Aminoethyl Glycine Research Articles

Extraction of Non-Protein Amino Acids from Cyanobacteria for Liquid Chromatography-Tandem Mass Spectrometry Analysis.

Non-protein amino acids (NPAAs) are a large class of amino acids (AAs) that are not genetically encoded for translation into proteins. The analysis of NPAAs can provide crucial information about cellular uptake and/or function, metabolic pathways, and potential toxicity. β-methylamino-L-alanine (BMAA) is a neurotoxic NPAA produced by various algae species and is associated with an increased risk for neurodegenerative diseases, which has led to significant research interest. There are numerous ways to extract AAs for analysis, with liquid chromatography-tandem mass spectrometry being the most common, requiring protein precipitation followed by acid hydrolysis of the protein pellet. Studies on the presence of BMAA in algal species provide contradictory results, with the use of unvalidated sample preparation/extraction and analysis a primary cause. Like most NPAAs, protein precipitation in 10% aqueous TCA and hydrolysis with fuming HCl is the most appropriate form of extraction for BMAA and its isomers aminoethylglycine (AEG) and 2,4-diaminobutyric acid (2,4-DAB). The present protocol describes the steps in a validated NPAA extraction method commonly used in research and teaching laboratories.

Mixture designs to investigate adverse effects upon co-exposure to environmental cyanotoxins

The goal of this study was to implement powerful mixture design techniques, commonly used in process optimization, to investigate enhanced adverse effects upon co-exposure to environmental cyanotoxins. Exposure to cyanobacteria, which are found ubiquitously in environmental water reservoirs, have been linked to several neurodegenerative diseases. Despite the known co-occurrence of various cyanotoxins, the majority of studies investigating this link have focused on the investigation of a single cyanotoxin, a noncanonical amino acid called β-methylamino-L-alanine (BMAA), which poorly recapitulates an actual environmental exposure. Interactions amongst cyanotoxic compounds is an area of great concern and remains poorly understood. To this end, we describe the use of a simplex axial mixture design to screen for interactive adverse effects of cyanotoxic mixtures. Using a combination of basic toxicity assays coupled with contemporary proteomic techniques, our results show the existence of a significant (p ≤ 0.01) interaction between BMAA and its isomers aminoethyl glycine (AEG) and 2,4-diaminobutyric acid (2,4DAB). Cyanotoxic mixtures significantly decreased cell viability by an average of 19% and increased caspases 3/7 activities by an average of 110% when compared to individual cyanotoxins (p ≤ 0.05). Cyanotoxic mixtures perturbed various biological pathways associated with neurodegeneration, including inhibition of protective autophagy and activation of mitochondrial dysfunction (z-score >|2|). Additionally, exposure to mixtures perturbed important upstream regulators involved in cellular dysfunction, morbidity, and development. Taken together, our results highlight: (1) the need to study combinations of cyanotoxins when investigating the link between cyanobacteria and neurodegenerative pathologies and (2) the application of design of experiment (DoE) as an efficient methodology to study mixtures of relevant environmental toxins.

Synthesis and evaluation of 99mTc-analogues of [123/131I]mIBG prepared via [99mTc][Tc(CO)3(H2O)3]+ synthon for targeting norepinephrine transporter

Introductionmeta-[123/131I]Iodobenzylguanidine (mIBG) is a clinical agent used for imaging neuroendocrine tumors, where uptake in tumor is via active transport mechanism through norepinephrine transporters (NET). Our group in past have evaluated a 99mTc-analogue of the above tracer, based on 99mTc-4 + 1 labeling approach, which exhibited significant affinity for NET but suffered from reduced specific uptake in comparison to reference standard no-carrier-added (n.c.a.) [125I]mIBG. The present work attempts to synthesize two new 99mTc-analogues of the radio-iodinated derivative following [99mTc]Tc(CO)31+ approach with an aim to improve the above specific uptake content. MethodsTwo different precursors, xylylenediamine and 1,3-bis(chloromethyl)benzene, were synthetically modified to yield meta-functionalized benzylguanidine derivatives bearing iminodiacetate (IDA) and aminoethylglycine (AEG) tridentate chelating moieties, respectively. These ligands were labeled with technetium-99m via [99mTc][Tc(CO)3(H2O)3]+ synthon to form desired radioactive complexes 9 and 10. The radiolabeling yields of the complexes obtained were >90% as confirmed by radio-HPLC. The HPLC purified complexes were used for in vitro and in vivo evaluation to understand the true biological efficacy. Structural characterization of the radiolabeled complexes was carried after synthesizing and characterizing their Re-analogues. ResultsCell uptake studies with the radiolabeled complexes in SK-N-SH neuroblastoma cell lines revealed reduced uptake in the cells (<1% of incubated radioactivity/106 cells) in comparison to n.c.a. [125I]mIBG (~12%). However, limited specificity (~60%) was observed for the complexes as ascertained through desmethylimipramine (DMI) inhibition. Biodistribution studies in normal Wistar rats exhibited desired non-target clearance pharmacokinetics for the complexes but in vivo NET efficacy in myocardium for the neutral complex 10 could not be established. ConclusionsTridentate [99mTc]Tc(CO)31+ chelation approach severely affects biological behavior of the present small bioactive molecule under study to a significant extent in comparison to monodentate ligation in 99mTc-4 + 1 strategy.

N-β-Methylamino-L-Alanine and Its Naturally Occurring Isomers in Cyanobacterial Blooms in Lake Winnipeg

Cyanobacterial blooms have affected Lake Winnipeg since the mid-1990s due to an increased phosphorus loading into the lake, which has been exacerbated by stressors such as climate change and eutrophication. Aquatic ecosystems involving cyanobacteria have been found to contain N-β-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB), non-protein amino acids that are associated with neurodegenerative disease, as well as two of the naturally occurring isomers, N-2(amino)ethylglycine (AEG) and β-amino-N-methylalanine (BAMA). We hypothesized that the cyanobacterial bloom in Lake Winnipeg produces BMAA and/or its naturally occurring isomers. Samples of cyanobacteria were collected by the Lake Winnipeg Research Consortium from standard sampling stations and blooms in July and September of 2016 and were analyzed for BMAA, DAB, AEG, and BAMA using previously published validated analytical methods. BMAA and BAMA were found in the highest concentration in the center of the north basin, the deepest and lowest-nitrogen zone of the lake, at an average concentration of 4μg/g (collected in July and September 2016) and 1.5mg/g (collected in July 2016), respectively. AEG and DAB were found in the highest concentration in cyanobacterial blooms from the nearshore region of the north basin, the slightly shallower and more nitrogen-rich zone of the lake, at 2.1mg/g (collected in July 2016) and 0.2mg/g (collected in July and September 2016), respectively. These findings indicate that the production of non-protein amino acids varies with the depth and nutrient contents of the bloom. It is important to note that we did not measure food or water samples directly and further study of the Lake Winnipeg food web is required to determine whether BMAA bioaccumulation represents an increased risk factor for neurodegenerative disease in the region.

Analysis of Neurotoxic Amino Acids from Marine Waters, Microbial Mats, and Seafood Destined for Human Consumption in the Arabian Gulf.

Human health risks associated with exposure to algal and cyanobacterial toxins (phycotoxins) have been largely concerned with aquatic habitats. People inhabiting desert environments may be exposed to phycotoxins present in terrestrial environments, where cyanobacterial crusts dominate. Seafood comprises a significant portion of the human diet in desert environments proximal to an ocean or sea. Consequently, in addition to terrestrial exposure to cyanotoxins, the potential exists that seafood may be an important exposure route for cyanotoxins in desert regions. Understanding the possible risk of exposure from seafood will help create cyanotoxin health guidelines for people living in environments that rely on seafood. Commonly-consumed local seafood products destined for human consumption were purchased from a fish market in Doha, Qatar. Organs were excised, extracted, and analyzed for the neurotoxic amino acid β-N-methylamino-L-alanine (BMAA) and the isomers 2,4-diaminobutyric acid (DAB) and N-2(aminoethyl)glycine (AEG). The presence and concentration of neurotoxic amino acids were investigated in organisms from various trophic levels to examine the potential for biomagnification. Although BMAA and isomers were detected in marine microbial mats, as well as in marine plankton net trawls associated with diatoms and dinoflagellates, in seafood, only AEG and DAB were present at low concentrations in various trophic levels. The findings of this study suggest that exposure to neurotoxic amino acids through seafood in the Arabian Gulf may be minor, yet the presence of BMAA in phytoplankton confirms the need for further monitoring of marine waters and seafood to protect human health.

Assessing the Combined Toxicity of BMAA and Its Isomers 2,4-DAB and AEG In Vitro Using Human Neuroblastoma Cells.

The non-protein amino acid (NPAA) ß-methylamino-L-alanine (BMAA) is produced by a diverse range of cyanobacteria, diatoms and dinoflagellates, and is present in both aquatic and terrestrial ecosystems globally. Exposure to BMAA has been implicated in the development of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD) and Parkinson's disease (PD). BMAA is often found in nature along with its structural isomers 2,4-diaminobutyric acid (2,4-DAB) and aminoethylglycine (AEG); however, the toxicity of these NPAAs in combination has not been examined. We have previously demonstrated that BMAA induces endoplasmic reticulum (ER) stress and increases caspase and cathepsin activity in human neuroblastoma cells (SH-SY5Y), effects consistent with proteotoxic stress due to disturbances in protein synthesis, folding or turnover. The current study investigates whether 2,4-DAB and AEG share a similar mechanism of toxicity to BMAA, and if simultaneous exposure of cells to BMAA and its isomers results in increased toxicity in vitro. We show that a 48-h treatment with both 500μM BMAA and 2,4-DAB decreases cell viability in vitro whereas AEG was not cytotoxic under the same conditions. Treatment of SH-SY5Y cells with 2,4-DAB did not increase expression of ER stress markers. Combined treatment of cells with BMAA and 2,4-DAB resulted in increased caspase activity and increased apoptosis above that of BMAA or 2,4-DAB on their own. These results suggest that 2,4-DAB does not share the same mechanism of toxicity as BMAA but the presence of 2,4-DAB increases the toxicity of BMAA to human cells in vitro.

Structural characteristics of cyclopentane-modified peptide nucleic acids from molecular dynamics simulations

A PNA molecule is a DNA strand where the sugar-phosphate backbone has been replaced by a structurally homomorphous pseudopeptide chain consisting of N (2-amino-ethyl)-glycine units. PNA binds strongly to both DNA and RNA. However, an analysis of the X-ray and NMR data show that the dihedral angles of PNA/DNA or PNA/RNA complexes are very different from those of DNA:DNA or RNA:RNA complexes. In addition, the PNA strand is very flexible. One way to improve the binding affinity of PNA for DNA/RNA is to design a more pre-organized PNA structure. An effective way to rigidify the PNA strand is to introduce ring structures into the backbone. In several experimental studies, the ethylenediamine portion of aminoethyl glycine peptide nucleic acids (aegPNA) has been replaced with one or more (S,S)-trans cyclopentyl (cpPNA) units. This substitution has met with varied success in terms of DNA/RNA recognition. In the present work, molecular modeling studies were performed to a PNA molecule. Detailed investigations on the conformational and dynamical properties of single-stranded aegPNA and cpPNA were undertaken to determine how the cyclopentane ring will improve binding and to determine the contributions of both entropy and dihedral angle preference to the observed stronger binding. The effects of single and multiple modifications of the PNA backbone were also analyzed to understand changes in conformational and dynamical properties.

New Typical Vector of Neurotoxin β-N-Methylamino-l-Alanine (BMAA) in the Marine Benthic Ecosystem.

The neurotoxin β-N-methylamino-l-alanine (BMAA) has been identified as an environmental factor triggering neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS) and Alzheimer’s disease (AD). We investigated the possible vectors of BMAA and its isomers 2,4-diaminobutyric acid (DAB) and N-2(aminoethyl)glycine (AEG) in marine mollusks collected from the Chinese coast. Sixty-eight samples of marine mollusks were collected along the Chinese coast in 2016, and were analyzed by an HILIC-MS/MS (hydrophilic interaction liquid chromatography with tandem quadrupole mass spectrometer) method without derivatization. BMAA was detected in a total of five samples from three species: Neverita didyma, Solen strictus, and Mytilus coruscus. The top three concentrations of free-form BMAA (0.99~3.97 μg·g−1 wet weight) were detected in N. didyma. DAB was universally detected in most of the mollusk samples (53/68) with no species-specific or regional differences (0.051~2.65 μg·g−1 wet weight). No AEG was detected in any mollusk samples tested here. The results indicate that the gastropod N. didyma might be an important vector of the neurotoxin BMAA in the Chinese marine ecosystem. The neurotoxin DAB was universally present in marine bivalve and gastropod mollusks. Since N. didyma is consumed by humans, we suggest that the origin and risk of BMAA and DAB toxins in the marine ecosystem should be further investigated in the future.

A study on nitroimidazole-99mTc(CO)3 complexes as hypoxia marker: some observations towards possible improvement in in vivo efficacy.

Hypoxia plays a negative role in the clinical management of cancer. Detection of hypoxic status of a cancer is important for selecting patients for hypoxia directed therapy. Though [(18)F]fluoromisonidazole ([(18)F]FMISO), a PET radiopharmaceutical, is presently being used in the clinic for the detection of hypoxia, considering the logistical advantages of (99m)Tc and wider availability of SPECT scanners, a radiopharmaceutical based on this isotope may find wider applicability. Nine nitroimidazole (2-, 4- and 5-nitroimidazole) ligands were synthesized and radiolabeled using [(99m)Tc(CO)3(H2O)3](+) precursor to obtain a group of complexes possessing different single electron reduction potential (SERP), overall charge and lipophilicity, the three attributes which decide the efficacy of the complex to detect hypoxic cells in vivo. The nitroimidazole-(99m)Tc(CO)3 complexes as well as [(18)F]FMISO were evaluated in fibrosarcoma tumor bearing mice. The (99m)Tc(CO)3 complexes of nitroimidazole iminodiacetic acid (IDA) showed better tumor uptake and retention than nitroimidazole diethylenetriamine (DETA) and nitroimidazole aminoethylglycine (AEG) complexes. Tumor uptake observed with [(18)F]FMISO was higher than any of the nitroimidazole-IDA- (99m)Tc(CO)3 complexes. However, [(18)F]FMISO clearance from tumor was found to be faster compared to 2-nitroimidazole-IDA-(99m)Tc(CO)3 complex. Observed tumor uptake and retention of the radiotracers evaluated could be correlated to its blood clearance pattern and SERP. Results of the present study indicated that uptake of the radiotracer in tumor is closely associated with its rate of clearance from blood. The study also indicated that along with SERP, clearance of radiotracer from blood (net effect of charge and lipophilicity) is a critical factor which decides the in vivo efficacy of the hypoxia detecting radiopharmaceutical.

Side chain modified peptide nucleic acids (PNA) for knock-down of six3in medaka embryos

BackgroundSynthetic antisense molecules have an enormous potential for therapeutic applications in humans. The major aim of such strategies is to specifically interfere with gene function, thus modulating cellular pathways according to the therapeutic demands. Among the molecules which can block mRNA function in a sequence specific manner are peptide nucleic acids (PNA). They are highly stable and efficiently and selectively interact with RNA. However, some properties of non-modified aminoethyl glycine PNAs (aegPNA) hamper their in vivo applications.ResultsWe generated new backbone modifications of PNAs, which exhibit more hydrophilic properties. When we examined the activity and specificity of these novel phosphonic ester PNAs (pePNA) molecules in medaka (Oryzias latipes) embryos, high solubility and selective binding to mRNA was observed. In particular, mixing of the novel components with aegPNA components resulted in mixed PNAs with superior properties. Injection of mixed PNAs directed against the medaka six3 gene, which is important for eye and brain development, resulted in specific six3 phenotypes.ConclusionsPNAs are well established as powerful antisense molecules. Modification of the backbone with phosphonic ester side chains further improves their properties and allows the efficient knock down of a single gene in fish embryos.

Coordination-Driven Inversion of Handedness in Ligand-Modified PNA

Peptide nucleic acid (PNA) is a synthetic analogue of DNA, which has the same nucleobases as DNA but typically has a backbone based on aminoethyl glycine (Aeg). PNA forms duplexes by Watson Crick hybridization. The Aeg-based PNA duplexes adopt a chiral helical structure but do not have a preferred handedness because they do not contain a chiral center. An L-lysine situated at the C-end of one or both strands of a PNA duplex causes the duplex to preferably adopt a left-handed structure. We have introduced into the PNA duplexes both a C-terminal L-lysine and one or two PNA monomers that have a γ-(S)-methyl-aminoethyl glycine backbone, which is known to induce a preference for a right-handed structure. Indeed, we found that in these duplexes the γ-methyl monomer exerts the dominant chiral induction effect causing the duplexes to adopt a right-handed structure. The chiral PNA monomer had a 2,2':6',2''-terpyridine (Tpy) ligand instead of a nucleobase and PNA duplexes that contained one or two Tpys formed [Cu(Tpy)(2)](2+) complexes in the presence of Cu(2+). The CD spectroscopy studies showed that these metal-coordinated duplexes were right-handed due to the chiral induction effect exerted by the S-Tpy PNA monomer(s) except for the cases when the [Cu(Tpy)(2)](2+) complex was formed with Tpy ligands from two different PNA duplexes. In the latter case, the metal complex bridged the two PNA duplexes and the duplexes were left-handed. The results of this study show that the preferred handedness of a ligand-modified PNA can be switched as a consequence of metal coordination to the ligand. This finding could be used as a tool in the design of functional nucleic-acid based nanostructures.

Unique Properties of Purine/Pyrimidine Asymmetric PNA·DNA Duplexes: Differential Stabilization of PNA·DNA Duplexes by Purines in the PNA Strand

PNA·DNA duplexes are significantly stabilized by purine nucleobases in the PNA strand. To elucidate and understand the effect of switching the backbone in a nucleic acid duplex, we now report a thermodynamics study along with a solution conformations study of two purine/pyrimidine strand asymmetric duplexes and a strand symmetrical control by comparing the behavior of all four possible PNA/DNA combinations. In essence, we are comparing an identical basepair stack connected by either an aminoethyl glycine PNA or a deoxyribose DNA backbone. We show that the PNA·DNA duplexes containing purine-rich PNA strands are stabilized with regard to the thermal melting temperature and free energy as well as enthalpy (and concomitantly relatively less entropically disfavored). Based on our data, we find it unlikely that differences in counterion binding (identical ionic-strength dependence was observed), hydration (identical and insignificant water release was observed), or single-strand conformation can be responsible for the difference in duplex stability. The only consistent difference observed between the purine-rich PNA versus the pyrimidine-rich PNA in isosequential PNA·DNA duplexes is the significant increase in both binding enthalpy and entropy for the PNA·DNA duplexes containing pyrimidine-rich PNA in organic solvent, which would indicate that these duplexes are relatively enthalpically disfavored in water. Although our results so far do not allow us to identify the origin of the different stabilities of homopurine/homopyrimidine PNA·DNA duplexes, the evidence does point to a significant structural component, which involves enthalpic contributions both within the duplex structure and also from bound water molecules.

Prediction of retention times of peptide nucleic acids during reversed-phase high-performance liquid chromatography

Peptide nucleic acids (PNAs) are synthetic biopolymers consisting of nucleobase side chains attached to an amino ethyl glycine backbone. At present this family of compounds enjoys a well deserved popularity in biomedical research, due to a number of favorable biological and chemical properties of PNAs compared to conventional synthetic oligonucleotides. PNAs are basically peptides, and are synthesized, purified and analyzed by traditional peptide chemistry, chromatography and mass spectrometry techniques. In the current report, we analyzed factors that influence the elution behavior of 29 PNAs on reversed-phase high-performance liquid chromatography using a water–acetonitrile–trifluoroacetic acid gradient elution system on C 18 columns. We found that increasing the temperature from 25°C to 55°C resulted in improved peak shape and resolution. The retention times of the PNA analogs were dependent upon the length of the polymers with longer PNAs eluting later. Mixtures of PNAs with varying length, originating from inefficient monomer couplings during the polymer assembly, could be separated by single chromatographic runs. The retention time also depended upon the cytosine, thymine, adenine and guanine content of the polymers. These differences in the contribution to the retention times could be explained by simple hydrophobicity features of the monomer side chains at pH 1.8. Based on all data, a linear equation was generated which predicted the retention time of any synthetic PNA based on composition and length. Comparison of the predicted and observed retention times showed a remarkable reliability of the algorithm.

Peptide Nucleic Acids with a Conformationally Constrained Chiral Cyclohexyl‐Derived Backbone

AbstractPeptide nucleic acid (PNA) is an achiral nucleic acid mimic with a backbone consisting of partly flexible aminoethyl glycine units. By replacing the aminoethyl portion of the backbone by an amino cyclohexyl moiety, either in the (S, S) or the (R, R) configuration, we have synthesized conformationally constrained PNA residues. PNA oligomers containing (S, S)‐cyclohexyl residues were able to form hybrid complexes with DNA or RNA, with little effect on the thermal stability (Tm = 1°C per (S, S) unit, depending on their number and the sequence). In contrast, incorporation of the (R, R) isomer resulted in a drastic decrease in the stability of the PNA‐DNA (or RNA) complex (Tm = −8°C per (R, R) unit). In PNA‐PNA duplexes, however, the (R, R)‐ and (S, S)‐cyclohexyl residues only exerted a minor effect on the stability, and the complexes formed with the two isomers are of opposite handedness, as evidenced from circular dichroism spectroscopy. In some cases the introduction of a single (S, S) residue in a PNA 15‐mer improves its sequence specificity for DNA or RNA. From the thermal stabilities and molecular modeling based on the solution structure of a PNA‐DNA duplex determined by NMR techniques, we conclude that the right‐handed helix can accommodate the (S, S) isomer more easily than the (R, R) isomer. Thermodynamic measurements of H and S upon PNA‐DNA duplex formation show that the introduction of an (S, S)‐cyclohexyl unit in the PNA does indeed decrease the entropy loss, indicating a more conformationally constrained structure. However, the more favorable entropic contribution is balanced by a reduced enthalpic gain, indicating that the structure constrained by the cyclohexyl group is not so well suited for DNA hybridization.

Peptide nucleic acid (PNA): A lead for gene therapeutic drugs

PNA (peptide nucleic acid) is a DNA mimic with a pseudopeptide backbone consisting of aminoethyl glycine units, to which the nucleobases are attached via methylene carbonyl linkers. Excellent RNA and DNA hybridization properties and biological stability, combined with convenient solid phase peptide synthesis, have made PNA a highly interesting lead for gene pharmaceutical drugs. The chemical and biochemical/biological properties in relation to the drug potential of PNA are reviewed.