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SYNTHESIS AND PURIFICATION OF 36 AROMATIC CHOLINE ESTERS AS STANDARDS

Jean-Paul Wathelet, Nicolas Mabon and Michel Marlier

FacultÚ universitaire des Sciences agronomiques, UnitÚ de chimie gÚnÚrale et organique
Passage des DÚportÚs, 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 BŘchi (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 (BŘchi) 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 Ám) 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 BŘchi (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 (BŘchi) 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

   

β 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

1. Andersen H. and Andersen P. (1982): Anvendelse af raps i foderblandiner til kvaeg. Production, 22, 23-26, in Danish

2. Andersen H., Varnum P., Andersen P., Klastrup S., S÷rensen S., S÷rensen H. and Olsen O. (1984): Dobbeltlav rapsskra i kraftfoderblander til kalve og ungtyre. Meddelelse Statens husdyrbrugsforsog. 4 pp, in Danish

3. Clausen S., Olsen O. and S÷rensen H. (1983): Separation of aromatic choline esters by high performance liquid chromatography. Journal of Chromatography, 260, 193-199

4. Fenwick G. (1981): Trimethylamine taint in eggs. In: Quality of eggs. Proc. Eur. Symp. 1st Apeldoorn, 18-23 May, 144-152

5. Hartman E. and Kilbinger H. (1974): Occurence of light-dependent acetylcholine concentrations in higher plants. Exp., 30, 1387-1388

6. Krygier K., Sosulski F. and Hogge L. (1982): Composition of phenolic acids in rapeseed flour and hulls. Journal of Agricultural and Food Chemistry, 30, 334-336

7. Kozlowska H., Rotkiewicz D., Zadernowski R. and Sosulski F. (1983): Phenolic acids in rapeseed and mustard. Journal of the American Oil Chemists Society. 60,1119-1123

8. Miura G. and Shih T.-M. (1984a): Cholinergic constituents in plants. Characterization and distribution of acetylcholine and choline. Physiol. Plant., 61, 417-421

9. Miura G. and Shih T.-M. (1984b): Identification of propionylcholine in higher plants. Physiol. Plant., 62, 341-343

10. Pl÷eger A., Larsen L., Olsen O., Clausen S., M÷eller P., Rasmussen K., Nielsen J. and S÷rensen H. (1985): Aromatic choline esters, aromatic choline esterase, glucosinolates and myrosinases in oilseed rape and other crucifers. Royal Veterinary and Agricultural University, Chemistry Department, Copenhagen, Denmark, thesis, 1-121

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

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