CZ714489A3 - method of binding biologically active substances containing an amine group to polysaccharides - Google Patents
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(57) Naviazanie biologicky aktívnych látok, obsahujúcich aminoskupinu, na polysacharid s karboxylovými skupinami a deje prosiredníctvom aktivácie karboxylových skupin izobutylchloroformiátom. Biologicky aktívna látka je k pólysacharidu naviazaná amidickou vazbou, ktorá je degradovateíná in vivo. Tento spósob. má uplalnenie vo farmacii, ochraně rastlín, humánnej a vctcrinárncj medici-(57) Binding of the amino-containing biologically active substances to the polysaccharide with carboxyl groups and proceeding via activation of the carboxyl groups with isobutyl chloroformate. The biologically active agent is bound to the polysaccharide by an amide bond that is degradable in vivo. This way. has application in pharmacy, plant protection, human and veterinary medicine-
Vynález sa týká spóspbu naviazania biologicky aktívnych látok, obsahujúcich aminoskupinu, na polysacharidy.The present invention relates to a method for coupling amino-containing biologically active substances to polysaccharides.
Jedným z hlavných problémov při využití biologicky aktívnych látok, či už ako lieciv alebo pesticídov, je spósob ich aplikácie. Má^okedy je totiž vhodné biologicky aktívnu látku aplikovat priamo d v koncentrovanéj formě. Prídavok róznych aditív zvýhodňuje vlastnosti ti aplikovanej biologicky aktívnej látky.One of the main problems in the use of biologically active substances, whether as active substances or pesticides, is the way they are applied. This is because it is advisable to apply the biologically active substance directly in concentrated form. The addition of different additives favors the properties of the biologically active agent applied.
Často je výhodné·,, ak sa. biologicky aktívna látka aplikuje· . v stave inaktívnom a do aktívneho stavu sa dostává postupné za fyziologických podmienok, napr. pósobením hydrolytíčkých enzýmov.Often, it is preferred that. the biologically active substance is applied. in the inactive state and into the active state it progresses gradually under physiological conditions, e.g. by the action of hydrolytic enzymes.
Postupné uvoTnovanie biologicky aktívnej látky sa dá v principe dosiahnuť dvoma spósobmi. Jednak enkapsuláciou - uzavretím . biologicky aktívnej látky do vhodného, biodegrádovateTného obalu, ktorý sa postupné za fyziologických podmienok rozloží. Druhý, vhodnější a čoraz častejšie používaný,je naviazanie biologicky aktívnej látky na vhodný nosič kovalentou vazbou. Tá sa volí tak, aby sa 2a fyziologických podmienok odštěpovala biologicky aktívna látka v aktívnom stave, pósobením hydrolytických alebo iných. enzýmov, štiepiacich mostík medzi nosičom a biologicky aktívnou látkou.The gradual release of a biologically active substance can in principle be achieved in two ways. First, by encapsulation - closing. of the biologically active substance into a suitable, biodegradable container which is gradually decomposed under physiological conditions. The second, more suitable and increasingly used, is the binding of the biologically active agent to a suitable carrier by a covalent bond. This is selected so that the biologically active substance in the active state is cleaved by physiological conditions by hydrolytic or other actions. enzymes, cleavage bridges between the carrier and the biologically active agent.
Ako nosiče biologicky aktívnych látok sa používajú predovšetkým inertné polymérne nosiče ako napr. pólyvinyIpyrolidon ÍGeiger,In particular, inert polymeric carriers such as e.g. pollyvinyIpyrolidone ÍGeiger,
Β., Von Specht, B. J., Arnon, R., Eur. J. Biochem., 73. 141 ( 1977)), polyetylénglykol ÍDavis, fí. Η., Abuchowski, A,, Van Es, T., Palesuk, N. C., Savoca, K., Chen, fí. Η., -L., Pytalak, Ρ., Am. Chem.Von., Von Specht, B.J., Arnon, R., Eur. J. Biochem., 73, 141 (1977)), polyethylene glycol Davis, et al. Abuchowski, A., Van Es, T., Palesuk, N. C., Savoca, K., Chen, et al. Ta., -L., Pytalak, Ρ., Am. Chem.
Soc. Polym. Prepr., 20, 357 (1979)], po^yvinylimidazol [KoTčova, S.Soc. Polym. Prepr., 20, 357 (1979)], polyvinyllimidazole [KoTčova, S.
C., Samsonov, G. V., Budovskaja, G. L., Sokolova, T. A., SU 422418 i (1974)] a i* Tieto polymery však vždy obsahujú časť nezreagovaných toxických monomérov, ktoré je problém kvantitativné odstrániť. I keá sú tieto nosiče spravidla neimunogénne, sú cudzie organizmu aC., Samson, G.V., Budovskaya, G.L., Sokolova, T.A., SU 422418 i (1974)] and i. However, these polymers always contain a portion of unreacted toxic monomers which is a quantitative problem. Although these carriers are generally non-immunogenic, they are foreign to the organism and
1. móžu vzniknúť problémy s ich odstraněním.1. problems may arise with their removal.
„ Inými nosičmi biologicky aktívnych látok sú bielkoviny [Bureš'Other carriers of biologically active substances are proteins [Bureš
Li , Boštík, J., Motyčka, K., Spundové, M.·, íehák, L., Neoplasma, . 329 ( 1988)] ,, ktoré sú zvačša imunogénne a vyvolávájú imunitnú odpoveň organizmu po ich aplikácii.Li, Boštík, J., Motyčka, K., Spundová, M., Iákák, L., Neoplasma,. 329 (1988)], which are largely immunogenic and elicit an immune response to the organism upon administration.
Z hladiska biokompatibility a imunitných vlastností sa zdá,, že najperspektívnejšími nosičmi biologicky aktívnych latok sú polysacharidy. Výhodou ich použitia je, že uvoTnovanie látky do prostredia sa dá regulovat kvalitou polysacharidu. Je možné vybrat od polysacharidu biodegrádovateTného ( škrob, dextrán, glykogén) až po stabilně polysacharidy (celulóza,, kvasinkový glukán).In terms of biocompatibility and immune properties, it appears that the most promising carriers of biologically active compounds are polysaccharides. The advantage of their use is that the release of the substance into the environment can be controlled by the quality of the polysaccharide. It is possible to choose from biodegradable polysaccharide (starch, dextran, glycogen) to stable polysaccharides (cellulose, yeast glucan).
Dosial’ najčastejšie používanou metodou k Vytvoreniu kovalentnej vazby medzi biologicky aktívnou látkou, obsahujúcou volné aminoskupiny a polysacharidickým nosičom je jodistanová oxidácia. Jodistan Specificky oxiduje vicinálne diolové skupiny po.lysacharidu na aldehydy, ktoré s aminoskupinou biologicky aktívnej látky tvoria Schifovu bázu. Tá sa stabilizuje na sekundárný amin redukciou borhydridom v bázickom prostředí. Nevýhodou tohoto spósobu naviazania, ako i příbuzných, využívájúcich bifunkčné činidla, reagujúce na jednej straně so sacharidom a na druhej straně s aminoskupinou biologicky aktívnej látky (napr, trichlórtriazín, epichlórhydrín a pod.) je predovšetkým to, že vazba biologicky aktívnej látky na sacharid je příliš silná, obtiašne štiepitelňé in vivo,. čo komplikuje uvolnenie látky do organizmu. Ďalšou nevýhodou je že polysacharid sa pri tomto spósobe naviazania příliš modifikuje,, stráca svoje povodně vlastnosti, móže sa degradovat na kratšie reťazce a pod. Náviac,. reakcia sa robí zvačša v hómogénnej fáze,; čo komplikuje odstránenie nežiadúcich produktov z reakčnej 2mesi a tiež umožňuje i priebeh'nežiadúcich bočných reakcií najma reakciu s hydroxylovými skupinami susedných polysacharidických reťazcov..The most commonly used method to date to form a covalent bond between a biologically active substance containing free amino groups and a polysaccharide carrier is periodate oxidation. Periodate Specifically oxidizes the vicinal diol groups of the polysaccharide to aldehydes, which form the Schif base with the amino group of the biologically active substance. This is stabilized to the secondary amine by reduction with borohydride in basic medium. The disadvantage of this binding method as well as of the relatives using bifunctional reagents reacting on the one hand with the saccharide and on the other hand with the amino group of the biologically active substance (e.g., trichlorotriazine, epichlorohydrin and the like) is mainly that the biologically active substance is too strong, difficult to cleave in vivo. which complicates the release of the substance into the body. A further disadvantage is that the polysaccharide is too modified by this binding method, loses its flood properties, can degrade to shorter chains and the like. In addition ,. the reaction is carried out mostly in the homogeneous phase ; which complicates the removal of unwanted products from the reaction 2 months and also allows undesired side reactions to take place, in particular the reaction with hydroxyl groups of adjacent polysaccharide chains.
Uvedené nedostatky odstraňuje spósob naviazania biologický aktívnej látky, obsahujúcej aminoskupiny, na polysacharid s karboxylovýgii skupinami,, prostredníctvom aktivácie karboxylov izobutylchloroformiátom v nevodnom prostředí. Podstata vynálezu spočívá v tom, že karboxylovaný polysacharid v Hi formě sa rozsuspenduje· v bezvodom dimetylformamide. Biologicky aktívna látka (ligand),, obsahujúca aminoskupinu,. sa rozpustí v rovnakom diele dimetylformamidu v molárnom pomere 1 až 1000 : 1 voči polysacharidu.. Suspenzia polysacharidu sa -ochladí minimálně na 0°G a přidá sa ekvimolárne množstvo (vzhladom na ligand) trietylamínu a izobutylchloroformiátu. Inkubuje sa 15 až 30 min. Potom sa přidá roztok biologicky -aktívnej látky v. dimetylformamide a inkubuje sa pri laboratórnej teplote 15 až 30 hod. Nezreagov.aná biologicky aktívna látka a v dimetylformamide rozpustné reakčné produkty sa odstránia filtráciou a premývanim vhodným nevodným rozpúšťadlomi (metanol, etanol, dioxán, aceton a poď.) alebo dialýzou. Výťažnosť je v závislosti od reaktivity aminu 10 až 50 %·These drawbacks overcome the method of binding an amino-containing biologically active substance to a carboxylic acid polysaccharide by activating the carboxyls with isobutyl chloroformate in a non-aqueous medium. The essence of the invention is that the carboxylated polysaccharide in H 1 form is suspended in anhydrous dimethylformamide. A biologically active substance (ligand) containing an amino group. The polysaccharide suspension is cooled to at least 0 DEG C. and an equimolar amount (relative to the ligand) of triethylamine and isobutyl chloroformate is added. Incubate for 15 to 30 min. Then, a solution of the biologically active substance in. dimethylformamide and incubated at room temperature for 15 to 30 hours. Unreacted biologically active substance and dimethylformamide soluble reaction products are removed by filtration and washing with a suitable non-aqueous solvent (methanol, ethanol, dioxane, acetone, etc.) or by dialysis. The yield is 10 to 50% depending on the reactivity of the amine ·
Viazanie biologicky aktívnych látfek, obsahujúcich aminosku- 3 piny na polysacharid,. sposobom pódia vynálezu., má oproti doteraz známým sposobom viacero výhod. Predovšetkým sa pracuje v heterogénnej fáze.,Biologicky aktívna látka je v kvapalnej fáze rozpuštěná v bezvodom dimetylf ormamidekým polysacharidostává v tom, to prostředí nerozpuštěný a tvoří druhá fázu. To znamená, že je možné jednoduché odstránenie(reakčných produktov od polysacharidu s 7 naviazanou biologicky aktívnou látkou v kvapalnej fáze jednoduchou 'j; filtráciou. Vytvořená amidická vazba medzi biologicky aktívnou ’ látkou a polysacharidom je štiepiteTná in vivo prostredníctvom hešpecifických esteráz a proteáz, nachádzajúcich sa na povrchu buniek, alebo v plazme vyšších organizmov.Binding of biologically active 3-amino-containing substances to the polysaccharide. The process according to the invention has several advantages over the prior art. In particular, the heterogeneous phase is employed. The biologically active substance is dissolved in the anhydrous dimethylformamide polysaccharide in the liquid phase in that it is undissolved and forms the second phase. This means that the simple removal ( reaction products of the 7-linked biologically active substance in the liquid phase by simple filtration) is possible. The amide bond formed between the biologically active substance and the polysaccharide is cleavable in vivo by means of the specific esterases and proteases found. on the surface of cells or in the plasma of higher organisms.
____ Uvedené.příklady ilustrujú, ale neobmedzujú predmet vynálezu.___These examples illustrate but do not limit the scope of the invention.
Příklad 1Example 1
Polygalakturónová kyselina Cl g)v H+ formě sa rozsuspenduje v 5 ml bezvodého dimetylformamidu a suspenzia sa ochladí na - 5°C. Přidá sa 1 ml trietylamínu a 1 ml izobutylchloroformiátu. Inkubuje sa za miešania 30 min. Přidá sa 500 mg aracinozyl cytozínu (araC) rozpuštěného v 5 ml·. bezvodého dimetylformamidu a inkubuje sa za miešania cez noc za laboratornej teploty. Reakčná zmes sa.dialyzuje 48 h oproti 3x 21 destilovanej vody pri 4°C. Množstvo araC naviazaného na polysacharid sa vyhodnotilo elementárnou a spektrálnou analýzou. Výťažok bol 100 mg araC/g nosiča (20 %)..Polygalacturonic acid C1 g) in H + form is suspended in 5 ml of anhydrous dimethylformamide and the suspension is cooled to -5 ° C. 1 ml of triethylamine and 1 ml of isobutyl chloroformate are added. Incubate with stirring for 30 min. Add 500 mg of aracinosyl cytosine (araC) dissolved in 5 ml ·. of anhydrous dimethylformamide and incubated with stirring overnight at room temperature. The reaction mixture was dialysed for 48 h against 3x21 distilled water at 4 ° C. The amount of araC bound to the polysaccharide was evaluated by elemental and spectral analysis. The yield was 100 mg araC / g carrier (20%).
' Příklad 2 „ ' jh'Example 2' 'jh
Postupovalo- sa ako v příklade 1 s tým rozdielom, že miesto l póly galak tur ónov.ej kyseliny sa použil 6-karboxymety 1-1,3-ft -glukán v H+ formě. Výťažok reakcie bol 110 mg/g /22 %).The procedure was as in Example 1 except that 6-carboxymethylene-1,3,3-glucan in the H + form was used instead of the 1-pole of the galactonic acid. Reaction yield was 110 mg / g / 22%).
iand
Příklad 3 Postupovalo sa ako v příklade 1 s tým rozdielom, že namiesto araC sa použil 1,6-diaminohexén. Výťažok reakcie bol 250 mg (50 %).Example 3 The procedure of Example 1 was followed except that 1,6-diaminohexene was used instead of araC. The yield of the reaction was 250 mg (50%).
Příklad 4Example 4
Postupovalo sa ako v přiklade 1 s tým rozdielom, že ako polysacharid sa.použil 6-karboxymetyl-l,4-alfa-glukan v H formě a ako ,ι biologicky aktívna látka s volnou aminoskupinou sa použil benzylamín. Výťažok reakcie 225 mg/g (45 %).The procedure was as in Example 1 except that 6-carboxymethyl-1,4-alpha-glucan in the H form was used as the polysaccharide and benzylamine was used as the biologically active compound with a free amino group. Reaction yield 225 mg / g (45%).
Příklad 5 'Example 5 '
-5 , f r< Pólygalakturonová kyselinami g/ v Ξί+ formě sa rozsuspenduje * v 10 ml bezvodého dimetylformamidu a suspenzia sa ochladí na 2°C.-5, f r <polygalacturonic acid g / in Ξί + form is slurried in 10 * ml of anhydrous DMF and the suspension was cooled to 2 ° C.
Přidá sa 100 ul trietylaminu a 100 ul izobutylchloroformiátu. Inkubuje sa za miešania 15 min. Přidá sa 50 mg benzylamínu a inkubuje sa 30 h za laboratórnej teploty a za miešania. fíeakčná zmes sa filtruje , premýva metanolem,, acetónom a vysuší sa. Výťažok 25 mg benzylamínu na gram polygalakturónovej kyseliny (50 %).Add 100 µl triethylamine and 100 µl isobutyl chloroformate. Incubate with stirring for 15 min. 50 mg of benzylamine is added and incubated for 30 h at room temperature with stirring. The reaction mixture was filtered, washed with methanol, acetone and dried. Yield 25 mg benzylamine per gram of polygalacturonic acid (50%).
Vynález má využitie pri ochraně polnohospodárskych rastlíh, humánněj a v.eterinárnej medicíně.The invention has utility in the protection of agricultural plants, human and veterinary medicine.
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SK7144-89A SK278783B6 (en) | 1989-12-18 | 1989-12-18 | Method for bonding biologically active substances containing amino group to polysaccharides |
CS897144A CZ714489A3 (en) | 1989-12-18 | 1989-12-18 | method of binding biologically active substances containing an amine group to polysaccharides |
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CS897144A CZ714489A3 (en) | 1989-12-18 | 1989-12-18 | method of binding biologically active substances containing an amine group to polysaccharides |
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CZ303485B6 (en) * | 1998-11-11 | 2012-10-17 | Sigmar Italia S.P.A. | Process for preparing crosslinked polysaccharides |
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CN105424267B (en) * | 2015-12-23 | 2019-05-14 | 太原航空仪表有限公司 | A kind of probe for gyroplane total pressure measurement |
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Cited By (1)
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CZ303485B6 (en) * | 1998-11-11 | 2012-10-17 | Sigmar Italia S.P.A. | Process for preparing crosslinked polysaccharides |
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