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Related Concept Videos

Nitrosation of Enols01:19

Nitrosation of Enols

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The nitrosation reaction is one of the methods of preparing 1,2-diketones. The enol tautomer of the starting ketone reacts with sodium nitrite in hydrochloric acid, generating the 1,2-diketone after hydrolysis.
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Synthesis of α-Substituted Carbonyl Compounds: The Stork Enamine Reaction01:26

Synthesis of α-Substituted Carbonyl Compounds: The Stork Enamine Reaction

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α-Substituted ketones or aldehydes can be synthesized from enamines by the Stork enamine reaction, named after its pioneer Gilbert Stork. Enamines are useful synthetic intermediates where the lone pair on nitrogen is in conjugation with the C=C bond. They resemble enolate ions, as the resonance forms of both species have a nucleophilic α carbon.
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Reactivity of Enols01:18

Reactivity of Enols

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Enols are a class of compounds where a hydroxyl group is attached to a carbon–carbon double bond, which implies that it is a vinyl alcohol. A carbonyl compound with an α hydrogen undergoes keto–enol tautomerism and remains in equilibrium with its tautomer, the enol form. Usually, the keto tautomer is present in a higher concentration than the enol tautomer due to the higher bond energy of C=O compared to C=C. Moreover, the direction of the keto–enol equilibrium is...
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Phase I Reactions: Reductive Reactions01:27

Phase I Reactions: Reductive Reactions

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Phase I biotransformation reductive reactions are chemical processes that modify drugs by introducing or revealing polar functional groups via reduction. Enzymes called reductases catalyze these reactions, playing a pivotal role in drug metabolism by transforming lipophilic drugs into more polar, water-soluble metabolites for easy excretion. An essential type of reductive reaction is the carbonyl group reduction, where aldehydes and ketones are reduced to alcohols. An example is the...
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Preparation of Amines: Reduction of Oximes and Nitro Compounds01:29

Preparation of Amines: Reduction of Oximes and Nitro Compounds

5.0K
Oximes can be reduced to primary amines using catalytic hydrogenation, hydride reduction, or sodium metal reduction. The reduction of aliphatic and aromatic nitro compounds to primary amines takes place by either catalytic hydrogenation or by using active metals like Fe, Zn, and Sn in the presence of an acid.
Though catalytic hydrogenation can reduce nitrobenzenes, the reduction is nonselective in the presence of other functional groups. For instance, if nitrobenzene contains an aldehyde group,...
5.0K
meta-Directing Deactivators: –NO2, –CN, –CHO, –⁠CO2R, –COR, –CO2H01:13

meta-Directing Deactivators: –NO2, –CN, –CHO, –⁠CO2R, –COR, –CO2H

7.1K
All meta-directing substituents are deactivating groups. These substituents withdraw electrons from the aromatic ring, making the ring less reactive toward electrophilic substitution. For example, the nitration of nitrobenzene is 100,000 times slower than that of benzene because of the deactivating effect of the nitro group. The first step in an electrophilic aromatic substitution is the addition of an electrophile to form a resonance-stabilized carbocation. The energy diagrams for...
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Related Experiment Video

Updated: Apr 17, 2026

Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells
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Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells

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Engineering towards nitroreductase functionality in ene-reductase scaffolds.

Jonathan T Park1, Lizzette M Gómez Ramos, Andreas S Bommarius

  • 1School of Chemical and Biomolecular Engineering, Parker H. Petit Institute for Bioengineering and Bioscience, 315 Ferst Drive, Atlanta, GA 30332-0363 (USA).

Chembiochem : a European Journal of Chemical Biology
|February 24, 2015
PubMed
Summary
This summary is machine-generated.

Modifying ene-reductase (ER) KYE1 by altering active site cavity size significantly enhanced nitroreductase (NR) activity. Specific mutations resulted in a 100-fold activity increase and complete conversion to NR functionality.

Keywords:
biocatalysisflavinsoxidoreductasesprotein engineeringsubstrate specificity

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Analytical Techniques for Assaying Nitric Oxide Bioactivity
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Area of Science:

  • Biochemistry
  • Enzymology
  • Protein Engineering

Background:

  • Nitroreductases (NRs) and ene-reductases (ERs) are flavin-dependent enzymes with overlapping yet distinct catalytic functions.
  • Previous work showed that modifying ER XenA can enhance its NR activity, suggesting potential for engineering these enzymes.

Purpose of the Study:

  • To investigate the role of active site cavity size in determining the functional specificity of ERs and NRs.
  • To engineer the ER KYE1 to enhance its NR activity through targeted mutations.

Main Methods:

  • Structural comparison of NRs and ERs to identify key residues for modification.
  • Site-directed mutagenesis of ER KYE1 to alter active site cavity size and hydrogen bonding patterns.
  • Enzyme activity assays to screen single variants and evaluate combined mutations for NR activity.

Main Results:

  • Single mutations were screened, and promising variants were combined to enhance NR activity.
  • Variant F296A/Y275A demonstrated a 100-fold increase in NR specific activity compared to wild-type.
  • Variant H191A/F296A/Y375A achieved complete conversion of activity towards nitroreduction.

Conclusions:

  • Active site cavity size is a critical determinant of enzyme functionality, influencing the substrate specificity between NR and ER activities.
  • Targeted engineering of ER KYE1 can successfully enhance its nitroreductase activity, demonstrating the potential for enzyme functional conversion.