WO2005037432A1

WO2005037432A1 - Activation and stabilization of sensitive catalysts in segmented amphiphilic networks

Info

Publication number: WO2005037432A1
Application number: PCT/EP2004/008846
Authority: WO
Inventors: Nico Bruns; Jörg TILLER
Original assignee: Universität Freiburg
Priority date: 2003-09-22
Filing date: 2004-08-06
Publication date: 2005-04-28
Also published as: EP1670587A1; DE10343794A1

Abstract

The invention relates to a catalyst system which can activate and stabilize sensitive catalysts, particularly enzymes and peptides, such that synthesis reactions, for example, such as the synthesis of active ingredients, can also be carried out efficiently in organic solvents. The invention relates in particular to an immobilized catalyst. The catalyst is introduced into a segmented amphiphilic polymer network with a specific nanophase separation.

Description

Activation and stabilization of sensitive catalysts in segmented amphiphilic networks

The present invention relates to a catalyst system which is capable of stabilizing and activating sensitive catalysts, such as, in particular, enzymes and peptides, so that, for example, synthesis reactions, such as in particular those for active substance synthesis, can also be carried out efficiently in organic solvents. In particular, the present invention relates to an immobilized catalyst, the catalyst being introduced into a segmented amphiphilic polymer network with a specific nanophase separation.

Enzymes and other enantioselective catalysts are particularly important in drug synthesis. However, many of these catalysts cannot be used on an industrial scale because they are sensitive to the solvent used or are not very active. In the amounts used, metal catalysts or organometallic catalysts are often highly toxic to the environment due to their high heavy metal content.

In addition, the natural environment of biocatalysts, such as enzymes in particular, is aqueous. However, water is a poor solvent for many preparative reactions in organic chemistry, since most organic compounds are insoluble in this medium. In addition, the removal of water from reaction mixtures is time and energy consuming due to its high boiling point and high evaporation enthalpy. In addition, side reactions such as hydrolysis, racemization or polymerizations increasingly occur in aqueous solution. Furthermore, the products formed are often unstable in the presence of water. To avoid these problems, most organic syntheses are carried out in organic solvents. However, insofar as the natural environment of biocatalysts, in particular enzymes, is aqueous, this complicates the use of such catalysts in organic ones Solvents, as enzymes and other proteins are usually less stable and less active in them. Enzymes are essentially insoluble in organic solvents, but some of them are also "immobilized" for use in these media. In this case one speaks of a support. It also facilitates the separation of the catalysts, since only larger particles have to be filtered off In addition, a larger surface area of the biocatalyst and, among other things, a higher activity is achieved by a support. Mostly, an adsorption of the enzyme on micro- or macroporous supports such as silica, celite or glass is used. In many cases, such leads However, immobilization does not achieve the desired success because the immobilized substance is no longer active or accessible due to conformal changes or diffusion problems.

Amphiphilic polymers have so far rarely been used in this connection. The literature describes only the immobilization of lipases by adsorption onto microporous amphiphilically modified polyallyl methacrylate-co-polyglycidyl methacrylate particles and onto copolymers of polystyrene crosslinked with divinyl and surfactant monomers; see. Yasuda et al., Macromolecular Chemistry and Physics, 2001, vol. 202 (16), pages 3189-3197, and 2002, vol. 203, pages 284-293; Journal of Polymer Science: Part A: Polymer Chemistry, 2002, vol. 40, pages 874-884, or Journal of Chemical Engineering of Japan, 2003, vol. 35 (6), pages 519-526.

Tetrahedron Letters 44 (2003), pages 2379-2382, describes a complex or aggregate of Pd ²⁺ and amphiphilic copolymers. Chemistry Letters (8) (1991), pages 1303-1306, describes a system composed of horseradish peroxidase and amphiphilic block copolymers, ie surfactants. However, the molecular structure of this system is rather an aggregate in the sense of inverse micelles. US-A-5,073,381 describes amphiphilic drug delivery networks.

The present invention is therefore based on the object of providing a catalyst system which is capable of stabilizing and activating enzymes, peptides and other enantioselective catalysts, so that organic Synthesis reactions, in particular those for the synthesis of active substances, can also be carried out efficiently in organic solvents, or if metal catalysts or organometallic catalysts are provided, even in aqueous solvents.

This object is achieved by the embodiments characterized in the claims.

In particular, an immobilized catalyst or catalyst system is provided, the catalyst, in particular an enzyme or a peptide, in a segmented amphiphilic network composed of at least one hydrophilic and one hydrophobic component, which separates the nanophases from the hydrophilic and the hydrophobic Component-formed units in the range from 5 nm to 500 nm, preferably 8 to 300 nm, particularly preferably 8 to 50 nm, is introduced. Such an immobilized catalyst can be produced by a method comprising the steps:

(i) Production of a segmented amphiphilic polymer network by copolymerization of two macromonomers with different hydrophilicity or a monomer and a macromonomer with different hydrophilicity and

(ii) Loading the polymer network with the catalyst.

The present invention is directed to the immobilization of sensitive catalysts, such as in particular enzymes or peptides, in a segmented amphiphilic polymer network. A segmented network is generally understood to mean network structures in which a polymer A acts as a macromolecular crosslinker for a polymer B. According to the invention, the segmented, amphiphilic polymer network of the present invention has a nanophase separation, so that hydrophilic and hydrophobic units of the polymer network are adjacent in the range from 5 nm to 500 nm. In this respect the phase separation is observed or determined via AFM measurements, the three-dimensional phase separation per se is translated into a one-dimensional quantity within the scope of the present invention. This nanophase separation occurs both on the surface and in the mass of the segmented, amphiphilic polymer network used according to the invention. This special network configuration enables free access to the interior thereof both for catalysts such as enzymes or peptides (in the aqueous phase) and substrates (in the organic phase), which is essential for the catalyst system concept according to the invention, for example when it is used for the synthesis of active substances.

Journal of Molecular Catalysis A: Chemical 129 (1998), pages 27-34, and Journal of Molecular Catalysis A: Chemical 130 (1998), pages 85-93, describes catalytically active Pd particles in an amphiphilic network which contains hydrophilic and hydrophobic domains. However, it is not a segmented network in which there is a phase separation of the units formed from the hydrophilic and the hydrophobic component in the order of 5 nm to 500 nm, since only low-molecular monomers are used to generate the network, so that only a very small-sized phase separation is achieved.

Basically, the nanophase separation is designed according to the invention such that the size of the domains is not smaller than the catalyst to be stored. The nanophase separation can be confirmed, for example, by AFM measurements. Depending on the type of solvent (aqueous, organic), either the hydrophilic or the hydrophobic regions swell in the polymer network used according to the invention. As a result, a catalyst, such as, in particular, an enzyme or a peptide, can diffuse into the amphiphilic network in a hydrophilic (hydrophobic) solvent and then develop catalytic activity in another hydrophobic (hydrophilic) solvent. The distribution of the catalyst in the amphiphilic polymer network used according to the invention is homogeneous. In addition, the close proximity of hydrophilic and hydrophobic areas ensures low diffusion paths. Both are prerequisites for an efficient catalytic reaction. 1 schematically illustrates the catalyst system concept according to the invention, in which, for example, an enzyme in a hydrophilic (aqueous) solvent diffuses into the hydrophilic phase of the amphiphilic, segmented network and then develops its catalytic activity in another hydrophobic (organic) solvent.

In a preferred embodiment of the present invention, the catalyst used is an enzyme or a peptide, preferably an enzyme. The class of the enzyme incorporated in the network is not specifically limited. In the context of the present invention, oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases are suitable as enzymes. In a particularly advantageous manner, peroxidases or, as their subgroup, heme enzymes, which contain the complex iron-protoporphyrin IX as a prosthetic group, and hydrolases can be used in the context of the present invention. In particular, horseradish peroxidase (HRP) and chloroperoxidase (CPO) should be mentioned as the corresponding peroxidases and trypsin as the corresponding hydrolase. Transport proteins such as hemoglobin and myoglobin are particularly suitable as peptides.

Peroxidases can catalyze a number of synthetically useful reactions, such as polymerizations of electron-rich aromatics, asymmetric sulfoxidations, and epoxidations. The potential of peroxidases in the area of (asymmetrical) biotransformations is currently hardly exploited in a commercial synthesis, since these enzymes have two major disadvantages. On the one hand, peroxidases such as heme enzymes are inactivated by peroxides. On the other hand, the poor water solubility of many synthetically interesting substrates in peroxidases, just like in most other enzymes, limits the commercial application of the enzymatic biotransformations. According to the invention, a solution to this general problem is achieved by immobilizing or embedding such enzymes, for example peroxidases, in a segmented amphiphilic polymer matrix with the above-mentioned nanophase separation. In addition to such enzymes, sensitive metal catalysts or organometallic catalysts can also be used in the context of the present invention, which are stabilized and activated by immobilization or embedding in an amphiphilic polymer matrix. Water-sensitive metal catalysts can thus be stabilized and activated by inclusion in the hydrophobic phases or regions of the segmented amphiphilic polymer networks used according to the invention. The sensitive metal catalysts which can be used in the context of the present invention are the Sharpless catalyst for the asymmetric epoxidation of allyl alcohols, Grubbs catalysts for the metathesis of alkenes or alkynes or else Pd nanoparticles for the Heck reaction or for hydrogenation, to call.

Polymer networks are polymers in which the polymer chains are linked three-dimensionally. In a network, all polymer chains are linked to one another, there is a single large molecule. In principle, networks can be synthesized by crosslinking linear polymer chains by means of a polymer-analogous reaction or by crosslinking copolymerization with a low molecular weight crosslinker. In the copolymerization, macromonomers can also be used instead of monomers. This term is understood to mean oligomers or polymers with polymerizable groups. The copolymerization of a macromonomer modified at both end groups with a polymerizable group and a low molecular weight monomer creates networks with a segmented topology which is between that of grafted or comb polymers and that of interpenetrating networks. Segmented polymer networks of this type, as provided in the context of the present invention, are usually referred to as poly (A) -linked-poly (B), where poly (A) is a polymeric crosslinker for the monomer B.

Chemically cross-linked polymers are generally insoluble in all solvents. However, if you put a polymer network in a solvent that is well compatible with the polymer segments, solvent will penetrate the sample. The Polymer swells until a balance is reached between the osmotic pressure of the solvent and the elastic restoring force of the chain segments. A swollen network is called a gel.

If, in a network composed of two components, one component is hydrophilic and the other hydrophobic, the network can be swellable in a wide range of compositions both in organic solvents and in aqueous media. One then speaks of an amphiphilic network. The strong incompatibility between the polar and non-polar components or chain segments leads to phase separation, but the covalent bonds between the segments prevent macroscopic phase separation, there is only a much smaller separation, and domains in the nanometer range are formed. The networks appear macroscopically homogeneous and optically clear. Amphiphilic networks are made up of immiscible, i.e. incompatible polymer chains built. The most common synthesis strategy in the prior art is the macromonomer method discussed above, which provides for the copolymerization of a macromonomer with a low molecular weight monomer and, viewed topologically, leads to segmented networks.

The segmented amphiphilic networks used according to the invention can be produced by copolymerizing a monomer (hydrophilic or hydrophobic) with a macromonomer (hydrophobic or hydrophilic) or by copolymerizing two macromonomers of different hydrophilicity. In step (i) of the process according to the invention, a hydrophobic macromonomer is preferably copolymerized with a hydrophilic monomer.

The hydrophobic component of the segmented, amphiphilic network is not subject to any specific restriction. For example, monomers or components based on alkyl (meth) acrylates (eg methyl methacrylate, n-butyl methacrylate, n-hexyl methacrylate), styrene, vinyl acetate, olefins (such as propylene, ethylene, isoprene, cyclooctadiene, etc.), Poly (ε-caprolactone), lactic acid, di-isocyanates, diols, diamines, diacids, polylactides, polypropylene fumarate, Diglycidyl ether of bisphenol A, poly-THF, polyalkyloxazolines (eg 2-phenyloxazoline), perfluoropolyethers or siloxanes such as dimethylsiloxane, etc., and copolymers of these monomers are used. In the context of the present invention, the hydrophobic component of the segmented, amphiphilic network is preferably a polydialkylsiloxane macromonomer, such as in particular a polydimethylsiloxane macromonomer. Again, the molecular weight of such a polydialkylsiloxane macromonomer is not specifically limited. For example, polydialkylsiloxane macromonomers such as, for example, polydimethylsiloxane macromonomers with a molecular weight M _n in the range from 500 to 1,000,000 g / mol ³ can be used. Usually, an α, ω-end group functionalized.es polydialkylsiloxane macromonomer, for example a bifunctional, ω-methacryloyloxypropyl-functionalized PDMS macromonomer (PDMS (MA) ₂ ) with a molecular weight M _n in the range from 500 to, is synthesized for the synthesis of the segmented, amphiphilic network 1,000,000 g / mol ³ used.

The hydrophilic component of the segmented, amphiphilic network is not subject to any specific restriction. For example, components based on monomers, such as (meth) acrylate and its derivatives, diisocyanates, diols, diamines, diacids, acylamide, N-isopropylacrylamide, N-vinylcaprolactam, 2,3-dihydroxypropyl methacrylate, maleic acid, fumaric acid or vinyl acetate, or on Based on macromonomers such as poly-1, 3-dioxolane, polyethyleneimine, polypropyleneimine, polyethylene glycol, polypropylene glycol and polyoxazolines with methacrylate, epoxy, amino, isocyanato or hydroxyl end groups, etc., are used. The hydrophilic component is preferably selected from (meth) acrylic acid, (meth) acrylate and their derivatives, in particular hydroxyethyl acrylate, hydroxyethyl methacrylate, dimethylacrylamide and dimethylaminoethyl methacrylate. The terms (meth) acrylic acid or (meth) acrylate mean acrylic acid and methacrylic acid or acrylate and methacrylate in the following. Of course, amphiphilic and non-amphiphilic Block copolymers can re, triblock copolymers (AB, ABA, BAB) are used as building blocks for the production of the segmented amphiphilic network etc., with blocks of the abovementioned monomers, for example, under the trade name Pluronic ^® Available polyethylene glycol-b-polypropylene glycol-b-polyethylene glycol.

In order to prevent the severe incompatibility between the different chain segments from leading to macroscopic phase separation during synthesis, the amphiphilic networks provided according to the invention are usually not polymerized in bulk, but rather from a solution as gels. If the monomers are not miscible with one another and the use of phase-mediating solvents is inadequate or if one of the monomers is already insoluble in them, monomers modified with protective groups can be used in an advantageous manner. These can then be polymerized from solution or in bulk. An example of this strategy is the replacement of the hydrophilic monomer methacrylic acid (MAA) by the hydrophobic trimethylsilyl methacrylate (TMSMA), which is well compatible with hydrophobic macromonomers and organic solvents. First of all, precursor networks are created whose polar chain segments carry trimethylsilyl protective groups. In order to obtain a segmented, amphiphilic network, the protective groups are then split off in a second step by hydrolysis with alcohol / water mixtures.

The segmented, amphiphilic networks which can be used in the present invention are preferably synthesized by thermally or UV-initiated free-radical copolymerization of PDMS macromonomers and (meth) acrylates or their derivatives, the preferred hydrophilic components being methacrylic acid (MAA), hydroxyethyl methacrylate ( HEMA), hydroxyethyl acrylate (HEA) and dimethylaminoethyl methacrylate (DMAEMA) are used. MAA, HEMA and HEA can be incorporated into the polymer network in the form of their hydrophobic trimethylsilyl-protected derivatives, after which the precursor network is subsequently converted into a segmented, amphiphilic network by removing the protective groups. Networks based on PHEMA-I-PIB or PMAA-I-PIB, for example, can also be produced using the synthesis strategy described above.

In a particularly preferred embodiment of the present invention, the segmented, amphiphilic networks can be prepared by radical copolymerization of a polydimethylsiloxane (PDMS) macromonomer with two methacrylate end groups and the monomer hydroxyethyl acrylate (HEA). Such a copolymer has the desired phase separation. While the hydrophilic phase (poly-HEA) swells selectively in water, the hydrophobic phase (PDMS) can be swollen in solvents such as toluene or n-heptane. According to the invention, such amphiphilic networks can be loaded with enzymes, such as horseradish peroxidase (HRP) or chloroperoxidase (CPO), by incubating them in an aqueous enzyme solution. After drying, the enzyme is in the hydrophilic phase of the network and, in an HRP-catalyzed reaction such as the oxidative coupling of phenol and N, N-dimethylphenylenediamine with t-butyl hydroperoxide in n-heptane, shows more than the free enzyme 40 to 120 times higher activity and one to three times higher stability. In the CPO-catalyzed reaction with regard to the oxidative coupling of phenol and N, N-dimethylphenylenediamine with t-butyl hydroperoxide in n-heptane, a more than 18-fold higher activity and a stability that was up to 20 times higher was observed compared to the free enzyme. However, such a surprising advantageous property is not observed when the respective enzymes are carried in networks which are composed of PHEA and a short-chain crosslinking agent and accordingly do not show any nanophase separation in the required range.

The segmented, amphiphilic networks used according to the invention can be provided in the form of films which are loaded with the catalyst, such as an enzyme, which in turn is stabilized and activated by inclusion in the segmented, amphiphilic polymer network and thus for reactions in organic solvents is made usable. The invented Immobilization of sensitive catalysts, such as enzymes, according to the invention through the use of segmented amphiphilic polymer networks in particular enables the hydrophobic phase to swell selectively in the organic solvent, while the enzyme molecules are in the hydrophilic phase and are surrounded by it in a protective manner. Thus, the hydrophilic phases and thus also the enzyme molecules are usually easily accessible to substrates dissolved in the organic solvent, which increases the enzyme activity.

When such enzymes are stored in the segmented, amphiphilic polymer networks used according to the invention, the loaded networks, if provided as a film, show no noticeable loss of activity after three weeks of storage at 4 ° C. The advantages of the incorporation of such catalysts, in particular enzymes, according to the invention over a support on microporous materials such as, for example, lies in the fact that it is an organic polymer which builds up the segmented, amphiphilic polymer network, which has any shape can enable. In addition, the catalyst distribution in the carrier material is much finer than with corresponding microporous materials, since domains are in the nanometer range and the catalyst is not only on an inner surface, but is directly embedded in one of the phases. In step (b) of the process according to the invention, the enzyme is enriched in the polymer network. This is due to the fact that enzymes have a tendency to attach to surfaces. If one considers the interfaces between the hydrophilic and the hydrophobic areas or phases of the network as a surface, the nanophase separation results in a very large “inner” surface on which the enzymes can concentrate compared to the solution from the enzyme concentration in the corresponding solution with which the polymer network is treated in step (b) and the specific chemical structure of the selected segmented amphiphilic polymer network, loads in the range of 1 to 100 μg enzyme, such as HRP, per cm ^{2 of} polymer network are usually obtained (corresponds to 0.5 mg / cm ³ to 500 mg / cm ³ ) in the form of a film reached. In the case of horseradish peroxidase, for example, the enzyme loading can be quantified by UV / VIS spectroscopy.

Most enzymes are usually insoluble in organic solvents. They are usually used as lyophilisate in crystalline form or supported on certain materials. The carrier materials are very often microporous materials such as silica. Takahashi et al., Chemical Materials, 2000, Vol. 12, pages 3301-3305, describes the support of horseradish peroxidase on silica, an increase in activity by a factor of 10 being achieved. In contrast, a horseradish peroxidase stabilized by the process according to the invention in organic solvents shows an activity which is 120 times higher than that of the pure enzyme. This is due in particular to the fact that the enzyme is “quasi-dissolved” due to the inclusion in the hydrophilic phase of the segmented, amphiphilic network.

Another object of the present invention relates to the use of the immobilized catalyst system according to the invention in organic synthesis, in particular the active ingredient synthesis.

Yet another object of the present invention relates to the use of segmented amphiphilic polymer networks which have a nanophase separation of the units formed in each case from the hydrophilic and the hydrophobic component in the range from 5 nm to 500 nm for the immobilization of catalysts, such as in particular enzymes.

The following examples illustrate the present invention.

Examples

1. Modification of glass surfaces with methacrylate groups

With a glass cutter, slides (with a matt edge, length: 7.6 cm) were cut to the width of a scotch tape (1.5 cm) and then pi ranha solution (45 ml conc. H ₂ S0 ₄ and H ₂ 0 ₂ solution (30%) in the ratio 3: 2) etched in a trough and under the influence of ultrasound.

They were then rinsed with water and air-dried. A solution of 3-methacryloyltrimethoxysilane (50 ml, 20 vol.% In toluene) was added to the slides pretreated in this way in a staining trough and shaken at room temperature for 2 hours. Then they were transferred to a further trough filled with water (50 ml), treated with ultrasound for 5 min, the water was exchanged (50 ml) and treated again with ultrasound (5 min). Finally, the slides were rinsed with water, dried in air and stored overnight at room temperature.

2. Production of thin films covalently bound to surfaces

A monomer mixture for polyhydroxyethyl acrylate-1-polydimethylsiloxane films (PHEA-I-PDMS) was prepared by dissolving the photoinitiator Irgacure 651 (5 mg / ml) in freshly distilled trimethylsilyloxyethyl acrylate (TMSOEA) with shaking, plus PDMS ₅ . ₂ (MA) _{2 was} given and shaken vigorously again.

A strip of scotch tape was stuck onto an unmodified slide free of bubbles and wrinkles and the monomer mixture (60 μl) was applied evenly to it. A “silanized” slide was then placed on the monomer mixture parallel to the scotch tape, avoiding the inclusion of air bubbles. The polymerization was carried out by irradiating with a commercially available UV polymerization lamp (Heraflash from Heraeus Kulzer) (2 × 180 s from each side, each with 30 s Cooling time between exposures (total exposure time: 720 s) The tesafilm and its support were then carefully removed from the amphiphilic film and the latter was added to a MeOH / H ₂ O mixture (9 ml per slide, 1: 1) Incubated at room temperature for at least 12 h with shaking and changing the solvent twice, the washed films were then washed with MeOH and dried in a drying cabinet (50 mbar, 60 ° C.) to constant weight. In this way, PHEA-I-PDMS films of the composition 91/9, 77/23, 50/50, 41/59 and 9/91 were produced.

3. Production of thin, unsupported PHEA-I-PDMS films

Unsupported PHEA-I-PDMS films with the composition 90/10, 77/23, 50/50, 30/70 and 10/90 were produced. The synthesis was carried out analogously to the method described above for the production of films covalently bound to supports. Instead of a surface-modified slide, an unmodified slide was used. After deprotection in MeOH / water, the film could be separated from it in a water bath. It was then transferred to a Teflon film and dried on it first in air, later in a drying cabinet (50 mbar, 60 ° C).

4. Loading of thin amphiphilic networks covalently bound to supports with enzymes

The matt edge end of the slide was removed from the films obtained above using a glass cutter, the slides with the film facing upwards in a specially made Teflon tub (internal dimensions: 5.7 x 1.7 cm, depth: 1.5 cm) enzyme solution (2 ml, c = 1 mg / ml or 25 μmol / l, horseradish peroxidase (HRP) in phosphate buffer (0.1 M, pH 7). Then at T = 4 ° C. with shaking for at least Incubated for 16 h, then the film was removed from the loading bath, dried with a paper towel, rinsed with phosphate buffer (3 x 5 ml) and dried again with a paper towel, and finally dried, the loaded film was left to stand in air (30 min) The films were stored at 4 ° C. 5. Activity determination of supported and suspended HRP using the example of the oxidative coupling of dimethylaminoaniline and phenol in heptane

Activity assays with dimethvlaminoaniline and phenol were performed in n-heptane. The following solutions were prepared: 1.dimethylaminoaniline solution (2.724 mg / ml, 20.00 mM in n-heptane) 2.phenol solution (1.882 mg / ml, 20.00 mM in n-heptane) 3 te / t-butyl hydroperoxide (10 mM in n-heptane): 9.10 μl of an fe / f BuOOH solution (approx. 5.5 M in dean) were made up to 5 ml with heptane.

The concentrations in the assay volume were (see below): c (dimethylaminoaniline) = 1.67 mM, c (phenol) = 1.67 mM, c (H ₂ O ₂ or f-BuOOH) = 0.83 mM.

Activity assay of HRP-loaded amphiphilic films:

Activity assays were carried out with HRP-loaded PHEA - / - PDMS film pieces with the composition 77/23 (A (film) = 0.7-1.3 cm ² ) in n-heptane. The area concentration of HRP was between 10 and 24 μg / cm ² . A trypsin-loaded PHEA - / - PDMS (77/23) film piece was used for the blind test. The film pieces were placed in a cuvette (d = 1 cm), solvent (1.8 ml), dimethylaminoaniline solution (200 μl) and phenol solution (200 μl) were added and the reaction was carried out by adding f-BuOOH solution ( 200 μl) started. The activity of the enzyme was measured by the increase in the absorption in the absorption maximum of the dye formed (λ _max = 546 nm) against pure solvent as the background. For this purpose, the reaction solution was stirred with a stirring fish and the absorption was recorded at regular intervals (60 s or 5 min).

The specific activity can be determined from the linear range of the reactions (tRxn = 30 - 70 min) taking into account the blind reaction according to the following equation: ^ S46 m ^ * Blind, 546nm Δt Δt ssppeezz 1000 [U / μg] A (film) • c area

One unit was defined as the increase in absorption at the absorption maximum of 0.001 per minute with a reaction volume of 2.4 ml and a layer thickness of 1 cm.

The specific activities of the supported HRP were 0.02 to 0.05 U / μg enzyme, while the enzyme showed no decrease in activity over a period of approx. 40 min.

Activity-free HRP assay

The activity of unsupported HRP was determined in n-heptane in two experiments with different amounts of enzyme (350 μg and 740 μg). For each of these experiments, the same amount of enzyme was weighed into several rolled rim glasses and 1.8 ml of heptane (tempered at 25 ° C.) were added in each case. The lyophilisate was then suspended by brief ultrasound treatment, dimethylaminoaniline solution (200 μl each) and phenol solution (200 μl each) were added and the reactions were started simultaneously by adding f-BuOOH solution (200 μl each). The mixture was stirred vigorously at 25 ° C. At intervals of initially 2 to 5 minutes and from a reaction time of 20 minutes, one of the batches was filtered through a syringe filter and the absorption of the filtrate was measured at 546 nm (layer thickness 1 cm).

The specific activity results from the linear range of the reactions according to the equation below, taking into account the blind reaction (see above)

^ 5.6κm blind, 546nm

The specific activity of suspended HRP was 0.0004 to 0.0005 U / μg enzyme, while the activity showed no decrease in activity over approx. 20 min.

Similar results were obtained when using chloroperoxidase (CPO) as a catalyst or enzyme.

Loading of PHEA-I-PDMS films with further enzymes or peptides:

Procedure: see exemplary embodiment for horseradish peroxidase solutions, each in acetate buffer (pH 5.0, 0.1 M) trypsin: 1 mg / ml, 42 μmol / ml myoglobin: 1 mg / ml, 53 μmol / mol hemoglobin: 1 mg / ml, 15.5 µmol / ml

Trypsin activity assay:

The activity of trypsin was determined by the aminolysis of Nα-benzoyl-L-arginine-p-nitroanilide (Bz-L-Arg-NH-Np) by L-leucinamide (L-LeuNH ₂ ) in toluene at room temperature. 7 μl of a 50 mM Bz-L-Arg-NH-Np hydrochloride solution in MeOH were added to toluene (875 μl) and a water content of 50 mM by adding 18 μl of a water-containing acetone solution (5% by volume water ) set. After the addition of the catalyst (trypsin in PHEA-I-PDMS or native trypsin), the reaction was started by adding 100 μl of a 100 mM L-LeuNH ₂ solution in toluene, which contained 2 vol% MeOH. The reaction was followed by the increase in absorption at 349 nm, a blind reaction served as background, to which an unloaded PHEA-I-PDMS polymer was added.

After 24 h, trypsin in PHEA-I-PDMS (77% by weight PHEA) showed a 3 order of magnitude higher conversion per amount of enzyme than unsupported native trypsin.

Claims

Expectations

1. Immobilized catalyst, the catalyst being introduced into a segmented amphiphilic network composed of at least one hydrophilic and a hydrophobic component, which has a nanophase separation of the units formed in each case from the hydrophilic and the hydrophobic component in the range from 5 nm to 500 nm ,

2. The catalyst of claim 1, wherein the catalyst is an enzyme or a peptide.

3. Catalyst according to claim 2, wherein the enzyme is a peroxidase, in particular horseradish peroxidase or chloroperoxidase, or a hydrolase, in particular trypsin

4. Catalyst according to claim 2, wherein the peptide is a transport protein, in particular myoglobin or hemoglobin.

5. Catalyst according to one of claims 1 to 4, wherein the hydrophobic component is a polydialkylsiloxane macromonomer with a molecular weight M _n in the range from 500 to 1,000,000 g / mol ³ .

6. Catalyst according to one of claims 1 to 5, wherein the hydrophilic component is selected from (meth) acrylic acid, (meth) acrylate or their derivatives.

7. A method for producing the immobilized catalyst according to one of the preceding claims 1 to 6, comprising the steps:

(i) Preparation of a segmented, amphiphilic polymer network by copolymerization of two macromonomers of different hydrophilic lie or a monomer and a macromonomer, each with different hydrophilicity and

(ii) Loading the polymer network with the catalyst.

8. The method according to claim 7, wherein in step (i) a hydrophobic macromonomer is reacted with a hydrophilic monomer.

9. The method according to claim 8, wherein the hydrophobic macromonomer is an α, ω-end-functionalized polydialkylsiloxane macromonomer with a molecular weight M _n in the range from 500 to 1,000,000 g / mol ³ .

10. The method according to claim 8 or 9, wherein the hydrophilic monomer is selected from (meth) acrylic acid, (meth) acrylate or their derivatives, in particular hydroxyethyl acrylate, hydroxyethyl methacrylate, dimethylacrylamide or dimethylaminoethyl methacrylate.

11. The method according to any one of the preceding claims 7 to 10, wherein the polymer network in step (i) is carried out in bulk by UV-initiated radical copolymerization.

12. Use of the immobilized catalyst according to one of the preceding claims 1 to 6 in organic synthesis, in particular active ingredient synthesis.

13. Use of segmented amphiphilic polymer networks which have a nanophase separation of the units formed in each case from the hydrophilic and the hydrophobic component in the range from 5 nm to 500 nm, for the immobilization of catalysts such as, in particular, enzymes and peptides.