Dihydrofolic acid preparation

Dihydrofolate reductase (DHFR) (EC 1.5.1.3) catalyzes the NADPH-depen-dent reduction of dihydrofolic acid, thereby restoring THFA as a central cofactor in Cl transfer reactions. Commonly the enzyme has been found to be monomeric, with a molecular mass around 20 kDa. It possesses an a/p fold and does not contain a cofactor. Phylogenetically, the relationship of the enzyme to eukaryotic homologs seems to be closer than to other bacterial DHFRs. Since the expression of the natural enzyme in Tm is extremely low, studies focused on the structure, stability, and folding of the recombinant protein. For its preparation and general characterization, cf. ref. 91a. 

In the context of preparing potential inhibitors of dihydrofolate reductase (DHFR), the group of Organ has developed a rapid microwave-assisted method for the preparation of biguanide libraries (Scheme 6.174) [330]. Initial optimization work was centered around the acid-catalyzed addition of amines to dicyandiamide. It was discovered that 150 °C was the optimum temperature for reaction rate and product recovery, as heating beyond this point led to decomposition. While the use of hydrochloric acid as catalyst led to varying yields of product, evaluation of trimethylsilyl chloride in acetonitrile as solvent led to improved results. As compared to the protic... 

Recently, the enzymatic formation of folinic acid has been utilized to synthesize radioactively labeled products.34 The preparation of 5-formyl tetrahydrofolate, 9,3, 5 -3H and 5-formyl-14C-tetrahydrofolate starts with tritiated folic acid, which is reduced to dihydrofolate, incubated in the presence of formaldehyde, dihydrofolate reductase, and NADPH, and finally incubated with 5,10-methylenetetrahydrofolate dihydrogenase. The product,... 

The cell-free system prepared from washed embryos has much higher translational activity than the conventional system (compare Fig. 3A and B). When 5 -capped dihydrofolate reductase (DHFR) mRNA containing 549 nt of 3 UTR with a pA tail was incubated with newly prepared as well as conventional extract, there was almost linear kinetics in DHFR synthesis over 4 h, compared with the regular system, which ceased to function after 1.5 h. Further, when washed extract in the reaction volume was increased to 48%, amino acid incorporation occurred initially at a rate twice that of 24% extract, and then stopped after 1 h. However, this pause was caused by a shortage of substrates rather than an irreversible inactivation of ribosomes or factors necessary for translation addition of amino acids, ATP, and GTP after cessation of the reaction (arrow) restarted... 

Fohc acid analogues containing amino acids other than glutamate, and also folate covalendy bound to a protein for the purposes of antibody production, have been prepared (58—60). Methotrexate is an analogue of fohc acid that is widely used in cancer chemotherapy (61) (see ChemotHERAPEUTICS, anticancer). Other analogues such as trimethoprim and pyrimethamine are used in the treatment of malaria and protozoal diseases (62). These analogues bind extremely tightly to dihydrofolate reductase. 

Atovaquone is an antimalarial preparation. It inhibits mitochondrial electron transport in parasites, causing inhibition of nucleic acid synthesis. Proguanil exerts its effect by means of the metabolite cycloguanil, which inhibits dihydrofolate reductase in the malarial parasite, disrupting deox-ythymidylate synthesis. It is indicated in prophylaxis of P. falciparum in patients with severe renal impairment (Ccr less than 30 mL/min) hypersensitivity to any component of the product. 

The former can be prepared by reductive alkylation of 2-acetamido-5-amino-4(3H)pyrimidone with dimethyl iV-(p-(A-(formylmethyl)formamido)ben-zoyl -L-glutamate, followed by reformylation, partial deblocking, and acid hydrolysis to (LXIII). Both (LXIII) and (LXIV) were reported to have inhibitory activity against S. faecalis equal to that of aminopterin. They both inhibited dihydrofolate reductase (source not named) at 4 x 10" m, for 50 per cent inhibition. Compound (LXIII) is a better inhibitor of thymidylate synthetase than (LXIV) (3 x 10 vj. 7 x I0 M for 50 per cent inhibition). 

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