Structure and dynamics of metallomacrocycles: recognition of zinc xylyl-bicyclam by an HIV coreceptor

As platforms for the design of metal-based therapeutic and diagnostic agents, macrocycles are rigid enough to provide strong metal binding sites and orient functional groups stereoselectively, yet flexible enough to accommodate structural changes required for induced-fit recognition of biological targets. We consider the recognition of the Zn(II) complex of the bis-tetraazamacrocycle xylyl-bicyclam, a potent anti-HIV agent, by the coreceptor CXCR4, a G-protein-coupled receptor used by HIV for membrane fusion and cell entry. NMR studies show that the macrocycles of Zn(II)(2)-xylyl-bicyclam perchlorate exist in aqueous solution as two major configurations, trans-I (nitrogen chirality R,S,R,S), and trans-III (S,S,R,R). Acetate addition induced a major structural change. X-ray crystallography shows that the acetate complex contains the unusual cis-V cyclam configuration (R,R,R,R and folded) with bidentate coordination of acetate to Zn(II) plus second-coordination-sphere double H-bond formation between diagonal NH protons on the opposite cyclam face and acetate carboxylate oxygens. Detailed 1D and 2D NMR studies show that the major configuration of Zn(II)(2)-xylyl-bicyclam acetate in aqueous solution is cis-V/trans-I. Molecular modeling shows that an analogous cis-V site can be formed when Zn(II)(2)-xylyl-bicyclam binds to CXCR4, involving the carboxylate groups of Asp262 (Zn(II) coordination) and Glu288 (double H-bonding). The second cyclam can adopt the trans-I (or trans-III) configuration with Zn(II) binding to Asp171. These interactions are consistent with the known structure-activity relationships for bicyclam anti-HIV activity and receptor mutation. Consideration of the anti-HIV activity of xylyl-bicyclam complexes of other metal ions suggests that affinity for carboxylates, configurational flexibility, and kinetic factors may all play roles in receptor recognition. For example, Pd(II) cyclam complexes interact only weakly with axial ligands and are inflexible and inactive, whereas Co(III) cyclams bind carboxylates strongly, are configurationally flexible, and yet have low activity. Our findings should aid the design of new generations of active macrocycles including highly specific chemokine receptor antagonists.     

Pulse Duration Dependent Asymmetry in Molecular Transmembrane Transport Due to Electroporation in H9c2 Rat Cardiac Myoblast Cells In Vitro

Electroporation (EP) is one of the successful physical methods for intracellular drug delivery, which temporarily permeabilizes plasma membrane by exposing cells to electric pulses. Orientation of cells in electric field is important for electroporation and, consequently, for transport of molecules through permeabilized plasma membrane. Uptake of molecules after electroporation are the greatest at poles of cells facing electrodes and is often asymmetrical. However, asymmetry reported was inconsistent and inconclusive—in different reports it was either preferentially anodal or cathodal. We investigated the asymmetry of polar uptake of calcium ions after electroporation with electric pulses of different durations, as the orientation of elongated cells affects electroporation to a different extent when using electric pulses of different durations in the range of 100 ns to 100 µs. The results show that with 1, 10, and 100 µs pulses, the uptake of calcium ions is greater at the pole closer to the cathode than at the pole closer to the anode. With shorter 100 ns pulses, the asymmetry is not observed. A different extent of electroporation at different parts of elongated cells, such as muscle or cardiac cells, may have an impact on electroporation-based treatments such as drug delivery, pulse-field ablation, and gene electrotransfection.     

Detection of hypophosphite, phosphite, and orthophosphate in natural geothermal water by ion chromatography

Current doctrine states that phosphorus is incorporated into cells in the pentavalent(V) oxidation state as orthophosphate. However, recent studies show that microorganisms contain enzymes used to metabolize reduced forms of phosphorous, including phosphite(III) and hypophosphite(I), which suggests that there is a natural source for these chemical species. This paper will discuss suppressed conductivity ion chromatography methods developed to detect hypophosphite, phosphite, and orthophosphate in a geothermal water matrix containing fluoride, chloride, bromide, nitrate, hydrogen carbonate and sulfate. All peaks were clearly resolved, and calibrations were linear with estimated 3sigma detection limits of 0.83, 0.39, and 0.35 microM for hypophosphite, phosphite, and orthophosphate, respectively.     

Development of an in situ fiber optic Raman system to monitor hydrothermal vents

The development of a field portable fiber optic Raman system modified from commercially available components that can operate remotely on battery power and withstand the corrosive environment of the hydrothermal vents is discussed. The Raman system is designed for continuous monitoring in the deep-sea environment. A 785 nm diode laser was used in conjunction with a sapphire ball fiber optic Raman probe, single board computer, and a CCD detector. Using the system at ambient conditions the detection limits of SO(4)(2-), CO(3)(2-) and NO(3)(-) were determined to be approximately 0.11, 0.36 and 0.12 g l(-1) respectively. Mimicking the cold conditions of the sea floor by placing the equipment in a refrigerator yielded slightly worse detection limits of approximately 0.16 g l(-1) for SO(4)(-2) and 0.20 g l(-1) for NO(3)(-). Addition of minerals commonly found in vent fluid plumes also decreased the detection limits to approximately 0.33 and 0.34 g l(-1) respectively for SO(4)(-2) and NO(3)(-).     

Formation and stability of N-heterocyclic carbenes in water: the carbon acid pKa of imidazolium cations in aqueous solution

We report second-order rate constants kDO (M-1 s-1) for exchange for deuterium of the C(2)-proton of a series of simple imidazolium cations to give the corresponding singlet imidazol-2-yl carbenes in D2O at 25 degrees C and I = 1.0 (KCl). Evidence is presented that the reverse protonation of imidazol-2-yl carbenes by solvent water is limited by solvent reorganization and occurs with a rate constant of kHOH = kreorg = 10(11) s-1. The data were used to calculate reliable carbon acid pK(a)s for ionization of imidazolium cations at C(2) to give the corresponding singlet imidazol-2-yl carbenes in water: pKa = 23.8 for the imidazolium cation, pKa = 23.0 for the 1,3-dimethylimidazolium cation, pKa = 21.6 for the 1,3-dimethylbenzimidazolium cation, and pKa = 21.2 for the 1,3-bis-((S)-1-phenylethyl)benzimidazolium cation. The data also provide the thermodynamic driving force for a 1,2-hydrogen shift at a singlet carbene: K12 = 5 x 10(16) for rearrangement of the parent imidazol-2-yl carbene to give neutral imidazole in water at 298 K, which corresponds to a favorable Gibbs free energy change of 23 kcal/mol. We present a simple rationale for the observed substituent effects on the thermodynamic stability of N-heterocyclic carbenes relative to a variety of neutral and cationic derivatives that emphasizes the importance of the choice of reference reaction when assessing the stability of N-heterocyclic carbenes.     

Ethynylphosphonamidates for the Rapid and Cysteine‐Selective Generation of Efficacious Antibody–Drug Conjugates

Requirements for novel bioconjugation reactions for the synthesis of antibody–drug conjugates (ADCs) are exceptionally high, since conjugation selectivity as well as the stability and hydrophobicity of linkers and payloads drastically influence the performance and safety profile of the final product. We report Cys‐selective ethynylphosphonamidates as new reagents for the rapid generation of efficacious ADCs from native non‐engineered monoclonal antibodies through a simple one‐pot reduction and alkylation. Ethynylphosphonamidates can be easily substituted with hydrophilic residues, giving rise to electrophilic labeling reagents with tunable solubility properties. We demonstrate that ethynylphosphonamidate‐linked ADCs have excellent properties for next‐generation antibody therapeutics in terms of serum stability and in vivo antitumor activity.     

Zinc(II) complexes of constrained antiviral macrocycles

The configurations of metallocyclams are of interest in relation to protein recognition and anti-HIV activity. We have synthesised four novel zinc(II) complexes with hexyl-Me(2)-cyclam (HMC; 3,14-dimethyl-2,6,13,17-tetraazatricyclo(16.4.0.0(7,12))docosane), 1, and naphthyl-hexyl-Me(2)-cyclam (NHMC; 2,13-bis(1-naphthylmethyl)-5,16-dimethyl-2,6,13,17-tetraazatricyclo(16.4.0.0(7,12))docosane), 2, as ligands. X-ray crystallographic data for Zn(II)-HMC diacetate, 3 show that zinc is six-coordinate in a distorted octahedral environment bound to four equatorial N atoms from the macrocycle and two axial acetato O atoms. The 14-membered metallo-macrocycle adopts a trans-III (RRSS) configuration with two six-membered rings in chair forms and two five-membered rings in gauche forms. In the chlorido Zn(II)-HMC complex 5, zinc appears to be 5-coordinate with square-pyramidal geometry. Interestingly, the chlorido Zn(II)-NHMC complex 6 crystallised in a trans-I configuration containing 4-coordinate tetrahedral zinc bound to three cyclam ring N atoms, a possible model for intermediates formed during the uptake and release of metals by cyclams. The ligand 1 and the zinc complex 3 were active towards viral strains HIV-1 (III(B)) (IC(50) values of 10.51 ± 0.23 and 3.50 ± 0.33 μM, respectively), and HIV-2 (ROD) (IC(50) values of 133.78 ± 14.10 and >110.67 μM, respectively). 2D [(1)H, (13)C] and [(1)H, (15)N] NMR spectroscopic studies suggested that the types of configurational isomers present in solution depend on the axial ligand.     

The Renner effect in triatomic molecules with application to CH+, MgNC and NH2

We have developed a computational procedure, based on the variational method, for the calculation of the rovibronic energies of a triatomic molecule in an electronic state that become degenerate at the linear nuclear configuration. In such an electronic state the coupling caused by the electronic orbital angular momentum is very significant and it is called the Renner effect. We include it, and the effect of spin-orbit coupling, in our program. We have developed the procedure to the point where spectral line intensities can be calculated so that absorption and emission spectra can be simulated. In order to gain insight into the nature of the eigenfunctions, we have introduced and calculated the overall bending probability density function f(p) of the states. By projecting the eigenfunctions onto the Born-Oppenheimer basis, we have determined the probability density functions f+(rho) and f-(rho) associated with the individual Born-Oppenheimer states phi(-)elec and phi(+)elec. At a given temperature the Boltzmann averaged value of the f(p) over all the eigenstates gives the bending probability distribution function F(rho), and this can be related to the result of a Coulomb Explosion Imaging (CEI) experiment. We review our work and apply it to the molecules CH2+, MgNC and NH2, all of which are of astrophysical interest.     

Biocompatible polymers for antibody support on gold surfaces

The elimination or minimization of non-specific protein adsorption from serum is critical for the use of surface plasmon resonance (SPR) sensors for in vitro and in vivo analysis of complex biological solutions. The ultimate goals in this application are to minimize non-specific adsorption of protein and to maximize analyte signal. A reduction of the non-specific protein adsorption from serum of up to 73% compared to carboxymethylated-dextran 500 kDa (CM-dextran) was achieved following a survey of eight biocompatible polymers and 10 molecular weights of CM-dextran. These coatings minimize non-specific adsorption on the sensor while also serving as immobilization matrices for antibody fixation to the probes. Polymers including polysaccharides: CM-dextrans, CM-hyaluronic acid, hyaluronic acid, and alginic acid were investigated. Humic acid, polylactic acid, polyacrylic acid, orthopyridyldisuldfide–polyethyleneglycol–N-hydroxysuccinimide (OPSS–PEG–NHS), and a synthesized polymer; polymethacrylic-acid-co-vinyl-acetate (PMAVA) were also used. The non-specific protein adsorption reduction was measured over a 14 day period at 0 °C for each polymer. Calibration curves using some of these polymers were constructed to show the performance and low detection limit possibilities of these new antibody supports. For many of the polymers, this is the first demonstration of employment as an antibody support for an optical or surface active sensor. CM-dextran is the polymer offering the largest signal for the antigen detection. However, the biocompatible polymers demonstrate a greater stability to non-specific binding in serum. These biocompatible polymers offer different alternatives for CM-dextran.