Application of Safirinium N-Hydroxysuccinimide Esters to Derivatization of Peptides for High-Resolution Mass Spectrometry, Tandem Mass Spectrometry, and Fluorescent Labeling of Bacterial Cells

Mass spectrometry methods are commonly used in the identification of peptides and biomarkers. Due to a relatively low abundance of proteins in biological samples, there is a need for the development of novel derivatization methods that would improve MS detection limits. Hence, novel fluorescent N–hydroxysuccinimide esters of dihydro-[1,2,4]triazolo[4,3-a]pyridin-2-ium carboxylates (Safirinium P dyes) have been synthesized. The obtained compounds, which incorporate quaternary ammonium salt moieties, easily react with aliphatic amine groups of peptides, both in solution and on the solid support; thus, they can be applied for derivatization as ionization enhancers. Safirinium tagging experiments with ubiquitin hydrolysate revealed that the sequence coverage level was high (ca. 80%), and intensities of signals were enhanced up to 8-fold, which proves the applicability of the proposed tags in the bottom–up approach. The obtained results confirmed that the novel compounds enable the detection of trace amounts of peptides, and fixed positive charge within the tags results in high ionization efficiency. Moreover, Safirinium NHS esters have been utilized as imaging agents for fluorescent labeling and the microscopic visualization of living cells such as E. coli Top10 bacterial strain.

Emulsifying Characteristics of Gelatin Hydrolysate from Tilapia Skin Covalently Attached with N-hydroxysuccinimide Esters of Fatty Acids

This study aims to combine two modification methods, namely enzymatic hydrolysis and covalent attachment with hydrophobic groups, to increase the emulsifying properties of gelatin. The experiment was conducted by using a completely randomized design with three replicates. Enzymatic hydrolysis of gelatin resulted in higher contents of free amino groups, which could be attached to hydrophobic groups. Gelatin hydrolysates covalently attached with the N-hydroxysuccinimide esters of C14:0 and C18:0 fatty acids at a molar ratio of 3.0 showed high emulsifying activity but low stability. Among the samples obtained, gelatin hydrolysate covalently attached with C18:0 at a molar ratio of 3.0 revealed the highest emulsifying activity; however, this sample cannot be considered the best emulsifier among the samples because of its low stability.

6-Oxyindan-1-ones with dipeptide chains

Through N-acylation of a- or b-amino acid units by 2-(3-oxo-2,3-dihydro-1H-inden-5-yloxy)acetic acid using the method of N-hydroxysuccinimide esters new dipeptide indan-1-one derivatives were obtained. In general, the direct interaction of the acetic carboxyl group of the substrate with the amino group of the a- or b-dipeptide is a more productive strategy than the sequential peptidic condensation of the two amino acids.

6-Oxyindan-1-ones with amino acid fragments

New indan-1-one derivatives (8 examples) with amino acid fragments were synthesized through the N-acylation of the amino acids by 2-(3-oxo-2,3-dihydro-1H-inden-5-yloxy)acetic acid using the method of activated N-hydroxysuccinimide esters. To obtain corresponding methyl esters (2 examples) two ways were possible: the N-acylation of the amino acid methyl esters by 2-(3-oxo-2,3-dihydro-1-inden-5-yloxy)acetic acid through the activated imidazole derivatives or methylation of the carboxylic function of preformed N-{[(1-oxoindan-6-yl)oxy]acetyl}amino acids.