Determination of 2-hydroxypropyl-gamma-cyclodextrin in plasma of cynomolgus monkeys after oral administration by gas chromatography-mass spectrometry.

This report describes a specific and highly sensitive gas chromatography-mass spectrometry (GC-MS) assay for the analysis of industrially produced 2-hydroxypropyl-gamma-cyclodextrin, a heterogeneous mixture of homologues and isomers, in plasma of cynomolgus monkeys. Instead of analyzing the polysaccharide mixture as a whole, in a first step the HP-gamma-cyclodextrin mixture, together with the internal standard (2,6-di-O-methyl-beta-cyclodextrin), was deuteromethylated, and in a second step hydrolyzed with hydrochloric acid to the respective monosaccharides. The resulting reaction mixture was trimethylsilylated to 1,4-bis(O-trimethylsilyl)-2,3-bis-O-deuteromethyl-6-O-2'-deuter omethoxypropylglucose (representative for HP-gamma-CD) and 1,4-bis-(O-trimethylsilyl)-bis-2,6-O-methyl-3-O-deuteromethylglucose+ ++ (representative for the internal standard), respectively, and analyzed by GC-MS. The limit of quantification of this assay was 20 nmol/l.

Primary structure and function of novel O-glycosylated hirudins from the leech Hirudinaria manillensis.

Hirudin from the leech Hirudo medicinalis is a most powerful anticoagulant, and many isoforms have been described. In the present work, the primary structure of two hirudins from the leech Hirudinaria manillensis has been elucidated. The antithrombotic activity is similar to that of H. medicinalis hirudins although the sequence identity is below 60%. Surprisingly, the hirudins were found to be glycosylated at one site. Sugar analysis after methanolysis yielded fucose, galactose, and N-acetylgalactosamine. These results combined with data from matrix-assisted laser desorption ionization mass spectrometry, plasma desorption mass spectrometry, capillary zone electrophoresis, and lectin-binding tests indicate that the sequence is Fuc-Gal beta 1-3GalNAc-(O-threonine). This structure shows an interesting similarity to human blood group H determinants.

Separation, purification, and sequence identification of TGF-beta 1 and TGF-beta 2 from bovine milk.

Cox and Bürk (Eur. J. Biochem., 1991) reported the partial characterization of Milk Growth Factor (MGF) which stimulated the migration of fibroblasts. We have fractionated the partially purified sample by RP-HPLC and obtained the separation of two peaks of activity. The two active components were isolated as pure MGF-a and MGF-b by RP-HPLC and preparative SDS-PAGE. The purified MGF-a, consisting of a single band by gel electrophoresis and a single peak on an HPLC reversed-phase C-4 column, has the same specific activity as TGF-beta 2 in the fibroblast migration assay. MGF-a was digested by endoprotease Asp-N and the cleaved peptides were analyzed by Edman degradation and plasma desorption mass spectrometry (PDMS). The whole sequence of MGF-a determined by automated sequenator and PDMS of S-pyridylethylated protein and selected fragments was found to be identical to that of TGF-beta 2. MGF-b protein mixture separated by SDS-PAGE was electrophoretically transferred onto a Biometra Glassybond membrane, and the blotted MGF-b protein was directly sequenced on an automated sequenator. The identified 29 amino acids sequence of MGF-b was identical to the amino-terminal sequence of TGF-beta 1. Our study demonstrates that MGF is composed of both TGF-beta 1 and TGF-beta 2. TGF-beta 2 (85%) is the predominant form.

Duramycins B and C, two new lanthionine containing antibiotics as inhibitors of phospholipase A2. Structural revision of duramycin and cinnamycin.

Duramycins B and C, two new lanthionine containing antibiotics, have been isolated from Streptoverticillium strain R2075 and Streptomyces griseoluteus (R2107). The known antibiotics duramycin and cinnamycin were reisolated from Streptoverticillium hachijoense (DSM 40114) and Streptomyces longisporoflavus (DSM 40165). The structures of these latter two compounds should be revised by changing amino acid residue 3 to glutamine and 17 to asparagine, respectively. Cinnamycin therefore seems to be identical to Ro 09-0198. Leucopeptin has been shown to be identical to duramycin. Physico-chemical data of these compounds provide evidence for a similar structure for all duramycin antibiotics. All compounds of this group inhibit human phospholipase A2 at a concentration of 10(-6) molar.

Pharmacokinetics and tissue distribution of the renin inhibitor N-(2-(R)-benzyl-3-tert-butyl-sulfonyl-propionyl)-His-ChacVal-n-butylami ne in marmosets.

The fate of CGP 38 560 [N-(2-(R)-benzyl-3-tert-butyl-sulfonyl-propionyl)-His-ChacVal-n-bu tylamine], a potent renin inhibitor, has been studied in marmosets. [3H]CGP 38 560 is rapidly cleared from the plasma. The elimination process is biphasic, with a t1/2 of 4.8 +/- 1.0 min (mean +/- SD) in the first phase and 26.6 +/- 8.4 min (mean +/- SD) in the second. The kinetics of elimination from plasma are similar when measured both in a radio-inhibitor binding assay and radiometrically using 3H-labeled substance. The drug is mainly eliminated in the bile, almost 73.8% of the i.v. administered dose being excreted within the first 60 min. It is detectable in bile in both unchanged (8.6%) and metabolized form. HPLC analysis of bile extracts showed at least five tritiated peaks representing constituents capable of binding human renin (A, B, C, D, and E). These fractions were isolated, purified, and analyzed by mass spectrometry. Peak E corresponded to unchanged CGP 38 560. Metabolites A, B, C, and D are more polar than the parent compound, as indicated by their retention times upon HPLC analysis. The metabolic pathways inferable from the respective molecular weights are hydroxylation, oxygenation, and, in one case, cleavage of the n-butylamino group located at the COOH-terminal. From comparisons of the pharmacokinetic parameters after iv (0.1 mg/kg) and oral (10 mg/kg) administration, it can be estimated that the bioavailability of CGP 38 560 in the marmoset is 0.3%.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and purification of novel hirudins from the leech Hirudinaria manillensis by high-performance liquid chromatography.

The isolation and purification of novel hirudins from a crude extract of the leech Hirudinaria manillensis and their analytical characterization are reported. Initial purification by gel permeation chromatography on Sephadex G50 and anion-exchange chromatography on Q Sepharose fast-flow removed most contaminants and yielded a highly active extract. Two isohirudins (designated hirudin P6 and P18) were isolated and purified by successive reversed-phase high-performance liquid chromatography on silica-based stationary phases and anion-exchange chromatography on Mono Q. The final products were characterized by reversed-phase high-performance liquid chromatography, 252Cf plasma desorption time-of-flight mass spectrometry and capillary zone electrophoresis. The molecular masses determined by 252Cf plasma desorption mass spectrometry were 7416 dalton for hirudin P6 and 7199 dalton for hirudin P18.

1H-NMR and mass spectrometric studies of tetrahydropterins. Evidence for the structure of 6-pyruvoyl tetrahydropterin, an intermediate in the biosynthesis of tetrahydrobiopterin.

The conversion of dihydroneopterin triphosphate in the presence of 6-pyruvoyl tetrahydropterin synthase was followed by 1H-NMR spectroscopy. The interpretation of the spectra of the product is unequivocal: they show formation of a tetrahydropterin system carrying a stereospecifically oriented substituent at the asymmetric C(6) atom. The spectra are compatible with formation of a (3')-CH3 function, and with complete removal of the 1' and 2' hydrogens of dihydroneopterin triphosphate. The fast-atom-bombardment/mass spectrometry study of the same product yields a [M + H]+ ion at m/z 238 compatible with the structure of 6-pyruvoyl tetrahydropterin. The data support the proposed structure of 6-pyruvoyl tetrahydropterin as a key intermediate in the biosynthesis of tetrahydrobiopterin.

Location of disulphide bonds in human insulin-like growth factors (IGFs) synthesized by recombinant DNA technology.

Natural human insulin-like growth factor (IGF) I is a relatively large single chain peptide (mol. wt 7649) with a known sequence of 70 amino acids. C6----C48, C47----C52 and C18----C61 assignments have been previously proposed for the three disulphide bonds linking six cysteine residues (C6, C18, C47, C48, C52 and C61), on the basis of analogy (and homology) with proinsulin. In this work, IGF I synthesized by recombinant DNA technology (r-IGF I, with identical biological activity and chromatographic behaviour) was subjected to a three-step mass spectrometric analysis in combination with degradation methods for structural verification. Firstly, the correct molecular weight of the intact peptide was determined by high-mass fast atom bombardment (FAB) analysis. Secondly, twofold enzymatic degradation (chymotrypsin followed by V8 protease, 'FAB mapping' of the cleavage products) was employed in order that fragments with 'isolated' S-S bonds would be produced which allow an unambiguous assignment. This immediately established the C18----C61 linkage as it was contained in a singly bridged two-chain peptide. The two other S-S bonds, which cross-link C6 and the 'tight' C47 to C52 segment, remained 'unresolved' within a more complex, doubly bridged triple-chain peptide. Thirdly, further degradation of this structural block, in which cleavage of the C47-C48 bond was required to discern these bonds, was carried out by using FAB tandem mass spectrometry and (for additional corroboration) manual Edman degradation. Both procedures confirmed the original C6----C48/C47----C52 prediction.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct stereochemical assignment of sugar subunits in naturally occurring glycosides by low energy collision induced dissociation. Application to papulacandin antibiotics.

A tandem mass spectrometric method is described which allows the assignment of stereochemistry to fragment ions comprising intact sugar subunits of larger glycosides without chemical degradation and product isolation by chromatography. The approach relies on the mass selection of the 'sugar ion' of interest followed by analysis of stereoselective fragmentation induced by low-energy collisional activation. The daughter ion spectra provide configurational fingerprints of the selected sugar ions which can be matched for identity with reference spectra obtained from suitable precursors of known stereochemistry. Glucose, mannose and galactose furnished the required set of the most important reference ions. By using peracetyl (and perdeuterioacetyl) derivatives, galactose was readily identified as the glycosidic sugar constituent of the (known) antibiotic papulacandin B and a further (unknown) congener.