Camptothecin (CPT), a quinoline alkaloid from *Camptotheca acuminata*, is a potent anticancer agent, but its clinical use is limited due to its hydrophobicity and side effects. Semi-synthetic derivatives, such as topotecan and irinotecan, overcome these limitations and are widely used in cancer treatment. However, their production relies on chemical synthesis, which often involves harsh conditions and toxic reagents. The limited natural abundance of CPT and its hydroxylated derivatives (HCPTs) further restricts the diversification of CPT analogues. This study aimed to discover and harness enzymes from *C. acuminata* that catalyze the regio-specific oxidation of CPT, providing a greener and more efficient route to producing clinically useful derivatives. The research hypothesized that enzymes within the cytochrome P450 monooxygenase family are responsible for the oxidative modifications of CPT observed in the plant, and that these enzymes could be leveraged for chemoenzymatic synthesis. This approach offers a significant improvement over current chemical synthesis methods by providing regio- and stereoselectivity under milder conditions, making it more efficient, cost-effective and environmentally friendly. The development of such a method would address the limitations of current production strategies, which are associated with purity, scalability, high costs, and reliance on toxic reagents. The successful isolation and characterization of these enzymes could unlock a new era of camptothecin-based drug development, providing access to a wider array of potent anticancer agents.

The literature extensively covers camptothecin's antitumor activity and its semi-synthetic derivatives, such as topotecan and irinotecan, which are clinically used to treat various cancers. Studies have highlighted the challenges associated with the chemical synthesis of these derivatives, which involves harsh conditions and toxic reagents. Previous research has focused on isolating CPT from plants and chemically modifying it to produce the desired derivatives. However, these chemical methods often have limitations in terms of scalability, purity, and regioselectivity. Prior work has also explored biocatalysis in the biosynthesis and biotransformation of CPT, showing the potential of enzyme-based processes for efficient and selective modifications under mild conditions. This prior work, therefore, sets the stage for the current study’s focus on identifying and exploiting specific enzymes in *C. acuminata* that can catalyze the regiospecific oxidation of CPT, resulting in a more sustainable and efficient means of creating clinically relevant camptothecin derivatives.

The researchers used a targeted metabolomics approach to analyze the distribution of CPT and its oxidative derivatives in different parts of the *C. acuminata* plant. They found higher levels of HCPTs in stems, fruits, and bark, suggesting that the enzymes responsible for their production are highly expressed in these tissues. Based on this, they focused their search for CPT oxidative enzymes within the cytochrome P450 monooxygenases (CYP450s) using transcriptome and genome data. Nine candidate CYP450 genes were identified based on their expression patterns. These genes were cloned into a yeast expression vector and expressed in yeast cells. The activity of the candidate enzymes was assessed by incubating the yeast cultures with CPT and analyzing the products using LC-MS. One candidate, C23236, showed the production of 10-hydroxy-CPT, confirmed by LC-MS and NMR. Another candidate, C23229, produced 11-hydroxy-CPT. To confirm the structure of the products, NMR spectroscopy (¹H, ¹³C, and 1D-TOCSY) was used. Subsequently, the substrate scope of the enzymes (C23236 and C23229, renamed CPT 10-hydroxylase (CPTI) and CPT 11-hydroxylase (CPTII), respectively) was investigated using a panel of 18 alkaloids. The chemoenzymatic synthesis of topotecan and irinotecan was demonstrated using the discovered enzymes. This involved enzymatic hydroxylation followed by chemical modification. LC-MS analysis was used to monitor the reaction progress and identify the products. The final products were characterized using various analytical techniques, including LC-MS and NMR, confirming the synthesis of topotecan and irinotecan via the chemoenzymatic approach.

The study successfully identified and characterized two cytochrome P450 monooxygenases from *C. acuminata* that catalyze the regio-specific oxidation of camptothecin at the C-10 and C-11 positions, respectively. These enzymes, named CPT 10-hydroxylase (CPTI) and CPT 11-hydroxylase (CPTII), showed high regioselectivity for their respective hydroxylations. CPTI produced 10HCPT and CPTII produced 11HCPT. Importantly, both enzymes accepted various CPT derivatives as substrates, enabling the chemoenzymatic synthesis of clinically relevant anticancer drugs such as topotecan and irinotecan. The chemoenzymatic synthesis of topotecan from CPT proceeded with 62-67% conversion rate under mild conditions (pH 7, 30°C). The synthesis of irinotecan from 7-ethylcamptothecin also showed high efficiency. The study also revealed that CPTI and CPTII produced additional, previously unreported, camptothecin derivatives, expanding the chemical space of potential anticancer agents. The substrate scope analysis showed that the enzymes had a limited substrate range; they primarily acted on the CPT scaffold and closely related derivatives. The findings also highlight the potential of this approach for developing more potent and less toxic anticancer drugs.

The discovery of these regio-specific CPT hydroxylases provides a significant advancement in the sustainable and efficient production of camptothecin analogues. The chemoenzymatic approach offers several advantages over traditional chemical synthesis, including higher regioselectivity, milder reaction conditions, and reduced reliance on toxic reagents. The ability to synthesize clinically relevant drugs such as topotecan and irinotecan using this approach opens up new avenues for drug development. The identification of novel camptothecin derivatives produced by the enzymes further expands the possibilities for creating more effective anticancer agents. This method offers considerable advantages over existing methods, highlighting the potential of biocatalysis for developing more effective and sustainable cancer therapies. The high conversion rates observed for the chemoenzymatic synthesis suggest that this method is scalable and suitable for industrial production. The study's findings have important implications for both basic research and drug development, paving the way for developing new therapeutic approaches to combat cancer.

This research successfully identified and characterized two novel cytochrome P450 enzymes from *C. acuminata* that catalyze the regio-specific hydroxylation of camptothecin, leading to a more efficient and environmentally friendly method for producing valuable anticancer drugs. The chemoenzymatic approach offers significant advantages over traditional chemical synthesis, paving the way for the development of novel and more effective anticancer therapies. Future research could focus on exploring the potential of these enzymes in synthesizing other camptothecin analogues and optimizing the chemoenzymatic process for industrial-scale production. Further investigation into the enzymes' structure and mechanism could also enhance their efficiency and broaden their substrate scope.

The study's substrate scope analysis showed that the enzymes exhibited a relatively limited substrate range, primarily accepting CPT and closely related derivatives. This limitation could be addressed through protein engineering to broaden substrate specificity. The study primarily focused on the synthesis of topotecan and irinotecan, and further investigation is needed to evaluate the potential of the enzymes in synthesizing a broader range of CPT analogues. The in vivo efficacy of the newly synthesized compounds needs further investigation in animal models and clinical trials.