Spying on Neuronal Membrane Potential with Genetically Targetable Voltage Indicators.

Methods for optical measurement of voltage dynamics in living cells are attractive because they provide spatial resolution surpassing traditional electrode-based measurements and temporal resolution exceeding that of widely used Ca2+ imaging. Chemically synthesized voltage-sensitive dyes that use photoinduced electron transfer as a voltage-sensing trigger offer high voltage sensitivity and fast-response kinetics, but targeting chemical indicators to specific cells remains an outstanding challenge. Here, we present a new family of readily functionalizable, fluorescein-based voltage-sensitive fluorescent dyes (sarcosine-VoltageFluors) that can be covalently attached to a genetically encoded cell surface receptor to achieve voltage imaging from genetically defined neurons. We synthesized four new VoltageFluor derivatives that possess carboxylic acid functionality for simple conjugation to flexible tethers. The best of this new group of dyes was conjugated via a polyethylene glycol (PEG) linker to a small peptide (SpyTag, 13 amino acids) that directs binding and formation of a covalent bond with its binding partner, SpyCatcher (15 kDa). The new VoltageSpy dyes effectively label cells expressing cell-surface SpyCatcher, display good voltage sensitivity, and maintain fast-response kinetics. In cultured neurons, VoltageSpy dyes enable robust, single-trial optical detection of action potentials at neuronal soma with sensitivity exceeding genetically encoded voltage indicators. Importantly, genetic targeting of chemically synthesized dyes enables VoltageSpy to report on action potentials in axons and dendrites in single trials, tens to hundreds of micrometers away from the cell body. Genetic targeting of synthetic voltage indicators with VoltageSpy enables voltage imaging with low nanomolar dye concentration and offers a promising method for allying the speed and sensitivity of synthetic indicators with the enhanced cellular resolution of genetically encoded probes.

Poly(Sarcosine) Surface Modification Imparts Stealth-Like Properties to Liposomes.

Circulation lifetime is a crucial parameter for a successful therapy with nanoparticles. Reduction and alteration of opsonization profiles by surface modification of nanoparticles is the main strategy to achieve this objective. In clinical settings, PEGylation is the most relevant strategy to enhance blood circulation, yet it has drawbacks, including hypersensitivity reactions in some patients treated with PEGylated nanoparticles, which fuel the search for alternative strategies. In this work, lipopolysarcosine derivatives (BA-pSar, bisalkyl polysarcosine) with precise chain lengths and low polydispersity indices are synthesized, characterized, and incorporated into the bilayer of preformed liposomes via a post insertion technique. Successful incorporation of BA-pSar can be realized in a clinically relevant liposomal formulation. Furthermore, BA-pSar provides excellent surface charge shielding potential for charged liposomes and renders their surface neutral. Pharmacokinetic investigations in a zebrafish model show enhanced circulation properties and reduction in macrophage recognition, matching the behavior of PEGylated liposomes. Moreover, complement activation, which is a key factor in hypersensitivity reactions caused by PEGylated liposomes, can be reduced by modifying the surface of liposomes with an acetylated BA-pSar derivative. Hence, this study presents an alternative surface modification strategy with similar benefits as the established PEGylation of nanoparticles, but with the potential of reducing its drawbacks.

Oxidation-Sensitive Polymersomes Based on Amphiphilic Diblock Copolypeptoids.

Stimuli-responsive polymersomes formed by amphiphilic block copolymers have attracted substantial attention as smart and robust containers for drug delivery and nano/microreactors. Biosourced amphiphilic diblock copolypeptoids were developed that can self-assemble into oxidation-responsive unilamellar vesicles. These vesicles can burst under the action of reactive oxygen species which can be the hydrogen peroxide or the singlet oxygen produced by light-activation of a photosensitizer with spatiotemporal control. Polysarcosine (PSar, also called poly(N-methyl glycine)) was selected as the hydrophilic block because of its resistance to protein adsorption and low toxicity, similar to poly(ethylene glycol) (PEG). We designed and synthesized poly(N-3-(methylthio)propyl glycine) as the hydrophobic block. Its polyglycine backbone is the same as that of PSar, and especially, its hydrophobic N-substituents, thioether side chains, can be oxidized to hydrophilic sulfoxides. These oxidation-responsive polymersomes entirely based on N-substituted poly(amino acid)s were biocompatible as confirmed by cell viability tests and may find applications in drug delivery, biosensing, biodetection, and nano/microreactors.

Synthesis and gelation of copolypept(o)ides with random and block structure.

Copolypept(o)ides of polysarcosine (PSar) and poly(N-isopropyl-L-glutamine) (PIGA) with random and block sequence structures were synthesized by ring-opening polymerization (ROP) of sarcosine N-carboxyanhydrides (Sar-NCA) and γ-benzyl-l-glutamate N-carboxyanhydrides (BLG-NCA) and post modification. With different distribution of Sar along the main chain, H-bonding pattern and secondary structure of polypeptides were turned, as well as aggregation and gelation behavior. Both copolypept(o)ides formed hydrogels above their critical gelation concentrations (CGCs) without thermo-sensitivity, which was normally reserved for PEG copolypeptides (eg, PEG-b-PIGA). In particular, a different mechanism from previously reported micellar percolation or fibrillar entanglement was suggested for gelation of the random copolypept(o)ide. Therefore, hydrogels from copolymers of PSar and PIGA represented a new approach to construct easy-handling, biocompatible, biodegradable and thermo-stable gels that could potentially be applied in biomedical fields.

Synthesis and sequential deprotection of triblock copolypept(o)ides using orthogonal protective group chemistry.

The synthesis of triblock copolymers based on polysarcosine, poly-N-ε-t-butyloxycarbonyl-l-lysine, and poly-N-ε-t-trifluoroacetyl-l-lysine by ring-opening polymerization of the corresponding α-amino acid N-carboxyanhydrides (NCAs) is described. For the synthesis of N-ε-t-butyloxycarbonyl-l-lysine (lysine(Boc)) NCAs, an acid-free method using trimethylsilylchloride/triethylamine as hydrochloric acid (HCl) scavengers is presented. This approach enables the synthesis of lysine(Boc) NCA of high purity (melting point 138.3 °C) in high yields. For triblock copolypept(o)ides, the degree of polymerization (Xn ) of the polysarcosine block is varied between 200 and 600; poly-N-ε-t-butyloxycarbonyl-l-lysine and poly-N-ε-t-trifluoroacetyl-l-lysine blocks are designed to have a Xn in the range of 10-50. The polypeptide-polypeptoid hybrids (polypept(o)ides) can be synthesized with precise control of molecular weight, high end group integrity, and dispersities indices between 1.1 and 1.2. But more important, the use of tert-butyloxycarbonyl- and trifluoroacetyl-protecting groups allows the selective, orthogonal deprotection of both blocks, which enables further postpolymerization modification reactions in a block-selective manner. Therefore, the presented synthetic approach provides a versatile pathway to triblock copolypept(o)ides, in which functionalities can be separated in specific blocks.

Polypept(o)ides: Hybrid Systems Based on Polypeptides and Polypeptoids.

Polypept(o)ides combine the multifunctionality and intrinsic stimuli-responsiveness of synthetic polypeptides with the "stealth"-like properties of the polypeptoid polysarcosine (poly(N-methyl glycine)). This class of block copolymers can be synthesized by sequential ring opening polymerization of α-amino acid N-carboxy-anhydrides (NCAs) and correspondingly of the N-substituted glycine N-carboxyanhydride (NNCA). The resulting block copolymers are characterized by Poisson-like molecular weight distributions, full end group integrity, and dispersities below 1.2. While polysarcosine may be able to tackle the currently arising issues regarding the gold standard PEG, including storage diseases in vivo and immune responses, the polypeptidic block provides the functionalities for a specific task. Additionally, polypeptides are able to form secondary structure motives, e.g., α-helix or ß-sheets, which can be used to direct self-assembly in solution. In this feature article, we review the relatively new field of polypept(o)ides with respect to synthesis, characterization, and first data on the application of block copolypept(o)ides in nanomedicine. The summarized data already indicates the great potential of polypept(o)ides.

Environmental nitroso compounds: reaction of nitrite with creatine and creatinine.

Creatine reacts with nitrite under acid conditions to produce first sarcosine and then N-nitrososarcosine, which is a weak carcinogen in the rat. Creatinine reacts with acidified nitrite to produce either creatinine-5-oxime or 1-methylhydantoin-5-oxime, depending on reaction conditions. The toxicity and environmental significance of these compounds is not yet known.

PeptoSomes for Vaccination: Combining Antigen and Adjuvant in Polypept(o)ide-Based Polymersomes.

In this work, the first vaccine is reported based on a PeptoSome, which contains a model antigen (SIINFEKL) and adjuvant (CpG). PeptoSomes are polypept(o)ide-based polymersomes built of a block-copolymer with polysarcosine (PSar) as the hydrophilic block (X n = 111) and poly(benzyl-glutamic acid) (PGlu(OBn)) as the hydrophobic one (X n = 46). The polypept(o)ide is obtained with low dispersity index of 1.32 by controlled ring-opening polymerization. Vesicle formation by dual centrifugation technique allows for loading of vesicles up to 40 mol%. PeptoSomes are characterized by multiangle dynamic light scattering, static light scattering, and cryogenic transmission electron microscopy (cryoTEM). The PeptoSomes have a hydrodynamic radius of 39.2 nm with a low dispersity (µ 2 = 0.1). The ρ-ratio R g /R h of 0.95 already indicates that vesicles are formed, which can be confirmed by cryoTEM. Loaded PeptoSomes deliver the antigen (SIINFEKL) and an adjuvant (CpG) simultaneously into dendritic cells (DCs). Upon cellular uptake, dendritic cells are stimulated and activated, which leads to expression of cluster of differentiation CD80, CD86, and MHCII, but induces excretion of proinflammatory cytokines (e.g., TNFα). Furthermore, DC-mediated antigen-specific T-cell proliferation is achieved, thus underlining the enormous potential of PeptoSomes as a versatile platform for vaccination.

Design and synthesis of novel sulfonamide-containing bradykinin hB2 receptor antagonists. 1. Synthesis and SAR of alpha,alpha-dimethylglycine sulfonamides.

We recently published the extensive in vivo pharmacological characterization of MEN 16132 (J. Pharmacol. Exp. Ther. 2005, 616-623; Eur. J. Pharmacol. 2005, 528, 7), a member of the sulfonamide-containing human B(2) receptor (hB(2)R) antagonists. Here we report, in detail, how this family of compounds was designed, synthesized, and optimized to provide a group of products with subnanomolar affinity for the hB(2)R and high in vivo potency after topical administration to the respiratory tract. The series was designed on the basis of indications from the X-ray structures of the key structural motifs A and B present in known antagonists and is characterized by the presence of an alpha,alpha-dialkyl amino acid. The first lead (17) of the series was submitted to extensive chemical work to elucidate the structural requirements to increase hB(2) receptor affinity and antagonist potency in bioassays expressing the human B(2) receptor (hB(2)R). The following structural features were selected: a 2,4-dimethylquinoline moiety and a piperazine linker acylated with a basic amino acid. The representative lead compound 68 inhibited the specific binding of [(3)H]BK to hB(2)R with a pKi of 9.4 and antagonized the BK-induced inositolphosphate (IP) accumulation in recombinant cell systems expressing the hB(2)R with a pA(2) of 9.1. Moreover, compound 68 when administered (300 nmol/kg) intratracheally in the anesthetized guinea pig, was able to significantly inhibit BK-induced bronchoconstriction for up to 120 min after its administration, while having a lower and shorter lasting effect on hypotension.

N-Nitrosarcosine: An Economic Precursor for the Synthesis of New Energetic Materials.

New energetic compounds have been synthesized starting from the readily available N-(cyanomethyl)-N-methylamine. From this, N-nitrosarcosine was prepared in few steps, which serves as a starting material for the synthesis of oxygen-rich compounds. The compounds were thoroughly characterized including multinuclear NMR and vibrational spectroscopy and also molecular structures by single X-ray diffraction were obtained. Their energetic properties were determined including the sensitivities towards impact and friction, their heat of formations were calculated and the detonation and combustion parameters were predicted using EXPLO5 V6.02.

Glucose-bearing biodegradable poly(amino acid) and poly(amino acid)-poly(ester) conjugates for controlled payload release.

The glucoseamine-initiated ring-opening polymerisation of amino acid N-carboxyanhydrides and O-carboxanhydrides to yield amphiphilic block copolymers that are capable of self-assembly in aqueous solution to form well-defined, glucose-presenting, particles is reported. The particles formed are susceptible to enzyme-mediated (lipase and protease) and pH-induced degradation, and can selectively bind the lectin concanavalin A. Consequently, such glycoparticles are of significance for the controlled release of payload molecules in response to an acidic environment, for instance cancerous tissue, and upon interaction with target enzymes.

Synthesis, characterization, and comparative in vitro cytotoxicity studies of platinum(II), palladium(II), and gold(III) methylsarcosinedithiocarbamate complexes.

This work reports on the synthesis, characterization, and in vitro cytotoxic activity of some new platinum(II), palladium(II), and gold(III) derivatives of methylsarcosinedithiocarbamate and its S-methyl ester, to study their behavior as potential antitumor agents. The biological activity of these compounds, as determined by growth inhibition and apoptosis induction, has been investigated in both human leukemic promyelocites HL60 and human squamous cervical adenocarcinoma HeLa cell lines, and their activity has been compared to the well-known platinum-based anticancer agent cisplatin. On the basis of these experimental results, [Pd(MSDT)X]n (MSDT = methylsarcosinedithiocarbamate; X = Cl, Br) complexes show a strong dose-dependent growth inhibition of both HL60 and HeLa cells, with IC(50) values slightly higher than those recorded for cisplatin; moreover, [Au(MSDT)X(2)] activity appears significantly higher or, at least, comparable to that of the reference drug. Exposure of both cell lines to [Pd(MSDT)X]n and [Au(MSDT)X(2)] complexes induces apoptosis, as determined by an Apo2.7 assay.

Sarcosine-Based Glycine Transporter Type-1 (GlyT-1) Inhibitors Containing Pyridazine Moiety: A Further Search for Drugs with Potential to Influence Schizophrenia Negative Symptoms.

We have synthesized a novel series of N-substituted sarcosines, analogues of NFPS (N-[3-(biphenyl-4- yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine), as type-1 glycine transporter (GlyT-1) inhibitors. Several compounds incorporated a diazine ring inhibited recombinant hGlyT-1b expressed permanently in CHO cells and GlyT-1 in rat brain synaptosomal preparations. A structure-activity relationship for the newly synthesized compounds was obtained and discussed on the ground of their GlyT-1 inhibitory potencies. Replacement of the biphenyl-4-yloxy moiety in NFPS with a 5-pyridazinylphenoxy moiety (compounds 3, 4, 5, and 6) or a 2-phenyl-5- pyridazinyloxy moiety (compounds 10, 11, and 12) afforded compounds exhibiting potent inhibition on GlyT-1 activity. The GlyT-1 inhibitory properties of NFPS analogues, in which sarcosine was closed into a ring forming (methylamino)pyridazine-3-(2H)-one, were markedly reduced (compounds 13 and 14). The pyridazine-containing GlyT-1 inhibitors with in vitro GlyT-1 inhibitory potency also enhanced extracellular glycine concentrations in conscious rat striatum as was measured by microdialysis technique. In contrast to NFPS, sarcosine-based pyridazine containing GlyT-1 inhibitors failed to evoke compulsive running behavior whereas they inhibited phencyclidine- induced hypermotility in mice. It is believed that increase of extracellular concentrations of glycine by inhibition of its reuptake may probably influence positively glutamate N-methyl-D-aspartate (NMDA)-type ionotropic receptors in the central nervous system. This may have importance in the treatment of neuropsychiatric disorders associated with hypofunctional NMDA receptor-mediated glutamatergic neurochemical transmission. Thus, impaired NMDA receptor functions have been shown to be involved in the development of the negative symptoms and the cognitive deficit of schizophrenia and the treatment of these symptoms is the possible clinical indication of GlyT-1 inhibitors including those containing pyridazine moiety.

Cenp-E inhibitor GSK923295: Novel synthetic route and use as a tool to generate aneuploidy.

Aneuploidy is a common feature of cancer, with human solid tumour cells typically harbouring abnormal chromosome complements. The aneuploidy observed in cancer is often caused by a chromosome instability phenotype, resulting in genomic heterogeneity. However, the role aneuploidy and chromosome instability play in tumour evolution and chemotherapy response remains poorly understood. In some contexts, aneuploidy has oncogenic effects, whereas in others it is anti-proliferative and tumour-suppressive. Dissecting fully the role aneuploidy plays in tumourigenesis requires tools and facile assays that allow chromosome missegregation to be induced experimentally in cells that are otherwise diploid and chromosomally stable. Here, we describe a chemical biology approach that induces low-level aneuploidy across a large population of cells. Specifically, cells are first exposed to GSK923295, an inhibitor targeting the mitotic kinesin Cenp-E; while the majority of chromosomes align at the cell's equator, a small number cluster near the spindle poles. By then driving these cells into anaphase using AZ3146, an inhibitor targeting the spindle checkpoint kinase Mps1, the polar chromosomes are missegregated. This results in, on average, two chromosome missegregation events per division, and avoids trapping chromosomes in the spindle midzone, which could otherwise lead to DNA damage. We also describe an efficient route for the synthesis of GSK923295 that employs a novel enzymatic resolution. Together, the approaches described here open up new opportunities for studying cellular responses to aneuploidy.

Study on the interaction of Co (III) DiAmsar with serum albumins: spectroscopic and molecular docking methods.

This study was designed to examine the interaction of cobalt-3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane-1,8-diamine (Co(III) DiAmsar) as a hexadentate ligand with human serum albumin (HSA) and bovine serum albumin (BSA) under physiological conditions in Tris-HCl buffer solution at pH 7.4. To this aim, at first, Co (III) DiAmsar was synthesized and characterized by nuclear magnetic resonance (NMR), and mass spectroscopy and then its interaction with HSA and BSA was investigated by means of various spectroscopic methods (Fourier transform infrared (FT-IR), UV-visible (UV-vis), fluorescence, and cyclic voltammetry (CV)) and molecular docking technique. The results of fluorescence titration revealed that the Co (III) DiAmsar strongly quench the intrinsic fluorescence of HSA and BSA through a static quenching procedure. Binding constants (Ka) and the number of binding sites (n∼1) were calculated using Stern-Volmer equations. The ΔG parameters at different temperatures were calculated. Subsequently, the values of ΔH and ΔS were also calculated, which revealed that the van der Waals and hydrogen bonding interaction splay a major role in Co (III) DiAmsar-HSA and Co (III) DiAmsar-BSA associations. The distance r between donor (HSA and BSA) and acceptor (Co (III) DiAmsar) was obtained according to fluorescence resonance energy transfer. The data obtained by the molecular modeling study revealed the surrounding residues of HSA and BSA around Co (III) DiAmsar.

Nitramino acids. Synthesis and biological evaluation of 1-nitroproline, 1-nitropipecolic acid, and N-nitrosarcosine.

The N-nitro derivatives of secondary alpha-amino acids, viz., 1-nitroproline (1a) (L and D), 1-nitro-DL-pipecolic acid (2a), and N-nitrosarcosine (3a), were prepared by the oxidation of the corresponding nitrosamino acids with peroxytrifluoroacetic acid. These nitramino acids (1a-3a) were not active against Escherichia coli, Candida albicans, Pseudomonas aeruginosa, or Mycobacterium smegmatis, and 1a and 2a did not show mutagenic activity in a Salmonella typhimurium TA-100 system, with or without added rat liver 9000g supernatant fraction. The marginal mutagenicity of 3a in this system suggests that additional work should be done to assess its carcinogenic-mutagenic potential.

Synthesis and characterization of 7-nitrobenzo-2-oxa-1,3-diazole (NBD)-labeled fluorescent opioids.

Alkylation of sarcosine with 4-chloro-nitrobenzo-2-oxa-1,3-diazole (NBD-chloride) furnished a fluorescent tag that was coupled with a tetrahydrothebaine derivative and beta-naltrexamine, respectively, to yield the fluorescent opioids 7 alpha-(1R)-1-hydroxy-1-methyl-3-(4-hydroxyphenyl)-propyl]-6,14- endoethenotetrahydrothebaine NBD-sarcosinate (ASM-5-10) and N-cyclopropylmethyl-3-hydroxy-14 beta-hydroxy-6 beta-(NBD sarcosinyl)-amino-epoxymorphinan (ASM-5-67). The fluorescence intensity of the novel opioids allowed their detection at subnanomolar concentrations, and was dependent on the polarity of the solvent. Maximum quantum yield was obtained in ethyl acetate and ethanol, and minimal fluorescence in heptane and water. Compounds ASM-5-10 and ASM-5-67 displaced the opioid receptor binding of [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol in monkey brain membranes with IC50 values of 8.4 and 1.5 nM, respectively. Whereas ASM-5-67 bound to mu, delta, and kappa receptors with comparable affinities, ASM-5-10 was mu-selective, with selectivity indices (ratio of respective IC50 values) of 0.04 for both mu/delta and mu/kappa. The sodium response ratio in binding revealed a pronounced agonist property of ASM-5-10. Both opioids were lipophilic, with octanol-water partition coefficients (log Papp) of 2.8 (ASM-5-10) and 1.0 (ASM-5-67). ASM-5-10 exhibited particularly strong membrane retention that was not reversible by four washes. Their favorable characteristics in fluorescence, receptor binding, and membrane interaction make these newly developed ligands useful molecular probes to study opioid receptor mechanisms.

Analysis of alcohols, as dimethylglycine esters, by electrospray ionization tandem mass spectrometry.

Dimethylglycine (DMG) esters are new derivatives for the rapid, sensitive and selective analysis of primary and secondary alcohols, in complex mixtures, by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Their development was inspired by the use of the complementary dimethylaminoethyl esters for the trace, rapid analysis of fatty acids. DMG esters are simply prepared by heating a dichloromethane solution of the imidazolide of dimethylglycine, containing triethylamine, and an alcohol. DMG esters of long-chain fatty alcohols, isoprenoidal alcohols and hydroxy-acids are analysed by electrospray ionization tandem mass spectrometry with a precursor ion of m/z 104 scan. Diols, glyceryl esters, glyceryl ethers and some sterols are analysed by a neutral loss of 103 Da scan. Trimethylglycine (TMG) ester iodides, prepared by alkylation of DMG esters with methyl iodide, are more sensitive derivatives for molecules containing secondary alcohol groups, such as cholesterol and gibberellic acid. They are analysed by a precursor ion of m/z 118 scan. DMG or TMG derivatives were shown to be at least comparable and sometimes an order of magnitude more sensitive than N-methylpyridyl ether derivatives for ESI-MS/MS analysis of the different classes of alcohols. Applications of these derivatives for the diagnosis of inherited disorders and the analysis of natural products are presented.

Synthesis, characterization and in vitro cytotoxicity of new organotin(IV) derivatives of N-methylglycine.

The synthesis and characterization of new coordination compounds of some diorganotins(IV) with N-methylglycine (sarcosine) are reported; all these derivatives mainly tend to assume a chelate structure. As single crystals were not obtained, a large number of experimental techniques were used to accomplish a definitive characterization and determination of their structure. Results obtained by (1)H/(119)Sn NMR, FT-IR and (119)mSn-Mössbauer spectroscopy and thermogravimetric analysis allow us to deduce the pentacoordination for 1:1 (Sn/sarcosine) derivatives [R(2)SnCl(2)(Sar)](+)Cl(-) (R=Me, n-Bu) in a trigonal-bipyramidal structure, and the hexacoordination for 1:2 complexes [R'(2)Sn(Sar)(2)](2+)2Cl(-) (R'=Me, n-Bu, Ph) in an octahedral structure; however, the probability of partially or totally non-chelate structures for some adducts increases with the steric hindrance of the R/R' groups and the number of the sarcosine molecules bound to the tin atom, so that they give rise to fluxional equilibria in solution. Finally, the synthesized compounds have been tested for in vitro cytotoxic activity against human adenocarcinoma HeLa cells showing, in some cases, strong activity even at low concentration.