Detection of major psychoactive compounds (safrole, myristicin, and elemicin) of nutmeg in human serum via GC-MS/MS using MonoSpin® extraction: Application in a nutmeg poisoning case.

Nutmeg is an inexpensive, readily available spice used in a variety of recipes. However, the use of nutmeg powder as a recreational drug for its hallucinogenic effects is resulting in an increase in overdose rates. We encountered a male patient being hospitalized after ingesting 75 g of commercially available nutmeg powder with the intent of committing suicide. There are no available reports documenting the toxic or comatose-fatal blood concentrations or time-course of drug action in cases of nutmeg poisoning. Therefore, to improve patient management, we endeavored to determine the blood serum levels and time-course of the major psychoactive compounds (safrole, myristicin, and elemicin) present in nutmeg. We designed a simple and reliable method using the MonoSpin® extraction kit and gas chromatography-tandem mass spectrometry to detect the presence of these psychoactive compounds in human serum. The method had detection and quantitation limits of 0.14-0.16 and 0.5 ng/mL (lowest calibration points), respectively. The calibration curves displayed excellent linearity (0.996-0.997) for all three compounds at 0.5-300 ng/mL blood concentrations. The intra- and inter-day precision values for quality assurance were in the ranges of 2.4-11 % and 2.5-11 %, respectively; bias ranged from - 2.6 % to 2.1 %. Blood serum levels of safrole, myristicin, and elemicin were measured at admission (approximately 8 h post-ingestion) and approximately 94 h after a post-admission fluid therapy to evaluate their biological half-lives. We developed this method to obtain information on the psychoactive constituents of nutmeg and, thereby, determine the toxicokinetic parameters of nutmeg in a case of nutmeg poisoning.

Developing a Targeted Quantitative Strategy for Sulfoxide-Containing MS-Cleavable Cross-Linked Peptides to Probe Conformational Dynamics of Protein Complexes.

In recent years, cross-linking mass spectrometry (XL-MS) has made enormous strides as a technology for probing protein-protein interactions (PPIs) and elucidating architectures of multisubunit assemblies. To define conformational and interaction dynamics of protein complexes under different physiological conditions, various quantitative cross-linking mass spectrometry (QXL-MS) strategies based on stable isotope labeling have been developed. These QXL-MS approaches have effectively allowed comparative analysis of cross-links to determine their relative abundance changes at global scales. Although successful, it remains challenging to consistently obtain quantitative measurements on low-abundant cross-links. Therefore, targeted QXL-MS is needed to enable MS "Western" analysis of cross-links to enhance sensitivity and reliability in quantitation. To this end, we have established a robust parallel reaction monitoring (PRM)-based targeted QXL-MS platform using sulfoxide-containing MS-cleavable cross-linker disuccinimidyl sulfoxide (DSSO), permitting label-free comparative analysis of selected cross-links across multiple samples. In addition, we have applied this methodology to study phosphorylation-dependent conformational dynamics of the human 26S proteasome. The PRM-based targeted QXL-MS analytical platform described here is applicable for all sulfoxide-containing MS-cleavable cross-linkers and can be directly adopted for comparative studies of protein-protein interactions in various cellular contexts.

Chemical Analysis and In Vitro Bioactivity of Essential Oil of Laurelia sempervirens and Safrole Derivatives against Oomycete Fish Pathogens.

In this study, the essential oil (EO) from Laurelia sempervirens was analyzed by GC/MS and safrole (1) was identified as the major metabolite 1, was subjected to direct reactions on the oxygenated groups in the aromatic ring and in the side chain, and eight compounds (4 to 12) were obtained by the process. EO and compounds 4-12 were subjected to biological assays on 24 strains of the genus Saprolegnia, specifically of the species 12 S. parasitica and 12 S. australis. EO showed a significant effect against Saprolegnia strains. Compound 6 presents the highest activity against two resistant strains, with minimum inhibitory concentration (MIC) and minimum oomyceticidal concentration (MOC) values of 25 to 100 and 75 to 125 µg/mL, respectively. The results show that compound 6 exhibited superior activities compared to the commercial controls bronopol and azoxystrobin used to combat these pathogens.

Synthesis and evaluation of novel 2,4-diaminopyrimidines bearing a sulfoxide moiety as anaplastic lymphoma kinase (ALK) inhibition agents.

Anaplastic lymphoma kinase (ALK) targeted therapies have demonstrated remarkable efficacy in ALK-positive lung adenocarcinomas. Here we synthesized and evaluated sixteen new 2,4-diaminopyrimidines bearing a sulfoxide moiety as anaplastic lymphoma kinase (ALK) inhibitors. The optimal compound 9e exhibited excellent antiproliferative activity against non-small cell lung cancer NCI-H2228 cells, which is better than that of Brigatinib and similar to Ceritinib. Mechanism study revealed that the optimal compound 9e decreased the mitochondrial membrane potential and arrested NCI-H2228 cells in the G0/G1 phase, finally resulting in cellular apoptosis. It is interesting that 9e could effectively inhibit the migration of NCI-H2228 cells and may be a promising leading compound for chemotherapy of metastatic cancer.

Biological evaluation of Safrole oil and Safrole oil Nanoemulgel as antioxidant, antidiabetic, antibacterial, antifungal and anticancer.

BACKGROUND: Safrole is a natural compound extracted from various plants, and has shown various biological activities. The current study aimed to investigate the antioxidant, antidiabetic, antimicrobial, and anticancer activity of safrole oil and to study the influence of safrole nanoemulgel on these activities. METHODS: The antioxidant and antidiabetic in-vitro assays were conducted using standard biomedical methods. The safrole oil nanoemulgel was developed using a self-emulsifying technique. Then the antimicrobial activity of the safrole oil and safrole nanoemulgel were performed on different microbial species, and cytotoxicity was determined against Hep3B cancer cell lines using the MTS assay. RESULTS: Safrole oil showed moderate antioxidant activity compared with standard Trolox, with IC50 value 50.28 ± 0.44 and 1.55 ± 0.32 µg/ml, respectively. Moreover, it had potent α-amylase inhibitory activity (IC50 11.36 ± 0.67 µg/ml) compared with Acarbose (IC50 value 5.88 ± 0.63). The safrole nanoemulgel had pseudo-plastic behaviour, droplet sizes below 200 nm, a polydispersity index (PDI) below 0.3, and a zeta potential of less than - 30 mV. Safrole oil has potential antimicrobial and anticancer activities, and these activities were improved with safrole nanoemulgel. CONCLUSION: The safrole oil may be applied for the prevention and treatment of oxidative stress, diabetes, different microbial species and cancer, and these activities could be improved by nano-carriers.

Pharmacokinetic Study of Safrole and Methyl Eugenol after Oral Administration of the Essential Oil Extracts of Asarum in Rats by GC-MS.

Asarum is a traditional medicine and has been widely used as remedies for inflammatory diseases, toothache, headache, local anesthesia, and aphthous stomatitis in China, Japan, and Korea. Our previous research found that safrole and methyl eugenol were vital compounds that contribute to distinguish the different species and raw Asarum and its processed products apart. The pharmacokinetics of safrole and methyl eugenol after oral administration of Asarum extract has not been reported yet. In this study, a rapid and simple gas chromatography-mass spectroscopy (GC-MS) method that has a complete run time of only 4.5 min was developed and validated for the simultaneous determination and pharmacokinetic study of safrole and methyl eugenol in rat plasma after administration of Asarum extracts. The chromatographic separation was realized on a DB-17 column (30 m × 0.25 mm × 0.25 µm). And detection was carried out under selected ion monitoring (SIM) mode. Plasma samples were pretreated by n-hexane. The pharmacokinetic parameters provided by this study will be beneficial for further developments and clinical applications of Asarum.

High Performance Liquid Chromatography versus Stacking-Micellar Electrokinetic Chromatography for the Determination of Potentially Toxic Alkenylbenzenes in Food Flavouring Ingredients.

Alkenylbenzenes, including eugenol, methyleugenol, myristicin, safrole, and estragole, are potentially toxic phytochemicals, which are commonly found in foods. Occurrence data in foods depends on the quality of the analytical methodologies available. Here, we developed and compared modern reversed-phase high performance liquid chromatography (HPLC) and stacking-micellar electrokinetic chromatography (MEKC) methods for the determination of the above alkenylbenzenes in food flavouring ingredients. The analytical performance of HPLC was found better than the stacking-MEKC method. Compared to other HPLC methods found in the literature, our method was faster (total run time with conditioning of 15 min) and able to separate more alkenylbenzenes. In addition, the analytical methodology combining an optimized methanol extraction and proposed HPLC was then applied to actual food flavouring ingredients. This methodology should be applicable to actual food samples, and thus will be vital to future studies in the determination of alkenylbenzenes in food.

A chemical covalent tactic for bio-thiol sensing and protein labeling agent design.

Discovering novel chemical reactions is important for bioanalysis. Herein, we report a tactic for bio-thiol sensing and protein labeling agent design by the installation of a sulfoxide group onto the skeleton of various fluorophores, and powerfully validate its abilities, which may shed light on the development of specific protein tags to give insight into their biological functions.

Molecular Dynamics and In Vitro Quantification of Safrole DNA Adducts Reveal DNA Adduct Persistence Due to Limited DNA Distortion Resulting in Inefficient Repair.

The formation and repair of N2-(trans-isosafrol-3'-yl)-2'-deoxyguanosine (S-3'-N2-dG) DNA adduct derived from the spice and herbal alkenylbenzene constituent safrole were investigated. DNA adduct formation and repair were studied in vitro and using molecular dynamics (MD) simulations. DNA adduct formation was quantified using liquid chromatography-mass spectrometry (LCMS) in wild type and NER (nucleotide excision repair) deficient CHO cells and also in HepaRG cells and primary rat hepatocytes after different periods of repair following exposure to safrole or 1'-hydroxysafrole (1'-OH safrole). The slower repair of the DNA adducts found in NER deficient cells compared to that in CHO wild type cells indicates a role for NER in repair of S-3'-N2-dG DNA adducts. However, DNA repair in liver cell models appeared to be limited, with over 90% of the adducts remaining even after 24 or 48 h recovery. In our further studies, MD simulations indicated that S-3'-N2-dG adduct formation causes only subtle changes in the DNA structure, potentially explaining inefficient activation of NER. Inefficiency of NER mediated repair of S-3'-N2-dG adducts points at persistence and potential bioaccumulation of safrole DNA adducts upon daily dietary exposure.

FEMA GRAS assessment of natural flavor complexes: Clove, cinnamon leaf and West Indian bay leaf-derived flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association initiated the safety re-evaluation of over 250 natural flavor complexes (NFCs) used as flavor ingredients. This publication, 4th in a series focusing on the safety evaluation of NFCs, presents an evaluation of NFCs rich in hydroxyallylbenzene and hydroxypropenylbenzene constituents using a procedure initially published in 2005 and updated in 2018 that evaluates the safety of naturally occurring mixtures for their intended use as flavoring ingredients. The procedure requires the characterization of the chemical composition for each NFC and subsequent organization of the constituents into defined congeneric groups. The safety of each NFC is evaluated using the conservative threshold of toxicological concern (TTC) approach together with studies on absorption, metabolism and toxicology of the NFC and its constituent congeneric groups. By the application of this procedure, seven NFCs, derived from clove, cinnamon leaf and West Indian bay leaf were affirmed as "generally recognized as safe (GRAS)" under their conditions of intended use as flavor ingredients. An eighth NFC, an oleoresin of West Indian bay leaf, was affirmed based on its estimated intake, which is below the TTC of 0.15 µg/person per day for compounds with structural alerts for genotoxicity.

Exploring Spacer Arm Structures for Designs of Asymmetric Sulfoxide-Containing MS-Cleavable Cross-Linkers.

Cross-linking mass spectrometry (XL-MS) has become a powerful structural tool for defining protein-protein interactions (PPIs) and elucidating architectures of large protein assemblies. To advance XL-MS studies, we have previously developed a series of sulfoxide-containing MS-cleavable cross-linkers to facilitate the detection and identification of cross-linked peptides using multistage mass spectrometry (MSn). While current sulfoxide-based cross-linkers are effective for in vivo and in vitro XL-MS studies at the systems-level, new reagents are still needed to help expand PPI coverage. To this end, we have designed and synthesized six variable-length derivatives of disuccinimidyl sulfoxide (DSSO) to better understand the effects of spacer arm modulation on MS-cleavability, fragmentation characteristics, and MS identification of cross-linked peptides. In addition, the impact on cross-linking reactivity was evaluated. Moreover, alternative MS2-based workflows were explored to determine their feasibility for analyzing new sulfoxide-containing cross-linked products. Based on the results of synthetic peptides and a model protein, we have further demonstrated the robustness and predictability of sulfoxide chemistry in designing MS-cleavable cross-linkers. Importantly, we have identified a unique asymmetric design that exhibits preferential fragmentation of cross-links over peptide backbones, a desired feature for MSn analysis. This work has established a solid foundation for further development of sulfoxide-containing MS-cleavable cross-linkers with new functionalities.

Selective Electroenzymatic Oxyfunctionalization by Alkane Monooxygenase in a Biofuel Cell.

Aliphatic synthetic intermediates with high added value are generally produced from alkane sources (e.g., petroleum) by inert carbon-hydrogen (C-H) bond activation using classical chemical methods (i.e. high temperature, rare metals). As an alternative approach for these reactions, alkane monooxygenase from Pseudomonas putida (alkB) is able to catalyze the difficult terminal oxyfunctionalization of alkanes selectively and under mild conditions. Herein, we report an electrosynthetic system using an alkB biocathode which produces alcohols, epoxides, and sulfoxides through bioelectrochemical hydroxylation, epoxidation, sulfoxidation, and demethylation. The capacity of the alkB binding pocket to protect internal functional groups is also demonstrated. By coupling our alkB biocathode with a hydrogenase bioanode and using H2 as a clean fuel source, we have developed and characterized a series of enzymatic fuel cells capable of oxyfunctionalization while simultaneously producing electricity.

A fluorescent turn-on probe for detection of hypochlorus acid and its bioimaging in living cells.

Hypochlorous acid has played several functions in the biological system. However, excess HOCl can cause damage to biomolecules and result in some diseases. Accordingly, a new fluorescent probe, BSP, has been developed for fast recognition of HOCl through the HOCl-induced oxidation of methyl phenyl sulfide to sulfoxide. The reaction of BSP with HOCl caused a 22-fold fluorescence enhancement (quantum yield increase from 0.006 to 0.133). The detection limit of HOCl is found to be 30 nM (S/N = 3). The fluorescence enhancement is due to the suppression of the photo-induced electron transfer from the methyl phenyl sulfide moiety to BODIPY. Eventually, the cellular fluorescence imaging experiment showed that BSP could be effectively used for monitoring HOCl in living cells.

Enantioselective Sulfoxidation of Thioanisole by Cascading a Choline Oxidase and a Peroxygenase in the Presence of Natural Deep Eutectic Solvents.

A bienzymatic cascade for selective sulfoxidation is presented. The evolved recombinant peroxygenase from Agrocybe aegeritra catalyses the enantioselective sulfoxidation of thioanisole whereas the choline oxidase from Arthrobacter nicotianae provides the H2 O2 necessary via reductive activation of ambient oxygen. The reactions are performed in choline chloride-based deep eutectic solvents serving as co-solvent and stoichiometric reductant at the same time. Very promising product concentrations (up to 15 mM enantiopure sulfoxide) and catalyst performances (turnover numbers of 150,000 and 2100 for the peroxygenase and oxidase, respectively) have been achieved.

A Click-Chemistry-Based Enrichable Crosslinker for Structural and Protein Interaction Analysis by Mass Spectrometry.

Mass spectrometry is the method of choice for the characterisation of proteomes. Most proteins operate in protein complexes, in which their close association modulates their function. However, with standard MS analysis, information on protein-protein interactions is lost and no structural information is retained. To gain structural and interactome data, new crosslinking reagents are needed that freeze inter- and intramolecular interactions. Herein, the development of a new reagent, which has several features that enable highly sensitive crosslinking MS, is reported. The reagent enables enrichment of crosslinked peptides from the majority of background peptides to facilitate efficient detection of low-abundant crosslinked peptides. Due to the special cleavable properties, the reagent can be used for MS2 and potentially for MS3 experiments. Thus, the new crosslinking reagent, in combination with high-end MS, should enable sensitive analysis of interactomes, which will help researchers to obtain important insights into cellular states in health and diseases.

Influence of Sulfoxide Group Placement on Polypeptide Conformational Stability.

The synthesis of a homologous series containing five new nonionic sulfoxide containing polypeptides was described. Sulfoxide groups bestowed water solubility for all homologues, which allowed their use as a model for study of helix-coil transitions in water while avoiding contributions from charged groups or phase separation. Polypeptides were found to adopt chain conformations in water that were dependent on distance of sulfoxides from chain backbones, overall side-chain lengths, and solvent. These results allow preparation of polypeptide segments with different chain conformations without changing chemical functionality for potential use in structural studies and functional applications.

Reactivity of Small Oxoacids of Sulfur.

Oxidation of sulfide to sulfate is known to consist of several steps. Key intermediates in this process are the so-called small oxoacids of sulfur (SOS)-sulfenic HSOH (hydrogen thioperoxide, oxadisulfane, or sulfur hydride hydroxide) and sulfoxylic S(OH)2 acids. Sulfur monoxide can be considered as a dehydrated form of sulfoxylic acid. Although all of these species play an important role in atmospheric chemistry and in organic synthesis, and are also invoked in biochemical processes, they are quite unstable compounds so much so that their physical and chemical properties are still subject to intense studies. It is well-established that sulfoxylic acid has very strong reducing properties, while sulfenic acid is capable of both oxidizing and reducing various substrates. Here, in this review, the mechanisms of sulfide oxidation as well as data on the structure and reactivity of small sulfur-containing oxoacids, sulfur monoxide, and its precursors are discussed.

Expanding the range of binding energies and oxidizability of biologically relevant S-aromatic interactions: imidazolium and phenolate binding to sulfoxide and sulfone.

Oxidation and protonation/deprotonation strongly impact intermolecular noncovalent interactions. For example, S-aromatic interactions are stabilized up to three-fold in the gas phase on oxidation of the sulfur ligand or protonation/deprotonation of the aromatic. To probe if such stabilizing effects are additive and to model interactions of oxidized methionine (MetOn) with protonated histidine and deprotonated tyrosine residues in proteins, we examined Me2SOn (n = 1, 2) binding to imidazolium, phenolate and their 4-methylated forms. Ab initio MP2(full)/6-311++G(d,p) gas-phase calculations reveal that the Me2SOn-imidazolium complexes adopt edge-on geometry with σ-type (N/C-HarO) H-bonding and interaction energies of -17.2 to -31.1 kcal mol-1. The less stable (-13.8 to -21.0 kcal mol-1) Me2SOn-phenolates possess en-face geometry stabilized by π-type (C-Hπar) H-bonding. Comparing these energies with those reported for the Me2S-neutral aromatics affirms the additive effects of ligand protonation/deprotonation and oxidation on gas-phase stability. However, this is not the case in water although the aqueous complexes retain their preferred gas-phase σ- and π-type H-bonded structures. Binding free energies (kcal mol-1) calculated from molecular dynamics simulations in bulk water (preceded by CHARMM36 force field calibration where necessary) reveal that Me2SO-imidazolium (-4.4) is more stable than Me2SO-phenolate (-2.4) but Me2SO2-imidazolium (-0.6) is less stable than Me2SO2-phenolate (-3.8). Vertical ionization potentials (IPV) calculated for the gas-phase complexes indicate that the Me2SOn-phenolates, but not the Me2SOn-imidazoles, are oxidizable under biological conditions. Charge transfer from the phenolate increases its IPV by ∼20%, decreasing its susceptibility to oxidation. Overall, this work provides fundamental data to predict the behaviour of protein-based MetOn-aromatic-ion interactions.

Shikimoyl-ligand decorated gold nanoparticles for use in ex vivo engineered dendritic cell based DNA vaccination.

Since mannose receptors (MRs) are expressed on the surfaces of dendritic cells (DCs), the most professional antigen presenting cells in our body, DNA vaccine carriers containing either covalently grafted mannosyl- or mannose-mimicking shikimoyl-ligands are being increasingly used in ex vivo DC-transfection based DNA vaccination. To this end, we have recently demonstrated that ex vivo immunization of mice with liposomes of shikimoylated cationic amphiphiles containing a 6-amino hexanoic acid spacer group in the head-group region in complexation with melanoma antigen (MART1) encoded DNA vaccine (pCMV-MART1) induces long lasting anti-melanoma immune responses (C. Voshavar, et al., J. Med. Chem., 2017, 60, 1605-1610). This finding prompted us to examine, in the present investigation, the efficacies of gold nanoparticles conjugated to the mannose-mimicking shikimoyl ligand (SL) via a 6-amino hexane thiol spacer (AuNPs-SL) for use in ex vivo DC-transfection based genetic immunization. Herein, we report on the design, synthesis, physico-chemical characterization and bioactivities of AuNPs-SL. Dynamic light scattering and transmission electron microscopy studies revealed the hydrodynamic diameters of theAuNPs-SL nanoconjugates to be within the range of 23-44 nm and their surface potentials within the range of 9-28 mV. MTT-assay showed the non-cytotoxic nature of AuNPs-SL and the findings in the electrophoretic gel retardation assays revealed strong DNA binding properties of the AuNPs-SL. Importantly, subcutaneous immunization of C57BL/6J mice with DCs ex vivo transfected with an electrostatic complex of AuNPs-SL & melanoma antigen (MART1) encoded DNA vaccine (p-CMV-MART1) induced a long lasting (100 days) anti-tumor immune response in immunized mice upon subsequent challenge with a lethal dose of melanoma. Notably, mice immunized with either autologous mbmDCs ex vivo pre-transfected with nanoplexes of shikimoylated AuNPs-SL & an irrelevant pCMV-SPORT-ß-gal plasmid (without having encoded melanoma antigen) or untransfected DCs showed no lasting protection against subsequent tumor challenge. The presently described shikimoyl-decorated gold nanoparticles (AuNPs-SL) are expected to find future use in ex vivo DC-transfection based genetic immunization against cancer and other infectious diseases.

Optimized Fragmentation Improves the Identification of Peptides Cross-Linked by MS-Cleavable Reagents.

Cross-linking mass spectrometry is becoming increasingly popular, and current advances are widening the applicability of the technique so that it can be utilized by nonspecialist laboratories. Specifically, the use of novel mass-spectrometry-cleavable (MS-cleavable) reagents dramatically reduces the complexity of the data by providing (i) characteristic reporter ions and (ii) the mass of the individual peptides rather than that of the cross-linked moiety. However, optimum acquisition strategies to obtain the best-quality data for such cross-linkers with higher energy C-trap dissociation (HCD) alone are yet to be achieved. Therefore, we have carefully investigated and optimized MS parameters to facilitate the identification of disuccinimidyl-sulfoxide-based cross-links on HCD-equipped mass spectrometers. From the comparison of nine different fragmentation energies, we chose several stepped-HCD fragmentation methods that were evaluated on a variety of cross-linked proteins. The optimal stepped-HCD method was then directly compared with previously described methods using an Orbitrap Fusion Lumos Tribrid instrument using a high-complexity sample. The final results indicate that our stepped-HCD method is able to identify more cross-links than other methods, mitigating the need for multistage MS-enabled (MSn) instrumentation and alternative dissociation techniques. Data are available via ProteomeXchange with identifier PXD011861.