Mechanism of extractive/oxidative desulfurization using the ionic liquid inimidazole acetate: a computational study

The dual role of the ionic liquid 1-butyl-3-methyl-imidazolium trifluoroacetic acid ([C₄mim]TFA) as an extractant for thiophene (TH) and a catalyst for the oxidation of TH was explored at the molecular level by performing density functional theory (DFT) calculations. The calculated interaction energies demonstrated why [C₄mim]TFA is a better extractant for thiophene sulfone (THO₂) than for TH. Two pathways were proposed for the oxidation of TH to THO₂ with [C₄mim]TFA acting as a catalyst. In the dominant pathway, a peracid is formed which then oxidizes TH to the sulfoxide and sulfones. The presence of [C₄mim]TFA was found to greatly reduce the barrier to the oxidative desulfurization (ODS) of TH using H₂O₂ as an oxidant.

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Graphical Abstract Possible reaction mechanisms of TH with the aid of [C4mim]TFA?

     

Studies on peptides CLVIII. Model experiments for the synthesis of open-chain unsymmetrical cystine-peptides

Treatment of a mixture of Cys(R)(O) and Cys(R) with an acid was found to generate cystine in fairly good yields, when suitable R, R, and an acid were selected. An unsymmetrical cystine peptide was prepared by treatment of a mixture of Z(OMe)-Cys(R) (0)-Ala-NH₂ (R=Acm or MBzl) and Z(OMe)-Cys(MBzl)-Gly-OBzl with TFA or 1 M TFMSA/TFA.³ Oxytocin was obtained in an excellent yield by TFA treatment of the protected peptide containing Cys(Acm)(0) and Cys(MBzl). Thus, formation of the disulfide bond was found feasible at the position of Cys(R) (0).The following abbreviations are used Boc t-butyloxycarbonyl - Z(OMe) p-methoxybenzyloxycarbonyl - MBzl p-methoxybenzyl - Acm acetamidomethyl - Bzl benzyl - Ad l-adamantyl - tBu t-butyl - TFA trifluoroacetic acid - TFMSA trifluoromethanesulfonic acid - TMSOTf trimethylsilyl trifluoromethane sulfonate     

Preferred conformations of protected homo-oligo-L-glutamate peptides in CDCl3 and CDCl3/TFA mixtures

A conformational analysis of protected glutamate homo-oligopeptides Z-[Glu(OEt)]_n-OEt (n = 2–7) was carried out in chloroform solution using high-resolution ¹H-nmr spectroscopy. At dilute peptide concentrations, the backbone NH and α-CH resonances are well resolved and can be assigned by combining extensive homonuclear decoupling experiments with data for co-oligopeptide derivatives. The structure of these peptides in solution was then assessed using information from chemical shifts, coupling constants, temperature coefficients, and titration of each oligomer with trifluoroacetic acid (TFA). The di- and tripeptides are found to be in disordered forms in deuterochloroform (CDCl₃) and CDCl₃/TFA mixtures. The tetrapeptide exhibits a folded structure with intramolecular hydrogen bonding at Glu² in CDCl₃ and undergoes a transition to increasingly disordered forms as TFA is added. The pentamer to heptamer show a folded structure with a strong intramolecular hydrogen bond at Glu² and a weaker hydrogen bond at Glu³, which are disrupted as these peptides go to random coils at high TFA/CDCl₃ ratios. In addition, the N-terminal portions of these glutamate peptides appear to be involved in side chain–main chain interactions. The results support the hypothesis that protected linear homo-oligopeptides may possess two or more segments of conformation with intramolecular folding preferred near the N-terminal portion.     

Investigation of an on-line two-dimensional chromatographic approach for peptide analysis in plasma by LC–MS–MS

Reversed phase and hydrophilic interaction chromatography (HILIC) were successfully coupled for the on-line extraction and quantitative analysis of peptides by ESI–LC–MS/MS. A total of 11 peptides were utilized to determine the conditions for proper focusing and separation on both dimensions. Minor modifications to the initial organic composition of the first reversed-phase dimension provided options between a comprehensive (generic) or more selective approach for peptide transfer to the second HILIC dimension. Ion-pairing with trifluoroacetic acid (TFA) provided adequate chromatographic retention and peak symmetry for the selected peptides on both C₁₈ and HILIC. The resulting signal suppression from TFA was partially recovered by a post-column “TFA fix” using acetic acid yielding improvements in sensitivity. Minimal sample preparation aligned with standard on-line extraction procedures provided highly reproducible and robust results for over 300 sequential matrix injections. Final optimized conditions were successfully employed for the quantitation of peptide PTHrP (1–36) in rat K₃EDTA plasma from 25.0 to 10,000 ng/mL using PTHrP (1–34) as the analog internal standard. This highly orthogonal two-dimensional configuration was found to provide the unique selectivity required to overcome issues with interfering endogenous components and minimize electrospray ionization effects in biological samples.     

High-sensitivity TFA-free LC-MS for profiling histones

The analysis of proteins by RPLC commonly involves the use of TFA as an ion‐pairing agent, even though it forms adducts and suppresses sensitivity. The presence of adducts can complicate protein molecular weight assignment especially when protein isoforms coelute as in the case of histones. To mitigate the complicating effects of TFA adducts in protein LC‐MS, we have optimized TFA‐free methods for protein separation. Protein standards and histones were used to evaluate TFA‐free separations using capillary (0.3 mm id) and nanoscale (0.1 mm id) C₈ columns with the ion‐pairing agents, formic acid or acetic acid. The optimized method was then used to examine the applicability of the approach for histone characterization in human cancer cell lines and primary tumor cells from chronic lymphocytic leukemia patients.     

Mechanistic aspects of CoII(HAPP)(TFA)2 in DNA bulge-specific recognition

A novel octahedral complex Co^II(HAPP)(TFA)₂ [hexaazaphenantholine-cyclophane (HAPP), trifluoroacetate (TFA)] is a DNA bulge-specific probe with single-strand DNA cleavage activity in the presence of H₂O₂. This complex exhibits low affinity towards double-stranded DNA and low reactivity toward single-stranded DNA. Metal–HAPP complexes with different coordination number and ring size were synthesized and their selectivity and reactivity for DNA bulges were compared. The DNA sequence at the bulge site influences the intensity of cleavage at the bulge and the flanking sites after piperidine treatment. Cleavage specificity of Co^II(HAPP)(TFA)₂ was characterized extensively using scavenger reagents to quench the cleavage reaction and high-resolution polyacrylamide gel electrophoresis. In addition, 3′-phosphoglycolate cleavage products were trapped and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. These data were used to deduce that the DNA cleavage pathway for Co^IIHAPP²⁺ in the presence of H₂O₂ involves 4′-H abstraction of the deoxyribose moiety.     

Environmental Risk Assessment of Trifluoroacetic Acid

The Montreal Protocol was developed in 1987 in response to concerns that the chlorofluorocarbons (CFCs) were releasing chlorine into the stratosphere and that this chlorine was causing a depletion of stratospheric ozone over Antarctica. This international agreement called for a phase out of these CFCs. Industry initiated a major effort to find replacements that are safe when properly used and safe to the environment. The toxicology and environmental fate of these first generation replacements has been studied extensively. It was determined that the new substances break down in the environment to give predominantly carbon dioxide, water and inorganic salts of chlorine and fluorine. The only exception is that some substances also break down to yield trifluoroacetic acid (HTFA), a substance resistant to further degradation. Recognizing this, industry embarked on a research and assessment program to study the potential effects of trifluoroacetate (TFA) on the environment and to investigate possible degradation pathways. The results of these recently completed studies are summarized below and described in further detail in this paper. Trifluoroacetic acid is a strong organic acid with a pKa of 0.23. It is miscible with water and its low octanol/water partition coefficient (log P_ow=?2.1) indicates no potential to bioaccumulate. Industrial use is limited and environmental releases are very low. Some additional TFA will be formed from the breakdown of a few halogenated hydrocarbons, most notably HFC-134a (CF₃CH₂F), HCFC-124 (CF₃CHFCl), and HCFC-123 (CF₃CHCl₂). As these substances have only been produced in limited commercial quantities, their contribution to environmental levels has been minimal. Surprisingly, environmental measurements in many of diverse locations show existing levels of 100 to 300?ng·l^?1 in water with one site (Dead Sea) having a level of 6400?ng·l^?1. These levels cannot be accounted for based on current atmospheric sources and imply a long-term, possibly pre-industrial source. Generally, soil retention of TFA is poor although soils with high levels of organic matter have been shown to have a greater affinity for TFA when contrasted to soils with low levels of organic matter. This appears to be an adsorption phenomenon, not irreversible binding. Therefore, TFA will not be retained in soil, but will ultimately enter the aqueous compartment. Modeling of emission rates and subsequent conversion rates for precursors has led to estimates of maximum levels of TFA in rain water in the region of 0.1?µ·^?1 in the year 2020. TFA is resistant to both oxidative and reductive degradation. While there had been speculation regarding the possibility of TFA being degraded into monofluoroacetic acid (MFA), the rate of breakdown of MFA is so much higher than for TFA that any MFA formed would rapidly degrade. Therefore, there would be no buildup of MFA regardless of the levels of TFA present in the environment. Although highly resistant to microbial degradation, there have been two reports of TFA degradation under anaerobic conditions. In the first study, natural sediments reduced TFA. However, even though this work was done in replicate, the investigators and others were unable to reproduce it in subsequent studies. In the second study, radiolabeled TFA was removed from a mixed anaerobic in vitro microcosm. Limited evidence of decarboxylation has also been reported for two strains of bacteria grown under highly specific conditions. TFA was not biodegraded in a semi-continuous activated sludge test even with prolonged incubation (up to 84 days). TFA does not accumulate significantly in lower aquatic life forms such as bacteria, small invertebrates, oligochaete worms and some aquatic plants including Lemna gibba (duckweed). Some bioaccumulation was observed in terrestrial higher plants, such as sunflower and wheat. This result appeared to be related to uptake with water and then concentration due to transpiration water loss. When transferred to clean hydroponic media, some elimination of TFA was seen. Also, more than 80% of the TFA in leaves was found to be water ex-tractable, suggesting that no significant metabolism of TFA had occurred. At an exposure level of 1200?mg·l^?1 of sodium trifluoroacetate (NaTFA) — corresponding to 1000?mg·l^?1 HTFA — no effects were seen on either Brachy-danio rerio (a fish) or Daphnia magna (a water flea). With duckweed, mild effects were seen on frond increase and weight increase at the same exposure level. At a concentration of 300?mg·l^?1 no effects were observed. Toxicity tests were conducted with 11 species of algae. For ten of these species the EC50 was greater than 100?mg·l^?1. In Selenastrum capricornutum the no-effect level was 0.12?mg·l^?1. At higher levels the effect was reversible. The reason for the unique sensitivity of this strain is unknown, but a recovery of the growth rate was seen when citric acid was added. This could imply a competitive inhibition of the citric acid cycle. The effect of TFA on seed germination and plant growth has been evaluated with a wide variety of plants. Application of NaTFA at 1000?mg·l^?1 to seeds of sunflower, cabbage, lettuce, tomato, mung bean, soy bean, wheat, corn, oats and rice did not affect germination. Foliar application of a solution of 100?mg·l^?1 of NaTFA to field grown plants did not affect growth of sunflower, soya, wheat, maize, oilseed rape, rice and plantain. When plantain, wheat (varieties Katepwa and Hanno) and soya were grown in hydroponic systems containing NaTFA, no effects were seen on plantain at 32?mg·l^?1, on wheat (Katepwa) and soya at 1?mg·l^?1, or on wheat (Hanno) at 10?mg·l^?1; some effects on growth were seen at, respectively, 100?mg·l^?1, 5?mg·l^?1, 5?mg·l^?1, and 10?mg·l^?1 and above. TFA is not metabolized in mammalian systems to any great extent. It is the major final metabolite of halothane, HCFC-123 and HCFC-124. The half-life of TFA in humans is 16 hours. As expected, the acute oral toxicity of the free acid is higher than the one of the sodium salt. The inhalation LC₅₀ (2 hour exposure) for mice was 13.5?mg·l^?1 (2900?ppm) and for rats it was 10?mg·l^?1 (2140?ppm). Thus, TFA is considered to have low inhalation toxicity. The irritation threshold for humans was 54?ppm. As one would expect of a strong acid, it is a severe irritant to the skin and eye. When conjugated with protein, it has been shown to elicit an immunolog-ical reaction; however, it is unlikely that TFA itself would elicit a sensitization response. Repeat administration of aqueous solutions have shown that TFA can cause increased liver weight and induction of peroxisomes. Relative to the doses (0.5% in diet or 150?mg·kg^?1·day^?1 gavage) the effects are mild. In a series of Ames assays, TFA was reported to be non-mutagenic. Its carcinogenic potential has not been evaluated. Although TFA was shown to accumulate in amniotic fluid following exposure of pregnant animals to high levels of halothane (1200?ppm), no fetal effects were seen. Likewise, a reproduction study that involved exposure of animals to halothane at levels up to 4000?ppm for 4 hours per day, 7 days per week, resulted in no adverse effects. Given the high levels of halothane exposure, it is unlikely that environmental TFA is a reproductive or developmental hazard. Overall the toxicity of TFA has been evaluated in stream mesocosms, algae, higher plants, fish, animals and humans. It has been found to be of very low toxicity in all of these systems. The lowest threshold for any effects was the reversible effect on growth of one strain of algae, Selenastrum capricornutum, which was seen at 0.12?mg·l^?1. There is a 1000-fold difference between the no-effect concentration and the projected environmental levels of TFA from HFCs and HCFCs (0.0001?mg·l^?1). Based on available data, one can conclude that environmental levels of TFA resulting from the breakdown of alternative fluorocarbons do not pose a threat to the environment.     

Side reactions in the synthesis of phosphotyrosine-containing peptides

Summary A series of phosphopeptides Tyr(PO₃H₂)-Val-Pro-Xxx-Leu (Xxx=Met, Met(O), Nle, Dab or Cys), derived from the native platelet-derived growth factor- receptor (PDGF-) sequence, has been prepared to study their interaction with the src-homology 2 (SH2) domains of the p85 subunit of PI3 kinase. The phosphopeptides were synthesized using Fmoc methodology incorporating N-Boc dibenzyl-protected phosphotyrosine (Boc-Tyr[PO₃(Bzl)₂]) as the N-terminal amino acid, since the benzyl groups can be removed during resin cleavage with TFA. Only peptides containing methionine were found to exist partially as S-benzyl sulfonium salts after TFA cleavage from the resin. The desired peptide could be obtained from the S-benzyl sulfonium salt by hydrogenolysis.     

Phosphate limitation promotes unsaturated fatty acids and arachidonic acid biosynthesis by microalgae <Emphasis Type="Italic">Porphyridium purpureum</Emphasis>

Polyunsaturated fatty acids (PUFAs) are highly appreciated on their nutritive value for human health and aquaculture. P. purpureum, one of the red microalgae acknowledged as a promising accumulator of ARA, was chosen as the target algae in the present research. Effects of sodium bicarbonate (0.04–1.2 g/L), temperature (25, 30 and 33 °C) and phosphate (0.00–0.14 g/L) on biomass yield, total fatty acids (TFA) and arachidonic acid (ARA) accumulation were investigated systemically. NaHCO₃ dose of 0.8 g/L and moderate temperature of 30 °C were preferred. In addition, TFA and ARA production were significantly enhanced by an appropriate concentration of phosphate, and the highest TFA yield of 666.38 mg/L and ARA yield of 159.74 mg/L were obtained at a phosphate concentration of 0.035 g/L. Interestingly, with phosphate concentration continuing to fall, UFA/TFA and ARA/EPA ratios were increased accordingly, suggesting that phosphate limitation promoted unsaturated fatty acids and arachidonic acid biosynthesis. Low concentration of phosphate may be favored to increase the enzymatic activities of ?6-desaturase, which played a key role in catalyzing the conversion of C16:0 to C18:2, and thus the selectivity of UFA increased. Meanwhile, the increase of ARA selectivity could be attributed to ω6 pathway promotion and ?17-desaturase activity inhibition with phosphate limitation. Phosphate limitation strategy enhanced unsaturated fatty acids and ARA biosynthesis in P. purpureum, and can be applied in commercial scale manufacturing and commercialization of ARA.     

Restricted rotation of the amino group in cytosine and 5-alkylcytosine derivatives caused by TFA complexation

The restricted rotation of the amino group in analogs of dinucleotides 5RCyt/C₃/5R′Cyt, where R,R′ = H, CH₃, C₂H₅, as well as in free bases, cytosine and 5-alkylcytosine, is observed by proton magnetic resonance in TFA and DMSO-TFA solutions.At low TFA concentration the complex bi-cytosine cations formation take place. The stability of these cations depends on basicity of cytosine derivatives and increases in the serie H < CH₃ < C₂H₅.     

Response of growth and fatty acid compositions of Nannochloropsis sp. to environmental factors under elevated CO2 concentration

Nannochloropsis sp. was grown with different levels of nitrate, phosphate, salinity and temperature with CO₂ at 2,800 μl l⁻¹. Increased levels of NaNO₃ and KH₂PO₄ raised protein and polyunsaturated fatty acids (PUFAs) contents but decreased carbohydrate, total lipid and total fatty acids (TFA) contents. Nannochloropsis sp. grew well at salinities from 22 to 49 g l⁻¹, and lowering salinity enhanced TFA and PUFAs contents. TFA contents increased with the increasing temperature but PUFAs contents decreased. The highest eicosapentaenoic acid (EPA, 20:5ω3) content based on the dry mass was above 3% under low N (150 μM NaNO₃) or high N (3000 μM NaNO₃) condition. Excessive nitrate, low salinity and temperature are thus favorable factors for improving EPA yields in Nannochloropsis sp.     

Sensitive and resistant of the homologous disulfide-bridged proteins α-lactalbumin and lysozyme to attack of hydrogen-atoms,dithiothreitol and trifluoroacetic acid,examined by matrix-assisted laser desorption/ionization mass spectrometry

BackgroundEvolutionarily homologous proteins bovine α-lactoalbumin (αLA) and hen egg-white lysozyme (HEL) are very similar in primary, secondary and tertiary structures involving the location of disulfide-bridges (S–S), and are resistant to the action of hydrolytic enzymes and reagents. It is of interest to examine and compare the difference in backbone cleavage characteristics, by using reductive and hydrolytic reagents.MethodsIn-source decay (ISD) combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), reductive treatment of αLA and HEL with dithiothreitol (DTT) and acid hydrolysis with trifluoroacetic acid (TFA) were employed to examine the difference in the backbone cleavage characteristics of αLA and HEL.ResultsThe treatment of αLA and HEL with DTT/AcOHNH₃ resulted in similar cleavage behaviors of the backbone N-Cα and S–S bonds, i.e., the enhancements of the intensity and m/z range of sequence-reflected fragment ions were very similar. However, the treatment of αLA with DTT/TFA resulted in unexpected residue-specific degradation at the peptide bond of the Asp-Xxx, Xxx-Ser/Thr, Gln-Xxx, Xxx-Gly and Gly-Xxx residues, while HEL did not occur such degradation.ConclusionsThe results obtained above indicate that acidic αLA is very sensitive to acidic additive such as TFA, while basic HEL is resistance to acidic additives.General significanceThe study demonstrates the sensitive and resistant of evolutionary homologous proteins αLA and HEL to the acid hydrolysis and these characters come from acidic and basic nature of the proteins.     

Effect of trifluoroacetate,a persistent degradation product of fluorinated hydrocarbons,on Phaseolus vulgaris and Zea mays

The aim of this study was to quantify the effect of the pollutant, trifluoroacetate (TFA), on growth and photosynthesis of Phaseolus vulgaris (C₃) and Zea mays (C₄) in order to elucidate the physiological and biochemical basis of its inhibitory action. In whole plant studies, photosynthetic gas exchange, fast phase fluorescence kinetics and Rubisco activity were measured in parallel over a 14-day period in plants cultivated in a water culture system with NaTFA added at concentrations ranging from 0.625 to 160 mg l^?1. Although initial stimulation of some photosynthetic parameters was observed at low TFA concentrations early on in the experiment, marked inhibition occurred at higher concentrations. In general Z. mays was affected more severely than P. vulgaris showing a large TFA-induced decrease in both apparent carboxylation efficiency (ACE) and in vitro Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) activity. Analysis of photosynthetic gas exchange revealed that besides constraints on mesophyll processes such as Rubisco activity, stomatal limitation also increased with increasing TFA concentration, especially in P. vulgaris. In depth analysis of the fast phase fluorescence transients pointed at TFA-induced uncoupling of the oxygen evolving complex (OEC) and inhibition of electron transport beyond Q_a including possible constraints on the reduction of end electron acceptors of photosystem I.     

Chemical and enzymatic oxidation of alkylated benzo[a]pyrenes

The chemical and enzymatic oxidations of 6-, 7- and 10-methylbenzo[a]pyrenes, 6,10- and 7,10-dimethylbenzo[a]pyrenes and benzo[a]pyrene (BP) itself have been investigated to study the effects of alkyl substitution on the pathways of oxidation. The chemical oxidizing systems employed were Fenton's reagent ($\text{FeSO}{}_4\text{H}{}_2\text{O}{}_2$); trifluoroacetic acid-hydrogen peroxide (TFA/H₂O₂); thallium tristrifluoracetate in trifluoroacetic acid (TTFA/TFA) and H₂SO₄. The enzymatic systems were horseradish peroxidase (HRP/H₂O₂) and rat liver microsomes. The oxidations were investigated by electron paramagnetic resonance (EPR) spectroscopy to detect radical intermediates and by high performance liquid chromatography (HPLC) to separate the products. All the compounds studied produced radicals, identified as cationic species, in both H₂SO₄ and TTFA/TFA. In addition the 7-methyl-, 10-methyl- and 7,10-dimethyl-BP's produced 6-oxy radicals in some or all of the remaining oxidizing systems. Both chemically and enzymatically these same three compounds were observed to produce quinones as stable products. Microsomal oxidations gave the broadest range of products exhibiting HPLC peaks in the diol, quinone and phenol regions of the chromatograms, however, there were considerable differences between products from the individual derivatives and those from the parent molecule. 6-Methyl and 6,10-dimethyl-BP's showed no evidence of oxy radical intermediates or quinones under any set of conditions, the 6-substituent effectively blocking this oxidation pathway. The observations are consistent with the expected effects of alkyl substituents at particular positions and indicate that studies such as this one are potentially useful in better understanding oxidation and possible activation pathways of polycyclic aromatic hydrocarbons.     

Circadian rhythms of folate and vitamin B12 concentration in relation to convulsive thresholds of mice

Circadian rhythms of folate and vitamin B₁₂ concentrations in the liver, brain and blood of Swiss mice have been determined. The relation of the changes in vitamin concentration to circadian rhythms in locomotor activity, drinking activity and convulsive thresholds (maximal electroshock seizure threshold) have been determined also. Both free folic acid (FFA) and total folic acid (TFA) of liver and blood showed minimum values at 21.00–24.00 h and maximum values at 06.00–09.00 h. Liver TFA declined at a steady rate from the peak value at 09.00 h whereas liver FFA and plasma folate maintained constant values from 09.00–18.00 h then declined rapidly. Brain folate (TFA and FFA) showed no rhythm. The concentrations of vitamin B₁₂ in plasma, liver and brain showed only minor fluctuations. Locomotor and drinking activities showed very similar rhythms with a sustained period of high activity between 12.00–21.00 h followed by a much shorter second period of high activity (23.00–03.00 h). Convulsive threshold declined during the first period of increased locomotor and drinking activities reaching a minimum value at 21.00 h thus just preceding the nadir in folate concentrations in liver and blood.     

Variations in HDL-carried miR-223 and miR-135a concentrations after consumption of dietary trans fat are associated with changes in blood lipid and inflammatory markers in healthy men - an exploratory study

A high consumption of trans fatty acids (TFAs) is associated with an increased risk of cardiovascular diseases (CVDs). High-density lipoproteins (HDLs) have many cardioprotective properties and transport functional microRNAs (miRNAs) to recipient cells. We hypothesized that dietary TFAs modify the HDL-carried miRNA profile, therefore modulating its cardioprotective properties. We assessed whether consumption of dietary TFAs modifies HDL-carried miR-223-3p and miR-135a-3p concentration and the inter-relationship between diet-induced changes in HDL-carried miRNA concentration and CVD risk markers. In a double blind, randomized, crossover, controlled study, 9 men were fed each of 3 experimental isoenergetic diets: 1) High in industrial TFA (iTFA; 3.7% energy); 2) High in TFA from ruminants (rTFA; 3.7% energy); 3) Low in TFA (control; 0.8% energy) for 4 weeks each. HDLs were isolated by ultracentrifugation and miRNAs were quantified by RT-qPCR. Variations in HDL-miR-223-3p concentration were negatively correlated with variations in HDL-cholesterol after the iTFA diet (r_s = 0.82; P = 0.007), and positively correlated with variations in C-reactive protein concentration after the rTFA diet (r_s = 0.75; P = 0.020). Variations in HDL-miR-135a-3p concentration were positively correlated with variations in total triglyceride (TG) concentration following the iTFA diet (r_s = ?0.82; P = 0.007), and with variations in low-density lipoprotein (LDL)-TG concentration following the rTFA diet (r_s = 0.83; P = 0.005), compared to the control diet. However, the consumption of dietary TFAs has no significant unidirectional impact on HDL-carried miR-223-3p and miR-135a-3p concentrations. Our results suggest that the variability in the HDL-carried miRNAs response to TFA intake, by being associated with variations in CVD risk factors, might reflect physiological changes in HDL functions.     

Comparison of the elution characteristics of individual forms of lovastatin in both isocratic and gradient modes and HPLC-PDA method development for pure and fermentation-derived lovastatin

The elution characteristics of lovastatin were studied by varying the composition of mobile phase in both isocratic and gradient elution modes to comprehend the role of organic modifier and acidifier on the overall analysis time and retention time of individual forms of lovastatin. Acetonitrile has influenced on the overall analysis time, whereas the acidifier determines the retention time of hydroxy acid form of lovastatin and the retention time gap between the individual forms. A combination of acetonitrile and 0.1% trifluoroacetic acid (TFA) (60:40, v/v) in isocratic elution mode eluted both hydroxy acid and lactone forms of lovastatin at 4.5 and 5.4?min, respectively. This appears to be a better approach for the separation of pharmaceutical and clinical lovastatin samples. At isocratic elution mode, a mixture of acetonitrile and either 0.05% TFA or 0.1% H₃PO₄ of 60:40 (v/v) has eluted both hydroxy acid and lactone forms of lovastatin at 10?±?0.5 and 17?±?0.5?min, respectively. This is suitable for the fermentation-derived samples or for the complex mixtures of structural analogs. The fermentation broth (pH not adjusted) extracted with ethyl acetate at a ratio of 1:1 (v/v) at 60°C for 30?min was the optimal extraction condition for lovastatin.     

Vibrational circular dichroism of polypeptides. VI. Polytyrosine alpha-helical and random-coil results

We have measured the VCD of polytyrosine in the amide I and II regions in dimethyl sulfoxide (DMSO) and in 80:20 mixtures of DMSO with trifluoroethanol, trifluoroacetic acid (TFA), and dichloroacetic acid and in 50:50 mixtures of DMSO and trimethyl phosphate (TMP). Additionally, VCD was obtained for deuterated polytyrosine in DMSO and DMSO:D₂O, DMSO:TFA(d₁), and DMSO:TMP mixtures as before. Amide A VCD was obtained in DMSO and DMSO:TMP mixtures. In the pure solvent, VCD of an opposite sign was seen as compared with that seen in the mixtures. The latter were characteristic in sign pattern and shape of right-handed α-helices for poly(L -tyrosine). The pure polytyrosine:DMSO results are similar to those of polylysine:D₂O at neutral pH and poly(β-benzyl-aspartate) in DMSO and may be characteristic of random-coil VCD.     

Unaided trifluoroacetic acid pretreatment solubilizes polyglutamine peptides and retains their biophysical properties of aggregation

Understanding the biophysical mechanism of polyglutamine (polyGln) aggregation is important to unravel the role of aggregates in the pathology of polyGln repeat disorders. To achieve this, synthetic polyGln peptides are widely used. Their disaggregation and solubilization is essential because it plays a crucial role in reproducing biophysical experimental data under in vitro conditions. Pretreatment with trifluoroacetic acid (TFA) and hexafluoroisopropanol (HFIP) at a 1:1 ratio is currently the method of choice to achieve solubility of polyGln peptides. Here we report that the disaggregation and solubilization of polyGln peptides can be achieved by TFA alone. We tested TFA due to the close similarity of it with HFIP in the nature of H-bond breakage and formation, higher cost, and the problems faced by us in the availability of HFIP. Our results demonstrate that the TFA disaggregated polyGln sequences give similar solubilization yield, aggregation kinetics, thioflavin T (ThT) binding, and structural features in comparison with the TFA/HFIP method. Furthermore, we show by limited validation studies that the proposed TFA method can replace the existing TFA/HFIP disaggregation method of polyGln sequences.