Structural characterization of small molecules is a crucial component of organic synthesis. In this work, we applied microcrystal electron diffraction (MicroED) to analyze the structure of the product of an enzymatic reaction that was intended to produce the unnatural amino acid 2,4-dihydroxyphenylalanine (24DHF). Characterization of our isolated product with nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) suggested that an isomer of 24DHF had been formed. Microcrystals present in the isolated product were then used to determine its structure to 0.62 Å resolution, which confirmed its identity as 2-amino-2-(2,4-dihydroxyphenyl)propanoic acid (24DHPA). Moreover, the MicroED structural model indicated that both enantiomeric forms of 24DHPA were present in the asymmetric unit. Notably, the entire structure determination process including setup, data collection, and refinement was completed in ~1 h. The MicroED data not only bolstered previous results obtained using NMR and MS but also immediately provided information about the stereoisomers present in the product, which is difficult to achieve using NMR and MS alone. Our results therefore demonstrate that MicroED methods can provide useful structural information on timescales that are similar to many commonly used analytical methods and can be added to the existing suite of small molecule structure determination tools in future studies.

小分子的结构表征是有机合成的重要组成部分。在这项工作中，我们应用微晶电子衍射 (MicroED) 来分析旨在产生非天然氨基酸 2,4-二羟基苯丙氨酸 (24DHF) 的酶促反应产物的结构。用核磁共振光谱 (NMR) 和质谱 (MS) 对我们分离的产物进行表征，表明已形成 24DHF 的异构体。然后使用分离产物中存在的微晶确定其结构，分辨率为 0.62 Å，证实其为 2-amino-2-(2,4-dihydroxyphenyl)propanoic acid (24DHPA)。此外，MicroED 结构模型表明 24DHPA 的两种对映体形式都存在于不对称单元中。尤其，包括设置、数据收集和改进在内的整个结构确定过程在约 1 小时内完成。MicroED 数据不仅支持以前使用 NMR 和 MS 获得的结果，而且还立即提供了有关产品中存在的立体异构体的信息，这很难单独使用 NMR 和 MS 获得。因此，我们的结果表明，MicroED 方法可以在时间尺度上提供有用的结构信息，这些信息类似于许多常用的分析方法，并且可以在未来的研究中添加到现有的小分子结构确定工具套件中。仅使用 NMR 和 MS 难以实现。因此，我们的结果表明，MicroED 方法可以在时间尺度上提供有用的结构信息，这些信息类似于许多常用的分析方法，并且可以在未来的研究中添加到现有的小分子结构确定工具套件中。仅使用 NMR 和 MS 难以实现。因此，我们的结果表明，MicroED 方法可以在时间尺度上提供有用的结构信息，这些信息类似于许多常用的分析方法，并且可以在未来的研究中添加到现有的小分子结构确定工具套件中。