Rapid sodium periodate cleavage of an unnatural amino acid enables unmasking of a highly reactive α-oxo aldehyde for protein bioconjugation

Brabham, Robin; Keenan, Tessa; Hüsken, Annika; Bilsborrow, Jacob; McBerney, Ryan; Kumar, Vajinder; Turnbull, W. Bruce; Fascione, M. A.

doi:10.1039/d0ob00972e

Cited by 10 publications

(16 citation statements)

References 26 publications

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“…This reaction typically reaches completion within 5 min in sodium phosphate, whereas the reaction does not reach completion in phosphate-buffered saline containing potassium ions, as these are known to hinder periodate reactivity. 67 Oxime ligation of the oxidized-W88E (450 μM) with 10 equiv linker 3- endo in the presence of aniline (1% v/v) at 37 °C reached completion within 16 h ( Figure 3 C). When the oxidized-W88E was used at a concentration below 200 μM, a mixture of labeled product and N-terminal cyclization was observed.…”

Section: Resultsmentioning

confidence: 99%

Bioorthogonal, Bifunctional Linker for Engineering Synthetic Glycoproteins

McBerney

Dolan

Cawood

et al. 2022

JACS Au

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Post-translational glycosylation of proteins results in complex mixtures of heterogeneous protein glycoforms. Glycoproteins have many potential applications from fundamental studies of glycobiology to potential therapeutics, but generating homogeneous recombinant glycoproteins using chemical or chemoenzymatic reactions to mimic natural glycoproteins or creating homogeneous synthetic neoglycoproteins is a challenging synthetic task. In this work, we use a site-specific bioorthogonal approach to produce synthetic homogeneous glycoproteins. We develop a bifunctional, bioorthogonal linker that combines oxime ligation and strain-promoted azide–alkyne cycloaddition chemistry to functionalize reducing sugars and glycan derivatives for attachment to proteins. We demonstrate the utility of this minimal length linker by producing neoglycoprotein inhibitors of cholera toxin in which derivatives of the disaccharide lactose and GM1os pentasaccharide are attached to a nonbinding variant of the cholera toxin B-subunit that acts as a size- and valency-matched multivalent scaffold. The resulting neoglycoproteins decorated with GM1 ligands inhibit cholera toxin B-subunit adhesion with a picomolar IC 50 .

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Section: Resultsmentioning

confidence: 99%

Bioorthogonal, Bifunctional Linker for Engineering Synthetic Glycoproteins

McBerney

Dolan

Cawood

et al. 2022

JACS Au

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“…Protein to be labeled was buffer exchanged into sodium phosphate buffer (pH 7.4); it is very important that the buffer does not contain potassium. Performing reactions in the presence of K + ions will result in oxidation failing to reach completion ( Brabham et al, 2020 ). Oxidation: the protein to be labeled (500 µl, 200 µM) in sodium phosphate buffer was mixed with L-methionine (4 µl of 250 mM stock, final concentration 2.0 mM, 10 eq) and NaIO4 (2 µl of 250 mM stock, final concentration 1.0 mM, 5 eq).…”

Section: Methodsmentioning

confidence: 99%

Synthesis of cholera toxin B subunit glycoconjugates using site-specific orthogonal oxime and sortase ligation reactions

Dolan

Machin

Dedola³

et al. 2022

Front. Chem.

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The chemoenzymatic synthesis of a series of dual N- and C-terminal–functionalized cholera toxin B subunit (CTB) glycoconjugates is described. Mucin 1 peptides bearing different levels of Tn antigen glycosylation [MUC1(Tn)] were prepared via solid-phase peptide synthesis. Using sortase-mediated ligation, the MUC1(Tn) epitopes were conjugated to the C-terminus of CTB in a well-defined manner allowing for high-density display of the MUC1(Tn) epitopes. This work explores the challenges of using sortase-mediated ligation in combination with glycopeptides and the practical considerations to obtain high levels of conjugation. Furthermore, we describe methods to combine two orthogonal labeling methodologies, oxime- and sortase-mediated ligation, to expand the biochemical toolkit and produce dual N- and C-terminal–labeled conjugates.

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“…is based on the imidazole ring, which has been linked together by a pi-bonds of the glycine-tyrosine dipeptide that represents in the neutral or anionic states [85][86][87] . So, the pH of the environment can be able to affect the fluorescence intensity of the EGFP/LDH directly, and this nanomaterial can be able to use as a suitable pH sen- www.nature.com/scientificreports/ sor in the range of pH of 4-10.…”

Section: Reversible Fluorescence Response Of the Egfp/ldh The Activementioning

confidence: 99%

ZnAl nano layered double hydroxides for dual functional CRISPR/Cas9 delivery and enhanced green fluorescence protein biosensor

Rabiee

Bagherzadeh

Ghadiri

et al. 2020

Sci Rep

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Evaluation of the effect of different parameters for designing a non-viral vector in gene delivery systems has great importance. In this manner, 2D crystals, precisely layered double hydroxides, have attracted the attention of scientists due to their significant adjustability and low-toxicity and low-cost preparation procedure. In this work, the relationship between different physicochemical properties of LDH, including pH, size, zeta potential, and synthesis procedure, was investigated and optimized for CRISPR/Cas9 delivery and reverse fluorescence response to the EGFP. In this manner, ZnAl LDH and ZnAl HMTA LDH were synthesized and characterized and applied in the HEK-293 cell line to deliver CRISPR/Cas9. The results were optimized by different characterizations as well as Gel Electrophoresis and showed acceptable binding ability with the DNA that could be considered as a promising and also new gold-standard for the delivery of CRISPR/Cas9. Also, the relationship of the presence of tertiary amines (in this case, hexamethylenetetramine (HMTA) as the templates) in the structure of the ZnAl LDH, as well as the gene delivery application, was evaluated. The results showed more than 79% of relative cell viability in most of the weight ratios of LDH to CRISPR/Cas9; fully quenching the fluorescence intensity of the EGFP/LDH in the presence of 15 µg mL−1 of the protoporphyrins along with the detection limit of below 2.1 µg mL−1, the transfection efficiency of around 33% of the GFP positive cell for ZnAl LDH and more than 38% for the ZnAl LDH in the presence of its tertiary amine template.

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Rapid sodium periodate cleavage of an unnatural amino acid enables unmasking of a highly reactive α-oxo aldehyde for protein bioconjugation

Abstract: A genetically incorporated ThrK unnatural amino acid can undergo rapid periodate oxidation to reveal a reactive internal α-oxo aldehyde.

Cited by 10 publications

References 26 publications

Bioorthogonal, Bifunctional Linker for Engineering Synthetic Glycoproteins

Bioorthogonal, Bifunctional Linker for Engineering Synthetic Glycoproteins

Synthesis of cholera toxin B subunit glycoconjugates using site-specific orthogonal oxime and sortase ligation reactions

ZnAl nano layered double hydroxides for dual functional CRISPR/Cas9 delivery and enhanced green fluorescence protein biosensor

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