Indirect detection in reversed-phase liquid chromatography: Ion-pair retention models for cations on polystyrene polymers

Summary The distribution equilibria of cationic compounds in reversed-phase chromatographic systems (ion-pair chromatography) have been studied on the basis of their effect on a detectable mobile phase component. The solid phase was a polystyrene-divinylbenzene copolymer and the detectable component, a quaternary ammonium ion, 1-methylpyridine. The solutes were mono- and divalent amines and quaternary ammonium ions. The cations can be retained by ion-pair adsorption and ion exchange. Expressions for the ion-pair retention of the solutes and the mobile phase cation (system peak) have been developed assuming Langmuir distribution of ion pairs to a solid phase with one kind of binding site. The validity of the expressions has been tested by evaluation of ion-pair distribution constants using non-linear curve fitting techniques. Good agreement for the constants of common ion pairs was obtained from different kinds of capacity ratio expressions. Ion exchange retention can appear beside ion-pair retention, and it has been observed in the pH range 1.6–6.1. The effect depends not only on cations in the mobile phase, but also on the nature of the buffering systems.     

Improved Synthesis of 1-Bromo-3-buten-2-one

A convenient procedure for the synthesis of 1-bromo-3-buten-2-one, 4, from commercially available 2-ethyl-2-methyl-1,3-dioxolane, 1, is described. The procedure involves three reaction steps: (1) The acetal 1 is converted to 2-(1-bromoethyl)-2-bromomethyl-1,3-dioxolane, 2, by reacting 1 with elemental bromine in dichloromethane to yield 98% of 2. (2) Dehydrobromination of 2 with potassium tert-butoxide in tetrahydrofuran gives 2-bromomethyl-2-vinyl-1,3-dioxolane, 3, in 84–93% yield. (3) Removal of the acetal protection from 3 by formolysis for 6–10 h afforded 1-bromo-3-buten-2-one, 4, in 85–94% yield. A more rapid method is acid hydrolysis of 3 under microwave activation (100 °C, 8–10 min), by which 4 was obtained in 75% yield. Full experimental details are given.

Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

Natural backbone-cyclized proteins have an increased thermostability and resistance towards proteases, characteristics that have sparked interest in head-to-tail cyclization as a method to stability-enhance proteins used in diagnostics and therapeutic applications, for example. In this proof-of principle study, we have produced and investigated a head-to-tail cyclized and HER2-specific Z_HER2:342 Affibody dimer. The sortase A-mediated cyclization reaction is highly efficient (>95%) under optimized conditions, and renders a cyclic Z_HER3:342-dimer with an apparent melting temperature, T_m, of 68 °C, which is 3 °C higher than that of its linear counterpart. Circular dichroism spectra of the linear and cyclic dimers looked very similar in the far-UV range, both before and after thermal unfolding to 90 °C, which suggests that cyclization does not negatively impact the helicity or folding of the cyclic protein. The cyclic dimer had an apparent sub-nanomolar affinity (K_d ~750 pM) to the HER2-receptor, which is a ~150-fold reduction in affinity relative to the linear dimer (K_d ~5 pM), but the anti-HER2 Affibody dimer remained a high-affinity binder even after cyclization. No apparent difference in proteolytic stability was detected in an endopeptidase degradation assay for the cyclic and linear dimers. In contrast, in an exopeptidase degradation assay, the linear dimer was shown to be completely degraded after 5 min, while the cyclic dimer showed no detectable degradation even after 60 min. We further demonstrate that a site-specifically DyLight 594-labeled cyclic dimer shows specific binding to HER2-overexpressing cells. Taken together, the results presented here demonstrate that head-to-tail cyclization can be an effective strategy to increase the stability of an Affibody dimer.     

Visible-Light Photoswitching by Azobenzazoles

Three visible-light responsive photoswitches are reported, azobis(1-methyl-benzimidazole) ( 1 ), azobis(benzoxazole) ( 2 ) and azobis(benzothiazole) ( 3 ). Photostationary distributions are obtained upon irradiation with visible light comprising approximately 80 % of the thermally unstable isomer, with thermal half-lives up to 8 min and are mostly invariant to solvent. On protonation, compound 1 H⁺ has absorption extending beyond 600 nm, allowing switching with yellow light, and a thermal half-life just under 5 minutes. The two isomers have significantly different pK_a values, offering potential as a pH switch. The absorption spectra of 2 and 3 are insensitive to acid, although changes in the thermal half-life of 3 indicate more basic intermediates that significantly influence the thermal barrier to isomerization. These findings are supported by high-level ab initio calculations, which validate that protonation occurs on the ring nitrogen and that the Z isomer is more basic in all cases.     

New methods for calculating \alpha BB-type underestimators

Most branch-and-bound algorithms in global optimization depend on convex underestimators to calculate lower bounds of a minimization objective function. The $\alpha $ BB methodology produces such underestimators for sufficiently smooth functions by analyzing interval Hessian approximations. Several methods to rigorously determine the $\alpha $ BB parameters have been proposed, varying in tightness and computational complexity. We present new polynomial-time methods and compare their properties to existing approaches. The new methods are based on classical eigenvalue bounds from linear algebra and a more recent result on interval matrices. We show how parameters can be optimized with respect to the average underestimation error, in addition to the maximum error commonly used in $\alpha $ BB methods. Numerical comparisons are made, based on test functions and a set of randomly generated interval Hessians. The paper shows the relative strengths of the methods, and proves exact results where one method dominates another.     

The synthetic utility of heteroaromatic azido compounds. Part VII. Preparation of some 2- and 4-substituted thieno[3,2-d]pyrimidines

Synthetically useful scission reactions of some 1H-1,2,3-triazolo[1,5-a]thieno[3,2-d]pyrimidines have been studied. The net result of these reactions is an elimination of nitrogen, and a 1,1-addition of the cleaving agent, forming 2-substituted thieno[3,2-d]pyrimidines. The mechanism of the scission is discussed, and a concomitant nucleophilic attack and expulsion of nitrogen is proposed. An unusual reaction between the unsubstituted triazole-fused parent compound ( 1 ) and bromine in alkaline medium is also reported.     

Electrophilic substitution of allylsilanes: synthesis of β,γ-unsaturated 1,3-dithianes

Regiospecific substitution of some allylsilanes using 1,3-dithienium tetrafluoroborate as a formyl cation equivalent is described.     

Influence of ignored and well-known zone distortions on the separation performance of proteins in capillary free zone electrophoresis with special reference to analysis in polyacrylamide-coated fused silica capillaries in various buffers. II. Experimental studies at acidic pH with on-line enrichment

The separation of acidic and basic model proteins was studied in capillary free zone electrophoresis in a polyacrylamide-coated, electroosmosis-free capillary at pH below their isoelectric points (pI) using various buffers at pH 2.7-4.8 with UV detection at 200 nm. The separation performance was significantly dependent on the coating quality, which may even differ within the same batch of capillaries. In addition, a washing step with 2 M HCl and the storage of the capillary in distilled water was essential for the performance. For high efficiency and resolution the choice of buffer constituents was extremely important which is discussed in quantitative terms in Part I. The most promising buffers were ammonium acetate and ammonium hydroxyacetate at pH 4 (ionic strengths: 0.12 and 0.15 M, respectively) with plate numbers up to 1,700,000 plates/m, corresponding to a zone width (2sigma) of only 1 mm in a capillary with 40 cm effective length, when the injected samples were dissolved in a 10-fold diluted background electrolyte (BGE), a zone even narrower than those obtained in polyacrylamide gel electrophoresis, the characteristic feature of which is remarkably thin zones. In the experiment giving this plate number, the calculated variance for longitudinal diffusion was larger than all the other calculated variances (those for the width of the starting zone, Joule heating, sedimentation and the curvature of the capillary). Interestingly, the effect of capillary curvature was significant. In addition, the sum of all other imaginable variances (corresponding to various types of slow on/off kinetics and hyper-sharp peaks) was in the most successful experiments only 28-50% of the variance for longitudinal diffusion. One hundred- to two hundred-fold dilution of the BGE improved the detection limits and provided high precision in both migration times and peak areas with ammonium hydroxyacetate and ammonium acetate as background electrolytes. However, that high dilution increased the variance 140-400% for these buffers, respectively, at least partly due to conductivity or pH differences between the sample and buffer zones (hyper-sharp peaks). Sedimentation of the enriched sample, a factor that has not previously been treated theoretically or experimentally, was probably another reason for our finding that peak heights did not increase when the sample was dissolved in a buffer diluted more than 200-fold, although pH changes and in some cases thermal expansion in the capillary also may contribute. Loss of protein may occur at the ionic strength 0.01 and lower due to precipitation. Limits of detection were in the range 4-17 pmol of proteins with ammonium acetate as BGE. No indication of denaturation of proteins at pH 4 was observed. However, the separation performance at pH 3 was not satisfactory and loss of proteins was observed, possibly indicating such problems. The protein mobilities decreased unexpectedly from pH 4 to 3--a further indication of conformation changes.     

Capillary electrochromatography of hydrophobic amines on continuous beds

The capillary electrochromatographic separation performance of hydrophobic amines and a related quaternary ammonium compound on continuous beds based on polymers of acrylamide has been studied. The chromatographic bed is polymerized in situ and the character of the polymers with regard to hydrophobicity and charge has been systematically changed by regulating its content of isopropyl and sulfonate ligands, respectively. The best performance was obtained for columns with a molar ratio of 1:80 for the sulfonate and isopropyl groups, and resulted in efficiencies up to 200000 plates per meter. The effects on retention, resolution and elution order by ionic strength, pH, and content of acetonitrile in the mobile phase have been investigated. The quaternary ammonium compound was always the least retained irrespective of pH. By increasing the pH, a reversal of the migration order between the tertiary and secondary amine was obtained. The results indicate a complex migration/retention mechanism where ion-exchange, adsorption and electrophoretic mobilities play a role. The concentration limit of detection could be lowered from 1.3 microg/mL to 50 pg/mL by using a high content of 2-propanol (96%) in the sample compared to dissolving the analytes in the mobile phase.