Iturinic acids

Iturins are a group of fungicidal cyclic lipopeptides produced by Bacillus subtilis m m m All members of this group are cyclic octapeptides with seven a-amino acids and a unique 13-amino fatty acid homologue of iturinic acid. The predominant iturin-A isomer, A2, contains the n-C14-isomer of iturinic acid (Itu) (Scheme 8). The synthesis of the molecule can be divided into two steps with the first stage consisting of the assembly of the linear octapeptide on solid phase followed by the backbone cyclization in solution. 

The synthesis of the linear octapeptide was performed applying Fmoc/tBu chemistry and starting from 0.82 g (0.39 mmol-g-1, 0.32 mmol) of Fmoc-Ser(tBu)-HMPAA-resin (Millipore). Iturinic acid was coupled as the jV-Boc derivative. After deprotection and cleavage with TFA/H20 (95 5), the product was purified by preparative RP-HPLC yield 37 mg (74%). 

Partial acid hydrolysis gave an amide of the P-amino fatty acid and serine, with an N-terminal position for the serine residue. The structures of several other peptides were established by chemical means and by mass spectrometry of their permethylated derivatives. From these results, the complete structure of iturin A was established as (57) (138). Circular dichroism of derivatives of the P-amino fatty acids (iturinic acids) showed their configurations to be R 139). 

The structures of three bacillomycins D, F and L have been completely established as (59), (58) and (60) respectively. All of them contained the characteristic iturinic acids and seven a-amino acids. The sequence of amino acids was studied using chemical methods, in particular Edman degradation of the product obtained by specific cleavage of the peptide moiety with N-bromosuccinimide. Confirmatory results were obtained by chemical ionisation (isobutane) mass spectrometry and various techniques of NMR spectroscopy. The exact molecular weights of the various species were obtained by FAB mass spectrometry 146, 147) (see Table 7). 

By using FAB mass spectrometry, two dimensional H-NMR spectroscopy and chemical degradations, structure (61) was established. This structure differs from that of the iturins by inversion of the two amino acid residues at the C-terminal end of the peptide moiety. D-Ser-L-Asn in mycosubtilin replaces D-Asn-L-Ser in the iturins (750). The 3-amino fatty acids are anteiso-C and fso-Cig-iturinic acids (see Table 7). 

This group is characterized by the presence of P-amino fatty acids, the iturinic acids. These acids are derivatives of the following fatty acids n-Ci3, n-Ci4, anteisO Ci5, iso-C, n-C, iso-Cig, n-Cj, anteiso-C and 1S0-C17. The ratios of the different amino fatty acids depend on the nature of the culture medium which can afford different precursors. 

Nagai, U., F. Besson, and F. Peypoux Absolute Configuration of an Iturinic Acid as Determined by CD Spectrum of its DNP-p-Methoxyanilide. Tetrahedron Letters 2359 (1979). 

Mycosubtilins were isolated by Peypoux et al. in 1976 as antifungal metabolites from B. subtil is. The constituent amino acids of mycosubtilin D are the same as those of iturin A, but the peptide sequences differ(Fig. 

Iturins, a family of antifungal peptides produced by various strains of Bacillus suh-tilis. The iturins comprise small cyclic pep-tidolipids characterized by a lipid-soluble -amino acid linked to a peptide containing D- and L-amino acids [F. Peypoux et al.. Tetrahedron 1973, 29, 3455 M. Klich et al., Mycopathology 1991, 226, 77]. 

B. suhtilis Rapeseed meal containing protein (38.87%), total nitrogen (62.19mg/g), total phosphorus (19.19mg/g), carbohydrates (by-product from edible oil production) Iturin A and poly-y-glutamic acid 89... 

Yao D, Ji Z.Wang C, Qi G, Zhang L, Ma X, et al. Co-producing iturin A and poly-y-glutamic acid from rapeseed meal under solid state fermentation by the newly isolated Bacillus subtilis strain 3-10. World J Microbiol Biotechnol 2012 1—7. 

A wide variety of microorganisms also produce many kinds of surface-active lipoproteins or lipopeptides [63]. Mostly they exhibit the typical amphiphilic character and are generally extracellular. Representative of such surface-active lipopeptides is surfactin produced by Bacillus subtilis. It is composed of a heptapeptide cycle closed by a C14.15 P-hydroxy fatty acid that forms a lactone ring system (Fig. 15) [64]. This structure resembles those of iturins, another class of lipopeptides also produced by Bacillus subtilis [65]. 

The B. subtilis lipopeptides are members of a particular antibiotic class formed by surfactin, iturin, and fengycin families. The general structure of these lipopeptides is a peptide cycle of seven (surfactin and iturin) or ten amino acids (fengycin) linked to a fatty add chain. The length of the fatty acid... 

B. subtilis produced a peptidolipid fraction named iturin because the bacterial strain had been isolated from the soil in a village called Ituri in Zaire. By chromatography on silicic acid three components (A, B and C) were obtained (136). In fact the mixture was more complex as it proved possible to resolve iturin A into seven components by HPLC (137). 

Iturin A behaved as a neutral compound on paper electrophoresis and alkalimetric titration it was thus likely that the aspartic and glutamic acid residues were present in the form of asparagine and... 

Chromic acid oxidation resulted in disappearance of serine and tyrosine likewise no ester absorption band was detected in the infrared spectrum of iturin A, thus precluding the presence of a lactone group. 

Structure (57) was in agreement with the results of mass spectrometric investigations of iturin A as a permethylated derivative. The interpretation of the mass spectra was made easier by the presence of pairs of peaks differing by 14 mass units due to the presence of the two homologous P-amino fatty acids 138). 

Iturin C (about 1 to 3% of the crude iturin fraction) contained one free carboxyl group as shown by its electrophoretic behaviour and by alkalimetric titration. The carboxyl group was reduced by diborane, causing the disappearance of one L-aspartic molecule in the hydrolyzate and the simultaneous appearance of one mole of homoserine. Mass spectrometry of the permethylated derivative of iturin C allowed localization of the aspartic acid residue in position 1 thus iturin C is Asp -iturin A 143). 

In addition to the differences in amino fatty acid composition, the various lactams that have been isolated exhibit some differences in a-amino acid composition (Table 7). At first it can be observed that the N-terminal part of the peptide moiety (sequence 1 3) is always constant except for the replacement of Asn and Asp. Frequent modifications are observed in the sequence of amino acids 4 - 7 which is the variable part of the molecule. What is most interesting is the fact that when a replacement occurs or an inversion in the sequence takes place the positions of the d or l residues remain quite stable. The most illustrative example is provided by iturin A and mycosubtilin with the respective sequences D-Asn-L-Ser and D-Ser-L-Asn (see Table 7). The configurations of the amino acids are more important than their structures for the role that they have to play in the bacterial environment. 

The cyanobacteria Anabaena BQ 16-1 produces a decapeptide linked to a p-amino fatty acid in the same manner as in the iturins. The p-amino fatty acid was identified as 2-hydroxy-3-amino-4-methyl-14-chloro-palmitic acid. This peptidolipid designated as puwainaphycine C exhibits cardiotonic activity. It is accompanied by puwainaphycine D which differs from the C-compound in that a threonine residue is replaced by a valine residue. This slight modification causes disappearance of the cardiotonic activity (222). 

Peypoux, F., M. Guinand, G. Michel, L. Delcambe, B.C. Das, P. Varenne, and E. Lederer Isolement de I acide 3-amino-12-methyl-tetradecanoi que et de I acide 3-amino-12-methyl-tridecanoYque a partir de I iturine, antibiotique de Bacillus subtilis. Tetrahedron 29, 3455 (1973). 

IsoGAi, A., S. Takayama, S. Murakoshi, and A. Suzuki Structures of P-amino Acids in Antibiotic Iturin A. Tetrahedron Letters 23, 3065 (1982). 

WiNKELMANN, G., H. Allgaier, R. Lupp, and G. Jung Iturin A, a New Long Chain Iturin A Possessing an Unusual High Content of Ci -P-Amino acid. J. Antibiotics 36, 1451 (1983). 

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