Fast atom bombardment mass spectrometry.

Fast atom bombardment mass spectrometry has become a powerful structural tool since the first reports of its use in 1981. Samples are ionized in the condensed state, usually in a glycerol matrix, by bombarding the matrix with xenon or argon atoms with energies of 5000 to 10,000 electron volts. This yields both positive and negative secondary ions, which are sputtered from the surface. The technique has been used to detect inorganic ion clusters to mass 25,800 and biologically active peptides to mass 5700, and it gives molecular ions of such highly polar or labile organic compounds as glycosphingolipids and polyene antibiotics. It can be especially valuable in determining the sequences of amino acids in polypeptides.

Didemnins: antiviral and antitumor depsipeptides from a caribbean tunicate.

Extracts of samples of a Caribbean tunicate (ascidian, sea squirt) of the family Didemnidae inhibit in vitro at low concentrations the growth of DNA and RNA viruses as well as L1210 leukemic cells. The active compounds isolated from the tunicate, didemnins A, B, and C, are depsipeptides, and didemnin B (a derivative of didemnin A) is the component active at the lowest concentration in inhibiting viral replication in vitro and P388 leukemia in vivo.

Effects of streptovaricins and their degradation products on RNA-directed DNA polymerase of Rauscher leukemia virus.

The activities of streptovaricin complexes, streptovaricins, streptovals, and streptovarinic degradation products were elevated against RNA-directed DNA polymerases of Rauscher leukemia virus, DNA-dependent DNA polymerase of bacterial and mammalian cells, and DNA-dependent RNA polymerases of mammalian origin. The activities of streptovaricins were also listed for comparison purposes. The effects of streptovaricin complexes on viral DNA polymerases varied significantly from lot to lot, and streptovaricin complex lot 7 was the most active. All the streptovals and streptovaricin degradation products except varicinal A showed a marked improvement (twofold to tenfold) in activity against the viral enzyme over the parent streptovaricins. None of these compounds, however, displayed any significant effect on either the DNA polymerase of L1210 leukemia cells and Escherichia coli or the RNA polymerase of isolated nuclei of mouse liver. As a result of tests in these systems, some specific inhibitors of RNA-directed DNA polymerases of Rauscher leukemia virus were selected.

Effects of streptovaricins and their degradation products on infectivity of Rauscher leukemia virus.

The virucidal effects of streptovaricin (Sv) A, SvC, SvD, streptoval (Sval) C, Sval Fc, and streptovarone were evaluated by incubation of the drug with Rauscher leukemia virus (RLV) at 37 degrees C for 60 minutes prior to dillution and addition to cells (in vitro assay) or before ip injection into animals (in vivo assay). The in vitro and in vivo assays were plaque formation and splenomegaly, respectively. A dose-related effect was observed with all six compounds with the in vitro assay. On an equimolar basis, the Sv degradation products, i.e., Sval C, Sval Fc, and streptovarone were most inhibitory, followed by SvD; SvA and SvC were least active. At 0.0625 mumoles, the three Sv degradation products inactivated over 90% of the RLV. Similar results were obtained through the in vivo assay. At 0.06 mumoles, streptovarone, Sval C, and SvD showed 78,62, and 29% inhibition of splenomegaly, respectively; SvA and SvC were essentially inactive. A direct relationship was observed between inhibition on RNA-directed DNA polymrase of RLV by these compounds and their virucidal effects. No drug given at the time of injection, however, showed any significant effect on virus infective processes in vitro or in vivo. The reason for the lack of therapeutic effects of these compounds is discussed.

Structure--activity relationships of the didemnins.

Bioactivities of 42 didemnin congeners, either isolated from the marine tunicates Trididemnun solidum and Aplidium albicans or prepared synthetically and semisynthetically, have been compared. The growth inhibition of various murine and human tumor cells and plaque reduction of HSV-1 and VSV grown on cultured mammalian cells were used to assess cytotoxicity and antiviral activity. Biochemical assays for macromolecular synthesis (protein, DNA, and RNA) and enzyme inhibition (dihydrofolate reductase, thymidylate synthase, DNA polymerase, RNA polymerase, and topoisomerases I and II) were also performed to specify the mechanisms of action of each analogue. Immunosuppressive activity of the didemnins was determined using a mixed lymphocyte reaction (MLR) assay. These assays revealed that the native cyclic depsipeptide core is an essential structural requirement for most of the bioactivites of the didemnins, especially for cytotoxicities and antiviral activities. The linear side-chain portion of the peptide can be altered with a gain, in some cases, of bioactivities. In particular, dehydrodidemnin B, tested against several types of tumor cells and in in vivo studies in mice, as well as didemnin M, tested for the mixed lymphocyte reaction and graft vs host reaction in murine systems, showed remarkable gains in their in vitro and in vivo activities compared to didemnin B.

Analysis of an egyptian mummy resin by mass spectrometry.

Fast atom bombardment combined with mass spectrometry (FAB/MS), high resolution FAB/MS, FAB tandem mass spectrometry (MS/MS), and gas chromatography/mass spectrometry (GC/MS) were used to determine the composition of the resinous material recovered from the wrappings of an Egyptian mummy believed to be from the Roman period (100-350 A.D.). FAB/MS and MS/MS studies identified several oxidation products of abietic acid as the principal resin components, indicating that one or more species of coniferous trees were used by the Egyptians as a source of the resin. GC/MS studies also identified several n-alkanes with carbon numbers from C19 to C33 in the sample. The relative amounts of these n-alkanes, along with characteristic trace metals, indicate that bitumen, an asphalt native to the region, was added to the resin. The presence of this ancient source of carbon in this sample explains the inconsistent date assigned to the mummy by carbon-14 analysis.

Electrospray ionization mass spectrometric analysis of microcystins, cyclic heptapeptide hepatotoxins: modulation of charge states and [M + H]+ to [M + Na]+ ratio.

Electrospray ionization mass spectrometry was used to develop a rapid, sensitive, and accurate method for determination and identification of hepatotoxic microcystins, cyanobacterial cyclic heptapeptides. To optimize the electrospray ionization conditions, factors affecting charge state distribution, such as amino acid components of sample, proton affinity of the additives, and additive concentration, were investigated in detail and a method for controlling charge states was developed to provide molecular-related ions for assignment of molecular weight and reasonably abundant precursor ions for MS/MS analysis. A procedure for identification of microcystins consisting of known amino acids was proposed: for microcystins giving abundant [M + 2H]2+ ions, the addition of nitrogen-containing bases to the aqueous sample solution is effective to obtain an increased intensity of [M + H]+ ions, whereas the addition of Lewis acids containing nitrogen can produce increased abundances of [M + 2H]2+ ions for microcystins giving weak [M + 2H]2+ ions. Microcystins possessing no arginine residue always give sodium adduct ions [M + Na]+ as the base peak, and these are difficult to fragment via low energy collision-induced dissociation to yield structurally informative products; the addition of oxalic acid increases [M + H]+ ion abundances, and these fragment readily.

Human fatalities from cyanobacteria: chemical and biological evidence for cyanotoxins.

An outbreak of acute liver failure occurred at a dialysis center in Caruaru, Brazil (8 degrees 17' S, 35 degrees 58' W), 134 km from Recife, the state capital of Pernambuco. At the clinic, 116 (89%) of 131 patients experienced visual disturbances, nausea, and vomiting after routine hemodialysis treatment on 13-20 February 1996. Subsequently, 100 patients developed acute liver failure, and of these 76 died. As of December 1996, 52 of the deaths could be attributed to a common syndrome now called Caruaru syndrome. Examination of phytoplankton from the dialysis clinic's water source, analyses of the clinic's water treatment system, plus serum and liver tissue of clinic patients led to the identification of two groups of cyanobacterial toxins, the hepatotoxic cyclic peptide microcystins and the hepatotoxic alkaloid cylindrospermopsin. Comparison of victims' symptoms and pathology using animal studies of these two cyanotoxins leads us to conclude that the major contributing factor to death of the dialyses patients was intravenous exposure to microcystins, specifically microcystin-YR, -LR, and -AR. From liver concentrations and exposure volumes, it was estimated that 19.5 microg/L microcystin was in the water used for dialysis treatments. This is 19.5 times the level set as a guideline for safe drinking water supplies by the World Health Organization.

Enantiospecific synthesis of N-Boc-Adda: a linear approach.

[structure: see text] Synthesis of the unusual amino acid (2S,3S,8S, 9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid (Adda), a unit of numerous cyanobacterial toxins, is described. Construction of the target molecule was achieved in 13 steps with an overall yield of 40%. The work is highlighted by a novel one-pot transformation from isoxazolidin-5-one intermediate 6 to the final product, a step that can also be used to form beta-amino acids.

Sequence analyses of two neuropeptides of the AKH/RPCH-family from the lubber grasshopper, Romalea microptera.

Two neuropeptides with adipokinetic activity in Locusta migratoria and hypertrehalosaemic activity in Periplaneta americana were purified by high-performance liquid chromatography from the corpus cardiacum of the lubber grasshopper, Romalea microptera. The sequences of both peptides, designated Ro I and Ro II, were determined by gas-phase sequencing employing Edman degradation after the N-terminal pyroglutamate residue was enzymatically deblocked, as well as by fast atom bombardment mass spectrometry. Ro I was found to be a decapeptide with the primary structure: pGlu-Val-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2, whereas Ro II is an octapeptide with the structure: pGlu-Val-Asn-Phe-Ser-Thr-Gly-Trp-NH2. Ro II is identical with AKH-G isolated from the cricket Gryllus bimaculatus. Synthetic materials having the assigned structures were found to be chromatographically, mass spectrometrically, and biologically indistinguishable from the natural peptides, confirming the sequences and establishing the Romalea peptides as members of the AKH/RPCH-family of peptides.

Isolation of Leu-Pro-Pro-Gly-Pro-Leu-Pro-Arg-Pro-NH2 (Antho-RPamide), an N-terminally protected, biologically active neuropeptide from sea anemones.

Using a radioimmunoassay against the C-terminal sequence Arg-Pro-NH2 (RPamide), we have isolated the peptide Leu-Pro-Pro-Gly-Pro-Leu-Pro-Arg-Pro-NH2 (Antho-RPamide) from an extract of the sea anemone Anthopleura elegantissima. Antho-RPamide is located in neurons of sea anemones. Application of low concentrations of Antho-RPamide to tentacle preparations of sea anemones strongly increased the frequency and duration of spontaneous contractions, suggesting that this peptide is involved in neurotransmission. Antho-RPamide has a free N-terminus, yet its X-Pro-Pro sequence makes it relatively resistant to degradation by nonspecific aminopeptidases. Thus, we have discovered another strategy by which sea anemones protect the N-termini of their bioactive neuropeptides.

Isolation of

Using a radioimmunoassay against the C-terminal sequence Arg-Pro-NH2 (RPamide) we have isolated the neuropeptide

Isolation of two novel neuropeptides from sea anemones: the unusual, biologically active L-3-phenyllactyl-Tyr-Arg-Ile-NH2 and its des-phenyllactyl fragment Tyr-Arg-Ile-NH2.

Using a radioimmunoassay for the carboxyl-terminal sequence Arg-Val-NH2, two novel peptides were purified from extracts of the sea anemone Anthopleura elegantissima. These peptides were L-3-phenyllactyl-Tyr-Arg-Ile-NH2 (name: Antho-RIamide I) and its des-phenyllactyl fragment Tyr-Arg-Ile-NH2 (Antho-RIamide II). Immunocytochemical staining showed that these peptides were localized in neurons of sea anemones. Application of low concentrations (10(-8) M) of Antho-RIamide I inhibited spontaneous contractions in several muscle groups of sea anemones, whereas Antho-RIamide II was inactive. Antho-RIamide I is the second neuropeptide from sea anemones that bears the unusual, amino-terminal L-3-phenyllactyl blocking group. We suggest that this group renders the peptide resistant agaist degradation by nonspecific aminopeptidases. In addition, the L-3-phenyllactyl residue might also play a role in receptor binding.

Low-energy collisionally activated decomposition and structural characterization of cyclic heptapeptide microcystins by electrospray ionization mass spectrometry.

Characteristics of electrospray ionization mass spectrometry/collision-induced dissociation (ESIMS/CID) mass spectra of microcystins, cyanobacterial cyclic heptapeptide hepatoxins, were examined. The collision conditions showed remarkable effects on the quality of the CID mass spectra, which were divided into three patterns according to the number of Arg residues. A characteristic cleavage reaction and neutral losses of MeOH, NH3 and guanidine group(s) from the (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4 E,6E-dienoic acid (Adda) and Arg residues were observed in the ESI and ESIMS/CID mass spectra, suggesting the most probable protonation sites in [M + H]+ and [M + 2H]2+ ions of microcystins. Microcystins with no Arg residue showed only [M + H]+ ions with a proton reacting at the methoxyl group in the Adda residue, and the ESIMS/CID/MS data revealed their structures unambiguously. The protonation site in [M + H]+ ions of microcystins with Arg residue(s) was the guanidine group. The [M + 2H]2+ ions of microcystins possessing one Arg residue had one proton on the Arg residue and probably another proton on the Adda residue, while the [M + 2H]2+ ions of microcystins having two Arg residues showed protonation at both Arg residues and the ESIMS/CID/MS data assigned their sequences. Structures of microcystins possessing one Arg residue can be assigned by ESIMS/CID/MS of [M + H]+ ions combined with those of [M + 2H]2+ ions.

Isolation of pyroGlu-Leu-Leu-Gly-Gly-Arg-Phe-NH2 (Pol-RFamide), a novel neuropeptide from hydromedusae.

The hydromedusa Polyorchis penicillatus is a good model system to study neurotransmission in coelenterates. Using a radioimmunoassay for the peptide sequence Arg-Phe-NH2 (RFamide), two peptides have now been purified from acetic acid extracts of this medusa. The structure of one of these peptides was established as pyroGlu-Leu-Leu-Gly-Gly-Arg-Phe-NH2, and was named Pol-RFamide. This peptide belongs to the same peptide family as a recently isolated neuropeptide from sea anemones (pyroGlu-Gly-Arg-Phe-NH2). Using antisera to Pol-RFamide, the peptide was found to be exclusively localized in neurones of Polyorchis, among them neurones associated with smooth-muscle fibres. This suggests that Pol-RFamide might be a transmitter or modulator at neuromuscular junctions.

Structure determination and toxicity of a new microcystin from Microcystis aeruginosa strain 205.

A new hepatotoxic microcystin was isolated from the cyanobacterium Microcystis aeruginosa strain 205. Its structure was found to be [Dha7]microcystin-RR as determined by amino acid analysis, mass spectrometry and 1H NMR spectroscopy. LD50 value (i.p. mouse) of this toxin was 180 micrograms/kg. The 48 hr lethal concentration (48-hr-LC50) of the toxin for larvae of the yellow fever mosquito, Aedes aegypti, was 14.9 micrograms/ml.

Isolation and identification of 12 microcystins from four strains and two bloom samples of Microcystis spp.: structure of a new hepatotoxin.

Sixteen microcystins, cyclic heptapeptide hepatotoxins, were isolated and purified by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) from four hepatotoxic strains and two Microcystis spp. bloom samples originating from five different lakes in Finland. The structures of a new [Dha7]MCYST-FR and 11 known microcystins MCYST-LR, [D-Asp3]MCYST-LR, [Dha7]MCYST-LR, [D-Asp3, Dha7] MCYST-LR, MCYST-RR, [D-Asp3]MCYST-RR, [Dha7]MCYST-RR, [D-Asp3,Dha7]MCYST-RR, [L-Ser7]MCYST-RR, MCYST-YR and [Dha7] MCYST-YR were assigned based on amino acid analysis, fast atom bombardment mass spectrometry (FABMS) and tandem FABMS. Four other new compounds allowed only determination of their molecular formulas and amino acid components because of inadequate amounts obtained. [Dha7]MCYST-RR was found most frequently in these samples as the main toxin.

Two methyl ester derivatives of microcystins, cyclic heptapeptide hepatotoxins, isolated from Anabaena flos-aquae strain CYA 83/1.

Cultured cells of Anabaena flos-aquae strain CYA 83/1, isolated from Lake Edlandsvatn, Norway, produced two microcystin mono-methyl ester derivatives (1 and 2) at the D-Glu unit in addition to microcystin-LR (3), [D-Asp3]microcystin-LR (4), microcystin-RR (5), and [D-Asp3]microcystin-RR (6). Structures of these compounds were assigned based on their amino acid analysis with a Waters Pico Tag HPLC system plus fast atom bombardment mass spectrometry (FABMS), including tandem FABMS, analysis on the two new microcystins, [D-Glu(OCH3)6]microcystin-LR (1) and [D-Asp3, D-Glu(OCH3)6]microcystin-LR (2). Toxicity data were not obtained for 1 and 2 because of the small amounts isolated from the cells.

Isolation and structures of five microcystins from a Russian Microcystis aeruginosa strain CALU 972.

Five microcystins were obtained from Microcystis aeruginosa strain CALU 972 isolated from a hepatotoxic water bloom collected in Lake Kroshnosero (Russia). The structure of a new toxin (1) was determined as [Dha7]microcystin-YR by amino acid analyses and fast atom bombardment mass spectrometry, and the toxins 2, 3, 4, and 5 were assigned the structures as [Dha7]microcystin-LR, [D-Asp3,Dha7]microcystin-LR, [Dha7]microcystin-RR, and [D-Asp3,Dha7]microcystin-RR, respectively, by direct comparison with authentic samples.

Isolation and structures of microcystins from a cyanobacterial water bloom (Finland).

A hepatotoxic cyanobacterial (blue-green algal) water bloom was collected from a constructed water reservoir in Finland. The water bloom contained two cyanobacterial species, Microcystis aeruginosa and Aphanizomenon flos-aquae. Two hepatotoxins, 1 and 2, were isolated from extracts of lyophilized cells. The structures of 1 and 2 were assigned based upon their amino acid analyses on a Waters Pico Tag HPLC system and a chiral GC capillary column (Chirasil Val III), fast atom bombardment mass spectrometry (FABMS), high resolution FABMS, and tandem FABMS data. Toxin 1 was identical to a previously reported compound, [D-Asp3]microcystin-RR. Toxin 2 was new and was assigned the structure [D-Asp3]microcystin-YR.