A study on the trastuzumab conjugation at tyrosine using diazonium salts

Griebenow, Nils; Greven, Simone; Lobell, Mario; Dilmaç, Alicia M.; Bräse, Stefan

doi:10.1039/c5ra18271a

Cited by 8 publications

(8 citation statements)

References 28 publications

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“…Interestingly, another study using a different stable diazonium reagent reported complete selectivity towards tyrosine at physiological pH when modifying the antibody trastuzumab. 22 Since the development of the first stable diazonium reagent for tyrosine modification, the variety of functional groups that can be introduced with such compounds has been expanded, enabling bioorthogonal click chemistry such as azide–alkyne cycloaddition 23 or tetrazine-ene reactions. 24 In addition, the method has been applied to many proteins, including the conjugation of the small-molecule inhibitor aplaviroc to a series of anti-HIV antibodies.…”

Section: Chemical Approaches For Tyrosine Modificationmentioning

confidence: 99%

Tyrosine bioconjugation – an emergent alternative

et al. 2020

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Section: Chemical Approaches For Tyrosine Modificationmentioning

confidence: 99%

Tyrosine bioconjugation – an emergent alternative

et al. 2020

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“…Aryl diazonium ions have undergone a renaissance in the bioconjugation literature, ,− but their chromophoric properties enabled them to be one of the first reliable tools to modify proteins dating back to the early 20th century. ,, While useful, many aryl diazonium ions suffer from the requirement of generation immediately prior to the experiment due to their short half-lives in solution and general instability. Recognizing the promise of aryl diazonium ions as robust small-molecule covalent probes, triazabutadienes were developed as a new biochemical tool for their generation .…”

Section: Introductionmentioning

confidence: 99%

Direct Intracellular Delivery of Benzene Diazonium Ions As Observed by Increased Tyrosine Phosphorylation

et al. 2022

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A challenge within the field of bioconjugation is developing probes to uncover novel information on proteins and other biomolecules. Intracellular delivery of these probes offers the promise of giving relevant context to this information, and these probes can serve as hypothesis-generating tools within complex systems. Leveraging the utility of triazabutadiene chemistry, herein, we discuss the development of a probe that undergoes reduction-mediated deprotection to rapidly deliver a benzene diazonium ion (BDI) into cells. The intracellular BDI resulted in an increase in global tyrosine phosphorylation levels. Seeing phosphatase dysregulation as a potential source of this increase, a tyrosine phosphatase (PTP1B) was tested and shown to be both inhibited and covalently modified by the BDI. In addition to the expected azobenzene formation at tyrosine side chains, key reactive histidine residues were also modified.

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“…However, our experiment showed that 229 Cys and 232 Cys on the same heavy chain were connected by modification with a (benzofuranyl)methine unit, presumably because 229 Cys and 232 Cys are close to each other. In addition, our experiment also showed a single S-arylation at some cysteine residues and azo coupling at some tyrosine residues , (Figure ). As the yield of the whole process in this preliminary experiment including photolabeling and protein digestion is low, no reliable quantitative analysis can be achieved.…”

Section: Resultsmentioning

confidence: 99%

Revisiting Disulfide–Yne and Disulfide–Diazonium Reactions for Potential Direct Modification of Disulfide Bonds in Proteins

et al. 2022

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To find their potential use in protein research, direct addition of a disulfide compound to alkyne (namely disulfide–yne reaction) and S-arylation with arenediazonium salt (namely disulfide–diazonium reaction) were investigated in aqueous or protic solutions. The reaction of dimethyl disulfide with 5-hexynol performed best under 300 nm irradiation in the presence of sodium acetate to afford 5,6-bis(methylthio)-5-hexenol in 60% yield. Without the prior reduction of a disulfide bond to thiols, the disulfide–yne reactions have the advantage of 100% atom economy. Disulfide–diazonium reaction was triggered by sodium formate and accelerated by photoirradiation with a 450 nm LED lamp (5 W). The reaction of 3,4-dihydroxy-1,2-dithiane with 2-(prop-2-yn-1-yloxy)benzene-1-diazonium tetrafluoroborate (8b) afforded 2-(benzofuran-3-yl)-1,3-dithiepane-5,6-diol (13), confirming that both S substituents originate from the same disulfide molecule. The trastuzumab antibody was incubated with diazonium 8b, followed by α-lytic protease digestion, LC-ESI-MS/MS analysis, and Mascot search, to verify that the proximal C229 and C232 residues on the same heavy chain were reconnected with a (benzofuranyl)methine moiety that originated from 8b, unlike the expected disulfide rebridging across two heavy chains. Nonetheless, disulfide–diazonium reactions still have potential for rebridging disulfide bonds if appropriate proteins and diazonium agents are chosen.

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A study on the trastuzumab conjugation at tyrosine using diazonium salts

Abstract: Herein, we report on the conjugation of trastuzumab with 2,5-difluorobenzene diazonium tetrafluoroborate.

Cited by 8 publications

References 28 publications

Tyrosine bioconjugation – an emergent alternative

Tyrosine bioconjugation – an emergent alternative

Direct Intracellular Delivery of Benzene Diazonium Ions As Observed by Increased Tyrosine Phosphorylation

Revisiting Disulfide–Yne and Disulfide–Diazonium Reactions for Potential Direct Modification of Disulfide Bonds in Proteins

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