4-Vinylphenol acts as a monomer and used in the preparation of poly-4-vinylphenol polymer, which is used in electronics as a dielectric layer in organic transistors in organic thin-film. 4-Vinylphenol solution has been used as an HPLC internal standard during the identification of 4-vinyl derivatives formed via decarboxylation of hydroxycinnamic acids extracted from corn cobs using phenolic acid decarboxylase (PAD) enzyme extracted from Lactobacillus plantarum.

Synthesis of 4-Vinylphenol

A mixture of methyl triphenylphosphonium bromide (1.1 g; 3mmol) in THF (4 mL) was treated with potassium tert-butoxide (562 mg, 5 mmol). After stirring for 10 min at room temperature, a solution of 4-hydroxybenzaldehyde (6c; 244 mg; 2 mmol) in THF (2 mL) was added in drops to the above suspension, and the resulting mixture was stirred at room temperature for 60 min., which was monitored by TLC. The resulting solution was quenched with saturated NH4Cl solution (10 mL), and concentrated in vacuum to remove THF. The concentrated mixture was extracted with CH2Cl2(40 mL). The organic layer was washed with brine(3 X 40 mL) and dried over Na2SO4 anhydrous, filtered, evaporated, and the residue was purified by chromatography column (ethylacetate/nhexane5:95) to give pure 4-Vinylphenol, 200.7 mg (1.97 mmol) 98 % yield, as a light yellow solid, mp 65 °C.20 1H NMR (400 MHz, CDCl3) δ 7.30 (d, J =8.5 Hz, 2H), 6.79 (d, J = 8.6 Hz, 2H), 6.65 (dd, J = 17.6, 10.9 Hz, 1H), 5.60 (d, J = 17.6 Hz, 1H), 5.12 (d, J = 10.9 Hz, 1H), 4.94 (s, 1H).[1]

Evaluation of bacterial hosts for conversion of lignin-derived p-coumaric acid to 4-vinylphenol

Hydroxycinnamic acids such as p-coumaric acid (CA) are chemically linked to lignin in grassy biomass with fairly labile ester bonds and therefore represent a straightforward opportunity to extract and valorize lignin components. In this work, we investigated the enzymatic conversion of CA extracted from lignocellulose to 4-vinylphenol (4VP) by expressing a microbial phenolic acid decarboxylase in Corynebacterium glutamicum, Escherichia coli, and Bacillus subtilis. The performance of the recombinant strains was evaluated in response to the substrate concentration in rich medium or a lignin liquor and the addition of an organic overlay to perform a continuous product extraction in batch cultures. We found that using undecanol as an overlay enhanced the 4-vinylphenol titers under high substrate concentrations, while extracting?>?97% of the product from the aqueous phase. C. glutamicum showed the highest tolerance to CA and resulted in the accumulation of up to 187 g/L of 4VP from pure CA in the overlay with a 90% yield when using rich media, or 17 g/L of 4VP with a 73% yield from CA extracted from lignin. These results indicate that C. glutamicum is a suitable host for the high-level production of 4-vinylphenol and that further bioprocess engineering strategies should be explored to optimize the production, extraction, and purification of 4VP from lignin with this organism.[2]

Three bacterial species were selected in this study for overexpression of a PAD enzyme: E. coli (Ec), B. subtilis (Bs) and C. glutamicum (Cg). Their ability to convert CA to 4VP was evaluated and compared to the species carrying the native gene, B. amyloliquefaciens (Ba). These organisms were chosen because they are common protein expression hosts with multiple engineering tools available and do not consume CA or 4-vinylphenol, with the exception of Cg that is capable of assimilating CA. The metabolic pathway for assimilation of CA and other phenylpropanoids in C. glutamicum has been described in detail by Kallscheuer et al., and interested readers are encouraged to consult that reference. Previous reports have found that the microbial production of 4-vinylphenol can be enhanced by using a biphasic cultivation system. One example is the extraction of 4VP from the aqueous phase with octanol in a continuous flow-bed reactor containing immobilized E. coli cells. Although the partition coefficient for 4VP in a mixture of octanol and water is predicted to be high towards octanol, this solvent is also known to cause significant toxicity to microbes. Therefore, we compared the performance of octanol to other mid-chain fatty alcohols with slightly longer alkane chains, decanol and undecanol, when added as overlays to the cultivation media (20% of the aqueous phase volume). To obtain high product concentrations and identify any limitations to the amount of 4-vinylphenol that can be extracted with the overlays, the four recombinant strains were incubated in the presence of 25 or 75 g/L of CA.

This work compared the performance of three organisms in a 4-vinylphenol production process and demonstrates its production from CA extracted from a solid lignin-rich corn stover bioprocess residue. We found that the plasmid expression of a PAD enzyme in C. glutamicum strains resulted in the highest 4-vinylphenol concentrations reported to date in any culture medium. In contrast, B. subtilis showed the lowest performance of the recombinant strains in all tested conditions. Continuous extraction of 4-vinylphenol from the liquid phase with an organic overlay enabled high product concentrations and yields in batch mode, and the best results (187 g/L of 4VP from pure CA in the overlay with a 90% yield) were obtained when using undecanol.

The natural agent 4-vinylphenol targets metastasis and stemness features

Cancer stem-like cells (CSC) are regarded as the source of tumour origins, metastasis and drug resistance, and limits current treatment regimens. Previously, we reported the first study of the anti-angiogenic and anti-tumour activities of 4-vinylphenol. To further examine the therapeutic role of 4-vinylphenol, the inhibitory effects of 4-vinylphenol on cancer stemness, drug resistance and metastasis in breast cancer were investigated in the present study.  We previously demonstrated the anti-angiogenic and anti-tumor activities of 4VP, the active compound isolated from HD water extract. 4VP inhibited blood vessels formation and suppressed invasive breast tumor growth in vivo. However, the role of 4VP on cancer stem-like cells still remains unknown. The aim of this study was to investigate the inhibitory effects of 4-Vinylphenol on the traits of CSCs in vitro and in vivo. This was the first time to report that 4VP inhibited cancer stem cell properties and suppressed the metastatic activities in CSC-enriched breast cancer cells.[3]

4VP-treated breast cancer cells became resistant to paclitaxel after 4-months incubation. Surprisingly, such long term incubation of breast cancer cells with 4-Vinylphenol could still sensitize breast cancer cells to 4VP, revealing that breast cancer cells were not resistant to 4VP, unlike other chemotherapeutic drugs. Importantly, 4VP suppressed breast CSC-induced metastasis in zebrafish model. We investigated the in vivo metastatic activity of 4VP in zebrafish model rather than mice model since the process is efficient with less than 72 h for breast cancer cell metastasis. Human cancer cells are capable of metastasizing once transplanted into zebrafish larvae, as it only develops an adaptive immune system after 14 days post-fertilization. Our study is also the first to report the inhibitory effects of 4-Vinylphenol on breast cancer stem-like cells.

CSC-enriched MDA-MB-231 cells demonstrated higher tumorigenic and metastatic potential. 4-Vinylphenol reduced spheroid formation and ALDH1 expression in CSC-enriched cultures, revealing its inhibitory effects on the traits of CSCs. 4-Vinylphenol suppressed colony formation and cell proliferation. 4VP also inhibited in vitro invasion and in vivo metastasis in zebrafish model. Our results showed that it reduced vimentin expression, suppressed cell migration, affected the expression and/or activity of MMPs, TIMPs and uPA. In addition, the expressions of caspases 3 and 9 were increased upon its treatment, and surprisingly, prolonged treatment did not confer cancer cells with drug resistance to 4-vinylphenol. 4-Vinylphenol probably exhibited its anti-cancer activities via beta-catenin, EGFR and AKT signaling pathways. 4-Vinylphenol was shown to inhibit metastasis and cancer stemness in CSC-enriched breast cancer cells. Since conventional therapies not targeting CSCs possibly lead to failure to eliminate cancer, 4-vinylphenol is a highly potential therapeutic agent for breast cancer patients.

References

[1]Demidoff, Felipe C.; De Souza, Fabrícia P.; Netto, Chaquip D.[Synthesis, 2017, vol. 49, # 23, p. 5217 - 5223]

[2]Rodriguez A, Meadows JA, Sun N, Simmons BA, Gladden JM. Evaluation of bacterial hosts for conversion of lignin-derived p-coumaric acid to 4-vinylphenol. Microb Cell Fact. 2021 Sep 15;20(1):181.

[3]Leung HW, Ko CH, Yue GG, Herr I, Lau CB. The natural agent 4-vinylphenol targets metastasis and stemness features in breast cancer stem-like cells. Cancer Chemother Pharmacol. 2018 Aug;82(2):185-197.