Nucleophilic Molecule Research Articles

Design of reactive-end DNA oligomers via incorporation of oxanine into oligonucleotides using terminal deoxynucleotidyl transferase

Abstract Oxanine (Oxa, O), a modified nucleobase, has a novel O-acylisourea structure. Oxa-incorporated oligodeoxynucleotides (ODNs) are reactive DNA oligomers that permit conjugation with various nucleophilic molecules in an activation-free manner. In this study, we developed a new procedure for enzymatic preparation of reactive-end DNA oligomers, using terminal deoxynucleotidyl transferase (TdT), in which a reactive Oxa base is incorporated into the 3′-end of ODNs. One limitation of TdT, an enzyme widely used for end labeling of DNA oligomers, is that it is difficult to control the number of incorporated labels, because it shows template-independent extension with random nucleotides. Notably, TdT showed a rate and efficiency of incorporation of the modified nucleobase, Oxa, different from that of natural bases. We investigated the conditions of TdT-mediated DNA incorporation of Oxa and achieved incorporation of Oxa at the 3′-end of ODNs by optimizing reaction parameters such as temperature and enzyme, cofactor, and substrate concentrations. We also confirmed the reactive functionality of Oxa after incorporation into ODNs by amide bonding conjugation with a polyamine (spermine) under physiological conditions, without need for an additional activation step.

Theoretical investigations on novel SiC5 siligraphene as gas sensor for air pollutants

The graphene-based gas sensors are limited by the weak physisorption to gas molecules, and the doping of silicon is an effective way to enhance its sensitivity. By density functional investigations, we reported a novel SiC5 siligraphene (g-SiC5) with great potential in detecting and sensing some specific air pollutants. The g-SiC5 is semimetallic as graphene is, possessing good structural and thermal stability. By comparing the adsorption performance of 12 common gas molecules, we have revealed that the nucleophilic gas molecules could form stable chemisorption with g-SiC5. Among the 12 gas molecules, NO, HCHO and SO2 exhibit moderate adsorption energies in the range of 0.4–0.6 eV when chemisorb on g-SiC5 and would open up a considerable band gap due to the orbital hybridization, thus changing the conductivity of g-SiC5 and making the sensing feasible. The further comparative research demonstrates that the g-SiC5 is superior to Si-doped graphene and other siligraphenes in gas sensing, enabling g-SiC5 to be a promising material as gas sensor for detecting NO, HCHO or SO2 from air mixture.

An easy method for the determination of active concentrations of cholinesterase reactivators in blood samples: Application to the efficacy assessment of non quaternary reactivators compared to HI-6 and pralidoxime in VX-poisoned mice

Organophosphorus nerve agents, like VX, are highly toxic due to their strong inhibition potency against acetylcholinesterase (AChE). AChE inhibited by VX can be reactivated using powerful nucleophilic molecules, most commonly oximes, which are one major component of the emergency treatment in case of nerve agent intoxication. We present here a comparative in vivo study on Swiss mice of four reactivators: HI-6, pralidoxime and two uncharged derivatives of 3-hydroxy-2-pyridinaldoxime that should more easily cross the blood-brain barrier and display a significant central nervous system activity. The reactivability kinetic profile of the oximes is established following intraperitoneal injection in healthy mice, using an original and fast enzymatic method based on the reactivation potential of oxime-containing plasma samples. HI-6 displays the highest reactivation potential whatever the conditions, followed by pralidoxime and the two non quaternary reactivators at the dose of 50 mg/kg bw. But these three last reactivators display equivalent reactivation potential at the same dose of 100 μmol/kg bw. Maximal reactivation potential closely correlates to surviving test results of VX intoxicated mice.

“Host–guest” binding of a luminescent dinuclear Au(i) complex based on cyclic diphosphine with organic substrates as a reason for luminescence tuneability

A dinuclear Au(i) complex with cyclic diphosphine is a versatile platform for different luminescence responses to nucleophilic and electrophilic molecules in solutions.

Obtaining graphene nanoplatelets from various graphite intercalation compounds

The work compares the exfoliation ability of different graphite materials (expanded graphite intercalation compound, thermally expanded and oxidatively intercalated graphites) and describes the properties of graphene nanoplatelets (GNPs) obtained dependently on intercalation/deintercalation conditions and reagents. Among the studied materials, the graphite intercalated with ammonium persulfate in sulfuric acid and expanded at 40 °C possesses the maximum ability for ultrasonic exfoliation in the presence of a surfactant. The exfoliation efficiency strongly depends on the content of water in sulfuric acid during the intercalation. The highest efficiency was achieved for the expanded graphite intercalation compound (EGIC) prepared in sulfuric acid containing diluted oleum, which may be explained by increased acidity of the medium and, correspondingly, redox potential of the persulfate compound. This is also related to increased amounts of oxygen groups in the GNPs obtained from the EGIC synthesized in 100% sulfuric acid and diluted oleum. Besides, the nature of surface groups on the GNPs strongly depends on the nature of a deintercalating reagent. Thus, the treatment of the EGIC with different nucleophilic molecules (such as water, ammonia, carbamide, hexamethylenetetramine, organic amines, etc.) can yield GNPs with various surface groups. The interaction between the EGIC and nucleophilic molecules does not only include the substitution of sulfate groups, but also redox reactions with participation of graphene layers. Depending on the nature of the nucleophile, those reactions can lead to the formation of different groups attached to the graphene surface. GNPs with almost pure surface were obtained when using ammonia and carbamide.

Unravelling the reaction mechanism of matrix metalloproteinase 3 using QM/MM calculations

The matrix metalloproteinase family (MMP) constitutes a family of zinc (Zn) proteases that catalyze the breaking of peptide bonds in proteins. These enzymes are very promising drug targets, since they are involved in remodeling and degradation of the extracellular matrix, which is a key process required for cancer metastasis, and thus, their reaction mechanism has been an area of intensive research. Early proposal based on acid base catalyzed hydrolysis, suggested that a conserved zinc bound water molecule acted as the nucleophile attacking the peptide bond carbon, after being activated by essential glutamate. The possibility of a direct nucleophilic attack by the enzyme, performed by the glutamate was also suggested. These are the key yet unsolved issues about MMP reaction mechanism.In the present work, we used hybrid quantum/classical calculations to analyze the structure and energetics of different possible hydrolysis reaction paths. The results support a water mediated mechanism, where both the nucleophile water molecule and the carbonyl oxygen of the scissile peptide bond are coordinated to zinc in the reactive configuration, while the essential glutamate acts as the base accepting the proton from the nucleophilic water. Formation of the carbon–oxygen bond and breaking of carbon–nitrogen bond were found to be concerted events, with a computed barrier of 14.8kcal/mol. Substrate polarization was found to be important for the observed reaction mechanism, and a substantial change in the metal coordination environment was observed, particularly, regarding the zinc–histidine coordination.

A First-Principles Study on Electron Donor and Acceptor Molecules Adsorbed on Phosphorene

Density functional theory calculations have been carried out to investigate single-layer phosphorene functionalized with two kinds of organic molecules, i.e., an electrophilic molecule tetracyano-p-quinodimethane (TCNQ) as electron acceptor and a nucleophilic molecule tetrathiafulvalene (TTF) as electron donor. The TCNQ molecule introduces shallow acceptor states in the gap of phosphorene close to the valence band edge, which makes the doped system become a p-type semiconductor. However, when the TTF molecule is adsorbed on the phosphorene, the occupied molecular states introduced into the gap are of deep donor states so that effective n-doping for transport cannot be realized. This disadvantageous situation can be amended by applying an external out-of-plane electric field with direction from phosphorene to TTF, or an in-plane tensile strain, or their combination, under which the conduction band edge of the phosphorene moves closer to the TTF-derived donor states, and then the TTF-adsorbed phosphorene system becomes an n-type semiconductor. It is also noted that the out-of-plane electric field and in-plane strain can modulate the band gap of the TTF-adsorbed phosphorene markedly. The effective bipolar doping of single-layer phosphorene via molecular adsorption, especially n-doping against its native p-doping propensity, and the good response of band gap in the infrared waveband of the TTF-adsorbed phosphorene to the out-of-plane electric field and in-plane strain would broaden the way to the application of this new type of two-dimensional material in nanoelectronic and optoelectronic devices.

Extending the chemistry of carbones: P–N bond cleavage via an SN2′-like mechanism

The reactivity of nucleophilic carbodiphosphorane (C(PPh3)2, 1) and carbodicarbene (C(C(NMe)2C6H4)2, 2) towards various dichlorophosphines has been explored. In most cases the expected carbone-for-chloride ligand exchange was observed. However, the use of MeN(PCl2)2 resulted in a unique P-N bond cleavage that, according to computational studies, occurred via an SN2'-like mechanism.

Complex micelles with the bioactive function of reversible oxygen transfer

A complex micelle as a hemoglobin functional model with the biaoactive function of reversible oxygen transfer has been constructed through the hierarchical assembly of the diblock copolymer poly(ethylene glycol)-blockpoly(4-vinylpyridine-co-N-heptyl-4-vinylpyridine) (PEG-b-P(4VP-co-4VPHep)), tetrakis(4-sulfonatophenyl)porphinato iron(II) (Fe(II)TPPS) and β-cyclodextrin (β-CD). The μ-oxo dimer of Fe(II)TPPS was successfully inhibited because the Fe(II)TPPS was included into the cavities of β-CDs through host-guest interaction. Fe(II)TPPS coordinated with pyridine groups functions as the active site to reversibly bind dioxygen. In adition, the host-guest inclusion (β-CD/Fe(II)TPPS) was encapsulated in the hydrophobic core of the complex micelle and tightly fixed by P4VP chains. The hydrophilic PEG blocks stretched in aqueous solution to constitute the shells which stabilize the structure of the complex micelle as well as endow the complex micelle with sufficient blood circulation time. Dioxygen can be bound to the Fe(II)TPPS located in the confined space and excellent reversibility of the binding-release process of dioxygen can be achieved. The quaternary amine N-heptyl-4-vinylpyridine can coerce abundant S2O4 2− ions into the core of the complex micelle to facilitate the self-reduction process. Dioxygen adducts (Fe(II)TPPS(O2)) were effectively protected by the double hydrophobic barriers constructed by the cavity of the cyclodextrin and the core of the complex micelle which enhances the ability to resist nucleophilic molecules. Therefore, the rationally designed amphiphilic structure can work as a promising artificial O2 carrier. Potentially, the complex micelle can be expected to improve the treatment of diseases linked with hypoxia.

Gastroprotective effects of goniothalamin against ethanol and indomethacin-induced gastric lesions in rats: Role of prostaglandins, nitric oxide and sulfhydryl compounds

Goniothalamin (GTN), a styryl-lactone, is a secondary metabolite naturally found in its enantiomeric form (R) in plants of the genus Goniothalamus (Annonaceae). The antiproliferative activity against human tumor cell lines reported in several studies suggest that the α,β-unsaturated δ-lactone moiety emerges as a key Michael acceptor for cysteine residues or other nucleophilic biological molecules. Our group reported on the in vivo activity of (R)- and (S)-GTN as well as its racemic form (rac-GTN) in both Ehrlich solid tumor and carrageenan-induced paw edema in mice, without side effects in the effective doses. Despite the rich body of data on the in vitro GTN biological activity, much less is known about its in vivo pharmacological action. Herein we describe the gastroprotective activity of rac-GTN on chemical-induced gastric ulcers models in rats. GTN has a potent gastroprotective effect on ethanol-induced ulcers (effective dose50=18mg/kg) and this activity is dependent on sulfhydryl compounds and prostaglandins generation, but independent of nitric oxide (NO), gastric secretion and mucus production. We hypothesize that goniothalamin may act as a mild irritant, inducing the production of sulfhydryl compounds and prostaglandins, in a process known as adaptive cytoprotection. This hypothesis is supported by the fact that Michael acceptors are the most potent inducers of antioxidant response (as activation of Nrf2 pathway) through generation of mild oxidative stress and that gastroprotective activity of goniothalamin is inhibited after pre-treatment with NEM (N-ethylmaleimide) and NSAID (non-steroidal anti-inflammatory drugs), highlighting the importance of sulfhydryl compounds and prostaglandins on GTN activity.

Evidence of co-operativity in the pre-micellar region in the hydrolytic cleavage of phenyl salicylate in the presence of cationic surfactants of CTAB, TTAB and CPC

The effects of cationic surfactants of cetyl trimethyl ammonium bromide (CTAB), tetradecyl trimethyl ammonium bromide (TTAB) and cetyl pyridinium chloride (CPC) on the kinetics of intramolecular general base catalyzed hydrolysis ([OH−] range 0.05–0.1molL−1) of phenyl salicylate have been studied at different temperatures. The rate is independent of [OH−] in the studied range. The anionic surfactant sodium dodecyl sulphate (SDS) has no effect on the rate. The presence of small amount of any of these cationic surfactants well below its critical micelle concentration markedly inhibits the rate of reaction suggesting a pre-micellar aggregation between the substrate and surfactant monomers. The kinetic data have been analyzed in terms of earlier reported models (Piszkiewicz's co-operativity model and Raghavan and Srinivasan's model) for micellar catalysis. The binding constants between the substrate and the surfactants evaluated from the two models are in good agreement. Three dimensional structure of the pre-micellar aggregate controls the approach of the nucleophile water molecule to the reaction center. The planar structure of the pyridinium head group of CPC provides less steric hindrance to the attacking water molecule that leads to the least enthalpy of activation for CPC among the three surfactants. The association between the negatively charged substrate and the cationic surfactant is favored owing to electrostatic as well as hydrophobic interactions. The binding between the substrate and pre-micelles follows the order: CPC>TTAB>CTAB.

Synthesis and characterization of trinuclear osmium clusters containing diallylphosphines as hemilabile ligands: Experimental and theoretical studies of their reactivity with nucleophilic molecules

A series of [Os3(CO)12] phosphine substituted derivatives containing alkenylphosphine ligands has been synthesized starting from the cluster [Os3(CO)10(MeCN)2] and RP(CH2CHCH2)2 where R = iPr2N, o‒MeOC6H4 or p−MeOC6H4. It was shown that the terminal alkenyl substituent in these phosphines undergoes coordination at the metal triangle to give the allyl fragment coordinated through the double bond. Solid–state structures of [Os3(CO)10{μ:κ3(P,C,C)–RP(CH2CCH2)2}] [R = iPr2N (1a), o–MeOC6H4 (1c)], [Os3(CO)10{κ1(P)–iPr2NP(CH2CCH2)2}L] [L = CO (3a), PPh3 (4a)], [Os3(CO)9(PPh3){μ:κ3(P,C,C)–iPr2NP(CH2CCH2)2}] (5a) and [Os3(CO)10(μ:η1:η3–C3H4){κ1(P)–p–MeOC6H4P(CH2CCH2)2}] (6b) were established by X–ray crystallography; all products obtained were also characterized by IR and NMR (1H, 13C{1H}, 31P{1H}, 1H–1H and 1H–13C correlations) spectroscopy. It was shown that [Os3(CO)10{μ:κ3(P,C,C)–(diallylphosphine)}] compounds in solution are present as a mixture of isomers; this isomerization is described as a reorientation of the coordinated double bond respect to the triosmium metal triangle. Also, reactivity studies were performed with CO, PPh3 and C3H4, establishing the hemilabile properties of the diallyphosphine ligands with the cleavage of the allyl π–coordination when the nucleophilic molecules are CO and PPh3; in contrast, the nucleophile C3H4 leads to cleavage and insertion of the allene into the Os–Os bond.

CrXTe3(X = Si, Ge) nanosheets: two dimensional intrinsic ferromagnetic semiconductors

Two-dimensional (2D) ferromagnetic semiconductors hold a great potential for nano-electronic and spintronic devices. Nevertheless, their experimental realization remains a big challenge. Through first-principles calculations, we here demonstrate the possibility of realizing 2D ferromagnetic semiconductors simply by exfoliating layered crystals of CrXTe3 (X = Si, Ge). The exfoliation of CrXTe3 is feasible due to its small cleavage energy, and CrXTe3 nanosheets can form free-standing membranes. Interestingly, upon exfoliation, the ferromagnetism and semiconducting character are well preserved from bulk to the nanosheet form. Long-range ferromagnetic order with a magnetization of 3 μB per Cr atom is confirmed in 2D CrXTe3 from classical Heisenberg model Monte Carlo simulations. Both bulk and 2D CrXTe3 are indirect-gap semiconductors with their valence and conduction bands fully spin-polarized in the same direction, which is promising for spin-polarized carrier injection and detection. We further demonstrate the tunability and enrichment of the properties of CrXTe3 nanosheets via external operations. Under moderate tensile strain, the 2D ferromagnetism can be largely enhanced. By pure electron doping or adsorbing nucleophilic molecules, CrXTe3 nanosheets become 2D half metals.

Hydrazinolysis of 3-R-[1,2,4]Triazino[2,3-c]quinazolin-2-ones. Synthetic and Theoretical Aspects

It has been shown that heating of 3-R-[1,2,4]triazino[2,3-c]quinazolin-2-ones with 5-fold excess of hydrazine hydrate in propanol-2 gave the corresponding 3-(2-aminophenyl)-6-R-1,2,4-triazin-5-ones with high yields. The mechanism of the reaction has been proposed based on the results of theoretical investigation at B3LYP and MP2 levels of theory. According to calculations, an attack of the hydrazine molecule on the C6═N7 double bond leads to ring-opening with N5-C6 bond cleavage. The process continues by addition of the second nucleophile molecule and elimination of the formazan fragment through a series of transformations that yield the target product.

Optimized fluorescent labeling to identify memory B cells specific for Neisseria meningitidis serogroup B vaccine antigens ex vivo.

Antigen-specific memory B cells generate anamnestic responses and high affinity antibodies upon re-exposure to pathogens. Attempts to isolate rare antigen-specific memory B cells for in-depth functional analysis at the single-cell level have been hindered by the lack of tools with adequate sensitivity. We applied two independent methods of protein labeling to sensitive and specific ex vivo identification of antigen-specific memory B cells by flow cytometry: stringently controlled amine labeling, and sortagging, a novel method whereby a single nucleophilic fluorochrome molecule is added onto an LPETG motif carried by the target protein. We show that sortagged NadA, a major antigen in the meningococcal serogroup B vaccine, identifies NadA-specific memory B cells with high sensitivity and specificity, comparable to NadA amine-labeled under stringent reaction parameters in a mouse model of vaccination. We distinguish NadA-specific switched MBC induced by vaccination from the background signal contributed by splenic transitional and marginal zone B cells. In conclusion, we demonstrate that protein structural data coupled with sortag technology allows the development of engineered antigens that are as sensitive and specific as conventional chemically labeled antigens in detecting rare MBC, and minimize the possibility of disrupting conformational B cell epitopes.

Toward Plasmonic Biosensors Functionalized by a Photoinduced Surface Reaction

We present a method for efficient coupling of amine nucleophilic molecules of choice to a nanostructured gold surface via photoinduced surface chemistry. The method is based on photoactive self-assembled monolayers and can be used to functionalize localized surface plasmon resonance (LSPR) based biosensors with biorecognition motifs while reducing nonspecific binding via introduction of hydrophilic units. The photoactive linker molecule, 5-bromo-7-nitroindoline, couples nucleophilic molecules such as biotin ethylenediamine to a surface when exposed to UV-light. The specific, noncovalent recognition between biotin and streptavidin is used for demonstration of a simple biorecognition assay based on the LSPR sensing principle. By doing so, one can envision that the binding of any streptavidin fusion protein, being attached to specific spots at the gold surface, is monitored by an LSPR peak shift. Since the surface functionalization is based on a photoinduced reaction, this method can be used to functionalize the surface in a local and site-specific way, and biomedical applications such as drug-screening platforms, microarrays, solid support protein synthesis, and even single molecule experiments can be envisioned.

Thiolactone Sulfoxides as New Reactive Metabolites Acting as Bis-Electrophiles: Implication in Clopidogrel and Prasugrel Bioactivation

The antithrombotics of the tetrahydrothienopyridine series, clopidogrel and prasugrel, are prodrugs that must be metabolized in two steps to become pharmacologically active. The first step is the formation of a thiolactone metabolite. The second step is a cytochrome P450 (P450)-dependent oxidation of this thiolactone resulting in the formation of a sulfenic acid that is eventually reduced into the corresponding active thiol. It has been postulated that the sulfenic acid metabolite resulted from a nucleophilic attack of water on a highly reactive thiolactone sulfoxide derived from P450-dependent oxidation of the thiolactone primary metabolite. The data described in the present article are in complete agreement with this proposition as they show that it was possible to trap these thiolactone sulfoxides by a series of nucleophiles such as amines, thiols, or cyclopentane-1,3-dione (CPDH), an equivalent of dimedone that is used as a sulfenic acid trapping agent. HPLC-MS studies showed that various bis-adducts having incorporated two nucleophile molecules were formed in these reactions. One of them that resulted from the oxidation of 2-oxo-prasugrel by human liver microsomes in the presence of ethanolamine and CPDH was isolated and completely characterized by (1)H and (13)C NMR spectroscopy in addition to MS and MS(2) spectrometry. All metabolites derived from an attack of H2O or an amine at the CO carbon of the intermediate thiolactone sulfoxide existed as a mixture of two diastereomers having a cis configuration of the double bond, whereas those formed in the presence of thiols appeared as a mixture of four diastereomers with a cis or trans configuration of the double bond. This led us to propose tentative mechanisms for the previously reported formation of trans isomers of the active thiol metabolite of clopidogrel upon microsomal metabolism of this antithrombotic in the presence of thiols. The results described in this article showed that thiolactone sulfoxides are formed as reactive metabolites during the metabolism of clopidogrel and prasugrel and are able to react as bis-electrophiles with a variety of nucleophiles. The possible implications of the formation of these reactive metabolites in the pharmacological and/or secondary toxic effects of these drugs remain to be studied.

Photoinitiated release of an aziridinium ion precursor for the temporally controlled alkylation of nucleophiles

A photo-activatable aziridinium precursor has been developed to investigate the possibility of a photo-initiated traditional nucleophilic reaction. The photolysis of a quaternary amine yields a tertiary amine and has allowed us to temporally control aziridinium formation and subsequent alkylation of a colorimetric nucleophilic reporter molecule. We have also used this photo-initiated reaction to alkylate a sulfhydryl group. This new photo-initiated alkylation strategy is water-soluble and expands the toolkit of photo-activated crosslinkers for protein labeling research.

Antibacterial Activity of 4-Oxo-(E)-2-hexenal from Adults and Nymphs of the Heteropteran,Dolycoris baccarum(Heteroptera: Pentatomidae)

We identified 4-oxo-(E)-2-hexenal (4-OHE) as a common component of the secretion from both Dolycoris baccarum nymphs (66.5 ± 34.7 µg/bug) and adults (87.4 ± 48.0 µg/bug) by GC/MS. We also found that this compound inhibited the growth of bacteria starting at 10 µg. The stronger antibacterial activity of 4-OHE than that of (E)-2-hexenal and (E)-2-octenal might be explained by the reactivity of α,β-unsaturated aldehydes with nucleophilic molecules.

Identification of novel phytotoxins from Australian range plants linked to severe mammalian digestive toxicity

We have used a digestive developmental in vivo zebrafish model to examine Australian rangeland plants for their involvement in specific digestive disorders in livestock. The model allows us to discover causative phytotoxins linked to a syndrome. Here, we report the isolation and structural elucidation of five isoflavonoid compounds 1-5 with specific biological activity. From the active fraction, we identified the causative phytotoxins as two novel isoflavonoids (1, 4), both with an unusual skeleton. In addition, we isolated an additional novel isoflavonoid (3), a novel pterocarpan derivative (5), and a known isoflavone, betavulgarin (2). Compound 4 contributed to the specific biological damage in the zebrafish. The compound is highly reactive and conjugates spontaneously with common nucleophilic biological molecules, indicating a strong electrophilic nature, suggesting a possible mechanism of action that explains death in the animals. The isolation and structural elucidation of 1- 5 , together with their biological data, will be presented.