Evidence for a novel pentyl radical adduct of the cyclic nitrone spin trap MDL 101,002.

3,4-Dihydro-3,3-dimethyl-isoquinoline-2-oxide (MDL 101,002) is a conformationally constrained cyclic analog of the known spin trap alpha-phenyl N-tert-butyl nitrone (PBN). Because of PBN's ability to scavenge free radicals, MDL 101,002 is currently being evaluated in stroke models as a means to ameliorate the oxidative insult associated with reperfusion injury. To augment our understanding of the radical scavenging mechanism of this potential drug, MDL 101,002 was incubated with soybean lipoxygenase in the presence of linoleic acid to study the interaction between MDL 101,002 and free radicals formed during lipid peroxidation. Analysis of the reaction mixture was performed by high performance liquid chromatography using normal phase conditions with detection by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Similar to the work by Iwahashi et al. [Arch. Biochem. Biophys., 1991, 285, 172], who studied the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (4-POBN), an adduct that suggested the trapping of pentyl radicals by MDL 101,002 was observed. However, the apparent molecular ion for this adduct (246 Da) was 1 Da lower than would be predicted if a pentyl radical had simply added to MDL 101,002. In addition, the adduct exhibited significant absorbance at 304 nm, consistent with the unsaturated nitrone structure of MDL 101,002. To account for these observations, it is postulated that, after the initial capture of a pentyl radical, subsequent abstraction of a hydrogen atom by a neighboring radical occurs to regenerate a nitrone (1-pentyl analog of MDL 101,002). We present evidence for this adduct and offer a mechanism for its formation. These findings indicate that mass spectroscopic analysis of stable nitrone radical adducts may be useful in the identification of radical-dependent damage in vivo and possibly in clinical development of MDL 101,002 as an antioxidant pharmaceutical.

Spectral analysis and structural identification of a major deflazacort metabolite in man.

1. The structure of a previously reported but uncharacterized major metabolite of deflazacort in man, designated V, has been characterized by nmr, MS and IR spectral techniques. 2. The major changes in V relative to deflazacort are deacetylation to form the 21-alcohol and A-ring modification to the 1,2-epoxy-3-hydroxy analogue. 3. Based on the spectral data and comparison with model compounds the structure, including relative stereochemistry, is (1 beta, 2 beta, 3 beta, 11 beta, 16 beta,)-1,2-epoxy-3,11,21-trihydroxy-2'-methyl-5H'-pregn-4-4-eno [17,16-d]oxazol-20-one.

A simple method for the isolation of zymosterol from a sterol mutant of Saccharomyces cerevisiae.

A simple method is described for the direct isolation of zymosterol (5 alpha-cholesta-8,24-dien-3 beta-ol) of high purity from a sterol mutant of Saccharomyces cerevisiae. This yeast strain, which is a double mutant of the ERG6 (sterol transmethylase) and ERG2 (C-8 sterol isomerase) genes, accumulates zymosterol as its major sterol component.

Evidence for a lysine-specific fragmentation in fast-atom bombardment mass spectra of peptides.

A fragmentation process observed for peptides that contain lysine, or other amino acids which possess a free amino group on their sidechain, is reported. The ions generated by this process are found 16 Da below the acylium-type B ions that result from fragmentation at the C-terminal side of lysine or other amine-containing residues in fast-atom bombardment (FAB) mass spectra. These ions, which are referred to as (B-16) ions, permit differentiation between the isobaric amino acids lysine and glutamine in peptide mass spectra. High resolution measurements indicate that (B-16) ions differ in composition from the corresponding B ions by the removal of one oxygen atom. Formation is believed to occur through a cyclization process initiated by nucleophilic attack by the free amino group of the lysine sidechain at the carbon of the acylium ion (B ion). A similar process initiated directly from the protonated peptide may also occur. Analogous cyclization processes are restricted for glutamine because this residue is comparatively less nucleophilic than lysine (i.e., amide vs amine). Although (B-16) ions have been detected under high energy collisionally induced dissociation, they are formed less readily than by FAB mass spectrometry. A mechanism consistent with this observation as well as other experimental evidence is presented to account for the formation of (B-16) ions.

Cloning and disruption of the yeast C-8 sterol isomerase gene.

The yeast ERG2 gene codes for the C-8 sterol isomerase, an enzyme required for the isomerization of the delta 8 double bond to the delta 7 position in ergosterol biosynthesis. The ERG2 gene was cloned by complementation of a C-8 sterol isomerase mutant strain (erg2). The complementing region of DNA required to restore ergosterol synthesis to erg2 was limited to a 1.0 kb StuI-BglII fragment. In order to determine whether the ERG2 gene was essential for yeast viability, a LEU2 gene was inserted into the NdeI site (made blunt) of this 1.0 kb fragment. Transformation of a wild type diploid strain with the ERG2 substituted DNA resulted in the generation of viable haploids containing the erg2 null allele (erg2-4::Leu2). These results suggest that the C-8 sterol isomerase activity is not essential for yeast cell viability. This disruption represents the second ergosterol biosynthetic gene in the distal portion of the pathway to be disrupted without adversely affecting cell viability.

Cloning, disruption and sequence of the gene encoding yeast C-5 sterol desaturase.

The ERG3 gene from Saccharomyces cerevisiae has been cloned by complementation of an erg3-2 mutation. ERG3 is the putative gene encoding the C-5 sterol desaturase required for ergosterol biosynthesis. The functional gene has been localized on a 2.5-kb HindIII-BamHI fragment containing an open reading frame comprising 365 amino acids. Gene disruption resulting from a deletion/substitution demonstrates that ERG3 is not essential for cell viability or the sparking function.

The use of thermospray liquid chromatography/tandem mass spectrometry for the class identification and structural verification of phytoestrogens in soy protein preparations.

The thermospray mass spectra of the phytoestrogens have intense protonated molecular ions but contain few or no ions indicative of structure. Tandem mass spectrometry (MS/MS) was used to obtain daughter ion spectra containing ions unique to the different structural characteristics of each phytoestrogen subclass and was used both to confirm identification and propose structures for unknowns. In addition to unique daughter ion spectra, MS/MS was used as a class identifier to detect phytoestrogens through the neutral loss of 56 (due to consecutive losses of CO) that is common to all members of this family. Several sources of soy protein were investigated to confirm the presence or absence of phytoestrogens. In one preparation investigated, daidzein and genistein were detected as well as an unknown phytoestrogen of the Biochanin A subclass. This unknown has been tentatively identified as 6,7-dihydroxy-4'-methoxyisoflavone using its daughter ion spectrum.

Characterization of a cytochrome P450 deficient mutant of Candida albicans.

A previously described Candida albicans nystatin resistant mutant blocked in 14 alpha-demethylation of lanosterol was shown to also lack all traces of cytochrome P450 as determined by carbon monoxide difference spectra. This strain does not require ergosterol for growth and reverted to an ergosterol producing, cytochrome P450 containing strain indicating no other lesions. Cytochrome P450 mutants described in Saccharomyces cerevisiae are auxotrophic for ergosterol or contain a second mutation in 5,6 desaturation of the sterol B ring. These results suggest that a cytochrome P450 lesion in these yeasts have different phenotypes and may reflect different sterol requirements for the two organisms.

Identification in man of urinary metabolites of a new bronchodilator, MDL 257, using triple stage quadrupole mass spectrometry-mass spectrometry.

The structure elucidation of drug metabolites directly from urine by tandem mass spectrometry (MS/MS) for a new bronchodilator is described. When urine samples from human subjects dosed with 400 mg of MDL 257 were examined by MS/MS, three major urinary metabolites previously characterized in animal studies were confirmed and two previously unsuspected metabolites were identified. Using the operational modes of a triple stage quadrupole mass spectrometer, it is possible both to detect and to identify possible metabolites. Since the pure drug and its metabolites often contain common structural daughter ions, the parent spectra of these common daughter ions should contain some or all of the molecular ions of possible metabolites. Daughter spectra of these suspected molecular ions were obtained and the resulting daughter spectra were interpreted for structural information of suspected metabolites. This study confirms the utility of MS/MS to do rapid metabolic profiling and identification directly from complex samples such as urine, with minimal time for sample preparation and analysis. This technique can provide unique and complimentary data when combined with the more classical approaches such as HPLC profiling, isolation, and off-line spectroscopy.