Snežana Miljanić

Lipophilic derivative of rhodamine 19: characterization and spectroscopic properties

A new octadecylamide derivative of rhodamine 19 was synthesized by reaction of rhodamine 19 ethyl ester (rhodamine 6 G) with octadecylamine. This lipophilic dye exists in two forms: as a cationic species containing a secondary amide group and as an electrically neutral molecule containing an additional ring closed between the oxygen of the amide group and the central carbon of the xanthene part. Both forms were isolated and characterized in detail by means of elemental analysis, mass spectrometry, IR, NMR and UV–VIS spectroscopy. Depending on the hydrogen ion activity in solution and the availability of solvent hydrogen for hydrogen bond formation, the cationic form easily converted into a neutral one and vice versa. On the basis of absorption and emission UV–VIS spectroscopic data, the equilibrium of the two forms was studied in various solvents. An increase in dye concentration in acidified methanol/water mixture (volume fraction of water, ϕ=80%) led to aggregation of the cationic molecules. Using the UV–VIS absorption data, a corresponding dimerization constant was evaluated.

Alteration of cholinesterase activity as possible mechanism of silver nanoparticle toxicity

Due to their broad-spectrum antimicrobial activity, silver nanoparticles (AgNPs) have been used in a large number of commercial and medical products. Such proliferated AgNP production poses toxicological and environmental issues which need to be addressed. The present study aimed to investigate the effects of AgNPs on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), important enzymes in areas of neurobiology, toxicology and pharmacology. Three different AgNPs, prepared by the chemical reduction using trisodium citrate, hydroxylamine hydrochloride (Cl-AgNPs), and borohydride following stabilization with poly(vinyl alcohol), were purified and characterised with respect to their sizes, shapes and optical properties. Their inhibition potential on AChE and BChE was evaluated in vitro using an enzyme assay with o-nitrophenyl acetate or o-nitrophenyl butyrate as substrates, respectively. All three studied AgNPs were reversible inhibitors of ChEs. Among tested nanoparticles, Cl-AgNP was found to be the most potent inhibitor of both AChE and BChE. Although the detailed mechanism by which the AgNPs inhibit esterase activities remains unknown, structural perturbation of the enzyme may be the common mode of ChE inhibition by AgNPs.

Adsorption mechanisms of RNA mononucleotides on silver nanoparticles

Surface-enhanced Raman scattering (SERS) of four RNA mononucleotides (AMP, GMP, CMP and UMP) has been studied on the citrate-reduced silver colloid aggregated with sodium sulfate. Concentration dependent spectra in the range of 1×10(-7)-1×10(-4) mol dm(-3) were obtained, assigned and interpreted according to the surface selection rules. For purine mononucleotides, AMP and GMP, adsorption onto the silver nanoparticles through the six-membered ring of the nitrogenous base was suggested. Concentration dependent splitting of the ring breathing band in the spectra of AMP indicated coexistence of two species on the silver surface, which differed in contribution of the adenine N1 atom and the exocyclic NH2 group in binding. Unlike the AMP spectra, the spectra of GMP implied only one mode of adsorption of the molecules onto the silver nanoparticles, taking place through the guanine N1H and C=O group. Weak SERS spectra of pyrimidine mononucleotides, CMP and UMP, pointed to involvement of carbonyl oxygen in adsorption process, whereby the molecules adopted the position on the nanoparticles with ribose close to the metal surface. Intense bands in the low wavenumber region, associated with stretching of the formed Ag-N and/or Ag-O bonds, supported chemical binding of the RNA mononucleotides with the silver surface.

Sorption of HDTMA cations on Croatian natural mordenite tuff.

Sorption of the cationic surfactant, hexadecyltrimethylammonium cations (HDTMA), on the solid/liquid interface of the natural mordenite tuff (MT) was studied. The examined tuff originated from Croatia consisting of 30% of mordenite. SEM observations confirmed the crystalline nature of mordenite which can be described in terms of aggregates of many small platelets with diameters in the range of 1 μm. Studying the porosity properties of MT, it was found that the average pore diameter (4.42 nm) between mordenites platelets allows penetration of HDTMA cations. The measurements of zeta potential indicated that in MT samples with surfactant concentration in the range between 0.013 and 0.25 mmol/g, HDTMA cations fill the mesopores of MT. By further increase in HDTMA concentration, the surfactant sorbs on the external zeolite surface, as revealed by the SEM micrographs. Vibrational (FTIR and FT Raman) spectra showed that in the MT samples with initial HDTMA concentration from 0.013 to 0.25 mmol/g, alkyl chains adopt mainly gauche conformation, whereas in the MT samples with higher initial HDTMA concentrations trans conformers are predominant and form a highly ordered structure on the mordenite surface.

In-line reaction monitoring of entacapone synthesis by Raman spectroscopy and multivariate analysis.

In-line Raman spectroscopy and multivariate analysis were used to monitor Knoevenagel condensation reaction, the final step in preparation of drug entacapone. By applying a fiber optical Raman probe immersed into a reaction vessel Raman spectra of the reaction mixture were recorded in situ during the entacapone synthesis in toluene, heptane and isobutyl acetate. Due to the complexity of the measured spectra, the obtained data were analyzed and interpreted by means of principal component analysis. It has been shown that progress of this reaction can be monitored in real-time and reaction end points can be determined in different solvents. The reaction was found to be the fastest in heptane due to the lower loss of the catalyst. For a comparison the reaction was independently monitored by off-line Raman spectroscopy and liquid chromatography which confirmed the results obtained in-line. The results presented here have shown that this in-line approach can be used as a fast, non destructive and reliable method to monitor the Knoevenagel reaction in real time. The knowledge gained in this study can further be exploited for the industrial process control.

Structural investigation of aroylhydrazones in dimethylsulphoxide/water mixtures.

Molecular structures of aroylhydrazones derived from salicylaldehyde, o-vanilin and nicotinic acid hydrazide in DMSO and DMSO/H(2)O mixtures have been studied by NMR, UV-Vis, ATR and Raman spectroscopy. The addition of water to the system did not induce the tautomeric conversion of the existing form constituted of the ketoamino hydrazide part and the enolimino aldehyde part, but it was involved in the formation of hydrated molecules. Vibrational spectra (ATR and Raman) clearly indicated hydrogen bonding of the studied hydrazones through the carbonyl, amino and hydroxyl groups with water molecules. Increasing the water content conversion from E to Z isomer was not observed.

Binding of a Phenanthridine-Biguanide Derivative with DNA/RNA Polynucleotides Studied by Surface-Enhanced Raman Spectroscopy (SERS)

Surface-enhanced Raman spectroscopy (SERS) in the near-infrared region has been applied to study interactions between a phenanthridine-biguanide derivative (PB) and polynucleotides. The PB molecules scatter radiation in the silver citrate colloid most intensively at concentration of 2.5 6 10−5 mol dm−3, at which they are optimally oriented towards the nanoparticles, with the phenanthridine plane placed perpendicularly to the silver surface. A band at 228 cm−1, attributed to the Ag–N stretching mode, implies that chemical mechanism, along with the electromagnetic mechanism, contributes to the total enhancement of the scattered radiation. A decrease in intensity and shifts of the biguanide-related bands in the SERS spectra of the mixtures of PB with DNA indicate binding of the small molecules with the nucleic acid. Studies with double-stranded DNA analogues reveal that the molecules of PB intercalate into the G–C base pairs by the phenanthridine system and bind within the minor groove of the A–T sequences through the biguanide substituent. Intensity decrease in the SERS spectrum of the PB/RNA mixture confirms insertion of the molecules between A–U base pairs, whereas an upward shift of the amino groups bending mode (1078 cm−1) as well as diminution of the CN stretching band at 1421 cm−1 imply hydrogen bonding of the biguanide moiety with the nucleobases. SERS spectra of the mixtures containing the single-stranded polynucleotides show that the polynucleotide secondary structure does not affect binding of PB with the polynucleotides but that affinity towards the polynucleotides depends solely on the molecular structure of the nucleic bases.

Quantitative Analysis of Entacapone Isomers Using Surface-Enhanced Raman Spectroscopy and Partial Least Squares Regression

Surface-enhanced Raman spectroscopy (SERS) and partial least squares (PLS) regression have been applied for the quantification of entacapone isomers E and Z in solution. Nine mixtures of isomers Z and E in ethanol ranging from 0% to 100% w/w were analyzed, for a total entacapone concentration of 1 times 10−3 mol L−1. Upon deposition onto commercially available Klarite® gold plates, highly intense and reproducible SERS spectra were obtained from the entacapone isomers. Based on the spectral measurements, a two-component PLS model for correlation of predicted and real content of the isomers mixtures was developed. Root-meansquare error of the predicted composition was found to be 8% of isomer Z in the isomers mixture, corresponding to the absolute concentration of 8 times 10−5 mol L−1 of isomer Z in solution.

Distinguishing binding modes of a new phosphonium dye with DNA by surface-enhanced Raman spectroscopy

To understand the disruption of the cell processes induced by small molecule binding to DNA, numerous structural studies of DNA complexes have been done, whereby a variety of methods has been applied. The use of various techniques is required, not only for the unique information on the structure provided by each of the methods, but also for limitations that arise during the sample preparation or measurement procedures. Here, surface-enhanced Raman scattering (SERS) spectroscopy has been employed to study binding of a new mitochondria-specific dye with ds-DNA polynucleotides. Concentration dependent differences in the SERS spectra of the dye were attributed to concentration induced changes in position of the dye molecules on the enhancing metal surface and assigned to aggregated molecules formed due to π-stacking interactions at higher dye concentration (5.0 × 10−5 mol L−1) and to single molecules at lower dye concentration (5.0 × 10−6 mol L−1). The characteristic SERS spectra were further on correlated with the spectra of the dye/DNA complexes, implying binding of the monomeric dye molecules at the excess of the adenine–thymine polynucleotide, [dye]/[AT] = 1/10, and in form of the stacked dye molecules at the higher [dye]/[AT] ratio of 1/2. In addition, the distinctive SERS spectra of the complexes with the AT alternating and homo-polymer indicated different placement of the dye molecules within the minor groove, affected by the polynucleotide groove width. Beside well-known quantitative, this study emphasized structural sensitivity of SERS, able to distinguish various molecular forms of the small molecules in the complex structures such as those formed with biomacromolecules.

Surface-enhanced Raman spectra of rhodamine 19 octadecylamide

Surface-enhanced Raman scattering (SERS) of the cationic and the neutral form of rhodamine 19 octadecylamide (R19OA) has been studied in the silver citrate colloid using NIR excitation at 1064 nm. Cationic molecules readily adsorb onto negatively charged silver nanoparticles through a positively charged xanthene part of the molecules resulting in surface enhancement of Raman scattering. Due to a lack of the positive charge in molecular structure, SERS spectrum of neutral molecules is not observed. Nevertheless, a broad band appearing at 1240 cm(-1) in the spectrum of the cationic form indicates conversion of the cationic into the neutral species occurring close to the silver surface. The observed band most likely arises from a vibration of the ring formed in the molecular structure after conversion, but before complete desorption of the neutral molecules from the metal surface. Upon addition of HCl and NaOH in the silver sol, equilibrium is shifted toward the cationic and the neutral form of R19OA, respectively, followed by corresponding changes in the Raman spectrum. In addition, FT-SERS spectra of two rhodamine dyes, rhodamine 19 (R19) and rhodamine 6G (R6G), that are structurally related to R19OA, have been studied under the same experimental conditions for comparison.