Field of Invention:

The present invention relates to an improved process for isomerization of Z-lycopene to E-lycopene substantially, wherein isomerization takes place in presence of catalytic amount of thiourea.

Background of the Invention:

Lycopene is a carotenoid substance naturally occurring in tomatoes and many other plant and microbial sources, as a mixture of geometrical isomers. It is an acyclic molecule of formula C40H56, with 11 conjugated and 2 isolated double bonds. (Carotenoids, G.Britton, S.Liaaen-Jensen and H.Pfander, Birkhauser Verlag, Basel, Several volumes, 1995 to 2004). Theoretically there can be many isomers possible. Some of them definitely occur naturally, although it is very difficult to distinguish them from each other by simple spectroscopic methods. For example the (5-Z) and the all-E isomers have identical UV-Vis spectrum and can be identified only when resolved by RP- HPLC. Eight (mono-Z) isomers were obtained by controlled stereospecific synthesis and six from isomerization mixtures. Four (di-Z) isomers and one (tetra-Z) isomer have also been reported. (Hengartner et al, Helv. Chim. Acta, 75, 1848, 1992). Lycopene from natural sources has been used as a coloring ingredient in foods. It has also been recommended as a useful anti-oxidant. There are no specific reports in literature on relative biological activities of the isomers of lycopene. Tomatoes are the major source of lycopene in human nutrition and are known to contain 71 to 90% of all-E- lycopene (all-trans) and 9 to 21% of Z-isomers (cis), mainly the 5-Z-isomer (Zumbrum et al, Helv. Chim.Acta, 68, 1540, 1985), depending on the source, season etc. Surprisingly in humans all-E lycopene accounted for only 12 to 21% whereas the Z-isomers accounted for 79 to 88% in benign or malignant prostate tissue. (Clinton et al, cancer Epidemiol. Biomarkers Prev., 5, 823, 1996). This means the human body is converting much of the all-E isomer to Z-isomers which may be influencing malignant growth. In beta-carotenes and retinoids the all-E isomers are definitely known to be more active than the Z-isomers. It is thus considered preferable to use all-E lycopene and avoid Z-lycopenes for human consumption. The USP-NF monograph on lycopene is a mixture of all-E lycopene containing up to 23% of 5-Z lycopene, the major isomer in natural lycopene. Synthetic lycopene is better controlled in terms of isomers compared to natural sources. Even so it is known that the (5-Z)- isomer predominates under certain conditions. A stereospecific synthesis of all-E lycopene suppressing the formation of Z-isomers would be very expensive. A process which enables isomerization of the Z-isomers to all-E isomer is attractive when a synthetic route for lycopene is adopted for its large scale production.

Isomerization of carotenoid compounds is known. Most studies deal with mechanics and mechanisms of photoisomerization and some with enzyme induced isomerizations. (See Dugave and Demange, Chem.Rev., 103, 2481, 2003). Mueller et al {Pure & Appl. Chem. 69, 2039, 1997) have reviewed the topic and reported that in solution (E/Z)-isomerization of carotenoids is promoted by heat, light, active surfaces and catalytic amounts of acids or iodine. It is believed that the Wittig and Homer condensation steps in the synthesis of carotenoids lead to isomeric mixtures and isomerization could be effected thermally in non-polar solvents. However the focus in this publication was on preparation of Z-isomers in pure form by isomerization of the all-E lycopene. US Patent 7126036 discloses a process of thermal isomerization of lycopene. The process essentially consists of first dissolving the mixture of all-E and Z-isomers in a non-polar solvent dichloromethane followed by addition of methanol, distilling off azeotropically dichloromethane to obtain a suspension in methanol which is then subjected to thermal isomerization by refluxing in methanol or under autogenous pressure raising the temperature to about 95°C. The yields of the enriched all-E isomer or the extent of isomerization did not improve with the autogenous pressure. Although the inventors claimed enrichment of the all-E isomer in mixtures of any proportion of the two isomers, all the examples indicate that the starting mixture consisted of 53% of all-E isomer. The Z-isomer content m the starting mixture is reported as 18%. The inventors achieved enrichment to about 76 to 87% of all-E isomer at the end of the process. The content of the Z-isomer in the enriched mixtures have not been revealed. The extent of isomerization of the Z-isomer to the all-E isomer is thus not clear. The example 7, without thermal isomerization, also achieved enrichment to 75% of all-E isomer in the crystallized sample indicating that solubility and crystallization steps also contributed to a large extent in the process.

We attempted to repeat the thermal isomerization process as described in the US7126036. We used samples containing about 56% of all-E and about 43% of Z-isomers (predominantly 5-Z-isomer) and also containing about 20 to 22% all-E and about 62 to 72% of Z-isomers. Divis product that is routinely obtained from the manufacturing process has this latter composition. The results obtained from these experiments are summarized in the Table 1 below.

Table 1

Notes: a: 43 g input; b: 25 g input, yield -22 g; c,: 20 g input, 120°C, 7 bar pressure; C2: 20 g input, 95°C, 1.8 bar pressure;

d: 43 kg input (pilot scale).

From the results it can be seen that thermal isomerization of lycopene does not take place when the Z-isomer content is higher than that of all-E isomer. Best result is seen at high temperature and pressure with some enrichment but not satisfying the USP-NF specification of less than 23% of the Z-isomer content. Since lycopene is used as additive to foods and medicaments it was felt desirable to avoid chlorinated hydrocarbon solvents in the final steps of synthesis.

We also attempted to use other solvents in the thermal isomerization experiments. The results are shown in Table 2 below:

Table 2
Thermal isomerization in different solvemts:
It can be seen that the best result was obtained in water as medium in respect of both yield and extent of isomerization. However this was still much below requirement.

Objectives of the Invention:

There is a need for an improved process of isomerization of Z-isomers present in the synthetic lycopene used as additive in foods or as antioxidant in medicaments, hi particular it was necessary to isomerize mixtures containing low levels of all-E isomer (less than 50%) and high levels of Z- isomers as obtained in commercial manufacture and to avoid halohydrocarbon solvents in the process.

Summary of the Invention:

The present invention reveals an improved process of isomerization of Z-isomers present in the lycopene product obtained during synthesis, wherein the all-E isomer content is less than 50% and the content of Z-isomers is more than the all-E-isomer. The process is carried out in presence of thiourea as catalyst in a polar solvent without pressure and halohydrocarbon solvents. The resulting product has a content of all-E isomer greater than 80% with less than 10% of Z-isomers.

Detailed Description of the Invention:

Lycopene is obtained by synthesis by any of the processes described in literature (e.g. Helv. Chim. Acta 75, 1848, 1992; Acta Chemica Scandinavica B 29, 1015, 1975; /. Chem. Soc. 2019, 1965). Lycopene is a symmetrical molecule and the strategy that is usually adopted consists of condensation of two molecules of the C-15 intermediate with one molecule of the C-10 intermediate. The C-15 intermediate is usually

the Wittig salt of 3,7,ll-trimethyl-l,4,6,10-tetraene-dodecan-3-ol and the C-10 intermediate is usually 3,7-dimethyl-2,4,6-octatetraene-l,8-dialdehyde (often called 'dial'). The reaction is generally carried out in a non-polar solvent like dichloromethane or hexane in presence of a strong base like sodium methoxide or potassium t-butoxide. The product may be isolated as a crude solid on work up or further purified by suitable recrystallisation procedures. For our experiments we used the crude solid. The crude lycopene product or the purified material contains a high proportion of Z- isomers ranging from about 40 to about 65% and the all-E isomer in the crude may be as low as about 25%.

An exhaustive search of literature revealed no attempts to preferentially increase the all-E isomeric content of isomeric mixmres of polyenes similar to carotenoids. We could find only a few references to isomerization of olefins in presence of some 'catalysts' like acids, sodium bisulfite, ammonium bromide, ammonium persulfate, potassium bromate, urea, thiourea etc., applied particularly to isomerization of maleic acid (cis-) to fumaric acid (trans-). (See GB2207915 and references cited therein). We attempted to isomerize lycopene product obtained by synthesis as outlined above with several of the 'catalysts' used in the reported isomerization of maleic acid to fumaric acid. Although most of them did not work, we were pleasantly surprised to find addition of thiourea induced satisfactory isomerization of 'lycopene' mixtures to enriched all-E lycopene.

We studied the effect of input quantity of thiourea on yield and extent of isomerization. The Table 3 below summarizes typical results obtained.

Table 3
Effect of thiourea on isomerization of lycopene

It can be seen that thiourea input should be about 25% of the input lycopene to achieve optimal isomerization and yield.

The crude lycopene as obtained by synthesis is suspended in a polar solvent like methanol or isopropanol, solid thiourea is added, the mixture heated to reflux temperature at atmospheric pressure and maintained for several hours. The isomerization process is monitored by in process checks. When the desired proportion of the all-E and Z-isomers is achieved, the reaction is stopped and suitable work up is adopted to recover and purify the lycopene.

The isomerisation process is monitored by HPLC analysis using YMC-Carotenoid (250 x 4.6mm) 5µm and YMC-Carotenoid (250 x 4.6mm) 3µm dual columns connected in series, detection at 472 rmi at flow rate of 1.0 ml/minute, tertiary butyl methyl ether: methanol: tetrahydrofuran as mobile phase at a ratio of 784:665:74, injection volume: 10 micro liter of sample solution and run time of 60 minutes.

Examples:

The following examples are for illustration purpose only and do not limit the invention in any way. The reagents and solvents mentioned in examples maybe replaced by other reagents and solvents known to those skilled in the art.

Example 1:

Lycopene (11.6 g, all E-content 29%) was charged to 250 ml three necked round bottomed flask containing 2-propanol (75 ml). To the resulting suspension, 2.9 g of thiourea was added and heated to maintain at 90-95°C for 15 hrs. The solvent was distilled at reduced pressure at 55-60''C. To the residue methanol (50 ml) was added, stirred at 55-60°C for 20 minutes and gradually cooled to 20- 25''C. The crystalline compound formed was filtered, washed with methanol (20 ml), dried under reduced pressure for 3 hours to obtain all-trans lycopene (9.86 g) having 86.46% all-E-isomer and 6.62% Z-isomer content.

Examples 2,3 & 4:

Few more experiments were conducted to demonstrate repeatability of the example 1 as above. The results are tabulated below:

Table 4

Example 5:

Lycopene having 63.13% of Z-isomer (43 Kg, all E-content: 29.83%) was charged to reactor containing 2-propanol (260 L). To the resulting suspension, 8 Kg of thiourea was added and heated to maintain 80±2°C for 15 hrs or until in-process HPLC indicated <15% content of Z-lycopene. The solvent was distilled off at atmospheric pressure followed by distillation at <10mm Hg. To the resulting mass, methanol (50 L) was added and the mass was distilled at reduced pressure at < 55 °C to remove traces of 2-propanol. Methanol (100 L) was added to reactor and the mass was stirred at 55°C for 15 minutes. The resulting crystalline mass was cooled to 23±2°C, stirred for 1 hr and filtered to recover solid. The wet material was stirred with water (100 L) to remove traces of thiourea, filtered, stirred with methanol (200 L), solid material filtered and dried at 30±2°C under reduced pressure to obtain 29.2 Kg all-E lycopene (trans) having 84% E-isomer and 9% Z-isomer content.

The procedure of example 5 was repeated with different batches of lycopene with the results obtained as shown in Table 5 below.

Table 5

We claim:

1. A process of isomerization of Z-lycopenes in a mixture containing less than 50% of all-E lycopene (all-trans) and more than 50% of the Z-lycopenes, in presence of thiourea as catalyst in a suitable polar solvent medium and at less than 100°C without pressure.

2. A process as in Claim l, in which the mixture of lycopenes is a purified or partially purified solid or a solution or suspension in an organic solvent, obtained by any synthetic process.

3. A process as in Claim 1, in which thiourea used may be added as solid or solution or suspension in a solvent medium.

4. A process as in Claim 1, in which the polar solvent used is a C1 to C4 alcohol or mixtures containing any of them.

5. A process as in Claim 1, in which the recovered enriched lycopene has an all-E isomer content of greater than 80% (HPLC area%) and Z-isomers less than 20% (HPLC area%).