Radical clock probes

Figure 6.5. The principle of radical clock probes of the cytochrome P450 mechanism based on the methyl-cyclopropyl radical rearrangement.

By using an identical radical clock substrate probe which did not rearrange upon hydroxylation with M. capsulatus (Bath), rearranged product was detected with MMO from AT. trichosporium 0B3b (59). From the ratio of unrearranged to rearranged products, a rebound rate constant was calculated to be 6 x 1012 s-1 at 30°C for this system. A separate study with another radical clock substrate probe with MMO from M. trichosporium 0B3b reported products consistent with both radical and cationic substrate intermediates (88). 

Since work with the radical clock substrate probes indicated important differences in the hydroxylation mechanisms for M. capsulatus (Bath) and M. trickosporium OB3b, work with (R) and (S)-[1-2H,1-3H]ethane with both enzymes was carried out (93, 94). With M. tri-chosporium OB3b, approximately 65% of the product displays retention of stereochemistry (93). A rebound rate constant of 2 - 6 x 1012 s-1 was calculated, assuming a free energy change of 0.5 kcal mole-1 for rotation about the C-C bond (94). This estimate approaches the value obtained from the radical clock substrate probe analysis (59). 

In summary, mechanistic studies have revealed intriguing differences between MMO from M. capsulatus (Bath) and MMO from M. triehosporium OB3b. With M. capsulatus (Bath), radical clock substrate probes indicated either that a substrate radical is not produced or that it reacts with a rate constant > 1013 s-1. With MMO from M. triehosporium OB3b, radical involvement was suggested from several experiments, and a rebound rate constant of 6 x 1012 s"1 was calculated for this system. 

Scheme 9.8 Radical clock-based mechanistic probe...

Rate constants for reactions of Bu3SnH with some a-substituted carbon-centered radicals have been determined. These values were obtained by initially calibrating a substituted radical clock on an absolute kinetic scale and then using the clock in competition kinetic studies with Bu3SnH. Radical clocks 24 and 25 were calibrated by kinetic ESR spectroscopy,88 whereas rate constants for clocks 26-31 were measured directly by LFP.19,89 90 For one case, reaction of Bu3SnH with radical 29, a rate constant was measured directly by LFP using the cyclization of 29 as the probe reaction.19... 

The reaction of bornyl and isobornyl bromides with the nucleophile (Scheme 18) is another case where the amount of inversion is small and the rate constant close to that observed with an aromatic anion radical of the same standard potential (Daasbjerg et al., 1989) it can therefore be rationalized along the same lines. Cyclizable radical-probe experiments carried out with the same nucleophile and 6-bromo-6-methyl-1-heptene, a radical clock presumably slower than the preceding one, showed no cyclized coupling product. It should be noted, on the other hand, that, unlike the case... 

There are numerous other examples of radical clock reactions in the literature used both for simple rate determinations to facilitate the quest for selectivity in synthesis and for more detailed probing of mechanistic pathways. 

Recently the ultrafast radical-clock technique (kr = 5xl012 -1013 s 1) has been developed (Newcomb et al, 2000 and references therein). Two probes, trans, trans-2-methoxy-3-phenylmethyl cyclopropane and methyl cubane were used to study the... 

Liu, K. E., Johnson, C. C., Newcomb, M., and Lippard, S. J., 1993, Radical clock substrate probes and kinetic isotope effect studies of the hydroxylation of hydrocarbons by methane monooxygenase, J. Am. Chem. Soc. 115 939n947. 

In the hydroxylahon of cyclopropyl probes, the cyclopropylcarbinyl radical clock can either rearrange to ring-opened 3-buten-l-yl radical before being trapped or rebound with the hydroxyl carrier to yield the alcohol product direchy (Scheme... 

In addition to their role in kinetics, radical clocks also serve an important mechanistic function in that the formation of the rearranged product provides evidence for radical involvement in the first place. The incorporation of a ring-opening probe into the substrate, followed by product analysis—specifically, a search for rearranged product—has been used in mechanistic studies of the action of some enzymes, such as cyt P450 [61] and methane monooxygenase [62]. 

The rearrangement of a-cyclopropyl radicals (162) to the ring-opened alkyl radical (163) has been recognized as an extremely rapid (ca. 10 s" ) and useful radical clock reaction (equation 30), with equilibrium greatly favoring the unstrained acyclic partner. In recent years this process has been used in enzymatic redox reactions to probe the nature of... 

Benzylic esters have been studied in considerable detail often as a continuation of the pioneering work by Zimmerman and co-workers (Scheme 2) in 1963 [44]. There are several reasons for this. First, the synthesis of compounds with the structural variables required to probe specific mechanistic questions is often straightforward. Second, products are usually formed from both ion pairs and radical pairs and, therefore, the structural variables that control this partitioning can be systematically studied. Third, the radical pair (ARCH2-O CO)— R) incorporates a built-in radical clock, the decarboxylation of the acyloxy radical, which serves as a useful probe for the reactivity of the radical pair. If the carbon of the acyloxy radical is sp hybridized, this decarboxylation rate is on the 1- to 1000-ps time scale, depending on R, so that decarboxylation will often occur within the solvent cage before diffusional escape. The topic of benzylic ester photochemistry has been recently reviewed twice by Pincock [5,98] and therefore only a brief summary will be given here. 

The probe is useful as a radical clock since it is us mentioned above possible lo measure the time spent between the HT and the radical recomhina (ion. Cyclizalion during the reaction is a proof of a radical mechanism (at least for the cyclized part), but that no cyclizalion lias taken place is mu a proof against a radical mechanism, but only tells that iI a free radical was produced, its lifetime wax signilicanlly less than ca. 10 s. 

When the probe reaction being calibrated is a unimolecular process, one measures the rate constant of a radical clock directly for the initial absolute kinetic values, and, thus, the method is inverted in approach from that used for alkyl radical kinetics. LFP studies of unimolecular process give more precise data than those of bimolecular processes, and the approach typically starts with inherently good kinetic data. The synthetic efforts necessary for production of appropriate radical precursors are a drawback to this method, but it is, nonetheless, useful for establishing absolute kinetics for some classes of radicals where little kinetic information was available, such as nitrogen-centered radicals discussed later. 

Atkinson, J.K. and K.U Ingold (1993). Cytochrome P450 hydroxylation of hydrocarbons Variation in the rate of oxygen rebound using cyclopropyl radical clocks including two new ultrafast probes. Biochemistry 32, 9209-9214. 

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