TY - JOUR
T1 - Rapid Structural Analysis of a Synthetic Non-canonical Amino Acid by Microcrystal Electron Diffraction
AU - Gleason, Patrick R.
AU - Nannenga, Brent L.
AU - Mills, Jeremy H.
N1 - Funding Information:
We would like to acknowledge support from the National Institutes of Health grants R21GM131299 (BN and JM), R01GM124152 (BN), and R01GM136996 (JM).
Publisher Copyright:
© Copyright © 2021 Gleason, Nannenga and Mills.
PY - 2021/1/8
Y1 - 2021/1/8
N2 - 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.
AB - 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.
KW - electron diffraction
KW - microcrystal electron diffraction (MicroED)
KW - non-canonical amino acid (ncAA)
KW - organic synthesis
KW - transmission electron microscope (TEM)
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U2 - 10.3389/fmolb.2020.609999
DO - 10.3389/fmolb.2020.609999
M3 - Article
AN - SCOPUS:85099756466
VL - 7
JO - Frontiers in Molecular Biosciences
JF - Frontiers in Molecular Biosciences
SN - 2296-889X
M1 - 609999
ER -