MONOMERS

FIELD OF THE INVENTION

[0001] This invention involves the production and purification of carboxylic betaine ^" zwitterionic monomers.

BACKGROUND OF THE INVENTION

[0002] The betaine compound having both a cationic group and an anionic group within its molecule has been attracting attention because of this structural feature. Conventional methods for producing betaine-type compounds are by reaction of a tertiary amine with beta-propiolactone (see, for example, Fiedorek, 1960, U.S. Pat. No. 2,548,428) and by reaction of a tertiary amine with an alkali-metal salt of a halogenated monocarboxylic acid (see, for example, Schuller et al, U.S. Pat. No. 2,958,682). For the first case, the use of toxic and expensive reagent such as beta-propiolactone makes it difficult for large-scale production. For the latter case, strict thermal and pH reaction conditions are required, making it hard to achieve higher polymer conversion and yield.

[0003] Japanese Kokai Publication Hei-5-32600 discloses a process for producing a betaine compound which comprises reacting an aliphatic primary amine with a quaternary ammonium compound having a defined structure at 6<pH<8 and further reacting the reaction product with a defined halogenated lower carboxylic acid at 6<pH<8. The betaine compound produced by this process has foaming and detergent properties so that it can be used as a surfactant for hair and body cleansing.

[0004] Japanese Kokai Publication Hei-5-294905 discloses a carbobetaine compound and a process for its production. This process for producing a carbobetaine compound comprises reacting a defined amino compound with a defined halogen-containing salt. The carbobetaine compound obtained by this process has an emolient action on hair and skin for hair care and skin care products. [0005] Patents US4012437 (1977) and US3689470 (1972) describe the synthesis of the carboxylic betaine methacrylate (CBMA) by reacting dimethylaminoethyl methacrylate (DMAEMA) with acrylic acid in aqueous media or in organic solvent. However, the reaction media was used directly for further polymerization, no monomer purification process was described.

SUMMARY OF THE INVENTION

[0006] One of the various aspects of the invention is a method for preparing a monomer comprising a carboxylic betaine, the monomer having the structure of Formula 2

The method comprises contacting a tertiary amine having the structure of Formula 1

with acrylic acid to form a reaction product comprising a monomer: acid complex; contacting the reaction product with a base to form the monomer; and isolating the monomer in a solid form, wherein A is oxygen, methylene, ester, amide, benzyl, pyridinyl, or imidazolyl; R is hydrogen, methyl, ethyl, propyl, butyl, pentyl, or hexyl; and n is an integer from 0 to 10.

[0007] Other objects and features will be in part apparent and in part pointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWING

[0008] Figure 1 is an ^X H NMR spectrum of the carboxybetaine methacrylate (CBMA) monomer. [0009] Figure 2 is a ^Χ Η NMR spectrum of the carboxybetaine acrylamide (CBAA) monomer.

[0010] Figure 3 is a ^X H NMR spectrum of the carboxybetaine methacrylamide

(CBMAA) monomer.

[0011] Figure 4 is a representative surface plasmon resonance (SPR) sensorgrams showing ultra low non-specific protein adsorption to gold surfaces coated with polyCBMA (monomer produced according to procedure in example 4) via atom-transfer radical

polymerization (ATRP) upon exposure to either fibrinogen or lysozyme, i.e., 0.2 ng/cm ² for fibrinogen and 0.1 ng/cm ² for lysozyme. Ultra low fouling is defined as <5 ng/cm ² adsorbed proteins.

[0012] Figure 5 is a representative SPR sensorgrams showing ultra-low non-specific protein adsorption to gold surfaces coated with polyCBAA (monomer produced according to procedure in example 6) via ATRP upon exposure to either undiluted blood plasma or serum, i.e., 3.6 ng/cm ² for plasma and 2.2 ng/cm ² for serum.

[0013] Figure 6 is a continuous synthesis flow chart.

DETAILED DESCRIPTION ON THE INVENTION

[0014] According to particular aspects, methods to produce and purify the carboxylic betaine monomer into a solid are herein disclosed. According to further particular aspects, the invention disclosed herein relates to purification methods that can separate carboxylic betaine monomer from its reaction mixture and a solid can be obtained.

[0015] The carboxylic betaine zwitterionic monomer can be synthesized via the reaction of acrylic acid and the corresponding tertiary amines.

Scheme 1 : Reaction scheme for the synthesis of the carboxylic betaine monomer.

[0016] More generally, the monomer comprising a carboxylic betaine having the structure of Formula 2 ··:: - ,

can be prepared by contacting a tertiary amine having the structure of Formula 1

with acrylic acid to form a reaction product comprising a monomenacid complex wherein A, R, and n are as defined above. The reaction product is contacted with a base to form the monomer and the monomer is isolated in a solid form, wherein A is oxygen, methylene, ester, amide, benzyl, pyridinyl, or imidazolyl; R is hydrogen, methyl, ethyl, propyl, butyl, pentyl, or hexyl; and n is an integer from 0 to 10.

[0017] In Formulae 1 and 2, A can be ester amide, benzyl, pyridinyl, or imidazolyl.

[0018] In Formulae 1 and 2, R can be hydrogen and methyl.

[0019] In Formulae 1 and 2, n can be an integer from 0 to 10. [0020] Also, for Formulae 1 and 2, A is ester, amide, benzyl, pyridinyl, or imidazolyl; R is hydrogen or methyl; the base is an inorganic base, an organic base, or a basic ion exchange resin; and the solid form is a powder.

[0021] Further, for Formulae 1 and 2, A can be ester or amide. When A is an ester, R can be hydrogen or methyl. When A is an ester, n can be 2 or 3. When A is an ester, R can be hydrogen or methyl and n can be 2 or 3.

[0022] For Formulae 1 and 2, when A is an amide, R can be hydrogen or methyl. Also, when A is an amide, n can be 3. Additionally, when A is an amide, R can be hydrogen or methyl and n can be 3.

[0023] The solid form can be a powder or a crystal form. Additionally, the solid form can be a powder form.

[0024] The reaction of a tertiary amine and acrylic acid can be carried out with or without solvent. The total concentration of acrylic acid and amine in the solvent medium can be anywhere from 5 to 99% by weight and is preferably about 60 to 95% by weight.

[0025] Generally, a relatively large ratio of acid to amine is preferred in order to favor the shift of the equilibrium toward the formation of the betaine. For practical purposes the ratio of acid to amine is from 1 : 10 to 10: 1, 1 :5 to 5: 1, 1 :2 to 2: 1, or 1 : 1 to 5: 1. Preferably, the ratio of acrylic acid to amine of formula 1 is 1 :2 to 2: 1. For example, 2: 1 to 3: 1 mole ratios can provide equilibrium conversions of amine to betaine as high as 80-90%.

[0026] The reaction of the tertiary amine of formula 1 with the acrylic acid can take place under acidic conditions. For example, this reaction can take place in the presence of an acid. Preferably, the acid comprises acetic acid.

[0027] For the production of polymerizable monomeric betaines, the use of two moles of acrylic acid for each mole of the tertiary amine (e.g., dimethylaminoethyl methacrylate) often produces what appears to be a complex of one mole of the betaine with one mole of the acid.

This betaine:acid complex can be separated into a white crystalline solid from the reaction media in the presence of organic solvent (e.g., hexane, acetone, or THF) with or without adding additional acids (e.g., HC1 or H ₂ SO ₄ ) to the reaction mixture. This betaine:acid complex

(where acid can be HC1, H2SO4, acrylic acid, or acetic acid (when acetic acid is present) can be used directly for further applications. The betaine:acid complex can be a betaine

(monomer): acrylic acid complex or a betaine (monomer): acetic acid complex when acetic acid is present.

[0028] Further purification can be applied to obtain pure betaine monomer of Formula 2. This purification process can be carried out using the reaction mixture directly or the separated betaine:acid as the starting materials. The acid can be removed from the reaction media or the betaine:acid complex by contacting the reaction mixture with a base. The base can be an inorganic base, organic base or base ion exchange resin.

[0029] When the base is an organic base, the organic base can be an amine having the structure NR R2R ₃ , where Ri, R2 and R ₃ are independently hydrogen or Ci to C12 alkyl. In particular, the amine can be a trialkylamine; specifically, trimethylamine, triethylamine, tripropylamine, tributylamine, tripentylamine, trihexylamine, triheptlyamine, trioctylamine, trinonylamine, tridecylamine, triundecylamine, tridodecylamine, ethyldimethylamine, methyldiethylamine, methyldipropylamine, propyldimethylamine, ethyldipropylamine, propyldiethylamine, or a combination thereof. Preferably, the amine comprises

trimethylamine.

[0030] Alternatively, an anhydrous organic solvent such as THF, dioxane, chloroform, dichloromethane, toluene, DMF, acetone, or a combination thereof can be mixed with the organic base, and then added to the reaction mixture. In this manner, the zwitterionic monomer can be precipitated from the reaction mixture.

[0031] The inorganic base can be sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium carbonate, potassium bicarbonate, potassium hydroxide, calcium carbonate, calcium bicarbonate, calcium hydroxide, magnesium carbonate, magnesium bicarbonate, magnesium oxide, or a combination thereof.

[0032] The basic ion exchange resin can be a resin that can remove the acid from the zwitterionic monomer: acid complex. The reaction mixture can be diluted with anhydrous solvent such as methanol or ethanol before passing through the base resin. The monomer concentration in the diluted reaction mixture can be in the range of 10-90%. The solutions after ion exchange resin treatment can be concentrated under vacuum, and then added into nonsolvent such as anhydrous tetrahydrofuran (THF), dioxane, chloroform, dichloromethane, toluene, dimethyl formamide (DMF), acetone, or a combination thereof for the precipitation of the monomer. A white solid can be obtained by this separation method.

[0033] These zwitterionic monomers can also be manufactured via a continuous process. The tertiary amine based monomer and the acrylic acid are pumped into a reactor from their corresponding stock solutions. The reaction mixture is then pumped into a precipitation flask, where a precipitation solvent (e.g., mixture of organic solvent and trialkylamine base) is also pumped in at the same time. A white solid can be formed quickly in the precipitation flask. These solid/liquid mixtures can be transferred into a filtration device to separate the solid from the liquid. All the above process can be set to run continuously. A general schematic of this process is depicted in Figure 6.

[0034] The obtained monomer purity can be assessed by ^X H NMR. Figures 1-3 show the NMR spectra of the obtained CBMA, CBAA, and CBMAA monomers. The monomer purity can be from 50% to 99% pure. The monomer purity can be greater than 93%. A monomer purity of greater than 99% can be achieved by further purification. The further purification step can include recrystallization.

[0035] The quality of these monomers can be further assessed using protein adsorption monitored by surface plasmon resonance (SPR) sensors when the monomers are polymerized onto SPR surfaces to form a coated surface or substrate. Nonspecific protein adsorption on these zwitterionic polymer coated gold surfaces are lower than about 30 ng/cm ² , particularly lower than about 5ng/cm ² and as low as 0.3 ng/cm ² for single-protein solution and undiluted blood plasma and serum.

[0036] Unless otherwise indicated, an alkyl group as described herein alone or as part of another group is an optionally substituted linear saturated monovalent hydrocarbon radical containing from one to twenty carbon atoms and preferably one to eight carbon atoms, or an optionally substituted branched saturated monovalent hydrocarbon radical containing three to twenty carbon atoms, and preferably three to eight carbon atoms. Examples of unsubstituted alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, s-pentyl, t-pentyl, and the like. [0037] The term "amide" as used herein represents a bivalent (i.e., difunctional) amido

O

v linkvage r (i.e., c ¹¹ N ¹ , ).

[0038] The term "ester" as used herein represents a bivalent (i.e., difunctional) ester linkage (i.e., -C(O)O-).

[0039] The term "methylene" as used herein represents a -CH ₂ - group.

[0040] Having described the invention in detail, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims.

[0041] The following modes of operation are suggested by way of illustration. EXAMPLES

[0042] The following non-limiting examples are provided to further illustrate the present invention.

EXAMPLE 1

[0043] DMAEMA (1.0 mol, 170 mL) was added to a 2.0 L flask equipped with a mechanical stirrer, the flask was then cooled to 0 C using an ice bath. Acrylic acid (2.0 mol, 140 mL) was added dropwise to the flask within 30 minutes. The reaction was carried out at 0 C for another 30 minutes, then at room temperature for 4 hours. The reaction solution became gradually more viscous indicating the reaction was ongoing. The above solution was then diluted with 100 mL ethanol and stirred overnight. Hexane (500mL) was then added under stirring into the reaction mixture. The flask containing the above mixture was sealed and left at room temperature. Within a few days, a lot of white solid precipitated from the mixture. The solid was then filtered and washed with hexane and dried under vacuum. NMR analysis showed that it was a betaine:acid complex. EXAMPLE 2

[0044] DMAEMA (1.0 mol, 170 mL) was added to a 2.0 L flask equipped with a mechanical stirrer, the flask was then cooled to 0 C using an ice bath. Acrylic acid (2.0 mol, 140 mL) was added dropwise to the flask within 30 minutes. The reaction was carried out at 0 C for another 30 minutes, then at room temperature for 4 hours. The reaction solution became gradually more viscous indicating the reaction was ongoing. The above solution was then diluted with 100 mL ethanol and stirred overnight.

[0045] Amberlyst@21 (650 grams; 4.6mmol/g; 3.0eq) (a weak ion exchange resin) suspended in 1.0 L ethanol was then added to the above monomer solution under stirring at room temperature to neutralize the excess amount of acrylic acid. After 3 hours, the resin was filtered off and washed with 100 mL ethanol. The solvent was removed using a rotary evaporator. The obtained viscous solution was then slowly added to dry acetone (10 times the volume of solution) under stirring. White CBMA crystals precipitated from the mixture. The solid was then filtered, washed with dry ether, and dried under vacuum. The total yield was about 70% (150grams).

EXAMPLE 3

[0046] DMAEMA (l.Omol, 170mL) together with 0.2 g radical inhibitor was added to a 2.0 L flask (Figure 1) equipped with a mechanical stirrer, the flask was then cooled to 0 C using an ice bath. Acrylic acid (2.0 mol, 140 mL) was added dropwise to the flask within 30 minutes. The reaction was carried out at 0 C for another 30 minutes, then at room temperature for 4 hours. The reaction solution became gradually more viscous indicating the reaction was ongoing. The above solution was then diluted with 100 mL ethanol and stirred overnight. The above solution was then diluted with another 200 mL methanol, 600 mL acetone, and 200 mL triethylamine was then added under stirring. After 30 minutes, a white solid precipitated from the mixture. The solid was then filtered, washed with dry acetone, and dried under vacuum. The total yield was about 75%. EXAMPLE 4

[0047] Dimethylaminoethyl methacrylate (DMAEMA, 0.48mol, 76g), together with 0.14 g radical inhibitor were added to a 2.0 L flask (Figure 1) equipped with a mechanical stirrer, the flask was then cooled to 0 C using an ice bath. Acetic acid (0.47 mol, 27 mL) and acrylic acid (0.95 mol, 65 mL) were sequentially added dropwise to the flask within 30 minutes. The reaction was carried out at 0 C for another 30 minutes, then at room temperature for 4 hours. The reaction solution became gradually more viscous indicating the reaction was ongoing.

Acetone (50 mL) was added to dilute the reaction mixture and the reaction mixture was stirred overnight. To the above solution was added over 1 hour a 1 : 1 volume ratio of

acetone/triethylamine solution under stirring. A white solid precipitated from the mixture. The solid was then filtered out, washed with dry acetone, and dried under vacuum. The total yield was about 75%.

EXAMPLE 5

[0048] Dimethylamino propyl acrylamide (DMAPA, 1.0 mol, 156 g) together with 0.2 g radical inhibitor was added to a 2.0 L flask (Figure 1) equipped with a mechanical stirrer, the flask was then cooled to 0 C using an ice bath. Acrylic acid (2.0 mol, 140 mL) was added drop wise to the flask within 30 minutes. The reaction was carried out at 0°C for another 30 minutes, then at room temperature for 4 hours. The reaction solution became gradually more viscous indicating the reaction was ongoing. The above solution was then diluted with 100 mL ethanol and stirred overnight. The above solution was then diluted with another 200 mL methanol, 600 mL acetone, and 200 mL triethylamine was then added under stirring. After 30 minutes, a white solid precipitated from the mixture. The solid was then filtered, washed with dry acetone, and dried under vacuum. The total yield was about 78%.

EXAMPLE 6

[0049] Dimethylamino propyl acrylamide (DMAPA, 1.0 mol, 156 g), and 200 mL methanol, together with 0.2 g radical inhibitor were added to a 2.0 L flask (Figure 1) equipped with a mechanical stirrer, and the flask was then cooled to 0 C using an ice bath. Acetic acid (1.0 mol, 57 mL) and acrylic acid (1.0 mol, 70 mL) were sequentially added drop wise to the flask within 40 minutes. The reaction was carried out at 0°C for another 30 minutes, then at room temperature for overnight. To the above solution was added over one hour a 1 : 1 volume ratio of acetone/triethylamine solution under stirring. A white solid precipitated from the solution. The solid was then filtered, washed with dry acetone, and dried under vacuum. The total yield was about 75%.

EXAMPLE 7

[0050] Dimethylamino propyl methacrylamide (DMAPMA, 1.0 mol, 168 g) together with 0.2 g radical inhibitor was added to a 2.0 L flask (Figure 1) equipped with a mechanical stirrer, the flask was then cooled to 0 C using an ice bath. Acrylic acid (2.0 mol, 140 mL) was added drop wise to the flask within 30 minutes. The reaction was carried out at 0 C for another 30 minutes, then at room temperature for 4 hours. The reaction solution became gradually more viscous indicating the reaction was ongoing. The above solution was then diluted with 100 mL ethanol and stirred overnight. The above solution was then diluted with another 200 mL methanol, 600mL acetone, and 200mL triethylamine was then added under stirring. After 30 minutes, a white solid precipitated from the mixture. The solid was then filtered, washed with dry acetone, and dried under vacuum. The total yield was about 70%.

EXAMPLE 8: Continuous synthesis

[0051] Stock solution A: 3.9 M DMAPA, 7.8 mM hydroquinone, in MeOH; Stock solution B: 3.9 M acetic acid, 3.9 M acrylic acid, in MeOH; Stock solution C: 1 : 1 volume TEA/acetone. DMAPA (1 mol) was added to a 2 liter flask, followed by 200 mL methanol and 0.23 g hydroquinone. The mixture was cooled with an ice bath. Acetic acid (1 mol) and 1 mol acrylic acid were slowly added to the flask and was stirred at room temperature for 24 hours.

[0052] Stock solution A and B were than pumped into the reaction flask at a speed of 0.15 mL/minute. At the same time, the reaction mixture was pumped out at a speed of 0.30 mL/minute to a precipitation flask (containing 60 mL stock solution C), stock solution C was added into the precipitation flask at a speed of 0.9 mL/minute via another pump. A schematic of the continuous process is depicted in Figure 6.

[0053] When the reaction mixture and precipitating agent are pumped into the flask, it gets full and the mixture overflows to the filtration funnel. CBAA monomer solid was collected in the filtration funnel.

[0054] For all the examples (Examples 1-8) shown above, the monomer purity could be increased by a further purification cycle such as dissolving the monomer into methanol, and precipitation in an inorganic solvent in the presence of organic base.

EXAMPLE 9: Protein adsorption measurements

[0055] Glass chips were first coated with an adhesion-promoting chromium layer (thickness 2 nm) and a surface plasmon active gold layer (48 nm) by electron beam evaporation under vacuum. Before self-assembling monolayer (SAM) preparation, the substrates were washed with pure ethanol, cleaned under UV light, and washed with water and pure ethanol. SAMs were formed by soaking gold-coated substrates in pure ethanol solution of

ω-mercaptoundecyl bromoisobutyrate at room temperature after careful cleaning.

[0056] The substrate with immobilized initiators was then placed in a reaction tube, sealed with rubber septum stoppers, and degassed with nitrogen for 30 minutes. Degassed CBMA solution (pure water and methanol in a 1 : 1 volume ratio) and bipyridine (bpy)/CuBr solution were then transferred to the tube using syringe under nitrogen protection. After the reaction, the substrate was removed and rinsed with ethanol and water, and the samples were kept in water overnight. Rinsing with phosphate buffered saline (PBS) buffer was also performed to remove unbound polymers before testing.

[0057] Protein adsorption was measured with a custom-built SPR sensor based on wavelength interrogation. A SPR chip was attached to the base of the prism, and optical contact was established using refractive index matching fluid (Cargille). A dual-channel flow cell with two independent parallel flow channels was used to contain the liquid sample during experiments. A peristaltic pump (Ismatec) was utilized to deliver the liquid sample to the two channels of the flow cell. A fibrinogen solution of 1.0 mg/mL in PBS (0.15 M, pH 7.4) was flowed over the surfaces at a flow rate of 0.05 mL/minute. Protein adsorption from lysozome, undiluted blood serum or plasma was also performed in a similar way. A surface-sensitive SPR detector was used to monitor protein-surface interactions in real time. In this study, wavelength shift was used to measure the change in surface concentration (mass per unit area). Figures 4 and 5 provide two examples of protein adsorption on surfaces covered with poly(CBAA) or poly(CBMA) polymers via ATRP using the monomers produced in this work. Protein adsorption was measured by SPR.

EXAMPLE 10

Comparison of plasma adsorption before and after antibody immobilization, antibody immobilization amount and film thickness from three batches of CBAA monomer produced

Batch Thickness Anti-TSH Plasma Plasma

(nm) 2 Pre-fouling Post-fouling

(ng/cm ) 2 2

2 23.9 366.6 3.2 6.3

3 22.1 336.5 5.1 8.3

[0058] In this study, three different batches of monomer (1, 2, and 3) were used in surface-initiated atom transfer radical polymerization (SI-ATRP) to form poly(carboxybetaine ) (pCB) polymer films on gold coated Surface Plasmon Resonance (SPR) biosensor chips. The synthesis of the three batches of monomer and their subsequent SI-ATRP polymerization were all performed under identical conditions. The monomer was synthesized as described above. For each batch shown in the Table, six SPR chips were coated using an SI-ATRP reactor and the average results from two of those six chips selected for testing are provided. The pCB film thickness was measured by ellipsometry. The initial fouling to undiluted human plasma on the non-antibody functionalized pCB surface (Plasma Pre-fouling), the antibody immobilization level (Anti-TSH), and the fouling to undiluted human plasma on the antibody functionalized surface (Plasma Post-Fouling) were all quantified using an SPR biosensor. The antibody immobilization was performed by first activating the pCB film using conventional

l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) amino-coupling agents followed by injecting an aqueous antibody solution and then subsequently by surface deactivation of residual amine-reactive agents. All three independent batches revealed high antibody immobilization levels and very low levels of non-specific binding to undiluted human plasma on both the non-functionalized and antibody functionalized pCB films. The exact results for different surface-initiated pCB films are dependent upon the polymerization conditions used.

[0059] When introducing elements of the present invention or the preferred embodiments(s) thereof, the articles "a", "an", "the" and "said" are intended to mean that there are one or more of the elements. The terms "comprising", "including" and "having" are intended to be inclusive and mean that there can be additional elements other than the listed elements.

[0060] In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results attained.

[0061] As various changes could be made in the above products and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.