Concentration effects on the self-assembly of tyrosine molecules.

Molecular self-assembly is a ubiquitous phenomenon in which individual atoms or molecules set up an ordered structure. It is of high interest for understanding the biology and a variety of diseases at the molecular level. In this work, we studied the self-assembly of tyrosine molecules via extensive molecular dynamics simulations. The formation of structures by self-assembly was systematically studied at various concentrations, from very low to very high. The temperature was kept constant, at which, in our former studies, we have already observed well-formed self-assembled structures. Depending on the concentration, the system displays a wide range of different structures, ranging from freely scattered monomers to very well formed four-fold structures. Different regimes of concentration dependence are observed. The results are proved by calculating the moments of inertia of the structures and the number of hydrogen bonds formed. Free energy landscapes calculated for the number of hydrogen bonds versus the number of contacts within a criterion provide insights into the structures observed.

Synthesis, structural, pharmacological and molecular docking simulations studies of Schiff base transition metal complexes procured from acetylacetonyl-4-iminoantipyrine and tyrosine.

Schiff base complexes of Cu(II), Ni(II), Co(II), VO(II) and Zn(II) ions have been synthesized by condensation of acetylaceto-4-iminoantipyrine and tyrosine. The structural characterization of compounds has been investigated using elemental analysis, molar conductance and magnetic susceptibility measurements, UV-Vis., FT-IR, ESI-mass, 1H-NMR, 13C-NMR, ESR spectroscopy, cyclic voltammetry, XRD and SEM techniques. The observed analytical data indicate that the metal chelates have the general formula of [ML] type. Powder XRD pattern of Schiff base and its complexes authenticate their crystalline nature. From SEM morphology studies, the reduction grain size of Schiff base in metal chelates was observed which is due to the chelation of the Schiff base. From the IR, electronic absorption spectra and magnetic measurement data, square planar geometry was proposed for the metal complexes except for the [VOL] complex which exhibits square pyramidal geometry. The X-band ESR spectra of [CuL] and [VOL] complexes in DMSO solution were recorded at 77 & 300 K and their spin Hamiltonian parameter values support the proposed geometry. The interactions of the [CuL] complex with CT-DNA were investigated by UV-Vis and absorption titrations. DNA binding interaction studies reveal the hydrophobic interaction between CT-DNA and complexes. Furthermore, analgesic, anti-inflammatory, CNS and antimicrobial studies of the Schiff base and its transition metal complexes were examined and resolved metal complexes have enhanced pharmacological and biological activity when compared to free Schiff base. Molecular docking studies of the complex with DNA and (PDB ID: 6COX) protein were investigated.

Synthesis of Isotopically Labeled, Spin-Isolated Tyrosine and Phenylalanine for Protein NMR Applications.

Isotopically labeled amino acids are widely used to study the structure and dynamics of proteins by NMR. Herein we describe a facile, gram-scale synthesis of compounds 1b and 2b under standard laboratory conditions from the common intermediate 7. 2b is obtained via simple deprotection, while 1b is accessed through a reductive deoxygenation/deuteration sequence and deprotection. 1b and 2b provide improved signal intensity using lower amounts of labeled precursor and are alternatives to existing labeling approaches.

Enhancement of the Antioxidant and Antimicrobial Activities of Porphyran through Chemical Modification with Tyrosine Derivatives.

The chemical modification of porphyran hydrocolloid is attempted, with the objective of enhancing its antioxidant and antimicrobial activities. Sulfated galactan porphyran is obtained from commercial samples of the red algae Porphyra dioica using Soxhlet extraction with water at 100 °C and precipitation with isopropyl alcohol. The extracted porphyran is then treated with modified L-tyrosines in aqueous medium in the presence of NaOH, at ca. 70 °C. The modified tyrosines L1 and L2 are prepared through a Mannich reaction with either thymol or 2,4-di-tert-butylphenol, respectively. While the reaction with 2,4-di-tert-butylphenol yields the expected tyrosine derivative, a mixture of products is obtained with thymol. The resulting polysaccharides are structurally characterized and the respective antioxidant and antimicrobial activities are determined. Porphyran treated with the N-(2-hydroxy-3,5-di-tert-butyl-benzyl)-L-tyrosine derivative, POR-L2, presents a noticeable superior radical scavenging and antioxidant activity compared to native porphyran, POR. Furthermore, it exhibited some antimicrobial activity against S. aureus. The surface morphology of films prepared by casting with native and modified porphyrans is studied by SEM/EDS. Both POR and POR-L2 present potential applicability in the production of films and washable coatings for food packaging with improved protecting characteristics.

Synthesis, antioxidant activity and bioinformatics studies of L-3-hydroxytyrosine templated N-alkyl/aryl substituted urea/thioureas.

A new series of urea/thiourea derivatives have been efficiently synthesized from the reaction of L-3-hydroxytyrosine with selective isocyanates/isothiocyanates and characterized by Infra-red, proton & carbon-13 nuclear magnetic resonance spectral and mass spectrometry studies. All the synthesized compounds have been screened for their antioxidant activity by 1,1-diphenyl1-2-picrylhydrazyl radical assay, ferric reducing antioxidant power assay and also studied their molecular docking interaction profiles against 1N8Q and 3NRZ enzymatic proteins. The in vitro antioxidant activity has further supported by quantitative structure activity relationship, absorption, distribution, metabolism, and excretion & toxicity studies, bioactivity studies & enzyme inhibition assay and identified that they were potentially bound to ASP490 & ASP361 aminoacid residue in chain A of 1N8Q protein and GLN1194 aminoacid residue in chain L of 3NRZ protein and are responsible for potential antioxidant activity. It is proved that urea derivatives linked with 4-fluoro & 4-nitro and thiourea derivatives linked with 3-chloro & 4-fluoro have exhibited promising antioxidant activity. In eventual synthesized compounds have been identified as potential blood-brain barrier penetrable compounds and proficient central nervous system active neuro-protective antioxidant agents as they have envisaged as easily penetrable to blood-brain barrier thresholds, a neuroprotective property.

Synthesis and antibacterial activities of marine natural product ianthelliformisamines and subereamine synthetic analogues.

Marine sponges of the genusSuberea produce variety of brominated tyrosine alkaloids which display diverse range of biological activities including antiproliferative, antimicrobial and antimalarial activities. In continuation of our search for biologically active marine natural products for antibacterial compounds, we report here the synthesis and evaluation of biological activity of panel of ianthelliformisamines and subereamine analogues using the literature known acid-amine coupling reaction. Several derivatives of Ianthelliformisamine were achieved by the coupling of Boc-protected polyamine chain with brominated aromatic acrylic acid derivatives by varying the bromine substituents on aromatic acid derivatives, amine spacer as well as geometry of the double bond, and then Boc-deprotection using TFA. Similarly, subereamine analogues were also synthesized employing coupling reaction between various brominated phenyl acrylic acids with commercially available chiral amino ester derivatives followed by ester hydrolysis. We screened these synthetic analogues for antibacterial activity against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) strains. One of the compound 7c showed bactericidal activity against Staphylococcus aureus with an IC50 value of 3.8 µM (MIC = 25 µM).

Sulfotyrosine-Mediated Recognition of Human Thrombin by a Tsetse Fly Anticoagulant Mimics Physiological Substrates.

Despite possessing only 32 residues, the tsetse thrombin inhibitor (TTI) is among the most potent anticoagulants described, with sub-picomolar inhibitory activity against thrombin. Unexpectedly, TTI isolated from the fly is 2000-fold more active and 180 Da heavier than synthetic and recombinant variants. We predicted the presence of a tyrosine O-sulfate post-translational modification of TTI, prompting us to investigate the effect of the modification on anticoagulant activity. A combination of chemical synthesis and functional assays was used to reveal that sulfation significantly improved the inhibitory activity of TTI against thrombin. Using X-ray crystallography, we show that the N-terminal sulfated segment of TTI binds the basic exosite II of thrombin, establishing interactions similar to those of physiologic substrates, while the C-terminal segment abolishes the catalytic activity of thrombin. This non-canonical mode of inhibition, coupled with its potency and small size, makes TTI an attractive scaffold for the design of novel antithrombotics.

Nickel(II) complexes based on L-amino-acid-derived ligands: synthesis, characterization and study of the role of the supramolecular structure in carbon dioxide capture.

The formation of the symmetrical µ3-carbonate-bridged self-assembled trinuclear NiII complex Na2{[Ni(LO)2(H2O)]3(µ3-CO3)} (LO is the carboxylate anion of a L-tyrosine derivative), involves atmospheric CO2 uptake. The asymmetric unit of the complex comprises an octahedral coordination for the NiII with two L-tyrosine-based ligands, a water molecule and one O atom of the carbonate bridge. The Ni3-µ3-CO3 core in this compound is the first reported of this kind according to the Cambridge Structural Database (CSD). The supramolecular structure is mainly sustained by hydrogen bonds developed by the phenolic functionality of the L-tyrosine moiety of one ligand and the carboxylate group of a neighbouring ligand. The crystal packing is then characterized by three interpenetrated supramolecular helices associated with a diastereoisomer of the type R-supP, which is essential for the assembly process. Magnetic susceptibility and magnetization data support weak ferromagnetic exchange interactions within the novel Ni3-µ3-CO3 core. The NiII complex obtained under the same synthetic conditions but using the analogous ligand derived from the amino acid L-phenylalanine instead of L-tyrosine gives rise to to a mononuclear octahedral system. The results obtained for the different complexes demonstrate the role of the supramolecular structure regarding the CO2 uptake property for these NiII-amino-acid-based systems.

Synthesis of a Unique Psammaplysin F Library and Functional Evaluation in Prostate Cancer Cells by Multiparametric Quantitative Single Cell Imaging.

The spirooxepinisoxazoline alkaloid psammaplysin F (1) was selected as a scaffold for the generation of a unique screening library for both drug discovery and chemical biology research. Large-scale extraction and isolation chemistry was performed on a marine sponge (Hyattella sp.) collected from the Great Barrier Reef in order to acquire >200 mg of the desired bromotyrosine-derived alkaloidal scaffold. Parallel solution-phase semisynthesis was employed to generate a series of psammaplysin-based urea (2-9) and amide analogues (10-11) in low to moderate yields. The chemical structures of all analogues were characterized using NMR and MS data. The absolute configuration of psammaplysin F and all semisynthetic analogues was determined as 6R, 7R by comparison of ECD data with literature values. All compounds (1-11) were evaluated for their effect on cell cycle distribution and changes to cancer metabolism in LNCaP prostate cancer cells using a multiparametric quantitative single-cell imaging approach. These investigations identified that in LNCaP cells psammaplysin F and some urea analogues caused loss of mitochondrial membrane potential, fragmentation of the mitochondrial tubular network, chromosome misalignment, and cell cycle arrest in mitosis.

Design, synthesis and biological evaluation of tyrosine derivatives as Mcl-1 inhibitors.

The upregulation of the protein myeloid cell leukemia-1 (Mcl-1) is closely associated with various human cancers, which can result in the evasion of apoptosis and a low survival rate. Therefore, developing Mcl-1 inhibitors has become a promising paradigm for cancer therapy. Herein, we designed and synthesized a novel series of tyrosine derivatives, among which compounds 5g, 6l and 6c exhibited very high binding affinity to Mcl-1 with Ki values of 0.18, 0.27 and 0.23 µM, respectively. Interestingly, compound 6l showed not only potent activity against Mcl-1 but also considerable selectivity over Bcl-2 and Bcl-xL, which was rationalized by molecular docking and fragment-centric topographical mapping (FCTM). It is worth noting that compounds 5g, 6l and 6c displayed potent antiproliferative activity against several cancer cell lines and could induce apoptosis of KM3 and HepG2 cells in a dose-dependent manner.

Copper, dityrosine cross-links and amyloid-ß aggregation.

Copper is involved in Alzheimer's disease (AD) where it appears to affect the aggregation of amyloid-ß (Aß) and to catalyze the production of reactive oxygen species (ROS). Oxidative stress apparently produces Aß dimers that are covalently linked through two tyrosine residues. Such dityrosine cross-links are considered as potential markers of the disease and seem to be implicated in the pathological disorder. In the present study, pure o,o'-dityrosine (diY) was prepared enzymatically (with horseradish peroxidase; HRP), which was subsequently used to construct calibration lines aimed at quantifying nanomolar amounts of diY in reaction mixtures by fluorescence spectroscopy. Hence, diY concentrations down to 67 nM could be determined, which allowed to find that ca. 3% of dityrosine-bridged dimers of Aß(1-40) were produced after 3 days at 37 °C in the presence of copper and dihydrogen peroxide. These cross-linked dimers in the presence of copper(II) ions completely inhibit the typical aggregation of Aß, since ß sheets could not be detected applying the usual Thioflavin T (ThT) method. Furthermore, the use of a potent Cu(II) chelator, such as the ATCUN tripeptide, L-histidyl-L-alanyl-L-histidine (HAH), efficiently prevented the copper-mediated generation of ROS and the associated dityrosine-bridged Aß dimers, suggesting that such metal chelators may find future applications in the field of anti-AD drug design.

Facile fully automated radiosynthesis and quality control of O-(2-[18F]fluoroethyl)-l-tyrosine ([18F]FET) for human brain tumor imaging.

O-(2-[18F]Fluoroethyl)-l-tyrosine ([18F]FET) has become one of the most successful amino acid tracers for human brain tumor imaging with positron emission tomography (PET). Facile fully automated radiosynthesis and quality control (QC) of [18F]FET using our home-built automated multi-purpose 18F-radiosynthesis module are described. [18F]FET was produced in 75-80 min overall synthesis time with 20-25% radiochemical yield decay corrected to end of bombardment (EOB), based on H[18F]F. The radiochemical and enantiomeric purities were >99%, and the molar activity (Am) was 189-411 GBq/µmol at EOB. The [18F]FET dose meets all QC criteria for clinical use, and is suitable for clinical PET study of brain tumor.

An improved automated one-pot synthesis of O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET) based on a purification by cartridges.

INTRODUCTION: O-(2-[18F]Fluoroethyl)-L-tyrosine ([18F]FET) is an established radiotracer used for oncology investigations by Positron Emission Tomography (PET). Main limitations to its widespread use are the synthesis itself (time; cost; radiochemical yield; complexity) and a troublesome and time-consuming HPLC purification. Aim of this work was to improve the preparation overall efficiency and, most important, to achieve an efficient and reliable purification by means of disposable cartridges. METHODS: [18F]FET was synthesized by direct nucleophilic radiofluorination of O-(2-tosyloxy-ethyl)-N-trityl-L-tyrosine t-butylester (TET) followed by acid hydrolysis with HCl. Several conditions and materials were tested for the synthesis and purification step. For the latter, a number of different commercial cartridges, varying in amount, particulate size and adsorbent, were examined. Best results were obtained by a combination of STRATA-X, tC18 and QMA cartridges. RESULTS: Starting from only 5 mg of TET, up to 11 GBq of injectable solutions of [18F]FET were produced within 36 min with 54-65% radiochemical yields and radiochemical purities >99%. No D-form was observed by chiral HPLC. Chemical purity was 1-2 order of magnitude below the limits imposed by the European Pharmacopoeia's monograph on [18F]FET. A radiochemical purity decrease by radiolysis, observed only on relatively large batches of [18F]FET, was efficiently suppressed by preloading in the receiving final vial a small amount of ethanol (<2% v/v). CONCLUSIONS: By combining improvements to a known synthetic route with a novel cartridge-based purification, [18F]FET was obtained in a very efficient and reproducible way. The whole process was easily implemented on a commercial automated module presently used for [18F]FDG production. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: A few drawbacks regarding the HPLC conditions recommended in the European Pharmacopoeia were highlighted. An alternative method able to cope with them is herein proposed The simplified preparation herein described is expected to encourage a more widespread clinical use of [18F]FET.

Nitration of Tyrosine Channels Photoenergy through a Conical Intersection in Water.

Nitration of tyrosine occurs under oxidative stress in vivo. The product, 3-nitrotyrosine (3NY), has a dramatically decreased quantum yield and can be used as a molecular ruler. In this study, femtosecond transient absorption spectroscopy and quantum calculations were implemented to elucidate the photoinduced relaxation processes of anionic 3NY in water. Upon 400 nm excitation into an excited electronic state with notable charge-transfer (CT) character, a barrierless nitro-twisting motion rapidly (<100 fs) guides the chromophore into an adjacent twisted intramolecular CT state, therein reaching a sloped S1/S0 conical intersection on the ∼100 fs time scale. Once in the hot ground state, excess energy is further released through vibrational cooling with biexponential time constants of ∼140 and 680 fs in water. Nitro back-twisting occurs on longer time scales (∼1.1 and 9 ps in water), returning the system to original ground state. Systematic evaluations of excited-state potential energies of anionic 3NY were performed by density functional theory (DFT) and time-dependent DFT calculations, showing that intersystem crossing (ISC) from the first singlet state (S1) to the first or second triplet state (T1 or T2) is unlikely. Inclusion of an explicit water molecule in calculations leads to improved mapping of the excited-state energy ordering of the second singlet state (S2) and T2, further diminishing ISC probability from S1 and favoring an ultrafast internal conversion to S0. These results provide deep insights into the highly efficient nonradiative decay of anionic 3NY in aqueous solution, with nitro-site-specific information that can help infer the characterization and potential optogenetic control of 3NY in protein environment.

Efficient Building Blocks for Solid-Phase Peptide Synthesis of Spin Labeled Peptides for Electron Paramagnetic Resonance and Dynamic Nuclear Polarization Applications.

Specific spin labeling allows the site-selective investigation of biomolecules by EPR and DNP enhanced NMR spectroscopy. A novel spin labeling strategy for commercially available Fmoc-amino acids is developed. In this approach, the PROXYL spin label is covalently attached to the hydroxyl side chain of three amino acids hydroxyproline (Hyp), serine (Ser) and tyrosine (Tyr) by a simple three-step synthesis route. The obtained PROXYL containing building-blocks are N-terminally protected by the Fmoc-protection group, which makes them applicable for the use in solid-phase peptide synthesis (SPPS). This approach allows the insertion of the spin label at any desired position during SPPS, which makes it more versatile than the widely used post synthetic spin labeling strategies. For the final building-blocks, the radical activity is proven by EPR. DNP enhanced solid-state NMR experiments employing these building-blocks in a TCE solution show enhancement factors of up to 26 for 1 H and 13 C (1 H→13 C cross-polarization). To proof the viability of the presented building-blocks for insertion of the spin label during SPPS the penta-peptide Acetyl-Gly-Ser(PROXYL)-Gly-Gly-Gly was synthesized employing the spin labeled Ser building-block. This peptide could successfully be isolated and the spin label activity proved by EPR and DNP NMR measurements, showing enhancement factors of 12.1±0.1 for 1 H and 13.9±0.5 for 13 C (direct polarization).

Indole-3-glyoxyl tyrosine: synthesis and antimalarial activity against Plasmodium falciparum.

AIM: More than 40% of the world's population, across 105 countries, live in malaria endemic areas. It is estimated that about 500 million cases of malaria and half a million deaths occur per year. RESULTS: Herein, we demonstrate the biological activity of indole-3-glyoxyl tyrosine against Plasmodium falciparum, which is the causal agent of the most virulent form of malaria in humans. We developed an efficient synthesis of indole-3-glyoxyl tyrosine derivatives, which were then used as key intermediates in the synthesis of functionalized indole-3-glyoxyl biphenyl tyrosines. CONCLUSION: In biological testing, the compounds exhibited a parasite growth inhibition of over 85%. A cell viability assay showed low cytotoxicity against human cells, with no significant changes in cell viability, making these compounds potential antimalarials.

Synthesis and Antiproliferative Activity of Marine Bromotyrosine Purpurealidin I and Its Derivatives.

The first total synthesis of the marine bromotyrosine purpurealidin I (1) using trifluoroacetoxy protection group and its dimethylated analog (29) is reported along with 16 simplified bromotyrosine derivatives lacking the tyramine moiety. Their cytotoxicity was evaluated against the human malignant melanoma cell line (A-375) and normal skin fibroblast cells (Hs27) together with 33 purpurealidin-inspired simplified amides, and the structure⁻activity relationships were investigated. The synthesized simplified analogs without the tyramine part retained the cytotoxic activity. Purpurealidin I (1) showed no selectivity but its simplified pyridin-2-yl derivative (36) had the best improvement in selectivity (Selectivity index 4.1). This shows that the marine bromotyrosines are promising scaffolds for developing cytotoxic agents and the full understanding of the elements of their SAR and improving the selectivity requires further optimization of simplified bromotyrosine derivatives.

Proteomic Identification of Protein Tyrosine Phosphatase and Substrate Interactions in Living Mammalian Cells by Genetic Encoding of Irreversible Enzyme Inhibitors.

Protein tyrosine phosphatases (PTPs) play critical roles in cell signaling pathways, but identification of unknown PTPs for a given substrate in live cells remain technically challenging. Here, we synthesized a series of tyrosine-based irreversible PTP inhibitors and characterized by site-specific encoding on substrate proteins in cells with an expanded genetic code. By fine-tuning the chemical reactivity, we identified optimal active amino acid probes to covalently cross-link a PTP and its substrate both in vitro and in mammalian cells. Using HER2 as an example, we provide first direct evidence of HER2 Y1023 and SHP2 cross-linking in situ in living human cells. Moreover, proteomic analysis using our approach identified PTP1B as a novel phosphatase for HER2 that specifically dephosphorylated pY1221 position, which may shed light on the puzzle of PTP1B's role in HER2 positive breast cancer. This novel method provides a useful tool for dissecting tyrosine phosphoregulation in living cells.

Site-Specific Incorporation of a Cu2+ Spin Label into Proteins for Measuring Distances by Pulsed Dipolar Electron Spin Resonance Spectroscopy.

Pulsed dipolar electron spin resonance spectroscopy (PDS) is a powerful tool for measuring distances in solution-state macromolecules. Paramagnetic metal ions, such as Cu2+, are used as spin probes because they can report on metalloprotein features and can be spectroscopically distinguished from traditional nitroxide (NO)-based labels. Here, we demonstrate site-specific incorporation of Cu2+ into non-metalloproteins through the use of a genetically encodable non-natural amino acid, 3-pyrazolyltyrosine (PyTyr). We first incorporate PyTyr in cyan fluorescent protein to measure Cu2+-to-NO distances and examine the effects of solvent conditions on Cu2+ binding and protein aggregation. We then apply the method to characterize the complex formed by the histidine kinase CheA and its target response regulator CheY. The X-ray structure of CheY-PyTyr confirms Cu labeling at PyTyr but also reveals a secondary Cu site. Cu2+-to-NO and Cu2+-to-Cu2+ PDS measurements of CheY-PyTyr with nitroxide-labeled CheA provide new insights into the conformational landscape of the phosphotransfer complex and have implications for kinase regulation.