Isolation and Characterization of Unusual Hydrazides fromStreptomyces sp. Impact of the Cultivation Support and ExtractionProcedureGeraldine Le Goff, Marie-Therese Martin, Bogdan I. Iorga, Emilie Adelin, Claudine Servy, Sylvie Cortial,and Jamal Ouazzani*
Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles ICSN, Centre National de la Recherche ScientifiqueCNRS, Avenue de la Terrasse 91198, Gif-sur-Yvette, France
*S Supporting Information
ABSTRACT: Three novel hydrazides, geralcins C−E (1−3),were isolated from Streptomyces sp. LMA-545, together withMH-031 and geralcins A and B. This unusual family ofcompounds was isolated from liquid-state and agar-supportedfermentation using Amberlite XAD-16 solid-phase extractionduring the cultivation step. The use of such neutral resinduring the cultivation step allowed the specific adsorption of microbial secondary metabolites, avoiding any contamination of thecrude extracts by the constituents of the culture medium. The trapped compounds were eluted from the resin with methanol, andtheir structures elucidated using 1H, 13C, and 15N NMR spectroscopic analysis and high-resolution mass spectrometry. Molecularmodeling calculations were applied in order to support structural attributions. No antimicrobial, cytotoxic, or DnaG-inhibitionactivities were detected for geralcins D and E. Geralcin C has no antimicrobial activity but exhibited an IC50 of 0.8 μM against KBand HCT116 cancer cell lines. Furthermore, geralcin C inhibited the E. coli DnaG primase, a Gram-negative antimicrobial target,with an IC50 of 0.7 mM.
Recently, a new scaffold of natural compounds was reportedconsisting of unusual alkyl hydrazides.1−4 Major repre-
sentatives of this family are montamine isolated from the seedsof Centaurea montana (Asteraceae),1 hydrazidomycins A, B, andC, also called elaiomycins, isolated from Streptomyces species,2,3
and recently geralcins A and B isolated from Streptomyces sp.LMA-545.4 The biological role of these compounds is stillunknown, while cytotoxic activities against tumor cell lines werereported for montamine1 (IC50 of 43.9 μM), hydrazidomycins2
(IC50 of 0.37 μM), and geralcin B4 (IC50 of 5 μM). Althoughnatural hydrazides are notably scarce, they are often found insynthetic therapeutics such as iproniazid, a monoamine oxidaseinhibitor used as an antidepressant.5
The actinomycete Streptomyces sp. LMA-545 was isolatedrecently as an α,β-unsaturated γ-lactono-hydrazides producerleading to geralcins A and B.4 In order to widen the panel ofsecondary metabolites isolated from this strain, we investigatedthe impact of the cultivation support and the extractionprocedure. We were encouraged by our recent developments inthe field of agar-supported fermentation (Ag-SF)6 and solid-phase extraction (SPE).4
In this paper, we report the structural characterization ofthree novel alkyl hydrazides produced by the bacterial strainStreptomyces sp. LMA-545, combining SPE with liquid-statefermentation (LSF) or SPE with agar-state fermentation (Ag-SF). The latter procedure had never been investigated by usand allowed the isolation of a novel hydrazide compound notproduced from LSF.
Antimicrobial, cytotoxic, and primase-inhibition activitieswere evaluated for the newly isolated compounds.
■ RESULTS AND DISCUSSION
The bacterial strain Streptomyces sp. LMA-545 was previouslyreported for the production of geralcins A and B.4 Thecompounds being studied were produced in liquid-statefermentation and agar-supported fermentation and recoveredvia in situ solid-phase extraction using Amberlite XAD-16neutral resin. Agar-supported cultivation was recently scaled-upin our laboratory in the specific device Platotex, offering 2 m2 ofcultivation surface.6 The crude extracts were eluted from theXAD-16 resin by methanol and analyzed by HPLC coupledwith PDA, ELS, and mass spectrometry detection. The HPLCprofile obtained for the LSF extract revealed the presence oftwo unknown compounds (1 and 2) together with thepreviously reported MH-031 and geralcins A and B. Thecrude extract obtained from Ag-SF showed the presence of oneunkown compound (3) together with MH-031 and geralcins Aand B (Figure 1).The structures of the new compounds were elucidated using
both 1D and 2D 1H and 13C NMR spectroscopic analysis andhigh-resolution mass spectrometry. 1H−15N NMR experimentswere required for full structural elucidation as well as molecular
Received: July 31, 2012Published: February 6, 2013
modeling calculations. The structures of these compounds areshown in Figure 2.Compound 1 was obtained as a yellowish oil. The HRESIMS
analysis gave the molecular formula C17H32N4O4. The NMRdata, recorded in DMF-d7, are listed in Table 1. According tothe molecular formula, four degrees of unsaturation should bepresent, corresponding to two carbonyl groups, two olefiniccarbons (δC 118.5 and 123.8), and an azoxy group revealed by acharacteristic IR band at 1506 cm−1 (NN+−O−). Acharacteristic IR band at 1692 cm−1 (OC−NR) suggestedthat the two carbonyl groups are from amides. Moreover, the1H−15N HMBC data revealed two nitrogen atoms at δN 148.0(N-A, N) and δN 142.4 (N-B, NH). The correlations of H-N(δH 11.00, s, NH) to N-A supported a N−N bondcorresponding of the hydrazide group. The 1H−15N HMBCspectra also showed the presence of two nitrogen atoms of anazoxy group at δN 345.1 (N-C, NO) and δN 348.0 (N-D, NN). The 13C NMR revealed the presence of 17 carbon atoms,which included two carbonyl groups at δC 168.9 (C-1) and δC
168.7 (C-16), two methine carbons at δC 123.8 (δC 126.9) (C-9) and δC 118.5 (δC 128.0) (C-10) (duplication of carbonchemical shifs were recorded corresponding to major andminor rotamers), nine methylene groups, which included oneoxygen-bound methylene at δC 60.7 (C-15) and one azoxy-bound methylene at δC 69.2 (C-3), one sp3 carbon atom at δC63.9 (C-2), linked to the azoxy moiety, and three methylcarbons at δC 13.6 (C-8), 13.7 (C-14), and 20.2 (C-17).Duplication of the 1H and 13C NMR signals for hydrogen andcarbon atoms connected to the hydrazide group was observedfor compound 1. This phenomenon was due to the equilibriumbetween amide rotamers and was previously described forgeralcin B.4 The nJ1H−13C connectivities given by HSQC andHMBC NMR, including 1H−15N correlations, are listed inTable 1.The key structural elements for compound 1 revealed by 1H
NMR were the singlet at δH 11.00 (H-N, s) associated with anitrogen atom at δN 148.0 (N-A, N) (15N-HSQC in theSupporting Information) and a broad multiplet at δH 5.18 (H-
Figure 1. HPLC analysis of LSF extract (chromatograms A and B) and Ag-SF extract (chromatogram C).
Figure 2. Compounds isolated from Streptomyces sp. LMA-545. MH-031 and geralcins A and B were previously reported.4 Compounds 1, 2, and 3are newly reported in this paper.
Journal of Natural Products Article
dx.doi.org/10.1021/np300527p | J. Nat. Prod. 2013, 76, 142−149143
O, m), linked to an oxygen atom, coupling with the twogeminal protons of the methylene group, at δH 3.77−3.95 (H-15, m) (Figure 3). The doublet at δH 6.41 (6.53) (H-9) and the
quadruplet at δH 4.88 (5.36) (H-10) were assigned to the twomethine proton signals. The coupling constant of 9.0 Hzbetween H-9 and H-10 of compound 1 indicated a cisconfiguration for the C-9/C-10 double bond. The 1H−1HCOSY correlations indicated that the single proton at δH 4.88(H-2, m) linked to the azoxy function was connected to thegeminal protons H-15. The triplet at δH 4.29 (H-3, t, 6.9) wasassigned to the methylene connected to the azoxy groupthrough the nitrogen at δN 345.1 (N-C, NO) and to themethylene protons H-4, which gave a broad signal between1.85 and 1.91 ppm. The 1H−15N HMBC correlations from H-3and H-4 to N-C (δN 345.1, NO) and H-3 to N-D (δN 348.0,NN) corroborated this observations. A broad signalcorresponding to two geminal protons H-11 at δH 2.08 and2.19 was connected to the methine H-10. A singlet at δH 2.95(2.05) was identified as the methyl group connected to thehydrazide function through C-16 (δC 168.7). A broad signal
Table 1. NMR Spectroscopic Dataa for Compounds 1, 2, and 3
geralcin C (1) geralcin D (2) geralcin E (3)
no. δC δH (mult; J in Hz) HMBC δCe δH (mult; J in Hz) HMBC δC δH (mult; J in Hz) HMBC
N-A 148.0,c N −, NN-B 142.4,c NH N-A, C-9, 10 241.2,c* N C-11 137.3c NH
138.6c,d
N-C 345.1,c NO C-3, 4
N-D 348.0,c
NNC-3
a1H chemical shifts were recorded at 600 MHz and 13C chemical shifts at 150 MHz in DMF-d7 for compounds 1 and 2, and CD2Cl2 for compound3. bSignals were not distinguishable. c15N chemical shift of the nitrogen atom δN in ppm. dNMR spectroscopic data recorded for the minor rotamerobserved for compounds 1 to 3. eCarbon chemical shifts were deduced from HSQC and HMBC data for compound 2. fData were recorded at 233 Kto observed H-O 1H NMR signal
dx.doi.org/10.1021/np300527p | J. Nat. Prod. 2013, 76, 142−149144
between 1.85 and 1.91 ppm identified as the methylene groupH-4 was connected to the methylene group H-3. A set of fivemethylene groups, with a broad signal between 1.26 and 1.31ppm, was assigned to two aliphatic methylene chains (C-5 to C-7 and C-12 to C-13). These chains were respectivly connectedto C-4 (δC 27.7) and C-11 (δC 26.1) and ended with methylgroups at δC 13.6 (C-8) and δC 13.7 (C-14). The two methylgroups were characterized by a broad signal at δH 0.87. The1H−13C HMBC connectivities allowed the construction of thealiphatic chain from C-7 to C-8. The 1H−15N HMBC datashowed the connection of this aliphatic chain to the azoxyfunction through N-C (δN 345.1, NO). The chemical shift forC-2 (δC 63.9) confirmed the presence of the azoxy group in thelocal environment, and 1H−13C HMBC supported theconnectivities from H-15 to C-2 and from H-2 to C-1 (δC168.9). C-1 is one of the two carbonyl groups involved in thehydrazide moiety. 1H−1H COSY and 1H−13C HMBC allowedthe construction of the second aliphatic chain from C-11 to C-14, which was connected to the C-10 position of the cis C-9/C-10 double bond. The connectivities from H-9 to N-A deducedfrom the 1H−15N HMBC data attached the unsaturated carbonchain to the hydrazide function through the nitrogen N-A. Theconnectivities from H-17 to C-16 and to N-B, given by both1H−13C HMBC and 1H−15N HMBC data, allowed to connectthe methyl C-17 to the second carbonyl group of the hydrazide.Finally, the 1H−15N HMBC experiments confirmed theposition of the two nitrogen atoms with the observation ofthe connectivity from H-N (δH 11.00) to N-A (δC 148.0, N).The overall structural assignments led to compound 1,
shown in Figure 2. This compound consists of a novel naturalscaffold of alkyl hydrazide with the notable presence of theazoxy function. Such an azoxy group was previously reportedfor similar structures3 but remains scarcely reported for naturalcompounds. Since the discovery of macrozamin in 1951,7 anumber of naturally occurring azoxy compounds have beenidentified, including cycasin,8 elaiomycin,9 maniwamycin I,10
and pyranadines B−G.15 Some of them exhibited antifungal orantibacterial activities.The physical properties and the small quantity obtained for
compound 1 did not allow the determination of the absoluteconfiguration for the carbon C-2.Compound 2 was obtained as a transparent oil. The
HRESIMS analysis gave the molecular formula C15H22N2O6.The NMR data, recorded for 2 in DMF-d7, are listed in Table 1and showed notable similarities to geralcins A and B,4 inparticular duplication of 1H and 13C signals. According to themolecular formula, six degrees of unsaturation should bepresent to account for an α,β-unsatured lactone, a carbonylgroup (δC 169.1), an imine group (δC 157.5), and aheterocyclic ring.A characteristic IR band at 1657 cm−1 (OC−NR)
suggested that the carbonyl group could be from an amide.Moreover, the 1H−15N HMBC data revealed a nitrogen atomat δN 241.2 (N-B, N). A notable IR band at 1448 cm−1
indicated the presence of a nitogen−oxygen bond. The 13CNMR revealed the presence of 15 carbon atoms, whichincluded two carbonyl groups at δC 174.3 (C-1) and 169.1 (C-7), an imine group at δC 157.5 (C-10), two olefinic carbons atδC 147.1 (C-3) and 131.6 (C-4), six methylene groups,including one oxygen-bound methylene at δC 70.7 (C-2), twosp3 carbon atoms at δC 83.0 (71.1) (C-8) and 90.5 (94.5) (C-9)both linked to oxyen atoms, and two methyl carbons at δC 13.5
(C-15) and 10.4 (C-11). Duplication of the 1H and 13C NMRsignals for hydrogen and carbon atoms connected to thehydrazide moiety was recorded for compound 2.The nJ1H−13C connectivities given by HSQC and HMBC
NMR, including 1H−15N correlations, are listed in Table 1.The key structural elements revealed by 1H NMR were the
broad and weak signal, recorded at 223 K, at δH 12.86 (5.86)(H-O, m) coupling with the proton at δH 6.49 (6.07) (H-9, d,1.9). The multiplet at δH 7.53 (H-3, m) was assigned to thedouble bond of the α,β-unsatured lactone and coupled to themethylene group at δH 4.87 (H-2, m). The 1H−1H COSYcorrelations indicated a pair of vicinal methylene groups, givinga broad signal at δH 2.53−2.58 (H-5, m) and 2.70−2.90 (H-6,m). The multiplet at δH 4.23 (3.83) was assigned to H-8 andcoupled with H-9. A set of three methylene groups, with abroad signal between 1.22 and 1.45 ppm, were attributed to thealiphatic methylene chain (C-12 to C-15). This chain wasconnected to the single proton H-8 and ended with a methylgroup characterized by the multiplet at δH 0.86 (H-15, m). Aduplicate singlet at δH 2.10 (2.07) was assigned to the methylgroup attached to the quaternary carbon at δC 157.5 (C-10).The 1H−13C HMBC connectivities of H-2 to C-1/C-3/C-4 andH-3 to C-1/C-2 allowed for the construction of the α,β-unsaturated γ-lactone moiety. Moreover, correlations from H-5to C-1/C-3/C-4/C-6 and from H-6 to C-4/C-5/C-7 showedthat the pair of vicinal methylene groups C-5/C-6 wereconnected to the α,β-unsaturated γ-lactone moiety and to thecarbonyl amide-type group at δC 169.1 (C-7). According to the1H−1H COSY and 1H−13C HMBC, H-8 was connected to thealiphatic chain (C-12 to C-15) through C-12 and C-9. Thechemical shift for C-8 (δC 83.0) and C-9 (δC 90.5) confirmedthe presence of oxygen atoms in the local environment.Moreover 1H−13C HMBC together with 1H−15N HMBC datagave connectivities from H-9 to C-10 and H-11 to C-10/N-B,allowing the construction of an original heteroheptacycle asproposed in Figure 4.
The characteristic IR band at 1448 cm−1 and the particularchemical shift of C-8 (δC 83.0) consolidated the existence ofthe nitrogen−oxygen bond between C-7 (δC 169.1) and C-8(δC 83.0) to close the seven-membered ring.As we did not observe any 1H−15N HMBC correlations
between H-8 and N-A to ensure this original heterocycle, and
Figure 4. 1H−13C HMBC and 1H−15N HMBC connectivitiesrecorded for compound 2.
Journal of Natural Products Article
dx.doi.org/10.1021/np300527p | J. Nat. Prod. 2013, 76, 142−149145
in the absence of any alternative structure that accounts for theNMR data, molecular modeling calculations were undertaken.Molecular modeling studies were carried out to evaluate the
conformational flexibility of different conformers for compound2 and to compare these results with the NMR data.As the relative configurations at C-8 and C-9 could not be
formally assigned by NMR, both 2A and 2B diastereoisomerswere included in the present study (Figure 5).
A conformational analysis of the diastereomers 2A and 2Bwas carried out using MacroModel,16 to generate 53 and 44conformers, respectively. For each diastereomer, threerepresentative conformations were selected, presenting anextended structure (a), an intramolecular hydrogen bondbetween the hydroxyl group and the carbonyl group at C-7 (b),and an intramolecular hydrogen bond between the hydroxylgroup and the carbonyl group at C-1 (c), respectively.The geometries of the six conformers selected in the first
step were optimized using DFT calculations, with Gaussian17 atthe B3LYP/6-31+G(d,p) level; then vibrational frequencycalculations confirmed that these structures are local minima.The relative energies calculated for these conformers (3−10kcal/mol) allow an easy conformer interconversion (in the caseof each diastereomer) to establish a thermodynamic equili-brium. The optimized structures of these conformers are shownin Figure 6.Dihedral angles H8−C8−C9−H9 were measured for the six
optimized structures presented above, and the 3JHH NMRcoupling constants were estimated using four different methods(Table 2 and Supporting Information). A comparison of the
experimental value for 3JH8−H9, which is about 1.86 Hz for bothspecies evidenced in the NMR spectra (Table 1), and the datafrom Table 2 suggests that the species present in solution are2A-a and 2A-b. Additionally, the Karplus method18 and theDFT calculations seem to give the best agreement with theexperimental data. Moreover, the conformer establishing ahydrogen bond involving the hydroxyl group and C-1 could beeliminated: duplication of the 13C signal was recorded only forC-7 (δC 169.1 for the major form and δC 169.5 for the minorone). This supported the existence of hydrogen bondingbetween the hydroxyl group and C-7 (2A-b) in equilibriumwith the 2A-a conformer, considering the NMR data.In conclusion, molecular modeling calculations allowed, by
comparison with the experimental NMR data, the assignmentof the relative stereochemistry at C-8 and C-9 and thusidentification of the naturally occurring cis diastereomer ofcompound 2. This conclusion is supported by the NOE effectsrecorded at 223 K. Concerning the major conformer (2A-b),correlations from H-9 (δH 6.49) to H-8 (δH 4.23) wererecorded, and for the minor conformer (2A-a), correlationsfrom H-9 (δH 6.07) to H-8 (δH 3.83) were recorded. NOESYdata were in accordance with molecular modeling calculations
Figure 5. Cis and trans diastereomers of 2 included in the molecularmodeling study.
Figure 6. Conformations of the diastereomers cis 2A and trans 2B presenting an extended structure (a), an intramolecular hydrogen bond betweenthe hydroxyl group and the carbonyl group at C-7 (b), and an intramolecular hydrogen bond between the hydroxyl group and the carbonyl group atC-1 (c). Relative energies (kcal/mol) calculated with Gaussian0917 at the B3LYP/6-31+G(d,p) level are shown in orange.
Table 2. Dihedral angles H8−C8−C9−H9 for theconformers included in this study and NMR couplingconstants estimated using 4 different methods: 1 - Karplus;18
2 - Haasnoot, as implemented online;19 3 - Karplusmodified, as implemented in Maestro;16 4 - DFT calculations(B3LYP/6-31+G(d,p)) using Gassian0917
dx.doi.org/10.1021/np300527p | J. Nat. Prod. 2013, 76, 142−149146
to establish the cis relative stereochemistry at C-8 and C-9 forcompound 2.Compound 3 was obtained as a colorless oil. The HRESIMS
analysis gave the molecular formula C10H18N2O3. The NMRdata, recorded in CD2Cl2, are listed in Table 1. According tothe molecular formula, three degrees of unsaturation should bepresent to account for two carbonyl groups and two olefiniccarbons (δC 123.6 and 124.5). A characteristic IR band at 1650cm−1 (OC−NR) suggested that the two carbonyl groups arefrom amides. Moreover, the 1H−15N HSQC data revealed thenitrogen atom δN 137.3 (138.6) (N-B, NH), showing signalduplication corresponding to the major and the minorrotamers. The small quantity of isolated compound did notallow 1H−15N HMBC, but the very similar NMR spectracompared to those recorded for the geralcins family suggestedthat compound 3 was a hydrazide. The 13C NMR revealed thepresence of 10 carbon atoms, which included two carbonylgroups at δC 169.1 (C-9) and 172.5 (C-1), two methinecarbons at δC 123.6 (δC 132.4) (C-4) and 124.5 (δC 124.3) (C-3), four methylene groups, which included one oxygen-boundmethylene at δC 60.9 (C-2), and two methyl carbons at δC 14.3(C-8) and 21.0 (C-10). As we found out for compounds 1 and2, duplication of the 1H and 13C NMR signals for hydrogen andcarbon atoms connected to the hydrazide group was recordedfor compound 3. This phenomenon was due to the equilibriumbetween amide rotamers and was previously described forgeralcin B.4 The nJ1H−13C connectivities given by HSQC andHMBC NMR, including 1H−15N correlations, are listed inTable 1.The key structural element revealed by 1H NMR was the
singlet at δH 8.17 (H-N, s) associated with a nitrogen atom atδN 137.3 (138.6) (N-A, N) (15N-HSQC in the SupportingInformation). The doublet at δH 6.31 (6.07) (H-3, d, 7.6) andthe quadruplet at δH 5.13 (5.44) (H-4, q, 7.6) were assigned tothe two methine proton signals. The coupling constant of 7.6Hz between H-3 and H-4 of compound 3 indicated a cisconfiguration for the C-3/C-4 double bond. A broad and largesignal at δH 4.12 was attributed to H-2 (2H, m), correspondingto the oxygen-bound methylene and to H-O. The twoquadruplets, at 2.07 for the major form and 2.14 ppm for theminor one, were assigned to H-5 (2H, bq, 6.7). The 1H−1HCOSY and 1H−13C HMBC correlations showed the con-nection from H-5 to the double bond C-3/C-4 through C-4. Asinglet at δH 2.02 was assigned to the methyl group H-10 (3H,s). The broad signal between 1.32 and 1.37 ppm was attributedto the two methylene protons H-6 and H-7. The aliphatic chainended with the methyl group at C-8, giving a triplet at δH 0.90(3H, t, 6.9). 1H−1H COSY correlations linked the aliphaticchain C-5 to C-8 to the double bond C-3/C-4 connected to N-B through C-3. 1H−13C HMBC data gave connectivities fromH-10 to C-9 and from H-2 to C-1. These correlations allowedconnecting the methylenoxy group C-2 (δC 60.9) to thecarbonyl C-1 (δC 172.5) and the methyl group C-10 (δC 21.0)to the carbonyl C-9 (δC 169.1) (Figure 7).Antimicrobial and cytotoxic activities of compounds 1, 2, and
3 were evaluated according to our previously reportedbioassays.4 Compounds were also screened for their ability toinhibit E. coli DnaG primase, a Gram-negative antimicrobialtarget.20−22 Geralcins D and E did not show any significantbioactivity. Geralcin C has no antimicrobial activity butexhibited an IC50 of 8 × 10−7 M against KB and HCT116cancer cell lines (IC50 for Taxotere KB 2.5 × 10−10 M, HCT1165 × 10−8 M). Furthermore, geralcin C inhibited the E. coli
DnaG primase, in a dose-dependent manner, with an IC50 of 7× 10−4 (Figure 8).Thus, combining SPE with LSF and Ag-SF allowed the
isolation of three novel hydrazides together with MH-031 andgeralcins A and B.4
■ EXPERIMENTAL SECTIONGeneral Experimental Procedures. The IR spectra were
obtained using a Perkin-Elmer Spectrum 100 model instrument.NMR experiments were performed using a Bruker Avance 600 MHzspectrometer equipped with a microprobe head (1.7 TXI, Bruker) forcompounds 1 to 3. The spectra for compounds 1 and 2 were acquiredin DMF-d7 (δH 2.75, 2.92, 8.03 ppm; δC 29.74, 34.89, 163.15 ppm) atroom temperature to observe all of the correlations present. Thespectra for compound 3 were obtained in methylene chloride-d2 (δH5.32 ppm; δC 54.0 ppm) at room temperature and at 233 K.
LC-MS experiments were performed using a Waters-MicromassZQ2000 simple-stage quadrupole mass spectrometer equipped with anESI (electrospray ionization) interface coupled to an Alliance Waters2695 HPLC instrument with PDA and ELS detection.
HRESIMS was conducted using a Waters-Micromass massspectrometer equipped with an ESI-TOF (electrospray-time-of-flight).
Biological Materials. Streptomyces sp. LMA-545 was isolated froma soil sample collected in La Reunion Island and grown on a PDB agar(potato dextrose broth, DIFCO) at 30 °C. The microorganism wasexamined for chemotaxonomic and morphological properties knownto be useful in the systematics of Streptomyces. A phylogenetic analysiswas performed using a fragment of the 16S rRNA gene amplified fromthe genomic DNA of Streptomyces sp. LMA-545. The 16S rRNA geneamplification and sequencing were performed, and the resultingmaterial was compared to the corresponding sequences in the related
Figure 7. 1H−13C HMBC and 1H−15N HMBC connectivitiesrecorded for compound 3.
Figure 8. Inhibition of E. coli DnaG primase by geralcin C (1).
Journal of Natural Products Article
dx.doi.org/10.1021/np300527p | J. Nat. Prod. 2013, 76, 142−149147
Streptomyces using the NCBI/BLAST Web site (GenBank). Theprimers used for PCR amplification were 16 S F 27, AGA GTT TGATC(AC) TGG CTC AG (Tm: 56.3 °C), and 16 S R 1492, TACGG(CT) TAC CTT GTT ACG ACT T (Tm: 57.5 °C). The GenBankaccession number for Streptomyces sp. LMA-545 sequence is BankIt-1535712 8404357.seq JX025158.Fermentation. Batch fermentation of Streptomyces sp. LMA-545
was conducted in a 15 L fermentor (Chemap 20 L unit) in PDBmedium (DIFCO) over 5 days at 30 °C with an aeration rate of 16volumes of air per volume per minute and 200 rpm agitation. Theinitial pH of 7.2 was progressively decreased to 4.3. Amberlite XAD-16(30 g/L) was added prior to sterilization to allow the in situ trappingof the microbial metabolites. Agar-state fermentation coupled withsolid-state fermentation was performed on PDB medium (DIFCO)supplemented with 2% agar (DIFCO) over 9 days at 30 °C. SterilizingAmberlite XAD-16 (20 g) mixed with 5 mL of inoculums was spreadon each of 10 agar plates (25 cm × 25 cm).Isolation. Concerning liquid-state fermentation, the XAD-16 resin
was separated from the broth culture via filtration and washed withwater before being eluted with MeOH (500 mL). The eluate wasconcentrated to dryness in vacuo (5.4 g) and extracted with MeOH.The crude extract (4.7 g) was subjected to flash chromatography on aCombiflash Companion using a Redisep 80 g silica column, with aheptane−ethyl acetate mixture serving as the eluent. The fractionscontaining compounds 1 and 2 were separated as pure compounds bypreparative RP-HPLC (Sunfire Prep C18 5 μm, 10 × 250 mm) elutedusing a linear H2O−CH3CN gradient supplemented with 0.1% formicacid. After concentrating in vacuo, compound 1 (4 mg) was obtainedas a yellowish oil, while compound 2 (0.7 mg) was obtained as acolorless oil. For agar-state fermentation, the XAD-16 resin wasrecovered by carefully scraping the agar plate surface. The recoveredresin was washed with water to eliminate the biomass before beingeluted with MeOH (500 mL). The eluate was concentrated to drynessin vacuo (2.9 g) and extracted with MeOH. The crude extract (1.2 g)was subjected to flash chromatography on a Combiflash Companionusing a Redisep 24 g silica column, with a heptane−ethyl acetatemixture serving as the eluent. The fractions containing compound 3were separated as pure compound by preparative RP-HPLC (SunfirePrep C18 5 μm, 10 × 250 mm) eluted using a linear H2O−CH3CNgradient supplemented with 0.1% formic acid (100→0 to 0→100).After concentrating in vacuo, compound 3 (0.5 mg) was obtained as acolorless oil.Geralcin C (1): yellowish oil; IR νmax 3457, 3264, 2958, 2928, 1692,
1680, 1506, 1373, 1051 cm−1; for complete NMR data see Table 1;HRESIMS m/z [M + H]+ 357.2488 (calcd for C17H33N4O4,357.2502).Geralcin D (2): translucent oil; IR νmax 3451, 3264, 2958, 2940,
2869, 1744, 1659, 1448, 1381, 1219, 1081, 1051 cm−1; for completeNMR data see Table 1; HRESIMS m/z [M + H]+ 327.1541 (calcd forC15H23N2O6, 327.1556).Geralcin E (3): translucent oil; IR νmax 3430, 3275, 2959, 2929,
2860, 1741, 1647, 1425, 1080, 1050 cm−1; for complete NMR data seeTable 1; HRESIMS m/z [M + H]+ 215.1392 (calcd for C10H19N2O3,215.1396).Antibacterial and Antitumor Cell Assays. The antibacterial
activity was measured using the disk inhibition zone method againstBacillus subtilis ATCC.6633, Micrococcus luteus ATCC.10240, andEscherichia coli ATCC.25922. Inhibition was compared for 10 μg ofgentamicin and 30 μg of chloramphenicol.Cytotoxicity Assays. A tetrazolium dye [3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide; MTT]-based colorimetric assaywas used to measure the inhibition of proliferation of the colonicepithelial cancer cell line HCT116, the hormone-responsive breastcancer cell line MCF7, the colon adenocarcinoma cell line HT29, thenaso-pharyngeal carcinoma KB cells, and the breast cancer cell lineMDA231, as previously reported.4 All of the test compounds wereformulated in DMSO and added to the cells such that the final DMSOconcentration ranged from 1% to 3%. Cells were grown in D-MEMmedium supplemented with 10% fetal calf serum (Invitrogen), in thepresence of penicillin, streptomycin, and fungizone, and plated in 96-
well microplates. After 24 h of growth, cells were treated with targetcompounds from 100 μM to 10 nM. After 72 h, MTS reagent(Promega) was added, and the absorbance was monitored (490 nm)to measure the inhibition of cell proliferation compared to untreatedcells. IC50 determination experiments were performed in separateduplicate experiments.
Primase Bioassay. E. coli DnaG primase cloning, purification, andbioassay conditions are fully described in the Supporting Information.
Molecular Modeling. The conformational analysis was carried outusing MacroModel v9.5, as implemented in the Schrodinger Suite.16
Default values were used except the allowed energy window (42 kJ/mol) and the number of evaluations per rotatable bond (500). Theresulting conformers were clustered using a 2.0 Å cutoff.
The geometries of all conformers were optimized in the gas phaseusing the Gaussian 09 package17 with Becke’s three-parameter hybridexchange functional (B3LYP)23,24 and the 6-31+G(d,p) basis set.Subsequent vibrational frequency calculations confirmed that theseconformations were local minima. For all calculations the IEFPCMand CPCM solvation models were also used, these results beingcompared with the ones obtained in the absence of solvation.However, no significant differences were observed, and only the resultsobtained without solvation are presented here.
■ ASSOCIATED CONTENT*S Supporting InformationExperimental section, physicochemical properties, 1D and 2DNMR spectroscopic data, IR data, and high-resolution Orbi-trap-ESIMS. This material is available free of charge via theInternet at http://pubs.acs.org.
(12) Tao, T.; Alemany, L. B.; Parry, R. J. Org. Lett. 2003, 5, 1213−1215.(13) Boschi, D.; Guglielmo, S.; Aiello, S.; Morace, G.; Borghi, E.;Fruttero, R. Bioorg. Med. Chem. Lett. 2011, 21, 3431−3434.(14) Kariya, Y.; Kubota, T.; Jane Fromont, J.; Kobayashi, J.Tetrahedron Lett. 2006, 47, 997−998.(15) Kariya, Y.; Kubota, T.; Fromont, J.; Kobayashi, J. Bioorg. Med.Chem. 2006, 14, 8415−8419.(16) http://www.schrodinger.com.(17) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci,B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H.P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima,T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery Jr., J. A.;Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin,K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.;Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega,N.; Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.;Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.;Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.;Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.;Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.;Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09,Revision A.2; Gaussian Inc.: Wallingford, CT, USA (http://www.gaussian.com).(18) Karplus, M. J. Am. Chem. Soc. 1963, 85, 2870−2871.(19) http://www.stenutz.eu/conf/jhh.html.(20) Agarwal, A.; Louise-May, S.; Thanassi, J. A.; Podos, S. D.;Cheng, J.; Thoma, C.; Liu, C.; Wiles, J. A.; Nelson, D. M.; Phadke, A.S.; Bradbury, B. J.; Deshpande, M. S.; Pucci, M. J. Bioorg. Med. Chem.Lett. 2007, 17, 2807−2810.(21) Maciag, M.; Kochanowska, M.; Lyzen, R.; Wegrzyn, G.;Szalewska-Palasz, A. Plasmids 2010, 63, 61−67.(22) Frick, D. N.; Richardson, C. C. Annu. Rev. Biochem. 2001, 70,39−80.(23) Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B 1988, 37, 785−789.(24) Becke, A. D. J. Chem. Phys. 1993, 98, 5648−5652.
Journal of Natural Products Article
dx.doi.org/10.1021/np300527p | J. Nat. Prod. 2013, 76, 142−149149
