Folic acid coupling

Theory Folic acid (I) undergoes cleavage by reduction with Zn-Hg in acidic medium to yield p-aminobenzoylglutamic acid (II). The primary aromatic amino group present in the latter is subsequently diazotized in the usual manner and coupled in acidic solution with N-(l-naphthyl)-ethylenediamine hydrochloride in the absence of light (caution). The colour thus produced has a maximum absorption at 550 nm and the extinction (E) is consequently compared with a calibration curve obtained from / -aminobcnzoic acid (PABA) that has been duly diazotized and coupled exactly in the same fashion as the/ -aminobcnzoylglutamic acid. 

The mode of action of the sulfonamides as antagonists of 4-aminobenzoic acid (PAB) is well documented, as is the effect of physicochemical properties of the sulfonamide molecule, e.g. pK, on potency (B-81MI10802). Sulfonamides compete with PAB in the biosynthesis of folic acid (44), a vital precursor for several coenzymes found in all living cells. Mammalian cells cannot synthesize folic acid (44), and rely on its uptake as an essential vitamin. However, bacteria depend on its synthesis from pteridine precursors, hence the selective toxicity of sulfonamides for bacterial cells. Sulfonamides may compete with PAB at an enzyme site during the assembly of folic acid (44) or they may deplete the pteridine supply of the cell by forming covalently-bonded species such as (45) or they may replace PAB as an enzyme substrate to generate coupled products such as (46) which are useless to the cell. 

An interesting example is die assay of clonidine hydrochloride in injections and tablets (British Pharmacopoeia, 1980). In 0.01 Mhydrochloric acid, clonidine exhibits two sharp maxima ne - 272 nm and 279 nm, which are not suitable for precise measurement. However, clonidine forms an ion pair with bromothymol blue, and this can be readily extracted into chloroform for subsequent measurement of the broad maximum near 420 nm. Because of the intiinsic variabihty of reagents used in such methods, a pharmacopoeial reference standard is employed for cahbration. A similar policy is adopted for assays involving chemical modification of the drug, as in the tetrazolium assay for corticosteroids, the assay for folic acid involving hydrolysis, diazotisation, and coupling with N-(l-naphthyl)ethylenediamine, and the reaction of penicillins with imidazole and mercuric salts. 

Thymidylate synthetase is an important enzyme, which is responsible for the reductive methylation of deoxyuridylic acid (dUMP, 7) to deoxythymidylic acid (dTMP, 8). The methylation of the uracil moiety (present in RNA) to 5-methyl uracil (thymine, present in DNA) requires participation of a folic acid coenzyme, N, methylenetetrahydrofolate as a methyl donor. The functioning of thymidylate synthetase is coupled with the activity of DHF reductase. That is why this biochemical target is usually referred to as thymidylate synthetase/ DHF reductase. 

A series of 5,8-dideaza analogs of folic acid such as isofolic acid aminopterine have been evaluated as inhibitors of thymidylate synthase. Coupling of 10-thia-5,8-dideaza-pteroic acid (208) with di-/-Bu-glutamate (209) using DEPC affords amide (210).78... 

Dideaza analogs of folic acid (214) are prepared via coupling of dideazapteroic acid (213) with di-/-Bu-Z-glutamate using DEPC.80 2-Desamino-5,8-dideazaisofolic acid is found to be 4-6 fold cytotoxic toward L1210 leukemia cells than their 2-NH2 counterparts and to be poor inhibitors of mammalian thymidylate synthase. 

The mechanism of the clastic cleavages of pyruvate, producing acetyl phosphate and formate or CO2 and Ha, is still obscure. Biotin (Schuster and Lynen, 1960), folic acid (Delavier-Klutchko, 1959), and vitamin B12 derivatives (Rabinowitz, 1960) have recently been implicated in these reactions. It seems possible that these may also be examples of tightly coupled systems in which 2-acetylthiamine pyrophosphate is an intermediate. 

The spectrophotometric determination of folic acid, either in pure form or in its pharmaceutical preparations, is based on the probable diazotization of p-aminobenzoylglutamic acid obtained after reductive cleavage of folic acid, followed by either coupling with iminodibenzyl (maximum absorbance at 580 nm) or with 3-aminophenol to produce an orange yellow colored product (460 nm). 

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