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Related Concept Videos

Acid Halides to Amides: Aminolysis01:07

Acid Halides to Amides: Aminolysis

Aminolysis is a nucleophilic acyl substitution reaction, where ammonia or amines act as nucleophiles to give the substitution product. Acid halides react with ammonia, primary amines, and secondary amines to yield primary, secondary, and tertiary amides, respectively.
In the first step of the aminolysis mechanism, the amine attacks the carbonyl carbon of the acyl chloride to form a tetrahedral intermediate. In the second step, the carbonyl group is re-formed with the elimination of a chloride...
Preparation of 1° Amines: Azide Synthesis01:22

Preparation of 1° Amines: Azide Synthesis

Direct alkylation of ammonia produces polyalkylated amines, along with a quaternary ammonium salt. To exclusively prepare primary amines, the azide synthesis method can be used.
Azide ions act as good nucleophiles and react with unhindered alkyl halides to form alkyl azides. Alkyl azides do not participate in further nucleophilic substitution reactions, thereby eliminating the chances of polyalkylated products. Alkyl azides are reduced by hydride-based reducing agents, like lithium aluminum...
Preparation of Amines: Alkylation of Ammonia and Amines01:30

Preparation of Amines: Alkylation of Ammonia and Amines

Alkylation is one of the methods used to prepare amines. Direct alkylation of ammonia or a primary amine with an alkyl halide gives polyalkylated amines along with a quaternary ammonium salt through successive SN2 reactions. This process of making the quaternary salt through the direct alkylation method is called exhaustive alkylation.
Each alkylation step makes the nitrogen center more nucleophilic, which triggers successive alkylations until a quaternary ammonium salt is formed. Considering...
Preparation of Amines: Reduction of Amides and Nitriles01:13

Preparation of Amines: Reduction of Amides and Nitriles

Nitriles can be reduced to primary amines using reducing agents like lithium aluminum hydride or catalytic hydrogenation. The reduction introduces an amino group with an extra carbon in the skeleton. Nitriles are formed from the reaction between alkyl halides and sodium cyanide through the SN2 mechanism. Primary alkyl halides are the preferred substrates to prepare nitriles.
Amides can be reduced to primary, secondary, and tertiary amines using catalytic hydrogenation, active metals like Fe,...
Preparation and Reactions of Thiols02:33

Preparation and Reactions of Thiols

Thiols are prepared using the hydrosulfide anion as a nucleophile in a nucleophilic substitution reaction with alkyl halides. For instance, bromobutane reacts with sodium hydrosulfide to give butanethiol.
Nitriles to Amines: LiAlH4 Reduction00:55

Nitriles to Amines: LiAlH4 Reduction

Nitriles are reduced to amines in the presence of strong reducing agents like lithium aluminum hydride through a typical nucleophilic acyl substitution. The reaction requires two equivalents of the reducing agent. The reducing agent acts as a source of hydride ions.
As shown below, the mechanism involves three steps. Firstly, the hydride ion acting as a nucleophile attacks the nitrile carbon to form an anion. In the second step, a second equivalent of the hydride ion attacks the anion to...

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Related Experiment Video

Updated: Jun 27, 2026

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF)
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Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF)

Published on: September 17, 2019

Practical guidelines for the AMI-Isonitrile ligation.

Ian Warm1, Athanasios Markos2, Helma Wennemers1

  • 1Laboratory of Organic Chemistry, D-CHAB, ETH Zürich, Zürich, Switzerland.

Methods in Enzymology
|June 25, 2026
PubMed
Summary
This summary is machine-generated.

This chapter details the azomethine imine (AMI)-isonitrile ligation, a rapid bioorthogonal reaction. Practical synthesis and installation protocols for isonitrile reagents and AMI conjugates are provided for protein and live-cell functionalization.

Keywords:
Azomethine iminesBioconjugationBioorthogonal chemistryCell labelingIsocyanidesIsonitrilespH dependence

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Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF)
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Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates
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Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Published on: March 6, 2019

Area of Science:

  • Bioorthogonal chemistry
  • Chemical biology
  • Organic synthesis

Background:

  • The azomethine imine (AMI)-isonitrile ligation is a bioorthogonal reaction known for its speed and pH-dependent kinetics.
  • Bioorthogonal reactions are crucial for studying biological processes in situ without interfering with native biochemical pathways.

Purpose of the Study:

  • To provide practical guidelines for synthesizing isonitrile reagents and functionalizable AMIs.
  • To detail protocols for installing isonitrile handles onto proteins and live cells.
  • To describe conditions for functionalizing introduced isonitriles using the AMI-isonitrile ligation.

Main Methods:

  • Synthesis of isonitrile reagents and AMI-fluorophore conjugates.
  • Installation of isonitrile handles onto proteins using thiol-maleimide or amine-reactive ester chemistries.
  • Installation of isonitrile handles onto live cells via reactive ester or antibody-antigen-mediated approaches.
  • Functionalization of isonitriles using the AMI-isonitrile ligation under specific conditions.

Main Results:

  • Established practical guidelines for isonitrile reagent and AMI conjugate synthesis.
  • Developed detailed protocols for isonitrile handle installation on proteins and live cells.
  • Optimized conditions for the AMI-isonitrile ligation for efficient functionalization.

Conclusions:

  • The AMI-isonitrile ligation offers a robust and efficient method for bioorthogonal functionalization.
  • The provided protocols facilitate the application of this ligation in protein and live-cell labeling.
  • This work enhances the utility of bioorthogonal chemistry in biological research.