Lajollamycins, Nitro Group-Bearing Spiro-Î²-lactone-Î³-lactams

Article pubs.acs.org/jnp

Lajollamycins, Nitro Group-Bearing Spiro-β-lactone-γ-lactams Obtained from a Marine-Derived Streptomyces sp. Keebeom Ko,† So-Hyoung Lee,‡ Seong-Hwan Kim,† Eun-Hee Kim,§ Ki-Bong Oh,‡ Jongheon Shin,† and Dong-Chan Oh*,† †

Natural Products Research Institute, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea ‡ Department of Agricultural Biotechnology, College of Agriculture and Life Science, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-921, Republic of Korea § Division of Magnetic Resonance, Korea Basic Science Institute, Ochang, Chungbuk 363-883, Republic of Korea S Supporting Information *

ABSTRACT: Lajollamycins (1−4), each of which bears a spiro-βlactone-γ-lactam ring and a nitro-tetraene moiety, were obtained from a marine-derived Streptomyces strain isolated from the southern area of Jeju Island, Republic of Korea. The planar structures of the lajollamycins were elucidated on the basis of spectroscopic analyses by NMR, UV, IR, and MS. The absolute configuration of lajollamycin (1), the planar structure of which has been previously reported, was determined using J-based configuration analysis based on 1H−1H and 1H−13C coupling constants, as well as ROESY correlations, followed by the modified Mosher’s method. The absolute configurations of lajollamycins B−D (2−4) were established by comparing their CD spectra with that of 1. The lajollamycins exhibited moderate inhibitory activity toward Candida albicans isocitrate lyase.

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using a combination of 1D and 2D NMR and mass spectroscopic analyses. We then established the absolute configuration of 1, which had been previously reported without its stereochemical information, and lajollamycins B−D (2−4). We also evaluated the biological activity of 1−4 as Candida albicans isocitrate lyase inhibitors.

ecent studies of marine microorganisms have revealed that the ocean is a large repository of microbes that produce diverse secondary metabolites with pharmaceutically valuable properties.1−3 Therefore, research on marine microbial secondary metabolites continues to increase. According to statistical data from 2011, the number of compounds isolated per year from marine microorganisms increased slowly between 1985 and 1996 but increased markedly during the 15-year period from 1997 to 2011. 4 Among novel secondary metabolites from marine microbes discovered during the 15year period, lajollamycin (1) is structurally unique by incorporating an unusual spiro fused β-lactone-γ-lactam.5 The spiro-β-lactone-γ-lactam moiety was originally discovered in the oxazolomycin class (only from Streptomyces spp. actinomycetes) with potent antiviral, antibacterial, and cytotoxic activity.6 However, lajollamycin (1) from a marine Streptomyces nodosus is the only compound further decorated with a nitro functional group at the end opposite from the spiro-β-lactoneγ-lactam ring.5 In our search for new bioactive compounds from marine microorganisms, we selectively isolated actinomycete strains from marine sediment samples from the southern area of Jeju Island in Republic of Korea and chemically screened their secondary metabolites by LC/MS. Through the screening process, we observed that strain SMC72, which belongs to the genus Streptomyces, produces the previously reported nitrotetraene spiro-β-lactone-γ-lactam lajollamycin (1)5 and three analogues, lajollamycins B−D (2−4),7 not previously reported. The planar structures of the lajollamycins were determined © 2014 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

Lajollamycin (1) was obtained as a yellow powder that was determined to have the molecular formula C36H53N3O10 on the basis of FAB-HRMS in combination with 1H and 13C NMR spectroscopic data (Table S1). The planar structure of 1 was confirmed to be the same as the previously reported lajollamycin5 on the basis of a comprehensive analysis of 1D and 2D NMR spectroscopic data, including COSY, HSQC, and HMBC NMR spectra. However, the absolute configuration of lajollamycin (1) was not previously determined. Therefore, we analyzed the ROESY NMR correlations around the spiro-βlactone-γ-lactam moiety and applied a J-based configuration analysis and the modified Mosher’s method to establish the absolute configuration of 1. Analysis of the ROESY NMR spectrum and molecular modeling with the calculation of minimum energy conformations of possible diastereomers determined the relative Received: June 19, 2014 Published: September 11, 2014 2099

dx.doi.org/10.1021/np500500t | J. Nat. Prod. 2014, 77, 2099−2104

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OH, establishing the orientation of 16-Me relative to the chain moiety. The ROESY correlation between H-16 and the N-Me protons established that H-16 faces away from the chain moiety. On the basis of the orientations of 16-Me and H-16 on the spiro-β-lactone ring, two possible diastereomers [(2R*, 3S*, 15S*, 16R*) and (2R*, 3S*, 15R*, 16S*)] could exist (Figure S31). These two diastereomers could be distinguished by the observed ROESY correlations between the protons of 16-Me and 3-OH. The minimum energy conformation of the 2R*, 3S*, 15S*, 16R* diastereomer supports this ROESY correlation by the calculated distance (2.09 Å) between 16-Me and 3-OH (Figure 1). However, the 2R*, 3S*, 15R*, 16S* diastereomer cannot explain this confinement with the calculated 16-Me/3OH distance (4.49 Å). Consequently, the relative configurations of the spiro-β-lactone-γ-lactam bicyclic ring were determined as 2R*, 3S*, 15S*, 16R* (Figure 1). To determine the relative configuration of the stereogenic centers at C-4, C-6, and C-7, we utilized a J-based configuration analysis8 with vicinal 1H−1H and two- and three-bond 13C−1H coupling constants. The 1H−1H coupling constants were measured in the 1 H NMR spectrum, and long-range heteronuclear coupling constants were acquired via a hetero half-filtered TOCSY (HETLOC) NMR experiment.9 The 3 JH6/H7 value (7.0 Hz) established an anti relationship between H-6 and H-7. The large (5.1 Hz) coupling between H-6 and C7, which bears an oxygen atom, indicated that the hydroxy group at C-7 is at a gauche position relative to H-6. Additional analysis of the small values of 3JC14/H7 (2.6 Hz), 3JC5/H7 (3.2 Hz), and 3JC8/H6 (1.8 Hz), the ROESY correlations of H-6 to 7OH and H-8, and the ROESY correlations of H-7 to H2-5 supported the relative configuration of 6R* and 7R* (Figure 2a). Because the chemical shifts of the methylene protons (H5a and H-5b) at C-5 were clearly separated (δH 1.98; 1.37), we consecutively applied J-based configuration analysis to relate the relative configuration of C-6 to C-8. The large vicinal coupling constant (7.5 Hz) between H-5a (δH 1.98) and H-6 assigned their anti relationship. Another anti relationship between H-5a and H-5b (δH 1.37) was deduced on the basis of the large 3JC15/H5 value (5.0 Hz). The ROESY couplings of H-5b to H-6 and H-7 and of H-5a to H-7 and H3-14 fully supported the configuration shown in Figure 2b. The coupling constants of 3JC4/H6 (2.7 Hz), 3JH5b/H6 (3.5 Hz), and 3JC7/H5 (3.1 Hz) were also consistent with the rotamer shown in Figure 2b. Although the combination of the observed coupling constants around C-4 and C-5 was most similar to that of the rotamer in Figure 2c, some of the J values were at the boundary of intermediate values.8 Therefore, we thoroughly analyzed the ROESY correlations. The strong ROESY correlation between 4-O-Me and H3-14 supported the rotamer in Figure 2c. Because C-3 lacks hydrogens, we analyzed the ROESY correlations between H-2 and H3-13. H-2 and H3-13 displayed strong ROESY correlations with H-5a, which also supported the rotamer in Figure 2c. Intermediate coupling constant values around C-4 and C-5 may have been due to a slight offset from the normal rotamer, resulting from the bulkiness of the β-lactone-γ-lactam ring connected to C-3. Therefore, the relative configuration of C-4 was deduced to be R*, which simultaneously proposed 2R*, 3S*, 4S*, 6R*, 7R*, 15S*, and 16R* relative configurations. The absolute configurations of the stereogenic centers at C-7 and C-3′, each bearing a secondary alcohol, were determined using the modified Mosher’s method.10 We derivatized the hydroxy groups at C-7 and C-3′ using (R)- and (S)-α-methoxy-

configuration of the spiro-β-lactone-γ-lactam ring system of lajollamycin (1) (Figure 1 and Figure S31). First, the ROESY correlation between H3-13 and 3-OH indicated that these groups are located on the same side. This assignment was supported by the ROESY correlation of H-2 to H-4. The protons of 16-Me correlated with the protons of 4-O-Me and 3-

Figure 1. Minimum energy conformation for the spiro-β-lactone-γlactam bicyclic ring of lajollamycin (1) with the 2R*, 3S*, 15S*, 16R* configuration. The key ROESY correlations and the distances between the protons are noted. 2100



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Figure 2. J-based configuration analysis of lajollamycin (1) at (a) C-6 and C-7, (b) C-5 and C-6, and (c) C-4 and C-5.

α-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl) to yield bis-S- and R-MTPA esters (5 and 6), respectively. The 1H chemical shifts of 5 and 6 were precisely assigned on the basis of their 1H and COSY NMR spectra. The calculation of the ΔδS−R values of 5 and 6 eventually established the absolute configuration of 7R and 3′S (Figure 3). On the basis of the

H and 10′-Me determined the 10′E configuration. The 4′E geometry of 2 differed from that of the 4′Z in 1. Lajollamycin C (3) was purified as a yellow powder. Its chemical formula was determined to be C36H53N3O10 on the basis of the analysis of FAB-HRMS and 1H and 13C NMR spectroscopic data (Table 1). The IR, UV, and mass spectra of 3 were analogous to those of 1. Further investigation of the 1H, 13 C, HSQC, HMBC, and ROESY NMR spectra revealed that, as expected, the gross structure of lajollamycin C (3) was identical to that of lajollamycin (1). Therefore, we carefully analyzed the geometries of the olefins with 1H−1H coupling constants and the ROESY NMR spectrum by comparing the data with those of 1. Our careful comparison of 3 with 1 revealed that the only difference between 1 and 3 is the doublebond geometry at C-7′. The coupling constant between H-6′ and H-7′ was 15.0 Hz, which is typical of a trans-geometry, revealing that lajollamycin C is a new geometric isomer of 1. The ROESY correlation between H-6′ and 4′-Me also supported the E configuration (Figure 4b). Along with the other lajollamycins (1−3), lajollamycin D (4) was isolated as a yellow powder. The molecular formula (C36H53N3O10) of 4 was determined to be identical to those of lajollamycins A and C on the basis of HR-FAB mass spectrometry (obsd [M + H]+ at 688.3815, calcd [M + H]+ 688.3807) as well as 1H and 13C NMR data (Table 1). Interpretation of the 1H, 13C, COSY, HSQC, and HMBC NMR spectra suggested that lajollamycin D (4) has a structure very similar to those of 1 and 3. Further careful analysis of the 1 H−1H coupling constants and ROESY spectroscopic data of 3 and 4 revealed that lajollamycin D is a new member of the lajollamycin family that differs geometrically because the ROESY correlation between 4′-Me and H-5 clearly supports the 4′Z configuration (Figure 4c). Lajollamycins B−D (2−4) exhibited NMR chemical shifts, coupling constants, physicochemical properties, and circular dichroism (CD) spectra (Figure 5) almost identical to those of lajollamycin (1), the absolute configuration of which was determined vide supra. Therefore, on the basis of the similarity of these data and a shared biogenesis, we propose that the absolute configurations of the stereogenic centers of 2, 3, and 4 are identical to those determined in 1. To examine the biological activities of the lajollamycins, the antimicrobial activities were first evaluated against various phylogenetically pathogenic bacterial strains such as Staphylococcus aureus ATCC 6538p, Bacillus subtilis ATCC 6633, Kocuria rhizophila NBRC 12708, Salmonella enterica ATCC 14028, Proteus hauseri NBRC 3851, and Escherichia coli ATCC

Figure 3. ΔδS−R values in ppm of 5 and 6 in chloroform-d.

relative configurations, the absolute configurations of C-4 and C-6 in the chain were simultaneously determined to be 4S and 6R. In addition, the establishment of the 4S configuration enabled us to assign the absolute configuration of the spiro-βlactone-γ-lactam bicyclic ring as 2R, 3S, 4S, 15S, and 16R. Lajollamycin B (2) was isolated as a yellow powder, the molecular formula of which was deduced to be C35H51N3O10 on the basis of FAB-HRMS as well as 1H and 13C NMR data (Table 1). The 1H and 13C NMR spectra of 2 in chloroform-d displayed a polyunsaturated and trihydroxylated feature that was very similar to that of lajollamycin (1). Through careful analysis of the 1H, 13C, and HSQC NMR spectra, we assigned all of the carbon-bound protons to the corresponding carbons and revealed that this compound lacks one doublet methyl group and one methine and bears an additional methylene (δC 65.9; δH 4.69, 4.38) compared to 1. The HMBC correlations between the methylene protons and C-3 (δC 79.9), C-15 (δC 86.3), and C-17 (δC 170.5) allowed for the assignment of the methylene in the β-lactone ring. Further analysis of COSY and HMBC NMR spectra successfully established the gross structure of lajollamycin B (2), which is a new member of the lajollamycin series. The double-bond geometries were determined on the basis of 1H−1H coupling constants and ROESY correlations (Figure 4a). First, trans coupling constants (15.0−15.5 Hz) were observed from H-8 through H-11, establishing 8E and 10E geometries of the diene. The 6′E and 8′E configurations were assigned on the basis of the large coupling constants at H-6′ to H-9′. With these geometries, further analyses of the ROESY correlations between 4′-Me and H-6′ led to the assignment of a 4′E geometry. Lastly, the ROESY correlations between 9′-H and 11′-Me and between 8′2101



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Table 1. NMR Data for Lajollamycins B−D (2−4) in Chloroform-da lajollamycin B (2) position 1 2 3 3-OH 4 5

175.0, C 42.6, CH 79.9, C

6 7 7-OH 8 9 10 11 12 NH 13 14 15 16

37.4, CH 77.1, CH

16-Me 17 1′ 2′ 2′-Mea 2′-Meb 3′ 3′-OH 4′ 4′-Me 5′ 6′ 7′ 8′ 9′ 10′ 10′-Me 11′ 11′-Me N-Me 4-O-Me a1

δC, type

82.7, CH 33.1, CH2

134.3, 131.5, 131.8, 130.1, 41.4,

CH CH CH CH CH2

10.2, 17.9, 86.3, 65.9,

CH3 CH3 C CH2

170.5, 178.0, 45.2, 26.0, 21.9, 84.2,

C C C CH3 CH3 CH

140.7, 14.0, 128.9, 132.3, 127.9, 135.8, 128.6, 133.3, 15.5, 145.0, 17.3, 26.5, 57.4,

C CH3 CH CH CH CH CH C CH3 C CH3 CH3 CH3

δH, mult (J in Hz) 2.44, q (7.5) 3.61, 3.58, 2.01, 1.38, 1.85, 3.95, 3.47, 5.67, 6.21, 6.18, 5.67, 3.89, 6.34, 1.21, 0.96,

s dd (4.5, 4.5) m m m dd (15.0, 7.0) s dd (15.0, 7.0) dd (15.0, 10.5) dd (15.5, 10.5) dd (15.5, 11.5) dd (11.5, 5.5) m d (7.5) d (7.0)

4.69, d (6.5) 4.38, d (6.5)

1.30, 1.10, 3.99, 4.28,

s s d (5.5) s

1.77, 6.05, 6.59, 6.73, 6.62, 6.42,

s d (11.0) dd (15.0, 11.0) dd (15.0, 11.0) dd (15.0, 11.0) d (15.0)

1.97, s 2.29, s 2.92, s 3.39, s

lajollamycin C (3) δC, type 175.4, C 43.4, CH 81.8, C

3.24, 3.69, 1.97, 1.38, 1.86, 3.97, 3.54, 5.68, 6.18, 6.20, 5.69, 3.92, 6.37, 1.20, 0.97,

37.3, CH 77.3, CH CH CH CH CH CH2

10.4, 18.1, 84.6, 77.7,

CH3 CH3 C CH

17.1, 170.1, 178.3, 44.9, 26.1, 21.8, 76.2,

CH3 C C C CH3 CH3 CH

141.0, 19.9, 130.1, 132.3, 132.5, 137.2, 128.5, 133.1, 15.4, 145.8, 15.9, 26.9, 57.6,


δC, type 175.6, C 43.3, CH 81.9, C

2.44, q (7.5)

82.3, CH 33.4, CH2

134.4, 131.7, 131.5, 129.7, 41.5,

lajollamycin D (4)

δH, mult (J in Hz)


4.87, q (6.5) 1.80, d (6.5)

1.36, 1.11, 4.63, 4.54,

s s d (4.5) s

1.78, 6.08, 6.70, 6.30, 6.72, 6.41,

s d (11.5) dd (15.0. 11.5) dd (15.0, 11.0) dd (15.5, 11.0) d (15.5)

2.04, s 2.29, s 2.91, s 3.41, s

82.3, CH 33.4, CH2 37.3, CH 77.2, CH 131.4, 129.8, 132.0, 134.4, 41.4,

CH CH CH CH CH2

10.4, 18.0, 84.6, 77.7,

CH3 CH3 C CH

17.0, 171.2, 178.6, 44.8, 25.9, 21.8, 76.3,

CH3 C C C CH3 CH3 CH

141.3, 19.8, 130.2, 131.9, 132.5, 137.2, 128.8, 133.1, 15.2, 145.9, 17.2, 26.7, 57.4,


δH, mult (J in Hz) 2.46, q (7.5) 3.49, 3.68, 1.97, 1.37, 1.88, 3.96, 3.50, 6.22, 5.69, 6.19, 5.69, 3.91, 6.57, 1.21, 0.98,


4.86, q (6.5) 1.79, d (6.5)

1.30, 1.10, 4.64, 4.63,

s s d (4.0) s

1.80, 6.10, 6.70, 6.30, 6.71, 6.41,

s d (11.5) dd (14.0, 11.5) dd (14.0, 10.0) dd (15.0, 10.0) d (15.0)

2.05, s 2.30, s 2.92, s 3.41, s

H and 13C data were recorded at 600 and 125 MHz, respectively.

35270 because a previous report5 indicated that lajollamycin inhibited drug-sensitive and -resistant bacteria, including S. aureus. However, the lajollamycins did not exhibit significant inhibitory activity against the tested microbial strains (MIC > 128 μg/mL). In addition, the lajollamycins did not exhibit cytotoxicity against the A549, HCT116, SNU638, K562, SKHEP1, or MDA-MB231 cell lines (IC50 > 100 μM). The lajollamycins also did not exhibit antifungal activity against Candida albicans or Aspergillus f umigatus. Interestingly, the lajollamycins displayed moderate activity against Candida albicans isocitrate lyase (ICL), which is closely related to fungal pathogenicity because of its diverse metabolic pathway.11 The lajollamycins (1−4) exhibited IC50 values of 42, 40, 50, and 120 μM, respectively.

Spiro-β-lactone-γ-lactam compounds are generally thought to be members of the oxazolomycin class,6 which was originally isolated from a terrestrial Streptomyces sp. actinomycetes. However, the lajollamycins represent the only class of natural products bearing both a nitro group and a spiro-β-lactone-γlactam. Our chemical investigation revealed new members of the lajollamycin class and determined the absolute configurations of these rare bacterial secondary metabolites.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Jasco P-1020 polarimeter using a 1 cm cell. UV spectra were acquired using a PerkinElmer Lambda 35 UV/vis spectrophotometer. CD spectra were recorded using an Applied Photophysics Chirascan-Plus circular dichroism detector at the

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medium (750 mL of seawater, 250 mL of distilled H2O, 18 g of agar, and 100 mg/L of cycloheximide), A6 medium (1 L of seawater, 18 g of agar, and 5 mg/L of polymyxin B sulfate), A7 medium (1 L of seawater, 18 g of agar, and 5 mg/L of kanamycin), and chitin-based agar. SMC72 was isolated on actinomycete isolation agar medium. Colonies were repeatedly inoculated onto fresh agar plates to obtain single strains. The 16S rDNA sequencing analysis data (GenBank accession number: KM117231) obtained from COSMO Co., Ltd. revealed that SMC72 is most closely related to Streptomyces qinglanensis (99% identity), identifying the strain as a Streptomyces sp. Cultivation and Extraction. The SMC72 strain was cultivated in 50 mL of YEME medium (4 g of yeast extract, 10 g of malt extract, and 4 g of glucose in 1 L of artificial seawater) in a 125 mL Erlenmeyer flask. After the strain was cultivated for 5 days on a rotary shaker at 190 rpm at 30 °C, 10 mL of the culture was inoculated in 1 L of YEME medium in 2.8 L Fernbach flasks (72 × 1 L for a total volume of 72 L). The large culture was incubated at 190 rpm at 30 °C. After 5 days, the whole culture was extracted twice using EtOAc. The EtOAc layer was separated and dried over anhydrous sodium sulfate. The EtOAc extract was enriched in vacuo to yield 5 g of dried material. Because of the light sensitivity of the lajollamycins, this procedure was repeated 12 times (6 L per each batch) under protection from light. The production of the lajollamycins was consistently observed regardless of light exposure (Figure S32). Isolation of the Lajollamycins (1−4). The extract was absorbed on Celite, loaded onto a 2 g Sep-Pak C18 cartridge, and fractionated with 20 mL each of 20%, 40%, 60%, 80%, and 100% MeOH in H2O and 1:1 MeOH/CH2Cl2. Lajollamycins (1−4) were detected in the 80% MeOH/H2O fraction. For the purification of 1−4, the 80% fraction was chromatographed using reversed-phase HPLC on a Kromasil column (5 μm, C18, 250 × 10 mm) under isocratic conditions (68:32 MeOH/H2O, UV 360 nm detection, flow rate: 2 mL/min). Four peaks were further purified using reversed-phase HPLC through the same column under 55% CH3CN in H2O over a 50 min period. The entire procedure was repeated six times. Finally, lajollamycin (1) (10 mg), lajollamycin B (2) (7 mg), lajollamycin C (3) (4 mg), and lajollamycin D (4) (3 mg) were isolated as pure compounds at retention times of 33, 27, 41, and 43 min, respectively. Lajollamycin B (2): yellow powder; [α]25 D +70 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 229 (3.82) nm, (log ε) 307 (3.72) nm, and (log ε) 378 (3.42) nm; CD (1.4 μM, MeOH) (Δε) 216 (1.26) nm; IR (neat) νmax 3369, 1826, 1736, 1646, and 1592 cm−1; 1H and 13C NMR data, see Table 1; HRFABMS m/z 674.3637 [M + H]+ (calcd for C35H52N3O10, 674.3647). Lajollamycin C (3): yellow powder; [α]25 D +110 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 229 (3.50) nm, (log ε) 308 (3.32) nm, and (log ε) 377 (3.04) nm; CD (1.4 μM, MeOH) (Δε) 215 (1.01) nm; IR (neat) νmax 3390, 1818, 1687, 1638, and 1518 cm−1; 1H and 13C NMR data, see Table 1; HRFABMS m/z 688.3804 [M + H]+ (calcd for C36H55N3O10, 688.3809). Lajollamycin D (4): yellow powder; [α]25 D +62 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 (4.27) nm, (log ε) 306 (4.15) nm, and (log ε) 378 (3.92) nm; CD (1.4 μM, MeOH) (Δε) 225 (0.57) nm; IR (neat) νmax 3344, 1819, 1689, 1646, and 1508 cm−1; 1H and 13C NMR data, see Table 1; HRFABMS m/z 688.3815 [M + H]+ (calcd for C36H55N3O10, 688.3809). MTPA Esterification of Lajollamycin (1). Lajollamycin (1) was prepared in two 40 mL vials (two 1 mg samples), which were dried completely under high vacuum for 6 h. First, freshly distilled anhydrous pyridine (1 mL) was added under argon gas. The reaction mixtures were stirred at room temperature for 10 min. After 10 min, (R)- and (S)-α-methoxy(trifluoromethyl)phenylacetic acid (MTPA) chloride (30 μL) were separately added. The reactions were quenched by adding 50 μL of MeOH over a 15 min period. The products were then purified using a reversed-phase C18 column (Kromasil, 5 μm, C18(2), 250 × 10.0 mm) under gradient conditions ranging from 40% to 100% aqueous acetonitrile. The S-MTPA ester (5) and R-MTPA ester (6) eluted at 43.2 and 42.8 min, respectively. The molecular formulas of the two derivatives were consistent with C56H67F6N3O14 via ESIMS analysis ([M + Na]+ m/z at 1142). The ΔδS−R values

Figure 4. Key ROESY correlations used to establish the double-bond geometries of (a) 2, (b) 3, and (c) 4.

Figure 5. CD spectra of the lajollamycins (1−4). NICEM (National Instrumentation Center for Environmental Management at the College of Agriculture & Life Sciences, Seoul National University). IR spectra were obtained on a Thermo N1COLET iS10 spectrometer. 1H, 13C, and 2D NMR spectra (referenced to the residual solvent signals of CDCl3; δH = 7.26, δC = 77.0) were acquired on a Bruker Avance 600-MHz spectrometer at the NCIRF (National Center for Inter-University Research Facilities) and on a Bruker Avance II 900-MHz NMR spectrometer at the KBSI (Korea Basic Science Institute at Ochang). Electrospray ionization (ESI) low-resolution LC/MS data were acquired on an Agilent Technologies 6130 quadrupole mass spectrometer coupled with an Agilent Technologies 1200-series HPLC using a reversed-phase C18 column (Phenomenex Luna, 100 × 4.6 mm). High-resolution fastatom bombardment (HR-FAB) mass spectra were obtained using a Jeol JMS-600 W high-resolution mass spectrometer at the NCIRF. Bacterial Isolation. A marine seashore sediment sample was collected from the southern area of Jeju Island, Republic of Korea. The sample (1 g) was diluted in 12 mL of sterilized artificial seawater (Aquarium Systems Inc.) (for a 1:3 dilution) and vortexed. The mixture was spread onto actinomycete isolation agar, A4 medium (1 L of seawater, 18 g of agar, and 100 mg/L of cycloheximide), A5 2103



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around the stereogenic centers of the derivatives were assigned by 1H NMR and COSY NMR experiments. Bis S-MTPA ester (5) of lajollamycin (1): 1H NMR (600 MHz, chloroform-d) δ 7.49−7.44 (m, 4H), 7.41−7.36 (m, 6H), 6.72 (dd, J = 15.0, 10.5, 1H), 6.59 (dd, J = 14.5, 11.5, 1H), 6.45 (d, J = 15.0, 1H), 6.44 (d, J = 11.5, 1H), 6.36 (dd, J = 14.5, 10.5, 1H), 6.20 (dd, J = 15.0, 10.5, 1H), 6.06 (dd, J = 15.0, 11.0, 1H), 5.91 (br, 1H), 5.58 (m, 1H), 5.55 (m, 1H), 5.51 (s, 1H), 5.46 (m, 1H), 4.86 (q, 13.0, 6.5, 1H), 3.87 (m, 1H), 3.79 (m, 1H), 3.53 (t, J = 5.0, 1H), 3.50 (s, 3H), 3.49 (m, 1H), 3.47 (s, 3H), 3.34 (s, 3H), 2.92 (s, 3H), 2.31 (s, 3H), 2.30 (m, 1H), 2.04 (s, 3H), 1.98 (m, 1H), 1.84 (d, J = 6.5, 3H), 1.81 (s, 3H), 1.25 (m, 2H), 1.18 (s, 3H), 1.14 (d, J = 7.5, 3H), 1.11 (s, 3H), and 1.00 (d, J = 6.0, 3H); ESIMS m/z 1142 [M + Na]+. Bis R-MTPA ester (6) of lajollamycin (1): 1H NMR (600 MHz, chloroform-d) δ 7.51−7.48 (m, 2H), 7.46−7.42 (m, 2H), 7.41−7.36 (m, 6H), 6.71 (dd, J = 14.5, 11.0, 1H), 6.58 (dd, J = 14.5, 11.5, 1H), 6.44 (d, J = 14.5, 1H), 6.30 (dd, J = 14.5, 11.0, 1H), 6.23 (m, 1H), 6.08 (m, 1H), 6.04 (d, J = 11.5, 1H), 6.01 (br, 1H), 5.59 (m, 1H), 5.58 (m, 1H), 5.53 (s, 1H), 5.51 (m, 1H), 4.86 (dd, J = 13.5, 6.5, 1H), 3.86 (m, 1H), 3,78 (m, 1H), 3.52 (s, 3H), 3.49 (t, J = 5.0, 1H), 3.47 (s, 3H), 3.46 (br, 1H), 3.32 (s, 3H), 2.91 (s, 3H), 2.32 (s, 3H), 2.26 (q, J = 7.5, 1H), 2.04 (s, 3H), 1.97 (m, 1H), 1.83 (d, J = 6.5, 3H), 1.80 (s, 3H), 1.21 (s, 3H), 1.19 (s, 3H), 1.18 (m, 2H), 1.08 (d, J = 7.5, 3H), and 0.91 (d, J = 7.0, 3H); ESIMS m/z 1142 [M + Na]+. Calculation of Minimum Energy Conformations of the Spiro-β-lactone-γ-lactam Bicyclic Ring of Lajollamycin (1). The lowest energy conformations were calculated for the truncated structures possessing the spiro-β-lactone-γ-lactam bicyclic ring of lajollamycin (1) with 2R*, 3S*, 15S*, 16R* and 2R*, 3S*, 15R*, 16S* configurations by Turbomole 6.5. In the calculation, the DFT settings (functional B3-LYP/Gridsize M3) and geometry optimization options (energy 10−6 hartree, gradient norm |dE/dxyz| = 10−3 hartree/bohr) were used. ICL Activity Assay. The 1 mL reaction volume consisted of 20 mM sodium phosphate buffer (pH 7.0), 1.27 mM threo-DL(+)-isocitrate, 3.75 mM MgCl2, 4.1 mM phenylhydrazine, and 2.5 μg/mL recombinant ICL. The reaction was started right after the addition of substrate with or without a prescribed concentration of the inhibitor dissolved in DMSO (final concentration, 0.5%). Glyoxylate phenylhydrazone formation was spectrophotometrically assessed at 324 nm after incubation at 37 °C for 30 min.12 Protein concentrations were measured by using the method of Bradford with the Bio-Rad protein assay kit (Bio-Rad) and bovine serum albumin as the standard. An inhibitor-free control was also prepared, and the percent inhibition of ICL enzyme activity caused by each compound was calculated relative to the inhibitor-free control. 3-Nitropropionic acid was used as a positive control, inhibiting ICL with an IC50 value of 4.2 μg/mL (35 μM).

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0083533 and M1A5A-2010-0020429) and by the grant from the High Field NMR Research Program of the Korea Basic Science Institute.

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REFERENCES

(1) Fenical, W.; Jensen, P. R. Nat. Chem. Biol. 2006, 2, 666−673. (2) Xiong, Z.-Q.; Wang, J.-F.; Hao, Y.-Y.; Wang, Y. Mar. Drugs 2013, 11, 700−717. (3) Bhatnagar, I.; Kim, S.-K. Environ. Toxicol. Pharmacol. 2012, 34, 631−643. (4) Hu, G.-P.; Yuan, J.; Sun, L.; She, Z.-G.; Wu, J.-H.; Lan, X.-J.; Zhu, X.; Lin, Y.-C.; Chen, S.-P. Mar. Drugs 2011, 9, 514−525. (5) Manam, R. R.; Teisan, S.; White, D. J.; Nicholson, B.; Grodberg, J.; Neuteboom, S. T. C.; Lam, K. S.; Mosca, D. A.; Lloyd, G. K.; Potts, B. C. M. J. Nat. Prod. 2005, 68, 240−243. (6) (a) Mori, T.; Takahashi, K.; Kashiwabara, M.; Uemura, D.; Iwadare, S.; Shizuri, Y.; Mitomo, R.; Nakano, F.; Matsuzaki, A. Tetrahedron Lett. 1985, 26, 1073−1076. (b) Moloney, M. G.; Trippier, P. C.; Yaqoob, M.; Wang, Z. Curr. Drug Discovery Technol. 2004, 1, 181−199. (7) Compounds 2−4 are named lajollamycins B−D to represent the second through fourth members of this structural class, with the original lajollamycin considered as the “A” member of the family, even though A was not assigned in the original paper. (8) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.; Tachibana, K. J. Org. Chem. 1999, 64, 866−876. (9) Kurz, M.; Schmieder, P.; Kessler, H. Angew. Chem., Int. Ed. 1991, 103, 1341−1342. (10) Seco, J. M.; Quiñoá, E.; Riguera, R. Tetrahedron: Asymmetry 2001, 12, 2915−2925. (11) Lorenz, M. C.; Fink, G. R. Nature 2001, 412, 83−86. (12) Hautzel, R.; Anke, H.; Sheldrick, W. S. J. Antibiot. 1990, 43, 1240−1244.

ASSOCIATED CONTENT

S Supporting Information *

NMR spectra of 1−4, NMR assignment of 1, molecular modeling results of 1, and detailed J-based configuration analyses of 1 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(D.-C. Oh) Tel: 82 2 880 2491. Fax: 82 2 762 8322. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Research Foundation of Korea (NRF) grants funded by the Korean government (Ministry of Science, ICT and Future Planning) (20092104


Lajollamycins, Nitro Group-Bearing Spiro-Î²-lactone-Î³-lactams

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