Synthesis, ocular effects, and nitric oxide donation of imidazole amidoximes.

Novel 1-R-imidazole-5-amidoximes and 1-R-5-cyano-imidazole-4-amidoximes (R: H, Me, Bn) were prepared from their corresponding nitriles and were tested for their efficacy to lower intraocular pressure (IOP) in rabbits. The ability of these compounds to donate nitric oxide (NO) was studied by observing the stimulation of formation of cyclic guanosine-3',5'-monophosphate (cGMP) in the incubation of porcine iris-ciliary body. In the incubation experiments, 1-methylimidazole-5-amidoxime and 1(H)-imidazole-4(5)-amidoxime stimulated formation of cGMP indicating NO donating ability of these compounds. 1-Methylimidazole-5-amidoxime lowered IOP significantly after intravitreal injection.

An electrospray ionization Fourier transform ion cyclotron resonance mass spectrometric study of the gas-phase stabilities and fragmentation of N-alkylammonium resorcarenes.

Five N-alkylated ammonium resorcarene derivatives, differing either in the nitrogen atom or in the lower rim alkyl chain, were studied using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. The main purpose was to clarify the fragmentation and the relative stabilities of the protonated molecules by collision-induced dissociation (CID) experiments. The results indicate that fragmentation of the upper rim alkyl amino substituents at the 2-position occurs readily. Variation in the gas-phase stabilities of these molecules was also observed. In addition, the proposed fragmentation pathways are depicted.

Mass spectrometric studies on small open-chain piperazine-containing ligands and their transition metal complexes.

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry was used to characterize the complexes formed between open-chain piperazine-containing ligands and transition metal salts (Cobalt, Copper, Zinc, and Cadmium as chlorides, nitrates, and acetates). Only single-charged complexes were observed, formed of one ligand (L) and mainly one metal ion (M). Since the net charge of the complexes was one, a counterion (X) was attached to some of the complexes, with formation of [L + M + X]+ complexes, and a proton was lost from others, as in [L - H + M]+ complexes. In most cases the composition of the complexes was more dependent on the ligand than the metal salt. Collision-induced dissociation measurements showed that complexes with related composition often differed in structure, or that interactions between the ligand and the metal ion were not alike. The metal ion influenced considerably the fragmentation pathways of the ligands, so that the fragmentation products could be used to deduce the binding sites of the metal. The variations observed in fragmentation behavior of complexes possessing the same ligand but different metal ions can mostly be explained by the ionic radius and electronic configuration of the metal ion. The results indicated a preference of the piperazine ring of the coordinated ligand for the boat conformation.

Mass spectrometric studies on pyridine-piperazine-containing ligands and their complexes with transition metals formed in solution.

Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) methods were used to study open-chain piperazine-containing ligands (L) and their complexes formed with transition-metal salts. ESI and MALDI measurements were performed with a Fourier transform ion cyclotron resonance (FT-ICR) and a time-of-flight (TOF) mass spectrometer, respectively. Only singly charged complexes, between one ligand and one or several metal ions, were formed in the ESI measurements. Because the net charge was always one, one or several counterions were attached to the complex. Under ESI conditions, the complexes formed between the ligands and metal (Co, Ni, Cu, and Cd) salts were [L + M + X](+), [L + H + M + X(2)](+) and [L + M(2) + X(3)](+) (M = metal ion, X = counterion). In collision induced dissociation reactions the [L + H + M + X(2)](+) complexes easily eliminated one proton and one counterion. Fragmentation pathways were more dependent on the metal ion than the ligand, and elimination of the second counterion occurred with one proton from copper and nickel complexes and with one proton and one hydrogen from cobalt complexes. Differences in the fragmentation of the complexes could be due to electronic configuration of the metal ion. In the MALDI measurements the ratio between the [L + H](+) and [L - H](+) ions varied with the matrix. Fragmentation of the ligands through elimination of 2-methylpyridine end groups occurred with the aromatic matrices containing carboxylic acid and hydroxyl substituents. Ionization of the complexes was not successful with MALDI as the matrix molecules were also attached to the complexes.

Thermostability of endo-1,4-beta-xylanase II from Trichoderma reesei studied by electrospray ionization Fourier-transform ion cyclotron resonance MS, hydrogen/deuterium-exchange reactions and dynamic light scattering.

Endo-1,4-beta-xylanase II (XYNII) from Trichoderma reesei is a 21 kDa enzyme that catalyses the hydrolysis of xylan, the major plant hemicellulose. It has various applications in the paper, food and feed industries. Previous thermostability studies have revealed a significant decrease in enzymic activity of the protein at elevated temperatures in citrate buffer [Tenkanen, Puls and Poutanen (1992) Enzyme Microb. Technol. 14, 566-574]. Here, thermostability of XYNII was investigated using both conventional and nanoelectrospray ionization Fourier-transform ion cyclotron resonance MS and hydrogen/deuterium (H/D)-exchange reactions. In addition, dynamic light scattering (DLS) was used as a comparative method to observe possible changes in both tertiary and quaternary structures of the protein. We observed a significant irreversible conformational change and dimerization when the protein was exposed to heat. H/D exchange revealed two distinct monomeric protein populations in a narrow transition temperature region. The conformational change in both the water and buffered solutions occurred in the same temperature region where enzymic-activity loss had previously been observed. Approx. 10-30% of the protein was specifically dimerized when exposed to the heat treatment. However, adding methanol to the solution markedly lowered the transition temperature of conformational change as well as increased the dimerization up to 90%. DLS studies in water confirmed the change in conformation observed by electrospray ionization MS. We propose that the conformational change is responsible for the loss of enzymic activity at temperatures over 50 degrees C and that the functioning of the active site in the enzyme is unfeasible in a new, more labile solution conformation.

Electrospray mass and tandem mass spectrometry identification of ozone oxidation products of amino acids and small peptides.

Aqueous ozonation of the 22 most common amino acids and some small peptides were studied by electrospray mass (ESI-MS) and tandem mass spectrometry. After 5 min of ozonation only His, Met, Trp, and Tyr form oxidation products clearly detectable by ESI-MS. For His, the main oxidation product is formed by the addition of three oxygen atoms, His + 30; for Met and Tyr by the addition of one oxygen atom, Met + O and Tyr + O, and for Trp by the addition of two oxygen atoms, Trp + 20. Ozone oxidation occurs rapidly, products are already detected after 30 s of ozonation, and the reactivity order is Met > Trp > Tyr > His. The structures of the oxygen addition products were investigated by electrospray product ion mass spectra, and by comparing these spectra to those of protonated intact amino acids, and when available, to those of model compounds. His + 30 was assigned as 2-amino-4-oxo-4-(3-formylureido)butanoic acid (1) formed by oxidation of the His imidazole ring, Met + O as methionine sulfoxide (2), Trp + 20 as N-formylkynurenine (4), and Tyr + O as a mixture of dihydroxyphenylalanines (7 and 8). Ozonation of peptides show that the same number of oxygen atoms are added as expected from the ozonation of the free amino acids. The product ion mass spectra of both the protonated intact peptides, MH+, and the main ozonation products (M + nO)H+ (n = 1-3) revealed b and y type ions as the main fragments, which allow one to assign the type and location of modified amino acid in the model peptides.

Qualitative and quantitative end-group analysis of a small molecular weight polyester by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used for qualitative and quantitative end-group analysis of a small molecular weight polyester, poly(2-butyl-2-ethyl-1,3-propylene phthalate). The presence of carboxyl-terminated linear and cyclic polyester oligomers was confirmed with the help of simple sample preparation methods. The presence of carboxyl end-groups in the polyester chains was verified through their formation of carboxylate salts with alkali metal cations. Cyclic oligomers were identified through deuterium exchange of the exchangeable protons of the polyester. Various inorganic salts were tested for salt formation of the carboxyl end-groups, but only the alkali metal salts proved effective. The influence of the alkali metal salts on the results of the quantitative end-group analysis was also studied. The relative amounts of differently terminated and cyclic oligomers were calculated when the alkali metal salts were used with different matrices. The results showed that both the salts and the matrices used in sample preparation can have a marked effect on the quantitative results of the end-group analysis. The measurements were carried out using 2,5-dihydroxybenzoic acid (DHB), 1,8, 9-trihydroxyanthracene (dithranol), and 2-(4-hydroxyphenylazo)benzoic acid (HABA) as matrix compounds. Dithranol and HABA repeatably exhibited similar results, and these results differed from those obtained with DHB probably because of the different ionization mechanisms in the MALDI process.

Ion-molecule reactions of 2-methoxyethanol with some amines, carboxylic acids and amino acids under chemical ionization conditions

2-Methoxyethanol chemical ionization of amines, carboxylic acids and amino acids has been found to produce numerous adduct ions. The most intense adduct ions for amines are [M + H](+) and [M + 77](+), for carboxylic acids [M + 27](+), [M + 59](+) and [M + 77](+), and for amino acids [M + H](+), [M + 13](+), [M + 27](+) and [M + 77](+). Either the adduct ion [M + H](+) or [M + 77](+) was the most abundant ion found for amino acids. The proton affinities of amino acids are noticed to control the formation of the [M + H](+) and [M + 77](+) ions. The relative abundance of [M + 13](+) and [M + 27](+) ions varied for different amino acids being most intense for phenylalanine and aspartic acid. Copyright 1999 John Wiley & Sons, Ltd.

Unusual fatty acids in the depot fat of the Canadian beaver (Castor canadensis).

Unusual fatty acids in the adipose tissue of the Canadian beaver were analysed by argentation thin-layer chromatography, gas-liquid chromatography and gas chromatography-mass spectrometry of several adducts and spectroscopic measurements. The total proportion of trans-octadecenoic fatty acids and the distribution of double bonds in these acids resembled those of ruminant fats. Because trans-11-18:1 was the main trans-18:1 isomer of beaver fat and a conjugated diene cis-9, trans-11-18:2 was also detected, it can be deduced that linoleic acid (cis-9, cis-12-18:2) is biohydrogenated in the beaver. The ideas that microbial processes intervene before normal absorption and fatty acid metabolism of the beaver is also supported by the larger amounts of anteiso odd-chain fatty acids with respect to the corresponding iso acids in the beaver depots. In addition, the distribution of double bonds in the cis-octadecenoic acids was wide, as it is in the depot fats of ruminants.

Methyl propionate radical cation.

Examination of the reactions of the long-lived (>0.5-s) radical cations of CD3CH2COOCH3 and CH3CH2COOCD3 indicates that the long-lived, nondecomposing methyl propionate radical cation CH3CH2C(O)OCH 3 (+·) isomerizes to its enol form CH3CH=C(OH)OCH 3 (+·) (ΔH isomerization ≃ -32 kcal/mol) via two different pathways in the gas phase in a Fourier-transform ion cyclotron resonance mass spectrometer. A 1,4-shift of a ß-hydrogen of the acid moiety to the carbonyl oxygen yields the distonic ion (·)CH2CH2C(+) (OH)OCH3 that then rearranges to CH3CH=C(OH)OCH 3 (+·) probably by consecutive 1,5- and 1,4-hydrogen shifts. This process is in competition with a 1,4-hydrogen transfer from the alcohol moiety to form another distonic ion, CH3CH2C(+)(OH)OCH 2 (·) , that can undergo a 1,4-hydrogen shift to form CH3CH=C(OH)OCH 3 (+·) . Ab initio molecular orbital calculations carried out at the UMP2/6-31G** + ZPVE level of theory show that the two distonic ions lie more than 16 kcal/mol lower in energy than CH3CH2C(O)OCH 3 (+·) . Hence, the first step of both rearrangement processes has a great driving force. The 1,4-hydrogen shift that involves the acid moiety is 3 kcal/mol more exothermic (ΔH isomerization=-16 kcal/mol) and is associated with a 4-kcal/mol lower barrier (10 kcal/mol) than the shift that involves the alcohol moiety. Indeed, experimental findings suggest that the hydrogen shift from the acid moiety is likely to be the favored channel.

Fatty acid composition in liver and blubber of the Saimaa ringed seal (Phoca hispida saimensis) compared with that of the ringed seal (Phoca hispida botnica) and grey seal (Halichoerus grypus from the Baltic.

1. The main differences in fatty acid composition of blubber between the Saimaa seal (Phoca hispida saimensis) and the Baltic seals (Phoca hispida botnica and Halichoerus grypus) were, that in fresh water the proportions of polyunsaturated C18 acids and arachidonic acid (20:4 omega 6) were high. 2. The trend became more evident when data on ringed seals from northern oceans were compared with the present data. 3. The proportions of exogenous 20:1 and 22:1 were extremely low in ringed seals from Lake Saimaa and in Baltic seals compared with seals living in oceans.

[The chemistry of immunosuppressive acting fractions of Meripilus giganteus (PERS ex. FR.) KARST., the giant spore]. / Untersuchungen zur Chemie immunsuppressiv wirksamer Fraktionen aus Meripilus giganteus (PERS. ex. FR.) KARST., dem Riesenporling.

In the methanolic extract of Meripilus giganteus a mixture of saturated and unsaturated fatty acids (among them palmitic, oleic and linoleic acid) and ergosterol peroxide were identified as immunosuppressive components. Furthermore a homologous series of straight-chained saturated hydrocarbons with 22 to 26 and 29 to 32 C-atoms could be found.

Effects of N-substitution on the fragmentations of some cyclohexene-fused 2-N-phenyliminoperhydro-3,1-oxazines and related thiazines.

Six cyclohexene-fused 2-N-phenyliminoperhydro-3,1-oxazines and four related thiazines were prepared and their mass spectrometric behavior was studied by means of metastable ion analysis and exact mass measurement. In most of the fragmentations, extensive rearrangements took place. The decompositions through the retro-Diels-Alder reaction initiated by the double bond dominated in the case of the unsubstituted compounds. The effect of the double bond, however, was greatly outweighed by the effect of the substituent on the ring nitrogen atom. In comparison with the unsubstituted compounds, both electron-releasing (methyl) and electron-withdrawing (benzyl) substituents increased the contribution of the ring cleavage reactions in the heterocyclic part of the molecule; in the case of the benzyl group the loss of the substituent also became important. For isomeric compounds, the relative peak intensities were so different that such compounds were easy to differentiate.