[This corrects the article DOI: 10.3389/fchem.2024.1415644.].

The ninhydrin reaction is commonly used for the detection of amino acids. However, in the literature, different conditions with respect to the buffer system, its pH and concentration, type of organic solvent, incubation time, and temperature, as well as the concentrations of the reagents, are described. To identify the most suitable conditions, colour development with reagents of varying compositions and different reaction temperatures and times were investigated using asparagine as a model amino acid. Asparagine was selected since it is one of the most abundant free amino acids in many types of samples. The optimal reaction mixture consisted of 0.8 mol L-1 potassium acetate, 1.6 mol L-1 acetic acid, 20 mg mL-1 ninhydrin and 0.8 mg mL-1 hydrindantin in DMSO/acetate buffer 40/60 (v/v) (final concentrations). The best reaction condition was heating the samples in 1.5 mL reaction tubes to 90 °C for 45 min. Afterwards, the samples were diluted with 2-propanol/water 50/50 (v/v) and the absorbance was measured at 570 nm. The proteinogenic amino acids showed a similar response except for cysteine and proline. The method was highly sensitive and showed excellent linearity as well as intra-day and inter-day reproducibility.

Bacteria are becoming increasingly resistant to antibiotics, therefore there is an urgent need for new classes of antibiotics to fight antibiotic resistance. Mammals do not express N ɑ -acetyl-L-ornithine deacetylase (ArgE), an enzyme that is critical for bacterial survival and growth, thus ArgE represents a promising new antibiotic drug target, as inhibitors would not suffer from mechanism-based toxicity. A new ninhydrin-based assay was designed and validated that included the synthesis of the substrate analog N 5, N 5-di-methyl N α-acetyl-L-ornithine (kcat/Km = 7.32 ± 0.94 × 104 M-1s-1). This new assay enabled the screening of potential inhibitors that absorb in the UV region, and thus is superior to the established 214 nm assay. Using this new ninhydrin-based assay, captopril was confirmed as an ArgE inhibitor (IC50 = 58.7 μM; Ki = 37.1 ± 0.85 μM), and a number of phenylboronic acid derivatives were identified as inhibitors, including 4-(diethylamino)phenylboronic acid (IC50 = 50.1 μM). Selected inhibitors were also tested in a thermal shift assay with ArgE using SYPRO Orange dye against Escherichia coli ArgE to observe the stability of the enzyme in the presence of inhibitors (captopril Ki = 35.9 ± 5.1 μM). The active site structure of di-Zn EcArgE was confirmed using X-ray absorption spectroscopy, and we reported two X-ray crystal structures of E. coli ArgE. In summary, we describe the development of a new ninhydrin-based assay for ArgE, the identification of captopril and phenylboronic acids as ArgE inhibitors, thermal shift studies with ArgE + captopril, and the first two published crystal structures of ArgE (mono-Zn and di-Zn).

Amino acid analysis is an accurate method for the composition and quantitation of polypeptides and among these synthetic peptides. Combined with mass spectrometry, it yields a reliable control of peptide quality and quantity prior to conjugation and immunization.Initially peptides are hydrolyzed, preferably in the gas phase, with 6-M HCl at 110 °C for 20-24 h and the resulting amino acids analyzed by chromatography, where the most reliable form is ion exchange chromatography with post-column ninhydrin derivatization. Depending on the hydrolysis conditions, tryptophan is destroyed, and likewise cysteine, unless derivatized, and the amides, glutamine, and asparagine are deamidated to glutamic acid and aspartic acid, respectively. Three different ways of calculating results are suggested, and taking the above limitations into account, a quantitation better than 5% can usually be obtained.

A ninhydrin-based colorimetric chemosensor (LH) was synthesized using 3-hydroxy-2-naphthoic hydrazide and 11H-indeno[1,2-b]quinoxalin-11-one. It was characterized by spectroscopic and single crystal X-ray diffraction techniques. In a semi-aqueous (MeOH/HEPES) system, LH displayed a characteristic chromogenic change from colorless to yellow upon adding Cu2+ ion, with the appearance of a new peak at λmax = 460 nm. A 1:1 binding stoichiometry between LH and Cu2+ ion has been found, with LOD = 2.3 μM (145 ppb) and LOQ = 8 μM (504 ppb). Based on experimental results the formula of [Cu(L)Cl(H2O)2] (1) was assigned and this in-situ generated 1 was found to exhibit a discoloration of upon gradual addition of cysteine (LOD = 60 nM) as well as ATP (LOD = 130 nM) having 1:2 and 1:1 stoichiometry respectively. The LH was useful for recognition of Cu2+ ion in real water samples and on filter paper strips. A two-input-two-output logic gate circuitry was also constructed by employing 1 and cysteine. The DFT/TDDFT calculations performed on LH and 1 were consistent with experimental findings. The binding affinity of LH towards HSA and BSA were determined with HSA having greater affinity than BSA, which was also supported by theoretical calculations.

The appearance of multidrug-resistant Gram-negative bacterial infections, along with the lack of newly discovered antibiotics, resulted in the return to old antimicrobial medications like Polymyxins. As a result, the suggested technique aims to develop a fast, environmentally friendly, and sensitive fluorimetric method for quantifying Polymyxin B. The investigated approach depends on generating a highly fluorescent derivative by a condensation pathway between the studied drug and ninhydrin in the presence of phenylacetaldehyde and then estimated spectrofluorimetrically. After the reaction conditions were well optimized, the fluorescent product was estimated at emission wavelength (λem) = 475.5 nm (following excitation at a wavelength (λex) = 386 nm. The developed calibration plot displayed rectilinear throughout the following range (0.2-3 µg mL- 1), and the calculated limit of detection and quantification were 0.062 µg mL- 1 and 0.187 µg mL- 1, respectively. As a consequence, the drug\'s ophthalmic and intravenous pharmaceutical forms were both successfully quantified with an excellent degree of recovery. Finally, the methodology\'s greenness was assessed utilizing Analytical Eco-Scale scores.

Mirabegron (MRB) is a β3-adrenoceptor agonist used for managing overactive bladder syndrome. A cost-effective, environmentally friendly, and highly sensitive spectrofluorimetric method was suggested to serve the purpose of quantifying MRB in its pure state, pharmaceutical tablets, spiked human plasma and urine, and testing content uniformity. In the present study, ninhydrin and phenylacetaldehyde react with the amino group moiety of MRB in Teorell-Stenhagen buffer (pH 7.5) to generate a strongly fluorescent diaryl pyrrolone compound that emits fluorescence at a wavelength of 477 nm upon excitation at 385 nm. The obtained calibration curve showed a linear relationship with a high correlation coefficient (r = 0.9997) in the concentration range of 0.25 to 5.0 µg mL-1. Limits of detection (LOD) and quantitation (LOQ) were 0.082 and 0.248 µg mL-1 respectively. The procedure was verified in accordance with the ICH guidelines. The suggested approach could be utilized for the selective analysis of MRB in its pharmaceuticals, either containing a single drug or co-formulated with solifenacin succinate. The greenness of the suggested method was confirmed using different green analytical metrics.

A number of solvents, (Solstice PF, Opteon SF33 and Amolea AS-300), are compared to the recommended carrier solvent of HFE7100 for the ninhydrin and 1,2-indandione formulations. As the supply of HFE7100 will cease by the end of 2025, suitable alternatives are required in the short-term to ensure the detection of latent fingermarks on porous surfaces is still effective. Although these solvents, with the exception of Amolea AS-300, are classified as per- and polyfluoroalkyl substances (PFAS); they are not classed as hazardous. The alternatives in this study have a low global warming potential and atmospheric lifetime and are volatile, non-flammable and non-ozone depleting, in addition to other desirable properties such as a high wetting-index. During Phase 2 trials with deposited fingermarks, HFE7100 provided the best performing results followed by Opteon SF33, Solstice PF and Amolea AS-300. A significant difference with a negligible effect size was observed for ninhydrin formulations (p-value 0.00179; ε2 0.00418) while a significant difference with a weak effect size was observed for 1,2-indanedione formulations (p-value 2.095 ×10-10; ε2 0.0167). Furthermore, HFE7100 provided the least ink diffusion and the brightest 1,2-indanedione luminosity (significant difference with a moderate effect size p-value 1.772 ×10-13; ε2 0.0434) but the HFE formulation turned cloudy more quickly and needed regular replacements. Phase 3 pseudo-operational trials of 100 porous items followed a similar trend whereby HFE7100 formulations detected the highest number of marks followed by Opteon SF33 and Solstice PF. Although HFE7100 is still the best performing carrier solvent, this study demonstrates that, in the short-term, Opteon SF33 and Solstice PF may have potential as non-flammable replacement carrier solvents while developing the long-term goal of solvent-less methods.

Acylaminoacyl peptidases (AAPs) play a pivotal role in various pathological conditions and are recognized as potential therapeutic targets. AAPs exhibit a wide range of activities, such as acylated amino acid-dependent aminopeptidase, endopeptidase, and less studied carboxypeptidase activity. We have determined the crystal structure of an AAP from Geobacillus stearothermophilus (S9gs) at 2.0 Å resolution. Despite being annotated as an aminopeptidase in the NCBI database, our enzymatic characterization proved S9gs to be a carboxypeptidase. Solution-scattering studies showed that S9gs exists as a tetramer in solution, and crystal structure analysis revealed adaptations responsible for the carboxypeptidase activity of S9gs. The findings present a hypothesis for substrate selection, substrate entry, and product exit from the active site, enriching our understanding of this rare carboxypeptidase.

Photodeposited TiO2/Ag nanocomposites were generally used to be a friendly catalyst for degrading organic contaminant in environmental field. However, electrochemiluminescence (ECL) sensing analysis based on photocatalysts remains a significant challenge. Herein, polyvinylimide (PEI)-TiO2/Ag nanocomposites (PEI-TiO2/AgNCPs) film with reduced graphene oxide(r-GO) was constructed as a sensing interface for copper(II) ECL detection. TiO2/Ag nanocomposites was prepared by reversed phase microemulsion method and photodeposition technique. Moreover, it was discovered that a small amount of Cu2+ could obviously boost the ECL signal of ninhydrin-hydrogen peroxide system. Signal amplification was achieved by using the synergistic effect between r-GO and TiO2/Ag nanocomposites, and the efficiently concentrated effect of PEI to Cu2+. Furthermore, the investigation showed that ECL mechanism of ninhydrin-hydrogen peroxide system was attributed to the generated hydroxyl radical and superoxide anion during the several type of reactions. Thus for the first time, an ultrasensitive ECL approach for detecting Cu2+ could be performed using ninhydrin as an ECL signal probe and hydrogen peroxide as a co-reaction reagent. Under the suitable circumstances, the proposed method showed an excellent linear relationship in the concentration range of Cu2+ from 1.0 fM to 5.0 nM. Detection limit was estimated to be as low as 0.26 fM. The sensing interface expanded the application of photodeposited TiO2/Ag nanocomposites in ultrasensitive ECL detection. It has potential applications in other components and biological analysis.