Influence of organic solvents on papain kinetics

Papain activity was studied in water-organic solvent mixtures using the fluorogenic substrate Dabcyl-Lys-Phe-Gly-Gly-Ala-Ala-Edans. The increase of organic solvent (MeOH, EtOH, iPrOH, TFE, MeCN, (MeO)2Et and DMF) concentration in the mixture caused a substantial decrease the initial rate of papain-catalyzed hydrolysis. Moreover, the number of papain active sites decreased with the increase of DMF and MeOH concentration.     

Liquid chromatographic separation of darunavir enantiomers on coated and immobilized amylose tris(3, 5-dimethylphenylcarbamate) chiral stationary phases

Liquid chromatographic separation of darunavir enantiomers on covalently bonded and physically adsorbed polysaccharide chiral stationary phases was studied at different temperatures. The separations were accomplished under normal-phase conditions by using different combinations of hexane, organic modifiers (2-propanol, 1-propanol and ethanol), and diethylamine as mobile phase solvents. The effect of organic modifiers and the column temperature on retention, separation, and resolution was investigated. The observed differences were explained in terms of the coated and immobilized nature of the two columns. Van't Hoff plots (ln k' vs. 1/T, ln α vs. 1/T) and apparent thermodynamic parameters were derived to understand the effect of temperature on separation.     

Solvent selectivity in chiral chromatography using a β-cyclodextrin-bonded phase

A β-cyclodextrin-bonded phase has been used to investigate the separation of the enantiomers of atenolol, oxprenolol, celiprolol, tertatolol, terbutaline, fluoxetine, norfluoxetine, and zopiclone, focusing on the importance of solvent selectivity. With cyclodextrin (CD)-bonded phases, chiral discrimination occurs because the two enantiomers of a racemate form inclusion complexes of different strengths within the CD cavity. The organic modifier molecules tend to compete with solutes for a definite number of adsorption sites on the stationary phase. Moreover, the ternary complex formation may play an important role in chiral recognition. In this study, it was of interest to estimate the influence of mobile phase modifiers with respect to solvent type (i.e., ACN, MeOH, EtOH, THF, i-PrOH, PrOH and t-BuOH), size and shape, and concentration. Solvent selectivity has been investigated by using different organic modifiers in mobile phases with the same polarity, and relationships were established between the logarithm of solvent partition coefficient (log P_s) and the three most important chromatographic parameters: retention time (t), resolution (R), and enantioselectivity (α). Thus, it seems that the hydrophobicity of the organic modifier becomes one of the dominant factors affecting the inclusion process phenomena. Further, the apparent partition coefficients of the compounds under study have been determined and a comparison has been attempted regarding the degree of their enantiomeric resolution. © 1995 Wiley-Liss, Inc.     

Extracting fumonisins from maize: efficiency of different extraction solvents in multi-mycotoxin analytics

The complete extraction of analytes is of utmost importance when analyzing matrix samples for mycotoxins. Mycotoxins consist of substances with widely different physicochemical properties; therefore, the loss of toxins that occurs in multi-mycotoxin methods due to compromises in the extraction solvent is currently a topic under discussion. With regard to fumonisins, several extractants from recently published multi-mycotoxin methods were investigated when analyzing unprocessed and processed maize matrices. All extractants were tested in a validated on-site method and the extraction yields were compared to those of an HPLC-FLD reference method (EN 14352). Most of the compared multi-mycotoxin methods that have been published were only for analyzing fumonisins in maize or maize-meal; we have applied the extractants of these methods to processed, complex maize matrices for the first time. Our results show that, for extractions with aqueous acetonitrile mixtures with the addition of acid, e.g. MeCN/H₂O/acetic acid (79/20/1, v/v/v), higher extraction yields are obtained than with MeCN/H₂O (80/20, v/v), in both spiked and naturally contaminated maize matrices. But compared to the results of the reference method EN 14352, the two extractants did not show a similar extraction efficiency. Overall, the extractant MeCN/MeOH/H₂O (1/1/2, v/v/v) turned out to be the most appropriate extractant applied in all experiments, obtaining the best and most comparable extraction yields and recoveries. Furthermore, our investigations showed that, with some of the tested extraction solvents, e.g. MeCN/H₂O (75/25) containing 50 mmol/l formic acid, stark differences occur when analyzing spiked and naturally contaminated matrices. With spiked matrices, recoveries of approximately 80–110 % were obtained, but with naturally contaminated matrices no results comparable to the EN method have been achieved. In contrast, a double extraction with MeCN/H₂O/formic acid (80/19,9/0,1, v/v/v), followed by a second polar extraction step with MeCN/H₂O/formic acid (20/79,9/0,1, v/v/v), led, for most naturally contaminated samples, to comparable results with the EN method. However, for spiked samples, the same extractant led to raised recoveries of between 120 and 140 %. For some processed matrices, like taco-chips, all tested extractants showed a poor extraction efficiency for fumonisins. By extending the extraction time from 1 to 15 min, a result comparable to that of the reference method could also be obtained for the extractant using MeCN/MeOH/H₂O (1/1/2, v/v/v). As this extractant has been used in our recently published method (Trebstein et al. Mycotoxin Res 25:201, 2009), this work also presents an update on this method with respect to the extended extraction time.     

Liquid and subcritical fluid chromatographic enantioseparation of Nα‐Fmoc proteinogenic amino acids on Quinidine‐based zwitterionic and anion‐exchanger type chiral stationary phases. A comparative study

Stereoselective high‐performance liquid chromatographic and subcritical fluid chromatographic separations of 19 N^α‐Fmoc proteinogenic amino acid enantiomers were carried out by using Quinidine ‐based zwitterionic and anion‐exchanger‐type chiral stationary phases Chiralpak ZWIX(−) and QD‐AX. For optimization of retention and enantioselectivity, the ratio of bulk solvent components (MeOH/MeCN, H₂O/MeOH, or CO₂/MeOH) and the nature and concentration of the acid and base additives (counter‐ and co‐ions) were systematically varied. The effect of column temperature on the enantioseparation was investigated and thermodynamic parameters were calculated from the van't Hoff plots ln α vs. 1/T. The thermodynamic parameters revealed that the enantioseparations were enthalpy‐driven. The elution sequence was determined in all cases and with the exception of Fmoc‐Cys(Trt)‐OH, it was identical on both chiral stationary phases whereby the L‐enantiomers eluted before the D‐enantiomers.     

Optimization of reversed-phase peptide liquid chromatography ultraviolet mass spectrometry analyses using an automated blending methodology.

The balance between chromatographic performance and mass spectrometric response has been evaluated using an automated series of experiments where separations are produced by the real-time automated blending of water with organic and acidic modifiers. In this work, the concentration effects of two acidic modifiers (formic acid and trifluoroacetic acid) were studied on the separation selectivity, ultraviolet, and mass spectrometry detector response, using a complex peptide mixture. Peptide retention selectivity differences were apparent between the two modifiers, and under the conditions studied, trifluoroacetic acid produced slightly narrower (more concentrated) peaks, but significantly higher electrospray mass spectrometry suppression. Trifluoroacetic acid suppression of electrospray signal and influence on peptide retention and selectivity was dominant when mixtures of the two modifiers were analyzed. Our experimental results indicate that in analyses where the analyzed components are roughly equimolar (e.g., a peptide map of a recombinant protein), the selectivity of peptide separations can be optimized by choice and concentration of acidic modifier, without compromising the ability to obtain effective sequence coverage of a protein. In some cases, these selectivity differences were explored further, and a rational basis for differentiating acidic modifier effects from the underlying peptide sequences is described.     

Chemical and seasonal controls on the dynamics of dissolved organic matter in a coniferous old-growth stand in the Pacific Northwest,USA

Soil organic matter (SOM) is the largest terrestrial C pool, and retention and release of dissolved organic matter (DOM) cause formation and loss of SOM. However, we lack information on how different sources of DOM affect its chemical composition, and how DOM chemical composition affects retention. We studied seasonal controls on DOM production and chemical controls on retention in soils of a temperate coniferous forest. The O horizon was not usually the dominant source for dissolved organic C (DOC) or N (DON) as has been reported for other sites. Rather, net production of both DOC and DON was often greater in the shallow mineral soil (0–10 cm) than in the O horizon. DOM production in the shallow mineral soil may be from root exudation as well as turnover of fine roots and microflora in the rhizosphere. In the field, the two acid fractions (hydrophobic and hydrophilic acids) dominated the soil solution at all depths. A major portion of net production and removal of total DOC within the soil column was explained by increases and decreases in these fractions, although a shift in chemical composition of DOM between the O and mineral soil horizons suggested different origins of DOM in these layers. A larger loss of the free amino fraction to deep soil water at this study site than at other sites suggested lower retention of labile DON. Field DOM removal measurements suggest that field-measured parameters may provide a good estimate for total DOM retained in mineral soil.     

Effect of guanidine and arginine on protein–ligand interactions in multimodal cation‐exchange chromatography

The addition of fluid phase modifiers provides significant opportunities for increasing the selectivity of multimodal chromatography. In order to optimize this selectivity, it is important to understand the fundamental interactions between proteins and these modifiers. To this end, molecular dynamics (MD) simulations were first performed to study the interactions of guanidine and arginine with three proteins. The simulation results showed that both guanidine and arginine interacted primarily with the negatively charged regions on the proteins and that these regions could be readily predicted using electrostatic potential maps. Protein surface characterization was then carried out using computationally efficient coarse‐grained techniques for a broader set of proteins which exhibited interesting chromatographic retention behavior upon the addition of these modifiers. It was shown that proteins exhibiting an increased retention in the presence of guanidine possessed hydrophobic regions adjacent to negatively charged regions on their surfaces. In contrast, proteins which exhibited a decreased binding in the presence of guanidine did not have hydrophobic regions adjacent to negatively charged patches. These results indicated that the effect of guanidine could be described as a combination of competitive binding, charge neutralization and increased hydrophobic interactions for certain proteins. In contrast, arginine resulted in a significant decrease in protein retention times primarily due to competition for the resin and steric effects, with minimal accompanying increase in hydrophobic interactions. The approach presented in this paper which employs MD simulations to guide the application of coarse‐grained approaches is expected to be extremely useful for methods development in downstream bioprocesses. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:435–447, 2017     

Reactivity and activation of dioxygen-derived species in aprotic media (a model matrix for biomembranes)

In aprotic media the electrochemical reduction of dioxygen yields superoxide ion (O2-), which is an effective Br?nsted base, nucleophile, one-electron reductant, and one-electron oxidant of reduced transition metal ions. With electrophilic substrates (organic halides and carbonyl carbons) O2- displaces a leaving group to form a peroxy radical (ROO.) in the primary process. Superoxide ion oxidizes the activated hydrogen atoms of ascorbic acid, catechols, hydrophenazines and hydroflavins. Combination of O2- with 1,2-diphenylhydrazine yields the anion radical of azobenzene, which reacts with O2 to give azobenzene and O2- (an example of O2--induced autoxidation). With phenylhydrazine, O2- produces phenyl radicals. The in situ formation of HO2. (O2- plus a proton source) results in H-atom abstraction from allylic and other groups with weak heteroatom--H bonds (binding energy (b.e.) less than 335 kJ). This is a competitive process with the facile second-order disproportionation of HO2. to H2O2 and O2 (kbi approximately equal to 10(4) mol-1 s-1 in Me2SO). Addition of [FeII(MeCN)4] (ClO4)2 to solutions of hydrogen peroxide in dry acetonitrile catalyses a rapid disproportionation of H2O2 via the initial formation of an adduct [FeII(H2O2)2+----Fe(O)(H2O)2+], which oxidizes a second H2O2 to oxygen. In the presence of organic substrates such as 1,4-cyclohexadiene, 1,2-diphenylhydrazine, catechols and thiols the FeII-H2O2/MeCN system yields dehydrogenated products; with alcohols, aldehydes, methylstyrene, thioethers, sulphoxides, and phosphines, the FeII(H2O2)2+ adduct promotes their monoxygenation. The product from the FeO2+-H2O2 reaction, [FeII(H2O2)22+], exhibits chemistry that is closely similar to that for singlet oxygen (1O2), which has been confirmed by the stoichiometric dioxygenation of diphenylisobenzofuran, 9,10-diphenylanthracene, rubrene and electron-rich unsaturated carbon-carbon bonds (Ph2C = CPh2, PhC = CPh and cis-PhCH = CHPh). In dry ligand-free acetonitrile (MeCN), anhydrous ferric chloride (FeIIICl3) activates hydrogen peroxide for the efficient epoxidation of alkenes. The FeIIICl3 further catalyses the dimerization of the resulting epoxides to dioxanes. These observations indicate that strong Lewis acids that are coordinatively unsaturated, [FeII(MeCN)4]2+ and [FeIIICl3], activate H2O2 to form an effective oxygenation and dehydrogenation agent.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential recovery of membrane proteins after extraction by aqueous methanol and trifluoroethanol

Cell membrane proteome analysis is limited by inherent membrane hydrophobicity. Conventional membrane protein extraction techniques use detergents, chaotropes and organic acids that require sample clean-up or pH adjustment, and are associated with significant sample loss. We extracted membrane proteins from red blood cells (RBCs) using methanol (MeOH), trifluoroethanol (TFE) and urea, and identified membrane proteins using 2-D LC coupled with MALDI-TOF/TOF-MS. We show that organic solvents MeOH- and TFE-based methods have membrane protein analysis efficiencies comparable to urea, and are complementary for the recovery of both hydrophilic and hydrophobic peptides. The mean grand average of hydropathicity (GRAVY) value of identified peptides from the TFE-based method (-0.107) was significantly higher than that of the MeOH-based method (-0.465) (p<0.001). Sequential and adjunctive use of the organic solvents MeOH and TFE increases the number of proteins identified, and the confidence of their identification. We show that this strategy is effective for shotgun membrane proteome analysis.     

On the stabilizing action of protein denaturants: acetonitrile effect on stability of lysozyme in aqueous solutions

Stability of hen lysozyme in the presence of acetonitrile (MeCN) at different pH values of the medium was studied by scanning microcalorimetry with a special emphasis on determination of reliable values of the denaturational heat capacity change. It was found that the temperature of denaturation decreases on addition of MeCN. However, the free energy extrapolation showed that below room temperature the thermodynamic stability increases at low concentrations of MeCN in spite of the general destabilizing effect at higher concentrations and temperatures. Charge-induced contribution to this stabilization was shown to be negligible (no pH-dependence was found); therefore, the most probable cause for the phenomenon is an increase of hydrophobic interactions at low temperatures in aqueous solutions containing small amounts of the organic additive. The difference in preferential solvation of native and denatured states of lysozyme was calculated from the stabilization free energy data. It was found that the change in preferential solvation strongly depends on the temperature in the water-rich region. At the higher MeCN content this dependence decreases until, at 0.06 mole fractions of MeCN, the difference in the preferential solvation between native and denatured lysozyme becomes independent of the temperature over a range of 60 K. The importance of taking into account non-ideality of a mixed solution, when analyzing preferential solvation phenomena was emphasized.     

Mobile phase modifier effects in multimodal cation exchange chromatography

This study examines protein adsorption behavior and the effects of mobile phase modifiers in multimodal chromatographic systems. Chromatography results with a diverse protein library indicate that multimodal and ion exchange resins have markedly different protein binding behavior and selectivity. NMR results corroborate the stronger binding observed for the multimodal system and provide insight into the structural basis for the observed binding behavior. Protein-binding affinity and selectivity in multimodal and ion exchange systems are then examined using a variety of mobile phase modifiers. Arginine and guanidine are found to have dramatic effects on protein adsorption, yielding changes in selectivity in both chromatographic systems. While sodium caprylate leads to slightly weaker chromatographic retention for most proteins, certain proteins exhibit significant losses in retention in both systems. The presence of a competitive binding mechanism between the multimodal ligand and sodium caprylate for binding to ubiquitin is confirmed using STD NMR. Polyol mobile phase modifiers are shown to result in increased retention for weakly bound proteins and decreased retention for strongly bound proteins, indicating that the overall retention behavior is determined by a balance between changes in electrostatic and hydrophobic interactions. This work provides an improved understanding of protein adsorption and mobile phase modifier effects in multimodal chromatographic systems and sets the stage for future work to develop more selective protein separation systems.     

Effects of organic solvents on the enzyme activity of Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase in calorimetric assays

In drug discovery programs, dimethyl sulfoxide (DMSO) is a standard solvent widely used in biochemical assays. Despite the extensive use and study of enzymes in the presence of organic solvents, for some enzymes the effect of organic solvent is unknown. Macromolecular targets may be affected by the presence of different solvents in such a way that conformational changes perturb their active site structure accompanied by dramatic variations in activity when performing biochemical screenings. To address this issue, in this work we studied the effects of two organic solvents, DMSO and methanol (MeOH), in the isothermal titration calorimetry (ITC) kinetic assays for the catalyzed reaction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Trypanosoma cruzi. The solvent effects on T. cruzi GAPDH had not yet been studied. This enzyme was shown here to be affected by the organic solvents content up to 5.0% for MeOH and up to 7.5% for DMSO. The results show that when GAPDH is assayed in the presence of DMSO (5%, v/v) using the ITC experiment, the enzyme exhibits approximately twofold higher activity than that of GAPDH with no cosolvent added. When MeOH (5%, v/v) is the cosolvent, the GAPDH activity is sixfold higher. The favorable effects of the organic solvents on the Michaelis-Menten enzyme-substrate complex formation ensure the consistency of the biological assays, structural integrity of the protein, and reproducibility over the measurement time. The reaction was also kinetically monitored by standard spectrophotometric assays to establish a behavioral performance of T. cruzi GAPDH when used for screening of potential inhibitors.     

Carvedilol,a beta adrenoceptor blocker with chelating properties. A copper 'superdimer' based on dimetal units

Copper(II) complexes of carvedilol molecule, (CARVH): 1-[carbazolyl-(4)-oxyl]-3-[(2-methoxyphenoxyethyl)-amino]-2-propanol, were synthesized and characterized with respect to their structural and spectroscopic properties. The crystal structure of [Cu(Carv)Cl(MeOH)](2).4MeOH complex revealed that the molecule chelates two Cu(II) ions via the N and O atoms belonging to the amino and propanol moiety, respectively. The coordination behaviour of carvedilol studied by 1H nuclear magnetic resonance (NMR, 1-D and 2-D-COSY) spectroscopy in dimethyl sulfoxide solution at room temperature, allowed us to obtain structural information and to identify the donor atoms involved in the coordination process in solution.     

Interaction of Hydrogen Peroxide with Ribulose-1,5-bisphosphate Carboxylase/Oxygenase from Rice

The properties of rice-derived ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) in different concentrations of hydrogen peroxide (H2O2) solutions have been studied. The results indicate that at low H2O2 concentrations (0.2-10 mM), the properties of rubisco (e.g., carboxylase activities, structure, and susceptibility to heat denaturation) change slightly. However, at higher H2O2 concentrations (10-200 mM), rubisco undergoes an unfolding process, including the loss of secondary and tertiary structure, forming extended hydrophobic interface, and leading to cross-links between large subunits. High concentrations of H2O2 can also result in an increase in susceptibility of rubisco to heat denaturation. Further pre-treatments with or without reductive reagents to rubisco show that the disulfide bonds in rubisco help to protect the enzyme from damage by H2O2 as well as other reactive oxygen species.     

Changes in dissolved lignin-derived phenols, neutral sugars, uronic acids, and amino sugars with depth in forested Haplic Arenosols and Rendzic Leptosols

A major part of the dissolved organic matter produced in the organic layers of forest ecosystems and leached into the mineral soil is retained by the upper subsoil horizons. The retention is selective and thus dissolved organic matter in the subsoils has different composition than dissolved organic matter leached from the forest floor. Here we report on changes in the composition of dissolved organic matter with soil depth based on C-to-N ratios, XAD-8 fractionation, wet-chemical analyses (lignin-derived CuO oxidation products, hydrolysable sugars and amino sugars) and liquid-state ¹³C nuclear magnetic resonance (NMR) spectroscopy. Dissolved organic matter was sampled directly beneath the forest floor using tension-free lysimeters and at 90cm depth by suction cups in Haplic Arenosols under Scots pine (Pinus sylvestris L.) and Rendzic Leptosols under European beech (Fagus sylvatica L.) forest. At both sites, the concentrations of dissolved organic carbon (DOC) decreased but not as strongly as reported for deeply weathered soils. The decrease in DOC was accompanied by strong changes in the composition of dissolved organic matter. The proportion of the XAD-8-adsorbable (hydrophobic) fraction, carboxyl and aromatic C, and the concentrations of lignin-derived phenols decreased whereas the concentrations of sugars, amino sugars, and nitrogen remained either constant or increased. A general feature of the compositional changes within the tested compound classes was that the ratios of neutral to acidic compounds increased with depth. These results indicate that during the transport of dissolved organic matter through the soils, oxidatively degraded lignin-derived compounds were preferentially retained while potentially labile material high in nitrogen and carbohydrates tended to remain dissolved. Despite the studied soils' small capacity to sorb organic matter, the preferential retention of potentially refractory and acidic compounds suggests sorption by the mineral soil matrix rather than biodegradation to govern the retention of dissolved organic matter even in soils with a low sorption capacity.     

Determination of the novel non-peptidic HIV-protease inhibitor tipranavir by HPLC-UV after solid-phase extraction

An HPLC method previously described for the assay of amprenavir (APV), ritonavir (RTV), indinavir (IDV), saquinavir (SQV), nelfinavir (NFV), lopinavir (LPV), atazanavir (ATV), nevirapine (NVP) and efavirenz (EFV) can be also conveniently applied, with minor gradient program adjustment, for the determination of the novel non-peptidic HIV protease inhibitor tipranavir (TPV) in human plasma, by off-line solid-phase extraction (SPE) followed by HPLC coupled with UV-diode array detection (DAD). After viral inactivation by heat, the plasma is diluted with phosphate buffer (pH 7), and subjected to a SPE on a C18 cartridge. Matrix components are eliminated with a solution of 0.1% H3PO4 solution neutralised to pH 7, and TPV is eluted with MeOH. The resulting eluate is evaporated and reconstituted in 100 microl MeOH/H2O 50/50. A 40 microl volume is injected onto a Nucleosil C18 AB column and TPV is analysed by UV detection at 201 nm using a gradient elution program constituted of MeCN and phosphate buffer adjusted to pH 5.12 and containing 0.02% sodium heptanesulfonate. The calibration curves are linear up to 75 microg/ml, with a lower limit of quantification of 0.125 microg/ml. The mean absolute recovery of TPV is 77.1+/-4.0%. The method is precise with mean inter-day coefficient of variations (CVs) within 2.2-3.4%, and accurate (range of inter-day deviations from 0.7 to 1.2%). The method has been validated and is currently applied to the monitoring of TPV plasma levels in HIV patients.     

Use of a Novel Sub‐2 µm Silica Hydride Vancomycin Stationary Phase in Nano‐Liquid Chromatography. II. Separation of Derivatized Amino Acid Enantiomers

A novel vancomycin silica hydride stationary phase was synthesized and the particles of 1.8 µm were packed into fused silica capillaries of 75 µm internal diameter (I.D.). The chiral stationary phase (CSP) was tested for the separation of some derivatized amino acid enantiomers by using nano‐liquid chromatography (nano‐LC). Some experimental parameters such as the type and the content of organic modifier, the pH, and the concentration of the buffer added to the mobile phase were modified and the effect on enantioselectivity, retention time, and enantioresolution factor was studied. The separation of selected dansyl amino acids (Dns‐AAs), e.g., Asp, Glu, Leu, and Phe in their enantiomers was initially achieved utilizing a mobile phase containing 85% (v/v) methanol (MeOH) and formate buffer measuring the enantioresolution factor and enantioselectivity in the range 1.74–4.17 and 1.39–1.59, respectively. Better results were obtained employing a more polar organic solvent as acetonitrile (ACN) in the mobile phase. Optimum results (Rs 1.41–6.09 and α 1.28–2.36) were obtained using a mobile phase containing formate buffer pH 2.5/water/MeOH/ACN 6:19:12.5:62.5 (v/v/v/v) in isocratic elution mode at flow rate of 130 nL/min. Chirality 27:767–772, 2015. © 2015 Wiley Periodicals, Inc.     

Combined effect of the DeltaPhe or DeltaAla residue and the p-nitroanilide group on a didehydropeptides conformation

Two series of dehydropeptides of the general formulae Boc-Gly-X-Phe-p-NA, Boc-Gly-Gly-X-Phe-p-NA, Gly-X-Gly-Phe-p-NA.TFA, and Boc-Gly-X-Gly-Phe-p-NA, with X = Delta(Z)Phe and DeltaAla, were studied with NMR in DMSO and CDCl(3)-DMSO, and with CD in MeOH, MeCN, and TFE. The NMR spectra measured in DMSO suggest that peptides with the DeltaPhe residue next to Phe are folded whereas peptides with Gly between DeltaPhe and Phe are less ordered. NMR spectra of DeltaAla-containing peptides indicate that these peptides are flexible and their conformational equilibria are populated by many different conformations. The CD spectra show that conformational properties of the peptides studied are distinctly influenced by a mutual position of the dehydroamino acid residue and the p-NA group. They indicate that all dehydropeptides with the DeltaPhe residue, Boc-Gly-DeltaAla-Phe-p-NA, and Boc-Gly-Gly-DeltaAla-Phe-p-NA adopt ordered conformations in all solvents studied, presumably of the beta-turn type. The last two peptides exhibit surprising chiroptical properties. Their spectra show exciton coupling-like couplets in the region of the p-NA group absorption. This shape of CD spectra suggests a rigid, chiral conformation with a fixed disposition of the p-NA group. The CD spectra indicate that Boc-Gly-DeltaAla-Gly-Phe-p-NA and Gly-DeltaAla-Gly-Phe-p-NA.TFA are unordered, independently of the solvent.     

High-performance liquid chromatographic study of the interactions between immobilized β-cyclodextrin polymers and hydrophobically end-capped polyethylene glycols

The formation of inclusion complexes between polyethylene glycols (PEGs) bearing hydrophobic ends (naphtyl and phenyladamantyl) and β-cyclodextrin polymers (polyβ-CD) immobilized onto silica particles was studied by high-performance liquid chromatography (HPLC). It was shown that hydrophobic interactions were involved in the retention mechanism of these compounds, since retention volumes decreased when organic solvents were added to the mobile phase while it was the contrary in the presence of salts. Moreover, the association could be reversed by adding a competitor (hydroxypropylβ-cyclodextrin) to the mobile phase. A theoretical model permitted the evaluation of affinity constants of 1:1 complexes formed between the modified PEGs and the immobilized polyβ-CD which depended on the type of hydrophobic groups grafted to the PEG.