E.s.r. study of spin-trapped radicals formed during the photolysis of aqueous solutions of acid amides and H2O2.

Free radicals formed by the reactions of OH radicals with amides and their N-methylated derivatives in aqueous solutions have been studied. The OH radicals were produced by U.V.-photolysis of H2O2, and the short-lived amide radicals were converted to more stable nitroxide radicals by addition to a spin-trap, tert-nitrosobutane. The spin-trapped radicals were identified by e.s.r. spectroscopy. For acetamide, chloroacetamide, malonamide, succinamide and propionamide, the observed radicals were formed by H-abstraction from the carbon atoms attached to the carbonyl group. The H atom attached to the carbonyl group was abstracted in formamide. For N-methyl acetamide, N,N-dimethyl acetamide and the corresponding formamide derivatives, H-abstraction occurred only from the N-methyl group. The non-equivalency of the amide protons was observed in the spin-trapped radicals for acetamide, formamide, malonamide, succinamide and propionamide. The identification of the site of OH attack on N-methyl amides is helpful for the study of radical formation in peptides and proteins.     

Experimental data and modelling of apparent molar volumes, isentropic compressibilities and refractive indices in aqueous solutions of glycine + NaCl

Experiments have been performed at 298.15 K to measure the density, sound velocity and refractive index of glycine in aqueous solutions of NaCl over a wide range of both glycine and NaCl concentrations. The values of apparent molar volume and isentropic compressibility of glycine were calculated from the measured data. The results show a positive transfer volume of glycine from an NaCl solution to a more concentrated NaCl solution. This indicates that the size of a glycine molecule is larger in a solution with higher NaCl concentration. The negative values of apparent isentropic compressibility imply that the water molecules around the glycine molecules are less compressible than the water molecules in the bulk solution. These effects are attributed to the doubly charged behaviour of glycine and to the formation of physically bonded ion-pairs between the charged groups of glycine and sodium and chloride ions. The formation of ion-pairs, whose extents of binding reactions depend on the concentrations of both NaCl and glycine, alter the hydration number of glycine. This also explains the reason for the increase in the size of glycine with an increase in the NaCl concentration. A model based on the Pitzer formalism has been developed to correlate the activity coefficient, apparent molar volume and isentropic compressibility of glycine in aqueous solutions of NaCl. The results show that the model can accurately correlate the interactions in aqueous solutions of glycine and NaCl.     

Conjugates of catecholamines. VII. Synthesis and beta-adrenergic activity of peptide catecholamine conjugates

A series of novel catecholamine derivatives has been prepared in which one of the N-methyl substituents of isoproterenol has been extended by a spacer consisting of a chain of four methylenes which terminates with an amide linkage to a peptide, the point of attachment being via the aromatic amino group of p-aminophenylalanine. In one of the derivatives, two catecholamines are attached to the same peptide in this manner. The peptides, which range in size from three to eight amino acid residues and contain phenylalanine, glycine, and L-alpha-amino-delta-hydroxyvaleric acid, were synthesized via stepwise and fragment condensation techniques. The beta-adrenergic agonist activities of the derivatives were evaluated in vitro by measuring the intracellular accumulation of cyclic AMP in S49 mouse lymphoma cells.     

An experimental study of potentiated aqueous solutions

A systematic study was undertaken of luminescent aqueous solutions of homeopathic preparation of sodium chloride at a dilution from D1 to D30, produced by "Weleda" company (Moscow) was carried out. It was shown that intensity of luminescence versus the degree of dilution is a non-monotonous function with several maxima, the main maximum corresponds to 13-14 decimal dilution. The dynamics of spectra was registered for several weeks. A systematic study of water samples (D1-D30) exposed to a similar procedure of potentization but without salt addition was also performed. The difference in the luminescence spectra of water of different stages of potentization was shown. The motility of infusoria Spirostoma ambiquum in solutions being examined was studied. A significant negative correlation between the infusoria motility and luminescence intensity was registered.     

Effect of cation and anion of an electrolyte on apparent molar volume, isentropic compressibility and refractive index of glycine in aqueous solutions

Experiments at 298.15 K have been performed to measure the density, velocity of sound and refractive index in three water+glycine+electrolyte systems. The electrolytes studied were KCl, KNO3 and NaNO3. The values of apparent molar volume and isentropic compressibility of glycine in aqueous electrolyte solutions were calculated from the measured data. The results obtained in this study and those reported previously for water+glycine+NaCl system have been comparatively studied. The results show that the nature of both the cation and the anion of an electrolyte influence the behaviour of glycine in aqueous solutions. For all four electrolytes studied, the comparison shows a positive volume transfer for glycine from an electrolyte solution to a more concentrated solution of the same electrolyte. The results also show a negative apparent isentropic compressibility for glycine in the presence of the electrolytes studied. These effects indicate that the volume of a glycine molecule is larger in solutions with higher electrolyte concentration and the water molecules around the glycine molecules are less compressible than the water molecules in the bulk solution. These effects were attributed to the doubly charged behaviour of glycine and to the formation of physically bonded ion-pairs between the charged groups of glycine and the cation and the anion of the electrolyte.     

Decomposition reactions of amino sugars: the dehydration of 2-amino-2-deoxy-D-glucose

The stability of the title compound (1) was investigated at 100° in acidified aqueous solutions containing, in some instances, glycine or pyridine. In strong acid (3M hydrochloric acid), the sugar was relatively stable, and no identifiable decomposition-products were observed. In less-acidic solutions (≤0.5M hydrochloric acid) in the presence of glycine, substantial decomposition occurred with the production of 5-(hydroxymethyl)-2-furaldehyde (2) in 0.5-5.2% yield. The major dehydration products, however (up to 18% of the starting sugar), were pyrazine derivatives bearing dissimilar, four-carbon, acyclic-sugar side-chains attached to C-2 and C-5 of the ring, respectively, arising, most probably, from C-3-C-6 of the original sugar molecules. When the conversions were performed in deuterium oxide solution, carbon-bound isotope was observed in 2 (at the aldehyde carbon and at C-3) and, in the pyrazine derivatives, on the ring (positions 3 and 6), and on the sugar-derived, side-chains.     

Luminescence of thymine aqueous solutions at the room temperature

The distinguishing features of luminescence of aqueous thymine solutions at room temperature (a broadening of luminescence spectra as compared with low-temperature spectra and differences between excitation and absorption spectra) are discussed. It is shown that these features are due to bimolecular photochemical reactions that lead to the formation of photoproducts (at the first stages, photoadducts) with a comparatively high luminescence ability.     

Rapid Gas Chromatographic Determination of Micro-quantities of N-Methyl Carbamates as their 2, 4-Dinitrophenyl Derivatives

A rapid and sensitive method is presented for the determination of micro-quantities (1 to 100 μg) of N-methyl carbamates as dinitrophenyl methylamine (DNP-MA) by electron capture gas liquid chromatography (GLC). The method described is characterized by rapidity and simplicity of procedure due to an improvement made in the present investigation, i.e., hydrolysis of an N-methyl carbamate and dinitrophenylation of the resulting methylamine with 2, 4-dinitro-1-fluorobenzene (DNFB) were accomplished simultaneously in a single reaction mixture. A novel approach was also made to eliminate unreacted DNFB by conversion to dinitrophenyl glycine (DNP-glycine).     

Calorimetric study of the interaction between amino acids and sugars in water at 298.15 K

The enthalpies of dissolving glycine and DL-alanine in water solutions of D-glucose, D-maltose, and sucrose at 298.15 K were determined by calorimetry. From the results obtained, the coefficients of enthalpy for pairwise interactions hxy of the amino acids and saccharides in water were calculated. It was found that the hxy values for glycine in solutions of all saccharides studied are negative; in the case of DL-alanine, the hxy values are positive for all saccharides except for sucrose solution. It was shown that the hxy values reflect the sum effect of interactions between the amino acids and saccharides in aqueous solutions and the contribution of hydration of the solutes.     

Photolysis of N-methyl and N-acetyl substituted amino acids

Summary Experiments on the production of free radicals in aqueous solutions of N-methyl and N-acetyl substituted amino acids by UV light are reported. The ESR spectra observed at 77 K show that the bond of the CH_3–group of these substances is ruptured to a great deal compared with other bond ruptures producing paramagnetic centres. But in N-methyl substituted amino acids the methyl radical is less predominant than in N-acetyl substituted ones. At temperatures higher than 200 K the ESR spectra of all substances studied in our experiments are similar. It is supposed that a radical is formed with an unpaired spin near an oxygen atom.A part of this paper was reported at a Congress in Freiburg i. Br. (Germany) organized by the Deutsche Gesellschaft für Biophysik, October 2–4, 1974.     

Time dependent inhibition of xanthine oxidase in irradiated solutions of folic acid, aminopterin and methotrexate

The xanthine oxidase catalyzed oxidation of hypoxanthine was followed by monitoring the formation of uric acid at 290 nm. Inhibition of xanthine oxidase occurs in aqueous solutions of folic acid methotrexate and aminopterin. These compounds are known to dissociate upon exposure to ultraviolet light resulting in the formation of their respective 6-formylpteridine derivatives. The relative rates of dissociation were monitored spectrophotometrically by determining the absorbance of their 2,4-dinitrophenylhydrazine derivatives at 500 nm. When aqueous solutions of folic acid, aminopterin and methotrexate were exposed to uv light, a direct correlation was observed between the concentrations of the 6-formylpteridine derivatives existing in solution and the ability of these solutions to inhibit xanthine oxidase. The relative potency of the respective photolysis products were estimated.     

Volume effects in aqueous solutions of macromolecules containing non-polar groups

Specific volumes at 25° of aqueous solutions of poly(N-methyl acrylamide) and poly-(ethylene oxide) have been measured, and the data obtained is compared with corresponding data on low-molecular-weight compounds. The results support the notion, that the volume changes following the interaction between aliphatic groups and water are positive. They are discussed with particular reference to pressure effects on protein conformations.     

Transfer free energies of peptide backbone unit from water to aqueous electrolyte solutions at 298.15 K

Transfer free energies (ΔG_tr) of amino acids from water to aqueous electrolyte solutions have been determined from the solubility measurements, as a function of salt concentration at 298.15 K under atmospheric pressure. The investigated aqueous systems contain amino acids of zwitterionic glycine peptides: glycine (Gly), diglycine (Gly₂), triglycine (Gly₃), and tetraglycine (Gly₄) and cyclic glycylglycine (c(GG)) with an electrolyte compound of potassium chloride (KCl), potassium bromide (KBr) or potassium acetate (KAc). The solubilities of glycine and diglycine in aqueous solution decrease with increasing the concentration of salts (salting-out effect), whereas those of triglycine and tetraglycine increase with increasing the concentration of salts (salting-in effect). Furthermore, salting-in effect was found in aqueous c(GG)/KBr system, while salting-out effect was observed in aqueous c(GG)/KCl or c(GG)/KAc system. The experimental results were used to estimate the transfer free energies (Δg_tr) of the peptide backbone unit (–CH₂CONH–) from water to the aqueous electrolyte solutions. We developed a new trail to determine the activity coefficients (γ) for aqueous and aqueous electrolyte solutions using an activity coefficient model, with which the total contribution of transfer free energy between solute and the solvent was calculated. We compared the difference between neglecting and using the activity coefficients term in predicting ΔG_tr. Since the transfer free energy contribution is negative, interactions between the ionic salts and the peptide backbone unit of zwitterionic glycine peptides are favorable and thus the ionic salts destabilize these amino acids. It was also found that KBr stabilizes c(GG), whereas KCl and KAc destabilize c(GG). These results provide evidence for the existence of interactions between the amide unit and ionic salts, in aqueous solution, which may be of importance in maintaining protein structure as well as in protein–solute and protein–solvent interactions.     

Cytotoxic effect of dihydroquercetin and its derivatives in liposomal form and in the form of fat nanoscale emulsions

We studied cytotoxic effects of dihydroquercetin (DHQ) and its derivatives, penta-O-acetylsalicylate dihydroquercetin (PAS DHQ), penta-O-acetate dihydroquercetin (PA DHQ), and penta-O-benzoate dihydroquercetin (PB DHQ), in aqueous solutions, liposomal form, and in the form of fat emulsions, on cultured tumor cells HL-60 and HEp-2. Solubility of the DHQ derivatives in aqueous solutions was much lower than that of DHQ. Modification of DHQ either did not alter its toxicity (PAS DHQ) or significantly reduced the cytotoxic effect of DHQ on tumor cells (PA DHQ, PB DHQ). DHQ and its derivative PAS DHQ were more toxic when dissolved in an aqueous solution than in the form of liposomes. Cytotoxic effect of DHQ applied in the form of fat emulsion was significantly higher than in the liposomal form and was close to the activity of DHQ in a solution. These results can be implicated for the development of new anticancer drugs based on liposomal and fat emulsions.     

Novel thermosensitive 5-fluorouracil-cyclotriphosphazene conjugates: synthesis, thermosensitivity, degradability, and in vitro antitumor activity

A novel thermosensitive macromolecular prodrug of 5-fluorouracil (5-FU) was synthesized using cyclotriphosphazene, and its thermosensitivity, degradability, and in vitro antitumor activity were studied. A series of alpha-substituted glycine derivatives of 5-FU containing carboxylic groups were prepared, and cyclotriphosphazenes with amino groups were synthesized via the stepwise substitution of hexachlorocyclotriphosphazene (NPCl(2))(3) with methoxy-poly(ethylene glycol) (MPEG) or alkoxy ethylene oxide and lysine ethyl ester (LysOEt). The coupling reaction of the two derivatives, and their subsequent deprotection, yielded a thermosenstive 5-FU-cyclotriphosphazene conjugate, which exhibited a unique octopus-shaped molecular structure, in which the three hydrophilic PEG groups (or alkoxy ethylene oxides) were oriented in one direction, opposing the other three hydrophobic groups containing 5-FU, with respect to the trimer ring plane. This conjugate exhibited a reversible and thermosensitive phase transition in an aqueous medium, from soluble to insoluble states. The lower critical solution temperature (LCST) of the conjugate was controlled by substitution with different hydrophilic/hydrophobic side groups, and a few of the conjugates displayed LCSTs which were just below body temperature. This, of course, implies possible applications for local drug delivery by direct intratumoral injection. The conjugate exhibited gradual degradation at 37 degrees C in both neutral and acidic buffer solutions, and high temperature significantly facilitated its hydrolytic degradation. All of the conjugates displayed dose-dependent cytotoxicity against the leukemia L1210 cell line and exhibited more pronounced cytotoxic effects than did 5-FU.     

Analytical quantification of low‐density lipoprotein using europium tetracycline indicator

Low‐density lipoprotein (LDL) is known as ‘bad’ cholesterol. If too much LDL circulates in the blood it can be retained in the walls of the arteries, causing atherosclerosis. In this paper we showed an alternative method to quantify LDL using the europium tetracycline (EuTc) indicator. The optical properties of the EuTc complex were investigated in aqueous solutions containing LDL. An enhancement was observed of the europium luminescence in the solutions with LDL compared those without the lipoprotein. A method to quantify the amount of LDL in a sample, based on EuTc enhanced luminescence, is proposed. The enhancement mechanism is also discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

6-Aminohexanoic acid-plasminogen interactions studied by fluorescence

Circular polarization of luminescence spectra of human plasminogen and of its derivatives were measured in solutions of ligand-free proteins and with saturating amounts of 6-aminohexanoic acid. Spectroscopic changes induced by the ligand reveal similar perturbations of the binding sites in all the protein derivatives. It is concluded that the gross conformational change induced by 6-aminohexanoic acid binding to the native plasminogen involves changes of sterical relations of entire protein domains.     

Proteinoid microspheres and the process of prebiological photophosphorylation

A chemical model of prebiological photophosphorylation with participation of hemoproteinoid microspheres, mixed microspheres containing bonded riboflavin and microspheres obtained from glycine rich proteinoids was studied. The illumination of aqueous solutions containing microspheres, K₂HPO₄, ADP and electron acceptor leads to an increase of ATP concentration and to a decrease of concentration of inorganic phosphate. Initial photochemical reactions with participations of proteinoid microspheres could have evolved in the course of chemical evolution and led to the emergence of the photophosphorylation in its modern biochemical form.     

Lipophilic ruthenium complexes with tuned cell membrane affinity and photoactivated uptake

Ruthenium dipyridophenazine (dppz) complexes are virtually non-emissive in aqueous solutions but show strong luminescence in hydrophobic environments, making them interesting as molecular probes in cellular imaging. We show by luminescence spectroscopy that by substituting the dppz ligand with alkyl ether chains of increasing length the complexes can be tuned from preferential intercalation into DNA to insertion in model phospholipid membranes. Confocal laser scanning microscopy (CLSM) on methanol fixed CHO-K1 cells show an analogous distribution in the cell, where the least hydrophobic complex exclusively stains the nucleus whereas the more hydrophobic ones seem to predominantly stain membrane structures in the cytoplasm. In live cells CLSM show that initially only the more hydrophobic derivatives stain the plasma membrane. However, brief further exposure to the laser light causes permeabilization of the membrane and accumulation of extracellular ruthenium complexes in internal cellular structures, similarly to the distribution found in fixed cells.