Egor A. Ulashchik, Yury V. Martynenko-Makaev
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GalNAc oligonucleotide conjugates demonstrate improved potency in vivo due to selective and efficient delivery to hepatocytes in the liver via receptor-mediated endocytosis. GalNAc-siRNA and GalNAc-antisense oligonucleotides are at various stages of clinical trials, while the first two drugs were already approved by FDA. Also, GalNAc conjugates are excellent tools for functional genomics and target validation in vivo. The number of GalNAc residues in a conjugate is crucial for delivery as cooperative interaction of several GalNAc residues with asialoglycoprotein receptor enhances delivery in vitro and in vivo. Here we provide a robust protocol for the synthesis of triple GalNAc CPG solid support and GalNAc phosphoramidite, synthesis and purification of RNA conjugates with multiple GalNAc residues either to 5′-end or 3′-end and siRNA duplex formation.
GalNAc 寡核苷酸偶联物通过受体介导的内吞作用，选择性且高效地传递至肝脏中的肝细胞，从而在体内表现出更强的效力。GalNAc-siRNA 和 GalNAc 反义寡核苷酸处于不同的临床试验阶段，其中前两种药物已获得 FDA 批准。此外，GalNAc 偶联物还是体内功能基因组学和靶点验证的优秀工具。一个偶联物中 GalNAc 残基的数量对于传递至关重要，因为多个 GalNAc 残基与脱唾液酸糖蛋白受体的协同作用可增强体内外的传递效率。本文提供了一个完善的方案，包括三价 GalNAc CPG 固体支持物和 GalNAc 磷酰胺的合成，多 GalNAc 残基 RNA 偶联物的合成与纯化，以及 5′端或 3′端的 siRNA 双链形成。
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RNA therapeutics demonstrated a tremendous progress in last years—already ten oligonucleotide drugs are approved by FDA and EMA, even more are coming soon. Proof-of-concept for therapeutic use of nucleic acids was shown more than 40 years ago, but many challenges were overcome since late 1970s to develop oligonucleotide drugs, like Nusinersen and Givosiran. Among these issues are poor stability of native oligonucleotides in vivo, innate immune response to single-stranded and double-stranded DNA and RNA, and limited targeted delivery to the specific organ and cell type. Until now the most important and challenging task is targeted delivery of oligonucleotides, including specific intracellular delivery. Initial success in intrahepatic delivery of naked phosphorothioate antisense oligonucleotides (ASO) and siRNA formulated in lipid nanoparticles, uncovered adverse effects—partial nonspecific delivery, some local inflammatory response due to ASO/lipid toxicity under chronic treatment and other issues. Some of them can be resolved by use of biodegradable lipids and more potent ASO/siRNA with prolonged activity that allows to reduce doses and frequency of administration. However, alternative solution to use less toxic RNA conjugates with the ligands of cellular receptors seems to be more straightforward. Among such targeting, moieties are peptides, folate, N-acetylgalactosamine (GalNAc), aptamers, and even antibodies. Also, we would like to emphasize recent progress of hydrophobic conjugates that allow functional delivery to muscles and CNS. Together with high stability of chemically modified siRNA, listed above properties of conjugates result in longevity of the effect and specificity of siRNA action. Thus, conjugates are a new paradigm in siRNA delivery as they provide improved targeting and biodistribution in comparison to nanoparticles and naked oligonucleotides.
近年来，RNA 疗法取得了巨大的进展——已有十种寡核苷酸药物获得了 FDA 和 EMA 的批准，更多的药物即将面世。早在 40 多年前就已经证明了核酸在治疗中的概念，但自 20 世纪 70 年代末以来，为开发如 Nusinersen 和 Givosiran 等寡核苷酸药物，克服了许多挑战。这些问题包括天然寡核苷酸在体内的稳定性差、单链和双链 DNA 和 RNA 引发的先天性免疫反应、以及有限的靶向传递到特定器官和细胞类型。时至今日，最重要且具有挑战性的任务仍是寡核苷酸的靶向传递，包括特定的细胞内传递。在最初的裸露磷酸硫代反义寡核苷酸（ASO）和脂质纳米颗粒包裹的 siRNA 肝内传递取得成功后，也暴露出了一些不良反应——部分非特异性传递、由于 ASO/脂质在慢性治疗中的毒性引发的一些局部炎症反应等问题。其中一些问题可以通过使用可降解脂质和更有效的、具有长效活性的 ASO/siRNA 来解决，这可以减少剂量和给药频率。然而，另一种解决方案是使用与细胞受体配体结合的毒性较小的 RNA 偶联物，这似乎更为直接。在这些靶向配体中，包括肽、叶酸、N-乙酰半乳糖胺（GalNAc）、适配子，甚至抗体。此外，我们还要强调最近在允许功能性传递到肌肉和中枢神经系统的疏水性偶联物方面的进展。与化学修饰的 siRNA 的高稳定性相结合，以上列出的偶联物特性导致了效果的持久性和 siRNA 作用的特异性。因此，偶联物在 siRNA 传递中代表了一种新的范例，因为它们在靶向性和生物分布方面比纳米颗粒和裸露的寡核苷酸具有更好的表现。
Among many successful conjugates we want to emphasize the development of unique triple GalNAc conjugates that drive selective and efficient oligonucleotide delivery to hepatocytes via asialoglycoprotein receptor (ASGPR). ASGPR is a C-type lectin involved in the metabolism of glycoproteins and seems to be the optimal receptor for targeted RNA delivery among known ones. ASGPR is highly represented at the surface of hepatocytes (0.5–1 M receptors/cell) and undergoes fast clathrin-mediated turnover into endosomes (~10–15 min−1). Despite these tremendous properties, single GalNAc residue binds to the receptor with relatively low affinity (high μM range). However, cooperative interactions of several GalNAcs with multimerized receptor subunits clusters demonstrated much higher affinity to ASGPR than mono-GalNAc. Thus, multivalent (di-, tri-, tetra-) GalNAc clusters enhance delivery in vitro and in vivo. Last year first GalNAc-siRNA conjugates was approved by FDA (Givosiran to treat acute hepatic porphyria and Lumasiran to treat primary hyperoxaluria type 1) plus several siRNA- and ASO-GalNAc conjugates are at different stages of clinical trials. This delivery system together with multiple chemical modifications of siRNA provides a unique opportunity to downregulate specific proteins in the liver by biannual subcutaneous administration of the drug.
在众多成功的偶联物中，我们特别强调了独特的三价 GalNAc 偶联物的开发，这些偶联物通过天门冬氨酸糖蛋白受体（ASGPR）将寡核苷酸选择性且高效地传递到肝细胞。ASGPR 是一种 C 型凝集素，参与糖蛋白的代谢，是目前已知的 RNA 靶向传递中最理想的受体。ASGPR 在肝细胞表面高度表达（每个细胞 0.5–1 百万个受体），并且具有快速的网格蛋白介导的内吞周转率（约 10–15 分钟）。尽管 ASGPR 具有这些优异的性质，单个 GalNAc 残基与受体的结合亲和力相对较低（高微摩尔范围）。然而，多 GalNAc 与多聚受体亚基簇的协同作用表现出比单一 GalNAc 更高的亲和力。因此，多价（双、三、四）GalNAc 簇在体外和体内显著增强了传递效果。去年，第一个 GalNAc-siRNA 偶联物获得了 FDA 的批准（Givosiran 用于治疗急性肝卟啉病和 Lumasiran 用于治疗 1 型原发性高草酸尿症），另外还有几个 siRNA 和 ASO-GalNAc 偶联物正处于不同的临床试验阶段。这种传递系统结合 siRNA 的多种化学修饰，为通过每半年一次的皮下注射药物下调肝脏中特定蛋白质提供了独特的机会。
Here we provide a robust protocol for the synthesis of triple GalNAc CPG solid support and GalNAc phosphoramidite that can be used in standard automated oligonucleotide synthesis to introduce multiple GalNAc residues either to 5′-end or 3′-end of native or chemically modified DNA and RNA oligonucleotides (Fig. 1). The first sections outline the procedures for the synthesis, purification, and characterization of triple GalNAc TEG Support and mono GalNAc TEG phosphoramidite. The phosphoramidite can be used to build GalNAc clusters either at 3′-end, 5′-end, or both. Last sections of the protocol describe synthesis, purification, and characterization of GalNAc modified RNA oligonucleotides and siRNA duplex formation.
这里我们提供了一种稳健的合成三价 GalNAc CPG 固体支撑物和 GalNAc 磷酰胺的方法，这些材料可以在标准的自动化寡核苷酸合成过程中使用，以在天然或化学修饰的 DNA 和 RNA 寡核苷酸的 5'端或 3'端引入多个 GalNAc 残基（如图 1 所示）。前几节内容详细介绍了三价 GalNAc TEG 支撑物和单一 GalNAc TEG 磷酰胺的合成、纯化和表征步骤。该磷酰胺可以用来在 3'端、5'端或两端构建 GalNAc 簇。最后几节则描述了 GalNAc 修饰的 RNA 寡核苷酸的合成、纯化和表征，以及 siRNA 双链体的形成。
Fig. 1 Synthesis of siRNA conjugates with different topology using GalNAc phosphoramidite and triple GalNAc TEG CPG
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2.1 Synthesis of Triple GalNAc TEG CPG Solid Support and GalNAc TEG Phosphoramidite 三价 GalNAc TEG CPG 固体载体和 GalNAc TEG 亚磷酰胺的合成
Reagents were purchased from commercial suppliers and used as is:
所用试剂购自商业供应商，直接使用
(a) N,N-Diisopropylethylamine (DIEA).
N,N-二异丙基乙胺 (DIEA)
(b) 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite.
2-氰乙基N,N-二异丙基氯膦酰胺
(c) Triethylamine (TEA).
三乙胺 (TEA)
(d) 40% Aqueous methylamine solution.
40% 甲胺水溶液
(e) EDC·HCl.
N-（3-二甲基氨基丙基）-N'-乙基碳二亚胺 盐酸盐
(f) HOBt.
1-羟基苯并三唑
(g) Trifluoroacetic acid (TFA).
三氟乙酸 (TFA)
(h) 10% (w/w) palladium on activated carbon.
10% (w/w) 钯/活性炭
(i) Hydrogen.
氢气
(j) Succinylated LCAA-CPG.
琥珀酰化 LCAA-CPG
(k) DMAP.
4-二甲氨基吡啶
(l) Diisopropylcarbodiimide.
二异丙基碳二亚胺
(m) Pentafluorophenol.
五氟苯酚
(n) Piperidine.
哌啶
(o) Acetic anhydride.
醋酸酐
(p) 2,6-Lutidine.
2,6-二甲基吡啶。
(q) 1-Methylimidazole.
1-甲基咪唑
(r) 2,2,2-Trifluoro-N-(4-hydroxy-1,1-bis(4-methoxyphenyl)-1-phenyl-2,6,9,12,15-pentaoxaoctadecan-18-yl)acetamide 9-use an available custom synthesis service (e.g., Primetech) to obtain this item.
2,2,2-三氟-N-(4-羟基-1,1-双(4-甲氧基苯基)-1-苯基-2,6,9,12,15-五氧杂十八烷-18-基)乙酰胺 9—通过使用可用的定制合成服务（例如 Primetech）获得此项
(s) Amino-tri-(t-butoxycarbonylethoxymethyl)-methane 1氨基-三-(t-丁氧羰基乙氧基甲基)-甲烷 1
(t) 12-(Benzyloxy)-12-oxododecanoic acid, 2-(2-(2-(2-(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-d-galactopyranosyloxy)ethoxy)ethoxy)ethoxy)ethylammonium trifluoroacetate 4.
12-(苄氧基)-12-氧代十二酸, 2-(2-(2-(2-(3,4,6-三-O-乙酰基-2-乙酰氨基-2-脱氧-β-d-半乳糖吡喃糖氧基)乙氧基)乙氧基)乙氧基)乙基铵三氟乙酸盐 4
(u) 1-Amino-3-O-(4,4′-dimethoxytrityl)propan-2-ol.
1-氨基-3-O-(4,4'-二甲氧基三苯甲基)丙-2-醇
(v) 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid 11—synthetize this item as described previously.
5-(((2R,3R,4R,5R,6R)-3-乙酰氨基-4,5-二乙酰氧基-6-(乙酰氧基甲基)四氢-2H-吡喃-2-基)氧基)戊酸 11—如前所述进行合成
(w) Iodine.
碘
(x) Anhydrous sodium sulfate.
无水硫酸钠
(y) Sodium bicarbonate.
碳酸氢钠
(z) Sodium chloride .
氯化钠。
(aa) Potassium carbonate.
碳酸钾
Solvents available from common commercial suppliers were high-performance liquid chromatography (HPLC) grade and were used without further purification unless otherwise noted:
溶剂由普通商业供应商提供，为高效液相色谱 (HPLC) 级别，除非另有说明，否则不进行进一步纯化
(a) Dichloromethane (DCM) was always used freshly distilled over calcium hydride.
始终使用经过氢化钙蒸馏新制的二氯甲烷
(b) Ethyl acetate (EtOAc).
乙酸乙酯 (EtOAc)
(c) Ethanol (EtOH).
乙醇 (EtOH)
(d) Methanol (MeOH).
甲醇 (MeOH)
(e) Dimethylformamide (DMF) was freshly distilled under reduced pressure.
减压蒸馏新制的二甲基甲酰胺
(f) Toluene (PhMe).
甲苯 (PhMe)
(g) Petroleum ether.
石油醚
(h) Diethyl ether (Et2O).
乙醚 (Et2O)
(i) Tetrahydrofuran (THF).
四氢呋喃 (THF)
(j) Hexane.
正己烷
(k) Acetone.
丙酮
(l) Benzene.
苯
(m) Pyridine.
吡啶
(n) Acetonitrile.
乙腈
Chromatography:
色谱条件
(a) Analytical thin-layer chromatography is performed on Kieselgel 60 F254 precoated aluminum plates (Merck); spots are visualized under UV light (254 nm). Phosphomolybdic acid stain solution preparation: dissolve 1 g of phosphomolybdic acid in 25 mL of absolute ethanol.
(a) 分析薄层层析 (TLC) 使用默克公司预涂层 Kieselgel 60 F254 铝板进行，斑点在紫外光 (254 nm) 下显色。磷钼酸染色液制备：将 1 克磷钼酸溶解在 25 毫升无水乙醇中。
(b) Column chromatography is performed on silica gel (Merck Kieselgel 60 0.040–0.063 mm).
柱层析填料为硅胶 (默克 Kieselgel 60 0.040–0.063 mm)
1H and 13C NMR spectra were recorded at 400 or 600 MHz and 101 or 151 MHz instruments, respectively. 31P NMR spectrum was recorded at 202 MHz instrument. Chemical shifts are reported in δ (ppm) units using residual 1H signals from deuterated solvents as references. The coupling constants (J) are given in Hz.
1H 和13C 核磁共振 (NMR) 光谱分别使用 400 或 600 MHz 以及 101 或 151 MHz 的仪器进行记录。31P 核磁共振光谱则使用 202 MHz 的仪器记录。化学位移以 δ (ppm) 为单位报告，参考氘代溶剂中的残余 1H 信号。偶合常数 (J) 以赫兹 (Hz) 为单位表示。
2.2 Oligonucleotide Synthesis 寡核苷酸合成
Standard reagents for oligonucleotide synthesis can be purchased from any commercial supplier:
用于寡核苷酸合成的标准试剂可从任何商业供应商处购买
(a) Protected 2′-O-methyl, 2′-deoxy, 2′-deoxy-2′-fluoro nucleoside phosphoramidites.
保护的 2'-O-甲基、2'-脱氧、2'-脱氧-2'-氟核苷磷酰胺
(b) Deprotection solution (3% trichloroacetic acid in DCM).
脱保护溶液（3%三氯乙酸溶于二氯甲烷中）
(c) Oxidizing solution (0.02 M iodine in THF/pyridine/water (80/10/10, v/v/v)).
氧化溶液（0.02 M 碘溶于四氢呋喃/吡啶/水（80/10/10, v/v/v）中）
(d) Capping solutions (Cap A: 10% acetic anhydride, 10% pyridine in THF; Cap B: 16% N-methylimidazole in THF).
封端溶液（封端 A：10%乙酸酐，10%吡啶溶于四氢呋喃中；封端 B：16% N-甲基咪唑溶于四氢呋喃中）
(e) Acetonitrile (DNA synthesis grade).
乙腈（用于 DNA 合成）
(f) Solution of activator (0.25 M 5-ethylthiotetrazole in acetonitrile).
活化剂溶液（0.25 M 5-乙基硫代四唑溶于乙腈中）
(g) Universal solid support.
通用固相载体
Standard reagents for oligonucleotide purification and characterization by IE-HPLC and PAGE can be purchased from any commercial supplier:
用于通过离子交换高效液相色谱（IE-HPLC）和聚丙烯酰胺凝胶电泳（PAGE）进行寡核苷酸纯化和表征的标准试剂可从任何商业供应商处购买
(a) Acrylamide.
丙烯酰胺
(b) Bis-acrylamide
双丙烯酰胺
(c) Tris.
三羟甲基氨基甲烷
(d) Boric acid.
硼酸
(e) Urea.
尿素
(f) Sodium perchlorate.
高氯酸钠
(g) Disodium EDTA.
乙二胺四乙酸二钠
(h) HPLC-grade water.
HPLC 级水
(i) HPLC gradient grade acetonitrile .
HPLC 梯度级乙腈👀
3.1 Preparation of Triple GalNAc TEG Solid Support for RNA Synthesis
用于 RNA 合成的三价半乳糖胺 (GalNAc) TEG 固体载体制备
Synthetic procedure is based on the synthesis of the key block—triple GalNAc TEG derivative 7 (Subheadings 3.1.1~3.1.4) followed by common transformations to obtain solid support 8 for automated oligonucleotide synthesis (Fig. 2). Before starting synthesis please read Notes 1 and 2.
本方法以关键构建单元 - 三价半乳糖胺 (GalNAc) TEG 衍生物 7 的合成 (3.1.1~3.1.4 小节) 为基础，然后通过常规转化步骤获得用于自动寡核苷酸合成的固体载体 8 (图 2)。开始合成之前，请阅读 注释 1 和 2。
Fig. 2 Synthesis of triple GalNAc TEG CPG solid support
3.1.1 Synthesis of 3,3′-((2-(12-(Benzyloxy)-12-oxododecanamido)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))-dipropionic acid
3 3,3′-((2-(12-(苄氧基)-12-氧代十二烷酰胺基)-2-((2-羧基乙氧基)甲基)丙烷-1,3-二基)双(氧基))-二丙酸 3的合成
Charge 2.15 g, 4.25 mmol amine 1 in a 50-mL round-bottom flask under dry argon atmosphere and add 20 mL of absolute DCM. Add 1.50 g, 4.89 mmol of 12-(Benzyloxy)-12-oxododecanoic acid and dissolve solid residues under magnetic stirring. Add sequentially 0.98 g, 5.1 mmol of EDC·HCl, 0.69 g, 5.1 mmol of HOBt, and 0.77 mL, 5.53 mmol of Et3N to the reaction mixture and stir it overnight. Monitor conversion of amine 1 (Rf 0.15) to the product 2 (Rf 0.85) by TLC in EtOAc–hexane, 1:1 v/v, to visualize spots—stain the plate with phosphomolybdic acid solution (see Note 3).
在干燥的氩气气氛下，于 50 mL 圆底烧瓶中加入 2.15 g (4.25 mmol) 的胺 1 和 20 mL 的无水 DCM。加入 1.50 g (4.89 mmol) 的 12-(苄氧基)-12-氧代十二烷酸，并用磁力搅拌溶解固体残留物。然后依次向反应混合物中加入 0.98 g (5.1 mmol) 的 EDC·HCl、0.69 g (5.1 mmol) 的 HOBt 和 0.77 mL (5.53 mmol) 的 Et3N，并搅拌过夜。通过薄层层析 (TLC) 用乙酸乙酯-己烷 (1:1 v/v) 溶液监测胺 1 (Rf 0.15) 转化成产物 2 (Rf 0.85)，用磷钼酸溶液染色显色斑点 (见 注释 3)。
Dilute reaction mixture with 100 mL of DCM, pour the reaction mixture into a 250-mL separatory funnel and wash it thrice with 50 mL of water, thrice with 50 mL of 10% citric acid in water, thrice with 50 mL of saturated sodium bicarbonate in water and once with 50 mL of brine. Dry organic layer with sodium sulfate and concentrate to dryness using a rotary evaporator under reduced pressure to obtain amide 2 as a colorless oil.
用 100 mL DCM 稀释反应混合物，将反应物倒入 250 mL 分液漏斗中，用 50 mL 水洗涤 3 次，用 50 mL 10% 的柠檬酸水溶液洗涤 3 次，用 50 mL 饱和碳酸氢钠水溶液洗涤 3 次，用 50 mL 饱和盐水洗涤 1 次。用硫酸钠干燥有机层，并使用旋转蒸发器在减压下浓缩至干燥，得到酰胺 2，为无色油状物。
Dissolve the colorless oil in 20 mL of 25% TFA in DCM and stir the reaction mixture overnight. Monitor reaction by TLC in EtOAc–hexane, 1:1 v/v until amide 2 (Rf 0.85) is gone (stain the plate with phosphomolybdic acid solution).
将无色油状物溶解在 20 mL 的 25% TFA/DCM 溶液中，并搅拌反应混合物过夜。通过薄层层析 (TLC) 用乙酸乙酯-己烷 (1:1 v/v) 溶液监测反应，直至酰胺 2 (Rf 0.85) 消失 (用磷钼酸溶液染色显色斑点)。
Concentrate the reaction mixture to dryness on a rotary evaporator under reduced pressure to obtain oil. Dissolve the oil in 20 mL of toluene and evaporate the solvent to remove residual TFA (repeat twice), then dissolve oil in 3 mL of ethyl acetate and precipitate the product with 16 mL of petroleum ether. Cool the mixture in a fridge (+4 °C for 2 h) and carefully decant the liquid phase. Dissolve the solid residue in 3 mL of ethyl acetate and precipitate the product again with 16 mL of petroleum ether. Decant liquid and dry residue in vacuum to obtain 2.36 g of pure product 3 (Rf 0.20) (EtOAc) as a white solid in 89% yield.
使用旋转蒸发器在减压下将反应混合物浓缩至干燥，得到油状物。将油状物溶解在 20 mL toluene 中，蒸发溶剂以除去残留的 TFA (重复两次)，然后将油状物溶解在 3 mL 乙酸乙酯中，用 16 mL 石油醚沉淀产物。将混合物冷却于冰箱中 (+4 °C，2 小时)，小心地倾出液体部分。将固体残留物溶解在 3 mL 乙酸乙酯中，并用 16 mL 石油醚再次沉淀产物。倾出液体，将残留物干燥抽真空，得到 2.36 g 纯净产物 3 (Rf 0.20) (乙酸乙酯) ，为白色固体，产率为 89%。
Confirm the purity of the compound 3 by 1H, 13C NMR spectroscopy and high-resolution mass-spectrometry (see Note 4). 1H NMR (500 MHz, DMSO-d6) δ 12.16 (s, 3H), 7.39–7.28 (m, 5H), 6.90 (s, 1H), 5.07 (s, 2H), 3.59–3.50 (m, 12H), 2.41 (t, J = 6.3 Hz, 6H), 2.33 (t, J = 7.4 Hz, 2H), 2.03 (t, J = 7.3 Hz, 2H), 1.57–1.47 (m, 2H), 1.46–1.38 (m, 2H), 1.26–1.19 (m, 12H); 13C NMR (126 MHz, DMSO-d6) δ 172.80, 172.63, 172.51, 136.33, 128.43, 127.98, 127.94, 68.17, 66.69, 65.29, 59.52, 35.89, 34.60, 33.49, 28.92, 28.91, 28.85, 28.68, 28.53, 28.45, 25.30, 24.50; ESI HRMS m/z calculated for m/z calculated for [C32H49NO12 + H]+ 640.3333, found 640.3321.
通过1H、13C NMR 光谱和高分辨质谱确认化合物3的纯度（见注释 4）。1H NMR (500 MHz, DMSO-d6) δ 12.16 (s, 3H), 7.39–7.28 (m, 5H), 6.90 (s, 1H), 5.07 (s, 2H), 3.59–3.50 (m, 12H), 2.41 (t, J = 6.3 Hz, 6H), 2.33 (t, J = 7.4 Hz, 2H, 13C NMR (126 MHz, DMSO-d6) δ 172.80, 172.63, 172.51, 136.33, 128.43, 127.98, 127.94, 68.17, 66.69, 65.29, 59.52, 35.89, 34.60, 33.49, 28.92, 28.91, 28.85, 28.68, 28.53, 28.45, 25.30, 24.50; ESI HRMS m/z calculated for [C32H49NO12 + H]+ 640.3333, found 640.3321.
3.1.2 N-(12-(Benzyloxy)-12-oxododecanamido)-tris[2,5,8,11,18-pentaoxa-14-aza-15-oxo-19-(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-d-galactopyranosyloxy)nonadecyl]methane 5
N-(12-(苄氧基)-12-氧代十二烷酰胺)-三[2,5,8,11,18-五氧杂-14-氮杂-15-氧代-19-(3,4,6-三-O-乙酰基-2-乙酰氨基-2-脱氧-β-D-半乳糖吡喃糖氧基)十九烷基]甲烷 5
harge 8.63 g, 13.56 mmol 2-(2-(2-(2-(((3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-d-galactopyranosyloxy)ethoxy)ethoxy)ethoxy)ethylammonium trifluoroacetate (4) and 2.36 g, 3.77 mmol of triacid (3) in 100-mL round-bottom flask under dry argon atmosphere and dissolve in 50 mL DCM under magnetic stirring. Add sequentially 4.19 mL, 30.16 mmol of Et3N, 2.89 g, 15.08 mmol of EDC·HCl and 2.04 g, 15.08 mmol HOBt to the reaction mixture and stir it overnight. Monitor conversion of triacid 3 (Rf 0.05) to product 5 (Rf 0.65) by TLC in DCM–MeOH, 87/13, v/v, to visualize spots—stain the plate with phosphomolybdic acid solution (see Note 5).
在干燥的氩气氛下，将 8.63 克（13.56 毫摩尔）的 2-(2-(2-(2-(3,4,6-三-O-乙酰基-2-乙酰氨基-2-脱氧-β-D-半乳糖吡喃糖氧基)乙氧基)乙氧基)乙基)铵三氟乙酸盐(4)和 2.36 克（3.77 毫摩尔）的三酸(3)加入 100 毫升的圆底烧瓶中，溶解在 50 毫升的二氯甲烷（DCM）中，磁力搅拌。依次加入 4.19 毫升（30.16 毫摩尔）的三乙胺（Et3N）、2.89 克（15.08 毫摩尔）的 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐（EDC·HCl）和 2.04 克（15.08 毫摩尔）的 1-羟基苯并三唑（HOBt），搅拌过夜。通过薄层色谱法（TLC）在 DCM 和甲醇（MeOH）的混合溶液（87:13，v/v）中监测三酸(3) (Rf 0.05)向产物5 (Rf 0.65)的转化情况，用磷钼酸溶液染色（见注释5）。
Dilute the reaction mixture with 100 mL of DCM, pour it into a 250-mL separatory funnel, and wash it thrice with 50 mL of water, thrice with 50 mL of 10% citric acid in water, thrice with 50 mL of saturated sodium bicarbonate in water, and once with 50 mL of brine. Dry organic layer with sodium sulfate and concentrate to dryness under reduced pressure using a rotary evaporator.
用 100 毫升的 DCM 稀释反应混合物，倒入 250 毫升的分液漏斗中，分别用 50 毫升的水洗三次，用 50 毫升的 10%柠檬酸水溶液洗三次，用 50 毫升的饱和碳酸氢钠水溶液洗三次，再用 50 毫升的盐水洗一次。用无水硫酸钠干燥有机层，并在减压下用旋转蒸发器浓缩至干燥。
Purify the residue by column chromatography on silica gel (use 4-cm diameter chromatography column filled with at least 125 g of silica gel in DCM) with a linear gradient of DCM–MeOH from 100:0 to 90:10 v/v. Combine and evaporate fractions that contain pure compound 5 (Rf 0.65 in DCM–MeOH, 87:13 v/v) to obtain 3.96 g of triamide 5 as a white solid in 78% yield.
用硅胶柱层析法纯化残留物（使用直径 4 厘米的层析柱，填充至少 125 克硅胶），以 DCM 和甲醇从 100:0 到 90:10（v/v）的线性梯度洗脱。合并并蒸发包含纯化合物5 (Rf 0.65 在 DCM–MeOH，87:13 v/v)的部分，得到 3.96 克白色固体的三酰胺5，产率为 78%。
Confirm the purity of compound 5 by 1H, 13C NMR spectroscopy and high-resolution mass-spectrometry. 1H NMR (500 MHz, DMSO-d6) δ 7.92 (t, J = 5.7 Hz, 3H), 7.81 (d, J = 9.3 Hz, 3H), 7.41–7.29 (m, 5H), 6.99 (s, 1H), 5.21 (d, J = 3.4 Hz, 3H), 5.07 (s, 2H), 4.96 (dd, J = 11.2, 3.4 Hz, 3H), 4.55 (d, J = 8.5 Hz, 3H), 4.05–3.98 (m, 9H), 3.88 (dt, J = 11.1, 8.8 Hz, 3H), 3.80–3.74 (m, 3H), 3.61–3.47 (m, 45H), 3.40–3.37 (m, 6H), 3.23–3.15 (m, 6H), 2.35–2.26 (m, 8H), 2.10 (s, 9H), 2.04 (t, J = 7.4 Hz, 2H), 1.99 (s, 9H), 1.88 (s, 9H), 1.77 (s, 9H), 1.55–1.48 (m, 2H), 1.45–1.39 (m, 2H), 1.26–1.16 (m, 12H). 13C NMR (126 MHz, DMSO-d6) δ 173.84, 172.63, 170.40, 170.11, 170.04, 169.74, 169.48, 136.36, 128.50, 128.06, 127.99, 101.02, 78.55, 70.59, 69.96, 69.88, 69.77, 69.64, 69.52, 69.16, 68.41, 68.36, 68.34, 67.36, 66.78, 65.35, 61.53, 59.57, 49.39, 38.57, 35.92, 33.53, 28.97, 28.96, 28.89, 28.74, 28.69, 28.50, 25.38, 24.55, 22.84, 20.59, 20.53, 20.50. ESI-HRMS m/z calculated for [C98H157N7O45 + H]+ 2153.0290, found 2153.0279.
通过1H 和13C 核磁共振（NMR）光谱和高分辨率质谱（HRMS）确认化合物 5 的纯度。1H NMR (500 MHz, DMSO-d6) δ 7.92 (t, J = 5.7 Hz, 3H), 7.81 (d, J = 9.3 Hz, 3H), 7.41–7.29 (m, 5H), 6.99 (s, 1H), 5.21 (d, J = 3.4 Hz, 3H), 5.07 (s, 2H), 4.96 (dd, J = 11.2, 3.4 Hz, 3H), 4.55 (d, J = 8.5 Hz, 3H), 4.05–3.98 (m, 9H), 3.88 (dt, J = 11.1, 8.8 Hz, 3H), 3.80–3.74 (m, 3H), 3.61–3.47 (m, 45H), 3.40–3.37 (m, 6H), 3.23–3.15 (m, 6H), 2.35–2.26 (m, 8H), 2.10 (s, 9H), 2.04 (t, J = 7.4 Hz, 2H), 1.99 (s, 9H), 1.88 (s, 9H), 1.77 (s, 9H), 1.55–1.48 (m, 2H), 1.45–1.39 (m, 2H), 1.26–1.16 (m, 12H)。13C NMR (126 MHz, DMSO-d6) δ 173.84, 172.63, 170.40, 170.11, 170.04, 169.74, 169.48, 136.36, 128.50, 128.06, 127.99, 101.02, 78.55, 70.59, 69.96, 69.88, 69.77, 69.64, 69.52, 69.16, 68.41, 68.36, 68.34, 67.36, 66.78, 65.35, 61.53, 59.57, 49.39, 38.57, 35.92, 33.53, 28.97, 28.96, 28.89, 28.74, 28.69, 28.50, 25.38, 24.55, 22.84, 20.59, 20.53, 20.50。ESI-HRMS m/z 计算值为 [C98H157N7O45 + H]+ 2153.0290，实测值为 2153.0279。
3.1.3 N-(11-Carboxy-undecanamido)-tris[2,5,8,11,18-pentaoxa-14-aza-15-oxo-19-(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-d-galactopyranosyloxy)nonadecyl]methane 6
N-(11-羧基-十一烷酰胺)-三[2,5,8,11,18-五氧杂-14-氮杂-15-氧代-19-(3,4,6-三-O-乙酰基-2-乙酰氨基-2-脱氧-β-D-半乳糖吡喃糖氧基)十九烷基]甲烷 6
Charge 3.96 g, 1.84 mmol compound 5 in a 50-mL round-bottom flask, co-evaporate twice with 25 mL of absolute methanol, and dissolve in 25 mL of absolute methanol .
在一个 50 毫升的圆底烧瓶中，加入 3.96 克（1.84 毫摩尔）的化合物5，用 25 毫升无水甲醇共蒸发两次，然后溶解在 25 毫升无水甲醇中。
Add 196 mg, 0.18 mmol of 10% (w/w) palladium on activated carbon, vacuum the flask and fill with hydrogen and stir mixture under 1 atm hydrogen pressure for 30 min. Monitor conversion of compound 5 (Rf 0.65) to product 6 (Rf 0.60) by TLC in DCM–MeOH, 87: 13 v/v, to visualize spots—stain the plate with phosphomolybdic acid solution (see Note 6).
加入 196 毫克（0.18 毫摩尔）的 10%（w/w）钯/活性炭，将烧瓶抽真空并充满氢气，在 1 个大气压的氢气压力下搅拌 30 分钟。通过薄层色谱法（TLC）在二氯甲烷和甲醇的混合溶液（87:13，v/v）中监测化合物5（Rf 0.65）向产物6（Rf 0.60）的转化情况，显现斑点—用磷钼酸溶液染色（见注释6）。
Filter off the catalyst using glass filter filled with silica gel (bed height ~1 cm), wash it twice with 15 mL of hot methanol and combine methanol fractions in 100-mL round-bottom flask. Concentrate the filtrate to dryness under reduced pressure using a rotary evaporator to obtain 3.68 g compound 6 as a white solid in 97% yield.
用填充有硅胶（床高度约 1 厘米）的玻璃滤器过滤催化剂，用 15 毫升热甲醇洗两次，并将甲醇部分合并于 100 毫升的圆底烧瓶中。用旋转蒸发器在减压下将滤液浓缩至干燥，得到 3.68 克白色固体化合物6，产率为 97%。
Confirm the purity of compound 6 by 1H, 13C NMR spectroscopy and high-resolution mass-spectrometry. 1H NMR (500 MHz, DMSO-d6) δ 7.92 (t, J = 5.7 Hz, 3H), 7.81 (d, J = 9.2 Hz, 3H), 6.99 (s, 1H), 5.21 (d, J = 3.4 Hz, 3H), 4.96 (dd, J = 11.2, 3.4 Hz, 3H), 4.55 (d, J = 8.5 Hz, 3H), 4.06–4.00 (m, 9H), 3.88 (dt, J = 11.2, 8.9 Hz, 3H), 3.80–3.75 (m, 3H), 3.61–3.46 (m, 45H), 3.39–3.36 (m, 6H), 3.22–3.16 (m, 6H), 2.29 (t, J = 6.4 Hz, 6H), 2.17 (t, J = 7.4 Hz, 2H), 2.10 (s, 9H), 2.04 (t, J = 7.4 Hz, 2H), 1.99 (s, 9H), 1.88 (s, 9H), 1.77 (s, 9H), 1.50–1.40 (m, 4H), 1.25–1.19 (m, 12H). 13C NMR (126 MHz, DMSO-d6) δ 174.60, 172.62, 170.38, 170.10, 170.03, 169.73, 169.46, 142.47, 101.01, 70.57, 69.96, 69.88, 69.76, 69.64, 69.52, 69.16, 68.41, 68.34, 68.33, 67.35, 66.78, 61.53, 59.56, 49.38, 38.57, 35.91, 33.73, 29.01, 28.99, 28.89, 28.82, 28.69, 28.64, 25.38, 24.58, 22.83, 20.58, 20.52, 20.50. ESI-HRMS m/z calculated for [C91H151N7O45 + H]+ 2062.9821, observed 2062.9832.
通过1H 和13C 核磁共振光谱及高分辨质谱确认化合物 6 的纯度。1H NMR (500 MHz, DMSO-d6) δ 7.92 (t, J = 5.7 Hz, 3H), 7.81 (d, J = 9.2 Hz, 3H), 6.99 (s, 1H), 5.21 (d, J = 3.4 Hz, 3H), 4.96 (dd, J = 11.2, 3.4 Hz, 3H), 4.55 (d, J = 8.5 Hz, 3H), 4.06–4.00 (m, 9H), 3.88 (dt, J = 11.2, 8.9 Hz, 3H), 3.80–3.75 (m, 3H), 3.61–3.46 (m, 45H), 3.39–3.36 (m, 6H), 3.22–3.16 (m, 6H), 2.29 (t, J = 6.4 Hz, 6H), 2.17 (t, J = 7.4 Hz, 2H), 2.10 (s, 9H), 2.04 (t, J = 7.4 Hz, 2H), 1.99 (s, 9H), 1.88 (s, 9H), 1.77 (s, 9H), 1.50–1.40 (m, 4H), 1.25–1.19 (m, 12H)。13C NMR (126 MHz, DMSO-d6) δ 174.60, 172.62, 170.38, 170.10, 170.03, 169.73, 169.46, 142.47, 101.01, 70.57, 69.96, 69.88, 69.76, 69.64, 69.52, 69.16, 68.41, 68.34, 68.33, 67.35, 66.78, 61.53, 59.56, 49.38, 38.57, 35.91, 33.73, 29.01, 28.99, 28.89, 28.82, 28.69, 28.64, 25.38, 24.58, 22.83, 20.58, 20.52, 20.50。ESI-HRMS m/z 计算值为 [C91H151N7O45 + H]+ 2062.9821，实测值为 2062.9832。
3.1.4 N-(12-((3-O-(4,4′-Dimethoxytrityl)-2-hydroxypropyl)-amino)-12-oxododecanamido)-tris[2,5,8,11,18-pentaoxa14-aza-15-oxo-19-(3,4,6-tri-O-acetyl-2-acetamido-2-deoxyβ-d-galactopyranosyloxy)nonadecyl]methane 7
N-(12-((3-O-(4,4′-二甲氧基三苯甲基)-2-羟丙基)氨基)-12-氧代十二烷酰胺)-三[2,5,8,11,18-五氧杂-14-氮杂-15-氧代-19-(3,4,6-三-O-乙酰基-2-乙酰氨基-2-脱氧-β-D-半乳糖吡喃糖氧基)十九烷基]甲烷 7
Charge 862 mg, 2.14 mmol 1-amino-3-O-(4,4′-dimethoxytrityl)propan-2-ol and 3.68 g, 1.78 mmol of acid 6 in 100-mL round-bottom flask and dissolve in 50 mL of DCM under magnetic stirring. Add 743 μL, 5.34 mmol of Et3N, 682 mg, 3.56 mmol of EDC·HCl and 480 mg, 3.56 mmol of HOBt to the reaction mixture and stir it overnight. Monitor conversion of acid 6 (Rf 0.60) to amide 7 (Rf 0.65) by TLC in DCM–MeOH, 87/13, v/v, to visualize spots—stain the plate with phosphomolybdic acid.
在 100 毫升的圆底烧瓶中，加入 862 毫克（2.14 毫摩尔）的 1-氨基-3-O-(4,4′-二甲氧基三苯甲基)丙-2-醇和 3.68 克（1.78 毫摩尔）的酸6，在磁力搅拌下溶解于 50 毫升二氯甲烷。加入 743 微升（5.34 毫摩尔）的三乙胺、682 毫克（3.56 毫摩尔）的 EDC·HCl 和 480 毫克（3.56 毫摩尔）的 HOBt，搅拌过夜。通过薄层色谱法（TLC）在二氯甲烷和甲醇的混合溶液（87:13，v/v）中监测酸6（Rf 0.60）向酰胺7（Rf 0.65）的转化情况，显现斑点—用磷钼酸溶液染色。
Dilute the reaction mixture with 400 mL of DCM, pour it into a 1000-mL separatory funnel, and wash it thrice with 150 mL of water, thrice with 150 mL of 10% citric acid in water, thrice with 150 mL of saturated sodium bicarbonate in water and with 150 mL of brine. Dry organic layer with sodium sulfate and concentrate to dryness under reduced pressure using a rotary evaporator.
用 400 毫升二氯甲烷稀释反应混合物，将其倒入 1000 毫升的分液漏斗中，先后用 150 毫升的水洗涤三次，用 150 毫升 10%柠檬酸水溶液洗涤三次，用 150 毫升饱和碳酸氢钠水溶液洗涤三次，最后用 150 毫升盐水洗涤。用硫酸钠干燥有机层，并用旋转蒸发器在减压下浓缩至干燥。
Purify the residue by column chromatography on silica gel (use 4-cm diameter chromatography column filled with silica gel (at least 85 g) using 1% Et3N in DCM) with a linear gradient of DCM–MeOH–Et3N from 100:0:1 to 95:5:1 v/v/v. Combine and evaporate fractions that contain pure compound 7 (Rf 0.65 in DCM–MeOH, 87:13 v/v) to obtain 3.30 g of amide 7 as a white solid in 76% yield.
用硅胶柱层析纯化残留物（使用 4 厘米直径的柱子，填充至少 85 克硅胶，用 1%三乙胺的二氯甲烷进行层析），以二氯甲烷-甲醇-三乙胺的线性梯度（从 100:0:1 到 95:5:1，v/v/v）洗脱。合并并蒸发含有纯化合物7（Rf 0.65 在二氯甲烷-甲醇，87:13，v/v）部分，得到 3.30 克白色固体酰胺7，产率为 76%。
Confirm the purity of compound 7 by 1H, 13C NMR spectroscopy and high-resolution mass-spectrometry. 1H NMR (500 MHz, DMSO-d6) δ 7.94 (t, J = 5.7 Hz, 3H), 7.84 (d, J = 9.2 Hz, 3H), 7.67 (t, J = 5.7 Hz, 1H), 7.41–7.36 (m, 2H), 7.33–7.16 (m, 7H), 7.00 (s, 1H), 6.91–6.81 (m, 4H), 5.21 (d, J = 3.3 Hz, 3H), 5.01–4.93 (m, 4H), 4.56 (d, J = 8.5 Hz, 3H), 4.05–3.99 (m, 9H), 3.88 (dt, J = 11.1, 8.9 Hz, 3H), 3.81–3.75 (m, 3H), 3.73–3.71 (m, 6H), 3.71–3.65 (m, 2H), 3.61–3.45 (m, 48H), 3.40–3.37 (m, 6H), 3.22–3.16 (m, 6H), 3.00–2.94 (m, 1H), 2.91–2.81 (m, 1H), 2.29 (t, J = 6.5 Hz, 6H), 2.10 (s, 9H), 2.05 (t, J = 7.7 Hz, 2H), 1.99 (s, 9H), 1.88 (s, 9H), 1.77 (s, 9H), 1.46–1.37 (m, 4H), 1.23–1.18 (m, 12H). 13C NMR (126 MHz, DMSO-d6) δ 172.61, 172.52, 170.37, 170.08, 170.01, 169.71, 169.45, 158.04, 145.15, 135.88, 129.78, 128.97, 127.80, 127.78, 127.69, 126.61, 113.12, 112.82, 101.00, 85.21, 70.58, 69.95, 69.87, 69.76, 69.63, 69.51, 69.14, 68.84, 68.40, 68.34, 67.35, 66.77, 65.78, 61.52, 59.55, 49.37, 42.64, 38.56, 35.97, 35.91, 35.42, 29.06, 29.03, 28.92, 28.88, 28.81, 28.72, 25.38, 22.83, 20.57, 20.50, 20.49. ESI-HRMS m/z calculated for [C115H176N8O48 + H]+2438.1650, observed 2438.1655.
通过1H、13C NMR 光谱及高分辨质谱确认化合物 7 的纯度。1H NMR (500 MHz, DMSO-d6) δ 7.94 (t, J = 5.7 Hz, 3H), 7.84 (d, J = 9.2 Hz, 3H), 7.67 (t, J = 5.7 Hz, 1H), 7.41–7.36 (m, 2H), 7.33–7.16 (m, 7H), 7.00 (s, 1H), 6.91–6.81 (m, 4H), 5.21 (d, J = 3.3 Hz, 3H), 5.01–4.93 (m, 4H), 4.56 (d, J = 8.5 Hz, 3H), 4.05–3.99 (m, 9H), 3.88 (dt, J = 11.1, 8.9 Hz, 3H), 3.81–3.75 (m, 3H), 3.73–3.71 (m, 6H), 3.71–3.65 (m, 2H), 3.61–3.45 (m, 48H), 3.40–3.37 (m, 6H), 3.22–3.16 (m, 6H), 3.00–2.94 (m, 1H), 2.91–2.81 (m, 1H), 2.29 (t, J = 6.5 Hz, 6H), 2.10 (s, 9H), 2.05 (t, J = 7.7 Hz, 2H), 1.99 (s, 9H), 1.88 (s, 9H), 1.77 (s, 9H), 1.46–1.37 (m, 4H), 1.23–1.18 (m, 12H)。13C NMR (126 MHz, DMSO-d6) δ 172.61, 172.52, 170.37, 170.08, 170.01, 169.71, 169.45, 158.04, 145.15, 135.88, 129.78, 128.97, 127.80, 127.78, 127.69, 126.61, 113.12, 112.82, 101.00, 85.21, 70.58, 69.95, 69.87, 69.76, 69.63, 69.51, 69.14, 68.84, 68.40, 68.34, 67.35, 66.77, 65.78, 61.52, 59.55, 49.37, 42.64, 38.56, 35.97, 35.91, 35.42, 29.06, 29.03, 28.92, 28.88, 28.81, 28.72, 25.38, 22.83, 20.57, 20.50, 20.49。ESI-HRMS m/z 计算值为 [C115H176N8O48 + H]+ 2438.1650，实测值为 2438.1655。
3.1.5 TRIS-(GalNAc-TEG)-CPG (8)
Charge 3.3 g, 1.35 mmol compound 7 in 25-mL round-bottom flask and dissolve in 8 mL of a 1:1 v/v pyridine-DMF mixture. Sequentially add 109 mg, 0.9 mmol of DMAP, 1.0 g of succinylated LCAA-CPG, and 2.16 mL, 13.8 mmol of diisopropylcarbodiimide under stirring. Leave the mixture at ambient temperature for 48 h and gently shake the mix daily.
将 3.3 g, 1.35 mmol 的化合物7置于 25 mL 的圆底烧瓶中，并溶解在 8 mL 1:1 v/v 的吡啶-DMF 混合物中。依次加入 109 mg, 0.9 mmol 的 DMAP，1.0 g 的琥珀酰化 LCAA-CPG，以及 2.16 mL, 13.8 mmol 的二异丙基碳二亚胺，在搅拌下进行反应。在室温下放置 48 小时，每天轻轻摇动混合物一次。
Gently shake the mix and immediately take a small portion ~150 μL of CPG suspension into 1.5 mL tube and separate the CPG by decanting. Wash CPG twice with 1 mL of DCM, twice with 2 mL of acetonitrile, and 5 times with 1 mL of diethyl ether. Dry CPG in high vacuo for 1 h. Accurately transfer ~10 mg of CPG 8 into a 25-mL flask and add to 15 mL 3% TFA in DCM. After 15 min exposure measure the absorbance of the mixture at 505 nm using UV-VIS spectrophotometer (please use 3% TFA in DCM as a blanc) and calculate the loading of triple GalNAc TEG CPG 8 according to the following equation: L (μmol/g) = (V × 1000 × Abs)/78 × m where m is the mass of 8 (in mg), Abs—the absorbance of the mixture at 505 nm, V the total volume of the solution (mL). The loading of the solid support 8 was calculated ~43 μmol/g CPG (see Note 7).
轻轻摇动混合物，立即取出约 150 μL 的 CPG 悬浮液放入 1.5 mL 的管中，通过倾析分离 CPG。分别用 1 mL 的二氯甲烷洗涤 CPG 两次，用 2 mL 的乙腈洗涤两次，再用 1 mL 的乙醚洗涤五次。将 CPG 在高真空下干燥 1 小时。准确转移约 10 mg 的 CPG8到 25 mL 的烧瓶中，加入 15 mL 3% TFA 在二氯甲烷中的溶液。经过 15 分钟的反应后，用紫外-可见分光光度计在 505 nm 处测量混合物的吸光度（请使用 3% TFA 在二氯甲烷中的溶液作为空白对照），并根据以下公式计算三重 GalNAc TEG CPG 8的负载量：L (μmol/g) = (V × 1000 × 吸光度)/78 × m，其中m是8的质量（mg），吸光度是混合物在 505 nm 处的吸光度，V是溶液的总体积（mL）。固体载体8的负载量约为 43 μmol/g CPG（见 注释 7）。
Add solution of 760 mg, 4.13 mmol pentafluorophenol in 2 mL pyridine and wait for 20 h.
加入 760 mg, 4.13 mmol 五氟苯酚的 2 mL 吡啶溶液，静置 20 小时
Separate CPG using glass filter and wash thoroughly with 15 mL of absolute pyridine. Charge CPG in a 25-mL round-bottom flask and add solution of 0.5 mL piperidine in 5 mL pyridine, leave it for 5 min to block unreacted carboxy groups (see Note 8).
通过玻璃滤器分离 CPG，用 15 mL 绝对吡啶彻底洗涤。将 CPG 置于 25 mL 的圆底烧瓶中，加入 0.5 mL 哌啶在 5 mL 吡啶中的溶液，放置 5 分钟以封闭未反应的羧基（见 注释 8）
Separate CPG through the glass filter, wash it with 20 mL pyridine, then with 20 mL acetonitrile and dry it in vacuo. Place CPG in a 25-mL round-bottom flask and suspend it in solution of 0.5 mL acetic anhydride, 0.9 mL 2,6-lutidine, and 0.5 mL 1-methylimidazole in 8.1 mL THF and leave for 2 h.
通过玻璃滤器分离 CPG，用 20 mL 吡啶洗涤，然后用 20 mL 乙腈洗涤，干燥后在真空中干燥。将 CPG 置于 25 mL 的圆底烧瓶中，悬浮在含有 0.5 mL 乙酸酐，0.9 mL 2,6-二甲基吡啶和 0.5 mL 1-甲基咪唑在 8.1 mL 四氢呋喃中的溶液中，放置 2 小时。
Separate the CPG through the glass filter, wash it sequentially with 20 mL of DCM, 20 mL of MeOH, 20 mL of acetonitrile, and 20 mL of diethyl ether. Dry CPG in vacuo overnight to yield 1.08 g modified CPG 8 (see Note 9).
通过玻璃滤器分离 CPG，依次用 20 mL 二氯甲烷，20 mL 甲醇，20 mL 乙腈和 20 mL 乙醚洗涤。将 CPG 在真空中干燥过夜，得到 1.08 g 修饰的 CPG 8（见 注释 9）。
