The Electrospray Ionization-Mass Spectra of Erythromycin A Obtained from a Marine Streptomyces sp . Mutant

Indian Journal of Pharmaceutical Sciences 310 May June 2008 In recent years, marine microorganisms have been given attention as a prodigious source of compounds with intriguing structures and interesting biological activity for drug development. Marine filamentous bacteria, belonging to the genus Streptomyces of Actinomycetes are an exceptionally rich source for a huge number of secondary metabolites. They are known as one of the most prospective natural sources for production of antibiotics and antitumor compounds1-3.

In recent years, marine microorganisms have been given attention as a prodigious source of compounds with intriguing structures and interesting biological activity for drug development.Marine filamentous bacteria, belonging to the genus Streptomyces of Actinomycetes are an exceptionally rich source for a huge number of secondary metabolites.They are known as one of the most prospective natural sources for production of antibiotics and antitumor compounds [1][2][3] .
Many members of the anthracyclin family are clinically very useful antineoplastic agents with a broad spectrum of activities extending to certain solid tumours that are normally resistant to most other modes of chemotherapy 4,5 .The clinical use of such drugs, however, is hampered by a number of undesired side effects, the most serious being the dose-related cardiotoxicity.There is therefore a great interest in related natural or synthetic compounds having improved therapeutic indices [4][5][6] .
Induction of mutations is one of the applied techniques which uses different types of chemical and physical mutagens, either individually or in different combinations, doses and times [7][8][9][10] .Wieczorek and Mordarski 11 treated S. olivaceus with UV irradiation leading to mutants different from the original strain, not only in their spectrum of antimicrobial activity, but also in the taxonomic properties, e.g.colour of the aerial mycelium, liquefaction of gelatine, growth on cellulose, production of ammonia and nitrate reduction.Lee and Rho 12 obtained tylosin hyper-producing mutants after treatment of S. fradiae NRRL 2702 with either n-nitroso-guanidine (NTG) or exposing to UV. Cheng et al. 13 have varied the productivity of S. hygroscopicus FC 904 (the producer of rapamycin) to 60-124% after mutagenesis by NTG and UV.Khattab and EL-Bondkly 14 used TLC technique to distinguish the produced bioactive components obtained by selected superior mutants compared with the wild type strain.In connection with our search for bioactive components with potential medical application, the marine Streptomyces sp.isolate AH2 was subjected to mutation by UV-light.During our biological screening, the most proliÞ c producer of bioactive constituents, sp.isolate AH2 and their mutants were cultivated at 28° on Trypticase Soy Broth (TSB) medium (Merck Co.) using 75% sea water and 25% distilled water.For seed culture preparation, a loopful of mycelium and spores were taken from the slope culture and used to inoculate 250 ml ß ask containing 25 ml of TSB medium and then incubated at 28° for 2 d.Five ml of seed culture were used to inoculate 250 ml ß asks containing 50 ml TSB medium, and the cultivated flasks were further incubated for additional 5 days at 28° on a rotary shaker (220 rpm).For cultivation of the test bacterial strains (Escherichia coli NRRL B-766, Bacillus subtilis NRRL B-543 and Micrococcus luteus NRRL B-287) Luria Broth (LB) medium was used.It contains (g/l):tryptone (10); yeast extract (5); NaCl (10); Agar (15) and the pH was adjusted to 7.0.Discs used for Antibiotic assay, Whatman product No. 2017, six mm diameter, were saturated with 25 µl supernatant extract of each isolate in comparison with the original strain.

Mutagenesis:
Marine Streptomyces sp.AH2 spores from old slants (7 days) were suspended in sterile distilled water and exposed to UV light (Philips T-UV-30 W lamp type number 57413 p/40) for 5, 10 and 15 min at a distance of 20 cm.After irradiation, the treated suspensions were kept in dark for ~ one hour.Appropriate dilutions were spread on TSB medium and incubated at 28º for 5 d.The growing colonies were transplanted on slants for further studies 14 .mutant 10/14 was selected, upscaled and the bioactive constituents isolated and identified.The potent antibacterial activity of mutant 10/14 was attributed to the major two components, erythromycin A (1) and an oily substance, HM1.We report here the detailed ESI-MS analysis of erythromycin A (1) combined with HRESI-MS along with 2D NMR spectra.

Microorganisms and culture conditions:
Marine Streptomyces species AH2 was isolated from Suez Canal, Ismailia, Egypt, and was identified according to Bergey ' s Manual of Systematic Bacteriology 1989 15 .The original marine Streptomyces (0.014-0.040 µm, 0.8×50 cm) using n-hexane/ethyl acetate, a crude white solid substance was obtained.
Re-crystallization of the substance from n-hexane/ ethyl acetate and from chloroform, respectively, yielded 12 mg of erythromycin A (1) as a white solid.Finally, puriÞ cation of the high polar fraction V by silica gel (0.014-0.040 µm, 0.8×50 cm) using a DCM/MeOH gradient followed by Sephadex LH-20 (DCM/40%MeOH) delivered 44 mg HM1 as an oily substance.

RESULTS AND DISCUSSION
Mutation was used as a major tool for the induction of a wide range of genetic variations for selection of higher antibiotic producers of marine streptomycetes.
After treatment of the marine Streptomyces sp.AH2 with UV irradiation for different exposure times; 5, 10, 15 min, respectively, 145 isolates were obtained, and their productivity of bioactive compounds was examined.

Fermentation of strain, extraction and separation:
Twenty litres of fermentation medium (TSB) were inoculated with 10% seeding of 10/14 mutant for five days at 28º.After fermentation, the pH of the culture broth had dropped to 3.9.The filtrate was extracted with ethyl acetate, and the organic extract was concentrated in vacuo to 500 ml, washed with brine (2×50 ml), then dried over Na 2 SO 4, and then evaporated in vacuo to dryness.The dark brown extract (748 mg) was ß ash chromatographed on silica gel of particle size (30-60 µm, 2×70 cm) eluting with an n-hexane/ethyl acetate gradient to deliver five fractions I, n-hexane; II, n-hexane-EtOAc (9:1); III, n-hexane-EtOAc (3:1); IV, n-hexane-EtOAc (1:1); V, EtOAc.Purification of the fast fraction I using silica gel (0.014-0.040 µm, 0.8×50 cm) and eluting with n-hexane/DCM gradient led to phenazine (2 mg), a greenish-yellow solid.PuriÞ cation of fraction II on silica gel (0.014-0.040 µm, 0.8×50 cm) eluting with petroleum ether/Et 2 O-gradient led to 1-acetylβ-carboline (5 mg) and perlolyrin (3 mg) as two pale yellow solids.On subjecting the middle polar fraction (III) to further purification on silica gel  Fermentation of a 20 L shaker culture using TSB medium at 28° was continued for 5 d.The culture broth was separated by centrifugation and extraction with ethyl acetate at pH 4. According to TLC monitoring, the mycelial cake did not contain interesting compounds and was discarded.The extract of filtrate was concentrated in vacuo followed by washing with brine and drying with anhydrous sodium sulphate.Finally, it was evaporated to dryness yielding a sticky brown extract.
Structures of the known compounds, phenazine 16 , 1-acetyl-β-carboline 17 and perlolyrin 18 were established on the bases of their chromatographic properties, NMR and MS spectroscopy as well as comparison with authentic spectra 19 .
Erythromycin A (1), a colourless solid, showed no UV absorbance (254 nm) or ß uorescence (366 nm), From the above study, the mutations of the marine Streptomyces sp AH2 genome by UV treatment, affected the production of antibiotics qualitatively and quantitatively.Chromatographic analysis of extracts of the selected 17 mutants using TLC in comparison with those of AH2, established that mutant 10/14 was the only one, which displayed more bands than the original one, and it was the most antibiotically active strain against all tested bacteria.
Chemical screening of the mutant 10/14 extract using TLC monitoring exhibited two major bands, which were not present in extracts of the parent strain.The Þ rst of them was Polar with no UV absorbance, which turned brown by spraying with anisaldehyde/sulphuric acid, and later to green.The other one showed an intensively blue UV ß uorescence polar band, which turned yellow by anisaldehyde/sulphuric acid after heating.
ESI MS of 1 showed three quasi molecular ion peaks at m/z 734.[m/z 734.4683850, (M+H)] established the molecular formula as C 37 H 67 NO 13 , bearing 5 double bond equivalents.Compound 1 was subjected to detailed ESI-MS 2 and MS 3 fragmentations combined with HRESI-MS, from which, 9 and 11 peak-fragments, respectively, were assigned (Table 3).
The common base peak fragment in both cases at m/z 158.1177 (C 8 H 16 NO 2 ) was attributed to desosamine (A), and the later was further established by the     27 , the structure of erythromycin A (1) was fully deduced, excluding also the initially suggested two macro-lactones with 13-and 12-memberd rings (6,7, fig.1).This was confirmed by the direct cross-signal from the oxymethine proton at δ 5.05 ppm (H-13) towards the lactone carbonyl (C-1, 175.7 ppm) in the HMBC spectrum establishing the ring closure between C-13 and C-1 via oxygen, excluding structure 6.Moreover, the methine proton at δ 3.79 ppm (H-11) exhibited no correlation to the same lactone carbonyl, negating also structure 7.
The aglycone, erythronolide A (5) and more than 40 derivatives are known from nature 28 .Erythromycin derivatives are well known with potent activity against Gram-positive bacteria, most of them showing additional DNA binding properties 29 .Recently, erthromycin A (1) was found to be of therapeutic use for treatment of inß ammatory immuneoreactions that may be the major cause of morbidity or mortality associated with "Bird Flue" inß uenza infection 30 .
3 (M+H), 756.4 (M+Na) and  1489.3 (2M+Na) in the (+)-ESI-MS mode, and one at m/z 778.3 (M+HCOO -) in the (-)-ESI-MS mode.This established the molecular weight as 733 Dalton, which is indicative of the existence of an odd number of nitrogen atoms.HRESI MS of 1

TABLE 4 : 13
of 1 displayed an absorption band at ν 3450 cm -1 (hydroxyl/amino groups).Two strong bands at ν 1740 and 1720 are indicative for carbonyls of ester and ketone systems.It was lacking oleÞ nic or aromatic signals, but displayed two strong bands between ν however, it exhibited a brown colour by spraying with anisaldehyde/sulphuric acid, which turned later to green.The UV spectra displayed a peak at λ max ~ 280, which is slightly bathochromic shifted in acidic solution to λ max 289 nm.The IR spectra C NMR AND 1 H NMR DATA OF ERYTHROMYCIN A(1) COMPARED WITH 6-O-METHYLERYTHROMYCIN A (2)Fig.1: Structural formula of the macrolides 1-7 beside to the sugars A and B.

Fig. 3: HMBC (→) and 1 H, 1 H COSY (↔) derived connectivities of erythromycin A (1)
oxygenated, and 4 methylene signals.Finally, 3 signals of quaternary sp 3 carbons were visible.By careful interpretation of the 1D and 2D NMR data (fig.3) in combination with the molecular formula and ESI-MS 2 and ESI-MS 3 fragmentations and comparison with related analogous23,