CS270819B1 - Method of net-like acidic polysaccharides preparation - Google Patents
Method of net-like acidic polysaccharides preparation Download PDFInfo
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- CS270819B1 CS270819B1 CS881656A CS165688A CS270819B1 CS 270819 B1 CS270819 B1 CS 270819B1 CS 881656 A CS881656 A CS 881656A CS 165688 A CS165688 A CS 165688A CS 270819 B1 CS270819 B1 CS 270819B1
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- 229920001284 acidic polysaccharide Polymers 0.000 title claims description 11
- 150000004805 acidic polysaccharides Chemical class 0.000 title claims description 11
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 238000000034 method Methods 0.000 title description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 16
- BRLQWZUYTZBJKN-UHFFFAOYSA-N Epichlorohydrin Chemical compound ClCC1CO1 BRLQWZUYTZBJKN-UHFFFAOYSA-N 0.000 claims abstract description 12
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims abstract description 12
- GYZLOYUZLJXAJU-UHFFFAOYSA-N diglycidyl ether Chemical compound C1OC1COCC1CO1 GYZLOYUZLJXAJU-UHFFFAOYSA-N 0.000 claims abstract description 9
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 5
- 239000005017 polysaccharide Substances 0.000 claims abstract description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 4
- 238000004132 cross linking Methods 0.000 claims description 13
- 239000012153 distilled water Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 3
- 150000004804 polysaccharides Chemical class 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims 1
- 102000053602 DNA Human genes 0.000 abstract description 4
- 108020004414 DNA Proteins 0.000 abstract description 4
- 102000004190 Enzymes Human genes 0.000 abstract description 4
- 108090000790 Enzymes Proteins 0.000 abstract description 4
- 239000002253 acid Substances 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 3
- 150000004676 glycans Chemical class 0.000 abstract 2
- 125000003147 glycosyl group Chemical group 0.000 abstract 1
- 229920002230 Pectic acid Polymers 0.000 description 14
- LCLHHZYHLXDRQG-ZNKJPWOQSA-N pectic acid Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)O[C@H](C(O)=O)[C@@H]1OC1[C@H](O)[C@@H](O)[C@@H](OC2[C@@H]([C@@H](O)[C@@H](O)[C@H](O2)C(O)=O)O)[C@@H](C(O)=O)O1 LCLHHZYHLXDRQG-ZNKJPWOQSA-N 0.000 description 14
- 239000010318 polygalacturonic acid Substances 0.000 description 14
- 230000008961 swelling Effects 0.000 description 11
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 7
- 229920002674 hyaluronan Polymers 0.000 description 7
- 229960003160 hyaluronic acid Drugs 0.000 description 7
- 229920001277 pectin Polymers 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000001814 pectin Substances 0.000 description 5
- 235000010987 pectin Nutrition 0.000 description 5
- 239000011541 reaction mixture Substances 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 4
- 229920000288 Keratan sulfate Polymers 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 239000000783 alginic acid Substances 0.000 description 4
- 229920000615 alginic acid Polymers 0.000 description 4
- 235000010443 alginic acid Nutrition 0.000 description 4
- 229960001126 alginic acid Drugs 0.000 description 4
- 150000004781 alginic acids Chemical class 0.000 description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 4
- 229920000669 heparin Polymers 0.000 description 4
- 229960002897 heparin Drugs 0.000 description 4
- KXCLCNHUUKTANI-RBIYJLQWSA-N keratan Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@H](COS(O)(=O)=O)O[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@H](O[C@@H](O[C@H]3[C@H]([C@@H](COS(O)(=O)=O)O[C@@H](O)[C@@H]3O)O)[C@H](NC(C)=O)[C@H]2O)COS(O)(=O)=O)O[C@H](COS(O)(=O)=O)[C@@H]1O KXCLCNHUUKTANI-RBIYJLQWSA-N 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 229920000045 Dermatan sulfate Polymers 0.000 description 3
- 229920001222 biopolymer Polymers 0.000 description 3
- 238000005886 esterification reaction Methods 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 229920002567 Chondroitin Polymers 0.000 description 2
- 229920001287 Chondroitin sulfate Polymers 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 239000005708 Sodium hypochlorite Substances 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- DLGJWSVWTWEWBJ-HGGSSLSASA-N chondroitin Chemical compound CC(O)=N[C@@H]1[C@H](O)O[C@H](CO)[C@H](O)[C@@H]1OC1[C@H](O)[C@H](O)C=C(C(O)=O)O1 DLGJWSVWTWEWBJ-HGGSSLSASA-N 0.000 description 2
- AVJBPWGFOQAPRH-FWMKGIEWSA-L dermatan sulfate Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@H](OS([O-])(=O)=O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](C([O-])=O)O1 AVJBPWGFOQAPRH-FWMKGIEWSA-L 0.000 description 2
- 229940051593 dermatan sulfate Drugs 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 2
- 238000005476 soldering Methods 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- OEANUJAFZLQYOD-CXAZCLJRSA-N (2r,3s,4r,5r,6r)-6-[(2r,3r,4r,5r,6r)-5-acetamido-3-hydroxy-2-(hydroxymethyl)-6-methoxyoxan-4-yl]oxy-4,5-dihydroxy-3-methoxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](OC)O[C@H](CO)[C@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](OC)[C@H](C(O)=O)O1 OEANUJAFZLQYOD-CXAZCLJRSA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N 2,3,4,5-tetrahydroxypentanal Chemical compound OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- OXIQCJDVMBRASN-UHFFFAOYSA-N 3-(3-hydroxypropylsulfinyl)propan-1-ol Chemical compound OCCCS(=O)CCCO OXIQCJDVMBRASN-UHFFFAOYSA-N 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- IAJILQKETJEXLJ-RSJOWCBRSA-N aldehydo-D-galacturonic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-RSJOWCBRSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000009194 citrus pectin Substances 0.000 description 1
- 229940040387 citrus pectin Drugs 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000007257 deesterification reaction Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 150000004679 hydroxides Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229920005615 natural polymer Polymers 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229910021653 sulphate ion Inorganic materials 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
Landscapes
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
Description
(57) Riešenia sa týká přípravy sletovaných kyslých polysacharidov postupom, kedy na 1 mol použitej anhydroglykozylovej jednotky kyslého polysacharidu sa aplikuje 0,1 až 1,5 molu homo- alebg hatarobifunkčného činidla, ako 2,2 ,-bis(oxiranylmetyljéteru alebo 2-chlororpetylooxiránu v prostředí 0,4 až 1,2 molu hydro-z xidu sodného alebo draselného pre 2-chlorom^tyloxirán alebo pri pH nad 11,2 pre 2,2 -bis(oxiranylmetyl)éter po dobu 1 až 3 hodin pri 40 až 80 °Č. RieŠenie má uplatnenie v analytickej biochémii v preparatívnej bioorganickej chémii, v klinickej biochémiiv enzýmovom lnžinierstve a v technologiach využívajúcich rekombinantnú dezoxyribonukleovú kyselinu.(57) The present invention relates to the preparation of fused acidic polysaccharides by the process of applying 0.1 to 1.5 moles of homo- or g-hatarobifunctional reagent, such as 2,2'-bis (oxiranylmethyl ether or 2-chloropolyloxirane) per mole of anhydroglycosyl unit of acidic polysaccharide used. in a 0.4 to 1.2 moles of hydroxypropyl sulfoxide, sodium or potassium for 2-chloro ^ tyloxirán or above pH 11.2 the 2,2-bis (oxiranylmethyl) ether for 1 to 3 hours at 40 to 80 The solution has application in analytical biochemistry in preparative bioorganic chemistry, in clinical biochemistry in enzyme engineering and in technologies using recombinant deoxyribonucleic acid.
CS 270 819 81CS 270 819 81
Vynález sa týká sposobu přípravy sletovaných kyslých polysacharidov pre využitie v analytickej biochémii, dioorganickej syntéze a enzýmovom inžinierstve.The invention relates to a process for the preparation of fused acidic polysaccharides for use in analytical biochemistry, diioorganic synthesis and enzymatic engineering.
Aj кеЭ v dosledku rozvoja biotechnologií dochádza к dynamickému rozvojů nových analytických a preparátivnych metod, založených na použití ionexov anorganického a organického syntetického povodu, biopolyméry, predovšetkým však polysacharidy kyslého a bazického povodu si ponechávajú svoj význam a to predovšetkým s ohl’adom na vysokú čistotu východiskových surovin - izolovaných pročištěných biopolymérov, ich pravidelní) štruktúru a vysoké zastúpenie funkčných skupin, karboxylových (pektin, kyselina pektová, kyselina alginová, kyselina hyaluronová, chondroitín, heparin a proteiny kyslého typu), sulfónových (chondroitín 6-sulfát, keratan sulfát, dermatan sulfát, hsparín) a primárných aminoskupin (chitozan, bazické proteiny). Ionexy připravené z prírodných polymérov sa nevyznačujú vysokými nespecifickými sorpciami a vzhladom к vysokej čistotě použitých surovin nedochádza к negativnému ovplyvneniu vlastností stanovovaných alebo izolovaných biologicky aktivnych látok.Even as a result of the development of biotechnology, there is a dynamic development of new analytical and preparative methods, based on the use of ion exchangers of inorganic and organic synthetic basins, biopolymers, but mainly acid polysaccharides and basic basins retain their importance, especially with regard to high purity of starting materials. raw materials - isolated purified biopolymers, their regular structure and high representation of functional groups, carboxylic (pectin, pectic acid, alginic acid, hyaluronic acid, chondroitin, heparin and acid type proteins), sulfonic (chondroitin 6-sulfate, keratan sulfate, dermatan sulfate , hsparin) and primary amino groups (chitosan, basic proteins). The ion exchangers prepared from natural polymers do not exhibit high non-specific sorptions and, due to the high purity of the raw materials used, do not adversely affect the properties of the assayed or isolated biologically active substances.
Přípravou pektínového katexu sa zaoberá čsl. patent 140 713, kde východiskovou surovinou pre reakciu sietovania js kyselina pektová (deesterifikovaný pektin). Přibližné ekvimolárne zastúpenie jednotlivých zložiek v reakčnej zmesi (pektin, hydroxid sodný a 2-chlórometyloxiran) neumožňuje přípravu sletovaného gélu o vhodných hydrodynamických vlasnostiach. Pri přípravě sietovaného gélu kyseliny pektovej podl'a A.O. 218 702 sa sice vychádza priamo z citrusového pektínu a deesterifikačná raakcia spolu s reakciou sietovania prebiehajú v jednom reakčnom stupni, připravený gél, vhodný po 3alšej aktivácii i imobilizácii enzýmov, nevyhovuje však к použitiu ako katex a to pre nízký stupeň sietovania a vysoký napučací objem. Oe potřebné však súčasna zdoraznit, že podmienky sietovania 2-chloromethyloxiránom vyhovujú len pre alkalistabilnejšie biopolyméry, zatial čo alkalilabilné sa v přítomnosti vysokej koncentrácie hydroxidov rozkládají.The preparation of the pectin cation exchanger is dealt with in the art. No. 140,713, wherein the starting material for the crosslinking reaction is pectic acid (deesterified pectin). The approximate equimolar representation of the individual components in the reaction mixture (pectin, sodium hydroxide, and 2-chloromethyloxirane) does not allow the preparation of a gelled gel with suitable hydrodynamic properties. In the preparation of the cross-linked pectic acid gel of A.O. 218 702 is based directly on citrus pectin and the de-esterification reaction together with the crosslinking reaction takes place in a single reaction step, the gel prepared after 3 further activation and immobilization of the enzymes is not suitable for use as a cation exchanger due to low crosslinking and high swelling volume. At the same time, however, it is necessary to make it clear that the cross-linking conditions of 2-chloromethyloxirane are only suitable for the more alkaline biopolymers, while the alkalilable decompose in the presence of a high concentration of hydroxides.
Uvedené nedostatky odstraňuje postup podlá vynálezu, podstata ktorého spočívá v tom, že kyslý polysachrid sa sletuje homo- alebo heterobifukčným činidlom, s výhodou 2,2-bis(oxiranylmetyl)éterom alebo 2-chlórometyloxiránom v pomere na 1 mol substituovanej anhydroglykozylovej jednotky kyslého polysacharidu 0,1 až 1,5 molu 2,2z-bis(oxiranylmetyl) éteru alebo 2-chlórometyloxiránu pri pH nad 11,2 pře sletováni© 2,2'-bis (oxiranylmetyl)éterom alebo v přítomnosti 0,4 až 1,2 molu hydroxidu sodného alebo draselného na 1 mól substituovanéj anhydroglykozylovej jednotky polysacharidu, pri sletovaní 2-chlórmetyloxiránom a 5 až 20 mólov destilovanej vody, pri teplote 40 až 80 °C po dobu 1 až 3 hodin.The above-mentioned drawbacks are overcome by the process according to the invention, characterized in that the acidic polysaccharide is fed off with a homo- or heterobifunctional agent, preferably 2,2-bis (oxiranylmethyl) ether or 2-chloromethyloxirane per 1 mol of substituted anhydroglycosyl unit of acidic polysaccharide. , 1 to 1.5 moles of 2,2-bis (oxiranylmethyl) ether or 2-chlórometyloxiránu above pH 11.2 disputes © soldering, 2,2'-bis (oxiranylmethyl) or ether in the presence of 0.4 to 1.2 mole of sodium or potassium hydroxide per mole of substituted anhydroglycosyl unit of the polysaccharide, when blending with 2-chloromethyl oxirane and 5 to 20 moles of distilled water, at a temperature of 40 to 80 ° C for 1 to 3 hours.
Po zosietení sa reakčná zmes zaleje nadbytkom destilovanej vody, rozvolni, neutralizuje pomocou kyseliny octovej alebo chlórovodikoVej, připadne vybieli účinkom zriedeného chlornanu sodného, odvodní postupným účinkom nadbytku 50% a potom 96 - 100% etanolu alebo acetonu a vysuší vo vákuovej sušiarni. Vysušený gél sa vytriedi na sitách na frakcie o potrebnom zrnění a upraví na vhodnú hodnotu pH pre ňalšie použitie.After cross-linking, the reaction mixture is quenched with excess distilled water, loosened, neutralized with acetic or hydrochloric acid, optionally bleached with dilute sodium hypochlorite, dehydrated in succession with an excess of 50% and then 96-100% ethanol or acetone and dried in a vacuum oven. The dried gel is screened for fractions of the necessary grain size and adjusted to a suitable pH for subsequent use.
Výhodou uvedeného postupu je skutočnost, že základný materiál, kyslý polysacharid je povaČŠine lahko dostupný a lačný, stupeň substitúcie DS kyslými fukčnými skupinami v případe kyseliny pektovej, alginovej, chondritín 6-sulfátu a dermetan sulfátu je okolo 1, v případe keratan sulfátu a hyaluronovej kyseliny Js okolo 0,5 a u heparínu je okolo 2. Pri sletovaní esterifikovaných kyslých polysacharidov predovšetkým pomocou 2-chlorometyloxiránu dochádza aj к súčasnej deesterifikácii· V případe sietovania pomocou 2,2'-bis(oxiranylmetyl)éteru možno reakciu sietovania zabezpečit aj bez deesterif ikácie. Zostavou reakčnej zmesi možno regulovat stupeň sietovania gélu a jeho napučaci objem v rozsahu od 3,8 až po 25 ml.g*.The advantage of this process is that the base material, the acidic polysaccharide, is generally readily available and fasting, and the degree of DS substitution by acidic functional groups in the case of pectic acid, alginic acid, chondritin 6-sulfate and dermethate sulfate is about 1, keratan sulfate and hyaluronic acid. It is about 0.5 and about 2 for heparin. When de-esterified acidic polysaccharides are fused together mainly with 2-chloromethyloxirane, simultaneous de-esterification also occurs. · In the case of crosslinking with 2,2'-bis (oxiranylmethyl) ether, the crosslinking reaction can be ensured without deesterification. The degree of crosslinking of the gel and its swelling volume in the range from 3.8 to 25 ml.g * can be controlled by the reaction mixture.
CS 270 819 B1CS 270 819 B1
Příklad 1Example 1
Ku kyselina pektovej (176 g; 1 mol anhydrogalakturónovej kyseliny) sa přidalo za miešania 40 ml destilovanej vody a potom postupné v priebehu dalších 30 minút za stálého miešania 1 mol NaOH, rozpuštěný v 50 ml destilovanej vody. Po dokonalom zvlhčeni a zhomogenizovaní reakčnej zmesi za stálého miešania a chladenia sa přidávalo postupné 0,5 molu 2-chlórometyloxiránu a po dokladnom premiešaní sa zvýšila teplota reakčnej zmesi na 40 °C. Po 3 hodinách priebehu sa vybielil účinkom 200 ml 0,5% chlornanu sodného, znovu vymyl v destilovanej vodě a adjustoval do H4 cyklu účinkom 400 ml 2% kyseliny fosforečnej. Nadbytok volnej kyseliny fosforečnej sa vymyl opakované 500 ml destilovanej vody a vysušil postupným odvodněním pomocou 50% etanolu alebo acetonu (1000 ml) a 96 až 100% etanolu alebo acetonu a dosušil vo vakuovej sušiarni. Získaný sletovaný gél kyseliny pektovej (204 g) mal výmennú kapacitu 5,2 mol.g'^ a napučaoí objem 12,5 ml.g~^o , Přiklad 2To pectic acid (176 g; 1 mol of anhydrogalacturonic acid) was added with stirring 40 ml of distilled water and then gradually over a further 30 minutes with stirring 1 mol of NaOH dissolved in 50 ml of distilled water. After thoroughly wetting and homogenizing the reaction mixture with stirring and cooling, successively 0.5 mol of 2-chloromethyloxirane was added and after thorough stirring the temperature of the reaction mixture was raised to 40 ° C. After 3 hours, it was bleached with 200 ml of 0.5% sodium hypochlorite, washed again in distilled water and adjusted to H 4 cycle with 400 ml of 2% phosphoric acid. The excess free phosphoric acid was washed repeatedly with 500 ml of distilled water and dried by successive dewatering with 50% ethanol or acetone (1000 ml) and 96-100% ethanol or acetone and dried in a vacuum oven. The soldering pectic gel (204 g) had an ion exchange capacity of 5.2 mol.g ^ napučaoí a volume of 12.5 ml g ~ ^ a, Example 2
Postupuje sa tak, ako Je uvedené v přiklade 1 s tým rozdielom, Že namiesto kyseliny pektovej sa použije 187 g jablčného pektinu o stupni esterifikácie 62%. Reakčným produktom bolo 205 g gélu sietovanej kyseliny pektovej o výmennej kapacitě 5,2 mól.g“^ a napučacom objeme 12 ml.g~^.The procedure is as described in Example 1 except that 187 g of apple pectin with an esterification degree of 62% is used instead of pectic acid. The reaction product was 205 g of cross-linked pectic acid gel having a exchange capacity of 5.2 mol / g and a swelling volume of 12 ml / g.
Příklad 3Example 3
Postupuje sa tak, ako je uvedené v přiklade 1 s tým rozdielom, že namiesto 40 ml destilovanej vody sa ku zvlhčeniu kyseliny pektovej použije 100 ml a 1,2 molz NaOH sa rozpustí v Salšich 100 ml destilovanej vody. К sietovaniu sa použilo 1,5 molu 2-chlórometyloxiránu. Reakcia sietovania prebiehala pri 80 °C po dobu 1 hodiny. Získaný gél kyseliny pektovej (211 g) mal výmennú kapacitu 4,6 mól.g^ a napučaci objem 6,0 ml.g1.The procedure is as described in Example 1 except that instead of 40 ml of distilled water, 100 ml is used to moisten the pectic acid and 1.2 mol of NaOH is dissolved in 100 ml of distilled water. 1.5 mol of 2-chloromethyloxirane was used for cross-linking. The crosslinking reaction was carried out at 80 ° C for 1 hour. The pectic gel (211 g) had an ion exchange capacity of 4.6 N, mól.g swelling volume 6.0 ml g 1st
Přiklad 4Example 4
Postupuje sa tak, ako v přiklade 1 s tým rozdielom, že sa к sietovaniu použije 0,1 mol 2-chlórometyloxiránu a 0,4 molu KOH. Získaný gél sietovanej kyseliny pektovej (145 g) mal výmennú kapacitu 5,4 mol.g1 a napučaci objem 25 ml.g 1.The procedure was as in Example 1 except that 0.1 mol of 2-chloromethyloxirane and 0.4 mol of KOH were used for crosslinking. The obtained cross-linked pectic acid gel (145 g) had a exchange capacity of 5.4 mol / l and a swelling volume of 25 ml / l .
Přiklad 5 ·Example 5 ·
Postupuje sa tak, ako je uvedené v přiklade 1, s tým rozdielom, že namiesto kyseliny pektovej sa použije kyselina alginová. Získaný gél sietovanej kyseliny alginovej * (145 g) mal výmennú kapacitu 4,8 mól.g 1 a napučaci objem 23 ml.g 1.The procedure is as described in Example 1 except that alginic acid is used instead of pectic acid. The obtained cross-linked alginic acid gel (145 g) had a exchange capacity of 4.8 mol / l and a swelling volume of 23 ml / l .
* Příklad 6* Example 6
Postupuje sa tak, ako v přiklade 1 s tým rozdielom, že kyselina pektová sa rozpustí v 150 ml borátového pufru (pH 11,2; 0,05 mol) а к sietovaniu sa použije 1 mol 2,2*-bis (oxiranylmetyl)éteru. Získaný gél sietovanej kyseliny pektovej (218 g) mal výmennú kapacitu 5,1 mol.g a napučaci objem 15,6 ml.g .The procedure is as in Example 1 except that pectic acid is dissolved in 150 ml of borate buffer (pH 11.2; 0.05 mol) and 1 mol of 2,2'-bis (oxiranylmethyl) ether is used for cross-linking. . The obtained cross-linked pectic acid gel (218 g) had a exchange capacity of 5.1 mol.g and a swelling volume of 15.6 ml.g.
Přiklad 7Example 7
Postupuje sa tak ako v příklade 6, s tým rozdielom, že namiesto kyseliny pektovej sa použije 0,01 molu substituovaných anhydroglykozylových skupin heparinu. Získaný gél sletovaného heparinu (210 g) mal výmennú kapacitu 7,1 mol.g 1 (SO3) a 2,1 mol.g karboxylových skupin. Napučaci objem získaného gélu bol 18,5 ml.g 1.The procedure is as in Example 6, except that 0.01 mol of substituted anhydroglycosyl groups of heparin are used instead of pectic acid. The recovered heparin gel (210 g) obtained had a capacity exchange of 7.1 mol.g 1 (SO 3) and 2.1 mol g carboxyl groups. Swelling volume of the gel obtained was 18.5 ml g 1st
Přiklad 8Example 8
Postupuje sa tak, ako je uvedené v přiklade 6, s tým rozdielom, že namiesto kyseliThe procedure is as described in Example 6 except that it is acidified instead
CS 270 819 81 ny pektovej sa použije kyselina hyaluronová (2 g; tj. cca 0,01 mol). Pri zachováni'· ostatných molárnych proporci! tak, ako je uvedené v přiklade 6 sa získal gél sietovanej kyseliny hyalurónovej v množstve 2,1 g o výmennej kapacita 2,8 mol.g 1 (karboxylová skupina) a napučacom objeme 17,7 ml.g 1.Hyaluronic acid (2 g; i.e. about 0.01 mol) was used in pectic acid. While keeping the other molar proportions! as in Example 6, a crosslinked hyaluronic acid gel was obtained in an amount of 2.1 g and a exchange capacity of 2.8 mol / l (carboxyl group) and a swelling volume of 17.7 ml / l .
Příklad 9Example 9
Postupuje sa tak, ako je uvedené v přiklade 8, s tým rozdielom. že sa namiesto kyseliny hyalurónovej použije chondroitín 6-sulfát. Získaný gél 2,4 g sletovaného chondroitin 6-sulfátu mal výmennú kapacitu 2,4 mol.g“1 (karboxylová skupina) a 2,1 mol.g“1 (-SO3).The procedure is as described in Example 8, with the difference. The method according to claim 1, wherein chondroitin 6-sulfate is used instead of hyaluronic acid. The gel obtained 2.4 g of cross-linked chondroitin 6-sulphate had a exchange capacity of 2.4 mol.g -1 (carboxyl group) and 2.1 mol.g -1 (-SO 3).
Příklad 10Example 10
Postupuje sa tak, ako je uvedené v přiklade 8, s tým rozdielom, že namiesto kyseliny hyalurónovej sa použije keratan sulfát. Získaný gél, 1,6 g sletovaného keratan sulfátu mal výmennú kapacitu 1,6 mol.g’1 (-SO3) a napučací objem 14,1 ml.g“1.The procedure is as described in Example 8 except that keratan sulfate is used instead of hyaluronic acid. The gel obtained, 1.6 g of cross-linked keratan sulfate had a exchange capacity of 1.6 mol.g -1 (-SO 3) and a swelling volume of 14.1 ml.g -1 .
Příklad 11Example 11
Postupuje sa tak, ako je uvedené v příklade 8, s tým rozdielom, že sa namiesto kyseliny hyalurónovej použije dermatan sulfát. Získaný gél, 1,8 g sletovaného dermatanu mal výmennú kapacitu 2,4 mol.g“1 (karboxylová skupina) a 2,2 mol.g“1 (-30Σ) a napučaci -1 J objem 19,2 ml.g .The procedure is as described in Example 8, except that dermatan sulfate is used instead of hyaluronic acid. The gel obtained, 1.8 g of cross-linked dermatan had a exchange capacity of 2.4 mol.g -1 (carboxyl group) and 2.2 mol.g -1 (-30 °) and a swelling -1 J volume of 19.2 ml.g.
Vynález má široké uplatnenie predovšetkým v analytickej biochémii, v bioorganickej chémii, v enzýmovom inžinierstve a v, technológiach využivajúcich rekombinantnú dezoxyribonukleovú kyselinu. \The invention has broad application in particular in analytical biochemistry, bioorganic chemistry, enzyme engineering and in technologies employing recombinant deoxyribonucleic acid. \
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