Dinitrophenyl Amino Acids Research Articles

Enantioselective Extraction of Dinitrophenyl Amino Acids Mediated by Lipophilic Deoxyguanosine Derivatives: Chiral Discrimination by Self-Assembly.

The transfer of potassium salts of dinitrophenyl amino acids from water to chloroform by the lipophilic guanosine derivative 1 takes place enantioselectively. Depending on the K(+):1 ratio, G-quartets of 1 self-assemble into octamers (O) or polymers.

Ligand Binding by Antibody IgE Lb4: Assessment of Binding Site Preferences Using Microcalorimetry, Docking, and Free Energy Simulations

Antibody IgE Lb4 interacts favorably with a large number of different compounds. To improve the current understanding of the structural basis of this vast cross-reactivity, the binding of three dinitrophenyl (DNP) amino acids (DNP-alanine, DNP-glycine, and DNP-serine) is investigated in detail by means of docking and molecular dynamics free energy simulations. Experimental binding energies obtained by isothermal titration microcalorimetry are used to judge the results of the computational studies. For all three ligands, the docking procedure proposes two plausible subsites within the binding region formed by the antibody CDR loops. By subsequent molecular dynamics simulations and calculations of relative free energies of binding, one of these subsites, a tyrosine-surrounded pocket, is revealed as the preferred point of complexation. For this subsite, results consistent with experimental observations are obtained; DNP-glycine is found to bind better than DNP-serine, and this, in turn, is found to bind better than DNP-alanine. The suggested binding mode makes it possible to explain both the moderate binding affinity and the differences in binding energy among the three ligands.

Centrifugal Countercurrent Partition Chromatography with a Chain Winding Column

A new kind of centrifugal countercurrent partition chromatographic column has been developed. The shape of the column looks like the chain of a bicycle. Using the column, retention ratio of the stationary phase was measured with three kinds of solvent systems under various experimental conditions. The chloroform solvent systems showed retention from 52 to 66% when the upper phase was used as the stationary phase. The performance of the column has been successfully demonstrated in the separation of dinitrophenyl (DNP) amino acids, Weimeisu and an extract of branch-leaves of Taxus cuspidate.

Toroidal coil counter-current chromatography: Achievement of high resolution by optimizing flow-rate, rotation speed, sample volume and tube length

This paper deals with optimization of a new seal-free compact toroidal coil centrifuge to achieve high resolution in analytical counter-current chromatography (CCC). Toroidal coil CCC (hydrostatic motion) has advantages compared with high-speed CCC (efficiently mixing solution with planetary motion) in the separation of protein or easily emulsified samples. A toroidal coil separation column of 0.4 mm I.D. PTFE tubing was accommodated around the periphery of the cylindrical centrifuge bowl. Using a two-phase solvent system composed of chloroform–acetic acid–0.1 M hydrochloric acid (2:2:1, v/v/v) and a set of dinitrophenyl-amino acids as test samples, a series of experiments was performed with parameters such as the column length, sample volume, flow-rate, elution mode of the mobile phase and rotation speed. The highest efficiency, over 10 000 theoretical plates, was achieved with a 100 m long coiled tube and an 11 ml total capacity at a flow-rate of 0.01 ml/min at 800 rpm.

Determination of available lysine in infant milk formulae by high-performance liquid chromatography

A high-performance liquid chromatography (HPLC) method to determine available lysine is proposed. Available lysine was measured by an optimised fluorodinitrobenzene method on the basis of the reactivity of the free ε-amino group of the lysine. The classical acid hydrolysis has been improved and shortened from the usual time of 12 h to 2 h 30 min using an oil bath. Optimal resolution and quantitation of N ε-dinitrophenyllysine was obtained with a Nova-Pak C 18 column using an isocratiic elution with 35% methanol and 65% 0.01 M sodium acetate buffer (pH 4.5) and a flow-rate of 1 ml/min. Satisfactory results were obtained for the reliability of the method in terms of linearity from 0.1 to 5.0 mg/l of lysine-free base, precision (R.S.D. values between 4.3% and 7.8%), recovery (91.5%) and sensitivity (detection limit of 0.02 mg/l). The proposed method has also been checked for lack of interferences from other dinitrophenyl-amino acids.

Enhanced capillary zone electrophoretic separation of dinitrophenyl-amino acid derivatives through control of electroosmotic flow by the buffer cation

The effect of Mg2+, Cd2+ and Zn2+ are evaluated as buffer additives to reduce electroosmotic flow (EOF) to facilitate the capillary zone electrophoretic (CZE) separation of 2,4-dinitrophenyl (DNP) derivatives of amino acids. As the divalent cation concentration increases EOF decreases and reduction of EOF follows the order Zn2+>Cd2+>Mg2+. The electrophoretic mobility of the DNP-amino acid derivatives remains constant for a given buffer and pH over a change in divalent cation concentration. The effect of the divalent cation buffer additive is consistent with cation-exchange at the silanol sites on the fused-silica wall. Higher buffer pH favors greater cation-exchange and reduced EOF. Separation of multicomponent mixtures of DNP-l-amino acid derivatives in the presence of Mg2+, Cd2+ and Zn2+ are compared at pH 7.00. Derivative migration time, resolution and peak shape are enhanced and are best for Mg2+ as the buffer additive although Mg2+ must be used at a higher concentration than either Cd2+ or Zn2+. Raising the buffer pH to 9.25 in the presence of Mg2+ increases migration time and resolution but analysis time is also increased.

Chiral interaction of a polyclonal anti-dinitrophenyl antibody with dinitrophenyl-amino acids determined by an enantioselective enzyme-linked immunosorbent assay

The enantioselectivity of the interaction of a polyclonal anti-DNP (dinitrophenyl) antiserum with a variety of DNP-amino acids has been determined by an indirect noncompetitive ELISA and the results obtained have been compared with those of an indirect competitive ELISA. The competitive ELISA offers an advantage to determine the I 50 values representing the relative affinities of the investigated DNP-amino acid enantiomers. In all cases, the I 50 values obtained were different for the individual DNP-amino acid enantiomers. While the l-enantiomers of ϵ-DNP-lysine, δ-DNP-ornithine, DNP-alanine and DNP-proline show a higher relative affinity for the antibody combining site than the d-enantiomers, an inverse chiral recognition was found for DNP-phenylalanine. On account of the high sensitivity of the ELISA system, the determination of small differences in the enantiomer recognition by the anti-DNP antibody is possible.

PH-Zone-refining counter-current chromatography: a displacement mode applied to separation of dinitrophenyl amino acids

A new mode of ph-zone-refining counter-current chromatography operates in a manner analogous to displacement chromatography. The method uses a retainer base acid in the stationary phase to retain analytes in the column and a displacer acid base to elute the analytes in the decreasing order of p K a and hydrophobicity. The elution produces a train of highly concentrated rectangular solute peaks with minimum overlap. Utility of the method is demonstrated in the separation of dinitrophenyl amino acids in a two-phase solvent system composed of methyl tert.-butyl ether and water. Compared with the original mode of ph-zone-refining counter-current chromatography, the present mode is more amenable to ligand-affinity separation which may cover a broader range of analytes including non-ionizable compounds.

Packed-capillary electrochromatographic separation of the enantiomers of neutral and anionic compounds using β-cyclodextrin as a chiral selector: Effect of operating parameters and comparison with free-solution capillary electrophoresis

Chiral separation by packed capillary electrochromatography was successfully achieved through the use of 50 μm I.D. fused-silica capillaries packed with a β-cyclodextrin chiral stationary phase. The properties of electroosmosis in these packed capillaries were studied by investigating the influence of buffer composition and pH on the velocity of electroosmotic flow. With the use of triethylammonium acetate (TEAA) as background electrolyte, the direction of electroosmotic flow was reversed compared to the direction of flow observed with a phosphate buffer system. The enantiomers of both neutral solutes (benzoin, hexobarbital) and anionic solutes (dansyl- and dinitrophenyl-amino acids) were resolved; the analysis of anions was facilitated by the reversal of electroosmotic flow with the TEAA buffer system. The effects of methanol concentration and the TEAA buffer concentration on the migration and resolution of the enantiomers were studied. In addition, separations were obtained using β-cyclodextrin as an additive in free-solution capillary electrophoresis using both phosphate and TEAA buffers. Comparison between the packed- and open-tubular systems was made. The same effect of electroosmotic flow reversal with TEAA buffer was observed in open-tubular fused-silica capillaries.

High-performance affinity chromatography of immunoglobulin E on a column of dinitrophenylamino acids covalently bound to a highly cross-linked polymeric micropellicular support

Coupling of different dinitrophenyl (DNP) amino acids to 2.5-μm highly cross-linked polystyrene—divinylbenzene beads was performed, using carbodiimide as catalyst. The binding capacity of affinity-purified monoclonal anti-DNP mouse immunoglobulin E (IgE) antibody to DNP-lysine-coated beads is ca. 4 nmols per mg of beads. The structure of the active coupled functional groups was investigated by X-ray photoelectron spectroscopy. The application of these ligand-carrying beads to the high-performance affinity chromatography of IgE antibody was demonstrated and the purity of IgE was confirmed by sodium dodecyl sulphate—polyacrylamide gel electrophoresis. This method is also suitable for coupling several other carboxyl compounds to a highly cross-linked polystyrene matrix.

Improved high-speed counter-current chromatography with the multilayer coils connected in series : IV. Evaluation of preparative capability with large multilayer coils

In order to further extend the preparative capability of the apparatus, we have constructed a new centrifuge which can hold much longer multilayer coils for the separation of multigram quantities of samples. The performance of the apparatus has been successfully demonstrated in the separation of dinitrophenyl (DNP) amino acids using a two-phase solvent system composed of chloroform-acetic acid 0.1 M hydrochloric acid at a volume ratio of 2:2:1

Influence of ionic and polymer composition on the properties of the phases of aqueous two-phase systems formed by non-ionic polymers

Partitioning of a homologous series of sodium salts of dinitrophenylated amino acids with aliphatic side-chains in aqueous dextran—poly(ethylene glycol), dextran–polyvinylpyrrolidone and dextran–Ficoll two-phase systems of different polymer concentrations and various ionic compositions was studied. The differences in the abilities of aqueous media in the coexisting phases of the systems used to participate in ionic and hydrophobic hydration interactions with a partitioned solute are estimated. The hydrophobic hydration ability appears to depend mostly on the polymer composition of the phases, while the ionic hydration ability is governed mostly by the ionic composition. In particular, it is found that the ionic hydration and hydrophobic hydration abilities of water are interrelated. Both these characteristics of water in the two phases are considered with regard to the distribution of hydrogen-bond strengths and the relative stability of the hydrogen-bond networks in the phases.

Systematic search for suitable two-phase solvent systems for high-speed counter-current chromatography

We have introduced two series of two-phase solvent systems which facilitate the systematic search for the solvent systems suitable for high-speed counter-current chromatography. The n-hexane—ethyl acetate— n-butanol—methanol—water systems provide a broad range of hydrophobicity, while the chloroform—methanol—water systems are extremely useful for separations of various natural products with moderate hydrophobicity. The practical use of these solvent series was demonstrated with several test samples which include dinitrophenyl amino acids, S-triazine herbicides, indole auxin, and non-ionic organic solvents.

Liquid chromatographic separation of the enantiomers of dinitrophenyl amino acids using a β-cyclodextrin-bonded stationary phase

A β-cyclodextrin-bonded chiral stationary phase was used for the liquid chromatographic resolution of racemic amino acid derivatives. Ten pairs of dinitrophenyl amino acid enantiomers were separated by this technique. Methanol—triethylammonium buffer solutions were used as the mobile phases. The effects of ph, methanol and triethylammonium acetate buffer concentration in the mobile phase, and the structural features of the solutes on the retention and enantio-selectivity were examined and discussed in terms of the overall retention mechanism.

Improved High-Speed Countercurrent Chromatograph with Three Multilayer Coils Connected in Series. III. Evaluation of Semianalytical Column with Various Biological Samples and Inorganic Elements

Abstract The partition efficiency of the counter-current chromatographic centrifuge was improved by mounting a set of three multilayer coils prepared from a 1.07 mm I.D. and ca 300 m length of PTFE (polytetrafluoroethylene) tube with a total capacity of 270 ml. The high performance of the present apparatus has been successfully demonstrated in separations of various testing samples which include DNP (dinitrophenyl) amino acids, indole auxins, tetracycline derivatives, flavonoids, bacitracin, triterpenoic acids, and lanthanoid chlorides.

Centrifugal couner-current partition chromatography with helical coil rotor : Simplified counter-current chromatography with a rotating face-seal

A centrifugal counter-current partition chromatograph has been developed and tested in order to simplify earlier counter-current chromatographic (CCC) procedures. It includes a helical coil rotor and a rotating face-seal. The rotor is designed to be adapted in an ordinary laboratory centrifuge for toroidal coil CCC. Twisting between the inlet and outlet tubing is avoided by using the rotating seal in the rotor. The seal is placed between the rotor, on which helical coils are mounted, and a newly designed centrifugde lid. This chromatographic rotor, rotating simply around its own axis, has simplified a previous CCC device in which a coil planet mechanism is used to avoid tube twisting whilst retaining the capability for chromatographic separations. Results for separations of nystatin, dinitrophenylamino acids and Poly I:C were comparable to those obtained by liquid chromatography and the previous CCC procedure.

Preliminary applications of cross-axis synchronous flow-through coil planet centrifuge for large-scale preparative counter-current chromatography

The cross-axis synchronous flow-through coil planet centrifuge with a 20-cm revolutional radius and a total capacity of 1600 ml was successfully applied to preparative counter-current chromatography of various biological samples, which include sea buckthorn extract, steroid reaction mixture, indole plant hormones, and dinitrophenylamino acids. The present system offers advantages of stable balance of the centrifuge, a large column capacity, and high resolution.

Cross-Axis Synchronous Flow-Through Coil Planet Centrifuge Free of Rotary Seals for Preparative Countercurrent Chromatography. Part II. Studies on Phase Distribution and Partition Efficiency in Coaxial Coils

Abstract Potential capability of the apparatus in performing countercurrent chroma-tography has been examined with three different types of coiled columns, all coaxially mounted around the holder. In single-layer coils, typical solvent systems display characteristic hydrodynamic distribution which ensures a stable retention of the stationary phase against heavy sample loading in preparative separations. Direct observation of the hydrodynamic motion in the rotating spiral column revealed vigorous mixing of the two solvent phases throughout the area which promises a high partition efficiency of the present method. Gram-quantity preparative separations were performed in the multilayer coil with two different sets of test samples: Isocratic elution of dinitrophenyl amino acids yielded high partition efficiency of 1600 theoretical plates while the versatility of the method was demonstrated on gradient elution of dipeptides including two pairs of sequential isomers.

Effect of polymer composition on the relative hydrophobicity of the phases of the biphasic system aqueous dextran-poly(ethylene glycol)

Partition coefficients for an homologous series of dinitrophenylated amino acids with aliphatic side-chains were determined in an aqueous Dextran-70-poly(ethylene glycol)-6000 ( PEG-6000) biphasic system at various concentrations of the polymers in the presence of 0.15 mol/kg sodium chloride in 0.01 mol/kg sodium phosphate buffer, pH 7.4. The free energy of transfer of a CH 2 group from one phase to the other was estimated and found to be directly proportional to the difference in the PEG concentrations between the phases. The results obtained imply that the effective concentration of PEG in a phase containing more than 11.4% (w/w) Dextran-70 differs from the real concentration of PEG in the phase, probably due to the hydration of dextran.

New angle rotor coil planet centrifuge for counter-current chromatography : II. Design of the apparatus and studies on phase retention and partition capability

A new angle rotor coil centrifuge (Ψ = 25°), which produces a Type J-L synchronous planetary motion, has been constructed to examine its capability in terms of stationary phase retention and solute partitioning. Studies on phase distribution diagrams obtained from various two-phase solvent systems indicated that the present system can be adapted to a wide variety of solvent systems by adjusting the centrifugal conditions. Excellent partition capability of the apparatus was successfully demonstrated in separations of dinitrophenyl amino acids samples with chloroform—acetic acid—0.1 N hydrochloric acid (2:2:1).