Development of a Polarizable Interatomic Potential for Molten Lithium, Sodium, and Potassium Nitrate.

A polarizable interatomic potential is developed for atomistic simulations of molten MNO3 (M = Li, Na, K) salts. The potential is parametrized using a force matching method relying on the adjustment of parameters such that density functional theory generated forces, stress tensors, and dipole moments are reproduced. Simulations conducted using the new potential are used to estimate physical parameters of the melt, which are then compared with available experimental results. The average calculated densities of NaNO3 and KNO3 are within 2% of the experimental value within the temperature range studied, while that of LiNO3 is within 3%. Thermal conductivities and viscosities are estimated using equilibrium calculations and the Green-Kubo method. The thermal conductivity values of NaNO3 and KNO3 are found to match well with experimental data, while that of LiNO3 is approximately 20% larger than experimentally determined values throughout the temperature ranges simulated. The calculated viscosities are also in good agreement with experimentally determined values. The (NaxK1-x)NO3 mixture is also investigated, with densities, thermal conductivities, and viscosities determined and compared with experimentally determined values where available. Additionally, radial and angular distribution function data is presented for all salts, revealing details of the atom-level structures present in the melts. We have found that the new interatomic potential is effective for atom scale modeling of the physical properties of molten nitrate salts.

Catalytic stimulation by restrained active-site floppiness--the case of high density lipoprotein-bound serum paraoxonase-1.

Despite the abundance of membrane-associated enzymes, the mechanism by which membrane binding stabilizes these enzymes and stimulates their catalysis remains largely unknown. Serum paraoxonase-1 (PON1) is a lipophilic lactonase whose stability and enzymatic activity are dramatically stimulated when associated with high-density lipoprotein (HDL) particles. Our mutational and structural analyses, combined with empirical valence bond simulations, reveal a network of hydrogen bonds that connect HDL binding residues with Asn168--a key catalytic residue residing >15Å from the HDL contacting interface. This network ensures precise alignment of N168, which, in turn, ligates PON1's catalytic calcium and aligns the lactone substrate for catalysis. HDL binding restrains the overall motion of the active site and particularly of N168, thus reducing the catalytic activation energy barrier. We demonstrate herein that disturbance of this network, even at its most far-reaching periphery, undermines PON1's activity. Membrane binding thus immobilizes long-range interactions via second- and third-shell residues that reduce the active site's floppiness and pre-organize the catalytic residues. Although this network is critical for efficient catalysis, as demonstrated here, unraveling these long-rage interaction networks is challenging, let alone their implementation in artificial enzyme design.

DFT computational study of the methanolytic cleavage of DNA and RNA phosphodiester models promoted by the dinuclear Zn(II) complex of 1,3-bis(1,5,9-triazacyclododec-1-yl)propane.

A density functional theory study of the cleavage of a DNA model [p-nitrophenyl methyl phosphate (2)] and two RNA models [p-nitrophenyl 2-hydroxypropyl phosphate (3) and phenyl 2-hydroxypropyl phosphate (4)] promoted by the dinuclear Zn((II)) complex of 1,3-bis(1,5,9-triazacyclododec-1-yl)propane formulated with a bridging methoxide (1a) was undertaken to determine possible mechanisms for the transesterification processes that are consistent with experimental data. The initial substrate-bound state of 2:1a or 3:1a has the two phosphoryl oxygens bridging Zn((II))1 and Zn((II))2. For each of 2 and 3, four possible mechanisms were investigated, three of which were consistent with the overall free energy for the catalytic cleavage step for each substrate. The computations revealed various roles for the metal ions in the three mechanisms. These encompass concerted or stepwise processes, where the two metal ions with associated alkoxy groups [Zn((II))1:((-)OCH3) and Zn((II))1:((-)O-propyl)] play the role of a direct nucleophile (on 2 and 3, respectively) or where Zn((II))1:((-)OCH3) can act as a general base to deprotonate an attacking solvent molecule in the case of 2 or the attacking 2-hydroxypropyl group in the case of 3. The Zn((II))2 ion can serve as a spectator (after exerting a Lewis acid role in binding one of the phosphates' oxygens) or play active additional roles in providing direct coordination of the departing aryloxy group or positioning a hydrogen-bonding solvent to assist the departure of the leaving group. An important finding revealed by the calculations is the flexibility of the ligand system that allows the Zn-Zn distance to expand from ~3.6 Å in 1a to over 5 Å in the transforming 2:1a and 3:1a complexes during the catalytic event.

Trifunctional metal ion-catalyzed solvolysis: Cu(II)-promoted methanolysis of N,N-bis(2-picolyl) benzamides involves unusual Lewis acid activation of substrate, delivery of coordinated nucleophile, powerful assistance of the leaving group departure.

The methanolyses of Cu(II) complexes of a series of N,N-bis(2-picolyl) benzamides (4a-g) bearing substituents X on the aromatic ring were studied under (s)(s)pH-controlled conditions at 25 °C. The active form of the complexes at neutral (s)(s)pH has a stoichiometry of 4:Cu(II):((-)OCH(3))(HOCH(3)) and decomposes unimolecularly with a rate constant k(x). A Hammett plot of log(k(x)) vs σ(x) values has a ρ(x) of 0.80 ± 0.05. Solvent deuterium kinetic isotope effects of 1.12 and 1.20 were determined for decomposition of the 4-nitro and 4-methoxy derivatives, 4b:Cu(II):((-)OCH(3))(HOCH(3)) and 4g:Cu(II):((-)OCH(3))(HOCH(3)), in the plateau region of the (s)(s)pH/log(k(x)) profiles in both CH(3)OH and CH(3)OD. Activation parameters for decomposition of these complexes are ΔH(++) = 19.1 and 21.3 kcal mol(-1) respectively and ΔS(++) = -5.1 and -2 cal K(-1) mol(-1). Density functional theory (DFT) calculations for the reactions of the Cu(II):((-)OCH(3))(HOCH(3)) complexes of 4a,b and g (4a, X = 3,5-dinitro) were conducted to probe the relative transition state energies and geometries of the different states. The experimental and computational data support a mechanism where the metal ion is coordinated to the N,N-bis(2-picolyl) amide unit and positioned so that it permits delivery of a coordinated Cu(II):((-)OCH(3)) nucleophile to the C═O in the rate-limiting transition state (TS) of the reaction. This proceeds to a tetrahedral intermediate INT, occupying a shallow minimum on the free energy surface with the Cu(II) coordinated to both the methoxide and the amidic N. Breakdown of INT is a virtually barrierless process, involving a Cu(II)-assisted departure of the bis(2-picolyl)amide anion. The analysis of the data points to a trifunctional role for the metal ion in the solvolysis mechanism where it activates intramolecular nucleophilic attack on the C═O group by coordination to an amidic N in the first step of the reaction and subsequently assists leaving group departure in the second step. The catalysis is very large; compared with the second order rate constant for methoxide attack on 4b, the computed reaction of CH3O(-) and 4b:Cu(II):(HOCH(3))(2) is accelerated by roughly 2.0 × 10(16) times.

RNA-sequencing analysis of 5' capped RNAs identifies many new differentially expressed genes in acute hepatitis C virus infection.

We describe the first report of RNA sequencing of 5' capped (Pol II) RNAs isolated from acutely hepatitis C virus (HCV) infected Huh 7.5 cells that provides a general approach to identifying differentially expressed annotated and unannotated genes that participate in viral-host interactions. We identified 100, 684, and 1,844 significantly differentially expressed annotated genes in acutely infected proliferative Huh 7.5 cells at 6, 48, and 72 hours, respectively (fold change ≥ 1.5 and Bonferroni adjusted p-values < 0.05). Most of the differentially expressed genes (>80%) and biological pathways (such as adipocytokine, Notch, Hedgehog and NOD-like receptor signaling) were not identified by previous gene array studies. These genes are critical components of host immune, inflammatory and oncogenic pathways and provide new information regarding changes that may benefit the virus or mediate HCV induced pathology. RNAi knockdown studies of newly identified highly upregulated FUT1 and KLHDC7B genes provide evidence that their gene products regulate and facilitate HCV replication in hepatocytes. Our approach also identified novel Pol II unannotated transcripts that were upregulated. Results further identify new pathways that regulate HCV replication in hepatocytes and suggest that our approach will have general applications in studying viral-host interactions in model systems and clinical biospecimens.

Cu(II)-promoted methanolysis of N,N-dipicolylacetamide. multistep activation by decoupling of > ̈̈N-C═O resonance via Cu(II)-N binding, delivery of the Cu(II):((-)OCH3) nucleophile, and metal ion assistance of the departure of the leaving group.

The methanolysis of the Cu(II) complex of N-acetyl-N,N-bis(2-picolyl)amine (2) was investigated by a kinetic study as a function of pH in methanol at 25 °C and computationally by DFT calculations. The active species is the basic form of the complex (3(-)), or (1:Cu(II))((-)OCH(3))(HOCH(3))), and the rate constant for its solvolysis is k(max) = 1.5 × 10(-4) s(-1). The mechanism involves Cu(II) binding to the amide N lone pair, decoupling it from >N-C═O resonance, concomitant with Cu(II):((-)OCH(3)) delivery to the adjacent >N-C═O unit, followed by Cu(II)-assisted departure of the N,N-bis(2-picolyl)amide from a tetrahedral intermediate.

Methanolysis of thioamide promoted by a simple palladacycle is accelerated by 10(8) over the methoxide-catalyzed reaction.

Palladacycle 1 catalyzes the methanolytic cleavage of N-methyl-N-(4-nitrophenyl)thiobenzamide (4) via a mechanism involving formation of a Pd-bound tetrahedral intermediate (TI). The rate constant for decomposition of the complex formed between 1, methoxide, and 4 is 9.3 s(-1) at 25 °C; this reaction produces methyl thiobenzoate and N-methyl-4-nitroaniline. The ratio of the second-order rate constant for the catalyzed reaction, given as k(cat)/K(d), relative to that of the methoxide-promoted reaction is 3 × 10(8), representing a very large catalysis of thioamide bond cleavage by a synthetic metal complex.

Evaluation of gastropexy and stoma tract maturation using a novel introducer kit for percutaneous gastrostomy in a porcine model.

BACKGROUND: Fluoroscopic placement of percutaneous gastrostomy (PG) requires the use of T-bar fasteners to affix the stomach to the anterior abdominal wall; the effect of T-fasteners on stoma tract maturation is unknown. The authors studied PG stoma tract maturation, comparing PG + gastropexy with standard percutaneous endoscopic gastrostomy (PEG). METHODS: Sixteen pigs underwent PG placement using a novel introducer kit. Three absorbable suture T-fasteners were placed around the stoma site, and PG was placed using the Russell method. A standard PEG was then placed using the Ponsky pull method, allowing each animal to serve as its own control. Gross and histopathological integrity of stoma tract formation was assessed at 1-3 weeks. RESULTS: At sacrifice, all PGs were intact with no evidence of infection, disruption, or significant leakage. Stoma tracts of all test and control sites were robust and histologically mature at all time points. Stoma tract diameters were also similar between test and control PGs (mean ± SEM: control 13.1 ± 0.7 mm, test 12.1 ± 0.4 mm; P = .2, n = 15). Histopathological evaluation demonstrated a generally comparable tissue response between test and control PGs, with slight decreases in fibrosis noted in test compared to control sites (P = .02, n = 15). CONCLUSIONS: Stoma tract maturation of PG with gastropexy provides similar results to standard PEG. Stoma tracts were mature at 1 week regardless of placement method. Placement and performance of PG using the new introducer kit with novel T-fasteners and absorbable suture yields effective gastric anchoring and has similar ease of use as standard PEG placement.

ENCODE tiling array analysis identifies differentially expressed annotated and novel 5' capped RNAs in hepatitis C infected liver.

Microarray studies of chronic hepatitis C infection have provided valuable information regarding the host response to viral infection. However, recent studies of the human transcriptome indicate pervasive transcription in previously unannotated regions of the genome and that many RNA transcripts have short or lack 3' poly(A) ends. We hypothesized that using ENCODE tiling arrays (1% of the genome) in combination with affinity purifying Pol II RNAs by their unique 5' m7GpppN cap would identify previously undescribed annotated and unannotated genes that are differentially expressed in liver during hepatitis C virus (HCV) infection. Both 5'-capped and poly(A)+ populations of RNA were analyzed using ENCODE tiling arrays. Sixty-four annotated genes were significantly increased in HCV cirrhotic as compared to control liver; twenty-seven (42%) of these genes were identified only by analyzing 5' capped RNA. Thirty-one annotated genes were significantly decreased; sixteen (50%) of these were identified only by analyzing 5' capped RNA. Bioinformatic analysis showed that capped RNA produced more consistent results, provided a more extensive expression profile of intronic regions and identified upregulated Pol II transcriptionally active regions in unannotated areas of the genome in HCV cirrhotic liver. Two of these regions were verified by PCR and RACE analysis. qPCR analysis of liver biopsy specimens demonstrated that these unannotated transcripts, as well as IRF1, TRIM22 and MET, were also upregulated in hepatitis C with mild inflammation and no fibrosis. The analysis of 5' capped RNA in combination with ENCODE tiling arrays provides additional gene expression information and identifies novel upregulated Pol II transcripts not previously described in HCV infected liver. This approach, particularly when combined with new RNA sequencing technologies, should also be useful in further defining Pol II transcripts differentially regulated in specific disease states and in studying RNAs regulated by changes in pre-mRNA splicing or 3' polyadenylation status.

Mechanistic and computational study of a palladacycle-catalyzed decomposition of a series of neutral phosphorothioate triesters in methanol.

The methanolytic cleavage of a series of O,O-dimethyl O-aryl phosphorothioates (1a−g) catalyzed by a C,N-palladacycle, (2-[N,N-dimethylamino(methyl)phenyl]-C1,N)(pyridine) palladium(II) triflate (3), at 25 °C and sspH 11.7 in methanol is reported, along with data for the methanolytic cleavage of 1a−g. The methoxide reaction gives a linear log k2−OMe vs sspKa (phenol leaving group) Brønsted plot having a gradient of ßlg = −0.47 ± 0.03, suggesting about 34% cleavage of the P−OAr bond in the transition state. On the other hand, the 3-catalyzed cleavage of 1 gives a Brønsted plot with a downward break at sspKa (phenol) 13, signifying a change in the rate-limiting step in the catalyzed reaction, with the two wings having ßlg values of 0.0 ± 0.03 and −1.93 ± 0.06. The rate-limiting step for good substrates with low leaving group sspKa values is proposed to be substrate/pyridine exchange on the palladacycle, while for substrates with poor leaving groups, the rate-limiting step is a chemical one with extensive cleavage of the P−OAr bond. DFT calculations support this process and also identify two intermediates, namely, one where substrate/pyridine interchange has occurred to give the palladacycle coordinated to substrate through the S═P linkage and to methoxide (6) and another where intramolecular methoxide attack has occurred on the P═S unit to give a five-coordinate phosphorane (7) doubly coordinated to Pd via the S− and through a bridging methoxide linked to P and Pd. Attempts to identify the existence of the phosphorane by 31P NMR in a d4-methanol solution containing 10 mM each of 3, trimethyl phosphorothioate (a very slow cleaving substrate), and methoxide proved unsuccessful, instead showing that the phosphorothioate was slowly converted to trimethyl phosphate, with the palladacycle decomposing to Pd0 and free pyridine. These results provide the first reported example where a palladacycle-promoted solvolysis reaction exhibits a break in the Brønsted plot signifying at least one intermediate, while the DFT calculations provide further insight into a more complex mechanism involving two intermediates.

Demonstration of prominent Cu(II)-promoted leaving group stabilization of the cleavage of a homologous set of phosphate mono-, di-, and triesters in methanol.

A series of phosphate mono-, di-, and triesters with a common leaving group (LG) (2'-(2-phenoxy)-1,10-phenanthroline) was prepared, and the kinetics of decomposition of their Cu(II) complexes was studied in methanol at 25 degrees C under (s)(s)pH-controlled conditions. The Cu(II) complexes of 2-[2'-phenanthrolyl]phenyl phosphate (Cu(II):6), 2-[2'-phenanthrolyl]phenyl methyl phosphate (Cu(II):7), and 2-[2'-phenanthrolyl]phenyl dimethyl phosphate (Cu(II):8) are tightly bound, having dissociation constants Kd < or = 3 x 10(-7) M, with the Cu(II) being in contact with the departing phenoxide. The (s)(s)pH/rate profile for cleavage of Cu(II):6 has a low (s)(s)pH plateau (k(o) = 6.3 x 10(-3) s(-1)), followed by a bell-shaped maximum (kcat(max) = 14.7 +/- 0.4 s(-1)) dependent on two ionizations with (s)(s)pKa(2) and (s)(s)pKa(3) = 7.8 +/- 0.1 and 11.8 +/- 0.2. The (s)(s)pH/rate profile for cleavage of Cu(II):7 has a broad plateau from (s)(s)pH 3 to (s)(s)pH 10 followed by a descending wing at higher (s)(s)pH with a gradient of -2. The (s)(s)pH/rate profile for cleavage of Cu(II):8 is sigmoidal with two plateaus (k1 = (2.0 +/- 0.2) x 10(-5) s(-1), k2 = (1.2 +/- 0.2) x 10(-6) s(-1)), connected by an ionization with a (s)(s)pKa of 6.03. Activation parameters are given for the reactions in the plateau regions: all three species show similar DeltaH(double dagger) terms of 21.4-21.6 kcal/mol, with major differences in the DeltaS(double dagger) terms, which vary from 18 to 2.3 to -7.4 cal/(mol x K) passing from the mono- to di- to triester. Detailed analyses of the kinetics indicate that the reactions involve spontaneous solvent-mediated cleavage of the Cu(II)-coordinated phosphate dianion [Cu(II):6b]0 and phosphate diester monoanion [Cu(II):7b]+ and, for the triester, complexes containing Cu(II) and Cu(II):(-)OCH3 designated as [Cu(II):8a]2+ and [Cu(II):8b]+. Reactions where methoxide is the active nucleophile are not observed. Comparisons of the rates of the decomposition of these species at their (s)(s)pH maxima in the neutral (s)(s)pH region with the estimated rates of the background reactions indicate that leaving group assistance provided by the coordinated Cu(II) accelerates the cleavage of the phosphate mono-, di-, and triesters by 10(14) to 10(15), 10(14), and 10(5). Detailed Hyperquad 2000 analysis of titration data indicates that phenoxide 9- is bound 23 kcal/mol stronger than the phosphate triester 8. It is the realization of part of this energy in the emerging products resulting from P-O(LG) cleavage that provides the driving force for the catalyzed reactions.

Kinetic resolution of esters via metal catalyzed methanolysis reactions.

Some chiral lanthanide complexes of the Schiff base adducts of: a) bis(2-pyridylcarboxaldehyde) and (1R),(2R)-trans-1,2-diaminocyclohexane (Pyr-R,R'-chxn: 3); b) 6-methyl-2-pyridylcarboxaldehyde and (1R),(2R)-trans 1,2-diaminocyclohexane (MePyr-chxn, 4); and c) 2,6-pyridyldicarboxaldehyde and (1R),(2R)-trans-1,2-diaminocyclohexane ((Pyr-R,R'-chxn)(2), 5) have been screened for their utility to promote kinetic resolution via metal catalyzed alcoholyses of the p-nitrophenyl esters of chiral D- and L-Boc-protected glutamine and phenylalanine. Solvents were varied to optimize the kinetic selectivity values, defined as k(2)(L)/k(2)(D) or k(2)(D)/k(2)(L), for the methanolysis and in some cases, ethanolysis of these substrates. At ambient temperature the greatest selectivity was found for the ethanolysis of Boc-Gln-OPNP, catalyzed by 3:Yb(3+):((-)OEt) (k(2)(L)/k(2)(D) = 7.2). The greatest selectivity for Boc-Phe-OPNP is k(2)(D)/k(2)(L) = 3.9 for its methanolysis promoted by 5:La(3+):((-)OMe). A kinetic method is introduced for the determination of both d and l rate constants for catalyzed alcoholysis from a single kinetic experiment. The activation parameters DeltaH(double dagger) and DeltaS(double dagger) were determined for the metal catalyzed methanolysis and ethanolysis of the Boc-Gln-OPNP substrates, and selectivity factors were found to increase at lower temperatures. A low temperature time course for the ethanolysis of racemic Boc-Gln-OPNP catalyzed by 3:Yb(3+):((-)OEt) at -15 degrees C indicated that after 3 hours 60% residual d-enantiomer was observed having an enantiomeric excess of >95% ee. The activation parameters for the ethanolysis of the same substrate catalyzed by (Pyr-R,R'-chxn)(2):La(3+):((-)OEt) predict a k(2)(D)/k(2)(L) = 40.4 at -40 degrees C with a large ee of >99% with approximately 80% of l isomer remaining at that temperature which has been experimentally confirmed.

Combination of a dinuclear Zn2+ complex and a medium effect exerts a 10(12)-fold rate enhancement of cleavage of an RNA and DNA model system.

The catalytic ability of a dinuclear Zn2+ complex of 1,3-bis-N1-(1,5,9-triazacyclododecyl)propane (3) in promoting the cleavage of an RNA model, 2-hydroxypropyl-p-nitrophenyl phosphate (HPNPP, 1), and a DNA model, methyl p-nitrophenyl phosphate (MNPP, 4), was studied in methanol solution in the presence of added CH3O- at 25 degrees C. The di-Zn2+ complex (Zn2 :3), in the presence of 1 equiv of added methoxide, exhibits a second-order rate constant of (2.75 +/- 0.10) x 10(5) M(-1) s(-1) for the reaction with 1 at s(s)pH 9.5, this being 10(8)-fold larger than the k2 value for the CH3O- promoted reaction (kOCH3 = (2.56 +/- 0.16) x 10(-3) M(-1) s(-1)). The complex is also active toward the DNA model 4, exhibiting Michaelis-Menten kinetics with a KM and kmax of 0.37 +/- 0.07 mM and (4.1 +/- 0.3) x 10(-2) s(-1), respectively. Relative to the background reactions at s(s)pH 9.5, Zn2 :3 accelerates cleavage of each phosphate diester by a remarkable factor of 1012-fold. A kinetic scheme common to both substrates is discussed. The study shows that a simple model system comprising a dinuclear Zn2+ complex and a medium effect of the alcohol solvent achieves a catalytic reactivity that approaches enzymatic rates and is well beyond anything seen to date in water for the cleavage of these phosphate diesters.