Nostocyclamide M: a cyanobacterial cyclic peptide with allelopathic activity from Nostoc 31

Phytochemistry 57 (2001) 613±619

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Nostocyclamide M: a cyanobacterial cyclic peptide with allelopathic activity from Nostoc 31 Friedrich JuÈttner a,*, Albena K. Todorova a, Nadja Walch b, Wolfgang von Philipsborn b a

Institute of Plant Biology, Limnology, Limnological Station, University of ZuÈrich, Seestrasse 187, CH-8802 Kilchberg, Switzerland b Institute of Organic Chemistry, University of ZuÈrich, Winterthurerstrasse 190, CH-8057 ZuÈrich, Switzerland Received 27 July 2000; received in revised form 13 November 2000

Abstract A cyclic peptide containing thiazole and oxazole moieties was isolated from Nostoc 31 and its structure determined by chemical degradation detailed NMR and mass spectroscopic analyses. The compound is stereochemically pure and closely related to nostocyclamide in which d-valine is replaced by d-methionine. Therefore, it di€ers from tenuecyclamide C reported to contain lmethionine. It shows allelopathic activity against Anabaena 7120, but is inactive against grazers. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Cyanobacteria; Nostoc; Cyclic peptide; Thiazole; Allelopathic compounds; NMR

1. Introduction Thiazole-containing cyclic and linear peptides have recently gained much pharmacological interest. Some of them turned out to be potent agents against multidrug resistance (Williams and Jacobs, 1993; Ogino et al., 1996) that develops in cancer cells resistant to chemotherapy. Marine seahares and ascidians were the ®rst sources of several structural variants, among them dolastatins, patellamides and tawicyclamides (Michael and Pattenden, 1993; McDonald et al., 1992). The extreme variability of concentration of these compounds in the animals was taken as an indication that in ascidians these compounds were accumulated from symbiotic prochlorophytes and in seahares from cyanobacterial food rather than synthesized by the animals. In recent years similar cyclopeptides have been isolated from cultured freshwater and terrestrial cyanobacteria. The importance of the diet for the accumulation of cyclic peptides became clear when the same compound was isolated from the ascidian Lissoclinum bistratum and a pure cyanobacterial culture (Westiellopsis proli®ca). The compounds were independently named * Corresponding author. Tel.: +41-1-716-1210; fax: +41-1-7161225. E-mail address: [email protected] (F. JuÈttner).

cycloxazoline (Hambley et al., 1992) and westiellamide (Prinsep et al., 1992) In Stigonema dendroideum, three cyclic peptides were found which were called dendroamides (Ogino et al., 1996). They exhibited a basic structure in which two thiazole rings and a methyloxazole moiety were connected via amide bonds and which di€ered only in their substituents. Nostoc spongiaeforme was the source of several tenuecyclamides (Banker and Carmeli, 1998) and raocyclamides were isolated from Oscillatoria raoi (Admi et al., 1996, Freeman and Pattenden, 1998). Recently we described nostocyclamide (2) that was isolated from a freshwater Nostoc strain and exhibited grazer toxicity and allelopathy against related cyanobacteria (Todorova et al., 1995). In this investigation we report on another cyclic peptide from this cyanobacterium that reveals anticyanobacterial activity. 2. Results and discussion During the investigation of nostocyclamide from the freshwater cyanobacterium Nostoc 31 (Todorova et al., 1995) a second component was observed that had the features of a cyclic peptide, but the small amounts available at that time did not allow the structural elucidation. In the present study this component was isolated

0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(00)00470-2

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in amounts that were sucient to determine its structure. Several features of this compound resembled those of nostocyclamide, such as the strong UV absorption and the intense sulfur signal shown by a sulfur speci®c detector during separation on a GC column, and some identical products that were obtained after acid hydrolysis. Mass spectrometric, chemical, and NMR studies showed that this compound has the same basic structure as the one for nostocyclamide, but the valine moiety is replaced by methionine. Using the one letter code of amino acids we named this compound nostocyclamide M (1). Nostocyclamide M exhibits a quasi-molecular ion of m/z 507 [M+H]+ when ESMS is applied in the positive mode. After GC separation of the underivatized molecule electron impact ionization a€ords a molecular ion (EIMS m/z 506 [M+]) and important fragment ions that are indicative for thiazole (m/z 139) and oxazole (m/z 136) fragments. Intense sulfur-containing fragment ions at m/z 47 (CH3±S), m/z 61 (CH3±S±CH2) and m/z 75 (CH3±S±CH2±CH2) are observed along with the corresponding cleavage products of the molecule, m/z 458 (M-48 [CH3±SH]), m/z 445 (M-61 [CH3±S±CH2]) and the base peak m/z 432 (M-74 [CH3±S±CHˆCH2]). The molecular ion and the base peak obtained with the 15N labelled molecule exhibit a shift of 6 mass units from m/z 506 to 512 and m/z 432 to 438, and indicate the presence of six nitrogen atoms in the molecule. The products of acid hydrolysis, that were tri¯uoroacetylated and transferred into the methyl ester for GC±MS analysis, a€ord alanine and two thiazole-containing amino acids

derived from ring B and C. The derivative of ring B is identical in the retention time and mass fragmentation pattern (m/z 268 [M+] and strong fragment ions at m/z 236, 208 and 139) with that obtained from nostocyclamide. The derivative of ring C (m/z 342 [M+] exhibits prominent sulfur-containing fragment ions at m/z 47 [CH3±S], m/z 61 [CH3±S±CH2], m/z 75 [CH3±S±CH2± CH2] and m/z 268 [M-74] as the base peak. The fragmentation pattern is consistent with the presence of a CH3±S±CH2±CH2 substituent. The pure compound is sparingly soluble in MeOH and MeCN but easily soluble in DMSO and CH2Cl2. It crystallizes from aqueous solutions in very thin needles. Suitable crystals for X-ray di€raction have not been obtained so far. The molecular formula C20H22N6O4S3 for nostocyclamide M was deduced from mass spectral data and NMR spectroscopy. Nostocyclamide M was subjected to a detailed 1H and 13 C NMR investigation. The 1D proton and carbon-13 and the 2D 1H,1H and 1H,13C correlated spectra were obtained in CDCl3 solution on a 600 MHz spectrometer. For further details see the experimental section. The proton spectrum exhibits signals for the following groups: 1 CH3(CH), 1 CH3(C), 1 CH3(S), 3 CHAHB, 1 CH(CH3), 1 CH(CH2), 2 H-C(sp2), and 3 NH signals in the 8.5-8.6 ppm region, corresponding to a total of 22 hydrogen atoms (Table 1). The carbon-13 spectrum obtained with proton decoupling shows eight signals in the aliphatic region and 12 signals in the sp2-carbon range. Based upon 1H,13C 2D correlation experiments (HSQC and HMBC) there are 3 CH3, 3 CH2, 2 (sp3)

F. JuÈttner et al. / Phytochemistry 57 (2001) 613±619

CH, 2 (sp2)CH, and 10 nonprotonated carbon atoms (Table 1). Thus, nostocyclamide M has a C20H22 skeleton just as nostocyclamide. Closer inspection of the 1D spectra reveals that there are no signals for an isopropyl group (valine) while the typical AB system of the glycine methylene group and the CH3CHNH group of alanine are still present. As a new feature, compared with nostocyclamide, the aliphatic region of the proton spectrum reveals the presence of a ±CH(CH2CH2SCH3) side chain for which we also have strong evidence from the mass spectrum (M74, M-75). On the other hand, the characteristic CH3C(O) signal from the oxazole methyl group and the two isolated thiazole CH signals of nostocyclamide are still present in the new compound which points to the same basic skeleton of both structures. The tentative conclusions are corroborated by 2D COSY and TOCSY 1H,1H-correlations and 1H,13C 2D HSQC and HMBC correlations. The most signi®cant correlation cross peaks are also listed in Table 1 and support the common skeleton for the two nostocyclamides. Moreover, from the TOCSY pattern the new side chain can be identi®ed as arising from methionine (HN(5)±C(12)H±C(18)HAHB±C(19)HAHB±S±CH3) that is replacing valine in nostocyclamide.

615

Nostocyclamide M (1) thus proves to have the same constitution as tenuecyclamide C (4) (Banker and Carmeli, 1998), and the 1H and 13C chemical shifts can be fully assigned. In fact, the 1H and 13C spectral data of the two compounds are virtually identical within experimental error limits. However, tenuecyclamide C di€ers in the UV absorption and was reported to contain l-methionine whereas our chemical degradation of nostocyclamide M gave d-methionine as the dominating sulfur containing cleavage product (vide infra). The excellent agreement of the NMR data of both compounds, in particular of the methionine side chain and its immediate surrounding is not what one would expect for a diastereoisomeric relationship. The open question remains to be settled by X-ray di€raction of suitable crystals. The absolute con®guration of the two chiral centers of nostocyclamide M was determined with the amino acids obtained after acid hydrolysis. The tri¯uoroacetylated methyl ester of alanine resulting after methanolysis from the oxazole ring was separated on a chiral GC column to give the pure l-stereoisomer. To analyze the con®guration at C(12), the thiazole ring was reacted with ozone that gave methionine sulfone upon acid hydrolysis. The d-enantiomer was found to represent 91% of the total

Table 1 1 H and 13C NMR chemical shifts (/ppm), 1H,1H coupling constants (J/Hz), and long-range C,H correlationsa  (H) C(1) C(2)H C(3) C(4) C(5) C(6) C(7)H2 C(8) C(9)H C(10) C(11) C(12)H C(13) C(14)H C(15) C(16)H3 C(17)H3 C(18)H2 C(19)H2 C(20)H3 N(1)H N(3)H N(5)H a

J (H,H) c

5.150 (dq)

HA:4.979 (dd) HB:4.814 (dd) 8.203 (s) 5.795 (dt) 8.187 (s) 1.695 (d) 2.700 (s) HA: 2.514 (ddt) HB: 2.203 (ddt) HA: 2.616 (ddd) HB: 2.347 (ddd) 2.075 (s) 8.62 (s) 8.51 (t) 8.61 (d)

From 1H-detected HMBC experiments. Refers to one-bond C,H coupling multiplicity. c,d,e Pairwise ambiguous. b

 (C) 160.03 45.03 161.60 153.88 128.34 160.91 41.05

b

(s) (d) (s) (s) (s) (s) (t)

165.34 (s) 124.63d (d) 148.74e (s) 159.77c (s) 51.03 (d) 169.34 (s) 124.24d (d) 149.11e (s) 20.24 (q) 11.59 (q) 37.37 (t) 28.99 (t) 15.42 (q)

5.6; 6.7

17.7; 4.7 17.7; 3.3

7.4; 5.4

N(1)H C(16)H N(1)H; C(2)H; C(16)H C(17)H C(17)H C(7)HA; C(17)H C(7)HAHB; C(9)H C(9)H N(5)H C(18)HAHB; C(19)HAHB C(12)H; C(14)H; C(18)HAHB C(14)H C(2)H

6.7 14.1; 14.1; 13.1; 13.1;

Correlation C±H

9.8; 5.2 10.0; 5.7 10.0; 5.1 10.0; 5.7

C(12)H C(12)H; C(18)HAHB, C(20)H C(19)HAHB; C(18)HB

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methionine, indicating that racemization has taken place only at a low rate. The presence of l-alanine and d-methionine sulfone upon ozone treatment and acid hydrolysis was also con®rmed by Marfey's analysis on a C18 HPLC column by superposition with authentic compounds. Stereochemically pure amino acids were only obtained when nostocyclamide M was analyzed that had undergone a short isolation procedure. Extracts separated on silica gels exhibited upon HPLC separation several isomers of nostocyclamide M that eluted closely to each other. Stereochemical analysis of these isomers gave mixture of the stereoisomeric alanines and methionine sulfones. Because these isomers turned out to be artifacts, they were not further investigated, and only freshly isolated material served for the ®nal analysis. In the HPLC chromatograms of cartridge-puri®ed extracts of Nostoc an additional peak was observed that eluted much earlier (8.2 min) than nostocyclamide M (12.4 min) and exhibited an ESMS that was consistent with the two diastereomeric nostocyclamide M sulfoxides. These compounds were present in much lower concentrations in fresh extracts that have not been concentrated in a rotary evaporator. When puri®ed nostocyclamide M was brought to dryness and redissolved, around 12% of the nostocyclamide M was oxidized to these sulfoxides. The same compounds were obtained by treatment of nostocyclamide M with hydrogen peroxide. Fresh extracts from Nostoc that were isolated under strict exclusion of oxygen under a nitrogen gas atmosphere in a closed apparatus (Wendel and JuÈttner, 1996) a€orded only a small signal in the HPLC chromatograms of which it was not clear whether it was the sulfoxides or another contaminant. These experiments indicate that the sulfoxides are not, or in only very small amounts, present in the live Nostoc and are in the observed amounts actually isolation artifacts. Small portions of 100 ml standing cultures of Nostoc (0.7 mg d.w.) were used to determine quantitatively nostocyclamide M and nostocyclamide by integration of the UV absorption peaks of HPLC chromatograms. For calculating the concentrations of the nostocyclamide M sulfoxides the same molar extinction coecient was used as for nostocyclamide M. The concentration of nostocyclamide M and nostocyclamide M sulfoxides, which were taken together, and nostocyclamide were 10 and 26 mg per mg chlorophyll a, respectively, in a four week old standing culture. The low solubility of nostocyclamide M in water did not allow us to perform bioassays in liquid media without addition of solubilizers. Even at low concentrations of around 4 nM white needles, the typical crystalline form of nostocyclamide M, were observed. The much lower water solubility in comparison to nostocyclamide caused by the (CH2)2±S±CH3 group may be the reason that toxicity was not observed in the Thamnocephalus

platyurus bioassay. However, allelopathic activity was easily detectable by the agar plate di€usion assay with cyanobacteria as the indicator organisms. Anabaena 7120 was more sensitive than Anabaena P-9, and cleared zones in the lawn of Anabaena 7120 were obtained when 217 nmol nostocyclamide M were spotted on the mineral ground agar. Nostocyclamide M is a member of the cyclic peptides which have been found in several structural variations in cyanobacteria and marine ascidians. Most likely the ascidians (Lissoclinum) accumulate these compounds from symbiotic prochlorophytes, a subgroup of cyanobacteria, and are not the producers. Peptides of this type are most probably synthesized nonribosomally by a multienzyme thiotemplate mechanism (Marahiel et al., 1997) on peptide synthetases. Amino acids are ®rst activated by ATP, then bound as a thioester, and ®nally a peptide bond formation takes place. In the state of the thioester, epimerases are known that convert l-amino acids into their d-con®guration. The strict control of the amino acid con®guration in the cyclic peptides is evident by the occurrence of compounds that are stereochemically pure and characteristic in their con®guration for a particular organism. Racemates which have also been observed are most likely produced during the rather lengthy isolation procedures. In the nostocyclamides 1 and 2, tenuecyclamide C (4), and raocyclamide A (3) the alanine moiety has the lcon®guration. Both valine and methionine, which replace each other in nostocyclamide and nostocyclamide M, exhibit d-con®guration, as does phenylalanine in raocyclamide A. An exception is methionine that is placed in the same position in tenuecyclamide C and has been reported to be in the l-con®guration. The dendroamides di€er essentially as all amino acids are in the d-con®guration, c.f. dendroamide B (5), while the structurally related bistratamide C (6) that was isolated from Lissoclinum (Foster et al., 1992) had the amino acids in the all l-con®guration. It is interesting to note that structurally closely related cyclic peptides are found in phylogenetically very distant cyanobacteria. The heterocyst-forming Nostoc 31 and N. spongiaeforme are closely related to each other but Oscillatoria raoi belongs to the very distant hormogonia-forming group of cyanobacteria. 3. Experimental 3.1. Physical measurements UV spectra were measured in MeOH on a spectrophotometer Cary 3 (Varian). Low-resolution EIMS were obtained on a combined GC±MS (Fisons MD 800), and ESMS in the positive mode on a mass spectrometer with an electrospray ion source (Vestec VT 201).

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The 1H and 13C NMR spectra were measured at 300 K on a Bruker DRX-600 spectrometer at 600 and 150 MHz, respectively. Solutions of 2±3 mg in 0.4 ml of CDCl3 were used. All chemical shifts are referred to TMS as internal standard. 2D spectra were obtained with standard pulse sequences i.e. double-quantum-®ltered phase-sensitive COSY, TOCSY, HSQC, and HMBC. For the latter experiments a delay time corresponding to nJ(C,H)=9 Hz was used and, therefore, not all conceivable C±H correlation peaks were actually observed. 3.2. Growth The origin of the axenic culture of Nostoc sp. 31 and the conditions of the small and large scale culture to obtain 100-g amounts of wet biomass were described recently (Todorova et al., 1995). 3.3. Analysis The procedure to obtain EI mass spectra after GC separation, the analysis of products of acid hydrolysis and 15N labeling of the molecule were published previously (Todorova et al., 1995). 3.4. Isolation The cyanobacterial biomass (160 g fr. wt) that was stored at 20 C was ®rst suspended in 90 ml H2O. 250 ml of ethanol were added for extraction in an ultrasound water bath (15 min). After centrifugation, the pellets were twice extracted with 125 ml 50% aq. EtOH. The combined extracts were partitioned between aq. EtOH and tert-butyl methyl ether (TBME), and the ether phase was washed several times with water and dried with dry Na2SO4. The TBME was replaced by 320 ml 90% aq. EtOH and supplied to three equilibrated reversed phase C18 cartridges (2 g, Mega Bond Elut, Varian). The ®rst 20 ml of the elutes were collected, brought to dryness in a rotary evaporator, and the residue was dissolved in a minimum of MeOH. 100± 300 ml were injected onto a semipreparative reversed phase HPLC column (25010 mm Spherisorb S 5 ODS, LC-18, 5 mm, Supelco). A gradient (MeOH/H2O; 50:50 in 12 min up to 100% MeOH and 10 min isocratic, ¯ow rate 3 ml/min) was used to separate (in about 50 runs) a mixture of nostocyclamide and nostocyclamide M (180 mg) at Rt 12.6 min. To remove the major part of nostocyclamide from nostocyclamide M, the crystalline residue was extracted four times with 0.5 ml portions of cold MeOH. The enriched residue of nostocyclamide M was dissolved in MeOH and a small amount of CH2Cl2 and separated on a semipreparative HPLC column (Grom-Sil ODS-4 HE, 5 mm, 8.0250 mm) in a 15 min linear gradient (aq. acetonitrile: 0.01% tri¯uoroacetic

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acid (15:85) to 100% acetonitrile). The ®rst eluting peak at Rt 12.4 min was nostocyclamide M and the second at Rt 12.8 min nostocyclamide. The yield of nostocyclamide M was ca. 25 mg and of nostocyclamide ca.100 mg. To obtain nostocyclamide M in high purity, the collected substance was rechromatographed (ca. 100 mg per run) under the same conditions. 3.5. Nostocyclamide M Nostocyclamide M (1): white thin needles, UVlmax (MeOH) 220 nm (" 41000); EIMS m/z (% rel. int.) 506 (27) [M+], 432 (100), 139 (84), 61 (79), 136 (61), 112 (56), 75 (54), 141 (49), 276 (49), 235 (45), 249 (37) and 445 (17). For 1H and 13C NMR data see Table 1. 3.6. Hydrolysis Nostocyclamide M was hydrolysed in 3 M HCl/ MeOH at 110 C for 48 h. The dried residue was treated with tri¯uoroacetic anhydride in CH2Cl2 for 60 min at 80 C. The reagent and solvent were blown o€ with He; the residue was dissolved in 50 ml MeOH. The tri¯uoroacetylated esters were separated on a DB-1301 (30 m, 0.32 mm i.d., 0.25 mm ®lm thickness) with a temperature program of 1 min 70 C, 10 C/min, 250 C isothermal for 20 min. Three tri¯uoroacetylated esters were obtained that could easily be deduced from the structure of the molecule: the alanine derivative (Rt 3.87 min) was identical to a reference compound, the amino acid B (Rt 14.35 min) exhibited ions at m/z 268 (M+, 58%), 236 (100%), 208 (96%), 139 (93%), 69 (82%), 57 (64%), 171 (54%), 45 (52%), 112 (51%), 237 (47%) and the amino acid C (Rt 17.80 min) at m/z 342 (M+, 1.3%), 268 (100%), 61 (69%), 45 (65%), 69 (63%), 208 (56%), 139 (38%), 236 (37%) and 171 (31%). Further indicative ions of this compound were 281 (M-61, 6.2%), 47 (CH3S, 18%) and 75 (CH3SCH2CH2, 13%). 3.7. Analysis of stereochemistry by chiral GC±MS The cleavage of the thiazole moieties by ozone and acid hydrolysis was conducted as stated by Ogino (Ogino et al., 1996). 0.7 mg of nostocyclamide M was dissolved in CH2Cl2 and bubbled for 10 min with a stream of ozone to give a bluish solution. The solvent was removed in a stream of nitrogen and the residue was treated with 200 ml 3 M HCl/MeOH for 24 h at 110 C. The sample was brought to dryness in a nitrogen gas stream and treated with 100 ml TFA in 100 ml CH2Cl2 for 1 h at 80 C. The sample was dried with nitrogen gas and the residue dissolved in 100 ml CH2Cl2. 1-ml samples were subjected to gas chromatography± mass spectrometry on a chiral column (25 m, 0.25 mm i.d., Permabond-l-Chirasil-Val, Macherey & Nagel, DuÈren, Germany) in a temperature program (1 min

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80 C, 5 C/min up to 120 C, 20 C/min at 190 C). The retention times were: d-alanine (Rt 4.71 min), l-alanine (Rt 4.98 min), d-methionine sulfone (Rt 25.54 min), and l-methionine sulfone (Rt 26.02 min). The identity of the stereoisomers was established by comparison with authentic compounds and con®rmed by superposition. Reference substances were obtained from Sigma. 3.8. Analysis of stereochemistry by Marfey's method Nostocyclamide M was treated with ozone as stated above for 10 min; the solvent was evaporated in a stream of nitrogen gas and the residue was subjected to vigorous hydrolysis in 6 M HCl. Marfey's analysis was conducted essentially as described by Ogino (Ogino et al., 1996) on a HPLC column (Grom-Sil 120 ODS 4 HE, 5 mm, 4.6250 mm) at 30 C using a linear gradient of 10±50% acetonitrile and 0.05% aq. triethylamine / phosphate bu€er pH 3. 5-Fluoro-2,4-dinitrophenyl-lalaninamide was used as the reagent. The typical absorption spectra of the amino acid derivatives with maxima at 341 nm and 417 nm were recorded on a Shimadzu LC 10 diode array detector SPDM 10 Avp. dAlanine and l-alanine were separated eciently to determine the l-con®guration by superposition with an authentic compound, however, separation of the methionine sulfone stereoisomers was not achieved with this and another acidic solvent gradient (acetonitrile/ 0.05% aq. TFA according to Fujii et al. (1997). Separation of the latter isomers was achieved applying an acetonitrile/0.05 M ammonium acetate gradient (10± 50% acetonitrile) as stated by Harrigan et al. (1998). lMethionine sulfone eluted at Rt 24.8 min and dmethionine sulfone at Rt 28.6 min. 3.9. Quantitative analysis Fresh wet biomass of 20 ml suspension of Nostoc was collected on a glass ®bre ®lter (Schleicher & Schuell GF 6) by vacuum ®ltration. The ®lter was extracted with 4 ml 85% aq. acetonitrile containing 0.05% tri¯uoroacetic acid. The extract was separated on a HPLC column. The integrated peak areas at 220 nm were used for quanti®cation of the compounds. The molar extinction coecient of nostocyclamide M was used to quantify the nostocyclamide M sulfoxides and a redetermined molar extinction coecient (UVlmax (MeOH) 220 nm (" 40000) for nostocyclamide. 3.10. Bioassays The growth of axenic cyanobacteria (Todorova and JuÈttner, 1996) and the cyanobacterial agar plate di€usion bioassays were essentially performed as described (Todorova et al., 1995). Samples of nostocyclamide M dissolved in CH2Cl2 were spotted on a ground agar

(50% cyanobacterial medium solidi®ed with 2% agar). After drying in a sterile bench, an overlay of Anabaena 7120 or Anabaena P-9 in 0.5% agar was poured over the ground agar and incubated at 25 C and 32 mmol quanta m 2 s 1 measured at the surface of the Petri dishes. Chlorophyll a was determined in methanolic solution using the molar extinction coecient given by Ogawa and Vernon (1971). Acknowledgements We thank Dr. S. Carmeli, Tel Aviv, for NMR spectral information and Professor R.E. Moore for help in HPLC separation of the optical isomers of methionine sulfone and E. Loher, H. LuÈthi, and A. Mechsner for valuable technical assistance. F.J. is grateful for ®nancial support from the Swiss National Science Foundation, Bern, and W.v.P. acknowledges generous funding by the Dr. Helmut-Legerlotz Stiftung. References Admi, V., Afek, U., Carmeli, S., 1996. Raocyclamides A and B, novel cyclic hexapeptides isolated from the cyanobacterium Oscillatoria raoi. Journal of Natural Products 59, 396±399. Banker, R., Carmeli, S., 1998. Tenuecyclamides A-D, cyclic hexapeptides from the cyanobacterium Nostoc spongiaeforme var. tenue. Journal of Natural Products 61, 1248±1251. Foster, M.P., ConcepcioÂn, G.P., Caraan, G.B., Ireland, C.M., 1992. Bistratamides C and D. Two new oxazole-containing cyclic hexapeptides isolated from a Philippine Lissoclinum bistratum ascidian. Journal of Organic Chemistry 57, 6671±6675. Freeman, D.J., Pattenden, G., 1998. Total synthesis and assignment of stereochemistry of raocyclamide cyclopeptides from cyanobacterium Oscillatoria raoi. Tetrahedron Letters 39, 3251±3254. Fujii, K., Ikai, Y., Oka, H., Suzuki, M., Harada, K., 1997. A nonempirical method using LC/MS for determination of the absolute con®guration of constituent amino acids in a peptide: combination of Marfey's method with mass spectrometry and its practical application. Analytical Chemistry 69, 5146±5151. Hambley, T.W., Hawkins, C.J., Lavin, M.F., Vandenbrenk, A., Watters, D.J., 1992. Cycloxazoline Ð a cytotoxic cyclic hexapeptide from the ascidian Lissoclinum bistratum. Tetrahedron 48, 341±348. . Harrigan, G.G., Luesch, H., Yoshida, W.Y., Moore, R.E., Nagle, D.G., Paul, V.J., Mooberry, S.L., Corbett, T.H., Valeriote, F.A., 1998. Symplostatin 1: a dolastatin 10 analogue from the marine cyanobacterium Symploca hydnoides. Journal of Natural Products 61, 1075±1077. Marahiel, M.A., Stachelhaus, T., Mootz, H.D., 1997. Modular peptide synthetases involved in nonribosomal peptide synthesis. Chemical Reviews 97, 2651±2673. McDonald, L.A., Foster, M.P., Phillips, D.R., Ireland, C.M., Lee, A.Y., Clardy, J., 1992. Tawicyclamides A and B, new cyclic peptides from the ascidian Lissoclinum patella: studies on the solution- and solid-state conformations. Journal of Organic Chemistry 57, 4616± 4624. Michael, J.P., Pattenden, G., 1993. Marine metabolites and metal-ion chelation Ð the facts and the fantasies. Angewandte Chemie International Edition in English 32, 1±23. Ogawa, T., Vernon, L.P., 1971. Increased content of cytochromes 554

F. JuÈttner et al. / Phytochemistry 57 (2001) 613±619 and 562 in Anabaena variabilis. Biochimica Biophysica Acta 226, 88±97. Ogino, J., Moore, R.E, Patterson, G.M.L., Smith, C.D., 1996. Dendroamides, new cyclic hexapeptides from the blue-green alga. Multidrug-resistance reversing activity of dendroamide A. Journal of Natural Products 59, 581±586. Prinsep, M.R., Moore, R.E., Levine, I.A., Patterson, G.M.L., 1992. Westiellamide, a bistratamide-related cyclic peptide from the blue-green alga Westiellopsis proli®ca. Journal of Natural Products 55, 140±142. Todorova, A.K., JuÈttner, F., Linden, A., PluÈss, T., von Philipsborn, W., 1995. Nostocyclamide: a new macrocyclic, thiazole-containing

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