Cytotoxic and Antifungal Amides Derived from Ferulic Acid: Molecular Docking and Mechanism of Action.

Amides derived from ferulic acid have a wide spectrum of pharmacological activities, including antitumor and antifungal activity. In the present study, a series of ten amides were obtained by coupling reactions using the reagents (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyBOP) and N,N'-dicyclohexylcarbodiimide (DCC). All the compounds were identified on the basis of their IR, 1H- and 13C-NMR, HRMS data, and with yields ranging from 43.17% to 91.37%. The compounds were subjected to cytotoxic tests by the alamar blue technique and antifungal screening by the broth microdilution method to determine the minimum inhibitory concentration (MIC). The amides 10 and 11 displayed the best result in both biological evaluations, and compound 10 was the most potent and selective in HL-60 cancer cells, with no cytotoxicity on healthy cells. This amide had antifungal activity in all strains and had the lowest MIC against Candida albicans and Candida tropicalis. The possible mechanism of antifungal action occurs via the fungal cell wall. Molecular modeling suggested that compounds 10 and 11 interact with the enzymes GWT1 and GSC1, which are essential for the development of C. albicans. The findings of the present study demonstrated that compounds 10 and 11 may be used as a platform in drug development in the future.

Aqueous suspension of biocompatible reduced graphene oxide- Au NPs composite as an effective recyclable catalyst in a Betti reaction.

Synthesis of noble metal nanoparticles (NPs) on modified graphene oxide with biocompatible polymers has attracted significant due to their unique properties and various applications. In this research, covalent-modified graphene oxide (MGO) with diacid terminated poly (ethylene glycol) (PEG) was used as a substrate and stabilizing of Au (ш). The reduction of MGO-Au (ш) complex with hydrazine monohydrate under reflux condition obtained biocompatible reduced MGO (rMGO)-Au NPs. Diacid terminated PEG obtained from the reaction of PEG with succinic anhydride in the presence of N,N- dicyclohexylcarbodiimide (DCC) and 4-methylamino pyridine (DMAP) was attached to GO sheets to prevent from the aggregation of rMGO sheets and Au NPs. The resulting aqueous suspension was characterized through UV-vis, FT-IR, Raman, XRD, DLS-zeta potential, SEM, EDX and TEM. Furthermore, nanocomposite showed good catalytic behavior in Betti reaction- synthesis of 1-(α-aminoalkyl)-2-naphthols. The favorable properties of colloidal nanocomposite were attributed to the stable and well distribution Au NPs on rMGO.

Preparation, Characterization, and Insecticidal Activity of Avermectin-Grafted-Carboxymethyl Chitosan.

Avermectin-grafted-N,O-carboxymethyl chitosan (NOCC) derivative was obtained by esterification reaction using dicyclohexylcarbodiimide (DCC) as dehydrating agent and 4-methylaminopyridine as catalyst. The structures of the conjugate were confirmed by FT-IR, (1)H NMR, and XRD. Insecticidal activities against armyworms, carmine spider mites, black bean aphids, and brown plant hoppers were investigated at concentrations ranging from 0.16 to 1000 mg/L. At the concentration of 1000 mg/L and 500 mg/L, the lethal rate was 100%. Good insecticidal activity at 4 mg/L was still shown, especially against the black bean aphids and brown plant hoppers. Moreover, the photostability of the conjugate was evaluated and showed an apparent improvement. At 300 mins, the residual rate of the conjugate was 11.22%, much higher than 0.2% of the avermectin technical material. The conjugate we developed showed potential for further study and application in crop protection.

Halogenated benzoate derivatives of altholactone with improved anti-fungal activity.

Altholactone exhibited the anti-fungal activity with a high MIC value of 128 µg ml(-1) against Cryptococcus neoformans and Saccharomyces cerevisiae. Fifteen ester derivatives of altholactone 1-15 were modified by esterification and their structures were confirmed by spectroscopic methods. Most of the ester derivatives exhibited stronger anti-fungal activities than that of the precursor altholactone. 3-Bromo- and 2,4-dichlorobenzoates (7 and 15) exhibited the lowest minimal inhibitory concentration (MIC) values against C. neoformans at 16 µg ml(-1), while the 4-bromo-, 4-iodo-, and 1-bromo-3-chlorobenzoates (11-13) displayed potent activity against S. cerevisiae with MIC values of 1 µg ml(-1). In conclusion, this analysis indicates that the anti-fungal activity of altholactone is enhanced by addition of halogenated benzoyl group to the 3-OH group.

Simultaneous refolding of denatured PsbS and reconstitution with LHCII into liposomes of thylakoid lipids.

The thylakoid membrane protein PsbS is critical for quenching excessive excitation energy in mechanisms that involve the light-harvesting complexes of photosystem II. Liposomes of thylakoid lipids have been shown to be a very good platform to study photosynthetic membrane proteins and their interactions. In this study, we simultaneously refolded and reconstituted functional pea PsbS into liposomes of thylakoid lipids starting from denatured expressed protein. Intrinsic fluorescence spectroscopy, trypsin digestion, and circular dichroism spectroscopy were used to characterize the native state of PsbS in the proteoliposomes. The functionality of refolded PsbS was further demonstrated by its effect on the fluorescence quenching of the major antenna system of photosystem II (LHCII) co-inserted into the liposomes. The fluorescence yield of native trimeric LHCII was lowered by PsbS by 50% at neutral pH and by a further 25% upon lowering the pH to 4.5. Furthermore, the acid-induced fluorescence reduction was completely reversed by addition of N,N'-dicyclohexylcarbodiimide, an inhibitor of protein protonation. These results indicate that reconstituted PsbS induces strong quenching of LHCII sensing changes in local pH via its protonation sites.

EPR Studies of V-ATPase with Spin-Labeled Inhibitors DCC and Archazolid: Interaction Dynamics with Proton Translocating Subunit†c.

Vacuolar-type H(+) -ATPases (V-ATPases) have gained recent attention as highly promising anticancer drug targets, and therefore detailed structural analyses and studies of inhibitor interactions are very important research objectives. Spin labeling of the V-ATPase holoenzyme from the tobacco hornworm Manduca sexta and V-ATPase in isolated yeast (Saccharomyces cerevisiae) vacuoles was accomplished by two novel methods involving the covalent binding of a (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) derivative of N,N'-dicyclohexylcarbodiimide (DCC) to the essential glutamate residue in the active site and the noncovalent interaction of a radical analogue of the highly potent inhibitor archazolid, a natural product from myxobacteria. Both complexes were evaluated in detail by electron paramagnetic resonance (EPR) spectroscopic studies and double electron-electron resonance (DEER) measurements, revealing insight into the inhibitor binding mode, dynamics, and stoichiometry as well as into the structure of the central functional subunit c of these medicinally important hetero-multimeric proton-translocating proteins. This study also demonstrates the usefulness of natural product derived spin labels as tools in medicinal chemistry.

Indole-based novel small molecules for the modulation of bacterial signalling pathways.

Gram-negative bacteria such as Pseudomonas aeruginosa use N-acylated L-homoserine lactones (AHLs) as autoinducers (AIs) for quorum sensing (QS), a major regulatory and cell-to-cell communication system for social adaptation, virulence factor production, biofilm formation and antibiotic resistance. Some bacteria use indole moieties for intercellular signaling and as regulators of various bacterial phenotypes important for evading the innate host immune response and antimicrobial resistance. A range of natural and synthetic indole derivatives have been found to act as inhibitors of QS-dependent bacterial phenotypes, complementing the bactericidal ability of traditional antibiotics. In this work, various indole-based AHL mimics were designed and synthesized via the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N,N'-dicyclohexylcarbodiimide (DCC) mediated coupling reactions of a variety of substituted or unsubstituted aminoindoles with different alkanoic acids. All synthesized compounds were tested for QS inhibition using a P. aeruginosa QS reporter strain by measuring the amount of green fluorescent protein (GFP) production. Docking studies were performed to examine their potential to bind and therefore inhibit the target QS receptor protein. The most potent compounds 11a, 11d and 16a showed 44 to 65% inhibition of QS activity at 250 µM concentration, and represent promising drug leads for the further development of anti-QS antimicrobial compounds.

Self-assembled polymeric nanoparticle of PEGylated chitosan-ceramide conjugate for systemic delivery of paclitaxel.

Chitosan has been widely explored as one of the most favorable biomaterials for various pharmaceutical applications due to its biodegradability and biocompatibility. Here, we report novel PEGylated-chitosan-ceramide (PEG-CS-CE) that forms stable polymeric nanoparticles capable of functioning as efficient carriers of hydrophobic drug molecules. The chitosan-ceramide conjugate (CS-CE) was linked with amine-polyethyleneglycol (NH2-PEG2000) by using dicyclohexylcarbodiimide/N-hydroxysuccinimide (DCC-NHS) to obtain PEG-CS-CE that could exhibit steric stabilization in biological environments. The structure of the conjugate was determined by proton ((1)H) NMR and FT-IR spectrometry. Under suitable conditions, the PEG-CS-CE self-assembled to form colloidally stable nanoparticles with a mean diameter of ∼ 200 nm. Further, hydrophobic anti-tumor agent paclitaxel (PTX) was incorporated into the polymeric nanoparticle with 90% loading efficiency and 11.3% loading capacity via an emulsion-solvent evaporation method. The PTX-loaded PEG-CS-CE nanoparticle showed sustained release and exhibited higher cellular uptake and a comparable cytotoxic efficacy to that of free PTX on B16F10 melanoma and MCF-7 human breast adenocarcinoma cell lines. The empty nanoparticle showed no toxicity, indicating that the co-polymer is safe to use in drug delivery. The polymeric nanoparticle PEG-CS-CE developed by us represent promising nanocarriers of hydrophobic drug molecules.

Synthesis and characterization of new derivatives of alginic acid and evaluation of their iron(III)-crosslinked beads as potential controlled release matrices.

CONTEXT: The excellent gelling and safety profiles of alginic acid combined, however, with drawbacks of its ionotropically crosslinked beads (i.e. their quick release of loaded drugs) prompted us to chemically modify alginic acid. OBJECTIVE: Alginic acid was chemically conjugated with four amines of varying hydrophilic-hydrophobic properties (i.e. tris(hydroxymethyl)methyl-, allyl-, benzyl- or pentyl-amines) in an attempt to enhance the drug release profiles from respective metal crosslinked beads. MATERIALS AND METHODS: Chemical conjugation procedures were performed using dicyclohexylcarbodiimide as a coupling agent and the resulting new derivatives were characterized using proton nuclear magnetic resonance ((1)H NMR), infrared (IR) spectroscopy and differential scanning calorimetry (DSC). These modified polymers were used to prepare iron (III)-crosslinked beads loaded with folic acid as model drug, which were tested in vitro to assess their folic acid release profiles. RESULTS AND DISCUSSION: Interestingly, the resulting beads accessed enteric release kinetics, with tris(hydroxymethyl)methyl-amide alginic conjugate producing most pronounced enteric profile. CONCLUSION: The results suggest the possibility of achieving controlled drug release from alginate-based beads via facile chemical modification of alginic acid.

Sensitive determination of the potential biomarker sarcosine for prostate cancer by LC-MS with N,N'-dicyclohexylcarbodiimide derivatization.

Sarcosine, a potential biomarker of prostate cancer, has drawn great attention in recent years. However, controversial research keeps arising about its role as a biomarker that might come from the two isomers (α-alanine and ß-alanine) of sarcosine due to their same molecular weight and similar properties, which could interfere with the accurate detection of sarcosine. In this study, a simple and sensitive method was developed for the detection of sarcosine and the two isomers by LC with ion-trap MS through a novel derivatization reagent N,N'-dicyclohexylcarbodiimide. N,N'-Dicyclohexylcarbodiimide is usually considered as a condensation reagent, however, it was directly used as a derivatization reagent through a rearrangement side reaction in this study. The proposed method not only improved the chromatographic retention behavior of sarcosine and its two isomers, which was a benefit to their separation, but also dramatically enhanced the detection sensitivity of sarcosine, which was more favorable for real sample analysis. The factors affecting the productivity of the derivatization reaction, such as reaction time and amount of derivatization reagent, were systematically optimized. The method shows good linearity (R(2) > 0.99), sensitivity with LODs of sarcosine as low as 1 ng/mL, and repeatability with the RSD < 6.07%. The developed method was applied to the analysis of urine.

A facile one-pot synthesis of a fluorescent agarose-O-naphthylacetyl adduct with slow release properties.

A microwave assisted facile synthesis of a fluorescent 6-O-naphthylacetyl agarose (NA-agarose) employing carbodiimide chemistry (dicyclohexylcarbodiimide/4-dimethylaminopyridine) has been described. NA-agarose was characterized by TGA, GPC, UV spectrophotometry, fluorescence spectroscopy, FT-IR, (1)H and (13)C NMR spectra, exhibiting that in NA-agarose the naphthylacetyl group was attached to the backbone of the agarose polymer. The hydrolysis of NA-agarose in heterogeneous aqueous phase showed that the 1-naphthyl acetic acid (NAA), a plant growth regulator, got released in a controlled manner, the release rate being dependent on the hydrophilicity of the polymer adduct as well as on pH and temperature. The fluorescence emission (λmax 332 nm) of NA-agarose (1×10(-3) M) in ethylene glycol was significantly higher (ca. 82%) than that of the molar equivalent of NAA content in the product i.e. 0.08 mg in 1×10(-3) M solution. The resulting polymer would be of potential utility as a sustained release plant growth regulator and sensory applications.

Single-molecule analysis of F0F1-ATP synthase inhibited by N,N-dicyclohexylcarbodiimide.

N,N-Dicyclohexylcarbodiimide (DCCD) is a classical inhibitor of the F0F1-ATP synthase (F0F1), which covalently binds to the highly conserved carboxylic acid of the proteolipid subunit (c subunit) in F0. Although it is well known that DCCD modification of the c subunit blocks proton translocation in F0 and the coupled ATP hydrolysis activity of F1, how DCCD inhibits the rotary dynamics of F0F1 remains elusive. Here, we carried out single-molecule rotation assays to characterize the DCCD inhibition of Escherichia coli F0F1. Upon the injection of DCCD, rotations irreversibly terminated with first order reaction kinetics, suggesting that the incorporation of a single DCCD moiety is sufficient to block the rotary catalysis of the F0F1. Individual molecules terminated at different angles relative to the three catalytic angles of F1, suggesting that DCCD randomly reacts with one of the 10 c subunits. DCCD-inhibited F0F1 sometimes showed transient activation; molecules abruptly rotated and stopped after one revolution at the original termination angle, suggesting that hindrance by the DCCD moiety is released due to thermal fluctuation. To explore the mechanical activation of DCCD-inhibited molecules, we perturbed inhibited molecules using magnetic tweezers. The probability of transient activation increased upon a forward forcible rotation. Interestingly, during the termination F0F1, showed multiple positional shifts, which implies that F1 stochastically changes the angular position of its rotor upon a catalytic reaction. This effect could be caused by balancing the angular positions of the F1 and the F0 rotors, which are connected via elastic elements.

Chitosan/alginate crosslinked hydrogels: preparation, characterization and application for cell growth purposes.

Chitosan hydrogels may be formed by various mechanisms. In this study, we aimed to form hybrid polymer networks of chitosan with alginate using a crosslinker which enabled the covalent binding of the two macromolecules. The structural and thermal characterization of these hydrogels was performed by using Fourier transform infrared (FTIR) and differential scanning calorimeter (DSC). The morphological analysis of the crosslinked material was investigated by scanning electron microscopy (SEM) and a scanning probe microscope with atomic force microscope (AFM) attachment. The swelling properties of these gels were analyzed in water and in phosphate buffered saline (PBS) solution. The presence of alginate in a chitosan/alginate hydrogel was shown to support the hydrogel stability. Compared to chitosan/alginate (1/2) hydrogel prepared with 1wt% DCC, the swelling of chitosan/alginate (1/2) hydrogels prepared with 3wt% DCC was limited. To measure the degree of cell proliferation, the hydrogels were seeded with L929 mouse fibroblasts and cell numbers measured by neutral red uptake assay. The cell attachment was also followed by (SEM) photography. It was observed that chitosan/alginate (1/2) hydrogels with 1wt% (DCC) provides a better environment for cell attachment and proliferation. This study presents functional hydrogel formation by crosslinked chitosan and alginate, a novel biomaterial which also supports cell growth.

Comparable effects of low-intensity electromagnetic irradiation at the frequency of 51.8 and 53 GHz and antibiotic ceftazidime on Lactobacillus acidophilus growth and survival.

The effects of low-intensity electromagnetic irradiation (EMI) with the frequencies of 51.8 and 53 GHz on Lactobacillus acidophilus growth and survival were revealed. These effects were compared with antibacterial effects of antibiotic ceftazidime. Decrease in bacterial growth rate by EMI was comparable with the inhibitory effect of ceftazidime (minimal inhibitory concentration-16 µM) and no enhanced action was observed with combined effects of EMI and the antibiotic. However, EMI-enhanced antibiotic inhibitory effect on bacterial survival. The kinetics of the bacterial suspension oxidation-reduction potential up to 24 h of the growth was changed by EMI and ceftazidime. The changes were more strongly expressed by combined effects of EMI and antibiotic especially up to 12 h. Moreover, EMI did not change overall energy (glucose)-dependent H(+) efflux across the membrane but it increased N,N'-dicyclohexylcarbodiimide (DCCD)-inhibited H(+) efflux. In contrast, this EMI in combination with ceftazidime decreased DCCD-sensitive H(+) efflux. Low-intensity EMI had inhibitory effect on L. acidophilus bacterial growth and survival. The effect on bacterial survival was more significant in the combination with ceftazidime. The H(+)-translocating F 0 F 1-ATPase, for which DCCD is specific inhibitor, might be a target for EMI and ceftazidime. The revealed bactericide effects on L. acidophilus can be applied in biotechnology, food producing and safety technology.

Enhancing sensitivity of liquid chromatographic/ion-trap mass spectrometric determination of jasmonic acid by derivatization with N,N'-dicyclohexylcarbodiimide.

A simple and sensitive method for the detection of jasmonic acid (JA) by liquid chromatography-ion trap mass spectrometry (LC-IT-MS/MS) after derivatization is described. The highlight of this method was that it involved a rearrangement reaction which was often considered as a side reaction in the formation of amides and esters using N,N'-dicyclohexylcarbodiimide (DCC) as a condensation reagent. While in this study, DCC was used as a derivatization reagent directly. Internal standard dehydrojasmonic acid (DHJA) was used to ensure the accuracy. Derivatives provided good signals corresponding to the protonated molecular ions [M + H](+) and product ions. Sensitivity had been improved 100-fold with the LOD being 0.1 ng mL(-1) in comparison with the LC-IT-MS analysis without derivatization. This proposed method was reliable with good recoveries found in rice floret and wintersweet, allowing quantification in real samples to be more accurate and efficient, thus offering a good way to further study the physiological and biological activities of jasmonic acid in plants.

Copper-free click biofunctionalization of silicon nitride surfaces via strain-promoted alkyne-azide cycloaddition reactions.

Cu-free "click" chemistry is explored on silicon nitride (Si(3)N(4)) surfaces as an effective way for oriented immobilization of biomolecules. An ω-unsaturated ester was grafted onto Si(3)N(4) using UV irradiation. Hydrolysis followed by carbodiimide-mediated activation yielded surface-bound active succinimidyl and pentafluorophenyl ester groups. These reactive surfaces were employed for the attachment of bicyclononyne with an amine spacer, which subsequently enabled room temperature strain-promoted azide-alkyne cycloaddition (SPAAC). This stepwise approach was characterized by means of static water contact angle, X-ray photoelectron spectroscopy, and fluorescence microscopy. The surface-bound SPAAC reaction was studied with both a fluorine-tagged azide and an azide-linked lactose, yielding hydrophobic and bioactive surfaces for which the presence of trace amounts of Cu ions would have been problematic. Additionally, patterning of the Si(3)N(4) surface using this metal-free click reaction with a fluorescent azide is shown. These results demonstrate the ability of the SPAAC as a generic tool for anchoring complex molecules onto a surface under extremely mild, namely ambient and metal-free, conditions in a clean and relatively fast manner.

(S)-Naproxen as a platform to develop chiral derivatizing reagent for reversed-phase high-performance liquid chromatographic enantioseparation of analytes having a carbonyl functional group.

(S)-Naproxen was used to synthesize a chiral reagent, (S)-2-(6-methoxynaphthalen-2-yl)propanehydrazide, by itsreaction with hydrazine hydrate in the presence of dicyclohexylcarbodiimide as coupling agent. The reagent was characterized and its chiral purity was established. It was used as a chiral derivatizing reagent for the synthesis of hydrazone diastereomers, under microwave irradiation, of certain chiral aldehydes and ketones. The respective diastereomers were separated by reversed-phase high-performance liquid chromatography using a binary solvent combination containing trifluoroacetic acid. The diastereomers were detected at 231 nm. The method was validated for accuracy, precision, and limit of detection (LOD). For a series of hydrazones the LOD was found to be in the range 1.62-1.65 pmol/mL.

An artificial photosensitizer drug network for mitochondria-selective photodynamic therapy.

We report a new class of photosensitizer drug networks that can home into mitochondria and provide geospatial phototoxicity for tumors. We take advantage of the cleavable chemical network between the photosensitizer drug and poly(ethylene glycol), and find a significant increase in the efficiency of the multimeric drug network in mitochondria uptake and tumor inhibition.

Low-intensity electromagnetic irradiation of 70.6 and 73 GHz frequencies enhances the effects of disulfide bonds reducer on Escherichia coli growth and affects the bacterial surface oxidation-reduction state.

Low-intensity electromagnetic irradiation (EMI) of 70.6 and 73 GHz frequencies (flux capacity - 0.06 mW cm(-2)) had bactericidal effects on Escherichia coli. This EMI (1h) exposure suppressed the growth of E. coli K-12(λ). The pH value (6.0-8.0) did not significantly affect the growth. The lag-phase duration was prolonged, and the growth specific rate was inhibited, and these effects were more noticeable after 73 GHz irradiation. These effects were enhanced by the addition of DL-dithiothreitol (DTT), a strong reducer of disulfide bonds in surface membrane proteins, which in its turn also has bactericidal effect. Further, the number of accessible SH-groups in membrane vesicles was markedly decreased by EMI that was augmented by N,N'-dicyclohexycarbodiimide and DTT. These results indicate a change in the oxidation-reduction state of bacterial cell membrane proteins that could be the primary membranous mechanism in the bactericidal effects of low-intensity EMI of the 70.6 and 73 GHz frequencies.

Structure of the rotor ring modified with N,N'-dicyclohexylcarbodiimide of the Na+-transporting vacuolar ATPase.

The prokaryotic V-ATPase of Enterococcus hirae, closely related to the eukaryotic enzymes, provides a unique opportunity to study the ion-translocation mechanism because it transports Na(+), which can be detected by radioisotope (22Na(+)) experiments and X-ray crystallography. In this study, we demonstrated that the binding affinity of the rotor ring (K ring) for 22Na(+) decreased approximately 30-fold by reaction with N,N(')-dicyclohexylcarbodiimide (DCCD), and determined the crystal structures of Na(+)-bound and Na(+)-unbound K rings modified with DCCD at 2.4- and 3.1-Å resolutions, respectively. Overall these structures were similar, indicating that there is no global conformational change associated with release of Na(+) from the DCCD-K ring. A conserved glutamate residue (E139) within all 10 ion-binding pockets of the K ring was neutralized by modification with DCCD, and formed an "open" conformation by losing hydrogen bonds with the Y68 and T64 side chains, resulting in low affinity for Na(+). This open conformation is likely to be comparable to that of neutralized E139 forming a salt bridge with the conserved arginine of the stator during the ion-translocation process. Based on these findings, we proposed the ion-translocation model that the binding affinity for Na(+) decreases due to the neutralization of E139, thus releasing bound Na(+), and that the structures of Na(+)-bound and Na(+)-unbound DCCD-K rings are corresponding to intermediate states before and after release of Na(+) during rotational catalysis of V-ATPase, respectively.