CS242287B1 - Ketosteroids' protein conjugates and method of their preparation - Google Patents
Ketosteroids' protein conjugates and method of their preparation Download PDFInfo
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- CS242287B1 CS242287B1 CS849698A CS969884A CS242287B1 CS 242287 B1 CS242287 B1 CS 242287B1 CS 849698 A CS849698 A CS 849698A CS 969884 A CS969884 A CS 969884A CS 242287 B1 CS242287 B1 CS 242287B1
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- protein conjugates
- solution
- formula
- ketosteroids
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- 102000004169 proteins and genes Human genes 0.000 title claims description 6
- 108090000623 proteins and genes Proteins 0.000 title claims description 6
- 238000000034 method Methods 0.000 title claims description 4
- 238000002360 preparation method Methods 0.000 title claims 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 9
- 108010088751 Albumins Proteins 0.000 claims description 4
- 102000009027 Albumins Human genes 0.000 claims description 4
- 229910052739 hydrogen Inorganic materials 0.000 claims description 4
- 239000003960 organic solvent Substances 0.000 claims description 4
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- PJWRZYGGTHXGQO-UHFFFAOYSA-N 4-cyanobenzohydrazide Chemical compound NNC(=O)C1=CC=C(C#N)C=C1 PJWRZYGGTHXGQO-UHFFFAOYSA-N 0.000 claims description 2
- 238000001704 evaporation Methods 0.000 claims description 2
- 230000008020 evaporation Effects 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims description 2
- LMBFAGIMSUYTBN-MPZNNTNKSA-N teixobactin Chemical compound C([C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H](CCC(N)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H]1C(N[C@@H](C)C(=O)N[C@@H](C[C@@H]2NC(=N)NC2)C(=O)N[C@H](C(=O)O[C@H]1C)[C@@H](C)CC)=O)NC)C1=CC=CC=C1 LMBFAGIMSUYTBN-MPZNNTNKSA-N 0.000 claims description 2
- QZLYKIGBANMMBK-UGCZWRCOSA-N 5α-Androstane Chemical compound C([C@@H]1CC2)CCC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CCC[C@@]2(C)CC1 QZLYKIGBANMMBK-UGCZWRCOSA-N 0.000 claims 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 239000011541 reaction mixture Substances 0.000 description 3
- 229910052938 sodium sulfate Inorganic materials 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 238000000921 elemental analysis Methods 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L sodium sulphate Substances [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 1
- SIHHLZPXQLFPMC-UHFFFAOYSA-N chloroform;methanol;hydrate Chemical compound O.OC.ClC(Cl)Cl SIHHLZPXQLFPMC-UHFFFAOYSA-N 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Steroid Compounds (AREA)
Description
242287 kde R je —SO3H, se nechá reagovat s hydrazidem kyseliny 4-kyanobenzoové v organickém rozpustidle při teplotě 10 až 70 °C po dobu 3 až 6 ho-din a, na izolovanou látku obecného vzor-ce II '
(«) kde R je —SO3H, se působí bezvodým chlorovodíkem v bez-vodém metanolu po dobu 1 až 2 hodin přiteplotě -—5 až +5 °C. načež po odpařeníorganického rozpouštědla za tlaku 1,5 až2 kPa se odparek rozpustí v dimethylforma-midu v poměru 1 : 100 dílů hmot. a tentoroztok se prikape do roztoku albuminu vborátovém pufru o pH 8,5, μ = 0,1 při tep-lotě místnosti v poměru 1 : 0,0005 až 0,001dílů hmot. nechá se reagovat dalších 10 až25 h, načež se konečný produkt vyčistí di-alýzou a ultrafiltrací. Dále jsou uvedeny příklady způsobu pří-právý”podle vynálezu. Příklad 1
Konjugát hovězího serumalbuminu s 3/3--hydroxy-5-androsten-17-on-3-natriumsul-fátem K roztoku 81 mg (0,5 mM) 4-kyanoben-zoylhydrazidu ve 4 ml absolutního etanolubylo přidáno 215 mg (0,55 mM) 5-andro-sten.-17-on-3/J-h.atriumsulfátu rozpuštěnéhove 2 ml 70% etanolu. Reakční směs byla za-hřívána při 60 °C po dobu 5 h a po přidáníněkolika kapek vody ponechána přes nocpři teplotě místnosti. Vzniklé krystaly bylyzfiltrovány a rekrystalovány z maléhomnožství vody 70% etanolem.
Bylo získáno 109 mg (37%) 3/S-hydroxy--5-androsten-17-on-3-natriumsulfát-17-(4--kyanobenzoylhydrazonu) s teplotou tání202 až 204 °G; elementární analýza je C59,98 %, H 6,01 %, N 7,52 %, S 5,85 %. Kon-trola čistoty byla prováděna tenkovrstev-nou chromatografií v soustavě metanol:: voda (8:2 obj.). Hodnoty RF: produkt-0,8,--4-kyanobenzoylhydrazid-0,65, 3^-hydroxy--5-androsten-17-on-3-natriumsulfát-0,l.
Do roztoku 10 mg uvedeného meziproduk-tu ve 4 ml bezvodého metanolu byl zaváděnpo dobu 2,5 h při teplotě 0 až -j-2 °C bez-vodý chlorovodík. Reakční směs byla odpa-řena za sníženého tlaku 1,5 až 2 kPa do-sucha, odparek promyt bezvodým diethyl-etherem, vysušen nad P2O5 a rozpuštěn v1 ml bezvodého dimethylformamidu. Tentoroztok byl přikapán do roztoku 6,5 mg ho- vězího serumalbuminu v 10 ml borátovéhopufru o pH 8,5 μ — 0,1 a míchán při teplo-tě místnosti po dobu 2 h. Výsledný bílko-vinný konjugát byl čištěn ultrafiltrací zapoužití ultrafiltrační membrány. Stanovenípoměru navázaného haptenu k bílkoviněbylo provedeno jednak UV spektrometrií,jednak izotopovou technikou a bylo naleze-no 1: 14 mol. Příklad 2
Konjugát hovězího sérumalbumlnu s 3ce--hydroxy-5-androsten-17-on-3-natriumsul-fátem. Bylo získáno 27,5 mg (21%) bílélátky o teplotě tání 198 až 200 °C; elemen-tární analýza: C 60,12 %, H 6,41 °/o, N 7,48procent. Kontrola čistoty byla prováděnastejným způsobem. Dále byla reakce prováděna jako v pří-padě 1. Poměr vázaného haptenu k bílko-vině byl 13 :1 mol. Příklad 3
Konjugát hovězího sérumalbuminu s 3--hydroxy-l,3,5-estratrien-17-on-3-natrium-sulfátem
Stejným způsobem jako v příkladu 1 by-la prováděna reakce se 102 mg (0,275 mM)sodné soli 3-hydroxy-l,3,5-estratrien-17-on--3-sulfátu rozpuštěným v 1 ml vody, 40,3miligramu (0,25 mM) 4-kyanobenzoylhyd-razidem rozpuštěným ve 2 ml etanolu s pří-davkem 1 ml kyseliny octové. Po zahřívá-ní 12 h při teplotě 80 °C byla reakční směsodpařena za sníženého tlaku 1,5 kP do su-cha. Separace produktu byla provedenapreparativní chromatografií na desce v sou-stavě chloroform — metanol — voda (80 :: 20 : 1 obj.). Produkt (RF = 0,7) byl vymytetanolem, směs odpařena za sníženého tla-ku do sucha a odparek překrystalován zesměsi etanol — voda (2:1 obj.). Bylo zís-káno 52,5 mg (37 %) nažloutlé látky o tep-lotě tání 161 až 164 °C; elementární analý-za: C 60,8%, H 4,94%, N 7,98%. Kontrolačistoty byla prováděna stejným způsobemjako v příkladu 1. Soustava chloroform —
Wherein R is -SO 3 H, is reacted with 4-cyanobenzoic acid hydrazide in an organic solvent at 10 to 70 ° C for 3-6 hours and on an isolated compound of formula II '
(R) wherein R is -SO 3 H, is treated with anhydrous hydrogen chloride in anhydrous methanol for 1-2 hours at -5 to + 5 ° C. after evaporation of the organic solvent at 1.5-2 kPa, the residue is dissolved in dimethylformamide (1: 100 parts by weight). and this solution is added dropwise to a solution of albumin in a borate buffer of pH 8.5, µ = 0.1 at room temperature in a ratio of 1: 0.0005 to 0.001 parts by weight. it is reacted for an additional 10-25 h, whereupon the final product is purified by di-alysis and ultrafiltration. The following are examples of the method of the invention. Example 1
Bovine Serum Albumin Conjugate with 3/3-Hydroxy-5-Androsten-17-one-3-Sodium Sulphate To a solution of 81 mg (0.5 mM) of 4-cyanobenzoylhydrazide in 4 mL of absolute ethanol was added 215 mg (0, 0). 55 mM) 5-androstan-17-one-3/4-thienesulfate dissolved in 2 ml of 70% ethanol. The reaction mixture was heated at 60 ° C for 5 h and after several drops of water was added over night at room temperature. The resulting crystals were filtered and recrystallized from a small amount of water with 70% ethanol.
109 mg (37%) of 3S-hydroxy-5-androsten-17-one-3-sodium sulphate-17- (4-cyanobenzoylhydrazone) with a melting point of 202 DEG-204 DEG C. were obtained; Elemental analysis C59.98%, H 6.01%, N 7.52%, S 5.85%. The purity was checked by thin layer chromatography in methanol: water (8: 2 v / v). RF values: product-0,8,4-cyanobenzoylhydrazide-0,65,3-hydroxy-5-androsten-17-one-3-sodium sulfate-0.1.
To a solution of 10 mg of the above intermediate in 4 ml of anhydrous methanol was fed 2.5 h at 0 to-2 ° C anhydrous hydrogen chloride. The reaction mixture was evaporated to dryness under reduced pressure (1.5 to 2 kPa), washed with anhydrous diethyl ether, dried over P2O5 and dissolved in 1 ml of anhydrous dimethylformamide. This solution was added dropwise to a solution of 6.5 mg of serum free albumin in 10 ml of borate buffer at pH 8.5 µ-0.1 and stirred at room temperature for 2 h. . The determination of the bound hapten to protein was performed by both UV spectrometry and isotope technique and 1: 14 moles were found. Example 2
Bovine Serumbumin Conjugate with 3ce-hydroxy-5-androsten-17-one-3-sodium sulphate. 27.5 mg (21%) of a white solid were obtained, m.p. 198-200 ° C; eluant: C 60.12%, H 6.41%, N 7.48%. The purity control was carried out in the same manner. Next, the reaction was carried out as in Example 1. The bound hapten to protein ratio was 13: 1 mol. Example 3
Bovine Serum Albumin Conjugate with 3-hydroxy-1,3,5-estratrien-17-one-3-sodium sulfate
In the same manner as in Example 1, the reaction was carried out with 102 mg (0.275 mM) of sodium salt of 3-hydroxy-1,3,5-estratrien-17-one-3-sulfate dissolved in 1 ml of water, 40.3 ml ( 0.25 mM) 4-cyanobenzoylhydrazide dissolved in 2 ml of ethanol with the addition of 1 ml of acetic acid. After heating at 80 ° C for 12 h, the reaction mixture was evaporated to dryness under reduced pressure (1.5 kP). Separation of the product was carried out by preparative plate chromatography in chloroform-methanol-water (80: 20: 1 by volume). The product (RF = 0.7) was eluted with ethanol, evaporated to dryness under reduced pressure and the residue recrystallized from ethanol-water (2: 1 by volume). 52.5 mg (37%) of a pale yellow substance were obtained, m.p. 161-164 ° C; Elemental analysis: C 60.8%, H 4.94%, N 7.98%. The counter-impurities were carried out in the same manner as in Example 1. Chloroform -
Claims (2)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS849698A CS242287B1 (en) | 1984-12-12 | 1984-12-12 | Ketosteroids' protein conjugates and method of their preparation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS849698A CS242287B1 (en) | 1984-12-12 | 1984-12-12 | Ketosteroids' protein conjugates and method of their preparation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CS969884A1 CS969884A1 (en) | 1985-08-15 |
| CS242287B1 true CS242287B1 (en) | 1986-04-17 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CS849698A CS242287B1 (en) | 1984-12-12 | 1984-12-12 | Ketosteroids' protein conjugates and method of their preparation |
Country Status (1)
| Country | Link |
|---|---|
| CS (1) | CS242287B1 (en) |
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1984
- 1984-12-12 CS CS849698A patent/CS242287B1/en unknown
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| Publication number | Publication date |
|---|---|
| CS969884A1 (en) | 1985-08-15 |
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