Regioselective Alcoholysis of Silychristin Acetates Catalyzed by Lipases

A panel of lipases was screened for the selective acetylation and alcoholysis of silychristin and silychristin peracetate, respectively. Acetylation at primary alcoholic group (C-22) of silychristin was accomplished by lipase PS (Pseudomonas cepacia) immobilized on diatomite using vinyl acetate as an acetyl donor, whereas selective deacetylation of 22-O-acetyl silychristin was accomplished by Novozym 435 in methyl tert-butyl ether/n-butanol. Both of these reactions occurred without diastereomeric discrimination of silychristin A and B. Both of these enzymes were found to be capable to regioselective deacetylation of hexaacetyl silychristin to afford penta-, tetra- and tri-acetyl derivatives, which could be obtained as pure synthons for further selective modifications of the parent molecule.


Introduction
Silymarin [1], a crude extract from fruits of the milk thistle (Silybum marianum), contains flavonolignans occurring as pairs of diastereoisomers [2] (silybin A and B, isosilybin A and B, silychristin A and B) and other related compounds (silydianin, taxifolin; only single stereoisomers). This unique mixture of flavonolignans is largely used in a plethora of nutraceutics due to their hepatoprotective activity and also to other beneficial effects. So far, silybins A and B, accounting for ca. 30% of silymarin, are the only readily-available pure components from silymarin. The compounds in the remaining fraction of silymarin have only been isolated, up to very recently, in small quantities using laborious procedures, e.g., repetitive preparatory HPLC, and so, they could only be used for analytical standards. In 2014, a novel method for the preparatory separation of the minority silymarin components using Sephadex LH-20 gel chromatography was reported. This protocol gives access mainly to pure silydianin and silychristin ( Figure 1) in multi-gram quantities [3]. The content of silychristin in milk thistle fruits varies according to the origin (region) where the milk thistle is grown and according to respective plant cultivars. Natural silychristin (1) exists as a mixture of two diastereoisomers: silychristin A (1a) and silychristin B (1b). Due to the high prevalence of silychristin A in silymarin [3], silychristin was initially considered to be a single diastereoisomer, with the absolute configuration 10R, 11S [4]. Later on, two diastereomeric forms of silychristin in a ratio 9:1 were observed [5], and in 2005, Smith and coworkers isolated a small amount of the second diastereoisomer of silychristin with the configuration 10S, 11R. Its structure was confirmed by NMR, and the compound was named silychristin B (1b) [6].
Silychristin has a number of beneficial properties, compared to the other silymarin flavonolignans. Its antioxidant and antiradical activity is twice as high as that of silybin [7]. This may be due to the higher polarity of this compound (six OH groups in the molecule), which is soluble in protic solvents (contrary to silybin) and, therefore, in principle, is more bioavailable (including water concoctions of Silybum fruits) [8].
The chemistry of flavonoids, similarly to other complex multifunctional natural products, is complicated by the need for complex protection/deprotection strategies due to the large number of chemo-equivalent groups. Further problems stem from the high sensitivity of flavonolignans towards oxidative agents and alkaline conditions and their tendency to complexate some metals. This limits the use of numerous protection/deprotection strategies. The application of enzymes may avoid these limitations, both in terms of selectivity and mild conditions [9].
Enzymatic protection/deprotection of primary OH groups has been reported to be useful in multi-step organic synthesis of flavonolignans. Acyl formations by lipases were used for the preparation of several derivatives of silymarin compounds, e.g., silybin [11]. For instance, acetylation at C-23 OH of silybin diastereomeric mixture and the following selective hydrolysis afforded optically pure diastereoisomers in multi-gram quantities [12][13][14].
In this work, the reaction protocols for the regioselective alcoholysis of silybin diastereoisomers catalyzed by lipase AK [14] have been extended to silychristin. Specifically, this paper presents the results obtained by screening a set of lipases for the enzymatic acetylation and alcoholysis at the C-22 OH group of silychristin. Subsequently, a basic kinetic study on the regioselective alcoholysis of peracetylated silychristin with the best performing lipases allowed the preparation of selectively-acetylated derivatives of silychristin suitable for further chemo-enzymatic modifications.

Enzymatic Acetylation of Silychristin (1)
This study deals with lipase-catalyzed reactions aimed at selectively modifying the molecule of silychristin. The idea of the preparation of 22-O-acetyl silychristin was inspired by our previous work engaged in the diastereomeric discrimination [13] of the mixture of silybin A and silybin B, which was accomplished by the combination of regioselective acetylation at C-23 OH and subsequent alcoholysis of 23-O-acetyl silybin.
A panel of lipases were screened (Table 1), and various solvents (acetone, tert-amyl alcohol, toluene, dioxane, THF) were tested for the acetylation of natural silychristin (1a/1b = 9:1; we use hereunder the term "silychristin (1)" for this mixture if not otherwise stated). The best result was achieved with lipase PS (Pseudomonas cepacia) immobilized on diatomite in acetone in the presence of vinyl acetate as the acyl donor (Scheme 1). This enzymatic acetylation proceeded to give a quantitative yield of 22-O-acetyl silychristin without any traces of side products and allowed a simple reaction mixture workup. However, virtually no diastereomeric discrimination of silychristin A and B was observed during time course study ( Figure 2).

Alcoholysis of 22-O-Acetyl Silychristin (2)
The alcoholysis of 22-O-acetyl silychristin (2) was attempted to achieve silychristin diastereomeric discrimination (Scheme 1). Alcoholysis in various solvents (MTBE, tert-amyl alcohol and toluene), previously found to be suitable for the discrimination of 22-O-acetyl silybin, was tested [14]. Among the two previously successful enzymes-Novozym 435 and lipase PS immobilized on diatomite-the only positive result was obtained with Novozym 435 in a mixture of solvents MTBE and n-butanol. The course of the reaction was monitored by HPLC, evaluating the formation of the diastereoisomers, 1a and 1b ( Figure 3). Once again, no diastereomeric discrimination good enough for the kinetic resolution of the mixture of 22-O-acetyl silychristin A and B (2) was observed.

Screening of Lipases for the Selective Alcoholysis of 3,5,7,15,19,22-Hexa-O-acetyl silychristin (3)
The goal of this set of experiments was to find an efficient method for the preparation of selectively protected silychristin synthons, suitable for further derivatization, from the easily available silychristin peracetate (3).
In this work, thirteen lipases were tested for the selective alcoholysis of 3, and three of them gave conversion >10% (Table 1). Sufficient conversion rates were observed with Novozym 435, lipase PS immobilized on celite and lipase PS immobilized on diatomite. The two lipase PS preparations gave a similar yield; thus, for large-scale reactions, only the commercially available lipase PS on diatomite and the well-known Novozym 435 were used. Once again, no diastereomeric discrimination good enough for kinetic resolution between silychristin A and silychristin B derivatives was observed.

Alcoholysis of 3,5,7,15,19,22-Hexa-O-acetyl silychristin (3)
Based on the above screening, Novozym 435 and lipase PS immobilized on diatomite were used for preparatory-scale alcoholysis experiments. Product formation during the regioselective alcoholysis of 3 was monitored by HPLC (Figures 4 and 5). The reactions catalyzed by Novozym 435 and by lipase PS immobilized on diatomite afforded different products (Scheme 2). All of the compounds were isolated and characterized by mass spectrometry and complex NMR investigation, allowing their unequivocal structural determination. Control experiments proved that no spontaneous alcoholysis of 3 occurred in the absence of the enzymes.   (Figure 4). A TLC of the reaction catalyzed by Novozym 435 is also reported in Figure S4. The three compounds, 6, 7 and 8, could be isolated in pure form. Unfortunately, not all OH signals were visible in the 1 H NMR spectra of the isolated acetyl silychristin derivatives in DMSO-d6. Therefore, it was impossible to use the presence of the signals of some of them (compared to the parent compound) as a proof of the deacetylation site. To overcome this, the four-bond heteronuclear correlation between methyl protons of the acetyl group and the acetylated carbon was used as direct evidence for its substitution. Moreover, the heteronuclear coupling between H-3 or H-22 and the CH3COO of acetyl was used as a direct proof of the acetylation at these positions.
Having the kinetic information in hand, it is now possible to terminate the alcoholysis reactions at specific times to obtain penta-, tetra-or tri-acetyl derivatives of silychristin in good yields.

General Methods
NMR spectra were recorded on a Bruker Avance III 700 MHz spectrometer (700.13 MHz for 1 H, 176.05 MHz for 13 C at 30 °C) (Bruker Daltonik, Bremen, Germany) and a Bruker Avance III 600 MHz spectrometer (600.23 MHz for 1 H, 150.93 MHz for 13 C at 30 °C) in DMSO-d6 (99.8% atom D, ARMAR Chemicals, Dӧttingen, Switzerland). The residual signal of the solvent was used as an internal standard (δH 2.500 ppm, δC 39.60 ppm). NMR experiments-1 H NMR, 13 C NMR, COSY, HSQC, HMBC and 1D TOCSY-were performed using the manufacturer's software. 1 H NMR and 13 C NMR spectra were zero filled to four-fold data points and multiplied by a window function before Fourier transformation. The two-parameter double-exponential Lorentz-Gauss function was applied for 1 H to improve resolution, and line broadening (1 Hz) was applied to get a better 13 C signal-to-noise ratio. Chemical shifts are given in δ-scale with digital resolution justifying the reported values to three (δH) or two (δC) decimal places.
Mass spectra were recorded on a Micromass Platform LC system (Waters, Milford, MA, USA) in methanol with the addition of formic acid.

Conclusions
A panel of lipases was screened for the selective acetylation and alcoholysis of silychristin and silychristin peracetate, respectively. Acetylation at the primary alcoholic group (C-22) of silychristin was accomplished by lipase PS immobilized on diatomite using vinyl acetate in acetone as an acetyl donor. Deacetylation of 22-O-acetyl silychristin (2) could be accomplished by Novozym 435 in MTBE/n-butanol. However, in both of these protocols, no diastereomeric discrimination good enough for the kinetic resolution of silychristin A and B was observed, and therefore, this approach cannot be used for the separation of the silychristin isomers. Both lipase PS immobilized on diatomite and Novozym 435 were found to catalyze the regioselective deacetylation of hexaacetyl silychristin to afford, in a good yield, 3,15,19,22-tetra-O-acetyl silychristin (5), 3,15,22-tri-O-acetyl silychristin (6), 3,5,7,15,22-penta-Oacetyl silychristin (7) and 3,5,15,22-tetra-O-acetyl silychristin (8). These compounds, as well as the silychristin monoacetate (2), can be used as pure synthons for further selective modifications of this valuable natural compound.