Aeruginosins from a Microcystis sp. Bloom Material Collected in

Jun 18, 2013 - water reservoir in Varanasi, India. Aeruginosins IN608 and IN652 are linear modified peptides containing four building blocks, one of w...
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Aeruginosins from a Microcystis sp. Bloom Material Collected in Varanasi, India Shira Elkobi-Peer,†,# Rahul Kunwar Singh,‡,# Tribhuban Mohan Mohapatra,‡ Shree Prakash Tiwari,§ and Shmuel Carmeli*,† †

Raymond and Beverly Sackler School of Chemistry and Faculty of Exact Sciences, Tel-Aviv University, Ramat Aviv, Tel-Aviv 69978, Israel ‡ Department of Microbiology, Institute of Medicinal Sciences, Banaras Hindu University, Varanasi-221005, India § Department of Microbiology, VBS Purvanchal University, Jaunpur-222001, India S Supporting Information *

ABSTRACT: Two novel biologically active short peptides, aeruginosins IN608 and IN652, were isolated from the cyanobacterium Microcystis sp. strain BHU006, which was collected from Durgakund water reservoir in Varanasi, India. Aeruginosins IN608 and IN652 are linear modified peptides containing four building blocks, one of which is the arogenate-derived modified amino acid 2-carboxy-6-hydroxyoctahydroindole. Aeruginosin IN608 and aeruginosin IN652 inhibit the activity of the proteolytic enzyme trypsin with IC50’s of 4.3 and 4.1 μM, respectively.

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inhibition of trypsin-type proteases and lowering the amounts of active GST in D. magna, a cyanobacteria grazer, may imply they are biosynthesized in order to prevent the detoxification of the microcystins by cyanobacteria competitors in the environment. A comprehensive review on the chemistry and biology of the aeruginosins, with an emphasis on their sources, structural revisions, and total syntheses was published several years ago by Hanessian et al.9 Herein, we report the isolation, structure elucidation, and biological activity of two new aeruginosins, aeruginosins IN608 (1) and IN652 (2), isolated from a Microcystis sp. water bloom material collected from Durgakund water reservoir in Varanasi, India. The hydrophilic crude extract of the cyanobacterium was separated on Sepahdex LH-20 and reversed-phase HPLC chromatography to afford two closely related pure compounds, aeruginosins IN608 (1) and IN652 (2). The 1H NMR spectrum (Table 1) of 1 and 2 in DMSO-d6 suggested that they exist in solution as a mixture of ca. 1:4 of the cis and trans rotamers of the amide bond between the Choi moiety and the adjacent leucine moiety.10 Aeruginosin IN608 (1) was obtained as a colorless, glassy material, which presented a positive HRESIMS molecular adduct ion at m/z 609.3166/611.3161 (3:1, [M + H]+), corresponding to the molecular formula C29H46ClN6O6 and 10 degrees of unsaturation. The 1H NMR spectrum of 1 in DMSO-d6 presented a broad singlet signal of a phenol, a pair of triplet amide protons, a doublet amide proton, a broad triplet proton, two very broad signals between 6.5 and 7.5 ppm

eruginosins belong to a group of 40 published linear modified peptides that are produced by water-bloomforming genera of cyanobacteria.1,2 They are characterized by the presence of a hydroxyphenyl lactic acid (Hpla) derivative at the N-terminus of the peptide, a variable amino acid (Leu, Ile, Phe, or Tyr) at the second position, a 2-carboxy-6hydroxyoctahydroindole (Choi) derivative at the third position, and an arginine derivative (if any) at the C-terminus of the peptide. These linear modified peptides are biosynthesized in cyanobacteria from amino acids or amino acid precursors by non ribosomal peptide synthase (NRPS) type enzyme assembly.3 The aeruginosins inhibit trypsin-type serine proteases such as trypsin and thrombin that hydrolyze the amide bond next to arginine or lysine. Microcin SF608, an aeruginosin metabolite isolated from a strain of Microcystis aeruginosa, inhibits trypsin with an IC50 of 0.5 μg/mL4 and lowers the cellular concentrations of soluble glutathione Stransferases (sGST) and microsomal GST (mGST) significantly in the water flea, Daphnia magna, possibly by inhibiting the serine protease responsible for transforming the s- and mGST to its active form.5 GST has been shown to be involved in the detoxification process of the hepatotoxic microcystins in plants, invertebrates, and mammals.6 The aeruginosins and other groups of protease inhibitors (i.e., micropeptins, anabaenopeptins, and microviridins) are biosynthesized in certain water-bloom-forming genera of cyanobacteria along with the hepatotoxic microcystins, and the crude extracts of these cyanobacteria have been shown to be more potent than the pure microcystins themselves.7,8 The trigger for the production of toxins and the accompanying protease inhibitors in cyanobacteria and the relationship between those groups of compounds are not fully understood. For the areuginosins, © 2013 American Chemical Society and American Society of Pharmacognosy

Received: February 5, 2013 Published: June 18, 2013 1187

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Table 1. NMR Data of the Major Rotamers of Aeruginosins IN608 (1) and IN652 (2) in DMSO-d6 aeruginosin IN608 (1)a δC, mult

position Cl/BrHpla

Leu

Choi

Agmd

a

1 2 3 3′ 4 5 6 7 8 9 2-OH 7-OH 1 2 3 3′ 4 5 6 NH 1 2 3 3′ 3a 4 4′ 5 6 6-OH 7 7′ 7a 1 1′ 2 3 4 1-NH 4-NH CN

c

171.9, C 71.5, CH 38.9, CH2 129.4, 130.6, 118.8, 151.3, 115.9, 129.2,

C CH C C CH CH

169.4, C 47.7, CH 42.1, CH2 23.8, CH 23.3, CH3 21.3, CH3 171.3, C 59.8, CH 30.5, CH2 35.9, CH 18.9, CH2 25.9, CH2 63.8, CH 33.4, CH 53.8 CH 37.8, CH2 25.7, CH2 26.3, CH2 40.2, CH2

δH, mult, J (Hz) 4.07, m 2.81, m 2.70, dd (14.0, 3.5) 7.12, s

6.82, 6.94, 5.84, 9.87,

d (8.4) d (8.4) m s

4.52, 1.31, 1.19, 1.17, 0.78, 0.84, 7.35,

m m m m d (5.6) d (5.6) d (8.4)

4.13, 1.98, 1.78, 2.26, 2.00, 1.40, 1.41, 3.91, 4.52, 2.01, 1.67, 4.01, 3.07, 3.00, 1.41, 1.38, 3.07, 7.85, 7.56,

t (8.8) m m m m m m brs m m m m m m m m m brt (5.2) brt (5.6)

156.7, C

aeruginosin IN652 (2)b δC, multc 172.2, C 71.8, CH 38.9, CH2 130.2, 133.9, 108.8, 152.7, 116.0, 129.2,

C CH C C CH CH

169.7, C 48.0, CH 42.5, CH2 24.1, CH 23.6, CH3 21.7, CH3 171.6, C 60.1, CH 30.9, CH2 36.3, CH 19.2, CH2 26.3, CH2 64.1, CH 33.8, CH 54.2 CH 38.1, CH2 26.1, CH2 26.5, CH2 40.2, CH2

δH, mult, J (Hz) 4.07, m 2.82, m 2.70, m 7.28, s

6.81, 6.98, 5.84, 9.94,

d (8.3) d (8.3) d (5.0) s

4.52, 1.31, 1.22, 1.20, 0.78, 0.84, 7.35,

m t (10.0) m m d (5.5) d (5.5) d (8.5)

4.11, 1.99, 1.78, 2.27, 2.03, 1.41, 1.41, 3.91, 4.49, 2.18, 1.67, 4.02, 3.05, 3.00, 1.41, 1.41, 3.07, 7.84, 7.52,

t (8.5) m m m m m m brs m m m m m m m m m brt (6.5) brt (6.5)

157.0, C

400 MHz for 1H, 100 MHz for 13C. b500 MHz for 1H, 125 MHz for 13C. cMultiplicity and assignment from HSQC experiment. dAgmatine.

Supporting Information). The aliphatic region was too complicated to be interpreted except for two pairs of doublet methyl groups that resonated between 0.6 and 0.9 ppm. The 13 C NMR spectrum of 1 revealed three pairs of amide/ester carbonyls, two guanidine/phenol sp2 carbons, five pairs of signals of aromatic sp2 carbons consistent with a pair of trisubstituted phenol moieties, five pairs of methines between 72 and 45 ppm (Figure S2 in the Supporting Information), and a handful of additional methine, methylene, and methyl signals in the aliphatic region. Both rotamers were fully characterized (Table 1 and Tables S1 and S1a in the Supporting Information, for the full data of the cis and trans rotamers), but for clarity the structure elucidation of only the major trans rotamer is discussed below. The interpretation of the data from the COSY, TOCSY, HSQC, and HMBC 2D NMR experiments allowed the assignment of the agmatine, Leu, and o-Cl-Hpla

presumably of guanidine protons, three pairs of signals indicating the presence of a trisubstituted phenol system in the aromatic region, two pairs of exchangeable hydroxy doublet signals, and five pairs of methine protons next to electronegative moieties in the 3.7−5.9 ppm region (Figure S1 in the 1188

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activity-guided purification of the trypsin-inhibiting components of the extract revealed that the new aeruginosins were responsible for the inhibition of trypsin. Aeruginosins IN608 (1) and IN652 (2) inhibited trypsin with IC50 values of 4.3 and 4.1 μM, respectively. These results indicated that the nature of the halogen substituent of the aromatic ring of the Hpla moiety does not influence the potency of the aeruginosins as in the case of aeruginosins GE686 and GE730.2 Substitution of the DLeu in aeruginosin IN608 with a D-alloIle, in aeruginosin KY608, however, results in a more potent trypsin inhibitor (IC50 = 2.8 μM).14

moieties (Table S1). The double doublet proton signal resonating at δH 4.13 and the broad singlet at δH 3.91 were characteristic of a Choi moiety.11 COSY correlations established the couplings between all of the Choi moiety protons, and NOESY correlations established its relative configuration (see Figure 1). A comparison of the chemical

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation values were obtained with a Jasco P-1010 polarimeter at the sodium D line (589 nm). UV spectra were recorded with an Agilent 8453 spectrophotometer. IR spectra were measured with a Bruker Tensor 27 instrument. NMR spectra were recorded on a Bruker DRX-500 spectrometer at 500.13 MHz for 1H and 125.76 MHz for 13C and a Bruker Avance 400 spectrometer at 400.13 MHz for 1H and 100.62 MHz for 13C. DEPT, COSY-45, gTOCSY, gROESY, gHSQC, gHMQC, and gHMBC spectra were recorded using standard Bruker pulse sequences. Mass spectra were recorded on a Waters MaldiSynapt instrument. HPLC separations were performed on an ISCO HPLC system (model 2350 pump and model 2360 gradient programmer) equipped with an Applied Biosystem Inc. diode-array detector. An ELx808 Absorbance Microplate Reader (BIO-TEK Instruments, Inc.) was used for protease inhibition assays. Biological Material. Microcystis sp., BHU strain BHU006, was collected in February 2010 from Durgakund water reservoir (25°28′90″ N; 82°99′95″ E) in Varanasi, India. Its evolutionary history was inferred using the UPGMA method (see Supporting Information). Samples of the cyanobacteria are deposited at the culture collection of Banaras Hindu University. Isolation Procedure. The freeze-dried cells (BHU006, 85 g) were extracted with 7:3 MeOH/H2O (3 × 3 L). The crude extract (11 g) was evaporated to dryness and separated on an ODS (YMC-GEL, 120A, 4.4 × 6.4 cm) flash column with increasing amounts of MeOH in water. Fraction 7/8 (6:4 to 7:3 MeOH/H2O, 0.5 g) was subjected to a Sephadex LH-20 column in 1:1 chloroform/MeOH to obtain 12 fractions. Fractions 6−8 from the Sephadex LH-20 column were separated on a reversed-phase HPLC (YMC-Pack C-8 250 mm × 20.0 mm, DAD at 210 nm, flow rate 5.0 mL/min) in 77:23 0.1% aqueous TFA/acetonitrile to obtain compounds 1 (8.9 mg, retention time 22.1 min, 0.08% yield based on the dry weight of the bacteria) and 2 (1.5 mg, retention time 24.8 min, 0.01% yield based on the dry weight of the bacteria). Aeruginosin IN608 (1): amorphous, colorless solid; [α]25D −8.2 (c 1.46, MeOH); UV (MeOH) λmax (log ε) 203 (4.29), 230 (3.61), 281 (3.15), 346 (2.88) nm; IR (ATR Diamond) 2936, 1635, 1200, 1135 cm−1; for NMR data see Table 1 and Tables S1 and S1a in the Supporting Information; HRESIMS m/z 609.3166/611.3161 (3:1 MH+, calcd for C29H4635ClN6O6 m/z 609.3167). Retention time of AA Marfey derivatives: D-leucine 53.7 min (L-Leu 50.1, D-Leu 53.7 min), Lchoi-6α-OH 41.8 min (L-choi-6α-OH 41.8, L-choi-6β-OH 40.7 min). Chiral HPLC retention times: D-Cl-Hpla 3.9 min (L-Cl-Hpla 3.6 min, D-Cl-Hpla 3.9 min). Aeruginosin IN652 (2): amorphous, colorless solid; [α]25D −29.6 (c 0.23, MeOH); UV (MeOH) λmax (log ε) 203 (4.30), 225 (3.72), 281 (2.90) nm; IR (ATR Diamond) 2934, 1652, 1201 cm−1; for NMR data see Table 1 and Tables S2 and S2a in the Supporting Information; HR ESI MS m/z 653.2663/655.2659 (1:1 MH+, calcd for C29H4679BrN6O6 m/z 653.2662)). Retention time of AA Marfey derivatives: D-leucine 52.9 min (L-Leu 49.4, D-Leu 52.9 min), L-Choi6α-OH 41.0 min (L-choi-6α-OH 41.0, L-choi-6β-OH 39.9 min). Chiral HPLC retention times: D-Br-Hpla 3.3 min (L-Br-Hpla 3.0 min, D-ClHpla 3.3 min).

Figure 1. Relative stereochemistry of L-Choi deduced from NOE correlations.

shifts and multiplicities of the Choi moiety of 1 with that of aeruginosin 298-A11 confirmed the suggested Choi structure. The sequence of the peptide was determined as o-Cl-Hpla-LeuChoi-agmatine, on the basis of the HMBC correlations of o-ClHpla-CO with Leu-NH, of Leu-CO with the Choi-H-2, and of Choi-CO with agmatine-1-NH. Applying Marfey’s method12 (L-FDAA) and chiral-phase HPLC chromatography (with the synthetic D,L-o-Cl-Hpla2) revealed the presence of L-Choi, DLeu, and D-o-Cl-Hpla moieties in 1. On the basis of the evidence discussed above the structure of aeruginosin IN608 (1) was established as D-o-Cl-Hpla-D-Leu-L-Choi-agmatine. Aeruginosin IN652 (2) was obtained as a yellowish glassy material. It exhibited a complex molecular quasimolecular adduct ion ([M + H]+) at m/z 653.2663/655.2659 (1:1), corresponding to the molecular formula C29H46BrN6O6 and 10 degrees of unsaturation. Aeruginosin IN652 (2) presented 1H and 13C NMR spectra similar to those of 1, except for some of the signals of the Hpla moiety (see Table 1). Hpla-H-5 resonated in 2 at 7.28 (s) ppm relative to 7.12 (s) ppm in 1, Hpla-C-5 resonated in 2 at 133.9 (CH) ppm relative to 130.6 (CH) ppm in 1, and Hpla-C-6 resonated in 2 at 108.8 (C) ppm relative to 118.8 (C) ppm in 1, suggesting the substitution of C-6 in 2 with a bromine instead of a chlorine in 1 and in accordance with an o-Br-Hpla moiety in aeruginosin 98-C.13 The interpretation of the data from the COSY, TOCSY, HSQC, and HMBC 2D NMR experiments allowed the full assignment of the agmatine, Choi, Leu, and o-Br-Hpla moieties (Table S2). The sequence of the peptide was determined as oBr-Hpla-Leu-Choi-agmatine, on the basis of the HMBC correlations of o-Cl-Hpla-CO with Leu-NH and of Choi-CO with agmatine-1-NH and NOE between Leu-H-2 and Choi-H-2 (Table S2). Applying Marfey’s method12 (L-FDAA) and chiralphase HPLC chromatography (with the synthetic D,L-o-BrHpla2) revealed the presence of L-Choi, D-Leu, and D-o-Br-Hpla moieties in 1. On the basis of the evidence discussed above, the structure of aeruginosin IN652 (2) was established as D-o-BrHpla-D-Leu-L-Choi-agmatine. The extracts of strain IN-1 exhibited significant inhibition of the serine protease trypsin at a concentration of 1 mg/mL. The 1189

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Determination of the Absolute Configurations of Amino and Hydroxy Acids. The procedures used to determine the absolute configurations of the amino and hydroxy acids of the new compounds were described in a previous paper.15 Protease Inhibition Assays. The procedure used to determine the inhibitory activity of the new compounds against trypsin was described in a previous paper.15

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ASSOCIATED CONTENT

S Supporting Information *

1D and 2D NMR spectra and HRMS data of compounds 1 and 2, tables of the full NMR data of the major and minor rotamers of compounds 1 and 2, and the evolutionary relationships of Microcystis sp. BHU006. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +972-3-6408550. Fax: +972-3-6409293. E-mail: carmeli@ post.tau.ac.il. Author Contributions #

S. Elkobi-Peer and R. K. Singh contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank N. Tal, the Mass Spectrometry Facility of the School of Chemistry, Tel Aviv University, for the measurements of the HRESI mass spectra. The authors thank J. Bonjoch, Faculty of Pharmacy, University of Barcelona, Spain, for the kind provision of the synthetic Choi isomers. This research was supported by the Israel Science Foundation grants 776/06 and Indian Council of Medical Research grant 45/98/PHA/BMS.

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REFERENCES

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