CN115605491A - 抗唾液酸酶的糖类、其制造方法及用途 - Google Patents
抗唾液酸酶的糖类、其制造方法及用途 Download PDFInfo
- Publication number
- CN115605491A CN115605491A CN202080022413.3A CN202080022413A CN115605491A CN 115605491 A CN115605491 A CN 115605491A CN 202080022413 A CN202080022413 A CN 202080022413A CN 115605491 A CN115605491 A CN 115605491A
- Authority
- CN
- China
- Prior art keywords
- saccharide
- sialic acid
- mmol
- fluoro
- acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/18—Acyclic radicals, substituted by carbocyclic rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/02—Monosaccharides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
- C07K2317/41—Glycosylation, sialylation, or fucosylation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Public Health (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Oncology (AREA)
- Cell Biology (AREA)
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明所揭示的是制备含3‑氟唾液酸的糖的方法,以及将其结合至均质性抗体的方法。同样在本发明范围内的是含有3‑氟唾液酸的化合物、与以α2,6连接3‑氟唾液酸糖苷为末端的N‑聚糖结合的单株抗体,以及用这类单株抗体治疗癌症的方法。
Description
背景技术
唾液酸是带负电荷的单糖,通常分布在糖脂和糖蛋白上的聚糖的最末端,其参与许多生理学的细胞内和细胞间过程,包括与细胞、病毒和细菌上其他生物分子和受体的相互作用1。此外,唾液酸化在调节分泌的糖蛋白和膜结合受体的功能和命运中扮演重要角色。例如,表皮生长因子受体(EGFR)的唾液酸化经证实可抑制EGFR二聚化,从而干扰EGF结合和磷酸化,这与肿瘤发生有关2。而且,唾液酸化调节血液循环中糖蛋白的半衰期,因为N-聚糖的去唾液酸化暴露出底下的半乳糖,其被肝脏脱唾液酸糖蛋白受体识别,导致糖蛋白从循环中快速去除3。因此,增加唾液酸化程度可以改善糖蛋白治疗剂的半衰期和不良作用。
近年来,糖基化对蛋白质结构和功能的作用已经有了深入的研究,激发了对蛋白质,特别是治疗性单株抗体(mAb)的聚糖合成(synthesicampillary)和糖工程(glycoengineering)的新方法开发5。例如,已证实具有末端α-2,6-连接唾液酸的无岩藻糖基化双触角(afucosylated biantennary)N-聚糖具有最佳的聚糖结构,可增强抗体依赖性细胞毒性(ADCC)、补体依赖性细胞毒性(CDC)和抗发炎活性6。
已知在C-3位置具有氟的唾液酸衍生物可抑制唾液酸转移酶和唾液酸酶(或称神经氨酸酶),并且更稳定7。然而,由于氟基团的强拉电子效应使糖基化反应失去活性,因此用氟化糖作为供体的糖基化常常带来重大挑战。尽管有一种唾液酸转移酶能够从相应的单磷酸胞苷-唾液酸转移3-氟唾液酸残基以形成α-2,3-连接唾液酸糖苷,却没有相应的α-2,6-唾液酸转移酶11。
因此,需要提供一种可靠地制备含α-2,6-连接3-氟唾液酸糖苷的糖的方法可用来开发糖蛋白治疗剂。
发明内容
本发明的一方面是制备含3-氟唾液酸的糖的方法。该方法包括使3-羟基唾液酸与糖反应进行糖基化反应,形成α2,6-连接3-羟基唾液酸糖苷,以及使该α2,6-连接3-羟基唾液酸糖苷与氟化剂反应进行氟化反应,形成含3-氟唾液酸的糖。
本发明的另一方面是制备与含3-氟唾液酸的糖结合的均质性抗体的方法。该方法是通过用该糖使单株抗体糖基化来进行。
本发明的另一方面是关于式(VI)化合物:
其中R1是Ac或H,R2是Bz或H,R3是甲基或H,R4是Bn或H,且X是OH、离去基或糖。注:“Ac”代表乙酰基,“Bz”代表苄酰基,“Bn”代表苄基,且糖可为单糖、双糖或寡糖。
本发明范围也包括一种与以α2,6连接3-氟唾液酸糖苷为末端的N-聚糖结合的单株抗体。
本发明更涵盖一种治疗癌症的方法。所述方法包括向有此需要的个体投予有效量的与以α2,6连接3-氟唾液酸糖苷为末端的N-聚糖结合的单株抗体。
以下发明说明将阐述本发明细节。通过以下几个具体实例的详细说明以及所附权利要求书,本发明的其他特征、目的和优点将更加明白。
附图说明
下面的说明参考附图,其中:
图1A显示两个图,描绘了三种唾液酸糖苷(即1、2和S27)的酶催化水解,其为酶浓度的函数;图1B显示两个图,描绘了唾液酸酶抑制作用,其为2、S27或2-脱氧-2,3-二去氢-N-乙酰基神经氨酸(DANA)(一种唾液酸酶抑制剂)浓度的函数;
图2是四种N-聚糖的染色电泳凝胶:(1)rituxan-G,(2)rituxan-Fax-SCT,(3)rituxan-SCT,及(4)市售rituxan;及
图3显示对三种N-聚糖,即rituxan-Fax-SCT、rituxan-SCT及rituxan所获得的质谱。
具体实施方式
本文首先详细揭示一种制备含3-氟唾液酸的糖的方法。
所述方法包括以下步骤:使3-羟基唾液酸与糖反应进行糖基化反应,形成α2,6连接3-羟基唾液酸糖苷,以及使该α2,6连接3-羟基唾液酸糖苷与氟化剂反应进行氟化反应,形成含3-氟唾液酸的糖。该糖可以是单糖、双糖或寡糖。在例示性方法中,该糖是单糖。
在本方法的某些具体实例中,该3-羟基唾液酸是2-溴-3-羟基唾液酸,且该糖基化反应是在三氟甲磺酸银(AgOTf)和磷酸氢二钠(Na2HPO4)的存在下进行。较佳地,该糖基化反应是在甲苯中进行。
在本方法的其他具体实例中,该氟化剂是全氟-1-丁烷磺酰氟(NfF),且该氟化反应是在催化剂存在下进行。例如,该催化剂是1,8-二吖双环[5,4,0]-十一-7-烯(DBU)。较佳地,该氟化反应是在甲苯中进行。值得注意的是,该反应可进一步在二氟三甲基硅酸三(二甲胺基)锍(TASF)的存在下进行。
在例示性方法中,3-羟基唾液酸具有式(I):
糖是式(II)化合物:
α2,6连接3-羟基唾液酸糖苷是式(III)化合物:
含3-氟唾液酸的糖是式(IV)化合物:
在另外的具体实例中,该方法进一步包括使用含3-氟唾液酸的糖与第二糖反应来进行另一个糖基化反应。
同样在本发明范围内的是一种制备与含3-氟唾液酸的糖结合的均质性抗体的方法。同样地,该方法包括用含3-氟唾液酸的糖使单株抗体糖基化。
在该方法的某些具体实例中,该含3-氟唾液酸的糖是通过上述的方法及其具体实例获得,其包括以下步骤:使3-羟基唾液酸与糖反应进行糖基化反应,形成α2,6连接3-羟基唾液酸糖苷,以及使该α2,6连接3-羟基唾液酸糖苷与氟化剂反应进行氟化反应,形成含3-氟唾液酸的糖。
在其他具体实例中,该含3-氟唾液酸的糖是一种以α2,6连接3-氟-唾液酸糖苷为末端的N-聚糖。其可具有式(V)的结构:
本发明的另一方面是关于式(VI)化合物:
R1、R2、R3、R4和X是如上述发明内容章节中所定义。
在一个具体实例中,该化合物具有式(IV)的结构:
在另一个具体实例中,式(VI)化合物各包括为N-聚糖的X。该具体实例的例示化合物具有式(V)的结构:
本发明还包括与含3-氟唾液酸的糖结合的单株抗体,该含3-氟唾液酸的糖是以α2,6方式连接3-氟唾液酸糖苷为末端的N-聚糖。较佳地,该以α2,6连接3-氟唾液酸糖苷为末端的N-聚糖具有上文所示的式(V)。
本发明进一步包括治疗癌症或自体免疫疾病的方法,其包括向有此需要的个体投予有效量的上述单株抗体。举例来说,该癌症可为白血病或淋巴瘤,而该自体免疫疾病可为类风湿性关节炎(rheumatoid arthritis)、自体免疫溶血性贫血(autoimmune hemolyticanemia)、纯红细胞发育不全(pure red cell aplasia)、栓塞性血小板低下紫斑(thrombotic thrombocytopenic purpura)、自发性血小板低下紫斑白血病(idiopathicthrombocytopenic purpura leukemia)、淋巴瘤、伊凡斯症候群(Evans syndrome)、血管炎、水疱性皮肤病(bullous skin disorders)、第1型糖尿病、修格兰氏症候群(Sjogren’ssyndrome)、抗NMDA受体脑炎、德维克氏病(Devic’s disease)、葛瑞夫兹氏眼病变(Graves’ophthalmopathy)、自体免疫性胰腺炎(autoimmune pancreatitis)、眼阵挛肌阵挛症候群肌(opsoclonus myoclonus syndrome)或IgG4相关疾病。
本文中的术语“治疗”是指将上述药学组合物投予患有上述疾病(即癌症)、有这种疾病症状或易患这种疾病的个体,目的在于赋予治疗或预防效果。术语“有效量”是指赋予这种效果所需的活性药物的量。如本领域技术人员所认知的,有效剂量将依据所治疗疾病的类型、给药途径、赋形剂使用以及与其他治疗处理共同使用的可能性而变化。
以下提供本发明某些具体方面的更详细说明。
与未氟化的唾液酸衍生物相比,3-氟取代的唾液酸衍生物对唾液酸酶更稳定7。通过将氟原子引入变旋异构和C-3位置,可发展出2,3-二氟唾液酸(DFSA)(流程1)8而作为生化探针9,和基于活性的蛋白质谱分析探针来研究唾液酸酶10。在DFSA的帮助下,氟原子在C-3轴向位置(3Fax)与3Feq衍生物相比,对减缓酶的去活化(ki)和再活化(khydr)的影响更大8b。受此观察的启发,看在含3-氟唾液酸的唾液酸糖苷在糖蛋白治疗中的潜在用途,我们想研究它们的稳定性。具体而言,我们打算研究若在mAb上的N-聚糖末端掺入一个3Fax-Neu5Ac模体(motif),是否可以增加其对唾液酸酶的稳定性并维持其效应物功能。
为了实现我们的目标,我们开发了一种制备方法,用来合成α2,6-连接3Fax-Neu5Ac寡糖,包括双触角N-聚糖。我们也证明了在唾液酸酶存在下,合成的3-FaxNeu5Ac-α2,6-Gal键联更稳定,而且带有相应双触角聚糖的抗体具有与非氟化对应物相同的结合亲和力(binding avidity)。
已经有报导出几种合成3F-Neu5Ac的方法,包括:(a)受保护聚糖与XeF2-BF3×OEt2 12、分子氟13及氟试剂(Selectfluor)7c的氟化作用;(b)唾液酸衍生物中C3位置赤道羟基(equatorial hydroxyl group)的反转7f;以及(c)醛醇缩酶催化的将ManNAc和3-氟-丙酮酸酯转变成3FeqNeu5Ac和3FaxNeu5Ac的酶催化转变作用7a,11a,14。然而,据我们所知,到目前为止尚无描述合成以3Fax-Neu5Ac为末端的N-聚糖的方法。唯一公开用3Fax-Neu5Ac制备双糖的方式限于3-FaxNeu5Ac-α2,3-Lac的酶催化合成,而不是所需的α2,6-键联11a。因此,我们将精力集中在该键联的化学合成15。
流程1
在使用3Fax-Neu5Ac基供体筛选各种糖基化条件后(流程2a)。我们研究了替代策略,包括了3OH-Neu5Ac-α2,6-Gal-STol中3OHeq变成3Fax的SN2反应(流程2b)。首先,我们使用Goto等人所报导的条件来研究O-6位置的唾液酸化作用(表1,表值1)16。该反应使用1当量AgOTf作为促进剂和Na2HPO4作为碱在甲苯中于-10℃下进行(流程3)。这些条件以低产率(15%)提供了双糖6,具有3:1(α:β)变旋异构比。将溶剂改成二氯甲烷并未改善非镜像选择性(表值2)。通过增加温度和反应时间,产率有改善,但选择性显著降低(表值3-5)。令人惊讶的是,使用的受体当量越多,观察到的立体选择性越好(表值3vs.6)。优化的条件(表值7)以35%(99%brsm)产率提供6,具有优异的α-选择性(α:β=13:1)。
流程2
流程3
表1.糖基化步骤的优化。
将OHeq反转成Fax证实是具挑战性的任务。使用TASF用氟取代OTf和OMs导致起始物的分解。为了使此转换作用优化,我们筛选了各种氟化剂,但均未成功。然而,在1,8-二吖双环[5,4,0]-十一-7-烯(DBU)存在下,于90℃下在无水甲苯中用全氟-1-丁烷磺酰氟(NfF)处理6(流程3)2天,以6%产率得到4(表2,表值1)17。进一步优化反应条件,例如降低反应温度(表值1、5及6)和增加反应时间(表值7),改善了4的总产率。由于C-3周围的立体阻碍,7到4的转换似乎是速率决定步骤。在许多情况下,我们能够在室温下在1天内将在NfF和DBU存在下形成的7单离出来。但是,将7转化成4(77%brsm产率)需要很长的反应时间。通过升高反应温度来改善转化率的尝试导致7的分解。于是,通过增加试剂量和反应时间到15天,我们获得最佳结果(表值8)。最后,我们发现到,添加TASF有助于改善反应效率,将反应时间缩短至仅2天(表值9)。完全保护的3Fax-Neu5Ac-α2,6-Gal-STol双糖(4)的立体化学是通过射线绕射分析来确认。
流程4
表2.氟化步骤的优化。
a试剂和条件:(a)NfF,DBU,甲苯,40℃,15d,49%(77%brsm)。b单离产物的产率。c未观察到产物。d逐份添加NfF(4当量/天)和DBU(4当量/天),然后搅拌14天。e逐份添加NfF(4当量/天)、DBU(4当量/天)及二氟三甲基硅酸三(二甲胺基)锍(TASF)(2当量/天)。
参考流程5,使用NIS/TMSOTf使3Fax-Neu5Ac-双糖供体4与受体(8)偶合以得到9。接着,通过在AcOH/NaOAc中用PdCl2异构化将变旋异构位置的O-烯丙基移除,并将变旋异构羟基(10)进一步转换成氟化物(11)和酰亚胺酸酯(12)。使用Cp2HfCl2/AgOTf条件,用3Fax-Neu5Ac为末端的氟化物供体(11)将O-3位置的核心双糖(13)糖基化,以85%产率得到六糖14。在移除亚苄基之后,使15在O-6位置糖基化,以70%产率得到所要的十糖(16),具有优异的位置选择性和α-立体选择性。我们还用N-苯基三氟乙酰亚胺酸酯供体(12)测试了TfOH促进的糖基化作用,但是产物产率不高。接着,在以下一系列步骤之后以40%总产率获得完全去保护的聚糖(17):(a)用LiOH皂化以除去酯和NHTroc基团;(b)自由胺与醇的乙酰化;(c)用甲醇钠除去OAc基团;以及(d)在MeOH/水/HCO2H的混合物中用Pd/C氢解O-苄基4b。
流程5
流程5中的试剂和条件:(a)8,TfOH,NIS,MS,CH2Cl2,-40℃,2h,64%.(b)PdCl2,CH3COONa,AcOH/H2O,82%.(c)DAST,CH2Cl2,-20℃,73%.(d)ClC(=NPh)CF3,Cs2CO3,CH2Cl2,0℃至r.t.,3h,56%;(e)11,AgOTf,Cp2HfCl2,甲苯,MS,0℃,3h,85%;(f)pTSA·H2O,CH3CN,r.t.,6h,75%;(g)11,AgOTf,Cp2HfCl2,甲苯,MS,-15℃,3h,70%(80%brsm);(h)12,TfOH,CH2Cl2,MS,-60至-20℃,3h,33%(55%brsm);(i)LiOH,二恶烷/H2O(4:1),90℃,16hrs;(j)Ac2O,Py,16h;(k)NaOMe,MeOH,16h;(l)Pd(OH)2,MeOH/H2O/HCOOH(6:3:1),H2,16h,40%(4步骤)。
建立了逐步合成的实验方案后,我们通过开发可程序的[2+2+2]一锅法合成六糖(14)(17的前驱物)来简化聚糖组装流程(流程6)。所设计的一锅实验方案包括使3Fax-Neu5Ac-双糖供体(4)(RRV=2053)与反应性较低的受体(18)(RRV=537)在-40℃下进行初始偶合,接着在-20℃下注入还原端受体13。在-10℃下1小时及标准纯化实验方案之后,以26%产率分离出六糖14。
流程6
为了收集有关3Fax-Neu5Ac-α2,6-Gal模体在唾液酸酶存在下的稳定性初步数据,我们制备了Neu5Ac-α2,6-Gal-pNP(1)和3Fax-Neu5Ac类似物(2)作为基质(流程1),以来自产气荚膜杆菌(C.perfingens)和霍乱弧菌(V.cholera)的市售唾液酸酶进行体外测定19。这两种酶对天然基质1均显示所预期的水解活性,但对3Fax-类似物2无活性(见图1A)。我们还观察到2并不如DANA般显著地抑制1的水解(见图1B)。
为了制备均质的mAb糖型,按照标准实验方案,在Endo S2(D184Q)的存在下将化合物17转化成恶唑啉供体并连接至GlcNAc引发的IgG(不含核心岩藻糖)6。均质3Fax-Neu5Ac-糖型对FcγRIIIa的结合亲和力是通过表面电浆共振来分析6,连同母体非氟化糖型(G2S2)和市售利妥昔单抗(rituximab)样品(主要糖型:G1F1,G0F1,G2F1)一起测量。相较于市售利妥昔单抗样品,带有经α2,6-唾液酸化且不含核心岩藻糖的双触角N-聚糖的IgG均质糖型,在结合亲和力方面提高了39.9倍(Neu5Ac-G2S2)和37.4-倍(3Fax-Neu5Ac-G2S2)(见表4)。发现3Fax-Neu5Ac修饰的糖型的亲和力在母体聚糖的亲和力同一范围内的事实,为3Fax-Neu5Ac-糖基化mAb的体内研究提供了前提。这些结果将在适当的时候报告。
综上所述,我们已经开发出一种3Fax-Neu5Ac-α2,6-Gal-STol的化学合成法,其可作为合成抗唾液酸酶寡糖和代表性具有以3Fax-Neu5Ac为末端的双触角N-聚糖的均质性抗体的建构单元。当与市售利妥昔单抗样品相比,经3Fax-Neu5Ac-聚糖修饰的均质糖型在与FcγRIIIa结合方面显示提高了37.4倍。此外,母体非氟化的和经3Fax-Neu5Ac修饰的抗体糖型表现出与Fc受体相似的结合亲和力。总体而言,我们的结果揭露了改善治疗性糖蛋白半衰期的新通用策略。
无需进一步阐述,相信本领域技术人员可以基于以上说明,最大程度地利用本发明。因此,以下具体实施例应被解释为仅仅是说明性的,而不以任何方式限制本揭示内容的其余部分。本文引用的所有出版物以引用全文方式并入本文中。
实施例1:合成含3-氟唾液酸的糖和前驱物
除非另有说明,否则所有反应均在惰性气氛下进行,并遵循标准的注射器-隔片技术。溶剂购自商业来源且未经进一步纯化即供使用。将粉碎的分子筛(EMD Millipore)磨成粉末,并在使用前活化。所有反应的进程是通过预涂有硅胶60F254(Merck KGaA)的TLC玻璃板监控。通过UV光(254nm)、对茴香醛及/或钼酸铵铈染色使TLC显像。在Across硅胶(粒径0.035–0.070mm,)上进行管柱层析。1H、13C及19F NMR光谱是用Bruker AVIII-600、DRX-500、AV-400、DPX-400、AVANCE 500AV及AVANCE 600光谱仪在25℃下记录,化学位移是用残留溶剂峰作为内标准品以δ(ppm)为单位做测量(δ,ppm:在1H NMR中为7.24(CHCl3)、4.80(H2O);在13C NMR中为77(CDCl3))。偶合常数(J)以Hz为单位做测量。数据表示如下:化学位移,多重性(s=单峰,d=双峰,t=三重峰,q=四重峰,m=多重峰,br=宽峰)。高解析质谱(HRMS)是通过电喷洒游离飞行时间(ESI-TOF)反射实验在Agilent LC/MSD TOF质谱仪上记录。单晶X射线绕射研究是在配备有Cu Kα辐射(=1.5478)的Bruker D8 Platinum-135CCD绕射仪上进行。HPLC测量是在Hitachi HPLC D-7000系统上进行。RRV测量是使用正相ZORBAX RX-SIL,5μm,4.6x250mm(Colloidal Silica,Agilent Technologies)使用溶剂系统EtOAc/己烷以1毫升/分的流速记录,并在254nm显像。
对甲苯基[5-乙酰胺基-4,7,8,9-四-O-乙酰基-5-脱氧-D-赤藻糖-α-L-葡萄糖-壬-2-酮糖吡喃糖酸甲酯]-(2→6)-2,3-二-O-苄酰基-4-O-苄基-1-硫基-β-D-半乳糖吡喃糖苷6α.将受体3(1.05g,1.80毫摩尔,1当量)与供体5(1.02毫克,1.80毫摩尔,1当量)在无水甲苯(60毫升)中的混合物在氩气氛下搅拌10分钟。然后,将反应混合物冷却到-50℃,并在搅拌下添加无水Na2HPO4(1.07g,7.54毫摩尔,4.2当量),接着添加AgOTf(692毫克,2.69毫摩尔,1.5当量,于甲苯18毫升中)。反应完成后(TLC显示起始物在24小时后消失),将反应混合物用EtOAc(80毫升)稀释,用20%Na2S2O3水溶液(10毫升)、饱和NaHCO3水溶液(5毫升)和盐水(5毫升)洗涤,然后将有机层经MgSO4脱水,过滤并浓缩。所得残余物通过硅胶管柱层析纯化,使用丙酮/甲苯(3:7)作为溶析液,得到化合物6(α:β=13:1),为白色粉末(680毫克,35%),伴随回收的受体3(670毫克,99%brsm)。α-变旋异构物6α:Rf=0.36(硅胶,CHCl3:MeOH=20:1);1H NMR(600MHz,CDCl3):δ7.96-7.94(m,2H,Ar-H),7.91-7.90(m,2H,Ar-H),7.50-7.47(m,2H,Ar-H),7.40-7.39(m,2H,Ar-H),7.37-7.32(m,5H,Ar-H),7.25-7.18(m,4H,Ar-H),7.06-7.04(m,2H,Ar-H),5.81(dd,J=10.2.9.6Hz,1H),5.59(d,J=10.2Hz,1H),5.38(dd,J=9.0,3.0Hz,1H),5.35-5.32(m,1H),5.25(dd,J=10.2,9.6Hz,1H),5.24(d,J=7.8,2.4Hz,1H),4.92(d,J=9.6Hz,1H,C1-Hβ),4.71(d,J=12.0Hz,1H),4.60(d,J=12.0Hz,1H),4.49(dd,J=10.8,1.8Hz,1H),4.30(dd,J=12.6,3.0Hz,1H),4.26(ddd,J=10.2,10.2,10.2Hz,1H),4.20(d,J=3.0Hz,1H),4.10(dd,J=10.2,7.2Hz,1H),4.04(dd,J=12.6,6.6Hz,1H),4.00(dd,J=6.6,6.6Hz,1H),3.86(d,J=9.6Hz,1H),3.82-3.73(m,2H),3.75(s,3H,-CH3),2.30(s,3H,-CH3),2.09(s,3H,-CH3),2.07(s,3H,-CH3),2.06(s,3H,-CH3),1.96(s,3H,-CH3),1.89(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ171.6,170.6,170.3,170.1,169.5,168.5,165.8,165.1,138.0,137.9,133.4,133.1,129.8,129.7,129.6,128.9,128.6,128.4,128.3,128.2,127.7,127.6,100.3,86.6,75.9,74.5,73.7,73.2,73.1,72.5,68.8,68.3,63.1,62.5,52.8,48.4,23.1,21.2,20.9,20.8,20.7,20.7;HRMS(ESI-TOF)m/e:C54H59NO20SNa[M+Na]+的计算值:1096.3243,实测值:1096.3241。
对甲苯基[5-乙酰胺基-4,7,8,9-四-O-乙酰基-5-脱氧-D-赤藻糖-β-L-葡萄糖-壬-2-酮糖吡喃糖酸甲酯]-(2→6)-2,3-二-O-苄酰基-4-O-苄基-1-硫基-β-D-半乳糖吡喃糖苷6β.β-变旋异构物6βRf=0.42(硅胶,CHCl3:MeOH=20:1);1H NMR(600MHz,CDCl3):δ7.94-7.92(m,4H,Ar-H),7.50-7.47(m,2H,Ar-H),7.43-7.40(m,6H,Ar-H),7.37-7.31(m,5H,Ar-H),7.13-7.12(m,2H,Ar-H),5.81(dd,J=10.2.10.2Hz,1H),5.51(dd,J=10.2,3.0Hz,1H),5.22(ddd,J=6.0,6.0,3.0Hz,1H),5.14(d,J=13.2Hz,1H),5.07(dd,J=6.0,2.4Hz,1H),4.90(d,J=10.2Hz,1H,C1-Hβ),4.66-4.62(m,2H),4.55(dd,J=12.6,2.4Hz,1H),4.29(d,J=1.8Hz,1H),4.14(dd,J=7.8,7.2Hz,1H),4.08(ddd,J=10.8,10.8,10.8Hz,1H),3.98(dd,J=12.6,6.6Hz,1H),3.92-3.88(m,2H),3.85-3.77(m,3H),3.83(s,3H,-CH3),3.53(dd,J=10.8,2.4Hz,1H),2.32(s,3H,-CH3),2.26(s,3H,-CH3),2.07(s,3H,-CH3),2.04(s,3H,-CH3),1.97(s,3H,-CH3),1.68(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ171.4,170.6,170.3,169.8,169.8,166.6,165.8,165.2,139.2,138.4,133.5,133.4,133.1,129.9,129.8,129.5,128.9,128.5,128.3,127.2,126.0,99.6,86.9,76.0,75.5,74.3,73.1,72.5,71.7,70.3,68.4,67.2,62.1,62.0,53.3,47.5,22.9,21.5,21.2,20.8,20.7,20.7;HRMS(ESI-TOF)m/e:C54H59NO20S[M+H]+的计算值:1074.3424,实测值:1074.3458。
对甲苯基[5-乙酰胺基-4,7,8,9-四-O-乙酰基-3,5-二脱氧-3-氟-D-赤藻糖-α-L-甘露糖-壬-2-酮糖吡喃糖酸甲酯]-(2→6)-2,3-二-O-苄酰基-4-O-苄基-1-硫基-β-D-半乳糖吡喃糖苷4.向置于有搅拌棒的旋盖小瓶中的化合物6α(500毫克,0.47毫摩尔,1当量)于甲苯(5毫升)中的溶液添加DBU(0.28毫升,1.86毫摩尔,4当量)、全氟-1-丁烷磺酰氟(0.33毫升,1.86毫摩尔,4当量)及TASF(256毫克,0.93毫摩尔,2当量)。将反应小瓶密封并于40℃搅拌。在24小时后,反应混合物用额外量的DBU(0.28毫升,1.86毫摩尔,4当量)、全氟-1-丁烷磺酰氟(0.33毫升,1.86毫摩尔,4当量)及TASF(256毫克,0.93毫摩尔,2当量)处理,然后于40℃再搅拌24小时。将反应混合物直接装载到硅胶管柱上,并用丙酮/甲苯(7:3)溶析。获得呈浅黄色粉末的双糖4(300毫克,60%),伴随呈浅黄色粉末的全氟-1-丁烷磺酰基化合物7(50毫克,8%)。
从7合成双糖4:向置于有搅拌棒的旋盖小瓶中的化合物7(730毫克,0.54毫摩尔,1当量)于甲苯(7毫升)中的溶液添加DBU(0.64毫升,4.31毫摩尔,8当量)和全氟-1-丁烷磺酰氟(0.77毫升,4.31毫摩尔,8当量)。将容器密封并于40℃搅拌15天。将反应混合物直接装载到硅胶管柱上,并用丙酮/甲苯(7:3)作为溶析液溶析;获得呈浅黄色粉末的双糖4(282毫克,49%),伴随呈浅黄色粉末的全氟-1-丁烷磺酰基化合物7(267毫克,77%brsm)。Rf=0.43(硅胶,丙酮/甲苯=2:3);1H NMR(600MHz,CDCl3):δ7.95-7.93(m,2H,Ar-H),7.87-7.85(m,2H,Ar-H),7.48-7.44(m,2H,Ar-H),7.41-7.39(m,2H,Ar-H),7.35-7.33(m,2H,Ar-H),7.31-7.29(m,2H,Ar-H),7.25-7.24(m,2H,Ar-H),7.21-7.19(m,2H,Ar-H),7.16-7.14(m,1H,Ar-H),7.04-7.03(m,2H,Ar-H),5.79(dd,J=10.2.9.6Hz,1H),5.49(ddd,J=9.0,5.4,2.4Hz,1H),5.42(dd,J=10.2,3.0Hz,1H),5.31-5.28(m,2H),5.20(dd,J=27.0,11.4Hz,1H,sia-C4-H),5.01(dd,J=51.6,1.8Hz,1H,sia-C3-H),4.97(d,J=10.2Hz,1H,C1-Hβ),4.66(d,J=11.4Hz,1H),4.58(d,J=11.4Hz,1H),4.37(dd,J=12.6,2.4Hz,1H),4.28-4.26(m,2H),4.17(dd,J=12.6,5.4Hz,1H),4.09-4.05(m,2H),3.98(dd,J=10.2,6.0Hz,1H),3.74(dd,J=10.2,8.4Hz,1H),3.72(s,3H,-CH3),2.29(s,3H,-CH3),2.17(s,3H,-CH3),2.16(s,3H,-CH3),2.09(s,3H,-CH3),1.98(s,3H,-CH3),1.91(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ170.9,170.7,170.4,170.2,169.8,165.6,165.3,138.3,137.6,133.2,133.0,132.6,129.8,129.8,129.7,129.5,129.3,129.1,128.4,128.3,128.1,127.5,127.3,98.3,98.2,88.1,86.8,86.4,76.2,75.6,74.6,74.0,71.4,69.2,69.0,68.5,68.0,67.3,63.4,62.5,53.2,45.5,23.4,21.2,21.2,20.8,20.7,20.7;19F NMR(376MHz,CDCl3):δ-215.9;HRMS(ESI-TOF)m/e:C54H58FNO19SNa[M+Na]+的计算值:1098.3200,实测值:1098.3212。
对甲苯基[5-乙酰胺基-4,7,8,9-四-O-乙酰基-5-脱氧-3-O-(全氟-1-丁烷)磺酰基-D-赤藻糖-α-L-葡萄糖-壬-2-酮糖吡喃糖酸甲酯]-(2→6)-2,3-二-O-苄酰基-4-O-苄基-1-硫基-β-D-半乳糖吡喃糖苷7.Rf=0.59(硅胶,丙酮:甲苯=2:3);1H NMR(600MHz,CDCl3):δ7.95-7.94(m,2H,Ar-H),7.90-7.89(m,2H,Ar-H),7.49-7.45(m,2H,Ar-H),7.40-7.39(m,2H,Ar-H),7.36-7.30(m,4H,Ar-H),7.27-7.26(m,2H,Ar-H),7.25-7.18(m,3H,Ar-H).7.04-7.03(m,2H,Ar-H),5.82(dd,J=10.2.9.6Hz,1H),5.62-5.38(m,4H),5.26(d,J=9.0Hz,1H),4.96-4.94(m,2H,C1-Hβ),4.70-4.64(m,3H),4.35-4.30(m,1H),4.22-4.20(m,2H),4.09(dd,J=9.6,6.0Hz,1H),4.02(dd,J=12.6,6.0Hz,1H),3.98(dd,J=6.6,6.0Hz,1H),3.93(dd,J=10.2,7.2Hz,1H),3.76(s,3H,-CH3),2.29(s,3H,-CH3),2.17(s,3H,-CH3),2.09(s,3H,-CH3),2.06(s,3H,-CH3),1.94(s,3H,-CH3),1.90(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ170.6,170.2,170.1,169.3,167.0,165.7,165.2,138.1,137.7,133.2,133.0,132.5,129.8,129.7,129.7,129.5,129.1,129.0,128.4,128.3,128.1,128.0,127.5,127.4,97.8,86.5,83.4,75.7,74.6,74.2,72.6,70.0,68.4,67.9,66.5,64.5,62.4,53.1,49.0,23.0,21.1,20.9.,20.7,20.6,20.3;19F NMR(376MHz,CDCl3):δ-126.2,-126.2,-121.5,-121.5,-110.4,-110.4,-81.0,-80.9,-80.9;HRMS(ESI-TOF)m/e:C58H58F9NO22S2Na[M+Na]+的计算值:1378.2640,实测值:1378.2644。
烯丙基[5-乙酰胺基-4,7,8,9-四-O-乙酰基-3,5-二脱氧-3-氟-D-赤藻糖-α-L-甘露糖-壬-2-酮糖吡喃糖酸甲酯]-(2→6)-[2,3-二-O-苄酰基-4-O-苄基-1-β-D-半乳糖吡喃糖苷基]-(1→4)-[3,6-二-O-苄基-2-脱氧-2-(2,2,2-三氯乙氧基)碳酰胺基-β-D-葡萄糖吡喃糖苷基]-(1→2)-3,4,6-三-O-苄基-α-D-甘露糖吡喃糖苷9.将受体8(1.04g,1.03毫摩尔,1当量)、供体4(1.39g,1.29毫摩尔,1.25当量)与活化的粉碎MS(0.70g)在无水CH2Cl2(7毫升)中的混合物在氩气下搅拌1小时。然后,将反应混合物冷却到-40℃,并在搅拌下添加NIS(465毫克,2.06毫摩尔,2当量),接着添加TfOH(0.5M于Et2O中,0.62毫升,0.31毫摩尔,0.3当量)。反应完成后(TLC显示起始物在~2小时后消失),将反应混合物用Et3N(0.4毫升)淬灭,并通过硅藻土垫过滤。将滤液用CH2Cl2(30毫升)稀释,用20%Na2S2O3水溶液(10毫升)、饱和NaHCO3水溶液(15毫升)和盐水(8毫升)洗涤。将分离的有机层经MgSO4脱水并浓缩。所得残余物通过硅胶管柱层析纯化,使用丙酮/甲苯(1:2)作为溶析液,得到化合物9,为白色粉末(1.30g,64%)。Rf=0.51(硅胶,丙酮:甲苯=2:3);1H NMR(600MHz,CDCl3):δ7.90-7.88(m,4H,Ar-H),7.48-7.45(m,2H,Ar-H),7.34-7.22(m,24H,Ar-H),7.18-7.13(m,10H,Ar-H),5.85-5.78(m,2H),5.47-5.44(m,1H),5.35-5.32(m,4H),5.24-5.16(m,2H),5.11(dd,J=10.2,1.2Hz,1H),5.06-4.97(m,3H,C1-Hβ),4.85-4.73(m,4H,C1-Hα,C1-Hβ),4.70-4.54(m,6H),4.53-4.42(m,4H),4.33-4.26(m,2H),4.23-4.18(m,3H),4.14-4.04(m,3H),3.97-3.93(m,1H),3.89-3.81(m,5H),3.73-3.58(m,10H),3.45-3.43(m,1H),3.32-3.30(m,1H),2.16(s,3H,-CH3),2.13(s,3H,-CH3),2.10(s,3H,-CH3),2.01(s,3H,-CH3),1.92(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ170.7,170.6,170.4,170.2,169.8,165.6,165.4,165.4,165.1,153.8,138.8,138.6,138.5,138.2,138.2,138.1,133.7,133.2,133.1,133.1,130.8,130.0,129.8,129.6,129.4,129.0,128.4,128.3,128.3,128.2,128.2,128.2,128.1,128.1,128.0,128.0,127.9,127.6,127.6,127.5,127.4,127.3,127.2,125.2,117.1,100.0,98.3,98.2,96.9,95.6,88.1,86.8,77.9,75.0,74.7,74.6,74.5,74.2,74.1,73.9,73.4,73.3,73.1,73.0,72.4,71.9,71.4,70.9,70.9,69.5,69.2,69.1,69.0,68.0,67.9,67.2,62.9,62.3,57.2,53.2,45.5,23.3,21.0,20.7,20.7,20.6;19F NMR(376MHz,CDCl3):δ-215.6;HRMS(ESI-TOF)m/e:C100H108Cl3FN2O31Na[M+Na]+的计算值:1979.5878,实测值:1979.5889。
[5-乙酰胺基-4,7,8,9-四-O-乙酰基-3,5-二脱氧-3-氟-D-赤藻糖-α-L-甘露糖-壬-2-酮糖吡喃糖酸甲酯]-(2→6)-[2,3-二-O-苄酰基-4-O-苄基-1-β-D-半乳糖吡喃糖苷基]-(1→4)-[3,6-二-O-苄基-2-脱氧-2-(2,2,2-三氯乙氧基)碳酰胺基-β-D-葡萄糖吡喃糖苷基]-(1→2)-3,4,6-三-O-苄基-α-D-甘露糖吡喃糖苷10.在室温下,在搅拌的四糖9(578毫克,0.30毫摩尔,1当量)于乙酸(6.0毫升,乙酸/水,10:1=v/v)的溶液中添加CH3COONa(121毫克,1.48毫摩尔,5当量),接着添加PdCl2(105毫克,0.59毫摩尔,2当量)。20小时之后,当TLC显示起始物消失,将反应混合物用乙酸乙酯(20毫升)稀释并倒入饱和NaHCO3水溶液(20毫升)中。水层用乙酸乙酯萃取(2×10毫升),然后将合并的有机相经MgSO4脱水并浓缩。所得残余物通过硅胶管柱层析纯化,使用丙酮/己烷(2:3)作为溶析液,得到化合物10,为白色粉末(465毫克,82%)。Rf=0.23(硅胶,丙酮:己烷=2:3);1H NMR(600MHz,CDCl3):δ7.90-7.87(m,4H,Ar-H),7.48-7.45(m,2H,Ar-H),7.34-7.20(m,24H,Ar-H),7.19-7.12(m,10H,Ar-H),5.80-5.75(m,1H),5.45-5.43(m,1H),5.33-5.28(m,3H),5.21-4.89(m,5H,man-C1-Hα,man-C1-Hβ,C1-Hβ),4.84-4.30(m,15H,C1-Hβ),4.27-3.79(m,12H),3.78-3.43(m,12H),2.13-2.12(m,6H,-2CH3),2.09-2.08(m,3H,-CH3),2.02-2.00(m,3H,-CH3),1.91(br,3H,-CH3);13C NMR(150MHz,CDCl3):δ170.8,170.7,170.7,170.7,170.6,170.5,170.4,170.3,170.3,170.2,169.9,169.8,165.6,165.6,165.4,165.2,165.2,154.0,138.6,138.5,138.3,138.2,138.1,133.2,133.1,129.8,129.6,129.5,129.0,128.4,128.4,128.3,128.3,128.2,128.2,128.2,128.1,128.0,127.9,127.9,127.7,127.7,127.6,127.5,127.5,127.4,127.4,127.3,100.0,99.5,98.3,98.2,98.1,95.8,95.7,92.3,88.2,88.1,86.8,76.1,74.8,74.7,74.6,74.6,74.3,74.2,74.2,73.9,73.8,73.3,73.2,73.1,73.1,72.7,72.6,72.4,72.3,71.6,71.4,71.2,70.9,70.8,69.9,69.5,69.3,69.2,69.1,69.0,68.1,68.0,67.2,67.1,63.0,62.3,62.2,62.1,53.3,53.3,45.5,45.4,26.3,23.3,21.1,20.8,20.7,20.7,20.6,20.6;19F NMR(376MHz,CDCl3):δ-215.2,-215.7;HRMS(ESI-TOF)m/e:C97H104Cl3FN2O31Na[M+Na]+的计算值:1939.5565,实测值:1939.5619。
[5-乙酰胺基-4,7,8,9-四-O-乙酰基-3,5-二脱氧-3-氟-D-赤藻糖-α-L-甘露糖-壬-2-酮糖吡喃糖酸甲酯]-(2→6)-[2,3-二-O-苄酰基-4-O-苄基-1-β-D-半乳糖吡喃糖苷基]-(1→4)-[3,6-二-O-苄基-2-脱氧-2-(2,2,2-三氯乙氧基)碳酰胺基-β-D-葡萄糖吡喃糖苷基]-(1→2)-3,4,6-三-O-苄基-α-D-甘露糖吡喃糖苷基氟11.在-20℃下,向充分搅拌的半缩醛10(395毫克,0.21毫摩尔,1当量)于无水CH2Cl2(12毫升)中的溶液添加DAST(81.6μL,0.62毫摩尔,3当量)。将反应混合物剧烈搅拌直到TLC显示起始物消失(3h)。将反应混合物用CH2Cl2(10毫升)稀释,用饱和NaHCO3水溶液(5毫升)和盐水(4毫升)洗涤。将有机相经MgSO4脱水,过滤并浓缩。所得残余物通过硅胶管柱层析纯化,使用丙酮/己烷(3:4)作为溶析液,得到化合物11,为白色粉末(289毫克,73%)。Rf=0.32(硅胶,丙酮:己烷=3:4);1HNMR(600MHz,CDCl3):δ7.89-7.88(m,4H,Ar-H),7.47-7.45(m,2H,Ar-H),7.33-7.22(m,22H,Ar-H),7.17-7.12(m,12H,Ar-H),5.78(dd,J=10.2,7.8Hz,1H),5.53-5.40(m,2H),5.33-5.29(m,3H,C1-Hα),5.21(dd,J=27.6,11.4Hz,1H,sia-C4-H),5.05-4.96(m,2H,C1-Hβ),4.94(d,J=11.4Hz,1H),4.82-4.79(m,4H,C1-Hβ),4.75-4.72(m,1H),4.68-4.59(m,3H),4.58-4.54(m,2H),4.50-4.48(m,2H),4.30(dd,J=12.6,3.0Hz,1H),4.26(d,J=10.2Hz,1H),4.23-4.15(m,4H),4.08-4.04(m,2H),3.95(dd,J=9.0,8.4Hz,1H),3.89-3.78(m,5H),3.72-3.57(m,9H),3.50-3.48(m,1H),3.35(d,J=0.6Hz,1H)2.14(s,3H,-CH3),2.12(s,3H,-CH3),2.09(s,3H,-CH3),2.00(s,3H,-CH3),1.91(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ170.8,170.7,170.4,170.2,169.9,165.6,165.4,165.2,153.9,138.2,138.1,137.8,133.2,133.1,129.8,129.6,129.4,129.0,128.4,128.3,128.2,128.1,128.0,127.9,127.7,127.6,127.6,127.4,127.3,127.3,106.9,105.4,99.8,99.2,98.3,98.2,95.6,88.1,86.8,74.9,74.8,74.2,74.0,73.9,73.8,73.3,73.2,72.5,71.4,71.3,70.9,69.1,69.1,68.9,68.0,67.3,62.8,62.3,56.9,53.3,45.6,23.4,21.1,20.8(2C),20.6;19F NMR(376MHz,CDCl3):δ-138.6,-215.7;HRMS(ESI-TOF)m/e:C97H103Cl3F2N2O30Na[M+Na]+的计算值:1941.5521,实测值:1941.5521。
(N-苯基)-2,2,2-三氟乙酰亚胺酸酯[5-乙酰胺基-4,7,8,9-四-O-乙酰基-3,5-二脱氧-3-氟-D-赤藻糖-α-L-甘露糖-壬-2-酮糖吡喃糖酸甲酯]-(2→6)-[2,3-二-O-苄酰基-4-O-苄基-1-β-D-半乳糖吡喃糖苷基]-(1→4)-[3,6-二-O-苄基-2-脱氧-2-(2,2,2-三氯乙氧基)碳酰胺基-β-D-葡萄糖吡喃糖苷基]-(1→2)-3,4,6-三-O-苄基-D-甘露糖吡喃糖苷12.向充分搅拌的半缩醛10(395毫克,0.21毫摩尔,1当量)于无水CH2Cl2(12毫升)中的溶液,伴随搅拌在0℃下添加Cs2CO3(83.2毫克,0.26毫摩尔,2当量)和2,2,2-三氟-N-苯基-亚胺乙酰氯(41μL,0.26毫摩尔,2当量),然后将混合物升温至室温并搅拌3小时。将反应混合物用CH2Cl2(10毫升)稀释,通过硅藻土垫过滤并浓缩。所得残余物通过硅胶管柱层析纯化,使用丙酮/己烷(1:2)作为溶析液,得到化合物12,为白色泡沫(150毫克,56%);变旋异构物混合物(α:β=1:1)。Rf=0.55(硅胶,丙酮:己烷=1:1);1H NMR(600MHz,CDCl3):δ7.93-7.88(m,4H,Ar-H),7.49-7.45(m,2H,Ar-H),7.40-7.12(m,38H,Ar-H),6.75-6.71(m,1H,Ar-H),5.82-5.78(m,1H),5.46-5.42(m,1H),5.36-5.30(m,3H),5.24-5.16(m,1H),5.08-4.81(m,5H,0.5man-C1-Hα,2C1-Hβ),4.78-4.74(m,1H),4.69-4.41(m,9H,0.5man-C1-Hβ),4.33-4.06(m,7H),3.95-3.82(m,5H),3.69-3.36(m,10H),2.16-2.10(m,9H,-3CH3),2.01-1.99(m,3H,-CH3),1.92(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ170.7,170.7,170.7,170.7,170.4,170.2,169.9,169.9,165.6,165.6,165.4,165.2,165.1,154.1,153.9,143.3,143.1,138.7,138.5,138.3,138.2,138.2,138.1,138.0,138.0,137.8,137.7,133.2,133.1,133.1,129.8,129.6,129.4,129.3,129.0,128.7,128.4,128.4,128.3,128.3,128.3,128.2,128.2,128.1,128.1,128.1,128.0,127.9,127.7,127.7,127.6,127.5,127.5,127.5,127.4,127.3,119.4,119.2,102.3,100.2,100.0,98.3,98.2,95.6,95.5,88.0,86.7,80.0,79.3,75.9,75.0,74.7,74.7,74.5,74.3,74.2,73.8,73.8,73.5,73.3,73.2,73.1,72.8,72.4,72.3,71.5,71.4,71.1,71.0,70.9,70.1,69.8,69.3,69.1,68.2,67.2,67.2,62.8,62.3,62.2,53.3,53.2,45.5,23.3,21.0,21.0,20.7,20.7,20.6;19F NMR(376MHz,CDCl3):δ-65.2,-65.4,-65.5,-65.6,-65.6,-65.7,-215.6,-215.7;HRMS(ESI-TOF)m/e:C105H108Cl3F4N3O31Na[M+Na]+的计算值:2110.5860,实测值:2110.5865。
苄基[5-乙酰胺基-4,7,8,9-四-O-乙酰基-3,5-二脱氧-3-氟-D-赤藻糖-α-L-甘露糖-壬-2-酮糖吡喃糖酸甲酯]-(2→6)-[2,3-二-O-苄酰基-4-O-苄基-1-β-D-半乳糖吡喃糖苷基]-(1→4)-[3,6-二-O-苄基-2-脱氧-2-(2,2,2-三氯乙氧基)碳酰胺基-β-D-葡萄糖吡喃糖苷基]-(1→2)-[3,4,6-三-O-苄基-α-D-甘露糖吡喃糖苷基]-(1→3)-[2-O-乙酰基-4,6-O-亚苄基-β-D-甘露糖吡喃糖苷基]-(1→4)-3,6-二-O-苄基-2-脱氧-2-(2,2,2-三氯乙氧基)碳酰胺基-β-D-葡萄糖吡喃糖苷14.将AgOTf(194毫克,0.75毫摩尔,相对于受体6当量)、双Cp2HfCl2(167毫克,0.44毫摩尔,相对于受体3.5当量)与新鲜活化的MS(1.50g)于无水甲苯(15毫升)中的混合物在氩气下于室温搅拌1小时。然后,将反应混合物冷却到-20℃,用供体11(290毫克,0.15毫摩尔,1.2当量)与受体13(115毫克,0.13毫摩尔,1当量)于无水甲苯(8毫升)中的溶液处理。将反应混合物在0℃下搅拌直到TLC显示起始物消失(3小时)。反应完成后,将反应混合物用Et3N(0.25毫升)淬灭,用EtOAc(25毫升)稀释,并通过硅藻土垫过滤。将滤液用饱和NaHCO3水溶液(10毫升)和盐水(6毫升)洗涤两次,将有机相经MgSO4脱水,过滤并浓缩。所得残余物通过硅胶管柱层析纯化,使用丙酮/甲苯(1:2)作为溶析液,得到化合物14,为白色粉末(299毫克,85%)。
一锅合成法:将受体18(40.0毫克,0.037毫摩尔,1当量)、供体4(60.1毫克,0.056毫摩尔,1.5当量)与活化粉碎的MS(0.25g)于无水CH2Cl2(1.25毫升)中的混合物在氩气下搅拌1小时。然后,将反应混合物冷却到-40℃并添加NIS(12.6毫克,0.056毫摩尔,1.5当量),接着添加TfOH(0.5M于Et2O中,22.3μL,0.011毫摩尔,0.3当量),然后让混合物搅拌至-20℃为时2小时。接着,起始物消失后(以TLC监测),将反应混合物中依序用受体13(34.2毫克,0.037毫摩尔,1当量)在无水CH2Cl2(1.25毫升)、NIS(16.8毫克,0.075毫摩尔,2当量)及TfOH(0.5M于Et2O中,22.3μL,0.011毫摩尔,0.3当量)处理,伴随在氩气下搅拌10分钟。添加所有试剂后,让混合物升温至-10℃并持续搅拌直到TLC显示起始物消失(1小时)。完成之后,将反应混合物用Et3N(0.4毫升)淬灭并通过硅藻土垫过滤。将滤液用CH2Cl2(10毫升)稀释,用20%Na2S2O3水溶液(3毫升)、饱和NaHCO3水溶液(2毫升)及盐水(2毫升)洗涤。将分离的有机层经MgSO4脱水,过滤并在真空中浓缩。所得残余物通过使用丙酮/甲苯(1:2)作为溶析液的硅胶管柱层析,接着通过使用丙酮/己烷(3:4)作为溶析液的第二硅胶管柱层析纯化,得到化合物14,为白色粉末(27毫克,26%)。Rf=0.43(硅胶,丙酮:甲苯=3:5);1H NMR(600MHz,CDCl3):δ7.96-7.95(m,2H,Ar-H),7.91-7.90(m,2H,Ar-H),7.51-7.47(m,2H,Ar-H),7.36-7.26(m,35H,Ar-H),7.23-7.10(m,19H,Ar-H),5.84(dd,J=10.2,7.8Hz,1H),5.45-5.42(m,1H),5.34-5.27(m,4H),5.21-5.15(m,2H),5.04-4.94(m,4H,C1-Hα,C1-Hβ),4.87-4.78(m,4H),4.71-4.50(m,14H,2C1-Hβ),4.46-4.34(m,5H),4.31-4.21(m,4H),4.16(dd,J=12.6,5.4Hz,1H),4.13-4.06(m,2H),3.98-3.95(m,4H,C1-Hβ),3.84-3.57(m,17H),3.51-3.41(m,4H),3.36-3.35(m,2H),3.15(br,1H),2.93(br,1H),2.68-2.67(m,1H),2.12(s,3H,-CH3),2.11(s,3H,-CH3),2.09(s,3H,-CH3),1.99(s,3H,-CH3),1.91(s,3H,-CH3),1.85(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ170.8,170.7,170.4,170.2,169.7,169.4,165.6,165.4,165.2,153.7,153.6,138.9,138.7,138.4,138.3,137.9,137.1,133.2,129.8,129.7,129.5,129.1,128.9,128.6,128.5,128.4,128.4,128.3,128.3,128.2,128.1,128.1,128.0,128.0,127.9,127.9,127.9,127.8,127.8,127.6,127.6,127.6,127.5,127.4,127.4,127.3,127.2,126.3,102.1,100.1,99.0,98.7,98.6,95.8,95.5,88.2,86.9,78.8,78.4,74.8,74.5,74.3,74.2,73.9,73.8,73.3,72.9,72.3,71.9,71.4,71.1,70.7,70.3,69.2,69.1,69.0,68.4,68.3,67.9,67.2,66.2,63.2,62.3,57.4,56.8,53.3,45.5,23.3,21.0,20.8,20.7,20.6,20.6;19F NMR(376MHz,CDCl3):δ-215.6;HRMS(ESI-TOF)m/e:C142H150Cl6FN3O43Na2[M+2Na]2+的计算值:1429.8771,实测值:1429.8864。
苄基[5-乙酰胺基-4,7,8,9-四-O-乙酰基-3,5-二脱氧-3-氟-D-赤藻糖-α-L-甘露糖-壬-2-酮糖吡喃糖酸甲酯]-(2→6)-[2,3-二-O-苄酰基-4-O-苄基-1-β-D-半乳糖吡喃糖苷基]-(1→4)-[3,6-二-O-苄基-2-脱氧-2-(2,2,2-三氯乙氧基)碳酰胺基-β-D-葡萄糖吡喃糖苷基]-(1→2)-[3,4,6-三-O-苄基-α-D-甘露糖吡喃糖苷基]-(1→3)-[2-O-乙酰基-β-D-甘露糖吡喃糖苷基]-(1→4)-3,6-二-O-苄基-2-脱氧-2-(2,2,2-三氯乙氧基)碳酰胺基-β-D-葡萄糖吡喃糖苷15.向搅拌的起始物14(318毫克,0.11毫摩尔,1当量)于乙腈(12毫升)中的溶液添加p-TsOH.H2O(21.5毫克,0.11毫摩尔,1当量)。将反应混合物剧烈搅拌直到TLC显示起始物消失(6小时)。完成之后,将反应混合物用Et3N(0.3毫升)淬灭并在高真空下浓缩。所得残余物通过硅胶管柱层析纯化,使用丙酮/甲苯(3:5)作为溶析液,得到化合物15,为白色粉末(231毫克,75%)。Rf=0.25(硅胶,丙酮:甲苯=1:2);1H NMR(600MHz,CDCl3):δ7.88-7.87(m,4H,Ar-H),7.46(dd,J=7.2,7.2Hz,1H,Ar-H),7.43(dd,J=7.2,7.2Hz,1H,Ar-H),7.33-7.13(m,47H,Ar-H),7.09-7.08(m,2H,Ar-H),6.01(br,1H),5.76(dd,J=10.2,7.8Hz,1H),5.46-5.43(m,1H),5.38(d,J=8.4Hz,1H),5.31-5.29(m,3H),5.19-5.13(m,2H,C1-Hα),5.05-4.96(m,3H,C1-Hβ),4.89-4.79(m,4H,C1-Hβ),4.69-4.60(m,8H,C1-Hβ),4.57-4.49(m,8H,C1-Hβ),4.45(d,J=12.0Hz,1H),4.40-4.36(m,2H),4.31(dd,J=12.6,2.4Hz,1H),4.24-4.16(m,4H),4.12-4.09(m,2H),4.02(br,1H),3.98-3.93(m,2H),3.91-3.86(m,2H),3.80(dd,J=7.8,6.0Hz,1H),3.76-3.69(m,6H),3.66-3.37(m,18H),2.96-2.93(m,1H),2.13(s,3H,-CH3),2.11(s,3H,-CH3),2.09(s,3H,-CH3),1.98(s,3H,-CH3),1.93(s,3H,-CH3),1.90(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ170.8,170.7,170.4,170.3,170.2,169.8,165.6,165.5,165.3,154.3,153.8,138.6,138.4,138.3,138.1,138.0,137.8,137.2,133.3,133.1,129.8,129.6,129.4,129.0,128.5,128.4,128.3,128.2,128.2,127.9,127.9,127.8,127.7,127.6,127.5,127.5,127.4,127.4,127.3,100.1,99.1,98.3,98.2,98.1,95.7,95.5,88.2,86.9,78.6,78.2,75.4,75.1,74.8,74.7,74.4,74.2,74.1,73.8,73.3,73.2,72.6,71.9,71.8,71.5,71.4,70.9,70.6,69.5,62.3,57.2,56.8,53.8,53.3,45.4,23.3,21.1,21.0,20.7(2C),20.7;19F NMR(376MHz,CDCl3):δ-215.6;HRMS(ESI-TOF)m/e:C135H146Cl6FN3O43Na[M+Na]+的计算值:2748.7337,实测值:2748.7373。
苄基[5-乙酰胺基-4,7,8,9-四-O-乙酰基-3,5-二脱氧-3-氟-D-赤藻糖-α-L-甘露糖-壬-2-酮糖吡喃糖酸甲酯-(2→6)-2,3-二-O-苄酰基-4-O-苄基-1-β-D-半乳糖吡喃糖苷基-(1→4)-3,6-二-O-苄基-2-脱氧-2-(2,2,2-三氯乙氧基)碳酰胺基-β-D-葡萄糖吡喃糖苷基-(1→2)-3,4,6-三-O-苄基-α-D-甘露糖吡喃糖苷基-(1→3)]-[5-乙酰胺基-4,7,8,9-四-O-乙酰基-3,5-二脱氧-3-氟-D-赤藻糖-α-L-甘露糖-壬-2-酮糖吡喃糖酸甲酯-(2→6)-2,3-二-O-苄酰基-4-O-苄基-1-β-D-半乳糖吡喃糖苷基-(1→4)-3,6-二-O-苄基-2-脱氧-2-(2,2,2-三氯乙氧基)碳酰胺基-β-D-葡萄糖吡喃糖苷基-(1→2)-3,4,6-三-O-苄基-α-D-甘露糖吡喃糖苷基-(1→6)]-[2-O-乙酰基-β-D-甘露糖吡喃糖苷基]-(1→4)-3,6-二-O-苄基-2-脱氧-2-(2,2,2-三氯乙氧基)碳酰胺基-β-D-葡萄糖吡喃糖苷16.
从氟化物供体:将AgOTf(142毫克,0.55毫摩尔,相对于受体8当量)、Cp2HfCl2(105毫克,0.28毫摩尔,相对于受体4当量)与新鲜活化的MS(1g)于无水甲苯(10毫升)中的混合物在氩气氛下于室温搅拌1小时。然后,将反应混合物冷却到-40℃,并添加供体11(232毫克,0.12毫摩尔,1.75当量)与受体15(188毫克,0.069毫摩尔,1当量)于无水甲苯(1毫升)中的溶液。在-15℃下持续搅拌3小时。将反应混合物用Et3N(0.20毫升)淬灭,用EtOAc(20毫升)稀释,并通过硅藻土垫过滤。将滤液用饱和NaHCO3水溶液(8毫升)和盐水(4毫升)洗涤两次。将有机相经MgSO4脱水,过滤并在真空中浓缩。所得残余物通过硅胶管柱层析纯化,使用丙酮/甲苯(2:3)作为溶析液,得到化合物16,为白色粉末(223毫克,70%),伴随回收的15(23.0毫克,80%brsm)。
从三氟乙酰亚胺酸酯供体:将受体15(32.0毫克,0.012毫摩尔,1当量)、供体12(42.9毫克,0.021毫摩尔,1.75当量)与活化的粉碎MS(200毫克)于无水CH2Cl2(2毫升)中的混合物在氩气下搅拌30分钟。然后,将反应混合物冷却到-60℃,并用TfOH(0.5M于Et2O中,5.86μL,2.93μmol,相对于受体0.25当量)处理。在-20℃下搅拌3小时之后,将反应混合物用Et3N(0.10毫升)淬灭,用CH2Cl2(10毫升)稀释,并通过硅藻土垫过滤。将滤液用饱和NaHCO3水溶液(4毫升)和盐水(3毫升)洗涤两次。将有机相经MgSO4脱水,过滤并浓缩。所得残余物通过硅胶管柱层析纯化,使用丙酮/甲苯(2:3)作为溶析液,得到化合物16,为白色粉末(18.0毫克,33%),伴随回收的受体(12.6毫克,55%brsm)。Rf=0.17(硅胶,丙酮:甲苯=1:2);1H NMR(600MHz,CDCl3):δ7.91-7.86(m,8H,Ar-H),7.48-7.42(m,4H,Ar-H),7.33-7.11(m,83H,Ar-H),5.81-5.75(m,2H),5.43-5.38(m,3H),5.31-5.07(m,11H,2C1-Hα),5.03-4.78(m,9H,2C1-Hβ),4.75-4.08(m,48H,4C1-Hβ),4.05-3.89(m,7H),3.87-2.99(m,40H),2.13-2.12(m,6H,-2CH3),2.10-2.07(m,15H,-5CH3),1.99-1.97(s,6H,-2CH3),1.90-1.88(m,6H,-2CH3);13C NMR(150 MHz,CDCl3):δ170.7,170.6,170.6,170.4,170.2,169.8,169.7,169.6,165.6,165.5,165.4,165.4,165.1,154.0,153.6,139.1,138.8,138.4,138.3,138.1,138.0,137.7,133.2,133.1,129.8,129.8,129.7,129.6,129.6,129.5,129.5,129.1,129.1,128.4,128.4,128.3,128.2,128.2,128.0,128.0,127.8,127.8,127.8,127.7,127.7,127.5,127.4,127.4,127.3,127.2,127.1,100.1,99.8,98.8,98.4,98.2,95.7,88.1,86.8,78.3,77.8,75.3,74.7,74.5,74.4,74.3,74.2,74.0,73.9,73.4,73.3,73.3,73.2,73.1,73.0,72.9,72.5,72.4,72.0,71.9,71.5,71.0,71.0,70.9,70.9,70.7,69.5,69.4,69.2,69.1,69.0,69.0,68.5,68.1,68.1,68.0,67.2,62.9,62.2,57.5,57.2,53.8,53.2,53.2,45.5,45.3,23.3,21.0,21.0,20.8,20.7,20.7,20.6;19F NMR(376MHz,CDCl3):δ-215.6(2F);HRMS(ESI-TOF)m/e:C232H255Cl9F2N6O74[M+H3O+NH4]2+的计算值:2330.6770,实测值:2330.6724。
[5-乙酰胺基-3,5-二脱氧-3-氟-D-赤藻糖-α-L-甘露糖-壬-2-酮糖吡喃糖酸酯-(2→6)-β-D-半乳糖吡喃糖苷基-(1→4)-2-乙酰胺基-2-脱氧-β-D-葡萄糖吡喃糖苷基-(1→2)-α-D-甘露糖吡喃糖苷基-(1→3)]-[5-乙酰胺基-3,5-二脱氧-3-氟-D-赤藻糖-α-L-甘露糖-壬-2-酮糖吡喃糖酸酯-(2→6)-β-D-半乳糖吡喃糖苷基-(1→4)-2-乙酰胺基-2-脱氧-β-D-葡萄糖吡喃糖苷基-(1→2)-α-D-甘露糖吡喃糖苷基-(1→6)]-[β-D-甘露糖吡喃糖苷基]-(1→4)-2-乙酰胺基-2-脱氧-D-葡萄糖吡喃糖苷17.将受保护聚糖16(103毫克,0.022毫摩尔,1当量)与LiOH(51.5毫克,50%by S.M.wt)于1,4-二恶烷/H2O(3毫升,4:1=v/v)混合物中的溶液在90℃下搅拌16小时。将反应混合物在高真空下浓缩并经受乙酰化条件(吡啶(2.5毫升),Ac2O(1.5毫升),r.t.,16h)。在移除溶剂之后,粗残余物通过RP-18逆相管柱层析纯化,使用H2O/MeOH(1:5)作为溶析液。将产物与NaOMe的MeOH溶液(3毫升,0.5M)一起搅拌16小时予以去乙酰化。将反应混合物用IR-120中和,过滤并在真空中浓缩。残余物通过RP-18逆相管柱层析纯化,使用H2O/MeOH(1:4)作为溶析液。将去乙酰化的粗产物溶解于MeOH/H2O/HCOOH(3毫升,6:3:1=v/v/v)混合物中,用Pd(OH)2(51.5毫克,50%by S.M.wt)处理20小时。将反应混合物通过硅藻土垫过滤并在真空中浓缩。残余物通过(BIO-RAD)Biogel P-2管柱层析(用水溶析)纯化,接着通过RP-18逆相管柱层析(用水溶析)纯化,得到化合物17,为变旋异构物混合物(α:β=0.65:0.35)的白色粉末(18.3毫克,40%)。1H NMR(600MHz,D2O):δ5.23(d,J=3.0Hz,0.65H,a-C1-Hα),5.14(d,J=52.8Hz,2H,sia-C3-H),5.14(br,1H,c-C1-Hα),4.96(br,1H,c’-C1-Hα),4.79(br,1H,b-C1-Hβ),4.73(br,0.35H,a-C1-Hβ),4.60(d,J=7.8Hz,2H,dd’-C1-Hβ),4.48(d,J=7.8Hz,2H,ee’-C1-Hβ),4.28-4.21(m,4H),4.13(br,1H),4.00-3.51(m,57H),2.09-2.08(m,9H,-3CH3),2.05(br,6H,-2CH3);13C NMR(150MHz,D2O):δ174.5,174.2,174.1,102.7,102.6,100.0,99.1,98.9,98.9,98.7,98.6,96.6,94.4,91.0,90.0,89.8,80.0,79.7,79.3,78.9,76.0,75.8,74.1,74.0,73.9,73.1,72.9,72.7,72.4,71.9,71.9,71.6,71.5,71.1,70.3,69.8,69.4,69.2,69.1,69.0,68.7,68.0,67.8,66.9,66.8,65.4,65.2,63.6,62.2,61.2,61.2,59.7,59.6,55.6,54.4,53.1,46.3,21.9,21.6;19F NMR(376MHz,D2O):δ-217.4,-217.4;HRMS(ESI-TOF)m/e:C76H121F2N5O57[M-2H]-2的计算值:1026.8351,实测值:1026.8299。
[5-乙酰胺基-3,5-二脱氧-3-氟-D-赤藻糖-α-L-甘露糖-壬-2-酮糖吡喃糖酸酯-(2→6)-β-D-半乳糖吡喃糖苷基-(1→4)-2-乙酰胺基-2-脱氧-β-D-葡萄糖吡喃糖苷基-(1→2)-α-D-甘露糖吡喃糖苷基-(1→3)]-[5-乙酰胺基-3,5-二脱氧-3-氟-D-赤藻糖-α-L-甘露糖-壬-2-酮糖吡喃糖酸酯-(2→6)-β-D-半乳糖吡喃糖苷基-(1→4)-2-乙酰胺基-2-脱氧-β-D-葡萄糖吡喃糖苷基-(1→2)-α-D-甘露糖吡喃糖苷基-(1→6)]-[β-D-甘露糖吡喃糖苷基]-(1→4)-(2-乙酰胺基-1,2-二脱氧-D-葡萄糖吡喃并)-[2,1-d]-2-甲基恶唑啉S1.聚糖恶唑啉的代表性合成。将聚糖17(14.5毫克,7.05μmol,1当量)、氯化2-氯-1,3-二甲基-1H-苯并咪唑-3-鎓(CDMBI)(12.2毫克,0.056毫摩尔,8当量)与Et3N(19.9μL)在水(121.1μL)中的溶液在4℃下搅拌1小时。让反应混合物在Sephadex G-25管柱上进行凝胶过滤层析,用0.05%Et3N水溶液溶析。将含有聚糖恶唑啉产物的流份合并并冻干,得到所要的产物S1,为白色粉末(12.6毫克,产率87%)。1H NMR(600MHz,D2O+0.05%Et3N):δ6.09(d,J=7.2Hz,1H,oxa-C1-Hα),5.12(d,J=52.2Hz,2H,sia-C3-H),5.11(br,1H,c-C1-Hα),4.95(br,1H,c’-C1-Hα),4.74(br,1H,b-C1-Hβ),4.60-4.57(m,2H,dd’-C1-Hβ),4.46-4.45(m,2H,ee’-C1-Hβ),4.39(br,1H),4.22-4.15(m,5H),3.98-3.49(m,55H),3.43-3.40(m,1H),2.07-2.02(m,15H,-5CH3);13C NMR(150MHz,D2O+0.05%Et3N):δ174.9,174.7,174.7,170.7,170.7,168.5,103.1,103.1,101.4,99.9,99.5,99.4,99.2,99.1,99.0,96.5,91.4,90.2,80.4,79.4,77.9,76.4,76.0,74.6,74.2,73.4,73.4,72.8,72.4,72.3,72.0,71.9,71.5,70.8,70.7,70.2,69.6,69.5,69.4,69.1,68.5,68.2,67.2,65.8,65.7,65.1,64.1,62.6,61.6,61.6,60.1,54.9,46.7,22.4,22.0,12.9;19F NMR(470MHz,D2O+0.05%Et3N):δ-217.3(d,JFH=51.3Hz),-217.3(d,JFH=51.3Hz);HRMS(ESI-TOF)m/e:C76H120F2N5O56[M-H]-的计算值:2036.6658,实测值:2036.6672.
流程S1.
流程S1中的试剂和条件:(a)TFA,MeOH,60℃,16h,然后AcCl,r.t.,2d,然后Na2HPO4,CH3CN,回流,20h,92%.(b)ref.3,80%(三步骤).(c)TiBr4,二氯乙烷,r.t.,10min.,92%.(d)BH3,Cu(OTf)2,THF,r.t.,8h,96%.
5-乙酰胺基-4,7,8,9-四-O-乙酰基-3,5-二脱氧-2,6-脱水-D-甘油基-D-半乳糖–壬-2-烯酸甲酯S3.19向搅拌的化合物S2(20g,64.7毫摩尔,1当量)于MeOH(600毫升)中的溶液添加TfOH(4.95毫升,64.7毫摩尔,1当量)。让反应混合物在60℃下搅拌16小时。将溶剂通过在减压下旋转蒸发除去并与甲苯共蒸发两次以除去水迹。在0℃下将所得残余物溶解于圆底烧瓶中的AcCl(200毫升)中,密封并搅拌至室温为时2天。移除溶剂后,将所得残余物用无水乙腈(200毫升)稀释,然后在氩气下添加Na2HPO4(19.6g,155毫摩尔,2.4当量)。将反应混合物在90℃下剧烈搅拌直到TLC显示起始物消失(16小时)。将溶液通过硅藻土垫过滤,经MgSO4脱水,并在真空中浓缩。所得残余物通过硅胶管柱层析纯化,使用EtOAc/己烷(4:1)作为溶析液,得到化合物S3,为棕色泡沫(28.2g,92%)。光谱数据与先前文献中所报导者一致。19Rf=0.30(硅胶,EtOAc);1H NMR(600MHz,CDCl3):δ5.95(d,J=3.0Hz,1H),5.81(d,J=9.0Hz,1H),5.47-5.44(m,2H),5.32-5.30(m,1H),4.59(dd,J=12.0,3.0Hz,1H),4.38-4.33(m,2H),4.15(d,J=12.0,7.2Hz,1H),3.76(s,3H,-CH3),2.08(s,3H,-CH3),2.03(s,3H,-CH3),2.02(s,3H,-CH3),2.01(s,3H,-CH3),1.88(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ170.7,170.5,170.1,170.1,170.0,161.6,145.0,107.9,70.6,67.8,67.6,61.9,52.5,46.5,23.1,20.8,20.7,20.7;HRMS(ESI-TOF)m/e:的计算值C20H27NO12Na[M+Na]+:496.1425,实测值:496.1435。
5-乙酰胺基-4,7,8,9-四-O-乙酰基-2-溴-2,5-二脱氧-D-赤藻糖-α-L-葡萄糖-壬-2-酮糖吡喃糖酸甲酯3.20利用经报导的程序从唾液酸进行环氧化物S4的合成。21向搅拌的环氧化合物S4(1.10g,2.25毫摩尔,1当量)于无水1,2-二氯乙烷(18毫升)中的溶液,在氩气下花10分钟添加TiBr4(0.91g,2.47毫摩尔,1.1当量)。通过在高真空下旋转蒸发除去溶剂。将所得残余物用EtOAc(30毫升)稀释,用饱和Na2SO4水溶液(10毫升)、5%NaHCO3水溶液(10毫升)及盐水(5毫升)洗涤。将分离的有机层经MgSO4脱水并在真空中浓缩,所得残余物通过硅胶管柱层析纯化,使用丙酮/己烷(2:1)作为溶析液,得到化合物3,为白色粉末(1.18g,92%)。光谱数据和实验方案与先前所报导者相同。20Rf=0.23(硅胶,丙酮:甲苯=1:1);1H NMR(600MHz,CDCl3):δ5.99(d,J=7.2Hz,1H),5.42(dd,J=7.2,1.2Hz,1H),5.21-5.18(m,1H),5.10(ddd,J=6.0,6.0,2.4Hz,1H),4.38(dd,J=12.6,2.4Hz,1H),4.32-4.30(m,2H),3.99(dd,J=12.6,6.0Hz,1H),3.86(s,3H,-CH3),3.78(d,J=9.0Hz,1H),3.66(br,1H,-OH),2.07(s,3H,-CH3),2.06(s,3H,-CH3),2.05(s,3H,-CH3),2.01(s,3H,-CH3),1.84(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ171.4,170.6,170.3,169.8,169.7,167.0,98.0,75.0,72.8,72.2,69.9,66.5,61.9,54.0,47.2,22.9,21.0,20.7,20.6;HRMS(ESI-TOF)m/e:C20H29BrNO13[M+H]+的计算值:570.0817,实测值:570.0822。
对甲苯基2,3-二-O-苄酰基-4-O-苄基-1-硫基-β-D-半乳糖吡喃糖苷5.22向搅拌的起始物S523(4.00g,6.86毫摩尔,1当量)于BH3-THF错合物(1M in THF,34.3毫升,34.3毫摩尔,5当量)中的溶液添加Cu(OTf)2(124毫克,0.34毫摩尔,0.05当量),然后将反应混合物在室温下搅拌8小时。完成之后,在0℃下将反应小心地用TEA(0.96毫升,6.86毫摩尔,1当量)中和,然后用MeOH(15毫升)稀释。在真空中移除溶剂之后,所得残余物通过硅胶管柱层析纯化,使用EtOAc/己烷(1:2)作为溶析液,得到化合物5,为白色粉末(3.85g,96%)。光谱数据与先前文献中所报导者一致22。Rf=0.45(硅胶,EtOAc:己烷=1:1);1H NMR(600MHz,CDCl3):δ7.97-7.94(m,4H,Ar-H),7.51-7.48(m,2H,Ar-H),7.38-7.34(m,6H,Ar-H),7.27-7.21(m,5H,Ar-H),5.85(dd,J=10.2,10.2Hz,1H),5.36(dd,J=9.6,3.0Hz,1H),4.87(d,J=9.6Hz,1H,C1-Hβ),4.74(d,J=12.0Hz,1H),4.47(d,J=12.0Hz,1H),4.16(d,J=2.4Hz,1H),3.91(d,J=11.4,7.2Hz,1H),3.76(dd,J=6.0,6.0Hz,1H),3.61(dd,J=11.4,5.4Hz,1H),2.30(s,3H,-CH3),1.83(br,1H);13C NMR(150MHz,CDCl3):δ165.9,165.2,138.1,137.4,133.4,133.1,133.0,129.8,129.7,129.6,129.5,128.9,128.7,128.5,128.4,128.3,128.1,127.9,86.8,79.0,76.0,74.6,73.7,68.5,61.9,21.1;HRMS(ESI-TOF)m/e:C34H32O7SNa[M+Na]+的计算值:607.1761,实测值:607.1770。
流程S2
流程S2中的试剂和条件:(a)TESOTf,BnCHO,TESH,甲苯,THF,2h,-20℃,90%.(b)NIS,TfOH,MS,-40℃,1.5h,CH2Cl2,75%.(c)NaBH3CN,HCl/醚,AW-300,THF,0℃tor.t.,16h,85%.
对甲苯基3-O-苄基-4,6-O-亚苄基-2-脱氧-1-硫基-2-(2,2,2-三氯乙氧基)胺甲酰基胺基-β-D-葡萄糖吡喃糖苷S7.在氩气下于-20℃,向起始物S624(5.17g,9.42毫摩尔,1当量)于THF/甲苯(45毫升,1:2=v/v)混合物中的溶液添加TESOTf(4.26毫升,18.8毫摩尔,2当量)。在-20℃下搅拌45分钟之后,将苯甲醛(4.79毫升,47.1毫摩尔,5当量)和三乙基硅烷(2.26毫升,14.1毫摩尔,1.5当量)伴随搅拌逐滴添加至该混合物中。在-20℃下2小时之后,TLC显示起始物消失,将反应用饱和Na2CO3水溶液淬灭,用EtOAc(100毫升)稀释并用饱和Na2CO3水溶液(30毫升)和盐水洗涤。将合并的有机层经MgSO4脱水,过滤并在真空中浓缩。所得残余物通过硅胶管柱层析纯化,使用CH2Cl2/甲苯(1:4)然后EtOAc/甲苯(1:10)作为溶析液,得到化合物S7,为白色固体(5.40g,90%)。Rf=0.50(硅胶,EtOAc:甲苯=1:25);1H NMR(600MHz,丙酮-d6):δ7.52-7.50(m,2H,Ar-H),7.42-7.36(m,4H,Ar-H),7.32-7.31(m,2H,Ar-H),7.29-7.22(m,4H,Ar-H),7.18-7.15(m,2H,Ar-H),5.72(s,1H,Ph-CH),5.03(d,J=10.8Hz,1H,C1-Hβ),4.90-4.82(m,3H),4.74(d,J=12.0Hz,1H),4.30(dd,J=10.2,5.4Hz,1H),3.95(dd,J=9.6,9.0Hz,1H),3.84-3.71(m,3H),3.56(ddd,J=9.6,9.6,4.8Hz,1H),2.83(s,2H),2.32(s,3H,-CH3);13C NMR(150MHz,丙酮-d6):δ155.3,139.8,139.4,138.6,133.3,130.7,130.5,129.8,129.6,129.1,129.0,129.0,128.5,128.2,127.1,101.8,97.1,88.6,82.7,80.8,75.0,75.0,71.2,69.1,57.4,21.1;HRMS(ESI-TOF)m/e:C30H30Cl3NO6SNa[M+Na]+的计算值:660.0752,实测值:660.0749。
烯丙基[3-O-苄基-4,6-O-亚苄基-2-脱氧-2-(2,2,2-三氯乙氧基)碳酰胺基-β-D-葡萄糖吡喃糖苷基]-(1→2)-3,4,6-三-O-苄基-α-D-甘露糖吡喃糖苷S9.将受体S825(523毫克,0.94毫摩尔,1当量)、供体S7(900毫克,1.41毫摩尔,1.5当量)与活化的粉碎MS(2.00g)于无水CH2Cl2(20毫升)中的混合物在氩气下搅拌1小时。然后,将反应混合物冷却到-40℃并用NIS(423毫克,1.88毫摩尔,2当量)和TfOH(0.5M于Et2O中,0.47毫升,0.23毫摩尔,0.25当量)处理。在该温度下搅拌1.5小时后,TLC分析显示起始物消失,将反应混合物用Et3N(0.2毫升)淬灭,通过硅藻土垫过滤。将滤液用CH2Cl2(20毫升)稀释,用20%Na2S2O3水溶液(5毫升)、饱和NaHCO3水溶液(10毫升)及盐水(5毫升)洗涤。将分离的有机层经MgSO4脱水并在真空中浓缩。所得残余物通过硅胶管柱层析纯化,使用EtOAc/甲苯(1:10)作为溶析液,得到化合物S9,为白色泡沫(710毫克,75%)。Rf=0.36(硅胶,EtOAc:甲苯=1:10);1H NMR(600MHz,CDCl3):δ7.50-7.48(m,2H,Ar-H),7.39-7.36(m,6H,Ar-H),7.33-7.25(m,15H,Ar-H),7.23-7.22(m,2H,Ar-H),5.84(ddd,J=16.2.10.8,5.4Hz,1H),5.55(s,1H,Ph-CH),5.26(d,J=1.2Hz,1H),5.23-5.20(m,1H),5.16(dd,J=10.8,1.2Hz,1H),5.01(d,J=7.8Hz,1H,C1-Hβ),4.90(d,J=10.8Hz,1H),4.83(d,J=11.4Hz,1H),4.75-4.62(m,6H,C1-Hα),4.55-4.51(m,3H),4.33-4.29(m,2H),4.13-4.10(m,1H),4.08(dd,J=2.4,2.4Hz,1H),4.00-3.97(m,1H),3.94(dd,J=9.6,3.0Hz,1H),3.89(dd,J=13.2,6.0Hz,1H),3.82-3.78(m,2H),3.72-3.66(m,3H),3.50-3.46(m,1H),3.12(d,J=6.0Hz,1H);13C NMR(150MHz,CDCl3):δ153.8,138.6,138.5,138.3,138.1,137.3,133.7,129.0,128.4,128.3,128.2,128.1,128.0,127.8,127.6,127.6,127.4,126.0,117.2,101.2,98.9,96.9,95.5,82.4,78.5,75.6,75.2,74.6,74.4,74.2,73.3,72.2,72.2,69.2,68.6,68.0,66.1,58.1;HRMS(ESI-TOF)m/e:C53H56Cl3NO12Na[M+Na]+的计算值:1026.2760,实测值:1026.2780。
烯丙基[3,6-二-O-苄基-2-脱氧-2-(2,2,2-三氯乙氧基)碳酰胺基-β-D-葡萄糖吡喃糖苷基]-(1→2)-3,4,6-三-O-苄基-α-D-甘露糖吡喃糖苷8.27在氩气下,向搅拌的起始物S9(500毫克,0.50毫摩尔,1当量)于无水THF(12毫升)中的溶液添加活化的粉碎AW 300MS(1.20克)。然后,将反应混合物冷却到0℃并添加NaCNBH3(313毫克,4.97毫摩尔,10当量),接着慢慢添加HCl·Et2O(2M于Et2O中,2.24毫升,4.48毫摩尔,9当量),然后将混合物持续搅拌直到TLC显示起始物消失(16小时)。完成之后,将反应混合物用饱和NaHCO3水溶液(0.5毫升)淬灭并通过硅藻土垫过滤。将滤液用CH2Cl2(20毫升)稀释,然后用饱和NaHCO3水溶液(8毫升)和盐水洗涤。将分离的有机层经MgSO4脱水并浓缩。所得残余物通过硅胶管柱层析纯化,使用EtOAc/甲苯(1:5)作为溶析液,得到化合物8,为白色泡沫(425毫克,85%)。光谱数据与文献中所报导者一致。27Rf=0.17(硅胶,EtOAc:甲苯=1:10);1H NMR(600MHz,CDCl3):δ7.36-7.23(m,23H,Ar-H),7.20-7.19(m,2H,Ar-H),5.84(ddd,J=16.2.10.2,6.0Hz,1H),5.35(br,1H),5.21(dd,J=16.2,1.2Hz,1H),5.14(dd,J=10.2,1.2Hz,1H),4.95(d,J=7.2Hz,1H,C1-Hβ),4.88(d,J=10.8Hz,1H),4.78(d,J=1.8Hz,1H,C1-Hα),4.75(d,J=11.4Hz,1H),4.70-4.63(m,5H),4.60(d,J=11.4Hz,1H),4.55-4.49(m,4H),4.14-4.07(m,3H),3.96-3.87(m,3H),3.77(dd,J=10.8,4.2Hz,1H),3.73-3.70(m,3H),3.66(dd,J=10.8,1.8Hz,1H),3.61(dd,J=9.6,8.4Hz,1H),3.54-3.51(m,1H),3.08(m,1H),2.71(m,1H,-OH);13C NMR(150MHz,CDCl3):δ154.0,138.5,138.5,138.4,138.1,137.6,133.7,128.5,128.5,128.3,128.3,128.3,128.2,128.0,128.0,127.8,127.7,127.7,127.6,127.6,127.5,117.3,79.2,78.2,75.1,74.6,74.3,74.2,73.8,73.7,73.4,73.3,72.0,71.7,70.9,69.3,68.0,57.5;HRMS(ESI-TOF)m/e:C53H58Cl3NO12Na[M+Na]+的计算值:1028.2917,实测值:1028.2927。
流程S3
流程S3中的试剂和条件:(a)NaBH3CN,HCl/醚,AW-300,THF,0℃to r.t.,16h,90%.(b)Ac2O,吡啶,r.t.,16h,98%.(c)NIS,TfOH,HOPO(OBu)2,MS,-30℃,2h,CH2Cl2,90%.(d)TMSOTf,MS,-50℃,1h,CH2Cl2,87%.(e)NaOMe,MeOH/CH2Cl2,0℃to r.t.,76%.
对甲苯基3,6-二-O-苄基-2-脱氧-1-硫基-2-(2,2,2-三氯乙氧基)胺甲酰基胺基-β-D-葡萄糖吡喃糖苷S10.28在氩气下,向搅拌的起始物S7(1.00克,1.56毫摩尔,1当量)于无水THF(30毫升)中的溶液添加活化的粉碎AW 300MS(3.00克)。然后,将反应混合物冷却到0℃,并添加NaCNBH3(0.98克,15.6毫摩尔,10当量),接着慢慢添加HCl·Et2O(2M于Et2O中,7.04毫升,14.1毫摩尔,9当量),将混合物搅拌直到TLC显示起始物消失(16小时)。完成之后,将混合物用饱和NaHCO3水溶液(0.5毫升)淬灭并通过硅藻土垫过滤。将滤液用CH2Cl2(50毫升)稀释,然后用饱和NaHCO3水溶液(15毫升)和盐水(10毫升)洗涤。将分离的有机层经MgSO4脱水并浓缩。所得残余物通过硅胶管柱层析纯化,使用EtOAc/己烷/CH2Cl2(1:3:1)作为溶析液,得到化合物S10,为白色泡沫(902毫克,90%)。光谱数据与文献中所报导者一致28。Rf=0.29(硅胶,EtOAc:己烷=1:3);1H NMR(600MHz,CDCl3):δ7.40-7.38(m,2H,Ar-H),7.36-7.27(m,10H,Ar-H),7.04-7.03(m,2H,Ar-H),5.20(d,J=7.8Hz,1H),4.84(d,J=10.2Hz,1H,C1-Hβ),4.77-4.72(m,4H),4.58-4.52(m,2H),3.76(d,J=4.8Hz,2H),3.71-3.64(m,2H),3.50-3.49(m,1H),3.42-3.38(m,1H),2.90(br,1H,-OH),2.29(s,3H,-CH3);13CNMR(150MHz,CDCl3):δ153.8,138.1,138.1,137.7,133.0,129.6,128.6,128.5,128.4,128.1,127.9,127.8,127.7,95.5,86.1,81.9,78.0,74.5,74.4,73.6,72.5,70.4,55.9,21.1;HRMS(ESI-TOF)m/e:C30H32Cl3NO6SNa[M+Na]+的计算值:662.0908,实测值:662.0919。
对甲苯基4-O-乙酰基-3,6-二-O-苄基-2-脱氧-1-硫基-2-(2,2,2-三氯乙氧基)胺甲酰基胺基-β-D-葡萄糖吡喃糖苷S11.向搅拌的起始物S10(0.97克,1.51毫摩尔,1当量)于吡啶(12毫升)中的溶液添加Ac2O(6毫升)。将反应混合物在室温下剧烈搅拌16小时,然后在真空中浓缩并通过硅胶管柱层析纯化,使用EtOAc/己烷/CH2Cl2(1:4:1)作为溶析液,得到化合物S11,为白色粉末(1.01克,98%)。Rf=0.47(硅胶,EtOAc:己烷=1:3);1H NMR(600MHz,CDCl3):δ7.41-7.40(m,2H,Ar-H),7.34-7.25(m,8H,Ar-H),7.22-7.21(m,2H,Ar-H),7.03-7.01(m,2H,Ar-H),5.29(d,J=7.2Hz,1H),5.04(d,J=10.2Hz,1H,C1-Hβ),4.98(dd,J=9.6,9.6Hz,1H),4.78(d,J=12.0Hz,1H),4.70(d,J=12.0Hz,1H),4.62(d,J=11.4Hz,1H),4.57(d,J=11.4Hz,1H),4.50(br,2H),4.05(dd,J=9.6,9.0Hz,1H),3.65-3.63(m,1H),3.59-3.55(m,2H),3.33(ddd,J=9.6,9.6,9.6Hz,1H),2.29(s,3H,-CH3),1.87(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ169.8,153.7,138.4,138.0,137.7,133.3,129.8,128.6,128.5,128.4,128.2,128.1,128.1,127.9,127.9,127.7,95.5,85.3,79.4,77.7,74.4,73.6,71.4,69.7,56.5,21.1,20.9;HRMS(ESI-TOF)m/e:C32H34Cl3NO7SNa[M+Na]+的计算值:704.1014,实测值:104.1031。
4-O-乙酰基-3,6-二-O-苄基-2-脱氧-1-硫基-2-(2,2,2-三氯乙氧基)胺甲酰基胺基-α或β-D-葡萄糖吡喃糖苷磷酸二丁酯S12a,b.将化合物S11(990毫克,1.45毫摩尔,1当量)、磷酸二丁酯(1.15毫升,5.80毫摩尔,3当量)与活化的粉碎MS(1.80克)于无水CH2Cl2(18毫升)中的混合物在氩气下搅拌1小时。然后将其随搅拌冷却到-30℃,添加NIS(978毫克,4.35毫摩尔,2当量),接着添加TfOH(0.5M in Et2O,0.87毫升,0.43毫摩尔,0.3当量)。2小时之后,TLC分析显示起始物消失,将反应混合物用饱和NaHCO3水溶液(0.5毫升)淬灭并通过硅藻土垫过滤。将滤液用CH2Cl2(50毫升)稀释,用20%Na2S2O3水溶液(10毫升)、饱和NaHCO3水溶液(5毫升)及盐水(5毫升)洗涤。将分离的有机层经MgSO4脱水并在真空中浓缩。所得残余物通过硅胶管柱层析纯化,使用EtOAc/甲苯(1:3)作为溶析液,得到化合物S12,为白色泡沫(1.00克,90%);变旋异构物混合物(α:β=1:3)。β-变旋异构物S12b:Rf=0.32(硅胶,EtOAc:甲苯=1:3);1H NMR(600MHz,CDCl3):δ7.30-7.22(m,10H,Ar-H),5.81(br,1H),5.35(dd,J=7.8,7.8Hz,1H,C1-Hβ),5.09(dd,J=9.6,9.0Hz,1H),4.74(d,J=12.0Hz,1H),4.65-4.58(m,3H),4.49-4.44(m,2H),4.06-3.97(m,4H),3.88(dd,J=9.6,9.0Hz,1H),3.77-3.74(m,1H),3.69(ddd,J=9.6,4.2,4.2Hz,1H),3.53-3.52(m,2H),1.83(s,3H,-CH3),1.61-1.54(m,4H),1.36-1.31(m,4H),0.93-0.85(m,6H);13C NMR(150MHz,CDCl3):δ169.5,154.2,137.6,137.6,128.5,128.4,128.3,127.9,127.9,127.7,127.7,96.5,95.3,78.3,74.5,73.9,73.6,73.5,70.6,69.2,68.1,68.1,68.1,68.0,57.2,57.2,32.1,32.0,32.0,20.7,18.6,18.6,13.5;HRMS(ESI-TOF)m/e:C33H45Cl3NO11PNa[M+Na]+的计算值:790.1688,实测值:790.1685。α-变旋异构物S12a:Rf=0.44(硅胶,EtOAc:甲苯=1:3);1H NMR(600MHz,CDCl3):δ7.31-7.22(m,10H,Ar-H),5.67(dd,J=6.0,3.6Hz,1H,C1-Hα),5.21-5.16(m,2H),4.75(d,J=12.0Hz,1H),4.62-4.56(m,3H),4.49-4.44(m,2H),4.11-3.99(m,6H),3.77(dd,J=10.2,9.6Hz,1H),3.50-3.49(m,2H),1.89(s,3H,-CH3),1.60-1.55(m,4H),1.35-1.31(m,4H),0.92-0.86(m,6H);13C NMR(150MHz,CDCl3):δ169.3,154.0,137.4,137.4,128.4,128.2,127.8,127.8,127.8,127.6,127.6,127.6,96.4,96.3,95.2,76.4,74.6,73.6,73.5,71.1,70.1,68.6,68.1,68.0,68.0,67.9,54.4,54.3,32.1,32.0,20.7,18.5,18.5,13.5;HRMS(ESI-TOF)m/e:C33H45Cl3NO11PNa[M+Na]+的计算值:790.1688,实测值:790.1688。
对甲苯基[4-O-乙酰基-3,6-二-O-苄基-2-脱氧-2-(2,2,2-三氯乙氧基)碳酰胺基-β-D-葡萄糖吡喃糖苷基]-(1→2)-3,4,6-三-O-苄基-1-硫基-α-D-甘露糖吡喃糖苷S14.将受体S1329(407毫克,0.73毫摩尔,1当量)、供体S12(900毫克,1.17毫摩尔,1.6当量)与活化的粉碎分子筛(200毫克)于无水CH2Cl2(2毫升)中的混合物在氩气下搅拌30min。然后将其冷却到-50℃,接着伴随搅拌添加TMSOTf(0.21毫升,1.17μmol,相对于受体1.6当量)直到TLC分析显示起始物消失(1小时)。将反应混合物用Et3N(0.30毫升)淬灭,用CH2Cl2(20毫升)稀释,然后通过硅藻土垫过滤。将滤液用饱和NaHCO3水溶液(8毫升)和盐水(4毫升)洗涤两次。将有机相经MgSO4脱水,过滤并在真空中浓缩。所得残余物通过硅胶管柱层析纯化,使用EtOAc/己烷(1:3)作为溶析液,得到化合物S14,为白色粉末(710毫克,87%)。Rf=0.26(硅胶,EtOAc:己烷=1:3);1H NMR(600MHz,CDCl3):δ7.37-7.36(m,2H,Ar-H),7.33-7.14(m,25H,Ar-H),7.05-7.03(m,2H,Ar-H),5.33(d,J=1.8Hz,1H,C1-Hα),5.26(d,J=4.8Hz,1H),5.07(d,J=9.0Hz,1H,C1-Hβ),4.93-4.90(m,2H),4.77(d,J=11.4Hz,1H),4.60-4.52(m,5H),4.46-4.34(m,5H),4.27(dd,J=8.4,8.4Hz,1H),4.19(dd,J=9.6,2.4Hz,1H),4.15(d,J=12.0Hz,1H),4.07(dd,J=9.6,9.0Hz,1H),3.84-3.81(m,2H),3.64-3.63(m,2H),3.57(dd,J=10.8,6.0Hz,1H),3.51(dd,J=11.4,3.0Hz,1H),3.01(dd,J=6.6Hz,1H),2.28(s,3H,-CH3),1.82(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ169.7,154.2,138.5,138.3,138.0,137.8,137.7,137.6,132.5,130.3,129.8,129.7,129.0,128.4,128.4,128.3,128.2,128.2,128.2,128.2,127.9,127.8,127.8,127.7,127.7,127.6,127.6,127.6,127.4,125.2,97.1,95.4,86.2,78.3,75.2,75.1,74.5,74.1,74.0,73.6,73.4,73.1,72.5,71.7,71.3,70.0,69.2,58.1,21.1,20.8;HRMS(ESI-TOF)m/e:C59H62Cl3NO12SNa[M+Na]+的计算值:1136.2950,实测值:1136.2978。
对甲苯基[3,6-二-O-苄基-2-脱氧-2-(2,2,2-三氯乙氧基)碳酰胺基-β-D-葡萄糖吡喃糖苷基]-(1→2)-3,4,6-三-O-苄基-1-硫基-α-D-甘露糖吡喃糖苷18.在0℃下,向充分搅拌的化合物S14(203毫克,0.18毫摩尔,1当量)于CH2Cl2/MeOH混合物(10毫升,1:1=v/v)中的溶液添加NaOMe(2.95毫克,0.055毫摩尔,0.3当量)。20分钟之后,移去冰浴并将反应混合物伴随搅拌升温至室温,直到TLC分析显示起始物消失(4小时)。完成之后,将反应混合物用IR-120中和,过滤并在真空中浓缩。所得残余物通过硅胶管柱层析纯化,使用EtOAc/己烷(1:2)作为溶析液,得到化合物化合物18,为白色泡沫(149毫克,76%)。Rf=0.14(硅胶,EtOAc:己烷=1:3);1H NMR(600MHz,CDCl3):δ7.39-7.37(m,2H,Ar-H),7.34-7.24(m,25H,Ar-H),7.06-7.05(m,2H,Ar-H),5.33(d,J=1.8Hz,1H,C1-Hα),5.24(d,J=4.8Hz,1H),5.00(d,J=7.8Hz,1H,C1-Hβ),4.93(d,J=10.8Hz,1H),4.78(d,J=11.4Hz,1H),4.69-4.64(m,2H),4.61(d,J=11.4Hz,2H),4.57-4.51(m,4H),4.43(d,J=11.4Hz,1H),4.34(dd,J=2.4,2.4Hz,1H),4.22-4.20(m,2H),4.07-4.04(m,2H),3.84-3.82(m,2H),3.77-3.72(m,2H),3.67-3.65(m,1H),3.59-3.54(m,2H),3.01(d,J=7.2Hz,1H),2.68(br,1H,-OH),2.30(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ154.2,138.5,138.4,138.3,137.9,137.7,137.6,132.5,130.3,129.8,128.5,128.5,128.4,128.3,128.1,128.0,127.9,127.8,127.8,127.7,127.6,127.6,127.5,97.5,95.4,86.2,79.1,78.4,75.3,75.2,74.6,74.4,74.2,73.8,73.8,73.2,73.1,72.5,71.4,70.9,69.3,57.8,21.1;HRMS(ESI-TOF)m/e:C57H60Cl3NO11SNa[M+Na]+的计算值:1094.2845,实测值:1094.2881。
流程S4
流程S4中的试剂和条件:(a)NIS,TfOH,MS,-40℃,1h,CH2Cl2,93%.(b)乙二胺/BuOH(1:4),90℃,2h,然后NaHCO3,TrocCl,CH2Cl2,0℃,3h,81%.(c)Tf2O,吡啶,CH2Cl2,0℃,4h.(d)Bu4NOAc,甲苯,超音波处理,r.t.,8h,然后NaHCO3,TrocCl,CH2Cl2,0℃,3h,61%(2步骤).(e)DDQ,CH2Cl2,磷酸盐缓冲液(pH=7),0℃至r.t.,3h,85%.(f)乙酸肼,THF,r.t.,16h,92%.(g)Tf2O,吡啶,CH2Cl2,0℃,2h.(h)Bu4NOAc,甲苯,超音波处理,r.t.,8h,93%(2步骤).(i)乙二胺/BuOH(1:4),90℃,2h,然后NaHCO3,TrocCl,CH2Cl2,0℃,3h.(j)Ac2O,吡啶,CH2Cl2,r.t.,16h,83%(2步骤).
苄基[4,6-O-亚苄基-3-O-p-甲氧基-苄基-2-O-乙酰丙酰基(levulinoyl)-β-D-葡萄糖吡喃糖苷基]-(1→4)-3,6-二-O-苄基-2-脱氧-2-酞酰亚胺基-β-D-葡萄糖吡喃糖苷S17.将受体S1630(1.59克,2.74毫摩尔,1当量)、供体S1526(2.11克,3.57毫摩尔,1.3当量)与活化的粉碎MS(5.00克)于无水CH2Cl2(50毫升)中的混合物在氩气下搅拌1小时。然后,将反应混合物冷却到-40℃并添加NIS(1.23克,0.49毫摩尔,2当量),接着添加TfOH(0.5M于Et2O中,1.37毫升,0.69毫摩尔,0.25当量)。反应持续直到TLC显示起始物消失(2小时)。完成之后,将混合物用Et3N(0.7毫升)淬灭并通过硅藻土垫过滤。将滤液用CH2Cl2(50毫升)稀释,用20%Na2S2O3水溶液(15毫升)、饱和NaHCO3水溶液(20毫升)和盐水(10毫升)洗涤。将分离的有机层经MgSO4脱水并在真空中浓缩。所得残余物通过硅胶管柱层析纯化,使用EtOAc/己烷(2:3)作为溶析液,得到化合物S17,为白色泡沫(2.68克,93%)。Rf=0.43(硅胶,丙酮:甲苯=1:6);1H NMR(600MHz,CDCl3):δ7.75(br,1H,Ar-H),7.63(br,2H,Ar-H),7.50(br,1H,Ar-H),7.46-7.45(m,2H,Ar-H),7.39-7.32(m,7H,Ar-H),7.29-7.26(m,1H,Ar-H),7.20-7.18(m,2H,Ar-H),7.07-7.05(m,1H,Ar-H),7.02-7.01(m,4H,Ar-H),6.98-6.97(m,2H,Ar-H),6.87-6.83(m,5H,Ar-H),5.44(s,1H,Ph-CH),5.10-5.08(m,1H,C1-Hβ),4.92(dd,J=9.0,8.4Hz,1H),4.81-4.71(m,4H),4.58(d,J=7.8Hz,1H,C1-Hβ),4.56(d,J=4.2Hz,1H),4.47(d,J=6.0Hz,1H),4.45(d,J=6.6Hz,1H),4.36(d,J=12.6Hz,1H),4.23(dd,J=10.2,4.8Hz,1H),4.20-4.19(m,2H),4.09-4.06(m,1H),3.87(dd,J=11.4,3.0Hz,1H),3.79-3.78(m,1H),3.78(s,3H,-CH3),3.59(dd,J=9.6,9.0Hz,1H),3.55-3.51(m,2H),3.43(dd,J=10.2,10.2Hz,1H),3.17(ddd,J=9.6,9.6,4.8Hz,1H),2.81-2.76(m,1H),2.70-2.65(m,1H),2.50-2.38(m,2H),2.20(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ206.2,171.1,159.2,138.6,138.1,137.3,133.5,131.6,130.4,129.4,129.0,128.5,128.2,128.1,127.9,127.8,127.7,127.5,127.5,127.0,126.0,123.1,113.6,101.1,100.6,97.4,81.7,78.0,77.9,76.6,74.8,74.5,73.7,73.6,70.7,68.6,67.7,65.8,55.7,55.3,37.7,29.9,27.8;HRMS(ESI-TOF)m/e:C61H61NO15Na[M+Na]+的计算值:1070.3933,实测值:1070.3933。
苄基[4,6-O-亚苄基-3-O-p-甲氧基-苄基-β-D-葡萄糖吡喃糖苷基]-(1→4)-3,6-二-O-苄基-2-脱氧-2-(2,2,2-三氯乙氧基)碳酰胺基-β-D-葡萄糖吡喃糖苷S18.将双糖S17(625毫克,0.596毫摩尔,1当量)于乙二胺/n-BuOH(7毫升,2:8=v/v)中的溶液在90℃下搅拌2小时。将溶剂通过在高真空下旋转蒸发除去并与甲苯共蒸发两次以除去水迹。将所得残余物溶解于无水CH2Cl2(15毫升)中并在氩气下于0℃用NaHCO3(250毫克,2.98毫摩尔,5当量)和2,2,2-氯甲酸三氯乙酯(0.41毫升,2.98毫摩尔,5当量)处理3小时。完成之后,将反应混合物用CH2Cl2(20毫升)稀释,用水(20毫升)和盐水(10毫升)洗涤。将有机层经MgSO4脱水并在真空中浓缩。所得残余物通过硅胶管柱层析纯化,使用EtOAc/甲苯(1:4)作为溶析液,得到化合物S18,为白色粉末(481毫克,81%)。Rf=0.60(硅胶,EtOAc:甲苯=1:2);1H NMR(600MHz,CDCl3):δ7.47-7.46(m,2H,Ar-H),7.40-7.25(m,20H,Ar-H),6.86-6.85(m,2H,Ar-H),5.46(s,1H,Ph-CH),5.12(br,1H),4.88-4.85(m,3H),4.71-4.55(m,9H,2C1-Hβ),4.06-4.03(m,2H),3.99(dd,J=11.4,3.6Hz,1H),3.87(br,1H),3.80(dd,J=11.4,1.8Hz,1H),3.78(s,3H,-CH3),3.56(dd,J=9.6,9.0Hz,1H),3.52-3.45(m,5H),3.15(ddd,J=9.6,9.6,4.8Hz,1H),3.03(br,1H,-OH);13C NMR(150MHz,CDCl3):δ159.3,153.8,138.4,137.7,137.3,137.2,130.4,129.6,128.9,128.4,128.4,128.3,128.2,128.0,127.8,127.8,127.6,127.4,126.0,113.8,103.2,101.1,99.2,95.5,81.3,79.9,79.2,77.7,74.9,74.5,74.3,74.2,73.5,70.7,68.6,68.2,66.2,57.6,55.2;HRMS(ESI-TOF)m/e:C51H54Cl3NO13Na[M+Na]+的计算值:1016.2553,实测值:1016.2554。
苄基[2-O-乙酰基-4,6-O-亚苄基-3-O-p-甲氧基-苄基-β-D-甘露糖吡喃糖苷基]-(1→4)-3,6-二-O-苄基-2-脱氧-2-(2,2,2-三氯乙氧基)碳酰胺基-β-D-葡萄糖吡喃糖苷S19.从S18:在氩气下于0℃向搅拌的双糖S18(1.02克,1.02毫摩尔,1当量)于无水CH2Cl2(13毫升)中的溶液添加无水吡啶(0.58毫升,7.17毫摩尔,7当量),接着逐滴添加Tf2O(0.30毫升,1.79毫摩尔,1.75当量)。将反应混合物在0℃下搅拌直到TLC显示起始物消失(4小时)。完成之后,将其用CH2Cl2(20毫升)稀释,用0.5N HCl(20毫升)和盐水(10毫升)洗涤。将分离的有机层经MgSO4脱水并在真空中浓缩。将粗残余物与Bu4NOAc(927毫克,3.07毫摩尔,3当量)混合并溶解于甲苯(20毫升)中。在真空中除去溶剂,将残余物与甲苯共蒸发两次;然后再溶解于无水甲苯(13毫升)中,将混合物用超音波处理8小时。在此期间,我们观察到NHTroc的部分去保护,将反应混合物在高真空下浓缩。将所得残余物溶解于CH2Cl2(13毫升)中并在氩气下于0℃用NaHCO3(430毫克,5.12毫摩尔,5当量)和氯甲酸2,2,2-三氯乙酯(0.71毫升,5.12毫摩尔,5当量)处理。在搅拌3小时后,将反应混合物用CH2Cl2(20毫升)稀释,用水(20毫升)和盐水(10毫升)洗涤。将分离的有机层经MgSO4脱水并在真空中浓缩。所得残余物通过硅胶管柱层析纯化,使用EtOAc/甲苯(1:5)作为溶析液,得到化合物S19,为白色粉末(650毫克,61%)。
从S21:将双糖S21(1.20克,1.21毫摩尔,1当量)于乙二胺/n-BuOH(15毫升,2:8=v/v)中的溶液在90℃下搅拌2小时。除去溶剂后,将粗产物与甲苯共蒸发两次。将所得残余物溶解于CH2Cl2(30毫升)中,并在氩气下于0℃用NaHCO3(508毫克,6.05毫摩尔,5当量)和氯甲酸2,2,2-三氯乙酯(0.83毫升,6.05毫摩尔,5当量)处理。3小时后,将反应混合物用CH2Cl2(45毫升)稀释,用水(30毫升)和盐水(15毫升)洗涤。将有机层经MgSO4脱水,浓缩,与甲苯共蒸发两次,然后在真空中蒸发。让所得残余物直接经受乙酰化条件Ac2O(7毫升)于吡啶(14毫升)中,温度在16小时内从0℃缓慢升高至室温。将反应混合物在高真空下浓缩,通过硅胶管柱层析纯化,使用丙酮/甲苯(1:5)作为溶析液,得到化合物S19,为白色粉末(1.04克,83%)。Rf=0.54(硅胶,EtOAc:甲苯=1:3);1H NMR(600MHz,CDCl3):δ7.48-7.46(m,2H,Ar-H),7.39-7.35(m,3H,Ar-H),7.33-7.25(m,14H,Ar-H),7.24-7.21(m,3H,Ar-H),6.84-6.82(m,2H,Ar-H),5.50(s,1H,Ph-CH),5.41(d,J=3.0Hz,1H),5.09(br,1H),4.92(d,J=10.8Hz,1H),4.88(d,J=12.0Hz,1H),4.72-4.62(m,5H,2C1-Hβ),4.58-4.56(m,3H),4.47-4.44(m,2H),4.08(dd,J=10.2,4.8Hz,1H),4.04(dd,J=9.0,8.4Hz,1H),3.87-3.84(m,2H),3.78-3.72(m,5H,-CH3),3.59(dd,J=10.2,10.2Hz,1H),3.45-3.43(m,2H),3.38(ddd,J=8.4,8.4,8.4Hz,1H),3.10(ddd,J=9.6,9.6,4.8Hz,1H),2.07(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ170.3,159.3,153.8,138.4,137.8,137.4,137.1,129.7,129.2,128.9,128.5,128.4,128.3,128.2,128.0,128.0,127.9,127.8,127.7,126.1,113.8,101.4,99.1,99.1,77.8,75.3,74.4,73.5,71.4,70.8,69.1,68.5,68.4,67.0,57.5,55.2,21.0;HRMS(ESI-TOF)m/e:C53H56Cl3NO14Na[M+Na]+的计算值:1058.2658,实测值:1058.2657。
苄基[2-O-乙酰基-4,6-O-亚苄基-β-D-甘露糖吡喃糖苷基]-(1→4)-3,6-二-O-苄基-2-脱氧-2-(2,2,2-三氯乙氧基)碳酰胺基-β-D-葡萄糖吡喃糖苷13.在0℃下,向搅拌的S19(120毫克,0.12毫摩尔,1当量)于CH2Cl2/磷酸盐缓冲液(pH=7)混合物(3毫升,9:1=v/v)中的溶液添加2,3-二氯-5,6-二氰基苯醌(52.5毫克,0.23毫摩尔,2.2当量)。将反应混合物剧烈搅拌直到TLC显示起始物消失(3小时)。完成之后,将反应混合物用CH2Cl2(10毫升)稀释,用饱和NaHCO3水溶液(8毫升)和盐水(4毫升)洗涤,然后将有机相经MgSO4脱水,过滤并浓缩。所得残余物通过硅胶管柱层析纯化,使用EtOAc/甲苯(1:4)作为溶析液,得到化合物13,为白色粉末(90毫克,85%)。Rf=0.29(硅胶,EtOAc:甲苯=1:3);1H NMR(600MHz,CDCl3):δ7.45-7.44(m,2H,Ar-H),7.39-7.34(m,7H,Ar-H),7.33-7.26(m,11H,Ar-H),5.47(s,1H,Ph-CH),5.23(d,J=3.6Hz,1H),5.06(br,1H),4.91(d,J=11.4Hz,1H),4.87(d,J=12.0Hz,1H),4.74(d,J=12.0Hz,1H),4.68-4.61(m,4H,2C1-Hβ),4.58-4.56(m,2H),4.48(d,J=12.0Hz,1H),4.08(dd,J=10.2,4.8Hz,1H),4.04(dd,J=9.0,9.0Hz,1H),3.83(br,1H),3.78(dd,J=11.4,3.0Hz,1H),3.73-3.70(m,2H),3.64(dd,J=9.6,3.6Hz,1H),3.56(dd,J=10.2,10.2Hz,1H),3.43-3.36(m,2H),3.11(ddd,J=9.6,9.6,4.8Hz,1H),2.21(d,J=3.6Hz,1H),2.11(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ170.5,153.8,138.5,137.8,137.1,137.0,129.3,128.6,128.6,128.4,128.4,128.3,128.1,128.0,128.0,127.9,127.8,127.7,126.2,102.1,99.1,99.0,78.5,78.2,74.4,74.4,73.6,71.2,70.8,69.7,68.4,68.4,66.7,57.5;HRMS(ESI-TOF)m/e:C45H49Cl3NO13[M+H]+的计算值:916.2264,实测值:916.2252。
苄基[4,6-O-亚苄基-3-O-p-甲氧基-苄基-β-D-葡萄糖吡喃糖苷基]-(1→4)-3,6-二-O-苄基-2-脱氧-2-酞酰亚胺基-β-D-葡萄糖吡喃糖苷S20.在氩气下于室温向搅拌的起始物S17(2.48克,2.37毫摩尔,1当量)于无水THF(55毫升)中的溶液添加乙酸肼(327毫克,3.55毫摩尔,1.5当量)。将反应混合物剧烈搅拌直到TLC显示起始物消失(16小时)。完成之后,将反应混合物用EtOAc(150毫升)稀释,用水(60毫升)和盐水(30毫升)洗涤。将有机相经MgSO4脱水,过滤并在真空中浓缩。所得残余物通过硅胶管柱层析纯化,使用EtOAc/己烷(1:2)作为溶析液,得到化合物S20,为白色泡沫(2.07克,92%)。Rf=0.54(硅胶,EtOAc:己烷=1:1);1H NMR(600MHz,CDCl3):δ7.79-7.54(m,4H,Ar-H),7.51-7.44(m,2H,Ar-H),7.38-7.33(m,7H,Ar-H),7.30-7.27(m,3H,Ar-H),7.08-7.04(m,1H,Ar-H),7.02-7.01(m,4H,Ar-H),6.98-6.97(m,2H,Ar-H),6.89-6.81(m,5H,Ar-H),5.46(s,1H,Ph-CH),5.09(d,J=8.4Hz,1H,C1-Hβ),4.85(d,J=11.4Hz,1H),4.77(d,J=12.6Hz,1H),4.75(d,J=4.2Hz,1H),4.73(d,J=4.2Hz,1H),4.68(d,J=11.4Hz,1H),4.62(d,J=7.2Hz,1H,C1-Hβ),4.56(d,J=12.0Hz,1H),4.46(d,J=12.0Hz,1H),4.38(d,J=12.0Hz,1H),4.32(dd,J=10.8,8.4Hz,1H),4.21(d,J=10.8,8.4Hz,1H),4.15-4.10(m,2H),4.04(dd,J=11.4,3.0Hz,1H),3.82(dd,J=11.4,1.8Hz,1H),3.78(s,3H,-CH3),3.61(ddd,J=9.6,3.0,3.0Hz,1H),3.56(dd,J=3.0,3.0Hz,1H),3.52-3.45(m,3H),3.18(ddd,J=9.6,9.6,4.8Hz,1H),2.98(br,1H,-OH);13C NMR(150MHz,CDCl3):δ167.9,159.3,138.4,137.8,137.3,137.2,133.6,131.6,130.5,129.7,128.9,128.4,128.2,128.1,128.0,127.9,127.8,127.5,127.4,127.1,126.0,123.2,113.8,103.4,101.1,97.5,81.3,79.5,78.8,77.8,75.0,74.7,74.5,74.2,73.6,70.8,68.6,68.2,66.2,55.8,55.3;HRMS(ESI-TOF)m/e:C56H55NO13Na[M+Na]+的计算值:972.3565,实测值:972.3567。
苄基[2-O-乙酰基-4,6-O-亚苄基-3-O-p-甲氧基-苄基-β-D-甘露糖吡喃糖苷基]-(1→4)-3,6-二-O-苄基-2-脱氧-2-酞酰亚胺基-β-D-葡萄糖吡喃糖苷S21.向搅拌的双糖S20(936毫克,0.985毫摩尔,1当量)于无水CH2Cl2(12毫升)中的溶液添加无水吡啶(0.56毫升,6.90毫摩尔,7当量),然后在氩气下于0℃逐滴添加三氟甲磺酸酐(0.29毫升,1.72毫摩尔,1.75当量),并将混合物在0℃下搅拌直到TLC显示起始物消失(2小时)。完成之后,将反应混合物用CH2Cl2(20毫升)稀释,用0.5N HCl(20毫升)和盐水(10毫升)洗涤。将分离的有机层经MgSO4脱水,在真空中浓缩,所得残余物原样供进一步反应之用。将以上获得的残余物添加Bu4NOAc(594毫克,1.97毫摩尔,2当量)并溶解于甲苯(18毫升)中。在真空中除去溶剂,将残余物与甲苯共蒸发两次以除去水迹。将残余物再溶解于无水甲苯(12毫升)中并将混合物用超音波处理8小时。完成之后,将反应混合物用EtOAc(20毫升)稀释,用水(20毫升)和盐水(10毫升)洗涤。将分离的有机层经MgSO4脱水并浓缩。所得残余物通过硅胶管柱层析纯化,使用EtOAc/己烷(1:2)作为溶析液,得到化合物S21,为白色粉末(913毫克,93%)。Rf=0.29(硅胶,EtOAc:己烷=1:2);1H NMR(600MHz,CDCl3):δ7.76-7.49(m,4H,Ar-H),7.48-7.46(m,2H,Ar-H),7.39-7.31(m,7H,Ar-H),7.26-7.22(m,3H,Ar-H),7.10-7.06(m,1H,Ar-H),7.04-7.03(m,4H,Ar-H),6.99-6.98(m,2H,Ar-H),6.90-6.84(m,5H,Ar-H),5.49(s,1H,Ph-CH),5.44(d,J=3.0Hz,1H),5.09(d,J=7.8Hz,1H,C1-Hβ),4.82-4.76(m,3H),4.68(s,1H,C1-Hβ),4.58(d,J=12.0Hz,1H),4.49-4.44(m,3H),4.38(d,J=12.6Hz,1H),4.26-4.19(m,2H),4.17-4.11(m,2H),3.86-3.82(m,2H),3.78(s,3H,-CH3),3.78-3.76(m,1H),3.57-3.54(m,2H),3.44(dd,J=10.2,3.6Hz,1H),3.13(ddd,J=9.6,9.6,4.8Hz,1H),2.15(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ170.2,167.8,159.2,138.5,137.8,137.4,137.1,133.6,131.5,129.7,129.2,128.9,128.5,128.1,128.1,127.9,127.9,127.8,127.7,127.6,127.5,127.1,126.0,123.1,113.8,101.4,99.5,97.3,79.1,77.7,76.9,75.4,74.5,74.3,73.5,71.3,70.7,69.1,68.4,68.3,66.9,55.6,55.2,21.0;HRMS(ESI-TOF)m/e:C58H57NO14Na[M+Na]+的计算值:1014.3671,实测值:1014.3699。
流程S5
流程S5中的试剂和条件:(a)Br2,CH2Cl2,0℃,10min.,然后对硝基酚,Ag2O,CH3CN,1h,73%.(b)NaBrO3,Na2S2O4,H2O,EtOAc,r.t.,1.5h,93%.(c)LiOH·H2O,MeOH,r.t.,16h,84%.(d)Ac2O,吡啶,r.t.,16h,90%.(e)Br2,CH2Cl2,0℃,10min.,然后对硝基酚,Ag2O,CH3CN,1h,74%.(f)NaBrO3,Na2S2O4,H2O,EtOAc,r.t.,1.5h,97%(g)LiOH·H2O,MeOH,r.t.,16h,94%.
对硝基苯基[5-乙酰胺基-4,7,8,9-四-O-乙酰基-3,5-二脱氧-3-氟-D-赤藻糖-α-L-甘露糖-壬-2-酮糖吡喃糖酸甲酯]-(2→6)-2,3-二-O-苄酰基-4-O-苄基-β-D-半乳糖吡喃糖苷S22.在0℃下向搅拌的硫苷4(115毫克,0.11毫摩尔,1当量)于无水CH2Cl2(2毫升)中的溶液添加溴(6.02μL,0.12毫摩尔,1.1当量)。在将反应混合物剧烈搅拌10分钟之后,在真空中除去溶剂,并将残余物与甲苯共蒸发两次以除去水迹。将如此获得的残余物溶解于无水CH3CN(2毫升)中,并用4-硝基酚(25.3毫克,0.18毫摩尔,1.7当量)和Ag2O(124毫克,0.53毫摩尔,5当量)处理。将反应混合物在N2下在黑暗中剧烈搅拌直到TLC显示起始物消失(1小时)。完成之后,将反应混合物用EtOAc(10毫升)稀释并通过硅藻土垫过滤。将滤液用饱和NaHCO3水溶液(4毫升)和盐水(3毫升)洗涤两次。将有机相经MgSO4脱水,过滤并在真空中浓缩。所得残余物通过硅胶管柱层析纯化,使用丙酮/甲苯(1:2)作为溶析液,得到化合物S22,为白色粉末(85毫克,73%)。Rf=0.46(硅胶,丙酮:甲苯=2:3);1H NMR(600MHz,CDCl3):δ8.13-8.11(m,2H,Ar-H),7.96-7.94(m,2H,Ar-H),7.91-7.89(m,2H,Ar-H),7.49-7.44(m,2H,Ar-H),7.35-7.30(m,6H,Ar-H),7.24-7.22(m,2H,Ar-H),7.20-7.18(m,2H,Ar-H),7.13-7.11(m,1H,Ar-H),6.09(dd,J=10.2,7.8Hz,1H),5.65-5.59(dd,J=10.2,3.0Hz,1H,C1-Hβ),5.35(d,J=9.6Hz,1H),5.28(dd,J=9.6,1.8Hz,1H),5.19(dd,J=27.0,10.8Hz,1H,sia-C4-H),5.04(dd,J=51.0,1.8Hz,1H,sia-C3-H),4.72-4.67(m,2H),4.44(dd,J=12.6,3.6Hz,1H),4.40(d,J=3.0Hz,1H),4.37(dd,J=8.4,6.6Hz,1H),4.31(d,J=10.8Hz,1H),4.18(ddd,J=4.2,4.2,4.2Hz,1H),4.08(dd,J=12.6,6.6Hz,1H),3.89-3.80(m,5H,-CH3),2.26(s,6H,-2CH3),2.10(s,3H,-CH3),1.93(s,3H,-CH3),1.92(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ171.1,170.8,170.4,170.3,170.2,165.5,165.5,165.4,165.3,161.8,142.6,138.0,133.2,133.1,129.8,129.7,129.4,129.0,128.4,128.3,128.2,128.1,128.0,127.5,125.6,116.8,98.8,98.7,98.2,88.1,86.8,75.0,73.9,73.7,73.2,71.6,69.7,69.1,69.0,67.3,67.2,63.4,63.3,53.4,45.1,23.3,21.2,20.8,20.7,20.6;19F NMR(376MHz,CDCl3):δ-215.5;HRMS(ESI-TOF)m/e:C53H55FN2O22Na[M+Na]+的计算值:1113.3123,实测值:1113.3131。
对硝基苯基[5-乙酰胺基-4,7,8,9-四-O-乙酰基-3,5-二脱氧-3-氟-D-赤藻糖-α-L-甘露糖-壬-2-酮糖吡喃糖酸甲酯]-(2→6)-2,3-二-O-苄酰基-β-D-半乳糖吡喃糖苷S23.向搅拌的化合物S22(63.0毫克,0.058毫摩尔,1当量)于EtOAc(0.8毫升)中的溶液添加溶于H2O(0.6毫升)中的NaBrO3(85%,39.2毫克,0.26毫摩尔,4.5当量),接着在室温下慢慢添加溶于H2O(1.2毫升)中的Na2S2O4(40.2毫克,0.23毫摩尔,4当量)。将反应混合物搅拌直到TLC显示起始物消失(1.5小时)。反应完成之后,将反应混合物用EtOAc(10毫升)稀释,用20%Na2S2O3水溶液(5毫升)和盐水(3毫升)洗涤。将有机相经MgSO4脱水,过滤并在真空中浓缩,然后将所得残余物通过硅胶管柱层析纯化,使用丙酮/甲苯(3:5)作为溶析液,得到化合物S23,为白色粉末(54毫克,93%)。Rf=0.40(硅胶,丙酮:甲苯=2:3);1H NMR(600MHz,CDCl3):δ8.13-8.12(m,2H,Ar-H),7.99-7.98(m,2H,Ar-H),7.95-7.94(m,2H,Ar-H),7.50-7.46(m,2H,Ar-H),7.37-7.32(m,4H,Ar-H),7.19-7.18(m,2H,Ar-H),6.05(dd,J=10.2,9.8Hz,1H),5.53(ddd,J=9.0,6.0,3.0Hz,1H),5.49-5.45(m,2H,C1-Hβ),5.36(d,J=9.0Hz,1H),5.27(dd,J=9.0,1.8Hz,1H),5.19(dd,J=27.0,11.4Hz,1H,sia-C4-H),5.05(dd,J=51.6,1.8Hz,1H,sia-C3-H),4.47(d,J=3.0Hz,1H),4.37(dd,J=12.6,3.0Hz,1H),4.26(dd,J=10.8,1.2Hz,1H),4.18(dd,J=6.6,6.6Hz,1H),4.13-4.07(m,1H),4.05(dd,J=12.6,6.6Hz,1H),3.99-3.98(m,2H),3.85(s,3H,-CH3),2.99(br,1H,-OH),2.17(s,3H,-CH3),2.08(s,3H,-CH3),2.07(s,3H,-CH3),1.93(s,3H,-CH3),1.88(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ170.9,170.8,170.5,170.3,165.6,165.6,165.3,161.8,142.9,133.4,133.3,129.8,129.7,129.2,129.1,129.0,128.4,128.4,128.2,125.6,125.3,117.1,99.0,98.1,98.0,88.3,87.0,73.9,73.3,71.6,69.2,69.1,69.0,67.9,67.2,66.5,63.6,62.9,53.4,45.1,23.3,21.1,20.7,20.6,20.6;19F NMR(376MHz,CDCl3):δ-216.0;HRMS(ESI-TOF)m/e:C46H50FN2O22[M+H]+的计算值:1001.2834,实测值:1001.2833。
对硝基苯基[5-乙酰胺基-3,5-二脱氧-3-氟-D-赤藻糖-α-L-甘露糖-壬-2-酮糖吡喃糖酸甲酯]-(2→6)-β-D-半乳糖吡喃糖苷2.向化合物S23(53毫克,0.053毫摩尔,1当量)于甲醇(5毫升)中的溶液添加容于水(1毫升)中的LiOH·H2O(22.2毫克,0.53毫摩尔,10当量)。在室温下搅拌16小时后,将反应混合物用IR-120中和,过滤并在真空中浓缩。所得残余物通过(BIO-RAD)Biogel P-2管柱层析(用水溶析)纯化,得到化合物2(27毫克,84%)。1HNMR(600MHz,D2O):δ8.29-8.28(m,2H,Ar-H),7.30-7.29(m,2H,Ar-H),5.21(dd,J=51.6,2.4Hz,1H,sia-C3-H),5.18(d,J=7.8Hz,C1-Hβ),4.17(dd,J=9.0,9.0Hz,1H),4.07-4.00(m,3H),3.91-3.77(m,7H),3.63(dd,J=12.0,6.6Hz,1H),3.57(dd,J=9.0,1.8Hz,1H),2.04(s,3H,-CH3);13C NMR(150MHz,D2O):δ175.0,170.8,161.9,142.5,126.1,116.5,100.0,98.9,98.9,91.6,90.3,73.9,72.4,72.4,71.6,70.3,69.7,69.5,68.4,68.1,63.7,62.6,46.8,46.8,22.0;19F NMR(376MHz,CDCl3):δ-217.6;HRMS(ESI-TOF)m/e:C23H32FN2O16[M+H]+的计算值:611.1730,实测值:611.1732。
对甲苯基[5-乙酰胺基-3,4,7,8,9-五-O-乙酰基-5-脱氧-D-赤藻糖-α-L-葡萄糖-壬-2-酮糖吡喃糖酸甲酯]-(2→6)-2,3-二-O-苄酰基-4-O-苄基-1-硫基-β-D-半乳糖吡喃糖苷S24.向搅拌的起始物6(174毫克,0.16毫摩尔,1当量)于吡啶(2毫升)中的溶液添加Ac2O(1毫升)。将反应混合物在室温下剧烈搅拌16小时,然后在高真空下浓缩。所得残余物通过硅胶管柱层析纯化,使用丙酮/甲苯(1:2)作为溶析液,得到化合物S24,为白色粉末(162毫克,90%)。Rf=0.49(硅胶,丙酮:甲苯=2:3);1H NMR(600MHz,CDCl3):δ7.94-7.92(m,2H,Ar-H),7.88-7.87(m,2H,Ar-H),7.48-7.44(m,2H,Ar-H),7.40-7.38(m,2H,Ar-H),7.35-7.27(m,6H,Ar-H),7.23-7.20(m,2H,Ar-H),7.18-7.16(m,1H,Ar-H),7.05-7.03(m,2H,Ar-H),5.80(dd,J=10.2.9.6Hz,1H),5.44-5.42(m,2H),5.35-5.29(m,3H),5.25(dd,J=8.4,1.8Hz,1H),4.95(d,J=10.2Hz,1H,C1-Hβ),4.67-4.61(m,3H),4.33(ddd,J=10.2,10.2,10.2Hz,1H),4.25-4.22(m,2H),4.03-3.97(m,3H),3.92-3.89(m,1H),3.75(s,3H,-CH3),2.29(s,3H,-CH3),2.15(s,3H,-CH3),2.07(s,3H,-CH3),2.00(s,3H,-CH3),1.95(s,3H,-CH3),1.94(s,3H,-CH3),1.89(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ171.0,170.5,170.3,170.0,169.3,168.5,168.0,165.7,165.2,138.4,137.6,133.1,132.9,132.5,129.8,129.7,129.7,129.5,129.2,129.1,128.3,128.2,128.0,127.4,127.2,98.9,86.5,76.9,75.5,74.6,74.3,72.7,71.6,71.5,68.5,68.3,66.9,62.6,62.5,52.7,48.4,23.0,21.1,20.8,20.7,20.6,20.6,20.5;HRMS(ESI-TOF)m/e:C56H61NO21SNa[M+Na]+的计算值:1138.3349,实测值:1138.3358。
对硝基苯基[5-乙酰胺基-3,4,7,8,9-五-O-乙酰基-5-脱氧-D-赤藻糖-α-L-葡萄糖-壬-2-酮糖吡喃糖酸甲酯]-(2→6)-2,3-二-O-苄酰基-4-O-苄基-β-D-半乳糖吡喃糖苷S25.向在0℃下搅拌的硫苷S24(132毫克,0.12毫摩尔,1当量)于无水CH2Cl2(2.5毫升)中的溶液添加溴(6.67μL,0.13毫摩尔,1.1当量)。在剧烈搅拌10分钟之后,在真空中除去溶剂,将残余物与甲苯共蒸发两次以除去水迹。将所得残余物溶解于无水CH3CN(2.5毫升)中并用4-硝基酚(28.0毫克,0.20毫摩尔,1.7当量)和Ag2O(137毫克,0.59毫摩尔,5当量)处理。将反应混合物在N2下在黑暗中剧烈搅拌直到TLC显示起始物消失(1小时)。反应完成后,将反应混合物用EtOAc(12毫升)稀释,并通过硅藻土垫过滤,将滤液用饱和NaHCO3水溶液(5毫升)和盐水(3毫升)洗涤两次。将有机相经MgSO4脱水,过滤并在真空中浓缩,所得残余物通过硅胶管柱层析纯化,使用丙酮/甲苯(3:5)作为溶析液,得到化合物S25,为白色粉末(99毫克,74%)。Rf=0.51(硅胶,丙酮:甲苯=2:3);1H NMR(600MHz,CDCl3):δ8.15-8.13(m,2H,Ar-H),7.95-7.92(m,4H,Ar-H),7.50-7.44(m,2H,Ar-H),7.36-7.31(m,6H,Ar-H),7.24-7.19(m,2H,Ar-H),7.17-7.15(m,3H,Ar-H),6.10(dd,J=10.2,7.8Hz,1H),5.54(dd,J=10.2,3.0Hz,1H),5.52(d,J=7.8Hz,1H,C1-Hβ),5.48(ddd,J=10.2,7.2,3.0Hz,1H),5.39(d,J=10.2Hz,1H),5.31-5.29(m,2H),5.23(dd,J=9.6,1.8Hz,1H),4.73(dd,J=10.8,2.4Hz,1H),4.71-4.65(m,2H),4.32-4.30(m,3H),4.19(dd,J=7.2,7.2Hz,1H),3.97(dd,J=12.6,7.2Hz,1H),3.88-3.86(m,2H),3.83(s,3H,-CH3),2.24(s,3H,-CH3),2.12(s,3H,-CH3),2.01(s,3H,-CH3),1.98(s,3H,-CH3),1.90(s,3H,-CH3),1.88(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ171.0,170.7(2C),170,0,169.6,168.5,168.3,165.7,165.3,161.7,142.7,137.9,133.3,133.2,129.9,129.7,129.3,128.9,128.4,128.3,128.2,128.0,127.6,125.7,116.8,99.7,98.4,75.1,74.1,74.0,73.9,72.8,71.6,71.1,69.7,67.6,67.0,63.8,63.3,52.9,48.4,23.0,20.9,20.8,20.7,20.6;HRMS(ESI-TOF)m/e:C55H59N2O24[M+H]+的计算值:1131.3452,实测值:1131.3440。
对硝基苯基[5-乙酰胺基-3,4,7,8,9-五-O-乙酰基-5-脱氧-D-赤藻糖-α-L-葡萄糖-壬-2-酮糖吡喃糖酸甲酯]-(2→6)-2,3-二-O-苄酰基-β-D-半乳糖吡喃糖苷S26.在室温下向搅拌的化合物S25(91.0毫克,0.080毫摩尔,1当量)于EtOAc(1.2毫升)中的溶液添加溶于H2O(0.9毫升)中的NaBrO3(54.6毫克,0.36毫摩尔,4.5当量),接着慢慢添加溶于H2O(1.8毫升)中的Na2S2O4(85%,56.0毫克,0.32毫摩尔,4当量)。将反应混合物搅拌直到TLC显示起始物消失(1.5小时)。完成之后,将反应混合物用EtOAc(15毫升)稀释,用20%Na2S2O3水溶液(7毫升)和盐水(4毫升)洗涤。将有机相经MgSO4脱水,过滤并在真空中浓缩。所得残余物通过硅胶管柱层析纯化,使用丙酮/甲苯(3:5)作为溶析液,得到化合物S26,为白色粉末(81毫克,97%)。Rf=0.23(硅胶,丙酮:甲苯=2:3);1H NMR(600MHz,CDCl3):δ8.18-8.16(m,2H,Ar-H),8.00-7.99(m,2H,Ar-H),7.94-7.93(m,2H,Ar-H),7.51-7.46(m,2H,Ar-H),7.38-7.32(m,4H,Ar-H),7.15-7.13(m,2H,Ar-H),6.07(dd,J=10.2,7.8Hz,1H),5.46-5.34(m,5H,C1-Hβ),5.30(d,J=10.2Hz,1H),5.22(dd,J=7.8,1.8Hz,1H),4.60(dd,J=10.8,1.8Hz,1H),4.43(d,J=3.0Hz,1H),4.33(dd,J=12.6,2.4Hz,1H),4.26(ddd,J=10.2,10.2,10.2Hz,1H),4.12(dd,J=6.6,6.6Hz,1H),4.05(dd,J=10.2,6.6Hz,1H),3.99(dd,J=10.2,6.6Hz,1H),3.93(dd,J=12.6,7.2Hz,1H),3.83(s,3H,-CH3),3.17(br,1H,-OH),2.14(s,3H,-CH3),2.05(s,3H,-CH3),2.02(s,3H,-CH3),2.00(s,3H,-CH3),1.87(s,3H,-CH3),1.85(s,3H,-CH3);13C NMR(150MHz,CDCl3):δ171.1,171.0,170.4,170.0,169.9,168.8,167.9,165.8,165.2,161.7,142.9,133.4,133.3,129.9,129.7,129.3,129.1,128.4,128.4,125.8,116.9,99.1,98.9,74.0,73.9,73.0,71.1,70.6,69.2,68.7,67.2,66.5,63.0,62.5,53.0,48.5,23.0,20.8,20.7,20.6,20.6;HRMS(ESI-TOF)m/e:C48H53N2O24[M+H]+的计算值:1041.2983,实测值:1041.2976。
对硝基苯基[5-乙酰胺基-5-脱氧-D-赤藻糖-α-L-葡萄糖-壬-2-酮糖吡喃糖酸酯]-(2→6)-β-D-半乳糖吡喃糖苷S27.向化合物S26(75毫克,0.072毫摩尔,1当量)于甲醇(5毫升)中的溶液添加溶于水(1毫升)中的LiOH·H2O(30毫克,0.72毫摩尔,10当量)。在室温下搅拌16小时之后,将反应混合物用IR-120中和,过滤并浓缩。所得残余物通过(BIO-RAD)Biogel P-2管柱层析纯化,使用水作为溶析液,得到化合物S27(41毫克,94%)。1H NMR(600MHz,D2O):δ8.29-8.28(m,2H,Ar-H),7.28-7.27(m,2H,Ar-H),5.20(d,J=7.2Hz,C1-Hβ),4.09-4.06(m,2H),4.01(dd,J=10.8,7.8Hz,1H),3.92-3.79(m,6H),3.71(d,J=10.8Hz,1H),3.64-3.60(m,2H),3.53(d,J=9.6Hz,1H),3.49(d,J=9.6Hz,1H),2.02(s,3H,-CH3);13C NMR(150MHz,D2O):δ174.8,173.0,161.9,142.5,126.1,116.4,99.9,98.1,76.1,73.9,73.5,72.4,72.2,71.5,70.3,68.4,68.0,63.0,62.6,50.7,21.9;HRMS(ESI-TOF)m/e:C23H33N2O17[M+H]+的计算值:609.1774,实测值:609.1774。
对硝基苯基[5-乙酰胺基-5-脱氧-D-甘油基-α-D-半乳糖-壬-2-酮糖吡喃糖酸酯]-(2→6)-β-D-半乳糖吡喃糖苷1.Neu5Ac-α2,6-Gal-pNP:通过将在15毫升Tris缓冲液(pH 7.0)中的pNP-β-Gal(1.0毫摩尔)、唾液酸(1.2毫摩尔)、三磷酸胞苷(1.2毫摩尔)、CMP-唾液酸合成酶(CSS,12U)、焦磷酸酶(PPA,1U)和α-2,6-唾液酸转移酶(SiaT,15U)与5mM毫克Cl2和5mM MnCl2混合来合成Neu5Ac-α2,6-Gal-pNP。在通过加热和离心除去蛋白质后,产物通过(BIO-RAD)Biogel P-2管柱层析纯化,使用水作为溶析液。将含有Neu5Ac-α2,6-Gal-pNP的流份收集起来并冻干,得到化合物1(50%)。1H NMR(600MHz,D2O):δ8.33-8.32(m,2H,Ar-H),7.31-7.29(m,2H,Ar-H),5.23(d,J=7.8Hz,C1-Hβ),4.06-4.04(m,2H),3.99(dd,J=10.2,8.4Hz,1H),3.90-3.86(m,3H),3.83(dd,J=10.2,3.6Hz,1H),3.80-3.76(m,1H),3.73-3.69(m,3H),3.65-3.62(m,2H),3.57(d,J=8.4Hz,1H),2.79(dd,J=12.6,4.8Hz,1H),2.04(s,3H,-CH3),1.68(dd,J=12.6,12.0Hz,1H);13C NMR(150MHz,D2O):δ174.6,173.1,161.4,142.1,125.7,116.0,99.8,99.4,73.6,72.1,71.9,71.3,69.8,68.0,67.7,67.7,62.5,62.2,51.4,39.8,21.6;HRMS(ESI-TOF)m/e:C23H33N2O16[M+H]+的计算值:593.1825,实测值:593.1825。
实施例2:抗唾液酸酶催化的水解的稳定性及唾液酸酶抑制作用的分析31
材料:
来自米曲菌(Aspergillus oryzae(G5160))的β-半乳糖苷酶和来自霍乱弧菌(11080725001)与产气荚膜杆菌(11585886001)的唾液酸酶得自Sigma Aldrich。
用于唾液酸酶的酶测定法:
本测定是在37℃下于96孔盘中重复进行,最终体积为50μL,含有基质(0-20mM)和β-半乳糖苷酶(100mU)。两种唾液酸酶的测定条件如下:产气荚膜杆菌(1mU)、醋酸钠缓冲液(50mM)pH 5.0及CaCl2(10mM);霍乱弧菌(2mU)、醋酸钠缓冲液(50mM)pH 5.5、CaCl2(10mM)及NaCl(150mM)。产气荚膜杆菌反应进行40分钟至2小时,霍乱弧菌反应则进行过夜。通过添加65μL的CAPS缓冲液(N-环己基-3-胺基丙烷磺酸(0.5M,pH 10.5)使测定停止。所形成对硝基酚盐的量是通过使用微盘读数仪测量反应混合物中的A405nm来确定。将三种化合物(Neu5Ac-α2,6-GalβpNP、3Fax-Neu5Ac-α2,6-GalβpNP和3OHeq-Neu5Ac-α2,6-GalβpNP)作为酶用基质来测试。在不存在唾液酸酶情况下,在37℃培育1小时后,三种化合物在20mM都有A405nm的背景吸亮度。三种化合物Neu5Ac-α2,6-GalβpNP、3Fax-Neu5Ac-α2,6-GalβpNP和3OHeq-Neu5Ac-α2,6-GalβpNP的吸亮度分别是0.044、0.129和0.072。pNP的标准曲线确定如下:连续两倍稀释0.35mM的pNP,然后将A405nm对pNP的浓度作图得到pNP的标准曲线。
唾液酸酶的抑制作用测定:
本测定是在37℃下于96孔盘中,最终体积为50μL,含有基质Neu5Ac-α2,6-GalβpNP(0.6mM)、β-半乳糖苷酶(100mU)及唾液酸酶,在不同浓度(0-20mM)抑制剂存在或不存在下重复进行。让产气荚膜杆菌反应进行40分钟至2小时,霍乱弧菌反应则进行过夜。通过添加CAPS缓冲液(65uL,0.5M,pH 10.5)使测定停止。所形成对硝基酚盐的量是通过使用微盘读数仪测量反应混合物中的A405nm来确定(图1)。
实施例3:制备经3Fax-Neu5Ac和Neu5Ac修饰的均质mAb,以表面电浆共振(SPR)分析来研究对FcγRIIIa结合的作用
酶的表现:
在大肠杆菌中表现内糖苷酶Endo-S、Endo-S2、Endo-S2突变体(D184Q)及来自脆弱类杆菌(Bacteroides fragilis)NCTC 9343的岩藻糖苷酶,并使用Ni-NTA琼脂糖珠进行酶的纯化。
单GlcNAc-利妥昔单抗的制备:
如先前所述32,将置于Tris-HCl缓冲液(50mM,pH 7.4,1.5毫升)中的利妥昔单抗(3.0毫克;Roche)用Endo-S(120μg)、Endo-S2(240μg)和BfFucH(4.5毫克)在37℃下培育24小时。LC-MS和SDS-PAGE分析显示重链上的N-聚糖完全断裂。让反应混合物在已经用Tris-HCl缓冲液(50mM,pH 7.4)预平衡的蛋白A-琼脂糖树脂管柱(1毫升;GEHealthcare)上进行亲和层析。然后,用Tris-HCl缓冲液(50mM,pH 7.4,20毫升)冲洗该管柱。用甘氨酸-HCl(100mM,pH 3.0,10毫升)释放结合的IgG,并用Tris-HCl缓冲液(1.0M,pH8.3)立即中和溶析流份。将含有抗体的流份合并并通过离心过滤(Amicon Ultra离心过滤器,Millipore,Billerica,MA)浓缩,得到单GlcNAc利妥昔单抗(2.4毫克)。将产物胰蛋白酶化,使用纳米喷雾LC/MS分析糖肽、TKPREEQYNSTYR(m/z=1391.58)和EEQYNSTYR(m/z=1873.88),以确认单GlcNAc的糖基化模式。
用聚糖恶唑啉使单利妥昔单抗转糖基化:
将聚糖恶唑啉添加到Endo-S2 D184Q与单GlcNAc利妥昔单抗于50mM Tris缓冲液(pH 7.4)的混合物中。将溶液在37℃下培育30分钟。然后,将反应混合物用蛋白-A亲和管柱、接着Capto Q阴离子交换管柱(GE Healthcare)纯化,以收集所要的产物。
糖工程贺癌平(Herceptin)抗体的SDS-PAGE检测:
糖工程mAb的MS光谱分析:
为分析胰蛋白酶化的糖肽,在配备有奈电喷洒离子源(New Objective,Inc.)、Agilent 1100系列二元高效液相层析泵(Agilent Technologies,Palo Alto,CA)和Famos自动取样器(LC Packings,San Francisco,CA)的LTQFT Ultra(线性四极离子阱傅立叶变换离子回旋共振(linear quadrupole ion trap Fourier transform ion cyclotronresonance))质谱仪(Thermo Electron,San Jose,CA)上进行高分辨率暨高质量准确度奈流LC-MS/MS实验。将消化液(6μL)以10μL/min流速注入自填充的预管柱(150μm I.D.x20mm,5μm,)。在自填充逆相C18纳米管柱(75μm I.D.x 300mm,5μm,)上进行层析分离,使用0.1%甲酸水溶液作为移动相A,0.1%甲酸于80%乙腈中的溶液作为移动相B,以300nL/min操作。全扫描MS条件的调查:质量范围m/z 320-2000,在m/z 400的分辨率100,000。以LTQ依次分离出对于MS2的十个最强离子。电喷洒电压维持在1.8kV,毛细管温度设定在200℃(图3和表3)。
表面电浆共振(SPR)分析
所有的SPR实验都是通过BIACORE T200在25℃使用HBS-EP(10mM HEPES pH7.4,0.15M NaCl,3mMEDTA,0.005%界面活性剂P20)作为电泳缓冲液,用单循环动力学方法进行。将FcγRIIIa转染至HEK-293细胞内以表现错合物型糖基化重组蛋白作为分析物。为分析利妥昔单抗与FcγRIIIa受体的结合,将人Fab捕获试剂盒(GE Healthcare)中的抗人Fab抗体固定在CM5传感器芯片的对照信道和活性信道上,然后将利妥昔单抗捕获在活性通道上,以供与连续稀释的FcγRIIIa分析物(对于2,6-FluoSCT和2,6-SCT为2.5,5,10,20,40nM;对于市售利妥昔单抗为8,24,72,216,648nM)于30μl/min进行交互作用,缔合240秒,接着解离时间420秒。利妥昔单抗数据用双重参考处理以供背景扣除。在BiaEvaluation软件(GE Healthcare)中将利妥昔单抗的数据拟合至1:1Langmuir结合模型以获得动力学/亲和力常数(表4)。将分析的抗体以人Fab捕获试剂盒捕获并用单循环动力学方法检测。
表3:N-聚糖相对丰度的校正33
表4:以SPR测量糖工程利妥昔单抗IgG1对FcγRIIIa的结合亲和力
样品 | ka(1/Ms) | kd(1/s) | KD(M) | Rmax(RU) | 倍数 |
利妥昔单抗 | 2.31E+05 | 0.07054 | 3.06E-07 | 32.33 | 1-倍 |
2,6-F<sup>ax</sup>SCT | 2.44E+05 | 0.001996 | 8.18E-09 | 71.28 | 37.4-倍 |
2,6-SCT | 2.68E+05 | 0.002059 | 7.67E-09 | 60.64 | 39.9-倍 |
实施例4:化合物4和18的相对反应值(RRV)
按照先前所报导的实验流程测量RRV三次。34双糖供体4的RRV(2053)是对竞争对照供体S3423(RRV=1791)做测量。双糖供体18的RRV(537)是对竞争对照供体S23(RRV=286)做测量。
其他具体实例
本说明书中所揭示的所有特征可以任何组合方式组合。本说明书中所揭示的每个特征可以由具有相同、等同或相似目的的替代特征所取代。因此,除非另有明确说明,否则所揭示的每个特征仅仅是一般系列等同或相似特征的例子。
此外,根据以上描述,本领域技术人员可以容易地确定本发明的基本特征,并且在不脱离本发明的精神和范围的情况下,可以对本发明进行各种改变和修改以使其适应各种用途和条件。因此,其他具体实例也在申请专利范围之内。
参考文献:
(1)Varki,A.Sialic acids in human health and disease.TrendsMol.Med.2008,14,351.
(2)(a)Liu,Y.-C.;Yen,H.-Y.;Chen,C.-Y.;Chen,C.-H.;Cheng,P.-F.;Juan,Y.-H.;Chen,C.-H.;Khoo,K.-H.;Yu,C.-J.;Yang,P.-C.;Hsu,T.-L.;Wong,C.-H.Sialylationand fucosylation of epidermal growth factor receptor suppress itsdimerization and activation in lung cancer cells.Proc.Natl.Acad.Sci.U.S.A.2011,108,11332.(b)Yen,H.-Y.;Liu,Y.-C.;Chen,N.-Y.;Tsai,C.-F.;Wang,Y.-T.;Chen,Y.-J.;Hsu,T.-L.;Yang,P.-C.;Wong,C.-H.Effect of sialylation on EGFRphosphorylation and resistance to tyrosine kinase inhibition.Proc.Natl.Acad.Sci.U.S.A.2015,112,6955.
(3)(a)Ashwell,G.;Harford,J.Carbohydrate-specific receptors of theliver.Annu.Rev.Biochem.1982,51,531.(b)Weigel,P.H.;Yik,J.H.Glycans asendocytosis signals:the cases of the asialoglycoprotein and hyaluronan/chondroitin sulfate receptors.Biochim.Biophys.Acta 2002,1572,341.
(4)(a)Wang,Z.;Chinoy,Z.S.;Ambre,S.G.;Peng,W.;McBride,R.;de Vries,R.P.;Glushka,J.;Paulson,J.C.;Boons,G.J.A general strategy for thechemoenzymatic synthesis of asymmetrically branched N-glycans.Science 2013,341,379.(b)Shivatare,S.S.;Chang,S.H.;Tsai,T.I.;Tseng,S.Y.;Shivatare,V.S.;Lin,Y.S.;Cheng,Y.Y.;Ren,C.T.;Lee,C.C.;Pawar,S.;Tsai,C.S.;Shih,H.W.;Zeng,Y.F.;Liang,C.H.;Kwong,P.D.;Burton,D.R.;Wu,C.Y.;Wong,C.H.Modular synthesis of N-glycans and arrays for the hetero-ligand binding analysis of HIVantibodies.Nat.Chem.2016,8,338.(c)Li,L.;Liu,Y.;Ma,C.;Qu,J.;Calderon,A.D.;Wu,B.;Wei,N.;Wang,X.;Guo,Y.;Xiao,Z.;Song,J.;Sugiarto,G.;Li,Y.;Yu,H.;Chen,X.;Wang,P.G.Efficient chemoenzymatic synthesis of an N-glycan isomerlibrary.Chem.Sci.2015,6,5652.
(5)Li,C.;Wang,L.-X.Chemoenzymatic Methods for the Synthesis ofGlycoproteins.Chem.Rev.2018,118,8359.
(6)(a)Lin,C.-W.;Tsai,M.-H.;Li,S.-T.;Tsai,T.-I.;Chu,K.-C.;Liu,Y.-C.;Lai,M.-Y.;Wu,C.-Y.;Tseng,Y.-C.;Shivatare,S.S.;Wang,C.-H.;Chao,P.;Wang,S.-Y.;Shih,H.-W.;Zeng,Y.-F.;You,T.-H.;Liao,J.-Y.;Tu,Y.-C.;Lin,Y.-S.;Chuang,H.-Y.;Chen,C.-L.;Tsai,C.-S.;Huang,C.-C.;Lin,N.-H.;Ma,C.;Wu,C.-Y.;Wong,C.-H.A commonglycan structure on immunoglobulin G for enhancement of effector functions.Proc.Natl.Acad.Sci.U.S.A.2015,112,10611.(b)Tsai,T.-I.;Li,S.-T.;Liu,C.-P.;Chen,K.Y.;Shivatare,S.S.;Lin,C.-W.;Liao,S.-F.;Lin,C.-W.;Hsu,T.-L.;Wu,Y.-T.;Tsai,M.-H.;Lai,M.-Y.;Lin,N.-H.;Wu,C.-Y.;Wong,C.-H.An Effective BacterialFucosidase for Glycoprotein Remodeling.ACS Chem.Biol.2017,12,63.(c)Liu,C.-P.;Tsai,T.-I.;Cheng,T.;Shivatare,V.S.;Wu,C.-Y.;Wu,C.-Y.;Wong,C.-H.Glycoengineering of antibody(Herceptin)through yeast expression and invitro enzymatic glycosylation.Proc.Natl.Acad.Sci.U.S.A.2018,115,720.
(7)(a)Gantt,R.;Millner,S.;Binkley,S.B.Inhibition of N-Acetylneuraminic Acid Aldolase by 3-Fluorosialic Acid.Biochemistry 1964,3,1952.(b)Hagiwara,T.;Kijima-Suda,I.;Ido,T.;Ohrui,H.;Tomita,K.Inhibition ofbacterial and viral sialidases by 3-fluoro-N-acetylneuraminicacid.Carbohydr.Res.1994,263,167.(c)Burkart,M.D.;Zhang,Z.;Hung,S.-C.;Wong,C.-H.A New Method for the Synthesis of Fluoro-Carbohydrates and Glycosides UsingSelectfluor.J.Am.Chem.Soc.1997,119,11743.(d)D.Burkart,M.;P.Vincent,S.;Wong,C.-H.An efficient synthesis of CMP-3-fluoroneuraminic acid.Chem.Commun.1999,1525.(e)Burkart,M.D.;Vincent,S.P.;Duffels,A.;Murray,B.W.;Ley,S.V.;Wong,C.-H.Chemo-enzymatic synthesis of fluorinated sugar nucleotide:usefulmechanistic probes for glycosyltransferases.Bioorg.Med.Chem.2000,8,1937.(f)Sun,X.-L.;Kanie,Y.;Guo,C.-T.;Kanie,O.;Suzuki,Y.;Wong,C.-H.Syntheses of C-3-Modified Sialylglycosides as Selective Inhibitors of Influenza Hemagglutininand Neuraminidase.Eur.J.Org.Chem.2000,2643.(g)Rillahan,C.D.;Antonopoulos,A.;Lefort,C.T.;Sonon,R.;Azadi,P.;Ley,K.;Dell,A.;Haslam,S.M.;Paulson,J.C.Globalmetabolic inhibitors of sialyl-and fucosyltransferases remodel theglycome.Nat.Chem.Biol.2012,8,661.
(8)Ishiwata,K.;Ido,T.;Nakajima,T.;Ohrui,H.;Kijima-Suda,I.;Itoh,M.Tumor uptake study of 18F-labeled N-acetylneuraminic acids.Internationaljournal of radiation applications and instrumentation.Int.J.Rad.Appl.Instrum.B 1990,17,363.
(9)(a)Watts,A.G.;Damager,I.;Amaya,M.L.;Buschiazzo,A.;Alzari,P.;Frasch,A.C.;Withers,S.G.Trypanosoma cruzi Trans-sialidase Operates through aCovalent Sialyl-Enzyme Intermediate:Tyrosine Is the CatalyticNucleophile.J.Am.Chem.Soc.2003,125,7532.(b)Buchini,S.;Gallat,F.X.;Greig,I.R.;Kim,J.H.;Wakatsuki,S.;Chavas,L.M.;Withers,S.G.Tuning Mechanism-BasedInactivators of Neuraminidases:Mechanisticand Structural Insights.Angew.Chem.Int.Ed.Engl.2014,53,3382.(c)Kim,J.H.;Resende,R.;Wennekes,T.;Chen,H.M.;Bance,N.;Buchini,S.;Watts,A.G.;Pilling,P.;Streltsov,V.A.;Petric,M.;Liggins,R.;Barrett,S.;McKimm-Breschkin,J.L.;Niikura,M.;Withers,S.G.Mechanism-BasedCovalent Neuraminidase Inhibitors with Broad-Spectrum Influenza AntiviralActivity.Science 2013,340,71.
(10)Tsai,C.S.;Yen,H.Y.;Lin,M.I.;Tsai,T.I.;Wang,S.Y.;Huang,W.I.;Hsu,T.L.;Cheng,Y.S.;Fang,J.M.;Wong,C.-H.Cell-permeable probe for identificationand imaging of sialidases.Proc.Natl.Acad.Sci.U.S.A.2013,110,2466.
(11)(a)Chokhawala,H.A.;Cao,H.;Yu,H.;Chen,X.Enzymatic Synthesis ofFluorinated Mechanistic Probes for Sialidases and Sialyltransferases.J.Am.Chem.Soc.2007,129,10630.(b)McArthur,J.B.;Yu,H.;Zeng,J.;Chen,X.ConvertingPasteurella multocidaα2–3-sialyltransferase 1(PmST1)to a regioselectiveα2,6-sialyltransferase by saturation mutagenesis and regioselectivescreening.Org.Biomol.Chem.,2017,15,1700.
(12)Petrie,C.R.;Sharma,M.;Simmons,O.D.;Korytnyk,W.Synthesis ofanalogs of N-acetylneuraminic acid and their effect on CMP-sialatesynthase.Carbohydr.Res.1989,186,326.
(13)Nakajima,T.;Hori,H.;Ohrui,H.;Meguro,H.;Ido,T.Synthesis of N-Acetyl-3-fluoro-neuraminic Acids Agric.Biol.Chem.1988,52,1209.
(14)Watts,A.G.;Withers,S.G.The synthesis of some mechanistic probesfor sialic acid processing enzymes and the labeling of a sialidase fromTrypanosoma rangeli.Can.J.Chem 2004,82,1581.
(15)Hayashi,T.;Kehr,G.;Gilmour R.Stereospecificα-sialylation by site-selective fluorination.Angew.Chem.Int.Ed.Engl.10.1002/anie.201812963.
(16)Okamoto,K.;Kondo,T.;Goto,T.An effective synthesis of α-glycosidesof N-acetylneuraminic acid derivatives by use of 2-deoxy-2β-halo-3β-hydroxy-4,7,8,9-tetra-O-acetyl-N-acetylneuraminic acid methyl ester.Tetrahedron 1987,43,5919.
(17)Bennua-Skalmowski,B.;Vorbrüggen,H.A facile conversion of primaryor secondary alcohols with n-perfluorobutane-sulfonyl fluoride/1,8-diazabicyclo[5.4.0]undec-7-ene into their corresponding fluorides.TetrahedronLett.1995,36,2611.
(18)Cao,H.;Li,Y.;Lau,K.;Muthana,S.;Yu,H.;Cheng,J.;Chokhawala,H.A.;Sugiarto,G.;Zhang,L.;Chen,X.Sialidase substratespecificity studies usingchemoenzymatically synthesized sialosides containing C5-modified sialicacids.Org.Biomol.Chem.,2009,7,5137.
(19)Orlova,A.V.;Shpirt,A.M.;Kulikova,N.Y.;Kononov,L.O.N,N-Diacetylsialyl chloride—a novel readily accessible sialyl donor in reactionswith neutral and charged nucleophiles in the absence of apromoter.Carbohydr.Res.2010,345,721.
(20)Okamoto,K.;Kondo,T.;Goto.T.Functionalization of 2-Deoxy-2,3-dehydro-N-acetylneuraminic Acid Methyl Ester.Bull.Chem.Soc.Jpn.1987,60,631.
(21)Pascolutti,M.;Madge,P.D.;Thomson,R.J.;von Itzstein,M.Access to 3-O-Functionalized N-Acetylneuraminic Acid Scaffolds.J.Org.Chem.2015,80,7746.
(22)Wang,C.-C.;Lee,J.-C.;Luo,S.-Y.;Kulkarni,S.S.;Huang,Y.-W.;Lee,C.-C.;Chang,K.-L.;Hung,S.-C.Regioselective one-pot protection ofcarbohydrates.Nature 2007,446,896.
(23)Zhang,Z.;Ollmann,I.R.;Ye,X.-S.;Wischnat,R.;Baasov,T.;Wong,C.-H.Programmable One-Pot Oligosaccharide Synthesis.J.Am.Chem.Soc.1999,121,734.
(24)Huang,L.;Huang,X.Highly Efficient Syntheses of Hyaluronic Acid Oligosaccharides.Chem.Eur.J.2007,13,529.
(25)Shivatare,S.S.;Chang,S.-H.;Tsai,T.-I;Ren,C.-T.;Chuang,H.-Y.;Hsu,L.;Lin,C.-W.;Li,S.-T.;Wu,C.-Yi;Wong,C.-H.Efficient Convergent Synthesis ofBi,Tri,and Tetra-antennary
(26)Complex Type N Glycans and Their HIV 1Antigenicity.J.Am.Chem.Soc.2013,135,15382.
(27)Shivatare,S.S.;Chang,S.-H.;Tsai,T.-I;Tseng,S.Y.;Shivatare,V.S.;Lin,Y.-S.;Cheng,Y.-Y.;Ren,C.-T.;Lee,C.-C.D.;Pawar,S.;Tsai,C.-S.;Shih,H.-W.;Zeng,Y.-F.;Liang,C.-H.;Kwong,P.D.;Burton,D.R.;Wu,C.-Y.;Wong,C.-H.Modularsynthesis of N-glycans and arrays for the hetero-ligand binding analysis ofHIV antibodies.Nat.Chem.2016,8,338.
(28)Mong,T.K.-K.;Huang,C.-Y.;Wong,C.-H.A New Reactivity-Based One-PotSynthesis of N-Acetyllactosamine Oligomers.J.Org.Chem.2003,68,2135.
(29)Dinkelaar,J.;Duivenvoorden,B.A.;Wennekes,T.;Overkleeft,H.S.;Boot,R.G.;Aerts,J.M.F.G.;Codée,J.D.C.;van der Marel,G.A.A Preparative Synthesis ofHuman Chitinase Fluorogenic Substrate (4’-Deoxychitobiosyl)-4-methylumbelliferone.Eur.J.Org.Chem.2010,2565.
(30)Chayajarus,K.;Chambers,D.J.;Chughtai,M.J.;Fairbanks,A.J.Stereospecific Synthesis of 1,2-cis Glycosides by Vinyl-MediatedIAD.Org.lett.2004,6,3797-3800.
(31)Cao,H.;Li,Y.;Lau,K.;Muthana,S.;Yu,H.;Cheng,J.;Chokhawala,H.A.;Sugiarto,G.;Zhang,L.;Chen,X.Sialidase substrate specificity studies usingchemoenzymatically synthesized sialosides containing C5-modified sialicacids.Org.Biomol.Chem.,2009,7,5137.
(32)Lin,C.-W.;Tsai,M.-H.;Li,S.-T.;Tsai,T.-I.;Chu,K.-C.;Liu,Y.-C.;Lai,M.-Y.;Wu,C.-Y.;Tseng,Y.-C.;Shivatare,S.S.;Wang,C.-H.;Chao,P.;Wang,S.-Y.;Shih,H.-W.;Zeng,Y.-F.;You,T.-H.;Liao,J.-Y.;Tu,Y.-C.;Lin,Y.-S.;Chuang,H.-Y.;Chen,C.-L.;Tsai,C.-S.;Huang,C.-C.;Lin,N.-H.;Ma,C.;Wu,C.-Y.;Wong,C.-H.A commonglycan structure on immunoglobulin G for enhancement of effector functions.Proc.Natl.Acad.Sci.U.S.A.2015,112,10611.
(33)Stavenhagen,K.;Hinneburg,H.;Thaysen-Andersen,M.;Hartmann,L.;Silva,D.V.;Fuchser,J.;Kaspar,S.;Rapp,E.;Seebergera,P.H.;KolarichaD.Quantitative mapping of glycoprotein micro-heterogeneity and macro-heterogeneity:an evaluation of mass spectrometry signal strengths usingsynthetic peptides and glycopeptides.J.Mass Spectrom.2013,48,627.
(34)Lee,J.-C.;Greenberg,W.A;Wong,C.-H.Programmable reactivity-basedone-pot oligosaccharide synthesis.Nat.Protoc.2006,1,3143.
Claims (32)
1.一种制备含3-氟唾液酸的糖的方法,其中,所述方法包含:
使3-羟基唾液酸与一糖反应进行糖基化反应,形成α2,6连接3-羟基唾液酸糖苷,以及
使所述α2,6连接3-羟基唾液酸糖苷与氟化剂反应进行氟化反应,形成含3-氟唾液酸的糖。
2.如权利要求1所述的方法,其中,所述糖是单糖。
3.如权利要求2所述的方法,其中,所述3-羟基唾液酸是2-溴-3-羟基唾液酸,且所述糖基化反应是在三氟甲磺酸银(AgOTf)和磷酸氢二钠(Na2HPO4)的存在下进行。
4.如权利要求3所述的方法,其中,所述糖基化反应是在甲苯中进行。
5.如权利要求2所述的方法,其中,所述氟化剂是全氟-1-丁烷磺酰氟(NfF),且所述氟化反应是在催化剂存在下进行。
6.如权利要求5所述的方法,其中,所述催化剂是1,8-二吖双环[5,4,0]-十一-7-烯(DBU)。
7.如权利要求6所述的方法,其中,所述氟化反应是在甲苯中进行。
8.如权利要求6所述的方法,其中,所述氟化反应是在二氟三甲基硅酸三(二甲胺基)锍(TASF)的存在下进行。
9.如权利要求2所述的方法,其中,所述3-羟基唾液酸是2-溴-3-羟基唾液酸,所述氟化剂是NfF,所述糖基化反应是在AgOTf和Na2HPO4的存在下进行,且所述氟化反应是在催化剂存在下进行。
10.如权利要求9所述的方法,其中,所述催化剂是DBU且所述糖基化和氟化反应都是在甲苯中进行。
12.如权利要求11所述的方法,其中,所述氟化反应是进一步在TASF存在下进行。
13.如权利要求2所述的方法,其中,所述方法更包含使含3-氟唾液酸的糖与一第二糖反应来进行另一个糖基化反应。
14.如权利要求13所述的方法,其中,所述3-羟基唾液酸是2-溴-3-羟基唾液酸,所述氟化剂是NfF,所述糖基化反应是在AgOTf和Na2HPO4的存在下进行,且所述氟化反应是在催化剂存在下进行。
15.如权利要求14所述的方法,其中,所述催化剂是DBU,且所述糖基化和氟化反应都是在甲苯中进行。
16.如权利要求15所述的方法,其中,所述氟化反应是在TASF存在下进行。
17.一种制备与含3-氟唾液酸的糖结合的均质性抗体的方法,其中,所述方法包括用含3-氟唾液酸的糖使单株抗体糖基化。
18.如权利要求17所述的方法,其中,所述含3-氟唾液酸的糖是以如下方式制得:
使3-羟基唾液酸与糖反应进行糖基化反应,形成α2,6连接3-羟基唾液酸糖苷,以及
使所述α2,6连接3-羟基唾液酸糖苷与氟化剂反应进行氟化反应,形成含3-氟唾液酸的糖。
19.如权利要求18所述的方法,其中,所述3-羟基唾液酸是2-溴-3-羟基唾液酸,所述氟化剂是NfF,所述糖基化反应是在AgOTf和Na2HPO4的存在下进行,且所述氟化反应是在催化剂存在下进行。
20.如权利要求19所述的方法,其中,所述催化剂是DBU,且所述糖基化和氟化反应都是在甲苯中进行。
22.如权利要求21所述的方法,其中,所述方法更包含在氟化反应之后,使所述含3-氟唾液酸的糖与第二糖反应进行另一个糖基化反应。
23.如权利要求17所述的方法,其中,所述含3-氟唾液酸的糖是以α2,6连接3-氟-唾液酸糖苷为末端的N-聚糖。
27.如权利要求25所述的化合物,其中,X是N-聚糖。
29.一种与含3-氟唾液酸的糖结合的单株抗体,其中,所述含3-氟唾液酸的糖是如权利要求27所述的化合物。
30.如权利要求29所述的单株抗体,其中,所述含3-氟唾液酸的糖是如权利要求28所述的化合物。
31.一种治疗癌症的方法,其中,所述方法包括向有此需要的个体投予有效量的如权利要求29所述的单株抗体。
32.如权利要求31所述的方法,其中,所述单株抗体是如权利要求30所述的单株抗体。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201962829736P | 2019-04-05 | 2019-04-05 | |
US62/829,736 | 2019-04-05 | ||
PCT/US2020/014608 WO2020205034A1 (en) | 2019-04-05 | 2020-03-10 | Sialidase-resistant saccharide and method of making and using the same |
Publications (1)
Publication Number | Publication Date |
---|---|
CN115605491A true CN115605491A (zh) | 2023-01-13 |
Family
ID=72666761
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202080022413.3A Pending CN115605491A (zh) | 2019-04-05 | 2020-03-10 | 抗唾液酸酶的糖类、其制造方法及用途 |
Country Status (9)
Country | Link |
---|---|
US (1) | US20220267363A1 (zh) |
EP (1) | EP3947406A4 (zh) |
JP (1) | JP2022529408A (zh) |
KR (1) | KR20210137485A (zh) |
CN (1) | CN115605491A (zh) |
CA (1) | CA3131096A1 (zh) |
IL (1) | IL286492A (zh) |
TW (1) | TWI749504B (zh) |
WO (1) | WO2020205034A1 (zh) |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
TW201442721A (zh) * | 2013-01-23 | 2014-11-16 | Daiichi Sankyo Co Ltd | 糖鏈修飾心房利尿鈉肽 |
KR20220151036A (ko) * | 2014-05-27 | 2022-11-11 | 아카데미아 시니카 | 항-cd20 글리코항체 및 이의 용도 |
-
2020
- 2020-03-10 JP JP2021558537A patent/JP2022529408A/ja active Pending
- 2020-03-10 EP EP20783332.8A patent/EP3947406A4/en active Pending
- 2020-03-10 CA CA3131096A patent/CA3131096A1/en active Pending
- 2020-03-10 US US17/598,064 patent/US20220267363A1/en active Pending
- 2020-03-10 WO PCT/US2020/014608 patent/WO2020205034A1/en unknown
- 2020-03-10 KR KR1020217031324A patent/KR20210137485A/ko unknown
- 2020-03-10 CN CN202080022413.3A patent/CN115605491A/zh active Pending
- 2020-04-01 TW TW109111324A patent/TWI749504B/zh active
-
2021
- 2021-09-19 IL IL286492A patent/IL286492A/en unknown
Also Published As
Publication number | Publication date |
---|---|
WO2020205034A1 (en) | 2020-10-08 |
JP2022529408A (ja) | 2022-06-22 |
CA3131096A1 (en) | 2020-10-08 |
IL286492A (en) | 2021-12-01 |
TWI749504B (zh) | 2021-12-11 |
EP3947406A4 (en) | 2022-12-28 |
TW202104272A (zh) | 2021-02-01 |
EP3947406A1 (en) | 2022-02-09 |
US20220267363A1 (en) | 2022-08-25 |
KR20210137485A (ko) | 2021-11-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10266502B2 (en) | Process for the cycloaddition of a halogenated 1,3-dipole compound with a (hetero)cycloalkyne | |
Kondo et al. | Glycosyl phosphites as glycosylation reagents: scope and mechanism | |
EP2981541B1 (en) | Synthesis and use of isotopically-labelled glycans | |
EP2935608A1 (en) | Modified glycoprotein, protein-conjugate and process for the preparation thereof | |
US9938312B2 (en) | Compounds and methods for chemical and chemo-enzymatic synthesis of complex glycans | |
Cattaneo et al. | Orthogonal cleavage of the 2-naphthylmethyl group in the presence of the p-methoxy phenyl-protected anomeric position and its use in carbohydrate synthesis | |
EP3097200A1 (en) | Process for the attachment of a galnac moiety comprising a (hetero)aryl group to a glcnac moiety, and product obtained thereby | |
Hribernik et al. | One pot synthesis of thio-glycosides via aziridine opening reactions | |
Wang et al. | Glycosylation of Nα-lauryl-O-(β-D-xylopyranosyl)-L-serinamide as a saccharide primer in cells | |
TWI749504B (zh) | 抗涎酸酶之醣類、其之製造方法及用途 | |
Xia et al. | Total synthesis of sialylated and sulfated oligosaccharide chains from respiratory mucins | |
WO2018098342A1 (en) | N-acetylated sialic acids and related sialosides | |
WO2022191313A1 (ja) | 糖鎖及び糖鎖を含む医薬品の製造方法 | |
Robina et al. | Glycosylation Methods in Oligosaccharide Synthesis. Part 1 | |
van Dorst et al. | Synthesis of Hexp-(1→ 4)-β-d-GlcpNac-(1→ 2)-α-d-Manp-(1→ O)(CH2) 7CH3 probes for exploration of the substrate specificity of glycosyltransferases: Part II, Hex= 3-O-methyl-β-d-Gal, 3-deoxy-β-d-Gal, 3-deoxy-3-fluoro-β-d-Gal, 3-amino-3-deoxy-β-d-Gal, β-d-Gul, α-l-Alt, or β-l-Gal | |
Ogawa et al. | Synthesis of Ether‐and Imino‐Linked Octyl N‐Acetyl‐5a′‐carba‐β‐lactosaminides and‐isolactosaminides: Acceptor Substrates for α‐(1→ 3/4)‐Fucosyltransferase, and Enzymatic Synthesis of 5a′‐Carbatrisaccharides | |
JP6198207B2 (ja) | 新規糖供与体及びそれを用いた糖鎖の合成方法 | |
Tanaka et al. | Synthesis of three deoxy-sophorose derivatives for evaluating the requirement of hydroxy groups at position 3 and/or 3’of sophorose by 1, 2-β-oligoglucan phosphorylases | |
WO2024053574A1 (ja) | 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 | |
Chu et al. | Efficient synthesis of a linear octyl pentaarabinofuranoside, a substrate for mycobacterial EmbA/EmbB proteins | |
Likhosherstov et al. | Synthesis of N-glycyl-β-glycopyranosyl amines, derivatives of natural oligosaccharides—glucose analogs of Le x antigen | |
Tricomi et al. | Stereoselective Synthesis of the Gal‐α‐(1→ 3)‐Gal‐β‐(1→ 3)‐GlcNAc Trisaccharide: a new Ligand for DCAR and Mincle C‐Type Lectin Receptors | |
Gagarinov | Modern approaches to the synthesis of diverse classes of oligosaccharides | |
Mała | Self-promoted Glycosylations with Trichloroacetimidate Glycosyl Donors: Synthesis of N-glycosides | |
Mała | Glikozylacje typu self-promoted z zastosowaniem trichloroacetoimidanów glikozylu: synteza N-glikozydów |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |