Dynamics of an enzymatic substitution reaction in haloalkane dehalogenase

Reactive flux molecular dynamics simulations have been carried out using a combined QM/MM potential to study the dynamics of the nucleophilic substitution reaction of dichloroethane by a carboxylate group in haloalkane dehalogenase and in water. We found that protein dynamics accelerates the reaction rate by a factor of 2 over the uncatalyzed reaction. Compared to the thermodynamic effect in barrier reduction, protein dynamic contribution is relatively small. However, analyses of the friction kernel reveal that the origins of the reaction dynamics in water and in the enzyme are different. In aqueous solution, there is significant electrostatic solvation effect, which is reflected by the slow reorganization relaxation of the solvent. On the other hand, there is no strong electrostatic coupling in the enzyme and the major effect on reaction coordinate motion is intramolecular energy relaxation.     

Direct, two-step synthetic pathway to novel dibenzo[a,c]phenanthridines

Novel dibenzo[a,c]phenanthridines are prepared regioselectively by the application of a straightforward synthetic pathway, starting from new 3,4-diaryl- and 3,4-dihydro-3,4-diarylisoquinolines prepared via Ritter-type heterocyclization and the more classical two-step reductive amination/Bischler-Napieralski cyclization of triarylethanones, respectively. A comparative study of nonphenolic oxidative coupling methodologies provides a highly efficient procedure, based on the hypervalent iodine reagent phenyliodine(III) bis(trifluoroacetate) (PIFA), to accomplish the final coupling step.     

Using second-order calibration to identify and quantify aromatic sulfonates in water by high-performance liquid chromatography in the presence of coeluting interferences

We used the Generalized Rank Annihilation Method (GRAM), a second-order calibration method, to quantify aromatic sulfonates in water with high-performance liquid chromatography (HPLC) when interferences coeluted with the analytes of interest. With GRAM, we can quantify in only two chromatographic analyses, one for a calibration sample and one for the unknown sample. The calculated concentrations were not statistically different to those obtained when the chromatographic separation of the unknown sample was modified in order to completely separate the analyte from the interferences before univariate calibration. With GRAM, the concentrations are determined much more quickly because a complete resolution is not required.     

Verification of gamma-spectroscopy programs: Accuracy and detectability

The newly revised ANSI N42.14^1.2 has provided analysis software developers with a set of well defined, consistent and unbiased procedures designed to evaluate the accuracy and limitations of peak search and peak area analysis programs. This work uses two of the procedures outlined in this standard to evaluate five peak analysis algorithms currently in use in Canberra and Nuclear Data software packages. The first procedure examines a program's behavior as the centroid separation and peak height ratio of a doublet are varied. A previous review of these data³ demonstrated significant peak area inaccuracies at peak separations at or below 1.5 FWHM. We will discuss improvements made to some of these programs and the impact on the doublet results. The second procedure examines a program's behavior as the Compton continuum beneath a fixed peak area is increased. For the same five algorithms we will discuss the dependence of peak area on Compton continuum and also explore the limits of peak detectability.     

The electronic spectrum of the triethylamine-perfluoro-tert-butanol complex

The far-ultraviolet absorption spectrum of the triethylamine-perfluoro-tert-butanol complex has been measured in the gas phase. The photoelectron band of lowest energy has also been determined. It is about 13000 cm^?1 higher than for free triethylamine. Up to at least 60000 cm^?1, the UV spectrum is readily interpreted as a triethylamine spectrum shifted to higher frequencies by about 13000 cm^?1. The bands of lowest frequency are the 3s and 3p Rydberg bands which follow the ionization potential. Up to 60000 cm^?1, no charge transfer type band has been found.     

Determination of weak (2.0–2.5) dissociation constants of non-UV absorbing solutes by capillary electrophoresis

Summary Capillary electrophoresis (CE) has proved to be a fast and convenient method for the determination of the dissociation constants of non-UV absorbing solutes in the acidic pK _A range (2.0–2.5). The electroosmotic flow was reversed by washing the capillary with 0.2% polybren aqueous solution. A series of background electrolytes was prepared with phenylphosphonic acid (pK _A=1.29) and β-alanine (pK _A=3.55) with the same ionic strength and a high buffer capacity in order to improve the repeatability (0.1–0.2 %) of the electrophoretic mobility and to determine the values of pK _A accurately. This procedure was applied to the determination of the dissociation constants of several alkyl-alkylphosphonic acids whose pK _A values have not yet been published in the literature. In this work, their dissociation constants have been found to vary between 1.91 and 2.34 for alkyl-methylphosphonic acids and between 2.10 and 2.38 for alkyl-ethylphosphonic acids.     

Target-templated de novo design of macrocyclic d-/l-peptides: discovery of drug-like inhibitors of PD-1

Peptides are a rapidly growing class of therapeutics with various advantages over traditional small molecules, especially for targeting difficult protein–protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing bioactive cyclic topologies that go beyond natural l-amino acids. Here, we report a generalizable framework that exploits the computational power of Rosetta, in terms of large-scale backbone sampling, side-chain composition and energy scoring, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we developed two new inhibitors (PD-i3 and PD-i6) of programmed cell death 1 (PD-1), a key immune checkpoint in oncology. A comprehensive biophysical evaluation was performed to assess their binding to PD-1 as well as their blocking effect on the endogenous PD-1/PD-L1 interaction. Finally, NMR elucidation of their in-solution structures confirmed our de novo design approach.

In silico design of heterochiral cyclic peptides that bind to a specific surface patch on the target protein (PD-1, in this case) and disrupt protein–protein interactions.     

Design and characterization of libraries of molecular fragments for use in NMR screening against protein targets

We have designed four generations of a low molecular weight fragment library for use in NMR-based screening against protein targets. The library initially contained 723 fragments which were selected manually from the Available Chemicals Directory. A series of in silico filters and property calculations were developed to automate the selection process, allowing a larger database of 1.79 M available compounds to be searched for a further 357 compounds that were added to the library. A kinase binding pharmacophore was then derived to select 174 kinase-focused fragments. Finally, an additional 61 fragments were selected to increase the number of different pharmacophores represented within the library. All of the fragments added to the library passed quality checks to ensure they were suitable for the screening protocol, with appropriate solubility, purity, chemical stability, and unambiguous NMR spectrum. The successive generations of libraries have been characterized through analysis of structural properties (molecular weight, lipophilicity, polar surface area, number of rotatable bonds, and hydrogen-bonding potential) and by analyzing their pharmacophoric complexity. These calculations have been used to compare the fragment libraries with a drug-like reference set of compounds and a set of molecules that bind to protein active sites. In addition, an analysis of the overall results of screening the library against the ATP binding site of two protein targets (HSP90 and CDK2) reveals different patterns of fragment binding, demonstrating that the approach can find selective compounds that discriminate between related binding sites.