**1. Introduction**

Nonribosomal and ribosomal cyanobacterial peptides, with their structural diversity and modified amino acid moieties, constitute one of the most interesting and biotechnologically promising groups of marine natural products [1–5]. Ribosomally synthesized and post-translationally modified (RiPPs) cyanobactins constitute a large family of compounds containing from three to twenty amino acids [6–9]. The biosynthesis of these metabolites starts with the encoding of a precursor peptide that undergoes multiple cleavages leading to a release of a core peptide that is subjected to further enzymatic modifications. The structure of cyanobactins is characterized by the presence of heterocyclized amino acids, mainly cysteine (cyclized to thiazole or oxidized thiazoline), threonine and serine (cyclized to oxazole or oxazoline) [6,7,10]. Cyanobactins can also contain prenyl or, more rarely, geranyl groups. Other modifications include carboxylation of glutamine, hydroxylation of proline, valine or lysine, bromination of tryptophan, acetylation of tyrosine, epimerization or formation of disulfate bridge [7,10,11].

Some cyanobactins, such as comoramides, keenamide A, patellamides and vineramides, exhibit cytotoxic activity against several cancer cell lines [12–17]. Venturamides, another class of the peptides, had strong in vitro antimalarial activity against *Plasmodium falciparum* [18]. Cyanobactins have been also described as allelopathic agents. Nostocyclamide from *Nostoc* 31 inhibited the growth of cyanobacterial strains representing other genera *Anabaena*, *Synechococcus* and *Synechocystis*, diatom *Navicula minima* and chlorophyceae *Nannochloris coccoides* [19,20].

The first cyanobactins, ulicyclamide and ulithiacyclamide with cytotoxic activity, were isolated from a tunicate *Lissoclinum patella* from Palau, Western Caroline Islands [21]. It was later established that some cyanobactins were in fact produced by the ascidians symbiont, *Prochloron* spp. [22,23]. Thus far, over a hundred cyanobactins have been detected in different free-living and symbiotic cyanobacteria. Amongst others, these compounds have been found and chemically characterized in *Anabaena* (anacyclamides) [24], *Arthrospira* (arthrospiramides) [25], *Lyngbya* (aesturamides) [26], *Microcystis* (aerucyclamides, aeruginosamides, kawaguchipeptins, microcyclamide, microphycin) [15,27–30], *Scytonema* (scytodecamide) [31] and *Sphaerospermopsis* (sphaerocyclamides) [32]. Cyanobactin gene clusters were found in up to 30% of cyanobacteria representing *Prochloron*, *Anabaena*, *Microcystis*, *Arthrospira* and other genera [6–8,24,33,34].

Initially, cyanobactins were described as cyclic peptides. Lawton et al. [28] reported the production of a linear aeruginosamide by *M. aeruginosa* from bloom sample collected in Rutland Water reservoir (Scotland). This peptide contained the diisoprenylamine and the carboxylated thiazole moieties and was later called aeruginosamide A (AEG-A) [34]. Further studies revealed the presence of modified linear cyanobactins: aeruginosamides B and C in *Microcystis aeruginosa* PCC 9432 and a virenamide A in *Oscillatoria nigro-viridis* PCC 7112 [34].

In the current study, the potential of *Limnoraphis* to produce cyanobactins has been explored for the first time. The non-targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of extract and fractions from *Limnoraphis* sp. CCNP1324, isolated from the brackish water Baltic Sea, led to the detection of eighteen new aeruginosamide variants. In cell viability assays some of the aeruginosamides produced by *Limnoraphis* sp. CCNP1324 showed cytotoxic activity against human breast cancer cells (T47D).

#### **2. Results and Discussion**

The existing knowledge about the structural diversity of aeruginosamides and aeruginosamide-producing cyanobacteria is limited. To date, only three aeruginosamides have been detected [28,34] (Table 1), and no reports on cyanobactins or genes involved in their biosynthesis in cyanobacteria of *Limnoraphis* genus have been published. In our work, the production and structural diversity of cyanobactins produced by *Limnoraphis* sp. CCNP1324 from the Baltic Sea were studied. As a result, eighteen new structural analogues of the linear aeruginosamides were characterized.

Of the eighteen AEGs produced by *Limnoraphis* sp. CCNP1324, the cell-bound content of AEG707, estimated on the basis of chromatographic peak area, was the highest. Ten peptides were produced in trace amounts and were only detected when a larger portion of cyanobacterial biomass was used for the extraction (Table 1). The structure elucidation of AEGs was based on the mass fragmentation spectra with characteristic immonium ions (e.g., at *m*/*z* 70 (proline Pro), 86 (isoleucine Ile/leucine Leu), 120 (phenylalanine Phe), 134 (homophenylalanine Hph/*N*-methyl-phenylalanine *N*-MePhe), 136 (tyrosine Tyr), 164 (*N*-methyl-homotyrosine *N*-MeHTyr)) and a series of other fragment ions. In addition, the collected product ion spectra were compared with the previously published spectra of AEG-A [28], AEG-B and AEG-C [34].


**Table 1.** Postulated structures of aeruginosamides (AEGs) described thus far, and identified in *Limnoraphis* sp. CCNP1324.

(1) Detected in the 10 mg extract. (2) Detected in the 20 g extract and flash fractions from CCNP1324.T traces of AEGs detected in 10 mg extract. Hph: homophenylalanine; MePhe: *N*-methy-phenylalanine; Ile/Leu: isoleucine/leucine; Phe: phenylalanine, Pre: prenyl group; Pro: proline; Pyr: pyrrolidine; Tyr: tyrosine; MeHTyr: *N*-methyl-homotyrosine; TzlCOOH: thiazolidyne-4-carboxylic acid; TzlCOOMe: methyl ester of thiazolidyne-4-carboxylic acid; Val: valine; 187, 205, 225: unknown residues.

Thiazole (Tzl) group, a characteristic element of numerous cyanobactins [6,7,25,35] was present in all AEGs produced by *Limnoraphis* sp. CCNP1324. In the fragmentation spectra, TzlCO gave a peak at *m*/*z* 112, while the ion at *m*/*z* 144 was indicative of methyl ester of thiazolidyne-4-carboxylic acid (TzlCOOMe) (Figures 1–3, Figures S1, S2, S5–S11 and S13). In the spectra of four AEGs, the ion at *m*/*z* 112 was present but instead of the *m*/*z* 144 ion, the ion at *m*/*z* 130 occurred, suggesting a modification in the ester group of TzlCOOMe. In the spectra of these peptides, instead of ions at *m*/*z* 213 (Pyr+TzlCOOMe) and *m*/*z* 310 (Pro+Pyr+TzlCOOMe), there were peaks at 14 units lower values, i.e., *m*/*z* 199 and 296. Pyr stands for pyrrolidine ring which constitutes a part of the proline structure. The 14-unit shift in the *m*/*z* value of the ions, compared to TzlCOOMe-containing peptides, and the ion at *m*/*z* 112 indicated the presence of thiazolidyne-4-carboxylic acid (TzlCOOH). Such modifications were observed in AEG625 (Figure 4), AEG657 (Figure S3), AEG693 (Figure 5) and AEG735 (Figure S12) (Table 1). The three *C*-terminal residues in aeruginosamides identified in CCNP1324, were found to be conserved. In other AEGs identified thus far the residues adjacent to TzlCOOMe were Val (valine)+Pyr [28], Phe+Pyr or Pro+Val [34] (Table 1).

**Figure 1.** Chemical structure and enhanced product ion mass spectrum of aeruginosamide AEG571 Tyr+Val+Pro+Pyr+TzlCOOMe identified based on the following fragment ions: 572 [M+H], 428 [M+H–TzlCOOMe], 409 [Val+Pro+Pyr+TzlCOOMe], 361 [M+H–Pyr+TzlCOOMe], 310 [Pro+Pyr+TzlCOOMe+H], 263 [Tyr+Val+H], 235 [Tyr+Val+H–CO], 213 [Pyr+TzlCOOMe+H], 144 [TzlCOOMe], 136 Tyr immonium ion, 112 TzlCO, 70 Pro immonium ion.

**Figure 2.** Chemical structure and enhanced product ion mass spectrum of aeruginosamide AEG639 Pre+Tyr+Val+Pro+Pyr+TzlCOOMe identified based on the following fragment ions: 640 [M+H], 572 [M+H–Pre], 409 [Val+Pro+Pyr+TzlCOOMe], 391 [Val+Pro+Pyr+TzlCOOMe–H2O], 360 [Tyr+Val+Pro+H], 331 [Pre+Tyr+Val+H], 310 [Pro+Pyr+TzlCOOMe+H], 303 [Pre+Tyr+Val+H–CO], 263 [Tyr+Val+H], 235 [Tyr+Val+H–CO], 213 [Pyr+TzlCOOMe+H], 204 [Pre+Tyr+H–CO], 144 [TzlCOOMe], 136 Tyr immonium ion, 112 TzlCO, 72 Val immonium ion, 70 Pro immonium ion.

**Figure 3.** Chemical structure and enhanced product ion mass spectrum of aeruginosamide AEG671 Pre+Phe+Phe+Pro+Pyr+TzlCOOMe identified based on the following fragment ions: 672 [M+H], 604 [M+H–Pre], 457 [M+H–(Pre+Phe)], 439 [M+H–(Pre+Phe)–H2O], 392 [Phe+Phe+Pro+H], 363 [Pre+Phe+Phe+H], 335 [Pre+Phe+Phe+H–CO], 310 [Pro+Pyr+TzlCOOMe+H], 295 [Phe+Phe+H], 267 [Phe+Phe+H–CO], 213 [Pyr+TzlCOOMe+H], 188 [Pre+Phe+H–CO], 144 [TzlOMe], 136 Tyr immonium ion, 120 Phe immonium ion; 112 TzlCO, 70 Pro immonium ion.

**Figure 4.** Chemical structure and enhanced product ion mass spectrum of aeruginosamide AEG625 Pre+Tyr+Val+Pro+Pyr+TzlCOOH identified based on the following fragment ions: 626 [M+H], 558 [M+H–Pre], 296 [Pro+Pyr+TzlCOOH+H], 263 [Tyr+Val+H], 332 [Tyr+Val+Pro+H–CO], 235 [Tyr+Val+H–CO], 199 [Pyr+TzlCOOH+H], 130 [TzlCOOH], 136 Tyr immonum ion, 112 TzlCO, 70 Pro immonium ion.

**Figure 5.** Chemical structure and enhanced product ion mass spectrum of aeruginosamide AEG693 (Pre)2+ Tyr+Val+Pro+Pyr+TzlCOOH identified based on the following fragment ions: 694 [M+H], 626 [M+H–Pre], 558 [M+H–(Pre)2], 477 [(Pre)2+Tyr+Val+Pro+H–H2O], 428 [Pre+Tyr+Val+Pro+H], 400 [Pre+Tyr+Val+ Pro+H–CO], 370 [(Pre)2+Tyr+Val+H–CO], 331 [Pre+Tyr+Val+H],303 [Pre+Tyr+Val+H–CO], 295 [Pro+Pyr+TzlCOOH+H], 272 [(Pre)2+Tyr+H–CO], 235 [Tyr+Val+H–CO], 204 [Pre+Tyr+H–CO], 199 [Pyr+TzlCOOH+H], 130 [TzlOH], 136 Tyr immonium ion, 112 TzlCO, 72 Val immonium ion, 70 Pro immonium ion.

Tyr1 was found to be the most frequent residue at the *N*-terminus and was present in six out of eighteen AEGs identified in this study. In other AEGs produced by *Limnoraphis* sp. CCNP1324, this position was occupied by MeHTyr1, Phe1 or Hph1 (Table 1, Figures 1–5 and Figures S1–S13). In the case of six AEGs (*m*/*z* [M+H] 596, 682a, 682b, 684, 736 and 750) we were not able to fully elucidate the structure and identify the *N*-terminal residue. Based on the fragmentation spectrum it was concluded that the residues gave strong immonium ions at *m*/*z* 160, 178, 180 and 198 and their residue masses were 187, 205, 207 and 225 respectively. In previously described linear cyanobactins such as virenamide A–C, aeruginosamide B and C, and viridisamide A, Phe1 was the most commonly identified *N*-terminal residue [12,34]. In other cyanobactins, position 1 was occupied by Ile [28,36] or Val [37]. The high residue masses of the unidentified amino acids and a frequent occurrence of aromatic amino acids at *N*-terminus of AEGs produced by *Limnoraphis* sp. indicated the presence of modified Tyr or Phe variants in this position. In some RiPPs, such as cyanobactins and microviridins, the presence of acetylated Tyr (AcTyr) was reported [11,38]. Based on the mass fragmentation spectrum, the presence of AcTyr1 in AEG681a is also possible (Figure S5). The position 2 in AEGs produced by *Limnoraphis* sp. CCNP1324 was least conserved and occupied by both aliphatic and aromatic amino acids: Val, Ile, Phe and Hph/MePhe (Table 1, Figures 1–5 and Figures S1–S13).

*Limnoraphis* sp. CCNP1324 synthesizes aeruginosamides with two, one and no prenyl groups at *N*-terminus (Table 1, Figures 1–5 and Figures S1–S13). The presence of prenyl was confirmed by the loss of one or two 68-Da fragments from the pseudomolecular ion of the analyzed peptides. The differences in retention times between AEGs without and with prenyl group (Table 1), indicate that the former ones are not the products of in-source degradation. In other cyanobactins, the number of Pre groups also varied depending on the peptide. Doubly prenylated cyanobactin, virenamide A, was reported from *D. virens* [12], while monoprenylated AEG-B, AEG-C, viridisamide A [34] and virenamide B and C [12] were identified in *M. aeruginosa* PCC9432, *O. nigro viridis* PCC7112 and *D. virens*, respectively. Prenyl groups at both C- and *N*-terminus were found in muscoride A and B from *N. muscorum* IAM M-14, *Nostoc* sp. PCC7906 and *Nostoc* sp. UMCC0398 [36,37].

Due to the chromatographic behaviour of AEG671, which allowed for the isolation of the peptide (1 mg) as a pure compound, the structural analyses with application of Nuclear Magnetic Resonance (NMR) were possible. Unfortunately, under the chromatographic conditions used in the current study, the majority of the detected aeruginosamides were poorly separated. They occurred in the chromatograms as broad peaks or/and co-eluted with other components of *Limnoraphis* extract. The NMR analyses of the isolated AEG671 confirmed the correctness of structure elucidation performed based on the MS/MS fragmentation pattern of pseudomolecular ion. The 1H NMR spectrum of the studied compound displayed a typical pattern of a peptide. The Correlation Spectroscopy COSY, Total Correlation Spectroscopy TOCSY and Heteronuclear Multiple Bond Correlation HMBC data (Figures S14–S19) allowed for the identification of the residues in AER671 as Dma (Dma = 1, 1-dimethylallyl), Phe, Phe, Pro, Pyr and TzlCOOMe (Table 2, Figure 6). Proton and carbon chemical shifts unambiguously showed that the prenyl group in the studied compound was in reverse prenyl, 1, 1-dimethylallyl form.

**Figure 6.** Key Rotation Frame Nuclear Overhauser Effect Spectroscopy ROESY and Heteronuclear Multiple Bond Coherence HMBC correlations for aeruginosamide AEG671.

The signals occurring in the aromatic region of the spectrum (δ<sup>H</sup> 7.1–7.5 ppm) and the TOCSY interaction between 19 (26), 20 (27) and 21 (28) protons were indicative of the presence of two aromatic phenylalanine residues in the molecule. The existence of proline residue and pyrrolidine ring was confirmed by their characteristic spin systems in the TOCSY spectrum. HMBC correlation of proton 6 (δ<sup>H</sup> 5.32 ppm) to thiazole carbon 5 (δ<sup>C</sup> 173.4 ppm) confirmed the connection of Pyr to Tzl ring. The presence of methyl thiazole-carboxylate was shown by characteristic proton (δ<sup>H</sup> 3.81 ppm) and carbon (δ<sup>C</sup> 51.1 ppm) chemical shifts and HMBC correlation of methyl protons 1 (δ<sup>H</sup> 3.81 ppm) to carbon 2 (δ<sup>C</sup> 160.3 ppm), and by HMBC and Heteronuclear Single Quantum Correlation HSQC of proton 4 (δ<sup>H</sup> 8.43 ppm) to carbons 2 (δ<sup>C</sup> 160.3 ppm), 5 (δ<sup>C</sup> 173.4 ppm), and 4 (δ<sup>C</sup> 128.0 ppm).


**Table 2.** Nuclear Magnetic Resonance NMR Spectroscopic Data (500 MHz, dimethyl sulfoxide-d6 DMSO-d6) for aeruginosamide AEG371 (Dma-Phe-Phe-Pro-Pyr-Tzl-COOMe).

<sup>a</sup> ROESY Rotation Frame Nuclear Overhauser Effect Spectroscopy; <sup>b</sup> HMBC correlations are given from proton(s) stated to the indicated carbon atom; <sup>c</sup> Dma: 1,1-dimethylallyl.

Apart from reversed prenyl as present in AEG671, cyanobactins can also contain a forward prenylated *N*-terminus (e.g., AEG-A [28] and virenamide A [12]), as well as, a forward C-, and reverse prenylated *N*-terminus (muscoride A [36]) or forward prenylated both C- and *N*-termini (muscoride B [37]).

Protein prenylation is an important posttranslational modification which increases the lipophilicity and affinity of compounds for biological membranes [39–41]. Prenylation also increases the biological activity of natural products [42,43]. The cytotoxic activities of prenylated licoflavone C and isobavachinas from plants, as well as their non-prenylated analogues (apigenin, liquiritigenin), were examined against glioma (C6) and rat hepatoma (H4IIE) cells. The prenylated compounds showed pronounced cytotoxicity against both types of cells while their non-prenylated analogues were weakly active [42].

The activity of cyanobactins and cyanobactin-like peptides has been tested against bladder carcinoma (T24), colon adenocarcinoma (HT29), lung carcinoma (A549) and murine leukemia (P388) cell lines, proving the pharmacological potential of these compounds [12–17]. Cyanobactins also showed multidrug-resistance reversing activity [44].

The existing knowledge about the activity of aeruginosamides is scarce. To date, only mild cytotoxic effects of aeruginosamide A against human ovarian tumor (A2780) and human leukemia (K562) cells have been reported [28]. In our work, the cytotoxic activity of three chromatographically separated samples labelled as A, B and C, was tested against T47D cancer cells. The sample marked as A contained AEG671, sample B contained partially separated AEG681a and, in sample C, a mixture of AEG681a and AEG667 was present. After 24-h exposure, sample B containing partially separated AEG681a with unknown residue in position 1 (residue mass 205) reduced the relative cell viability to 4.2% <sup>±</sup> 0.5% at 200 <sup>μ</sup>g mL<sup>−</sup>1. Sample C, containing a mixture of AEG681a and AEG667 (with MeHTyr1), reduced the relative cell viability to 21% <sup>±</sup> 1.2% at 200 <sup>μ</sup>g mL<sup>−</sup>1. These effects were dose dependent. No activity was observed for Phe1 containing AEG671 present in sample A. Unfortunately, the cytotoxic peptides with the unidentified residues are produced by *Limnoraphis* sp. CCNP1324 in minute amounts (Table 1), which seriously restricts the ability to perform more detailed structural analyses with the application of NMR technique.

The vast structural diversity of AEGs, as well as the cytotoxic activity of some of the variants, create an opportunity for more detailed studies on the structure-activity relationship. Several cyanobacterial peptides are already in clinical or pre-clinical trials as potent anti-cancer agents [45]. The most successful was the development of Auristatine (brentuximab vedotin), a synthetic analogue of dolastatin 10 isolated from *Dolabella auricularia*, but actually produced by the cyanobacterium *Symploca* sp. [46]. This microtubule-impacting agent was approved by the Food and Drug Administration (FDA), and is globally used in the treatment of Hodgkin's lymphoma [47].
