Aminosterols from the dogfish shark Squalus acanthias.

Seven new aminosterols related to squalamine (8) were isolated from the liver of the dogfish shark Squalus acanthias. Their structures (1-7) were determined using spectroscopic methods, including 2D NMR and HRFABMS. These aminosterols possess a relatively invariant cholestane skeleton with a trans AB ring junction, a spermidine or spermine attached equatorially at C3, and a steroidal side-chain that may be sulfated. The structure of the lone spermine conjugate, 7 (MSI-1436), was confirmed by its synthesis from (5alpha,7alpha, 24R)-7-hydroxy-3-ketocholestan-24-yl sulfate. Some members of this family of aminosterols exhibit a broad spectrum of antimicrobial activity comparable to squalamine.

Oxidation of the N-terminal gly-residue of peptides: stress study of pexiganan acetate in a drug formulation.

PURPOSE: The purpose of this study was to identify four major degradation products, which were formed during a stress study of pexiganan (a 22-mer peptide) in a 1% formulation. METHODS: The degradation products were isolated and characterized by LC/MS, tryptic and aminopeptidase digests. RESULTS: One of the degradation products was shown to be des-glyl-pexiganan. The other three are structural isomers of N-glyoxylyl-desgly1-pexiganan. These isomers undergo reversible inter-conversions, as well as decompose irreversibly to des-gly1-pexiganan. Thus, all the impurities were formed from a single oxidation product of pexiganan, N-glyoxylyl-des-gly1-pexiganan. The aldehyde group of the glyoxylyl residue and the NH-amide of the adjacent isoleucine residue form a piperazinedione derivative of des-gly1-pexiganan. This heterocyclic compound rearranges to other tautomers or back to the N-glyoxylyl compound (see Fig. 3). Tryptic digests of the three degradation products showed that their N-terminal segment produced N-glyoxylyl-I-G-K whereas the N-terminal segment of pexiganan produced G-I-G-K. All the other tryptic-digest segments were identical to those formed in pexiganan. The LC/MS of the N-terminal segment and of synthetic N-glyoxylyl-I-G-K were identical. The enzymatic resistance of the three impurities to undergo aminopeptidase-M cleavage further supported the conclusion that their N-terminal amino residues are substituted. CONCLUSIONS: After a year under stress conditions 1% pexiganan cream lost about 15% of the active component to oxidative-deamination, where the N-terminal glycine residue was oxidized to N-glyoxylyl-desgly1-pexiganan. The other nine epsilon-amino lysine-residues of the peptide stayed intact. This oxidation product inter-converted and formed two additional impurities, tautomers of piperazinedionyl-des-gly-pexiganan, and decomposed to des-gly1-pexiganan, the forth impurity.

Mechanistic aspects of chiral discrimination on modified cellulose.

Cellulose and cellulose derivatives are biopolymers which are often used as stationary phases for the separation of enantiomers. Describing the mechanism of such separations is a difficult task due to the complexity of these phases. In the present study, we attempt to elucidate the types of interactions occurring between a diol intermediate for a LTD(4) antagonist and a tris(4-methylbenzoate)-derivatized cellulose stationary phase. Thermodynamic studies indicate that, at low temperatures, the enantioselectivity is entropy driven. At higher temperatures, the separation is enthalpy driven. DSC and IR experiments reveal that the transitions between the enthalpic and the entropic regions of the van't Hoff plots are a result of a change in conformation of the stationary phase. Investigation of chromatographic kinetic parameters reveals that, at low temperature, the second eluted enantiomer undergoes sluggish inclusion interactions. Subtle changes in the structure of the analyte indicates that π-π interactions do not contribute to enantioselectivity. Finally, molecular modeling of (R)- and (S)-diol and the stationary phase suggests that hydrogen bonding is a primary factor in the separation, and the calculated energy values obtained from the molecular modeling correlate well with the chromatographic elution order.

Automated HPLC analyses of drugs of abuse via direct injection of biological fluids followed by simultaneous solid-phase extraction and derivatization with fluorescence detection.

An automated system is described for the simultaneous extraction and derivatization of nucleophilic compounds from various biological media. The method includes the use of a solid-phase reagent containing a 9-fluorenylacetate activated ester. The reagent is based on a controlled pore, polystyrene divinylbenzene support prepared through a silica template procedure. An X-Y-Z robotic arm equipped with a needle is used in conjunction with a syringe pump for aspirating and dispensing samples and standards into the HPLC system. A precolumn cartridge containing the solid-phase reagent is put on-line in place of the fixed-volume injection loop. Injections of biological fluids such as urine or plasma with minimal sample treatment and handling are made directly into this reactor. The analytes are derivatized as they are extracted, allowing virtually unlimited sample volumes to be injected. The polymeric cartridge can be used for up to 100 injections without accruing unacceptable reductions in sensitivity. A detection limit of 500 p.p.t. (parts per trillion) of amphetamine in urine was achieved with this system.

Stabilization of reactive species within polystyrene divinylbenzene polymer networks.

The effect of cross-linking, surface area, and porous nature of modified polystyrene-divinylbenzene (STY-DVB) reagents has been investigated. The supports were prepared via two techniques and modified to contain various chemical functionalities. These reagents were used in an on-line reactor for automated derivatization of amines in HPLC. The reproducibility of the response vs the physical nature of the porous support and the chemical functionality was determined. The ability to stabilize highly reactive acylating reagents toward high concentrations of aqueous base was found to be a complex interaction of pore size distribution, percent cross-linking, surface area, and absolute loading of the analytical reagent on the porous support.

Direct determination of adamantanamine in plasma and urine with automated solid phase derivatization.

A simple, highly sensitive and selective method is described for adamantanamine determination in plasma and urine by high-performance liquid chromatography with fluorescence detection. The method involved a simultaneous extraction and derivatization of biological fluids with a 9-fluoreneacetate (9-FA) solid-phase derivatization reagent. This approach eliminated tedious sample preparation steps and provided automatic derivatization with selective and efficient sample clean-up for direct injection of biological fluids. Derivatized adamantanamine was separated under conventional reversed-phase conditions and determined by fluorescence detection. The optimization and validation of the derivatization method with the 9-FA solid-phase reagent is described.

9-Fluoreneacetyl-tagged, solid-phase reagent for derivatization in direct plasma injection.

We describe here a resin-based derivatization reagent, containing a 9-fluoreneacetyl tag on a controlled-pore substrate, for direct injection analysis of amphetamine in plasma. On-line, pre-column derivatization was performed by direction injection of diluted plasma sample into an sodium dodecyl sulfate-containing mobile phase. Amphetamine was trapped in the hydrophobic derivatization column and derivatized at elevated temperature by the activated solid-phase reagent. The derivatized 9-fluoreneacetyl amphetamide was separated by reversed-phase high-performance liquid chromatography with a step gradient and determined by fluorescence detection. The synthesis scheme, characterization, and optimization of the derivatization conditions for the solid-phase reagent are described. The method was evaluated by reproducibility tests and single blind spiking analysis. This solid-phase reagent combined with a surfactant containing mobile phase provided a sensitive and simple procedure for on-line derivatization in direct injection analysis of biological fluids.

Hydrophilic shielding of hydrophobic, cation- and anion-exchange phases for separation of small analytes: direct injection of biological fluids onto high-performance liquid chromatographic columns.

Shielded hydrophobic phases (SHPs) have an external hydrophilic network that prevents larger protein molecules from interacting with hydrophobic zones. Smaller analytes are not sterically hindered from interacting with the hydrophobic zones and are retained. This mechanism allows the separation of the proteins from the analytes of interest. SHPs have been shown to be useful for the direct-injection analysis of drugs in biological fluids. In this paper, two kinds of shielded phases are discussed: bonded micellar phases and embedded polymeric phases. For some compounds of interest, the hydrophobic retention and selectivity is insufficient to obtain the desired resolution. To increase selectivity without affecting protein exclusion, shielded phases were prepared with ion-exchange groups added to the hydrophobic zones. Such modified phases with cation-exchange and anion-exchange capabilities were examined for additional selectivities. Usage of these additional selectivities will be demonstrated to achieve better analysis and resolution for basic, acidic, and neutral compounds.

High-performance immobilized-metal affinity chromatography of proteins on iminodiacetic acid silica-based bonded phases.

High-performance separations of proteins, based on immobilized-metal affinity chromatography (HPIMAC), are described. The stationary phase consisted of iminodiacetic acid (IDA) chelate groups, bonded to small particle, wide pore silica gel by means of a polyether hydrophilic leash. After loading the column with metal, retention of proteins was achieved by protein-metal complexation at high concentrations of sodium sulfate. Elution was accomplished by addition of competitive complexing ligands, such as ammonia at constant pH, and/or by a decreasing pH gradient of a specially designed buffer system to maintain buffer capacity constant throughout the gradient. Selective separations, based on differences in the number of histidine residues present on the surface of the proteins, are described. The application of HPIMAC in the separation and purification of structurally similar proteins is presented. The potential application of IDA columns in three chromatographic modes (HPIMA, hydrophobic interaction, and cation exchange) is also described.

High-performance hydrophobic interaction chromatography: purification of rat liver carbamoylphosphate synthetase I and ornithine transcarbamoylase.

The applicability of high-performance hydrophobic interaction chromatography using newly developed silica-based ether-bonded phases is demonstrated in the purification of the rat liver enzymes carbamoylphosphate synthetase I and ornithine transcarbamoylase from crude mitochondrial extracts. As a result of the mild adsorption/elution conditions in this high-performance chromatographic mode, the enzymes are recovered in 20 min with 3- to 15-fold increases in specific activity. Since the enzymes are labile and may aggregate in solution, in one case up to Mr 330,000, this rapid purification demonstrates the potential of hydrophobic interaction chromatography in complex biological systems.

Wide-pore silica-based ether-bonded phases for separation of proteins by high-performance hydrophobic-interaction and size-exclusion chromatography.

This paper examines the use of wide-pore silica-based hydrophilic ether-bonded phases for the chromatographic separation of proteins under mild elution conditions. In particular, ether phases of the following structure identical to Si-(CH2)3-O-(CH2-CH2-O)n-R, where n = 1, 2, 3 and R = methyl, ethyl or n-butyl, have been prepared. These phases can be employed either in high-performance hydrophobic-interaction or size-exclusion chromatography, depending on mobile phase conditions. In the hydrophobic-interaction mode, a gradient of decreasing salt concentration, e.g., from 3 M ammonium sulfate (pH 6.0, 25 degrees C), yields sharp peaks with high mass recovery of active proteins. In this mode, retention can be controlled by salt type and concentration, as well as by column temperature. In the size-exclusion mode, use of medium ionic strength, e.g., 0.5 M ammonium acetate (pH 6.0) yields linear calibration of log (MW[eta]) vs. retention volume. Even at 0.05 M salt concentration, no stationary phase charge effects on protein elution are observed. These bonded-phase columns exhibit good column-to-column reproducibility and constant retention for at least five months of continual use. Examples of the high-performance separation of proteins in both modes are illustrated.

Interaction between asymmetric solutes and solvents. Diamides derived from L-valine as stationary phases in gas-liquid partition chromatography.

The resolution of enantiomers of (+/-)-a-amino acid derivatives on optically active diamides derived from L-valine, R1CONHCH[CH(CH3)2]CONHR2, as gas chromatographic stationary phases was studied. The effects of the structure of R1 and R2 in the stationary phases, the structure of the racemic a-amino acid derivatives, temperature, etc., on the separation factors are reported. In the light of these and related results, a more probable mechanism of resolution is presented.

Separation of enantiomers on packed columns containing optically active diamide phases.

Chromatography of fourteen protein amino acids has been studied on 2-4- m long packed columns containing chiral diamide phases. Twelve of the thirteen optically active compounds examined could be resolved, with R greater than or equal to 1 in many instances. Two of the phases, N-docosanoyl-L-valine tert.-butylamide and N-lauroyl-L-valine 2-methyl-2-hepatadecylamide, can be employed at temperatures as high as 190 degrees and 180 degrees, respectively, without losing their efficiency, even after prolonged use. The problem of peak overlap in the analysis of mixtures of different amino acids was examined and partially solved.