Hydantoanabaenopeptins from Lake Kinneret Microcystis Bloom, Isolation, and Structure Elucidation of the Possible Intermediates in the Anabaenopeptins Biosynthesis

Anabaenopeptins are common metabolites of cyanobacteria. In the course of reisolation of the known aeruginosins KT608A and KT608B for bioassay studies, we noticed the presence of some unknown anabaenopeptins in the extract of a Microcystis cell mass collected during the 2016 spring bloom event in Lake Kinneret, Israel. The 1H NMR spectra of some of these compounds presented a significant difference in the appearance of the ureido bridge protons, and their molecular masses did not match any one of the 152 known anabaenopeptins. Analyses of the 1D and 2D NMR, HRMS, and MS/MS spectra of the new compounds revealed their structures as the hydantoin derivatives of anabaenopeptins A, B, F, and 1[Dht]-anabaenopeptin A and oscillamide Y (1, 2, 3, 6, and 4, respectively) and a new anabaenopeptin, 1[Dht]-anabaenopeptin A (5). The known anabaenopeptins A, B, and F and oscillamide Y (7, 8, 9, and 10, respectively) were present in the extract as well. We propose that 1–4 and 6 are the possible missing intermediates in the previously proposed partial biosynthesis route to the anabaenopeptins. Compounds 1–6 were tested for inhibition of the serine proteases trypsin and chymotrypsin and found inactive at a final concentration of ca. 54 μM.


Introduction
Anabaenopeptins are common metabolites of cyanobacteria, especially water-bloomforming genera, but have been isolated from terrestrial and marine (Anabeana sp. and Nudularia spumigena) cyanobacteria and marine sponges (Theonella spp. and Melophlus sp.) as well [1]. They are nonribosomally synthesized cyclic-peptides that, in cyanobacteria, are composed of five variable L-amino acids and a conserved D-lysine. Their general structure is 1 X-CO-[ 2 Lys-3 X-4 X-NMe-5 X-6 X], where their macrocycle is derived from the cyclization of Lys-e-NH 2 to the carboxyl of the sixth amino acid. The Lys-a-amine is connected through an ureido bridge to the a-amine of the first amino acid. The chemical shifts of the latter ureaamide protons are distinctive and commonly allocated between 6 and 7 ppm in the 1 H NMR in DMSO-d 6 . The latest report sums the number of known anabaenopeptins to 152, many of which resulted from LC-MS/MS studies, which, according to our findings, sometimes fail to report the correct structure of the compounds (see below) [2]. The anabaenopeptins were reported to exhibit inhibitory activity toward serine proteases, carboxypeptidases, and phosphatases [1]. Their biosynthesis was studied by the analyses of gene clusters isolated from different strains of cyanobacteria, Anabaena sp., Nodularia spumigena [3], Nostoc punctiforme [4], Sphaerospermopsis torques-reginae [5], Planktothrix agardhii [6], and Snowella sp. [7], and three strains of Microcystis aeruginosa [8,9], all showing great similarity. However, no explanation was given in these studies to the transformation of the amide bond between the N-terminal amino acid and 2 Lys to an ureido bridge between the a-amines of the latter

Results and Discussion
Hydantoanabaenopeptin A (1) presented a molecular ion (negative HR ESI MS, [M-H] − ) at m/z 824.4016 corresponding to the molecular formula C 44 H 55 N 7 O 9 and 21 degrees of unsaturation. The IR spectrum of 1 presented stretching bands at 3274 (amide-NH), 1766 (CO), 1710 (CO), 1640 (amide-CO), and 1592 (aromatic-C=C) cm −1 , of which those at 1766 (C-4) and 1710 (C-2) cm −1 are characteristic of five-membered carbonyls of hydantoin [12]. The 1 H NMR spectrum of 1 in DMSO-d 6 (see Table 1 for the signals of the major signals and Figure S1 and Table S1 in supporting material for both major and minor signals) presented doubling of signals at a ratio of 2:1 of all of the amide protons and a-protons and many other signals in the spectrum. In the 13 C NMR spectrum of 1, most of the carbonyls and α-carbons were doubled at a ratio of 2:1, as well as few of the amino acid side-chain carbons ( Figure S2). Analysis of the 2D NMR data (COSY, TOCSY, HSQC, and HMBC) of 1 in DMSO-d 6 revealed the presence of the following amino acid residues: Phe, NMe-Ala, Hty, Val, Tyr, and a trisubstituted Lys (in which the expected a-amide proton was missing) and a urea-type carbonyl, similar to the one incorporated in the anabaenopeptin skeleton (Table S1) [13,14]. The latter composition of amino acids resembled those of anabaenopeptin A (7) [13]. The assembly of the amino acids by HMBC and ROESY correlations revealed the same amino acid sequence, cyclo-(Phe-NMeAla-Hty-Val-Lys-ε-NH)-, as in anabaenopeptin A, for the macrocycle of 1. The carbon chemical shifts of Lys C-1 to C-4 were different from the regular chemical shifts of these carbons in the anabaenopeptins [13]. Lys C-1 and C-3 were up-field-shifted (γ substitution effect), while C-2 and C-4 were down-fieldshifted (β and δ substitution effects, respectively), in accordance with an extra substitution of the α-nitrogen of Lys. The diastereotopic character of Lys β-protons (Lys-3a and Lys-3b) appears to be much more pronounced in 1 (δ H 2.12 and 0.84, respectively) than in anabaenopeptin A (7, δ H 1.28 and 1.18, respectively). The chemical shifts of the carbons of the substituted Tyr residue of 1 presented some differences from those of anabaenopeptin A (7) [13]. Although Tyr-CO (Tyr-1) and the ureido-CO presented chemical shifts similar to those of anabaenopeptin A (7), Tyr-C-2 to C-4 presented significant differences, with C-2 up-field-shifted and C-3 and C-4 down-field-shifted relative to those of anabaenopeptin A (7) [13]. The chemical shifts of Tyr-H-2 (Dd 0.5 ppm) and especially that of Tyr-NH (Dd 2.6 ppm) were significantly down-field-shifted relative to the latter in anabaenopeptin A (7). In the HMBC spectrum, Tyr-CO presented correlations with Lys-H-2, Tyr-H-2, Tyr-NH, and Tyr-H-3a, while "urea"-CO presented correlations with Lys-H-2, Tyr-H-2, and Tyr-NH, suggesting an extra hydantoin-type cycle for 1, in accordance with the 21 degrees of unsaturation. This finding was supported by the strong fragment ion in the MS/MS spectrum of 1, m/z 621.3406 ([C 34 H 47 N 5 O 6 ] + ), which resulted from the fragmentation of the C-N bond between Lys-C-2 and Lys-nitrogen (which is part of the hydantoin moiety, Figure S10). The strongest fragment ion in the spectrum, m/z 649.3352 (B in Scheme 1, [C 35 H 47 N 5 O 7 ] + ), for which we suggest a migration process of ureido-CO to Lys-C-2, and the strong M+H + -CO fragment ion at m/z 798.4196 are, as well, in accordance with the proposed hydantoin moiety. We assumed that the doubling in the NMR spectra of 1 resulted from the restricted rotation of the hydantoin moiety around the Lys C-N bond. However, when we tried to confirm it by running the 1 H NMR spectrum at an elevated temperature, the ratio of the doubled signals changed to 1:1 when the temperature reached 390 • K, and the ratio remained unchanged after lowering the temperature back to room temperature, suggesting that the doubling in the spectra is a result of the epimerization of a chiral center of one of the amino acids in 1. Application of Marfey's method [15] coupled with HR ESI LCMS established the absolute configuration of the amino acids as L-NMeAla, L-Hty, L-Phe, L-Val, D-Lys, and 1:1 D,L-Tyr ( Figure S11). We suspect that the tyrosine epimerization occurs due to the easy keto/enol tautomerism of the hydantoin moiety during the compound separation [16]. Based on these arguments, structure 1 was proposed for hydantoanabaenopeptin A. Scheme 1. Proposed mechanism of the migration process of the ureido carbonyl in the mass spectrometer.
Hydantoanabaenopeptin B (2) presented a negative HR ESI MS molecular ion, [M-H] − ion, at m/z 817.4361 corresponding to the molecular formula C42H58N10O8 and 18 degrees of unsaturation. Like for 1, the 1 H and 13 C NMR spectra of 2 presented doubling at a ratio of ca. 1:1 of all of the amide protons, two carbonyl carbons, two a-carbons, and few side-chain carbons (see Figures S12 and S13, Tables 1 and S2). Analysis of the 2D NMR data (COSY, TOCSY, HSQC, HMBC) of 2 revealed that it is composed of the amino acids Phe, NMeAla, Hty, Val, Lys, and Arg, where the last two amino acids presented the most pronounced doubling of proton and carbon signals (Table S2). The amino acid composition of 2 was thus found to be identical with those of anabaenopeptin B (8) [13]. Based on the HMBC and NOE correlations (Table S2), the structure of 2 was assigned to be the hydantoin derivative of anabaenopeptin B. The proposed structure was supported by the MS/MS fragment ion at m/z 200.1147 ([C7H14N5O2] + ), which derived from the cleavage of the Lys C-N bond and the preferred allocation of the positive charge on the arginine moiety, and the strong M+H + -CO fragment ion at m/z 791.4575 ( Figure S21). As for 1, the Lys β-protons presented a pronounced diastereotopic difference. The application of Marfey's method [15], coupled with HR ESI LCMS, established the absolute configuration of the amino acids as L-NMeAla, L-Hty, L-Phe, L-Val, D-Lys, and 1:2 D,L-Tyr ( Figure S22), confirming the structure of hydantoanabaenopeptin B as 2.
Hydantoanabaenopeptin F (3) presented a positive HR ESI MS molecular cluster ion, M+H + , at m/z 833.4688 corresponding to the molecular formula C42H60N10O8 and 18 degrees of unsaturation. The 1 H and 13 C NMR spectra of 3 presented doubling at a ratio of ca. 1:1 of all of the amide protons, three carbonyl carbons, three α-carbons, and a few side-chain carbons (see Figures S23 and S24, Table 1 and Table S3), similar to those of 2. Analysis of the 2D NMR data (COSY, TOCSY, HSQC, HMBC) of 3 revealed that it is composed of the amino acids Phe, NMeAla, Hty, Ile, Lys, and Arg, where the last three amino acids presented the most pronounced doubling of proton and carbon signals (Figures S23 and S24  and Table S3). The sequence of the amino acids in the peptide, Phe, NMeAla, Hty, Ile, Lys, Scheme 1. Proposed mechanism of the migration process of the ureido carbonyl in the mass spectrometer.
Hydantoanabaenopeptin B (2) presented a negative HR ESI MS molecular ion, [M-H] − ion, at m/z 817.4361 corresponding to the molecular formula C 42 H 58 N 10 O 8 and 18 degrees of unsaturation. Like for 1, the 1 H and 13 C NMR spectra of 2 presented doubling at a ratio of ca. 1:1 of all of the amide protons, two carbonyl carbons, two a-carbons, and few side-chain carbons (see Figures S12 and S13, Tables 1 and S2). Analysis of the 2D NMR data (COSY, TOCSY, HSQC, HMBC) of 2 revealed that it is composed of the amino acids Phe, NMeAla, Hty, Val, Lys, and Arg, where the last two amino acids presented the most pronounced doubling of proton and carbon signals (Table S2). The amino acid composition of 2 was thus found to be identical with those of anabaenopeptin B (8) [13]. Based on the HMBC and NOE correlations (Table S2) Table S3). The sequence of the amino acids in the peptide, Phe, NMeAla, Hty, Ile, Lys, and Arg, was elucidated based on the correlations from the HMBC and ROESY 2D spectra of 3 (Table S3) to be identical with that of anabaenopeptin F (9) [14] except of the extra correlations of both Lys-H-2 and Arg-2-NH with Arg-CO and ureido-CO, which suggest the closure of a hydantoin moiety similar to that of 2. The isoleucine moiety was identified by its characteristic 1 H and 13 C chemical shifts (5-Me: δ H 0.89 t, δ C 11.8 CH 3 : 6-Me, δ H 0.89 d, δ C 14.8 CH 3 ) to be allo-isoleucine in accordance with the chemical shifts of L-allo-isoleucine in ferintoic acid A [17] (5-Me: δ H 0.88 t, δ C 11.5 CH 3 : 6-Me, δ H 0.87 d, δ C 14.9 CH 3 ) and different from those of L-Ile (5-Me: δ H 0.73-0.82 t, δ C 10.3-10.8 CH 3 : 6-Me, δ H 0.88-1.00 d, δ C 14.9-15.5 CH 3 ) in anabaenopeptin F [14], lyngbyaureidamide A [18], and schizopeptin 791 [19] and those of D-Ile  Figure S32). The application of Marfey's method [15] in conjugation with HR ESI LCMS established the absolute configuration of the amino acids as L-NMeAla, L-Hty, L-Phe, L-alloIle, D-Lys, and 1:2 D,L-Arg ( Figure S33), verifying the structure of hydantoanabaenopeptin F as 3.
Hydanto-oscillamide Y (4) gave a negative HR ESI MS molecular ion at m/z 838.4143 corresponding to a molecular formula of C 45 H 57 N 7 O 9 and 21 degrees of unsaturation. The 1 H and 13 C NMR spectra of 4 (Table 1, Figures S34 and S35) presented, in contrast to 1-3, a single rotamer; however, the chemical shifts of the protons and carbons of the Lys and the Tyr moieties were shifted similarly to those of 1. Analysis of the 2D NMR data (COSY, TOCSY, ROESY, HSQC, HMBC, Table S4) of 4 revealed that it is composed of the amino acids Phe, NMeAla, Hty, Ile, Lys, and Tyr as in oscillamide Y (10) [21] and established its structure as hydanto-oscillamide Y. The  Figure S45) presented a typical anabaenopeptin spectrum [13] with the ureido bridge amide protons appearing as doublets at 6.48 and 6.27 ppm, 13 aromatic protons corresponding to a phenyl and two p-phenol rings, a singlet N-Me group at 1.76 ppm, and three doublet methyl groups at 1.05, 1.02, and 0.92 ppm, suggesting that 5 contains N-MeAla and valine residues. Analysis of the 2D NMR data (COSY, TOCSY, ROESY, HSQC, HMBC, Table S5) of 5 revealed that the five amino acids of the cycle in it are identical to those of anabaenopeptin A, Phe, NMeAla, Hty, Val, and Lys, while the sixth amino acid turned to be an amino acid first described in the anabaenopeptins, which we named dihomotyrosine (Dht) based on the analogy to the known biosynthesis of homophenylalanine and homotyrosine in cyanobacteria [22]. The 2D spectra failed to assign the two carboxy carbons resonating at 174.6 and 171.2 ppm, which were assigned by comparison with those of anabaenopeptin A [13], as Dht-C-1 and Phe-C-1, respectively.  Figure S54). Dihomotyrosine is homologous to PNV (5-phenylnorvaline) previously described in anabaenopeptins SA4 and SA7 [23]. The sequence of the amino acids in 5 was established as Dht-CO-cyclo-[Lys-Val-Hty-NMeAla-Phe] based of the HMBC correlations of Phe-NH with NMeAla-CO, NMeAla-H-2, and -N-Me with Hty-CO, Hty-NH with Val-CO, and the NOE correlations between Dth-NH and Lys-2-NH, Lys-6-NH and Phe-NH, Phe-NH and NMeAla-H-2, NMeAla-H-2 and Hty-NH, and Hty-H-2 and Val-H-2, leaving only the connection of Val-NH and Lys-CO unproved. However, the strong HR ESI MS/MS fragment ions at m/z 775.1253, 695.3779, and 637.3714 support the cyclic structure of 5 ( Figure S54). The application of Marfey's method [15] in conjunction with HR ESI LCMS established the absolute configuration of the amino acids as L-NMeAla, L-Hty, L-Phe, L-Val, D-Lys, and L-Dht ( Figure S55), establishing the structure of 1 [Dht]-anabaenopeptin A as 5.  The anabaenopeptin E [14] structure was established based on analysis of its NMR and MS data to contain 7-methylhomotyrosine at the fourth position. However, MS/MS studies on bloom material or compounds accumulated in animal organs cannot distinguish 7-methylhomotyrosine (MeHty) from dihomotyrosine (due to the identical mass and molecular formula) unless a dipper study based on MS n is utilized. We believe that many of the publications in which MS/MS studies (i.e., Riba et al. [24]) were used to report the structure of cyanobacteria metabolites erroneously report the presence of MeHty instead of Dht.  [15] in conjunction with HR ESI LCMS to be L-NMeAla, L-Hty, L-Phe, L-Val, D-Lys, and D,L-Dht ( Figure S66).
Compounds 1 and 4-6 were tested at concentrations of 55.1, 54.2, 53.3, and 53.3 µM, respectively, for the inhibition of the serine protease, chymotrypsin, and compounds 2 and 3 were tested at concentrations of 55.6 and 54.6 µM, respectively, for the inhibition of the serine protease and trypsin, but all were found nonactive at those concentrations.
The biosynthetic route proposed by genome mining and gene analyses [3][4][5][6][7][8][9] suggested a synthesis of a linear hexapeptide that contains in the second position a D-lysin, while the other five amino acids are varied L-amino acids of which the amino acid at the fifth position is N-methylated. The assembly of the six amino acids is followed by a release of the peptide from the enzyme and cyclization to the mature anabaenopeptin. However, no explanation was given in these studies to the transformation of the amide bond between the N-terminal amino acid and the D-2 Lys to an ureido bridge between the a-amines of the latter two amino acids. Considering our proposal that the hydantoanabaenopeptins 1-4 and 6 are intermediates in the biosynthesis of 7-10 and 5, respectively, several alternative routes are possible. The first one, route A in Scheme 2, suggests that the carboxylation of the N-terminus amine, cyclization to the hydantoin derivative, opening of the hydantoin to the ureido derivative, cleavage of the C-terminus thioester, and cyclization to the mature anabaenopeptin are mediated by the enzymes encoded in the last module of the biosynthetic cluster. Such a route would require the expression of at least one additional condensing enzyme in the last module of the gene cluster. The latter route is less reasonable due to the composition of enzymatic functions encoded in the last module of the biosynthetic cluster, C, A, T, and Te [3][4][5][6][7][8][9]. Routes B-D (Scheme 2) suggest loading of a presynthesized N-carboxy or N-carboxy-phosphate amino acid to the initial module of the biosynthetic cluster, explaining why no additional condensing enzyme is encoded in the biosynthetic cluster. The loading of a presynthesized N-carboxy-amino acid to the first adenylation domain of the anabaenopeptin biosynthetic enzyme cluster might explain its low specificity for certain amino acid [3][4][5][6][7][8][9], if the specificity is for the N-carboxy-moiety. Such specificity may lead to the production of a set of anabaenopeptins that differ in the starting unit but have a conserved sequence of amino acids in the ring, as in the case of the current study. In route B (Scheme 2), except for the loading of N-carboxy-amino acid, all of the other steps leading to the ureido bridge are facilitated by the last module of the biosynthetic cluster. In route C (Scheme 2), the hydantoin is proposed to be produced by the condensing enzyme encoded in the first module. The hydrolysis of the hydantoin to the ureido product is proposed to happen as the last step of the biosynthesis by the thioesterase (Te). The fourth possible route D suggests that both the cyclization to the hydantoin and its hydrolysis to the ureido moiety occur during the condensation process of lysine to the N-carboxy-amino acid. The hydantoin formation and opening might be catalyzed by a single enzyme of the cyclic amidohydrolase family (allantoinase, dihydropyrimidinase, dihydrooratase, hydantoinase, urease, and imidase) [25] that can reversibly catalyze the concession of N-carboxy-peptide to the hydantoin and its stereoselective hydrolysis to the ureido-bridge-containing peptide [26]. Alternatively, such conversion might be spontaneous in a similar fashion to the nonenzymatic isomerization of Asp in protein under physiological conditions. The latter isomerization proceeds through a succinimide intermediate and leads to racemization of the Asp and mixture of Asp and iso-linked-Asp, a total of four isomers [27]. However, the possibility that such transformation occurs spontaneously is low due to the stability toward hydrolysis of the hydantoin moiety of 1-4 and 6 in the isolation process (acidic aqueous solution) and even at 390 • K in DMSO-d 6 . Thus, if such a process occurs in the biosynthesis of the anabaenopeptins, it should occur on the condensation domain (of the N-carboxy-Lamino acid, the donor substrate, and L-Lys, the acceptor substrate) possibly with the aid of the donor pantetheinyl arm (PPE) and the catalytic triad of the acceptor condensation domain. The catalysis might prevent the racemization of the donor amino acid prior to the hydantoin opening and explain the L-configuration of the starter amino acid in the anabaenopeptin despite the tendency of the immature hydantoanabaenopeptins, 1-4 and 6, to racemize [28]. A biosynthetic route to the anabaenopeptins that encompass a mechanism similar to the one proposed for the ureido bridge formation in syringolin A is less feasible due to the occurrence, in the anabaenopeptin gene cluster, of two distinct adenylation and condensation domains for the amino acids involved in the construction of the ureido bridge production, unlike the single one for the syringolins [29]. The involvement of the epimerization domain in conversion of the hydantoin moiety to the mature anabaenopeptin is possible as well [30]. However, in the case of a presynthesized N-carboxy-amino acid loading to the first adenylation domain and its linking to the thiol through the N-carboxy moiety, one cannot exclude a direct biosynthesis of the ureido bridge. In such a case, the N-carboxy moiety might be phosphorylated (activated) and transferred to the thiol of the condensation domain and condensed with the amine of Lys to produce the ureido moiety in one biosynthetic step. Iso-linked amino acids are known in cyanobacterial peptides, i.e., the iso-linked Glu, Asp and MeAsp in the microcystins [31]. Further biosynthetic studies, which are beyond our scientific skills, are required in order to conclude on this issue. domains for the amino acids involved in the construction of the ureido bridge production, unlike the single one for the syringolins [29]. The involvement of the epimerization domain in conversion of the hydantoin moiety to the mature anabaenopeptin is possible as well [30]. However, in the case of a presynthesized N-carboxy-amino acid loading to the first adenylation domain and its linking to the thiol through the N-carboxy moiety, one cannot exclude a direct biosynthesis of the ureido bridge. In such a case, the N-carboxy moiety might be phosphorylated (activated) and transferred to the thiol of the condensation domain and condensed with the amine of Lys to produce the ureido moiety in one biosynthetic step. Iso-linked amino acids are known in cyanobacterial peptides, i.e., the iso-linked Glu, Asp and MeAsp in the microcystins [31]. Further biosynthetic studies, which are beyond our scientific skills, are required in order to conclude on this issue. Scheme 2. Possible routes to the ureido bridge of the anabaenopeptins.

General Experimental Procedure
Optical rotation values were obtained on a Jasco P-1010 polarimeter at the sodium D line (589 nm). UV spectra were recorded on an Agilent 8453 spectrophotometer (Agilent, Santa Clara, CA, USA). IR spectra were recorded on a Bruker Tensor 27 FT-IR instrument (Bruker, Billerica, MA, USA). NMR spectra were recorded on Bruker Avance III spectrometers (Bruker Karlsruhe, Germany) at 500.13 MHz for 1 H and 125.76 MHz for 13 C; chemical shifts were referenced to TMS δH and δC = 0 ppm. DEPT, COSY-45, gTOCSY, gROESY, gHSQC, and gHMBC spectra were recorded using standard Bruker pulse sequences. ESI low-and high-resolution mass spectra and MS/MS spectra were recorded on a Waters (Waters, Milford, MA, USA) Xevo G2-XS QTOP instrument equipped with Acquity Hi Scheme 2. Possible routes to the ureido bridge of the anabaenopeptins.

General Experimental Procedure
Optical rotation values were obtained on a Jasco P-1010 polarimeter at the sodium D line (589 nm). UV spectra were recorded on an Agilent 8453 spectrophotometer (Agilent, Santa Clara, CA, USA). IR spectra were recorded on a Bruker Tensor 27 FT-IR instrument (Bruker, Billerica, MA, USA). NMR spectra were recorded on Bruker Avance III spectrometers (Bruker Karlsruhe, Germany) at 500.13 MHz for 1 H and 125.76 MHz for 13 C; chemical shifts were referenced to TMS δ H and δ C = 0 ppm. DEPT, COSY-45, gTOCSY, gROESY, gHSQC, and gHMBC spectra were recorded using standard Bruker pulse sequences. ESI low-and high-resolution mass spectra and MS/MS spectra were recorded on a Waters (Waters, Milford, MA, USA) Xevo G2-XS QTOP instrument equipped with Acquity Hi Class UPLC (binary solvent manager) with an FTN sample manager, column manager, and PDA detector, using a 2.1 × 50 mm BEH C18 (1.7 mm) column and a flow of 0.1-0.3 mL/min. HPLC separations were performed on an Agilent 1100 Series HPLC system (Agilent, Santa Clara, CA, USA).

Biological Material
Microcystis biomass, TAU collection number IL-444, was collected in February 2016 from Lake Kinneret, Israel. The cell mass was frozen and lyophilized. A sample of the cyanobacteria is deposited at the culture collection of Tel Aviv University.

Determination of the Absolute Configuration of the Amino Acids by Marfey's Method [15]
Compounds 1-6 and authentic samples of anabaenopeptins A (7), B (8) and F (9) and oscillamide Y (10) were hydrolyzed in 6 N HCl (1 mL). The reaction mixture was maintained in a sealed glass bomb at 104 • C for 18 h. The acid was removed in vacuo, and the residue was suspended in 250 µL of H 2 O. A solution of 1-fluoro-2,4-dinitrophenyl-5-Lalanine amide (FDAA) in acetone (0.03 M, 20 µL per each amino acid in the peptide) and NaHCO 3 (1 M, 20 µL per each amino acid) was added to the reaction vessel. The reaction mixture was stirred at 40 • C for 2.5 h in the dark. HCl (2 M, 10 µL per each amino acid) was added to the reaction vessel, and the solution was evaporated in vacuo. The samples of L-FDAA derivatives were analyzed by ESI LC MS. The analysis was performed on a Waters (USA) Xevo G2-XS QTOP instrument equipped with Acquity Hi Class UPLC (binary solvent manager) with an FTN sample manager, column manager, and PDA detector, using a 2.1 × 50 mm BEH C18 (1.7 mm) column and a flow of 0.1-0.3 mL/min. The mobile phase compositions were (A) Water containing 0.1% formic acid and (B) MeCN containing 0.1% formic acid. The elution gradient was as follows: 1 min of 100% A, linear gradient to 30% B over 60 min, and then recycling by linear gradient to 50% B over 10 min and to 100% A over additional 5 min. Samples of 10 µL were injected, and the flow rate was 0.1-0.3 mL/min. The UV detector was set to 340 nm, and the mass spectrometer was operated in both negative and positive ion modes, scanning between 100 and 1000 mass units. The interpretation of the data was conducted after the run on both positive and negative ion modes using Waters MassLynx software (v.4.1).

Protease Inhibition Assays
The samples for biological assays were dissolved in DMSO at a concentration of 1 mg/mL and were tested for inhibition of the proteases at a single concentration: 1 (55.1 mM), 2 (55.6 mM), 3 (54.6 mM), 4 (54.2 mM), 5 (53.3 mM), and 6 (53.3 mM). Assays were performed in a 96-well plate format.

Trypsin
The assay was performed in a Tris buffer (0.6 g Tris HCl in 100 mL H 2 O, pH 7.5). Benzoyl-L-arginine-p-nitroanilide hydrochloride (BAPNA), the trypsin substrate, was dissolved at 1 mg/mL in 1:9 DMSO/buffer. The enzyme was dissolved in buffer at 1 mg/mL. To each well were added 100 µL of buffer, 10 µL of enzyme, and 10 µL of sample. The plate was placed in the spectrophotometer at 37 • C. After 5 min, 100 µL of substrate solution was added to each well, and the plate was placed in the spectrophotometer for the kinetic measurement of the absorbance intensity over 30 min at a wavelength of 405 nm.

Chymotrypsin
The assay was performed in a Tris buffer (0.6 gr TRIS HCl/100 mL H 2 O, pH 7.5). The enzyme and the substrate Suc-Gly-Gly-phenylalanine-p-nitroanilide (SGGPNA) were dissolved in the buffer at a concentration of 1 mg/mL. To each well were added 100 µL of buffer, 10 µL of enzyme, and 10 µL of sample. Then, the plate was placed in the spectrophotometer for a 5 min incubation at 37 • C. Thereafter, to each well, 100 µL of substrate solution was added, and the plate was placed in the spectrophotometer for the kinetic measurement of the absorbance intensity over 30 min at a wavelength of 405 nm.

Conclusions
We are monitoring the seasonal blooms of cyanobacteria in Lake Kinneret for almost three decades [32][33][34]. Along the years, the spring Microcystis bloom was shifted from green to brown in color (dominant by the Microcystis aeruginosa strain marked Mic B due to its dominant pigmentation, brown color) [10,11]. The brown bloom material contains a large amount of the aeruginosins KT608A and KT608B, which we use for an ongoing study on their ecological role. While isolating the aeruginosins for our study, we noticed several unknown masses in the LCMS of some fractions. The latter masses turned to be the new hydantoanabaenopeptins described above. The occurrence of hydantoanabaenopeptins may explain how the hexapeptide, encoded in the biosynthetic genes responsible for the biosynthesis of the anabaenopeptins in cyanobacteria, is converted to the ureido bridge containing anabaenopeptin. We propose several possible routes that include the formation and opening of the hydantoin moiety to the mature anabaenopeptin. The accumulation of the hydantoanabaenopeptins in this bloom material and the fact that it was not noticed in other studies in the past might result from the misfunction of the enzyme responsible for the production and opening of the hydantoin moiety, or misfunction of one of the enzymes that produces the hydantoin, as a by-product. Further genetic and enzymatic studies, which are beyond our capabilities, are needed to clarify this issue.  Table S3. NMR Data of Hydanto-anabaenopeptin F (3) in DMSO-d6; Figure S36. LCMS Chromatogram and Mass spectrum of Hydanto-anabaenopeptin F (3); Figure S37. 1H NMR Spectrum of Hydanto-oscillamide Y (4) in DMSO-d6; Figure S38. 13C NMR Spectrum of Hydanto-oscillamide Y (4) in DMSO-d6; Figure S39. HSQC Spectrum of Hydanto-oscillamide n Y (4) in DMSO-d6; Figure S40. HMBC Spectrum of Hydanto-oscillamide Y (4) in DMSO-d6; Figure S41. COSY Spectrum of Hydanto-oscillamide Y (4) in DMSO-d6; Figure S42. TOCSY Spectrum of Hydanto-oscillamide Y (4) in DMSO-d6; Figure S43. ROESY Spectrum of Hydanto-oscillamide Y (4) in DMSO-d6; Figure S44. Negative HR ESI MS of Hydanto-oscillamide Y (4); Figure S45. Positive HR ESI MS/MS Spectrum of Hydanto-oscillamide Y (4); Figure S46. Scheme of Fragmentation in the Positive HR ESI MS/MS Spectrum of Hydanto-oscillamide Y (4) Figure S47. LCMS Traces of Marfey's Method Amino Acids Analysis of Hydanto-oscillamide Y (4); Table S4. NMR Data of Hydanto-oscillamide Y (4) in DMSO-d6; Figure S48. LCMS Chromatogram and Mass spectrum of Hydanto-oscillamide Y (4); Figure