Tripropeptin E, a new tripropeptin group antibiotic produced by Lysobacter sp. BMK333-48F3.

Since the late 20th century, the prevalence of infectious diseases caused by the drug-resistant bacteria, especially by the Gram-positive ones has become a serious problem in the medical treatments, rendering the major antimicrobial drugs less active or ineffective against many important bacterial infections1,2). Thus the newer drugs effective against the multidrug-resistant bacteria are being requested. Recently, we have discovered novel compounds designated tripropeptins (TPPs); particularly tripropeptin C (TPPC) and tripropeptin D (TPPD) show the excellent activities against Gram-positive bacteria including both methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE)3). The antimicrobial activities of these tripropeptins correlate well to the length of fatty acyl side-chains, indicating that the longer was the more active3). Thus, we are greatly interested in the study on the structure-activity relationship regarding fatty acyl side-chains. The present paper described the isolation of a new type of tripropeptin-antibiotic, tripropeptin E (TPPE), its chemical and physicochemical characterization and also its anti-bacterial activities. Furthermore, the structure-activity relationship of tripropeptin-like lipopeptides will be discussed. Fermentations were carried out likewise as reported previously3) except that the culture medium consists of 1.5% glycerol, 1.5% cotton seed meal, 0.3% NaCl, 0.5% sodium L-glutamate monohydrate, 0.2% L-leucine in deionized water (pH 7.4 before sterilization). A fermentation broth (15 liters) was poured into 15 liters of acetone. Mixing the above suspension well, 30 liters of deionized water and then Diaion HP20 (Mitsubishi Chemical Co., 1 liter wet volume) were added subsequently. The mixture was filtered, and the residues were washed with 3 liters of deionized water and 50% aqueous methanol, successively. The active principles were then eluted with 3 liters of acetone. The acetone eluate was concentrated in vacuo to yield a brownish oil (30g), which was chromatographed on a column of silica gel (1000 ml wet volume) with 3000ml each of CHCl3:MeOH:H2O=10:5:1 and BuOH:MeOH:H2O=4:1:2 successively. Active fractions eluted with the latter solvent mixture were concentrated in vacuo to give a yellowish brown oil (5.1g). The oil was dissolved in a small volume of 30% aqueous methanol, adjusted to pH 2.6 with 1M HCl, and was applied to column chromatography using 250ml wet volume of CHP20P (Mitsubishi Chemical Co.). The elution with stepwise gradients of acetone and H2O (750ml each of 12:28, 14:26, 16:24, 18:22, 20:20, 22:18, 23:17, 24:16, 25:15, 26:14, 27:13 and 28:12 V/V), gave active components; Tripropeptin A (45.7mg, 18:22 and 20:20), TPPC (918.4mg, 20:20, 22:18 and 23:17), and a mixture of TPPC and TPPE (288.9mg, 23:17-26:14) in the ratio of acetone and H2O in parentheses. The mixture of TPPC and TPPE was dissolved in a small volume of distilled water, adjusted to pH 2.0 with 1M HCl and subjected to column chromatography using 30ml wet volume of CHP20P (Mitsubishi Chemical Co.). The elution was carried out with stepwise gradients of acetonitrile and H2O (100ml each of 30:70, 31:69, 32:68, 33:67, 34:66, 35:65, 36:64, 37:63, 38:62, 39:61 and 40:60 V/V). The fractions eluted with a mixture of acetonitrile and H2O (33:67 and 34:66), and a mixture of acetonitrile and H2O (36:64 and 37:63) gave 198.5mg of TPPC and 17.5mg of TPPE, respectively. The chemical structure of TPPE as shown in Fig. 1 was determined on the basis of the spectroscopic and mass spectrometric data. The IR spectrum displayed the characteristic absorption of peptide bonds (1635 and 1538cm-1) and of lactone linkage (1741cm-1). The molecular weight of 1181 and the molecular formula of TPPE (C53H87N11O19: see Table 1) were suggested by m/z 1202.5901 in the HRESI-MS ([M-2H+Na]-, calcd