Reactivity and Selectivity Principles in Native Protein Bioconjugation

Adakkattil, Ramesh; Thakur, Kalyani; Rai, Vishal

doi:10.1002/tcr.202100108

Cited by 11 publications

(6 citation statements)

References 87 publications

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“…Generating compDNA-biomolecule conjugates requires careful design to preserve the activity of the biomolecule during conjugation and surface attachment [43][44][45] . For example, in antibody conjugation, tris(2-carboxyethyl)phosphine hydrochloride (TCEP) is used to selectively reduce hinge-region disul de bonds to free thiol groups for thiol-DNA conjugation 46 .…”

Section: Development Of the Protocolmentioning

confidence: 99%

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Precise surface functionalization of PLGA particles for human T cell modulation

Hadley,

Chen,

Cline

et al. 2023

Nat Protoc

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Biofunctionalization of synthetic materials has broad utility in various biomedical applications but was limited by insu cient e ciency and controllability for bioconjugation. Therefore, we developed a new platform of building synthetic DNA-scaffolds on material surfaces to assemble and organize functional cargos, allowing for more precise control over cargo density and ratio. The adaptation of this technology for biomedical applications requires diverse expertise ranging from materials to bioconjugation chemistry to cell biology, and there are many critical checkpoints to ensure the quality of the platform for the expected biological function. In this protocol, we describe the three key fabrication procedures involved: 1) fabrication of polymeric particles engrafted with DNA-scaffolds (3 days), 2) attachment of functional cargos with complementary DNA strands (3-4 days), and 3) surface assembly control and quanti cation (<1 day). We have also provided additional experimental design considerations for modifying the platform-for example, varying the material composition, size, or cargo types-which may be required for different biological needs. An area of increasing interest is immunomodulation, where immune cells have been recognized for their ability to sense extracellular cues to shape their phenotypic adaptation. We have reported how their modulation can be advanced by this precision biomaterial platform. Here, we exemplify the protocol of primary human T cells activation and identi ed various parameters that can impact T cell ex vivo manufacturing. This protocol will equip investigators with the necessary fabrication procedures and validation assays to design high-delity DNA-scaffolded biomaterials for uses in diverse biomedical applications.

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Section: Development Of the Protocolmentioning

confidence: 99%

“…There are many protein bioconjugation chemistries available which should be balanced with conjugation e ciency, cost, and maintenance of biomolecule activity 43,44,48,58,59 . Alternatively, a protein tag (e.g.…”

Section: Protein-dna Conjugationmentioning

confidence: 99%

Precise surface functionalization of PLGA particles for human T cell modulation

Hadley,

Chen,

Cline

et al. 2023

Nat Protoc

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“…These developments could arguably be extended to the scope of enzyme functionalization. In this context, the research group from Vishal Ray has provided inventive and valuable tools to site-specific protein modification [149]. Beside their previously referenced reviews on bioconjugation strategies [27,30,32] and their linchpin directed modification [83], the group has developed different aldehyde-based two-step methods for site-selective modification of lysines.…”

Section: Perspectivesmentioning

confidence: 99%

Protein Modifications: From Chemoselective Probes to Novel Biocatalysts

2021

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Chemical reactions can be performed to covalently modify specific residues in proteins. When applied to native enzymes, these chemical modifications can greatly expand the available set of building blocks for the development of biocatalysts. Nucleophilic canonical amino acid sidechains are the most readily accessible targets for such endeavors. A rich history of attempts to design enhanced or novel enzymes, from various protein scaffolds, has paved the way for a rapidly developing field with growing scientific, industrial, and biomedical applications. A major challenge is to devise reactions that are compatible with native proteins and can selectively modify specific residues. Cysteine, lysine, N-terminus, and carboxylate residues comprise the most widespread naturally occurring targets for enzyme modifications. In this review, chemical methods for selective modification of enzymes will be discussed, alongside with examples of reported applications. We aim to highlight the potential of such strategies to enhance enzyme function and create novel semisynthetic biocatalysts, as well as provide a perspective in a fast-evolving topic.

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“…As one of the essential amino acids, tryptophan is key in the (bio)synthesis of peptides, proteins, and many small indole-containing molecules of biological relevance. − In synthetic chemistry routes, harnessing this abundant and biobased building block can be a large asset for their overall sustainability and cost efficiency . Moreover, tryptophan residues are often tied to specific protein functions and possess intrinsic fluorescence, which results in a widespread use of tryptophan analogues in biochemical research. − Along with the rise of bioconjugation methods for the site-selective synthetic modification of peptides or proteins, the catalytic conversion of the C–H bonds in native tryptophan residues is a promising approach to further expand such synthetic possibilities. − Rather than reacting on functional groups, such as −NH 2 or −SH, in lysine or cysteine or relying on the labeling with non-native amino acid residues (e.g., azide or alkyne functional groups), a recent research focus is to translate the advances in Pd-catalyzed C–H bond activation on the C2 position of indole scaffolds − toward the direct C–H coupling of tryptophan residues. Along with the diversification of the individual amino acid, the strategy has also been demonstrated in the postsynthetic modification of tryptophan residues in peptide chains, − for the formation of peptide macrocycles, − or the direct bioconjugation of proteins by cleaving the C–H bonds in native tryptophan residues.…”

Section: Introductionmentioning

confidence: 99%

Overcoming Pd Catalyst Deactivation in the C–H Coupling of Tryptophan Residues in Water Using Air as the Oxidant

Beckers,

Vos,

Van Dessel

et al. 2024

ACS Catal.

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The Pd-catalyzed C−H activation of natural tryptophan residues has emerged as a promising approach for their direct synthetic modification. While using water as the solvent and harnessing air as the oxidant is enticing, these conditions induce catalyst deactivation by promoting the formation of inactive Pd(0) clusters. In this work, we have studied optimized Pd-based catalytic systems via nonsteady state kinetic analysis and in situ Xray absorption spectroscopy (XAS) to overcome catalyst deactivation, which enables the effective use of lower Pd loadings.

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Reactivity and Selectivity Principles in Native Protein Bioconjugation

Cited by 11 publications

References 87 publications

Precise surface functionalization of PLGA particles for human T cell modulation

Precise surface functionalization of PLGA particles for human T cell modulation

Protein Modifications: From Chemoselective Probes to Novel Biocatalysts

Overcoming Pd Catalyst Deactivation in the C–H Coupling of Tryptophan Residues in Water Using Air as the Oxidant

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