Hydrophobic Amines Research Articles

Stabilization of clathrin coated vesicles by amantadine, tromantadine and other hydrophobic amines

Amantadine and related compounds stabilized the structure of purified pig brain clathrin coated vesicles (CCV) at biologically relevant concentrations. Incubation of purified CCV for 30 min at 25 degrees C or 37 degrees C caused the release of clathrin, as determined by a centrifugation assay, and a reduction in the number of coated vesicles, by electron microscopy. Amantadine (10 mM), tromantadine (1 mM), amidine D295 (cyclohexylcarboximidamide-(N-benzyl)hydrochloride (10 mM), chloroquine (0.1 mM) and monodansylcadaverine (10 mM) significantly reduced the extent of dissociation.

Type C Niemann-Pick Disease: Use of Hydrophobic Amines to Study Defective Cholesterol Transport

Niemann-Pick Type C (NPC) disease is a cholesterol lipidosis resulting from defective postlysosomal cholesterol transport. In normal cells this segment of cholesterol trafficking is inhibited by treatment with either U18666A or imipramine. Other compounds are also capable of blocking postlysosomal cholesterol transport: stearylamine, RV-538, and sphinganine inhibit low-density lipoprotein-induced esterification of cholesterol and cause unesterified cholesterol to accumulate in perinuclear vesicles. These vesicles can be stained with filipin to give a staining pattern indistinguishable from that seen in NPC fibroblasts. Because all of these compounds are hydrophobic amines, we conclude that most, if not all, hydrophobic amines block the postlysosomal transport of cholesterol. These results also raise the possibility that an endogenous amine, e.g., sphinganine, may inhibit cholesterol transport in NPC.

An HPLC method using FMOC-ADAM for determination of hydroxyproline in muscle

An HPLC method using 9-fluorenylmethyl chloroformate-1-amino-adamantane (FMOC-ADAM) for the determination of hydroxyproline (HYP) in muscle hydrolyzates is described. The HYP was converted to a stable fluorescent derivative with FMOC after removal of all primary amino acids with ortho-phthaldialdehyde and 2-mercaptoethanol. Excess FMOC was reacted with a hydrophobic amine, ADAM, thus eliminating the need for pentane or diethyl ether extraction. The method was performed without the use of controlled temperature derivatizations or extra equipment (e.g. column switching valves). Analysis of HYP was complete after 3 min, with a total analysis time of 7·5 min. The HYP content of various muscle samples obtained with the HPLC procedure compared very well with values obtained using a colorimetric assay.

Studies on system peaks in ion-pair adsorption chromatography : IV. Optimization of peak compression

Optimization of a chromatographic system in order to obtain extremely narrow analyte peaks was investigated. The system consisted of silanized silica as a solid phase and an acidic eluent containing an UV-detectable hydrophobic amine (the probe). Large probe deficiency peaks (system peaks) were generated by injecting high concentrations of an organic anion together with the analyte. In the rear part of the large system peaks the probe concentration increased steeply. Strong peak compression effects were obtained when small amounts of cationic analystes were eluted together with this co-ion gradient. Variation of the probe concentration in the eluent was an efficient way of obtaining peak matching. Peak deformations developed at high analyte concentrations and/or when the analyte was eluted in other parts of the system peak. Guidelines for increasing the peak compression effect and to avoid the deformations are presented.

( Z)-5-Methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421), calcium ions and ethylenediamine as the K +-site directed probe for Na +/K +-ATPase

Recently, we have shown that a hydrophobic amine (AU-1421) produces an irreversible inactivation of Na +/K +-ATPase activity. This inactivation was prevented by K + and its congeners. In this study, we examined the possibility of Ca 2+ or ethylenediamine as a probe of the K + occlusion center of Na +/K +-ATPase. The inactivation by AU-1421 was prevented by Ca 2+ with an apparent high affinity (≈0.1 mM). Ca 2+ protection was cancelled by high concentrations of ATP, ADP or Mg 2+. Ca 2+ and K + were similar in these respects. Kinetic analyses of the above data indicated the presence of two AU-1421 occlusion sites on the enzyme, either one of which is susceptible to Ca 2+ occlusion. Ethylenediamine also prevented the inactivation by AU-1421 or by C 12E 8 solubilization of the enzyme, suggesting that ethylenediamine, like K +, stabilized the enzyme. However, an apparent affinity of ethylenediamine (≈1.4 mM) was one order of magnitude lower than that of K + (≈0.2 mM), and the protective manner did not show a simple competition. In addition, ethylenediamine binding was unaffected by ATP or ADP at a low affinity site, and antagonized K + binding. From these results we concluded that ethylenediamine does not act like K + or Ca 2+ in protecting AU-1421 inactivation, since it can't stabilize the enzyme conformation as an E 2(K +-bound form).

Synthesis and Characterization of a Series of Hydrophobic Antitumor Amine Platinum(II) Carboxylates

Abstract As part of a systematic study, a series of hydrophobic amine platinum(II) carboxylate complexes has been prepared as potential antitumor agents in a liposomal-entrapped form. These complexes have been characterized by elemental analysis, 1R, 13C and 195Pt NMR spectroscopic techniques. Effect of ligand donors on NMR chemical shift is briefly discussed.

Mechanism of penetration and of action of local anesthetics in Escherichia coli cells

Escherichia coli cells were used to study the mechanism of penetration of local anesthetics and the relationship between permeation and functional properties. We show that both the neutral and the protonated form of dibucaine can be accumulated in the cells. Accumulation of the protonated form occurs in response to a transmembrane electrical potential (negative inside) and results in high trapped concentrations (70 mM). Accumulation can lead to an alkalinization of the internal pH. Low concentrations of dibucaine stimulate the respiration, increase the transmembrane electrical potential and raise the accumulation of solutes. Inhibition of these functions occurs at higher concentrations of the drug. Furthermore, the drug concentration required to inhibit these functions is smaller at alkaline external pH than at acidic external pH, suggesting that the inhibition is mainly due to the neutral form of the anesthetics. Other hydrophobic amines also stimulate and inhibit different membrane functions, their efficiency being correlated to their lipophilicity.

Regulation of Gi and Go by mastoparan, related amphiphilic peptides, and hydrophobic amines. Mechanism and structural determinants of activity.

Mastoparan (MP), a cationic, amphiphilic tetradecapeptide, stimulates guanine nucleotide exchange by GTP-binding regulatory proteins (G proteins) in a manner similar to that of G protein-coupled receptors. 1) MP stimulated exchange by isolated G protein alpha subunits and alpha beta gamma trimers. Relative stimulation was greater with alpha beta gamma trimers and beta gamma subunits could increase net MP-stimulated activity. 2) MP action was enhanced by reconstitution of trimeric G protein into phospholipid vesicles. Hill coefficients for activation were 2-4. The membrane-bound alpha-helical conformation of MP appeared to be the activating species. 3) MP blocked the ability of Go to increase the affinity of muscarinic receptors for agonist ligands, suggesting that MP and the receptor may compete for a common binding site on Go. 4) MP stimulated steady state GTPase activity at less than 1 microM Mg2+ and stimulated the dissociation of both GDP and guanosine 5'-O-(3-thiotriphosphate) at less than 1 nM Mg2+. Millimolar Mg2+ blocked the stimulatory effect of MP. Both high and low affinity Mg2+ binding sites are on the alpha subunit. 5) Increasing the amphiphilicity or hydrophobicity of MP enhanced its regulatory activity more than 2-fold and lowered the EC50 more than 10-fold. Several natural amphiphilic peptides also displayed modest stimulatory activity. 6) Benzalkonium chloride competitively antagonized the stimulation of Gi by MP but potently stimulated nucleotide exchange on Go. Because cationic, amphiphilic sequences on the cytoplasmic faces of receptors are required for G protein regulation, these findings suggest that nucleotide exchange on G proteins is regulated by the presentation of multiple cationic structures on the inner face of the plasma membrane.

Studies on system peaks in ion-pair adsorption chromatography : III. Regulation of system peak gradient retention for obtaining analyte peak compression

Peak compression can be obtained when an analyte is eluted in the co-ion gradient in a large system peak. Matching of the analyte retention with this gradient was investigated. The chromatographic system was based on silanized silica and an acidic eluent, containing acetonitrile and a probe, which was an UV-detectable hydrophobic amine, protriptyline. Large system peaks, or zones, containing probe deficiencies were induced by injecting high concentrations of a hydrophobic organic anion together with cationic analytes. The back part of these system zones consisted of a steep gradient of increasing probe (co-ion) concentration, giving strongly compressed peaks when low analyte amounts were also eluted with this back-gradient. It was found that variation of the probe concentration was an efficient way of matching the retention of the system peak with analytes of widely varying retentions.

A novel hydrophobic amine, ( Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine, as a probe of the K + occlusion center of Na +/K +-ATPase

A hydrophobic amine, ( Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421), was examined as a probe of the K + occlusion center of Na +/K +-ATPase. Treatment of the enzyme with AU-1421 at 37°C and pH 7.0 produced irreversible inactivation of the enzyme. This inactivation was prevented, with simple competitive kinetics, by K + or its congeners in the order of Tl + > Rb + > K + > NH 4 + > Cs +. The concentrations of these cations required for the protection, were consistent with the affinities for transport and ATPase activity. The apparent binding constant for K + was calculated to be 0.03 mM, from the competition with AU-1421. This protection was cancelled by a high concentration of ATP or ADP. A high concentration of Na + ( K d = 6.5–6.9 mM), as a substitute for K +, also prevented the inactivation by AU-1421. Thus, the enzyme was protected from AU-1421 when the occlusion center was occupied by a monovalent cation, irrespective of the enzyme conformation, E 1 (Na +-bound form) or E 2 (K +-bound form). On the other hand, the enzyme was most sensitive to AU-1421 in the presence of low concentration of Na + (0.4–0.8 mM) or a high concentration of ATP. Tris, imidazole or choline, which favors the E 1 state, also accelerated the inactivation by AU-1421. These suggest that AU-1421 reacts with the occlusion center through the E 1 state.

Liquid chromatographic separation of acidic phosphoserine peptides on macroporous copoly(styrene-divinyl-benzene) using amines to regulate retention

Using phosphoserine peptides as model substances, we have studied the separation of acidic peptides as substituted ammonium ion-pairs on a macroporous copoly(styrene-divinylbenzene) ( PLRP-S) chromatographic column. Various hydrophobic amines were examined, of which long-chain N-methyl- and N,N-dimethyl- n-alkylamines proved to be the best with respect to selectivity and peak symmetry. Excellent separations were obtained in ammonium bicarbonate buffer (p H 7.5) with ethanol as organic modifier under isocratic conditions using the corresponding heptylto nonylamines.

Studies on System Peaks in Ion-Pair Adsorption Chromatography. II. Effects on Analyte Peak Compression and Deformation

Abstract Peak compression and deformation effects were studied in a reversed phase ion-pair adsorption chromatographic system, comprising silanized (C18) silica as solid phase and an acidic mobile phase. An indirect detection technique was used, protriptyline (a secondary hydrophobic amine) being the probe, and substituted benzamides the analytes.

Inhibition of gastric H+,K+-ATPase and acid secretion by SCH 28080, a substituted pyridyl(1,2a)imidazole.

A hydrophobic amine, SCH 28080, 2-methyl-8-(phenylmethoxy)imidazo(1,2a)pyridine-3-acetonitrile, previously shown to inhibit gastric acid secretion in vivo and in vitro, was also shown to inhibit basal and stimulated aminopyrine accumulation in isolated gastric glands when histamine, high K+ concentrations, or dibutyryl cAMP were used as secretagogues. Stimulated, but not basal, oxygen consumption was also inhibited. Neutralization of the acid space of the parietal cell by high concentrations of the weak base, imidazole, reduced the potency of the drug, suggesting that SCH 28080 was active when protonated. Studies on the isolated H+,K+-ATPase showed that the compound inhibited the enzyme competitively with K+, whether ATP or p-nitrophenyl phosphate were used as substrates. In contrast, the inhibition was mixed with respect to p-nitrophenyl phosphate and uncompetitive with respect to ATP. The drug reduced the steady state level of the phosphoenzyme but not the observed rate constant for phosphoenzyme formation in the absence of K+ nor the quantity of phosphoenzyme reacting with K+. The drug quenched the fluorescence of fluorescein isothiocyanate-modified enzyme and also inhibited the ATP-independent K+ exchange reaction of the H+,K+-ATPase. Its action on gastric acid secretion can be explained by inhibition of the H+,K+-ATPase by reversible complexation of the enzyme. This class of compound, therefore, acts as a reversible inhibitor of gastric acid secretion.

Effects of tricyclic antidepressant drugs on energy-linked reactions in mitochondria

The effects of impramine and chlorimipramine on energy-linked reactions in mitochondria were characterized. Both compounds exhibited some characteristics of classical uncouplers of oxidative phosphorylation, i.e. they released respiratory control, hindered ATP synthesis, and enhanced ATPase activity of isolated rat liver mitochondria. Unlike classical uncouplers, however, these compounds only weakly stimulated proton uptake in intact mitochondria. They also exhibited unusual effects on energy-linked reactions in beef heart submitochondrial particles (SMP). Both compounds inhibited NADH oxidation in SMP in an “oligomycin-like” manner, and inhibited ATPase activity of SMP and the soluble F 1-ATPase. In contrast, the drugs weakly inhibited ATPase activities of bovine adrenal gland chromaffin granules and resealed granule ghosts. The mechanisms responsible for the multiple effects on mitochondrial energy-linked processes are unclear. They may be related to the hydrophobicity of the drugs, as has been shown for other hydrophobic amines.

Polar and hydrophobic effects on ester aminolysis in dimethyl sulfoxide

Aminolysis of various carboxyl-containing esters by several tetra-, tri-, and diamines was kinetically studied in dimethyl sulfoxide. Rates were measured in the presence or absence of added sulfuric acid with the amine concentration being much greater than the ester concentration. In some reactions, pseudo-first-order rate constants manifested saturation kinetic behavior with respect to the total amine concentration, indicating the formation of complexes between the amines and the esters. The complex formation was most efficient when the carboxyl group of the ester substrate was located at the meta position to the ester group. In addition, the complex formation was facilitated by hydrophobic amines. In order to explain the positional stereoselectivity and the hydrophobic effects observed in the kinetic study, a structure of the complex is proposed. In this structure of the complex, two nitrogen atoms of the amine are linked by an intramolecular hydrogen bond and then further interact with the two carbonyl oxygen atoms of the substrate. In addition, the hydrophobic interaction between the hydrophobic portion of the amine and the benzene ring of the substrate stabilizes the complex. Dimethyl sulfoxide accommodates both the polar and the hydrophobic interactions that are needed in the formation of the complex, mimicking the microenvironment of enzyme active sites.

Direct liquid chromatographic resolution of racemic drugs by means of α 1-acid glycoprotein as the chiral complexing agent in the mobile phase

The plasma protein α 1-acid glycoprotein (orosomucoid) has been used as a chiral complexing agent in the mobile phase in combination with a non-chiral diol silica column for the resolution of racemic drugs (neuroleptics and tricyclic antidepressants). The retention behaviour of the solutes has been investigated and a model for the retention has been developed. From the retention data it has been possible to calculate the affinity constants for the binding of the enantiomers of promethazine, alimemazine and trimipramine to the protein. The capacity factors of the solutes can be regulated by the protein concentration or by adding a tertiary amine to the mobile phase. Increasing the protein concentration had little influence on the separation factor but decreased the capacity factors. The addition of the tertiary amine to the mobile phase affected the stereo-selectivity, and hydrophobic amines caused drastic effects.

Studies on the mechanism of uncoupling by amine local anesthetics. Evidence for mitochondrial proton transport mediated by lipophilic ion pairs.

The hydrophobic weak acid, carbonyl cyanide m-chlorophenylhydrazone (CCCP), induced rapid swelling of mitochondria suspended in NH4SCN, as predicted from the protonophoretic property of CCCP. Butacaine and other hydrophobic amines produced the same response as CCCP. As expected of a protonophore, CCCP (a) induced swelling in mitochondria suspended in potassium acetate and valinomycin, (b) induced rapid K+ efflux from mitochondria treated with valinomycin, and (c) caused an 8-fold stimulation of respiration. Butacaine alone was unable to mimic these effects of CCCP; however, all of these effects were observed with butacaine when SCN- or tetraphenylboron were added to the assay medium. We conclude that amine local anesthetics are not protonophores; rather, their uncoupler-like activity derives from their ability to form lipophilic ion pairs with certain anions. Depending on conditions, ion pair transport may result in electroneutral anion uptake or in electrophoretic proton transport. Uncoupling results from transmembrane cycling of neutral amine, charged anion, and neutral ion pair. Dose response studies were carried out for several pharmacologically important amines with local anesthetic properties. Their relative potencies in the NH4SCN swelling assay followed the order: chlorpromazine much greater than quinine greater than dibucaine greater than quinidine greater than butacaine greater than propranolol greater than tetracaine much greater than lidocaine, procaine, procainamide. Neutral anesthetics (progesterone and benzocaine) and an amine with low anesthetic potency (timolol) were found ineffective. Among hydrophobic drugs, swelling rates in NH4SCN correlate poorly with octanol:water partition coefficients, suggesting that the rate of ion pair transport is determined by the formation constant for lipophilic ion pairs.

Binding of hydrophobic drugs to lipid bilayers and to the [formula omitted

Microelectrophoretic studies of the binding of a number of commonly used hydrophobic amine drugs to liposomes demonstrated the existence of relatively large surface potentials associated with binding of the protonated forms of the drugs. A theoretical treatment based on Langmuir adsorption isotherms and the Gouy-Chapman theory of the diffuse double layer allows estimation of drug-binding constants from electrophoretic mobility data. Such constants allow calculation of the charge effects arising from drug binding in more complex membrane systems, and it is shown that shifts in the apparent Ca 2+ affinity of the ( Ca 2+ + Mg 2+)-ATPase of sarcoplasmic reticulum in the presence of hydrophobic amine drugs are readily explicable in terms of the electrostatic effects of drug binding.

On-line HPLC method for clean-up and analysis of hydrocortisone and sulconazole nitrate in a cream

The application of a new on-line high-performance liquid Chromatographic method for the clean-up and analysis of drugs in semi-solid formulations was investigated. A short column of a chemically bonded stationary phase (10 μm octadecylsilane) mounted into the sample loop position of the high pressure injection valve was used as the primary clean-up step. The elution behavior of commonly used cream excipients on the loop column as a function of various combinations of water, methanol and THF was studied using a refractive index detector. Typically, samples of the cream were dissolved in methanol-THF and injected onto the loop column. The drug substances were eluted onto the analytical column with predetermined volumes of the mobile phase. The relatively non-polar cream excipients were retained on the loop column. At this time the injection valve was switched, isolating the loop column and the retained excipients were eluted off to waste with a strong solvent (methanol-THF). Loss of efficiency due to the insertion of the loop column was negligible. This technique was applied to the simultaneous analysis of a corticosteroid (hydrocortisone) and a hydrophobic amine (sulconazole nitrate) in a topical cream formulation.

Derivatization of amines with chloroformate esters for gas chromatographic analysis

The gas chromatographic analysis of amines after conversion to electron capture sensitive carbamates in two-phase systems has been studied. Hydrophilic compounds, for instance methylamine, are reacted with 2,2,2-trichloro-tert. butyl chloroformate. Quantitation below 10−7 M can be made by thermionic or electron capture detection. A hydrophobic amine, namely N,N-dimethyl-n-octylamine, was derivatized with 2,4,6-tribromophenyl chloroformate with addition of iodide ion to the aqueous phase. The favorable effect of iodide ion as well as the choice of pH and chloroformate ester is discussed.