Synthesis of (6S)-5,6,7,8-tetrahydrofolic acid
A process for the large-scale chemoenzymatic production of (6S)-5-methyl-5,6,7,8-tetrahydrofolic acid, also known as (6S)-5-methylTHFA, the process comprising the steps of: (1) reducing folic acid (FA) so as to yield dihydrofolic acid (DHFA); (2) stereoselectively reducing DHFA with dihydrofolate reductase (DHFR) in the presence of NADP/NADPH, glucose and GluDH so as to yield (6S)-THFA; (3) converting (6S)-THFA to (6S)-5-methlTHFA; and (4) isolating (6S)-5-methylTHFA.
1. A process for the large-scale chemoenzymatic production of (6S)-5-methyl-5,6,7,8-tetrahydrofolic acid, also known as (6S)-5-methylTHFA, the process comprising the steps of:
(1) reducing folic acid (FA) with zinc powder under basic conditions to yield dihydrofolic acid (DHFA);
(2) stereoselectively reducing DHFA with dihydrofolate reductase (DHFR) in the presence of an NADP/NADPH recycling system, glucose and GluDH so as to yield (6S)-THFA;
(3) converting (6S)-THFA to (6S)-5-methylTHFA in situ by conventional methods; and
(4) isolating (6S)-5-methylTHFA as its calcium salt;
wherein (6S)-5-methyl-THFA is produced without isolation of an intermediate.
2. A chemoenzymatic process for producing calcium (6S)-5-methylTHFA, comprising the steps of:
(1) allowing dihydrofolate reductase (DHFR) to act upon dihydrofolic acid (DHFA) in the presence of NADP, NADPH glucose and GIuDH;
(2) accumulating (6S)-THFA in aqueous solution and converting it in situ to (6S)-5-methylTHFA; and
(3) recovering (6S)-5-methylTHFA as its calcium salt therefrom;
wherein (6S)-5-methyl-THFA is produced without isolation of an intermediate.
3. A process for the large-scale chemoenzymatic production of (6S)-5-methyl-5,6,7,8-tetrahydrofolic acid, also known as (6S)-5-methylTHFA, the process comprising the steps of:
(1) reducing folic acid (FA) so as to yield dihydrofolic acid (DHFA);
(2) stereoselectively reducing DHFA with dihydrofolate reductase (DHFR) in the presence of NADP/NADPH, glucose and GluDH so as to yield (6S)-THFA;
(3) converting (6S)-THFA to (6S)-5-methylTHFA; and
(4) isolating (6S)-5-methylTHFA;
wherein (6S)-5-methyl-THFA is produced without isolation of an intermediate.
4. The process according to claim 3 wherein step 1 further comprises stirring a solution of FA, NaOH and zinc powder for approximately two hours at approximately 40° C.
5. The process according to claim 3 wherein at least one step of the process is performed in an inert atmosphere.
6. The process according to claim 5 wherein the inert atmosphere comprises N 2 .
7. The process according to claim 5 wherein the inert atmosphere comprises Ar.
8. The process according to claim 3 wherein step 1 further comprises:
(i) suspending folic acid in water and adding an NaOH solution so as to create a first solution;
(ii) creating a solution of ascorbic acid and water and adding an NaOH solution so as to create a second solution; and
(iii) combining the second solution and the first solution, adding sodium dithionite and stirring until the folic acid has been converted to DHFA.
9. The process according to claim 3 wherein step 2 further comprises stirring the DHFA, glucose, GluDH, DHFR and NADP/NADPH for 2 hours at 40° C.
10. The process according to claim 3 wherein step 2 further comprises stirring the DHFA, glucose, GluDH, DHFR and NADP/NADPH for 2 hours at a temperature between 22° C. −30° C.
11. The process according to claim 3 wherein step 2 further comprises:
(i) stirring the DHFA, glucose, GluDH, DHFR, NADP/NADPH and water for 1 hour; and
(ii) adding additional amounts of GluDH, DHFR and water and stirring for approximately 3.5 hours.
12. The process according to claim 3 wherein step 3 comprises adding a suspension of NaBH 4 in NaOH.
13. The process according to claim 3 wherein step 4 comprises adding a solution containing CaC1 2 .
14. The process according to claim 3 wherein step 4 comprises adding a solution containing calcium chloride dihydrate in methanol.