Some properties of diastereomers formed in the reactions of N-ethylmaleimide with biological thiols.

Kuninori, Toyo; Nishiyama, Junko

doi:10.1271/bbb1961.49.2453

Cited by 13 publications

(16 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3c ) was prepared from DM1 according to literature procedures 22 (for details, see Supplementary Information). The reaction produced two high-performance liquid chromatography (HPLC)-separable, but slowly interconverting, C9’-diastereoisomers (designated as M9A and M9B ) in an approximately 1:1 ratio, with full equilibration in phosphate buffer pH 7.2 being observed after 24 h. This finding is in line with previous data suggesting that the thiol addition products of N -ethyl maleimide with different biological thiols (including cysteine and glutathione) readily interconvert even at neutral pH 24 . The method used to prepare M9 inevitably leads to two diastereoisomeric products, due to the formation of an additional stereocenter at C9’ in the 1,4-addition of DM1 to the maleimido part of the linker precursor and the inherently non-selective nature of the reaction.…”

Section: Resultssupporting

confidence: 90%

A fluorescence anisotropy assay to discover and characterize ligands targeting the maytansine site of tubulin

et al. 2018

View full text Add to dashboard Cite

Microtubule-targeting agents (MTAs) like taxol and vinblastine are among the most successful chemotherapeutic drugs against cancer. Here, we describe a fluorescence anisotropy-based assay that specifically probes for ligands targeting the recently discovered maytansine site of tubulin. Using this assay, we have determined the dissociation constants of known maytansine site ligands, including the pharmacologically active degradation product of the clinical antibody-drug conjugate trastuzumab emtansine. In addition, we discovered that the two natural products spongistatin-1 and disorazole Z with established cellular potency bind to the maytansine site on β-tubulin. The high-resolution crystal structures of spongistatin-1 and disorazole Z in complex with tubulin allowed the definition of an additional sub-site adjacent to the pocket shared by all maytansine-site ligands, which could be exploitable as a distinct, separate target site for small molecules. Our study provides a basis for the discovery and development of next-generation MTAs for the treatment of cancer.

show abstract

Section: Resultssupporting

confidence: 90%

A fluorescence anisotropy assay to discover and characterize ligands targeting the maytansine site of tubulin

et al. 2018

View full text Add to dashboard Cite

show abstract

“…Thus, derivatizing reduced thiols with NEM clearly prolongs their retention time on the column, particularly when the derivatization product involves two succinimide groups, as with sulfide. For some compounds more than others double peaks were observed, a phenomenon previously described for Cys and GSH [41] , [42] . This has been attributed to the formation of two diasteromers that differ in position of the sulfur atom in relation to the maleimide nitrogen, depending on where the reduced thiol adds to the double bond of the pyrrole ring.…”

Section: Resultssupporting

confidence: 58%

“…Since the sulfur can add to either side of the double bond it gives rise to different diastereomeres (see Fig. 1 B), sometimes complicating the chromatographic separation of thiols [41] by leading to double peaks. Formation of the maleimide-thiol adduct was long thought to be irreversible (unless subjected to electrolysis).…”

Section: Discussionmentioning

confidence: 99%

A robust and versatile mass spectrometry platform for comprehensive assessment of the thiol redox metabolome

et al. 2018

View full text Add to dashboard Cite

Several diseases are associated with perturbations in redox signaling and aberrant hydrogen sulfide metabolism, and numerous analytical methods exist for the measurement of the sulfur-containing species affected. However, uncertainty remains about their concentrations and speciation in cells/biofluids, perhaps in part due to differences in sample processing and detection principles. Using ultrahigh-performance liquid chromatography in combination with electrospray-ionization tandem mass spectrometry we here outline a specific and sensitive platform for the simultaneous measurement of 12 analytes, including total and free thiols, their disulfides and sulfide in complex biological matrices such as blood, saliva and urine. Total assay run time is < 10 min, enabling high-throughput analysis. Enhanced sensitivity and avoidance of artifactual thiol oxidation is achieved by taking advantage of the rapid reaction of sulfhydryl groups with N-ethylmaleimide. We optimized the analytical procedure for detection and separation conditions, linearity and precision including three stable isotope labelled standards. Its versatility for future more comprehensive coverage of the thiol redox metabolome was demonstrated by implementing additional analytes such as methanethiol, N-acetylcysteine, and coenzyme A. Apparent plasma sulfide concentrations were found to vary substantially with sample pretreatment and nature of the alkylating agent. In addition to protein binding in the form of mixed disulfides (S-thiolation) a significant fraction of aminothiols and sulfide appears to be also non-covalently associated with proteins. Methodological accuracy was tested by comparing the plasma redox status of 10 healthy human volunteers to a well-established protocol optimized for reduced/oxidized glutathione. In a proof-of-principle study a deeper analysis of the thiol redox metabolome including free reduced/oxidized as well as bound thiols and sulfide was performed. Additional determination of acid-labile sulfide/thiols was demonstrated in human blood cells, urine and saliva. Using this simplified mass spectrometry-based workflow the thiol redox metabolome can be determined in samples from clinical and translational studies, providing a novel prognostic/diagnostic platform for patient stratification, drug monitoring, and identification of new therapeutic approaches in redox diseases.

show abstract

“…S4 Fig is an example of an unformatted graphic, showing the distribution of putative intact NESyl groups. Note how some of the DPs in Fig 2 occur as pairs of putative diastereoisomers [ 43 ] (i.e., modified peptides with identical m / z values and fragmentation patterns but different retention times).…”

Section: Resultsmentioning

confidence: 99%

Visualisation tools for dependent peptide searches to support the exploration of in vitro protein modifications

et al. 2020

View full text Add to dashboard Cite

Dependent peptide searching is a method for discovering covalently-modified peptides-and therefore proteins-in mass-spectrometry-based proteomics experiments. Being more permissive than standard search methods, it has the potential to discover novel modifications (e.g., post-translational modifications occurring in vivo, or modifications introduced in vitro). However, few studies have explored dependent peptide search results in an untargeted way. In the present study, we sought to evaluate dependent peptide searching as a means of characterising proteins that have been modified in vitro. We generated a model data set by analysing N-ethylmaleimide-treated bovine serum albumin, and performed dependent peptide searches using the popular MaxQuant software. To facilitate interpretation of the search results (hundreds of dependent peptides), we developed a series of visualisation tools (R scripts). We used the tools to assess the diversity of putative modifications in the albumin, and to pinpoint hypothesised modifications. We went on to explore the tools' generality via analyses of public data from studies of rat and human proteomes. Of 19 expected sites of modification (one in rat cofilin-1 and 18 across six different human plasma proteins), eight were found and correctly localised. Apparently, some sites went undetected because chemical enrichment had depleted necessary analytes (potential 'base' peptides). Our results demonstrate (i) the ability of the tools to provide accurate and informative visualisations, and (ii) the usefulness of dependent peptide searching for characterising in vitro protein modifications. Our model data are available via PRIDE/ProteomeXchange (accession number PXD013040).

show abstract

Some properties of diastereomers formed in the reactions of N-ethylmaleimide with biological thiols.

Cited by 13 publications

References 0 publications

A fluorescence anisotropy assay to discover and characterize ligands targeting the maytansine site of tubulin

A fluorescence anisotropy assay to discover and characterize ligands targeting the maytansine site of tubulin

A robust and versatile mass spectrometry platform for comprehensive assessment of the thiol redox metabolome

Visualisation tools for dependent peptide searches to support the exploration of in vitro protein modifications

Contact Info

Product

Resources

About