scholarly journals A Tale of Two Bioconjugations: pH Controlled Divergent Reactivity of Protein A-Oxo Aldehydes in Competing A-Oxo-Mannich and Catalyst-Free Aldol Ligations

2021 ◽  
Author(s):  
Tessa Keenan ◽  
Richard Spears ◽  
Saeed Akkad ◽  
Clare Mahon ◽  
Natasha E. Hatton ◽  
...  
Keyword(s):  
Chemical Biology ◽  
Protein A ◽  
Acidic Ph ◽  
Chemical Methods ◽  
New Methods ◽  
Catalyst Free ◽  
Selective Chemical ◽  
Site Selective ◽  
Novel Protein ◽  
Protein Enzyme

<div><div><div><p>Site-selective chemical methods for protein bioconjugation have revolutionised the fields of cell and chemical biology through the development of novel protein/enzyme probes bearing fluorescent, spectroscopic or even toxic cargos. Herein we report two new methods for the bioconjugation of a-oxo aldehyde handles within proteins using small molecule aniline and/or phenol probes. The ‘a-oxo-Mannich’ and ‘catalyst-free aldol’ ligations both compete for the electrophilic a-oxo aldehyde which displays pH divergent reactivity proceeding through the “Mannich” pathway at acidic pH to afford bifunctionalised bioconjugates, and the “catalyst-free aldol” pathway at neutral pH to afford monofunctionalised bioconjugates. We explore the substrate scope and utility of both these bioconjugations in the construction of neoglycoproteins, in the process formulating a mechanistic rationale for how both pathways intersect with each other at different reaction pH.</p></div></div></div>

scholarly journals A Tale of Two Bioconjugations: pH Controlled Divergent Reactivity of Protein A-Oxo Aldehydes in Competing A-Oxo-Mannich and Catalyst-Free Aldol Ligations

2021 ◽  
Author(s):  
Tessa Keenan ◽  
Richard Spears ◽  
Saeed Akkad ◽  
Clare Mahon ◽  
Natasha E. Hatton ◽  
...  
Keyword(s):  
Chemical Biology ◽  
Protein A ◽  
Acidic Ph ◽  
Chemical Methods ◽  
New Methods ◽  
Catalyst Free ◽  
Selective Chemical ◽  
Site Selective ◽  
Novel Protein ◽  
Protein Enzyme

<div><div><div><p>Site-selective chemical methods for protein bioconjugation have revolutionised the fields of cell and chemical biology through the development of novel protein/enzyme probes bearing fluorescent, spectroscopic or even toxic cargos. Herein we report two new methods for the bioconjugation of a-oxo aldehyde handles within proteins using small molecule aniline and/or phenol probes. The ‘a-oxo-Mannich’ and ‘catalyst-free aldol’ ligations both compete for the electrophilic a-oxo aldehyde which displays pH divergent reactivity proceeding through the “Mannich” pathway at acidic pH to afford bifunctionalised bioconjugates, and the “catalyst-free aldol” pathway at neutral pH to afford monofunctionalised bioconjugates. We explore the substrate scope and utility of both these bioconjugations in the construction of neoglycoproteins, in the process formulating a mechanistic rationale for how both pathways intersect with each other at different reaction pH.</p></div></div></div>

scholarly journals Selenomethionine as an Expressible Handle for Bioconjugations

2020 ◽  
Author(s):  
Dillon T. Flood ◽  
Jordi C.J. Hintzen ◽  
Chenxi Lu ◽  
Philip A. Cistrone ◽  
Jason Chen ◽  
...  
Keyword(s):  
Rate Constant ◽  
Chemical Biology ◽  
Organic Molecules ◽  
Protein Ligation ◽  
On Demand ◽  
Physical Organic ◽  
Mechanistic Analysis ◽  
Selective Chemical ◽  
Site Selective ◽  
Complex Organic

<b>Site-selective chemical protein ligation reactions are enabling tools for chemical biology. Herein, we employ a physical organic study to refine the selenomethionine (SeM) benzylation as a practical protein bioconjugation strategy. SeM is readily introduced through auxotrophic expression and exhibits unique nucleophilic properties that allow it to be selectively modified even in the presence of cysteine. The resulting benzylselenonium adduct is stable at physiological pH, selectively labile to glutathione and embodies a broadly tuneable reactivity profile. Guided by a mechanistic analysis of the reaction, a 4-bromomethylphenylacetyl linker is identified for efficient conjugations of complex organic molecules to SeM containing proteins. This optimized benzyl linker exhibits a rate constant of 3x10<sup>-1</sup> M<sup>-1</sup>s<sup>-1</sup>, facilitating efficient conjugation at micromolar concentrations. The selenonium conjugate is further advanced through a linker that can be selectively photo-locked or reductively cleaved on demand. This tool-kit of selenonium forming reagents have broad potential in the development of chemically enhanced proteins.</b>

scholarly journals Selenomethionine as an Expressible Handle for Bioconjugations

2020 ◽  
Author(s):  
Dillon T. Flood ◽  
Jordi C.J. Hintzen ◽  
Chenxi Lu ◽  
Philip A. Cistrone ◽  
Jason Chen ◽  
...  
Keyword(s):  
Rate Constant ◽  
Chemical Biology ◽  
Organic Molecules ◽  
Protein Ligation ◽  
On Demand ◽  
Physical Organic ◽  
Mechanistic Analysis ◽  
Selective Chemical ◽  
Site Selective ◽  
Complex Organic

<b>Site-selective chemical protein ligation reactions are enabling tools for chemical biology. Herein, we employ a physical organic study to refine the selenomethionine (SeM) benzylation as a practical protein bioconjugation strategy. SeM is readily introduced through auxotrophic expression and exhibits unique nucleophilic properties that allow it to be selectively modified even in the presence of cysteine. The resulting benzylselenonium adduct is stable at physiological pH, selectively labile to glutathione and embodies a broadly tuneable reactivity profile. Guided by a mechanistic analysis of the reaction, a 4-bromomethylphenylacetyl linker is identified for efficient conjugations of complex organic molecules to SeM containing proteins. This optimized benzyl linker exhibits a rate constant of 3x10<sup>-1</sup> M<sup>-1</sup>s<sup>-1</sup>, facilitating efficient conjugation at micromolar concentrations. The selenonium conjugate is further advanced through a linker that can be selectively photo-locked or reductively cleaved on demand. This tool-kit of selenonium forming reagents have broad potential in the development of chemically enhanced proteins.</b>

scholarly journals Selenomethionine as an Expressible Handle for Bioconjugations

2020 ◽  
Author(s):  
Dillon T. Flood ◽  
Jordi C.J. Hintzen ◽  
Chenxi Lu ◽  
Philip A. Cistrone ◽  
Jason Chen ◽  
...  
Keyword(s):  
Rate Constant ◽  
Chemical Biology ◽  
Organic Molecules ◽  
Protein Ligation ◽  
On Demand ◽  
Physical Organic ◽  
Mechanistic Analysis ◽  
Selective Chemical ◽  
Site Selective ◽  
Complex Organic

<b>Site-selective chemical protein ligation reactions are enabling tools for chemical biology. Herein, we employ a physical organic study to refine the selenomethionine (SeM) benzylation as a practical protein bioconjugation strategy. SeM is readily introduced through auxotrophic expression and exhibits unique nucleophilic properties that allow it to be selectively modified even in the presence of cysteine. The resulting benzylselenonium adduct is stable at physiological pH, selectively labile to glutathione and embodies a broadly tuneable reactivity profile. Guided by a mechanistic analysis of the reaction, a 4-bromomethylphenylacetyl linker is identified for efficient conjugations of complex organic molecules to SeM containing proteins. This optimized benzyl linker exhibits a rate constant of 3x10<sup>-1</sup> M<sup>-1</sup>s<sup>-1</sup>, facilitating efficient conjugation at micromolar concentrations. The selenonium conjugate is further advanced through a linker that can be selectively photo-locked or reductively cleaved on demand. This tool-kit of selenonium forming reagents have broad potential in the development of chemically enhanced proteins.</b>

scholarly journals Rational Generation of Lasso Peptides Based on Biosynthetic Gene Mutations and Site-Selective Chemical Modifications

2021 ◽  
Author(s):  
Tan Liu ◽  
Xiaojie Ma ◽  
Jiahui Yu ◽  
Wensheng Yang ◽  
guiyang wang ◽  
...  
Keyword(s):  
Natural Products ◽  
Gene Mutations ◽  
Proteolytic Degradation ◽  
Biosynthetic Gene ◽  
Chemical Modifications ◽  
Lasso Peptides ◽  
Selective Chemical ◽  
Site Selective

Lasso peptides are a unique family of natural products whose structures feature a specific threaded fold, which confers these peptides the resistance to thermal and proteolytic degradation. This stability gives...

scholarly journals Comparison of protein A resins and novel protein A matrices

2020 ◽  
Author(s):  
Talaial Alina ◽  
Paul Randolph ◽  
John Schreffler
Keyword(s):  
Protein A ◽  
Novel Protein

scholarly journals Site-Selective C-C Modification of Proteins at Neutral pH Using Organocatalyst-Mediated Cross Aldol Ligations

2018 ◽  
Author(s):  
Martin A. Fascione ◽  
Richard J. Spears ◽  
Robin L. Brabham ◽  
Darshita Budhadev ◽  
Tessa Keenan ◽  
...  
Keyword(s):  
Small Molecules ◽  
Stable Carbon ◽  
Biological Mechanisms ◽  
Large Excess ◽  
Reaction Conditions ◽  
Chemical Methods ◽  
Neutral Ph ◽  
Chemical Modification Of Proteins ◽  
A Site ◽  
Site Selective

The bioconjugation of proteins with small molecules has proved an invaluable strategy for probing and perturbing dynamic biological mechanisms. The general use of chemical methods for the functionalisation of proteins remains limited however by the frequent requirement for complicated reaction partners to be present in large excess, and harsh reaction conditions which are incompatible with many protein scaffolds. Herein we describe a site-selective organocatalyst-mediated protein aldol ligation (OPAL) that affords stable carbon-carbon linked bioconjugates at neutral pH under biocompatible conditions. OPAL enables rapid chemical modification of proteins within an hour using simple aldehyde probes in minimal excess, and is utilised here in the selective affinity tagging of proteins in cell lysate. Furthermore we demonstrate that the b-hydroxy aldehyde product of the OPAL can be functionalised a second time at neutral pH in a subsequent organocatalyst-mediated oxime ligation. This tandem strategy is showcased in the ‘chemical mimicry’ of a previously inaccessible natural dual post-translationally modified protein integral to the pathogenesis of the neglected tropical disease Leishmaniasis. <br>

scholarly journals REST/NRSF-Interacting LIM Domain Protein, a Putative Nuclear Translocation Receptor

2003 ◽  
Vol 23 (24) ◽  
pp. 9025-9031 ◽  
Author(s):  
Masahito Shimojo ◽  
Louis B. Hersh
Keyword(s):  
Nuclear Localization ◽  
Nuclear Translocation ◽  
Protein A ◽  
Lim Domain ◽  
Nuclear Targeting ◽  
Lim Domains ◽  
Lim Domain Protein ◽  
Domain Protein ◽  
Nuclear Localization Sequences ◽  
Novel Protein

ABSTRACT The transcriptional repressor REST/NRSF (RE-1 silencing transcription factor/neuron-restrictive silencer factor) and the transcriptional regulator REST4 share an N-terminal zinc finger domain structure involved in nuclear targeting. Using this domain as bait in a yeast two-hybrid screen, a novel protein that contains three LIM domains, putative nuclear localization sequences, protein kinase A phosphorylation sites, and a CAAX prenylation motif was isolated. This protein, which is localized around the nucleus, is involved in determining the nuclear localization of REST4 and REST/NRSF. We propose the name RILP, for REST/NRSF-interacting LIM domain protein, to label this novel protein. RILP appears to serve as a nuclear receptor for REST/NRSF, REST4, and possibly other transcription factors.

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