Resonance Raman characterization of the peroxo and hydroperoxo intermediates in cytochrome P450

Resonance Raman (RR) studies of intermediates generated by cryoreduction of the oxyferrous complex of the D251N mutant of cytochrome P450(cam) (CYP101) are reported. Owing to the fact that proton delivery to the active site is hindered in this mutant, the unprotonated peroxo-ferric intermediate is observed as the primary species after radiolytic reduction of the oxy-complex in frozen solutions at 77 K. In as much as previous EPR and ENDOR studies have shown that annealing of this species to approximately 180 K results in protonation of the distal oxygen atom to form the hydroperoxo intermediate, this system has been exploited to permit direct RR interrogation of the changes in the Fe-O and O-O bonds caused by the reduction and subsequent protonation. Our results show that the nu(O-O) mode decreases from a superoxo-like frequency near approximately 1130 cm(-1) to 792 cm(-1) upon reduction. The latter frequency, as well as its lack of sensitivity to H/D exchange, is consistent with heme-bound peroxide formulation. This species also exhibits a nu(Fe-O) mode, the 553 cm(-1) frequency of which is higher than that observed for the nonreduced oxy P450 precursor (537 cm(-1)), implying a strengthened Fe-O linkage upon reduction. Upon subsequent protonation, the resulting Fe-O-OH fragment exhibits a lowered nu(O-O) mode at 774 cm(-1), whereas the nu(Fe-O) increases to 564 cm(-1). Both modes exhibit a downshift upon H/D exchange, as expected for a hydroperoxo-ferric formulation. These experimental RR data are compared with those previously acquired for the wild-type protein, and the shifts observed upon reduction and subsequent protonation are discussed with reference to theoretical predictions.     

Pigmentation Chemistry and Radical‐Based Collagen Degradation in Alkaptonuria and Osteoarthritic Cartilage

Alkaptonuria (AKU) is a rare disease characterized by high levels of homogentisic acid (HGA); patients suffer from tissue ochronosis: dark brown pigmentation, especially of joint cartilage, leading to severe early osteoarthropathy. No molecular mechanism links elevated HGA to ochronosis; the pigment's chemical identity is still not known, nor how it induces joint cartilage degradation. Here we give key insight on HGA‐derived pigment composition and collagen disruption in AKU cartilage. Synthetic pigment and pigmented human cartilage tissue both showed hydroquinone‐resembling NMR signals. EPR spectroscopy showed that the synthetic pigment contains radicals. Moreover, we observed intrastrand disruption of collagen triple helix in pigmented AKU human cartilage, and in cartilage from patients with osteoarthritis. We propose that collagen degradation can occur via transient glycyl radicals, the formation of which is enhanced in AKU due to the redox environment generated by pigmentation.     

Ethynylation of Cysteine Residues: From Peptides to Proteins in Vitro and in Living Cells

Current approaches to introduce terminal alkynes for bioorthogonal reactions into biomolecules still present limitations in terms of either reactivity, selectivity, or adduct stability. We present a method for the ethynylation of cysteine residues based on the use of ethynylbenziodoxolone (EBX) reagents. The acetylene group is directly introduced onto the thiol group of cysteine and can be used for copper‐catalyzed alkyne‐azide cycloaddition (CuAAC) without further processing. Labeling proceeded with reaction rates comparable to or higher than the most often used iodoacetamide on peptides or maleimide on the antibody trastuzumab, and high cysteine selectivity was observed. The reagents were also used in living cells for cysteine proteomic profiling and displayed improved coverage of the cysteinome compared to previously reported iodoacetamide or hypervalent iodine reagents. Fine‐tuning of the EBX reagents allows optimization of their reactivity and physical properties.     

Metal-assisted in situ formation of a tridentate acetylacetone ligand for complexation of fac-Re(CO)3+ for radiopharmaceutical applications

Reaction of [NEt4]2[ReBr3(CO)3] with 2,4-pentanedione (acac) yields a complex of the type fac-Re(acac)(OH2)(CO)3 (1) under aqueous conditions. 1 was further reacted with a monodentate ligand (pyridine) to yield a fac-Re(acac)(pyridine)(CO)3 complex (2). Complex 1 was found to react with primary amines to generate a Schiff base (imine) in aqueous solutions. When a mixed-nitrogen donor bidentate ligand, 2-(2-aminoethyl)pyridine, that has different coordination affinities for fac-Re(acac)(OH2)(CO)3 was utilized, a unique tridentate ligand was formed in situ utilizing a metal-assisted Schiff base formation to yield a complex fac-Re(CO)3(3[(2-phenylethyl)imino]-2-pentanone) (3). Tridentate ligand formation was found to occur only with the Re-coordinated acac ligand. Reactions of acac with fac-Re(CO)3Br(2-(2-aminoethyl)pyridine) (4) or a mixture of [NEt4]2[ReBr3(CO)3], acac, and 2-(2-aminoethyl)pyridine did not yield the formation of complex 3 in water.     

Lanthanide distribution in some doped alkaline earth aluminates and gallates

High-resolution neutron and synchrotron X-ray powder diffraction studies are reported for the six oxides AB₂O₄ (A=Ca²⁺, Sr²⁺ and Ba²⁺ and B=Al³⁺ and Ga³⁺). These oxides all adopt a stuffed tridymite type structure, the precise nature of which depends on both the A- and B-type cations. Bond valence calculations reveal a range of values for the various A-type cations, in all cases at least one site is significantly underbonded. Conversely the tetrahedral B-type sites invariably exhibit unexceptional bond valencies. Attempts to dope the oxides with various lanthanides to the 1% level invariably resulted in some segregation into alternate phases located at the grain boundaries. The identity of the impurity phases is presented and the importance of bond valencies in understanding this segregation is highlighted.     

The nature of halogen...halogen synthons: crystallographic and theoretical studies

A study of the halogen...halogen contacts in organic compounds using ab initio calculations and the results of previously reported crystallographic studies show that these interactions are controlled by electrostatics. These contacts can be represented by the geometric parameters of the C--X1...X2--C moieties (where theta1=C--X1...X2 and theta2=X1...X2--C; ri=X1...X2 distance). The distributions of the contacts within the sum of van der Waals radii (rvdW) versus thetai (theta1=theta2) show a maximum at theta approximately 150 degrees for X=Cl, Br, and I. This maximum is not seen in the distribution of F...F contacts. These results are in good agreement with our ab initio calculations. The theoretical results show that the position of the maximum depends on three factors: 1) The type of halogen atom, 2) the hybridization of the ipso carbon atom, and 3) the nature of the other atoms that are bonded to the ipso carbon atom apart from the halogen atom. Calculations show that the strength of these contacts decreases in the following order: I...I>Br...Br>Cl...Cl. Their relative strengths decrease as a function of the hybridization of the ipso carbon atom in the following order: sp2>sp>sp3. Attaching an electronegative atom to the carbon atom strengthens the halogen...halogen contacts. An electrostatic model is proposed based on two assumptions: 1) The presence of a positive electrostatic end cap on the halogen atom (except for fluorine) and 2) the electronic charge is anisotropically distributed around the halogen atom.     

Ligand-promoted solvent-dependent ionization and conformational equilibria of Re(CO)3Br[CH2(S-tim)2] (tim = 1-methylthioimidazolyl). Crystal structures of Re(CO)3Br[CH2(S-tim)2] and {Re(CO)3(CH3CN)[CH2(S-tim)2]}(PF6)

The compounds Re(CO)3Br[CH2(S-tim)2] (1) and {Re(CO)3(CH3CN)[CH2(S-tim)2]}(PF6) (2), where tim is 1-methylthioimidazolyl, were prepared in high yields and characterized both in the solid state and in solution. The solid-state structures show that the ligand acts in a chelating binding mode where the eight-member chelate ring adopts twist-boat conformations in both compounds. A comparison of both solid-state IR data for CO stretching frequencies and the solution-phase voltammetric measurements for the Re(1+/2+) couples between 1, 2, and related N,N-chelates of the rhenium tricarbonyl moiety indicate that the CH2(S-tim)2 ligand is a stronger donor than even the ubiquitous dipyridyl ligands. A combination of NMR spectroscopic studies and voltammetric studies revealed that compound 1 undergoes spontaneous ionization to form {Re(CO)3(CH3CN)[CH2(S-tim)2]+}(Br-) in acetonitrile. Ionization does not occur in solvents such as CH2Cl2 or acetone that are less polar and Lewis basic (less coordinating). The equilibrium constant at 293 K for the ionization of 1 in CH3CN is 4.3 x 10(-3). The eight-member chelate rings in each 1 and 2 were found to be conformationally flexible in all solvents, and boat-chair conformers could be identified. Variable-temperature NMR spectroscopic studies were used to elucidate the various kinetic and thermodynamic parameters associated with the energetically accessible twist-boat to twist-boat and twist-boat to boat-chair interconversions.     

Incorporation of a tripodal ligand with a (N,O,O)-donor set into a new family of nickel and cobalt spin clusters

The reaction of Ni(OAc)₂, NiX₂ (X = Cl, Br) or CoCl₂ with the proligand 2-amino-2-methyl-1,3-propanediol (ampdH₂) affords a new family of tetranuclear complexes. The syntheses of [Ni₄(OAc)₄(ampdH)₄] (1) and [M₄X₄(ampdH)₄] (M = Ni, X = Cl, 2; M = Ni, X = Br, 3; M = Co, X = Cl, 4) are reported, together with the single crystal X-ray structures of 1, 2 and 4 and the magnetochemical characterization of 1, 3 and 4. Each member of this family of complexes displays a low symmetry structure that incorporates a {M₄O₄} core unit based on a distorted cubane. Magnetic measurements reveal ferromagnetic exchange interactions for 1, 3 and 4. These give rise to S = 4 ground state spins for the tetranuclear Ni complexes and an anisotropic effective S′ = 2 ground state for the Co complex.