7.8- Dihydrofolate

En2ymatic reduction of folic acid leads to the 7,8-dihydrofolic acid (H2 folate) (2), a key substance in biosynthesis. Further reduction, cataly2ed by the en2yme dihydrofohc acid reductase, provides (65)-5,6,7,8-tetrahydrofohc acid (H folate) (3). The folate (3) is the key biological intermediate for the formation of other folates (4—8) (Table 2). 

Catalytic reduction of folic acid to 5,6,7,8-tetrahydrofolic acid (225) proceeds fast in trifluoroacetic acid (66HCA875), but a modified method using chemical reductants leads with sodium dithionite to 7,8-dihydrofolic acid (224). Further treatment with sodium borohydride gives (225) which has been converted into 5-formyl-(6i ,S)-5,6,7,8-tetrahydro-L-folic acid (leucovorin) (226) by reaction with methyl formate (equation 70) (80HCA2554). 

This interesting conversion of a five- into a six-membered heterocyclic ring was proven by the isolation of the enzyme GTP-cyclohydrolase from E. coli (71MI21600) and a similar one from Lactobacillus platarum (B-71MI21601) which catalyzes the reaction (300)(303). Dephosphorylation leads to 7,8-dihydro-D-neopterin (304), which is then cleaved in the side-chain to 6-hydroxymethyl-7,8-dihydropterin (305), the direct precursor of 7,8-dihy-dropteroic acid and 7,8-dihydrofolic acid (224). The alcohol (305) requires ATP and Mg " for the condensation with p-aminobenzoic and p-aminobenzoylglutamic acid, indicating pyrophosphate formation to (306) prior to the substitution step. 

DHFR has been the object of intense research for the last few decades. The enzyme catalyses the NADPH-dependent reduction of 7,8-dihydrofolate to 5,6,7,8 tetrahydrofolate, a chemical which participates in the thymidilate synthesis cycle. Thus, the enzyme is crucial in the synthesis of thymidine monophosphate as well as in various one-carbon unit transfer reactions. 

The reduction of 7,8-dihydrofolate (H2F) to 5,6,7,8-tetrahydrofolate (H4F) has been analyzed extensively14 26-30 and a kinetic scheme for E. Coli DHFR was proposed in which the steady-state kinetic parameters as well as the full time course kinetics under a variety of substrate concentrations and pHs were determined. From these studies, the pKa of Asp27 is 6.5 in the ternary complex between the enzyme, the cofactor NADPH and the substrate dihydrofolate. The second observation is that, contrary to earlier results,27 the rate determining step involves dissociation of the product from the enzyme, rather than hydride ion transfer from the cofactor to the substrate. 

Figure 1. Tautomeric equilibrium of the pteridine ring and the structure of 7,8-dihydrofolate.

Absorption interferences would arise from compounds other than leucovorin that convert to anhydroleucovorin at acidic pH. An example of such a compound is 10fH F, although it is not expected to contribute to absorption due to its instability. However, 10-formyl-7,8-dihydrofolate is a possible contaminant. Additional absorption could also result from unsaturated pteridines. 

This enzyme [EC 1.5.1.3], also called tetrahydrofolate dehydrogenase, catalyzes the reversible reaction of 7,8-dihydrofolate with NADPH to produce 5,6,7,8-tetrahy-drofolate and NADP+. The enzyme isolated from mammals and some microorganisms can also slowly catalyze... 

Folic acid derivatives folate, 7,8-dihydrofolate, Bu N--l>0< Me0H-H,0 (35 65) Ocnidccyl 238... 

I I 3. The answer is c. (Hardman, pp 1243-1247.) Antimetabolites of folic acid such as methotrexate, which is an important cancer chemotherapeutic agent, exert their effect by inhibiting the catalytic activity of the enzyme dihydrofolate reductase. The enzyme functions to keep folic acid in a reduced state. The first step in the reaction is the reduction of folic acid to 7,8-dihydrofolic acid (FH2), which requires the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). The second step is the conversion of FH2 to 5,6,7,8-tetrahydrofolic acid (FH ). This part of the reduction reaction requires nicotinamide adenine dinucleotide (NADH) or NADPH. The reduced forms of folic acid are involved in one-carbon transfer reactions that are required during the synthesis of purines and pyrimidine thymidylate. The affinity of methotrexate for dihydrofolate reductase is much greater than for the substrates of folic acid and FH2. The action of... 

FH2—7,8-dihydrofolic acid FH4—5,6,7,8-tetrahydrofolic acid FSH—follicle-stimulating hormone FU—fluorouracil... 

H FIGURE 22-49 Thymidylate synthesis and folate metabolism as targets of chemotherapy, (a) During thymidylate synthesis, /V5,N10-methylenetetrahydrofolate is converted to 7,8-dihydrofolate the N5,N10-methylenetetrahydrofolate is regenerated in two steps (see Fig. 22-44). This cycle is a major target of several chemotherapeutic agents, (b) Fluorouracil and methotrexate are important chemotherapeutic agents. In cells, fluorouracil is converted to FdUMP, which... 

Folic acid [225 R = NHC6H4CONHCH(COOHXCH2)2COOH] in acidic solution gives 7,8-dihydrofolic acid (227).359 In neutral medium with low... 

A key step in DNA biosynthesis, that of conversion of deoxyuridylate (dUMP) to deoxythymidylate (dTMP), is catalyzed by thymidylate synthetase which uses (25) as cofactor. This reaction involves both the transfer of a one carbon unit at the formaldehyde level and hydride transfer (from C-6 of (25)) to produce 7,8-dihydrofolate (27) and dTMP... 

This is a small monomeric enzyme (molecular weight around 20000) that catalyses reduction of a double bond between carbon and nitrogen, converting 7,8-dihydrofolate into 5,6,7,8-tetrahydrofolate (Fig. 14). Hydride is transferred from the 4-pro-R position of NADPH to C-6, N-5 acquiring a proton [64-66]. 

A-2) It is common to combine trimethoprim with mlfonamide in the treatment of certain infections. The sulfonamide acts on bacteria to inhibit folate fonnation in bacteria by competitively inhibiting PABA incorporation (fig. 10.4). The trimethoprim acts on the dihydrofolate reductase step (between 7,8 dihydrofolate and 5,6,7,8 THF). Apparently, trimethoprim has a more damaging effect on bacteria and protozoans than it has on humans. 

Folic acid [LXVII R = NHC6H4CONHCH(COOH)(CH2)2COOH] is in the first step reduced as LXVII (R = H) in acidic solution 7,8-dihydrofolic acid is reduced with cleavage of the C-9/N-10 bond to LXIX (R = H) [365]. Besides the electrochemical cleavage, a nonelectrochemical cleavage with loss of the 4-aminobenzoylglutamate side chain from the 5,8-dihydrofolic acid has been observed [368]. In neutral medium with low buffer capacity, it is possible to reduce 7,8-dihydrofolic acid to the 5,6,7,8-tetrahydrofolic acid [369]. 

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