Discovery and Identification of Three Homocysteine Metabolites by Chemical Derivatization and Mass Spectrometry Fragmentation.

Discovery and identification of a new endogenous metabolite are typically hindered by requirements of large sample volumes and multistage purifications to guide synthesis of the standard. Presented here is a metabolomics platform that uses chemical tagging and tandem mass spectrometry to determine structure, direct synthesis, and confirm identity. Three new homocysteine metabolites are reported: N-succinyl homocysteine, 2-methyl-1,3-thiazinane-4-carboxylic acid (MTCA), and homolanthinone.

Discovery of a novel homocysteine thiolactone hydrolase and the catalytic activity of its natural variants.

Homocysteine thiolactone (HTL), a toxic metabolite of homocysteine (Hcy) in hyperhomocysteinemia (HHcy), is known to modify protein structure and function, leading to protein damage through formation of N-Hcy-protein. HTL has been highly linked to HHcy-associated cardiovascular and neurodegenerative diseases. The protective role of HTL hydrolases against HTL-associated vascular toxicity and neurotoxicity have been reported. Although several endogeneous enzymes capable of hydrolyzing HTL have been identified, the primary enzyme responsible for its metabolism remains unclear. In this study, three human carboxylesterases were screened to explore new HTL hydrolase and human carboxylesterase 1 (hCES1) demonstrates the highest catalytic activity against HTL. Given the abundance of hCES1 in the liver and the clinical significance of its single-nucleotide polymorphisms (SNPs), six common hCES1 nonsynonymous coding SNP (nsSNPs) variants were examined and characterized for their kinetic parameters. Variants E220G and G143E displayed 7.3-fold and 13.2-fold lower catalytic activities than its wild-type counterpart. In addition, the detailed catalytic mechanism of hCES1 for HTL hydrolysis was computational investigated and elucidated by Quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) method. The function of residues E220 and G143 in sustaining its hydrolytic activity of hCES1 was analyzed, and the calculated energy difference aligns well with experimental-derived results, supporting the validity of our computational insights. These findings provide insights into the potential protective role of hCES1 against HTL-associated toxicity, and warrant future studies on the possible association between specific genetic variants of hCES1 with impaired catalytic function and clinical susceptibility of HTL-associated cardiovascular and neurodegenerative diseases.

Pilot Evaluation of S-(3-[18F]Fluoropropyl)-D-Homocysteine and O-(2-[18F]Fluoroethyl)-D-Tyrosine as Bacteria-Specific Radiotracers for PET Imaging of Infection.

PURPOSE: There is currently no ideal radiotracer for imaging bacterial infections. Radiolabelled D-amino acids are promising candidates because they are actively incorporated into the peptidoglycan of the bacterial cell wall, a structural feature which is absent in human cells. This work describes fluorine-18 labelled analogues of D-tyrosine and D-methionine, O-(2-[18F]fluoroethyl)-D-tyrosine (D-[18F]FET) and S-(3-[18F]fluoropropyl)-D-homocysteine (D-[18F]FPHCys), and their pilot evaluation studies as potential radiotracers for imaging bacterial infection. PROCEDURES: D-[18F]FET and D-[18F]FPHCys were prepared in classical fluorination-deprotection reactions, and their uptake in Staphylococcus aureus and Pseudomonas aeruginosa was evaluated over 2 h. Heat killed bacteria were used as controls. A clinically-relevant foreign body model of S. aureus infection was established in Balb/c mice, as well as a sterile foreign body to mimic inflammation. The ex vivo biodistribution of D-[18F]FPHCys in the infected and inflamed mice was evaluated after 1 h, by dissection and gamma counting. The uptake was compared to that of [18F]FDG. RESULTS: In vitro uptake of both D-[18F]FET and D-[18F]FPHCys was specific to live bacteria. Uptake was higher in S. aureus than in P. aeruginosa for both radiotracers, and of the two, higher for D-[18F]FPHCys than D-[18F]FET. Blocking experiments with non-radioactive D-[19F]FPHCys confirmed specificity of uptake. In vivo, D-[18F]FPHCys had greater accumulation in S. aureus infection compared with sterile inflammation, which was statistically significant. As anticipated, [18F]FDG showed no significant difference in uptake between infection and inflammation. CONCLUSIONS: D-[18F]FPHCys uptake was higher in infected tissues than inflammation, and represents a fluorine-18 labelled D-AA with potential to detect a S. aureus reference strain (Xen29) in vivo. Additional studies are needed to evaluate uptake of this radiotracer in clinical isolates.

A metal-phenolic coordination framework nanozyme exhibits dual enzyme mimicking activity and its application is effective for colorimetric detection of biomolecules.

Biomolecules play vital roles in many biological processes and diseases, making their identification crucial. Herein, we present a colorimetric sensing method for detecting biomolecules like cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). This approach is based on a reaction system whereby colorless 3,3',5,5'-tetramethylbenzidine (TMB) undergoes catalytic oxidation to form blue-colored oxidized TMB (ox-TMB) in the presence of hydrogen peroxide (H2O2), utilizing the peroxidase and catalase-mimicking activities of metal-phenolic coordination frameworks (MPNs) of Cu-TA, Co-TA, and Fe-TA nanospheres. The Fe-TA nanospheres demonstrated superior activity, more active sites and enhanced electron transport. Under optimal conditions, the Fe-TA nanospheres were used for the detection of biomolecules. When present, biomolecules inhibit the reaction between TMB and H2O2, causing various colorimetric responses at low detection limits of 0.382, 0.776 and 0.750 µM for Cys, Hcy and GSH. Furthermore, it was successfully applied to real water samples with good recovery results. The developed sensor not only offers a rapid, portable, and user-friendly technique for multi-target analysis of biomolecules at low concentrations but also expands the potential uses of MPNs for other targets in the environmental field.

A two-photon fluorescent probe for highly selective detection of Cys over GSH and Hcy based on the Michael addition and transcyclization mechanism and its application in bioimaging and protein straining in SDS-PAGE.

BACKGROUND: Cysteine (Cys), glutathione (GSH), and homocysteine (Hcy), as three major biothiols are involved in a variety of physiological processes and play a crucial role in plant growth. Abnormal levels of Cys can cause plants to fail to grow properly. To date, although a very large number of fluorescent probes have been reported for the detection of biothiols, very few of them can be used for the selective discrimination of Cys from GSH and Hcy due to their structural similarity, and only a few of them can be used for plant imaging. RESULTS: Here, three fluorescent probes (o-/m-/p-TMA) based on TMN fluorophore and the ortho-/meta-/para-substituted maleimide recognition groups were constructed to investigate the selective response effect of Cys. Compared to the o-/m-TMA, p-TMA can selectively detect Cys over GSH and Hcy with a rapid response time (10 min) and a low detection limit (0.26 µM). The theoretical calculation confirmed that the intermediate p-TMA-Cys-int has shorter interatomic reaction distances (3.827 Å) compared to o-/m-TMA-Cys (5.533/5.287 Å), making it more suitable for further transcyclization reactions. Additionally, p-TMA has been employed for selective tracking of exogenous and endogenous Cys in Arabidopsis thaliana using both single-/two-photon fluorescence imaging. Furthermore, single cell walls produced obvious two-photon fluorescence signals, indicating that p-TMA can be used for high-concentration Cys analysis in single cells. Surprisingly, p-TMA can be used as a fluorescent dye for protein staining in SDS-PAGE with higher sensitivity (7.49 µg/mL) than classical Coomassie brilliant blue (14.11 µg/mL). SIGNIFICANCE: The outstanding properties of p-TMA make it a promising multifunctional molecular tool for the highly selective detection of Cys over GSH and Hcy in various complex environments, including water solutions, zebrafish, and plants. Additionally, it has the potential to be developed as a fluorescent dye for a simple and fast SDS-PAGE fluorescence staining method.

Synthesis and performance of a nanosensing platform for homocysteine detection: A series of iridium(III) complexes containing aldehyde group as probe and MOF as supporting substrate.

The low cost and simple detection method for Hcy (homocysteine) is highly desired in analytical and biological fields since Hcy has been regarded as a bio-marker for multiple diseases. In this work, five Ir(C^N)2(N^N)+ compounds having -CHO group in their C^N or N^N ligand were synthesized and tried for Hcy sensing. Electron-donating groups such as -NH2 and -CH3 were incorporated into the C^N or N^N ligand. Their geometric structure, electronic structure, and optical parameters (with or without Hcy) were analyzed and compared carefully to explore their Hcy sensing potential. The sensing mechanism was revealed by NMR titration and theoretical simulation as a cyclization reaction between the -CHO group and Hcy. The optimal compounds, which showed increased emission quantum yield (2.5-fold) and emission blue-shift (by âˆ¼ 100 nm) upon Hcy, were then covalently grafted into a porous host bio-MOF-1. Linear working plots were fitted, with good selectivity, LOD of 0.15 µM, and response time of 33 s. The novelty of this work was the eye-sensitive emission color change of this nanosensing platform from red (without Hcy) to green (with Hcy).

Catalytic specificity and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa.

The escalating drug resistance among microorganisms underscores the urgent need for innovative therapeutic strategies and a comprehensive understanding of bacteria's defense mechanisms against oxidative stress and antibiotics. Among the recently discovered barriers, the endogenous production of hydrogen sulfide (H2S) via the reverse transsulfuration pathway, emerges as a noteworthy factor. In this study, we have explored the catalytic capabilities and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa (PaCGL), a multidrug-opportunistic pathogen chiefly responsible for nosocomial infections. In addition to a canonical L-cystathionine hydrolysis, PaCGL efficiently catalyzes the production of H2S using L-cysteine and/or L-homocysteine as alternative substrates. Comparative analysis with the human enzyme and counterparts from other pathogens revealed distinct structural features within the primary enzyme cavities. Specifically, a distinctly folded entrance loop could potentially modulate the access of substrates and/or inhibitors to the catalytic site. Our findings offer significant insights into the structural evolution of CGL enzymes across different pathogens and provide novel opportunities for developing specific inhibitors targeting PaCGL.

A lateral flow strip for on-site detection of homocysteine based on a truncated aptamer.

An elevated level of homocysteine (Hcy) in serum is closely related to the development of various diseases. Therefore, homocysteine has been widely employed as a biomarker in medical diagnosis and the on-site detection of homocysteine is highly desired. In this study, a truncated highly specific aptamer for homocysteine was screened and used to design a lateral flow strip (LFS) for the detection of homocysteine. The aptamer was derived from a previously reported sequence. Based on the result of molecular docking, the original sequence was subjected to truncation, resulting in a reduction of the length from 66 nt to 55 nt. Based on the truncated aptamer, the LFS was designed for the detection of homocysteine. In the presence of homocysteine, the aptamer selectively binds to it, releasing cDNA from the aptamer/cDNA duplex. This allows cDNA to bind to the capture probe immobilized on the T zone of the strip, resulting in a red signal on the T zone from gold nanoparticles (AuNPs). The strip enables the visual detection of homocysteine in 5 min. Quantitative detection can be facilitated with the aid of ImageJ software. In this mode, the linear detection range for homocysteine is within 5-50 µM, with a detection limit of 4.18 µM. The strip has been effectively utilized for the detection of homocysteine in human serum. Consequently, the combination of the truncated aptamer and the strip offers a method that is sensitive, quick, and economical for the on-site detection of homocysteine.

Homocystamide Conjugates of Human Serum Albumin as a Platform to Prepare Bimodal Multidrug Delivery Systems for Boron Neutron Capture Therapy.

Boron neutron capture therapy is a unique form of adjuvant cancer therapy for various malignancies including malignant gliomas. The conjugation of boron compounds and human serum albumin (HSA)-a carrier protein with a long plasma half-life-is expected to extend systemic circulation of the boron compounds and increase their accumulation in human glioma cells. We report on the synthesis of fluorophore-labeled homocystamide conjugates of human serum albumin and their use in thiol-'click' chemistry to prepare novel multimodal boronated albumin-based theranostic agents, which could be accumulated in tumor cells. The novelty of this work involves the development of the synthesis methodology of albumin conjugates for the imaging-guided boron neutron capture therapy combination. Herein, we suggest using thenoyltrifluoroacetone as a part of an anticancer theranostic construct: approximately 5.4 molecules of thenoyltrifluoroacetone were bound to each albumin. Along with its beneficial properties as a chemotherapeutic agent, thenoyltrifluoroacetone is a promising magnetic resonance imaging agent. The conjugation of bimodal HSA with undecahydro-closo-dodecaborate only slightly reduced human glioma cell line viability in the absence of irradiation (~30 µM of boronated albumin) but allowed for neutron capture and decreased tumor cell survival under epithermal neutron flux. The simultaneous presence of undecahydro-closo-dodecaborate and labeled amino acid residues (fluorophore dye and fluorine atoms) in the obtained HSA conjugate makes it a promising candidate for the combination imaging-guided boron neutron capture therapy.

Thiolactomide: A New Homocysteine Thiolactone Derivative from Streptomyces sp. with Neuroprotective Activity.

A new homocysteine thiolactone derivative, thiolactomide (1), was isolated along with a known compound, N-acetyl homocysteine thiolactone (2), from a culture extract of soil-derived Streptomyces sp. RK88-1441. The structures of these compounds were elucidated by detailed NMR and MS spectroscopic analyses with literature study. In addition, biological evaluation studies revealed that compounds 1 and 2 both exert neuroprotective activity against 6-hydroxydopamine (6-OHDA)-mediated neurotoxicity by blocking the generation of hydrogen peroxide in neuroblastoma SH-SY5Y cells.

A simple and accurate HFCF-UF method for the analysis of homocysteine, cysteine, cysteinyl-glycine, and glutathione in human blood.

The presence of reduced aminothiols, including homocysteine (Hcy), cysteine (Cys), cysteinyl-glycine (CG), and glutathione (GSH), is significantly increased in the pathological state. However, there have been no reports on the relationship between reduced aminothiols (Hcy, Cys, CG, and GSH) and different genders, ages, and drug combinations in human blood. The accurate quantification of these reduced thiols in biological fluids is important for monitoring some special pathological conditions of humans. However, the published methods typically not only require cumbersome and technically challenging processing procedures to ensure reliable measurements, but are also laborious and time-consuming, which may disturb the initial physiological balance and lead to inaccurate results. We developed a hollow fiber centrifugal ultrafiltration (HFCF-UF) method for sample preparation coupled with a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method and used it to determine four reduced aminothiols (Hcy, Cys, CG, and GSH) in human blood for the first time. A total of 96 clinical patients were enrolled in our study. The influence of different genders, ages, and drug combinations on the levels of four reduced thiols in human blood was also discussed by SPSS 24.0. The sample preparation was simplified to a single 5 min centrifugation step in a sealed system that did not disturb the physiological environment. The validation parameters for the methodological results were excellent. The procedure was successfully applied to monitoring the concentrations of four reduced aminothiols (Hcy, Cys, CG, and GSH) in 96 clinical blood samples. There were no significant differences in Hcy, Cys, CG, or GSH for the different genders, ages, or combinations with methotrexate or vancomycin (P > 0.05). However, there was a significant increase in Hcy concentration in patients treated with valproic acid who were diagnosed with epilepsy (p=0.0007). It is advisable to measure reduced Hcy level in patients taking valproic acid. The developed HFCF-UF method was simple and accurate. It can be easily applied in clinical research to evaluate oxidative stress in further study.

Headspace study of chiral interconversion of N-acetyl-homocysteine thiolactones.

The rate constants for (L)-N-acetyl homocysteine thiolactone enantiomerization have been obtained from batch-wise studies and by dynamic gas chromatography of racemic mixtures. Results from the batch-wise experiments show that the kinetics of racemization at 150 °C is the same for vials made of glass, silanized glass or Teflon-coated glass so that the vial surface exhibited no effect on the kinetics of racemization. From the temperature dependence of the rate constants the preexponential factor, activation energy, the activation Gibbs energy and activation entropy have been obtained from transition state theory. The catalytic effect of G-DP, G-BP and B-DP GC chiral stationary phases on racemization has been observed and quantified by the values of rate constants; B-DP exhibited the greatest activity. The Eyring activation parameters obtained from batch-wise experiment were compared with theoretical values acquired from quantum chemical modelling. Agreement between the experimental and calculated values of activation Gibbs energy, activation enthalpy and activation entropy is good. The dynamic gas chromatography of racemic mixture on chiral B-DP, G-DP and G-BP capillary columns indicate that the rate constants of forward and reverse reactions are different in chiral environments. The greatest accelerating effect in the process of enantiomerization has been identified for G-BP both in the batch-wise experiments and by the dynamic gas chromatography.

Homocysteine fibrillar assemblies display cross-talk with Alzheimer's disease ß-amyloid polypeptide.

High levels of homocysteine are reported as a risk factor for Alzheimer's disease (AD). Correspondingly, inborn hyperhomocysteinemia is associated with an increased predisposition to the development of dementia in later stages of life. Yet, the mechanistic link between homocysteine accumulation and the pathological neurodegenerative processes is still elusive. Furthermore, despite the clear association between protein aggregation and AD, attempts to develop therapy that specifically targets this process have not been successful. It is envisioned that the failure in the development of efficacious therapeutic intervention may lie in the metabolomic state of affected individuals. We recently demonstrated the ability of metabolites to self-assemble and cross-seed the aggregation of pathological proteins, suggesting a role for metabolite structures in the initiation of neurodegenerative diseases. Here, we provide a report of homocysteine crystal structure and self-assembly into amyloid-like toxic fibrils, their inhibition by polyphenols, and their ability to seed the aggregation of the AD-associated ß-amyloid polypeptide. A yeast model of hyperhomocysteinemia indicates a toxic effect, correlated with increased intracellular amyloid staining that could be rescued by polyphenol treatment. Analysis of AD mouse model brain sections indicates the presence of homocysteine assemblies and the interplay between ß-amyloid and homocysteine. This work implies a molecular basis for the association between homocysteine accumulation and AD pathology, potentially leading to a paradigm shift in the understanding of AD initial pathological processes.

Homocysteine: Biochemistry, Molecular Biology and Role in Disease.

Homocysteine is a non-proteinogenic sulfhydryl-containing amino acid derived from methionine and is a homologue of cysteine [...].

Human Serum Albumin Labelling with a New BODIPY Dye Having a Large Stokes Shift.

BODIPY dyes are photostable neutral derivatives of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene. These are widely used as chemosensors, laser materials, and molecular probes. At the same time, BODIPY dyes have small or moderate Stokes shifts like most other fluorophores. Large Stokes shifts are preferred for fluorophores because of higher sensitivity of such probes and sensors. The new boron containing BODIPY dye was designed and synthesized. We succeeded to perform an annulation of pyrrole ring with coumarin heterocyclic system and achieved a remarkable difference in absorption and emission maximum of obtained fluorophore up to 100 nm. This BODIPY dye was equipped with linker arm and was functionalized with a maleimide residue specifically reactive towards thiol groups of proteins. BODIPY residue equipped with a suitable targeting protein core can be used as a suitable imaging probe and agent for Boron Neutron Capture Therapy (BNCT). As the most abundant protein with a variety of physiological functions, human serum albumin (HSA) has been used extensively for the delivery and improvement of therapeutic molecules. Thiolactone chemistry provides a powerful tool to prepare albumin-based multimodal constructions. The released sulfhydryl groups of the homocysteine functional handle in thiolactone modified HSA were labeled with BODIPY dye to prepare a labeled albumin-BODIPY dye conjugate confirmed by MALDI-TOF-MS, UV-vis, and fluorescent emission spectra. Cytotoxicity of the resulting conjugate was investigated. This study is the basis for a novel BODIPY dye-albumin theranostic for BNCT. The results provide further impetus to develop derivatives of HSA for delivery of boron to cancer cells.

Identifying a role for the interaction of homocysteine and copper in promoting cardiovascular-related damage.

Observations that copper and homocysteine levels are simultaneously elevated in patients with cardiovascular disease has generated interest in investigating the interactions between copper and homocysteine. Several prior studies have shown that complexes of copper and homocysteine are toxic, leading to cardiovascular damage in vitro. It is not clear, however, why related effects do not occur with other structurally similar, more abundant cellular thiols such as glutathione and cysteine. Herein, a mechanism for a selective redox interaction between copper and homocysteine is demonstrated. It involves a kinetically favored intramolecular hydrogen atom transfer that results in an alpha-amino carbon-centered radical known to promote biomolecular damage.

A novel rosamine-based fluorescent probe for the rapid and selective detection of cysteine in BSA, water, milk, cabbage, radish, apple, and pear.

A novel fluorescent probe (RA), based on the rosamine skeleton bearing acrylate group, has been reasonably designed and prepared, which employed an addition-cyclization-elimination sequence reaction mechanism to detect cysteine. RA displayed rapid response to cysteine within 1.5 min, and exhibited satisfactory selectivity for cysteine over H2S, glutathione (Glu), and homocysteine (Hcy), due to the formation of seven-membered lactam favored kinetically. Fluorescence ratio was utilized to detect cysteine from 6.0 to 20.0 µM with a detection limit of 0.29 µM. More, RA was used to monitor cysteine in BSA, water, milk, milk powder, cabbage, radish, apple, and pear.

Colorimetric detection of homocysteine by a pyridylazo dye-based Cu2+ complex via indicator displacement mechanism.

A BrPAPS based Cu2+ complex has been developed as a colorimetric probe for the selective recognition of homocysteine (Hcy) over cysteine (Cys) and glutathione (GSH) in an aqueous solution via the indicator displacement assay. BrPAPS formed a complex with Cu2+ in a 1:1 ratio (BrPAPS-Cu2+) accompanied by the color change from yellow to red. Detecting Hcy is based on high affinity of Hcy for Cu2+. The addition of Hcy to BrPAPS-Cu2+ caused the complex formation of Hcy with Cu2+ in a 2:1 stoichiometry, resulting a hypsochromic shift with change back of color from red to yellow by the release of BrPAPS from BrPAPS-Cu2+. The absorption response is linear with the Hcy concentration in the range of 0-20 µM with a detection limit of 1.46 µM. Moreover, the detection of Hcy was not significantly affected by other amino acids from the competition experiments. Thus, BrPAPS-Cu2+ can be used as a simple probe for Hcy in aqueous solution.

Four families of folate-independent methionine synthases.

Although most organisms synthesize methionine from homocysteine and methyl folates, some have "core" methionine synthases that lack folate-binding domains and use other methyl donors. In vitro, the characterized core synthases use methylcobalamin as a methyl donor, but in vivo, they probably rely on corrinoid (vitamin B12-binding) proteins. We identified four families of core methionine synthases that are distantly related to each other (under 30% pairwise amino acid identity). From the characterized enzymes, we identified the families MesA, which is found in methanogens, and MesB, which is found in anaerobic bacteria and archaea with the Wood-Ljungdahl pathway. A third uncharacterized family, MesC, is found in anaerobic archaea that have the Wood-Ljungdahl pathway and lack known forms of methionine synthase. We predict that most members of the MesB and MesC families accept methyl groups from the iron-sulfur corrinoid protein of that pathway. The fourth family, MesD, is found only in aerobic bacteria. Using transposon mutants and complementation, we show that MesD does not require 5-methyltetrahydrofolate or cobalamin. Instead, MesD requires an uncharacterized protein family (DUF1852) and oxygen for activity.

Simple Turn-On Fluorescent Sensor for Discriminating Cys/Hcy and GSH from Different Fluorescent Signals.

As a kind of bioactive sulfur species, biothiols (Cys, Hcy, and GSH) play an irreplaceable role in regulating the redox balance of life processes. Because of their similar chemical structures and properties, a sulfydryl group, and an amino group, it is an important challenge to distinguish two or more of them at the same time. Herein, a fluorescent sensor (NTPC) based on the coumarin structure was developed to discriminate Cys/Hcy and GSH simultaneously. The sensor has no fluorescence due to the d-PET effect but displays strong fluorescence after its reaction with biothiols. There are two potential reaction sites (nitrophenyl sulfide group and aldehyde group) in the structure of NTPC, resulting in different fluorescent signal changes after reacting with biothiols (green for Cys and Hcy and red for GSH). Under double-wavelength excitation, the sensor shows low background fluorescence, high selectivity, and low detection limits toward biothiols. Moreover, the sensor can be used to discriminate different biothiols (Cys/Hcy and GSH) in cells and zebra fish by different fluorescence signals with low toxicity and might provide a promising tool for studying the roles of different biothiols in various physiological and pathological processes.