CN111991552B - 一种基于角蛋白巯基—二硫键交换反应的硫化氢供体及其合成方法和应用 - Google Patents
一种基于角蛋白巯基—二硫键交换反应的硫化氢供体及其合成方法和应用 Download PDFInfo
- Publication number
- CN111991552B CN111991552B CN202010787120.6A CN202010787120A CN111991552B CN 111991552 B CN111991552 B CN 111991552B CN 202010787120 A CN202010787120 A CN 202010787120A CN 111991552 B CN111991552 B CN 111991552B
- Authority
- CN
- China
- Prior art keywords
- keratin
- hydrogen sulfide
- reaction
- sulfydryl
- exchange reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- A61K38/1748—Keratin; Cytokeratin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/04—Sulfur, selenium or tellurium; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/18—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing inorganic materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/42—Use of materials characterised by their function or physical properties
- A61L15/44—Medicaments
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/025—Other specific inorganic materials not covered by A61L27/04 - A61L27/12
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/02—Inorganic materials
- A61L31/028—Other inorganic materials not covered by A61L31/022 - A61L31/026
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/04—Macromolecular materials
- A61L31/043—Proteins; Polypeptides; Degradation products thereof
- A61L31/047—Other specific proteins or polypeptides not covered by A61L31/044 - A61L31/046
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L31/16—Biologically active materials, e.g. therapeutic substances
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
- A61L2300/602—Type of release, e.g. controlled, sustained, slow
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
本发明公开了一种基于角蛋白巯基—二硫键交换反应的硫化氢供体及其合成方法和应用,该合成包括:从人发中提取角蛋白,得到含巯基的角蛋白溶液;将含二硫键的小分子溶于有机溶剂中,并将其加入角蛋白溶液中,得到反应液A,反应后透析并冷冻干燥得固体样品B;将固体样品B溶于有机溶剂中,加入含硫代羧基的小分子后,继续反应得到反应液C;将反应液C透析后,冷冻干燥得到基于角蛋白巯基交换反应的硫化氢供体。本发明首次采用基于角蛋白的巯基交换反应合成还原性响应的硫化氢供体,合成的基于角蛋白巯基—二硫键交换反应的硫化氢供体具有良好的生物相容性,稳定性高能够溶于水,并具有靶向性释放等特点,有利于制备多功能的硫化氢释放复合材料。
Description
技术领域
本方法涉及硫化氢供体,具体涉及一种通过还原性角蛋白的巯基与二硫键交换反应制备新型硫化氢供体及其应用。
背景技术
近年来硫化氢被证实在人体中不可或缺,其在心血管系统、神经系统中发挥着重要的调节作用,是继一氧化氮和一氧化碳之后出现的第三类内源性气体信号分子。硫化氢具有广泛的生物学作用,例如舒张血管、保护心脏、抗炎、抗氧化和抗肿瘤等。大量的研究表明,硫化氢具有广泛的生物学活性,且活性具有一定的剂量依赖关系,特别是在肿瘤生物学方面。已知低浓度的硫化氢具有促癌作用,而较高浓度的硫化氢具有明显的抑制癌细胞生长的作用。所以,硫化氢供体药物研究的关键科学问题在于如何使H2S供体分子选择性地在靶部位(一般为病变部位)释放一定浓度的H2S,而在正常组织器官中不释放或者仅释放少量的H2S,在发挥治疗作用的同时最大限度地避免H2S的不良反应。常见的硫化氢供体有硫化物无机盐如亚硫化钠(NaHS)和硫化钠(Na2S),有机多硫化物包括大蒜中含量较高的二烯丙基三硫化物(DATS)及二烯丙基二硫化物(DADS),Lawesson试剂水溶性衍生物GYY4137以及1,2-二硫杂-3-硫酮(DTT)等。传统的H2S供体虽然为H2S基础生物学研究方面提供了易得的工具分子,但缺乏生物相容性并且H2S释放的选择性及释放速率的可控性较差。
角蛋白是一种生物相容性优异的生物材料,可以由人体毛发或者动物的羽毛、角、趾中提取获得,具有pH、还原性和酶响应等优点,被广泛应用于组织工程的各个领域中,例如药物载体、组织工程支架材料、创伤敷料等。通过还原法制备的角蛋白有较多的巯基,易于发生巯基交换反应,但是目前基于角蛋白的巯基交换反应制备还原性响应的硫化氢供体没有相关报道。
发明内容
发明目的:针对现有技术存在的问题,本发明提供一种基于角蛋白巯基—二硫键交换反应的硫化氢供体的合成方法。本发明是一种全新的、简单的合成基于角蛋白巯基—二硫键交换反应的硫化氢供体的方法,本发明合成的硫化氢供体具有优异的稳定性和生物相容性、较好的水溶性以及选择性释放等优点,可以广泛应用于组织工程支架、伤口敷料和药物载体等领域。本发明制备的硫化氢供体解决了现有技术中供体释放快,生物相容性差的缺点,弥补了基于角蛋白巯基交换反应制备还原性响应的硫化氢供体的空白。
技术方案:为了实现上述目的,本发明所述一种基于角蛋白巯基—二硫键交换反应的硫化氢供体的合成方法,包括如下步骤:
(1)采用还原法提取角蛋白,得到含巯基的角蛋白溶液,并测定其巯基的含量;
(2)将含二硫键的小分子溶于有机溶剂中,并将其加入上述步骤(1)中的角蛋白溶液中,得到反应液A,反应后透析并冷冻干燥得固体样品B;
(3)将步骤(2)中的固体样品B溶于有机溶剂中,加入含硫代羧基的小分子后,并加入几滴冰醋酸作为催化剂,继续反应得到反应液C;
(4)将步骤(3)中得到的反应液C透析后,冷冻干燥得到基于角蛋白巯基交换反应的硫化氢供体。
其中,步骤(1)所述的人发角蛋白的提取按照文献:Extraction,characterization,and NO release potential of keratin from humanhair.Materials Letters,2016,175:188-190.,从人发中采用还原法提取角蛋白,步骤(1)测定巯基含量的方法是Ellman法。
作为优选,所述含二硫键的小分子为二硫二吡啶或二苯基二硫醚。
作为优选,步骤(2)所述反应时间为10-12h,温度为15-25℃,条件为避光。
其中,步骤(2)和步骤(3)中所述有机溶剂为DMSO或者DMF。
其中,步骤(3)中所述的含硫代羧基的小分子为硫代苯甲酸或4-甲基硫代苯甲酸。
作为优选,步骤(3)中所述反应温度为15-25℃,反应时间为4-6h,条件为通氮气、避光。
其中,步骤(2)和步骤(4)中所述透析采用透析袋透析48-60h,截留分子量为3500Da。
作为优选,步骤(2)所述含有二硫键的小分子与角蛋白上的巯基浓度的摩尔比为5-15:1,步骤(3)所述含硫代羧基的小分子与角蛋白上的巯基的摩尔比为3-10:1。
本发明所述的基于角蛋白巯基—二硫键交换反应的硫化氢供体的合成方法所合成的基于角蛋白巯基—二硫键交换反应的硫化氢供体。
本发明所述的基于角蛋白巯基—二硫键交换反应的硫化氢供体在制备心血管支架、伤口敷料、人造血管或治疗心肌细胞缺血性损伤的药物或材料、组织工程支架或药物载体中的应用。
本发明的设计原理是通过角蛋白上的巯基和二硫键进行了两次巯基-二硫键交换反应,制备了一种新型硫化氢供体。该供体具有还原性响应、缓释、生物相容性优异、易于溶于水等优点。
有益效果:与现有技术相比,本发明具有如下优点:
(1)本发明首次采用基于角蛋白的巯基交换反应合成还原性响应的硫化氢供体,本发明所制备合成的基于角蛋白巯基—二硫键交换反应的硫化氢供体稳定性较高且能够溶于水,并具有靶向性释放等特点。
(2)本发明所使用的合成方法操作简单,条件温和,易于实现。
(3)本发明制备合成的硫化氢供体能够有效释放硫化氢,且释放时间较长,解决了现有技术中供体的气体释放过快的问题。
(4)本发明合成的硫化氢供体由于角蛋白的存在有着优异的生物相容性、可降解性,可以有效促进细胞生长,其释放时间得到明显延长,达到了缓释的效果,并具有还原性敏感等独特的性质,可以实现靶向释放等目标,可以用于制备硫化氢释放复合材料,广泛应用到血管组织工程,伤口敷料等生物组织工程领域。
附图说明
图1为硫化氢催化释放曲线图;
图2为对比例中硫化氢供体的释放曲线示意图;
图3为硫化氢供体促进细胞生长的示意图。
具体实施方式
以下结合附图和实施例对本发明作进一步说明。
本发明中的原料如无特殊说明均市售可得,其中人发角蛋白的提取和巯基含量测定按现有技术中方法。
实施例1
按照Ellman方法测定还原法新提取人发角蛋白溶液的巯基含量,取该角蛋白溶液50mL(巯基含量为3.748mM)放入三口烧瓶中,将五倍巯基摩尔量的二硫二吡啶(0.2067g)溶于20mL DMSO中,然后将其倒入装有角蛋白溶液的三口烧瓶中。用N2作为保护气,在25℃下避光反应12h,将其用3500Da的透析袋透析48h后,将透析液冷冻干燥得到固体样品改性角蛋白0.2176g。随后,将该改性角蛋白全部溶解在DMSO中(每1g改性角蛋白溶于50mLDMSO),完全溶解后加入硫代苯甲酸(100μL,0.8496mmol),并加入3-4滴冰醋酸作为催化剂,用N2作为保护气,避光在25℃下反应4h后,用3500Da的透析袋透析60h,将透析液冷冻干燥得到角蛋白巯基交换反应的硫化氢供体。
将25mg角蛋白巯基交换反应的硫化氢供体溶于5mL含4mM半胱氨酸的PBS缓冲液中(pH 7.4),采用亚甲基蓝显色法检测H2S在溶液中的释放量。其释放曲线如图1所示,从图中可以看到有明显的硫化氢释放,且释放时间可达220min,而图2中对比例硫化氢供体释放时间为5min左右(文献:Arylthioamides as H2S Donors:L Cysteine-Activated ReleasingProperties and Vascular Effects in Vitro and in Vivo.ACS Med.Chem.Lett.2013,4(10):904-908),证明采用本发明角蛋白巯基—二硫键交换反应制备的新型硫化氢供体具有缓释作用。本发明合成的角蛋白巯基交换反应的硫化氢供体可以由还原性物质催化释放硫化氢,因此其对还原性物质敏感,可以应用到组织工程领域。
实施例2
按照Ellman方法测定还原法新提取人发角蛋白溶液的巯基含量,取该角蛋白溶液150mL(巯基含量为7.486mM)放入三口烧瓶中,将十五倍巯基摩尔量的二硫二吡啶(2.067g)溶于60mL DMSO中,然后将其倒入装有角蛋白溶液的三口烧瓶中。用N2作为保护气,在25℃下避光反应12h后,将反应液用3500Da的透析袋透析48h,将透析液冷冻干燥得到改性角蛋白0.6782g。随后,将该改性角蛋白全部溶解在DMSO中(每1g改性角蛋白溶于50mLDMSO),完全溶解后加入硫代苯甲酸(720μL,6.1167mmol),用N2作为保护气,并滴入3-4滴醋酸作为催化剂,在25℃下避光反应4h后,用3500Da的透析袋透析60h,将透析液冷冻干燥得到角蛋白巯基交换反应的硫化氢供体。
实施例3
按照Ellman方法测定还原法新提取人发角蛋白溶液的巯基含量,取该角蛋白溶液50mL(巯基含量为6.523mM)放入三口烧瓶中,将十倍巯基摩尔量的二硫二吡啶(0.7185g)溶于20mL DMSO中,然后将其倒入装有角蛋白溶液的三口烧瓶中。N2作为保护气,在25℃下避光反应12h后,用3500Da的透析袋透析48h,将透析液冷冻干燥得到改性角蛋白0.2287g。随后,将该改性角蛋白全部溶解在DMSO中(每1g改性角蛋白溶于50mL DMSO),完全溶解后加入硫代苯甲酸(200μL,1.6991mmol),用N2作为保护气,并滴入3-4滴醋酸作为催化剂,在25℃下避光反应4h后,最后用3500Da的透析袋透析60h,将透析液冷冻干燥得到角蛋白巯基交换反应的硫化氢供体。
实施例4
按照Ellman方法测定还原法新提取人发角蛋白溶液的巯基含量,取该角蛋白溶液100mL(巯基含量为6.255mM)放入三口烧瓶中,将六倍巯基摩尔量的二硫二吡啶(0.8268g)溶于40mL DMSO中,然后将其倒入装有角蛋白溶液的三口烧瓶中。N2作为保护气,在25℃下避光反应12h,将其用3500Da的透析袋透析48h后,将透析液冷冻干燥得到改性角蛋白0.4877g。随后,将该改性角蛋白全部溶解在DMSO中(每1g改性角蛋白溶于50mLDMSO),完全溶解后加入硫代苯甲酸(720μL,6.1167mmol),用N2作为保护气,并滴入3-4滴冰醋酸催化,在25℃下避光反应4h后,用3500Da的透析袋透析60h,将透析液冷冻干燥得到角蛋白硫化氢供体。
实施例5
实施例5与实施例1的合成方法相同,不同之处在于,将步骤(2)中的二硫二吡啶替换成二苯基二硫醚,步骤(2)和(3)中的DMSO替换成DMF,步骤(3)中硫代苯甲酸替换成4-甲基硫代苯甲酸,与角蛋白上的巯基的摩尔比为3:1;步骤(2)为避光15℃反应10h;步骤(3)为避光15℃反应6h,步骤(2)和步骤(4)中,用3500Da的透析袋透析48h。
实施例6
取对数生长期中的L-929细胞,用胰蛋白酶消化后,配成1×105cells/mL的细胞悬液。取100μL细胞悬浮液分别接种于96孔板。将用上述实施例1制备的硫化氢供体溶于培养液,用0.22μm无菌滤头过滤后,配成不同浓度的供体溶液(10μg/mL-1mg/mL)。将100μL供体溶液分别加入孔板中,并加入10μL半胱氨酸溶液(半胱氨酸的终浓度为500μM),阴性对照组不加半胱氨酸。将96孔板放入37℃、5%CO2的培养箱中培养3天,然后每孔加入20μL浓度为5mg/mL的MTT溶液,培养箱中继续培养4h。吸弃MTT溶液和细胞培养液,加入100μL DMSO,避光震荡30min,用酶标仪测其细胞活力。如图3所示,当该供体浓度高于100μg/mL时,加入半胱氨酸催化硫化氢释放的组的细胞活性比没有硫化氢释放的组有显著性差异,因此基于角蛋白巯基—二硫键交换反应的硫化氢供体具有良好的生物相容性,且在半胱氨酸的催化下能够释放硫化氢,具有有效促进L-929细胞生长的作用,该促进作用与硫化氢的浓度成正比,当浓度较低时,对细胞生长的促进作用不显著,而当溶液中含有较高浓度的硫化氢时对细胞有明显促进的作用。综上,根据角蛋白巯基-二硫键交换反应制备的硫化氢供体具有良好的生物相容性,有明显促进细胞生长的作用。同时,其释放时间得到明显延长,达到了缓释的效果,并且其对还原性物质有一定的响应性,可以用于制备硫化氢释放复合材料,广泛应用到血管组织工程,伤口敷料等生物组织工程领域。
Claims (2)
1.一种基于角蛋白巯基—二硫键交换反应的硫化氢供体的合成方法,其特征在于,包括如下步骤:
(1)采用还原法提取角蛋白,得到含巯基的角蛋白溶液,并测定其巯基的含量;
(2)将含二硫键的小分子溶于有机溶剂中,并将其加入上述步骤(1)中的角蛋白溶液中,得到反应液A,反应后透析并冷冻干燥得固体样品B;
(3)将步骤(2)中的固体样品B溶于有机溶剂中,加入含硫代羧基的小分子后,再加入冰醋酸,继续反应得到反应液C;
(4)将步骤(3)中得到的反应液C透析后,冷冻干燥得到基于角蛋白巯基交换反应的硫化氢供体;
步骤(2)所述含二硫键的小分子为二硫二吡啶或二苯基二硫醚;步骤(2)所述反应时间为10-12h,温度为15-25℃,条件为避光;
步骤(3)所述的含硫代羧基的小分子为硫代苯甲酸或4-甲基硫代苯甲酸;步骤(3)中所述反应温度为15-25℃,反应时间为4-6h,条件为通氮气、避光;
步骤(2)和步骤(3)中所述有机溶剂包括DMSO或DMF;
步骤(2)所述含有二硫键的小分子与角蛋白上的巯基的摩尔比为5-15:1,步骤(3)所述含硫代羧基的小分子与角蛋白上的巯基的摩尔比为3-10:1。
2.根据权利要求1所述的基于角蛋白巯基—二硫键交换反应的硫化氢供体的合成方法,其特征在于,步骤(2)和步骤(4)中所述透析采用透析袋透析48-60h,截留分子量为3500Da。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010787120.6A CN111991552B (zh) | 2020-08-06 | 2020-08-06 | 一种基于角蛋白巯基—二硫键交换反应的硫化氢供体及其合成方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010787120.6A CN111991552B (zh) | 2020-08-06 | 2020-08-06 | 一种基于角蛋白巯基—二硫键交换反应的硫化氢供体及其合成方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111991552A CN111991552A (zh) | 2020-11-27 |
CN111991552B true CN111991552B (zh) | 2023-04-07 |
Family
ID=73462895
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010787120.6A Active CN111991552B (zh) | 2020-08-06 | 2020-08-06 | 一种基于角蛋白巯基—二硫键交换反应的硫化氢供体及其合成方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111991552B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113855851B (zh) * | 2021-09-24 | 2023-03-31 | 广东省科学院健康医学研究所 | 一种水凝胶及其制备方法和应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104784758A (zh) * | 2015-05-09 | 2015-07-22 | 南京师范大学 | 一种聚合物/角蛋白复合抗凝血血管组织工程支架的制备方法 |
CN107141345A (zh) * | 2017-06-07 | 2017-09-08 | 南京师范大学 | 一种角蛋白生物大分子一氧化氮供体及其合成与应用 |
CN111000979A (zh) * | 2019-12-04 | 2020-04-14 | 南京师范大学 | 一种基于角蛋白的硫化氢供体及其合成方法和应用 |
-
2020
- 2020-08-06 CN CN202010787120.6A patent/CN111991552B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104784758A (zh) * | 2015-05-09 | 2015-07-22 | 南京师范大学 | 一种聚合物/角蛋白复合抗凝血血管组织工程支架的制备方法 |
CN107141345A (zh) * | 2017-06-07 | 2017-09-08 | 南京师范大学 | 一种角蛋白生物大分子一氧化氮供体及其合成与应用 |
CN111000979A (zh) * | 2019-12-04 | 2020-04-14 | 南京师范大学 | 一种基于角蛋白的硫化氢供体及其合成方法和应用 |
Non-Patent Citations (2)
Title |
---|
二硫代羧酸和其硫代酰胺基衍生物的化学;蒋尚信;《化学通报》;19621231(第6期);29-37 * |
江宁主编.第六章 微生物酶工程.《微生物生物技术》.北京化学工业出版社,2008,211. * |
Also Published As
Publication number | Publication date |
---|---|
CN111991552A (zh) | 2020-11-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP2087910B1 (en) | Polymeric prodrugs | |
WO1992001476A1 (en) | Novel drug delivery systems for proteins and peptides using albumin as a carrier molecule | |
CN107141345B (zh) | 一种角蛋白生物大分子一氧化氮供体及其合成与应用 | |
CN111991552B (zh) | 一种基于角蛋白巯基—二硫键交换反应的硫化氢供体及其合成方法和应用 | |
CN103626920B (zh) | 一种吲哚-3-乙酸分子印迹磁性纤维素微球及其制备方法和应用 | |
EP3613792B1 (en) | Multi-arm targeted anti-cancer conjugate | |
CN102382332B (zh) | 液相合成银、壳聚糖和/或其衍生物纳米复合物的方法 | |
CN109517162B (zh) | 可注射水凝胶及制备方法 | |
CA3163070A1 (en) | Sulfhydryl modified hyaluronic acid, preparation method therefor and use thereof | |
CN111000979B (zh) | 一种基于角蛋白的硫化氢供体及其合成方法和应用 | |
CN105963703B (zh) | 一种抗肿瘤药物的制备方法 | |
CN1978462B (zh) | 咪唑啉修饰的氨基酸,其合成方法及在多肽标记中的应用 | |
CN110755638A (zh) | 一种具有骨靶向性的药物载体及其制备方法与应用 | |
CN114057830B (zh) | 寡肽-1衍生物及其制备方法和用途 | |
CN114668745A (zh) | 一种葡萄糖和h2o2双重响应的双层交联聚合物纳米递药系统及其制备方法和应用 | |
CN108721636B (zh) | 联硒键连接的具有双重响应性的药物递送材料及其制备方法和应用 | |
CN106668932A (zh) | 一种止血材料及其制备方法与应用 | |
CN112851756A (zh) | 一种酚酸多肽偶联物及其制备方法和应用 | |
CN104926710A (zh) | 卡络磺钠及其制法 | |
CN107759804B (zh) | 可定点结合含组氨酸标签蛋白的明胶衍生材料及其制备方法 | |
CN114262391B (zh) | 一种tempo修饰透明质酸及其制备方法和应用 | |
CN109438387A (zh) | 一种taem活性酯的制备方法 | |
CN116283648B (zh) | 一种取代的苯丙烯酰基或苯丙酰基苯乙胺类化合物及其制备方法和应用 | |
CN113321633B (zh) | 槲皮素-3-o-乙酸-(3-氯-4-硫代氨基)-苯酯及在制备糖尿病药物中的应用 | |
CN115554272B (zh) | 一种纳米颗粒的制备方法及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |