Optimization of normal-phase chromatographic separation of compounds with primary, secondary and tertiary amino groups.

The retention behavior of primary, secondary and tertiary amines was studied using normal-phase-HPLC on silica, diol, and cyano stationary phases. Several classes of amines, including benzylamines, anilines, ephedrines, tryptamines, and azatryptamines were chromatographed using mixtures of hexane and ethoxynonafluorobutane with methylene chloride and methanol. Peak tailing, diminished selectivity and low plate count were minimized by the addition of volatile amines to the mobile phase. The optimal additive was n-propylamine at 0.1% concentration. On diol columns, the elution order of free primary, N-N-methyl, and N,N-dimethylamines was predictable, while the elution order of primary and secondary amines on cyano columns varied depending on the alcohol modifier concentration. The feasibility of preparative normal-phase chromatography was demonstrated by the separation of a mixture of primary, secondary and tertiary amines obtained by direct methylation of norephedrine. The procedures described may provide a practical alternative to traditional methods of analysis and purification of potential drug candidates.

Species-specific in vitro pharmacological effects of the cannabinoid receptor 2 (CB2) selective ligand AM1241 and its resolved enantiomers.

BACKGROUND AND PURPOSE: Racemic (R,S) AM1241 is a cannabinoid receptor 2 (CB(2))-selective aminoalkylindole with antinociceptive efficacy in animal pain models. The purpose of our studies was to provide a characterization of R,S-AM1241 and its resolved enantiomers in vitro and in vivo. EXPERIMENTAL APPROACH: Competition binding assays were performed using membranes from cell lines expressing recombinant human, rat, and mouse CB(2) receptors. Inhibition of cAMP was assayed using intact CB(2)-expressing cells. A mouse model of visceral pain (para-phenylquinone, PPQ) and a rat model of acute inflammatory pain (carrageenan) were employed to characterize the compounds in vivo. KEY RESULTS: In cAMP inhibition assays, R,S-AM1241 was found to be an agonist at human CB(2), but an inverse agonist at rat and mouse CB(2) receptors. R-AM1241 bound with more than 40-fold higher affinity than S-AM1241, to all three CB(2) receptors and displayed a functional profile similar to that of the racemate. In contrast, S-AM1241 was an agonist at all three CB(2) receptors. In pain models, S-AM1241 was more efficacious than either R-AM1241 or the racemate. Antagonist blockade demonstrated that the in vivo effects of S-AM1241 were mediated by CB(2) receptors. CONCLUSIONS AND IMPLICATIONS: These findings constitute the first in vitro functional assessment of R,S-AM1241 at rodent CB(2) receptors and the first characterization of the AM1241 enantiomers in recombinant cell systems and in vivo. The greater antinociceptive efficacy of S-AM1241, the functional CB(2) agonist enantiomer of AM1241, is consistent with previous observations that CB(2) agonists are effective in relief of pain.

Normal-phase chiral liquid chromatography-mass spectrometry of non-UV-active compounds: applications for pharmaceutically relevant racemates.

Mixtures of hexane-like ethoxynonafluorobutane with alcohols were used as MS-friendly mobile phases for separation and efficient detection of non-UV-active enantiomers and diastereomers using normal-phase HPLC-APCI-MS. Racemic muscone, camphorsulfonamide, camphorsultam, BOC-protected 1-(3-aminopropyl)-2-pipecoline and diastereomeric 2-methylhexanoyl camphorsultams were resolved on Chiralpak AS and AD and achiral Luna CN columns. The responses of UV and APCI-MS detectors were compared under separation conditions studied, with MS detection achieving lowest detectable quantity in the range of 0.5-2 ng per chromatographic peak. The absolute configuration of crystalline derivatives of racemic 2-methylhexanoic acid with (S)-(-)-2,10-camphorsultam was determined by X-ray analysis after their automatic purification by preparative LC-MS. The technique described can be used to purify and determine the absolute stereochemistry of compounds of unknown structure which contain free carboxy group and lack sufficient UV absorbance.

Normal-phase high-performance liquid chromatographic separations using ethoxynonafluorobutane as hexane alternative. II. Liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry applications with methanol gradients.

We have reported recently that high-speed normal-phase (NP) HPLC separations of a broad range of organic compounds can be performed on cyano columns using gradients of methanol in hexane-like solvent-ethoxynonafluorobutane (ENFB), available commercially. In this communication, we demonstrate that atmospheric pressure chemical ionization (APCI) in combination with mass spectrometry (MS) can be effectively used for detection in such separations. The efficiency of APCI under conditions studied has also been compared to the efficiency of traditional electrospray ionization (ESI) in combination with MS for reversed-phase (RP) HPLC of the same compounds. The compounds included in this study were steroids, benzodiazepines, and other central nervous system-active substances, nonsteroidal anti-inflammatory drugs, tricyclic antidepressants, and beta-adrenergic blocking agents. Non-polar compounds were found to respond stronger when APCI-MS technique was used, whereas APCI and ESI ionization efficiencies were comparable when polar substances were studied. The combination of normal-phase HPLC separation conditions with mass spectral detection may expand the range of LC-MS applications traditionally associated with reversed-phase HPLC and ESI-MS detection.

Dissection of two hallmarks of the open promoter complex by mutation in an RNA polymerase core subunit.

Deletion of 10 evolutionarily conserved amino acids from the beta subunit of Escherichia coli RNA polymerase leads to a mutant enzyme that is unable to efficiently hold onto DNA. Open promoter complexes formed by the mutant enzyme are in rapid equilibrium with closed complexes and, unlike the wild-type complexes, are highly sensitive to the DNA competitor heparin (Martin, E., Sagitov, V., Burova, E., Nikiforov, V., and Goldfarb, A. (1992) J. Biol. Chem. 267, 20175-20180). Here we show that despite this instability, the mutant enzyme forms partially open complexes at temperatures as low as 0 degrees C when the wild-type complex is fully closed. Thus, the two hallmarks of the open promoter complex, the stability toward a challenge with DNA competitors and the sensitivity toward low temperature, can be uncoupled by mutation and may be independent in the wild-type complex. We use the high resolution structure of Thermus aquaticus RNA polymerase core to build a functional model of promoter complex formation that accounts for the observed defects of the E. coli RNA polymerase mutants.

ATP-dependent activation of the atrial acetylcholine-induced K+ channel does not require nucleoside diphosphate kinase activity.

Prior reports by others have shown that cytoplasmically applied ATP can activate the acetylcholine-induced K+ channel in inside-out atrial membrane patches when no guanine nucleotides are present in the solution bathing the cytosolic face of the membrane. A nucleoside diphosphate kinase mechanism was proposed to explain the activation by ATP. We show in the present study that cytoplasmic adenylylimidodiphosphate mimics the activation by ATP. Unlike ATP, the activation by adenylylimidodiphosphate does not subside on washout. Although commercially available adenylylimidodiphosphate is contaminated by guanylylimidodiphosphate, the activation by adenylylimidodiphosphate still occurs after HPLC purification to remove guanine nucleotide contamination. Adenylylimidodiphosphate does not support phosphotransferase activity by nucleoside diphosphate kinase. Therefore, nucleoside diphosphate kinase activity cannot explain the activation of atrial acetylcholine-induced K+ current by ATP and adenylylimidodiphosphate. We hypothesize that the activation by millimolar concentrations of ATP is due to binding of adenine nucleotide to the guanine nucleotide binding site of the G protein(s) responsible for stimulating the acetylcholine-induced K+ current.

New approaches to chromatographic purification of bovine dopamine-beta-hydroxylase.

The use of the traditional scheme for the isolation of bovine dopamine-beta-hydroxylase (bDBH) from bovine adrenal medulla resulted in active but not pure bDBH, containing about 50% of admixtures. Immobilized metal chelate affinity chromatography on agarose modified with iminodiacetic acid residues and charged with cobalt ions was applied in the final stage to obtain more than 90% pure and active bDBH. Final purification of bDBH using step elution with 0-0.5 M methyl-D-mannoside in buffer solution from concanavalin A-Sepharose was studied. The determination of bDBH in various samples was performed using size-exclusion chromatography.

High-performance liquid chromatography of human glycoprotein hormones.

The chromatographic behavior of the glycoprotein hormones from human pituitary glands and of placental origin [thyroid-stimulating hormone, luteinizing hormone and chorionic gonadotropin (CG)] was studied. It was shown that hydrophobic interaction chromatography on a microparticulate packing and anion-exchange HPLC can be applied for the purification of these hormones. Reversed-phase HPLC on wide-pore C4-bonded silica at neutral pH can be applied for the determination of the above hormones and for the isolation of pure CG and its subunits.

[Study of the fractions and fibrinolytic activity of fungal mannan]. / Izuchenie fraktsii i fibrinoliticheskoi aktivnosti drozhzhevogo mannana.

Characteristics of the fraction composition of extracellular mannan produced by Rhodotorula rubra are presented. Various lots of the polysaccharide mainly contained two fractions similar by their chemical structures and differing in the solution relative viscosity. HPLC was used for determining the molecular weight of the samples. In the isolated fractions it differed 3-5 fold. Relationship between the fibrinolytic activity of the polysaccharide and its molecular weight was revealed. The samples of mannan with the molecular weight of 400-500 kD had the highest capacity for lowering the fibrinogen blood levels in rats. The polysaccharide with the molecular weight of less than 100 kD had practically no fibrinolytic activity.

[Determination of the molecular weight of microbial polysaccharides using high performance exclusion chromatography]. / Opredelenie molekuliarnykh mass mikrobnykh polisakharidov s pomoshch'iu vysokoéffektivnoi ékskliuzionnoi khromatografii.

An automatic method of determining the molecular weight parameters (Mw, Mn) of microbial polysaccharides such as dextran, pullulan was developed based on the use of high performance size-exclusion chromatography on the two types of columns: Zorbax PSM 60 + 300 + 1000 and SynChropack GPC 100 + 500 + 1000. The Mw and Mn values were determined for a number of domestic and foreign dextran preparations. Changes in the molecular weight of pullulan and hydroxyethylstarch resulted from acid and enzymatic hydrolysis were estimated.

[Physico-chemical and biological properties of a new plasma substitute based on hydroxyethylated starch]. / Fiziko-khimicheskie i biologicheskie svoistva novogo plazmozamenitelia na osnove oksiétilirovannogo krakhmala.

Samples of hydroxyethylstarch of M 100-160 kDa, Mn 50-60 kDa and substitution degree 0.6-0.7 were prepared and characterized. Hydroxyethylstarch was shown to be susceptible to cleavage by amylolytic enzymes. All samples of hydroxyethylstarch at 2-4% concentration were compatible with perfluorohydrocarbon emulsion "Perftoran" and exhibited high haemodynamic efficiency. The 6 and 10% solutions of hydroxyethylstarch in 0.9% aqueous sodium chloride normalized haemodynamics in massive blood losses. Hydroxyethylstarch was completely removed from blood-stream. The preparations were shown to be nontoxic.

[Analysis of protected peptides--ACTH(1-24) fragments using high performance liquid chromatography]. / Analiz zashchishchennykh peptidov--fragmentov ACTH-(1-24) metodom vysokoéffektivnoi zhidkostnoi khromatografii.

The applicability of reversed-phase ion-pair HPLC for separating the ACTH-(1-24) fragments obtained at the final stage of the synthesis has been examined. Optimization of chromatographic conditions (selection of proper packing material, eluent and concentration of ion-pairing reagent) for separation of protected peptides has been performed. A mixture of the ACTH fragments 1-4, 5-12, 13-24, 5-24, and 1-24 has been resolved on a Zorbax C8 column using methanol - 0,01 M tetrabutylammonium bromide (94:6) as mobile phase.