Oligomerization of optically active N-(4-hydroxyphenyl)mandelamide in the presence of β-cyclodextrin and the minor role of chirality

Summary The oxidative oligomerization of a chiral mandelamide derivative (N-(4-hydroxyphenyl)mandelamide, 1) was performed in the presence of horseradish peroxidase, laccase and N,N'-bis(salicylidene)ethylenediamine-iron(II) to obtain chiral oligophenols 2. The low enantioselectivity of the enzymatic catalyzed asymmetric enantiomer-differentiating oligomerizations was investigated. In addition, the poor influence of cyclodextrin on the enantioselectivity of enzymatic catalyzed asymmetric enantiomer-differentiating oligomerizations was studied.


Introduction
Lignin consists of mechanical stabilizing polyphenols and thus plays an important role in many plants [1,2]. For the in vitro synthesis of polyphenols via oxidative coupling reactions, laccases and peroxidases are suitable enzymes to catalyze the oligomerization of substituted electron-rich phenols in the presence of oxidizing agents [3,4]. In addition to that, N,N'-bis(salicylidene)ethylenediamine-iron(II) (iron(II)-salen) represents an alternative catalyst for oxidative coupling reactions of phenol derivatives [5]. The use of β-cyclodextrin (CD) allows the oxidative coupling of poor water soluble phenol derivatives via complexation without using of organic solvents [6][7][8]. However, to the best of our knowledge, there were no studies published dealing with the potential enantioselective control of enzyme catalyzed oligomerization reaction of chiral phenol derivatives, respectively. Thus, the enantioselectivity of enzymatic asymmetric enantiomer-differentiating oligomerizations of a chiral mandeloamide-phenol derivative as model compound and the influence of cyclodextrin is a main subject of the present study.

Results and Discussion
The chiral N-(4-hydroxyphenyl)mandelamide (1) was synthesized through condensation of p-aminophenol with (R)-or (S)mandelic acid, respectively in presence of dicyclohexylcarbodiimide as condensing agent. For oligomerization of 1 via oxidative coupling laccase from Pleurotus ostreatus, peroxidase from horseradish or iron(II)-salen were used as catalysts. The obtained yellow powdery oligomers 2 show high solubility in many commonly used organic solvents like acetone, THF, ethanol, methanol, acetonitrile and 1,4-dioxan. Because of the broad signals of the oligomers 2 in the 1 H NMR spectra the ratio of the phenylene and oxyphenylene units (Scheme 1) could not be clearly determined.
The oligomerization of 1 in water could be easily performed through complexation of the monomer 1 with randomly methylated β-cyclodextrin (RAMEB-CD). The formation of the complex was verified with 2D ROESY NMR spectroscopy. The magnetic interaction of the monomer with the cavity of RAMEB-CD is obvious in the 2D ROESY NMR spectra as shown in Figure 1 (marked areas). Principally, cyclodextrins and their derivatives are able to discriminate enantiomeric com- pounds [9,10]. Such chirality recognition is provable with 1 H NMR spectroscopy because of the different induced shift of the protons which became diastereotopic through complexation [11,12]. Actually, the chirality discrimination of 1 with RAMEB-CD is evident from the different induced shift of the protons 8 at 5.2 ppm (zoomed out in Figure 1).
The MALDI-TOF MS measurements indicate the formation of oligomers 2 from the monomer 1 as shown in Figure 2. As expected the repetitive unit has a molecular mass of 241 g/mol, which confirms the linkage of the monomers via a formal abstraction of two hydrogen atoms. The highest molecular weight oligomers 2 obtained through enzymatic oligomerization consists of up to 10 repetitive units which could be detected by MALDI-TOF MS measurements. Furthermore comparable molecular weights are accessible through oligomerization of 1 with iron(II)-salen as catalyst. Here oligomers 2 with up to 8 repetitive units are detectable.
The conversion of the enantiomers of 1 during the enzymatic oligomerization has been studied using chiral HPLC. Accordingly, the racemate of 1 was oligomerized three times with each enzyme in the absence of RAMEB-CD or in the presence of RAMEB-CD, respectively to evaluate the reproducibility. The isolated monomeric residual of each oligomerization was measured twice. The obtained enantiomeric excess (ee) values of the monomeric residual are given in Table 1. Because of the rapid conversion of the monomer 1 during the oligomerization with highly active peroxidase-H 2 O 2 system at room temperature, the reaction time was limited to one minute at 0 °C. In the presence of the lower active laccase-O 2 system, the reaction was carried out for 4 h at room temperature. In the absence of RAMEB-CD it is apparent that laccase shows no enantioselectivity. However it can be established that during the oligomerization with peroxidase the (S)-enantiomer 1 slightly enriches the reaction solution. Additionally to that, it was of some interest to verify, whether the complexation of the enantiomers with RAMEB-CD affects the conversion of the enantiomers. Therefore, the relatively slow oligomerizations in the presence of laccase were carried out in pH 5 buffer at room temperature for 4 hours. The rapid oligomerizations in the presence of peroxidase were carried out in pH 7 buffer at 0 °C for 1 min. It was found that, in the presence of RAMEB-CD, the (R)enantiomer of 1 slightly enriches the reaction mixture with laccase as well as with peroxidase. As already mentioned above, the opposite effect was observed when the oligomerization was carried out with peroxidase without using RAMEB-CD. However, the obtained data show that the degree of enantioselectivity during conversion of 1 is generally very low.

Conclusion
Oligomers of both enantiomers of N-(4-hydroxyphenyl)mandelamide (1) were obtained though oxidative coupling with peroxidase and laccase and also via oligomerization with iron(II)salen and hydrogen peroxide as a catalyzing system. In the presence of RAMEB-CD it was possible to oligomerize the poorly water soluble enantiomers of 1 without using organic solvents.
In the case of oligomerization with laccase the monomeric residual keeps racemic. In contrast to this, with peroxidase the (S)-enantiomer of 1 slightly enriches the monomeric residual. RAMEB-CD promotes the (S)-enantiomer of 1 which was more readily converted with both enzymes. However this preference is very low in all cases.

Experimental Materials
Horseradish peroxidase practical grade I (370,2 U/mg) was purchased from Appli Chem and Laccase from pleurotus ostreatus (9,4 U/mg) from Sigma-Aldrich. p-Aminophenol was bought from Grüssing, N,N'-dicyclohexylcarbodiimide from Appli Chem, N-hydroxysuccinimide from Fluka and RAMEB-CD from Wacker Chemie AG. Racemic-and (S)-mandelic acid was obtained from Merck and (R)-mandelic acid from TCI. The used buffers were ordered from Carl Roth. The solvents used for synthesis were used in p.a. quality. Technical solvents used for column chromatography were distilled before usage.