Synthesis of a Novel Cysteine-Incorporated Anthraquinone Derivative and Its Structural Properties

A novel cysteine-incorporated anthraquinone derivative was synthesized, and its molecular structure was determined by X-ray crystal analysis. Each mercapto group was located separately and did not form a disulfide bond, and hydrogen bondings and π-π interaction were observed from the packing structure.


Introduction
In the field of artificial molecular modeling, cysteine is an attractive compound because of its unique properties. In living organisms, cysteine plays important roles such as protein folding or antioxidant activity depending on the sulfur moiety it contains [1][2][3]. The protein morphology related OPEN ACCESS to cysteine can change drastically because of the formation of a disulfide bond by oxidation. Therefore, the construction of artificial molecules containing mercapto moieties is difficult, because a multi-step synthes is required owing to the reactivities of the mercapto group. For example, in the case of two adjacent cysteine residues, a disulfide bond is easily formed by oxidation, and occasionally, enzymes work well by exploiting such reactivities of cysteine.
From these viewpoints, the construction of cysteine-containing artificial molecules is important for opening up new possibilities for elucidating the functionalities of cysteine. Herein, we report the synthesis and structural properties of a novel cysteine-incorporated anthraquinone derivative.

Results and Discussion
According to the reported method [21], cysteine was converted to S-trityl-L-cysteine methyl ester (2) as shown in Scheme 1. At first, the mercapto group of cysteine was protected by a trityl group. S-Trityl-L-cysteine methyl ester (2) was prepared by esterification by using SOCl2.  After the coupling reaction of 2 and 5, the reaction mixture was subjected to silica-gel column chromatography to afford anthraquinone bearing S-trityl-L-cysteine methyl esters 6. Deprotection was conducted using triethylsilane and trifluoroacetic acid, and product anthraquinone bearing L-cysteine methyl esters 7 was also purified by silica-gel column chromatography (Scheme 3). From the 1 H-and 13 C-NMR, IR, and mass spectral measurements, 7 was synthesized successfully.  To apply this synthetic protocol to other chalcogen-containing amino acids, L-methionine methyl ester and L-selenomethionine methyl ester were also introduced into the anthraquinone system, successfully affording methionine-anthraquinone (8) and selenomethionine-anthraquinone (9), respectively (Scheme 4). L-Methionine methyl ester hydrochloride and L-selenomethionine methyl ester hydrochloride were prepared in the same manner as that of S-trityl-L-cysteine (shown in Scheme 1) and treated with 5. After purification of the respective reaction mixtures by column chromatography, the corresponding anthraquinone bearing L-methionine methyl esters 10 and anthraquinone bearing L-selenomethionine methyl esters 11 were obtained. We also attempted to apply this protocol to selenocysteine-incorporated anthraquinone, however, the corresponding trityl-group-protected selenocysteine could not be obtained from commercially available L-selenocystine (Se(Cys)2). To clarify the molecular structure, recrystallization of each anthraquinone derivatives was carried out and a single crystal was successfully obtained by recrystallization of compound 7 from acetonitrile. The molecular structure as determined by X-ray crystal analysis is shown in Figure 1. The two cysteine chains of 7 were oriented in a similar manner, and the two carbonyl groups at the 1,8-positions were incorporated perpendicularly to the anthraquinone rings with torsion angles of 61.09° and 83.04°, respectively [23][24][25][26][27][28][29]. About the anthraquinone moiety, in contrast to the reported structure, both benzene rings were twisted with a torsion angle of 8.25°. Each mercapto group was located individually, and the distance between them was 7.205 Å in the molecule. Table 1 summarizes the selected bond lengths and angles.

D-H ••• A D-H H ••• A D ••• A D-H •••
The photochemical properties were elucidated from the UV-vis and fluorescence spectra. Compound 7 was dissolved in acetonitrile (2.0 × 10 −3 M) and measurements were carried out (Figure 4a). In the UV-vis spectrum, an absorbance peak was observed at 335 nm (ε = 110) [31]. The solid-state spectrum was also measured by depositing onto Pyrex glass (Figure 4b). Although a broadened peak was observed around 400-500 nm, there was no shift in the absorbance peak, thus each amide group is oriented in a perpendicular manner. The fluorescence excited at 350 nm appeared at 400-600 nm in Supplementary Materials (Fluorescence spectra of 7), which corresponds to the anthraquinone moiety as reported [23][24][25][26].

Crystal Structures of 7
Of the 23,904 reflections were collected, 5463 were unique (Rint = 0.0368); equivalent reflections were merged. The linear absorption coefficient, µ, for Mo-Kα radiation is 2.771 cm −1 . An empirical absorption correction was applied which resulted in transmission factors ranging from 0.823 to 0.992. The data were corrected for Lorentz and polarization effects.
The structure was solved by direct methods1 and expanded using Fourier techniques. The non-hydrogen atoms were refined anisotropically. Hydrogen atoms were refined using the riding model.

Synthesis of 10
Anthraquinone-1,8-dicarbonyl chloride (0.24 g, 0.72 mmol) was prepared as described in the synthesis of 7 and dissolved in dry CH2Cl2 (20 mL) and methionine methylester hydrochloride (0.24 g, 1.44 mmol) was added into the flask, the solution was stirred at 0 °C in the ice bath under an inert atomosphere with dropwise addition of diisopropylethylamine (0.5 mL, 2.88 mmol). The resulting mixture was allowed to warm to ambient temperature and was stirred overnight. After evaporation of solvents, the resulting brown oil was purified by silica gel column chromatography without extraction (Rf = 0.11, eluant hexane/EtOAc = 1:1) to yield 49% of anthraquinone-Met(OMe) (yellow solid). Mp 78-80 °C; 1

Synthesis of 11
Anthraquinone-1,8-dicarbonyl chloride (0.409 g, 1.22 mmol) was prepared as described in the synthesis of 7 and dissolved in dry CH2Cl2 (30 mL) and selenomethionine methylester hydrochloride (0.606 g, 2.45 mmol) was added into the flask, the solution was stirred at 0 °C in the ice bath under inert atomosphere with dropwise addition of diisopropylethylamine (0.855 mL, 4.88 mmol). The resulting mixture was allowed to warm to ambient temperature and was stirred overnight. After evaporation of solvents, the resulting brown oil was purified by silica gel column chromatography without extraction (Rf = 0.11, eluant hexane/EtOAc = 1:1) to yield 75% of anthraquinone-SeMet(OMe) (yellow solid).

Conclusions
In summary, we synthesized novel cysteine-, methionine-, and selenomethionine-incorporated anthraquinone derivatives. A single crystal of cysteine-incorporated anthraquinone was obtained, and its crystal structure was successfully confirmed. Intermolecular hydrogen bonds were observed in the crystal structure of 7, and the molecules are linked into an infinite chain by hydrogen bonding. Although the results of UV-vis. spectra in solution and in the solid state indicated that the intermolecular interaction does not seem to be so strong, this molecule may act as a metal receptor based on the cysteine moieties, as the mercapto groups are individually detected, without the formation of disulfide and hydrogen bonds. The investigation of the electrochemical and coordination properties of this derivative is currently underway.