Computational Insights into the Mechanism of Nitric Oxide Generation from S-Nitrosoglutathione Catalyzed by a Copper Metal-Organic Framework.

The controlled generation of nitric oxide (NO) from endogenous sources, such as S-nitrosoglutathione (GSNO), has significant implications for biomedical implants due to the vasodilatory and other beneficial properties of NO. The water-stable metal-organic framework (MOF) Cu-1,3,5-tris[1H-1,2,3-triazol-5-yl]benzene has been shown to catalyze the production of NO and glutathione disulfide (GSSG) from GSNO in aqueous solution as well as in blood. Previous experimental work provided kinetic data for the catalysis of the 2GSNO → 2NO + GSSG reaction, leading to various proposed mechanisms. Herein, this catalytic process is examined using density functional theory. Minimal functional models of the Cu-MOF cluster and glutathione moieties are established, and three distinct catalytic mechanisms are explored. The most thermodynamically favorable mechanism studied is consistent with prior experimental findings. This mechanism involves coordination of GSNO to copper via sulfur rather than nitrogen and requires a reductive elimination that produces a Cu(I) intermediate, implicating a redox-active copper site. The experimentally observed inhibition of reactivity at high pH values is explained in terms of deprotonation of a triazole linker, which decreases the structural stability of the Cu(I) intermediate. These fundamental mechanistic insights may be generally applicable to other MOF catalysts for NO generation.

Is it homogeneous or heterogeneous catalysis derived from [RhCp*Cl2]2? In operando XAFS, kinetic, and crucial kinetic poisoning evidence for subnanometer Rh4 cluster-based benzene hydrogenation catalysis.

Determining the true, kinetically dominant catalytically active species, in the classic benzene hydrogenation system pioneered by Maitlis and co-workers 34 years ago starting with [RhCp*Cl(2)](2) (Cp* = [η(5)-C(5)(CH(3))(5)]), has proven to be one of the most challenging case studies in the quest to distinguish single-metal-based "homogeneous" from polymetallic, "heterogeneous" catalysis. The reason, this study will show, is the previous failure to use the proper combination of: (i) in operando spectroscopy to determine the dominant form(s) of the precatalyst's mass under catalysis (i.e., operating) conditions, and then crucially also (ii) the previous lack of the necessary kinetic studies, catalysis being a "wholly kinetic phenomenon" as J. Halpern long ago noted. An important contribution from this study will be to reveal the power of quantitiative kinetic poisoning experiments for distinguishing single-metal, or in the present case subnanometer Rh(4) cluster-based catalysis, from larger, polymetallic Rh(0)(n) nanoparticle catalysis, at least under favorable conditions. The combined in operando X-ray absorption fine structure (XAFS) spectroscopy and kinetic evidence provide a compelling case for Rh(4)-based, with average stoichiometry "Rh(4)Cp*(2.4)Cl(4)H(c)", benzene hydrogenation catalysis in 2-propanol with added Et(3)N and at 100 °C and 50 atm initial H(2) pressure. The results also reveal, however, that if even ca. 1.4% of the total soluble Rh(0)(n) had formed nanoparticles, then those Rh(0)(n) nanoparticles would have been able to account for all the observed benzene hydrogenation catalytic rate (using commercial, ca. 2 nm, polyethyleneglycol-dodecylether hydrosol stabilized Rh(0)(n) nanoparticles as a model system). The results--especially the poisoning methodology developed and employed--are of significant, broader interest since determining the nature of the true catalyst continues to be a central, often vexing issue in any and all catalytic reactions. The results are also of fundamental interest in that they add to a growing body of evidence indicating that certain, appropriately ligated, coordinatively unsaturated, subnanometer M(4) transition-metal clusters can be relatively robust catalysts. Also demonstrated herein is that Rh(4) clusters are poisoned by Hg(0), demonstrating for the first time that the classic Hg(0) poisoning test of "homogeneous" vs "heterogeneous" catalysts cannot distinguish Rh(4)-based subnanometer catalysts from Rh(0)(n) nanoparticle catalysts, at least for the present examples of these two specific, Rh-based catalysts.

In situ formed "weakly ligated/labile ligand" iridium(0) nanoparticles and aggregates as catalysts for the complete hydrogenation of neat benzene at room temperature and mild pressures.

"Weakly ligated/labile ligand" nanoparticles, that is nanoparticles where only weakly coordinated ligands plus the desired catalytic reactants are present, are of fundamental interest. Described herein is a catalyst system for benzene hydrogenation to cyclohexane consisting of "weakly ligated/labile ligand" Ir(0) nanoparticles and aggregates plus dry-HCl formed in situ from commercially available [(1,5-COD)IrCl](2) plus 40 +/- 1 psig (approximately 2.7 atm) H(2) at 22 +/- 0.1 degrees C. Multiple control and other experiments reveal the following points: (i) that this catalyst system is quite active with a TOF (turnover frequency) of 25 h(-1) and TTO (total turnovers) of 5250; (ii) that the BF(4)(-) and PF(6)(-) iridium salt precursors, [(1,5-COD)Ir(CH(3)CN)(2)]BF(4) and [(1,5-COD)Ir(CH(3)CN)(2)]PF(6), yield inferior catalysts; (iii) that iridium black with or without added, preformed HCl cannot achieve the TOF of 25 h(-1) of the in situ formed Ir(0)/dry-HCl catalyst. However and importantly, CS(2) poisoning experiments yield the same activity per active iridium atom for both the Ir(0)/dry-HCl and Ir black/no-HCl catalysts (12.5 h(-1) Ir(1-)), but reveal that the Ir(0)/dry-HCl system is 10-fold more dispersed compared to the Ir(0) black catalyst. The simple but important and key result is that "weakly ligated/labile ligand" Ir(0) nanoparticles and aggregates have been made in situ as demonstrated by the fact that they have identical, per exposed Ir(0) activity within experimental error to Ir(0) black and that they have no possible ligands other than those desired for the catalysis (benzene and H(2)) plus the at best poor ligand HCl. As expected, the in situ catalyst is poorly stabilized, exhibiting only 60% of its initial activity in a second run of benzene hydrogenation and resulting in bulk metal precipitation. However, stabilization of the Ir(0) nanoparticles with a ca. 2-fold higher catalytic activity and somewhat longer lifetime for the complete hydrogenation of benzene was accomplished by supporting the Ir(0) nanoparticles onto zeolite-Y (TOF of 47 h(-1) and 8600 TTO under otherwise identical conditions). Also reported is the interesting result that Cl(-) (present as Proton Sponge x H(+)Cl(-)) completely poisons benzene hydrogenation catalysis, but not the easier cyclohexene hydrogenation catalysis under otherwise the same conditions, results that suggest different active sites for these ostensibly related hydrogenation reaction. The results suggest that synthetic routes to "weakly ligated/labile ligand" nanoparticles exhibiting improved catalytic performance is an important goal worthy of additional effort.

Fitting neurological protein aggregation kinetic data via a 2-step, minimal/"Ockham's razor" model: the Finke-Watzky mechanism of nucleation followed by autocatalytic surface growth.

The aggregation of proteins has been hypothesized to be an underlying cause of many neurological disorders including Alzheimer's, Parkinson's, and Huntington's diseases; protein aggregation is also important to normal life function in cases such as G to F-actin, glutamate dehydrogenase, and tubulin and flagella formation. For this reason, the underlying mechanism of protein aggregation, and accompanying kinetic models for protein nucleation and growth (growth also being called elongation, polymerization, or fibrillation in the literature), have been investigated for more than 50 years. As a way to concisely present the key prior literature in the protein aggregation area, Table 1 in the main text summarizes 23 papers by 10 groups of authors that provide 5 basic classes of mechanisms for protein aggregation over the period from 1959 to 2007. However, and despite this major prior effort, still lacking are both (i) anything approaching a consensus mechanism (or mechanisms), and (ii) a generally useful, and thus widely used, simplest/"Ockham's razor" kinetic model and associated equations that can be routinely employed to analyze a broader range of protein aggregation kinetic data. Herein we demonstrate that the 1997 Finke-Watzky (F-W) 2-step mechanism of slow continuous nucleation, A --> B (rate constant k1), followed by typically fast, autocatalytic surface growth, A + B --> 2B (rate constant k2), is able to quantitatively account for the kinetic curves from all 14 representative data sets of neurological protein aggregation found by a literature search (the prion literature was largely excluded for the purposes of this study in order provide some limit to the resultant literature that was covered). The F-W model is able to deconvolute the desired nucleation, k1, and growth, k2, rate constants from those 14 data sets obtained by four different physical methods, for three different proteins, and in nine different labs. The fits are generally good, and in many cases excellent, with R2 values >or=0.98 in all cases. As such, this contribution is the current record of the widest set of protein aggregation data best fit by what is also the simplest model offered to date. Also provided is the mathematical connection between the 1997 F-W 2-step mechanism and the 2000 3-step mechanism proposed by Saitô and co-workers. In particular, the kinetic equation for Saitô's 3-step mechanism is shown to be mathematically identical to the earlier, 1997 2-step F-W mechanism under the 3 simplifying assumptions Saitô and co-workers used to derive their kinetic equation. A list of the 3 main caveats/limitations of the F-W kinetic model is provided, followed by the main conclusions from this study as well as some needed future experiments.

Adenosylcobinamide plus exogenous, sterically hindered, putative axial bases: a reinvestigation into the cause of record levels of Co-C heterolysis.

A reinvestigation of an earlier Ph.D. thesis (Sirovatka, J. M. Ph.D. Thesis, Colorado State University, Fort Collins, CO, 1999) is reported herein. That thesis examined the thermolysis reaction of AdoCbi(+)BF(4)(-) in ethylene glycol solution with exogenous bases, N-methylimidazole (N-Me-Im) and the sterically hindered 1,2-dimethylimidazole, (1,2-Me(2)-Im), 2-methylpyridine (2-Me-py), and 2,6-dimethylpyridine (2,6-Me(2)-py). In the present work, multiple purities of each base have been utilized as a check to see if impurities in the nitrogenous bases are causing the observed homolysis and heterolysis product distributions as others have implied (Trommel, J. S.; Warncke, K.; Marzilli, L. G. J. Am. Chem. Soc. 2001, 123, 3358). The "impurity hypothesis" is disproven by a series of results, including the following: N-Me-Im displays an invariant 52 +/- 10% heterolysis and the 1,2-Me(2)-Im system displays an invariant 83 +/- 7% heterolysis as a function of different base purification methods. Moreover, 2-Me-py and 2,6-Me(2)-py also display an invariant approximately 16 +/- 5% heterolysis as a function of different purification methods. What is responsible for the high levels of Co-C heterolysis in the AdoCbi(+) plus sterically bulky base thermolyses was uncovered via a revisitation of our four, earlier alternative hypotheses for the enhanced Co-C heterolysis (Sirovatka, J. M.; Finke, R. G. Inorg. Chem. 1999, 38, 1697). Our prior number one alternative hypothesis is shown to be correct: the added bases simply deprotonate the ethylene glycol solvent, forming ethylene glycolate anion and base-H(+)() as the key agents behind the previously ill-understood Co-C heterolyses. Also reported are Co(II)Cbi(+) titrations with five bases (1,2-Me(2)-Im, N-Me-Im, pyridine, 2-MePy, and 2,6-Me(2)-py). These experiments confirm Marzilli and co-workers' (op. cit.) results by showing that sterically hindered bases do not bind to Co(II)Cbi(+); therefore, Co(II)Cbi(+) EPR literature showing binding of bulky pyridines is erroneous as is the previously reported binding of bulky pyridine bases to Co(II)Cbi(+) by UV-vis spectroscopy (Sirovatka, J. Ph.D. Thesis, op. cit.). Also reported is our current best synthesis and purification of AdoCbi(+)BF(4)(-), work that builds off our 1987 synthesis of AdoCbi(+)BF(4)(-) (Hay, B. P.; Finke, R. G. J. Am. Chem. Soc. 1987, 109, 8012). Finally, the multiple, compounding errors which have caused problems in this project are listed, notably errors in the protein X-ray crystallography literature, the EXAFS literature, the Co(II)Cbi(+) plus bulky-bases EPR literature, the misleading B(12)-model literature, the erroneous experimental work (Sirovatka, op. cit.) and thus incorrect conclusions in one of our prior papers, as well as the erroneous implications in parts of the Marzilli and co-workers paper (op. cit.). It is hoped that a forthright reporting of these errors will help others avoid similar mistakes in the future when studying complex, bioinorganic systems.

The first experimental test of the hypothesis that enzymes have evolved to enhance hydrogen tunneling.

The literature hypothesis that "the optimization of enzyme catalysis may entail the evolutionary implementation of chemical strategies that increase the probability of quantum-mechanical tunneling" is experimentally tested herein for the first time. The system employed is the key to being able to provide this first experimental test of the "enhanced hydrogen tunneling" hypothesis, one that requires a comparison of the three criteria diagnostic of tunneling (vide infra) for the same, or nearly the same, reaction with and without the enzyme. Specifically, studied herein are the adenosylcobalamin (AdoCbl, also known as coenzyme B(12))-dependent diol dehydratase model reactions of (i). H(D)(*) atom abstraction from ethylene glycol-d(0) and ethylene glycol-d(4) solvent by 5'-deoxyadenosyl radical (Ado(*)) and (ii.) the same H(*) abstraction reactions by the 8-methoxy-5'-deoxyadenosyl radical (8-MeOAdo(*)). The Ado(*) and 8-MeOAdo(*) radicals are generated by Co-C thermolysis of their respective precursors, AdoCbl and 8-MeOAdoCbl. Deuterium kinetic isotope effects (KIEs) of the H(*)(D(*)) abstraction reactions from ethylene glycol have been measured over a temperature range of 80-120 degrees C: KIE = 12.4 +/- 1.1 at 80 degrees C for Ado(*) and KIE = 12.5 +/- 0.9 at 80 degrees C for 8-MeOAdo(*) (values ca. 2-fold that of the predicted maximum primary times secondary ground-state zero-point energy (GS-ZPE) KIE of 6.4 at 80 degrees C). From the temperature dependence of the KIEs, zero-point activation energy differences ([E(D) - E(H)]) of 3.0 +/- 0.3 kcal mol(-)(1) for Ado(*) and 2.1 +/- 0.6 kcal mol(-)(1) for 8-MeOAdo(*) have been obtained, both of which are significantly larger than the nontunneling, zero-point energy only maximum of 1.2 kcal mol(-)(1). Pre-exponential factor ratios (A(H)/A(D)) of 0.16 +/- 0.07 for Ado(*) and 0.5 +/- 0.4 for 8-MeOAdo(*) are observed, both of which are significantly less than the 0.7 minimum for nontunneling behavior. The data provide strong evidence for the expected quantum mechanical tunneling in the Ado(*) and 8-MeOAdo(*)-mediated H(*) abstraction reactions from ethylene glycol. More importantly, a comparison of these enzyme-free tunneling data to the same KIE, (E(D) - E(H)) and A(H)/A(D) data for a closely related, Ado(*)-mediated H(*) abstraction reaction from a primary CH(3)- group in AdoCbl-dependent methylmalonyl-CoA mutase shows the enzymic and enzyme-free data sets are identical within experimental error. The Occam's Razor conclusion is that at least this adenosylcobalamin-dependent enzyme has not evolved to enhance quantum mechanical tunneling, at least within the present error bars. Instead, this B(12)-dependent enzyme simply exploits the identical level of quantum mechanical tunneling that is available in the enzyme-free, solution-based H(*) abstraction reaction. The results also require a similar, if not identical, barrier width and height within experimental error for the H(*) abstraction both within, and outside of, the enzyme.

Is it homogeneous or heterogeneous catalysis? Identification of bulk ruthenium metal as the true catalyst in benzene hydrogenations starting with the monometallic precursor, Ru(II)(eta 6-C6Me6)(OAc)2, plus kinetic characterization of the heterogeneous nucleation, then autocatalytic surface-growth mechanism of metal film formation.

A reinvestigation of the true catalyst in a benzene hydrogenation system beginning with Ru(II)(eta(6)-C(6)Me(6))(OAc)(2) as the precatalyst is reported. The key observations leading to the conclusion that the true catalyst is bulk ruthenium metal particles, and not a homogeneous metal complex or a soluble nanocluster, are as follows: (i) the catalytic benzene hydrogenation reaction follows the nucleation (A --> B) and then autocatalytic surface-growth (A + B --> 2B) sigmoidal kinetics and mechanism recently elucidated for metal(0) formation from homogeneous precatalysts; (ii) bulk ruthenium metal forms during the hydrogenation; (iii) the bulk ruthenium metal is shown to have sufficient activity to account for all the observed activity; (iv) the filtrate from the product solution is inactive until further bulk metal is formed; (v) the addition of Hg(0), a known heterogeneous catalyst poison, completely inhibits further catalysis; and (vi) transmission electron microscopy fails to detect nanoclusters under conditions where they are otherwise routinely detected. Overall, the studies presented herein call into question any claim of homogeneous benzene hydrogenation with a Ru(arene) precatalyst. An additional, important finding is that the A --> B, then A + B --> 2B kinetic scheme previously elucidated for soluble nanocluster homogeneous nucleation and autocatalytic surface growth (Widegren, J. A.; Aiken, J. D., III; Ozkar, S.; Finke, R. G. Chem. Mater. 2001, 13, 312-324, and ref 8 therein) also quantitatively accounts for the formation of bulk metal via heterogeneous nucleation then autocatalytic surface growth. This is significant for three reasons: (i) quantitative kinetic studies of metal film formation from soluble precursors or chemical vapor deposition are rare; (ii) a clear demonstration of such A --> B, then A + B --> 2B kinetics, in which both the induction period and the autocatalysis are continuously monitored and then quantitatively accounted for, has not been previously demonstrated for metal thin-film formation; yet (iii) all the mechanistic insights from the soluble nanocluster system (op. cit.) should be applicable to metal thin-film formations which exhibit sigmoidal kinetics and, hence, the A --> B, then A + B --> 2B mechanism.

Synthesis of adenosylcobinamide 2-chlorophenyl phosphate, a zwitterionic cobinamide phosphate analog of adenosylcobalamin en route to crystallizable cobinamides.

The design and implementation of a new, higher yield synthetic method for synthesizing zwitterionic cobinamide phosphates is described. Adenosylcobinamide 2-chlorophenyl phosphate, beta-AdoCbi-PAr -- a 5,6-dimethylbenzimidazole-free adenosylcobalamin analog, where a 2-chlorophenyl group replaces the ribofuranose and 5,6-dimethylbenzimidazole moieties -- is prepared in tens of milligram quantities, quantities sufficient for crystallization and enzyme trials, amounts 100-fold greater than previously available. The use of (31)P NMR spectroscopy to follow reactions directly, the use of control reactions to learn how to reduce reactant water content, and the use of reaction solvents that completely dissolved the corrinoid reactants were crucial for developing this new synthetic route. beta-AdoCbi-PAr was synthesized in 10% overall isolated yield from cyanocobinamide. Cyanocobinamide was converted to cyanocobinamide 2-chlorophenyl phosphate by direct phosphorylation with 2-chlorophenyl phosphodi-(1,2,4-triazolide) in 25% isolated yield and > or = 98% purity. Sodium borohydride reduction of cyanocobinamide 2-chlorophenyl phosphate and reaction with 5'-chloro-5'-deoxy-adenosine produced beta-AdoCbi-PAr in 42% yield and > or = 98% purity. These compounds were characterized by HPLC, (1)H and (31)P NMR, UV-visible spectroscopy, and liquid secondary ionization mass spectroscopy.

The synthesis and characterization of 8-methoxy-5'-deoxyadenosylcobalamin: a coenzyme B(12) analog which, following Co-C bond homolysis, avoids cyclization of the 8-methoxy-5'-deoxyadenosyl radical.

The compound 8-methoxy-5'-deoxyadenosylcobalamin (8-MeOAdoCbl), has been synthesized in 37% yield and > or = 95% purity by HPLC, monitored at both 254 and 525 nm, or 90+/-2% purity as judged by the (1)H NMR spectrum of the aromatic cobalamin region. This is the first synthesis of this complex in which sufficient details are reported, where a yield and purity are reported, and where key problems in the synthesis and purification are overcome, so that 8-MeOAdoCbl can actually be obtained for use in other studies. Also demonstrated is the clean Co-C bond homolysis of 8-MeOAdoCbl to give initially 8-MeOAdoCbl and Co(II)Cbl in a UV-visible thermolysis experiment at 110 degrees C, results which show that the 8-MeO moiety suppresses the cyclization to the 8,5'-anhydro-adenosine otherwise seen for the adenosyl radical (Ado)*. Suppression of this cyclization pathway makes 8-MeOAdoCbl invaluable for studying the kinetic isotope effect (KIE) of the Ado* plus substrate H* abstraction reaction, a component of the first definitive test of Klinman's hypothesis that the optimization of enzyme catalysis may entail strategies that increase the probability of tunneling and thereby accelerate H* atom abstraction reaction rates.

Coenzyme B(12) Axial-Base Chemical Precedent Studies. Adenosylcobinamide Plus Sterically Hindered Axial-Base Co-C Bond Cleavage Product and Kinetic Studies: Evidence for the Dominance of Axial-Base Transition-State Effects and for Co-N(Axial-Base) Distance-Dependent, Competing sigma and pi Effects.

Adenosylcobinamide (AdoCbi(+)) plus the sterically hindered bases 1,2-dimethylimidazole, 2-methylpyridine, and 2,6-dimethylpyridine, as well as control experiments with imidazolate and 4-methylimidazolate, have been investigated to provide chemical precedent for the benzimidazole base-off, protein histidine imidazole base-on form of adenosylcobalamin (AdoCbl, also coenzyme B(12)). This imidazole base-on form of AdoCbl was observed in the recent X-ray crystallographic structural study of methylmalonyl-CoA (MMCoA) mutase; of interest to the present work is the fact that MMCoA mutase contains a long, ca. 2.5 Å, Co-N(imidazole) axial bond, at least in the enzyme's crystallographically characterized Co(II)/Co(III) state and conformation. In the present studies, upper limits for the axial-base binding K(assoc) parameters to form [AdoCbi.bulky base](+) BF(4)(-) have been obtained; these thermodynamic studies reveal that sterically hindered bases do not bind detectably to AdoCbi(+) in the ground state, which results in negligible ground-state free-energy stabilization via the formation of [AdoCbi.bulky base](+). The sterically hindered bases do, however, bind to Co(II)Cbi(+), a good energetic model of the [Ado. - - -.CoCbi](+) homolysis transition state. Kinetic studies demonstrate that the sterically hindered bases are involved in the rate-determining step of Co-C bond homolysis, accelerating it by 200-fold; hence, Co-C cleavage does occur via the low-level and otherwise nondetectable amount of [AdoCbi.bulky base](+) formed in solution. Product studies reveal (i) that both Co-C heterolysis and homolysis occur, and (ii) that there is no simple correlation between the ratio of Co-C heterolysis to homolysis and the Co-N(axial-base) bond length. Overall, the results provide strong evidence for the dominance of axial-base transition-state effects on Co-C bond cleavage, and reveal a subtle interplay of sigma and pi effects as a function of the Co-N(axial-base) bond length.