Effects of the Protonation State of Titratable Residues and the Presence of Water Molecules on Nocodazole Binding to β‐Tubulin

Regulation of microtubule assembly by antimitotic agents is a potential therapeutic strategy for the treatment of cancer, parasite infections, and neurodegenerative diseases. One of these agents is nocodazole (NZ), which inhibits microtubule polymerization by binding to β‐tubulin. NZ was recently co‐crystallized in Gallus gallus tubulin, providing new information about the features of interaction for ligand recognition and stability. In this work, we used state‐of‐the‐art computational approaches to evaluate the protonation effects of titratable residues and the presence of water molecules in the binding of NZ. Analysis of protonation states showed that residue E198 has the largest modification in its pK_a value. The resulting E198 pK_a value, calculated with pH‐REMD methodology (pK_a=6.21), was higher than the isolated E amino acid (pK_a=4.25), thus being more likely to be found in its protonated state at the binding site. Moreover, we identified an interaction between a water molecule and C239 and G235 as essential for NZ binding. Our results suggest that the protonation state of E198 and the structural water molecules play key roles in the binding of NZ to β‐tubulin.     

Experimental pKa Value Determination of All Ionizable Groups of a Hyperstable Protein

Electrostatic interactions significantly contribute to the stability and function of proteins. The stabilizing or destabilizing effect of local charge is reflected in the perturbation of the pK_a value of an ionizable group from the intrinsic pK_a value. Herein, the charge network of a hyperstable dimeric protein (ribbon–helix–helix (rhh) protein from plasmid pRN1 from Sulfolobus islandicus) is studied through experimental determination of the pK_a values of all ionizable groups. Transitions were monitored by multiple NMR signals per ionizable group between pH 0 and 12.5, prior to a global analysis, which accounted for the effects of neighboring residues. It is found that for several residues involved in salt bridges (four Asp and one Lys) the pK_a values are shifted in favor of the charged state. Furthermore, the pK_a values of residues C40 and Y47, both located in the hydrophobic dimer interface, are shifted beyond 13.7. The necessary energy for such a shift is about two-thirds of the total stability of the protein, which confirms the importance of the hydrophobic core to the overall stability of the rhh protein.     

Preclinical Characterization of Acyl Sulfonimidamides: Potential Carboxylic Acid Bioisosteres with Tunable Properties

Herein we present the preclinical characterization of novel compounds containing the linear acyl sulfonimidamide functionality. Specifically, we studied the pK_a, lipophilicity, in vitro metabolic stability, plasma protein binding, Caco‐2 permeability, and aqueous solubility for nine aryl acyl sulfonimidamides. In comparison with widely used carboxylic acid bioisosteres, the acyl sulfonimidamides were found to be less acidic and more lipophilic depending on the substitution pattern in the studied compounds. Importantly, the pK_a values (5.9–7.6) were significantly influenced by substituents on the nitrogen atom and the aryl substituents. Moreover, the acyl sulfonimidamides displayed membrane permeabilities ranging from moderate to very high, which correlated with decreased pK_a and low to negligible efflux ratios. We foresee that the chiral sulfur center and the two handles for structural diversity of linear acyl sulfonimidamides will offer new opportunities for drug design and for improving the oral bioavailability of acidic drug candidates.     

Dissociation constants of long chain fatty acids in methanol-water and ethanol-water mixtures

The pK_a values of lauric acid, myristic acid, palmitic acid and stearic acid in methanol-water and ethanol-water mixtures of different compositions at 303 K and ionic strength, I=0.1M(KNO₃) have been determined. The pK_a values of the fatty acids have been found to increase with the alcohol content of the solvent. It has also been found that, in general, the pK_a values in the ethanolic solvent are greater than those in the methanolic solvent. In a given solvent mixture the observed pK_a value decreases in the order of stearic acid > palmitic acid > myristic acid > lauric acid. Departure from the predicted linearity of the plots of pK_a against the inverse of the dielectric constant, 1/D indicates that the electrostatic effects taken into account by the Born theory are not adequate enough to represent the total medium effects on pK_a.     

Phosphor‐in‐fluorescent‐glasses for high color rendering white light emitting diodes

Fluorescent glass frits were prepared and used to synthesize phosphor‐in‐fluorescent glass composites (PiFGs) to realize stable white light emitting diodes with high color‐rendering properties. Commercial red, green, and blue phosphors were co‐sintered and red phosphors were partially replaced by Eu³⁺ in glass frits. Phosphor‐in‐glass composites were placed on UV‐light emitting diodes (UV‐LEDs) to generate white light. Pure white light with a luminous efficacy=58.4 lm/W, general color rendering index R_a=87 and special color rendering index for strong red R₉=73 was realized with glass frits containing 7 mol% Eu₂O₃ and RGB ratio of 35:20:15. Luminous efficacy, R_a and R₉ increased as red phosphors were replaced by red‐fluorescent glass frits.     

Optochemical Control of Protein Degradation

Optochemical approach has been successfully utilized to regulate cellular protein degradation with a high resolution of spatiotemporal control. In this highlight, we discuss two recent developments of combining reversible optochemical functionalities with the bifunctional proteolysis targeting chimeras, or PROTACs to achieve light-controlled degradation of protein targets of interest. PHOTACs are azobeneze-containing molecules that are inactive as trans forms and active as cis forms, switchable upon pulse-irradiation with either an activating 390 nm light or a deactivating 525 nm light. In contrast, photoPROTACs are o-F₄-azobenzene-containing molecules that can be switched between active trans isomers and inactive cis isomers by a single irradiation event using 415 or 530 nm light. Combining these two optochemically controlled PROTAC systems has the potential to achieve orthogonal control on protein degradation.     

Directed Evolution of a Formate Dehydrogenase for Increased Tolerance to Ionic Liquids Reveals a New Site for Increasing the Stability

The formate dehydrogenase (FDH) from Candida boidinii is a well‐known enzyme in biocatalysis for NADH regeneration. Nevertheless, it has low activity in a water‐miscible ionic liquid (1,3‐dimethylimidazolium dimethyl phosphate, [MMIm][Me₂PO₄]). In this work, this enzyme was subjected to directed evolution by using error‐prone PCR, and a mutant (N187S/T321S) displaying higher activity was obtained following selection based on the formazan‐based colorimetric assay. The mutation N187S is responsible for improved activity both in aqueous solution and in [MMIm][Me₂PO₄], through an enhancement of the k_cat value by a factor of 5.8. Fluorescence experiments performed in the presence of a quenching agent revealed that the mutant does not unfold in the presence of 50 % (v/v) [MMIm][Me₂PO₄] whereas the wild‐type enzyme does. Molecular modelling revealed that the mutation is located at the monomer–monomer interface and causes an increase in the pK_a of residue E163 from 4.8 to 5.5. Calculation of the pK_a of this residue in other microbial FDHs showed that thermostable FDHs have a highly basic glutamate at this position (pK_a up to 6.2). We have identified a new site for improving FDH thermostability and tolerance to ionic liquids, and it is linked to the local charge of the enzymes in this class.     

Reversible luminescence modulation of Ho‐doped K0.5Na0.5NbO3 piezoelectrics with high luminescence contrast

A significant luminescence modulation behavior based on photochromic reactions was observed in Ho³⁺‐doped (Na_0.52K_0.48)_0.92Li_0.08NbO₃ ceramics, fabricated by the conventional solid‐state reaction method. Under visible light irradiation (407 nm) for 20 second, the samples changed pale gray from initial pale green, and returned to their original color by a thermal stimulus of 230°C for 10 minutes, showing typical photochromic phenomenon. Under 453 nm excitation, the samples exhibited strong green emission at 551 nm. Interestingly, their green emission intensity can be effectively tailored by controlling photochromic reaction processes (irradiation wavelength and time), and the luminescent modulation ratio (ΔR_t) reaches up to 77%. And, the ΔR_t value has no any obvious degradation after 10 cycles by alternating visible light irradiation and thermal stimulus, showing excellent reversibility. These results make it potential applications in many fields as a kind of multifunctional material.     

Zur physikalisch‐chemischen Charakterisierung von 5‐Amino‐1‐aryl‐1H‐tetrazolen: Elektronische Molekülparameter aus der thermischen Isomerisierung zu 5‐Arylamino‐1H‐tetrazolen

The known thermal isomerization of 5‐amino‐1‐aryl‐1H‐tetrazoles ( A ) into corresponding 5‐arylamino‐1H‐tetrazoles ( HB ) was used to derive physicochemical parameters characterizing the electronic substituent effect on isomerism and dissociation equilibria. For a series of 26 tetrazoles A as starting materials the equilibrium constants (pK_i) of isomerization in boiling ethylene glycol at 197 °C and the dissociation constants (pK_a) of the NH‐acidic tetrazoles HB were determined by potentiometric titration of rapidly cooled equilibrium mixtures in water and ethanol/water with KOH at 25 °C. The pK values are closely correlated with Hammett′s electronic substituent constants σ and can be used as electronic molecule parameters in QSAR or QSPR (QSAR = quantitative structure‐activity relationship; QSPR = quantitative structure‐property relationship) studies.     

Interfacial chemistry on carboxylate-functionalized monolayer assemblies

Deposition of trichlorosilanes with ester groups at their remote termini provides a convenient entry to carboxylic acid-bearing siloxane-anchored self-assembled monolayers. The de-esterification of these esters has been optimized to minimize monolayer damage, and their quantitative re-esterification provides clear evidence for the stability of these systems. Both the structure of the ester-terminated monolayer and its de-esterification/esterification chemistry can be easily monitored by FTIR-ATR measurements. This spectroscopic tool, together with a liquid cell that enables IR spectra to be measured in an aqueous environment, enables a detailed structural analysis of the carboxylic acid-bearing siloxane-anchored self-assembled monolayers and an assessment of their acid/base behavior (by in situ titration). The use of D₂O instead of H₂O for the in situ titration experiments also improves the available IR window. Both monomeric and dimeric/oligomeric acid groups are seen, and the relative ease of deprotonation of these various species can be directly monitored. Monomers of alkyl carboxylic acids that are hydrogen bonded only to surrounding water molecules have a pK_a = 4.9, while the pK_a for the aggregated molecules is 9.3. Similar behavior is seen for surface-bound benzoic acids, where the two pK_a values are 4.7 and 9.0. The influence of temperature on these structures and their chemistry has been explored to a limited extent as well. When the alkylcarboxylic acid system is cooled to 10 °C, the pK_a value for the acid monomers is reduced from 4.9 to 4.5 and increases from 9.3 to 10.3 for the aggregates.     

Selective Covalent Protein Modification by 4‐Halopyridines through Catalysis

We have investigated 4‐halopyridines as selective, tunable, and switchable covalent protein modifiers for use in the development of chemical probes. Nonenzymatic reactivity of 4‐chloropyridine with amino acids and thiols was ranked with respect to common covalent protein‐modifying reagents and found to have reactivity similar to that of acrylamide, but could be switched to a reactivity similar to that of iodoacetamide upon stabilization of the positively charged pyridinium. Diverse, fragment‐sized 4‐halopyridines inactivated human dimethylarginine dimethylaminohydrolase‐1 (DDAH1) through covalent modification of the active site cysteine, acting as quiescent affinity labels that required off‐pathway catalysis through stabilization of the protonated pyridinium by a neighboring aspartate residue. A series of 2‐fluoromethyl‐substituted 4‐chloropyridines demonstrated that the pK_a and k_inact/K_I values could be predictably varied over several orders of magnitude. Covalent labeling of proteins in an Escherichia coli lysate was shown to require folded proteins, indicating that alternative proteins can be targeted, and modification is likely to be catalysisdependent. 4‐Halopyridines, and quiescent affinity labels in general, represent an attractive strategy to develop reagents with switchable electrophilicity as selective covalent protein modifiers.     

Two reduction waves of oximes and imine formation in acidic media

The separation of the two two-electron waves in acidic media in solutions of some aromatic aldoximes and ketoximes is attributed to differences between the pK_a-values of oximes (which are smaller than about 1.0) and those of protonated forms of corresponding imines. Dependences of half-wave potentials on pH show that the lower limit pK_a of imine varies from −0.25 for p-formylbenzaldehyde oxime to 2.2 for p-fluorobenzaldehyde oxime. For the benzaldehyde and acetophenone oximes bearing substituents, which have pK_a higher than −1.50, a good agreement was observed for pK_a-values obtained spectrophotometrically and pK_ox values obtained from the change in the slope of plots. Past misrepresentations of the electroreduction mechanism of protonated forms of imines is attributed to insufficiently controlled compositions of supporting electrolytes. The reduction potential of protonated forms of oximes in the more positive wave i₁ is affected by the concentration of halides in the supporting electrolyte. This is attributed to an ion-pair formation, between these anions and protonated forms of oximes. The reduction of the protonated form of imines at acidity, where in the bulk predominates the same protonated form that is reduced, is independent of the nature and concentration of anions of the supporting electrolyte.     

Biophysical Features of Bacillithiol,the Glutathione Surrogate of Bacillus subtilis and other Firmicutes

Bacillithiol (BSH) is the major low‐molecular‐weight (LMW) thiol in many low‐G+C Gram‐positive bacteria (Firmicutes). Evidence now emerging suggests that BSH functions as an important LMW thiol in redox regulation and xenobiotic detoxification, analogous to what is already known for glutathione and mycothiol in other microorganisms. The biophysical properties and cellular concentrations of such LMW thiols are important determinants of their biochemical efficiency both as biochemical nucleophiles and as redox buffers. Here, BSH has been characterised and compared with other LMW thiols in terms of its thiol pK_a, redox potential and thiol–disulfide exchange reactivity. Both the thiol pK_a and the standard thiol redox potential of BSH are shown to be significantly lower than those of glutathione whereas the reactivities of the two compounds in thiol–disulfide reactions are comparable. The cellular concentration of BSH in Bacillus subtilis varied over different growth phases and reached up to 5 mM , which is significantly greater than previously observed from single measurements taken during mid‐exponential growth. These results demonstrate that the biophysical characteristics of BSH are distinctively different from those of GSH and that its cellular concentrations can reach levels much higher than previously reported.     

Mass Transfer Characteristics in a Rotor‐Stator Reactor

Mass transfer characteristics in a rotor‐stator reactor in terms of the overall volumetric mass‐transfer coefficient (K_xa) using the N₂‐H₂O‐O₂ system were investigated. The effects of various operating parameters including rotation speed, liquid volumetric flow rate, and gas volumetric flow rate on K_xa were systematically examined, and a gas‐liquid mass transfer model was established to predict K_xa. Results reveal that K_xa increased with higher rotation speed, liquid volumetric flow rate, and gas volumetric flow rate. The results also confirm that the predicted values of K_xa were in agreement with the experimental values with deviation within 15 %. The results contribute to a better understanding of mass transfer characteristics in rotor‐stator reactors.     

The Dynamic Range of Acidity: Tracking Rules for the Unidirectional Penetration of Cellular Compartments

Labeled ammonium cations with pK_a∼7.4 accumulate in acidic organelles because they can be neutralized transiently to cross the membrane at cytosolic pH 7.2 but not at their internal pH<5.5. Retention in early endosomes with less acidic internal pH was achieved recently using weaker acids of up to pK_a 9.8. We report here that primary ammonium cations with higher pK_a 10.6, label early endosomes more efficiently. This maximized early endosome tracking coincides with increasing labeling of Golgi networks with similarly weak internal acidity. Guanidinium cations with pK_a 13.5 cannot cross the plasma membrane in monomeric form and label the plasma membrane with selectivity for vesicles embarking into endocytosis. Self-assembled into micelles, guanidinium cations enter cells like arginine-rich cell-penetrating peptides and, driven by their membrane potential, penetrate mitochondria unidirectionally despite their high inner pH. The resulting tracking rules with an approximated dynamic range of pK_a change ∼3.5 are expected to be generally valid, thus enabling the design of chemistry tools for biology research in the broadest sense. From a practical point of view, most relevant are two complementary fluorescent flipper probes that can be used to image the mechanics at the very beginning of endocytosis.     

Photochromic polyacylsemicarbazides based on azobenzene containing dihydrazide

Polyacylsemicarbazides exhibiting photochromic behaviour were synthesised by solution polymerisation of azobenzene chromophore containing dihydrazide and various diisocyanates (aliphatic, cycloaliphatic and aromatic). A model compound was also prepared from o‐tolylene isocyanate and dihydrazide. The structures were confirmed by ¹H NMR and FTIR techniques. The polymers were found to exhibit λ_max values around 330 nm corresponding to the trans isomer which reduced with irradiation of the solutions of polymers in DMAc due to trans→cis isomerisation as observed by UV‐VIS spectrometry. The reverse reaction (cis→trans isomerisation) was found to take place photochemically when irradiated with visible light or when kept in the dark. The rates of isomerisation reactions were found to be independent of the chromophore concentration and the nature of the polymer backbone. The inherent viscosity of the solutions of polymers in DMAc reduced upon irradiation with UV light and was regained in the dark. Thermal studies showed that the polymer degradation started with the simultaneous cleavage of imino (N? N) and aromatic azo (? N?N? ) bond and that recrystallisation occurred on heating the samples. Copyright © 2004 Society of Chemical Industry     

Determination of the pKa of the Aci-Nitro Intermediate in o-Nitrobenzyl Systems

The structural volume changes following photoexcitation of o-nitrobenzyl systems are used to estimate the excited state pK_a of the aci-nitro intermediate in aqueous solutions. The rather large contractions induced in solution by UV excitation are due to the rapid deprotonation of the short-lived aci-nitro intermediate, which leads to the formation of two charged species. The magnitude of the measured contraction as a function of the pH in acidic solutions follows a sigmoidal curve, from which it is possible to extract the pK_a of the aci-nitro intermediate. This method is generally applicable to short-lived intermediates with stronger acid character than the parent compound, provided they undergo irreversible chemical transformations to a product that cannot rebind the photodetached proton. The reaction volume for water formation from its ionic constituents at basic pH allows the determination of the deprotonation quantum yields.     

Measurement of the overall volumetric oxygen transfer coefficient in alkane‐aqueous dispersions

BACKGROUND: A reliable procedure for the determination of the volumetric oxygen transfer coefficient (K_La) in hydrocarbon–aqueous dispersions is critical to the assessment of oxygen transfer in hydrocarbon‐based bioprocesses. The pressure step procedure (PSP) and gassing out procedure (GOP) for K_La determination were evaluated in n‐C_10–13 alkane–aqueous dispersions over agitation rates (600 to 1200 rpm) and alkane concentrations (up to 20%) typical of hydrocarbon‐based bioprocesses. RESULTS: K_La from the procedures diverged progressively as agitation increased, with K_La underprediction attenuated at high alkane. At 1200 rpm and 5% alkane, K_La from the GOP was underpredicted by 49%. Compared with K_La from the PSP, K_La from the PSP without nitrogen transport was the same, but K_La from the PSP without response lag (PSP‐lag) was underpredicted by approximately 50% at 1200 rpm. Moreover, K_La from the PSP‐lag was lower than K_La from the GOP, except at 1200 rpm and alkane concentration up to 5%. CONCLUSION: The PSP was superior to the GOP in alkane–aqueous dispersions, particularly at extreme agitation and low alkane concentrations. This was attributable to the probe response lag and the non‐uniformity of gas composition inherent in the GOP and independent of the rate of coincident nitrogen transport. Copyright © 2009 Society of Chemical Industry     

Preparation of poly(tert‐butyl acrylate)‐poly(acrylic acid) amphiphilic copolymers via radiation‐induced reversible addition–fragmentation chain transfer mediated polymerization of tert‐butyl acrylate

Poly(tert‐butyl acrylate) (PtBA) is a versatile hydrophobic macromolecule usually preferred in the development of new materials for a host of applications. PtBA homopolymers with well‐defined structure and controlled molecular weight in a wide range were successfully synthesized via radiation‐induced reversible addition–fragmentation chain transfer (RAFT) polymerization in the presence of a trithiocarbonate type RAFT agent. The polymerization of tBA was performed under ⁶⁰Co γ‐irradiation in the presence of 2‐(dodecylthiocarbonothioylthio)‐2‐methylpropionic acid (DDMAT) as the RAFT agent in toluene at room temperature with three [tBA]/[DDMAT] ratios (400, 600 and 1000) and different irradiation times. Radiation‐induced polymerization of tBA displayed controlled free radical polymerization characteristics: a narrow molecular weight distribution (M_w/M_n ~ 1.1), pseudo first order kinetics and controlled molecular weights. The system followed the RAFT polymerization mechanism even at very low amounts of RAFT agent ([tBA]/[DDMAT] = 1000), and molecular weights up to 113 900 with narrow dispersity (Ð =1.06) were obtained. PtBA was further hydrolysed into different amphiphilic PtBA‐co‐poly(acrylic acid) (PAA) copolymers by low (27.5%) and high (77.3%) degrees of hydrolysis. The pH sensitivity of the two copolymers was investigated by dynamic light scattering at pH 2 and pH 9 (above and below the pK_a value of PAA) and their hydrodynamic diameters and zeta potential values were determined. © 2020 Society of Chemical Industry     

Stabilizing p-Dithiobenzyl Urethane Linkers without Rate-Limiting Self-Immolation for Traceless Drug Release

Exploiting the redox sensitivity of disulfide bonds is a prevalent strategy in targeted prodrug designs. In contrast to aliphatic disulfides, p-thiobenzyl-based disulfides have rarely been used for prodrug designs, given their intrinsic instability caused by the low pK_a of aromatic thiols. Here, we examined the interplay between steric hindrance and the low-pK_a effect on thiol–disulfide exchange reactions and uncovered a new thiol–disulfide exchange process for the self-immolation of p-thiobenzyl-based disulfides. We observed a central leaving group shifting effect in the α,α-dimethyl-substituted p-dithiobenzyl urethane linkers (DMTB linkers), which leads to increased disulfide stability by more than two orders of magnitude, an extent that is significantly greater than that observed with typical aliphatic disulfides. In particular, the DMTB linkers display not only high stability, but also rapid self-immolation kinetics due to the low pK_a of the aromatic thiol, which can be used as a general and robust linkage between targeting reagents and cytotoxic drugs for targeted prodrug designs. The unique and promising stability characteristics of the present DMTB linker will likely inspire the development of novel targeted prodrugs to achieve traceless release of drugs into cells.