GalNAc偶联siRNA的合成（下）

3.2 Preparation of a Mono GalNAc TEG Phosphoramidite 

单 GalNAc TEG 亚磷酰胺的制备

The procedure includes the synthesis of the GalNAc TEG carboxy block 12 (Subheadings 3.2.1 and 3.2.2) followed by common transformations into phosphoramidite 13 (Fig. 3).

该过程包括 GalNAc TEG 羧基嵌段 12 的合成（子标题 3.2.1 和 3.2.2），然后通过常见转化成亚磷酰胺 13（图 3）。

Fig. 3 Synthesis of a mono GalNAc TEG phosphoramidite

3.2.1 Synthesis of 16-Amino-1-O-(4,4′-dimethoxytrityl)-4,7,10,13-tetraoxahexadecan-1,2-diol 10 

合成 16-氨基-1-O-(4,4′-二甲氧基三苯甲基)-4,7,10,13-四氧杂十六烷-1,2-二醇 10

1. Charge 1.90 g, 2.80 mmol protected amine 9 in a 100-mL round-bottom flask and dissolve in 38 mL of methanol. Add 1.93 g, 13.98 mmol of potassium carbonate in 12 mL of water and stir the reaction mixture overnight under an argon atmosphere. Monitor conversion of trifluoroacetamide 9 (Rf 0.65) to amine 10 (Rf 0.10) by TLC in DCM–MeOH, 10/1, v/v, to visualize spots—stain the plate with phosphomolybdic acid solution.

   将 1.90 克，2.80 毫摩尔的保护氨基9加入 100 毫升的圆底烧瓶中，并溶解在 38 毫升甲醇中。加入 1.93 克，13.98 毫摩尔的碳酸钾溶解在 12 毫升水中，并在氩气环境下搅拌过夜。通过薄层色谱（TLC）在二氯甲烷-甲醇，10/1，v/v 中监测三氟乙酰胺9（Rf 0.65）向氨基10（Rf 0.10）的转化情况，以磷钼酸溶液显色。

2. Concentrate the mixture approximately to one third by volume under reduced pressure using a rotary evaporator. Add 40 mL of DCM and 25 mL of water to the mixture and pour the mixture into a 100-mL separatory funnel. Separate organic layer and wash the water fraction thrice with 50 mL DCM, combine organic fractions and dry with anhydrous sodium sulfate. Concentrate solution to dryness under reduced pressure using a rotary evaporator to obtain 1.61 g of amine 10 as a colorless oil and use it for the next step without purification .

   使用旋转蒸发仪在减压下将混合物体积浓缩至约三分之一。向混合物中加入 40 毫升二氯甲烷和 25 毫升水，将混合物倒入 100 毫升的分液漏斗中。分离有机层，并用 50 毫升二氯甲烷洗涤水层三次，合并有机层并用无水硫酸钠干燥。在减压下使用旋转蒸发仪将溶液浓缩至干，得到 1.61 克无色油状的氨基10，无需纯化直接用于下一步反应。

3.2.2 Synthesis of 5-(3,4,6-Tri-O-acetyl-2-acetamido-2-deoxy-β-d-galactopyranosyloxy)-N-(1-O-(4,4′-dimethoxytrityl)-1,2-dihydroxy-4,7,10,13-tetraoxahexadec-16-yl)pentylamide 12 

合成 5-(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)-N-(1-O-(4,4′-二甲氧基三苯甲基)-1,2-二羟基-4,7,10,13-四氧杂十六烷基)戊酰胺 12

1. Charge 1.61 g, 2.76 mmol amine 10 in 50-mL round-bottom flask and dissolve in 15 mL DCM under magnetic stirring. Add 1.16 mL, 8.28 mmol of Et3N, 1.24 g, 2.76 mmol of acid 11, 1.06 g, 5.52 mmol of EDC·HCl, and 0.75 g, 5.52 mmol of HOBt to the reaction mixture and stir it overnight. Monitor conversion of amine 10 (Rf 0.10 in DCM–MeOH, 10:1, v/v) and acid 11 (Rf 0.55 in DCM–MeOH, 10:1 v/v) to amide 12 (Rf 0.20 in DCM–acetone, 1:1 v/v) by TLC. To visualize spots—stain the plate with phosphomolybdic acid solution.

   将 1.61 克，2.76 毫摩尔氨基10加入 50 毫升的圆底烧瓶中，并在磁力搅拌下溶解于 15 毫升二氯甲烷中。依次加入 1.16 毫升（8.28 毫摩尔）三乙胺，1.24 克（2.76 毫摩尔）酸11，1.06 克（5.52 毫摩尔）EDC·HCl 和 0.75 克（5.52 毫摩尔）HOBt 到反应混合物中，搅拌过夜。通过薄层色谱（TLC）监测氨基10（Rf 0.10，在二氯甲烷-甲醇，10:1，v/v 中）和酸11（Rf 0.55，在二氯甲烷-甲醇，10:1，v/v 中）向酰胺12（Rf 0.20，在二氯甲烷-丙酮，1:1，v/v 中）的转化情况。用磷钼酸溶液显色。

2. Dilute the reaction mixture with 35 mL of DCM, pour it into a 100-mL separatory funnel, and wash it sequentially: twice with 25 mL of water, twice with 25 mL of 10% citric acid in water, twice with 25 mL of saturated sodium bicarbonate in water and with 25 mL of brine. Dry organic layer with anhydrous sodium sulfate, filtrate, and concentrate to dryness under reduced pressure using a rotary evaporator.

   将反应混合物用 35 毫升二氯甲烷稀释，倒入 100 毫升的分液漏斗中，按顺序进行以下洗涤：两次用 25 毫升水洗涤，两次用 25 毫升 10%柠檬酸水溶液洗涤，两次用 25 毫升饱和碳酸氢钠水溶液洗涤，最后用 25 毫升盐水洗涤。用无水硫酸钠干燥有机层，过滤后在减压下使用旋转蒸发仪将溶液浓缩至干，得到粗品。

3. Purify the residue by column chromatography on silica gel (use 3-cm diameter chromatography column filled with at least 45 g silica gel using 1% Et3N in DCM) with a linear gradient of DCM–acetone–Et3N from 100:0:1 to 50:50:1 v/v/v. Combine and evaporate fractions that contain pure compound 12 (Rf 0.2, DCM–acetone, 1:1 v/v) to obtain 2.01 g of amide 12 as a white solid in 72% yield.

   使用硅胶柱色谱法纯化残留物（使用直径 3 厘米的色谱柱，填充至少 45 克硅胶，用 1%三乙胺的二氯甲烷洗脱），用 DCM-丙酮-三乙胺从 100:0:1 逐渐到 50:50:1 的线性梯度洗脱。合并并蒸发含有纯化合物12（Rf 0.2，DCM-丙酮，1:1，v/v）的馏分，得到 2.01 克白色固体酰胺12，产率为 72%。

4. Confirm the purity of compound 12 by ¹H, ¹³C NMR spectroscopy and high-resolution mass-spectrometry. ¹H NMR (500 MHz, DMSO-d6) δ 7.81 (d, J = 9.2 Hz, 1H), 7.72 (t, J = 5.6 Hz, 1H), 7.42–7.39 (m, 2H), 7.32–7.28 (m, 2H), 7.28–7.24 (m, 4H), 7.23–7.19 (m, 1H), 6.90–6.86 (m, 4H), 5.21 (d, J = 3.4 Hz, 1H), 4.97 (dd, J = 11.2, 3.5 Hz, 1H), 4.86 (d, J = 5.4 Hz, 1H), 4.48 (d, J = 8.5 Hz, 1H), 4.05–3.99 (m, 3H), 3.87 (dt, J = 11.2, 8.8 Hz, 1H), 3.79–3.65 (m, 8H), 3.51–3.42 (m, 13H), 3.42–3.35 (m, 4H), 3.06 (q, J = 6.6 Hz, 2H), 2.92 (d, J = 5.5 Hz, 2H), 2.10 (s, 3H), 2.03 (t, J = 7.1 Hz, 2H), 1.99 (s, 3H), 1.89 (s, 3H), 1.77 (s, 3H), 1.59 (p, J = 6.7 Hz, 2H), 1.53–1.39 (m, 4H). ¹³C NMR (126 MHz, DMSO-d6) δ 171.82, 169.98, 169.89, 169.61, 169.30, 157.97, 145.12, 135.87, 129.71, 127.75, 127.70, 126.52, 113.06, 100.96, 85.09, 72.73, 70.45, 70.11, 69.81, 69.79, 69.77, 69.75, 69.74, 69.51, 68.76, 68.65, 68.08, 66.69, 64.94, 61.43, 55.00, 49.36, 35.70, 35.02, 29.41, 28.57, 22.75, 21.83, 20.51, 20.45, 20.44. ESI-HRMS m/z calculated for [C₅₂H₇₂N₂O₁₈ + H]⁺ 1013.4853, observed 1013.4859.

   通过¹H，¹³C NMR 光谱和高分辨质谱（HRMS）确认化合物 12 的纯度。¹H NMR (500 MHz, DMSO-d6) δ 7.81 (d, J = 9.2 Hz, 1H), 7.72 (t, J = 5.6 Hz, 1H), 7.42–7.39 (m, 2H), 7.32–7.28 (m, 2H), 7.28–7.24 (m, 4H), 7.23–7.19 (m, 1H), 6.90–6.86 (m, 4H), 5.21 (d, J = 3.4 Hz, 1H), 4.97 (dd, J = 11.2, 3.5 Hz, 1H), 4.86 (d, J = 5.4 Hz, 1H), 4.48 (d, J = 8.5 Hz, 1H), 4.05–3.99 (m, 3H), 3.87 (dt, J = 11.2, 8.8 Hz, 1H), 3.79–3.65 (m, 8H), 3.51–3.42 (m, 13H), 3.42–3.35 (m, 4H), 3.06 (q, J = 6.6 Hz, 2H), 2.92 (d, J = 5.5 Hz, 2H), 2.10 (s, 3H), 2.03 (t, J = 7.1 Hz, 2H), 1.99 (s, 3H), 1.89 (s, 3H), 1.77 (s, 3H), 1.59 (p, J = 6.7 Hz, 2H), 1.53–1.39 (m, 4H). ¹³C NMR (126 MHz, DMSO-d6) δ 171.82, 169.98, 169.89, 169.61, 169.30, 157.97, 145.12, 135.87, 129.71, 127.75, 127.70, 126.52, 113.06, 100.96, 85.09, 72.73, 70.45, 70.11, 69.81, 69.79, 69.77, 69.75, 69.74, 69.51, 68.76, 68.65, 68.08, 66.69, 64.94, 61.43, 55.00, 49.36, 35.70, 35.02, 29.41, 28.57, 22.75, 21.83, 20.51, 20.45, 20.44. ESI-HRMS m/z 计算得出 [C₅₂H₇₂N₂O₁₈ + H]⁺ 1013.4853，观测到 1013.4859。

3.2.3 Synthesis of 5-(3,4,6-Tri-O-acetyl-2-acetamido-2-deoxy-β-d-galactopyranosyloxy)-N-(1-O-(4,4′-dimethoxytrityl)-2-O-(N,N-diisopropylamino-(2-cyanoethyloxy)-phosphonamidic)-1,2-dihydroxy-4,7,10,13-tetraoxahexadec-16-yl)pentylamide 13 

合成 5-(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)-N-(1-O-(4,4′-二甲氧基三苯甲基)-2-O-(N,N-二异丙氨基-(2-氰基乙氧基)-磷酰胺基)-1,2-二羟基-4,7,10,13-四氧杂十六烷基)戊酰胺 13

1. Charge 1.68 g, 1.66 mmol of compound 12 in 50-mL round-bottom flask and dissolve in 11 mL of anhydrous DCM under magnetic stirring in argon atmosphere. Add 300 μL, 2.16 mmol of Et3N followed by 471 mg, 1.99 mmol of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite to the reaction mixture and stir for 2 h at room temperature. Monitor conversion of alcohol 12 (Rf 0.20) to phosphoramidite 13 (0.50) by TLC in DCM–acetone, 1:1 v/v; to visualize spots—stain the plate with phosphomolybdic acid solution.

   将 1.68 克，1.66 毫摩尔的化合物12加入 50 毫升的圆底烧瓶中，在氩气气氛下溶解于 11 毫升无水二氯甲烷中，并进行磁力搅拌。加入 300 微升（2.16 毫摩尔）的三乙胺，再加入 471 毫克（1.99 毫摩尔）的 2-氰基乙基N,N-二异丙基氯磷酰胺到反应混合物中，在室温下搅拌 2 小时。通过薄层色谱（TLC）监测化合物12（Rf 0.20）向磷酰胺基13（Rf 0.50）的转化，使用二氯甲烷-丙酮（1:1，v/v）。用磷钼酸溶液显色

2. Dilute the reaction mixture with 25 mL of DCM , pour it into a 100-mL separatory funnel, and wash it twice with 25 mL of saturated sodium bicarbonate in water and then with 25 mL of brine. Dry organic layer with sodium sulfate.

   将反应混合物用 25 毫升二氯甲烷稀释，倒入 100 毫升的分液漏斗中，用 25 毫升饱和碳酸氢钠溶液洗两次，然后用 25 毫升盐水洗。用无水硫酸钠干燥有机层。

3. Purify the residue by column chromatography on silica gel (use 3-cm diameter chromatography column filled with at least 35 g of silica gel) using 1% Et3N in DCM with a linear gradient of DCM–acetone–Et3N from 100:0:1 to 50:50:1 v/v/v. Combine and evaporate fractions that contain pure compound 13 (Rf 0.50 in DCM–acetone, 1:1 v/v) to obtain 1.23 g of phosphoramidite 13 as a white solid in 61% yield.

   使用硅胶柱色谱法纯化残留物（使用直径 3 厘米的色谱柱，填充至少 35 克硅胶），用 1%三乙胺的二氯甲烷洗脱，使用二氯甲烷-丙酮-三乙胺从 100:0:1 到 50:50:1（v/v/v）的线性梯度洗脱。合并并蒸发含有纯化合物13（Rf 0.50，二氯甲烷-丙酮，1:1，v/v）的馏分，得到 1.23 克白色固体磷酰胺基13，产率为 61%。

4. Confirm the purity of compound 13 by ³¹P NMR spectroscopy and high-resolution mass-spectrometry. ³¹P NMR (202 MHz, CD3CN) δ 149.08, 148.58. ESI-HRMS m/z calculated for[C₆₁H₈₉N₄O₁₉P + H]⁺ 1213.5931, observed 1213.5935 (see Note 10).

   通过³¹P NMR 光谱和高分辨质谱（HRMS）确认化合物13的纯度。³¹P NMR（202 MHz, CD3CN）δ 149.08, 148.58。ESI-HRMS m/z 计算得出 [C₆₁H₈₉N₄O₁₉P + H]⁺ 1213.5931，观测到 1213.5935（参见 注释 10）。

3.3 Synthesis of GalNAc-siRNA Conjugates 

合成 GalNAc-siRNA 共轭物

This section describes the preparation of 3′- and 5′-(triple GalNAc) modified siRNA, including automated oligonucleotide synthesis and purification of sense and antisense RNA strands (Table 1) using solid support 8 and GalNAc TEG phosphoramidite 13.

本部分介绍 3′和 5′（三重 GalNAc）修饰的 siRNA 的制备，包括使用固体支持物8和 GalNAc TEG 磷酰胺基13的自动寡核苷酸合成以及正义链和反义链 RNA 链的纯化（表 1）。

Table 1 Modified RNA and GalNAc-RNA conjugates synthesized in this study

Chemical modifications: mN—2′-O-methyl nucleotides, fN—2′-deoxy-2′-fluoronucleotides, N—2′-deoxynucleotides, *—phosphorothioate linkages

化学修饰：mN—2′-O-甲基核苷酸、fN—2′-脱氧-2′-氟核苷酸、N—2′-脱氧核苷酸、*—硫代磷酸酯键

3.3.1 Synthesis of GalNAc-RNA Conjugates 合成 GalNAc-RNA 偶联物

1. RNA should be assembled in DNA/RNA synthesizer at 1 μmol scale (e.g., ABI 3400 or ASM-2000) using phosphoramidite method according to the manufacturer’s recommendations DMT-off mode using triple GalNAc TEG solid support 8 or DMT-on mode using mono GalNAc TEG phosphoramidite (see Note 11).

   使用 DNA/RNA 合成仪（如 ABI 3400 或 ASM-2000），按照制造商的建议，在 1 μmol 规模下通过磷酰胺方法组装 RNA。在使用三重 GalNAc TEG 固体支持物8时，选择 DMT-off 模式；在使用单一 GalNAc TEG 磷酰胺基时，选择 DMT-on 模式（见 注释 11）

2. After completion of the synthesis dry column in vacuo and transfer CPG with protected oligonucleotide into the screw-capped tube (1.5 mL). Add 750 μL of 40% aqueous methylamine solution and perform deprotection for 1 h at 45 °C under gentle shaking. Place the tubes with solution for 15 min at −20 °C, filter off solids, wash them thoroughly by water (2 × 200 μL), and evaporate combined washings using centrifuge vacuum concentrator to dryness.

   合成完成后，将柱子在真空中干燥，并将含有保护性寡核苷酸的 CPG 转移到螺旋盖试管（1.5 mL）中。加入 750 μL 的 40%水相甲胺溶液，在 45°C 下轻轻摇动进行脱保护 1 小时。将装有溶液的试管在-20°C 下放置 15 分钟，过滤掉固体，分别用水洗涤两次（2 × 200 μL），然后使用离心真空浓缩器蒸发联合洗涤液至干燥。

3. Dissolve crude oligonucleotides in 1 mL pure water.

   将粗寡核苷酸溶解在 1 mL 纯水中

3.3.2 Purification of GalNAc-RNA Conjugates 纯化 GalNAc-RNA 偶联物

1. Purify the crude GalNAc-RNA conjugates by ion exchange HPLC equipped with TSKGEL SUPERQ-5PW 7.5 × 75 column using a linear gradient from 10–60% of buffer B for 40 min at a flow rate of 1 mL/min. Buffer A: 50 mM Tris–HCl pH 7.6 in sterile water/acetonitrile (9:1, v/v), buffer B: 600 mM NaClO4 and 50 mM Tris–HCl pH 7.6 in sterile water/acetonitrile (9:1, v/v).

   通过配备 TSKGEL SUPERQ-5PW 7.5 × 75 柱的离子交换 HPLC，在流速为 1 mL/min 的条件下，以 10–60%的缓冲液 B 的线性梯度，持续 40 分钟，纯化粗 GalNAc-RNA 共轭物。缓冲液 A：50 mM Tris–HCl pH 7.6 在无菌水/乙腈（9:1，v/v）中的溶液，缓冲液 B：600 mM 高氯酸钠和 50 mM Tris–HCl pH 7.6 在无菌水/乙腈（9:1，v/v）中的溶液。

2. Pool the appropriate fractions with the conjugate, evaporate to the final volume ~100 μL, and add 1.2 mL of acetone (molecular biology grade) to precipitate RNA conjugate and to remove most of sodium perchlorate.

   将适当的部分合并，蒸发至最终体积约 100 μL，然后加入 1.2 mL 丙酮（分子生物学级）以沉淀 RNA 共轭物并去除大部分高氯酸钠。

3. A mixture is cooled in a freezer at −20 °C for 1 h and then RNA conjugate is isolated by centrifugation for 5 min at 10,000 × g followed by final drying of acetone traces.

   将混合物在-20°C 下冷却 1 小时，然后通过以 10,000 × g离心 5 分钟，分离 RNA 共轭物，并最终去除丙酮痕迹。

4. Dissolve solid residue in 100 μL and add 1.2 mL of acetone (molecular biology grade) to precipitate RNA conjugate. Repeat step 3.

   将固体残留物溶解在 100 μL 中，并加入 1.2 mL 丙酮（分子生物学级）沉淀 RNA 共轭物。重复步骤 3。

5. Dissolve the product in water, and quantify the oligonucleotide by measuring the UV absorbance at 260 nm. Store the solution frozen.

   将产品溶解在水中，通过在 260 nm 处测量 UV 吸光度定量寡核苷酸。将溶液冷冻保存。

6. Check the purity of the product by PAGE as described below or ESI-MS (see Note 12).

   通过 PAGE 或 ESI-MS 检查产品的纯度（见 注释 12）

3.3.3 Analysis of GalNAc-RNA Conjugates by Polyacrylamide Gel Electrophoresis (Fig. 4b)通过聚丙烯酰胺凝胶电泳分析 GalNAc-RNA 共轭物

The denaturing gel electrophoresis of RNA conjugates is performed in 15% PAGE containing 7 M urea in Tris-borate buffer (50 mM Tris, 50 mM boric acid, 1 mM EDTA, pH 8.3).

在含有 7 M 尿素的 15%聚丙烯酰胺凝胶（PAGE）中，以 Tris-硼酸盐缓冲液（50 mM Tris，50 mM 硼酸，1 mM EDTA，pH 8.3）进行 RNA 共轭物的变性电泳分析。

1. Prepare large vertical gel chamber (gel size 200 × 200 × 1 mm) and pre-run the gel for 3 h at 200 V to remove salts. Dissolve an aliquot of RNA conjugates (4–5 nmol) in 20 μL of 80% formamide, denature the sample by heating at 90 °C for 3 min, then rapidly cool on ice.

   准备大型垂直凝胶室（凝胶尺寸 200 × 200 × 1 毫米），并在 200 伏特下预运行凝胶 3 小时以去除盐分。将一份 RNA 共轭物（4-5 纳摩尔）溶解在 20 微升的 80%甲酰胺中，样品在 90 摄氏度下加热 3 分钟进行变性，然后迅速在冰上冷却。

2. Flush the wells of the gel with Tris-borate buffer to remove precipitated urea and load the solution of RNA conjugates. Run PAGE at constant voltage (500 V) and stop electrophoresis when the marker dyes have migrated an appropriate distance. For RNA conjugates a running time of 2 h is sufficient.

   用 Tris-硼酸盐缓冲液冲洗凝胶孔以去除沉淀的尿素，然后加载 RNA 共轭物的溶液。以恒定电压（500 伏特）运行 PAGE，当标记染料迁移到适当距离时停止电泳。对于 RNA 共轭物，运行时间为 2 小时。

3. Slab PAGE can be visualized under 254 nm UV lamp using fluorescent TLC plate (TLC Silica gel 60G F254 or analog) as a substrate (see Note 13).

   在 254 纳米的紫外灯下使用荧光薄层色谱板（TLC 硅胶 60G F254 或类似产品）作为基底来可视化 PAGE 板（参见 注释 13）

Fig. 4 Analysis of GalNAc conjugated modified RNA purified by ion-exchange oligonucleotides. (a) Ion exchange HPLC profiles of conjugates. (b) Image of RNA conjugates separation using denaturative polyacrylamide gel electrophoresis

3.3.4 Annealing of GalNAc Modified siRNA Duplexes GalNAc 修饰的 siRNA 双链的退火

Combine solutions with equimolar amounts (~30 nmol) of complementary RNA—ON1–ON7, ON3–ON7, ON5–ON7, ON2–ON8, ON4–ON8, ON6–ON8 in individual 1.5-mL microcentrifuge tubes and evaporate solutions to dryness using a centrifugal vacuum concentrator. Add 200 μL of TE buffer to each tube and heat tubes to 95 °C, then turn off heating and slowly cool tubes to room temperature (at least 2–3 h) for duplex formation (see Note 14).

将等摩尔量（约 30 纳摩尔）的互补 RNA 溶液——ON1–ON7、ON3–ON7、ON5–ON7、ON2–ON8、ON4–ON8、ON6–ON8分别放入各自的 1.5 毫升微量离心管中，并使用离心真空浓缩器将溶液蒸干。向每个管中加入 200 微升的 TE 缓冲液，加热至 95 摄氏度，然后关闭加热，并缓慢冷却至室温（至少 2-3 小时）以形成双链（参见 注释 14）

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4 Notes

1. All reactions must be carried out in a fume hood with switched on ventilation. Personal protective glasses and lab coats must be used.

   所有反应必须在通风良好的化学通风罩下进行，必须佩戴个人防护眼镜和实验服。

2. All glassware should be oven dried, and all reactions should be performed under anhydrous conditions unless otherwise specified.

   所有玻璃器皿必须经过烘干处理，并且除非另有规定，所有反应都必须在无水条件下进行。

3. If you still see a dot of amine 1, add sequentially EDC·HCl (0.1 g, 0.5 mmol), HOBt (0.07 g, 0.5 mmol), and Et3N (0.07 mL, 0.5 mmol) to the reaction mixture and stir it overnight. Then repeat TLC analysis.

   如果仍然看到胺的小点 1，请依次向反应混合物中加入 EDC·HCl (0.1 g, 0.5 mmol), HOBt (0.07 g, 0.5 mmol) 和 Et3N (0.07 mL, 0.5 mmol)，并在室温下搅拌过夜。然后重复薄层色谱分析。

4. If the purity is below 95%, repeat step 4, Subheading 3.1.1 or carefully dry out the residual solvents.

   如果纯度低于 95%，请重复 步骤 4，3.1.1 小标题或仔细干燥残留溶剂。

5. Before termination reaction, you should be sure that intermediate amide and diamide are also fully converted into a final triamide.

   在终止反应之前，必须确保中间酰胺和二酰胺完全转化为最终的三酰胺。

6. If you observe partial conversion into the product, please repeat step 2, Subheading 3.1.3.

   如果观察到部分转化成产物，请重复 步骤 2，3.1.3 小标题。

7. If loading is below 40 μmol/g, then add diisopropylcarbodiimide (216 μL, 1.38 mmol) and wait for additional 12 h.

   如果负载量低于 40 μmol/g，则添加二异丙基碳二亚胺 (216 μL, 1.38 mmol)，等待额外 12 小时。

8. Do not exceed 5 min reaction time; otherwise partial loss of the triple GalNAc residue occurs.

   反应时间不得超过 5 分钟；否则会部分丢失三重 GalNAc 残基。

9. Thorough washing of the CPG support is the must; otherwise partial loss of the triple GalNAc residue occurs during storage. CPG product should be stored in a fridge (−20 °C) and warmed to RT before opening to avoid degradation.

   必须彻底清洗 CPG 支持物；否则会在储存过程中部分丢失三重 GalNAc 残基。CPG 产物应存放在冰箱中 (−20 °C)，并在开封前将其升温至室温以避免降解。

10. Purity of the phosphoramidite is crucial for successful synthesis of RNA conjugates. First, admixtures of H-phosphonate or phosphoramide (31P NMR (202 MHz, CD3CN) δ 10–25) should be accurately removed by column chromatography. Second, traces of triethylamine should be removed by repetitive co-evaporation with acetonitrile, especially if you scale up the protocol. Residual triethylamine will dramatically drop down the yield of RNA conjugates.

    磷酰胺基团的纯度对于成功合成 RNA 共轭物至关重要。首先，必须通过柱层析精确去除 H-磷酸酯或磷酰胺的混合物 (31P NMR (202 MHz, CD3CN) δ 10–25)。其次，尤其在扩展协议时，必须通过重复共蒸发与乙腈去除三乙胺的微量。残留的三乙胺会大幅降低 RNA 共轭物的产率。

11. Synthesis of RNA ON5 and ON6 (or other with 5′-GalNac modification) requires DMT-on mode. If you remove DMT group before ammonia-based deprotection, partial degradation of the conjugate will occur via reaction of the primary hydroxy group with the phosphate residue. We observed several degradation products after DMT-off synthesis of ON5 and ON6 that can be separated either by ion exchange HPLC or PAGE (Fig. 5, peaks (A) or bands (B) a, b, c). These products correspond to the GalNac-cleavaged oligonucleotides: a—with two GalNac residues, b—with one and c—without GalNAc as confirmed by ESI-MS. Oligonucleotide with the same sequence as ON5, but without 5′ modifications (ON9) was used as reference. To avoid the loss of 5′ GalNAc modifications, one should always use DMTr-ON mode and remove DMTr group after deblock.

    合成带有 5′-GalNAc 修饰的 RNA ON5 和 ON6 (或其他修饰) 需要使用 DMT-on 模式。如果在基于氨基的去保护之前去除 DMT 基团，将会导致共轭物的部分降解，主要是由于主要羟基与磷酸残基的反应。我们在对 ON5 和 ON6 进行 DMT-off 合成后观察到几种降解产物，可以通过离子交换 HPLC 或 PAGE 分离 (图 5，峰 (A) 或带 (B) a、b、c)。这些产物对应于经 GalNac 裂解的寡核苷酸：a—带有两个 GalNAc 残基，b—带有一个 GalNAc 残基，c—没有 GalNAc，经 ESI-MS 确认。具有与 ON5 相同序列但没有 5′修饰的寡核苷酸 (ON9) 用作参考。为避免丢失 5′ GalNAc 修饰，应始终使用 DMTr-ON 模式，并在去保护后去除 DMTr 基团。

12. PAGE usually provide better resolution than IE HPLC—one can compare HPLC traces at Fig. 4awith PAGE image at Fig. 4b. In case of ON6 (and in part for ON5) IE HPLC failed to uncover n-1 admixture.

    PAGE 通常比 IE HPLC 提供更好的分辨率——可以比较 HPLC 示踪图 (图 4a) 与 PAGE 图像 (图 4b)。对于 ON6 (以及部分 ON5)，IE HPLC 未能揭示 n-1 杂质。

13. Each lane should contain only one individual band—presence of the ladder or smears confirm low purity of the GalNAc-RNA conjugate. Depending on the purity and the amount, the GalNAc-RNA conjugate should be repurified or resynthesized.

    每条带中应仅包含一个单独的条带——阶梯或模糊带的存在确认了 GalNAc-RNA 共轭物的低纯度。根据纯度和数量的不同，GalNAc-RNA 共轭物应重新纯化或重新合成。

14. Store the solution of siRNA without freezing to maintain it as a duplex. If you need long-term storage at −20 °C or −80 °C, repeat annealing stage after unfreezing before further use.

    存储 siRNA 溶液时避免冷冻以保持其作为双链体的状态。如果需要长期存储在−20 °C 或−80 °C，请在解冻后重复退火阶段，然后再进行进一步使用。

Fig. 5 Analysis of crude modified 5′-(GalNAc)3-RNA conjugate ON5 and RNA ON9 . (A) Ion exchange HPLC profiles. (B) Image of RNA separation using denaturative polyacrylamide gel electrophoresis