Radical clock substrates

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). 

Allylic rearrangements with 3,3,6,6-dj-cyclohexene occurred in 20% of the MMO hydroxylation products compared to 33% for cytochrome P-450. These two experiments suggest that, with M. trichospor-ium OB3b, a rebound reaction must occur with a greater rate constant than with cytochrome P-450, in accord with the radical clock substrate work. 

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. 

Formation of a protein radical, for example Cys 151, RS, which promotes the synchronous insertion of oxygen atoms across the substrate C-H bond (Waller and Limscomb, 1996, Shilov, 1997). The absence of rearranged products of the radical clock substrates for MMOH isolated from M. capsulatus raises the possibility in principle, of such a mechanism. 

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. 

Figure 20 (a) An ultrafast radical clock substrate capable of distinguishing between radical and carbocation intermediates (b) The proposed two-oxidant pathway of C-H bond oxidation by P450 enzymes in which Compound 1 follows a concerted mechanism with no intermediate while the ferric-hydroperoxo attacks substrates by insertion of HO+ leading to carbocation intermediates which rearrange to give the alcohol product... 

The rate of the ring-opening reaction of 5, " and other substrates have been determined using an indirect method for the calibration of fast radical reactions, applicable for radicals with lifetimes as short as 1 ps/ This radical clock method is based on the use of Barton s use of pyridine-2-thione-Al-oxycarbonyl esters as radical precursors and radical trapping by the highly reactive thiophenol and benzeneselenol/ A number of radical clock substrates are known/ Other radical clock processes include racemization of radicals with chiral conformations, one-carbon ring expansion in cyclopentanones, norcarane and sprro[2,5]octane, a-and p-thujone radical rearrangements, and cyclopropylcarbinyl radicals or... 

Table 18-1 Experimentally determined recombination rates, ikoH) for various radical clock substrates...

Substrate probes have aided mechanistic understanding of the key C— H activation step in the MMOH reaction cycle. Chiral alkanes and radical-clock substrate probes " " were used to discriminate between radical recoil/rebound and nonsynchronous concerted insertion pathways. A short lifetime (< 150 fs) estimated for the putative radical species derived from cyclopropane-based radical-clock substrates favors the latter process,whereas partial racemization of chiral ethane substrate is consistent with the former scenario. A unifying model was proposed, in which both recoil/rebound and concerted reaction channels are available for a bound radical intermediate and the partitioning between each trajectory is dependent on the substrate. Formation of carboca-tion-derived products from certain probes implicates yet another route involving a formal OH+ insertion.Participation of multiple species capable of oxygen transfer is an emerging mechanistic view in both heme and nonheme systems, as exemplified by the studies of cP450s and their synthetic models.Scheme 3 depicts various density functional theory (DFT) models of MMOHq and their computed reaction pathways, which are reviewed in detail elsewhere. 

Kumar D, de Visser SP, Sharma PK, Cohen S, Shaik S (2004) Radical clock substrates, their C-H hydroxylation mechanism by cytochrome P450, other reactivity patterns what does theory reveal about the clocks behavior J Am Chem Soc 126 1907-1920... 

Furthermore, the C—H insertion with N-tosyloxycarbamates appears also to involve a singlet rhodium nitrene species, as the reaction is sterospecific, and no ring opening product is observed with a radical clock substrate (Scheme 5.11). 

Newcomb and Lippard reported the sMMO-catalyzed oxygenation of methylcyclo-propane derivatives [77]. In their study, four cyclopropyl derivatives shown in Fig. 4 were used as radical clock substrates ... 

Figure 4. Radical clock substrates used in the sMMO-catalyzed oxygenation of methycyclopropane [77].

Valentine AM, LeTadic-Biadatti MH, Toy PH, Newcomb M, Lippard SJ. 1999. Oxidation of ultrafast radical clock substrate probes by the soluble methane monooxygenase from Methylococcus capsulatus (Bath). JBiol Chem 274 10771-10776. 

One important line of investigation which has supported the radical rebound hypothesis is the use of radical clock substrate probes. These probes rearrange in a diagnostic way on a very rapid and calibrated time scale when a hydrocarbon radical is formed. In the case of P450, rearranged products have been isolated after oxidation and have been used as evidence of an intermediate substrate radical. In this way, even though the lifetime of the radical is too short for it to be observed directly, its character can be explored by the judicious choice of substrate analogues. 

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