Theoretical study of chloroperoxidase catalyzed chlorination of beta-cyclopentanedione and role of water in the chlorination mechanism

Chloroperoxidase (CPO) is a potential biocatalyst for use in asymmetric synthesis. The mechanisms of CPO catalysis are therefore of interest. The halogenation reaction, one of several chemical reactions that CPO catalyzes, is not fully understood and is the subject of this dissertation. The mechanism by which CPO catalyzes halogenation is disputed. It has been postulated that halogenation of substrates occurs at the active site. Alternatively, it has been proposed that hypochlorous acid, produced at the active site via oxidation of chloride, is released prior to reaction, so that halogenation occurs in solution. The free-solution mechanism is supported by the observation that halogenation of most substrates often occurs non-stereospecifically. On the other hand, the enzyme-bound mechanism is supported by the observation that some large substrates undergo halogenation stereospecifically. The major purpose of this research is to compare chlorination of the substrate β-cyclopentanedione in the two environments. One study was of the reaction with limited hydration because such a level of hydration is typical of the active site. For this work, a purely quantum mechanical approach was used. To model the aqueous environment, the limited hydration environment approach is not appropriate. Instead, reaction precursor conformations were obtained from a solvated molecular dynamics simulation, and reaction of potentially reactive molecular encounters was modeled with a hybrid quantum mechanical/molecular mechanical approach. Extensive work developing parameters for small molecules was pre-requisite for the molecular dynamics simulation. It is observed that a limited and optimized (active-site-like) hydration environment leads to a lower energetic barrier than the fully solvated model representative of the aqueous environment at room temperature, suggesting that the stable water network near the active site is likely to facilitate the chlorination mechanism. The influence of the solvent environment on the reaction barrier is critical. It is observed that stabilization of the catalytic water by other solvent molecules lowers the barrier for keto-enol tautomerization. Placement of water molecules is more important than the number of water molecules in such studies. The fully-solvated model demonstrates that reaction proceeds when the instantaneous dynamical water environment is close to optimal for stabilizing the transition state.

Magnetic nanoformulation of azidothymidine 5’-triphosphate for targeted delivery across the blood–brain barrier

Despite significant advances in highly active antiretroviral therapy (HAART), the prevalence of neuroAIDS remains high. This is mainly attributed to inability of antiretroviral therapy (ART) to cross the blood–brain barrier (BBB), thus resulting in insufficient drug concentration within the brain. Therefore, development of an active drug targeting system is an attractive strategy to increase the efficacy and delivery of ART to the brain. We report herein development of magnetic azidothymidine 5′-triphosphate (AZTTP) liposomal nanoformulation and its ability to transmigrate across an in vitro BBB model by application of an external magnetic field. We hypothesize that this magnetically guided nanoformulation can transverse the BBB by direct transport or via monocyte-mediated transport. Magnetic AZTTP liposomes were prepared using a mixture of phosphatidyl choline and cholesterol. The average size of prepared liposomes was about 150 nm with maximum drug and magnetite loading efficiency of 54.5% and 45.3%, respectively. Further, magnetic AZTTP liposomes were checked for transmigration across an in vitro BBB model using direct or monocyte-mediated transport by application of an external magnetic field. The results show that apparent permeability of magnetic AZTTP liposomes was 3-fold higher than free AZTTP. Also, the magnetic AZTTP liposomes were efficiently taken up by monocytes and these magnetic monocytes showed enhanced transendothelial migration compared to normal/non-magnetic monocytes in presence of an external magnetic field. Thus, we anticipate that the developed magnetic nanoformulation can be used for targeting active nucleotide analog reverse transcriptase inhibitors to the brain by application of an external magnetic force and thereby eliminate the brain HIV reservoir and help to treat neuroAIDS.

Water Quality and Wet Season Diatom Assemblage Characteristics from the Tamiami Trail Pilot Swales Sites (Everglades National Park, Florida, USA)

A pivotal component of hydrological restoration of the Florida Everglades is the improvement of water conveyance to Everglades National Park by the degradation of the current network of canals, roadways and levees. The Tamiami Trail (L29) road/canal complex represents a major barrier to natural water flows into the park and a variety of modification options for flow improvement are currently being explored, including the installation of spreader swales immediately downstream of culverts conveying water under Tamiami Trail from the L29 canal into Everglades National Park. In this study, we evaluated water column chemistry and wet-season diatom community structure to provide baseline information for use in future monitoring activities related to the proposed Tamiami Trail modifications. Water chemistry showed pronounced fluctuations in response to precipitation and anthropogenically mediated hydrological events. Differences in water quality variables among sites were dampened during periods of inundation, and became more pronounced during periods of low canal stage, suggesting the importance of small-scale mechanisms related to isolation of habitat patches. Diatom assemblages were unexpectedly speciose (127 taxa in 40 samples) compared to typical Everglades assemblages, and spatially heterogeneous in sites associated with concentric areas of dense vegetation immediately downstream of culverts. We also observed significant compositional dissimilarities among transects, indicating that culvert pool and north transect assemblages were substantially influenced by propagule input from the canal and areas to the north, while south transect sites were compositionally similar to typical sawgrass prairie diatom communities. Central transect sites were compositionally intermediate to their north and south counterparts. We propose that the position and spatial extent of this “transitional assemblage” is a sensitive indicator of subtle environmental change related to Tamiami Trail modifications.