Lipase-catalyzed preparation of chromomycin A3 analogues and biological evaluation for anticancer activity.

Several acyl derivatives of the aureolic acid chromomycin A(3) were obtained via lipase-catalyzed acylation. Lipase B from Candida antarctica (CAL-B) was found to be the only active biocatalyst, directing the acylation regioselectively towards the terminal secondary hydroxyl group of the aglycone side chain. All new chromomycin A(3) derivatives showed antitumor activity at the micromolar or lower level concentration. Particularly, chromomycin A(3) 4'-vinyladipate showed 3-5 times higher activity against the four tumor cell lines assayed as compared to chromomycin A(3).

Deoxysugar transfer during chromomycin A3 biosynthesis in Streptomyces griseus subsp. griseus: new derivatives with antitumor activity.

Chromomycin A3 is an antitumor drug produced by Streptomyces griseus subsp. griseus. It consists of a tricyclic aglycone with two aliphatic side chains and two O-glycosidically linked saccharide chains, a disaccharide of 4-O-acetyl-D-oliose (sugar A) and 4-O-methyl-D-oliose (sugar B), and a trisaccharide of D-olivose (sugar C), D-olivose (sugar D), and 4-O-acetyl-L-chromose B (sugar E). The chromomycin gene cluster contains four glycosyltransferase genes (cmmGI, cmmGII, cmmGIII, and cmmGIV), which were independently inactivated through gene replacement, generating mutants C60GI, C10GII, C10GIII, and C10GIV. Mutants C10GIV and C10GIII produced the known compounds premithramycinone and premithramycin A1, respectively, indicating the involvement of CmmGIV and CmmGIII in the sequential transfer of sugars C and D and possibly also of sugar E of the trisaccharide chain, to the 12a position of the tetracyclic intermediate premithramycinone. Mutant C10GII produced two new tetracyclic compounds lacking the disaccharide chain at the 8 position, named prechromomycin A3 and prechromomycin A2. All three compounds accumulated by mutant C60GI were tricyclic and lacked sugar B of the disaccharide chain, and they were named prechromomycin A4, 4A-O-deacetyl-3A-O-acetyl-prechromomycin A4, and 3A-O-acetyl-prechromomycin A4. CmmGII and CmmGI are therefore responsible for the formation of the disaccharide chain by incorporating, in a sequential manner, two D-oliosyl residues to the 8 position of the biosynthetic intermediate prechromomycin A3. A biosynthetic pathway is proposed for the glycosylation events in chromomycin A3 biosynthesis.

Biosynthesis of the antitumor chromomycin A3 in Streptomyces griseus: analysis of the gene cluster and rational design of novel chromomycin analogs.

The biosynthetic gene cluster of the aureolic acid type antitumor drug chromomycin A3 from S. griseus subsp. griseus has been identified and characterized. It spans 43 kb and contains 36 genes involved in polyketide biosynthesis and modification, deoxysugar biosynthesis and sugar transfer, pathway regulation and resistance. The organization of the cluster clearly differs from that of the closely related mithramycin. Involvement of the cluster in chromomycin A3 biosynthesis was demonstrated by disrupting the cmmWI gene encoding a polyketide reductase involved in side chain reduction. Three novel chromomycin derivatives were obtained, named chromomycin SK, chromomycin SA, and chromomycin SDK, which show antitumor activity and differ with respect to their 3-side chains. A pathway for the biosynthesis of chromomycin A3 and its deoxysugars is proposed.

The solution conformation of the antibiotic anticancer chromomycin A3 by two-dimensional NMR spectroscopy.

The solution conformations of chromomycin A3 (CRA) and dechromose-A chromomycin A3 (CRA-B) in dichloromethane and methanol were studied by two-dimensional (2D) NMR techniques. In dichloromethane, the drugs are found in a compact wedged-like conformation, with the phenolic hydroxyls at the tip and the side chains folded back to one side of the aglycon plane, oriented parallel to each other. The overall structure is stabilised by intramolecular hydrogen bonds and by the formation of a hydrophobic pocket, enclosed by the three side chains. In methanol, the drugs have the expected open conformation with extended side chains. Like its parent drug, CRA-B binds to d(ATGCAT)2 duplex with a major groove orientation, a 2:1 drug/duplex ratio and a two-fold symmetry of the resultant complex. The drug molecule is suggested to reside diagonally across the two strands of the duplex and to span 3 base pairs, while all three side chains of the drug are folded away from the major groove of the DNA. The observed nonequivalent positions of CRA and CRA-B within the major groove of the duplex results from the different conformation adopted by the sugar side-chains in the two complexes.

Solution conformation of the antitumor antibiotic chromomycin A3 determined by two-dimensional NMR spectroscopy.

A conformational analysis and a complete assignment of the nonexchangeable proton resonances of chromomycin A3, dechromose-A chromomycin A3, and deacetylchromose-B chromomycin A3 were carried out in organic solvents. The resulting conformation in methanol has the three side chains of chromomycin A3 fully extended, away from one another and from the aglycon. In dichloromethane on the other hand, the drug was shown to adopt a highly compact conformation in which most of the 26 oxygen atoms in the molecule point out toward the solvent. The two carbohydrate side chains extend parallel to each other on the same side of the aglycon. Two intramolecular nuclear Overhauser enhancement contacts have been observed between different sugar units on these side chains, indicating close proximity for these moieties. In addition, the aliphatic side chain is folded toward the aglycon, parallel to the two oligosaccharide side chains. The overall conformation has a wedge-like shape with the two phenoxy groups exposed at the pointed edge. The presence of some exchange cross-peaks in the NOESY spectra suggests the presence of intramolecular hydrogen bonds that probably help to maintain the compact conformation. The derivatives of chromomycin A3 have qualitatively similar conformations, though their respective conformations are not as compact as the parent drug. The significance of these results is discussed in terms of a model of chromomycin A3 binding to DNA in the major groove.