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¹H NMR of Conformationally Flexible Molecules: Temporal Resolution00:52

¹H NMR of Conformationally Flexible Molecules: Temporal Resolution

807
At room temperature, the chair conformer of cyclohexane undergoes rapid ring flipping between two equivalent chair conformers at a rate of approximately 105 times per second. These two chair conformers are in equilibrium. The rapid ring flipping results in the interconversion of the axial proton to an equatorial proton and an equatorial to the axial proton. Such interconversions are too rapid and cannot be detected on the NMR timescale. Hence, the NMR spectrometer cannot distinguish between the...
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¹H NMR of Conformationally Flexible Molecules: Variable-Temperature NMR01:15

¹H NMR of Conformationally Flexible Molecules: Variable-Temperature NMR

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The axial and equatorial protons in cyclohexane can be distinguished by performing a variable-temperature NMR experiment. In this process, except for one proton, the remaining eleven protons are replaced by deuterium. The deuterium substitution avoids the possible peak splitting caused by the spin-spin coupling between the adjacent protons. The remaining proton flips between the axial and equatorial positions.
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NMR Spectroscopy of Aromatic Compounds01:14

NMR Spectroscopy of Aromatic Compounds

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Aromatic compounds can be identified or analyzed using proton NMR and carbon‐13 NMR. Typically, aromatic hydrogens or hydrogens directly bonded to the aromatic rings are strongly deshielded by the aromatic ring current. Therefore, they absorb in the range of 6.5–8.0 ppm in proton NMR spectra. For instance, aromatic hydrogens directly bonded to the benzene ring absorb at 7.3 ppm. However, aromatic hydrogens of larger rings absorb farther upfield or downfield than the ideal range.
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NMR Spectroscopy: Spin–Spin Coupling01:08

NMR Spectroscopy: Spin–Spin Coupling

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The spin state of an NMR-active nucleus can have a slight effect on its immediate electronic environment. This effect propagates through the intervening bonds and affects the electronic environments of NMR-active nuclei up to three bonds away; occasionally, even farther. This phenomenon is called spin–spin coupling or J-coupling. Coupling interactions are mutual and result in small changes in the absorption frequencies of both nuclei involved. While nuclei of the same element are involved...
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NMR Spectroscopy Of Amines01:19

NMR Spectroscopy Of Amines

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In proton NMR spectroscopy, primary amines and secondary amines showcase their N–H protons as a broad signal in the chemical shift range between δ 0.5 and 5 ppm. The exact position in this range depends on several factors, including sample concentration, hydrogen bonding, and the type of solvent used. Since amine protons undergo fast proton exchange in solution, the protons are labile and therefore do not participate in any splitting with adjacent protons. Thus, the observed peak is...
8.5K
¹H NMR of Labile Protons: Temporal Resolution01:10

¹H NMR of Labile Protons: Temporal Resolution

1.1K
Protons bonded to heteroatoms such as nitrogen and oxygen exhibit a range of chemical shift values. This is due to the varying degree of hydrogen bonding between the proton and the heteroatom in other molecules. The extent of hydrogen bonding affects the electron density around the proton, thereby giving different chemical shift values for the protons in the proton NMR spectrum.
The –OH proton in alcohols typically appears in the range of δ 2 to 5 ppm but can vary depending on the specific...
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Characterizing Y224 conformational flexibility in FtmOx1-catalysis using 19F NMR spectroscopy.

Xinye Wang1, Lingyun Yang2, Shenlin Wang1

  • 1State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology Shanghai 200237 China liuxueting@ecust.edu.cn lizhang@ecust.edu.cn.

Catalysis Science & Technology
|December 13, 2024
PubMed
Summary

This study reveals how active site dynamics in α-ketoglutarate-dependent non-haem iron (αKG-NHFe) enzymes enable multifunctional catalysis. Fluorine-19 NMR and crystallography show how enzyme conformations influence catalytic activity for potential biocatalyst development.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Natural Product Biosynthesis

Background:

  • α-Ketoglutarate-dependent non-haem iron (αKG-NHFe) enzymes are vital in natural product synthesis.
  • Some αKG-NHFe enzymes exhibit multifunctional catalytic capabilities.
  • FtmOx1 is an αKG-NHFe enzyme involved in verruculogen biosynthesis, catalyzing multiple reactions.

Purpose of the Study:

  • To investigate the role of active site tyrosine (Y224) conformational dynamics in FtmOx1's multifunctional catalysis.
  • To elucidate the mechanism by which specific enzyme-substrate conformations affect catalytic activities.

Main Methods:

  • Site-directed mutagenesis using amber codon suppression to create a Y224-to-3,5-difluorotyrosine mutant.
  • 19F NMR spectroscopy to assess enzyme structural flexibility.
  • Biochemical assays and X-ray crystallography to analyze enzyme-substrate complexes and catalytic outcomes.

Main Results:

  • The Y224 substitution provided insights into FtmOx1's structural flexibility via 19F NMR.
  • Biochemical and crystallographic data revealed correlations between FtmOx1 conformations and substrate-specific catalytic activities.
  • The study demonstrates the utility of 19F NMR for studying enzyme dynamics.

Conclusions:

  • Conformational dynamics of active site residues, like Y224, are critical for the multifunctional catalysis of FtmOx1.
  • Understanding these dynamics can guide the engineering of novel biocatalysts.
  • 19F NMR is a valuable tool for mechanistic studies of complex enzymes.