Aqueous dispersions (U) containing:

Textile material which is used in an environment in which ultraviolet (UV) light acts on the textile material, especially UV light filtering through glass, as for the interior fittings in automobiles (carpets, belts, trim and seat covers), shop windows or glass-enclosed verandas (wall-to-wall carpets), are advantageously finished with a UV absorber in order that the fibres, especially synthetic and cellulosic fibres, and the dyeings too to some extent, may be protected against the damaging action of the UV rays. A preferred procedure for textile material used in an environment of filtered UV light involves applying the UV absorbers to the substrate, in particular to the yarns, together with the respective dyes. Since the yarns, according to a technically preferred procedure, are dyed on packages, in particular cheeses, in doing so it is also desirable to apply the UV absorbers at the same time. However, when dyeing the packages with disperse dyes, a problem is that, in the presence of UV absorbers which have been formulated with surfactants, the liquors may, under the action of the high shearing forces which occur in the cheese, alter to such an extent that the dyeings of the cheeses become unlevel, in particular due to the dye being filtered off, so that the yarn when further processed, for example into knits or wovens, provides an unlevel appearance, or/and, if the dispersions change in their flow behaviour under the action of the high shearing forces, the liquor flow through the cheeses may gradually lead to a troublesome build-up of pressure, whichxe2x80x94especially depending on yarn quality and type of windxe2x80x94leads to correspondingly more pronounced changes in the shearing forces in the package and may even lead to a complete standstill of the flow of liquor through the package. The situation can be analogous with the use of dispersed optical brighteners.
EP-A-468921, 474595 and 490819 describe certain aqueous UV-absorber dispersions containing UV-absorbers and anionic surface active compounds or non-ionogenic and anionic surface active compounds and also such dispersions additionally containing polysaccharides or further linear polymeric compounds as stabilisers and/or thickening agents. EP-A-328485 describes aqueous dispersions of certain dyes, optical brighteners or UV-absorbers containing, besides dispersant, an alkylene-bisamide defoamer and in some examples also certain linear polymers (polyvinyl alcohol or a propyleneoxide/ethyleneoxide 3000 blockpolymer) or a polysaccharide.
It has now been found, surprisingly, that by employing the hereinbelow-defined (C)-containing formulations the above-described problems may be solved or avoided, so that it becomes possible as a result also to treat cheeses with disperse dyes and UV absorber liquors or with disperse brightener liquors without fear of a troublesome pressure build-up or dye or brightener filtration deposits.
The invention relates to the defined compositions, their production and their use.
The invention thus firstly provides aqueous dispersions (U) containing:
(A) a UV-light-absorbing textile treatment agent,
(B) a dispersant system and
(C) a carboxyl-containing crosslinked copolymer.
The UV-light-absorbing textile treatment agents (A) are UV-active agents, i.e. their fundamental property is the absorbing of UV light, whether to convert it into visible wavelengths, as in the case of optical brighteners, or to convert it into other energy forms or other wavelengths, as is the case with typical UV absorbers. Thus, the UV-active agents (A) are in particular
(A1) UV absorbers or
(A2) optical brighteners.
Suitable UV absorbers (A1) include generally known substances as are usually employed for the UV-light-absorbing finishing of synthetic and semisynthetic textile fibres from an aqueous dispersion, chiefly those of the benzophenone, triazine or benzotriazole series, in particular phenols which bear a substituent of the triazine, benzoyl or benzotriazole series in ortho-position relative to the hydroxyl group and which may bear further substituents, in particular non-chromophoric and non-water-solublilizing substituents.
Suitable UV absorbers of the benzophenone series include generally 2-hydroxybenzophenones in which the two benzene rings may optionally be substituted with substituents customary in UV absorbers, in particular non-chromophoric and non-water-solubilizing substituents, for example with hydroxyl, alkyl, alkoxy, aryl, aryloxy, aralkoxy or halogen, aryl (in aryl, aryloxy and aralkoxy) being naphthyl or preferably phenyl, the alkyl and alkoxy radicals containing for example 1 to 4 carbon atoms, phenyl as aryl optionally being further substituted with alkyl, halogen and/or alkoxy, and halogen signifying preferably chlorine, and the alkoxy bridge in aralkoxy containing e.g. 1 or 2 carbon atoms. There may be mentioned as examples 2-hydroxybenzophenones of the following formulae
wherein
R1 signifies hydrogen, hydroxyl, phenoxy, aralkoxy or C1-14-alkoxy,
R2 signifies hydrogen, halogen or C1-4-alkyl,
R3 signifies hydrogen or hydroxyl,
R4 signifies hydrogen, hydroxyl, phenoxy, aralkoxy or C1-4-alkoxy,
R5 signifies hydrogen or C1-4-alkyl,
R6 signifies hydrogen or C1-4-alkyl and
X signifies a hydrocarbon bridge member having 2 to 12 carbon atoms or hydroxyalkylene.
In the compounds of the formula (I), R2, R3 and R4 stand each preferably for hydrogen. R1 stands preferably for hydrogen, hydroxyl, phenoxy or C1-14-alkoxy.
In the formula (II), the two substituents R5 and R6 are advantageously in the positions ortho and para to the carbonyl group. Preferably, R5 and also R6 signify hydrogen. When X stands for hydroxyalkylene, it advantageously contains at least 3 carbon atoms and the hydroxyl group is advantageously not attached to the first or last carbon atom of the bridge. X may e.g. stand for 1,4-phenylene, 1,4-naphthylene or C2-4-alkylene or also for 2-hydroxy-1,3-propylene.
The following may be mentioned in particular:
2-hydroxy-4-methoxybenzophenone,
2,4-dihydroxybenzophenone,
2-hydroxy-5-chlorobenzophenone,
1,3-bis-(3xe2x80x2-hydroxy-4xe2x80x2-benzoylphenyl)-2-hydroxypropane.
Suitable UV absorbers of the triazinc series include in general 2-(2xe2x80x2-hydroxyphenyl)-s-triazines in which the positions 4 and 6 are substituted by hydrocarbon radicals which in turn may be further substituted, in which case the substituents are neither chromophoric nor water-solubilizing. The hydrocarbon radicals in 4-position and in 6-position arm for example aryl, alkyl or aralkyl, aryl standing e.g. for naphthyl or preferably phenyl, aralkyl preferably standing for benzyl and alkyl containing e.g. 1 to 18 carbon atoms. The substituents occurring on phenyl rings are for example hydroxyl, halogen, low molecular weight alkyl, alkoxy or alkylthio, and the substituents occurring on the alkyl radicals are e.g. hydroxyl, low molecular weight alkoxy, alkylthio or dialkylamino. There may be mentioned for example the 2-(2xe2x80x2-hydroxyphenyl)-s-triazines of the following formula
wherein
R7 signifies hydroxyl, halogen, C1-4-alkyl or C1-4-alkoxy,
R8 signifies C1-18-alkyl, which is optionally substituted with hydroxyl, halogen, C1-4-alkoxy, C1-4-alkylthio or di-(C1-4-alkyl)-amino, or phenyl, which is optionally substituted with hydroxyl, halogen, C1-4-alkyl or C1-4-alkoxy,
R9 signifies C1-18-alkyl, which is optionally substituted with hydroxyl, halogen, C1-4-alkoxy, C1-4-alkylthio or di-(C1-4-alkyl)-amino, or phenyl, which is optionally substituted with hydroxyl, halogen, C1-4-alkyl or C1-4-alkoxy, and
m signifies 0, 1 or 2.
The alkyl radicals which occur may be linear or, if they contain three or more carbon atoms, may also be branched or, if they contain six or more carbon atoms, may also be cyclic.
R7 stands preferably for chlorine or C1-4-alkyl. When m signifies 2, the two R7 substituents may have equal or different significances, in which latter case it is preferable for the second R7 to signify methyl.
When m signifies 1 or 2, one R7 is preferably in para-position relative to the 2-positioned hydroxyl group.
There may be mentioned by way of example:
2-(2xe2x80x2-hydroxyphenyl)-s-triazine in which R8 and R9 have the same significance and signify each propyl or t.butyl,
4,6-dimethyl-2-(2xe2x80x2-hydroxy-5xe2x80x2-chloro-, -methyl- or -t.butyl-phenyl)-s-triazine,
4,6-dimethyl-2-(2xe2x80x2-hydroxy4xe2x80x2,5xe2x80x2- or -3xe2x80x2,5xe2x80x2-dimethylphenyl)-s-triazine,
4,6-diethyl-2-(2xe2x80x2-hydroxy4xe2x80x2,5xe2x80x2-dimethylphenyl)-s-triazine,
4,6-diphenyl-2-(2xe2x80x2-hydroxy-4xe2x80x2-methoxy-, -ethoxy- or -isopropyl-phenyl)-s-triazine.
Suitable UV absorbers of the benzotriazole series include generally 2-(2xe2x80x2-hydroxyphenyl)-benzotriazoles wherein the two benzene rings may optionally be substituted in particular with substituents customary in UV absorbers, in particular non-chromophoric and non-water-solubilizing substituents, for example hydroxyl, alkyl, alkoxy, aryl, aralkyl, alkylaryl, alkoxycarbonyl or halogen, aryl signifying naphthyl or preferably phenyl, the alkyl radicals containing for example from 1 to 12 carbon atoms, the alkoxy radicals containing for example from 1 to 4 carbon atoms, phenyl as aryl being optionally further substituted with halogen, C1-4-alkyl and/or C1-4-alkoxy and halogen standing preferably for chlorine. There may be mentioned for example 2-(2xe2x80x2-hydroxyphenyl)-benzotriazoles of the following formula
wherein
R10 signifies C1-12-alkyl, C1-5-alkoxy, aryl, aralkyl, (C1-12-alkyl)-aryl, (C1-5-alkoxy)-carbonyl or halogen
and the rings A and B are each optionally substituted with one or more of the substituents hydroxyl, C1-5-alkyl, C1-5-alkoxy, C1-5-alkoxycarbonyl or halogen, and B may also bear a C1-12-alkyl radical in ortho position to the hydroxyl group.
The alkyl radicals which occur may be linear or, if they contain three or more carbon atoms, may also be branched or, if they contain six or more carbon atoms, may also be cyclic. Aralkyl stands preferably for benzyl. Halogen stands preferably for chlorine. In the ring B, the position 6 is preferably unsubstituted; in the benzotriazole ring, the positions 4 and 7 are preferably unsubstituted.
Preferred UV absorbers of the benzotriazole series conform to the formula
wherein
Rxe2x80x2 signifies hydrogen, C1-5-alkyl, C1-5-alkoxy, (C1-5-alkoxy)-carbonyl or chlorine,
Rxe2x80x3 signifies hydrogen or chlorine,
Rxe2x80x2xe2x80x3 signifies hydrogen, C1-12-alkyl, phenyl, (C1-5-alkyl)-phenyl, benzyl or chlorine,
Rxe2x80x3xe2x80x3 signifies hydrogen, hydroxyl, C1-5-alkyl, C1-5-alkoxy or chlorine, and
R10xe2x80x2 signifies C1-12-alkyl, C1-5-alkoxy, phenyl, (C1-5-alkyl)-phenyl, benzyl, (C1-5-alkoxy)-carbonyl or chlorine,
especially to the formula
wherein
R0 signifies C1-5-alkyl, C1-5-alkoxy or chlorine, preferably C1-5-alkyl,
R0xe2x80x2 signifies hydrogen, chlorine, C1-5-alkyl or C1-5-alkoxy, preferably hydrogen or C1-5-alkyl, and
R0xe2x80x3 signifies hydrogen, chlorine, C1-5-alkyl or C1-5-alkoxy, preferably hydrogen, chlorine or methyl.
There may be mentioned by way of example:
2-(2xe2x80x2-hydroxy-5xe2x80x2-methylphenyl)-benzotriazole,
2-(2xe2x80x2-hydroxy-3xe2x80x2,5xe2x80x2-dimethylphenyl)-benzotriazole,
2-(2xe2x80x2-hydroxy-5xe2x80x2-t.butylphenyl)-benzotriazole,
2-(2xe2x80x2-hydroxy-3xe2x80x2-t.butyl-5xe2x80x2-methylphenyl)-5-chlorobenzotriazole,
2-(2xe2x80x2-hydroxy-3xe2x80x2,5xe2x80x2-di-t.butylphenyl)-benzotriazole,
2-(2xe2x80x2-hydroxy-3xe2x80x2,5xe2x80x2-di-t.butylphenyl)-5-chlorotriazole,
2xe2x80x2-(2xe2x80x2-hydroxy-3xe2x80x2,5xe2x80x2-di-t-amylphenyl)-benzotriazole.
Useful optical brighteners (A2) are generally known substances of the type usually employed for the optical brightening of synthetic and cellulosic textile fibres from an aqueous dispersion, in particular those of the coumarin, naphthalimide, benzoxazole, stilbene, bisstyrylbenzene, thiophene or pyrene series and which may bear any substituents usual in optical brighteners, especially non-chromophoric and non-water-solubilizing substituents.
Suitable optical brighteners (A2) are for example those of the following formulae (V) to (XI)
wherein
R11 signifies C1-4-alkyl,
R12 signifies C1-4-alkyl and
R13 signifies hydrogen or C1-4-alkoxy,
wherein
R14 signifies hydrogen, chlorine or C1-4-alkyl,
R15 signifies hydrogen or C1-4-alkyl,
Y signifies a radical of the formula
R16 signifies hydrogen or C1-4-alkyl,
R17 signifies hydrogen, chlorine or C1-4-alkyl,
R18 signifies xe2x80x94CN, (C1-4-alkoxy)-carbonyl, phenyl or a radical of the formula
R19 signifies C1-4-alkyl and
Z signifies xe2x80x94CHxe2x95x90CHxe2x80x94, 3,4-thiophenylene or 1,4-naphthylene,
wherein
R20 signifies xe2x80x94CN or (C1-4-alkoxy)-carbonyl,
wherein
R21 signifies hydrogen, chlorine, xe2x80x94CN or C1-4-alkyl,
wherein
R22 signifies 1-pyrazolyl which is substituted with chlorine or C1-4-alkyl in position 3 or 4, or signifies phenyl and
R23 signifies 2-triazolyl or 1-pyrazolyl which are substituted with C1-4-alkyl and/or phenyl, or signifies 2-naphthotriazolyl, and
wherein
R24 signifies C1-4-alkyl and
R25 signifies C1-4-alkyl.
The dispersant system (B) is used for dispersing the UV-active textile treatment agents (A) in water and consequently contains appropriate, suitable surfactants and optionally suitable auxiliaries. (B) thus expediently consists of
(B1) at least one surfactant and optionally
(B2) at least one dispersing auxiliary.
The surfactants (B1) are surfactants suitable for dispersing (A) in water and they consequently have dispersant characteristics; they include inter alia also such surfactants as are known in the art also as emulsifiers. Advantageously as (B1) there are employed
(B11) anionic dispersants and/or
(B12) non-ionogenic dispersants.
As (B11) there may be employed any anion-active surfactants having dispersant or emulsifier properties, in particular compounds of distinctly anionic character which contain at least one lipophilic radical, in particular at least one lipophilic hydrocarbon radical, and at least one distinctly anionic group (sulpho group, phosphoric acid group or carboxyl group), the lipophilic hydrocarbon radical advantageously containing at least 9 carbon atoms, for example 9 to 30 carbon atoms. The following may be mentioned by way of example:
condensation products of formaldehyde with sulphonated aromatic compounds (for example sulphonated naphthalene, mono- or di-(C1-4-alkyl)-naphthalene, biphenyl, diphenyl ether, ditolyl ether, phenol, C1-4-alkylphenol and/or corresponding sulphones) and/or with ligninsulphonate, optionally in a mixture with dihydroxydiphenyl sulphone;
ligninsulphonates;
C12-24-alkyl sulphonates;
sulphonation products of paraffins (prepared for example by sulphochlorination or sulphoxidation), of xcex1-olefins, of alkylbenzenes and of unsaturated fatty acids;
C12-24-fatty acid salts;
esterification products of non-ionogenic surfactants as described below, in particular of oligoethylene glycol ethers of fatty alcohols, alkylphenols or di- or tristyrylphenols, with sulphuric acid, phosphoric acid or aminosulphonic acid, or alkylation products of the mentioned non-ionogenic surfactants with chloroacetic acid or chloropropionic acid, to form the corresponding esters or carboxymethylated or carboxyethylated products.
The anionic surfactants are advantageously present, at least partly, in the form of salts, chiefly in the form of alkali metal salts (preferably sodium or potassium salts) or ammonium salts. It is possible to use single compounds or also mixtures of compounds, including especially technical grade mixtures.
As non-ionogenic surfactants (B12) there may be employed those which act directly as emulsifiers or dispersants or also which are employed as cosurfactants to (B11). Generally known substances are suitable, principally those containing 9 to 30, preferably 12 to 24, carbon atoms in the lipophilic moiety, or also those having an oligomeric or polymeric basic structure, and whose hydrophilic moieties contain ethylene glycol radicals, glycerol radicals and/or sorbitan radicals. As examples there may be mentioned:
adducts of ethylene oxide to fatty alcohols, alkylphenols, di- or tristyrylphenols, novolaks or fatty acid N,N-diethanolamide or diisopropanolamide; fatty acid monoesters of polyethylene glycols. Polypropylene glycol chains may occasionally also occur in the molecule as lipophilic radicals, and accordingly the non-ionogenic surfactants used can also comprise block copolymers which contain propyleneoxy and ethyleneoxy units, and also optionally styreneoxy and/or butyleneoxy units (for example those known under the designation xe2x80x9cPluronicxe2x80x9d). The non-ionogenic surfactants are advantageously selected such that they can display an adequate dispersing action on (A). The HLB value of the non-ionogenic surfactants is advantageously  greater than 10, preferably in the range from 12 to 18, while those having an HLB value  greater than 15, preferably xe2x89xa716, may also be used as protective colloids.
As dispersants there may e.g. be employed (B11) alone [i.e. without (B12)] or (B12) alone [i.e. without (B11)] or mixtures of (B11) and (B12). Preferably at least one non-ionogenic surfactant (B12) is employed either as dispersant or, when (B11) is used as dispersant, also as cosurfactant to (B11). In this the weight ratio of (B11) to (B12) is e.g. in the range from 30/70 to 95/5, preferably 40/60 to 90/10. To disperse (A) in water, a suitable amount of (B1) is advantageously selected, which may vary according to the kind of the components and their concentration. The weight ratio of the total dispersants (B1) to (A), i.e. of [(B11)+(B12)] to (A), is advantageously in the range from 5/100 to 150/100, preferably 6/100 to 80/100, particularly preferably 10/100 to 70/100. When (B12) is used as cosurfactant to (B11), the weight ratio of (B12)/(B11) is advantageously 1/100 to 40/100, preferably 5/100 to 20/100.
Preferably, (B1) includes at least one polymeric surfactant, especially at least one of the mentioned condensation products of formaldehyde with sulphonated aromatic compounds or at least one of the mentioned block copolymers which contain propyleneoxy and ethyleneoxy units and optionally also styreneoxy and/or butyleneoxy units.
Dispersing auxiliaries (B2) may be employed in addition in order that the dispersing effect of (B1) may be sustained and/or the appearance of the dispersion improved.
As (B2) there are particularly worth mentioning:
(B21) protective colloids,
(B22) wetting agents
(B23) solubilizers and/or
(B24) dispersion stabilizers
If desired, polymeric protective colloids (B21) may be employed, preferably (B21) is at least one non-ionogenic and/or anionic protective colloid. Known substances are suitable as polymeric protective colloids (B21), whichxe2x80x94in contrast to the surface-active protective colloids of high hydrophilicity mentioned above under (B12)xe2x80x94have the character of polymers capable of forming a protective sheath around the dispersed colloid particles. Chiefly they are polyvinylpyrrolidones, polyvinyl alcohols, copolymers of N-vinylpyrrolidone and vinyl acetate or propionate, polyethylene glycols having for example an average molecular weight in the range from 300 to 10,000 or carboxymethylcellulose.
Among the protective colloids (B21), preference is given to the non-ionogenic ones. Advantageously a protective colloid (B21) is used. The weight ratio of (B21)/(A) is for example in the range from 1/100 to 200/100, preferably 2/100 to 150/100.
(B22) may be any wetting agent known per se, preferably at least one non-ionogenic or anionic wetting agent. As non-ionogenic wetting agents come generally into consideration surfactants of the abovementioned categories which, however, have a correspondingly lower HLB value or contain a correspondingly lower number of added ethyleneoxy units. As anionic wetting agents there may be mentioned e.g. mono- and dialkyl esters of sulphosuccinic acid. The anionic groups, analogously as described above for (B11), may advantageously be present as alkali metal salts or ammonium salts.
The use of wetting agents (B22) is possible; however, very effective dispersions can be prepared even without wetting agents. If a wetting agent is used, the weight ratio of (B22)/(A) is e.g. xe2x89xa610/100, e.g. in the range from 0/100 to 5/100.
If desired or required, solubilizers (B23) may be used, in particular non-ionogenic solubilizers, e.g. mono-, di- or oligoethylene glycols or their mono- or di-(C1-4-alkyl) ethers or glycerol.
A solubilizer (B23) may optionally be used, if desired. However, very good dispersions can be prepared even without solubilizer. If a solubilizer is used, the weight ratio of (B23)/(A) is advantageously xe2x89xa610/100, for example in the range from 1/100 to 8/100.
A dispersion stabilizer (B24) may be added, if desired. By the addition of a dispersion stabilizer (B24) the storage and transport stability of (U) may optionally also be influenced. As dispersion stabilizers (B24) there may be employed for example polysaccharides, polysaccharide derivatives, (co)poly(meth)acrylic acids and also such thickening substances as are also useful as protective colloids, for example xanthan gum, cellulose gum, guar gum, dextrins, gum arabic, carboxymethylcellulose, acrylic-modified polysaccharides, and also the above-mentioned polyvinylpyrrolidones and/or polyvinyl alcohols or polyethylene glycols e.g. with an average molecular weight in the range from 200 to 6000. Advantageously, the acid groups, particularly the carboxylic acid groups, are at least partly present in the form of salts (so that the respective products are water-soluble), e.g. as alkali metal salts (principally sodium salts). When thickeners are used, they are advantageously used in such amounts that the viscosity of the aqueous concentrated dispersion is  less than 1000 mPaxc2x7s, in particular at values xe2x89xa6300 mPaxc2x7s, preferably in the range from 50 to 200 mPaxc2x7s.
The weight ratio of the entire dispersing system (B) to (A) is for example in the range from 5/100 to 250/100, advantageously in the range from 10/100 to 150/100, preferably in the range from 50/100 to 150/100.
The dispersant system (B) serves for dispersing (A) and consists essentially of the mentioned components (B1) and optionally (B2), i.e. at least one surfactant (B11) or (B12) having dispersing properties, in which (B12) may optionally also serve as cosurfactant to (B11), and optionally further dispersing auxiliary substances (B21), (B22), (B23) and/or (B24).
The copolymer (C) is carboxyl-containing and crosslinked. It may be prepared in a manner conventional per se, by copolymerization of corresponding monomers which contain an ethylenic double bond, of which at least a portion are carboxyl-containing monomers and at least one portion is a crosslinking comonomer. Non-ionogenic comonomers may also have been incorporated in the polymer, if desired. The carboxyl-containing monomers are advantageously monomers which contain at least two carboxyl groups and 4 to 6 carbon atoms and are preferably used as anhydride, especially as cyclic dicarboxylic anhydride, for example itaconic acid or especially maleic acid or respectively its anhydride. The monomers or comonomers preferred for preparing (C) are in particular
(C1) maleic acid or maleic anhydride,
(C2) at least one monoethylenically unsaturated non-ionogenic comonomer and
(C3) a crosslinking comonomer.
As comonomers (C2) are suitable in particular low molecular weight comonomers, especially alkyl methacrylates wherein alkyl advantageously contains 1 to 4 carbon atoms and preferably is methyl or ethyl, alkyl vinyl ethers wherein alkyl advantageously contains 1 to 4 carbon atoms and preferably is methyl, vinyl esters of low molecular weight aliphatic carboxylic acids, in particular of those having 2 to 4 carbon atoms, chiefly vinyl acetate, and/or vinylpyrrolidone.
As crosslinking comonomers (C) are suitable especially non-ionogenic compounds which contain at least two mutually nonconjugated ethylenic double bonds, advantageously at least two terminal double bonds, for example N,Nxe2x80x2-methylenediacrylamide, xcex1,xcfx89-alkanedienes having 5 to 12 carbon atoms or divinyl ethers of xcex1,xcfx89-alkanediols having for example 2 to 12 carbon atoms in the alkane moiety or of polyethylene glycols, or also divinylbenzene. Of the crosslinking comonomers mentioned, the xcex1,xcfx89-alkanedienes are preferred.
The molar ratio (C1)/(C2)/(C3) is advantageously in the range of 1/(0.2-3)/0.001-0.1), preferably 1/(0.5-1.8)/(0.004-0.04).
The copolymers (C) are known or may be prepared analogously to known methods, expediently in the presence of a suitable initiator of free-radical polymerization [e.g. azobisisobutyronitrile, benzoyl peroxide, acetylbenzoyl peroxide, di-t.butyl peroxide, t.butyl peroxypivalate, azobis-(2,4-dimethyl-valeronitrile), for example in concentrations of 0.001 to 1%] and advantageously in the presence of a suitable aprotic solvent, for example benzene, toluene, xylene, acetone, methyl ethyl ketone, methylene chloride, cyclohexane and/or ethyl acetate; it is particularly advantageous to use a mixture of cyclohexane and ethyl acetate. The polymerization is advantageously carried out at temperatures in the range from 50 to 100xc2x0 C., preferably from 60 to 80xc2x0 C. The production of the polymer is advantageously carried out in such a way that crosslinking takes place at least in part intramolecularly, especially in such a way that the linear sequence of the monomers forms loops in the course of the polymerization owing to the copolymerized crosslinking monomer (C3). Since the carboxyl-containing monomer, especially (C1), is advantageously used in the form of the anhydride, especially maleic anhydride, the polymer (Cxe2x80x2) prepared therefrom still contains the anhydride groups. This polymer (Cxe2x80x2) may if desired, after completion of the polymerization and removal of the solvent, be hydrolysed to the carboxyl-containing polymer (C) in salt form. In a preferred procedure, this hydrolysis takes place in the presence of at least a portion of the surfactants (B1).
The polymers (Cxe2x80x2) and (C) may be produced for example by the process described in U.S. Pat. No. 5,024,779; the content of this US patent is incorporated herein by reference.
The dispersions (U) of the invention may be produced by mixing (C) with the other components in aqueous medium, and preferably grinding. It is advantageous to precharge water and at least a portion of the components (B1) and to add the other components thereto; the component (C) may be added before or also after the grinding and optionally even after an optional dilution with water and/or optionall addition of further components. Preferably, the component (C) is added before the component (A). In a preferred procedure, (Cxe2x80x2) is added before (A) and hydrolysed in the presence of a base (e.g. alkali metal hydroxide) so that (C) is formed therefrom in the corresponding salt form, and then the remaining components (A) and optionally further (B) and optionally formulation additives (D) are mixed in.
The weight ratio of (C)/(A) is e.g. in the range from 0.1/100 to 15/100, advantageously in the range from 0.1/100 to 5/100, preferably in the range from 0.2/100 to 2/100.
The amount of water employed is in principle freely choosable; advantageously, sufficient water is added for the dispersion to be stirrable and pourable or respectively pumpable. During grinding the amount of water present in the dispersion is advantageously such that the concentration of (A) in (U) is e.g. 2 to 50% by weight, preferably 5 to 45% by weight. More dilute dispersions are for example application liquors and stock dispersions which may optionally also contain further application-dictated additives. Concentrated to dilute dispersions may thus be produced, for example those having an (A) content in the range from 0.01 to 50% by weight. In application liquors the (A) concentrations are for example 0.01 to 20% by weight, preferably 0.05 to 1% by weight, in stock dispersions for example 0.1 to 20% by weight, preferably from 0.5 to 10% by weight, in concentrated dispersions for example 2 to 50% by weight, preferably 5 to 40% by weight. The concentrated dispersions (U) are a particular aspect of the invention.
The relative amount of the respective components and the amount of water employed are advantageously chosen so that even the concentrated dispersion (U), in particular those in which the content of (A)+(B)+(C) is advantageously in the range from 10 to 70, preferably 20 to 60% by weight, are fluent.
By the addition of suitable bases (for example of alkali metal hydroxide or carbonate and/or of an amine as described above for salt formation) or/and acids (for example of a mineral acid, such as hydrochloric acid, sulphuric acid and phosphoric acid, or of a low molecular weight aliphatic carboxylic acid, for example having 1 to 4 carbon atoms) and optionally with buffer salts, in particular phosphoric acid mono- and/or di-sodium salt and/or -potassium salt, the pH of the dispersion may be set e.g. to values in the range from 3.5 to 8.5, advantageously 3.5 to 7, preferably from 4 to 6.
Further formulation additives (D) may be used if desired, to further adapt the properties of the dispersions (U) to particular requirements or wishes concerning transport, storage and/or use. Such additives are in particular at least one of the following:
(D1) a defoamer
(D2) an agent to protect against the damaging action of microorganisms and/or
(D3) a dye.
Any defoamers are suitable as (D1), for example paraffins or mineral oils in dispersed form, silicone defoamers, silica, ethylenebisstearamide and/or mixtures of two or more thereof. Commercially available products may be used in particular. The amounts of defoamer which can be used in the compositions of the invention, are in the ranges conventional per se and are in general dependent on the nature and amount of the surfactants. In general, very small amounts of defoamer are sufficient, e.g. xe2x89xa61% by weight based on the entire aqueous composition (U).
As components (D2) are suitable in general known substances, essentially fungi- or bacteriostatic substances and/or microbicides, as are commercially available, and the concentrations used may conform to those recommended for each, for example xe2x89xa61% by weight based on the entire aqueous dispersion (U).
If as (A) there is employed (A2), i.e. an optical brightener, a component (D3) may also be employed. (D3) is advantageously a disperse dye of the type usable in general for shading disperse brighteners. Disperse dyes are a generally known category of dyes and are known in the art and extensively described in the technical literature, for example in the Colour Index. Suitable components (D3) include any disperse dyes which possess a colour suitable for shading optical brighteners, for example blue, violet and red disperse dyes. They are advantageously added in low concentrations, for example xe2x89xa60.1% by weight based on (A2), as is generally sufficient in order, for example, that a yellowish, greenish or brownish shade of the substrate may be partially compensated or/and a, for example, greenish self-colour of the optical brightener, which may become evident at a certain application concentration, may be balanced out and thereby the visual impression created by the optical brightening may be improved.
The (A) content of the concentrated aqueous dispersions (U) is for example in the range from 2 to 50% by weight, advantageously 5 to 45% by weight, preferably 10 to 40% by weight. The water content of the concentrated aqueous dispersions (U) of the invention is advantageously in the range from 90 to 30%, preferably 70 to 33%, by weight:
The dispersions (U) of the invention can be produced as described above, in particular with grinding, in which case with the aid of suitable mills, for example ball mills or bead mills (glass or porcelain beads), the dispersed particles may be ground to a particle size of the desired fineness, and, if desired, a portion of the components, particularly of the water-soluble or water-thinnable components, particularly for example a protective colloid (B1), a solubilizer (B23), a component (D2) and/or (D3), may also not be mixed in until after the grinding. The average particle size of the dispersed particles in the dispersions (U) of the invention is advantageously in the range from 0.05 to 10 xcexcm, preferably 0.2 to 6 xcexcm, particularly preferably 0.4 to 4 xcexcm. Preferably, the dispersions are filtered through a suitable sieve or a suitable filter in such a way that the dispersed particles are not larger than 20 xcexcm, preferably not larger than 10 xcexcm, particularly preferably not larger than 6 xcexcm.
The aqueous dispersions (U) prepared in this manner, even the concentrated dispersions, are surprisingly stable and fluent. The concentrated dispersions have in particular a viscosity which is  less than 1000 mPaxc2x7s; preferably, they have a viscosity in the range from 20 to 300, preferably 50 to 200, mPaxc2x7s. They may be handled, stored and/or transported and also be used directly for metered addition, so as they have been prepared. They are notable for surprisingly good stability in storage and transport.
The dispersions (U) of the invention serve for the corresponding UV-active finishing of textile material, in particular textile material which can be dyed with disperse dyes or optically brightened with disperse brighteners. Suitable textile material includes in particular textile material composed of synthetic and semisynthetic materials, chiefly of polyester, polyamides, polyurethanes and cellulose acetates, and also of fibre blends comprising such synthetic or semisynthetic materials (e.g. polyester/cotton, polyester/viscose, polyester/cotton/elastane). The textile material may be in any desirable processed state suitable for the particular application, for example in the form of loose fibres filaments, yarns, hanks, wovens, knits, carpets, ready-made and half-ready-made goods. Particular preference is given to using the dispersions (U) of the invention in the HT dyeing of yarns, which are wound on packages, especially in the form of cheeses, or in the optical brightening by the HT or thermosol processes.
The dispersions (U) of the invention are highly compatible with disperse dyes (F) and, when (A) is a UV absorber (A1), are advantageously formulated together with a suitable disperse dye (F) to form an aqueous liquor which includes both the disperse dye (F) and the UV absorber (A1). The disperse dye (F) may be any desired disperse dye or disperse dye mixture as are otherwise used for dyeing synthetic or semisynthetic textile material. Disperse dyes are generally known and extensively described in the technical literature, for example in the Colour Index under the heading xe2x80x9cDisperse Dyesxe2x80x9d. Disperse dyes are generally formulated with suitable dispersants in order that they may be dispersed in the dyeing liquor; the dispersants used for this purpose are usually anionic and optionally non-ionogenic dispersants, for example such as described under (B1). Disperse dyes formulated in this way customarily include 30 to 60% of dye and about 70 to 40% by weight of dispersant besides, optionally, traces of by-products.
When (A) is an optical brightener (A2), generally either no dyes will be employed at all or dyes will be employed as described under (D3) for shading.
The concentrations of dyes (F) in the respective liquor may vary within wide limits, depending on the substrate, the choice of dyeing method and the choice of depth of shade. The concentration of (U) in the liquor is generally dependent on the nature and the content of (A) and on the desired UV-active effect. Concentrations are employed, for example, such that the concentration of (A1) based on the substrate is in the range from 0.1 to 4% by weight, preferably 0.2 to 2% by weight. The concentration of (A2) based on the substrate is advantageously chosen so as to obtain a good white effect and may also vary according to substrate, application method and constitution of (A2); it is for example within the range from 0.01 to 4%, advantageously 0.05 to 3%, preferably 0.1 to 2%, by weight. The liquor pH is advantageously in the distinctly acidic to nearly neutral range, for example in the pH range from 4 to 6.5, preferably 5 to 6.
The liquors may be applied to the textile material by conventional methods, for example by exhaust processes or impregnation processes, and at appropriate suitable liquor lengths and temperature conditions. For impregnation processes, the liquors may be applied to the substrate by customary methods, e.g. by padding, dipping or spraying, and the impregnated substrate, optionally after intermediate drying, may then be thermofixed at elevated temperature, e.g. in the range from 150 to 220xc2x0 C., in which case a suitable fixation temperature can be chosen according to the material (for polyester e.g. from 160 to 220xc2x0 C., for other synthetic fibres and for semisynthetic fibres e.g. from 150 to 180xc2x0 C.). For exhaust processes short or also long liquors may be employed, e.g. liquor-to-goods ratios in the range from 2:1 to 100:1, usually 3:1 to 60:1. For HT processes and especially for the preferred treatment of cheeses, liquor-to-goods ratios of 4:1 to 40:1, in particular 5:1 to 20:1, are preferred; the temperatures may also vary as desired in the respectively suitable ranges, for example from 95 to 180xc2x0 C., HT conditions being preferred, e.g. in the range from 105 to 180xc2x0 C., preferably xe2x89xa7125xc2x0 C., e.g. in the range from 125 to 135xc2x0 C.
To dye or optically brighten the cheeses, according to a preferred procedure the packages are immersed into the liquor or have the liquor poured over them and then the liquor is pumped through the cheese (from in to out or vice versa or also alternately) for the entire dyeing time. As the liquor is pumped through the package, the temperature is slowly raised, for example at a rate of 0.5 to 5xc2x0 C. per minute, preferably 1 to 4xc2x0 C. per minute, until the desired dyeing temperature (HT conditions) is reached, at which dyeing may be carried out e.g. for 20 to 60 minutes, and then it is brought back down slowly, for example at a rate of 1 to 10xc2x0 C. per minute, preferably 2 to 6xc2x0 C. per minute. The overpressure upstream of the package may be for example in the range from 0.02 to 0.5 bar, preferably 0.5 to 0.2 bar, whilexe2x80x94using the dispersions (U) of the invention, especially the preferred onesxe2x80x94practically no pressure build-up takes place during the entire dyeing or brightening operation.
In the following Examples, parts are parts by weight and percentages are percentages by weight; the temperatures are reported in degrees Celsius; the disperse dyes and optical brighteners employed in the Examples are commercially available products containing about 50% of pure dye or pure brightener and about 50% of dispersant; the other additives, apart from the products of Examples 1 to 5, are commercial products. Product (C31) employed in the Examples is a copolymer of maleic anhydride, methyl vinyl ether and 1,9-decadiene in a molar ratio of 1:1:0.0125, prepared analogously to example 1 of U.S. Pat. No. 5,024,779, which as a 0.5% aqueous suspension at pH 7.0 has an average particle size  less than 75 xcexcm and a rotation viscosity of 45000 to 70000 mPaxc2x7s (spindle no. 7, 20 rpm).

Aqueous compositions of UV-active agents, their production and use

Abstract: Aqueous dispersions (U) containing:

Abstract: (B) a dispersant system and

Abstract: (C) a carboxyl-containing crosslinked copolymer, and optionally

Abstract: (D) at least one formulation additive,

Abstract: are useful for the corresponding UV-active finishing of textile material, in particular textile material which can be dyed with disperse dyes or optically brightened with disperse brighteners, especially for treating cheeses with liquors comprising disperse dyes and UV absorbers or liquors comprising disperse brighteners, without fear of troublesome pressure build-up or dye or brightener deposits.


The present invention relates to a sulfonated polymer membrane and to a process for the preparation thereof. According to the process, a polymer film is irradiated, and the irradiated polymer film is sulfonated in order to link sulfonic acid groups thereto. According to the invention, the sulfonation is continued until the total concentration of sulfonic acid groups in the membrane is 0.4-3.0 meq/g and they are homogeneously distributed in the membrane material in such a manner that their concentration in the middle of the membrane is at minimum 0.2 meq/g. With the help of the invention, it is possible by a rapid and simple process to prepare membranes the chemical and mechanical properties of which can be regulated by means of the selection of the initial film, by irradiation and by the sulfonation process.

The present invention relates to a process according to the preamble of claim 1 for the preparation of a sulfonated polymer membrane.
The invention also relates to the polymer membrane according to the preamble of claim 12, its use according to claim 18 in electrochemical cells, and the electrochemical cell according to the preamble of claim 19.
Conductivity is one of the most essential properties in any application of a membrane such as the membrane according to the present invention. The membranes are also required to have mechanical strength, chemical stability, and good barrier properties against the permeation of non-desirable components from one side of the membrane to the other.
Ion-conductive membranes can be used in numerous applications. Some examples are their use as proton conductors in fuel cells or electrolytic cells. In a fuel cell, the energy released in the reaction is converted to electric current at a conversion rate of approximately 60-80%. Fuels suitable for such cells include hydrogen, natural gas and methanol. Fuel cells which have polymer membranes as electrolytes are regarded as one of the most interesting options for relatively small-scale energy production applications in which the power source is less than 150 kW. Such applications include vehicles and certain electric appliances.
At present, many polymer membranes are known which are suitable for use for the purposes mentioned above. In the state of the art (compiled in the work Davis, T. A., Genders, J. D. and Pletcher, D., A First Course in Ion Permeable Membranes, pp. 35-57), two principal preparation processes are disclosed, of which, of course, several variations have been developed.
In the first prior art process, an unsubstituted alkene is copolymerized with a functionalized alkene which contains ionizable groups or, more probably, precursors of ionizable groups. It has been observed that perfluorinated membranes have the best properties in particular as regards stability and the chemical and physical properties. The first step in the preparation of a membrane such as this is monomer synthesis, whereupon the result obtained is a perfluorinated, substituted alkene having an ion-exchange group at the end of a side chain. Sulfonate or carboxylate groups are used commercially. The length of the side chain usually varies from 1 to 4 carbon atoms. This ionomer is thereafter copolymerized with polytetrafluoroethylene (PTFE). PTFE forms the backbone in almost all membranes prepared by the technique described above. The polymer is brought to film form before the conversion of the precursors to ion-exchange active groups.
According to another prior art process, the alkene is polymerized, whereafter ion groups are introduced into the polymer. Usually the membranes prepared in this manner are based on copolymers of styrene and divinyl benzene. There are numerous different alternative embodiments, for example, it is possible to irradiate a stable, inert polymer in order to enable this polymer to be grafted with some aromatic polymer. Ion-exchange groups, which become linked to the aromatic ring, are usually introduced into the structure by means of a strong sulfuric acid solution.
Furthermore, there are a number of membranes of special production, the best known of them being probably Gore Select. It is based on filling the pores of a material like Gore Tex, known as a weatherproof material and being based on PTFE material, with a ion-exchange active polymer, such as commercial Nafion(copyright). The conductivity of Gore Select is, of course, not in the order of that of Nafion(copyright).
The performance of membranes according to the first technique, one example being specifically Nafion(copyright), is quite fair. The problem involved with these membranes is their difficult preparation process. For this reason the price of the product remains high and the amounts used remain low. It is also to be noted that the properties of the membrane are largely determined already during the monomer stage, and thus the modification of the properties in membrane form is nearly impossible. The processing of the membrane is also cumbersome. On the other hand, membranes prepared by the grafting process are seldom chemically resistant.
In prior art there is also disclosed a process in which reactive sites are provided in a polyvinyl fluoride film (PVF film) by electron or proton irradiation (Paronen, M., Sundholm, F., Rauhala, E., Lehtinen, T. and Hietala, S., Effects of Irradiation on Sulfonation of Poly(vinyl fluoride), J. Mater. Chem., 1997, 7(12), 2401-2406). An irradiated film was treated with chlorosulfonic acid having a concentration of 2.5% by volume. It is noted in the publication that both the absorbed radiation dose and the mass of the irradiating particle affect the sulfonation. The conductivity of proton-irradiated membranes was at best 10-20 mS/cm when the absorbed doses were 400-1000 kGy. However, the distribution of sulfonic acid groups in the membrane is not discussed in the publication. Furthermore, it was observed in laboratory experiments that the membranes according to the publication, when treated, were not self-supporting or their conductivity was very low.
The object of the present invention is to eliminate the disadvantages associated with prior art and to provide a new process for the preparation of sulfonated polymer membranes.
According to the invention, a polymer film is irradiated with ions or gamma radiation in order to produce reactive sites. The irradiated membrane material is sulfonated in order to link sulfonic acid groups to it. The sulfonation is continued until the total concentration of sulfonic acid groups in the membrane is 0.4-3.0 meq/g and they are homogeneously distributed in the membrane in such a manner that their concentration in the middle of the membrane is at least 0.2 meq/g.
By the process according to the invention it is possible to prepare a membrane in which the sulfonic acid groups are linked directly to the repeating unit of the polymer chain and not to a side chain as in prior-art options, when the membrane material is non-aromatic.
More specifically, the process according to the invention is characterized by what is stated in the characterizing part of claim 1.
The membrane according to the invention, for its part, is characterized by what is stated in the characterizing part of claim 12.
The electrochemical cell according to the invention is characterized by what is stated in the characterizing part of claim 19.
Considerable advantages are achieved by means of the invention. By the process according to the invention, a membrane is obtained which is self-supporting and which can be used in various applications, such as fuel cells. The membrane can also be used as an ion-exchange active material in ion exchange, in the coating of material, in ion-selective purification, in applications exploiting filter or separator membranes, or in applications exploiting semi-permeable membranes. The homogeneous distribution of the sulfonic acid groups in such a manner that their concentration in the middle of the membrane is above 0.2 meq/g ensures that conductivity will not decrease too much. On the other hand, the homogeneous distribution of sulfonic acid groups in the membrane improves the mechanical properties of the membrane as compared with a situation in which the sulfonic acid groups are mainly located on the membrane surface.
By the process according to the invention it is possible to prepare, by a rapid and simple process, membranes the chemical and mechanical properties of which can be regulated by the selection of the starting membrane, by irradiation and by the sulfonation process. Owing to the simple process, the production costs are also much lower, and thus the selling price of the membrane is also significantly lower. This is of special significance considering the use of the electrochemical cell according to the invention, in particular a fuel cell, as a source of energy. Prior art membranes are so expensive that their use, for example, in cells intended for sources of energy for automobiles would considerably increase the costs of manufacture of automobiles. The best known and most researched membrane on the market for the fuel cell application is Nafion(copyright). The price of Nafion(copyright) per square meter is, however, so high that the use of a fuel cell as the source of energy would almost double the automobile manufacturing costs.
Compared with the technology of prior art, the special feature of the membrane material is that the sulfonic acid groups are linked directly to the linear carbon backbone of the polymer chain and not to the end of a side chain. In other words, the sulfonic acid groups are linked to a polymer chain carbon atom which, together with the other carbon atoms, forms the carbon backbone of the polymer chain. Through this, the advantage is gained that a greater crystallinity of the material is made possible, in which case the density of the material is higher and its macro structure more rigid. From these, it follows that the permeability of the membrane is lower, its solubility is lower, and its dimensional stability is better.
The invention is discussed below with the help of the accompanying drawing. The figure shows the polarization curves of a commercial material and a membrane according to the invention in a fuel cell test.
In order that the product should have the desired mechanical and chemical properties, the membrane material must be selected with these factors in mind. The membrane material should be such that irradiation will cause a change which promotes functionalisation, and it should be sufficiently sensitive to irradiation. Thus, according to the first preferred embodiment of the invention, the membrane material used is a non-aromatic polymer. In the context of the present invention, xe2x80x9cnon-aromaticxe2x80x9d means that the material consists mainly of non-aromatic units. Possibly the material may, however, contain aromatic groups, but so that the presence of the aromatic groups will not substantially affect the irradiation. According to another preferred embodiment of the invention, the membrane material used is an aromatic polymer.
It is important that, before treatment, the membrane material is sufficiently tough and strong and that it is chemically resistant. In particular, because of the applications, the mechanical strength and self-supporting quality of the membrane material even after treatment is to be taken into account. By self-supporting quality is meant here not only the capability of the membrane material to cohere without support but also that it must withstand the strain caused by the various uses, in other words, that it will not crumble, crack or break even when acted upon by relatively small, partly non-homogeneous forces. For example, in a fuel cell the gas diffusion electrodes creep, whereupon the ion-exchange membrane is subjected to non-homogeneous forces. The self-supporting membrane according to the invention withstands these forces.
The permeability of the membrane material is especially important in a fuel cell application, in which case the permeability must be sufficiently low in order to prevent the permeation of the fuel from one side of the membrane to the other. Thus, in the present invention, membranes are preferably made from materials which are not substantially porous.
The most preferable of the non-aromatic materials which fulfill the above-mentioned requirements is polyvinyl fluoride (PVF). Other especially suitable materials include other fluorinated materials such as polyhexafluoropropylene, polychlorotrifluoroethylene, polytetrafluoroethylene (PTFE) and polyvinylene fluoride (PVDF). Polyethylene (PE) and polypropylene (PP) can also be used. In addition, it is possible to use mutual copolymers or blends of any of the above-mentioned polymers. It is also possible to use a copolymer or blend of any polymer of the group with a polymer selected from outside the group.
The aromatic membrane material is selected from a group which includes polyesters, polysulfones, polyphenylene sulfides, polyketones, polyether sulfones, polybenzimidazoles, polyimides, polyetherether ketones and polyarylether sulfones. Preferably polyimide is used. In addition, it is possible to use mutual copolymers, graft polymers or blends of any of the above-mentioned polymers. It is also possible to use a copolymer, graft polymer or blend of any one or several of the polymers with a polymer or polymers selected from outside the group. By blends is meant here both homogeneous and micro- or macro-phase separated blends. Sandwich-structured materials can also be used.
Alternatively, the membrane material used may also be a multiple-layer film. In this case the membrane material is made up of at least two layers which may be mutually of the same material or of different materials selected from the group including the materials listed in the preceding paragraph. However, the materials must be compatible in such a manner that they can be processed at an elevated temperature, i.e. be hot pressed, glued or otherwise joined tightly together. Preferably these materials are selected so that the film layers with a higher chemical resistance (e.g. PTFE) are placed outermost and the most readily sulfonating film layers in the middle. One preferred option is to place PTFE layers outermost and a PE layer between them.
The membrane material is irradiated with ion or gamma radiation. Irradiation causes in the material a change which controls the sulfonation position, either directly through the reaction product or through a change in crystallinity. The irradiation speed should be such that it does not cause excessive heating up of the film being irradiated. The radiation dose (absorbed dose) is typically 50-1500 kGy. The irradiation time depends largely on the thickness of the film being irradiated and on the apparatus used, and thus the time consumed by the providing of the desired absorbed dose varies greatly. The irradiation is carried out while the film is in an inert atmosphere or a vacuum. After the irradiation the film is stored preferably in substantially oxygen-free conditions until its sulfonation.
By ion irradiation, a linear track of reaction products and structural changes is produced. In ion irradiation it is possible to use, for example, any of the following ions: H+, He+, He2+, Li+, Li2+, Li3+. In ion irradiation, radiation absorption forms a linear track across the film, whereupon a passage of an amorphous phase is formed across the film. The sulfonation reaction is more rapid in the amorphous phase. Thus a site-selective and more rapid sulfonation is achieved. Under the effect of ion irradiation, micropores are formed in the membrane material. The sulfonic acid groups also attach to the walls of the micropores.
In gamma radiation, the irradiation time is quite long; it may be up to 10 days. It should, however, be noted that by gamma radiation it is possible to treat considerably thicker samples than by, for example, conventional electron irradiation. The use of gamma irradiation is based on the fact that it increases crystallinity and cross-linking. When crystallinity increases, sulfonation is directed to the amorphous phase.
The relatively even distribution of sulfonic acids or their derivatives in the film relative to the cross-section of the membrane is essential in sulfonation. According to the invention, the concentration of sulfonic acid groups in the middle of the membrane is at minimum 0.2 meq/g, which ensures that conductivity will not decrease too much because of a less conductive middle area. Preferably the concentration of acid groups in the middle of the membrane is at minimum 0.4 meq/g. The concentration of sulfonic acid groups quite on the surface of the membrane may vary widely. At their highest the concentrations may be up to approximately 7-10 meq/g, but when the topmost surface layer is not taken into account, the concentration in the vicinity of the surface is typically 1-3 meq/g.
Another important quantity is sufficient total sulfonation degree, since the total sulfonation degree is in practice the same as the ion exchange capacity; sulfonic acid groups are specifically ion-exchange active groups. The total concentration of sulfonic acid groups in the membrane is 0.4-3.0 meq/g, preferably 0.5-1.5 meq/g. If the total concentration in the membrane is lower than 0.4 meq/g, the conductivity of the membrane is low. On the other hand, if the concentration of sulfonic acid groups in the membrane exceeds 3 meq/g, the structure begins to dissolve in water or the membrane begins to lose its mechanical strength.
The sulfonation, which is preferably carried out at minimum 10 minutes after the irradiation, can be carried out either as solution sulfonation or as gas-phase sulfonation. In each case, the sulfonation is carried out in substantially oxygen-free conditions. By this is meant that oxygen is present in so small an amount that secondary reactions detrimental to sulfonation are not started. Thus the processing takes place in the presence of an inert gas (e.g. N2).
In gas phase sulfonation the reagent is a sulfur compound which is in the gas phase at the sulfonation temperature. For example, it is possible to use oleum, chlorosulfonic acid, sulfur dioxide (SO2) or sulfur trioxide (SO3). The sulfonation is carried out in a pressure-resistant container, in which the gas-forming reagent is placed first or it is fed into the container after the irradiated film has been placed in the container. The pressure in the container is approximately 0.5-20 bar, preferably 0.5-1.5 bar, and it is regulated by means of an inert gas. The temperature in the container is approximately 0xc2x0 C. The time consumed by the gas sulfonation varies according to the film thickness and the desired degree of sulfonation; typically it is approximately from 15 minutes to 3 hours. In solution sulfonation it has been observed in connection with the present invention that, when dilute sulfonation solutions are used, i.e. when the concentration of the sulfonation reagent in the solution is low, a relatively even sulfonation relative to the cross-section of the film is attained. The concentration of the solution is preferably 0.1-1.5% by volume, especially preferably 0.5-1.5% by volume. In solution sulfonation, there is preferably used chlorosulfonic acid dissolved in some chlorinated hydrocarbon, which may be, for example, dichloroethane.
The sulfonation temperature is preferably between 0xc2x0 C. and 25xc2x0 C. The sulfonation is typically carried out at room temperature. In this case the sulfonation time is approximately 0.5-2 h. It is also possible to operate at a lowered temperature. The reaction rate of sulfonation at 0xc2x0 C is, however, very low and, thus, the sulfonation is preferably carried out by allowing the film to stand at a lowered temperature in order to cause the sulfonic acid to diffuse into the film, and by raising the temperature after approximately 1-2 h, whereupon the reaction starts properly.
After both gas sulfonation and solution sulfonation the membrane must be cleaned in order to remove the water-soluble polymer and the free acid present in the membrane. Suitable cleaning methods include, for example, evaporation, which can be carried out under normal pressure or under a reduced pressure, and a multiple-step cleaning, which can be carried out in part or entirely under pressurized conditions or under normal pressure. Preferably these methods are used combined.
According to one preferred embodiment, the membrane is first washed with a solvent of the sulfonation reagent at a lowered temperature for approximately 10-60 min, the temperature is thereafter raised, typically to room temperature, and the wash is continued for approximately 10-60 min. Thereafter the solvent is evaporated and the washing is continued for 1-3 h by using some other solvent, for example, THF, acetone or ethanol. The solvent is allowed to evaporate or the membrane is dried by means of heat and is transferred to water-vapor humidification in order to saturate the membrane gradually with water. Finally the membrane is washed with water.
The thickness of the non-aromatic sulfonated polymer membrane prepared by the process described above is approximately 5-200 xcexcm, preferably approximately 30-60 xcexcm. The total concentration of sulfonic acid groups in the membrane is 0.4-3.0, preferably 0.5-1.5 meq/g. The concentration of sulfonic acid groups in the middle of the membrane is at minimum 0.2 meq/g, preferably at minimum 0.4 meq/g. It is to be noted that in the membrane the ion exchange groups are linked directly to the linear carbon backbone of the polymer chain and not to the end of a side chain as in prior-known options.
A preferred application of the invention is an electrochemical cell, in particular a fuel cell. The electrochemical cell according to the invention comprises a first plate, a second plate placed at a distance, and at least one membrane-electrode assembly fitted between the first and the second plates. By an assembly is meant here a combination which comprises a first porous electrode, which is called a fuel electrode and which serves as the anode in the cell. At a distance from the first electrode there is located a second porous electrode, which is an oxidant electrode and which serves as the cathode in the cell. Between the first and the second electrodes there is fitted an ion-conducting electrolyte membrane. This membrane comprises a membrane according to the invention, Furthermore, each assembly has first feed units for the feeding fuel into the first electrode and second feed units for feeding an oxidant to the second electrode, and electric switching elements which enable switching between the first and the second electrodes.
The electrodes are interconnected by means of an external circuit in such a manner that there is a load (e.g. an electric motor) between them. The electric current in the external circuit is a stream of electrons, whereas it is ions that travel in the electrolyte, protons (H+) in acid electrolytes and hydroxyl ions (OHxe2x88x92) in alkaline ones. In the case of an acid electrolyte, the fuel cell works so that the fuel(typically hydrogen gas) arriving at the anode becomes ionized, producing ions and electrons. The electrons travel via the external circuit, and the ions travel via the proton-conducting electrolyte to the cathode. In the cathode reaction, on the other band, the oxidant (typically oxygen gas) reacts with the ions which have traveled via the electrolyte and the electrons coming from the external circuit.
The invention is described below with the help of embodiment examples.

Polymer membrane and a process for the production thereof


The present invention provides an isolated IxcexaB kinase binding protein designated Y2H14 and functional equivalents therof. The amino acid sequence of Y2H14, the nucleotide sequence encoding Y2H14, and other related protein and nucleic acid molecules are also provided.

The NF-xcexaB family of transcription factors are involved in the regulation of a wide variety of cellular responses. These transcription factors mediate extracellular signals that induce expression of genes involved in such diverse processes as cell division, inflammation, and apoptosis. See, for example, Baldwin, Annu. Rev. Immunol. 12, 141-179 (1996); Beg and Baltimore, Science 274, 782-274 (1996); Gilmore et al., Oncogene 13, 1267-1378 (1996); Mayo, et al, Science 278, 1812-1815 (1997); and Van Antwerp et al., Science 274, 787-789 (1996); Ashkenazi and Dixit, Science 281, 1305-1308 (1998).
NF-xcexaB is anchored in the cytoplasm of most non-stimulated cells by a non-covalent interaction with one of several inhibitory proteins known as IxcexaBS. See for example, Baeuerle and Baltimore, Science 242, 540-546 (1988). Cellular stimuli associated with immune and inflammatory responses, for example inflammatory cytokines such as tumor necrosis factor xcex1 (TNFxcex1) or interleukin-1 (IL-1), activate kinases, which in turn activate NF-xcexaB by phosphorylating IxcexaBs. The kinases that phosphorylate IxcexaBs are called IxcexaB kinases (IKKs).
Phosphorylation marks the IxcexaBs for ubiquitination and proteosome mediated degradation. The degradation and dissociation of IxcexaBs from NF-xcexaB unmasks the NF-xcexaB nuclear localization signal, and facilitates the nuclear translocation of active NF-xcexaB to the nucleus, thereby upregulating NF-xcexaB responsive target genes. See, for example, Baeuerle and Henkel, Annu. Rev. Immunol. 12, 141-179 (1994); Baldwin, Annu.Rev. Immunol. 14, 649-683 (1996); Siebenlist et al., Annu.Rev.Cell Biol. 12, 405-455 (1994); and Verma et al, Genes Dev. 9, 2723-2735 (1995). Thus, this phosphorylation of IxcexaBs is a key regulatory step for NF-xcexaB mediated processes.
The determination and characterization of kinases that directly phosphorylate IxcexaBs are instrumental in the delineation of signaling pathways involving NF-xcexaB activation. Recently, an IxcexaB kinase, designated IKKxcex1 but also referred to as CHUK (conserved helix-loop-helix ubiquitous kinase), was identified in a yeast-two-hybrid screen with NIK as bait. Regnier et al., Cell 90, 373-383 (1997). IKKxcex1 was determined to be responsible for the major IxcexaB kinase activity induced by TNF stimulation of HeLa cells. DiDonato et al., Nature 388, 548-554 (1997). The identification of IKKxcex1 as a cytoplasmic kinase which phosphorylates IxcexaB family members at their physiologically relevant sites and targets them for proteosome-mediated degradation was a major breakthrough.
The IKKxcex1 gene encodes a 745 amino-acid polypeptide (having a molecular mass of approximately 85 kDa). Murine and human IKKxcex1 cDNA clones were found to be almost identical. Connelly and Marcu, Cellular and Molecular Biology Research 41, 537-549 (1995).
Another kinase, termed IKKxcex2, homologous to IKKxcex1, has also been reported. Stancovski and Baltimore, Cell 91, 299-302 (1997); Woronicz et al., Science 278, 866-869 (1997); and Zandi et al., Cell 91, 243-252 (1997). IKKxcex1 and IKKxcex2 have 52% overall similarity to each other and 65% identity in the kinase domain. Zandi et al., Cell 91, 243-252 (1997). An IxcexaB kinase termed T2K has also been described in U.S. Pat. No. 5,776,717 to Cao.
The known IxcexaB protein kinases generally phosphorylate IxcexaBs at specific serine residues. For example, they specifically phosphorylate serines 32 and 36 of IxcexaBxcex1. Phosphorylation of both sites is required to efficiently target IxcexaBxcex1 for destruction in vivo. Moreover, activation of IKKxcex1 and IKKxcex2 occurs in response to NF-xcexaB activating agents and mutant IKKxcex1 and IKKxcex2 that are catalytically inactive block NF-xcexaB stimulation by cytokines. These results highlight the important role played by IxcexaB protein kinases in NF-xcexaB activation processes. See Stancovski and Baltimore, Cell 91, 299-302 (1997) for a recent discussion of IxcexaB kinases.
IKKxcex1 and IKKxcex2 have structural motifs characteristic of the IKK kinases. This includes an amino terminal serine-threonine kinase domain separated from a carboxyl proximal helix-loop-helix (H-L-H) domain by a leucine zipper domain. These structural characteristics are unlike other kinases, and the domains are thought to be involved in protein-protein interactions.
Numerous proteins are involved in the signaling pathways that lead to immune, inflammatory, and apoptotic responses. A complete elucidation of these processes requires the identification of additional proteins that are involved and a determination of the protein interactions.
The discovery of additional proteins involved in these processes is important for controlling immune, apoptotic, and inflammatory processes. Thus, there is a great need for the identification and characterizion of additional proteins involved in IKK mediated cellular processes.
The present invention provides an isolated IKK binding protein comprising the amino acid sequence set forth in SEQ ID NO:1 and functional equivalents. Also included are isolated nucleic acid molecules that encode the IKK binding protein comprising the amino acid sequence set forth in SEQ ID NO: 1 and its functional equivalents, methods of making the IKK binding proteins comprising expressing nucleic acid molecules encoding the proteins, and antibodies directed to the IKK binding proteins.
The invention is directed to an IKK binding protein, designated Y2H14, and its functional equivalents. Y2H14 will hereinafter refer to the protein defined by SEQ ID NO:1, which is found in humans.
In this specification, functional equivalents are proteins or fragments that are substantially homologous to SEQ ID NO:1 and that specifically bind to an IKK protein, such as IKKxcex1 or IKKxcex2. The term IKK is used herein to refer to all kinases that phosphorylate any IxcexaB and that have helix-loop-helix and leucine zipper domains. Y2H14 and its functional equivalents bind to the region of the IKK proteins made up of the contiguous helix-loop-helix and leucine zipper domains.
In order to determine whether the sequence of a first protein, or fragment thereof, is substantially homologous to the sequence of a second protein, such as Y2H14, or fragment thereof, the sequences are first aligned so as to optimize the percent of amino acid residues that are identical, or that are identical or equivalent, at corresponding positions. Gaps may be introduced in the sequences, if necessary, to achieve optimization.
Amino acids generally considered to be equivalent are indicated below in separate rows (a) through (e):
(a) Ala (A), Ser (S), Thr (T), Pro (P), Gly (G)
(b) Asn (N), Asp (D), Glu (E), Gln (Q)
(c) His (H), Arg (R), Lys (K)
(d) Met (M), Leu (L), Ile (I), Val (V)
(e) Phe (F), Tyr (Y), Trp (W)
The amino acid sequences of highly homologous proteins can usually be aligned by visual inspection. If visual inspection is insufficient, the proteins are aligned in accordance with any of the methods described by George, D. G. et al, in Macromolecular Sequencing and Synthesis, Selected Methods and Applications, pages 127-149, Alan R. Liss, Inc. (1988), such as the formula described at page 137 using a match score of 1, a mismatch score of 0, and a gap penalty of xe2x88x921.
In a first embodiment, the sequence of a protein or fragment thereof is considered substantially homologous to the sequence of Y2H14 or a fragment thereof if the amino acid sequences, after alignment, are at least about 25% identical, preferably at least about 35% identical, more preferably at least about 50% identical, even more preferably at least about 65% identical, still more preferably at least about 75% identical, most preferably at least about 85% identical, and, ideally at least about 95% identical.
In a second embodiment, the sequence of a protein or fragment thereof is considered substantially homologous to the sequence of Y2H14 or a fragment thereof if the amino acid sequences, after alignment, are at least about 50% identical or equivalent, preferably at least about 65% identical or equivalent, most preferably at least about 85% identical or equiavalent, and ideally at least about 95% identical or equivalent.
Functional equivalents include all modifications of the polypeptide set forth in SEQ ID NO:1. These modifications may be introduced deliberately, as by site-directed mutagenesis, or may be natural variations and mutations. Such modifications include substitutions, additions, and/or deletions in the protein sequences as long as substantial homology and specific binding to IKK proteins are maintained.
For example, functional equivalents include variant Y2H14 proteins that are expressed by naturally occurring alleles. Alleles are alternative forms of a gene that occupy a given locus of a chromosome within a species.
Functional equivalents of Y2H14 also include proteins from non-human mammalian species (species orthologs) as well as proteins expressed by alleles of such species orthologs. Non-human mammals include, for example, primates, pet animals such as dogs and cats, laboratory animals such as rats and mice, and farm animals such as horses, sheep, and cows.
The proteins of the invention that do not occur in nature are isolated. The term xe2x80x9cisolatedxe2x80x9d as used herein, in the context of proteins, refers to a polypeptide which is unaccompanied by at least some of the material with which it is associated in its natural state. The isolated protein constitutes at least 0.5%, preferably at least 5%, more preferably at least 25% and still more preferably at least 50% by weight of the total protein in a given sample.
Most preferably the xe2x80x9cisolatedxe2x80x9d protein is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated, and yields a single major band on a non-reducing polyacrylamide gel. Substantially free means that the protein is at least 75%, preferably at least 85%, more preferably at least 95% and most preferably at least 99% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
The invention also provides novel and/or isolated nucleic acid molecules that encode SEQ ID NO:1 and the functional equivalents thereof Nucleic acid molecules (nucleic acids) of the invention include deoxyribonucleic acid (DNA), complementary DNA (cDNA), and ribonucleic acid (RNA) sequences.
For example, the invention includes an isolated nucleic acid molecule as set forth in SEQ ID NO:2, isolated nucleic acid molecules that are substantially homologous with SEQ ID NO:2, isolated nucleic acid molecules that hybridize with SEQ ID NO:2 under stringent conditions, nucleic acid sequences that are degenerate as a result of the genetic code, and the complements and fragments thereof
In order to determine whether the sequence of a first nucleic acid molecule, or fragment thereof, is substantially homologous to the sequence of a second nucleic acid molecule, such as SEQ ID NO: 2, or fragment thereof, the sequences are first aligned so as to optimize the percent of nucleotides that are identical at corresponding positions. Gaps may be introduced in the sequences if necessary to achieve optimization.
The nucleic acid sequences of highly homologous nucleic acid molecules can usually be aligned by visual inspection. If visual inspection is insufficient, the nucleic acid molecules are aligned in accordance with any of the methods described by George, D. G. et al, in Macromolecular Sequencing and Synthesis, Selected Methods and Applications, pages 127-149, Alan R. Liss, Inc. (1988), such as the formula described at page 137 using a match score of 1, a mismatch score of 0, and a gap penalty of xe2x88x921.
In the present specification, the sequence of a nucleic acid molecule or fragment thereof is considered substantially homologous to SEQ ID NO:2, or a fragment thereof, if the nucleic acid sequences, after alignment, are at least about 40% identical, preferably at least about 50% identical, more preferably at least about 60% identical, even more preferably at least about 70% identical, still more preferably at least about 80% identical, most preferably at least about 90% identical, and, ideally at least about 95% identical.
The invention also includes nucleic acid molecules that hybridize to SEQ ID NO:2, a fragment of SEQ ID NO:2, a complement of SEQ ID NO:2, or a complement of a fragment of SEQ ID NO:2 under stringent conditions.
Also included in the invention are a preferred group of protein functional equivalents of SEQ ID NO:1 encoded by nucleic acid molecules that hybridize under stringent conditions to a sequence complementary to SEQ ID NO:2 or a fragment of SEQ ID NO:2.
The term xe2x80x9cstringent conditions,xe2x80x9d as used herein, is equivalent to xe2x80x9chigh stringent conditionsxe2x80x9d and xe2x80x9chigh stringency.xe2x80x9d These terms are used interchangeably in the art.
Stringent conditions are defined in a number of ways. In one definition, stringent conditions are selected to be about 50xc2x0 C. lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched sequence. Typical stringent conditions are those in which the salt concentration is about 0.02 M at pH 7 and the temperature is at least about 60xc2x0 C. Further examples of stringent conditions can be found in U.S. Pat. No. 5,789,550 to Goeddel et al. (1998). The description of stringent conditions in U.S. Pat. No. 5,789,550 is herein incorporated by reference.
Stringent conditions, in the nucleic acid hybridization context, include a combination of conditions, such as the nature and concentration of salts and organic solvents, temperature, and other parameters that are typically known to control hybridization reactions. The combination of parameters is more important than the measure of any single parameter. See U.S. Pat No. 5,786,210; Wetmur and Davidson, J. Mol. Biol. 31, 349-370 (1968). Generally, stringent conditions are obtained at higher temperatures and lower ionic strength. Control of hybridization conditions, and the relationships between hybridization conditions and degree of homology are understood by those skilled in the art. See, for example, Bej, A. K., Nucleic Acid Hybridizations: Principles and Strategies, in Dangler, C. A. ed, Nucleic Acid Analysis: Principles and Bioapplications, Wiley-Liss, Inc., pp. 1-29 (1996); Adams et al., The Biochemistry of the Nucleic Acids, pp. 605-606, Chapman and Hall (1992); Sambrook J, Fritsch EF, and Maniatis T, Molecular Cloning. A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor (1989).
Fragments of nucleic acid molecules of the invention include primers and probes which are useful as tools in molecular biology and biotechnology. The fragment may or may not encode a polypeptide that binds to IKKxcex1 or IKKxcex2. Even if the encoded polypeptide does not bind, the fragment can be used, for example, as a primer (xe2x80x9camplimerxe2x80x9d) to selectively amplify nucleic acid, such as genomic DNA or total RNA. Primers can also be used in nucleic acid amplification procedures such as the polymerase chain reaction (PCR), ligase chain reaction (LCR), repair chain reaction (RCR), PCR oligonucleotide ligation assay (PCR-OLA), and the like.
Fragments of the nucleic acid molecules of the invention can also be oligonucleotides complementary to a target nucleic acid molecule, i.e., the fragment can be a probe. Such oligonucleotides can be DNA or RNA. Oligonucleotides useful as probes in hybridization studies, such as in situ hybridization, can also be constructed.
The length of the oligonucleotide probe is not critical, as long as it is capable of hybridizing to the target molecule. The oligonucleotide should contain at least 6 nucleotides, preferably at least 10 nucleotides, and more preferably, at least 15 nucleotides. There is no upper limit to the length of the oligonucleotide probes. Longer probes are more difficult to prepare and require longer hybridization times. Therefore, the probe should not be longer than necessary. Normally, the oligonucleotide probe will not contain more than 50 nucleotides, preferably not more than 40 nucleotides, and, more preferably, not more than 30 nucleotides.
Numerous methods for detectably labeling such probes with radioisotopes, fluorescent tags, enzymes, binding moieties (e.g., biotin), and the like are known, so that the probes of the invention can be adapted for easy detectability. Methods for making and using nucleic acid probes are understood by those skilled in the art. See, for example, Keller G H and Manak M M, DNA Probes, 2d ed., Macmillan Publishers Ltd., England (1991) and Hames B D and Higgins S J, eds., Gene Probes I and Gene Probes II, IRL Press, Oxford (1995).
The nucleic acid molecules may contain synthetic sequences that do not occur in nature and/or they are isolated. The term xe2x80x9cisolated,xe2x80x9d as used herein, in the context of nucleic acids, includes nucleic acid molecules unaccompanied by at least some of the material with which they are associated in their natural state. The isolated nucleic acid may constitute at least 0.5%, preferably at least 5%, more preferably at least 25% and still more preferably at least 50% by weight of the total nucleic acid in a given sample. Most preferably the xe2x80x9cisolatedxe2x80x9d nucleic acid is substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which it is naturally associated. Substantially free means that the nucleic acid is at least 75%, preferably at least 85%, more preferably at least 95% and most preferably at least 99% free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which it is naturally associated. The nucleic acid molecules of the invention can also be recombinant, meaning that they comprise a non-natural sequence or a natural sequence joined to nucleotide(s) other those in which they are joined on the natural chromosome.
Y2H14 was shown, using yeast two-hybrid screens, to bind to the carboxyl terminal domain regions of IKKxcex1 and IKKxcex2. See example 1. Y2H14 specifically binds with the region of the IKK proteins made up of the contiguous helix-loop-helix and leucine zipper domains. The binding of the Y2H14 protein to the IKKs is about the same as the binding of IKKxcex1 to a natural substrate of IKKxcex1, viz. IxcexaBxcex22.
The Y2H14 protein and the other IKK binding proteins described in example 1 are useful for elucidating and controlling pathways leading to inflammation and apoptosis. These processes are mediated by receptors such as tumor necrosis factor (TNF) receptors. The IKK binding proteins can also be used to detect IKK complexes and modulate IKK activity in cells undergoing signalling by inflammatory mediators such as TNFxcex1 and Il-1.
The Y2H14 and its functional equivalents are also useful for identifying therapeutically active agents that modulate the binding or interaction of Y2H14 and either IKKxcex1 or IKKxcex2. Such agents can either prevent the formation of Y2H14/IKK complexes or prevent or inhibit the dissociation of Y2H14/IKK complexes. Molecules that prevent the formation of Y2H14/IKK complexes or inhibit the dissociation of these complexes are useful for boosting the immune system, or as immunosuppresants, or as antiinflammatory agents.
Complex formation or dissociation can be determined by methods well known in the art. Such methods include, for example, gel filtration, sucrose density gradient centrifugation, crosslinking, and immunoprecipitation.
The proteins and variants of the proteins can be prepared by methods known in the art. Such methods include isolating the protein directly from cells, and synthesizing the protein chemically from individual amino acids. Preferably, the proteins of the invention can be prepared by providing DNA that encodes the protein, amplifying or cloning the DNA, expressing the DNA in a suitable host, and harvesting the protein.
DNA encoding the proteins of the invention can be synthesized or isolated. The DNA of the invention can be synthesized chemically from the four nucleotides in whole or in part by methods known in the art. Such methods include those described by Caruthers, Science 230, 281-285 (1985). DNA can also be synthesized by preparing overlapping double-stranded oligonucleotides, filling in the gaps, and ligating the ends together. See, generally, Sambrook et al. (1989) and Glover D M and Hames B D, eds., DNA Cloning, 2d ed., Vols. 1-4, IRL Press, Oxford (1995).
DNA expressing functional homologs of the protein can be prepared from wild-type DNA by site-directed mutagenesis. See, for example, Zoller and Smith, Nucleic Acids Res 10, 6487-6500 (1982); Zoller, Methods Enzymol 100, 468-500 (1983); Zoller, DNA 3(6), 479-488 (1984); and McPherson, ed., Directed Mutagenesis: A Practical Approach, IRL Press, Oxford (1991).
DNA encoding the protein of the invention can be isolated from different species by using the human sequence, SEQ ID NO:2, to prepare one or more oligonucleotide probes. The probe is labeled and used to screen a genomic or cDNA library in a suitable vector, such as phage lambda. The homology between the DNA of the Y2H14 of the species being screened and that of the human DNA is taken into account in determining the conditions of hybridization. The cDNA library may be prepared from mRNA by known methods, such as those described in Gubler and Hoffman, Gene 25, 263-270 (1983). Oligonucleotide probes can be used to screen cDNA libraries from different species and tissues. The oligonucleotide probe should be labeled so that it can be detected upon hybridization to DNA in the library being screened. These methods are well known in the art.
The DNA isolated is sequenced, and the sequence used to prepare additional oligonucleotide probes. This procedure may be repeated to obtain overlapping fragments until a complete open reading frame is produced.
The nucleic acids of the invention may be amplified by methods known in the art. One suitable method is the polymerase chain reaction (PCR) method described by Saiki et al., Science 239, 487 (1988), Mullis et al in U.S. Pat. No. 4,683,195 and by Sambrook et al. (1989). It is convenient to amplify the clones in the lambda-gt10 or lambda-gt11 vectors using lambda-gt10 or lambda-gt11-specific oligomers as the amplimers (available from Clontech, Palo Alto, Calif.). Other amplification procedures that are well known in the art such as ligase chain reaction (LCR), repair chain reaction (RCR), and PCR oligonucleotide ligation assay (PCR-OLA) can also be used to amplify the nucleic acids of the invention.
DNA encoding the proteins of the invention, or unique fragments thereof, may also be cloned in a suitable host cell and expressed by methods well known in the art. The DNA and protein may be recovered from the host cell. See, generally, Sambrook et al. (1989), for methods relating to the manufacture and manipulation of nucleic acids. The entire gene or additional fragments of the gene can be isolated by using the known DNA sequence or a fragment thereof as a probe. To do so, restriction fragments from a genomic or cDNA library may be identified by Southern hybridization using labeled oligonucleotide probes derived from SEQ ID NO:2.
The amplified or cloned DNA can be expressed in a suitable expression vector by methods known in the art. See, generally, Sambrook et al. (1989).
A variety of expression vectors and host cell systems can be used. These include, for example, microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA containing the Y2H14 coding region. Other expression vectors and host cell systems that can be used include yeast transformed with recombinant yeast expression vectors containing the Y2H14 coding sequence, insect cells infected with recombinant virus expression vectors containing the Y2H14 coding sequence, plant cells infected with recombinant virus expression vectors containing the Y2H14 coding sequence, or animal cells infected with recombinant virus expression vectors (e.g., retroviruses, adenovirus, vaccinia virus) containing the Y2H14 coding sequence.
The expression vectors useful in the present invention contain at least one expression control sequence that is operatively linked to the DNA sequence or fragment to be expressed. The control sequence is inserted in the vector in order to control and to regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences are the lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of fd coat protein, the glycolytic promoters of yeast, e.g., the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, e.g., Pho5, the promoters of the yeast alpha-mating factors, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, e.g., the early and late promoters or SV40, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells and their viruses or combinations thereof.
Useful expression hosts include well-known prokaryotic and eukaryotic cells. Some suitable prokaryotic hosts include, for example, E. coli, such as E. coli SG-936, E. coli HB 101, E. coli W3 110, E. coli X1776, E. coli X2282, E. coli DHI, and E. coli MRCl, Pseudomonas sp., Bacillus sp., such as B. subtilis, and Streptomyces sp. Suitable eukaryotic cells include yeasts and other fungi, insect, animal cells, such as COS cells and CHO cells, human cells and plant cells in tissue culture.
Preferably, Y2H14 and its functional equivalents are expressed using baculoviral vectors in insect cell cultures. In general, the transformation of insect cells and production of foreign proteins therein is disclosed in Guarino et al., U.S. Pat. No. 5,162,222.
Proteins can be isolated from a solubilized fraction by standard methods. Some suitable methods include precipitation and liquid chromatographic protocols such as ion exchange, hydrophobic interaction, and gel filtration. See, for example, Methods Enzymol (Guide to Protein Chemistry, Deutscher, ed., Section VII) pp. 182:309 (1990) and Scopes, Protein Purification, Springer-Verlag, New York (1987), which are herein incorporated by reference.
Alternatively, purified material is obtained by separating the protein on preparative SDS-PAGE gels, slicing out the band of interest and electroeluting the protein from the polyacrylamide matrix by methods known in the art. The detergent SDS is removed from the protein by known methods, such as by dialysis or the use of a suitable column, such as the Extracti-Gel column from Pierce. Mixtures of proteins can be separated by, for example, SDS-PAGE in accordance with the method of Laemmli, Nature 227, 680-685 (1970). Such methods are well known in the art.
The proteins of the invention can also be chemically synthesized by methods known in the art. Suitable methods for synthesizing proteins are described by Stuart and Young, Solid Phase Peptide Synthesis, 2d ed., Pierce Chemical Company (1984).
The invention also includes an antibodies or antibody fragments that specifically bind to epitopes of the proteins of the invention defined by the amino acid sequence set forth in SEQ ID NO:1. An xe2x80x9cantibodyxe2x80x9d in accordance with the present specification is defined broadly as a protein that binds specifically to an epitope. The antibodies of the invention can be monoclonal antibodies, polyclonal antibodies, chimerized antibodies, humanized antibodies, single chain antibodies, or a fragment thereof For use in in vivo applications with human subjects, the antibody is preferably chimerized or humanized, containing an antigen binding region from, e.g., a rodent, with the bulk of the antibody replaced with sequences derived from human immunoglobulin.
Antibodies further include recombinant polyclonal or monoclonal Fab fragments prepared in accordance with the method of Huse et al., Science 246, 1275-1281 (1989).
Polyclonal antibodies are isolated from mammals that have been inoculated with the protein or a functional analog in accordance with methods known in the art. Briefly, polyclonal antibodies may be produced by injecting a host mammal, such as a rabbit, mouse, rat, or goat, with the protein or a fragment thereof capable of producing antibodies that distinguish between mutant and wild-type protein. The peptide or peptide fragment injected may contain the wild-type sequence or the mutant sequence. Sera from the mammal are extracted and screened to obtain polyclonal antibodies that are specific to the peptide or peptide fragment.
The antibodies are preferably monoclonal. Monoclonal antibodies may be produced by methods known in the art. These methods include the immunological method described by Kohler and Milstein, Nature 256:495-497 (1975) and by Campbell, in Burdon et al., eds, Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Elsevier Science Publishers, Amsterdam (1985); as well as the recombinant DNA method described by Huse et al.(1989).
To produce monoclonal antibodies, a host mammal is inoculated with a peptide or peptide fragment as described above, and then boosted. Spleens are collected from inoculated mammals a few days after the final boost. Cell suspensions from the spleens are fused with a tumor cell in accordance with the general method described by Kohler and Milstein (1975). See also Campbell (1985). To be useful, a peptide fragment must contain sufficient amino acid residues to define the epitope of the molecule being detected.
If the fragment is too short to be immunogenic, it may be conjugated to a carrier molecule. Some suitable carrier molecules include keyhole limpet hemocyanin and bovine serum albumin. Conjugation may be carried out by methods known in the art. One such method is to combine a cysteine residue of the fragment with a cysteine residue on the carrier molecule.
Methods for making chimeric and humanized antibodies are also known in the art. For example, antibodies can be engineered using genetic techniques to produce chimeric antibodies including protein components from two or more species.
For example, methods for making chimeric antibodies include those described in U.S. patents by Boss (Celltech) and by Cabilly (Genentech). See U.S. Pat. Nos. 4,816,397 and 4,816,567, respectively. Methods for making humanized antibodies are described, for example, in Winter, U.S. Pat. No. 5,225,539, Co et al., Nature 351, 501-502 (1992); Queen et al., Proc. Natl. Acad. Sci. 86, 10029-1003 (1989) and Rodrigues et al., Int. J. Cancer, Supplement 7, 45-50 (1992).
Methods are also known for inducing expression of engineered antibodies in various cell types, such as mammalian and microbial cell types. Numerous techniques for preparing engineered antibodies are described, for example, in Owens and Young, xe2x80x9cThe genetic engineering of monoclonal antibodies,xe2x80x9d J. Immunol. Meth. 168, 149-165 (1994).
Methods for making single chain antibodies are also known in the art. Some suitable examples include those described by Wels et al. in European patent application 502 812 and Int. J. Cancer 60, 137-144 (1995).
Assays for directly detecting the presence of Y2H14 and its functional equivalents with antibodies follow known formats, such as, fluorescent activated flow cytometry, fluorescent microscopy, and immuno-electron microscopy. Moreover, assays for detecting the presence of proteins with antibodies have been previously described and follow known formats, such as standard blot and ELISA formats. These formats are normally based on incubating an antibody with a sample suspected of containing the protein and detecting the presence of a complex between the antibody and the protein. The antibody is labeled either before, during, or after the incubation step. The protein is preferably immobilized prior to detection. Immobilization may be accomplished by directly binding the protein to a solid surface, such as a microtiter well, or by binding the protein to immobilized antibodies.
Suitable assays are known in the art, such as the standard ELISA protocol described by R. H. Kenneth, xe2x80x9cEnzyme-linked antibody assay with cells attached to polyvinyl chloride platesxe2x80x9d in Kenneth et al., Monoclonal Antibodies, Plenum Press, New York, pp. 376 et seq. (1981).

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