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-phenylethan 1 胺骨干。在这条路线, 羟基取代 stereogenic 中心由 aldolisation 介绍苯甲醛和衍生物, 由苏氨酸醛缩酶催化。该第一步的低 diastereoselectivity 和中等产量克服了随后的立体脱羧反应催化的 l-酪氨酸 DC, 导致 enantioenriched 芳香β氨基醇11, 12.
我们报告了一个简单的协议, 以合成各种氨基醇从现成的 l-赖氨酸开始。尽管这项协议非常有效, 但由于αKAO 和中、中、中、中、中、下酶的基板范围有限, 因此存在缺陷。然而, 催化合成各种氨基醇可以考虑使用不同的组合, 其他αKAO 和氨基酸 DCs29,30,31,32。值得注意的是, 两步顺序是关键的梯级, 因为进工党-DCs 也积极对 l-赖氨酸。必须注意确保所有的 l-赖氨酸在第一氧化步骤中消耗, 然后再运行由脱羧反应催化的反应。通过生色剂对反应介质的衍生化后的 HPLC 对反应进行仔细监测, 确保了这一点。酶对高基质浓度的耐受性低, 是进一步发展的局限。为了解决这个问题, 酶进化策略可用于优化酶的性质, 如基质浓度耐受33。此外, 酶固定化可以考虑, 以确保酶的重用。
该协议易于执行, 其最具吸引力的特点之一是净化步骤的效率。在我们之前的工作中, 直接从复杂的酶反应混合物中提取极性分子是一个问题, 而疏水性基团衍生的化合物是必要的16。直接从反应不衍生化的氨基醇的纯化需要广泛的工作。在本协议中, 将离子交换树脂与固相萃取相结合的两步纯化法除去甘油 (含在酶溶液中) 和 HEPES 缓冲液, 两个极性分子与物理化学性质接近的靶向氨基醇, 因此很难与这些化合物分离。这种纯化协议可用于从复杂反应混合物中提取各种极性分子, 如酶反应。

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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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>(5S)-hydroxy-L-lysine</td>
			<td>Sigma Aldrich</td>
			<td>GPS NONH</td>
			<td>Out sourcing</td>
		</tr>
		<tr>
			<td>&#945;-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&#181;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&#160;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&#252;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 &#181;L</td>
			<td>Eppendorf</td>
			<td>3121000031</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette 100 &#181;L</td>
			<td>Eppendorf</td>
			<td>3121000074</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette 500 &#181;L</td>
			<td>Eppendorf</td>
			<td>3121000112</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette 1000 &#181;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的活动, 向商用 (5R)-羟基-l-赖氨酸。然后, 我们测定了 DC 的活动朝向单衍生物, 3-羟基-l-赖氨酸 (1) 和 4-羟基-l-赖氨酸 (2), 在级联与氧化步骤催化的适当的αKAO。表 1显示了单羟基 l-lysines 的催化 decarboxylations 的结果。用高效液相色谱法测定反应混合物与 DNFB 后的转化, 给出相应的 DNB 衍生物基材和产物。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>项</td>
			<td>基体</td>
			<td>进工党-DC</td>
			<td>产品</td>
			<td>转换</td>
		</tr>
<tr>
<td>1</td>
			<td>(5R)-羟基-l-赖氨酸</td>
			<td>最不发达国家Srumb)
</td>
			<td>4</td>
			<td>100%</td>
		</tr>
<tr>
<td>2</td>
			<td>(5R)-羟基-l-赖氨酸</td>
			<td>DCCpinb)
</td>
			<td>4</td>
			<td>北达科他州</td>
		</tr>
<tr>
<td>3</td>
			<td>
1a)
</td>
			<td>最不发达国家Srumb)
</td>
			<td>5</td>
			<td>100%</td>
		</tr>
<tr>
<td>4</td>
			<td>
1a)
</td>
			<td>DCCpinb)
</td>
			<td>5</td>
			<td>29%</td>
		</tr>
<tr>
<td>5</td>
			<td>
2a)
</td>
			<td>最不发达国家Srumc)
</td>
			<td>6</td>
			<td>60%</td>
		</tr>
<tr>
<td>6</td>
			<td>
2a)
</td>
			<td>DCCpinc)
</td>
			<td>6</td>
			<td>100%</td>
		</tr>
</tbody></table>表 1: monohydroxy lysines 的催化脱羧反应。反应条件: 基板 (10 毫米), 进给 DC (0.1 至0.15 毫克 mL-1), HEPES 缓冲器 (50 毫米, pH 7.5), 进工党 (1 毫米), 德勤 (1 毫米; 未与最不发达国家Srum一起使用), 隔夜, RT, 300 rpm。(a) 合成酶就地。(b) 0.1 毫克/毫升。(c) 0.15 毫克/毫升;北达科他州: 未检测到
rumirantium (最不发达国家Srum) 中的 DC 显示了对所有单羟基 lysines 的活动, 并将3和5衍生物的最佳转换转化为相应的手性羟基二胺化学 (条目1、3和 5)。正如预期的那样, dcCpin, αKAO 基因组上下文的 dc, 结果最适合脱羧反应 (4R)-羟基-l-赖氨酸(2) (6)。在标准反应条件下, (3S)-羟基-l-赖氨酸 (1) 与 DCCpin的转换为 decarboxylated 对应 (5) 低 (进入 4), 没有观察到任何活动 (5R)-羟基-l-赖氨酸 (入口 2)。
最后, 我们研究了两个 DCs 对二羟基-l-赖氨酸衍生物3、 8和9的活动, 由 KDO1、KDO2 或两者的组合所催化的一个或两个羟基化步骤合成原位(图2). 脱羧反应-羟基-l-lysines 的催化的结果概述在表2中。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>项</td>
			<td>基体</td>
			<td>进工党-DC</td>
			<td>产品</td>
			<td>转换</td>
		</tr>
<tr>
<td>1</td>
			<td>3</td>
			<td>最不发达国家Srumb)
</td>
			<td>7</td>
			<td>北达科他州</td>
		</tr>
<tr>
<td>2</td>
			<td>3</td>
			<td>DCCpinb)
</td>
			<td>7</td>
			<td>100%</td>
		</tr>
<tr>
<td>3</td>
			<td>
8a)
</td>
			<td>最不发达国家Srumb)
</td>
			<td>10</td>
			<td>19%</td>
		</tr>
<tr>
<td>4</td>
			<td>
8a)
</td>
			<td>DCCpinb)
</td>
			<td>10</td>
			<td>12%</td>
		</tr>
<tr>
<td>5</td>
			<td>
9a)
</td>
			<td>最不发达国家Srumc)
</td>
			<td>11</td>
			<td>北达科他州</td>
		</tr>
<tr>
<td>6</td>
			<td>
9a)
</td>
			<td>DCCpinc)
</td>
			<td>11</td>
			<td>北达科他州</td>
		</tr>
</tbody></table>表 2: 二羟基 lysines 催化脱羧反应。反应条件: 基板 (10 毫米), 进给-DC (0.1 至0.15 毫克/毫升), HEPES 缓冲 (50 毫米, pH 7.5), 进工党 (1 毫米), 德勤 (1 毫米; 未与最不发达国家Srum一起使用), 隔夜, RT, 300 rpm。(a) 合成酶就地。(b) 0.1 毫克/毫升。(c) 0.15 毫克/毫升;北达科他州: 未检测到。
关于二羟基 lysines 3、 8和9的脱羧反应, 结果并不完全令人满意。在标准反应条件下, 仅 (3r、4r)-二羟基-l-赖氨酸 (3) 被定量转化为相应的二羟基二胺7 (条目 1-2)。45-二羟基-l-赖氨酸 (8) 的转化是适度的 (条目 3-4), 但值得注意的是, 它可以通过大大增加酶的负荷来改善。两个经测试的进工党-DCs 均未发现对 35-二羟基-l-赖氨酸 (9) 有积极作用 (条目 5-6)。
通过高效液相色谱监测确定了定量转化的酶联反应 (图 4)。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56926/56926fig4v2.jpg"> 图 4: 三步酶级联的代表性分析数据.(A) 高效液相色谱监测 l-赖氨酸的催化转化为 (2r, 3R)-15-diaminopentan-23 二醇 (7)。RT = 保留时间。(B) 1H 核磁共振和 (c) 13c 核磁共振的氨基醇 (7) 纯化后。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56926/56926fig4v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
本发明提出的纯化协议允许从复杂的酶反应混合物中高效提取氨基醇。在优异的产量 (图 5) 中获得了氨基醇4、 5、 6和7 。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56926/56926fig5.jpg"> 图 5: 用于合成氨基醇的酶叶栅.合成 (R)-15-diaminopentan-2-ol (4), (S)-15-diaminopentan-2 ol (5), 15-diaminopentan-3 ol (6),和 (2r, 3R)-15-diaminopentane-23-二醇 (7)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56926/56926fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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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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

Microwave-assisted Intramolecular Dehydrogenative Diels-Alder Reactions for the Synthesis of Functionalized Naphthalenes/Solvatochromic Dyes

Functionalized naphthalenes have applications in a variety of research fields ranging from the synthesis of natural or biologically active molecules to the preparation of new organic dyes. Although numerous strategies have been reported to access naphthalene scaffolds, many procedures still present limitations in terms of incorporating functionality, which in turn narrows the range of available substrates. The development of versatile methods for direct access to substituted naphthalenes is therefore highly desirable.
The Diels-Alder (DA) cycloaddition reaction is a powerful and attractive method for the formation of saturated and unsaturated ring systems from readily available starting materials. A new microwave-assisted intramolecular dehydrogenative DA reaction of styrenyl derivatives described herein generates a variety of functionalized cyclopenta[b]naphthalenes that could not be prepared using existing synthetic methods. When compared to conventional heating, microwave irradiation accelerates reaction rates, enhances yields, and limits the formation of undesired byproducts.
The utility of this protocol is further demonstrated by the conversion of a DA cycloadduct into a novel solvatochromic fluorescent dye via a Buchwald-Hartwig palladium-catalyzed cross-coupling reaction. Fluorescence spectroscopy, as an informative and sensitive analytical technique, plays a key role in research fields including environmental science, medicine, pharmacology, and cellular biology. Access to a variety of new organic fluorophores provided by the microwave-assisted dehydrogenative DA reaction allows for further advancement in these fields.

Microwave-assisted intramolecular dehydrogenative Diels-Alder (DA) reactions provide concise access to functionalized cyclopenta[b]naphthalene building blocks. The utility of this methodology is demonstrated by one-step conversion of the dehydrogenative DA cycloadducts into novel solvatochromic fluorescent dyes via Buchwald-Hartwig palladium-catalyzed cross-coupling reactions.

微波辅助分子内Diels-Alder反应的功能化萘/变色染料的合成脱氢

Functionalized naphthalenes have applications in a variety of research fields ranging from the synthesis of natural or biologically active molecules to ...

科研

化学

Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions

Isothermal titration calorimetry (ITC) is a well-described technique that measures the heat released or absorbed during a chemical reaction, using it as an intrinsic probe to characterize virtually every chemical process. Nowadays, this technique is extensively applied to determine thermodynamic parameters of biomolecular binding equilibria. In addition, ITC has been demonstrated to be able of directly measuring kinetics and thermodynamic parameters (kcat, KM, &#916;H) of enzymatic reactions, even though this application is still underexploited. As heat changes spontaneously occur during enzymatic catalysis, ITC does not require any modification or labeling of the system under analysis and can be performed in solution. Moreover, the method needs little amount of material. These properties make ITC an invaluable, powerful and unique tool to study enzyme kinetics in several applications, such as, for example, drug discovery.
In this work an experimental ITC-based method to quantify kinetics and thermodynamics of enzymatic reactions is thoroughly described. This method is applied to determine kcat and KM of the enzymatic hydrolysis of urea by Canavalia ensiformis (jack bean) urease. Calculation of intrinsic molar enthalpy (&#916;Hint) of the reaction is performed. The values thus obtained are consistent with previous data reported in literature, demonstrating the reliability of the methodology.

Isothermal titration calorimetry measures heat flow released or absorbed in chemical reactions. This method can be used to quantify enzyme-catalysis. In this paper, the protocol for instrumental setup, experiment running, and data analysis is generally described, and applied to the characterization of enzymatic urea hydrolysis by jack bean urease.

热生物催化:等温滴定量热法表征酶促反应

Isothermal titration calorimetry (ITC) is a well-described technique that measures the heat released or absorbed during a chemical reaction, using it as ...

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of trifluoromethoxylated aromatic compounds remains a formidable challenge in organic synthesis. Conventional approaches often suffer from poor substrate scope, or require use of highly toxic, difficult-to-handle, and/or thermally labile reagents. Herein, we report a user-friendly protocol for the synthesis of methyl 4-acetamido-3-(trifluoromethoxy)benzoate using 1-trifluoromethyl-1,2-benziodoxol-3(1H)-one (Togni reagent II). Treating methyl 4-(N-hydroxyacetamido)benzoate (1a) with Togni reagent II in the presence of a catalytic amount of cesium carbonate (Cs2CO3) in chloroform at RT afforded methyl 4-(N-(trifluoromethoxy)acetamido)benzoate (2a). This intermediate was then converted to the final product methyl 4-acetamido-3-(trifluoromethoxy)benzoate (3a) in nitromethane at 120 &#176;C. This procedure is general and can be applied to the synthesis of a broad spectrum of ortho-trifluoromethoxylated aniline derivatives, which could serve as useful synthetic building blocks for the discovery and development of new pharmaceuticals, agrochemicals, and functional materials.

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

An operationally simple procedure for the synthesis of ortho-trifluoromethoxylated aniline derivatives via a two-step sequence of O-trifluoromethylation of N-aryl-N-hydroxyacetamide followed by thermally induced intramolecular OCF3-migration is reported.

协议的合成<em&gt;邻</em&gt; -trifluoromethoxylated苯胺类化合物

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

蛋白质生物缀合物的合成<em&gt;通过</em&gt;半胱氨酸马来酰亚胺化学

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins with the synthesis of a hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) homopolymer using reversible addition-fragmentation chain-transfer (RAFT) polymerization. Under mild visible light irradiation (&#955; = 460 nm, 0.7 mW/cm2), this macro-chain transfer agent (macro-CTA) in the presence of a ruthenium based photoredox catalyst, Ru(bpy)3Cl2 can be chain extended with a second monomer to form a well-defined block copolymer in a process known as Photoinduced Electron Transfer RAFT (PET-RAFT). When PET-RAFT is used to chain extend POEGMA with benzyl methacrylate (BzMA) in ethanol (EtOH), polymeric nanoparticles with different morphologies are formed in situ according to a polymerization-induced self-assembly (PISA) mechanism. Self-assembly into nanoparticles presenting POEGMA chains at the corona and poly(benzyl methacrylate) (PBzMA) chains in the core occurs in situ due to the growing insolubility of the PBzMA block in ethanol. Interestingly, the formation of highly pure worm-like micelles can be readily monitored by observing the onset of a highly viscous gel in situ due to nanoparticle entanglements occurring during the polymerization. This process thereby allows for a more reproducible synthesis of worm-like micelles simply by monitoring the solution viscosity during the course of the polymerization. In addition, the light stimulus can be intermittently applied in an ON/OFF manner demonstrating temporal control over the nanoparticle morphology.

This article describes a process for producing polymeric self-assembled nanoparticles using visible light mediated dispersion polymerization. Using low energy visible light to control the polymerization allows for the reproducible formation of self-assembled worm-like micelles at high solids content.

使用Photoredox催化剂蠕虫状胶束的简便合成可见光介导的分散聚合

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins ...

Palladium N-Heterocyclic Carbene Complexes: Synthesis from Benzimidazolium Salts and Catalytic Activity in Carbon-carbon Bond-forming Reactions

Detailed and generalized protocols are presented for the synthesis and subsequent purification of four palladium N-heterocyclic carbene complexes from benzimidazolium salts. Detailed and generalized protocols are also presented for testing the catalytic activity of such complexes in arylation and Suzuki-Miyaura cross-coupling reactions. Representative results are shown for the catalytic activity of the four complexes in arylation and Suzuki-Miyaura type reactions. For each of the reactions investigated, at least one of the four complexes successfully catalyzed the reaction, qualifying them as promising candidates for catalysis of many carbon-carbon bond-forming reactions. The protocols presented are general enough to be adapted for the synthesis, purification and catalytic activity testing of new palladium N-heterocyclic carbene complexes.

Palladium N-Heterocyclic Carbene Complexes: Synthesis from Benzimidazolium Salts and Catalytic Activity in Carbon-carbon Bond-forming Reactions

Detailed and generalized protocols are presented for the synthesis and subsequent purification of four palladium N-heterocyclic carbene complexes from benzimidazolium salts. The complexes were tested for catalytic activity in arylation and Suzuki-Miyaura reactions. For each reaction investigated, at least one of the four complexes successfully catalyzed the reaction.

钯<em&gt;ñ</em&gt; - 杂环碳烯配合物:苯并咪唑盐的合成和碳 - 碳键合反应中的催化活性

Detailed and generalized protocols are presented for the synthesis and subsequent purification of four palladium N-heterocyclic carbene complexes from ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

洞察氨基酸和多肽与无机材料的使用相互作用的单分子力谱

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

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 ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

硫醇反应试剂的合成与生物共轭作用--用于原位选择性修饰免疫共轭的生成

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

Enzymatic Synthesis of Epoxidized Metabolites of Docosahexaenoic, Eicosapentaenoic, and Arachidonic Acids

The epoxidized metabolites of various polyunsaturated fatty acids (PUFAs), termed epoxy fatty acids, have a wide range of roles in human physiology. These metabolites are produced endogenously by the cytochrome P450 class of enzymes. Because of their diverse and potent biological effects, there is considerable interest in studying these metabolites. Determining the unique roles of these metabolites in the body is a difficult task, as the epoxy fatty acids must first be obtained in significant amounts and with high purity. Obtaining compounds from natural sources is often labor intensive, and soluble epoxide hydrolases (sEH) rapidly hydrolyze the metabolites. On the other hand, obtaining these metabolites via chemical reactions is very inefficient, due to the difficulty of obtaining pure regioisomers and enantiomers, low yields, and extensive (and expensive) purification. Here, we present an efficient enzymatic synthesis of 19(S),20(R)- and 16(S),17(R)-epoxydocosapentaenoic acids (EDPs) from DHA via epoxidation with BM3, a bacterial CYP450 enzyme isolated originally from Bacillus megaterium (that is readily expressed in Escherichia coli). Characterization and determination of purity is performed with nuclear magnetic resonance spectroscopy (NMR), high-performance liquid chromatography (HPLC), and mass spectrometry (MS). This procedure illustrates the benefits of enzymatic synthesis of PUFA epoxy metabolites, and is applicable to the epoxidation of other fatty acids, including arachidonic acid (AA) and eicosapentaenoic acid (EPA) to produce the analogous epoxyeicosatrienoic acids (EETs) and epoxyeicosatetraenoic acids (EEQs), respectively.

We present a method useful for large-scale enzymatic synthesis and purification of specific enantiomers and regioisomers of epoxides of arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) with the use of a bacterial cytochrome P450 enzyme (BM3).

多克沙六烯、环氧体和阿拉奇酮酸的环氧代谢物酶合成

The epoxidized metabolites of various polyunsaturated fatty acids (PUFAs), termed epoxy fatty acids, have a wide range of roles in human physiology. These ...