Thomas Braumann

Determination of hydrophobic parameters by reversed-phase liquid chromatography: theory, experimental techniques, and application in studies on quantitative structure-activity relationships

The use of RPLC retention parameters as descriptors of the hydrophobic nature of bioactive compounds has been evaluated. The relationship between the capacity factor, measured on n-alkyl-bonded stationary phases using binary eluents, and the n-octanol-water partition coefficient has been illustrated experimentally and theoretically. It is suggested that retention parameters, in particular the capacity factor (log kw) obtained by extrapolation of retention data from binary eluents to 100% water, could successfully replace the n-octanol-water partition coefficient in studies on quantitative structure-activity relationship, and that their use may result in a better correlation with biological data.

Quantitative structure—activity relationships for herbicides : Reversed-phase liquid chromatographic retention parameter, log kw, versus liquid-liquid partition coefficient as a model of the hydrophobicity of phenylureas, s-triazines and phenoxycarbonic acid derivatives

The retention behaviour of phenylureas, s-triazines and phenoxycarbonic acid derivatives in a reversed-phase high-performance liquid chromatographic (RPLC) system has been examined. Using methanol—water or acetonitrile—water as the mobile phase, a linear relationship between the volume fraction of the organic modifier, ϕ, and the logarithm of the capacity factor, log k′, is established for each solute. The different correlation curves for each compound indicate selective effects upon retention due to solute—solvent and solute—stationary phase interactions. It is shown that log kw, a theoretical capacity factor obtained by extrapolation of retention data in binary solvent systems to pure aqueous eluent, is suitable for eliminating the selective effects and thereby for quantitatively describing the hydrophobic nature of solutes in a way which is strongly related to the partition coefficient, log P, of the standard n-octanol—water partitioning system. The dependence of log kw on the nature of the organic modifier and an analysis of functional group behaviour in different eluents reveal that log P and log kw are not completely interchangeable, because certain substituents, i.e., methylthio and trifluoromethyl groups, behave differently in RPLC and a true liquid—liquid partitioning system. The consequences of this non-polar group selectivity in RPLC on the quality of quantitative structure—activity relationships for electron transport-inhibiting herbicides are demonstrated. The results suggest that log kw might be a better model for the assessment of the hydrophobicity of drugs in biological systems.

Reversed-phase high-performance liquid chromatography of chlorophylls and carotenoids

Abstract Reversed-phase high-performance liquid chromatography with octadecyl- or octylsilylated silica gel as the stationary phase provides a powerful tool in the analysis of chloroplast pigments from higher plants and green algae. Chromatographic columns packed with 10 μm chemically bonded silica gel particles allow the simultaneous separation of chlorophylls a and b , chlorophyll isomers, pheophytins a and b , α-carotene, β-carotene, lutein, violaxanthin, lutein-5,6-epoxide, antheraxanthin, neoxanthin and several minor carotenoids from a single sample within a short analysis time. The quantitative analysis requires a minimum of 1–5 pmol for carotenoids and 5–10 pmol for chlorophylls. Pigment degradation products, formed on polar stationary phases, are not found in reversed-phase high-performance liquid chromatography due to the weak hydrophobic forces on which the separation mechanism is based. The production of altered pigments however, either induced by various treatments or generated during the isolation, can be monitored as the reversed-phase system is selective enough to separate cis -isomers and oxidation products from their parent compounds. The reproducibility of the individual retention time for each pigment is better than ±1.5% which facilitates the identification of unknown pigments. The method is applied to the analysis of the pigment composition of Chlorella fusca , spinach ( Spinacia oleracea ) chloroplasts, and to the rapid determination of the ratio of chlorophyll a to chlorophyll b .

Fate of Cyclic Nucleotides in PC12 Cell Cultures: Uptake, Metabolism, and Effects of Metabolites on Nerve Growth Factor‐Induced Neurite Outgrowth

Abstract: The fate of cyclic AMP (cAMP), dibutyryl‐cAMP (Bt2‐cAMP), and the (Sp)‐isomer of adenosine 3′,5′‐monophosphorothioate [(Sp)‐cAMPS] was studied in the PC12 culture medium by means of HPLC. In the absence of PC12 cells, cAMP and Bt2‐cAMP were rapidly degraded by nonspecific esterases and cyclic nucleotide phosphodiesterase both originating from the serum commonly used as a culture medium ingredient, whereas (Sp)‐cAMPS was completely stable. Since 5′‐AMP, adenosine, inosine, and hypoxanthine appeared in the culture medium after incubation with cAMP or Bt2‐cAMP, we have determined their effect on nerve growth factor (NGF)‐induced neurite outgrowth. 5′‐AMP, adenosine, and inosine were indeed potent agents in producing a potentiating effect on NGF‐induced early neurite outgrowth at a concentration of 1 mM. Thus, cAMP metabolites had the capacity to induce an effect that has been described as cAMP‐specific. In serum‐free culture medium and in the presence of cells, all cyclic nucleotides were taken up by PC12 cells. Uptake was highly correlated with the hydrophobic nature of the compounds, and was accompanied by a simultaneous excretion of metabolites. On incubation with cAMP, NGF had a pronounced effect on the metabolic pattern found in the culture medium. In particular, dephosphorylation of 5′‐AMP was specifically enhanced. This effect of NGF on the degradation of cAMP was also apparent when cAMP metabolites were incubated with PC12 cells. Whereas 5′‐AMP degradation was greatly increased, NGF had no effect on the metabolism of the other purine compounds.

Physico-chemical characterization of cyclic nucleotides by reversed-phase high-performance liquid chromatography: II. Quantitative determination of hydrophobicity

Retention data for 42 adenosine 3′,5′-monophosphate derivatives were measured in a reversed-phase high-performance liquid chromatographic (RPLC) system. Using methanol—water and methanol—phosphate buffer as the mobile phase, the relationship between the volume fraction or organic modifier, φ, and the logarithm of the capacity factor, log k′, can be adequately described by a linear regression equation. The resulting log kw values, obtained by extrapolation of retention to φ = 0, are correlated with other physico-chemical solute properties that are commonly used to described quantitatively the hydrophobic nature of solutes. The results indicate that the retention behaviour of complex molecular structures, including ionic, hydrogen bonding and hydrophobic functions, is only moderately described by the standard Hansch π substituent constant, although the underlying distribution mechanism of both RPLC and a true liquid—liquid system is the solvophobic effect. This additionally demonstrated by the strong dependence of log kw on the molecular volume of the substituents. It is concluded that, rather than attempt to establish correlations between RPLC retention data and distribution coefficients, log kw values can be used directly as descriptors of a solutes hydrophobicity and may therefore be used in studies of quantitative structure—activity relationships for cyclic nucleotides.

Pigment composition of the photosynthetic membrane and reaction center of the green bacterium Prosthecochloris aestuarii

Abstract We have performed a quantitative analysis of the pigment composition of different pigment-protein complexes present in the membrane of the green sulfur bacterium Prosthecochloris aestuarii, using the resolving power of reversed-phase high-performance liquid chromatography. The most purified photochemically active complexes contained only carotenoids (OH-chlorobactene and rhodopin), bacteriochlorophyll a and a chlorophyllous pigment with absorption maxima at 663 and 433 nm, like bacteriochlorophyll c. However, the lipophilicity of this pigment, labeled BChl 663, is quite high and indicates that it contains 5–6 additional methylene groups compared to the BChl c homologue known as most lipophilic. Comparison of the BChl 663 content of various pigment-protein complexes indicates that BChl 663 is present in an amount of 10–15 molecules per reaction center. BChl 663 absorbs at 670 nm in vivo, with a specific extinction coefficient of 85 (±10) mM−1 · cm−1. In view of the evidence that the primary electron acceptor in P. aestuarii is a pigment with absorption maximum at 670 nm (Nuijs, A.M., Vasmel, H., Joppe, H.L.P., Duysens, L.N.M. and Amesz, J. (1985) Biochim. Biophys. Acta 807, 24–34) a direct consequence of these experiments is the fact that only BChl 663 can be a likely candidate for the role of primary electron acceptor as no other pigments absorbing around 670 nm (e.g., bacteriopheophytin c) are present in a photochemically active pigment-protein complex derived from the membrane of this green bacterium.

Pigment—protein complexes of green algae: Improved methodological steps for the quantification of pigments in pigment—protein complexes derived from the green algae Chlorella and Chlamydomonas

Abstract An improved method for the determination of pigments within pigment—protein complexes (PPCS) of thylakoid membranes is presented. It comprises (a) a fast high resolution SDS — polyacrylamide gel electrophoresis (SDS ≡ sodium dodecylsulphate), (b) a new approach for the recovery of PPCs from the gel with an additional purification step and (c) a one-step separation of the chlorophylls and carotenoids by reversed-phase high performance liquid chromatography. This three-step procedure is applied to the analysis of thylakoid membranes derived from synchronized cells of the green alga Chlorella fusca , and to PPCs from Chlamydomonas reinhardii , parent strain 11/23f and the Chl- b -less mutant pg 113. The results show that a varying pattern of pigment—protein complexes exists during the life cycle in Chlorella and that there are changes in the ratio of pigments arranged in reaction centre and antenna PPCS. The pigment composition of Chlamydomonas appears to be identical with that of Chlorella ; however, the pattern of PPCs is different. Thylakoids from the Chl- b -less mutant of Chlamydomonas reveal two distinct PPCs representing photosystems I and II, both enriched in Chl a and β-carotene. About 90% of the xanthophylls migrate as free pigments, indicating an improper binding of xanthophylls to the light-harvesting complex(es) within the mutant.

Physico-chemical characterization of cyclic nucleotides by reversed-phase high-performance liquid chromatography : I. Cation binding in the mobile phase

Abstract The retention of nucleoside monophosphoric acids and cyclic nucleotide derivatives was measured on LiChrosorb RP-18 as a function of the cation concentration in the mobile phase. The retention increased upon cation binding due to the reduction in electronic charge on the solute molecule. The hyperbolic dependence of the capacity factor on the concentration of K+ or Mg2+ in the eluent allowed the calculation of apparent stability constants for cyclic nucleotide—metal ion complexes. The results indicate large differences with respect to cation binding, depending both on the nature of the cation and the structure of the phosphate group. Base modifications, on the other hand, have only minor effects on the stability constants. Among the cations studied, Mg2+ shows the highest affinity for the cyclic phosphate group. It is concluded that Mg2+ may be complexed to cyclic nucleotides within living systems.

Uptake and metabolism of the phenylpyridazinone herbicide metflurazon during the bleaching and regeneration process of the green alga, Chlorella fusca

Abstract The uptake and metabolism of the pyridazinone herbicide metflurazon by the green alga Chlorella fusca was studied using a new and sensitive technique which is based on reverse-phase high-performance liquid chromatography. During the metflurazon-induced bleaching process, the agent is rapidly taken up and metabolized to the monomethyl derivative, norflurazon, by N -demethylation. Norflurazon accumulates during the treatment in the culture medium because Chlorella cells demethylate norflurazon to the less-phytotoxic SAN 9774 only to a very minor extent. It is shown that only 10% of the initial 1 μ M metflurazon is sufficient to induce the bleaching process. This amount is much too small to directly inhibit the photosynthetic electron transport, so that a contribution of this site of action to the bleaching process is not likely. Treated Chlorella cells are able to regenerate to photosynthetically active cells. This phenomenon is based on the inability of the regenerating algae to further take up and demethylate metflurazon. Fully regenerated cells are resistant to metflurazon because a treatment with 1 μ M metflurazon neither affects growth nor inhibits photosynthetic activity. This resistance results from an altered permeability of the cell membrane with respect to phenylpyridazinones and an inhibited N -demethylation of metflurazon. These results clearly show that metflurazon itself is nonphytotoxic, and has to be demethylated to norflurazon to exert its bleaching activity.