Diversity analysis on secondary metabolites of Antarctic microbes, Rhodococcus sp. NJ-008 and Pseudomonas sp. NJ-011, together with the structural elucidation of some purified compounds, has been carried out for understanding of their chemical constituents. The methanol extracts of Rhodococcus sp. NJ-008 and Pseudomonas sp. NJ-011 were subjected to HPLC-TOF MS test for diversity analysis on secondary metabolites, respectively. The chemical constituents of NJ-011 are mainly N-containing compounds including some alkaloids and short polypeptides, while those of NJ-008 are not N-containing ones. Three compounds were also isolated and identified from extract of NJ011 by different column chromatography and preparative HPLC, and their structures were elucidated as b-carboline (1), 3-benzylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione (2) and 3-isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione (3) by comparison of TOF MS, 1Hand 13C-NMR data with those reported. More microbial material of Pseudomonas sp. NJ-011 should be needed for exploration of the minor constituents with complicated structures.
Marine microbes from Antarctic area were well-known as bioactive compounds producers. In the past few years, some Chinese scientists have done fine chemical investigation from Antarctic fungui and led to the isolation of many interesting secondary metabolites, including new structures of two epipolythiodioxopiperazines and five diketopiperazines [
All common solvents used were of anal. grade (Shanghai Chemical Plant). Solvents for HPLC and HPLC-TOF MS: Methanol and acetonitrile (Tedia, USA). Thin-layer chromatography (TLC): pre-coated silica gel GF254 plates (Qingdao Haiyang Chemical Co. Ltd.). Column chromatography (CC): HP-20 macroporos resin (Mitsu- bishi Chemical Industries Co., Ltd.) and Sephadex LH-20 (Pharmacia Biotech, Sweden). Semi-preparative HPLC was performed on a Shimadazu LC-20AT pump equipped with a Shimadazu SPD-M20A PDA detector and a YMC-Pack ODS-AQ column (250 ´ 10 mm, S-5 mm, 12 nm), flow rate: 2.5 mL×min−1. NMR spectra were recorded on a Bruker AM-400 spectrometer; d in ppm rel. to Me4Si, J in Hz. HR-ESI-MS were carried out on a Agilent 1290 HPLC-6224 TOF MS instrument; in m/z (rel. %).
Rhodococcus sp. NJ-008 and Pseudomonas sp. NJ-011 were isolated from the Antarctic krill Euphausia superba, collected in Jan 2010 in FAO 48.1 area, and were identified by Dr. Yang Qiao. The voucher specimens (No. DHS-NJ-008 and DHS-NJ-011) were deposited in East China Sea Fisheries Research Institute, Chinese Acade- my of Fishery Sciences.
Rhodococcus sp. NJ-008 in 2.0 L R2A liquid medium was shared with three 2000 mL Erlenmeyer flasks, and the flasks were incubated on a rotary shaker at 150 rpm for 7 d (28˚C). Pseudomonas sp. NJ-011 in 2.0 L GAUZE’s Medium NO.1 also in three 2000 mL flasks was incubated on the same rotary shaker as one batch culture.
Pseudomonas sp. NJ-011 in 8.0 L GAUZE’s Medium NO.1 in twelve 2000 mL flasks was incubated on the same rotary shaker at 150 rpm for 7 d (28˚C) as another batch culture.
The culture broth (2 L) of Rhodococcus sp. NJ-008 (or Pseudomonas sp. NJ-011) was centrifuged at 4000 rpm. The bacterial cells were frozen at −78˚C for 3 h, and then were extracted with 100 mL acetone and 100 mL me- thanol, successively. The organic solutions were concentrated under reduced pressure to afford a residue. The supernatant was evaporated to a salty dryness in vacuum. And these two parts were combined in 100 mL me- thanol, taking 1.0 mL supernatant for HPLC-TOF MS tests.
The culture broth (8 L) of Pseudomonas sp. NJ-011was extracted with ethyl acetate (1500 mL) for three times, and the mixture was treated with ultrasonic cleaner for 30 min each time. The organic solutions were combined and concentrated under reduced pressure to afford a residue (1.4 g).
HPLC column: YMC ODS-AA12S03-L546WT (4.6 ´ 75 mm, S-5 μm, 12 nm); injection volume: 10 mL; flow rate: 0.5 mL×min−1; mobile phase (CH3CN in H2O containing 0.1% HCOOH): 0 - 1.2 min 5%, 14 min 95%, 17 min 95%, 17.5 min 5%, 20 min 5%; TOF MS: positive ion mode ESI; gas temperature 340˚C, N2 8.0 L×min−1, nebulizer 40 psi. After test, the data of ion peaks with volume > 100,000 were extracted in TIC scan. Possible formula for each peak and the calculated exact mass for [M + H]+ were given by DNP database and ChemBio Draw Ultra version 12.0, respectively.
Total extract (1.3 g) from 8 L culture broth of Pseudomonas sp. NJ-011 was absorbed by HP-20 macroporos re- sin (600 mL), and eluted with H2O (1500 mL) and methanol/H2O (10:90, 1500 mL) to remove some salts, sugars, amino acids and proteins, and then eluted with methanol/H2O (50:50 ® 80:20 ® 100:0, 2500 mL each)to afford three sections NJ-011-A (0.5 g), NJ-011-B (0.1 g) and NJ-011-C (0.4 g), successively. The section NJ-011-A was passed through a Sephadex LH-20 column, eluted with ethanol/H2O (70:30), to yield three fractions A1 to A3. Finally, A1 were purified by repeated semi-preparative HPLC with mobile phase of CH3CN/H2O-0.1%TFA (25:75 ® 70:30, 2.5 mL/min), and three compounds 1 (7.6 mg), 3 (2.3 mg), and 2 (3.5 mg) were obtained.
b-carboline (1): positive ion mode ESI m/z: 169.0758 [M + H]+; 1H-NMR (CD3OD, 400 MHz) d: 8.80 (1H, br s), 8.28 (1H, br s), 8.20 (1H, dt, J = 1.0, 8.0 Hz), 8.10 (1H, br d, J = 5.5 Hz), 7.57 (1H, m), 7.56 (1H, m), 7.27 (1H, ddd, J = 3.1, 5.1, 8.0 Hz); 13C-NMR (CD3OD, 100 MHz) d: 143.5, 138.8, 134.6, 130.9, 130.3, 123.3 (C ´ 2), 122.7, 121.4, 116.7, 113.3.
3-benzylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione (2): positive ion mode ESI m/z: 245.1283 [M + H]+; 1H-NMR (CD3OD, 400 MHz) d: 7.28 (5H, m), 4.47 (1H, s), 4.08 (1H, m), 3.55 (1H, m), 3.39 (1H, m), 3.20 (2H, m), 2.11 (1H, m), 1.82 (2H, m), 1.34 (1H, m); 13C-NMR (CD3OD, 100 MHz) d: 169.9, 166.0, 135.3, 129.9 (2C), 128.3 (2C), 127.1, 58.4, 57.7, 44.8, 39.6, 28.4, 21.1.
3-isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione (3): positive ion mode ESI m/z: 211.1438 [M + H]+; 1H-NMR (CD3OD, 400 MHz) d: 4.27 (1H, m), 4.14 (1H, m), 3.54 (2H, m), 2.63 (2H, m), 2.32 (1H, m), 2.02 (1H, m), 1.92 (1H, m), 1.67 (1H, m), 1.54 (1H, m), 0.99 (3H, d, J = 6.4 Hz), 0.98 (3H, d, J = 6.3 Hz); 13C-NMR (CD3OD, 100 MHz) d: 171.4, 167.5, 58.8, 53.2, 45.0, 37.9, 27.7, 24.3, 22.2, 21.9, 20.8.
By high-resolution HPLC-TOF MS spectra (
The methanol extract solution of Pseudomonas sp. NJ-011 was also subjected to HPLC-TOF MS test. Among the total 53 observed peaks (
Three compounds were obtained from the high polarity extract section of Pseudomonas sp. NJ-011, by different column chromatography and repeated preparative HPLC, and their structures were elucidated by comparison of
. LC-TOF MS data of the NJ-008 methanol extract.
Cpd | Possible formula | Cacld. for [M + H]+ (m/z) | Base peak (m/z) | RT (min) | Vol% |
---|---|---|---|---|---|
1 | -a | 132.04055 | 1.677 | 1.25 | |
2 | C4H6O2S3 | 182.9603 | 182.96213 | 1.679 | 1.81 |
3 | - | 223.98907 | 1.681 | 1.55 | |
4 | C8H6O3 | 151.0390 | 151.03505 | 1.684 | 1.9 |
5 | - | 265.0155 | 1.686 | 1.01 | |
6 | C26H42N4O4 | 475.3279 | 475.32547 | 5.984 | 0.46 |
7 | C20H36O4 | 341.2686 | 341.26267 | 6.665 | 9.83 |
8 | C26H47NO5 | 454.3527 | 454.34701 | 7.044 | 4.62 |
9 | C29H44N2O2 | 453.3476 | 453.3437 | 7.279 | 4.47 |
10 | C30H55N5O5 | 566.4276 | 566.42768 | 7.298 | 1.01 |
11 | C9H18N2O | 171.1492 | 171.14896 | 9.521 | 4.47 |
12 | C26H43NO6 | 466.3163 | 466.31663 | 10.182 | 0.28 |
13 | - | 199.18021 | 11.461 | 3.18 | |
14 | C13H24N2O | 225.1961 | 225.19591 | 12.309 | 0.99 |
15 | C19H22O | 267.1743 | 267.17183 | 15.316 | 0.45 |
16 | C19H26O6 or C15H30N2O3S2 | 351.1802 351.1771 | 351.17785 | 15.513 | 0.18 |
17 | C20H34N2O6 or C25H34O4 | 399.2490 399.2530 | 399.25088 | 15.84 | 0.45 |
18 | C18H35NO | 282.2791 | 282.27894 | 15.985 | 0.79 |
19 | plasticizerb | 301.14378 | 16.339 | 0.67 | |
20 | C23H38O4 | 379.2843 | 379.28203 | 16.381 | 3 |
21 | C18H37NO | 284.2948 | 284.29486 | 16.554 | 8.57 |
22 | - | 344.27943 | 16.716 | 1.03 | |
23 | C21H32O4 | 349.2373 | 349.23495 | 16.735 | 0.83 |
24 | C24H38O4 | 391.2843 | 391.2845 | 17.96 | 4.64 |
25 | - | 280.26352 | 18.319 | 0.48 | |
26 | C24H40O4 | 393.2999 | 393.29749 | 18.433 | 11.2 |
27 | C6H10O4 | 147.0652 | 147.06501 | 18.459 | 3.48 |
28 | C22H42O4 | 371.3156 | 371.31557 | 18.461 | 9.28 |
29 | C6H8O3 | 129.0546 | 129.05452 | 18.462 | 6.66 |
30 | C14H26O4 | 259.1904 | 259.19034 | 18.472 | 4.25 |
31 | - | 402.35806 | 18.479 | 1.03 | |
32 | C46H82O8 | 763.6082 | 763.6059 | 18.521 | 6.2 |
aNo possible formula given; bIon peak of plasticizer dibutyl phthalate [M + Na]+.
. LC-TOF MS data of the NJ-011 methanol extract.
Cpd | Possible formula | Cacld. for [M + H]+ (m/z) | Base peak (m/z) | RT (min) | Vol% |
---|---|---|---|---|---|
1 | C10H16N2O2 | 197.1285 | 197.1282 | 6.377 | 0.88 |
2 | C23H33NO | 340.2635 | 340.25938 | 6.596 | 16.15 |
3 | C28H45NO7 | 508.3269 | 508.32833 | 6.613 | 0.33 |
4 | C11H8N2 | 169.0760 | 169.07582 | 6.615 | 3.12 |
5 | - | 453.54518 | 7.025 | 0.36 | |
6 | C29H44N2O2 | 453.3476 | 453.34386 | 7.025 | 7.45 |
7 | C29H44N2O2 | 453.3476 | 453.34377 | 7.261 | 6.12 |
8 | C30H55N5O5 | 566.4276 | 566.42761 | 7.287 | 1.96 |
9 | C11H18N2O2 | 211.1441 | 211.14378 | 7.465 | 0.63 |
10 | C14H16N2O2 | 245.1285 | 245.12826 | 7.747 | 0.77 |
11 | C9H18N2O | 171.1492 | 171.14889 | 9.449 | 7.17 |
12 | C31H49NO4 | 500.3734 | 500.37952 | 10.786 | 0.49 |
13 | C11H19NO | 182.1539 | 182.15379 | 11.277 | 0.91 |
14 | - | 199.1802 | 11.427 | 4.48 | |
15 | C13H24N2O | 225.1961 | 225.19595 | 12.289 | 1.75 |
16 | C9H19NO | 158.1539 | 158.15366 | 12.398 | 0.87 |
17 | - | 337.28505 | 12.648 | 1.13 | |
18 | C12H25NO | 200.2009 | 200.20057 | 14.672 | 1.57 |
19 | C18H33NO | 280.2635 | 280.26367 | 14.954 | 0.77 |
20 | C39H69N5O9 | 752.5168 | 752.51521 | 15.252 | 0.17 |
21 | C19H22O | 267.1743 | 267.17211 | 15.314 | 1.35 |
22 | C43H65NO7 | 708.4834 | 708.48923 | 15.355 | 0.37 |
23 | - | 664.46319 | 15.453 | 0.59 | |
24 | C17H28O6 | 329.1959 | 329.19595 | 15.5 | 0.34 |
25 | C16H24O5 | 297.1697 | 297.16966 | 15.505 | 0.47 |
26 | C16H33NO | 256.2635 | 256.26346 | 15.527 | 3.05 |
27 | - | 620.43719 | 15.555 | 0.73 | |
28 | C18H33NO2 | 296.2584 | 296.25842 | 15.591 | 3.05 |
29 | - | 576.41077 | 15.653 | 0.8 | |
30 | - | 532.38476 | 15.748 | 0.77 | |
31 | - | 488.35824 | 15.835 | 0.59 | |
32 | C20H34N2O6 | 399.2490 | 399.25077 | 15.835 | 0.87 |
33 | - | 444.33208 | 15.895 | 0.38 | |
34 | C18H35NO | 282.2791 | 282.27922 | 16.023 | 9.17 |
35 | C18H32O7 | 361.2221 | 361.22235 | 16.158 | 0.15 |
36 | plasticizera | 579.29763 | 16.374 | 6.77 | |
37 | C30H47NO3 | 470.3629 | 470.3638 | 16.912 | 0.59 |
38 | C15H32N4O4 | 333.2496 | 333.24722 | 17.503 | 0.3 |
39 | C11H10N2O3S | 251.0485 | 251.04837 | 17.614 | 1.87 |
40 | C18H30N4O8 | 431.2136 | 431.21082 | 17.615 | 1.01 |
41 | - | 385.15449 | 17.616 | 0.64 | |
42 | C25H34O8 | 463.2326 | 463.23608 | 17.617 | 0.35 |
43 | C23H32O7 | 421.2221 | 421.22551 | 17.618 | 0.55 |
44 | C24H30N4O | 391.2492 | 391.25225 | 17.845 | 0.56 |
45 | C30H40O3 | 449.3050 | 449.30534 | 17.846 | 0.48 |
46 | C31H38O3 or C24H43O6P | 459.2894 459.2870 | 459.28978 | 17.848 | 0.37 |
47 | C24H40O5 | 409.2949 | 409.29372 | 17.926 | 0.6 |
48 | C24H38O5 | 407.2792 | 407.2777 | 18.149 | 0.67 |
49 | C31H36 | 409.2890 | 409.29303 | 18.183 | 0.49 |
50 | C18H36N6O3 | 385.2922 | 385.29356 | 18.301 | 0.41 |
51 | C18H33NO | 280.2635 | 280.26444 | 18.349 | 3.75 |
52 | C20H36O4 | 341.2686 | 341.26662 | 18.355 | 0.21 |
53 | C24H38O5 | 407.2792 | 407.27805 | 18.4 | 0.59 |
aIon peak of plasticizer dibutyl phthalate [2M + Na]+.
HPLC-TOF MS, 1H- and 13C-NMR data with those reported. Their structures were identified as b-carboline (1) [
Diversity analysis on secondary metabolites by HPLC-TOF MS tests exhibited that the chemical constituents of Pseudomonas sp. NJ-011 were mainly N-containing compounds including some alkaloids and short polypep- tides, while those of Rhodococcus sp. NJ-008 were not N-containing ones. One alkaloid and two cyclic dipep- tides were also isolated and identified from extract of Pseudomonas sp. NJ-011, which confirmed the chemical diversity analysis. There have been more than 200 compounds reported from the Pseudomonas genus, but only a small part of them were from marine derived Pseudomonas spp. Some known cyclic dipeptides [
We thank financial support from the National High Technology Research and Development Program (No. 2012AA092105), Chinese Polar Environment Comprehensive Investigation & Assessment Programmes (CHINARE2014-01-06), and the Natural Science Foundation of Shanghai (11ZR1450000).