WO2008092628A1 - Method for the production of biocompatible hybrimers for optical applications - Google Patents

Abstract

The invention relates to inorganic-organic hybrid materials (hybrimers) which can be obtained by condensing and/or cross-linking organometallic compounds containing one or more reactive groups and using condensed metal oxides containing cross-linked and optionally non-cross-linked groups. Said hybrimers contain photochromic and/or fluorescent proteins or polyamino acids. The invention further relates to methods for producing said hybrid materials and the use thereof.

Description

Process for the preparation of biocompatible hybrimers for optical applications

The present invention relates to inorganic-organic hybrid materials, the photochromic and / or fluorescent

Contain proteins available through condensation as well

Crosslinking of one or more reactive groups containing organometallic compounds and by the use of

Metal oxide condensates containing already crosslinked and optionally uncrosslinked groups, processes for their preparation and their use.

Hybrimers are a class of inorganic-organic composites made up of atomic ceramic and plastic meshes that interconnect and interpenetrate. The term "hybrimer" is an abbreviation for "hybrid polymers". Corresponding materials are known in the art as Ormocere (organically modified ceramics) or, in special cases, as Ormosile (organically modified silicon compounds). These polymers are prepared by a sol-gel process in the presence of acidic or basic catalysts (Wu et al., Chem. Mater., 1993, 5, 115-120; Shamansky et al., Bios. Bioelectr., 2002, 17 , 227-231; Innozenzi et al., Chem. Mater., 1999, 11, 1672-1679; Piana et al., Chem. Mater., 1994, 6, 1504-1508; Yoda et al., J. Non. Cryst. Solids, 1996, 208, 191-198; Ochi et al., J. Pol. Sci., 2001, 39, 1071-1084; Jones et al., J. Non-Cryst., Solids, 2001, 291, 206 -210).

The combination of novel materials with biological molecules has become one of the most innovative areas of research in recent years. During the last decades has been the development of biomaterials driven mainly by medical applications in the form of implants. Such materials had to have good mechanical properties and be biocompatible in order to be implanted into the human body and replace damaged tissue or bone. The industrial advancement of biotechnology was another important factor in the search for new materials, since the immobilization of bioactive molecules such. As enzymes, often offers advantages on solid carriers. However, bioencapsulation, ie, entrapment of biomolecules in a polymer backbone, has long been restricted to organic polymers because processing of glass at high temperatures is incompatible with susceptible biomolecules. However, metal oxide compounds offer some advantages, such as improved mechanical strength and chemical resistance. Furthermore, leaching of biomolecules can be prevented since metal oxide compounds do not swell in aqueous or organic solvents. In addition, metal oxide compounds have the advantage that they are not a source of nutrients for microorganisms, non-toxic and biologically inert. For this reason, techniques have been developed in the past for the immobilization of enzymes on glass surfaces. However, this covalent coupling often requires coupling reagents and chemical modifications that can adversely affect the bioactivity of enzymes and cells. Therefore, physical encapsulation in sol-gel materials can offer new opportunities for biotechnology.

The so-called sol-gel process is a process that allows the synthesis of glassy metal oxide compounds at room temperature. First experiments have already shown that biomolecules can be encapsulated while maintaining their activity in metal oxide compounds, in particular silicate compounds (Livage et al., J. Phys .: Condens.Matter, 2001, 13, R673-R691, Giill & Ballesteros, J. Am. Chem Soc., 1998, 120, 8587-8598; Weetall, Biosensors & Bioelectronics, 1996, 11 (3), 327-333; Zink et al., New J. Chem., 1994, 18, 1109-1115; Reetz, Adv Mater., 1997, 9 (12), 943-954).

The sol-gel method is similar to biomineralization. Biomineralization occurs in water under mild conditions, at neutral pH and room temperature. When silicates are used as the metal oxide, silicic acid molecules polymerize Si (OH) ₄ with elimination of water to form a network of silicon atoms connected by oxygen atoms.

At present, however, alkoxysilanes, which condense with the elimination of alcohols, are used predominantly in chemical applications instead of silica.

As the condensation progresses, the silica particles grow, resulting in pH dependent formation of colloidal solutions (sols) or gels. These gels may be partially dried at room temperature to obtain a porous network of hydrated amorphous silica SiCVnH ₂ O (xerogel).

The sol-gel chemistry offers a versatile method for the synthesis of metal oxides at low temperature. Thin films can be applied to any type of support material (glass, ceramics, metals, polymers) by dip-coating, spin coating, knife coating, printing, spraying, etc. Furthermore, can fibers are drawn from viscous gels and nanoparticles are synthesized from colloidal dispersions.

The mild chemical conditions in the sol-gel synthesis of metal oxides allow access to inorganic-organic hybrid materials. The mixture of organic molecules and alkoxides in the precursor solution makes it possible to combine organic and inorganic components at the molecular level. Organic molecules can be easily embedded in the metal oxide matrix or covalently linked to the matrix via reactive groups.

The hybrid materials thus obtained have a number of advantageous properties. In particular, unlike the solid and brittle pure metal oxide materials, their optical quality, flexibility and moldability can be controlled by the type and amount of organic radicals, depending on the intended application.

The finding that biomolecules, especially proteins, can be encapsulated in inorganic glass-like polymers without losing their functionality and the above-described progress in the field of inorganic-inorganic hybrid materials, in particular the ability to produce these materials in monoliths, thin films, powders and fibers has the combination of novel hybrid materials with biomolecules has made it attractive for demanding applications in bioorganic synthesis, medicine, biotechnology and environmental technology.

Therefore, great efforts have been made to sol-gel methods for the efficient immobilization of sensitive biomolecules, especially proteins and whole Cells to develop in physico-chemically robust composites.

Despite the undeniable potential of sol-gel technology, there are still major problems, particularly in the entrapment of biomolecules. These are presumably due to the alkyl, alkoxy and alkylalkoxy silanes generally used for sol-gel materials. The poor water solubility and reactivity of this compound usually requires solvents and other additives, e.g. Surfactants or catalysts that adversely affect biological materials. Furthermore, the hydrolysis of the alkoxysilanes releases alcohols which are detrimental to bioactivity, and biomolecules, e.g. Proteins irreversibly damage, i. can denature.

For this reason, there is still a need for improved methods of making biocompatible hybrid inorganic-inorganic materials which, on the one hand, allow the functionality of biomolecules immobilized in the polymer material to be maintained and, on the other hand, retain their advantageous material properties.

In particular, there is a need for methods of making hybrimer materials that permit the functional encapsulation of proteins.

The object of the present invention was therefore to provide a corresponding process for the preparation of biocompatible inorganic-organic hybrid materials, which on the one hand allows biomolecules, in particular photochromic or fluorescent proteins, to be incorporated into these polymers without the functionality of the biomolecule lost and, on the other hand, to maintain the beneficial material properties.

This object is achieved by the method according to claim 1.

Photochromic proteins are relatively rare in nature. The term "photochromic" refers to the property of being able to change the color when exposed to light. Examples of photochromic proteins are, for example, rhodopsins, for example bacteriorhodopsin or proteorhodopsin, or proteins such as phytochromes, cryptochromes, and complexes of retinal or retinal derivatives or chomophores which perform trans-isomerizations on illumination (eg azo-chromophores) with other proteins or polyamino acids (artificial proteins) as rhodopsin. In addition to photochromic proteins, fluorescent proteins, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP) or yellow fluorescent protein (YFP) can also be used.

Bacteriorhodopsin is an integral membrane protein found in the cell membrane of the halophilic organism Halobacterium salinarum (halobacteria). The polypeptide portion of the bacteriorhodopsin consists of 248 amino acids that form a pore in the form of seven antiparallel alpha helices. In this pore is the cofactor retinal, which acts as a chromophore. Retinal is bound via an imide bond (Schiff base) to the amino group of the side chain of Lys216. Retinal is present under physiological conditions as an all-trans or 13-cis isomer. The isomerization takes place under the action of visible light. In the cell membrane of the halobacteria lies bacteriorhodopsin as a trimer and forms two-dimensional crystalline (hexagonal) regions. These membrane areas, which are up to five micrometres in size, are called the purple membrane (PM). In this form bacteriorhodopsin has a high stability against physicochemical influences. Thus, the color and photochemical activity of the purple membrane are retained even in the presence of oxygen, under high salt concentrations and over a wide range of humidity.

The bacteriorhodopsin is the light energy converter of the photosynthetic energy production of Halobacterium salinarum. The photosynthesis of Halobacterium salinarum is fundamentally different from the photosynthesis of the plants. The light energy is not used here for splitting water, but serves via the energy converter bacteriorhodopsin to build up a proton gradient between the interior (cytoplasm) of the bacterium and the external environment. This proton gradient represents the energy source for ATP synthase synthesized from ADP ATP.

The proton gradient is generated through a multi-step process initiated by light-induced isomerization of the retina and driven by changes in proton affinities of amino acid functions. The chromophore, which is present in the unexposed state as an all-trans retinal isomerizes after exposure to 13-cis retinal. This results in conformational changes of the protein due to the binding of the chromophore to the protein, which directly affects the initially protonated state of the Schiff base. After isomerization, this proton is in an energetically unfavorable Environment and is delivered to the immediate interaction partner of Schiff's base, Asp85, in extracellular direction. This is followed by a series of four further unidirectional proton shifts, before finally the initial state of the protein is restored and a new cycle can be run through. This light-driven pumping of protons is more spectroscopically distinguishable to a cyclic sequence

States of the protein linked. This episode is called a photocycle. Passing through the photocycle as a result of

Exposure is with a reversible color change of violet

(B-state, absorption maximum 570nm) to yellow (M-state,

Absorption maximum 410nm).

Due to its exceptional functionality, the protein is of great scientific interest, and a number of different technical applications have been proposed for bacteriorhodopsin. Of particular interest is its use as a security pigment for the protection of personal documents, value documents and products against imitation and counterfeiting as well as optical data storage.

In the case of use as a security pigment, the high-contrast color change of the molecule is exploited on exposure to visible light, namely from violet to yellow. This feature of switchability between two colors with visible light provides high visibility by the user, as well as effective copy protection for suitably equipped documents. The imitation of such a security feature is easy to recognize because of the lack of optical switchability. By suitable modifications of the bacteriorhodopsin, the origin of a material can be reliably identified, for example via recognition tags or mutations in the amino acid sequence of the protein as well as chemical modification of the retina, and also by means of suitable laboratory methods. (Birge, Annu., Rev. Phys., Chem., 1990, 41, 683; Oesterhelt et al., Quarterly Rev. Biophys., 1991, 24, 425; Brauchle et al., Adv. Mater., 1991, 3, 420, Miyasaka et al., Science 1992, 255, 342).

It has already been demonstrated that bacteriorhodopsin in glassy silicates prepared by a sol-gel process retains its functionality (Weetall, supra). The glass allows the transport of small molecules, but immobilizes the protein in its pores. It has been shown that the properties of the encapsulated bacteriorhodopsin in the glass are comparable to those of the dissolved protein. However, these glassy materials have the disadvantage of high brittleness and porosity, which involves disadvantages in handling and optical quality (scattering). Furthermore, due to the porosity penetrated water weaken the network by cleavage (hydrolysis) of Si-O-Si bonds and thus additionally adversely affect the transparency and stability of the material and the immobilization of the protein.

The encapsulation of functional bacteriorhodopsin or other photochromic and fluorescent proteins in inorganic-organic hybrid materials, however, has not previously been described for lack of suitable methods.

Thus, for the first time, the present invention discloses a process for functional photochromic or fluorescent proteins, for example, bacteriorhodopsin, in an inorganic-organic hybrid material, a Hybrimer store. The method is preferably a sol-gel method.

The invention furthermore relates to the hybrimers produced by the process according to the invention and to their use.

As mentioned earlier, "hybrimers", sometimes referred to as ormocers or ormosils, are composite materials consisting of a network of organic and inorganic polymers. The term "network" refers to a three-dimensional arrangement of covalently linked compounds. In this case, the organic network fills in vacancies of the inorganic network, which as a rule is produced by means of condensation, so that both networks penetrate one another or are firmly connected to one another.

In this context, inorganic means that the main chains are formed from metal oxide bonds, which may be both linear and branched, in particular -Si-O bonds. In addition to the use of Si atoms for the construction of the inorganic network, other metal or semimetal atoms, such as, for example, Al, B, Zr, Y, Ba and Ti or combinations thereof, may optionally also be used. Further, combinations of Si with other atoms such as Al, B, Zr, Y, Ba and Ti or combinations thereof may also be used to construct the inorganic network. An example of this is the combined use of Si and Zr compounds to build up the inorganic part of the network. The organic network is obtained by polymerization or polycondensation of organic radicals. The polymerization reaction may involve mechanisms such as polyaddition or cycloaddition or may be radical in nature. The organic radicals used contain reactive groups which can be crosslinked via a corresponding chemical reaction. These groups may, for example, be polymerizable with one another and / or copolymerizable with other reactive groups. For example, the organic radicals may contain epoxide radicals as reactive groups, it also being possible for additional radicals which are polymerizable with one another and / or copolymerizable with epoxide radicals to be present. Another example is the use of isocyanate and hydroxy groups on the organic radicals to produce urethanes.

The process according to the invention therefore comprises the formation of an inorganic network by condensation of at least one metal oxide compound, wherein the metal oxide compound has the formula

Ri R ₄ M-

R ^s

(D has, where M is Si, Al, B, Zr, Y, Ba or Ti, wherein each of the radicals R _if R ₂ , R3 and R _{4 are} independently an unsubstituted or substituted aliphatic, cycloaliphatic or aromatic radical having 1 to 20 Carbon atoms, wherein one or more CH ₂ - groups of the aliphatic or cycloaliphatic radical by O, C = O, C (O) O-, NR ', Si (R') ₂ , Si (R ') ₂ O, CR' ₂ and / or S, and / or optionally one or more CH ₃ groups of the aliphatic or cycloaliphatic radical may be replaced by OR ', C (O) R', COOR ', NR ₂ ', Si (R ') ₃ , Si (IV) ₂ OR' and / or SR ', where each R' independently H, Ci-C ₂₀ alkyl, alkenyl, alkynyl or alkoxy, OH or halogen, wherein at least two of R ₁ , R ₂ , R ₃ and R _{4 is} a hydrolyzable alkoxide, in particular methoxy, ethers of glycerol or glycol or other complexing Compounds such as acetylacetonate, wherein at least one of R ₁ , R ₂ , R ₃ and R _{4 is} at least one reactive crosslinkable group, for example an acrylate, vinyl, allyl, methacrylate, oxetane, epoxy acrylamido, hydroxy , Nitrile, isonitrile, cyanate, isocyanate, amino, alkylamino, dialkylamino, mercapto, halogen, silane or cyanoalkyl group, and depending on the valence of MR ₃ and / or R ₄ present or may not exist, or wherein the metal oxide compound is a cyclic, branched or linear oligo- or polymetal oxide comprising structural units of the formula (II)

Ri

-0 M-0-

R ₂

(II)

wherein M, R ₁ and R _{2 have} the abovementioned meaning, and wherein MR ₂ may be present or absent, and at least one of R ₁ and R _{2 is} at least one reactive crosslinkable group, for example an acrylate, Vinyl, allyl, methacrylate, oxetane, epoxy, acrylamido, hydroxy, Nitrile, isonitrile, cyanate, isocyanate, amino, alkylamino, dialkylamino, mercapto, halogen, silane or cyanoalkyl group carries.

"Reactive groups" are those chemical groups that are crosslinkable, ie, polymerizable or polycondensable, according to methods known in the art, "crosslinking" therefore means polymerization and / or polycondensation, wherein the term "polymerization" also includes polyaddition or cycloaddition reactions as well as radical reactions which are suitable for cross-linking several molecules. In this case, two identical or two different reactively crosslinkable groups can be crosslinked with one another. Examples of identical reactive crosslinkable groups which can be crosslinked with one another are, for example, acrylic, vinyl or epoxide groups. Examples of different reactive crosslinkable groups, however, are the combinations hydroxy / isocyanate, vinyl / silane and amino / hydroxy. For the crosslinking of the reactive groups it is also possible to use crosslinking compounds (crosslinkers). Examples of such crosslinkers are, for example, 3-amino-1-propanol, glycerol, bisphenol A, bisepoxides, succinic anhydride, succinimide, and isophoronediamine.

In one embodiment, glycerin may be used as a crosslinking compound to crosslink epoxide groups. Alternatively, 3-amino-1-propanol can be used as a crosslinker to crosslink isocyanate groups.

The aliphatic radicals are alkynyl, alkenyl and / or alkyl radicals having 1-20 carbon atoms, preferably 1-6 Carbon atoms which may be straight-chain, branched or cyclic.

Examples of suitable alkyl radicals are C 1 -C 6 -alkyl radicals, in particular methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl, isobutyl, n-pentyl and n-hexyl.

The alkenyl radicals include vinyl, allyl, 2-butenyl, cyclopentenyl and cyclohexenyl, among others.

Examples of alkynyl radicals include ethynyl, propynyl and 2-butynyl.

Cycloaliphatic radicals include cycloalkyl radicals which have one or more ring systems. Examples are C1-C2 ₀ mono- or bicyclic cycloalkyl, for example cyclopentyl, cyclohexyl and norbornyl.

In addition, the metal oxides of formulas (I) or (II) may also include aromatic radicals. Among the preferred

Aryl radicals include phenyl, diphenylyl and naphthyl. The aromatic radicals also include alkylaryl radicals such as benzyl. Furthermore, the aromatic radicals may also be heteroaromatic radicals which contain 1-3 heteroatoms, for example selected from O, N and S.

In certain embodiments, the metal oxides of formulas (I) or (II) may also include amino acid residues, steroid residues or polyethylene oxide residues.

All the radicals mentioned may be substituted or unsubstituted, ie, if appropriate, one or more substituents, for example halogen, C 1 -C ₂₀ -alkyl, C 1 -C ₂₀ , instead of an H atom Hydroxyalkyl, C ₂ O alkenyl, Ci-C ₂₀ -alkoxy, C ₂ O aryl, C _X -C ₂₀ aryloxy, Ci-C ₂₀ aralkyl, C ₂₀ acyloxy, _Ci-C20 alkylcarbonyl, Ci-C ₂₀ alkoxycarbonyl, amino, Ci-C ₂₀ alkylamino, Ci-C ₂₀ dialkylamino, Ci-C ₂₀ trialkylammonium, amido, hydroxy, formyl, carboxy, mercapto, cyano, nitro and epoxy wear. Particular preference is given to radicals which are substituted by one or more epoxy, hydroxyl or isocyanate groups.

The alkoxy, aryloxy, acyloxy and alkylcarbonyl radicals are derived inter alia from the abovementioned alkyl and aryl radicals. These include, inter alia, methoxy, ethoxy, n- and i-propoxy, n-, i-, s- and t-butoxy, acetyloxy, propionyloxy,

Methylcarbonyl, ethylcarbonyl, methoxycarbonyl, benzyloxy, 2-

Phenylethyloxy and tolyloxy. These radicals may also have the abovementioned substituents.

Specific examples of metal oxide compounds of the formula (I) are silicon compounds such as glycidoxypropyltrimethoxysilane. These silicon compounds can also be used as mixtures. Another example of metal oxide compounds of the formula (I) are zirconium compounds, which can also be used as a mixture with silicon compounds.

Suitable silanes or siloxanes are for the most part commercially available; moreover, they can be obtained synthetically in a manner known per se. The expert also learns helpful hints, for example, from Ullmann's Encyclopedia of Industrial Chemistry, 5th ed.

To the metal oxides of the formulas (I) or (II) or corresponding mixtures of metal oxides of the formulas (I) or (II), from which according to the inventive method Inorganic networks can be obtained by hydrolytic condensation, further organic metalloid and metal compounds can be added, which are incorporated into the inorganic network during the hydrolysis. These include, but are not limited to, silanes, zirconium compounds, and other metal oxides, particularly siloxanes, that do not have reactive crosslinkable groups.

Examples of these are R "-M-Cl _x , R" -M- (OCH ₃ ) _x , (R ") ₂ -M-Cl _x , (R") 2-M-Br _x , (R ") 2-M- (OR ") x, M- (R") _x or M- (OR ") _X , where

M is Si, Al, B, Zr, Y, Ba or Ti, each R "is independently H or a straight-chain or branched C _{1 -C 20} -alkyl, alkenyl,

Alkynyl or cycloalkyl and x is 1, 2 or 3 depending on the valency of M. Suitable compounds are therefore, for example, CH ₃ -Si-Cl ₃ , CH ₃ -Si (OCH ₃ ) ₃ , C ₂ H ₅ -Si-Cl ₃ , C ₂ H ₅ -Si

(OCH ₃ ) ₃ , (CH ₃ J ₂ -Si-Cl ₂ , (CH ₃ ) ₂ -Si-Br ₂ and Si (OCH ₃ ) ₄ ) Another suitable compound is, for example

Zirconium ethoxide or a complex of zirconium ethoxide and

Triethanolamine. When using such a zirconium compound as an additional constituent of

Reaction, over the amount of the zirconium compound the

Refractive index of the resulting film or monoliths are controlled.

In one embodiment, this additional metal oxide is a silane, preferably tetramethoxysilane. In another embodiment, this additional metal oxide is zirconium ethoxide.

The mechanical and optionally also optical properties of the hybrimers obtainable by the process according to the invention can be determined by type and the Affect the ratio of metal oxide compounds used.

Furthermore, the higher the proportion of hydrolyzable groups, for example methoxy groups, compared to the non-hydrolyzable groups, the harder but also more brittle the material becomes.

In some embodiments, the ratio of these groups, which is hydrolyzable to non-hydrolyzable, is in the range of 1: 2 to 100: 1, for example in the range 1: 1 to 20: 1, preferably in the range 1: 1 to 8: 1 , These values are to be understood as the mean value of the hydrolyzable compounds which can optionally be used as a mixture.

The preparation of the inorganic networks can be carried out in the manner customary in the field of poly (hetero) condensation. For the (hydrolytic) condensation of silicon compounds in most cases admixing of water or, in the case of anhydrous hydrolysis, of boric acid (B (OH) ₃ ) or similar compounds, at room temperature or under slight cooling, after which the resulting mixture for some time is stirred. The possible hydrolysis of the silicon compounds can also be effected by adding an acid or base, if appropriate also with the exclusion of water, ie anhydrous.

In one embodiment of the invention, the hydrolysis is carried out in the presence of a substoichiometric amount of H ₂ O and optionally an acid, for example HCl, HF, HBr, tetrabutylammonium fluoride (TBAF), B (OH) ₃ or Al (OH) ₃ . In the case of using glycidoxypropyltrimethoxysilane and Depending on the concentration of the acid and the acid strength used, tetramethoxysilane gives a low-viscosity mixture which remains stable for several months, ie the viscosity does not change significantly.

In general, the hydrolysis is carried out at temperatures between -20 degrees Celsius and 130 degrees Celsius, preferably between 0 and 30 degrees Celsius. The reaction can be carried out both in bulk and in a solvent such as methanol.

Since the reactive groups, for example epoxide groups, can also be acidic or basic crosslinkable, for example polymerizable, the pH for the hydrolysis and condensation of the inorganic polymer matrix in a medium becomes dependent on the rate of hydrolysis of the alkoxides and the stability of the reactively crosslinkable groups selected.

In the use of various easily hydrolyzable compounds, it has proven to be particularly advantageous not to present all starting compounds at the beginning of the hydrolysis, but to contact only a portion of these compounds with water and then add other compounds. The same applies to the addition of water, which can be done in several stages, after each addition of water, the mixture is stirred for a certain time. This procedure may be required if parts of the hydrolyzed compounds tend to precipitate, segregate or phase separate or the entire system tends to gel fast. This can also be done with complexing compounds to slow the rate of hydrolysis. Suitable complexing compounds are, for example, acetylacetonate and ethanolamines such as triethanolamine.

The condensation time depends on the particular starting components and their proportions, the pH, the concentration, optionally the catalyst used, such as Ti (OPr) ₄ , the reaction temperature and the desired final viscosity, etc. In general, the polycondensation is carried out at atmospheric pressure. However, it can also be performed at elevated or reduced pressure. The polycondensate thus obtained can be used either as such or after removal of volatile substances used or formed, such as solvents.

Since the formation of the organic network can take place by basic, acidic, cationic, anionic or free-radical polymerization or polycondensation, polymerization or polycondensation initiators are added to the polycondensates or the mixtures from which they are obtained. If, for example, when using epoxy or allyl groups as reactive crosslinkable groups, a cationic polymerization is carried out, initiators are preferably used, the Lewis or Brönstedt acids or compounds which release such acids, such as BF ₃ or its ethereal adducts (BF ₃ -THF, BF ₃ -Et ₂ O, etc.), AlCl ₃ , TiCl ₄ , Ti (OEt) ₄ , Ti (OiPr) ₄ , FeCl ₃ , NbCl ₅ , HPF ₆ , HAsF ₆ , HSbF ₆ , HBF ₄ , are. Furthermore, an epoxide opening can also be carried out by base catalysis. Suitable for this purpose are primary, secondary or tertiary amines and diamines as well as amidines. Particularly suitable are amidine bases such as imidazoles and bicyclic amidines such as 1,8- Diazabicyclo [5.4.0] undec-7-ene (DBU). Furthermore, initiators may also be tetrabutylammonium fluoride (TBAF), Ti (OPr) ₄ (titanium tetraisopropylate), photoacids and / or onium salts and mixtures of the abovementioned compounds.

The term "initiator" with respect to the polymerization or polycondensation reaction, therefore, includes both catalyst substances as well as other means of initiating the reaction, for example by UV light, free radical generator, etc.

For this reason, one aspect of the invention also includes the photochemical crosslinking of the reactive groups of the organic radicals. Another aspect of the invention involves the thermal crosslinking of the reactive groups of the organic radicals.

For the formation of the organic network one or more can be used depending on the reactively crosslinkable groups

Compounds which initiate and / or catalyze the crosslinking reaction can be used. In particular, too

Mixtures of suitable catalyst substances, which are for example commercially available, can be used.

For crosslinking of the reactive groups it is also possible to use suitable crosslinking compounds, such as bi- or multivalent crosslinkers. For the crosslinking of epoxy groups via crosslinkers, for example, glycerol, bisphenol A, succinic anhydride, isophoronediamine and bisepoxides are suitable. For the crosslinking of isocyanate groups with the formation of urethanes, for example, 3-amino-1-propanol is suitable as crosslinker. The time sequence of (hydrolytic) condensation and crosslinking, for example by means of polymerization, is not fixed and can be carried out in a suitable manner simultaneously or in any desired sequence.

Before or after the condensation and / or crosslinking, further polymerisable monomers or precondensates / polymers or mixtures thereof may be added to the hydrolyzable metal oxide compound or the condensate or network prepared therefrom.

These may be monofunctional as well as polyfunctional monomers or polymers with crosslinkable, i. polymerizable or polycondensable groups, such as, for example, acrylate, vinyl, allyl, methacrylate, oxetane, epoxy or acrylamido groups or hydroxyl, nitrile, isonitrile, cyanate, isocyanate, amino, alkylamino, Dialkylamino, mercapto, halogen, silane or cyanoalkyl groups, act.

Further, before, after or during the condensation and / or crosslinking or even after the addition of the protein, further non-functional monomers or polymers may be added to the hybrimer solution which impart further properties, such as conductivity, to the material obtainable therefrom. Examples of such polymers are poly (p-phenylene-vinylene) (PPV), polyp, 4-

Ethylenedioxythiophene) (PEDOT) and polyaniline (PANI).

In one embodiment of the process according to the invention, the hybrimers are prepared by using glycidoxypropyltrimethoxysilane and tetramethoxysilane as Starting components mixed, the silicon compounds hydrolyzed and the epoxy radicals cationically polymerized, wherein hydrolysis, condensation and polymerization can be carried out simultaneously or in any order sequentially.

The cationic polymerization of the epoxide groups is carried out by adding a Lewis acid, preferably boron trifluoride diethyl etherate (BF ₃ -Et ₂ O), in a solvent, preferably the alcohol formed in the hydrolysis. The ratio of epoxide groups: Lewis acid determines the rate of polymerization and thus also the rate of viscosity increase of the solution.

The polymerization time depends on the starting components, their molar ratios, the initiator used and the reaction temperature.

In addition, before or after the crosslinking step or even during the hydrolysis, anti-fouling agents (UV blockers), for example siloxane-modified diphenyl ketones, preservatives, such as antibacterial additives, antioxidants, free-radical scavengers, hindered amine light stabilizers (HALS), organic dyes, such as laser dyes, inorganic Semiconductor particles, such as CdS or CdSe nanoparticles, and / or oxide or metallic nanoparticles are added to the sol. Further, fluoropolymers such as siloxanes and / or titanium / zirconia compounds may be added to adjust the refractive index of the resulting film or monolith. Furthermore, moisture regulators such as glycerol may be added. When using glycerin as a moisture regulator, if it is added before or during the crosslinking reaction, it may additionally act as cross-linking agent.

If acidic polymerization of the reactive crosslinkable groups is carried out with a Lewis acid catalyst (initiator), the Lewis acid may then be hydrolyzed with water and the pH of the gel with a base in a range of 4.0 to 13.0, preferably 4 , 0 to 11.0, more preferably 5.0 to 10.0, still more preferably 5.5 to 9.5. For bacteriorhodopsin a pH range of 6.0 to 10.0 and for proteorhodopsin a pH range of 6.0 to 11.5 is optimal. The type and amount of base added determine the type and rate of gelation and, optionally, further crosslinking of the epoxide network. Examples of suitable bases are NaOH, KOH, Ca (OH) ₂ , Mg (OH) ₂ , primary, secondary and tertiary amines (see J. Pol. Sei., 2001, 39, 1071-1084), for example imidazoles, in particular methylimidazole , Pyridines, pyrimidines, pyrazoles, piperidines, ammonia, ethanolamines, especially triethanolamine, but also DBU (1, 8-diazabicyclo [5.4.0] undec-7-ene) and DABCO (1, 4-diazabicyclo [2.2.2] octane ), as well as mixtures thereof. Furthermore, these bases may also be buffer substances or mixtures of the above bases with buffer substances. In this way, a specific pH range can be set in a targeted manner, as a result of which the properties of the protein to be included can be optimized. Suitable buffer substances include TRIS, HEPES, BICINE, CAPS. Furthermore, inorganic buffers such as phosphate, borate, etc. can be used.

If, on the other hand, polymerisation is carried out under basic conditions, the pH of the gel must be adjusted correspondingly with an acid to the above-indicated pH range. The thus adjusted pH, which is advantageously close to the physiological pH, is particularly important for the functionality of the later added photochromic or fluorescent proteins.

In the following, the alcohol optionally formed from the alcoholate in the hydrolysis, e.g. Methanol, and other volatile components usually gently and slowly under (compared to normal pressure) reduced pressure and optionally also at elevated temperature, e.g. removed in a rotary evaporator. The gentle procedure in the removal of the alcohol formed serves to avoid the further gelation of the mixture.

To the resulting gel may now be added the desired photochromic or fluorescent protein in the form of a solid or as a solution / dispersion in a suitable solvent, for example water or mixtures of water with other solvents and / or additives such as salts, e.g. chaotropic salts, glycerol, sugars, ureas, hydroxylamine, guanidine, amino acids such as arginine, other protein stabilizers, surfactants such as cationic, anionic and nonionic surfactants, ethylene glycols or other water-soluble polymers which serve to provide optimal conditions for the protein and / or to influence the photocycle. In this case, the pH of the gel and the viscosity can be adjusted again by adding water and / or a base, an acid or a buffer.

In one embodiment of the invention, the photochromic or fluorescent protein obtained by means of the inventive method is incorporated into an inorganic-organic hybrid material, a rhodopsin, for example bacteriorhodopsin or proteorhodopsin. Other suitable proteins are, for example, phytochromes, cryptochromes and complexes of retinal or chemically modified retinal or chomophores, which perform trans-cis isomerizations on illumination (eg azo-chromophore), and other proteins or polyamino acids (artificial proteins). Also suitable are fluorescent proteins such as Green Fluorescent Protein (GFP), Enhanced Green Fluorescent Protein (EGFP) or Yellow Fluorescent Protein (YFP).

The use of chemically modified variants of these proteins as well as mutants thereof is also contemplated herein. By such modifications / mutations, for example, properties of the protein, e.g. the absorption properties or half-lives of individual states of the photocycle vary. An example of such a mutation is the D96N mutant of bacteriorhodopsin, which causes the return of the excited M state to be greatly retarded with an absorption maximum of 410 nm into the ground state with an absorption maximum of 570 nm. This leads to an extended life of the M state, since the mutation after photon absorption and isomerization of the retina leads to a much slower reprotonation of the Schiff base by the more basic amino acid Asn 96.

Likewise, other amino acid substitutions in the sequence of the proteins used can alter the properties. Furthermore, chemical modifications of the proteins, for example, the use of artificial or modified amino acids or amino acid analogs, further advantageous properties, such as increased stability, condition. Other possibilities are the chemical modification of the cofactor, for example, the retina in rhodopsins or bilin in phytochromes, for example, to change the absorption properties or half-lives. In addition, other chromophores, such as azo dyes, can be directly attached to the protein.

Furthermore, the absorption maxima can be optimized by addition of chemical additives, for example of glycerol as moisture regulator, of amino acids to achieve half-lives, of hydroxylamine to realize permanent storage media from the hybrimers of the invention, UV blocking agents and oxidic nanoparticles as fillers or for other applications become.

The gel can then be applied to appropriate substrates such as glass, ceramics, metals, polymers, wood, composites and papers. For the application on

Substrates are a number of suitable in the art

Methods are known, which include, among others, "dip coating", "spray coating", "spin coating", printing, knife coating and extruding.

The resulting film is then dried, optionally leaving any remaining silanols condensed to Si-O-Si. This step is catalyzed by a base such as methylimidazole or other primary, secondary or tertiary amines as well as diamines as well as amidines or base mixtures. Particularly suitable are amidine bases such as imidazoles and bicyclic amidines such as 1,8- Diazabicyclo [5.4.0] undec-7-ene (DBU). Furthermore, these amines can catalyze the opening of possibly existing epoxy functions. However, the temperature must be maintained within a range that prevents denaturation of the photochromic or fluorescent proteins. The drying can be carried out, for example, over silica gel, also known as blue gel, or over other drying agents under vacuum or in air at temperatures up to 60 ^° C., ideally at room temperature.

Silanol functions which may still be present in the resulting film can also be saturated with reactive silanes, for example hexamethyldisilazane, (CH ₃ ) ₃ SiCl, HSiCl ₃ , HSi (OMe) ₃ or other haloalkylsilanes, for example perfluorinated compounds. Furthermore, the surface of the films can be modified with reactive group-bearing silanes. As a result, the surface is optionally hydrophobized depending on the silanes used and thus water repellent. This can be advantageous, for example, in the coating of papers. Alternatively, the surface can also be hydrophilized by treatment with ozone, plasma, SiCl ₄ and subsequent water treatment. Further, by selecting suitable Si-based coupling reagents, the films may be coated with other materials such as Si ₃ N ₄ , SiO ₂ or ITO, with metals such as Al, Au, Cr, Ag, Pt and Ni, metal nitrides, Metal oxides, such as indium tin oxide, or suitable organic molecules are vapor-deposited, or cast or laminated in epoxies, siloxanes, etc. The resulting materials are suitable for the production of optical components, such as resonators or variable waveguides. Alternatively, monoliths can also be produced from the hybrid systems according to the invention.

The controlled multi-stage synthesis process of the present invention enables the production of a long-term stable hybrimer system. The selection of materials, the precise adjustment of the pH value (buffering) and the careful removal of any pollutants, such as methanol, make the method according to the invention suitable for processing extremely sensitive materials such as proteins, for example rhodopsin, and thus, for example, water-repellent hybrid films. which contain photochromic or fluorescent proteins.

The following examples serve to explain the invention in more detail, without this being intended to be limited thereto. The following percentages are based on the total weight, unless otherwise noted.

Example 1. Preparation of a bacteriorhodopsin-containing hybrid film

1767 μl (8 mmol) of glycidoxypropyltrimethoxysilane were mixed while stirring with 595 μl (4 mmol) of tetramethoxysilane. With constant stirring, then 684 μl of 10 mM HCl (38 mmol) were added slowly. The mixture was then stirred at room temperature for 24 hours.

Then, with vigorous stirring and under a protective gas atmosphere (argon), 25 μl of boron trifluoride etherate (0.19 mmol) were slowly added and stirred again for 24 hours. 1 ml of the resulting solution was added with stirring 1 ml of water and then the pH by addition of 2 M methylimidazole in water to about 6.0 to 8.0 increased (about 70 to 250 ul 2 M methylimidazole);

Methanol and other volatile substances were gently removed at 2O ⁰ C and 100 mbar for one hour in a rotary evaporator.

The resulting solution was mixed 1: 1 with a solution of 3 wt .-% bacteriorhodopsin (in the form of purple membrane) in water and applied by knife coating on a glass substrate, pre-dried at room temperature and then dried at room temperature and atmospheric pressure in a desiccator over silica gel for 24 h ,

Example 2. Preparation of a bacteriorhodopsin-containing, refractive index-free adjustable Zr-epoxide film

In order to obtain a Zr-epoxide film which is freely adjustable in the refractive index, 200 μl of 10 mM HCl were slowly added to 200 μl of (10 mM) glycidoxypropyltrimethoxysilane with stirring and stirred at room temperature for 24 h.

Then, with vigorous stirring and under a protective gas atmosphere (Ar), 5 μl of boron trifluoride etherate (0.19 mM) were added slowly, and the reaction mixture was stirred for a further 24 hours.

Parallel to this, with stirring, 1.328 ml (10 mM) of triethanolamine were added to 2,715 g of zirconium ethoxide (10 mM), and 1 h implemented. Thereafter, the Zr-TEA complex was slowly added with 1 ml H ₂ O and again stirred for 1 h.

To prepare films, 2.0 ml of the Zr-TEA solution was diluted with 2.0 ml of water. To the resulting slightly cloudy solution was added 2.0 ml of the epoxide solution with stirring, resulting in the formation of a clear, slightly viscous solution having a pH of about 8.5.

After removal of volatiles in vacuo, the protein (bacteriorhodopsin) was added to the solution.

After the solution has been applied to substrates, hard films with optimum optical properties are obtained after drying.

Example 3. Use of glycerol as cross-linker in epoxy films as well as moisture regulator

2200 .mu.l (10 mM) glycidoxypropyltrimethoxysilane, 241 .mu.l (3.3 .mu.M) glycerol and 744 .mu.l (5 mM) TMOS were added while stirring slowly with 880 .mu.l of 1OmM HCl and stirred for 24 h at room temperature.

Then, with vigorous stirring and under a protective gas atmosphere (Ar), 25 μl of boron trifluoride etherate (0.19 mM) were added slowly, and the reaction mixture was stirred again for 24 h.

The solution was further processed as in Example 1. Example 4. Preparation of a siloxane-urethane-urea composite

Under ice-cooling and stirring, 225 μl (3 mM) of 3-aminopropanol were added to 1125 μl (4.5 mM) of isocyanatopropyltrimethoxysilane. After the reaction, the mixture was heated at 110 ⁰ C under reflux for 45 min. After cooling to room temperature, 300 .mu.l of TMOS, then 3000 .mu.l of H ₂ O and then 100 .mu.l of HCl were added to 1000 .mu.l of the substance with vigorous stirring. given. After the solution is clear, 800 μl of 2 M TEA were also added with stirring. After removing volatile components in vacuo, the protein (bacteriorhodospin) was added to the viscous solution.

The resulting solution can be used excellently for printing processes.

Claims

claims A process for the preparation of a metal oxide based hybrimer containing one or more photochromic or fluorescent proteins or polyamino acids, the process comprising: Formation of an inorganic network by condensation of at least one metal oxide compound, wherein the metal oxide compound has the formula Ri R ₄ MR ₂ R ₃ (D has, where M is Si, Al, B, Zr, Y, Ba or Ti, wherein each of Ri, R ₂ , R ₃ and R _{4 is} independently an unsubstituted or substituted aliphatic, cycloaliphatic or aromatic radical having 1 to 20 carbon atoms, one or more CH ₂ groups of the aliphatic or cycloaliphatic radical being O, C =O, C (O) O-, NR ', Si (R ') _{2 /} Si (R') ₂ O, CR ' ₂ and / or S, and / or one or more CH ₃ groups of the aliphatic or cycloaliphatic radical by OR', C (O) R ', C (O) OR ', N (R') ₂ , Si (R ') ₃ , Si (R') ₂ OR 'and / or SR' may be replaced, each R 'being independently H, Ci-C _2O alkyl, alkene, alkyne or alkoxy, OH or halogen, wherein at least two of Ri, R ₂ , R ₃ and R _{4 are} a hydrolyzable alkoxide, wherein at least one of Ri, R ₂ , R ₃ and R ₄ carries at least one reactive crosslinkable group, and wherein depending on the valence of MR ₃ and / or R ₄ may be present or absent, or wherein the metal oxide compound is a cyclic, branched or linear oligo- or polymetal oxide comprising structural units of the formula (II) O-M-O R ₂ (II) wherein M, R ₁ and R ₂ are as defined above, and wherein, depending on the valency of MR _2, may be present or absent, and wherein at least one of Ri and R ₂ carries at least one reactive crosslinkable group; Crosslinking of the reactively crosslinkable groups of the metal oxide compound by polymerization or polycondensation such that a stable sol or gel forms; and adding a photochromic or fluorescent protein or the polyamino acid such that the protein is dissolved, emulsified or dispersed in the sol. 2. The method of claim 1, wherein M is Si or Zr. 3. The method of claim 1 or 2, wherein the hydrolyzable alcoholate is methoxy, ethoxy, a glycol or glycerol ether or acetylacetonate. 4. The method of claim 3, wherein the hydrolyzable alcoholate is methoxy. 5. The method according to any one of claims 1-4, wherein the at least one reactive crosslinkable group is an acrylate, vinyl, allyl, methacrylate, oxetane, epoxy, Acrylamido, hydroxy, nitrile, isonitrile, cyanate, isocyanate, amino, alkylamino, dialkylamino, mercapto, halogen, silane or cyanoalkyl group. 6. The method of claim 5, wherein at least one reactive crosslinkable group is an epoxy group. A process according to any one of claims 1-6, wherein the metal oxide compound of formula (I) is glycidoxypropyltrimethoxysilane. 8. The method of claim 5, wherein at least one reactive crosslinkable group is an isocyanate group. The process of claim 8 wherein the metal oxide compound of formula (I) is isocyanatopropyltrimethoxysilane. 10. The method according to any one of claims 1-9, wherein the photochromic or fluorescent protein or the Polyamino acid as a solution or dispersion in a suitable solvent such as water or mixtures of Water is added with other solvents or optional additives or in the form of a solid. 11. The method according to any one of claims 1-10, wherein when cross-linking the reactively crosslinkable groups, a crosslinking agent (crosslinker) is added. 12. The method of claim 11, wherein the cross-linking agent is selected from 3-amino-l-propanol, glycerol, bisphenol A, bisepoxides, Succinic anhydride, succinimide, and isophoronediamine. 13. The method according to any one of claims 1-12, wherein additionally at least one metal oxide compound containing no reactive crosslinkable group is used. 14. The method of claim 13, wherein the additional metal oxide compound R '' -M-Cl _x, R "-M- (OCH _{3) x,} (R") ₂ - M (OR ^') x, (R ") 2-M-Cl _x , (IV) ₂ -M-Br _x , M- (R ") _x or M- (OR") _x where M is Si, Al, B, Zr, Y, Ba or Ti wherein each R "is independently H or a straight or branched C ₁ -C ₂₀ alkyl, alkenyl, alkynyl or cycloalkyl, and wherein x is 1, 2 or 3 depending on the valency of M. 15. The method of claim 14, wherein M is Si or Zr. 16. The method of claim 15, wherein the additional metal oxide compound is tetramethoxysilane (H ₃ CO) ₄ Si. 17. The method of claim 15, wherein the additional metal oxide compound is zirconium ethoxide. 18. The method according to any one of claims 1-17, wherein after or during the condensation and / or crosslinking or even after the addition of the protein conductive polymers selected from poly (p-phenylene-vinylene) (PPV), poly (3, 4- Ethylenedioxythiophene) (PEDOT) and polyaniline (PANI). 19. The method according to any one of claims 1-18, wherein the molar ratio of hydrolyzable groups to non-hydrolyzable groups in the / used Metal oxide (s) is in the range of 1: 2 to 100: 1. The process of claim 19, wherein the molar ratio of hydrolyzable groups to nonhydrolyzable groups in the metal oxide compound (s) used is in the range of 1: 1 to 8: 1. 21. The method according to any one of claims 1-20, wherein the hydrolytic condensation is carried out additionally in the presence of an acid or base or an acid or base mixture. 22. The method of claim 21, wherein the acid is selected from HCl, HF, HBr, B (OH) ₃ and Al (OH) ₃ . 23. The method according to any one of claims 1 to 22, wherein the crosslinking of the reactively crosslinkable groups by addition of an initiator or photochemically or thermally. 24. The method of claim 23, wherein the initiator is an acid, a base or a free radical generator. 25. The method of claim 24, wherein the initiator is an acid. 26. The method of claim 25, wherein the initiator is a Lewis acid. 27. The method of claim 26, wherein the Lewis acid is selected from BF ₃ or its ether adducts BF ₃ -THF or BF ₃ ¹ Et ₂ O, AlCl ₃ , TiCl ₄ , Ti (OEt) ₄ , Ti (OiPr) ₄ , FeCl ₃ , NbCl ₅ , HPF ₆ , HAsF ₆ , HSbF ₆ and HBF ₄ . 28. The process of claim 27, wherein the Lewis acid is boron trifluoride diethyl etherate (BF ₃ OEt ₂ ). 29. The method according to any one of claims 1-28, wherein after the condensation and the crosslinking, the pH is adjusted to 4.0 to 13.0. 30. The method of claim 29, wherein the adjustment of the pH is carried out by adding a base, wherein the added base is selected from NaOH, KOH, Ca (OH) ₂ , Mg (OH) ₂ , primary, secondary and tertiary amines such as Imidazoles, pyridines, pyrimidines, pyrazoles, piperidines, ammonia, ethanolamines or mixtures thereof, amidines such as DBU (1, 8-diazabicyclo [5.4.0] undek-7-ene) and DABCO (1, 4-diazabicyclo [2.2.2] octane) and mixtures thereof. The process of claim 30, wherein the base is methylimidazole or triethanolamine. 32. The method according to any one of claims 29-31, wherein the pH is adjusted to 5.0 to 10.0. -33. A method according to any one of claims 1-32, wherein alcohol formed by the hydrolysis of alcoholate is removed prior to addition of the protein. 34. The method of claim 33, wherein the alcohol is removed under reduced pressure. 35. The method according to any one of claims 1-34, wherein prior to the addition of the protein, an anti-fouling agent, a preservative, nanoparticles, hindered amine light stabilizers HALS, antioxidants, an organic dye, a moisture-regulating agent and / or semiconductor particles are added. 36. The method according to any one of claims 1-35, wherein the photochromic or fluorescent protein is selected from rhodopsins, phytochromes, cryptochromes, complexes of retinal or retinal derivatives or chomophores that perform trans-cis isomerizations on illumination, with other proteins or Polyamino acids, GFP, EGFP and YFP or chemically modified derivatives or mutants thereof. The method of claim 36, wherein the photochromic or fluorescent protein is a rhodopsin. 38. The method of claim 37, wherein the rhodopsin is bacteriorhodopsin or proteorhodopsin or a chemically modified derivative or a mutant thereof. 39. The method according to any one of claims 1-38, wherein the hybrimer is applied as a film on a support material or used for the production of a monolith. 40. The method of claim 39, wherein prior to the application, the viscosity of the Hybrimer is adjusted by the addition of water / base. 41. The method according to any one of claims 39 or 40, wherein after the application, a curing takes place. 42. The method of claim 41, wherein the curing is carried out in the presence of a base. The process of claim 42, wherein the base is methylimidazole. 44. The method according to any one of claims 39-43, wherein the cured film or monolith by reactive Coupling reagents functionalized, laminated, cast or vaporized with metals, metal nitrides, metal oxides or organic molecules. 45. A metal oxide-based hybrid comprising one or more photochromic or fluorescent proteins. 46. Hybrimer according to claim 45, wherein the protein (s) is present in a sol or gel of the hybrimer dissolved, emulsified or dispersed. 47. Hybrimer according to claim 45 or 46, wherein the protein (s) are encapsulated in the hybrimer. 48. Hybrimer according to any one of claims 45 to 47, wherein the photochromic or fluorescent protein is selected from rhodopsins, phytochromes, cryptochromes, complexes of retinal or retinal derivatives or chomophores, the trans-cis isomerizations at Illumination, with other proteins or polyamino acids, GFP, EGFP and YFP or chemically modified derivatives or mutants thereof. A hybridiser according to any one of claims 45 to 47, wherein the photochromic or fluorescent protein is a rhodopsin. 50. The hybrimer according to claim 49, wherein the rhodopsin is bacteriorhodopsin or proteorhodopsin or a chemically modified derivative or a mutant thereof. 51. A biocompatible coating film obtainable by a process according to any one of claims 1 to 44.