Carbon Skeleton Rearrangement Research Articles

The role and source of 5′-deoxyadenosyl radical in a carbon skeleton rearrangement catalyzed by a plant enzyme

The last step in the biosynthesis of tropane alkaloids is the carbon skeleton rearrangement of littorine to hyoscyamine. The reaction is catalyzed by a cell-free extract prepared from cultured hairy roots of Datura stramonium. Adenosylmethionine stimulated the rearrangement 10–20-fold and showed saturation kinetics with an apparent K m of 25 μM. It is proposed that S-adenosylmethionine is the source of a 5′-deoxyadenosyl radical which initiates the rearrangement in a similar manner as it does in analogous rearrangements catalyzed by coenzyme B 12-dependent enzymes. Possible roles of S-adenosylmethionine as a radical source in higher plants are discussed.

Coupling of cobalt-carbon bond homolysis and hydrogen atom abstraction in adenosylcobalamin-dependent glutamate mutase.

Adenosylcobalamin-dependent glutamate mutase catalyzes an unusual carbon skeleton rearrangement that proceeds through the formation of free radical intermediates generated by the substrate-induced cleavage of the coenzyme cobalt-carbon bond. The reaction was studied at 10 degrees C with various concentrations of L-glutamate and L-threo-3-methylaspartate and with use of stopped-flow spectroscopy to follow the formation of cob(II)alamin. Either substrate induces rapid formation of cob(II)alamin, which accumulates to account for about 25% of the total enzyme species in the steady state when substrate is saturating. Measurements of the rate constant for the formation of cob(II)alamin demonstrate that the enzyme accelerates the rate of homolysis of the cobalt-carbon bond by at least 10(12)-fold. Very large isotope effects on cob(II)alamin formation, of 28 and 35, are observed with deuterated L-glutamate and deuterated L-threo-3-methylaspartate, respectively. This implies a mechanism in which Co-C bond homolysis is kinetically coupled to substrate hydrogen abstraction. Therefore, adenosyl radical can only be formed as a high-energy intermediate only at very low concentrations on the enzyme. The magnitude of the isotope effects suggests that hydrogen tunneling may play an important role catalysis.

Hydrophobic vitamin B 12. Part 15. Carbon-skeleton rearrangement reactions mediated by hydrophobic vitamin B 12 covalently bound to an anionic lipid species in aqueous media

A novel artificial vitamin B 12 holoenzyme was prepared in aqueous media by combination of a hydrophobic vitamin B 12 covalently bound to an anionic lipid species and a bilayer matrix of sodium N,N-dihexadecyl- N α-(6-sulfohexanoyl)-L-alaninamide. Microenvironmental properties around a hydrophobic vitamin B 12 placed in the bilayer membrane were examined by electronic spectroscopy and fluorescence polarization measurements. The hydrophobic vitamin B 12 was well separated from a bulk aqueous phase, and its molecular motion was markedly suppressed. A reaction mimicking catalytic functions of methylmalonyl-CoA mutase was carried out by using hydrophobic vitamin B 12 derivatives having a diethyl 2,2-bis(ethoxycarbonyl)propyl group at an axial site of the nuclear cobalt. A carbon-skeleton rearrangement of the alkyl ligand bound to a hydrophobic vitamin B 12 was markedly promoted in the bilayer matrix, relative to the reaction in methanol and benzene, via formation of a radical intermediate. A reaction simulating catalysis by α-methyleneglutarate mutase was also carried out. The cyanide ion enhanced a carbon-skeleton rearrangement of the 2,3-bis(ethoxycarbonyl)-1-butene moiety bound to hydrophobic vitamin B 12 derivatives in the bilayer membrane under photolysis conditions via formation of an anionic intermediate. As an extension of such mimicking reactions, a carbon-skeleton rearrangement reaction of diethyl 2-acetylamino-2-methylpropanedioate coordinated to a hydrophobic vitamin B 12 covalently bound to a lipid species, which afforded diethyl 2-acetylaminobutanedioate, was also examined in the bilayer membrane under photolysis conditions. The motional repression and desolvation effects operated on the substrate-bound hydrophobic vitamin B 12 were found to be responsible for enhancement of the rearrangement reactions of the substrate radicals formed under photolysis conditions.

Identification of the 4-Glutamyl Radical as an Intermediate in the Carbon Skeleton Rearrangement Catalyzed by Coenzyme B12-Dependent Glutamate Mutase from Clostridium cochlearium

A series of 2H- and 13C-labeled glutamates were used as substrates for coenzyme B12-dependent glutamate mutase, which equilibrates (S)-glutamate with (2S,3S)-3-methylaspartate. These compounds contained the isotopes at C-2, C-3, or C-4 of the carbon chain: [2-2H], [3,3-2H2], [4,4-2H2], [2,3,3,4,4-2H5], [2-13C], [3-13C], and [4-13C]glutamate. Each reaction was monitored by electron paramagnetic resonance (EPR) spectroscopy and revealed a similar signal characterized by g'xy = 2.1, g'z = 1.985, and A' = 5.0 mT. The interpretation of the spectral data was aided by simulations which gave close agreement with experiment. This approach underpinned the idea of the formation of a radical pair, consisting of cob(II)alamin interacting with an organic radical at a distance of 6.6 +/- 0.9 A. Comparison of the hyperfine couplings observed with unlabeled glutamate with those from the labeled glutamates enabled a principal contributor to the radical pair to be identified as the 4-glutamyl radical. These findings support the currently accepted mechanism for the glutamate mutase reaction, i.e., the process is initiated through hydrogen atom abstraction from C-4 of glutamate by the 5'-deoxyadenosyl radical, which is derived by homolysis of the Co-C sigma-bond of coenzyme B12.

Mechanism of Action of Vitamin B12. Ultrafast Radical Clocks Provide No Evidence for Radical Intermediates in Cyclopropane Models for the Methylmalonyl-CoA to Succinyl-CoA Carbon Skeleton Rearrangement

To probe for free radical intermediates in the model studies for the coenzyme B12-dependent, methylmalonyl-CoA to succinyl-CoA carbon-skeleton rearrangement, new models incorporating cyclopropane rings (unsubstituted 23 and 2-phenyl-substituted 28) at the 2-position were developed. The reaction of 23 or 28 with vitamin B12s gives only rearranged succinate 24 or 29, respectively, with the cyclopropyl group intact. When this reaction was carried out in EtOD/D2O, a monodeuterided product, 24-d1 or 29-d1, was obtained and the deuterium was incorporated at the 2-position. Control reactions of the 2-phenylselenylsuccinate with tri-n-butyltin hydride yielded the ring-opened 2-propanylidenesuccinate via a free radical pathway. The results suggest that the skeletal rearrangement step in the B12-catalyzed isomerization of methylmalonyl-CoA to succinyl-CoA occurs not by a radical pathway but by an anionic or organocobalt pathway.

Bond-shift rearrangement during hydrogenolysis of neopentyl iodide on group VIII metal catalysts. An analogue of the mechanism of action of vitamin B 12

The hydrogenolysis of neopentyl iodide on reduced Co or Pt dispersed on MgO support yields increasing amounts of 2-metylbutane and 2-methylbutenes in addition to neopentane, as the temperature is raised from 50 to 150°C. These results support the idea that a π-complexed half-reaction state is simultaneously involved in activation of the RI bond and in the evident 1,2-methyl shift. This π-complex mechanism is the same as that first suggested almost a quarter of a century ago for neopentane isomerism during hydrogenolysis at ⩾ 200°C on Pt catalysts. The present results also support the contention that similar π-complexes are responsible for analogous carbon skeleton rearrangement reactions mediated by coenzyme B 12.

Structure-based perspectives on B12-dependent enzymes.

Two X-ray structures of cobalamin (B12) bound to proteins have now been determined. These structures reveal that the B12 cofactor undergoes a major conformational change on binding to the apoenzymes of methionine synthase and methylmalonyl-coenzyme A mutase: The dimethylbenzimidazole ligand to the cobalt is displaced by a histidine residue from the protein. Two methyltransferases from archaebacteria that catalyze methylation of mercaptoethanesulfonate (coenzyme M) during methanogenesis have also been shown to contain histidine-ligated cobamides. In corrinoid iron-sulfur methyltransferases from acetogenic and methanogenic organisms, benzimidazole is dissociated from cobalt, but without replacement by histidine. Thus, dimethylbenzimidazole displacement appears to be an emerging theme in cobamide-containing methyltransferases. In methionine synthase, the best studied of the methyltransferases, the histidine ligand appears to be required for competent methyl transfer between methyl-tetrahydrofolate and homocysteine but dissociates for reductive reactivation of the inactive oxidized enzyme. Replacement of dimethylbenzimidazole by histidine may allow switching between the catalytic and activation cycles. The best-characterized B12-dependent mutases that catalyze carbon skeleton rearrangement, for which methylmalonyl-coenzyme A mutase is the prototype, also bind cobalamin cofactors with histidine as the cobalt ligand, although other cobalamin-dependent mutases do not appear to utilize histidine ligation. It is intriguing to find that mutases, which catalyze homolytic rather than heterolytic cleavage of the carbon-cobalt bond, can use this structural motif. In methylmalonylCoA mutase a significant feature, which may be important in facilitating homolytic cleavage, is the long cobalt-nitrogen bond linking histidine to the co-factor. The intermediate radical species generated in catalysis are sequestered in the relatively hydrophilic core of an alpha/beta barrel domain of the mutase.

ビタミンＢ１２モデル錯体を触媒とした有機電解反応

Vitamin B12-dependent enzymes, involving the cobalt species as a catalytic center, mediate various isomerization reactions accompanied by carbon-skeleton rerragements. In order to simulate the catalytic functions of vitamin B12 as excerted in the hydrophobic active sites of enzymes concerned, we have been dealing with hydrophobic vitamin B12 derivatives which have ester groups in place of the peripheral amide moieties of the naturally occurring vitamin B12. In this work, the carbon-skeleton rearrangements as mediated by hydrophobic vitamin B12 derivatives were investigated under elec-trochemical conditions. The controlled-potential electrolyses of alkyl halides with various electronwithdrawing groups were carried out, and the electrochemical carbon-skeleton rearrangements proceeded effectively via formation of anionic intermediates. These reactions can be also applied to the ring-expansin reactins. We have carried out the electrolysis of an alkyl halide having two ester groups and one phenyl group on the β-carbon atom in the presence of a catalytic amount of the hydrophobic vitamin B12. The migration of the pheny group was observed under the conditions forming a radical intermediate. The ester-migrated product was detected under the conditions forming an anionic intermediate. This is the first successful example in selecting a migrating group by electroylsis potential. We have also prepared a strapped hydrophobic vitamin B12 in order to change the enantioselectivity. The controlled-potential electrolysis of a racemic alkyl halide having phenyl, methoxy, and carboxylic ester groups on the same carbon atom was carried out. The simple hydrophobic vitamin B12 tends to bind S-enantiomers more favorably. On the other hand, strapped hydrophobic vitamin B12 acts to bind R-enantiomers more favorably. These results suggested that the stability of alkylated complexes was dominated the enantioselectivity of reduction products.

Mechanism of Action of Vitamin B12. A New Test for Free Radical Intermediates Using a Cyclopropane Model for the Methylmalonyl-CoA to Succinyl-CoA Carbon Skeleton Rearrangement

Mechanism of Action of Vitamin B₁₂. A New Test for Free Radical Intermediates Using a Cyclopropane Model for the Methylmalonyl-CoA to Succinyl-CoA Carbon Skeleton Rearrangement

Carbon-skeleton rearrangement of an amino acid derivative as mediated by hydrophobic vitamin B12 covalently bound to a lipid species in a bilayer membrane

Diethyl 2-acetylamino-2-methylpropanedioate co-ordinated to a hydrophobic vitamin B12 Covalently bound to a peptide lipid underwent a carbon-skeleton rearrangement to afford diethyl 2-acetylaminobutanedioate in the single-walled vesicle of N,N-bis(hexadecyl)-Nα-(6-sulfohexanoyl)-L-alaninamide under photolysis conditions.

Metal-directed syntheses of dithiadiazacyclotetradecane macrocycles with pendant alcohol and nitro or carboxylate groups involving macrocycle ring expansion

Copper(II)-directed condensation of 4-hydroxymethyl-3,6-dithiaoctane-1,8-diamine with nitroethane or diethyl malonate and formaldehyde yielded (anti-6-methyl-6-nitro-1,11-dithia-4,8-diazacyclotetradecan-13-ol)copper(II) and (diethyl 13-hydroxy-1,11-dithia-4,8-diazacyclotetradecane-6,6-dicarboxylate)copper(II) respectively in good yields. The latter was readily converted by decarboxylation and hydrolysis in aqueous base to the copper(II) complex of anti-13-hydroxy-1,11-dithia-4,8-diazacyclotetradecane-6-carboxylic acid from which the free ligand can be obtained by zinc–acid reduction of the copper ion, whereas the former can be converted by zinc–acid reduction directly to the hydrochloride salt of the free anti-6-amino-6-methyl-1,11-dithia-4,8-diazacyclotetradecan-13-ol ligand. The condensations should yield a thirteen-membered macrocycle with a pendant hydroxymethyl group fused to a macrocyclic ring carbon, but the molecules undergo a Wagner–Meerwein or alternate carbon skeleton rearrangement to yield the fourteen-membered macrocycle with a pendant alcohol group. The pendant alcohol can act as an axial donor group, as illustrated in the crystal structure analyses of (anti-6-methyl-6-nitro-1,11-dithia-4,8-diazacyclotetradecan-13-ol)copper(II) and (anti-13-hydroxy-1,11-dithia-4,8-diazacyclotetradecane-6-carboxylato)copper(II) as perchlorate salts. The former complex crystallizes in the monoclinic P21/c space group, a= 8.703(2), b= 18.699(2), c= 13.293(2)A, β= 105.15(1)° and the latter in the monoclinic P21/n space group, a= 7.450(1), b= 15.325(2), c= 16.772(1)A, β= 98.38(1)°. In both complexes the alcohol is disposed anti to the nitro or carboxylate group. In the former, the copper ion lies in a distorted-octahedral environment of two sulfur donors (Cu–Sav 2.333 A), two nitrogen donors (Cu–Nav2.016 A), the pendant alcohol donor [Cu–O 2.365(3)A] and a perchlorate anion [Cu–O 2.544(3)A], whereas in the latter a dimer with each copper in a very distorted octahedral environment exists where each carboxylate bridges to the alternate copper ion [Cu–O 2.293(3)A] and the alcohol is weakly bound [Cu–O 2.520(3)A] in addition to the macrocycle heteroatoms (Cu–Nav 2.035, Cu–Sav 2.353 A).

Characterization of the Coenzyme‐B12–Dependent Glutamate Mutase from Clostridium cochlearium Produced in Escherichia coli

The glutamate mutase dependent on adenosylcobalamin (coenzyme B12) catalyzes the carbon skeleton rearrangement of (S)-glutamate to (2S,3S)-3-methylaspartate, the first step of the glutamate fermentation pathway of the anaerobic bacterium Clostridium cochlearium. The enzyme consists of two protein components, E, a dimer epsilon 2 (epsilon, 53.5 kDa) and S, a monomer (sigma, 14.8 kDa). The corresponding genes (glmE and glmS) were cloned, sequenced and over-expressed in Escherichia coli. The genes glmS and glmE are separated by glmL encoding a protein of unknown function. The deduced amino acid sequence of GlmL contains an ATP-binding motif which is common to chaperones of the HSP70-type, actin and procaryotic cell-cycle proteins. Both components of glutamate mutase were purified with excellent yields from cell-free extracts of E. coli carrying the corresponding genes. In contrast to component E, component S was shown to bind coenzyme B12. This observation strongly supports the idea that significant similarities of the amino acid sequences of component S and several other cobamide-dependent enzymes represent a common binding motif. Incubation of pure components E and S with coenzyme B12 resulted in the formation of a fully active glutamate mutase heterotetramer (epsilon 2 sigma 2) containing one molecule of coenzyme B12. EPR spectra of recombinant glutamate mutase, now available in sufficiency large amounts, were recorded after incubation of the enzyme with coenzyme B12 and (S)-glutamate. The EPR signals (gx,y approximately 2.1, gz = 1.985) were of much better resolution than observed earlier with the clostridial enzyme. Their typical hyperfine splitting is clearly derived from Co(II), which is involved in the formation of the paramagnetic species but is different from cob(II)alamin (gx,y = 2.25). The spin concentration was 34-50% of the concentration of the enzyme (epsilon 2 sigma 2) coenzyme complex. The competitive inhibitors (2S, 4S)-4-fluoroglutamate and 2-methyleneglutarate induced similar but not identical signals with spin concentrations of 134-148% of the enzyme concentration. Even (S)-[2,3,3,4,4-2H5]glutamate induced a signal significantly different to that of (S)-glutamate with an intensity of only 7%. These data suggest an involvement of the Co(II)-containing paramagnetic species in catalysis, the concentration of which reflects a steady state between its formation and decomposition. The large difference in the spin concentrations observed with (S)-glutamate as compared to the predeuterated glutamate is probably due to a kinetic isotope effect and indicates a cleavage of a C-H bond during formation of the paramagnetic species.(ABSTRACT TRUNCATED AT 400 WORDS)

Isomerization of an axial ligand coordinated to hydrophobic vitamin B 12 as effected by cyanide ion and microenvironment

The cyanide ion enhanced a carbon-skeleton rearrangement of the 2,3-bis(ethoxycarbonyl)-1-butene moiety bound to hydrophobic vitamin B 12 derivatives in single-compartment bilayer vesicles under photolysis conditions via formation of the anionic substrate intermediate.

Stereospecific annulation and sequential ring-opening of (R)-carvone: Formation of a novel tricyclic dione

Free radical annulation followed by trimethylsilyl iodide-promoted ring-opening of (R)-carvone-dichloroketene adducts leads to an unusual carbon skeleton rearrangement. The carbonyl group of carvone enhances the reactivity of the radical cyclization, changes the ring-opening pathway and leads to the formation of a new tricyclic dione product.

Cloning, sequencing and expression inEscherichia coliof the gene encoding component S of the coenzyme B12-dependent glutamate mutase fromClostridium cochlearium

Adenosylcobalamin (coenzyme B12) dependent glutamate mutase catalyzes the carbon skeleton rearrangement of (S)-glutamate to (2S,3S)-methylaspartate. This is the first step of the fermentation of glutamate by the strict anaerobic bacterium Clostridium cochlearium. The enzyme consists of the two protein components E and S. The gene encoding component S (glmS) was cloned in Escherichia coli and its nucleotide sequence was determined. The nucleotide sequence and the deduced amino acid sequence showed very strong identities to the sequence of the glmS (also called mutS) gene (80%) and to component S (82%) from the related C. tetanomorphum, respectively. Cell-free extracts of E. coli carrying the glmS gene showed glutamate mutase activity which was strictly dependent on the addition of coenzyme B12 and component E purified from C. cochlearium. Enzyme activity of the recombinant protein was achieved up to 2200 nkat/g wet cells which is due to a ten-fold overexpression compared with the activities determined in cell-free extracts of C. cochlearium. This is the first report of overexpression of an active component of glutamate mutase. A rapid purification procedure consisting only of ammonium sulfate precipitation and a gel filtration step was developed to obtain large amounts of pure component S in a short time.

Unimolecular fragmentations of long‐chain aliphatic iminium ions

AbstractThe metastable ion characteristics of N‐alkyl‐N‐methylmethyleneiminium ions (\documentclass{article}\pagestyle{empty}\begin{document}$ {\rm H}_2 {\rm C}=\!=\mathop {\rm N}\limits^ + ({\rm CH}_3){\rm R} $\end{document}, R = n‐CmH2m + 1, m = 3, 4, 5, 6, 8, 10, 14, 18) are reported and discussed. For R = n‐propyl, alkene loss by onium reaction and alkene loss by McLafferty rearrangement occur, whereas for the higher homologues only the latter reaction is observed. As a result of 2H and 13C labelling experiments, the mechanism of alkene loss by γ‐H transfer and β‐cleavage does not change with increasing chain length and the iminium ions do not isomerize prior to decomposition, neither by H–D scrambling nor by carbon skeleton rearrangement. Whereas the sequence of elementary steps during fragmentation is not affected, the energetics of the reaction change as the chain length increases. Resulting from thermodynamic estimations, the enthalpy of reaction ΔHr, critical energy E0 and reverse critical energy E0r diminish markedly as R increases from n‐propyl to n‐octadecyl. The knowledge of the reaction energetics including kinetic energy release data allows information about partitioning of excess energy into internal and translational degrees of freedom to be deduced.

Epimerization of aldoses catalysed by self-organized metallomicelles in an aqueous solution

Nickel(II) complexes coordinated with long chain N-alkylated ethylenediamine ligands were synthesized. It was found that the complexes possessed a marked C-2 epimerization activity for aldoses in aqueous media. In contrast, a short chain diamine nickel(II) complex had little activity. The agreement between the formation of micelles and the enhancement of epimerization was clearly recognized. Amphiphilic complexes formed ‘metallomicelles’ which coordinated the aldoses and produced a new ternary complex composed of nickel, diamine and sugar. The stereospecific epimerization occurred in this aggregate accompanied by carbon skeleton rearrangement. One explanation for the high epimerizing ability of the amphiphilic nickel complex is the accumulation effect of nickel ion and ethylenediamine at the micelle surface.

First hydrogen abstraction-rearrangement model for the coenzyme B12-dependent methylmalonyl-CoA to succinyl-CoA carbon skeleton rearrangement reaction

First hydrogen abstraction-rearrangement model for the coenzyme B12-dependent methylmalonyl-CoA to succinyl-CoA carbon skeleton rearrangement reaction

Catalytic Functions of Artificial Enzyme Composed of Simple Vitamin B12 Model and Synthetic Bilayer Membrane

Abstract An artificial enzyme was constructed with a combination of the single-walled vesicle of N,N-dihexadecyl-Nα-[6-(trimethylammonio)hexanoyl]-l-alaninamide bromide (N+C5Ala2C16) and a simple vitamin Bi12 model; (11-hydroxyimino-4,10-dipropyl-5,9-diazatrideca-4,9-dien-3-one oximato)cobalt, [Co(C2C3)(DO)(DOH)pn], or [N-(2-hydroxyiminocyclohexylidene)-N′-(cyclohexyliden-2-one oximato)-1,3-diaminopropane]cobalt, [Co(ch)(DO)(DOH)pn]. Carbon-skeleton rearrangement reactions of alkyl ligands bound to the cobalt complex were markedly favored in the single-walled vesicle, relative to the reactions in methanol and benzene, under anaerobic photolysis conditions at ordinary temperatures. The cobalt complex incorporated into the intramembrane domain is subjected to significant repression of its molecular motion and to desolvation as confirmed by fluorescence and fluorescence polarization measurements. The 1,2-migration of cyano and carboxylic ester groups involved in the alkyl ligands is plausibly enhanced by such effects.

Hydrophobic Vitamin B12. X. Steric Effect in Electrochemical Carbon-Skeleton Rearrangement Catalyzed by Hydrophobic Vitamin B12 in Nonaqueous Media

Abstract A steric effect in the carbon-skeleton rearrangement catalyzed by heptamethyl cobyrinate perchlorate, [Cob(II)7C1ester]C104, was investigated under electrochemical conditions. The controlled-potential electrolyses of alkyl halides having two carboxylic ester groups of different bulkiness on the same carbon atom, such as 2,2-bis(ethoxycarbonyl)-l-bromopropane, l-bromo-2-tert-butoxycarbonyl-2-ethoxy-carbonylpropane, l-bromo-2-cyclohexyloxycarbonyl-2-ethoxycarbonylpropane, and l-bromo-2-ethoxy-carbonyl-2-phenoxycarbonylpropane, were carried out in N,N-dimethylformamide, as catalyzed by [Cob(II)7C1ester]CIO4, to give the corresponding ester-migrated products in the dark at—1.5 V vs SCE in the presence of acetic acid and at—2.0 V vs SCE without acetic acid. As regards a correlation between bulkiness of an ester group and a migration aptitude, a smaller ester group tends to migrate to the adjacent carbon atom more readily than a larger one. The origin of such a steric effect is discussed with attention...