Bloom Material Collected at a Water Reservoir near Kibbutz

May 29, 2013 - Metabolites from Microcystis aeruginosa Bloom Material Collected at ... cyanobacterial blooms in water bodies,7 a biomass of Microcysti...
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Metabolites from Microcystis aeruginosa Bloom Material Collected at a Water Reservoir near Kibbutz Hafetz Haim, Israel Anat Lodin-Friedman and Shmuel Carmeli* Raymond and Beverly Sackler School of Chemistry and Faculty of Exact Sciences, Tel-Aviv University, Ramat Aviv, Tel-Aviv 69978, Israel S Supporting Information *

ABSTRACT: An aqueous MeOH extract of Microcystis aeruginosa (IL-399) afforded three new protease inhibitors, micropeptin HH978 (1), micropeptin HH960 (2), and micropeptin HH992 (3), as well as the known aeruginosin GH553 and microguanidine AL772. The structures of the compounds were elucidated using 1D and 2D NMR techniques, as well as high-resolution mass spectrometry. The absolute configurations of 1−3 were determined using Marfey’s method for amino acid and chiral-phase HPLC for hydroxy acids. The inhibitory activity of the compounds was determined for the serine proteases trypsin, thrombin, elastase, and chymotrypsin. The structure elucidation and biological activities of the new natural products are discussed.

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enera of cyanobacteria that inhabit fresh water bodies tend to produce massive blooms, of which about 50% are toxic. The toxins produced by these genera of cyanobacteria might be alkaloids, predominated by the neurotoxic anatoxins and paralytic shellfish poisons, or hepatotoxic cyclic peptides, usually microcystins.1 Many of these genera of cyanobacteria produce an array of structurally diverse metabolites that inhibit different types of proteases, irrespective of whether they produce microcystins.2 Of the five major groups of protease inhibitors, micropeptins, aeruginosins, anabaenopeptins, microginins, and microviridins, the 3-amino-6-hydroxypiperidone (Ahp)-containing cyclic depsipeptides (micropeptins) are the most abundant, with 128 isolated members.3 Evidence accumulated over the past 30 years suggests that these groups of protease inhibitors affect the ability of these cyanobacteria to survive in their ecological niche by preventing the detoxification of microcystins,4 thus having a negative impact on population growth and on survival of zooplankton species.5 The involvement of the micropeptins and anabaenopeptins in the lysis of cultured cyanobacteria cells was demonstrated and may imply their role in strains dynamic in water blooms.6 As part of our ongoing research on the chemistry and chemical ecology of cyanobacterial blooms in water bodies,7 a biomass of Microcystis aeruginosa (TAU IL-399) was collected in November 2008 from a water reservoir near Kibbutz Hafetz Haim, Israel. The extract of this bloom material afforded three new metabolites, micropeptin HH978 (1), micropeptin HH960 (2), and micropeptin HH992 (3), and two known metabolites, aeruginosin GH5538 and microguanidine AL772.9 The isolation and structure elucidation of the new secondary metabolites and their biological activities are discussed below. The field-collected bloom material was frozen immediately after collection and freeze-dried. The freeze-dried biomass was extracted with 70% MeOH in H2O. The extract, which inhibited trypsin and chymotrypsin, was flash chromatographed © 2013 American Chemical Society and American Society of Pharmacognosy

Received: April 2, 2013 Published: May 29, 2013 1196

dx.doi.org/10.1021/np400281q | J. Nat. Prod. 2013, 76, 1196−1200

Journal of Natural Products

Note

Table 1. 1H and 13C NMR Data of Micropeptins HH978 (1), HH960 (2), and HH992 (3) in DMSO-d6 micropeptin HH978 (1)a position

δC, multc

Val 1 2 3 4 5 NH NMePhe 1 2 3

172.4, 56.1, 30.9, 17.4, 19.3,

4 5,5′ 6,6′ 7 NMe 1 Leu 1 2 3

137.7, 129.4, 128.7, 126.6, 30.7, 170.9, 47.7, 38.5,

4 5 6 NH Ahp/Apo 2/Mpc 1 3/3/2 4/4/3

C CH CH CH3 CH3

169.3, C 60.8, CH 33.9, CH2 C CH CH CH CH3 C CH CH2

23.7, CH 24.0, CH3 22.4, CH3 169.4, C 49.1, CH 21.9, CH2

5/5/4

29.9, CH2

6/6/5 6-OH/−/5-OMe NH/NH/− 2 Leu 1 2 3

73.6, CH

4 5 6 NH Thr 1 2 3 4 NH Asn 1 2 3

170.8, C 50.9, CH 39.1, CH2 24.3, CH 21.1, CH3 23.5, CH3 169.2, 55.1, 72.1, 17.9,

C CH CH CH3

171.8, C 49.3, CH 36.9, CH2

4 NH 4-NH2

172.1, C

Hpla 1 2 2-OH 3

174.1, C 72.8, CH

4

129.0, C

δH, mult., J (Hz) 4.70, 2.01, 0.69, 0.78, 7.51,

dd (9.8, 4.7) m d (6.7) d (7.6) d (9.5)

5.00, brd (11.8) 2.80, dd (11.8, 12.4) 3.20, brd (12.4) 7.12, 7.24, 7.19, 2.73,

d (7.4) t (7.4) t (7.4) s

4.57, d (9.9) 0.25 t (10.2) 1.50, m 0.92, m 0.64, d (6.4) 0.40, d (6.4)

4.38, 2.54, 1.68, 1.68, 1.69, 4.86, 6.04, 7.36,

brq (7.2) m m m m brs brs d (8.9)

4.29, 1.38, 1.81, 1.53, 0.76, 0.86, 8.44,

m brt (11.2) brt (11.2) m d (7.0) d (6.4) d (8.5)

4.53, 5.47, 1.21, 7.79,

m brq (7.0) d (7.0) d (9.1)

4.73, m 2.54, m

micropeptin HH960 (2)a δC, mult.c 171.2, 56.6, 31.3, 18.0, 18.5,

C CH CH CH3 CH3

168.7, C 62.7, CH 33.6, CH2 138.1, 129.7, 129.3, 126.9, 30.4, 170.3, 47.3, 35.8,

C CH CH CH CH3 C CH CH2

23.7, CH 23.3, CH3 20.7, CH3

40.0, CH2

δH, mult., J (Hz) 4.51, 2.17, 0.80, 0.75, 6.27,

dd (7.0, 3.8) m d (6.7) d (7.6) d (7.0)

C CH CH CH3 CH3

170.2, C 62.5, CH 33.5, CH2

d (7.3) t (7.3) t (7.3) s

4.86, dd (11.4, 3.2) −0.31, ddd (14.4, 10.3, 3.2) 1.64, ddd (14.4, 11.4, 4.1) 1.08, m 0.65, d (6.4) 0.44, d (6.4)

139.0, 130.2, 128.8, 126.5, 29.6, 173.4, 47.4, 37.3,

C CH CH CH CH3 C CH CH2

23.4, CH 19.4, CH3 23.5, CH3

4.45, m 2.36, m

104.8, CH

5.15, dt (8.5, 4.3)

28.8, CH2

125.0, CH

6.19, d (8.5)

87.4, CH 55.3, CH3

171.2, C 58.8, CH 23.9, CH2

δH, mult., J (Hz) 4.57, 2.25, 0.66, 0.78, 8.70,

dd (9.7, 3.4) m d (6.8) d (7.6) d (9.5)

4.88, dd (11.3, 2.0) 2.95, m 3.60, brd (11.9) 7.26, 7.28, 7.20, 2.73,

d (7.4) t (7.4) t (7.4) s

4.52, m 0.91 brdd (14.4, 11.9) −0.67 brdd (14.4, 11.9) 1.59, m 0.76, d (6.8) 0.56, d (6.6) 8.58, d (3.6) 4.30, d (9.0) 2.26,d m 1.86,e dd (12.5, 7.5) 1.62,e dd (12.5, 6.5) 1.41,d m 5.22, d (5.2) 3.13, s

7.06, d (8.8) 171.7, C 50.9, CH 40.2, CH2 24.5, CH 21.3, CH3 23.5, CH3 166.4, 54.2, 72.8, 16.0,

C CH CH CH3

171.8, C 49.6, CH 37.0, CH2

4.31, 1.38, 1.73, 1.51, 0.76, 0.86, 8.44,

ddd (11.4, 8.8, 3.5) ddd (14.4, 11.4, 4.1) m m d (6.7) d (6.4) d (8.8)

4.51, 5.09, 1.04, 7.60,

dd (10.1, 1.8) brq (5.6) d (5.6) d (9.9)

4.68, m 2.54, m

172.2, C

brdd (8.8, 2.8) brd (2.8) m brdd (13.8, 2.0)

169.9, 56.0, 28.7, 17.2, 19.7,

5.28, dd (11.4, 2.3) 2.80, dd (14.1, 11.4) 3.23, brdd (14.1, 2.3) 7.19, 7.29, 7.25, 2.75,

δC, mult.c

168.5, C 48.5, CH 26.1, CH2

8.23, d (8.0) 6.91, brs 7.42, brs 4.00, 5.63, 2.54, 2.93,

micropeptin HH992 (3)b

174.2, C 48.4, CH 42.9, CH2 24.3, CH 21.2, CH3 23.5, CH3 166.4, 54.2, 72.8, 16.0,

C CH CH CH3

171.4, C 49.4, CH 35.4, CH2

40.5, CH2 129.1, C 1197

td (9.7, 3.4) m dt (13.3, 3.4) m m d (6.5) d (9.7)

4.51, 5.09, 1.04, 7.60,

dd (10.1, 1.8) brq (5.6) d (5.6) d (9.9)

4.76, dt (7.9, 6.5) 2.38, m 2.61, dd (15.5, 6.5)

172.1, C 8.24, d (7.9) 6.91, brs 7.39, brs

174.2, C 73.0, CH

4.39, 1.38, 1.15, 1.41, 0.79, 0.81, 8.10,

3.99, 5.64, 2.54, 2.89,

m d (5.9) m dd (14.1, 2.6)

8.43, d (7.9) 6.11, brs 7.35, brs 175.0, C 73.2, CH 40.0, CH2

3.95, 5.32, 2.44, 2.95,

m d (6.3) m m

129.2, C dx.doi.org/10.1021/np400281q | J. Nat. Prod. 2013, 76, 1196−1200

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Table 1. continued micropeptin HH978 (1)a position 5,5′ 6,6′ 7 7-OH a

δC, multc 130.4, CH 114.9, CH 155.8, C

δH, mult., J (Hz) 7.05, d (8.5) 6.65, d (8.5)

micropeptin HH960 (2)a δC, mult.c 130.5, CH 115.1, CH 156.0, C

9.17, brs

micropeptin HH992 (3)b

δH, mult., J (Hz) 7.03, d (8.5) 6.65, d (8.5) 9.12, brs

δC, mult.c 130.5, CH 114.9, CH 155.8, C

δH, mult., J (Hz) 7.08, d (8.5) 6.62, d (8.5) 9.08, brs

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

unsaturation. The 1H and 13C NMR spectrum of 2 in DMSO-d6 (Table 1) resembled that of 1, except for the Ahp moiety. In the 1H NMR spectrum of 2 the Ahp hydroxy and aminal protons were absent and two new protons, a doublet at δH 6.19 and a double-double-doublet at δH 5.15, were observed. In the 13C NMR spectrum an aliphatic methylene and the aminal carbon were absent and two doublet vinyl carbons, δC 125.0 and 104.8, were observed. The interpretation of the NMR (COSY, TOCSY, HSQC, and HMBC) data of 2 (Table S2) established the later vinyl moiety as part of a 3-amino-5piperiden-2-one (Apo), probably derived from the elimination of H2O from the Ahp-aminal. The chemical shifts of the protons and carbons of this moiety were similar to those of the elimination product of microcystilide A.13 The rest of the residues composing 2, their sequence, Val-NMePhe-Leu-ApoLeu-Thr-Asn-Hpla, and the cyclization of the valine carbonyl to the threonine oxymethine were assigned as described above for 1 (Table S2). Marfey’s analysis11 established the L-configuration of Asp (from Asn), Leu × 2, NMePhe, Thr, and Val, while an analysis on a chiral-phase HPLC column established the absolute configuration of Hpla as L. Assuming that Apo is an H2O elimination product of Ahp suggests that the absolute configuration of position 3 of Apo is S, as well. On the basis of these arguments the structure of micropeptin HH960 was established as 2. Micropeptin HH992 (3) displayed a positive HRESIMS quasimolecular ion at m/z 1015.5118, in agreement with the molecular formula C50H72N8NaO13 and 19 degrees of unsaturation. Similar to 2, the NMR spectra of 3 in DMSOd6 (Table 1) were comparable to that of 1. Micropeptin HH992 (3) presented an additional methoxy residue (δH 3.13, s; δC 55.3, CH3), and the characteristic signals of the Ahp were replaced by a new set of signals (Table 1) assigned by interpretation of the 2D NMR (COSY, TOCSY, HSQC, and HMBC, Table S3) data as a 5-methoxypyrrolidine-2-carboxyamide (Mpc) moiety. COSY and TOCSY correlations established the connectivity of H-2 through H-5, and HMBC correlations of H-2 with the carboxyamide C-1 and the aminal carbon C-5 and of H-5 with the methoxy carbon completed the structure elucidation of the Mpc residue. The relative configuration of the asymmetric centers of Mpc was deduced from the coupling constants and NOEs of the proton signals in this spin system. Mpc-H-2 exhibited a large (9 Hz) coupling constant and strong NOE with H-3 that resonated at 2.26 ppm, thus assigned as syn to H-2. Assuming a 2S-configuration for Mpc-C-2 (confirmed by Marfey’s procedure, which established the absolute L-configuration of Glu, which resulted from hydrolysis and oxidation of Mpc), the proton resonating at 2.26 ppm was established as H-3ProS. H-3ProR, which resonated at 1.86 ppm, had a very small coupling constant and a weak NOE correlation with H-2, suggesting a dihedral angle of ca. 90° between these two protons. H-3ProS presented a NOE correlation only with H-4, which resonated at 1.62 ppm,

on a reversed-phase C18 column, and the fractions that exhibited protease inhibitory activity were further separated on a Sephadex LH-20 column and reversed-phase HPLC columns to afford five pure natural products, micropeptins HH978 (1, 13.2 mg, 0.005% yield), HH960 (2, 1.7 mg, 0.0007% yield), and HH992 (3, 5.6 mg, 0.002% yield), aeruginosin GH553 (8.3 mg, 0.003% yield), and a relatively large quantity of the known microguanidine AL772 (294.0 mg, 0.11% yield). Micropeptin HH978 (1) presented a positive quasimolecular ion in the HRESI mass spectrum at m/z 1001.4955, corresponding to the molecular formula C49H70N8NaO13 and 19 degrees of unsaturation. The NMR spectra of 1 in DMSO-d6 (Table 1) revealed its peptide nature and that it belongs to the micropeptins.10 Micropeptin HH978 (1) differed from micropeptin HM978 in the third position of the macrocycle, presenting Leu instead of Ile.10 Interpretation of the COSY, TOCSY, HSQC, and HMBC data (Table S1) enabled the assignment of the structure of eight residues: Val, NMePhe, 1 Leu, Ahp, 2 Leu, Thr, Asn, and Hpla. The structure determination of the 1Leu moiety started with the doublet proton that resonated at δH 4.57 and was assigned as the αproton of a leucine moiety based on the COSY and TOCSY correlations, although the proton signals for positions 3, 4, 5, and 6 were shifted upfield. This could be explained as an anisotropic effect of the neighboring NMePhe moiety and was supported by the relevant NOEs (Table S1). The α-proton of 1 Leu failed to present any correlation with either an amine or amide proton. The carboxyamide of 1Leu δC 170.9 was assigned on the basis of its HMBC correlation with 1Leu-H-2. The correlation of the latter proton with the aminal carbon (Ahp-C6) supported the structure of this moiety as 1Leu-Ahp. The sequence of the later residues in 1, Val-NMePhe-1LeuAhp-2Leu-Thr-Asn-Hpla, and the cyclization of the valine carbonyl to the threonine oxymethine were independently assigned by the inter-residual HMBC or ROESY correlations (Table S1). The relative configuration of the Ahp residue, 3S*, 6R*, was assigned on the basis of the similarity of its proton and carbon chemical shifts with those of micropeptin HM978 and the NOE of the pseudoaxial-H-4 (H-4pax, δH 2.54) with the 6-OH.10 Marfey’s analysis11 (using L-FDAA as the derivatizing reagent), preceded by Jones’ oxidation,12 established the L-configuration of Asp (from Asn), Glu (from 3S,6RAhp), Leu × 2, NMePhe, Thr, and Val, while analysis on a chiral-phase HPLC column established the absolute configuration of Hpla as L and the structure of micropeptin HH978 as 1. Micropeptins HH978 (1), HH960 (2), and HH992 (3) share the same amino acid sequence but differ in the structure of the amino acid in the fourth position of the lactone ring. Micropeptin HH960 (2) exhibited a positive ESIMS quasimolecular ion at m/z 983.4856, in agreement with the molecular formula C49H68N8NaO12 and 20 degrees of 1198

dx.doi.org/10.1021/np400281q | J. Nat. Prod. 2013, 76, 1196−1200

Journal of Natural Products

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HPLC system (L-7000A intelligent pump and model L-6200 UV−vis detector). An ELISA ELx808 reader (BIO-TEK Instruments, Inc) was used for protease inhibition assays. Biological Material. Microcystis aeruginosa, TAU IL-399, was collected in November 2008 from a water reservoir near Kibbutz Hafetz Haim, Israel. The cell mass was frozen and lyophilized. A sample of the cyanobacteria is deposited at the culture collection of Tel Aviv University. Isolation Procedure. The lyophilized cell mass of IL-399 (258 g) was extracted with 7:3 MeOH/H2O (3 × 2 L), and evaporation yielded 30 g of extract. The extract was chromatographed, in 5 g portions, on a reversed-phase (ODS) flash column (YMC-GEL, 120A, 4.4 × 6.4 cm) eluted with an increasing percentage of MeOH in H2O to afford 12 fractions. Fraction 8, eluted from the ODS column with 7:3 MeOH/H2O (0.7 g), was separated on Sephadex LH-20 (MeOH/ H2O, 1:1) and reversed-phase HPLC columns (YMC Pack C8, 5 μm, 250 × 20 mm, DAD at maximum absorbance, 4:6 H2O/MeOH, flow rate 5.0 mL/min) to obtain pure micropeptin HH978 (1) (13.2 mg, 0.0051% yield) and micropeptin HH960 (2) (1.7 mg, 0.0004% yield). Fraction 7 (3:2 MeOH/H2O, total of 0.42 g) was separated on a Sephadex LH-20 column eluted with 1:1 MeOH/CHCl3 to obtain 21 fractions. Fractions 7−12 (0.21 g) were combined and separated repeatedly on a reversed-phase HPLC column (YMC-Pack C18, 5 μm, 250 × 20 mm, DAD at maximum absorbance, 9:11 H2O/MeOH, flow rate 5.0 mL/min) to obtain a semipure fraction (72 mg, tR 30.15 min) that was further purified on a reversed-phase HPLC column (YMC Pack C8, 5 μm, 250 × 20 mm, DAD at maximum absorbance, 9:11 0.1% TFA in H2O/MeOH, flow rate 5.0 mL/min) to obtain pure micropeptin HH992 (3) (5.6 mg, 0.0022% yield). Fraction 6 from the initial RP-18 chromatography (1.5 g) was separated on Sephadex LH20 (MeOH/H2O, 7:3) to afford 20 fractions. Fractions 5−12 (1.1 g) were further purified on a Sephadex LH-20 column (MeOH/H2O, 1:1) to afford 10 fractions. Fractions 4−9 (1.1 g) were further purified by RP-18 chromatography to afford the known microguanidine AL772 (294 mg, 0.11% yield). Fractions 13 and 14 from the Sephadex LH-20 column were separated on a reversed-phase HPLC column (YMC Pack C8, 5 μm, 250 × 20 mm, DAD at maximum absorbance, 1:1 H2O/MeOH, flow rate 5.0 mL/min) to obtain pure aeruginosin GH553 (8.3 mg, 0.0032% yield). Micropeptin HH978 (1): glassy, white solid; [α]23D −53 (c 0.94, MeOH); UV (MeOH) λmax (log ε) 202 (4.56), 228 (4.00), 278 (3.04) nm; 1H and 13C NMR (Table 1 and Table S1 in Supporting Information (SI)); HRESIMS m/z 1001.4956 [M + Na]+ (calcd for C49H70N8NaO13, 1001.4960). Retention times of amino acid (AA) Marfey’s derivatives: L-Asp 30.1 min (D-Asp 30.7 min), L-Glu 32.3 min (D-Glu 32.7 min), L-Leu 43.9 min (D-Leu 46.7 min), L-NMePhe 44.0 min, L-Thr 29.8 min (D-Thr 32.3 min), L-Val 39.9 min (D-Val 43.0 min). Retention time of L-hydroxyphenyllactic acid (Hpla) on a chiralphase column 3.4 min (D-Hpla 4.3 min). Micropeptin HH960 (2): glassy, white solid; [α]23D −70 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 202 (4.39), 227 (3.89), 271 (3.17) nm; 1H and 13C NMR (Table 1 and Table S2 in SI); HRESIMS m/z 983.4856 [M + Na]+ (calcd for C49H68N8NaO12, 983.4854). Retention times of AA Marfey’s derivatives: L-Asp 30.4 min (D-Asp 31.0 min), LLeu 44.0 min (D-Leu 46.8 min), L-NMePhe 43.9 min, L-Thr 29.8 min (D-Thr 32.4 min), L-Val 39.9 min (D-Val 43.0 min). Retention time of L-hydroxyphenyllactic acid (Hpla) on a chiral-phase column 3.9 min (D-Hpla 4.1 min). Micropeptin HH992 (3): glassy, white solid; [α]23D −62 (c 0.42, MeOH); UV (MeOH) λmax (log ε) 203 (4.36), 227 (3.82), 278 (2.90) nm; 1H and 13C NMR (Table 1 and Table S3 in SI); HRESIMS m/z 1015.5118 [M + Na]+ (calcd for C50H72N8NaO13, 1015.5117). Retention times of AA Marfey’s derivatives: L-Asp 30.0 min (D-Asp 30.6 min), L-Glu 32.4 min (D-Glu 33.1 min), L-Leu 44.0 min (D-Leu 46.8 min), L-NMePhe 43.7 min, L-Thr 29.4 min (D-Thr 31.9 min), LVal 39.6 min (D-Val 42.7 min). Retention time of L-hydroxyphenyllactic acid (Hpla) on a chiral-phase column 3.9 min (D-Hpla 4.2 min). Aeruginosin GH553: 8 glassy, white solid; [α]23D 17 (c 1.02, MeOH), reported [α]23D −88 (c 0.20, MeOH);8 1H and 13C NMR

while H-3ProR presented a NOE correlation only with H-4, which resonated at 1.41. This suggested that H-3ProS and the proton resonating at 1.62 ppm, and H-3ProR and the proton resonating at 1.41 ppm, respectively, are situated on the same faces of the Mpc ring. The proton resonating at 1.62 ppm was thus assigned as H-4ProR, while the other one as H-4ProS. H4ProS presented a 5.2 Hz coupling constant and a strong NOE correlation with H-5, while H-4ProS had a coupling constant of ca. 0 Hz and a weak NOE correlation with H-5, suggesting that H-5 is situated syn to H-4ProS, and thus the configuration of C5 is R. The structure elucidation of the rest of the acid residues that compose 3 was achieved by interpretation of the 2D NMR data (Table S3), Marfey’s analysis,11 and chiral-phase HPLC, which established the L-configuration of all amino acids and Hpla, as described for 1. On the basis of these arguments the structure of micropeptin HH992 was established as 3. The extracts of strain IL-399 exhibited significant inhibition of the serine proteases trypsin and chymotrypsin at a concentration of 1 mg/mL.14 The activity-guided purification of the protease-inhibiting components of the extract revealed that micropeptins HH978 (1) HH960 (2), and HH992 (3) were responsible for the inhibition of chymotrypsin, while aeruginosin GH553 was responsible for the inhibition of trypsin. The inhibitory activities of 1−3 were determined for the serine proteases trypsin, thrombin, chymotrypsin, and elastase. Micropeptin HH978 (1) inhibited chymotrypsin with an IC50 of 4.3 μM and elastase with an IC50 of 17.6 μM, but not trypsin and thrombin at a concentration of 45.5 μM. Micropeptin HH960 (2) inhibited chymotrypsin with an IC50 of 48.5 μM and elastase with an IC50 of 55.5 μM, but not trypsin and thrombin at a concentration of 45.5 μM. Micropeptin HH992 (3) inhibited chymotrypsin with an IC50 of 45.9 μM and elastase with an IC50 of 16.9 μM, but not trypsin and thrombin at a concentration of 45.5 μM. Aeruginosin GH553 inhibited trypsin with an IC50 value of 45.5 μM. Comparison of the inhibitory activities of micropeptins HH978 (1) and HH960 (2) against chymotrypsin and elastase suggests that the Ahp-6-OH is essential for the interaction and inhibition with both enzymes. In contrast, comparison of the inhibitory activities of micropeptins HH978 (1) and HH992 (3) against chymotrypsin and elastase reveals that the Ahp is essential for the interaction and inhibition of chymotrypsin, but elastase is more tolerant to the structure variation and accepts the Mpc moiety as well. These variations in the potency of 1−3 against chymotrypsin and elastase suggest that bridging the Ahp-6-O with the Ahp-amide-nitrogen may result in inhibitors that present better selectivity toward elastase.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined on a JASCO P-1010 polarimeter. UV spectra were recorded on an Agilent 8453 spectrophotometer. NMR spectra were recorded on a Bruker DMX-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 (mixing time 60 ms), gROESY (spinlock pulse 0.2 s), gHSQC, and gHMBC spectra were recorded using standard Bruker pulse sequences. High-resolution MS were recorded on a Waters MALDISynapt instrument. HPLC separations were performed on a JASCO HPLC system (model PU-2080 Plus pump, model LG-208004 Quaternary Gradient unit, and model PU-2010 Plus multiwavelength detector), a Merck HPLC system (model L-6200A pump and model L-4200 UV−vis detector), and a Merck Hitachi 1199

dx.doi.org/10.1021/np400281q | J. Nat. Prod. 2013, 76, 1196−1200

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(Tables S4 and S4a in SI); HRESIMS m/z 576.2325 [M + Na]+ (calcd for C29H35N3NaO8, 576.2322). Microguanidine AL772: 9 glassy, white solid; [α]23D 7 (c 0.28, H2O) reported [α]22D 16.0 (c 1.0, MeOH);9 1H and 13C NMR (Table S5 in SI); HRESIMS m/z 635.1002 [M − H]− (calcd for C19H31N4O14S3, 635.0999). 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.14 Protease Inhibition Assays. The procedures used to determine the inhibitory activity of the new compounds against trypsin, thrombin, elastase, and chymotrypsin were described in a previous paper.14

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ASSOCIATED CONTENT

* Supporting Information S

1D and 2D NMR spectra, NMR tabulated data, and HRMS data of compounds 1−3, as well as 1H and 13C NMR spectra and NMR tabulated data of aeruginosin GH553 and microguanidine AL772. 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: [email protected]. 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. This research was supported by the Israel Science Foundation grant 776/06.

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REFERENCES

(1) Wiegand, C.; Pflugmacher, S. Toxicol. Appl. Pharmacol. 2005, 203, 201−218. (2) Welker, M.; Marsalek, B.; Sejnohova, L.; von Dohren, H. Peptides 2006, 27, 2090−2103. (3) Kang, H.-S.; Krunic, A.; Orjala, J. J. Nat. Prod. 2012, 75, 807−811. (4) Wiegand, C.; Peuthert, A.; Pflugmacher, S.; Carmeli, S. Environ. Toxicol. 2002, 17, 400−406. (5) Rohrlack, T.; Christoffersen, K.; Hansen, P. E.; Zhang, W.; Czarnecki, O.; Henning, M.; Fastner, J.; Erhard, M.; Neilan, B. A.; Kaebernick, M. J. Chem. Ecol. 2003, 29, 1757−1770. (6) Sedmak, B.; Carmeli, S.; Elersek, T. Microb. Ecol. 2008, 56, 201− 209. (7) Elkobi-Peer, S.; Faigenbaum, R.; Carmeli, S. J. Nat. Prod. 2012, 75, 2144−2151. (8) Lifshits, M.; Carmeli, S. J. Nat. Prod. 2012, 75, 209−219. (9) Gesner-Apter, S.; Carmeli, S. Tetrahedron 2008, 64, 6628−6634. (10) Lifshits, M.; Zafrir-Ilan, E.; Raveh, A.; Carmeli, S. Tetrahedron 2011, 67, 4017−4024. (11) Marfey’s reagent: 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide. Marfey, P. Carlsberg Res. Commun. 1984, 49, 591−596. (12) Bowden, K.; Heilbron, I. M.; Jones, E. R. H.; Weedon, B. C. L. J. Chem. Soc. 1946, 39−45. (13) Tsukamoto, S.; Painuly, P.; Young, K. A.; Yang, X. M.; Shimizu, Y.; Cornell, L. J. Am. Chem. Soc. 1993, 115, 11046−11047. (14) Zafrir-Ilan, E.; Carmeli, S. Tetrahedron 2010, 66, 9194−9202.

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dx.doi.org/10.1021/np400281q | J. Nat. Prod. 2013, 76, 1196−1200