FR2728897A1 - Prodn. of frambinone - Google Patents

Abstract

A new process for the prodn. of frambinone by the catalytic oxidn. of frambinol using horse liver alcohol des-hydrogenase (HLADH) in the presence of co-factor NAD<+>.

Description

DH PRODUCTION PROCESS DB FRAKBINONB
The present invention relates to a process for the simple production of aroma by enzymatic oxidation catalyzed by an alcohol dehydrogenase. The present invention relates more particularly to a process for the production of frambinone from frambinol.

The flavors correspond to a growing sector of the food industry. In fact, consumer requirements relating to the reproducibility of taste and aroma make it necessary to use natural and artificial flavors in food products.

Artificial flavors, said to be synthetic, have certain advantages over natural flavors. On the one hand, there is no supply constraint and the cost of producing an artificial flavor is considerably lower than that of a natural flavor.

There are therefore needs for processes making it possible to obtain artificial flavors on an industrial scale, in particular artificial rambinone.

In addition, in recent years, the public has turned more and more to natural products, in particular so-called natural flavors or so-called identical to nature.

Obtaining these products is difficult, and their extraction is often expensive. Manufacturers are therefore also led to seek precursors of these products which are easily accessible and inexpensive in order to obtain natural products with high added value. But to keep the natural label, these products must not be obtained by organic synthesis reactions from these precursors. There is therefore also a need for a process making it possible, from a natural precursor, to obtain a natural flavor or one identical to nature, in particular a fruit flavor and more particularly of raspberry, in a simple and inexpensive manner.

Thus, a search is made for a process for preparing aromas which can be applied both to natural and synthetic precursors. In particular, a process is sought for the preparation of raspberry flavor, known under the name of rambinone.

Frambinone is a known compound, and is also referred to in the literature as 4- 4- (para-hydroxyphenyl) -2-butanone, or raspberry ketone in English.

Two routes are known at the present time allowing the synthesis of this ketone. The first route consists of the condensation of p-hydroxy-benzaldehyde with acetone, followed by the hydrogenation of the intermediate produced to frambinone. The second route consists of the condensation of methyl vinyl ketone with phenol. Only the first route is currently used on an industrial scale.

Frambinol, a natural precursor of rambinone, is also a compound known per se. Frambinol is also identified in the literature under the names of rhododendrol or betuligenol.

Virinder S. Parmar et al (J. Chem. Soc. Perkin Trans 1, pp 2687-2689, 1991) describe the determination of the absolute configuration of epi-rhododendrin and of its aglucone form (-) rhododendrol and it is indicated that these compounds were initially isolated from leaves of
Rhododendron Chrysanthym. The isolation from raspberries of 4 '-0 - D-glucopyranoside of 4- (4. -hydroxy-phenyl) butan-2-one and of 3-0-ss-D-glucopyranoside of 4- (3' , 4'-dihydroxyphenyl) butan-2-one is described by Anni Pabst et al. In Phytochemistry, Vol 29, No. 12, pp 3853-3858, 1990.

The enzymatic synthesis of rhododendrol from frambinol has also been described in JP 90188641 relating to the production of (S) rhododendrol by use of baker's yeast, in JP 90-45287 relating to the production of (S) rhododendrol by use of callus from Acer nikoense, and in JP 91-311663 relating to the preparation of (S) rhododendrol by use of a dehydrogenase from callus from Acer nikoense.

The enzymatic catalysis of an oxidation reaction of an alcohol to a ketone is known per se. Indeed, the use of enzymes and in particular of an alcohol dehydrogenase to carry out redox reactions is described in numerous documents of the prior art, although the research has been more particularly focused on the reactions of reduction.

The article by JB Jones et al (CAN. J. CHEM., Vol. 55, pp 2685-2691,1977) represents a general study carried out on alcohol dehydrogenases and it is more particularly described that liver alcohol dehydrogenase from horse, hereinafter referred to as HLADH, catalyzes the oxidation-reduction reactions of a broad spectrum of substrates including alcohols, aldehydes, ketones and promotes certain stereospecific reactions.

The article by Hans w. Adolph et al (Eur J. Biochem. 201, pp 615-625, 1991) relates to the study of the substrate specificity and of the stereoselectivity of HLADH.

It is also known that the oxidation-reduction reactions catalyzed by alcohol dehydrogenases require a specific cofactor of the enzyme which is consumed during the reaction. It may therefore prove to be advantageous to regenerate this cofactor.

The systems allowing the regeneration of cofactors are widely described in the literature. While the regeneration of reduced cofactors is relatively widespread, that of oxidized cofactors remains less studied (Chenault and Whitesides Appl. Biochem. Biotechnol, 14, pp 147-197, 1985; LGLee et al J. Am. Chem. Soc., 107, pp 6999-7008, 1985).

LGLee et al in J. Am. Chem. Soc., 107, pp 6999-7008, 1985 describe a comparative study of three types of enzymatic systems for regeneration of NAD + from
NADH for use in organic synthesis on an industrial scale catalyzed by an enzyme. The first and most commonly used system involves the use of an organic oxidant 2-oxoglutarate with catalysis by glutamate dehydrogenase. The second comprises dioxygen used as a terminal oxidant with a dye corresponding to an intermediate electron acceptor and diaphorase as a catalyst. The third is based on the use of an inorganic oxidant ferricyanide and diaphorase as a catalyst. The use of these systems in the conversion of cis-cyclohexanedimethanol to (+) - [1R, 6Si-cis-8- oxabicyclo [4.3.0] nonan-7-one by HLADH is in particular described.

H. Keith Chenault et al in Bioorganic Chemestry 17, pp 400-409, 1989 describe the use of lactate dehydrogenase (LDH) coupled to pyruvic and glyoxylic acids as an oxidant as a system for regeneration of NAD + in situ, for example. in the case of the conversion of cis-1,2-bis (hydroxymethyl) cyclohexane to (+) - (1R-6S) -cis-8- oxabicyclo- [4-3-0] nonan-7-one by HLADH .

Dale G. Drueckhammer et al in J. Org. Chem., 50, pp 5387-5389, 1985 describe an efficient NAD (P) regeneration system based on FMN reductase for use in enzymatic syntheses. Its use in the HLADH-catalyzed oxidation of 3-methyl-1,5-diol is in particular described.

VDWesterhausen et al (Angew. Chem. Int. Ed. Engl, 31, n011, pp 1529-1531, 1992) describe a non-enzymatic regeneration system for NADH and NADPH with [(C5Me5) Rh111 (bpy) (H2O )] C12 and similar complexes used as homogeneous redox catalysts and either an electrode or formate as a donor. This system is applied to the reduction of 4-phenyl-2-butanone by
HLADH, and it is indicated that it leads to better results than the systems using a coupling of the substrate with the co-substrates such as methanol.

None of the documents of the prior art describes or suggests the present invention as defined below.

Thus, the present invention relates to a process for the production of rambinone, comprising the step of catalytic oxidation of frambinol by HLADH, in the presence of NAD + cofactor.

According to one embodiment, in the process according to the invention, the oxidation reaction is carried out with a ratio of frambinol / cofactor molar concentrations of 1 to 104, preferably from 1 to 100, and a ratio of molar concentrations frambinol / HLADH from 103 to 109, preferably from 103 to 105.

According to another embodiment, in the process according to the invention, the oxidation reaction is carried out in a buffered aqueous solution at a pH of between 4 and 10, preferably between 8 and 9, at a temperature of 4 to 600C, preferably 25 to 400C.

According to a first embodiment of the process according to the invention, the frambinol is a frambinol of natural origin, as obtained by hydrolysis of a rhododendron extract or of a birch bark extract by ss-D - glucosidase.

According to a second embodiment of the process according to the invention, the frambinol is a synthetic frambinol.

According to one embodiment, in the process according to the invention, the frambinol is chosen from the group consisting of the R and S isomers, and the racemic mixture.

According to one embodiment, in the process according to the invention, said step of catalytic oxidation by
HLADH in the presence of NAD + is coupled to a step of regeneration of the NAD + cofactor using acetaldehyde as co-substrate.

It would not be departing from the scope of the present invention if, in addition to the continuous or discontinuous additions to the reaction mixture of frambinol, and / or acetaldehyde, the enzyme or NAD + was added discontinuously.

The present invention will now be described in more detail, with reference to the figures.

Figure 1 shows the oxidation reaction of FOH in
FC = O according to the present invention;
FIG. 2 represents the mechanism of the reaction of oxidation of frambinol to frambinone by HLADH coupled to the NAD + regeneration system according to the present invention;
Figure 3 shows a comparison of the activity of
HLADH on two frambinols, one being in the R configuration (natural origin) and the other being a racemic (synthetic);
Figure 4 shows the influence of buffer and pH;
Figures 5 (a), 5 (b), and 5 (c) respectively represent the activity of HLADH in borate, phosphate and triethanolamine buffers;
Figure 6 shows a comparison of the activity of
HLADH in phosphate and triethanolamine buffers, for a pH of 8.5. The curve also shows that additions of cofactor or acetaldehyde during the recation make it possible to improve the percentage of conversion of frambinol, and that various buffers can be used.

The starting products and operating conditions are more particularly described below.

Preparation and purification of frambinol.

Natural frambinol is extracted from birch bark and rhododendron according to the extraction process described in a note by A. SOSA, report of the Academy of Sciences, 196, 1827 (1933). 1 kg of fresh bark of white birch (Betula alba L.) is exhausted with ethyl alcohol at a temperature of 800C for 20 minutes. The spent bark is crushed and treated with new alcohol. The combined organic phases are evaporated and the residue is taken up in water at 45 ° C. in order to precipitate a large quantity of a product essentially consisting of betulin. After filtration, the aqueous solution is washed with ethyl ether and then added with Calcined magnesia (100 g per kg of fresh bark) The paste obtained is dried in an oven and then exhausted with boiling ethyl alcohol.

After concentration of the alcohol, the residue is taken up in boiling ethyl acetate. Carbohydrate crystallizes on cooling. It is purified by crystallization in water. The yield is about 3 g per kg of fresh bark. Frambinol is conventionally obtained by hydrolysis with B-D-glucosidase.

The extract obtained is then purified by “flash chromatography” under the following conditions. 2 g of frambinol (10 to 20 ml of methanolic solution) obtained as above are loaded onto a column of 200 g of silica gel 60. Elution is carried out with a dichloromethane / methanol mixture = 98/2 (vol / vol). The yield is 62%. The specific optical rotation of natural frambinol purified from birch bark, determined on a 100% pure fraction, is: [(Z] 20 = - 15.8 while (a] 20 D theoretical = - 170.

Racemic chemical frambinol is obtained by conventional chemical synthesis.

Conversion of frambinol to frambinone.

The enzymes used correspond to the HLADH of
Boehringer (102741) and Sigma's HLADH (A6128). The first source is preferred. NAD + is available from
Boehringer. Natural frambinol comes from birch bark; synthetic frambinol is obtained by chemical reduction of frambinone with sodium borohydride according to a conventional reduction protocol. Ethyl acetate is used as the solvent for recovering the frambinol.

The reaction according to the invention is carried out in a medium buffered at a pH of 7.8 to 9.6. Any buffer system exhibiting buffering power in this pH range can be used, for example: borate, phosphate, triethanolamine, etc. buffer. The reaction temperature used is 25 to 400C.

Usually the frambinol / HLADH molar ratio is 7.1 x 103 and the frambinol / cofactor molar ratio is 25.

It is also possible to use less purified forms of the HLADH enzyme such as, for example, powders obtained from acetone precipitations of ground horse liver.

To reduce the cost of the process, it is preferable to regenerate the NAD + in situ to avoid using it in a stoichiometric amount.

Regeneration of the NAD + cofactor can optionally be coupled to the oxidation reaction. It includes the use of acetaldehyde as a co-substrate.

Acetaldehyde is reduced to ethanol by HLADH.

The reaction can be carried out in any suitable reactor system in a continuous or batch mode, with a continuously operating system being preferred.

The acetaldehyde is preferably added continuously or in portions so as to avoid any possible problem of inhibiting the reaction. Frambinol can be added in portions or continuously to the reaction mixture. It may be advantageous to add more enzyme and / or NAD + during the reaction. The reaction product can also be withdrawn continuously or in parts of the reaction mixture by withdrawing part of the reaction medium and separated from the frambinol by any conventional and appropriate separation process, for example by isocratic HPLC. The presence of ethanol and acetaldehyde do not pose any particular problem, these products being much more volatile. Recycling after separation of the various products is also possible. The enzyme can be used in an immobilized form or in a free form, the latter corresponding to the preferred form.

Particular embodiments of the present invention will now be described in the examples which follow, the latter being given for purely illustrative and non-limiting purposes. In these examples, frambinol is referred to as FOH, while frambinone is referred to as FC = O.

Example 1
Conversion of R-frambinol and racemic mixture of frambinol, without regeneration system.

The operating conditions used are as follows:
HLADH Boehringer = 0.5U / ml; frambinol = 5 mM; NAD + = 5mN; 50 mM phosphate buffer pH 7.8, at 250C.

The oxidation reaction is carried out in stoppered hemolysis tubes, with a stirring system. The total volume is 2 ml. The reaction is initiated by the addition of enzyme.

The enzymatic activity is determined by taking 100 gl of reaction medium regularly and diluting in a solution which stops the reaction (water / acetonitrile / acetic acid mixture = 75/25 / 0.5, vol / vol / vol) then analysis by HPLC of the mixture obtained under the following conditions:
KROMASIL HPLC C 18 column 0.46 x 22 cm;
Precolumn RP 18;
Flow rate = 1.5ml / min;
Volume injected = 20R1; Eluent: water / acetonitrile / acetic acid, 75/25 / 0.5, by volume;
Retention time:
Frambinol (FOH) (166.22g / mol): 5 min
Frambinone (FC = O) (164.20g / mol: 7min e = 1662 cm 1 M-1;
Detection wavelength = 275 nm;
The percentage of conversion is then obtained as follows
Area of FC = O Conversion (%) = ----------------------------- x 100
(Area of FC = O + Area of FOH)
The results of this experiment are represented graphically in FIG. 3. It is observed that the R isomer is oxidized but less rapidly than the racemic mixture, which means that the S isomer is a better substrate for the.
HLADH.

Example 2
Conversion carried out at different pH, without a regeneration system.

The percent conversion of frambinol was determined for different buffers under the following conditions:
HLADH Boerhinger = 0.5U / ml, frambinol = 3mM, NAD + = 5mM, 50mM buffer, 250C, reaction time 21 hours. The buffer is glycine buffer, phosphate buffer, or buffer
Sort.

The activity of the enzyme is determined according to the methods described in Example 1. The results obtained are presented in FIG. 4. It will be necessary to take into account, by adjusting the pH, the variation in the stability of the enzyme. depending on the pH.

example 3 study of the conversion process according to the present invention, under different operating conditions.

The conversion reactions are carried out under the following conditions:
Conditions 1: 0.2U HLADH / ml, NAD + = 400pM, pH = 8.5, 0.1M phosphate buffer, 400C, 45 hours.

Conditions 2: 0.4U HLADH added in 2 times, NAD + = 400M, acetaldehyde: 24mM added in 12 times, 0.1M triethanolamine buffer pH = 8.5, 400C, 150 hours.

Conditions 3: 0.4 U HLADH added in 2 times, acetaldehyde added continuously using a syringe at a rate of 2 mM / h, NAD + = 400 M, phosphate buffer pH = 8.5, 0.1M, 350C.

The enzymatic activity determined according to the methods described in Example 1 leads to the following results:

<tb> conditions <SEP> FOH <SEP> mg / ml <SEP> FC = O <SEP> mg / ml <SEP>% <SEP> of <SEP> con
<tb> Operations <SEP> to <SEP> ion
<tb> 1 <SEP> 1.7 <SEP> 1.1 <SEP> 65
<tb> 2 <SEP> 1.2 <SEP> 0.96 <SEP> 80
<tb> 3 <SEP> ~ <SEP><SEP> 1.7 <SEP> 1.24 <SEP> ~ <SEP><SEP> 73
<tb>
These results show that up to 1g / l of rambinone can be obtained in solution.

Example 4
Conversion with NAD + regeneration system carried out at different pH.

The process for converting frambinol to frambinone according to the present invention is carried out in the presence of the NAD + regeneration system for different buffers: borate buffer, phosphate buffer and triethanolamine buffer, the three pHs considered are 8, 8.5 and 9 .

The operating conditions are as follows: - HLADH sigma = 0.2U / ml in a 100 mM buffer at 40 C, frambinol = 7 mM, NAD + = 400WM, acetaldehyde = 2 mM then 2 are added after each sample (16 mM in total) .

The results are shown in Figures 5 (a), 5 (b) and 5 (c).

It is observed that for a pH = 8.5 the conversion conditions are good in phosphate and triethanolamine buffers (30% conversion in 6 h).

A second experiment is carried out at this pH with these two buffers for a longer period. The reaction conditions are as follows: - HLADH sigma = 0.2U / ml in a 100 mM buffer at 400C, pH = 8.5; frambinol = 7mM, NAD + = 400M; acetaldehyde = 2 mM then 2 mM are added after each sample (24 mM in total).

The measurement of the enzymatic activity according to the methods described in Example 1 shows that, after 30 h, 50% of frambinol are transformed in these two buffers. An addition of enzyme and cofactor was carried out.

These results are illustrated in FIG. 6. They show that after about 25 hours, the enzyme and the cofactor are limiting components for the oxidation of frambinol.

Claims (7)

1. Process for the production of frambinone, comprising the step of catalytic oxidation of frambinol by HLADH, in the presence of NAD + cofactor. 2. The method of claim 1, wherein the oxidation reaction is carried out with a ratio of molar concentrations of frambinol / cofactor of 1 to 1041, preferably of 1 to 100, and a ratio of molar concentrations of frambinol / HLADH of 103. to 109, preferably from 103 to 105. 3. The method of claim 1 or 2, wherein the oxidation reaction is carried out in buffered aqueous solution at a pH between 4 and 10, preferably between 8 and 9, at a temperature of 4 to 600C, of preferably from 25 to 400C. 4. Method according to any one of claims 1 to 3, in which the frambinol is a frambinol of natural origin. 5. Method according to any one of claims 1 to 3, in which the frambinol is a synthetic frambinol. 6. A method according to any one of claims 1 to 5, wherein the frambinol is selected from the group consisting of the R and S isomers, and the racemic mixture. 7. Method according to any one of the preceding claims, wherein said step of catalytic oxidation by HLADH in the presence of NAD + is coupled to a step of regenerating the NAD + cofactor using acetaldehyde as co-substrate.