FUNCTIONALISED CYCLIC DITHIOCARBAMATE SYNTHESIS METHOD

Abstract

Provided is a process for synthesizing a functionalized cyclic dithiocarbamate.

Claims

1. A process for synthesizing a functionalized cyclic dithiocarbamate of formula (I): ##STR00008## in which R.sub.1 is a hydrogen or an aromatic or nonaromatic, linear or cyclic, saturated or unsaturated, branched or unbranched, hydrocarbon-based chain including from 1 to 20 carbon atoms, and which may, optionally, include one or more heteroatoms chosen from O, S, N, P and Si; X represents C(O) or CH.sub.2 or CN; R.sub.2 is (i) either nonexistent (when X represents CN), (ii) or a hydrogen, (iii) or OR.sub.3, R.sub.3 being a hydrogen or an aromatic or nonaromatic, linear or cyclic, saturated or unsaturated, branched or unbranched, hydrocarbon-based chain including from 1 to 20 carbon atoms, and which may, optionally, comprise or more heteroatoms chosen from O, S, N, P and Si, (iv) or NR.sub.4R.sub.5, with R.sub.4 and R.sub.5, which are the same or different being a hydrogen or an aromatic or nonaromatic, linear or cyclic, saturated or unsaturated, branched or unbranched, hydrocarbon-based chain including from 1 to 20 carbon atoms, and which may, optionally, include one or more heteroatoms chosen from O, S, N, P and Si; n is equal to 0, 1 or 2; and * represents an asymmetric carbon; said process comprising the steps of: a/ providing at least one compound of formula (II):
G-(CH.sub.2).sub.nC*H(NHR.sub.1)XR.sub.2(II) in which n, R.sub.1, R.sub.2, X and * are as defined previously, G represents either (i) R.sub.6C(O)OCH.sub.2, or (ii) (R.sub.7O)(R.sub.8O)P(O)OCH.sub.2, or (iii) R.sub.7OSO.sub.2OCH.sub.2; R.sub.6 is a hydrogen or an aromatic or nonaromatic, linear or cyclic, saturated or unsaturated, branched or unbranched, hydrocarbon-based chain including from 1 to 20 carbon atoms, and which may, optionally, include one or more heteroatoms chosen from O, S, N, P and Si; R.sub.7 and R.sub.8, which may be identical or different, are chosen, independently of each other, from a proton H, an alkali metal, an alkaline-earth metal or an ammonium; b/ providing at least one inorganic trithiocarbonate; c/ reaction between said at least compound of formula (II) and said at least inorganic trithiocarbonate in the presence of at least one enzyme chosen from sulfhydrylases; d/ production of at least one functionalized cyclic dithiocarbamate of formula (I); e/ separation and isolation of said at least one functionalized cyclic dithiocarbamate of formula (I); f/ optionally, additional functionalization of the functionalized cyclic dithiocarbamate of formula (I) obtained in step d/ or e/; steps a/ and b/ optionally being performed simultaneously.

2. The process as claimed in claim 1, in which the functionalized cyclic dithiocarbamate of formula (I) is enantiomerically pure.

3. The process as claimed in claim 1, in which the functionalized cyclic dithiocarbamate of formula (I) is l-raphanusamic acid or l-homoraphanusamic acid.

4. The process as claimed in claim 1, in which the compound of formula (II) is chosen from l-serine derivatives and l-homoserine derivatives.

5. The process as claimed in claim 4, in which the l-serine derivative is chosen from O-phospho-l-serine, O-succinyl-l-serine, O-acetyl-l-serine, O-acetoacetyl-l-serine, O-propio-l-serine, O-coumaroyl-l-serine, O-malonyl-l-serine, O-hydroxymethylglutaryl-l-serine, O-pimelyl-l-serine and O-sulfato-l-serine.

6. The process as claimed in claim 4, in which the l-homoserine derivative is chosen from O-succinyl-l-homoserine, O-acetyl-l-homoserine, O-acetoacetyl-l-homoserine, propio-l-homoserine, O-coumaroyl-l-homoserine, O-malonyl-l-homoserine, O-hydroxymethylglutaryl-l-homoserine, O-pimelyl-l-homoserine, O-phospho-L-homoserine and O-sulfato-l-homoserine.

7. The process as claimed in claim 1, in which the sulfhydrylase is chosen from the sulfhydrylases associated with the l-serine derivatives and the sulfhydrylases associated with the l-homoserine derivatives.

8. The process as claimed in claim 7, in which the sulfhydrylase associated with the l-serine derivative is chosen from O-phospho-l-serine sulfhydrylase, O-succinyl-l-serine sulfhydrylase, O-acetyl-l-serine sulfhydrylase, O-acetoacetyl-l-serine sulfhydrylase, O-propio-l-serine sulfhydrylase, O-coumaroyl-l-serine sulfhydrylase, O-malonyl-l-serine sulfhydrylase, O-hydroxymethylglutaryl-l-serine sulfhydrylase, O-pimelyl-l-serine sulfhydrylase and O-sulfato-l-serine sulfhydrylase.

9. The process as claimed in claim 7, in which the sulfhydrylase associated with the l-homoserine derivative is chosen from O-phospho-l-homoserine sulfhydrylase, O-succinyl-l-homoserine sulfhydrylase, O-acetyl-l-homoserine sulfhydrylase, O-acetoacetyl-l-homoserine sulfhydrylase, O-propio-l-homoserine sulfhydrylase, O-coumaroyl-l-homoserine sulfhydrylase, O-malonyl-l-homoserine sulfhydrylase, O-hydroxymethylglutaryl-l-homoserine sulfhydrylase, O-pimelyl-l-homoserine sulfhydrylase and O-sulfato-l-homoserine sulfhydrylase, preferably O-phospho-homoserine sulfhydrylase, O-succinyl-l-homoserine sulfhydrylase, O-acetyl-l-homoserine sulfhydrylase and O-sulfato-l-homoserine sulfhydrylase.

10. The process as claimed in claim 1, in which the inorganic trithiocarbonate is chosen from an alkali metal trithiocarbonate, an alkaline-earth metal trithiocarbonate and an ammonium trithiocarbonate.

11. The process as claimed in claim 1, including an optional step f/ of additional functionalization of the functionalized cyclic dithiocarbamate of formula (I) obtained in step d/ or in step e/.

12. A functionalized cyclic dithiocarbamate of formula (I) prepared according to the process described in claim 1.

13. An L-Raphanusamic acid or L-homoraphanusamic acid prepared according to the process described in claim 1.

14. The homocysteine sodium trithiocarbonate of formula (IV):
X.sub.2.sup.+S.sup.C(S)SCH.sub.2(CH.sub.2).sub.nC*H(NHR.sub.1)XR.sub.2(IV) in which R.sub.1, R.sub.2, X, * and n are as defined in claim 1 and X.sub.2 represents an alkali metal, an alkaline-earth metal or an ammonium group.

Description

EXAMPLES

Example 1: Enzymatic Synthesis of L-Homoraphanusamic Acid

Step 1:

[0078] O-Acetyl-L-homoserine (OAHS) was synthesized from L-homoserine and acetic anhydride according to Sadamu Nagai, Synthesis of O-acetyl-L-homoserine, Academic Press (1971), vol. 17, pages 423-424.

Step 2:

[0079] 10 g (62 mmol) of OAHS, synthesized beforehand, are placed in 140 ml of distilled water in a thermostatically controlled 250 mL glass reactor. The solution is brought to 35 C. with mechanical stirring. The pH of the reaction medium is 4.8. Before adding the enzyme, the pH is set at 6.5 with a few drops of sodium trithiocarbonate solution (4.78 g; 31 mmol, dissolved in 20 mL of distilled water). A sample of 1 mL of the reaction medium is taken (at t=0). A solution of pyridoxal 5-phosphate (10 mmol, 0.4 g) and the enzyme O-acetyl-L-homoserine sulfhydrylase (0.6 g) are dissolved in 10 mL of water and then added to the reactor.

[0080] The reaction begins, which brings about a lowering of the pH. The reaction medium is maintained at a pH of 6.5 by slow addition of sodium trithiocarbonate via the dropping funnel. Samples (1 mL) are taken during the reaction. The analyses by potentiometric titration, TLC, HPLC and UPLC/UV-mass show a gradual disappearance of the reagents (OAHS and Na.sub.2CS.sub.3) and the gradual appearance, in increasingly large amounts, of the following compound:

##STR00005##

[0081] This intermediate compound in turn gradually disappears to give in equimolar amounts: [0082] L-homoraphanusamic acid (the functionalized cyclic dithiocarbamate)

##STR00006## [0083] and L-homocysteine

##STR00007##

[0084] The only other products observed after the complete disappearance of the OAHS are traces of homoserine (hydrolysis of the OAHS).

Step 3: Separation and Isolation of the Dithiocarbamate:

[0085] The reaction medium is concentrated by partial evaporation of the water (so as to avoid the precipitation of the sodium acetate present in the reaction medium) under reduced pressure at 30 C. A precipitate forms since the dithiocarbamate proves to be the least soluble of the compounds present in the reaction medium. After filtration and drying, 4.9 g of dithiocarbamate are obtained. The overall isolated yield of dithiocarbamate is 45% (4.9 g obtained out of 11 g theoretically expected). Additional analyses on this dry product showed that this solid contains only traces of homocysteine.

Example 2: Synthesis of Dithiocarbamate (without Enzyme or Coenzyme)

[0086] Example 1 was repeated, the only difference being that the solution of pyridoxal 5-phosphate (10 mmol; 0.4 g) and the enzyme O-acetyl-L-homoserine sulfhydrylase (0.6 g) dissolved in 10 mL of water were not added to the reactor. It turns out that the reaction does not start and that it is impossible to continually add the solution of trithiocarbonate while attempting to conserve a pH of 6.5. On increasing to pH 8 and then to pH 12 by addition of sodium trithiocarbonate solution, the only reaction observed is the start of hydrolysis of the OAHS to homoserine. This example shows that the synthesis of dithiocarbonate has to be catalyzed with an enzyme to be effective.

Example 3: Enzymatic Synthesis of Dithiocarbamate (with Addition of CS.SUB.2 .at the End of the Reaction)

Step 1:

[0087] O-Acetyl-L-homoserine (OAHS) was synthesized from L-homoserine according to a protocol taken from the literature (Sadamu Nagai, Synthesis of O-acetyl-l-homoserine, Academic Press (1971), vol. 17, pages 423-424).

Step 2:

[0088] 10 g (62 mmol) of OAHS are placed in 140 mL of distilled water in a thermostatically controlled 250 mL glass reactor. The solution is brought to 35 C. with mechanical stirring. The pH of the reaction medium is 4.8. Before adding the enzyme, the pH is set at 6.5 by adding a few drops of sodium trithiocarbonate solution (the total amount added throughout the reaction is equal to 4.78 g, i.e. 31 mmol, dissolved in 20 mL of distilled water). A sample of 1 mL of the reaction medium is taken (at t=0).

[0089] A solution of 10 mL of distilled water containing 400 L of a solution of pyridoxal 5-phosphate (10 mmol/L) and of 0.6 g of enzyme (O-acetyl-L-homoserine sulfhydrylase) is prepared. A reduction in the pH indicating the formation of acetic acid makes it possible to state that the reaction has begun. It is necessary to maintain the reaction medium at a pH equal to 6.5. To do this, the sodium trithiocarbonate solution is added slowly via the dropping funnel. Samples (1 mL) are taken during the reaction.

[0090] When the analyses by potentiometric titration indicate a 50% conversion of the OAHS to homocysteine, 1.87 mL of carbon disulfide (31 mmol) are added to the reaction medium. The pH of the reaction medium is adjusted to 10 with 1M sodium hydroxide solution. The reaction medium is then brought to 50 C. Disappearance of the cysteine by potentiometric analysis is observed. Hydrochloric acid solution (2N) is then used to lower to 5 the pH of the reaction medium.

[0091] The additional analyses by TLC, HPLC and UPLC/UV-mass show the formation of a main product, L-homoraphanusamic acid.

[0092] The only other products observed after the complete disappearance of the OAHS are traces of homoserine (hydrolysis of the OAHS) and also traces of homocysteine.

Step 3: Separation and Isolation of the Dithiocarbamate

[0093] The reaction medium is concentrated by partial evaporation of the water (so as to avoid the precipitation of the sodium acetate and of the other salts present in the reaction medium) under reduced pressure at 30 C. A precipitate thus forms since the dithiocarbamate proves to be the species that is the least soluble in water. After filtration and drying, 9.2 g of dithiocarbamate are obtained. The overall isolated yield of dithiocarbamate is 9.2 g out of the theoretical 11 g, i.e. 84%.

Example 4: Enzymatic Synthesis of the Dithiocarbamate (with Addition of CS.SUB.2 .During the Reaction

Step 1:

[0094] O-Acetyl-L-homoserine (OAHS) was synthesized from L-homoserine according to a protocol taken from the literature (source: Sadamu Nagai, Synthesis of O-acetyl-homoserine, Academic Press, (1971), vol. 17, pages 423-424).

Step 2:

[0095] 10 g (61 mmol) of OAHS, synthesized beforehand, are placed in 140 ml of distilled water in a thermostatically controlled 250 mL glass reactor. The solution is brought to 35 C. with mechanical stirring. The pH of the reaction medium is 4.8. Before adding the enzyme, the pH is set at 7.2 by adding a few drops of the solution of trithiocarbonate and of carbon disulfide (4.78 g of trithiocarbonate, 31 mmol, 1.87 mL of carbon disulfide; 31 mmol dissolved in 20 mL of distilled water).

[0096] A sample of 1 mL of the reaction medium is taken (at t=0). A solution of 10 mL of distilled water containing 400 L of a solution of pyridoxal 5-phosphate (10 mmol/L) and of 0.6 g of enzyme (O-acetyl-L-homoserine sulfhydrylase) is prepared. A reduction in the pH indicating the formation of acetic acid makes it possible to state that the reaction has begun. It is necessary to maintain the reaction medium at a pH equal to 7.2. To do this, the sodium trithiocarbonate solution is added slowly via the dropping funnel. Samples (1 mL) are taken during the reaction. The analyses by potentiometric titration, TLC, HPLC and UPLC/UV-mass (after derivatization) show a gradual disappearance of the reagents (OAHS and Na.sub.2S.sub.3) and the gradual appearance, in increasingly large amounts, of L-homoraphanusamic acid. When all of the OAHS has reacted, the pH of the medium is lowered to 5 using 2M hydrochloric acid solution.

[0097] L-homoraphanusamic acid (the dithiocarbamate) is obtained.

[0098] Derivatization for the UPLC/UV-mass method was performed via the same method described in Example 1.

[0099] The only other products observed after the complete disappearance of the OAHS are traces of homoserine (hydrolysis of the OAHS) and also traces of homocysteine.

Step 3: Separation and Isolation of the Dithiocarbamate

[0100] The reaction medium is concentrated by partial evaporation of the water (so as to avoid the precipitation of the sodium acetate and of the other salts present in the reaction medium) under reduced pressure at 30 C. A precipitate thus forms since the dithiocarbamate proves to be the species that is the least soluble in water. After filtration and drying, 8.3 g of dithiocarbamate are obtained. The overall isolated yield of dithiocarbamate is 8.3 g out of the theoretical 11 g, i.e. 75.4%.