Hydroxybenzoate, hydroxylation

In the weakly acidic preservatives, activity resides primarily in the unionized molecules and they only have significant efficacy at pHs where ionization is low. Thus, benzoic and sorbic acids (pKa = 4.2 and 4.75, respectively) have limited preservative usefulness above pH 5, while the 4(p)-hydroxybenzoate esters with their non-ionizable ester group and poorly ionizable hydroxyl substituent (pKa ca. 8.5) have moderate protective effect even at neutral pH levels. The activity of quaternary ammonium preservatives and chlorhexidine probably resides with their cations and are effective in products of neutral pH. Formulation pH can also directly influence the sensitivity of microorganisms to preservatives (see Chapter 11). 

Hydroxybenzoates can nndergo hydroxylation with or withont concomitant loss of CO2. For example, salicylate —> catechol + CO2 (salicylate-l-hydroxylase) (Fignre 3.9a) (White-Stevens et al. 1972) and 4-hydroxybenzoate —> 1,4-dihydroxybenzene + CO2 [(4-hydroxy-benzoate 1-hydroxylase (decarboxylating)] in Candida parapsilosis (Fignre 3.9b) (Eppink etal. 1997). 

The degradation of bisphenol-A by Sphingomonas sp. strain AOl is initiated by hydroxylation to intermediates that undergo fission to 4-hydroxyacetophenone and 4-hydroxybenzoate. The components have been purified, and consist of cytochrome P450, ferredoxin reductase, and ferredoxin (Sasaki et al. 2005). 

The anaerobic degradation of some hydroxybenzoates and phenols involves reductive removal of the phenolic hydroxyl group. The enzyme that dehydroxylates 4-hydroxybenzoyl-CoA in Thauera aromatica is a molybdenum-flavin-iron-sulfur protein (Breese and Fuchs 1998), and is similar to the enzyme from the nonsulfur phototroph Rhodopseudomonas palustris that carries out the same reaction (Gibson et al. 1997). 

For hydroxybenzoates with hydroxyl groups at the ortho or meta positions, degradation is initiated by decarboxylation. 

The fermentation of 3-hydroxybenzoate by Sporotomaculum hydroxybenzoicum produces acetate, butyrate, and CO2, with benzoate as a transient intermediate (Brauman et al. 1998). However, although the degradation of 3-hydroxybenzoate by Thauera aromatica begins with the formation of the CoA-ester, this is followed by the reduction of the ring with retention of the original hydroxyl group (Laempe et al. 2001). 

Alcaligenes denitrificans strain NTB-1 is able to use 4-chloro-, 4-bromo-, and 4-iodoben-zoates as sole sources of carbon and energy. The pathway involves hydrolytic dehalogenation to 4-hydroxybenzoate followed by hydroxylation to 3,4-dihydroxybenzoate (van den Tweel et al. 1987). 

In strain RH025, a hydrolytic mechanism brought about the loss of fluoride to produce 4-hydroxybenzoate, which was further hydroxylated to 3,4-dihydroxybenzoate before intradiol ring fission (Figure 9.33a). 

While oxidation of p-coumaric acid was occurring, the major intermediates formed are p-hydroxybenzaldehyde and p-hydroxybenzoic acid. Low concentrated aromatic intermediates such as phenol and p-hydroxybenzyl alcohol and traces of hydroxylation products such as, 3,4- dihydroxybenzaldehyde and 3,4-dihydroxybenzoic acid were detected. Furthermore, formic and oxalic acids were the ring-cleavage compounds detected. 

On the basis of the obtained results and the literature[17-18], we assume that during the oxidative decomposition of p-coumaric acid 1, three kind of reaction can happen before the opening of the aromatic ring (Scheme 1) cleavage of the very reactive exocyclic double bond to give p-hydroxybenzaldehyde 2, hydroxylation of the aromatic ring to yield 3,4-dihydroxycinnamic acid 3 and oxidation of aldehydes to carboxylic acids such as oxidation of 2 to p-hydroxybenzoic acid 4. Compound 2 can be hydroxylated to yield 3,4- dihydroxybenzaldehyde 6. and Compound 4 can also be hydroxylated to yield 3,4-dihydroxybenzoic acid 5. 

Replacement of the hydroxyl group on the phenyl ring with a carboxyl group forms a molecule of benzoic acid. Addition of a hydroxyl at the 2-position on a benzoic acid molecule forms 2-hydroxybenzoic acid or salicylic acid. The slightly more complex phenylpropanoid skeleton contains a linear three-carbon chain (the propanoic group) added to the benzene ring (the phenyl group). Addition of ammonia to carbon 2 of this three-carbon side chain yields the amino acid phenylalanine (Fig. 3.3). Phenylalanine... 

Van der Bolt FJT, van den Heuvel RHH, Vervoort J, van Berkel WJH (1997) 19F NMR study on the regiospecificity of hydroxylation of tetrafluoro-4-hydroxybenzoate by wild-type and Y385F p-hydroxybenzoate hydroxylase evidence for a consecutive oxygenolytic dehalogenation. Biochemistry 36 14192-14201... 

Biological. Benzoic acid may degrade to catechol if it is the central metabolite whereas, if protocatechuic acid (3,4-dihydroxybenzoic acid) is the central metabolite, the precursor is 3-hydroxybenzoic acid (Chapman, 1972). Other compounds identified following degradation of benzoic acid to catechol include cis,c/5-muconic acid, (+)-muconolactone, 3-oxoadipate enol lactone, and 3-oxoadipate (quoted, Verschueren, 1983). Pure microbial cultures hydroxylated benzoic acid to 3,4-dihydroxybenzoic acid, 2- and 4-hydroxybenzoic acid (Smith and Rosazza, 1974). In activated sludge, 65.5% mineralized to carbon dioxide after 5 d (Freitag et al., 1985). 

The following is review on the molecular and physical properties of this class of monooxygenases, which are also known as hydroxylases. A typical monooxygenase reaction is the hydroxylation of an alkane to an alcohol which involves a reduced cosubstrate that reduces a second atom within the O2 molecule to form water. Flavin-containing monooxygenases include lysine oxygenase and 4-hydroxybenzoate hydroxylase. Reduced pteri-dines are involved in the phenylalanine hydroxylase and tryptophan hydroxylase reactions. See also Cytochrome P-450... 

With the benzene-derived Ugands 26a,b the Cu-catalyzed orfho-hydroxyl-ation of methyl-4-hydroxybenzoate was only highly selective at low temperature, as at room temperature bound catecholate was shown to undergo a formal Michael addition with unreacted phenolate to yield methyl 2-[4-(carbomethoxy)phenoxy]-3,4-dihydroxybenzoate [181,270]. With the amine-based ligand 27, no such reactivity was observed and the catechol product methyl 3,4-dihydroxybenzoate was formed selectively. Interestingly, this latter ligand system also displayed some selective reactivity with the neutral 4-hydroxybenzoic acid substrate. 

The hydroxybenzoic acids have both hydroxyl and the carboxyl groups and, therefore, participate in chemical reactions characteristic of each of these moieties. In addition, these acids can undergo electrophilic ring substitution. The following reactions are discussed in terms of salicylic acid, but are characteristic of all the hydroxybenzoic acids. 

Decarboxylation of salicylic acid takes place with slow heating because of the presence of the electronic configuration of the carboxyl group ortho to the hydroxyl group, but does not occur in the other isomers of hydroxybenzoic acid. On rapid heating, salicylic acid sublimes because of its low vapor pressure. This property allows commercial separation from the other isomers as a means of purification analogous to distillation. The differences in the vapor pressures are shown in Table 4. 

Reactions. -Hydroxybenzoic acid undergoes the typical reactions of the carboxyl and hydroxyl moieties. When heated above its melting point, it decomposes almost completely into phenol and carbon dioxide. It reacts with electrophilic reagents in the predicted manner and does not undergo the Friedel-Crafts reaction. Nitration, halogenation, and sulfonation afford the 3-substituted products. Heating -hydroxybenzoic acid with 8 IV-nitric acid results in a 95% yield of picric acid. In a similar fashion, treatment with chlorine water yields 2,4,6-trichlorophenol (50). 

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