Name: enzymatic cascade reactions for the synthesis of chiral amino alcohols from l-lysine
Uploaded: 2018-02-16T12:30:00.000+00:00
Duration: 9 min 14 s
Description: Watch this Scientific Journal Video about Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine at JoVE.com

作者感谢 Véronique de Berardinis 为卓有成效的讨论和阿兰佩雷, 克里斯汀 Pellé和佩奇 Sirvain 的技术支持。

1. 酶制剂
<ol><li>表达和纯化先前描述的26中的蛋白质。 注: 重组蛋白获得的最终浓度: αKAO 从Catenulispora acidiphila, UniProtKB ID: C7QJ42 (KDO1), 1.35 毫克/毫升;αKAO 来自C. pinensis, UniProtKB ID: C7PLM6 (KDO2), 2.29 毫克/毫升;从rumirantium的进工党-DCs (UniProtKB ID: O50657 (最不发达国家Srum), 无细胞提取物, 总酶为12.44 毫克/毫升;从pinensis, UniProtKB ID: C7PLM7 (DCCpin), 2.62 毫克/毫升的进工党 DC。</li>
</ol>2. 解决方案的准备工作
注意: 下面的所有解决方案都是在去离子水中制备的。
<ol><li>准备1米 HEPES 缓冲液, pH 值 7.5;用5米的氢氧化钠调节。 注: 在 pH 调节步骤中, 佩戴防护手套、防护服、眼部防护和脸部防护 (P280)。如与眼睛接触 (P305、P351、P338), 请小心用水冲洗几分钟. 如果可能, 请取出隐形眼镜。继续冲洗, 并立即致电毒中心或医生 (P310)。P 代码是指预防性陈述 (危险类和类别; 参见,例如, pubchem. ncbi. nlm. nih/全球统一制度/)。</li>
	<li>准备100毫米 l-赖氨酸, 100 毫米 (5S)-羟基-l-赖氨酸, 150 毫米α ketoglutaric 酸, 100 毫米抗坏血酸钠, 10 毫米铵铁 (II) 硫酸盐六水合物 (摩尔盐), 100 毫米吡哆醛 5-磷酸盐 (进工党) 和100毫米 dithiothreitol (德勤)。</li>
	<li>按照与2.1 中描述的相同的安全指示, 从 37% HCl 溶液 (近似 12 M 溶液) 中制备1米、2M 和6M 的盐酸 (hcl)。 注: ketoglutaric 酸、抗坏血酸钠和硫酸铵 (II.) 的溶液在使用之日必须新鲜。赖氨酸溶液可以在室温下储存几个月。进工党的解决方案可以储存一个月在摄氏4摄氏度。该方法可存储在摄氏-20 摄氏度的一个月。</li>
</ol>3. 分析尺度反应
<ol><li>(5R)-羟基-l-赖氨酸脱羧反应反应的方法发展<ol>
<li>添加11µL 1 M 溶液的 HEPES 缓冲 pH 7.5 (最终浓度50毫米) 到2毫升微细。然后, 添加22µL 100 毫米 (5S)-羟基-l-赖氨酸 (最终浓度10毫米), 2.2 µL 的毫米进工党 (最后浓度100毫米), 1 µL 2.2 毫米 (最终浓度100毫米)。 注: 在与最不发达国家的Srum发生反应的情况下, 不需要加用德勤。</li>
		<li>添加纯化的进给-DC (最终浓度0.1 毫克/毫升)。完成与 H2O 的最终卷220µL。</li>
		<li>在室温下摇动反应混合物, 打开空气打开盖子, 在 300 rpm 3 小时。 注: 在开放空气中用打开的盖子摇动反应混合物, 可确保反应介质有良好的氧合性。因此, 没有必要在反应介质中泡氧。</li>
		<li>根据步骤6中描述的反应监测程序, 收集10µL 反应混合物的分析结果。样品优选衍生物, 并在取样后直接进行分析。</li>
	</ol>
</li>
	<li>αKAO 催化脱羧反应催化的酶联反应的方法开发<ol>
<li>添加11µL 1 米 HEPES 缓冲 pH 7.5 (最终浓度50毫米) 到2毫升微细。然后, 添加22µL 150 毫米α ketoglutaric 酸 (最终浓度15毫米), 5.5 µL 100 毫米的抗坏血酸钠 (最终浓度2.5 毫米), 22 µL 的10毫米的摩尔的盐 (最终浓度1毫米), 22 µL 100 毫米的 l-赖氨酸 (最终浓度10毫米)。</li>
		<li>完成与 H2O 的最终体积为220µL, 考虑到在步骤3.2.3 中添加的酶解的体积。</li>
		<li>根据靶向选择性添加所需的纯化αKAO 酶: KDO1 位置 C-3 或 KDO2 对 l-赖氨酸位置 C-4 的羟基化。最后浓度的纯化酶 (0.05-0, 5 毫克/毫升), 以使完全转化为近似3小时。</li>
		<li>在室温下摇动反应混合物, 打开空气打开盖子, 在 300 rpm 3 小时。</li>
		<li>加入2.2 µL 100 毫米的进工党 (最后浓度 ~ 1 毫米) 和2.2 µL 100 毫米的德勤 (最后浓度 ~ 1 毫米)。 注: 在与最不发达国家的Srum发生反应的情况下, 不需要加用德勤。</li>
		<li>在浓度为 18 h: 最不发达国家Srum , 在0.1 毫克/毫升与 KDO1 的级联反应的近似最终浓度, 和 DCCpin在一个近似的最终浓度为0.5 毫克/毫升时, 加入纯净的中和中的进给-dcKDO2 的级联反应。</li>
		<li>在室温下摇动反应混合物, 在300转18小时的开放空气中打开盖子。</li>
		<li>按照步骤3.1.4 运行监视过程。</li>
	</ol>
</li>
	<li>脱羧反应催化αKAOs 催化双羟基化反应的酶促叶变换方法研究进展<ol>
<li>运行步骤 3.2. 1-3. 2.2。</li>
		<li>添加 KDO1 在最终浓度为0.05 毫克/毫升。在室温下摇动反应混合物, 打开空气打开盖子, 在 300 rpm 3h。</li>
		<li>添加 KDO2 在大约最终浓度为0.5 毫克/毫升, 计算使用初始反应量。在室温下摇动反应混合物, 打开空气打开盖子, 在 300 rpm 18 小时。</li>
		<li>运行步骤3.2.5。</li>
		<li>在大约最终浓度为0.5 毫克/毫升的情况下, 添加纯化的中、直流 dcCpin , 用初始反应量计算。在室温下摇动反应混合物, 打开空气打开盖子, 在 300 rpm 18 小时。</li>
		<li>按照步骤3.1.4 运行监视过程。</li>
	</ol>
</li>
</ol>4. 半制备一锅催化反应
注: 酶反应是在0.1 毫摩尔的 l-赖氨酸在一个开放空气250毫升玻璃锥形瓶的总体积10毫升。
<ol><li>monohydroxylated 衍生物的合成<ol>
<li>添加0.5 毫升的1米 HEPES 缓冲器, pH 7.5 (最终浓度50毫米) 的烧瓶。然后, 添加1毫升的100毫米 l-赖氨酸 (最终浓度10毫米), 1 毫升150毫米α ketoglutaric 酸 (最后浓度15毫米), 0.25 毫升100毫米抗坏血酸钠 (最终浓度2.5 毫米), 1 毫升的10毫米摩尔的盐 (最终浓度1毫米)。</li>
		<li>完成与 H2O 的最终体积为10毫升, 考虑到在步骤4.1.3 中添加的酶溶液的体积。</li>
		<li>根据目标选择性添加所需的纯化αKAO 酶: KDO1 在最终浓度为0.075 毫克/毫升的羟基化的位置 C-3 或 KDO2 在最终浓度为0.5 毫克/毫升的位置 C-4 的 l-赖氨酸。最后浓度的纯化酶被确定, 以使完全转化为近似3小时。</li>
		<li>在室温下, 在适当的持续时间内摇动反应混合物 300 rpm。当反应监测表明已完成的羟化反应 (运行步骤6中详细的反应监测协议步骤), 继续步骤4.1.5。一个典型的反应时间是3小时。</li>
		<li>加入100µL 100 毫米进给αKAO 反应混合物 (最后浓度 ~ 1 毫米)。然后, 在使用最不发达国家Srum时, 添加100µL 100 毫米的 (最终浓度为1毫米)。</li>
		<li>添加纯化的进给-DC: 最不发达国家Srum在大约最终浓度为0.05 毫克/毫升的级联反应与 KDO1 或 DCCpin在一个近似最终浓度为0.5 毫克/毫升的级联反应与 KDO2。</li>
		<li>在室温下摇动反应混合物, 300 rpm, 18 小时, 并直接继续执行步骤4.3 中详细说明的步骤。</li>
	</ol>
</li>
	<li>dihydroxylated 衍生物的合成<ol>
<li>加入0.5 毫升的1米 HEPES 缓冲器, pH 值 7.5 (最终浓度50毫米), 1 毫升100毫米 l-赖氨酸 (最终浓度10毫米), 1 毫升的150毫米α ketoglutaric 酸 (最终浓度15毫米), 0.25 毫升的100毫米钠抗坏血酸 (最终浓度2.5 毫米), 1 毫升10毫米摩尔的盐 (最后浓度1毫米)。完成与 H2O 的最终体积为10毫升, 考虑到在步骤4.2.2 中添加的酶溶液的体积。</li>
		<li>添加 KDO1 到最终浓度为0.075 毫克/毫升。在室温下摇动反应混合物, 在适当的持续时间为 300 rpm。</li>
		<li>当反应监测表明已完成的羟基化反应 (见步骤6的反应监测协议), 继续步骤4.2.5。一个典型的反应时间是3小时。</li>
		<li>加入1毫升150毫米α ketoglutaric 酸 (最终浓度15毫米), 0.25 毫升100毫米抗坏血酸钠 (最终浓度2.5 毫米), 1 毫升的10毫米摩尔的盐 (最终浓度1毫米)。</li>
		<li>将 KDO2 添加到大约最终浓度为0.5 毫克/毫升, 计算使用初始反应量。在室温下将反应混合物摇动300转18小时。</li>
		<li>当反应监测表明已完成的 dihydroxylation 反应 (见步骤6的反应监测协议), 继续步骤4.2.7。一个典型的反应时间是18小时。</li>
		<li>在使用最不发达国家Srum时, 添加100µL 100 毫米的德勤 (最终浓度为1毫米)。</li>
		<li>将纯化的 DCCpin添加到大约最终浓度为0.5 毫克/毫升, 用初始反应量计算。</li>
		<li>在室温下将反应混合物摇动300转18小时。</li>
	</ol>
</li>
	<li>淬火<ol>
<li>在冰浴中放置250毫升玻璃锥形瓶, 冷却反应混合物。</li>
		<li>小心地添加, 超过近似1分钟, 0.25 毫升6米盐酸通过手动轻轻摇动冷却反应混合物。按照步骤2.1 中描述的安全说明进行操作。</li>
		<li>使用装有灯泡的玻璃巴斯德吸管将酸性混合物转化为50毫升锥形底离心管, 然后离心机在 1680 x g 和4摄氏度处进行15分钟。</li>
		<li>取出上清, 并保持在250毫升的圆底烧瓶。</li>
		<li>将10毫升的去离子水添加到含有颗粒的锥形底离心管中。涡旋重新悬浮颗粒。</li>
		<li>离心机在 1680 x g, 4 °c, 15 分钟。</li>
		<li>取出上清, 并将其添加到含有第一上清的250毫升的圆底烧瓶中 (步骤 4.3.4)。</li>
		<li>将所收集的上清液通过浸泡到液氮中, 并将其固定在手里旋转, 然后立即将烧瓶转移到台式歧管冷冻干燥器中, 以防止材料解冻。</li>
		<li>冷冻干燥过程完成后 (隔夜), 从冷冻干燥器中取出烧瓶。</li>
	</ol>
</li>
</ol>5. 粗酶反应混合物中氨基醇的纯化
<ol><li>离子交换树脂<ol>
<li>将冷冻干燥的产品溶解在最低量的盐酸0.1 米 (近似4毫升), 直到固体颗粒不再可见肉眼。</li>
		<li>条件 a 磺酸官能团阳离子交换树脂, 200-400 目, 在玻璃柱 (20 x 250 毫米) 通过洗脱在大气压 1M hcl (4 列容量) 跟随 0.1 M HCl (4 个专栏容量; 列容量 = 10 mL)。</li>
		<li>使用1000µL 微在玻璃柱壁上的树脂顶部仔细地装载样品。然后, 用1毫升0.1 米的 HCl 水溶液冲洗所含原油的锥形底部离心管三次, 并以同样的方式将产生的漂洗量加载到柱上。</li>
		<li>具有非线性渐变的洗脱: 4 列容量 0.1 M HCl, 4 列容量水, 4 列容量 5% nh4oh, 4 列容量 10% nh4oh, 4 列容量 15% nh4oh, 4 列容量 20% nh4oh, 4 列卷 25% nh4oh 和最后4列容量 28% NH4oh。</li>
		<li>用 HPLC 监测纯化 (见步骤 6)。</li>
		<li>将包含复合物 (NH4OH 20-28%) 和冻干的分数池按步骤 4.3. 8-4. 3.9 中所述进行。 注: 离子交换柱可在洗脱用去离子水后再使用, 以中和洗脱水 (近似50毫升), 然后再用50毫升1米的盐酸洗脱。</li>
	</ol>
</li>
	<li>spe<ol>
<li>溶解冻干化合物在近似2毫升的 H3PO4/水 (20 µL/毫升)。</li>
		<li>将混合模式阳离子交换 SPE 6 毫升墨盒放在连接到水泵的萃取歧管上。在流形所提供的减压压力下, 通过4毫升甲醇的拉伸, 使墨盒状态良好。通过绘制4毫升的 H3PO4/水 (20 µL/毫升), 平衡墨盒。</li>
		<li>使用1000µL 微在玻璃柱壁上的树脂顶部, 将样品装入墨盒, 然后用0.75 毫升 H3PO4/水 (20 µL/毫升) 冲洗含有该产品三次的瓶子。以相同的方式将生成的冲洗卷加载到列上。</li>
		<li>在减少压力下洗脱墨盒, 由歧管提供, 4 毫升2% 甲酸在水中, 然后4毫升水。洗脱与一个梯度的 NH 4 oh 在水 (4 毫升为每洗脱, 从 4% NH 4 OH: 10%, 15%, 15%, 20% 和 28%).</li>
		<li>根据步骤6中描述的反应监测协议, 对每一个分数进行分析, 以高效液相色谱法监测纯化。根据紫外 (紫外) 色谱保持所有含有纯理想导数的分数, 并进行步骤5.2.6。</li>
		<li>将含有所需化合物的馏分放入100毫升圆底烧瓶中, 用水冲洗含有馏分的管子, 并将冲洗量添加到100毫升圆底烧瓶中。</li>
		<li>使用装有恒温水浴水壶的旋转蒸发器, 在40摄氏度的情况下将溶剂移除, 并连接至10毫巴的真空泵组。</li>
		<li>溶解在最低体积的水中的固体, 并把溶液转化成一个重25毫升的圆底烧瓶。用最少的水量冲洗烧瓶, 将冲洗量加到25毫升烧瓶中, 然后进行步骤5.2.9。</li>
		<li>称量纯化产物, 计算出产量。</li>
		<li>将纯化后的产品溶解在盐酸2M 的最小体积内, 直到固体颗粒不再可见于肉眼和冻干, 如步骤4.3 中所述. 8-4. 3.9。 注意: 氨基醇是敏感的化合物, 并作为其盐酸盐保存。</li>
	</ol>
</li>
</ol>6. 反应监测和产品分析
<ol><li>液相色谱分析前的基质和产物的衍生化 注: 基材和产品需要衍生物为芳香衍生物, 以增加其紫外色谱 detectivity。我们用了 1-氟-24-硝基苯 (DNFB)。在衍生化后立即将每个样品注射到 HPLC 中。<ol>
<li>采取酶反应的10µL 和转移到1.5 毫升微细。添加10µL 0.25 米的 NaHCO3水溶液, 100 µL 乙醇, 30 毫克/毫升 DNFB 溶液在乙醇的µL。</li>
		<li>关闭微细, 并在65摄氏度, 在1000转每分钟的1小时震动结果解决方案。用10µL 1 米的 HCl 淬火。</li>
		<li>用1毫升鲁尔注射器, 用0.22 µm 孔径亲水性聚偏氟乙烯 (PVDF) 膜过滤4毫米直径的无菌注射器过滤器。</li>
	</ol>
</li>
	<li>使用任何能够分离衍生物氨基酸的系统对酶反应进行监测。该协议涉及紫外检测的氨基酸衍生物由硝基苯 (DNB) 生色组27。<ol>
<li>C18 与三甲基硅 endcapping 反相柱 (5 µm, 3 x 150 毫米) 配合 HPLC。</li>
		<li>平衡 C18 列 (30:70) 淋洗 B (MeCN + 0.1% TFA) 淋洗 a (H2O + 0.1% TFA), 其流速为1毫升/分钟, 35 °c 为15分钟 (对应于近似20列卷)。</li>
		<li>注入10µL 的衍生物反应混合物 (见步骤 6.1) 到 HPLC C18 柱上。使用 MeCN 的混合物, 0.1% TFA/小时2O 和 0.1% TFA 作为淋洗与线性梯度 (比率30:70 到70:30 在9分钟, 温度35°c, 流动1毫升/分钟)。</li>
		<li>使用 400 nm 的紫外线检测, 对色谱柱上的洗脱产品进行分析。DNB 赖氨酸通常 elutes 约6.5 分钟, 羟基 DNB 赖氨酸约5.5 分钟, dihydroxylated DNB 赖氨酸约4.2 分钟, DNB aminodiol 约5.3 分钟左右。</li>
	</ol>
</li>
</ol>7. 纯化氨基醇的核磁共振分析
<ol><li>在 D2O (700 µL) 中溶解纯化的氨基醇, 并将溶液转移到核磁共振 (NMR) 管上。</li>
	<li>根据适当的核磁共振设备协议28获取1H 和13C 核磁共振谱。</li>
</ol>

<ol>
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作者没有什么可透露的。

手性氨基醇和衍生物具有广泛的应用范围, 从手学助剂到有机合成到药物治疗。常规有机合成制备氨基醇的多步法合成方法很多, 但由于繁琐的保护/脱步骤以及对立体16的敏感控制, 可能并不总是有效的。一个催化的方法, 免除了保护/脱步骤, 通常是高度立体是一个很好的选择。
以往的研究报告了通过动态动力学不对称转化 (DYKAT) 合成各种β氨基醇的 2-phenyletha...

尽管生物催化提供了好处, 催化步骤在合成通路或总催化路线中的整合仍然主要限于酶动力学的决议。这些路线被广泛地用作不对称的化学酶合成的第一步, 但生物催化在功能组 interconversions 中提供了更多的可能性高立体选择性 1, 2, 3.此外, 由于催化反应是在类似的条件下进行的, 因此在一壶式的4,5中执行级联反应是可行的。
手性氨基醇是在有机合成中用作辅助剂或支架的多功能分子6。氨基醇基团经常见于次生代谢物和活性药物成分 (API)。通过常规的化学合成, 可以从相应的α氨基酸中获得初级β氨基醇, 但接触手性γ氨基醇或二次氨基醇通常需要繁琐的合成通路以及敏感控制立体7,8,9,10。由于其高立体选择性, 生物催化可能提供了一个优越的合成路线, 这些手性构建块 11, 12, 13, 14.
我们以前报告了由铁 (II)/α ketoacid 依赖氧合酶家族 (αKAO) (图 1)15dioxygenases 催化的 diastereoselective 酵素羟基化合成单羟基-l-lysines。特别是, 从 l-赖氨酸开始, KDO1 酶催化形成 (3S)-羟基衍生物 (1), 而 (4R)-衍生物 (2) 由反应形成与 KDO2 酶。连续的 regiodivergent hydroxylations 由 KDO1 和 KDO2 导致形成 (3r, 4r)-二羟基-l-赖氨酸 (3) 在光学上纯净的形式。然而, 这些酶的有限基底范围阻碍了它们在化学合成中的大量使用, 特别是在简单胺的羟基化中, 因为在氨基基团α位置的羧酸基团对于活动16是必不可少的。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56926/56926fig1.jpg"> 图 1: l-赖氨酸的催化转换.转换成 (3S)-羟基-l-赖氨酸 (1) 由 KDO1 酶催化;(4R)-羟基-l-赖氨酸 (2) 由 KDO2 酶催化;和 (3r, 4R)-二羟基-l-赖氨酸 (3) 由 KDO1 和 KDO2 dioxygenases 连续催化的级联反应。<a href="//cloudflare.jove.com/files/ftp_upload/56926/56926fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
脱羧反应是新陈代谢的一个共同的反应17。特别是, 氨基酸 DCs (EC 4.1.1) 是无余子的 (pyruvoyl 依赖) 或与进工党相关的酶, 并催化氨基酸脱羧反应到相应多胺的细菌和更高的有机体18, 19,20,21,22. 单和二羟基化合物 (图 3) 4-7, 10-11对应于羟基尸胺, 由 l-赖氨酸脱羧反应获得的二胺。尸胺是化学工业的关键构件, 特别是它是聚酰胺和聚氨酯聚合物的组成部分。因此, 从可再生资源中以生物为基础生产这二胺, 已引起人们的注意, 作为一种替代石油的途径, 并为此目的设计了各种微生物。在这些代谢通路中, 赖氨酸 DC (最不发达国家) 是关键酶。土发公司是属于丙氨酸 racemase (AR) 结构家族23的一项与工党相关的酶。与工党相关的 dcs (进工党-dcs) 被称为高度基底特异。然而, 一些酶拥有轻微的滥交的能力, 积极地对 l-鸟氨酸和 l-赖氨酸氨基酸, 例如土发公司从Selenomonas rumirantium (最不发达国家的Srum), 有相似的动力学常数为赖氨酸和鸟氨酸脱羧反应24, 25.这种扩展的基质特异性使这种酶成为一个良好的候选脱羧反应的单和二羟基-l-赖氨酸。此外, 为了找到对赖氨酸羟基衍生物有积极作用的 DCs, 我们研究了αKAO 酶编码基因的遗传学背景。事实上, 在原核基因组中, 同一生物合成通路中所涉及的酶的基因在基因簇中通常是相互定位的。KDO2 (从Chitinophaga pinensis) 基因被发现与一个基因编码的假定的中工党 (图 2) 联合本地化。相比之下, 在分析 KDO1 酶的基因组背景时, 没有发现 DC 的基因编码。因此, 从C. pinensis (dcCpin) 中的中进-dc 蛋白被选为一个有希望的候选分子, 以催化级联反应的脱羧反应步骤。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56926/56926fig2.jpg"> 图 2: KDO2 基因的基因组上下文C. pinensis.<a href="//cloudflare.jove.com/files/ftp_upload/56926/56926fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
因此, 我们设计了涉及 dioxygenases 和 DCs 的酶联反应, 以实现从氨基酸中合成脂肪族手性β和γ氨基醇 (图 3)。如前所述, αKAO 催化的 C H 氧化反应引入了全 diastereoselectivity 的羟基取代 stereogenic 中心;Cβ/γ手性将保留在 decarboxylative 步骤, 这只影响 cα-cα碳的氨基酸基团16。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56926/56926fig3.jpg"> 图 3: Retrosynthetic 分析.(A) Retrosynthesis β和γ氨基醇 (R)-15-diaminopentan-2-ol (4) (5R)-羟基-l-赖氨酸, 并且 (S)-15-diaminopentan-2 ol (5) 和 15-diaminopentan-3 ol (6) 从l-赖氨酸。(B) Retrosynthesis β、γ和β、δ氨基二醇 (2 S, 3S)-15-diaminopentane-23-二醇 (10) 和 (2R, 4S)-15-diaminopentane-24-二醇 (11) 从 (5R) 开始-羟基-l-赖氨酸和 (2r, 3r)-15-diaminopentane-23-二醇 (7) 从 l-赖氨酸开始。<a href="//cloudflare.jove.com/files/ftp_upload/56926/56926fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
从 l-赖氨酸及其 (5R)-羟基衍生物开始, 本文报告了两个/三步, 一锅, 酶法结合 dioxygenases 和进工党-DCs, 以获得目标氨基醇。在靶分子的实验室尺度合成之前, 该方法是在分析尺度上发展起来的, 以调整反应条件,例如, 酶浓度, 要求允许完全转化的起始材料;我们也提出这个程序。

<table><tbody><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H3375</td><td></td></tr><tr><td>L-lysine hydrochloride</td><td>Sigma Aldrich</td><td>L5626</td><td></td></tr><tr><td>(5&lt;em&gt;S&lt;/em&gt;)-hydroxy-L-lysine</td><td>Sigma Aldrich</td><td>GPS NONH</td><td>Out sourcing</td></tr><tr><td>&amp;alpha;-ketoglutaric acid</td><td>Sigma Aldrich</td><td>75892</td><td></td></tr><tr><td>Sodium ascorbate</td><td>Sigma Aldrich</td><td>A7631</td><td></td></tr><tr><td>Ammonium Iron(II) sulfate hexahydrate</td><td>Acros</td><td>201370250</td><td></td></tr><tr><td>Pyridoxal phosphate (PLP)</td><td>Sigma Aldrich</td><td>82870</td><td></td></tr><tr><td>3,4-dimercaptobutane-1,2-diol (DTT)</td><td>Sigma Aldrich</td><td>D0632</td><td></td></tr><tr><td>1-fluoro-2,4-dinitrobenzene (DNFB)</td><td>Sigma Aldrich</td><td>D1529</td><td></td></tr><tr><td>Ethanol</td><td>VWR</td><td>20825.290</td><td></td></tr><tr><td>Sodium hydrogen carbonate</td><td>Sigma Aldrich</td><td>71631</td><td></td></tr><tr><td>HCl 37%</td><td>Sigma Aldrich</td><td>435570</td><td></td></tr><tr><td>HCl 0.1M</td><td>Fluka</td><td>35335</td><td></td></tr><tr><td>Acetonitrile HiPerSolv CHROMANORM for LC-MS</td><td>VWR</td><td>83640.320</td><td></td></tr><tr><td>2,2,2-trifluoroacetic acid</td><td>VWR</td><td>153112E</td><td></td></tr><tr><td>Ammonia 28%</td><td>VWR</td><td>21182.294</td><td></td></tr><tr><td>Methanol HiPerSolv CHROMANORM for LC-MS</td><td>VWR</td><td>83638.32</td><td></td></tr><tr><td>Formic acid</td><td>Acros</td><td>270480010</td><td></td></tr><tr><td>Phosphoric acid 85%</td><td>Acros</td><td>201145000</td><td></td></tr><tr><td>Deuterium oxide</td><td>Acros</td><td>320,710,075</td><td></td></tr><tr><td>NaOH</td><td>Sigma Aldrich</td><td>S5881</td><td></td></tr><tr><td>C18 HPLC column</td><td>Phenomenex</td><td>00F-4601-Y0</td><td></td></tr><tr><td>Accela UHPLC System</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>Accela PDA detector</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>4mm syringe filters - 0,22&amp;micro;m - PVDF</td><td>Merck</td><td>SLGVR04NL</td><td></td></tr><tr><td>Single-use tuberculin syringe with ml graduation, Luer tip</td><td>VWR</td><td>HSWA5010.200V0</td><td></td></tr><tr><td>Cation exchange resin 100-200&amp;nbsp;mesh</td><td>Sigma Aldrich</td><td>217506</td><td></td></tr><tr><td>Mixed mode cation-exchange solid-phase extraction cartridge 6 mL</td><td>Waters</td><td>186000776</td><td></td></tr><tr><td>Extraction manifold</td><td>Waters</td><td>WAT200609</td><td></td></tr><tr><td>Rotary evaporator</td><td>B&amp;uuml;chi</td><td>531-0103</td><td></td></tr><tr><td>Lyophilizer alpha 1-2 LDplus</td><td>Christ</td><td>L083302</td><td></td></tr><tr><td>Micropipette 20 &amp;micro;L</td><td>Eppendorf</td><td>3121000031</td><td></td></tr><tr><td>Micropipette 100 &amp;micro;L</td><td>Eppendorf</td><td>3121000074</td><td></td></tr><tr><td>Micropipette 500 &amp;micro;L</td><td>Eppendorf</td><td>3121000112</td><td></td></tr><tr><td>Micropipette 1000 &amp;micro;L</td><td>Eppendorf</td><td>3121000120</td><td></td></tr><tr><td>300 MHz spectrometer</td><td>Bruker</td><td></td><td></td></tr><tr><td>2 mL microtube</td><td>CLEARLine</td><td>CL20.002.0500</td><td></td></tr><tr><td>50 mL conical-bottom centrifuge tube</td><td>Fischer Scientific</td><td>05-539-8</td><td></td></tr><tr><td>25 mL round-bottom flask 14/23</td><td>Fischer Scientific</td><td>10353331</td><td></td></tr><tr><td>100 mL round-bottom flask 29/32</td><td>Fischer Scientific</td><td>11786183</td><td></td></tr><tr><td>250 mL round-bottom flask 29/32</td><td>Fischer Scientific</td><td>11786183</td><td></td></tr><tr><td>250 mL erlenmeyer flask</td><td>Fischerbrand</td><td>15496143</td><td></td></tr></tbody></table>

enzymatic cascade reactions for the synthesis of chiral amino alcohols from l-lysine

氨基醇是具有广泛应用范围的多功能化合物。例如, 它们在有机合成中被用作手性支架。它们通过常规有机化学合成通常需要繁琐的多步法合成过程, 难以控制立体化学结果。我们提出了一个协议, 酶合成氨基醇开始从现成的 l-赖氨酸在48小时。该协议结合了两种化学反应, 这是很难进行常规有机合成。在第一步, diastereoselective 氧化处于草稿的赖氨酸侧链的 c-H 键, 由酶催化;regiodivergent 酶催化的第二个 diastereoselective 氧化作用可能导致 12-二醇的形成。在最后一步, 阿尔法氨基酸的羧酸组被吡哆醛磷酸脱羧酶 (DC) 所劈裂。这一 decarboxylative 步骤只影响氨基酸的α碳, 保留羟基取代 stereogenic 中心在β/伽玛位置。由此产生的氨基醇因此光学上丰富。该协议成功地应用于四氨基醇的 semipreparative 级合成。采用高效液相色谱 (HPLC) 对 1-氟-24-硝基苯进行衍生反应监测。固相萃取 (SPE) 直接纯化, 为氨基醇提供了优良的产量 (93% 至 > 95%)。

我们以前报道过由铁 (II)/αKAO 家族 (图 1)16dioxygenases 催化的 diastereoselective 酶羟基化合成单羟基-l-lysines。为了优化这里提出的整个叶栅的协议, 将αKAO 催化的一个或两个羟基化步骤结合起来, 然后由脱羧反应催化的反应条件进行调整, 以满足两者的要求。酶反应。我们首先调查了两个 DCs、最不发达国家Srum和 DCCpin的活?...

Watch this Scientific Journal Video about Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine at JoVE.com

Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine

手性氨基醇是在有机合成中用作支架的多功能分子。从 l-赖氨酸开始, 用酶催化的酶联反应合成氨基醇, 然后将相应羟基氨基酸的羧酸基团 diastereoselective羧.

酶联反应合成 l-赖氨酸手性氨基醇的研究

Amino alcohols are versatile compounds with a wide range of applications. For instance, they have been used as chiral scaffolds in organic synthesis. ...

enzymatic-cascade-reactions-for-synthesis-chiral-amino-alcohols-from

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Chemistry

Chemical Kinetics

SCIENTISTS IN ACTION

12.0K 次观看.  CEA, CNRS, Univ Evry, Univ Paris-Saclay. 手性氨基醇是在有机合成中用作支架的多功能分子。从 l-赖氨酸开始, 用酶催化的酶联反应合成氨基醇, 然后将相应羟基氨基酸的羧酸基团 diastereoselective羧.

视频: Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical probe that targets the enzyme&#39;s active sites. A reporter tag introduced into the probe allows for the detection of the labeled enzyme by in-gel fluorescence scanning, protein blot, fluorescence microscopy, or liquid chromatography-mass spectrometry. Here, we describe the preparation and use of the compound ARN14686, a click chemistry activity-based probe (CC-ABP) that selectively recognizes the enzyme N-acylethanolamine acid amidase (NAAA). NAAA is a cysteine hydrolase that promotes inflammation by deactivating endogenous peroxisome proliferator-activated receptor (PPAR)-alpha agonists such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). NAAA is synthesized as an inactive full-length proenzyme, which is activated by autoproteolysis in the acidic pH of the lysosome. Localization studies have shown that NAAA is predominantly expressed in macrophages and other monocyte-derived cells, as well as in B-lymphocytes. We provide examples of how ARN14686 can be used to detect and quantify active NAAA ex vivo in rodent tissues by protein blot and fluorescence microscopy.

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Here, we describe the preparation and use of an activity-based probe (ARN14686, undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl]carbamate) that allows for the detection and quantification of the active form of the proinflammatory enzyme N-acylethanolamine acid amidase (NAAA), both in vitro and ex vivo.

制备及<em&gt;在体内</em&gt;使用基于活动的探针对<em&gt;ñ</em&gt; -acylethanolamine酸酰胺酶

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

科研

生物化学

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

可逆二硫键交联胶束生产的简便和有效途径

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this procedure amino functionalized 'lysine' and guanido functionalized 'arginine' peptoid monomers can be included within the same peptoid sequence. This procedure uses on-resin (N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) or Dde protection, orthogonal conditions to the Boc protection of lysine monomers. Subsequent deprotection allows an efficient on-resin guanidinylation reaction to form the arginine residues. The procedure is compatible with the commonly used submonomer method of peptoid synthesis, allowing simple peptoids to be made using common laboratory equipment and commercially available reagents. The representative synthesis, purification and characterization of two mixed peptoids is described. The evaluation of these compounds as potential anti-infectives in screening assays against Leishmania mexicana is also described. The protozoan parasite L. mexicana is a causative agent of cutaneous leishmaniasis, a neglected tropical disease that affects up to 12 million people worldwide.

A protocol to synthesize peptoids with mixed cationic functionality in the same sequence is presented (lysine- and arginine-type monomers). Subsequent testing of these compounds against Leishmania mexicana, the protozoan parasites that cause cutaneous leishmaniasis, is also described.

对于拟肽与他们抗利什曼原虫活性混合赖氨酸型/精氨酸型单体及评价合成的有效方法

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this ...

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide sequences. Synthesis of some unnatural amino acids often includes the use of a precursor consisting of a Schiff-base stabilized by a nickel cation. Unnatural side-chains can be installed on an amino acid backbone found in this Schiff-base complex. The resulting unnatural amino acid can then be isolated from this complex using hydrolysis of the Schiff-base, typically by employing reflux in strongly acidic solution. These highly acidic conditions may remove acid-labile side-chain protecting groups necessary for the unnatural amino acids to be used in microwave-assisted solid-phase peptide synthesis. In this work, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff base complex. Hydrolysis conditions presented in this work are suitable for retention of acid-labile side-chain protecting groups and may be adaptable to a variety of unnatural amino acid substrates.

Here, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff-base complex. Hydrolysis conditions presented here are suitable for use when retention of acid-labile side-chain protecting groups is required. This technique may be adaptable to a variety of unnatural amino acid substrates.

镍 - 席夫碱的水解复杂环境条件适宜酸不稳定保护基团的保留

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide ...

Highly Stereoselective Synthesis of 1,6-Ketoesters Mediated by Ionic Liquids: A Three-component Reaction Enabling Rapid Access to a New Class of Low Molecular Weight Gelators

In organic chemistry ionic liquids (ILs) have emerged as safe and recyclable reaction solvents. In the presence of a base ILs can be deprotonated to form catalytically active N-Heterocyclic Carbenes (NHCs). Here we have used ILs as precatalysts in the addition of &#945;,&#946;-unsaturated aldehydes to chalcones to form 1,6-ketoesters, incorporating an anti-diphenyl moiety in a highly stereoselective fashion. The reaction has a broad substrate scope and several functional groups and heteroaromatics can be integrated into the ketoester backbone in generally good yields with maintained stereoselectivity. The reaction protocol is robust and scalable. The starting materials are inexpensive and the products can be obtained after simple filtration, avoiding solvent-demanding chromatography. Furthermore, the IL can be recycled up to 5 times without any loss of reactivity. Moreover, the 1,6-ketoester end product is a potent gelator in several hydrocarbon based solvents. The method enables rapid access to and evaluation of a new class of low molecular weight gelators (LMWGs) from recyclable and inexpensive starting materials.

Ionic liquids (ILs) mediate fast, simple and cheap access to 1,6-ketoesters in high diastereoselectivities and good yields. The reaction protocol is robust and the 1,6-ketoesters can be obtained in gram scale after a simple filtration protocol. Moreover, the 1,6-ketoesters are potent gelators in hydrocarbon solvents.

1,6-酮酯高立体选择性合成离子液体介导:三组分反应启用快速进入低分子量胶凝剂的新型类

In organic chemistry ionic liquids (ILs) have emerged as safe and recyclable reaction solvents. In the presence of a base ILs can be deprotonated to form ...

化学

Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene hydrothiolation. In addition, this protocol describes the preparation of vinyl-sulfide-tethered peptides using in-solution intramolecular thiol-yne hydrothiolation between amino acids that possess alkene/alkyne side chains and cysteine residues at i, i+4 positions. Linear peptides were synthesized using a standard Fmoc-based solid-phase peptide synthesis (SPPS). Thiol-ene hydrothiolation is carried out using either an intramolecular thio-ene reaction or an intermolecular thio-ene reaction, depending on the peptide length. In this research, an intramolecular thio-ene reaction is carried out in the case of shorter peptides using on-resin deprotection of the trityl groups of cysteine residues following the complete synthesis of the linear peptide. The resin is then set to UV irradiation using photoinitiator 4-methoxyacetophenone (MAP) and 2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone (MMP). The intermolecular thiol-ene reaction is carried out by dissolving Fmoc-Cys-OH in an N,N-dimethylformamide (DMF) solvent. This is then reacted with the peptide using the alkene-bearing residue on resin. After that, the macrolactamization is carried out using benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 1-hydroxybenzotriazole (HoBt), and 4-Methylmorpholine (NMM) as activation reagents on the resin. Following the macrolactamization, the peptide synthesis is continued using standard SPPS. In the case of the thio-yne hydrothiolation, the linear peptide is cleaved from the resin, dried, and subsequently dissolved in degassed DMF. This is then irradiated using UV light with photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA). Following the reaction, DMF is evaporated and the crude residue is precipitated and purified using high-performance liquid chromatography (HPLC). These methods could function to simplify the generation of thioether-tethered cyclic peptides due to the use of the thio-ene/yne click chemistry that possesses superior functional group tolerance and good yield. The introduction of thioether bonds into peptides takes advantage of the nucleophilic nature of cysteine residues and is redox-inert relative to disulfide bonds.

We present a protocol for the construction of thioether/vinyl sulfide-tethered helical peptides using photo-induced thiol-ene/thiol-yne hydrothiolation.

利用光致硫醇-烯/yne Hydrothiolation 构造硫醚类/乙烯基硫化物栓系螺旋肽

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene ...

Efficient Construction of Drug-like Bispirocyclic Scaffolds Via Organocatalytic Cycloadditions of &#945;-Imino &#947;-Lactones and Alkylidene Pyrazolones

Bispirocyclic scaffolds are one of the important structural subunits in many natural products that exhibit diverse and attractive biological activities. Recently, we have developed an efficient organocatalytic strategy, which provides facile access to a variety of enantiomerically enriched bispiro[&#947;-butyrolactone-pyrrolidin-4,4&#39;-pyrazolone] skeletons. In this paper, we demonstrate a detailed protocol for the asymmetric synthesis of drug-like bispirocyclic compounds with two spirocyclic carbon centers via an organocatyltic 1,3-dipolar cycloaddition reaction. Spirocyclization synthons &#945;-imino &#947;-lactones and alkylidene pyrazolones are prepared first, which are then subjected to a cycloaddition reaction in the presence of a bifunctional squaramide organocatalyst to afford the desired bispirocycles in high yields and excellent stereoselectivities. Chiral high-performance liquid chromatography (HPLC) is carried out to determine the enantiomeric purity of the products, and the d.r. value is examined by proton nuclear magnetic resonance (1H NMR). The absolute configuration of the product is assigned according to an X-ray crystallographic analysis. This synthetic strategy allows scientists to prepare a diversity of bispirocyclic scaffolds in high yields and excellent diastereo- and enantioselectivities.

Enantiomerically enriched bispiro[&#947;-butyrolactone-pyrrolidin-4,4&#39;-pyrazolone] skeletons are asymmetrically synthesized through a simple organocatalytic 1,3-dipolar cycloaddition reaction.

α-伊米诺γ-乳胶酮和阿基地利酮吡唑酮有机催化环加花法高效构建药物样双环脚手架

Bispirocyclic scaffolds are one of the important structural subunits in many natural products that exhibit diverse and attractive biological activities. ...

Preparation of Enantiopure Non-Activated Aziridines and Synthesis of Biemamide B, D, and epiallo-Isomuscarine

Nitrogen-containing heterocycle aziridines are synthetically very valuable for the preparation of azacyclic and acyclic molecules. However, it is very difficult and laborious to make aziridines in optically pure forms on a large scale to apply asymmetric synthesis of aza compounds. Fortunately, we successfully achieved both enantiomers (2R)- and (2S)-aziridine-2-carboxylates with the electron-donating &#945;-methylbenzyl group at the ring nitrogen as non-activated aziridines. These starting aziridines have two distinct functional groups-highly reactive three-membered ring and versatile carboxylate. They are applicable in ring-opening or ring-transformation with aziridine and in functional group transformation to others from carboxylate. Both of these enantiomers were utilized in the preparation of biologically important amino acyclic and/or aza-heterocyclic compounds in an asymmetric manner. Specifically, this report describes the first expedient asymmetric synthesis of both enantiomers of 5, 6-dihydrouracil-type marine natural products biemamide B and D as potential TGF-&#946; inhibitors. This synthesis consisted of regio- and the stereoselective ring-opening reaction of aziridine-2-carboxylate and subsequent formation of 4-aminoteterahydropyrimidine-2,4-dione. One more example in this protocol dealt a highly stereoselective Mukaiyama reaction of aziridine-2-carboxylate and silyl enol ether, following intramolecular aziridine ring-opening to provide easy and facile access to (-)-epiallo-isomuscarine.

Preparation of Enantiopure Non-Activated Aziridines and Synthesis of Biemamide B, D, and epiallo-Isomuscarine

In this study, we prepare both enantiomers of aziridine-2-carboxylate, which are used in the asymmetric synthesis of alkaloids, including biemamide B and D, and (-)-epiallo-isomuscarine.

对映草非活化氮丙啶的制备及比咪咪唑B、D和 表皮异毒羯碱的合成

Nitrogen-containing heterocycle aziridines are synthetically very valuable for the preparation of azacyclic and acyclic molecules. However, it is very ...

Organocatalysis

Source: Vy M. Dong and Faben Cruz, Department of Chemistry, University of California, Irvine, CA
This experiment will demonstrate the concept of organocatalysis by illustrating the proper setup of a reaction that utilizes enamine catalysis. Organocatalysis is a form of catalysis that uses substoichiometric amounts of small organic molecules to accelerate reactions. This type of catalysis is complementary to other forms of catalysis such as transition metal or biocatalysis. Transition metal catalysis involves transition metals as catalysts and biocatalysis uses enzymes as catalysts. Some advantages of organocatalysis include the low toxicity and cost of the organocatalysts in comparison to many metal catalysts. In addition, most organocatalysts are not sensitive to air and moisture, unlike metal catalysts. In contrast to enzymes found in living organisms, the small molecules that act as organocatalysts are typically easy to access. Furthermore, organocatalysis offers complementary and new reactivity not observed with other forms of catalysis.

Organocatalysis: Principles and Applications

催化

Source: Vy M. Dong and Faben Cruz, Department of Chemistry, University of California, Irvine, CA
This experiment will demonstrate the concept of ...

教育

JoVE Science Education

有机化学 (2)

Acid Halides to Esters: Alcoholysis

Alcoholysis is a nucleophilic acyl substitution reaction in which an alcohol functions as a nucleophile. Acid halides react with alcohol to produce esters. The mechanism proceeds in three steps:
<ol>
	<li>First, the alcohol acts as a nucleophile and attacks the acyl carbon to form a tetrahedral intermediate.</li>
	<li>Next, the carbonyl re-forms with the loss of a chloride ion.</li>
	<li>Lastly, the positively charged intermediate loses a proton to give an ester as the final product along with H3O+, making HCl an overall byproduct of the reaction.</li>
</ol>
<img alt="Figure1" src="/files/ftp_upload/12362/12362_PT_Figure_1.svg" style="text-align: center; height: 200px;" />
Pyridine is used as a base to neutralize the acidic reaction mixture.
Alcoholysis of sulfonyl chlorides follows a similar pattern and forms sulfonic acid esters.
<img alt="Figure2" src="/files/ftp_upload/12362/12362_PT_Figure_2.svg" style="text-align: center; height: 75px;" />

酸卤化物到酯类:醇解

Alcoholysis is a nucleophilic acyl substitution reaction in which an alcohol functions as a nucleophile. Acid halides react with alcohol to produce ...

酶联反应合成 l-赖氨酸手性氨基醇的研究

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