Synthesis of (6S)-5-methyl-5,6,7,8-tetrahydrofolic acid

Abstract

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.

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 commercially available 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.    
   
   
       2 . A chemoenzymatic process for producing calcium (6S)-5-methylTHFA, comprising the steps of: 
 allowing DHFR to act upon DHFA in the presence of NADP, NADPH glucose and GluDH;    accumulating (6S)-THFA in aqueous solution and converting it in situ to (6S)-5-methylTHFA; and    recovering (6S)-5-methylTHFA as its calcium salt therefrom.    
   
   
       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.    
   
   
       4 . A 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 . A process according to  claim 3  wherein at least one step of the process is performed in an inert atmosphere.  
   
   
       6 . A process according to  claim 5  wherein the inert atmosphere comprises N 2 .  
   
   
       7 . A process according to  claim 5  wherein the inert atmosphere comprises Ar.  
   
   
       8 . A 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 . A 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 . A 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 . A 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 . A process according to  claim 3  wherein step 3 comprises adding a suspension of NaBH 4  in NaOH.  
   
   
       13 . A process according to  claim 3  wherein step 4 comprises adding a solution containing CaCl 2 .  
   
   
       14 . A process according to  claim 3  wherein step 4 comprises adding a solution containing calcium chloride dihydrate in methanol.