New Mediators

As discussed in Section 2.2.1.7, improved mediators are needed to further develop viable laccase catalytic systems. To do this, extensive work has been carried out to test derivatives of promising known mediators, such as ABTS, HBT, and phe-nothiazine-10-propionic acid, to systematically study their structure-function relationship [30, 31, 136, 144, 175]. Phenols with extended conjugation and/or steric structure causing low pKa have been studied to better balance their reactivity towards laccase/target with their stability at the oxidized form [137]. Pyrazolones have been investigated and found to be inferior to ABTS as potential laccase mediators [176]. Many redox-active metal complexes have been studied [29]. The Mn3+/Mn2+ couple has been exploited, and the oxidation potency of Mn3+ depends on its chelator [177]. Polyoxometalates (like tungsto-vanado-silicates, E ° ~0.74 V) have previously been used alone as robust redox catalysts for delignification. Their slow reoxidation by O2 is often rate-limiting, but may be accelerated under laccase catalysis [178, 179].

The most significant advance in the field comes from the discovery and characterization of TEMPO and its analogs as novel laccase mediators. The cyclic structure of TEMPO hosts a very stable N—O^, making the radical less active in H-abstraction than the N—O^ of oxidized HBT or its derivatives. The BDE of the NO—H in N-hydroxy-2,2,6,6-tetramethylpiperidine is ~290 kJ mol-1, which is lower than that in N-hydroxyacetanilide (~330) and N-hydroxyphthalimide (~370) [35, 180]. Since the BDE of a benzylic C—H is ~340 kJ mol-1, abstracting this H by TEMPO would be unfavorably endothermic, in contrast to the H-abstraction by N-hydroxyphthalimide radical. Oxidized TEMPO, an oxoammonium (>N+= O) species and a more potent oxidant, is well known for its potency/efficacy in oxidizing various molecules [180-182, 182a]. The oxoammonium has an E° of ~0.9 V, and may be readily made by an appropriate laccase [181, 183]. Thus TEMPO can serve as a mediator for laccase biocatalysis.

Unlike the electron-transfer or H-abstraction mechanisms for (laccase-) oxidized ABTS or N-hydroxy/oxide/oxime mediators (as discussed in Section 2.2.1.7), the mechanism of TEMPO oxoammonium in oxidizing target molecule is believed to be of a two-electron, "ionic oxidation" type. For an alcohol target, nucleophilic attack of the alcohol O on the N + of the oxoammonium forms a tet-rahedral intermediate, followed by removal of an Ha in the alcohol (by TEMPO's N—O- or a solute base under acidic or basic conditions, respectively), resulting in N-hydroxyl-2,2,6,6-tetramethylpiperidine and an aldehyde product. The hy-droxylamine can be reoxidized by laccase to first TEMPO and then the oxoammonium form to start another round of alcohol oxidation [35, 144, 144a, 184]. Hence, TEMPO-mediated laccase catalysis may be applied on targets with ioniza-tion potential too high for a direct oxidation by laccase [2]. The involvement of a tetrahedral intermediate in the oxoammonium's interaction with the target may also give the catalysis more stereoselective than other laccase oxidations. The TEMPO-mediated laccase biocatalysis has recently been exploited for delignifica-tion [39], dye bleaching [42], degradation of chlorinated aromatics [185], and phenolic oligomerization [38].

Despite TEMPO's ability to mediate this unique type of laccase catalytic process, there is still a need to find better TEMPO analogs. TEMPO is a poor laccase substrate, mainly due to its slow kcat (-10 s-1) [183]. Oxidized TEMPO can be degraded/deoxygenated to the inactive 2,2,6,6-tetramethylpiperidine, significantly reducing the catalytic turnover efficiency. The problem may be addressed by systematically evaluating piperidine/pyrrolidine nitroxyls and other TEMPO analogs [144, 144a, 181]. A dual-mediator system may be used, in which a good laccase substrate could quickly mediate the oxidation/recycling of TEMPO by the laccase, while the TEMPO oxoammonium effectively oxidizes the target [125, 183], although likely at higher cost.

In addition to TEMPO and analogs, polyunsaturated hydrocarbons (including fatty acids/lipids) and derivatives may also be applied in laccase catalysis. In linoleate, the "doubly" allylic C site, flanked by two > C=C< bonds, may form an allylic C readily after a C—H bond homolysis (by H-abstraction). The radical may be oxygenated to a peroxy radical or delocated to another C site (via >C=C< bond rearrangement), and these radicals or their descendants may serve as potent oxidants. A mediated laccase oxidation may be used to generate the allylic radicals, which may in turn oxidize a target substance. An example of such a dual-mediator laccase biocatalysis has been reported, in which HBT and linoleate mediate a laccase-catalyzed oxidative degradation of phenanthrene [129]. The effect might also contribute to the often observed benefit (for laccase biooxidation) from Tween 80 or other surfactants containing unsaturated moieties.

It has long been suspected that some of the natural delignification microbial systems employ endogenous mediators for their laccases. For Pycnoporus cinna-barinus, the native metabolite 3-hydroxyanthranilic acid has been shown to act as a laccase mediator (see Section 2.2.1.7) [133]. For Petriellidium fusoideum, a 1.6 kDa Fe-containing compound has been speculated to assist its extracellular laccase

[186]. Identifying naturally occurring mediators is of interest for the development of highly efficient, inexpensive, and safe laccase biocatalysis systems [132].

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Heal Yourself With Qi Gong

Qigong also spelled Ch'i Kung is a potent system of healing and energy medicine from China. It's the art and science of utilizing breathing methods, gentle movement, and meditation to clean, fortify, and circulate the life energy qi.

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