Corallorazines from the Myxobacterium Corallococcus coralloides

Note pubs.acs.org/jnp

Corallorazines from the Myxobacterium Corallococcus coralloides Alexander Schmitz,†,‡ Stefan Kehraus,† Till F. Schab̈ erle,†,‡ Edith Neu,† Celso Almeida,§ Martin Roth,‡,⊥ and Gabriele M. König*,†,‡ †

Institute for Pharmaceutical Biology, University of Bonn, Nussallee 6, D-53115 Bonn, Germany German Centre for Infection Research (DZIF), partner sites Bonn-Cologne and Jena, Germany § Centro de Biodiversidade, Genómica Integrativa e Funcional (BioFIG), Faculdade de Ciências, Edifício ICAT, Campus da FCUL, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal ⊥ Leibniz Institute for Natural Product Research and Infection Biology e.V., Hans Knöll Institute, Adolf-Reichwein-Strasse 23, D-07745 Jena, Germany ‡

S Supporting Information *

ABSTRACT: The myxobacterium Corallococcus coralloides is the producer of the antibiotic compound corallopyronin A, which is currently in preclinical evaluation. To obtain suitable amounts of this antibiotic, the production strain C. coralloides B035 was cultured in large volumes, which in the addition to the isolation of the target molecule facilitates the detection of additional metabolites of this myxobacterial strain (corallorazines A−C). Corallorazine A is a new structural type of dipeptide composed of a dehydroalanine and a glycine moiety that are linked via a semiaminal bond, thus forming a piperazine ring. The latter is further connected via an amide bond to an unusual aliphatic acyl chain.

M

C15H22N2O3. IR data suggested the presence of a hydroxy (broad absorption at 3285 cm−1) and a carbonyl group (1709 cm−1). The 15 carbons of the molecule were attributed by 13C NMR and DEPT spectra to three methyl (CH3-1, CH3-2, and CH3-17), three methylene (CH2-4, CH2-14, and CH2-16), and six methine groups (CH-3, CH-5 to CH-8, and CH-15) as well as three quaternary carbons (C-9, C-11, and C-12). From 13C NMR data it was obvious that CH-15 (δ 72.4) is linked to a hydroxy group, whereas CH3-17 (δ 35.3) had to be connected to a nitrogen atom. The chemical shift of C-16 (δ 116.6) in the 13 C NMR spectrum and that of the corresponding protons (δ 6.33 and δ 5.41) in the 1H NMR spectrum indicated the presence of an exomethylene group. The downfield shift of the resonance signals for H-5 to H-8 (δ 5.95, 6.30, 6.49, and δ 7.72) implied two further carbon−carbon double bonds. With the help of COSY NMR data the molecule was deduced to have two 1H−1H spin systems. COSY correlations of H3-1 and H3-2 to H-3, and further to H-8, revealed that an isobutyryl moiety is linked to the two conjugated double bonds. This first 1H−1H NMR spin system thus comprised C-1 to C-8 as part of the molecule. The double bond Δ5,6 is Z-configured, as proven by a 1 H−1H coupling constant of J = 11.0 Hz and NOESY correlation between the resonance signal for proton H-5 with that for proton H-6, while Δ7,8 is E-configured as evidenced by a 1H−1H coupling constant of J = 15.0 Hz. The second 1H−1H spin system consists merely of CH2-14 and CH-15, which are

yxobacteria are a rich source of bioactive secondary metabolites.1−3 Corallopyronin A (5) (Figure 1), isolated from Corallococcus coralloides, is one of these fascinating myxobacterial natural products that was shown to have superior antibiotic activity.4 This compound is a specific inhibitor of bacterial DNA-dependent RNA-polymerase (RNAP), but shows no cross-resistance with rifampicin, another RNAP inhibitor, due to its unique binding site deep inside the RNAP clamp head domain. In addition to the antibacterial effect, in vivo antifilarial activity was found with an effective concentration in the range of doxycycline.5 In a time of increasing bacterial resistance against many established antibiotic classes, we initiated a preclinical evaluation of corallopyronin A. To obtain suitable amounts of corallopyronin A, we cultivated C. coralloides B035 in large volumes, which enabled us to isolate the target molecule and to identify additional metabolites, i.e., the corallorazines (1−4). Corallorazine A (1) (Figure 1) is a piperazine-containing dipeptide composed of a dehydroalanine and a glycine building block and further connected via an amide bond to an unusual aliphatic acyl chain. Interestingly the amino acids are linked via a semiaminal functionality giving rise to stereoisomers. During the purification of corallopyronin A from C. coralloides B035, a compound with a prominent UV maximum at λmax = 267 nm was detected, pointing toward an extended chromophore (Figures S1 and S2, Supporting Information). A 12 mg amount of the new compound 1, named corallorazine A (Figure 1), was isolated from a 40 L culture (0.3 mg L−1). An HRMS measurement gave a signal at m/z 301.1509 [M + Na]+, leading together with NMR data to a molecular formula of © 2014 American Chemical Society and American Society of Pharmacognosy

Received: September 10, 2013 Published: January 14, 2014 159

dx.doi.org/10.1021/np400740u | J. Nat. Prod. 2014, 77, 159−163

Journal of Natural Products

Note

Figure 1. Structures of corallorazine A, B, and C and corallopyronin A.

Figure 2. Origin of corallorazine A piperazine ring and conversion of 1-13C glycine into 1-13C serine. Black points illustrate the 13C-labeled carbon atoms.

linked as shown by a COSY correlation between the respective resonance signals. Heteronuclear couplings as detected in an 1H−13C HMBC spectrum allowed further delineation of the structure of corallorazine A. Thus, the correlation between H-8 and the quaternary C-9 gave evidence for the complete acyl moiety (C1 to C-9 part of the molecule). The HMBC correlation of H216 to C-11 (strong correlation) and C-12 (weak correlation) implied that the exomethylene group C-16 is directly connected with quaternary C-11, which is in the vicinity of the carbonyl group C-12 with a 13C NMR chemical shift of δ 161.8. 1H−15N HMBC correlations between both Hb-14 and Ha-16 and the amide nitrogen N-10 and from Hb-14, H-15, and H3-17 to N-13 as well as 1H−13C HMBC correlations of methyl protons H3-17 to C-14 and C-12 led to the identification of a piperazine ring. The acyl side chain had to be connected to this piperazine ring at nitrogen N-10, as indicated by the 13C NMR chemical shift of carbonyl carbon C-9 (δ 167.6). Thus, corallorazine A is a reduced and methylated diketopiperazine, connected with a rare acyl residue (iso-octadienoic acid) (Figure 1). Corallorazine A has one asymmetric carbon atom at position C-15. After failure of the modified Mosher’s method,6 it was concluded that the hemiaminal functionality was indeed unstable as was suspected (Figures S3 and S4, Supporting Information). A chiral HPLC separation on a cellulose column with 2-propanol−hexane finally proved this. The resulting HPLC chromatogram showed two peaks, one for each enantiomer (Figure S3, Supporting Information). The value of the specific optical rotation (−4.2°) indicated that the ratio

of enantiomers is not 50:50. Indeed, the HPLC chromatogram showed a ratio of 44:56. The structure of corallorazine A suggests that two amino acids, one of which should be glycine, form the heterocyclic ring. Thus, a feeding experiment with 1-13C-labeled glycine was performed. Carbons C-12 and C-15, resonating at δ 161.8 and 72.4, respectively, were significantly enhanced in the 13C NMR spectrum (Figure S11, Supporting Information). Enrichment of 1-13C glycine was 38.6% for C-15 and 34.9% for C-12, as judged from comparison of 13C NMR spectra of labeled and unlabeled compound (Table S2, Supporting Information). Labeling of C-15 was expected and provided proof for glycine incorporation. Enhancement of the 13C NMR signal for C-12 was surprising, because we expected that this carbon is derived from serine. However, due to the shunt in primary metabolism between serine and glycine mediated by serine-hydroxymethyltransferase, glycine is converted into serine within only one biosynthetic step (Figure 2). The experiment with 1-13C glycine is consequently an indirect proof of serine incorporation. The exomethylene group presumably arises by dehydration of serine. Corallorazine B is a very minor metabolite of C. coralloides B035 and was detected in the same VLC fraction as 1 (Figures S1 and S2, Supporting Information), but only present at 0.0016 mg L−1. Thus, from a 120 L culture only 200 μg was isolated. Analysis of the NMR data showed that the structure of corallorazine B contains the same iso-octadienoic acid moiety as corallorazine A. Resonance signals in the 1H NMR spectrum at δ 5.92 and 6.41 arose from the exomethylene group CH2-12. 160



Note

However, in the 1H NMR spectrum of corallorazine B resonance signals for H2-14 and H-15, as well as a resonance signal for a N-methyl group, were missing. Instead a resonance signal at δ 3.87 indicated a methoxy function. The latter was confirmed by 13C NMR data, with a corresponding signal at δ 53.2. Proton signals of the exomethylene group CH2-12 correlated in the HMBC with the resonance signal for the quaternary carbons C-11 and C-13. The latter is a carbonyl group resonating at δ 165.6, while C-11 (δ 133.9) is connected to the exomethylene group. C-15, the O-methyl group, is part of the carbonic acid function (C-13) as deduced from HMBC correlations from H3-15 to C-13. Thus, the acyl chain (C-1 to C-9) is linked to a dehydroalanine via the nitrogen N-10, while the methyl group CH3-15 is connected to the carboxylic function of the dehydroalanine moiety. Corallorazine B (Figure 1) could be a biosynthetic intermediate of corallorazine A, even though we propose acylation as a late step in the biosynthesis (Figure 4). The presence of the O-methyl group suggests that it is not a degradation product of corallorazine A.

isolated compound was extremely similar to that of corallorazine B, and the main difference was the lack of a resonance signals for an O-methyl group. Even though signals for the quaternary carbons C-9, C-11, and C-13 were not detectable due to the low amounts of compound, all other spectroscopic data supported compound 3 having the structure as depicted in Figure 1, containing a carboxylic acid function instead of a methyl ester as in 2. The free acyl residue iso-octadienecarbonic acid of corallorazines A, B, and C (Figure 1) was also obtained when C. coralloides was fermented under continuous control at pH 7.4. The crude extract was fractionated according to the scheme in Figure S1 (Supporting Information). During this experiment no corallopyronin A was detected, but a peak for an unknown compound with resonances between δ 6.0 and 7.0 in the 1H NMR spectrum was found in VLC fraction 6. Purification via HPLC using UV detection yielded a single compound (4.1 mg, 0.6 mg L−1). 1H and 13C NMR data (Table S5, Supporting Information) of the compound led to the conclusion that its structure is that of the acyl side chain of the corallorazines. ESIMS measurement yielded a signal at m/z 154 [M + H]+ for C9H15O2 and thus confirmed the molecule to be (2E,4Z)-isoocta-2,4-dienoic acid (4). Antimicrobial activity of corallorazine A (1) was tested, but it was found to be inactive in agar diffusion assays against bacteria and fungi. Also no affinity to cannabinoid receptors and no inhibition of human leucocyte elastase were detected. The major metabolite of C. coralloides B035 is corallopyronin A.7 Scaled-up cultivation of the myxobacterium in order to obtain sufficient material for the development of the antibiotic corallopyronin A resulted in the identification of the corallorazines. The most intriguing compound of this series is corallorazine A (Figure 1), a cyclic dipeptide connected to an aliphatic acyl residue. According to our literature searches, this is a new group of natural products. Some structural resemblance of corallorazine A (1) to diketopiperazines has to be acknowledged, and a closely related synthetic compound that is described as a side product of phenylahistin synthesis has been reported.8 The structure of this synthetic product (Figure 3) has a dipeptide core, which in corallorazine A is reduced to create an aminal functional group. Also, the acyl chain being bound to one of the ring nitrogens is

Figure 3. Structures of phenylahistin (A), a side product of its synthesis (B), as well as cyrmenin A (C).

One of the more polar fractions of the C. coralloides extract yielded 250 μg of corallorazine C (0.003%) (Figure 1). HRMS data displayed a signal for a sodium adduct (m/z 246.1101 [M + Na]+) and established the molecular formula as C12H17NO3. The signal pattern in the 1H and 13C NMR spectra of the

Figure 4. Proposed biosynthesis of corallorazine A. 161



Note

missing in the synthesized material.9 Cyrmenin A (Figure 3), a myxobacterial metabolite isolated from Cystobacter armeniaca strain Cb a24,10 also shares some structural features with corallorazine A. Cyrmenin A, like corallorazine A, is a dipeptide linked via an amide bond to a lipophilic chain. One of the amino acids is dehydroalanine as found in corallorazine A, and the second amino acid is an O-methylated dihydroserine. Further differences concern the acyclic nature of the dipeptide core of cyrmenin A. During the current study the biosynthetic origin of corallorazine A was investigated. On the basis of these studies we assume that a nonribosomal peptide synthetase assembles and connects the amino acids glycine and serine. Reductive release from the biosynthetic enzyme would give rise to an aminal. N-Methylation and dehydration are then responsible for the modification of the cyclic core structure, to which at some stage the branched carbonic acid is transferred (Figure 4). The latter may result from isovaleryl-CoA, which is extended with two units of malonyl-CoA. Formerly it was described that myxobacteria are able to form isovaleryl-CoA from leucine through a transaminase reaction or from 3-hydroxy-3methylglutaric-acid-CoA similar to isoprenoid biosynthesis.11

■

(Macherey-Nagel, Dü ren, Germany)) (Figure S1, Supporting Information). For the fractionation of the ethyl actetate extract a 6.5 cm diameter column was used. The sorbent was poured as a methanolic suspension into the column and compressed under vacuum. Afterward the system was equilibrated with 100 mL of methanol−water (20:80). The sample was dissolved in a small volume of the same solvent mixture and applied onto the top of the column. Nine fractions were obtained through elution with nine different methanol−water mixtures beginning with methanol−water in a ratio of 20:80. In each step the methanol content was increased by 10%, up to 100% methanol for the ninth fraction. It was eluted with 100 mL of solvent for each fraction, except for fraction 9 (100% methanol), where 200 mL was used. VLC fractions 4, eluting with 50% methanol (containing corallorazine C), and 6, eluting with 70% methanol (containing corallorazines A and B and corallopyronin A), were further purified via HPLC (Figures S1 and S2, Supporting Information). Corallorazines A and B (1, 2) were isolated from VLC fraction 6 using the Merck-HPLC system. A Knauer Eurospher 100 RP18 column (5 μm, 250 × 8 mm) was used with methanol−water (70:30) as solvent and a flow of 1.5 mL min−1. Using these conditions a peak was detected at 17.5 min with a prominent UV absorption maximum at λmax = 267 nm (Figure S2, Supporting Information). This peak was identified as corallorazine A (1), of which 12 mg was isolated from a 40 L culture (0.3 mg L−1). Corallorazine B (2) was isolated from the same VLC fraction 6 using the same HPLC system and conditions. The retention time for 2 was 24.5 min (Figure S2, Supporting Information). From a 120 L culture 200 μg of corallorazine B was obtained (0.0016 mg L−1). Corallorazine C (3) was found in VLC fraction 4 (Figure S1, Supporting Information) of the C. coralloides B035 extract. The fraction was purified using the Agilent HPLC system with a Knauer Eurospher 100 RP18 column (5 μm, 250 × 8 mm) and methanol−water (35:65) as solvent (flow of 1.5 mL min−1). Corallorazine C eluted at 14.5 min from the HPLC column. For the isolation of iso-octadienoic acid (4) C. coralloides B035 was alternatively cultivated in a Labfors 3 fermenter (Infors) containing 7 L of MD1 + G medium. The fermenter was inoculated with seven precultures and subsequently cultivated for 12 days at 30 °C and 150 rpm. The pH was adjusted at 7.4 using 2 M citric acid. The workup was as described above. A 3.5 g amount of crude acetone extract and 2.2 g of ethylacetate extract were obtained from this 7 L culture. After VLC fractionation, the 1H NMR spectrum of VLC fraction 6 was clearly different from that of the equivalent fraction when flask cultivation was employed. VLC fraction 6 of the fermenter cultivation was thus further purified using the Merck HPLC system with a Waters Xterra RP18 analytical column (250 mm × 4.6 mm, 5 μm) and methanol−water (48:52) as eluent. Using a flow of 0.9 mL min−1 the compound eluted at 11.0 min from the column. Corallorazine A (1): yellow oil (12.0 mg, 0.3 mg/L); [α]20D −4.2 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 267 (3.86) nm; IR (ATR) νmax 3285, 2955, 2860, 1709, 1651, 1613, 1503, 1409, 1277, 1090, 997, 969, 867, 804, 644, 607 cm−1; 1H and 13C NMR (Table1); HRESIMS m/z 301.1509 [M + Na]+ (calcd for C15H22N2O3Na, 301.1528). Corallorazine B (2): yellow oil (200 μg, 0.0016 mg/L); UV (MeOH) λmax (log ε) 280 (4.15) nm; IR (ATR) νmax 2923, 1722, 1666, 1631, 1514, 1337, 1192, 1088 cm−1; 1H NMR (MeOH-d4, 300 MHz) δ 7.62 (1H, ddd, J = 1.1, 11.6, 15.0, H-7); 6.41 (1H, s, Ha-12); 6.30 (1H, t, J = 11.6, H-6); 6.27 (1H, d, J = 15.0, H-8); 5.96 (1H, m, H-5); 5.92 (1H, s, Hb-12); 3.87 (3H, s, H3-15); 2.26 (2H, t, J = 7.5, H4); 1.75 (1H, m, H-3); 0.99 (6H, d, J = 6.9, H3-1, H3-2); 13C NMR (MeOH-d4, 75 MHz) δ 167.7 (C, C-9); 165.6 (C, C-13); 140.9 (CH, C-5); 138.5 (CH, C-7); 133.9 (C, C-11); 128.4 (CH, C-6); 124.7 (CH, C-8); 111.2 (CH2, C-12); 53.2 (CH3, C-15); 38.1 (CH2, C-4); 29.9 (CH, C-3); 22.7 (CH3, C-1, C-2); HRESIMS m/z 260.1257 [M + Na]+ (calcd for C13H19NO3Na, 260.1263). Corallorazine C (3): yellow oil (250 μg, 0.003 mg/L); UV (MeOH) λmax (log ε) 280 (4.15) nm; IR (film) νmax 2923, 1722, 1666, 1631, 1514, 1337, 1192, 1088 cm−1; 1H NMR (MeOH-d4, 300 MHz) δ 7.60 (1H, ddd, J = 1.1, 11.6, 15.0, H-7); 6.29 (1H, t, J = 11.6, H-6); 6.28 (1H, s, Ha-12); 6.20 (1H, d, J = 15.0, H-8); 5.92 (1H, dt, J = 11.6, 8.1, H-5); 5.74 (1H, s, Hb-12); 2.27 (2H, td, J = 8.1, 1.5, H-4); 1.73 (1H,

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a Jasco DIP 140 polarimeter. UV and IR spectra were obtained employing Perkin-Elmer Lambda 40 and Perkin-Elmer Spectrum BX instruments, respectively. 1H, 13C, COSY, HSQC, and HMBC NMR spectra were recorded in MeOH-d4 using a Bruker Avance 300 DPX spectrometer operating at 300 MHz for proton and at 75 MHz for 13C, respectively. Spectra were calibrated to residual solvent signals with resonances at δH/C 3.35/49.0 (MeOH-d4). The 1 H−15N HMBC spectrum was measured at a Bruker Avance 500 DRX spectrometer and was referenced externally to urea (15N chemical shift is reported relative to liquid ammonia). HPLC was performed either on a Merck-Hitachi system equipped with an L-6200A pump, an L4500 A photodiode array detector, a D-6000A interface with D-7000 HSM software, and a Rheodyne 7725i injection system or on an Agilent system 1200 with a G1322 degasser, a G1311A quaternary pump, and a G1315B DAD detector and Chemstation software. LRESIMS measurements were performed employing an API 2000 triple quadrupole LC/MS/MS (Applied Biosystems/MDS Sciex) with ESI source. HRESIMS were recorded on a Bruker Daltonik micrOTOF-Q time-of-flight mass spectrometer with ESI source. Cultivation, Extraction, and Isolation. Corallococcus coralloides B035 was isolated by one of the authors (E.N.) from a soil sample from Remonchamps, Belgium, 2001. The strain is stored as a cryoculture at −80 °C. For the isolation of corallopyronin A and corallorazines A−C (1−3) the myxobacterium was cultivated in 5 L Erlenmeyer flasks with 1.5 L of MD1 + G medium (3.0 g L−1 casitone, 0.7 g L−1 CaCl2·2H2O, 2.0 g L−1 MgSO4·7H2O, and 2.2 g L−1 glucose· H2O). The adsorber resin Amberlite XAD 16 was added in a concentration of 2% to each flask to avoid end product inhibition. For a 40 L cultivation 28 flasks were prepared. The flasks were inoculated with precultures (300 mL flasks each with 100 mL of MD1 + G medium) and were cultivated for seven days at 30 °C and 140 rpm on an HT Orbitron rotary shaker (Infors, Bottmingen, Switzerland) under light-free conditions. Cells and Amberlite XAD 16 were separated from the medium by filtration with a glass filter size 2. Cells and Amberlite XAD 16 were further processed. For desorption of the compounds the adsorber resin (including the bacterial pellet) was placed into a size 2 glass filter and extracted with 6 × 500 mL of acetone (for a 40 L culture). The organic phase was evaporated to result in approximately 25 g of extract, which was then dissolved in 250 mL of water and extracted with 5 × 250 mL of ethyl acetate. The ethylacetate extract (approximately 4 g) was evaporated and fractionated with RP18-VLC (Polygoprep 60-50 RP18 silica gel 162



■

Table 1. NMR Spectroscopic Data of Corallorazine A (1) in MeOH-d4 (1H, 300 MHz; 13C, 75 MHz) pos.

δC/N, mult

1 2 3 4 5 6 7

22.7, 22.7, 29.9, 38.2, 141.1, 128.6, 139.9,

CH3 CH3 CH CH2 CH CH CH

8 9 10 11 12 13 14

122.9, 167.6, 149.0, 136.3, 161.8, 100.5, 55.0,

CH C N C C N CH2

15 16

72.4,CH 116.6, CH2

17

35.3, CH3

COSY

HMBC (H to C/ N)

0.99, d (6.8) 0.99, d (6.8) 1.76, m 2.27, td (7.9, 1.0) 5.95, dt (11.0, 7.9) 6.30, br t (11.3) 7.72, ddd (1.0, 11.6, 15.0) 6.49, d (15.0)

3 3 1, 3, 4, 5, 6,

3, 3, 1, 1, 4, 4, 5,

7

6, 9

a: 3.81, dd (3.1, 13.2) b: 3.45, dd (1.1, 13.2) 6.17, dd (1.1, 3.1) a: 6.33, s b: 5.41, s 3.07, s

14b, 15 14a, 15 14a/b

15, 17 15, 17, N-10/13 N-13 12, N-10 11, 12 12, 14, N-13

δH (J in Hz)

2, 4 5 6 7 8

4 4 2, 2, 6, 7, 6,

Note

ASSOCIATED CONTENT

S Supporting Information *

Spectroscopic data and other relevant information are included for the new compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

■

4, 5 3, 5, 6 7 8 8, 9

AUTHOR INFORMATION

Corresponding Author

*Tel: +49228733747. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors wish to thank M. Gütschow and S. Hautmann for performing the enzyme assays, and C. E. Müller and co-workers for the cannabinoid receptor tests. Financial support came from the Deutsche Forschungsgemeinschaft (FOR 854).

■

REFERENCES

(1) Gerth, K.; Pradella, S.; Perlova, O.; Beyer, S.; Müller, R. J. Biotechnol. 2003, 106, 233−253. (2) Ohlendorf, B.; Leyers, S.; Krick, A.; Kehraus, S.; Wiese, M.; König, G. M. ChemBioChem 2008, 9, 2997−3003. (3) Felder, S.; Dreisigacker, S.; Kehraus, S.; Neu, E.; Bierbaum, G.; Wright, P. R.; Menche, D.; Schäberle, T. F.; König, G. M. Chem.Eur. J. 2013, 19, 9319−9324. (4) Erol, Ö .; Schäberle, T. F.; Schmitz, A.; Rachid, S.; Gurgui, C.; El Omari, M.; Lohr, F.; Kehraus, S.; Piel, J.; Müller, R.; König, G. M. ChemBioChem 2010, 11, 1253−1265. (5) Schiefer, A.; Schmitz, A.; Schäberle, T. F.; Specht, S.; Lämmer, C.; Johnston, K. L.; Vassylyev, D. G.; König, G. M.; Hörauf, A.; Pfarr, K. J. Inf. Dis. 2012, 206, 249−257. (6) Seco, J. M.; Quinoa, E.; Riguera, R. Chem. Rev. 2004, 104, 17− 117. (7) Jansen, R.; Irschik, H.; Reichenbach, H.; Höfle, G. Liebigs Ann. Chem. 1985, 822−836. (8) Kanoh, K.; Kohno, S.; Katada, J.; Takahashi, J.; Uno, I. J. Antibiot. 1999, 52, 134−141. (9) Chai, C. L. L.; Elix, J. A.; Huleatt, P. B. Tetrahedron 2005, 61, 8722−8739. (10) Leibold, T.; Sasse, F.; Reichenbach, H.; Höfle, G. Eur. J. Org. Chem. 2004, 431−435. (11) Bode, H. B.; Ring, M. W.; Schwär, G.; Kroppenstedt, R. M.; Kaiser, D.; Müller, R. J. Bacteriol. 2006, 188, 6524−6528. (12) Schulz, B.; Boyle, C.; Draeger, S.; Rommert, A. K.; Krohn, K. Mycol. Res. 2002, 106, 996−1004. (13) Schulz, B.; Sucker, J.; Aust, H.-J.; Krohn, K.; Ludewig, K.; Jones, P. G.; Döring, D. Mycol. Res. 1995, 99, 1007−1015. (14) Elsebai, M. F.; Rempel, V.; Schnakenburg, G.; Kehraus, S.; Müller, C. E.; König, G. M. ACS Med. Chem. Lett. 2011, 2, 866−869. (15) Elsebai, M. F.; Kehraus, S.; Lindequist, U.; Sasse, F.; Shaaban, S.; Gütschow, M.; Josten, M.; Sahl, H.-G.; König, G. M. Org. Biomol. Chem. 2011, 9, 802−808.

m, H-3); 0.99 (6H, d, J = 6.6, H3-1, H3-2); 13C NMR (MeOH-d4, 75 MHz) 140.2 (CH, C-5); 137.5 (CH, C-7); 128.5 (CH, C-6); 125.8 (CH, C-8); 104.7 (CH2, C-12); 38.1 (CH2, C-4); 29.9 (CH, C-3); 22.6 (CH3, C-1, C-2); C-9, C-11, and C-13 were not detectable; HRESIMS m/z 246.1101 [M + Na]+ (calcd for C12H17NO3Na, 246.1106). (2E,4Z)-Iso-octa-2,4-dienoic acid (4): yellow oil (4.1 mg, 0.6 mg/ L); UV (MeOH) λmax (log ε) 260 nm (3.58); IR (film) νmax 3193, 2955, 1660, 1599, 1415, 1381, 996, 869, 649 cm−1; 1H NMR (MeOHd4, 300 MHz) δ 7.58 (1H, ddd, J = 1.1, 11.0, 15.0, H-7); 6.24 (1H, t, J = 11.0, H-6); 6.05 (1H, d, J = 15.0, H-8); 5.92 (1H, dt, J = 11.0, 8.1, H-5); 2.25 (2H, td, J = 8.1, 1.5, H-4); 1.75 (1H, m, H-3); 0.98 (6H, d, J = 6.6, H3-1, H3-2); 13C NMR (MeOH-d4, 75 MHz) 171.5 (C, C-9); 140.2 (CH, C-5); 137.8 (CH, C-7); 128.4 (CH, C-6); 124.5 (CH, C8); 38.1 (CH2, C-4); 29.9 (CH, C-3); 22.7 (CH3, C-1, C-2); ESIMS m/z 154.2 [M + H]+. Bioassays. Corallorazine A was tested in antibacterial (Escherichia coli DSM 498, Bacillus megaterium DSM 32), antifungal (Mycotypha microspora, Eurotium rubrum DSM62631, Microbotryum violaceum), and antialgal (Chlorella f usca) assays.12,13 It showed no activity up to a concentration of 1 mg mL−1. Corallorazine A was also tested at cannabinoid receptors CB1 and CB2.14 The compound showed no affinity to the receptors at a concentration of 10 μg mL−1. Finally, corallorazine A was tested for inhibitory activity against several proteases including human leucocyte elastase, chymotrypsin, trypsin, human thrombin, cathepsin S, cathepsin L, and cathepsin B.15 The compound was inactive in concentrations up to 10 μg mL−1. Chiral HPLC. Investigation of interconversion of corallorazine A stereoisomers was done using the Merck HPLC system with a Phenomenex Cellulose 3 column (250 mm × 4.6 mm, 5 μm). The solvent 2-propanol−hexane (10:90) was used with a flow rate of 1.0 mL min−1. Using these conditions the enantiomers of corallorazine A had retention times of 14.5 and 22.4 min, respectively (Figure S3). Labeling Experiment. A 500 mg amount 1-13C-labeled glycine (Cambridge Isotope Laboratories (Andover, USA)) dissolved in water was added to two 1.5 L cultures of C. coralloides B035, two days after inoculation. Corallorazine A (1) was isolated as described above. 163


Corallorazines from the Myxobacterium Corallococcus coralloides

Recommend Documents