WO2021087709A1 - 一种(1-5)-Gal-(1-6)-Man糖蛋白及其制备方法和应用 - Google Patents

一种(1-5)-Gal-(1-6)-Man糖蛋白及其制备方法和应用 Download PDF

Info

Publication number
WO2021087709A1
WO2021087709A1 PCT/CN2019/115506 CN2019115506W WO2021087709A1 WO 2021087709 A1 WO2021087709 A1 WO 2021087709A1 CN 2019115506 W CN2019115506 W CN 2019115506W WO 2021087709 A1 WO2021087709 A1 WO 2021087709A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
glycoprotein
reaction
dichloromethane
antigen
Prior art date
Application number
PCT/CN2019/115506
Other languages
English (en)
French (fr)
Inventor
李文举
邬晓乐
高健
唐鹏飞
庄永忠
李秀珍
Original Assignee
济南山目生物医药科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 济南山目生物医药科技有限公司 filed Critical 济南山目生物医药科技有限公司
Publication of WO2021087709A1 publication Critical patent/WO2021087709A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • 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
    • A61K39/0002—Fungal antigens, e.g. Trichophyton, Aspergillus, Candida
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides

Definitions

  • the invention relates to the field of glycoprotein vaccines, in particular to a (1-5)-Gal-(1-6)-Man glycoprotein against fungal infection and a preparation method thereof.
  • Invasive fungal infections are also called deep fungal infections.
  • the main pathogens are Candida, Aspergillus and Cryptococcus.
  • Candida Aspergillus
  • Cryptococcus the proportion of fungal infections in hospitalized patients has increased, and due to geographical differences, the prevalent strains are different.
  • invasive aspergillosis is a more common infectious disease in severely immunocompromised patients, and its incidence has increased significantly in recent years.
  • the present invention provides a (1-5)-Gal-(1-6)-Man-Linker-KLH glycoprotein, which can effectively resist Aspergillus infection.
  • Another object of the present invention is to provide an application of the aforementioned glycoprotein in anti-fungal infections.
  • the present invention adopts the following technical solutions.
  • a glycoprotein whose structure is:
  • the immunogenic carrier protein is a non-human protein; selected from KLH (keyhole limpet hemocyanin), BSA (bovine serum albumin), OVA (chicken ovalbumin), BluecarrierTM (mollusk-derived highly soluble hemocyanin) Protein), non-toxic variant of diphtheria toxin, tetanus toxin/toxoid, high molecular weight protein (HMP) isolated from unacquired Haemophilus influenzae, detoxified Pseudomonas toxin A, cholera toxin/toxoid, Pertussis toxin/toxoid, Clostridium perfringens exotoxin/toxoid, hepatitis B surface antigen, hepatitis B core antigen, rotavirus VP7 protein, diphtheria toxin mutant CRM, CRM191, CRM3201, respiratory syncytial virus F and At least one of G protein; preferably KLH, BSA.
  • the preparation method of the aforementioned glycoprotein includes the following steps:
  • TMSOTf catalyzes the reaction of compound 2 and compound 3 in dichloromethane. After the reaction is terminated, compound 4 is obtained by separation and purification:
  • TMSOTf catalyzes the reaction of compound 5 and compound 6 in dichloromethane. After the reaction is terminated, compound 7 is separated and purified:
  • TMSOTf catalyzes the reaction of compound 5 and compound 8 in dichloromethane. After the reaction is terminated, compound 9 is separated and purified:
  • the glycoprotein of the present invention is based on the fungal surface polysaccharide antigen structure to design and prepare a structured oligosaccharide antigen by chemical synthesis, and then covalently couple it with an immunogenic protein to form a new semi-synthetic glycoprotein conjugate vaccine .
  • an immunogenic protein to form a new semi-synthetic glycoprotein conjugate vaccine .
  • Preliminary animal experiments show that the glycoconjugate exhibits good activity. After immunization, the antibody titer in the blood of mice increases significantly, and the titer of IgG antibody accounts for a large proportion of the total antibody. This result shows that The glycoconjugate synthesized in the present invention is a very promising anti-fungal sugar vaccine.
  • Figure 1 is a 1 H NMR chart of the oligosaccharide fragment obtained in Example 1;
  • Figure 2 is a 13 C NMR chart of the oligosaccharide fragment obtained in Example 1;
  • Figure 3 is a graph showing the immunogenic antibody titer of (1-5)-Gal-(1-6)-Man-7 carbon chain Linker-KLH glycoprotein;
  • Figure 4 is a graph of immunogenic antibody titers of (1-5)-Gal-(1-6)-Man-3 carbon chain Linker-KLH glycoprotein.
  • the activated oligosaccharide fragment and KLH protein were dissolved in 0.1M phosphate buffer (PBS, 1 ml) according to the molar ratio of 25:1, the reaction solution was stirred overnight, the glycoprotein was purified by dialysis, and the aqueous solution was lyophilized to obtain a white powder Glycoprotein (1-5)-Gal-(1-6)-Man-7 Carbon Linker-KLH glycoprotein.
  • PBS 0.1M phosphate buffer
  • oligosaccharide fragments and glycoproteins were prepared according to the method and raw material ratio of Example 1, except that the structural formula of compound 6 was replaced with:
  • Dissolve oligosaccharide fragment 1 (8 mg) in DMF (1 ml) and 0.1M phosphate buffer (PBS, 0.25 ml), add disuccinimidyl glutarate (5 mg) to the mixture while stirring The reaction solution was stirred at room temperature overnight, and the solvent was distilled off under high vacuum under reduced pressure to obtain a white solid. The solid was washed with ethyl acetate, filtered and dried to obtain an activated oligosaccharide fragment.
  • PBS 0.1M phosphate buffer
  • the activated oligosaccharide fragment and BSA were dissolved in 0.1M phosphate buffer (PBS, 1ml) according to the molar ratio of 25:1, the reaction solution was stirred overnight, the glycoprotein was purified by dialysis, and the aqueous solution was lyophilized to obtain a white powder.
  • PBS 0.1M phosphate buffer
  • the (1-5)-Gal-(1-6)-Man-3 carbon Linker-BSA glycoprotein was prepared according to the same method and raw material ratio as in Example 2.
  • the blood was taken 1 day before the immunization and the second day after the last immunization to prepare antibody serum, respectively, with the corresponding oligosaccharide BSA glycoprotein (1-5)-Gal-(1-6)-Man-7 carbon Linker-BSA And (1-5)-Gal-(1-6)-Man-3 carbon Linker-BSA was used as the fixed antigen, and the specific antibody titer was detected by ELISA.
  • the results are shown in Figure 3 and Figure 4.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Mycology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)

Abstract

一种抗真菌感染的(1-5)-Gal-(1-6)-Man糖蛋白及其制备方法。该糖蛋白是基于真菌表面多糖抗原结构以设计并通过化学合成方法制备结构明确的寡糖抗原，随后将其与免疫原性蛋白共价偶联形成新型的半合成糖缀合物疫苗，以增强糖抗原免疫原性，使其转化为T细胞依赖性抗原，同时可以克服由天然多糖导致的疫苗制备和质量控制困难等问题。通过初步的动物实验表明该糖蛋白表现了良好的活性，小鼠经免疫后血液中的抗体滴度明显增高，IgG抗体的滴度在总抗体中占有较大的比例，是一种非常有前景的抗真菌糖疫苗。

Description

一种(1-5)-Gal-(1-6)-Man糖蛋白及其制备方法和应用 技术领域
本发明涉及糖蛋白疫苗领域，具体涉及一种抗真菌感染的(1-5)-Gal-(1-6)-Man糖蛋白及其制备方法。
背景技术
侵袭性真菌感染又称深部真菌感染，病原菌主要为念珠菌、曲霉和隐球菌。近年来住院患者的真菌感染比例有所上升，并且由于地域差别导致流行菌株不同，其中侵袭性曲霉病在严重免疫受损患者群体是较普遍的传染病，它的发病率近年来上升趋势明显，仅次于念珠菌感染。虽然新的抗真菌药物不断涌现，它的病死率依然很高(≥50％)，是引起干细胞移植和实体器官移植患者相关感染死亡的主要原因。
由于侵袭性真菌感染临床表现没有特征性，容易被原发病或其他并发症所掩盖，加上实验室诊断方法费时、敏感性差、阴性结果不能排除诊断，有些临床标本即使分离出真菌也很难确立是定植还是侵袭或污染，因此导致很多患者得不到及时、准确的诊断。另外，侵袭性真菌感染的病情比较凶险，治疗也比较困难，虽然新的抗真菌药不断增多，但真菌本身也在不断发生变异，导致耐药菌株不断增多，加上部分药物的毒副反应使得患者无法耐受，因此抗真菌治疗依然面临严峻的挑战。研发抗侵袭性真菌疫苗，对真菌感染进行早期预防是对特殊病患者真正有效的生命健康保障。然而，到目前为止还没有一种成熟有效的真菌疫苗。
发明内容
针对目前缺乏有效的真菌疫苗的问题，本发明提供一种(1-5)-Gal-(1-6)-Man-Linker-KLH糖蛋白，能够有效抗曲霉感染。
本发明的另一目的是提供一种上述糖蛋白在抗真菌感染中的应用。
为实现上述目的，本发明采用如下技术方案。
一种糖蛋白，其结构为：
Figure PCTCN2019115506-appb-000001
所述免疫原性载体蛋白为非人源蛋白；选自KLH(匙孔血蓝蛋白)，BSA(牛血清白蛋白)，OVA(鸡卵白蛋白)，Blue carrierTM(软体动物源性高可溶的血蓝蛋白)，白喉毒素无毒变异体，破伤风毒素/类毒素，分离自非获得型流感嗜血杆菌的高分子量蛋白(HMP)，脱毒后的绿脓杆菌毒素A，霍乱毒素/类毒素，百日咳毒素/类毒素，产气荚膜梭状芽胞杆菌外毒素/类毒素，乙肝表面抗原，乙肝核心抗原，轮状病毒VP7蛋白，白喉毒素突变体CRM、CRM191、CRM3201，呼吸合胞病毒F和G蛋白中的至少一种；优选为KLH、BSA。
上述糖蛋白的制备方法，包括以下步骤：
(1)氮气保护、分子筛存在下，TMSOTf催化化合物2和化合物3在二氯甲烷中反应，终止反应后分离纯化得化合物4：
Figure PCTCN2019115506-appb-000002
(2)化合物4溶解于甲醇:二氯甲烷5:1混合溶剂中在PdCl 2存在下于室温中反应，过滤后滤液用碱中和，分离获得油状物；油状物溶解于二氯甲烷，加入碳酸铯和CF 3C(NPh)Cl，TLC跟踪反应，室温搅拌至反应完全，体系浓缩后用硅胶柱分离得到化合物5：
Figure PCTCN2019115506-appb-000003
(3)氮气保护、分子筛存在下，TMSOTf催化化合物5和化合物6于二氯甲烷中反应，终止反应后分离纯化得化合物7：
Figure PCTCN2019115506-appb-000004
(4)化合物7与吡咯烷在二氯甲烷中反应，再用稀酸洗涤，获得的有机相分离纯化，得化合物8：
Figure PCTCN2019115506-appb-000005
(5)氮气保护、分子筛存在下，TMSOTf催化化合物5和化合物8于二氯甲烷中反应，终止反应后分离纯化得化合物9：
Figure PCTCN2019115506-appb-000006
(6)隔氧条件下，化合物9的甲醇溶液加入10％钯碳，以氢气加氢，在氮气保护下过滤，滤液中加入甲醇钠的甲醇溶液反应后再加入水反应，分离纯化后得到寡糖片段1：
Figure PCTCN2019115506-appb-000007
(7)将免疫原性载体蛋白、寡糖片段和双琥珀酰亚胺戊二酸酯溶于偶联液，孵育，然后分离纯化、纯化获得糖蛋白：
Figure PCTCN2019115506-appb-000008
一种上述糖蛋白在制备抗真菌感染药物中的应用。
一种包含上述糖蛋白的药物。
本发明具有以下优点：
本发明的糖蛋白是基于真菌表面多糖抗原结构以设计并通过化学合成方法制备结构明确的寡糖抗原，随后将其与免疫原性蛋白共价偶联形成新型的半合成糖蛋白缀合物疫苗，以增强糖抗原免疫原性，使其转化为T细胞依赖性抗原，同时可以克服由天然多糖导致的疫苗制备和质量控制困难等问题。通过初步的动物实验表明该糖缀合物表现了良好的活性，小鼠经免疫后血液中的抗体滴度明显增高，IgG抗体的滴度在总抗体中占有较大的比例，这一结果说明本发明合成的糖缀合物是一种非常有前景的抗真菌糖疫苗。
附图说明
图1是实施例1中获得的寡糖片段的 1H NMR图；
图2是实施例1中获得的寡糖片段的 13C NMR图；
图3是(1-5)-Gal-(1-6)-Man-7碳链Linker-KLH糖蛋白免疫原性抗体滴度图；
图4是(1-5)-Gal-(1-6)-Man-3碳链Linker-KLH糖蛋白的免疫原性抗体滴度图。
具体实施方式
下面结合实施例和附图对本发明做进一步说明，但本发明不受下述实施例的限制。
实施例1(1-5)-Gal-(1-6)-Man-7碳链-KLH糖蛋白的制备
Figure PCTCN2019115506-appb-000009
(1)经干燥除水的化合物2(213mg,0.40mmol)和化合物3(440mg,0.5mmol)溶解于干燥的二氯甲烷(8毫升)中，体系中加入预活化的分子筛(MS300AW，400mg)。室温氮气保护下体系搅拌10分钟，然后降温至-80℃，加入TMSOTf(16μL,0.09mmol)。50分钟后体系升温至-10℃然后加入一滴三乙胺淬灭反应。混合物用硅胶柱纯化(甲苯/乙酸乙酯＝20/1洗脱，RF＝0.6)得到油状化合物4(467mg，95％收率)，其 1H NMR(400MHz,CDCl 3)：δ8.06-7.18(m,38H,Ar),5.99(m,1H,H-5II),5.95-5.84(m,1H,OCH2CHCH2),5.83(dd,J3,4＝5.3Hz,J3,2＝1.4Hz,1H,H-3I),5.78(s,1H,H-1II),5.65(d,J2,3＝1.3Hz,1H,H-2II),5.58(dd,J3,4＝5.2Hz,J3,2＝1.3Hz,1H,H-3II),5.53(d,J2,3＝1.4Hz,1H,H-2I),5.34-5.28(m,1H,OCH2CHCHaHb),5.29(s,1H,H-1I),5.17(dq,J＝10.4,1.4Hz,1H,OCH2CHCHaHb),4.97(dd,J4,3＝5.2Hz,J4,5＝3.4Hz,1H,H-4II),4.76(dd,J6a,6b＝8.4Hz,J6a,5＝6.3Hz,1H,H-6aI),4.72-4.63(m,3H,H-6bI,H-5I,H-6aII),4.58–4.51(m,2H,H-4I,H-6bII),4.29(dd,J＝10.3,7.4Hz,1H,Fmoc-CHaHb),4.26-4.16(m,2H,OCHaHbCHCH2,Fmoc-CHaHb),4.14-4.04(m,2H,Fmoc-CH OCH aH bCHCH 2)。 13C NMR(100MHz,CDCl 3)：δ＝165.7(PhCO),165.2(PhCO),154.8(C＝O),143.4(Cq Fmoc),141.2(Cq Fmoc),133.7(OCH 2CHCH 2),133.4-133.0(Ar),130.0- 119.9(Ar),117.4(OCH2CHCH2),105.5(C-1II),104.8(C-1I),82.4(C-4I),82.1(C-2II),82.0(C-2I),81.8(C-4II),77.7(C-3II),77.1(C-3I),73.4(C-5I),70.1(2C,Fmoc-CH 2,C-5II),67.9(OCH 2CHCH 2),66.5(C-6II),64.5(C-6I),46.6(Fmoc-CH).HRMS(ESI):M＝C 72H 60O 19.Calcd m/z for[M+NH4]+1246.4067,found 1246.4036.Calcd m/z for[M+Na]+1251.3621,found 1251.3586.Calcd m/z for[M+K]+1267.3360,found 1267.3332。
(2)化合物4(467mg mg,0.48mmol)溶解于体积比为5:1的甲醇:二氯甲烷混合溶剂(21毫升)中，体系中加入PdCl 2(63mg,0.44mmol)，在室温下搅拌3小时，然后过滤通过硅藻土，滤液用一滴三乙胺中和，加压浓缩，油状物溶解于二氯甲烷(15毫升)，加入碳酸铯(515mg,2.18mmol)和CF 3C(NPh)Cl(260μL,1.635mmol)，TLC跟踪反应，室温搅拌至反应完全。体系浓缩后用硅胶柱分离(甲苯/乙酸乙酯＝10:1v/v淋洗，RF＝0.3)得到化合物5(320mg,α:β＝1:0.75，62％收率)；其 1H NMR(400MHz,CDCl 3)：δ8.08-7.12(m,84H),7.04(t,J＝7.5Hz,0.75H,p-Ph),6.99(t,J＝7.4Hz,1H,p-Ph),6.82(d,J＝8.0Hz,2.5H,o-Ph,H-1αI),6.59(d,J＝7.8Hz,2H,o-Ph),6.35(dd,J3,2＝7.2Hz,J3,4＝5.9Hz,1H,H-3αI),6.05(m,1H,H-5αII),5.95-5.91(m,1.5H,H-5βII,H-3βI),5.89(dd,J2,3＝7.2Hz,J2,1＝4.7Hz,1H,H-2αI),5.79(s,1H,H-1αII),5.78(s,0.75H,H-2βI),5.7(s,0.75H,H-1βII),5.65(dd,J3,4＝5.6Hz,J3,2＝1.8Hz,1H,H-3αII),5.63(d,J2,3＝1.8Hz,1H,H-αII),5.61(d,J2,3＝1.4Hz,0.75H,H-2βII),5.58(dd,J3,4＝5.2Hz,J3,2＝1.4Hz,0.75H,H-3βII),5.03(dd,J4,3＝5.6Hz,J4,5＝3.2Hz,1H,H-4αII),4.91(dd,J4,3＝5.2Hz,J4,5＝3.7Hz,0.75H,H-4βII),4.8-4.59(m,9.75H,2×H-6αII,H-4βI,H-6aβI,H-5βI,H-4αI,H-5αI,2×H-6αI),4.52(dd,J6b,6a＝11.8Hz,J6b,5＝7.8Hz,1H,H-6bβI),4.32-4.15(m,3.5H,Fmoc-CH 2),4.15-4.08(m,1.75H,Fmoc-CH)。 13C NMR(100MHz,CDCl 3)δ＝166.0(PhCO),165.6(PhCO),165.5(PhCO),165.2(PhCO),143.3(Cq Ph),133.7(Ar),133.2(Ar),129.9-124.1(Ar),119.9-119.3(Ar),105.5(C-1βII),105.3(C-1αII),96.3(C-1αI),85.6(C-4βI),82.5(C-2αII),81.9(C-2βII),81.7(C-4βII,C-4αI),81.4(C-4αII),80.5(C-2βI),77.7(C-3αII),77.5(C-3βII),76.4(C-2αI),76.2(C-3βI),74.0(C-3I),73.8(C-5αI),73.2(C-5βI),70.1(C-5βII,Fmoc-CH 2,C-5αII),66.6(C-6βI,C-6αII),64.2(C-6βII),63.5(C-6αI),46.6(Fmoc-CH).HRMS(ESI):M＝C 77H 60F 3NO 19.Calcd m/z for[M+Na]+1382.3604,found1382.3606.Calcd m/z for[M+K]+1398.3343,found 1398.3338。
(3)经真空干燥的化合物5(163mg,0.120mmol)和化合物6(85mg,0.132mmol)溶解于干燥的二氯甲烷(4毫升)中，然后加入预活化的分子筛(MS300AW，200mg)。体系在氮气保护下室温搅拌15分钟，然后降温至-80℃，加入TMSOTf(6.5μL,0.036mmol)。体系在-80℃下搅拌50分钟后升温至-10℃，然后加入一滴三乙胺淬灭反应。体系浓缩后用硅 胶柱纯化(甲苯：乙酸乙酯＝5:1v/v洗脱，RF＝0.25)，得到油状化合物7(188mg,收率86％)；其 1H NMR(600MHz,CDCl 3)：δ8.07-7.14(m,53H,Ar),5.97(m,1H,H-5III),5.85(dd,J3,4＝9.9Hz,J3,2＝3.1Hz,1H,H-3I),5.85(m,1H,H-3II),5.81(t,J4,3＝J4,5＝9.9Hz,1H,H-4I),5.77(s,1H,H-1III),5.66(d,J2,3＝1.2Hz,1H,H-2III),5.65(dd,J2,3＝3.1Hz,J2,1＝1.6Hz,1H,H-2I),5.58(dd,J3,4＝5.0Hz,J3,2＝1.2Hz,1H,H-3III),5.55(d,J2,3＝1.5Hz,1H,H-2II),5.47(s,1H,H-1II),5.03(d,J1,2＝1.6Hz,1H,H-1I),4.95(m,J4,3＝5.0Hz,J4,5＝3.6Hz,1H,H-4III),4.76-4.71(m,1H,H-6aII),4.69-4.63(m,3H,H-6bII,H-6aIII,H-5II),4.55-4.50(m,2H,H-4II,H-6bIII),4.31(ddd,J5,4＝9.3Hz,J5,6a＝7.3Hz,J5,6b＝2.0Hz,1H,H-5I),4.26(dd,J＝10.4,7.5Hz,1H,Fmoc-CHaHb),4.16(dd,J＝10.4,7.7Hz,1H,Fmoc-CH aH b),4.09(t,J＝7.5Hz,1H,Fmoc-CH),3.99-3.93(m,2H,H-6aI,OCH aH bCH 2CH 2N),3.91(dd,J6a,6b＝12.4,J6b,5＝2.0Hz,1H,H-6bI),3.63(td,J＝12.8,6.1Hz,1H,OCH 2CH 2CH aH bN),3.5＝3.45(m,2H,OCH aH bCH 2CH 2N,OCH 2CH 2CH aH bN),2.04-1.92(m,2H,OCH 2CH 2CH 2N)。 13C NMR(150MHz,CDCl 3)：δ＝166.1(PhCO),165.8(PhCO),165.7(PhCO),165.6(PhCO),165.5(PhCO),165.4(PhCO),165.3(PhCO),165.1(PhCO),154.8(C＝O),143.4(Cq Fmoc),141.1(CqFmoc),133.6-133.0(Ar),130.0-127.1(Ar),125.2-119.9(Ar),106.4(C-1II),105.7(C-1III),97.6(C-1I),82.4(C-4II),82.0(C-2III),81.9(2C,C-2II,C-4III),77.7(C-3III),77.1(C-3II),73.0(C-5II),71.4(C-5I),70.4(C-2I),70.2(C-5III),70.1(Fmoc-CH 2),69.9(C-3I),67.2(C-4I),66.6(C-6III),66.2(OCH 2CH 2CH 2N),66.1(C-6I),65.00(C-6II),46.6(Fmoc-CH),37.6(OCH 2CH 2CH 2N),28.5(OCH 2CH 2CH 2N).HRMS(ESI):M＝C 101H 84F 3NO 28.Calcd m/z for[M+NH 4]+1833.5470,found 1833.5451.Calcd m/z for[M+Na]+1838.5024,found 1838.4993。
(4)化合物7(127mg,0.070mmol)溶解于干燥的二氯甲烷(2毫升)，加入吡咯烷(200μL)，室温下搅拌30分钟，然后体系用稀盐酸洗，分出有机相浓缩，混合物用硅胶柱纯化(甲苯/乙酸乙酯＝7/1v/v洗脱，RF＝0.25)，得到油状化合物8(77mg,69％)，其 1H NMR(400MHz,CDCl 3)：δ8.08-7.14(m,45H,Ar),5.87-5.78(m,3H,H-3I,H-3II,H-4I),5.74(s,1H,H-1III),5.71(d,J2,3＝1.7Hz,1H,H-2III),5.67-5.64(m,2H,H-3III,H-2I),5.53(d,J2,3＝1.6Hz,1H,H-2II),5.46(s,1H,H-1II),5.04(d,J1,2＝1.3Hz,1H,H-1I),4.74(dd,J4,3＝4.8Hz,J4,5＝3.2Hz,1H,H-4III),4.72-4.64(m,2H,H-6aII,H-6bII),4.62-4.58(m,1H,H-5II),4.54(dd,J4,3＝5.5Hz,J4,5＝3.1Hz,1H,H-4II),4.52-4.44(m,2H,H-6aIII,H-6bIII),4.39-4.32(m,1H,H-5III),4.31(ddd,J5,4＝9.3Hz,J5,6a＝6.7Hz,J5,6b＝2.4Hz,1H,H-5I),4.00-3.93(m,2H,OCH aH bCH 2CH 2N,H-6aI),3.90(dd,J6a,6b＝12.3,J6b,5＝2.4Hz,1H,H-6bI),3.69-3.60(m,1H,OCH 2CH 2CH aH bN),3.58-3.45(m,2H,OCH aH bCH 2CH 2N,OCH 2CH 2CH aH bN),2.75(d,JH5OH＝7.5Hz,1H),2.07-1.91(m,2H,OCH 2CH 2CH 2N)。 13C NMR(100MHz,CDCl 3)：δ＝166.4(PhCO),166.1(PhCO),165.8(PhCO),165.7(PhCO),165.5(PhCO),165.4(PhCO), 165.1(PhCO),133.6-132.9(Ph),129.8-128.2(Ph),106.4(C-1II),105.8(C-1III),97.6(C-1I),83.7(C-4III),82.2(C-4II),81.9(C-2II),81.6(C-2III),78.0(C-3III),76.9(C-3II),73.1(C-5II),71.3(C-5I),70.4(C-2I),69.9(C-3I),69.5(C-5III),67.2(C-4I),66.2(OCH 2CH 2CH 2N),66.15(C-6III),66.1(C-6I),64.9(C-6II),37.6(OCH 2CH 2CH 2N),28.5(OCH 2CH 2CH 2N)。HRMS(ESI):M＝C 86H 74F 3NO 26.Calcd m/z for[M+NH 4]+1611.4789,found 1611.4768.Calcd m/z for[M+Na]+1616.4343,found 1616.4322.Calcd m/z for[M+K]+1632.4083,found 1632.4056。
(5)经真空干燥的化合物5(80mg,0.058mmol)和化合物8(77mg,0.048)溶解于二氯甲烷(4mL)，加入预活化的分子筛(MS300AW，200mg)。体系在氮气保护下室温搅拌15分钟，然后降温至-80℃，加入TMSOTf(6.5μL,0.036mmol)。体系在-80℃下搅拌50分钟后升温至-10℃，然后加入一滴三乙胺淬灭反应。体系浓缩后用硅胶柱纯化(甲苯：乙酸乙酯＝5:1v/v洗脱，RF＝0.20)，得到油状化合物9(120mg,90％)；其 1H NMR(600MHz,CDCl 3)：δ8.09-7.10(m,83H,Ar),5.99-5.96(m,1H,H-5V),5.90-5.86(m,4H,H-3I,H-3II,H-3III,H-3IV),5.83(t,J4,3＝J4,5＝10.0Hz,1H,H-4I),5.80(s,1H,H-1),5.77(s,1H,H-1),5.75(s,1H,H-1),5.73(d,J2,1＝1.2Hz,1H,H-2),5.71(d,J2,3＝1.9Hz,1H,H-2),5.69–5.67(m,2H,H-2I,H-2V),5.59(dd,J3,4＝5.2Hz,J3,2＝1.4Hz,1H,H-3V),5.55(d,J2,1＝1.2Hz,1H,H-2II),5.50(s,1H,H-1II),5.06(d,J1,2＝1.5Hz,1H,H-1I),4.92(dd,J4,3＝5.1Hz,J4,5＝3.5Hz,1H,H-4V),4.88(dd,J4,3＝4.7Hz,J4,5＝3.2Hz,1H,H-4),4.86(dd,J4,3＝5.3Hz,J4,5＝2.6Hz,1H,H-4),4.80-4.66(m,9H,4×H-6,H-5II,2×H-6II,2×H-5),4.65(dd,J6a,6b＝12.0Hz,J4,5＝4.1Hz,1H,H-6aV),4.554.50(m,2H,H-4II,H-6bV),4.35(m,1H,H-5I),4.24(dd,J＝10.1,7.4Hz,1H,Fmoc-CH aH b),4.15(dd,J＝10.1,7.7Hz,1H,Fmoc-CH aH b),4.10(t,J＝7.5Hz,1H,Fmoc-CH),4.02-3.97(m,2H,H-6aI,OCH aH bCH 2CH 2N),3.92(dd,J6b,6a＝12.4Hz,J6b,5＝2.0Hz,1H,H-6bI),3.70-3.64(m,1H,OCH 2CH 2CH aH bN),3.59-3.50(m,2H,OCH aH bCH 2CH 2N,OCH 2CH 2CH aH bN),2.07-1.96(m,2H,OCH 2CH 2CH 2N)。 13C NMR(150MHz,CDCl 3)：δ＝166.2(PhCO),166.0(PhCO),165.6(PhCO),165.2(PhCO),154.7(C＝O),143.3(Cq Fmoc),141.2(Cq Fmoc),133.6-132.8(Ar),130.0-127.1(Ar),125.3(Ar),125.2(Ar),119.9(Ar),106.3(C-1II),105.7(C-1),105.5(C-1),105.2(C-1),97.6(C-1I),83.5(C-4),83.1(C-4),82.8(C-4II),82.0(C-2),81.9(C-2V),81.8(3C,C-2,C-2II,C-4V),77.6(C-3V),77.0(3C,C-3II,C-3,C-3),73.2(C-5),73.0(C-5II),72.9(C-5),71.5(C-5I),70.4(C-2I),70.2(C-5V),70.0(Fmoc-CH2),69.9(C-5I),67.2(C-4I),66.7(C-6V),66.2(OCH 2CH 2CH 2N),65.9(C-6I),65.5(C-6),64.8(2C,C-6II,C-6),46.5(Fmoc-CH),37.6(OCH 2CH 2CH 2N),28.5(OCH 2CH 2CH 2N)。HRMS(ESI):M＝C 125H 128F 3NO 44.Calcd m/z for[M+NH 4]+2781.8100,found 2781.8066。
(6)化合物9(97mg,0.035mg)的甲醇溶液加入Pd/C(10％负载量，20mg)，高纯氮 气和氢气置换3次，体系在10atm压力氢气条件下室温搅拌过夜，在氮气保护下过滤硅藻土。滤液中加入甲醇钠0.1M甲醇溶液(1毫升)，室温下搅拌过夜，然后加入0.1毫升纯化水，体系升温至60℃搅拌过夜。体系浓缩后直接用Bio Gel P2纯化，纯净水淋洗，HPLC跟踪收集淋洗液。浓缩淋洗液至2毫升，冻干得到白色粉状寡糖片段1(11mg,89％)。 1H NMR(600MHz,D 2O):5.22(d,J12＝2.0Hz,1H,H-1V),5.19(m,2H,H-1III,H-1IV),5.03(d,J12＝1.5Hz,1H,H-1II),4.85(d,J12＝1.8Hz,1H,H-1I),4.17–4.14(m,5H,H-4III,H-4IV,H-2III,H-2IV,H-2V),4.13–4.08(m,5H,H-3II,H-3III,H-3IV,H-2II,H-4II),4.08–4.06(m,2H,H-3V,H-4V),4.02(m,1H,H-6aI),3.98–3.92(m,4H,H-5II,H-5III,H-5IV,H-2I),3.87–3.83(m,2H,OCH 2,H-5V),3.82–3.78(m,7H,H-6aIII,H-6bIII,H-6aIV,H-6bIV,H-6aII,H-6bII,H-3I),3.74–3.69(m,4H,H-6bI,H-6aV,H-5I,H-4I),3.67(dd,2J6a6b＝11.7Hz,J56b＝7.3Hz,1H,H-6bV),3.61(ddd,2Jab＝10.2Hz,J＝6.8Hz,J＝5.4Hz,1H,OCH 2CHCH 2),3.17–3.09(m,2H,OCH 2CH 2CH 2N),2.02–1.96(m,2H,OCH 2CH 2CH 2N)。 13C NMR(150MHz,D 2O):δ108.7(C-1II),108.0(C-1V)107.9,107.9(C-1III,C-1IV),100.8(C-1I),83.6(C-4V),82.8(C-4II),82.5(2C,C-4III,C-4IV),82.3,82.3,82.2(C-2III,C-2IV,C-2V),82.0(C-2II),77.7(C-3II),77.5(2C,C-3III,C-3IV),77.3(C-3V),77.0(C-5II),76.6,76.5(C-5III,C-5IV),72.7(C-5I),71.5(C-3I),71.5(C-5V),70.9(C-2I),67.8(C-6I),67.6(C-4I),66.0(OCH2),63.8(C-6V),62.1(2C,C-6III,C-6IV),61.9(C-6II),38.5(OCH 2CH 2CH 2N),27.6(OCH 2CH 2CH 2N)。HRMS(ESI):Calcd m/z for[M+H]+C 33H 59NO 26 886.3398,found 886.3396.Calcd m/z for[M+Na]+C 33H 59NO 26 908.3218,found 908.3209。
(7)将步骤(6)所得寡糖片段1(8毫克)溶解于DMF(1毫升)和0.1M磷酸盐缓冲液(PBS，0.25毫升)，搅拌下向混合液中加入双琥珀酰亚胺戊二酸酯(5毫克)，反应液室温搅拌过夜，高真空度减压蒸馏除去溶剂，得到白色固体，用乙酸乙酯搅洗固体，过滤干燥，得到活化的寡糖片段。按照25:1的摩尔比取活化的寡糖片段和KLH蛋白溶解于0.1M磷酸盐缓冲液(PBS，1毫升)，反应液搅拌过夜，用透析法纯化糖蛋白，水溶液冻干，得到白色粉末状糖蛋白(1-5)-Gal-(1-6)-Man-7碳Linker-KLH糖蛋白。
对比例1(1-5)-Gal-(1-6)-Man-3碳Linker-KLH糖蛋白的制备
按照实施例1的方法和原料比例制备寡糖片段和糖蛋白，不同在于，化合物6的结构式替换为：
Figure PCTCN2019115506-appb-000010
实施例2(1-5)-Gal-(1-6)-Man-7碳Linker-BSA糖蛋白的制备
将寡糖片段1(8毫克)溶解于DMF(1毫升)和0.1M磷酸盐缓冲液(PBS，0.25毫升)，搅拌下向混合液中加入双琥珀酰亚胺戊二酸酯(5毫克)，反应液室温搅拌过夜，高真空度减压蒸馏除去溶剂，得到白色固体，用乙酸乙酯搅洗固体，过滤干燥，得到活化的寡糖片段。按照25:1的摩尔比取活化的寡糖片段和BSA溶解于0.1M磷酸盐缓冲液(PBS，1毫升)，反应液搅拌过夜，用透析法纯化糖蛋白，水溶液冻干，得到白色粉末状(1-5)-Gal-(1-6)-Man-7碳Linker-BSA糖蛋白。
对比例2(1-5)-Gal-(1-6)-Man-3碳Linker-BSA糖蛋白的制备
按照实施例2一样的方法和原料比例制备得到(1-5)-Gal-(1-6)-Man-3碳Linker-BSA糖蛋白。
实施例3(1-5)-Gal-(1-6)-Man-Linker-KLH糖蛋白糖含量的测定
分别取1毫克实施例1获得的(1-5)-Galla-(1-6)-Man-7碳-KLH和对比例1获得的(1-5)-Gla-(1-6)-Man-3碳-KLH，用通用硫酸-苯酚法(苯酚-硫酸比色法测定多糖含量，食品与药品，2004(6):7)测定糖蛋白的含糖量分别为5.2％和4.5％，对比表明7碳链(1-5)-Gal-(1-6)-Man-Linker-KLH糖蛋白载糖量高于3碳链(1-5)-Gal-(1-6)-Man-Linker-KLH糖蛋白。更高的糖含量会有更强的免疫原性，其抗菌效果更好。
实施例4不同糖蛋白免疫原性
将实施例1和对比例1制备的(1-5)-Gal-(1-6)-Man-7碳Linker-KLH和(1-5)-Gal-(1-6)-Man-3碳Linker-KLH在小鼠(C57/BL，七周大，每组10只)体内进行免疫实验。对实验小鼠进行皮下注射方式免疫，每只小鼠每次2μg寡糖注射量，分别在第1、14、21和28天进行注射免疫。分别在免疫前1天和最后一次免疫后第2天取血制备抗体血清，分别以相应寡糖的BSA糖蛋白(1-5)-Gal-(1-6)-Man-7碳Linker-BSA和(1-5)-Gal-(1-6)-Man-3碳Linker-BSA作为固定抗原，以酶联免疫法ELISA检测特异性抗体滴度，结果见图3、图4。由图3和图4可知，经过(1-5)-Gal-(1-6)-Man-Linker-KLH糖蛋白免疫后，小鼠血液中的抗体滴度明显增高，IgG抗体的滴度在总抗中占有较大比例，说明化合物诱导产生的免疫响应主要是IgG型。IgG型抗体属于T细胞参与的免疫响应，该响应能够使宿主细胞产生免疫记忆，促进抗体产生起到抗真菌感染的作用。图3和图4数据对比表明(1-5)-Gal-(1-6)-Man-7C-KLH比3碳链类似物(1-5)-Gal-(1-6)-Man-3C-KLH有更好的抗体免疫活性。该动物免疫实验说明(1-5)-Gal-(1-6)-Man-7C-KLH糖蛋白是一种可以被继续开发为抗真菌感染的疫苗。

Claims (6)

  1. 一种糖蛋白，其结构为：
    Figure PCTCN2019115506-appb-100001
  2. 根据权利要求1所述的糖蛋白，其特征在于，所述免疫原性载体蛋白为非人源蛋白；选自KLH，BSA，OVA，Blue carrier TM，白喉毒素无毒变异体，破伤风毒素/类毒素，分离自非获得型流感嗜血杆菌的高分子量蛋白，脱毒后的绿脓杆菌毒素A，霍乱毒素/类毒素，百日咳毒素/类毒素，产气荚膜梭状芽胞杆菌外毒素/类毒素，乙肝表面抗原，乙肝核心抗原，轮状病毒VP7蛋白，白喉毒素突变体CRM、CRM191、CRM3201，呼吸合胞病毒F和G蛋白中的至少一种。
  3. 根据权利要求1所述的糖蛋白，其特征在于，所述免疫原性载体蛋白为KLH或BSA。
  4. 一种如权利要求1-3任一所述的糖蛋白的制备方法，其特征在于，包括以下步骤：
    (1)氮气保护、分子筛存在下，TMSOTf催化化合物2和化合物3在二氯甲烷中反应，终止反应后分离纯化得化合物4：
    Figure PCTCN2019115506-appb-100002
    (2)化合物4溶解于甲醇:二氯甲烷5:1混合溶剂中在PdCl 2存在下于室温中反应，过滤后滤液用碱中和，分离获得油状物；油状物溶解于二氯甲烷，加入碳酸铯和CF 3C(NPh)Cl，TLC跟踪反应，室温搅拌至反应完全，体系浓缩后用硅胶柱分离得到化合物5：
    Figure PCTCN2019115506-appb-100003
    (3)氮气保护、分子筛存在下，TMSOTf催化化合物5和化合物6于二氯甲烷中反应，终止反应后分离纯化得化合物7：
    Figure PCTCN2019115506-appb-100004
    (4)化合物7与吡咯烷在二氯甲烷中反应，再用稀酸洗涤，获得的有机相分离纯化，得化合物8：
    Figure PCTCN2019115506-appb-100005
    (5)氮气保护、分子筛存在下，TMSOTf催化化合物5和化合物8于二氯甲烷中反应，终止反应后分离纯化得化合物9：
    Figure PCTCN2019115506-appb-100006
    (6)隔氧条件下，化合物9的甲醇溶液加入10％钯碳，以氢气加氢，在氮气保护下过滤，滤液中加入甲醇钠的甲醇溶液反应后再加入水反应，分离纯化后得到寡糖片段：
    Figure PCTCN2019115506-appb-100007
    (7)将免疫原性载体蛋白、寡糖片段和双琥珀酰亚胺戊二酸酯溶于偶联液，孵育，然后分离纯化、纯化获得糖蛋白：
    Figure PCTCN2019115506-appb-100008
  5. 一种如权利要求1-3任一所述的糖蛋白在制备抗真菌感染药物中的应用。
  6. 一种包含如权利要求1-3任一所述的糖蛋白的药物。
PCT/CN2019/115506 2019-11-04 2019-11-05 一种(1-5)-Gal-(1-6)-Man糖蛋白及其制备方法和应用 WO2021087709A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911065356.2 2019-11-04
CN201911065356.2A CN110724189B (zh) 2019-11-04 2019-11-04 一种(1-5)-Gal-(1-6)-Man糖蛋白及其制备方法和应用

Publications (1)

Publication Number Publication Date
WO2021087709A1 true WO2021087709A1 (zh) 2021-05-14

Family

ID=69223708

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/115506 WO2021087709A1 (zh) 2019-11-04 2019-11-05 一种(1-5)-Gal-(1-6)-Man糖蛋白及其制备方法和应用

Country Status (2)

Country Link
CN (1) CN110724189B (zh)
WO (1) WO2021087709A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113861246B (zh) * 2021-11-02 2024-01-05 中山大学 一种β -D-***呋喃糖苷键的立体选择性合成方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104004085A (zh) * 2014-05-28 2014-08-27 山东大学 一种结核杆菌lam寡糖缀合物及其制备方法与应用
CN106390113A (zh) * 2016-08-31 2017-02-15 天津科技大学 一种结核杆菌PGL‑tb1寡糖缀合物及其制备方法与应用
CN106432371A (zh) * 2016-10-31 2017-02-22 中国人民解放军第二军医大学 β‑1,2‑D‑寡聚甘露糖蛋白缀合物及其制备方法和应用
CN107224577A (zh) * 2017-05-10 2017-10-03 山东大学 一种基于3型肺炎链球菌荚膜多糖的寡糖缀合物及其制备方法与应用
CN108864277A (zh) * 2017-05-12 2018-11-23 北京大学 卡他莫拉菌los核心寡糖缀合物及其制备方法与应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8846411B2 (en) * 2009-06-11 2014-09-30 Microgenics Corporation Derivatives, reagents, and immunoassay for detecting levetiracetam
CN106084037B (zh) * 2016-06-08 2020-01-07 中国人民解放军第二军医大学 一种炭疽杆菌荚膜表面三糖缀合物及其制备方法和应用
CN107184972B (zh) * 2017-06-20 2020-12-01 康希诺生物股份公司 一种结核杆菌OS-tb寡糖缀合物及其制备方法与应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104004085A (zh) * 2014-05-28 2014-08-27 山东大学 一种结核杆菌lam寡糖缀合物及其制备方法与应用
CN106390113A (zh) * 2016-08-31 2017-02-15 天津科技大学 一种结核杆菌PGL‑tb1寡糖缀合物及其制备方法与应用
CN106432371A (zh) * 2016-10-31 2017-02-22 中国人民解放军第二军医大学 β‑1,2‑D‑寡聚甘露糖蛋白缀合物及其制备方法和应用
CN107224577A (zh) * 2017-05-10 2017-10-03 山东大学 一种基于3型肺炎链球菌荚膜多糖的寡糖缀合物及其制备方法与应用
CN108864277A (zh) * 2017-05-12 2018-11-23 北京大学 卡他莫拉菌los核心寡糖缀合物及其制备方法与应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ARGUNOV DMITRY A., KRYLOV VADIM B., NIFANTIEV NIKOLAY E.: "The Use of Pyranoside-into-Furanoside Rearrangement and Controlled O(5) → O(6) Benzoyl Migration as the Basis of a Synthetic Strategy To Assemble (1→5)- and (1→6)-Linked Galactofuranosyl Chains", ORGANIC LETTERS ., AMERICAN CHEMICAL SOCIETY, US, vol. 18, no. 21, 4 November 2016 (2016-11-04), US, pages 5504 - 5507, XP055810483, ISSN: 1523-7060, DOI: 10.1021/acs.orglett.6b02735 *

Also Published As

Publication number Publication date
CN110724189A (zh) 2020-01-24
CN110724189B (zh) 2022-01-25

Similar Documents

Publication Publication Date Title
AU616670B2 (en) Saponin adjuvant
JP3717511B2 (ja) サポニン−抗原複合物とその用途
US5057540A (en) Saponin adjuvant
JP6524218B2 (ja) 肺炎連鎖球菌血清型８に対するワクチン
US20180346503A1 (en) Baicalin magnesium compound and its preparation method and application
US9815889B2 (en) Antibodies for prevention and treatment of diseases caused by clostridium difficile
KR102250099B1 (ko) 스트렙토코커스 뉴모니에 1형에 대한 합성 백신
US9238669B2 (en) Oligosaccharides and oligosaccharide-protein conjugates derived from Clostridium difficile polysaccharide PS-I, methods of synthesis and uses thereof, in particular as vaccines and diagnostic tools
JPH01157995A (ja) 鎮痛−抗炎症活性を有するガングリオシドの内部エステル
CN107224577B (zh) 一种基于3型肺炎链球菌荚膜多糖的寡糖缀合物及其制备方法与应用
CN106390113B (zh) 一种结核杆菌PGL-tb1寡糖缀合物及其制备方法与应用
WO2021087709A1 (zh) 一种(1-5)-Gal-(1-6)-Man糖蛋白及其制备方法和应用
CN101014698A (zh) 作为多种疫苗候选物的保守性内核脂多糖表位
AU651319B2 (en) Lipid A analog/immunogenic carrier conjugates and the use thereof as vaccines
JP2018537428A (ja) カンピロバクター・ジェジュニに対する合成抗原コンストラクト
CN111467368B (zh) 含庚糖链的寡糖化合物在制备幽门螺旋杆菌疫苗中的应用
WO2021169100A1 (zh) 三糖重复单元寡糖链在制备金黄色葡萄球菌疫苗中的应用
CN106084037B (zh) 一种炭疽杆菌荚膜表面三糖缀合物及其制备方法和应用
WO2016104647A1 (ja) 糖鎖結合ワクチン抗原及び糖鎖導入剤
US20050153057A1 (en) Haemophilus influenzae lipopolysaccharide inner-core oligosaccharide epitopes as vaccines for the prevention of haemophilus influenzae infections
CN113663065B (zh) 一种肌醇甘露寡糖缀合物、制备方法及作为抗结核糖疫苗的应用
JPH09104693A (ja) グリコシド類、グリコシドの無糖分解産物およびその誘導体
EP0575517A1 (en) Lipid a analog/immunogenic carrier conjugates, the use thereof as vaccines and polyvalent hyperimmune gammaglobulins
CN118420796A (zh) 空肠弯曲杆菌cg8486荚膜寡糖及其糖蛋白缀合物的合成方法和应用
EP3000820A1 (en) Synthetic vaccines against Streptococcus pneumoniae serotype 8

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19951499

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19951499

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 03/11/2022)

122 Ep: pct application non-entry in european phase

Ref document number: 19951499

Country of ref document: EP

Kind code of ref document: A1