Reaction of Folin-Ciocalteau phenol reagent with purines, pyrimidines, and pteridines and its relationship to structure

Abstract

Purines containing hydroxyl and/or amino groups at both the 2-and 6-positions react strongly with Folin-Ciocalteau phenol reagent. Methylation at either the 3or 7-positions greatly reduces the reactivity. Analogously substituted pteridines containing hydroxyl and/or amino groups at both the 2-and 4-positions do not react strongly unless additional hydroxyl groups are present at the 6and/or 7-positions. Analogously substituted pyrimidines containing hydroxyl and/or amino groups at both the 2-and 4-positions do not react. The evidence agrees with voltammetric interpretations that the 4-and 5-positions of the purine nucleus at the ring junctions are uniquely involved in the oxidation of the appropriately substituted purines. Folin-Ciocalteau reagent is a useful chromatographic spray reagent for the detection of small amounts (1 Mg or less) or the strongly reacting purines.It is generally recognized that Folin-Ciocalteau phenol reagent, which is used in the well-known Lowry et al. (1951) method for the estimation of protein, also reacts strongly with substances other than phenols (Peterson, 1979;Box, 1983). Lowry et al. ( 1951) also had recognized that uric acid, guanine, and xanthine reacted with the reagent, while adenine, hypoxanthine, cytosine, uracil, thymine, guanosine, adenosine, cytidine, and thymidine showed little or no reaction. In a study on the determination of protein contamination in DNA using the Lowry et al. (1951) procedure, Shepherd and Hopkins (1963) reported that guanine and xanthine gave an intense color with Folin-Ciocalteau reagent, that guanosine, guanylic acid, and hypoxanthine gave significant color, and that adenine, cytosine, uracil, and thymine and their nucleosides and nucleotides and orotic acid gave little or no color. The authors cautioned that the high color-yielding purines must be removed before ascertaining the protein contamination of DNA preparations. We have recently found that Folin-Ciocalteau reagent is useful for the detection of paralytic shellfish poisons (red tide poisons) (Mosley et al., 1985). The presence of a partially reduced purine ring system in these poisons prompted us to examine in more detail the effects of substituents on the reactivity of purines and related substances with Folin-Ciocalteau reagent and to establish a rationale for the reaction of the reagent with purines and related compounds. EXPERIMENTAL SECTION Materials. The adenine A^-oxide, 2-hydroxypurine, 2aminopurine, xanthine A^-oxide, 2-(dimethylamino)-6hydroxypurine, isoguanine hemisulfate, isoguanine N1oxide, 2,6-diaminopurine hemisulfate, 1-methylxanthine, 3methylxanthine, 7-methylxanthine, theophylline, 1,7dimethylxanthine, theobromine, caffeine, 1,3,9-trimethylxanthine, pterine, lumazine, xanthopterin, and leucopterin were purchased from Sigma Chemical Co., St. Louis, MO.

Utilization of Folin−Ciocalteu Phenol Reagent for the Detection of Certain Nitrogen Compounds

“…That a strain imposed by the five-membered fused ring system in saxitoxin may contribute to its reaction with Folin-Ciocalteu reagent is made likely by the fact that guanine is highly reactive to the reagent, whereas similarly substituted pterine and isocytosine (Figure 1) hardly react. 2-Aminopurine is also highly reactive (8). The 2-amino group of saxitoxin may be key to its reaction with Folin-Ciocalteu reagent.…”

“…The method described by Ikawa et al (8) using the optimum conditions described by Box (4) was used. To 2.5 mL of test solution were added 0.375 mL of a 20% (w/v) solution of anhydrous sodium carbonate and 0.125 mL of 2 N Folin-Ciocalteu phenol reagent (Fisher Scientific Co., Pittsburgh, PA) with a pipet.…”

“…The ability to utilize the reagent for this purpose appears to be somewhat remarkable when one considers the variety of compounds that may react with the reagent and the fact that PSPs occur at very low concentrations. Because of the similarity of the PSP ring system to the purine ring system, we have previously also studied the structural requirements for purines to react with Folin-Ciocalteu reagent (8) and shown the importance of an amino or hydroxyl group at the 2-position.…”

Hypothetical Standard Screening Bioassays

“…The F-C reactive compounds are surrogates for the promoter/modifier/inhibitor complex in leachates. The Folin-Ciocalteau reagent is reduced by substances with phenolic hydroxyl groups (e.g., cinnamic acids, hydroxybenzoic acids, gallic acid, tannic acids, and tyrosine), some substances without phenolic hydroxyl groups (e.g., pyridines, purines, alanine, cysteine, tryptophan, ascorbic acid, fructose, and proteins), some inorganic substances [e.g., iron sulphate, manganese sulphate, and potassium nitrate], and some other compounds (Anderson and Todd 1968;McAllister 1969;Box 1983;Ikawa et al 1988;Blum et al 1991Blum et al , 1992. F-C equivalent values could be expressed in terms of any of these compounds although substances with phenolic hydroxyl groups have been the primary reference compound used in the literature.…”