Discovering and harnessing oxidative enzymes for chemoenzymatic synthesis and diversification of anticancer camptothecin analogues

Semi-synthetic derivatives of camptothecin, a quinoline alkaloid found in the Camptotheca acuminata tree, are potent anticancer agents. Here we discovered two C. acuminata cytochrome P450 monooxygenases that catalyze regio-specific 10- and 11-oxidations of camptothecin, and demonstrated combinatorial chemoenzymatic C-H functionalizations of the camptothecin scaffold using the new enzymes to produce a suite of anticancer drugs, including topotecan (Hycamtin®) and irinotecan (Camptosar®). This work sheds new light into camptothecin metabolism, and represents greener approaches for accessing clinically relevant camptothecin derivatives.


Plants and chemicals
Camptotheca acuminata cuttings were obtained from Quarryhill Botanical Garden (California, USA) and the Huntington Library, Art Collections, and Botanical Gardens (California, USA). The cuttings were snap-frozen upon receipt for RNA isolation. Secologanin, ajmaline, tetrahydroalstonine, serpentine, and yohimbine were purchased from Northernchem Inc. (Ontario, Canada). All other chemicals were of analytical grade from Sigma-Aldrich.

Phylogenetic analysis
Unrooted neighbour-joining phylogenetic tree for CYP450 candidates from this study and other reported CYP450s from other organisms were performed using the Geneious Tree Builder program in the Geneious software package (Biomatters). The names, abbreviations and GenBank accession numbers of the included sequences are: C. acuminata CPT 10-hydroxylase, CaCPT10H, OK631678; C. acuminata CPT 11-hydroxylase, CaCPT11H, OK631675; C.

Yeast culture, microsome preparation and immunoblot analysis
For routine yeast culture, the transgenic yeast strain was inoculated in 2 mL of synthetic complete (SC) medium lacking leucine (SC-Leu) containing 2% (w/v) glucose and cultured overnight at 30 o C and 250 rpm. The culture was subsequently diluted 100-fold to an OD600 of 0.05 in SC-Leu supplemented with 2% (w/v) glucose and cultured for 16 hr. Yeast was then harvested and sub-cultured for 24 hr in YPA medium containing 2% (w/v) galactose to induce the production of recombinant CYP450s. Yeast cells were harvested by centrifugation and lysed for 2 min using a micro-bead beater (VWR) and 500-μm diameter glass beads in TES (0.6 M sorbitol in TE) buffer. The resulting lysate was subsequently centrifuged at 10,000 g for 15 min at 4 o C. The supernatant was then transferred to a new tube and centrifuged at 40,000 g for 60 min at 4 o C. Finally, the pellet containing microsomes was resuspended with TEG buffer (20% (v/v) glycerol in TE). Expression of Ca32229 and Ca32236 was confirmed by immunoblot analysis of microsomal fractions prepared from S. cerevisiae cultures harbouring the pESC-Leu2d::CPR/Ca32229 and pESC-Leu2d::CPR/Ca32236 vectors using α-FLAG M2 antibodies (ThermoFisher Scientific) detectable with SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher Scientific) to probe epitope-tagged recombinant proteins (Supplementary Figure 3).

LC-MS/MS analysis
Enzyme assays were analyzed by ultra-performance liquid chromatography (UPLC) on a Xevo TQ-S Cronos Triple Quadrupole Mass Spectrometry (Waters). For all studies, chromatography was performed on an XBridge BEH XP (10 × 2.1 mm, 1.7 μm) column at a flow rate of 0.6 mL.min -1 . The column was equilibrated in solvent A (0.1% formic acid) and the following elution conditions were used: 0 min, 5% B (100% acetonitrile); from 0 to 3.5 min, 35% B; from 3.5 min to 3.75 min, 100%B; 3.75 min to 4.75, 100%B; 4.75 to 6 min, 5% B to re-equilibrate the column. Data were analyzed with MassLynx and TargetLynx (Waters) For high-resolution MS (HRMS) analysis, new compounds were subjected to the Agilent 1290 Infinity system connected to the Agilent 6530 Quadrupole Time-of-Flight (QTOF). Chromatography was performed on an XBridge BEH XP (10 x 2.1 mm, 1.7 μm) column at a flow rate of 0.6 mL.min -1 . The column was equilibrated in solvent A (0.1% formic acid) and the following elution conditions were used: 0 min, 5% B (100% acetonitrile); from 0 to 3.5 min, 35% B; from 3.5 min to 3.75 min, 100%B; 3.75 min to 4.75, 100%B; 4.75 to 6 min, 5% B to re-equilibrate the column. Data were analyzed with Mass Hunter (Agilent Technologies)

Conversion rate and yield calculation
A calibration curve using camptothecin from 0-50 nM was made for quantification. Peaks areas of LC-MS chromatograms were calculated using MassLynx and TargetLynx from Waters and normalized. The amount of substrate consumption, product formation, conversion, and total product yield was quantified using corresponding calibration curves.

Semi-preparative HPLC and NMR analyses for structure elucidation
A scaled-up yeast in vivo assay with CPT and 7-ethyl-CPT substrates were performed to produce sufficient product quantities of HCPTs and 7-ethyl-HCPT for NMR analysis. The supernatant of the assays was obtained by centrifugation. The crude containing HCPT and 7-ethyl-HCPT in the supernatant were collected by liquid-liquid extraction with ethyl acetate and chloroform, respectively. Product purification from the concentrated sample was performed by a semi-preparative HPLC system with Kinetex ® 5 μm EVO C18 100 Å, 10 x 250 mm column at a flow rate of 1.5 mL.min -1 . The column was equilibrated in solvent A (water, 0.1 % formic acid) and solvent B (0.1% formic acid in acetonitrile). Then, the following elution conditions were used: 0 min, 10 % B; from 0 to 5 min, 20 % B; from 5 to 25 min, 70 % B; from 25 to 27 min, 90 % B; from 27 to 30 min, 90 % B; from 30 to 31 min; 10 % B; from 31 to 34 min, 10 % B to re-equilibrate the column. Approximately 1 mg of each product was independently dissolved in 600 μL DMSO-d6 and subjected to 1 H NMR analysis on Bruker Avance 600 NMR spectrometer. 1D-TOCSY NMR technique (50 ms spin-lock time) were used afterwards to analyze the overlapped aromatic protons signals with irradiation frequency set at 8.02 ppm. The 1 H NMR spectra were analyzed and compared with those of standards and literature for known compounds.

Scale-up and purification of new compounds for chemoenzymatic synthesis of hydroxycamptothecin derivatives
To generate sufficient amounts of HCPTs (10 and 11HCPT) and 7-ethyl-HCPT (7-ethyl-10 and 11HCPT) for the synthesis of topotecan, irinotecan and other compounds, we scaled up the enzymatic reactions. The transgenic yeast strain was inoculated in 2 mL of synthetic complete medium lacking leucine (SC-Leu) containing 2% (w/v) glucose and cultured overnight at 30 o C and 275 rpm. The culture was subsequently diluted to an OD600 of 0.05 in SC-Leu supplemented with 2% (w/v) glucose and cultured for 16 hr. The yeast was then harvested and sub-cultured for 48 hr in YPA medium containing 2% (w/v) galactose, and 10% glycerol to induce the production of recombinant CYP450s. CPT or 7-ethyl-CPT substrate was fed directly into the culture to reach a final concentration of 50 μM as soon as the yeast was switched from SC-Leu to YPA medium. After 48-hr inoculation, a conversion rate of approximately 70% from CPT or 7-ethyl-CPT to its hydroxylated product was obtained and confirmed by LCMS analysis. The supernatant was collected by centrifugation at 4000 rpm, for 5 minutes. HCPT and 7-ethyl-HCPT were extracted out of reaction matrix by liquid-liquid extraction with ethyl acetate and chloroform, respectively. The solvent was removed by using a rotary evaporator to obtain crude HCPT and 7-ethyl-HCPT substrates for chemical synthesis to topotecan and irinotecan. HCPT and 7-ethyl-HCPT were purified by semi-preparative HPLC prior to the synthesis of derivatives.

Semi-synthesis of topotecan and topotecan-11 (12-[(dimethylamino)methyl]-11HCPT)
Fifteen mg of solid N,N-dimethylmethyleneiminium chloride was added into an empty 4 mL reaction flask. Six mg of HCPT substrates from the enzymatic reaction was dissolved by 1 mL isopropanol:chloroform (1:1) and transferred into the reaction flask. Two μL triethylamine was added into the mixture then the reaction mixture was magnetically stirred at room temperature for 24 hr. Then, the mixture was acidified to pH 3-4 with 1 N HCl 1 . The reaction mixture was analyzed by LC-MS/MS method to identify the topotecan product. The solvent in the reaction mixture was removed to dryness in vacuo. The dried reaction mixture was dissolved in methanol and the final product was purified by semi-prep HPLC to yield approximately 4 mg dried product. The dried product was dissolved in DMSO-d6 and subjected to 1 H NMR analysis on Bruker Avance 600 NMR spectrometer in order to elucidate the structure of the final product.

Semi-synthesis of irinotecan and irinotecan-11 (7-ethyl-11-[4-(1-piperidino)-1-piperidino]carbonyloxyCPT)
Six mg of solid 4-piperidinopiperidine-1-carbonyl chloride was added into an empty 4 mL reaction flask. One mg of 7-ethyl-HCPT substrates from the enzymatic reaction was dissolved by 200 μL pyridine and transferred into the reaction flask. The reaction mixture was magnetically stirred at room temperature for 2 hr. The reaction mixture was analyzed by LC-MS/MS method to detect the irinotecan product. Pyridine was removed by rotatory evaporator after 2 hr. The dried crude mixture was dissolved in 300 μL water. Then 1.5 mL dichloromethane was used to extract the irinotecan product out of the mixture. Dichloromethane layer was dried in vacuo to obtain 1.5 mg dried product. The dried product was dissolved in DMSO-d6 and subjected to 1 H NMR analysis on a Bruker Avance 600 NMR spectrometer in order to elucidate the structure of the final product.

Semi-synthesis of brominated HCPTs
An amount of 15 mg of solid N-bromosuccinimide (NBS) was added into an empty 4 mL reaction flask. 3 mgs of dried HCPT substrates from the enzymatic reaction were dissolved by 200 μL DMSO (pre-cooled at 4 o C). After that, the substrate was transferred into the flask containing N-bromosuccinimide on ice. The mixture was magnetically stirred at room temperature in the dark for 2 hr. The reaction progress was analyzed by LC-MS/MS method to detect the brominated HCPT product. Then, the reaction mixture was transferred into 5 mL cold water, the pH of the mixture was adjusted to 3-4 with 1 N HCl 2 . Water and organic solvent were removed by GeneVac evaporator with a temperature below 40 o C. The dried reaction mixture was then dissolved in methanol, and the pure brominated product was purified by semi-prep HPLC to obtain 1.1 mg dried product. The dried product was dissolved in DMSO-d6 and subjected to 1 H NMR analysis on a Bruker Avance 600 NMR spectrometer to determine the position of the bromine substituent position.