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Covalently Linked Protein Regulators02:04

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Proteins can undergo many types of post-translational modifications, often in response to changes in their environment. These modifications play an important role in the function and stability of these proteins. Covalently linked molecules include functional groups, such as methyl, acetyl, and phosphate groups, and also small proteins, such as ubiquitin. There are around 200 different types of covalent regulators that have been identified.
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Acetylation, a phase II biotransformation reaction, introduces an acetyl group to drugs or their metabolites. Acetyltransferase enzymes facilitate this reaction, which resembles α-amino acid conjugation due to the addition of a functional group to the drug molecule.
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Histone Modification02:32

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The histone proteins have a flexible N-terminal tail extending out from the nucleosome. These histone tails are often subjected to post-translational modifications such as acetylation, methylation, phosphorylation, and ubiquitination. Particular combinations of these modifications form “histone codes” that influence the chromatin folding and tissue-specific gene expression.
Acetylation
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Acetoacetic ester synthesis is a method to obtain ketones from alkyl halides and β-keto esters. The reaction occurs in the presence of an alkoxide base that abstracts the acidic proton of the β-keto esters. The step results in an enolate ion which is doubly stabilized. The enolate then reacts with an alkyl halide via the SN2 process to produce an alkylated ester intermediate with a new C–C bond. The hydrolysis of the intermediate, followed by acidification, results in an...
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Ketones with α protons are deprotonated by strong bases like lithium diisopropylamide (LDA) to form enolate ions. The anion is stabilized by resonance, and its hybrid structure exhibits negative charges on the carbonyl oxygen and the α carbon. This ambident nucleophile can attack an electrophile via two possible sites: the carbonyl oxygen, known as O-attack, or the α carbon, known as C-attack. The nucleophilic attack via the carbanionic site is preferred. This is due to the...
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Updated: Dec 25, 2025

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli
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Selective Lysine Modification Enabled by Intramolecular Acyl Transfer.

David Hymel1, Fa Liu1

  • 1Novo Nordisk Research Center, 530 Fairview Avenue North, Seattle, Washington 98109, United States.

Organic Letters
|April 2, 2020
PubMed
Summary
This summary is machine-generated.

We developed a novel method for modifying proteins using a cysteine residue to attach reagents, enabling precise lysine acylation. This traceless technique regenerates the original protein sequence after modification, aiding biologic therapeutic development.

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Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry
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Area of Science:

  • Biochemistry
  • Protein Engineering
  • Medicinal Chemistry

Background:

  • Selective modification of recombinant proteins is crucial for developing biologic therapeutics.
  • Existing methods may lack efficiency or introduce unwanted alterations.

Purpose of the Study:

  • To introduce a novel strategy for site-specific protein modification.
  • To achieve lysine acylation via a cysteine-mediated intramolecular reaction.
  • To demonstrate a traceless modification approach.

Main Methods:

  • A strategy engaging a cysteine (Cys) residue to covalently tether reagents to a target protein.
  • Utilizing proximity-driven intramolecular O-to-N acyl-transfer for modification.
  • Employing a case study with Glucagon-Like Peptide-1 (GLP-1).
  • Performing desulfurization of the Cys mutation to alanine (Ala) for sequence regeneration.

Main Results:

  • Successful covalent tethering of reagents to the target protein via Cys engagement.
  • Efficient generation of desired lysine-acylated products through intramolecular acyl-transfer.
  • Demonstrated traceless modification of GLP-1, regenerating the native sequence.

Conclusions:

  • The reported Lys modification strategy is effective for site-specific protein acylation.
  • The method offers a traceless approach, preserving protein integrity.
  • This technique holds promise for the discovery and development of biologic therapeutics.