Atom Isotope Effects Research Articles

Incorporation of deuterated coronene into cage-like sodalite-type porous organic salts and improvement of room-temperature phosphorescence properties

Abstract Host materials with external heavy atom effects do not change the chemical structures of incorporated luminescent molecules but promote intersystem crossing from the excited singlet state to the excited triplet state, which induces room-temperature phosphorescence (RTP). The deuteration of luminescent molecules suppresses non-radiative deactivation via C–H stretching vibration; therefore, the improvement of both phosphorescence lifetime and quantum efficiency (i.e. isotope effect) is expected. Although a combination of the external heavy atom effect and isotope effect could be expected to improve phosphorescent performances dramatically, an environment with a strong external heavy atom effect (density of iodine atoms ≥0.65 gcm−3) increases non-radiative deactivation via spin-orbit coupling; therefore, the isotope effect is hindered, and the phosphorescent lifetime and quantum efficiency are not usually improved. In the current work, we constructed cage-like sodalite-type porous organic salts (s-POSs) where the density of iodine atoms (0.55 gcm−3) was moderate (0.13 ̶ 0.65 gcm−3). Incorporation of a deuterated representative luminescent molecule such as coronene (coronene-d12) into s-POSs enabled the exerting of both the external heavy atom effect and isotope effect, which successfully improved both RTP lifetime (1.1 times) and quantum efficiency (1.6 times) over those of an incorporated ordinary coronene (coronene-h12).

A general and efficient approach to synthesize the phosphoramidites of 5′-18 O labeled purine nucleosides

5′-18 O labeled RNA oligos are important probes to investigate the mechanism of 2′-O-transphosphorylation reactions. Here we describe a general and efficient synthetic approach to the phosphoramidite derivatives of 5′-18 O labeled nucleosides starting from the corresponding commercially available 5′-O-DMT protected nucleosides. Using this method, we prepared 5′-18 O-guanosine phosphoramidite in 8 steps (13.2% overall yield), 5′-18 O-adenosine phosphoramidite in 9 steps (10.1% overall yield) and 5′-18 O-2′-deoxyguanosine phosphoramidite in 6 steps (12.8% overall yield). These 5′-18 O labeled phosphoramidites can be incorporated into RNA oligos by solid phase synthesis for determination of heavy atom isotope effects in RNA 2′-O-transphosphorylation reactions.

Structural Modifications as Tools in Mechanistic Studies of the Cleavage of RNA Phosphodiester Linkages.

The cleavage of RNA phosphodiester bonds by RNase A and hammerhead ribozyme at neutral pH fundamentally differs from the spontaneous reactions of these bonds under the same conditions. While the predominant spontaneous reaction is isomerization of the 3',5'-phosphodiester linkages to their 2',5'-counterparts, this reaction has never been reported to compete with the enzymatic cleavage reaction, not even as a minor side reaction. Comparative kinetic measurements with structurally modified di-nucleoside monophosphates and oligomeric phosphodiesters have played an important role in clarification of mechanistic details of the buffer-independent and buffer-catalyzed reactions. More recently, heavy atom isotope effects and theoretical calculations have refined the picture. The primary aim of all these studies has been to form a solid basis for mechanistic analyses of the action of more complicated catalytic machineries. In other words, to contribute to conception of a plausible unified picture of RNA cleavage by biocatalysts, such as RNAse A, hammerhead ribozyme and DNAzymes. In addition, structurally modified trinucleoside monophosphates as transition state models for Group I and II introns have clarified some features of the action of large ribozymes.

Mechanistic Aspects of Phosphate Diester Cleavage Assisted by Imidazole. A Template Reaction for Obtaining Aryl Phosphoimidazoles.

Phosphoimidazole-containing compounds are versatile players in biological and chemical processes. We explore catalytic and mechanistic criteria for the efficient formation of cyclic aryl phosphoimidazoles in aqueous solution, viewed as a template reaction for the in situ synthesis of related compounds. To provide a detailed analysis for this reaction a series of o-(2'-imidazolyl)naphthyl (4-nitrophenyl) phosphate isomers were examined to provide a basis for analysis of both mechanism and the influence of structural factors affecting the nucleophilic attack of the imidazolyl group on the phosphorus center of the substrate. Formation of the cyclic aryl phosphoimidazoles was probed by NMR and ESI-MS techniques. Kinetic experiments show that cyclization is faster under alkaline conditions, with an effective molarity up to 2900 M for the imidazolyl group, ruling out competition from external nucleophiles. Heavy atom isotope effect and computational studies show that the reaction occurs through a SN2(P)-type mechanism involving a pentacoordinated phosphorus TS, with apical positions occupied by the incoming imidazolyl nucleophile and the p-nitrophenolate leaving group. The P-O bond to the leaving group is about 50-60% broken in the transition state.

W. W. “Mo” Cleland: A Catalytic Life

Professor W. Wallace Cleland, the architect of modern steady-state enzyme kinetics, died on March 6, 2013, from injuries sustained in a fall outside of his home. He will be most remembered for giving the enzyme community Ping-Pong kinetics and the invention of dithiothreitol (DTT). He pioneered the utilization of heavy atom isotope effects for the elucidation of the chemical mechanisms of enzyme-catalyzed reactions. His favorite research journal was Biochemistry, in which he published more than 135 papers beginning in 1964 with the disclosure of DTT.

Oxygen Isotope Effects as Probes of Electron Transfer Mechanisms and Structures of Activated O2

Competitively determined oxygen ((18)O) isotope effects can be powerful probes of chemical and biological transformations involving molecular oxygen as well as superoxide and hydrogen peroxide. They play a complementary role to crystallography and spectroscopy in the study of activated oxygen intermediates by forging a link between electronic/vibrational structure and the bonding that occurs within ground and transition states along the reaction coordinate. Such analyses can be used to assess the plausibility of intermediates and their catalytic relevance in oxidative processes. This Account describes efforts to advance oxygen kinetic isotope effects ((18)O KIEs) and equilibrium isotope effects ((18)O EIEs) as mechanistic probes of reactive, oxygen-derived species. We focus primarily on transition metal mediated oxidations, outlining both advances over the past five years and current limitations of this approach. Computational methods are now being developed to probe transition states and the accompanying kinetic isotope effects. In particular, we describe the importance of using a full-frequency model to accurately predict the magnitudes as well as the temperature dependence of the isotope effects. Earlier studies have used a "cut-off model," which employs only a few isotopic vibrational modes, and such models tend to overestimate (18)O EIEs. Researchers in mechanistic biological inorganic chemistry would like to differentiate "inner-sphere" from "outer-sphere" reactivity of O(2), a designation that describes the extent of the bonding interaction between metal and oxygen in the transition state. Though this problem remains unsolved, we expect that this isotopic approach will help differentiate these processes. For example, comparisons of (18)O KIEs to (18)O EIEs provide benchmarks that allow us to calibrate computationally derived reaction coordinates. Once the physical origins of heavy atom isotope effects are better understood, researchers will be able to apply the competitive isotope fractionation technique to a wide range of pressing problems in small molecule chemistry and biochemistry.

Energy transfer of highly vibrationally excited naphthalene. II. Vibrational energy dependence and isotope and mass effects

The vibrational energy dependence, H and D atom isotope effects, and the mass effects in the energy transfer between rare gas atoms and highly vibrationally excited naphthalene in the triplet state were investigated using crossed-beam/time-sliced velocity-map ion imaging at various translational collision energies. Increase of vibrational energy from 16 194 to 18 922 cm(-1) does not make a significant difference in energy transfer. The energy transfer properties also remain the same when H atoms in naphthalene are replaced by D atoms, indicating that the high vibrational frequency modes do not play important roles in energy transfer. They are not important in supercollisions either. However, as the Kr atoms are replaced by Xe atoms, the shapes of energy transfer probability density functions change. The probabilities for large translation to vibration/rotation energy transfer (T-->VR) and large vibration to translation energy transfer (V-->T) decrease. High energy tails in the backward scatterings disappear, and the probability for very large vibration to translation energy transfer such as supercollisions also decreases.

Efficient Synthesis of [2‘-18O]Uridine and Its Incorporation into Oligonucleotides: A New Tool for Mechanistic Study of Nucleotidyl Transfer Reactions by Isotope Effect Analysis

Lack of sufficient quantities of isotopically labeled materials has precluded the use of heavy atom isotope effects to investigate mechanisms of nucleotidyl transfer reactions in nucleic acids. Here we achieve regioselective opening of 2,2'-cyclouridine with [(18)O2]benzoic acid/potassium hydride, allowing an efficient "one-pot" synthesis of [2'-18O]uridine in 88% yield. Conversion to the corresponding phosphoramidite enables solid-phase synthesis of [2'-(18)O] RNA substrates for isotope effect studies with nucleotidyl transferases and hydrolases.

Investigating the roles of putative active site residues in the oxalate decarboxylase from Bacillus subtilis

Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into CO 2 and formate using a catalytic mechanism that remains poorly understood. The Bacillus subtilis enzyme is composed of two cupin domains, each of which contains Mn(II) coordinated by four conserved residues. We have measured heavy atom isotope effects for a series of Bacillus subtilis OxDC mutants in which Arg-92, Arg-270, Glu-162, and Glu-333 are conservatively substituted in an effort to define the functional roles of these residues. This strategy has the advantage that observed isotope effects report directly on OxDC molecules in which the active site manganese center(s) is (are) catalytically active. Our results support the proposal that the N-terminal Mn-binding site can mediate catalysis, and confirm the importance of Arg-92 in catalytic activity. On the other hand, substitution of Arg-270 and Glu-333 affects both Mn(II) incorporation and the ability of Mn to bind to the OxDC mutants, thereby precluding any definitive assessment of whether the metal center in the C-terminal domain can also mediate catalysis. New evidence for the importance of Glu-162 in controlling metal reactivity has been provided by the unexpected observation that the E162Q OxDC mutant exhibits a significantly increased oxalate oxidase and a concomitant reduction in decarboxylase activities relative to wild type OxDC. Hence the reaction specificity of a catalytically active Mn center in OxDC can be perturbed by relatively small changes in local protein environment, in agreement with a proposal based on prior computational studies.

Analysis of solvent nucleophile isotope effects: evidence for concerted mechanisms and nucleophilic activation by metal coordination in nonenzymatic and ribozyme-catalyzed phosphodiester hydrolysis.

Heavy atom isotope effects are a valuable tool for probing chemical and enzymatic reaction mechanisms; yet, they are not widely applied to examine mechanisms of nucleophilic activation. We developed approaches for analyzing solvent (18)O nucleophile isotope effects ((18)k(nuc)) that allow, for the first time, their application to hydrolysis reactions of nucleotides and nucleic acids. Here, we report (18)k(nuc) for phosphodiester hydrolysis catalyzed by Mg(2+) and by the Mg(2+)-dependent RNase P ribozyme and deamination by the Zn(2+)-dependent protein enzyme adenosine deaminase (ADA). Because ADA incorporates a single solvent molecule into the product inosine, this reaction can be used to monitor solvent (18)O/(16)O ratios in complex reaction mixtures. This approach, combined with new methods for analysis of isotope ratios of nucleotide phosphates by whole molecule mass spectrometry, permitted determination of (18)k(nuc) for hydrolysis of thymidine 5'-p-nitrophenyl phosphate and RNA cleavage by the RNase P ribozyme. For ADA, an inverse (18)k(nuc) of 0.986 +/- 0.001 is observed, reflecting coordination of the nucleophile by an active site Zn(2+) ion and a stepwise mechanism. In contrast, the observed (18)k(nuc) for phosphodiester reactions were normal: 1.027 +/- 0.013 and 1.030 +/- 0.012 for the Mg(2+)- and ribozyme-catalyzed reactions, respectively. Such normal effects indicate that nucleophilic attack occurs in the rate-limiting step for these reactions, consistent with concerted mechanisms. However, these magnitudes are significantly less than the (18)k(nuc) observed for nucleophilic attack by hydroxide (1.068 +/- 0.007), indicating a "stiffer" bonding environment for the nucleophile in the transition state. Kinetic analysis of the Mg(2+)-catalyzed reaction indicates that a Mg(2+)-hydroxide complex is the catalytic species; thus, the lower (18)k(nuc), in large part, reflects direct metal ion coordination of the nucleophilic oxygen. A similar value for the RNase P ribozyme catalyzed reaction provides support for nucleophilic activation by metal ion catalysis.

Understanding the transition states of phosphodiester bond cleavage: insights from heavy atom isotope effects.

The nucleotides of DNA and RNA are joined by phosphodiester linkages whose synthesis and hydrolysis are catalyzed by numerous essential enzymes. Two prominent mechanisms have been proposed for RNA and protein enzyme catalyzed cleavage of phosphodiester bonds in RNA: (a) intramolecular nucleophilic attack by the 2'-hydroxyl group adjacent to the reactive phosphate; and (b) intermolecular nucleophilic attack by hydroxide, or other oxyanion. The general features of these two mechanisms have been established by physical organic chemical analyses; however, a more detailed understanding of the transition states of these reactions is emerging from recent kinetic isotope effect (KIE) studies. The recent data show interesting differences between the chemical mechanisms and transition state structures of the inter- and intramolecular reactions, as well as provide information on the impact of metal ion, acid, and base catalysis on these mechanisms. Importantly, recent nonenzymatic model studies show that interactions with divalent metal ions, an important feature of many phosphodiesterase active sites, can influence both the mechanism and transition state structure of nonenzymatic phosphodiester cleavage. Such detailed investigations are important because they mimic catalytic strategies employed by both RNA and protein phosphodiesterases, and so set the stage for explorations of enzyme-catalyzed transition states. Application of KIE analyses for this class of enzymes is just beginning, and several important technical challenges remain to be overcome. Nonetheless, such studies hold great promise since they will provide novel insights into the role of metal ions and other active site interactions.

2H, 13C, and 15N kinetic isotope effects on the reaction of the ammonia-rescued K258A mutant of aspartate aminotransferase.

Deuterium isotope effects at C2 of aspartate and heavy atom isotope effects at C2, C3, and the amino group of aspartate were determined for the reaction of the lysine-258 to alanine mutant of Escherichia coli rescued with exogenous ammonia. We were able to calculate an (15)N intrinsic isotope effect of 1.034. The intrinsic (13)C isotope effect at C3 is 1.0060, and the (13)C isotope effect at C2 is 1.0016. These isotope effects reveal that collapse of the carbinolamine (or gem-diamine) to give the final product is the rate-determining step in this system. Furthermore, these results indicate that lysine-258 is critical to the catalysis of the final breakdown to give product, and in fact this step is more strongly affected by mutation of lysine-258 than the deprotonation of the external aldimine.

13C and (15)N kinetic isotope effects on the reaction of aspartate aminotransferase and the tyrosine-225 to phenylalanine mutant.

Heavy atom isotope effects at C-2, C-3, and the amino nitrogen of aspartate were determined for the reaction of porcine heart cytosolic aspartate aminotransferase and the tyrosine-225 to phenylalanine mutant of Escherichia coli aspartate aminotransferase. The effects of deuteration at C-2 of aspartate and of D(2)O on the observed heavy atom isotope effects were determined. The multiple isotope effects support the contribution of C(alpha)-H cleavage, ketimine hydrolysis, and oxaloacetate dissociation to the rate limitation with the wild-type enzyme. The existence of a quinonoid intermediate could not be determined due to the kinetic complexity of the enzyme. For the tyrosine-225 to phenylalanine mutant, we are able to conclude that ketimine hydrolysis is the major rate-determining step.

A remarkable heavy atom isotope effect in the dissociative chemisorption of nitrogen on Ru(001)

An extremely large isotope effect [Ieff=Pdiss(15N2)/Pdiss(14N2)], has been measured in the dissociative chemisorption of nitrogen molecules over Ru(001). It varies from unity at kinetic energies above 2 eV to 0.2 at Ek=1.4 eV. These observations are consistent with a barrier for direct dissociation of 1.8 eV, in agreement with previous experiments and recent ab initio density functional theory calculations. It supports earlier studies that proposed tunneling as the dissociation dynamics mechanism.

The Transition State of the Phosphoryl-Transfer Reaction Catalyzed by the Lambda Ser/Thr Protein Phosphatase

The catalytic reaction of the Mn2+ form of the native bacteriophage λ phosphatase and the H76N mutant was studied with the substrate p-nitrophenyl phosphate using heavy atom isotope effects and pH-...

Isotope effects on the equilibrium of p-benzoquinone and its radical anion: ab initio and DFT studies

The isotope effect on the equilibrium between p-benzoquinone and its radical anion has been quantitatively estimated using Hartree–Fock (3-21G, 6-31G ∗ and 6-31+G ∗∗ basis sets) as well as pure and hybrid density functional methods (BP86 and B3LYP with 6-31G ∗∗ and 6-31+G ∗∗ basis sets). Equilibrium constants involving 2 H , 13 C , 17 O and 18 O isotopes have been calculated. At all levels, the deuterium isotope effect is substantial, while heavy atom isotope effects are small. The trend remains the same even when the presence of a counterion (Na +) is taken into account explicitly in the calculations. These conclusions differ qualitatively from an earlier experimental study.

Heavy Atom Isotope Effects Reveal a Highly Polarized Transition State for Chorismate Mutase

Heavy Atom Isotope Effects Reveal a Highly Polarized Transition State for Chorismate Mutase

Leaving–atom isotope effect in the reactions of N(22D) with isotopic hydrogens

The nascent internal state distributions of NH(ND) radicals produced in the reaction of N(2DJ) with HD were measured and compared with those for analogous reactions with H2 and D2. There was no leaving-atom isotope effect in the rotational distributions of NH(v′′=0) and ND(v′′=0), except at highly rotationally excited levels near the thermochemical limits. The observed isotope effect at high rotational levels can be explained by the conservation of both energy and angular momentum. The absence of isotope effect at low and medium rotational levels suggests that the transition state is a bent H(D)–N–H(D) type where the H(D)–H(D) bond is broken.

Mutagenesis and Chemical Rescue Indicate Residues Involved in β-Aspartyl-AMP Formation by Escherichia coli Asparagine Synthetase B

Site-directed mutagenesis and kinetic studies have been employed to identify amino acid residues involved in aspartate binding and transition state stabilization during the formation of beta-aspartyl-AMP in the reaction mechanism of Escherichia coli asparagine synthetase B (AS-B). Three conserved amino acids in the segment defined by residues 317-330 appear particularly crucial for enzymatic activity. For example, when Arg-325 is replaced by alanine or lysine, the resulting mutant enzymes possess no detectable asparagine synthetase activity. The catalytic activity of the R325A AS-B mutant can, however, be restored to about 1/6 of that of wild-type AS-B by the addition of guanidinium HCl (GdmHCl). Detailed kinetic analysis of the rescued activity suggests that Arg-325 is involved in stabilization of a pentacovalent intermediate leading to the formation beta-aspartyl-AMP. This rescue experiment is the second example in which the function of a critical arginine residue that has been substituted by mutagenesis is restored by GdmHCl. Mutation of Thr-322 and Thr-323 also produces enzymes with altered kinetic properties, suggesting that these threonines are involved in aspartate binding and/or stabilization of intermediates en route to beta-aspartyl-AMP. These experiments are the first to identify residues outside of the N-terminal glutamine amide transfer domain that have any functional role in asparagine synthesis.

Far-wing excitation study of the reactions in the Hg–H2 collisional quasimolecules. II. Rovibrational distributions of HgH, HgD (X 2Σ+) formed from Hg–H2, D2, HD

We have applied the laser-pump/probe and double-beam absorption/dispersion approaches to the far wings of the Hg 3P1–1S0 resonance line broadened by collisions with H2, D2, and HD. Absolute reduced absorption coefficients of the Hg–D2 quasimolecules have been determined as a function of the wave-number shift Δ from the resonance-line center both in the red and blue wings. The nascent rotational distributions have been determined for the v=0 and 1 levels of HgH (X 2Σ+) and the v=0 level of HgD (X 2Σ+) formed from the Hg*(3P1)–H2, D2, and HD collisional–quasimolecular states à and B̃ attained by the red- and blue-wing excitation, respectively. Both of the intermediate states à and B̃ give quite similar rotational distributions peaking around N≂18 for HgH and N≂25 for HgD insensitive to the excitation-wave-number shift Δ. However, a small difference is found: the red-wing excitation gives larger populations in the low-N levels than the blue-wing one. The departing atom isotope effect is observed in these low-N populations of HgD from Hg–D2 and Hg–HD. The absolute ratio of the nascent yields of v=1 to 0 has been measured to be 0.3, being nearly constant against Δ in both the red and blue wings. These observations indicate that HgH is formed predominantly from a bent H–Hg–H configuration on both the pathways via the à and B̃ states. The different type of transition state, however, may be encountered on the pathways producing the minor components in the low-N levels.