Abstract
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.
Highlights
The ability to unambiguously characterize the products of chemical reactions is of paramount importance in organic synthesis
We were drawn to an enzymatic semi-synthesis of this compound using tyrosine phenol lyase (TPL) from Citrobacter intermedius because the desired product could be synthesized in a single step from very inexpensive, commercially available starting materials
In previous reports, tyrosine phenol-lyase (TPL) was found to catalyze the synthesis of the 24DHF using only resorcinol, pyruvic acid and ammonia (Yamada et al, 1972; Sawada et al, 1975; Nagasawa et al, 1981) as reactants
Summary
The ability to unambiguously characterize the products of chemical reactions is of paramount importance in organic synthesis. A suite of analytical tools including mass spectrometry (MS) and spectroscopic techniques including ultraviolet and visible (UV-vis), infrared (IR) and nuclear magnetic resonance (NMR) are commonly used to characterize organic molecules. These methods are rapid, highly sensitive and—with the exception of MS—non-destructive to the sample. Molecular structures provide the three-dimensional coordinates of each atom in the molecule, and allow inferences about atom connectivity (i.e., bond number) and molecular packing interactions to be made Despite these benefits, structural techniques suffer from limitations including the requirement of a large quantity of the small molecule and that large, well-diffracting crystals are formed. X-ray crystallographic methods are often more time consuming than other analytical methods and are not commonly viewed as high-throughput for rapid small molecule analysis
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