Protonation Of Groups Research Articles

Dynamics of solid alanine by means of nuclear magnetic resonance relaxometry.

1H nuclear magnetic resonance relaxometry was applied to investigate the dynamics of l-alanine in the solid phase (powder). The experimental studies were carried out in a very broad frequency range, covering four orders of magnitude-from 4 kHz to 40 MHz (referring to the 1H resonance frequency) in order to probe motional processes of much different time scales by a single experiment. To get access to the dynamics of different proton groups of alanine, the 1H spin-lattice relaxation measurements were performed for non-deuterated and partially deuterated alanine. The experiments were carried out in the temperature range of 293 K-370 K (non-deuterated alanine) and 318 K-370 K (partially deuterated alanine). As a result of a thorough theoretical analysis of the extensive set of experimental results, three motional processes occurring on different time scales are identified and quantitatively described. The slowest process occurs on a time scale of μs and it is attributed to the collective dynamics of a 3D hydrogen bond network of alanine, while the intermediate, attributed to the dynamics of the NH3 group, corresponds to the range of tenths of ns. The fast process describes the rotation of the CH3 group.

Magnetization Transfer Contrast and Chemical Exchange Saturation Transfer MRI. Features and analysis of the field-dependent saturation spectrum

Magnetization Transfer Contrast (MTC) and Chemical Exchange Saturation Transfer (CEST) experiments measure the transfer of magnetization from molecular protons to the solvent water protons, an effect that becomes apparent as an MRI signal loss (“saturation”). This allows molecular information to be accessed with the enhanced sensitivity of MRI. In analogy to Magnetic Resonance Spectroscopy (MRS), these saturation data are presented as a function of the chemical shift of participating proton groups, e.g. OH, NH, NH2, which is called a Z-spectrum. In tissue, these Z-spectra contain the convolution of multiple saturation transfer effects, including nuclear Overhauser enhancements (NOEs) and chemical exchange contributions from protons in semi-solid and mobile macromolecules or tissue metabolites. As a consequence, their appearance depends on the magnetic field strength (B0) and pulse sequence parameters such as B1 strength, pulse shape and length, and interpulse delay, which presents a major problem for quantification and reproducibility of MTC and CEST effects.The use of higher B0 can bring several advantages. In addition to higher detection sensitivity (signal-to-noise ratio, SNR), both MTC and CEST studies benefit from longer water T1 allowing the saturation transferred to water to be retained longer. While MTC studies are non-specific at any field strength, CEST specificity is expected to increase at higher field because of a larger chemical shift dispersion of the resonances of interest (similar to MRS). In addition, shifting to a slower exchange regime at higher B0 facilitates improved detection of the guanidinium protons of creatine and the inherently broad resonances of the amine protons in glutamate and the hydroxyl protons in myoinositol, glycogen, and glucosaminoglycans. Finally, due to the higher mobility of the contributing protons in CEST versus MTC, many new pulse sequences can be designed to more specifically edit for CEST signals and to remove MTC contributions.

EXCI-CEST: Exploiting pharmaceutical excipients as MRI-CEST contrast agents for tumor imaging

Chemical Exchange Saturation Transfer (CEST) approach is a novel tool within magnetic resonance imaging (MRI) that allows visualization of molecules possessing exchangeable protons with water. Many molecules, employed as excipients for the formulation of finished drug products, are endowed with hydroxyl, amine or amide protons, thus can be exploitable as MRI-CEST contrast agents. Their high safety profiles allow them to be injected at very high doses. Here we investigated the MRI-CEST properties of several excipients (ascorbic acid, sucrose, N-acetyl-d-glucosamine, meglumine and 2-pyrrolidone) and tested them as tumor-detecting agents in two different murine tumor models (breast and melanoma cancers). All the investigated molecules showed remarkable CEST contrast upon i.v. administration in the range 1–3ppm according to the type of mobile proton groups. A marked increase of CEST contrast was observed in tumor regions up to 30min post injection. The combination of marked tumor contrast enhancement and lack of toxicity make these molecules potential candidates for the diagnosis of tumors within the MRI-CEST approach.

Interaction between DNA and Drugs Having Protonable Basic Groups: Characterization through Affinity Constants, Drug Release Kinetics, and Conformational Changes

This paper reports the in vitro characterization of the interaction between the phosphate groups of DNA and the protonated species of drugs with basic groups through the determination of the affinity constants, the reversibility of the interaction, and the effect on the secondary structure of the macromolecule. Affinity constants of the counterionic condensation DNA–drug were in the order of 106. The negative electrokinetic potential of DNA decreased with the increase of the proportion of loading drugs. The drugs were slowly released from the DNA–drug complexes and had release kinetics consistent with the high degree of counterionic condensation. The circular dichroism profile of DNA was not modified by complexation with atenolol, lidocaine, or timolol, but was significantly altered by the more lipophilic drugs benzydamine and propranolol, revealing modifications in the secondary structure of the DNA. The in vitro characterization of such interactions provides a physicochemical basis that would contribute to identify the effects of this kind of drugs in cellular cultures, as well as side effects observed under their clinical use. Moreover, this methodology could also be projected to the fields of intracellular DNA transfection and the use of DNA as a carrier of active drugs.

Effects of Benzothiazolamines on Voltage-Gated Sodium Channels.

Benzothiazole is a versatile fused heterocycle that aroused much interest in drug discovery as anticonvulsant, neuroprotective, analgesic, anti-inflammatory, antimicrobial, and anticancer. Two benzothiazolamines, riluzole and lubeluzole, are known blockers of voltage-gated sodium (Nav) channels. Riluzole is clinically used as a neuroprotectant in amyotrophic lateral sclerosis. Inhibition of Nav channels by riluzole is voltage-dependent due to preferential binding to inactivated sodium channels. Yet the drug exerts little use-dependent block, probably because it lacks protonable amine. One important property is riluzole ability to inhibit persistent Na+ currents, which likely contributes to its neuroprotective activity. Lubeluzole showed promising neuroprotective effects in animal stroke models, but failed to show benefits in acute ischemic stroke in humans. One important concern is its propensity to prolong the cardiac QT interval, due to hERG K+ channel block. Lubeluzole very potently inhibits Nav channels in a voltage- and use-dependent manner, due to its great preferential affinity for inactivated channels and the presence of a protonable amine group. Patch-clamp experiments suggest that the binding sites of both drugs overlap the local anesthetic receptor within the ion-conducting pathway. Riluzole and lubeluzole displayed very potent antimyotonic activity in a rat model of myotonia, a pathological skeletal muscle condition characterized by high-frequency runs of action potentials. Such results well support the repurposing of riluzole as an antimyotonic drug, allowing the launch of a pilot study in myotonic patients. Riluzole, lubeluzole, and new Nav channel blockers built on the benzothiazolamine scaffold will certainly continue to be investigated for possible clinical applications.

Simultaneous measurement ofβ-delayed proton andγdecay ofP27

This is the first study of $^{27}\mathrm{P}$ to measure both the $\ensuremath{\beta}$-delayed proton and $\ensuremath{\beta}$-delayed $\ensuremath{\gamma}$ decays. While no new proton groups in the astrophysically interesting energy region of 300--400 keV were observed, a new upper limit on the proton branching of 0.16% was estimated. Several new $\ensuremath{\gamma}$-ray lines were observed, mainly coming from the isobaric analog state in $^{27}\mathrm{Si}$, which has been assigned a more accurate energy value of 6638(1) keV.

The Mg2+-containing Water Cluster of Mammalian Cytochrome c Oxidase Collects Four Pumping Proton Equivalents in Each Catalytic Cycle

Bovine heart cytochrome c oxidase (CcO) pumps four proton equivalents per catalytic cycle through the H-pathway, a proton-conducting pathway, which includes a hydrogen bond network and a water channel operating in tandem. Protons are transferred by H3O+ through the water channel from the N-side into the hydrogen bond network, where they are pumped to the P-side by electrostatic repulsion between protons and net positive charges created at heme a as a result of electron donation to O2 bound to heme a3 To block backward proton movement, the water channel remains closed after O2 binding until the sequential four-proton pumping process is complete. Thus, the hydrogen bond network must collect four proton equivalents before O2 binding. However, a region with the capacity to accept four proton equivalents was not discernable in the x-ray structures of the hydrogen bond network. The present x-ray structures of oxidized/reduced bovine CcO are improved from 1.8/1.9 to 1.5/1.6 Å resolution, increasing the structural information by 1.7/1.6 times and revealing that a large water cluster, which includes a Mg2+ ion, is linked to the H-pathway. The cluster contains enough proton acceptor groups to retain four proton equivalents. The redox-coupled x-ray structural changes in Glu198, which bridges the Mg2+ and CuA (the initial electron acceptor from cytochrome c) sites, suggest that the CuA-Glu198-Mg2+ system drives redox-coupled transfer of protons pooled in the water cluster to the H-pathway. Thus, these x-ray structures indicate that the Mg2+-containing water cluster is the crucial structural element providing the effective proton pumping in bovine CcO.

Identifying involvement of Lys251/Asp252 pair in electron transfer and associated proton transfer at the quinone reduction site of Rhodobacter capsulatus cytochrome bc1

Describing dynamics of proton transfers in proteins is challenging, but crucial for understanding processes which use them for biological functions. In cytochrome bc1, one of the key enzymes of respiration or photosynthesis, proton transfers engage in oxidation of quinol (QH2) and reduction of quinone (Q) taking place at two distinct catalytic sites. Here we evaluated by site-directed mutagenesis the contribution of Lys251/Asp252 pair (bacterial numbering) in electron transfers and associated with it proton uptake to the quinone reduction site (Qi site). We showed that the absence of protonable group at position 251 or 252 significantly changes the equilibrium levels of electronic reactions including the Qi-site mediated oxidation of heme bH, reverse reduction of heme bH by quinol and heme bH/Qi semiquinone equilibrium. This implicates the role of H-bonding network in binding of quinone/semiquinone and defining thermodynamic properties of Q/SQ/QH2 triad. The Lys251/Asp252 proton path is disabled only when both protonable groups are removed. With just one protonable residue from this pair, the entrance of protons to the catalytic site is sustained, albeit at lower rates, indicating that protons can travel through parallel routes, possibly involving water molecules. This shows that proton paths display engineering tolerance for change as long as all the elements available for functional cooperation secure efficient proton delivery to the catalytic site.

Kinetics of H+/Me+ (Me = Na, K) ion exchange in polyantimonic acid

The kinetics of ion exchange in polyantimonic acid (PAA) with the composition of Sb2O5 · 3.2H2O are studied. It is shown that sodium and potassium ions replace mobile proton groups and are located in channels of the PAA crystal structure. Diffusion coefficients are calculated in the approximation of the spheric grain model on the basis of the ion exchange data.

PH Dependence of Absorption and Emission Spectra of Ru(phen)2(phenOH)2+(PF6)2 Complex

A ruthenium complex of the 4-hydroxy- 1,10-phenanthroline ligand was synthesized, and the variation of its absorption and emission intensity and litetime with pH characterized. Excited state lifetime, luminescence intensity, and emission properties were determined. The complex exhibits a maximum at 460nm, and a small red shift at higher pH. The spectra show a well defined isobestic point. Luminescence intensity exhibited a sigmoidal relationship with pH, a behaviour that is similar to those of other ruthenium complexes carrying protonable functional groups. This characteristic is suggestive of the suitability of this complex for pH sensor design for medical practices as well as industrial processes. pH dependence is more evident in the emission than the absorption spectra, a behaviour characteristic of higher pH dependence on the excited than the ground state of the complex. Emission lifetimes of 165.4ns and 3.08ns for the protonated and deprotonated states respectively, were determined and the pK * a value calculated as 3.68.

Classical and quantum theories of proton disorder in hexagonal water ice

It has been known since the pioneering work of Bernal, Fowler and Pauling that common, hexagonal (Ih) water ice is the archetype of a frustrated material : a proton-bonded network in which protons satisfy strong local constraints - the "ice rules" - but do not order. While this proton disorder is well established, there is now a growing body of evidence that quantum effects may also have a role to play in the physics of ice at low temperatures. In this Article we use a combination of numerical and analytic techniques to explore the nature of proton correlations in both classical and quantum models of ice Ih. In the case of classical ice Ih, we find that the ice rules have two, distinct, consequences for scattering experiments - singular "pinch points", reflecting a zero-divergence condition on the uniform polarization of the crystal, and broad, asymmetric features, coming from its staggered polarisation. In the case of the quantum model, we find that the collective quantum tunnelling of groups of protons can convert states obeying the ice rules into a quantum liquid, whose excitations are birefringent, emergent photons. We make explicit predictions for scattering experiments on both classical and quantum ice Ih, and show how the quantum theory can explain the "wings" of incoherent inelastic scattering observed in recent neutron scattering experiments [Bove et al., Phys. Rev. Lett. 103, 165901 (2009)]. These results raise the intriguing possibility that the protons in ice Ih could form a quantum liquid at low temperatures, in which protons are not merely disordered, but continually fluctuate between different configurations obeying the ice rules.

Foam dried composite glass: An alternative to lyophilization for stability enhancement of bendamustine hydrochloride

Bendamustine hydrochloride, a thermolabile biopharmaceutical, in lyophilized form is unsteady proving difficult to maintain activity and shelf life. To counterbalance the thermo-fragile nature of this product and eliminate limitations of lyophilization an alternative method with rational selection of stabilizers is developed. The present research work was aimed to screen sugar-phosphate composite mixtures to enhance storage stability of drug using evaporative foam drying. The effect of foaming agents and their strengths, pH and temperature on product features were investigated. The physicochemical stability of products prepared using sugars, polymer and inorganic water soluble phosphates alone and in combination was studied. The products were characterized for residual moisture, reconstitution time and drug content. The amount of drug retained after processing and at storage was analyzed by HPLC. Foam dried products were tested for short term stability as per ICH guidelines for biologicals. Pluronic® F108 at 0.6% exhibited optimum foamability. Foams were stable at 0.4% polyvinyl pyrrolidone K30. The enhancement in drug stability by evaporative foam drying attributed to electrostatic interaction between protonable groups of drug and deprotonable groups of composite stabilizer mixture during dehydration. The processing and storage stability of drug in composite glass was superior at normal storage. Proper selection of stabilizers for this technique is a key to successful stabilization of drug at ambient temperatures without loss of their activity during shipping and storage without refrigeration. Keywords:.

Arylpiperidines as a new class of oxidosqualene cyclase inhibitors

The cyclization of oxidosqualene to lanosterol, catalyzed by the enzyme oxidosqualene cyclase (OSC), goes through a number of carbocationic high energy intermediates (HEI), and mimicking these intermediates is a promising approach for the development of OSC inhibitors. 3-Arylpiperidines (or tetrahydropyridines) were designed as steroidomimetic rings A + C equivalents containing two protonable amino groups for mimicking both the pro-C4 HEI and the pro-C20 HEI of the OSC-mediated cyclization cascade. Inhibitory activity is strongly dependent on the nature of the lipophilic substituent representing an equivalent of the sterol side chain. Here aromatic residues (substituted benzyl, cinnamyl, naphthylmethyl) were found to be most suitable. Docking experiments on a first optimized 3-arylpiperidine compound led to an isomeric 4-arylpiperidine with submicromolar activity on human OSC. This inhibitor reduced total cholesterol biosynthesis in a cellular assay with an IC50 value of 0.26 μM.

Multifunctional cationic polyurethanes designed for non-viral cancer gene therapy

Nano-polyplexes from bioreducible cationic polymers have a massive promise for cancer gene therapy. However, the feasibility of cationic polyurethanes for non-viral gene therapy is so far not well studied. In this work, a linear cationic polyurethane containing disulfide bonds, urethane linkages and protonable tertiary amino groups was successfully generated by stepwise polycondensation reaction between 2,2′-dithiodiethanol bis(p-nitrophenyl carbonate) and 1,4-bis(3-aminopropyl)piperazine (BAP). We confirmed that the cationic polyurethane (denoted as PUBAP) displayed superior gene delivery properties to its cationic polyamide analogue, thus causing higher in vitro transfection efficiency in MCF-7 and SKOV-3 cells. Besides, further folate-PEGylation and hydrophobic deoxycholic acid (DCA) conjugation to amino-containing PUBAP can be conducted to afford multifunctional polyurethane gene delivery system. After optimization, folate-decorated nano-polyplexes from the PUBAP conjugated with 8 folate-PEG chains and 12 DCA residues exhibited superb colloidal stability under physiological conditions, and performed rapid uptake via folate receptor-mediated endocytosis, efficient intracellular gene release and nucleus translocation into SKOV-3 cells in vitro and in vivo. Importantly, PUBAP based polyplexes possess low cytotoxicity as a result of PUBAP biodegradability. Therefore, marked growth inhibition of SKOV-3 tumor xenografted in Balb/c nude mice was achieved with negligible side effects on the mouse health after intravenous administration of PUBAP based polyplexes with a therapeutic plasmid encoding for TNF-related apoptosis-inducing ligand. This work provides a new insight into biomedical application of bio-responsive polyurethanes for cancer therapy. Statement of significanceIn this study, we have confirmed that disulfide-based cationic polyurethane presents a new non-viral vector for gene transfer and cancer gene therapy. The significance of this work includes: (1) design and synthesis of a group of novel disulfide-based cationic polyurethane by non-isocyanate chemistry; (2) comparative study of transfection activity between cationic polyurethanes and cationic polyamides; (3) feasibility of bioreducible cationic polyurethanes for in vivo cancer gene therapy.

Strong Quantum Coupling in the Vibrational Signatures of a Symmetric Ionic Hydrogen Bond: The Case of (CH3OH)2H(.).

Vibrational coupling between proton and flanking group motions in the ionic hydrogen bond (IHB) of (CH3OH)2H(+) were studied by solving reduced-dimension vibrational Schrödinger equations. Potential energy and dipole surfaces along a few key normal modes were constructed with high-level ab initio methods. It was found that the IHB stretch parallel to O-O axis strongly couples with the out-of-phase C-O stretch and out-of-phase in-plane CH3 rock with COH deformation. Such strong quantum coupling leads to a complex triplet at 850-1100 cm(-1) region. Furthermore, we have investigated the possible active role of torsional motion in intensity redistribution.

Pcetk: A pDynamo-based Toolkit for Protonation State Calculations in Proteins.

Pcetk (a pDynamo-based continuum electrostatic toolkit) is an open-source, object-oriented toolkit for the calculation of proton binding energetics in proteins. The toolkit is a module of the pDynamo software library, combining the versatility of the Python scripting language and the efficiency of the compiled languages, C and Cython. In the toolkit, we have connected pDynamo to the external Poisson-Boltzmann solver, extended-MEAD. Our goal was to provide a modern and extensible environment for the calculation of protonation states, electrostatic energies, titration curves, and other electrostatic-dependent properties of proteins. Pcetk is freely available under the CeCILL license, which is compatible with the GNU General Public License. The toolkit can be found on the Web at the address http://github.com/mfx9/pcetk. The calculation of protonation states in proteins requires a knowledge of pKa values of protonatable groups in aqueous solution. However, for some groups, such as protonatable ligands bound to protein, the pKa aq values are often difficult to obtain from experiment. As a complement to Pcetk, we revisit an earlier computational method for the estimation of pKa aq values that has an accuracy of ± 0.5 pKa-units or better. Finally, we verify the Pcetk module and the method for estimating pKa aq values with different model cases.

Long‐Range Residual Dipolar Couplings: A Tool for Determining the Configuration of Small Molecules

AbstractTogether with NOE and J coupling, one‐bond residual dipolar coupling (RDC), which reports on the three‐dimensional orientation of an internuclear vector in the molecular frame, plays an important role in the conformation and configuration analysis of small molecules in solution by NMR spectroscopy. When the molecule has few CH bonds, or too many bonds are in parallel, the available RDCs may not be sufficient to obtain the alignment tensor used for structure elucidation. Long‐range RDCs that connect nuclei over multiple bonds are normally not parallel to the single bonds and therefore complement one‐bond RDCs. Herein we present a method for extracting the long‐range RDC of a chosen proton or group of protons to all remotely connected carbon atoms, including non‐protonated carbon atoms. Alignment tensors fitted directly to the total long‐range couplings (T=J+D) enabled straightforward analysis of both the long‐range and one‐bond RDCs for strychnine.

Long-Range Residual Dipolar Couplings: A Tool for Determining the Configuration of Small Molecules.

Together with NOE and J coupling, one-bond residual dipolar coupling (RDC), which reports on the three-dimensional orientation of an internuclear vector in the molecular frame, plays an important role in the conformation and configuration analysis of small molecules in solution by NMR spectroscopy. When the molecule has few C-H bonds, or too many bonds are in parallel, the available RDCs may not be sufficient to obtain the alignment tensor used for structure elucidation. Long-range RDCs that connect nuclei over multiple bonds are normally not parallel to the single bonds and therefore complement one-bond RDCs. Herein we present a method for extracting the long-range RDC of a chosen proton or group of protons to all remotely connected carbon atoms, including non-protonated carbon atoms. Alignment tensors fitted directly to the total long-range couplings (T=J+D) enabled straightforward analysis of both the long-range and one-bond RDCs for strychnine.

Photochemistry of imidazolium cations. Water addition to methylimidazolium ring induced by UV radiation in aqueous solution

The UV-C induced photoaddition of water to N-alkyl-N′-methylimidazolium cations was studied. The main photoreaction products exhibit chemical additions of a proton and a hydroxyl group to either positions 4 or 5 of the imidazolium ring. For unsymmetrical imidazolium cations, two positional isomers were obtained as products. In these cases, the most abundant isomer is the one in which the hydroxyl group adds at the side of the ring having the longer alkyl substituent. Experiments performed in D2O solutions reveal that the additions of proton and hydroxyl group never take place at the same carbon atom, in a reaction that produces equal amounts of diastereoisomers. Moreover, the formation of diastereoisomers at equal proportions suggests that the reaction proceeds in an unconcerted fashion.

Temperature dependence of the oxidation kinetics of TyrZ and TyrD in oxygen-evolving photosystem II complexes throughout the range from 320 K to 5 K

The photo-induced oxidation of TyrZ and TyrD by P680•+, that involves both electron and proton transfer (PCET), has been studied in oxygen-evolving photosystem II from Thermosynechococcus elongatus. We used time-resolved absorption spectroscopy to measure the kinetics of P680•+ reduction by tyrosine after the first flash given to dark-adapted PS II as a function of temperature and pH. The half-life of TyrZ oxidation by P680•+ increases from 20ns at 300K to about 4μs at 150K. Analyzing the temperature dependence of the rate, one obtains a reorganization energy of about 770meV. Between 260K and 150K, the reduction of P680•+ by TyrZ is increasingly replaced by charge recombination between P680•+ and QA•−. We propose that the driving force for TyrZ oxidation by P680•+ decreases upon lowering the temperature. TyrZ oxidation cannot be excluded in a minority of PS II complexes at cryogenic temperatures.TyrD oxidation by P680•+ with a half-life of about 30ns was observed at high pH. The pH dependence of the yield of TyrD oxidation can be described by a single protonable group with a pK of approximately 8.4. The rate of TyrD oxidation by P680•+ is virtually identical upon substitution of solvent exchangeable protons with deuterons indicating that the rate is limited by electron transfer. The rate is independent of temperature between 5K and 250K. It is concluded that TyrD donates the electron to P680•+ via PD2.