Kinetics and activation parameters for oxidations of styrene by Compounds I from the cytochrome P450(BM-3) (CYP102A1) heme domain and from CYP119.

Cytochrome P450 (CYP or P450) enzymes are ubiquitous in nature where they catalyze a vast array of oxidation reactions. The active oxidants in P450s have long been assumed to be iron(IV)-oxo porphyrin radical cations termed Compounds I, but P450 Compounds I have proven to be difficult to prepare. The recent development of an entry to these transients by photo-oxidation of the corresponding iron(IV)-oxo neutral porphyrin species (Compounds II) permits spectroscopic and kinetic studies. We report here application of the photo-oxidation method for production of Compound I from the heme domain of CYP102A1 (cytochrome P450(BM-3)), and product and kinetic studies of reactions of styrene with this Compound I transient and also Compound I from CYP119. The studies were performed at low temperatures in 1:1 (v:v) mixtures of glycerol and phosphate buffer. Single-turnover reactions at 0 degrees C gave styrene oxide in good yields. In kinetic studies conducted between -10 and -50 degrees C, both Compounds I displayed saturation kinetics permitting determinations of binding constants and first-order oxidation rate constants. Temperature-dependent functions for the binding constants and rate constants were determined for both Compounds I. In the temperature range studied, the Compound I transient from the CYP102A1 heme domain bound styrene more strongly than Compound I from CYP119, but the rate constants for oxidations of styrene by the latter were somewhat larger than those for the former. The temperature-dependent functions for the first-order oxidation reactions are as follows: log k = 13.2-15.2/2.303RT and log k = 13.3-14.6/2.303RT (kilocalories per mole) for Compounds I from the CYP102A1 heme domain and CYP119, respectively.

N-heterocyclic carbene boryl radicals: a new class of boron-centered radical.

Reduction of xanthates by N-heterocyclic carbene boranes (NHC-boranes) has been suggested to occur by a radical chain mechanism involving heretofore unknown NHC-boryl radicals. In support of this suggestion, both the expected borane dithiocarbonate product and an unexpected borane xanthate product have now been isolated. These are the first NHC-boranes with boron-sulfur bonds, and their structures have been secured by spectroscopic and crystallographic means. The first rate constants for H-atom transfer from an NHC borane complex were determined by using the ring opening of a substituted cyclobutylcarbinyl radical as a clock reaction. The rate constant for reaction of the NHC-borane with a secondary alkyl radical at ambient temperature is 4 x 10(4) M(-1) s(-1), and the Arrhenius function displayed an entropic term (log A term) that was typical for a bimolecular reaction. The B-H bond dissociation energy of an NHC-borane complex has been estimated at 88 kcal/mol. The putative NHC-boryl radical in these transformations has been detected by EPR spectroscopy. Spectral analysis suggests that it is a pi-radical, analogous to the benzyl radical.

Development of highly regioselective amidyl radical cyclization based on lone pair-lone pair repulsion.

The substituent effect on the reactivity and regioselectivity of N-(4-pentenyl)amidyl radical cyclization was investigated. Exclusive 6- endo cyclization was observed for N-(4-pentenyl)amidyl radicals with internal vinylic heteroatom substitution (Cl, Br, I, OMe, SEt). The substituent on the carbonyl group also showed a significant influence on the reactivity of amidyl radicals, which increases in the order of Ph < Me < OEt. As a result, the photostimulated reactions of N-(4-halopent-4-enyl)amides and carbamates (X = Cl, Br, I) with DIB/I 2 or Pb(OAc) 4/I 2 led to the efficient and exclusive formation of the corresponding piperidines while those of N-(5-halopent-4-enyl)amides afforded the pyrrolidine products only. The halogen-substitution effect also allowed the 6- exo and 7- endo amidyl radical cyclization to proceed in a highly regioselective manner. The above experimental results, in combination with theoretical analyses, revealed that the lone pair-lone pair repulsion between the nitrogen radical and the vinylic heteroatom played an important role in controlling the regioselectivity of cyclization.

Copper-catalyzed intramolecular N-vinylation of sulfonamides: general and efficient synthesis of heterocyclic enamines and macrolactams.

With the catalysis of CuI/N,N'-dimethylethylenediamine, intramolecular C-N coupling between sulfonamides and vinyl halides was successfully implemented, leading to the efficient synthesis of 5-, 6-, 7-, and even 8-membered heterocyclic enamines in both exo and endo modes. The bicyclic enamines thus formed provided a convenient entry to the corresponding 9- to 12-membered lactams by oxidative C=C bond cleavage.

Low temperature photo-oxidation of chloroperoxidase Compound II.

Oxidation of the heme-thiolate enzyme chloroperoxidase (CPO) from Caldariomyces fumago with peroxynitrite (PN) gave the Compound II intermediate, which was photo-oxidized with 365 nm light to give a reactive oxidizing species. Cryo-solvents at pH ≈ 6 were employed, and reactions were conducted at temperatures as low as -50° C. The activity of CPO as evaluated by the chlorodimedone assay was unaltered by treatment with PN or by production of the oxidizing transient and subsequent reaction with styrene. EPR spectra at 77K gave the amount of ferric protein at each stage in the reaction sequence. The PN oxidation step gave a 6:1 mixture of Compound II and ferric CPO, the photolysis step gave an approximate 1:1 mixture of active oxidant and ferric CPO, and the final mixture after reaction with excess styrene contained ferric CPO in 80% yield. In single turnover reactions at -50°C, styrene was oxidized to styrene oxide in high yield. Kinetic studies of styrene oxidation at -50°C displayed saturation kinetics with an equilibrium constant for formation of the complex of K(bind)=3.8 x 10(4)M(-1) and an oxidation rate constant of k(ox)=0.30s(-1). UV-Visible spectra of mixtures formed in the photo-oxidation sequence at ca. -50° C did not contain the signature Q-band absorbance at 690 nm ascribed to CPO Compound I prepared by chemical oxidation of the enzyme, indicating that different species were formed in the chemical oxidation and the photo-oxidation sequence.

5-Exo versus 6-endo cyclization of primary aminyl radicals: an experimental and theoretical investigation.

The cyclization of neutral primary pent-4-enylaminyl radicals was investigated experimentally and theoretically. Unlike the corresponding secondary aminyl radicals, primary pent-4-enylaminyl radicals underwent efficient cyclization to afford the pyrrolidine and/or piperidine products in good to high yields. While the simple pent-4-enylaminyl radical gave predominately the 5-exo cyclization product, 4-chloropent-4-enylaminyl radicals led to the formation of the corresponding 6-endo cyclization products in excellent regioselectivity. Theoretical calculations revealed that the 5-exo cyclization rate of primary aminyl radicals is about 3-4 orders of magnitude higher than that of secondary aminyl radicals.

O-attack versus N-attack: electrophilic halocyclization of unsaturated amides with vinylic halogen substitution.

Electrophilic iodocyclization of unsaturated amides with an internal vinylic halogen (Cl, Br, or I) substitution afforded exclusively the corresponding cyclic iminoketones via iodolactamization. On the other hand, amides having a terminal vinylic halogen substituent underwent iodolactonization only. Theoretical calculations revealed that the iodocyclization proceeds via the intramolecular iodonium ion transfer from the amide nitrogen to the C=C double bond.