Almiramide D, cytotoxic peptide from the marine cyanobacterium Oscillatoria nigroviridis

Accepted Manuscript Almiramide D, cytotoxic peptide from the marine cyanobacterium Oscillatoria nigroviridis Jairo Quinta, Lina M. Bayona, Leonardo Castellanos, Mónica Puyana, Paola Camargo, Fabio Aristizábal, Christine Edwards, Jioji N. Tabudravu, Marcel Jaspars, Freddy A. Ramos PII: DOI: Reference:

S0968-0896(14)00770-6 http://dx.doi.org/10.1016/j.bmc.2014.10.039 BMC 11885

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

15 August 2014 18 October 2014 29 October 2014

Please cite this article as: Quinta, J., Bayona, L.M., Castellanos, L., Puyana, M., Camargo, P., Aristizábal, F., Edwards, C., Tabudravu, J.N., Jaspars, M., Ramos, F.A., Almiramide D, cytotoxic peptide from the marine cyanobacterium Oscillatoria nigroviridis, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/ 10.1016/j.bmc.2014.10.039

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O

O

O H N

H N

N N H

R2

O

almiramide almiramide almiramide almiramide

NH 2

N

O

D (1) E (3) F (4) H (6)

R1

O

R1 = CH 3 R2 = Me R 1 = CH3 R2 = H R 1 = CH2OH R 2 = Me R1 = H R2 = Me

O

O

O H N

H N

N N

NH 2

N

H

O

O

O

almiramide G (5)

9 O

7

N 1

1

O

N 1

N H

3

O H N

1

1

N

NH 2

8 O

O 4

O

3

5 4

5 5

almiramide B (2)

.

7

Bioorganic & Medicinal Chemistry jo u r n a l h o m e p a g e : w w w .e ls e v ie r .c o m

Almiramide D, cytotoxic peptide from the marine cyanobacterium Oscillatoria nigroviridis Jairo Quintaa , Lina M. Bayonaa, Leonardo Castellanosa, Mónica Puyanab, Paola Camargoc, Fabio Aristizábalc, Christine Edwardsd, Jioji N. Tabudravue, Marcel Jasparse, Freddy A. Ramosa* a

Departamento de Química, Universidad Nacional de Colombia. Carrera 30 Nº 45- 03. Bogotá, Colombia Departamento de Ciencias Biológicas y Ambientales, Programa de Biología Marina, Universidad Jorge Tadeo Lozano, Carrera 4 Nº 22-61, Modulo 4, Oficina 439. Bogotá, Colombia c Departamento de Farmacia, Universidad Nacional de Colombia. Carrera 30 Nº 45- 03. Bogotá, Colombia d School of Life Sciences, The Robert Gordon University, AB10 1FR Aberdeen, UK e Marine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Aberdeen AB24 3UE, Scotland, UK b

A R T IC LE IN F O

A B S TR A C T

Article history: Received Received in revised form Accepted Available online

Marine benthic cyanobacteria are widely known as a source of toxic and potentially useful compounds. These microorganisms have been studied from many Caribbean locations, which recently include locations in the Colombian Caribbean Sea. In the present study, six lipopeptides named almiramides D to H, together with the known almiramide B are identified from a mat characterized as Oscillatoria nigroviridis collected at the Island of Providence (Colombia, S.W. Caribbean Sea).The most abundant compounds, almiramides B and D were characterized by NMR and HRESIMS, while the structures of the minor compounds almiramides E to H were proposed by the analysis of their HRESIMS and MS2 spectra. Almiramides B and D were tested against six human cell lines including a gingival fibroblast cell line and five human tumor cell lines (A549, MDA-MB231, MCF-7, HeLa and PC3) s howing a strong but not selective toxicity.

Keywords: Marine natural products Marine benthic cyanobacteria Oscillatoria nigroviridis Almiramides Lipopeptides Toxicity against human cell

2009 Elsevier Ltd. All rights reserved.

1. Introduction Marine benthic cyanobacteria are a prolific source of biologically active compounds, particularly peptides with cytotoxic activity [1]. Several compounds isolated from cyanobacteria are, in their natural form or as synthetic derivatives, in clinical trials particularly against cancer [2]. Cyanobacteria as a group are currently of major interest for biologists and ecologists because they produce under varying ecological regimes, a suite of cytotoxic peptides and related compounds with severe ecological and economical implications [3]. Species of the marine genus Oscillatoria have yielded the polyketides debromoaplysiatoxin and oscillatoxin A and three additional brominated toxins structurally related to these two molecules [4]. These compounds were found to be the causative agents of severe contact dermatitis that may occasionally affect swimmers [5]. Debromoaplysiatoxin also displays antineoplastic activity, while aplysiatoxins and oscillatoxin A, are powerful tumor promoters [6]. Other Oscillatoria species have yielded the peptides largamides A-H from an Oscillatoria species collected in Florida [7], ve nturamides A-B from Oscillatoria sp. from Panamá [8], viridamides A and B from Oscillatoria nigroviridis from Curacao [9], coibacines A-D from an Oscillatoria species

collected at the Coiba Island at the Panamanian pacific sea [10] as well as the PKS-NRPS hybrids jamaicamides [11], tumonoic acids and its derivatives, among others [12]. Most of these compounds have been characterized and show promising activity against human tumor cell lines, and parasites. In our continued search for bioactive molecules from Colombian marine organisms [13], we recently evaluated the toxicity and cytotoxicity of several extracts from marine benthic cyanobacteria collected in the Colombian Caribbean. The organic extract of a cyanobacterium identified as Oscillatoria nigroviridis showed high toxicity against Artemia salina and cytotoxicity against the human cancer cell line HeLa. This extract also showed feeding deterrence against the opisthobranch Bursatella leachii and the sea urchin Lytechinus variegatus [14]. In this paper, we present the isolation and structural elucidation of the new linear peptide almiramide D, and the known compound almiramide B, based on their 1D and 2D NMR and MS data, together with the identification of almiramides E to I based on their MS data. The isolated compounds were further evaluated for their toxic effect on human cell lines cultures in vitro.

*Corresponding author. Tel.: +57-1-3165000 ext 14451; fax: +57-1-3165220; e-mail: [email protected] (F.A. Ramos)

2. Results and Discussion 2.1Structural elucidation of compounds 1-7 In our previous work we assessed the toxicity of marine benthic cyanobacterial mats collected at the Colombian Caribbean Sea, as a contribution to the study of these organisms in our coast (Data not showed). The organic extract of a sample of Oscillatoria nigroviridis (Figure S1 supplementary data) was selected for chemical studies based on its high toxicity against A. salina (LC50 4.5 µg/mL) and cytotoxicity against HeLa tumor cell line (IC50 9.9 µg/mL). After column chromatography of the crude extract, fraction F7 showed the strongest activities against A. salina (LC50 3.5 µg/mL), and the tumor cell lines A549 (IC50 62.3 µg/mL), MCF‐7 (IC50 102.2 µg/mL) HeLa (IC50 41.66 µg/mL) and PC3 (IC50 102.2 µg/mL) (Table S2 supplementary data), suggesting that this fraction contained the compounds responsible for the extract-observed toxicity. From this fraction, after successive RPHPLC separations (Figure S3 supplementary data), pure compounds 1 and 2, and fractions P1, P2 and P3 containing compounds 3-6 were obtained. Compound 1 (7.2 mg) was an optically active yellow amorphous solid [α]D20 = -134.6 (c 0.49, MeOH), with a molecular formula of C37 H66N6O6 determined by the ions observed in the HRESIMS spectrum (observed [M + H]+ at m/z 691.5092, calculated [M+H]+ 691.5117; and observed [M + Na]+ at m/z 713.4906, calculated [M + Na]+ 713.4942). Analysis of the 1H-NMR spectrum in CD3OD (Table 1, Figure S4.1 supplementary data) revealed the pattern of chemical shifts typical for a peptide with a single conformer that allows NMR structural elucidation as follows: five α-methine proton signals at δH 4.36 (1H, q, J = 7.1 Hz), 4.84 (1H, d), 4.67 (1H, d, J = 9.7 Hz), 4.70 (1H, d, J = 11.2 Hz) and 5.22 (1H, d, J = 10.7 Hz), suggesting that 1 was comprised of five amino acid residues. Additionally, 1H-NMR spectrum showed three sharp singlets at δH 3.04 (3H, s), 3.08 (3H, s) and 3.19 (3H, s) indicating the presence of three N-CH3 groups and signals for ten C-CH3 groups between δH 0.70-1.40, and signals for aliphatic methines and methylenes ranging from 0.95 to 2.89 ppm. Phase-sensitive multiplicity-edited gHSQC spectrum (Figure S4.2 supplementary data) confirmed the peptidic character of the compound 1 by the five α-CH groups at δC 49.5, 54.1, 59.6, 61.4 and 63.0 and six amide carboxyl resonances at δC 171.2, 171.4, 172.6, 174.6, 176.8 and 179.1. Additionally, the three N-CH3 groups at δC 30.8, 31.0 and 31.3, together with ten aliphatic methyls at δC 10.5, 10.6, 14.9, 15.4, 18.1, 18.2, 18.6, 18.6, 19.4, and 19.7 were confirmed. Six methylenes at δC 18.6, 25.3, 25.5, 27.5, 29.4 and 34.3, together with five methines at δC 27.5, 28.0, 33.1, 37.0 and 37.8 were also identified, accounting for 35 of the 37 carbons. Analysis of gHSQC and gHMBC spectra (Figure S4.2 and S4.3, respectively at the supplementary data) established the presence of an acetylene terminal group with carbon resonances at δC 69.3 (CH) and δC 84.5 (C), completing the 37 carbons found in the molecular formula and suggesting that compound 1 was a linear peptide. Interpretation of 2D-NMR spectra (gHMBC, gCOSY and gTOCSY, Figure S4.3, S4.4 and S4.5 at the supplementary data) afforded the identification of the amino acids Ala, Ile, N-Me-Ile and two N-Me-Val together with an additional acyl moiety identified as 2-methyl-oct-7-ynioc acid (Moya), a PKS-derived moiety that accounted for all the atoms predicted by the molecular formula (Figure 1).

Table 1: 1D and 2D NMR data of compound 1 in CD3OD ( 1H-NMR at 600 MHz; 13C-NMR at 150 MHz)

Residue Ala

Position

δH

NH2

n/o*

mult. (J in hz)

δC

1

Ile1

176.8 (C)

2

4.36

q (7.1)

49.5 (CH)

3

1.30

d (7.1)

18.2 (CH3)

NH

n/o*

d**

61.4 (CH)

1

171.2 (C)

2

4.84

3

2.06

M

33.1 (CH)

4

0.95/1.36

M

25.3 (CH2)

5

0.86

m

10.6 (CH3)

6

0.92

d (5.6)

15.4 (CH3)

NMe

3.19

s

31.3 (CH3)

2

4.67

d (9.7)

3

1.92

m

37.8 (CH)

4

1.16/1.52

m

25.5 (CH2)

5

0.85

m

10.5 (CH3)

6

0.87

d

14.9 (CH3)

NH

n/o*

m

Ile2

Val1

Val2

Moya

1

174.6 (C)

1

54.1 (CH)

171.4 (C)

2

4.70

d (11.2)

63.0 (CH)

3

2.22

m

27.5 (CH)

4

0.76

d (6.5)

18.6 (CH3)

5

0.86

d

19.4 (CH3)

NMe

3.08

s

31.0 (CH3)

2

5.22

d (10.7)

59.6 (CH)

3

2.40

m

28.0 (CH)

4

0.93

d (5.6)

19.7 (CH3)

5

0.87

d (6.2)

18.6 (CH3)

NMe

3.04

s

30.8 (CH3)

2

2.89

m

37.0 (CH)

3

1.39/1.72

m

34.3 (CH2)

4

1.40

m

27.5 (CH2)

5

1.50

m

29.4 (CH2)

6

2.16

m

18.6 (CH2)

1

172.6 (C)

1

179.1 (C)

7

84.5 (C)

8

n/o*

9

1.08

* n/o = not observed. ** Overlapped with HOD signal.

69.3 (CH) d (6.8)

18.1 (CH3)

c 0.3, MeOH) with those published for almiramide B indicated that both compounds have the same absolute stereochemistry, and in the same way, assume that compound 1 could share the same stereochemistry (Figure 3). Furthermore, analysis of derivatized amino acids in hydrolyzed peptide 1 and 2 compared to D- and L- configuration standards show coincidence with those retention times of L-Val-FDVA, L-Ile-FDVA and L-Ala-FDVA, for compound 1, and L-Val-FDVA, L-Phe-FDVA and L-AlaFDVA, for compound 2, establishing the stereochemistry presented in Figure 3 for the compounds 1 and 2. It is likely that related variants, as could be the case of N-Me-amino acids have similar configuration, as is typically the case in mutli-variant groups of natural products [16].

Figure 3: Structures of the identified compounds 1- 6

O

O

O

H N

H N

N N

Figure 1: Substructures of the amino acid and fatty acid residues for compound 1 showing COSY/TOCSY and HMBC correlations The HMBC correlations from the α-CH, β-H and N-methyl protons to carbonyl carbons established the linear sequence as Ala, N-Me-Ile1, Ile2, N-Me-Val1, N-Me-Val2, Moya (Figure 2). Additionally, this sequence was confirmed by the HRESIMS fragmentation pattern where the observed ions correspond to B-type ions in the peptide sequences, as follows: pseudomolecular ions [M + Na]+ at m/z 713.4906 and [M + H]+ at m/z 691.5092, sequential losses of each amino acid residue: [M - Ala]+ at m/z 603.4456, [M - Ala - NMeIle]+ at m/z 476.3463, [M - Ala - NMeIle-Ile]+ at m/z 363.2628 and finally [M – Ala NMeIle - Ile - NMeVal1]+ at m/z 250.1793. This last ion accounting for the weight of the N-Me-Val2 - Moya moiety (Figure 2).

H

O

almiramide almiramide almiramide almiramide

O

D (1) E (3) F (4) H (6)

R1

R1 = CH3 R2 = Me R1 = CH3 R2 = H R1 = CH2OH R2 = Me R1 = H R2 = Me

O

O

O H N

H N

N N H

NH2

N

O

R2

NH 2

N

O

O

O

almiramide G (5)

9 O

7

1

8

O

1

1

N O

4

O

N 1

N H

3

O H N

N 1

NH 2 O

3

5 4

5

5

almiramide B (2)

Due to the minimal amounts obtained of P1 to P3 fractions (1.3, 0.9 and 1.5 mg, respectively), the structure of compounds 3 to 6 is proposed on the basis of the HPLC-HRESIMS data obtained for these compounds.

Figure 2: Linear structure of 1 established from key HMBC correlations and HRESIMS fragmentation analysis. The structure of this compound is related to almiramide B, a lipopeptide with five amino acid and Moya residues previously isolated from a Lyngbya majuscula strain collected on mangrove roots at Bocas del Toro Marine Park in the Panamanian Caribbean Sea [15]. The sequence of compound 1 however, differs in the first three amino acids changing from N-Me-Phe, NMe-Ala and Val in almiramide B to Ala, N-Me-Ile and Ile at the carboxyl terminus of lipopeptide 1. Therefore this new compound is named almiramide D. The analysis of 1D- and 2D-NMR spectra (Table S5 supplementary data) and HRESIMS (Figure S6 supplementary data) of compound 2 indicated the sequence as N-Me-Phe, N-MeAla, Val, N-Me-Val, N-Me-Val and Moya, the same as that of almiramide B [15]. A careful comparison of 1H- and 13 C-NMR data and the optical rotation value for compound 2 ([α]D20 -127.5,

In the P1 HPLC chromatogram, observed as a single peak, showed to be a mixture of two different compounds. In the LCMS spectrum, as confirmed by the analysis of the TIC (Figure S7a supplementary data), these compounds presented two different and recognizable fragmentation patterns (Figures S7b and S7c supplementary data). Analysis of the left part of the TIC (Figures S7a and S7b supplementary data), clearly reveals the mass spectrum of one of the compounds, numbered as 3, with a fragmentation pattern quite similar to that observed for compounds 1 and 2 and allowing its identification as follows: The molecular formula for compound 3 is proposed as C36H64 N6O6 based on the observed ion [M + H]+ at m/z 677.4946 (calculated [M + H]+ 677.4966) and the ion [M + Na]+ at m/z 699.4764 (calculated [M + Na]+ 699.4785). The most abundant ion in the spectrum was observed at m/z 589.4312 corresponding to a [M - Ala]+ fragment. The MS2 analysis of this ion (Figure S8 supplementary data) showed daughter ions at m/z 462.3312 [M - Ala - NMeIle]+, 349.2477 [M - Ala - NMeIle - Ile]+ and 250.1796 [M - Ala - NMeIle - Ile -

7

Val]+ showing a similar fragmentation pattern to that of almiramide D (1), with a difference of 14 u in the third fragment, suggesting the absence of the N-methyl in the Val1 residue. Hence, compound 3 consists of the amino acid sequence Ala, NMe-Ile, Ile, Val, N-Me-Val and Moya, named almiramide E (Figure 3). This proposal is in agreement with the expected exact mass of the proposed fragments, where the observed differences between the experimental and calculated fragments are less than 20 ppm (Figure S8 supplementary data). In a similar way, the later eluting portion of the TIC peak obtained for P1, showed the fragmentation pattern of a second compound different to that assigned for almiramide E (3) (Figures S7a and S7c, supplementary data). The structure of this second compound, named almiramide F (4) is proposed as follows: The molecular formula for compound 4 is assigned as C37H66N6 O7 based on the observed ion [M + H]+ at m/z 707.5046 (calculated [M + H]+ 707.5071) and the ion [M + Na]+ at m/z 729.4864 (calculated [M + Na]+ 729.4885). The spectrum presents predominant ions at m/z 603.4464 [M - Ser]+, and the previously described ions observed in almiramide D (1) [M - Ser -NMeIle - Ile]+ at m/z 363.2632 and [M - Ser - NMeIle - Ile NMeVal1]+ at 250.1796. These ions were also observed in the MS2 spectra for the ion 603.45, where additionally the ion 476.3463 [M - Ser - NMeIle]+ was also observed. The analysis for these ions allowed proposing the sequence of almiramide F (4) as Ser, N-Me-Ile1, Ile2, N-Me-Val1, N-Me-Val2, and Moya (Figure 3). The presence of the Ser amino acid instead of Ala is proposed for the difference of +15.99 compared to almiramide D (1) in the first residue. Again, the difference between the obtained and calculated fragments did not exceed 20 ppm supporting the structure herein proposed (Figure S9 supplementary data). Based on its HPLC purity profile P2 was initially believed to be a single compound, as occurred with P1. However, when analyzed by LC-MS (Figure S10a supplementary data), P2 revealed to be a mixture of two different peptide like compounds similar to the almiramides. A predominant series of ions at m/z 699.4766, 677.4950, 589.4315, 462.3315, 363.2637 and 250.1798 was observed in the MS spectrum for the left half of the peak observed in the TIC (Figure S10b supplementary data). These were assigned to compound 5, named almiramide G. The molecular formula for 5 is proposed as C36H64N6O6 based on the observed ion [M + H]+ at m/z 677.4950 (calculated [M + H]+ 677.4966) and the ion [M + Na]+ at m/z 699.4766 (calculated [M + Na]+ 699.4785). The observed ion at m/z 589.4312 corresponds to the [M - Ala]+ fragment. The MS2 analysis for m/z 589.43 gave daughter ions at m/z 462.3316 [M -Ala - NMeIle]+, 363.2634 [M Ala - NMeIle - Val]+ and 250.1797 [M - Ala - NMeIle - Val NMeVal]+ (Figure S11 supplementary data). This analysis allowed proposing the sequence of 5 as Ala, N-Me-Ile, Val, NMe-Val, N-Me-Val and Moya (Figure 3). The difference observed with respect to almiramide D (1) was the -14 u after the second amino acid fragmentation, suggesting that the amino acid Ile2 had been replaced by a valine residue in almiramide G. TIC analysis for P3 (Figure S12, supplementary data) showed a complex mixture of at least 3 different compounds, where only one cluster of fragments could be fully identified by its MS fragmentation pattern, leading to the proposed structure for almiramide H (6, Figure 3 and Figure S13). Again, this compound resembled mostly the structure of almiramide D, but showed a difference of -14 u in the first amino acid, suggesting that the alanine residue was replaced by glycine. Hence, the sequence for 6 is proposed as Gly, N-Me-Ile1, Ile2, N-Me-Val1, N-Me-Val2, and Moya (Figure 3).

Almiramides D to H represent new members of the almiramide cyanobacterial peptides. These compounds are characterized by the presence of 2-methyl-7-octynoic acid as the acyl residue, based on data reported in the literature [17]. This triple bond terminus is a characteristic feature of many cyanobacterial compounds including carmabin A [18], yanucamides [19], malevamide C [20], georgamide [21], pitipeptolide A [22], antapeptins A and [23], wewakpeptins A and C [24], trungapeptin A [25], dragonamides C,D [26], viridamides A and B [9], hantupeptin A [27], and E [17], and veraguamides [28]. This particular Moya moiety has been found with two possible configurations for its stereocenter as (2S)-Moya in dragonamide A [29], dragonamide B and dragomabine [17], and as (2R)-Moya in almiramide B [15]. Due to the low amount of the isolated compounds, absolute stereochemistry for the carbon at the position 2 of the Moya residue could not be assigned. However, based on the comparison of our NMR spectra and optical rotation data for almiramide B (2), isolated in this study, with those reported for the same compound by [15] we propose that the compounds described here have the same stereochemistry as almiramides A-C (Figure 3). The absolute configuration for the amino acids of almiramides E to H was established by Marfey´s analysis of the mixtures P1, P2, and P3. Analysis of P1 shows the presence of L-Ser-FDVA, L-Ala-FDVA, L-Val-FDVA, L-Ile-FDVA, L-NMe Ile-FDVA, and L-NMe Val-FDVA, confirming that almiramides E and F contain only L amino acids. In the same way, Marfeys analysis for mixture P2 showed the presence of L-Ala-FDVA L-Val-FDVA, L-NMe Ile-FDVA, L-NMe Val-FDVA, confirming only the presence of L amino acids for almiramides G. In the analysis of P3, the presence of L-NMe Ile-FDVA, L-NMe Val-FDVA, L-IleFDVA, among others amino acids of the non identified peptides in the mixture. This analysis confirms the structures presented in Figure 3. Almiramides A-C were isolated from a sample originally identified as Lyngbya majuscula (PAB-04-NOV-05-7) identified based on morphologic and morphometric characteristics [15]. Almiramide B was also obtained from a sample identified as Lyngbya sp. (in our previous work, data not showed). In this study however, almiramide B and almiramides D - H were obtained from a cyanobacterial sample identified as Oscillatoria nigroviridis (Figure S1, supplementary data). Recently, many of the samples collected in the Caribbean sea initially identified as Lyngbya and Oscillatoria have been classified as the recently described genera Moorea spp. and Okeania spp. [30]. Among them, Okeania comitata represents samples previoulsy known as Oscillatoria nigroviridis. Therefore, the identification of the almiramides producing strains should be confirmed in further studies. 2.2 Toxicity evaluation All six compounds found in the O. nigroviridis sample were obtained from F7 fraction, which at that time showed the greatest toxicity against A. salina nauplii (LC50 3.5 µg/mL). The in vitro toxicity assessment of the isolated compounds against the six evaluated human cell lines, showed mild toxicity against cancer cells for almiramide B (2) and D (1), but high toxicity against the gingival fibroblast cell line used here as reference to establish selectivity against tumor cell lines compared with primary cell line (Table 2).

column, and a Waters XTerra® RP-18 (250 × 4.6 mm) column, with detection at 210 nm. All used solvents were HPLC grade. 4.2 Cyanobacterium collection

Table 2. Toxicity against human cell lines for the isolated compounds almiramide D (1) and B (2) IC50 (µ µM) Cell line

Almiramide D

Almiramide B

(1)

Doxorubicin

(2)

Fibroblast

1.5 x 10 -4

2.7 x 10-9

1,3 x 10-11

A 549

59

76

14

HT – 29

91

100

NTa

HeLa

17

53

NT

PC3

62

107

17

MDA

821

13

NT

a

NT = Not tested.

3. Conclusion In conclusion, this study allowed the identification of the proposed new peptides almiramides E to H, together with the isolation of the new almiramide D and the known almiramide B isolated from a cyanobacterial mat identified as Oscillatoria nigroviridis collected from the Colombian Caribbean Sea. The isolated compounds showed only high toxicity against the gingival fibroblast cell line and mild activity against human tumor cell lines. 4. Experimental 4.1 General experimental procedures Optical rotations were measured on a Polartronic E, Schmidt + Haensch polarimeter. NMR data of fractions were recorded on a Bruker Avance 400 MHz spectrometer (400 and 100 MHz for 1H and 13C NMR, respectively) in CDCl 3, with solvent residual signals as internal standard (δH 7.26). NMR spectra of pure compounds were acquired on Varian VNMRS 400 MHz and Varian VNMRS600 MHz spectrometers in methanol-d4, using residual solvent signals as internal standards (δH 3.31 and 4.87, δC 49.1). The carbon resonances were determined by gHSQC spectra. High-resolution electrospray ionization mass spectra (HRESIMS) data in positive mode were obtained using a LTQ Orbitrap XL Hybrid Fourier Transform mass spectrometer Discovery system (Thermo Scientific Instruments) coupled to a Thermo Instruments HPLC system (Accela PDA detector, Accela autosampler, and Accela pump, Thermo Scientific Instruments). The following conditions were used: capillary voltage 4.5 kV, capillary temperature 260 °C, auxiliary gas flow rate 10-20 arbitrary units, sheath gas flow rate 40-50 arbitrary units, spray voltage 4.5 kV, mass range 100 - 1000 u (maximum resolution 30000). Column chromatography (CC) was performed using silica gel (0.043-0.060 µm, Merck®). Solid phase extraction was made using LiChrolut® RP-18 cartridges (40-63 µm, Merck®). HPLC separation was carried out on a Merck-Hitachi 6000 instrument equipped with a Merck-Hitachi L-6200A pump and a PDA L4500 detector. HPLC profiles were obtained on a Phenomenex Lichrosphere® RP-18 (250 × 4.6 mm) column. Compounds were purified using a LiChroCART® LiChrospher® (250 × 10 mm)

A sample of Oscillatoria nigroviridis was collected in October 2009 at Lawrence Reef (Old Providence Island, Colombia, SW Caribbean Sea) by SCUBA diving. The cyanobacterium grew as a dark flimsy mat on coralline sand at a depth of 5 m. The cyanobacterial mat was collected by hand and frozen for chemical analyses. A small portion was stored in sea water with 4% formalin for further taxonomic analyses following [31] (Figure S1 supplementary data). A voucher of the studied sample is kept at the Bioprospecting and Biotechnology Laboratory at Universidad Jorge Tadeo Lozano, Bogotá, Colombia under the code BB-PNM-CB08. 4.3 Extraction, purification and structural elucidation The frozen sample was freeze-dried prior to extraction. The freeze-dried material (185 g) was then extracted with a mixture of CH2Cl2/MeOH 1:1, three times in 24 h intervals. The extracts were filtered, pooled and concentrated to dryness in vacuo to yield the crude extract (11.3 g). From that 7.14 g of the extract were fractionated by silica gel column chromatography (CC) using a step gradient solvent system of increasing polarity starting with n-hexane (100%), then n-hexane/EtOAc (9:1), nhexane/EtOAc (8:2), n-hexane/EtOAc (1:1), n-hexane/EtOAc (2:8), EtOAc (100%), EtOAc/MeOH (8:2), EtOAc/MeOH (7:3) and MeOH (100%), obtaining 9 fractions (F1-F9). The toxicity of crude extract (LC50 5 µg/mL) as well as the chromatographic fractions (Table S2 supplementary data) were evaluated for their toxicity against Artemia salina (see numeral 2.4). Fraction 7 (480 mg), eluted with AcOEt/MeOH (8:2), showed an LC50 of 3.5 µg/mL against Artemia salina. This fraction was further separated by C-18 SPE cartridges employing a step gradient solvent system starting with MeOH/H2O (1:9), to MeOH, and finally MeOH/CH2Cl2 (1:1) to yield 10 subfractions (F7.1-F7.10). Fractions F7.6 to F7.8 eluted with MeOH/H2O (6:4), (7:3) and (8:2), respectively, were pooled and further purified by RPHPLC using a LiChroCART® LiChrospher® RP-18 column, with a gradient from 60% MeOH/H2O to 100% MeOH over 20 minutes. In this way, we obtained enriched fractions of the compounds, which were subsequently purified by RP-HPLC employing a XTerra® reversed phase column, in isocratic mode 50% ACN/H2O acidified with 0.05% formic acid to yield fractions P1 (1.3 mg, Rt 8.2 min), P2 (0.9 mg, Rt 8.9 min), P3 (1.5 mg, Rt 9.7 min), P4(7.2 mg, Rt 11 min), and P5 (3.7 mg, Rt 12.6 min). Compounds 1 and 2 were obtained pure from fractions P4 and P5, respectively. Structural elucidation of these compounds was achieved by extensive 1D and 2D NMR analyses. The HRMS spectra were used to confirm the structures and the peptide sequences proposed here by NMR. Fractions P1 to P3 yielded minor compounds 3 to 6. The structure of these compounds was proposed by careful analysis of their MS data. For the amino acid absolute stereochemistry analysis using Marfey´s method [32], compounds and fractions were hydrolysed with 6 N HCl at 150°C for1 h. Samples were then dried under nitrogen at 45°C. D- and L- reference amino acids were obtained from Sigma-Aldrich (Poole, UK). Aqueous solutions (50 mM) of reference amino acid were prepared. N-(2,4-Dinitro-5fluorophenyl)-D-valinamide (FDVA;100 µL of 1% in acetone) was added to each amino acid (50 µL) and the hydrolysed peptide in an Eppendorf vial, followed by 20 µL of 1.0 M NaHCO3. The reaction mixture was vortexed and heated over a hot plate at 40°C for 1 h. After cooling to room temperature, 20 µL of 2 M HCl was added to each reaction and the volume was

made up to 1 mL with methanol. The samples were separated by UHPLC-MS using a modified version of Marfey’s chromatography conditions [32]. Samples were separated on a BEH C18 column (2.1 mm I.D. 100 mm long; 1.7 µm particle size), which was maintained at 40 ºC. Mobile phase was MilliQWater plus 0.1% formic acid (A) and acetonitrile plus 0.1% formic acid (B). Separation was achieved using a gradient increasing from 20% B to 80% B over 10 min, followed by a 100% B step and re-equilibration. Data were obtained in positive ion electrospray MS mode (ESI+) scanning from m/z 50-2000 u with a scan time of 0.25 s and inter-scan delay of 0.03 s. Ion source parameters: sprayer voltage, 3 kV; cone voltage, 30 V; desolvation temperature, 300°C; and source temperature, 80 °C. Instrument control, data acquisition and processing were achieved using MassLynx v 4.1. Using this method D-amino acids eluted before L-amino acids. Mass data of derivatives and retention times were compared withthose of standard amino acids. For compound 1, chromatographic peaks for NMe L-Ala (Rt 5,06), NMe L-Val (Rt 6,46) and NMe L-Ile (Rt 6,63) were observed. For compound 2, chromatographic peaks for NMe LAla (Rt 5,08), NMe L-Val (Rt 6,46), L-Val (Rt 6,39) and NMe-LPhe (Rt 6,91) were observed. For compound 3 and 4 chromatographic peaks for of L-Ser-FDVA (Rt 3.62), L-AlaFDVA (Rt 5.02), L-Val-FDVA (Rt 6.32), L-Ile-FDVA (Rt 6.96), L-NMe-Ile-FDVA (Rt 7.07), and L-NMe-Val-FDVA (Rt 6.46) were observed. For compounds 5 the chromatographic peaks for L-Ala-FDVA (Rt 5.02) L-Val-FDVA (Rt 6.28), L-NMe-Ile-FDVA (Rt 7.07), L-NMe-Val-FDVA (Rt 6.46), were observed. For compound 6, the chromatographic peaks for L-NMe-Ile-FDVA (Rt 7.06), L-NMe-Val-FDVA (Rt 6.46), L-Ile-FDVA (Rt 6.98), were observed 4.4 Toxicity evaluation 4.4.1 Evaluation of toxicity by the Artemia salina lethality assay The brine shrimp Artemia salina toxicity assay was performed with the O. nigroviridis crude extract and all chromatographic fractions following [33]. Artemia cysts were hatched in artificial sea water at 26 ‐ 30 °C for 48 h. Assays were performed in 24 ‐ well plates. 10 to 15 nauplii were placed in 2 or 3 drops of artificial sea water in each well. Fractions to be tested were resuspended in a mixture of 30 µL of DMSO and 20 µL of acetone and then mixed with artificial seawater. Two milliliters of the fraction solutions were placed in each well. Fractions were evaluated at concentrations of 1000, 500, 100, 10, 1 and 0.1 µg/mL. Each fraction was evaluated by triplicate. After 24 h the live and dead shrimp were counted and the LC50 was calculated following Reed‐Munchen graphic method. The results are presented in Table S2 in the supplementary data. 4.4.2 Evaluation of cytotoxicity against human cancer cell lines Cell cultures 4.4.2.1 Culture conditions Our previous results showed that crude extract of O. nigroviridis exhibited mild cytotoxicity against the ATCC human tumor cell lines A549 (lung cancer, IC50 37 µg/mL), MDA MB231 (breast cancer, IC50 62 µg/mL), MCF-7 (breast cancer, IC50 49 µg/mL), HeLa (cervix carcinoma, IC50 37 µg/mL), and PC3 (prostate cancer, IC50 76 µg/mL). Therefore further cytoxicity evaluation of chromatographic fractions and purified compounds was performed against a gingival fibroblast cell line from the Department of Pharmacy Universidad Nacional de Colombia and the six ATCC human tumor cell lines above mentioned. All cell lines were maintained in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS), gentamicin (50 µg/mL), in culture cell flasks of 75 or 150 cm2 at 37°C, in a 5% CO2

atmosphere and 100% of relative humidity. The culture media was renewed 3 times per week [34]. 4.4.2.2 Cytotoxicity assays Culture flasks in 90% confluence were tripsinized and counted in a Neubauer chamber using the trypan-blue exclusion procedure. Human tumor cells were inoculated into 96 well microtiter plates at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of individual cell lines according with our previous work [34-35]. For the fibroblast cell line, a density of 4,500 cells/well was used. After cell inoculation, the microtiter plates were incubated at 37 °C, in a constant humidified atmosphere of 5:95 CO2/air during 24 h for attachment. Test compounds were re-suspended in DMSO and evaluated at 100, 50, 10 and 1 µM. Doxorubicin hydrochloride was used as positive control at concentrations of 0.1, 1.0 and 10 µg/mL. A solution of RPMI 1640 and DMSO 0,1 % was used as blank. Treatments were added and plates were incubated for 48 h at 37 °C, 5% CO2, 95% air and 100 % relative humidity. After treatment remotion, 100 µL/well of a medium solution with AB and Alamar Blue (resazurin 10 %) and without FBS was added to each well, and incubated again for 4 h. After that, plate fluorescence derived from resazurin was read using a TECAN GENios spectrofluorometer. Inhibition was determined as decrease of fluorescence relative to fluorescence of control cultures and plotted against compound concentration and IC50 calculatedvalues. The GraphPad Prism 5.0 software was used to calculate IC50 values by nonlinear regression and compare them, by ANOVA analysis followed by a Tukey Test, with 95 % confidence. 4.5.1 Almiramide D (1) Amorphous solid; [α]D20 -134.62 (c 0.49, MeOH); For 1H NMR and 13C NMR spectroscopic data, see Table 1 and Figures S4.1 to S4.5 supplementary data; HR-ESIMS m/z: 691.5092 [M + H]+ (calcd. for C37 H66N6O6, 691.5117), 713.4906 [M + Na]+ (calcd for C37H66N6O6Na, 713.4942), 603.4456 [M - Ala]+, 476.3463 [M - Ala - NMeIle]+, 363.2628 [M - Ala - NMeIle - Ile]+, 250.1793 [M - Ala - NMeIle - Ile NMeVal1]+. Figure 2. 4.5.2 Almiramide E (3) HRESIMS m/z: 677.4946 [M + H]+ (calcd. for C36H64N6O6, 677.4966), 699.4764 [M + Na]+ (calcd for C36 H64N6O6Na, 699.4785), 589.4312 [M - Ala]+. HRESIMS2 for 589.43 m/z showed ions at 462.3312 [M - Ala - NMeIle]+, 349.2477 [M - Ala - NMeIle - Ile]+ and 250.1796 [M - Ala NMeIle - Ile - Val]+. (Figure S8 supplementary data) 4.5.3 Almiramide F (4) HRESIMS m/z: 707.5046 [M + H]+ (calcd. for C37H66N6O7, 707.5071), 729.4864 [M + Na]+ (calcd for C37H66N6O7 Na, 729.4885), 603.4464 [M - Ser]+, 363.2632 [M - Ser - NMeIle - Ile]+, 250.1796 [M - Ser - NMeIle - Ile NMeVal1]+. HRESIMS2 for 603.45 showed the ion at m/z 476.3463 [M - Ser - NMeIle]+. (Figure S9 supplementary data). 4.5.4 Almiramide G (5) HRESIMS m/z: 677.4950 [M + H]+ (calcd. for C36H64N6 O6, 677.4966), 699.4766 [M + Na]+ (calcd. for C36 H64N6O6Na, 699.4785), 589.4312 [M - Ala]+. HRESIMS2 for 589.43 showed the ion at m/z 462.3316 [M - Ala - NMeIle]+, 363.2634 [M - Ala - NMeIle - Val]+, 250.1797 [M - Ala - NMeIle - Val - NMeVal]+ (Figure S11 supplementary data) 4.5.5. Almiramide H (6) HRESIMS m/z: 677.4946 [M + H]+ (calcd. for C36H65N6 O6, 677.4966), 699.4764 [M + Na]+ (calcd. for C36H65N6 O6Na, 699.4785), 603.4468 [M - Gly]+, 476.3468 [M - Gly - NMeIle]+, 363.2635 [M - Gly - NMeIle - Ile]+, 250.1797 [M - Gly - NMeIle - Ile - NMeVal1]+. (Figure S13 supplementary data). ACKNOWLEDGMENTS

This research was supported by grants from COLCIENCIAS Proyecto 1216-452-21241, Universidad Jorge Tadeo Lozano, Universidad Nacional de Colombia sede Bogotá (DIB and Facultad de Ciencias), Fundación para la Promoción de la Investigación y la Tecnología del Banco de la República and Fundación Mariano Ospina Pérez-ICETEX. We want to acknowledge Jerónimo Vásquez, Rafael Barragán and Enrique Pomare for field assistance. The Ministerio de Ambiente, Vivienda y Desarrollo Territorial granted permission for scientific research on biological diversity (permission No. 4 of 10/02/2010)

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