In 1979, pyrroloquinoline quinone** (Figure 8.6) was identified as a novel coenzyme in methanol dehydrogenase from a methylotrophic bacterium.19,20 PQQ also may be impor-
tant in plants, where a role has been suggested for it in diamine oxidase and in N-methylpu- trescine oxidase.21 An important role has been suggested for PQQ and perhaps for some of
its closely related analogs as growth and nutritional factors in eukaryotes.22 In addition,
PQQ may act as a tissue-protective agent mediated through tissue flavin reductases,23 as
well as through electron-transfer reactions with biological reducing agents mediated non- enzymatically. PQQ may be used by methemoglobin reductase in place of flavin. Indeed, the Km of the enzyme for PQQ (2 µM) is lower than that of riboflavin23 (25 µM). Deducing a specific role for PQQ in eukaryotes is complicated by the apparently facile biosynthesis of both PQQ and its isomeric analogs. Further, it is possible that not only PQQ but PQQ
FIGURE 8.6
PQQ and its analogs.
* It should be noted that as these agents bear a permanently charged quaternary ammonium group they would not be expected to penetrate the blood–brain barrier. Thus, the study of the ability to inactivate central cholinergic receptors selectively and produce an Alzheimer’s-like experimental dementia would have to be studied in iso- lated brain preparations in vitro and with the aid of push–pull cannula strategies for in vivo studies.
** Methoxatin, 4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3f]quinoline-2,7,9-tricarboxylic acid.
Reactive Quinones: From Chemical Defense Mechanisms in Plants to Drug Design 115
isomers may be formed during the turnover of amine oxidases that utilize an integral topaquinone residue as a redox-enabling cofactor.24 The actual formation of PQQ isomers*
and their function in nature, if any, is not well documented. Thus, it seemed to us at the out- set that synthesis and study of the catalytic potential of isomeric PQQs were required prior to more general examination of the pharmaceutical potential of PQQ analogs.
We synthesized25 each of the PQQ isomers shown in Figure 8.6 via a strategy requiring
the formation of the intermediate indole in a multistep procedure from suitably trisubsti- tuted methoxynitroanilines followed by regioselective addition of the pyridine ring in a Doebner–von Miller quinoline synthesis. All isomers have similar pH-dependent oxida- tion–reduction behavior. From pH-dependent cyclic voltammograms, the pKa of each of the
five independently protonated sites in each molecule may be estimated.26 While there are
some similarities between each of the isomers in the way they carry out the nonenzymatic catalytic oxidation of some substrates, the catalytic properties of both isomers 2 and 3 are poor in relation to PQQ, strongly suggesting that if either isomer were formed in nature it would be of relatively little use as an enzyme catalytic cofactor. This finding was consistent with earlier studies on nonisomeric analogs. On the other hand, isomer 1 is as potent a cat- alyst as PQQ, but undergoes a rapid inactivation reaction in the course of catalyzing amine oxidative deamination. This suggests that if a cell were to attempt to use isomer 1 as a cat- alyst in amine oxidase reactions, an unacceptable level of catalyst turnover would make its effectiveness as an enzyme cofactor problematic. Furthermore, the accelerated inactivation reaction, illustrated for benzylamine oxidative deamination catalysis in Figure 8.7, results in formation of a tetracyclic aromatic oxazole, which is probably genotoxic.
It is interesting that such a nominally small change as movement of the pyrrole nitrogen from one side of the ring to the other (in PQQ vs. isomer 1) should result in a catalyst with an unacceptable turnover problem. The oxazole-forming reaction also occurs in PQQ , albeit at a very much lower rate. A cyclic oxazole is formed only once in several hundred catalytic reactions in PQQ as compared with once in every 4 to 5 catalytic turnovers in iso- mer 1. Thus, formation of the cyclic oxazole from isomer 1 is far more facile in comparison with cyclic oxazole formation from PQQ. This is also true for isomer 3 relative to isomer 2; however, due to inverted incorporation of the pyridine ring in the molecule, neither rates of catalysis nor cyclic oxazole formation are nearly as rapid as they are with isomer 1 or PQQ. The origin of the enhanced rate of oxazole formation is certainly due to the presence of a pyrrole ring nitrogen on the reactive side of the molecule in isomer 1 (and isomer 2), where a role for a pyrrole NH as a participant in intramolecular catalysis to facilitate for- mation of the oxazole is postulated. While the explicit mechanism has yet to be elucidated, a step involving intramolecular acid catalysis exploiting the well-positioned pyrrole NH in
FIGURE 8.7
Proposed mechanism for intramolecular catalysis of oxazole formation by pyrrole NH.
* The mechanisms for the nonenzymatic formation of PQQ or any of its isomers are by no means obvious. These reactions must proceed from tyrosine, or partially oxidized tyrosines, and glutamic acid or glutamine liberated from proteins. From tyrosine and glutamic acid, a ring alkylation and two successive ring closures are required together with a net 12-electron oxidation.
© 2000 by CRC Press LLC
isomer 1 is potentially attractive. The pKa of the pyrrole NH in each of these molecules is
9.5 to 10, and thus the normally very weakly acidic pyrrole NH more nearly resembles a phenolic group. A possible scheme involving intramolecular NH-assisted formation of the cyclic oxazole intervening during the course of the catalytic oxidative deamination reaction is suggested in Figure 8.7.
These preliminary studies, focusing on the three isomeric PQQs discussed above, dem- onstrate two important points:
1. Nature’s design of PQQ was not frivolous. Even subtle changes in the structure of PQQ can result in an alternative redox cofactor with little utility in any cell. In isomer 1, where catalytic redox functions are retained, a facilitated inactivation reaction such as oxazole formation, which can take place even nonenzymatically in any cell, results in potential toxicity.
2. The design and evaluation of pharmaceuticals based on PQQ will be more difficult than might have been imagined given a lack of knowledge of the mech- anisms of action of the agent upon which a suggested extensive analog synthesis and testing program would be based.
In the present case, for example, isomer 1 is an excellent alternative to flavin as a flavin reductase substrate. The Km for isomer 1 is 1.6 µM. This value compares favorably with that
for PQQ, which has a Km of 2 µM. However, attempted use of isomer 1 in protection against
reoxygenation injury would likely result in complete conversion to the noncatalytic and probably genotoxic oxazole before isomer 1 had any chance to protect against reoxygen- ation injury. Thus, as isomer 1 attempted to deaminate oxidatively simple amines and amino acids encountered in the tissue, the deamination intermediates would be converted at unacceptable rates into genotoxic oxazoles.