Selective Displacement Research Articles

Determining Protease Substrate Selectivity and Inhibition by Label-Free Supramolecular Tandem Enzyme Assays

An analytical method has been developed for the continuous monitoring of protease activity on unlabeled peptides in real time by fluorescence spectroscopy. The assay is enabled by a reporter pair comprising the macrocycle cucurbit[7]uril (CB7) and the fluorescent dye acridine orange (AO). CB7 functions by selectively recognizing N-terminal phenylalanine residues as they are produced during the enzymatic cleavage of enkephalin-type peptides by the metalloendopeptidase thermolysin. The substrate peptides (e.g., Thr-Gly-Ala-Phe-Met-NH(2)) bind to CB7 with moderately high affinity (K ≈ 10(4) M(-1)), while their cleavage products (e.g., Phe-Met-NH(2)) bind very tightly (K > 10(6) M(-1)). AO signals the reaction upon its selective displacement from the macrocycle by the high affinity product of proteolysis. The resulting supramolecular tandem enzyme assay effectively measures the kinetics of thermolysin, including the accurate determination of sequence specificity (Ser and Gly instead of Ala), stereospecificity (d-Ala instead of l-Ala), endo- versus exopeptidase activity (indicated by differences in absolute fluorescence response), and sensitivity to terminal charges (-CONH(2) vs -COOH). The capability of the tandem assay to measure protease inhibition constants was demonstrated on phosphoramidon as a known inhibitor to afford an inhibition constant of (17.8 ± 0.4) nM. This robust and label-free approach to the study of protease activity and inhibition should be transferable to other endo- and exopeptidases that afford products with N-terminal aromatic amino acids.

Stereoselective Synthesis of MLN4924, an Inhibitor of NEDD8-Activating Enzyme

MLN4924 (1), which is in clinical trials as an anticancer agent, was stereoselectively synthesized from d-ribose via a route involving stereoselective reduction, regioselective cleavage of an isopropylidene moiety, and selective displacement of a cyclic sulfate moiety as key steps.

Alkyl Based Selective Displacers for Protein Purification in Ion Exchange Chromatography

Robotic high-throughput batch screening was carried out to examine the efficacy of positively charged, alkyl based molecules as chemically selective displacers. Steric mass action (SMA) dynamic affinity plots were constructed, identifying some of the displacers as SMA selective. Saturation transfer difference NMR experiments were conducted, identifying other displacers as chemically selective. These analyses identified molecules which gave selective separations, but did not conform to either the SMA or chemically selective model. Finally, dynamic light scattering experiments were conducted to directly detect micelle formation of the displacers. The use of micelles potentially adds an orthogonal dimension of selectivity to the displacement process.

Unique selectivity windows using selective displacers/eluents and mobile phase modifiers on hydroxyapatite

A detailed study was carried out to combine the unique selectivity of ceramic hydroxyapatite (CHA) with the separation power of selective displacement chromatography. A robotic liquid handling system was employed to carry out a parallel batch screen on a displacer library made up of analogous compounds. By incorporating positively charged, metal chelating and/or hydrogen bonding groups into the design of the displacer, specific interaction sites on CHA were targeted, thus augmenting the selectivity of the separation. The effect of different mobile phase modifiers, such as phosphate, sulfate, lactate and borate, were also investigated. Important functional group moieties and trends for the design of CHA displacers were established. Selective batch separations were achieved between multiple protein pairs which were unable to be resolved using linear gradient techniques, demonstrating the applicability of this technique to multiple protein systems. The specific interaction moieties used on the selective displacer were found to dictate which protein was selectively displaced in the separation, a degree of control not possible using a mono-interaction type resin in displacement chromatography. Mobile phase modifiers were also shown to play a crucial role, augmenting the selectivity of a displacer in a synergistic fashion. Column separations were carried out using selective displacers and mobile phase modifiers identified in the batch experiments, and baseline separation of the previously unresolved protein pairs was achieved. Further, the elution order in these systems was able to be reversed while still maintaining baseline separations. This work establishes a new class of separations which combine the selectivities of multi-modal resins, displacers/eluents, and mobile phase modifiers to create unique selectivity windows unattainable using traditional modes of operation.

Synthesis of 5-hydroxyquinolines

A series of 5-hydroxyquinolines has been prepared via the Skraup reaction. Several regioisomers were made either by selective displacement of a leaving group or by using a bromo substituent as a blocking group. The bromo group was found to be an excellent blocking group due to its stability during the Skraup reaction and easy removal thereafter. Halides at the 5-position of quinoline were found to be much more reactive than those at the 7- and 8-positions. Finally, we have also found a unique method to reduce the pyridyl ring on quinolines, leaving a halogen substituent untouched.

A Synthesis of 3,5-Disubstituted Phenols

Robust and scalable syntheses of some synthetically useful 3,5-disubstituted phenols are presented. The process involves the selective displacement of a halogen by nucleophilic aromatic substitution using a preformed mixture of potassium tert-butoxide and p-methoxybenzyl alcohol (PMB−OH), followed by deprotection of the PMB ether with acid in the presence of 1,3-dimethoxybenzene. These processes have been demonstrated on kilogram scale, providing crystalline phenols in good yield and high purity.

Simultaneous and selective preconcentration of trace Cu and Ag by one-step displacement cloud point extraction for FAAS determination

A simple, rapid and selective one-step displacement cloud point extraction (D-CPE) procedure was developed for the simultaneous preconcentration of trace Cu and Ag followed by flame atomic absorption spectrometric (FAAS) measurement. In this proposed procedure, Ni-DDTC complex instead of DDTC was used to achieve one-step extraction. Since the stability of Cu-DDTC and Ag-DDTC is higher than that of Ni-DDTC, simultaneous preconcentration of Cu and Ag can be achieved in a single step. Up to 15,000 mg L(-1) Co(2+), 10,000 mg L(-1) Zn(2+), 5000 mg L(-1) Cd(2+) and 5000 mg L(-1) Al(3+) did not cause significant interferences for the determination of 50 microg L(-1) Cu by FAAS, while 8000 mg L(-1) Zn(2+), 4000 mg L(-1) Co(2+), 4000 mg L(-1) Cd(2+) and 5000 mg L(-1) Al(3+) had no distinct effect on the determination of 80 microg L(-1) Ag. The limits of detection (LODs) of 0.5 microg L(-1) for Cu and 1.0 microg L(-1) for Ag, respectively, were obtained by FAAS detection. The accuracy of the proposed method is validated by analyzing certified reference materials (GBW 07405, GBW 07311 and GBW 07260) with satisfactory results. Furthermore, the one-step D-CPE was also applied for the preconcentration/separation of Cu and Ag in high-purity salts and artificial seawater, followed by FAAS or inductively coupled plasma-mass spectrometry (ICP-MS) determination.

Silver-Catalyzed Diallylation and Dibenzylation of gem-Dibromoalkanes with Grignard Reagents

Treatment of gem-dibromoalkanes with allylic Grignard reagents in the presence of a catalytic amount of silver triflate in diethyl ether leads to diallylation to afford 1,6-heptadiene derivatives. Benzylic Grignard reagents also undergo a similar transformation to yield 1,3-diphenylpropanes. Controlled sequential treatment of gem-dibromoalkane with two different Grignard reagents results in selective displacement of the two bromine atoms with allylic and benzylic moieties.

The effect of feed composition on the behavior of chemically selective displacement systems

In this paper we examine whether adding a more retained protein to the feed will mitigate displacer–protein interactions in the column, thus affecting the displacement modality that occurs (chemically selective vs. traditional displacement chromatography). STD-NMR experiments were carried out to probe displacer–protein interactions for the chemically selective displacer chloroquine diphosphate and the results indicated that this displacer only had measurable interactions with the protein α-chymotrypsinogen A. For a two component feed mixture containing ribonuclease A and α-chymotrypsinogen A, the separation resulted in the displacement of ribonuclease A, with the more hydrophobic α-chymotrypsinogen A remaining on the column. On the other hand, when the experiment was repeated with cytochrome c added to the feed, all three feed proteins were displaced. Column simulations indicated that the combination of sample self-displacement occurring during the introduction of the feed, along with the dynamics of the initial displacement process at the column inlet was responsible for this behavior. These results indicate that for this class of hydrophobic-based selective displacers, in order for the protein to be selectively retained, the protein should be the most strongly retained feed component.

An anion sensor based on calix[4]arene-Reichardt’s dye

A highly selective colorimetric displacement assay for anions, F− and H2PO4−, based on calix[4]arene-Reichardt’s dye was studied in DMSO. When calix[4]arene forms a complex with Reichardt’s, the absorption peak at 386, 419 and 632 nm disappeared. Addition of anions to solution of calix[4]arene-Reichardt’s dye causes the blue color of Reichardt’s dye to return as it is displaced from the calix[4]arene. These results demonstrate that this displacement assay can be favorably utilized for the development of potential sensors for the detection of anion such as F− and H2PO4− in relation to the other anions.

Mesoporous Hybrid Materials Containing Nanoscopic “Binding Pockets” for Colorimetric Anion Signaling in Water by using Displacement Assays

Mesoporous solids functionalized with anion-binding groups have proved to be suitable anion hosts and have been used in selective colorimetric displacement assays. The material UVM-7, a mesoporous MCM41-type support characterized by the presence of nanometric mesoporous particle conglomerates, was selected as inorganic scaffolding. Reaction of the template-free UVM-7 solid with 3-aminopropyltriethoxysilane (1) yielded solid S1, from which the derivatives S2 and S3 were obtained by reaction with 2-methylthio-2-imidazoline hydroiodide (2) and butyl isocyanate (3), respectively. Solids S4 and S5 were prepared by reaction of the starting mesoporous UVM-7 scaffolding with N-methyl-N'-propyltrimethoxysilyl imidazolium chloride (4) and with 3-(trimethoxysilyl)propyl-N,N,N-trimethylammonium chloride (5), respectively. The solids synthesized contain mesoporous binding pockets that can interact with anions through electrostatic attractive forces (S1, S2, S4, S5) and hydrogen-bonding interactions (S1, S2, S3, S4). These functionalized solids were loaded with a dye (d) capable of interacting coordinatively with the anchored binding sites, in our case 5-carboxyfluorescein, to yield the hybrid materials S1d, S2d, S3d, S4d and S5d. These dye-containing solids are the signaling reporters. Their sensing ability towards a family of carboxylates, namely acetate, citrate, lactate, succinate, oxalate, tartrate, malate, mandelate, glutamate and certain nucleotides, has been studied in pure water at pH 7.5 (Hepes, 0.01 mol dm(-3)). In the sensing protocol, a particular analyte may be bonded preferentially by the nanoscopic functionalized pocket, leading to delivery of the dye to the solution and resulting in colorimetric detection of the guest. The response to a given anion depends on the characteristics of the binding pockets and the specific interaction of the anion with the binding groups in the mesopores. We believe that the possibility of using a wide variety of mesoporous supports that can easily be functionalized with anion-binding sites, combined with suitable dyes as indicators, make this approach significant for opening new perspectives in the design of chromogenic assays for anion detection in pure water.

Job Displacement and Mortality: An Analysis Using Administrative Data*

We use administrative data on the quarterly employment and earnings of Pennsylvanian workers in the 1970s and 1980s matched to Social Security Administration death records covering 1980–2006 to estimate the effects of job displacement on mortality. We find that for high-seniority male workers, mortality rates in the year after displacement are 50%–100% higher than would otherwise have been expected. The effect on mortality hazards declines sharply over time, but even twenty years after displacement, we estimate a 10%–15% increase in annual death hazards. If such increases were sustained indefinitely, they would imply a loss in life expectancy of 1.0–1.5 years for a worker displaced at age forty. We show that these results are not due to selective displacement of less healthy workers or to unstable industries or firms offering less healthy work environments. We also show that workers with larger losses in earnings tend to suffer greater increases in mortality. This correlation remains when we examine predicted earnings declines based on losses in industry, firm, or firm-size wage premiums.

Evaluation of Chemically Selective Displacer Analogues for Protein Purification

A library of molecular analogues to the selective displacer, N'1'-(4-methylquinolin-2-yl)ethane-1,2-diamine dinitrate, was employed to study the effects of changes in displacer chemistry on their efficacy for selective separations. High throughput screens were carried out using a robotic liquid handling system to examine the ability of these compounds to selectively displace proteins in batch adsorption systems. Experiments were conducted using the model protein pairs ribonuclease A/alpha-chymotrypsinogen A and cytochrome C/lysozyme on a strong cation exchanger. Selectivity pathway and DC-50 plots were constructed from the analogue screen data, and results indicated that minor changes in the molecular design of the displacer can have a significant impact on the separation behavior. Specifically, charge density and spacing of resin and protein interaction moieties were found to be important. The screen also identified a new displacer, 4-methyl-2-piperazin-1-yl-quinoline, which produced a more selective displacement than previously reported with the original compound. A steric mass action dynamic affinity plot was constructed to validate that this new displacer was acting as a chemically selective, rather than a steric mass action selective displacer. Finally, saturation transfer difference NMR experiments were conducted to examine protein-displacer interactions with these displacers and protein pairs. These results demonstrate how subtle changes in displacer design can be employed to fine-tune the separation performance of chemically selective displacers.

Inhibitory Inputs to Hippocampal Interneurons Are Reorganized in Lis1 Mutant Mice

Epilepsy and brain malformation are commonly associated with excessive synaptic excitation and decreased synaptic inhibition of principal neurons. However, few studies have examined the state of synaptic inhibition of interneurons in an epileptic, malformed brain. We analyzed inhibitory inputs, mediated by gamma-aminobutyric acid (GABA), to hippocampal interneurons in a mouse model of type 1 lissencephaly, a neurological disorder linked with severe seizures and brain malformation. In the disorganized hippocampal area CA1 of Lis1(+/-) mice, we initially observed a selective displacement of fast-spiking, parvalbumin-positive basket-type interneurons from stratum oriens (SO) locations to s. radiatum and s. lacunosum-moleculare (R/LM). Next, we recorded spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) onto visually identified interneurons located in SO or R/LM of Lis1(+/-) mice and age-matched littermate controls. We observed significant, layer-specific reorganizations in GABAergic inhibition of interneurons in Lis1 mutant mice. Spontaneous IPSC frequency onto SO interneurons was significantly increased in hippocampal slices from Lis1(+/-) mice, whereas mIPSC mean amplitude onto these interneurons was significantly decreased. In addition, the weighted decay times of sIPSCs and mIPSCs were significantly increased in R/LM interneurons. Taken together, these findings illustrate the extensive redistribution and reorganization of inhibitory connections between interneurons that can take place in a malformed brain.

Characterization and design of chemically selective cationic displacers using a robotic high‐throughput screen

A robotic high-throughput displacer screen was developed and employed to identify chemically selective displacers for several protein pairs in cation exchange chromatography. This automated screen enabled the evaluation of a wide range of experimental conditions in a relatively short period of time. Displacers were evaluated at multiple concentrations for these protein pairs, and DC-50 plots were constructed. Selectivity pathway plots were also constructed and different regimes were established for selective and exclusive separations. Importantly, selective displacement was found to be conserved for multiple protein pairs, demonstrating the technique to be applicable for a range of protein systems. Although chemically selective displacers were able to separate protein pairs that had similar retention in ion exchange but different surface hydrophobicities, they were not able to distinguish protein pairs with similar surface hydrophobicities. This corroborates that displacer-protein hydrophobic interactions play an important role for this class of selective displacers. Important functional group moieties were established and efficient displacers were identified. These results demonstrate that the design of chemically selective displacers requires a delicate balance between the abilities to displace proteins from the resin and to bind to a selected protein. The use of robotic screening of displacers will enable the extension of chemically selective displacement chromatography beyond hydrophobic displacer-protein interactions to other secondary interactions and more selective displacement systems.

Synthesis of novel pyrrolidine 3,4-diol derivatives as inhibitors of α-L-fucosidases

The stereoselective synthesis of new 3,4-dihydroxypyrrolidine derivatives starting from D-mannose, D-ribose and L-fucose is presented. Two synthetic strategies employing organometallic addition to hemiacetalic sugars followed by selective nucleophilic displacement or conjugate addition of ammonia to conjugate aldonic esters as key steps, are used. The new compounds were assayed for their inhibitory activity towards 13 commercially available glycosidases. Compounds that share the absolute configuration at C(2,3,4,5) of L-fucopyranosides and incorporate aromatic moieties are potent and selective inhibitors of alpha-L-fucosidases in the nM range.

Selective wetting in a hydrocarbon liquid-perfluorocarbon liquid-solid system

Selective (displacement) wetting is, for the first time, investigated in a hydrocarbon liquid-perfluorocarbon liquid-fluorinated polyethylene system. Contact angles upon the selective wetting are shown to be quite sensitive to the presence of fluorocarbon groups on the surface of the polymer, and wetting inversion takes place when the surface concentration of fluorine is increased above 10−4 kg/m2.

Mechanistic studies of displacer–protein binding in chemically selective displacement systems using NMR and MD simulations

A parallel batch screening technique was employed to identify chemically selective displacers which exhibited exclusive separation behavior for the protein pair alpha-chymotrypsin/ribonuclease A on a strong cation exchange resin. Two selective displacers, 1-(4-chlorobenzyl)piperidin-3-aminesulfate and N'1'-(4-methyl-quinolin-2-yl)-ethane-1,2-diamine dinitrate, and one non-selective displacer, spermidine, were selected as model systems to investigate the mechanism of chemically selective displacement chromatography. Saturation transfer difference (STD) NMR was used to directly evaluate displacer-protein binding. The results indicated that while binding occurred between the two chemically selective displacers and the more hydrophobic protein, alpha-chymotrypsin, no binding was observed with ribonuclease A. Further, the non-selective displacer, spermidine, was not observed to bind to either protein. Importantly, the binding event was observed to occur primarily on the aromatic portion of the selective displacers. Extensive molecular dynamic simulations of protein-displacer-water solution were also carried out. The MD results corroborated the NMR findings demonstrating that the binding of selective displacers occurred primarily on hydrophobic surface patches of alpha-chymotrypsin, while no significant long term binding to ribonuclease A was observed. The non-selective displacer did not show significant binding to either of the proteins. MD simulations also indicated that the charged amine group of the selective displacers in the bound state was primarily oriented towards the solvent, potentially facilitating their interaction with a resin surface. These results directly confirm that selective binding between a protein and displacer is the mechanism by which chemically selective displacement occurs. This opens up many possibilities for future molecular design of selective displacers for a range of applications.

Synthesis and Characterization of Fluorescent Displacers for Online Monitoring of Displacement Chromatography

One of the major impediments to the implementation of displacement chromatography for the purification of biomolecules is the need to collect fractions from the column effluent for time-consuming offline analysis. The ability to employ direct online monitoring of displacement chromatography would have significant implications for applications ranging from analytical to preparative bioseparations. To this end, a set of novel fluorescent displacers were rationally designed using known chemically selective displacers as a template. Fluorescent cores were functionalized with different charge moieties, creating a homologous library of displacers. These compounds were then tested on two protein pairs, alpha-chymotrypsinogen A/ribonuclease A and cytochrome c/lysozyme, using batch and column displacement experiments. Of the synthesized displacers, two were found to be highly selective while one was determined to be a high-affinity displacer. Column displacements using one of the selective displacers yielded complete separation of both protein pairs while facilitating direct online detection using UV and fluorescence detection. Saturation transfer difference NMR was also carried out to investigate the binding of the fluorescent displacers to proteins. The results indicated a selective binding between the selective displacers and alpha-chymotrypsinogen A, while no binding was observed for ribonuclease A, confirming that protein-displacer binding is responsible for the selectivity in these systems. This work demonstrates the utility of fluorescent displacers to enable online monitoring of displacer breakthroughs while also acting as efficient displacers for protein purification.

Reversible Carboxamide-Mediated Internal Activation at C(6) of 2-Chloro-4-anilino-1H-pyrrolo[2,3-d]pyrimidines

A synthetic route to bisanilino-1H-pyrrolo[2,3-d]pyrimidines has been discovered, wherein the C(6)-chloride reactivity is necessarily enhanced via reversible acid-catalyzed internal activation of the pyrimidine ring by a C(1')-carboxamide moiety. Subsequent selective nucleophilic displacements at C(6) and C(1') constitute a one-pot tandem protocol for the rapid assembly of bisanilino-1H-pyrrolo[2,3-d]pyrimidines.