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In the present study, a comparative Raman vibrational analysis of alpha-cyano-4-hydroxycinnamic acid (4CHCA) and its derivative, alpha-cyano-3-hydroxycinnamic acid (3CHCA), was performed. The Raman spectra of the 4CHCA and 3CHCA in solid form were obtained and analyzed to determine differences between the two structurally similar derivatives. For comparison, the CHCA derivatives cyanocinnamic acid (CCA) and coumaric acid (CA) were also studied. The plausible vibrational assignments were made and matched with those obtained theoretically using density functional theory (DFT) based method employing a 6-31 g basis set. The computational wavenumbers obtained were in good agreement with the observed experimental results. This was the first reported Raman study of CCA, 3CHCA and 4CHCA.

Tetracyclines (TCs), broad spectrum antibiotics widely used in the prevention and treatment of infectious diseases, are amphoteric molecules containing several ionizable functional groups that exist predominantly as zwitterions at a given pH value. TCs are reported to undergo a wide variety of reactions at different pH values i.e. TCs form to anhydrotetracyclines at low pH, 4-epitetracyclines at pH 3-5 and isotetracyclines at high pH values. The pH-dependent absorbance and emission properties of tetracycline and its 10 analogs (4-epitetracycline, doxycyline, oxytetracycline, chlortetracycline, 4-epichlortetracycline, isochlortetracycline, methacycline, rolitetracycline, minocycline, and demeclocycline) were investigated and reported in this paper. The main focus of the study was on the pH dependent transformation of epichlortetracycline, chlortetracycline and isotetracycline at basic pH. Absorption, emission and time resolved spectroscopy were used to determine the behavior of the three TC derivatives at this condition. Increasing the buffer's ionic concentration leads to faster transformation to iCTC. A pH dependent transformation of CTC to iCTC was observed and the lifetimes of CTC and iCTC were determined to be 3.0 and 5.89 ns respectively. The distribution factor of CTC to iCTC at basic pH was also reported for the first time.

An automated stochastic docking program with a graphical user interface, RANDOMDOCK (RD), has been developed to aid the development of molecularly imprinted polymers and xerogels. RD supports computations with ab initio and semiempirical quantum chemistry programs. The RD algorithms have been tested by searching for the most stable geometries of a varying number of methacrylic acid molecules interacting with nicotinamide. The optimal structures found are either as stable or more stable than those previously proposed for this molecularly imprinted polymer, illustrating that RD is capable of identifying the lowest-energy structures out of a potentially vast number of possible configurations. RD was subsequently applied to determine the most favorable binding sites between silane molecules and tetracycline (TC) as well as TC analogues. Hydrogen bonding between the templates and a silane is an important determinant of stability. Dispersion interactions are also sizable, sometimes dominant, especially between the largest silane and TC analogues not possessing a site readily available for hydrogen bonding. We highlight the importance of exploring the full intermolecular potential energy landscape when studying systems which may not afford highly specific interactions.

Different computational models were used and screened to find a rational way in selecting the appropriate functional silane monomer for the best molecular imprinted xerogel (MIX) formulation. Several functional silane monomers were used and allowed to react with a template model, tetracycline (TC). The resulting template-monomer complex molecules were first optimized and their interaction energies (IEs) were calculated using different computational methods such as semi-empirical methods, ab-initio methods, density functional theory (DFT) methods and solvent model method. The formulations used for calculation were also prepared and their performance in binding with TC was determined using tritium labeled sample. Results showed that the rankings of the different formulations varied with the different computational methods. However, rankings of the IEs of the xerogels are similar to that of the imprinting factor (IF) when HF and B3LYP at SV(P) and SVP basis set levels were used. The best imprinted xerogel, allyltriethoxysilane (AtEOS) ranked first in ten out of the 26 computational models that were screened and at all computational methods at tetramer system.

The role aromatic amino acids play in the formation of amyloid is a subject of controversy. In an effort to clarify the contribution of aromaticity to the self-assembly of human islet amyloid polypeptide (hIAPP)22-29 , peptide analogs containing electron donating groups (EDGs) or electron withdrawing groups (EWGs) as substituents on the aromatic ring of Phe-23 at the para position have been synthesized and characterized using turbidity measurements in conjunction with Raman and fluorescence spectroscopy. Results indicate the incorporation of EDGs on the aromatic ring of Phe-23 virtually abolish the ability of hIAPP22-29 to form amyloid. Peptides containing EWGs were still capable of forming aggregates. These aggregates were found to be rich in β-sheet secondary structure. Transmission electron microscopy images of the aggregates confirm the presence of amyloid fibrils. The observed difference in amyloidogenic propensity between peptides containing EDGs and those with EWGs appears not to be based on differences in peptide hydrophobicity. Fluorescence and Raman spectroscopic investigations reveal that the environment surrounding the aromatic ring becomes more hydrophobic and ordered upon aggregation. Furthermore, Raman measurements of peptide analogs containing EWGs, conclusively demonstrate a distinct downshift in the CC ring mode (ca. 1600 cm(-1) ) upon aggregation that has previously been shown to be indicative of π-stacking. While previous work has demonstrated that π-stacking is not an absolute requirement for fibrillization, our findings indicate that Phe-23 also contributes to fibril formation through π-stacking interactions and that it is not only the hydrophobic nature of this residue that is relevant in the self-assembly of hIAPP22-29 . © Proteins 2013. © 2012 Wiley Periodicals, Inc., (Copyright © 2013 Wiley Periodicals, Inc.)

Plant responses to natural stresses have been the focus of numerous studies; however less is known about plant responses to artificial (i.e., man-made) stress. Chlortetracycline (CTC) is widely used in agriculture and becomes an environmental contaminant when introduced into soil from manure used as fertilizer. We show here that in the model plant Arabidopsis (Arabidopsis thaliana), root uptake of CTC leads to toxicity, with growth reductions and other effects. Analysis of protein accumulation and in vivo synthesis revealed numerous changes in soluble and membrane-associated proteins in leaves and roots. Many representative proteins associated with different cellular processes and compartments showed little or no change in response to CTC. However, differences in accumulation and synthesis of NAD-malic enzyme in leaves versus roots suggest potential CTC-associated effects on metabolic respiration may vary in different tissues. Fluorescence resonance energy transfer (FRET) analysis indicated reduced levels of intracellular calcium are associated with CTC uptake and toxicity. These findings support a model in which CTC uptake through roots leads to reductions in levels of intracellular calcium due to chelation. In turn, changes in overall patterns and levels of protein synthesis and accumulation due to reduced calcium ultimately lead to growth reductions and other toxicity effects.

The creation of tetracycline (TC) responsive molecularly imprinted xerogels (MIXs) was investigated using electronic absorbance, liquid chromatography-ion-trap mass spectrometry (LC-ITMS), and first-principles theory. Experimental results show that the template molecule converts to its epimer, 4-epitetracycline (ETC), during the imprinting process. Additionally, end capping of the MIX surface silanols transforms TC into anhydrotetracycline (ATC) and 4-epianhydrotetracycline (EATC). Hence, despite aiming to imprint for a single analyte (TC), one simultaneously imprints for up to four analogs (TC, ETC, EATC and ATC) within a MIX. Binding studies using LC-MS showed the binding of the prepared xerogels with the four analogs. In some formulations, preferential uptake of ETC, EATC and ATC relative to the template molecule (TC) was observed. Computations of the interaction energies between silane monomers and the four analogs reveal that ETC, EATC and ATC have higher interaction energies and are more likely to be imprinted in comparison to TC.

A series of silica-based tetracycline (TC)-imprinted xerogel sorbents were prepared by sol-gel processing and were characterized for TC binding. Molecularly imprinted xerogels (MIXs) formed from allyltriethoxysilane (AtEOS) and tetraethoxysilane (TEOS) and end capped with trimethylchlorosilane exhibited the best analytical performance (imprinting factor, IF, of 7.46±0.13). Computational modeling was used to estimate the interaction energy (IE) between TC and each type of silane to evaluate our ability to predict the analytical performance of a given MIX. Rankings from the computations agreed with the experimental data showing the AtEOS having the highest IE in comparison to the other formulations. Together, these results demonstrate the potential and limitations of using theoretical calculations to guide the development of analyte selective MIXs in comparison to arbitrary trial and error approaches traditionally used to produce MIXs as sorbents for solid phase extraction., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Tetracycline antibiotics, such as chlortetracycline (CTC) and tetracycline (TC), are introduced into agricultural lands through the application of manure as fertilizer. These compounds are phytotoxic to certain crop plants, including pinto beans (Phaseolus vulgaris), the species used for this investigation. While the mechanism of this toxicity is not yet understood, CTC is known to be a calcium chelator. We describe here a novel method to show that CTC is taken up by pinto bean plants and chelates calcium in leaves. Cameleon fusion proteins can provide qualitative and quantitative imaging of intracellular calcium levels, but current methodology requires stable transformation. Many plant species, including pinto beans, are not yet transformable using standard Agrobacterium-based protocols. To determine the role of calcium chelation in this plant, a rapid, biolistic method was developed to transiently express the cameleon protein. This method can easily be adapted to other plant systems. Our findings provide evidence that chelation of intracellular calcium by CTC is related to phytotoxic effects caused by this antibiotic in pinto beans. Root uptake of CTC and TC by pinto beans and their translocation to leaves were further verified by fluorescence spectroscopy and liquid chromatography/mass spectrometry, confirming results of the biolistic method that showed calcium chelation by tetracyclines in leaves.

The extracellular and intracellular metabolites formed upon exposure of activated sludge microorganisms to a sublethal concentration of N-ethylmaleimide were monitored by liquid chromatography with ion trap mass spectrometry. The metabolite N-ethylsuccinimido-S-glutathione (m/z 433) was converted rapidly to N-(2-oxoethyl)-2,2-(propionylamino)propanamide (m/z 187) and N-ethylmaleamic acid (m/z 144).