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Cereusitin A, a cyclic tetrapeptide from a Bacillus cereus strain isolated from the soft coral Antillogorgia (syn. Pseudopterogorgia) elisabethae
Accepted Manuscript Cereusitin A, a cyclic tetrapeptide from a Bacillus cereus strain isolated from the soft coral Antillogorgia (syn. Pseudopterogorgia) elisabethae Angela Pinzón, Diana Martinez-Matamoros, Leonardo Castellanos, Carmenza Duque, Jaime Rodríguez, Carlos Jiménez, Freddy A. Ramos PII: DOI: Reference:
S0040-4039(17)30002-3 http://dx.doi.org/10.1016/j.tetlet.2017.01.002 TETL 48505
To appear in:
Tetrahedron Letters
Received Date: Revised Date: Accepted Date:
12 October 2016 28 December 2016 2 January 2017
Please cite this article as: Pinzón, A., Martinez-Matamoros, D., Castellanos, L., Duque, C., Rodríguez, J., Jiménez, C., Ramos, F.A., Cereusitin A, a cyclic tetrapeptide from a Bacillus cereus strain isolated from the soft coral Antillogorgia (syn. Pseudopterogorgia) elisabethae, Tetrahedron Letters (2017), doi: http://dx.doi.org/10.1016/ j.tetlet.2017.01.002
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Cereusitin A, a cyclic tetrapeptide from a Bacillus cereus strain isolated from the soft coral Antillogorgia (syn. Pseudopterogorgia) elisabethae a
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a,b
Angela Pinzón, Diana Martinez-Matamoros, a a Leonardo Castellanos, Carmenza Duque, b b a Jaime Rodríguez, Carlos Jiménez, and Freddy A. Ramos * a
Universidad Nacional de Colombia – Bogotá - Facultad de Ciencias - Departamento de Química - Laboratorio de Productos Naturales Marinos, Carrera 30 Nº 45-03, Bogotá, 111321 - Colombia b Departamento de Química Fundamental, Facultade de Ciencias e Centro de Investigacións Científicas Avanzadas (CICA), Universidade da Coruña, A Coruña E-15071, Spain
1
Tetrahedron Letters
Cereusitin A, a cyclic tetrapeptide from a Bacillus cereus strain isolated from the soft coral Antillogorgia (syn. Pseudopterogorgia) elisabethae Angela Pinzón,a Diana Martinez-Matamoros,a,b Leonardo Castellanos,*a Carmenza Duque,a Jaime Rodríguez,b Carlos Jiménez,b and Freddy A. Ramosa* Universidad Nacional de Colombia – Bogotá - Facultad de Ciencias - Departamento de Química - Laboratorio de Productos Naturales Marinos, Carrera 30 Nº 45-03, Bogotá, 111321 – Colombia. b Departamento de Química Fundamental, Facultade de Ciencias e Centro de Investigacións Científicas Avanzadas (CICA), Universidade da Coruña, A Coruña E-15071, Spain. a
A RT I C L E I N F O
A BS T RA C T
Article history: Received Received in revised form Accepted Available online
Two new compounds, cereusitin A (1), the tetrapeptide cyclo-(L-phenylalanyl-trans-4-hydroxyL-prolyl-L-leucyl-trans-4-hydroxy-L-proline and 4-(R)-hydroxysattabacin (2), along with the methyl, isopropyl, and propyl esters of p-hydroxybenzoic acid (3–5) were isolated from the organic extracts of the culture of the B. cereus RKHC-09 strain in two culture media (MOLP and B1), recovered from the sea fan Antillogorgia elisabethae (syn. Pseudopterogorgia elisabethae). Cereusitin A (1) showed mild antifungal activity against Colletotrichum gloeosporoides C26 (yam pathogen) but was inactive against Fusarium oxysporum f.sp. dianthi (carnation pathogen). The methyl and propyl esters of p-hydroxybenzoic acid (4 and 5) showed antimicrobial activity against S. aureus ATCC 33591 and S. cerevisiae, with an MIC of 2 M.
Keywords: Bacillus cereus Cyclopeptide 4-hydroxyproline Marfey’s method Marine Natural Products
In the last five decades, marine natural products have represented one of the most important sources of novel and biologically active molecules, yielding at least seven compounds on the market as well as a high number of chemical entities in phase II and III clinical trials.1,2 Although the most common sources are sponges, cnidarians and macroalgae, there has been a significant increase in the number of molecules isolated from marine microorganisms in recent years. 3–5 In the present work, our starting point was the soft coral Antillogorgia elisabethae (syn. Pseudopterogorgia elisabethae), which has been recognized as a rich source of bioactive compounds.6,7 The organic extracts from a collection of bacterial symbionts from A. elisabethae collected in Providencia Island (Colombia)8 were evaluated for antimicrobial activity (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 33591, and Saccharomices cereviceae). As a result of this evaluation, the strain identified as Bacillus cereus RKHC-09 was selected for chemical studies on the basis of its activity. Bacteria belonging to the Bacillus genus are present in numerous environments and they are considered as microbial factories for the production of a large number of biologically active molecules.9 In the case of Bacillus subtilis, it has been postulated that around 4% of its genome is devoted to the production of antimicrobial compounds.10 Cyclic lipopeptides surfactines, iturines, and fengicines have been isolated from several strains of Bacillus usually associated with plant rhizomes and some of these compounds have shown activity in the control
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pathogenic infections in the host plants.10 Furthermore, the emetic toxins valinomycin and cereulide, isolated from Bacillus cereus strains, are remarkable compounds due to their pHdependence antimicrobial activity against Gram-positive bacteria associated to their ecological role.11,12 In addition to peptiderelated compounds that comprise diketopiperazines and ribosomal and non-ribosomal peptides, compounds such as terpenes, aminosugar, polyketides, and phospholipids have also been isolated from strain cultures belonging to this genus13. In this paper, and as part of our continued search for bioactive compounds from marine sources,14 we describe the isolation and structural elucidation of two new metabolites from the organic extracts obtained from the strain B. cereus RKHC-09 in two different culture media (MOLP and B1): the new cyclopeptide cereusitin A (1), from MOLP culture medium, and 4-(R)hydroxysattabacin (2) along with three known aromatic compounds (3-5), from the B1 medium. They were tested in antimicrobial assays against human pathogenic strains. The organic extract of a bacterial strain identified as B. cereus RKHC-09, which is part of the 13 Firmicutes strains recovered from the soft coral A. elisabethae (syn. Pseudopterogorgia elisabethae) collected from the Colombian Caribbean Sea,8 was selected on the basis of its antimicrobial activity (against E. coli and S. cereviciae) and on the results of a UPLC-MS dereplication study on its organic extracts (data not shown).
2
Tetrahedron
As an approach to OSMAC (one strain many compounds) strategies15, this strain was grown in eight different culture media, chosen by their composition (rich and poor media, chemically defined and complex media whit different sources of carbon and nitrogen - Supplementary data). Comparison of the HPLC profiles and the antibacterial activity displayed by the corresponding organic extracts allowed us to identify MOLP and B1 as the culture media that showed activity against the above mentioned pathogenic strains and enhanced production of compounds, which were not found in sterile culture medium extract. Thus, compound 1 was isolated from the organic extracts obtained from culture of B. cereus RKHC-09 in MOLP medium, while compounds 2 - 5 were isolated when the B1 medium was used.
by the following key HMBC correlations: From the -proton of γHyp-2 at δH3.69 and 3.49 correlated to the Leu carbonyl group at δC 166.2 and from the NH amide proton of the Leu unit at δH 6.14 to the carbonyl carbon of the Hyp-1 (δC 170.5) (Figure 1).
Figure 1. Key NMR COSY and HMBC correlations in 1.
The molecular formula of compound 1, which was obtained as a pale yellow gum, was established as C25H34N4O6 (11 degrees of unsaturation) on the basis on its NMR data and the [M + Na]+ ion peak observed at m/z 509.2375 (calculated for C25H34N4O6Na m/z 509.2370, ∆ 0.5 ppm) in its (+)-ESI-HRMS. The 1H and 13C NMR spectral data, along with the results of an extensive 2D NMR analysis (1H-1H COSY, HSQC and HMBC), suggested a cyclic tetrapeptide structure for compound 1.16 The 1H NMR spectrum (500 MHz, CDCl3, Table 1) showed signals corresponding to one monosubstituted aromatic ring at H 7.28– 7.16 (5H), which is characteristic of a phenylalanine (Phe) unit, two NH amide protons at H 6.14 and 5.79 (1H, brs each), and two methine protons at H 4.49, which are characteristic of two γhydroxyproline (γHyp) units, four α peptide protons at H 4,50, 4.42, 4.38, and 3.99, and two aliphatic methyls at H 0.93 (3H, d, J = 6.5 Hz) and H 0.89 (3H, d, J = 6.5 Hz), which are indicative of a leucine (Leu) unit. The 13C NMR spectrum (125 MHz, CDCl3, Table 1) and the DEPT experiment showed the presence of four carbonyl signals at C 170.5, 169.6, 166.2, and 165.1, aromatic carbon signals in the range C 135.6–127.5, four α peptide carbons at C 57.3, 57.2, 56.0, and 53.3, and two aliphatic methyl carbons at C 23.1 and 21.1. The HSQC experiment allowed us to assign each proton to its corresponding carbon. The analysis of both, COSY and HMBC spectra showed that compound 1 is a tetrapeptide, composed by one Phe, one Leu, and two γHyp residues. The carbon signals at C 68.3 and 68.1 correspond to carbons on the γHyp residues. The amino acid sequence was determined from the observed key HMBC correlations showed in Figure 1 as follows: long range correlations between the proton signal at δH 5.79, assigned as the NH Phe amide proton and the carbonyl carbons of Phe at δC 165.1 and γHyp-2 at δC 169.6 and the α carbon of γHyp-2 at δC 57.2 (Figure 1). On the other hand, the -protons of γHyp-1 at δH 3.63 and 3.48 correlated to the Phe carbonyl group at δC 165.1. All of these long range correlations indicate a cyclic structure for compound 1, were the Phe unit was flanked by the two γHyp units. The Leu unit was also placed between the two γHyp units
The data outlined above allowed us to propose that the amino acid sequence of compound 1 is cyclo-[Phe-Hyp-Leu-Hyp]. The complete amino acid sequence of 1 was confirmed by (+)ESI-HRMS (see Figure 2), which showed an ion at m/z 283.1053 (calculated for C14H16N2O3Na m/z 283.1053, ∆ 0.0 ppm) corresponding to [M – Leu-Hyp + Na]+ and m/z 249.1220 (calculated for C11H18N2O3Na m/z 249.1209, ∆ 1.1 ppm) corresponding to the fragment [M – Phe-Hyp + Na]+.
Figure 2. Key fragments found in the (+)-ESI-HRMS of 1. The absolute configurations of the -amino acids present in 1 were determined by Marfey’s method. 17 Analysis of FDAAderivatized amino acids in hydrolysed peptide 1, compared to amino acid standards r with D- and L-configurations, showed the L-configuration for all of the residues (Suplementary Data). Based on these data, the structure of compound 1, named as cereusitin A, was established as cyclo-(L-Phe-trans-L-Hyp-LLeu-trans-L-Hyp). The structure of cereusitin A 1 is closely related to that of the peptide cyclo-(Phe-Pro-Leu-Pro), isolated from a Pseudoalteromonas sp. obtained from the marine sponge Halisarca ectofibrosa18 and cyclo-(L-leucyl-D-cis-4-hydroxy-D-
3 prolyl-L-leucyl-trans-4-hydroxy-L-proline) isolated from a from the exo-cellular extract of Pseudomonas sp., a bacterium associated with the sponge Ircinia muscarum.19,20 In general terms, cyclic tetrapeptides are well known fungal and bacterial metabolites from both, terrestrial and marine environments.21,22 Table 1: 1D NMR data of cereusitin (1) in CDCl3 (1H-NMR at 500 MHz; 13C-NMR at 125 MHz) Amino acid Hyp-1
Phe
Hyp-2
Leu
δC, mult.
δH, mult. (J in Hz)
57.3, CH 37.3, CH2
68.3, CH 54.3, CH2
4.42, dd (6.3, 11.1) 2.31, dd (6.3, 13.3) 2.06, m 4.49, m 3.63, dd (4.3, 13.0) 3.48, m
CO
170.5, C
NH
56.0, CH 36.5, CH2
5.79, br s 4.50, dd (3.9, 10.1) 3.51, m 2.73, 1H (10.3, 14.5)
1 2/6 3/5 4 CO
135.6, C 129.0 × 2, CH 129.1 × 2, CH 127.5, CH 165.1, C
7.16, d (7.1) 7.28, t, (7.3) 7.21, t, (7.3)
position
57.2, CH 37.6, CH2
68.1, CH 54.3, CH2
CO
169.6, C
NH
53.3, CH 38.4, CH2
(CH3) (CH3) CO
24.6, CH 23.1, CH3 21.1, CH3 166.2, C
4.38, dd, (6.3, 11.2) 2.26, dd, (6.8, 13.2) 2.06, m 4.49, m 3.69, dd, (4.5, 13.1) 3.49, m
drugs, and cosmetics.26 Although there are examples of the production of parabens by other microorganisms,27 the results described here represent the first evidence of the production of this class of compound from strains of the Bacillus genera. Evaluation of the antimicrobial activity of the isolated compounds (1, 3–5) showed that only compounds 3 and 5 were active against Staphylococcus aureus ATCC 33591 and Saccharomices cereviceae with a MIC value of ~ 2 M (values of 0.3 and 0.4 mg/mL for compounds 3 and 5, respectively). These values are lower than the reported LD50 data when these parabens were tested against different mammalian species.28-31 Further evaluation of the antifungal activity for compound 1 in the agar diffusion test showed a weak inhibition of growth of Colletotrichum gloeosporoides C26 (yam pathogen) compared with clotrimazole, which was used as a positive control, but this compound was inactive against Fusarium oxysporum f.sp. dianthi (carnation pathogen). A more detailed evaluation of compound 1 as a phytopathogen control agent is currently underway. In summary, two new compounds, namely cereusitin A (1) and 4-(R)-hydroxysattabacin (2), along with four known compounds 3–5, were isolated from the strain Bacillus cereus RKHC-09 obtained from the soft coral Antillogorgia elisabethae (syn. Pseudopterogorgia elisabethae). Cereusitin A (1) showed a mild antifungal activity against Colletotrichum gloeosporoides C26 (yam pathogen) but was inactive against Fusarium oxysporum f.sp. dianthi (carnation pathogen). The same strain cultured in B1 medium allowed the isolation of the new compound 4-(R)-hydroxysattabacin (2), along with the known methyl, propyl, and isopropyl esters of p-hydroxybenzoic acid (3–5). An antibacterial test on the isolated compounds showed activity for compounds 3 and 5 against S. cerevisiae and S. aureus.
Acknowledgments 6.14, br s 3.99, dd (3.7, 9.6) 1.94, m 1.45, m 1.70, m 0.93, d, (6.5) 0.89, d, (6.5)
Compound 2 was obtained from a culture of B. cereus RKHC09 in B1 medium and was isolated as a white amorphous solid. Comparison of 1D and 2D NMR and the (+)-ESI-LRMS data of 223 with literature values allowed compound 2 to be identified as 4-hydroxysattabacin, previously isolated from a Bacillus strain obtained from soil samples Bacillus sp. from Sardina (Italy) by Satta and co-workers and it exhibits antiviral activity, most notably against herpes simplex virus type 1 (HSV1) and 2 (HSV2).23 However, the optical rotation of compound 2 ([α]25D = –33.2, c 0.3, CHCl3) was opposite in sign to that previously reported for natural ([α]25D = +14, c 0.3, CHCl3)23 and synthetic ([α]25D = +18.3, c 0.21, CH3OH)22 4-(S)- hydroxysattabacin, indicating that compound 2 must be the R enantiomer of 4hydroxysattabacin. In addition, the NMR and MS data for the methyl ester (3), isopropyl ester (4), and propyl ester (5) of p-hydroxybenzoic acid,24,25 isolated from the extract of the culture in B1 medium, matched those reported respectively. These compounds, also known as parabens, are commonly used as preservatives in foods,
This work was partially financed by grants from Colciencias, Universidad Nacional de Colombia, and IFS (Grant F/5023-1) of Colombia, and from Ministry of Economy and Competitiveness (MINECO) (Grant AGL2015-63740-C2-2-R) of Spain. The Ministerio de Ambiente y Desarrollo Sostenible granted permission (permission No. 4 of 10/02/2010, contrato de acceso a recurso genético No 108 to collect samples and perform this research on A. elisabethae at the Archipelago of San Andrés and Providencia, Colombian Caribbean. The authors are grateful to Prof. Edelberto Silva, Departamento de Farmacia, Prof. Angélica Kundson, Facultad de Medicina and Prof. Nubia Moreno Instituto de Biotecnología, Universidad Nacional de Colombia, who kindly donated the phatogenic bacterial and fungal strains for antimicrobial assays. Supplementary Data 1
H, 13C, HSCQ, HMBC NMR spectra and (+)-ESI-LRMS and (+)-ESI-HRMS of cereusitin A 1, Along with its analysis by Marfey’s method. References and notes 1. 2. 3.
Gerwick, W. H.; Moore, B. S. Chem. Biol. 2012, 19, 85–98. Mayer, A. M.; Nguyen, M.; Newman, D. J.; Glasser, K. B.. Faseb J. 2015, 30, 932.7-932.7 Cragg, G. M.; Newman, D. J. Biochim. Biophys. Acta 2013, 1830, 3670–3695.
4
Tetrahedron 4. Penesyan, A.; Kjelleberg, S.; Egan, S. Mar. Drugs 2010, 8, 438– 459. 5. Imhoff, J. F.; Labes, A.; Wiese, J. Biotechnol. Adv. 2011, 29, 468– 482. 6. Duque, C.; Puyana, M.; Narváez, G.; Osorno, O.; Hara, N.; Fujimoto, Y. Tetrahedron 2004, 60, 10627–10635. (b) Duque, C.; Puyana, M.; Castellanos, L.; Arias, A.; Correa, H.; Osorno, O.; Asai, T.; Hara, N.; Fujimoto, Y. Tetrahedron 2006, 62, 4205– 4213. 7. Lei, H. Chem. Biodiversity 2016, 13, 345 – 365 8. Correa, H.; Haltli, B.; Duque, C.; Kerr, R. Microb. Ecol. 2013, 66, 972–985 9. Rahman, H.; Austin, B.; Mitchell, W. J.; Morris, P. C.; Jamieson, D. J.; Adams, D. R.; Spragg, A. M.; Schweizer, M. Mar. Drugs 2010, 8, 498–518. 10. Ongena, M.; Jacques, P. Trends Microbiol. 2008, 16, 115–125. 11. Kroten, M.A.; Bartoszewicz, M.; Swiecicka I. Pol. J. Microbiol. 2010, 59, 3–10. 12. M.H. Tempelaars, S. Rodrigues, T. Abee, Appl. Environ. Microbiol. 77 (2011) 2755–62. 13. Mondol. M.A.; Shin, H.J.; Islam, M.T. Mar. Drugs 2013, 11, 2846 - 2872. 14. (a) Quintana, J.; Bayona, L.M.; Castellanos, L.; Puyana, M.; Camargo, P.; Aristizabal, F.; Edwards, C.; Tabudravu, J.N.; Jaspars M.; Ramos, F.A. Bioorg & Med. Chem. 2014, 22, 67896795. (b) Martinez-Matamoros, D.; Laiton, F.M.; Duque, C.; Ramos, F.A.; Castellanos, L. Vitae 2016. 23, 30 – 47. (c) PardoVargas, A.; de Barcelos Oliveira, I.; Stephens, P. R. S.; CirneSantos, C. C.; de Palmer Paixão, I. C. N.; Ramos, F. A.; Jiménez, C.; Rodríguez, J.; Resende, J. A. L. C.; Teixeira, V. L.; Castellanos, L. Mar. Drugs 2014, 12, 4247–4259. 15. Bode, H. B.; Bethe, B.; Höfs, R.; Zeeck, A. ChemBioChem 2002, 3, 619–627. 16. Compound 1: Pale yellow gum. [α]25D –33.2 (c 0.3, CHCl3). 1HNMR (500 MHz, CDCl3) and 13C-NMR (125 MHz, CDCl3), see Table 1. 1H-NMR (300 MHz, CD3OD) γHyp-1 δH: 4.47 (1H, m, H), 4.38 (1H, t, J = 4.6 Hz, Hα), 2.06 (1H, dd, J = 5.9, 13.2 Hz, Hβ), 2.29 (1H, dt, J = 4.6, 12.4 Hz, Hβ), 3.71 (1H, dd, J = 5.0, 13.1 Hz, H), 3.49 (H, m, H); γHyp-2 δH: 4.46 (1H, m, H), 4.36 (1H, t, J = 4.6 Hz, Hα), 2.10 (2H, dd, J = 5.9, 13.2 Hz, Hβ), 2.28 (1H, dt, J = 4.6, 12.4 Hz, Hβ), 3.66 (1H, dd, J = 5.0, 13.1 Hz, H), 3.44 (1H, m, H); Phe δH: 4.50 (1H, m, Hα), 3.17 (2H, m), 7.22– 7.29 (5H, m), Leu δH:4.17 (1H, m, Hα), 1.93 (1H, m), 1.51 (1H, t, J = 8.0), 1.89 (1H, m), 0.97 (3H, d, J = 2.5 Hz), 0.96 (3H, d, J = 2.5 Hz). 13C-NMR (75 MHz, CD3OD) γHyp-1 δC:67.1 (C), 56.7 (Cα), 37.4 (Cβ), 53.8 (C), 170.1 (CO), γHyp-2 δC: 67.7 (C), 57.1 (Cα), 36.6 (Cβ), 53.7 (C), 171.9 (CO); Phe δC :56.1 (Cα), 36.5 (Cβ), 136.2 (C), 128.1 × 2, 129.6 × 2, 126.7 (Aromatic carbons), 165.6 (CO); Leu δC: 53.1 (Cα), 37.9 (Cβ), 24.3 (C), 20.7 (C), 21.9 (C), 167.6 (CO). (+)-ESI-LRMS m/z (rel. int.) 509 [M + Na]+ (7), 283 [M-Phe – Hyp + Na]+ (100), 249 [M – Leu-Hyp + Na]+ (25). (+)-ESI-HRMS m/z 509.2375 (calcd for C25H34N4O6Na, 509.2370), 283.1053 (calcd for C14H16N2O3Na m/z 283.1053), 249.1220 (calcd for C11H18N2O3Na, 249.1209). Melting point 134 ± 1 °C. 17. (a) Bhushan, R.; Brückner, H. Amino Acids 2004, 27, 231–247. (b) Harada, K.; Fujii, K.; Hayashi, K.; Suzuki, M.; Ikai, Y.; Oka, H. Tetrahedron Lett. 1996, 37, 3001–3004. 18. Rungprom, W.; Siwu, E. R. O.; Lambert, L. K.; Dechsakulwatana, C.; Barden, M. C.; Kokpol, U.; Blanchfield, J. T.; Kita, M.; Garson, M. J. Tetrahedron 2008, 64, 3147–3152. 19. Mitova, M.; Tommonaro, G.; De Rosa. S.; Z. Naturforsch. - Sect. C J. Biosci. 2003, 58, 740–745. 20. Zhou, H.; Yang, Y.; Yang, X.; Li, W.; Xiong, Z.; Zhao, L.; Xu, L.; Ding, Z. Nat. Prod. Res. 2014, 28, 318–23. 21. Gao, C.-H.; Chen, Y.-N.; Pan, L.-X.; Lei, F.; Long, B.; Hu, L.-Q.; Zhang, R.-C.; Ke, K.; Huang, R.-M. J. Antibiot. 2014, 67, 541– 543. 22. Pérez-Victoria, I.; Martín, J.; González-Menéndez, V.; de Pedro, N.; El Aouad, N.; Ortiz-López, F. J.; Tormo, J. R.; Platas, G.; Vicente, F.; Bills, G. F.; Genilloud, O.; Goetz, M. A; Reyes, F. J. Nat. Prod. 2012, 75, 1210–1214. 23. Compound 2: White amorphous solid, [α]25D –33.2 (c 0.3, CHCl3). 1H-NMR (300 MHz, CD3OD) δH 7.07 (2H, d, J = 8.6 Hz), 6.71 (2H, d, J = 8.6 Hz), 4.23 (1H, dd, J = 4.8, 7.8 Hz), 2.94 (1H, dd, J = 4.8, 14.0 Hz), 2.71 (1H, dd, J = 7.8, 14.0 Hz), 2.40 (1H, d, J = 1.6 Hz), 2.37 (1H, d, J = 1.6 Hz), 0.91 (3H, d, J = 1.1 Hz), 0.88 (3H, d, J = 1.1 Hz). 13C-NMR (300 MHz, CD3OD) δC 214.6,
24. 25. 26. 27. 28. 29. 30. 31.
156.1, 129.3 (× 2), 128.1, and 114.3 (× 2), 77.5, 47.2, 38.7, 23.6, 21.7 × 2. (+)-ESI- LRMS m/z 223 [M + H]+. Lampis, G.; Deidda, D.; Maullu, C.; Madeddu, M. A.; Pompei, R.; Monache, F.D.; Satta, G. J. Antibiot. 1995, 48, 967–972. Huang, S.-X.; Powell, E.; Rajski, S. R.; Zhao, L.-X.; Jiang, C.-L.; Duan, Y.; Xu, W.; Shen, B. Org. Lett. 2010, 12, 3525–3527. Soni, M. G.; Carabin, I. G.; Burdock, G. A. Food Chem. Toxicol. 2005, 43, 985–1015. Quévrain, E.; Domart-Coulon, I.; Pernice, M.; BourguetKondracki, M.-L. Environ. Microbiol. 2009, 11, 1527–1539. Peng, X.; Adachi, K.; Chen, C.; Kasai, H.; Kanoh, K.; Shizuri, Y.; Misawa, N. Appl. Environ. Microbiol. 2006, 72, 5556–5561. Al-Zereini, W.; Schuhmann, I.; Laatsch, H.; Helmke, E. J. Antibiot. 2007, 60, 301–308. Valgas, C.; Souza, S. Brazilian J. Microbiol. 2007, 38, 369–380. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disk susceptibility test. Fourteenth Supplementary data Supplement; CLSI/NCCLS document M100-S14: Wayne, Pennsylvania, 2004
5 Highlights
A cyclic tetrapeptide (1) was isolated from a marine strain Bacillus cereus. 4-(R)-hydroxysattabacin (2), and three parabens were also isolated and identified. Absolute configuration of Cereusitin A (1) was established by Marfey method. The compounds were tested against human patogens and phytopatogenic fungi.
S0040-4039(17)30002-3 http://dx.doi.org/10.1016/j.tetlet.2017.01.002 TETL 48505
To appear in:
Tetrahedron Letters
Received Date: Revised Date: Accepted Date:
12 October 2016 28 December 2016 2 January 2017
Please cite this article as: Pinzón, A., Martinez-Matamoros, D., Castellanos, L., Duque, C., Rodríguez, J., Jiménez, C., Ramos, F.A., Cereusitin A, a cyclic tetrapeptide from a Bacillus cereus strain isolated from the soft coral Antillogorgia (syn. Pseudopterogorgia) elisabethae, Tetrahedron Letters (2017), doi: http://dx.doi.org/10.1016/ j.tetlet.2017.01.002
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Cereusitin A, a cyclic tetrapeptide from a Bacillus cereus strain isolated from the soft coral Antillogorgia (syn. Pseudopterogorgia) elisabethae a
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a,b
Angela Pinzón, Diana Martinez-Matamoros, a a Leonardo Castellanos, Carmenza Duque, b b a Jaime Rodríguez, Carlos Jiménez, and Freddy A. Ramos * a
Universidad Nacional de Colombia – Bogotá - Facultad de Ciencias - Departamento de Química - Laboratorio de Productos Naturales Marinos, Carrera 30 Nº 45-03, Bogotá, 111321 - Colombia b Departamento de Química Fundamental, Facultade de Ciencias e Centro de Investigacións Científicas Avanzadas (CICA), Universidade da Coruña, A Coruña E-15071, Spain
1
Tetrahedron Letters
Cereusitin A, a cyclic tetrapeptide from a Bacillus cereus strain isolated from the soft coral Antillogorgia (syn. Pseudopterogorgia) elisabethae Angela Pinzón,a Diana Martinez-Matamoros,a,b Leonardo Castellanos,*a Carmenza Duque,a Jaime Rodríguez,b Carlos Jiménez,b and Freddy A. Ramosa* Universidad Nacional de Colombia – Bogotá - Facultad de Ciencias - Departamento de Química - Laboratorio de Productos Naturales Marinos, Carrera 30 Nº 45-03, Bogotá, 111321 – Colombia. b Departamento de Química Fundamental, Facultade de Ciencias e Centro de Investigacións Científicas Avanzadas (CICA), Universidade da Coruña, A Coruña E-15071, Spain. a
A RT I C L E I N F O
A BS T RA C T
Article history: Received Received in revised form Accepted Available online
Two new compounds, cereusitin A (1), the tetrapeptide cyclo-(L-phenylalanyl-trans-4-hydroxyL-prolyl-L-leucyl-trans-4-hydroxy-L-proline and 4-(R)-hydroxysattabacin (2), along with the methyl, isopropyl, and propyl esters of p-hydroxybenzoic acid (3–5) were isolated from the organic extracts of the culture of the B. cereus RKHC-09 strain in two culture media (MOLP and B1), recovered from the sea fan Antillogorgia elisabethae (syn. Pseudopterogorgia elisabethae). Cereusitin A (1) showed mild antifungal activity against Colletotrichum gloeosporoides C26 (yam pathogen) but was inactive against Fusarium oxysporum f.sp. dianthi (carnation pathogen). The methyl and propyl esters of p-hydroxybenzoic acid (4 and 5) showed antimicrobial activity against S. aureus ATCC 33591 and S. cerevisiae, with an MIC of 2 M.
Keywords: Bacillus cereus Cyclopeptide 4-hydroxyproline Marfey’s method Marine Natural Products
In the last five decades, marine natural products have represented one of the most important sources of novel and biologically active molecules, yielding at least seven compounds on the market as well as a high number of chemical entities in phase II and III clinical trials.1,2 Although the most common sources are sponges, cnidarians and macroalgae, there has been a significant increase in the number of molecules isolated from marine microorganisms in recent years. 3–5 In the present work, our starting point was the soft coral Antillogorgia elisabethae (syn. Pseudopterogorgia elisabethae), which has been recognized as a rich source of bioactive compounds.6,7 The organic extracts from a collection of bacterial symbionts from A. elisabethae collected in Providencia Island (Colombia)8 were evaluated for antimicrobial activity (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 33591, and Saccharomices cereviceae). As a result of this evaluation, the strain identified as Bacillus cereus RKHC-09 was selected for chemical studies on the basis of its activity. Bacteria belonging to the Bacillus genus are present in numerous environments and they are considered as microbial factories for the production of a large number of biologically active molecules.9 In the case of Bacillus subtilis, it has been postulated that around 4% of its genome is devoted to the production of antimicrobial compounds.10 Cyclic lipopeptides surfactines, iturines, and fengicines have been isolated from several strains of Bacillus usually associated with plant rhizomes and some of these compounds have shown activity in the control
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pathogenic infections in the host plants.10 Furthermore, the emetic toxins valinomycin and cereulide, isolated from Bacillus cereus strains, are remarkable compounds due to their pHdependence antimicrobial activity against Gram-positive bacteria associated to their ecological role.11,12 In addition to peptiderelated compounds that comprise diketopiperazines and ribosomal and non-ribosomal peptides, compounds such as terpenes, aminosugar, polyketides, and phospholipids have also been isolated from strain cultures belonging to this genus13. In this paper, and as part of our continued search for bioactive compounds from marine sources,14 we describe the isolation and structural elucidation of two new metabolites from the organic extracts obtained from the strain B. cereus RKHC-09 in two different culture media (MOLP and B1): the new cyclopeptide cereusitin A (1), from MOLP culture medium, and 4-(R)hydroxysattabacin (2) along with three known aromatic compounds (3-5), from the B1 medium. They were tested in antimicrobial assays against human pathogenic strains. The organic extract of a bacterial strain identified as B. cereus RKHC-09, which is part of the 13 Firmicutes strains recovered from the soft coral A. elisabethae (syn. Pseudopterogorgia elisabethae) collected from the Colombian Caribbean Sea,8 was selected on the basis of its antimicrobial activity (against E. coli and S. cereviciae) and on the results of a UPLC-MS dereplication study on its organic extracts (data not shown).
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Tetrahedron
As an approach to OSMAC (one strain many compounds) strategies15, this strain was grown in eight different culture media, chosen by their composition (rich and poor media, chemically defined and complex media whit different sources of carbon and nitrogen - Supplementary data). Comparison of the HPLC profiles and the antibacterial activity displayed by the corresponding organic extracts allowed us to identify MOLP and B1 as the culture media that showed activity against the above mentioned pathogenic strains and enhanced production of compounds, which were not found in sterile culture medium extract. Thus, compound 1 was isolated from the organic extracts obtained from culture of B. cereus RKHC-09 in MOLP medium, while compounds 2 - 5 were isolated when the B1 medium was used.
by the following key HMBC correlations: From the -proton of γHyp-2 at δH3.69 and 3.49 correlated to the Leu carbonyl group at δC 166.2 and from the NH amide proton of the Leu unit at δH 6.14 to the carbonyl carbon of the Hyp-1 (δC 170.5) (Figure 1).
Figure 1. Key NMR COSY and HMBC correlations in 1.
The molecular formula of compound 1, which was obtained as a pale yellow gum, was established as C25H34N4O6 (11 degrees of unsaturation) on the basis on its NMR data and the [M + Na]+ ion peak observed at m/z 509.2375 (calculated for C25H34N4O6Na m/z 509.2370, ∆ 0.5 ppm) in its (+)-ESI-HRMS. The 1H and 13C NMR spectral data, along with the results of an extensive 2D NMR analysis (1H-1H COSY, HSQC and HMBC), suggested a cyclic tetrapeptide structure for compound 1.16 The 1H NMR spectrum (500 MHz, CDCl3, Table 1) showed signals corresponding to one monosubstituted aromatic ring at H 7.28– 7.16 (5H), which is characteristic of a phenylalanine (Phe) unit, two NH amide protons at H 6.14 and 5.79 (1H, brs each), and two methine protons at H 4.49, which are characteristic of two γhydroxyproline (γHyp) units, four α peptide protons at H 4,50, 4.42, 4.38, and 3.99, and two aliphatic methyls at H 0.93 (3H, d, J = 6.5 Hz) and H 0.89 (3H, d, J = 6.5 Hz), which are indicative of a leucine (Leu) unit. The 13C NMR spectrum (125 MHz, CDCl3, Table 1) and the DEPT experiment showed the presence of four carbonyl signals at C 170.5, 169.6, 166.2, and 165.1, aromatic carbon signals in the range C 135.6–127.5, four α peptide carbons at C 57.3, 57.2, 56.0, and 53.3, and two aliphatic methyl carbons at C 23.1 and 21.1. The HSQC experiment allowed us to assign each proton to its corresponding carbon. The analysis of both, COSY and HMBC spectra showed that compound 1 is a tetrapeptide, composed by one Phe, one Leu, and two γHyp residues. The carbon signals at C 68.3 and 68.1 correspond to carbons on the γHyp residues. The amino acid sequence was determined from the observed key HMBC correlations showed in Figure 1 as follows: long range correlations between the proton signal at δH 5.79, assigned as the NH Phe amide proton and the carbonyl carbons of Phe at δC 165.1 and γHyp-2 at δC 169.6 and the α carbon of γHyp-2 at δC 57.2 (Figure 1). On the other hand, the -protons of γHyp-1 at δH 3.63 and 3.48 correlated to the Phe carbonyl group at δC 165.1. All of these long range correlations indicate a cyclic structure for compound 1, were the Phe unit was flanked by the two γHyp units. The Leu unit was also placed between the two γHyp units
The data outlined above allowed us to propose that the amino acid sequence of compound 1 is cyclo-[Phe-Hyp-Leu-Hyp]. The complete amino acid sequence of 1 was confirmed by (+)ESI-HRMS (see Figure 2), which showed an ion at m/z 283.1053 (calculated for C14H16N2O3Na m/z 283.1053, ∆ 0.0 ppm) corresponding to [M – Leu-Hyp + Na]+ and m/z 249.1220 (calculated for C11H18N2O3Na m/z 249.1209, ∆ 1.1 ppm) corresponding to the fragment [M – Phe-Hyp + Na]+.
Figure 2. Key fragments found in the (+)-ESI-HRMS of 1. The absolute configurations of the -amino acids present in 1 were determined by Marfey’s method. 17 Analysis of FDAAderivatized amino acids in hydrolysed peptide 1, compared to amino acid standards r with D- and L-configurations, showed the L-configuration for all of the residues (Suplementary Data). Based on these data, the structure of compound 1, named as cereusitin A, was established as cyclo-(L-Phe-trans-L-Hyp-LLeu-trans-L-Hyp). The structure of cereusitin A 1 is closely related to that of the peptide cyclo-(Phe-Pro-Leu-Pro), isolated from a Pseudoalteromonas sp. obtained from the marine sponge Halisarca ectofibrosa18 and cyclo-(L-leucyl-D-cis-4-hydroxy-D-
3 prolyl-L-leucyl-trans-4-hydroxy-L-proline) isolated from a from the exo-cellular extract of Pseudomonas sp., a bacterium associated with the sponge Ircinia muscarum.19,20 In general terms, cyclic tetrapeptides are well known fungal and bacterial metabolites from both, terrestrial and marine environments.21,22 Table 1: 1D NMR data of cereusitin (1) in CDCl3 (1H-NMR at 500 MHz; 13C-NMR at 125 MHz) Amino acid Hyp-1
Phe
Hyp-2
Leu
δC, mult.
δH, mult. (J in Hz)
57.3, CH 37.3, CH2
68.3, CH 54.3, CH2
4.42, dd (6.3, 11.1) 2.31, dd (6.3, 13.3) 2.06, m 4.49, m 3.63, dd (4.3, 13.0) 3.48, m
CO
170.5, C
NH
56.0, CH 36.5, CH2
5.79, br s 4.50, dd (3.9, 10.1) 3.51, m 2.73, 1H (10.3, 14.5)
1 2/6 3/5 4 CO
135.6, C 129.0 × 2, CH 129.1 × 2, CH 127.5, CH 165.1, C
7.16, d (7.1) 7.28, t, (7.3) 7.21, t, (7.3)
position
57.2, CH 37.6, CH2
68.1, CH 54.3, CH2
CO
169.6, C
NH
53.3, CH 38.4, CH2
(CH3) (CH3) CO
24.6, CH 23.1, CH3 21.1, CH3 166.2, C
4.38, dd, (6.3, 11.2) 2.26, dd, (6.8, 13.2) 2.06, m 4.49, m 3.69, dd, (4.5, 13.1) 3.49, m
drugs, and cosmetics.26 Although there are examples of the production of parabens by other microorganisms,27 the results described here represent the first evidence of the production of this class of compound from strains of the Bacillus genera. Evaluation of the antimicrobial activity of the isolated compounds (1, 3–5) showed that only compounds 3 and 5 were active against Staphylococcus aureus ATCC 33591 and Saccharomices cereviceae with a MIC value of ~ 2 M (values of 0.3 and 0.4 mg/mL for compounds 3 and 5, respectively). These values are lower than the reported LD50 data when these parabens were tested against different mammalian species.28-31 Further evaluation of the antifungal activity for compound 1 in the agar diffusion test showed a weak inhibition of growth of Colletotrichum gloeosporoides C26 (yam pathogen) compared with clotrimazole, which was used as a positive control, but this compound was inactive against Fusarium oxysporum f.sp. dianthi (carnation pathogen). A more detailed evaluation of compound 1 as a phytopathogen control agent is currently underway. In summary, two new compounds, namely cereusitin A (1) and 4-(R)-hydroxysattabacin (2), along with four known compounds 3–5, were isolated from the strain Bacillus cereus RKHC-09 obtained from the soft coral Antillogorgia elisabethae (syn. Pseudopterogorgia elisabethae). Cereusitin A (1) showed a mild antifungal activity against Colletotrichum gloeosporoides C26 (yam pathogen) but was inactive against Fusarium oxysporum f.sp. dianthi (carnation pathogen). The same strain cultured in B1 medium allowed the isolation of the new compound 4-(R)-hydroxysattabacin (2), along with the known methyl, propyl, and isopropyl esters of p-hydroxybenzoic acid (3–5). An antibacterial test on the isolated compounds showed activity for compounds 3 and 5 against S. cerevisiae and S. aureus.
Acknowledgments 6.14, br s 3.99, dd (3.7, 9.6) 1.94, m 1.45, m 1.70, m 0.93, d, (6.5) 0.89, d, (6.5)
Compound 2 was obtained from a culture of B. cereus RKHC09 in B1 medium and was isolated as a white amorphous solid. Comparison of 1D and 2D NMR and the (+)-ESI-LRMS data of 223 with literature values allowed compound 2 to be identified as 4-hydroxysattabacin, previously isolated from a Bacillus strain obtained from soil samples Bacillus sp. from Sardina (Italy) by Satta and co-workers and it exhibits antiviral activity, most notably against herpes simplex virus type 1 (HSV1) and 2 (HSV2).23 However, the optical rotation of compound 2 ([α]25D = –33.2, c 0.3, CHCl3) was opposite in sign to that previously reported for natural ([α]25D = +14, c 0.3, CHCl3)23 and synthetic ([α]25D = +18.3, c 0.21, CH3OH)22 4-(S)- hydroxysattabacin, indicating that compound 2 must be the R enantiomer of 4hydroxysattabacin. In addition, the NMR and MS data for the methyl ester (3), isopropyl ester (4), and propyl ester (5) of p-hydroxybenzoic acid,24,25 isolated from the extract of the culture in B1 medium, matched those reported respectively. These compounds, also known as parabens, are commonly used as preservatives in foods,
This work was partially financed by grants from Colciencias, Universidad Nacional de Colombia, and IFS (Grant F/5023-1) of Colombia, and from Ministry of Economy and Competitiveness (MINECO) (Grant AGL2015-63740-C2-2-R) of Spain. The Ministerio de Ambiente y Desarrollo Sostenible granted permission (permission No. 4 of 10/02/2010, contrato de acceso a recurso genético No 108 to collect samples and perform this research on A. elisabethae at the Archipelago of San Andrés and Providencia, Colombian Caribbean. The authors are grateful to Prof. Edelberto Silva, Departamento de Farmacia, Prof. Angélica Kundson, Facultad de Medicina and Prof. Nubia Moreno Instituto de Biotecnología, Universidad Nacional de Colombia, who kindly donated the phatogenic bacterial and fungal strains for antimicrobial assays. Supplementary Data 1
H, 13C, HSCQ, HMBC NMR spectra and (+)-ESI-LRMS and (+)-ESI-HRMS of cereusitin A 1, Along with its analysis by Marfey’s method. References and notes 1. 2. 3.
Gerwick, W. H.; Moore, B. S. Chem. Biol. 2012, 19, 85–98. Mayer, A. M.; Nguyen, M.; Newman, D. J.; Glasser, K. B.. Faseb J. 2015, 30, 932.7-932.7 Cragg, G. M.; Newman, D. J. Biochim. Biophys. Acta 2013, 1830, 3670–3695.
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Tetrahedron 4. Penesyan, A.; Kjelleberg, S.; Egan, S. Mar. Drugs 2010, 8, 438– 459. 5. Imhoff, J. F.; Labes, A.; Wiese, J. Biotechnol. Adv. 2011, 29, 468– 482. 6. Duque, C.; Puyana, M.; Narváez, G.; Osorno, O.; Hara, N.; Fujimoto, Y. Tetrahedron 2004, 60, 10627–10635. (b) Duque, C.; Puyana, M.; Castellanos, L.; Arias, A.; Correa, H.; Osorno, O.; Asai, T.; Hara, N.; Fujimoto, Y. Tetrahedron 2006, 62, 4205– 4213. 7. Lei, H. Chem. Biodiversity 2016, 13, 345 – 365 8. Correa, H.; Haltli, B.; Duque, C.; Kerr, R. Microb. Ecol. 2013, 66, 972–985 9. Rahman, H.; Austin, B.; Mitchell, W. J.; Morris, P. C.; Jamieson, D. J.; Adams, D. R.; Spragg, A. M.; Schweizer, M. Mar. Drugs 2010, 8, 498–518. 10. Ongena, M.; Jacques, P. Trends Microbiol. 2008, 16, 115–125. 11. Kroten, M.A.; Bartoszewicz, M.; Swiecicka I. Pol. J. Microbiol. 2010, 59, 3–10. 12. M.H. Tempelaars, S. Rodrigues, T. Abee, Appl. Environ. Microbiol. 77 (2011) 2755–62. 13. Mondol. M.A.; Shin, H.J.; Islam, M.T. Mar. Drugs 2013, 11, 2846 - 2872. 14. (a) Quintana, J.; Bayona, L.M.; Castellanos, L.; Puyana, M.; Camargo, P.; Aristizabal, F.; Edwards, C.; Tabudravu, J.N.; Jaspars M.; Ramos, F.A. Bioorg & Med. Chem. 2014, 22, 67896795. (b) Martinez-Matamoros, D.; Laiton, F.M.; Duque, C.; Ramos, F.A.; Castellanos, L. Vitae 2016. 23, 30 – 47. (c) PardoVargas, A.; de Barcelos Oliveira, I.; Stephens, P. R. S.; CirneSantos, C. C.; de Palmer Paixão, I. C. N.; Ramos, F. A.; Jiménez, C.; Rodríguez, J.; Resende, J. A. L. C.; Teixeira, V. L.; Castellanos, L. Mar. Drugs 2014, 12, 4247–4259. 15. Bode, H. B.; Bethe, B.; Höfs, R.; Zeeck, A. ChemBioChem 2002, 3, 619–627. 16. Compound 1: Pale yellow gum. [α]25D –33.2 (c 0.3, CHCl3). 1HNMR (500 MHz, CDCl3) and 13C-NMR (125 MHz, CDCl3), see Table 1. 1H-NMR (300 MHz, CD3OD) γHyp-1 δH: 4.47 (1H, m, H), 4.38 (1H, t, J = 4.6 Hz, Hα), 2.06 (1H, dd, J = 5.9, 13.2 Hz, Hβ), 2.29 (1H, dt, J = 4.6, 12.4 Hz, Hβ), 3.71 (1H, dd, J = 5.0, 13.1 Hz, H), 3.49 (H, m, H); γHyp-2 δH: 4.46 (1H, m, H), 4.36 (1H, t, J = 4.6 Hz, Hα), 2.10 (2H, dd, J = 5.9, 13.2 Hz, Hβ), 2.28 (1H, dt, J = 4.6, 12.4 Hz, Hβ), 3.66 (1H, dd, J = 5.0, 13.1 Hz, H), 3.44 (1H, m, H); Phe δH: 4.50 (1H, m, Hα), 3.17 (2H, m), 7.22– 7.29 (5H, m), Leu δH:4.17 (1H, m, Hα), 1.93 (1H, m), 1.51 (1H, t, J = 8.0), 1.89 (1H, m), 0.97 (3H, d, J = 2.5 Hz), 0.96 (3H, d, J = 2.5 Hz). 13C-NMR (75 MHz, CD3OD) γHyp-1 δC:67.1 (C), 56.7 (Cα), 37.4 (Cβ), 53.8 (C), 170.1 (CO), γHyp-2 δC: 67.7 (C), 57.1 (Cα), 36.6 (Cβ), 53.7 (C), 171.9 (CO); Phe δC :56.1 (Cα), 36.5 (Cβ), 136.2 (C), 128.1 × 2, 129.6 × 2, 126.7 (Aromatic carbons), 165.6 (CO); Leu δC: 53.1 (Cα), 37.9 (Cβ), 24.3 (C), 20.7 (C), 21.9 (C), 167.6 (CO). (+)-ESI-LRMS m/z (rel. int.) 509 [M + Na]+ (7), 283 [M-Phe – Hyp + Na]+ (100), 249 [M – Leu-Hyp + Na]+ (25). (+)-ESI-HRMS m/z 509.2375 (calcd for C25H34N4O6Na, 509.2370), 283.1053 (calcd for C14H16N2O3Na m/z 283.1053), 249.1220 (calcd for C11H18N2O3Na, 249.1209). Melting point 134 ± 1 °C. 17. (a) Bhushan, R.; Brückner, H. Amino Acids 2004, 27, 231–247. (b) Harada, K.; Fujii, K.; Hayashi, K.; Suzuki, M.; Ikai, Y.; Oka, H. Tetrahedron Lett. 1996, 37, 3001–3004. 18. Rungprom, W.; Siwu, E. R. O.; Lambert, L. K.; Dechsakulwatana, C.; Barden, M. C.; Kokpol, U.; Blanchfield, J. T.; Kita, M.; Garson, M. J. Tetrahedron 2008, 64, 3147–3152. 19. Mitova, M.; Tommonaro, G.; De Rosa. S.; Z. Naturforsch. - Sect. C J. Biosci. 2003, 58, 740–745. 20. Zhou, H.; Yang, Y.; Yang, X.; Li, W.; Xiong, Z.; Zhao, L.; Xu, L.; Ding, Z. Nat. Prod. Res. 2014, 28, 318–23. 21. Gao, C.-H.; Chen, Y.-N.; Pan, L.-X.; Lei, F.; Long, B.; Hu, L.-Q.; Zhang, R.-C.; Ke, K.; Huang, R.-M. J. Antibiot. 2014, 67, 541– 543. 22. Pérez-Victoria, I.; Martín, J.; González-Menéndez, V.; de Pedro, N.; El Aouad, N.; Ortiz-López, F. J.; Tormo, J. R.; Platas, G.; Vicente, F.; Bills, G. F.; Genilloud, O.; Goetz, M. A; Reyes, F. J. Nat. Prod. 2012, 75, 1210–1214. 23. Compound 2: White amorphous solid, [α]25D –33.2 (c 0.3, CHCl3). 1H-NMR (300 MHz, CD3OD) δH 7.07 (2H, d, J = 8.6 Hz), 6.71 (2H, d, J = 8.6 Hz), 4.23 (1H, dd, J = 4.8, 7.8 Hz), 2.94 (1H, dd, J = 4.8, 14.0 Hz), 2.71 (1H, dd, J = 7.8, 14.0 Hz), 2.40 (1H, d, J = 1.6 Hz), 2.37 (1H, d, J = 1.6 Hz), 0.91 (3H, d, J = 1.1 Hz), 0.88 (3H, d, J = 1.1 Hz). 13C-NMR (300 MHz, CD3OD) δC 214.6,
24. 25. 26. 27. 28. 29. 30. 31.
156.1, 129.3 (× 2), 128.1, and 114.3 (× 2), 77.5, 47.2, 38.7, 23.6, 21.7 × 2. (+)-ESI- LRMS m/z 223 [M + H]+. Lampis, G.; Deidda, D.; Maullu, C.; Madeddu, M. A.; Pompei, R.; Monache, F.D.; Satta, G. J. Antibiot. 1995, 48, 967–972. Huang, S.-X.; Powell, E.; Rajski, S. R.; Zhao, L.-X.; Jiang, C.-L.; Duan, Y.; Xu, W.; Shen, B. Org. Lett. 2010, 12, 3525–3527. Soni, M. G.; Carabin, I. G.; Burdock, G. A. Food Chem. Toxicol. 2005, 43, 985–1015. Quévrain, E.; Domart-Coulon, I.; Pernice, M.; BourguetKondracki, M.-L. Environ. Microbiol. 2009, 11, 1527–1539. Peng, X.; Adachi, K.; Chen, C.; Kasai, H.; Kanoh, K.; Shizuri, Y.; Misawa, N. Appl. Environ. Microbiol. 2006, 72, 5556–5561. Al-Zereini, W.; Schuhmann, I.; Laatsch, H.; Helmke, E. J. Antibiot. 2007, 60, 301–308. Valgas, C.; Souza, S. Brazilian J. Microbiol. 2007, 38, 369–380. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disk susceptibility test. Fourteenth Supplementary data Supplement; CLSI/NCCLS document M100-S14: Wayne, Pennsylvania, 2004
5 Highlights
A cyclic tetrapeptide (1) was isolated from a marine strain Bacillus cereus. 4-(R)-hydroxysattabacin (2), and three parabens were also isolated and identified. Absolute configuration of Cereusitin A (1) was established by Marfey method. The compounds were tested against human patogens and phytopatogenic fungi.