Identification of Fredericamycin E from Streptomyces griseus: Insights into Fredericamycin A Biosynthesis Highlighting Carbaspirocycle Formation

Abstract

Fredericamycin (FDM) A ( 1), a pentadecaketide featuring two sets of peri-hydroxy tricyclic aromatic moieties connected through a unique asymmetric carbaspiro center, exhibits potent cytotoxicity and represents a novel anticancer drug lead. We have localized previously the fdm gene cluster to a 33 kb DNA segment of Streptomyces griseus ATCC49344, the involvement of which in the biosynthesis of 1 was confirmed by gene inactivation, complementation, and heterologous expression experiments. We now report the isolation and characterization of FDM E ( 5), a heretofore undetected intermediate for 1 biosynthesis from S. griseus, shedding new insight into the mechanism of carbaspirocycle formation. The structure of 5 was elucidated through the combination of spectroscopic methods and isotope-labeling experiments. The core spiro[4.5]decane scaffold of 5 is characterized by a unique cyclohexa-1,2,4-triketone moiety. Transformation of the spiro[4.5]decane 5 into the spiro[4.4]nonane 1 can be rationalized by a biosynthetic benzilic acid-like rearrangement. This unusual rearrangement can be mimicked in vitro by proceeding under aerobic conditions in the absence of enzyme. FDM E displays cytotoxic activity on par with 1 against a selected set of cancer cells, a finding that further supports the unique molecular topology, resulting from the unprecedented carbaspirocycle as exemplified by 1 and 5, as a novel pharmacophore for this family of anticancer agents.

Characterization of FdmV as an Amide Synthetase for Fredericamycin A Biosynthesis in Streptomyces griseus ATCC 43944

“…FDM C was eluted mainly with a 70:30 ratio of CHCl 3 -CH 3 OH. These fractions containing FDM C were then concentrated and subjected to silica gel chromatography (230 -400 mesh; Natland Inc., Morrisville, NC) eluted with CHCl 3 -CH 3 The FDM C-containing fractions were finally concentrated and purified by semipreparative HPLC on an Alltima C-18 column (5 m, 250 ϫ 10 mm; Alltech., Deerfield, IL) as described previously (19).…”

“…Chemical Synthesis of FDM B-FDM C (40 mg) was mixed with 1 g of CH 3 CO 2 NH 4 in 3 ml of acetic acid and heated at 60°C for 12 h to afford crude FDM B. FDM B was then purified by semipreparative HPLC and confirmed by spectroscopic analyses as described previously (19).…”

“…Analytical HPLC was performed on a Microsorb-MV 100-5 C-18 column (5 m, 250 ϫ 4.6 mm, Varian), and the semipreparative HPLC was on an Alltima C-18 column (5 m, 250 ϫ 10 mm, Alltech). The HPLC conditions have been described previously (19).…”

“…2). Finally, the lactam ring F can be found in both FDM A and FDM E, indicating that the spirocyclic moiety is installed following amide formation (19). Bioinformatics analysis, however, failed to reveal any typical amide synthetase gene within the cloned fdm biosynthetic gene cluster.…”

“…We previously cloned and sequenced the fdm gene cluster, setting the stage to study the biosynthetic pathway responsible for FDM A production (17). Reexamination of S. griseus wild-type fermentation recently resulted in the isolation of three FDM analogs FDM B, FDM C, and FDM E (18,19) (Fig. 2).…”