SURVEY AND SUMMARY: Recent advances in the elucidation of the mechanisms of action of ribozymes

The cleavage of RNA can be accelerated by a number of factors. These factors include an acidic group (Lewis acid) or a basic group that aids in the deprotonation of the attacking nucleophile, in effect enhancing the nucleophilicity of the nucleophile; an acidic group that can neutralize and stabilize the leaving group; and any environment that can stabilize the pentavalent species that is either a transition state or a short-lived intermediate. The catalytic properties of ribozymes are due to factors that are derived from the complicated and specific structure of the ribozyme–substrate complex. It was postulated initially that nature had adopted a rather narrowly defined mechanism for the cleavage of RNA. However, recent findings have clearly demonstrated the diversity of the mechanisms of ribozyme-catalyzed reactions. Such mechanisms include the metal-independent cleavage that occurs in reactions catalyzed by hairpin ribozymes and the general double-metal-ion mechanism of catalysis in reactions catalyzed by the Tetrahymena group I ribozyme. Furthermore, the architecture of the complex between the substrate and the hepatitis delta virus ribozyme allows perturbation of the pKa of ring nitrogens of cytosine and adenine. The resultant perturbed ring nitrogens appear to be directly involved in acid/base catalysis. Moreover, while high concentrations of monovalent metal ions or polyamines can facilitate cleavage by hammerhead ribozymes, divalent metal ions are the most effective acid/base catalysts under physiological conditions.

COOH-terminal processing of nascent polypeptides by the glycosylphosphatidylinositol transamidase in the presence of hydrazine is governed by the same parameters as glycosylphosphatidylinositol addition.

Proteins anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) moiety are found in all eukaryotes. After NH2-terminal peptide cleavage of the nascent protein by the signal peptidase, a second COOH-terminal signal peptide is cleaved with the concomitant addition of the GPI unit. The proposed mechanism of the GPI transfer is a transamidation reaction that involves the formation of an activated carbonyl intermediate (enzyme-substrate complex) with the ethanolamine moiety of the preassembled GPI unit serving as a nucleophile. Other nucleophilic acceptors like hydrazine (HDZ) and hydroxylamine have been shown to be possible alternate substrates for GPI. Since GPI has yet to be purified, the use of readily available nucleophilic substitutes such as HDZ and hydroxylamine is a viable alternative to study COOH-terminal processing by the putative transamidase. As a first step in developing a soluble system to study this process, we have examined the amino acid requirements at the COOH terminus for the transamidation reaction using HDZ as the nucleophilic acceptor instead of GPI. The hydrazide-forming reaction shows identical amino acid requirement profiles to that of GPI anchor addition. Additionally, we have studied other parameters relating to the kinetics of the transamidation reaction in the context of rough microsomal membranes. The findings with HDZ provide further evidence for the transamidase nature of the enzyme and also provide a starting point for development of a soluble assay.

Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases.

The regions surrounding the catalytic amino acids previously identified in a few "retaining" O-glycosyl hydrolases (EC 3.2.1) have been analyzed by hydrophobic cluster analysis and have been used to define sequence motifs. These motifs have been found in more than 150 glycosyl hydrolase sequences representing at least eight established protein families that act on a large variety of substrates. This allows the localization and the precise role of the catalytic residues (nucleophile and acid catalyst) to be predicted for each of these enzymes, including several lysosomal glycosidases. An identical arrangement of the catalytic nucleophile was also found for S-glycosyl hydrolases (myrosinases; EC 3.2.3.1) for which the acid catalyst is lacking. A (beta/alpha)8 barrel structure has been reported for two of the eight families of proteins that have been grouped. It is suggested that the six other families also share this fold at their catalytic domain. These enzymes illustrate how evolutionary events led to a wide diversification of substrate specificity with a similar disposition of identical catalytic residues onto the same ancestral (beta/alpha)8 barrel structure.

Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinity.

We have inserted a fourth protein ligand into the zinc coordination polyhedron of carbonic anhydrase II (CAII) that increases metal affinity 200-fold (Kd = 20 fM). The three-dimensional structures of threonine-199-->aspartate (T199D) and threonine-199-->glutamate (T199E) CAIIs, determined by x-ray crystallographic methods to resolutions of 2.35 Angstrum and 2.2 Angstrum, respectively, reveal a tetrahedral metal-binding site consisting of H94, H96, H119, and the engineered carboxylate side chain, which displaces zinc-bound hydroxide. Although the stereochemistry of neither engineered carboxylate-zinc interaction is comparable to that found in naturally occurring protein zinc-binding sites, protein-zinc affinity is enhanced in T199E CAII demonstrating that ligand-metal separation is a significant determinant of carboxylate-zinc affinity. In contrast, the three-dimensional structure of threonine-199-->histidine (T199H) CAII, determined to 2.25-Angstrum resolution, indicates that the engineered imidazole side chain rotates away from the metal and does not coordinate to zinc; this results in a weaker zinc-binding site. All three of these substitutions nearly obliterate CO2 hydrase activity, consistent with the role of zinc-bound hydroxide as catalytic nucleophile. The engineering of an additional protein ligand represents a general approach for increasing protein-metal affinity if the side chain can adopt a reasonable conformation and achieve inner-sphere zinc coordination. Moreover, this structure-assisted design approach may be effective in the development of high-sensitivity metal ion biosensors.

Solvent-Free Enantioselective Organocatalyzed Aldol Reactions

The use of proline as catalyst for the aldol process has given a boost to the development of organocatalysis as a research area. Since then, a plethora of organocatalysts of diverse structures have been developed for this and other organic transformations under different reaction conditions. The use of an organic molecule as catalyst to promote a reaction meets several principles of Green Chemistry. The implementation of solvent-free methodologies to carry out the aldol reaction was soon envisaged. These solvent-free processes can be performed using conventional magnetic stirring or applying ball milling techniques and are even compatible with the use of supported organocatalysts as promoters, which allows the recovery and reuse of the organocatalysts. In addition, other advantages such as the reduction of the required amount of nucleophile and the acceleration of the reaction are accomplished by using solvent-free conditions leading to a “greener” and more sustainable process.

Inhibition studies of purple acid phosphatases: Implications for the catalytic mechanism

Purple acid phosphatases (PAPs) belong to the family of binuclear metallohydrolases and catalyse the hydrolysis of a large group of phosphoester substrates at acidic pH. Despite structural conservation in their active sites PAPs appear to display mechanistic versatility. Here, aspects of the catalytic mechanism of two PAPs are investigated using the inhibitors vanadate and fluoride as probes. While the magnitude of their vanadate inhibition constants are similar the two enzymes differ with respect to the mode of inhibition; vanadate interacts in a non-competitive fashion with pig PAP (K-i = 40 mu mol L-1) while it inhibits red kidney bean PAP competitively (K-i = 30 mu mol L-1). Similarly, fluoride also acts as a competitive inhibitor for red kidney bean PAP, independent of pH, while the inhibition of pig PAP by fluoride is uncompetitive at low pH and non-competitive at higher pH, independent of metal ion composition. Furthermore, while fluoride acts as a slow-binding inhibitor in pig PAP it binds rapidly to the catalytic site of the red kidney bean enzyme. Since vanadate and fluoride are proposed to act as transition state and nucleophile mimics, respectively, the observed differences in inhibition kinetics indicate subtle but distinct variations in the reaction mechanism of these enzymes.

Aryliminopropadienone-C-amidoketenimine-amidinoketene-2-aminoquinolone cascades and the ynamine-isocyanate reaction

Imidoylketenes 11 and oxoketenimines 12 are generated by flash vacuum thermolysis of Meldrum's acid derivatives 9, pyrrolediones 17 and 18, and triazole 19 and are observed by IR spectroscopy. Ketenimine-3-carboxylic acid esters 12a are isolable at room temperature. Ketenes 11 and ketenimines 12 undergo rapid interconversion in the gas phase, and the ketenes cyclize to 4-quinolones 13. When using an amine leaving group in Meldrum's acid derivatives 9c, the major reaction products are aryliminopropadienones, ArN=C=C=C=O (15). The latter react with 1 equiv of nucleophile to produce ketenimines 12 and with 2 equiv to afford maIonic acid imide derivatives 16. N-Arylketenimine-C-carboxamides 12c cyclize to quinolones 13c via the transient amidinoketenes 11c at temperatures of 25-40 degrees C. This implies rapid interconversion of ketenes and ketenimines by a 1,3-shift of the dimethylamino group, even at room temperature. This interconversion explains previously poorly understood outcomes of the ynamine-isocyanate reaction. The solvent dependence of the tautomerism of 4-quinolones/4-quinolinols is discussed. Rotational barriers of NMe2 groups in amidoketenimines 12c and malonioc amides and amidines 16 (24) are reported.

The metabolism of n-methyl compounds

The metabolism of compounds containing the N-methyl group is discussed with particular consideration being made to the possible role of the product of oxidative metabolism, the N-hydroxymethyl moiety, in the generation of potentially toxic, reactive electrophiles. Particular pathways which are considered are: (i), the production of formaldehyde; (ii), the generation of iminium ions or imines; and (iii), the formation of N-formyl compounds which might act as formylating agents. 4-Chloro-N-(hydroxymethyl)benzamide and 3-(4-chlorophenyl)-1-hydroxy-methyl-1-methylurea (the product of oxidative metabolism of 3-(4-chlorophenyl)-1,1-dimethylurea) are model carbinolamides which do not readily release formaldehyde. The electrophilic properties of these model carbinolamides were investigated: neither reacted with nucleophiles such as cyanide or glutathione under physiological conditions. In contrast, N-(acetoxymethyl)-4-chlorobenzamide yielded the cyanomethylamide with potassium cyanide and S-(4-chlorobenzamidomethyl)glutathione with glutathione. 4-Chloro-N-(hydroxymethyl)benzamide and 3-(4-chlorophenyl)-1,1-dimethylurea were not biotransformed to electrophilic moieties when incubated with mouse hepatic 9000 x g supernatant and Acetyl-CoA or PAPS-generating system. N-(Acetoxymethyl)-4-chlorobenzamide was non-mutagenic to Salmonella typhimurium in the short term bacterial assay; but toxicity to the bacteria was observed. 4-Chloro-N-(hydroxymethyl)benzamide and 3-(4-chlorophenyl)-1,1-dimethylurea showed no mutagenicity or toxicity in the mutagenicity assay including an Aroclor-induced rat hepatic 9000 x g supernatant. Addition of Acetyl-CoA or a PAPS-generating system did not produce a mutagenic response. 4-Chloro-N-formlbenzamide did not act as a formylating agent towards the weak nucleophile aniline. However, 4-chloro-N-formylbenzamide, N-formylbenzamide, 3-(4-chlorophenyl)-1-formyl-1-methylurea and 3-(4-chlorophenyl)-1-formylurea are all metabolised by mouse hepatic mirosomes and post-microsomal supernatant. The results demonstrate the potential for N-hydroxymethyl compounds to generate highly reactive species if these are substrates for conjugation with sulphate (or acetate). The model compounds employed here, apparently do not show any ability to be conjugated themselves, however, other N-hydroxymethyl compounds might be readily conjugated. The formation of N-formyl compounds does not appear to be toxicologically significant, as adjudged on limited experiments performed, but rather represent a detoxification pathway.

Residue-specific investigation of the relationship between oxidation and activity of a dual specificity phosphatase by mass spectrometry

Redox regulation of signalling pathways is critical in proliferation and apoptosis; redox imbalance can lead to pathologies such as inflammation and cancer. Vaccinia H1-related protein (VHR; DUSP3) is a dual-specificity phosphatase important in controlling MAP kinase activity during cell cycle. the active-site motif contains a cysteine that acts as a nucleophile during catalysis. We used VHR to investigate the effect of oxidation in vitro on phosphatase activity, with the aim of determining how the profile of site-specific modification related to catalytic activity. Recombinant human VHR was expressed in E. coli and purified using a GST-tag. Protein was subjected to oxidation with various concentrations of SIN-1 or tetranitromethane (TNM) as nitrating agents, or HOCl. the activity was assayed using either 3-O-methylfluorescein phosphate with fluorescence detection or PIP3 by phosphate release with malachite green. the sites of oxidation were mapped using HPLC coupled to tandem mass spectrometry on an ABSciex 5600TripleTOF following in-gel digestion. More than 25 different concentration-dependent oxidative modifications to the protein were detected, including oxidations of methionine, cysteine, histidine, lysine, proline and tyrosine, and the % oxidized peptide (versus unmodified peptide) was determined from the extracted ion chromatograms. Unsurprisingly, methionine residues were very susceptible to oxidation, but there was a significant different in the extent of their oxidation. Similarly, tyrosine residues varied greatly in their modifications: Y85 and Y138 were readily nitrated, whereas Y38, Y78 and Y101 showed little modification. Y138 must be phosphorylated for MAPK phosphatase activity, so this susceptibility impacts on signalling pathways. Di- and tri- oxidations of cysteine residues were observed, but did not correlate directly with loss of activity. Overall, the catalytic activity did not correlate with redox state of any individual residue, but the total oxidative load correlated with treatment concentration and activity. This study provides the first comprehensive analysis of oxidation modifications of VHR, and demonstrates both heterogenous oxidant effects and differential residue susceptibility in a signalling phosphatase.

Intrinsic gas-phase electrophilic reactivity of Ccclic N-alkyl- and N-acyliminium ions

he intrinsic gas-phase reactivity of cyclic N-alkyl- and N-acyliminium ions toward addition of allyltrimethylsilane (ATMS) has been compared using MS2 and MS3 pentaquadrupole mass spectrometric experiments. An order of electrophilic reactivity has been derived and found to agree with orders of overall reactivity in solution. The prototype five-membered ring N-alkyliminium ion 1a and its N-CH3 analogue 1b, as well as their six-membered ring analogues 1c and 1d, lack N-acyl activation and they are, accordingly, inert toward ATMS addition. The five- and six-membered ring N-acyliminium ions with N-COCH3 exocycclic groups, 3a and 3b, respectively, are also not very reactive. The N-acyliminium ions 2a and 2c, with s-trans locked endocyclic N-carbonyl groups, are the most reactive followed closely by 3c and 3d with exocyclic (and unlocked) N-CO2CH3 groups. The five-membered ring N-acyliminium ions are more reactive than their six-membered ring analogues, that is:  2a > 2c and 3c > 3d. In contrast with the high reactivity of 2a, its N-CH3 analogue 2b is inert toward ATMS addition. For the first time, the transient intermediates of a Mannich-type condensation reaction were isolatedthe β-silyl cations formed by ATMS addition to N-acyliminium ionsand their intrinsic gas-phase behavior toward dissociation and reaction with a nucleophile investigated. When collisionally activated, the β-silyl cations dissociate preferentially by Grob fragmentation, that is, by retro-addition. With pyridine, they react competitively and to variable extents by proton transfer and by trimethylsilylium ion abstractionthe final and key step postulated for α-amidoalkylation. Becke3LYP/6-311G(d,p) reaction energetics, charge densities on the electrophilic C-2 site, and AM1 LUMO energies have been used to rationalize the order of intrinsic gas-phase electrophilic reactivity of cyclic iminium and N-acyliminium ions.

Diversity in Catalytic Reactions of Propargylic Diazoesters

Abstract Title of Document: Diversity in Catalytic Reactions of Propargylic Diazoesters Huang Qiu, Doctor of Philosophy, 2016 Directed By: Professor Michael P. Doyle, Department of Chemistry and Biochemistry Propargylic aryldiazoesters, which possess multiple reactive functional groups in a single molecule, were expected to undergo divergent reaction pathways as a function of catalysts. A variety of transition metal complexes including rhodium(II), palladium(II), silver(I), mercury(II), copper(I and II), and cationic gold (I) complexes have been examined to be effective in the catalytic domino reactions of propargylic aryldiazoesters. An unexpected Lewis acid catalyzed pathway was also discovered by using FeCl3 as the catalyst. Under the catalysis of selected gold catalysts, propargylic aryldiazoesters exist in equilibrium with 1-aryl-1,2-dien-1-yl diazoacetate allenes that are rapidly formed at room temperature through 1,3-acyloxy migration. The newly formed allenes further undergo a metal-free rearrangement in which the terminal nitrogen of the diazo functional group adds to the central carbon of the allene initiating a sequence of bond forming reactions resulting in the production of 1,5-dihydro-4H-pyrazol-4-ones in good yields. These 1,5-dihydro-4H-pyrazol-4-ones undergo intramolecular 1,3-acyl migration to form an equilibrium mixture or quantitatively transfer the acyl group to an external nucleophile with formation of 4-hydroxypyrazoles. In the presence of a pyridine-N-oxide, both E- and Z-1,3-dienyl aryldiazoacetates are formed in high combined yields by Au(I)-catalyzed rearrangement of propargyl arylyldiazoacetates at short reaction times. Under thermal reactions the E-isomers form the products from intramolecular [4+2]-cycloaddition with H‡298 = 15.6 kcal/mol and S‡298= -27.3 cal/ (mol•degree). The Z-isomer is inert to [4+2]-cycloaddition under these conditions. The Hammett relationships from aryl-substituted diazo esters ( = +0.89) and aryl-substituted dienes ( = -1.65) are consistent with the dipolar nature of this transformation. An unexpected reaction for the synthesis of seven-membered conjugated 1,4-diketones from propargylic diazoesters with unsaturated imines was disclosed. To undergo this process vinyl gold carbene intermediates generated by 1,2-acyloxy migration of propargylic aryldiazoesters undergo a formal [4+3]-cycloaddition, and the resulting aryldiazoesters tethered dihydroazepines undergo an intricate metal-free process to form observed seven-membered conjugated 1,4-diketones with moderate to high yields.