Partition Experiments Research Articles

Polyethylene glycol: Catalytic effect on the crystallization of phosphoglucomutase at high salt concentration

A cold, aqueous solution containing (NH 4) 2SO 4 at 53% of saturation and 5.9% w/v polyethylene glycol-400 (PEG) produces PEG-rich coacervate droplets (16%(NH 4) 2SO 4 and 37% PEG) when warmed to 25°C. In partition experiments conducted at low protein concentration, phosphoglucomutase and several other common proteins concentrate at least 20-fold in the PEG-rich phase. A temperature-induced phase separation similar to that above, but conducted in the presence of 5 mg/ml of phosphoglucomutase, can produce coacervate droplets in which the concentration of protein is about 500 mg/ml and thus approaches that in the crystal phase. The nucleation and subsequent conversion of such droplets into micrometer-size crystals of phosphoglucomutase were studied by light microscopy. Nucleation usually occurs in the periphery of these droplets, and neither phase nucleates efficiently by itself, although both support growth. Most droplets do not nucleate and subsequently dissolve as the protein concentration in the surrounding medium is depleted by incorporation into growing srystals. A major role of PEG in the nucleation/crystallization process is to repress the formation of salt-induced, disordered aggregates whose non-lattice protein-protein interactions presumably are less mobile than those in the droplet phase. In this sense, PEG acts as a nucleation catalyst. Such a mode of action is supported by studies on the effect of PEG in the conversion of salt-induced aggregates of phosphoglucomutase into protein crystals. An analogous role in the nucleation and slow growth of much larger crystals under somewhat different conditions is inferred. Such a PEG-induced effect may be general for proteins that crystallize from concentrated salt solutions.

Über die Enantiomerentrennung durch Verteilung zwischen flüssigen Phasen. 4. Mitteilung. Enantioselektivität der diastereoisomeren Weinsäure‐dimenthylester gegenüber α‐Aminoalkohol‐Salzen

Separation of Enantiomers by Partition between Liquid Phases. Enantioselectivity of Diastereoisomeric Dimenthyl Tartarates towards α‐Aminoalcohol SaltsThe enantioselectivity of the easily obtainable diastereoisomeric dimenthyl tartarates I and II towards the salts of a series of α‐aminoalcohols 1‐10 was investigated by partition between aqueous and lipophilic phase. The measured enantiomer distribution constants Q (=KA/KB) confirm the previously observed configurational relationships between the lipophilic tartarates and the preferentially extracted enantiomer of the hydrophilic ammonium slat. However, the (R,R)‐ester I is appreciably more selective than its (S,S)‐diastereoisomer II or other previously investigated esters. The tartarates I‐III transport norephedrin salts through bulk lipophilic membranes with enantioselectivity comparable to that observed in partition experiments. The most enantioselective ester I can be used as an efficient mobile phase for preparative separation of norephedrine enantiomers by flash partition chromatography.

Isolation and fractionation of CHO chromosomes in aqueous two phase systems using charged polymers and base specific macroligands

Chromosomes were isolated in a preparative scale by synchronisation of CHO cells with a double Thymidine block followed by an arrest in the metaphase by addition of Colcemid. Under proper cultivation conditions a mitotic index of 77% total cells could be routinely achieved. Bulk chromosome preparations free of nuclei and other subcellular particles have been obtained by low speed centrifugation followed by a 60 transfer countercurrent distribution using aqueous two phase systems composed of polyethylenglycol and dextran. The partition of CHO chromosomes previously purified in aqueous two phase systems were studied further to develop a protocol for the separation and isolation of individual chromosomes. Partition experiments with chromosomes changing the electrostatic phase potential by addition of charged PEG-derivatives suggest the existence of relatively highly charged chromosome groups. Most promising results with regard to separation were obtained using two PEG-derivatives, which interact specifically with the bases in DNA. For this affinity partitioning a GC- and AT-specific macroligand were employed. Comparing CCD's using each of these ligands information on the GC and AT content of exposed DNA in the chromosomes groups could be derived, demonstrating that specific sequences of DNA are accessible at the surface of metaphase chromosomes.

Golgi-derived membranes that contain an acylated viral polypeptide are used for vaccinia virus envelopment.

A 37,000-dalton polypeptide (p37K) present on purified extracellular vaccinia virus but absent from intracellular virus particles of classical morphology (G. Hiller et al., J. Virol. 39:903-913, 1981; L. G. Payne, J. Virol. 27:28-37, 1978) was further characterized. The polypeptide was only expressed in infected cells after onset of viral DNA replication. Phase partition experiments showed that it is relatively hydrophobic. Although p37K apparently is not a glycoprotein, in vivo radioisotope labeling detected tightly associated palmitic acid. Antibodies to p37K were used to monitor its distribution within infected cells at the light and electron microscopic levels. After synthesis p37K first accumulated in the Golgi region due to a tight membrane association. During progressing infection p37K-carrying membranes were used to form double-walled envelopes around brick-shaped vaccinia particles. Within these specialized vesicles vaccinia particles were moved through the cytoplasm toward the cell's surface, presumably along cellular routes for certain secretory products. Finally, single enveloped viruses were released into the extracellular space by an exocytotic process.

Prodrugs of 5-fluorouracil. IV. Hydrolysis kinetics, bioactivation and physicochemical properties of various N-acyloxymethyl derivatives of 5-fluorouracil

The kinetics and mechanism of hydrolysis of various 1-, 3- and 1,3-acyloxymethyl derivatives of 5-fluorouracil were studied to assess their potential as prodrugs with the aim of enhancing the delivery characteristics of the parent drug. All the derivatives hydrolyzed to yield 5-fluorouracil in quantitative amounts, passing through an unstable N-hydroxymethyl-5-fluorouracil intermediate. The pH-rate profiles obtained revealed the occurrence of specific acid and base catalysis as well as of a water-catalyzed reaction. The rates of hydrolysis were accelerated markedly in the presence of human plasma or rat liver homogenate, suggesting the utility of the derivatives as prodrugs. The derivatives were all more lipophilic than 5-fluoro-uracil as determined by partition experiments in octanol-aqueous buffer systems but the aqueous solubility was only slightly reduced or, for some derivatives, even greater than that of 5-fluorouracil. This behaviour was attributed to differences in the crystal lattice energy, and relationships between melting points, partition coefficients and water-solubilities for these and 11 other prodrug derivatives of 5-fluoro-uracil were established.

Prodrugs of 5-fluorouracil. III. Hydrolysis kinetics in aqueous solution and biological media, lipophilicity and solubility of various 1-carbamoyl derivatives of 5-fluorouracil

The kinetics of hydrolysis of five 1-carbamoyl-5-fluorouracil derivatives (methyl-, ethyl-, butyl-, phenyl- and N,N-dimethylcarbamoyl derivatives) was studied in aqueous solution of pH 1–14, human plasma solutions and in the presence of rat liver homogenate at 37°C. All the derivatives hydrolyzed to yield 5-fluorouracil in quantitative amounts. The decomposition rates in aqueous solution (pH 1–10) showed a sigmoid pH-dependence which could be ascribed to water-catalyzed or spontaneous hydrolysis of the neutral and anionic forms (pK a 6.1–6.7). The greater reactivity observed for the anionic form of the derivatives was attributed to intramolecular general base catalysis. The half-lives of hydrolysis of the 1-alkylcarbamoyl derivatives at pH 7.40 and 37°C were 8–11 min and that of the 1-phenyl-carbamoyl derivative 5 s, whereas the N,N-dimethylcarbamoyl derivative proved to be highly stable. The hydrolysis was not catalyzed by rat liver homogenates, and human plasma surprisingly showed a pronounced inhibition. This deceleration of the hydrolysis rate was attributed to a non-productive binding to or inclusion of the compounds by plasma proteins. The derivatives were more lipophilic than 5-fluorouracil as determinded by partition experiments in octanol-aqueous buffer systems and possessed a lower water-solubility.

Allopurinol prodrugs. I. Synthesis, stability and physicochemical properties of various N 1-acyl allopurinol derivatives

Seven novel N 1-acyl derivatives of allopurinol were synthesized and evaluated as potential prodrugs with the purpose of developing preparations suitable for rectal and parenteral administration. The kinetics of hydrolysis of the derivatives was studied in aqueous solutions at various pH-values and in human plasma solutions at 37° C. All the compounds hydrolyzed to yield allopurinol in quantitative amounts and rate expressions were derived to account for the pH-rate profiles observed. The rates of hydrolysis were accelerated by plasma enzymes, the half-lives of hydrolysis in 80% human plasma solutions at 37°C being 6, 4, 2.5 and 4 min for the N 1-acetyl, N 1-propionyl, N 1-butyryl and N 1-benzoyl derivatives, respectively. These N-acyl derivatives were more lipophilic than allopurinol as determined by partition experiments in octanol-water but the solubility in water was even greater (the N 1-acetyl derivative) or only slightly reduced (the other derivatives) as compared with allopurinol. This behaviour was attributed to a decreased intermolecular hydrogen bonding in the crystal lattice achieved by blocking the 1-NH group by acylation. It is suggested that N 1-acylation may be a promising means of obtaining prodrug forms of allopurinol with the aim of enhancing the rectal delivery characteristics of the drug. This was confirmed in preliminary animal experiments. Two highly water-soluble derivatives, 1-(N,N-dimethylglycyl)allopurinol hydrochloride and 1-(4-N,N-di-methylaminobutyryl)allopurinol hydrochloride, were prepared but found to be less suitable as parenteral delivery forms of allopurinol due to a very limited stability in aqueous solution, arising from intramolecular catalysis by the dimethylamino group in the acyl moiety.

Environmental chemistry of 1,2-propanediol dinitrate: azeotrope formation, photolysis and biodegradability.

Investigation of the biodegradability of 1,2-propanediol dinitrate, commonly called propylene glycol dinitrate (PGDN), the major component of the Naval torpedo fuel, Otto fuel II, has shown that this compound is resistant to biodegradation by microorganisms found in sewage sludge, a pure culture ofPseudomonas aeruginosa (previously shown to biodegrade nitroaromatic explosives), and a commercially available inoculum, Phenobac®, employed for degradation of nitrogen-containing organic wastes. However, PGDN is photolabile in ultraviolet, decomposing to pyruvic and lactic acids. In biodegradation experiments, PGDN rapidly evaporated from aqueous solutions. This unexpected volatility of PGDN, which poses a potential health hazard, was attributed to the formation of a PGDN-water azeotrope, whose presence was demonstrated by partition experiments.

Prodrugs of 5-fluorouracil. I. Hydrolysis kinetics and physicochemical properties of various N-acyl derivatives of 5-fluorouracil

The kinetics and mechanism of decomposition of nine N 1-acyl, N 3-acyl and N 1, N 3-diacyl derivatives of 5-fluorouracil in aqueous solution and in human plasma at 37°C were studied to assess their suitability as prodrugs for the parent compound. All the derivatives hydrolyzed to yield 5-fluorouracil in quantitative amounts. N 1-Deacylation proceeded much faster than N 3-deacylation and showed a pH-dependence which could be ascribed to water-catalyzed and hydroxide ion-catalyzed reactions. The hydrolysis of the N 1,N 3-diacyl derivatives passed through the corresponding N 3-acyl compound. The pH-rate profiles obtained for the N 3-deacylation were accounted for in terms of spontaneous and hydroxide ion-catalyzed hydrolysis of undissociated N 3-acyl derivative (pK a~7) as well as hydroxide ion-catalyzed hydrolysis of anionic derivative. The rate of N 3-deacylation was accelerated in human plasma, the half-life of hydrolysis in 80% plasma solutions at 37°C being 4.6, 20, 28 and 110 min for the N 3-acetyl, N 3-propionyl, N 3-butyryl and N 3-benzoyl derivatives, respectively. The N 3-acyl derivatives were more lipophilic than 5-fluorouracil as determined by partition experiments in octanol-aqueous buffer systems but their aqueous solubilities were even greater or only slightly reduced as compared with 5-fluorouracil. This behaviour was attributed to a decreased intermolecular hydrogen bonding in the crystal lattice achieved by blocking the 3-NH group by the acylation and manifested in a pronounced melting point decrease. The results suggested that N 3-acylation may be a promising means of obtaining prodrug forms of 5-fluorouracil with the aim of enhancing the delivery characteristics of the drug, e.g. by prodrugs exhibiting increased lipophilicity and/or affording protection against first-pass metabolism.

Prodrugs as drug delivery systems. XXX. 4-Imidazolidinones as potential bioreversible derivatives for the α-aminoamide moiety in peptides

The hydrolysis kinetics of five 4-imidazolidinones derived from acetone and the dipeptides Ala-Gly, Ala-Ala, Phe-Leu, Leu-Gly and Asp-Phe methyl ester were studied to assess their suitability as prodrug forms for the α-aminoamtde moiety occurring in peptides. The imidazolidinyl peptides were found to undergo a complete hydrolysis in the pH range 1–10 at 37°C and most of them showed a sigmoidal pH-rale profile with maximum rates at pH > 4. The stability of the derivatives varied widely, the following half-lives being obtained at pH 7.40 and 37°C: 0.9 h (Asp-Phe methyl ester), 3.4 h (Phe-Leu), 24.6 h (Ala-Ala). 410 h (Ala-Gly) and 530 h (Leu-Gly). The major structural factor influencing the stability appeared to be the stcric properties within the C-terminal amino acid residue. The 4-imidazolidinones are much weaker bases (pK a about 3.1) than the parent dipeptides and, as determined by partition experiments in octanol-aqueous buffer systems with the Phe-Leu derivative, they are more Hpophilic than the parent compounds. It is suggested that 4-imidazolidinone formation in principle may Become a useful approach to bioreversible dehvatization of dipeptides or other peptides containing an α aminoamide function with the aim of solving delivery problems fur peptide drugs.

Gastrointestinal Absorption of the Strongly Acidic Drug Proxicromil

Gastrointestinal absorption of the strongly acidic drug proxicromil has been studied with respect to its physical organic chemistry. The lipophilicity of the drug above pH 6 in octanol-buffer partition experiments is dependent on ion pair formation. Similar trends were demonstrated for the in vitro partition of the compound into GI tissue. The absorption of the compound from the perfused GI tract of rats in vivo was not consistent with classical un-ionized drug absorption theories and indicated the operation of other processes, especially ion pair formation, as major mechanisms of proxicromil absorption.

141. Chlorine compounds in volcanic gases : on the basis of partition experiments between gas phase and silicate melt(Abstracts of Papers Presented at the Spring Meeting of the Society)

141. Chlorine compounds in volcanic gases : on the basis of partition experiments between gas phase and silicate melt(Abstracts of Papers Presented at the Spring Meeting of the Society)

Über die Stereoselektivität der 9,9′‐Spirobifluoren‐kronenäther gegenüber α‐Aminoalkoholen

Stereoselectivity of 9,9′‐Spirobifluorene Crown Ethers towards α‐Amino AlcoholsCrown ethers I‐VI were tested by partition experiments for their stereoselectivity towards α‐amino alcohols 1–10. The stereoselectivity depends in a regular way on both the absolute and relative configuration of the crown ether and α‐aminoalcohol. Comments are made on some high stereoselectivities.

Pro-drugs as drug delivery systems XX. Oxazolidines as potential pro-drug types for β-aminoalcohols, aldehydes or ketones

The kinetics of hydrolysis of oxazolidines derived from (-)-ephedrine or (+)- pseudoephedrine and benzaldehyde or salicylaldehyde was studied at 37°C to assess their suitability as pro-drug forms for β-aminoalcohols and carbonyl-containing compounds. The oxazolidines were found to undergo a facile and complete hydrolysis in the pH range 1–11. The oxazolidines derived from benzaldehyde showed bell-shaped pH-rate profiles with rate maxima occurring at pH 4–5.5. The oxazolidine from salicylaldehyde showed a sigmoidal pH-rate profile and was 100-fold more reactive than the corresponding benzaldehyde derivative at pH > 7. At pH 7.4 and 37°C half-lives of hydrolysis from 5s to 28 min were observed for the 3 oxazolidines studied. The oxazolidines are weaker bases than the parent β-aminoalcohols and are more lipophilic than these as determined by partition experiments in an octanol-phosphate buffer system. It is suggested that oxazolidines be considered as potentially useful pro-drug candidates for β-aminoalcohols or drugs containing carbonyl groups.

Substituent interaction effects in aromatic molecules in reversed-phase liquid chromatograhy

Retention volumes of monosubstituted benzenes, chlorobenzenes, benzoic acids, phenols, anilines and pyridines have been measured in the reversed-phase mode of a high-performance liquid chromatographic system. Buffered methanol—water and acetonitrile—water eluents were used with an octadecylsilylsilica adsorbent. From the net retention volumes a substituent interaction effect was derived, and described with the Taft equation. The resultant values of the ρ-parameters were compared with values holding for gas-liquid chromatography and for normal-phase liquid chromatography, derived in previous investigations, and with values holding for batch partition coefficients. They were interpreted in terms of hydrogen bonding between the solutes and the eluent. Batch partition experiments with monosubstituted benzenes, phenols and pyridines were carried out with an n-hexadecane/methanol—water system, in order to obtain more information concerning the retention mechanism. A general discussion is given on retention mechanisms in reversed-phase chromatography.

Evaluation of lipophilic properties for a series of phenols, using reversed-phase high-performance liquid chromatography and high-performance thin-layer chromatography

Experimental R M o and log k o values for 48 mono- to penta-substituted phenols were determined using reversed-phase high-performance liquid chromatography and high-performance thin-layer chromatography. Octadecylsilylated silica served as “lipophilic” stationary phase. R M o and log k o values were calculated by extrapolation of the plot of the methanol percentage composition of the mobile phase (methanol-0.01 N aqueous HCl mixtures) versus R M or log k values to the intercept with the y-axis ( i.e., 0% methanol). R M o and log k o values obtained under these standardized conditions are correlated significantly to the log P values reported for shake-flask partition experiments. Substituent constants derived from the chromatographic lipophilicity data, in accordance to π, lead to values that can be used to estimate the hydrophobic properties of other compounds.

Pro-drugs as drug delivery systems XVII. Esters of 4-hydroxybutyric acids as potential pro-drug types for γ-lactones

The methyl and ethyl ester of 4-hydroxybutyric acid were prepared and evaluated as pro-drug models for the γ-lactone moiety occurring in several drugs such as pilocarpine. The esters were found to undergo a quantitative cyclization in neutral and alkaline aqueous solution at 37°C to γ-butyrolactone. The rates of lactonization were directly proportional to the hydroxide ion activity over the pH range studied (7.4–9.8) and the reactions appear to proceed by an intramolecular nucleophilic attack of alkoxide ion on the ester carbonyl moiety. Lactone formation proceeded rather slowly at pH 7.4 but was greatly accelerated in case of the corresponding phenyl ester according to a previous report. The ester derivatives are more lipophilic than the parent γ-butyrolactone as determined by partition experiments in a L-octanol-water system. It is suggested that esters, in particular aryl esters, of 4-hydroxybutyric acids may be potentially useful pro-drug candidates for agents containing a γ-lactone moiety, e.g. to facilitate biomembrane penetration through increased lipophilicity of the transport forms.

Effect of specific binding of human albumin, fibrinogen, and immunoglobulin G on surface characteristics of bacterial strains as revealed by partition experiments in polymer phase systems.

Four strains of gram-positive cocci with different combinations of positive binding of human proteins were investigated with respect to changes in physicochemical surface properties after specific protein binding. Staphylococcus aureus Cowan I, two group A beta-hemolytic streptococci, and one group G streptococcal strain were studied; they represented three different combinations of reactivity for human serum albumin, human immunoglobulin G, and fibrinogen. Using single-tube partition of bacterial cells in a dextran-polyethylene glycol system of constant polymer concentration but varying ionic compositions, it was possible to detect changes in the partition of bacteria after specific protein binding. There was a correlation between the binding of radiolabled human proteins to the bacterial strains and the effect of human proteins on the partition of the bacteria in the phase systems. Thus, the specific binding of proteins to the bacteria changes their physicochemical surface properties. These types of bacteria-protein interactions may play an important role in modulating host-parasite relationships.

Thermodynamics of caffeine in aqueous denaturant solutions

The effects of urea, guanidinium chloride, and potassium chloride on the solution properties of caffeine have been investigated by solubility and partition experiments and by measuring heats of dilution, solution, and mixing. Thermodynamic transfer properties are used to discuss the possible mechanism of action of the two denaturants. The ability of caffeine to form stacked aggregates is taken as possible evidence for a direct interaction of this solute with urea and guanidinium ion. The data are discussed both in terms of an ideally associating system and in terms of the virial expansion following the McMillan-Mayer solution theory.

Hydrophobic characterization of powder Some criteria and experimental evidence

The immersion times, contact angle and also the partition between solvents, have been measured experimentally for a series of powdered solids. According to experimental results obtained here a characterization comparison of the solids studied is undertaken with other solids taken as model systems. Thus, a classification of substances is proposed in terms of qualitative differences in free energies of immersion in water 1. Solids having a positive free energy of immersion or solids intrinsically non-wetted by water. This group would comprise the substances normally referred to as hydrophobic, i. e. paraffins, organic polymers etc., which could be ordered in terms of their critical surface tension. 2. Solids having a negative free energy of immersion. This group can be subdivided in two further groups according to the affinity for a specific phase (partition experiments).- Substances concentrated in organic phase;- Substances concentrated in aqueous phase.