Alkylation of histidine with maleimido‐compounds

Papini, Anna Maria; Rudolph, S.; Siglmüller, Gabriele; Musiol, Hans-Jürgen; Göhring, Walter; Moröder, Luis

doi:10.1111/j.1399-3011.1992.tb01594.x

Cited by 18 publications

(4 citation statements)

References 17 publications

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“…A small amount of adduct at the expected mass was observed only in the case of NEM, although subsequent digestion and nanoLC-MS analysis showed that the succinimide modification occurred at histidine sites and not at C46. Indeed, slow alkylation of non-cysteine amino acid residues by NEM has been reported at longer reaction times [28][29][30][31]. AhpC-SNO was generated via trans-nitrosation by S-nitrosocysteine (CySNO), then incubated at pH 7.5 in the presence of each electrophile after CySNO removal.…”

Section: Kinetic Analysismentioning

confidence: 99%

Thiol‐blocking electrophiles interfere with labeling and detection of protein sulfenic acids

et al. 2013

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Cellular exposure to reactive oxygen species induces the rapid oxidation of DNA, proteins, lipids, and other biomolecules. At the proteome level, cysteine thiol oxidation is a prominent post-translational process implicated in normal physiology and numerous pathologies. Methods for investigating protein oxidation include direct labeling with selective chemical probes and indirect tag-switch techniques. Common to both approaches is a chemical blocking of free thiols with reactive electrophiles to prevent post-lysis oxidation or other thiol-mediated cross-reactions. These reagents are used in large excess and their reactivity with cysteine sulfenic acid, a critical oxoform in numerous proteins, has not been investigated. Here we report the reactivity of three thiol-blocking electrophiles, iodoacetamide, N-ethylmaleimide, and methyl methanethiosulfonate, with protein sulfenic acid and dimedone, the structural core of many sulfenic acid probes. We demonstrate that covalent cysteine -SOR (product) species are susceptible to reduction by DTT, TCEP, and ascorbate, regenerating protein thiols, or in the case of ascorbate, more highly oxidized species. The implications of this reactivity on detection methods for protein sulfenic acids and S-nitrosothiols are discussed.

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Section: Kinetic Analysismentioning

confidence: 99%

Thiol‐blocking electrophiles interfere with labeling and detection of protein sulfenic acids

et al. 2013

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“…Without CTAB, the strong counter EOF in the cathodic direction caused broadening of the fast migrating anions and did not allow the slow anions (organic acids) to reach the detector. Additionally, the BGE co‐ion HIS is alkylated by maleimides , that are present in excess as derivatization agent (EMA) and this BGE cannot be used.…”

Section: Resultsmentioning

confidence: 99%

Optimization of background electrolyte composition for simultaneous contactless conductivity and fluorescence detection in capillary electrophoresis of biological samples

et al. 2019

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In this article, optimization of BGE for simultaneous separation of inorganic ions, organic acids, and glutathione using dual C4D‐LIF detection in capillary electrophoresis is presented. The optimized BGE consisted of 30 mM 2‐[4‐(2‐hydroxyethyl)piperazin‐1‐yl]ethanesulfonic acid, 15 mM 2‐amino‐2‐hydroxymethyl‐propane‐1,3‐diol, and 2 mM 18‐crown‐6 at pH 7.2 and allowed simultaneous separation of ten inorganic anions and cations, three organic acids and glutathione in 20 min. The samples were injected hydrodynamically from both capillary ends using the double‐opposite end injection principle. Sensitive detection of anions, cations, and organic acids with micromolar LODs using C4D and simultaneously glutathione with nanomolar LODs using LIF was achieved in a single run. The developed BGE may be useful in analyses of biological samples containing analytes with differing concentrations of several orders of magnitude that is not possible with single detection mode.

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“…Side-reaction between maleimide groups and imidazole (contained in the initial buffer) was expected as previously reported by several groups showing the histidine side-chain alkylation. 50,51 Nonetheless, further investigation revealed that 4 could be directly conjugated to 3 in the affinity column elution buffer, with limited excess of the maleimide reagent and despite a large molar excess of imidazole. After a final diafiltration to remove the imidazole and excess reagent, the chemically-defined conjugate R3VQ-S-(DOTA/Gd) 3 5 was obtained with a yield of 83% and a high quality, as demonstrated by MS ( Fig.…”

Section: Site-specific Conjugationmentioning

confidence: 99%

Chemically-defined camelid antibody bioconjugate for the magnetic resonance imaging of Alzheimer's disease

Vandesquille

et al. 2017

mAbs

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Today, molecular imaging of neurodegenerative diseases is mainly based on small molecule probes. Alternatively, antibodies are versatile tools that may be developed as new imaging agents. Indeed, they can be readily obtained to specifically target any antigen of interest and their scaffold can be functionalized. One of the critical issues involved in translating antibody-based probes to the clinic is the design and synthesis of perfectly-defined conjugates. Camelid single-domain antibody-fragments (VHHs) are very small and stable antibodies that are able to diffuse in tissues and potentially cross the blood brain barrier (BBB). Here, we selected a VHH (R3VQ) specifically targeting one of the main lesions of Alzheimer's disease (AD), namely the amyloid-beta (Aß) deposits. It was used as a scaffold for the design of imaging probes for magnetic resonance imaging (MRI) and labeled with the contrastophore gadolinium using either a random or site-specific approach. In contrast to the random strategy, the site-specific conjugation to a single reduced cysteine in the C-terminal part of the R3VQ generates a well-defined bioconjugate in a high yield process. This new imaging probe is able to cross the BBB and label Aß deposits after intravenous injection. Also, it displays improved r1 and r2 relaxivities, up to 30 times higher than a widely used clinical contrast agent, and it allows MRI detection of amyloid deposits in post mortem brain tissue of a mouse model of AD. The ability to produce chemically-defined VHH conjugates that cross the BBB opens the way for future development of tailored imaging probes targeting intracerebral antigens.

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Alkylation of histidine with maleimido‐compounds

Cited by 18 publications

References 17 publications

Thiol‐blocking electrophiles interfere with labeling and detection of protein sulfenic acids

Thiol‐blocking electrophiles interfere with labeling and detection of protein sulfenic acids

Optimization of background electrolyte composition for simultaneous contactless conductivity and fluorescence detection in capillary electrophoresis of biological samples

Chemically-defined camelid antibody bioconjugate for the magnetic resonance imaging of Alzheimer's disease

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