To determine in more detail the reaction of Folin-Ciocalteu phenol reagent with nitrogen compounds, a number of hydroxylamine-related compounds and a large number of guanidine-containing compounds were tested. In general, guanidine compounds did not react strongly unless they were hydroxyamino or hydrazino derivatives. The non-hydroxyamino paralytic shellfish poison saxitoxin, however, reacted to a significant extent. This may be due to the presence of a five-membered ring structure and its analogy to 2-aminopurines, which react strongly. A number of simpler amines were also tested. Tertiary aliphatic amines, but not primary, secondary, or quaternary amines, reacted strongly with the reagent. Primary, secondary, and tertiary aromatic amines all reacted strongly with the reagent. Reactivity was extended to pyrroles and indole derivatives but not to imidazole and benzimidazole derivatives. Defining the reactivity of Folin-Ciocalteau phenol reagent with nitrogen compounds extends the usefulness of the reagent for the detection and determination of certain nitrogen compounds in basic extracts by colorimetric means and by thin-layer chromatography.

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.