P-aminobenzoylglutamic acid

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. 

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. 

Folate is a relatively unstable nutrient processing and storage conditions that promote oxidation are of particular concern since some of the forms of folate found in foods are easily oxidized. The reduced forms of folate (dihydro- and tetrahydrofolate) are oxidized to p-aminobenzoylglutamic acid and pterin-6-carboxylic acid, with a concomitant loss in vitamin activity. 5-Methyl-H4 folate can also be oxidized. Antioxidants (particularly ascorbic acid in the context of milk) can protect folate against destruction. The rate of the oxidative degradation of folate in foods depends on the derivative present and the food itself, particularly its pH, buffering capacity and concentration of catalytic trace elements and antioxidants. 

An early synthesis of folic acid developed by chemists at American Cyanamid involved condensation of 2,4,5-triamino-6-hydroxypyrimidine, 2,3-dibromopropionaldehyde and p-aminobenzoylglutamic acid (Scheme 3.9) [55a-d].This three-component procedure has become known as the Waller condensation, and has been widely employed for the synthesis of analogues because of the ease with which the various components of the condensation can be varied. Products from this three-component reaction, however, are mixtures of 6- and 7-substituted regioisomers. 

AzaFA (567a) has been synthesized as outlined in Scheme 3.113 [230]. Nitrosation of p-aminobenzoylglutamic acid followed by reduction afforded... 

One of the earliest attempts to prepare analogues of FA as potential inhibitors involved the synthesis of 2-amino-4,7-dihydroxypteridine-6-carboxylyl-p-ami-nobenzoic acid (612) (in which the change from the structure of FA itself is exchange of the methylene bridge for a carbonyl group, and oxidation of position 7 to a lactam). This compound, which was a surprisingly effective inhibitor, was prepared from isoxanthopterin carboxylic acid (611) by in situ conversion to its acid chloride with a mixture of phosphorus oxychloride and phosphorus pentachloride, followed by addition of p-aminobenzoylglutamic acid (Scheme 3.132) [115]. 

This activity (FAS) cleaves folic acid into pterin-6-aldehyde and p-aminobenzoylglutamic acid, and the HPLC assay developed for it involves the separation of the two. 

The separation was carried out on a reversed phase HPLC column (Li-Chrosorb 10 RP-18) or on an anion exchanger (Partisil PXS 10/25 SAX). For pterin-6-aldehyde, the mobile phase was a 15 mM phosphate buffer (pH 6.0) with 10% methanol. For p-aminobenzoylglutamic acid, a 0.1M NH3 solution containing 0.2M NaCl, 20% (v/v) 1-propanol, and 10% (v/v) acetonitrile adjusted to pH 5.32 with acetic acid was used. Both columns were eluted isocrati-cally, and the detection was by UV at 254 nm and by liquid scintillation counting. 

Figure 9.130 Identification of the products of folic acid C9-N10 cleavage. (A) [2-l4C]folic acid was incubated with FAS and coinjected with pterin-6-aldehyde. Radioactivity was measured in 0.5 niL fractions. (B) [7,9,3, 5 -3H]folic acid was incubated with FAS and coinjected with pterin-6-aldehyde and p-aminobenzoylglutamic acid. Radioactivity was determined in 0.5 mL fractions. Arrows indicate retention volumes of related compounds. Abbreviations P-6-COOH, pterin-6-carboxylic acid 6-HMP, 6-hydroxymethylpterin P-6-CHO, pterin-6-aldehyde FA, folic acid p-ABGA, p-aminobenzoylglutamic acid. (From De Witt et al., 1983.)...

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). 

Unconjugated pteridines, which lack a p-aminobenzoylglutamic acid residue, are of interest because they can be formed by the oxidative transformation of reduced folate derivatives moreover, pteridines in biological fluids are elevated under certain pathological conditions (McCormack and Newman, 1985). 

Figure 3 Chromatogram of folate derivatives with UV absorbance detection at 280 nm ethanol gradient in lOmM TBAP reagent (pH 7.55) X-Bondapak Cij column (3.9 X 300 mm, 10 im) flow rate 1 mL/min. Peaks (1) p-aminobenzoylglutamic acid (2) lO-HCO-H4-folate (3) H4-folate, (4) 5-HCO-H4-folate (5) H2-folate (6) 5-CH3-H4-folate (7) folic acid (3-5 nmol of each derivative). (From Ref. 70.)...

Bacteria use p-aminobenzoic acid only for conversion to 7,8-dihydrofolic acid (Woods, 1962 Griffin and Brown, 1964). Thus, E, coli condenses p-aminobenzoic acid (and, alternatively, p-aminobenzoylglutamic acid) with 2-amino-4-oxo-6-hydroxymethyl-7,8-dihydropteridine (9.12) (as the 6-pyrophosphate) to give dihydropteroic acid (and alternatively, dihydrofolic acid) (Jaenicke and Chan, i960). The sulphonamides competitively inhibit the isolated enzyme dihydrofolate synthetase which catalyses these steps (G. Brown, 1962). From Lactobacillus plantarum two enzymes responsible for this synthesis have been isolated in a pure state (Shiota, Baugh, Jackson and Dillard, 1969). The first of these catalyses the esterification of 2-amino-4-oxo-6-hydroxymethyl-7,8-dihydropteridine (9.12) to its pyrophosphoryl derivative. The second is Brown s dihydrofolate synthetase. This second enzyme has also been isolated from several strains of... 

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