Antinociceptive and adverse effects of morphine:ketamine mixtures in rats.

Prescription opioids are the gold standard for treating moderate to severe pain despite their well-documented adverse effects. Of all prescription medications, opioids are abused most widely, and fatal overdoses have reached epidemic levels. One strategy for improving the margin of safety of opioids is combining them with non-opioid drugs to decrease the opioid dose needed for pain relief, thereby reducing adverse effects that occur with larger doses. The N-methyl-D-aspartate receptor antagonist ketamine has been used safely as an analgesic but only under a very limited range of conditions. The current studies characterized the antinociceptive, behavioral suppressant, and gastrointestinal effects of morphine and ketamine alone and in mixtures to determine their interaction in 24 adult male Sprague-Dawley rats (n = 8 per assay). Given alone, both morphine and ketamine produced antinociception, decreased responding for food, and reduced gastrointestinal transit (i.e. produced constipation). The effects of morphine:ketamine mixtures generally were additive, except for the antinociceptive effects of 1:1 mixtures for which the difference in slope (i.e. non-parallel shift) between the observed and predicted effects suggested synergy at smaller doses and additivity at larger doses. The potency of morphine to produce constipation was not enhanced by administration of morphine:ketamine mixtures with antinociceptive effects. The nature of the interaction between morphine and ketamine for adverse effects such as dependence, withdrawal, abuse, or respiratory depression remains unknown but also might be related to the ratio of each drug in mixtures. It will be important to identify conditions that produce the largest potential therapeutic window in humans.

Quantum Suppression of Intramolecular Deuterium Kinetic Isotope Effects in a Pericyclic Hydrogen Transfer Reaction.

It is generally accepted that hydrogen tunneling enhances both primary and secondary H/D kinetic isotope effects (KIEs) over what would be expected under the assumptions of classical barrier transition. Previous studies have exclusively shown that the effects of tunneling upon primary H/D KIEs have been much larger than those observed for secondary H/D KIEs. Here we present a series of experimental H/D KIE results associated with the Chugaev elimination of methyl xanthate derived from ß-phenylethanol over the temperature range of 180 to 290 °C. Intramolecular H/D KIEs computed according to classical transition state theory (TST) are markedly overestimated relative to experimentally measured values. Experimental intermolecular H/D KIEs and direct dynamic calculations based on canonical variational transition state theory (CVT) with small-curvature tunneling correction (SCT) reveal that this result is largely the consequence of extraordinary tunneling enhancement of the secondary H/D KIE. This unexpected behavior is examined in the context of other similar hydrogen transfer reactions.

Formation of Aza-ortho-quinone Methides Under Room Temperature Conditions: Cs2CO3 Effect.

Since the first report of a facile, room temperature process to access aza-ortho-quinone methides (aoQMs) by Corey in 1999, this chemistry has remained dormant until our report of an enantioselective catalytic example in 2014. We report a theoretical and experimental study of the key to success behind these successful examples to enable broader exploitation of this useful intermediate. We have discovered that transformations involving the aoQM are remarkably facile with barriers <17 kcal/mol. The main difficulty of exploiting aoQM in synthesis is that they are unstable (ΔG > 30 kcal/mol), precluding their formation under mild conditions. The use of Cs2CO3 as base is critical. It provides a thermodynamically and kinetically favorable means to form aoQMs, independent of the salt solubility and base strength. The exothermic formation of salt byproducts provides a driving force (average ΔG = -30.8 kcal/mol) compensating for the majority of the inherent unfavorable thermodynamics of aoQM formation.

A general approach to mechanism in multiproduct reactions: product-specific intermolecular kinetic isotope effects.

Here we report a general method for the measurement of (13)C kinetic isotope effects at natural abundance for reactions that yield two or more products concurrently. We use, as an example, a recently reported Co-catalyzed reaction between cyclopentene and 1-phenyl-1-propyne. High-precision intermolecular (13)C isotope effects are reported for both the formal [2+2] cycloaddition (major) and Alder-ene (minor) reaction products. Mechanistic possibilities that are in accord with observed isotope effect measurements are discussed.

Enthalpy/entropy contributions to conformational KIEs: theoretical predictions and comparison with experiment.

Previous theoretical studies of Mislow's doubly-bridged biphenyl ketone 1 and dihydrodimethylphenanthrene 2 have determined significant entropic contributions to their normal (1) and inverse (2) conformational kinetic isotope effects (CKIEs). To broaden our investigation, we have used density functional methods to characterize the potential energy surfaces and vibrational frequencies for ground and transition structures of additional systems with measured CKIEs, including [2.2]-metaparacyclophane-d (3), 1,1'-binaphthyl (4), 2,2'-dibromo-[1,1'-biphenyl]-4,4'-dicarboxylic acid (5), and the 2-(N,N,N-trimethyl)-2'-(N,N-dimethyl)-diaminobiphenyl cation (6). We have also computed CKIEs in a number of systems whose experimental CKIEs are unknown. These include analogs of 1 in which the C=O groups have been replaced with CH2 (7), O (8), and S (9) atoms and ring-expanded variants of 2 containing CH2 (10), O (11), S (12), or C=O (13) groups. Vibrational entropy contributes to the CKIEs in all of these systems with the exception of cyclophane 3, whose isotope effect is predicted to be purely enthalpic in origin and whose Bigeleisen-Mayer ZPE term is equivalent to DDH‡. There is variable correspondence between these terms in the other molecules studied, thus identifying additional examples of systems in which the Bigeleisen-Mayer formalism does not correlate with DH/DS dissections.

Extreme oxatriquinanes and a record C-O bond length.

Oxatriquinanes are fused, tricyclic oxonium ions that are known to have exceptional stability compared to simple alkyl oxonium salts. C-O bonds in ethers are generally ∼1.43 Å in length, but oxatriquinane has been found to have C-O bond lengths of 1.54 Å. A search of the Cambridge Structural Database turned up no bona fide C-O bond length exceeding this value. Computational modelling of oxatriquinane alongside other alkyl oxonium ions indicated that the electronic consequences of molecular strain were primarily responsible for the observed bond elongation. We also show that substitution of the oxatriquinane ring system with alkyl groups of increasing steric demand pushes the C-O bond to unheard of distances, culminating in a tert-butyl derivative at a predicted 1.60 Å. Chemical synthesis and an X-ray crystallographic study of these compounds validated the results of the modelling work and, finally, an extraordinary 1.622 Å C-O bond was observed in 1,4,7-tri-tert-butyloxatriquinane.

Investigating inner-sphere reorganization via secondary kinetic isotope effects in the C-H cleavage reaction catalyzed by soybean lipoxygenase: tunneling in the substrate backbone as well as the transferred hydrogen.

This work describes the application of NMR to the measurement of secondary deuterium (2° (2)H) and carbon-13 ((13)C) kinetic isotope effects (KIEs) at positions 9-13 within the substrate linoleic acid (LA) of soybean lipoxygenase-1. The KIEs have been measured using LA labeled with either protium (11,11-h2-LA) or deuterium (11,11-d2-LA) at the reactive C11 position, which has been previously shown to yield a primary deuterium isotope effect of ca. 80. The conditions of measurement yield the intrinsic 2° (2)H and (13)C KIEs on k(cat)/K(m) directly for 11,11-d2-LA, whereas the values for the 2° (2)H KIEs for 11,11-h2-LA are obtained after correction for a kinetic commitment. The pattern of the resulting 2° (2)H and (13)C isotope effects reveals values that lie far above those predicted from changes in local force constants. Additionally, many of the experimental values cannot be modeled by electronic effects, torsional strain, or the simple inclusion of a tunneling correction to the rate. Although previous studies have shown the importance of extensive tunneling for cleavage of the primary hydrogen at C11 of LA, the present findings can only be interpreted by extending the conclusion of nonclassical behavior to the secondary hydrogens and carbons that flank the position undergoing C-H bond cleavage. A quantum mechanical method introduced by Buhks et al. [J. Phys. Chem. 1981, 85, 3763] to model the inner-sphere reorganization that accompanies electron transfer has been shown to be able to reproduce the scale of the 2° (2)H KIEs.

Cationic intermediates in Friedel-Crafts acylation: structural information from theory and experiment.

Experimentally-determined intramolecular and intermolecular deuterium kinetic isotope effects and computational studies imply three possible kinetic scenarios for the Friedel-Crafts acylation of xylene. The influence of the structural and energetic properties of the π-complexes and σ-complex upon the observed isotope effects is explored.

Mechanism of the Swern oxidation: significant deviations from transition state theory.

Deprotonation of the alkoxysulfonium intermediate has been shown to be rate-determining in the Swern oxidation of benzyl alcohol. Directly following this rate-determining step is the intramolecular syn-ß-elimination of the ylide. In the present study, intramolecular (2)H kinetic isotope effects (KIEs) are used to gain insight into this syn-ß-elimination step. As a result of the stereogenic sulfur center in the ylide intermediate, two diastereomeric transition states (endo-TS1 and exo-TS1) must be assumed to contribute to the intramolecular KIE. The intramolecular (2)H KIE determined at -78 °C is 2.82 ± 0.06. Attempts to reproduce this measurement computationally using transition state theory and a Bell tunneling correction yielded a value (1.58) far below that determined experimentally. Computational analysis is complicated by the existence of two distinct transition structures owing to the stereogenic center. Two extremes of Curtin-Hammett kinetics are explored using energies, vibrational frequencies, and moments of inertia from computed transition structures. Neither Curtin-Hammett scenario can reproduce the observed KIE to any acceptable degree of fidelity. Evidence based upon previous kinetics measurements and calculations upon a model system suggests that the stereogenic sulfur center is not likely to undergo inversion to a significant degree at the temperatures at which the Swern oxidation is performed here. Proceeding under the assumption of no stereoinversion at the sulfur center, calculations predict a nearly linear Arrhenius plot for the KIE--even with the inclusion of a one-dimensional tunneling correction. By contrast, the experimentally determined temperature dependence shows a significant concave upward curvature indicative of the influence of tunneling. Notably, KIEs measured in CCl(4), CHCl(3), CH(2)Cl(2), dichloroethane, and chlorobenzene at -23 °C showed little variance. This finding discounted the possible influence from dynamical effects due to incomplete vibrational relaxation. An ad hoc amplification of the imaginary frequencies corresponding to the first-order saddle points corresponding to endo-TS1 and exo-TS1 allowed us to reproduce the experimental temperature dependence of the KIE using only two adjustable parameters applied to a kinetic scenario that involves four isotopomeric transition states. The cumulative data and computational modeling strongly suggest that, even though the intramolecular (2)H KIE observed in these experiments is small, this reaction requires a multidimensional description of the tunneling phenomenon to accurately reproduce experimental trends.

Kinetic isotope effects in asymmetric reactions.

Kinetic isotope effects are exquisitely sensitive probes of transition structure. As such, kinetic isotope effects offer a uniquely useful probe for the symmetry-breaking process that is inherent to stereoselective reactions. In this Concept article, we explore the role of steric and electronic effects in stereocontrol, and we relate these concepts to recent studies carried out in our laboratory. We also explore the way in which kinetic isotope effects serve as useful points of contact with computational models of transition structures. Finally, we discuss future opportunities for kinetic isotope effects to play a role in asymmetric catalyst development.

Nonbonding interactions and stereoselection in the Corey-Bakshi-Shibata reduction.

Deuterium kinetic isotope effect measurements upon enantiotopic methyl groups for the Corey-Bakshi-Shibata reduction of 2',5'-dimethylphenyl isopropyl ketone suggest a complex role for nonbonding interactions in the mediation of stereoselection.

Rate limiting step precedes C-C bond formation in the archetypical proline-catalyzed intramolecular aldol reaction.

The archetypical proline-catalyzed intramolecular aldol reaction, the Hajos-Parrish-Eder-Sauer-Wiechert reaction, has served as a model reaction for the mechanistic study of the ever-growing class of proline-catalyzed conversions. Experimental measurements of the (13)C kinetic isotope effects for this reaction show conclusively that carbon-carbon bond formation is not rate-limiting.

Computational and experimental structure-reactivity relationships: evidence for a side reaction in Alpine-Borane reductions of d-benzaldehydes.

Extraordinary stereoselectivity, approaching 100%, has been reported in the reductions of d-benzaldehydes by B-isopinocampheyl-9-borabicyclo[3.3.1]nonane (Alpine-Borane). This is likely because of the extreme size disparity of groups on either side of the carbonyl. Here, we present a structure-reactivity study whereby the reductions of variably substituted d-benzaldehydes are explored using highly sensitive measures for enantiomeric excess and relative reactivity. These results are compared to the relative rates predicted from density functional calculations. The results indicate that 2,6-disubstitution adversely affects the stereoselectivity by means of a non-selective reduction via the dehydroboration product of Alpine-Borane, 9-borabicyclo[3.3.1]nonane.

A mechanistic probe for asymmetric reactions: deuterium isotope effects at enantiotopic groups.

The development of a mechanistic probe that is especially suitable for the study of asymmetric reactions is presented. Chemically innocuous enantiotopic methyl groups are utilized as probes for the distinct environments that develop at the transition state for the (-)-B-chlorodiisopinocampheylborane reduction of 4'-methylisobutyrophenone. 2H kinetic isotope effects (KIEs) are determined for both enantiotopic methyl groups using two types of competition reactions. One competition is that between the d3-methyl enantiomeric isotopomers. The other competition reaction is that between the d6-dimethyl and perprotiated isotopologues. The rate constant ratios can be converted into kinetic isotope effects upon each of the individual enantiotopic methyl groups by invoking the rule of the geometric mean. The resulting isotope effect measurements yield highly precise values and contribute further understanding to the transition structure for this stereoselective reduction. The results are discussed in the context of steric isotope effects and the origins of these effects, which arise from the impact of steric crowding upon the anharmonicity of C-H bonds in the transition structure relative to the reactant state.

Susceptibility of Chlamydia trachomatis to the excipient hydroxyethyl cellulose: pH and concentration dependence of antimicrobial activity.

Hydroxyethyl cellulose (HEC) is used as a neutral excipient in microbicides used against sexually transmitted pathogens. However, HEC inhibits the infection of cervical epithelial cells by Chlamydia trachomatis at pH 5 in a concentration-dependent manner. At pH 7, infection is inversely dependent on the concentration of HEC, possibly due to pH-dependent calcium sequestration.

High rate of mobilization for blaCTX-Ms.

We constructed a phylogenetic analysis of class A beta-lactamases and found that the blaCTX-Ms have been mobilized to plasmids approximately 10 times more frequently than other class A beta-lactamases. We also found that the blaCTX-Ms are descended from a common ancestor that was incorporated in ancient times into the chromosome of the ancestor of Kluyvera species through horizontal transfer. Considerable sequence divergence has occurred among the descendents of that ancestral gene sequence since that gene was inserted. That divergence has mainly occurred in the presence of purifying selection, which indicates a slow rate of evolution for blaCTX-Ms in the pre-antimicrobial drug era.

Enzyme structure and dynamics affect hydrogen tunneling: the impact of a remote side chain (I553) in soybean lipoxygenase-1.

This study examines the impact of a series of mutations at position 553 on the kinetic and structural properties of soybean lipoxygenase-1 (SLO-1). The previously uncharacterized mutants reported herein are I553L, I553V, and I553G. High-resolution x-ray studies of these mutants, together with the earlier studied I553A, show almost no structural change in relation to the WT-enzyme. By contrast, a progression in kinetic behavior occurs in which the decrease in the size of the side chain at position 553 leads to an increased importance of donor-acceptor distance sampling in the course of the hydrogen transfer process. These dynamical changes in behavior are interpreted in the context of two general classes of protein motions, preorganization and reorganization, with the latter including the distance sampling modes [Klinman JP (2006) Philos Trans R Soc London Ser B 361:1323-1331; Nagel Z, Klinman JP (2006) Chem Rev 106:3095-3118]. The aggregate data for SLO-1 show how judicious placement of hydrophobic side chains can influence enzyme catalysis via enhanced donor-acceptor hydrogenic wave function overlap.

Synthesis of linoleic acids combinatorially-labeled at the vinylic positions as substrates for lipoxygenases.

Mammalian lipoxygenases have been implicated in a number of inflammation-related human diseases. Soybean lipoxygenase-1 is the archetypical example of known lipoxygenases. Here we report the synthesis of linoleic acid and (11,11)-d2-linoleic acid which are combinatorially labeled at the vinylic positions (9, 10, 12, and 13). Combinatorial labeling schemes allow for the simultaneous determination of KIEs in enzymatic reactions using NMR. Substrates are, thus, available as probes of detailed mechanism in kinetic isotope effect (KIE) studies of lipoxygenases.