Matrine-imprinted monolithic stationary phase for extraction and purification of matrine from Sophorae flavescentis Ait

A matrine-imprinted monolithic stationary phase (MIP monolith) was prepared by in situ polymerization for extraction and purification of matrine from Sophorae flavescentis Ait. Matrine was used as the template molecule, methacrylic acid as the function monomer, ethylene glycol dimethacrylate as the cross-linking agent, and toluene and dodecanol as the porogenic solvents. Scanning electron microscope study revealed that a monolithic structure with mesopores and 36 μm diameter nodules was obtained. The molecular recognition process and the effect of varying chromatographic conditions on separation were examined by high-performance liquid chromatography (HPLC). Hydrogen bonding, electrostatic, hydrophobic interactions and the molecular shape matching in MIP monolith cavities were proposed to be responsible for the recognition mechanism. The use of MIP monolith as a solid-phase extraction (SPE) sorbent for extraction and purification of matrine from S. flavescentis Ait was investigated. The extraction yield was 89.2% (for 3.0 mmol l(-1) matrine) with enrichment factor 29.     

Molecularly imprinted polymers from nicotinamide and its positional isomers

Imprinted polymers were prepared for nicotinamide and its positional isomers. The influence of porogenic solvent and functional monomer on recognition properties of the polymer was compared. The results indicated that two functional groups, the heterocyclic nitrogen and the amide group, in the nicotinamide or isonicotinamide molecule have a synergistic effect in binding to the polymer. The polymers prepared with nicotinamide and isonicotinamide can be used as HPLC stationary phase for the separation of positional isomers of nicotinamide or isonicotinamide, while the polymer prepared with picolinamide showed no specificity toward the template. The mechanisms for the differences in recognition are discussed. In addition to the retention of polymers to their templates the polymers also displayed excellent retention to nicotinic acid and isonicotinic acid, compounds structurally similar to the template. This dual recognition property of the polymer may be useful in circumstances where the preparation of a polymer for a specific template may be problematic because of poor stability or solubility.     

Gas chromatography-mass spectrometry identification of photoproducts of hexahydroquinoline derivatives: potential calcium channel antagonists

Photodegradation products of hexahydroquinoline derivatives (HHQ) have been analysed with gas chromatography-mass spectrometry (GC-MS). The photodegradation was carried out under the conditions recommended in the first version of the document issued by the International Conference on Harmonization (ICH), currently in force in the studies of photochemical stability of drugs and therapeutic substances. The study was performed on the compounds having two chlorine atoms at different positions of the phenyl ring. Photodegradation of dichlorophenyl derivatives of HHQ resulted in formation of one or three photoproducts. The main product of their decomposition was aromatic compound formed as a result of dehydrogenation of the dihydropyridine ring. The most often observed fragmentation pathway of the photoproducts formed was elimination of methyl and methoxy radicals from the ester groups. The fragmentation of the photoproducts containing one chlorine atom at the ortho-position of the phenyl ring occurred through elimination of chlorine radical.     

Stereoselective binding of 2-(4-biphenylyl)-3-substituted-3-hydroxypropionic acids on an immobilised human serum albumin chiral stationary phase

A series of 2-(4-biphenylyl)-3,3'-hydroxy-substituted phenyl propionic acid, with anti-inflammatory properties, bearing two chiral centres, were studied by HPLC upon HSA-CSP (human serum albumin-based chiral stationary phase). The compounds were analysed in their stereoisomeric erythro and threo forms. The study involved the enantioselective analysis on HSA-CSP, the determination of the racemate lipophilicity (log k'(w)), a QSRR (quantitative structure-retention relationship) analysis and CD study for the assessment of the absolute configuration of the most retained enantiomer. Lipophilicity was found to be an important factor affecting the affinity of the compounds for the HSA stationary phase, but electronic properties seemed to play a role. The position of the substituent of the phenyl group on carbon 3 was found important to modulate stereoselective interaction, the highest value of enantioselectivities being found for the erythro ortho-substituted phenyl derivatives. The previously proposed two steps mechanism of enantiodiscrimination for cyclohexylphenyl substituted derivatives was confirmed for this series of derivatives bearing the biphenylyl moiety.     

Synthesis and evaluation of molecularly imprinted polymers for enalapril and lisinopril, two synthetic peptide anti-hypertensive drugs

Molecularly imprinted polymers (MIPs) for the recognition of enalapril and lisinopril were prepared using 4-vinylpyridine as the functional monomer. Following thermal polymerisation the resulting materials were crushed, ground and sieved. First generation MIPs were produced in protic polar porogenic solvents (mixture of methanol (MeOH) and acetonitrile (ACN)). These MIPs were used and validated as sorbents for solid phase extraction and binding assays. Second generation MIPs were produced with polar aprotic porogenic solvent (DMSO). These polymers were packed in HPLC columns in order to investigate their molecular recognition properties in a dynamic mode. The study of the mobile phase composition included two major parameters: organic modifier content and pH value. Retention factors illustrate selective binding of the template from the imprinted polymers, compared to structurally related compounds.     

Development and characterization of an immobilized human organic cation transporter based liquid chromatographic stationary phase

Membranes from a stably transfected cell line that expresses the human organic cation 1 transporter (hOCT1) have been immobilized on the immobilized artificial membrane (IAM) liquid chromatographic stationary phase to form the hOCT1(+)-IAM stationary phase. Membranes from the parent cell line that does not express the hOCT1 were also immobilized to create the hOCT1(-)-IAM stationary phase. Columns were created using both stationary phases, and frontal displacement chromatography experiments were conducted using [(3)H]-methyl phenyl pyridinium ([(3)H]-MPP(+)) as the marker ligand and MPP(+), verapamil, quinidine, quinine, nicotine, dopamine and vinblastin as the displacers. The K(d) values calculated from the chromatographic studies correlated with previously reported K(i) values (r(2)=0.9987; p<0.001). The data indicate that the hOCT1(+)-IAM column can be used for the on-line determination of binding affinities to the hOCT1 and that these affinities are comparable to those obtained using cellular uptake studies. In addition, the chromatographic method was able to identify a previously undetected high affinity binding site for MPP(+) and to determine that hOCT1 bound (R)-verapamil to a greater extent than (S)-verapamil.     

Evaluation of "click" binaphthyl chiral stationary phases by liquid chromatography

Two "click" binaphthyl chiral stationary phases were synthesized and evaluated by liquid chromatography. Their structures incorporate S-(-)-1,1'-binaphthyl moiety as the chiral selector and 1,2,3-triazole ring as the spacer. These chiral stationary phases (CSPs) allowed the efficient resolution for a wide range of racemic BINOL derivatives, particularly for nonpolar diether derivatives and 3-phenyl indolin-2-one analogs. The chromatographic data showed that the π-π interaction was crucial for enantiorecognition of these CSPs. Loss of enantioselectivity observed on CSP3, which are lacking the triazole ring linkage, indicated that the triazole ring linkage took part in the enantioseparation process, although it was remote from the chiral selector of the CSP. The substitution of the phenyl group at 6 and 6' positions can significantly improve the separation ability of the CSP. The chiral recognition mechanism was also investigated by tracking the elution orders and studying the thermodynamic parameters.     

Design, synthesis and evaluation of novel molecules with a diphenyl ether nucleus as potential antitubercular agents

A series of compounds with a diphenyl ether nucleus were synthesized by incorporating various amines into the diphenyl ether scaffold with an amide bond. Their antitubercular activities were evaluated against Mycobacterium tuberculosis H(37)Rv by a microdilution method, with MIC values ranging from 4 to 64μg/mL. Through structure-activity relationship studies, the two chlorine atoms at 3 and 4 positions in the phenyl ring of R(2) group were found to play a significant role in the antitubercular activity. The most potent compound 6c showed an MIC value of 4μg/mL and a good safety profile in HepG2 cell line by the MTT assay. Compound 6c was further found to be effective in a murine model of BCG infection, providing a good lead for subsequent optimization.     

The substrate specificity of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase. Analysis of myristic acid analogs containing oxygen, sulfur, double bonds, triple bonds, and/or an aromatic residue

We have explored the acyl-CoA substrate specificity of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (NMT) by synthesizing 81 fatty acid analogs and surveying their activity in a coupled in vitro assay containing Pseudomonas acyl-CoA synthetase and Escherichia coli-derived yeast NMT. Single oxygen or sulfur substitution for C-3 through C-13 is well tolerated by both enzymes. Detailed kinetic analyses suggest that the acyl-CoA and peptide-binding sites of NMT are relatively insensitive to placement of single group 6B heteroatoms. By contrast, di-oxygen-substituted analogs were very poor substrates, producing dramatic reductions in the affinity of NMTs peptide-binding site for a synthetic octapeptide substrate derived from the NH2-terminal sequence of a known N-myristoylprotein, the gag poly-protein precursor of human immunodeficiency virus 1 (HIV-1). This observation provides an example of binding site cooperativity in NMT. Replacement of one oxygen with sulfur at either the 6, 9, or 12 position of dioxatetradecanoic acids results in a general increase in peptide catalytic efficiency (Vmax/Km). An analysis of five fatty acids from octanoic to dodecanoic having terminal phenyl groups indicated that the best substrate was 10-phenyldecanoic acid even though Corey-Pauling-Koltun molecular models indicate that it has a length equivalent to that of tridecanoic acid. Six analogs having an equivalent length of 13 carbon atoms were subsequently prepared in which the phenyl group was systematically moved one methylene group closer to carboxyl. Movement of the phenyl just one carbon closer to carboxyl (producing 9-(p-methylphenyl) nonanoic acid) decreases peptide catalytic efficiency (Vmax/Km) severalfold compared to 10-phenyldecanoic acid. 10-(4-Tolyl)decanoic acid has the same relative positions of phenyl and carboxyl as 10-phenyldecanoic acid even though a methyl group is present on the phenyl ring. It produces peptide Km and Vmax values that are the same as 10-phenyldecanoic acid. Substitution of either oxygen or sulfur for a methylene group fails to override the effects noted when the phenyl group position is altered in the C-14 equivalent fatty acid series. Several fatty acids of differing chain lengths with cyclohexyl-, 2-furyl, and 2-thienyl groups at their omega termnius had activity profiles that paralleled those of the comparable phenyl-substituted compounds. Myristic acid analogs with triple bonds (beginning at positions 2 through 13), cis-double bonds (positions 3 through 13) and trans-double bond isomers (E5, E6, and E7) were also tested.(ABSTRACT TRUNCATED AT 400 WORDS)     

Face-to-face porphyrin moieties assembled with spacing for pyrazine recognition in molecularly imprinted polymers

A strategy for arranging two porphyrin moieties in a face-to-face fashion in polymeric material was demonstrated by molecular imprinting, whereby porphyrin Zn(II) complex monomers were cross-linked with ethylene glycol dimethacrylate in the presence of pyrazine or 1,5-naphthyridine as a template molecule. In chromatographic studies using the resultant imprinted polymers as stationary phase, both the polymers showed selectivity for the original template molecule, suggesting that two zinc porphyrin moieties were immobilized in the face-to-face fashion, and were center-aligned for pyrazine recognition and offset-arranged for 1,5-naphthyridine recognition. The imprinted polymer with porphyrin moieties also showed a decrease in its fluorescence intensity in response to the concentration of the target molecule, suggesting the potential utility as sensing material.     

Immobilization of (1S,2R)-(+)-2-amino-1,2-diphenylethanol derivates on aminated silica gel with different linkages as chiral stationary phases and their enantioseparation evaluation by HPLC

A chiral selector was prepared through the reaction between (1S,2R)-(+)-2-amino-1,2-diphenylethanol and phenyl isocyanate. This selector was immobilized on aminated silica gel, respectively, with bifunctional group linkers of 1,4-phenylene diisocyanate, methylene-di-p-phenyl diisocyanate, and terephthaloyl chloride to produce corresponding three chiral stationary phases. The prepared compounds and chiral stationary phases were characterized by FT-IR, elemental analysis, (1)H NMR, and solid-state (1)H NMR. The enantioseparation ability of these chiral stationary phases was evaluated with structurally various chiral compounds. The chiral stationary phase prepared with 1,4-phenylene diisocyanate as linker showed excellent enantioseparation ability. The influence of different linkages on the enantioseparation was discussed.     

Immobilized horse liver alcohol dehydrogenase as an on‐line high‐performance liquid chromatographic enzyme reactor for stereoselective synthesis

Horse liver alcohol dehydrogenase (HLADH) has been non‐covalently immobilized on an immobilized artificial membrane (IAM) high‐performance liquid chromatography (HPLC) stationary phase. The resulting IAM‐HLADH retained the reductive activity of native HLADH as well as the enzyme's enantioselectivity and enantiospecificity. HLADH was also immobilized in an IAM HPLC stationary phase prepacked in a 13 × 4.1 mm ID column to create an immobilized enzyme reactor (HLADH‐IMER). The reactor was connected through a switching valve to a column containing a chiral stationary phase (CSP) based upon p‐methylphenylcarbamate derivatized cellulose (Chiralcel OJR‐CSP). The results from the combined HLADH‐IMER/CSP and chromatographic system demonstrate that the enzyme retained its activity and stereoselectivity after immobilization in the column and that the substrate and products from the enzymatic reduction could be transferred to a second column for analytical or preparative separation. The combined HLADH‐IMER/CSP system is a prototype for the preparative on‐line use of cofactor‐dependent enzymes in large‐scale chiral syntheses. Chirality 11:39–45, 1999. © 1999 Wiley‐Liss, Inc.     

Ion Exclusion Chromatography: Parameters Influencing Retention

Ion Exclusion Chromatography (IEC) finds application in the separation of a wide range of small, neutral or partially ionized molecules. In IEC, the strong as well as weak electrolytes are eluted unseparated, the first at the beginning and the latter at the end of the elution. The retention volumes of the remaining electrolytes are found to be proportional to their dissociation constant values. The dead and inner volumes of the chromatographic column can be determined from the observed dependence of retention volumes on dissociation constant values. The retention mechanism is described by the analytical equations and by the results obtained from the computer simulation of the column performance (using global thermodynamic and chromatographic equations or the Craig method). The mixed retention mechanism involving hydrophobic adsorption and screening effect is observed for weak electrolytes and aromatic compounds. Aromatic compounds are found to be retained almost solely by a reverse-phase mechanism involving interaction of the solute with the unfunctionalized regions of the stationary phase. The purpose of this paper is to survey the field. Using theoretical and experimental approaches, I show how different parameters can influence ion-exclusion solute retention. Although this retention is affected by the physicochemical parameters of the sorbent, stationary and mobile phases especially, this study primarily deals with the structural solute parameters, stationary phase form and column temperature, that have had little or no discussion in literature.     

A chromatographic approach to the determination of relative free energies of interaction between hydrophobic and amphiphilic amino acid side chains.

A liquid chromatographic stationary phase was prepared by covalently binding to the surface of microparticulate silica gel functionality (benzylsilane), which mimics the side chain of the amino acid phenylalanine. The chromatographic retentions of the N-acetyl C-(N'-methyl) amides of various hydrophobic and amphiphilic amino acids on this stationary phase were measured using an aqueous mobile phase. A retention order of Gly < Ala < Cys < Val < Met < Pro < Ile < Leu < Tyr < Phe < Trp is seen at room temperature. Chromatographic retentions were used to derive free energies of adsorption of the amino acid derivatives on the chromatographic support relative to that of the glycine derivative. The temperature dependencies of the retention of aromatic and aliphatic amino acid derivatives differ in curvature, indicating a qualitative difference in the absorption mechanism. An adsorption model for retention is proposed, and arguments are made as to the suitability of an adsorption model for describing the contacts between amino acid side chains during the initial steps of protein folding.     

Chiral separation and determination of amino acid enantiomers in fruit juice by open‐tubular nano liquid chromatography

A novel chiral porous‐layer stationary phase was developed for use in open‐tubular nano liquid chromatography. The stationary phase was prepared by an in‐situ polymerization of 3‐chloro‐2‐hydroxypropylmethacrylate (HPMA‐Cl) and ethylene dimethacrylate (EDMA). The reactive chloro groups at the surface of the porous stationary phase were reacted with β‐Cyclodextrin (β‐CD). The resulting morphology was characterized by using scanning electron microscopy (SEM) and Fourier‐transform infrared spectroscopy (FT‐IR). The chromatographic performance of the column was evaluated by hydrophilic interaction chromatography (HILIC). Amino acids were used as test solutes. The running buffer conditions for the enantioseparation were found to be 85% acetonitrile (ACN):10%MeOH: 5% H₂O at 0.1% v/v trifluoro acetic acid (TFA) (flow rate: 800 nL/min). The enantioseparation provided high theoretical plate numbers up to 26 000 platesm^?1. A good retention capacity within satisfactory retention times was also achieved. Real sample applicability of this column to the separation of amino acid enantiomers in fruit juice sample was demonstrated.     

Application of chiral technology in a pharmaceutical company. Enantiomeric separation and spectroscopic studies of key asymmetric intermediates using a combination of techniques. Phenylglycidols

Phenylglycidols substituted in the 2-, 3-, and 4- positions with fluorine, chlorine, and trifluoromethyl, and with methoxy in the 3- position, were synthesized from the corresponding E-cinnamic acids and separated into their (R,R)- and (S,S)- enantiomers using subcritical fluid chromatography with mixtures of MeOH in CO(2), on either a Chiralpak AD or AS chiral stationary phase. These compounds and commercially-available (R,R)- and (S,S)-phenylglycidol were analyzed for their vibrational circular dichroism (VCD), electronic circular dichroism (ECD), and optical rotation (OR) properties to exemplify a strategy whereby the absolute stereochemistry of common and key chiral intermediates is established early in the structure-activity and structure-property relationship phase of a drug discovery program in a pharmaceutical company. From this study, substituents in the phenyl group of the synthesized molecules were found not to grossly alter spectroscopic features, and therefore, diagnostic absorption bands in the respective VCD spectra, and the sign and shape of the measured ECD curves could be used to determine and track the absolute stereochemistry of analogs without necessarily requiring time-consuming ab initio calculations of all low energy conformers for all compounds. VCD, OR, and ECD calculations for the determination of absolute configuration carried out at the DFT level with the hybrid B3PW91 functional and the TZVP basis set were found to be especially useful in this study.     

Physiological properties of a Pseudomonas strain which grows with p-xylene in a two-phase (organic-aqueous) medium.

Pseudomonas putida Idaho utilizes toluene, m-xylene, p-xylene, 1,2,4-trimethylbenzene, and 3-ethyltoluene as growth substrates when these hydrocarbons are provided in a two-phase system at 5 to 50% (vol/vol). Growth also occurs on Luria-Bertani medium in the presence of a wide range of organic solvents. The ability of the organism to grow in the presence of organic solvents is correlated with the logarithm of the octanol-water partition coefficient, with dimethyl-phthalate (log P(OCT) = 2.3) being the most polar solvent tolerated. During growth with p-xylene (20% [vol/vol]), there was an initial lag period accompanied by cell death, which was followed by a period of exponential growth. The stationary phase of growth was characterized by a dramatic decrease in cell viability, although cell dry weight and turbidity measurements slowly increased. Electron micrographs revealed that during growth in the presence of p-xylene, the outer cell membrane becomes convoluted and membrane fragments are shed into the culture medium. At the same time, the cytoplasmic membrane invaginates, forming vesicles, and becomes disorganized. Electron-dense intracellular inclusions were observed in cells grown with p-xylene (20% [vol/vol]) and p-xylene vapors, which are not present in cells grown with succinate. Attempts to demonstrate the presence of plasmid DNA in P. putida Idaho were negative. However, polarographic studies indicated that the organism utilizes the same pathway for the degradation of toluene, m-xylene, and p-xylene as that used by P. putida mt-2 which contains the TOL plasmid pWWO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effective separation of peptides using highly dense thermo-responsive polymer brush-grafted porous polystyrene beads

For the development of well-defined highly dense thermo-responsive polymer grafted surface as an improved stationary phase for thermo-responsive chromatography, poly(N-isopropylacrylamide) (PIPAAm) brush-grafted porous polystyrene beads were prepared by surface-initiated atom transfer radical polymerization (ATRP). The PIPAAm grafted region of polystyrene beads was adjusted by the addition of isooctane as a poor solvent for polystyrene upon the reaction of ATRP initiator immobilization. Using a thermo-responsive HPLC column containing the prepared beads with PIPAAm brush grafted on the inside pores nearby the outer surfaces, angiotensin subtypes were effectively separated with aqueous mobile phase, because the densely grafted PIPAAm on nearby the outer surface effectively interacted with the peptides hydrophobically. Retention of basic peptide was achieved by the beads with basic mobile phase. These results indicated that the prepared beads with grafted PIPAAm nearby the outer surface became an effective chromatographic stationary phase for retaining basic peptides using wide pH range of mobile phase.     

Physiological properties of a Pseudomonas strain which grows with p-xylene in a two-phase (organic-aqueous) medium.

Pseudomonas putida Idaho utilizes toluene, m-xylene, p-xylene, 1,2,4-trimethylbenzene, and 3-ethyltoluene as growth substrates when these hydrocarbons are provided in a two-phase system at 5 to 50% (vol/vol). Growth also occurs on Luria-Bertani medium in the presence of a wide range of organic solvents. The ability of the organism to grow in the presence of organic solvents is correlated with the logarithm of the octanol-water partition coefficient, with dimethyl-phthalate (log P(OCT) = 2.3) being the most polar solvent tolerated. During growth with p-xylene (20% [vol/vol]), there was an initial lag period accompanied by cell death, which was followed by a period of exponential growth. The stationary phase of growth was characterized by a dramatic decrease in cell viability, although cell dry weight and turbidity measurements slowly increased. Electron micrographs revealed that during growth in the presence of p-xylene, the outer cell membrane becomes convoluted and membrane fragments are shed into the culture medium. At the same time, the cytoplasmic membrane invaginates, forming vesicles, and becomes disorganized. Electron-dense intracellular inclusions were observed in cells grown with p-xylene (20% [vol/vol]) and p-xylene vapors, which are not present in cells grown with succinate. Attempts to demonstrate the presence of plasmid DNA in P. putida Idaho were negative. However, polarographic studies indicated that the organism utilizes the same pathway for the degradation of toluene, m-xylene, and p-xylene as that used by P. putida mt-2 which contains the TOL plasmid pWWO.(ABSTRACT TRUNCATED AT 250 WORDS)     

1,3-Diarylcycloalkanopyrazoles and diphenyl hydrazides as selective inhibitors of cyclooxygenase-2

Novel 1,3-diarylcycloalkanopyrazoles 1, and diphenyl hydrazides 2 were identified as selective inhibitors of cyclooxygenase-2. The 1,3-diaryl substitution pattern of the pyrazole ring in 1 differentiates these compounds from most of the known selective COX-2 inhibitors that contain two aryl rings at the adjacent positions on a heterocyclic or a phenyl ring. Similarly, the two phenyl rings in 2 are also separated by three atoms. SAR of both phenyl rings in 1 and 2, and the aliphatic ring in 1 will be discussed.