The Hydroxyl Radical - Free-Radical-Induced DNA Damage and Its Repair - page 57

35%. Again, side reactions [reactions (49) and (50)] are the reason for its lower
yields (Chap. 8). The detection limit of the f fluorescing 2-hydroxyterephthalate
has been given as 5

10 −8 mol dm −3 (Saran and Summer 2000).
Aromatic hydroxylation is also the basis of the formation of f fluorescent 7-
hydroxycoumarine-3-carboxylic acid from coumarine-3-carboxylic acid. Bio-
molecule-conjugates with coumarin-3-carboxylic acid can be made, and this
permits ex vivo probing of • OH in the vicinity of a polymeric conjugate such as
proteins or DNA (Makrigiorgos et al. 1993, 1995; Parker 1998; Chakrabarti et al.
1996, 1998; for a review: Makrigiorgos 1999). However, the 7-position in is not
an activated position, and hence the yield of 7-hydroxycoumarine-3-carboxylic
is very low (

×

5% of • OH yield; von Sonntag et al. 2000). Despite this fact, this
• OH-probing system rates among the most sensitive ones (detection limit 1.5

∼

×

10 −8 mol dm −3 • OH, taking data reported by Makrigiorgos 1999).

A widely used system is salicylic acid, which was first studied by Grinstead
(1960). Being a phenol, the rate of reaction of salicylic acid with • OH is faster
[ k ( • OH + salicylate ion) = 2

10 10 dm 3 mol −1 s −1 ] than those of the other aro-
matic acids discussed above. Its main products, 2,3-dihydroxybenzoic acid
and 2,5-dihydroxybenzoic acid, are very sensitively determined by HPLC using
electrochemical detection (Coolen et al. 1998) or by LC/MS methods (detection
limit near 2

×

10 −7 mol dm −3 ; Tabatabaei and Abbott 1999). Due to its phenolic
function, the chemistry in the presence of O 2 is different (see below) to that of
the other aromatic acids, yielding in the presence of this oxidant high yields of
the desired products. The same holds for its isomer, 4-hydroxybenzoic acid. A
potential disadvantage is that in a cellular environment salicylic acid may be
oxidized by other oxidizing agents.
Since • OH is strongly electrophilic, the OH group directs • OH into its or-
tho - and para -positions [reactions (51)−(53)]. One of the ortho -positions is oc-
cupied by the somewhat bulky carboxylate group which renders reaction (51)
less likely than reaction (52). An addition to the meta -position can be largely
neglected. Upon oxidation of the • OH-adduct radicals, cyclohexadienones are
formed [reactions (54)−(56)] which either decarboxylate [reaction (57)] or rear-
range into the corresponding phenols [reactions (58) and (59); e.g., Bausch et al.
1976]. Product yields from hydroxybenzoic acids are compiled in Table 3.5; from
salicylic acid in Table 3.6.
The • OH-adducts of phenols behave differently as compared to those of other
aromatic compounds. The parent compound, phenol, has been investigated in
quite some detail (Land and Ebert 1967; Raghavan and Steenken 1980; Roder
et al. 1999; Mvula et al. 2001). Its ortho - and para - • OH adducts undergo rapid
H + /OH − -catalyzed water elimination thereby yielding the (thermodynamically

×