Weiqing He

Identification of 4,5-Dihydro-4-hydroxygeldanamycins As Shunt Products of Geldanamycin Biosynthesis

Two new geldanamycin (GDM) analogues, (4S)-4,5-dihydro-4-hydroxygeldanamycin (1) and (4R)-4,5-dihydro-4-hydroxygeldanamycin (2), were identified from Streptomyces hygroscopicus 17997. Compounds 1 and 2 were not normal intermediates of GDM biosynthesis but shunt products of C-4,5 oxidation catalyzed by GdmP, a cytochrome P450 oxidase acting as a desaturase in GDM biosynthesis. Preliminary assays implied that, compared with GDM, 1 and 2 exhibited decreased cytotoxicity.

A pair of sulfur-containing geldanamycin analogs, 19-S-methylgeldanamycin and 4,5-dihydro- 19-S-methylgeldanamycin, from Streptomyces hygroscopicus 17997

Geldanamycin (GDM, Figure 1) is a benzoquinone ansamycin produced by Streptomyces hygroscopicus.1, 2, 3 It is a specific inhibitor of human heat shock protein 90 (Hsp90), which is a molecular chaperone assisting in protein folding, cell signaling and tumor repression, and a potential cellular target for anti-tumor agent. GDM is only used as a promising lead compound for anti-cancer drug development because of its poor water solubility and severe liver toxicity.4, 5, 6

A new post-PKS modification process in the carbamoyltransferase gene inactivation strain of Streptomyces hygroscopicus 17997.

Genetic manipulation of geldanamycin (GDM) producer Streptomyces species is a rational approach to understand biosynthesis processes and create new analogues. In this study, the carbamoyltransferase gene gdmN was inactivated by insertion of an apramycin-resistance gene aac3 (IV) into the genome of the geldanamycin-producing strain Streptomyces hygroscopicus 17997. GDM analogues produced by this mutant strain were isolated and characterized, such as new compound 4,5-dihydro-7-O-descarbamoyl-7-hydroxy-19-O-glycylgeldanamycin. This compound could be converted to compound 4,5-dihydro-19-O-glycylgeldanamycin, another new GDM analogue, by a strain of Streptomyces hygroscopicus 17997 in which the GDM-pks was inactivated. These new compounds exhibited reductions of cytotoxicity against HepG2 cancer cells, but increases of aqueous solubility. These results suggest that a new post-polyketide synthase modification was involved in this process to produce new GDM analogues.

7-O-descarbamoyl-7-hydroxygeldanamycin, a minor component from the gdmN disruption mutant of Streptomyces hygroscopicus 17997.

7- O -descarbamoyl-7-hydroxygeldanamycin, a minor component from the gdmN disruption mutant of Streptomyces hygroscopicus 17997

Identification of 6-demethoxy-6-methylgeldanamycin and its implication of geldanamycin biosynthesis.

Geldanamycin (1, Figure 1), the first member of benzoquinone ansamycins, was isolated from Streptomyces hygroscopicus in 1970.1 Although exhibiting potent cytotoxicity against various cancer cells, 1 is not a clinical compound due to its severe hepatotoxicity and poor water solubility.2 17-AAG (17-allylamino-17-demethoxygeldanamycin) as a semisynthetic derivative of 1 with much improved water solubility is currently under clinical trial for breast cancer treatment.3 Many new analogs or derivatives of 1 have been created or discovered in the past few years.4–8 We are interested in natural 1 analogs and understanding their synthetic mechanisms. We identified such analogs as 4,5-dihydro-4hydroxygeldanamycins, thiazinogeldanamycin and 19-S-methylgeldanamycin from S. hygroscopicus 17997 and characterized their synthetic mechanisms.9–13 We also discovered a minor component 7-descarbamoyl-7-hydroxygeldanamycin from a gdmN disruption mutant of S. hygroscopicus 17997, which presented an additional proof for C-7 carbamoylation taking place before C-4,5 oxidation in 1 biosynthesis.14 Recently, as a result of our continued efforts for natural 1 analogs, we discovered 6-demethoxy-6-methylgeldanamycin (2) in 1 preparation from S. hygroscopicus 17997. In this paper, we reported the structure of 2 and its implication of 1 biosynthesis. Some preparations of 1 were found to contain 1 analogs as small or trace impurities.15,16 The HPLC of our 1 preparation (with a purity of about 90%; see Supplementary material: a brief description of 1 preparation from S. hygroscopicus 17997) from S. hygroscopicus 17997 displayed a small peak at 24.7 min (about 1.7% of the principle 1 peak at 22.3 min; Figure 2). The peak revealed a molecular ion at m/z 567 ([MþNa]þ ), which exhibited a typical MS2 fragment pattern of 1 (Supplementary Figure S1). The m/z 567 aroused our interests, as we could not assign a reasonable structure for it from MS data and current understanding of 1 biosynthesis.17–19 To elucidate the structure of the analog with m/z 567 (2) by NMR, a total amount of 1070 mg 1 preparation, dissolved in 10 ml dimethyl sulfoxide, was used to make a pure preparation of 2 by reversed-phase HPLC (Shimadzu LC-20AP, SHIMADZU, Kyoto, Japan; YMC ODSA, 21.2 150 mm, mobile phase MeOH-H2O, 62–100% in 21 min, 12.5 ml min-1, wavelength 254 nm; Supplementary Figure S2). After evaporation, an amount of 5.6 mg of 2 as yellow amorphous powder was obtained. Analytical HPLC indicated that it displayed an UV absorption profile very similar to that of 1 (Supplementary Figure S3). The molecular formula of 2 was established as C29H40N2O8 by HR-ESI(þ )-MS (m/z 567.26596, calculated 567.26769 for C29H40N2O8Na, Supplementary Figure S4), which is one oxygen atom less than 1 (C29H40N2O9). The 1H and 13C NMR spectra of 2 (Supplementary Figures S5 and S6) were very similar to those of 1.18 Comparison of the NMR data of 2 with those of 1 revealed that the only difference between the two compounds was replacement of the 6-methoxy group in 1 by the 6-methyl group in 2, which was confirmed by the 2D NMR data analysis of 2. In particular, the 1H-1H COSY correlations of H-5/H-6/H-7, H-6/H3-23 and HMBC correlations of H3-23/C-4, C-6, C-5, in combination with the shifts of these proton and carbon resonances established the CH3-6 in 2. Therefore, the structure of 2 was determined to be 6-demethoxy-6methylgeldanamycin (Figure 1). The NMR chemical shifts of 2 were assigned completely by HSQC, COSY and HMBC spectroscopic data (Supplementary Figures S7–S10) as indicated in Table 1. Compound 2 is a shunt product in 1 biosynthesis. Compound 1’s biosynthesis consists of a starter unit (3-amino-5-hydroxybenzoic acid) assembly, extender units (one malonyl, two 2-methoxymalonyl and four 2-methylmalonyl units for polyketide chain building) condensation and tailoring modifications.17–19 Obviously, 2 is derived from mis-incorporation of a 2-methylmalonyl unit in place

17- O -demethylreblastatin, a subnormal intermediate in geldanamycin biosynthesis

Geldanamycin (GDM), produced by Streptomyces hygroscopicus, is a 19-membered macrocyclic lactam related to benzoquinone ansamycins. Interest in GDM increased greatly upon the discovery of its remarkable antitumor properties, but severe hepatotoxicity made it unfitting for direct clinical uses.1 At present, GDM is a promising lead compound for antitumor drug development. A clear understanding of GDM biosynthesis is of great importance in creating novel GDM analogs by genetic manipulation or combinatorial biosynthesis. The biosynthesis of GDM involves the assembly of one starter unit (3-amino-5-hydroxybenzoic acid), with seven extender units (one acetate, four propionates and two methoxyacetates). The assembly process forms a polyketide backbone, which then undergoes a postPKS tailoring process that includes C-7 carbamoylation, C-17 hydroxylation, C-17 methylation, C-21 hydroxylation, C-4,5 oxidation and oxidation of hydroquinone to quinone, to form GDM finally.2,3 The genes required for GDM biosynthesis have been cloned, sequenced and analyzed from several strains of Streptomyces.3–6 Up to now, the order of post-PKS tailoring process in GDM biosynthesis is only defined partially.3,6,7 The C-7 carbamoylation must take place before C-4,5 oxidation. The C-4,5 oxidation takes place at a later time in the post-PKS tailoring process. The order of benzoquinone modifications of the post-PKS tailoring process, in particular, the order of C-17 hydroxylation and C-21 hydroxylation remains unclear. The isolation of reblastatin from S. hygroscopicus subsp. hygroscopicus SANK61995,8 a GDM producer, and the isolation of reblastatin-like ansamycins (17-O-demethylreblastatin and autolytimicin) from Streptomyces sp. S6699 and S. autolyticus JX-47,9,10 seem to suggest that C-17 hydroxylation (and C-17 methylation) take place before C-21 hydroxylation, but biological evidences are needed to prove this point. We are interested in understanding the biosynthetic details of GDM production. Recently, we reported the characterization of a minor component, 7-O-descarbamoyl-7-hydroxygeldanamycin, from the gdmN (encoding the enzyme for C-7 carbamoylation) disruption mutant of S. hygroscopicus 17997, and provided evidences that C-7 carbamoylation must take place before C-4,5 oxidation in GDM biosynthesis.7 Recently, in our monitoring of secondary metabolites of S. hygroscopicus 17997, a wild-type GDM (300–400 mg l 1) producer, we found a GDM analog from the mid-stage (40–60 h) fermentation broth(s) of S. hygroscopicus 17997 (Supplementary Figures S1 and S2). It was found to exist also in the gdmP (encoding the cytochrome P450 monooxygenase for C-4,5 oxidation) disruption mutant of S. hygroscopicus 17997 (Figures 1 and 2). The compound disappeared from the fermentation broths in older cultures of S. hygroscopicus 17997 or its gdmP disruption mutant, coinciding with the slowdown or stop of GDM or 4,5-dihydrogeldanamycin biosynthesis. Besides, we detected the descarbamoylated form of the compound from the gdmN disruption mutant of S. hygroscopicus 17997 (Supplementary Figure S3). The compound aroused our attention. We isolated the compound and determined its chemical structure, which is 17-O-demethylreblastatin (or 17-hydroxyautolytimicin, compound 3 in Figure 4), a non-benzoquinone GDM analog. In this paper, we reported the re-discovery of 17-O-demethylreblastatin and its verification as a subnormal intermediate of GDM biosynthesis in S. hygroscopicus 17997; besides, we proposed an order of benzoquinone modifications of the post-PKS tailoring process in GDM biosynthesis. Frozen stock spores of the gdmP disruption mutant of S. hygroscopicus 17997 were thawed and spread onto ISPII (0.4% yeast extract, 1.0% malt extract, 0.4% glucose and 1.5% agar power) plates, incubated at 28 1C for 8–10 days, for mycelium growth and sporulation, then slices of the plate culture were picked up as seed and inoculated into the fermentation medium (starch 2 1C, 0.5% cottonseed meal, 0.5% glucose, 1.0% corn steep liquor, 0.5% yeast