Enhancing protein backbone binding--a fruitful concept for combating drug-resistant HIV

The evolution of drug resistance is one of the most fundamental problems in medicine. In HIV/AIDS, the rapid emergence of drug-resistant HIV-1 variants is a major obstacle to current treatments. HIV-1 protease inhibitors are essential components of present antiretroviral therapies. However, with these protease inhibitors, resistance occurs through viral mutations that alter inhibitor binding, resulting in a loss of efficacy. This loss of potency has raised serious questions with regard to effective long-term antiretroviral therapy for HIV/AIDS. In this context, our research has focused on designing inhibitors that form extensive hydrogen-bonding interactions with the enzyme's backbone in the active site. In doing so, we limit the protease's ability to acquire drug resistance as the geometry of the catalytic site must be conserved to maintain functionality. In this Review, we examine the underlying principles of enzyme structure that support our backbone-binding concept as an effective means to combat drug resistance and highlight their application in our recent work on antiviral HIV-1 protease inhibitors.     

Konformation und Vernetzung von (CuCN)6‐Ringen in polymeren Cyanocupraten(I) [Cu2(CN)3—] (n = 2, 3)

Conformation and Cross Linking of (CuCN)₆‐Rings in Polymeric Cyanocuprates(I) equation/tex2gif-stack-8.gif [Cu₂(CN)₃^—] (n = 2, 3) The alkaline‐tricyano‐dicuprates(I) Rbequation/tex2gif-stack-9.gif[Cu₂(CN)₃] · H₂O ( 1 ) and Csequation/tex2gif-stack-10.gif[Cu₂(CN)₃] · H₂O ( 2 ) were synthesized by hydrothermal reaction of CuCN and RbCN or CsCN. The dialkylammonium‐tricyano‐dicuprates(I) [NH₂(Me)₂]equation/tex2gif-stack-11.gif[Cu₂(CN)₃] ( 3 ), [NH₂(iPr)₂]equation/tex2gif-stack-12.gif[Cu₂(CN)₃] ( 4 ), [NH₂(Pr)₂]equation/tex2gif-stack-13.gif[Cu₂(CN)₃] ( 5 ) and [NH₂(secBu)₂]equation/tex2gif-stack-14.gif[Cu₂(CN)₃] ( 6 ) were obtained by the reaction of dimethylamine, diisopropylamine, dipropylamine or di‐sec‐butylamine with CuCN and NaCN in the presence of formic acid. The crystal structures of these compounds are built up by (CuCN)₆‐rings with varying conformations, which are connected to layers ( 1 ) or three‐dimensional zeolite type cyanocuprate(I) frameworks, depending on the size and shape of the cations ( 2 to 6 ). Crystal structure data: 1 , monoclinic, P2₁/c, a = 12.021(3)Å, b = 8.396(2)Å, c = 7.483(2)Å, β = 95.853(5)°, V = 751.4(3)ų, Z = 4, d_c = 2.728 gcm^—1, R₁ = 0.036; 2 , orthorhombic, Pbca, a = 8.760(2)Å, b = 6.781(2)Å, c = 27.113(5)Å, V = 1610.5(5)ų, Z = 8, d_c = 2.937 gcm^—1, R₁ = 0.028; 3 , orthorhombic, Pna2₁, a = 13.504(3)Å, b = 7.445(2)Å, c = 8.206(2)Å, V = 825.0(3)ų, Z = 4, d_c = 2.023 gcm^—1, R₁ = 0.022; 4 , orthorhombic, Pbca, a = 12.848(6)Å, b = 13.370(7)Å, c = 13.967(7)Å, V = 2399(2)ų, Z = 8, d_c = 1.702 gcm^—1, R₁ = 0.022; 5 , monoclinic, P2₁/n, a = 8.079(3)Å, b = 14.550(5)Å, c = 11.012(4)Å, β = 99.282(8)°, V = 1277.6(8)ų, Z = 4, d_c = 1.598 gcm^—1, R₁ = 0.039; 6 , monoclinic, P2₁/c, a = 16.215(4)Å, b = 13.977(4)Å, c = 14.176(4)Å, β = 114.555(5)°, V = 2922(2)ų, Z = 8, d_c = 1.525 gcm^—1, R₁ = 0.070.     

Structural modifications and adsorption capability of C18-silica/binary solvent interphases studied by EPR and RP-HPLC

The structure of the octadecyl (C18) chain layer attached to a silica surface in the presence of binary solvents (acetonitrile/water; methanol/water) was investigated by electron paramagnetic resonance (EPR) and reverse-phase high-performance liquid chromatography (RP-HPLC), using 4-hydroxy-2,2,6,6 tetramethylpiperidine-N-oxyl (Tempol) to mimic the behavior of pollutants with medium-low polarity. The computer-aided analysis of the EPR spectra provided structural and dynamical information of the probe and its environments which clarified the modifications of the chain conformations that occur at different solvent compositions. Capacity factors, k', were calculated as a function of the percentage of water/organic solvent (mobile phase), and the retention behavior of the C18-functionalized silica surface (stationary phase) was compared with the results obtained with EPR analysis under static conditions. In particular, EPR analysis showed that, at percentages of ACN equal or higher than 50%, the chain layer assume a quite ordered structure, whereas at percentages lower than 50% the chains tend to collapse and fold on the silica surface. In this latter situation, the hydrophobic net of the C18 chains strongly limits Tempol mobility. In methanol/water mixtures, both EPR and RP-HPLC analysis showed that the probe was adsorbed into a poorly ordered interphase. If the residual silanols at the silica surface were bonded to a sililating agent (endcapping), both EPR and RP-HPLC analysis showed a decreased adsorption of the probe with respect to the non-endcapped silica at the same mobile phase composition.     

Chemoselective addition of in situ prepared lithium alkynyl borates to aldehydes: a practical and transition metal free approach toward the synthesis of propargylic alcohols

A convenient synthesis of functionalized propargylic alcohols arising from the 1,2-addition of lithium alkynyl-trimethyl borate onto aldehydes under transition metal free mild conditions is reported. The reaction tolerates a wide range of functional groups, is highly chemoselective and the propargylic alcohols are isolated in good to excellent yields.     

A porous graphitized carbon column HPLC method for the quantification of paracetamol, pseudoephedrine, and chlorpheniramine in a pharmaceutical formulation

A simple, rapid, and stability-indicating HPLC method has been developed, fully validated, and applied to the quantification of paracetamol, pseudoephedrine hydrochloride, and chlorpheniramine maleate in a pharmaceutical formulation, using hydrochlorothiazide as an internal standard. Chromatographic separation was achieved isocratically on an RP porous graphitized carbon analytical column (125 x 2.1 mm id, particle size 5 microm) using 5.0 mM ammonium acetate-acetonitrile (35 + 65, v/v) mobile phase at a flow rate of 0.50 mL/min. UV spectrophotometric detection at 220 nm was used. The method had linear calibration curves over the range of 30-70 microg/mL for paracetamol, 1.8-4.2 microg/mL for pseudoephedrine hydrochloride, and 120-280 ng/mL for chlorpheniramine maleate. The intraday and interday RSD values were less than 3.2% for all compounds, while the relative error was less than 2.9%. Accelerated stability studies performed under various stress conditions proved the selectivity of the method. The developed method was applied successfully to QC and content uniformity tests of commercial tablets.     

Sensitive liquid chromatography/tandem mass spectrometry assay for absolute quantification of ITIH4‐derived putative biomarker peptides in clinical serum samples

To explore the potential of peptide fragments derived from inter‐α‐trypsin inhibitor heavy chain‐4 (ITIH₄) as serum markers for different cancer types, sensitive and specific analytical assays are required. Liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) would be suitable; however, a previously developed method for quantification of eight ITIH₄ fragments (ITIH₄‐21, ‐22, ‐25, ‐26, ‐27, ‐28, ‐29 and ‐30) was found to be insensitive for clinical use. A more sensitive LC/MS/MS assay has now been developed and validated, which was further optimized to facilitate analyses of large sets of clinical serum samples. Benefits compared to the previous method include reduction of sample volume (100 µL), omission of protein precipitation and evaporation and transferring solid‐phase extraction (SPE) to a 96‐well format. Chromatographic separation on an XBridge BEH300 C₁₈ column, using a water/methanol gradient containing acetic acid, was coupled to triple quadrupole mass spectrometric detection, applying heated electrospray ionization. Method validation revealed deviations from nominal concentrations below 10.1% and intra‐ and inter‐assay precisions below 17.4 and 20.0%, respectively, at the lower limit of quantification (LLOQ) for all peptides. The reported changes resulted in more rapid and efficient analyses and reduced LLOQs for the six less abundant peptides (1.2; 1.0; 1.2; 2.0; 2.0 and 2.0 ng/mL vs. 2.1; 2.0; 2.5; 2.6; 2.2 and 2.4 ng/mL for ITIH₄‐21, ‐22, ‐25, ‐27, ‐28 and ‐29, respectively). The method has shown its applicability by quantifying all peptides in appropriate concentration ranges in serum from healthy volunteers and application to clinical samples from breast cancer patients. Copyright © 2010 John Wiley & Sons, Ltd.     

Quenched Instationary Polymerization Systems, 6. The Direct Determination of Axial Dispersion in Size Exclusion Chromatography by Comparison of Theoretical and Ideal Peak Widths

For the direct determination of axial dispersion in size exclusion chromatography a simple method is presented which makes use of the measured and ideal peak widths. The peak width can be defined in two ways: either absolute as the difference of successive points of inflection or relative as the ratio of these points. If the absolute peak width is invariant for the number, molar mass and hyper distribution then this distribution can unambiguously be classified as Poissonian. The relative peak width for such distributions is strictly determined by the experimental parameters. It is demonstrated that axial dispersion only leads to an additive increase in the peak variances for peaks with a relative peak width smaller than 1.25. Thus, it is possible to determine directly the axial dispersion of an experimental size exclusion chromatography set‐up by the use of Poisson distributions prepared by quenched instationary polymerization techniques or any other technique leading to ideal Poisson distributions.     

Biodegradation of a medium-chain-length polyhydroxyalkanoate in tropical river water

The medium-chain-length polyhydroxyalkanoate (PHA_MCL) produced by Pseudomonas putida PGA1 using saponified palm kernel oil as the carbon source could degrade readily in water taken from Kayu Ara River in Selangor, Malaysia. A weight loss of 71.3% of the PHA film occurred in 86 d. The pH of the river water medium fell from 7.5 (at d 0) to 4.7 (at d 86), and there was a net release of CO₂. In sterilized river water, the PHA film also lost weight and the pH of the water fell, but to lesser extents. The C8 monomer of the PHA was completely removed after 6 d of immersion in the river water, while the proportions of the other monomers (C10, C12, and C14) were reversed from that of the undegraded PHA. By contrast, the monomer composition of the PHA immersed in sterilized river water did not change significantly from that of the undegraded PHA. Scanning electron microscopy showed physical signs of degradation on the PHA film immersed in the river water, but the film immersed in sterilized river water was relatively unblemished. The results thus indicate that the PHA_MCL was degraded in tropical river water by biologic as well as nonbiologic means. A significant finding is that shorter-chain monomers were selectively removed throughout the entire PHA molecule, and this suggests enzymatic action.     

Association patterns of platinated purine nucleobases in metal-modified pairs and triples

Blocking of Watson-Crick or Hoogsteen edges in purine nucleobases by a metal entity precludes involvement of these sites in interbase hydrogen bonding, thereby leaving the respective other edge or the sugar edge as potential H bonding sites. In mixed guanine, adenine complexes of trans-a2PtII (a = NH3 or CH3NH2) of composition trans-[(NH3)2Pt(9-EtA-N1)(9-MeGH-N7)](NO3)2 (1a), trans-[(NH3)2Pt(9-EtA-N1)(9-MeGH-N7)](ClO4)2 (1b), and trans,trans-[(CH3NH2)2(9-MeGH-N7)Pt(N1-9-MeA-N7)Pt(9-MeGH-N7)(CH3NH2)2](ClO4)4*2H2O (2) (with 9-EtA = 9-ethyladenine, 9-MeA= 9-methyladenine, 9-MeGH = 9-methylguanine), this aspect is studied. Thus, in 1b pairing of two adenine ligands via Hoogsteen edges and in 2 pairing of two guanine bases via sugar edges is realized. These situations are compared with those found in a series of related complexes.