Unexpectedly Stable (Chlorocarbonyl)(N-ethoxycarbonylcarbamoyl)disulfane, and Related Compounds That Model the Zumach-Weiss-Kühle (ZWK) Reaction for Synthesis of 1,2,4-Dithiazolidine-3,5-diones.

The Zumach-Weiss-Kühle (ZWK) reaction provides 1,2,4-dithiazolidine-3,5-diones [dithiasuccinoyl (Dts)-amines] by the rapid reaction of O-ethyl thiocarbamates plus (chlorocarbonyl)sulfenyl chloride, with ethyl chloride and hydrogen chloride being formed as coproducts, and carbamoyl chlorides or isocyanates generated as yield-diminishing byproducts. However, when the ZWK reaction is applied with (N-ethoxythiocarbonyl)urethane as the starting material, heterocyclization to the putative "Dts-urethane" does not occur. Instead, the reaction directly provides (chlorocarbonyl)(N-ethoxycarbonylcarbamoyl)disulfane, a reasonably stable crystalline compound; modified conditions stop at the (chlorocarbonyl)[1-ethoxy-(N-ethoxycarbonyl)formimidoyl]disulfane intermediate. The title (chlorocarbonyl)(carbamoyl)disulfane cannot be converted to the elusive Dts derivative, but rather gives (N-ethoxycarbonyl)carbamoyl chloride upon thermolysis, or (N-ethoxycarbonyl)isocyanate upon treatment with tertiary amines. Additional transformations of these compounds have been discovered, providing entries to both known and novel species. X-ray crystallographic structures are reported for the title (chlorocarbonyl)(carbamoyl)disulfane; for (methoxycarbonyl)(N-ethoxycarbonylcarbamoyl)disulfane, which is the corresponding adduct after quenching in methanol; for [1-ethoxy-(N-ethoxycarbonyl)formimidoyl](N'-methyl-N'-phenylcarbamoyl)disulfane, which is obtained by trapping the title intermediate with N-methylaniline; and for (N-ethoxycarbonylcarbamoyl)(N'-methyl-N'-phenylcarbamoyl)disulfane, which is a short-lived intermediate in the reaction of the title (chlorocarbonyl)(carbamoyl)disulfane with excess N-methylaniline. The new chemistry and structural information reported herein is expected to contribute to accurate modeling of the ZWK reaction trajectory.

Near-IR single fluorophore quenching system based on phthalocyanine (Pc) aggregation and its application for monitoring inhibitor/activator action on a therapeutic target: L1-EN.

Controlled H-aggregation of single Pc-labeled oligonucleotides is utilized as a fluorescence quenching system to discern changes in enzyme activity for the discovery of inhibitors for Long Interspersed Element 1 endonuclease (L1-EN), which is involved in genome instability and implicated in many different diseases.

Reversibility of beta-amyloid self-assembly: effects of pH and added salts assessed by fluorescence photobleaching recovery.

The 40-residue peptide isoform beta-amyloid (Abeta(1-40)) is associated with Alzheimer's disease. Although found in the tangles and fibrous mats that characterize the brain in advanced stages of the disease, the toxic form of Abeta is believed to be oligomers or "protofibrils". Characterization of these fairly small structures in solution, especially in the presence of the much larger assemblies they also form, is a daunting task. Additionally, little is known about the rate of Abeta assembly or whether it can be triggered easily. Perhaps most importantly, the conditions for reversing assembly are not fully understood. Fluorescence photobleaching with modulation detection of the recovery profile is a sensitive and materials-efficient way to measure diffusers over a wide range of hydrodynamic sizes. The method does require attachment of a fluorescent label. Experiments to validate the use of 5-carboxyfluorescein-labeled Abeta(1-40) as a representative of the unlabeled, naturally occurring material included variation of photobleaching time and mixture of labeled and unlabeled materials. A dialysis cell facilitated rapid in situ changes in pH and salt conditions. Multiple steps and complex protocols can be explored with relative ease. Oligomeric aggregates were found by fluorescence photobleaching recovery to respond readily to pH and salt conditions. Changing these external cues leads to formation or disassembly of aggregates smaller than 100 nm within minutes.

Side chain and flexibility contributions to the Raman optical activity spectra of a model cyclic hexapeptide.

A model peptide, cyclo-(Phe-d-Pro-Gly-Arg-Gly-Asp), with a distinct folded structure containing short beta-hairpin and beta-sheet patterns was studied by Raman and Raman optical activity (ROA) spectroscopies. Unlike for previously analyzed vibrational circular dichroism of the same compound (Chirality 2008, 20, 1104), the Raman spectrum is dominated by side chain contributions and is more sensitive to their geometry fluctuations. The spectra and molecular motion were analyzed with the aid of the density functional theory simulations combined with molecular dynamics (MD). The side chain geometry fluctuations were found to significantly contribute to the broadening of the spectral bands, while dynamics of the backbone is rather restricted. According to our MD results, the side chains do not move freely but largely oscillate around preferred conformations. Averaging of computed spectra for many structures derived from the MD trajectories provided better spectral profiles than did a fixed geometry. The Raman and ROA scattering is dominated by the more polarizable phenylalanine and proline groups, as could be verified both by the computations and by comparison to experiments with a model Phe-d-Pro dipeptide. Computational analyses suggest that the ROA spectrum mostly senses local side chain conformation, whereas a vibrational coupling between different side chains contributes less. The coupling is mostly mediated by the peptide backbone and is restricted to specific vibrational region. The ROA spectroscopic technique thus provides important local structural information that needs, however, to be extracted by multiscale (QM/MM) simulation techniques.

Phthalocyanine dimerization-based molecular beacons using near-IR fluorescence.

Herein we demonstrate the use of a novel dimerization-based molecular beacon (MB) probe consisting of two metallo-phthalocyanine (Pc) fluorophores that use near-IR fluorescence, appropriate for highly specific and sensitive in vivo and/or in vitro DNA/RNA detection. Pc's possess a propensity to form nonfluorescent H-dimers that is utilized as the molecular "off" switch in the closed MB conformation. The "on" switch, which is generated when the solution target binds to the loop of the MB forming the open form, also provides two fluorophores for transduction resulting in a doubling of the extinction coefficient and improving the resulting fluorescence yield compared to a classical single-fluorophore/quencher MB system. In addition, the Pc-based MBs possess high thermal, photo, and chemical stabilities that are essential for many highly sensitive applications, such as molecular imaging. The dimer-based MBs were obtained using a simple single-step synthesis procedure and demonstrated excellent quenching efficiencies (98%) as well as a high signal-to-background ratio (approximately 60) exceeding the performance characteristics of many conventionally available MB probes.

Peptide targeting of platinum anti-cancer drugs.

Besides various side effects caused by platinum anticancer drugs, they are not efficiently absorbed by the tumor cells. Two Pt-peptide conjugates; cyclic mPeg-CNGRC-Pt (7) and cyclic mPeg-CNGRC-Pten (8) bearing the Asn-Gly-Arg (NGR) targeting sequence, a malonoyl linker, and low molecular weight miniPEG groups have been synthesized. The platinum ligand was attached to the peptide via the carboxylic end of the malonate group at the end of the peptide. The pegylated peptide is nontoxic and highly soluble in water. Platinum conjugates synthesized using the pegylated peptides are also water-soluble with reduced or eliminated peptide immunogenicity. The choice of carboplatin as our untargeted platinum complex was due to the fact that the malonate linker chelates platinum in a manner similar to that of carboplatin. Cell toxicity assay and competition assay on the PC-3 cells (CD13 positive receptors) revealed selective delivery and destruction of PC-3 cells using targeted Pt-peptide conjugates 7 and 8 significantly more than untargeted carboplatin. Platinum uptake on PC-3 cells was 12-fold more for conjugate 7 and 3-fold more for conjugate 8 compared to that of the untargeted carboplatin, indicating selective activation of the CD13 receptors and delivery of the conjugates to CD13 positive cells. Further analysis on effects of conjugates 7 and 8 on PC-3 cells using caspase-3/7, fluorescence microscopy, and DNA fragmentation confirmed that the cells were dying by apoptosis.

Mono-amine functionalized phthalocyanines: microwave-assisted solid-phase synthesis and bioconjugation strategies.

Phthalocyanines (Pcs) are excellent candidates for use as fluors for near-infrared (near-IR) fluorescent tagging of biomolecules for a wide variety of bioanalytical applications. Monofunctionalized Pcs, having two different types of peripheral substitutents, one for covalent conjugation of the Pc to biomolecules and others to improve the solubility of the macrocycle, are ideally suited for the desired applications. To date, difficulties faced during the purification of monofunctionalized Pcs limited their usage in various types of applications. Herein are reported a new synthetic method for rapid synthesis of the target Pcs and bioconjugation techniques for labeling of the oligonucleotides with the near-IR fluors. A novel synthetic route was developed utilizing a hydrophilic, poly(ethylene glycol) (PEG)-based support with an acid-labile Rink Amide linker. The Pcs were functionalized with an amine group for covalent conjugation purposes and were decorated with short PEG chains, serving as solubilizing groups. Microwave-assisted solid-phase synthetic method was successfully applied to obtain pure asymmetrically substituted monoamine functionalized Pcs in a short period of time. Three different bioconjugation techniques, reductive amination, amidation, and Huisgen cycloaddition, were employed for covalent conjugation of Pcs to oligonucleotides. The described microwave-assisted bioconjugation methods give an opportunity to synthesize and isolate the Pc-oligonucleotide conjugate in a few hours.

Contrasting in vivo effects of two peptide-based amyloid-beta protein aggregation inhibitors in a transgenic mouse model of amyloid deposition.

Previous studies have shown that 17,19,21-tri-N-methyl-Abeta16-22 peptide (Abeta16-22m), and a peptide analogue containing alpha,alpha-disubstituted amino acids (alphaalpha AA) in the hydrophobic core domain of Abeta, termed AMY-1, effectively inhibited full-length Abeta aggregation in vitro. To investigate the amyloid-modifying effects of these agents in vivo, we injected these compounds into the hippocampus of 13-month-old amyloid precursor protein (APP) transgenic mice, a model of amyloid deposition. After 7 days, brain tissues were stained for immunohistochemistry to detect total Abeta and thioflavine-S (THIO-S) to measure Abeta compact plaques. Both diffuse Abeta deposits and compact amyloid plaques were significantly increased when injecting 0.3 nmol Abeta16-22m compared to the PBS vehicle. The amyloid aggregation-modifying peptide AMY-1 showed a slight reduction of Abeta deposition in the injection area at a dose of 0.3 nmol, but neuronal toxicity, measured by Fluoro-Jade and Nissl stains, appeared when higher doses (3 nmol) were tested. Our data indicate that, unlike observations reported in vitro, the Abeta16-22m increased deposition of Abeta in the brain of APP transgenic mice in vivo. Possible explanations for this outcome include unique influences of the brain environment and/or modification of Abeta production or clearance by the administered agent. The AMY-1 peptide showed a trend for reducing Abeta deposits, but led to toxicity at higher doses. These data emphasize the need for evaluating potential Abeta aggregation inhibitors with in vivo models of amyloid deposition before assuming they will have benefit in treating Alzheimer's disease patients.

Solid-phase synthesis of asymmetrically substituted "AB3-type" phthalocyanines.

Synthesis of phthalocyanines with asymmetrical substitution on the periphery is often difficult due the problems in purification of the phthalocyanine mixtures obtained. Using a poly(ethylene glycol) (PEG)-based support with a Wang-type linker, we have developed the synthesis of monohydroxylated, oligoethylene glycol substituted phthalocyanines utilizing an amidine-base-promoted phthalonitrile tetramerization reaction. The use of a hydrophilic support allows symmetrical phthalocyanine product formed in solution to be readily and completely removed by washing while leaving the "AB3" product on the support. Acid cleavage with 10% trifluoroacetic acid provides the pure unsymmetrically substituted Pc. This method was applied to several metallo Pcs. Additionally, methods to avoid premature reactions on-resin that give A2B2 products are provided.

Photoinduced RNA interference using DMNPE-caged 2'-deoxy-2'-fluoro substituted nucleic acids in vitro and in vivo.

Various chemical modifications to RNA have been incorporated in attempts to improve their pharmacological properties for RNAi interference (RNAi). Recent studies have shown that small interfering RNA (siRNA) containing 2'-fluoro modifications can elicit gene silencing through RNAi. Despite developments in using chemical modifications for increased stability, safety, and efficiency of these therapeutics, they still face challenges of spatial and temporal targeting. One potential targeting strategy is to use photocaging techniques, which involve the covalent attachment of photolabile compounds to the effector nucleic acid species that block bioactivity until exposed to near UV light. In this study we demonstrate that fully 2'-fluorinated nucleic acids (FNAs) can be caged for photoactivated gene silencing in cell culture and in zebrafish embryos. This strategy combines the improvement in chemical and enzymatic stability associated with 2'-substitutions with the targeting ability of a photoinducible trigger. Statistical alkylation of FNAs with 1-(4,5-dimethoxy-2-nitrophenyl)diazoethane (DMNPE) improved resistance to enzymatic degradation, reduced RNAi effectiveness, and protected the biological system from toxic doses of the effector. Photo-exposure to 365 nm light partially restored the silencing activity of the 2'-fluoro siRNAs. These results suggest that photocaging may offer control over RNAi therapeutics for spatially and temporally directed activation, while improving enzymatic stability and potentially enabling therapeutic dosing via light dose intensity.

Vibrational circular dichroism and IR spectral analysis as a test of theoretical conformational modeling for a cyclic hexapeptide.

A model cyclohexapeptide, cyclo-(Phe-(D)Pro-Gly-Arg-Gly-Asp) was synthesized and its IR and VCD spectra were used as a test of density functional theory (DFT) level predictions of spectral intensities for a peptide with a nonrepeating but partially constricted conformation. Peptide structure and flexibility was estimated by molecular dynamics (MD) simulations and the spectra were simulated using full quantum mechanical (QM) approaches for the complete peptide and for simplified models with truncated side chains. After simulated annealing, the backbone conformation of the ring structure is relatively stable, consisting of a normal beta-turn and a tight loop (no H-bond) which does not vary over short trajectories. Only in quite long MD runs at high temperatures do other conformations appear. MD simulations were carried out for the cyclic peptide in water and in TFE, which match experimental solvents, as well as with and without protonation of the Asp carboxyl group. DFT spectral simulations were made using the annealed structure and were extended to include basis set variation, to determine an optimal computational approach, and solvent simulation with a polarized continuum model (PCM). Stepwise full DFT simulation of spectra was done for various sequences with the same backbone geometry but based on (1) solely Gly residues, (2) Ala substitution except Gly and Pro, and (3) complete sequences with side chains. Additionally, a selection of structures was used to compute IR and VCD spectra with the optimal method to determine structural variation effects. The side chains, especially the Asp-COOH and Arg-NH(2) transitions, had an impact on the computed amide frequencies, IR intensities and VCD pattern. Since experimentally these groups would have little chirality, due to conformational variation, they do not impact the observed VCD spectra. Correcting for frequency shifts, the Ala model for the cyclopeptide gives the clearest representation of the amide VCD. The experimental sign pattern for the amide I' band in D(2)O and also the sharper, more intense amide I VCD band in TFE was seen to some degree in one conformer with Type II' turns, but the data favor a mix of structures.

Inter-conversion of neuregulin2 full and partial agonists for ErbB4.

The EGF family hormone NRG2beta potently stimulates ErbB4 tyrosine phosphorylation and coupling to IL3 independence. In contrast, the NRG2alpha splicing isoform has lower affinity for ErbB4, does not potently stimulate ErbB4 phosphorylation, and fails to stimulate ErbB4 coupling. Here we investigate these differences. The NRG2beta Q43L mutant potently stimulates ErbB4 phosphorylation but not ErbB4 coupling to IL3 independence. This failure to stimulate ErbB4 coupling is not due to differential ligand purity, glycosylation, or stability. The NRG2alpha K45F mutant potently stimulates ErbB4 phosphorylation but not ErbB4 coupling to IL3 independence. Thus, this failure to stimulate ErbB4 coupling is not due to inadequate affinity for ErbB4. In contrast, the NRG2alpha L43Q/K45F mutant stimulates ErbB4 coupling, even though it does not have greater affinity for ErbB4 than does NRG2alpha/K45F. Collectively, these data indicate that Gln43 of NRG2beta is both necessary and sufficient for NRG2 stimulation of ErbB4 coupling to IL3 independence.

Beta-amyloid protein aggregation.

The beta-amyloid peptide aggregates via a nucleation pathway where micellar aggregates propagate to form oligomers (protofibrils), which then polymerize into insoluble fibrils. This fibrillogenic process has been linked to the pathogenesis associated with Alzheimer's disease. One purpose of this chapter is to provide a protocol for reliably producing monomeric Abeta as a starting point for physical and biological studies. Many research groups have used organic solvents to disaggregate pre-seeded Abeta in an attempt to acquire monomeric starting materials. Others have used instrumental techniques such as size exclusion chromatography to isolate monomer, structural intermediates, and fibrils and study their affects on Abeta nucleation. This chapter discusses a modified method of Abeta preparation using organic solvents followed by dissolution into aqueous phosphate buffer systems that renders monomeric Abeta starting solutions for kinetic experiments. Additionally, this chapter details a number of physical techniques such as scanning force microscopy, circular dichroism spectroscopy, transmission electron microscopy, fluorescence spectroscopy, fluorescence photobleaching recovery, and dynamic light scattering, together with physiological techniques such as cell viability assays to characterize Abeta nucleation, aggregation, and fibrillization and the potential biological activity of the various Abeta particles.

Peptide-mediated cell transport of water soluble porphyrin conjugates.

Five new porphyrin-peptide conjugates bearing a nuclear localizing sequence SV40 or a fusogenic peptide (HIV-1Tat 40-60 or octa-arginine) linked by low molecular weight poly(ethylene glycol) have been synthesized. In vitro studies using human HEp2 cells show that the cellular uptake of the conjugates depends significantly on the nature and sequence of amino acids in the peptide and on the nature of the substituents on the porphyrin macrocycle. The fusogenic peptide sequences HIV-1Tat 40-60 and octa-arginine were the most effective in delivering the conjugates to the cells. The subcellular distribution of the conjugates was found to be dependent on the nature of substituents on the porphyrin macrocycle. The conjugates bearing a hydrophobic porphyrin localized preferentially in the endoplasmic reticulum and were significantly more phototoxic to HEp2 cells than the carboxylic acid functionalized porphyrin conjugates, which localized mainly in the lysosomes.

Five carboxyl-terminal residues of neuregulin2 are critical for stimulation of signaling by the ErbB4 receptor tyrosine kinase.

The neuregulins (NRGs) are members of the epidermal growth factor (EGF) family of peptide growth factors. These hormones are agonists for the ErbB family of receptor tyrosine kinases, a family that includes the epidermal growth factor receptor (EGFR/ErbB1), ErbB2/Neu/HER2, ErbB3/HER3, and ErbB4/HER4. We recently observed that the EGF family hormone NRG2beta is a potent agonist for ErbB4. In contrast, NRG2alpha, a splicing isoform of the same gene that encodes NRG2beta, is a poor ErbB4 agonist. We hypothesized that carboxyl-terminal residues of NRG2beta are critical for stimulation of ErbB4 tyrosine phosphorylation and coupling to downstream signaling events. Here, we demonstrate that the substitution of a lysine residue for Phe45 in NRG2beta results in reduced ligand potency. We also demonstrate that substitution of a phenylalanine for Lys45 in NRG2alpha results in increased ligand potency. Finally, analyses of the gain-of-function NRG2alpha Chg5 mutant demonstrate that Gln43, Met47, Asn49, and Phe50 regulate ligand efficacy. Thus, these data indicate that carboxyl-terminal residues of NRG2beta are critical for activation of ErbB4 signaling. Moreover, these NRG2alpha and NRG2beta mutants reveal new insights into models for ligand-induced ErbB family receptor tyrosine phosphorylation and coupling to downstream signaling events.

Neuregulin isoforms exhibit distinct patterns of ErbB family receptor activation.

During the last decade, several novel members of the Epidermal Growth Factor family of peptide growth factors have been identified. Most prominent among these are the Neuregulins or Heregulins. To date, four different Neuregulin genes have been identified (Neuregulin1-4) and several different splicing isoforms have been identified for at least two of these genes (Neuregulin1 and Neuregulin2). While Neuregulin1 isoforms have been extensively studied, comparatively little is known about Neuregulin3, Neuregulin4, or the Neuregulin2 isoforms. Indeed, there has been no systematic comparison of the activities of these molecules. Here we demonstrate that Neuregulin2alpha and Neuregulin2beta stimulate ErbB3 tyrosine phosphorylation and coupling to biological responses. In contrast, Neuregulin3 and Neuregulin4 fail to activate ErbB3 signaling. Furthermore, Neuregulin2beta, but not Neuregulin2alpha, stimulates ErbB4 tyrosine phosphorylation and coupling to biological responses. Finally, both Neuregulin3 and Neuregulin4 stimulate modest amounts of ErbB4 tyrosine phosphorylation. However, whereas Neuregulin3 stimulates a modest amount of ErbB4 coupling to biological responses, Neuregulin4 fails to stimulate ErbB4 coupling to biological responses. This suggests that there are qualitative as well as quantitative differences in ErbB family receptor activation by Neuregulin isoforms.

Synthesis and cellular studies of nonaggregated water-soluble phthalocyanines.

Water-soluble phthalocyanines are promising photosensitizers for application in cancer therapy and in the photoinactivation of viruses. The water-soluble zinc(II) phthalocyanines 5 and 6 were synthesized by converting the corresponding ester derivative 4 into the sodium carboxylate and carboxylic acid species. Compound 5 can be solubilized in water as a monomeric species, as demonstrated by UV/vis and fluorescence spectroscopy. These compounds were characterized by analytical and spectroscopic methods and, in the case of 4, by X-ray crystallography. The water-soluble phthalocyanines were found to have low dark cytotoxicity toward V79 hamster fibroblasts and human HEp2 cells, to be phototoxic at low light and drug doses, to be taken up by cells in culture, and to localize intracellularly, mainly in the cell lysosomes. Conjugation of the anionic phthalocyanines with positively charged LipoGen liposomes resulted in effective delivery of these compounds into the nuclei of cells. It is concluded that these highly water-soluble phthalocyanines are promising sensitizers for the photodynamic therapy of tumors.

BioMEMS using electrophoresis for the analysis of genetic mutations.

Biomedical microelectromechanical systems (BioMEMS) are rapidly emerging in many areas of genetic analysis. These devices demonstrate potential for rapid analysis using modular components capable of sample purification, amplification, mutation discrimination and detection on small, portable point-of-care instruments. Here, various approaches to genetic mutation detection and the modern analysis platform, capillary electrophoresis, will be briefly reviewed. Microfluidic devices will be discussed in relation to fabrication techniques, mutation detection using simple electrophoretic separations, multiplexed designs and modular functionalities, as well as challenges and issues surrounding this technology.

Efficient acylation of the N-terminus of highly hindered C(alpha)(alpha)-disubstituted amino acids via amino acid symmetrical anhydrides.

[reaction: see text] Fmoc amino acid symmetrical anhydrides are efficient and readily available reagents for acylation of the N-terminus of highly hindered C(alpha)(alpha)-dialkylated alpha-amino acids. Comparison of a variety of coupling protocols showed that the symmetrical anhydride method always provided the superior results. This method was successfully applied to the solid-phase synthesis of a peptide containing three alpha(alpha)AAs at alternating positions.

Synthesis of oligonucleotides containing thiazole and thiazole N-oxide nucleobases.

[reaction: see text] The thiazole C-nucleoside analogue was synthesized by the Hantzsch cyclization method to form the thiazole ring and was then converted to the thiazole N-oxide C-nucleoside analogue by peracid oxidation of the heterocycle nitrogen. Incorporation of the thiazole and thiazole N-oxide phosphoramidites into DNA was successful though significant deoxygenation of the N-oxide occurred during DNA assembly. The mechanism proposed for the reduction of the thiazole N-oxide to thiazole involves the formation of an N-oxide phosphite ester.