Aureobasidins A to R were isolated from the fermentation broth of Aureobasidium pullulans R106. Aureobasidins are cyclic depsipeptide antibiotics with MW's ranging from 1,070 to 1,148. Aureobasidins showed high in vitro antifungal activity against Candida albicans.

Aureobasidin A, a new antifungal antibiotic, was isolated from the culture mediumof Aureobasidium pullulans R106. Aureobasidin A was a cyclic depsipeptide consisting of eight a-amino acid units and one hydroxy acid unit. The structures of the units were found by acid hydrolysis of the antibiotic to be 2 and L-phenyl-alanine. The sequence of the units was identified by NMRand FAB-MSof the products from the alkaline hydrolysis of aureobasidin A. 925Aureobasidins are complexes of antifungal antibiotics produced by Aureobasidium pullulans R106. The production, isolation, characterization, and antifungal activity of aureobasidins have been described in the preceding paper.1) In this paper, we report on the structure of aureobasidin A.Aureobasidin A (1, Fig. 1 The IR spectrum of 1 showed the presence of ester (1750cm"1) and amido carbonyls (1640cm"1). Mild alkaline hydrolysis (0.25 n NaOHin aqueous MeOH, room temperature, 25 hours) of 1 gave 2 and 3. Compounds 1 and 3 were negative in the ninhydrin color reaction and did not react with diazomethane or acetic anhydride in pyridine at room temperature. Compound 2 was also negative in the ninhydrin color reaction, but gave the methyl ester derivative 2a by treatment with diazomethane, and O-acetyl derivative 2b by treatment with acetic anhydride in pyridine. Further, 2a gave derivative 2c with O-acetyl and methyl ester groups when treated with acetic anhydride in pyridine.

The structures of OF4949-I, II, III and IV were identified by analysis of the products of their chemical degradation and by 1H NMR, 13C NMR, and mass spectrometry. These compounds were new cyclic peptides containing diphenyl ether as a chromophore.OF4949-I had two amino acids, ~-hydroxy-L-asparagine and 4-methylisodityrosine.The structural differences between I and II and between III and IV lay solely in the diphenyl ether moiety; the phenolic hydroxyl group in II and IV was methylated in I and III. OF4949-III and IV contained L-asparagine instead of the 13-hydroxy-L-asparagine moiety of I and II.

Three kinds of trisaccharides were prepared by digesting fucoidan from the brown alga Kjellmaniella crassifolia, with the extracellular enzymes of the marine bacterium Fucobacter marina. Their structures were determined as delta 4,5GlcpUA1-2(L-Fucp(3-O-sulfate)alpha 1-3)D-Manp, delta 4,5GlcpUA1-2(L-Fucp(3- O-sulfate)alpha 1-3)D-Manp(6-O-sulfate), and delta 4,5GlcpUA1-2(L-Fucp(2,4-O-disulfate)alpha 1-3)D-Manp(6-O-sulfate), which indicated the existence of a novel polysaccharide in the fucoidan and a novel glycosidase in the extracellular enzymes. In order to determine the complete structure of the polysaccharide and the reaction mechanism of the glycosidase, the fucoidan was partially hydrolyzed to obtain glucuronomannan, which is the putative backbone of the polysaccharide, and its sugar sequence was determined as (-4-D-GlcpUAbeta 1-2D-Manpalpha 1-)n, which disclosed that the main structure of the polysaccharide is (-4-D-GlcpUAbeta 1-2(L-Fucp(3-O-sulfate)alpha 1-3)D-Manpalpha 1-)n. Consequently, the glycosidase was deduced to be an endo-alpha-D-mannosidase that eliminatively cleaves the alpha-D-mannosyl linkage between D-Manp and D-GlcpUA residues in the polysaccharide and produces the above trisaccharides. The novel polysaccharide and glycosidase were tentatively named as sulfated fucoglucuronomannan (SFGM) and SFGM lyase, respectively.

Optically active 1 5-deoxyspergualin (II) and 1 5-deoxy-l l-O-methylspergualin (Ha) were synthesized, and their antitumor activities were examined. The (-)-enantiomers of both II and Ila were active against mouse leukemia LI210, while the (H-)-enantiomers were almost inactive. The optical resolution of the key intermediate, (±)-7V-(7-guanidinoheptanoyl)-a:alkoxyglycine (VI) was achieved by use of an exopeptidase, serine (acid) carboxypeptidase [EC 3.4.16.1] and (±)-N-(7-guanidinoheptanoyl)-a-alkoxyglycyl-L-amino acid (VIII) as the substrate. Considering the enzymatic susceptibility of the substrate (VIH), we deduced that the absolute configuration of the carbon at ll (C-ll) of the bioactive (-)-enantiomer, and so that of natural spergualin (I), is S. This is, to our knowledge, the first report of the use of carboxypeptidase for the resolution of 7V-acyl amino acid. The carbons at ll (C-ll) and 15 (C-15) of natural spergualin (I) are asymmetric (Fig. 1). The configuration at C-15 is S with respect to L-lysine,^and the configuration here has some slight effect on the antitumor activity of the compound. The configuration at C-ll has remained unknown,

Structures of 17 minor forms of aureobasidins (Abs), Abs B~R, were elucidated by mass fragmentation and amino acid analysis. The fragmentation patterns by FAB-MSspectroscopy of Abs A~Eseemed to follow predictable rules, so we used the rules to elucidate the 13 other Abs. All Abs consisted of eight amino acids and one hydroxy acid. 1187A new antifungal antibiotic, aureobasidin (Ab) A (Fgi. 1),1} has been isolated from the fermentation broth ofAureobasidiumpullulans R1 06. This strain also produces many antifungal antibiotics with structures that might be related to Ab A. We isolated 17 of these antibiotics and named them Abs B~R.1} The structures ofAbs B~E were elucidated by chemical degradation. Acid hydrolysis ofAbs B~E gave amino acids and hydroxy acids. These amino acids were analyzed by HPLCor NMR. The structures of the hydroxy acids were confirmed by comparison with synthetic compounds. Alkaline hydrolysis of Abs B~E gave linear chain peptides. The fragmentation patterns of their derivatives by FAB-MSwere used to deduce the amino acid sequences, from which the structures of Abs B~E were identified. Wefound that the fragmentation patterns by FAB-MSof Abs A~Eseemed to follow predictable rules, so we applied the rules to elucidate the structure of the other Abs. The structures of Abs A~Rare shownin 2) The hydroxy acid was purified from the hydrolysate by Dowex 50W. By NMR,its structure was found to be 2-hydroxyisovaleric acid (Hiv). Hiv from Ab B coincided with the R form of the synthetic Hiv3) on chiral HPLC(Chiralpak WH, Daicel Chemical Industries, Ltd.). Thus, Ab B had the same amino acids as Ab A, and Hiv instead of the 2-hydroxy-3-methylpentanoic acid (Hmp) of Ab A. Alkaline hydrolysis (0.25 n NaOH in aqueous MeOH, room temperature, 15 hours) of Ab B gave Bl and B2. By FAB-MS, the MWofBl was 1,046 and that ofB2 was 1,068. Compound Bl was methylated to give Bla. The acid hydrolysate of Bla contained sarcosine (Sar) instead of the jSHOMeValand methylamine found in the acid hydrolysate of Ab B.

No abstract

Aureobasidins are a group of cyclic depsipeptides with antifungal activity and are produced by Aureobasidium pullulans. Aureobasidins are composed of eight amino acids and one hydroxy acid such as 2-hydroxy-3-methylpentanoic acid (Hmp), and highly lipophilic. Five new aureobasidins, si> S2a> S2b, S3 and S4, which have higher hydrophilicity in reversed phase HPLC than the known aureobasidins A~R, were discovered in a fermentation broth of A. pullulans R106 by means of on-line liquid chromatography/mass spectrometry with electrospray ionization. We identified the structures of the compounds and studied their antifungal activities. Three of the newaureobasidins, S2b, S3 and S4, which have hydroxylated Hmpas the hydroxy acid, were highly active against Candida spp. and Cryptococcus neoformans.