Synthesis,characterization, and curing of hyperbranched allyl ether–maleate functional ester resins

A model two-step synthesis of a saturated hyperbranched hydroxyl-terminated ester has been developed to show a synthesis route. Three different series of hyperbranched esters with different terminations have been synthesized to relate some of their properties to their structures. This route has then been used to synthesize three different allyl ethermaleate functional hyperbranched ester resins in a two-step procedure. The resins have been characterized with respect to rheology, structure, and properties, and the differences are discussed. The allyl ether-maleate functional resins have also been studied with respect to curing performance and final film properties. © 1993 John Wiley & Sons, Inc.     

Analytical characterization of a facile porous polymer monolithic trypsin microreactor enabling peptide mass mapping using mass spectrometry

A simple and rapid single-step method is presented to fabricate an enzyme reactor using trypsin immobilized on a macroporous polymer monolith. A reactor produced in a capillary format is ready to use within 1 h of preparation. The monomers making up the monolith, including N-acryloxysuccinimide for covalent immobilization of the enzyme, are mixed with trypsin and introduced into the column by capillary force for polymerization/immobilization. The enzyme activity from column-to-column is reproducible below 5% relative standard deviation (RSD), while the reactor is durable for at least 20 weeks when stored at room temperature. The apparent kinetic constants V(max) and K(m) are of value similar to those obtained by free trypsin in solution. Enzymatic digestion of proteins was shown to be feasible on a time-scale of seconds and submicromolar concentrations enabling peptide mass mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.     

Photometric titrations with indicators

Various types of photometric titration curves are discussed. If a metal M is titrated conipleximetrically using a metal indicator and the absorbance is plotted vs. the titrant consumed, the inflection point appears at a pM value defined by the equation 3 pM_infl = pM_trans + 2 pM_eqThis expression is valid when M combines in a 1 : 1 ratio with the complexing agent and the indicator and when the indicator concentration is small compared to the total metal concentration.The difference between the pM values at the inflection and equivalence points can be calculated from the equation ΔpM = pM_infl — pM_eq = $\text{1}\text{3}$(pM_trans — pM_eq) = $\text{1}\text{6}$log(C_MK²_MI/K_MY)If the inflection point is taken as the equivalence point, the error arising can be calculated from ΔpM, or more simply, read from a diagram.If transmittance, instead of absorbancc, is plotted as a function of the titrant volume, the inflection point depends on the added amount of indicator. However, at high transmittance values, i.e., at low indicator concentrations, the inflection point of a transmittance curve occurs practically at the same volume of added titrant as the inflection point of an absorbance curve. Rules are given for applying an indicator correction for the amount of metal bound to the indicator at the end-point.The derived equations and discussions can also be applied to acid-base titrations.     

Combinatorial synthesis of benztropine libraries and their evaluation as monoamine transporter inhibitors

A combinatorial synthesis of benztropine analogues is presented. Radical azidonation of 3-benzyloxy-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester 3 to 3-(1-azidobenzyloxy)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester 4 was used as a key step in the synthesis. This step was optimized by adding 10% DMF to the reaction. Reaction of 4 with phenyl magnesium bromide followed by Boc removal and N-methylation gave benztropine 1. Reaction of five-component Grignard reagents with 4 was used to create a two-dimensional library of 25 N-normethylbenztropine analogues. Further reaction of this library with five alkyl bromides was carried out to create a three-dimensional library containing 125 compounds. Screening of the libraries towards binding and inhibition of uptake of the human dopamine (hDAT), serotonin (hSERT) and norepinephrine transporters (hNET) was carried out. None of the synthesized compounds were found to be stronger than benztropine, and none were selective for inhibition of binding over monoamine uptake.     

Mechanism and kinetics of protein transport in chromatographic media studied by confocal laser scanning microscopy. Part I. The interplay of sorbent structure and fluid phase conditions

An experimental study on the interplay of sorbent structure and fluid phase conditions (pH) has been carried out examining adsorption and transport of bovine serum albumin (BSA) and a monoclonal antibody (IgG 2a) on SP Sepharose Fast Flow and SP Sepharose XL. SP Sepharose Fast Flow is characterised by a relatively open pore network, while SP Sepharose XL is a composite structure with ligand-carrying dextran chains filling the pore space. Both adsorbents have similar ionic capacity. Protein transport and adsorption profiles were evaluated using confocal laser scanning microscopy. Under all investigated conditions, BSA uptake could be adequately explained by a pore diffusion mechanism. The adsorption profiles obtained for IgG 2a, however, indicated that changes in fluid phase conditions as well as a change in the solid phase structure could result in a more complex uptake mechanism as compared to pore diffusion alone. This mechanism results in a fast transport of proteins into the adsorbent, followed by an overshoot of protein in the center of the sorbent and a setback towards a homogeneous adsorption profile.     

Capillary isoelectric focusing of proteins utilizing poly(vinylpyrrolidone)- and plexiglas-coated columns

Fused-silica capillaries chemically derivatized with silane/poly(vinylpyrrolidone) (PVP) or dynamically modified with plexiglas [poly(methyl methacrylate)] were prepared and evaluated with regard to column stability and separation performance for capillary isoelectric focusing of standard proteins. The PVP coating showed the better stability and was good for at least 100 runs while the plexiglas coating started to deteriorate after about 30 runs. The time spent for the plexiglas coating is about 40 minutes while the PVP coating requires two days. The migration time reproducibility was better with the PVP capillary (RSD 0.7-1.6%, n = 5) compared to the plexiglas-coated column (RSD 1.2-2.9%, n = 5) while peak area and height varied over a similar interval (RSD 2-28.1% area; 0.9-22.7% height, n = 5). The two most consistent proteins in this evaluation, viz. myoglobin A and carbonic anhydrase II, showed linear dynamic ranges between 5-150 and 5-50 microg/mL, and limits of detections at 2 and 1 microg/mL, respectively, employing UV detection at 280 nm.     

Chemically-assisted fragmentation combined with multi-dimensional liquid chromatography and matrix-assisted laser desorption/ionization post source decay, matrix-assisted laser desorption/ionization tandem time-of flight or matrix-assisted laser desorption/ionization tandem mass spectrometry for improved sequencing of tryptic peptides

Derivatization of tryptic peptides using an Ettan CAF matrix-assisted laser desorption/ionization (MALDI) sequencing kit in combination with MALDI-post source decay (PSD) is a fast, accurate and convenient way to obtain de novo or confirmative peptide sequencing data. CAF (chemically assisted fragmentation) is based on solid-phase derivatization using a new class of water stable sulfonation agents, which strongly improves PSD analysis and simplifies the interpretation of acquired spectra. The derivatization is performed on solid supports, ZipTip(microC18, limiting the maximum peptide amount to 5 microg. By performing the derivatization in solution enabled the labeling of tryptic peptides derived from 100 microg of protein. To increase the number of peptides that could be sequenced, derivatized peptides were purified using multidimensional liquid chromatography (MDLC) prior to MALDI sequencing. Following the first dimension strong cation exchange (SCX) chromatography step, modified peptides were separated using reversed-phase chromatography (RPC). During the SCX clean up step, positively charged peptides are retained on the column while properly CAF-derivatized peptides (uncharged) are not. A moderately complex tryptic digest, prepared from six different proteins of equimolar amounts, was CAF-derivatized and purified by MDLC. Fractions from the second dimension nano RPC step were automatically sampled and on-line dispensed to MALDI sample plates and analyzed using MALDI mass spectrometry fragmentation techniques. All proteins in the derivatized protein mixture digest were readily identified using MALDI-PSD or MALDI tandem mass spectrometry (MS/MS). More than 40 peptides were unambiguously sequenced, representing a seven-fold increase in the number of sequenced peptides in comparison to when the CAF-derivatized protein mix digest was analyzed directly (no MDLC-separation) using MALDI-PSD. In conclusion, MDLC purification of CAF-derivatized peptides significantly increases the success rate for de novo and confirmative sequencing using various MALDI fragmentation techniques. This new approach is not only applicable to single protein digests but also to more complex digests and could, thus, be an alternative to electrospray ionization MS/MS for peptide sequencing.     

Synthesis of Acyclic Nitroazole Nucleosides and Their Incorporation into Oligonucleotides,and Their Duplex and Triplex Formation

Acyclic nucleosides of 4‐nitro‐1H‐imidazole and 4‐nitropyrazole have been synthesized by nucleophilic addition of the appropriate 4‐nitroazole to (?)‐(S)‐(hydroxymethyl)oxirane in the presence of a catalytic amount of potassium carbonate. (+)‐(R)‐3‐(4‐nitro‐1H‐imidazol‐1‐yl)propane‐1,2‐diol and (+)‐(R)‐3‐(2‐methyl‐4‐nitro‐1H‐imidazol‐1‐yl)propane‐1,2‐diol were also obtained in an independent reaction starting from appropriate 1,4‐dinitro‐1H‐imidazole and (+)‐(R)‐3‐aminopropane‐1,2‐diol. (+)‐(R)‐3‐(4‐Nitropyrazol‐1‐yl)propane‐1,2‐diol was also obtained by direct noncatalyzed addition of 4‐nitropyrazole to (?)‐(S)‐(hydroxymethyl)oxirane, whereas the (S)‐enantiomer was obtained by reaction of 4‐nitropyrazole with (+)‐(S)‐1,2‐O‐isopropylideneglycerol under Mitsunobu reaction conditions, followed by a cleavage of the isopropylidene group with 80% AcOH. Racemization during any of these syntheses has not been observed. 3‐(4‐Nitroazol‐1‐yl)propane‐1,2‐diols were incorporated into a 26‐mer oligonucleotide. UV Thermal melting studies of duplexes of the oligonucleotides with 4‐nitropyrazole or 4‐nitro‐1H‐imidazole paired with four natural bases showed moderately decreased stabilities of the duplexes. A narrow range of melting temperatures, typically being within 2° for each acyclic nucleoside, fulfill one of the requirements of using acyclic 4‐nitroazoles as general bases. Single incorporation of 4‐nitroazoles into a 14‐mer triplex forming oligonucleotide resulted in considerably decreased triplex stabilities.     

HPLC Method for Determination of Rosiglitazone in Plasma

A fast and sensitive high performance liquid chromatography method for quantitative determination of rosiglitazone in human plasma has been developed. The extraction from plasma was performed using solid-phase extraction (SPE) on C4 silica (100 mg) disposable extraction cartridges (DEC). The separation of rosiglitazone and two metabolites was achieved on a Phenomenex® Synergi 4 µm MAX-RP (150 × 4.6 mm) column, protected by a guard column. The mobile phase was 0.01 M ammonium acetate, pH 7.0 - acetonitrile (65:35, v/v). (3S)-3-OH-quinidine was used as internal standard. The analytes were detected using fluorescence detection. The method was validated. The limit of quantitation was 1 ng mL⁻¹ and the detection limit was 0.25 ng mL⁻¹ for rosiglitazone in human plasma. The recovery was 90% for rosiglitazone. Linearity was observed over a range of 1-1000 ng mL⁻¹ (r²=0.9959). The intra- and inter-day precision (C.V.) did not exceed 8.7 %. Applicability of the method was demonstrated by a clinical pharmacokinetic study. A healthy volunteer received in two separate phases 4 mg and 8 mg rosiglitazone maleate as a single oral dose. Plasma concentrations were measured for 24 h in both phases.     

Selective Inversion of the Proximal or Distal Hydroxyl Groups in syn,syn-3-[N-(Alkoxycarbonyl)amino] 1,2-Diols via Cyclic Sulfates

The formation of cyclic sulfates (4) from syn,syn-3-[N-(benzyloxycarbonyl)amino] 1,2-diols provides a common intermediate to access other diastereomers via two inversion procedures. Thermolysis of the cyclic sulfates in acetonitrile normally leads to inversion of the distal hydroxyl group to form a 1,3-oxazin-2-one (6). Catalytic hydrogenation of the cyclic sulfates under basic conditions (NEt(3)) results in inversion at the proximal hydroxyl group to form a 1,3-oxazolidin-2-one (5).