Novel 1,4-homofragmentation via an alpha-lactone.

Conversion of an alpha,alpha-dichloroester to the corresponding alpha-keto acid was unexpectedly complicated by a novel 1,4-homofragmentation. Investigation of the kinetics of this reaction revealed a mechanism involving an alpha-lactone intermediate, which can lead to both the desired alpha-keto acid and the 1,4-homofragmentation, with the product distribution being dependent upon reaction conditions. This information allowed development of a process that affords the alpha-keto acid exclusively and should be generally applicable to the preparation of alpha-keto acids from alpha,alpha-dichloroesters or acids.

Addition of benzylic and allylic organozinc and Grignard reagents to resin-bound imines to provide alpha-branched secondary amines bearing a wide variety of functional groups. Utility in the synthesis of beta-3 adrenergic receptor agonists.

Benzylic and allylic organozinc and Grignard reagents have been added to resin-bound imines to provide alpha-branched secondary amines. Many functional groups, including electrophilic groups, were compatible with this methodology. Three modules--a resin-bound primary amine, an aromatic aldehyde, and the organometallic--were independently varied to produce a combinatorial library of alpha-branched secondary amines designed as beta-3 adrenergic receptor agonists.

N-Amination of pyrrole and indole heterocycles with monochloramine (NH2Cl).

A survey of several electrophilic ammonia reagents for the N-amination of indole- and pyrrole-containing heterocycles revealed that monochloramine (NH(2)Cl) is an excellent reagent for this transformation. Pyrroles and indoles containing a variety of substitution were aminated on nitrogen with isolated yields ranging from 45% to 97%.

Efficient asymmetric synthesis of the vasopeptidase inhibitor BMS-189921.

[reaction: see text] An efficient asymmetric synthesis of the vasopeptidase inhibitor BMS-189921 was accomplished. Two short enantioselective syntheses of the common key intermediate (S)-alpha-aminoazepinone 6b were developed. Olefin 3 was converted to 6b via asymmetric hydrogenation. Alternatively, enyne 12 was converted to racemic alpha-aminoazepinone 15b, which was transformed to 6b by a practical dynamic resolution.

Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I: cysteine ligation of the [4Fe-4S] cluster with protein rearrangement is preferred over serine ligation.

The [4Fe-4S] cluster of Azotobacter vinelandii ferredoxin I receives three of its four ligands from a Cys-Xaa-Xaa-Cys-Xaa-Xaa-Cys sequence at positions 39-45 while the fourth ligand, Cys20, is provided by a distal portion of the sequence. Previously we reported that the site-directed mutation of Cys20 to Ala (C20A protein) resulted in the formation of a new [4Fe-4S] cluster that obtained its fourth ligand from Cys24, a free cysteine in the native structure. That ligand exchange required significant protein rearrangement. Here we report the conversion of Cys20 to Ser (C20S protein), which gives the protein the opportunity either to retain the native structure and use the Ser20 O gamma as a ligand or to rearrange and use Cys24. X-ray crystallography demonstrates that the cluster does not use the Ser20 O gamma as a ligand; rather it rearranges to use Cys24. In the C20S protein the [4Fe-4S] cluster has altered stability and redox properties relative to either C20A or the native protein.

Mössbauer and EPR studies of Azotobacter vinelandii ferredoxin I.

Azotobacter vinelandii ferredoxin I (FdI) is a small protein that contains one Fe4S4 cluster and one Fe3S4 cluster. Previous studies of FdI have shown that the redox potential of the Fe3S4 cluster and the MCD and CD spectra of the reduced Fe3S4 cluster are pH-dependent. Using Mössbauer and EPR spectroscopy, we have studied FdI in different oxidation states and at different pH values. Here, we report the spin Hamiltonian parameters of the oxidized (S = 1/2) Fe3S4 cluster at pH 7.4 and the reduced (S = 2) Fe3S4 cluster at pH 6.0 and 8.5. The pH dependence observed by MCD is also evident in the Mössbauer spectra which show a change of the magnetic hyperfine tensor for one Fe site of the valence-delocalized pair. The Fe4S4 cluster is ligated by cysteines 20, 39, 42, and 45, but not by the adjacent cysteine 24. Treatment of FdI with 3 equiv of ferricyanide alters the Fe4S4 cluster, yielding a new species, [Fe4S4]'. The S = 1/2 EPR signal of [Fe4S4]' has previously been attributed to the formation of a cysteine disulfide radical from Cys24 and cluster sulfide. Here we show that the EPR signal is broadened by 57Fe, indicating that the electronic spin is significantly coupled to the cluster iron. Consistent with this, substantial magnetic hyperfine interactions are observed by Mössbauer spectroscopy. In addition, the average isomer shift of the four Fe sites is smaller for [Fe4S4]' than for [Fe4S4]2+, indicating that the oxidation is iron-based to at least some extent. Incubation of FdI with excess ferricyanide destroys the Fe4S4 cluster but leaves the Fe3S4 cluster intact. Our studies of (3Fe)FdI show that the S = 1/2 spin of the Fe3S4 cluster interacts with another paramagnet, presumably a radical generated at the site left vacant by the removal of the Fe4S4 cluster.

Azotobacter vinelandii ferredoxin I. Alteration of individual surface charges and the [4FE-4S]2+/+ cluster reduction potential.

The structures of Azotobacter vinelandii ferredoxin I (AvFdI) and Peptococcus aerogenes ferredoxin (PaFd), near their analogous [4e-4S]2+/+ clusters, are highly conserved (Backes, G., Mino, Y., Loehr, T.M., Meyer, T.E., Cusanovich, M.A., Sweeney, W.V., Adman, E.T., and Sanders-Loehr, J. (1991) J. Am. Chem. Soc. 11, 2055-2064). Despite these similarities, the reduction potential (E0') of the AvFdI [4Fe-4S]2+/+ cluster is more than 200 mV more negative than that of PaFd. We have tested the contribution that individual amino acid residues make to the control of E0' by converting residues in AvFdI into the corresponding residue in PaFd. Four mutations involved substitutions of negatively charged surface residues with neutral residues and two involved substitution of buried hydrophobic residues. All AvFdI variants were characterized by x-ray crystallography, absorption, CD, EPR, and 1H NMR spectroscopies and by electrochemical methods. For the F25I mutation, significant structural changes occurred that affected the EPR and 1H NMR spectroscopic properties of AvFdI and had a minor influence on E0'. For all other mutations there were no changes in reduction potential. Thus we conclude, that variations in charged surface residues do not account for the observed differences in E0' between the analogous [4Fe-4S]2+/+ cluster of PaFd and AvFdI. These differences are therefore most likely to be due to differences in solvent accessibility.

Formate dehydrogenase from Methylosinus trichosporium OB3b. Purification and spectroscopic characterization of the cofactors.

NAD(+)-coupled formate dehydrogenase has been purified to near-homogeneity from the obligate methanotroph Methylosinus trichosporium OB3b. The inclusion of stabilizing reagents in the purification buffers has resulted in a 3-fold increase in specific activity (98 microM/min/mg; turnover number 600 s-1) and as much as a 25-fold increase in yield over previously reported purification protocols. The enzyme, (molecular weight 400,000 +/- 20,000) is composed of four subunit types (alpha, 98,000; beta, 56,000; gamma, 20,000; delta, 11,500) apparently associated as 2 alpha beta gamma delta protomers. The holoenzyme contains flavin (1.8 +/- 0.2), iron (46 +/- 6), inorganic sulfide (38 +/- 4), and molybdenum (1.5 +/- 0.1). The flavin is optically similar to the common flavin cofactors, but it is chromatographically distinct. Anaerobic incubation of the enzyme with formate, NADH, or sodium dithionite, resulted in approximately 50% reduction of the iron and elicited an electron paramagnetic resonance (EPR) spectrum (approximately 2.5 spins/protomer) from which the spectra of five distinct EPR-active centers could be resolved in the g = 1.94 region. Four of these spectra were characteristic of [Fe-S]x clusters. The fifth (gave = 1.99; approximately 0.1 spins/protomer) was similar to that observed for the molybdenum cofactor of xanthine oxidase, and it exhibited the expected hyperfine splitting when the enzyme was enriched with 95Mo (I = 5/2). Mössbauer spectroscopy showed that all of the iron in the enzyme became reduced upon the addition of a redox mediator, proflavin, to the dithionite reduced enzyme at pH 8.0. Nevertheless, a decrease in the EPR-active spin concentration in the g = 1.94 region of the spectrum occurred and was attributed to the reduction of the molybdenum center to the EPR-silent Mo(IV) state (S = 1). The fully reduced enzyme also exhibited a new species with an S = 3/2 ground state (1-2 spins/protomer). Addition of 50% ethylene glycol to the fully reduced enzyme revealed no new species, but caused an increase in the EPR-detectable spin quantitation to 5-6 spins/protomer. This suggests that cluster spin-spin interactions may occur in both the partially and fully reduced native enzyme.

Purification and characterization of salicylate hydroxylase from Pseudomonas putida PpG7.

The salicylate hydroxylase from P. putida PpG7 was purified and characterized. The enzyme appears to be monomeric, and it showed one major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 45 kDa. The sequence of the first 25 amino acids of salicylate hydroxylase (PpG7) was determined. Also, the total amino acid composition of salicylate hydroxylase (PpG7) was obtained and compared with that of the known salicylate hydroxylase from P. putida.

Purification and Characterization of Microsomal Cytochrome b(5) and NADH Cytochrome b(5) Reductase from Pisum sativum.

In this communication we document the reproducible protocols for the purification of milligram quantities of cytochrome b(5) and NADH-cytochrome b(5) reductase from the microsomal fraction of Pisum sativum. The cytochrome b(5) component of this NADH linked electron transport chain was found to have a molecular mass of 16,400 daltons and the reductase a molecular mass of 34,500 daltons. These components could be reconstituted into a functional NADH oxidase activity active in the reduction of exogenous cytochrome c or ferricyanide. In the latter assay the purified reductase exhibited a turnover number of 22,000 per minute. The amino-terminal amino acid sequence of the cytochrome b(5) component was determined by sequential Edmund degredation, thus providing crucial information for the efficient cloning of this central protein of plant microsomal electron transfer.

Synthesis, bacterial expression, and mutagenesis of the gene coding for mammalian cytochrome b5.

We have totally synthesized a gene that codes for rat hepatic cytochrome b5. The 5' flanking region was designed for efficient expression of this gene in Escherichia coli by incorporating an optimum ribosome binding site and spacer region. Both a soluble form, analogous to the protease-treated microsomal protein, as well as the complete cytochrome with hydrophobic membrane anchor, was constructed and expressed. Transformants with the gene for the soluble protein overproduce authentic cytochrome b5 to a level of 8% of the total cell protein. The complete cytochrome is expressed to a lesser extent with most of the protein found in the cell membrane fraction. This represents complete synthesis and bacterial expression of a mammalian metalloprotein gene. Cytochrome b5 is normally a six-coordinate low spin heme protein with histidine-39 and histidine-63 as axial ligands. We have replaced histidine-63 with a methionine residue by cassette mutagenesis, utilizing specific restriction enzyme sites engineered into the synthetic gene. The resultant protein has histidine-39 as sole axial ligand and is five-coordinate high spin in the ferric resting state, as indicated by optical and electron spin resonance spectroscopy. The ability to generate mutant cytochrome b5 in high yield is a crucial step in understanding heme protein folding, protein-protein recognition and binding, and biological electron transfer processes.

Resonance Raman detection of bound dioxygen in cytochrome P-450cam.

We have used resonance Raman spectroscopy and isotopic labeling techniques to unambiguously assign the dioxygen stretching frequency (vo-o) in the substrate-bound oxygenated complex of cytochrome P-450cam. The frequency found for Vo-o in the P-450cam system (1140 cm-1) is in remarkable agreement with recent studies of thiolate heme model compounds. The general features of the oxy-P-450cam Raman spectra are tabulated and comparisons are made with the oxy complexes of hemoglobin, myoglobin, and various model compounds. Most of the results are qualitatively explained by consideration of electron donation into the pi g (O2)/d pi (M) orbitals of the oxygenated complex (M = Fe or Co). It is also noted that the effect of the "extra" electron in the nitrogen base Co(II) oxy complexes, in some ways, parallels the effect of the lone pair electrons of thiolate in the oxy-P-450cam complex. This is evidenced by the enhanced resonance Raman activity of vo-o in both the Co(II) and P-450 systems as well as by the similarity of the vo-o frequencies.

Effect of cis-platinum on kidney cytochrome P-450 and heme metabolism: evidence for the regulatory role of the pituitary hormones.

A novel action of the gonadotropic hormones of the adenohypophysis on the regulation of kidney heme metabolism and cytochrome P-450 concentrations is described. The treatment of rats with cis-platinum for 7 days caused a greater than twofold increase in the microsomal cytochrome P-450 and heme concentrations in the kidney. The sodium dodecyl sulfate-gel electrophoresis of the microsomal preparation revealed increased levels of both apocytochrome P-450 and heme in the molecular weight region corresponding to cytochrome P-450. In hypophysectomized rats, similar increases in heme and the cytochrome contents in the kidney were observed. Conversely, the treatment of rats with human chorionic gonadotropin (hCG) fully reversed the effect of cis-platinum on heme and cytochrome P-450 concentrations. The cellular basis of increases in concentrations of heme and the hemoprotein was explored by measuring the incorporation of [14C]glycine-labeled hemoglobin heme into the kidney microsomal heme fractions. In comparison with the control rats, the specific 14C activity of heme in microsomal fraction was not increased. Moreover, the effect of cis-platinum on kidney cytochrome P-450 appeared to be unrelated to alterations in the activities of the rate-limiting enzymes of heme biosynthesis and degradation pathways, delta-aminolevulinate synthetase, and heme oxygenase, respectively. On the other hand, ferrochelatase activity and the concentration of total porphyrins in the kidney were profoundly altered by cis-platinum treatment; a twofold increase in ferrochelatase activity and a marked reduction (40%) in the total porphyrin concentration were observed. Also, the activities of uroporphyrinogen-I synthetase and delta-aminolevulinate dehydratase were decreased in cis-platinum-treated animals. The latter effects reflect a direct inhibitory action of cis-platinum. It appears that the cis-platinum-mediated increase in the microsomal heme concentrations involves an accelerated rate of heme production as a consequence of increased ferrochelatase activity. This, in turn, could increase the production of cytochrome P-450. It is suggested that the anterior pituitary hormones control the concentration of the cytochrome P-450 in the kidney, and this process may be interrupted by cis-platinum.

Dissociation of heme metabolic activities from the microsomal cytochrome P-450 turnover in testis of hypophysectomized rats.

In the rat testis, 7 days after hypophysectomy, the microsomal content of cytochrome P-450 decreased to a negligible level. The sodium dodecyl sulfate gel electrophoresis of the microsomal preparation did not reveal a decrease in apocytochrome P-450; however, in this preparation, heme was undetectable. The latter did not reflect decreases in the activities of the heme biosynthesis enzymes. Also, an increase in heme oxygenase activity did not appear responsible for the suppression of the cytochrome levels. The cellular basis for the depression of the cytochrome was explored by measuring the incorporation of [14C]delta-aminolevulinate into the testicular microsomal and mitochondrial hemoproteins, and determining the relative affinity of microsomal heme for the endoplasmic reticulum membranes. In comparison with the sham-operated animals, in hypophysectomized rats, the specific 14C activity of heme in mitochondrial fraction was not decreased; however, that of the microsomal fraction was markedly reduced. The latter appeared to reflect a lowered binding affinity of the apoprotein moiety of cytochrome P-450 for heme. The treatment of hypophysectomized rats with human chorionic gonadotropin partially restored the normal level of the cytochrome. It is suggested that the anterior pituitary hormones control the level of cytochrome P-450 in the testis through factors which do not involve the production of heme; rather, the control appears to involve the processes of assembly of the hemoprotein and the association of the heme molecule with the apoprotein.