P-aminobenzoylglutamic Acid Research Articles

Improved photostability of folic acid by the radical-scavenging effect of tannic acid

Folic acid (FA) is sensitive to ultraviolet (UV) light and decomposes into photoproducts with higher fluorescence quantum yield: 6-formylpterin (Fop) and p-aminobenzoylglutamic acid (PABA-Glu). Fop is subsequently oxidized into 6-carboxypterin (Cap). The increased fluorescence intensity of FA solution with increasing irradiation time proved the degradation. Addition of tannic acid (TA) at the TA/FA molar ratio of 0.025/1 could protect 99% FA compared to that no FA could be detected without addition of TA after 120 min of UV irradiation. The retention of FA without TA at pH 5–6 was below 0.4% and above 93% at pH 3–4, but the retention of FA with TA was over 97% for pH 3–6. Moreover, the retention of FA with TA in gummy was over 81% which was significantly higher than that of the one without TA. No covalent bonds were formed between TA and FA. Ethylenediaminetetraacetic acid could not protect FA against photodegradation. Ascorbic acid (Vc) could protect FA to the same extent as TA, but with more addition. It agreed with the results that the DPPH radical-scavenging effect of Vc is lower than TA. These results demonstrated that TA could improve the photostability of FA by the radical-scavenging effect.

Synthesis of p-Aminophenyl Aryl H-Phosphinic Acids and Esters via Cross-Coupling Reactions: Elaboration to Phosphinic Acid Pseudopeptide Analogues of Pteroyl Glutamic Acid and Related Antifolates

The synthesis of suitably protected p-aminophenyl H-phosphinic acids and esters from the corresponding para-substituted aryl halides has been accomplished via the Pd-catalyzed cross-coupling reaction of anilinium hypophosphite, either in the absence or presence of a tetraalkyl orthosilicate, to provide the free H-phosphinic acid or the corresponding ester, respectively. Subsequent conjugate addition of either a PIII species or phosphorus anion, generated in situ from either the free H-phosphinic acid or ester, to a 2-methylene glutaric acid ester provided the aryl phosphinic acid analogue of p-aminobenzoyl glutamic acid. Alkylation of these suitably protected p-aminophenyl phosphinic acid esters with a 6-(bromomethyl)pteridine or the corresponding (bromomethyl)pyridopyrmidine, followed by hydrolytic removal of protecting groups, provided the target aryl phosphinic acid analogues of folic acid and related antifolates. Alternatively, for the synthesis of the folate or 5-deazafolate analogues on a slightly larger scale, reductive amination with either N2-acetyl or N2-pivaloyl-6-formylpterin or the corresponding formylpyridopyrmidine and the same suitably protected p-aminophenyl phosphinic acid esters, followed by removal of protecting groups, is preferred. In the course of this research, it was observed that the nucleophilicity of both the aniline nitrogen and various PIII species derived from p-aminophenyl phosphinic acid derivatives is significantly reduced compared to that of the unsubstituted counterpart.

Increased folate catabolism following total body gamma-irradiation in mice.

Concentrations of total folates and their oxidative degradate, para-aminobenzoyl glutamic acid, were determined in mouse after 2-7 Gy total body gamma-irradiation (TBI). Total liver folate levels were drastically reduced by almost 47% over a period of 120 h after TBI with 7 Gy. Oxidative damage, splitting the folate molecule into pterin and p-aminobenzoylglutamic acid (p-ABG), was observed after TBI. p-ABG levels, which 24 h after irradiation were raised by 15%, were further elevated to more than three-fold over the control after 120 h. A dose-dependent increase in the oxidative degradation of folate was observed. The oxidative cleavage of folate may be one factor contributing to folate deficiency in radiation stress.

Spectrophotometric determination of folic acid in pharmaceutical preparations by coupling reactions with iminodibenzyl or 3-aminophenol or sodium molybdate–pyrocatechol

Novel coupling reagents are used for the simple and sensitive spectrophotometric determination of folic acid either in pure form or in its pharmaceutical preparations. The methods are based on the probable diazotization of the p-aminobenzoylglutamic acid obtained after reductive clevage of folic acid, followed by either coupling with iminodibenzyl to give a violet product with λ max of 580 nm or coupling with 3-aminophenol to produce an orange yellow-colored product with λ max of 460 nm. Sodium molybdate and pyrocatechol are used in the third method and the pale red-colored product formed has a λ max of 490 nm. The methods are highly reproducible and have been applied to the determination of folic acid in tablets and the results compare favorably with the official method. Common excipients used as additives in pharmaceutical preparations do not interfere in the proposed methods.

Validity of Dual-Label Stable Isotopic Protocols and Urinary Excretion Ratios to Determine Folate Bioavailability from Food

We tested the hypothesis that some cereal-product vehicles may reduce fortificant bioavailability below 85% and examined the feasibility of using single dose, dual-label, non-saturation protocols for studying bioavailability based on urinary excretion ratios (UER; % oral 13C6 isotope dose excreted in intact folate/% i.v. 2H4 isotope dose excreted). Fifteen females received 225 micrograms oral folate (capsules, fortified bran flakes, and fortified white bread), mainly as 13C6-PteGlu, followed by i.v. injection of 100 micrograms 2H4-PteGlu. UERs were used as the primary index of absorption. Urinary folate was cleaved to p-aminobenzoylglutamic acid, dervivatized and determined by gas chromatography/mass spectroscopy (GC/MS). The UER mean (95%CI) for folic acid was 2.18 (1.2-3.8) at 48 hours and as these were greater than 1.0, it was concluded that oral and i.v. isotopes of folic acid are handled differently by the body. Compared to the 48 hour UER for folic acid, UERs for white bread and bran flakes were 0.71 and 0.37, respectively, thus indicating some matrix inhibition of absorption. Consideration should be given to the choice of cereal-based fortification vehicles in order to maximize bioavailability. Plasma enrichment of folate can be measured using LC/MSMS (liquid chromatography/mass spectroscopy-mass spectroscopy) but seems unfeasible with the GC/MS method.

Study of the photolysis of folic acid and 6-formylpterin in acid aqueous solutions

Folic acid and 6-formylpterin solutions of pH in the range 4.5–6.0 were photolysed at 350 nm at room temperature. The photochemical reactions were followed by UV–VIS spectrophotometry, thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). In the presence of oxygen, folic acid is photochemically cleavaged yielding 6-formylpterin and p-aminobenzoylglutamic acid. As the photolysis proceeds, 6-carboxypterin arises from 6-formylpterin, as observed when solutions only containing the latter compound are irradiated in the presence of oxygen. In the absence of oxygen, folic acid is not light-sensitive. However, when oxygen free solutions of 6-formylpterin are irradiated, a ‘red intermediate’ is observed, which is thermally oxidised producing 6-carboxypterin on admission of oxygen. The quantum yields of substrates disappearance and of photoproducts formation are reported.

Epidermal N-acetylation of p-aminobenzoyl glutamic acid: difference in response to ultraviolet B irradiation

Arylamine N-acetyltransferases (EC 2.3.1.5) are conjugating drug-metabolizing enzymes and consist of two major isozymes, NAT1 and NAT2. As they have different substrate specificity and expression of polymorphism, distribution of the isozymes may be detected by investigating acetylation. p-Aminobenzoyl glutamic acid (pABG), one of the specific substrates for NAT1 in human pro-monocytic cell-line, was metabolized through acetylation by 9000 × g supernatant fraction from human epidermal keratinocytes and the effect of ultraviolet B (UVB) irradiation on the acetylation was also studied. Forty-eight hours after irradiation of UVB (200 J/m 2), the activity was not increased (114 ± 8.3%, n = 3), while increase in N-acetylating capacity for 2-aminofluorene (substrate for NAT1 and NAT2) amounted to 201 ± 16% ( n = 3). These results suggest that there are at least two isozymes for N-acetylationin the human epidermic and NAT2 may be affected by UVB irradiation.

Pteridines, XCVI. Synthesis and Reactions of 6-(1,2,3-Trihydroxypropyl)pteridines

Summary Various hydropyrano [3.2-g] pteridines (7, 10, 12, 21 , 33) were oxidized to the corresponding 6-(1,2,3- trihydroxypropyl)pteridine derivatives (8,9, 11, 13,32, 34), which are valuable intermediates for the synthesis of new 6-substituted pteridines (15 - 46). Periodate oxidation led to pteridine-6-carboxaldehydes (15, 16, 17, 38), which easily formed oximes (19 - 22, 39, 40). The aldehydes were further oxidized to 6-carboxylic acids (25,26,42) or reduced to 6-hydroxymethyl derivatives (27, 28, 43). Reductive condensations of the aldehydes (15, 16, 38) with p-aminobenzoylglutamic acid afforded folic acid analogues (29, 30, 44). Dehydration of the oximes (19, 20, 39) resulted in the formation of 6-carbonitriles (23, 24, 41, 46). The newly synthesized compounds were characterized by elemental analysis and by UV and NMR spectra.

Monitoring the effect of triamterene and hydrochlorothiazide on dihydrofolate reductase activity using a new spectrophotometric method

A new spectrophotometric method is developed and applied for the study of the inhibitory effect of triamterene, hydrochlorothiazide and their combinations on the in vitro activity of dihydrofolate reductase enzyme. The method is based on incubating the drug (0.1–1.0 μM) or a buffer control with a solution containing reduced nicotinamide adenine dinucleotide phosphate (0.5 mM), magnesium chloride (1.29 mM), and folic acid as a substrate (0.01–0.1 mM) with the dihydrofolate reductase (0.25 unit). The resulting tetrahydrofolic acid is determined by first hydrolysing it by a methanol—hydrochloric acid mixture to produce p-aminobenzoyl glutamic acid, then adding p-dimethylaminocinnamic aldehyde reagent to form a stable pink coloured product. The colour is found to develop within 5 min and is stable over 12 h, with a maximum absorption at 545 nm. A linear calibration curve is formed by using standard solutions of tetrahydrofolic acid. The presence of the studied drugs did not interfere with the determination. Lineweaver—Burk plots of the reaction kinetics, in the presence of triamterene and/or hydrochlorothiazide showed a competitive inhibition of the dihydrofolate reductase in the presence of triamterene with or without hydrochlorothiazide. A 100% inhibition is obtained by 1 μM solution of triamterene at a folic acid concentration of 0.01 mM. No measurable effect of hydrochlorothiazide at the studied concentration range is demonstrated.

3 Synthesis of Analogues of Folic Acid, Aminopterin and Methotrexate as Antitumour Agents

Publisher Summary This chapter discusses the syntheses of folic acid, aminopterin, and methotrexate analogs in which an intact L-glutamic acid side-chain is present while simultaneously varying the pteridine ring moiety, the C-9, N- 10 bridge and/or the benzoyl group. Several analogs of folic acid are known in which the only remaining structural feature of the folic acid molecule is the p-aminobenzoylglutamic acid moiety. Biological results are discussed in those cases where the analog has significant activity in tumor systems relative to methotrexate (MTX) or aminopterin (AP). Of the many classes of deazaFA derivatives investigated, none has received more attention or resulted in more promising candidate drugs than 5,8-dideaza (quinazoline) analogs. Methylation of folic acid (FA) with a 4-molar excess of methyl iodide gave a low yield of the tetramethyl derivative. A Dimroth rearrangement brought about by base at room temperature then gave hydrolysis of the methyl esters having taken place under the conditions of the rearrangement.

Receptor-mediated folate accumulation is regulated by the cellular folate content.

Cultured monkey kidney cells are shown to possess a cell-surface receptor that binds 5-methyltetrahydro[3',5',7,9-3H4]folic acid (5-methyltetrahydro[3H]folic acid) with high affinity (Kd = 3 nM at 4 degrees C). The cell-surface binding capacity for [3H]folic acid or [3H]methotrexate is equivalent to that for 5-methyltetrahydro[3H]folate, but the Kd values are 0.4 and 20 nM, respectively. These nonlabeled folates also inhibit cellular binding and accumulation of 5-methyltetrahydro[3H]folate, whereas specific moieties of folic acid (i.e., p-aminobenzoylglutamic acid, pteroic acid, and glutamic acid) had no effect. Surface-bound folate(s) can be released by incubation of the cells at pH 3.5. At 37 degrees C the bound material is slowly transferred into cells, where it becomes resistant to acid release. Within 4 hr the cells internalize about 3-4 times as much folate as is bound to the surface in the steady state. The amount of receptor activity increases markedly when cells are depleted of folate through growth in folate-depleted medium. Binding of folate was inhibited by an antibody raised against a soluble plasma folate-binding protein, suggesting that the cell-surface receptor and the circulating folate-binding protein are immunologically related. These data indicate that cells possess a high-affinity, high-specificity folate receptor whose expression is regulated by the folate content of the cell. They also suggest that a small molecule such as folate can enter cells by receptor-mediated endocytosis.

Antagonism of sulfadoxine and pyrimethamine antimalarial activity in vitro by p-aminobenzoic acid, p-aminobenzoylglutamic acid and folic acid

The activity of pyrimethamine and sulfadoxine against two strains of Plasmodium falciparum has been studied in vitro by a radioisotopic technique. Low level antagonism of pyrimethamine resulted from the inclusion of p-aminobenzoic acid, p-aminobenzoylglutamic acid or folic acid in the test medium. Sulfadoxine activity was antagonised slightly by p-aminobenzoic but not by p-aminobenzoylglutamic acid, and antagonised markedly by folic acid at concentrations above 4 × 10 −8 M. At 10 −7 M folic acid, a concentration lower than that of normal RPMI medium 1640, sulfadoxine activity was reduced 7000 to 9000-fold in comparison with controls. These results are of importance in terms of the utilisation of folates by P. falciparum, the susceptibility of the parasite to antifolate drugs and the in vitro determination of parasite susceptibility.

Electrochemistry of the C(9)N(10) bond in folic acid and related compounds

An electrochemical synthesis of isomer-free 6-methylpterin from folic acid has been developed. Folic acid is reduced to 7,8-dihydrofolic acid via 5,8-dihydrofolic acid. Under acidic conditions the C(9)N(10) bond in 7,8-dihydrofolic acid is reduced electrochemically, resulting in 6-methyl-7,8-dihydropterin and p-aminobenzoylglutamic acid. 6-Methylpterin can be obtained by oxidation of the corresponding dihydropterin. 7,8-Dihydrofolic acid is stable under anaerobic conditions between pH 4 and 9. Under strongly acidic conditions hydrolysis of the C(9)N(10) bond takes place and 6-hydroxymethyl-7,8-dihydropterin and p-aminobenzoylglutamic acid are obtained. Under aerobic conditions oxidative cleavage of the C(9)N(10) bond results in various pterin derivatives. Some 6-azomethinpterins were synthesized. Voltammetric studies of these compounds show that the reduction of the pterin nucleus follows the same general pathway described for the reduction of pterins. Reduction of the C(9)N(10) double bond proceeds through a reversible two-electron step, followed by a transformation, resulting in saturation of the bond.

The degradation of folic acid in a model food system and in beer

The rate of degradation of folic acid in the light and in the dark is investigated in a model buffer solution and in beer, with and without the addition of sulphur dioxide. In the model solution, held in the light, sulphur dioxide markedly accelerates the rate of degradation of folic acid whereas, in the dark, it exhibits a protective effect. In beer, the presence of sulphur dioxide has no effect on the degradation of folic acid, whether in the light or the dark. In the model solution, in the presence of sulphur dioxide, only the degradation product p-aminobenzoyl glutamic acid was identified. In the absence of sulphur dioxide and in the beer samples, pterin-6-carboxylic acid was additionally identified as a degradation product.

Two improved high-performance liquid chromatographic separations of biologically significant forms of folate

Two new separations of folate derivatives by in situ paired-ion high-performance liquid chromatography are described. The first utilizes two C 18 phenyl columns and the second utilizes a C 8 radial compression cartridge. Folic acid, dihydrofolic acid, folinic acid, tetrahydrofolic acid, 5-methyltetrahydrofolic acid and a decomposition product, p-aminobenzoylglutamic acid are separated. A comparison is made between these two methods and with the standard microbiological Lactobacillus casei assay for folates. The first method offers improved resolution and in situ sample clean-up. The second method accomplishes this in one-fifth the time. Both methods provide information not obtained by the microbiological assay.

Determination of three different pools of reduced one-carbon-substituted folates: I. A study of the fundamental chemical reactions

The reduced one-carbon-substituted derivatives of folic acid can be grouped in three pools according to their response to acid treatment. Pool 1 is made up of N 5, N 10-methylene-tetrahydrofolic acid and unsubstituted dihydro- and tetrahydrofolic acid which at pH 1.0 and subsequent exposure to air cleave to p-aminobenzoylglutamic acid. Pool 2 is made up by the acid-stable N 5-methyl-tetrahydrofolic acid, and pool 3 includes N 5, N 10-methenyl-tetrahydrofolic acid, N 10-formyltetrahydrofolic acid, N 5-formyltetrahydrofolic acid, and N 5-formiminotetrahydrofolic acid, all of which convert to the stable N 5, N 10-methenyl-tetrahydro form when acid treated. Conditions are described to selectively cleave the C 9-N 10 bond of the folates of pool 1, pools 1 + 2, and pools 1 + 2 + 3. The cleaved pools are quantitated as the Bratton-Marshall azo dyes of p-aminobenzoylglutamate. The uncleaved pools are converted to Bratton-Marshall-negative products. Pool 1 is determined by converting pool 2 to 4a-hydroxy-5-methyltetrahydrofolic acid and pool 3 to N 10-formylfolic acid, both Bratton-Marshall negative, by 10% hydrogen peroxide oxidation at pH 6.0. Pools 1 + 2 are cleaved with 0.015% hydrogen peroxide and 0.1% potassium permanganate at pH 9.0 which convert the N 5-methyltetrahydrofolic acid to the acid-cleavable N 5-methyl-dihydrofolic acid. Pool 3 oxidizes to the Bratton-Marshall-negative N 10-formylfolic acid. Pools 1 + 2 + 3 are cleaved by first reducing pool 3 to N 5-methyltetrahydrofolic acid with sodium borohydride followed by oxidation at pH 9.0 to its acid-labile dihydro form. Determination of the poly-γ-glutamyl chain length of each pool is possible by chromatographing the azo- p-aminobenzoylpolyglutamates with authentic synthetic markers.

A novel chemotaxis regulating enzyme that splits folic acid into 6-hydroxymethylpterin and P-aminobenzoylglutamic acid

A novel chemotaxis regulating enzyme that splits folic acid into 6-hydroxymethylpterin and P-aminobenzoylglutamic acid

58] Separation of folic acid derivatives and pterins by high-performance liquid chromatography

This chapter discusses the separation of folic acid derivatives and pterins, by high-performance liquid chromatography. For this study, folic acid, 7,8-dihydrofolic acid, 5,6,7,8-tetrahydrofolic acid, 5-methyltetrahydrofolic acid, and p-aminobenzoylglutamic acid were purchased from Sigma Chemical Co. 5,10-Methenyltetrahydrofolic acid and 10-formyltetrahydrofolic acid were prepared by the method of Rabinowitz. The 5,10-methylenetetrahydrofolic acid was prepared by the method of Huennekens et al. N(10)‑nitrosofolic acid was prepared by the method of Cosulich and Smith. 2‑Hydroxy-N(10)-nitrosofolic acid and 2-hydroxyfolic acid were prepared by the method of Angier et al. Xanthopterin, pterin, and pterin-6-carboxylic acid were purchased from Sigma. Pterin-6-carboxaldehyde was prepared by the method of Waller et al. Because most reduced derivatives of folic acid are extremely labile to oxidation, by air, 2-mercaptoethanol is normally added to the solutions, containing 7,8-dihydro and 5,6,7,8-tetrahydro derivatives. 2-Mercaptoethanol absorbs at 254 nm and interferes with the detection of chromatographic peaks. This chapter discusses the separation of the three oxidation states of folic acid. Separation of N(5)-and N(10)-substituted tetrahydrofolates is described in the chapter. Separation of the products of nitrosation of folic acid is also elaborated in the chapter in detail.

Characterization of mutationally altered dihydropteroate synthase and its ability to form a sulfonamide-containing dihydrofolate analog

Among spontaneous mutants of Escherichia coli selected for resistance against sulfonamides, thermosensitive strains were found. These were shown to possess a changed dihydropteroate synthase (EC 2.5.1.15), which had a substantially higher Km value for its normal substrate, p-aminobenzoic acid, and an about 150-fold higher Km for sulfonamides. The mutationally changed dihydropteroate synthase was found to be thermosensitive by in vitro assays. The thermosensitivity was used as an enzyme marker to demonstrate the complex formation between 2-amino-4-hydroxy-6-pyrophosphorylmethyl pteridine and sulfonamides by partially purified dihydropteroate synthase. The formation of folate from 2-amino-4-hydroxy-6-pyrophosphorylmethyl pteridine and p-aminobenzoylglutamic acid by dihydropteroate synthase was found to be very sensitive to inhibition by sulfonamides and very inefficient with the mutationally changed enzyme.

Crystal Structure of Monoclinic Ribonuclease-S at 4 Å Resolution

A four-A electron density map was calculated for the monoclinic crystal of ribonuclease-S (RNase-S) based on two heavy-atom derivatives. Close geometrical similarity was found between the two crystallographically independent RNase-S molecules (called molecules ZA and ZB) in this crystal and that (called molecule Y) in the trigonal crystal. Using the rotational and translational parameters relating these three molecules, it was established that the crystallographic two-fold symmetry between the two molecules ZA in the monoclinic crystal was exactly identical to that between the two molecules Y in the trigonal crystal, suggesting the tendency of RNase-S molecules to associate in this way although the interaction is weak. The 4-A difference Fourier maps calculated for the monoclinic crystal established the following conclusions. (1) 4-Thiouridine-2'(3')-monophosphates binds to the B1 and R1 sites like other pyrimidine nucleoside-2'(3')-monophosphates as expected from previous spectrophotometric studies, but not to the B2 site even at the concentration of 20 mM. An attempt to visualize the photoproduct generated by irradiation of near-ultraviolet light in this complex failed. (2) p-Aminobenzoylglutamic acid, a fragment of folic acid, seems to bind to RNase-S with its benzene ring close to the B2 site and the alpha-carboxylate group close to the p1 site. The model is compatible with most of the chemical results obtained by Sawada et al. ((1977) Biochim. Biophys. Acta 479, 188-197).