A novel class of macrolides for which the name sanglifehrins is proposed, has been discovered from actinomycete strains based on their high affinity binding for cyclophilin A (CypA), an immunophilin originally identified as a cytosolic protein binding cyclosporin A (CsA). The sanglifehrins were produced by Streptomyces sp. A92-3081 10. They were isolated and purified by extraction and several chromatographic, activity-guided steps.

A novel class of macrolides, the sanglifehrins, was discovered by screening of actinomycete strains with a cyclophilin-binding assay. The chemical structures and absolute stereochemistries of the sanglifehrins A, B, C and D were determined unambiguously by NMR-techniques and by X-ray crystallography of the complex with cyclophilin A.Sanglifehrin A consists of a 22-membered macrocycle containing a tripeptide subunit and features in position 23 a chain of nine carbon atoms bearing a spirocyclic substituent. Sanglifehrins A and B are genuine metabolites whereas sanglifehrins C and D are artefacts.Screening for cyclophilin binding metabolites from actinomycete strains led to the discovery of a novel class of compounds, which were named sanglifehrins. The taxonomy, fermentation, isolation and biological activity are described in the foregoing paper1}.In this part we describe the structure elucidation mainly done by NMR-analysis, and give the chemical characteristics of these newmicrobial compounds.Methods Spectroscopy The XHand 13C NMRspectra of the sanglifehrins were recorded in T>MSO-d6 on a Bruker Avance DMX-500spectrometer with TMSas internal standard. The 1H, 13C and 15N NMRshifts are listed in Table 2 and image, at a crystal-to-detector distance of 130mm.Data processing was done with the programs DENZO1.5.ll &Scalepack2), and the CCP43.0-package3). The overall Rsymon intensities for a total of 179130 measurements of 41 552 independent reflections in the resolution range 15A-1.6A (completeness 99.2%) was 7.5%.The structure was solved by molecular replacement and refined with the program X-PLOR, Version 3.14).

Rifamycin B biosynthesis in Amycolatopsis mediterranei N/813 was inactivated by introducing a small deletion in the rifF gene situated directly downstream of the rifamycin polyketide synthase (PKS) gene cluster. The corresponding mutant strain produced a series of linear intermediates of rifamycin B biosynthesis that are most probably generated by obstruction of the normal release of the end product of the rifamycin PKS. This result provides evidence that the rifF gene product catalyses the release of the completed linear polyketide from module 10 of the PKS and the intramolecular macrocyclic ring closure by formation of an amide bond, as indicated by sequence similarity of this protein to amide synthases. The chemical structures of the new rifamycin polyketide synthase intermediates released from modules 4 to 10 were determined by spectroscopic methods (UV, IR, NMR and MS) and gave insight into the reaction steps of rifamycin ansa chain biosynthesis and the timing of the formation of the naphthoquinone ring. The intermediates released from modules 6 and 8 were isolated as lactones formed by the terminal carboxyl group ; proton NMR double resonance and ROESY(rotated frame nuclear Overhauser enhancement spectroscopy) experiments enabled the deduction of the relative configurations in the linear chain which correspond to the known absolute stereochemistry of rifamycin B.Keywords : antibiotic biosynthesis, ansamycins, amide synthase, gene replacement, pathway engineering INTRODUCTIONRifamycins are clinically important ansamycin antibiotics, composed of a naphthalenic chromophore spanned by a long aliphatic ansa chain. The rifamycins and the semisynthetic drugs derived from them exert their antibiotic activity by specific inhibition of bacterial DNA-dependent RNA polymerase (Wehrli, 1977). At higher concentrations, these antibiotics also inhibit the RNA-dependent DNA polymerase of retroviruses (Szabo et al., 1976 bacterium leprae, causative agents of tuberculosis and leprosy, respectively, they are also active against a variety of other organisms, including bacteria and viruses (Szabo et al., 1976 ;Oppenheim et al., 1986 ;Barakett et al., 1993 ;Bachs et al., 1992). Furthermore, it was shown recently that rifampicin-containing regimens are able to cure staphylococcal implant-related infections (Zimmerli et al., 1998).The identification and sequencing of the rifamycin polyketide synthase (PKS) gene cluster by our group (Schupp et al., 1998) and by August et al. (1998) Our interest in further studying rifamycin B biosynthesis led us to undertake the inactivation of the rifF gene, situated directly downstram of the PKS genes, and to analyse the effect of this mutation on rifamycin biosynthesis. The rifF gene product has been characterized as rifamycin amide synthase by sequence homologies to different arylamine N-acetyltransferases (August et al., 1998) and the putative function of the RifF protein in rifamycin biosynthesis was suggested to be a cyclase, catalysing the formation of an intramolecular amide bond between ...

Proansamycin B, the formerly postulated intermediate of rifamycin B biosynthesis, was isolated from cultures of the Amycolatopsis mediterranei mutant F1/24. The structure was determined using UV, IR, NMR and MS techniques. Biotransformation studies demonstrate that proansamycin B is an intermediate of a shunt pathway, a 8-deoxy variant, of rifamycin B biosynthesis leading to 8-deoxy-rifamycin B as the final product.In addition, 34a-deoxy-rifamycin W, the direct precursor of rifamycin W, could be isolated representing the earliest macrocyclic intermediate obtained so far in the biosynthetic route to rifamycin B. Furthermore, the new rifamycin W-28-desmethyl-28-carboxy and rifamycin Whemiacetal, intermediates in, the transformation sequence of rifamycin W to rifamycin S, were isolated. Application of proton NMR measurements (double resonance and ROESY experiments) on the latter compound indicated that the stereochemistry at the chiral center C-28 is R.

Twonovel metabolites, cymbimicins A and B, were isolated from the culture broth of a strain of Micromonospora sp. by screening for cyclophilin binding metabolites from actinomycete strains. Cymbimicin A binds to cyclophilin A with a high affinity six fold lower than to that of cyclosporin A. The binding affinity of cymbimicin B is about 100 times lower. The taxonomy of the producing strain, fermentation, isolation, physical and biological properties and structure elucidation are described.