Azide-functionalized copolymers

Abstract

The present invention relates to a copolymer having several azide functionalities that can be prepared by cationic ring opening polymerisation. The copolymer comprises a first monomer unit and a second monomer unit which are different from each other.

Claims

1. A copolymer comprising at least a first and a second monomer unit, wherein: the first and the second monomer unit are different from each other; the first monomer unit is selected from the group consisting of compounds 202, 203, 204, 205, 206, 207, 302, 303, 304, 305, 306, and 307 TABLE-US-00010 No. Monomer unit No. Monomer unit 202 302 203 303 204 304 205 305 206 306 207 307 and the second monomer unit is selected from the group consisting of 2-methyl-4,5-dihydro-1,3-oxazole, 2-ethyl-4,5-dihydro-1,3-oxazole, 2-methyl-5,6-dihydro-4H-1,3-oxazine and 2-ethyl-5-6-dihydro-4H-1,3-oxazine with the proviso that if the second monomer unit is 2-ethyl-4,5-dihydro-1,3-oxazole, the first monomer unit is selected from the group consisting of compounds 202, 204 and 205, and if the second monomer unit is 2-ethyl-5-6-dihydro-4H-1,3-oxazine, the first monomer unit is selected from the group consisting of compounds 302, 304 and 305; and the copolymer is protein resistant.

2. The copolymer according to claim 1, wherein the second monomer unit is 2-methyl-4,5-dihydro-1,3-oxazole.

3. The copolymer according to claim 1, wherein the second monomer unit is 2-methyl-5,6-dihydro-4H-1,3-oxazine.

4. The copolymer according to claim 1, wherein the first monomer unit is selected from the group consisting of compounds 202, 203, 204, 205, 302, 303, 304, and 305.

5. The copolymer according to claim 1, wherein the first monomer unit is selected from the group consisting of compounds 202, 203, 204, 205, and 206.

6. The copolymer according to claim 5, wherein the first monomer unit is compound 203.

7. The copolymer according to claim 5, wherein the copolymer comprises 1 to 30% of the first monomer unit and 70 to 99% of the second monomer unit.

8. The copolymer according to claim 1 comprising 25 to 150 monomer units in total.

9. The copolymer according to claim 1 further comprising a terminal linker group.

10. The copolymer according to claim 1, wherein the copolymer is hydrophilic.

11. The copolymer according to claim 1, wherein the copolymer is not amphiphilic.

12. A functional polymer comprising a polymer backbone and a plurality of side chains, wherein at least a part of said side chains are copolymers according to claim 1.

13. The functional polymer according to claim 12, wherein all side chains are said copolymers.

14. The functional polymer according to claim 12, wherein said functional polymer comprises at least two different types of side chains, and wherein at least one type of side chain is said copolymers.

15. The functional polymer according to claim 14, wherein said functional polymer comprises a further type of side chain D which is intended to reversible bind to a substrate or has a coating function, and the side chain D is selected from the group consisting of a short chain side chain D1 having a linear or branched, substituted or unsubstituted C1 to C12 alkylene group which optionally comprises heteroatoms selected from the group consisting of oxygen and nitrogen, and which carries at least one functional end or side group K1 selected from the group consisting of amines, carboxy, poly(propylene sulfide), and thioethers; a side chain D2 having a long chain D2 comprising more than 15 carbon or silicium atoms in the chain, wherein said long chain D2 is selected from the group consisting of polydimethylsiloxane, perfluoroethers, perfluoroalkyls, polyisobutene, polyethylene glycol, polydimethylacrylamide, polyvinylpyrrolidone, polyalkyloxazolines, dextran, carboxymethyl dextran, poly(N-isopropylacrylamide), poly(N-hydroxyethylacrylamide, poly(2-hydroxyethyl methacrylate), poly-hydroxypropylmethacrylate), poly-(methacryloyloxylethyl phosphorylcholine), poly-(sulfobetaine methacrylate), polyalkylene residues having more than 20 carbon atoms, peptide chains, DNA fragments and poly-(sulfobetaine acrylamide), whereby D2 has no functional end group or side group; and a side chain D3 having a long chain D3 selected from the group consisting of a polydimethylsiloxane, perfluoroethers, perfluoroalkyls, polyisobutene, polyethylene glycol, polydimethylacrylamide, polyvinylpyrrolidone, polyalkyloxazolines, dextran, carboxymethyl dextran, poly(N-isopropylacrylamide), poly(N-hydroxyethylacrylamide, poly(2-hydroxyethyl methacrylate), poly-hydroxypropylmethacrylate), poly-(methacryloyloxylethyl phosphorylcholine), poly-(sulfobetaine methacrylate), polyalkylene residues having more than 20 carbon atoms, peptide chains, DNA fragments and poly-(sulfobetaine acrylamide) whereby D3 carries at least one functional end or side group K3 selected from the group consisting of amines, carboxy, and nitrilotriacetic acid (NTA), biotin, azide, terminal alkene groups, terminal alkine groups, tetrazine.

16. The functional polymer according to claim 14, wherein said functional polymer comprises a further type of side chain E which is intended to irreversibly bind to a substrate, said side chain E having a linear or branched, substituted or unsubstituted C1 to C12 alkylene group which optionally comprises heteroatoms selected from the group consisting of oxygen and nitrogen, and which carries at least one functional end or side group K4 selected from the group of alkoxy silanes, chloro silanes, catechols, nitrocatechols, bromocatechols, chlorocatechols, phosphates, phosphonates, mimosine derivatives, anacheline, gallols, thiols, N-heterocyclic carbenes, perfluorophenyl azides, benzophenon, diaryldiazomethane, aryltrifluoromethyldiazomethane, and organoboron.

17. The functional polymer according to claim 12, wherein the polymer backbone is a polyacrylamide, wherein the polymer backbone and the side chains are linked by amide bonds.

18. A conjugate of a copolymer according to claim 1 and at least one active moiety selected from a therapeutic moiety and a targeting moiety.

19. The copolymer according to claim 1 formulated for use as a coating agent.

20. A method for coating a surface comprising a surface active headgroup, the method comprising coating the surface with the copolymer according to claim 9.

Description

FIGURES

(1) FIG. 1 shows a polymer comprising a copolymer according to the present invention successfully coated on a silicon oxide surface;

(2) FIGS. 2, 3A and 3B show a comparison between the copolymer of the present invention and a copolymer of the state of the art.

EXAMPLES

Preparation of methyl-4-azidobutanoate

(3) To a solution of methyl-4-Bromobutanoate (35.95 g, 0.20 mol) in dimethylformamide (200 ml) was added sodium azide (32.32 g, 0.50 mol). The resulted mixture was stirred at 60 C. for 17 h under argon. Then ultrapure water (460 ml) was added to the mixture. The resulted solution was extracted with diethyl ether three times. The combined organic phases were washed with ultrapure water and brine, dried over MgSO.sub.4 and concentrated by evaporation to yield 24.14 g methyl-4-azidobutanoate as a yellow oil. Yield: 84.9% .sup.1HNMR (400 MHz, CDCl.sub.3) 3.71 (s, 3H), 3.37 (t, 2H, J=6.7 Hz), 2.44 (t, 2H, J=7.3 Hz) 1.93 (p, 2H, 7 Hz) ppm.

Preparation of 4-azidobutanoic acid

(4) To a solution of methyl-4-azidobutanoate (24.14 g, 0.17 mol) in methanol (130 ml) was added 6M NaOH solution (60 ml, 0.36 mol). The resulted mixture was stirred at room temperature for 21 h. Then 2M HCl (270 ml, 0.52 mol) was added to the mixture. The resulted solution was extracted with dichloromethane three times. The combined organic phases were dried over MgSO.sub.4 and concentrated by evaporation to yield 20.46 g 4-azidobutanoic acid as a yellow oil. Yield: 94% .sup.1HNMR (400 MHz, CDCl.sub.3) 3.38 (t, 2H, J=6.7 Hz), 2.48 (t, 2H, J=7.25), 1.92 (p, 2H, J=6.85).

Preparation of 4-azidobutanoyl chloride

(5) To 4-azidobutanoic acid (20.29 g, 0.16 mol) was added thionyl chloride (13.9 ml, 0.19 mol) dropwise under nitrogen. The resulted solution was stirred at room temperature for 1.5 h. Excessive thionyl chloride was removed by evaporation to purify the intermediate 4-azidobutanoyl chloride. .sup.1HNMR (400 MHz, CDCl.sub.3) 3.42 (t, 2H, J=6.48 Hz), 3.04 (t, 2H, J=7.08 Hz), 2.00 (p, 2H, J=6.95)

Preparation of N-2-Chloroethyl-4-azidobutyramide

(6) To a solution of 4-azidobutanoyl chloride (23.19 g, 0.16 mol) in dichloromethane (175 ml) was added 2-Chloroethylamine (20.05 g, 0.17 mol) under nitrogen. Then triethylamine (54.8 ml, 0.39 mol) was added dropwise while cooling with an ice bath. The mixture was stirred at room temperature for 64 h. The resulted mixture was washed with diethyl ether and the supernatant was filtered and concentrated by evaporation to afford 14.3 g N-2-Chloroethyl-4-azidobutyramide as a yellow-brown oil. Yield: 47.7% .sup.1HNMR (400 MHz, CDCl.sub.3) 3.62 (m, 4H), 3.37 (t, 2H, J=6.52 Hz), 2.32 (t, 2H, J=7.22 Hz), 1.95 (p, 2H, J=6.88 Hz) ppm.

(7) ##STR00055##

Preparation of 2-(3-Azidopropyl)-4,5-dihydro-1,3-oxazole (APOXA)

(8) To a solution of N-2-Chloroethyl-4-azidobutyramide (13.52 g, 0.07 mol) in dry MeOH (45 ml) was added a solution of KOH (3.79 g, 0.068 mol) in dry MeOH (22 ml) under nitrogen. The resulted mixture was stirred at 50 C. for 15 h. Then the solution was cooled to room temperature and filtered. The solvent was removed by evaporation. The resulted mixture was dissolved in dichloromethane (100 ml) and filtered through celite. The solvent was removed by evaporation to yield 9.9 g 2-(3-Azidopropyl)-4,5-dihydro-1,3-oxazole. Yield: 91% .sup.1HNMR (400 MHz, CDCl.sub.3) 4.13 (t, 2H, J=9.52 Hz), 3.73 (t, 2H, J=9.48 Hz), 3.28 (t, 2H, J=6.73 Hz), 2.26 (t, 2H, J=7.32 Hz), 1.83 (p, 2H, J=7.02 Hz) ppm.

(9) ##STR00056##

Preparation of N-2-chloroethyl-3-azidopropionamide

(10) Azido propionic acid NHS is dissolved in dichloromethane. 1.5 equivalent of 1-Chloro ethylamine hydrochloride was added and the mixture is cooled to 0 C. using an ice bath. 2.5 eq of triethylamine is added dropwise at 0 C. and then the mixture is warmed to room temperature and stirred for 16 h at room temperature during which time triethylamine hydrochloride precipitates. Water was added to the mixture and the organic phase is collected, washed with HCl, a saturated NaHCO.sub.3 solution, a saturated NaCl solution, dried with MgSO.sub.4 and evaporated to dryness to obtain N-2-chloroethyl-3-azidpropionamide as a yellow oil. Yield: 76% .sup.1HNMR (400 MHz, DMSO-d.sub.6) 8.29 (s, 1H), 3.61 (t, 2H, J=6.10 Hz), 3.51 (t, 2H, J=6.36 Hz), 3.40 (q, 2H, J=5.99 Hz) ppm.

(11) ##STR00057##

Preparation of 2-(2-Azidoethyl)-4,5-dihydro-1,3-oxazole (AEOXA)

(12) 2-(2-Azidoethyl)-4,5-dihydro-1,3-oxazole is synthesized equivalent to 2-(3-Azidopropyl)-4,5-dihydro-1,3-oxazole starting from 1-Chloro ethylamine hydrochloride and Azidopropanoic acid NHS.

(13) .sup.1HNMR (400 MHz, CDCl.sub.3) 4.25 (t, 2H, J=9.54 Hz), 3.84 (t, 2H, J=9.48 Hz), 3.58 (t, 2H, J=6.78 Hz), 2.54 (t, 2H, J=6.82 Hz) ppm.

(14) .sup.13CNMR (400 MHz, CDCl.sub.3) 166.72, 67.90, 54.88, 48.06, 28.43 ppm

(15) IR, /cm.sup.1: 2105 (azide)

(16) ##STR00058##

Preparation of 2-(2-Azidoethyl)-5,6-dihydro-4H-1,3-oxazine

(17) 2-(2-Azidoethyl)-5,6-dihydro-4H-1,3-oxazine is synthesized equivalent to 2-(3-Azidopropyl)-4,5-dihydro-1,3-oxazole starting from 1-Bromo propylamine hydrobromide and Azidopropanoic acid NHS.

(18) ##STR00059##

Preparation of 2-(3-Azidopropyl)-5,6-dihydro-4H-1,3-oxazine

(19) 2-(3-Azidopropyl)-5,6-dihydro-4H-1,3-oxazine is synthesized following the protocol for 2-(3-Azidopropyl)-4,5-dihydro-1,3-oxazole. Instead of 1-chloroethylamine hydrochloride, 1-chloro-propylamine hydrochloride is used.

(20) ##STR00060##

Preparation of 2-(Axidodifluoromethyl)-4,5-dihydro-1,3-oxazole

(21) Sodium azide (1.2 eq) is added to a stirred solution of 2-bromo-2,2-difluoroacetate in DMSO. After stirring at room temperature for 18 h, the mixture is poured into water and extracted with dichloromethane. The combined extracts were washed with water, dried with MgSO.sub.4, filtered and dichloromethane was evaporated using a rotary evaporator. The product was distilled at 133 mbar at a boiling point of 86 C. to obtain pure ethyl difluoroazidoacetate.

(22) Ethyl difluoroazidoacetate was dissolved in DMF, cooled to 0 C. and 1 equivalent of 1-bromo-ethylamine hydrobromide was added. After addition of 2.2 equivalents of triethylamine, the reaction mixture was stirred first at 0 C. for 1 h and then at room temperature for 20 h. The intermediate N-2-Bromoethylazidodifluoroacetamide is not isolated and cyclized spontaneously. After distillation at reduced pressure, 2-(Azidodifluoromethyl)-4,5-dihydro-1,3-oxazole was obtained as a colorless liquid.

(23) ##STR00061##

Preparation of 2-(Azidodifluoromethyl)-5,6-dihydro-4H-1,3-oxazine

(24) Ethyl difluoroazidoacetate was dissolved in DMF, cooled to 0 C. and 1 equivalent of 1-bromo-propylamine hydrobromide was added. After addition of 2.2 equivalents of triethylamine, the reaction mixture was stirred first at 0 C. for 1 h and then at room temperature for 20 h. The intermediate N-2-Bromopropylazidodifluoroacetamide is not isolated and cyclized spontaneously. After distillation at reduced pressure, 2-(Azidodifluoromethyl)-5,6-dihydro-4H-1,3-oxazine was obtained as a colorless liquid.

(25) ##STR00062##

Preparation of 2-(Azidofluoromethyl)-4,5-dihydro-1,3-oxazole

(26) 2-(Azidofluoromethyl)-4,5-dihydro-1,3-oxazole can be synthesized analog to 2-(Azidodifluoromethyl)-4,5-dihydro-1,3-oxazole starting from Ethyl bromofluoroacetate.

(27) ##STR00063##

Preparation of 2-(Azidofluoromethyl)-5,6-dihydro-4H-1,3-oxazine

(28) 2-(Azidofluoromethyl)-5,6-dihydro-4H-1,3-oxazine can be synthesized analog to 2-(Azidodifluoromethyl)-5,6-dihydro-4H-1,3-oxazine starting from Ethyl bromofluoroacetate.

(29) ##STR00064##

Preparation of 2-(4-Azido-2,3,5,6-tetrafluorophenyl)-4,5-dihydro-1,3-oxazole

(30) 4-Azido-2,3,5,6-tetrafluorobenzoic acid-NHS is reacted with 1.1 equivalent of 1-bromo-ethylamine hydrobromide and 1.1 eq of triethylamin in ethanol at room temperature for 24 h. After removal of the solvent with a rotary evaporator, the intermediate N-3-Bromoethyl-4-azido-2,3,5,6-tetrafluorobenzamide is obtained as a yellow oil. The crude oil is dissolved in methanol and 1.1 eq of KOH is added. After stirring for 24 h at room temperature, the solution is filtered, evaporated to dryness and the product recrystallized in hexane to obtain 2-(4-Azido-2,3,5,6-tetrafluorophenyl)-4,5-dihydro-1,3-oxazole as a white powder.

(31) ##STR00065##

Preparation of 2-(4-Azido-2,3,5,6-tetrafluorophenyl)-5,6-dihydro-4H-1,3-oxazine

(32) 2-(4-Azido-2,3,5,6-tetrafluorophenyl)-5,6-dihydro-4H-1,3-oxazine can be obtained analog to 2-(4-Azido-2,3,5,6-tetrafluorophenyl)-4,5-dihydro-1,3-oxazole using 1-bromo-propylamine hydrobromide instead of 1-bromo-ethylamine hydrobromide.

(33) ##STR00066##

Preparation of 2-(2-Azidobutyl)-4,5-dihydro-1,3-oxazole (ABOXA)

(34) 2-(2-Azidobutyl)-4,5-dihydro-1,3-oxazole is synthesized equivalent to 2-(2-Azidopropyl)-4,5-dihydro-1,3-oxazole starting from 1-Chloro ethylamine hydrochlorid and Azidopentanoicoic acid NHS. Yield: 42.8% .sup.1HNMR (400 MHz, DMSO-d.sub.6) 4.18 (t, 2H, J=9.75 Hz), 3.78 (t, 2H, J=9.76 Hz), 3.25 (t, 2H, J=6.68 Hz), 2.25z (t, 2H, J=7.30 Hz), 1.72-1.56 (m, 4H) ppm.

(35) ##STR00067##

Preparation of Poly(MOXA-Co-APOXA)-Amine with 10% APOXA Content DP=50

(36) To a solution of 2-methyl-4,5-dihydrooxazole (4.61 g, 54.2 mmol) and 2-(3-azidopropyl)-4,5-dihydro-1,3-oxazole (0.93 g, 6.02 mmol) in dry acetonitrile (17 ml) was added methyl trifluoromethanesulfonate (0.1975 g, 1.2 mmol) under nitrogen. The solution was stirred at 70 C. for 20 h. The resulting solution was cooled to room temperature. Then Bis(trimethylsilyl)amine (0.5828 g, 3.6 mmol) was added to terminate the reaction. The solution was stirred at room temperature for 23 h. The polymer was precipitated in diethyl ether (150 ml). The supernatant was removed and the polymer dried by evaporation. After dissolving in mQ-water (25 ml) Silicagel (7.5 g) and 2N HCl (2.17 ml) was added to the polymer solution. The mixture was stirred for 23 h at room temperature. The supernatant was filtered and lyophilized to yield 4.00 g poly(MOXA-co-APOXA)-amine as a white solid. Yield: 47.6% 1HNMR (400 MHz, D2O) 3.7-3.47 (m, 300H), 3.44-3.34 (m, 15H), 3.11-2.91 (m, 3H), 2.60-2.35 (m, 15H), 2.20-1.93 (m, 220H), 1.91-1.79 (m, 15H) ppm.

Synthesis of Poly(MOXA-Co-APOXA)-Amine with 10% APOXA Content DP=100

(37) This polymer was synthesized equivalent to poly(MOXA-co-APOXA)-amine with 10% APOXA content DP=50 by changing the ratio of total monomer to methyl trifluoromethanesulfonate initiator ratio to 100:1.

Synthesis of Poly(MOXA-Co-ABOXA)-Amine with 10% ABOXA Content and a DP=50

(38) To a solution of 2-methyl-4,5-dihydrooxazole (3.60 g, 42.3 mmol) and 2-(4-azidobutyl)-4,5-dihydro-1,3-oxazole (0.79 g, 4.70 mmol) in dry acetonitrile (13 ml) was added methyl trifluoromethanesulfonate (0.1543 g, 0.94 mmol) under nitrogen. The solution was stirred at 70 C. for 16 h. The resulting solution was cooled to room temperature. Then Bis(trimethylsilyl)amine (0.4552 g, 2.8 mmol) was added to terminate the reaction. The solution was stirred at room temperature for 23 h. The polymer was precipitated in diethyl ether (150 ml). The supernatant was removed and the polymer dried by evaporation. After dissolving in mQ-water (30 ml) Silicagel (13 g) and 2N HCl (2.8 ml) was added to the polymer solution. The mixture was stirred for 23 h at room temperature. The supernatant was filtered and lyophilized to yield 1.62 g poly(MOXA-co-APOXA)-amine as a white solid. Yield: 32.3% 1HNMR (400 MHz, D2O) 3.85-3.38 (m, 260H), 3.38-3.34 (m, 18H), 3.09-2.90 (m, 3H), 2.52-2.89 (m, 15H), 2.20-1.92 (m, 180H), 1.73-1.53 (m, 28H) ppm.

Synthesis of Poly(MOXA-Co-ABOXA)-Amine with 10% APOXA Content DP=100

(39) This polymer was synthesized equivalent to poly(MOXA-co-ABOXA)-amine with 10% ABOXA content DP=50 by changing the ratio of total monomer to methyl trifluoromethanesulfonate initiator ratio to 100:1.

Synthesis of Poly(MOXA-b-APOXA)-Amine

(40) To a solution of 2-methyl-4,5-dihydrooxazole (4.61 g, 54.2 mmol) in dry acetonitrile (17 ml) was added methyl trifluoromethanesulfonate (0.1975 g, 1.2 mmol) under nitrogen. The solution was stirred at 70 C. for 20 h. Then 2-(3-Azidopropyl)-4,5-dihydro-1,3-oxazole (0.93 g, 6.02 mmol) was added to the solution under nitrogen. The solution was stirred at 70 C. for 20 h. The resulting solution was cooled to room temperature. Then Bis(trimethylsilyl)amine (0.5828 g, 3.6 mmol) was added to terminate the reaction. The solution was stirred at room temperature for 23 h. The polymer was precipitated in diethyl ether (150 ml). The supernatant was removed and the polymer dissolved in mQ-water (25 ml). To the polymer solution was added Silicagel (7.5 g) and 2N HCl (2.17 ml). The mixture was stirred for 23 h at room temperature. The supernatant was filtered and lyophilized to yield poly(MOXA-b-APOXA)-amine.

Synthesis of PAcrAm-g-(poly(MOXA-co-APOXA), NH.SUB.2., Si)

(41) A solution of poly(MOXA-co-APOXA)-amine (500 mg, 0.07 mmol) in dimethylformamide (5 ml) was added to a solution of poly(pentafluorophenyl acrylate) (171.8 mg, 0.0071 mmol) in DMF (1.7 ml) and triethylamine (10.7 l, 0.08 mmol). The solution was stirred at 50 C. for 18 h. After the addition of a solution of N-Boc-1,6-hexanediamine (82 mg, 0.32 mmol) and triethylamine (90 l, 0.65 mmol) in DMF (0.82 ml) the solution was stirred at 50 C. for additional 23 h. Then a solution of 3-(ethoxydimethylsilyl)propylamine (104.7 mg, 0.65 mmol) triethylamine (181 l, 1.3 mmol) in DMF (1.05 ml) and was added and the solution stirred at 50 C. for additional 30 h. The resulted mixture was concentrated by evaporation, followed by dissolving in dichloromethane (4.45 ml). To the solution was added trifluoroacetic acid (1.11 ml 14.4 mmol). The solution was stirred at room temperature for 19 h. The solvent was removed by evaporation. The resulted brown oil was washed with diethyl ether (15 ml) three times. The brown oil was concentrated by evaporation, followed by dissolving in ultrapure water (15 ml). The solution was dialyzed against ultrapure water, filtered (0.22 m) and lyophilized to yield 326.96 mg PAcrAm-g-(poly(MOXA-co-APOXA), NH.sub.2, Si) as a white solid. .sup.1HNMR (400 MHz, D2O) 3.75-3.46 (m, 3000H), 3.45-3.33 (m, 150H), 3.14-2.91 (m, 30H), 2.58-2.36 (m, 150H), 2.28-1.92 (m, 2200H), 1.92-1.82 (m, 150H), 1.74-1.28 (m, 300H), 0.16 (s, 270H) ppm.

(42) Coating Experiments on Silicon-Oxide Wafer:

(43) Silicon oxide wafers were cleaned with oxygen plasma for 2 minutes. The wafers were immersed each in 1 ml of a solution of 0.5 mg/ml PAcrAm-g-(poly(MOXA-co-APOXA), NH.sub.2, Si) in HEPES 0 for 30 min. The wafers were washed with mQ-water and blow-dried with nitrogen. The wafers were immersed in a HEPES 2 solution for 15 h, washed with mQ-water and blow-dried with nitrogen. Then the first half of the wafers were immersed in 0.1 mM solution of DBCO-Biotin (DBCO=Dibenzocyclooctyne) in HEPES 2 for 1 h followed by washing with mQ-water and blow-drying with nitrogen. These wafers and the other half of the coated wafers were then immersed in a 0.1 mg/ml solution of streptavidin in HEPES 2 for 30 min.

(44) As shown in FIG. 1, the polymer was successfully coated on a silicon oxide surface. This coating showed a sufficient stability to retain 1.29 nm adlayer thickness after ageing in a polar buffer solution (H2). The coating's azide groups were successfully utilised to modify the surface with DBCO-Biotin via strain promoted azide alkyne click reaction. This enabled the functionalisation of the surface with the protein streptavidin, which was confirmed with an increase in adlayer thickness to 3.34 rm. The specificity of this reaction was proven by the inability of streptavidin to adsorb on the surface without a previous functionalisation with DBCO-Biotin, where the adlayer thickness remained at 1.29 nm.

Synthesis of PAcrAm-g-(poly(MDXA-co-APOXA), NH.SUB.2., ND)

(45) A solution of poly(MOXA-co-APOXA)-amine (300 mg, 0.06 mmol) in dimethylformamide (3 ml) was added to a solution of poly(pentafluorophenyl acrylate) (101.3 mg, 0.0041 mmol) in DMF (1 ml) and triethylamine (35 l, 0.26 mmol). The solution was stirred at 50 C. for 18 h. After the addition of a solution of N-Boc-1,6-hexanediamine (45.7 mg, 0.18 mmol) and triethylamine (50 l, 0.36 mmol) in DMF (0.46 ml) the solution was stirred at 50 C. for 23 h. Then a solution of nitro-dopamine (107.2 mg, 0.36 mmol) and triethylamine (151.2 l, 1.08 mmol) in DMF (1.07 ml) was added and the solution stirred at 50 C. for 22 h. The resulted mixture was concentrated by evaporation. Water (1.8 ml) and trifluoroacetic acid (1.8 ml 23.36 mmol) were added to the mixture. The solution was stirred at room temperature for 20.5 h. The solvent was removed by evaporation, followed by mixing with ultrapure water (3.5 ml). The mixture was dialyzed against ultrapure water, filtered (0.22 m) and lyophilized to yield 326.96 mg PAcrAm-g-(poly(MOXA-co-APOXA), NH2, ND) as a brownish-orange solid. .sup.1HNMR (400 MHz, D.sub.2O) 3.75-3.46 (m, 5080H), 3.45-3.33 (m, 150H), 3.14-2.91 (m, 30H), 2.58-2.36 (m, 150H), 2.28-1.92 (m, 3429H), 1.92-1.82 (m, 150H), 1.74-1.28 (m, 300H) ppm.

Synthesis of PAcrAm-g-(poly(MOXA-co-ABOXA), NH.SUB.2., ND)

(46) A solution of poly(MOXA-co-ABOXA)-amine (500 mg, 0.09 mmol) in dimethylformamide (5 ml) was added to a solution of poly(pentafluorophenyl acrylate) (141.3 mg, 0.59 mmol) in DMF (1.4 ml) and triethylamine (49 l, 0.36 mmol). The solution was stirred at 50 C. for 18 h. After the addition of a solution of N-Boc-1,6-hexanediamine (63.7 mg, 0.25 mmol) and triethylamine (70 l, 0.5 mmol) in DMF (0.64 ml) the solution was stirred at 50 C. for 23 h. Then a solution of nitro-dopamine (149.4 mg, 0.50 mmol) and triethylamine (211 l, 1.51 mmol) in DMF (1.49 ml) was added and the solution stirred at 50 C. for 20 h. The resulted mixture was concentrated by evaporation. Water (3 ml) and trifluoroacetic acid (3 ml 38.94 mmol) were added to the mixture. The solution was stirred at room temperature for 20.5 h. The solvent was removed by evaporation. The brown oil was concentrated by evaporation, followed by mixing in ultrapure water (15 ml). The mixture was dialyzed against ultrapure water, filtered (0.22 m) and lyophilized to yield 135.79 mg PAcrAm-g-(poly(MOXA-co-ABOXA), NH.sub.2, ND) as a brownish-orange solid. .sup.1HNMR (400 MHz, D.sub.2O) 3.82-3.41 (m, 3900H), 3.41-3.28 (m, 188H), 3.14-2.89 (m, 85H), 2.53-2.31 (m, 135H), 2.26-1.94 (m, 2838H), 1.73-1.25 (m, 460H) ppm.

(47) Coating Experiments on Gold Wafer Comparing PAcrAm-g-(Poly(MOXA-Co-ABOXA), NH.sub.2, ND), PAcrAm-g-(Poly(MOXA-Co-APOXA), NH.sub.2, ND) and PAcrAm-g-(poly(MOXA), NH.sub.2, ND):

(48) Gold wafers were cleaned with oxygen plasma for 2 minutes followed by immersion in Ethanol for 10 minutes and blow dried with nitrogen.

(49) Polymer Self Assembly on Gold Wafers:

(50) The wafers were immersed each in either a solution of 1. 0.5 mg/ml PAcrAm-g-(poly(MOXA-co-APOXA), NH.sub.2, ND), 2. 0.5 mg/ml PAcrAm-g-(poly(MOXA-co-ABOXA), NH.sub.2, ND), 3. 0.5 mg/ml PAcrAm-g-(poly(MOXA), NH.sub.2, ND), 4. 1.0 mg/ml PAcrAm-g-(poly(MOXA-co-APOXA), NH.sub.2, ND) or 5. 1.0 mg/ml PAcrAm-g-(poly(MOXA-co-ABOXA), NH.sub.2, ND)
in HEPES 0 for 18.5 h at room temperature. The wafers were washed with mQ-water and blow-dried with nitrogen.
Stability Testing:

(51) The coated wafers were immersed in a HEPES 2 solution (10 mM HEPES, 150 mM NaCl) or NaCl solution (2 M)
for 15 h, washed with mQ-water and blow-dried with nitrogen.

(52) As shown in FIG. 2, when immersed at 0.5 mg/mL PAcrAm-g-(poly(MOXA-co-ABOXA), NH.sub.2, ND) has the thinnest adlayer thickness of 1.1 nm while both PAcrAm-g-(poly(MOXA-co-APOXA), NH.sub.2, ND) and PAcrAm-g-(poly(MOXA), NH.sub.2, ND) form adlayers exceeding 2 nm. After immersion in HEPES 2 or NaCl the adlayer thickness drops below 0.5 nm in case of PAcrAm-g-(poly(MOXA-co-ABOXA), NH.sub.2, ND) while the other two polymers show a much higher resistance towards HEPES 2 and NaCl equal or bigger to 2 nm adlayer thickness.

(53) It is known that generally a thicker layer improves the protein resistance of a coating, therefore experiments at higher concentration were conducted. When immersed at 1.0 mg/mL, the adlayer thickness for PAcrAm-g-(poly(MOXA-co-ABOXA), NH.sub.2, ND) direct after assembly and after ageing in HEPES 2 and NaCl did increases to bigger than 1 nm. At the same conditions (1.0 mg/ml) PAcrAm-g-(poly(MOXA-co-APOXA), NH.sub.2, ND) has a total adlayer thickness of above 2.5 nm. This clearly shows the superior properties of the APOXA containing polymer (n=3) over the ABOXA containing polymer (n=4), which is due to better solubility and smaller steric hindrance of the formed poly(MOXA-co-APOXA) brushes compared to the poly(MOXA-co-ABOXA) brushes leading to a denser and better packed coating.

(54) Bio-Functional Testing:

(55) Polymer coated gold wafers that were either aged in HEPES 2 or NaCl solution are used for this test. The samples are reacted via strain promoted azide-alkyne click chemistry (SPAAC) with DBCO-biotin (0.1 mM in HEPES 2) for 1 hours followed by immersion in a 0.1 mg/mL solution of streptavidin in HEPES 2 for 30 min.

(56) The following 4 sets of coated PAcrAm-g-(poly(MOXA-co-ABOXA), NH.sub.2, ND) and PAcrAm-g-(poly(MOXA-co-APOXA), NH.sub.2, ND) samples were prepared and compared: 1. Aged in HEPES 2 and immersed in a DBCO-biotin solution 2. Aged in HEPES 2 only (Reference no biotin) 3. Aged in NaCl and immersed in a DBCO-biotin solution 4. Aged in NaCl only (Reference no biotin)

(57) As shown in FIG. 3a (HEPES 2 aged samples), both polymers were successfully coated on a gold surface and remained with a significant adlayer thickness after ageing in HEPES 2. Both polymers react with DBCO-biotin as is shown by an increase of adlayer thickness of 0.51 nm for the ABOXA polymer and 0.55 nm for the APOXA polymer. After immersion in the streptavidin solution a further increase in adlayer thickness is observed with 2.34 nm for the ABOXA polymer and 1.47 nm for the APOXA polymer. When no DBCO-biotin was present, no increase in adlayer thickness is observed for the APOXA polymer while a significant non-specific binding of 0.66 nm of streptavidin is detected for the ABOXA polymer. This experiment shows the superior performance of the APOXA polymer with only specific targeted binding compared to the high non-specific binding of the ABOXA version.

(58) As shown in FIG. 3b (NaCl aged samples), both polymers were successfully coated on a gold surface and remained with a significant adlayer thickness after ageing in NaCl. Both polymers react with DBCO-biotin as is shown by an increase of adlayer thickness of 0.57 nm for the ABOXA polymer and 0.72 nm for the APOXA polymer. After immersion in the streptavidin solution a further increase in adlayer thickness is observed with 2.52 nm for the ABOXA polymer and 1.56 nm for the APOXA polymer. When no DBCO-biotin was present, no increase in adlayer thickness is observed for the APOXA polymer while a significant non-specific binding of 0.84 nm of streptavidin is detected for the ABOXA polymer. This experiment shows again the superior performance of the APOXA polymer with only specific targeted binding compared to the high non-specific binding of the ABOXA version.

(59) Protein Resistance Testing:

(60) Polymer coated gold wafers that were either aged in HEPES 2 or NaCl solution are used for this test. The following 6 sets of coated PAcrAm-g-(poly(MOXA-co-ABOXA), NH.sub.2, ND), PAcrAm-g-(poly(MOXA-co-APOXA), NH.sub.2, ND) and PAcrAm-g-(poly(MOXA), NH.sub.2, ND) samples were prepared and compared: 1. PAcrAm-g-(poly(MOXA-co-ABOXA), NH.sub.2, ND) coated aged in HEPES 2 2. PAcrAm-g-(poly(MOXA-co-ABOXA), NH.sub.2, ND) coated aged in NaCl 3. PAcrAm-g-(poly(MOXA-co-APOXA), NH.sub.2, ND) coated aged in HEPES 2 4. PAcrAm-g-(poly(MOXA-co-APOXA), NH.sub.2, ND) coated aged in NaCl 5. PAcrAm-g-(poly(MOXA), NH.sub.2, ND) coated aged in HEPES 2 6. PAcrAm-g-(poly(MOXA), NH.sub.2, ND) coated aged in NaCl 7. Bare Gold surface as reference

(61) All samples were immersed in full human serum for 30 min, washed first with HEPES 2 buffer followed by washing with ultrapure water. Protein resistance is expressed in % as reduction of protein uptake on polymer coated sample compared to uncoated reference sample.

(62) Result:

(63) TABLE-US-00009 Polymer: Ageing: Protein resistance 1. ABOXA HEPES 2 55 18% 2. ABOXA NaCl 46 15% 3. APOXA HEPES 2 94 4.8% 4. APOXA NaCl 91 8.2% 5. MOXA HEPES 2 98 0.96% 6. MOXA NaCl 97 10.2% 7. 0%

(64) This experiment again shows the superior performance of the APOXA (n=3) and MOXA (reference) polymers in respect to resistance to protein fouling compared to the ABOXA (n=4) polymer. The shorter (n=3) spacer in case of the APOXA polymer increases its hydrophilicity and reduces possible hydrophobic interactions with proteins leading to very low protein fouling (>90% protein resistance) close to the non-functionalized MOXA reference while the ABOXA version does show a significant fouling with proteins due to the longer more hydrophobic alkyl spacer group.