Dataset Information

Photoredox-Catalyzed Labeling of Hydroxyindoles with Chemoselectivity (PhotoCLIC) for Site-Specific Protein Bioconjugation.

ABSTRACT: We have developed a novel visible-light-catalyzed bioconjugation reaction, PhotoCLIC, that enables chemoselective attachment of diverse aromatic amine reagents onto a site-specifically installed 5-hydroxytryptophan residue (5HTP) on full-length proteins of varied complexity. The reaction uses catalytic amounts of methylene blue and blue/red light-emitting diodes (455/650 nm) for rapid site-specific protein bioconjugation. Characterization of the PhotoCLIC product reveals a unique structure formed likely through a singlet oxygen-dependent modification of 5HTP. PhotoCLIC has a wide substrate scope and its compatibility with strain-promoted azide-alkyne click reaction, enables site-specific dual-labeling of a target protein.

SUBMITTER: Singha Roy SJ

PROVIDER: S-EPMC10330600 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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Photoredox-Catalyzed Labeling of Hydroxyindoles with Chemoselectivity (PhotoCLIC) for Site-Specific Protein Bioconjugation.

Singha Roy Soumya Jyoti SJ Loynd Conor C Jewel Delilah D Canarelli Sarah E SE Ficaretta Elise D ED Pham Quan A QA Weerapana Eranthie E Chatterjee Abhishek A

Angewandte Chemie (International ed. in English) 20230523 27

We have developed a novel visible-light-catalyzed bioconjugation reaction, PhotoCLIC, that enables chemoselective attachment of diverse aromatic amine reagents onto a site-specifically installed 5-hydroxytryptophan residue (5HTP) on full-length proteins of varied complexity. The reaction uses catalytic amounts of methylene blue and blue/red light-emitting diodes (455/650 nm) for rapid site-specific protein bioconjugation. Characterization of the PhotoCLIC product reveals a unique structure forme ...[more]

PMID: 37219923

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Project description:Precise control of covalent bond formation in the presence of multiple functional groups is pertinent in the development of many next-generation bioconjugates and materials. Strategies derived from bioorthogonal chemistries are contributing greatly in that regard; however, the gain of chemoselectivity is often compromised by the slow rates of many of these existing chemistries. Recent work on a variation of the classical aldehyde/ketone condensation based on ortho-carbonylphenylboronic acids has uncovered markedly accelerated rates compared to those of the simple carbonyl counterparts. The products of these reactions are distinct, often in the form of boron-nitrogen heterocycles. In particular, we have shown that 2-formylphenylboronic acid (2fPBA), when coupled with an α-amino-hydrazide, produces a unique zwitterionic and stable 2,3,1-benzodiazaborine derivative. In this work, we apply this chemistry to generate chemically defined and functional bioconjugates, herein illustrated with immunoconjugates. We show that an antibody and a fluorophore (as payload) equipped with the relevant reactive handles undergo rapid conjugation at near-stoichiometric ratios, displaying a reaction half-life of only ∼5 min with 2 equiv of the linker payload. Importantly, the reaction can be extended to multicomponent labeling by partnering with the popular strain-promoted azide-alkyne cycloaddition and tetrazine- trans-cyclooctene (Tz-TCO) ligation. The mutual orthogonality to both of these chemistries allows simultaneous triple bioorthogonal conjugations, a rare feat thus far that will widen the scope of various multilabeling applications. Further collaboration with the Tz-TCO reaction enables rapid one-pot synthesis of a site-specific dual-payload antibody conjugate. Altogether, we envision that the 2fPBA-α-amino-hydrazide ligation will facilitate efficient assembly of diverse bioconjugates and materials, enabling access to more complex modalities via partnership with other orthogonal chemistries.

Project description:Site-specific protein labeling is an important technique in protein chemistry and is used for diverse applications ranging from creating protein conjugates to protein immobilization. Enzymatic reactions, including protein prenylation, have been widely exploited as methods to accomplish site-specific labeling. Enzymatic prenylation is catalyzed by prenyltransferases, including protein farnesyltransferase (PFTase) and geranylgeranyltransferase type I (GGTase-I), both of which recognize C-terminal CaaX motifs with different specificities and transfer prenyl groups from isoprenoid diphosphates to their respective target proteins. A number of isoprenoid analogues containing bioorthogonal functional groups have been used to label proteins of interest via PFTase-catalyzed reaction. In this study, we sought to expand the scope of prenyltransferase-mediated protein labeling by exploring the utility of rat GGTase-I (rGGTase-I). First, the isoprenoid specificity of rGGTase-I was evaluated by screening eight different analogues and it was found that those with bulky moieties and longer backbone length were recognized by rGGTase-I more efficiently. Taking advantage of the different substrate specificities of rat PFTase (rPFTase) and rGGTase-I, we then developed a simultaneous dual labeling method to selectively label two different proteins by using isoprenoid analogue and CaaX substrate pairs that were specific to only one of the prenyltransferases. Using two model proteins, green fluorescent protein with a C-terminal CVLL sequence (GFP-CVLL) and red fluorescent protein with a C-terminal CVIA sequence (RFP-CVIA), we demonstrated that when incubated together with both prenyltransferases and the selected isoprenoid analogues, GFP-CVLL was specifically modified with a ketone-functionalized analogue by rGGTase-I and RFP-CVIA was selectively labeled with an alkyne-containing analogue by rPFTase. By switching the ketone-containing analogue to an azide-containing analogue, it was possible to create protein tail-to-tail dimers in a one-pot procedure through the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. Overall, with the flexibility of using different isoprenoid analogues, this system greatly extends the utility of protein labeling using prenyltransferases.

Dataset Information

Photoredox-Catalyzed Labeling of Hydroxyindoles with Chemoselectivity (PhotoCLIC) for Site-Specific Protein Bioconjugation.

Publications

Photoredox-Catalyzed Labeling of Hydroxyindoles with Chemoselectivity (PhotoCLIC) for Site-Specific Protein Bioconjugation.

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