A) Shaker culture
4.11.1 Phenazine-1- carboxylic acid; Tubermycin B
The first fast moving fraction I showed a yellowish-green non polar band which turned to red by sulphuric acid. A yellowish-green middle polar amorphous com-pound 188 was isolated by further applying the fraction to PTLC and Sephadex
LH-20. The 1H NMR spectrum showed seven aromatic protons, the first four of them were exhibited as dd each of 1H at δ 8.92, 8.61, 8.52 and 8.41, and the residual three protons were shown as multiplet between δ 8.20-8.08. Hence, two aromatic 1,2-disubstituted and 1,2,3-trisubstituted rings could be derived. The displayed deep field shift pointed to their fusion with a hetero-aromatic ring.
The (+)-ESI mass spectrum of compound 188 fixed its molecular weight as 224 Dalton. The EI mass spectrum exhibited in addition to the molecular ion a base peak at m/z 180 resulting from the loss of a carboxy group, and the mass m/z 180 is indica-tive for the phenazine skeleton. A search in AntiBase led to phenazine-1-carboxylic acid (tubermycin B, 188). The compound was further confirmed by comparing the data with authentic spectra and the literature[234].
Tubermycin B (188) was isolated previously from microorganisms Pseudomo-nas., Streptomyces cinnamonensis, Streptomyces misakiensis and Actinomadura das-sonvillei. It exhibits a weak activity against Gram-positive bacteria, and a moderate activity against both Mycobacterium tuberculosis BCG and Mycobacterium tubercu-losis H37Rv (streptomycin resistant)[235].
N N
OH O
1 10
6 4 9
5
188 4.11.2 Surfactin C
Further separation of fraction II afforded a bulky zone of compound 189 which became brown after spraying with anisaldehyde/sulphuric and heating. Moreover, the compound showed a blue colour with the chlorine/o-anisidine reaction, however, was yellow with ninhydrin, as an indication of a cyclic or N-acylated peptide. The com-pound showed, however, no UV absorbance, referring to 189 as a non aromatic or olefinic compound which was isolated as a colourless solid by chromatography on silica gel and Sephadex LH-20. HPLC/MS analysis revealed it as a mixture of homo-logues.
NMR spectrum showed broad singlets of NH protons between δ 8.4-7.30 with an intensity of 6H and multiplets between δ 5.25-4.06 of 8H in α-position to nitrogen or oxygen. In the region between δ 2.88-1.15, it exhibited multiplets of methine and methylene protons with an intensity of 36 H. In addition, multiplets of 30H between δ 1.05-0.85 could be assigned as ten methyl groups. The 13C NMR spectrum dis-played 10 overlapped carbonyl signals between δ 178-169. Besides, 8 CH of oxy- and/or nitrogenous methines, which were displayed between δ 75-48. Overlapping methylene carbon signals (17 CH2) were observed between δ 42∼28, and possibly it showed two methine carbons between δ 27.32 and 24.22 of 2 isopropyl moieties. It exhibited numerous overlapped signals between δ 25∼12, probably due to 10 CH3
groups.
The ESI mass spectra of peptide 189 indicted a mixture of six homologues, the main of which showed quasi-molecular ions of [M+H]+, [M + Na]+ and [M-H]- at m/z 1036.7, 1058.8 and 1034.8, respectively. Therefore the molecular mass was es-tablished as 1036 Dalton.
By applying the above spectral data to AntiBase, 7 possible structures were re-vealed: Esperin, four surfactins C (189), daitocidin A1 and pumilacidin B. All are cyclic ß-hydroxyacyl peptides with 10 carbonyl groups, which fits well on the spec-tra and the TLC colour reactions. Pumilacidins were isolated in our research group previously by Huth[48]. They are described as a complex of antiviral antibiotics iso-lated from the culture broth of Bacillus pumilus[236]. All pumilacidins possessed no inhibitory effects against bacteria and fungi. Surfactins[237] (obtained from Bacillus subtilis natto) are most efficient biosurfactants, and exhibit potent antifungal, and antitumor activities against Ehrlich ascites carcinoma cells and inhibit fibrin clot formation, as well as cyclic adenosine 3',5'-monophosphate phosphodiesterase[238,239]. Both pumilacidins (A) and surfactins (B) are structurally closely related. They com-prise the same amino acids, but they differ in the sequence of the amino acids.
R CHCH2CO-Glu-Leu-Leu
O Val-Leu-Asp-Leu
R CHCH2CO-Glu-Leu-Leu O Leu-Leu-Asp-Val
A B
The structure of the peptide was elucidated by detailed MS studies. ESI MS2 and MS3 of 1036.70 [M+H]2+ showed sequential losses of amino acids, con-firming the sequence as Leu-Leu-Val-Asp-Leu-Leu (Figure 83). The observed intensive peak at m/z 685.3 in MS2 confirmed the cleavage of the lactone bond and the loss of an hydroxyacyl glutamic acid fragment. MS sequencing of m/z 685 as above gave again the amino acid sequence Leu-Leu-Val-Asp-Leu-Leu-OH (Table 8)[240], thus confirming the surfactin C sequence with Leu as the first C-terminal amino acid, which is involved in the lactone ring. The fragment ions at m/z 370.0 [FA-Glu ] (Table 8) and 339.4 [FA-Glu - CO]- (
Table 9) correspond to the β-hydroxy fatty acid (FA) linked to Glu. Addition-ally, most of the observed fragment ions were accompanied by the loss of CO and CO2, which is attributed to the presence of Glu and Asp in the peptide skeleton of 189.
In accordance, the whole sequence of the cyclic peptide directed to surfactin C (189), cyclo[FA-Glu-Leu-Leu-Val-Asp-Leu-Leu] (Figure 84). The alternative struc-ture of pumilacidine B (A) can be excluded. The strucstruc-ture of esperin (Figure 85) with its lactone between aspartic acid and the acyl residue was also excluded: MS2 of es-perin should show an intense peak at m/z 245, which was not observed.
a b
Figure 83: ESI MS2 (a) and ESI MS3 (b) fragmentation of 1036.7 [M+H]+ of surfac-tin C (189)
Table 8: Fragment ions of surfactin C (189) observed in ESI MS2 and MS3 of [M + H]+
MS2 Sequence of amino acids MS3 Sequence of amino acids 370 FA-Glu 198.8 Leu-Leu – CO
596.2 FA-Glu-Leu-Leu 709.3 [M+H – Leu-Val-Asp]
649.2 FA-Glu-Leu-Leu-Val - CO – H2O 937.4 [M+H – Val]+
667.3 FA-Glu-Leu-Leu-Val - CO 413.2 Val-Asp-Leu-Leu – CO 677.2 FA-Glu-Leu-Leu-Val – H2O 423.0 Val-Asp-Leu-Leu – H2O 695.2 FA-Glu-Leu-Leu-Val 441.1 Val-Asp-Leu-Leu 774.3 FA-Glu-Leu-Leu-Val-Asp – 2H2O 536.1 Leu-Val-Asp-Leu-Leu – H2O 792.2 FA-Glu-Leu-Leu-Val-Asp – H2O 554.2 Leu-Val-Asp-Leu-Leu 810.2 FA-Glu-Leu-Leu-Val-Asp 667.3 Leu-Leu-Val-Asp-Leu-Leu 877.3 FA-Glu-Leu-Leu-Val-Asp-Leu - H2O
– CO
685.3 Leu-Leu-Val-Asp-Leu-Leu-OH 905.3 FA-Glu-Leu-Leu-Val-Asp-Leu –
H2O
923.3 FA-Glu-Leu-Leu-Val-Asp-Leu 1018.4 FA-Glu-Leu-Leu-Val-Asp-Leu-Leu
– H2O
1036.5 cyclo[FA-Glu-Leu-Leu-Val-Asp-Leu-Leu + H]+
Table 9: Fragment ions of 189 observed in MS2 and MS3 of [M + Na]+ and [M - H]-
MS2& MS3 [M +Na]
Sequence of amino acids MS2&MS3 [M-H]
Sequence of amino acids
391.3 Val-Asp-Leu-Leu - CO – CO2 339.4 FA-Glu - CO 435.2 Val-Asp-Leu-Leu -CO 452.5 FA-Glu-Leu - CO
463.2 Val-Asp-Leu-Leu 552.4 Leu-Val-Asp-Leu-Leu
481.2 Val-Asp-Leu-Leu-OH 665.6 Leu-Leu-Val-Asp-Leu-Leu-OH 594.4 Leu-Val-Asp-Leu-Leu-OH 692.7 FA-Glu-Leu-Leu-Val
618.4 FA-Glu-Leu-Leu 776.6 [M-H -FA - H2O]
-671.4 FA-Glu-Leu-Leu-Val - CO – H2O 790.7 FA-Glu-Leu-Leu-Val-Asp - H2O 707.4 Leu-Leu-Val-Asp-Leu-Leu-OH 794.6 [M-H -FA]
-742.5 FA-Glu-Leu-Leu-Val-Asp –H2O – CO – CO2
859.7 FA-Glu-Leu-Leu-Val-Asp-Leu - CO2
– H2O
814.4 FA-Glu-Leu-Leu-Val-Asp – H2O 903.7 FA-Glu-Leu-Leu-Val-Asp-Leu - H2O
832.4 FA-Glu-Leu-Leu-Val-Asp 885.6 FA-Glu-Leu-Leu-Val-Asp-Leu - 2H2O
899.6 FA-Glu-Leu-Leu-Val-Asp-Leu - H2O – CO
954.6 [M-H - 2H2O – CO2] -927.5 FA-Glu-Leu-Leu-Val-Asp-Leu – 998.8 [M-H - 2H2O]
-H2O
Leu Leu Val Asp Leu Leu
O O
Figure 84: Fragment ions observed in [M+H]+ mass spectrum of surfactin C (189)
FA CH CH2C GLu O
Leu Leu Val Asp Leu LeuOH
O O
m/z 245 +2H
Figure 85: A characteristic ESI MS fragment should be existent in esperin.
N