SYNTHESIS AND PURIFICATION OF 36 AROMATIC CHOLINE ESTERS AS STANDARDS

Jean-Paul Wathelet, Nicolas Mabon and Michel Marlier

Faculté universitaire des Sciences agronomiques, Unité de chimie générale et organique

Passage des Déportés, n°2, B-5030 Gembloux, Belgium

e-mail: wathelet.jp@fsagx.ac.be

ABSTRACT

Natural aromatic choline esters occurring in seeds of Brassicaceae are determined, after extraction with a mixture of methanol and acetic acid 0.01N (70/30), by high performance liquid chromatography. So, from a qualitative and a quantitative point of view, it is very useful to have in the laboratory pure standards for confirm retention times, response factors and for the constitution of u.v., infra-red or mass spectra libraries. As choline esters are not available by a commercial way, we decided to synthesise different aromatic choline esters (36). The structure of the acidic part of all the choline esters prepared are derived from benzoic or cinnamic acids with hydroxy or methoxy substituants in ortho, para or meta position. Pure aromatic choline esters were synthesised according to a fast method using bromocholine bromide. An aromatic acid (in excess) in methanol is first neutralised by NaOH (0.1 N). Then bromocholine bromide in methanol is added in the evaporation flask. Solvent is evaporated to dryness using a rotative evaporator Büchi (40°C). The flask containing the dried mixture is placed in an oven (107°C) during 5 hours, avoiding carbonisation. The choline ester is taken up with 2 x 3 ml of distilled water and purified on a SP Sephadex C25-120 resin. After washing with distilled water, the ester is eluted HCl (1N). The elution is followed by measuring u.v. absorbance at 280 nm. The eluate is evaporated to dryness with a rotative evaporator (Büchi) and the crystals obtained are washed with acetone. The purity obtained for all the choline esters produced was close to 98% and total amount between 5 to 100 mg. An u.v. spectra library of each aromatic choline esters has been constituted for rapid identification when a diode array detector is coupled with the HPLC.

KEYWORDS

Brassicaceae, rapeseed, sinapine, nutrition, reference material, antinutritional factor

INTRODUCTION

Choline esters are present in the living cells. Acetylcholine (Hartman et Kilbinger, 1974; Miura and Shih, 1984a) has an important physiological role in nervous tissues. Propionylcholine has been also identified in plants (Miura and Shih, 1984b). Aromatic choline esters (quaternary ammonium compounds) are found especially in plants. About 13 natural esters have been identified until now in vegetals (Ploeger et al., 1985). Sinapine is the major compound in a lot of Brassicaceae. In rapeseed, sinapine represents more than 80% of choline esters (Krygier et al., 1982; Kozlowska et al., 1983, Pokorny and Reblova, 1995). Choline esters are easily broken down into trimethylamine who restrict utilisation of high quality rapeseed protein as food and feed: a "fishy" odour in eggs (Fenwick et al., 1981), a disagreeable taste in the meat of calves (Anderson et al., 1984) and in the milk of cows (Andersen and Andersen, 1982). That is the reason why the measurement of these choline esters is important. Natural aromatic choline esters are determined after extraction by high performance liquid chromatography. So, it is very useful to get in the laboratory pure standards in order to confirm the retention times, the response factors and for the constitution of u.v., infra-red or mass spectra libraries. As choline esters are not available by a commercial way, we decided to synthesise different aromatic choline esters from available aromatic acids (36). Synthesis method with bromocholine bromide used by Clausen et al. (1983) is improved.

MATERIALS and METHODS

Reagents

All chemicals were analytical grade obtained from commercial sources.

Purity determination

The purity of choline esters is determined by HPLC (Hewlett Packard, serie 1050) using an Inertsil 5 ODS-2 (3 x 250 mm, 5 µm) column and a ternary solvent gradient (water-acetonitrile-phosphate buffer: NaH₂PO₄, 20 mM at pH 2 with o-phosphoric acid) (wavelength: 280 or 335 nm).

RESULTS and DISCUSSION

Choline esters are prepared by reaction between an aromatic acid and the bromocholine bromide (Figure 1).

Figure 1: Reaction of a cinnamic acid with the bromocholine bromide

The acidic part of the 36 choline esters prepared is derived from benzoic or cinnamic acids with hydroxy or methoxy substituants in ortho, para or meta position (Table 1).

Synthesis

The pure aromatic choline esters were synthesised according to a fast method using bromocholine bromide. The aromatic acid in methanol (in excess, 1.1 mmol) is first neutralised by NaOH (0.1 N). Then bromocholine bromide in methanol (0.85 mmol) is added in a 100 ml flask. Solvent is evaporated to dryness using a rotative evaporator Büchi (40°C). The flask containing the dried mixture is placed in an oven (107°C +/- 1°C) for maximum 5 hours in order to avoid carbonisation. The temperature used for the synthesis is different from Clausen et al. (1983) who suggest 104°C. At this temperature we observed that the synthesis is partially realised after 10 hours and that remaining intact crystals cause problems during purification. At 107°C, we avoid carbonisation and improve the yield.

Purification

The choline ester is taken up with 2 x 3 ml of distilled water and purified on a SP Sephadex C25‑120 resin (500 mg). After washing the resin with distilled water, the ester is eluted with 10 ml of HCl (1N). The elution is followed by measuring the u.v. absorbance at 280 nm. The eluate is evaporated to dryness with a rotative evaporator (Büchi) and crystals obtained are washed with acetone (3 x 2 ml). Acetone removes a lot of brown impurities. The HPLC purity obtained for all the choline esters produced was close to 98 %. The general yield was approximately 50%.

Table 1: List of aromatic acids used for the synthesis of the choline esters

Acid	R 2	R 3	R 4	R 5	R 6	Name	Formula
Benzoic							C7H6O2
Benzoic	OH					salicylic acid	C7H6O3
Benzoic		OH					C7H6O3
Benzoic			OH				C7H6O3
Benzoic	OH	OH					C7H6O4
Benzoic	OH		OH			b resorcylic acid	C7H6O4
Benzoic	OH			OH		gentisic acid	C7H6O4
Benzoic	OH				OH		C7H6O4
Benzoic		OH	OH			protocatechic acid	C7H6O4
Benzoic		OH		OH		resorcylic acid	C7H6O4
Benzoic		OH	OH	OH		gallic acid	C7H6O5
Benzoic	OMe					o-anisic acid	C8H8O3
Benzoic		OMe				m-anisic acid	C8H8O3
Benzoic			OMe			p-anisic acid	C8H8O3
Benzoic		OMe	OMe				C9H10O4
Benzoic		OMe		OMe			C9H10O4
Benzoic		OMe	OMe	OMe			C10H12O5
Benzoic	OH	OMe					C8H8O4
Benzoic	OH		OMe				C8H8O4
Benzoic	OH			OMe			C8H8O4
Benzoic		OH	OMe			isovanillic acid	C8H8O4
Benzoic		OMe	OH			vanillic acid	C8H8O4
Benzoic		OMe	OH	OMe		syringic acid	C9H10O5
Cinnamic						cinnamic acid	C9H8O2
Cinnamic	OH					o-coumaric acid	C9H8O3
Cinnamic		OH				m-coumaric acid	C9H8O3
Cinnamic			OH			p-coumaric acid	C9H8O3
Cinnamic		OH	OH			cafeic acid	C9H8O4
Cinnamic	OMe	OMe					C11H12O4
Cinnamic	OMe		OMe				C11H12O4
Cinnamic	OMe			OMe			C11H12O4
Cinnamic		OMe	OMe				C11H12O4
Cinnamic		OMe	OMe	OMe			C12H14O5
Cinnamic		OH	OMe			isoferulic acid	C10H10O4
Cinnamic		OMe	OH			ferulic acid	C10H10O4
Cinnamic		OMe	OH	OMe		sinapic acid	C11H12O5

u.v. library

The u.v. spectra libraries of each aromatic choline ester and the corresponding aromatic acid are constituted for rapid identification of peaks when a diode array detector is coupled with the HPLC .

CONCLUSIONS

36 aromatic choline esters were successfully synthesised with a high purity (> 98%) by reaction of the corresponding acid on bromocholine bromide. These compounds, not available by a commercial way, can be used as references to determine retention time or calculate response factors in HPLC (Reference method for choline esters measurements). The libraries created are also useful for the confirmation of chromatographic peaks.

ACKNOWLEDGEMENTS

This work has been supported by the General Office of Research and Development of the Belgian Agricultural Ministery and by the General Direction of Technologies, Research and Energy of Ministery of "Region wallonne" in Belgium.

REFERENCES

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Pokorny J. and Reblova Z.(1995): Sinapines and other phenolics of Brassicaceae seeds. Potrav. Vedy, 13(2), 155-168