In Situ Stable Generation of Reactive Intermediates by Open Microfluidic Probe for Subcellular Free Radical Attack and Membrane Labeling.

Subcellular stimulation by free radicals is crucial for deeper insight of cell behaviors. However, it remains a tough challenge due to the high spatial precision requirement and short life of radicals. Herein, we report a versatile open microfluidic probe for stable generation of free radical and subcellular stimulation. By optimizing parameters, the chemical reaction can be confined in a microregion with a diameter of several µm, and the real-time produced reactive radicals can attack the desired subcellular region of a single cell. In order to reveal the attacked region, fluorescent cyanine 3 labeled tyramide free radicals are synthesized, and the target microregion on a single cell is successfully stained by the covalent linking reaction between radicals and membrane proteins, which proves the feasibility of our method. We believe this method will open new avenues for short-lived reactive intermediates stimulation at the single-cell/sub-cell level and selective membrane labeling.

Design of a Multiuse Photoreactor To Enable Visible-Light Photocatalytic Chemical Transformations and Labeling in Live Cells.

Despite the growing use of visible-light photochemistry in both chemistry and biology, no general low-heat photoreactor for use across these different disciplines exists. Herein, we describe the design and use of a standardized photoreactor for visible-light-driven activation and photocatalytic chemical transformations. Using this single benchtop photoreactor, we performed photoredox reactions across multiple visible light wavelengths, a high-throughput photocatalytic cross-coupling reaction, and in vitro labeling of proteins and live cells. Given the success of this reactor in all tested applications, we envision that this multi-use photoreactor will be widely used in biology, chemical biology, and medicinal chemistry settings.

Extracellular matrix-mimetic composite hydrogels of cross-linked hyaluronan and fibrillar collagen with tunable properties and ultrastructure.

A platform of enzymatically-crosslinked Collagen/Tyramine hyaluronan derivative (Col/HA-Tyr) hydrogels with tunable compositions and gelation conditions was developed to evaluate the impact of the preparation conditions on their physical, chemical and biological properties. At low HA-Tyr content, hydrogels exhibited a fibrillar structure, with lower mechanical properties compared to pure Col hydrogels. At high HA-Tyr and Horse Radish Peroxydase (HRP) content, a microfibrillar network was formed beside the banded Col fibrils and a synergistic effect of the hybrid structure on mechanical properties was observed. These hydrogels were highly resistant against enzymatic degradation while keeping a high degree of hydration. Unlike HA-Tyr hydrogels, encapsulation of human dermal fibroblasts within Col/HA-Tyr hydrogels allowed for high cell viability. These results showed that high HA-Tyr and HRP concentrations are required to positively impact the physical properties of hydrogels while preserving collagen fibrils. Those Col/HA-Tyr hydrogels appear promising for novel tissue engineering applications following a biomimetic approach.

Tyramine Derivatives as Potent Therapeutics for Type 2 Diabetes: Synthesis and In Vitro Inhibition of α-Glucosidase Enzyme.

BACKGROUND: Tyramine derivatives 3-16 were prepared and tested first time for their α- glucosidase (Sources: Saccharomyces cerevisiae) inhibitory activity by using an in vitro mechanismbased biochemical assay. All the compounds were found to be new, except compounds 3, 10-12 and 16. OBJECTIVE: In continuation of our research to synthesize and identify potent inhibitors of α-glucosidase enzyme, we intended to synthesize new inhibitors of α-glucosidase enzyme with enhanced efficacy in order to provide the basis for the better treatment of the type-II diabetic. METHODS: Tyramine (1) was allowed to react with a variety of aryl chlorides (2) to yield the corresponding amides. Synthesized compounds were then purified through normal phase column chromatography. Compounds 3-16 were then assessed for their α-glucosidase inhibitory activity in an in vitro biochemical assay. The cytotoxicity of compounds 3-16 was determined by using 3T3 mouse fibroblast cell lines. RESULTS: Compounds 3-5, 8, 13, and 15-16 were found to be more active (IC50 = 103.1±0.46, 37.3±4.51, 56.7±4.2, 23.9±2.31, 43.6±2.88, 55.8±1.73, and 38.2±0.86 µM, respectively) than the acarbose, the standard inhibitor of α-glucosidase enzyme, (IC50= 840.0±1.73 µM). To determine the dissociation constants and mode of inhibition, the kinetic studies were also performed for compounds 4 and 8 (the most potent inhibitors). It was observed that compounds 4 and 8 possess noncompetitive properties as the inhibitors of α-glucosidase. All the compounds were found to be noncytotoxic, except 5 and 12 (IC50= 14.7± 0.24 and 6.6± 0.38 µM, respectively). CONCLUSION: The current study gives the facile synthesis and identification of potent inhibitors of α- glucosidase. The new inhibitors reported here may be investigated further for the designing and development of novel anti-diabetic agents.

Enzymatic syntheses of 3'-halotyramines, selectively 2H- and 3H-labeled in the side chain.

3'-Halotyramines, stereo-selectively labeled with deuterium and tritium, in the (1R) or (1S) position were synthesized using enzymatic methods. The isotopomers labeled in the (1R) position were synthesized by enzymatic decarboxylation of an appropriate 3'-halo-L-tyrosine in deuterated or tritiated incubation medium. The isotopomers labeled in the (1S) position were synthesized by decarboxylation of an appropriate 3'-halo-L-tyrosine, labeled with deuterium or tritium in the α-position of the side chain.

Solid-State Form Characterization of Riparin I.

Riparin I is an alkamide with potential anxiolytic activity in preclinical studies. The characterization and understanding of solid-state properties play an importance role in drug development. For this work, the solid state of five riparin I batches (RIP-1, RIP-2, RIP-3, RIP-4, and RIP-5), obtained by the same synthesis process, were characterized by Scanning Electron Microscopy (SEM), Differential Scanning Calorimetry (DSC), DSC-photovisual, Thermogravimetry (TG), Fourier Transform Infrared (FTIR), Pyrolysis (Pyr-GC/MS), X-ray Powder Diffraction (PXRD), and Solid-State Nuclear Magnetic Resonance (ssNMR) techniques. Batches of riparin I with different crystal habits resulting in crystallization impurities were observed, which can be attributed to the presence of triethylamine. The main differences were observed by DSC, PXRD, and ssNMR analysis. DSC curves of RIP-2 and RIP-3 presented endothermic peaks at different temperatures of fusion, which can be attributed to the mixture of different crystalline forms. PXRD and ssNMR results confirmed crystallinity differences. The results offer evidence of the importance of controlling the reproducibility of the synthesis in order to obtain the adequate morphology for therapeutic efficacy and avoiding future problems in quality control of riparin I products.

Ultrasonic synthesis of tyramine derivatives as novel inhibitors of α-glucosidase in vitro.

Tyramine derivatives 3-27 were synthesized by using conventional and environmental friendly ultrasonic techniques. These derivatives were then evaluated for the first time for their α-glucosidase (Sources: Saccharomyces cerevisiae and mammalian rat-intestinal acetone powder) inhibitory activity by using in vitro mechanism-based biochemical assays. Compounds 7, 14, 20, 21 and 26 were found to be more active (IC50 = 49.7 ± 0.4, 318.8 ± 3.7, 23.5 ± 0.9, 302.0 ± 7.3 and 230.7 ± 4.0 µM, respectively) than the standard drug, acarbose (IC50 = 840.0 ± 1.73 µM (observed) and 780 ± 0.028 µM (reported)) against α-glucosidase obtained from Saccharomyces cerevisiae. Kinetic studies were carried out on the most active members of the series in order to determine their mode of inhibition and dissociation constants. Compounds 7, 20 and 26 were found to be the competitive inhibitors of α-glucosidase. These compounds were also screened for their protein antiglycation, and dipeptidyl peptidase-IV (DPP-IV) inhibitory activities. Only compounds 20, 22 and 27 showed weak antiglycation activity with IC50 values 505.27 ± 5.95, 581.87 ± 5.50 and 440.58 ± 2.74 µM, respectively. All the compounds were found to be inactive against DDP-IV enzyme. Inhibition of α-glucosidase, DPP-IV enzymes and glycation of proteins are valid targets for the discovery of antidiabetic drugs. Cytotoxicity of compounds 3-27 was also evaluated by using mouse fibroblast 3T3 cell lines. All the compounds were found to be noncytotoxic. The current study describes the synthesis α-glucosidase inhibitory activity of derivatives, based on a natural product tyramine template. The compounds reported here may serve as the starting point for the design and development of novel α-glucosidase inhibitors as antidiabetic agents.

3-Chlorotyramine Acting as Ligand of the D2 Dopamine Receptor. Molecular Modeling, Synthesis and D2 Receptor Affinity.

We synthesized and tested 3-chlorotyramine as a ligand of the D2 dopamine receptor. This compound displayed a similar affinity by this receptor to that previously reported for dopamine. In order to understand further the experimental results we performed a molecular modeling study of 3-chlorotyramine and structurally related compounds. By combining molecular dynamics simulations with semiempirical (PM6), ab initio and density functional theory calculations, a simple and generally applicable procedure to evaluate the binding energies of these ligands interacting with the D2 dopamine receptors is reported here. These results provided a clear picture of the binding interactions of these compounds from both structural and energetic view points. A reduced model for the binding pocket was used. This approach allowed us to perform more accurate quantum mechanical calculations as well as to obtain a detailed electronic analysis using the Quantum Theory of Atoms in Molecules (QTAIM) technique. Molecular aspects of the binding interactions between ligands and the D2 dopamine receptor are discussed in detail. A good correlation between the relative binding energies obtained from theoretical calculations and experimental IC50 values was obtained. These results allowed us to predict that 3-chlorotyramine possesses a significant affinity by the D2 -DR. Our theoretical predictions were experimentally corroborated when we synthesized and tested 3-chlorotyramine which displayed a similar affinity by the D2 -DR to that reported for DA.

Synthesis of poly(glutamic acid)-tyramine hydrogel by enzyme-mediated gelation for controlled release of proteins.

An in situ-formed hydrogel was synthesized by enzymatic cross-linking of poly(γ-glutamic acid)-tyramine conjugates (PGA-Tyr) using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). The gelation time ranged from 25 s to 5 min was accomplished by tuning the concentration of HRP, H2O2/Tyr molar ratio and the degree of substitution (DS) of Tyr groups. The storage modulus (G'), cross-link density, and mesh size can be tailored by controlling the H2O2/Tyr ratio and DS. The rheological analysis indicated that the storage modulus (G') can be tailored from approximately 40 to over 1100 Pa with the increasing H2O2/Tyr ratio and DS. The bovine serum albumin (BSA) was used as model protein and encapsulated into the hydrogel during the enzyme-mediated cross-linking reaction. Controlled release of BSA in vitro from the PGA-Tyr hydrogel was obtained. The release rate and cumulative release amount of encapsulated BSA were manipulated by controlling the H2O2/Tyr ratio and DS. More than 90% of encapsulated BSA was released from the hydrogel with low cross-link density and lager mesh size in 60 h, while only 68% of BSA was released from the hydrogel with high cross-link density and small mesh size. The results indicated that the PGA-Tyr hydrogel is a promising material for the controlled release of therapeutic protein or peptides.

Synthesis, HPLC measurement and bioavailability of the phenolic amide amkamide.

Amkamide is a phenolic amide whose analogues were recently reported to have potent mitochondria protective activity, although their bioavailability is still unknown. Therefore, in this study, amkamide was synthesized and confirmed by nuclear magnetic resonance, and a high-performance liquid chromatography (HPLC) method was developed for analyzing the amides in biological samples. HPLC separation was performed on a Nova-Pak C18 column using a gradient condition and a coulometric electrochemical detector. The HPLC method was able to produce excellent and reproducible separations of amkamide from other amides (oretamide, becatamide, enferamide and veskamide). All five amides yielded outstanding peak resolutions with detection limits as low as 100 fmol. Therefore, the bioavailability of amkamide together with becatamide was determined in a mouse model. Their plasma concentrations were measured following two oral administrations (2 and 4 mg per 30 g body weight) using the HPLC method. As expected, relatively high amounts of amkamide and becatamide (51-58 µM) were detected at the time after administration when the maximum concentration was reached (approximately 20 and 25 min) at the high dose. The data indicate that HPLC methods with amide standards may be utilized to measure amkamide and its analogues in biological samples with excellent resolution and detection limits.

Sensitive whole-mount fluorescent in situ hybridization in zebrafish using enhanced tyramide signal amplification.

Whole-mount in situ hybridization is the preferred method for detecting transcript distributions in whole embryos, tissues, and organs. We present here a sensitive fluorescent in situ hybridization method for colocalization analysis of different transcripts in whole embryonic zebrafish brains. The method is based on simultaneous hybridization of differently hapten-labeled RNA probes followed by sequential rounds of horseradish peroxidase (POD)-based transcript detection. Sequential detection involves enhancement of fluorescent signals by tyramide signal amplification (TSA) and effective inactivation of the antibody-POD conjugate prior to the following detection round. We provide a detailed description of embryo preparation, hybridization, antibody detection, POD-TSA reaction, and mounting of embryos for imaging. To achieve high signal intensities, we optimized key steps of the method. This includes improvement of embryo permeability by hydrogen peroxide treatment and efficacy of hybridization and TSA-POD reaction by addition of the viscosity-increasing polymer dextran sulfate. The TSA-POD reaction conditions are further optimized by application of substituted phenol compounds as POD accelerators and use of highly efficient bench-made tyramide substrates. The obtained high signal intensities and cellular resolution of our method allows for co-expression analysis and generation of three-dimensional models. Our protocol is tailored to optimally work in zebrafish embryos, but can surely be modified for application in other species as well.

First total synthesis of dioxepine bastadin 3.

The synthesis of dioxepine bastadin 3, a tyrosine-tyramine derivative with a dibenzo-1,3-dioxepine scaffold that is rarely present among natural products, is described. The dibenzo-1,3-dioxepine ring was formed early in the sequence and the (E)-2-(hydroxyimino)-N-alkylamide was generated in the last step by oxidation of the 2-amino-N-alkylamide precursor. The presumably biogenetic late-stage ring formation starting from congener bastadin 3 failed. A new synthesis of this alkaloid was also developed. This new route requires a minimal use of protecting groups and the order of the two key steps was reversed relative to the route to dioxepine bastadin 3.

Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain.

BACKGROUND: In recent years, mapping of overlapping and abutting regulatory gene expression domains by chromogenic two-color in situ hybridization has helped define molecular subdivisions of the developing vertebrate brain and shed light on its basic organization. Despite the benefits of this technique, visualization of overlapping transcript distributions by differently colored precipitates remains difficult because of masking of lighter signals by darker color precipitates and lack of three-dimensional visualization properties. Fluorescent detection of transcript distributions may be able to solve these issues. However, despite the use of signal amplification systems for increasing sensitivity, fluorescent detection in whole-mounts suffers from rapid quenching of peroxidase (POD) activity compared to alkaline phosphatase chromogenic reactions. Thus, less strongly expressed genes cannot be efficiently detected. RESULTS: We developed an optimized procedure for fluorescent detection of transcript distribution in whole-mount zebrafish embryos using tyramide signal amplification (TSA). Conditions for hybridization and POD-TSA reaction were optimized by the application of the viscosity-increasing polymer dextran sulfate and the use of the substituted phenol compounds 4-iodophenol and vanillin as enhancers of POD activity. In combination with highly effective bench-made tyramide substrates, these improvements resulted in dramatically increased signal-to-noise ratios. The strongly enhanced signal intensities permitted fluorescent visualization of less abundant transcripts of tissue-specific regulatory genes. When performing multicolor fluorescent in situ hybridization (FISH) experiments, the highly sensitive POD reaction conditions required effective POD inactivation after each detection cycle by glycine-hydrochloric acid treatment. This optimized FISH procedure permitted the simultaneous fluorescent visualization of up to three unique transcripts in different colors in whole-mount zebrafish embryos. CONCLUSIONS: Development of a multicolor FISH procedure allowed the comparison of transcript gene expression domains in the embryonic zebrafish brain to a cellular level. Likewise, this method should be applicable for mRNA colocalization studies in any other tissues or organs. The key optimization steps of this method for use in zebrafish can easily be implemented in whole-mount FISH protocols of other organisms. Moreover, our improved reaction conditions may be beneficial in any application that relies on a TSA/POD-mediated detection system, such as immunocytochemical or immunohistochemical methods.

Protective effects of veskamide, enferamide, becatamide, and oretamide on H2O2-induced apoptosis of PC-12 cells.

Veskamide, enferamide, becatamide, and oretamide are phenolic amides whose analogues are found in plants. In this study, the four amides were prepared by chemical synthesis and their protective effects on H(2)O(2)-induced apoptosis in PC-12 cells were investigated. The syntheses were relatively simple and the yields were more than 43%. Using NMR spectroscopic methods, the chemical structures of veskamide, enferamide, becatamide, and oretamide were confirmed. The decreasing order of the protective effects on H(2)O(2)-induced apoptosis was becatamide>enferamide≥oretamide>veskamide. In fact, becatamide suppressed H(2)O(2)-induced mitochondrial membrane depolarization in a dose-dependent manner. At the concentration of 10 µM, becatamide maintained mitochondrial membrane depolarization at 16% compared to 51% in H(2)O(2)-treated PC-12 cells (P<0.05). Also, at the same concentration, becatamide inhibited H(2)O(2)-induced caspase-9 activation and caspase-independent chromatin condensation by 68% (P<0.05) and 73% (P<0.05), respectively. This is the first report about the chemical synthesis of becatamide and its potential biological activity to inhibit H(2)O(2)-induced apoptosis of PC-12 cells via protecting mitochondrial membrane integrity, thereby suppressing caspase-9 activation and chromatin condensation.

Injectable hyaluronic acid-tyramine hydrogels for the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease caused by inflammation of the synovial membrane, leading in turn to articular cartilage destruction. In this work, injectable tyramine modified hyaluronic acid (HA-Tyr) hydrogels were developed for the treatment of RA. HA-Tyr conjugate was synthesized by amide bond formation between carboxyl groups of HA and amine groups of tyramine. Then, HA-Tyr hydrogels were prepared by radical crosslinking reaction using H(2)O(2) and horse-radish peroxidase. Intra-articular injection of HA-Tyr hydrogels encapsulating dexamethasone (DMT) as a model drug resulted in successful treatment of RA with reduced interlukine-6, prostaglandin E2 and four types of cytokine levels in collagen-induced arthritis animal models. Histological analysis with hematoxylin and eosin (H&E) staining also confirmed the therapeutic effect of injectable HA-Tyr hydrogels with DMT. Taken together, the injectable HA-Tyr hydrogels were thought suitable to be developed as a therapeutically effective drug carrier for the treatment of RA.

Isolation, synthesis, and biological activity of aphrocallistin, an adenine-substituted bromotyramine metabolite from the Hexactinellida sponge Aphrocallistes beatrix.

A new adenine-substituted bromotyrosine-derived metabolite designated as aphrocallistin (1) has been isolated from the deep-water Hexactinellida sponge Aphrocallistes beatrix. Its structure was elucidated on the basis of spectral data and confirmed through a convergent, modular total synthetic route that is amenable toward future analogue preparation. Aphrocallistin inhibits the growth of a panel of human tumor cell lines with IC(50) values ranging from 7.5 to >100 microM and has been shown to induce G1 cell cycle arrest in the PANC-1 pancreatic carcinoma cell line. Aphrocallistin has been fully characterized in the NCI cancer cell line panel and has undergone in vitro ADME pharmacological profiling.

Synthesis, spectroscopic and structural elucidation of sympathomimetic amine, tyraminium dihydrogenphosphate.

The synthesis, isolation, spectroscopic and structural elucidation of sympathomimetic amine, tyramine dihydrogenphosphate are of interest due to its biological activity and the establishing correlation between spectroscopic properties and structure. The complex approach for investigation included single crystal X-ray diffraction, new technique in linear-polarized IR-spectroscopy in solid state and quantum chemical calculations with a view to predict the electronic structure and vibrational data of interacting species in entitled compound, the correlation structure-spectroscopic properties as well as the influence of intermolecular interaction on IR-characteristic bands are carried out.

Both ionically and enzymatically crosslinkable alginate-tyramine conjugate as materials for cell encapsulation.

The swelling behavior of the structural material of cell-enclosing capsules is a key factor for the successful transplantation of these capsules in the treatment of diseases. The present study aimed to develop cell-enclosing capsules displaying minimal swelling under physiological conditions. We investigated the use of an alginate-tyramine conjugate synthesized by a carbodiimide/active-ester coupling reaction. The conjugate gel crosslinked by calcium ions and peroxidase-catalyzed oxidative coupling of phenols swelled less in saline than in unmodified alginate. The degree of swelling could be controlled by conjugate preparation conditions. The conjugate gel showed no obvious cytotoxicity for cells, including the process of oxidative coupling generation. Further, encapsulated cells could be cultured for up to 2 months and achieve approximately 5.2-fold greater mitochondrial activity after 51 days of encapsulation than after 1 day. These results show that this alginate-tyramine conjugate is a promising material for use in cell-enclosing capsules for cell therapy.

Enzyme-mediated fast in situ formation of hydrogels from dextran-tyramine conjugates.

Dextran hydrogels were formed in situ by enzymatic crosslinking of dextran-tyramine conjugates and their mechanical, swelling and degradation properties were evaluated. Two types of dextran-tyramine conjugates (M(n,dextran)=14k, M(w)/M(n)=1.45), i.e. dextran-tyramine linked by a urethane bond (denoted as Dex-TA) or by an ester-containing diglycolic group (denoted as Dex-DG-TA), with different degrees of substitution (DS) were prepared. Hydrogels were rapidly formed under physiological conditions from Dex-TA DS 10 or 15 and Dex-DG-TA DS 10 at or above a concentration of 2.5 wt% in the presence of H(2)O(2) and horseradish peroxidase (HRP). The gelation time ranged from 5s to 9 min depending on the polymer concentration and HRP/TA and H(2)O(2)/TA ratios. Rheological analysis showed that these hydrogels are highly elastic. The storage modulus (G'), which varied from 3 to 41 kPa, increased with increasing polymer concentration, increasing HRP/TA ratio and decreasing H(2)O(2)/TA ratio. The swelling/degradation studies showed that under physiological conditions, Dex-TA hydrogels are rather stable with less than 25% loss of gel weight in 5 months, whereas Dex-DG-TA hydrogels are completely degraded within 4-10d. These results demonstrate that enzymatic crosslinking is an efficient way to obtain fast in situ formation of hydrogels. These dextran-based hydrogels are promising for use as injectable systems for biomedical applications including tissue engineering and protein delivery.