CN109195996A - N-聚醣及其阵列的模组化合成方法 - Google Patents
N-聚醣及其阵列的模组化合成方法 Download PDFInfo
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- CN109195996A CN109195996A CN201780027439.5A CN201780027439A CN109195996A CN 109195996 A CN109195996 A CN 109195996A CN 201780027439 A CN201780027439 A CN 201780027439A CN 109195996 A CN109195996 A CN 109195996A
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- glycan
- base
- benzyl
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- 125000004797 2,2,2-trichloroethoxy group Chemical group ClC(CO*)(Cl)Cl 0.000 claims description 36
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Abstract
本发明涉及用于产生多样化高甘露糖型、混合型及复合型N‑聚醣的新颖模组化方法。本发明亦关于点样至涂有氧化铝的载片上的合成的N‑聚醣的例示性阵列。这些阵列可用以侦测及分析这些合成的N‑聚醣与诸如HIV‑1中和抗体的聚醣结合分子之间的结合相互作用。本发明亦关于用于鉴别结合至这些阵列上的各种类型的分子的试剂的方法及关于限定这些阵列上的这些分子的结合至那些试剂的结构要素。本文所提供的阵列及方法可以用于一般的抗原决定基鉴别、药物发现及用作分析工具。本发明亦提供适用于侦测、诊断、复发监测及预防诸如HIV的病理性疾病的适用的聚醣及抗原决定基决定子。
Description
相关申请案
本申请主张于2016年3月8日申请的美国临时专利申请案案号62/305,441的优先权。其内容以全文引用方式并入本文。
技术领域
本发明大体上关于用于合成高甘露糖型、混合型及复合型高度多样化N-聚醣的化学及化学酶促方法、由这些聚醣制成的阵列及这些阵列的用途。特定言之,本发明涉及使用新颖的模组化N-聚醣合成途径在涂有氧化铝的基板上合成N-聚醣阵列的方法,及这些N-聚醣阵列于侦测及分析诸如广谱中和HIV抗体的聚醣结合分子的结合反应的用途。
背景技术
蛋白质的N-连接型醣基化对于在真核生物及原核生物两者中所发现的转译后修饰而言均为基本的且重要的。此蛋白质修饰使寡醣共价连接至多肽链的天冬酰胺残基上。聚醣通常为细胞与其环境之间最重要的界面。作为所有生命***的重要组成,聚醣参与大部分必需的生物过程,诸如蛋白质折迭、细胞信号传导、受精、胚胎发生以及细胞增殖及其组织成特定组织。异常的细胞表面醣基化及/或聚醣图谱通常与诸如癌症及动脉粥样硬化的疾病相关。因此,醣基化改变在人类病理学发展过程中可能是早期关键点的指示。由此深刻理解了碳水化合物相关的生物及病理过程,且需要发展针对人类疾病的诊断学及治疗学。
复合寡醣的生物合成一般复杂性及多样性极大,主要是因为聚醣构建块(单醣)的可变的且多重的连接性。这些鉴别细胞的醣基化分子包括醣蛋白及醣脂,且由诸如凝集素、抗聚醣抗体、化合物的各种聚醣结合分子以及其他聚醣及醣脂等特异性识别。然而,这些相互作用的复杂性极大且缺少定义明确的聚醣库及分析方法已被证实为醣体学发展的主要障碍。此外,天然产生的聚醣通常以极少的量分离且仅以异构体的混合物形式存在。因此,这些天然产生的聚醣的有限的可获得性及有限的纯度使其无法作为能充分表征的寡醣的可靠来源。因此,新颖的合成方法适用于制备用于生物及结构应用的多样化聚醣库。
核苷酸及蛋白质微阵列的发展澈底改变了基因组、基因表现及蛋白质体研究。类似地,聚醣微阵列的发展允许以前所未有的高产量探索各种各样的聚醣结合分子的特异性。聚醣在阵列上的***性排列允许经由多价呈现来有效探测低亲和力蛋白质-聚醣相互作用。自从阵列的建立以来,已研发出各种类型的“阵列”,包括可自功能性醣体学联盟(Consortium of Functional Glycomics;CFG)获得的阵列,其在N-羟基丁二酰亚胺(NHS)活化的玻璃载片上含有超过600个寡醣。然而,不同阵列版本中所用的间隔基及固定化学无疑导致聚醣呈现的密度、分布及位向方面的差异,其显著影响聚醣-蛋白质相互作用的结合亲和力且甚至影响特异性。因此,在不同的阵列平台中交叉比较且研发新的聚醣阵列以增进例如杂配位体结合的侦测的灵敏度尤为重要。此外,医药公司及研究机构将大大受益于用于各种筛选及药物发现应用的聚醣阵列,包括便于分析聚醣中有助于与聚醣结合分子结合的结构要素的阵列,这些聚醣结合分子包括抗体、受体及其他生物分子。
发明内容
本发明提供制备高甘露糖型、混合型及复合型多样化N-连接型寡醣(N-聚醣)的组合物及方法。本发明方法包括化学合成及/或酶促合成。在某些实施例中,将这些合成的N-聚醣中的一或多者点样至基板上的指定区域(称为“斑点(spot)”)中,其中将这些斑点中的一或多者合并,产生N-聚醣阵列,其用于由诸如抗体及病毒的聚醣结合分子识别的最佳聚醣的例如结合图谱分析、快速筛选及鉴别。
在一个态样中,本发明提供如下方法,其中称为“D1及D2/D3臂模组”的战略上经保护的构建块可在经正交保护的Manβ1-4GlcNAcβ1-4GlcNAcβ1核心的3-O及/或6-O位置处发生区域及立体特异性醣基化。在某些实施例中,可以脱除所得聚醣的保护基及/或去掩蔽以便得到最终寡醣产物,或者在某些实施例中,可使其以酶促方式选择性延长,得到最终复合寡醣产物。在某些实施例中,最终寡醣产物在还原端具有胺基烷基连接基团以便恢复其结构完整性及充当用于固定在阵列上的手柄,或者在某些其他实施例中,这些产物或可结合至载体蛋白。
在一个态样中,较佳的经正交保护的Manβ1-4GlcNAcβ1-4GlcNAcβ1核心三醣含有至少两个正交保护基,且具有式(I)
其中R1、R2、R3、R4、R5、R6、R7及R8中的每一者独立地选自由正交或永久保护基组成的群;其中,在R6处的正交保护基中的每一者较佳选自对甲氧基苄基醚(PMB)、甲氧基苯基醚(PMP)、乙酰丙酰基(Lev)、苄酰基(Bz)、烯丙醚(烯丙基)及硅烷基醚;且R7及R8稠合形成亚苄基环,其可以裂解形成4-OH及/或6-OH;
R2、R3、R4及R5中的每一者独立地为在用于移除正交保护基的条件下稳定且较佳为苄基(Bn)或乙酰基(Ac)的永久保护基;
R1为在用于移除正交保护基的条件下稳定且较佳为苄基(Bn)或乙酰基(Ac)的永久保护基;或经由α1,6键联连接的经保护的海藻糖苷残基;
X为-OR9;其中R9为-H或经由-N3或NR10组成的基团封端的视情况经取代的C3-C10烷基链;且R10为-H或苄基(Bn)或苄氧羰基(Cbz);
Y及Z为-NHR11;且R11较佳选自由以下组成的群:9-茀基甲氧基羰基(Fmoc)、烯丙氧基羰基(Alloc)、[2,2,2-三氯乙氧基羰基](Troc)、乙酰基(Ac)、邻苯二酰亚胺基(Phth)、苄氧羰基(Cbz)或第三丁氧羰基(Boc)。
如本文所用,“永久保护基”可为当暴露于通常将使官能基官能化的条件中时阻止所述官能基官能化的化学部分。可以选择性移除永久保护基以暴露出所得分子中的官能基。在一些态样中,暴露的官能基可以进一步与另一部分反应,或可以保持未官能化。
在第二态样中,较佳的D1及D2/D3臂模组具有通式(II)
其中,R1、R2、R3及R4中的每一者为-H;或在用于移除正交保护基的条件下稳定且较佳为苄基(Bn)或乙酰基(Ac)或苄酰基(Bz)的永久保护基;或独立地选自
其中,R5、R6、R7及R8中的每一者为-H或独立地为在用于移除正交保护基的条件下稳定且较佳为苄基(Bn)或乙酰基(Ac)或苄酰基(Bz)的永久保护基;
R9及R10中的每一者为-H或独立地为在用于移除正交保护基的条件下稳定且较佳为苄基(Bn)或乙酰基(Ac)或苄酰基(Bz)的永久保护基;或经由α1,3键联连接至GlcNAc及/或经由α1,2键联连接至半乳糖的经保护的海藻糖苷残基;
R11为-H或Me、Et;
R12为-H;或独立地为在用于移除正交保护基的条件下稳定且较佳为苄基(Bn)或乙酰基(Ac)或苄酰基(Bz)的永久保护基;或经由α2,6键联连接的经保护的Neu5Ac残基;
R13为-H;或独立地为在用于移除正交保护基的条件下稳定且较佳为苄基(Bn)或乙酰基(Ac)或苄酰基(Bz)的永久保护基;或独立地选自
X为-OR14或-SR15
其中,R14为H、烷基、烯基、炔基、芳基或经取代的芳基;保护基,诸如硅烷基或经取代的硅烷基,较佳为第三己基二甲基硅烷基(TDS)、第三丁基二甲基硅烷基(TBS)、第三丁基二苯基硅烷基(TBDPS)、三异丙基硅烷基(TIPS)、三甲基硅烷基(TMS)或三乙基硅烷基(TES);甲基(Me)、乙酰基(Ac)、苄基(Bn)、2-萘甲基(Nap)或1-萘甲基(1-Nap);对甲氧基苄基醚(PMB)、甲氧基苯基醚(PMP)、烯丙醚(烯丙基);或变旋异构离去基,诸如氟基-F、三氯乙酰亚胺酸酯基-C(NH)-CCl3、苯基三氟乙酰亚胺酯基-C(NPh)-CF3、三氟乙酰亚胺酯基-C(NH)-CF3;硫烷基、苯硫基;
R15为H、烷基、芳基或经取代的芳基,较佳为甲基、乙基、苯基、甲苯磺酰基或甲苯基。
Y为-NHR16;且R16较佳选自由以下组成的群:9-茀基甲氧基羰基(Fmoc)、烯丙氧基羰基(Alloc)、[2,2,2-三氯乙氧基羰基](Troc)、乙酰基(Ac)、邻苯二酰亚胺基(Phth)、苄氧羰基(Cbz)或第三丁氧羰基(Boc)。
根据所述第二态样,通式(II)的D1及D2/D3臂模组以化学方式或较佳以化学酶促方式制备。
在所述第二态样的另一实施例中,以化学方式合成的D1及D2/D3臂模组的范例选自由以下组成的群:
在所述第二态样的另一实施例中,D1及D2/D3臂模组的化学酶促合成从以化学方式制备的式(III)接受体受质开始
其中,
R1及R2中的每一者独立地选自-H或保护基乙酰基(Ac)或苄酰基(Bz);
X为-OR3且R3为H、苄基(Bn)、2-萘甲基(Nap)或1-萘甲基(1-Nap);对甲氧基苄基醚(PMB)、甲氧基苯基醚(PMP)、烯丙醚(烯丙基)。
在所述第二态样的另一实施例中,以化学酶促方式制备的式(III)接受体受质经历酶促延长,其中酶独立地选自由以下组成的群:β(1→4)半乳醣苷基转移酶、α(1→3)海藻醣苷基转移酶、α(1→2)海藻醣苷基转移酶、β(1→3)N-乙酰基葡糖胺转移酶、α(2→3)唾液酸转移酶及α(2→6)唾液酸转移酶。
在另一实施例中,以化学酶促方式制备的式(II)模组可以转化成其乙酸酯或苯甲酸酯形式。
在另一实施例中,完全乙酰化或苄酰化的式(II)模组可以经历还原端修饰以形成变旋异构离去基,诸如三氯乙酰亚胺酸酯基-C(NH)-CCl3、苯基三氟乙酰亚胺酯基-C(NPh)-CF3、三氟乙酰亚胺酯基-C(NH)-CF3;硫烷基、苯硫基,且更佳为氟基-F。
在第三态样中,以化学方式或以化学酶促方式制备的式(II)的D1及D2/D3臂模组经独立地选择以便在式(I)的Manβ1-4GlcNAcβ1-4GlcNAcβ1核心三醣的3-O及/或6-O位置处化学醣基化。
本发明亦提供一种经正交保护的Manβ1-4GlcNAcβ1核心,其含有至少两个正交保护基,且具有式(IV)
其中R1、R2、R3、R4、R5及R6中的每一者独立地选自由正交或永久保护基组成的群;其中,在R4处的正交保护基中的每一者较佳选自对甲氧基苄基醚(PMB)、甲氧基苯基醚(PMP)、乙酰丙酰基(Lev)、苄酰基(Bz)、烯丙醚(烯丙基)及硅烷基醚;且R5及R6稠合形成亚苄基环,其可以裂解形成4-OH及/或6-OH;
R1、R2及R3中的每一者独立地为在用于移除正交保护基的条件下稳定且较佳为苄基(Bn)或乙酰基(Ac)的永久保护基;
X为-OR7;其中R7为-H或苄基(Bn)或烯丙基(All);
Y为-NHR8;且R8较佳选自由以下组成的群:9-茀基甲氧基羰基(Fmoc)、烯丙氧基羰基(Alloc)、[2,2,2-三氯乙氧基羰基](Troc)、乙酰基(Ac)、邻苯二酰亚胺基(Phth)、苄氧羰基(Cbz)或第三丁氧羰基(Boc)。
在一些实施例中,可有效引起不同正交保护基中的每一者裂解的条件较佳不引起寡醣构建块上所存在的其他保护基裂解。
N-聚醣核心结构视情况具有间隔基或连接基团且包括在还原端的-(CH2)5NH2以促进有效固定在阵列上及与载体蛋白结合。本发明亦提供诸如-(CH2)nNH-CO-O-[(CH2)2-O-(CH2)2]2-3-O-PO(OH)2的间隔基以促进经由膦酸酯化学有效固定在氧化铝阵列上。
在另一态样中,本发明提供一种用于制造经正交保护的Manβ1-4GlcNAcβ1-4GlcNAcβ1核心的方法。
在另一态样中,本发明提供用于制造D1及D2/D3臂模组的化学及化学酶促方法。
在一个态样中,本发明提供用于制备数千个在结构上不同的复合N-连接型寡醣(N-聚醣)的策略,其中材料可用于(1)研发聚醣阵列,从而测定诸如凝集素、半乳糖结合蛋白(半乳糖凝集素)、抗聚醣抗体、HIV广谱中和抗体的碳水化合物结合分子,且较佳流感血球凝集素、化合物以及其他聚醣及醣脂的结合特异性;(2)使用质谱进行聚醣定序的标准品;(3)制备在诸如美罗华(Rituxan)、赫赛汀(Herceptin)、修美乐(Humira)的治疗性抗体的Fc区的多样化糖型以便增强其效应子功能;及(4)用于糖生物学研究实验室的寡醣标准品。
在一个态样中,本发明包含固定在基板上的碳水化合物部分的阵列,阵列包含:多数个聚醣,其中一或多个聚醣部分沈积在基板上的离散位置(例如斑点),其中多样化聚醣包含高甘露糖型、混合型及复合型同质及/或混合N-聚醣,其中阵列适用于侦测杂聚醣结合行为。
在一个态样中,本发明包含一种用于侦测杂聚醣结合行为的方法,所述方法包含使阵列与一个或多数个杂聚醣接触。在一些态样中,杂聚醣可为广谱中和HIV-1抗体的一部分。
在某些实施例中,包含高甘露糖型、混合型及复合型同质及/或混合N-聚醣的聚醣是通过模组化化学酶促方法制备。在某些实施例中,使用阵列侦测杂聚醣结合行为包含侦测广谱中和HIV-1抗体的结合特异性的步骤。
在某些实施例中,聚醣包含如图S11中所阐述的化合物G1至G33中的一或多者及/或如图S17中所阐述的化合物I至XI中的任何一或多者。
在某些实施例中,聚醣可以包括或不包括如图S11中所阐述的化合物G1至G33中的一或多者且包括或不包括如图S17中所阐述的化合物I至XI中的任何一或多者。
在另一态样中,提供一种用于合成如技术方案1至6中任一项的聚醣部分的方法,其中所述合成方法是部分地或完全地基于或衍生自如图1至图4中所阐述的合成流程。
在另一态样中,提供一种用于产生疫苗标靶的方法,其包含筛选及鉴别由HIV-1中和抗体识别的最佳细胞表面聚醣,其中所述方法进一步包含使HIV抗体与如技术方案1至6中任一项的阵列接触。
在一个态样中,提供一种聚醣微阵列,其在涂有氧化铝的玻璃(ACG)载片或涂有氧化铟锡(ITO)的玻璃载片上,含有一组多样化聚醣,包括通过模组化化学酶促方法制备的高甘露糖型、混合型及复合型同质及混合N-聚醣。在某些实施例中,阵列适用于侦测杂聚醣结合行为。
在一个相关态样中,提供产生/制造所述聚醣微阵列的方法。在某些实施例中,可以通过组装所谓的“D1及D2/D3臂模组”,随后在常见核心三醣的甘露糖残基的3-O及/或6-O位置处进行α-特异性甘露醣基化,从而产生以多样性为基础的合成策略。
在某些其他实施例中,将醣苷氟化物策略与酶促唾液酸化组合来构建对称的二触角、三触角及四触角复合型聚醣50的库。在某些实施例中,提供用于制备高甘露糖、混合及不对称唾液酸化的多触角聚醣的模组化合成方法(例如图1b)。
在一个态样中,提供一种用于快速筛选及鉴别由中和抗体识别的最佳聚醣的方法。在某些实施例中,筛选直接有助于研发靶向细胞表面聚醣的HIV-1疫苗。
在一个态样中,本发明涉及聚醣结合搭配物的筛选库,其用于鉴别结合至包含一或多个如本文中所揭示的N-聚醣的阵列的N-聚醣结合搭配物。在一些态样中,这些库中的分子可以包含例如抗体、奈米抗体、抗体片段、适体、凝集素、肽、生物分子或组合库分子。在一个实施例中,所述筛选包含侦测步骤,其中侦测步骤包含用HIV抗体侦测N-聚醣结合。在一个实施例中,侦测所述HIV抗体包含使用N-聚醣阵列,其包含一或多个如本文中所揭示的N-聚醣。
在一个态样中,本发明涉及一种侦测N-聚醣结合的方法,其中通过使阵列与一个或多数个N-聚醣接触形成复合物,且使复合物与标签接触。在一些态样中,随后可侦测标签。在一些态样中,标签可为经标记抗体。在一些态样中,经标记抗体可以进一步包含标签,其包含酶、萤光团、化学发光部分或奈米粒子。
在一个态样中,本发明涉及一种侦测HIV抗体的方法,所述方法包含:提供如本文所述的N-连接型聚醣的阵列,使阵列与HIV抗体接触;在HIV抗体与阵列上的聚醣之间形成复合物;使HIV抗体-聚醣阵列复合物与标签接触;及侦测标签。在一些态样中,标签可为经标记抗体,其进一步包含酶、萤光团、化学发光部分或奈米粒子。
在一些实施例中,阵列包含基板及在固体载体上的众多限定的聚醣探针位置,各聚醣探针位置限定固体载体的一个区域,所述区域具有与其连接的一或多个类型的类似聚醣分子的多个复本。在一些实施例中,各聚醣探针位置限定固体载体的一个区域,所述区域具有与其连接的超过一种类型的类似聚醣分子的多个复本。
在一些实施例中,A与X之间的相互作用可为共价键、凡得瓦相互作用(Van derWaals interaction)、氢键、离子键或疏水相互作用。
本发明的一或多个实施例的详情阐述于以下说明中。本发明的其他特征或优点将自附图及以下几个实施例的详细说明且亦自所附申请专利范围为显而易见的。
图式简单说明
图1显示用于模组化合成N-聚醣的例示性一般策略。因为糖苷键联可能数目庞大且产生各种各样的结构,尤其是自GlcNAc残基至非还原末端,所以使用模组化方法来最小化反应步骤且产生足以反映N-醣基化的性质的多样性。图1A)经由用一组模组化多样化醣苷供体使经正交保护的核心三醣在3-O及6-O位置处区域及立体选择性醣化来合成高甘露糖型、混合型及复合型N-聚醣。图1B)可由此策略产生的代表性N-聚醣。图1C)高甘露糖型、混合型及复合型聚醣的还原合成拆分,示出了组装所需的构建块。
图2显示D1及D2/D3臂构建块的例示性结构。通过全化学合成制备且用于组装寡醣的一组模组化构建块。
图3显示模组的例示性化学酶促合成。用于合成N-聚醣组装所必需的图3A线性模组、图3B对称分支模组及图3C不对称分支模组的代表性化学酶促途径。试剂及条件:i)UDP-半乳糖,β-1,4-GalT;ii)GDP-海藻糖,α-1,3-FucT;iii)CMP-Neu5Ac,α-2,6-SiaT;iv)CMP-Neu5Ac,α-2,3-SiaT;v)GDP-海藻糖,α-1,2-FucT;vi)NaOH。
图4为用于N-聚醣合成的化学酶促策略的代表性概念验证(proof-of-concept)论证。试剂及条件:i)乙酸酐、吡啶;ii)(1)CAN、甲苯:ACN:H2O:甲苯;(2)DAST、CH2Cl2,-30℃;iii)AgOTf、Cp2HfCl2、甲苯、MS,0℃至室温;iv)p-TSA、乙腈,室温;v)(1)LiOH、1,4-二恶烷:H2O;90℃,隔夜;(2)Ac2O、吡啶,隔夜;(3)NaOMe、MeOH,隔夜;(4)Pd(OH)2、MeOH:H2O:HCOOH(5:3:2)、H2。CAN:硝酸铈铵;DAST:三氟化二乙基胺基硫;AgOTf:三氟甲磺酸银;Cp2HfCl2:二氯化双(环戊二烯基)铪;MS:分子筛。
图5显示在ACG阵列上的HIV-1bNAb的例示性聚醣特异性。图5A)合成的N-聚醣经化学修饰具有膦酸尾部,以便经由膦酸酯化学共价连接至涂有氧化铝的玻璃(ACG)载片。图5B)、图5C)PG9、PG16及PGT 141至144与印刷在ACG载片上的结构I至XI的结合。图5D)、图5E)评估PG9及PG16对聚醣混合物中的每一者的结合以判定相邻聚醣对结合亲和力的影响。阵列是通过将100μM的Man5GlcNAc2或复合型聚醣与自I至XI的每一种结构以1:1的比率混合来印刷。在图例中以μM计的莫耳浓度给出抗体。示出了关于阵列上的五个独立复本计算的平均信号强度及标准误差。
图6A及6B显示PG9(A部分-图6A)及PG16(B部分-图6B)对各种比率的Man5与XI的混合物的例示性聚醣特异性。通过100μM的连接基团Man5GlcNAc2(IV)、复合型聚醣(XI)、以及比率为1:1/2/3/4/5的(IV+XI)或(XI+IV)的混合物中的每一者印刷阵列。在图例中以μM计的莫耳浓度给出抗体。示出了关于阵列上的八个独立复本计算的平均信号强度及标准误差。
图7显示本发明的例示性结构实施例。
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图11A、11B及11C:图11A显示本发明的例示性结构实施例。图11B显示本发明的例示性结构实施例。图11C显示本发明的例示性结构实施例。
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图14:高甘露糖型、混合型及复合型N-聚醣的例示性模组化合成。
图15A至图15C:高甘露糖型、混合型及复合型寡醣的例示性糖残基。(图15A=高甘露糖型;图15B=混合型;图15C=复合型)。
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图58:高甘露糖型聚醣及其片段的例示性结构
图59A及59B:图59A:本发明的例示性结构实施例及合成流程实施例。图59B:本发明的例示性结构实施例及合成流程实施例。
图60A及60B:图60A:本发明的例示性结构实施例及合成流程实施例。图60B:本发明的例示性结构实施例及合成流程实施例。
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图74:混合型聚醣及其片段的例示性结构。
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图93A、93B及93C:图93A:复合型聚醣的例示性结构。图93B:复合型聚醣的例示性结构。图93C:复合型聚醣的例示性结构。
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图152流程S23|线性模组的例示性制备流程。
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图160流程S24|对称分支模组的例示性制备流程。
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图165流程S25|不对称模组的例示性制备流程。
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图176显示本发明的例示性结构实施例及合成流程实施例。
图177显示本发明的例示性结构实施例及合成流程实施例。
图178显示本发明的例示性结构实施例及合成流程实施例。
图179显示本发明的例示性结构实施例及合成流程实施例。
图180显示本发明的例示性结构实施例及合成流程实施例。
图181显示本发明的例示性结构实施例及合成流程实施例。
图182显示本发明的例示性结构实施例及合成流程实施例。
图183A及183B:图183A:本发明的例示性结构实施例及合成流程实施例。图183B:本发明的例示性结构实施例及合成流程实施例。
图184显示本发明的例示性结构实施例及合成流程实施例。
图185显示本发明的例示性结构实施例及合成流程实施例。
图186显示本发明的例示性结构实施例及合成流程实施例。
图187显示本发明的例示性结构实施例及合成流程实施例。
图188显示本发明的例示性结构实施例及合成流程实施例。
图189显示本发明的例示性结构实施例及合成流程实施例。
图190显示本发明的例示性结构实施例及合成流程实施例。
图191图S4|制造阳极化氧化铝(AAO)玻璃基板的示意图。
图192图S5|图192A)AFM图像-表面粗糙度分析.Img.Rms(Rq)2.319nm及图192B)AAO玻璃基板的截面SEM图像。
图193图S6|Cy5-膦酸连接基团及Cy5-胺连接基团的结构。
图194图S8|在AAO玻璃基板及NHS玻璃载片上的Cy5-膦酸及Cy5-胺的代表性共焦显微镜图像。在斑点内的选择性900μm2面积。
图195A、195B及195C图S7|1mM的(图195A)在AAO玻璃基板上的Cy5-膦酸、(图195B)在NHS玻璃载片上的Cy5-胺的GenePix扫描(在PMT 450下),及(图195C)其平均20个斑点
图196图S9|AAO玻璃基板上的对比NHS玻璃载片上的ConA488/甘露糖结合的共焦显微镜。
图197A及197B图S10|原子力显微镜影像,示出了在图197A)涂有氧化铝的玻璃载片上及图197B)涂有NHS的玻璃载片上的糖分布。
图198图S11|印刷在涂有NHS的玻璃载片上的N-聚醣的示意性图示。
图199图S12使用涂有NHS的玻璃载片得到的PG16的结合行为。PG16与一组N-聚醣的结合用条形图表示。
图200图S17草图表示印刷在ACG阵列上的聚醣。连接基团的结构示出在左上角。
图201A及201B(图S18)用条形图显示PGT 141-144与ACG阵列上的一组N-聚醣的结合。
图202A及202B(图S19)聚醣X、XI及混合物V+XI及X+XII在100μM浓度下观察到的抗体PG16结合曲线。曲线是通过使用DyLight649结合的驴抗人类IgG二级抗体获得。
具体实施方式
在以下描述中参考了附图,附图形成其一部分,且在附图中以说明的方式显示了可以实施的特定实施例。详细地描述这些实施例,使得熟习此项技术者能够实施本发明,且应理解,可利用其他实施例,且可在不偏离本发明的范畴的情况下进行结构、逻辑及电改变。因此,不应将例示性实施例的以下描述理解为具有限制性意义,且本发明范畴由随附申请专利范围界定。
除非另外定义,否则本文中所用的所有技术及科学术语具有与本发明所属领域的一般技术者通常所理解相同的含义。虽然类似于或等效于本文所述者的任何方法及材料可用于实施或测试本发明,但现在要描述较佳的方法及材料。本文特别提及的所有出版物及专利以引用的方式并入本文中用于所有目的,包括描述及揭示出版物中所报导、可联合本发明使用的化学品、细胞株、载体、动物、仪器、统计学分析及方法。本说明书中所引用的所有参考文献视为此项技术的技能水准的指标。本文不应理解为承认本发明无权凭借先前发明而先于此类揭示内容。
在描述本发明材料及方法之前,应理解本发明不限于所述特定方法论、方案、材料及试剂,因为其可变化。亦应了解,本文中所用的术语仅出于描述特定实施例的目的而并不意欲限制本发明的范畴,本发明的范畴将仅由所附申请专利范围限制。
当提供值范围时,除非上下文另外明确指明,否则应了解彼范围的上限与下限之间的各中间值,直至下限单位的十分之一以及彼所陈述的范围内的任何其他所陈述的值或中间值均涵盖在本发明内。这些较小范围的上限及下限可独立地包括于较小范围内,且亦涵盖在本发明中,属于在所陈述的范围中任何明确排除在外的限值。当所陈述的范围包括限值中的一者或两者时,排除那些所包括的限值中的任一者或两者的范围亦包括于本发明中。
除非另外指明,否则本发明的实施将采用属于此项技术的技能范围内的习知分子生物学、微生物学、重组DNA及免疫学技术。此类技术完整解释于文献中。参见例如Molecular Cloning A Laboratory Manual,第2版,由Sambrook、Fritsch及Maniatis编(Cold Spring Harbor Laboratory Press,1989);DNA Cloning,第I卷及第II卷(D.N.Glover编,1985);Culture Of Animal Cells(R.I.Freshney,Alan R.Liss,Inc.,1987);Immobilized Cells And Enzymes(IRL Press,1986);B.Perbal,A PracticalGuide To Molecular Cloning(1984);专著Methods In Enzymology(Academic Press,Inc.,N.Y.);Gene Transfer Vectors For Mammalian Cells(J.H.Miller及M.P.Calos编,1987,Cold Spring Harbor Laboratory);Methods In Enzymology,第154卷及第155卷(Wu等人编),Immunochemical Methods In Cell And Molecular Biology(Mayer及Walker编,Academic Press,London,1987);Antibodies:A Laboratory Manual,Harlow及Lanes(ColdSpring Harbor Laboratory Press,1988);及Handbook Of Experimental Immunology,第I-IV卷(D.M.Weir及C.C.Blackwell编,1986)。
化学定义
下文更详细地描述特定官能基及化学术语的定义。化学元素是根据元素周期表(Periodic Table of the Elements),CAS版本,Handbook of Chemistry and Physics,第75版,内封面来鉴别,且特定官能基一般如其中所描述来定义。另外,有机化学的一般原理以及特定官能部分及反应性描述于以下各者中:Thomas Sorrell,Organic Chemistry,University Science Books,Sausalito,1999;Smith及March,March's Advanced OrganicChemistry,第5版,John Wiley&Sons,Inc.,New York,2001;Larock,ComprehensiveOrganic Transformations,VCH Publishers,Inc.,New York,1989;及Carruthers,SomeModern Methods of Organic Synthesis,第3版,Cambridge University Press,Cambridge,1987。
本文所述的化合物可包含一或多个不对称中心,且因此可以各种异构形式存在,例如对映异构体及/或非对映异构体。举例而言,本文所述的化合物可呈个别对映异构体、非对映异构体或几何异构体形式,或可呈立体异构体的混合物的形式,包括外消旋混合物及富含一或多种立体异构体的混合物。可利用熟习此项技术者已知的方法(包括对掌性高压液相层析(HPLC)以及对掌性盐的形成及结晶)而自混合物分离异构体;或可通过不对称合成来制备偏好的异构体。参见例如Jacques等人,Enantiomers,Racemates andResolutions(Wiley Interscience,New York,1981);Wilen等人,Tetrahedron 33:2725(1977);Eliel,Stereochemistry of Carbon Compounds(McGraw-Hill,NY,1962);及Wilen,Tables of Resolving Agents and Optical Resolutions第268页(E.L.Eliel编,Univ.of Notre Dame Press,Notre Dame,IN 1972)。本发明另外涵盖呈基本上不含其他异构体的个别异构体形式及或者呈各种异构体的混合物形式的本文所述的化合物。
当列出值的范围时,意欲在所述范围内涵盖各值及子范围。举例而言,“C1-6”意欲涵盖C1、C2、C3、C4、C5、C6、C1-6、C1-5、C1-4、C1-3、C1-2、C2-6、C2-5、C2-4、C2-3、C3-6、C3-5、C3-4、C4-6、C4-5及C5-6。
“烷基”是指具有1至20个碳原子的直链或分支链饱和烃基(“C1-20烷基”)。在一些实施例中,烷基具有1至10个碳原子(“C1-10烷基”)。在一些实施例中,烷基具有1至9个碳原子(“C1-9烷基”)。在一些实施例中,烷基具有1至8个碳原子(“C1-8烷基”)。在一些实施例中,烷基具有1至7个碳原子(“C1-7烷基”)。在一些实施例中,烷基具有1至6个碳原子(“C1-6烷基”)。在一些实施例中,烷基具有1至5个碳原子(“C1-5烷基”)。在一些实施例中,烷基具有1至4个碳原子(“C1-4烷基”)。在一些实施例中,烷基具有1至3个碳原子(“C1-3烷基”)。在一些实施例中,烷基具有1至2个碳原子(“C1-2烷基”)。在一些实施例中,烷基具有1个碳原子(“C1烷基”)。在一些实施例中,烷基具有2至6个碳原子(“C2-6烷基”)。C1-6烷基的实例包括甲基(C1)、乙基(C2)、正丙基(C3)、异丙基(C3)、正丁基(C4)、第三丁基(C4)、第二丁基(C4)、异丁基(C4)、正戊基(C5)、3-戊基(C5)、戊基(C5)、新戊基(C5)、3-甲基-2-丁基(C5)、第三戊基(C5)及正己基(C6)。烷基的其他实例包括正庚基(C7)、正辛基(C8)及其类似者。除非另外说明,否则烷基的各实例独立地视情况经取代,亦即未经取代(“未经取代的烷基”)或经一或多个取代基取代(“经取代的烷基”)。在某些实施例中,烷基为未经取代的C1-10烷基(例如,-CH3)。在某些实施例中,烷基为经取代的C1-10烷基。
“烯基”是指具有2至20个碳原子、一或多个碳-碳双键且无参键的直链或分支链烃基(“C2-20烯基”)。在一些实施例中,烯基具有2至10个碳原子(“C2-10烯基”)。在一些实施例中,烯基具有2至9个碳原子(“C2-9烯基”)。在一些实施例中,烯基具有2至8个碳原子(“C2-8烯基”)。在一些实施例中,烯基具有2至7个碳原子(“C2-7烯基”)。在一些实施例中,烯基具有2至6个碳原子(“C2-6烯基”)。在一些实施例中,烯基具有2至5个碳原子(“C2-5烯基”)。在一些实施例中,烯基具有2至4个碳原子(“C2-4烯基”)。在一些实施例中,烯基具有2至3个碳原子(“C2-3烯基”)。在一些实施例中,烯基具有2个碳原子(“C2烯基”)。一或多个碳-碳双键可位于内部(诸如在2-丁烯基中)或末端(诸如在1-丁烯基中)。C2-4烯基的实例包括乙烯基(C2)、1-丙烯基(C3)、2-丙烯基(C3)、1-丁烯基(C4)、2-丁烯基(C4)、丁二烯基(C4)及其类似者。C2-6烯基的实例包括前述C2-4烯基以及戊烯基(C5)、戊二烯基(C5)、己烯基(C6)及其类似者。烯基的其他实例包括庚烯基(C7)、辛烯基(C8)、辛三烯基(C8)及其类似者。除非另外说明,否则烯基的各实例独立地视情况经取代,亦即未经取代(“未经取代的烯基”)或经一或多个取代基取代(“经取代的烯基”)。在某些实施例中,烯基为未经取代的C2-10烯基。在某些实施例中,烯基为经取代的C2-10烯基。
“炔基”是指具有2至20个碳原子、一或多个碳-碳参键及视情况存在的一或多个双键的直链或分支链烃基(“C2-20炔基”)。在一些实施例中,炔基具有2至10个碳原子(“C2-10炔基”)。在一些实施例中,炔基具有2至9个碳原子(“C2-9炔基”)。在一些实施例中,炔基具有2至8个碳原子(“C2-8炔基”)。在一些实施例中,炔基具有2至7个碳原子(“C2-7炔基”)。在一些实施例中,炔基具有2至6个碳原子(“C2-6炔基”)。在一些实施例中,炔基具有2至5个碳原子(“C2-5炔基”)。在一些实施例中,炔基具有2至4个碳原子(“C2-4炔基”)。在一些实施例中,炔基具有2至3个碳原子(“C2-3炔基”)。在一些实施例中,炔基具有2个碳原子(“C2炔基”)。一或多个碳-碳参键可位于内部(诸如在2-丁炔基中)或末端(诸如在1-丁炔基中)。C2-4炔基的实例包括(但不限于)乙炔基(C2)、1-丙炔基(C3)、2-丙炔基(C3)、1-丁炔基(C4)、2-丁炔基(C4)及其类似者。C2-6炔基的实例包括前述C2-4炔基以及戊炔基(C5)、己炔基(C6)及其类似者。炔基的其他实例包括庚炔基(C7)、辛炔基(C8)及其类似者。除非另外说明,否则炔基的各实例独立地视情况经取代,亦即未经取代(“未经取代的炔基”)或经一或多个取代基取代(“经取代的炔基”)。在某些实施例中,炔基为未经取代的C2-10炔基。在某些实施例中,炔基为经取代的C2-10炔基。
“碳环基”或“碳环”是指在非芳环***中具有3至10个环碳原子(“C3-10碳环基”)及零个杂原子的非芳族环烃基。在一些实施例中,碳环基具有3至8个环碳原子(“C3-8碳环基”)。在一些实施例中,碳环基具有3至6个环碳原子(“C3-6碳环基”)。在一些实施例中,碳环基具有3至6个环碳原子(“C3-6碳环基”)。在一些实施例中,碳环基具有5至10个环碳原子(“C5-10碳环基”)。例示性C3-6碳环基包括(但不限于)环丙基(C3)、环丙烯基(C3)、环丁基(C4)、环丁烯基(C4)、环戊基(C5)、环戊烯基(C5)、环己基(C6)、环己烯基(C6)、环己二烯基(C6)及其类似者。例示性C3-8碳环基包括(但不限于)前述C3-6碳环基以及环庚基(C7)、环庚烯基(C7)、环庚二烯基(C7)、环庚三烯基(C7)、环辛基(C8)、环辛烯基(C8)、羟基[2.2.1]庚基(C7)、羟基[2.2.2]辛基(C8)及其类似者。例示性C3-10碳环基包括(但不限于)前述C3-8碳环基以及环壬基(C9)、环壬烯基(C9)、环癸基(C10)、环癸烯基(C10)、八氢-1H-茚基(C9)、十氢萘基(C10)、螺[4.5]癸基(C10)及其类似者。如前述实例所说明,在某些实施例中,碳环基为单环(“单环碳环基”)或含有稠合、桥连或螺环***,诸如双环***(“双环碳环基”),且可为饱和的或可为部分不饱和的。“碳环基”亦包括以下环***,其中如上文所定义的碳环基环与一或多个芳基或杂芳基稠合,其中连接点在碳环基环上,且在这些情况下,碳的编号继续指示碳环基环***中碳的编号。除非另外说明,否则碳环基的各实例独立地视情况经取代,亦即未经取代(“未经取代的碳环基”)或经一或多个取代基取代(“经取代的碳环基”)。在某些实施例中,碳环基为未经取代的C3-10碳环基。在某些实施例中,碳环基为经取代的C3-10碳环基。
在一些实施例中,“碳环基”为具有3至10个环碳原子的单环饱和碳环基(“C3-10环烷基”)。在一些实施例中,环烷基具有3至8个环碳原子(“C3-8环烷基”)。在一些实施例中,环烷基具有3至6个环碳原子(“C3-6环烷基”)。在一些实施例中,环烷基具有5至6个环碳原子(“C5-6环烷基”)。在一些实施例中,环烷基具有5至10个环碳原子(“C5-10环烷基”)。C5-6环烷基的实例包括环戊基(C5)及环己基(C6)。C3-6环烷基的实例包括前述C5-6环烷基以及环丙基(C3)及环丁基(C4)。C3-8环烷基的实例包括前述C3-6环烷基以及环庚基(C7)及环辛基(C8)。除非另外说明,否则环烷基的各实例独立地未经取代(“未经取代的环烷基”)或经一或多个取代基取代(“经取代的环烷基”)。在某些实施例中,环烷基为未经取代的C3-10环烷基。在某些实施例中,环烷基为经取代的C3-10环烷基。
“杂环基”或“杂环”是指具有环碳原子及1至4个环杂原子的3员至10员非芳环***的基团,其中各杂原子独立地选自氮、氧、硫、硼、磷及硅(“3-10员杂环基”)。在某些实施例中,杂原子独立地选自氮、硫及氧。在含有一或多个氮原子的杂环基中,在价数允许时,连接点可为碳或氮原子。杂环基可为单环(“单环杂环基”)或稠合、桥连或螺环***,诸如双环***(“双环杂环基”),且可为饱和的或部分不饱和的。杂环基双环***可在一个或两个环中包括一或多个杂原子。“杂环基”亦包括如下环***,其中如上文所定义的杂环与一或多个碳环基稠合,其中连接点在碳环基或杂环上;或如下环***,其中如上文所定义的杂环与一或多个芳基或杂芳基稠合,其中连接点在杂环上,且在这些情况下,环成员的编号继续表示杂环***中环成员的编号。除非另外说明,否则杂环基的各实例独立地视情况经取代,亦即未经取代(“未经取代的杂环基”)或经一或多个取代基取代(“经取代的杂环基”)。在某些实施例中,杂环基为未经取代的3-10员杂环基。在某些实施例中,杂环基为经取代的3-10员杂环基。
在一些实施例中,杂环基为具有环碳原子及1-4个环杂原子的5-10员非芳环***,其中各杂原子独立地选自氮、氧、硫、硼、磷及硅(“5-10员杂环基”)。在一些实施例中,杂环基为具有环碳原子及1-4个环杂原子的5-8员非芳环***,其中各杂原子独立地选自氮、氧及硫(“5-8员杂环基”)。在一些实施例中,杂环基为具有环碳原子及1-4个环杂原子的5-6员非芳环***,其中各杂原子独立地选自氮、氧及硫(“5-6员杂环基”)。在一些实施例中,5-6员杂环基具有1-3个选自氮、氧及硫的环杂原子。在一些实施例中,5-6员杂环基具有1-2个选自氮、氧及硫的环杂原子。在一些实施例中,5-6员杂环基具有一个选自氮、氧及硫的环杂原子。
含有一个杂原子的例示性3员杂环基包括(但不限于)氮丙啶基(azirdinyl)、环氧乙基及环硫乙基(thiorenyl)。含有一个杂原子的例示性4员杂环基包括(但不限于)氮杂环丁烷基、氧杂环丁烷基及硫杂环丁烷基。含有一个杂原子的例示性5员杂环基包括(但不限于)四氢呋喃基、二氢呋喃基、四氢噻吩基、二氢噻吩基、吡咯啶基、二氢吡咯基及吡咯基-2,5-二酮。含有两个杂原子的例示性5员杂环基包括(但不限于)二氧戊环基(dioxolanyl)、氧硫呋喃基(oxasulfuranyl)、二硫呋喃基(disulfuranyl)及恶唑啶-2-酮。含有三个杂原子的例示性5员杂环基包括(但不限于)***啉基(triazolinyl)、恶二唑啉基(oxadiazolinyl)及噻二唑啉基(thiadiazolinyl)。含有一个杂原子的例示性6员杂环基包括(但不限于)哌啶基、四氢哌喃基、二氢吡啶基及噻烷基。含有两个杂原子的例示性6员杂环基包括(但不限于)哌嗪基、吗啉基、二噻烷基及二恶烷基。含有两个杂原子的例示性6员杂环基包括(但不限于)三氮杂环己烷基。含有一个杂原子的例示性7员杂环基包括(但不限于)氮杂环庚烷基、氧杂环庚基及硫杂环庚烷基。含有一个杂原子的例示性8员杂环基包括(但不限于)氮杂环辛基、氧杂环辛基及硫杂环辛基。与C6芳环稠合的例示性5员杂环基(在本文中亦称为5,6-双环杂环)包括(但不限于)吲哚啉基、异吲哚啉基、二氢苯并呋喃基、二氢苯并噻吩基、苯并恶唑啉酮基及其类似者。与芳环稠合的例示性6员杂环基(在本文中亦称为6,6-双环杂环)包括(但不限于)四氢喹啉基、四氢异喹啉基及其类似者。
“芳基”是指芳环***中具有6-14个环碳原子及零个杂原子的单环或多环(例如双环或三环)4n+2芳环***(例如,环状阵列中共用6、10或14个π电子)的基团(“C6-14芳基”)。在一些实施例中,芳基具有六个环碳原子(“C6芳基”;例如苯基)。在一些实施例中,芳基具有十个环碳原子(“C10芳基”;例如萘基,诸如1-萘基及2-萘基)。在一些实施例中,芳基具有十四个环碳原子(“C14芳基”;例如蒽基)。“芳基”亦包括如下环***,其中如上文所定义的芳环与一或多个碳环基或杂环基稠合,其中连接基团或连接点在芳环上,且在这些情况下,碳原子的编号继续指示芳环***中碳原子的编号。除非另外说明,否则芳基的各实例独立地视情况经取代,亦即未经取代(“未经取代的芳基”)或经一或多个取代基取代(“经取代的芳基”)。在某些实施例中,芳基为未经取代的C6-14芳基。在某些实施例中,芳基为经取代的C6-14芳基。
“芳基烷基”为如本文所定义的烷基及芳基的子集且是指经视情况经取代的芳基取代的视情况经取代的烷基。在某些实施例中,芳烷基为视情况经取代的苄基。在某些实施例中,芳烷基为苄基。在某些实施例中,芳烷基为视情况经取代的苯乙基。在某些实施例中,芳烷基为苯乙基。
“杂芳基”是指芳环***中设置有环碳原子及1-4个环杂原子的5-10员单环或双环4n+2芳环***(例如,环状阵列中共用6或10个π电子)的基团,其中各杂原子独立地选自氮、氧及硫(“5-10员杂芳基”)。在含有一或多个氮原子的杂芳基中,在价数允许时,连接点可为碳或氮原子。杂芳基双环***可在一个或两个环中包括一或多个杂原子。“杂芳基”包括如下环***,其中如上文所定义的杂芳基环与一或多个碳环基或杂环基稠合,其中连接点在杂芳基环上,且在这些情况下,环成员的编号继续指示杂芳基环***中环成员的编号。“杂芳基”亦包括如上文所定义的杂芳基环与一或多个芳基稠合的环***,其中连接点在芳基或杂芳基环上,且在这些情况下,环成员的编号指示稠合(芳基/杂芳基)环***中环成员的编号。一个环不含杂原子的双环杂芳基(例如吲哚基、喹啉基、咔唑基及其类似者),连接点可在任一环上,亦即携带杂原子的环(例如2-吲哚基)或不含杂原子的环(例如5-吲哚基)。
在一些实施例中,杂芳基为芳环***中设置有环碳原子及1-4个环杂原子的5-10员芳环***,其中各杂原子独立地选自氮、氧及硫(“5-10员杂芳基”)。在一些实施例中,杂芳基为芳环***中设置有环碳原子及1-4个环杂原子的5-8员芳环***,其中各杂原子独立地选自氮、氧及硫(“5-8员杂芳基”)。在一些实施例中,杂芳基为芳环***中设置有环碳原子及1-4个环杂原子的5-6员芳环***,其中各杂原子独立地选自氮、氧及硫(“5-6员杂芳基”)。在一些实施例中,5-6员杂芳基具有1-3个选自氮、氧及硫的环杂原子。在一些实施例中,5-6员杂芳基具有1-2个选自氮、氧及硫的环杂原子。在一些实施例中,5-6员杂芳基具有1个选自氮、氧及硫的环杂原子。除非另外说明,否则杂芳基的各实例独立地视情况经取代,亦即未经取代(“未经取代的杂芳基”)或经一或多个取代基取代(“经取代的杂芳基”)。在某些实施例中,杂芳基为未经取代的5-14员杂芳基。在某些实施例中,杂芳基为经取代的5-14员杂芳基。
含有一个杂原子的例示性5员杂芳基包括(但不限于)吡咯基、呋喃基及噻吩基。含有两个杂原子的例示性5员杂芳基包括(但不限于)咪唑基、吡唑基、恶唑基、异恶唑基、噻唑基及异噻唑基。含有三个杂原子的例示性5员杂芳基包括(但不限于)***基、恶二唑基及噻二唑基。含有四个杂原子的例示性5员杂芳基包括(但不限于)四唑基。含有一个杂原子的例示性6员杂芳基包括(但不限于)吡啶基。含有两个杂原子的例示性6员杂芳基包括(但不限于)哒嗪基、嘧啶基及吡嗪基。含有三个或四个杂原子的例示性6员杂芳基分别包括(但不限于)三嗪基及四嗪基。含有一个杂原子的例示性7员杂芳基包括(但不限于)氮呯基(azepinyl)、氧呯基(oxepinyl)及噻呯基(thiepinyl)。例示性5,6-双环杂芳基包括(但不限于)吲哚基、异吲哚基、吲唑基、苯并***基、苯并噻吩基、异苯并噻吩基、苯并呋喃基、苯并异呋喃基、苯并咪唑基、苯并恶唑基、苯并异恶唑基、苯并恶二唑基、苯并噻唑基、苯并异噻唑基、苯并噻二唑基、吲哚嗪基及嘌呤基。例示性6,6-双环杂芳基包括(但不限于)啶基、喋啶基、喹啉基、异喹啉基、啉基、喹喏啉基、酞嗪基及喹唑啉基。
“杂芳烷基”为如本文所定义的烷基及杂芳基的子集且是指经视情况经取代的杂芳基取代的视情况经取代的烷基。
如本文所定义的作为二价桥连基团的烷基、烯基、炔基、碳环基、杂环基、芳基及杂芳基另外使用后缀-伸基(-ene)提及,例如伸烷基、伸烯基、伸炔基、伸碳环基、伸杂环基、伸芳基及伸杂芳基。
如本文所用,术语“视情况经取代”是指经取代或未经取代的部分。
如本文所定义的烷基、烯基、炔基、碳环基、杂环基、芳基及杂芳基视情况经取代(例如“经取代”或“未经取代”的烷基、“经取代”或“未经取代”的烯基、“经取代”或“未经取代”的炔基、“经取代”或“未经取代”的碳环基、“经取代”或“未经取代”的杂环基、“经取代”或“未经取代”的芳基或“经取代”或“未经取代”的杂芳基)。一般而言,术语“经取代”,不论之前是否有术语“视情况”,均意指存在于基团(例如碳或氮原子)上的至少一个氢经容许的取代基置换,所述容许的取代基例如在取代后产生稳定化合物,例如不会自发经历诸如重排、环化、消除或其他反应的转化的化合物的取代基。除非另外指明,否则“经取代”的基团在所述基团的一或多个可取代位置具有取代基,且当任何既定结构中超过一个位置经取代时,各位置上的取代基相同或不同。术语“经取代”预期包括经有机化合物的所有容许取代基、促使形成稳定化合物的本文所述任何取代基取代。本发明涵盖任何及所有此类组合以便获得稳定化合物。出于本发明的目的,诸如氮的杂原子可具有氢取代基及/或满足杂原子价数且促使形成稳定部分的如本文所述的任何合适的取代基。
例示性碳原子取代基包括(但不限于)卤素、-CN、-NO2、-N3、-SO2H、-SO3H、-OH、-ORaa、-ON(Rbb)2、-N(Rbb)2、-N(Rbb)3 +X-、-N(ORcc)Rbb、-SH、-SRaa、-SSRcc、-C(=O)Raa、-CO2H、-CHO、-C(ORcc)2、-CO2Raa、-OC(=O)Raa、-OCO2Raa、-C(=O)N(Rbb)2、-OC(=O)N(Rbb)2、-NRbbC(=O)Raa、-NRbbCO2Raa、-NRbbC(=O)N(Rbb)2、-C(=NRbb)Raa、-C(=NRbb)ORaa、-OC(=NRbb)Raa、-OC(=NRbb)ORaa、-C(=NRbb)N(Rbb)2、-OC(=NRbb)N(Rbb)2、-NRbbC(=NRbb)N(Rbb)2、-C(=O)NRbbSO2Raa、-NRbbSO2Raa、-SO2N(Rbb)2、-SO2Raa、-SO2ORaa、-OSO2Raa、-S(=O)Raa、-OS(=O)Raa、-Si(Raa)3、-Osi(Raa)3-C(=S)N(Rbb)2、-C(=O)SRaa、-C(=S)SRaa、-SC(=S)SRaa、-SC(=O)SRaa、-OC(=O)SRaa、-SC(=O)ORaa、-SC(=O)Raa、-P(=O)2Raa、-OP(=O)2Raa、-P(=O)(Raa)2、-OP(=O)(Raa)2、-OP(=O)(ORcc)2、-P(=O)2N(Rbb)2、-OP(=O)2N(Rbb)2、-P(=O)(NRbb)2、-OP(=O)(NRbb)2、-NRbbP(=O)(ORcc)2、-NRbbP(=O)(NRbb)2、-P(Rcc)2、-P(Rcc)3、-OP(Rcc)2、-OP(Rcc)3、-B(Raa)2、-B(ORcc)2、-BRaa(ORcc)、C1-10烷基、C1-10全卤烷基、C2-10烯基、C2-10炔基、C3-10碳环基、3-14员杂环基、C6-14芳基及5-14员杂芳基,其中各烷基、烯基、炔基、碳环基、杂环基、芳基及杂芳基独立地经0、1、2、3、4或5个Rdd基团取代;
或碳原子上的两个氢经基团=O、=S、=NN(Rbb)2、=NNRbbC(=O)Raa、=NNRbbC(=O)ORaa、=NNRbbS(=O)2Raa、=NRbb或=NORcc置换;
Raa的各实例独立地选自C1-10烷基、C1-10全卤烷基、C2-10烯基、C2-10炔基、C3-10碳环基、3-14员杂环基、C6-14芳基及5-14员杂芳基,或两个Raa基团接合形成3-14员杂环基或5-14员杂芳基环,其中各烷基、烯基、炔基、碳环基、杂环基、芳基及杂芳基独立地经0、1、2、3、4或5个Rdd基团取代;
Rbb的各实例独立地选自氢、-OH、-ORaa、-N(Rcc)2、-CN、-C(=O)Raa、-C(=O)N(Rcc)2、-CO2Raa、-SO2Raa、-C(=NRcc)ORaa、-C(=NRcc)N(Rcc)2、-SO2N(Rcc)2、-SO2Rcc、-SO2ORcc、-SORaa、-C(=S)N(Rcc)2、-C(=O)SRcc、-C(=S)SRcc、-P(=O)2Raa、-P(=O)(Raa)2、-P(=O)2N(Rcc)2、-P(=O)(NRcc)2、C1-10烷基、C1-10全卤烷基、C2-10烯基、C2-10炔基、C3-10碳环基、3-14员杂环基、C6-14芳基及5-14员杂芳基,或两个Rbb基团接合形成3-14员杂环基或5-14员杂芳基环,其中各烷基、烯基、炔基、碳环基、杂环基、芳基及杂芳基独立地经0、1、2、3、4或5个Rdd基团取代;
Rcc的各实例独立地选自氢、C1-10烷基、C1-10全卤烷基、C2-10烯基、C2-10炔基、C3-10碳环基、3-14员杂环基、C6-14芳基及5-14员杂芳基,或两个Rcc基团接合形成3-14员杂环基或5-14员杂芳基环,其中各烷基、烯基、炔基、碳环基、杂环基、芳基及杂芳基独立地经0、1、2、3、4或5个Rdd基团取代;
Rdd的各实例独立地选自卤素、-CN、-NO2、-N3、-SO2H、-SO3H、-OH、-ORee、-ON(Rff)2、-N(Rff)2、-N(Rff)3 +X-、-N(ORee)Rff、-SH、-SRee、-SSRee、-C(=O)Ree、-CO2H、-CO2Ree、-OC(=O)Ree、-OCO2Ree、-C(=O)N(Rff)2、-OC(=O)N(Rff)2、-NRffC(=O)Ree、-NRffCO2Ree、-NRffC(=O)N(Rff)2、-C(=NRff)ORee、-OC(=NRff)Ree、-OC(=NRff)ORee、-C(=NRff)N(Rff)2、-OC(=NRff)N(Rff)2、-NRffC(=NRff)N(Rff)2、-NRffSO2Ree、-SO2N(Rff)2、-SO2Ree、-SO2ORee、-OSO2Ree、-S(=O)Ree、-Si(Ree)3、-Osi(Ree)3、-C(=S)N(Rff)2、-C(=O)SRee、-C(=S)SRee、-SC(=S)SRee、-P(=O)2Ree、-P(=O)(Ree)2、-OP(=O)(Ree)2、-OP(=O)(ORee)2、C1-6烷基、C1-6全卤烷基、C2-6烯基、C2-6炔基、C3-10碳环基、3-10员杂环基、C6-10芳基、5-10员杂芳基,其中各烷基、烯基、炔基、碳环基、杂环基、芳基及杂芳基独立地经0、1、2、3、4或5个Rgg基团取代,或两个Rdd取代基可以接合形成=O或=S;
Ree的各实例独立地选自C1-6烷基、C1-6全卤烷基、C2-6烯基、C2-6炔基、C3-10碳环基、C6-10芳基、3-10员杂环基及3-10员杂芳基,其中各烷基、烯基、炔基、碳环基、杂环基、芳基及杂芳基独立地经0、1、2、3、4或5个Rgg基团取代;
Rff的各实例独立地选自氢、C1-6烷基、C1-6全卤烷基、C2-6烯基、C2-6炔基、C3-10碳环基、3-10员杂环基、C6-10芳基及5-10员杂芳基,或两个Rff基团接合形成3-14员杂环基或5-14员杂芳基环,其中各烷基、烯基、炔基、碳环基、杂环基、芳基及杂芳基独立地经0、1、2、3、4或5个Rgg基团取代;且
Rgg的各实例独立地为卤素、-CN、-NO2、-N3、-SO2H、-SO3H、-OH、-OC1-6烷基、-ON(C1-6烷基)2、-N(C1-6烷基)2、-N(C1-6烷基)3 +X-、-NH(C1-6烷基)2 +X-、-NH2(C1-6烷基)+X-、-NH3 +X-、-N(OC1-6烷基)(C1-6烷基)、-N(OH)(C1-6烷基)、-NH(OH)、-SH、-SC1-6烷基、-SS(C1-6烷基)、-C(=O)(C1-6烷基)、-CO2H、-CO2(C1-6烷基)、-OC(=O)(C1-6烷基)、-OCO2(C1-6烷基)、-C(=O)NH2、-C(=O)N(C1-6烷基)2、-OC(=O)NH(C1-6烷基)、-NHC(=O)(C1-6烷基)、-N(C1-6烷基)C(=O)(C1-6烷基)、-NHCO2(C1-6烷基)、-NHC(=O)N(C1-6烷基)2、-NHC(=O)NH(C1-6烷基)、-NHC(=O)NH2、-C(=NH)O(C1-6烷基)、-OC(=NH)(C1-6烷基)、-OC(=NH)OC1-6烷基、-C(=NH)N(C1-6烷基)2、-C(=NH)NH(C1-6烷基)、-C(=NH)NH2、-OC(=NH)N(C1-6烷基)2、-OC(NH)NH(C1-6烷基)、-OC(NH)NH2、-NHC(NH)N(C1-6烷基)2、-NHC(=NH)NH2、-NHSO2(C1-6烷基)、-SO2N(C1-6烷基)2、-SO2NH(C1-6烷基)、-SO2NH2、-SO2C1-6烷基、-SO2OC1-6烷基、-OSO2C1-6烷基、-SOC1-6烷基、-Si(C1-6烷基)3、-Osi(C1-6烷基)3-C(=S)N(C1-6烷基)2、C(=S)NH(C1-6烷基)、C(=S)NH2、-C(=O)S(C1-6烷基)、-C(=S)SC1-6烷基、-SC(=S)SC1-6烷基、-P(=O)2(C1-6烷基)、-P(=O)(C1-6烷基)2、-OP(=O)(C1-6烷基)2、-OP(=O)(OC1-6烷基)2、C1-6烷基、C1-6全卤烷基、C2-6烯基、C2-6炔基、C3-10碳环基、C6-10芳基、3-10员杂环基、5-10员杂芳基;或两个Rgg取代基可以接合形成=O或=S;其中X-为相对离子。
“卤基”或“卤素”是指氟(氟基、-F)、氯(氯基、-Cl)、溴(溴基、-Br)或碘(碘基、-I)。
如本文所用的“酰基”是指选自由以下组成的群的部分:-C(=O)Raa、-CHO、-CO2Raa、-C(=O)N(Rbb)2、-C(=NRbb)Raa、-C(=NRbb)ORaa、-C(=NRbb)N(Rbb)2、-C(=O)NRbbSO2Raa、-C(=S)N(Rbb)2、-C(=O)SRaa及-C(=S)SRaa,其中Raa及Rbb如本文所定义。
价数允许时,氮原子可经取代或未经取代,且包括一级、二级、三级及四级氮原子。例示性氮原子取代基包括(但不限于)氢、-OH、-ORaa、-N(Rcc)2、-CN、-C(=O)Raa、-C(=O)N(Rcc)2、-CO2Raa、-SO2Raa、-C(=NRbb)Raa、-C(=NRcc)ORaa、-C(=NRcc)N(Rcc)2、-SO2N(Rcc)2、-SO2Rcc、-SO2ORcc、-SORaa、-C(=S)N(Rcc)2、-C(=O)SRcc、-C(=S)SRcc、-P(=O)2Raa、-P(=O)(Raa)2、-P(=O)2N(Rcc)2、-P(=O)(NRcc)2、C1-10烷基、C1-10全卤烷基、C2-10烯基、C2-10炔基、C3-10碳环基、3-14员杂环基、C6-14芳基及5-14员杂芳基,或连接至氮原子的两个Rcc基团接合形成3-14员杂环基或5-14员杂芳基环,其中各烷基、烯基、炔基、碳环基、杂环基、芳基及杂芳基独立地经0、1、2、3、4或5个Rdd基团取代,且其中Raa、Rbb、Rcc及Rdd如上文所定义。
在某些实施例中,存在于氮原子上的取代基为氮保护基(亦称为胺基保护基)。氮保护基包括(但不限于)-OH、-ORaa、-N(Rcc)2、-C(=O)Raa、-C(=O)N(Rcc)2、-CO2Raa、-SO2Raa、-C(=NRcc)Raa、-C(=NRcc)ORaa、-C(=NRcc)N(Rcc)2、-SO2N(Rcc)2、-SO2Rcc、-SO2ORcc、-SORaa、-C(=S)N(Rcc)2、-C(=O)SRcc、-C(=S)SRcc、C1-10烷基(例如芳烷基、杂芳烷基)、C2-10烯基、C2-10炔基、C3-10碳环基、3-14员杂环基、C6-14芳基及5-14员杂芳基,其中各烷基、烯基、炔基、碳环基、杂环基、芳烷基、芳基及杂芳基独立地经0、1、2、3、4或5个Rdd基团取代,且其中Raa、Rbb、Rcc及Rdd如本文所定义。氮保护基为此项技术中熟知的且包括Protecting Groups in OrganicSynthesis,T.W.Greene及P.G.M.Wuts,第3版,John Wiley&Sons,1999中所述的氮保护基,所述文献以引用的方式并入本文中。
举例而言,诸如酰胺基团(例如,-C(=O)Raa)的氮保护基包括(但不限于)甲酰胺、乙酰胺、氯乙酰胺、三氯乙酰胺、三氟乙酰胺、苯基乙酰胺、3-苯基丙酰胺、吡啶酰胺、3-吡啶基甲酰胺、N-苄酰基***酰基衍生物、苄酰胺、对苯基苄酰胺、邻硝基苯基乙酰胺、邻硝基苯氧基乙酰胺、乙酰乙酰胺、(N'-二硫苯甲氧基酰胺基)乙酰胺、3-(对羟苯基)丙酰胺、3-(邻硝基苯基)丙酰胺、2-甲基-2-(邻硝基苯氧基)丙酰胺、2-甲基-2-(邻苯基偶氮基苯氧基)丙酰胺、4-氯丁酰胺、3-甲基-3-硝基丁酰胺、邻硝基肉桂酰胺、N-乙酰基甲硫胺酸衍生物、邻硝基苄酰胺及邻(苄酰氧基甲基)苄酰胺。
诸如胺基甲酸酯基(例如-C(=O)ORaa)的氮保护基包括(但不限于)胺基甲酸甲酯、胺基甲酸乙酯、胺基甲酸9-茀基甲酯(Fmoc)、胺基甲酸9-(2-磺酸基)茀基甲酯、胺基甲酸9-(2,7-二溴)茀基甲酯、胺基甲酸2,7-二第三丁基-[9-(10,10-二侧氧基-10,10,10,10-四氢噻基)]甲酯(DBD-Tmoc)、胺基甲酸4-甲氧基苄酰甲酯(Phenoc)、胺基甲酸2,2,2-三氯乙酯(Troc)、胺基甲酸2-三甲基硅烷基乙酯(Teoc)、胺基甲酸2-苯乙酯(hZ)、1-(1-金刚烷基)-1-甲基乙基(Adpoc)、胺基甲酸1,1-二甲基-2-卤基乙酯、胺基甲酸1,1-二甲基-2,2-二溴乙酯(DB-t-BOC)、胺基甲酸1,1-二甲基-2,2,2-三氯乙酯(TCBOC)、胺基甲酸1-甲基-1-(4-联苯基)乙酯(Bpoc)、胺基甲酸1-(3,5-二第三丁基苯基)-1-甲基乙酯(t-Bumeoc)、胺基甲酸2-(2'-及4'-吡啶基)乙酯(Pyoc)、胺基甲酸2-(N,N-二环己基甲酰胺基)乙酯、胺基甲酸第三丁酯(BOC)、胺基甲酸1-金刚烷酯(Adoc)、胺基甲酸乙烯酯(Voc)、胺基甲酸烯丙酯(Alloc)、胺基甲酸1-异丙基烯丙酯(Ipaoc)、胺基甲酸桂皮酯(Coc)、胺基甲酸4-硝基桂皮酯(Noc)、胺基甲酸8-喹啉酯、胺基甲酸N-羟基哌啶酯、烷基二硫基胺基甲酸酯、胺基甲酸苄酯(Cbz)、胺基甲酸对甲氧基苄酯(Moz)、胺基甲酸对硝基苄酯、胺基甲酸对溴苄酯、胺基甲酸对氯苄酯、胺基甲酸2,4-二氯苄酯、胺基甲酸4-甲基亚磺酰基苄酯(Msz)、胺基甲酸9-蒽基甲酯、胺基甲酸二苯甲酯、胺基甲酸2-甲硫基乙酯、胺基甲酸2-甲基磺酰基乙酯、胺基甲酸2-(对甲苯磺酰基)乙酯、胺基甲酸[2-(1,3-二噻烷基)]甲酯(Dmoc)、胺基甲酸4-甲硫基苯酯(Mtpc)、胺基甲酸2,4-二甲基噻吩酯(Bmpc)、胺基甲酸2-磷鎓基乙酯(Peoc)、胺基甲酸2-三苯基磷鎓基异丙酯(Ppoc)、胺基甲酸1,1-二甲基-2-氰基乙酯、胺基甲酸间氯-对酰氧基苄酯、胺基甲酸对(二羟基氧硼基)苄酯、胺基甲酸5-苯并异恶唑基甲酯、胺基甲酸2-(三氟甲基)-6-色酮基甲酯(Tcroc)、胺基甲酸间硝基苯酯、胺基甲酸3,5-二甲氧基苄酯、胺基甲酸邻硝基苄酯、胺基甲酸3,4-二甲氧基-6-硝基苄酯、胺基甲酸苯基(邻硝苯基)甲酯、胺基甲酸第三戊酯、硫胺基甲酸S-苄酯、胺基甲酸对氰基苄酯、胺基甲酸环丁酯、胺基甲酸环己酯、胺基甲酸环戊酯、胺基甲酸环丙基甲酯、胺基甲酸对癸氧基苄酯、胺基甲酸2,2-二甲氧基酰基乙烯酯、胺基甲酸邻(N,N-二甲基甲酰胺基)苄酯、胺基甲酸1,1-二甲基-3-(N,N-二甲基甲酰胺基)丙酯、胺基甲酸1,1-二甲基丙炔酯、胺基甲酸二(2-吡啶基)甲酯、胺基甲酸2-呋喃基甲酯、胺基甲酸2-碘乙酯、胺基甲酸异冰片酯、胺基甲酸异丁酯、胺基甲酸异烟碱酯、胺基甲酸对(对'-甲氧基苯基偶氮基)苄酯、胺基甲酸1-甲基环丁酯、胺基甲酸1-甲基环己酯、胺基甲酸1-甲基-1-环丙基甲酯、胺基甲酸1-甲基-1-(3,5-二甲氧基苯基)乙酯、胺基甲酸1-甲基-1-(对苯基偶氮基苯基)乙酯、胺基甲酸1-甲基-1-苯乙酯、胺基甲酸1-甲基-1-(4-吡啶基)乙酯、胺基甲酸苯酯、胺基甲酸对(苯偶氮基)苄酯、胺基甲酸2,4,6-三-第三丁基苯酯、胺基甲酸4-(三甲铵)苄酯及胺基甲酸2,4,6-三甲基苄酯。
诸如磺酰胺基(例如,-S(=O)2Raa)的氮保护基包括(但不限于)对甲苯磺酰胺(Ts)、苯磺酰胺、2,3,6,-三甲基-4-甲氧基苯磺酰胺(Mtr)、2,4,6-三甲氧基苯磺酰胺(Mtb)、2,6-二甲基-4-甲氧基苯磺酰胺(Pme)、2,3,5,6-四甲基-4-甲氧基苯磺酰胺(Mte)、4-甲氧基苯磺酰胺(Mbs)、2,4,6-三甲基苯磺酰胺(Mts)、2,6-二甲氧基-4-甲基苯磺酰胺(iMds)、2,2,5,7,8-五甲基色满-6-磺酰胺(Pmc)、甲磺酰胺(Ms)、β-三甲基硅烷基乙磺酰胺(SES)、9-蒽磺酰胺、4-(4',8'-二甲氧基萘基甲基)苯磺酰胺(DNMBS)、苄基磺酰胺、三氟甲基磺酰胺及苄酰甲基磺酰胺。
其他氮保护基包括(但不限于)啡噻嗪基-(10)-酰基衍生物、N'-对甲苯磺酰基胺酰基衍生物、N'-苯基胺基硫代酰基衍生物、N-苄酰基苯基丙胺酰基衍生物、N-乙酰基甲硫胺酸衍生物、4,5-二苯基-3-恶唑啉-2-酮、N-邻苯二甲酰亚胺、N-二硫杂丁二酰亚胺(Dts)、N-2,3-二苯基顺丁烯二酰亚胺、N-2,5-二甲基吡咯、N-1,1,4,4-四甲基二硅烷基氮杂环戊烷加合物(STABASE)、5-取代的1,3-二甲基-1,3,5-三氮杂环己-2-酮、5-取代的1,3-二苄基-1,3,5-三氮杂环己-2-酮、1-取代的3,5-二硝基-4-羟基、N-甲胺、N-烯丙胺、N-[2-(三甲基硅烷基)乙氧基]甲胺(SEM)、N-3-乙酰氧基丙胺、N-(1-异丙基-4-硝基-2-侧氧基-3-吡咯啉-3-基)胺、四级铵盐、N-苄胺、N-二(4-甲氧基苯基)甲胺、N-5-二苯并环庚胺、N-三苯基甲胺(Tr)、N-[(4-甲氧基苯基)二苯甲基]胺(MMTr)、N-9-苯基茀基胺(PhF)、N-2,7-二氯-9-茀基亚甲基胺、N-二茂铁基甲基胺基(Fcm)、N-2-吡啶甲基胺基N'-氧化物、N-1,1-二甲基硫基亚甲基胺、N-亚苄基胺、N-对甲氧基亚苄基胺、N-二苯基亚甲基胺、N-[(2-吡啶基)基]亚甲基胺、N-(N',N'-二甲基胺基亚甲基)胺、N,N'-亚异丙基二胺、N-对硝基亚苄基胺、N-亚柳基胺、N-5-氯亚柳基胺、N-(5-氯-2-羟苯基)苯基亚甲基胺、N-亚环己基胺、N-(5,5-二甲基-3-侧氧基-1-环己烯基)胺、N-硼烷衍生物、N-二苯基硼酸衍生物、N-[苯基(五酰基铬-或钨)酰基]胺、N-铜螯合物、N-锌螯合物、N-硝基胺、N-亚硝基胺、N-氧化胺、二苯基膦酰胺(Dpp)、二甲基硫基膦酰胺(Mpt)、二苯基硫基膦酰胺(Ppt)、胺基磷酸二烷酯、胺基磷酸二苄酯、胺基磷酸二苯酯、苯亚磺酰胺、邻硝基苯亚磺酰胺(Nps)、2,4-二硝基苯亚磺酰胺、五氯苯亚磺酰胺、2-硝基-4-甲氧基苯亚磺酰胺、三苯基甲基亚磺酰胺及3-硝基吡啶亚磺酰胺(Npys)。
在某些实施例中,存在于氧原子上的取代基为氧保护基(在本文中亦称为“羟基保护基”)。氧保护基包括(但不限于)-Raa、-N(Rbb)2、-C(=O)SRaa、-C(=O)Raa、-CO2Raa、-C(=O)N(Rbb)2、-C(=NRbb)Raa、-C(=NRbb)ORaa、-C(=NRbb)N(Rbb)2、-S(=O)Raa、-SO2Raa、-Si(Raa)3、-P(Rcc)2、-P(Rcc)3、-P(=O)2Raa、-P(=O)(Raa)2、-P(=O)(ORcc)2、-P(=O)2N(Rbb)2及-P(=O)(NRbb)2,其中Raa、Rbb及Rcc如本文所定义。氧保护基为此项技术中熟知的且包括ProtectingGroups in Organic Synthesis,T.W.Greene及P.G.M.Wuts,第3版,John Wiley&Sons,1999中所述的氧保护基,所述文献以引用的方式并入本文中。
例示性氧保护基包括(但不限于)甲基、甲氧基甲基(MOM)、甲硫基甲基(MTM)、第三丁硫基甲基、(苯基二甲基硅烷基)甲氧基甲基(SMOM)、苄氧基甲基(BOM)、对甲氧基苄氧基甲基(PMBM)、(4-甲氧基苯氧基)甲基(p-AOM)、愈创木酚甲基(guaiacolmethyl)(GUM)、第三丁氧基甲基、4-戊烯氧基甲基(POM)、硅烷氧基甲基、2-甲氧基乙氧基甲基(MEM)、2,2,2-三氯乙氧基甲基、双(2-氯乙氧基)甲基、2-(三甲基硅烷基)乙氧基甲基(SEMOR)、四氢哌喃基(THP)、3-溴四氢哌喃基、四氢硫哌喃基、1-甲氧基环己基、4-甲氧基四氢哌喃基(MTHP)、4-甲氧基四氢硫哌喃基、4-甲氧基四氢硫哌喃基S,S-二氧化物、1-[(2-氯-4-甲基)苯基]-4-甲氧基哌啶-4-基(CTMP)、1,4-二恶烷-2-基、四氢呋喃基、四氢硫呋喃基、2,3,3a,4,5,6,7,7a-八氢-7,8,8-三甲基-4,7-甲醇苯并呋喃-2-基、1-乙氧基乙基、1-(2-氯乙氧基)乙基、1-甲基-1-甲氧基乙基、1-甲基-1-苄氧基乙基、1-甲基-1-苄氧基-2-氟乙基、2,2,2-三氯乙基、2-三甲基硅烷基乙基、2-(苯基氢硒基)乙基、第三丁基、烯丙基、对氯苯基、对甲氧基苯基、2,4-二硝基苯基、苄基(Bn)、对甲氧基苄基、3,4-二甲氧基苄基、邻硝基苄基、对硝基苄基、对卤基苄基、2,6-二氯苄基、对氰基苄基、对苯基苄基、2-吡啶甲基、4-吡啶甲基、3-甲基-2-吡啶甲基N-氧离子基、二苯甲基、对,对'-二硝基二苯甲基、5-二苯并环庚基、三苯甲基、α-萘基二苯甲基、对甲氧基苯基二苯甲基、二(对甲氧基苯基)苯甲基、三(对甲氧基苯基)甲基、4-(4'-溴苄酰甲基氧基苯基)二苯甲基、4,4',4″-参(4,5-二氯邻苯二甲酰亚胺基苯基)甲基、4,4',4″-参(乙酰丙酰氧基苯基)甲基、4,4',4″-参(苄酰氧基苯基)甲基、3-(咪唑-1-基)双(4',4″-二甲氧基苯基)甲基、1,1-双(4-甲氧基苯基)-1'-芘基甲基、9-蒽基、9-(9-苯基)基、9-(9-苯基-10-侧氧基)蒽基、1,3-苯并二硫杂环戊-2-基、苯并异噻唑基S,S-二氧离子基、三甲基硅烷基(TMS)、三乙基硅烷基(TES)、三异丙基硅烷基(TIPS)、二甲基异丙基硅烷基(IPDMS)、二乙基异丙基硅烷基(DEIPS)、二甲基第三己基硅烷基、第三丁基二甲基硅烷基(TBDMS)、第三丁基二苯基硅烷基(TBDPS)、三苄基硅烷基、三-对二甲苯基硅烷基、三苯基硅烷基、二苯基甲基硅烷基(DPMS)、第三丁基甲氧苯基硅烷基(TBMPS)、甲酸酯、苄酰基甲酸酯、乙酸酯、氯乙酸酯、二氯乙酸酯、三氯乙酸酯、三氟乙酸酯、甲氧基乙酸酯、三苯基甲氧基乙酸酯、苯氧基乙酸酯、对氯苯氧基乙酸酯、3-苯基丙酸酯、4-侧氧基戊酸酯(乙酰丙酸酯)、4,4-(伸乙基二硫基)戊酸酯(乙酰丙酰基二硫缩醛)、新戊酸酯、金刚酸酯、巴豆酸酯、4-甲氧基巴豆酸酯、苯甲酸酯、对苯基苯甲酸酯、2,4,6-三甲基苯甲酸酯(均三甲苯甲酸酯)、碳酸甲酯、碳酸9-茀基甲酯(Fmoc)、碳酸乙酯、碳酸2,2,2-三氯乙酯(Troc)、碳酸2-(三甲基硅烷基)乙酯(TMSEC)、碳酸2-(苯磺酰基)乙酯(Psec)、碳酸2-(三苯基磷鎓基)乙酯(Peoc)、碳酸异丁酯、碳酸乙烯酯、碳酸烯丙酯、碳酸第三丁酯(BOC)、碳酸对硝基苯酯、碳酸苄酯、碳酸对甲氧基苄酯、碳酸3,4-二甲氧基苄酯、碳酸邻硝基苄酯、碳酸对硝基苄酯、硫代碳酸S-苄酯、碳酸4-乙氧基-1-萘酯、二硫代碳酸甲酯、2-碘苯甲酸酯、4-迭氮基丁酸酯、4-硝基-4-甲基戊酸酯、邻(二溴甲基)苯甲酸酯、2-甲酰基苯磺酸酯、2-(甲硫基甲氧基)乙基、4-(甲硫基甲氧基)丁酸酯、2-(甲硫基甲氧基甲基)苯甲酸酯、2,6-二氯-4-甲基苯氧基乙酸酯、2,6-二氯-4-(1,1,3,3-四甲基丁基)苯氧基乙酸酯、2,4-双(1,1-二甲基丙基)苯氧基乙酸酯、氯二苯基乙酸酯、异丁酸酯、单丁二酸酯、(E)-2-甲基-2-丁烯酸酯、邻(甲氧基酰基)苯甲酸酯、α-萘甲酸酯、硝酸酯、N,N,N',N'-四甲基二胺基磷酸烷基酯、N-苯基胺基甲酸烷基酯、硼酸酯、二甲基膦基硫酰基、2,4-二硝基苯基亚磺酸烷基酯、硫酸酯、甲烷磺酸酯(甲磺酸酯)、苄基磺酸酯及甲苯磺酸酯(Ts)。
在某些实施例中,存在于硫原子上的取代基为硫保护基(亦称为硫醇保护基)。硫保护基包括(但不限于)-Raa、-N(Rbb)2、-C(=O)SRaa、-C(=O)Raa、-CO2Raa、-C(=O)N(Rbb)2、-C(=NRbb)Raa、-C(=NRbb)ORaa、-C(=NRbb)N(Rbb)2、-S(=O)Raa、-SO2Raa、-Si(Raa)3、-P(Rcc)2、-P(Rcc)3、-P(=O)2Raa、-P(=O)(Raa)2、-P(=O)(ORcc)2、-P(=O)2N(Rbb)2及-P(=O)(NRbb)2,其中Raa、Rbb及Rcc如本文所定义。术语“保护基”,且尤其是硫保护基,为此项技术中熟知的且包括Protecting Groups in Organic Synthesis,T.W.Greene及P.G.M.Wuts,第3版,JohnWiley&Sons,1999中所述的那些保护基,所述文献以全文引用的方式并入本文中。
如本文所用,术语“离去基”具有合成有机化学技术中的一般含义且是指能够经亲核试剂置换的原子或基团。适合离去基的实例包括(但不限于)卤素(诸如F、Cl、Br或I(碘))、烷氧基羰氧基、芳氧基羰氧基、烷磺酰基氧基、芳烃磺酰基氧基、烷基-羰氧基(例如乙酰氧基)、芳基羰氧基、芳氧基、甲氧基、N,O-二甲基羟胺基、苯基呫吨基(pixyl)及卤基甲酸酯基。在一些情况下,离去基为磺酸酯基,诸如甲苯磺酸酯基(toluenesulfonate/tosylate,-OT)、甲烷磺酸酯基(甲磺酸酯基,-OM)、对溴苯磺酰基氧基酯基(溴苯磺酸酯基,-OB)或三氟甲磺酸酯基(三氟甲磺酸酯基,-OTf)。在一些情况下,离去基为溴苯磺酸酯基,诸如对溴苯磺酰基氧基。在一些情况下,离去基为硝基苯磺酸酯基,诸如2-硝基苯磺酰基氧基。在一些实施例中,离去基为含磺酸酯的基团。在一些实施例中,离去基为甲苯磺酸酯基。离去基亦可为氧化膦(例如在光延反应(Mitsunobu reaction)期间形成)或内部离去基,诸如环氧化物或环状硫酸酯。离去基的其他非限制性实例为水、氨、醇、醚部分、硫醚部分、卤化锌、镁部分、重氮盐及铜部分。
定义:
如本文所用,术语“聚醣”是指多醣、寡醣或单醣。聚醣可为糖残基的单体或聚合物且可为线性的或分支的。聚醣可以包括天然糖残基(例如,葡萄糖、N-乙酰葡糖胺、N-乙酰神经胺酸、半乳糖、甘露糖、海藻糖、己糖、***糖、核糖、木糖等)及/或经修饰的糖(例如,2'-氟核糖、2'-去氧核糖、磷酸甘露糖、6'磺酸基N-乙酰葡糖胺等)。聚醣在本文中亦用于指醣结合物的碳水化合物部分,诸如醣蛋白、醣脂、醣肽、醣蛋白质体、肽聚醣、脂多醣或蛋白聚醣。聚醣通常仅由单醣之间的O-糖苷键联组成。举例而言,纤维素为由β-1,4连接型D-葡萄糖构成的聚醣(或更特定而言,葡聚醣),且甲壳素为由β-1,4连接型N-乙酰基-D-葡糖胺构成的聚醣。聚醣可为单醣残基的均聚物或杂聚物,且可为线性的或分支的。可发现聚醣连接至蛋白质,如在醣蛋白及蛋白聚醣中。其通常于细胞外表面上发现。O连接型及N连接型聚醣在真核生物中很常见,而且可于原核生物中发现,虽然并不常见。
如本文所用,术语“海藻糖”、“核心海藻糖”及“核心海藻糖残基”可互换使用且是指在α1,6位置连接至N-乙酰葡糖胺的海藻糖。
如本文所用,术语“N-聚醣”、“N-连接型聚醣”、“N-连接型醣基化”、“Fc聚醣”及“Fc醣基化”可互换使用且是指连接至含Fc多肽中的天冬酰胺残基的酰胺氮的通过N-乙酰葡糖胺(GlcNAc)连接的N-连接型寡醣。术语“含Fc多肽”是指包含Fc区的多肽,诸如抗体。
如本文所用,术语“醣基化模式”及“醣基化图谱”可互换使用且是指以酶促方式或以化学方式自醣蛋白或抗体释放,且随后例如使用LC-HPLC或MALDI-TOF MS及其类似者分析其碳水化合物结构所得的N-聚醣物种的特征性“指纹”。参见例如Current AnalyticalChemistry,第1卷,第1期(2005),第28-57页中的综述;所述文献以全文引用的方式并入本文中。
如本文所用,“广谱中和HIV-1抗体”为中和多个HIV-1病毒株的中和抗体且可包括或不包括以下抗体范例中的任何一或多者:
如本文所用的“限定的聚醣探针位置”为固体载体上连接有某个密度的聚醣分子的预定区域,这些聚醣分子全部具有类似的聚醣结构。术语“聚醣区域”或“所选区域”或简单地“区域”在本文中可与术语限定的聚醣探针位置互换地使用。限定的聚醣探针位置可以具有任何适宜形状,例如圆形、矩形、椭圆形、楔形及其类似者。在一些实施例中,限定的聚醣探针位置小于约1cm2,或小于1mm2,或小于0.5mm2,且因此,上面连接有各个不同聚醣类型或一组各不相同的在结构上相关的聚醣的区域亦如此。在一些实施例中,聚醣探针位置的面积小于约10,000μm2或小于100μm2。连接在各限定的聚醣探针位置内的聚醣分子基本上相同。另外,在各限定的聚醣探针位置内存在有各聚醣类型的多个复本。在各限定的聚醣探针位置内的各聚醣类型的复本数目可为数千至数百万。
如本文所用,本发明的阵列具有限定的聚醣探针位置,各自具有“一种类型的聚醣分子”。所用的“一种类型的聚醣分子”可为结构基本上相同的一组聚醣分子或结构类似的一组聚醣分子。限定的聚醣探针位置内的每一个聚醣分子不需具有相同结构。在一些实施例中,在单一限定的聚醣探针位置内的聚醣为结构异构体,具有可变数目的糖单元或分支方式略有不同。然而,一般而言,在限定的聚醣探针位置内的聚醣具有基本上相同类型的糖单元及/或大致相同比例的各类型糖单元。限定的聚醣探针位置内的聚醣的糖单元上的取代基类型亦基本上相同。
结合的侦测可为直接的,例如通过侦测直接连接至测试分子的标签。或者,侦测可为间接的,例如通过侦测经标记二级抗体或其他可以结合至测试分子的经标记分子。所结合的标签可以使用任何可用的侦测方法观察。举例而言,可以采用阵列CCD分析仪来侦测结合至阵列的经化学发光标记的分子。
如本文所用,术语“抗原”定义为任何能够诱发免疫反应的物质。
如本文所用,术语“免疫原性”是指免疫原、抗原或疫苗刺激免疫反应的能力。
如本文所用,术语“抗原决定基”定义为抗原分子中接触抗体或T细胞受体的抗原结合位点的部分。
如本文所用,术语“疫苗”是指含有抗原的制剂,其由完整致病生物体(杀死或减毒)或这些生物体的组分(诸如蛋白质、肽或多醣)组成,用于赋予针对这些生物体所致的疾病的免疫力。疫苗制剂可为天然的、合成的或通过重组DNA技术获得。
如本文所用,术语“抗原特异性”是指细胞群体的一种特性,使得特定抗原或抗原片段的供应引起特异性细胞增殖。
如本文所用,术语“特异性结合”是指结合对(例如抗体与抗原)之间的相互作用。在各种情形下,特异性结合可体现为约10-6莫耳/公升、约10-7莫耳/公升或约10-8莫耳/公升或更小的亲和力常数。
“经分离”抗体为已鉴别且自其天然环境的组分分离及/或回收的抗体。其天然环境的污染物组分为会干扰抗体的研究、诊断或治疗用途的物质,且可包括酶、激素及其他蛋白质或非蛋白质溶质。在一个实施例中,抗体将纯化(1)至大于95重量%的抗体,如通过例如洛瑞法(Lowry method)所测定,且在一些实施例中超过99重量%;(2)至足以通过使用例如旋杯式定序仪获得N端或内部氨基酸序列的至少15个残基的程度;或(3)至均质,此是通过使用例如库马斯蓝(Coomassie blue)或银染色、在还原或非还原条件下进行SDS-PAGE所达成。经分离抗体包括在重组细胞内的原位抗体,因为抗体的天然环境的至少一种组分将不存在。然而,经分离抗体通常将通过至少一个纯化步骤来制备。
如在本文中可互换地使用的术语“载体”或“基板”是指一种材料或一组材料,其包含一种或多数种组分,一或多个分子与所述一种或多数种组分直接地或间接地结合、连接、合成、键联或以其他方式缔合。载体可以自生物材料、非生物材料、无机材料、有机材料或这些材料的组合构筑。载体可基于其在特定实施例内的用途而呈任何适当大小或组态。
如本文所用的术语“标靶”是指分析中感兴趣的物种。标靶可为天然产生的或合成的,或组合。标靶可不经更改(例如,直接用于生物体或其样品内),或以适合于分析的方式更改(例如,纯化、扩增、过滤)。在某些分析内,标靶可以经由适合手段结合至结合成员。标靶的非限制性实例包括(但不限于)抗体或其片段、细胞膜受体、可与特异性抗原决定子(诸如在病毒、细胞或其他物质上)反应的单株抗体及抗血清、药物、寡核苷酸、核酸、肽、辅因子、糖、凝集素、多醣、细胞、细胞膜及胞器。标靶可视分析而具有任何适合大小。
如本文所用,片语“基本上相似”、“基本上相同”、“等效”或“基本上等效”表示两个数值(例如一个与一种分子相关且另一个与参考/比较分子相关)之间具有足够高的相似度,使得在依据这些值(例如Kd值、抗病毒作用等)量度的生物学特征的背景下,熟习此项技术者认为所述两个值之间的差异具有极小或不具有生物学及/或统计学显著性。所述两个值之间的差异为例如小于约50%、小于约40%、小于约30%、小于约20%及/或小于约10%,随参考/比较分子的值而变。
如本文所用,片语“基本上降低”或“基本上不同”表示两个数值(通常,一个与一种分子相关且另一个与参考/比较分子相关)之间有足够大的差异程度,使得在依据这些值(例如Kd值)所量度的生物学特征的背景下,熟习此项技术者认为两个值之间的差异具有统计学显著性。所述两个值之间的差异为例如大于约10%、大于约20%、大于约30%、大于约40%及/或大于约50%,随参考/比较分子的值而变。
“结合亲和力”一般指分子(例如抗体)的单一结合位点与其结合搭配物(例如抗原)之间的非共价相互作用的强度总和。除非另外指明,否则如本文所用,“结合亲和力”是指反映结合对成员(例如抗体与抗原)之间1:1相互作用的固有结合亲和力。分子X对其搭配物Y的亲和力一般可由解离常数(Kd)表示。可通过此项技术中已知的常用方法(包括本文所述的那些方法)来量测亲和力。低亲和力抗体一般缓慢结合抗原且倾向于容易分解,而高亲和力抗体一般较快结合抗原且倾向于保持结合状态更久。此项技术中已知多种量测结合亲和力的方法,其中任一者可用于本发明之目的。特定说明性实施例描述于下文中。
如在本文中可互换地使用的“聚核苷酸”或“核酸”是指任何长度的核苷酸的聚合物且包括DNA及RNA。核苷酸可为去氧核糖核苷酸、核糖核苷酸、经修饰核苷酸或碱基及/或其类似物,或可通过DNA或RNA聚合酶或通过合成反应并入至聚合物中的任何受质。聚核苷酸可包含经修饰核苷酸,诸如甲基化核苷酸及其类似物。若存在,则可在聚合物组装之前或之后赋予对核苷酸结构的修饰。核苷酸的序列可杂有非核苷酸组分。聚核苷酸可在合成之后进一步修饰,诸如与标签结合。其他类型的修饰包括例如“封端”;用类似物取代天然产生的核苷酸中的一或多者;核苷酸间修饰,诸如那些具有不带电键联(例如膦酸甲酯、磷酸三酯、胺基磷酸酯、胺基甲酸酯等)及具有带电键联(例如硫代磷酸酯、二硫代磷酸酯等)的修饰,那些含有诸如蛋白质(例如核酸酶、毒素、抗体、讯息肽、聚-L-离胺酸等)的侧接部分的修饰,那些具有嵌入剂(例如吖啶、补骨脂素等)的修饰,那些含有螯合剂(例如金属、放射性金属、硼、氧化金属等)的修饰,那些含有烷基化剂的修饰,那些具有经修饰的键联(例如α变旋异构核酸等)的修饰,以及聚核苷酸的未经修饰的形式。此外,一般存在于糖中的任何羟基可例如由膦酸酯基、磷酸酯基置换,由标准保护基保护,或经活化以制备与额外核苷酸的额外键联,或可结合至固体或半固体载体。5'及3'端OH可经磷酸化或经胺或1至20个碳原子的有机封端基团部分取代。其他羟基亦可衍生成标准保护基。聚核苷酸亦可含有此项技术中通常已知的类似形式的核糖或去氧核糖,包括例如2'-O-甲基-、2'-O-烯丙基、2'-氟-或2'-迭氮基-核糖、碳环糖类似物、α-变旋异构糖、差向异构糖(诸如***糖、木糖或来苏糖)、哌喃醣、呋喃醣、景天庚酮糖、非环类似物及碱基核苷类似物(诸如甲基核糖苷)。一或多个磷酸二酯键可通过替代性键联基团替换。这些替代性键联基团包括(但不限于)如下实施例,其中磷酸酯经P(O)S(“硫代酸酯”)、P(S)S(“二硫代酸酯”)、(O)NR2(“酰胺化物”)、P(O)R、P(O)OR'、CO或CH2(“甲缩醛”)置换,其中各R或R'独立地为H或视情况含有醚(-O-)键的经取代或未经取代的烷基(1-20个C)、芳基、烯基、环烷基、环烯基或芳醛基。聚核苷酸中所有的键不需均相同。前述描述适用于本文中所提及的所有聚核苷酸,包括RNA及DNA。
如本文所用,“寡核苷酸”一般指短的、一般为单股的、一般性合成的聚核苷酸,其长度通常(但不一定)小于约200个核苷酸。术语“寡核苷酸”及“聚核苷酸”并非相互排斥的。以上关于聚核苷酸的描述同样且完全适用于寡核苷酸。
“抗体”(Ab)及“免疫球蛋白”(IG)为具有相同结构特征的醣蛋白。虽然抗体对于特定抗原展现结合特异性,但免疫球蛋白包括抗体及通常缺乏抗原特异性的其他抗体样分子。免疫球蛋白例如由淋巴***以低水准产生且由骨髓瘤以增加的水准产生。
术语“抗体”及“免疫球蛋白”可以最广泛含义互换使用,且包括单株抗体(例如全长或完整单株抗体)、多株抗体、单价抗体、多价抗体、多特异性抗体(例如双特异性抗体,只要其展现所要生物活性即可),且亦可包括某些抗体片段(如本文中更详细描述)。抗体可为嵌合、人类、人类化及/或经亲和力成熟的。
抗体的“可变区”或“可变域”是指抗体的重链或轻链的胺基端结构域。这些结构域通常为抗体最为多变的部分且含有抗原结合位点。
术语“可变”是指如下事实,在各抗体中可变域的某些部分在序列方面相差极大,且用于各特定抗体对于其特定抗原的结合及特异性。然而,可变性并非平均分布于抗体的整个可变域中。其集中于三个片段中,这些片段称为互补决定区(CDR)或轻链及重链可变域两者中的高变区。可变域的更高度保守部分称为框架(FR)。天然重链及轻链的可变域各自包含由三个CDR连接的四个FR区,这些FR区大体上采用β-褶板组态,这些CDR形成连接β-褶板结构的环且在一些情况下形成β-褶板结构的一部分。各链中的CDR通过FR区紧密地固定在一起,且通过其他链的CDR,有助于形成抗体的抗原结合位点(参见Kabat等人,Sequencesof Proteins of Immunological Interest,第五版,National Institute of Health,Bethesda,Md.(1991))。恒定域不直接参与抗体与抗原的结合,但展现各种效应子功能,诸如使抗体参与抗体依赖性细胞毒性。
以木瓜酶消化抗体会产生两个相同的抗原结合片段,称为“Fab”片段,各自具有单一抗原结合位点;以及残余“Fc”片段,其名字反映其容易结晶的能力。胃蛋白酶处理会产生F(ab')2片段,其具有两个抗原组合位点且仍能够交联抗原。
“Fv”为含有完整抗原识别及结合位点的最小抗体片段。在双链Fv形式中,此区由紧密、非共价缔合的一个重链可变域及一个轻链可变域的二聚体组成。在单链Fv形式中,一个重链可变域及一个轻链可变域可通过可挠性肽连接子共价连接,以便轻链及重链可以类似于双链Fv形式中的结构的“二聚”结构缔合。在此组态中,各可变域的三个CDR相互作用以界定VH-VL二聚体表面上的抗原结合位点。六个CDR共同地赋予抗体以抗原结合特异性。然而,即使单一可变域(或仅包含三个特异性针对抗原的CDR的Fv的一半)能够识别及结合抗原,但亲和力比完整的结合位点低。
Fab片段亦含有轻链的恒定域及重链的第一恒定域(CH1)。Fab'片段因为在包括一或多个来自抗体铰链区的半胱胺酸的重链CH1结构域的羧基端处添加几个残基而与Fab片段不同。Fab'-SH在本文中为恒定域的半胱胺酸残基携带游离硫醇基的Fab'的名称。F(ab')2抗体片段最初是以其间具有铰链半胱胺酸的Fab'片段对形式产生。抗体片段的其他化学偶联亦已知。
来自任何脊椎动物物种的抗体(免疫球蛋白)的“轻链”可基于其恒定域的氨基酸序列而归类为两种明显不同类型(称为κ及λ)中的一种。
视其重链恒定域的氨基酸序列,抗体(免疫球蛋白)可归为不同类别。免疫球蛋白存在五种主要类别:IgA、IgD、IgE、IgG及IgM,且其中若干者可进一步分成子类(同型),例如IgG1、IgG2、IgG3、IgG4、IgA1及IgA2。对应于不同类别的免疫球蛋白的重链恒定域分别称为α、δ、ε、γ及μ。不同类别的免疫球蛋白的亚单元结构及三维组态已熟知且大体描述于例如Abbas等人Cellular and Mol.Immunology,第4版(2000)中。抗体可为由抗体与一或多种其他蛋白质或肽共价或非共价结合所形成的较大融合分子的一部分。
术语“全长抗体”、“完整抗体”及“全抗体”在本文中可互换地用以指呈其基本上完整形式、不为如下文所定义的抗体片段的抗体。这些术语尤其是指具有含有Fc区的重链的抗体。
“抗体片段”包含完整抗体的仅一部分,其中所述部分保持与所述部分存在于完整抗体中时通常相关的功能中的至少一种功能,及多达大多数或全部功能。在一个实施例中,抗体片段包含完整抗体的抗原结合位点且因此保持结合抗原的能力。在另一实施例中,抗体片段(例如包含Fc区的抗体片段)保持与所述Fc区存在于完整抗体中时通常相关的生物学功能中的至少一者,诸如FcRn结合、抗体半衰期调节、ADCC功能及补体结合。在一个实施例中,抗体片段为活体内半衰期基本上类似于完整抗体的单价抗体。举例而言,此类抗体片段可包含连接至Fc序列的抗原结合臂,其能够赋予所述片段活体内稳定性。
如本文所用的术语“单株抗体”是指自基本上均质的抗体群体获得的抗体,亦即构成所述群体的个别抗体除了可以少量存在的可能天然产生的突变之外相同。因此,修饰语“单株”表示抗体的特征为并非离散抗体的混合物。所述单株抗体通常包括包含结合标靶的多肽序列的抗体,其中标靶结合多肽序列是通过包括自多数个多肽序列中选择单一标靶结合多肽序列的方法获得。举例而言,选择方法可为自多数个纯系,诸如一组融合瘤纯系、噬菌体纯系或重组DNA纯系选择独特纯系。应理解,所选标靶结合序列可经进一步改变,例如以增进对于标靶的亲和力、使标靶结合序列人类化、增进其于细胞培养物中的产量、降低其活体内免疫原性、产生多特异性抗体等,且包含经改变标靶结合序列的抗体亦为本发明的单株抗体。相比于通常包括针对不同决定子(抗原决定基)的不同抗体的多株抗体制剂,单株抗体制剂的各单株抗体是针对抗原上的单一决定子。除了其特异性之外,单株抗体制剂的有利的处在于其通常未受到其他免疫球蛋白污染。修饰语“单株”指示抗体的特征为自基本上均质的抗体群体获得,且不应理解为需要通过任何特定方法产生抗体。举例而言,待根据本发明使用的单株抗体可以通过各种技术制得,包括例如融合瘤方法(例如Kohler等人,Nature,256:495(1975);Harlow等人,Antibodies:A Laboratory Manual,(Cold SpringHarbor Laboratory Press,第2版1988);Hammerling等人,Monoclonal Antibodies andT-Cell hybridomas 563-681(Elsevier,N.Y.,1981))、重组DNA法(参见例如美国专利第4,816,567号)、噬菌体呈现技术(参见例如Clackson等人,Nature,352:624-628(1991);Marks等人,J.Mol.Biol.222:581-597(1992);Sidhu等人,J.Mol.Biol.338(2):299-310(2004);Lee等人,J.Mol.Biol.340(5):1073-1093(2004);Fellouse,Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004);及Lee等人,J.Immunol.Methods 284(1-2):119-132(2004)),以及用于在具有编码人类免疫球蛋白序列的部分或所有人类免疫球蛋白基因座或基因的动物中产生人类或人类样抗体的技术(参见例如WO98/24893;WO96/34096;WO96/33735;WO91/10741;Jakobovits等人,Proc.Natl.Acad.Sci.USA 90:2551(1993);Jakobovits等人,Nature 362:255-258(1993);Bruggemann等人,Year in Immunol.7:33(1993);美国专利第5,545,807号;第5,545,806号;第5,569,825号;第5,625,126号;第5,633,425号;第5,661,016号;Marks等人,Bio.Technology 10:779-783(1992);Lonberg等人,Nature 368:856-859(1994);Morrison,Nature368:812-813(1994);Fishwild等人,Nature Biotechnol.14:845-851(1996);Neuberger,Nature Biotechnol.14:826(1996);以及Lonberg及Huszar,Intern.Rev.Immunol.13:65-93(1995))。
本文中的单株抗体特别包括“嵌合”抗体,其中重链及/或轻链的一部分与源自特定物种或属于特定抗体类别或子类别的抗体中的对应序列相同或同源,而链的其余部分与源自另一物种或属于另一抗体类别或子类别的抗体中的对应序列相同或同源;以及此类抗体的片段,只要其展现所需生物活性即可(美国专利第4,816,567号;及Morrison等人,Proc.Natl.Acad.Sci.USA 81:6851-6855(1984))。
非人类(例如鼠类)抗体的“人类化”形式为含有源自非人类免疫球蛋白的最小序列的嵌合抗体。在一个实施例中,人类化抗体为人类免疫球蛋白(接受者抗体),其中来自接受者的高变区的残基经来自具有所需特异性、亲和力及/或能力的非人类物种(诸如小鼠、大鼠、兔或非人类灵长类动物)的高变区的残基(供体抗体)置换。在一些情况下,人类免疫球蛋白的构架区(FR)残基置换为相应非人类残基。此外,人类化抗体可包含在接受者抗体或供体抗体中未发现的残基。进行这些修饰以进一步改进抗体效能。一般而言,人类化抗体将包含至少一个且通常两个可变域中的基本上所有可变域,其中所有或基本上所有高变环对应于非人类免疫球蛋白的高变环且所有或基本上所有FR为人类免疫球蛋白序列的FR。人类化抗体视情况亦将包含免疫球蛋白恒定区(Fc)的至少一部分,通常是人类免疫球蛋白的至少一部分。关于其他细节,参见Jones等人,Nature 321:522-525(1986);Riechmann等人,Nature 332:323-329(1988);及Presta,Curr.Op.Struct.Biol.2:593-596(1992)。亦参见以下综述文章及其中所引用的参考文献:Vaswani及Hamilton,Ann.Allergy,Asthma&Immunol.1:105-115(1998);Harris,Biochem.Soc.Transactions 23:1035-1038(1995);Hurle及Gross,Curr.Op.Biotech.5:428-433(1994)。
如本文所用,“正常含量”可为例如基于来自正常患者或正常患者群体的样品中TACA结合的抗体的含量的量测结果的参考值或参考范围。“正常含量”亦可为例如基于来自正常患者或正常患者群体的样品中TACA的量测结果的参考值或参考范围。
如本文所用,“个体”为哺乳动物。此类哺乳动物包括驯养动物、农场动物、实验中所用的动物、动物园动物及其类似者。在一些实施例中,个体为人类。
“病症”为将受益于用本发明抗体治疗的任何病状。此病症包括慢性及急性病症或疾病,包括使哺乳动物易患所讨论的病症的那些病理学病状。本文中欲治疗的病症的非限制性实例包括HIV。
以下定义为本申请案中所用的较一般术语。
除非上下文另外明确指示,否则如本文中及所附申请专利范围中所使用,单数形式“一(a/an)”及“所述”包括多数个指示物。因此,举例而言,“输送增强剂”涵盖多数个输送增进剂以及单一输送增强剂。对“螯合剂”的提及包括对两种或更多种螯合剂以及单一螯合剂等的提及。在本说明书及随后的申请专利范围中,将提及多个术语,这些术语定义成具有以下含义:
如本文所用,术语“治疗(treating/treatment)”是指向罹患不良病状、病症或疾病的有临床症状的个体投与药剂或调配物,以便实现症状的严重程度及/或频率降低、消除症状及/或其潜在病因及/或有助于损伤好转或修复。术语“预防(preventing/prevention)”是指向易罹患特定不良病状、病症或疾病的无临床症状的个体投与药剂或组合物,且因此涉及预防症状出现及/或其潜在病因。除非本文中另外明确地或通过暗示指明,否则若在未提及可能的预防的情况下使用术语“治疗”,则亦意欲涵盖预防。
如“视情况选用的取代基”或“视情况存在的添加剂”中的“视情况选用”或“视情况存在”意谓随后所述的组分(例如取代基或添加剂)可以存在或可以不存在,使得描述包括组分存在的情况及组分不存在的情况。
“医药学上可接受”意谓材料在生物学上或在其他方面无不当,例如可以将材料并入至本发明调配物中而不会引起任何不当的生物效应或不会以有害方式与剂型调配物的其他组分中的任一者相互作用。然而,当术语“医药学上可接受”用以指医药赋形剂时,其暗示所述赋形剂已满足毒理学及制造测试所要求的标准及/或其包括在由美国食品药物管理局(the U.S.Food and Drug Administration)准备的非活性成分指南(InactiveIngredient Guide)上。如下文进一步详细解释,如“药理活性”衍生物或类似物中的“药理活性”(或简单地“活性”)是指衍生物或类似物具有与母本药剂相同类型的药理活性。
如本文所用,术语“抗原”定义为任何能够诱发免疫反应的物质。
如本文所用,术语“免疫原”是指抗原或能够诱导抗原产生的物质,诸如DNA疫苗。
本发明提供可用于鉴别哪些类型的蛋白质、受体、抗体、脂质、核酸、碳水化合物及其他分子及物质可以结合至指定聚醣结构的聚醣库及阵列。
本发明的阵列及方法亦提供高度准确的结果。本发明的库及阵列提供较大数目的且各种各样的聚醣。举一非限制性实例,本发明的库及阵列具有至少一个、至少两个、至少三个、至少十个或至少100个聚醣。在一些实施例中,本发明的库及阵列每阵列具有约1至约100,000、或约1至约10,000、或约1至约1,000、或约1至约100、或约2至约100、或约2至约10、或约1至约10个不同的聚醣。如此大数目的聚醣允许同时分析众多聚醣类型。如本文所述,本发明阵列已成功地用于筛选各种聚醣结合蛋白。本发明阵列上的聚醣的组成可以视情况而不同。可以将多种不同的聚醣结合物并入至本发明阵列中,包括例如天然产生的或合成的聚醣、醣蛋白、醣肽、醣脂、细菌及植物细胞壁聚醣及其类似者。
根据本发明的各种实施例经适当组态的例示性阵列包括例如美国专利第US8383554B2号“Quantitative Microarray of Intact Glycolipid CD1d Interactionand Correlation with Cell-Based Cytokine Production”;美国专利第US 8906832B2号“QUANTITATIVE ANALYSIS OF CARBOHYDRATE-PROTEIN INTERACTIONS USING GLYCANMICROARRAYS:DETERMINATION OF SURFACE AND SOLUTION DISSOCIATION CONSTANTS”、美国专利第US8680020B2号“Glycan arrays on PTFE-like aluminum coated glass slidesand related methods”、美国专利第US8507660B2号“Alpha-Selective Sialyl PhosphateDonors For Preparation Of Sialosides And Sialoside Arrays For Influenza VirusDetection”、美国公开案第US20150160217号(亦公开为WO2011130332A1)“Glycan ArraysFor High Throughput Screening Of Viruses”,其全部以其全文引用的方式并入本文中。
在一些实施例中,基板可以选自表面、固体表面、不透明固体、对可见或非可见光的所选波长透明的固体、粒子、阵列、微泡或珠粒。在一些实施例中,珠粒可以在表面上、嵌入表面中或连接到表面。在一些实施例中,基板可经涂布。
本发明的基板可为表面。表面可为平整的、有特征的、圆形、弯曲的、粗糙的、多孔的、固体、凝胶状、聚合物、寡聚物或珠粒。基板可以由玻璃、聚合物或塑胶构成。珠粒可为圆形、圆柱形、卵形、椭圆形、大致圆形、圆盘形、正方形、六角形或任何多面体形实体。在一些实施例中,基板可以经化学改质,从而在表面呈现能够结合至另一分子的反应性基团。在一些实施例中,反应性基团可为羧酸。在一些实施例中,珠粒可为二氧化硅珠粒。在一些实施例中,珠粒可为经二氧化硅官能化涂布的二氧化硅珠粒。在一些实施例中,二氧化硅官能化可为通过使二氧化硅珠粒与官能化硅烷分子接触达成的官能化。在一些实施例中,官能化硅烷分子可为官能化三氯硅烷、官能化二氯硅烷或官能化单氯硅烷分子。在一些实施例中,二氧化硅官能化可为通过使二氧化硅珠粒与官能化三烷氧基官能化硅烷接触达成的官能化。在一些实施例中,二氧化硅官能化可为通过使二氧化硅珠粒与官能化二烷氧基官能化硅烷接触达成的官能化。在一些实施例中,二氧化硅官能化可为通过使二氧化硅珠粒与官能化单烷氧基官能化硅烷接触达成的官能化。官能化可为胺基、醛、卤化物、环氧基、NHS(N-羟基丁二酰亚胺)、顺丁烯二酰亚胺、炔基、乙炔基、羰基(包括羧基)或羟基官能基。在一些实施例中,官能化可为经由C1-C6烷基、烷氧基或芳基与硅烷接触。
在某些态样中,用于使不同聚醣连接至本发明阵列的固定程序为易于控制的,从而容易构筑阵列。在一些实施例中,各聚醣可以黏附至特定的珠粒类型,从而与彼珠粒类型形成聚醣特异性结合。在一些实施例中,珠粒可以进一步包含区分特定珠粒类型与其他珠粒类型的独特标记物。在一些实施例中,各自黏附至独特珠粒类型的多数个不同聚醣可以在多重反应中混合。珠粒类型及多重反应侦测方法可为美国专利第6,696,304号及PCT专利申请案第PCT/US2004/038416号中所述者,其均以引用的方式并入本文中。
在一些实施例中,基板可用一种材料涂布,所述材料可以在表面呈现能够结合至另一分子的反应性基团。在一些实施例中,涂布基板的材料为硝化纤维素膜或聚合物。此类涂层呈现具有高表面积的3D表面,能够具有比平整表面更低的侦测极限。在一些实施例中,卵白素、抗生蛋白链菌素或中性抗生物素蛋白可以呈递至经涂布表面,诸如硝化纤维素膜涂层,以便连接生物素标记分子。在一些实施例中,化学连接基团可以呈递至表面,直接呈递至表面或呈递至先前已呈递至表面的涂层。
在一个实施例中,本发明提供可用于中各种应用中的连接基团。举例而言,本发明的连接基团可以用于使分子连接至基板,诸如表面、固体表面、粒子、阵列或珠粒。
在一个实施例中,本发明涉及一种用于疾病诊断及药物发现的珠粒,珠粒包含:(a)在各珠粒上或在各珠粒内的独特识别符;及(b)经由连接基团部分连接至珠粒表面的聚醣。聚醣可为本文所述的多样化聚醣中的任一者。在一些态样中,可以用某一聚醣类型的均质群体涂布珠粒。
在一些实施例中,本发明涉及制造聚醣-连接基团-珠粒的方法,包含(a)提供包含在各珠粒上或在各珠粒内的独特识别符的珠粒;(b)使聚醣-连接基团与珠粒接触;及(c)在聚醣-连接基团与珠粒之间形成结合物。在一些实施例中,在各珠粒上或在各珠粒内的独特识别符可为浸渍在二氧化硅或玻璃珠粒内的全像影像、连接至各珠粒的特异性氨基酸序列或寡核苷酸序列,或连接至各珠粒的特异性萤光团。在一些实施例中,连接至各珠粒的特异性氨基酸序列或寡核苷酸序列可以另外连接于连接基团部分,连接基团部分另外与连接至各珠粒的特异性聚醣连接。
在一些实施例中,本发明涉及通过以下方法制得的聚醣-连接基团珠粒,所述方法包含:(a)提供包含在各珠粒上或在各珠粒内的独特识别符的珠粒;(b)使聚醣-连接基团与珠粒接触;及(c)在聚醣-连接基团与珠粒之间形成结合物。
用于使不同聚醣连接至本发明阵列的固定程序为易于控制的,从而容易构筑阵列。在一些实施例中,各聚醣可以黏附至特定的珠粒类型,从而与彼珠粒类型形成聚醣特异性结合。在一些实施例中,珠粒可以进一步包含区分特定珠粒类型与其他珠粒类型的独特标记物。在一些实施例中,独特标记物可为氨基酸序列、寡核苷酸序列、萤光团或接触各珠粒类型或在各珠粒类型内的染料。在一些实施例中,萤光团或染料可为此项技术中已知的那些萤光团或染料。在一些实施例中,氨基酸序列或寡核苷酸序列可通过抗体或与接触珠粒的寡核苷酸序列互补的另一寡核苷酸序列侦测。在一些实施例中,与接触珠粒表面的寡核苷酸序列互补的寡核苷酸序列可以经一个或多数个萤光团标记。萤光团可为此项技术中已知的那些萤光团中的任一者(例如,FAM、Cy3、Cy5、Cy 7、Cy3.5、Cy5.5、BHQ猝灭剂、TAMRA、ROX、德克萨斯红(Texas Red)、Alexa fluor,截至本申请案的申请日在Molecular Probes/Invitrogen/ThermoFisher目录中所述的那些萤光团等)。在一些实施例中,各自黏附至独特珠粒类型的多数个不同聚醣可以在多重反应中混合。
包含黏附至阵列表面的固体载体上的限定区域的不同聚醣的独特库的阵列可以通过任何可用程序黏附。一般而言,通过以下制得阵列:获得本文所述的聚醣连接型醣肽分子的库,获得表面经改质以与存在于库的聚醣连接型醣肽分子上的特定连接部分反应的基板,且通过在聚醣连接型醣肽分子的连接部分与经改质的基板表面之间形成凡得瓦相互作用,使聚醣分子连接至固体载体。
改质试剂可以经由碳-碳键连接至基板,举一非限制性实例,使用硅氧烷键(例如使用玻璃或氧化硅或经活化的二氧化硅作为固体基板,其中经活化的二氧化硅为硅烷分子(例如,截至本申请案的申请日,可自Geleste,Inc.目录获得的那些硅烷分子,其以引用的方式并入本文中)的反应结果)。在一些实施例中,经由携带单、二或三氯硅烷基或者单、二或三烷氧基硅烷基的衍生化试剂的反应与基板表面形成硅氧烷键。硅烷上的非离去基(氯-或烷氧基-)可为烃。在一些实施例中,非离去基可为直链或分支链烷基链,从而与聚醣连接型醣肽的肽链形成凡得瓦相互作用。
改质试剂可以经由熟习此项技术者已知的其他用于施加涂层的沈积法而施加于基板。此类方法包括化学气相沈积、溶液相沈积、朗谬-布洛杰膜形成(Langmuir-Blodgettfilm formation)、用以暴露反应性表面分子的化学电浆处理、旋涂、喷雾干燥或电纺丝。在一些实施例中,改质试剂可为聚合物。聚合物可选自聚苯乙烯、聚丙烯、聚乙烯、聚乙亚胺、聚己内酰胺(polycaprolactine)、经改质的聚己内酯、聚甲基丙烯酸甲酯、聚丙烯酰胺、聚-N,N-烷基丙烯酰胺、聚甲基丙烯酸烷基酯、聚丙烯酸烷基酯、多醣或其共聚物。多醣可为纤维素、硝化纤维素、几丁聚醣、直链淀粉、乙酸纤维素、三仙胶、聚葡萄糖、威伦胶(welangum)、瓜尔胶(guar gum)、结冷胶(gellan gum)、代尤坦胶(diutan gum)或普鲁兰(pullulan)。多醣可以通过氧化基团的反应进一步官能化。在一些实施例中,氧化基团可为过碘酸钠。在一些实施例中,聚醣可以在还原剂存在下反应成多醣。在一些实施例中,还原剂可为官能化硼氢化物部分。在一些实施例中,官能化硼氢化物部分可为氢-硼氢化物(例如硼氢化钠或硼氢化钾、氰基硼氢化物或烷基硼氢化物部分)。
在一些实施例中,各类型的聚醣可以接触或印刷至固体载体上限定的聚醣探针位置。可使用微阵列基因印表机将各种聚醣连接型醣肽施加至限定的聚醣探针位置。举例而言,每限定的聚醣探针位置或珠粒可以施用约0.1微升至约10奈升(nL)或约0.5nL的聚醣溶液。各种浓度的聚醣连接型醣肽溶液可以接触或印刷至固体载体上。举例而言,可以采用约0.1至约1000微莫耳(μM)聚醣连接型醣肽或约1.0至约500μM聚醣连接型醣肽或约10至约100μM聚醣连接型醣肽的聚醣连接型醣肽溶液。一般而言,向若干(例如三个至六个)限定的聚醣探针位置的复本施加各浓度是适当的。此类复本提供内部对照,内部对照确认聚醣连接型醣肽与测试分子之间的结合反应是否为真正的结合相互作用。
侦测分子阵列适用于平行侦测样品中多个分析物的存在情况。侦测分子阵列的要素包含基板、在基板上呈现生物活性分子涂层、向经涂布基板呈现一个或多数个分析物、在基板上在分析物与生物活性分子之间形成复合物以及侦测机构。如本文所用,术语“生物活性分子”意谓其在此项技术中的普通含义及存在或模拟生物学或化学上已知的分子且能够经由静电、凡得瓦相互作用、疏水相互作用、共价键及/或氢键结合至另一分子的任何分子。
本发明的基板可为表面。表面可为平整的、有特征的、圆形、弯曲的、粗糙的、多孔的、固体、凝胶状、聚合物、寡聚物或珠粒。基板可以由玻璃、聚合物或塑胶构成。珠粒可为圆形、圆柱形、卵形、椭圆形、大致圆形、圆盘形、正方形、六角形或任何多面体形实体。在一些实施例中,基板可以经化学改质,从而在表面呈现能够结合至另一分子的反应性基团。在一些实施例中,反应性基团可为羧酸。
在一些实施例中,基板可用一种材料涂布,所述材料可以在表面呈现能够结合至另一分子的反应性基团。在一些实施例中,涂布基板的材料为硝化纤维素膜或聚合物。此类涂层呈现具有高表面积的3D表面,能够具有比平整表面更低的侦测极限。在一些实施例中,化学连接基团可以呈递至表面,直接呈递至表面或呈递至先前已呈递至表面的涂层。
在一些实施例中,基板可为官能化珠粒,如由Illumina,Inc.商业化的BeadXpress,或Luminex***,截至本发明的申请日。
本发明的一般态样
模组化合成高甘露糖型、混合型及复合型N-聚醣
如本文中所揭示的例示性方法实施例是基于以下设计的:可以通过组装所谓的“D1及D2/D3臂模组”,随后在常见核心三醣的甘露糖残基的3-O及/或6-O位置处进行特异性甘露醣基化而产生多样性。
基于N-聚醣结构的还原合成拆分(图1c),发明人意外发现,可以使用具有重要的β-甘露糖苷键联的一组模组化构建块1-13及核心三醣14-15(图2)作为起始物质来制备各种N-聚醣(G1-33,补充图S11)。为证明此策略的有效性及效用,首先以多公克规模以化学方式合成寡甘露糖型(单醣至五醣,1-5)、复合型(双醣至七醣,6-13)及核心三醣(14-15)构建块,其中在转化成氟化物之前安装临时变旋异构保护基。对于高甘露糖系列(Man3/Man4/Man5/Man9GlcNAc2)聚醣而言,将供体1及2立体选择性连接于14的3-O位置;且对于混合系列聚醣而言,则为供体6及7。随后,移除亚苄基环以得到4,6-二醇,且最后在6-O位置用供体1-7达成区域选择性醣基化。在醣基化反应过程中,视醣苷供体的选择采用各种启动子。将所有葡糖胺残基处的邻苯二甲酰亚胺保护修饰为乙酰胺,且去乙酰化且最后进行脱苄基以获得游离聚醣(图S1:聚醣1、2、4-9、12)。利用高特异性,使混合聚醣的复合型D1臂以酶促方式唾液酸化,得到α-2,3/6-Neu5Ac同功异型物(图S2-聚醣10-11、13-14)。
在确立了用于快速合成复合型聚醣(聚醣15-17、20-23、26-28及32-33,补充图S3)的方式之后,发明人开始使用以公克数量制备的构建块9-13产生更多样化的不对称聚醣且集中于由PG9及PG16识别的α-2,6-唾液酸化抗原(补充流程S4-S8)。随后使用氟化物供体8-13在AgOTf/Cp2HfCl2的促进下使核心15醣化。出人意料地,发现所有这些复合物结合非常澈底(补充流程S14-S19),且立体选择性极佳。8的醣基化产生异构体混合物。最后,整体脱除保护基得到二触角、三触角及四触角不对称N-聚醣的天然产生的位置异构体(补充图S3聚醣18、24、25、29-31)。为了研究核心海藻糖的作用,亦制备实例3及19。
通过此原位汇集合成策略,实施用于合成D1及D2/D3N-聚醣臂供体的化学酶促途径以快速组装多样化N-聚醣。使用各种醣苷基转移酶,通过酶促延长以化学方式合成的接受体16-20来制备线性及分支模组(图3),醣苷基转移酶包括β-1,4-半乳醣苷基转移酶、α-2,3/2,6唾液酸转移酶及α-1,3/1,2海藻醣苷基转移酶。通过使用β-1,4-半乳醣苷基转移酶及尿苷5'-二磷酸半乳糖(UDP-Gal)将接受体16的GlcNAc部分转化成LacNAc以形成21,其在胞苷-5'-单磷酸-N-乙酰神经胺酸(CMP-Neu5Ac)存在下通过α-2,6/2,3唾液酸转移酶进一步延长,分别得到标靶23及24。随后,用来自幽门螺旋杆菌(Helicobacter pylori)的α-1,3-海藻醣苷基转移酶(Hpα1,3FT)处理21、23-24,从而修饰LacNAc及α-2,3-唾液酸基LacNAc,但不修饰2,6-唾液酸基LacNAc,得到22及25。另外,发现α-1,3-海藻醣基化LacNAc限制末端半乳糖的酶促α-2,3/6-唾液酸化。在鸟苷5'-二磷酸-β-L-海藻糖(GDP-海藻糖)存在下,用来自HEK293细胞的α-1,2-海藻醣苷基转移酶修饰接受体21,得到26(图3a)。观察到,α-2,6唾液酸转移酶接受α-1,2海藻醣基化模组26,得到27,但α-2,3唾液酸转移酶不能接受此受质(图3a及补充流程S23)。随后,由接受体17-18制备对称模组28-33(图3b)。
在不对称模组的情况下,需要将唾液酸或海藻糖选择性并入触角中的一者。因此,按如下方式设计接受体19-20:通过乙酰化掩蔽4-羟基以防止酶促半乳醣基化同时保留其水溶性,从而将在甘露糖2-O位置处的GlcNAc与在4或6-O位置处的GlcNAc区分开来。此策略允许选择性延长一个臂,同时保持其他臂完整。如图3c中所描绘,通过β-1,4-GalT向β-1,4/1,6甘露糖分支处的GlcNAc残基添加Gal残基,然而在β-1,2分支处的GlcNAc残基保持完整。通过利用其特异性,使用α-1,3-FucT及α-2,6-SiaT制备不对称模组36-43及45-47(图3c及补充流程S25),其经纯化及完全表征(补充III)。
为了说明通过化学酶促法制备的模组于进一步醣基化的用途,选择模组21及22进行概念验证实验(图4)。使模组21及22全乙酰化,随后转化成醣苷氟化物,分别得到供体50及51。在AgOTf/Cp2HfCl2存在下,用氟化物50醣基化实际上以70%产率得到所期望的六醣52。随后,在对甲苯磺酸催化剂存在下裂解亚苄基,且在6-位置处立体特异性安装供体51,得到十醣54,脱除其保护基,得到聚醣55(补充流程S26及S27)。
综合而言,发明人已论证了一种有效的方式,经由恰当选择一组以化学方式以及以化学酶促方式产生的限定模组来制备感兴趣的复合型N-聚醣。寡醣苷氟化物供体的通用性允许高度分支模组与核心以极佳立体及区域选择性澈底结合。在还原端预安装烷基胺连接基团的寡醣可直接用于与NHS(N-羟基丁二酰亚胺,但环氧基官能化载片亦可)载片经由形成酰胺键而反应,或经进一步修饰或其他阵列形式,或结合至蛋白质以进行结构及功能研究。
在经NHS活化且涂有氧化铝的玻璃载片上的聚醣微阵列
据报导,PG9、PG16及PGT 128、141-145能够以很强的活性中和70-80%的循环HIV-1分离株20,表明所靶向的抗原决定基在HIV-1变异体中高度保守,且可指导免疫原的设计。为了深入了解这些抗体的聚醣特异性,发明人使用吾等新研发的阵列对HIV-1bNAb的配位体进行图谱分析。将合成的N-聚醣配位体经由与各100μM聚醣1-33形成酰胺键而印刷在经NHS活化的玻璃载片上(补充图S11)。各样品印刷五个复本且在与DyLight649结合的驴抗人类IgG抗体一起培育之后自萤光扫描获得载片影像。吾等的结果揭示,PG16结合至α-2,6-唾液酸化复合型寡醣,与吾等先前结果一致50,且结合亲和力与末端唾液酸残基的数目成比例(补充图S12)。另外,发明人发现,PG16结合不受核心海藻糖的存在情况所影响(聚醣19对比16)。令人感兴趣的是,PG16与不对称聚醣29-33的结合表明D1臂上的唾液酸的重要性。最后,发明人未观察到与高甘露糖型聚醣Man5GlcNAc2的结合(补充图S12)。另外,未侦测到PG9及PGT 141-145结合NHS阵列上的聚醣中的任一者,此可能是因为其结合极弱。(补充图S13-15)。在吾等的结合研究中,发明人观察到针对聚醣5Man4GlcAc2的萤光信号强烈,稍后证实其来自二级抗体的非特异性结合(补充图S16)。
为进一步理解为何结合至这些bNAb的聚醣不容易在涂有NHS的玻璃载片上观察到,发明人用ACG阵列进行特异性测试。ACG载片与涂有NHS的玻璃载片之间的均质性比较显示,ACG载片表面上的聚醣分布更平均(补充图S4-S9),且基于原子力显微镜(AFM),聚醣在ACG载片上的结构位向呈更为伸展的构形(补充图S10)。因此,发明人可以简单地调整聚醣的浓度来控制聚醣在ACG表面上的密度及距离。为制备代表性ACG阵列,将聚醣I-XI(图5a)连接于膦酸尾部以便自发性共价固定于ACG载片上(补充流程S21)。在与二级抗体一起培育之后,发明人测定与感兴趣抗体相互作用的聚醣的解离常数(KD.surf.)59。使用ACG载片,信号强度增强,如通过在ACG载片上使用1μg/mL的PG16相比于在涂有NHS的玻璃载片上使用25μg/mL所示(图5b及补充图S12)。PG16与二触角复合型N-聚醣(XI)的结合亲和力(KD=0.320μM)高于与混合型聚醣(X)的结合亲和力(KD=0.935μM)(补充图S19及表S2),支持所提出的在Asn173处存在这些聚醣,如与gp120复合的PG16的结构研究所提出30。
为评估ACG阵列形式是否可增强侦测灵敏度,发明人用各种浓度的PG9进行配位体特异性图谱分析。有趣的是,在ACG阵列上,PG9显示出对混合型聚醣(X)的表观特异性(图5b)以及与Man5GlcNAc2(IV)及α2,6-唾液酸化二触角复合型寡醣(XI)的可侦测的结合。先前显示,PG9在主要(Asn160)及次要(Asn156或Asn173)结合位点需要Man5GlcNAc2以及在gp120识别中需要一股短肽26,然而在Asn156或Asn173处的聚醣组成在随后的研究中限定为复合型聚醣30,31。在本发明研究中,相较于Man5GlcNAc2及复合型聚醣两者而言,PG9与混合型结构的强相互作用表明存在紧密相邻的混合型聚醣或寡甘露糖及复合型聚醣作为配位体。尽管如此,据我们所知,这些结果代表PG9结合至不含蛋白质或肽结构域的碳水化合物的第一证据。
为理解由抗体PGT 141-145识别的确切聚醣抗原决定基20,在ACG载片上制备一组聚醣I-XI进行分析。结果揭示,PGT 141-144可识别寡甘露糖聚醣Man3/5/9GlcNAc2,且观察到结合亲和力趋势是PGT142>PGT144>PGT141>PGT143(图5c及补充图S18);然而,所述组中最有效的PGT145却未显示可侦测到结合。PGT 141-144与Man9GlcNAc2的亲和力显著降低可能是因为末端甘露糖残基屏蔽了内核(Man3/5GlcNAc2)。综合而言,这些结果论证了ACG阵列形式在侦测最近分离的HIV-1bNAb的低亲和力相互作用方面的效率。
PG9及PG16的杂聚醣结合
因为与混合型聚醣复合的PG9不存在共晶结构,所以难以判定相互作用的分子详情。结构特征表明,PG9可容纳混合型聚醣中所存在的高甘露糖型D2/D3臂及复合型D1臂,或具有可以容纳在Asn160处的Man5GlcNAc2及在Asn156或173处的复合型聚醣的结合位点30,31;然而,复合型及混合型聚醣均含有α-2,6-NeuAc-Gal-GlcNAc臂。
为评估在gp120的Asn160及Asn156/Asn173处的最适合PG9结合袋的聚醣组合,发明人印刷两个不同的混合聚醣阵列。在一个阵列中,Man5GlcNAc2(IV)与I-XI的每一个聚醣以1:1的莫耳比混合(图5d),而在另一阵列中,二触角复合型结构(XI)与I-XI的每一个聚醣混合(图5e)。PG9与各种混合物的结合概况表明,含有Man5GlcNAc2及二触角聚醣的混合物[(IV+XI)或(XI+IV)]与PG9的相互作用比单独的IV或XI强。另外,发明人亦观察到和Man5与X及XI的组合的类似结合,表明在Asn160处的Man5GlcNAc2为主要结合位点,而结构IX、X及XI则用于复合型D1臂以与次要结合位点相互作用。基于PG9的均匀阵列结果(图5b),单独的Man5GlcNAc2IV或复合型聚醣XI似乎提供不了足够的结合亲和力;另一方面,混合型聚醣X显示结合显著增强。然而,在混合聚醣研究(图5c)中,发现IV与XI的组合与PG9达成了最强结合,其次为Man5与混合型的组合。发明人以类似方式研究PG16的结合特异性(图5e),且发现Man5GlcNAc2与复合型N-聚醣的组合(IV+XI)(KD=0.827μM)或混合与复合聚醣的组合(X+XI)(KD=0.988μM)比单独的复合型聚醣(KD=0.320μM)还弱(补充图S19及表S2)。这些结果表明唾液酸化触角在PG16结合位点中的重要性,可能包括先前报导的三触角及四触角复合型N-聚醣50。
为进一步理解混合物中IV与XI的确切比率,发明人进行了连续稀释实验。将IV与XI以各种比率(1:1/2/3/4/5)混合且反之亦然。将这些混合物中的每一者的100μM溶液与连接基团一起印刷在ACG表面上,且印刷单独的聚醣作为对照。有趣的是,在IV固定的情况下,随着混合物中的XI比例增加,PG9结合逐渐降低。通过在XI固定的情况下改变IV比例,PG9在1:1及2:1的IV:XI比下达成最强结合(图6a)。这些结果表明1:1的聚醣比率为PG9的最佳配位体。然而,PG16以不同于这些混合物中的每一者的方式反应,其中因为在混合物中存在复合型聚醣,相互作用大大增强(图6b)。然而,发明人不能侦测到PG16与Man5GlcNAc2的结合。发明人推断,PG9识别Man5与复合型聚醣的混合物,然而,单独的复合型聚醣就足以引起PG16反应。
结论
总之,发明人已成功地研发出一种模组化合成策略,用于以极高的纯度及足够的量快速产生高甘露糖型、混合型及复合型N-连接型寡醣的多样化阵列,使得有可能研究各种N-聚醣及研发用于测定新发现的HIV-1bNAb的聚醣特异性的新聚醣阵列平台。ACG阵列与以高产量方式获得的结合量测结果一起提供一种用于侦测HIV-1bNAb与聚醣的极微弱结合的有效手段,且能够发现并理解由抗体识别的必需抗原决定基及杂配位体。这些发现可以帮助快速设计针对HIV-1的基于碳水化合物的有效疫苗。
方法及概述
除非另外指出,否则所有反应均在惰性氛围下,使用无水溶剂在无水条件中进行。所有新化合物的全部实验详情、聚醣微阵列分析及表征资料(1H及13C核磁共振、高解析度质谱及Rf值)均包括在补充资讯I-III中。
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已采用的术语及表述用作描述而非限制的术语,且在使用这些术语及表述时不意欲排除所显示及描述的特征或其部分的任何等效语,但应认知到在如所主张的本发明的范畴内,各种修改为可能的。因此,应了解虽然本发明已由较佳实施例及视情况选用的特征进行具体揭示,但是熟习此项技术者可以对本文所揭示的概念进行修改及改变,且这些修改及改变视为在如由随附申请专利范围所限定的本发明的范畴内。
已在本文中广泛地且上位性地描述本发明。属于上位揭示内容内的较狭义物种及亚属组中的每一者亦形成本发明的一部分。此包括本发明的上位描述,其限制条件或负面限制为自所述类属中移除任何主题,无论所删除的材料是否在本文中具体叙述。
其他实施例在以下申请专利范围内。此外,当以马库什群组(Markush group)描述本发明的特征或态样时,熟习此项技术者应认识到,由此亦通过所述马库什群组的任何个别成员或成员亚群描述本发明。其他实施例在以下申请专利范围内。此外,当以马库什群组描述本发明的特征或态样时,熟习此项技术者应认识到,由此亦通过所述马库什群组的任何个别成员或成员亚群描述本发明。
实例:
本发明及本文中的实例证明以下发现:以阵列方式合成及产生N-聚醣具有出人意料的功效及效率,从而在疾病病况(例如HIV-1)测定、预测及/或诊断中达成出人意料的功效。
下表内容阐述本发明各种属性的实施例的范例:
化学合成
材料及方法
所有试剂均购自Sigma Aldrich,Across且不经进一步纯化即使用。无水溶剂购自商业来源,无需进一步蒸馏。用于醣基化的粉状分子筛(Aldrich)通过在350℃下加热3h进行活化。反应通过分析型薄层层析(TLC)在EM硅胶60F254盘中监测且在UV(254nm)下及/或通过用酸性钼酸铈铵或对茴香醛染色而可视化。急骤层析是在40-63μm粒度的硅胶(Merck)上执行。在25℃下在Bruker AVANCE 600(600MHz)光谱仪上记录1H NMR谱。所有1H化学位移(以ppm为单位)均根据CDCl3(δ=7.24ppm)及D2O(δ=4.80ppm)分配。13C NMR谱是通过Bruker AVANCE 600光谱仪获得且用CDCl3(δ=77.00ppm)校准。以赫兹(Hz)为单位报告耦合常数(J)。***模式是通过使用以下缩写来描述:s,单峰;brs,宽单峰;d,双重峰;t,三重峰;q,四重峰;dd,双重峰的双重峰;m,多重峰。1H NMR谱按以下次序报告:化学位移、多重性、耦合常数及质子数。所有NMR信号是基于1H NMR、COSY、HSQC、HMQC、TOCSY及13C实验分配。在Bruker Daltonics光谱仪上记录高解析度ESI质谱。涂有NHS的玻璃载片购自SCHOTT(Nexterion H)。广谱中和HIV抗体PG9、PG16及PGT141-145由NIH的Peter Kwong教授友情提供(PG9/PG16亦购自Polymun,Vienna Austria)。PGT128由TSRI的Dennis Burton教授友情提供。二级抗体DyLight649结合的驴抗人类IgG购自Jackson Immuno Research。胞苷5'-三磷酸(CTP)、N-乙酰神经胺酸(Neu5Ac)、UDP半乳糖、L-海藻糖及磷酸(烯醇)丙酮酸(PEP)购自Sigma-Aldrich。
NMR命名法.高甘露糖型、混合型及复合型寡醣的个别糖残基已如下所示进行标记。
通用程序S
酶表现、纯化及反应:
根据吾等先前报导3获得α-2,3唾液酸转移酶(JTFAJ-16)1及α-2,6唾液酸转移酶(JT-ISH-224)2的功能域。来自牛乳的酶β-1,4半乳糖苷转移酶购自Sigma。酶α-1,2海藻醣苷基转移酶(源自HEK-293细胞的人类Fut2)购自R&D SYSTEMS。此研究中所用的酶包括脆弱类杆菌(Bacteroides fragilis)L-海藻糖激酶/GDP-海藻糖焦磷酸化酶(FKP)、丙酮酸激酶(PK)、焦磷酸酶(PPA)、胞苷单磷酸激酶(CMK)、CMP-唾液酸合成酶(CSS)及来自幽门螺旋杆菌的α1-3-海藻醣苷基转移酶(Hp1-3FTΔ26695),为适合在我们的实验室中表现及纯化的重组性酶的实例。酶促反应与辅因子再生是根据自我们的团队4先前报导的程序进行。
整体脱除保护基:
方法1(关于在所有葡糖胺残基处经-N邻苯二甲酰胺保护的聚醣):将经保护的聚醣(50mmol)及10mL的乙二胺:nBuOH(1:4)的混合物在90℃下搅拌隔夜。随后蒸发挥发物且使粗产物与10mL Ac2O/吡啶(1:2)反应隔夜。使用高真空移除溶剂且通过急骤管柱层析(丙酮:甲苯,2/8,v/v)纯化产物。使用MeOH(10mL)中的甲醇钠使产物去乙酰化隔夜。使用IR-120中和反应混合物,过滤且在真空中浓缩。通过急骤管柱层析(丙酮:甲苯,3/7,v/v)纯化残余物。将产物溶解于10mL MeOH:H2O:HCOOH(6:3:1)中,添加Pd(OH)2(50重量%)且使反应混合物氢化隔夜。经由硅藻土过滤反应混合物且在真空中浓缩。通过Bio-Gel P-2(BIO-RAD)管柱层析,使用水作为溶离剂纯化残余物,且冻干产物,得到呈白色粉末状的所要寡醣。
方法2(关于在所有葡糖胺残基处经-NHTroc保护的聚醣):将经保护的聚醣(50mmol)及氢氧化锂(250mmol)于10mL的1,4二恶烷:H2O(4:1)中的混合物在90℃下搅拌隔夜。随后蒸发挥发物且使粗产物与10mL Ac2O:吡啶(1:2)反应隔夜。使用高真空移除溶剂且通过C18凝胶管柱层析(MeOH:H2O作为溶离剂)纯化产物。使用MeOH(10mL)中的甲醇钠使产物去乙酰化隔夜。使用IR-120中和反应混合物,过滤且在真空中浓缩。通过C18凝胶管柱层析(MeOH:H2O作为溶离剂)纯化残余物。将产物溶解于10mL MeOH:H2O:HCOOH(6:3:1)中,添加Pd(OH)2(50重量%)且使混合物氢化隔夜。经由硅藻土过滤反应混合物且在真空中浓缩。通过Bio-Gel P-2(BIO-RAD)管柱层析,使用水作为溶离剂纯化残余物。冻干产物,得到呈白色粉末状的所要寡醣。
酶促唾液酸化与辅因子再生:将聚醣(5μmol)、Neu5Ac(10μmol)、ATP(0.05μmol)、CTP(1μmol)、磷酸烯醇丙酮酸(10μmol,一钾盐)、胞苷单磷酸激酶(CMK,80个单位)、CMP-唾液酸合成酶(CSS,120个单位)、丙酮酸激酶(PK,40个单位)、焦磷酸酶(PPA,40个单位)及α2,6/2,3唾液酸转移酶(150个单位)溶解于50μmol Tris缓冲液(25mM,pH 7.5)中。将反应保温在37℃下且轻轻搅拌。通过质谱分析证实起始物质完全耗尽。使反应混合物离心且上清液经P2-Biogel凝胶过滤(溶离剂水)。将含有产物的部分合并且冻干,得到呈非晶白色固体状的各别产物。
酶促β-1,4-半乳醣基化:
将聚醣(1当量)及UDP半乳糖(2当量/半乳糖)溶解于含有MnCl2(10mM)的Tris缓冲液(25mM,pH 7.5)中。添加酶β-1,4-GalT-1(150个单位)以达成在2-5mM范围内的最终聚醣浓度。将所得反应混合物在37℃下保温24h。在二半乳醣基化情况下,当TLC显示单半乳醣基化中间体时,再添加UDP-半乳糖(2当量)、β-1,4-GalT(100个单位)且在37℃下保温直至中间体完全耗尽。使反应混合物离心且上清液经P2-Biogel凝胶过滤(溶离剂水)。将含有产物的部分合并且冻干,得到呈非晶白色固体状的各别产物。
酶促α-1,2/3-海藻醣基化:向聚醣(5μmol)、L-海藻糖(5μmol)、ATP(0.5μmol)、GTP(0.5μmol)、PEP(10μmol)及10mM MnCl2于25mM Tris缓冲液(pH 7.4)中的溶液中添加L-海藻糖激酶/GDP-海藻糖焦磷酸化酶(FKP,200个单位)、PK(200个单位)、PPA(200个单位)及α-1,2/1,3-海藻醣苷基转移酶(200个单位),且将混合物在37℃下保温隔夜。使反应混合物离心且上清液经P2-Biogel凝胶过滤(溶离剂水)。将含有产物的部分合并且冻干,得到呈非晶白色固体状的各别产物。
合成构建块1-13S
合成单醣、三醣及五醣构建块1-5。
通过所报导的程序5获得甘露糖基三氯乙酰亚胺酸酯构建块1。Man9GlcNAc2的D1臂三醣2的制备开始如下:氯化甘露糖基S1a6及硫苷接受体S1b7在2,6-二第三丁基吡啶(DTBP)及三氟甲磺酸银(AgOTf)处理下醣基化,获得双醣S1c(流程S1)。使S1c在2-O位置Zemplén去乙酰化,随后通过采用DTBP及AgOTf,用S1a醣基化,以93%产率得到所要三醣S1e。在N-溴代丁二酰亚胺(NBS)及三氟化二乙基胺基硫(DAST)的作用下,对S1e进行自硫苷至醣苷氟化物2的离去基修饰。就用壳二糖三醣醣基化期间的α-选择性及极佳产率而言,氟化物转化为吾人提供较佳结果。在AgOTf的活化下,发生供体S1a6与接受体S1f7的缩合,得到三甘露糖3,其随后去乙酰化且用S1a进一步二醣基化,以71%产率得到D2/D3臂五醣5。接着使用NBS及DAST使化合物3经历离去基修饰,以68%产率得到三甘露醣苷氟化物4。仔细观察吾人的策略,发现采用单一甘露糖基氯化物供体S1a及独特的DTBP/AgOTf介导的醣基化条件可以极佳产率得到D1、D2/D3臂三醣及五醣中间体。
流程S1|制备化合物1-5.i,DTBP、AgOTf、MS、CH2Cl2,-30℃至室温,隔夜;S1c:93%,S1e:91%,3:66%,5:74%;ii,NaOMe、MeOH:CH2Cl2=1/1;S1d:89%,S1g:90%;iii,N-溴代丁二酰亚胺(NBS)、DAST、CH2Cl2,-30℃至-10℃;2:58%,4:61%。DTBP:2,6-二第三丁基吡啶;DAST:三氟化二乙基胺基硫;AgOTf:三氟甲磺酸银。
对甲苯基-2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→2)-3,4,6-三-O-苄基-1-硫基-α-D-甘露哌喃糖苷(S1c):向接受体S1b(1.70g,3.05mmol)及供体S1a(2.34g,4.58mmol)于20mL CH2Cl2中的溶液中添加经活化的分子筛且在室温下搅拌1h。在另一个烧瓶中,将10mL CH2Cl2中的AgOTf(1.19g,4.58mmol)及DTBP(1.03mL,4.58mmol)与MS一起搅拌1h。将含有AgOTf/DTBP的烧瓶冷却至-30℃且历经5min添加含有供体与接受体的混合物的溶液。搅拌溶液,且历经24h逐渐升温至室温。TLC(乙酸乙酯:己烷,2/8)指示产物形成且起始物质耗尽,用Et3N淬灭反应,经由硅藻土过滤,用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层,且在真空中浓缩。通过硅胶管柱层析(0%→15%EA/己烷)纯化残余物,得到呈无色泡沫状的S1c(2.90g,93%)。TLC(乙酸乙酯:己烷=2/8v/v):Rf=0.35;1H NMR(600MHz,CDCl3):δ7.38-7.16(m,30H,Ar-H),7.11-7.09(m,2H,Ar-H),7.00(d,J=8.4Hz,2H,Ar-H),5.44(s,1H,H-1a),5.51(d,J=2.4Hz,1H),5.06(s,1H,H-1b),4.88(d,J=11.8Hz,1H),4.81(d,J=11.2Hz,1H),4.73-4.37(m,10H),4.29(t,J=8.9Hz,1H),4.22(s,1H),3.96-3.88(m,4H),3.83-3.71(m,2H),3.69-3.63(m,2H),3.55(d,J=12.1Hz,1H),2.26(s,3H,-C(O)CH3),2.13(s,3H,STol的-CH3);13C NMR(150MHz,CDCl3):δ170.49,138.75,138.67,138.62,138.42,138.32,138.24,137.87,132.55,130.51,128.77,128.64,128.61,128.56,128.52,128.39,128.29,128.20,128.15,128.03,127.99,127.94,127.78,127.63,99.99,87.78,80.19,78.35,76.88,75.48,75.32,75.03,74.56,73.46,73.08,72.47,72.21,72.18,68.48,69.02,68.85,21.42,21.36;ESI-MS:m/zC63H66O11S计算值1030.4218;实验值1053.4228(M+Na)+。
对甲苯基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→2)-3,4,6-三-O-苄基-1-硫基-α-D-甘露哌喃糖苷(S1d):向化合物S1c(1.01g,0.970mmol)于20mL的甲醇:CH2Cl2(1/1)中的溶液中添加甲醇钠(0.024g,0.42mmol),在室温下搅拌直至TLC(乙酸乙酯:己烷,3/7)指示产物形成且起始物质耗尽。用IR-120中和反应混合物,过滤且在真空中浓缩且通过硅胶管柱层析(0%→30%EA/己烷)纯化残余物,得到呈无色油状的S1d(0.859g,89%)。TLC(乙酸乙酯:己烷=3/7v/v):Rf=0.29;1H NMR(600MHz,CDCl3):δ7.33-7.10(m,32H,Ar-H),6.96(d,J=7.8Hz,1H,Ar-H),5.56(s,1H,H-1),5.11(s,1H,H-1),4.85(d,J=10.8Hz,1H),4.75(d,J=10.8Hz,1H),4.69-4.62(m,3H),4.52-4.38(m,6H),4.37(d,J=12.1Hz,1H),4.25(d,J=12Hz,2H),4.09(s,1H),3.90-3.77(m,4H),3.70(d,J=10.8Hz,1H),3.62(dd,J=4.8,8.2Hz,1H),3.54(d,J=10.2Hz,1H),2.23(s,3H,CH3,STol);13C NMR(150MHz,CDCl3):δ138.61,138.42,138.33,138.24,137.97,137.51,132.18,130.38,129.73,128.55,128.47,128.38,128.29,128.24,127.93,127.91,127.89,127.86,127.85,127.78,127.68,127.57,127.48,127.45,127.33,101.23,87.62,80.06,79.99,76.52,75.18,75.01,74.95,94.35,73.21,73.18,72.87,72.38,72.18,71.69,69.28,68.70,68.56,19.45;ESI-MS:m/z C61H64O10S计算值988.4112;实验值1011.4125(M+Na)+。
对甲苯基-2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→2)-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→2)-3,4,6-三-O-苄基-1-硫基-α-D-甘露哌喃糖苷(S1e):向接受体S1d(2.6g,2.62mmol)及供体S1a(2.01g,3.94mmol)于10mL CH2Cl2中的溶液中添加经活化的分子筛且在室温下搅拌1h。在另一个烧瓶中,将30mL CH2Cl2中的AgOTf(1.02g,3.94mmol)及DTBP(893μL,3.94mmol)与经活化的分子筛一起搅拌1h。将AgOTf/DTBP的混合物冷却至-30℃且历经5min添加供体及接受体的溶液。搅拌溶液,且历经24h逐渐升温至室温,直至TLC(乙酸乙酯:己烷,2/8)指示产物形成且起始物质耗尽。用Et3N淬灭反应,经由硅藻土过滤。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层,过滤且在真空中浓缩。通过硅胶管柱层析(0%→20%EA/己烷)纯化残余物,得到呈无色泡沫状的S1e(3.50g,91%)。TLC(乙酸乙酯:己烷=2/8v/v):Rf=0.41;1H NMR(600MHz,CDCl3):δ7.34-7.11(m,45H,Ar-H),7.05-7.03(m,2H,Ar-H),6.93(d,J=7.2Hz,1H,Ar-H),5.65(s,1H,H-1),5.50(d,J=3.0Hz,1H,H-1),5.10(d,J=3.2Hz,1H,H-1),4.82(dd,J=3.1,10.2Hz,2H),4.75(d,J=12.1Hz,1H),4.68(s,2H),4.60-4.54(m,8H),4.35-4.19(m,9H),3.90-3.84(m,5H),3.78-3.72(m,2H),3.66-3.63(m,3H),3.55-3.52(m,1H),3.46(d,J=4.2Hz,1H),3.41(dd,J=3.4,9.2Hz,1H),3.31(d,J=9.8Hz,1H),2.20(s,3H,-C(O)CH3),2.05(s,3H,CH3,STol);13C NMR(150MHz,CDCl3):δ170.84,139.12,138.85,138.79,138.65,138.60,138.52,138.37,138.25,137.94,137.60,132.30,130.75,129.97,129.90,128.98,128.63,128.52,128.33,128.20,128.03,127.88,127.64,100.79,96.52,88.06,80.83,80.45,76.45,76.42,76.04,75.65,75.21.75.14,74.69,73.63,73.05,73.01,72.95,71.99,71.87,71.43,69.66,69.51,69.35,68.40,68.34,21.40,21.36,21.31;ESI-MS:m/z C90H94O16S计算值1462.6155;实验值1485.6190(M+Na)+。
2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→2)-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→2)-3,4,6-三-O-苄基-1-α-D-甘露哌喃醣苷氟化物(2):在-30℃下,向三醣S1e(0.600g,0.410mmol)于CH2Cl2(10mL)中的溶液中添加NBS(0.218mg,1.23mmol),搅拌10分钟。缓慢地添加DAST(324μL,2.46mmol)且在-10℃下搅拌所得反应混合物6h。TLC(乙酸乙酯:己烷,3/7)指示产物形成且起始物质耗尽,用NaHCO3水溶液淬灭反应,且用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层,过滤且在真空中浓缩。通过硅胶管柱层析(0%→25%EA/己烷)纯化残余物,得到呈白色泡沫状的氟化物2(0.320g,58%)及0.200g副产物醇(变旋异构-OH)。TLC(乙酸乙酯:己烷=3/7,v/v):Rf=0.31;1H NMR(600MHz,CHCl3):δ7.32-7.12(m,45H,Ar-H),5.67(d,J=50.4Hz,1H,Ar-H),5.49(s,1H,H-1),5.16(s,1H,H-1),4.99(s,1H,H-1),4.82-4.77(m,3H),4.65-4.38(m,15H),4.30(d,J=12.1Hz,1H),4.03(s,1H),3.97-3.94(m,3H),3.90-3.64(m,11H),3.54(d,J=10.2Hz,1H),2.11(s,3H,-C(O)CH3);13C NMR(150MHz,CDCl3):δ170.44,138.77,138.64,138.60,138.54,138.46,138.41,138.28,128.76,128.64,128.61,128.54,128.44,128.31,128.19,128.13,128.09,128.04,128.01,127.95,127.90,127.87,127.84,127.79,127.73,107.65,106.19,101.04,99.72,78.56,78.31,75.45,75.41,75.31,74.86,74.59,74.31,74.10,73.86,73.68,73.65,73.57,72.69,72.49,72.38,72.17,69.51,69.31,69.01,68.95,21.44;ESI-MS:m/z C83H87FO16计算值1358.5870;实验值1381.5891(M+Na)+。
对甲苯基-2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)-2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→6)-2,4-二-O-苄基-1-硫基-α-D-甘露哌喃糖苷(3):向接受体S1f(0.545g,1.23mmol)及供体S1a(1.57g,3.07mmol)于CH2Cl2(10mL)中的溶液中添加经活化的MS且在室温下搅拌1h。在另一个烧瓶中,将10mL CH2Cl2中的AgOTf(0.787g,3.07mmol)及DTBP(690μL,3.07mmol)与MS一起搅拌。搅拌1h后,将含有AgOTf/DTBP的烧瓶冷却至-30℃且历经5min添加含有供体与接受体的混合物的溶液。搅拌溶液,且历经24h逐渐升温至室温。TLC(乙酸乙酯:己烷,2/8)指示产物形成且起始物质耗尽。用Et3N淬灭反应,经由硅藻土过滤,用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层,过滤且在真空中浓缩。通过硅胶管柱层析(0%→20%EA/己烷)纯化残余物,得到呈无色泡沫状的3(1.10g,66%)。TLC(乙酸乙酯:己烷=2/8,v/v):Rf=0.36;1H NMR(600MHz,CDCl3):δ7.35-7.10(m,32H,Ar-H),7.05(d,J=8.0Hz,2H,Ar-H),5.51(s,1H),5.47(s,1H,H-1),5.45(dd,J=2.0,2.8Hz,1H),5.21(s,1H,H-1),4.91(d,J=1.5Hz,1H,H-1),4.86(t,J=10.2Hz,2H),4.75(d,J=11.2Hz,1H),4.67-4.58(m,5H),4.54-4.36(m,8H),4.23(dd,J=3.9,9.6Hz,1H),4.12-4.06(m,2H),4.01(dd,J=3.2,9.2Hz,1H),3.97-3.86(m,5H),3.82(t,J=9.2Hz,1H)3.79-3.75(m,1H),3.72(dd,J=3.9,10.7Hz,1H),3.70-3.56(m,4H),2.17(s,3H,-C(O)CH3),2.13(s,3H,-C(O)CH3),2.09(s,3H,CH3,STol);13C NMR(150MHz,CDCl3):δ170.27,170.10,138.57,138.53,138.18,138.16,137.81,137.77,137.42,131.46,130.85,129.83,128.44,128.42,128.36,128.27,128.21,128.06,127.81,127.72,127.68,127.63,127.53,127.48,99.83,98.17,85.23,78.98,78.08,77.76,75.15,74.98,74.89,74.32,74.15,73.52,73.30,72.23,72.05,71.85,71.48,71.43,71.36,69.05,68.72,68.60,68.41,66.60,60.39,21.15,21.00,20.94;ESI-MS:m/zC85H90O17S计算值1414.5791;实验值1437.5821(M+Na)+。
对甲苯基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→6)]-2,4-二-O-苄基-1-硫基-α-D-甘露哌喃糖苷(S1g):向三醣3(1.30g,0.918mmol)于10mL甲醇:CH2Cl2(1/1)中的溶液中添加甲醇钠(0.024g,0.459mmol),在室温下搅拌直至TLC(乙酸乙酯:己烷,3/7)指示产物形成且起始物质耗尽。用IR-120中和反应混合物,过滤且在真空中浓缩且通过硅胶管柱层析(0%→25%EA/己烷)纯化残余物,得到呈无色油状的S1g(1.10g,90%)。TLC(乙酸乙酯:己烷=3/7,v/v):Rf=0.26;1H NMR(600MHz,CDCl3):δ7.36-7.12(m,42H,Ar-H),7.06(d,J=8.0Hz,2H,Ar-H),5.46(s,1H,H-1),5.24(s,1H,H-1),5.01(d,J=1.1Hz,1H,H-1),4.82(t,J=11.1Hz,2H),4.69(d,J=11.6Hz,1H),4.65-4.44(m,13H),4.23(dd,J=4.1,9.6Hz,1H),4.16(s,1H),4.07(dd,J=2.8,9.4Hz,1H),4.03(d,J=10.1Hz,1H),3.96-3.88(m,4H),3.87-3.81(m,3H),3.80-3.75(m,1H),3.73-3.64(m,4H),3.62(dd,J=1.6,10.8Hz,1H),2.38(s,1H),2.34(s,1H,-C(O)CH3),2.19(s,3H,CH3,STol);13C NMR(150MHz,CDCl3):δ138.12,138.11,137.69,131.75,130.12,128.82,128.76,128.73,128.67,128.63,128.57,128.54,128.53,128.41,128.35,128.29,128.19,128.12,128.08,128.03,127.95,128.92,127.82,124.43,99.90,85.55,80.32,80.00,78.49,79.46,75.46,75.41,75.27,75.19,75.13,74.66,74.50,74.39,73.85,73.81,73.62,72.32,72.24,71.85,71.71,71.40,71.33,69.48,69.00,68.96,68.90,68.32,66.51,21.15;ESI-MS:m/z C81H86O15S计算值1330.5580;实验值1353.5614(M+Na)+。
2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)-2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→6)-2,4-二-O-苄基-α-D-甘露哌喃醣苷氟化物(4):在-30℃下,向化合物3(0.270g,0.197mmol)于CH2Cl2(10mL)中的溶液中添加NBS(0.052g,0.296mmol),搅拌10min。随后缓慢地添加DAST(52μL,0.395mmol)且在-10℃下搅拌所得反应混合物4h。TLC(乙酸乙酯:己烷,3/7)指示产物形成且起始物质耗尽。用NaHCO3水溶液淬灭反应混合物,且用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过硅胶管柱层析(0%→8%EA/甲苯)纯化残余物,得到呈白色泡沫状的4(0.150g,61%)。TLC(乙酸乙酯:甲苯=1/9,v/v):Rf=0.19;1H NMR(600MHz,CHCl3):δ7.29-7.10(m,40H,Ar-H),5.50(s,1H,H-1),5.47(d,J=50Hz,1H,H-1),5.46(s,1H,H-1),5.18(s,1H),4.92(s,1H),4.86(d,J=11.2Hz,1H),4.83(d,J=10.8Hz,1H),4.72(d,J=10.0Hz,1H),4.72-4.56(m,6H),4.48-4.40(m,7H),4.10(d,J=8.9Hz,1H),4.00-3.92(m,4H),4.90-3.77(m,5H),3.71-3.64(m,5H),3.57(d,J=7.2Hz,1H),2.13(s,3H,-C(O)CH3),2.06(s,3H,-C(O)CH3);13C NMR(150MHz,CHCl3):δ170.56,170.36,138.80,138.74,138.50,138.39,138.11,138.04,137.89,137.88,129.70,128.81,128.70,128.68,128.65,128.58,128.56,128.54,128.43,128.27,128.18,128.12,128.07,128.04,128.00,127.97,127.89,127.82,126.55,106.66,105.18,100.02,98.84,98.81,78.31,77.70,76.31,76.03,75.42,75.37,75.23,74.62,74.41,74.27,73.90,73.80,73.65,73.16,72.66,72.14,71.92,71.58,69.42,68.96,68.70,68.61,66.53,42.20,32.21,29.98,29.65,22.98,21.63,21.44,21.27,14.71,14.41;ESI-MS:m/z C78H83O17F计算值1310.5507;实验值1333.5536(M+Na)+。
对甲苯基-2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→2)-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)-[2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→2)-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→6)]-2,4-二-O-苄基-1-硫基-α-D-甘露哌喃糖苷(5):向接受体S1g(1.01g,0.739mmol)及供体S1a(0.947g,1.84mmol)于20mLCH2Cl2中的溶液中添加经活化的MS且在室温下搅拌1h。在另一个烧瓶中,将10mLCH2Cl2中的AgOTf(0.472g,1.85mmol)及DTBP(415μL,1.85mmol)与MS一起搅拌。搅拌1h后,将含有AgOTf/DTBP的烧瓶冷却至-30℃且历经5min添加含有供体及接受体于CH2Cl2中的混合物的溶液。搅拌溶液,且历经24h逐渐升温至室温。TLC(乙酸乙酯:己烷,3/7)指示产物形成且起始物质耗尽。随后用Et3N淬灭反应,经由硅藻土过滤,且用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层,过滤且在真空中浓缩。通过硅胶管柱层析(0%→25%EA/己烷)纯化残余物,得到呈白色固体状的5(1.25g,74%)。TLC(乙酸乙酯:己烷=3/7,v/v):Rf=0.33;1H NMR(600MHz,CDCl3):δ7.27-7.04(m,72H,Ar-H),7.02(d,J=8.0Hz,2H,Ar-H),5.52(d,J=1.5Hz,2H,2x H-1),5.46(s,1H,H-1),5.18(s,1H,H-1),5.04(d,J=8.2Hz,1H),4.85(t,J=11.0Hz,2H),4.78(t,J=11.1Hz,2H),4.72-4.44(m,18H),4.41-4.30(m,8H),4.27(d,J=12.3Hz,2H),4.20(s,2H),4.05-3.92(m,6H),3.91-3.73(m,8H),3.68-3.56(m,6H),3.48(d,J=10.1Hz,1H),3.40(d,J=10.3Hz,1H),3.34(d,J=10.5Hz,1H),2.10(s,6H,-C(O)CH3),2.01(s,3H,CH3,STol);13C NMR(150MHz,CDCl3):δ170.22,152.99,152.78,152.57,138.43,138.38,138.24,138.05,138.02,137.86,137.81,131.15,129.80,128.44,128.30,128.20,128.05,127.95,127.85,127.72,127.70,127.62,127.55,127.53,127.47,127.41,127.38,127.35,127.23,126.94,101.16,99.37,98.86,84.97,79.24,78.07,78.02,75.22,74.99,74.87,74.77,74.55,74.47,74.29,74.17,73.94,73.82,73.38,73.24,73.18,73.01,72.58,71.93,71.85,71.77,71.60,71.55,71.47,70.99,69.41,68.64,68.57,68.46,68.01,29.69,21.22,20.91;ESI-MS:m/zC139H146O27S计算值2279.9698;实验值2302.9747(M+Na)+。
合成双醣构建块6.
通过NIS及TfOH介导的接受体S2a8与供体S2b9的缩合以64%产率得到含唯一的β-键联(J1',2'=8.5Hz)的双醣S2c。接着经由变旋异构去烯丙基化、随后在DAST存在下将游离-OH转化为-F而将化合物S2c修饰为氟化物6(流程S2)。
流程S2|制备化合物6.i,NIS、TfOH、CH2Cl2、MS,-40℃,2h,64%;ii,(1)PdCl2、MeOH:CH2Cl2=1/1,(2)DAST、CH2Cl2,-30℃至-10℃,3-5h,61%。
烯丙基-O-4-O-乙酰基-3,6-O-二苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基-(1→2)-O-3,4,6-三-O-苄基-α-D-甘露哌喃糖苷(S2c):将硫苷供体S2b(2.33g,3.65mmol)、接受体S2a(1.50g,3.05mmol)及经活化的分子筛(3g)于30mL无水CH2Cl2中的混合物在室温下搅拌1h。在-40℃下缓慢地添加NIS(1.35g,6.01mmol)及TfOH(66.2μL,0.75mmol)且搅拌2h直至TLC(乙酸乙酯:甲苯,1/9)指示产物形成且起始物质耗尽。用Et3N淬灭反应混合物,经由硅藻土过滤,且用NaHCO3水溶液(2×50mL)、Na2S2O3水溶液(2×50mL)洗涤滤液,且最后用盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→10%EA/甲苯)纯化残余物,得到呈白色固体状的S2c(2.10g,64%)。TLC(乙酸乙酯:甲苯=1/9,v/v):Rf=0.29;1H NMR(600MHz,CDCl3):δ7.52-7.50(m,4H,Ar-H),7.33-7.10(m,16H,Ar-H),7.10-7.09(m,4H,Ar-H),7.00-6.90(m,2H,Ar-H),6.89-6.81(m,3H,Ar-H),5.73-5.71(m,1H,-OCH2-CH=CH2),5.26(d,J=8.4Hz,1H,H-1),5.14-5.06(m,3H),4.71(t,J=7.8Hz,2H),4.58(d,J=12.1Hz,1H),4.50-4.39(m,6H),4.34(d,J=4.8Hz,1H),4.32(d,J=6.6Hz,1H),4.10(t,J=8.6Hz,1H),4.07-4.00(q,2H),3.78(dd,J=3.2,6.1Hz,1H),3.81-3.78(m,2H),3.71-3.70(dd,J=3.2,6.3Hz,1H),3.65-3.60(m,1H),3.58(dd,J=3.1,6.5Hz,1H),3.56(t,J=8.9Hz,1H),3.48(t,J=10.2Hz,1H),3.39(d,J=8.4Hz,1H),2.97-2.96(m,1H),1.93(s,3H,-C(O)CH3);13C NMR(150MHz,CDCl3):δ169.9,138.69,138.6,138.5,137.9,137.9,134.,133.8,133.7,131.9,130.3,128.5,128.4,128.4,128.3,128.2,128.0,127.9,127.7,127.7,127.6,127.5,127.5,123.3,117.5,97.1,96.4,77.9,76.9,75.1,74.8,73.9,73.8,73.8,73.05,72.8,71.9,71.0,70.4,70.1,68.1,55.5,28.7,21.5,21.1;ESI-MS:m/z C60H61NO13计算值1003.4035;实验值1026.4043(M+Na)+。
4-O-乙酰基-3,6-O-二苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基-(1→2)-O-3,4,6-三-O-苄基-α-D-甘露哌喃醣苷氟化物(6):向S2c(0.750g,0.747mmol)于10mLCH2Cl2:MeOH(1/1)中的溶液中添加PdCl2(0.02g)且在室温下搅拌隔夜直至TLC(乙酸乙酯:甲苯,2/8)指示产物形成且起始物质耗尽。随后在真空中浓缩反应混合物,且通过急骤管柱层析纯化残余物,得到呈无色泡沫状的醇(0.6g,80%)。在-30℃下,向醇(0.270g,0.197mmol)于CH2Cl2(10mL)中的溶液中添加DAST(52μL,0.395mmol)且将所得反应混合物在-10℃下搅拌8h直至TLC指示产物形成且起始物质耗尽。用NaHCO3水溶液淬灭反应,且用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过硅胶管柱层析(0%→25%EA/己烷)纯化残余物,得到呈白色泡沫状的6(0.150g,61%)。TLC(乙酸乙酯:己烷=3/7,v/v):Rf=0.45;1H NMR(600MHz,CDCl3):δ7.57-7.55(m,4H,Ar-H),7.35-7.21(m,11H,Ar-H),7.07(d,J=6.4Hz,4H,Ar-H),6.99(d,J=7.2Hz,2H,Ar-H),6.88-6.82(m,3H,Ar-H),5.30(d,J=50.4Hz,1H,H-1Man),5.30(d,J=12.2Hz,1H,H-1GlcNAc),4.88(d,J=11.2Hz,1H),4.80-4.71(m,3H),4.64(d,J=11.3Hz,1H),4.49-4.46(m,2H),4.43(d,J=11.8Hz,2H),4.34-4.28(m,3H),4.23(dd,J=6.1,11.3Hz,1H),4.16(s,1H),4.06(d,J=12.1Hz,1H),4.02(d,J=12.3Hz,1H),3.76-3.57(m,5H),3.32(d,J=10.8Hz,1H),3.02(dd,J=4.2,11.3Hz,1H),1.97(s,3H,-C(O)CH3);13C NMR(150MHz,CDCl3):δ170.99,138.40,138.28,138.16,137.94,137.70,133.94,131.82,128.90,128.63,128.57,128.47,128.40,128.31,128.27,128.04,127.89,127.89,127.76,123.54,106.33,104.85,97.45,79.55,78.48,75.43,75.23,74.09,73.84,73.70,73.16,72.78,72.54,71.36,69.18,63.40,55.80,31.21,29.98,21.11;ESI-MS:m/z C57H56FNO12计算值965.3679;实验值988.3690(M+Na)+。
合成三醣构建块7及8.
根据先前报导3获得构建块7(图2a)。葡糖胺残基处的N-邻苯二甲酰胺保护经修饰为NH-Troc,以制备构建块8(流程S3)。
流程S3|制备化合物8.i,(1)EDA、n-BuOH,90℃,(2)Troc-Cl、NaHCO3、CH2Cl2,ii,Ac2O、吡啶,室温,隔夜,72%,历经3个步骤;iii,(1)PdCl2、MeOH:CH2Cl2,(2)DAST、CH2Cl2,-30℃,66%,历经2个步骤。
烯丙基-O-2-O-乙酰基-3,4,6-O-三-苄基-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→2)-O-3,4,6-三-O-苄基-α-D-甘露哌喃糖苷(S3b):将化合物S3a(1g,0.693mmole)及10mL的乙二胺:n-BuOH(1:4)的混合物在90℃下搅拌隔夜。蒸发挥发物,且使用高真空干燥粗产物。随后将其溶解于CH2Cl2(20mL)中,在0℃下添加NaHCO3(0.376g,6.93mmol)及氯甲酸2,2,2-三氯乙酯(0.665mL,6.93mmol),使其升温至室温且搅拌隔夜。TLC(乙酸乙酯:甲苯,2/8)指示产物形成且起始物质耗尽。用CH2Cl2(100mL)稀释反应混合物,用水(2×50mL)及盐水(50mL)溶液洗涤。经Na2SO4干燥有机层且在真空中浓缩。通过硅胶管柱层析(0%→15%EA/甲苯)纯化残余物。随后使用10mL的吡啶/乙酸酐(6:4)使产物乙酰化,直至TLC(乙酸乙酯:甲苯,2/8)指示起始物质完全耗尽。随后在真空中浓缩反应混合物且通过硅胶管柱层析纯化,得到呈白色泡沫状的S3b(0.760g,72%)。TLC(乙酸乙酯:甲苯=2/8,v/v):Rf=0.64;1H NMR(600MHz,CDCl3):δ7.34-7.13(m,40H,-Ph),5.86-5.77(m,1H,烯丙基-CH),5.30-5.28(t,J=8.2Hz,1H,H2gal),5.25(bd,1H,-NHTroc),5.16(d,J=17.6Hz,1H,Troc),5.10(d,J=10.2Hz,1H),4.90(t,3H),4.82-4.76(m,3H),4.74(s,1H),4.65-4.61(dd,J=8.4&2.8Hz,3H),4.55-4.41(m,8H),4.30(d,J=12.3Hz,2H),4.20(d,J=12.2Hz,2H),4.10-4.01(m,3H),3.90-3.84(m,5H),3.76-3.61(m,5H),3.55-3.40(m,3H),3.35-3.30(m,3H),3.13(d,J=7.2Hz,1H),1.92(s,3H,-C(O)CH3);13C NMR(150MHz,CDCl3):δ170.23,154.11,139.17,138.98,138.75,138.25,138.20,134.04,127.41,100.7,97.5,96.80,80.55,78.53,75.36,72.87,73.55,72.78,72.49,72.10,71.96,69.98,68.90,68.65,57.64,41.51,21.30。ESI-MS:m/z C82H88Cl3N1O18计算值1458.4960;实验值1504.4956(M+Na)+。
2-O-乙酰基-3,4,6-O-三-苄基-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→2)-O-3,4,6-三-O-苄基-α-D-甘露哌喃醣苷氟化物(8):向S3b(1.3g,0.942mmol)于10mL的CH2Cl2:MeOH(1:1)中的溶液中添加PdCl2(0.030g)。在室温下搅拌反应混合物2h直至TLC(乙酸乙酯:甲苯,2/8)指示产物形成且起始物质耗尽。随后经由硅藻土过滤反应混合物且在真空中浓缩。通过急骤管柱层析纯化残余物,得到呈白色泡沫状的1-OH化合物(0.980g)。在-30℃下,将残余物(0.850g,0.608mmol)溶解于CH2Cl2(10mL)中,随后缓慢地添加DAST(160μL,1.21mmol)。搅拌所得反应混合物1h。当TLC(乙酸乙酯:甲苯,2/8)指示产物形成且起始物质耗尽时,用NaHCO3水溶液淬灭反应。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过硅胶管柱层析(0%→15%EA/甲苯)纯化残余物,得到呈白色泡沫状的8(0.700g,66%,历经2个步骤)。TLC(乙酸乙酯:甲苯=2/8,v/v):Rf=0.64;1H NMR(600MHz,CDCl3):δ7.35-7.10(m,40H),4.57(d,J=51.4Hz,1H),5.33(t,J=10.2Hz,1H),5.22(d,J=8.2Hz,1H),4.99-4.91(m,3H),4.82(d,J=7.8Hz,1H),4.75(t,J=10.3Hz,1H),4.64-4.44(m,12H),4.38(dd,J=3.4&10.2Hz,2H),4.26(s,2H),4.06-4.33(m,5H),3.76-3.64(m,4H),3.54-3.46(m,2H),3.40-3.34(m,3H),3.12(d,J=8.4Hz,1H),1.65(S,3H);13C NMR(150MHz,CDCl3):δ169.5,154.0,139.0,138.8,138.7,138.5,138.4,138.3,138.1,129.4,128.7,128.6,128.4,128.2,128.0,127.9,127.7,127.5,127.4,127.2,127.1,125.5,107.6,105.4,100.7,98.7,95.8,80.5,77.4,74.3,73.6,73.3,73.1,72.3,72.1,72.0,71.9,68.9,68.7,68.3,57.4,41.4,21.7,21.2;ESI-MS:m/z C79H83Cl3FN1O17计算值1443.4602;实验值1466.4608(M+Na)+。
合成复合型D1臂四醣9.
经由两个主要构建单元S4c及S4d的偶合达成唾液酸化D1臂的制备。使用唾液酸磷酸酯供体S4a进行Gal S4b的α-2,6醣基化,产生完全α-选择性10。将S2c的葡糖胺处的N-Phth保护修饰为NHTroc,在这么做的同时移除4-OAc基团,得到所要S4d。最后,S4c与S4d偶合得到所要四醣,其进一步经历变旋异构修饰,得到供体9。
流程S4|制备化合物9.i,TMSOTf、CH2Cl2,-50℃,64%;ii,(1)EDA、n-BuOH,90℃,(2)Troc-Cl、NaHCO3、CH2Cl2,78%,历经2个步骤;iii,NIS、TfOH、CH2Cl2,-50℃,65%;iv,(1)PdCl2、MeOH:CH2Cl2,(2)DAST、CH2Cl2,-30℃,55%,历经2个步骤。
甲基-5-乙酰胺基-7,8,9-三-O-乙酰基-3,5-二去氧基-D-甘油-α-D-半乳糖-壬-2-酮哌喃糖苷酸酯-(2→6)-对甲苯基-4-O-苄基-2,3-二-O-苄酰基-1-硫基-β-D-半乳-哌喃糖苷(S4c):将供体S4a(0.113g,0.178mmol)、接受体S4b(0.220g,0.119mmol)及经活化的分子筛于无水CH2Cl2(10mL)中的混合物在室温下搅拌1h。将反应冷却至-50℃,缓慢地添加三氟甲磺酸三甲基硅烷酯(12μL,0.06mmol)且将所得反应混合物搅拌2h。通过添加Et3N淬灭反应,用CH2Cl2稀释,经由硅藻土过滤,用饱和NaHCO3萃取,经硫酸钠干燥且在真空中浓缩。通过急骤管柱层析(0%→25%EA/己烷)纯化残余物,得到呈无色泡沫状的S4c(0.190g,64%)。TLC:(乙酸乙酯:己烷=3/7,v/v):Rf=0.46;1H NMR(600MHz,CDCl3):δ7.93(d,J=7.8Hz,2H),7.89(d,J=8.4Hz,2H),7.50-7.45(m,2H),7.42-7.30(m,6H),7.24-7.13(m,5H),7.04(d,J=8.2Hz,2H),5.80(t,J=10.3Hz,1H),5.47(d,J=8.9Hz,1H),5.37(s,3H),5.32(dd,J=4.2&7.8Hz,1H),5.12(dd,J=4.1&7.2Hz,1H),4.90(d,J=12.2Hz,1H),4.65(d,J=12.1Hz,1H),4.20(dd,J=3.2&7.8Hz,1H),4.18(d,J=6.8Hz,1H),3.94-3.91(m,3H),3.74-3.72(m,3H),3.71(s,3H),3.65(dd,J=3.2&7.8Hz,1H),3.06(t,J=10.2Hz,2H),2.87(dd,J=3.6&8.6Hz,1H),2.33(s,3H),2.17(s,3H),2.15(t,J=7.8Hz,1H),2.13(s,3H);13C NMR(150MHz,CDCl3):δ171.9,170.8,169.8,168.2,166.8,165.2,159.2,138.1,133.7,133.3,130.0,129.8,129.4,129.3,129.2,128.7,128.6,128.5,128.4,127.8,127.7,125.5,100,5,86.8,76.8,76.1,74.7,73.8,68.9,68.5,68.3,67.2,63.7,62.0,58.1,53.3,37.7,25.88,21.7,21.5,21.3,21.0,20.9;ESI-MS:m/zC51H53NO18S计算值999.2876;实验值1022.2882(M+Na)+。
烯丙基-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄-哌喃糖基-(1→2)-O-3,4,6-三-O-苄基-α-D-甘露哌喃糖苷(S4d):将化合物S2c(2g,1.99mmol)及20mL的乙二胺:n-BuOH(1:4)的混合物在90℃下搅拌隔夜。蒸发挥发物,且使用高真空干燥粗产物。随后将其溶解于CH2Cl2(20mL)中,在0℃下添加NaHCO3(1.05g,19.9mmol)及氯甲酸2,2,2-三氯乙酯(1.9mL,19.9mmol),使其升温至室温且搅拌隔夜,直至TLC(乙酸乙酯:甲苯,1.5/8.5)指示产物形成且起始物质耗尽。用CH2Cl2(100mL)稀释反应混合物,用水(2×50mL)及盐水(50mL)溶液洗涤。经Na2SO4干燥有机层且在真空中浓缩。通过硅胶管柱层析(0%→10%EA/甲苯)纯化残余物,得到呈白色泡沫状的S4d(1.5g,78%)。TLC(乙酸乙酯:甲苯=1.5/8.5,v/v):Rf=0.44;1H NMR(600MHz,CDCl3):δ7.30-7.15(m,25H),5.87-7.78(m,1H),5.33(d,J=5.2Hz,1H),5.21(d,J=3.2Hz,1H),5.14(d,J=7.2Hz,1H),4.94(d,J=5.8Hz,1H),4.80(d,J=8.5Hz,1H),4.73-4.44(m,10H),4.12-4.06(m,3H),3.97-3.86(m,3H),3.78-3.49(m,7H);13C NMR(150MHz,CDCl3):δ153.97,138.45,138.37,138.28,137.98,137.45,133.63,128.94,128.43,128.40,128.26,128.16,127.97,127.91,127.78,127.65,127.53,127.41,125.20,117.23,97.93,96.88,95.50,79.33,79.08,75.08,74.47,74.37,73.53,73.19,73.00,71.98,71.65,70.83,69.16,67.91,57.41,44.53;ESI-MS:m/z C53H58Cl3NO12计算值1005.3025;实验值1006.3089(M+H)+。
烯丙基-[甲基-5-乙酰胺基-7,8,9-三-O-乙酰基-3,5-二去氧基-D-甘油-α-D-半乳糖-壬-2-酮哌喃糖苷酸酯]-(2→6)-4-O-苄基-2,3-二-O-苄酰基-1-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→2)-O-3,4,6-三-O-苄基-α-D-甘露哌喃糖苷(S4e):向接受体S4d(0.208g,0.169mmol)及供体S4c(0.310g,0.199mmol)于无水CH2Cl2(10mL)中的溶液中添加经活化的分子筛(1g)。在室温下搅拌混合物1h。将反应混合物冷却至-50℃,缓慢地添加NIS(0.076g,0.338mmol)及TfOH(3.7μL,0.042mmol)。搅拌所得反应混合物2h。当TLC(乙酸乙酯:甲苯,1.5/8.5)指示产物形成且起始物质耗尽时,通过添加Et3N淬灭反应且经由硅藻土过滤。用NaHCO3水溶液(2×50mL)、Na2S2O3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→10%EA/甲苯)纯化残余物,得到呈透明泡沫状的S4e(390g,65%)。TLC(乙酸乙酯:甲苯=1.5/8.5,v/v):Rf=0.51;1H NMR(600MHz,CDCl3):δ7.91-7.85(m,4H,Ph),7.50-7.46(m,1H,Ph),7.35-7.19(m,35H),5.82-5.76(m,2H),5.39-5.36(m,3H),5.24(d,J=6.4Hz,1H),5.22(d,J=7.2Hz,1H),5.19(d,J=3.4Hz,1H),5.15(d,J=3.4Hz,1H),5.11-5.08(dd,J=6.3&3.1Hz,2H),4.96(d,J=12.1Hz,2H),4.83(d,J=12.1Hz,2H),4.73-4.57(m,10H),4.47-4.42(m,6H),4.25-4.15(m,8H),4.08-3.93(m,10H),3.91(s,3H,),3.39(d,J=7.8Hz,1H),3.21(bd,1H),2.93(t,J=7.8Hz,1H),2.78(dd,J=3.6&7.8Hz,1H),2.11(s,3H),2.01(s,3H),1.66(s,3H);13C NMR(150MHz,CDCl3):δ171.83,170.84,169.91,168.03,166.15,165.43,159.57,154.17,139.14,138.85,138.36,134.08,133.78,132.66,13.54,129.67,128.78,128.43,128.23,127.78,127.67,127.56,127.43,127.23,127.10,117,53,100.67,100.32,98.94,97.18,75.90,78.31,75.32,74.95,7.81,74.43,73.99,73.58,73.45,73.33,73.29,73.23,73.12,69.90,69.56,68.66,67.56,58.70,53.43,21.28,21.07,20,98;ESI-MS:m/z C97H103Cl3N2O13计算值1982.5562;实验值1905.5558(M+Na)+。
甲基-5-乙酰胺基-7,8,9-三-O-乙酰基-3,5-二去氧基-D-甘油-α-D-半乳糖-壬-2-酮哌喃糖苷酸酯-(2→6)-4-O-苄基-2,3-二-O-苄酰基-1-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→2)-O-3,4,6-三-O-苄基-α-D-甘露哌喃醣苷氟化物(9):向S4e(2.7g,1.42mmol)于20mL的CH2Cl2:MeOH(1:1)中的溶液中添加PdCl2(0.050g)。在室温下搅拌反应混合物5h直至TLC(乙酸乙酯:甲苯,2/8)指示产物形成且起始物质耗尽。随后经由硅藻土过滤反应混合物且在真空中浓缩。通过急骤管柱层析(0%→15%EA/甲苯)纯化残余物,得到呈白色泡沫状的1-OH化合物(2.0g)。在-30℃下,将残余物(2g,1.07mmol)溶解于CH2Cl2(10mL)中,随后缓慢地添加DAST(284μL,2.14mmol)。搅拌所得反应混合物1h。当TLC(乙酸乙酯:甲苯,2/8)指示产物形成且起始物质耗尽时,用NaHCO3水溶液淬灭反应。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过硅胶管柱层析(0%→15%EA/甲苯)纯化残余物,得到呈白色泡沫状的9(1.45g,55%,历经2个步骤)。TLC(乙酸乙酯:甲苯=2/8,v/v):Rf=0.64;1H NMR(600MHz,CDCl3):δ7.96(m,4H),7.55(m,2H),7.40-7.17(m,34H),5.86(t,J=10.2Hz,1H),5.50(d,J=52Hz,1H),5.44-5.40(m,2H),5.29(dd,J=1.6&7.8Hz,1H),5.15(dd,J=1.7&7.2Hz,1H),5.05-4.99(m,2H),4.84-4.44(m,10H),4.33-4.20(m,3H),4.27-4.20(m,4H),4.03-3.90(m,3H),3.72-3.65(s,3H),3.70-3.06(m,12H),3.46-3.30(m,1H),3.00(t,J=10.2Hz,1H),2.86(dd,J=3.2&12.3Hz,1H),2.19(s,3H),2.16(s,3H),2.12(t,J=8.4Hz,1H),2.0(s,3H);13C NMR(150MHz,CDCl3):δ170.8,170.0,168.0,166.1,165.2,159.5,154.1,138.9,138.4,138.2,138.1,133.7,133.6,133.4,130.0,129.9,129.6,129.3,128.8,128.6,128.5,128.4,128.3,128.1,127.9,127.5,127.2,125.5,107.7,100.7,100.1,99.2,95.8,78.1,77.1,76.6,75.3,74.9,74.6,74.4,74.1,73.9,73.7,73.5,73.3,73.0,72.6,72.5,69.2,69.0,67.3,63.2,61.8,58.1,57.3,53.4,37.3,21.7,21.3,21.0,20.9;ESI-MS:m/z C94H98Cl3FN2O29计算值1844.5204;实验值1867.5192(M+Na)+。
合成构建块10及11.
根据吾人的先前报导3获得五醣S5a及S5b,其中官能基自N-邻苯二甲酰胺经进一步修饰为N-Troc。最后移除变旋异构对甲氧基苯基且将所得-OH在DAST存在下改为氟基。
流程S5|制备化合物10及11.i,(1)EDA、n-BuOH,90℃;(2)Troc-Cl、NaHCO3、CH2Cl2;(3)Ac2O、吡啶;ii,(1)CAN、ACN:甲苯:H2O;(2)DAST、CH2Cl2,-30℃。
步骤i的一般程序:将化合物S5a及S5b(0.500g,0.212mmol)及10mL的乙二胺:n-BuOH(1:4)的混合物在90℃下搅拌隔夜。蒸发挥发物,且使用高真空干燥粗产物。随后将其溶解于CH2Cl2(20mL)中,在0℃下添加NaHCO3(0.114g,2.12mmol)及氯甲酸2,2,2-三氯乙酯(0.2mL,2.12mmol),使其升温至室温且搅拌隔夜。TLC(乙酸乙酯:甲苯,2/8)指示产物形成且起始物质耗尽。用CH2Cl2(100mL)稀释反应混合物,用水(2×50mL)及盐水(50mL)溶液洗涤。经Na2SO4干燥有机层且在真空中浓缩。通过硅胶管柱层析(0%→15%EA/甲苯)纯化残余物。随后使用10mL的吡啶/乙酸酐(6:4)使产物乙酰化,直至TLC(乙酸乙酯:甲苯,2/8)指示起始物质完全耗尽。随后在真空中浓缩反应混合物且通过硅胶管柱层析(0%→15%EA/甲苯)纯化,得到呈无色泡沫状的S5c(0.332g,65%)及S5d(0.340g,68%)。
对甲氧基苯基-O-2-O-乙酰基-3,4,6-O-三-苄基-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→2)-[2-O-乙酰基-3,4,6-O-三-苄基-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→4)]-3,6-O-二苄基-α-D-甘露哌喃糖苷(S5c):根据以上所提及的一般程序制备化合物S5c。TLC(乙酸乙酯:甲苯=2/8,v/v):Rf=0.64;1H NMR(600MHz,CDCl3):δ7.40-7.08(m,50H,Ar-H),6.92-6.72(m,10H,Ar-H),6.63-6.53(m,2H,PMP-H),5.31-5.24(m,2H'),5.22(d,J=7.8Hz,1H),5.15(d,J=8.4Hz,1H,),4.94(d,J=3Hz,),4.88(d,J=3.6Hz,1H),4.86(d,J=3.6Hz,1H),4.80-4.71(m,7H),4.64-4.56(m,3H),4.49-4.44(m,10H,),4.39-4.10(m,14H),4.15-4.05(m,2H),4.03(t,J=8.4Hz,1H),3.98-3.86(m,3H),3.79-3.72(m,1H),3.69(s,3H),3.58-3.56(m,1H),3.49-3.25(m,13H),3.08(d,J=8.4Hz,1H),3.05(d,J=8.9Hz,1H),2.90(bs,1H),1.93(s,3H),1.92(s,3H);13C NMR(150MHz,CDCl3):δ169.5,169.4,168.3,167.8,154.8,150.3,139.2,139.1,139.0,138.9,138.8,138.5,138.3,138.2,138.0,134.0,133.6,132.1,131.9,131.7,129.2,128.6,128.5,128.3,128.2,128.1,128.0,127.9,127.9,127.8,127.7,127.6,127.5,127.4,127.4,127.3,127.1,127.0,126.8,123.5,123.4,123.3,118.1,114.4,108.9,100.9,100.7,98.5,97.6,97.1,80.5,50.5,79.7,77.9,77.8,77.7,77.4,77.2,75.4,74.9,74.6,74.5,73.7,73.6,73.6,73.4,73.3,73.0,72.8,72.7,72.2,71.9,71.8,71.7,71.2,69.2,68.5,68.3,68.2,67.6,67.3,56.4,55.9,55.7,21.3,21.2,21.2;ESI-MS:m/z C131H138Cl6N2O31计算值2449.7492;实验值2449.7272(M+H)+。
对甲氧基苯基-O-2-O-乙酰基-3,4,6-O-三-苄基-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→2)-[2-O-乙酰基-3,4,6-O-三-苄基-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→6)]-3,6-O-二苄基-α-D-甘露哌喃糖苷(S5d):根据以上所提及的一般程序制备化合物S5d。TLC(乙酸乙酯:甲苯=2/8,v/v):Rf=0.61;1H NMR(600MHz,CDCl3):δ7.42-7.05(m,60H),7.02(d,J=9.2Hz,2H),6.85(d,J=9.0Hz,2H),5.59(s,1H),5.42(bs,1H),5.39-5.29(m,2H),5.15(d,J=3.8Hz,1H),5.00-4.84(m,7H),4.80-4.25(m,20H),4.10(dd,J=3.8&7.8Hz,2H),4.00-3.83(m,6H),3.76(s,3H),3.62-3.20(m,23H),1.99(s,3H),1.96(s,3H);13C NMR(150MHz,CDCl3):δ169.9,169.5,168.5,155.0,154.6,150.1,138.9,138.4,138.2,138.1,134.3,132.0,129.2,128.6,128.4,128.3,128.2,128.1,128.0,127.9,127.8,127.6,127.5,127.3,125.5,123.6,117.8,117.2,114.9,106.1,101.5,100.9,100.7,98.3,96.4,95.7,80.3,80.1,79.4,78.3,77.9,78.3,75.6,74.8,74.5,74.4,74.3,73.6,73.4,73.2,73.0,72.6,72.3,71.8,68.9,68.1,67.9,67.5,57.9,56.7,55.8,41.3,40.4,37.7,29.9,21.6,21.2,21.1;ESI-MS:m/z C131H138Cl6N2O31计算值2448.7492;实验值2449.7414(M+H)+。
步骤ii的一般程序:向化合物S5c或S5d(0.350g,0.145mmol)于10mL的乙腈:甲苯:H2O(4:2:1)中的溶液中添加硝酸铈铵(0.616g,0.725mmol)。在0℃下搅拌所得反应混合物2h。用EtOAc(100mL)稀释反应物且用H2O(30×2mL)及盐水(30mL)洗涤。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→15%EA/甲苯)纯化产物,得到呈透明泡沫状的1-OH化合物(0.180m)。在-30℃下,将残余物(0.180g,0.078mmol)溶解于CH2Cl2(10mL)中。随后,缓慢地添加DAST(30μL,0.234mmol),且将所得反应混合物搅拌1h。当TLC(乙酸乙酯:甲苯,1/9)指示产物形成且起始物质耗尽时,用NaHCO3水溶液淬灭反应。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过硅胶管柱层析(0%→8%EA/甲苯)纯化残余物,得到10(0.120g,34%,历经2个步骤)及11(0.150g,42%,历经2个步骤)。
2-O-乙酰基-3,4,6-O-三-苄基-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→2)-[2-O-乙酰基-3,4,6-O-三-苄基-β-D-半乳糖-哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→4)]-3,6-O-二苄基-α-D-甘露哌喃醣苷氟化物(10):TLC(乙酸乙酯:甲苯=1/9,v/v):Rf=0.34;1H NMR(600MHz,CDCl3):δ7.34-7.13(m,60H),5.41(s,1H),5.50(d,J=51.1Hz,1H),5.30-5.25(m,2H),5.12(d,J=8.4Hz,1H),4.92-4.85(m,5H),4.69-4.38(m,20H),4.32-4.14(m,8H),3.91-3.77(m,7H),3.70(dd,J=3.8及7.8Hz,1H),3.60-3.56(m,3H),3.49-3.24(m,14H),2.03(s,3H),1.98(s,3H);13C NMR(150MHz,CHCl3):δ169.68,169.58,154.31,154.09,139.06,138.97,138.93,138.32,138.29,138.27,138.20,138.17,129.32,128.99,128.69,128.63,128.50,128.49,128.45,128.27,128.24,128.16,128.15,128.07,128.05,128.00,127.97,127.94,127.91,127.82,127.72,127.60,127.57,127.44,125.58,100.64,100.45,95.86,80.54,80.51,76.81,76.70,76.03,75.34,75.08,86,74.77,74.66,74.57,74.47,73.74,73.70,73.67,73.64,73.58,73.46,73.42,73.27,72.90,72.19,72.17,71.99,71.95,68.49,68.27,68.14,68.01,57.53,21.40,21.27;ESI-MS:m/z C124H131Cl6FN2O29计算值2340.6953;实验值2363.7200(M+Na)+。
2-O-乙酰基-3,4,6-O-三-苄基-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→2)-[2-O-乙酰基-3,4,6-O-三-苄基-β-D-半乳糖-哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→4)]-3,6-O-二苄基-α-D-甘露哌喃醣苷氟化物(11):TLC(乙酸乙酯:甲苯=1/9,v/v):Rf=0.38;1H NMR(600MHz,CDCl3):δ7.33-7.09(m,60H),5.60(d,J=52.1Hz,1H),5.32-5.28(m,2H),5.10(d,J=2.1Hz,1H),4.93-4.80(m,5H),4.78(d,J=8.4Hz,1H),4.74(d,J=8.2Hz,1H),4.65-4.40(m,28H),3.95(dd,J=8.4Hz,1H),3.79-3.69(m,10H),3.50-3.40(m,4H),3.39-3.20(m,8H),1.91(s,6H);13C NMR(150MHz,CHCl3):δ169.69,169.56,154.72,154.66,139.55,139.02,139.00,138.43,138.34,138.27,138.20,138.19,128.93,128.80,128.76,128.72,128.65,128.45,128.41,128.30,128.28,128.21,127.67,127.34,127.10,106.45,105.33,101.82,101.21,100.00,97.86,97.46,95.60,75.46,75.27,74.86,74.58,74.26,74.10,73.50,73.47,73.10,72.90,72.88,72.37,72.30,71.40,71.37,71.09,70.57,70.35,70.45,69.78,69.46,29.99,25.78,22.87,21.36,21.29,15.57,14.49,144.42;ESI-MS:m/z C124H131Cl6FN2O29计算值2340.6953;实验值2362.7225(M+Na)+。
合成构建块12.
二唾液酸化触角的制备始于用供体S2b使甘露糖基接受体S6a发生β-1,2及β-1,4醣基化。随后将化合物S6b修饰为S6c且随后用S4c醣基化,得到所要七醣S6d。最后,对还原端进行修饰,得到供体12。
流程S6|制备化合物12.i,NIS、TfOH、CH2Cl2,-50℃,76%;ii,(1)EDA、n-BuOH,90℃,(2)Troc-Cl、NaHCO3、CH2Cl2,72%,历经2个步骤;iii,NIS、TfOH、CH2Cl2,-50℃,86%;iv,(1)CAN、ACN:甲苯:H2O,(2)DAST、CH2Cl2,-30℃,40%,历经2个步骤。
对甲氧基苯基-O-4-O-乙酰基-3,6-O-二苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基-(1→2)-[4-O-乙酰基-3,6-O-二苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基-(1→4)]-3,6-O-二苄基-α-D-甘露哌喃糖苷(S6b):向接受体S6a(0.560g,1.19mmol)及供体S2b(1.74g,2.74mmol)于无水CH2Cl2(10mL)中的溶液中添加经活化的分子筛(1g)。将反应混合物在室温下搅拌1h,随后冷却至-50℃。缓慢地添加NIS(1.07g,4.75mmol)及TfOH(52μL,0.595mmol),且将所得反应混合物搅拌2h。当TLC(乙酸乙酯:甲苯,1.5/8.5)指示产物形成且起始物质耗尽时,通过添加Et3N淬灭反应,随后经由硅藻土过滤。用NaHCO3水溶液(2×50mL)、Na2S2O3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→10%EA/甲苯)纯化残余物,得到呈透明泡沫状的S6b(1.27g,76%)。TLC(乙酸乙酯:甲苯=1.5/8.5,v/v):Rf=0.57;1H NMR(600MHz,CDCl3):δ7.66-7.16(m,18H),6.97(m,12H),6.66-6.49(m,4H),5.25(t,J=10Hz,2H),5.08-5.00(dd,J=9.6&3.4Hz,2H),4.90(d,J=4.6Hz,1H),4.69(d,J=12Hz,1H),4.57(t,3H),4.42-4.21(m,10H),4.12(dd,J=3.2&8.4Hz,2H),3.90(dd,J=3.2&7.8Hz,1H),3.84-3.60(m,7H),3.66(s,3H),3.54-3.31(m,7H),3.01(d,J=7.8Hz,1H),1.84(s,3H),1.80(s,3H);13C NMR(150MHz,CDCl3):δ169.61,169.12,154.49,150.07,138.69,138.21,137.99,137.77,137.60,133.52,131.29,128.28,128.19,127.99,127.94,127.91,127.83,127.68,127.59,127.47,127.25,127.08,127.94,123.17,117.55,114.08,98.10,97.37,96.54,78.89,74.56,73.56,73.27,72.98,72.42,72.32,71.91,71.43,71.02,69.91,69.25,68.90,55.73,55.45,55.26,20.80;ESI-MS:m/z C77H82Cl6N2O21计算值1585.3589;实验值1586.5687(M+H)+。
对甲氧基苯基-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→2)-[3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→4)]-3,6-O-二苄基-α-D-甘露哌喃糖苷(S6c):将化合物S6b(1.9g,1.27mmol)及20mL的乙二胺:n-BuOH(1:4)的混合物在90℃下搅拌隔夜。蒸发挥发物,且使用高真空干燥粗产物。随后将其溶解于CH2Cl2(20mL)中,在0℃下添加NaHCO3(0.687g,12.7mmol)及氯甲酸2,2,2-三氯乙酯(1.75mL,12.7mmol),使其升温至室温且搅拌隔夜,直至TLC(丙酮:甲苯,1/9)指示产物形成且起始物质耗尽。用CH2Cl2(100mL)稀释反应混合物,用水(2×50mL)及盐水(50mL)溶液洗涤。经Na2SO4干燥有机层且在真空中浓缩。通过硅胶管柱层析(0%→8%EA/甲苯)纯化残余物,得到呈无色固体状的S6c(1.35g,72%)。TLC(丙酮:甲苯=1/9,v/v):Rf=0.54;1H NMR(600MHz,CDCl3):δ7.35-7.14(m,30H),6.90(d,J=8.4Hz,2H),6.79(d,J=8.6Hz,2H),5.32(d,J=3.2Hz,1H),5.27(d,J=7.2Hz,1H),4.93(d,J=8.4Hz,1H),4.73-4.58(m,10H),4.48-4.43(m,7H),4.19(d,J=7.2Hz,1H),4.05-3.96(m,3H),3.68(s,3H),3.62-3.56(m,8H),3.26(q,1H),3.26-3.15(m,3H),2.93(m,3H),2.96(s,1H),2.12(s,1H);13C NMR(150MHz,CDCl3):δ155.26,154.37,154.27,150.53,138.93,138.63,138.48,137.83,137.74,131.18,128.96,128.76,128.67,128.36,128.21,128.15,127.89,127.78,118.00,114,85,101.31,98.79,97.79,95.81,81.39,79.59,75.90,74.70,74.47,74.01,73.93,73.53,73.02,72.83,71.63,71.37,70.97,68.99,57.73,57.40,55.94;ESI-MS:m/z C73H78Cl6N2O19计算值1499.3338;实验值1499.3419。
对甲氧基苯基-O-[甲基-5-乙酰胺基-7,8,9-三-O-乙酰基-3,5-二去氧基-D-甘油-α-D-半乳糖-壬-2-酮哌喃糖苷酸酯]-(2→6)-4-O-苄基-2,3-二-O-苄酰基-1-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→2)-[甲基-5-乙酰胺基-7,8,9-三-O-乙酰基-3,5-二去氧基-D-甘油-α-D-半乳糖-壬-2-酮哌喃糖苷酸酯]-(2→6)-4-O-苄基-2,3-二-O-苄酰基-1-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→4)-O-3,4,6-三-O-苄基-α-D-甘露哌喃糖苷(S6d):向接受体S6c(0.500g,0.341mmol)及供体S4c(0.772g,0.751mmol)于无水CH2Cl2(10mL)中的溶液中添加经活化的分子筛(1g)。将混合物在室温下搅拌1h,且随后冷却至-50℃。缓慢地添加NIS(0.383g,1.70mmol)及TfOH(15μL,0.170mmol)。搅拌所得反应混合物2h。当TLC(丙酮:甲苯,2/8)指示产物形成且起始物质耗尽时,通过添加Et3N淬灭反应且经由硅藻土过滤。用NaHCO3水溶液(2×50mL)、Na2S2O3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→15%EA/甲苯)纯化残余物,得到呈透明泡沫状的S6d(0.950g,86%)。TLC(丙酮:甲苯=2/8,v/v):Rf=0.48;1H NMR(600MHz,CDCl3):δ7.90-7.87(m,7H),7.81-7.79(m,3H),7.50-7.07(m,50H),6.98(d,2H),6.40(d,2H),5.78-5.73(m,2H),5.36-5.26(m,4H),5.20(d,J=8.6Hz,1H),5.19-5.15(dd,J=3.2&8.4Hz,2H),5.05(dd,J=4.3&8.4Hz,2H),4.94-4.78(m,4H),4.68-4.15(m,30H),3.96-3.86(m,6H),3.71(S,6H),3.70-3.59(m,15H),3.57(s,3H),3.34-3.26(m,5H),2.98-2.76(m,4H),2.15(s,3H),2.09(s,3H),2.00(S,3H),1.98(s,3H),1.89(S,3H),1.89(S,3H);13C NMR(150MHz,CDCl3):δ171.56,170.89,169.56,167.20,165.28,165.00,159.11,154.79,138.78,138.56,138.21,137.29,137.00,133,67,133.24,130.79,129.70,129.60,129.26,128.78,128.56,128.42,128.32,128.11,127.69,127.56,127.44,127.32,127.14,117.78,100.89,100.56,99.87,99.68,95.41,79.13,75.56,74.18,73.48,73.26,73.12,71.00,72.90,72.78,71.76,68.78,68.40,68.42,61.78,57.78,57.33,55.89,53.80,38.90,38.7,30.98,30.76,28.87,23.89,22.65,20.87,20.65,20.54,14.08,10.86;ESI-MS:m/z C161H168Cl6N4O55计算值3251.8695;实验值3251.8657(M+Na)+。
[甲基-5-乙酰胺基-7,8,9-三-O-乙酰基-3,5-二去氧基-D-甘油-α-D-半乳糖-壬-2-酮哌喃糖苷酸酯]-(2→6)-4-O-苄基-2,3-二-O-苄酰基-1-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→2)-[甲基-5-乙酰胺基-7,8,9-三-O-乙酰基-3,5-二去氧基-D-甘油-α-D-半乳糖-壬-2-酮哌喃糖苷酸酯]-(2→6)-4-O-苄基-2,3-二-O-苄酰基-1-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→4)-O-3,4,6-三-O-苄基-α-D-甘露哌喃醣苷氟化物(12):向化合物S6d(0.300g,0.092mmol)于10mL的乙腈:甲苯:H2O(4:2:1)中的溶液中添加硝酸铈铵(0.470g,0.554mmol)。在室温下搅拌所得反应混合物3h。用EtOAc(100mL)稀释反应物且用H2O(30×2mL)及盐水(30mL)洗涤。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→20%EA/甲苯)纯化产物,得到呈泡沫状的1-OH化合物(0.180g)。在-30℃下,将残余物(0.180g,0.057mmol)溶解于CH2Cl2(10mL)中。随后,缓慢地添加DAST(22μL,0.171mmol),且将所得反应混合物搅拌1h。当TLC(丙酮:甲苯,2/8)指示产物形成且起始物质耗尽时,用NaHCO3水溶液淬灭反应。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过硅胶管柱层析(0%→15%EA/甲苯)纯化残余物,得到呈白色固体状的13(0.120g,41%,历经2个步骤)。TLC(丙酮:甲苯=2/8,v/v):Rf=0.24;1H NMR(600MHz,CDCl3):δ7.92-7.83(m,10H),7.54-7.47(m,5H),7.40-6.93(m,45H),5.83(m,2H),5.40-5.35(m,6H),5.2(dd,J=3.2&7.8Hz,2H),5.11(dd,J=4.3&8.4Hz,2H),4.98-4.89(m,4H),4.76-4.56(m,15H),4.28-4.10(m,11H),4.00-3.89(m,6H),3.79-3.72(m,6H),3.65(s,3H),3.67(s,3H),3.62-3.43(m,6H),3.36-3.21(m,5H),3.02-2.98(m,4H),2.16(s,3H),2.14(s,3H),2.11(s,3H),2.06(s,3H),2.01(s,3H),1.98(s,3H),1.89-1.76(m,2H);13C NMR(150MHz,CDCl3):δ171.80,170.85,170.06,168.03,166.12,165.41,159.56,154.16,138.75,138.34,133.73,130.11,129.94,129.57,129.19,128.79,128.72,128.50,128.38,128.03,127.75,127.56,100.69,100.00,96.67,95,78,95.35,95.12,74.91,74.65,74.29,73.00,70.91,69.01,67.35,61.87,58.15,57.60,53.42,37.17,21.28,20.97;ESI-MS:m/z C154H161Cl6FN4O53计算值3147.6554;实验值3169.8022(M+Na)+。
合成构建块13.
部分唾液酸化触角13的总化学合成显示于流程S7中。
流程S7|制备化合物13.i,NIS、TfOH、CH2Cl2,-50℃,86%;ii,BH3.THF、Bu2BOTf、CH2Cl2,65%;iii,NIS、TfOH、CH2Cl2,-50℃,76%;iv,(1)EDA、n-BuOH,90℃,(2)Troc-Cl、NaHCO3、CH2Cl2;(3)Ac2O、吡啶,65%,历经3个步骤;v,三乙基硅烷、TFA、CH2Cl2,58%;vi,NIS、TfOH、CH2Cl2,-50℃,73%;vii,(1)CAN、ACN:甲苯:H2O,(2)DAST、CH2Cl2,-30℃,30%,历经2个步骤。
对甲氧基苯基-O-2-O-乙酰基-3,4,6-O-三-苄基-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基-(1→2)-O-3,4-O-苄基-α-D-甘露哌喃糖苷(S7c):向接受体S7a(0.500g,1.07mmol)及供体S7b(1.21g,1.28mmol)于无水CH2Cl2(10mL)中的溶液中添加经活化的分子筛(1g)。将反应混合物在室温下搅拌1h,随后冷却至-50℃。缓慢地添加NIS(0.481g,2.14mmol)及TMSOTf(48μL,0.267mmol),且将所得反应混合物搅拌3h。当TLC(乙酸乙酯:甲苯,1/9)指示产物形成且起始物质耗尽时,通过添加Et3N淬灭反应,随后经由硅藻土过滤。用NaHCO3水溶液(2×50mL)、Na2S2O3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→10%EA/甲苯)纯化残余物,得到呈浅黄色固体状的S7c(1.30g,86%)。TLC(乙酸乙酯:甲苯=1/9,v/v):Rf=0.59;1H NMR(600MHz,CDCl3):δ7.75-7.65(m,4H),7.37-7.15(m,30H),6.98(d,J=8.1Hz,2H),6.82(d,J=8.4Hz,3H),6.70(d,J=8.4Hz,2H),6.60(d,J=9.2Hz,2H),5.39(s,1H),5.34(t,J=10.2Hz,1H),5.22(dd,J=7.2&2.8Hz,1H),5.06(d,J=3.2Hz,1H),4.89(d,J=11.4Hz,1H),4.82(d,J=12.1Hz,1H),4.75(d,J=11.8Hz,1H),4.66-4.62(m,3H),4.54-4.38(m,6H),4.32(d,J=11.2Hz,1H),4.29(dd,J=3.1&7.2Hz,2H),4.27(d,J=10.8Hz,1H),4.19(t,J=8.3Hz,1H),4.00-3.89(m,4H),3.72(s,3H),3.58-3.33(m,8H),3.0(t,J=8.7Hz,1H),2.07(s,3H);13C NMR(150MHz,CDCl3):δ169.3,154.9,149.6,138.8,138.7,138.6,138.5,138.1,138.0,137.9,137.5,133.6,131.9,128.7,128.4,128.4,128.3,128.2,128.1,128.1,128.1,128.0,127.8,127.8,127.7,127.7,127.7,127.6,127.6,127.5,127.4,127.3,127.2,126.8,126.0,123.0,117.03,114.5,101.4,100.9,97.0,96.2,96.2,80.3,78.1,78.0,75.6,74.9,74.5,74.4,73.7,73.4,73.3,72.6,72.6,72.0,71.7,71.3,68.4,68.1,68.1,64.2,55.6,55.6,55.6,21.0;ESI-MS:m/z C84H83NO19计算值1409.5559;实验值1432.5499(M+Na)+。
对甲氧基苯基-O-2-O-乙酰基-3,4,6-O-三-苄基-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基-(1→2)-O-3,4-O-二苄基-α-D-甘露哌喃糖苷(S7d):在0℃下,向化合物S7c(1.30g,1.02mmol)及经活化的分子筛(1g)于无水CH2Cl2(15mL)中的混合物中添加硼烷.THF复合物(0.978mL的1M THF溶液,10.2mmol)及Bu2BOTf(0.439mL的1M CH2Cl2溶液,10.2mmol)。将反应混合物在室温下搅拌4h。TLC(丙酮:甲苯,1/9)指示产物形成且起始物质耗尽。在0℃下,向反应混合物中添加三乙胺,随后缓慢添加甲醇。当不再产生氢气时,经由硅藻土过滤反应混合物,用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→7%EA/甲苯)纯化残余物,得到呈透明泡沫状的S7d(0.850g,65%)。TLC(丙酮:甲苯=1/9,v/v):Rf=0.31;1H NMR(600MHz,CDCl3):δ7.84-7.66(m,4H),7.42-7.02(m,30H),7.03(d,J=8.4Hz,2H),6.87(t,J=7.8Hz,3H),6.67(d,J=12.1Hz,2H),6.48(d,J=12.1Hz,2H),5.39(t,J=10.2Hz,1H),5.17(d,J=8.4Hz,1H),4.93(d,J=12Hz,2H),4.88(d,J=7.2Hz,1H),4.85(d,J=12.1Hz,2H),4.67(dd,J=4.3&8.4Hz,2H),4.59-4.30(m,11H),4.18(t,J=7.4Hz,1H),4.01-3.96(m,3H),3.84-3.78(m,3H),3.76(s,3H),3.68(dd,J=3.2&7.8Hz,1H),3.54-3.34(m,6H),3.25-3.20(m,1H),2.01(s,3H);13C NMR(150MHz,CDCl3):δ169.3,167.9,154.7,149.6,138.9,138.7,138.4,138.2,138.0,138.0,137.9,134.1,133.6,131.9,131.5,129.0,128.4,128.4,128.4,128.3,128.2,128.2,128.1,128.1,127.9,127.8,127.8,127.7,127.7,127.7,127.6,127.6,127.5,127.3,127.3,126.8,123.2,123.0,116.8,114.4,100.9,97.6,95.7,80.3,78.0,76.9,75.2,75.1,74.4,74.4,73.9,73.6,73.4,73.3,72.5,72.3,72.0,71.6,70.7,68.6,68.1,62.2,55.9,55.6,21.0;ESI-MS:m/z C84H85NO19计算值1411.5716;实验值1412.5726(M+H)+。
对甲氧基苯基-O-[2-O-乙酰基-3,4,6-O-三-苄基-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基]-(1→2)-O-[3-O-苄基-4,6-O-亚苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基]-(1→6)-3,4-O-二苄基-α-D-甘露哌喃糖苷(S7f):向接受体S7d(0.600g,0.470mmol)及供体S7e(0.557g,0.940mmol)于无水CH2Cl2(10mL)中的溶液中添加经活化的分子筛(1g)。将反应混合物在室温下搅拌1h,随后冷却至-50℃。缓慢地添加NIS(0.211g,0.940mmol)及TfOH(10.4μL,0.117mmol),且将所得反应混合物搅拌2h。当TLC(乙酸乙酯:甲苯,2/8)指示产物形成且起始物质耗尽时,通过添加Et3N淬灭反应,随后经由硅藻土过滤。用NaHCO3水溶液(2×50mL)、Na2S2O3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→15%EA/甲苯)纯化残余物,得到呈透明泡沫状的S7f(0.610g,76%)。TLC(乙酸乙酯:甲苯=2/8,v/v):Rf=0.49;1H NMR(600MHz,CDCl3):δ7.66(m,8H),7.38-7.15(m,29H),7.10(d,J=8.4Hz,2H),6.95-6.91(m,5H),6.88-6.78(m,5H),6.63(d,J=8.4Hz,2H),6.50(d,J=8.8Hz,2H),5.41(s,1H),5.32(t,J=10.2Hz,1H),5.11(d,J=8.4Hz,1H),4.89(d,J=4.3Hz,1H),4.87(s,1H),4.80(t,J=8.8Hz,2H),4.73(s,1H),4.70(s,1H),4.60(t,J=10.8Hz,3H),4.57-4.40(m,5H),4.35-4,16(m,8H),4.08(t,J=10.2Hz,1H),4.00-3.89(m,4H),3.85(dd,J=3.6&7.8Hz,1H),3.78(s,3H),3.70-3.61(m,3H),3.52-3.41(m,4H),3.40-3.28(m,4H),3.20(t,J=10.2Hz,2H),2.98(m,1H),1.98(s,3H);13C NMR(150MHz,CDCl3):δ168.5,164.8,158.0,153.0,152.8,151.6,151.5,150.6,150.5,149.2,139.6,138.9,138.4,138.1,138.0,137.4,129.3,129.2,128.9,128.9,128.8,128.9,128.7,128.5,128.4,128.2,128.1,128.0,128.0,127.9,127.9,127.7,127.5,127.4,126.2,125.5,117.3,114.9,114.2,102.2,101.4,100.9,98.8,96.5,95.8,95.7,95.5,95.3,95.3,95.0,82.3,80.6,78.3,76.2,76.0,75.8,75.5,75.4,75.2,74.9,74.8,74.7,74.7,74.2,74.1,73.8,73.7,73.5,72.9,72.3,71.9,68.9,68.8,68.2,68.0,66.4,58.0,57.1,55.9,44.3,42.9,40.8,40.4,40.3,40.0,21.7,21.2;ESI-MS:m/z C111H108N2O24计算值1882.7247;实验值1882.7343。
对甲氧基苯基-O-[2-O-乙酰基-3,4,6-O-三-苄基-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基]-(1→2)-O-[3-O-苄基-4,6-O-亚苄基-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基]-(1→6)-3,4-O-二苄基-α-D-甘露哌喃糖苷(S7g):将化合物S7f(0.950g,0.545mmol)及10mL的乙二胺:n-BuOH(2:8)的混合物在90℃下搅拌隔夜。蒸发挥发物,且使用高真空干燥粗产物。随后将其溶解于CH2Cl2(20mL)中,在0℃下添加NaHCO3(0.363g,5.45mmol)及氯甲酸2,2,2-三氯乙酯(0.86mL,5.45mmol),使其升温至室温且搅拌隔夜。TLC(乙酸乙酯:甲苯,2/8)指示产物形成且起始物质耗尽。用CH2Cl2(100mL)稀释反应混合物,用水(2×50mL)及盐水(50mL)溶液洗涤。经Na2SO4干燥有机层且在真空中浓缩。通过硅胶管柱层析(0%→10%EA/甲苯)纯化残余物。随后使用10mL的吡啶/乙酸酐(6:4)使产物乙酰化,直至TLC(乙酸乙酯:甲苯,2/8)指示起始物质完全耗尽。随后在真空中浓缩反应混合物且通过硅胶管柱层析(0%→10%EA/甲苯)纯化,得到呈白色泡沫状的S7g(0.650g,65%)。TLC(乙酸乙酯:甲苯=2/8,v/v):Rf=0.64;H NMR(600MHz,CDCl3):δ7.48(d,J=7.8Hz,2H),7.39(d,J=8.2Hz,2H),7.35-7.12(m,41H),6.91(d,J=10.2Hz,2H),6.87(d,J=10.3Hz,2H),5.55(s,1H),5.50(s,1H),5.32(t,J=10.2Hz,1H),4.94-4.89(m,8H),4.83-4.67(m,10H),4.64-4.66(m,5H),4.54-4.50(m,3H),4.36-4.22(m,6H),4.06-3.87(m,6H),3.83-3.46(m,10H),3.38-3.34(m,3H),3.20-3.10(m,2H),2.33(s,3H);13C NMR(150MHz,CDCl3):δ169.5,162.8,155.0,155.0,154.8,154.6,154.51,150.6,150.5,150.2,139.6,138.9,138.4,138.3,138.3,138.2,138.1,137.5,129.3,129.2,128.7,128.6,128.6,128.5,128.5,128.4,128.3,128.2,128.1,128.0,128.0,127.9,127.9,127.7,127.5,127.4,126.2,125.5,117.3,114.9,114.2,102.2,101.4,100.8,98.3,9 6.5,95.8,95.7,95.5,95.3,95.3,95.0,82.3,80.6,78.3,76.2,76.0,75.8,75.5,75.4,75.2,74.9,74.8,74.7,74.7,74.2,74.1,73.8,73.7,73.5,72.9,72.3,71.9,68.9,68.8,68.2,68.0,66.4,58.0,57.1,55.9,44.3,42.9,40.8,40.4,40.3,40.0,21.7,21.2;ESI-MS:m/z C102H106Cl6N2O25计算值1962.6590;实验值1973.5278(M+H)+。
对甲氧基苯基-O-[2-O-乙酰基-3,4,6-O-三-苄基-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基]-(1→2)-O-[3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基]-(1→6)-3,4-O-二苄基-α-D-甘露哌喃糖苷(S7h):在0℃下,向S7g(0.600g,0.328mmol)于无水CH2Cl2(70mL)中的溶液中相继添加三乙基硅烷(2.10mL,13.2mmol)、三氟乙酸(0.953mL,13.2mmol)。搅拌所得反应混合物2h。2h后,TLC(乙酸乙酯:甲苯,1.5/8.5v/v)指示产物形成且起始物质耗尽。用饱和NaHCO3(2×50mL)洗涤反应混合物。进一步用CH2Cl2(3×30mL)萃取水层,且用盐水溶液(100mL)洗涤经合并的有机层,经MgSO4干燥,过滤且在真空中浓缩。通过急骤管柱层析(0%→10%EA/甲苯)纯化残余物,得到呈透明油状的S7h(0.350g,58%)。TLC(乙酸乙酯:甲苯=1.5/8.5,v/v):Rf=0.35;1H NMR(600MHz,CDCl3):δ7.33-7.04(m,45H),6.92(d,J=8.2Hz,2H),6.80(d,J=8.3Hz,2H),5.55(s,1H),5.31(t,J=10.1Hz,1H),5.06(d,J=3.2Hz,1H),4.93-4.80(m,4H),4.47-4.35(m,13H),4.35-4.18(m,8H),4.05(dd,J=3.2&7.8Hz,2H),3.82(d,J=8.4Hz,2H),3.80-3.62(m,14H),3.49-3.20(m,6H),3.10(bs,2H),2.08(s,3H);13C NMR(150MHz,CDCl3):δ169.5,168.0,155.3,155.0,154.7,150.2,138.9,138.4,138.3,138.2,137.8,132.7,131.1,129.0,128.7,128.7,128.6,128.6,128.6,128.5,128.4,128.3,128.2,128.1,128.1,128.0,127.9,127.8,127.7,127.5,127.4,117.3,114.9,101.5,100.8,95.7,80.6,78.5,75.8,75.3,74.9,74.8,74.6,74.1,74.0,73.8,73.7,73.64,73.5,72.8,72.3,71.9,71.1,68.9,68.8,68.4,68.2,67.6,58.0,56.5,55.9,41.5,39.0,37.3,33.7,32.0,31.4,30.6,29.9,29.20,26.6,24.0,23.2,22.9,21.2,20.1,14.6,14.4,14.3,14.3,11.2;ESI-MS:m/z C102H108Cl6N2O25计算值1974.6750;实验值1975.5438(M+H)+。
对甲氧基苯基-O-[2-O-乙酰基-3,4,6-O-苄基-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→2)-[甲基-5-乙酰胺基-7,8,9-三-O-乙酰基-3,5-二去氧基-D-甘油-α-D-半乳糖-壬-2-酮哌喃糖苷酸酯]-(2→6)-4-O-苄基-2,3-二-O-苄酰基-1-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→6)-O-3,4,-二-O-苄基-α-D-甘露哌喃糖苷(S7i):将供体S4c(0.187g,0.182mmol)、接受体S7h(0.300g,0.151mmol)及经活化的分子筛(0.700g)于无水CH2Cl2(10mL)中的混合物在室温下搅拌1h。将反应混合物冷却至-50℃,缓慢地添加NIS(0.067g,0.302mmol)及TfOH(4μL,0.037mmol),且将所得反应混合物搅拌2h。通过添加Et3N淬灭反应,用CH2Cl2稀释,经由硅藻土过滤,相继用饱和Na2S2O3、NaHCO3萃取,经硫酸钠干燥且在真空中浓缩。通过急骤管柱层析(0%→10%EA/甲苯)纯化残余物,得到呈无色泡沫状的S7i(0.317g,73%)。TLC:(乙酸乙酯:甲苯=1.5/8.5,v/v):Rf=0.61;1H NMR(600MHz,CDCl3):δ7.90(m,4H),5.52-7.45(m,2H),7.37-7.08(m,59H),6.90(d,J=8.3Hz,2H),6.80(d,J=8.3Hz,2H),5.72(t,J=10.2Hz,1H),5.53(s,1H),5.35-5.32(m,3H),5.20(dd,J=3.2&8.3Hz,1H),5.08(dd,J=2.8&8.4Hz,2H),4.99-4.79(m,7H),4.70-4.18(m,26H),4.06(dd,J=3.2&7.8Hz,1H),3.94(m,2H),3.90-3.84(m,10H),3.74(s,3H),2.60(s,3H),2.59-2.57(m,3H),3.45-3.32(m,6H),3.20(t,J=9.8Hz,1H),2.90(t,J=10.2Hz,2H),2.83(dd,J=3.2&7.8Hz,1H),2.08(s,3H),2.06(s,3H),2.02(t,J=10.1Hz,1H),1.99(s,3H),1.91(s,3H);13C NMR(150MHz,CDCl3):δ171.4,170.5,169.7,169.1,167.4,165.7,165.1,159.1,154.6,159.8,138.5,138.2,138.1,137.9,137.4,133.3,133.1,132.3,130.2,129.8,129.6,129.4,128.7,128.5,128.4,128.1,127.9,127.5,127.4,127.1,127.08,116.9,114.5,101.3,100.6,100.4,100.0,95.3,80.2,79.9,77.4,75.5,75.3,74.5,74.3,74.1,73.7,73.4,73.2,73.1,72.5,72.1,71.5,71.2,70.8,68.6,68.5,68.3,67.8,67.5,67.3,63.1,61.5,57.8,56.6,55.5,53.1,38.6,37.0,36.8,33.3,32.0,31.0,30.9,28.8,26.2,25.5,23.6,22.8,22.5,20.8,20.6,19.7,14.3,13.9,10.8;ESI-MS:m/z C146H153Cl6N3O43计算值2850.5080;实验值2850.8248。
[2-O-乙酰基-3,4,6-O-苄基-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→2)-[甲基-5-乙酰胺基-7,8,9-三-O-乙酰基-3,5-二去氧基-D-甘油-α-D-半乳糖-壬-2-酮哌喃糖苷酸酯]-(2→6)-4-O-苄基-2,3-二-O-苄酰基-1-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基-(1→6)-O-3,4,-二-O-苄基-α-D-甘露哌喃醣苷氟化物(13):向化合物S7i(0.430g,0.150mmol)于10mL的乙腈:甲苯:H2O(4:2:1)中的溶液中添加硝酸铈铵(0.641g,0.754mmol),且将所得反应混合物在0℃下搅拌2h。用EtOAc(100mL)稀释反应物且用H2O(30×2mL)及盐水(30mL)洗涤。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→20%EA/甲苯)纯化产物,得到呈透明泡沫状的1-OH化合物(0.240g,57%)。在-30℃下,将残余物(0.200g,0.072mmol)溶解于CH2Cl2(10mL)中。随后,缓慢地添加DAST(29μL,0.216mmol),且将所得反应混合物搅拌2h。当TLC(乙酸乙酯:甲苯,2/8)指示产物形成且起始物质耗尽时,用NaHCO3水溶液淬灭反应。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过硅胶管柱层析(0%→10%EA/甲苯)纯化残余物,得到呈白色泡沫状的13(0.104g,50%)。TLC(乙酸乙酯:甲苯=2/8,v/v):Rf=0.44;1H NMR(600MHz,CDCl3):δ7.95-7.90(m,4H),7.58-7.49(m,2H),7.39-7.18(m,59H),5.82(t,J=10.2Hz,1H),5.65(d,J=51Hz,1H),5.34(t,J=10.2Hz,2H),5.21(dd,J=3.6&7.8Hz,1H),5.13-4.19(m,30H),4.10-4.09(m,5H),4.02-3.74(m,12H),3.65(s,3H),3.64-3.27(m,14H),3.02(t,J=10.2Hz,2H),2.87(dd,J=3.2&7.8Hz,1H),2.12(s,3H),2.10(s,3H),2.09(t,J=10.2Hz,1H),2.00(s,3H),1.97(s,3H);13C NMR(150MHz,CDCl3):δ171.4,170.2,169.7,169.1,167.4,165.6,165.1,159.1,154.4,139.0,138.8,138.2,138.1,138.0,137.7,133.3,129.8,129.7,129.2,128.7,128.4,128.1,127.9,127.7,127.6,127.5,127.4,127.3,127,1,127.0,125.2,101.4,100.7,100.4,9.9,97.3,95.180.2,79.6,78.6,75.5,74.5,74.3,73.8,73.5,73.3,73.1,72.8,71.3,71.3,70.7,70.1,68.6,67.8,66.9,63.1,61.5,57.8,53.1,37.1,25.5,21.3,20.9,20.5;ESI-MS:m/z C139H146Cl6FN3O41计算值2746.3454;实验值2746.7604。
制备核心三醣14-15S
根据吾人的先前报导3获得具有N-邻苯二甲酰胺保护的还原端三醣(Man-β-1,4-GlcNAc-β-1,4-GlcNAc-β-连接基团)14。接着如流程S8中所示将化合物14修饰为15。
流程S8|制备化合物15.i,(1)EDA、n-BuOH,90℃,(2)Troc-Cl、NaHCO3、CH2Cl2,72%,历经2个步骤;ii,Ac2O、吡啶,79%;iii,DDQ、CH2Cl2H2O,58%。
5-迭氮基戊基-O-2-O-乙酰基-3-O-对甲氧基-苄基-4,6-O-亚苄基-β-D-甘露-哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基)-(1→4)-O-3,6-二-O-苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖苷(S8b):将化合物S8a(0.950g,0.545mmol)及10mL的乙二胺:n-BuOH(2:8)的混合物在90℃下搅拌隔夜。蒸发挥发物,且使用高真空干燥粗混合物。随后将其溶解于CH2Cl2(20mL)中,在0℃下添加NaHCO3(0.363g,5.45mmol)及氯甲酸2,2,2-三氯乙酯(0.86mL,5.45mmol),使其升温至室温且搅拌隔夜。TLC(乙酸乙酯:甲苯,2/8)指示产物形成且起始物质耗尽。用CH2Cl2(100mL)稀释反应物,用水(2×50mL)及盐水(50mL)溶液洗涤。经Na2SO4干燥有机层且在真空中浓缩。通过硅胶管柱层析(0%→10%EA/甲苯)纯化残余物。随后使用10mL的吡啶/乙酸酐(6:4)使产物乙酰化,直至TLC(乙酸乙酯:甲苯,2/8)指示起始物质完全耗尽。随后在真空中浓缩反应混合物且通过硅胶管柱层析(0%→10%EA/甲苯)纯化,得到呈白色泡沫状的S8b(0.650g,65%)。TLC(乙酸乙酯:甲苯=2/8,v/v):Rf=0.64;1H NMR(600MHz,CDCl3):δ7.62-7.20(m,34H),5.89(s,1H),4.56(d,J=3.2Hz,1H),4.23(d,J=8.9Hz,2H),4.20-4.10(m,12H),4.00-3.86(m,5H),3.69-3.30(m,14H),3.29(s,3H),3.22-3.20(m,4H),3.20-3.00(m,1H),2.66(s,3H,-C(O)CH3),1.56-1.38(m,4H,-CCH2C-,连接基团),1.21-1.23(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,CDCl3):δ177.7,169.6,167.0,138.5,138.4,138.3,138.2,137.2,137.8,137.5,134.2,133.2,133.6,131.7,131.1,131.45,129.8,129.06,128.9,128.2,128.4,128.2,128.4,128.7,127.8,127.87,127.83,127.4,127.4,127.0,127.2,127.29,127.6,127.20,126.88,126.24,125.1,123.6,123.3,101.5,102.7,99.7,98.0,97.1,78.13,78.55,78.05,75.82,74.57,75.38,75.27,74.25,74.30,72.78,72.26,70.89,66.85,68,43,68.25,67.75,66.87,55.57,53.71,52.10,28.97,28.87,23.80,22.77,20.12;ESI-MS:m/z C74H83Cl6N5O20计算值1575.1930;实验值1576.235(M+H)+。
5-迭氮基戊基-O-2-O-乙酰基-4,6-O-亚苄基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基)-(1→4)-O-3,6-二-O-苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖苷(15):在0℃下,向S8a(1.0g,0.673mmol)于10mL(CH2Cl2:H2O,10/1)中的溶液中添加DDQ(0.183g,0.808mmol),且搅拌所得反应混合物直至TLC(乙酸乙酯:甲苯,2/8)指示产物形成且起始物质耗尽。随后过滤反应混合物且用H2O(2×30mL)洗涤有机层。进一步用CH2Cl2(2×50mL)萃取水层。用盐水溶液(40mL)洗涤经合并的有机层,经Na2SO4干燥且在真空中浓缩。通过急骤管柱层析(0%→15%EA/甲苯)纯化残余物,得到呈无色泡沫状的15(0.652g,70%)。TLC(乙酸乙酯:甲苯=2/8v/v):Rf=0.24;1H NMR(600MHz,CDCl3):δ7.44-7.16(m,30H),5.44(s,1H),5.15(d,J=3.2Hz,1H),4.88(t,J=8.9Hz,2H),4.54-4.40(m,12H),4.08-3.58(m,3H),3.56-3.40(m,14H),3.25-3.20(m,4H),3.02-3.00(m,1H),2.16(s,3H,-C(O)CH3),1.51-1.47(m,4H,-CCH2C-,连接基团),1.30-0.99(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,CDCl3):δ171.5,168.6,167.0,138.7,138.4,138.38,138.2,137.2,137.8,137.5,134.2,133.2,133.6,131.7,131.1,131.45,129.8,129.06,128.9,128.2,128.4,128.2,128.4,128.7,127.8,127.87,127.83,127.4,127.4,127.0,127.2,127.29,127.6,127.20,126.88,126.24,125.1,123.6,123.3,101.5,102.7,99.7,98.0,97.1,78.13,78.55,78.05,75.82,74.57,74.37,74.27,73.25,73.30,72.78,71.26,69.89,68.85,69,43,68.25,67.73,66.67,56.57,55.71,51.10,28.67,28.27,23.00,21.47,21.02;ESI-MS:m/zC66H75Cl6N5O19计算值1455.0420;实验值1476.3082(M+Na)+。
合成高甘露糖型寡醣(G1-G6)S
图S1|高甘露糖型聚醣及其片段的结构
通过供体1-5,吾人研究其通过核心三醣接受体14达成的醣基化及偶合产物(流程S9)。用已知的酰亚胺酯供体1使14醣基化,以70%产率11得到四醣S9a。在S9a的选择性亚苄基开环之后,所得6″-OH S9b经1醣基化,以60%产率得到所要经完全保护的Man3GlcNAc2五醣S9d,N-聚醣生物合成中的早期中间体,且由此得到所有N-聚醣中的保守基元。在另一途径中,还原性打开亚苄基S9a产生二醇S9c,通过使用Cp2HfCl2及AgOTf,其进一步与三甘露糖氟化物4缩合,以52%产率11获得七醣S9e,高基氏体(Golgi apparatus)中GlcNAc介导分支的中间体。
流程S9|制备Man3及Man5GlcNAc2.a,BF3.OEt2、CH2Cl2,MS,-40℃,2h,70%;b,三乙基硅烷、PhBCl2、CH2Cl2,MS,-78℃,1h,82%;c,1,BF3.OEt2、CH2Cl2,MS,-60℃至-20℃,2h,60%;d,pTsOH、CH3CN,2h,66%;e,4,AgOTf、Cp2HfCl2、甲苯,MS,-40℃,2h,52%;f,(1)NH2CH2CH2NH2、nBuOH,90℃,隔夜;(2)Ac2O、吡啶,隔夜;(3)NaOMe、MeOH,隔夜;(4)Pd(OH)2、MeOH:H2O:HCOOH(5:3:2)、H2;G1:65%;G2:52%;G4:29%;Cp2HfCl2:二氯化双(环戊二烯基)铪。为合成Man9GlcNAc2,其为HIV-1gp120表面上所存在的主要糖型及由广谱中和抗体识别的抗原决定基的重要组分,在Cp2HfCl2/AgOTf的促进下用氟化物2使化合物14经历3″-O醣基化,以65%产率得到六醣S10a。S10a的对甲苯磺酸介导的还原性开环得到二醇S10b,其经5进一步醣基化,得到十一醣S10c(流程S10)。
流程S10|制备Man4(G5)及Man9GlcNAc2(G6).a,2,AgOTf、Cp2HfCl2、甲苯,MS,-40℃,2h,58%;b,p-TsOH、CH3CN,2h,60%;c,5,(BrC6H4)3NSbCl6、CH3CN,MS,-10℃至室温,4h,52%;d,(1)NH2CH2CH2NH2、nBuOH,90℃,隔夜;(2)Ac2O、吡啶,隔夜;(3)NaOMe、MeOH,隔夜;(4)Pd(OH)2、MeOH:H2O:HCOOH(5:3:2)、H2;G5:42%;SG6:26%;(BrC6H4)3NSbCl6:参(4-溴苯基)铵基六氯锑酸盐。
5-胺基戊基-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(G1):通过以下一般程序2(方法1)脱除化合物14(0.150g,0.110mmol)的保护基,得到呈白色固体状的所要三醣G1(0.045g,65%)。1H NMR(600MHz,D2O):δ4.72(S,1H,与D2O重迭,H-1c),4.60(d,J=7.8Hz,1H,H-1a),4.49(d,J=7.8Hz,1H,H-1b),4.06(d,J=3Hz,1H),3.94-3.89(m,4H),3.86-3.58(m,14H),3.50-3.49(m,1H),3.42-3.42(m,1H),2.97(t,J=10.8Hz,2H,-CH2NH2,连接基团),2.07(s,3H,-C(O)CH3),2.03(s,3H,-C(O)CH3),1.68-1.65(m,2H,-CCH2C-,连接基团),1.60-1.58(m,2H,-CCH2C-,连接基团),1.40-1.39(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,D2O):δ177.32,177.17,173.76,104.12(C-1a,1J C,H=163.1),103.80(C-1b,1J C,H=162.2),102.80(C-1c,1J C,H=160.2),82.07,81.35,79.15,77.31,77.25,75.49,75.13,74.68,73.23,72.84,69.34,63.65,62.83,62.77,57.75,57.72,42.05,30.78,29.10,24.86,24.84,24.83;ESI-MS:m/z C27H49N3O16计算值671.3005;实验值694.3115(M+Na)+。
5-迭氮基戊基-O-(2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)-2-O-乙酰基-4,6-O-亚苄基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基)-(1→4)-O-3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄-哌喃糖苷(S91):将三氯乙酰亚胺酸酯1(0.278g,0.437mmol)、壳二糖接受体14(0.300g,0.219mmol)及经活化的分子筛于无水CH2Cl2(10mL)中的混合物在室温下搅拌1h。将反应物冷却至-40℃,随后缓慢地添加三氟化硼合乙基醚(12μL,0.109mmol)且将所得反应混合物搅拌2h。通过添加Et3N淬灭反应,用CH2Cl2稀释,经由硅藻土过滤且在真空中浓缩。通过急骤管柱层析(0%→15%EA/甲苯)纯化残余物,得到呈白色泡沫状的S9a(0.310g,70%)。TLC(乙酸乙酯:甲苯=2/8,v/v):Rf=0.46;1H NMR(600MHz,CDCl3):δ7.84-7.65(m,8H,Ar-H),7.38-7.31(m,2H,Ar-H),7.30-7.18(m,28H,Ar-H),6.98-6.90(m,7H,Ar-H),6.75-6.71(m,3H,Ar-H),5.45(s,1H,Ph-CH,亚苄基),5.43-5.43(d,J=3.4Hz,1H,H-2d),5.34(d,J=3.1Hz,1H,H-2c),5.21(d,J=8.1Hz,1H,H-1a),5.19(d,J=2.1Hz,1H,H-1d),4.90(d,J=8.1Hz,1H,H-1a),4.83-4.81(m,3H),4.67(s,1H),4.67-4.61(m,2H),4.53-4.43(m,7H),4.38-4.23(q,2H),4.22-4.16(m,1H),4.13-4.06(m,6H),3.87-3.75(m,6H),3.66-3.60(m,3H),3.53-3.45(m,3H),3.38(dd,J=4.2&12.0Hz,1H),3.26-3.17(m,3H),3.09-3.07(m,1H),2.87-2.82(m,2H),2.06(s,3H,-C(O)CH3),2.03(s,3H,-C(O)CH3),1.35-1.23(m,4H,-CCH2C-,连接基团),1.07-1.01(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,CDCl3):δ169.87,168.75,167.83,138.97,138.47,138.17,137.36,134.30,134.10,131.72,129.12,128.90,128.64,128.55,128.51,128.32,128.23,128.09,127.84,127.46,128.14,126.24,123.40,101.44,99.38,98.90,98.37(C-1d,1J C,H=172Hz),97.31,79.05,78.84,77.89,76.14,7 5.02,74.78,73.70,73.40,73.06,73.04,72.37,72.03,71.00,69.11,69.85,69.11,68.85,68.59.68.50,67.84,66.70,56.85,55.99,51.38,28.95,28.55,23.28,21.33,21.16;ESI-MS:m/z C105H107N5O25计算值1838.7180;实验值1861.7223(M+Na)+。
5-迭氮基戊基-O-(2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)-2-O-乙酰基-4-O-苄基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基)-(1→4)-O-3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖苷(S9b):将四醣S9a(0.280g,0.152mmol)及经活化的分子筛于CH2Cl2(10mL)中的混合物在室温下搅拌1h。将反应物冷却至-78℃,添加三乙基硅烷(73μL,0.456mmol)及二氯苯基硼烷(69μL,0.532mmol)且搅拌1h。通过添加Et3N淬灭反应,经由硅藻土过滤且在真空中浓缩。将残余物与甲醇共蒸馏2-3次,随后通过急骤管柱层析(0%→15%EA/甲苯)纯化,得到6″-OH S9b(0.230g,82%)。TLC:(乙酸乙酯:甲苯=2/8,v/v):Rf=0.36;1H NMR(600MHz,CDCl3):δ7.85-7.52(m,8H,Ar-H),7.52-7.12(m,29H,Ar-H),7.00-6.88(m,9H,Ar-H),6.74-6.70(m,3H,Ar-H),5.39(d,J=1.8Hz,1H,H-2d),5.32(d,J=3.6Hz,1H,H-2c),5.21(d,J=8.4Hz,1H,H-1a),5.10(s,1H,H-1d),4.90-4.81(m,4H),4.67-4.61(m,2H,重迭的H-2b),4.59(s,1H,H-1c),4.59-4.44(m,9H),4.37(d,J=12Hz,2H),4.24-4.12(m,3H),4.10-4.05(m,3H),3.90-3.77(m,4H),3.69-3.58(m,7H),3.49(t,J=11.5Hz,2H),3.41-3.36(dd,J=6.1,11.2Hz,2H),3.26(dd,J=6.0,9.2Hz,1H),3.26-3.18(m,2H),3.06-2.98(m,1H),2.88-2.81(m,2H,连接基团),2.06(s,3H,-C(O)CH3),2.04(s,3H,-C(O)CH3),1.37-1.22(m,4H,连接基团),1.07-1.00(m,2H,连接基团);13C NMR(150MHz,CDCl3):δ170.45,169.88,168.78,168.24,167.86,138.95,138.92,138.77,138.58,138.21,138.11,137.95,137.37,134.35,134.16,133.89,132.09,131.70,128.90,128.76,128.71,128.64,128.58,128.51,128.41,128.31,128.08,127.96,127.81,127.64,127.13,123.97,123.46,100.14,98.40,98.36,97.36,76.24,75.52,75.30,74.96,74.71,74.51,74.29,73.41,73.09,72.55,72.12,69.41,69.20,68.85,68.48,67.93,67.71,61.87,56.78,56.00,51.38,28.96,28.55,23.28,21.37;ESI-MS:m/z C105H109N5O25计算值1840.7337;实验值1863.7383(M+Na)+。
5-迭氮基戊基-O-(2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)-2-O-乙酰基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄-哌喃糖基)-(1→4)-O-3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖苷(S9c):在室温下,向四醣S9a(0.220g,0.119mmol)于乙腈(10mL)中的搅拌溶液中添加对甲苯磺酸单水合物(0.045g,0.239mmol)。8h后,用Et3N淬灭反应混合物且在真空中浓缩。通过急骤管柱层析(0%→15%EA/甲苯)纯化残余物,得到呈白色固体状的标题二醇S9c(0.140g,66%)。TLC:(乙酸乙酯:甲苯=2/8,v/v):Rf=0.26;1H NMR(600MHz,CDCl3):δ7.85-7.52(m,8H,Ar-H),7.32-7.17(m,25H,Ar-H),6.98-6.91(m,7H,Ar-H),6.75-6.71(m,3H,Ar-H),5.30(d,J=3.6Hz,1H,),5.21(d,J=8.6Hz,1H,H-1a),5.20(s,2H,重迭的H-1d),4.89(d,J=8.4Hz,1H,H-1b),4.85-4.82(m,3H),4.64-4.60(m,3H),4.544.46(m,7H),4.38(d,J=12.0Hz,1H),4.34(d,J=12.2Hz,1H),4.21-4.04(m,5H),3.92(m,1H),3.87-3.79(m,3H),3.70-3.59(m,5H),3.52-3.43(m,4H),3.39(dd,J=3.2,8.3Hz,1H),3.28(dd,J=3.2,8.2Hz,1H),3.29-3.19(m,2H),2.29(m,1H),2.86-2.83(m,3H),2.11(s,3H,-C(O)CH3),2.03(s,3H,-C(O)CH3),1.35-1.23(m,4H,-CCH2C-,连接基团),1.06-1.01(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,CDCl3):δ170.76,169.88,138.92,138.70,138.62,138.52,138.12,138.06,134.27,134.07,133.81,131.95,131.64,128.82,128.64,128.59,128.52,128.50,128.39,138.33,128.20,128.09,128.06,128.03,128.01,127.94,127.81,127.75,127.54,127.42,127.29,127.06,123.90,123.36,98.51,98.30,98.24(C-1d,1J,C,H=173Hz),97.30,79.07,78.38,77.60,76.10,75.52,75.03,74.79,74.71,74.59,74.51,73.39,73.03,72.07,72.01,71.30,69.53,69.27,69.05,68.44,67.70,67.17,62.44,56.74,55.94,51.32,28.89,28.49,23.22,21.31,21.22;ESI-MS:m/z C98H103N5O25计算值1750.6868;实验值1773.6913(M+Na)+。
5-迭氮基戊基-O-二-(2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3),(1→6)-2-O-乙酰基-4-O-苄基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基)-(1→4)-O-3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖苷(S9d):将供体1(0.113g,0.178mmol)、接受体S9b(0.220g,0.119mmol)及经活化的分子筛于无水CH2Cl2(10mL)中的混合物在室温下搅拌1h。将反应物冷却至-60℃,随后缓慢地添加三氟化硼合乙基醚(12μL,0.109mmol)且将所得反应混合物在-20℃下搅拌2h。通过添加Et3N淬灭反应,用CH2Cl2稀释,经由硅藻土过滤且在真空中浓缩。通过急骤管柱层析(0%→20%EA/己烷)纯化残余物,得到呈无色泡沫状的S9d(0.170g,60%)。TLC:(乙酸乙酯:己烷=3/7,v/v):Rf=0.46;1H NMR(600MHz,CDCl3):δ7.77-7.59(m,8H,Ar-H),7.32-7.16(m,43H,Ar-H),7.14-7.08(m,2H,Ar-H),6.91-6.89(m,4H,Ar-H),6.73-6.67(m,6H,Ar-H),5.45(s,1H,H-2d),5.34(d,J=3.1Hz,1H,H-2c),5.32(s,1H,H-2d'),5.17(d,J=8.4Hz,1H,H-1a),5.09(s,1H,H-1d),5.0(s,1H,H-1d'),4.87(d,J=8.4Hz,1H,H-1b),4.83-4.78(q,2H),4.74-4.71(q,2H),4.65-4.61(m,4H),4.56-4.42(m,15H),4.31(d,J=9.2Hz,1H),4.12-4.03(m,5H),4.03-3.76(m,9H),3.68-3.43(m,9H),3.30(dd,J=3.2,9.1Hz,1H),3.21-3.15(m,2H),3.13(d,J=9.2Hz,1H),3.09(d,J=8.4Hz,1H),2.89-2.79(m,2H,-CH2N3-,连接基团),2.12(s,3H,-C(O)CH3),2.07(s,3H,-C(O)CH3),1.69(s,3H,-C(O)CH3),1.37-1.12(m,4H,-CCH2C-,连接基团),1.07-1.01(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,CDCl3):δ170.45,170.22,170.18,168.21,167.71,138.93,138.72,138.68,138.50,138.24,138.22,138.07,133.83,133.75,128.87,128.79,128.67,128.63,128.58,128.42,128.21,128.06,127.99,127.96,127.81,127.76,127.70,127.46,127.08,123.36,100.13(C-1a,1J C,H=159.3Hz),99.39(C-1d',1J C,H=171.5Hz),98.35(C-1b,1J C,H=162.3Hz),98.31(C-1d,1J C,H=170Hz),97.16,76.20,76.05,75.58,75.21,74.97,74.89,74.70,74.47,74.28,73.94,73.72,73.00,72.25,72.20,71.70,71.50,69.15,69.07,69.04,68.99,68.50,68.45,67.80,65.45,56.83,51.37,29.94,23.27,21.31,21.28,20.83;ESI-MS:m/z C134H139N5O31计算值2314.9379;实验值2337.9461(M+Na)+。
5-胺基戊基-二-(α-D-甘露哌喃糖基)-(1→3),(1→6)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄-哌喃糖苷(G2):通过以下一般程序2(方法1)脱除五醣S9d(0.090g,0.038mmol)的保护基,得到呈白色粉末状的化合物G2(0.020g,52%)。1H NMR(600MHz,D2O):δ5.10(d,J=1.3Hz,1H,H-1d),5.09(d,J=1.5Hz,1H,H-1d'),4.78(s,1H,与D2O重迭,H-1c),4.59(d,J=8.4Hz,1H,H-1a),4.49(d,J=7.8Hz,1H,H-1b),4.25(s,1H),4.01(d,J=1.5Hz,1H),4.00(d,J=1.3Hz,1H),3.97-3.77(m,8H),3.75-3.65(m,20H),3.52-3.48(m,2H),2.98(t,J=11.2Hz,2H),2.06(s,3H),2.02(s,3H),1.68-1.65(m,2H),1.601.57(m,2H),1.49-1.40(m,2H);13C NMR(150MHz,D2O):δ181.17,174.69,174.42,102.51(C-1d,1J C,H=171.5Hz),101.34,101.04,100.35(C-1d',1J C,H=173.5Hz),99.60,80.48,79.61,79.30,74.49,74.36,74.16,73.43,72.66,72.37,71.93,70.39,70.31,70.13,70.09,69.98,69.85,66.84,66.76,65.80,61.12,90.93,60.07,59.97,54.97,54.85,39.30,28.03,26.35,23.07,22.17,22.07;ESI-MS:m/z C39H69N3O26计算值995.4067;实验值1018.4106(M+Na)+。
5-迭氮基戊基-O-(2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)-{2-O-乙酰基3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)}-[2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→6)]-2,4-二-O-苄基-α-D-甘露哌喃糖基-(1→6)}-2-O-乙酰基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基)(1→4)-O-3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖苷(S9e):将三氟甲磺酸银(0.127g,0.497mmol)、二氯化双(环戊二烯基)铪(0.121g,0.319mmol)及经活化的分子筛于无水甲苯(10mL)中的混合物在室温下搅拌1h。随后将反应混合物冷却至-40℃,添加供体4(0.103g,0.085mmol)及接受体S9c(0.125g,0.071mmol)于5mL甲苯中的溶液。将混合物搅拌2h,用Et3N淬灭,用CH2Cl2稀释且经由硅藻土过滤。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→15%EA/甲苯)纯化残余物,得到呈无色泡沫状的S9e(0.110g,52%)。TLC:(丙酮:甲苯=2/8,v/v):Rf=0.36;1H NMR(600MHz,CDCl3):δ7.77-7.40(m,8H,Ar-H),7.32-7.07(m,85H,Ar-H),6.99-6.88(m,4H,Ar-H),6.79-6.68(m,6H,Ar-H),5.44(s,1H,H-2d),5.43(s,1H,H-2e),5.41(s,1H,H-2e'),5.29(d,J=3.4Hz,1H,H-2c),5.18(s,2H,H-1d,d'),5.16(s,1H,H-1e),5.09(s,1H,H-1e'),4.92(s,1H,H-1c),4.91(d,J=8.5Hz,1H,H-1a),4.83-4.78(q,1H),4.84-4.73(m,6H),4.65-4.61(m,2H),4.60-4.57(m,5H),4.524.35(m,17H),4.27(d,J=12.3Hz,1H),4.20-4.19(m,1H),4.10-3.99(m,6H),3.97-3.70(m,13H),3.69-3.57(m,8H),3.56-3.31(m,6H),3.30(dd,J=3.2,9.2Hz,1H),3.30-3.18(m,5H),2.97-2.96(m,1H),2.89-2.79(m,2H,-CCH2C-,连接基团),2.12(s,3H,-C(O)CH3),2.07(s,3H,-C(O)CH3),2.01(s,6H,-C(O)CH3),1.33-1.23(m,4H,-CCH2C-,连接基团),1.06-0.99(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,CDCl3):δ170.56,170.42,170.26,170.02,168.45,168.69,138.95,138.93,138.72,138.68,138.58,138.24,138.22,138.07,133.83,132.09,131.76,128.87,128.79,128.67,128.63,128.61,128.58,128.52,128.42,128.13,128.06,127.99,127.92,127.81,127.76,127.74,127.46,127.08,123.71,123.36,100.13(C-1e,e',1J C,H=169.2Hz),99.39(C-1b,1J C,H=162.2Hz),98.35(C-1d,1J C,H=171.5Hz),98.31(C-1a,1J C,H=160.2Hz),97.16(C-1d',1J C,H=170.5Hz),76.20,76.05,75.42,74.97,74.89,74.80,74.70,74.65,74.47,74.28,73.94,73.68,73.26,72.60,72.25,72.20,71.70,71.50,69.15,69.07,69.04,68.99,68.50,68.45,67.80,65.54,56.73,55.90,51.29,31.14,29.91,28.87,28.47,23.19,21.33,21.27,21.19l;HRMS(MALDI-TOF):m/z C176H185N5O42计算值3042.2400;实验值3065.2210(M+Na)+。
5-胺基戊基-α-D-甘露哌喃糖基(1→3),[二-(α-D-甘露哌喃糖基)-(1→3),(1→6)-α-D-甘露哌喃糖基](1→6)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(G4):通过以下一般程序2(方法1)脱除七醣S9e(0.140g,0.024mmol)的保护基,获得呈白色固体状的标题化合物G4(0.039g,29%)。1H NMR(600MHz,D2O):δ5.09(s,1H,H-1d),4.89(s,1H,H-1d'),4.86(d,J=1.5Hz,1H,H-1e),4.75(d,J=1.5Hz,1H,H-1e'),4.76(s,1H,与D2O重迭,H-1c),4.50(d,J=7.8Hz,1H,H-1a),4.35(d,J=7.9Hz,1H,H-1b),4.17(d,J=1.7Hz,1H),4.07-4.05(m,1H),4.00-3.97(m,2H),3.94-3.37(m,40H),2.90-2.85(m,2H,-CH2N连接基团),1.98(s,3H,-C(O)CH3),1.94(s,3H,-C(O)CH3),1.61-1.46(m,4H,-CCH2C-,连接基团),1.35-1.26(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,D2O):δ177.2,172.3,102.44,101.44,101.01,100.37,99.85,99.25,80.54,79.49,79.28,78.58,74.45,74.43,72.50,71.93,70.64,70.24,70.15,70.04,70.01,69.87,69.46,69.39,66.69,66.66,65.94,65.91,65.56,65.13,65.12,60.93,60.91,60.43,60.13,60.04,59.97,54.90,54.86,39.28,27.98,26.48,22.08,22.02,21.99;ESI-MS:m/z C51H89N3O36计算值1319.5123;实验值1342.5165(M+Na)+。
5-迭氮基戊基-O-(2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→2)-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→2)-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)-2-O-乙酰基-4,6-O-亚苄基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基)-(1→4)-O-3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖苷(S10a):将三氟甲磺酸银(0.328g,1.28mmol)、二氯化双(环戊二烯基)铪(0.340g,0.897mmol)及经活化的分子筛于无水甲苯(10mL)中的混合物在室温下搅拌1h。随后将反应混合物冷却至40℃,添加供体2(0.383g,0.282mmol)及接受体14(0.350g,0.256mmol)于5mL甲苯中的溶液。将混合物搅拌2h,用Et3N淬灭,用CH2Cl2稀释且经由硅藻土过滤。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→15%EA/甲苯)纯化残余物,得到呈无色泡沫状的S10a(0.405g,58%)。TLC:(乙酸乙酯:甲苯=2/8,v/v):Rf=0.46;1H NMR(600MHz,CDCl3):δ7.89-7.35(m,8H,Ar-H),7.38-7.12(m,56H,Ar-H),7.12-7.08(m,3H,Ar-H),7.03-6.88(m,7H,Ar-H),6.78-6.69(m,4H,Ar-H),5.50(s,1H,H-1f),5.37(d,J=1.4Hz,1H,H-2c),5.25(s,1H,Ph-CH-,亚苄基),5.23-5.17(m,3H,重迭的,H-1d,e,f),4.98(s,1H,H-1c),4.95(d,J=7.5Hz,1H,H-1a),4.91-4.53(m,5H),4.69-4.58(m,3H),4.554.28(m,16H),4.25-4.00(m,13H),3.95-3.91(m,1H),3.91-3.60(m,14H),3.58(d,J=12Hz,1H),3.51-3.45(m,4H),3.42-3.38(m,3H),3.29-3.10(m,3H),2.98-2.78(m,3H),2.08(s,3H,-C(O)CH3),1.99(s,3H,-C(O)CH3),1.35-1.23(m,4H,-CCH2C-,连接基团),1.07-1.00(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,CDCl3):δ170.38,169.81,168.76,168.26,139.06,138.95,138.92,138.87,138.60,137.36,128.85,128.66,128.64,128.58,128.55,128.48,128.43,128.39,128.35,128.26,128.10,128.02,127.82,127.71,101.48,100.08,99.75,99.39,98.36,97.27,77.80,78.75,76.10,75.38,74.93,74.82,74.70,73.58,73.28,73.21,73.06,72.79,72.65,72.57,72.17,71.01,69.60,69.10,68.96,68.63,66.74,56.85,55.97,51.37,28.95,28.54,23.27,21.43,21.09;ESI-MS:m/z C159H163N5O35计算值2703.1054;实验值2726.1214(M+Na)+。
5-胺基戊基-α-D-甘露哌喃糖基-(1→2)-α-D-甘露哌喃糖基-(1→2)-α-D-甘露-哌喃糖基-(1→3)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(G5):通过以下一般程序2(方法1)脱除六醣S10a(0.140g,0.051mmol)的保护基,获得呈白色固体状的标题化合物G5(0.025g,42%)。1HNMR(600MHz,D2O):δ5.32(s,1H,H-1d),5.27(s,1H,H-1e),5.01(s,1H,H-1f),4.74(s,1H,H-1c),4.56(d,J=7.8Hz,1H,H-1a),4.63(d,J=7.8Hz,1H,H-1b),4.18(d,J=3.3Hz,1H),4.07(s,1H),4.04(d,J=3.2Hz,1H),4.03(d,J=3.1Hz,1H),3.32-3.33(m,35H),2.05(t,J=11.2Hz,2H,-CH2NH2,连接基团),2.03(s,3H,-C(O)CH3),2.00(s,3H,-C(O)CH3),1.64(m,2H,-CCH2C-,连接基团),1.50(m,2H,-CCH2C-,连接基团),1.36(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,D2O):δ174.54,174.38,102.17(C-1e,1J C,H=170.2Hz),101.34(C-1b,1J C,H=161.2Hz),101.014(C-1d,1J C,H=169.8Hz),100.70(C-1f,1J C,H=172.2Hz),100.56(C-1a,1J C,H=160.2Hz),99.85,80.50,79.25,78.66,78.55,78.47,76.12,74.48,73.37,73.13,72.33,71.88,70.24,70.17,70.05,69.95,69.89,66.95,66.81,66.71,65.81,61.01,60.97,60.88,60.76,60.02,59.92,54.92,39.25,27.99,26.32,23.19,22.06,22.04;ESI-MS:m/z C45H79N3O31计算值1157.4590;实验值1180.4591(M+Na)+。
5-迭氮基戊基-O-(2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→2)-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→2)-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)-2-O-乙酰基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基)-(1→4)-O-3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖苷(S10b):向S10a(0.205g,0.075mmol)于10mL乙腈中的溶液中添加对甲苯磺酸单水合物(0.020g,0.113mmol),在室温下搅拌9h。用Et3N淬灭反应且在真空中浓缩。通过急骤管柱层析(0%→20%EA/甲苯)纯化残余物,得到二醇S10b(0.120g,60%)。TLC:(丙酮:甲苯=2/8,v/v):Rf=0.26;1H NMR(600MHz,CDCl3):δ7.83-7.62(m,8H,Ar-H),7.41-7.06(m,57H,Ar-H),6.95-6.89(m,6H,Ar-H),6.73-6.71(m,2H,Ar-H),5.50(d,J=4.8Hz,1H,H-2f),5.23(d,J=1.8Hz,1H,H-1d),5.22(d,J=3.8Hz,1H,H-1c),5.19(d,J=3.2Hz,1H,H-1e),5.18(s,1H,H-1f),5.03(d,J=7.8Hz,1H,H-1a),4.88(t,J=7.8Hz,1H),4.82-4.74(m,5H),4.68-4.38(m,21H),4.33(d,J=7.8Hz,1H),4.29(d,J=7.9Hz,1H),4.20-4.00(m,8H),3.95-3.71(m,9H),3.69-3.53(m,6H),3.52-3.33(m,8H),3.29-3.15(m,5H),2.90-2.79(m,2H),2.08(s,3H,-C(O)CH3),1.92(s,3H,-C(O)CH3),1.35-1.20(m,4H,-CCH2C-,连接基团),1.081.01(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,CDCl3):δ170.40,169.88,168.68,138.20,138.94,138.78,138.71,138.56,138.48,138.38,138.21,138.02,134.30,133.84,132.02,128.78,128.65,128.63,128.43,128.40,128.38,128.25,128.15,128.06,127.94,127.88,127.84,127.77,127.69,127.65,127.56,127.10,123.90,123.41,100.98,99.83,98.34,97.32,78.31,78.17,78.11,78.00,76.22,75.52,75.28,75.05,74.82,74.40,73.98,73.67,73.58,73.36,73.06,72.59,72.48,72.42,72.09,71.32,70.52,69.46,69.09,68.83,68.72,68.42,68.20,67..61,62.57,56.77,55.98,51.37,29.99,29.65,28.94,28.54,23.27,21.44,21.13,14.41;ESI-MS:m/z C152H159N5O35计算值1615.0741;实验值2638.0754(M+Na)+。
5-迭氮基戊基-O-(2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→2)-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→2)-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)-{2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→2)-3,4,6-三-O-苄基-α-D-甘露-哌喃糖基-(1→3)-[2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→2)-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→6)]-2,4-二-O-苄基-α-D-甘露哌喃糖基-(1→6)}-2-O-乙酰基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄-哌喃糖基)-(1→4)-O-3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖苷(S10c):将六醣接受体S10a(0.1g,0.038mmol)、硫基甘露糖苷供体5(0.104g,0.045mmol)及经活化的分子筛(0.5g)于CH3CN(10mL)中的混合物在室温下搅拌1h。将所得混合物冷却至-10℃,添加六氯锑酸参(4-溴苯基)铵(0.096g,0.114mmol)且在室温下搅拌6h。TLC(乙酸乙酯:甲苯,2/8)指示产物形成且起始物质耗尽,通过Et3N淬灭反应。用CH2Cl2稀释反应混合物且经由硅藻土过滤。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→10%EA/甲苯)纯化残余物,得到呈无色泡沫状的S10c(0.095g,52%)。TLC:(丙酮:甲苯=2/8,v/v):Rf=0.41;1H NMR(600MHz,CDCl3):δ7.77-7.50(m,8H,Ar-H),7.29-6.98(m,126H,Ar-H),6.95-6.89(m,4H,Ar-H),6.746.64(m,5H,Ar-H),5.56(s,1H,H-2f),5.53(s,1H,H-2h),5.51(s,1Hh'),5.34(s,1H,H-2c),5.32(d,J=12.2Hz,2H),5.18(d,J=7.8Hz,1H),5.12(s,1H),5.08(s,1H),5.04(s,2H),4.59-3.75(m,116H),3.26-3.16(m,4H),3.08-3.02(m,2H),2.93-2.83(m,2H),2.12(s,3H),2.06(s,3H),2.06(s,3H),1.97(s,3H),1.39-1.26(m,4H),1.07-1.05(m,2H);13C NMR(150MHz,CDCl3):δ170.12,170.03,169.76,168.14,167.36,138.64,138.54,138.45,138.41,138.27,138.07,133.68,133.57,131.74,129.05,128.62,128.57,128.51,128.33,128.23,128.14,128.06,127.99,127.94,127.81,127.78,127.73,127.45,101.24,99.81,99.59,99.48,99.34,99.24,99.05,98.02,96.95,79.82,79.23,78.33,78.24,78.14,75.86,75.22,75.12,75.02,74.93,74.77,74.57,74.47,74.38,74.30,74.17,73.29,73.11,72.50,72.20,71.94,71.35,71.25,69.13,69.03,68.79,68.53,68.48,68.35,68.17,67.40,56.56,55.69,51.09,28.66,28.26,22.99,21.17,21.12;HRMS(MALDI-TOF):m/z C284H297N5O62计算值4772.0232;实验值4794.9783(M+Na)+。
5-胺基戊基-α-D-甘露哌喃糖基-(1→2)-α-D-甘露哌喃糖基-(1→2)-α-D-甘露-哌喃糖基-(1→3)-{α-D-甘露哌喃糖基-(1→2)-α-D-甘露哌喃糖基-(1→3)-[α-D-甘露-哌喃糖基-(1→2)-α-D-甘露哌喃糖基-(1→6)]-α-D-甘露哌喃糖基-(1→6)}-β-D-甘露-哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(G6):通过以下一般程序2(方法1)脱除化合物S10c(0.120g,0.024mmol)的保护基,得到呈白色固体状的Man9GlcNAc2G6(0.012g,26%)。1H NMR(600MHz,D2O):δ5.40(s,1H,H-1d),5.33(s,1H,H-1d'),5.30(s,1H,H-1e),5.14(s,1H,H-1),5.05(s,1H,H-1),5.03(d,J=2.4Hz,2H,H-1),4.86(s,1H,H-1),4.58(d,J=7.8Hz,1H,H-1a),4.48(d,J=7.8Hz,1H,H-1b),4.22(d,J=2.4Hz,1H),4.14(s,1H),4.14-3.95(m,12H),3.94-3.59(m,54H),3.50-3.48(m,1H),3.02(t,J=9.2Hz,2H),2.06(s,3H,-C(O)CH3),2.02(s,3H,-C(O)CH3),1.68-1.63(m,2H,-CCH2C-,连接基团),1.59-1.56(m,2H,-CCH2C-,连接基团),1.41-1.37(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,D2O):δ174.47,171.01,102.24(C-1d,1J C,H=171.8Hz),102.21(C-1d',1J C,H=169.0Hz),102.19(C-1e,1J C,H=173.8Hz),101.43,101.03,100.82,100.59(C-1a,1J C,H=162.8Hz),100.21,99.61,97.97(C-1b,1J C,H=159.8Hz),81.86,79.32,78.88,78.63,78.45,74.50,74.48,74.14,73.39,73.27,73.20,73.17,72.69,72.36,71.90,71.61,70.21,70.03,69.93,69.37,67.01,66.94,66.90,66.85,66.81,66.76,65.42,65.40,65.07,64.90,61.11,61.06,61.01,60.91,60.06,59.94,54.95,39.29,38.61,28.02,26.34,22.16,22.10,22.06;ESI-MS:m/z C75H129N3O56计算值1967.7231;实验值1990.7279(M+Na)+。
合成混合型寡醣(G7-G14)S
混合型糖的合成始于通过AgOTf/Cp2HfCl2催化的14与6的缩合,以63%产率得到五醣S11a(流程S11)。还原性亚苄基打开产生二醇S11b,其在6″-O处经三甘露糖硫苷3进一步醣基化,以55%产率得到S11c。Cp2HfCl2/AgOTf介导的接受体S11b与6的缩合造成以58%产率形成S11d。
流程S11|制备八醣G7及七醣G8.a,6,AgOTf、Cp2HfCl2、甲苯,MS,-40℃,4h,63%;b,pTsOH、CH3CN,5h,78%;c,3,(BrC6H4)3NSbCl6、CH3CN,MS,-10℃至室温,4h,55%;d,6,AgOTf、Cp2HfCl2、甲苯,MS,-40℃,4h,58%;e,(1)NH2CH2CH2NH2、nBuOH,90℃,隔夜;(2)Ac2O、吡啶,隔夜;(3)NaOMe、MeOH,隔夜;(4)Pd(OH)2、MeOH:H2O:HCOOH(5:3:2)、H2;G7:26;G8:32%。
5-迭氮基戊基-O-2-O-乙酰基-3,6-O-二苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄-哌喃糖基-(1→2)-O-(3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)-2-O-乙酰基-4,6-O-亚苄基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基)-(1→4)-O-3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖苷(S11a):将三氯乙酰亚胺酸酯供体6(0.341g,0.308mmol)、壳二糖接受体14(0.350g,0.256mmol)及经活化的分子筛于无水CH2Cl2(10mL)中的混合物在室温下搅拌1h。将反应物冷却至-40℃,随后缓慢地添加三氟化硼合乙基醚(14.8μL,0.129mmol)且将所得反应混合物搅拌2h。通过添加Et3N淬灭反应,用CH2Cl2稀释,经由硅藻土过滤且在真空中浓缩。通过急骤管柱层析(0%→10%EA/甲苯)纯化残余物,得到呈白色泡沫状的S11a(0.360g,63%)。TLC:(乙酸乙酯:甲苯=2/8,v/v):Rf=0.56;1H NMR(600MHz,CDCl3):δ7.84-7.55(m,12H,Ar-H),7.45-7.10(m,28H,Ar-H),7.06-6.90(m,18H,Ar-H),6.75-6.71(m,4H,Ar-H),5.37(d,J=8.4Hz,1H,H-1),5.21(d,J=8.4Hz,1H,H-1),5.19(s,1H,Ph-CH,亚苄基),5.09(t,J=9.0Hz,1H),4.93-4.76(m,6H),4.57(s,1H),4.54(d,J=12.0Hz,1H),4.45-4.07(m,20H),3.69-3.15(m,22H),3.02-3.00(m,1H),2.86-2.81(m,2H,连接基团),2.25(s,3H,-C(O)CH3),1.84(s,3H,-C(O)CH3),1.35-1.23(m,4H,-CCH2C-,连接基团),1.07-1.02(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,CDCl3):δ170.68,170.05,167.88,138.98,138.89,138.85,138.71,138.63,138.14,138.07,137.78,134.08,133.90,133.46,132.06,131.81,129.32,128.90,128.83,128.67,128.55,128.50,128.34,128.27,127.95,127.79,127.70,127.60,101.00,99.41,98.38,97.91,97.29,80.38,79.02,76.82,76.76,76.52,76.14,75.25,74.90,74.83,74.67,74.51,74.44,73.68,73.52,73.38,73.06,73.02,72.93,71.46,70.65,70.40,70.03,69.66,69.14,68.52,67.56,56.83,55.98,55.85,51.37,29.99,28.95,28.55,23.28,21.67,21.19;ESI-MS:m/z C133H132N6O31计算值2309.8862;实验值2332.8861(M+Na)+。
5-迭氮基戊基-O-2-O-乙酰基-3,6-O-二苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄-哌喃糖基(1→2)-O-(3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)-2-O-乙酰基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基)-(1→4)-O-3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖苷(S11b):向S11a(0.220g,0.095mmol)于乙腈(20mL)中的溶液中添加对甲苯磺酸单水合物(0.020g,0.143mmol)且将所得反应混合物在室温下搅拌5h。通过添加Et3N淬灭反应且在真空中浓缩。通过急骤管柱层析(0%→15%EA/甲苯)纯化残余物,得到二醇S11b(0.165g,78%)。TLC:(乙酸乙酯:甲苯=2/8,v/v):Rf=0.32;1H NMR(600MHz,CDCl3):δ7.89-7.43(m,12H,Ar-H),7.35-7.16(m,28H,Ar-H),7.09-7.05(m,5H,Ar-H),7.03-6.89(m,9H,Ar-H),6.75-6.72(m,3H,Ar-H),5.30(d,J=8.4Hz,1H,H-1a),5.23(d,J=8.4Hz,1H,H-1b),5.12(d,J=9.0Hz,1H),5.09(t,J=9.8Hz,1H),4.95(d,J=12.0Hz,1H),4.90(d,J=8.4Hz,1H,H-1e),4.87-4.80(m,2H),4.58-4.55(m,2H),4.50-4.41(m,10H,重迭的,H-1d),4.37-4.34(m,3H),4.27-4.24(m,2H),4.18-4.15(m,2H),4.10-3.92(m,5H),3.73-3.70(m,2H),3.65-3.61(m,2H),3.59-3.51(m,5H),3.47-3.38(m,6H),3.23-3.17(m,4H),3.04(dd,J=3.6,9.0Hz,1H),3.01-3.98(m,1H),2.83-2.78(m,3H),2.38(s,3H,-C(O)CH3),1.90(s,3H,-C(O)CH3),1.36-1.22(m,4H,-CCH2C-,连接基团),1.06-1.01(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,CDCl3):δ170.93,170.05,168.77,167.86,167.69,138.90,138.83,138.65,138.47,138.23,138.14,138.07,138.04,134.35,134.13,133.91,132.04,131.84,131.72,128.81,128.65,128.64,128.62,128.54,128.53,128.48,128.46,128.36,128.17,128.09,128.02,127.99,127.97,127.92,127.91,127.83,127.77,127.74,127.63,127.13,124.73,123.97,123.43,123.08,118.73,100.25(C-1e),98.37(C-1d),98.21(C-1c),97.86(C-1b),97.30(C-1a),82.86,80.05,78.38,76.92,76.15,75.72,75.60,75.45,74.86,74.83,74.70,74.42,74.24,73.75,73.42,73.38,72.82,70.81,70.60,70.27,69.99,69.50,69.15,69.13,68.53,67.88,66.28,62.98,56.78,55.99,55.88,51.37,32.21,29.98,28.94,28.54,23.27,21.69,21.18;ESI-MS:m/z C126H128N6O31计算值2221.8549;实验值2244.8529(M+Na)+。
5-迭氮基戊基-O-{4-O-乙酰基-3,6-O-二苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄-哌喃糖基-(1→2)}-O-{3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)-{2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)}-[2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露-哌喃糖基(1→6)]-2,4-二-O-苄基-α-D-甘露哌喃糖基-(1→6)}-2-O-乙酰基-β-D-甘露-哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基)-(1→4)-O-3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖苷(S11c):将五醣接受体S11b(0.150g,0.067mmol)、硫基甘露糖苷供体3(0.110g,0.080mmol)及经活化的分子筛(0.500g)于CH3CN(10mL)中的混合物在室温下搅拌1h。将所得混合物冷却至-10℃,添加六氯锑酸参(4-溴苯基)铵(0.170g,0.201mmol)且将所得反应物在室温下搅拌4h。TLC(乙酸乙酯:甲苯,2/8)指示产物形成且起始物质耗尽,通过Et3N淬灭反应。用CH2Cl2稀释反应混合物且经由硅藻土过滤。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→10%EA/甲苯)纯化残余物,得到呈无色泡沫状的S11c(0.130g,55%)。TLC:(乙酸乙酯:甲苯=1/9,v/v):Rf=0.42;1H NMR(600MHz,CDCl3):δ7.71-7.30(m,8H,Ar-H),7.28-7.10(m,70H,Ar-H),7.97-6.86(m,12H,Ar-H),6.69(m,7H,Ar-H),5.47(d,J=8.4Hz,2H),5.44(s,1H),5.32(d,J=8.4Hz,1H,H-1a),5.15(d,J=8.4Hz,1H,H-1b),5.13-5.10(m,2H),5.00(s,1H,H-1d),4.95(s,1H,H-1f),4.91(s,2H,H-1g,H-1g'),4.87-4.73(m,8H),4.62(d,J=8.7Hz,1H),4.57-4.28(m,26H),4.15-3.70(m,20H),3.68-3.40(m,15H),3.38-3.25(m,6H),3.24-3.09(m,5H),2.98(dd,J=2.3,7.8Hz,1H),2.89-2.79(m,2H),2.73-2.71(m,1H),2.27(s,3H,-C(O)CH3),2.05(s,3H,-C(O)CH3),1.99(s,3H,-C(O)CH3),1.88(s,3H,-C(O)CH3),1.32-1.28(m,4H,-CCH2C-,连接基团),1.07-0.98(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,CDCl3):δ170.26,168.89,138.38,128.47,128.37,128.32,128.30,128.26,128.24,128.19,128.08,128.04,127.91,127.86,127.73,127.56,127.49,127.39,127.26,100.35,100.23,99.89,99.56,99.23,98.54,98.33,98.23,78.43,76.09,75.37,75.07,74.08,74.44,74.04,73.47,73.31,72.63,68.81,51.08,29.71,29.37,28.66,28.25,22.98;HRMS(MALDI-TOF):m/z C204H210N6O48计算值3513.4134;实验值3536.4050(M+Na)+。
5-胺基戊基-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖基-(1→3),[二-(α-D-甘露哌喃糖基)-(1→3),(1→6)-α-D-甘露哌喃糖基](1→6)-β-D-甘露-哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(G7):通过以下一般程序2(方法1)脱除化合物S11c(0.105g,0.030mmol)的保护基,得到呈白色固体状的标题化合物G7(0.012g,26%)。1H NMR(600MHz,D2O):δ5.09(d,J=1.2Hz,1H,H-1d),5.02(d,J=8.4Hz,1H,H-1a),4.95(s,1H,H-1f),4.91(s,1H,H-1g),4.85(s,1H,H-1g'),4.78(S,1H,H-1c),4.61(d,J=7.8Hz,1H,H-1e),4.51(d,J=7.8Hz,1H,H-1b),4.22(t,J=10.2Hz,2H),4.18(dd,J=1.8,3.0Hz,1H),4.00(dd,J=1.8,3.1Hz,1H),4.08-3.38(m,47H),3.03(t,J=8.2Hz,2H,-NCH2-,连接基团),2.09(s,3H,-C(O)CH3),2.08(s,3H,-C(O)CH3),2.01(s,3H,-C(O)CH3),1.71-1.66(m,2H,-CCH2C-,连接基团),1.62-1.59(m,2H,-CCH2C-,连接基团),1.44-1.40(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,D2O):δ174.91,174.42,171.02,110.64(C-1d,1J C,H=174Hz),102.32,101.48,100.51(C-1a,1J C,H=161Hz),100.44,100.15,99.26,97.58(C-1g,1J C,H=169.6Hz),79.48,78.86,78.72,76.64,76.04,75.72,74.65,74.51,74.42,73.31,72.69,72.35,72.09,71.96,70.86,70.57,70.34,70.12,70.09,69.94,69.42,67.39,66.70,66.36,65.54,65.14,61.68,61.02,60.93,39.31,38.62,28,03,26.39,22.58,22.20,22.11,22.08,21.82;ESI-MS:m/z C59H102N4O41计算值1522.6092;实验值1523.6148(M+H)+。
5-迭氮基戊基-O-二-[{4-O-乙酰基-3,6-O-二苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄-哌喃糖基-(1→2)}-O-{3,4,6-三-O-苄基-α-D-甘露哌喃糖基}]-(1→3),(1→6)-2-O-乙酰基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基)-(1→4)-O-3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖苷(S11d):将三氟甲磺酸银(0.080g,0.315mmol)、二氯化双(环戊二烯基)铪(0.084g,0.220mmol)及经活化的分子筛于无水甲苯(10mL)中的混合物在室温下搅拌1h。随后将反应混合物冷却至-40℃,添加供体6(0.091g,0.094mmol)及接受体S11b(0.140g,0.063mmol)于5mL甲苯中的溶液。将混合物搅拌4h,用Et3N淬灭,用CH2Cl2稀释且经由硅藻土过滤。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→15%EA/甲苯)纯化残余物,得到呈无色泡沫状的S11d(0.110g,58%)。TLC:(丙酮:甲苯=2/8,v/v):Rf=0.39;1H NMR(600MHz,CDCl3):δ7.62-7.32(m,16H,Ar-H),7.27-6.87(m,41H,Ar-H),6.85-6.59(m,29H,Ar-H),5.27(d,J=8.4Hz,1H),5.16(s,1H),5.14(d,J=3.6Hz,1H),5.10(d,J=1.8Hz,1H),4.98(d,J=3.6Hz,1H),4.89-4.68(m,10H),4.59-4.21(m,21H),4.19-3.87(m,7H),3.80(dd,J=3.2,1.8Hz,1H),3.70-3.52(m,7H),3.51-3.28(m,8H),3.27-3.18(m,5H),3.15-3.03(m,3H),3.01(dd,J=3.2,1.8Hz,1H),2.912.75(m,5H),2.73-2.69(m,1H),2.27(s,3H),1.98(s,3H),1.86(s,3H),1.32-1.23(m,4H),1.03-0.99(m,2H);13C NMR(150MHz,CDCl3):δ171.09,171.04,170094,167.91,167.57,139.19,138.86,138.63,138.54,138.42,138.31,138.21,138.14,137.93,133.84,133.71,132.04,131.80,131.69,128.80,128.64,128.39,128.32,128.13,128.08,127.79,127.70,127.60,100.18,100.00,98.36,97.88,97.61,97.30,97.22,80.14,79.97,79.31,75.85,75.56,75.39,75.30,75.04,74.71,74.59,73.95,73.64,73.24,72.86,72.14,71.55,71.37,71.04,70.53,70.40,70.15,69.71,69.12,68.12,65.40,63.29,56.34,55.92,51.45,51.37,51.27,28.95,28.54,23.26,21.13,21.02;HRMS(MALDI-TOF):m/z C183H183N7O43计算值3168.2306;实验值3191.2375(M+Na)+。
5-胺基戊基-二-[2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖基]-(1→3),(1→6)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(G8):通过以下一般程序2(方法1)脱除化合物S11d(0.130g,0.041mmol)的保护基,得到呈白色固体状的所要七醣G8(0.015g,29%)。1H NMR(600MHz,D2O):δ5.99(d,J=8.5Hz,1H,H-1a),4.93(s,1H,H-1d),4.83(s,1H,H-1d'),4.76(s,1H,H-1c),4.60(d,J=7.8Hz,1H,H-1b),4.56(d,J=7.9Hz,1H,H-1e),4.49(d,J=7.9Hz,1H,H-1e'),4.27(s,2H),4.11(d,J=1.8Hz,1H),3.96-3.41(m,41H),2.98(t,J=8.9Hz,2H,-NCH2-,连接基团),2.13(s,3H,C(O)CH3),2.09(s,3H,-C(O)CH3),2.06(s,3H,-C(O)CH3),2.01(s,3H,-C(O)CH3),1.69-1.64(m,2H,-CCH2C-,连接基团),1.60-1.57(m,2H,-CCH2C-,连接基团),1.42-1.38(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,D2O):δ174.88,174.78,174.65,174.42,171.0,101.39,101.05,100.61(C-1c),100.39(C-1a),99.55(C-1e'),97.60(C-1d'),97.20(C-1d),79.41,79.28,78.92,76.63,76.25,75.79,74.50,73.64,72.84,71.95,70.09,69.89,69.48,67.39,67.35,66.20,65.39,61.66,61.61,60.75,60.61,59.96,55.68,54.98,54.94,39.30,28.04,26.35,22.55,22.31,22.21,22.12,22.08;ESI-MS:m/z C55H95N5O36计算值1402.5830;实验值1403.5862(M+H)+。
制备聚醣G12-G14
在Cp2HfCl2/AgOTf的促进下,在核心三醣接受体14的3-O位置立体选择性安装三醣触角7,得到六醣S12a。S12a的对甲苯磺酸介导的还原性开环得到二醇S12b。考虑到一级羟基的反应性较高,用三甘露糖硫苷3使S12b在6-O位置进一步醣基化,且随后通过稳定的自由基阳离子参(4-溴苯基)铵基六氯锑酸盐在单电子转移反应中活化,得到壬醣S12c。通过化合物S12c,进行一系列官能基转换,得到所要完全脱除保护基的聚醣G12。前驱体寡醣G12为通过源自海洋细菌的α-2,6或2,3唾液酸转移酶唾液酸化的适当起始物质,因其较宽的接受体特异性且无固有的唾液酸酶活性而出名。G12的唾液酸转移酶(SiaT)介导的酶促末端唾液酸化1-4分别可有效地获得α-2,6及α-2,3唾液酸化聚醣G13及G14(流程S12)。
流程S12|制备G12-G14.i)7,Cp2HfCl2、AgOTf,MS,-40℃,2h,63%;ii)p-TsOH、CH3CN,5h,78%;iii)3,(BrC6H4)3NSbCl6、CH3CN,MS,-10℃至室温,4h,55%;iv)(1)NH2CH2CH2NH2、n-BuOH,90℃,隔夜;(2)Ac2O、吡啶,隔夜;(3)NaOMe、MeOH,隔夜;(4)Pd(OH)2、MeOH:H2O:HCOOH(5:3:2)、H2;62%;v)CMP-β-D-唾液酸、α-2,6-或2,3-唾液酸转移酶,G13:60%;G14:56%;Cp2HfCl2:二氯化双(环戊二烯基)铪;AgOTf:三氟甲磺酸银;(BrC6H4)3NSbCl6:参(4-溴苯基)铵基六氯锑酸盐。
5-迭氮基戊基-O-2-O-乙酰基-3,4,6-O-三-苄基-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基-(1→2)-O-(3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)-2-O-乙酰基-4,6-O-亚苄基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基)-(1→4)-O-3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖苷(S12a):将三氟甲磺酸银(0.327g,1.28mmol)、二氯化双(环戊二烯基)铪(0.339g,0.896mmol)及经活化的分子筛于无水甲苯(10mL)中的混合物在室温下搅拌1h。随后将反应混合物冷却至-40℃,添加供体7(0.430g,0.307mmol)及接受体壳二糖三醣14(0.35g,0.256mmol)于5mL甲苯中的溶液。将混合物搅拌2h,用Et3N淬灭,用EtOAc稀释且经由硅藻土过滤。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→15%EA/甲苯)纯化残余物,得到呈白色泡沫状的S12a(0.500g,71%)。TLC:(乙酸乙酯:甲苯=2/8,v/v):Rf=0.62;1H NMR(600MHz,CDCl3):δ7.85-7.49(m,8H,Ar-H),7.02-7.39(m,44H,Ar-H),7.19-7.02(m,11H,Ar-H),6.99-6.90(m,6H,Ar-H),6.83-6.78(m,3H,Ar-H),6.75-6.70(m,5H,Ar-H),5.310(t,J=7.5Hz,1H,H-2f),5.27(d,J=8.1Hz,1H,H-1a),5.25(d,J=8.4Hz,1H,H-1e),5.15(d,J=2.3Hz,1H),5.01(s,1H,Ph-CH,亚苄基),4.90-4.80(m,7H),4.62(d,J=8.5Hz,1H),4.58-4.53(m,3H),4.52-4.38(m,10H),4.32-4.13(m,11H),4.12-4.05(m,5H),4.02(m,1H),3.89(d,J=3.2Hz,1H),3.75-3.73(m,2H),3.72-3.49(m,8H),3.48-3.37(m,5H),3.34-3.29(m,4H),3.29-3.18(m,4H),2.98(m,1H),2.89-2.79(m,2H,连接基团),2.29(s,3H,-C(O)CH3),1.99(s,3H,-C(O)CH3),1.49-1.30(m,4H,-CCH2C-),1.10-1.00(m,2H,-CCH2C-);13C NMR(150MHz,CDCl3):δ170.8,169.4,168.7,167.7,139.1,139.9,138.9,138.8,138.8,138.5,138.2,138.2,138.1,137.9,137.6,134.2,133.8,133.1,131.9,131.8,131.6,129.1,128.8,128.6,128.5,128.4,128.4,128.4,128.3,128.3,128.2,128.1,128.0,128.0,127.9,127.8,127.8,127.7,127.7,127.5,127.5,127.4,127.3,127.0,126.7,126.1,123.8,123.3,122.8,100.88,100.83,99.94,99.38,98.43,98.30,97.26,80.31,79.00,78.05,77.61,76.42,76.31,76.1,.75.1,74.8,74.7,74.6,74.6,74.4,74.4,74.2,73.6,73.4,73.3,72.9,72.8,72.2,71.8,71.7,70.4,70.0,69.8,69.0,68.8,68.4,68.2,67.7,67.6,56.7 56.0,55.9,51.3,28.8,28.4,23.2,21.7,21.2;ESI-MS:m/z C160H160N6O36计算值2742.0799;实验值2765.0952(M+Na)+。
5-迭氮基戊基-O-2-O-乙酰基-3,4,6-O-三-苄基-β-D-半乳哌喃糖基-(1→4)-O-3,6-O-二苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基-(1→2)-O-(3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)-2-O-乙酰基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基)-(1→4)-O-3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖苷(S12b):向S12a(0.601g,0.219mmol)于乙腈(10mL)中的溶液中添加对甲苯磺酸单水合物(0.046g,0.328mmol),在室温下搅拌2h。用Et3N淬灭反应且在真空中浓缩。通过急骤管柱层析(0%→15%EA/甲苯)纯化残余物,得到二醇S12b(0.460g,78%)。TLC:(丙酮:甲苯=2/8,v/v):Rf=0.32;1H NMR(600MHz,CDCl3):δ7.91-7.42(m,12H,Ar-H),7.39-7.12(m,41H,Ar-H),7.10-6.93(m,13H,Ar-H),6.85-6.81(m,3H,Ar-H),6.78-6.73(m,3H,Ar-H),5.33(t,J=7.8Hz,1H,H-2f),5.23(t,J=8.4Hz,2H,H-1),5.08(d,J=6.2Hz,1H),4.97(d,J=8.9Hz,1H,H-1),4.94-4.80(m,5H),4.64-4.60(m,2H),4.49-4.21(m,21H),4.19-4.15(m,2H),4.15-3.89(m,8H),3.89(d,J=6.2Hz,1H),3.78(d,J=5.8Hz,1H),3.69(dd,J=6.3,12.1Hz,1H),3.65-3.64(m,1H),3.56-3.32(m,15H),3.29(dd,J=6.6,12.5Hz,1H),3.29-3.23(m,3H),3.00-2.99(m,2H),2.87-2.82(m,2H),2.74(t,J=9.5Hz,1H),2.28(s,3H,-C(O)CH3),1.98(s,3H,-C(O)CH3),1.37-1.22(m,4H,-CCH2C-),1.07-1.00(m,2H,-CCH2C-);13C NMR(150MHz,CDCl3):δ171.29,169.57,168.78,168.58,168.03,167.90,139.01,138.92,138.84,138.68,138.45,138.39,138.36,138.22,138.14,137.98,134.35,134.15,134.02,133.89,132.04,131.95,131.68,130.10,129.54,129.46,128.80,128.70,128.66,128.62,128.60,128.52,128.47,128.46,128.39,128.30,128.15,128.07,128.01,127.98,127.94,127.91,127.83,127.72,127.60,127.39,127.20,126.96,123.90,123.46,122.88,101.11,100.87,98.87,98.33,83.75,80.57,80.14,78.58,78.29,76.15,75.78,75.45,74.11,74.96,74.83,74.70,74.61,74.44,74.10,73.77,73.73,73.59,73.43,73.38,73.03,72.30,71.97,70.61,70.29,69.82,69.14,69.08,68.54,68.38,67.94,66.94,66.47,63.21,56.80,56.09,56.01,51.37,31.71,31.21,30.97,30.59,29.99,29.65,28.55,27.87,27.29,26.96,26.24,25.59,25.36,24.26,23.27,22.98,21.86,21.32,20.80,20.49,18.94,16.76,15.95,15.44,15.10,14.40,14.07,13.67,13.47;ESI-MS:m/z C153H156N6O36计算值1327.0189;实验值1350.0207(M+Na)+。
5-迭氮基戊基-O-{2-O-乙酰基-3,4,6-O-三-苄基-β-D-半乳哌喃糖基-(1→4)}-O-{3,6-O-二苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基-(1→2)}-O-{3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)-{2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)}-[2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→6)]-2,4-二-O-苄基-α-D-甘露-哌喃糖基-(1→6)}-2-O-乙酰基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基)-(1→4)-O-3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖苷(S12c):将六醣接受体S12b(0.40g,0.15mmol)、硫基甘露糖苷供体3(0.229g,0.226mmol)及经活化的分子筛(0.50g)于CH3CN(10mL)中的混合物在室温下搅拌1h。将所得混合物冷却至-10℃,添加六氯锑酸参(4-溴苯基)铵(0.254g,0.30mmol)且在室温下搅拌3h。TLC指示产物形成且起始物质耗尽,随后通过Et3N淬灭反应。用CH2Cl2稀释反应混合物且经由硅藻土过滤。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→10%EA/甲苯)纯化残余物,得到呈无色泡沫状的S12c(0.335g,55%)及接受体9(0.103g)。TLC:(乙酸乙酯:甲苯=1/9,v/v):Rf=0.46;1H NMR(600MHz,CDCl3):δ7.72-7.45(m,11H,Ar-H),7.37-7.28(m,5H,Ar-H),7.28-7.12(m,67H,Ar-H),7.09-7.02(m,12H,Ar-H),6.94-6.90(m,7H,Ar-H),6.80-6.78(m,4H,Ar-H),6.69-6.68(m,3H,Ar-H),6.62-6.61(m,3H,Ar-H),5.49(s,1H),5.48(s,1H),5.35(t,J=8.4Hz,1H),5.28(d,J=8.4Hz,1H,H-1),5.17(d,J=8.4Hz,1H,H-1),5.09(d,J=6.2Hz,1H),5.00(s,1H,H-1),4.92(d,J=3.1Hz,1H),4.90-4.72(m,12H),4.69-4.72(m,23H),4.26-4.17(m,5H),4.133.97(m,12H),3.89-3.82(m,12H),3.72(t,1H),3.68-3.62(m,1H),3.65-3.55(m,7H),3.54-3.45(m,4H),3.43-3.40(m,4H),3.40-3.29(m,9H),3.23-3.19(m,3H),3.17-3.09(m,2H),2.91-2.79(m,3H),2.68(s,1H),2.28(s,3H,-C(O)CH3),2.05(s,3H,-C(O)CH3),2.01(s,3H,-C(O)CH3),1.99(s,3H,-C(O)CH3),1.34-1.22(m,4H,连接基团),1.06-1.00(m,2H,连接基团);13C NMR(150MHz,CDCl3):δ171.37,170.38,170.29,169.54,168.49,168.23,167.97,167.53,139.28,139.11,139.03,138.97,138.94,138.67,138.63,138.58,138.53,138.41,138.38,138.25,138.10,128.74,128.70,128.67,128.63,128.58,128.55,128.53,128.51,128.48,128.44,128.40,128.37,128.34,128.30,128.15,128.03,128.00,127.95,127.92,127.84,127.77,127.75,127.70,127.64,127.61,101.17,100.92,98.84,98.28,97.71,97.45,75.49,75.36,75.26,75.17,75.06,74.96,74.82,74.75,74.65,74.60,74.38,74.30,73.99,73.77,73.73,73.59,73.25,73.25,73.06,72.99,72.91,72.26,72.18,71.98,71.82,71.57,71.45,70.52,70.03,69.92,69.56,69.10,68.90,68.85,68.57,68.48,68.37,66.64,66.06,56.75,56.09,55.99,51.37,34.25,29.99,28.94,28.54,25.90,23.27,21.86,21.42,21.33;HRMS(MALDI-TOF):m/z C231H238N6O53计算值3945.6071;实验值3968.6006(M+Na)+。
5-胺基戊基-β-D-半乳哌喃糖基-(1→4)-[2-乙酰胺基-2-去氧-β-D-葡萄-哌喃糖基-(1→2)-α-D-甘露哌喃糖基-(1→3),[二-(α-D-甘露哌喃糖基)-(1→3),(1→6)-α-D-甘露-哌喃糖基](1→6)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄-哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(G12):通过以下一般程序2(方法1)脱除化合物S12c(0.16g,0.048mmol)的保护基,得到所要壬醣G12(0.052g,62%)。1H NMR(600MHz,D2O):δ5.10(s,1H,H-1d),5.07(d,J=9.5Hz,1H,H-1a),4.91(s,2H,H-1h,h'),4.84(s,1H,H-1f),4.61(s,1H,H-1c),4.50(d,J=9.2Hz,1H,H-1b),4.49(d,J=8.1Hz,1H,H-1e),4.31(s,2H),4.19(bs,1H),4.07(bs,1H),4.02-3.51(m,53H),3.40-3.30(m,2H)2.99(t,J=11.2Hz,2H,-NCH2-,连接基团),2.08(s,3H,-C(O)CH3),2.07(s,3H,-C(O)CH3),2.04(s,3H,-C(O)CH3),1.70-1.65(m,2H,-CCH2C-,连接基团),1.62-1.58(m,2H,-CCH2C-,连接基团),1.48-1.38(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,D2O):δ177.60,177.17,105.68(C-1b),105.05(C-1d),104.23,104.04,103.79,103.37,103.18,102.88,102.23(C-1h'),102.03(C-1h),100.24(C-1g),82.20,82.06,82.01,81.58,81.51,81.40,79.38,78.70,78.08,77.43,77.37,77.18,77.03,76.07,75.66,75.43,75.23,75.11,75.06,74.96,74.83,74.71,73.72,73.65,73.33,73.20,73.11,72.95,72.89,72.84,72.69,72.18,71.28,70.15,68.50,69.46,69.36,69.01,68.31,67.90,67.86,64.45,63.75,63.69,63.61,62.87,62.82,62.75,57.94,57.72,42.06,30.78,29.10,25.35,25.30,24.96,24.87,24.83;ESI-MS:m/z C65H112N4O46计算值1684.6620;实验值1685.6968(M+H)+。
5-胺基戊基-5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-[2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖基-(1→3),[二-(α-D-甘露哌喃糖基)-(1→3),(1→6)-α-D-甘露-哌喃糖基](1→6)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(G13):通过以下一般程序3用α-2,6-唾液酸转移酶使化合物G12(5mg,0.0029mmol)唾液酸化2天,得到呈白色固体状的所要标题化合物G13(3.5mg,60%)。1H NMR(600MHz,D2O):δ5.07(s,1H,H-1d),5.06(d,J=8.5Hz,1H,H-1a),4.92(s,1H,H-1h),4.90(s,1Hi),4.89(s,1H,H-1i'),4.78(s,1H,H-1c),4.58(d,J=7.2Hz,1H,H-1f),4.46(d,J=7.8Hz,1H,H-1b),4.41(d,J=8.4Hz,1H,H-1e),4.25-4.24(m,2H),4.12(s,1H),4.04(d,J=4.7Hz,1H),4.04-3.42(m,61H),3.40(t,J=10.8Hz,1H),3.39-3.35(m,1H),2.95(t,J=10.9Hz,2H),2.64(dd,J=4.2,12.2Hz,1H,H-3赤道g),2.04(s,3H,-C(O)CH3),2.04(s,3H,-C(O)CH3),2.00(s,3H,-C(O)CH3),2.00(s,3H,-C(O)CH3),1.71-1.61(m,3H,-CCH2C-,连接基团及H-3轴向g),1.58-1.54(m,2H,-CCH2C-,连接基团),1.39-1.35(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,D2O):δ174.90,174.79,174.38,173.47,103.42,102.29(C-1d,1J C,H=170.9Hz),101.48,101.01,100.41,100.18(C-1a,1J C,H=162.9Hz),100.15,99.24((C-1h,1J C,H=171.3Hz)),97.87,97.27,80.78,79.59,79.20,78.72,78.36,76.61,75.81,74.79,74.48,74.36,73.63,73.28,72.63,72.52,72.35,72.30,72.09,71.94,71.65,70.82,70.69,70.53,70.31,70.12,70.05,69.89,69.41,68.33,68.17,67.32,67.17,66.66,65.47,65.09,63.30,62.62,61.66,60.98,60.89,60.32,60.02,59.94,57.95,54.91,51.81,40.01,39.27,28.00,26.37,23.62,23.19,22.59,22.17,22.05,22.00;ESI-MS(负模式):m/z C76H129N5O54计算值1975.7418;实验值1974.7600(M-H)-。
5-胺基戊基-5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→3)-β-D-半乳哌喃糖基-(1→4)-[2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖基-(1→3),[二-(α-D-甘露哌喃糖基)-(1→3),(1→6)-α-D-甘露-哌喃糖基](1→6)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(G14):通过以下一般程序3用α-2,3-唾液酸转移酶使化合物G12(5mg,0.0029mmol)唾液酸化8天,得到呈白色固体状的所要标题化合物G14(3.1mg,56%)。1H NMR(600MHz,D2O):δ5.01(s,1H,H-1d),4.97(dd,J=3.2,7.2Hz,1H,H-1a),4.84(s,1H,H-1h),4.81(s,1H,H-1i),4.75(s,1H,H-1i'),4.62(s,1H,H-1c),4.52(d,J=7.8Hz,1H,H-1f),4.97(d,J=9.2Hz,1H,H-1b),4.42(d,J=8.3Hz,1H,H-1e),4.39(d,J=8.3Hz,1H),4.22(t,J=7.8Hz,2H),4.09(s,1H),4.06(d,J=7.4Hz,1H),4.03(d,J=7.3Hz,1H),4.00(d,J=3.2Hz,1H),3.96-3.85(m,10H),3.85-3.71(m,19H),3.72-3.52(m,26H),3.51-3.48(m,2H,),3.38-3.370(m,2H),2.90(t,J=7.9Hz,2H,-NCH2-,连接基团),2.68(dd,J=4.8,12.0Hz,1H,H-3赤道g),2.01(s,3H,-C(O)CH3),1.99(s,3H,-C(O)CH3),1.96(s,3H,-C(O)CH3),1.95(s,3H,-C(O)CH3),1.73(t,J=8.6Hz,1H,H-3轴向g),1.61-1.56(m,2H,-CCH2C-,连接基团),1.54-1.46(m,2H,-CCH2C-,连接基团),1.34-1.33(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,D2O):δ171.94,171.81,171.37,170.80,113.44,99.86,99.53,99.27,98.42,97.99,97.38,97.08,96.74,96.21,75.74,75.66,75.52,73.56,72.85,72.41,72.10,71.62,71.45,71.36,70.27,69.81,69.62,69.28,69.14,69.02,68.90,68.69,67.91,67.82,67.50,67.20,67.08,66.87,66.36,65.46,65.29,65.01,64.39,64.32,63.68,62.47,62.09,61.02,59.50,59.39,58.64,57.96,57.87,57.10,56.91,52.12,51.91,48.60,42.85,40.32,36.54,36.25,35.04,25.27,24.98,23.34,20.60,19.53,19.13,19.03,18.95,13.70;ESI-MS(负模式):m/z C76H129N5O54计算值1975.7418;实验值1974.7671(M-H)-。
制备聚醣G9-G11
流程S13|制备G9-G11.i)(1)NH2CH2CH2NH2、n-BuOH,90℃,隔夜;(2)Ac2O、吡啶,隔夜;(3)NaOMe、MeOH,隔夜;(4)Pd(OH)2、MeOH:H2O:HCOOH(5:3:2)、H2;51%;ii)CMP-β-D-唾液酸、α-2,6/2,3-唾液酸转移酶,G10:77%;G11:52%
5-胺基戊基-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基(1→2)-α-D-甘露哌喃糖基]-(1→3)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-βD-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(G10):根据一般程序2(方法1)脱除六醣S12a(0.110g,0.041mmol)的保护基,得到呈白色固体状的所要化合物G10(0.025g,51%)。1H NMR(600MHz,D2O):δ5.04(d,J=8.4Hz,1H,H-1a),4.84(s,1H,H-1d),4.78(s,1H,H-1c),4.60(d,J=8.2Hz,1H,H-1e),4.51(d,J=8.1Hz,1H,H-1b),4.46(d,J=7.8Hz,1H,H-1f),4.28(d,J=3.3Hz,1H),4.26(d,J=3.6Hz,1H),4.10-3.82(m,9H),3.82-3.56(m,23H),3.52-3.38(m,5H),2.97(t,J=7.8Hz,2H,-NCH2-,连接基团),2.07(s,3H,-C(O)CH3),2.05(s,3H,-C(O)CH3),2.03(s,3H,-C(O)CH3),1.701.65(m,2H,-CCH2C-,连接基团),1.62-1.58(m,2H,-CCH2C-,连接基团),1.48-1.41(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,D2O):δ177.64,177.32,177.17,173.74,105.65,104.13,103.80,103.18,102.70,100.08,82.05,81.50,81.37,79.38,79.04,78.85,78.09,77.42,77.28,77.24,75.23,75.13,75.06,74.80,74.67,73.69,72.84,71.27,70.14,70.07,68.12,64.43,63.74,63.61,62.85,62.82,62.75,57.91,57.78,57.73,42.05,30.78,29.10,25.27,24.87,24.85,24.83;ESI-MS:m/z C47H82N4O3计算值1198.4855;实验值1221.5223(M+Na)+。
5-胺基戊基-[5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖基]-(1→3)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄-哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(G10):通过以下一般程序3用α-2,6-唾液酸转移酶使化合物G9(10mg,0.0083mmol)唾液酸化2天,得到呈白色固体状的所要标题化合物G10(9.5mg,77%)。1H NMR(600MHz,D2O):δ5.06(d,J=7.8Hz,1H,H-1a),4.80(s,1H,H-1d),4.78(s,1H,H-1c),4.60(d,J=7.8Hz,1H,H-1e),4.49(d,J=7.8Hz,1H,H-1b),4.43(d,J=7.8Hz,1H,H-1f),4.27(d,J=3.6Hz,1H),4.25(d,J=3.6Hz,1H),4.08-3.38(m,45H),2.98(t,J=11.2Hz,2H,-NCH2-,连接基团),2.60(dd,J=4.8,12.0Hz,1H,H-3赤道g),2.08(s,6H,-C(O)CH3)2.05(s,3H,-C(O)CH3),2.03(s,3H,-C(O)CH3),1.71(t,J=7.8Hz,1H,H-3轴向g),1.69-1.69(m,2H,-CCH2C-,连接基团),1.611.56(m,2H,-CCH2C-,连接基团),1.41-1.37(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,D2O):δ174.94,174.83,174.56,174.40,103.50,101,37,101.03,100.31,100.12,99.92,97.31,80.99,79.28,78.63,78.59,76.61,76.32,76.07,74.51,74.48,74.38,73.67,72.52,72.38,72.36,72.08,71.90,71.66,70.70,70.06,68.37,68.34,67.33,63.35,62.63,61.67,60.88,60.09,59.98,54.99,54.87,51.85,40.02,39.30,28.01,26.36,22.56,22.10,22.08,21.99;ESI-MS(负模式):m/z C58H99N5O39计算值1489.5833;实验值1488.5949(M-H)-。
5-胺基戊基-[5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→3)-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖基]-(1→3)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄-哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(G11):通过以下一般程序3用α-2,3-唾液酸转移酶使化合物G9(7mg,0.0058mmol)唾液酸化4天,得到呈白色固体状的所要标题化合物G11(4.6mg,52%)。1H NMR(600MHz,D2O):δ5.06(d,J=8.4Hz,1H,H-1a),4.79(s,1H,H-1d),4.75(s,1H,H-1c),4.62(d,J=6.6Hz,1H,H-1e),4.56(d,J=7.8Hz,1H,H-1b),4.51(d,J=7.8Hz,1H,H-1f),4.28(d,J=8.4Hz,2H),4.13(d,J=9.6Hz,1H),3.99-3.83(m,12H),3.82-3.57(m,27H),3.53-3.39(m,4H),2.99(t,J=7.8Hz,2H),2.77(dd,J=4.8,2.0Hz,1H,H-3赤道g),2.12(s,3H,-C(O)CH3)2.09(s,3H,-C(O)CH3),2.05(s,3H,-C(O)CH3),2.03(s,3H,-C(O)CH3),1.80(t,J=7.8Hz,1H,H-3轴向g),1.71-1.66(m,2H,-CCH2C-,连接基团),1.62-1.59(m,2H,-CCH2C-,连接基团),1.44-1.41(m,2H,-CCH2C-,连接基团);13C NMR(150MHz,D2O):δ174.88,174.53,174.46,174.40,102.50,102,37,101.65,101.31,100.12,99.86,98.31,80.32,79.67,78.78,78.56,76.66,75.32,75.07,74.51,74.48,74.38,73.67,72.52,72.38,72.36,72.08,71.90,71.66,70.70,70.06,68.37,68.34,67.33,63.35,62.63,61.67,60.88,60.09,59.98,54.99,54.87,51.85,40.02,39.30,28.01,26.36,22.56,22.10,22.08,21.27 20.27;ESI-MS(负模式):m/z C58H99N5O39计算值1489.5833;实验值1488.5944(M-H)-。
合成复合型寡醣S
先前,吾人已研发出一种用于快速产生二触角(G15、G16及G17)、三触角(G20-G22、G23、G25及G26)及四触角(G28、G32及G33)复合型N-聚醣的有效的化学酶促策略,含或不含经由α-2,6或α-2,3键联连接的末端N-乙酰神经胺酸残基。另外,吾人已研发出一种全化学合成策略,其采用一组模组化的醣苷供体(1-13)对核心三醣15进行立体选择性及区域选择性醣基化。然而,通过酶达成的不对称唾液酸化聚醣的合成因其特异性而复杂。出于此原因,吾人证明醣苷氟化物制备不对称复合型聚醣的实用性。首先,吾人从触角及核心三醣(14)的所有葡糖胺残基的C2-胺处的邻苯二甲酰胺保护开始,然而,在预安装的唾液酸存在下,发现整体脱除保护基制程很复杂。为克服这个困难,用碳酸三氯乙酯(troc)替换所有葡糖胺残基的C2-胺处的邻苯二甲酰胺保护。
合成聚醣G18.
用核心15使唾液酸化触角9在3-O位点醣基化,以63%产率得到所要七醣S14a。在p-TSA存在下移除亚苄基,得到4,6-二醇S14b,其在6-O位置经8进一步α醣基化,得到呈α及β形式(7/3分离产率)的混合物状的所要S14c。通过管柱层析分离主要α异构体且最后脱除保护基,得到呈白色固体状的G18。
流程S14|制备G18.i,9,AgOTf、Cp2HfCl2、甲苯,-20℃至0℃,63%;ii,pTSA、乙腈,59%;iii,8,AgOTf、Cp2HfCl2、甲苯,-40℃至-20℃,58%;iv,(1)LiOH、1,4-二恶烷:H2O,90℃,(2)Ac2O、吡啶,(3)NaOMe、MeOH,(4)Pd(OH)2、MeOH:H2O、H2,25%。
化合物S14a:将三氟甲磺酸银(0.087g,0.34mmol)、二氯化双(环戊二烯基)铪(0.090g,0.23mmol)及经活化的分子筛于无水甲苯(10mL)中的混合物在室温下搅拌1h。随后将反应混合物冷却至-20℃,添加供体9(0.153g,0.082mmol)及接受体壳二糖三醣15(0.100g,0.068mmol)于5mL甲苯中的溶液。将混合物在0℃下搅拌2h,用Et3N淬灭,用EtOAc稀释且经由硅藻土过滤。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→10%丙酮/甲苯)纯化残余物,得到呈白色泡沫状的S14a(0.140g,63%)。TLC:(丙酮:甲苯=1.5/8.5,v/v):Rf=0.52;1H NMR(600MHz,CDCl3):δ7.89(d,J=7.8Hz,4H),7.49-7.46(m,2H),7.37-7.06(m,59H),5.77(t,J=10.8Hz,1H),5.35-5.37(m,2H),5.26(s,1H),5.18(dd,J=3.8&7.7Hz,1H),5.08(d,J=3.2Hz,1H),5.07(dd,J=3.2&8.4Hz,1H),4.97(d,J=8.4Hz,1H),4.91(d,J=9.1Hz,1H),4.86(d,J=8.7Hz,3H),4.73-4.13(m,32H),4.02-3.30(m,29H),3.20(t,J=10.8Hz,2H),3.10(d,J=8.7Hz,1H),2.98(t,J=9.2Hz,1H),2.87-2.85(m,1H),2.80(dd,J=3.2&7.8Hz,1H),2.10(s,6H),2.08(t,J=9.3Hz,1H),2.00(s,6H),1.40-1.17(m,4H),0.87-0.82(m,2H);13C NMR(150MHz,CDCl3):δ171.9,170.8,170.2,170.0,167.9,166.1,165.5,159.5,154.5,154.3,154.1,138.2,130.1,129.8,129.6,129.4,128.9,128.8,128.7,128.6,128.5,128.4,128.3,128.2,128.1,127.9,127.8,127.6,127.0,100.8,100.5,98.0,95.8,76.8,76.7,75.5,74.8,74.6,74.4,74.3,74.1,73.8,73.7,73.4,73.2,69.9,69.7,69.5,68.7,67.5,66.7,61.9,58.9,57.6,53.4,37.4,29.9,29.2,28.8,23.4,21.3,21.2,21.0,20.9,14.3;ESI-MS:m/z C160H172Cl9N7O48计算值3278.8343;实验值32788127。
化合物S14b:向S14a(0.150g,0.045mmol)于乙腈(20mL)中的溶液中添加对甲苯磺酸单水合物(0.004g,0.022mmol)且将所得反应混合物在室温下搅拌5h。通过添加Et3N淬灭反应且在真空中浓缩。通过急骤管柱层析(0%→15%EA/甲苯)纯化残余物,得到二醇S14b(0.085g,59%)。TLC:(丙酮:甲苯=2/8,v/v):Rf=0.32;NMR(600MHz,CDCl3):δ7.89-7.86(m,4H),7.49-7.37(m,2H),7.30-7.09(m,54H),5.78(t,J=10.2Hz,1H),5.51(d,J=7.8Hz,1H),5.37(dt,J=2.4&7.2Hz,1H),5.26(s,1H),5.19(dd,J=2.4&10.8Hz,1H),5.12(d,J=3.6Hz,1H),4.95-4.85(m,5H),4.75-4.12(m,25H),4.08-4.03(m,3H),3.96-3.89(m,7H),3.74-3.67(m,8H),3.63-3.30(m,20H),3.20(t,J=10.2Hz,2H),3.10(d,J=8.9Hz,2H),2,89(s,1H),2.76(dd,J=3.6&7.9Hz,1H),2.08(s,3H),2.05(s,3H),1.99(s,3H),1.95(s,3H),1.41-1.31(m,5H),1.27-1.17(m,2H);13C NMR(150MHz,CDCl3):δ172.4,172.3,171.3,171.2,170.7,170.3,170.0,168.4,168.3,166.1,165.5,154.5,154.3,154.1,153.9,138.5,138.4,138.1,138.0,133.7,133.3,130.1,129.8,129.7,129.5,129.3,128.9,128.8,128.7,128.6,128.6,128.5,128.4,128.3,128.2,128.1,128.0,127.9,101.1,100.8,100.6,100.4,99.7,99.2,96.0,95.8,76.3,76.1,75.7,75.5,74.9,74.8,74.5,74.3,74.0,73.7,73.4,73.3,73.2,73.1,73.0,72.6,72.3,72.1,71.3,71.0,69.9,69.6,69.4,69.3,68.4,67.8,66.1,66.2,62.8,62.2,59.2,57.7,53.4,51.5,36.9,29.9,29.2,28.8,25.0,23.4,21.4,21.3,21.2,21.1,15.5,14.4;ESI-MS(负模式):m/zC153H168Cl9N7O48计算值3191.8105;实验值3236.8033(M+2Na)-。
化合物S14c:将三氟甲磺酸银(0.067g,0.27mmol)、二氯化双(环戊二烯基)铪(0.071g,0.18mmol)及经活化的分子筛于无水甲苯(10mL)中的混合物在室温下搅拌1h。随后将反应混合物冷却至-40℃,添加供体8(0.097g,0.068mmol)及接受体S14b(0.175g,0.054mmol)于5mL甲苯中的溶液。将混合物在-20℃下搅拌2h,用Et3N淬灭,用EtOAc稀释且经由硅藻土过滤。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→10%丙酮/甲苯)纯化残余物以分离α及β异构体的混合物,得到呈白色泡沫状的S14c(0.149g,58%)。TLC:(丙酮:甲苯=1.5/8.5,v/v):Rf=0.52;1H NMR(600MHz,CDCl3):δ7.90(d,J=7.2Hz,2H),7.83(d,J=7.8Hz,2H),7.51-7.41(m,4H),7.34-7.06(m,92H),5.83(t,J=10.2Hz,1H),5.48-5.03(m,3H),5.00-4.75(m,13H),4.60-4.08(m,41H),3.99-3.07(m,53H),2.96(t,J=10.2Hz,2H),2.80(dd,J=3.8&7.9Hz,1H),2.15(s,3H),2.10(s,3H),2.07(s,3H),2.00(s,3H),1.93(s,3H),1.40-1.17(m,5H),0.91-0.81(m,2H);13C NMR(150MHz,CDCl3):δ170.9,170.6,170.4,169.9,169.4,166.5,159.5,154.7,154.4,139.8,138.8,138.7,138.6,138.4,138.2,133.7,130.9,130.7,129.7,129.6,129.4,129.2,129.0,128.7,128.6,128.6,128.4,128.3,128.2,127.9,127.6,127.3,100.4,100.2,99.8,99.4,74.9,74.7,74.5,73.3,73.2,66.1,60.6,51.5,32.1,29.8,29.7,29.6,29.3,28.7,23.7,22.8,22.4,22.0,21.6,21.4,21.2,20.9,15.4,14.3,14.2,14.0,11.3,10.2;ESI-MS:m/z C232H250Cl12N8O65计算值4607.2765;实验值2330.6261(M+Na)2+。
5-胺基戊基-[5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖基]-(1→3),-[β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖基]-(1→6)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷G18:通过以下一般程序2(方法2)脱除化合物S14c(0.135g,0.029mmol)的保护基,得到所要聚醣G18(0.015g,25%)。1H NMR(600MHz,D2O):δ5.11(s,1H,H-1d),4.89(s,1H,H-1d'),4.73(s,1H,H-1c),4.57(dt,J=10.2Hz,2H,H-1f,f'),4.47(d,J=7.8Hz,2H,H-1a,b),4.47(d,J=8.4Hz,2H,H-1e,e'),4.23(s,1H),4.17(s,1H),4.09(d,J=1.3Hz,1H),4.03-3.48(m,61H),2.98(t,J=10.2Hz,2H,-CH2-连接基团),2.65(dd,J=4.8&12.6Hz,1H,H-3赤道g),2.10(s,3H),2.09(s,3H),2.07(s,3H),2.02(s,6H),1.72-1.64(m,3H,H-3轴向g,连接基团-CH2-),1.63-1.54(m,2H),1.39-1.35(m,2H);13C NMR(150MHz,D2O):δ177.7,177.5,177.4,177.1,176.2,176.1,106.2,106.1,104.1,103.7,103.5,103.1,102.9,102.9,99.5,83.6,83.1,82.2,82.1,81.7,79.4,79.1,78.9,77.3,77.2,76.8,76.4,75.3,75.1,74.8,74.5,73.5,73.4,72.8,71.2,70.9,70.0,67.6,66.0,65.6,65.4,64.5,62.8,57.7,57.3,55.0,54.6,54.0,42.8,42.3,42.0,41.78,30.7,30.5;ESI-MS(负模式):m/z C78H132N6O54计算值2016.7767;实验值1007.3799(M-H)2-。
合成聚醣G24.
化合物S14b充当G18及G24两者的共同接受体。S14b在6-O位点处的醣基化以10%接受体回收率得到所要S15a。最后,整体脱除保护基产生所要聚醣G24。
流程S15|制备G24.i,12,AgOTf、Cp2HfCl2、甲苯,-40℃,63%;ii,(1)LiOH、1,4-二恶烷,90℃,(2)Ac2O、吡啶,(3)NaOMe、MeOH,(4)Pd(OH)2、MeOH:H2O、H2,42%。
化合物S15a:将三氟甲磺酸银(0.058g,0.23mmol)、二氯化双(环戊二烯基)铪(0.061g,0.23mmol)及经活化的分子筛于无水甲苯(10mL)中的混合物在室温下搅拌1h。随后将反应混合物冷却至-40℃,添加供体12(0.135g,0.056mmol)及接受体S14b(0.150g,0.046mmol)于5mL甲苯中的溶液。将混合物在-20℃下搅拌2h,用Et3N淬灭,用EtOAc稀释且经由硅藻土过滤。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→10%丙酮/甲苯)纯化残余物,得到呈白色泡沫状的S15a(0.165g,63%)。TLC:(丙酮:甲苯=1.5/8.5,v/v):Rf=0.58;1H NMR(600MHz,CDCl3):δ8.00-7.80(m,4H),7.50-6.80(m,116H),5.80(t,J=10.2Hz,1H),5.40-5.30(m,7H),5.10-4.08(m,65H),4.00-3.05(m,65H),2.98(dd,J=3.2&8.5Hz,1H),2.15(s,6H),2.10(s,6H),2.00(s,6H),1.37-1.17(m,5H),0.87-0.84(m,2H);13C NMR(150MHz,CDCl3):δ170.8,167.9 154.1,139.1,139.0,138.9,138.8,138.6,138.3,138.1,138.0,129.8,129.6,129.3,128.9,128.8,128.7,128.7,128.6,128.5,128.4,128.2,128.0,127.7,127.4,127.3,127.2,127.1,127.0,100.8,100.0,74.9,74.8,74.5,74.4,73.6,73.3,72.6,53.5,29.9,29.2,23.4,21.3,21.2,21.0,20.9,15.5,14.4;ESI-MS(负模式):m/z C277H298Cl15N9O77计算值5517.1670;实验值2803.7442(M+2Na)2-。
5-胺基戊基-[5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖基]-(1→3),-[β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2),(1→6)-α-D-甘露哌喃糖基]-(1→6)-β-D-甘露哌喃糖基-(1→4)2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷G24:通过以下一般程序2(方法2)脱除化合物S15a(0.090g,0.016mmol)的保护基,得到呈白色固体状的所要聚醣G24(0.016g,42%)。1H NMR(600MHz,D2O):δ5.14(s,1H,H-1d),4.88(s,1H,H-1d'),4.60(t,J=10.2Hz,3H,H-1e,f,e'),4.55(d,J=8.4Hz,1H),4.48(dd,J=3.2&8.6Hz,4H),4.45(d,J=7.8Hz,1H),4.26(s,1H),4.20(s,1H),4.09(s,1H),4.01-3.50(m,71H),2.97(t,J=10.2Hz,2H,连接基团-CH2-),2.68(dd,J=3.8&8.3Hz,1H,H-3赤道g),2.08(s,3H,-C(O)CH3),2.07(s,3H,-C(O)CH3),2.04(s,3H,-C(O)CH3),2.03(s,6H,-C(O)CH3),1.72(t,J=10.2Hz,1H,H-3轴向g),1.67-1.65(m,2H),1.60-1.56(m,2H,连接基团-CH2-),1.40-1.39(m,2H,连接基团-CH2-);13C NMR(150MHz,D2O):δ174.9,174.7,174.6,174.5,174.4,174.1,173.5,103.6,103.5,102.9,102.8,101.5,101.4,101.0,100.3,100.1,99.5,99.3,80.7,80.4,79.5,79.3,79.2,78.5,76.5,75.4,75.2,74.7,74.5,74.4,74.3,73.7,73.4,72.5,72.4,72.3,72.1,71.9,71.6,71.3,70.9,70.8,70.5,70.3,69.5,69.4,68.5,68.3,68.2,67.5,67.3,65.4,63.3,62.5,61.7,61.0,60.2,60.1,59.8,55.08,54.9,54.8,54.6,51.0,40.9,39.4,28.0,26.7,22.5,22.4,22.3,22.2,22.1,22.0,21.09;ESI-MS(负模式):m/z C92H155N7O64计算值2383.2370;实验值1213.4483(M+Na)2-。
合成聚醣G25.
流程S16|制备G25.i,12,AgOTf、Cp2HfCl2、甲苯,-40℃,64%;ii,pTSA、乙腈,56%;iii,8,AgOTf、Cp2HfCl2、甲苯,-78℃,49%;iv,(1)LiOH、1,4-二恶烷,90℃,(2)Ac2O、吡啶,(3)NaOMe、MeOH,(4)Pd(OH)2、MeOH:H2O、H2,38%。
化合物16a:将三氟甲磺酸银(0.087g,0.34mmol)、二氯化双(环戊二烯基)铪(0.090g,0.23mmol)及经活化的分子筛于无水甲苯(10mL)中的混合物在室温下搅拌1h。随后将反应混合物冷却至-40℃,添加供体12(0.259g,0.082mmol)及接受体15(0.100g,0.068mmol)于5mL甲苯中的溶液。将混合物在-10℃下搅拌3h,用Et3N淬灭,用EtOAc稀释且经由硅藻土过滤。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→20%丙酮/甲苯)纯化残余物,得到呈白色泡沫状的S16a(0.201g,64%)。TLC:(丙酮:甲苯=2.5/7.5,v/v):Rf=0.50;1H NMR(600MHz,CDCl3):δ7.92-7.83(m,9H),7.69-7.67(m,2H),7.52-7.44(m,6H),7.37-6.96(m,63H),5.34-5.32(m,2H),5.26-5.24(m,4H),5.20-5.10(m,3H),5.08(d,J=9.8Hz,3H),4.59-4.40(m,8H),4.75-4.50(m,20H),4.49-4.00(m,20H),3.98-3.60(m,16H),3.48-3.06(m,16H),2.98(t,J=10.7Hz,2H),2.76(dd,J=3.8&7.8Hz,2H),2.08(s,3H),2.07(s,3H),2.06(s,6H),1.99(s,9H),1.37-1.17(m,6H),0.89-0.84(m,2H);13C NMR(150MHz,CDCl3):δ171.8,171.7,171.6,169.8,169.4,168.0,167.6,166.6,166.4,166.2,165.4,165.3,159.7,159.4,154.3,154.2,139.8,139.7,139.6,139.4,139.2,138.7,138.6,138.5,138.4,138.3,138.2,133.9,132.7,131.9,131.2,130.6,129.8,129.6,129.4,129.3,129.2,129.0,128.8,128.7,128.6,128.6,128.4,128.2,128.1,128.0,127.9,127.8,127.7,127.6,127.5,127.4,127.3,127.2,127.0,102.7,102.4,101.9,101.8,100.8,100.7,100.4,100.2,100.0,99.2,99.4,98.4,96.5,95.9,74.3,74.1,74.0,73.8,73.7,73.5,73.4,73.2,73.1,72.7,72.5,72.1,71.3,70.9,70.4,69.5,69.4,68.5,68.4,68.3,68.1,67.9,67.4,53.4,51.6,37.5,33.9,32.2,31.8,29.9,28.8,26.5,23.4,22.9,21.2,21.0,20.0,14.6,14.3;ESI-MS:m/z C220H235Cl12N9O72计算值4582.6910;实验值915.6684(M+H)5+。
化合物16b:向S14a(0.200g,0.043mmol)于乙腈(10mL)中的溶液中添加对甲苯磺酸单水合物(0.002g,0.008mmol)且将所得反应混合物在室温下搅拌隔夜。通过添加Et3N淬灭反应且在真空中浓缩。通过急骤管柱层析(0%→20%丙酮/甲苯)纯化残余物,得到二醇S16b(0.110g,56%)。TLC:(丙酮:甲苯=2.5/7.5,v/v):Rf=0.32;1H NMR(600MHz,CDCl3):δ7.90-7.80(m,7H),7.79(d,J=8.4Hz,2H),7.69-7.46(m,5H),7.41-7.04(m,65H),5.78(t,J=10.1Hz,1H),5.73(t,J=10.7Hz,1H),5.35-5.25(m,7H),5.20(dd,J=3.2&7.9Hz,1H),5.15(dd,J=3.8&8.4Hz,1H),5.08(t,J=10.8Hz,2H),4.90-4.80(m,5H),4.79-4.50(m,15H),4.48-4.30(m,8H),4.20-4.10(m,15H),3.92-3.20(m,35H),3.2(t,J=8.8Hz,2H),3.18-3.05(m,3H),3.0-2.95(m,3H),2.81-2.70(m,3H),2.08(s,6H),2.07(s,6H),2.03(s,6H),1.92(s,6H),1.32-1.17(m,6H),0.89-0.83(m,2H);13C NMR(150MHz,CDCl3):δ171.8,170.7,168.0,166.1,159.6,138.1,133.7,132.7,131.1,130.1,129.9,129.7,129.4,128.9,128.8,128.7,128.6,128.4,128.3,128.2,127.8,127.7,127.6,74.9,74.7,74.6,73.9,68.8,66.1,53.4,51.6,37.3,32.4,31.4,29.9,29.8,29.7,28.7,28.4,23.4,22.3,20.9,20.7,20.4,15.5,14.6,14.2;ESI-MS:m/z C213H231Cl12N9O72计算值4494.5820;实验值681.2953(M+K)7+。
化合物16c:将三氟甲磺酸银(0.068g,0.34mmol)、二氯化双(环戊二烯基)铪(0.065g,0.17mmol)及经活化的分子筛于无水甲苯(10mL)中的混合物在室温下搅拌1h。随后将反应混合物冷却至-78℃,添加供体8(0.109g,0.076mmol)及接受体16b(0.230g,0.051mmol)于5mL甲苯中的溶液。将混合物在-20℃下搅拌3h,用Et3N淬灭,用EtOAc稀释且经由硅藻土过滤。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→20%丙酮/甲苯)纯化残余物,得到呈白色泡沫状的S16c(0.148g,49%)。TLC:(丙酮:甲苯=2.5/7.5,v/v):Rf=0.50;1H NMR(600MHz,CDCl3):δ7.90(m,10H),7.52-6.80(m,110H),5.79(t,J=10.2Hz,2H),5.48(dd,J=4.2&8.7Hz,2H),5.34-5.14(m,10H),4.98-3.10(m,129H),2.85(t,J=10.2Hz,2H),2.30(dd,J=3.4&8.2Hz,2H),2.01(s,3H),2.00(s,6H),1.95(s,6H),1.94(s,6H),1.91(s,3H),1.39-1.29(m,6H),1.26-1.91(m,2H);13C NMR(150MHz,CDCl3):δ172.1,170.8,170.3,170.1,170.0,169.3,169.2,168.0,167.3,166.5,165.8,165.1,165.0,154.0,153.8,153.7,153.7,139.0,138.9,138.8,138.7,138.6,138.3,138.2,138.1,137.9,137.4,133.4,133.2,132.0,130.3,129.8,129.7,129.4,129.3,129.2,129.1,128.9,128.8,128.5,128.3,128.2,128.1,128.0,127.9,100.4,100.2,99.3,75.8,75.6,75.4,75.3,75.2,75.1,74.8,74.7,74.6,74.5,74.4,74.3,74.2,74.2,74.1,73.5,73.4,73.3,73.2,73.1,73.0,72.9,72.6,72.2,71.9,71.7,71.6,71.3,71.1,70.1,69.9,69.8,68.5,68.3,68.2,63.4,58.8,58.3,58.0,53.4,51.3,31.9,30.3,30.0,29.8,29.7,29.5,28.7,28.5,28.3,27.1,24.5,24.1,23.8,22.7,22.1,21.7,20.9,20.7,20.6,14.1,14.0,10.9;ESI-MS(负模式):m/z C292H313Cl15N10O89计算值5918.4470;实验值1486.4746(M+6H)4+。
5-胺基戊基-二-[5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2),(1→4)-α-D-甘露哌喃糖基]-(1→3),-[β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖基]-(1→6)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷G25:通过以下一般程序2(方法2)脱除化合物S16c(0.115g,0.019mmol)的保护基,得到所要聚醣G25(0.020g,38%)。1H NMR(600MHz,D2O):δ5.15(d,1H,H-1d),4.94(s,1H,H-1d'),4.63(d,J=8.4Hz,2H,H-1a,b),4.58(d,J=7.8Hz,2H,H-1e,e'),4.52(d,J=7.8Hz,2H,H-1e",f'),4.50(s,1H,H-1c),4.42(dd,J=3.2&10.2Hz,2H,H-1f,f"),4.23(s,2H),4.08(s,1H),3.92(dd,J=3.2&7.8Hz,2H),3.90-3.45(m,75H),3.01(t,J=10.2Hz,2H,连接基团CH2),2.69(dd,J=3.6&12.1Hz,2H,H-3赤道g,g'),2.12(s,3H),2.10(s,3H),2.09(s,3H),2.07(s,3H),2.05(s,9H),1.77-1.66(m,4H,H-3轴向.g,g'及连接基团CH2),1.62-1.60(m,2H),1.42-1.40(m,2H);13C NMR(150MHz,D2O):δ177.6,177.4,177.3,177.1,176.1,106.3,105.6,104.2,104.1,103.7,103.1,102.8,102.1,101.8,99.8,78.7,78.0,77.4,77.2,77.176.4,75.5,75.2,75.1,75.0,74.8,74.4,74.0,73.7,73.4,73.0,72.8,72.1,71.5,70.9,70.8,70.0,68.4,66.0,65.4,64.3,64.0,63.7,63.1,63.8,62.6,57.7,57.6,57.5,57.3,54.6,42.7,42.0,30.7,29.0,25.3,25.0,24.9,24.8,24.7;ESI-MS(负模式):m/zC103H172N8O72计算值2674.4930;实验值1335.9948(M-2H)2-。
合成聚醣G29.
聚醣G29为单唾液酸化的四触角复合型结构。合成始于通过供体11在15的3-O位置处的醣基化构筑D1臂,得到化合物S17a。通过TLC检查反应进程,如流程S17中所示。移除亚苄基,随后6-O醣基化,得到所要十四醣S17c。最后,整体脱除保护基,得到所要聚醣G29。
流程S17|制备G29.i,11,AgOTf、Cp2HfCl2、甲苯,-40℃,77%;ii,pTSA、乙腈,73%;iii,13,AgOTf、Cp2HfCl2、甲苯,-40℃,57%;iv,(1)LiOH、1,4-二恶烷,90℃,(2)Ac2O、吡啶,(3)NaOMe、MeOH,(4)Pd(OH)2、MeOH:H2O、H2,34%。
化合物S17a:将三氟甲磺酸银(0.173g,0.675mmol)、二氯化双(环戊二烯基)铪(0.177g,0.469mmol)及经活化的分子筛于无水甲苯(10mL)中的混合物在室温下搅拌1h。随后将反应混合物冷却至-50℃,添加供体11(0.345g,0.470mmol)及接受体15(0.195g,0.134mmol)于5mL甲苯中的溶液。将混合物在-10℃下搅拌2h,用Et3N淬灭,用EtOAc稀释且经由硅藻土过滤。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→10%丙酮/甲苯)纯化残余物,得到呈白色泡沫状的S17a(0.380g,77%)。TLC:(丙酮:甲苯=1/9,v/v):Rf=0.60;NMR(600MHz,CDCl3):δ7.30-7.12(m,60H),7.08-6.72(m,25H),5.39(t,J=10.2Hz,1H),5.29(t,J=10.3Hz,1H'),5.16(d,J=4.2Hz,1H),5.09(d,J=8.4Hz,1H),4.91-4.72(m,18H,重迭的),4.63-4.56(m,5H),4.01-4.80(m,30H),3.80-3.95(m,10H),3.60-3.75(m,4H),3.52-3.48(m,6H),3.41-3.15(m,20H),2.98(d,J=9.6Hz,1H),2.90-2.76(m,3H),2.66-2.62(m,1H),1.95(s,3H),1.93(s,3H),1.87(s,3H),1.38-1.23(m,4H,-CCH2C-),1.08-1.01(m,2H,-CCH2C-);13C NMR(150MHz,CDCl3):δ172.1,171.9,170.5,156.7,156.6,142.0,141.8,141.6,141.5,141.4,141.3,141.3,141.1,140.8,140.6,140.5,139.8,135.2,133.6,133.2,131.8,131.5,131.3,131.1,130.6,130.5,130.2,130.1,129.8,129.0,127.3,104.8,104.8,103.2,102.9,77.7,77.5,77.3,77.2,77.1,76.9,76.2,76.1,76.0,75.9,75.5,75.4,74.8,74.6,74.5,74.4,74.3,71.0,70.8,70.6,54.0,31.7,31.3,25.9,23.9,23.8,23.7,23.6,17.2,17.1,16.8,16.7,13.7,13.6;HRMS(负模式):m/zC190H205Cl12N7O48计算值3778.9981;实验值3824.9969(M+2Na)-。
化合物S17b:向S17a(0.377g,0.102mmol)于乙腈(10mL)中的溶液中添加对甲苯磺酸单水合物(0.004g,0.02mmol)且将所得反应混合物在室温下搅拌隔夜。通过添加Et3N淬灭反应且在真空中浓缩。通过急骤管柱层析(0%→10%丙酮/甲苯)纯化残余物,得到二醇S17b(0.270g,73%)。TLC:(丙酮:甲苯=1.5/8.5,v/v):Rf=0.32;1H NMR(600MHz,CDCl3):δ7.34-7.11(m,80H),5.37(s,1H),5.29(t,J=10.2Hz,1H),5.22(t,J=9.8Hz,1H),5.02(d,J=3.6Hz,2H),4.92-4.74(m,7H),4.70-4.21(m,34H),3.93-3.51(m,17H),3.65-3.20(m,30H),3.20(t,J=10.2Hz,4H),2.98(d,J=3.8Hz,2H),2.89(s,1H),2.11(s,3H),1.94(s,3H),1.90(s,3H),1.40-1.17(m,4H),091-0.84(m,2H);13C NMR(150MHz,CDCl3):δ170.1,170.0,169.6,168.0,154.3,154.1,139.6,139.1,139.0,138.9,138.7,138.5,138.3,138.1,138.0,132.7,131.1,129.6,129.4,129.3,129.0,128.7,128.6,128.5,128.4,128.3,128.2,128.1,128.0,127.9,127.6,127.5,127.3,127.1,127.0,100.9,100.6,100.5,98.6,95.9,95.8,76.75,76.3,75.6,74.8,74.6,74.5,74.3,74.2,73.8,73.7,73.5,73.2,72.8,72.5,72.1,69.6,68.8,68.5,68.4,68.2,68.1,62.8,62.1,57.6,39.0,37.3,33.7,32.5,31.4,30.6,29.9,29.2,28.8,26.6,24.0,23.4,23.2,22.9,21.5,21.3,20.1,15.5,14.6,14.4,14.3,11.2;HRMS(负模式):m/z C183H201Cl12N7O48计算值3692.0222;实验值3737.9645(M+2Na)2-。
化合物S17c:将三氟甲磺酸银(0.046g,0.180mmol)、二氯化双(环戊二烯基)铪(0.048g,0.126mmol)及经活化的分子筛于无水甲苯(5mL)中的混合物在室温下搅拌1h。随后将反应混合物冷却至-50℃,添加供体13(0.100g,0.36mmol)及接受体S17b(0.118g,0.032mmol)于5mL甲苯中的溶液。将混合物在-10℃下搅拌3h,用Et3N淬灭,用EtOAc稀释且经由硅藻土过滤。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→10%丙酮/甲苯)纯化残余物,得到呈白色泡沫状的S17c(0.120g,57%)。TLC:(丙酮:甲苯=1/9,v/v):Rf=0.60;1HNMR(600MHz,CDCl3):δ7.58-7.54(m,6H),7.50-6.98(m,134H),5.57(t,J=10.3Hz,4H),5.42-5.30(m,24H),5.01-4.70(m,35H),4.65-4.10(m,50H),4.0-2.90(m,52H),2.80(d,J=9.8Hz,2H),2.15(s,3H),2.09(s,3H),2.07(s,3H),1.96(s,3H),1.93(s,3H),1.88(s,3H),1.86(s,3H),1.34-1.17(m,5H),0.89-0.81(m,2H);13C NMR(150MHz,CDCl3):δ139.1,139.0,138.3,138.2,138.1,130.1,129.9,129.1,129.0,128.9,128.8,128.7,128.6,128.4,128.3,128.1,128.0,127.9,127.8,127.6,127.4,74.8,74.6,73.7,73.6,71.9;HRMS(负模式):m/z C322H346Cl18N10O89计算值6418.3910;实验值3253.3555(M+2Na)2-。
5-胺基戊基-二-[β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2),(1→4)-α-D-甘露哌喃糖基]-(1→3),-[5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→6)-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖基]-(1→6)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷G29:通过以下一般程序2(方法2)脱除化合物S17c(0.095g,0.015mmol)的保护基,得到呈白色粉末状的所要聚醣G29(0.014g,34%)。1H NMR(600MHz,D2O):δ4.99(s,1H,H-1d),4.75(s,1H,H-1d'),4.72(d,J=8.4Hz,1H,H-1e),4.62(d,1H,J=7.8Hz,1H,H-1e'),4.63(s,1H,H-1c),4.45(dd,J=3.6&7.8Hz,4H,H-1e″,e″',f,f'),4.36-4.31(m,4H),4.09(s,3H),3.98-3.04(m,85H),2.83(t,J=10.2Hz,2H,连接基团),2.56(dd,J=3.2&8.4Hz,1H,H-3赤道g'),1.95(s,3H,-C(O)CH3),1.94(s,3H,-C(O)CH3),1.93(s,3H,-C(O)CH3),1.92(s,3H,-C(O)CH3),1.92(s,3H,-C(O)CH3),1.90(s,3H,-C(O)CH3),1.89(s,3H,-C(O)CH3),1.61-1.42(m,5H,H-3轴向g'及连接基团CH2),1.30-1.24(m,2H,连接基团);13C NMR(150MHz,D2O):δ186.6,174.7,174.3,174.2,103.3,102.7,101.8,100.8,99.9,98.5,97.3,78.9,78.1,77.8,75.1,74.4,74.2,74.1,72.2,71.7,71.5,70.7,69.8,68.3,68.1,63.1,62.4,60.8,60.2,59.7,54.9,54.6,51.6,39.9,39.1,27.8,26.1,22.2,21.8,17.6;ESI-MS(负模式):m/z C106H178N8O74计算值2747.0411;实验值1396.0177(M+Na)2-。
合成聚醣G30.
如流程S18中所描绘,使用来自流程S16的前驱体S16b作为接受体,在三氟甲磺酸银及二氯二茂铪(hafnocene dichloride)的促进下用供体11进行6-O醣基化,获得所要S18a。最终脱除保护基,得到二唾液酸化四触角复合聚醣G30。
流程S18|制备G30.i,11,AgOTf、Cp2HfCl2、甲苯,-40℃,37%;ii,(1)LiOH、1,4-二恶烷,90℃,(2)Ac2O、吡啶,(3)NaOMe、MeOH,(4)Pd(OH)2、MeOH:H2O、H2,45%。
化合物S18a:将三氟甲磺酸银(0.051g,0.200mmol)、二氯化双(环戊二烯基)铪(0.053g,0.140mmol)及经活化的分子筛于无水甲苯(5mL)中的混合物在室温下搅拌1h。随后将反应混合物冷却至-50℃,添加供体11(0.144g,0.060mmol)及接受体S16b(0.180g,0.040mmol)于5mL甲苯中的溶液。将混合物在-10℃下搅拌3h,用Et3N淬灭,用EtOAc稀释且经由硅藻土过滤。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→10%丙酮/甲苯)纯化残余物,得到呈白色泡沫状的S18a(0.103g,37%)。TLC:(丙酮:甲苯=2/8,v/v):Rf=0.53;1HNMR(600MHz,CDCl3):δ7.69-7.52(m,20H),7.33-7.06(m,130H),5.60(m,17H),4.80-4.30(m,40H),4.20-4.00(m,50H),3.99-3.02(m,25H),2.08(s,9H),2.07(s,6H),2.05(s,6H),2.02(s,6H),1.78-1.74(m,6H),0.91-0.84(m,2H);13C NMR(150MHz,CDCl3):δ170.9,170.4,170.2,169.8,169.4,169.3,132.7,131.1,129.0,128.7,128.4,128.3,128.2,128.0,127.8,127.7,127.6,127.5,68.8,68.4,39.0,37.3,33.7,31.4,30.6,29.9,29.2,26.6,26.0,24.0,23.2,22.9,20.1,14.6,14.2,11.40;ESI-MS(负模式):m/zC337H361Cl18N11O101计算值6806.6710;实验值723.5969(M+K)10-。
5-胺基戊基-二-[5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2),(1→4)-α-D-甘露哌喃糖基]-(1→3),-二-[-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→6),(1→2)-α-D-甘露哌喃糖基]-(1→6)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷G30:通过以下一般程序2(方法2)脱除化合物S18a(0.075g,0.010mmol)的保护基,得到所要聚醣G30(0.015mg,45%)。1H NMR(600MHz,D2O):δ5.12(s,1H,H-1d),4.86-4.74(m,3H,H-1d',c,b,与D2O重迭),4.59-4.55(m,5H,H-1e,e',e",e'",a),1.48-4.42(m,4H,H-1f,f',f",f'"),4.21-4.06(m,5H),3.98-3.3.20(m,91H),2.98(t,J=10.7Hz,2H,连接基团CH2),2.66(dd,J=3.7&10.1Hz,2H,2x H-3赤道g,g'),2.12(s,3H),2.09(s,3H,-C(O)CH3),2.06(s,3H,-C(O)CH3),2.05(s,3H,-C(O)CH3),2.03(s,12H,-C(O)CH3),1.75-1.54(m,6H,2x H-3轴向g,g'及连接基团CH2),1.42-1.36(m,2H,连接基团);13C NMR(150MHz,D2O):δ174.7,174.2,174.1,103.4,102.7,101.3,101.2,101.0,100.8,100.6,99.9,98.9,97.9,78.1,75.1,74.3,73.4,72.3,72.1,71.7,70.7,70.4,69.8,68.3,68.0,59.9,59.7,54.7,51.6,39.9,27.8,22.7,22.0,21.9,21.8;ESI-MS(负模式):m/zC117H195N9O82计算值3039.8280;实验值1518.5585(M-H)2-。
合成聚醣G31.
使用源自流程S16的中间体S16b,使用供体13在AgOTf/Cp2HfCl2存在下进行α-选择性醣基化,以74%产率得到S19a。然而,新形成的产物在TLC上与接受体重迭(流程S19)。随后进一步脱除S19a的保护基,得到聚醣G31。
流程S19|制备G31.i,13,AgOTf、Cp2HfCl2、甲苯,-50℃,74%;ii,(1)LiOH、1,4-二恶烷,90℃,(2)Ac2O、吡啶,(3)NaOMe、MeOH,(4)Pd(OH)2、MeOH:H2O,H2,47%。
化合物S19a:将三氟甲磺酸银(0.042g,0.165mmol)、二氯化双(环戊二烯基)铪(0.043g,0.115mmol)及经活化的分子筛于无水甲苯(5mL)中的混合物在室温下搅拌1h。随后将反应混合物冷却至-50℃,添加供体13(0.100g,0.036mmol)及接受体S16b(0.150g,0.033mmol)于5mL甲苯中的溶液。将反应混合物在-20℃下搅拌2h,用Et3N淬灭,用EtOAc稀释且经由硅藻土过滤。用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→15%丙酮/甲苯)纯化残余物,得到呈白色泡沫状的S19a(0.183g,74%)。TLC:(丙酮:甲苯=2/8,v/v):Rf=0.50;1HNMR(600MHz,CDCl3):δ7.90-7.83(m,9H),7.69-7.67(m,3H),7.57-7.50(m,3H),7.38-7.04(m,125H),5.77(t,J=10.2Hz,4H),5.54-5.40(m,20H),5.39(t,J=8.4Hz,4H),5.08(t,J=7.8Hz,4H),4.98-4.60(m,25H),4.53-4.42(m,31H),4.30-4.10(m,35H),4.00-3.80(m,42H),3.21-3.18(m,64H),2.93-2.78(m,4H),2.08(s,6H),2.07(s,6H),2.05(s,3H),2.02(s,3H),1.98(s,6H),1.94(s,6H),1.74-1.18(m,7H),0.91-0.84(m,2H);13C NMR(150MHz,CDCl3):δ171.8,171.7,170.8,169.9,169.8,168.5,168.0,166.1,165.4,159.5,132.7,131.7,130.1,129.9,129.2,128.9,128.8,128.7,128.6,128.4,128.3,128.2,128.1,128.0,127.9,127.8,127.7,127.6,127.5,100.7,100.6,100.4,95.8,74.9,74.6,74.2,73.9,73.7,73.6,73.2,72.9,72.2,71.9,71.0,70.5,69.3,68.5,68.3,68.0,67.3,66.1,65.3,62.3,61.2,53.4,51.5,39.0,37.3,33.7,32.4,32.2,30.6,29.9,29.6,29.4,29.3,28.8,26.6,24.0,23.9,23.5,23.4,23.1,21.2,20.1,15.5,14.6,14.4,14.3,11.2;HRMS(负模式):m/z C352H376Cl18N12O113计算值7220.9510;实验值3654.9220(M+2Na)2-。
5-胺基戊基-二-[5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2),(1→4)-α-D-甘露哌喃糖基]-(1→3),-[5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→6)-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖基]-(1→6)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷G31:通过以下一般程序2(方法2)脱除化合物S19a(0.180g,0.024mmol)的保护基,得到所要聚醣G31(0.039g,47%)。1H NMR(600MHz,D2O):δ5.12(s,1H,H-1d),4.87(s,2H,H-1d',c),4.75(d,J=8.4Hz,2H,H-1b,e),5.59-4.55(m,5H,重迭的H-1a,e',e'",e"",f),4.49-4.42(m,5H,重迭的H-1f'-f""),4.21-4.19(m,4H),4.06-3.54(m,95H),2.97(t,J=10.2Hz,2H,连接基团-CH2),2.66(dd,J=3.2&7.8Hz,3H,H-3g,g',g'"赤道),2.09(s,3H),2.06(s,9H),2.04(s,3H),2.02(s,12H),1.74(m,2H,连接基团),1.64(t,J=10.2Hz,3H,H-3g,g',g'"轴向),1.59-1.57(m,2H,连接基团),1.39-1.37(m,2H,连接基团);13C NMR(150MHz,D2O):δ174.8,174.7,174.5,174.4,174.3,174.1,173.3,103.5,102.9,101.7,101.4,101.2,100.2,100.0,99.8,99.6,99.3,80.8,80.6,80.3,79.3,78.4,78.3,76.7,75.3,74.3,74.2,74.1,74.0,73.8,72.9,72.8,72.6,72.4,72.3,72.2,72.1,72.0,71.9,71.7,71.3,70.8,70.4,70.3,70.2,68.4,68.4,63.4,62.9,60.4,60.3,60.2,59.8,54.8,54.3,54.2,51.8,51.0,43.7,43.3,39.8,39.5,34.4,27.5,26.3,22.6,22.4,22.4,22.2,22.0,21.9;ESI-MS(负模式):m/zC128H212N10O90计算值3331.0840;实验值1664.1108(M-H)2-。
合成核心海藻醣基化聚醣G3及G19 S
经由供体S20i使二醇接受体S20j海藻醣基化,自还原端双醣S20k组装携带核心海藻糖G312的少甘露糖型结构(流程S20)。通过首先由Lemieux与同事研发的原位变旋异构化方案14(CuBr2、溴化四丁基铵)增强α-选择性,以67%产率得到双醣S20k,且通过NMR分析证实其区域化学及立体化学。使用亚砜供体S20a13及接受体S20b,使用DTBP及Tf2O形成关键的β-甘露糖基键联,以67%产率得到双醣S20c,且α/β比为1:7。通过使用DDQ移除在3"处的对甲氧基苄基醚保护,得到3"-OH S20d,通过使用酰亚胺酯1使其在BF3.OEt2的促进下进一步经历3"-Oα-甘露醣基化,获得三醣S20e。选择性打开S20e的亚苄基产生S20f,其进一步在6"-O位置处经历α-甘露醣基化,得到四醣S20g。随后通过用PdCl2处理,且随后用三氯乙酰亚胺酸酯及DBU处理,将化合物S20g转化成醣苷基酰亚胺酯S20h。最后,使酰亚胺酯S20h及双醣S20k12在BF3.OEt2存在下缩合,得到所要六醣S20i。整体脱除保护基得到少甘露糖型寡醣G3。
流程S20|制备聚醣G3.i,DTBP、Tf2O、CH2Cl2,-60℃,3h,67%(α/β1:7);ii,DDQ、CH2Cl2:H20=10/1,3h,70%;iii,1,BF3.OEt2、CH2Cl2, MS,-40℃,1h,81%;iv,三乙基硅烷、PhBCl2、CH2Cl2,1h,73%;v,BF3.OEt2、CH2Cl2,MS,-20℃,2h,65%;vi,(i)PdCl2、MeOH:CH2Cl2,室温,6h,(ii)DBU、三氯乙腈、CH2Cl2,63%,历经2个步骤;vii,Cu(II)Br、TBAB、DMF:CH2Cl2,MS,0℃至室温,隔夜,76%;viii,BF3.OEt2、CH2Cl2,MS,-70℃,2h,45%;ix,(i)NH2CH2CH2NH2、nBuOH,100℃;(ii)Ac2O、吡啶,0℃至室温;(iii)NaOMe、MeOH;(iv)Pd(OH)2、MeOH:H2O:HCOOH(5:3:2)、H2,22%,历经4个步骤。TBAB=溴化四丁基铵。
烯丙基-O-2-O-苄基-3-O-对甲氧基-苄基-4,6-O-亚苄基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖苷(S20c):将供体S20a(0.272g,0.483mmol)及经活化的分子筛(0.5g)于CH2Cl2(10mL)中的混合物在室温下搅拌1h。将反应混合物冷却至-60℃,相继添加2,6-二-第三丁基吡啶(268μL,1.24mmol)、Tf2O(79μL,0.483mmol),且搅拌40分钟。缓慢地添加接受体S20b(0.2g,0.378mmol)于CH2Cl2(5mL)中的溶液且将所得反应混合物搅拌1h,直至TLC(乙酸乙酯:甲苯,1/9)指示产物形成且起始物质耗尽。用Et3N淬灭反应混合物,用CH2Cl2稀释,经由硅藻土过滤且在真空中浓缩。通过急骤管柱层析(0%→10%EA/甲苯)纯化残余物,得到呈白色固体状的S20c(0.230g,67%)。TLC:(乙酸乙酯:甲苯=1/9,v/v):Rf=0.52;1H NMR(600MHz,CDCl3):δ7.65-7.45(m,8H,Ar-H),7.35-7.21(m,13H,Ar-H),6.91-6.90(m,2H,Ar-H),6.90-6.81(m,5H,Ar-H),5.67-5.65(m,1H,烯丙基),5.49(s,1H,Ph-CH,亚苄基),5.13(d,J=8.4Hz,1H,H-1a),5.07(d,J=8.8Hz,1H),4.99(d,J=10.4Hz,1H),4.87-4.82(m,3H),4.65(d,J=5.4Hz,1H),4.63(d,J=5.4Hz,1H),4.53(s,1H),4.50(d,J=12.0Hz,1H),4.41(d,J=6.0Hz,1H),4.39(d,J=6.0Hz,1H),4.24-4.21(m,2H),4.05-3.96(m,3H),3.81(s,3H,PMB的OMe),3.71(d,J=3.0Hz,1H),3.64(d,J=10.8Hz,1H),3.56-3.52(m,2H),3.45-3.47(d,J=10.0Hz,1H),3.40(dd,J=3.0,6.6Hz,1H),3.13-3.12(m,1H);13C NMR(150MHz,CDCl3):δ159.46,139.08,138.99,138.12,138.00,134.05,130.93,129.37,129.22,128.84,128.60,128.54,128.47,128.45,128.26,128.12,128.09,128.07,127.83,127.21,126.41,117.60,114.04,102.28,101.63,97.70,79.85,78.99,78.33,77.44,75.29,75.03,74.95,73.86,72.61,69.99,68.86,68.84,67.66,55.96,55.58;ESI-MS:m/zC59H59NO13计算值989.3879;实验值1012.3888(M+Na)+。
烯丙基-O-2-O-苄基-4,6-O-亚苄基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖苷(S20d):在0℃下,向S20c(0.5g,0.505mmol)于10mL CH2Cl2:H2O(10:1)中的溶液中添加DDQ(0.219g,1.01mmol),且将所得反应混合物搅拌3h。随后过滤反应混合物,用H2O(2×30mL)洗涤有机层。进一步用CH2Cl2(2×50mL)萃取水层。用盐水溶液(40mL)洗涤经合并的有机层,经Na2SO4干燥且在真空中浓缩。通过急骤管柱层析(0%→10%EA/甲苯)纯化残余物,得到呈无色泡沫状的S20d(0.308g,70%)。TLC:(乙酸乙酯:甲苯=2/8,v/v):Rf=0.42;1H NMR(600MHz,CDCl3):δ7.65-7.44(m,6H,Ar-H),7.43-7.28(m,13H,Ar-H),6.92-6.90(m,2H,Ar-H),6.83-6.81(m,3H,Ar-H),5.68-5.64(m,1H,烯丙基),5.42(s,1H,Ph-CH,亚苄基),5.14(d,J=8.4Hz,1H,H-1a),5.08(d,J=12.8Hz,1H),5.01-4.98(m,2H),4.85(d,J=12.6Hz,1H),4.74(d,J=12.0Hz,1H),4.65(d,J=12.0Hz,1H),4.62(s,1H,H-1b),4.84(d,J=12.0Hz,1H),4.40(d,J=12.0Hz,1H),4.25-4.16(t,J=9.6Hz,1H),3.99(dd,J=13.0&6.3Hz,1H),3.73-3.65(m,4H),3.54-3.47(m,3H),3.13-3.09(m,1H);13C NMR(150MHz,CDCl3):138.97,138.44,137.93,137.56,133.98,129.37,128.92,128.79,128.53,128.43,128.35,128.26,128.21,128.10,127.99,127.27,126.58,117.64,102.33,102.25,97.66,79.91,79.42,79.15,77.43,76.00,75.03,74.89,74.01,71.24,69.99,68.78,68.67,67.17,55.90;ESI-MS:m/zC51H51NO12计算值869.3303;实验值892.3314(M+Na)+。
烯丙基-O-(2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)-2-O-苄基-4,6-O-亚苄基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖苷(S20e):将供体1(0.537g,0.863mmol)、接受体S20d(0.50g,0.574mmol)及经活化的分子筛于无水CH2Cl2(10mL)中的混合物在室温下搅拌30分钟。将反应物冷却至-40℃,随后缓慢地添加三氟化硼合乙基醚(32μL,0.288mmol)且将所得反应混合物搅拌1h。通过添加Et3N淬灭反应,用CH2Cl2稀释,经由硅藻土过滤且在真空中浓缩。通过急骤管柱层析(0%→20%EA/己烷)纯化残余物,得到呈无色油状的S20e(0.630g,81%)。TLC:(乙酸乙酯:己烷=3/7,v/v):Rf=0.62;1H NMR(600MHz,CDCl3):δ7.41-7.39(m,4H,Ar-H),7.39-7.25(m,30H,Ar-H),7.16-6.82(m,5H,Ar-H),5.57-5.57(m,1H,烯丙基),5.59(d,J=3.0Hz,1H),5.47(s,1H,Ph-CH,亚苄基),5.29(d,J=2.1Hz,1H,H-1c),5.13(d,J=8.4Hz,1H,H-1a),5.08(dd,J=6.0&12.2Hz,1H),5.00(dd,J=6.0,10.2Hz,1H),4.87-4.82(m,2H),4.79-4.78(m,2H),4.66-4.59(m,3H),4.54(s,1H,H-1b),4.46-4.44(m,3H),4.22-4.20(m,2H),4.17-4.14(m,2H),4.03-3.93(m,4H),3.82-3.71(m,5H),3.64-3.62(m,2H),3.60-3.52(m,2H),3.50(d,J=8.2Hz,1H),3.19(m,1H),2.34(s,3H,-C(O)CH3);13CNMR(150MHz,CDCl3):δ170.26,163.58,162.83,138.98,138.77,138.60,138.40,138.09,138.04,137.58,133.97,131.97,129.01,128.79,128.71,128.64,128.62,128.56,128.52,128.38,128.32,128.24,128.12,128.05,127.97,127.94,127.84,127.18,126.26,123.57,117.58,101.76,101.35,98.96,97.63,79.24,78.91,78.56,78.22,75.76,75.62,75.03,74.77,74.44,73.75,72.55,71.83,69.94,69.32,68.64,68.54,68.39,67.16,55.88,36.75,31.70,21.27;ESI-MS:m/z C80H81NO18计算值1343.5346;实验值1366.5369(M+Na)+。
烯丙基-O-(2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3)-2,4-O-二苄基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖苷(S20f):将化合物S20e(0.630g,0.468mmol)及经活化的分子筛于20mLCH2Cl2中的混合物在室温下搅拌1h。将反应混合物冷却至-78℃,相继添加三乙基硅烷(148μL,0.946mmol)、二氯苯基硼烷(146μL,1.18mmol),且搅拌1h。通过添加Et3N、甲醇淬灭反应,经由硅藻土过滤且在真空中浓缩。将残余物与甲醇共蒸馏2-3次,随后通过急骤管柱层析(0%→10%EA/甲苯)纯化,得到醇S20f(0.460g,73%)。TLC:(乙酸乙酯:甲苯=2/8,v/v):Rf=0.42;1H NMR(600MHz,CDCl3):δ7.73-7.57(m,4H,Ar-H),7.50-7.08(m,31H,Ar-H),6.83-6.81(m,4H,Ar-H),5.76-5.69(m,1H,烯丙基),5.44(t,J=3.0Hz,1H),5.13(s,1H,H-1c),5.12(s,1H),5.08(d,J=7.8Hz,1H,H-1a),5.01(d,J=11Hz,1H),4.93(d,J=11.0Hz,1H),4.87(d,J=8.6Hz,1H),4.83(d,J=8.0Hz,1H),4.76(d,J=12.0Hz,1H),4.69(d,J=11.0Hz,1H),4.62(t,J=8.6Hz,1H),4.57(t,J=7.8Hz,2H),4.50(d,J=10.8Hz,1H),4.44(d,J=2.5Hz,1H),4.42(s,1H),4.40(d,J=8.0Hz,1H),4.35(d,J=12.0Hz,1H),4.22-4.181(m,3H),4.00-3.99(m,2H),4.90-3.91(s,1H),3.83-3.82(m,1H),3.83(d,J=2.4Hz,1H),3.77-3.78(m,2H),3.64-3.54(m,6H),3.41-3.39(m,2H),3.19-3.18(m,1H),2.08(s,3H);13C NMR(150MHz,CDCl3):δ170.34,138.90,138.77,138.73,138.36,138.14,138.07,138.03,135.91,134.50,134.03,133.86,132.97,131.94,131.40,129.31,128.86,128.68,128.66,128.61,128.57,128.50,128.45,128.38,128.26,128.20,128.12,128.11,128.03,127.97,127.94,127.79,127.76,127.72,127.47,127.35,123.50,117.59,101.04,99.89,97.61,80.92,78.64,78.48,78.41,78.21,75.93,75.26,75.20,75.19,75.16,74.82,74.59,74.52,73.78,73.75,72.61,72.11,69.91,69.50,68.99,68.43,62.25,55.86,21.29;ESI-MS:m/z C80H83NO18计算值1345.5502;实验值1368.5527(M+Na)+。
烯丙基-O-二-(2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3),(1→6)-2,4-O-二苄基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖苷(S20g):将供体1(0.429g,0.690mmol)、接受体S20f(0.460g,0.345mmol)及经活化的分子筛于无水CH2Cl2(10mL)中的混合物在室温下搅拌30分钟。将反应物冷却至-20℃,随后缓慢地添加三氟化硼合乙基醚(19μL,0.173mmol)且将所得反应混合物搅拌2h。通过添加Et3N淬灭反应,用CH2Cl2稀释,经由硅藻土过滤且在真空中浓缩。通过急骤管柱层析(0%→20%EA/己烷)纯化残余物,得到呈无色泡沫状的S57(0.380g,65%)。TLC:(乙酸乙酯:己烷=3/7,v/v):Rf=0.56;1H NMR(600MHz,CDCl3):δ7.60-7.29(m,4H,Ar-H),7.28-7.08(m,37H,AR-H),7.07-7.08(m,8H,Ar-H),7.07-6.93(d,J=8.0Hz,1H,Ar-H),6.82-6.59(m,3H,Ar-H),5.61-5.59(m,1H,烯丙基),5.43(d,J=6.0Hz,1H,H-2c),5.31(d,J=6.0Hz,1H,H-2d),5.10(d,J=3.0Hz,1H,H-1c),5.06(d,J=8.0Hz,1H,H-1a),4.99(d,J=10.0Hz,1H),4.85(d,J=8.0Hz,1H),4.83(s,1H,H-1d),4.67(d,J=8.0Hz,1H),4.64-4.62(q,4H),4.56(d,J=3.0Hz,1H,H-1b),4.55-4.45(m,8H),4.45-4.39(m,9H),4.38(d,J=10.0Hz,1H),4.21(d,J=9.0Hz,1H),4.19-4.10(m,2H),4.08-3.70(m,10H),3.69-3.49(m,7H),3.39(d,J=9.1Hz,1H),3.19(d,J=9.1Hz,1H),2.07(s,3H,-C(O)CH3),1.82(s,3H,-C(O)CH3);13C NMR(150MHz,CDCl3):δ170.31,170.11,163.43,139.07,138.93,138.83,138.80,138.65,138.35,138.32,138.21,138.09,134.08,133.77,132.00,128.87,128.76,128.66,128.56,128.43,128.37,128.16,127.99,127.91,127.82,127.73,127.13,123.46,117.45,102.07,99.92,99.74,98.62,97.55,81.40,79.81,78.31,78.23,76.84,75.87,75.77,75.66,74.87,74.65,74.44,74.38,31.21,21.29,21.23;ESI-MS:m/zC109H113NO24计算值1820.7578;实验值1843.7676(M+Na)+。
二-(2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3),(1→6)-2,4-O-二苄基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄-哌喃糖基三氯乙酰亚胺酸酯(S20h):向S20g(0.2g,0.109mmol)于10mL CH2Cl2:MeOH(1/1)中的溶液中添加PdCl2(20mg)且在室温下搅拌6h,直至TLC(乙酸乙酯:甲苯,2/8)指示产物形成且起始物质耗尽。随后在真空中浓缩反应混合物。通过急骤管柱层析(0%→10%EA/甲苯)纯化残余物,得到呈无色泡沫状的醇(0.140g,60%)。在0℃下,向醇(0.150g,0.084mmol)于CH2Cl2(10mL)中的溶液中相继添加三氯乙酰胺酸酯(33μL,0.336mmol)、DBU(12μL,0.033mmol),且在室温下搅拌8h。用Et3N淬灭反应且在真空中浓缩。通过急骤管柱层析(0%→10%EA/甲苯)纯化残余物,得到呈无色油状的S20h(0.121g,67%)。TLC:(乙酸乙酯:甲苯=2/8,v/v):Rf=0.47;1H NMR(600MHz,CDCl3):δ8.51(s,1H,C=NH),7.53-7.42(m,4H,Ar-H),7.40-7.00(m,49H,Ar-H),6.58(d,J=6.6Hz,2H,Ar-H),6.52-6.50(m,3H,Ar-H),5.43(s,1H,H-1a),5.32(s,1H,H-1c),5.03(s,1H),5.01(t,J=7.8Hz,1H),4.90-4.78(m,6H),4.67-4.45(m,7H),4.43-4.30(m,9H),4.28-4.20(m,2H),4.10(t,J=7.8Hz,1H),3.39-3.42(m,18H),3.19(d,J=9.3Hz,1H),2.09(s,3H,-C(O)CH3),1.83(s,3H,-C(O)CH3)。
5-迭氮基戊基-O-2,3,4-三-O-苄基-α-L-海藻哌喃糖基-(1→6)-3-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖苷(S20k):将CuBr2(0.450g,2.01mmol)、Bu4NBr(0.655g,2.03mmol)及经活化的分子筛于10mL的CH2Cl2:DMF(2/1)中的混合物搅拌且在冰水浴中冷却。逐滴添加供体S20i(0.5g,0.925mmol)及接受体S20j(0.450g,0.882mmol)于CH2Cl2(10mL)中的溶液,且将所得反应混合物在室温下搅拌隔夜。TLC(乙酸乙酯:己烷,4/6)指示产物形成且起始物质耗尽。用NaHCO3水溶液淬灭反应,用乙酸乙酯(50mL)稀释且经由硅藻土过滤。用NaHCO3水溶液、盐水(50mL)洗涤滤液,经硫酸钠干燥。在真空中浓缩有机层。通过急骤管柱层析(0%→30%EA/己烷)纯化残余物,得到呈无色油状的S20k(0.604g,76%)。TLC:(乙酸乙酯:己烷=4/6,v/v):Rf=0.47;1H NMR(600MHz,CDCl3):δ7.80-7.64(m,4H,Ar-H),7.41-7.23(m,15H,Ar-H),7.00-6.98(m,2H,Ar-H),6.90-6.87(m,3H,Ar-H),5.06(d,J=8.4Hz,1H,H-1a),4.96(d,J=11.4Hz,1H),4.86(d,J=11.4Hz,1H),4.82-4.72(m,4H),4.67(d,J=12.0Hz,1H),4.64(d,J=12.0Hz,1H),4.48(d,J=12.6Hz,1H),4.16-4.13(m,1H),4.08-4.05(m,2H),3.39-3.95(m,2H),3.90-3.83(m,3H),3.78(m,1H),3.73-3.69(m,2H),3.56-3.53(m,1H),3.35-3.21(m,1H),2.00-2.03(m,2H,-CCH2C-,连接基团),1.38-1.30(m,4H,-CCH2C-,连接基团),1.12(d,J=8.4Hz,3H,海藻糖的Me),1.00-0.96(m,2H,-CCH2C-,连接基团);13C NMR(150,MHz,CDCl3):δ138.83,138.77,138.36,134.13,128.90,128.77,128.67,128.60,128.52,128.31,128.26,128.17,127.93,127.88,127.52,123.64,98.81,98.59,79.60,78.07,77.77,76.67,75.22,74.54,74.51,74.24,73.35,73.20,69.37,68.79,67.15,55.88,51.39,29.00,28.58,23.30,16.89;ESI-MS:m/zC53H58N4O11计算值926.3994;实验值949.3998(M+Na)+。
5-迭氮基戊基-O-2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→6)-(2-O-乙酰基-3,4,6-三-O-苄基-α-D-甘露哌喃糖基-(1→3))-2,4-二-O-苄基-β-甘露哌喃糖基-(1→4)-O-3,6-二-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基-(1→4)-(2,3,4-三-O-苄基-α-L-海藻哌喃糖基-(1→6))-3-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖苷(S20l):将供体S20h(0.050g,0.025mmol)、接受体S20k(0.028g,0.031mmol)及经活化的分子筛于无水CH2Cl2(10mL)中的混合物在室温下搅拌30分钟。将反应物冷却至-70℃,随后缓慢地添加三氟化硼合乙基醚(0.725μL,0.0062mmol)且将所得反应混合物搅拌2h。通过添加Et3N淬灭反应,用CH2Cl2稀释,经由硅藻土过滤且在真空中浓缩。通过急骤管柱层析(0%→20%EA/己烷)纯化残余物,得到呈白色泡沫状的S20l(0.027g,45%)。TLC:(乙酸乙酯:己烷=3/7,v/v):Rf=0.50;1H NMR(600MHz,CDCl3):δ7.83-7.62(m,7H,Ar-H),7.49-7.30(m,8H,Ar-H),7.30-7.10(m,47H,Ar-H),7.09-7.03(m,10H,AR-H),7.03-6.99(m,1H,Ar-H),6.93(d,J=8.9Hz,2H,AR-H),6.79-6.63(m,6H,AR-H),6.55(t,J=7.8Hz,2H,H-2d,d'),5.40(s,1H,H-1d),5.29-5.29(m,1H),5.09(d,J=1.3Hz,1H,H1d'),4.88-4.76(m,13H),4.67-4.65(m,2H),4.58-5.48(m,9H),4.42-4.40(m,3H),4.36-4.30(m,5H),4.20-4.10(m,5H),4.05-4.00(m,2H),3.92-3.80(m,7H),3.79-3.60(m,7H),3.62-3.41(m,10H),3.25(dd,J=3.2,8.2Hz,1H),3.18-3.17(m,2H),2.85-2.79(m,2H),2.06(s,3H,-C(O)CH3),1.76(s,3H,-C(O)CH3,1.42-1.34(m,4H,-CCH2C-,连接基团),1.03-0.99(m,5H,-CCH2C-,连接基团及海藻糖的CH3);13C NMR(150MHz,CDCl3):δ170.30,169.91,168.15,167.76,139.16,138.99,138.88,138.82,138.77,138.54,138.28,138.21,138.10,138.08,138.05,133.86,132.02,131.72,128.80,128.62,128.60,128.57,128.55,128.33,128.23,128.40,127.98,127.92,127.70,127.55,127.26,127.06,123.33,101.97,99.62,98.41,97.95,97.09,97.05,81.42,79.71,78.36,74.98,74.75,74.68,73.65,72.57,72.51,72.03,71.38,68.93,68.86,68.12,66.63,66.12,56.76,56.01,51.26,28.82,28.56,23.20,21.23,20.91;ESI-MS:m/z C159H165N5O34计算值2689.1261;实验值2712.1338(M+Na)+。
5-胺基戊基-二-(α-D-甘露哌喃糖基)-(1→3),(1→6)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-(α-L-海藻哌喃糖基-(1→6)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(G3):通过以下一般程序2(方法1)脱除化合物S20h(0.030g,0.011mmol)的保护基,获得呈白色粉末状的标题化合物G3(0.007g,22%)。1H NMR(600MHz,D2O):δ5.09(s,1H,H-1d),4.91(s,1H,H-1d'),4.89(d,J=3.2Hz,1H,H-1e),4.78(s,1H,H-1c),4.65(d,J=7.8HZ,1H,H-1a),4.49(d,J=7.8Hz,1H,H-1b),4.25(s,1H),4.15-4.10(m,1H),4.05(s,1H),3.95(s,1H),3.95-3.80(m,8H),3.80-3.50(m,23H),3.00(m,2H),2.10(s,3H,-C(O)CH3),2.09(s,3H,-C(O)CH3,1.67-1.60(m,2H,-CCH2C-),1.60-1.57(m,2H,-CCH2C-),1.41-1.37(m,2H,-CCH2C-),1.25(d,J=7.8Hz,3H,海藻糖的-CH3);13C NMR(150MHz,D2O):δ174.67,174.39,170.97,102.51,101.10,101.04,100.38,99.60,99.39,80.46,79.72,78.60,74.33,74.15,73.42,72.65,72.30,71.95,71.85,70.29,70.12,70.00,69.97,69.85,69.45,68.15,66.77,66.14,65.85,65.80,61.11,60.92,59.97,55.05,54.86,46.66,39.28,38.61,28.05,26.36,22.27,22.15,21.08,21.50,15.37,15.34;ESI-MS:m/z C45H80N3O30计算值1141.4821;实验值1142.4827(M+H)+。
5-胺基戊基-二-[5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖基]-(1→3),(1→6)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-(α-L-海藻哌喃糖基-(1→6)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(G19):通过使用所报导的程序11、12制备唾液酸化双触角核心海藻糖聚醣G19。1HNMR(600MHz,D2O):δ4.90(s,1H,H-1d),4.72(s,1H,H-1d'),4.66(d,J=3.6Hz,1H,H-1c),4.56(s,1H),4.43(d,J=7.2Hz,1H,H-1b),4.37(d,J=7.2Hz,2H,H-1e,e'),4.25(d,J=7.8Hz,1H,H-1a),4.21(d,J=7.8Hz,2H,H-1f),4.04(s,1H),3.97(s,1H),3.90(t,J=10.1Hz,2H),3.76-3.25(m,65H),2.74(t,J=7.8Hz,2H,连接基团),2.43(dd,J=3.8&7.2Hz,1H,H-3赤道g,g'),1.87(s,3H),1.84(s,3H,-C(O)CH3),1.83(s,3H,-C(O)CH3),1.80(s,6H,-C(O)CH3),1.51(t,J=12H,2H,H-3轴向g,g'),1.46-1.41(m,2H),1.37-1.34(m,2H),1.18-1.15(m,2H),1.00(d,J=6.6Hz,3H,海藻糖的-CH3);13C NMR(150MHz,D2O):δ174.5,174.3,174.1,173.3,103.3,100.9,100.7,100.2,99.7,99.6,99.3,99.1,98.9,96.5,80.2,79.5,77.8,75.9,75.6,74.0,73.3,73.2,73.1,72.4,72.2,71.8,71.7,71.3,70.3,69.9,69.7,69.3,69.1,68.0,67.9,67.0,66.9,66.5,65.7,65.4,63.0,62.2,61.4,61.3,59.8,59.4,54.7,54.4,54.2,51.5,39.7,38.9,27.7,26.1,22.1,22.0,22.0,21.8,21.7,15.1;ESI-MS:m/z C95H159N7O66计算值2451.9121;实验值1226.4513(M-H)2-。
合成含膦酸尾部的聚醣S
一般程序:向含胺尾部的糖(3-5μmol)于DMF(400μL)中的溶液中添加连接基团[2(2(2(双(苄氧基)磷酰基)乙氧基乙氧基)乙基(2,5-二侧氧基吡咯啶-1-基)碳酸酯](15-25μmol),且将所得反应混合物在室温下搅拌5h。通过使用高真空移除DMF且通过使用Bio-GelP-2层析(溶离剂H2O)纯化产物。将固体溶解于2mL H2O中,添加Pd(OH)2(50重量%)且氢化隔夜。经由硅藻土过滤反应混合物且在真空中浓缩。通过Bio-Gel P-2(BIO-RAD)管柱层析,使用水作为溶离剂纯化残余物。冻干产物,获得呈白色粉末状的所要具有膦酸尾部的糖。
流程S21|制备具有膦酸连接基团的糖.i,(1)连接基团、DMF,室温,5h;(2)Pd(OH)2、H2O、H2,室温,隔夜。
β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(I):通过以上一般程序修饰化合物S23(3.5mg,5.4μmol),得到呈白色固体状的化合物I(2.2mg,61%)。TLC(MeOH:EA:AcOH:H2O,7/1/1/1,v/v)。1H NMR(600MHz,D2O):δ4.60(d,J=8.4Hz,1H),4.48(d,J=7.8Hz,1H),4.19(s,2H),4.05(d,J=3.2Hz,1H),3.93-3.49(m,33H),1.75(t,J=7.8Hz,2H),2.06(s,3H),1.95(s,3H),1.95-1.92(m,2H),1.58-1.54(m,2H),1.50-1.47(m,2H),1.33-1.30(m,2H)。HRMS(MALDI-TOF):m/z C34H59N3O23P计算值908.3067;实验值954.3099(M+2Na)+。
(膦酸酯-三-(乙二醇)-羰基-胺基)-戊基-α-D-甘露-哌喃糖基(1→3)-α-D-甘露哌喃糖基]-(1→6)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(II):通过以上一般程序修饰聚醣G2(4mg,2.3μmol),得到呈白色固体状的化合物II(3.2mg,71%)。TLC(MeOH:EA:AcOH:H2O,7/1/1/1,v/v)。1H NMR(600MHz,D2O):δ5.01(s,1H),4.83(s,1H),4.51(d,J=6.1Hz,1H),4.40(d,J=6.2Hz,1H),4.17(s,1H),4.11(s,2H),3.98(s,1H),3.88-3.39(m,52H),3.03(t,J=7.8Hz,2H),1.99(s,3H),1.95(s,3H),1.95-1.91(m,2H),1.50-1.45(m,2H),1.42-1.38(m,2H),1.26-1.22(m,2H)。HRMS(MALDI-TOF)负模式:m/z C58H102N3O43P计算值1234.4484;实验值1278.5001(M+2Na-2H)-。
(膦酸酯-三-(乙二醇)-羰基-胺基)-戊基-α-D-甘露-哌喃糖基(1→3)-α-D-甘露哌喃糖基]-(1→6)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-)-(α-L-海藻哌喃糖基-(1→6)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷)(III):通过以上一般程序修饰聚醣G3(1.2mg,1.0μmol),得到呈白色固体状的化合物III(1.0mg,91%)。TLC(MeOH:EA:AcOH:H2O,7/1/1/1,v/v)。1H NMR(600MHz,D2O):δ5.09(s,1H),4.90(s,1H),4.88(d,J=7.4Hz,1H),4.66(d,J=7.5Hz,1H),4.48(d,J=7.2Hz,1H),4.25(s,1H),4.19(s,2H),4.12-4.10(m,1H),4.06(s,1H),3.95(s,1H),3.92-3.84(m,10H),3.79-3.69(m,31H),3.11-3.09(m,2H),2.08(s,3H),2.02(s,3H),1.99-1.93(m,2H),1.58-1.53(m,2H),1.49-1.47(m,2H),1.34-1.30(m,2H),1.22(d,3H)。HRMS(MALDI-TOF):m/zC34H59N3O23P计算值1381.5709.3067;实验值1450.5709(M+3Na)+。
(膦酸酯-三-(乙二醇)-羰基-胺基)-戊基-α-D-甘露-哌喃糖基(1→3),[二-(α-D-甘露哌喃糖基)-(1→3),(1→6)-α-D-甘露哌喃糖基]-(1→6)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(IV):通过以上一般程序修饰聚醣G4(4mg,3.1μmol),得到呈白色固体状的化合物IV(3.7mg,78%)。TLC(MeOH:EA:AcOH:H2O,7/1/1/1,v/v)。1H NMR(600MHz,D2O):δ5.10(d,J=9.6Hz,2H),4.93(s,1H),4.70(s,1H),4.57(d,J=7.2Hz,1H),4.48(d,J=7.8Hz,1H),4.24(d,J=3.2Hz,2H),4.19-4.13(m,4H),4.06-4.04(m,3H),3.95-3.40(m,45H),3.38-3.37(m,1H),2.10(t,J=7.8Hz,2H),2.07(s,6H),2.02(s,6H),1.98-1.93(m,2H),1.58-1.47(m,2H),1.34-1.29(m,2H),1.27-1.25(m,2H);HRMS(MALDI-TOF):m/z C58H102N3O43P计算值1560.3985;实验值1607.0452(M+2Na+H)+。
(膦酸酯-三-(乙二醇)-羰基-胺基)-戊基-α-D-甘露哌喃糖基-(1→2)-α-D-甘露哌喃糖基-(1→2)-α-D-甘露-哌喃糖基-(1→3)-β-D-甘露-哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(V):通过以上一般程序修饰聚醣G5(5mg,4.4μmol),得到呈白色固体状的化合物V(3.5mg,58%)。TLC(MeOH:EA:AcOH:H2O,7/1/1/1,v/v)。1H NMR(600MHz,D2O):δ5.34(s,1H),5.29(s,1H),5.03(s,1H),4.58(d,J=7.8Hz,1H),4.47(d,J=7.8Hz,1H),4.20(bs,3H),4.09-4.05(m,3H),3.90-3.20(m,41H),3.10(t,J=7.8Hz,2H),2.05(s,3H),2.01(s,3H),2.00-1.92(m,2H),1.56-1.53(m,2H),1.49-1.46(m,2H),1.33-1.30(m,2H);HRMS(MALDI-TOF):m/zC52H92N3O38P计算值1398.2579;实验值1444.5470(M+2Na)+。
(膦酸酯-三-(乙二醇)-羰基-胺基)-戊基-α-D-甘露哌喃糖基-(1→2)-α-D-甘露哌喃糖基-(1→2)-α-D-甘露-哌喃糖基-(1→3)-{α-D-甘露哌喃糖基-(1→2)-α-D-甘露哌喃糖基-(1→3)-[α-D-甘露-哌喃糖基-(1→2)-α-D-甘露哌喃糖基-(1→6)]-α-D-甘露哌喃糖基-(1→6)}-β-D-甘露-哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(VI):通过以上一般程序修饰聚醣G6(4mg,2.0μmol),得到呈白色固体状的化合物VI(3.7mg,66%)。TLC(MeOH:EA:AcOH:H2O,7/1/1/1,v/v)。1H NMR(600MHz,D2O):δ5.53(s,1H),5.39(d,J=10.2Hz,1H),5.26(d,J=9.8Hz,1H),5.23(s,1H),5.16(d,J=4.8Hz,1H),5.08-5.05(m,5H),4.60(d,J=7.8Hz,1H),4.50(d,J=7.8Hz,1H),2.08(s,3H),2.02(s,3H),1.38-1.03(m,2H),1.58-1.56(m,2H),1.52-1.48(m,2H),1.35-1.32(m,2H);HRMS(MALDI-TOF):m/z C82H142N3O63P计算值2209.0310;实验值2257.0812(M+2Na)+。
(膦酸酯-三-(乙二醇)-羰基-胺基)-戊基-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖基]-(1→3)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(VII):通过以上一般程序修饰聚醣G9(6.0mg,5.1μmol),得到呈白色固体状的化合物VII(3.5mg,50%)。TLC(MeOH:EA:AcOH:H2O,7/1/1/1,v/v)。1H NMR(600MHz,D2O):δ5.03(d,J=8.4Hz,2H),4.58(d,J=7.2Hz,1H),4.48(d,J=7.2Hz,1H),4.45(d,J=7.8Hz,1H),4.26(dd,J=3.0&9.2Hz,2H),4.19(bs,2H),4.00-3.38(m,48H),3.11(t,J=7.8Hz,2H),2.06(s,3H),2.05(s,3H),2.02(s,3H),1.99-1.93(m,2H),1.56-1.54(m,2H),1.50-1.48(m,2H),1.31-1.27(m,2H);HRMS(MALDI-TOF):m/z C54H92N4O38P计算值1435.4917;实验值1481.5069(M+2Na)+。
(膦酸酯-三-(乙二醇)-羰基-胺基)-戊基-β-D-半乳哌喃糖基-(1→4)-[2-乙酰胺基-2-去氧-β-D-葡萄-哌喃糖基-(1→2)-α-D-甘露哌喃糖基-(1→3),[二-(α-D-甘露哌喃糖基)-(1→3),(1→6)-α-D-甘露-哌喃糖基](1→6)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄-哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(VIII):通过以上一般程序修饰聚醣G12(5.0mg,3.0μmol),得到呈白色固体状的化合物VIII(3.1mg,54%)。TLC(MeOH:EA:AcOH:H2O,7/1/1/1,v/v)。1H NMR(600MHz,D2O):δ5.11(s,2H),5.02(d,J=7.8Hz,2H),4.68(d,J=3.2Hz,2H),4.48(d,J=7.2Hz,1H),4.45(d,J=7.2Hz,1H),4.27(s,2H),4.22(s,2H),4.15(s,1H),4.08(s,2H),4.00-3.30(m,60H),3.10(t,J=7.8Hz,2H),2.08(s,3H),2.05(s,3H),2.02(s,3H),1.93-1.87(m,2H),1.56-1.54(m,2H),1.51-1.47(m,2H),1.33-1.30(m,2H);HRMS(MALDI-TOF):m/z C72H122N4O53P计算值1921.6502;实验值1967.6622(M+2Na)+。
膦酸酯-三-(乙二醇)-羰基-胺基)-戊基-[5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖基]-(1→3)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄-哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(IX):通过以上一般程序修饰聚醣G10(5.0mg,2.5μmol),得到呈白色固体状的化合物IX(3.8mg,63%)。TLC(MeOH:EA:AcOH:H2O,7/1/1/1,v/v)。1H NMR(600MHz,D2O):δ5.08(d,J=8.4Hz,2H),4.93(s,1H),4.62(d,J=7.2Hz,1H),4.51(d,J=7.8Hz,1H),4.45(d,J=7.8Hz,1H),4.30(d,J=3.2Hz,1H),4.28(s,1H),4.22(s,2H),4.02-3.30(m,53H),3.13(t,J=7.8Hz,2H),2.68(dd,J=3.2,7.8Hz,1H),2.09(s,6H),2.05(s,6H),2.01-1.93(m,2H),1.74(t,J=12.1Hz,1H),1.60-1.57(m,2H),1.54-1.51(m,2H),1.35-1.32(m,2H);HRMS(MALDI-TOF):m/z C65H109N5O46P计算值1726.5871;实验值1772.5817(M+2Na)+。
(膦酸酯-三-(乙二醇)-羰基-胺基)-戊基-5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-[2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖基-(1→3),[二-(α-D-甘露哌喃糖基)-(1→3),(1→6)-α-D-甘露-哌喃糖基](1→6)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(X):通过以上一般程序修饰聚醣G13(4.0mg,2.3μmol),得到呈白色固体状的化合物X(3.2mg,71%)。TLC(MeOH:EA:AcOH:H2O,7/1/1/1,v/v)。1H NMR(600MHz,D2O):δ5.08(d,J=10.2Hz,1H),5.03(s,1H),4.94(s,2H),4.91(s,1H),4.62(s,1H),4.51(d,J=6.6Hz,1H),4.46(d,J=7.8Hz,1H),4.30(s,2H),4.25(s,2H),4.17(s,1H),4.06(s,1H),4.00-3.20(m,70H),2.70(dd,J=3.2,7.8Hz,1H),2.11(s,6H),2.05(s,6H),2.03-1.97(m,2H),1.14(t,J=12.6Hz,1H),1.58-1.51(m,4H),1.36-1.31(m,2H)。HRMS(MALDI-TOF):m/z C83H144N5O61P计算值1102.2301;实验值1148.2946(M+2Na)2-。
膦酸酯-三-(乙二醇)-羰基-胺基)-戊基-二-[5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖基]-(1→3),(1→6)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(XI):通过以上一般程序修饰聚醣G16(3.5mg,1.5μmol),得到呈白色固体状的化合物XI(2.6mg,69%)。TLC(MeOH:AcOH:H2O,8/1/1,v/v)。1H NMR(600MHz,D2O):δ5.05(d,J=8.4Hz,1H),4.63(d,J=6.6Hz,1H),4.51(s,1H),4.46(t,J=7.8Hz,2H),4.32(s,1H),4.29(s,2H),4.22(s,2H),4.09-3.48(m,79H),3.13(t,J=7.8Hz,2H),2.74-2.68(m,2H),2.10(s,9H),2.05(s,9H),2.12-2.00(m,2H),1.76-1.73(m,2H),1.58-1.55(m,2H),1.51-1.50(m,2H),1.35-1.29(m,2H);HRMS(MALDI-TOF负模式):m/z C96H162N7O69P计算值2547.9096;实验值1294.9685(M+Na)2-。
化学酶促合成D1及D2/D3臂模组S
吾人的化学酶促策略从制备接受体受质16-20开始。如流程S22中所描绘,在NIS/TfOH存在下,用供体S22a使甘露糖基接受体S22b醣基化,以60%产率得到双醣S22c。在不同的反应条件下,在4-OH或6-OH处打开S22c的亚苄基环,分别得到S22d及S22g。分别在S22d及S22g的4-O及6-O位置处单独安装供体S22e。最后,进行中间体S22c、S22f及S22h的整体脱除保护基。在化合物S22j及S22l的情况下,在Man 4及6-O位置处的GlcNAc残基经由4-OH的乙酰化而与2-O GlcNAc产生差异,用于制备不对称模组。
流程S22|制备接受体受质.i,NIS、TfOH、CH2Cl2,-50℃,3h;ii,三乙基硅烷、TFA,MS,CH2Cl2,0℃,2h,58%;iii,BH3.THF、Bu2BOTf、CH2Cl2,MS,-10℃,1h,71%;iv,(1)NH2CH2CH2NH2、nBuOH,100℃;(2)Ac2O、吡啶,0℃至室温;(3)Pd(OH)2、MeOH:H2O:HCOOH(5:3:2)、H2;
对甲氧基苯基-O-[4-O-乙酰基-3,6-O-二苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄-哌喃糖基-(1→2)-O-4,6-O-亚苄基-3-O-苄基-α-D-甘露哌喃糖苷(S22c):向接受体S22b(0.250g,0.536mmol)及供体S22a(0.410g,0.643mmol)于无水CH2Cl2(10mL)中的溶液中添加经活化的分子筛(1g)。将反应混合物在室温下搅拌1h,随后冷却至-50℃。缓慢地添加NIS(0.241g,1.07mmol)及TfOH(11.8μL,0.134mmol),且将所得反应混合物搅拌1h。当TLC(乙酸乙酯:甲苯,2/8)指示产物形成且起始物质耗尽时,通过添加Et3N淬灭反应,随后经由硅藻土过滤。用NaHCO3水溶液(2×50mL)、Na2S2O3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→10%EA/甲苯)纯化残余物,得到呈白色泡沫状的S22c(0.403g,76%)。TLC(乙酸乙酯:甲苯=2/8,v/v):Rf=0.49;1H NMR(400MHz,CDCl3):δ7.69-3.56(m,4H),7.36-7.22(m,15H),7.20-6.96(m,5H),6.53(d,J=8.4Hz,2H),6.50(d,J=8.2Hz,2H),5.41(s,1H),5.24(d,J=7.8Hz,1H),5.10(t,1H),4.93(s,1H),4.73-4.43(m,10H),4.00-3.20(m,5H),1.98(s,3H);13C NMR(150MHz,CHCl3):δ169.2,152.3,135.1,134.9,133.9,128.7,128.6,128.4,128.1,128.1,128.0,127.7,127.6,127.5,117.8,114.7,97.1,96.3,74.0,73.9,73.1,72.9,72.8,72.8,72.5,72.2,71.4,65.7,55.4,54.2,22.5;ESI-MS:m/z C57H55NO14计算值977.3623;实验值1000.3490(M+Na)+。
对甲氧基苯基-O-[4-O-乙酰基-3,6-O-二苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄-哌喃糖基-(1→2)-O-3,6-O-二苄基-α-D-甘露哌喃糖苷(S22d):在0℃下,向S22c(1.01g,1.02mmol)于无水CH2Cl2(10mL)中的溶液中相继添加三乙基硅烷(1.63mL,10.2mmol)、三氟乙酸(0.758mL,10.2mmol)。搅拌所得反应混合物3h。3h后,TLC(乙酸乙酯:甲苯,1.5/8.5v/v)指示产物形成且起始物质耗尽。用饱和NaHCO3(2×50mL)洗涤反应混合物。进一步用CH2Cl2(3×30mL)萃取水层,且用盐水溶液(100mL)洗涤经合并的有机层,经MgSO4干燥,过滤且在真空中浓缩。通过急骤管柱层析(0%→10%EA/甲苯)纯化残余物,得到呈透明油状的S22d(0.580g,65%)。TLC(乙酸乙酯:甲苯=1.5/8.5,v/v):Rf=0.35;1HNMR(400MHz,CDCl3):δ7.63-7.35(m,4H),7.30-7.10(m,14H),7.02-6.97(m,3H),6.93-6.86(m,3H),6.72-6.66(m,4H),5.27(d,J=8.4Hz,1H),5.11(t,J=10.2Hz,1H),5.02(d,J=2.8Hz,1H),4.88(s,2H),4.80(s,1H),4.60(d,J=10.2Hz,2H),4.58-4.24(m,6H),4.01(s,2H),3.79-3.74(m,2H),3.72(s,3H),3.64-3.56(m,3H),3.35(dd,J=2.8&8Hz,1H),2.93(dd,J=2.3&7.8Hz,1H),1.94(s,3H);13C NMR(150MHz,CHCl3):δ150.33,138.18,137.98,133.96,128.70,128.65,128.42,128.18,128.12,128.02,127.76,127.62,127.52,117.89,114.71,97.19,96.33,74.08,73.96,73.10,72.95,72.80,71.86,70.96,70.75,70.48,67.72,55.89,55.69,21.21;ESI-MS:m/z C57H57NO14计算值979.3779;实验值1002.3660(M+Na)+。
对甲氧基苯基-O-4-O-乙酰基-3,6-O-二苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基-(1→2)-[-O-4,6-O-亚苄基-3-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基-(1→4)]-3,6-O-二苄基-α-D-甘露哌喃糖苷(S33f):向接受体S22d(0.580g,0.590mmol)及供体S22e(0.525g,0.880mmol)于无水CH2Cl2(10mL)中的溶液中添加经活化的分子筛(1g)。将反应混合物在室温下搅拌1h,随后冷却至-50℃。缓慢地添加NIS(0.265g,1.14mmol)及TfOH(13μL,0.147mmol),且将所得反应混合物搅拌2h。当TLC(乙酸乙酯:甲苯,1.5/8.5)指示产物形成且起始物质耗尽时,通过添加Et3N淬灭反应,随后经由硅藻土过滤。用NaHCO3水溶液(2×50mL)、Na2S2O3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→10%EA/甲苯)纯化残余物,得到呈浅黄色固体状的S22f(0.730g,85%)。TLC(乙酸乙酯:甲苯=1.5/8.5,v/v):Rf=0.60;1H NMR(400MHz,CDCl3):δ7.69-6.67(m,8H),7.59-7.18(m,20H),6.96-6.75(m,10H),6.57-6.52(m,4H),5.44(s,1H),5.27(d,J=8.4Hz,1H),5.21(d,J=8.8Hz,1H),5.11(t,J=10.7Hz,1H),3.29-3.24(m,1H),4.94(d,J=8.4Hz,1H),4.76-4.69(m,2H),4.57(d,J=7.8Hz,1H),4.53(d,J=7.2Hz,1H),4.45-4.19(m,9H),4.10-4.06(m,2H),3.99-3.94(m,2H),3.63(s,3H),3.56-3.48(m,8H),3.00(dd,J=2.3及7.8Hz,2H),1.99(s,3H);13C NMR(150MHz,CHCl3):δ169.98,168.04,138.94,138.46,138.14,137.96,133.90,131.78,129.24,128.69,128.52,128.34,128.25,128.18,128.08,127.72,127.33,126.38,123.56,117.90,114.47,101.43.99.38,83.11,78.85,74.79,74.27,73.94,73.76,72.80,72.34,71.43,70.26,69.23,68.90,66.00,56.49,55.85,55.70,21.70;ESI-MS:m/z C85H80N2O20计算值1448.5153;实验值1471.5156(M+Na)+。
对甲氧基苯基-O-[4-O-乙酰基-3,6-O-二苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄-哌喃糖基-(1→2)-O-3,4-O-二苄基-α-D-甘露哌喃糖苷(S22g):在0℃下,向化合物S22c(0.800g,0.816mmol)及经活化的分子筛(1g)于无水CH2Cl2(10mL)中的混合物中添加硼烷.THF复合物(0.781mL的1M THF溶液,8.15mmol)及Bu2BOTf(0.351mL的1M CH2Cl2溶液,1.63mmol)。将反应混合物在室温下搅拌3h。TLC(丙酮:甲苯,1/9)指示产物形成且起始物质耗尽。在0℃下,向反应混合物中添加三乙胺,随后缓慢添加甲醇。当不再产生氢气时,经由硅藻土过滤反应混合物,用NaHCO3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→10%EA/甲苯)纯化残余物,得到呈透明泡沫状的S22g(0.500g,62%)。TLC(丙酮:甲苯=2/8,v/v):Rf=0.41;1H NMR(400MHz,CDCl3):δ7.84-7.55(m,4H),7.40-6.90(m,10H),6.87-6.80(m,7H),6.93-6.86(m,3H),6.72-6.66(m,4H),5.27(d,J=8.4Hz,1H),5.11(t,J=10.2Hz,1H),5.02(d,J=2.8Hz,1H),4.88(s,2H),4.80(s,1H),4.60(d,J=10.2Hz,2H),4.58-4.24(m,6H),4.01(s,2H),3.79-3.74(m,2H),3.72(s,3H),3.64-3.56(m,3H),3.35(dd,J=2.8&8Hz,1H),2.93(dd,J=2.3&7.8Hz,1H),1.94(s,3H);13C NMR(150MHz,CHCl3):δ154.2,138.6,136.5,133.5,128.0,127.5127.4,128.18,127.1,127.0,126.7,126.6,126.5,117.89,114.71,97.19,96.33,74.08,73.96,73.10,72.9,72.8,72.5,72.6,71.9,71.4,65.7,56.8,56.6,21.9;ESI-MS:m/z C57H57NO14计算值979.2640;实验值979.2012。
对甲氧基苯基-O-4-O-乙酰基-3,6-O-二苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基-(1→2)-[-O-4,6-O-亚苄基-3-O-苄基-2-去氧-2-邻苯二酰亚胺基-β-D-葡萄哌喃糖基-(1→6)]-3,6-O-二苄基-α-D-甘露哌喃糖苷(S22h):向接受体S22g(0.500g,0.509mmol)及供体S22e(0.452g,0.763mmol)于无水CH2Cl2(10mL)中的溶液中添加经活化的分子筛(1g)。将反应混合物在室温下搅拌1h,随后冷却至-50℃。缓慢地添加NIS(0.229g,1.01mmol)及TfOH(22μL,0.250mmol),且将所得反应混合物搅拌1h。当TLC(乙酸乙酯:甲苯,1/9)指示产物形成且起始物质耗尽时,通过添加Et3N淬灭反应,随后经由硅藻土过滤。用NaHCO3水溶液(2×50mL)、Na2S2O3水溶液(2×50mL)及盐水(50mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→10%EA/甲苯)纯化残余物,得到呈浅黄色固体状的S22h(0.610g,82%)。TLC(乙酸乙酯:甲苯=1/9,v/v);1H NMR(400MHz,CDCl3):δ7.75-7.50(m,8H),7.47-7.05(m,25H),7.02-6.82(m,10H),6.66(d,J=8.5Hz,2H),6.48(d,J=8.6Hz,2H),5.38(s,1H),5.17(d,J=8.4Hz,1H),5.21(t,J=10.3Hz,1H),4.85(d,J=8.6Hz,1H),3.29-3.24(m,1H),4.94(d,J=8.4Hz,1H),4.76-4.69(m,2H),4.57(d,J=7.8Hz,1H),4.53(d,J=7.2Hz,1H),4.45-4.19(m,9H),4.10-4.06(m,2H),3.99-3.94(m,2H),3.63(s,3H),3.56-3.55(m,6H),3.44-3.25(m,2H),3.00(t,J=10.3Hz,2H),1.99(s,3H);13C NMR(150MHz,CHCl3):δ170.0,154.9,150.7,146.8,138.6,137.9,137.4,137.1,136.9,133.9,132.7,129.24,128.69,128.52,128.34,128.25,127.9,127.72,127.33,126.38,123.56,117.90,114.47,101.43.99.38,83.11,78.85,74.79,74.27,73.94,73.76,72.80,72.34,71.43,70.26,69.23,68.90,66.00,58.49,56.8,56.7,55.90,55.8,21.8;ESI-MS:m/z C85H80N2O20计算值1448.5153;实验值1449.5678(M+H)+。
对甲氧基苯基-O-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖苷(16):通过以下一般程序2(方法1)脱除化合物S22c(0.105g,0.107mmol)的保护基,得到呈白色固体状的标题化合物16(0.035g,66%)。1H NMR(400MHz,D2O):δ7.08(d,J=9.6Hz,2H),6.92(d,J=9.1Hz,2H),5.43(s,1H),4.60(d,J=8.4Hz,1H),4.25(t,J=2.3Hz,1H),4.01(dd,J=3.2&10.1Hz,1H),3.87(dd,J=3.1&12.2Hz,1H),3.80(dd,J=3.0&12.8Hz,1H),3.78(s,3H),3.72-3.67(m,3H),3.61-3.52(m,3H),3.46-3.40(m,2H),1.99(s,3H);13C NMR(150MHz,D2O):δ174.64,170.85,154.46,149.57,118.60,114.83,99.57,96.50,76.24,75.67,73.35,73.10,69.67,69.28,66.90,61.14,60.40,55.55,55.20;ESI-MS:m/z C21H31NO1计算值489.1738;实验值512.1715(M+Na)+。
对甲氧基苯基-O-二-(2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→2),(1→4)-α-D-甘露哌喃糖苷(17):通过以下一般程序2(方法1)脱除化合物S22f(0.125g,0.086mmol)的保护基,得到呈白色固体状的标题化合物17(0.42g,66%)。1H NMR(400MHz,D2O):δ7.10(d,J=8.8Hz,2H),7.08(d,J=8.2Hz,2H),5.39(s,1H),4.53(d,J=8.2Hz,1H),4.50(d,J=7.8Hz,1H),4.25(s,1H),4.20(d,J=7.8Hz,1H),4.00(dd,J=3.2及7.2Hz,1H),3.71(s,3H),3.67-3.74(m,9H),2.02(s,3H),1.91(s,3H);13C NMR(150MHz,D2O):δ174.48,147.04,154.49,149.86,118.53,114.97,100.80,99.64,96.55,76.40,75.71,75.66,73.74,73.01,72.63,69.74,69.27,69.19,67.32,60.72,60.60,60.45,59.05,55.62,55.34,55.22,22.24,22.01;ESI-MS:m/z C29H44N2O17计算值693.2713;实验值693.2688(M+H)+。
对甲氧基苯基-O-二-(2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→2),(1→6)-α-D-甘露哌喃糖苷(18):通过以下一般程序2(方法1)脱除化合物S22h(0.200g,0.138mmol)的保护基,得到呈白色固体状的标题化合物18(0.60g,63%)。1H NMR(400MHz,D2O):δ7.10(d,J=8.8Hz,2H),6.96(d,J=8.2Hz,2H),5.44(s,1H),4.60(d,J=7.5Hz,1H),4.52(d,J=7.3Hz,1H),4.31(s,1H),4.18(d,J=2.8Hz,1H),3.90(t,J=10.7Hz,2H),3.80(s,3H),3.77-3.70(m,7H),3.56-3.44(m,8H),2.02(s,6H);13C NMR(150MHz,CHCl3):δ172.7,171.9,157.9,150.3,118.5,114.9,100.2,99.4,99.0,75.74,74.2,73.9,72.1,70.3,68.7,68.3,68.2,55.9,55.6,55.1,20.1;ESI-MS:m/z C29H44N2O17计算值692.2532;实验值715.2518(M+Na)+。
对甲氧基苯基-O-(4-O-乙酰基-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→2)-O-(2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→4)-α-D-甘露哌喃糖苷(19):将S22f(0.610g,0.421mmol)及10mL的乙二胺:nBuOH(1:4)的混合物在90℃下搅拌隔夜。随后蒸发挥发物且使粗产物与10mL Ac2O/吡啶(1:2)反应隔夜。使用高真空移除溶剂且通过急骤管柱层析(丙酮:甲苯,2/8,v/v)纯化产物。将产物溶解于10mL MeOH:H2O:HCOOH(6:3:1)中,添加Pd(OH)2(50重量%)且将反应混合物氢化隔夜。经由硅藻土过滤反应混合物且在真空中浓缩。通过Bio-Gel P-2(BIO-RAD)管柱层析,使用水作为溶离剂纯化残余物,且冻干产物,得到呈白色粉末状的19(0.210g,67%)。1H NMR(400MHz,D2O):δ7.01(d,J=9.2Hz,2H),6.90(d,J=9.8Hz,2H),5.40(s,1H),4.61(d,J=8.0Hz,1H),4.52(d,J=8.4Hz,1H),4.25(d,J=2.1Hz,1H),4.73(dd,J=1.2&7.2Hz,1H),3.92(d,J=12.3Hz,1H),3.74(s,3H),3.72-3.67(m,8H),3.60-3.41(m,7H),2.12(s,3H),2.03(s,6H);13C NMR(150MHz,D2O):δ174.67,174 17,172.91,154.43,149.58,118.53,118.37,114.81,101.48,99.70,96.04,77.51,76.00,75.75,73.50,73.39,71.74,71.06,70.93,69.57,69.34,68.04,60.58,60.47,60.00,55.55,55.39,55.13,22.24,21.95,20.17,20.03;ESI-MS:m/z C31H46N2O18计算值735.2818;实验值735.2780(M+H)+。
对甲氧基苯基-O-(4-O-乙酰基-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→2)-O-(2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→6)-α-D-甘露哌喃糖苷(20):将S22h(0.350g,0.241mmol)及10mL的乙二胺:nBuOH(1:4)的混合物在90℃下搅拌隔夜。随后蒸发挥发物且使粗产物与10mL Ac2O/吡啶(1:2)反应隔夜。使用高真空移除溶剂且通过急骤管柱层析(丙酮:甲苯,2/8,v/v)纯化产物。将产物溶解于10mL MeOH:H2O:HCOOH(6:3:1)中,添加Pd(OH)2(50重量%)且将反应混合物氢化隔夜。经由硅藻土过滤反应混合物且在真空中浓缩。通过Bio-Gel P-2(BIO-RAD)管柱层析,使用水作为溶离剂纯化残余物,且冻干产物,得到呈白色粉末状的20(0.120g,70%)。1H NMR(400MHz,D2O):δ7.08(d,J=8.4Hz,2H),6.91(d,J=8.2Hz,2H),5.38(s,1H),4.66(d,J=8.0Hz,1H),4.48(d,J=8.2Hz,1H),4.38-4.27(m,2H),4.19(t,J=2.8Hz,1H),4.09(d,J=12.2Hz,1H),3.98(dd,J=2.8及7.8Hz,1H),3.84(d,J=12.3Hz,2H),3.75(s,3H),3.67-3.33(m,11H),1.93(s,3H),1.91(s,3H),1.78(s,3H);13C NMR(150MHz,D2O):δ174.41,173.93,173.75,154.47,149.86,118.53,118.48,114.93,100.84,99.71,96.50,76.76,75.66,73.75,73.27,72.74,72.60,69.76,69.47,69.24,69.12,67.28,62.94,60.62,55.60,55.32,55.22,22.30,22.06,20.03;ESI-MS:m/z C31H46N2O18计算值735.2818;实验值735.2769(M+H)+。
制备线性模组.如流程S23中所描绘,线性模组的制备从接受体16的GlcNAc残基的酶促β-1,4-醣基化开始,从而形成LacNAc部分21。模组21随后经历α-1,3-海藻醣苷基转移酶、α-2,6-唾液酸转移酶、α-2,3-唾液酸转移酶及α-1,3-海藻醣苷基转移酶的作用,分别获得模组22、23、24及25。α-1,3海藻糖残基于GlcNAc上的存在限制了在相邻半乳糖上于3位及6位添加唾液酸。有趣的是,发现经α-2,3-唾液酸化的LacNAc 24是α-1,3-海藻醣苷基转移酶的受质,得到25,但经α-2,6-唾液酸化的LacNAc 23不是。更让吾人感到震惊的是,经α-1,2-海藻醣基化的LacNAc 26是α-2,6-唾液酸转移酶的受质,而非α-2,3-唾液酸转移酶的受质。FucTs及SiaTs的独特受质特异性让吾人能够建立一种快速获得更多样化类型的模组用于N-聚醣合成的方式。
流程S23|制备线性模组.i,UDP-半乳糖、β1,4-GalT;ii,GDP-海藻糖、α1,3-FucT;iii,CMP-Neu5Ac、α2,6-SiaT;iv,CMP-Neu5Ac、α2,3-SiaT;v,GDP-海藻糖、α1,2-FucT。
对甲氧基苯基-O-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖苷(21):通过使用一般程序4使化合物16(100mg,0.204mmol)半乳醣基化,得到呈非晶白色固体状的21(115mg,86%)。1H NMR(400MHz,D2O):δ7.10(d,J=8.8Hz,2H),6.96(d,J=8.1Hz,2H),5.44(s,1H),4.59(d,J=8.4Hz,1H),4.49(d,J=8.2Hz,1H),4.45(s,1H),4.00-3.75(m,17H),3.60(s,3H),2.01(s,3H);ESI-MS:m/zC27H41NO17计算值652.2447;实验值674.2262(M+H)+。
对甲氧基苯基-O-β-D-半乳哌喃糖基-(1→4)-[α-L-海藻哌喃糖基-(1→3)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→2)-α-D-甘露哌喃糖苷(22):通过使用一般程序5使化合物21(80mg,0.122mmol)海藻醣基化,在冻干之后得到呈白色固体状的22(82mg,84%)。1H NMR(400MHz,D2O):δ7.15(d,J=9.2Hz,2H),7.00(d,J=9.1Hz,2H),5.45(s,1H),5.12(d,J=4.1Hz,1H),4.67(d,J=7.8Hz,2H),4.44(d,J=8.4Hz,1H),4.05(s,1H),4.01(dd,J=3.2及7.8Hz,1H),3.98(t,J=10.2Hz,2H),3.80-3.70(m,6H),3.79(s,3H),3.70-3.40(m,11H),2.06(s,3H),1.18(d,J=6.4Hz,3H);ESI-MS:m/z C33H51NO21计算值797.7570;实验值820.2837(M+Na)+。
对甲氧基苯基-O-[5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖苷(23):通过使用一般程序3使化合物21(150mg,0.184mmol)进行α2,6-唾液酸化,在冻干之后得到呈白色固体状的23(169mg,90%)。1H NMR(400MHz,D2O):δ7.10(d,J=9.2Hz,2H),7.08(d,J=9.1Hz,2H),5.49(s,1H),4.64(d,J=8.1Hz),1H,4.40(d,J=8.2Hz,1H),4.26(t,J=2.1Hz,1H),4.01-3.80(m,7H),3.74(s,3H),3.66-3.58(m,20H),2.63(dd,J=4.4及12.8Hz,1H),2.07(s,3H),1.97(s,3H),1.68(t,1H);ESI-MS:m/zC38H58N2O25计算值942.3245;实验值941.3269(M-H)-。
对甲氧基苯基-O-[5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→3)-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖苷(24):通过使用一般程序3使化合物21(10mg,15.3μmol)进行α2,3-唾液酸化,在冻干之后得到呈白色固体状的24(11.5mg,80%)。1H NMR(400MHz,D2O):δ7.16(d,J=9.2Hz,2H),7.03(d,J=9.5Hz,2H),5.49(s,1H),4.67(d,J=8.4Hz,1H),4.56(d,J=8.4Hz,1H),4.32(t,J=2.1Hz,1H),4.15(dd,J=3.2&7.5Hz,1H),4.10-3.98(m,4H),3.95-3.85(m,6H),3.84(s,3H),3.80-3.50(m,13H),2.79(dd,J=4.8及12.1Hz,1H),2.09(s,3H),2.07(s,3H),1.85(t,1H);ESI-MS:m/z C38H58N2O25计算值942.3245;实验值941.3312(M-H)-。
对甲氧基苯基-O-[5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→3)-β-D-半乳哌喃糖基-(1→4)-[α-L-海藻哌喃糖基-(1→3)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→2)-α-D-甘露哌喃糖苷(25):通过使用一般程序5使化合物24(8mg,8.5μmol)海藻醣基化,在冻干之后得到呈白色固体状的25(6.5mg,70%)。1H NMR(400MHz,D2O):δ7.14(d,J=6.8Hz,2H),6.98(d,J=7.2Hz,2H),5.46(s,1H),5.12(d,J=4.2Hz,1H),4.67(d,J=8.4Hz,1H),4.30(t,J=2.3Hz,1H),4.10(dd,J=3.0&7.8Hz,1H),4.00-3.80(m,14H),3.79(s,3H),3.75-3.50(m,13H),2.75(dd,J=4.0及12.0Hz,1H),2.03(s,3H),2.01(s,3H),1.76(t,1H),1.17(d,3H,Fuc-Me);ESI-MS:m/z C44H68N2O2 9计算值1088.3857;实验值1087.3850(M-H)-。
对甲氧基苯基-O-[α-L-海藻哌喃糖基-(1→2)-β-D-半乳哌喃糖基]-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→2)-α-D-甘露哌喃糖苷(26):通过使用一般程序5使化合物21(3mg,4.6μmol)海藻醣基化,在冻干之后得到呈白色固体状的26(2.1mg,53%)。1H NMR(400MHz,D2O):δ7.17(d,J=7.2Hz,2H),7.01(d,J=10.1Hz,2H),5.49(s,1H),5.33(s,1H),4.67(d,J=8.2Hz,1H),4.56(d,J=8.2Hz,1H),4.33(m,1H),4.25(q,1H),3.98(dd,J=3.1及7.2Hz,1H),3.96(dd,J=1.8及7.2Hz,1H),3.85-3.50(m,20H),3.40(m,1H),1.97(s,3H),1.14(d,J=6.4Hz,3H);ESI-MS:m/z C33H51NO21计算值797.2954;实验值820.2922(M+Na)+。
对甲氧基苯基-O-[5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-[α-L-海藻哌喃糖基-(1→2)-β-D-半乳哌喃糖基]-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→2)-α-D-甘露哌喃糖苷(27):通过使用一般程序3使化合物26(2.0mg,2.7μmol)进行α2,6-唾液酸化,在冻干之后得到呈白色固体状的27(1.8mg,66%)。1H NMR(400MHz,D2O):δ7.08(d,J=9.5Hz,2H),6.91(d,J=9.2Hz,2H),5.49(s,1H),5.27(s,1H),4.59(d,J=8.2Hz,1H),4.47(d,J=8.1Hz,1H),4.22(bs,1H),4.12(d,J=6.8Hz,1H),4.00-3.49(m,30H),2.63(dd,J=3.2及12.0Hz,1H),2.01(s,3H),1.97(s,3H),1.67(t,1H),1.19(d,3H,Fuc-Me);ESI-MS:m/z C44H68N2O29计算值1088.3857;实验值1087.3814(M-H)-。
制备对称分支模组.
i,β-l,4GalT、UDP-Gal;ii,α1,3-海藻醣苷基转移酶、GDP-海藻糖;iii,α2,6-唾液酸转移酶、CMP-β-D-唾液酸;
化合物28及29:通过使用一般程序4使化合物17(10mg,14.3μmol)及18(10mg,14.3μmol)半乳醣基化,得到呈非晶白色固体状的28(10mg,71%)及29(9mg,61%)。
对甲氧基苯基-O-二-[β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→2),(1→4)-α-D-甘露哌喃糖苷(28):1H NMR(400MHz,D2O):δ7.11(d,J=9.2Hz,2H),6.95(d,J=8.5Hz,2H),5.44(s,1H),4.65(d,J=8.9Hz,1H),4.58(d,J=9.2Hz,1H),4.45(dd,J=3.8&9.2Hz,2H),4.30(t,J=2.1&6.5Hz,1H),4.18(dd,J=3.2&7.8Hz,1H),4.00(t,J=10.8Hz,2H),3.90(d,J=8.2Hz,2H),3.78(s,3H),3.75-3.64(m,20H),3.58-3.49(m,4H),2.02(s,3H),2.01(s,3H);ESI-MS:m/z C41H64N2O27计算值1016.9564;实验值1039.3607(M+Na)+。
对甲氧基苯基-O-二-[β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→2),(1→6)-α-D-甘露哌喃糖苷(29):1H NMR(400MHz,D2O):δ7.02(d,J=9.0Hz,2H),6.92(d,J=9.0Hz,2H),5.32(s,1H),4.55(d,J=4.3Hz,1H),4.41(d,J=8.2Hz,1H),4.34(dd,J=13.1,7.9Hz,2H),4.16(s,1H),4.00(d,J=11.0Hz,1H),3.843.95(m,2H),3.70-3.81(m,9H),3.49-3.68(m,16H),3.37-3.44(m,4H),1.94(s,3H),1.82(s,3H);ESI-MS:m/z C41H64N2NaO27计算值1039.3594;实验值1039.3551。
化合物30及31:通过使用一般程序3使化合物28(8mg,7.3μmol)及29(8mg,7.3μmol)唾液酸化,在冻干之后得到呈白色固体状的30(9mg,78%)及31(7.5mg,65%)。
对甲氧基苯基-O-二-[5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→2),(1→4)-α-D-甘露哌喃糖苷(30):1H NMR(400MHz,D2O):δ7.12(d,J=9.8Hz,2H),6.98(d,J=9.3Hz,2H),5.46(s,1H),4.63(d,J=8.4Hz,1H),4.58(d,J=8.2Hz,1H),4.42(d,J=7.8Hz,2H),4.32(t,J=3.2Hz,1H),4.20(dd,J=3.2&7.2Hz,1H,4.03-3.85(m,4H),3.82-3.40(m,43H),2.58(dd,2H,J=3.2&10.3Hz),2.05(s,3H),2.04(s,3H),2.02(s,3H),2.00(s,3H),1.53(m,2H);ESI-MS:m/z C63H98N4O43计算值1599.4620;实验值798.2775(M-H)2-。
对甲氧基苯基-O-二-[5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→2),(1→5)-α-D-甘露哌喃糖苷(31):1H NMR(600MHz,D2O):δ7.05(d,J=9.2Hz,2H),6.95(d,J=9.2Hz,2H),5.36(d,J=1.6Hz,1H),4.59(d,J=8.2Hz,1H),4.46(d,J=8.1Hz,1H),4.36(d,J=7.9Hz,1H),4.32(d,J=7.9Hz,1H),4.20(dd,J=3.4,1.8Hz,1H),4.06(d,J=10.3Hz,1H),3.86-3.98(m,4H),3.64-3.86(m,19H),3.53-3.62(m,13H),3.37-3.50(m,9H),2.58(ddd,J=12.0,4.4,2.1Hz,2H),1.98(s,3H),1.94(s,3H),1.94(s,3H),1.87(s,3H),1.63(td,J=12.2,8.6Hz,2H);ESI-MS:m/z C63H98N4NaO43计算值1621.5479;实验值1621.5473。
对甲氧基苯基-O-二-{β-D-半乳哌喃糖基-(1→4)-[α-L-海藻哌喃糖基-(1→3)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]}-(1→2),(1→4)-α-D-甘露哌喃糖苷(32):通过使用一般程序5使化合物28(15mg,14.7μmol)海藻醣基化,得到呈非晶白色固体状的32(14mg,73%)。1H NMR(400MHz,D2O):δ7.13(d,J=9.2Hz,2H),6.99(d,J=9.0Hz,2H),5.44(d,J=3.2Hz,1H),5.12(d,J=4.1Hz,1H),5.10(d,J=4.0Hz,1H),4.81(d,J=8.4Hz,2H),4.68(d,J=7.4Hz,1H),4.58(d,J=7.2Hz,1H),4.45(dd,J=3.2&8.4Hz,2H),4.31(t,J=3.2Hz,1H),4.20(dd,J=3.2&8.4Hz,1H),4.09-3.84(m,14H),3.82(s,3H),3.80-3.40(m,14H),2.06(s,6H),1.17(d,J=6.4Hz,6H);ESI-MS:m/z C53H84N2O35计算值1308.4855;实验值1309.4911(M+H)+。
对甲氧基苯基-O-二-{β-D-半乳哌喃糖基-(1→4)-[α-L-海藻哌喃糖基-(1→3)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]}-(1→2),(1→6)-α-D-甘露哌喃糖苷(33):通过使用一般程序5使化合物29(15mg,14.7μmol)海藻醣基化,得到呈非晶白色固体状的33(15.5mg,81%)。1H NMR(400MHz,D2O):δ7.17(d,J=8.2Hz,2H),7.06(d,J=8.0Hz,2H),5.42(s,1H),5.14(d,J=4.0Hz,1H),5.09(d,J=4.0Hz,1H),4.87-4.85(m,2H),4.60(d,J=7.2Hz,1H),4.56(q,2H),4.30(m,1H),4.15(d,J=12.2Hz,1H),4.06(dd,J=3.2&7.8Hz,1H),4.00-3.45(m,37),2.06(s,3H),1.95(s,3H),1.19(d,J=6.5Hz,6H);ESI-MS:m/zC53H84N2O35计算值1308.4855;实验值1331.3561(M+Na)+。
制备不对称分支模组.
流程S25|制备不对称模组.UDP-半乳糖、β1,4-GalT;ii,GDP-海藻糖α1,3-FucT;iii,CMP-Neu5Ac、α2,6-SiaT;vi,NaOH。
对甲氧基苯基-O-(4-O-乙酰基-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→2)-O-(β-D-半乳哌喃糖基-(1→4)-O-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→4)-α-D-甘露哌喃糖苷(34):使用一般程序4使化合物19(15mg,20.4μmol)半乳醣基化,得到呈非晶白色固体状的34(12.5mg,68%)。1H NMR(600MHz,D2O)δ7.01(d,J=9.2Hz,2H),6.88(d,J=9.2Hz,1H),5.34(d,J=1.8Hz,1H),4.56(d,J=8.5Hz,1H),4.45(d,J=8.1Hz,1H),4.37(d,J=7.7Hz,1H),4.20-4.33(m,2H),4.16-4.20(m,1H),4.07(dd,J=8.7,3.0Hz,1H),3.86-3.94(m,1H),3.81(d,J=3.4Hz,1H),3.31-3.78(m,21H),2.03(s,3H),1.93(s,3H),1.92(s,3H);ESI-MS:m/z C37H56N2NaO23计算值919.9166;实验值919.3156。
对甲氧基苯基-O-(4-O-乙酰基-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→2)-O-(β-D-半乳哌喃糖基-(1→4)-O-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→6)-α-D-甘露哌喃糖苷(S27a):通过使用一般程序4使化合物20(8mg,10.9μmol)半乳醣基化,得到呈非晶白色固体状的35(5mg,75%)。1H NMR(600MHz,D2O):δ7.12(d,J=9.1Hz,2H),7.01(d,J=9.8Hz,2H),5.40(s,1H),4.67(d,J=8.4Hz,1H),4.49(d,J=8.4Hz,1H),4.40(d,J=8.3Hz,2H),4.31(dd,J=3.1&7.8Hz,1H),4.21(t,J=3.1Hz,1H),4.08(d,J=12.3Hz,2H),4.00(dd,J=3.2&8.1Hz,1H),3.90-3.48(m,3H),3.80(s,3H),3.75-3.47(m,16H),2.15(s,3H),2.0(s,3H),1.98(s,3H);ESI-MS:m/z C37H56N2O23计算值896.3162;实验值919.3164(M+Na)+。
对甲氧基苯基-O-(4-O-乙酰基-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→2)-O-(5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-O-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→4)-α-D-甘露哌喃糖苷(36):通过使用一般程序3使化合物34(10mg,11.1μmol)进行α2,6-唾液酸化,在冻干之后得到呈白色固体状的36(11.2mg,84%)。1H NMR(600MHz,D2O):δ7.02(d,J=9.2Hz,2H),6.89(d,J=9.2Hz,2H),5.35(d,J=2.0Hz,1H),4.55(dd,J=18.4,8.4Hz,1H),4.48(d,J=7.9Hz,1H),4.17-4.37(m,4H),4.05-4.12(m,1H),3.29-3.97(m,36H),2.56(dd,J=12.4,4.7Hz,1H),2.04(s,3H),1.96(s,3H),1.92(s,3H),1.92(s,3H),1.60(t,J=12.2Hz,1H);ESI-MS:m/z C48H72N3O31计算值1186.4155;实验值1186.4175。
对甲氧基苯基-O-(4-O-乙酰基-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→2)-O-(5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-O-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→6)-α-D-甘露哌喃糖苷(37):通过使用一般程序3使化合物35进行α2,6-唾液酸化,在冻干之后得到呈白色固体状的37(5.1mg,77%)。1H NMR(600MHz,D2O):δ7.11(d,J=9.1Hz,2H),7.00(d,J=9.2Hz,2H),5.38(s,1H),4.50(d,J=8.4Hz,1H),4.36(d,J=8.2Hz,1H),4.22(t,J=2.1Hz,1H),4.10(dd,J=3.1&7.8Hz,1H),3.97-3.85(m,4H),3.77(s,3H),3.73-3.46(m,25H),2.63(dd,J=3.2及7.8Hz,1H),2.09(s,3H),2.01(s,3H),1.99(s,3H),1.94(s,3H),1.65(t,1H);ESI-MS:m/z C48H73N3O31计算值1187.4144;实验值1186.4133(M-H)-。
对甲氧基苯基-O-(β-D-半乳哌喃糖基-(1→4)-O-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→2)-O-(5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-O-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→4)-α-D-甘露哌喃糖苷(40):向化合物36(7mg,5.8μmol)于0.5mL H2O中的溶液中添加NaOH(9.4mg,23.6μmol)且搅拌4h。中和反应且用Bio-Gel P-2层析(溶离剂H2O)纯化产物,在冻干之后得到呈白色固体状的38(6.1mg,89%)。将化合物38(6mg,5.3μmol)及UDP半乳糖(6.4mg,10.6μmol)溶解于Tris缓冲液(25mM,pH 7.5)及MnCl2(20mM)中。添加GalT-1(150个单位)以达成5mM的最终聚醣浓度。将所得反应混合物在37℃下保温48h。使反应混合物离心且上清液经P2-Biogel凝胶过滤(溶离剂水)。将含有产物的部分合并且冻干,得到呈非晶白色固体状的40(5mg,73%)。1H NMR(400MHz,D2O)δ7.02(d,J=9.2Hz,2H),6.88(d,J=9.2Hz,2H),5.35(d,J=1.8Hz,1H),4.56(d,J=7.9Hz,1H),4.48(d,J=7.9Hz,1H),4.35(dd,J=9.6,7.7Hz,2H),4.27-4.30(m,2H),4.21-4.24(m,1H),4.02-4.20(m,6H),3.87-3.92(m,3H),3.39-3.85(m,28H),2.56(dd,J=12.4,4.6Hz,1H),1.96(s,3H),1.93(s,3H),1.92(s,3H),1.60(t,J=12.2Hz,1H);ESI-MS:m/z C52H80N3O35计算值1306.4578;实验值1306.4617。
对甲氧基苯基-O-(β-D-半乳哌喃糖基-(1→4)-O-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→2)-O-(5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→6)-O-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→4)-α-D-甘露哌喃糖苷(41):向化合物37(5mg,4.2μmol)于0.5mL H2O中的溶液中添加NaOH(6.7mg,16.8μmol)且搅拌4h。中和反应且用Bio-Gel P-2层析(溶离剂H2O)纯化产物,在冻干之后得到呈白色固体状的39(3.8mg,79%)。将化合物39(3.5mg,3.0μmol)及UDP半乳糖(3.9mg,6.1μmol)溶解于Tris缓冲液(25mM,pH 7.5)及MnCl2(20mM)中。添加GalT-1(150个单位)以达成5mM的最终聚醣浓度。将所得反应混合物在37℃下保温48h。使反应混合物离心且上清液经P2-Biogel凝胶过滤(溶离剂水)。将含有产物的部分合并且冻干,得到呈非晶白色固体状的41(3.3mg,75%)。1H NMR(400MHz,D2O):δ7.11(d,J=9.1Hz,2H),7.00(d,J=9.2Hz,2H),5.38(s,1H),4.64(d,J=8.1Hz,1H),4.50(d,J=8.2Hz,1H),4.23(d,J=8.3Hz,1H),4.38(d,J=8.0Hz,1H),4.22(t,J=2.1Hz,1H),4.10(d,J=12.2Hz,1H),4.01-3.95(m,4H),3.87-3.63(m,26H),3.52-3.45(m,8H),2.63(dd,J=2.1及7.8Hz,1H),2.63(dd,J=3.2及7.8Hz,1H),2.01(s,3H),1.99(s,3H),1.93(s,3H),1.67(t,1H);ESI-MS:m/zC52H81N3O35计算值1307.4567;实验值1306.4609(M-H)-。
对甲氧基苯基-O-(β-D-半乳哌喃糖基-(1→4)-O-[α-L-海藻哌喃糖基-(1→3)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→2)-O-(5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-O-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→4)-α-D-甘露哌喃糖苷(42):通过使用一般程序5使化合物40(2mg,1.52μmol)进行α1,3-海藻醣基化,得到呈非晶白色固体状的42(2mg,85%)。1H NMR(400MHz,D2O):δ7.16(d,J=8.8Hz,2H),7.06(d,J=8.1Hz,2H),5.45(s,1H),5.08(d,J=4.1Hz,1H),4.60(d,J=4.2Hz,1H),4.44(dd,J=3.5&7.9Hz,2H),4.30(bs,1H),4.15(d,J=8.4Hz,1H),4.09-3.40(m,40H),2.70(d,J=11.2Hz,1H),2.08(s,3H),2.05(s,3H),1.93(s,3H),1.74(t,1H),1.20(d,J=6.4Hz,3H);ESI-MS:m/z C58H91N3O39计算值1453.5245;实验值1452.5157(M-H)-。
对甲氧基苯基-O-(β-D-半乳哌喃糖基-(1→4)-O-[α-L-海藻哌喃糖基-(1→3)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→2)-O-(5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-O-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→6)-α-D-甘露哌喃糖苷(43):通过使用一般程序5使化合物41(4mg,3.05μmol)进行α1,3-海藻醣基化,得到呈非晶白色固体状的43(3.2mg,72%)。1H NMR(400MHz,D2O):δ7.21(d,J=8.2Hz,2H),7.06(d,J=8.0Hz,2H),5.74(s,1H),5.72(d,J=8.1Hz,2H),5.52(dd,J=3.1&7.2Hz,1H),5.40(s,1H),5.01(d,J=4.2Hz,1H),4.50-3.25(m,42H),2.50(dd,J=3.1&7.2Hz,1H),2.01(s,3H),1.99(s,6H),1.56(t,1H),1.05(d,3H,Fuc-Me);ESI-MS:m/z C58H91N3O39计算值1453.5245;实验值1452.5445(M-H)-。
对甲氧基苯基-O-(2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→2)-O-(β-D-半乳哌喃糖基-(1→4)-O-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→6)-α-D-甘露哌喃糖苷(44):向化合物35(20mg,22.3μmol)于0.5mL H2O中的溶液中添加NaOH(3.57mg,89.2μmol)且搅拌4h。中和反应且用Bio-Gel P-2层析(溶离剂H2O)纯化产物,在冻干之后得到呈白色固体状的44(15.3mg,80%)。1H NMR(400MHz,D2O):δ7.15(d,J=9.2Hz,2H),7.06(d,J=9.8Hz,2H),5.42(s,1H),4.67(d,J=8.2Hz,1H),4.54(d,J=8.0Hz,1H),4.47(d,J=8.2Hz,1H),4.32(t,J=2.1Hz,1H),4.10(d,J=12.2Hz,1H),4.09(dd,J=3.2&8.2Hz,1H),4.01-3.91(m,4H),3.85(s,3H),3.80-3.40(m,21H),2.03(s,3H),1.98(s,3H);ESI-MS:m/zC35H54N2O22计算值854.3125;实验值877.3062(M+Na)+。
对甲氧基苯基-O-(2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→2)-O-(β-D-半乳哌喃糖基-(1→4)-O-[α-L-海藻哌喃糖基-(1→3)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→6)-α-D-甘露哌喃糖苷(45):通过使用一般程序5使化合物44(8mg,9.3μmol)进行α1,3-海藻醣基化,在冻干之后得到呈白色固体状的45(6mg,66%)。1H NMR(400MHz,D2O):δ7.14(d,J=8.2Hz,2H),7.02(d,J=8.1Hz,2H),5.41(s,1H),5.07(d,J=4.0Hz,1H),4.64(d,J=8.2Hz,2H),4.58(d,J=7.4Hz,1H),4.44(d,J=8.5Hz,1H),4.28(t,J=2.1Hz,1H),4.10(d,J=12.2Hz,1H),4.05(dd,J=3.1&7.2Hz,1H),3.95-3.85(m,8H),3.82(s,3H),3.80-3.33(m,17H),2.04(s,3H),1.93(s,3H),1.17(d,J=6.8Hz,3H);ESI-MS:m/zC41H64N2O26计算值1000.2356;实验值1023.3599(M+Na)+。
对甲氧基苯基-O-(β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→2)-O-(β-D-半乳哌喃糖基-(1→4)-O-[α-L-海藻哌喃糖基-(1→3)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→6)-α-D-甘露哌喃糖苷(46):通过使用一般程序4使化合物45(7mg,7.2μmol)发生β1,4-半乳醣基化,得到呈非晶白色固体状的46(6.5mg,80%)。1H NMR(400MHz,D2O):δ7.16(d,J=9.4Hz,2H),7.05(d,J=9.3Hz,2H),5.43(s,1H),5.09(d,J=4.2Hz,1H),4.83(d,J=7.5Hz,1H),4.78(d,J=7.2Hz,1H),4.58(d,J=8.2Hz,1H),4.48(d,J=9.5Hz,1H),4.42(d,J=8.5Hz,1H),4.30(dd,J=2.1&3.4Hz,1H),4.14(s,1H),4.09-3.92(m,14H),3.85(s,3H),3.84-3.50(m,17H),2.06(s,3H),1.95(s,3H),1.20(d,J=6.5Hz.3H);ESI-MS:m/z C47H74N2O31计算值1162.4309;实验值1185.4076(M+Na)+。
对甲氧基苯基-O-(β-D-半乳哌喃糖基-(1→4)-O-[α-L-海藻哌喃糖基-(1→3)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→6)-O-(5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯-(2→6)-β-D-半乳哌喃糖基-(1→4)-O-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→2)-α-D-甘露哌喃糖苷(47):通过使用一般程序3使化合物46(5mg,4.31μmol)进行α2,6-唾液酸化,得到呈非晶白色固体状的47(5.2mg,83%)。1H NMR(400MHz,D2O):δ7.08(d,J=8.1Hz,2H),6.97(d,J=8.0Hz,2H),5.48(s,1H),5.10(d,J=5.1Hz,1H),4.50(d,J=7.2Hz,1H),4.43(dd,J=3.1&8.2Hz,2H),4.32(bs,1H),4.08(d,J=8.1Hz,1H),4.09-3.40(m,41H),2.60(dd,J=3.2&9.2Hz,1H),1.97(s,3H),1.95(s,3H),1.90(s,3H),1.54(t,1H),1.18(d,J=6.4Hz,3H);ESI-MS:m/z C58H91N3O39计算值1453.5245;实验值1452.5101(M-H)-。
以化学酶促方式制备的模组的化学衍生化.随后在Ac2O/吡啶存在下使通过化学酶促方式产生的模组21及22全乙酰化。随后使用硝酸铈铵裂解还原端对甲氧基苯基醚保护且将游离羟基改为氟基,获得供体50及51(流程S28)。
流程S26|试剂及条件.i,Ac2O、吡啶,室温,隔夜;ii,(1)CAN、ACN:甲苯:H2O,(2)DAST、CH2Cl2,-30℃。CAN:硝酸铈铵;DAST:三氟化二乙基胺基硫。
对甲氧基苯基-O-[2,3,4,6-O-四乙酰基-β-D-半乳哌喃糖基]-(1→4)-[3,6-O-二乙酰基-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→2)-3,4,6-O-三乙酰基-α-D-甘露哌喃糖苷(48):在0℃下,向21(0.230g,0.360mmol)于10mL吡啶中的溶液中添加乙酸酐(6mL)且在室温下搅拌隔夜。随后浓缩反应混合物,用50mL的CH2Cl2稀释且用饱和NaHCO3萃取。蒸发经合并的有机层且通过硅胶管柱层析(0%→20%丙酮/甲苯)纯化产物,得到所要48(0.260g,72%)。TLC(丙酮:甲苯=3/7,v/v):Rf=0.34;1H NMR(400MHz,CHCl3):δ7.00(d,J=9.1Hz,2H),6.82(d,J=9.1Hz,2H),5.74(d,J=8.7Hz,1H),5.33-5.35(m,2H),5.27-5.30(m,2H),5.22(dd,J=9.6,8.2Hz,1H),5.10(dd,J=10.5,7.9Hz,1H),4.96(dd,J=10.5,3.4Hz,1H),4.72(d,J=7.5Hz,1H),4.47(d,J=7.9Hz,1H),4.41(dd,J=11.8,2.7Hz,1H),4.31(t,J=2.2Hz,1H),3.98-4.21(m,6H),3.81-3.91(m,2H),3.72-3.79(m,4H),3.63(ddd,J=8.6,5.6,2.7Hz,1H),2.14(s,3H),2.11(s,3H),2.08(s,3H),2.06(s,2H),2.03(s,3H),2.03(s,6H),2.02(s,3H),1.96(s,6H);ESI-MS:m/z C45H59NO26计算值1029.9480;实验值1030.4587(M+H)+。
[2,3,4,6-O-四乙酰基-β-D-半乳哌喃糖基]-(1→4)-[3,6-O-二乙酰基-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→2)-3,4,6-O-三乙酰基-α-D-甘露哌喃醣苷氟化物(50):向化合物48(0.200g,0.203mmol)于10mL的乙腈:甲苯:H2O(4:2:1)中的溶液中添加硝酸铈铵(0.2g,0.554mmol)。在室温下搅拌所得反应混合物3h。用EtOAc(50mL)稀释反应物且用H2O(30×2mL)及盐水(30mL)洗涤。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→25%丙酮/甲苯)纯化产物,得到呈泡沫状的-OH化合物(0.120g)。在-30℃下,将残余物(0.120g,0.130mmol)溶解于CH2Cl2(10mL)中。随后,缓慢地添加DAST(34μL,0.260mmol),且将所得反应混合物搅拌1h。当TLC(丙酮:甲苯,3/7)指示产物形成且起始物质耗尽时,用NaHCO3水溶液淬灭反应。用NaHCO3水溶液(2×30mL)及盐水(20mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过硅胶管柱层析(0%→20%丙酮/甲苯)纯化残余物,得到呈白色固体状的50(0.080g,51%,历经2个步骤)。TLC(丙酮:甲苯=3/7,v/v):Rf=0.34;1H NMR(600MHz,CHCl3):δ5.50(d,J=49.6Hz,1H),5.30(d,J=3.8Hz,1H),2.26(t,J=7.8Hz,1H),5.05-4.89(m,4H),4.55(d,J=8.4Hz,1H),4.40(d,J=8.2Hz,1H),4.32(s,2H),4.18(dd,J=3.2及7.8Hz,1H),4.05-4.01(m,6H),3.81(t,J=7.8Hz,1H),3.71(t,J=7.1Hz,1H),3.50(t,J=3.1Hz,1H),2.29(s,3H),2.08(s,3H),2.07(s,3H),2.06(s,3H),2.02(s,3H),1.99(s,3H),1.98(s,3H),1.96(s,3H),1.91(s,3H),1.82(s,3H);13C NMR(150MHz,CHCl3):171.29,171.10,170.66,170.56,170.35,170.30,169.50,129.21,128.40,125.47,106.54,104.31,101.05,100.31,76.07,72.82,72.64,72.36,71.16,70.95,70.71,69.41,69.28,66.86,64.92,62.38,62.13,61.04,53.12,23.13,21.64,21.22,20.95,20.87,20.77,20.68,20.62;ESI-MS:m/z C38H52FNO24计算值925.8145;实验值925.8354(M+H)+。
对甲氧基苯基-O-[2,3,4,6-O-四乙酰基-β-D-半乳哌喃糖基]-(1→4)-[2,3,4-O-三乙酰基-α-L-海藻哌喃糖基-(1→3)-3,6-O-二乙酰基-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→2)-3,4,6-O-三乙酰基-α-D-甘露哌喃糖苷(49):在0℃下,向22(0.100g,0.125mmol)于6mL吡啶中的溶液中添加乙酸酐(4mL)且在室温下搅拌隔夜。随后浓缩反应混合物,用50mL的CH2Cl2稀释且用饱和NaHCO3萃取。蒸发经合并的有机层且通过硅胶管柱层析(0%→10%丙酮/CH2Cl2)纯化产物,得到所要49(0.135g,85%)。TLC(丙酮:CH2Cl2=1/9,v/v):Rf=0.31;1H NMR(400MHz,CHCl3):δ7.00(d,J=6.8Hz,2H),6.83(d,J=6.8Hz,2H),5.43(d,J=3.2Hz,1H),4.42(d,J=3.2Hz,1H),3.98(d,J=2.1Hz,2H),5.25(dd,J=3.2&8.2Hz,2H),5.19(dd,J=3.8&8.5Hz,2H),5.10(t,J=9.8Hz,2H),4.89(d,J=6.3Hz,1H),4.85(d,J=2.2Hz,1H),4.70(d,J=9.8Hz,2H),4.61(d,J=1.2Hz,1H),4.40(dd,J=3.2&7.2Hz,1H),4.31(m,3H),4.22-3.89(m,6H),3.80(m,2H),3.78(s,3H),3.60(m,1H),2.10(s,3H),2.08(s,3H),2.08(s,3H),2.02(s,3H),2.00(s,3H),1.99(s,3H),1.98(s,3H),1.96(s,3H),1.93(s,3H),1.90(s,3H),1.89(s,3H),1.88(s,3H),1.11(d,3H);ESI-MS:m/zC55H73NO32计算值1259.4116;实验值1282.3974(M+Na)+。
[2,3,4,6-O-四乙酰基-β-D-半乳哌喃糖基]-(1→4)-[2,3,4-O-三乙酰基-α-L-海藻哌喃糖基-(1→3)-3,6-O-二乙酰基-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→2)-3,4,6-O-三乙酰基-α-D-甘露哌喃醣苷氟化物(50):向化合物49(0.090g,0.071mmol)于7mL的乙腈:甲苯:H2O(4:2:1)中的溶液中添加硝酸铈铵(0.121g,0.142mmol)。在室温下搅拌所得反应混合物3h。用EtOAc(40mL)稀释反应物且用H2O(10×2mL)及盐水(10mL)洗涤。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→15%丙酮/CH2Cl2)纯化产物,得到呈泡沫状的-OH化合物(0.065g)。在-30℃下,将残余物(0.060g,0.052mmol)溶解于CH2Cl2(5mL)中。随后,缓慢地添加DAST(11.6μL,0.104mmol),且将所得反应混合物搅拌1h。当TLC(丙酮:CH2Cl2,1/9)指示产物形成且起始物质耗尽时,用NaHCO3水溶液淬灭反应。用NaHCO3水溶液(2×10mL)及盐水(10mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过硅胶管柱层析(0%→10%丙酮/CH2Cl2)纯化残余物,得到呈白色固体状的51(0.055g,67%,历经2个步骤)。TLC(丙酮:CH2Cl2=1/9,v/v):Rf=0.44;1H NMR(400MHz,CHCl3):δ5.55(d,J=52.1Hz,1H),5.40(dt,J=3.2&7.8Hz,3H),5.21(dd,J=2.1&8.2Hz,2H),5.18-5.04(m,3H),5.0(bs,1H),4.60(s,2H),4.40(dd,J=5.6&8.5Hz,1H),4.38-4.00(m,7H),3.80(m,2H),3.60(m,1H),2.16(s,3H),2.14(s,3H),2.13(s,3H),2.10(s,3H),2.09(s,3H),2.07(s,3H),2.06(s,3H),2.06(s,3H),2.05(s,3H),2.04(s,3H),2.00(s,3H),1.95(s,3H),1.94(s,3H),1.17(d,J=6.4Hz,3H);ESI-MS:m/z C48H66FNO30计算值1155.0314;实验值1178.3491(M+Na)+。
对核心三醣的化学醣基化:在此阶段,吾人致力于研究氟化物供体50与51对核心三醣15的偶合效率。如流程S29中所描绘,在AgOTf/Cp2HfCl2存在下,50对核心的3-O位置的立体选择性结合以56%产率得到预期六醣52。使用催化对甲苯磺酸还原性裂解亚苄基环,得到4,6-二醇53。利用其反应性,随后用51使53的一级羟基选择性醣基化,以中等产率得到经完全保护的十醣54。最后,整体脱除保护基,得到所要经选择性海藻醣基化的二触角复合型聚醣55。
流程S27|试剂及条件.i,AgOTf、Cp2HfCl2、甲苯,MS,0℃至室温;ii,p-TSA、乙腈,室温;iii,(1)LiOH、1,4-二恶烷:H2O;90℃,隔夜;(2)Ac2O、吡啶,隔夜;(3)NaOMe、MeOH,隔夜;(4)Pd(OH)2、MeOH:H2O:HCOOH(5:3:2)、H2。
AgOTf:三氟甲磺酸银;Cp2HfCl2:二氯化双(环戊二烯基)铪;MS:分子筛。
5-迭氮基戊基-O-{[2,3,4,6-O-四乙酰基-β-D-半乳哌喃糖基]-(1→4)-[3,6-O-二乙酰基-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→2)-[3,4,6-O-三乙酰基-α-D-甘露哌喃糖基]}-(1→3)-[2-O-乙酰基-4,6-O-亚苄基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基)-(1→4)-O-3,6-二-O-苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖苷(52).将三氟甲磺酸银(0.039g,0.155mmol)、二氯化双(环戊二烯基)铪(0.041g,0.108mmol)及经活化的分子筛于无水甲苯(3mL)中的混合物在室温下搅拌1h。随后将反应混合物冷却至0℃,添加供体50(0.043g,0.046mmol)及接受体15(0.045g,0.031mmol)于3mL甲苯中的溶液。将混合物在室温下搅拌3h,用Et3N淬灭,用CH2Cl2稀释且经由硅藻土过滤。用NaHCO3水溶液(2×20mL)及盐水(20mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→10%丙酮/CH2Cl2)纯化残余物,得到呈无色泡沫状的52(0.051g,70%)。TLC:(丙酮:CH2Cl2=1.5/8.5,v/v):Rf=0.46;1H NMR(400MHz,CHCl3):δ7.46-7.16(m,25H),5.41(s,1H),5.32(d,J=9.8Hz,1H),5.29(d,J=7.2Hz,1H),5.20(t,J=10.2Hz,1H),5.09(t,J=10.3Hz,1H),4.93-4.87(m,5H),4.82-4.57(m,5H),4.50(d,J=12.2Hz,2H),4.41-4.22(m,4H),4.13-3.56(m,8H),3.43-3.36(m,4H),3.20(t,J=10.3Hz,4H),3.19(t,J=10.2Hz,1H),2.20(s,3H),2.18(s,3H),2.13(s,3H),2.06(s,3H),2.05(s,3H),2.04(s,3H),2.03(s,3H),1.98(s,3H),1.96(s,3H),1.90(s,3H),1.81(s,3H),1.58-1.51(m,4H),1.40-1.34(m,2H);13C NMR(150MHz,CHCl3):δ171.28,170.80,170.69,170.49,170.43,170.36,169.97,169.68,169.41,154.30,154.13,138.83,138.18,137.44,131.28,129.25,129.11,128.91,128.63,128.54,128.37,128.18,128.10,128.00,127.79,127.71,126.47,102.55,101.37,100.84,100.44,98.60,95.82,76.07,74.74,74.61,74.51,74.19,73.83,73.59,72.35,71.24,70.95,70.79,70.03,69.62,69.52,69.37,68.66,68.42,66.92,66.41,65.51,62.42,62.02,61.15,57.88,57.23,53.60,51.58,29.27,28.82,23.46,23.29,21.10,20.96,20.82;ESI-MS:m/z C104H126Cl6N6O41计算值2360.8510;实验值2361.6131(M+H)+。
5-迭氮基戊基-O-{[2,3,4,6-O-四乙酰基-β-D-半乳哌喃糖基]-(1→4)-[3,6-O-二乙酰基-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→2)-[3,4,6-O-三乙酰基-α-D-甘露哌喃糖基]}-(1→3)-[2-O-乙酰基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基)-(1→4)-O-3,6-二-O-苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖苷(53):向52(0.040g,0.016mmol)于乙腈:MeOH 2/1(3mL)中的溶液中添加对甲苯磺酸单水合物(0.001g,0.008mmol)且将所得反应混合物在室温下搅拌5h。通过添加Et3N淬灭反应且在真空中浓缩。通过急骤管柱层析(0%→15%丙酮/CH2Cl2)纯化残余物,得到二醇53(0.022g,57%)。TLC:(丙酮:CH2Cl2=1.5/8.5,v/v):Rf=0.32;1H NMR(400MHz,CDCl3):δ7.43-7.17(m,20H),5.33(d,J=3.2Hz,1H),5.25(d,J=3.2Hz,1H),5.19-5.04(m,4H),4.98(d,J=3.2Hz,1H),4.95(d,J=3.1Hz,1H),4.94-4.89(m,2H),4.72-4.55(m,8H),4.50-4.28(m,9H),4.20-3.30(m,30H),3.20(t,5H),2.15(s,3H),2.14(s,3H),2.12(s,3H),2.09(s,3H),2.07(s,3H),2.06(s,3H),2.04(s,3H),2.03(s,3H),2.02(s,3H),2.01(s,3H),2.00(s,3H),1.95(s,3H),1.94(s,3H),1.56-1.51(m,4H),1.40-1.34(m,2H);ESI-MS:m/z C97H122Cl6N6O43计算值2272.7420;实验值2295.5522(M+Na)+。
5-迭氮基戊基-O-{[2,3,4,6-O-四乙酰基-β-D-半乳哌喃糖基]-(1→4)-[3,6-O-二乙酰基-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→2)-[3,4,6-O-三乙酰基-α-D-甘露哌喃糖基]}-(1→3)-{2,3,4,6-O-四乙酰基-β-D-半乳哌喃糖基]-(1→4)-[2,3,4-O-三乙酰基-α-L-海藻哌喃糖基-(1→3)-3,6-O-二乙酰基-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→2)-3,4,6-O-三乙酰基-α-D-甘露哌喃糖基}-(1→6)-[2-O-乙酰基-β-D-甘露哌喃糖基-(1→4)-O-(3,6-二-O-苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖基)-(1→4)-O-3,6-二-O-苄基-2-去氧-2-(2,2,2-三氯乙氧基)羰基胺基-β-D-葡萄哌喃糖苷(53):将三氟甲磺酸银(0.011g,44.1μmol)、二氯化双(环戊二烯基)铪(0.012g,30.8μmol)及经活化的分子筛于无水甲苯(3mL)中的混合物在室温下搅拌1h。随后将反应混合物冷却至0℃,添加供体50(0.015g,13.2μmol)及接受体15(0.020g,8.80μmol)于2mL甲苯中的溶液。将混合物在室温下搅拌5h,用Et3N淬灭,用CH2Cl2稀释且经由硅藻土过滤。用NaHCO3水溶液(2×10mL)及盐水(20mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过急骤管柱层析(0%→15%丙酮/CH2Cl2)纯化残余物,得到呈无色泡沫状的52(0.010g,34%)。TLC:(丙酮:CH2Cl2=1.5/8.5,v/v):Rf=0.41;1H NMR(400MHz,CHCl3):δ7.48-7.20(m,20H),5.40-4.51(m,24H),4.50-4.00(m,10H),3.98-3.50(m,30H),3.49-3.10(m,20H),2.18(s,3H),2.16(s,3H),2.15(s,3H),2.14(s,3H),2.13(s,3H),2.12(s,3H),2.11(s,3H),2.10(s,3H),2.09(s,3H),2.07(s,3H),2.06(s,3H),2.05(s,3H),2.04(s,3H),2.03(s,3H),2.02(s,3H),2.01(s,3H),2.00(s,3H),1.99(s,3H),1.97(s,3H),1.95(s,3H),1.94(s,3H),1.93(s,3H),1.33-1.23(m,4H),1.20-1.15(m,2H);ESI-MS:m/zC145H187Cl6N7O73计算值3408.7670;实验值1176.3855(M+K)3+。
5-胺基戊基-β-D-半乳哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→2)-α-D-甘露哌喃糖基]-(1→3),-[β-D-半乳哌喃糖基-(1→4)-(α-L-海藻哌喃糖基-(1→3)-2-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基)-(1→2)-α-D-甘露哌喃糖基]-(1→6)-β-D-甘露哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基-(1→4)-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖苷(55):将54(0.010g,2.9μmol)及氢氧化锂(0.005g,50重量%)于2mL的1,4-二恶烷:H2O(4:1)中的混合物在90℃下搅拌隔夜。随后蒸发挥发物且使粗产物与3mL Ac2O:吡啶(1:2)反应隔夜。使用高真空移除溶剂且通过C18凝胶管柱层析(MeOH:H2O作为溶离剂)纯化产物。使用MeOH(3mL)中的甲醇钠使产物去乙酰化隔夜。使用IR-120中和反应混合物,过滤且在真空中浓缩。通过C18凝胶管柱层析(MeOH:H2O作为溶离剂)纯化残余物。将产物溶解于3mL MeOH:H2O:HCOOH(6:3:1)中,添加Pd(OH)2(50重量%)且将混合物氢化隔夜。经由硅藻土过滤反应混合物且在真空中浓缩。通过Bio-Gel P-2(BIO-RAD)管柱层析,使用水作为溶离剂纯化残余物。冻干产物,得到呈白色粉末状的所要寡醣55(0.002g,36%)。TLC:(n-BuOH:AcOH:H2O=1/1/1,v/v):Rf=0.32;1H NMR(400MHz,D2O):δ5.15(d,J=4.2Hz,1H),4.94(s,1H),4.60(m,3H),4.45(q,3H),4.30(s,1H),4.21(d,J=2.1Hz,1H),4.13(d,J=2.0Hz,1H),4.05-3.40(m,58H),3.01(t,2H,连接基团),2.10(s,3H,-Ac),2.07(s,3H,-Ac),2.06(s,3H,-Ac),2.05(s,3H,-Ac),1.76-1.58(m,4H,连接基团),1.45-1.36(m,2H,连接基团),1.21(d,J=6.5Hz,3H,Fuc-Me);13C NMR(100MHz,CHCl3):δ174.6,174.4,102.9,101.8,101.0,99.4,99.1,98.5,96.8,80.3,79.6,79.3,78.4,76.4,75.3,75.2,74.7,74.6,74.5,74.3,73.5,72.8,72.4,71.9,71.0,70.9,70.0,69.5,69.4,69.1,68.5,68.3,67.7,67.2,66.7,65.8,61.4,60.9,59.6,59.5,55.6,54.9,54.8,39.3,28.0,26.3,22.3,22.2,22.0,15.3;ESI-MS:m/z C73H125N5O50计算值1871.7545;实验值1872.7477(M+H)+。
唾液酸化模组的化学衍生化.唾液酸化触角的制备尤其重要,此是因为与唾液酸化学有关的复杂性。在确立了非唾液酸化模组的合成方案(流程S28)之后,吾人将我们的注意力转向唾液酸化模组22。使羧酸官能性在三甲基硅烷基重氮甲烷存在下酯化,且不影响游离羟基。随后,使用乙酸酐及吡啶进行全乙酰化,得到S30a。全乙酰化步骤允许在移除变旋异构基团之前纯化寡醣物质。最后,进行还原端转化,形成变旋异构氟化物供体S30b。
流程S28|试剂及条件.i,(1)三甲基硅烷基重氮甲烷、MeOH;(2)Ac2O、吡啶,室温,隔夜;ii,(1)CAN、ACN:甲苯:H2O,(2)DAST、CH2Cl2,-30℃,34%,历经2个步骤。
对甲氧基苯基-O-[3,7,8,9-O-四乙酰基-5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯]-(2→6)-O-[2,3,4,6-O-四乙酰基-β-D-半乳哌喃糖基]-(1→4)-[3,6-O-二乙酰基-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→2)-3,4,6-O-三乙酰基-α-D-甘露哌喃糖苷(S28a):向化合物23(30mg,0.031mmol)于MeOH(3mL)中的溶液中添加三甲基硅烷基重氮甲烷(1M己烷溶液,0.046mL,0.31mmol)。所得反应混合物在室温下搅拌隔夜。通过NMR及质量证实起始物质完全耗尽。将反应混合物浓缩至干燥且通过C18(溶离剂MeOH:H2O)纯化。TLC:(正丁醇:H2O:乙酸=2/1/1,v/v):Rf=0.45;1H NMR(400MHz,MeOD):δ7.14(d,J=9.5Hz,2H),6.97(d,J=9.8Hz,2H),5.50(s,1H),4.68(d,J=8.5Hz),1H,4.45(d,J=8.0Hz,1H),4.36(s,1H),4.20-3.90(m,10H),3.84(s,3H),3.76-3.58(m,17H),3.51(s,3H),2.66(dd,J=3.8及11.5Hz,1H),2.00(s,3H),1.96(s,3H),1.60(t,1H);ESI-MS:m/z C39H60N2O25计算值942.3245;实验值987.3033(M+Na)+。
将甲酯(0.025mg,0.025mmol)于吡啶(2mL)及乙酸酐(1.5mL)中的溶液在室温下搅拌隔夜。随后将反应物浓缩至干燥,溶解于二氯甲烷中,用饱和NaHCO3溶液萃取,干燥且浓缩。通过C18管柱(MeOH:水溶离剂)纯化产物,得到呈白色固体状的S28a(0.030mg,74%)。TLC:(丙酮:CH2Cl2 4/6,v/v):Rf=0.42;1H NMR(400MHz,MeOD):δ7.07(d,J=8.5Hz,2H),6.88(d,J=8.9Hz,2H),5.45(s,1H),5.40(d,J=7.2Hz,1H),5.36(t,J=10.3Hz,1H),5.30(d,J=7.5Hz,1H),5.28(d,J=7.0Hz,1H),5.26(s,1H),5.23(s,1H),5.19(d,J=8.6Hz,1H),5.17(t,J=10.3Hz,1H),5.11-5.07(m,5H),5.00-4.95(m,3H),4.70(dd,J=4.2&7.8Hz,2H),4.46-4.35(m,4H),4.30-4.13(m,3H),4.08-3.97(m,8H),3.76(s,3H),3.75(s,3H),3.60-3.56(m,3H),2.58(dd,J=4.2&8.5Hz,1H),2.13(s,3H),2.12(s,3H),2.11(s,3H),2.10(s,6H),2.07(s,3H),2.05(s,6H),2.02(s,6H),1.99(s,3H),1.98(s,3H),1.91(s,6H),1.84(t,J=10.2Hz,1H);ESI-MS:m/z C63H84N2O37计算值1461.3420;实验值1486.4864(M+2H+Na)+。
对甲氧基苯基-O-[3,7,8,9-O-四乙酰基-5-乙酰胺基-3,5-二去氧基-D-甘油-α-D-半乳糖-2-壬酮哌喃糖苷酸酯]-(2→6)-O-[2,3,4,6-O-四乙酰基-β-D-半乳哌喃糖基]-(1→4)-[3,6-O-二乙酰基-2-乙酰胺基-2-去氧-β-D-葡萄哌喃糖基]-(1→2)-3,4,6-O-三乙酰基-α-D-甘露哌喃醣苷氟化物(S28b):向化合物S28a(0.020mg,0.013mmol)于2mL的乙腈:甲苯:H2O(4:2:1)中的溶液中添加硝酸铈铵(0.037g,0.065mmol)。在室温下搅拌所得反应混合物3h。用EtOAc(10mL)稀释反应物且用H2O(10×2mL)及盐水(10mL)洗涤。经Na2SO4干燥有机层且在真空中浓缩。通过C18管柱层析(MeOH:H2O溶离剂)纯化产物,得到1-OH化合物(11mg)。在-30℃下,将残余物(10mg,0.007mmol)溶解于CH2Cl2(5mL)中。随后,缓慢地添加DAST(3μL,0.021mmol),且将所得反应混合物搅拌1h。当TLC(丙酮:CH2Cl2,4/6)指示产物形成且起始物质耗尽时,用NaHCO3水溶液淬灭反应。用NaHCO3水溶液(2×5mL)及盐水(5mL)溶液洗涤滤液。经Na2SO4干燥有机层且在真空中浓缩。通过C18管柱层析(MeOH:H2O溶离剂)纯化残余物,得到S30b(0.007g,38%,历经2个步骤)。TLC(丙酮:CH2Cl2=6/6,v/v):Rf=0.24;1H NMR(400MHz,CHCl3):δ5.99(d,J=10.2Hz,1H),5.50(d,J=52.2Hz,1H),5.39(d,J=10.3Hz,1H),5.33-5.17(m,7H),4.87-4.81(m,2H),4.61(d,J=10.2Hz,1H),4.52(d,J=10.3Hz,1H),4.40-4026(m,5H),4.21-3.83(m,4H),3.70(s,3H),3.49-3.47(m,2H),2.54(dd,J=3.8&8.1Hz,1H),2.19(s,6H),2.17(s,6H),2.14(s,6H),2.10(s,3H),2.05(s,6H),2.02(s,6H),1.98(s,3H),1.97(s,6H),1.23(t,J=10.1Hz,1H);ESI-MS:m/z C56H77FN2O35计算值1357.2094;实验值1339.3978(M+Na)+。
聚醣阵列分析
微阵列制造
a)制造经N-羟基丁二酰亚胺涂布的玻璃载片:所有单价聚醣(G1-33)个别地以10mM浓度制备且充当母体溶液,用印刷缓冲液稀释母体溶液以制备工作溶液。通过机器人顶针(SMP3:TeleChem International)印刷(BioDot Cartesian Technologies)微阵列,将约0.6nL于印刷缓冲液中的浓度为100μM的含胺聚醣自384孔盘沈积至涂有NHS的玻璃载片上。使经印刷的载片在80%湿度的氛围中反应一小时,随后干燥隔夜。这些载片是设计成一个载片有16个网格,且在使用之前在室温下将其储存于干燥器中。在结合分析之前,用阻断溶液阻断这些载片3h。随后在使用之前用PBST-BSA缓冲液洗涤载片。除非另外说明,否则试剂自商业供应商获得且不经纯化即使用。所有水溶液皆由经蒸馏的去离子水制备,所述去离子水经Milli-Q纯化***过滤且经由0.2μm过滤器无菌过滤。实验中所用的缓冲液包括印刷缓冲液(pH 8.5,300mM磷酸盐缓冲液,含有0.05%(v/v)Tween-20)、阻断缓冲液(于PBS中的superblock阻断缓冲液,Pierce)、洗涤缓冲液(PBST缓冲液;PBS及0.05%Tween-20)及结合缓冲液(PBST-BSA缓冲液;PBST缓冲液及3%BSA)。印刷缓冲液、阻断缓冲液及结合缓冲液均在使用之前新鲜制备。
b)制造经氧化铝涂布的玻璃载片.氧化铝玻璃基板提供能够通过质谱及萤光扫描评定的优点。另外,发现这些基板上的糖-蛋白质结合的萤光强度比在透明玻璃载片15上还要灵敏。许多报导描述了阳极化氧化铝的微米至奈米细孔结构的表面。然而,仅一些文章提及氧化铝16在表面阳极化处理期间的初始平面层。在AAO玻璃基板的研发过程中,使用电脑实验设计程式(Design Expert 8.0)使聚醣微阵列17中所用的表面阳极化氧化铝的平面层最佳化。
如图S4中所示,由实验研究院(National Applied Research Laboratories)仪器科技研究中心(Instrument Technology Research Center)的薄膜技术分部(Thin FilmTechnology Division)(Hsinchu Science Park,Hsinchu,Taiwan,R.O.C.)使用电子束VDP涂布机制造较大的、上面涂有约300nm铝的玻璃载片(1×75.5×24.5mm)。在Al涂布之后,紧接着包装载片(每容器一个载片)且将其真空密封于气密式层压箔袋中直至进行表面阳极化的时刻。涂有铝的玻璃载片的表面阳极化在4℃腔室中经由于0.3M草酸水溶液中的电化学反应内部进行。通过电压及反应时间控制阳极化反应。在恰当的反应条件下,可以在涂有铝的玻璃载片的顶部一致地生成一层约50至65nm(厚度)的光滑阳极化氧化铝(AAO)。不同于含孔的习知阳极氧化铝表面,如图S5A中所示的AFM图像,吾人研发出一种Rsm表面粗糙度(2.3nm)类似于玻璃载片的表面(<3nm)的光滑AAO玻璃基板。典型的AAO玻璃基板的截面图像显示于图S5B中。为获得具有图S5中所示的物理性质的经最佳化AAO玻璃基板,使用电脑程式产生的因子设计及反应表面方法进行详细实验且将在另一篇文章中报导。
图S5|(A)AFM图像-表面粗糙度分析.Img.Rms(Rq)2.319nm及(B)AAO玻璃基板的截面SEM图像。
AAO玻璃基板对比经NHS活化的玻璃载片S的表面比较
在我们团队及其他团队中,NHS-玻璃载片已广泛用于聚醣微阵列。NHS官能基与糖衍生物的胺的酰胺化反应发生在表面上。其第一化学反应为共价键结至官能化玻璃表面的羧基。AAO玻璃基板的表面上含有一层非晶氧化铝的稳定的聚合物网状物,其准备与聚醣衍生物的膦酸尾部发生化学反应,形成膦酸酯。相较于在NHS载片上的反应,膦酸在金属氧化物(氧化铝或氧化钛)上的界面反应为自发的且所得阵列较为均匀,且可以容易地控制密度及分布。为鉴别可用于界面共价键结的反应性位点,将Cy5-膦酸及Cy5-胺直接用于表面上的微阵列。这些染料(图S6)已自Cy5-NHS酯(Lumiprobe Corp.项目编号63020)开始内部合成。
将染料Cy5-膦酸及Cy5-胺溶解于比率为7:3的乙二醇/水混合物(1mM)中,分别将pH调整至6及8.5。用5%甲醇于水中的混合物充分洗涤阵列式载片,旋转干燥,然后进行GenePix(4300A)扫描。图S7显示在AAO玻璃基板上的Cy5-膦酸(图S7A)、在NHS-玻璃载片上的Cy5-胺(图S7B)的微阵列图像及其20个斑点的平均萤光强度(图S7C)。这些阵列式斑点的代表性共焦显微镜图像提供于图S8中。已观察到负载容量更大且分布更均匀的Cy5-分子,类似于通常在聚醣微阵列及糖/蛋白质结合中所见到的情况。
图S7|1mM的(A)在AAO玻璃基板上的Cy5-膦酸、(B)在NHS玻璃载片上的Cy5-胺的GenePix扫描(在PMT 450下),及(C)其平均20个斑点的萤光强度。
图S8|在AAO玻璃基板及NHS玻璃载片上的代表性共焦显微镜图像Cy5-膦酸及Cy5胺。在斑点内的选择性900μm2面积。
使用共焦显微镜比较ACG及NHS载片的表面均匀性
为评估在ACG及经NHS活化的玻璃载片上的聚醣阵列的密度及均匀性,吾人使用甘露糖单醣形成的聚醣阵列作为模型。使用100μM的甘露糖溶液用于阵列形成,且使用50μg/mL的ConA488溶液用于糖/蛋白质结合。自共焦显微镜观察到的ConA488/甘露糖结合的影像进一步证实,AAO玻璃基板具有更密集且分布更均匀的共价键结糖。图S9显示,自共焦显微镜(Leica SP5)观察到的在AAO玻璃基板上对比在NHS玻璃载片上的ConA488/甘露糖结合的影像。
图S9|AAO玻璃基板上的对比NHS玻璃载片上的ConA488/甘露糖结合的共焦显微镜。
在涂有氧化铝的玻璃载片上及在涂有NHS的玻璃载片上的糖分布的原子力显微镜影像表明,AAO玻璃基板(图S10a)提供比NHS玻璃载片(图S10a)更为均匀的共价键结糖分布。通过计数超过粒子高度的最大数目的宽度分布的二分之一的高度的粒子获得这些影像的粒子计数。甘露糖衍生物可以仅共价键结于载片上,其中可在表面上获得经活化的官能基。不论玻璃基底材料的Rms的差异,AAO玻璃基板提供比NHS玻璃载片更为均匀的分布。
图S10|原子力显微镜影像,显示a)涂有氧化铝的玻璃载片及b)涂有NHS的玻璃载片上的糖分布
抗体结合分析
抗体PG9、PG16及PGT 141-145在使用之前用结合缓冲液稀释至100μg/mL。随后将DyLight649结合的驴抗人类IgG抗体与一级抗体预复合。用结合缓冲液将预复合溶液的最终浓度调整至50μg/mL。将所印刷的玻璃载片组装成框架载片支架随后相应地向各孔中施加80μL预复合抗体溶液。在轻轻震荡下,在4℃下维持抗体结合制程,且随后在培育6小时后,小心地用移液管移取抗体溶液。用100μL PBST洗涤缓冲液轻轻地洗涤玻璃载片,且随后旋转干燥3分钟。
影像处理及资料分析
用微阵列萤光晶片读取器(4300A,Molecular Devices Corporation)在635nm下扫描载片。用GenePix Pro 6.0分析软体(Molecular Devices Corporation)分析扫描影像。将影像解析度设定为每像素5μm。斑点定义为直径限定为100μm的圆形特征。选择总强度值,用Graphpad6.0进行资料处理。计算强度且取平均值。误差杠表示所报导的所有资料点的标准差。
分析在涂有NHS的玻璃载片上的HIV-1bNAb的聚醣结合特异性
用于研究PG9、PG16及PGT 141-145的载片经由形成共价键将聚醣G1-33(图S11)印刷在涂有N-羟基丁二酰亚胺的表面上来制备。在二级抗体培育之后,通过萤光扫描获得载片影像。
图S11|印刷在涂有NHS的玻璃载片上的N-聚醣的示意性图示。
在吾人的阵列中并入多样化HIV-1gp120相关的N-聚醣之后,接着表征PG16对那些聚醣的结合行为。吾人的结果与吾人先前报导的资料一致,其中,PG16结合与末端的α2,6-Neu5Ac计数成比例(图S12)。根据吾人的研究的简短结论是限定位向的二唾液酸化触角的存在对PG16结合袋中的结合具有决定性。
图S12.使用涂有NHS的玻璃载片得到的PG16的结合行为。PG16与一组N-聚醣的结合用条形图表示。
结合至抗体PG9及PGT 141-45的一组限定聚醣的结合。吾人在吾人的涂有NHS的玻璃载片阵列上筛选PG9及PGT 141-145。
图S13.使用涂有NHS的玻璃载片,PG9的结合行为。
图S14.使用涂有NHS的玻璃载片,PGT 141-143的结合行为。此处所用的抗体浓度为25μg/mL
图S15.使用涂有NHS的玻璃载片,PGT 14-145的结合行为。此处所用的抗体浓度为25μg/mL
吾人观察到,抗体PG9(图S13)及PGT 141-145(图S14-15)与阵列上所印刷的聚醣中的任一者的低程度结合。
分析在ACG载片上的HIV-1bNAb的聚醣结合特异性
在证明了就聚醣密度及信号强度增强而言ACG载片优于涂有NHS的载片的适用性之后,吾人使用膦酸酯化学将聚醣I-XI印刷于ACG阵列上(图S17)。PGT 141-144的结合特异性如下所示。
图S17.草图表示印刷在ACG阵列上的聚醣。连接基团的结构示出在左上角。
PGT 141-144的结合特异性显示于图S18中。然而,吾人未发现PGT145在ACG阵列上的结合,最可能是因为其微弱的聚醣结合亲和力。
图S18.PGT 141-144与ACG阵列上的一组N-聚醣的结合用条形图显示。
测定ACG载片上的表面解离常数(KD.surf)
用于测定解离常数的涂有氧化铝的玻璃载片用100及50μM浓度的聚醣Man5GlcNAc2(V)、混合型(XI)、二触角复合型(XII)、V+XI(1:1莫耳比)、V+XII及XI+XII点样。用3%BSΑ-PBST缓冲液将抗体PG16连续稀释至3.32、1.66、0.833、0.415、0.207、0.103、0.051及0.025μM。将DyLight649结合的驴抗人类IgG抗体与一级抗体PG16预复合(按重量计1:1)。向各孔施加预复合溶液(100μL)且在暗处在4℃下培育6h。最后,用PBST洗涤载片,旋转干燥且用微阵列萤光晶片读取器在450nm下扫描。用GenePix Pro 6.0分析软体分析扫描影像。PG16与Man5GlcNAc2(V)及V+XI(1:1莫耳比)的混合物的结合的信号强度过弱以致于无法测定KD。印刷在阵列上的其余样品的结合曲线显示于图S19中,且KD值概述于表S2中。
在PG9的情况下,因为其聚醣结合亲和力非常弱,所以吾人不能达成信号饱和度来量测结合常数,然而,将PG9浓度增加至700μg/mL会在阵列表面上导致沈淀。
图S19.聚醣X、XI及混合物V+XI及X+XII在100μM浓度下观察到的抗体PG16结合曲线。曲线是通过使用DyLight649结合的驴抗人类IgG二级抗体获得。
表S2.抗体PG16与聚醣X、XI及混合物V+XI及X+XI的KD,surf(μM)值。
额外实例
额外实例作为单独文件附上,标记为P3-1至P3-7。
额外实例:(P3-1)
化合物42为混合的化合物4及化合物20的转换物,其中,其亦由抗HIV-1广谱中和单株抗体(bnMAb)识别及结合,其中结合且识别化合物4及化合物20的混合物,例如PG9及PG16
额外实例:(P3-2)
印刷在涂有NHS的玻璃载片上的N-聚醣的示意性图示
额外实例:(P3-3)
合成化合物42
额外实例:(P3-4)
用于合成在3臂含Man3/Man5聚醣且在6臂含复合聚醣的混合聚醣的例示性通用程序
额外实例:(P3-5)
用于合成在6臂含Man3/Man5聚醣且在3臂含复合聚醣的混合聚醣的例示性通用程序
额外实例:(P3-6)
经由合成得到的例示性混合聚醣
额外实例:(P3-7)
经由合成得到的例示性混合聚醣
参考文献
1.Barouch,D.H.Challenges in the development of an HIV-1vaccine.Nature 455,613-619,(2008).
2.Gaschen,B.et al.AIDS-Diversity considerations in HIV-1 vaccineselection.Science
3.296,2354-2360,(2002).
4.Astronomo,R.D.&Burton,D.R.Carbohydrate vaccines:developing sweetsolutions to sticky situations?Nat.Rev.Drug.Discov.9,308-324,(2010).
5.Craigo,J.K.,Montelaro,R.C.Lessons in AIDS vaccine developmentlearned from studies of equine infectious,anemia virus infection andimmunity.Viruses 5,2963-76,(2013)
6.Scanlan,C.N.,Offer,J.,Zitzmann,N.&Dwek,R.A.Exploiting the defensivesugars of HIV-1for drug and vaccine design.Nature 446,1038-1045,(2007).
7.Kwong,P.D.,Mascola,J.R.&Nabel,G.J.Rational Design of Vaccines toElicit Broadly Neutralizing Antibodies to HIV-1.Cold SpringHarb.Perspect.Med.1,(2011).
8.Burton,D.R.,Mascola,J.R.Antibody responses to envelopeglycoproteins in HIV-1infection.Nature Immunol.16,571-6,(2015).
9.Pritchard,L.K.,Harvey,D.J.,Bonomelli,C.,Crispin,M.,Doores,K.J.Cell-and Protein-Directed Glycosylation of Native Cleaved HIV-1Envelope.J.Virol.89,8932-44,(2015).
10.Pritchard,L.K.et al.Structural Constraints Determine theGlycosylation of HIV-1 Envelope Trimers.Cell Rep.11,1604-13,(2015).
11.Doores,K.J.et al.Envelope glycans of immunodeficiency virions arealmost entirely oligomannose antigens.Proc.Natl.Acad.Sci.U.S.A.107,13800-13805,(2010).
12.Pabst,M.,Chang,M.,Stadlmann,J.&Altmann,F.Glycan profiles of the27N-glycosylation sites of the HIV envelope protein CN54gp140.Biol.Chem.393,719-730,(2012).
13.Go,E.P.et al.Comparative Analysis of the Glycosylation Profiles ofMembrane-Anchored HIV-1 Envelope Glycoprotein Trimers and Solublegp140.J.Virol.89,8245-57,(2015).
14.Wang,W.et al.A systematic study of the N-glycosylation sites ofHIV-1envelope protein on infectivity and antibody-mediatedneutralization.Retrovirology,10,14,(2014).
15.Calarese,D.A.et al.Antibody domain exchange is an immunologicalsolution to carbohydrate cluster recognition.Science 300,2065-2071,(2003).
16.Wang,L.X.Carbohydrate-based vaccines against HIV/AIDS.AcsSym.Ser.932,133-160(2006).
17.Joyce,J.G.et al.An oligosaccharide-based HIV-1 2G12 mimotopevaccine induces carbohydrate-specific antibodies that fail to neutralize HIV-1virions.Proc.Natl.Acad.Sci.U.S.A.105,15684-15689,(2008).
18.Wang,L.X.Synthetic carbohydrate antigens for HIV vaccinedesign.Curr.Opin.Chem.Biol.17,997-1005,(2013).
19.Horiya,S.,MacPherson,I.S.,Krauss,I.J.Recent strategies targetingHIV glycans in vaccine design.Nat.Chem.Biol.10,990-999,(2014).
20.Fernandez-Tejada,A.,Haynes,B.F.,Danishefsky,S.J.Designingsynthetic vaccines for HIV.Expert Rev.Vaccines 14,815-31,2015.
21.Walker,L.M.et al.Broad neutralization coverage of HIV by multiplehighly potent antibodies.Nature 477,466-470,(2011).
22.Sok,D.,Moldt,B.,Burton,D.R.SnapShot:broadly neutralizingantibodies.Cell 155,728-728,(2013).
23.Garces,F.et al.Structural evolution of glycan recognition by afamily of potent HIV antibodies.Cell 159,69-79,(2014).
24.Wu,X.et al.Rational design of envelope identifies broadlyneutralizing human monoclonal antibodies to HIV-1.Science 329,856-861,(2010).
25.Pancera,M.et al.Crystal structure of PG16 and chimeric dissectionwith somatically related PG9:structure-function analysis of two quaternary-specific antibodies that effectively neutralize HIV-1.J.Virol.84,8098-8110,(2010).
26.Doores,K.J.&Burton,D.R.Variable Loop Glycan Dependency of theBroad and Potent HIV-1-Neutralizing Antibodies PG9 and PG16.J.Virol.84,10510-10521,(2010).
27.McLellan,J.S.et al.Structure of HIV-1 gp120 V1/V2 domain withbroadly neutralizing antibody PG9.Nature 480,336-343,(2011).
28.Pejchal,R.et al.A potent and broad neutralizing antibodyrecognizes and penetrates the HIV glycan shield.Science 334,1097-1103,(2011).
29.Mouquet,H.et al.Complex-type N-glycan recognition by potentbroadly neutralizing HIV antibodies.Proc.Natl.Acad.Sci.U.S.A.109,E3268-E3277,(2012).
30.Falkowska,E.et al.Broadly neutralizing HIV antibodies define aglycan-dependent epitope on the prefusion conformation of gp41 on cleavedenvelope trimers.Immunity 40,657-68,2014.
31.Pancera,M.et al.Structural basis for diverse N-glycan recognitionby HIV-1-neutralizing V1-V2-directed antibody PG16.Nat.Struct.Mol.Biol.20,804-813,(2013).
32.Amin,M.N.et al.Synthetic glycopeptides reveal the glycanspecificity of HIV-neutralizing antibodies.Nat.Chem.Biol.9,521-526,(2013).
33.Murphy,C.I.et al.Enhanced expression,secretion,and large-scalepurification of recombinant HIV-1 gp120 in insect cell using the baculovirusegt and p67 signal peptides.Protein Expres.Purif.4,349-357(1993).
34.Kong,L.et al.Expression-system-dependent modulation of HIV-1envelope glycoprotein antigenicity and immunogenicity.J.Mol.Biol.403,131-147,(2010).
35.Go,E.P.et al.Characterization of glycosylation profiles of HIV-1transmitted/founder envelopes by mass spectrometry.J.Virol.85,8270-8284,(2011).
36.Eggink,D.et al.Lack of complex N-glycans on HIV-1 envelopeglycoproteins preserves protein conformation and entry function.Virology 401,236-247,(2010).
37.Zhu,X.,Borchers,C.,Bienstock,R.J.&Tomer,K.B.Mass spectrometriccharacterization of the glycosylation pattern of HIV-gp120expressed in CHOcells.Biochemistry 39,11194-11204(2000).
38.Raska,M.et al.Glycosylation patterns of HIV-1 gp120 depend on thetype of expressing cells and affect antibody recognition.J.Biol.Chem.285,20860-20869,(2010).
39.De Paz,J.L.,Horlacher,T.&Seeberger,P.H.Oligosaccharide microarraysto map interactions of carbohydrates in biological systems.MethodsEnzymol.415,269-292,(2006).
40.Oyelaran,O.&Gildersleeve,J.C.Glycan arrays:recent advances andfuture challenges.Curr.Opin.Chem.Biol.13,406-413,(2009).
41.Rillahan,C.D.&Paulson,J.C.Glycan microarrays for decoding theglycome.Annu.Rev.Biochem.80,797-823,(2011).
42.Blixt,O.et al.Printed covalent glycan array for ligand profilingof diverse glycan binding proteins.Proc.Natl.Acad.Sci.U.S.A.101,17033-17038,(2004).
43.Fukui,S.,Feizi,T.,Galustian,C.,Lawson,A.M.&Chai,W.Oligosaccharidemicroarrays for high-throughput detection and specificity assignments ofcarbohydrate-protein interactions.Nat.Biotechnol.20,1011-1017,(2002).
44.Wang,D.,Liu,S.,Trummer,B.J.,Deng,C.&Wang,A.Carbohydratemicroarrays for the recognition of cross-reactive molecular markers ofmicrobes and host cells.Nat.Biotechnol.20,275-281,(2002).
45.Fazio,F.,Bryan,M.C.,Blixt,O.,Paulson,J.C.&Wong,C.-H.Synthesis ofsugar arrays in microtiter plate.J.Am.Chem.Soc.124,14397-14402,(2002).
46.Paulson,J.C.,Blixt,O.&Collins,B.E.Sweet spots in functionalglycomics.Nat.Chem.Biol.2,238-248,(2006).
47.Scurr,D.J.et al.Surface characterization of carbohydratemicroarrays.Langmuir 26,17143-17155,(2010).
48.Chang,S.H.et al.Glycan array on aluminum oxide-coated glass slidesthrough phosphonate chemistry.J.Am.Chem.Soc.132,13371-13380,(2010).
49.Tseng,S.Y.et al.Glycan arrays on aluminum-coated glassslides.Chem.-Asian J.3,1395-1405,(2008).
50.Wang,Z.et al.A general strategy for the chemoenzymatic synthesisof asymmetrically branched N-glycans.Science 341,379-383,(2013).
51.Shivatare,S.S.et al.Efficient convergent synthesis of bi-,tri-,andtetra-antennary complex type N-glycans and their HIV-1antigenicity.J.Am.Chem.Soc.135,15382-15391,(2013).
52.Li,L.et al.Efficient chemoenzymatic synthesis of an N-glycanisomer library.Chem.Sci.6,5652-5661(2015).
53.Takakura,Y.,Tsukamoto,H.&Yamamoto,T.Molecular cloning,expressionand properties of an alpha/beta-Galactoside alpha 2,3-sialyltransferase fromVibrio sp.JT-FAJ-16.J.Biochem.142,403-412,(2007).
54.Tsukamoto,H.,Takakura,Y.,Mine,T.&Yamamoto,T.Photobacterium sp.JT-ISH-224 produces two sialyltransferases,alpha-/beta-galactoside alpha2,3-sialyltransferase and beta-galactoside alpha2,6-sialyltransferase.J.Biochem.143,187-197,(2008).
55.Toshima,K.Glycosyl fluorides in glycosidations.Carbohydr.Res.327,15-26(2000).
56.Rabbani,S.,Schwardt,O.&Ernst,B.Glycosyltransferases:An efficienttool for the enzymatic synthesis of oligosaccharides and derivatives as wellas mimetics thereof.Chimia 60,23-27,(2006).
57.Muthana,S.,Yu,H.,Huang,S.,and Chen,X.Chemoenzymatic synthesis ofsize-defined polysaccharides by sialyltransferase-catalyzed block transfer ofoligosaccharides.J.Am.Chem.Soc.129,11918-11919,(2007).
58.Lau,K.et al.Highly efficient chemoenzymatic synthesis ofβ1-4-linked galactosides with promiscuous bacterialβ1-4-galactosyltransferases.Chem.Commun.46,6066-6068,(2010).
59.Soriano del Amo,D.et al.Chemoenzymatic synthesis of the sialylLewis X glycan and its derivatives.Carbohydr.Res.345,1107-13,(2010).
60.Liang,P.H.,Wang,S.K.&Wong,C.-H.Quantitative analysis ofcarbohydrate-protein interactions using glycan microarrays:Determination ofsurface and solution dissociation constants.J.Am.Chem.Soc.129,11177-11184,(2007).
61.Takakura,Y.,Tsukamoto,H.&Yamamoto,T.Molecular cloning,expressionand properties of an alpha/beta-Galactoside alpha2,3-sialyltransferase fromVibrio sp.JT-FAJ-16.J.Biochem.142,403-412,(2007).
62.Tsukamoto,H.,Takakura,Y.,Mine,T.&Yamamoto,T.Photobacterium sp.JT-ISH-224 produces two sialyltransferases,alpha-/beta-galactoside alpha2,3-sialyltransferase and beta-galactoside alpha2,6-sialyltransferase.J.Biochem.143,187-197,(2008).
63.Shivatare,S.S.et al.Efficient convergent synthesis of bi-,tri-,andtetra-antennary complex type N-glycans and their HIV-1antigenicity.J.Am.Chem.Soc.135(41),15382-15391,(2013).
64.TSai,T.I.et al.Effective sugar nucleotide regeneration for thelarge-scale enzymatic synthesis of Globo H and SSEA4.J.Am.Chem.Soc.135(39),14831-9,(2013).
65.Doores KJ,et al.A nonself sugar mimic of the HIV glycan shieldshows enhanced antigenicity.Proc.Natl.Acad.Sci.U.S.A.107(40),17107-17112,(2010).
66.Mandal,M.,Dudkin,V.Y.,Geng,X.&Danishefsky,S.J.In pursuit ofcarbohydrate-based HIV vaccines,part 1:The total synthesis of hybrid-type gp120fragments.Angew.Chem.Int.Ed.43,2557-2561,(2004).
67.Lee,H.K.et al.Reactivity-based one-pot synthesis of oligomannoses:defining antigens recognized by 2G12,a broadly neutralizing anti-HIV-1antibody.Angew.Chem.Int.Ed.43,1000-1003,(2004).
68.John,F.&Hendrickson,T.L.Synthesis of Truncated Analogues forStudying the Process of Glycosyl PhosphatidylinositolModification.Org.Lett.12,2080-2083,(2010).
69.Pratt,M.R.&Bertozzi,C.R.Chemoselective ligation applied to thesynthesis of a biantennary N-linked glycoform of CD52.J.Am.Chem.Soc.125,6149-6159,(2003).
70.Hsu,C,H.et al.Highly alpha-selective sialyl phosphate donors forefficient preparation of natural sialosides.Chem.Eur.J.16-6,1754-1760,(2010).
71.Serna,S.,Etxebarria,J.,Ruiz,N.,Martin-Lomas,M.&Reichardt,N.C.Construction of N-Glycan Microarrays by Using Modular Synthesis and On-Chip Nanoscale Enzymatic Glycosylation.Chem.Eur.J.16,13163-13175,(2010).
72.Sun,B.,Srinibasan,B.,Huang,X.,Pre-activation-based one-potsynthesis of an alpha-(2,3)-sialylated core-fucosylated complex type bi-antennary N-glycan dodecasaccharide.Chem.Eur.J.14(23),7072-81,(2008).
73.Kanie,O.,Ito,Y.&Ogawa,T.Orthogonal glycosylation strategy insynthesis of extended blood group B determinant.Tetrahedron Lett.37,4551-4554(1996).
74.Lemieux,R.U.,Hendriks,K.B.,Stick,R.V.,James,K.Halide ion catalyzedglycosidation reactions.Syntheses of a-linked disaccharides.J.Am.Chem.Soc.97(14),4056-62,(1975).
75.S.Y.Tseng,C.-C.Wang,C.-W.Lin,C.-L.Chen,W.-Y.Yu,C.-H.Chen,C.-Y.Wu,C.-H.Wong“Glycan Arrays on Aluminum Coated Glass Slides”.Chem.Asian J,2008,3,1395-1405
76.Z.Su,G.W.Zhou“Investigation of the pore formation in anodicaluminum oxide”J.Mater.Chem.2008,18 5787-5795.
77.K.Rana,G.Kucukayan-Dogu,E.Bengu“Growth of vertically alignedcarbon nanotubes over self-ordered nano-porous alumina films and theirsurface properties”Applied Surface Science,2012,258 7112-7117.
Claims (22)
1.一种经正交保护的Manβ1-4GlcNAcβ1-4GlcNAcβ1核心三醣,其具有式(I):
其中R1、R2、R3、R4、R5、R6、R7及R8中的每一者独立地选自由正交或永久保护基组成的群;其中,在R6处的正交保护基中的每一者较佳选自由对甲氧基苄基醚(PMB)、甲氧基苯基醚(PMP)、乙酰丙酰基(Lev)、苄酰基(Bz)、烯丙醚(烯丙基)及硅烷基醚组成的群;且R7及R8稠合形成亚苄基环,其可以裂解形成4-OH及/或6-OH聚醣结构;
R2、R3、R4及R5中的每一者独立地为在用于移除正交保护基的条件下稳定且较佳为苄基(Bn)或乙酰基(Ac)的永久保护基;
R1为在用于移除正交保护基的条件下稳定且较佳为苄基(Bn)或乙酰基(Ac)的永久保护基;或经由α1,6键联连接的经保护的海藻糖苷残基;
X为-OR9;其中R9为-H或经由-N3及NR10组成的基团封端的视情况经取代的C3-C10烷基链;且R10为-H或苄基(Bn)或苄氧羰基(Cbz);
Y及Z为-NHR11;且R11较佳选自由以下组成的群:9-茀基甲氧基羰基(Fmoc)、烯丙氧基羰基(alloc)、[2,2,2-三氯乙氧基羰基](troc)、乙酰基(Ac)、邻苯二酰亚胺基(Phth)、苄氧羰基(Cbz)及第三丁氧羰基(Boc)。
2.一种D1及D2/D3臂模组,其具有通式(II):
其中,R1、R2、R3及R4中的每一者为-H;或在用于移除正交保护基的条件下稳定且较佳为苄基(Bn)或乙酰基(Ac)或苄酰基(Bz)的永久保护基;或独立地选自
其中,R5、R6、R7及R8中的每一者为-H或独立地为在用于移除正交保护基的条件下稳定且较佳为苄基(Bn)或乙酰基(Ac)或苄酰基(Bz)的永久保护基;
R9及R10中的每一者为-H或独立地为在用于移除正交保护基的条件下稳定且较佳为苄基(Bn)或乙酰基(Ac)或苄酰基(Bz)的永久保护基;或经由α1,3键联连接至GlcNAc及/或经由α1,2键联连接至半乳糖的经保护的海藻糖苷残基;
R11为-H、Me(甲基)或Et(乙基);
R12为-H;或独立地为在用于移除正交保护基的条件下稳定且较佳为苄基(Bn)或乙酰基(Ac)或苄酰基(Bz)的永久保护基;或经由α2,6键联连接的经保护的Neu5Ac残基;
R13为-H;或独立地为在用于移除正交保护基的条件下稳定且较佳为苄基(Bn)或乙酰基(Ac)或苄酰基(Bz)的永久保护基;或独立地选自
X为-OR14或-SR15或离去基;
其中,R14为H、烷基、烯基、炔基、芳基或经取代的芳基;保护基,诸如硅烷基或经取代的硅烷基,较佳为第三己基二甲基硅烷基(TDS)、第三丁基二甲基硅烷基(TBS)、第三丁基二苯基硅烷基(TBDPS)、三异丙基硅烷基(TIPS)、三甲基硅烷基(TMS)或三乙基硅烷基(TES);甲基(Me)、乙酰基(Ac)、苄基(Bn)、2-萘甲基(nap)或1-萘甲基(1-Nap);对甲氧基苄基醚(PMB)、甲氧基苯基醚(PMP)、烯丙醚(烯丙基);或变旋异构离去基,诸如三氯乙酰亚胺酸酯基-C(NH)-CCl3、苯基三氟乙酰亚胺酯基-C(NPh)-CF3、三氟乙酰亚胺酯基-C(NH)-CF3;硫烷基、苯硫基;且更佳为氟基;
R15为H、烷基、芳基或经取代的芳基,较佳为甲基、乙基、苯基、甲苯磺酰基或甲苯基;
Y为-NHR16;且R16较佳选自由以下组成的群:9-茀基甲氧基羰基(Fmoc)、烯丙氧基羰基(Alloc)、[2,2,2-三氯乙氧基羰基](Troc)、乙酰基(Ac)、邻苯二酰亚胺基(Phth)、苄氧羰基(Cbz)及第三丁氧羰基(Boc);
其中「D1及D2/D3臂模组」是指N-聚醣。
3.一种用于合成适合产生聚醣阵列的多样化聚醣的方法,其中所述方法包含:
a)产生如权利要求1的经正交保护的核心三醣及如权利要求2的D1及D2/D3臂模组,
b)使如权利要求2的D1及D2/D3臂模组立体选择性及区域选择性醣基化至如权利要求1的经正交保护的核心三醣的3-O或/及6-O位置,
c)去掩蔽或移除保护基,获得高甘露糖型、混合型及二触角、三触角及四触角复合型聚醣,及
d)进行酶促唾液酸化,产生唾液酸化聚醣库。
4.一种制备如权利要求2的D1及D2/D3臂模组的方法,其中所述方法包含化学及化学酶促合成。
5.如权利要求2的D1及D2/D3臂模组,其中这些模组选自由以下组成的群:
6.如权利要求4的方法,其进一步包含:
具有式(III)的化学合成的接受体受质的逐步酶促延长
其中,
R1及R2中的每一者独立地选自-H或乙酰基(Ac)或苄酰基(Bz);
X为-OR3且R3为H、苄基(Bn)、2-萘甲基(Nap)、1-萘甲基(1-Nap);对甲氧基苄基醚(PMB)、甲氧基苯基醚(PMP)或烯丙醚(烯丙基)。
7.如权利要求6的方法,其中酶独立地选自由以下组成的群:β(1→4)半乳醣苷基转移酶、α(1→3)海藻醣苷基转移酶、α(1→2)海藻醣苷基转移酶、β(1→3)N-乙酰基葡糖胺转移酶、α(2→3)唾液酸转移酶及α(2→6)唾液酸转移酶。
8.一种根据如权利要求3的方法制备的碳水化合物部分的阵列,所述阵列包含:
复数个聚醣,其中各聚醣部分沈积在基板上的离散位置,其中这些多样化聚醣包含高甘露糖型、混合型及复合型同质及/或混合N-聚醣。
9.如权利要求8的阵列,其中这些聚醣包含通过如权利要求3、4、6及7中任一项的方法制备的高甘露糖型、混合型及复合型同质及/或混合N-聚醣。
10.一种侦测杂聚醣结合行为的方法,其包含使如权利要求8的阵列与一个或复数个杂聚醣接触。
11.如权利要求9的阵列,其中这些聚醣进一步包含由广谱中和HIV-1抗体识别的聚醣。
12.如权利要求8的阵列,其中所述基板包含涂有氧化铝的玻璃(ACG)表面。
13.如权利要求8或11中任一项的阵列,其中这些聚醣包含图S11中所阐述的化合物G1至G33中的一或多者及/或图S17中所阐述的化合物I至XI中的任何一或多者。
14.如权利要求9的阵列,其中这些聚醣可以包括或不包括图S11中所阐述的化合物G1至G33中的一或多者且包括或不包括图S17中所阐述的化合物I至XI中的任何一或多者。
15.一种用于产生疫苗标靶的方法,其包含:
筛选且鉴别由HIV-1中和抗体识别的最佳细胞表面聚醣,其中所述方法进一步包含使这些HIV抗体与如权利要求8、9、11、12或13中任一项的阵列接触。
16.如权利要求8的阵列,其中所述基板选自表面、固体表面、不透明固体、对可见或非可见光的所选波长透明的固体、粒子、微泡或珠粒。
17.如权利要求8的阵列,其中所述复数个N-聚醣通过凡得瓦相互作用(vander Waalsinteraction)或通过疏水相互作用涂布在所述基板上或黏附至所述基板。
18.一种固定在基板上的N-聚醣阵列,其中所述阵列是通过以下方法制造,所述方法包含:
(a)提供基板;
(b)经由连接基团将所述N-聚醣连接至所述基板;及
(c)将复数个N-聚醣部分固定在所述基板表面上的离散位置。
19.一种表征如权利要求18的阵列的方法,其包含使所固定的含有经标记抗体的N-聚醣部分与这些聚醣接触,在所述抗体与所述聚醣之间形成复合物,且侦测这些复合物。
20.如权利要求19的方法,其中所述经标记抗体包含标签,所述标签包含酶、萤光标签、化学发光标签或奈米粒子标签。
21.一种侦测HIV抗体的方法,所述方法包含:
(a)提供如权利要求18的阵列;
(b)使所述阵列与HIV抗体接触;
(c)在这些HIV抗体与所述阵列上的这些聚醣之间形成复合物;
(d)使所述HIV抗体-聚醣阵列复合物与标签接触;及
(e)侦测所述标签。
22.如权利要求21的方法,其中所述标签为经标记抗体,其进一步包含酶、萤光团、化学发光部分或奈米粒子。
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109069523A (zh) * | 2016-12-10 | 2018-12-21 | 扬州蓝色生物医药科技有限公司 | 甘露寡聚糖类化合物及其作为抗rna病毒药物的应用 |
Families Citing this family (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10087236B2 (en) | 2009-12-02 | 2018-10-02 | Academia Sinica | Methods for modifying human antibodies by glycan engineering |
US11377485B2 (en) | 2009-12-02 | 2022-07-05 | Academia Sinica | Methods for modifying human antibodies by glycan engineering |
CN105682666B (zh) | 2013-09-06 | 2021-06-01 | 中央研究院 | 使用醣脂激活人类iNKT细胞 |
US10005847B2 (en) | 2014-05-27 | 2018-06-26 | Academia Sinica | Anti-HER2 glycoantibodies and uses thereof |
AU2015267047A1 (en) | 2014-05-27 | 2017-01-05 | Academia Sinica | Anti-CD20 glycoantibodies and uses thereof |
EP3904388A1 (en) | 2014-05-27 | 2021-11-03 | Academia Sinica | Fucosidase from bacteroides and methods using the same |
WO2015184001A1 (en) | 2014-05-28 | 2015-12-03 | Academia Sinica | Anti-tnf-alpha glycoantibodies and uses thereof |
US10495645B2 (en) | 2015-01-16 | 2019-12-03 | Academia Sinica | Cancer markers and methods of use thereof |
JP7213549B2 (ja) | 2016-08-22 | 2023-01-27 | シーエイチオー ファーマ インコーポレイテッド | 抗体、結合性断片、および使用の方法 |
TW202334175A (zh) * | 2021-10-29 | 2023-09-01 | 日商第一三共股份有限公司 | 新穎寡糖、該寡糖之製造中間體及其等之製造方法 |
WO2024053574A1 (ja) * | 2022-09-09 | 2024-03-14 | 第一三共株式会社 | 新規なオリゴ糖、該オリゴ糖の製造中間体、及びそれらの製造方法 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1524180A (zh) * | 2001-04-10 | 2004-08-25 | 纽约市哥伦比亚大学信托人 | 新型微列阵及其使用方法 |
WO2014161960A1 (en) * | 2013-04-03 | 2014-10-09 | Asociación Centro De Investigación Cooperativa En Biomateriales | Synthesis and use of isotopically-labelled glycans |
Family Cites Families (344)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3773919A (en) | 1969-10-23 | 1973-11-20 | Du Pont | Polylactide-drug mixtures |
US3896111A (en) | 1973-02-20 | 1975-07-22 | Research Corp | Ansa macrolides |
US4151042A (en) | 1977-03-31 | 1979-04-24 | Takeda Chemical Industries, Ltd. | Method for producing maytansinol and its derivatives |
US4137230A (en) | 1977-11-14 | 1979-01-30 | Takeda Chemical Industries, Ltd. | Method for the production of maytansinoids |
USRE30985E (en) | 1978-01-01 | 1982-06-29 | Serum-free cell culture media | |
US4307016A (en) | 1978-03-24 | 1981-12-22 | Takeda Chemical Industries, Ltd. | Demethyl maytansinoids |
US4265814A (en) | 1978-03-24 | 1981-05-05 | Takeda Chemical Industries | Matansinol 3-n-hexadecanoate |
JPS5562090A (en) | 1978-10-27 | 1980-05-10 | Takeda Chem Ind Ltd | Novel maytansinoid compound and its preparation |
US4256746A (en) | 1978-11-14 | 1981-03-17 | Takeda Chemical Industries | Dechloromaytansinoids, their pharmaceutical compositions and method of use |
JPS5566585A (en) | 1978-11-14 | 1980-05-20 | Takeda Chem Ind Ltd | Novel maytansinoid compound and its preparation |
JPS55164687A (en) | 1979-06-11 | 1980-12-22 | Takeda Chem Ind Ltd | Novel maytansinoid compound and its preparation |
JPS55102583A (en) | 1979-01-31 | 1980-08-05 | Takeda Chem Ind Ltd | 20-acyloxy-20-demethylmaytansinoid compound |
JPS55162791A (en) | 1979-06-05 | 1980-12-18 | Takeda Chem Ind Ltd | Antibiotic c-15003pnd and its preparation |
JPS55164685A (en) | 1979-06-08 | 1980-12-22 | Takeda Chem Ind Ltd | Novel maytansinoid compound and its preparation |
JPS55164686A (en) | 1979-06-11 | 1980-12-22 | Takeda Chem Ind Ltd | Novel maytansinoid compound and its preparation |
US4309428A (en) | 1979-07-30 | 1982-01-05 | Takeda Chemical Industries, Ltd. | Maytansinoids |
JPS5645483A (en) | 1979-09-19 | 1981-04-25 | Takeda Chem Ind Ltd | C-15003phm and its preparation |
JPS5645485A (en) | 1979-09-21 | 1981-04-25 | Takeda Chem Ind Ltd | Production of c-15003pnd |
EP0028683A1 (en) | 1979-09-21 | 1981-05-20 | Takeda Chemical Industries, Ltd. | Antibiotic C-15003 PHO and production thereof |
US4270537A (en) | 1979-11-19 | 1981-06-02 | Romaine Richard A | Automatic hypodermic syringe |
US4376110A (en) | 1980-08-04 | 1983-03-08 | Hybritech, Incorporated | Immunometric assays using monoclonal antibodies |
WO1982001188A1 (en) | 1980-10-08 | 1982-04-15 | Takeda Chemical Industries Ltd | 4,5-deoxymaytansinoide compounds and process for preparing same |
US4450254A (en) | 1980-11-03 | 1984-05-22 | Standard Oil Company | Impact improvement of high nitrile resins |
US4419446A (en) | 1980-12-31 | 1983-12-06 | The United States Of America As Represented By The Department Of Health And Human Services | Recombinant DNA process utilizing a papilloma virus DNA as a vector |
US4315929A (en) | 1981-01-27 | 1982-02-16 | The United States Of America As Represented By The Secretary Of Agriculture | Method of controlling the European corn borer with trewiasine |
US4313946A (en) | 1981-01-27 | 1982-02-02 | The United States Of America As Represented By The Secretary Of Agriculture | Chemotherapeutically active maytansinoids from Trewia nudiflora |
JPS57192389A (en) | 1981-05-20 | 1982-11-26 | Takeda Chem Ind Ltd | Novel maytansinoid |
US4596792A (en) | 1981-09-04 | 1986-06-24 | The Regents Of The University Of California | Safe vaccine for hepatitis containing polymerized serum albumin |
US4741900A (en) | 1982-11-16 | 1988-05-03 | Cytogen Corporation | Antibody-metal ion complexes |
US4601978A (en) | 1982-11-24 | 1986-07-22 | The Regents Of The University Of California | Mammalian metallothionein promoter system |
US4560655A (en) | 1982-12-16 | 1985-12-24 | Immunex Corporation | Serum-free cell culture medium and process for making same |
US4657866A (en) | 1982-12-21 | 1987-04-14 | Sudhir Kumar | Serum-free, synthetic, completely chemically defined tissue culture media |
US4767704A (en) | 1983-10-07 | 1988-08-30 | Columbia University In The City Of New York | Protein-free culture medium |
US4599231A (en) | 1984-03-09 | 1986-07-08 | Scripps Clinic And Research Foundation | Synthetic hepatitis B virus vaccine including both T cell and B cell determinants |
US4599230A (en) | 1984-03-09 | 1986-07-08 | Scripps Clinic And Research Foundation | Synthetic hepatitis B virus vaccine including both T cell and B cell determinants |
US4965199A (en) | 1984-04-20 | 1990-10-23 | Genentech, Inc. | Preparation of functional human factor VIII in mammalian cells using methotrexate based selection |
US4970198A (en) | 1985-10-17 | 1990-11-13 | American Cyanamid Company | Antitumor antibiotics (LL-E33288 complex) |
US4596556A (en) | 1985-03-25 | 1986-06-24 | Bioject, Inc. | Hypodermic injection apparatus |
US4601903A (en) | 1985-05-01 | 1986-07-22 | The United States Of America As Represented By The Department Of Health And Human Services | Vaccine against Neisseria meningitidis Group B serotype 2 invasive disease |
GB8516415D0 (en) | 1985-06-28 | 1985-07-31 | Celltech Ltd | Culture of animal cells |
US4676980A (en) | 1985-09-23 | 1987-06-30 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Target specific cross-linked heteroantibodies |
US4927762A (en) | 1986-04-01 | 1990-05-22 | Cell Enterprises, Inc. | Cell culture medium with antioxidant |
US5567610A (en) | 1986-09-04 | 1996-10-22 | Bioinvent International Ab | Method of producing human monoclonal antibodies and kit therefor |
US5075109A (en) | 1986-10-24 | 1991-12-24 | Southern Research Institute | Method of potentiating an immune response |
US5811128A (en) | 1986-10-24 | 1998-09-22 | Southern Research Institute | Method for oral or rectal delivery of microencapsulated vaccines and compositions therefor |
US4886499A (en) | 1986-12-18 | 1989-12-12 | Hoffmann-La Roche Inc. | Portable injection appliance |
IL85035A0 (en) | 1987-01-08 | 1988-06-30 | Int Genetic Eng | Polynucleotide molecule,a chimeric antibody with specificity for human b cell surface antigen,a process for the preparation and methods utilizing the same |
US5079233A (en) | 1987-01-30 | 1992-01-07 | American Cyanamid Company | N-acyl derivatives of the LL-E33288 antitumor antibiotics, composition and methods for using the same |
GB8704027D0 (en) | 1987-02-20 | 1987-03-25 | Owen Mumford Ltd | Syringe needle combination |
DE3883899T3 (de) | 1987-03-18 | 1999-04-22 | Sb2 Inc | Geänderte antikörper. |
US4849222A (en) | 1987-03-24 | 1989-07-18 | The Procter & Gamble Company | Mixtures for treating hypercholesterolemia |
US4790824A (en) | 1987-06-19 | 1988-12-13 | Bioject, Inc. | Non-invasive hypodermic injection device |
US4941880A (en) | 1987-06-19 | 1990-07-17 | Bioject, Inc. | Pre-filled ampule and non-invasive hypodermic injection device assembly |
US4940460A (en) | 1987-06-19 | 1990-07-10 | Bioject, Inc. | Patient-fillable and non-invasive hypodermic injection device assembly |
US4975278A (en) | 1988-02-26 | 1990-12-04 | Bristol-Myers Company | Antibody-enzyme conjugates in combination with prodrugs for the delivery of cytotoxic agents to tumor cells |
US5004697A (en) | 1987-08-17 | 1991-04-02 | Univ. Of Ca | Cationized antibodies for delivery through the blood-brain barrier |
US5606040A (en) | 1987-10-30 | 1997-02-25 | American Cyanamid Company | Antitumor and antibacterial substituted disulfide derivatives prepared from compounds possessing a methyl-trithio group |
US5770701A (en) | 1987-10-30 | 1998-06-23 | American Cyanamid Company | Process for preparing targeted forms of methyltrithio antitumor agents |
US5053394A (en) | 1988-09-21 | 1991-10-01 | American Cyanamid Company | Targeted forms of methyltrithio antitumor agents |
JP2670680B2 (ja) | 1988-02-24 | 1997-10-29 | 株式会社ビーエムジー | 生理活性物質含有ポリ乳酸系微小球およびその製造法 |
US5339163A (en) | 1988-03-16 | 1994-08-16 | Canon Kabushiki Kaisha | Automatic exposure control device using plural image plane detection areas |
JPH01287029A (ja) | 1988-05-13 | 1989-11-17 | Mect Corp | 新規抗ウィルス剤 |
ATE135397T1 (de) | 1988-09-23 | 1996-03-15 | Cetus Oncology Corp | Zellenzuchtmedium für erhöhtes zellenwachstum, zur erhöhung der langlebigkeit und expression der produkte |
GB8823869D0 (en) | 1988-10-12 | 1988-11-16 | Medical Res Council | Production of antibodies |
FR2638359A1 (fr) | 1988-11-03 | 1990-05-04 | Tino Dalto | Guide de seringue avec reglage de la profondeur de penetration de l'aiguille dans la peau |
US5175384A (en) | 1988-12-05 | 1992-12-29 | Genpharm International | Transgenic mice depleted in mature t-cells and methods for making transgenic mice |
US5530101A (en) | 1988-12-28 | 1996-06-25 | Protein Design Labs, Inc. | Humanized immunoglobulins |
DE3920358A1 (de) | 1989-06-22 | 1991-01-17 | Behringwerke Ag | Bispezifische und oligospezifische, mono- und oligovalente antikoerperkonstrukte, ihre herstellung und verwendung |
DE69029036T2 (de) | 1989-06-29 | 1997-05-22 | Medarex Inc | Bispezifische reagenzien für die aids-therapie |
US5690938A (en) | 1989-07-07 | 1997-11-25 | Oravax, Inc. | Oral immunization with multiple particulate antigen delivery system |
US5518725A (en) | 1989-09-25 | 1996-05-21 | University Of Utah Research Foundation | Vaccine compositions and method for induction of mucosal immune response via systemic vaccination |
US5208020A (en) | 1989-10-25 | 1993-05-04 | Immunogen Inc. | Cytotoxic agents comprising maytansinoids and their therapeutic use |
CA2026147C (en) | 1989-10-25 | 2006-02-07 | Ravi J. Chari | Cytotoxic agents comprising maytansinoids and their therapeutic use |
US5238843A (en) | 1989-10-27 | 1993-08-24 | Genencor International, Inc. | Method for cleaning a surface on which is bound a glycoside-containing substance |
US5312335A (en) | 1989-11-09 | 1994-05-17 | Bioject Inc. | Needleless hypodermic injection device |
US5064413A (en) | 1989-11-09 | 1991-11-12 | Bioject, Inc. | Needleless hypodermic injection device |
EP1690935A3 (en) | 1990-01-12 | 2008-07-30 | Abgenix, Inc. | Generation of xenogeneic antibodies |
US5061620A (en) | 1990-03-30 | 1991-10-29 | Systemix, Inc. | Human hematopoietic stem cell |
US5268164A (en) | 1990-04-23 | 1993-12-07 | Alkermes, Inc. | Increasing blood-brain barrier permeability with permeabilizer peptides |
US5112596A (en) | 1990-04-23 | 1992-05-12 | Alkermes, Inc. | Method for increasing blood-brain barrier permeability by administering a bradykinin agonist of blood-brain barrier permeability |
US5643577A (en) | 1990-04-24 | 1997-07-01 | The University Of Newcastle Research Associates Limited | Oral vaccine comprising antigen surface-associated with red blood cells |
AP249A (en) | 1990-04-24 | 1993-03-17 | Biota Scient Management Pty Ltd | Anti-viral compounds. |
US5229275A (en) | 1990-04-26 | 1993-07-20 | Akzo N.V. | In-vitro method for producing antigen-specific human monoclonal antibodies |
US5427908A (en) | 1990-05-01 | 1995-06-27 | Affymax Technologies N.V. | Recombinant library screening methods |
KR0149181B1 (ko) | 1990-06-29 | 1998-08-17 | 데이비드 알, 맥지 | 형질전환된 미생물에 의한 멜라닌의 제조방법 |
US5190521A (en) | 1990-08-22 | 1993-03-02 | Tecnol Medical Products, Inc. | Apparatus and method for raising a skin wheal and anesthetizing skin |
US5625126A (en) | 1990-08-29 | 1997-04-29 | Genpharm International, Inc. | Transgenic non-human animals for producing heterologous antibodies |
US5661016A (en) | 1990-08-29 | 1997-08-26 | Genpharm International Inc. | Transgenic non-human animals capable of producing heterologous antibodies of various isotypes |
KR100272077B1 (ko) | 1990-08-29 | 2000-11-15 | 젠팜인터내셔날,인코포레이티드 | 이종 항체를 생산할 수 있는 전이유전자를 가진 인간이외의 동물 |
US5545806A (en) | 1990-08-29 | 1996-08-13 | Genpharm International, Inc. | Ransgenic non-human animals for producing heterologous antibodies |
US5633425A (en) | 1990-08-29 | 1997-05-27 | Genpharm International, Inc. | Transgenic non-human animals capable of producing heterologous antibodies |
US5714374A (en) | 1990-09-12 | 1998-02-03 | Rutgers University | Chimeric rhinoviruses |
US5122469A (en) | 1990-10-03 | 1992-06-16 | Genentech, Inc. | Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins |
WO1992006691A1 (en) | 1990-10-19 | 1992-04-30 | Biota Scientific Management Pty. Ltd. | Anti-viral compounds that bind the active site of influenza neuramidase and display in vivo activity against orthomyxovirus and paramyxovirus |
US5508192A (en) | 1990-11-09 | 1996-04-16 | Board Of Regents, The University Of Texas System | Bacterial host strains for producing proteolytically sensitive polypeptides |
US5264365A (en) | 1990-11-09 | 1993-11-23 | Board Of Regents, The University Of Texas System | Protease-deficient bacterial strains for production of proteolytically sensitive polypeptides |
ATE164395T1 (de) | 1990-12-03 | 1998-04-15 | Genentech Inc | Verfahren zur anreicherung von proteinvarianten mit geänderten bindungseigenschaften |
US5527288A (en) | 1990-12-13 | 1996-06-18 | Elan Medical Technologies Limited | Intradermal drug delivery device and method for intradermal delivery of drugs |
US5571894A (en) | 1991-02-05 | 1996-11-05 | Ciba-Geigy Corporation | Recombinant antibodies specific for a growth factor receptor |
WO1992019971A1 (en) | 1991-04-30 | 1992-11-12 | Alkermes, Inc. | Cationized antibodies against intracellular proteins |
DK0590058T3 (da) | 1991-06-14 | 2004-03-29 | Genentech Inc | Humaniseret heregulin-antistof |
GB9114948D0 (en) | 1991-07-11 | 1991-08-28 | Pfizer Ltd | Process for preparing sertraline intermediates |
GB9118204D0 (en) | 1991-08-23 | 1991-10-09 | Weston Terence E | Needle-less injector |
SE9102652D0 (sv) | 1991-09-13 | 1991-09-13 | Kabi Pharmacia Ab | Injection needle arrangement |
WO1993006217A1 (en) | 1991-09-19 | 1993-04-01 | Genentech, Inc. | EXPRESSION IN E. COLI OF ANTIBODY FRAGMENTS HAVING AT LEAST A CYSTEINE PRESENT AS A FREE THIOL, USE FOR THE PRODUCTION OF BIFUNCTIONAL F(ab')2 ANTIBODIES |
DK0605522T3 (da) | 1991-09-23 | 2000-01-17 | Medical Res Council | Fremgangsmåde til fremstilling af humaniserede antistoffer |
ES2136092T3 (es) | 1991-09-23 | 1999-11-16 | Medical Res Council | Procedimientos para la produccion de anticuerpos humanizados. |
US5362852A (en) | 1991-09-27 | 1994-11-08 | Pfizer Inc. | Modified peptide derivatives conjugated at 2-hydroxyethylamine moieties |
US5587458A (en) | 1991-10-07 | 1996-12-24 | Aronex Pharmaceuticals, Inc. | Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof |
US5288502A (en) | 1991-10-16 | 1994-02-22 | The University Of Texas System | Preparation and uses of multi-phase microspheres |
WO1993008829A1 (en) | 1991-11-04 | 1993-05-13 | The Regents Of The University Of California | Compositions that mediate killing of hiv-infected cells |
WO1993009764A1 (en) | 1991-11-19 | 1993-05-27 | Center For Innovative Technology | Combined virustatic antimediator (covam) treatment of common colds |
JPH0826057B2 (ja) | 1992-01-16 | 1996-03-13 | 株式会社ディ・ディ・エス研究所 | シアル酸オリゴ糖誘導体及び微粒子キャリヤー |
US5667988A (en) | 1992-01-27 | 1997-09-16 | The Scripps Research Institute | Methods for producing antibody libraries using universal or randomized immunoglobulin light chains |
EP1997894B1 (en) | 1992-02-06 | 2011-03-30 | Novartis Vaccines and Diagnostics, Inc. | Biosynthetic binding protein for cancer marker |
US5328483A (en) | 1992-02-27 | 1994-07-12 | Jacoby Richard M | Intradermal injection device with medication and needle guard |
US5733743A (en) | 1992-03-24 | 1998-03-31 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
US5326856A (en) | 1992-04-09 | 1994-07-05 | Cytogen Corporation | Bifunctional isothiocyanate derived thiocarbonyls as ligands for metal binding |
ZA932522B (en) | 1992-04-10 | 1993-12-20 | Res Dev Foundation | Immunotoxins directed against c-erbB-2(HER/neu) related surface antigens |
JP2904647B2 (ja) | 1992-06-12 | 1999-06-14 | 株式会社蛋白工学研究所 | 5−ブロム−4−クロロインド−3−イル−2−シアル酸の製造方法 |
ATE348175T1 (de) | 1992-07-17 | 2007-01-15 | Dana Farber Cancer Inst Inc | Method eder intrazellularen bindung von zielgerichteten molekülen |
US5383851A (en) | 1992-07-24 | 1995-01-24 | Bioject Inc. | Needleless hypodermic injection device |
WO1994002178A1 (en) | 1992-07-27 | 1994-02-03 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Targeting of liposomes to the blood-brain barrier |
AU668423B2 (en) | 1992-08-17 | 1996-05-02 | Genentech Inc. | Bispecific immunoadhesins |
US5569189A (en) | 1992-09-28 | 1996-10-29 | Equidyne Systems, Inc. | hypodermic jet injector |
DE69328435T2 (de) | 1992-10-22 | 2000-09-14 | Kirin Brewery | Neues sphingolglycolipid und verwendung davon |
US5334144A (en) | 1992-10-30 | 1994-08-02 | Becton, Dickinson And Company | Single use disposable needleless injector |
US5807722A (en) | 1992-10-30 | 1998-09-15 | Bioengineering Resources, Inc. | Biological production of acetic acid from waste gases with Clostridium ljungdahlii |
US5736137A (en) | 1992-11-13 | 1998-04-07 | Idec Pharmaceuticals Corporation | Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma |
WO1994011026A2 (en) | 1992-11-13 | 1994-05-26 | Idec Pharmaceuticals Corporation | Therapeutic application of chimeric and radiolabeled antibodies to human b lymphocyte restricted differentiation antigen for treatment of b cell lymphoma |
US5635483A (en) | 1992-12-03 | 1997-06-03 | Arizona Board Of Regents Acting On Behalf Of Arizona State University | Tumor inhibiting tetrapeptide bearing modified phenethyl amides |
US5780588A (en) | 1993-01-26 | 1998-07-14 | Arizona Board Of Regents | Elucidation and synthesis of selected pentapeptides |
US5374541A (en) | 1993-05-04 | 1994-12-20 | The Scripps Research Institute | Combined use of β-galactosidase and sialyltransferase coupled with in situ regeneration of CMP-sialic acid for one pot synthesis of oligosaccharides |
US20020037517A1 (en) | 1993-05-28 | 2002-03-28 | Hutchens T. William | Methods for sequencing biopolymers |
CA2163345A1 (en) | 1993-06-16 | 1994-12-22 | Susan Adrienne Morgan | Antibodies |
WO1995011010A1 (en) | 1993-10-22 | 1995-04-27 | Genentech, Inc. | Methods and compositions for microencapsulation of antigens for use as vaccines |
US5369017A (en) | 1994-02-04 | 1994-11-29 | The Scripps Research Institute | Process for solid phase glycopeptide synthesis |
CA2181237A1 (en) | 1994-02-25 | 1995-08-31 | Subramaniam Sabesan | 4-n-substituted sialic acids and their sialosides |
WO1995024176A1 (en) | 1994-03-07 | 1995-09-14 | Bioject, Inc. | Ampule filling device |
US5466220A (en) | 1994-03-08 | 1995-11-14 | Bioject, Inc. | Drug vial mixing and transfer device |
US5773001A (en) | 1994-06-03 | 1998-06-30 | American Cyanamid Company | Conjugates of methyltrithio antitumor agents and intermediates for their synthesis |
US5622701A (en) | 1994-06-14 | 1997-04-22 | Protein Design Labs, Inc. | Cross-reacting monoclonal antibodies specific for E- and P-selectin |
ATE306930T1 (de) | 1994-08-12 | 2005-11-15 | Immunomedics Inc | Für b-zell-lymphom und leukämiezellen spezifische immunkonjugate und humane antikörper |
US5639635A (en) | 1994-11-03 | 1997-06-17 | Genentech, Inc. | Process for bacterial production of polypeptides |
EP0794792A1 (en) | 1994-12-02 | 1997-09-17 | Chiron Corporation | Method of promoting an immune response with a bispecific antibody |
US5663149A (en) | 1994-12-13 | 1997-09-02 | Arizona Board Of Regents Acting On Behalf Of Arizona State University | Human cancer inhibitory pentapeptide heterocyclic and halophenyl amides |
US5599302A (en) | 1995-01-09 | 1997-02-04 | Medi-Ject Corporation | Medical injection system and method, gas spring thereof and launching device using gas spring |
US5731168A (en) | 1995-03-01 | 1998-03-24 | Genentech, Inc. | Method for making heteromultimeric polypeptides |
US5840523A (en) | 1995-03-01 | 1998-11-24 | Genetech, Inc. | Methods and compositions for secretion of heterologous polypeptides |
US6673533B1 (en) | 1995-03-10 | 2004-01-06 | Meso Scale Technologies, Llc. | Multi-array multi-specific electrochemiluminescence testing |
US5641870A (en) | 1995-04-20 | 1997-06-24 | Genentech, Inc. | Low pH hydrophobic interaction chromatography for antibody purification |
US5869046A (en) | 1995-04-14 | 1999-02-09 | Genentech, Inc. | Altered polypeptides with increased half-life |
WO1996036436A1 (en) | 1995-04-25 | 1996-11-21 | Irori | Remotely programmable matrices with memories and uses thereof |
DE69637481T2 (de) | 1995-04-27 | 2009-04-09 | Amgen Fremont Inc. | Aus immunisierten Xenomäusen stammende menschliche Antikörper gegen IL-8 |
EP0823941A4 (en) | 1995-04-28 | 2001-09-19 | Abgenix Inc | HUMAN ANTIBODIES DERIVED FROM IMMUNIZED XENO MOUSES |
US5730723A (en) | 1995-10-10 | 1998-03-24 | Visionary Medical Products Corporation, Inc. | Gas pressured needle-less injection device and method |
US5714586A (en) | 1995-06-07 | 1998-02-03 | American Cyanamid Company | Methods for the preparation of monomeric calicheamicin derivative/carrier conjugates |
US5837234A (en) | 1995-06-07 | 1998-11-17 | Cytotherapeutics, Inc. | Bioartificial organ containing cells encapsulated in a permselective polyether suflfone membrane |
US5712374A (en) | 1995-06-07 | 1998-01-27 | American Cyanamid Company | Method for the preparation of substantiallly monomeric calicheamicin derivative/carrier conjugates |
US6265150B1 (en) | 1995-06-07 | 2001-07-24 | Becton Dickinson & Company | Phage antibodies |
JPH11510164A (ja) | 1995-07-26 | 1999-09-07 | マキシム ファーマシューティカルズ | ポリヌクレオチドの粘膜送達 |
DE19544393A1 (de) | 1995-11-15 | 1997-05-22 | Hoechst Schering Agrevo Gmbh | Synergistische herbizide Mischungen |
US5893397A (en) | 1996-01-12 | 1999-04-13 | Bioject Inc. | Medication vial/syringe liquid-transfer apparatus |
NZ331189A (en) | 1996-03-01 | 1999-06-29 | Biota Scient Management | Method of detection of influenza virus using neuraminidase binders |
WO1997038123A1 (en) | 1996-04-05 | 1997-10-16 | Board Of Regents, The University Of Texas System | Methods for producing soluble, biologically-active disulfide bond-containing eukaryotic proteins in bacterial cells |
GB9607549D0 (en) | 1996-04-11 | 1996-06-12 | Weston Medical Ltd | Spring-powered dispensing device |
US5922845A (en) | 1996-07-11 | 1999-07-13 | Medarex, Inc. | Therapeutic multispecific compounds comprised of anti-Fcα receptor antibodies |
US6340702B1 (en) | 1996-07-22 | 2002-01-22 | Sankyo Company, Limited | Neuraminic acid derivatives, their preparation and their medical use |
US6506564B1 (en) | 1996-07-29 | 2003-01-14 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
IL129762A0 (en) | 1996-11-14 | 2000-02-29 | Biota Scient Management | Novel macromolecules methods for their preparation pharmaceutical compositions thereof and their use as anti-influenza agents |
EP2305027B1 (en) | 1996-12-03 | 2014-07-02 | Amgen Fremont Inc. | Transgenic mammals having human Ig loci including plural VH and Vkappa regions and antibodies produced therefrom |
TW555562B (en) | 1996-12-27 | 2003-10-01 | Kirin Brewery | Method for activation of human antigen-presenting cells, activated human antigen-presenting cells and use thereof |
AU733282B2 (en) | 1997-04-18 | 2001-05-10 | Novartis Ag | Neoglycoproteins |
US5993412A (en) | 1997-05-19 | 1999-11-30 | Bioject, Inc. | Injection apparatus |
US6083715A (en) | 1997-06-09 | 2000-07-04 | Board Of Regents, The University Of Texas System | Methods for producing heterologous disulfide bond-containing polypeptides in bacterial cells |
JPH1135593A (ja) | 1997-07-18 | 1999-02-09 | Daikin Ind Ltd | 2−フルオロフコシル−n−アロイルグルコサミン誘導体及びその中間物、並びにそれらの製造方法 |
TW477783B (en) | 1997-12-12 | 2002-03-01 | Gilead Sciences Inc | Novel compounds useful as neuraminidase inhibitors and pharmaceutical compositions containing same |
IT1298087B1 (it) | 1998-01-08 | 1999-12-20 | Fiderm S R L | Dispositivo per il controllo della profondita' di penetrazione di un ago, in particolare applicabile ad una siringa per iniezioni |
JP2002507410A (ja) | 1998-03-27 | 2002-03-12 | プロルーム・リミテッド | ルシフェラーゼ、蛍光タンパク質、ルシフェラーゼおよび蛍光タンパク質をコードする核酸および、診断、高処理スクリーニングおよび新規アイテムにおけるその使用 |
JP2002510481A (ja) | 1998-04-02 | 2002-04-09 | ジェネンテック・インコーポレーテッド | 抗体変異体及びその断片 |
EP2261229A3 (en) | 1998-04-20 | 2011-03-23 | GlycArt Biotechnology AG | Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity |
US6455571B1 (en) | 1998-04-23 | 2002-09-24 | Abbott Laboratories | Inhibitors of neuraminidases |
JP4469933B2 (ja) | 1998-05-06 | 2010-06-02 | ジェネンテック, インコーポレイテッド | イオン交換クロマトグラフィによるタンパク質精製 |
JP3773153B2 (ja) | 1998-05-29 | 2006-05-10 | 独立行政法人理化学研究所 | シアル酸誘導体 |
US6528286B1 (en) | 1998-05-29 | 2003-03-04 | Genentech, Inc. | Mammalian cell culture process for producing glycoproteins |
EP2306195A3 (en) | 1998-09-18 | 2012-04-25 | Massachusetts Institute of Technology | Biological applications of semiconductor nanocrystals |
FR2783523B1 (fr) | 1998-09-21 | 2006-01-20 | Goemar Lab Sa | Fuco-oligosaccharides, enzyme pour leur preparation a partir des fucanes, bacterie productrice de l'enzyme et applications des fuco-oligosaccharides a la protection des plantes |
US6994966B2 (en) | 2000-02-17 | 2006-02-07 | Glycominds Ltd. | Combinatorial complex carbohydrate libraries and methods for the manufacture and uses thereof |
US6696304B1 (en) | 1999-02-24 | 2004-02-24 | Luminex Corporation | Particulate solid phase immobilized protein quantitation |
AUPP913999A0 (en) | 1999-03-12 | 1999-04-01 | Biota Scientific Management Pty Ltd | Novel chemical compounds and their use |
US7090973B1 (en) | 1999-04-09 | 2006-08-15 | Oscient Pharmaceuticals Corporation | Nucleic acid sequences relating to Bacteroides fragilis for diagnostics and therapeutics |
US7854934B2 (en) | 1999-08-20 | 2010-12-21 | Sloan-Kettering Institute For Cancer Research | Glycoconjugates, glycoamino acids, intermediates thereto, and uses thereof |
US6824780B1 (en) | 1999-10-29 | 2004-11-30 | Genentech, Inc. | Anti-tumor antibody compositions and methods of use |
AUPQ422399A0 (en) | 1999-11-24 | 1999-12-16 | University Of New South Wales, The | Method of screening transformed or transfected cells |
US6727356B1 (en) | 1999-12-08 | 2004-04-27 | Epoch Pharmaceuticals, Inc. | Fluorescent quenching detection reagents and methods |
US7019129B1 (en) | 2000-05-09 | 2006-03-28 | Biosearch Technologies, Inc. | Dark quenchers for donor-acceptor energy transfer |
US7863020B2 (en) | 2000-06-28 | 2011-01-04 | Glycofi, Inc. | Production of sialylated N-glycans in lower eukaryotes |
US6514221B2 (en) | 2000-07-27 | 2003-02-04 | Brigham And Women's Hospital, Inc. | Blood-brain barrier opening |
US20020065259A1 (en) | 2000-08-30 | 2002-05-30 | Schatzberg Alan F. | Glucocorticoid blocking agents for increasing blood-brain barrier permeability |
AUPR001000A0 (en) | 2000-09-08 | 2000-10-05 | Biota Scientific Management Pty Ltd | Novel chemical compounds and their use |
US7034036B2 (en) | 2000-10-30 | 2006-04-25 | Pain Therapeutics, Inc. | Inhibitors of ABC drug transporters at the blood-brain barrier |
US20030083299A1 (en) | 2000-11-04 | 2003-05-01 | Ferguson Ian A. | Non-invasive delivery of polypeptides through the blood-brain barrier |
JP2002153272A (ja) | 2000-11-24 | 2002-05-28 | Inst Of Physical & Chemical Res | 生体分子マイクロアレイ |
US7754208B2 (en) | 2001-01-17 | 2010-07-13 | Trubion Pharmaceuticals, Inc. | Binding domain-immunoglobulin fusion proteins |
WO2002086096A2 (en) | 2001-01-23 | 2002-10-31 | University Of Rochester Medical Center | Methods of producing or identifying intrabodies in eukaryotic cells |
US6884869B2 (en) | 2001-04-30 | 2005-04-26 | Seattle Genetics, Inc. | Pentapeptide compounds and uses related thereto |
DE10121982B4 (de) | 2001-05-05 | 2008-01-24 | Lts Lohmann Therapie-Systeme Ag | Nanopartikel aus Protein mit gekoppeltem Apolipoprotein E zur Überwindung der Blut-Hirn-Schranke und Verfahren zu ihrer Herstellung |
WO2003009812A2 (en) | 2001-07-25 | 2003-02-06 | New York University | Use of glycosylceramides as adjuvants for vaccines against infections and cancer |
ATE445838T1 (de) | 2001-07-25 | 2009-10-15 | Raptor Pharmaceutical Inc | Zusammensetzungen und verfahren zur modulation des transports durch die blut-hirn-schranke |
EP1423510A4 (en) | 2001-08-03 | 2005-06-01 | Glycart Biotechnology Ag | ANTIBODY GLYCOSYLATION VARIANTS WITH INCREASED CELL CYTOTOXICITY DEPENDENT OF ANTIBODIES |
BRPI0213207B1 (pt) | 2001-10-10 | 2021-06-15 | Novo Nordisk A/S | Processo in vitro, isento de células, para a remodelagem de um peptídeo e processos para formar um conjugado entre peptídeos e um grupo de modificação |
KR20040077655A (ko) | 2001-10-19 | 2004-09-06 | 슈페리어 마이크로파우더스 엘엘씨 | 전자 형상 증착용 테잎 조성물 |
AUPR879601A0 (en) | 2001-11-09 | 2001-12-06 | Biota Scientific Management Pty Ltd | Novel chemical compounds and their use |
US20040018501A1 (en) | 2001-11-21 | 2004-01-29 | Keith Allen | Methods and systems for analyzing complex biological systems |
EP2295468B1 (en) | 2002-02-14 | 2015-07-08 | Immunomedics, Inc. | Anti-CD20 antibodies and fusion proteins thereof and methods of use |
US20030162695A1 (en) | 2002-02-27 | 2003-08-28 | Schatzberg Alan F. | Glucocorticoid blocking agents for increasing blood-brain barrier permeability |
US7317091B2 (en) | 2002-03-01 | 2008-01-08 | Xencor, Inc. | Optimized Fc variants |
AU2003219277A1 (en) | 2002-03-14 | 2003-09-29 | Medical Research Council | Intracellular antibodies |
MXPA04011249A (es) | 2002-05-14 | 2005-06-06 | Chiron Srl | Vacunas mucosales con adyuvante de quitosano y antigenos meningococicos. |
WO2004005313A2 (en) | 2002-07-08 | 2004-01-15 | Pliva - Istrazivacki Institut D.O.O. | Hybrid molecules of macrolides with steroid/non-steroid anti-inflammatory, antineoplastic and antiviral active molecules |
US20080070324A1 (en) | 2002-07-15 | 2008-03-20 | Floyd Alton D | Quantity control device for microscope slide staining assays |
EP1391213A1 (en) | 2002-08-21 | 2004-02-25 | Boehringer Ingelheim International GmbH | Compositions and methods for treating cancer using maytansinoid CD44 antibody immunoconjugates and chemotherapeutic agents |
US20040062682A1 (en) | 2002-09-30 | 2004-04-01 | Rakow Neal Anthony | Colorimetric sensor |
LT3284753T (lt) | 2002-10-17 | 2020-06-10 | Genmab A/S | Žmogaus monokloniniai antikūnai prieš cd20, skirti naudoti gydant išsėtinę sklerozę |
AU2003302676A1 (en) | 2002-12-03 | 2004-06-23 | Blanchette Rockefeller Neurosciences Institute | Artificial low-density lipoprotein carriers for transport of substances across the blood-brain barrier |
PL212899B1 (pl) | 2002-12-16 | 2012-12-31 | Genentech Inc | Humanizowane przeciwcialo, kompozycja zawierajaca to przeciwcialo, wyrób fabryczny, przeciwcialo lub jego fragment do zastosowania w sposobie indukowania apoptozy, izolowany kwas nukleinowy, wektor ekspresji, komórka gospodarza, sposób wytwarzania przeciwciala lub jego fragmentu, plynny preparat i zastosowanie przeciwciala do wytwarzania leku |
AU2004204494B2 (en) | 2003-01-09 | 2011-09-29 | Macrogenics, Inc. | Identification and engineering of antibodies with variant Fc regions and methods of using same |
US8088387B2 (en) | 2003-10-10 | 2012-01-03 | Immunogen Inc. | Method of targeting specific cell populations using cell-binding agent maytansinoid conjugates linked via a non-cleavable linker, said conjugates, and methods of making said conjugates |
AR044388A1 (es) | 2003-05-20 | 2005-09-07 | Applied Molecular Evolution | Moleculas de union a cd20 |
US20040259142A1 (en) | 2003-06-04 | 2004-12-23 | Imperial College Innovations Limited | Products and methods |
EP2003196A3 (en) | 2003-06-09 | 2009-01-07 | The Regents of the University of Michigan | Compositions and methods for treating and diagnosing cancer |
JP4148844B2 (ja) | 2003-06-11 | 2008-09-10 | ソニー・エリクソン・モバイルコミュニケーションズ株式会社 | 情報端末装置及び音声付画像ファイルの出力方法 |
WO2005025511A2 (en) | 2003-09-10 | 2005-03-24 | Cedars-Sinai Medical Center | Potassium channel mediated delivery of agents through the blood-brain barrier |
WO2005027990A2 (en) | 2003-09-15 | 2005-03-31 | Univ Jefferson | Implants with attached silylated therapeutic agents |
CA2539914A1 (en) | 2003-09-22 | 2005-04-07 | Acidophil Llc | Small molecule compositions and methods for increasing drug efficiency using compositions thereof |
WO2005033663A2 (en) | 2003-09-30 | 2005-04-14 | Sequenom, Inc. | Methods of making substrates for mass spectrometry analysis and related devices |
US20050221337A1 (en) | 2003-10-02 | 2005-10-06 | Massachusetts Institute Of Technology | Microarrays and microspheres comprising oligosaccharides, complex carbohydrates or glycoproteins |
HUE031632T2 (en) | 2003-11-05 | 2017-07-28 | Roche Glycart Ag | Antigen binding molecules with enhanced Fc receptor binding affinity and effector function |
BR122018071808B8 (pt) | 2003-11-06 | 2020-06-30 | Seattle Genetics Inc | conjugado |
US20050255491A1 (en) | 2003-11-13 | 2005-11-17 | Lee Frank D | Small molecule and peptide arrays and uses thereof |
EP1723422A2 (en) | 2004-03-05 | 2006-11-22 | The Scripps Research Institute | High throughput glycan microarrays |
US20050221397A1 (en) | 2004-03-30 | 2005-10-06 | Northern Advancement Center For Science & Technology | RM2 antigen (beta1,4-GalNAc-disialyl-Lc4) as prostate cancer-associated antigen |
EP1740946B1 (en) | 2004-04-20 | 2013-11-06 | Genmab A/S | Human monoclonal antibodies against cd20 |
ITMI20040928A1 (it) | 2004-05-07 | 2004-08-07 | Uni Di Bologna Dipartiment O D | Procedura per la preparazione di coniugati della doxorubicina con l'albumina umana lattosaminata |
JP2008504531A (ja) | 2004-06-24 | 2008-02-14 | ザ スクリップス リサーチ インスティテュート | 切断可能なリンカーを有するアレイ |
RU2361880C2 (ru) | 2004-07-22 | 2009-07-20 | Дженентек, Инк. | Композиция антител к her2 |
US8022043B2 (en) | 2004-08-27 | 2011-09-20 | Albert Einstein College Of Medicine Of Yeshiva University | Ceramide derivatives as modulators of immunity and autoimmunity |
WO2006055925A2 (en) | 2004-11-19 | 2006-05-26 | Swiss Federal Institute Of Technology | Microarrays for analyte detection |
WO2006064983A1 (en) | 2004-12-14 | 2006-06-22 | Korea Research Institute Of Bioscience And Biotechnology | Monoclonal antibody specific human embryonic stem cell |
EP1833489A4 (en) | 2004-12-28 | 2011-08-03 | Univ Rockefeller | GLYCOLIPIDS AND RELATED ANALOGUES AS ANTIGENS OF NKT LYMPHOCYTES |
US7923013B2 (en) | 2004-12-28 | 2011-04-12 | The Rockefeller University | Glycolipids and analogues thereof as antigens for NKT cells |
ATE517183T1 (de) | 2005-03-31 | 2011-08-15 | Biomedics Inc | Monoklonaler antikörper gegen cd20 |
AU2006250888A1 (en) | 2005-05-24 | 2006-11-30 | Avesthagen Limited | A method for the production of a monoclonal antibody to CD20 for the treatment of B-cell lymphoma |
CN101282993A (zh) | 2005-06-02 | 2008-10-08 | 阿斯利康公司 | 针对cd20的抗体和其用途 |
US7781203B2 (en) | 2005-12-29 | 2010-08-24 | Corning Incorporated | Supports for assaying analytes and methods of making and using thereof |
WO2007133855A2 (en) | 2006-03-27 | 2007-11-22 | University Of Maryland Biotechnology Institute | Glycoprotein synthesis and remodeling by enzymatic transglycosylation |
PL2018564T3 (pl) | 2006-05-18 | 2010-12-31 | Veterinaermedizinische Univ Wien | Sposób wykrywania wirusów grypy |
EP2035034A4 (en) | 2006-06-09 | 2009-11-18 | Univ Maryland | GLYCOSYLATION-CONTROLLED ANTIBODY THERAPY |
WO2008006373A1 (en) | 2006-07-12 | 2008-01-17 | Merck Patent Gmbh | Solid-phase detection of terminal monosaccharides cleaved from glycosylated substrates |
EP2059593A2 (en) | 2006-08-18 | 2009-05-20 | Oncotherapy Science, Inc. | Treating or preventing cancers over-expressing reg4 or kiaa0101 |
EP2962697A1 (en) | 2006-11-27 | 2016-01-06 | diaDexus, Inc. | Ovr110 antibody compositions and methods of use |
EP2115461A4 (en) | 2007-01-18 | 2010-01-13 | Suomen Punainen Risti Veripalv | NEW SPECIFIC CELL BINDING AGENTS |
WO2008087257A1 (en) | 2007-01-18 | 2008-07-24 | Suomen Punainen Risti, Veripalvelu | Novel methods and reagents directed to production of cells |
SI2123271T1 (sl) | 2007-03-07 | 2012-05-31 | Daiichi Sankyo Co Ltd | Zdravilo za zdravljenje influence |
WO2008153615A2 (en) | 2007-03-07 | 2008-12-18 | Ada Technologies, Inc. | Preparing carbohydrate microarrays and conjugated nanoparticles |
US7960139B2 (en) | 2007-03-23 | 2011-06-14 | Academia Sinica | Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells |
EP2308514B1 (en) | 2007-03-23 | 2013-06-05 | to-BBB Holding B.V. | Conjugates for targeted drug delivery across the blood-brain barrier |
US7943330B2 (en) | 2007-03-23 | 2011-05-17 | Academia Sinica | Tailored glycoproteomic methods for the sequencing, mapping and identification of cellular glycoproteins |
WO2008128207A1 (en) | 2007-04-13 | 2008-10-23 | Academia Sinica | Alpha-galactosyl ceramide analogs and their use as immunotherapies |
JP2010526066A (ja) | 2007-04-23 | 2010-07-29 | シェーリング コーポレイション | 抗mdl−1抗体 |
US20110306049A1 (en) | 2007-08-24 | 2011-12-15 | Tokyo Institute Of Technology | Method for detecting gynecologic cancer |
JP5509080B2 (ja) | 2007-08-31 | 2014-06-04 | アカデミア シニカ | 抗インフルエンザ活性を有するオセルタミビル含有ホスホネートコンジナーの合成 |
FR2921387B1 (fr) | 2007-09-26 | 2012-04-20 | Sanofi Pasteur | Procede de production du virus de la grippe |
US8647626B2 (en) | 2007-10-02 | 2014-02-11 | Avaxia Biologics, Incorporated | Compositions comprising TNF-specific antibodies for oral delivery |
US20090123439A1 (en) | 2007-11-09 | 2009-05-14 | The Jackson Laboratory | Diagnostic and prognosis methods for cancer stem cells |
CA2710779C (en) | 2007-12-31 | 2017-06-20 | Bayer Schering Pharma Aktiengesellschaft | Antibodies to tnf.alpha. |
BRPI0908968A8 (pt) | 2008-03-21 | 2016-06-28 | Danisco Us Inc | composição enriquecidas com hemicelulase para potencializar a hidrólise de biomassa |
EP2272854B1 (en) | 2008-03-25 | 2015-08-05 | Riken | Novel glycolipid and use thereof |
US8383554B2 (en) | 2008-04-14 | 2013-02-26 | Academia Sinica | Quantitative microarray of intact glycolipid CD1d interaction and correlation with cell-based cytokine production |
US8906832B2 (en) | 2008-04-30 | 2014-12-09 | Academia Sinica | Quantitative analysis of carbohydrate-protein interactions using glycan microarrays: determination of surface and solution dissociation constants |
WO2009140853A1 (en) | 2008-05-23 | 2009-11-26 | The University Of Hong Kong | Combination therapy for the treatment of influenza |
US20110137570A1 (en) | 2008-05-30 | 2011-06-09 | Anthony Lapadula | Methods for structural analysis of glycans |
JP2011524375A (ja) | 2008-06-16 | 2011-09-01 | アカデミア シニカ | GloboHおよびSSEA3に特異的な免疫反応を誘起する組成物および癌治療におけるその使用 |
NZ590064A (en) | 2008-06-16 | 2012-05-25 | Academia Sinica | Cancer diagnosis based on levels of antibodies against globo h and its fragments |
JP2010014691A (ja) | 2008-06-20 | 2010-01-21 | Igaku Seibutsugaku Kenkyusho:Kk | 腹水中のメソテリン及び/又は巨核球増強因子を検出するための方法、キット、試薬及び装置 |
US7928077B2 (en) | 2008-07-11 | 2011-04-19 | Academia Sinica | Alpha-galactosyl ceramide analogs and their use as immunotherapies |
DK2318832T3 (da) | 2008-07-15 | 2014-01-20 | Academia Sinica | Glycan-arrays på PTFE-lignende aluminiumcoatede objektglas og relaterede fremgangsmåder |
US20100022916A1 (en) | 2008-07-24 | 2010-01-28 | Javanbakhsh Esfandiari | Method and Apparatus for Collecting and Preparing Biological Samples for Testing |
WO2010111703A1 (en) | 2009-03-27 | 2010-09-30 | Academia Sinica | Alpha-selective sialyl phosphate donors for preparation of sialosides and sialoside arrays for influenza virus detection |
WO2011005756A1 (en) | 2009-07-06 | 2011-01-13 | Puretech Ventures, Llc | Delivery of agents targeted to microbiota niches |
WO2011006237A1 (en) | 2009-07-15 | 2011-01-20 | The University Of British Columbia | 2,3-fluorinated glycosides as neuraminidase inhibitors and their use as anti-virals |
US20120171201A1 (en) | 2009-07-22 | 2012-07-05 | Enzon Pharmaceuticals, Inc. | Methods of treating her2 positive cancer with her2 receptor antagonist in combination with multi-arm polymeric conjugates of 7-ethyl-10-hydroxycamptothecin |
US20120172329A1 (en) | 2009-09-14 | 2012-07-05 | Thailand Excellence Center For Tissue Engineering | Phytochemical compositions including xanthones for anti-inflammatory, anti-cytokine storm, and other uses |
US10087236B2 (en) | 2009-12-02 | 2018-10-02 | Academia Sinica | Methods for modifying human antibodies by glycan engineering |
WO2011074621A1 (ja) | 2009-12-18 | 2011-06-23 | 株式会社医学生物学研究所 | メソセリン(msln)に対する抗体及びその用途 |
EP2347769A1 (en) | 2010-01-20 | 2011-07-27 | Glycotope GmbH | Cancer stem cell markers and uses thereof |
JP2013519682A (ja) | 2010-02-11 | 2013-05-30 | アレクシオン ファーマシューティカルズ, インコーポレイテッド | 抗cd200抗体を使用する治療方法 |
AR080513A1 (es) | 2010-03-12 | 2012-04-11 | Inmunogen Inc | Moleculas de union cd37 y sus inmunoconjugados |
WO2011130332A1 (en) | 2010-04-12 | 2011-10-20 | Academia Sinica | Glycan arrays for high throughput screening of viruses |
WO2011130624A2 (en) | 2010-04-16 | 2011-10-20 | Immune Disease Institute, Inc. | Sustained polypeptide expression from synthetic, modified rnas and uses thereof |
US9403855B2 (en) | 2010-05-10 | 2016-08-02 | Academia Sinica | Zanamivir phosphonate congeners with anti-influenza activity and determining oseltamivir susceptibility of influenza viruses |
WO2011145957A1 (en) | 2010-05-20 | 2011-11-24 | Auckland Uniservices Limited | Agents and methods for detection and/or imaging of hypoxia |
CN103080130B (zh) | 2010-05-27 | 2016-08-17 | 默沙东公司 | 制备具有改进特性的抗体的方法 |
GB201015569D0 (en) | 2010-09-16 | 2010-10-27 | Medical Res Council | Blood assay for prions |
WO2012082635A1 (en) | 2010-12-13 | 2012-06-21 | Ancora Pharmaceuticals, Inc. | Synthetic oligosaccharide group a streptococcus |
CN103748103A (zh) | 2011-01-05 | 2014-04-23 | 台湾大学 | 制备鞘糖脂的方法及其应用 |
US10851174B2 (en) | 2011-03-03 | 2020-12-01 | University Of Maryland, Baltimore | Core fucosylated glycopeptides and glycoproteins: chemoenzymatic synthesis and uses thereof |
WO2012135049A1 (en) * | 2011-03-25 | 2012-10-04 | University Of Georgia Research Foundation, Inc. | Compounds and methods for chemical and chemo-enzymatic synthesis of complex glycans |
AU2012258907A1 (en) | 2011-05-25 | 2013-11-07 | Merck Sharp & Dohme Corp. | Method for preparing Fc-containing polypeptides having improved properties |
EP3418300B1 (en) | 2011-07-18 | 2020-10-28 | Institute for Research in Biomedicine | Neutralizing anti-influenza a virus antibodies and uses thereof |
KR102021159B1 (ko) | 2011-08-12 | 2019-09-11 | 닛산 가가쿠 가부시키가이샤 | 트리시클릭 헤테로시클릭 화합물 및 jak 저해제 |
WO2013074598A1 (en) | 2011-11-18 | 2013-05-23 | Merck Sharp & Dohme Corp. | Fc CONTAINING POLYPEPTIDES HAVING INCREASED ANTI-INFLAMMATORY PROPERTIES AND INCREASED FcRN BINDING |
EP2604281B1 (en) | 2011-12-14 | 2014-07-30 | Centre National de la Recherche Scientifique (CNRS) | Clicked somatostatin conjugated analogs for biological applications |
EP2812442B9 (en) | 2012-02-10 | 2023-02-15 | University of Maryland, Baltimore | Chemoenzymatic glycoengineering of antibodies and fc fragments thereof |
WO2013130603A1 (en) | 2012-02-27 | 2013-09-06 | Board Of Regents, The University Of Texas System | Ganglioside gd2 as a marker and target on cancer stem cells |
JP2015514113A (ja) | 2012-04-02 | 2015-05-18 | メリマック ファーマシューティカルズ インコーポレーティッド | 単一特異性および二重特異性抗igf−1rおよび抗erbb3抗体の用法および用量 |
WO2013151649A1 (en) | 2012-04-04 | 2013-10-10 | Sialix Inc | Glycan-interacting compounds |
US10130714B2 (en) | 2012-04-14 | 2018-11-20 | Academia Sinica | Enhanced anti-influenza agents conjugated with anti-inflammatory activity |
WO2013181585A2 (en) | 2012-06-01 | 2013-12-05 | Momenta Pharmaceuticals, Inc. | Methods related to adalimumab |
EP2885311B1 (en) | 2012-08-18 | 2020-01-01 | Academia Sinica | Cell-permeable probes for identification and imaging of sialidases |
TWI563092B (en) | 2012-08-20 | 2016-12-21 | Academia Sinica | Large scale enzymatic synthesis of oligosaccharides |
US9547009B2 (en) | 2012-08-21 | 2017-01-17 | Academia Sinica | Benzocyclooctyne compounds and uses thereof |
KR102460297B1 (ko) | 2012-10-30 | 2022-10-28 | 에스퍼란스 파마슈티컬스, 인코포레이티드 | 항체/약물 컨쥬게이트 및 이의 사용 방법 |
US20150284452A1 (en) | 2012-11-13 | 2015-10-08 | Iogenetics, Llc | Antimicrobial compositions |
US10086054B2 (en) | 2013-06-26 | 2018-10-02 | Academia Sinica | RM2 antigens and use thereof |
EP3013347B1 (en) | 2013-06-27 | 2019-12-11 | Academia Sinica | Glycan conjugates and use thereof |
TWI599370B (zh) | 2013-07-26 | 2017-09-21 | 中央研究院 | 靈芝多醣誘發之抗體介導抗腫瘤活性 |
CN105682666B (zh) | 2013-09-06 | 2021-06-01 | 中央研究院 | 使用醣脂激活人类iNKT细胞 |
EP3043647A4 (en) | 2013-09-12 | 2017-05-10 | Teva Pharmaceutical Industries Ltd. | Gene expression biomarkers of laquinimod responsiveness |
WO2015054039A1 (en) | 2013-10-08 | 2015-04-16 | Merck Sharp & Dohme Corp. | Fc CONTAINING POLYPEPTIDES HAVING INCREASED BINDING TO FcGammaRIIB |
US10150818B2 (en) | 2014-01-16 | 2018-12-11 | Academia Sinica | Compositions and methods for treatment and detection of cancers |
EP3094352B1 (en) | 2014-01-16 | 2020-09-23 | Academia Sinica | Compositions and methods for treatment and detection of cancers |
TWI687428B (zh) | 2014-03-27 | 2020-03-11 | 中央研究院 | 反應性標記化合物及其用途 |
US10005847B2 (en) | 2014-05-27 | 2018-06-26 | Academia Sinica | Anti-HER2 glycoantibodies and uses thereof |
EP3904388A1 (en) | 2014-05-27 | 2021-11-03 | Academia Sinica | Fucosidase from bacteroides and methods using the same |
US10118969B2 (en) | 2014-05-27 | 2018-11-06 | Academia Sinica | Compositions and methods relating to universal glycoforms for enhanced antibody efficacy |
AU2015267047A1 (en) | 2014-05-27 | 2017-01-05 | Academia Sinica | Anti-CD20 glycoantibodies and uses thereof |
WO2015184001A1 (en) | 2014-05-28 | 2015-12-03 | Academia Sinica | Anti-tnf-alpha glycoantibodies and uses thereof |
KR102003138B1 (ko) | 2014-08-22 | 2019-07-23 | 아카데미아 시니카 | 신규한 글리칸 접합체 및 그의 용도 |
KR102422375B1 (ko) | 2014-09-08 | 2022-07-18 | 아카데미아 시니카 | 당지질을 사용한 인간 iNKT 세포 활성화 |
US10495645B2 (en) | 2015-01-16 | 2019-12-03 | Academia Sinica | Cancer markers and methods of use thereof |
US9975965B2 (en) | 2015-01-16 | 2018-05-22 | Academia Sinica | Compositions and methods for treatment and detection of cancers |
WO2016118090A1 (en) | 2015-01-23 | 2016-07-28 | Agency For Science, Technology And Research | Cancer specific antigen-binding proteins |
US20170283878A1 (en) | 2015-12-11 | 2017-10-05 | Academia Sinica | Modulation of globoseries glycosphingolipid synthesis and cancer biomarkers |
-
2017
- 2017-03-08 WO PCT/US2017/021454 patent/WO2017156192A1/en active Application Filing
- 2017-03-08 CN CN201780027439.5A patent/CN109195996A/zh active Pending
- 2017-03-08 AU AU2017231749A patent/AU2017231749A1/en not_active Abandoned
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- 2017-03-08 TW TW106107652A patent/TW201808978A/zh unknown
- 2017-03-08 CA CA3016170A patent/CA3016170A1/en not_active Abandoned
- 2017-03-08 EP EP17764050.5A patent/EP3426693A4/en not_active Withdrawn
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- 2017-03-08 KR KR1020187028421A patent/KR20180114210A/ko not_active Application Discontinuation
-
2018
- 2018-09-02 IL IL261517A patent/IL261517A/en unknown
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1524180A (zh) * | 2001-04-10 | 2004-08-25 | 纽约市哥伦比亚大学信托人 | 新型微列阵及其使用方法 |
WO2014161960A1 (en) * | 2013-04-03 | 2014-10-09 | Asociación Centro De Investigación Cooperativa En Biomateriales | Synthesis and use of isotopically-labelled glycans |
Non-Patent Citations (7)
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109069523A (zh) * | 2016-12-10 | 2018-12-21 | 扬州蓝色生物医药科技有限公司 | 甘露寡聚糖类化合物及其作为抗rna病毒药物的应用 |
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