CN113166164A - 将膝沟藻毒素制备性规模转化为新石房蛤毒素 - Google Patents

将膝沟藻毒素制备性规模转化为新石房蛤毒素 Download PDF

Info

Publication number
CN113166164A
CN113166164A CN201980055613.6A CN201980055613A CN113166164A CN 113166164 A CN113166164 A CN 113166164A CN 201980055613 A CN201980055613 A CN 201980055613A CN 113166164 A CN113166164 A CN 113166164A
Authority
CN
China
Prior art keywords
neostx
gtx1
dithiol
amount
saxitoxin
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.)
Granted
Application number
CN201980055613.6A
Other languages
English (en)
Other versions
CN113166164B (zh
Inventor
A·I·塞尔伍德
R·范金克尔
C·A·沃
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Trusteeship Committee Of Caldwell Institute
Original Assignee
Trusteeship Committee Of Caldwell Institute
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Trusteeship Committee Of Caldwell Institute filed Critical Trusteeship Committee Of Caldwell Institute
Priority to CN202410100344.3A priority Critical patent/CN117964629A/zh
Publication of CN113166164A publication Critical patent/CN113166164A/zh
Application granted granted Critical
Publication of CN113166164B publication Critical patent/CN113166164B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
    • C07D487/14—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
    • C07D487/20—Spiro-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
    • C07D487/16—Peri-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C12N1/125—Unicellular algae isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/02—Photobioreactors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/14—Bags
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/22—Transparent or translucent parts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/26—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/02—Separating microorganisms from the culture medium; Concentration of biomass
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/89—Algae ; Processes using algae

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Botany (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

本申请描述了以足以使该化合物被用作活性药物成分(API)的一定量纯度制备新石房蛤毒素的方法。该方法包括在pH 7.4‑7.6的溶液中使用二硫醇(诸如DTT或DTBA)对膝沟藻毒素1(GTX1)和膝沟藻毒素4(GTX4)的未溶解混合物进行还原脱磺化。使用基于二氧化硅的弱阳离子交换吸附剂纯化反应产物，以使新石房蛤毒素与二硫醇分离。

Description

将膝沟藻毒素制备性规模转化为新石房蛤毒素
技术领域
本发明涉及将膝沟藻毒素1(GTX1)和膝沟藻毒素4(GTX4)的混合物以制备性规模转化为新石房蛤毒素(neoSTX)以及纯化neoSTX以用作活性药物成分(API)。
背景技术
正如Garcia-Altares(2017)的出版物所述，海洋微藻毒素构成了最多样化和最复杂的天然产物组之一。实例是麻痹性贝类毒素(PST)(诸如石房蛤毒素(STX))、其类似物和衍生物。膝沟藻毒素(GTX)是STX的硫酸化类似物，并且海洋细菌可以通过还原消除将GTX转化为STX。在海洋环境中，STX的主要生产者是真核鞭毛藻类。
STX是一种单萜吲哚生物碱，其含有三环3,4-丙炔基全氢化嘌呤(3,4-propinoperhydropurine)系统，该系统带有在还原嘌呤的C2和C8位置由NH2基团形成的2个胍基部分：
Figure BDA0002949104050000011
STX不仅阻止电压门控钠通道(VGSC)，而且也与钙和钾通道结合。取代基的性质极大地影响了石房蛤毒素类似物的整体毒性。N1的羟基化(例如如新石房蛤毒素(neoSTX)一样)在结合亲和力中不发挥主要作用，但似乎增加了效力。
现有技术中充斥着PST的美容和治疗应用的公开内容，包括其作为局部麻醉剂和止痛剂的用途。Mezher(2018)的出版物公开了美国食品药品监督管理局(FDA)计划制定指导文件，以鼓励开发缓释局部麻醉剂，其可在某些情况下取代全身性口服阿片类药物的需求。美国FDA的期望是开发新的非阿片类药物来治疗慢性疼痛，这可以为需要长期使用止痛药的患者提供更安全的选择。Kohane等人(2000)、Rogriguez-Navarro等人(2011)、Templin等人(2015)和Wylie等人(2012)的出版物公开了在这些应用中使用neoSTX。对这些应用的开发和广泛采用的限制在于提供足够数量和足够纯度的PST使其在商业上可用于制造药物制剂的可行性。
以下出版物公开膝沟藻毒素1(GTX1)、膝沟藻毒素4(GTX4)或新石房蛤毒素(neoSTX)的制备。制剂通常为分析规模，或者没有提供将该制剂用作活性药物成分(API)所需的数量和纯度。
Hall等人(1984)的出版物公开了通过X射线晶体学证实三个取代基的位置和同一性，所述三个取代基与母体化合物一起形成在protogonyaulax中发现的十二种毒素系列。
Daigo等人(1985年)的出版物公开了从螃蟹标本中提取和分离新石房蛤毒素(neoSTX)。分离的neoSTX所获得的剂量-死亡时间曲线与石房蛤毒素(STX)的曲线明显不同。
Laycock等人(1994)的出版物公开了用于纯化一些足以用作分析标准品的常见麻痹性贝类中毒(PSP)毒素的方法。PSP毒素纯化自鞭毛藻Alexandrium excavatum、巨型海扇贝(Plagopecten magellanicus)肝胰腺和蓝藻水华束丝藻(Aphanizomenon flos-aquae)。
Ravn等人(1995)的出版物公开了据认为是从塔玛亚历山大藻(Alexandriumtamarense)的克隆中提取麻痹性贝类毒素的最佳条件。用浓度为0.01-1.0N的乙酸和盐酸提取麻痹性贝类毒素。观察到0.03-1.0N的盐酸浓度导致C1和C2毒素的量急剧下降，而膝沟藻毒素2(GTX2)和3(GTX3)的量随之增加。
Tsai等人(1997)的出版物公开了通过河豚毒素生物测定法对螃蟹标本进行麻痹毒性检测。毒素的部分纯化和鉴定表明，主要毒素是河豚毒素，其中含有少量的膝沟藻毒素(GTX)和新石房蛤毒素(neoSTX)。
Siu等人(1997)的出版物公开了环境和营养因素对链状亚历山大藻(Alexandriumcatenella)种群动态和毒素产量的影响的研究。公开了该鞭毛藻物种的最佳生长条件以及在这些条件下生长的物种的毒素谱。发现通过HPLC检测到的毒素谱包括降序排列的GTX4、GTX3、GTX1、B2、新石房蛤毒素(neoSTX)和石房蛤毒素(STX)。
Sato等人(2000)的出版物公开了将GTX1和GTX4的O-硫酸基转化为亚甲基以形成新石房蛤毒素。使用硫醇(诸如谷胱甘肽)实现转化，并分离了转化的中间体。
Parker等人(2002)的出版物公开了对有毒的鞭毛藻，微小亚历山大藻(Alexandrium minutum)在三种不同的高生物量培养系统中自养生长的研究，以评估其生长、产率和毒素产生。该生物体在曝气和非曝气的2升锥形烧瓶、0.5升玻璃充气管和4升实验室规模的牙槽板光生物反应器中生长。观察到响应曝气的生物量和产率显著增加。据报道，每周收获的最大细胞浓度为3.3x105个细胞/mL，平均产率为0.4x104个细胞/mL/天，且毒素产量约为20μg/L/天。
Baker等人(2003)的出版物公开了从很容易被误认为是膝沟藻毒素4(GTX4)的产生毒素的化合物中分离的细菌菌株的生产。
Miao等人(2004)的出版物公开了从微小亚历山大藻(Alexandrium minutumHalim)的两个菌株中分离膝沟藻毒素(GTX1、GTX2、GTX3和GTX4)。据称被称为Amtk4的微小亚历山大藻(Alexandrium minutum Halim)菌株适合于制备膝沟藻毒素。
Jiang和Jiang(2008)的出版物公开了从华贵栉孔扇贝(Chlamys nobilis)的性腺提取麻痹性贝类毒素的最佳条件的建立。萃取使用浓度为0.04-1.0mol/L的乙酸和盐酸。结果表明，使用浓度为0.25-1.0mol/L的盐酸会导致毒素C1、C2和GTX5显著减少，并伴随毒素GTX2,3的增加。萃取中使用乙酸时，三种不稳定毒素的量未显示任何变化。
Liu等人(2010)的出版物公开了在实验室中培养产生毒素的链状亚历山大藻。培养八天之内获得的最大细胞密度为0.4x104个细胞/mL。高效液相色谱(HPLC)分析的粗提物显示主要毒性成分为C1/2、GTX4、GTX5和neoSTX，其浓度分别约为0.04550、0.2526、0.3392、0.8275和0.1266μmol/L。
Foss等人(2012)的出版物公开了从丝状蓝藻Lyngbya wollei提取麻痹性贝类毒素(PST)的方法的比较。在缺乏由该蓝细菌产生的独特毒素的可商购标准品的情况下，不可能对提取的毒素进行定量。
Li等人(2013)的出版物公开了一种快速筛选和鉴定赤潮藻中的麻痹性贝类中毒(PSP)毒素的方法。该方法利用结合准确的质量数据库的亲水相互作用色谱-高分辨率质谱法(HILIC-HR-MS)。十种常见PSP化合物的检出极限(LOD)为10-80nmol/L。据称，开发的方法是快速筛选和定性鉴定有害藻类中常见PSP毒素的有用工具。
Bernardi Bif等人(2013)的出版物公开了海胆对含有石房蛤毒素的有毒细胞提取物的敏感性。
Poyer等人(2015)的出版物公开了开发一种分析方法以表征和区分石房蛤毒素类似物，包括硫酸化的(膝沟藻毒素)和未硫酸化的类似物。使用亲水相互作用液相色谱法(HILIC)分离硫酸化的类似物。离子迁移质谱(IM-MS)用作基于离子气相确认的新分离维度，以区分石房蛤毒素类似物。正和负离子化模式用于膝沟藻毒素。正离子化模式用于非硫酸化类似物。三种互补技术的耦合(HILIC-IM-MS)允许分离和鉴定石房蛤毒素类似物，在HILIC维度实现了异构体的区分，而在IM-MS维度实现了未硫酸化类似物的分离。
Rubio等人(2015)的出版物公开了一种使用基于离子对的液相色谱方法纯化石房蛤毒素的方法。使用盐酸提取石房蛤毒素，并在用三氯乙酸和己烷/乙醚(97/3)处理后，用硫酸铵处理。通过半制备性HPLC分析样品，以收集石房蛤毒素的纯级分，并将这些级分在固相阳离子交换STX萃取柱中洗脱。据报道，纯化的石房蛤毒素是稳定且均质的，并且通过LC-MS-MS证实了其身份。据报道，纯化的石房蛤毒素中不存在脱氨基甲酰基石房蛤毒素的类似物，诸如新石房蛤毒素。
Chen等人(2016)的出版物公开了反相液相色谱(RPLC)和亲水相互作用色谱(HILIC)结合高分辨率质谱(HR-MS)的串联耦合以同时筛选和鉴定有害赤潮(HAB)的藻类中已知的亲脂性和亲水性毒素。通过使用超声辅助提取(UAE)同时提取亲脂性和亲水性毒素。该出版物证明，HPLC/HILIC-HR-MS与已知海藻毒素的准确质量列表相结合可以用作筛选海洋有害藻类中不同类别已知毒素的有力工具。
Cho等人(2016)的出版物公开了使用亲水相互作用色谱仪(HILIC)结合质谱通过色谱柱切换和两步梯度洗脱法，分析了产毒素的鞭毛藻的粗提取物。该出版物指出，所获得的数据支持以下假设：在鞭毛藻和蓝藻中，石房蛤毒素生物合成和分流途径的早期阶段是相同的。
Beach等人(2018)的出版物公开了使用毛细管电泳(CE)-串联质谱(MS/MS)方法对海鲜中的麻痹性贝类毒素、河豚毒素和海藻酸进行灵敏的多类分析。包含5M甲酸的新型高酸性背景电解质用于最大化分析物的质子化，并且据称通常可用于同时分析具有不同pKa值的其他种类的极性小分子。
Kellmann and Neilan(2007)的出版物公开了重组大肠杆菌菌株中新石房蛤毒素及其类似物的发酵生产。
Lagos Gonzáles(2010、2015a、2015b和2016)的出版物公开了从连续培养产生的蓝藻中纯化藻毒素的方法。藻毒素主要分离自细菌，但是也可以分离自培养基。在所公开的方法的一个实施方案中，仅产生新石房蛤毒素(neoSTX)和石房蛤毒素(STX)。在所公开的方法的另一个实施方案中，仅产生膝沟藻毒素2(GTX2)和膝沟藻毒素3(GTX3)。
Wang等人(2010年)的出版物公开了麻痹性贝类毒素(PSP)标准溶液的制备。该标准溶液的制备方法是：从贝类材料中去除杂质，收集贝类的肉，添加蒸馏水和0.1-0.3mol/L盐酸溶液，将pH值调节至1.5-5.0，然后均质化以获得匀浆，在-20℃下预冷30分钟-24小时，然后冻干以获得核心样品，研磨并过筛，在-20℃下预冷却10分钟-6小时，并冻干以获得标准样品。据称该制备方法具有原料成本低、制备工艺简单的优点。
Xiong和Qiu(2009)的出版物公开了双胍基嘌呤衍生物及其盐和酯用于改进抗肿瘤药的治疗效果和减少副作用的应用。双胍嘌呤衍生物是石房蛤毒素类似物。
本发明的目的是提供制备足够量和足够纯度的新石房蛤毒素的方法，以使其能够用于制备药物制剂。该目的应被解读为替代至少提供有用的选择的目的。
发明内容
在第一方面，本发明提供制备一定量的neoSTX的方法，包括以下步骤：
1.使一定量的纯化GTX1,4和一定量的二硫醇在反应溶剂的溶液中接触一段时间并处于足以提供反应产物的温度下，其中将超过97.5％(w/w)的GTX1,4转化为neoSTX；和然后
2.将所述反应产物施加到基于二氧化硅的弱阳离子交换吸附剂上并用弱有机酸水溶液洗脱，以将所述neoSTX与所述二硫醇分离并提供所述量的neoSTX，
其中所述溶液的pH为7.4-7.6。
优选地，所述反应溶剂是缓冲乙酸水溶液。
优选地，所述二硫醇选自下组：二硫苏糖醇(DTT)和二硫丁胺(DTBA)。更优选地，所述二硫醇是二硫苏糖醇(DTT)。
优选地，neoSTX的量大于100mg，纯度大于99.5％(w/w)。
优选地，该量的GTX1,4的纯度为至少97.5％(w/w)。更优选地，该量的GTX1,4的纯度为至少98.75％(w/w)。更优选地，该量的GTX1,4的纯度为至少99％(w/w)。
优选地，制备一定量的neoSTX的方法是近定量方法。
本发明的方法提供了以一定量和之前无法获得的纯度分批制备neoSTX(参见Lagos Gonzáles(2010,2015a,2015b和2016))。
在本说明书的说明书和权利要求书中，下列缩写、首字母缩写词、短语和术语具有所提供的含义：“分批制备”是指不连续制备；“生物合成”是指在活生物体或细胞内制备；“CAS RN”是指化学文摘社(CAS，Columbus,Ohio)注册号；“包含”是指“包括”、“含有”或“其特征在于”，并且不排除任何其他要素、成分或步骤；“由……组成”是指排除未指定的任何要素、成分或步骤，杂质和其他附带物除外；“基本上由……组成”是指排除任何构成实质限制的要素、成分或步骤；“GTX”是指膝沟藻毒素；“GTX1”是指膝沟藻毒素1[CAS RN 60748-39-2]；“GTX4”是指膝沟藻毒素4[CAS RN 64296-26-0]；“GTX1,4”是指包含膝沟藻毒素1和膝沟藻毒素4的未溶解的混合物(作为固体或在溶液中)；“GTX2,3”是指包含膝沟藻毒素2和膝沟藻毒素3的未溶解的混合物(作为固体或在溶液中)；“近定量”是指将大于97.5％(w/w)的底物(例如GTX1,4)转化为产物，例如neoSTX；“NeoSTX”是指(3aS,4R,10aS)-2-氨基-4-[[(氨基羰基)氧基]甲基]-3a,4,5,6,8,9-六氢-5-羟基-6-亚氨基-1H,10H-吡咯并[1,2-c]嘌呤-10,10-二醇[CAS RN 64296-20-4]；“制备性规模”是指以大于100mg的批次制备；“半合成”是指通过化学转化至少部分纯化的生物合成前体而制备。任何定义的术语的同义词都具有相应的含义。
参照发明内容和权利要求书中限定的事项的要素、特征或整数，或者参照本发明的替代实施方案，术语“第一”、“第二”、“第三”等并非意在暗示优先顺序。指定试剂的浓度或比例时，指定的浓度或比例为试剂的初始浓度或比例。如果值以一个或多个小数位表示，则采用标准舍入法。例如，1.7涵盖1.650循环-1.749循环的范围。分离的neoSTX的纯度根据方法3[F.分析]确定。
现在将参考以下实施例和随附附图页面的图来描述本发明。
附图简要说明
图1.膝沟藻毒素(GTX 1(◆)和GTX 4(■))浓度与时间的图。
具体实施方式
Laycock等人(1994)的出版物公开了从扇贝(Placopecten magallanicus)的肝胰腺提取和纯化GTX1和GTX4：
使用Polytron组织匀浆器(型号PT10/35，Brinkman Instruments Canada Ltd，Rexdale，ON)在1L的0.1M HCl中匀浆组织(1kg)。将浆液加热至80℃30分钟，然后冷却并离心(5,000g，20分钟)以去除沉淀的蛋白质。将上清液流体用二氯甲烷萃取两次(每次500ml)。通过旋转蒸发将水层浓缩至200ml，然后倒入活性炭(Norite,A,500g,BDH Ltd.)和硅藻土(500g,Johns-Manville)混合物的柱(10cm ID x 15cm)上。用20％乙醇和1％乙酸的溶液洗涤该柱。收集几个一升级分，并通过HPLC-FD监测毒素浓度。通过旋转蒸发浓缩含毒素的级分并冻干。
该出版物进一步公开了对于任何膝沟藻毒素，Bio-Rex-70的分离并不完全。但是，通过反复去除GTX2并重新平衡混合物，逐渐降低了污染GTX1和GTX4级分的GTX2和GTX3的比例。
Laycock等人(1995)的出版物公开了在pH 8.5的水溶液中浓度为100mM的二硫苏糖醇迅速将GTX1,4转化为NeoSTX和少量(少于10％)的新石房蛤毒素(通过毛细管电泳测定)。相比之下，现已确定，当使用二硫苏糖醇(DTT)作为还原剂时，当以制备性(而不是分析性)规模进行转化时，最佳pH值较低且范围为7.2-7.8，更优选7.4-7.6。
在溶液中，GTX1和GTX4被认为是一对差向异构体，其中GTX1在热力学上最受青睐。差向异构被认为在大多数条件下经由在C-12处的酮-烯醇平衡发生。在根据方案I的假定的两步反应机理的第一步中，二硫醇(R-SH)的硫醇基团攻击酮型(I)的亲电子C-12以形成硫代半缩酮(II)。当离去基团(O-硫酸盐)相对于硫原子定向时(如在反应性差向异构体GTX1中)，经由表硫鎓离子中间体(III)转化为硫醚(IV)。在提出的反应机理的第二步中，二硫醇的硫醇基与硫醚(IV)的硫反应形成二硫键，从而生成易于水合为neoSTX(V)的烯醇化物。
Figure BDA0002949104050000091
在以下实施例中描述的用于将GTX1,4转化为neoSTX的最佳pH已经确定为约7.5，并且不希望受到理论的束缚，据信该pH确保(i)膝沟藻毒素差向异构体之间最佳的差向异构速率，以及(ii)C-12处的亲电性以及用作还原剂的二硫醇的去质子化的最佳程度。与使用一硫醇(诸如谷胱甘肽(GSH)和巯基乙醇(ME))相比，优选使用二硫醇(诸如二硫苏糖醇(DTT)和二硫丁胺(DTBA))(参见Sakamoto et al(2000)和Sato et al(2000))。使用二硫醇时可获得较高的转化率，使其更适合用于制备性规模的neoSTX生产。
已经发现，通过使用阳离子交换色谱，可以最方便地从含有neoSTX的转化产物中去除过量的二硫醇、磷酸钠缓冲液和未反应的GTX1,4。基于二氧化硅的弱阳离子交换吸附剂SepraTM WCX被确定为合适的吸附剂，因为已确定其不保留DTT。基于聚合的弱阳离子交换吸附剂Strata-XTM CW(Phenomenex)的试验确定该吸附剂不适合以制备性规模从转化产物中纯化neoSTX。当使用这种吸附剂时，过量的二硫醇通过离子交换和反相机理被保留。尽管一部分过量的DTT被有机溶剂(诸如乙腈/水)洗脱，但是另一部分被1M乙酸洗脱，这阻碍了使用该吸附剂的neoSTX的纯化。
A.分析
将GTX1,4或neoSTX样品在10mM乙酸中稀释至200μg/mL的浓度。然后，将稀释的样品在8％乙腈/0.25％乙酸中进一步稀释100倍，以提供浓度为20mg/mL的产物溶液用于定量分析。还可以在相同溶剂中制备含有许多参考麻痹性贝类毒素(PST)的混合标准品。通过保持在40℃的自动进样器将2μL稀释产物的溶液(20ng/mL)注入到1.7μmWaters AcquityUPLC BEH酰胺柱(2.1x100mm)上，以0.6mL/min的流速洗脱，同时保持在60℃的温度下。进样后，将柱用80％流动相B/20％流动相C逐步洗脱6分钟，然后再用55％流动相B/45％流动相C洗脱0.50分钟，然后恢复为80％流动相B/20％流动相C。通过质谱监测以ESI-和ESI+电离模式监测洗脱液。
B.转化
将183mg(作为游离碱)的量的GTX1,4溶于总体积为5mL的稀乙酸中，并在100mL圆底烧瓶中与pH值为7.5的体积为45mL的0.2M磷酸盐缓冲液混合。将混合物置于冰上，并在搅拌的同时添加固体碳酸钠将pH从6.8调节至7.5。将1.5g二硫苏糖醇(DTT)的量添加至pH调节的混合物中，并且通过将含有反应混合物的圆底烧瓶置于超声浴中来促进其溶解，然后转移至保持在50℃的温度的水浴中。从反应混合物中取出体积为10μL的等分试样，转移到水浴中(T＝0)，然后定期(每15分钟)转移。从反应混合物中移出后，立即通过添加490μL80％乙腈、0.25％乙酸来稀释等分试样50倍，并按照(A.分析)所述进行LC-MS分析，以近实时监测反应进程(图1)。在50℃下孵育45分钟后，通过将圆底烧瓶转移至冰浆中来冷却反应混合物。在这些条件下，观察到GTX1,4近定量转化为neoSTX，收率接近100％。
C.分离
将该转化产物加载到装在空闪蒸滤芯(Grace)中的39g SepraTM的量的WCX上，并先以体积为250mL的50％(w/w)乙腈、然后再以体积为250mL的去离子水进行预处理。用去离子水洗涤将转化产物加载到装好的滤芯中，并收集流出物(约200mL)。通过添加最少量的去离子水，实现了在4℃下储存转化产物过程中形成的所有晶体的溶解。然后，以50mL/min的速度用1.5L的总体积洗脱加载的填充滤芯，然后在20分钟内以连续梯度洗脱至1M乙酸，并顺次收集体积为10mL洗脱液作为级分，同时监控205nm和254nm的紫外线吸收。
在80％的乙腈、0.25％的乙酸中将证明在205nm处的紫外线吸收的体积为5μL的级分稀释100,000倍，并通过LC-MS分析。将确认包含neoSTX的级分合并，在-70℃下冷冻并冻干。将干燥的neoSTX溶解为10mM的小体积，并转移到预先称重的10mL玻璃小瓶中，并分析10μL的体积。表1提供了根据上述方法制备的一批(CNC00063)neoSTX的纯度和数量。
成分 量(mg) ％(w/w)
neoSTX 118 99.58
L-精氨酸 0.444 0.37
STX 0.0546 0.05
DTT <0.005 <0.004
总计 119 100
表1.根据所述的半合成方法制备的一批(CNC00063)neoSTX的规格。
尽管已经参考实施方案或实施例描述了本发明，但是应当理解，可以在不脱离本发明的范围的情况下对这些实施方案或实施例进行变化和修改。在特定要素、特征或整数存在已知等同物的情况下，将这些等同物并入，就如同在本说明书中特别提及一样。除非特别声明，对包括在参考出版物中公开的和从参考出版物中选择的要素、特征或整数的实施方案或实施例的变型和修改都在本发明的范围内。在本发明的这些不同实施方案中，可以替代地或组合地提供由本发明提供并在说明书中讨论的优点。
出版物
Baker et al(2003)GTX4 imposters:characterization of fluorescentcompounds synthesized by Pseudomonas stutzeri SF/PS and Pseudomonas/Alteromonas PTB-1,symbionts of saxitoxin-producing Alexandrium spp.Toxicon,41(3),339-347.
Beach et al(2018)Capillary electrophoresis-tandem mass spectrometryfor multiclass analysis of polar marine toxins Analytical and BioanalyticalChemistry,410(22),5405-5420.
Bernardi Bif et al(2013)Evaluation of mysids and sea urchins exposedto saxitoxins Environmental Toxicology and Pharmacology,36(3),819-825.
Chen et al(2016)Simultaneous screening for lipophilic and hydrophilictoxins in marine harmful algae using a serially coupled reversed-phase andhydrophilicinteraction liquid chromatography separation system with high-resolution mass spectrometry Analytica Chimica Acta,914,117-126.
Cho et al(2016)Column switching combined with hydrophilic interactionchromatography-tandem mass spectrometry for the analysis of saxitoxinanalogues,and their biosynthetic intermediates in dinoflagellates Journal ofChromatography A,1474,109-120.
Daigo et al(1985)Isolation and some properties of neosaxitoxin from axanthid crab Zosimus aeneus Nippon Suisan Gakkaishi,51(2),309-13.
Foss et al(2012)Investigation of extraction and analysis techniquesfor Lyngbya wollei derived Paralytic Shellfish Toxins Toxicon,60(6),1148-1158.
Garcia-Altares(2017)Structural diversity of microalgal marine toxinsComprehensive Analytical Chemistry,78,35-75.
Hall et al(1984)Dinoflagellate neurotoxins related to saxitoxin:structures of toxins C3 and C4,and confirmation of the structure ofneosaxitoxin Tetrahedron Letters,25(33),3537-8.
Jiang and Jiang(2008)Investigation of extraction method for paralyticshellfish poisoning toxins in shellfish Fenxi Huaxue,36(11),1460-1464.
Kellmann and Neilan(2007)Fermentative production of neosaxitoxin andits analogs in recombinant Escherichia coli strains International applicationno.PCT/EP2017/053077[publ.no.WO 2017/137606 A1].
Kohane et al(2000)The local anesthetic properties and toxicity ofsaxitoxin homologues for rat sciatic nerve block in vivo Regional Anesthesiaand Pain Medicine,25,1,52-59.
Lagos Gonzáles(2010)Methods for purifying phycotoxins,pharmaceuticalcompositions containing purified phycotoxins,and methods of use thereofInternational application nos.PCT/IB2010/051187[publ.no.WO 2010/109386 A1]andPCT/IB2010/051188[publ.no.WO 2010/109387 A1].
Lagos Gonzáles(2015a)Methods for producing phycotoxins United Statespatent no.8,957,207 B2.
Lagos Gonzáles(2015b)Methods for purifying phycotoxins,pharmaceuticalcompositions containing purified phycotoxins and methods of use thereofUnited States patent no.8,952,152 B2.
Lagos Gonzáles(2016)Methods for purifying phycotoxins,pharmaceuticalcompositions containing purified phycotoxins and methods of use thereofUnited States patent no.9,249,150 B2.
Laycock et al(1994)Isolation and purification procedures for thepreparation of paralytic shellfish poisoning toxin standards Natural Toxins,2(4),175-83.
Laycock et al(1995)Some in vitro chemical interconversions ofparalytic shellfish poisoning(PSP)toxins useful in the preparation ofanalytical standards Journal of Marine Biotechnology(Proceedings of the ThirdInternational Marine Biotechnology Conference,1994),3(1-3),121-125.
Li et al(2013)Rapid screening,identification of paralytic shellfishpoisoning toxins in red tide algae using hydrophilic interactionchromatography-high resolution mass spectrometry with an accurate-massdatabase Fenxi Huaxue,41(7),979-985.
Liu et al(2010)Cultivation of Alexandrium catenella and extractionand detection of paralytic shellfish poisoning toxins Shuichan Xuebao,34(11),1783-1788.
Mezher(2018)FDA to replace analgesic drug development guidance withnew documents Regulatory Focus News Article(www.raps.org/news-and-articles/news-articles/2018/8/fda-2-replace-analgesic-drug-development-guidance).
Miao et al(2004)Isolation and purification of gonyautoxins from twostrain of Alexandrium minutum Halim Journal of Chinese PharmaceuticalSciences,13(2),103-105.
Parker et al(2002)Growth of the toxic dinoflagellate Alexandriumminutum(Dinophyceae)using high biomass culture systems Journal of AppliedPhycology,14(5),313-324.
Poyer et al(2015)Identification and separation of saxitoxins usinghydrophilic interaction liquid chromatography coupled to traveling wave ionmobility-mass spectrometry Journal of Mass Spectrometry,50(1),175-181.
Ravn et al(1995)Standardized extraction method for paralyticshellfish toxins in phytoplankton Journal of Applied Phycology,7(6),589-94.
Rogriguez-Navarro et al(2011)Comparison of neosaxitoxin versusbupivacaine via port infiltration for postoperative analgesia followinglaparoscopic cholecystectomy,a randomized,double-blind trial RegionalAnesthesia and Pain Medicine,36,2,pp.103.
Rubio et al(2015)Purification and characterization of saxitoxin fromMytilus chilensis of southern Chile Toxicon,108,147-153.
Rutman et al(2011)Treatment of loss of sense of touch with saxitoxinderivatives International application no.PCT/EP2011/051992[Publ.no.WO 2011/098539 A1].
Rutman et al(2013)Paralytic shellfish poison Internationalapplication no.PCT/EP2013/055448[Publ.no.WO 2013/135884 A1].
Sakamoto et al(2000)Formation of intermediate conjugates in thereductive transformation of gonyautoxins to saxitoxins by thiol compoundsFisheries Science,66,136-141.
Sato et al(2000)Identification of thioether intermediates in thereductive transformation of gonyautoxins into saxitoxins by thiols Bioorganicand Medicinal Chemistry Letters 10,1787-1789.
Siu et al(1997)Environmental and nutritional factors which regulatepopulation dynamics and toxin production in the dinoflagellate Alexandriumcatenella Hydrobiologia,352,117-140.
Templin et al(2015)Improved therapeutic index using combinations withbupivacaine,with and without epinephrine Anesthesiology,123,4,886.
Tsai et al(1997)Toxicity and toxic components of two xanthid crabs,Atergatis floridus and Demania reynaudi,of Taiwan Toxicon,35(8),1327-1335.
Wang et al(2010)Paralytic shellfish poison or PSP standard substancesand preparations for monitoring sea food industries Chinese patentapplication no.2010-10179534[publ.no.CN 101858833 A].
Wylie et al(2012)Respiratory,neuromuscular,and cardiovascular effectsof neosaxitoxin in isoflurane-anesthetized sheep Regional Anesthesia and PainMedicine,37,2,pp.103.
Xiong and Qui(2009)Application of biguanido purine derivatives forimproving therapeutic effect and reducing side effect of antitumor agentsChinese patent application no.2008-10007215[publ.no CN101513408].

Claims (5)

1.制备一定量的neoSTX的方法，包括以下步骤：
(a)使一定量的纯化GTX1,4和一定量的二硫醇在反应溶剂的溶液中接触一段时间并处于足以提供反应产物的温度下，其中将超过97.5％(w/w)的GTX1,4转化为neoSTX；和然后
(b)将所述反应产物施加到基于二氧化硅的弱阳离子交换吸附剂上并用弱有机酸水溶液洗脱，以将所述neoSTX与所述二硫醇分离并提供所述量的neoSTX，
其中所述溶液的pH为7.4-7.6。
2.权利要求1所述的方法，其中所述反应溶剂是缓冲乙酸水溶液。
3.权利要求1或2所述的方法，其中所述二硫醇选自下组：二硫苏糖醇(DTT)和二硫丁胺(DTBA)。
4.权利要求1所述的方法，其中所述二硫醇是二硫苏糖醇(DTT)。
5.根据前述权利要求中任一项的所述方法制备的一定量的neoSTX，其纯度大于99.5％(w/w)，其中所述量大于100mg。
CN201980055613.6A 2018-09-21 2019-09-23 将膝沟藻毒素制备性规模转化为新石房蛤毒素 Active CN113166164B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202410100344.3A CN117964629A (zh) 2018-09-21 2019-09-23 将膝沟藻毒素制备性规模转化为新石房蛤毒素

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
IBPCT/IB2018/057274 2018-09-21
PCT/IB2018/057274 WO2020058750A1 (en) 2018-09-21 2018-09-21 A semisynthetic method of preparing neosaxitoxin
PCT/IB2019/058019 WO2020058949A1 (en) 2018-09-21 2019-09-23 Preparative scale conversion of gonyautoxins to neosaxitoxin

Related Child Applications (1)

Application Number Title Priority Date Filing Date
CN202410100344.3A Division CN117964629A (zh) 2018-09-21 2019-09-23 将膝沟藻毒素制备性规模转化为新石房蛤毒素

Publications (2)

Publication Number Publication Date
CN113166164A true CN113166164A (zh) 2021-07-23
CN113166164B CN113166164B (zh) 2024-01-30

Family

ID=69888393

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201980055613.6A Active CN113166164B (zh) 2018-09-21 2019-09-23 将膝沟藻毒素制备性规模转化为新石房蛤毒素

Country Status (8)

Country Link
US (2) US10822344B2 (zh)
JP (1) JP7110403B2 (zh)
CN (1) CN113166164B (zh)
CA (1) CA3113727A1 (zh)
CL (1) CL2020003015A1 (zh)
GB (1) GB2587997B (zh)
SG (1) SG11202102830SA (zh)
WO (2) WO2020058750A1 (zh)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020058750A1 (en) * 2018-09-21 2020-03-26 The Cawthron Institute Trust Board A semisynthetic method of preparing neosaxitoxin
US12018034B2 (en) 2018-09-21 2024-06-25 The Cawthron Institute Trust Board Semisynthetic methods of preparing GTX1,4 and neosaxitoxin
CN112461913B (zh) * 2020-11-06 2022-02-22 华南农业大学 一种提高同分异构体化合物鉴别能力的方法
FR3130842A1 (fr) 2021-12-22 2023-06-23 CarbonWorks Procede de captation des phytotoxines dans un reacteur biologique

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2412714B1 (en) * 2009-03-24 2015-11-11 Proteus S.a. Method for the industrial purification of biologically active phycotoxins
US9249150B2 (en) * 2009-03-24 2016-02-02 Proteus S.A. Methods for purifying phycotoxins, pharmaceutical compositions containing purified phycotoxins, and methods of use thereof

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003012699A (ja) * 2001-07-04 2003-01-15 Japan Science & Technology Corp 抗麻酔性貝毒抗体の製法、新規抗体、該抗体を用いるｅｌｉｓａ測定キット、該製法による系標識毒標品
JP2006098293A (ja) * 2004-09-30 2006-04-13 Japan Science & Technology Agency 麻痺性貝毒成分の変換方法
JP6422814B2 (ja) * 2015-04-16 2018-11-14 学校法人北里研究所 デカルバモイルサキシトキシン及びその類縁体の製造方法
WO2020058750A1 (en) * 2018-09-21 2020-03-26 The Cawthron Institute Trust Board A semisynthetic method of preparing neosaxitoxin

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2412714B1 (en) * 2009-03-24 2015-11-11 Proteus S.a. Method for the industrial purification of biologically active phycotoxins
US9249150B2 (en) * 2009-03-24 2016-02-02 Proteus S.A. Methods for purifying phycotoxins, pharmaceutical compositions containing purified phycotoxins, and methods of use thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LINCOLIN MACKENZIE A.等: "ICHA New Zealand marine and fresh-water harmful algae 2014", 《INTERNATIONAL CONFERENCE ON HARMFUL ALGAE》 *
MAURICE V.LAYCOK等: "Some in vitro chemical interconversions of paralytic shellfish poisoning (PSP) toxins useful in the preparation of analytical standards", 《J MAR BIOTECHNOL》 *
SETSUKO SAKAMOTO等: "Formation of intermediate conjugates in the reductive transformation of gonyautoxins to saxitoxins by thiol compounds", 《FISHERIES SCIENCE》 *
SHIGERU SATO,等: "Identification of Thioether Intermediates in the Reductive Transformation of Gonyautoxins into Saxitoxins by Thiols", 《BIOORGANIC & MEDICINAL CHEMISTRY》 *

Also Published As

Publication number Publication date
US10822344B2 (en) 2020-11-03
US20200255441A1 (en) 2020-08-13
SG11202102830SA (en) 2021-04-29
US20200407367A1 (en) 2020-12-31
GB2587997B (en) 2023-05-10
GB2587997A (en) 2021-04-14
CN113166164B (zh) 2024-01-30
JP7110403B2 (ja) 2022-08-01
JP2021526539A (ja) 2021-10-07
CL2020003015A1 (es) 2021-06-25
WO2020058750A1 (en) 2020-03-26
CA3113727A1 (en) 2020-03-26
GB202018189D0 (en) 2021-01-06
US11028094B2 (en) 2021-06-08
WO2020058949A1 (en) 2020-03-26

Similar Documents

Publication Publication Date Title
CN113166164B (zh) 将膝沟藻毒素制备性规模转化为新石房蛤毒素
JP5728774B2 (ja) 生物学的に活性な藻類毒素の工業的精製法
RU2743715C1 (ru) Способ получения целастрола и пентациклических тритерпеновых производных
EP3077522A1 (en) Compositions and methods for antibody production
Loader et al. Convenient large-scale purification of yessotoxin from Protoceratium reticulatum culture and isolation of a novel furanoyessotoxin
US20230145895A1 (en) Preparation of neosaxitoxin
CN117964629A (zh) 将膝沟藻毒素制备性规模转化为新石房蛤毒素
AU2020202129A1 (en) Preparative scale conversion of gonyautoxins to neosaxitoxin
US12018034B2 (en) Semisynthetic methods of preparing GTX1,4 and neosaxitoxin
Mosin et al. Studying the influence of heavy and deuterium depleted types of water on biological objects
DE60216616T2 (de) Enzymatisches verfahren zur herstellung von organofluorverbindungen
Hakobyan et al. Synthesis of (S)-alanyl-(S)-β-(thiazol-2-yl-carbamoyl)-α-alanine, dipeptide containing it and in vitro investigation of the antifungal activity
Mosin et al. Research the Influence of Deuterium Depleted and Heavy Types of Water on Biological Objects
CN107949567B (zh) 三种芋螺毒素肽、其制备方法及应用
JPS6253157B2 (zh)
Desjardine B. Sc. The University of Manitoba, 2001
CN103374057A (zh) 一种对甘氨酸转运子具有抑制活性的新化合物

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