8.1.1 Introduction
Many secondary metabolites accumulate in plants as a chemical defensive strategy against microbes and other threats. These systems have evolved generally through Darwinian selection and provide us with significant clues to understand (1) ways in which plants have developed a survival strategy in a harsh environment and (2) a rationale for understanding some aspects of the chemistry and biochemistry of higher plants. Further, as it becomes clear how plants defend themselves, some of their defensive strategies may become useful directly or indirectly in the design of pharmaceuticals for human use.
Numerous catechols and hydroquinones in both glycoside-masked and -unmasked forms are useful metabolites in plant chemical defense. Many such metabolites are present in concentrations that can prove detrimental due to oxygenation of the tissue accompany- ing wounding of the plant in the infection process or in other direct physical injury. Some agents are also synthesized subsequent to enzyme induction in association with infection to mediate chemical defense, as in the broad class of defensive substances known as phyto- alexins.1-2 Some of these induced substances are oxidizable polyphenols, while others are
not (Figure 8.1).
The presence of catechols and more complex, oxidizable polyphenols in nature is wide- spread, and their functions are not limited to chemical defense. However, biological control of their oxidation is usually a feature of their function, as it is (1) in melanin synthesis,3
(2) in immunologically mediated delayed-type hypersensitivity responses,4 (3) in the hard-
ening or curing of arthropod secretions (for example, as in the surface attachment adhe- sives of the barnacle and in tanning of the cuticle in insects),5 as well as (4) in defensive
mechanisms in higher plants, particularly in the unleashing of immediate necrotrophic responses.6
FIGURE 8.1
Some naturally occurring catechols and polyphenols implicated in chemical defenses in plants.
Reactive Quinones: From Chemical Defense Mechanisms in Plants to Drug Design 111
The intermediate o- and p-quinones formed during catechol oxidation are often unstable and react through water adducts to produce autocatalytic polymerizing mixtures via elec- trophilic attack on protein nucleophiles, or, alternatively, are inactivated through internal cyclization with appropriately positioned nucleophiles (Figure 8.2). The underlying oxida- tion reactions of urushiols (Figure 8.3), which constitute the active agents in delayed-type hypersensitivity skin reactions of poison ivy, oak, and sumac, were characterized by Daw- son and Tarpley4 and Symes and Dawson.7 Modified proteins of the skin are immobilized,
inducing subsequent complex cellular immune responses to activate the subject toward future contact with the plant.
While in nature the oxidative reactions of catechols are often controlled through com- partmentation of metabolites and through specific activation schemes, the site-directing inactivating capacity of intermediates in the oxidation reactions of catechols requires fur- ther study. Thus, we incorporated site-directing functionality into simple small molecules bearing catechol or hydroxycatechol functionality, where reactivities could be modulated for site-directed and specific inactivation studies of well-characterized biological receptors. These systems represent useful models for pharmaceutical targeting.
8.1.2 Quaternary Ammonium Catechols in Acetylcholine Receptor Site-Directed Reactions
We controlled the chemistry of these catechol-quinone reactive species through the affinity- directing reactions of quaternary-ammonium groups attached to catechol derivatives. This allowed us to direct such agents to purified cholinergic receptors as drug design model sys- tems. We were interested in both simple ammonium-substituted catechols as well as inter- mediate, more reactive species produced from cycles of oxidation and hydroxylation. Here, we imagined that reactive quinones could intervene as labeling species in biological sites
FIGURE 8.2
Fate of quinone intermediates formed by pH-dependent oxidation of catechols by molecular oxygen.
FIGURE 8.3
Structures of urushiols.
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to which the catechols were directed. Inactivating reactions could also be observed from reduced oxygen species produced via oxidation of the catechol with molecular oxygen.
In the major investigations of catechol-containing quaternary ammoniums we used the nicotinic acetylcholine receptor8-11 as a model biological target. A nicotinic-type acetylcho-
line receptor (nAChR), which has actions quite analogous to those associated with the mammalian neuromuscular junction, is easily isolated and purified12 from the electric
organ of the ray Torpedo californica. Initially, we studied the receptor-directed reactions of a simple molecule containing both a tetra-alkylammonium group and a catechol. Additional model reagents were synthesized and studied subsequently in greater detail, as they rep- resented either more reactive analogs of simpler compounds,12-15 analogs of biologically
active metabolites, or drug or druglike molecules16,17 (Figure 8.4). Of special interest were
some of the compounds representing quaternary ammonium analogs of naturally occur- ring catecholamines or their hydroxyl derivatives.
The synthesis of trimethylammoniomethyl catechol (TMC) was devised to facilitate introduction of a high-specific-activity radio label using 3H-methyl iodide.12 In the presence
of molecular oxygen the reaction of receptor with TMC was slow and concentration depen- dent. An equilibrium dissociation constant (28 µM) could be measured directly from the competing effect of 125I-neurotoxin* (α-bungarotoxin) binding. TMC reacted rapidly with
half the acetylcholine binding sites. This so-called half-of-sites reactivity was reminiscent of other affinity agents reported to label the nAChR.8-10 In those cases half-of-sites labeling
was also observed, but only after prereduction with low concentrations of dithiothreitol (DTT). The interpretation was that half the sites were easily reduced with DTT, whereas the other half were either not easily reduced or were easily reoxidized after rapid removal of excess DTT leading to half-of-sites alkylation of the free cysteine sulfhydryl. The specificity and efficiency of labeling were consistent with an affinity-dependent oxidation–reduction reaction of one of the early oxidation products with the receptor; e.g., 4- or 5-HTMC (hydroxy-3-trimethylammoniomethyl catechol), as shown in Figure 8.5. Indeed, specific and efficient half-of-sites labeling could be demonstrated by chromatographic isolation of the 3H-labeled receptor product subsequent to incubation of nAChR with 3H-TMC.12 Half-
of-sites labeling via one or both of the hydroxy intermediates was consistent with the redox
FIGURE 8.4
Quaternary ammonium analogs of catecholamines.
* A high-affinity competing ligand for the acetylcholine binding site in the nAChR.
Reactive Quinones: From Chemical Defense Mechanisms in Plants to Drug Design 113
potential of a disulfide present at the nAChR site.15 This redox-assisted affinity reaction of
the reactive disulfide affords a novel targeting mechanism in affinity labeling of receptors. We characterized and further studied this basic mechanism of covalent affinity labeling using spectroelectrochemical techniques. The kinetics and stability of quinone oxidation products at high dilution and low pH were consistent with the proposed mechanism, as was the concentration dependence of rapid labeling reactions of the more reactive catechol with the receptor.12,15 Spectroelectrochemical and direct cyclic voltametric determination of
the half-potentials of the hydroxylated quinones were further consistent with their inter- mediacy in the labeling reactions of TMC.15 The quinone oxidation products of 4- and
5-HTMC were characterized in part as cyclopentadiene Diels–Alder adducts.15 The instan-
taneous reactions of these hydroxy TMCs with receptor were consistent with their interme- diacy in the TMC reactions. From the concentration dependence of the half-of-sites labeling reactions we could deduce Kd for each isomer: Kd(4-HTMC) = 224 ± 98 µM, Kd(5-HTMC) = 39 ± 17 µM.
We also prepared both hexamethonium and decamethonium analogs, each containing two catechol rings.16 Hexamethonium and decamethonium were originally prepared as FIGURE 8.5
Proposed reaction sequences occurring in the oxidation of TMC with the nAChR.
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simpler models of d-tubocurarine but were unreliable as neuromuscular blocking agents in thoracic surgery (for a brief review of these agents see Reference 16). We demonstrated a new feature in these synthetic analogs that may be representative of affinity-directed mol- ecules containing two catechol rings. With such compounds it has been possible to demon- strate metal ion-induced receptor inactivation. These reactions are apparently affinity directed, because reactivity parallels the high affinity of the reagents for the receptor (18 and 230 nM for the hexamethonium and decamethonium analogs, respectively).16 As
with the earlier agents, we again demonstrated half-of-sites reactivity. However, metal ion–induced inactivation may be associated with site-specific Fenton reactions similar to those suggested by Godinger et al.18 to explain the ascorbate-enhanced cytotoxic reactions
of metalloproteins. At present, we do not know whether the nAChR is covalently labeled by the reagent during these metal ion–induced inactivations.
In summary, we observed complex but selective reactions with oxidizable catechols into which affinity-directing functionality has been built. The current class of reagents could be useful in the study of disorders in which selective cholinergic degradation is a feature (e.g., in Alzheimer’s disease),* or as a starting point in the discovery of pharmaceutical agents in which the selective oxidative destruction of a targeted receptor would provide a drug effect, such as in deleting targetable activated oncogenic receptors.