Oxocarbenium Ion Intermediates Research Articles

Structural Basis for Nicotinamide Inhibition and Base Exchange in Sir2 Enzymes

The Sir2 family of proteins consists of broadly conserved NAD(+)-dependent deacetylases that are implicated in diverse biological processes, including DNA regulation, metabolism, and longevity. Sir2 proteins are regulated in part by the cellular concentrations of a noncompetitive inhibitor, nicotinamide, that reacts with a Sir2 reaction intermediate via a base-exchange reaction to reform NAD(+) at the expense of deacetylation. To gain a mechanistic understanding of nicotinamide inhibition in Sir2 enzymes, we captured the structure of nicotinamide bound to a Sir2 homolog, yeast Hst2, in complex with its acetyl-lysine 16 histone H4 substrate and a reaction intermediate analog, ADP-HPD. Together with related biochemical studies and structures, we identify a nicotinamide inhibition and base-exchange site that is distinct from the so-called "C pocket" binding site for the nicotinamide group of NAD(+). These results provide insights into the Sir2 mechanism of nicotinamide inhibition and have important implications for the development of Sir2-specific effectors.

Stereochemical Control in the Reduction of 2-Chromanols

[reaction: see text] Reduction of C5-substituted 2-hydroxychromans selectively provides 2,4-cis-chromans using large silane reductants and 2,4-trans-chromans using the smaller silane PhSiH(3). The stereochemical outcome has been rationalized on the basis of a Curtin-Hammett kinetic situation arising from hydride delivery to two different conformations of an intermediate oxocarbenium ion. This method provides a powerful way to control the relative stereochemistry of these substructures which are prevalent in bioactive natural products.

Racemization in Prins cyclization reactions.

Isotopic labeling experiments were performed to elucidate a new mechanism for racemization in Prins cyclization reactions. The loss in optical activity for these reactions was shown to occur by 2-oxonia-Cope rearrangements by way of a (Z)-oxocarbenium ion intermediate. Reaction conditions such as solvent, temperature, and the nucleophile employed played a critical role in whether an erosion in enantiomeric excess was observed. Additionally, certain structural features of Prins cyclization precursors were also shown to be important for preserving optical purity in these reactions.

Tetrahydropyran Rings from a Mukaiyama−Michael Cascade Reaction

A new annulation reaction leading to tetrahydropyrans has been discovered. The reaction of homoallylic enol ethers (e.g., 1) with alpha,beta-unsaturated ketones or esters begins with a Mukaiyama-Michael addition. The intermediate oxocarbenium ion undergoes a rapid 2-oxonia-Cope rearrangement, and the resulting zwitterion collapses to form a tetrahydropyran. The reaction is stereoselective with 3-butene-2-one, but leads to diastereomeric mixtures with ethyl acrylate. More complex enones, such as cyclohexenone, also undergo the reaction to produce fused ring products. The optical activity of the substrates is relayed in the tetrahydropyran products.

BiBr3 initiated cyclization–addition reactions: effect of π-nucleophile on oxocarbenium ion addition and total syntheses of (+)-(S,S)-(cis-6-methyltetrahydropyran-2-yl)acetic acid and its trans-diastereomer

For BiBr3 initiated tandem cyclization–additions of very reactive silyl ketene acetal nucleophiles with δ-silyloxy aldehydes to afford 2,6-disubstituted THP products, diastereoselectivities range from 5–6:1 (trans-/cis-). The selectivity for axial attack on the intermediate oxocarbenium ion is inversely proportional to π-nucleophilicity. We have utilized this chemistry to convert a common starting material to both cis- and trans-diastereomers of an acid first isolated from the secretions of Viverra civetta.

Transformation of Glycals into 2,3‐Unsaturated Glycosyl Derivatives

AbstractFor Abstract see ChemInform Abstract in Full Text.

An oxocarbenium-ion intermediate of a ribozyme reaction indicated by kinetic isotope effects.

Many of the enzymes that catalyze reactions at nucleotide glycosidic linkages proceed through either a reactive oxocarbenium-ion intermediate or a transition state with considerable oxocarbenium character. To investigate how an RNA active site deals with the catalytic challenge of nucleotide synthesis, we probed the transition state of a ribozyme able to promote the formation of a pyrimidine nucleotide. Primary and secondary kinetic isotope effects indicate that this ribozyme stabilizes a highly dissociative reaction with considerable sp2 hybridization and negligible bond order between the departing pyrophosphate leaving group and the anomeric carbon. The small primary 13C isotope effect of 1.002 +/- 0.003 indicates that the reaction is likely to be less concerted than that observed for protein nucleotide synthesis enzymes, which typically have primary 13C isotope effects of 1.02-1.03. The dissociative nature of the ribozyme reaction most resembles the reaction of some hydrolytic enzymes, such as uracil DNA glycosylase, which uses the negative charges found in the phosphodiester backbone of its DNA substrate to transiently stabilize an oxocarbenium ion during hydrolysis. The detectable hydrolysis observed in the ribozyme reaction indicates that shielding of this reactive intermediate from water is a significant challenge for RNA, which protein enzymes that synthesize nucleotides have managed to overcome during evolution, apparently by the utilization of more concerted chemistry.

TRNA−Guanine Transglycosylase from Escherichia coli: Molecular Mechanism and Role of Aspartate 89

The enzyme tRNA-guanine transglycosylase (TGT, EC 2.4.2.29) catalyzes a posttranscriptional transglycosylation reaction involved in the incorporation of the modified base queuine [Q, 7-(4,5-cis-dihydroxy-2-cyclopenten-1-ylaminomethyl)-7-deazaguanine] into tRNA. Previously, the crystal structure of the TGT from Zymomonas mobilis was solved in complex with preQ(1) (the substrate for the eubacterial TGT) [Romier et al. (1996) EMBO J. 15, 2850-2857]. An aspartate residue at position 102 (position 89 in the Escherichia coli TGT) was proposed to play a nucleophilic role in an associative catalytic mechanism. Although this is an attractive and precedented mechanism, a dissociative mechanism is equally plausible. In a dissociative mechanism, aspartate 89 would provide electrostatic stabilization of an oxocarbenium ion intermediate that is formed by dissociation of guanine. To clarify the nature of the catalytic mechanism of TGT, we have generated and characterized four mutations of aspartate 89 in the E. coli TGT (alanine, asparagine, cysteine, and glutamate). All four mutant TGTs were able to noncovalently bind tRNA, but only the glutamate mutant was able to form a stable complex with the RNA substrate under denaturing conditions that was comparable to wild type. Furthermore, the glutamate mutant was the only mutant TGT that demonstrated significant activity. Kinetic parameters were determined for this enzyme and shown to be comparable to wild type, revealing that the enzyme is considerably tolerant of the positioning of the carboxylate. Under conditions of high enzyme concentrations and long time courses, the alanine, asparagine, and cysteine mutants showed very low levels (ca. 10(3)-fold lower than wild type) of activity that were linear with respect to enzyme concentration and dependent upon pH in a fashion similar to that of the wild type. However, the observed initial velocities were too low to accurately determine k(cat) and K(m) values. We hypothesize that the activity observed for these mutants is most likely derived from host strain TGT (wt) contamination. These results are most consistent with aspartate 89 acting as a nucleophile in an associative catalytic mechanism.

1,4-anti induction in C-glycosylation of pentose glycals

Abstract The C-glycosylation of pentose glycals with silylacetylenes or allylsilanes via oxocarbenium ion intermediates proceeded under high regio- and stereoselectivity giving the 1,4-anti products as the predominant products.

The N-Acetyl Neuraminyl Oxecarbenium Ion Is an Intermediate in the Presence of Anionic Nucleophiles

Solvolysis of CMP N-acetyl neuraminate (CMP-NeuAc) in 1.8 M acetate buffer at pH 5 containing 0.9 M azide results in the formation of both anomers of 2-deoxy-2-azido N-acetyl neuraminic acid in addition to N-acetyl neuraminic acid as determined by 1H-NMR product analysis. A rate dependence on [azide] was observed with an apparent bimolecular rate constant of (2.1 ± 0.3) × 10-3 M-l min-l which could only account for half of the azido-NeuAc formed. Comparison of rate, product ratio, and stereochemical data indicate that concurrent pathways for formation of N3-NeuAc are operative, with 17% of product forming from reaction of azide and the tight ion pair, 12% via the solvent separated ion pair, and 6% from the free NeuAc oxocarbenium ion. From the corrected product ratio data, the lifetime of the oxocarbenium ion was estimated to be ≥3 × 10-11 s. Solvolysis of CMP-NeuAc at pL = 5.0 afforded an observed solvent deuterium isotope effect (SDIE) kH2O/kD2O = 0.45, consistent with specific acid catalysis of glycosidic bond cleavage. A SDIE of 0.66 for the apparent bimolecular azide trapping pathway was also observed. An apparent isotope effect of ∼1.1 for trapping of the N-acetyl neuraminyl oxocarbenium ion by water was determined by product analysis of azide trapping in H2O and D2O. An ab initio transition state for attack of water on an N-acetyl neuraminyl oxocarbenium ion model was located which featured a hydrogen bond between the oxocarbenium ion carboxylate and water; proton transfer was not part of the reaction coordinate. It is proposed that the N-acetyl neuraminate carboxylate group stabilizes an intermediate oxocarbenium ion, but the barrier for capture by water is lowered by a transition state hydrogen bond.

The Ratio between Endocyclic and Exocyclic Cleavage of Pyranoside Acetals Is Dependent upon the Anomer, the Temperature, the Aglycon Group, and the Solvent

Several cis-fused decalin pyranosides with intramolecular nucleophiles of high effective molarity were studied to determine the ratio between endocyclic and exocyclic cleavage in specific-acid-catalyzed solvolysis reactions. The molecular design that allows a differentiation between endo- or exocyclic cleavage is the symmetry and asymmetry of the respective oxocarbenium ion intermediates. The synthesis of the molecular probes involves eight steps from a known compound, and proceeds via a key intermediate functionalized with three different oxidation states. A crystal structure confirmed the relative stereochemistry of the probes. A quantifiable percentage of endocyclic cleavage for β-pyranosides was found for all reaction conditions, whereas α-pyranosides show exclusively exocyclic cleavage. The percent of endocyclic cleavage for β-pyranosides is dependent upon the temperature, the aglycon group, and the solvent. At lower temperatures endocyclic cleavage increases. The ΔH⧧ and ΔS⧧ for endocyclic and exocy...

First Nonenzymatic Synthesis of Kdo8P through a Mechanism Similar to That Suggested for the Enzyme Kdo8P Synthase

The mechanism of Kdo8P synthase, the enzyme that catalyzes the unusual condensation of d-arabinose 5-phosphate (A5P) with phosphoenolpyruvate (PEP) to form Kdo8P, remains a fascinating subject for bioorganic research. This paper describes the synthesis of two intramolecular models (1 and 2) bearing an enolpyruvate moiety at C-3 of the arabinose fraction. This means that their open-chain aldehyde forms closely mimic the proposed situation, whereby two substrates A5P and PEP evolve into a ternary complex with the synthase. Examination of 1 (in organic solvent) and 2 (in a water solution) under Lewis acid conditions establishes that they both undergo highly stereospecific intramolecular condensation of the enolpyruvate double bond with the carbonyl of sugar. This results in the required Kdo structure possessing the desired stereochemistry. Mechanistic studies suggest that the observed intramolecular condensation process takes place via a stepwise mechanism involving the formation of a transient oxocarbenium ion intermediate. The results obtained, uniquely demonstrate enzyme-like chemistry in the stereospecific synthesis of the Kdo system. Further investigation is certainly warranted, in order to facilitate the construction of other 3-deoxy-2-ulosonoc acids and sialic acids on the basis of the same general model. This is illustrated here in the case of Kdo. Furthermore, the results support the validity of the mechanism suggested for the Kdo8P synthase action, in particular, the possible role of the enzyme in the catalysis of the initial condensation step.

24] Preparation of cyclic ADP-ribose, 2′-phospho-cyclic ADP-ribose, and nicotinate adenine dinucleotide phosphate: Possible second messengers of calcium signaling

The metabolism of cyclic ADP-ribose (cADPR) and 2'-phospho-cADPR (P-cADPR) in mammals is apparently achieved by enzymes known as nicotinate adenine dinucleotide phosphate (NADP) glycohydrolases. NAD(P)ases catalyze the release of nicotinamide from NAD or NADP with the formation of an enzyme-bound oxocarbenium ion intermediate. This intermediate has three possible fates—(1) it can react via an intermolecular reaction with nicotinamide to regenerate the original dinucleotide or with a variety of other nucleophiles with retention of configuration to form analogs of either NAD or NADP; (2) it can undergo an intramolecular reaction to form cADPR or P-cADPR; or (3) it can react with water to form ADPR or P-ADPR. The methodology described in the chapter uses an NAD(P)ase from the marine mollusk Aplysia californica . The strategy described in the chapter achieves the synthesis of cyclic nucleotides by simple passage of NAD, NADP, or a structural analog through a column of immobilized ADPR cyclase. Conversely, to prepare NAD or NADP analogs, cADPR or P-cADPR can be mixed with the nucleophile of choice and passed through a column of immobilized enzyme. Because the approach involves few reactants and products, purification of the products can be achieved easily.

Synthesis of Alkyl Fructosides Using Solid Acid Catalysts. Part I: Silica-Alumina Cracking Catalysts

Abstract Silica-alumina cracking catalysts and acid clays efficiently catalyze the 2-O-alkylation of d-fructose with long chain alcohols. Under the conditions applied virtually no degradation of fructose is observed. l-Sorbose and the aldopentoses also undergo silica-alumina-catalyzed alkylation. The rate of conversion is related to the solubility of the monosaccharide and the stability of the intermediate oxocarbenium ion. Best results in fructose alkylation are obtained by applying a recirculation method with butyl fructoside as soluble intermediate.

Highly Diastereoselective Intramolecular Allylation Reactions of Mixed Silyl-Substituted Acetals

The reaction of preformed mixed acetals derived from (α-hydroxyalkyl)dimethylallylsilane with a number of aromatic and aliphatic aldehydes in the presence of Lewis acids results in a highly diastereoselective intramolecular allylation reaction. The reaction proceeds through a cyclic synclinal SE‘ addition of the allylsilane to an intermediate oxocarbenium ion. The reaction occurs exclusively by an intramolecular process as determined by means of a cross-over experiment. The relative stereochemistry was determined by the conversion of one of the allylation products to a known (stereodefined) aldol-type product. A greater degree of diastereoselectivity is obtained by in-situ formation of an oxocarbenium ion from (α-hydroxyhexyl)dimethylallylsilane and an aldehyde in the presence of boron trifluoride etherate. The diastereoselectivity of the in-situ allylation reaction typically exceeds 100:1 in favor of the syn adduct. However, reactions with electron rich aryl aldehydes resulted in a diminished degree of d...

Chemoselective Protection of Aldehydes as Dithioacetals in Lithium Perchlorate-Diethyl Ether Medium. Evidence for the Formation of Oxocarbenium Ion Intermediate from Acetals.

Aldehydes and acetals were very efficiently converted to acyclic and cyclic dithioacetals in 5 M lithium perchlorate/diethyl ether (LPDE) medium at ambient temperature in high yields. Spectroscopic and other experimental evidences strongly suggest the formation of oxocarbenium ion intermediates from acetals in 5 M LPDE which subsequently reacted with thiols to give the dithioacetals. Under the same conditions keto- nes and their acetals also reacted, albeit very slowly compared to al- dehydes and acetals, to yield dithioacetals. The difference in their reactivity was successfully employed in the chemoselective dithioace- talization of aldehydes and acetals in the presence of ketones and their acetals

Additional evidence for the exceptional mechanism of the acid-catalysed hydrolysis of 4-oxopyrimidine nucleosides: hydrolysis of 1-(1-alkoxyalkyl)uracils, seconucleosides, 3′-C-alkyl nucleosides and nucleoside 3′,5′-cyclic monophosphates

The rate constants for the acid-catalysed hydrolysis of 1-(1 -alkoxyethyl)uracils and 1-alkoxymethyluracils have been determined. With both series of compounds, the hydrolysis rate is rather insensitive to the polar nature of the alkoxy group, in striking contrast with the hydrolysis of the corresponding analogues of adenine and cytosine nucleosides, which react via rate-limiting formation of an oxocarbenium ion intermediate. Furthermore, it has been shown that the 3′,5′-cyclic monophosphates of thymidine and uridine undergo the hydrolysis of the N-glycosidic bond 760 and 260 times as fast as their parent nucleosides, while the cyclic monophosphates of 2′-deoxyadenosine and adenosine are depurinated much more slowly than the corresponding nucleosides. On this basis it is suggested that 4-oxopyrimidine nucleosides are hydrolysed by opening of the sugar ring. To obtain further evidence for this exceptional mechanism, comparative kinetic measurements with some seco- and 3′-C-alkyl nucleosides of uracil and adenine have been carried out.

Mechanisms for the solvolytic decompositions of nucleoside analogues—VIII: Acidic Hydrolysis of 5-substituted 1-(alkoxyethyl)cytosines and cytidines

Several 5-substituted 1-(l-alkoxyethyl)cytosines have been prepared and the rate constants for their hydrolysis determined at various concentrations of oxonium ion. The acidity constants for the monoprotonated substrates and the rate constants for their decomposition have been calculated from the pH-rate profiles obtained. The effecs that varying the polar nature of the l-alkoxyethyl group exerts on the heterolysis of the monoprotonated substrates are interpreted to indicate that the acidic hydrolysis of l-(l-alkoxyethyl)cytosines proceeds by rate-limiting departure of the protonated base moiety with formation of an oxocarbenium ion intermediate. The same mechanism is extended to the hydrolysis of cytidines by comparing the influences that the 5-substituents have on the heterolysis of protonated 5-substituted l-(l-ethocyethyl)cytosines and correspondingly substituted cytidines.