这项研究由日本教育、文化、体育、科学和技术部 (下个15K00408、24659011、24640156、245900425和22390005号为新青木) 提供的助学金资助, 由东京生物化学研究资助。基金会, 东京, 日本, 和由土族 (东京科学大学) 基金的战略研究领域。我们要感谢纪子 Sawabe (东京理工大学药科学院) 测量核磁共振谱, 璐子长谷川 (美国东京理工大学药学系) 为质量的测量光谱和友子松 (东京理工大学科技研究院) 对元素分析的测量。

注: 所有实验数据 [核磁共振, 红外线 spectroscopies (IR), 质量 spectroscopies (MS), 光学旋转, 和元素分析数据] 的合成化合物被报告在前51篇。
1. O-糖基化反应的程序
<ol><li>复方α/β-12 的合成 (表1中的进入 12) 注:表 1中的条目 1-13 是使用类似的程序进行的。<ol>
<li>
2 ', 3 '-二醇 ribonucleoside40的临时保护
		<ol>
<li>在10毫升梨形烧瓶 (烧瓶 1), 溶解 mannosyl 捐赠α9 (28.4 毫克, 0.0486 毫摩尔)52, 苷10 (7.9 毫克, 0.0324 毫摩尔) 和 4-(三氟甲基) 苯硼酸11c (9.3 毫克, 0.0490 毫摩尔) 在无水吡啶 (0.40毫升)。 注: 建议使用10毫升梨形烧瓶, 因为, 在步骤 1.1.3.1, 反应混合物将转移到烧瓶 2 (一个10毫升的双颈圆底烧瓶附在它的隔膜) 含有分子筛粉。</li>
			<li>将反应混合物 (在步骤1.1.1.1 中获得) 与无水吡啶 (0.40 毫升, 3x) 和无水 14-二恶烷 (0.40 毫升, 3x) 在室温下至约40 °c, 以除去任何水。</li>
			<li>将残留物 (在步骤1.1.1.2 中获得) 溶解于无水 14-二恶烷 (0.32 毫升) 中, 并在其回流温度下搅动反应混合物 1 h 形成硼酸酯 (临时保护)。</li>
			<li>使用旋转蒸发器, 然后用真空泵除去溶剂。</li>
		</ol>
</li>
		<li>
分子筛的活化
		<ol>
<li>在一个10毫升的双颈圆底烧瓶与隔膜附加到它 (烧瓶 2), 添加4Å分子筛粉 (64 毫克)。 注: 应根据用于糖基化的溶剂 (乙腈3Å和4Å 14-二恶烷、二氯甲烷和-丙腈) 选择合适的分子筛。</li>
			<li>在气压下加热分子筛, 在真空泵 (3x) 减压下冷却它们, 然后用热枪在减压的同时用氩气代替空气, 然后将其烘干几次。</li>
		</ol>
</li>
		<li>
糖化
		<ol>
<li>溶解步骤1.1.1.4 的残留物。在瓶1在-丙腈 (0.64 毫升) 或其他溶剂和转移这个解答到烧瓶2。 注: 乙腈, 14-二恶烷, 氯甲烷和-丙腈用于条目 1-7 和 9, 入口 10, 入口 11, 和条目 8, 12 和 13, 分别。</li>
			<li>搅拌反应混合物在瓶2在室温0.5 小时之后冷却它到-40 °c。 注: 温度是根据用于糖基化的溶剂 (40 °c 为二氯甲烷和-丙腈, 室温为14二恶烷,-20 °c 为乙腈) 而改变的。</li>
			<li>添加银三氟 (49.9 毫克, 0.194 毫摩尔) 和对 toluenesulfenyl 氯 (12.8 µL, 0.0968 毫摩尔) 的反应混合物在相同的温度, 在步骤1.1.3.2 使用。</li>
			<li>将反应混合物在相同温度下搅拌1.5 小时。</li>
			<li>用薄层层析 (TLC) 与己烷/乙酸乙酯检查反应 [3/1 (v/v)] 检查糖捐赠者 [保留因子 (Rf) (捐赠者α-9) = 0.63] 并且与氯仿或甲醇 [10/1 (v/v)]] 检查糖受众和产品 [rf (承兑人10) = 0.03, Rf (期望的产品) = 0.50]。</li>
			<li>用饱和水碳酸氢钠 (1.0 毫升) 淬火反应混合物, 用氯仿 (2.0 毫升) 稀释, 用硅藻土去除不溶物, 并用氯仿 (20 毫升) 仔细冲洗硅藻土。</li>
			<li>使用100毫升部位漏斗, 用饱和水碳酸氢钠 (20 毫升, 3x) 和盐水 (20 毫升) 洗涤滤液 (有机层)。</li>
			<li>用硫酸钠干燥产生的有机层, 过滤不溶性物质, 用旋转蒸发器将滤液浓缩。</li>
			<li>用柱层析法对剩余残留物进行大致净化 [硅胶, 氯仿/甲醇 = 1/0-50/1 (v/v)] 以提供粗 5 'o(6 "-acetyl-2", 3 ", 4"-三O苄基α/β-ᴅ-mannopyranosyl) 苷含有少量的副产品 (15.2 毫克, 无色糖浆)。</li>
		</ol>
</li>
		<li>
乙酰 化
		<ol>
<li>在5毫升的小瓶中, 溶解在无水吡啶 (0.20 毫升) 中的步进1.1.3.9 中制备的粗化合物。</li>
			<li>添加n,n-dimethyl-4-aminopyridine (催化量) 和醋酸酐 (20.4 µL, 0.0216 毫摩尔:10 当量根据原油化合物) 的溶液在0摄氏度。</li>
			<li>将反应混合物在相同温度下搅拌0.5 小时, 然后加热至室温。</li>
			<li>搅拌一夜后, 用氯仿/甲醇 (30/1 (v/v)][Rf (α/β-12) = 0.45], 用薄层色谱法检查反应。</li>
			<li>用氯仿稀释反应混合物 (20 毫升)。</li>
			<li>用1米盐酸 (20 毫升, 3x), 饱和水碳酸氢钠 (20 毫升, 3x) 和盐水 (20 毫升) 洗涤有机层, 使用100毫升部位漏斗。</li>
			<li>用硫酸钠干燥产生的有机层, 过滤不溶性物质, 用旋转蒸发器将滤液浓缩。</li>
			<li>用柱层析法净化剩余残留物 [硅胶, 氯仿/甲醇 = 1/0-90/1 (v/v)] 给α/β-12 (15.8 毫克, 61%, α/β = 1.6/1, 无色无定形固体)。</li>
		</ol>
</li>
	</ol></li>
	<li>化合物的合成β-22 到β-30 (表 2) 和β-33 (表 3) 注: 合成β-22β30和β-33是用类似的程序进行的。<ol><li>复方β-22 的合成 (表2中的入口 1)
		<ol>
<li>
ribonucleoside 2 '、3 '-二醇的临时保护
			<ol>
<li>在10毫升梨形烧瓶 (烧瓶 3), 溶解腺苷13 (20.4 毫克, 0.0763 毫摩尔), 半乳糖捐助者β21 (80.4 毫克, 0.114 毫摩尔)53, 和 4-(三氟甲基) 苯硼酸11c (21.7 毫克, 0.114 毫摩尔) 在无水吡啶 (0.76 毫升)。 注: 建议使用10毫升梨形烧瓶, 因为反应混合物将转移到烧瓶 4 (一个10毫升的双颈圆底烧瓶附在它的隔膜) 包含分子筛粉在步骤 1.2. 1.3. 1。</li>
				<li>用无水吡啶 (0.76 毫升, 3x) 和无水 14-二恶烷 (0.76 毫升, 3x) 在室温下, 将反应混合物 (在步骤 1.2. 1.1. 1) 中蒸发。</li>
				<li>在无水14二恶烷 (0.76 毫升) 中溶解残留物 (在步骤 1.2 1.1. 2 中获得), 并在其回流温度下搅拌反应混合物, 以形成硼酸酯 (临时保护)。</li>
				<li>使用旋转蒸发器, 然后用真空泵除去溶剂。</li>
			</ol>
</li>
			<li>
分子筛的活化
			<ol>
<li>在一个10毫升的双颈圆底烧瓶与隔膜附加到它 (烧瓶 4), 添加4Å分子筛粉 (150 毫克)。</li>
				<li>在气压下加热分子筛, 在真空泵 (3x) 减压下冷却它们, 然后用热枪在减压的同时用氩气代替空气, 然后将其烘干几次。</li>
			</ol>
</li>
			<li>
糖化
			<ol>
<li>溶解步骤1.2 的残留物 1.1. 4。在瓶3在-丙腈 (1.50 毫升) 和转移这个解答到烧瓶4。</li>
				<li>将反应混合物在室温下搅拌0.5 小时, 然后冷却到-40 摄氏度。</li>
				<li>添加银三氟 (117.6 毫克, 0.458 毫摩尔) 和对 toluenesulfenyl 氯 (30.3 µL, 0.229 毫摩尔) 的反应混合物在相同的温度, 如步骤 1.2. 1.3. 2。</li>
				<li>搅拌反应混合物, 在相同的温度为1.5 小时。</li>
				<li>用己烷/乙酸乙酯薄层色谱法检查反应 [2/1 (v/v)] 检查糖捐赠者 [Rf (捐助者β-21) = 0.62] 并且与氯仿或甲醇 [10/1 (v/v)] 检查糖接受物和产品 [Rf (承兑13) = 0.05, Rf (想要的产品) = 0.30]。</li>
				<li>用饱和水碳酸氢钠 (2.0 毫升) 淬火反应混合物, 用氯仿 (3.0 毫升) 稀释, 通过硅藻土去除不溶性物质, 并用氯仿 (30 毫升) 仔细冲洗硅藻土。</li>
				<li>使用100毫升部位漏斗, 用饱和水碳酸氢钠 (30 毫升, 3x) 和盐水 (30 毫升) 洗涤滤液 (有机层)。</li>
				<li>用硫酸钠干燥产生的有机层, 过滤不溶性物质, 用旋转蒸发器将滤液浓缩。</li>
				<li>用柱层析法净化剩余残留物 [硅胶, 氯仿/甲醇 = 1/0-30/1 (v/v)], 以支付β-22 (27.4 毫克, 42%, 无色固体)。</li>
			</ol>
</li>
		</ol></li>
		<li>复方β-23 的合成 (表2中的入口 2)
		<ol>
<li>使用14 (28.4 毫克, 0.0765 毫摩尔)54, β21 (80.5 毫克, 0.115 毫摩尔), 11c (21.8 毫克, 0.115 毫摩尔), 对 toluenesulfenyl 氯化物 ( 30.3 µL, 0.229 毫摩尔), 银三氟 (117.8 毫克, 0.458 毫摩尔), 无水的反应14-二恶烷 (0.76 毫升), 无水-丙腈 (1.50 毫升) 和4Å分子筛 (150 毫克)。用柱层析法净化产生的残渣 [硅胶, 氯仿/甲醇 = 1/0-50/1 (v/v)] 给β-23 (21.9 毫克, 30%, 无色固体)。薄层色谱: Rf (β-23) = 0.37 [氯仿/甲醇 = 10/1 (v/v)]。</li>
		</ol></li>
		<li>复方β-24 的合成 (表2中的入口 3)
		<ol>
<li>进行反应使用15 (21.6 毫克, 0.0763 毫摩尔), β-21 (80.5 毫克, 0.115 毫摩尔), 11c (21.8 毫克, 0.115 毫摩尔), 对toluenesulfenyl 氯化物 (30.3 µL, 0.229 毫摩尔), 银三氟 (117.6 毫克, 0.458 毫摩尔), 无水 14-二恶烷 (0.76 毫升), 无水-丙腈 (1.50 毫升), 4 Å分子筛 (150 毫克)。用柱层析法净化产生的残渣 [硅胶, 氯仿/甲醇 = 1/0-8/1 (v/v)] 给β-24 (8.1 毫克, 12%, 无色固体)。薄层色谱: Rf (β-24) = 0.20 [氯仿/甲醇 = 10/1 (v/v)]。</li>
		</ol></li>
		<li>复方β-25 的合成 (表2中的入口 4)
		<ol>
<li>使用16 (27.0 毫克, 0.0764 毫摩尔)55, β21 (80.5 毫克, 0.115 毫摩尔), 11c (21.8 毫克, 0.115 毫摩尔), 对 toluenesulfenyl 氯化物 ( 30.3 µL, 0.229 毫摩尔), 银三氟 (117.8 毫克, 0.458 毫摩尔), 无水的反应14-二恶烷 (0.76 毫升), 无水-丙腈 (1.50 毫升) 和4Å分子筛 (150 毫克)。用柱层析法净化产生的残渣 [硅胶, 氯仿/甲醇 = 1/0-20/1 (v/v)] 给β-25 (31.4 毫克, 44%, 无色固体)。薄层色谱: Rf (β-25) = 0.27 [氯仿/甲醇 = 10/1 (v/v)]。</li>
		</ol></li>
		<li>复方β-26 的合成 (表2中的入口 5)
		<ol>
<li>进行反应使用10 (18.6 毫克, 0.0762 毫摩尔), β-21 (80.4 毫克, 0.114 毫摩尔), 11c (21.7 毫克, 0.114 毫摩尔), 对toluenesulfenyl 氯化物 (30.3 µL, 0.229 毫摩尔), 银三氟 (117.6 毫克, 0.458 毫摩尔), 无水 14-二恶烷 (0.76 毫升), 无水-丙腈 (1.50 毫升), 4 Å分子筛 (150 毫克)。用柱层析法净化产生的残渣 [硅胶, 氯仿/甲醇 = 1/0-40/1 (v/v)] 给β-26 (26.1 毫克, 42%, 无色固体)。薄层色谱: Rf (β-26) = 0.45 [氯仿/甲醇 = 10/1 (v/v)]。</li>
		</ol></li>
		<li>复方β-27 的合成 (表2中的入口 6)
		<ol>
<li>进行反应使用17 (19.7 毫克, 0.0763 毫摩尔), β-21 (80.5 毫克, 0.115 毫摩尔), 11c (21.8 毫克, 0.115 毫摩尔), 对toluenesulfenyl 氯化物 (30.3 µL, 0.229 毫摩尔), 银三氟 (117.6 毫克, 0.458 毫摩尔), 无水 14-二恶烷 (0.76 毫升), 无水-丙腈 (1.50 毫升), 4 Å分子筛 (150 毫克)。用柱层析法净化产生的残渣 [硅胶, 氯仿/甲醇 = 1/0-40/1 (v/v)] 给β-27 (33.8 毫克, 53%, 无色固体)。薄层色谱: Rf (β-27) = 0.50 [氯仿/甲醇 = 10/1 (v/v)]。</li>
		</ol></li>
		<li>复方β-28 的合成 (表2中的入口 7)
		<ol>
<li>进行反应使用18 (20.0 毫克, 0.0763 毫摩尔), β-21 (80.4 毫克, 0.114 毫摩尔), 11c (21.7 毫克, 0.114 毫摩尔), 对toluenesulfenyl 氯化物 (30.3 µL, 0.229 毫摩尔), 银三氟 (117.6 毫克, 0.458 毫摩尔), 无水 14-二恶烷 (0.76 毫升), 无水-丙腈 (1.50 毫升), 4 Å分子筛 (150 毫克)。用柱层析法净化产生的残渣 [硅胶, 氯仿然后乙酸乙酯/氯仿 = 1/1 (v/v)] 给β-28 (38.8 毫克, 61%, 无色固体)。薄层色谱: Rf (β-28) = 0.33 [氯仿/甲醇 = 10/1 (v/v)]。</li>
		</ol></li>
		<li>复方β-29 的合成 (表2中的入口 8)
		<ol>
<li>进行反应使用19 (18.5 毫克, 0.0761 毫摩尔), β-21 (80.4 毫克, 0.114 毫摩尔), 11c (21.7 毫克, 0.114 毫摩尔), 对toluenesulfenyl 氯化物 (30.3 µL, 0.229 毫摩尔), 银三氟 (117.6 毫克, 0.458 毫摩尔), 无水 14-二恶烷 (0.76 毫升), 无水-丙腈 (1.50 毫升), 4 Å分子筛 (150 毫克)。用柱层析法净化产生的残渣 [硅胶, 氯仿/甲醇 = 1/0-10/1 (v/v)] 给β-29 (34.1 毫克, 55%, 无色固体)。薄层色谱: Rf (β-29) = 0.25 [氯仿/甲醇 = 10/1 (v/v)]。</li>
		</ol></li>
		<li>复方β-30 的合成 (表2中的入口 9)
		<ol>
<li>使用20 (26.6 毫克, 0.0766 毫摩尔)56, β21 (80.6 毫克, 0.115 毫摩尔), 11c (21.8 毫克, 0.115 毫摩尔), 对 toluenesulfenyl 氯化物 ( 30.3 µL, 0.229 毫摩尔), 银三氟 (117.8 毫克, 0.458 毫摩尔), 无水的反应14-二恶烷 (0.76 毫升), 无水-丙腈 (1.50 毫升) 和4Å分子筛 (150 毫克)。用柱层析法净化产生的残渣 [硅胶, 氯仿/甲醇 = 1/0-50/1 (v/v)] 给β-30 (28.0 毫克, 40%, 无色固体)。薄层色谱: Rf (β-30) = 0.48 [氯仿/甲醇 = 10/1 (v/v)]。</li>
		</ol></li>
		<li>复方β-33 的合成 (表3中的入口 1)
		<ol>
<li>使用18 (20.0 毫克, 0.0762 毫摩尔), β-31 (80.4 毫克, 0.114 毫摩尔)57, 11c (21.7 毫克, 0.114 毫摩尔), 对 toluenesulfenyl 氯化物(30.3 µL, 0.229 毫摩尔), 银三氟 (117.6 毫克, 0.458 毫摩尔), 无水的反应14-二恶烷 (0.76 毫升), 无水-丙腈 (1.50 毫升) 和4Å分子筛 (150 毫克)。用柱层析法净化产生的残渣 [硅胶, 氯仿/甲醇 = 1/0-30/1 (v/v)] 给β-33 (34.5 毫克, 54%, 无色固体)。薄层色谱: Rf (β-33) = 0.33 [氯仿/甲醇 = 10/1 (v/v)]。</li>
		</ol></li>
	</ol></li>
</ol>2. β-28 的脱 (图 2)
<ol><li>在5毫升瓶中, 加入β-28 (25.2 毫克, 0.0300 毫摩尔) 和 10 M 甲胺在甲醇 (2.0 毫升)58。</li>
	<li>搅拌的反应混合物在0°c 2 小时后, 升温到室温。</li>
	<li>搅拌后的混合物为13小时, 检查反应用氯仿/甲醇的薄层色谱 [10/1 (v/v)][Rf (β-35) = 0.20]。</li>
	<li>使用旋转蒸发器将反应混合物浓缩。</li>
	<li>用50毫升部位漏斗溶解在水中产生的残渣 (15 毫升), 并用二氯甲烷 (15 毫升, 3x) 冲洗水层。</li>
	<li>使用旋转蒸发器将水层浓缩。</li>
	<li>制备高效液相色谱 (HPLC) [柱: ODS (octadecylsilane) 柱 (20Φ x 250 毫米), 淋洗: 水 (含 0.1% [v/v] 三氟乙酸酸), 流速: 8.0 毫升/分, 检测: 266 nm,温度:25 °c, 保留时间:20 分钟] 给β-35 (7.9 毫克, 62%, 无色无定形固体)59。</li>
</ol>3. 循环硼酸酯的核磁共振研究 (图 3和4)
<ol><li>
36的制备和测量
	<ol>
<li>在10毫升梨形烧瓶中, 溶解苷10 (34.3 毫克, 0.140 毫摩尔) 和 4-三氟甲基苯硼酸11c (40.0 毫克, 0.211 毫摩尔) 在无水吡啶 (1.00 毫升)。</li>
		<li>将与无水吡啶 (1.00 毫升, 3x) 和无水14二恶烷 (1.00 毫升, 3x) 的反应混合物在室温下与40摄氏度共蒸发, 以除去任何水。</li>
		<li>在无水 14-二恶烷 (1.40 毫升) 中溶解残渣, 在其回流温度下搅拌反应混合物, 以形成硼酸酯 (临时保护)。</li>
		<li>将反应混合物 (0.14 毫升) 分配给5毫升瓶。</li>
		<li>使用旋转蒸发器, 然后用真空泵将溶剂从5毫升瓶中取出。</li>
		<li>溶解产生的残留物36在乙腈-d3 (0.64 毫升)。</li>
		<li>测量1H, 11B 和19F 核磁共振 spectroscopies 使用石英核磁共振管在25°c。</li>
	</ol>
</li>
	<li>
38的制备和测量
	<ol>
<li>使用与步骤3.1 类似的步骤, 从11c (40.0 毫克, 0.211 毫摩尔) 中制备反应混合物38 。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

本手稿的目的是展示一个方便的合成方法, 以制备双糖苷使用无保护的 ribonucleosides, 而不繁琐的保护组操作。我们在此报告的选择性O-glycosylations 的苷通过临时 2 ', 3 '-二醇保护的循环硼酸酯 (图 1B)51。
循环硼酸酯中间体的制备是其中的重要步骤之一。无水溶剂应用于反应混合物的共同蒸发 (步骤1.1.1.2 和 1.2. 1.1....

双糖苷, 是核苷的共轭物和通过 O型糖苷键连接的碳水化合物基团, 构成了一类自然发生的碳水化合物衍生物1,2 ,3,4,5,6,7。例如, 它们被纳入生物大分子, 如 tRNA (转移核糖核酸) 和聚 (adp-核糖) (adp = 磷酸腺苷), 以及一些抗菌剂和其他生物活性物质 (例如,adenophostins, amicetins, ezomycin)5,6,8,9,10,11,12,13, 14,15,16,17,18,19。因此, 双糖苷及其衍生物有望成为药物发现研究的铅化合物。双糖苷的合成方法分为三类;酶法O-糖基化20,21, 化学n-糖基化5,9,16,22,23, 24、化学O-糖基化7、9、14、16、18、19、24、 25,26,27,28,29,30,31,32,33, 34,35,36,37。化学O-糖基化是一种有效的立体合成和双糖苷的大规模合成方法。以往的研究表明, 2 '-deoxyribonucleoside 2与 thioglycosyl 捐助者1的O糖基化, 采用 toluenesulfenyl 氯和银三氟的结合, 提供了理想的双糖核苷3 (图 1A;Ar = 芳和 PG = 保护组)38。
根据这些结果, 我们决定应用 toluenesulfenyl 氯化/银三氟启动子系统, 开发 ribonucleosides 的邻糖基化。虽然部分保护 ribonucleosides 的O-糖基化的几个例子已经证明了 7,9,14,16,18,19 、24、32、33、34、35、36、37、使用无保护或暂时保护ribonucleosides 作为糖受体的O-糖基化已微乎其微报道。因此, 不受保护的或暂时保护的 ribonucleosides 的选择性O-糖基化的发展将提供一种更有益的合成方法, 而不需要对 ribonucleosides 的群操作进行防护。为了实现 ribonucleosides 的选择性O-糖基化, 我们重点研究了硼化合物, 因为几个例子的内和/或立体酰化, 烷基, 硅烷化和糖基性的碳水化合物39,40,41,42,43,44,45 , 被硼酸或 borinic 酸辅助的衍生物报告了,46,47,48,49,50。在本文中, 我们展示了通过硼酸酯中间体在 ribonucleosides 的 5 '-羟基双糖苷中利用选择性邻糖基化合成的方法。在这里提出的战略, 硼酸酯中间体6将提供的酯化的 ribonucleoside 4与硼酸5, 这使得选择性O-糖基化在5 '-羟基基团与 thioglycosyl 捐赠者7给双糖核苷8 (图 1B)51。通过核磁共振 (NMR) 光谱学研究了 ribonucleoside 和硼酸的相互作用, 观察了硼酸酯的形成。酯化使硼酸酯和糖基化反应要求无水条件, 以防止硼酸酯和糖的水解。在这篇文章中, 我们展示了为研究人员和学生不仅在化学, 而且在其他研究领域获得成功的糖基化反应提供无水条件的典型程序。

<table><tbody><tr><td>Silver trifluoromethanesulfonate</td><td>Nacalai Tesque</td><td>34945-61</td><td></td></tr><tr><td>Phenylboronic acid (contains varying amounts of anhydride)</td><td>Tokyo Chemical Industry</td><td>B0857</td><td></td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-Methoxyphenylboronic acid</td><td>Wako Pure Chemical Industries</td><td>321-69201</td><td></td></tr><tr><td>4-(Trifluoromethyl)phenylboronic acid (contains varying amounts of anhydride)</td><td>Tokyo Chemical Industry</td><td>T1788</td><td></td></tr><tr><td>2,4-Difluorophenylboronic acid (contains varying amounts of anhydride)</td><td>Tokyo Chemical Industry</td><td>D3391</td><td></td></tr><tr><td>Cyclopentylboronic acid (contains varying amounts of Anhydride)</td><td>Tokyo Chemical Industry</td><td>C2442</td><td></td></tr><tr><td>4-Nitrophenylboronic acid (contains varying amounts of anhydride)</td><td>Tokyo Chemical Industry</td><td>N0812</td><td></td></tr><tr><td>4-Hexylphenylboronic acid (contains varying amounts of anhydride)</td><td>Tokyo Chemical Industry</td><td>H1489</td><td></td></tr><tr><td>Adenosine</td><td>Merck KGaA</td><td>862.</td><td></td></tr><tr><td>Guanosine</td><td>Acros Organics</td><td>411130050</td><td></td></tr><tr><td>Cytidine</td><td>Tokyo Chemical Industry</td><td>C0522</td><td></td></tr><tr><td>Uridine</td><td>Tokyo Chemical Industry</td><td>U0020</td><td></td></tr><tr><td>5-Fluorouridine</td><td>Tokyo Chemical Industry</td><td>F0636</td><td></td></tr><tr><td>5-Methyluridine</td><td>Sigma</td><td>M-9885</td><td></td></tr><tr><td>Methylamine (40% in Methanol,&amp;nbsp;ca. 9.8mol/L)</td><td>Tokyo Chemical Industry</td><td>M1016</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-dimethyl-4-aminopyridine</td><td>Wako Pure Chemical Industries</td><td>044-19211</td><td></td></tr><tr><td>Acetic anhydride</td><td>Nacalai Tesque</td><td>00226-15</td><td></td></tr><tr><td>Pyridine, Dehydrated</td><td>Wako Pure Chemical Industries</td><td>161-18453</td><td></td></tr><tr><td>Acetonitrile</td><td>Kanto Chemical</td><td>01031-96</td><td></td></tr><tr><td>1,4-Dioxane</td><td>Nacalai Tesque</td><td>13622-73</td><td></td></tr><tr><td>Dichloromethane</td><td>Wako Pure Chemical Industries</td><td>130-02457</td><td></td></tr><tr><td>Propionitrile</td><td>Wako Pure Chemical Industries</td><td>164-04756</td><td></td></tr><tr><td>Molecular sieves 4A powder</td><td>Nacalai Tesque</td><td>04168-65</td><td></td></tr><tr><td>Molecular sieves 3A powder</td><td>Nacalai Tesque</td><td>04176-55</td><td></td></tr><tr><td>Celite 545RVS</td><td>Nacalai Tesque</td><td>08034-85</td><td></td></tr><tr><td>Acetonitrile-D3 (D,99.8%)</td><td>Cambridge Isotope Laboratories</td><td>DLM-21-10</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Nacalai Tesque</td><td>34831-25</td><td></td></tr><tr><td>TLC Silica gel 60 F&lt;sub&gt;254&lt;/sub&gt;</td><td>Merck KGaA</td><td>1.05715.0001</td><td></td></tr><tr><td>Chromatorex</td><td>Fuji Silysia Chemical</td><td>FL100D</td><td></td></tr><tr><td>Sodium hydrogen carbonate</td><td>Wako Pure Chemical Industries</td><td>191-01305</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Wako Pure Chemical Industries</td><td>080-01061</td><td></td></tr><tr><td>Sodium sulfate</td><td>Nacalai Tesque</td><td>31915-96</td><td></td></tr><tr><td>Chloroform</td><td>Kanto Chemical</td><td>07278-81</td><td></td></tr><tr><td>Sodium chloride</td><td>Wako Pure Chemical Industries</td><td>194-01677</td><td></td></tr><tr><td>Methanol</td><td>Nacalai Tesque</td><td>21914-74</td><td></td></tr><tr><td>JEOL Always 300</td><td>JEOL</td><td></td><td>Measurement of NMR</td></tr><tr><td>Lamda 400</td><td>JEOL</td><td></td><td>Measurement of NMR</td></tr><tr><td>PerkinElmer Spectrum 100 FT-IR Spectrometer</td><td>Perkin Elmer</td><td></td><td>Measurement of IR</td></tr><tr><td>JEOL JMS-700</td><td>JEOL</td><td></td><td>Measurement of MS</td></tr><tr><td>PerkinElmer CHN 2400 analyzer</td><td>Perkin Elmer</td><td></td><td>Measurement of elemental analysis</td></tr><tr><td>JASCO P-1030 digital polarimeter</td><td>JASCO</td><td></td><td>Measurement of optical rotation</td></tr><tr><td>JASCO PU-2089 Plus intelligent HPLC pump</td><td>JASCO</td><td></td><td>For HPLC</td></tr><tr><td>Jasco UV-2075 Plus Intelligent UV/Vis Detector</td><td>JASCO</td><td></td><td>For HPLC</td></tr><tr><td>Rheodyne Model 7125 Injector</td><td>Sigma-Aldrich</td><td>58826</td><td>For HPLC</td></tr><tr><td>Chromatopac C-R8A</td><td>Shimadzu</td><td></td><td>For HPLC</td></tr><tr><td>Senshu Pak Pegasil ODS</td><td>Senshu Scientific</td><td></td><td>For HPLC</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-Toluenesulfenyl&amp;nbsp;chloride</td><td></td><td></td><td>Prepared&amp;nbsp; Ref. 38</td></tr><tr><td>Phenyl 6-O-acetyl-2,3,4-tri-&lt;em&gt;O&lt;/em&gt;-benzyl-1-thio-a-D-mannopyranoside (a-&lt;strong&gt;9&lt;/strong&gt;)</td><td></td><td></td><td>Prepared&amp;nbsp; Ref. 52</td></tr><tr><td>4-Metylphenyl 2,3,4,6-tetra-&lt;em&gt;O&lt;/em&gt;-benzoyl-1-thio-b-D-galactopyranoside (b-&lt;strong&gt;21&lt;/strong&gt;)</td><td></td><td></td><td>Prepared&amp;nbsp; Ref. 53</td></tr><tr><td>4-Metylphenyl 2,3,4,6-tetra-&lt;em&gt;O&lt;/em&gt;-benzoyl-1-thio-b-D-glucopyranoside (b-&lt;strong&gt;31&lt;/strong&gt;)</td><td></td><td></td><td>Prepared&amp;nbsp; Ref. 57</td></tr><tr><td>4-Metylphenyl 2,3,4,6-tetra-&lt;em&gt;O&lt;/em&gt;-benzoyl-1-thio-a-D-Mannopyranoside (a-&lt;strong&gt;32&lt;/strong&gt;)</td><td></td><td></td><td>Prepared&amp;nbsp; Ref. 67</td></tr><tr><td>6-&lt;em&gt;N&lt;/em&gt;-Benzoyladenosine (&lt;strong&gt;14&lt;/strong&gt;)</td><td></td><td></td><td>Prepared&amp;nbsp; Ref. 54</td></tr><tr><td>2-&lt;em&gt;N&lt;/em&gt;-Isobutyrylguanosine (&lt;strong&gt;16&lt;/strong&gt;)</td><td></td><td></td><td>Prepared&amp;nbsp; Ref. 55</td></tr><tr><td>4-&lt;em&gt;N&lt;/em&gt;-Benzoylcytidine (&lt;strong&gt;20&lt;/strong&gt;)</td><td></td><td></td><td>Prepared&amp;nbsp; Ref. 56</td></tr></tbody></table>

regioselective o-glycosylation of nucleosides via the temporary 2',3'-diol protection by a boronic ester for the synthesis of disaccharide nucleosides

由双糖和碱基基团组成的双糖苷被称为具有多种生物活性的有价值的天然产物。虽然化学O-糖基化是一种普遍有益的战略, 以合成双糖苷, 制备基质如糖捐赠者和受体需要繁琐的保护组操作和净化每个合成步骤。同时, 一些研究小组报告说, 硼酸和 borinic 酯作为一种保护或活化的碳水化合物衍生物, 以实现立体酰化、烷基化、硅烷化和糖基性。本文介绍了利用硼酸对无保护 ribonucleosides 进行选择性邻糖基化的过程。以硼酸为 ribonucleosides 的 2 ', 3 '-二醇的酯化, 使二醇的临时保护, 并在 toluenesulfenyl 氯和银三氟存在后, 与糖的捐助者进行邻糖基化, 允许5 '-羟基的选择性反应, 以支付双糖苷。该方法可应用于各种苷, 如鸟嘌呤、腺苷、胞苷、苷、5-metyluridine 和 5-fluorouridine。这篇文章和随附的视频代表了有用的 (视觉) 信息的O-糖基化的无保护苷及其类似物的合成不仅双糖苷, 而且还有各种生物相关衍生物。

在表 160,61中总结了苷10与 thiomannoside α-9的邻糖基化的结果。在进入 1, O-糖基化10与α-9在没有硼酸衍生物导致形成一个复杂混合物。进入 2, 10和苯硼酸11a混合, 并与吡啶和14二恶烷共蒸发, 然后, 在其回流温度下, 在14二恶烷中搅?...

Watch this Scientific Journal Video about Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides at JoVE

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

在这里, 我们提出了合成双糖苷的协议, 选择性O-糖基化的 ribonucleosides通过临时保护其 2 ', 3 '-二醇的基团利用循环硼酸酯。该方法适用于几种不受保护的苷, 如腺苷、鸟嘌呤、胞苷、苷、5-methyluridine 和 5-fluorouridine, 给予相应的双糖苷。

苷 2 ', 3 ' -二醇酯对双糖苷合成的选择性O-糖基化

Disaccharide nucleosides, which consist of disaccharide and nucleobase moieties, have been known as a valuable group of natural products having ...

regioselective-o-glycosylation-nucleosides-via-temporary-2-3-diol

Research

JoVE Journal

Chemistry

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

8.6K 次观看.  Tokyo University of Science. 在这里, 我们提出了合成双糖苷的协议, 选择性O-糖基化的 ribonucleosides通过临时保护其 2 ', 3 '-二醇的基团利用循环硼酸酯。该方法适用于几种不受保护的苷, 如腺苷、鸟嘌呤、胞苷、苷、5-methyluridine 和 5-fluorouridine, 给予相应的双糖苷。

视频: Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.
	
	In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.

The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.

核苷三磷酸 - 从合成到生化特征

The  traditional strategy for the introduction of chemical functionalities is the  use of solid-phase synthesis by appending suitably modified ...

科研

化学

Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are rapidly oxidized, for example by atmospheric oxygen, to indigo type dyes. This reaction enables fast and easy screening in vivo without isolation or purification of enzymes, as well as rapid tests on agar plates or in solution (e.g., blue-white screening, micro-wells) and is used in biochemistry, histochemistry, bacteriology and molecular biology. Unfortunately the synthesis of such substrates proved to be difficult, due to various side reactions and the low reactivity of the indoxyl hydroxyl function. Especially for glucose type structures low yields were observed. Our novel approach employs indoxylic acid ester as key intermediates. Indoxylic acid esters with varied substitution patterns were prepared on scalable pathways. Phase transfer glycosylations with those acceptors and peracetylated glycosyl halides can be performed under common conditions in high yields. Ester cleavage and subsequent mild silver mediated glycosylation yields the peracetylated indoxyl glycosides in high yields. Finally deprotection is performed according to Zempl&#233;n.

Indoxyl glycosides are well-established and widely used tools for enzyme screening and enzyme activity monitoring. Especially for glucose type structures previous syntheses proved to be challenging and low yielding. Our novel approach employs indoxylic acid esters as precious intermediates to yield a considerable number of indoxyl glycosides in good yields.

的吲哚酚苷的检测糖苷酶活动的合成

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

碳水化合物和聚酸酐纳米粒子功能化的高通量合成

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

生物工程

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.1,2
The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically1 or analytically,2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.1,2,7
In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.
In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein &#x2013; strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.

通过Bioorthogonal点击化学化学选择性病毒表面改性

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

免疫与感染

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise &#945;-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial &#946;-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man5GlcNAc2 (Man5) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

用于阵列发育和 HIV 抗体分析的糖的化学酶法合成

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are ...

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5&#39;-[CUA CGG AAU CAU]-3&#39; containing zero, one, or two modifications located at the C2&#39;-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2&#39;-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2&#39;-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

用于固相合成含有2&#39;-<em&gt; 0</em通过圆二色性修饰和表征

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...

Hierarchical and Programmable One-Pot Oligosaccharide Synthesis

This article presents a general experimental protocol for programmable one-pot oligosaccharide synthesis and demonstrates how to use Auto-CHO software for generating potential synthetic solutions. The programmable one-pot oligosaccharide synthesis approach is designed to empower fast oligosaccharide synthesis of large amounts using thioglycoside building blocks (BBLs) with the appropriate sequential order of relative reactivity values (RRVs). Auto-CHO is a cross-platform software with a graphical user interface that provides possible synthetic solutions for programmable one-pot oligosaccharide synthesis by searching a BBL library (containing about 150 validated and &#62;50,000 virtual BBLs) with accurately predicted RRVs by support vector regression. The algorithm for hierarchical one-pot synthesis has been implemented in Auto-CHO and uses fragments generated by one-pot reactions as new BBLs. In addition, Auto-CHO allows users to give feedback for virtual BBLs to keep valuable ones for further use. One-pot synthesis of stage-specific embryonic antigen 4 (SSEA-4), which is a pluripotent human embryonic stem cell marker, is demonstrated in this work.

This protocol demonstrates how to use the Auto-CHO software for hierarchical and programmable one-pot synthesis of oligosaccharides. It also describes the general procedure for RRV determination&#160;experiments and one-pot glycosylation of SSEA-4.

分层和可编程单罐寡糖合成

This article presents a general experimental protocol for programmable one-pot oligosaccharide synthesis and demonstrates how to use Auto-CHO software for ...

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded oligomeric strands mediated by paired dynamic covalent interactions in a manner mimicking the thermal cycling commonly employed for the self-assembly of complementary nucleic acid sequences. Primary amine monomers bearing aldehyde and amine pendant moieties are functionalized with orthogonal protecting groups for use as dynamic covalent reactant pairs. Using a modified automated peptide synthesizer, the primary amine monomers are encoded into oligo(peptoid) strands through solid-phase submonomer synthesis. Upon purification by high-performance liquid chromatography (HPLC) and characterization by electrospray ionization mass spectrometry (ESI-MS), sequence-specific oligomers are subjected to high-loading of a Lewis acidic rare-earth metal triflate which both deprotects the aldehyde moieties and affects the reactant pair equilibrium such that strands completely dissociate. Subsequently, a fraction of the Lewis acid is extracted, enabling annealing of complementary sequence-specific strands to form information-encoded molecular ladders characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The simple procedure outlined in this report circumvents kinetic traps commonly experienced in the field of dynamic covalent assembly and serves as a platform for the future design of robust, complex architectures.

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

信息承载Peptoids及其序列定向动态共价自组装的合成

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded ...

Chemical Triphosphorylation of Oligonucleotides

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of oligonucleotides in biotechnology and synthetic biology. Oligonucleotide 5&#8242;-triphosphates have historically been prepared in vitro by enzymatic methods. However, these methods are limited to natural RNA oligonucleotides, have strong sequence preferences, and tend to produce heterogeneous products. New methods of chemical triphosphorylation complement both the reduced cost of automated oligonucleotide synthesis by phosphoramidite chemistry and the diverse range of nucleotide modifications now available. Thus, the synthesis of oligonucleotide triphosphates of arbitrary sequence and length, and optionally containing various nonnatural modifications, is now accessible.
This paper presents the appropriate methods and techniques for chemical triphosphorylation of oligonucleotides using salicyl phosphorochloridite and pyrophosphate. This method uses commercially available reagents, is compatible with most oligonucleotides prepared by standard solid-phase synthesis methods, and can be completed in 2 h following oligonucleotide synthesis, before deprotection and purification. Two uses of chemically triphosphorylated oligonucleotides as substrates for catalytic RNA enzymes are demonstrated, including the synthesis of a mirror-image version of the hammerhead ribozyme from nonbiological L-RNA triphosphates.

Oligonucleotide 5&#8242;-triphosphates are ubiquitous components in essential biological pathways and have seen increasing use in biotechnology applications. Here, we describe techniques for the routine synthesis and purification of oligonucleotide 5&#8242;-triphosphates, starting from oligonucleotides prepared by standard automated synthesis techniques.

寡核苷酸的化学三磷酸化

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of ...

<meta charset="utf8"></head><body>通过 MOE 观察细胞内唾液酸化聚糖的扩增显微镜

Visualizing Intracellular Sialylation with Click Chemistry and Expansion Microscopy

Metabolic labeling techniques allow the incorporation of bioorthogonal reporters into glycans, enabling the targeted bioconjugation of molecular dyes within cells through click and bioorthogonal chemistry. Metabolic oligosaccharide engineering (MOE) has attracted considerable interest due to the essential role of glycosylation in numerous biological processes that involve molecular recognition and its impact on pathologies ranging from cancer to genetic disorders to viral and bacterial infections.
Although MOE is better known for the detection of cell surface glycoconjugates, it is also a very important methodology for the study of intracellular glycans in physiological and pathological contexts. Such studies greatly benefit from high spatial resolution. However, super-resolution microscopy is not readily available in most laboratories and poses challenges for daily implementation. Expansion microscopy is a recent alternative that enhances the resolution of microscopy by physically enlarging biological specimens labeled with fluorescent markers. By embedding the sample in a swellable gel and causing it to expand isotropically through chemical treatment, subcellular structures can be visualized with enhanced precision and resolution without the need for super-resolution techniques.
In this work, we illustrate the capacity of expansion microscopy to visualize intracellular sialylated glycans through the combined use of MOE and click chemistry. Specifically, we propose a procedure for bioorthogonal labeling and expansion microscopy that employs a reporter targeting sialylation, which may be associated with immunofluorescence for co-localization studies. This protocol enables localization studies of sialoconjugate biosynthesis, intracellular trafficking, and recycling.

<meta charset="utf8"></head><body>使用 Click 化学和扩增显微镜观察细胞内唾液酸化

Here, we propose a simple protocol combining metabolic oligosaccharide engineering, click chemistry, and expansion microscopy that allows bioimaging of intracellular sialylated N-glycoproteins with improved resolution using routine microscopy equipment.

使用 Click 化学和扩增显微镜观察细胞内唾液酸化

Metabolic labeling techniques allow the incorporation of bioorthogonal reporters into glycans, enabling the targeted bioconjugation of molecular dyes ...