BIOCATALYSIS OF PRECURSORS TO NEW-GENERATION SB-T-TAXANES EFFECTIVE AGAINST PACLITAXEL-RESISTANT CANCER CELLS
Taxus acyltransferases of the BAHD acyltransferases plant superfamily were used as an alternative method for the biocatalytic production of the next-generation paclitaxel analog precursors. A Taxus 10-deacetylbaccatin III: 10-O-acetyltransferase (DBAT) was used to install cyclopropane carbonyl and propionyl groups at the C10 position of 10-deacetylbaccatin III (10-DAB). The kcat and KM of the acyltransferase for cyclopropanecarbonyl CoA (0.83 s−1, 0.15 M) and n-propionyl CoA (1.2 s−1, 0.15 M) guided scale-up efforts. The 10-acyl-10-O-deacetylbaccatin III analogs (∼45 mg each) were made by the acyltransferase when incubated with the commercial taxane 10-O-deacetylbaccatin III and synthesized cyclopropanecarbonyl or n-propionyl CoA. The structures of the 10-acyl products were verified by NMR analyses that confirmed C10 acylation of the taxane substrate. LC/ESI-MS/MS analysis also supported the identities of the 10-O-n-propionyl-10-O-deacetylbaccatin III and 10-O-cyclopropanecarbonyl-10-O-deacetylbaccatin III biocatalyzed products. This effort provides a biocatalysis framework to produce new-generation taxane precursors. A Taxus taxane-2-O-benzoyltransferase (mTBT) biocatalyzed the de-aroylation and re-aroylation of next-generation taxane precursors of drugs effective against multidrug-resistant cancer cells. Various taxanes bearing an acyl, hydroxyl, or oxo group at C13 were screened to assess their turnover by mTBT catalysis. The 13-oxotaxanes were the most productive where 2-O-debenzoylation of 13-oxobaccatin III was turned over faster compared to 13-oxo-10-O-(n-propanoyl)-10-O-deacetylbaccatin III and 13-oxo-10-O-(cyclopropane carbonyl)-10-O-deacetylbaccatin III, yielding ~20 mg of each. mTBT catalysis was likely affected by an intramolecular hydrogen bond with the C13-hydroxyl; oxidation to the 13-oxo recovered catalysis. The experimental data for the debenzoylation reaction was supported by Gaussian-accelerated molecular dynamics simulations that evaluated the conformational changes caused by different functional groups at C13 of the substrate. These findings also helped postulate where the 2-O-benzoylation reaction occurs on the paclitaxel pathway in nature. mTBT rearoylated the debenzoylated 13-oxobaccatin III acceptors fastest with a non-natural 3-fluorobenzoyl CoA among the other aroyl CoA thioesters evaluated, yielding ~10 mg of each with excellent regioselectivity at laboratory scale. Reducing the 13-oxo group to a hydroxyl yielded key modified baccatin III precursors (~10 mg at laboratory scale) of new-generation taxoids. The role of Mg2+ ions in the Taxus baccatin III: 3-amino-3-phenylpropanoyltransferase (BAPT) catalysis has been studied. This hypothesis was tested by screening phenylisoserine CoA with baccatin III. The results suggested that Mg2+ ions are critical for the BAPT catalysis by interrupting the intramolecular hydrogen bond between C13 hydroxyl group and OAc, organizing the amino acid active site, and act as oxyanion hole by stabilizing the negative charge form in the tetrahedral intermediate. Further, the BAPT, Mg2+ independent catalysis was able to transfer the isobutenylisoserinyl at C13 position of taxnne cores selectively and product the next generation paclitaxel precursors (~10 mg at laboratory scale).
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- In Collections
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Electronic Theses & Dissertations
- Copyright Status
- In Copyright
- Material Type
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Theses
- Authors
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Al-Hilfi, Aimen Sabah
- Thesis Advisors
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Walker, Kevin
- Committee Members
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Hong, Heedeok
Wang, Tuo
Draths, Karen
- Date Published
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2024
- Subjects
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Chemistry
- Program of Study
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Chemistry - Doctor of Philosophy
- Degree Level
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Doctoral
- Language
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English
- Pages
- 375 pages
- Permalink
- https://doi.org/doi:10.25335/80pf-2a40