Kinetics of lipase-catalyzed esterification in supercritical CO(2)

This study compares two solvents for enzymatic reactions: supercritical carbon dioxide (SCCO(2)) and organic solvent (n-hexane). The model reaction that was chosen was the esterification of oleic acid by ethanol catalyzed by an immobilized lipase from Mucor miehei (Lypozyme). The stability of the enzyme appeared to be quite good and similar in both media but was affected by the water content. Partition of water between solvents and immobilized enzyme has been calculated from experimental adsorption isotherms. The water content of the solid phase has a dramatic influence on the activity of the enzyme and its optimum value for activity was about 10% (w/w) in both media. A kinetic study enabled a Ping-Pong Bi-Bi reaction mechanism with inhibition by ethanol to be suggested. Despite some differences in kinetic constants, activity was in the same range in both media. Hypotheses for explaining the kinetic constant variations have been proposed and particular attention has been paid to the pH effects.     

A two-phase model for water and heat transfer within an intermittently-mixed solid-state fermentation bioreactor with forced aeration

A two-phase dynamic model is developed that describes heat and mass transfer in intermittently-mixed solid-state fermentation bioreactors. The model predicts that in the regions of the bed near the air inlet there can be significant differences in the air and solid temperatures, while in the remainder of the bed the gas and solid phases are much closer to equilibrium, although there can be differences in water activity of around 0.05. The increase in the temperature of the gas as it flows through the bed means that it is impossible to prevent the bed from drying out, even if saturated air is used at the air inlet. The substrate can dry to water activities that severely limit growth, unless the bed is intermittently mixed, with the addition of water to bring the water activity back to the desired value. Under the conditions assumed for the simulation, which was designed to mimic the growth of Aspergillus niger on corn, two mixing events were necessary, one at 17.4 and the other at 27.9 h. Even though such a strategy can minimize the restriction of growth by water-limitation, temperature-limitation remains a problem due to the rapid heating dynamics. The model is obviously a useful tool that can be used to guide scale-up and to test control strategies. Such a model, describing the non-equilibrium situation between the gas and solid phases, has not previously been proposed for solid-state fermentation bioreactors. Models in the literature that assume gas-solid temperature and moisture equilibrium cannot describe the large temperature differences between the gas and solid phase which occur within the bed near the air inlet.     

Efficient solid phase peptide synthesis. Use of methanesulfonic acid alpha-amino deprotecting procedure and new coupling reagent, 2-(benzotriazol-1-yl)oxy-1,3-dimethylimidazolidinium hexafluorophosphate (BOI).

An efficient method for solid phase peptide synthesis was developed, which consists of N alpha-selective deprotection by dilute methanesulfonic acid, in situ neutralization and rapid coupling reaction using benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) or 2-(benzotriazol-1-yl)oxy-1,3- dimethylimidazolidinium hexafluorophosphate (BOI) reagent. Selective removal of the N alpha-Boc group by dilute methanesulfonic acid was of more advantage than removal by TFA in terms of stability of semipermanent protecting groups and suppression of undesired side reactions. The use of in situ neutralization and rapid coupling method reduced intramolecular aminolytic cyclization by shortening exposure of the deprotected nucleophilic amino group. A successful synthesis of porcine brain natriuretic peptide (pBNP) has been achieved using this efficient solid phase peptide synthesis scheme.     

Preparation of tri(ethylene glycol) grafted core‐shell type polymer support for solid‐phase peptide synthesis

A core‐shell type polymer support for solid‐phase peptide synthesis has been developed for high coupling efficiency of peptides and versatile applications such as on‐bead bioassays. Although various kinds of polymer supports have been developed, they have their own drawbacks including poor accessibility of reagents and incompatibility in aqueous solution. In this paper, we prepared hydrophilic tri(ethylene glycol) (TEG) grafted core‐shell type polymer supports (TEG SURE) for efficient solid‐phase peptide synthesis and on‐bead bioassays. TEG SURE was prepared by grafting TEG derivative on the surface of AM PS resin via biphasic diffusion control method and subsequent acetylation of amine groups which are located at the core region of AM PS resin. The performance of TEG SURE was evaluated by synthesizing several peptides. Three points can be highlighted: (1) easy control of loading level of TEG, (2) improved efficiency of peptide synthesis compared with the conventional resins, and (3) applicability of on‐bead bioassays.     

Blue green algal (cyanobacterial) toxins, surface drinking water, and liver cancer in Florida

The blue green algae or cyanobacteria represent a diverse group of organisms that produce potent natural toxins. There have been case reports of severe morbidity and mortality in domestic animals through drinking water contaminated by these toxins. Microcystins, in particular, have been associated with acute liver damage and possibly liver cancer in laboratory animals. Although, there has been little epidemiologic research on toxin effects in humans, a study by Yu (1995) found an association between primary liver cancer and surface water. Surface water drinking supplies are particularly vulnerable to the growth of these organisms; current US drinking water treatment practices do not monitor or actively treat for blue green algal toxins including the microcystins.After a monitoring survey in Florida found organisms and microcystins (among other cyanobacterial toxins) in surface water drinking sources, a pilot ecological study was performed using a Geographic Information System (GIS) to evaluate the risk of primary hepatocellular carcinoma (HCC) and proximity to a surface water treatment plant at cancer diagnosis. The study linked all HCC cancers diagnosed in Florida from 1981 to 1998 with environmental databases.A significantly increased risk for HCC with residence within the service area of a surface water treatment plant was found compared to persons living in areas contiguous to the surface water treatment plants. However, this increased risk was not seen in comparison to persons living in randomly selected ground water treatment service areas or compared to the Florida cumulative incidence rate for the study period, using various comparison and GIS methodologies. Furthermore, these findings must be interpreted in light of significant issues of latency, high population mobility, and the lack of individual exposure information. Nevertheless, the issue of acute and chronic human health effects associated with the consumption of surface waters possibly contaminated by blue green algal toxins merits further investigation.     

Synthesis and spectroscopic properties of 4-ethoxymethylene-2-(1)-naphthyl-5(4H)-oxazolone-labeled fluorescent peptides

Strategies for the preparation of new fluorescent oligopeptide conjugates labeled with 4-ethoxymethylene-2-[1]-naphthyl-5(4H)-oxazolone (naOx-OEt) at the N-terminal on solid support or in solution have been devised. These procedures are simple and easy to carry out by reacting naOx-OEt or N(alpha)-naOx-amino acid with side chain protected peptide chains attached to resins. The integrity of the N-alkyl bond was maintained even after the trifluoracetic acid or HF based cleavages procedures. Our data show that the naOx fluorophore is compatible with both Fmoc/tBu and Boc/Bzl methods and also suggest that naOx-amino acid could be utilized as building blocks for solid phase peptide synthesis. Comparative analysis of fluorescence properties of naOx-conjugates indicated that the spectral properties of the fluorophore do not change after incorporating into peptides. The compact size, the definite chemical reaction for its introduction in combination with the appropriate spectral features (e.g., intense emission, pH independent fluorescent characteristics, and beneficial photobleaching dose constant and rates) and with chemical and spectral stability, naOx-based labeling could be attractive for novel cellular fluorescent techniques (e.g., in laser scanning confocal FRET) to study peptide-protein and protein-protein interactions even in biological matrices.     

Storage and export of mercury from a contaminated bay (Ria de Aveiro,Portugal)

Mercury has been determined in water, suspended particulate matter (SPM)and sediments (solid fraction and pore water) collected in a heavilycontaminated confined area (Largo do Laranjo) of the Ria de Aveiro.Mercury has been analysed in seabass (Dicentrarchus labrax) capturedin several locations of the lagoon. The levels of mercury in water and SPMin the contaminated sampled places fluctuated daily with the tide (from 10to 53 ng.dm^-3 and 3.5 to 26 g.g^-1, respectively),indicating the export at the tidal rhythm. Bottom sediments exhibitedhigher concentrations, the maximum (48 g.g^-1) occurringnear the anthropogenic source at depths corresponding to the periods ofhigher industrial discharges. Mercury concentrations in pore waters (max.80 ng.dm^-3) exceed the values found in the water column and werehigher at depths where Acid Volatile Sulphides (AVS) presented broadmaximums. This implies a remobilization at anoxic conditions. However theexport across the sediment-water interface predicted through moleculardiffusion is low (between 1 to 4 × 10^-3 ng.cm^-2.h^-1) incomparison to the industrial input. The anthropogenic mercury appears tobe rapidly accumulated in seabass, a predator fish that enter seasonally inLargo do Laranjo.     

On‐resin conversion of Cys(Acm)‐containing peptides to their corresponding Cys(Scm) congeners

The Acm protecting group for the thiol functionality of cysteine is removed under conditions (Hg²⁺) that are orthogonal to the acidic milieu used for global deprotection in Fmoc‐based solid‐phase peptide synthesis. This use of a toxic heavy metal for deprotection has limited the usefulness of Acm in peptide synthesis. The Acm group may be converted to the Scm derivative that can then be used as a reactive intermediate for unsymmetrical disulfide formation. It may also be removed by mild reductive conditions to generate unprotected cysteine. Conversion of Cys(Acm)‐containing peptides to their corresponding Cys(Scm) derivatives in solution is often problematic because the sulfenyl chloride reagent used for this conversion may react with the sensitive amino acids tyrosine and tryptophan. In this protocol, we report a method for on‐resin Acm to Scm conversion that allows the preparation of Cys(Scm)‐containing peptides under conditions that do not modify other amino acids. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Molecular mobility and phase structure of biodegradable poly(butylene succinate) and poly(butylene succinate-co-butylene adipate)

Molecular mobility and phase structure of biodegradable poly(butylene succinate) (PBS) and poly(butylene succinate-co-20 mol % butylene adipate) [P(BS-co-20 mol % BA)] have been investigated by high-resolution solid-state (13)C NMR. For both samples, two components with different (13)C spin-lattice relaxation time (T(1C)) values have been observed in the crystalline region. The crystalline component with shorter T(1C) value is assignable to the interface near amorphous phase. The crystalline component with longer T(1C) value is ascribed to the inside of the crystalline region. On the basis of T(1C), it has been concluded that the BA units are not included in the crystalline region of P(BS-co-20 mol % BA). Molecular mobility and higher-ordered structure of amorphous phase have been also compared between the melt and solid state. Variable-temperature high-resolution (13)C NMR measurements for the amorphous phase have revealed the remarkable difference in dynamics and structure between the melt and solid state.     

Synthesis,characterization, and biological activity of poly(arginine)‐derived cancer‐targeting peptides in HepG2 liver cancer cells

The solid‐phase synthesis, structural characterization, and biological evaluation of a small library of cancer‐targeting peptides have been determined in HepG2 hepatoblastoma cells. These peptides are based on the highly specific Pep42 motif, which has been shown to target the glucose‐regulated protein 78 receptors overexpressed and exclusively localized on the cell surface of tumors. In this study, Pep42 was designed to contain varying lengths (3–12) of poly(arginine) sequences to assess their influence on peptide structure and biology. Peptides were effectively synthesized by 9‐fluorenylmethoxycarbonyl‐based solid‐phase peptide synthesis, in which the use of a poly(ethylene glycol) resin provided good yields (14–46%) and crude purities >95% as analyzed by liquid chromatography–mass spectrometry. Peptide structure and biophysical properties were investigated using circular dichroism spectroscopy. Interestingly, peptides displayed secondary structures that were contingent on solvent and length of the poly(arginine) sequences. Peptides exhibited helical and turn conformations, while retaining significant thermal stability. Structure–activity relationship studies conducted by flow cytometry and confocal microscopy revealed that the poly(arginine) derived Pep42 sequences maintained glucose‐regulated protein 78 binding on HepG2 cells while exhibiting cell translocation activity that was contingent on the length of the poly(arginine) strand. In single dose (0.15 mM) and dose‐response (0–1.5 mM) cell viability assays, peptides were found to be nontoxic in human HepG2 liver cancer cells, illustrating their potential as safe cancer‐targeting delivery agents. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

The determination of (-)-(S)- and (+)-(R)-ifosfamide in plasma using enantioselective gas chromatography: a validated assay for pharmacokinetic and clinical studies

An enantioselective gas chromatographic method has been developed and validated for the determination of the plasma concentration of the enantiomers of the anticancer drug ifosfamide (IFF). In this approach, the IFF enantiomers are separated from the plasma matrix by solid phase extraction, chromatographically resolved by gas chromatography on a chiral stationary phase, and detected by mass selective detection using selective ion monitoring. The assay has been validated for routine clinical and pharmacokinetic use and has a limit of detection in plasma of 250 ng/ml of each isomer.     

Determination of thiram in natural waters using flow‐injection with cerium(IV)–quinine chemiluminescence system

A simple and rapid flow‐injection chemiluminescence method has been developed for the determination of dithiocarbamate fungicide thiram based on the chemiluminescence reaction of thiram with ceric sulfate and quinine in aqueous sulfuric acid. The present method allowed the determination of thiram in the concentration range of 7.5–2500 ng/mL and the detection limit (signal‐to‐noise ratio = 3) was 7.5 ng/mL with sample throughput of 120/h. The relative standard deviation was 2.5% for 10 replicate analyses of 500 ng/mL thiram. The effects of foreign species including various anions and cations present in water at environmentally relevant concentrations and some pesticides were also investigated. The proposed method was applied to determine thiram in spiked natural waters using octadecyl bonded phase silica (C₁₈) cartridges for solid‐phase extraction. The recoveries were in the range 99 ± 1 to 104 ± 1%. Copyright © 2009 John Wiley & Sons, Ltd.     

Formation of truncated peptide by‐products via sequence‐specific formyl group transfer from Trp(For) residues to Nα in the course of Boc‐SPPS

(N^In)‐Formyl protective group of tryptophan has been introduced as a base/nucleophile‐labile protective group. It has long been known that a free Nα‐amino group of the peptide can serve as a nucleophile: an irreversible formyl N^In → NH₂ transfer is consistently observed when deformylation is performed last on an otherwise deprotected peptide that possesses free Nα‐amino group. Obviously, this particular side reaction should be expected any time free amino group is exposed to Trp(For), but, at the best of our knowledge, has never been reported in the course of Boc‐SPPS. In the present communication, we describe a set of appropriately designed model experiments that permitted to detect the title side reaction both in solution and in solid‐phase reactions. We observed intermolecular formyl group transfer with a model compound, Trp(For)‐NH₂. Importantly, we also observed this migration on solid support with the rate roughly estimated to be up to 1% of residues per minute. We also observed that the formyl‐group transfer reaction occurred in a sequence‐dependent manner and was suppressed to a non‐detectable level using ‘in situ neutralization’ technique. Because this side reaction is sequence dependent, there might be situations when the rate of the formation of N_α‐formyl termination by‐products is significant. In other cases, the N_α‐For truncated by‐products would not contaminate the final peptide significantly but still could be a source of microheterogeneity. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Practical access to the proline analogs (S,S,S)- and (R,R,R)-2-methyloctahydroindole-2-carboxylic acids by HPLC enantioseparation

An efficient methodology for the preparation of the α‐tetrasubstituted proline analog (S,S,S)‐2‐methyloctahydroindole‐2‐carboxylic acid, (S,S,S)‐(αMe)Oic, and its enantiomer, (R,R,R)‐(αMe)Oic, has been developed. Starting from easily available substrates and through simple transformations, a racemic precursor has been synthesized in excellent yield and further subjected to HPLC resolution using a cellulose‐derived chiral stationary phase. Specifically, a semipreparative (250 mm × 20 mm ID) Chiralpak® IC column has allowed the efficient resolution of more than 4 g of racemate using a mixture of n‐hexane/tert‐butyl methyl ether/2‐propanol as the eluent. Multigram quantities of the target amino acids have been isolated in enantiomerically pure form and suitably protected for incorporation into peptides. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

(α-Pyridyl) methyl phosphoro-bis-triazolide as a new phosphorylating reagent for internucleotide bond formation

(α-Pyridyl)methyl phosphoro-bis-triazolide has been found to be a reagent of choice for phosphate protection in oligodeoxyribonucleotide synthesis. The reagent has been used successfully to phosphorylate all the four 5’-and N-protected deoxynucleosides. The resulting 3′-phosphorylated derivatives were found to be fairly stable as either triethyl ammonium salts or cyanoethyl derivatives. The phosphorylated derivatives were used in the preparation of the dimers T_pT and d(A_pT) in solution phase and a tetramer, TTTT, and a hexamer, d(ATATAT), on solid phase using glass support. The method gave excellent yields. Considerably reduced condensation time (6–9 min) and practically no cleavage of the internucleotide bond during the removal of the group are the advantages. Presented at the 3rd National Symposium on Bioorganic Chemistry, 1987, Hyderabad.     

Phospholipid lateral phase separation and the partition of cis-parinaric acid and trans-parinaric acid among aqueous, solid lipid, and fluid lipid phases.

The partition of cis-parinaric acid (9,11,13,15-cis, trans, trans,cis-octadecatetraenoic acid, cis-PnA) and trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid, trans-PnA) among aqueous, solid lipid, and fluid lipid phases has been measured by three spectroscopic parameters: absorption spectral shifts, fluorescence quantum yield, and fluorescence polarization. The solid lipid was dipalmitoylphosphatidylcholine (DPPC); the fluid lipid was palmitoyldocosahexaenoylphosphatidylcholine (PDPC). Mole fraction partition coefficients between lipid and water were determined by absorption spectroscopy to be for ci--PnA, 5.3 X 10(5) with a solid lipid and 9 X 10(5) with fluid lipid and, for trans-PnA, 5 X 10(6) with solid lipid and 1.7 X 10(6) with fluid lipid. Ratios of the solid to the fluid partition coefficients (Kps/f) are 0.6 +/- 0.2 for cis-PnA and 3 +/- 1 for trans-PnA. A phase diagram for codispersions of DPPC and PDPC has been constructed from the measurements of the temperature dependence of the fluorescence quantum yield and polarization of cis-PnA and trans-PnA and their methyl ester derivatives. A simple analysis based on the phase diagram and fluorescence data allows additional calculations of Kps/f's which are determined to be 0.7 +/- 0.2 for the cis probes and 4 +/- 1 for the trans probes. The relative preference of trans-PnA for solid phase lipids and its enhanced quantum yield in solid phase lipids make it sensitive to a few percent solid. The trans probes provide evidence that structural order may persist in dispersions of these phospholipids 10 degrees C or more above their transition temperature. It is concluded that measurements of PnA fluorescence polarization vs. temperature are better suited than measurements of quantum yield vs. temperature for determining phospholipid phase separation.     

Water uptake in germinating pecan (Carya illinoinensis) seed

• Water uptake is the fundamental and essential requirement for seed germination. Pecan seed has a hard woody endocarp that plays an important role during water uptake.
• To explore water uptake during germination, the spatiotemporal pattern of water and effect of the endocarp were analysed using high-field MRI, dye-tracing, wax blocking and SEM of water uptake.
• Isolated seeds completed water uptake in 8 h while whole seeds required 6 days, hence, cracking the endocarp plays an important role. The hilum is the channel through which water enters the seed, while the remainder of the seed coat consist of cells covered with a waxy layer that act as a barrier to water absorption. The region with the highest water content in pecan seed is the edge of the U-shaped region, and water can progressively diffuse from this U-shaped region into the whole kernel.
• We report a new water absorption stage between phase II and phase III of the triphasic model of water uptake of pecan seeds. Cracking the endocarp changed water distribution in pecan seeds, which may trigger further water absorption and radicle elongation.

     

Synthesis and application of N‐[1‐(4‐(4‐fluorophenyl)‐2,6‐dioxocyclohexylidene)ethyl] (Fde)‐protected amino acids for optimization of solid‐phase peptide synthesis using gel‐phase 19F NMR spectroscopy

N‐[1‐(4‐(4‐fluorophenyl)‐2,6‐dioxocyclohexylidene)ethyl] (Fde) protected amino acids have been prepared and applied in solid‐phase peptide synthesis monitored by gel‐phase ¹⁹F NMR spectroscopy. The Fde protective group could be cleaved with 2% hydrazine or 5% hydroxylamine solution in DMF as determined with gel‐phase ¹⁹F NMR spectroscopy. The dipeptide Ac‐L ‐Val‐L ‐Val‐NH₂ 12 was constructed using Fde‐L ‐Val‐OH and no noticeable racemization took place during the amino acid coupling with N,N′‐diisopropylcarbodiimide and 1‐hydroxy‐7‐azabenzotriazole or Fde deblocking. To extend the scope of Fde protection, the hydrophobic nonapeptide LLLLTVLTV from the signal sequence of mucin MUC1 was successfully prepared using Fde‐L ‐Leu‐OH at diagnostic positions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Thermotropic phase properties of 1,2-di-O-tetradecyl-3-O-(3-O-methyl- beta-D-glucopyranosyl)-sn-glycerol.

The hydration properties and the phase structure of 1,2-di-O-tetradecyl-3-O(3-O-methyl-beta-D-glucopyranosyl)-sn-glycerol (3-O-Me-beta-D-GlcDAIG) in water have been studied via differential scanning calorimetry, 1H-NMR and 2H-NMR spectroscopy, and x-ray diffraction. Results indicate that this lipid forms a crystalline (Lc) phase up to temperatures of 60-70 degrees C, where a transition through a metastable reversed hexagonal (Hll) phase to a reversed micellar solution (L2) phase occurs. Experiments were carried out at water concentrations in a range from 0 to 35 wt%, which indicate that all phases are poorly hydrated, taking up < 5 mol water/mol lipid. The absence of a lamellar liquid crystalline (L alpha) phase and the low levels of hydration measured in the discernible phases suggest that the methylation of the saccharide moiety alters the hydrogen bonding properties of the headgroup in such a way that the 3-O-Me-beta-D-GlcDAIG headgroup cannot achieve the same level of hydration as the unmethylated form. Thus, in spite of the small increase in steric bulk resulting from methylation, there is an increase in the tendency of 3-O-Me-beta-D-GlcDAIG to form nonlamellar structures. A similar phase behavior has previously been observed for the Acholeplasma laidlawii A membrane lipid 1,2-diacyl-3-O-(6-O-acyl-alpha-D-glucopyranosyl)-sn-glycerol in water (Lindblom et al. 1993. J. Biol. Chem. 268:16198-16207). The phase behavior of the two lipids suggests that hydrophobic substitution of a hydroxyl group in the sugar ring of the glucopyranosylglycerols has a very strong effect on their physicochemical properties, i.e., headgroup hydration and the formation of different lipid aggregate structures.     

Molecular phylogeny of the subgenus Holothuria (Selenkothuria) Deichmann, 1958 (Holothuroidea: Aspidochirotida)

The subgenus Selenkothuria comprises 12 species of tropical shallow water sea cucumbers that share morphological features, such as rods in the body wall and tube feet, modified tentacles for suspension feeding, and cryptic colours. The taxonomic status of this taxon has been controversial, but currently it is accepted as a subgenus of the genus Holothuria. Phylogenetic analyses of mitochondrial genes [cytochrome c oxidase subunit 1 (COI), 16S RNA] of ten species of Selenkothuria and related subgenera showed the polyphyly of this subgenus; monophyly was rejected by a likelihood ratio test. A geographical split divides the species of this subgenus into three different groups: one Indo‐West‐Pacific (IWP) group and two American groups. The IWP group is more closely related to Holothuria (Semperothuria) cinerascens and to other subgenera such as Roweothuria, Holothuria, and Vaneyothuria, whereas the two American groups are more closely related to each other and to some species of the subgenus Halodeima. These results suggest multiple parallel originations and diversification of ossicle morphology within the subgenus Selenkothuria. The current scheme of subgenera for the genus Holothuria is not supported, suggesting the need for a new classification. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 165 , 109–120.