CHEMISTRY AND METABOLISM OF ASCORBIC ACID AND ASCORBATE SULFATE * B. M. Tolbert, M. Downing, R. W. Carlson, M. K. Knight, and E. M. Baker Department of Chemistry University of Colorado Boulder, Colorado 80302
INTRODUCTION The purpose of this review is to summarize developments in the chemistry and metabolism of ascorbic acid during the past 14 years. Particular emphasis has been placed on information concerning ascorbate sulfate. Ofttimes the development of new chemistry related to ascorbic acid has appeared to progress slowly, yet in retrospect considerable progress has been made during the past 14 years. The work has been done in many laboratories and by many people. The major thrust of chemical work has been (1) to prepare chemical derivatives with biological activity but resistant to air oxidation, ( 2 ) to understand oxidative decomposition of ascorbic acid, and (3) to develop easier or better analytical methods for assay of ascorbic acid. In this report no coverage has been made of chemical developments to be reviewed by other speakers at this conference, such as the chemistry of monodehydroascorbic acid, or the enzyme ascorbic acid oxidase.
DERIVATIVES OF ASCORBIC ACID Ascorbic acid has a variety of reactive positions that can be used to syn1). TABLE 1 summarizes the thesize a number of simple derivatives (FIGURE derivatives of ascorbic acid that have been prepared in the past 14 years. The 6-0 atom of ascorbic acid is readily derivatized with a variety of electrophylic reagents in aprotic solvents such as dimethyl formamide or dimethylsulfoxide. Somewhat less reactive under these conditions is the 2-0 atom, and derivatization has usually been made through the 5,6-O-isopropylidene derivative. Only in recent years has it been realized that in aqueous solutions at pH 9-10 the 2-0 position is more reactive than the 6 - 0 atom. Under these conditions there is a significant concentration of the 2-0- ion with a high charge density, leading to rapid nucleophylic attack in substitution reactions. An efficient synthesis of ascorbate sulfate is based on this fact and is described later in this paper. Only a few 3-0 derivatives of ascorbic acid have been prepared. The 3-0 position is not very reactive because the ene-diol system provides extensive delocalization of the ascorbate monoionic charge. In addition, many of the possible derivatives are probably unstable and rapidly hydrolyze in the presence of H,O. Ascorbic acids derivatized at the 2-0 position have often been called
* This work was supported, in part, by the United States Army Medical Research and Development Command. 48
Tolbert et al. : Ascorbic Acid
As = Ascorbate dAs = dehydroascorbic acid
exchanges a t high pH 7
acyl and methyl deriva t i v e s a f t e r 2 and/or 6 substitutions
2- benzyl dAs,
Indolylmethyl dAs (Ascorbigen 1
b methyl and acyl derivatives
As-6-S04, acyl and methyl derivatives
FIGURE 1. Derivatives of ascorbic acid: reactive positions.
3-0 derivatives, and early work should be critically evaluated. The ferric chloride test is inadequate because both the 2-OH and 3-OH groups give a positive result. 5-0-Acyl derivatives of ascorbic acid are formed after substitution reactions on the 2 and/or 6 oxygens block these more reactive sites. Thus this secondary alcohol is the least reactive of the four 0's. No monosubstitute 5 - 0 derivatives of ascorbic acid have been prepared, although they have potential as antimetabolites of ascorbic acid. There are no known derivatives of ascorbic acid on the 1-0. A study of the ascorbate molecule does not provide clear reasons why such derivatives should not be formed and have a reasonable stability. Brenner et showed that ascorbate could be racemized at the C-4 position in solution at pH 13, and 70" by ionization of the 4-H. This indicates a pka for the 4-hydrogen of 16-1 7. This exchangc reaction was later used to prepare 4-D- and 4-'iH-labeled ascorbic acid (24). There is no evidence for exchange of this hydrogen in biological systems. The ionization of this hydrogen may be useful in synthetic reactions leading to 4-derivatives. The reactivity of the 2-C position is demonstrated by only 2 derivatives, the 2-C-benzyl dehydroascorbic acid 1 and ascorbigen, which is 2-( 3-indolylmethyl) dehydroascorbic acid.', :1 Both of these compounds display geometrical B R was isomerism.2 The question of the antiscorbutic activity of ascorbigen restudied by Imai,29who found only weak activity. The antiscorbutic activity of a number of the ascorbic acid derivatives listed in TABLE 1 has been studied in guinea pigs8. 8, Very little work has been published on the synthesis of ascorbic acid analogues that might possess antimetabolite activities related to the biochemical role of ascorbic acid. Roushdi et aL3: