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2,3-Seco-2,3-dioxo-lyngbyatoxin A from a Red Sea strain of the marine cyanobacterium Moorea producens

Diaa T.A. Youssefâ, Lamiaa A. Shaala^bc, Gamal A. Mohamedâd, Sabrin R.M. Ibrahimê, Zainy M. Banjar^f, Jihan M. Badrâg, Kerry L.

McPhail^h, April L. Risingerⁱ & Susan L. Mooberryⁱ

a Department of Natural Products, Faculty of Pharmacy, King Abdulaziz University, Jeddah21589, Kingdom of Saudi Arabia

b Natural Products Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah21589, Kingdom of Saudi Arabia

c Suez Canal Hospital, Suez Canal University, Ismailia41522, Egypt

d Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut71524, Egypt

e Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut71526, Egypt

f Department of Clinical Biochemistry, Faculty of Medicine, King Abdulaziz University, Jeddah21589, Kingdom of Saudi Arabia

g Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, Ismailia41522, Egypt

h Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR97331, USA

i Department of Pharmacology and Cancer Therapy & Research Center, University of Texas Health Science Center, San Antonio, TX78229, USA

Published online: 25 Nov 2014.

To cite this article: Diaa T.A. Youssef, Lamiaa A. Shaala, Gamal A. Mohamed, Sabrin R.M.

Ibrahim, Zainy M. Banjar, Jihan M. Badr, Kerry L. McPhail, April L. Risinger & Susan L. Mooberry (2014): 2,3-Seco-2,3-dioxo-lyngbyatoxin A from a Red Sea strain of the marine cyanobacterium Moorea producens, Natural Product Research: Formerly Natural Product Letters, DOI:

10.1080/14786419.2014.982647

To link to this article: http://dx.doi.org/10.1080/14786419.2014.982647

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2,3-Seco-2,3-dioxo-lyngbyatoxin A from a Red Sea strain of the marine cyanobacterium Moorea producens

Diaa T.A. Youssefâ*, Lamiaa A. Shaala^bc, Gamal A. Mohamedâd, Sabrin R.M. Ibrahimê, Zainy M. Banjar^f, Jihan M. Badrâg, Kerry L. McPhail^h, April L. Risingerⁱand

Susan L. Mooberryⁱ

aDepartment of Natural Products, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia;^bNatural Products Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia;^cSuez Canal Hospital, Suez Canal University, Ismailia 41522, Egypt;^dDepartment of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut 71524, Egypt;^eDepartment of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt;^fDepartment of Clinical Biochemistry, Faculty of Medicine, King Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia;^gDepartment of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt;^hDepartment of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR 97331, USA;ⁱDepartment of Pharmacology and Cancer Therapy & Research Center, University of Texas Health Science Center, San Antonio, TX 78229, USA

(Received 26 July 2014; final version received 26 October 2014)

Chemical investigation of the organic extract of a Red Sea strain of the cyanobacterium Moorea producenshas afforded 2,3-seco-2,3-dioxo-lyngbyatoxin A (1). Five known compounds including lyngbyatoxin A (2), majusculamides A and B (3 and 4), aplysiatoxin (5) and debromoaplysiatoxin (6) were also isolated. Their structures were elucidated by using HR-FAB-MS, 1D and 2D NMR analyses. The compounds were evaluated for antiproliferative activity against HeLa cancer cells. Lyngbyatoxin A (2) showed potent activity, with an IC50of 9.2 nM, while5and6displayed modest activity with IC50values of 13.3 and 3.03mM, respectively. In contrast, compounds1,3and4 were inactive, with IC50values greater than 50mM. The lack of cytotoxicity for 2,3- seco-2,3-dioxo-lyngbyatoxin A (1) demonstrates that the indole moiety in lyngbyatoxin (2) is essential for its cytotoxicity, and suggests that detoxification of2 may be carried out by biological oxidation of the indole moiety to yield1.

Keywords:Red Sea cyanobacterium;Moorea producens; 2,3-seco-2,3-dioxolyngbya- toxin A; lyngbyatoxin; HeLa cells; antiproliferative activity

q2014 Taylor & Francis

*Corresponding author. Email:[email protected] Natural Product Research, 2014

http://dx.doi.org/10.1080/14786419.2014.982647

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1. Introduction

The pan-tropical marine cyanobacterial genusMoorea(previously classified asLyngbya) (Engene et al. 2012) has been investigated widely as a prolific source of diverse biologically active secondary metabolites, including harmful toxins in the environment (Taylor et al. 2014) and biomedically relevant agents (Nunnery et al.2010). In the course of our studies to compare the biosynthetic capabilities of Red Sea cyanobacteria with those collected pantropically, we have reported new cytotoxic grassypeptolides (Thornburg et al.2011), apratoxins (Thornburg et al.

2013) and malyngamide 4 (Shaala et al. 2013), together with structurally diverse known compounds. The subject of this report is a new collection ofMoorea producensmade at a different site but from the same region as that which previously yielded malyngamide 4 and known aplysiatoxins. We present the isolation, structural elucidation and antiproliferative activity testing of 2,3-seco-2,3-dioxo-lyngbyatoxin A (1) (Figure 1), an oxidative cleavage product of lyngbyatoxin A (2) (Cardellina et al. 1979), which was also isolated, together with majusculamides A and B (3and4) (Marner et al.1977), aplysiatoxin (5) and debromoaplysiatoxin (6) (Yoshinori & Scheuer 1974) (Figure 1) from the cytotoxic fraction ofM. producens. It is noteworthy that lyngbyatoxins, debromoaplysiatoxin, majusculamides A and B, and malyngamides A and B were previously co-isolated fromM. producenscollected around the Hawaiian Islands (Aimi et al.1990). As recently summarised by Jiang et al. (2014), the indolactam alkaloid lyngbyatoxin A is a well-known activator of protein kinase C that is a causative agent of

‘swimmer’s itch’ (seaweed dermatitis). It is identical with the Streptomyces mediocidicus metabolite teleocidin A-1, which co-occurs with its C-19 epimer teleocidin A-2 (Sakai et al.

1986). The latter metabolite is epimeric in the pendant linalyl side chain and has only been

N H N

HN

OH O

1 2 3

4 5

6 7

3a 8

10 9 12 11

13 14

15 16 17

18

19

20 21

22 23

24 25 26 27

28

O O

7a

1

O NH₂ N N

O

O O O

R₂ R₁

R₁ R₂ 3 CH₃ H 4 H CH₃

R 5 Br 6 H

OH O

O O

O O HO

O O

OH

R

NH N

HN

OH O

2

3 2 5

6 19

14 12

8 9

25 18

20 21

Figure 1. Structures of isolated compounds1–6.

2 D.T.A. Youssefet al.

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reported fromStreptomycesbacteria, whereas 12-epi-lyngbyatoxin A, containing anN-methyl-D- valine residue has been reported from M. producens as a mixture of cis and trans amide conformers (as for lyngbyatoxin A itself) (Jiang et al.2014). Conformers around the C-7/C-19 bond of the linalyl substituent have also been observed. Given their wide-ranging biological functions, these and related indolactam alkaloids have been the target of numerous chemical syntheses (e.g. Nathel et al.2014) which support the assignment of their absolute configuration.

2. Results and discussion

Chromatographic separation of the EtOAc-soluble extract of the marine cyanobacterium M. producenson silica gel, Sephadex LH-20, and final HPLC purification resulted in the isolation of 2,3-seco-2,3-dioxo-lyngbyatoxin A (1) together with five known compounds (2–6). The isolated compounds were evaluated for their antiproliferative activity against HeLa cancer cells.

Compound1was isolated as a colourless solid. Initial inspection of the dispersed¹H NMR spectrum for1(Supplementary Figure S1) showed a sharp downfield¹H singlet (dH8.05, 1H), aromatic¹H doublets (dH7.37 – 6.11), multiple prominent mid-field multiplets (dH5.21 – 3.34), a deshielded methyl singlet (dH 2.96), methine multiplets (dH 2.53 – 1.52), methyl singlets (dH

1.60 – 1.43) and shielded methyl doublets (dH0.76 and 0.55). These data were reminiscent of those for lyngbyatoxin A (2), which was isolated concurrently from the same cyanobacterial extract. Furthermore, HR-FAB-MS analysis revealed a molecular ion peak at m/z 470.3020 [MþH]^þ, which is 32 mass units more than that for lyngbyatoxin A (2), and consistent with a molecular formula of C₂₇H₃₉N₃O₄. Thus, these data indicated the presence of two additional oxygen atoms and 10 degrees of unsaturation. The¹³C (Supplementary Figure S2) and edited HSQC (Supplementary Figure S4) NMR data for1and2were comparable, with signals for six methyls, five methylenes, eight methines and eight non-protonated carbons. However, formamide methine (dC160.0, Shaala et al.2012) and ketone carbon (dC 207.5) signals were unique to the spectrum for1, and closer inspection further revealed the absence of indole C-2 and C-3 signals. In agreement with these data, the IR spectrum for1showed a sharp absorbance at 1700 (amidic carbonyl) and a broad absorbance around 1722 (ketone carbonyls) cm²¹. Close inspection of the¹H NMR spectrum for1 showed two deshieldedortho-coupled aromatic ¹H doublets atdH6.94 (J¼8.1 Hz, H-5) and 7.37 (J¼8.1 Hz, H-6), which correlated with the¹³C signals atdC116.1 and 131.0, respectively, in the HSQC spectrum. These data are consistent with the presence of a 1,2,3,4-tetrasubstituted phenyl moiety, which was confirmed by³JHMBC correlations (Supplementary Figures S5 and S6) from the H-6 doublet to C-4 (dC149.5) and C- 7a (dC130.9) signals, and the H-5 doublet to C-3a (dC113.6) and C-7 (dC116.5) signals. The anticipated formamide moiety comprising NH-1 and CH-2 (dH8.05,dC160.0) was located at C- 7a due to an HMBC correlation from H-2 to C-7a. Three additional substructures (A – C) (Supplementary Figure S6) could be delineated as follows. The¹H NMR spectrum showed two methyl doublets atdH0.55 (d,J¼6.6 Hz, H₃-16) and 0.76 (d,J¼6.6 Hz, H₃-17) correlated with a methine multiplet at dH 2.53 (m, H-15) in the COSY spectrum (Supplementary Figure S3), consistent with an isopropyl moiety. The H-15 multiplet was in turn coupled to an amino methine doublet (dH 3.45, H-12) (substructure A) (Supplementary Figure S6). An N- methylvaline substructure A was supported by HMBC correlations from signals for H-12 to C- 15 and C-17, H₃-16 and H₃-17 to C-12 and C-15, and H-15 to amide carbonyl C-11 (dC171.9), and fromN-methyl H₃-18 (dH 2.96) to C-12. Substructure B (Supplementary Figure S6) was delineated by COSY correlations between the NH-10 doublet (dH 6.05) and methine H-9 (dH

3.87), and between H-9 and oxymethylene H₂-14 (dH3.82 and 3.70) as well as H₂-8 (dH3.61 and 3.34) signals, and was secured by HMBC correlations from H-9 to C-3 and C-14, and H-8 to C- 14. The connectivity of substructures A and B was established by HMBC correlations from H-9 and H-10 to C-11, and these could be connected to the phenyl core on the basis of HMBC Natural Product Research 3

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correlations from H₃-18 to C-4 and from H-8 to C-3a, providing the 9-membered lactam ring in 1. A linalyl terpene substructure C (Supplementary Figure S6) in 1identical to that found in lyngbyatoxin A (2) was readily deduced by comparison of the¹H and¹³C NMR data for1and2 and observed COSY and HMBC correlations (Supplementary Figure S6). The attachment of substructure C at C-7 in1was confirmed by HMBC correlations from H₃-20 to C-7 and C-7a.

Thus, the planar structure of 1 could be assigned as 2,3-seco-2,3-dioxo-lyngbyatoxin A. A 9S, 12S, 19Rconfiguration for1was assigned by consideration of optical rotation values for both1 (½a²⁵D 211.8,c0.5, CHCl₃) and2isolated here (compared with the literature reports), and also due to their consistent ¹H and¹³C NMR data, again compared with the literature (Cardellina et al.1979; Aimi et al.1990; Gallimore et al.2000). Lyngbyatoxin (2) was isolated as a mixture of conformers, as reported previously for lyngbyatoxins (Jiang et al. 2014) and the related teleocidins (Sakai et al. 1986). The ¹H and ¹³C NMR data for 2 were consistent with the originally reported (9S, 12S, 19R) lyngbyatoxin A, as was the optical rotation value obtained:

½a²⁵_D 298.08,c0.1, CHCl₃, compared with½a¹⁸_D 2102.48(c0.13, MeOH, Jiang et al.2014).

This is in contrast to ½a¹⁸D þ85.58for 12-epi-lyngbyatoxin A (9S, 12R, 19R,c0.12, MeOH, Jiang et al.2014) and½a¹⁸D 2185.18for teleocidin A-2 (¼9S, 12S, 19S-lyngbyatoxin A,c0.18, MeOH, Sakai et al.1986).

The remaining compounds were identified as majusculamides A and B (3 and4) (Marner et al. 1977), aplysiatoxin (5) (Yoshinori & Scheuer 1974) and debromoaplysiatoxin (6) (Yoshinori & Scheuer1974) by the analysis of the spectroscopic data (1D, 2D NMR and MS) and comparison of their data with those in the literature.

Compounds 1–6 were evaluated for their antiproliferative effects against HeLa cervical cancer cells (Risinger et al.2008) (Table 1). The most active compound was lyngbyatoxin A (2) with an IC₅₀of 9.2 nM, while aplysiatoxin (5) and debromoaplysiatoxin (6) displayed modest activity (IC50¼13.3 and 3.03mM, respectively), and 2,3-seco-2,3-dioxo-lyngbyatoxin A (1) and majusculamides A and B (3and4) were essentially inactive (IC50.50mM).

3. Experimental

3.1. General experimental procedures

Melting point determination was carried out using an Electrothermal 9100 Digital Melting Point apparatus (Electrothermal Engineering Ltd, Essex, England). Optical rotations were measured on a JASCO DIP-370 digital polarimeter (Jasco Co., Tokyo, Japan) at 258C at the sodium D line (589 nm). UV spectra were measured on a Hitachi 300 Spectrophotometer (Hitachi High- Technologies Corporation, Kyoto, Japan). IR spectra were measured on a Shimadzu Infrared- 400 spectrophotometer (Shimadzu, Kyoto, Japan). Positive mode HR-FAB-MS data were obtained on a Finnigan MAT-312 spectrometer (ThermoFinnigan GmbH, Tokyo, Japan). NMR spectra were obtained in CDCl₃on Varian Inova 500 spectrometers (Varian Inc., Palo Alto, CA,

Table 1. Antiproliferative potency of compounds1–6against HeLa cells.

Compound HeLa cells (IC50,mM) Mean^SEM

2,3-Seco-2,3-dioxo-lyngbyatoxin A (1) .50

Lyngbyatoxin A (2) 0.0092^0.0013

Majusculamide A (3) .50

Majusculamide B (4) .50

Aplysiatoxin (5) 13.3^0.5

Debromoaplysiatoxin (6) 3.03^0.35

Paclitaxel^a 0.0017^0.0001

aPositive control.

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USA) at 500 MHz for¹H NMR and 125 MHz for¹³C NMR. NMR chemical shifts are expressed in parts per million (ppm) referenced to residual CDCl₃solvent signals (dH7.25 for¹H anddC

77.23 for ¹³C). For column chromatography, silica gel (70 – 230 mesh, Merck, Darmstadt, Germany) and Sephadex LH-20 (0.25 – 0.1 mm, Merck, Darmstadt, Germany) were used. Pre- coated SiO₂60 F₂₅₄plates (Merck, Darmstadt, Germany) were used for TLC.

3.2. Biological material

The marine cyanobacteriumM. producenswas collected by hand in March 2013 from the Red Sea at 3 m depth near Obhur, Saudi Arabia. The cyanobacterium was identified by Dr Ali Gab- Alla, Faculty of Science, Suez Canal University, Ismailia, Egypt. A voucher sample has been deposited at the Department of Natural Products, Faculty of Pharmacy, King Abdulaziz University under the registration code No. 2013-MP3.

3.3. Extraction and purification of compounds 1 – 6

The lyophilised cyanobacteriumM. producens(43 g) was extracted with MeOH/CH₂Cl₂(1:1, 3 mL£500 mL) at room temperature. The organic extracts were combined and evaporated under reduced pressure to yield an oily residue (520 mg). The residue was suspended in 150 mL H₂O and extracted with EtOAc (3 mL£150 mL). The organic layers were combined and concentrated, and the resulting extract (430 mg) was chromatographed on a silica gel column using n-hexane/CH₂Cl₂– MeOH gradient. The silica gel column fraction eluted with MeOH/

CH₂Cl₂(10:90, 67 mg) was subjected to Sephadex LH-20 (0.25 – 0.1 mm, Merck, Darmstadt, Germany) using CH₂Cl₂/MeOH (1:1). The major LH-20 column fraction was then purified on a semi-preparative HPLC column (Cosmosil AR II, 5 mm, 250 mm£10 mm) (Nacalai Tesque, Kyoto, Japan) using 50% CH3CN/H2O at a flow rate of 2 mL/min to yield compounds1(3.1 mg), 2(6.2 mg),3(7.9 mg),4(3.1 mg) and5(4.4 mg). Similarly, the silica gel column fraction eluted with MeOH/CH₂Cl₂(20:80, 43 mg) was subjected to Sephadex LH-20 (0.25 – 0.1 mm, Merck, Darmstadt, Germany) using CH₂Cl₂/MeOH (1:1) and the major LH-20 column fraction was purified by HPLC (Cosmosil AR II, 5 mm, 250 mm£10 mm, 60% ACN-H₂O, 2 mL/min) (Nacalai Tesque, Kyoto, Japan) to yield compound6(4.5 mg).

3.4. Spectral data

2,3-Seco-2,3-dioxo-lyngbyatoxin A (1): colourless solid (3.1 mg); m.p. 110 – 1118C; ½a²⁵D

211.8 (c0.5, CHCl₃); UV (lmax, MeOH) (log 1): 226 (4.31), 285 (2.54) nm; IRnmax(KBr):

3380, 2958, 1722, 1700, 1075, 849 cm²¹; ¹H NMR (CDCl₃, 500 MHz): dH 8.05 (1H, d, J¼1.5 Hz, H-2), 7.46 (1H, br s, H-1), 6.94 (1H, d,J¼8.1 Hz, H-5), 7.37 (1H, d,J¼8.1 Hz, H- 6), 3.61 (1H, dd,J¼14.3, 5.5 Hz, H-8a), 3.34 (1H, dd,J¼14.3, 10.4 Hz, H-8b), 3.87 (1H, m, H-9), 6.05 (1H, d,J¼5.8 Hz, H-10), 3.45 (1H, d,J¼9.6 Hz, H-12), 3.82 (1H, dd,J¼13.6, 6.2 Hz, H-14a), 3.70 (1H, dd, J¼13.6, 3.7 Hz, H-14b), 2.53 (1H, m, H-15), 0.55 (3H, d, J¼6.6 Hz, H₃-16), 0.76 (3H, d,J¼6.6 Hz, H₃-17), 2.96 (3H, s, H₃-18), 1.43 (3H, s, H₃-20), 6.11 (1H, dd, J¼17.5, 10.9 Hz, H-21), 5.21 (1H, d, J¼10.9 Hz, H-22a), 5.07 (1H, d, J¼17.5 Hz, H-22b), 1.94 (1H, m, H-23a), 1.58 (1H, m, H-23b), 1.84 (1H, m, H-24a), 1.52 (1H, m, H-24b), 4.94 (1H, t,J¼6.8 Hz, H-25), 1.47 (3H, s, H₃-27), 1.60 (3H, s, H₃-28);¹³C NMR (CDCl₃, 125 MHz):dC160.0 (C-2), 207.5 (C-3), 113.6 (C-3a), 149.5 (C-4), 116.1 (C-5), 131.0 (C-6), 116.5 (C-7), 130.9 (C-7a), 42.1 (C-8), 52.9 (C-9), 171.9 (C-11), 71.1 (C-12), 64.1 (C-14), 28.9 (C-15), 19.4 (C-16), 20.0 (C-17), 32.9 (C-18), 44.2 (C-19), 17.1 (C-20), 147.9 (C-21), 112.4 (C-22), 37.5 (C-23), 23.2 (C-24), 124.1 (C-25), 131.6 (C-26), 17.5 (C-27), 25.6 (C-28); HR- FAB-MSm/z470.3020 (calcd for C₂₇H₄₀N₃O₄[MþH]^þ, 470.3018).

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3.5. Antiproliferative activity

The antiproliferative effects of compounds1–6 against HeLa cells were evaluated using the sulforhodamine B (SRB) assay as previously described (Risinger et al.2008). HeLa cells were cultured in basal medium Eagle (BME) containing Earle’s salts with 10% FBS and 50mg/mL gentamicin sulphate. Cells were plated in a 96-well plate (2500 cells/well) and allowed to grow for 24 h, at which point the test compounds were added. The cells were then incubated with test compounds or DMSO vehicle for 48 h after which the cell density was determined by using the SRB assay. The IC₅₀values, the concentrations that caused 50% inhibition of proliferation, were calculated from the log concentration – response curves. The values inTable 1 represent the average of 3 – 4 independent experiments, each conducted in triplicate^SEM, with paclitaxel as a positive control.

4. Conclusions

In conclusion, investigation of the EtOAc-soluble extract of M. producensafforded the new metabolite 2,3-seco-2,3-dioxo-lyngbyatoxin A (1), together with lyngbyatoxin A (2) and four other known compounds, majusculamides A and B (3 and 4), aplysiatoxin (5) and debromoaplysiatoxin (6). In antiproliferative assays using HeLa cancer cells, the lack of activity (IC₅₀.50mM) for 2,3-seco-2,3-dioxo-lyngbyatoxin A (1) was in sharp contrast to the potent action of lyngbyatoxin A (2, IC₅₀¼9.2 nM). We propose that 2,3-seco-2,3-dioxo- lyngbyatoxin A (1) is a biological oxidation product formed by the action of a dioxygenase on lyngbyatoxin A (Figure 2), analogous to the transformation of tryptophan to N- formylkynurenine catalysed by tryptophan 2,3-dioxygenase. Such an oxygenase may be produced for inactivation (detoxification) of lyngbyatoxin A by a heterotrophic bacterium, or even fungus, associated with the field-collected M. producens. While this type of oxidation product has not been detected previously alongside the lyngbyatoxins, teleocidins or closely related olivoretins (first reported from Streptomyces olivoreticuli, Sakai et al. 1984), 2-oxo- teleocidin-A1 (JBIR-31), an oxidised intact indole was reported from an obligate marine Streptomycesspecies (Izumikawa et al.2010).

Supplementary material

Supplementary material relating to this article is available online, alongside Figures S1 – S6.

Acknowledgements

The authors also gratefully acknowledge the Science and Technology Unit, King Abdulaziz University for technical support. The award from the President’s Council Research Excellence (to SLM) is also acknowledged. We are thankful to Lauren Holmgren for her technical assistance.

N HN O N

His Fe O O OH

H

N HN O N

His Fe O O OH

H H

Base

N HN O NH

O OH

O

2+ 2+

N HN O NH

O OH

O

2 1

Figure 2. Proposed biological oxidation of lyngbyatoxin A (2) to 2,3-seco-2,3-dioxo-lyngbyatoxin A (1).

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Funding

This project was supported by the NSTIP strategic technologies program in the Kingdom of Saudi Arabia- Project No. (11-BIO1555-03).

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