Two new l -serine variants of microcystins-LR and -RR from Anabaena sp. strains 202 A1 and 202 A2

0041-0101/92 /5.00 + .00 ® 1992 Pmpmon Press Ltd

Toxiaon, Vol . 30, No. 11, pp. 1457-1464, 1992. Printed in Great Britain.

TWO NEW L-SERINE VARIANTS OF MICROCYSTINS-LR AND -RR FROM ANABAENA SP. STRAINS 202 A1 AND 202 A2 MicHio NAMmosm,' KAARINA SIVONEN,2' WILLIAM R. EVANS ,3 WAYNE W. CARMICHAEL,3 FURONG SUN,' LEo RouHiArNEN,2 RAIJA LuUKKAINEN 2 and KENNETH L. RINEHART' 'Roger Adams Laboratory, University of Illinois, Urbana, IL 61801, U.S .A. ; =Department of Microbiology, University of Helsinki, SF-00710 Helsinki, Finland; and 3 Department of Biological Sciences, Wright State University, Dayton, OH 45435, U .S.A. (Received 4 March 1992; accepted 23 lune 1992)

M. NAMIKOSHI, K. SIvoNEN, W. R. EVANS, W. W. CARMICHAEL, F. SUN, L. ROUHIAINEN, R. LUUKKATNEN and K. L. RINEHART . Two new L-serine variants of microcystins-LR and -RR from Anabaena sp . strains 202 Al and 202 A2 . Toxicon 30, 1457-1464, 1992 .-Two new microcystins, [L-Serlmicrocystin-LR (1) and [L-Serlmicrocystin-RR (2), were isolated from a filamentous fresh water cyanobacterium (blue-green alga), Anabaena sp . strain 202 Al, along with the two major toxins, [Dhalmicrocystin-LR (3) and [Dhalmicrocystin-RR (4) and their minor components the D-Asp variants [D-Asp3,Dhalmicrocystin-LR (5) and [D-Asp 3,Dhalmicrocystin-RR (6). Anabaena sp . strain 202 A 1 also produced another new toxin, whose structure is tentatively proposed as [D-Asp 3,L-SerJmicrocystin-XR (7), where X is a leucine homologue. Anabaena sp . strain 202 A2 produced one new microcystin, 1, and three known microcystins, 3, 4, and 5. The structures of the toxins were assigned based on their amino acid analyses, and fast atom bombardment mass spectrometry data . INTRODUCTION

MoRE than 30 microcystins have been chemically identified from cyanobacteria (bluegreen algae) such as coccoid Microcystis and filamentous Anabaena, Nostoc, and Oscillatoria species (CARMICHAEL, 1992 ; HARADA et al., 1991a,b; KIVIRANTA et al., 1992 ; KRISHNAMuRTHY et al., 1989; MERILuoTo et al., 1989 ; NAwKosFu et al., 1990, 1992a,b; SIvoNEN et al ., 1990a,b, 1992a,b) . A filamentous Nodularia spwnigena produces another type of hepatotoxin, nodularin, a cyclic pentapeptide (RINEHART et al., 1988 ; $IvoNEN et al., 1989). The general structure of microcystins is cyclo(-D-Ala-X-D-MeAsp-Z-Adda-D-GluMdha-), where X and Z are variable L-amino acids, D-MeAsp is D-erythro-ß-methylaspartic acid, and Mdha is N-methyldehydroalanine (CARMICHAEL et al., 1988). Adda, (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid, is the

" Author to whom correspondence should be addressed. 1457

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M. NAMIKOSHI et al.

remarkable structural feature of these toxins which confers toxicity (BoTEs et al., 1984 ; RINEHART et al., 1988). We have reported two modified Adda units in microcystins which retain hepatotoxicity-from Nostoc sp. strain 152 (NAMIKOSHI et al., 1990 ; SIvoNEN et al., 19906, 1992x) and from a waterbloom of Microcystis spp. collected from Homer Lake (Illinois, U.S .A.) (NAmncosm et al., 1992x) . Demethyl variants at the D-MeAsp and/or Mdha residues have been reported as the most common modifications (CARMICHAEL, 1989 ; DIERsTEIN et al., 1990 ; HARADA et al., 1991a,b; KIVIRANTA et al., 1992 ; KRmNAMuRTHY et al., 1989; MERILuoTo et al., 1989 ; NAMmosHI et al ., 1990, 1992a,b; SIvoNEN et al., 1990b) . We have recently described a D-Ser variant at the D-Ala residue, which was previously thought to be an invariable amino acid component, from Nostoc sp . 152 (SIvoNEN et al., 1992x) . We have been investigating new sources of hepatotoxin producers and new toxins and have recently reported the isolation of hepatotoxic strains of Anabaena spp. and their toxin components (NAamosm et al., 1992b; SIvoNEN et al., 1990x, 1992b). In this paper, we assign the structures of two new microcystins and a tentative structure for a new microcystin, together with four known microcystins, all obtained from Anabaena sp . strains 202 Al and 202 A2 . Structures were determined using amino acid analysis and fast atom bombardment mass spectrometry (FABMS), including tandem FABMS (FABMS/ MS) (NAMIKOSHI et al., 1990, 1992a,b; SrvoNEN et al., 1992x) . MATERIALS AND METHODS Organisms and culturing Two hepatotoxic Anabaena sp. strains, 202 AI and 202 A2, were isolated from a hepatotoxic water bloom sample collected on 23 July 1987 at Lake Vesijiirvi (southern Finland) (Stvomer et al., 1992b) . Strains were isolated and cultured in the laboratory using the inorganic nutrient medium Z8 minus nitrogen as described previously (Sworn et at., 1990x, 1992b). Isolation of toxins Lyophilized cells were extracted twice (2hr and overnight) with 1-butanol:methanol:water (1 :4:15) . Supernatants were combined and evaporated to one-half of the original volume, and applied on preconditioned C18 silica gel columns (Bond, Elut, Analytichem, Harbor City, CA, U.S .A .). The toxic fractions were eluted with 80% methanol and evaporated to dryness. The toxins were then separated and purified by HPLC as follows. The first HPLC system consisted of a Varian Vista model 5560 solvent delivery system with a Varian model 200 u.v . detector plus a semipreparative C18 silica gel column (l9 x 150mm, #Bondspak, Waters Associates, Milford MA, U.S .A .) . The mobile phase acetonitrile :l0 mM ammonium acetate (26:74) was used at a flow rate of 4 ml/min and toxins were detected by u.v. at 238 nm . The second purification step was accomplished with a Beckman model 421 solvent delivery system with a Beckman model 165 u.v. detector plus a semipreparative C18 silica gel column (l9 x 300mm, #Bondspak, Waters Associates, Milford, MA, U.S .A .) using a methanol gradient from 0-50% over 25 min in 10 mM phosphate buffer (pH 6.8). The fractions were further purified with a Beckman model 406 HPLC with a model 167 u.v . detector and an analytical C18 silica gel column (4 .6 x 250 mm, Alltech Associates, Deerfiel i, IL, U.S .A.) with acetonitrile :20 mM ammonium acetate (pH 5, 25 :75) and a flow rate of 1 ml/min . The purity of the toxins was monitored and toxins identified in the isolated fractions after the second purification step by their u.v. spectra using a Waters 600E HPLC system with a 990 photodiode array detector. The column used was ISRP-column (4.6 x 250 mm, Regis Pinkerton, Regis Chemical Co ., Morton Grove, IL, .-) .S and mobile phase acetonitrile :0 .1M phosphate buffer (pH 6.8,15:85) and a flow rate U of 1 ml/min. Seven toxins were isolated and purified from 10g of dried cells of Anabaena sp . 202 A1 and four from 1 .4 g of Anabaena sp. 202 A2 . The pure compounds were desalted and stored at -20°C. Amino acid analysis Isolated compounds were hydrolyzed with 6 N HCI at 110°C for 21 hr, and the phenyl isothiocyanate derivatized amino acids were analyzed with a Waters Pico Tag HPLC system. The derivatized amino acids were separated on an ODS column (3 .9 x 150 mm) using Pico Tag eluents A and B detected by u.v. absorption at 254 nm .

Two New Microcystins from Anabaena app. TABLE

I . HIGH-R00LUTION

Compound 1

2 3 4 5 6 7

FABMS

1-7'

1459

DATA POR COMPOUNDS

(M+H), m/z

Composition

At

999.5533 1042 .5702 981 .5423 1024 .5602 967.5269 1010 .5439 999.5523

C4,H 73N, 00, 3 C4gH7,14 13013 C4&H7.1N10O12 C4g H74Nu0,2 C47 H7,N, 00, 2 C47 H72N, 30, 2 C4,H7,N, 00, 3

-1 .8 -1 .6 -1 .4 -2 .2 -1 .6 -1 .6 -0 .8

* Obtained by a VG 70-SE4F mass spectrometer . t Difference (mmu) from the calculated value for each composition . Gas chromatography (GC) Capillary GC analyses were carried out on a Varian 3700 gas chromatograph, using a Chirasil Val III column (FRANK et al., 1977) (0.32mm x 25 m, Alltech Associates, Deerfield, IL, U.S.A.) with helium as the carrier gas (flow rate : 37 ml/min, split ratio: 20:1). The program rate for the analysis of amino acid derivatives, except arginine, was 90°C (2 min) to 170°C at 8°C/min. The arginine derivative was detected at 200°C. The other conditions were as follows: injector temperature, 210°C; detector temperature, 220°C; make-up gas, helium (20 ml/min). Each authentic amino acid (100 ug) was treated with 300 pl of 4 N HCl/MeOH at l10°C for 15 min in a screw-capped vial . The reaction mixture was evaporated in a stream of N2, dichloromethane (CH2Cl2, 200 pl) and trifluoroacetic anhydride (TFAA, 200 pl) were added, and the mixture was heated at I l0°C for 5 min, then evaporated by N2. The residue was dissolved in CH 2Cl2 for GC analysis. Arginine was converted to a dimethylpyrimidine derivative prior to the above treatment. The amino acid (100 ,ug) was heated at l 10°C for 4hr with H2O (25 pl), EtOH (50pl), triethylamine (Et3N, 25 pl), and acetylacetone (50pl) (MORRIS et al., 1973). The reaction mixture was evaporated to dryness by N2.

Acid hydrolysis of toxins and derivatization of the hydrolyzatefor GC analysis Compounds 1 (30 leg/ 100 pl), 2 (30,ug/ 100 id), 3 (100 leg/200pl), 4 (100 pg/200 pl), 5 (40pg/100 pl), 6 (40pg/100 id), and 7 (10 pg/501d) in 6N HC I were heated separately at l 10°C for 22-24 hr . The reaction mixture was cooled to room temperature and divided into two portions, except the reaction mixture of 7. Each portion was evaporated to dryness by N2. One portion was treated with 4 N HCI/MeOH followed by CH 2Cl 2 and TFAA as above. Another portion was treated with a mixture of acetylacetone, H2O, EtOH, and Et3N followed by esterification and acylation as before. The residue was dissolved in CH2C1 2 for GC .

FABMS analysis FAB mass spectra were obtained on either a VG ZAB-SE or a VG 70-SE4F mass spectrometer using xenon atoms accelerated to 8 kV and a matrix of dithiothreitol/dithioerythritol ('magic bullet') (WITTEN et al., 1984). Collisionally induced tandem mass spectra in the FAB mode were obtained on a four-sector tandem mass spectrometer (70-SE4F) using helium as a collision gas. High-resolution (HR) FABMS and FABMS/MS data are summarized in Tabks 1 and 2, respectively. Approximately 5- 101+8 of each sample was applied as a methanol solution .

Toxicity testing The cells and the fractions from the first HPLC run were tested for hepatotoxicity by mouse bioassay. Samples in water solution were injected intraperitoneally into mice (20-25 g, female NMRI-mice, University of Helsinki, Finland) . Mice were observed for 4 hr and the signs of poisoning typical for hepatotoxins from cyanobacteria were noted and recorded .

M . NAMIKOSHI er al.

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TABLE 2. FABMS/MS DATA POR COMPOUNDS 1, 2 AND 7"

Fragment ion, m/z Sequence M+H M-135 C  H O-Glu-ert CO-Glu-Ser-Ala-2H Arg-McAsp+H CO-Glu-Ser-H Glu -S er +H Ser-A1a+H PhCH2CH(OCH3)

1

2

999 863 379 313 286 243 wk: 159 13511

1042 906 wk : 313 286 243 217 159 13511

7

999 963 379 313 272§ 243 217 . 159 13511

*Obtained by a VG 70-SE4F mass spectrometer. tCH3 CH : CHC(CH 3) : CHCH : CHCH(CH3)CO-Glu-Ser-H . $Intensity of a corresponding peak was weak . §Contains Asp instead of McAsp (14mu less than that of I and

2).

III peak of daughter ions . RESULTS AND DISCUSSION

Organisms, culturing and toxin isolation Anabaena sp. strains, 202 A1 and 202 A2, were isolated from a hepatotoxic water bloom (S1vONEN et al., 1992b) and both strains were found to be hepatotoxic by intraperitoneal mouse bioassay. Identification at the genus level and specification of the strains by number minimized possible taxonomic problems . The cells of lyophilized Anabaena strains 202 A 1 and 202 A2 were hepatotoxic in mouse bioassay, causing hepatocyte necrosis, pooling of blood in the liver and death of the test animals within 1-3 hr. These typical signs were also observed using several peaks collected at the first HPLC separation, which correspond to isolated and purified compounds 1-7. Although the amounts of purified new compounds 1, 2 and 7 were insufficient to obtain toxicity data, the crude toxins showed symptoms consistent with hepatotoxin poisoning caused by the other microcystins. Compounds 1 (0.1 mg), 2 (0.1 mg), 3 (1 .0 mg), 4 (1 .3 mg), 5 (0.1 mg), 6 (0.2 mg), and 7 (0.05 mg) were isolated from 10 g of Anabaena strain 202 Al, and 1(0.05 mg), 3 (0.1 mg), 4 (0.2 mg), and 5 (0.05 mg) were obtained from 1 .4 g of Anabaena strain 202 A2. Since the amounts of cells used for extraction of the toxins were different, the number and amounts of toxins found from these strains were different. However, the main toxins produced by both strains were the same . Amino acid components Toxins 1-7 were hydrolyzed with 6 N HC1 at 110°C for 21-24 hr, and the amino acid components of the hydrolysates were first analyzed by a Waters Pico Tag HPLC system, which showed the quantitative contents of the amino acid components . The stereochemistries of the amino acids were determined by GC analysis using a chiral capillary column (Chirasil Val III) (FRANK et al., 1977). The acid hydrolysates of toxins 17 were derivatized as their N-trifluoroacetyl methyl esters (NAMIKOSHi et al., 1992b;

Two New Microcystins from Anabaena spp. TABLE 3. FABMS AND AMINO ACID ANALYSIS DATA Compound 1 2 3 4 5 6 7

(M+H), m/z' 999 1042 981 1024 967 1010 999

FOR

146 1

COMPOUNDS 1-7

Amino acidt D-Ala, D-Ala, D-Ala, D-Ala, D-Ala, D-Ala, D-Ala,

L-Leu, D-McAsp, L-Arg, D-GIU, L-Ser L-Arg, D-McAsp, L-Arg, D-Glu, L-Ser L-Leu, D-McAsp, L-Arg, D-Glu L-Arg, D-MeMp, L-Arg, D-Glu L-Leu, D-Asp, L-Arg, D-Glu L-Arg, D-Asp, L-Arg, D-Glu unk,t D-Asp, Argh D-Glu, L-Der

*Obtained by a VG ZAB-SE mass spectrometer . twaters Pico Tag HPLC system and GC on a chiral capillary column (Chirasil Val HI). $Unknown amino acid (would be a Leu homologue) . §Stereochemistry not determined . SwONEN et al., 1992x) . Arg was converted to the 4,6-dimethylpyrimidine derivative of the

guanido group (MORRis et al., 1973) then esterified and acylated . The results of the amino acid analyses are summarized in Table 3.

Mol. wis and molecular formulas The mol. wts of toxins 1-7 were deduced from protonated molecular ions [(M + H)+] using FABMS and a magic bullet matrix (W=N et al., 1984) (Table 3) . HRFABMS data on the (M + H)+ ions revealed the molecular formulas of 1 (C48H,4NIO013), 2 (C4gH75N13013), 3 (C4gH72NI001), 4 (C48H73N1301)+ 5 (C47H70N10012), 6 (C47H71N13012), and 7 (C4sH74N1u013) with the assistance of amino acid analysis data (Table 1). The amino acid analysis and HRFABMS data showed that toxins 1, 2, and 7 were new compounds. The HRFABMS data and amino acid components of 3, 4, 5, and 6 were the same as [Dha 7]microcystin-LR (HARADA et al ., 19916; NAhaxosw et al., 1992x), [Dhalmicrocystin-RR (KIVMANTA et al., 1992), [D-ASP 3,Dhalmicrocystin-LR (HARADA et al., 19916), and [D-Asp 3,Dhalmicrocystin-RR (KRISHNAMURTHY et al., 1989), respectively . The assignments were confirmed by direct comparison with authentic samples (KIVIRANTA et al., 1992; NAMIIcosiu et al., 1992x) .

Structure of toxin 1 The amino acid components of 1 were similar to those of 3, except for the presence of LSer in 1 (Table 3) . The difference between 1 and 3 was also observed in their mol. wts and molecular formulas (Tables 3 and 1) . The mol. wt (formula) of 1 was 18 mu (H20) higher than that of 3, which is ascribable to the replacement of L-Ser in 1 by Dha in 3. The structure of 1 was confirmed by its FABMS/MS data as shown below. The (M + H)+ ion of 1 was subjected to collisionally induced FABMS/MS to give the product ion peaks (NAM1KOsm et al., 1990, 1992). The N-methylserine (Mser) and N-demethyl (Dha) variants at the Mdha unit of microcystins gave rather weaker fragment ions than those of Mdha variants (NAMmosm et al., 1992x; SrvoNEN et al., 1992x) except for the ions at m/z 135 [PhCH2CH(OCH3)+] and M-135 . The L-Ser variant at this unit gave much weaker fragment ions than those of Mser and Dha variants (NAMIKOsFu et al., 1992b) . Although toxins 1, 2, and 7 did not give good FABMS/MS for the above reason

1462

M . NAMIKOSHI et al.

a

DOu

X [L-s~ oK~R (L-SrrBmloro ~ [Dha7>da~n-) .R [Dha7)Mao~R

(1) : (2) : (3) : (4) :

[D-A@O,Dha7~ayän-LR (a) : [D-~,Dha7Jmlao~R (a) : [D-Mp3,l-Ser7Si~XR (7) :

7

L8W (Ohm)

R1

R2

L-LOU L-kg L-Leu L-11rp

as

K CH~ H, CHZOH

CFb CH3

L-Leu L-AM C7H13N0

H H H

CH2 CH2 CH2 CH2

CHb

H, CH2CH

FiG . 1 . Srnucrune oF rima 1-6 isouim meom culruRED Anabaena sp. sTRAna 202 Al AND 202 A2 .

and because of inadequate sample sizes, several fragment ions, listed in Table 2, were detected . The fragment ion peaks at m/z 135 and M-135 showed the presence of the Adda unit (NAMmosm et al., 1992, 1992a,b) . The sequence Adda-Glu-Ser-Ala was revealed from the fragment ion peaks at m/z 379, 313, 243, and 159, and Arg-MeAsp was suggested by the peak at m/z 286. The structure of 1 is, therefore, assigned as the L-Ser variant at the Dha unit of [Dhalmicrocystin-LR (3), that is, [L-Serlmicrocystin-LR as shown in Fig. 1 . Structure of toxin 2 The amino acid analysis data of 2 showed two L-Arg and suggested the structure of 2 as an L-Ser variant of 4 (Table 3). The differences in the mol. wt and molecular formula observed between 1 and 3 (18 mu, H2O) were also detected between 2 and 4 (Table 1). The presence ofthe Adda unit was confirmed by fragment ion peaks at m/z 135 and M -135 in the FABMS/MS of 2 (Table 2). The fragment ion peaks in the FABMS/MS of 2 at m/z 286 and at m/z 313, 243, 217, and 159 showed the sequences Arg-MeAsp and Adda-Glu--Ser-Ala, respectively (Table 2). These observations revealed the structure of 2 to be [L-Ser 7]microcystin-RR, as shown in Fig. 1 . Structure of toxin 7 The mol. wt and molecular formula of 7 were the same as those of 1 (Table 1). The amino acid analysis data for 7 showed an unknown amino acid, which had a longer

Two New Microcystins from Anabaena spp .

146 3

retention time than other amino acid components on Waters Pico Tag HPLC (retention time, 10.0 min), together with Ala, Asp, Arg, Glu, and Ser (Table 3). The fragment ion peaks at m/z 135 and M -135 in the FABMS/MS of 7 showed the Adda unit as the seventh amino acid component (Table 2). The total mass of the six identified amino acid residues; Adda, Ala, Asp, Arg, Glu, and Ser was 871, which is 127 mu less than the mol. wt of 7. The residual weight (formula) 127 (C7H,3NO) is 14mu (CH 7) higher than that of a Leu or Ile residue (113, C'H ,NO). The differences in the amino acid components between 1 and 7 were observed with the replacement of McAsp and Leu in 1 by Asp and the unknown amino acid (14 mu higher than Leu) in 7, respectively, which results in 7 having the same mol. wt (formula) as that of 1 . The sequence Arg-Asp was deducted from the FABMS/MS fragment ion peak at m/z 272, which is 14 mu less than the corresponding peak of 1 and 2 (286, Arg-McAsp), and Adda-Glu-Ser-Ala was confirmed by peaks at m/z 379, 313, 243, 217, and 159 (Table 2). Thus, the structure of 7 is proposed to be [D-ASp 3,L-SerJmicrocystin-XR, where X would be a Leu homologue, whose structure has not been assigned because of inadequate sample size and the lack of an authentic amino acid sample . The assignment of the structure of the leucine homologue and the stereochemistry of Arg will be made as more material becomes available. Conclusion

Two new L-Ser variants at the Dha unit of [Dha7]microcystins-LR (3) and -RR (4), that is, [L-Serlmicrocystins-LR (1) and -RR (2) were isolated from cultured Anabaena sp. strain 202 Al and assigned structures using amino acid analyses on a Waters Pico Tag HPLC system and a chiral GC capillary column (Chirasil Val III), FABMS, HRFABMS and FABMS/MS . The other strain, 202 A2, also produced toxin 1. We have previously reported Mser and Ser variants including [Mserlmicrocystin-LR from a water bloom of Microcystis spp. collected from Homer Lake (NAMIKOSHI et al., 1992x), [ADMAddas,Mserlmicrocystin-LR from cultured Nostoc sp. strain 152 (SIvoNEN et al., 1992x), and [L-Serlmicrocystin-HtyR from cultured Anabaena sp. strain 66 (NAmncosm et al., 1992b). Since serine is the biosynthetic precursor of dehydroalanine (PEARCE and RINEHART, 1979), toxins 1 and 2 described in this paper as well as the above compounds just listed would be biosynthetic precursors of the corresponding dehydroamino acid derivatives. Acknowledgement.-This study was supported in part by grants from the National Institutes of Allergy and Infectious Diseases (AI 04769) and of General Medical Sciences (GM 27029) to K .L.R. and by a subcontract from the former grant to W.W.C . Research at the University of Helsinki was supported by grants from the Academy of Finland, the Maj and Tor Nessling Foundation, and University of Helsinki. REFERENCES Borm, D . P ., Tu~, A. A ., Wtmt& P. L ., VtLtDEN, C . C., Kauom H., WtLLtAtrs, D . H ., SANnKAaN, S ., Scant, R . J . and HAioroND, S . J . (1984) The structure of cyanoginosin-LA, a cyclic heptapeptide toxin from the eyanobacterium Microcystis aerugirwsa, J. Chem . Soc. Perkier Trans. 1, 2311-2318 . CAawcHAE., W . W . (1989) Freshwater cyanobacteria (blue-green algae) toxins. In: Natural Toxins: Characterization, Pharmacology and Therapeutics, pp. 3-16 (OwNBY, C. L. and ODELL, G. V ., Eds). Oxford : Pergamon Press. CARu cHAEL, W . W. (1992) Cyanobacterial sooondary metabolites-the cyanotoxins . J. Appl. Bact. 72,445-459 .

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