Comparative analysis of viscosity of complex liquids and cytoplasm of mammalian cells at the nanoscale

We present a scaling formula for size-dependent viscosity coefficients for proteins, polymers, and fluorescent dyes diffusing in complex liquids. The formula was used to analyze the mobilities of probes of different sizes in HeLa and Swiss 3T3 mammalian cells. This analysis unveils in the cytoplasm two length scales: (i) the correlation length ξ (approximately 5 nm in HeLa and 7 nm in Swiss 3T3 cells) and (ii) the limiting length scale that marks the crossover between nano- and macroscale viscosity (approximately 86 nm in HeLa and 30 nm in Swiss 3T3 cells). During motion, probes smaller than ξ experienced matrix viscosity: η(matrix) ≈ 2.0 mPa·s for HeLa and 0.88 mPa·s for Swiss 3T3 cells. Probes much larger than the limiting length scale experienced macroscopic viscosity, η(macro) ≈ 4.4 × 10(-2) and 2.4 × 10(-2) Pa·s for HeLa and Swiss 3T3 cells, respectively. Our results are persistent for the lengths scales from 0.14 nm to a few hundred nanometers.     

One‐Step C‐Terminal Deprotection and Activation of Peptides with Peptide Amidase from Stenotrophomonas maltophilia in Neat Organic Solvent

Chemoenzymatic peptide synthesis is a rapidly developing technology for cost effective peptide production on a large scale. As an alternative to the traditional C→N strategy, which employs expensive N‐protected building blocks in each step, we have investigated an N→C extension route that is based on activation of a peptide C‐terminal amide protecting group to the corresponding methyl ester. We found that this conversion is efficiently catalysed by Stenotrophomonas maltophilia peptide amidase in neat organic media. The system excludes the possibility of internal peptide cleavage as the enzyme lacks intrinsic protease activity. The produced peptide methyl ester was used for peptide chain extension in a kinetically controlled reaction by a thermostable protease.

     

A single line linearly polarized source for Mössbauer spectroscopy

A single line source of linearly polarized radiation in a conventional Mössbauer setup working at room temperature with ⁵⁷Co in Cr matrix was constructed. The ordered Fe₃Si was used as a filter. The estimated degree of polarization of the source depends on the preparation and thickness of the polarizer. The best polarization degree (92 ± 8)% was achieved. The measurements with polarized radiation lead to significant simplification of the complex spectra and allow to study components arising from various polarizations separately. In particular, it is possible to extract Δm = (1, −1) and Δm = 0 transitions by use of a linearly polarized beam. Moreover polarized beam can be used for determination of texture parameters related to spatial arrangements of spins in the plane perpendicular to the radiation beam. Such parameters are not easily measurable by other methods. The Mössbauer spectra of invar Fe₆₅Ni₃₅ measured by monochromatic linearly polarized radiation are shown as an example.     

Enzymatic Synthesis of Enantiopure α‐ and β‐Amino Acids by Phenylalanine Aminomutase‐Catalysed Amination of Cinnamic Acid Derivatives

The phenylalanine aminomutase (PAM) from Taxus chinensis catalyses the conversion of α‐phenylalanine to β‐phenylalanine, an important step in the biosynthesis of the N‐benzoyl phenylisoserinoyl side‐chain of the anticancer drug taxol. Mechanistic studies on PAM have suggested that (E)‐cinnamic acid is an intermediate in the mutase reaction and that it can be released from the enzyme's active site. Here we describe a novel synthetic strategy that is based on the finding that ring‐substituted (E)‐cinnamic acids can serve as a substrate in PAM‐catalysed ammonia addition reactions for the biocatalytic production of several important β‐amino acids. The enzyme has a broad substrate range and a high enantioselectivity with cinnamic acid derivatives; this allows the synthesis of several non‐natural aromatic α‐ and β‐amino acids in excellent enantiomeric excess (ee >99 %). The internal 5‐methylene‐3,5‐dihydroimidazol‐4‐one (MIO) cofactor is essential for the PAM‐catalysed amination reactions. The regioselectivity of amination reactions was influenced by the nature of the ring substituent.     

Experimental and Computational Studies on Structure and Energetic Properties of Halogen Derivatives of 2-Deoxy-D-Glucose

The results of structural studies on a series of halogen-substituted derivatives of 2-deoxy-D-glucose (2-DG) are reported. 2-DG is an inhibitor of glycolysis, a metabolic pathway crucial for cancer cell proliferation and viral replication in host cells, and interferes with D-glucose and D-mannose metabolism. Thus, 2-DG and its derivatives are considered as potential anticancer and antiviral drugs. X-ray crystallography shows that a halogen atom present at the C2 position in the pyranose ring does not significantly affect its conformation. However, it has a noticeable effect on the crystal structure. Fluorine derivatives exist as a dense 3D framework isostructural with the parent compound, while Cl- and I-derivatives form layered structures. Analysis of the Hirshfeld surface shows formation of hydrogen bonds involving the halogen, yet no indication for the existence of halogen bonds. Density functional theory (DFT) periodic calculations of cohesive and interaction energies (at the B3LYP level of theory) have supported these findings. NMR studies in the solution show that most of the compounds do not display significant differences in their anomeric equilibria, and that pyranose ring puckering is similar to the crystalline state. For 2-deoxy-2-fluoro-D-glucose (2-FG), electrostatic interaction energies between the ligand and protein for several existing structures of pyranose 2-oxidase were also computed. These interactions mostly involve acidic residues of the protein; single amino-acid substitutions have only a minor impact on binding. These studies provide a better understanding of the structural chemistry of halogen-substituted carbohydrates as well as their intermolecular interactions with proteins determining their distinct biological activity.