then the filtrate was concentrated to give a residue which was then purified by chromatography over silica gel (cyclohexane.fwdarw.cyclohexane/ethyl acetate (70/30)) to give a white solid.

(148) The characteristics of the product obtained were the following:

(149) Mass obtained: 3.85 g

(150) Yield: 74%

(151) .sup.1H NMR (200 MHz; CDCl.sub.3): 1.30 (10H; m; H.sup.711); 1.64 (2H; m; H.sup.6); 2.03 (2H; m; H.sup.12); 2.30 (2H; t; H.sup.5; J.sub.HH=7.4 Hz); 3.72 (3H; s; H.sup.1); 3.77 (3H; s; H.sup.1); 4.16 (2H; s; H.sup.3); 4.19 (2H; s; H.sup.3); 4.96 (2H; m; H.sup.14); 5.81 (1H; m; H.sup.13)

(152) .sup.13C NMR (200 MHz; CDCl.sub.3): 25.18 (C.sup.6); 29.28; 29.45; 29.59; 29.69; 29.74; 33.06 (C.sup.5); 34.18 (C.sup.12); 48.18 (C.sup.3); 50.34 (C.sup.3); 52.53 (C.sup.1); 52.90 (C.sup.1); 114.52 (C.sup.14); 139.58 (C.sup.13); 169.88 (C.sup.2); 170.30 (C.sup.2); 174.02 (C.sup.4)

(153) Melting point: 25-30 C.

b) Step 2: Synthesis of 1-trimethoxysilanyl-10-amidoundecyl-11-iminodiacetic acid methyl ester

(154) ##STR00036##

(155) 11-amidoundec-1-eneiminodiacetic acid methyl ester (3.34 g; 10 mmol) was mixed with trimethoxysilane (95%) (2.05 g; 2.1 ml; 16 mmol; 1.6 eq.). The Karstedt catalyst (0.024 g; 0.025 mmol; 0.0025 eq.) was added very slowly. The reaction took place at ambient temperature over 16 hours. The crude reaction product was purified by extraction with pentane to give a viscous yellowy liquid.

(156) The characteristics of the product obtained were the following:

(157) Mass obtained: 3 g

(158) Yield: 66%

(159) .sup.1H NMR (200 MHz; CDCl.sub.3): 0.65 (2H; m; H.sup.14); 1.26 (14H; m; H.sup.713); 1.61 (2H; m; H.sup.6); 2.30 (2H; t; H.sup.5; .sup.3J.sub.HH=7.4 Hz); 3.57 (9H; s; H.sup.15); 3.73 (3H; s; H.sup.1); 3.78 (3H; s; H.sup.1); 4.16 (2H; s; H.sup.3); 4.19 (2H; s; H.sup.3)

(160) .sup.13C NMR (200 MHz; CDCl.sub.3): 9.52 (C.sup.14); 23.00 (C.sup.13); 25.23 (C.sup.6); 29.65 (2C); 29.82; 29.88; 29.90; 33.12 (C.sup.5); 33.56 (C.sup.12); 48.20 (C.sup.3); 50.38 (C.sup.3); 50.91 (C.sup.15); 52.57 (C.sup.1); 52.93 (C.sup.1); 169.90 (C.sup.2); 170.34 (C.sup.2); 174.05 (C.sup.4)

c) Silanization of a Silicon Support by the Compound (VII)

(161) First, the silicon support, covered with a 5000 thick layer of thermal oxide, is subjected to a hydroxylation by bringing into contact with a 3.5M sodium hydroxide solution over two hours.

(162) A solution comprising the silane compound prepared above at a concentration of 10.sup.2M in anhydrous trichloroethylene was used, and the silanization reactions were carried out at a controlled temperature of 2 C. over 24 hours.

(163) The grafted support had the following configuration:

(164) ##STR00037##

(165) The iminodiacetic acid functional group was then released by reaction of the modified support with 10.sup.2M AlI.sub.3/CH.sub.3CN, then the thus treated support was reacted with an aqueous copper sulphate solution in order to enable the copper complexation according to the following reaction scheme:

(166) ##STR00038##

(167) Such a complex may advantageously be used to ensure the retention of a protein comprising a sequence known as Histidine-Tag. Histidine-Tag is a sequence corresponding to a consecutive linking of 5 to 6 histidines placed in the terminal C or N position of a protein and comprising an imidazole functional group in its side chain. This functional group is capable of chelating with the free coordination site of a metallic ion that is itself chelated to an iminodiacetic acid group (in this case the metallic ion is Cu.sup.2+). Metallic ions that can also be envisaged may be Ni.sup.2+, Zn.sup.2+ or Co.sup.2+.

(168) The grafted silane compounds described above may therefore be used for the separation and purification of proteins bearing the Histidine-Tag sequence.