技术领域Technical Field
本发明涉及药物化学和药理学技术领域，具体涉及一种天然Toll-like Receptor2激动剂CaLGL-1的合成方法。The invention relates to the technical field of medicinal chemistry and pharmacology, and in particular to a method for synthesizing a natural Toll-like Receptor 2 agonist CaLGL-1.
背景技术Background technique
固有免疫反应是人体内的第一道防线,对宿主有效抵抗外来病原微生物的入侵至关重要。模式识别受体(pattern recognition receptors,PRRs)通过检测病原体相关分子模式(pathogen-associated molecular patterns,PAMPs)和损伤相关分子模式(damage-associated molecular patterns,DAMPs),启动下游对I型干扰素(type I interferons,IFNs)和促炎细胞因子反应。Toll样受体(TLRs)、C型凝集素受体(CLRs)、视黄酸诱导基因(RIG)-I样受体(RLRs)和NOD样受体(NLRs)是已知的PRR家族。不同的PAMPs对特定的微生物具有特异性。脂多糖(来自革兰氏阴性菌)、肽聚糖和脂质胆酸(来自革兰氏阳性菌)、鞭毛蛋白和脂蛋白(存在于革兰氏阳性菌和革兰氏阴性菌中)、酵母菌(来自酵母)、DNA和RNA核酸(来自细菌和病毒)均是一些最广为人知的PAMPs。除部分NLRs外，其他PRRs均检测PAMPs信号并诱导炎症基因转录，编码促炎细胞因子、I型干扰素(IFN)、趋化因子以及抗菌蛋白等。The innate immune response is the first line of defense in the human body and is essential for the host to effectively resist the invasion of foreign pathogenic microorganisms. Pattern recognition receptors (PRRs) initiate downstream responses to type I interferons (IFNs) and proinflammatory cytokines by detecting pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Toll-like receptors (TLRs), C-type lectin receptors (CLRs), retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) and NOD-like receptors (NLRs) are known PRR families. Different PAMPs are specific to specific microorganisms. Lipopolysaccharides (from Gram-negative bacteria), peptidoglycans and lipid bile acids (from Gram-positive bacteria), flagellins and lipoproteins (present in Gram-positive and Gram-negative bacteria), yeasts (from yeast), DNA and RNA nucleic acids (from bacteria and viruses) are some of the most well-known PAMPs. Except for some NLRs, other PRRs detect PAMPs signals and induce the transcription of inflammatory genes, encoding proinflammatory cytokines, type I interferons (IFNs), chemokines, and antimicrobial proteins.
在所有PRR家族中，Toll样受体(TLRs)是最具特征的，在先天免疫中起关键作用。在各种TLR中，TLR2具有特殊的地位，由于其与TLR1或TLR6的异源二聚化，使其能够感知最广泛的PAMPs，从而扩大了其抗病原体的配体多样性。总的来说，在过去的20年里，各种骨架被报道可以激活TLR2，其中包括天然存在的脂蛋白、合成的脂肽和几种杂环小分子，显示了该受体在各种疾病以及疫苗佐剂开发中的重要性。因此，开发新型TLR-2激动剂备受关注。Among all PRR families, Toll-like receptors (TLRs) are the best characterized and play a key role in innate immunity. Among the various TLRs, TLR2 has a special status, as its heterodimerization with TLR1 or TLR6 enables it to sense the widest range of PAMPs, thus expanding its ligand diversity against pathogens. In general, over the past 20 years, various scaffolds have been reported to activate TLR2, including naturally occurring lipoproteins, synthetic lipopeptides, and several heterocyclic small molecules, showing the importance of this receptor in various diseases as well as in the development of vaccine adjuvants. Therefore, the development of novel TLR-2 agonists has attracted much attention.
天然存在和合成的TLR激动剂可以利用这些内源性免疫信号通路来增强和调节疫苗反应，从而使其成为优良的疫苗佐剂。这不仅对开发针对传染病的疫苗具有重要意义，而且对针对癌症、过敏、阿尔茨海默病和其他疾病的免疫疗法也具有重要意义。每种TLR都有自己的特定组织定位和下游基因信号通路，TLR激动剂可以与其他TLR或替代佐剂组合，以产生具有协同或调节作用的组合佐剂，这为研究人员提供了精确定制具有特定免疫效果的佐剂的机会。Naturally occurring and synthetic TLR agonists can exploit these endogenous immune signaling pathways to enhance and modulate vaccine responses, making them excellent vaccine adjuvants. This is not only important for the development of vaccines against infectious diseases, but also for immunotherapy against cancer, allergies, Alzheimer's disease and other diseases. Each TLR has its own specific tissue localization and downstream gene signaling pathways, and TLR agonists can be combined with other TLRs or alternative adjuvants to produce combination adjuvants with synergistic or modulatory effects, which provides researchers with the opportunity to precisely customize adjuvants with specific immune effects.
人类肠道微生物群的一些成员深刻地影响宿主的生理、健康和治疗反应，但负责的分子和机制在很大程度上是未知的。作为鉴定肠道微生物产生的免疫调节剂项目的一部分。2023年，Jon Clardy课题组在许多关联研究中分析了占据重要地位的放线菌(Collinsella aerofaciens)的代谢组分。利用树突状细胞(mBMDCs)和细胞因子数据进行表型分析指导筛选，鉴定出活性化合物CaLGL-1，其结构特征为半乳糖甘油脂，头部有一个缩醛的β-半乳糖呋喃糖基团。最后，通过全合成进行结构归属，并用tlr2-/-、tlr4-/-和野生型的mBMDCs的细胞检测发现了TLR-2的依赖性信号(EC50＝3.2uM)。该课题组对其进行合成时，发现在脱除硅基保护基时伴随着酰基1，2-迁移，造成体系复杂，给分离提纯带来了困难；同时，由于糖苷键耐酸性弱，目标产物的的缩醛基团很难合成，只能在苛刻的条件下以1％的产率得到目标产物，这些都极大的限制了CalGL-1后续的应用研究。Some members of the human gut microbiota profoundly influence host physiology, health, and therapeutic responses, but the responsible molecules and mechanisms are largely unknown. As part of a project to identify immunomodulators produced by gut microbes. In 2023, Jon Clardy's group analyzed the metabolites of Collinsella aerofaciens, which plays an important role in many association studies. Phenotypic analysis guided screening using dendritic cells (mBMDCs) and cytokine data identified the active compound CaLGL-1, which is structurally characterized as a galactosylglycerol lipid with an acetal β-galactofuranose group on the head. Finally, the structure was assigned by total synthesis, and TLR-2-dependent signals (EC50 = 3.2uM) were found using cell assays of tlr2-/-, tlr4-/-, and wild-type mBMDCs. When the research group synthesized it, they found that the removal of the silicon-based protecting group was accompanied by 1,2-migration of the acyl group, which made the system complicated and brought difficulties to separation and purification; at the same time, due to the weak acid resistance of the glycosidic bond, the acetal group of the target product was difficult to synthesize, and the target product could only be obtained with a yield of 1% under harsh conditions. All these greatly limited the subsequent application research of CalGL-1.
CaLGL-1的结构式The structural formula of CaLGL-1
本发明的目的是解决一种天然TLR2激动剂CaLGL-1的合成方法，为后续的TLR-2激动剂在疫苗佐剂、癌症免疫疗法等相关的药物研究提供技术路线和物质基础。The purpose of the present invention is to solve a method for synthesizing a natural TLR2 agonist CaLGL-1, and to provide a technical route and material basis for subsequent drug research on TLR-2 agonists in vaccine adjuvants, cancer immunotherapy, etc.
发明内容Summary of the invention
本发明所述的一种天然的TLR2激动剂CaLGL-1，其结构式如下(I)：The natural TLR2 agonist CaLGL-1 described in the present invention has the following structural formula (I):
本发明的目的在于提供一种天然TLR2激动剂CaLGL-1的合成，其技术路线如下：The purpose of the present invention is to provide a synthesis of a natural TLR2 agonist CaLGL-1, and the technical route is as follows:
所述一种新型TLR2激动剂的设计合成方法，技术路线其特征在于，包括以下步骤：The design and synthesis method of the novel TLR2 agonist is characterized by comprising the following steps:
将商业获得的D-半乳糖在无水的N,N-二甲基甲酰胺做溶剂，咪唑做碱，室温下经过TBS保护得到产物(II)。Commercially obtained D-galactose was treated with TBS protection in anhydrous N,N-dimethylformamide as solvent and imidazole as base at room temperature to obtain product (II).
将[0011]中所得(II)在无水的二氯甲烷作溶剂，N-乙基二异丙胺做碱，(S)-(2,2-二甲基-1，3-二氧戊环-4-基)甲醇和活化的4A分子筛为添加物，氩气氛围，零下六十度加料，室温反应得到产物(III)。The obtained compound (II) in [0011] was reacted in anhydrous dichloromethane as solvent, N-ethyldiisopropylamine as base, (S)-(2,2-dimethyl-1,3-dioxolan-4-yl)methanol and activated 4A molecular sieve as additives, under argon atmosphere, at -60 degrees Celsius, and at room temperature to obtain the product (III).
将[0012]中所得(III)在三氟乙酸：水：二氯甲烷＝2.5：2.5：95的比例中，从零度到室温反应30分钟，便可以得到大规模产物(IV)。The compound (III) obtained in [0012] is reacted in a mixture of trifluoroacetic acid: water: dichloromethane = 2.5:2.5:95 at a temperature from zero to room temperature for 30 minutes to obtain a large-scale product (IV).
将[0013]中所得(IV)在甲苯做溶剂，1-(3-二甲氨基丙基)-3-乙基碳二亚胺作为缩合剂，对应的酸作为反应原料，催化量的4-二甲氨基吡啶为添加物，室温反应12小时，得到大规模的产物(V)。The obtained compound (IV) in [0013] was reacted in toluene as solvent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as condensation agent, the corresponding acid as reaction raw material, and a catalytic amount of 4-dimethylaminopyridine as additive at room temperature for 12 hours to obtain a large-scale product (V).
将[0014]中所得(V)在无水的四氢呋喃做溶剂，零度条件下将氟化氢吡啶缓慢滴加其中，然后室温反应2小时，用薄层色谱检测反应完全后，将其放置冰浴中，缓慢滴加饱和碳酸氢钠溶液淬灭反应，然后，用乙酸乙酯萃取三次，无水硫酸钠干燥有机相，将有机相减压浓缩，最后柱层析得到大规模产物(VI)。The obtained (V) in [0014] is used as a solvent in anhydrous tetrahydrofuran, and hydrofluoride pyridine is slowly added dropwise thereto under zero degree condition, and then reacted at room temperature for 2 hours. After the reaction is completed by thin layer chromatography, it is placed in an ice bath, and a saturated sodium bicarbonate solution is slowly added dropwise to quench the reaction. Then, it is extracted three times with ethyl acetate, the organic phase is dried over anhydrous sodium sulfate, and the organic phase is concentrated under reduced pressure, and finally column chromatography is performed to obtain a large-scale product (VI).
将[0015]中所得(VI)在无水的二氯甲烷作溶剂，用对应的对甲苯硫酚活化的缩醛为反应原料，N-碘代丁二酰亚胺为氧化剂，在氩气氛围，-30摄氏度反应3小时，薄层色谱检测反应完全后，将其移置冰浴中，缓慢滴加10％硫代硫酸钠淬灭反应，用二氯甲烷萃取，无水硫酸钠干燥有机相，减压浓缩有机相，最后柱层析得到大规模产物(VII)。The compound (VI) obtained in [0015] was treated in anhydrous dichloromethane as solvent, the corresponding acetal activated by p-toluene thiophenol as reaction raw material, and N-iodosuccinimide as oxidant, under argon atmosphere at -30 degrees Celsius for 3 hours. After the reaction was complete as detected by thin layer chromatography, it was placed in an ice bath, 10% sodium thiosulfate was slowly added dropwise to quench the reaction, and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure. Finally, the large-scale product (VII) was obtained by column chromatography.
将[0015]中所得(VI)在无水的二氯甲烷作溶剂，用对应的对甲苯硫酚活化的缩醛为反应原料，N-溴代丁二酰亚胺为氧化剂，在氩气氛围，-20～室温摄氏度反应3小时，薄层色谱检测反应完全后，将其移置冰浴中，缓慢滴加10％硫代硫酸钠淬灭反应，用二氯甲烷萃取，无水硫酸钠干燥有机相，减压浓缩有机相，最后柱层析得到大规模产物(VII)。The compound (VI) obtained in [0015] is treated in anhydrous dichloromethane as solvent, the corresponding acetal activated by p-toluene thiophenol as reaction raw material, and N-bromosuccinimide as oxidant, and reacted at -20 to room temperature for 3 hours in an argon atmosphere. After the reaction is complete as detected by thin layer chromatography, it is placed in an ice bath, 10% sodium thiosulfate is slowly added dropwise to quench the reaction, and extracted with dichloromethane. The organic phase is dried over anhydrous sodium sulfate, and the organic phase is concentrated under reduced pressure. Finally, the large-scale product (VII) is obtained by column chromatography.
将[0015]中所得(VI)在无水的二氯甲烷作溶剂，用对应的对甲苯硫酚活化的缩醛为反应原料，二溴海因为氧化剂，在氩气氛围，-20～室温摄氏度反应3小时，薄层色谱检测反应完全后，将其移置冰浴中，缓慢滴加10％硫代硫酸钠淬灭反应，用二氯甲烷萃取，无水硫酸钠干燥有机相，减压浓缩有机相，最后柱层析得到大规模产物(VII)。The compound (VI) obtained in [0015] was treated with anhydrous dichloromethane as solvent, the corresponding acetal activated by p-toluene thiophenol as reaction material, and dibromohydroxyin as oxidant, and reacted at -20 to room temperature for 3 hours in an argon atmosphere. After the reaction was complete as detected by thin layer chromatography, it was transferred to an ice bath, and 10% sodium thiosulfate was slowly added dropwise to quench the reaction. The compound was extracted with dichloromethane, the organic phase was dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure. Finally, the large-scale product (VII) was obtained by column chromatography.
本发明与现有技术相比具有以下优点：Compared with the prior art, the present invention has the following advantages:
本发明具有操作安全、环境友好、反应条件温和和收率高的优点。The invention has the advantages of safe operation, environmental friendliness, mild reaction conditions and high yield.
本发明所用原料和添加物廉价易得，环保压力小。The raw materials and additives used in the invention are cheap and readily available, and have little environmental pressure.
本发明符合绿色合成的要求，具有较大的实际应用价值。The invention meets the requirements of green synthesis and has great practical application value.
下面通过实施例对本发明技术方案做进一步的详细说明The technical solution of the present invention is further described in detail below through embodiments.
具体实施方式Detailed ways
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现对本发明的技术方案进行以下具体实施例的说明，是对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明的可实施范围的限定。凡基于本发明上述内容所实现的技术均属于本发明的范围。In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in the following specific embodiments, which further describes the above content of the present invention in detail. However, this should not be understood as a limitation on the scope of implementation of the present invention. All technologies realized based on the above content of the present invention belong to the scope of the present invention.
下述实施例中所述的实验方法，如果无特殊说明，均为常规方法；所述试剂和材料，如无特殊说明，均可从商业途径获得。The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.
下述实施例中所述的实验方法，所用溶剂和药品均为分析纯或化学纯；无水溶剂按照标准方法处理；柱层析和硅胶板皆为常规型号。所用萃取得到的有机相干燥剂未经说明，均用无水硫酸钠。LC-MS用Agilent公司1260高效液相-离子阱质谱(ESI源)，二极管阵列检测器(DAD)，检测波长210nm和254nm。系统型号如果无特殊说明，均为常规方法；所述试剂和材料，如无特殊说明，均可从商业途径获得。The experimental methods described in the following examples are all analytically pure or chemically pure solvents and drugs; anhydrous solvents are treated according to standard methods; column chromatography and silica gel plates are all conventional models. The organic phase drying agent obtained by extraction is not specified, and anhydrous sodium sulfate is used. LC-MS uses Agilent's 1260 high-performance liquid phase-ion trap mass spectrometer (ESI source), diode array detector (DAD), and detection wavelengths of 210nm and 254nm. If there is no special description of the system model, it is a conventional method; the reagents and materials, if not otherwise specified, can be obtained from commercial channels.
所述一种天然Toll-like Receptor 2激动剂CaLGL-1的合成方法，具体实施方式如下：The synthesis method of the natural Toll-like Receptor 2 agonist CaLGL-1 is specifically implemented as follows:
第一步：first step:
将商业可得的D-半乳糖(I)(30mmol)加入到带有磁子的250mL圆底烧瓶中并向其体系加入无水的N,N-二甲基甲酰胺(DMF，150mL)且将其放置于0℃，接着将咪唑(Imidazole,6.6当量)和叔丁基二甲基氯硅烷(TBSCl,7.25当量)分批缓慢加入该体系中。然后，移置室温反应48小时，用薄层色谱检测反应完全后，将反应体系分批缓慢倒入冰水中，然后用二氯甲烷(100mL×3)萃取水相合并有机相，再用5％盐酸、饱和碳酸氢钠、饱和食盐水依次洗涤有机相，无水硫酸钠干燥有机相，减压浓缩有机相，然后向其中加入无水甲醇(20mL)，析出白色固体，过滤以80％的产率得到产物(II)。1H NMR(400MHz,Chloroform-d)δ5.15(d,J＝2.4Hz,1H),4.09(t,J＝3.8Hz,1H),4.00(t,J＝4.1Hz,1H),3.92(t,J＝2.8Hz,1H),3.78–3.70(m,1H),3.67(dd,J＝9.9,6.3Hz,1H),3.55(dd,J＝9.8,5.6Hz,1H),0.88(m,45H),0.14–0.02(m,30H).[M+Na]+＝773.7.Commercially available D-galactose (I) (30 mmol) was added to a 250 mL round-bottom flask with a magnetic rod, and anhydrous N,N-dimethylformamide (DMF, 150 mL) was added to the system and placed at 0°C, and then imidazole (Imidazole, 6.6 equivalents) and tert-butyldimethylsilyl chloride (TBSCl, 7.25 equivalents) were slowly added to the system in batches. Then, the reaction was moved to room temperature for 48 hours. After the reaction was complete by thin layer chromatography, the reaction system was slowly poured into ice water in batches, and then the aqueous phase was extracted with dichloromethane (100 mL×3) and the organic phase was combined. The organic phase was then washed with 5% hydrochloric acid, saturated sodium bicarbonate, and saturated brine in sequence, and the organic phase was dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure, and then anhydrous methanol (20 mL) was added thereto, and a white solid was precipitated, which was filtered to obtain the product (II) with a yield of 80%.1 H NMR (400 MHz, Chloroform-d) δ5.15 (d, J = 2.4 Hz, 1H), 4.09 (t, J = 3.8 Hz, 1H), 4.00 (t, J = 4.1 Hz, 1H), 3.92 (t, J = 2.8 Hz, 1H), 3.78–3.70 (m, 1H), 3.67 (dd, J = 9.9, 6.3 Hz, 1H), 3.55 (dd, J = 9.8, 5.6 Hz, 1H), 0.88 (m, 45H), 0.14–0.02 (m, 30H). [M+Na]+ ＝773.7.
第二步：Step 2:
将[0028]所得产物(II)(10mmol)和活化的4A分子筛加入到带有磁子的250mL圆底烧瓶中且用氩气保护，再向其中加入无水的二氯甲烷(DCM,100mL),室温下搅拌30分钟,然后将其放置于-40℃，向其中加入三甲基碘硅烷(TMSI,1.2当量),反应30分钟后，分别向其中缓慢加入N-乙基二异丙胺(DIPEA,2.0当量)，(S)-(2,2-二甲基-1，3-二氧戊环-4-基)甲醇((S)-(+)-1,2-Isopropylideneglycerol,1.2当量),然后室温反应2小时，用薄层色谱检测反应完全后，滤掉分子筛所得液体，用饱和碳酸氢钠、饱和食盐水洗涤有机相，无水硫酸钠干燥有机相，减压浓缩有机相，经柱层析分离纯化以74％的产率得到产物(III)。1H NMR(400MHz,Chloroform-d)δ4.81(d,J＝2.2Hz,1H),4.28(m,1H)4.11(dd,J＝5.8,3.3Hz,1H),4.05(m,1H),4.01(m,1H),3.93(dd,J＝5.8,3.0Hz,1H),3.81(dd,J＝8.3,6.2Hz,1H),3.77(dd,J＝10.0Hz,4.7Hz,1H),3.75(m,1H),3.65(dd,J＝9.9,6.7Hz,1H),3.57(dd,J＝9.8,5.8Hz,1H),3.41(dd,J＝10.2,6.7Hz,1H),1.39(s,3H),1.34(s,3H),0.87(m,36H),0.10–0.03(m,24H).[M+Na]+＝773.6.The obtained product (II) (10 mmol) and activated 4A molecular sieves were added to a 250 mL round-bottom flask with a magnetic rod and protected by argon. Anhydrous dichloromethane (DCM, 100 mL) was added thereto. The mixture was stirred at room temperature for 30 minutes. The mixture was then placed at -40°C. Trimethylsilyl iodide (TMSI, 1.2 equivalents) was added thereto. After reacting for 30 minutes, N-ethyldiisopropylamine (DIPEA, 2.0 equivalents) and (S)-(2,2-dimethyl-1,3-dioxolan-4-yl)methanol ((S)-(+)-1,2-Isopropylideneglycerol, 1.2 equivalents) were slowly added thereto respectively. The mixture was reacted at room temperature for 2 hours. After the reaction was complete as detected by thin layer chromatography, the liquid obtained from the molecular sieve was filtered off. The organic phase was washed with saturated sodium bicarbonate and saturated brine. The organic phase was dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure. The product (III) was separated and purified by column chromatography to obtain a yield of 74%.1 H NMR (400 MHz, Chloroform-d) δ 4.81 (d, J = 2.2 Hz, 1H), 4.28 (m, 1H) 4.11 (dd, J = 5.8, 3.3 Hz, 1H), 4.05 (m, 1H), 4.01 (m, 1H), 3.93 (dd, J = 5.8, 3.0 Hz, 1H), 3.81 (dd, J = 8.3, 6.2 Hz, 1H), 3.77 (dd, J = 10.0Hz,4.7Hz,1H),3.75(m,1H),3.65(dd,J＝9.9,6.7Hz,1H),3.57(dd,J＝9.8,5.8Hz,1H),3.41(dd,J＝10.2,6.7Hz,1H),1.39(s,3H),1.34(s,3H),0.87(m,36H),0.10–0.03(m,24H).[M+Na]+ ＝773.6.
第三步：third step:
将[0029]所得产物(III)(8.5mmol)加入到带有磁子的500mL圆底烧瓶中向其中加入400mL二氯甲烷(DCM),然后移置冰浴中，再向其中加入三氟乙酸(TFA,10.6mL),水(H2O,10.6mL),将其移置室温反应30分钟，用薄层色谱检测反应完全后，将其放置冰浴中，缓慢滴加饱和碳酸氢钠溶液淬灭反应，然后，用二氯甲烷萃取三次，无水硫酸钠干燥有机相，将有机相减压浓缩，柱层析以83％的产率得到产物(IV)。1H NMR(600MHz,Chloroform-d)δ4.83(d,J＝2.3Hz,1H),4.12(dd,J＝4.7,3.0Hz,1H),4.02–3.96(m,2H),3.82(q,J＝4.6Hz,1H),3.74(td,J＝6.1,3.9Hz,1H),3.67(d,J＝4.8Hz,2H),3.63(dd,J＝10.2,6.6Hz,1H),3.58(m,2H),3.33(d,J＝4.3Hz,1H),2.41(t,J＝6.1Hz,1H),0.90–0.85(m,36H),0.12–0.03(m,24H).[M+Na]+＝733.6.The product (III) (8.5 mmol) obtained in [0029] was added to a 500 mL round-bottom flask with a magnetic rod, 400 mL of dichloromethane (DCM) was added thereto, and then placed in an ice bath. Trifluoroacetic acid (TFA, 10.6 mL) and water (H2 O, 10.6 mL) were added thereto, and the mixture was placed at room temperature to react for 30 minutes. After the reaction was completed as detected by thin layer chromatography, the mixture was placed in an ice bath, and a saturated sodium bicarbonate solution was slowly added dropwise to quench the reaction. Then, the mixture was extracted three times with dichloromethane, the organic phase was dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure. The product (IV) was obtained by column chromatography with a yield of 83%.1 H NMR (600 MHz, Chloroform-d) δ 4.83 (d, J = 2.3 Hz, 1H), 4.12 (dd, J = 4.7, 3.0 Hz, 1H), 4.02–3.96 (m, 2H), 3.82 (q, J = 4.6 Hz, 1H), 3.74 (td, J = 6.1, 3.9 Hz, 1H), 3.67 (d, J = 4.8 Hz, 2H), 3.63 (dd, J = 10.2, 6.6 Hz, 1H), 3.58 (m, 2H), 3.33 (d, J = 4.3 Hz, 1H), 2.41 (t, J = 6.1 Hz, 1H), 0.90–0.85 (m, 36H), 0.12–0.03 (m, 24H). [M+Na]+ ＝733.6.
第四步：the fourth step:
将[0030]所得产物(IV)(5mmol)加入到带有磁子的100mL圆底烧瓶中，向其中加入无水甲苯(toluene，60mL)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDCI,2.0当量)，4-二甲氨基吡啶(DMAP,0.1当量)和月桂酸(Lauric acid，2.0当量)，然后室温反应12小时，用薄层色谱检测反应完全后，移置室温，向其中加入乙酸乙酯(100mL),用饱和碳酸氢钠、饱和食盐水洗涤有机相，无水硫酸钠干燥有机相，减压浓缩有机相，经柱层析分离纯化以92％的产率得到产物(V)。1H NMR(600MHz,Chloroform-d)δ4.84(d,J＝2.5Hz,1H),4.13(dd,J＝5.0,3.2Hz,1H),4.12–4.09(m,2H),4.02–3.94(m,3H),3.74(m,1H),3.66–3.60(m,2H),3.60–3.54(m,2H),3.22(d,J＝3.3Hz,1H),2.32(t,J＝7.6Hz,2H),1.61(m,2H),1.26(m,16H),0.88(m,39H),0.11–0.03(m,24H).[M+Na]+＝915.8.The obtained product (IV) (5 mmol) was added into a 100 mL round-bottom flask with a magnetic rod, and anhydrous toluene (60 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 2.0 equivalents), 4-dimethylaminopyridine (DMAP, 0.1 equivalents) and lauric acid (2.0 equivalents) were added thereto, and then reacted at room temperature for 12 hours. After the reaction was completed by thin layer chromatography, the reaction was returned to room temperature, and ethyl acetate (100 mL) was added thereto. The organic phase was washed with saturated sodium bicarbonate and saturated brine, and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and separated and purified by column chromatography to obtain the product (V) with a yield of 92%.1 H NMR (600 MHz, Chloroform-d) δ 4.84 (d, J = 2.5 Hz, 1H), 4.13 (dd, J = 5.0, 3.2 Hz, 1H), 4.12–4.09 (m, 2H), 4.02–3.94 (m, 3H), 3.74 (m, 1H), 3.66–3.60 (m, 2H), 3.60–3.54 (m, 2H), 3.22 (d, J = 3.3 Hz, 1H), 2.32 (t, J = 7.6 Hz, 2H), 1.61 (m, 2H), 1.26 (m, 16H), 0.88 (m, 39H), 0.11–0.03 (m, 24H). [M+Na]+ = 915.8.
第五步：the fifth step:
将[0031]所得产物(V)(5mmol)加入到带有磁子的100mL塑料反应瓶中，向其中加入无水的四氢呋喃(THF)，0℃条件下将氟化氢吡啶(6.0当量)缓慢滴加其中，然后室温反应2小时，用薄层色谱检测反应完全后，将其放置冰浴中，缓慢滴加饱和碳酸氢钠溶液淬灭反应，然后，用乙酸乙酯萃取三次，无水硫酸钠干燥有机相，将有机相减压浓缩，经柱层析以84％的产率得到产物(VI)。1H NMR(600MHz,Chloroform-d)δ5.22(s,2H),4.96(s,1H),4.86(d,J＝2.6Hz,1H),4.81–4.70(m,1H),4.58(s,1H),4.08(d,J＝10.8Hz,3H),3.99(d,J＝15.1Hz,2H),3.76(m,5H),3.59–3.43(m,1H),2.32(t,J＝7.6Hz,2H),1.58(p,J＝7.2Hz,2H),1.26(d,J＝15.8Hz,16H),0.86(t,J＝7.0Hz,3H).[M+Na]+＝459.3.The obtained product (V) (5 mmol) was added to a 100 mL plastic reaction bottle with a magnetic rod, anhydrous tetrahydrofuran (THF) was added thereto, and hydrofluoric acid pyridine (6.0 equivalents) was slowly added dropwise at 0°C, and then reacted at room temperature for 2 hours. After the reaction was completed by thin layer chromatography, it was placed in an ice bath, and a saturated sodium bicarbonate solution was slowly added dropwise to quench the reaction. Then, it was extracted three times with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure. The product (VI) was obtained by column chromatography with a yield of 84%.1 H NMR (600 MHz, Chloroform-d) δ5.22 (s, 2H), 4.96 (s, 1H), 4.86 (d, J = 2.6 Hz, 1H), 4.81–4.70 (m, 1H), 4.58 (s, 1H), 4.08 (d, J = 10.8 Hz, 3H), 3.99 (d, J = 15.1 Hz, 2H), 3.76 (m, 5H), 3.59–3.43 (m, 1H), 2.32 (t, J = 7.6 Hz, 2H), 1.58 (p, J = 7.2 Hz, 2H), 1.26 (d, J = 15.8 Hz, 16H), 0.86 (t, J = 7.0 Hz, 3H). [M+Na]+ ＝459.3.
第六步：Step 6:
将[0032]中所得(VI)(3.6mmol)和N-碘代丁二酰亚胺(NIS,2.4当量)加入到带有磁子的100mL圆底烧瓶中，氩气下，向其中加入无水的二氯甲烷(40mL)且置于-30℃，然后向其中缓慢加入VII(1.2当量),-30℃反应3小时，薄层色谱检测反应完全后，将其移置冰浴中，缓慢滴加10％硫代硫酸钠淬灭反应，用二氯甲烷萃取，无水硫酸钠干燥有机相，减压浓缩有机相，经柱层析以72％的产率得到GalGL-1(I)及其差向异构体。CaLGL-1(I)1H NMR(600MHz,Methanol-d4)δ4.90(t,J＝4.8Hz,1H),4.88(d,J＝1.5Hz,1H),4.19–4.08(m,3H),4.00–3.92(m,3H),3.92–3.85(m,2H),3.82(m,1H),3.75(m,1H),3.54–3.44(m,1H),2.35(t,J＝7.2Hz,2H),1.70–1.58(m,4H),1.43(m,2H),1.34–1.26(m,36H),0.90(t,J＝7.2Hz,6H).13C NMR(151MHz,Methanol-d4)δ175.5,109.9,106.5,85.5,83.5,79.45,77.8,70.0,69.7,67.1,66.5,35.1,35.0,33.2,30.91,30.89,30.87,30.84,30.81,30.8,30.7,30.6,30.5,30.3,26.1,25.1,23.8,14.5.[M+Na]+＝653.6.The (VI) (3.6 mmol) obtained in [0032] and N-iodosuccinimide (NIS, 2.4 equivalents) were added to a 100 mL round-bottom flask with a magnetic rod. Anhydrous dichloromethane (40 mL) was added thereto under argon and placed at -30°C. Then VII (1.2 equivalents) was slowly added thereto. The reaction was carried out at -30°C for 3 hours. After the reaction was completed by thin layer chromatography, it was moved to an ice bath, 10% sodium thiosulfate was slowly added dropwise to quench the reaction, and the reaction was extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure. GalGL-1 (I) and its diastereomer were obtained by column chromatography with a yield of 72%. CaLGL-1(I)1 H NMR (600 MHz, Methanol-d4) δ4.90 (t, J = 4.8 Hz, 1H), 4.88 (d, J = 1.5 Hz, 1H), 4.19–4.08 (m, 3H), 4.00–3.92 (m, 3H), 3.92–3.85 (m, 2H), 3.82 (m, 1H), 3.75 (m, 1H), 3.54–3.44 (m, 1H), 2.35 (t, J = 7.2 Hz, 2H), 1.70–1.58 (m, 4H), 1.43 (m, 2H), 1.34–1.26 (m, 36H), 0.90 (t, J = 7.2 Hz, 6H).13 C NMR (151 MHz, Methanol-d4) δ 175.5, 109.9, 106.5, 85.5, 83.5, 79.45, 77.8, 70.0, 69.7, 67.1, 66.5, 35.1, 35.0, 33.2, 30.91, 30.89, 30.87, 30.84, 30.81, 30.8, 30.7, 30.6, 30.5, 30.3, 26.1, 25.1, 23.8, 14.5. [M+Na]+ ＝653.6.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
附图为CaLGL-1核磁共振谱，包括1H NMR和13C NMR。The attached figure shows the nuclear magnetic resonance spectrum of CaLGL-1, including1 H NMR and13 C NMR.
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