Isolation of two toxic heptapeptide microcystins from an axenic strain of Microcystis aeruginosa, K-139

ToslcoN Vol . 29, No. 4/5, pp . 479-489, 1991 . Prioted in Oreat B~tain .

0041-0101/91 53.00+ .00 Q c 1991 Pagrmon Prw pk

ISOLATION OF TWO TOXIC HEPTAPEPTIDE MICROCYSTINS FROM AN AXENIC STRAIN OF MICROCYSTIS AERUGINOSA, K-139

KEN-ICHI HARADA,1 KIYOSHI OGAWA, I KENJI MATSUURA,I HIDETOSHI NAGAI, 1 HIDEAKI MURATA,1 MAKOTO SUZUKI,1 YOSHIKO ITEZON0,2 NOBORU NAKAYAMA,Z MAKOTO SHIRAI 3 and MASAYASU NAKAN04 'Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468, Japan, =Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247, Japan, 'Department of Agricultural Chemistry, Ibaraki University, Ami, Ibaraki 300-03, Japan and'Department of Microbiology, Jichi Medical School, Tochigi 329-04, Japan (Received

6 August 1990 ;

accepted

18

October

1990)

K. HARADw, K. OGAWA, K. MATSUURA, H. NAGAI, H. MURATA, M. $UZUKI, Y. ITEZONO, N. NAKAYAMA, M. SHiRAI and M. NAKANO . Isolation of two toxic heptapeptide microcystins from an axenic strain of Microcystis aeruginosa, K139. Toxicon 29, 479-489, 1991 .-Two toxic heptapeptides were isolated from an axenic Microcystis aeruginosa strain, K-139. Using mainly a non-destructive NMR method, we determined the structure of the major toxin to be 7-desmethylmicrocystin LR which lacks an N-methyl group of the dehydroalanine moiety of microcystin LR . The minor toxin was deduced to be 3,7-didesmethylmicrocystin LR . The chromatographic and NMR analyses of 7-desmethylmicrocystin LR were compared with those of 3-desmethylmicrocystin LR .

INTRODUCTION OVER 10 hepatotoxic peptides have been isolated from cyanobacteria, Microcystis, Anabaena and Oscillatoria (CARMICHAEL, 1988, 1989 ; GATHERCOLE et al., 1987 ; STONER et al., 1989). They are called microcystins and have the general structure cyclo(-o-Ala-t,-Xn-erythro-ß-methyl-Asp-L-Z-Adda-D-Glu-Mdha-), where Adda is 3-amino-9-methoxy10-phenyl-2,3,8-trimethyldeca-4,6-dienoic acid and Mdha is N-methyldehydroalanine. The letters X and Z refer to two variable z-amino acids. X is arginine (R), leucine (L) or tyrosine (Y) and Z is alanine (A), arginine (R), tyrosine (Y) or methionine (M) (CARMICHAEL et al., 1988). We have established an isolation and analysis method for these peptides (HARADw et al., 1988a, b) and isolated microcystins LR, YR, RR and LA (HARADA et al., 1988a; WATANABE et al ., 1988), and geometrical isomers of microcystins LR and RR (HARADw et al., 1990a, b) . We reported (OHTAKE et al., 1989) that six out of twenty Microcystis strains isolated from toxic waterblooms in Lake Kasumigaura became axenic by our method (SHIRAI et al., 1989). We also reported that a toxic and axenic strain of six of Microcystis aeruginosa, K-139 produced at least two toxins which show hepatotoxicity similar to that of 479

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microcystins, but neither toxin corresponds stucturally to known microcystins, LR, YR, RR and LA. In the present paper we describe the isolation and structural determination of the toxins from K-139 . MATERIALS AND METHODS Organism Microcystis aeruginosa, K-139 was purified from waterblooms that were collected from Lake Kasumigaura on 24 September 1985 (OxrNCt: et al., 1989) . Cells were lyophilized prior to toxin extraction . Purification of toxins Dried cells (4.29 g) were extracted with 5% aqueous acetic acid three times for 30 min while stirring . The combined extracts were centrifuged at 9300a for 1 hr, and the supernatant was collected and applied directly to a preconditioned reversed phase silica gel column (12 x 4.3 cm LD., Chromatorex ODS, 100-200 mesh, FujiDavison Chemical Ltd, Tokyo, Japan) . The column was washed with water (150m1), followed by water :methanol (8 :2, 500 ml) and then eluted with methanol (400 ml) to give 72.2 mg of the toxin in the fraction . The fraction was chromatographed on a preconditioned reversed phase silica gel (9l x 1 .1 cm LD ., Chromatorex ODS) with methano1 :0.05 M sodium sulfate (6 :4) as the mobile phase; consequently, 22.7 mg of semipure Toxin A and 7 .0 mg of semipure Toxin B were obtained . Both fractions containing toxins were desalted with ODS cartridges (Baker, Phillipsburgh, NJ, U .S.A .). Toxin A (14.3 mg) was finally purified by TOYOPEARL HW-40 column chromatography (91 x 1 .2 cm LD ., TOSOH, Tokyo, Japan) . Toxin B was purified by preparative HPLC with methano1:0 .05 M sodium sulfate (6 :4) as the mobile phase . Desalting of the desired fraction with an ODS cartridge gave almost pure toxin B (1 .5 mg) . HPLC The following two systems were used : (1) JASCO 880-PU pump and 875-UV u.v . detector and (2) Hitachi 665 pump and 665A u.v . detector. Separation was carried out under reversed phase isocratic conditions with a Nucleosil SC 18 column (150 x 4 .6 mm, Chemco Scientific Co ., Osaka, Japan) for analysis and a Cosmosil SC18-P column (250 x l0 mm, Nacalai Tesque, Kyoto, Japan) for preparative separation and mobile phases of methano1:0 .05 M phosphate buffer (pH 3) (58 :42) and methano1:0.05 M sodium sulfate (6 :4) . The flow rate was 1 ml/min for analysis and 2 ml/min for the preparative separation, and these toxins were detected with u.v. absorbance at 238 nm .

TLC A silica gel plate (Kieselgel 60 F~ E . Merck, Darmstadt, F.R .G.) was used . Two solvent systems, chloroform :metbanol:water = 65 :35:10 (lower phase) and ethyl acetate :isopropanol :water = 4:3 :7 (upper phase) were employed. The samples were detected with iodine and short wavelength u.v . light .

Amino acid analysis Purified peptides were hydrolyzed in 6 N HCl at 105°C for 24 hr prior to amino acid analysis. The liberated amino acids were derivatived with phenylisothiocyanate before putting on the column and the phenylthiocarbamyl amino acids analyzed with a Waters (Milford, MA, U .S .A .) Pico Tag HPLC system . The derivatives were loaded onto a C18 (150 x 4 .6 mm) column and eluted over 8 min with a 0 to 60% gradient of acetonitrile in 0.138 M aqueous sodium acetate. The flow rate was maintained at 1 ml/min . The eluted compounds were detected by u .v. absorption at 254 nm .

Chemical analysis Fast atom bombardment (FAB) mss spectrometry was a double focusing JEOL (Tokyo, Japan) JMS-HXl 10 mass spectrometer was carried out. The fast atom beam was operated at 6 kV using xenon gas and the spectrometer was operated at 10 kV accelerating potential . A mixture of glycerol and 1 N hydrochloric acid was used as the matrix . The sample solution was adjusted to lOpg/ml . Nuclear magnetic resonance (NMR) spectra were recorded on a 400 MHz JEOL JNM-GSX 400 and a GX400 spectrometers equipped with 'Hf ~ dual probe . The samples were dissolved" in 0.7 ml of CD,OD with

7-Desmethylmicrocystin LR from Cyanobacteria

481

Toxin A

FIG. 1. HIGH PERFORMANCE LIQUID CHROMATOGRAM OF THE TOXIC FRACTION AFTER PASSAGE THROUGH THE ODS SILICA GEL. Column, Nucleosil SC18 (150 x 4.6 mm); mobile phase, methano1 :0.05 M phosphate buffer, pH 3 (58:42); flow rate, 1 ml/min ; detection, 238 nm .

tetramethylsilane as an internal standard. Operating conditions were described in our previous paper (HARADA et al., 19906) . u.v. spectra and specific rotations were measured with a Shimadzu (Kyoto, Japan) u.v .-vis. spectrophotometer and a JASCO (Tokyo, Japan) DIP-18l polarimeter, respectively.

RESULTS AND DISCUSSION

The cell samples were treated according to our isolation method (HARADA et al., 1988a) . The resulting toxic fraction contained two toxic peaks, abbreviated as Toxins A and B, in the high performance liquid chromatrogram (Fig. 1). After chromatography on an ODSsilica gel the two crude toxins were obtained and the main toxin (14.3 mg) was finally purified by TOYOPEARL HW-40 chromatography . Toxin B was further purified by preparative HPLC, but was not in a pure state. Comparison by HPLC of the toxins obtained with the known microcystins LA, LR, YR and RR suggested that they are new microcystins . Amino acid analysis of Toxin A gave equimolar amounts of Glu, ß-Me-Asp, Arg, Ala and Leu, which is the same as the amino acid composition of microcystin LR except for the absence of N-methylamine . Fast atom bombardment (FAB) mass spectrum of Toxin A indicated that the toxin has a mol. wt of 980, which is 14 mass units less than that of microcystin LR. Table 1 shows the physicochemical properties of Toxin A together with those of microcystin LR and 3desmethyhnicrocystin LR which has the same molecular weight as Toxin A, and has Asp instead of ß-Me-Asp in microcystin LR (Fig. 2). In the DEPT (distortionless enhancements by polarization techniques) NMR spectrum of Toxin A, 46 carbon signals were observed, indicating that Toxin A possesses one carbon less than microcystin LR and, in fact, the N-methyl signal of the Mdha moiety was TABIE 1. PHYSICOCFIEMICAL PROPERTII75 OF MICROCYSfIN LR, 3-DESMEfHYLMICROCYSTIN LR AND TOXIN A

Appearance [a]n u.v . .im,°H(log s) FAB MS (m/z) (Gly+ 1 N HCI)

Microcystin LR

3-Desmethylmicrocystin LR

Toxin A

white powder -93.0° (c 0.50, McOH) 238 nm (4.60) 995 (M+H)'

white powder -78.6° (c 0.30, McOH) 238 nm (4.50) 98l (M+H)'

white powder -87.6° (c 0.94, McOH) 238 nm (4.67) 981 (M+H)'

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Mlaocyatln lA : &De~m~CyhnfonooyaCn LR : 7-Deem~UyY~ia~o~ LR : 3,7-DMmrt~tfylmkrocysbn LR :

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LR Arro 3,7-DIDFSS~LAncROCYSrtN LR .

not found (data not shown) . This result was also supported by the 'H-NMR spectra of Toxin A, microcystin LR and 3-desmethylmicrocystin LR. Table 2 summarizes the 'HNMRspectral data of the olefin region, N-methyl and ß-Me-Asp of the three toxins . The two olefinic protons of Mdha in Tôxin A appear with an upper field shift compared with those of microcystin LR and 3-desmethylmicrocystin LR, and no N-methyl signal is observed . These results strongly suggest that Toxin A is 7-desmethylmicrocystin LR which lacks an N-methyl group in the dehydroalanine moiety of the microcystin LR (CARbIICHAEL et al ., 1988). We have established a non-destructive method using a combination of these 2D-NMR techniques, DQF-COSY (double quantum filter correlation spectroscopy) (PIANTIIdI et al ., 1982), HMQC ('H-detected multiple quantum coherence) (BAx and SusRAMANU,x, 1986) and HMBC (heteronuclear multiple bond correlation) (BAx and SUMMERS, 1986) for structure determination of microcystins . The method was successfully applied to assign all carbons and protons of microcystins so that the segence of constituent amino acids in TABLE 2. 'H-NMR SPECTRAL DATA OF THE OLEFIIY REGION, N-e~IxsrL GROUP wND ß-Me-Asp of MlcaocvsnN LR, 3-DFS,èsETHYLAIICROCYSTIN LR AND TOXiN A Microcystin LR

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microcystins were definitely confirmed (HARADA et al., 1990b). Thus, this procedure was also used for sequencing of amino acids in Toxin A. The DQF-COSY spectrum (400 MHz) of Toxin A is shown in Fig . 3. In the lower field region the olefinic and aromatic protons from Adda and Dha (dehydroalanine) appear . The connectivity of the cross peaks A, B, C and D indicates a coupling network, 6.23 ppm (Adda H-55.51 ppm (Adda H-4}-4.52 ppm (Adda H-3}3.01 ppm (Adda H-21 .05 ppm (Adda H-11). Similarly, the connectivities of cross peaks E and F, and G, H, I and J indicate other coupling networks, 1 .63 ppm (Adda H-125.40 ppm (Adda H-72.58 ppm (Adda H-8) and 1 .00 ppm (Adda H-132;58 ppm (Adda H-83.26 ppm (Adda H-9}-2 .68 ppm (Adda H-l0a), 2.81 ppm (Adda H-lOb), respectively . These spectral data for the Adda moiety are almost identical to those of microcystin LR, so we concluded that

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Toxin A also has Adda . There are six signals due to H-2s of the constituent amino acids at 4.80-4.10 ppm (H-3 only in the case of Adda). Each H-2 was assigned by connecting the protons of the side chains in the upper field region as follows: Glu, 4.22 ppm; Ala, 4.67 ppm; Leu, 4.31 ppm; ß-Me-Asp, 4.40 ppm; Arg, 4.45 ppm and Adda (H-3), 4.52 ppm (Fig. 4). The'H-NMR spectral data of Toxin A are shown in Table 3 together with those of microcystin LR. The HMQC method is quite effective for determining one bond coupling relation between carbon and hydrogen atoms when the sample amount is limited . The HMQC spectrum of Toxin A enabled us definitely to assign all carbon signals except for the carbonyl carbons (Table 3). The sequence of the constituent amino acids in Toxin A was determined by the HMBC method which can sensitively detect the long range couplings (two (2Jce) and three (3Jce) bond couplings) between carbon and hydrogen atoms. First, the carbonyl carbons were assigned by correlation of the carbonyl carbon and its own H-2 assigned via the zJcH coupling . Next, the relationship between the carbonyl carbon and H-2 of the neighboring amino acid was confirmed by 'JcH coupling . There are nine carbonyl carbon signals at 180-165 ppm in the HMBC spectrum . The signal with the highest chemical shift (166 .5 ppm) was assigned to the carbonyl carbon of the Dha moiety because it has the cross peaks due to'JcH with two Dha H-3s . The connectivity between Dha and Ala was confirmed by the presence of the cross peak from the carbonyl carbon of Dha to Ala H-2. Figure 5 shows the HMBC spectrum of Toxin A and the following carbonyl carbons are assigned from the cross peaks due to the ZJcH: C (Ala, 175.1 ppm), E (Glu-1, 179.2 ppm); G, (Leu, 175.5 ppm); I (ß-Me-Asp-1, 176.7 ppm); M (ß-Me-Asp-4, 178.8 ppm) and N (Glu-5, 176.2 ppm). Also the cross peak F of Adda H-3 and Adda C-1 (176 .6 ppm) is observed. The remaining signal (172 .2 ppm), therefore, was assigned to the carbonyl carbon of Arg. The cross peaks due to the'JcH couplings, GZ (Ala C-1 and Leu H-2), H (ß-Me-Asp C-4 and Arg H-2), J (Leu C-1 and ß-Me-Asp H-2) and L (Adda C-1 and Glu H-2) were observed, so that the following sequences, Adda-Glu and Dha-Ala-Leu-ß-Me-Asp-Arg were confirmed . It was impossible to correlate Glu with Dha by the HMBC method because of the lack of an N-methyl group in Toxin A (HARADA et al ., 1990b) and the cross peak due to the 3Jctt coupling between Arg and Adda was ambiguous. However, since the assigned 'H and "C NMR signals are essentially identical with those of microcystins LR (fable 3), we conclude that toxin A has the same sequence and monocyclic structure as known microcystins . Additionally, both a-carboxylic acids of Glu and ß-Me-Asp are found to be free because the H-2s were shifted to a lower field in the NMR measurement under acidic conditions (Oor et al ., 1989). These results show that Toxin A is 7-desmethylmicrocystin LR and this is the second case of desmethylated microcystin LR (3-desmethylmicrocystin LR is the first case) (KRLSrnvnMURTx~t et al ., 1989). Toxin B was obtained as an amorphous powder . It shows an absorption maximum at 238 nm (log e 4.42) in the u.v. spectrum and the protonated molecule (M + H)+ at m/z 967 in the FAB mass spectrum . These data suggest that Toxin B corresponds to 3,7-didesmethylmicrocystin LR whose constituent amino acids, Asp and Dha are replaced by ßMe-Asp and Mdha in microcystin LR, respectively (KRISHNAMURTHY et al., 1989). However, because only a limited amount was obtained, the structure determination remains to be solved . Although we have so far only determined the LDP valves for both toxins, they seem to possess similar biological activities to the conventional microcystins .

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Recently desmethylated microcystins have been isolated from cyanobacteria. KRISHNAet al. (1989) structurally characterized 3-desmethylmicrocystin LR and 3.7-didesmethylmicrocystin LR by tandem mass spectrometry. MERILUOTO et al. (1989) determined the structure of 3-desmethylmicrocystin RR produced by Oscillatoria agardhü . We also purified 3-desmethylmicrocystin LR from a Japanese bloom sample (HARAI)A et al., 1990a) and 7-desmethylmicrocystin LR in the present study. Although the physicochemical properties of both desmethylmicrocystins LR are similar, they can be differentiated from each other by the chemical shifts of the exomethylene signals in the Mdha (Dha) and the presence or absence of an N-methyl signal (Table 2). Figure 6 shows the thin layer chromatogram (normal phase) and high performance liquid chromatogram (reversed phase) of both toxins . They can also be chromatographically separated with the following mobile phases, ethyl acetate:isopropanol :water (upper phase, 4:3:7) for TLC and methano1 :0.05 M sodium sulfate (55:45) for HPLC (HARADA et al., 19ßßa, b). In the present paper we describe the isolation of 7-desmethylmicrocystin LR from an axenic Microcystis aeruginosa, K-139, and we succeeded in the structural determination by our non-destructive NMR method . Additionally we established the chromatographic method using normal phase TLC and reversed phase HPLC for separation of 3- and 7-desmethylmicrocystins LR. Probably desmethylated microcystins other than microcystins LR and RR will be found and the results presented in this paper will be effectively applied to the structural characterization of unknown desmethylmicrocystins . MuRTHV

Acknowledgement-We

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are grateful to Professor WAYNE W. CARMICHAEL, Wright State University, U .S .A . for

7-Desmethylmicrocystin LR from Cyanobacteria

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