Peracetylation of polyphenols under rapid and mild reaction conditions

Abstract Structural modifications are an important tool for studying the properties of naturally occurring polyphenols. Regarding the preparation of acetyl esters, the presence of hydroxyl groups stabilized by intramolecular hydrogen bonds may pose an obstacle for the peracetylation of these compounds. In this paper, we present a facile protocol for the acetylation of selected polyphenols under mild reaction conditions by using acetic anhydride, catalytic amount 4-dimethylaminopyridine (DMAP) and dimethylformamide (DMF) as solvent. Reaction conditions were adjusted for optimal formation of peracetylated polyphenols while minimizing the formation of byproducts. Butyric anhydride was employed as an alternative acylating agent and showed similar results. Reaction yields varied from 78–97%, and products were obtained in high purity, as determined by LCMS(ESI+), 1H NMR and 13C NMR. Graphical Abstract


Introduction
Polyphenols are an important family of natural substances that configure one of the most numerous and widely distributed classes of secondary metabolites of plant origin. In general, phenolic compounds can be defined as substances that have at least one hydroxyl group (OH) bound to a carbon atom at a benzene ring. Phenolic compounds of natural origin can be classified as flavonoid and non-flavonoid polyphenols (Durazzo et al. 2019). They are present in different types of human consumption foods, commonly found as glycosides (Cory et al. 2018). One of the most striking characteristics of such biologically active compounds is related to their ability to act as antioxidants blocking the action of reactive oxygen species (ROS) (Scalbert et al. 2005;Te sanovi c et al. 2017). In addition, many examples are found in the literature describing the bioactivity of naturally occurring polyphenols, such as anti-inflammatory (Kırmı zı bekmez et al. 2019), anti-neurodegenerative (Uddin et al. 2020), anti-carcinogenic (Villota et al. 2021), protective of the cardiovascular system (Sanches-Silva et al. 2020), anti-biofilm, anti-quorum sensing (Pejin et al. 2013), and anti-viral activities (Singh et al. 2020;Mehany et al. 2021). However, the extrapolation of several of the biological activities observed for polyphenols in in vitro to in vivo models conflicts with some physicochemical characteristics of these substances, such as their high polarity. Therefore, the bioavailability of polyphenols is reduced, and their therapeutic efficacy is impaired by their low absorption and reduced ability to cross lipophilic membranes. A good alternative to tackle this problem is to prepare phenyl esters by acetylation of phenolic hydroxyl groups (Munin and Edwards-L evy 2011). This strategy enables an increase in lipophilicity, and esters may be hydrolyzed in the intracellular medium, releasing free polyphenols and thus acting as pro-drugs of the corresponding phenols (Ahmed et al. 2016).
Different methodologies are described in the literature for the preparation of acetylated polyphenol derivatives using both chemical and biochemical reactions (Theodosiou et al. 2009;Selvi and Nagarajan 2018). The peracetylation of polyphenols, however, could be challenging if phenolic OH groups make intramolecular hydrogen bonds with neighboring carbonyl ketone groups, which increases their stability (Lucarini et al. 2004). The chemical acetylation of polyphenols is usually carried out in the presence of acetic anhydride and pyridine (Mattarei et al. 2010). However, pyridine is a toxic solvent (Schultz and Allison 1979), and many experimental procedures describe its use at reflux temperature, especially when the goal is the peracetylation of polyphenolic derivatives (Vogl et al. 2011), which could lead to thermic degradation of the starting polyphenol. In this work, we present a rapid and mild method for the peracetylation of polyphenols using acetic anhydride (Ac 2 O) and dimethylformamide (DMF) as solvent and 4-dimethylaminopyridine (DMAP) as a catalyst. To assess the generality of this methodology, it was evaluated in a range of natural polyphenols, including flavonoids and xanthones, glycosylated or not.

Results and discussion
The use of DMF as a solvent in these acetylation reactions was planned considering its high dielectric constant (e ¼ 37.51 at 25 C). Dimethylformamide is an important organic solvent with a large range of applications. Due to its high polarity, it acts as an aprotic protophilic medium with a large dipole moment (p ¼ 3.86 D at 25 C) and strong solvating power (Corradini et al. 1992). Several studies described in the literature report the DMF's ability to interfere in binary systems formed mainly by hydrogen bonding interactions (Yang et al. 2021). In addition, DMAP was chosen for this protocol due its known effectiveness as a catalyst for acylation reactions and its ease of separation from apolar products (Berry et al. 2001). The reaction optimization was carried out using xanthone a-mangostin (1) as a model polyphenol due to the steric hindrance around its OH groups and the presence of an OH group involved in intramolecular hydrogen bonding. Reaction conditions are shown in Table S1 (see supplementary material), and product formation was monitored by LCMS. We identified two interesting reaction conditions in this study: 18 equivalents of Ac 2 O in the absence of DMAP (entry iv) and in the presence of 0.3 equivalents of DMAP (entry vii), which led to the formation of bis-acetylated and peracetylated products respectively. Both reactions were kept at 45 C. After 1 h for both cases, maximum conversion was achieved (98% and 91%, respectively), while after 2 h, byproducts were formed at higher quantities. Additionally, higher amounts of DMAP (0.5 and 1.0 equivalents) increased the rate of formation of byproducts both after 1 h or 2 h. Thus, we established the optimal conditions for the peracetylation protocol as 6 equivalents of Ac 2 O per OH group and 0.3 equivalents of DMAP at 45 C.
After reaction completion (monitored by TLC), the products were isolated from the reaction medium by liquid-liquid extraction using ethyl acetate and aqueous solutions: 10% NH 4 Cl (3Â), 10% NaHCO 3 (3Â) and 10% NaCl (3Â). The products were recovered from the organic phase and characterized by 1 H and 13 C NMR. The reaction conditions and yields for the peracetylated polyphenols 1a-8a are shown in Table 1.
Additionally, some polyphenols (2, 4, 6) reacted under the same conditions in the presence of butyric anhydride to assess the feasibility of introducing a different acyl group. The corresponding perbutyrylated esters (2b, 4b, 6b) were obtained after 1 h, and yields for isolated products matched those found for acetyl esters (88-97%).

Experimental
The full experimental data, spectra and additional images are in the supplementary material.

Conclusions
By using DMF as the solvent and catalytic amounts of DMAP, it was possible to obtain peracetylated derivatives (1a-8a) from naturally occurring polyphenols (1-8) at all OH groups, including the phenolic hydroxyls involved in intramolecular hydrogen bonds. The esters were completely formed after 0.5 to 2 h and, after purification by liquid-liquid extraction, were isolated in high yields and high purity grades. The same protocol was employed to obtain the butyric esters of selected polyphenols (2b, 4b, 6b), and the results matched those observed for acetylation reactions.