Chem. Res. Toxicol. 1998, 11, 143-149
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Seven New Microcystins Possessing Two L-Glutamic Acid Units, Isolated from Anabaena sp. Strain 186 Michio Namikoshi,*,† Moucun Yuan,† Kaarina Sivonen,‡ Wayne W. Carmichael,§ Kenneth L. Rinehart,| Leo Rouhiainen,‡ Furong Sun,| Scott Brittain,§ and Akira Otsuki† Department of Ocean Sciences, Tokyo University of Fisheries, Konan, Minato-ku, Tokyo 108, Japan, Department of Applied Chemistry and Microbiology, P.O. Box 56, University of Helsinki, Helsinki 00014, Finland, Department of Biological Sciences, Wright State University, Dayton, Ohio 45435, and Department of Chemistry, University of Illinois, Urbana, Illinois 61801 Received July 14, 1997
Electrospray ionization mass spectrometry has been applied to the structure assignment of seven new microcystins (1-7), obtained from cultured Anabaena sp. strain 186. The seven new microcystins contain the dehydroalanine (Dha) or L-Ser unit instead of the N-methyldehydroalanine unit and the L-Glu and/or its δ-methyl ester [E(OMe)] units at the two variable L-amino acid units, and the structures were assigned as [Dha7]microcystin-E(OMe)E(OMe) (1), [D-Asp3,Dha7]microcystin-E(OMe)E(OMe) (2), [L-Ser7]microcystin-E(OMe)E(OMe) (3), [D-Asp3,LSer7]microcystin-E(OMe)E(OMe) (4), [Dha7]microcystin-EE(OMe) (5), [D-Asp3,Dha7]microcystinEE(OMe) (6), and [L-Ser7]microcystin-EE(OMe) (7). These microcystins are the first examples containing dicarboxylic amino acids at the two variable L-amino acid units in microcystins.
Introduction Microcystins are well-known cyclic heptapeptide hepatotoxins produced by certain genera of cyanobacteria (blue-green algae) such as Anabaena, Microcystis, Nostoc, and Oscillatoria (1, 2). These cyanobacteria often form hepatotoxic waterblooms in eutrophicated fresh and brackish water bodies. Hepatotoxic cyanobacteria cause fatal poisonings of mammals, birds, and fish (1) and also cause adverse effects on human health (3). These hepatotoxicoses caused by toxic cyanobacteria are attributed to the production of the microcystins. Microcystins show liver toxicity by inhibiting protein phosphatases type 1 and type 2A (4, 5) and act as potent tumor promoters (6-8). More than 50 structural variations have been characterized from natural bloom and cultured cell materials (9, 10). The structures of the microcystins differ primarily in the two L-amino acids and secondarily in the presence or absence of the methyl groups on D-erythroβ-methylaspartic acid (D-MeAsp1) and/or N-methyldehydroalanine (Mdha). Microcystin-LR (8; Chart 2), containing Leu and Arg as variable L-amino acids, is one of the most potent and commonly found compounds among * Corresponding author. Phone: +81-3-5463-0454. Fax: +81-35463-0398. E-mail:
[email protected]. † Tokyo University of Fisheries. ‡ University of Helsinki. § Wright State University. | University of Illinois. 1 Abbreviations: Adda, (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-(4E,6E)-dienoic acid; CID, collisionally induced dissociation; Dha, dehydroalanine; E(OMe), L-glutamic acid δ-methyl ester; ESI, electrospray ionization; ESIMS, ESI mass spectrometry; ESIMS/CID/MS, tandem ESIMS; FAB, fast atom bombardment; FABMS/CID/MS, tandem FAB mass spectrometry; Glu(OMe), Lglutamic acid δ-methyl ester; HRFABMS, high-resolution FABMS; Mdha, N-methyldehydroalanine; MeAsp, erythro-β-methylaspartic acid; ODS, octadecylsilanated.
Chart 1
the microcystins. The microcystins have a unique C20 amino acid, (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-(4E,6E)-dienoic acid (Adda), as the common and unusual structural feature, which plays an important role in their activity (10-12). The general structure of microcystins is cyclo(-D-Ala-X-D-MeAsp-ZAdda-D-Glu-Mdha-) (2). X and Z are the two variable L-amino acids, and their one letter abbreviations are used as the suffix of names (2). The amino acids found at the variable L-amino acid units are Leu, Tyr, Arg, Phe, homotyrosine, Ala, methionine S-oxide, Trp, Val, homoisoleucine, tetrahydrotyrosine, and homophenylalanine at the 2 position and Ala, Arg, methionine S-oxide, ami-
S0893-228x(97)00120-3 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/07/1998
144 Chem. Res. Toxicol., Vol. 11, No. 2, 1998 Chart 2
Namikoshi et al.
with very small amounts. We describe here the application of ESIMS/CID/MS to the structure assignment of seven new microcystins (1-7, Chart 1) obtained from Anabaena sp. strain 186. The seven new microcystins have the L-Glu and its methyl ester residues at the variable L-amino acid units (positions 2 and 4), which is the first example of a dicarboxylic amino acid at these positions.
Experimental Procedures
nobutyric acid, Phe, homoarginine, Tyr, and Trp at the 4 position (9, 10). Demethyl variants of microcystins contain the Asp and/or dehydroalanine (Dha) residues instead of the MeAsp and/or Mdha residues, respectively (10). Progress in studies on microcystins has recently been published in the December issue (Suppl. 6) of Phycologia (1996, 35). There is threat of human poisonings by trace amounts of microcystins, since cyanobacterial waterblooms producing the microcystins sometimes grow in drinking water reservoirs and recreational lakes and ponds and structurally unknown microcystins are still detected in natural waterblooms and cultured cyanobacteria. It is therefore important to develop sensitive and reliable detection methods for these toxins to characterize the structures with very small amounts, and we have examined the use of electrospray ionization (ESI) MS and tandem ESIMS (ESIMS/CID/MS) for this purpose. Collisionally induced dissociation (CID) mass spectra are useful for determining amino acid sequences of peptides and proteins (13). The introduction of fast atom bombardment (FAB) MS in the early 1980s opened the door to the routine analysis of peptides and proteins. This technique allowed the application of mass spectrometric techniques to underivatized peptides, and the high yield of molecular ions permitted the application of different tandem MS techniques. We have also applied the FABMS technique to microcystins and reported the general method for the characterization of microcystins combined with accurate mass measurement and tandem MS (10, 12, 14, 15). Since the introduction of the ESI technique (16), ESIMS has become a powerful method for the analysis of peptides and proteins. The extremely high sensitivity obtained by ESI is due to the very high ionization efficiency (17). ESIMS has, therefore, been applied to the analysis of the microcystins (9, 18-20). Edwards et al. described ESIMS/CID/MS of microcystinsLR and -RR (18), and Bateman et al. reported the combination of ESIMS with capillary electrophoresis and analyzed microcystin-LR analogues (9). We have reported the detection of several unknown microcystins in FAB mass spectra of hepatotoxic fractions separated from cultured Anabaena sp. strain 186, isolated from Lake Vaaranlampi in Finland (21). Since the obtained sample amounts were quite small, ESIMS and ESIMS/CID/MS were applied to analyze these new microcystins, which showed that ESIMS is a powerful method for characterizing the structures of microcystins
Caution: The microcystins are toxic compounds and should be handled carefully. Organism and Separation of Toxins. Anabaena sp. strain 186 was isolated from Lake Vaaranlampi, Finland, and batchcultured in Z8 medium minus nitrogen at 22 °C with continuous illumination of about 50 microeinsteins/m2/s (21, 22). The cells were harvested after 12-14 days of incubation and lyophilized prior to toxicity testing and toxin separation. Toxins were extracted with 1-butanol-methanol-water (1: 4:15, v/v) and separated by repeated high-performance liquid chromatography (HPLC) on octadecylsilanated (ODS) columns in a manner similar to that reported for other Anabaena spp. strains (23). Fractions which showed the characteristic UV absorption maximum at 238 nm by a photodiode array detector were tested toxic to mice and the amino acid components analyzed by a Waters Pico-Tag amino acid analyzer and/or gas chromatography on a chiral capillary column. The toxicity test and amino acid analyses were done by procedures similar to those reported previously (15). Mass Spectrometry. FAB mass spectra were obtained on either a VG ZAB-SE or a VG 70-SE4F mass spectrometer using Xe atoms (8-keV energy) and a matrix of dithiothreitol/dithioerythritol (“magic bullet”). Tandem FABMS (FABMS/CID/MS) (linked scan at constant B/E) were run on a four-sector tandem mass spectrometer (70-SE4F) using He as the collision gas: resolution of the first and second mass spectrometers, both 1000; accelerating potential, 8 keV; collision energy, 4 keV; attenuation, 90%. High-resolution (HR) FAB mass spectra were acquired at a resolving power of 10 000 (10% valley). ESI mass spectra were recorded on a Finnigan TSQ 700 triple quadrupole mass spectrometer with ESIF mode equipped with a DEC 2100 data system. Full-scan spectra were acquired in the positive ion peak centroid or profile modes over the mass range 50-1200 at 1.2-2.0 s. Samples were dissolved in methanol-water (1:1) containing 5% acetic acid and introduced into the ion source by direct infusion or flow injection. ESIMS/ CID/mass spectra were measured using Ar as the collision gas (collision energy, 10-50 eV) in the range of 1.2-3.0 mTorr and scanned at a rate of 2-3 s/decade through the required mass range, and 10-25 scans were accumulated and averaged.
Results and Discussion The cultured cells of Anabaena sp. strain 186 were extracted with 1-butanol-methanol-water (1:4:15, v/v), and after the solvents were evaporated, the aqueous residue was subjected to solid-phase extraction by ODS silica gel. The toxin fraction was then separated by repeated HPLC using ODS columns. The UV spectra of the HPLC fractions were measured by a photodiode array detector, and the characteristic absorption maximum (238 nm) for microcystins was observed in several fractions which showed hepatotoxicity to mice (ip). The FABMS analysis of the fractions revealed the presence of five compounds (1-5). Compound 5 was detected as a minor component in the fraction containing 1 (1:5 ) ca. 9:1) and separated by HPLC (ODS column, 4.6 mm × 25 cm) using methanol-0.05% trifluoroacetic acid (6: 4). The analytical HPLC separation detected 6 and 7 as
New Microcystins from Anabaena sp. 186
Chem. Res. Toxicol., Vol. 11, No. 2, 1998 145
Table 1. FABMS/CID/MS Data for 1 and ESIMS/CID/MS Data for 2-4a ion composition
1
2
3
4
M+H Ph-CH2CH(OMe) CH(Me)-CHdC(Me)-CHdCH-CH2-CH(Me)-CO 163 - Glu 163 - Glu-Dha 163 - Glu-Dha-Ala 163 - Glu-Dha-Ala-Glu(OMe) 163 - Glu-Dha-Ala-Glu(OMe)-MeAsp-Glu(OMe)-NH2 + H Glu(OMe) - CO + H MeAsp-Glu(OMe) - CO + H MeAsp-Glu(OMe) + H Ala-Glu(OMe) + H Dha-Ala-Glu(OMe) + H Dha-Ala-Glu(OMe)-MeAsp + H Dha-Ala-Glu(OMe)-MeAsp-Glu(OMe) + H Glu-Dha-Ala-Glu(OMe)-MeAsp-Glu(OMe) + H MeAsp-Glu(OMe)-NH2 + 2H Glu(OMe)-MeAsp-Glu(OMe)-NH2 + 2H Ala-Glu(OMe)-MeAsp-Glu(OMe)-NH2 + 2H Dha-Ala-Glu(OMe)-MeAsp-Glu(OMe)-NH2 + 2H
998 135 163 292 361 432 575 864 116 245 273 215 284 413 556 685 290 433 504 573
984 135 163 292 361 432 575 850* 116 231* 259* 215 284 399* 542* 671* 276* 419* 490* 559*
1016 135 163 292 379† 450† 593† 882† 116 245 273 215 302† 431† wk 703† 290 433 504 591†
1002 135 163 292 379† 450† 593† 868‡ 116 231* 259* 215 wk 417‡ 560‡ 689‡ 276* 419* 490* 577‡
a Legend: *contains Asp instead of MeAsp; †contains Ser instead of Dha; ‡contains Asp and Ser instead of MeAsp and Dha, respectively; wk, peak intensity was small.
the minor components of 2 and 3, respectively. Since the amounts of these fractions were not enough to isolate 6 and 7, ESIMS was applied to the fractions without further purification. The structure assignment was first carried out with the main component, 1, by the established method using FABMS/CID/MS (high-energy CID), and the results were compared with those obtained by ESIMS/CID/MS (lowenergy CID) to examine the applicability of ESIMS to the structure assignment of 2-7. Structure Assignment of 1 by FABMS. Compound 1 was obtained as the main component among seven microcystins from Anabaena sp. strain 186. The molecular formula (C48H67N7O16) was determined based on HRFABMS [(M + H)+, m/z 998.4731 (∆ -0.8)] and amino acid analysis data, which showed the presence of D-Ala, two L-Glu, D-MeAsp, and D-Glu. FABMS/CID/MS data for 1 (Table 1) revealed the Adda unit as an amino acid component of 1 by the characteristic fragment ion peaks at m/z 135 and 163 (12, 15). The seventh amino acid unit of 1 was deduced to be Dha from the fragment ion peaks at m/z 361, 432, etc. (Table 1). The sum of the unit weights of the above seven amino acid units (969 Da) was 28 Da less than the molecular weight of 1. Subtraction of mass numbers of the fragment ion peaks in the FABMS/CID/mass spectrum of 1 at m/z 432 [163 - Glu-Dha-Ala], 290 [MeAsp-Glu(OMe)NH2 + 2H], and 413 [Dha-Ala-Glu(OMe)-MeAsp + H] from the peaks at m/z 575 [163 - Glu-Dha-Ala-Glu(OMe)], 433 [Glu(OMe)-MeAsp-Glu(OMe)-NH2 + 2H], and 556 [Dha-Ala-Glu(OMe)-MeAsp-Glu(OMe) + H], respectively, gave the same mass number, 143 Da, for the two variable L-amino acid units, each of which is 14 Da (CH2) more than the unit weight of Glu. The amino acid analysis of 1 showed two L-Glu. These data suggested that the L-amino acid units in 1 are methylene or methyl derivatives of L-Glu, such as homoglutamic acid, N- or C-methylglutamic acid, and glutamic acid methyl ester (methyl glutamate). Since only the methyl ester among the above derivatives can give glutamic acid by the acid hydrolysis used for amino acid analysis, the two L-amino acid units were assigned as the methyl ester derivative of Glu.
Figure 1. Three characteristic fragmentation patterns (A-C) in the FABMS/CID/mass spectrum of 1 (fragment ion peaks shown in m/z).
FABMS/CID/MS data listed in Table 1 showed the complete sequence of the seven amino acid components of 1. Three remarkable fragmentation patterns were observed as shown in Figure 1. The fragment ion peak at m/z 135 was derived from the Adda residue and is the characteristic ion for microcystins possessing the ordinary Adda unit (12, 15). Cleavages of the above unit and C-N bond in the Adda residue gave the fragment CH(Me)-CHdC(Me)-CHdCH-CH2-CH(Me)-CO (Figure 1A). This fragment ion peak was detected in the FABMS/CID/ mass spectrum of 1 at m/z 163, and the fragment ion peaks containing this unit were observed at m/z 292, 361, 432, and 575. These fragment ion peaks revealed the sequence of Adda-Glu-Dha-Ala-Glu(OMe) (Figure 1A). The second pattern is the C” series fragmentation as shown in Figure 1B, which gives an amide moiety at the C-terminus (13). The fragment ion peaks of this pattern at m/z 290, 433, 504, and 573 showed the sequence of Dha-Ala-Glu(OMe)-MeAsp-Glu(OMe) (Figure 1B). The third pattern is the B series fragmentation (13) showing the whole sequence except the Adda unit of 1 by the fragment ion peaks at m/z 284, 413, 556, 273, and 685
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Figure 2. FABMS/CID/mass spectrum (A) and ESIMS/CID/mass spectrum (B) of 1.
(Figure 1C). Consequently, the structure of 1 was assigned as [Dha7]microcystin-E(OMe)E(OMe), in which E(OMe) is the glutamic acid δ-methyl ester unit, as shown in Chart 1, and the accepted abbreviations apply (2).
ESIMS Analysis of 1. The fragment ion peaks useful for the structure assignment (Table 1) observed in the FABMS/CID/mass spectrum of 1 (Figure 2A) were also detected in its ESIMS/CID/mass spectrum (Figure 2B). The other interpretable fragment ion peaks in the
New Microcystins from Anabaena sp. 186
FABMS/CID/mass spectrum of 1 were also observed in its ESIMS/CID/mass spectrum. Accordingly, the ESIMS/ CID/mass spectrum (low-energy CID) of 1 was very similar to the FABMS/CID/mass spectrum (high-energy CID) as shown in Figure 2. The ESIMS/CID/mass spectra of 1 showed high reproducibility. Papayannopoulos reported the significant differences between high- and low-energy CID mass spectra of linear peptides (24). High-energy CID spectra showed all possible fragmentation types, although different collision conditions are required for one sample to observe all the types, while a few fragmentation types were observed in low-energy CID spectra. This difference shows the different spectra between high- and low-energy CID spectra of linear peptides. However, compound 1 did not show such a difference. The ESIMS/CID/mass spectra of 2-4 were also very similar to their FABMS/CID/mass spectra. The major CID fragment ions generated from the (M + H)+ ion of a cyclic peptide can be explained in terms of the mechanism in which protonation occurs on an amide nitrogen of a peptide bond (25, 26). If this amide nitrogen is in the ring of the cyclic peptide, the N-acyl bond may then be cleaved generating a linear peptide acylium ion. Further cleavages will occur by the loss of amino acid residues to give a series of fragment ions. It may, therefore, be suggested that the cleavage of the cyclic structure of microcystins to linear peptide ions is the rate-determining step in both high- and lowenergy CID processes, which resulted in the similar fragmentation patterns obtained from the generated linear peptide ions. It was proved that ESIMS/CID/MS can be applied to the same extent as FABMS/CID/MS to assign the structures of the other new microcystins (2-7). Structure of 2. The molecular ion peak at m/z 984 [(M + H)+] detected in FABMS and ESIMS of 2 was 14 Da less than the molecular ion peak of 1 (m/z 998). The difference was observed in the amino acid components. Compound 2 gave D-Asp instead of D-MeAsp detected for 1 in the amino acid analysis. ESIMS/CID/MS data for 2 showed a difference of 14 Da for the fragment ion peaks containing the Asp unit as marked by an asterisk (*) in Table 1 from the corresponding ion peaks of 1 (containing MeAsp). The fragment ion peaks at m/z 361, 432, 575, 116, 215, and 284 in the ESIMS/CID/mass spectrum of 2 were observed at the same positions in that of 1. Thus, the structure of 2 was assigned as the Asp variant of 1, that is, [D-Asp3,Dha7]microcystin-E(OMe)E(OMe), as shown in Chart 1. Structure of 3. The molecular formula C48H69N7O17 for 3 was assigned from HRFABMS data [(M + H)+, m/z 1016.4841 (∆ -1.3)] combined with amino acid analysis data. Compound 3 gave Ser in the amino acid analysis, and the other five amino acids were identical with those of 1. The difference in molecular weight and formula (18 Da, H2O) between 1 and 3 was, therefore, ascribable to the Ser unit in 3 instead of the Dha unit in 1. The ESIMS/CID/mass spectrum of 3 showed an 18-Da difference for the fragment ion peaks marked by a dagger (†) in Table 1 relative to the corresponding peaks in that of 1 and confirmed the sequence of all amino acid components of 3. Accordingly, the structure of 3 was assigned as [L-Ser7]microcystin-E(OMe)E(OMe) (Chart 1). L-Ser variants of microcystins at the Dha unit have been isolated from Anabaena spp. (27, 28) and Microcystis aeruginosa (29) collected in Finland, and L-N-meth-
Chem. Res. Toxicol., Vol. 11, No. 2, 1998 147 Table 2. ESIMS/CID/MS Data for 5-7a ion composition
5
6
7
M+H CH(Me)-CHdC(Me)-CHdCH-CH2-CH(Me)-CO 163 - Glu 163 - Glu-Dha 163 - Glu-Dha-Ala 163 - Glu-Dha-Ala-Glu 163 - Glu-Dha-Ala-Glu-MeAsp-Glu(OMe) 163 - Glu-Dha-Ala-Glu-MeAsp-Glu(OMe)-NH2 + H Glu(OMe) - CO + H MeAsp-Glu(OMe) - CO + H MeAsp-Glu(OMe) + H Ala-Glu + H Dha-Ala-Glu + H Dha-Ala-Glu-MeAsp-Glu(OMe) + H Glu-Dha-Ala-Glu-MeAsp-Glu(OMe) + H MeAsp-Glu(OMe)-NH2 + 2H Glu-MeAsp-Glu(OMe)-NH2 + 2H Ala-Glu-MeAsp-Glu(OMe)-NH2 + 2H Dha-Ala-Glu-MeAsp-Glu(OMe)-NH2 + 2H
984 163 292 361 432 561 833 850 116 245 273 201 270 542 671 290 419 490 559
970 163 292 361 432 561 819* 836* 116 231* 259* 201 270 528* 657* 276* 405* 476* 545*
1002 163 292 379† wk 579† 851† 868† 116 245 273 wk wk 560† 689† 290 419 490 wk
a Legend: *contains Asp instead of MeAsp; †contains Ser instead of Dha; wk, peak intensity was small.
ylserine variants instead of Mdha have been isolated from a waterbloom (M. aeruginosa dominant) collected in Illinois (12), Oscillatoria agardhii (30), and Nostoc sp. strain 152 (31). Structure of 4. The HRFAB mass spectrum [(M + H)+, m/z 1002.4678 (∆ -0.6)] and amino acid analysis data for 4 assigned the molecular formula C47H67N7O17. The molecular weight (formula) of 4 was 18 Da (H2O) more than that of 2 and 14 Da (CH2) less than that of 3. The differences were detected in amino acid components of these compounds, that is, 4 has Ser and Asp in lieu of Dha in 2 and MeAsp in 3, respectively. The ESIMS/CID/ mass spectrum of 4 showed the above differences with the masses of fragment ion peaks at m/z 379, 450, 593, 868, 231, 259, 417, 560, 689, 276, 419, 490, and 577 compared with the corresponding peaks in the spectra of 2 and 3 (Table 1). These data revealed the structure of 4 as [D-Asp3,L-Ser7]microcystin-E(OMe)E(OMe), as shown in Chart 1. Structure of 5. The molecular weight (983 Da) determined from the ESI mass spectrum of 5 was 14 Da less than that of 1. The amino acid analysis data for 5 were essentially the same as those for 1. The difference of 14 Da between 5 and 1 was detected in the fragment ion peaks at m/z 561, 850, 201, 270, 542, 671, 419, 490, and 559 of 5 (Table 2) and the corresponding peaks at m/z 575, 864, 215, 284, 556, 685, 433, 504, and 573 of 1 (Table 1). These fragment ion peaks revealed that the L-amino acid unit between the Ala and MeAsp units in 5 is a demethylated form of the Glu(OMe) unit in 1. Thus, the structure of 5 was assigned as [Dha7]microcystin-EE(OMe) (Chart 1), and the accepted abbreviations apply (2). Structures of 6 and 7. Compounds 6 and 7 were detected by ESIMS in the fractions containing 2 and 3, respectively. The analytical HPLC separation showed a small peak in each fraction (about 10% of the main peak). Since the amounts of these fractions were small, purification of 6 and 7 was not practical. The structure analysis of 6 and 7 was, therefore, done by ESIMS/CID/MS without further separation. ESIMS/CID/MS data for 6 showed a 14-Da difference for the fragment ion peaks marked by an asterisk (*) in Table 2 relative to the corresponding peaks of 5, which was consistent with the difference in molecular weights
148 Chem. Res. Toxicol., Vol. 11, No. 2, 1998
between 5 and 6. The same difference was detected between 1 and 2 (Table 1). These data suggested that the structure of 6 is an Asp variant of 5, and the ESIMS/ CID/MS data for 6 listed in Table 2 confirmed the sequence of the seven amino acid components of 6. Consequently, the structure of 6 was assigned as [D-Asp3,Dha7]microcystin-EE(OMe) (Chart 1). The molecular weight of 7 (1001 Da), determined by the ESI mass spectrum, was 18 Da more than that of 5. The difference (18 Da) between 5 and 7 was observed in the fragment ion peaks of 7 marked by a dagger (†) and the corresponding peaks of 5 in the ESIMS/CID/mass spectra of 5 and 7 (Table 2). Although several fragment ion peaks in the ESIMS/CID/mass spectrum of 7 were observed in insufficient intensity because of the small sample size, the amino acid sequence of 7 can be assigned from the fragment ion peaks listed in Table 2. The structure of 7 was, therefore, deduced to be [L-Ser7]microcystin-EE(OMe), as shown in Chart 1. The seven microcystins mentioned in this paper have the L-Glu and its δ-methyl ester derivative units at the variable L-amino acid units. These variations have not previously been detected in the microcystins, i.e., these compounds are the first examples possessing a dicarboxylic amino acid at these positions. The FABMS/CID/mass spectra of 1-4 showed more abundant fragment ion peaks than those of microcystinsLR (8), -YR (9), and -RR (10). These fragment ion peaks detected in the FABMS/CID/mass spectra of 1-4 were useful to assign the complete sequences of these compounds, while microcystins-LR, -YR, and -RR did not give enough fragment ion peaks to determine the sequences, and the linearization by ozonolysis prior to FABMS/CID/ MS analysis was required to assign their structures (14, 15). Since the FABMS/CID/mass spectrum of microcystin-LA (11) gave similar fragment ion peaks to those listed in Table 1,2 the fewer fragment ion peaks observed in the FABMS/CID/mass spectra of microcystins-LR (8), -YR (9), and -RR (10) appear to be ascribable to the presence of the Arg unit between the MeAsp and Adda units. This study showed that ESIMS/CID/MS is applicable to the analysis of microcystins in the same manner as FABMS/CID/MS. It is notable that the fragmentation patterns observed in the ESIMS/CID/MS (low-energy CID) of 1-4 were very similar to those of FABMS/CID/ MS (high-energy CID), although significant differences were reported for linear peptides (24). Compounds 2-4 did not give good-quality FABMS/CID/mass spectra because of the small sample sizes obtained; on the other hand, ESIMS/CID/MS of 2-4 showed better spectra with lower amounts of samples than those used in the FABMS analysis. The CID mass spectra of 5-7 were obtained only by the ESI method. Since the sensitivity of ESIMS/ CID/MS is superior to that of FABMS/CID/MS and no matrix is required for ionization, ESIMS analysis is more useful than FABMS analysis for the characterization of microcystins.
Acknowledgment. This study was supported in part by grants from the National Institute of General Medical Sciences (GM 27029) and the National Institute of Allergy and Infectious Diseases (AI 04769) to K.L.R. and by a subcontract from the latter grant to W.W.C. The 2
M. Namikoshi, F. Sun, and K. L. Rinehart, unpublished data.
Namikoshi et al.
research in Japan was partly supported by the Environmental Research Fund from the Sumitomo Foundation (94-104-596), the Fund for Research Encouragement in Celebration of the Centennial Anniversary of the Founding of Tokyo University of Fisheries, and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (08680628) to M.N. Supporting Information Available: FABMS/CID/mass spectra of 2-4, ESIMS/CID/mass spectra of 2-7, and tables of assigned ESIMS/CID/MS data for 1-7 (16 pages). Ordering information is given on any current masthead page.
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