Isothiocyanate Function Research Articles

Synthesis and characterization of two novel red-shifted isothiocyanate BODIPYs and their application in protein conjugation

Fluorescent organic small molecules are very important tools for researchers in the fields of biochemistry and biotechnology, and boron-dipyrromethene (BODIPY) class of organic fluorophores gained much attention in recent years due to their remarkable photophysical properties and chemical versatility. Their application often involves conjugation with biomolecules, which can be accomplished by inserting a reactive functionality to the fluorescent dye. In this paper we present the preparation and characterization of two unpublished BODIPYs bearing the electrophilic isothiocyanate group, widely used for conjugation with proteins and biological amines. These dyes were prepared by inserting a nitrobenzene group to the BODIPY core via direct arylation, which was later converted to the isothiocyanate functionality. The arylated BODIPYs retained their fluorescence and showed red-shifted absorption and fluorescence spectra, when compared to previously published isothiocyanate-BODIPYs. Their application in bioconjugation was tested with bovine serum albumin (BSA) and was confirmed from spectroscopic and electrophoretic analysis.

Diverted Natural Lossen-type Rearrangement for Bioconjugation through in Situ Myrosinase-Triggered Isothiocyanate Synthesis.

Fluorescein isothiocyanate (FITC) is one of the most extensively used fluorescent probes for the labeling of biomolecules. The isothiocyanate function reacts with lysine residues of proteins to provide a chemically stable thiourea linkage without releasing any byproduct. However, diversification of isothiocyanate-based reagents is still hampered by the lack of mild conditions to generate isothiocyanate chemical functions, as well as by their poor stability and limited solutions available to increase water solubility, restricting the use of isothiocyanate labeling to highly water-soluble fluorophores. Inspired by plant biological processes, we report a safe and biocompatible myrosinase-assisted in situ formation of isothiocyanate conjugates from a highly water-soluble and stable glucosinolate precursor. This method was applied for the fluorescence labeling of a plasmatic protein and fluorescence imaging of living cells.

Memantine prodrug as a new agent for Alzheimer\u2019s Disease

Hydrogen sulphide has recently drawn much attention due to its potent anti-inflammatory and neuroprotective roles in brain functions. The purpose of the current study was to exploit these beneficial properties of H2S to design a new agent for the treatment of Alzheimer’s disease (AD). To pursue our aims, we replaced the free amine group of memantine with an isothiocyanate functionality as a putative H2S-donor moiety. The new chemical entity, named memit, was then tested in vitro to determine whether it retains the pharmacological profile of the “native drug”, while also providing a source of H2S in the CNS. Indeed, Memit showed the ability to release H2S through a cysteine-mediated mechanism, thus generating memantine. Moreover, the new hybrid molecule exerts protective effects against neuronal inflammation and induces a drastic fall in ROS production. In addition, memit was also able to reduce the Aβ(1-42) self-induced aggregation and exerted cytoprotective effect against Aβ oligomers-induced damage in both human neurons and rat microglia cells. Finally, similarly to memantine, the new compound promotes autophagy, a complex process required for cellular homeostasis in cell survival that results to be altered in neurodegenerative diseases. In conclusion, our study revealed that memit is a prodrug of memantine. Further in vivo studies will be necessary to fully investigate the synergic or cumulative effects due to the H2S-releasing moiety and the native drug.

Human Cannabinoid Receptor 2 Ligand-Interaction Motif: Transmembrane Helix 2 Cysteine, C2.59(89), as Determinant of Classical Cannabinoid Agonist Activity and Binding Pose.

Cannabinoid receptor 2 (CB2R)-dependent signaling is implicated in neuronal physiology and immune surveillance by brain microglia. Selective CB2R agonists hold therapeutic promise for inflammatory and other neurological disorders. Information on human CB2R (hCB2R) ligand-binding and functional domains is needed to inform the rational design and optimization of candidate druglike hCB2R agonists. Prior demonstration that hCB2R transmembrane helix 2 (TMH2) cysteine C2.59(89) reacts with small-molecule methanethiosulfonates showed that this cysteine residue is accessible to sulfhydryl derivatization reagents. We now report the design and application of two novel, pharmacologically active, high-affinity molecular probes, AM4073 and AM4099, as chemical reporters to interrogate directly the interaction of classical cannabinoid agonists with hCB2R cysteine residues. AM4073 has one electrophilic isothiocyanate (NCS) functionality at the C9 position of its cyclohexenyl C-ring, whereas AM4099 has NCS groups at that position and at the terminus of its aromatic A-ring C3 side chain. Pretreatment of wild-type hCB2R with either probe reduced subsequent [3H]CP55,940 specific binding by ∼60%. Conservative serine substitution of any hCB2R TMH cysteine residue except C2.59(89) did not affect the reduction of [3H]CP55,940 specific binding by either probe, suggesting that AM4073 and AM4099 interact irreversibly with this TMH2 cysteine. In contrast, AM841, an exceptionally potent hCB2R megagonist and direct AM4073/4099 congener bearing a single electrophilic NCS group at the terminus of its C3 side chain, had been demonstrated to bind covalently to TMH6 cysteine C6.47(257) and not C2.59(89). Molecular modeling indicates that the AM4073-hCB2R* interaction at C2.59(89) orients this classical cannabinoid away from TMH6 and toward the TMH2-TMH3 interface in the receptor's hydrophobic binding pocket, whereas the AM841-hCB2R* interaction at C6.47(257) favors agonist orientation toward TMH6/7. These data constitute initial evidence that TMH2 cysteine C2.59(89) is a component of the hCB2R binding pocket for classical cannabinoids. The results further demonstrate how interactions between classical cannabinoids and specific amino acids within the hCB2R* ligand-binding domain act as determinants of agonist pharmacological properties and the architecture of the agonist-hCB2R* conformational ensemble, allowing the receptor to adopt distinct activity states, such that interaction of classical cannabinoids with TMH6 cysteine C6.47(257) favors a binding pose more advantageous for agonist potency than does their interaction with TMH2 cysteine C2.59(89).

Synthesis and biological activity of diisothiocyanate-derived mercapturic acids.

This Letter deals with new non-natural diisothiocyanates, their mercapturic acid derivatives-conjugated with N-acetylcysteine as well as their antiproliferative activity towards human colon cancer cell lines and their inhibitory potency towards histone deacetylase activity. The activity of analysed isothiocyanates is not significantly different than their N-acetylcysteine conjugates. In comparison to simple mono-isothiocyanate analogues, aliphatic diisothiocyanates and their conjugates are much more active than the simple presence of two isothiocyanate functionalities could indicate.

Molecular-interaction and signaling profiles of AM3677, a novel covalent agonist selective for the cannabinoid 1 receptor.

The cannabinoid 1 receptor (CB1R) is one of the most abundant G protein-coupled receptors (GPCRs) in the central nervous system. CB1R involvement in multiple physiological processes, especially neurotransmitter release and synaptic function, has made this GPCR a prime drug discovery target, and pharmacological CB1R activation has been demonstrated to be a tenable therapeutic modality. Accordingly, the design and profiling of novel, drug-like CB1R modulators to inform the receptor's ligand-interaction landscape and molecular pharmacology constitute a prime contemporary research focus. For this purpose, we report utilization of AM3677, a designer endocannabinoid (anandamide) analogue derivatized with a reactive electrophilic isothiocyanate functionality, as a covalent, CB1R-selective chemical probe. The data demonstrate that reaction of AM3677 with a cysteine residue in transmembrane helix 6 of human CB1R (hCB1R), C6.47(355), is a key feature of AM3677's ligand-binding motif. Pharmacologically, AM3677 acts as a high-affinity, low-efficacy CB1R agonist that inhibits forskolin-stimulated cellular cAMP formation and stimulates CB1R coupling to G protein. AM3677 also induces CB1R endocytosis and irreversible receptor internalization. Computational docking suggests the importance of discrete hydrogen bonding and aromatic interactions as determinants of AM3677's topology within the ligand-binding pocket of active-state hCB1R. These results constitute the initial identification and characterization of a potent, high-affinity, hCB1R-selective covalent agonist with utility as a pharmacologically active, orthosteric-site probe for providing insight into structure-function correlates of ligand-induced CB1R activation and the molecular features of that activation by the native ligand, anandamide.

ChemInform Abstract: The Chemistry of Hapalindoles, Fischerindoles, Ambiguines, and Welwitindolinones

AbstractReview: 118 refs.

Hydrogen sulfide releasing capacity of natural isothiocyanates: is it a reliable explanation for the multiple biological effects of Brassicaceae?

Hydrogen sulfide is an endogenous pleiotropic gasotransmitter, which mediates important physiological effects in the human body. Accordingly, an impaired production of endogenous hydrogen sulfide contributes to the pathogenesis of important disorders. To date, exogenous compounds, acting as hydrogen sulfide-releasing agents, are viewed as promising pharmacotherapeutic agents. In a recent report, the hydrogen sulfide-releasing properties of some synthetic aryl isothiocyanate derivatives have been reported, indicating that the isothiocyanate function can be viewed as a suitable slow hydrogen sulfide-releasing moiety, endowed with the pharmacological potential typical of this gasotransmitter. Many isothiocyanate derivatives (deriving from a myrosinase-mediated transformation of glucosinolates) are well-known secondary metabolites of plants belonging to the family Brassicaceae, a large botanical family comprising many edible species. The phytotherapeutic and nutraceutic usefulness of Brassicaceae in the prevention of important human diseases, such as cancer, neurodegenerative processes and cardiovascular diseases has been widely discussed in the scientific literature. Although these effects have been largely attributed to isothiocyanates, the exact mechanism of action is still unknown. In this experimental work, we aimed to investigate the possible hydrogen sulfide-releasing capacity of some important natural isothiocyanates, studying it in vitro by amperometric detection. Some of the tested natural isothiocyanates exhibited significant hydrogen sulfide release, leading us to hypothesize that hydrogen sulfide may be, at least in part, a relevant player accounting for several biological effects of Brassicaceae.

Chapter Two - The Chemistry of Hapalindoles, Fischerindoles, Ambiguines, and Welwitindolinones

Isolated from diverse cyanobacteria, the hapalindole family of indole alkaloids comprises over 80 interrelated natural products. The family is further divided into several subclasses including the hapalindoles, fischerindoles, ambiguines, and welwitindolinones, each sharing one or more of the many common structural motifs, including an indole or oxindole core, a highly functionalized cyclohexane unit fused to the indole at C-3, and the uncommon isonitrile or isothiocyanate functionalities. Reported here is a comprehensive review covering the isolation of all currently known hapalindole-type alkaloids, the biological activity for several members of the family, and the proposed biosyntheses that give rise to the intricate structural diversity within the family. Finally, the major thrust of this chapter is a comprehensive outline of decades of cutting edge synthetic chemistry toward these complex alkaloids. To date, only a small fraction of the hapalindole-type alkaloids have yielded to chemical synthesis, yet the strategies and methodologies developed showcase the state of the art in synthetic chemistry and provide access to new compounds that can be evaluated for their biological activities.

Pharmacological characterization of the vascular effects of aryl isothiocyanates: Is hydrogen sulfide the real player?

Hydrogen sulfide (H2S) is an endogenous gasotransmitter, which mediates important physiological effects in the cardiovascular system. Accordingly, an impaired production of endogenous H2S contributes to the pathogenesis of important cardiovascular disorders, such as hypertension. Therefore, exogenous compounds, acting as H2S-releasing agents, are viewed as promising pharmacotherapeutic agents for cardiovascular diseases. Thus, this paper aimed at evaluating the H2S-releasing properties of some aryl isothiocyanate derivatives and their vascular effects. The release of H2S was determined by amperometry, spectrophotometry and gas/mass chromatography. Moreover, the vascular activity of selected isothiocyanates were tested in rat conductance (aorta) and coronary arteries. Since H2S has been recently reported to act as an activator of vascular Kv7 potassium channels, the possible membrane hyperpolarizing effects of isothiocyanates were tested on human vascular smooth muscle (VSM) cells by spectrofluorescent dyes. Among the tested compounds, phenyl isothiocyanate (PhNCS) and 4-carboxyphenyl isothiocyanate (PhNCS–COOH) exhibited slow-H2S-release, triggered by organic thiols such as l-Cysteine. These compounds were endowed with vasorelaxing effects on conductance and coronary arteries. Moreover, these two isothiocyanates caused membrane hyperpolarization of VSM cells. The vascular effects of isothiocyanates were strongly abolished by the selective Kv7-blocker XE991. In conclusion, the isothiocyanate function can be viewed as a suitable slow H2S-releasing moiety, endowed with vasorelaxing and hypotensive effects, typical of this gasotransmitter. Thus, such a chemical moiety can be employed for the development of novel chemical tools for basic studies and promising cardiovascular drugs.

A one-pot conversion of di-substituted thiourea to O-organyl arylthiocarbamate using FeCl3

Unsymmetrical thiourea, which on demand can generate isothiocyanate in the presence of FeCl3, can serve as a latent isothiocyanate functionality and circumvent the difficulties associated with the direct use of reactive isothiocyanate functionality. An unusual and unorthodox reactivity has been achieved during a one-pot reaction of an unsymmetrically di-substituted thiourea with an alcohol in the presence of FeCl3 leading to an expeditious synthesis of O-organyl arylthiocarbamates. In this reaction, a thiono-ester (C‒O) bond is formed at the expense of a thioamidic (C‒N) bond and works over a wide range of structurally diverse thioureas and alcohols without affecting the other functional groups.

Design, synthesis and biological evaluation of new naphtalene diimides bearing isothiocyanate functionality

The synthesis and the biological activities of new derivatives 1– 3, characterized by the isothiocyanate (ITC) functionalities coming from sulforaphane (SFN), a well-known anticancer natural product, were reported. The most interesting compound of the series was 2. It was chemically characterized by two ITC functionalities mounted on the 1,4,5,8-naphthalentetracarboxylic diimide (NDI) scaffold through two polymethylene chains, each constituted by three carbon units. It demonstrated an IC 50 value in the submicromolar range, more potent than SFN, displaying also the ability to trigger apoptotic induction in the same range by eliciting both extrinsic and intrinsic apoptotic pathways. Finally, it was observed that 2 inhibited the cell growth by blocking the cell cycle in G1 phase.

Abstract 2131: Synthesis and biological evaluation of novel N-alkyl derivatives of 5, 7-dibromoisatin as potent anticancer agents

Abstract Microtubules display an important role in a variety of cellular process, including mitosis and cell division. Various anti-mitotic agents interfering with the natural dynamics of tubulin, polymerization and depolymerization inhibit cancer cell growth. The isatin is found as an endogenous molecule in humans and other mammals, and its analogues display diverse types of biological activities including anti-cancer. Recently, it has been reported that N-alkylation of 5, 7-dibromoisatin increased its cytotoxicity against a range of human cancer cell lines. In this context, it was of interest to further investigate the cytotoxicity of N-alkylated 5,7-dibromoisatin analogues like isothiocyanates and selenocyantes. Isothiocyanates are one of the most effective naturally occurring cancer chemopreventive agents that inhibit carcinogenicity in animal models. Selenium compounds have been found capable of inhibiting and/or retarding tumorigenesis in a variety of experimental animal models. Several synthetic alkyl and aryl selenocyanates have been evaluated for anticarcinogenicity in animal models. The combined literature survey of isatin derivatives, isothiocyantes and selenocyanates have prompted us to develop a novel series of 5,7-dibromoisatin containing thiocyanate, isothiocyanate and selenocyante functionalities. The cytotoxicity of a series of new N-substituted derivatives of 5,7-dibromoisatin was evaluated against a panel of four different human cancer cell lines e.g. a colon (HT29), breast (MCF-7), lung (A549) and melanoma (UACC903) and compared with the 5,7-dibromoisatin. The results showed that the cytotoxicity of the 5,7-dibromoisatin significantly increased through N-alkylation, as reported previously for the 5,7-dibromoisatin derivatives. Introduction of isothiocyante or thiocyanate groups to alkyl chain yielded the most active compounds in this series against HT29 cells. MCF-7 cell line was susceptible to compounds with substitution of selenocyanate group in the alkyl chain, indicating that selenium is playing a role in the antiproliferative activity. These selenium substituted compounds also showed good inhibitory activity against HT29 and A549 cell lines, but poor inhibitory activity in UACC903 cells. Apoptosis assay showed that at 5μM concentration, compounds containing isothiocyanates and thiocyanates induced 99% apoptosis making them the most potent compounds in HT29 cells. The selected compounds were screened for their inhibition of tubulin polymerization in a cell-free in vitro assay. The test compounds significantly inhibited the rate of microtubule polymerization at 10 μM, as compared to vinblastine, a well known microtubule destabilizer. Examination of efficacy of isothiocyanates, thiocyanates, and selenocyanates suggests that these functional groups contribute in enhancing the anticancer activity of novel isatin analogues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2131. doi:10.1158/1538-7445.AM2011-2131

Optical Properties of Fluorescein‐labeled Organoclay

We report the preparation of aminopropyl-functionalized magnesium phyllosilicate (Mg-organoclay) conjugated to fluorescein isothiocyanate (FITC) functionality (FITC-organoclay) by one-pot sol-gel synthesis. The physical characteristics of the Mg- and FITC-organoclays were examined by scanning electron microscopy (SEM), X-ray diffraction (XRD), FT-IR and UV-Vis spectrophotometry. X-ray fluorescence and elemental analysis were conducted to confirm the composition of the Mg-organoclay. The FITC-organoclay particles were polydispersed with an average particle size of approximately 50 nm, as determined from SEM images. The XRD patterns of FITC-organoclay exhibited broad peaks and reduced basal d(001) indicating a less condensed and more disordered structure than was observed for Mg-organoclay. The conjugation between FITC- and Mg-organoclay was characterized by FT-IR spectroscopy. Fluorescence excitation and emission spectral data demonstrated the successful conjugation of FITC dye molecules to the Mg-organoclay. The time-resolved fluorescence measurements revealed that FITC had a lifetime of 4.58 ns, whereas the lifetime of FITC-organoclay required a double exponential fit (tau(1,2) = 0.72 and 2.68 ns). As a result, the lifetime of the FITC-organoclay was shorter than that of FITC in ethanol and indicated moderate photostability in the solution state. The cellular uptake of FITC-organoclay in human lung alveolar carcinoma epithelial cells (A549) was quantified and characterized.

Molecular Mapping of An IKS Channel Opener Reveals Crucial Interactions Between KCNE1 and the Kv7.1 Voltage Sensor Paddle

Voltage-gated K+ channels co-assemble with accessory subunits to form macromolecular complexes. In heart, assembly of Kv7.1 pore-forming subunits with KCNE1 auxiliary subunits generates the repolarizing K+ current IKS. We and others, recently suggested a strategic location of KCNE1 wedged close to helices S1 and S4 of two adjacent Kv7.1 voltage sensing domains (VSD) and nearby helix S6 of another Kv7.1 subunit. Here we show that the IKS channel opener, diisothiocyanostilbene-2′,2′-disulfonic acid (DIDS) acts on IKS as a gating-modifier, thereby converting the time- and voltage-dependent channels into almost voltage- and time-independent currents. While DIDS activates Kv7.1, it does not affect Kv7.2. The two isothiocyanate functionalities are crucial for the potent activating effect of DIDS on IKS, since 4′-acetamido-4′-isothiocyanostilbene-2′,2′-disulfonic acid (SITS) that has only one of these groups and 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS), which lacks isothiocyanate groups and thus cannot form covalent bonds with amino acids, do not activate IKS currents. Mutagenesis and modeling data indicate that DIDS activates IKS by docking to an externally-accessible pocket, formed at the interface between the superficial N-terminal boundary of the KCNE1 transmembrane segment and the VSD paddle motif of Kv7.1. DIDS does not activate the channel complex formed by co-expression of KCNE1 and a chimeric Kv7.1 endowed with a Kv7.2 VSD paddle. DIDS binding at the Kv7.1 VSD-KCNE1 interface reveals that two lysine residues, K41 in KCNE1 and K218 in Kv7.1 S3-S4 linker are distant to about 10 [[Unable to Display Character: A]]. Thus, KCNE1 affects Kv7.1 channel gating by closely interacting with the VSD paddle motif.

Bridgehead enolates and bridgehead alkenes in a welwistatin model series

Bridgehead metallation is possible in a ketone having the welwistatin skeleton, and this facilitates installation of the isothiocyanate function present in the natural product, and also enables synthesis of remarkable bridgehead alkenes.

N-(3-Triethoxysilylpropyl)-4-(isothiocyanatomethyl)-cyclohexane-1-carboxamide (TPICC): A heterobifunctional reagent for immobilization of biomolecules on glass surface

An efficient heterobifunctional reagent, N-(3-triethoxysilylpropyl)-4-(isothiocyanatomethyl)cyclohexane-1-carboxamide (TPICC) has been developed by a simple ‘two step reaction’ for the preparation of bioconjugates and immobilization of biomolecules such as oligonucleotides, peptides and proteins on the glass surface. The isothiocyanate functionality at one end of the reagent, TPICC was found specific for the ligands having either aminoalkyl (RNH 2) or mercaptoalkyl (R-SH) functionality. The synthesis of bioconjugates with the reagent was achieved through its isothiocyanate functionality at one end via the formation of stable thiourea linkage with aminoalkyl and dithiocarbamate linkage with mercaptoalkyl derivatives. The triethoxysilyl functionality of the reagent has been utilized for specific coupling with the virgin glass surface by a very simple methodology.

Synthesis of Thiourea-Linked Glycooligomers that Mimic the Branching Patterns of Natural Oligosaccharides

: A general blockwise strategy for the synthesis of accurately sequenced unnatural glycooligomers that can reproduce the branching patterns encountered in native oligosaccharides, based on the use of thiourea groups as intersaccharide bridges, is reported. The linear pseudooligosaccharide backbone is built from monosaccharide building blocks bearing a latent amino group and an isothiocyanate functionality through an iterative process in three steps per cycle that include: (i) thiourea-forming reaction, (ii) deprotection of the hydroxyl groups in the adduct and (iii) generation of a new amino group in the growing chain. Branching points are incorporated by inserting links bearing orthogonal amine functionalities at specific locations in the chain. The approach is illustrated by the preparation of glucopyranosylthiourea pseudoheptasaccharides that mimic the branching patterns of a phytoalexin elicitor oligosaccharine and of lentinan.

Label-free detection of proteins using SERS-based immuno-nanosensors

This paper describes the development and optimization of a novel class of SERS-based immuno-nanosensors for the label-free detection of specific proteins in complex environments (e.g., cell culture matrices and intracellular environments). These SERS-based nanosphere sensors are fabricated by depositing multiple layers of silver on silica nanospheres, followed by binding of the antibody of interest to the silver surface via a short rigid crosslinker. In these studies, several different crosslinkers were characterized and evaluated for optimal nanosensor activity. The crosslinkers evaluated contained either thiol or isothiocyanate functionalities, which bind to the silver surface on one end, while the other end of the crosslinker contained either a carboxylic or primary amine group, which reacts readily with the antibodies. These SERS-based nanosensors were also optimized for underlying silica sphere diameters, silica sphere coating conditions during silver deposition, number of silver layers applied, and silver surface coverage with crosslinkers. Upon optimization, the nanosensors were evaluated by monitoring their response to various antigens (e.g., human insulin or interleukin II) in complex environments.

A Convenient Synthesis of An Original, Bifunctional Chelator Based on an ON2S Ligand and its Coordination Chemistry with the Oxorhenium(V) Core

AbstractA semi‐rigid, tetradentate, bifunctional chelator (BFC) for the oxorhenium core has been synthesised in five steps with an overall yield of 46 %. An aromatic amine is available for conjugation to a peptide through an isothiocyanate function. This BFC is the first amine‐functionalised tetradentate ligand containing both amido and thiol functionalities. Monooxorhenium complexes were readily prepared from both the bifunctional chelator and a β‐alanine‐conjugated system. These complexes were obtained in good yield and were fully characterised. Moreover, they were found to be stable to ligand‐exchange reactions.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)